MULTI-SUBSTITUENT PSILOCYBIN DERIVATIVES AND METHODS OF USING
20230270768 · 2023-08-31
Inventors
Cpc classification
C12N9/1029
CHEMISTRY; METALLURGY
A61P25/18
HUMAN NECESSITIES
A61K31/675
HUMAN NECESSITIES
A61K31/4045
HUMAN NECESSITIES
A61K31/7028
HUMAN NECESSITIES
C12Y401/01028
CHEMISTRY; METALLURGY
C12Y205/01
CHEMISTRY; METALLURGY
International classification
Abstract
Disclosed are novel multi-substituent psilocybin derivative compounds and pharmaceutical and recreational drug formulations containing the same. The compounds may be produced by reacting a reactant psilocybin derivative with a substituent containing compound.
Claims
1. A chemical compound or a salt thereof having a formula (I): ##STR00361## wherein, at least two of R.sub.2, R.sub.4, R.sub.5, R.sub.6, or R.sub.7 are substituents independently selected from at least two of (i) a halogen atom, (ii) a hydroxy group, (iii) a nitro group, (iv) a glycosyloxy group, (v) an amino group or an N-substituted amino group, (vi) a carboxyl group or a carboxylic acid derivative, (vii) an aldehyde or a ketone group, (viii) a prenyl group, and (ix) a nitrile group, and wherein each nonsubstituted R.sub.2, R.sub.5, R.sub.6, or R.sub.7 is a hydrogen atom and when R.sub.4 is not substituted with any of the foregoing substituents, R.sub.4 is a hydrogen atom, an O-alkyl group, an O-acyl group, or a phosphate group, and wherein R.sub.3a and R.sub.3b are each independently a hydrogen atom, an alkyl group, a cycloalkyl group, an acyl group, or an aryl group, and R.sub.3c is a hydrogen atom or a carboxyl group.
2. A chemical compound according to claim 1, wherein the at least two substituents are selected from a halogen atom and an amino group or substituted amino group.
3. A chemical compound according to claim 1, wherein the at least two substituents are selected from a halogen atom and an amino group or substituted amino group, and wherein at least one of R.sub.4, R.sub.5, and R.sub.6 is substituted.
4. A chemical compound according to claim 1, wherein the at least two substituents are selected from a halogen atom and an amino group or substituted amino group, and wherein either R.sub.4 and R.sub.6 or R.sub.5 and R.sub.6 are substituted, and wherein R.sub.2, and each non substituted R.sub.4, R.sub.5 and R.sub.7 are a hydrogen atom.
5. A chemical compound according to claim 4, wherein the substituted amino group has the formula (VI): ##STR00362## wherein R′ and R″ are each independently selected from a hydrogen atom or an alkyl group.
6. A chemical compound according to claim 5, wherein R″ is a hydrogen atom.
7. A chemical compound according to claim 5, wherein R″ is a hydrogen atom and R′ is an alkyl group.
8. A chemical compound according to claim 5, wherein R″ is a hydrogen atom and R′ is an alkyl group.
9. A chemical compound according to claim 8, wherein R″ is a hydrogen atom and R′ is a (C.sub.1-C.sub.6)-alkyl group.
10. A chemical compound according to claim 8, wherein R″ is a hydrogen atom and R′ is a (C.sub.1-C.sub.3)-alkyl group.
11. A chemical compound according to claim 8, wherein R″ is a hydrogen atom and R′ is a methyl group (–CH.sub.3).
12. A chemical compound according to claim 4, wherein R.sub.4 and R.sub.6 are substituted, R.sub.4 is an amino group or substituted amino group and R.sub.6 is a halogen atom.
13. A chemical compound according to claim 4, wherein R.sub.4 and R.sub.6 are substituted, R.sub.6 is an amino group or substituted amino group and R.sub.4 is a halogen atom.
14. A chemical compound according to claim 4, wherein R.sub.4 and R.sub.6 are substituted, R.sub.4 is an amino group and R.sub.6 is a fluorine atom.
15. A chemical compound according to claim 4, wherein R.sub.4 and R.sub.6 are substituted, R.sub.4 is a fluorine group and R.sub.6 is a substituted alkyl group.
16. A chemical compound according to claim 15, wherein the substituted amino group has the formula (VI): ##STR00363## wherein R′ and R″ are each independently selected from a hydrogen atom or an alkyl group.
17. A chemical compound according to claim 16, wherein R″ is a hydrogen atom.
18. A chemical compound according to claim 16, wherein R″ is a hydrogen atom and R′ is an alkyl group.
19. A chemical compound according to claim 16, wherein R″ is a hydrogen atom and R′ is an alkyl group.
20. A chemical compound according to claim 19, wherein R″ is a hydrogen atom and R′ is a (C.sub.1-C.sub.6)-alkyl group.
21. A chemical compound according to claim 19, wherein R″ is a hydrogen atom and R′ is a (C.sub.1-C.sub.3)-alkyl group.
22. A chemical compound according to claim 19, wherein R″ is a hydrogen atom and R′ is a methyl group (–CH.sub.3).
23. A chemical compound according to claim 1 wherein R.sub.3a and R.sub.3b are a hydrogen atom and an acyl group, and R.sub.3c is a hydrogen atom.
24. A chemical compound according to claim 1 wherein R.sub.3a, R.sub.3b and R.sub.3c are each a hydrogen atom.
25. A chemical compound according to claim 23 wherein the acyl group is a —(C═O)—CH.sub.3 group.
26. A chemical compound according to claim 1, wherein chemical compound (I) is selected from a compound having a chemical formula (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), (XXIII), (XXIV), (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), (XXX), (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), (XXXVI), (XXXVII), (XXXVIII), (XXXIX), (XL), (XLI), (XLII), (XLIII), (XLIV), (XLV), (XLVI), (XLVII), (XLVIII), (XLIX), (L), (LI), (LII), (LIII), (LIV), or (LV): ##STR00364## ##STR00365## ##STR00366## ##STR00367## ##STR00368## ##STR00369## ##STR00370## ##STR00371## ##STR00372## ##STR00373## ##STR00374## ##STR00375## ##STR00376## ##STR00377## ##STR00378## ##STR00379## ##STR00380## ##STR00381## ##STR00382## ##STR00383## ##STR00384## ##STR00385## ##STR00386## ##STR00387## ##STR00388## ##STR00389## ##STR00390## ##STR00391## ##STR00392## ##STR00393## ##STR00394## ##STR00395## ##STR00396## ##STR00397## ##STR00398## ##STR00399## ##STR00400## ##STR00401## ##STR00402## ##STR00403## ##STR00404## ##STR00405## ##STR00406## ##STR00407## ##STR00408## ##STR00409## ##STR00410## and ##STR00411## .
27. A chemical compound according to claim 1, wherein chemical compound (I) is selected from a compound having a chemical formula (IX), (X), (XV), (XVI), ( (XXI), (XXII), (XXV), or (XXVI): ##STR00412## ##STR00413## ##STR00414## ##STR00415## ##STR00416## ##STR00417## ##STR00418## and ##STR00419## .
28. A chemical compound according to claim 1, wherein the compound is at least about 95% (w/w) pure.
29. A pharmaceutical or recreational drug formulation comprising an effective amount of a chemical compound according to claim 1, together with a pharmaceutically acceptable excipient, diluent, or carrier.
30. A method for treating a psychiatric disorder, the method comprising administering to a subject in need thereof a pharmaceutical formulation comprising a chemical compound according to claim 1, wherein the pharmaceutical formulation is administered in an effective amount to treat the psychiatric disorder in the subject.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0323] The disclosure is in the hereinafter provided paragraphs described, by way of example, in relation to the attached figures. The figures provided herein are provided for a better understanding of the example embodiments and to show more clearly how the various embodiments may be carried into effect. The figures are not intended to limit the present disclosure.
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[0384] The figures together with the following detailed description make apparent to those skilled in the art how the disclosure may be implemented in practice.
DETAILED DESCRIPTION
[0385] Various compositions, systems or processes will be described below to provide an example of an embodiment of each claimed subject matter. No embodiment described below limits any claimed subject matter and any claimed subject matter may cover processes, compositions or systems that differ from those described below. The claimed subject matter is not limited to compositions, processes or systems having all of the features of any one composition, system or process described below or to features common to multiple or all of the compositions, systems or processes described below. It is possible that a composition, system, or process described below is not an embodiment of any claimed subject matter. Any subject matter disclosed in a composition, system or process described below that is not claimed in this document may be the subject matter of another protective instrument, for example, a continuing patent application, and the applicant(s), inventor(s) or owner(s) do not intend to abandon, disclaim or dedicate to the public any such subject matter by its disclosure in this document.
[0386] As used herein and in the claims, the singular forms, such “a”, “an” and “the” include the plural reference and vice versa unless the context clearly indicates otherwise. Throughout this specification, unless otherwise indicated, “comprise,” “comprises” and “comprising” are used inclusively rather than exclusively, so that a stated integer or group of integers may include one or more other non-stated integers or groups of integers.
[0387] Various compositions, systems or processes will be described below to provide an example of an embodiment of each claimed subject matter. No embodiment described below limits any claimed subject matter and any claimed subject matter may cover processes, compositions or systems that differ from those described below. The claimed subject matter is not limited to compositions, processes or systems having all of the features of any one composition, system or process described below or to features common to multiple or all of the compositions, systems or processes described below. It is possible that a composition, system, or process described below is not an embodiment of any claimed subject matter. Any subject matter disclosed in a composition, system or process described below that is not claimed in this document may be the subject matter of another protective instrument, for example, a continuing patent application, and the applicant(s), inventor(s) or owner(s) do not intend to abandon, disclaim or dedicate to the public any such subject matter by its disclosure in this document.
[0388] When ranges are used herein for physical properties, such as molecular weight, or chemical properties, such as chemical formulae, all combinations and sub-combinations of ranges and specific embodiments therein are intended to be included. Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range may vary between 1% and 15% of the stated number or numerical range, as will be readily recognized by context. Furthermore any range of values described herein is intended to specifically include the limiting values of the range, and any intermediate value or sub-range within the given range, and all such intermediate values and sub-ranges are individually and specifically disclosed (e.g., a range of 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). Similarly, other terms of degree such as “substantially” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of the modified term if this deviation would not negate the meaning of the term it modifies.
[0389] Unless otherwise defined, scientific and technical terms used in connection with the formulations described herein shall have the meanings that are commonly understood by those of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.
[0390] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
Terms and Definitions
[0391] The term “psilocybin”, refers to a chemical compound having the structure set forth in
[0392] The term “indole prototype structure” refers to the chemical structure shown in
[0393] The terms “psilocybin derivative”, as used herein, refers to compounds that can be derivatized from psilocybin, wherein such compounds include an indole prototype structure and a C.sub.3 ethylamine or ethylamine derivative group having the formula (LXXVIII):
##STR00151##
wherein R.sub.3a and R.sub.3b are each independently a hydrogen atom, an alkyl group, a cycloalkyl group, an acyl group, or an aryl group, and wherein and R.sub.3c is a hydrogen atom or a carboxyl group. Psilocybin derivatives include compounds containing one or more substituents at each of C.sub.2, C.sub.4, C.sub.5, C.sub.6 and C.sub.7. Thus, in formula (LXXVIII), R.sub.2, R.sub.4, R.sub.5, R.sub.6 and R.sub.7 can each be, for example, any of (i) a halogen atom, (ii) a hydroxy group, (iii) a nitro group, (iv) a glycosyloxy group, (v) an amino group or an N-substituted amino group, (vi) a carboxyl group or a carboxylic acid derivative, (vii) an aldehyde or a ketone group, (viii) a prenyl group, and (ix) a nitrile group, (x) an O-alkyl group, (xi) an (xii) O-acyl group, (xiii) a phosphate group, or (xiv) a hydrogen atom.
[0394] The term “multiple-substituent psilocybin derivative” refers to a psilocybin derivative compound wherein two or more substituent entities have been bonded to psilocybin or a psilocybin derivative. Reference may be made to specific carbon atoms which may be substituted. Furthermore the substituent entities may be referred to as S1, S2, S3 or S4, wherein each of S1, S2, S3 and S4 refer to a different substituent entity. For example, a 5,7-S1,S2-di-psilocybin derivative refers to a psilocybin derivative in which carbon atom number 5 and carbon number 7 (as identified in the indole prototype structure) each possess a different substituent entity, or, similarly, a 2,5,6-tri-S1,S2,S3-psilocybin derivative refers to a psilocybin derivative in which carbon atom number 2,5,6 (as identified in the indole prototype structure) possess a different substituent entity (or at least two of the three substituents are different). By way of another example, a 2,5,6-tri-S1,S2,S2-psilocybin derivative refers to a psilocybin derivative in which carbon atom number 2,5,6 (as identified in the indole prototype structure) each possess a substituent entity, the substituent entity possessed by carbon atom number 5 and 6 being the same. It is noted that S1, S2, S3 and S4 can herein additionally include numerical subscripts, such as S1.sub.5, S3.sub.6, S4.sub.7 etc. Where such numerical values are included, they reference the numbered C atom of the prototype indole structure. Thus, for example, S1.sub.5 is a substituent entity extending from the C.sub.5 atom of the indole ring structure, S3.sub.7 is a substituent entity extending the C.sub.7 atom of the indole ring structure, and so forth. The term multiple-substituent psilocybin derivatives further includes chemical compounds having a chemical formula (I):
##STR00152##
wherein, at least two of R.sub.2, R.sub.4, R.sub.5, R.sub.6, or R.sub.7 are substituents independently selected from at least two of (i) a halogen atom, (ii) a hydroxy group, (iii) a nitro group, (iv) a glycosyloxy group, (v) an amino group or an N-substituted amino group, (vi) a carboxyl group or a carboxylic acid derivative, (vii) an aldehyde or a ketone group, (viii) a prenyl group, and (ix) a nitrile group, and wherein each non-substituted R.sub.2, R.sub.5, R.sub.6, or R.sub.7 is a hydrogen atom and when R.sub.4 is not substituted with any of the foregoing substituents, R.sub.4 is a hydrogen atom, O-alkyl group, an O-acyl group, or a phosphate group, wherein R.sub.3a and R.sub.3b are each independently a hydrogen atom, an alkyl group, a cycloalkyl group, an acyl group, or an aryl group, and wherein and R.sub.3c is a hydrogen atom or a carboxyl group. The term multiple-substituent psilocybin derivatives, further also includes compounds having a formula (IV):
##STR00153##
wherein, at least two of R.sub.2, R.sub.5, R.sub.6, or R.sub.7 are substituents independently selected from at least two of (i) a halogen atom, (ii) a hydroxy group, (iii) a nitro group, (iv) a glycosyloxy group, (v) an amino group or an N-substituted amino group, (vi) a carboxyl group or a carboxylic acid derivative, (vii) an aldehyde or a ketone group, (viii) a prenyl group, and (ix) a nitrile group, and wherein each non-substituted R.sub.2, R.sub.5, R.sub.6, or R.sub.7 is a hydrogen atom, wherein R.sub.3a and R.sub.3b are each independently a hydrogen atom, an alkyl group, a cycloalkyl group, an acyl group, or an aryl group, and wherein and R.sub.3c is a hydrogen atom or a carboxyl group. The term further includes salts of multiple-substituent psilocybins, such as a sodium salt, a potassium salt etc.
[0395] The terms “halogen”, “halogenated” and “halo-”, as used herein, refer to the class of chemical elements consisting of fluorine (F), chlorine (CI), bromine (Br), and iodine (I). Accordingly, halogenated compounds can refer to “fluorinated”, “chlorinated”, “brominated”, or “iodinated” compounds.
[0396] The terms “phosphate group” or “phospho group”, as used herein, is a molecule containing one atom of phosphorus, covalently bound to four oxygen atoms (three single bonds and one double bond). Of the four oxygen atoms one oxygen atom may be a hydroxy group, and one of the non-hydroxylated oxygen atom may be chemically bonded to another entity.
[0397] The terms “hydroxy group”, and “hydroxy”, as used herein refers to a molecule containing one atom of oxygen bonded to one atom of hydrogen, and having the formula —OH. A hydroxy group through its oxygen atom may be chemically bonded to another entity.
[0398] The term “nitro group” and “nitro”, as used herein refers to a molecule containing one atom of nitrogen bonded to two atoms of oxygen and having the formula —NO.sub.2. A nitro group through its nitrogen atom may be chemically bonded to another entity. Furthermore, it is noted that an entity attached to a nitro group may be referred to herein as a “nitrated” entity, e.g., a nitrated psilocybin derivative is a psilocybin derivative possessing a nitro group.
[0399] The term “amino group” and “amino”, as used herein refers to a molecule containing one atom of nitrogen bonded to hydrogen atoms and having the formula —NH.sub.2. An amino group also may be protonated and having the formula —NH.sub.3.sup.+. An amino group through its nitrogen atom may be chemically bonded to another entity. Furthermore, it is noted that an entity attached to an amino group may be referred to herein as an “aminated” entity, e.g., an aminated psilocybin derivative is a psilocybin derivative possessing either an amino group or a N-substituted amino group.
[0400] The term “N-substituted amino group”, as used herein, refers to an amino group wherein at least one of the hydrogen atoms has been substituted by another atom or group, such as, for example, an alkyl group, an acyl group, an aryl group a sulfonyl group etc. An N-substituted amino group also may be protonated, and the amino group through its nitrogen atom may be chemically bonded to another entity. Thus, N-substituted amino group may be represented herein as:
##STR00154##
Furthermore N-substituted amino groups include: [0401] chemical group (IV) (an alkyl group, an aryl group): wherein R′ and R″ are each independently selected from a hydrogen atom, an alkyl group, and an aryl group, provided however that at least one of R′, and R″ is not a hydrogen atom; [0402] chemical group (V): wherein R′, R″ and R‴ are each independently selected from a hydrogen atom, an alkyl group, and an aryl group, provided however that at least one of R′, R″, and R‴ is not a hydrogen atom; [0403] chemical group (VI) (an acyl group): wherein R′ and R″ are each independently selected from a hydrogen atom, an alkyl group and an aryl group; [0404] chemical group (VII) (a sulfonyl group): wherein R′, and R″ are each independently selected from a hydrogen atom, an alkyl group and an aryl group; and [0405] chemical group (VIII) (a sulfonate group): wherein R″ is selected from a hydrogen atom, an alkyl group, and an aryl group. The nitrogen atom of chemical groups (VI), (VII) and (VIII) can also be positively charged and be further substituted with H, or R‴. It is noted that R′, R″ and R‴ can herein additionally include numerical subscripts, such as .sub.5a, .sub.6b, .sub.7b etc., and be represented, for example, as R′.sub.5a, R″.sub.6b or R‴.sub.7a, respectively. Where such numerical values are included, they reference chemical entity extending from the amino group extending in turn from the thus numbered C atom of the prototype indole structure. Thus, for example, R′.sub.5a is a chemical entity extending from an aminated group attached to the C.sub.5 atom of the indole ring structure, R′.sub.2a is a chemical entity extending from an aminated group attached to the C.sub.2 atom of the indole ring structure, and so forth. Furthermore, it is noted that an entity attached to an N-substituted amino group may be referred to herein as an “aminated” entity, e.g., an aminated psilocybin derivative is a psilocybin derivative possessing either an amino group or a N-substituted amino group.
[0406] The terms “carboxyl group”, “carboxyl”, and “carboxy”, as used herein, refer to a molecule containing one atom of carbon bonded to an oxygen atom and a hydroxy group and having the formula —COOH. A carboxyl group includes a deprotonated carboxyl group, i.e., a carboxyl ion, having the formula —COO.sup.-. In its deprotonated form a carboxyl group may form a carboxyl salt, for example, a sodium or potassium carboxyl salt, or an organic carboxyl salt, all of which may be represented herein as COO.sup.-M.sup.+. It is further to be understood that a carboxyl group through its carbon atom may be chemically bonded to another entity. Furthermore, it is noted that an entity attached to a carboxyl group may be referred to herein as a “carboxylated” entity, e.g., a carboxylated psilocybin derivative is a psilocybin derivative possessing either a carboxyl group or a OH-substituted carboxyl group.
[0407] The term “carboxylic acid derivative”, as used herein, refers to a carboxyl group wherein the hydroxy group of the carboxyl group has been substituted by another atom or group, such as, for example, an —OR″ group or an —NR′R″ group. Thus a carboxylic acid derivative includes chemical group (III):
##STR00160##
wherein, R′, is an alkyl group, an aryl group and a hydrogen atom. It is noted that chemical group (III) is an ester. It is noted that R′ can herein additionally include numerical subscripts, such as .sub.3c, .sub.6b, .sub.7b etc., and be represented, for example, as R′.sub.3c, R′.sub.6b or R′.sub.7a, respectively. Where such numerical values are included, they reference chemical entity extending from the carboxyl group extending in turn from the thus numbered C atom of the prototype indole structure. Thus, for example, R′.sub.5a is a chemical entity extending from a carboxylated group attached to the C.sub.5 atom of the indole ring structure, R′.sub.2a is a chemical entity extending from a carboxylated group attached to the C.sub.2 atom of the indole ring structure, and so forth. Furthermore, it is noted that an entity attached to a carboxylic acid derivative may be referred to herein as an “carboxylated” entity, e.g., a carboxylated psilocybin derivative is a psilocybin derivative possessing either a carboxyl group or an OH-substituted carboxyl group.
[0408] The terms “aldehyde” or “aldehyde group”, as used herein, refers to a molecule containing one atom of carbon double bonded to an oxygen atom, and bonded to a hydrogen atom, and having the chemical formula:
##STR00161##
which may, further alternatively be represented herein as —CHO. A —CHO group may also by referred to herein as a formyl group. It is to be understood that an aldehyde through its carbon atom may be chemically bonded to another entity.
[0409] The terms “ketone” or “ketone group”, as used herein, refer to a molecule containing two atoms of carbon, a first carbon atom double bonded to an oxygen atom, and the first carbon further bonded to a second carbon atom, the molecule having the chemical formula:
##STR00162##
wherein R is any entity or plurality of entities which taken together allow the carbon atom bonded to R to achieve its ordinary valency. Thus, for example, R may represent 3 hydrogen atoms, or R may represent 2 hydrogen atoms and a methyl group. It is to be understood that a ketone through its first carbon atom may be chemically bonded to another entity, such as an alkylene group (C.sub.1-C.sub.6)-alkylene.
[0410] The term “nitrile group” and “nitrile”, as used herein, refer to a molecule containing one atom of carbon bonded to a nitrogen atom and having the formula
##STR00163##
It is to be understood that a nitrile group through its carbon atom may be chemically bonded to another entity. Furthermore, it is noted that an entity attached to a nitrile group may be referred to herein as a “nitrilated” entity, e.g., a nitrilated psilocybin derivative is a psilocybin derivative possessing a nitrile group.
[0411] The terms “glycosylated” or “glycosyl”, as used herein, refer to a saccharide group, such as a mono-, di-, tri- oligo- or a poly-saccharide group, which can be or has been bonded from its anomeric carbon either in the pyranose or furanose form, either in the α or the β conformation. When bonded through its anomeric carbon via an oxygen atom to another entity, the bonded saccharide group, inclusive of the oxygen atom, may be referred to herein as a “glycosyloxy” group. Example monosaccharide groups include, but are not limited to, a pentosyl, a hexosyl, or a heptosyl group. The glycosyloxy group may also be substituted with various groups. Such substitutions may include lower alkyl, lower alkoxy, acyl, carboxy, carboxyamino, amino, acetamido, halo, thio, nitro, keto, and phosphatyl groups, wherein the substitution may be at one or more positions on the saccharide, Included in the term glycosyl are further stereoisomers, optical isomers, anomers, and epimers of the glycosyloxy group. Thus, a hexose group, for example, can be either an aldose or a ketose group, can be of D- or L-configuration, can assume either an α or β conformation, and can be a dextro- or levo-rotatory with respect to plane-polarized light. Example glycosyloxy groups further include, without limitation, glucosyl groups, glucuronic acid groups, galactosyl groups, fucosyl groups, xylose groups, arabinose groups, and rhamnose groups.
[0412] The terms “prenyl group”, and “prenyl”, as used herein refers to a chemical group having the structure (LVI):
##STR00164##
and further includes poly-prenyl compounds having the structure:
##STR00165##
Wherein n is an integer having a value of 2 or more, e.g., 2, 3, 4, 5, etc. Furthermore, the term “prenyl compound” refers to a chemical compound being, substantially being, or possessing a reactive prenyl group, i.e., a prenyl group that may be received by another entity. Prenyl compounds include, for example, geranyl pyrophosphate (GPP), dimethylallyl diphosphate (DMAPP), farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP).
[0413] The term “alkyl group”, as used herein, refers to a straight and/or branched chain, saturated alkyl radical containing from one to “p” carbon atoms (“C.sub.1-C.sub.p-alkyl”) and includes, depending on the identity of “p”, methyl, ethyl, propyl, isopropyl, n-butyl, s-butyl, isobutyl, t-butyl, 2,2-dimethylbutyl, n-pentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, n-hexyl, and the like, where the variable p is an integer representing the largest number of carbon atoms in the alkyl radical. Alkyl groups further include hydrocarbon groups arranged in a chain having the chemical formula —CnH.sub.2n+1, including, without limitation, methyl groups (—CH.sub.3), ethyl groups (—C.sub.2H.sub.5), propyl groups (—C.sub.3H.sub.7), and butyl groups (—C.sub.4H.sub.9), further also includes cyclic alkyl groups, including cyclo-propane, cyclo-butane, cyclo-pentane, cyclo-hexane, and cyclo-heptane.
[0414] The term “cycloalkyl” refers to cyclic alkyl groups, including (C.sub.3-C.sub.20), (C.sub.3-C.sub.10), and (C.sub.3-C.sub.6) cycloalkyl groups, and further including cyclo-propane, cyclo-butane, cyclo-pentane, cyclo-hexane, and cyclo-heptane.
[0415] The term “O-alkyl group”, as used herein, refers to a hydrocarbon group arranged in a chain having the chemical formula —O—C.sub.nH.sub.2n+.sub.1. O-alkyl groups include, without limitation, O-methyl groups (—O—CH.sub.3), O-ethyl groups (—O—C.sub.2H.sub.5), O-propyl groups (—O—C.sub.3H.sub.7) and O-butyl groups (—O—C.sub.4H.sub.9).
[0416] The term “aryl group”, as used herein, refers to a hydrocarbon group arranged in an aromatic ring and can, for example, be a C.sub.6-C.sub.14-aryl, a C.sub.6-C.sub.10-aryl. Aryl groups further include phenyl, naphthyl, tetrahydronaphthyl, phenanthrenyl, biphenylenyl, indanyl, tolyl, xylyl, or indenyl groups, and the like.
[0417] The term “acyl group”, as used herein, refers to a carbon atom double bonded to an oxygen and single bonded to an alkyl group. The carbon atom further can be bonded to another entity. An acyl group can be described by the chemical formula: —C(═O)—C.sub.nH.sub.2n+1.
[0418] The term “O-acyl group”, as used herein, refers to an acyl group in which the carbon atom is single bonded to an additional oxygen atom. The additional oxygen atom can be bonded to another entity. An O-acyl group can be described by the chemical formula: —O—C(═O)—C.sub.nH.sub.2n+1. Furthermore, depending on the carbon chain, length specific O-acyl groups may be termed an acetoxy group (n=1), a propanoyloxy group (n=2), butyryloxy group (n=3), a pentanoyloxy group (n=4) etc.
[0419] The term “alcohol group” or “hydroxylalkyl”, as used herein, refers to a hydrocarbon group arranged in a chain having the chemical formula C.sub.nH.sub.n+.sub.1OH. Depending on the carbon chain, length specific alcohol groups may be termed a methanol group (n=1) or hydroxymethyl, an ethanol group (n=2) or hydroxyethyl, a propanol group (n=3) or hydroxypropyl, a butanol group (n=4) or hydroxybutyl etc.
[0420] The term “5-HT.sub.2A receptor”, as used herein, refers to a subclass of a family of receptors for the neurotransmitter and peripheral signal mediator serotonin. 5-HT.sub.2A receptors can mediate a plurality of central and peripheral physiologic functions of serotonin. Central nervous system effects can include mediation of hallucinogenic effects of hallucinogenic compounds.
[0421] The term “modulating 5-HT.sub.2A receptors”, as used herein, refers to the ability of a compound disclosed herein to alter the function of 5-HT.sub.2A receptors. A 5-HT.sub.2A receptor modulator may activate the activity of a 5-HT.sub.2A receptor, may activate or inhibit the activity of a 5-HT.sub.2A receptor depending on the concentration of the compound exposed to the 5-HT.sub.2A receptor, or may inhibit the activity of a 5-HT.sub.2A receptor. Such activation or inhibition may be contingent on the occurrence of a specific event, such as activation of a signal transduction pathway, and/or maybe manifest only in particular cell types. The term “modulating 5-HT.sub.2A receptors,” also refers to altering the function of a 5-HT.sub.2A receptor by increasing or decreasing the probability that a complex forms between a 5-HT.sub.2A receptor and a natural binding partner to form a multimer. A 5-HT.sub.2A receptor modulator may increase the probability that such a complex forms between the 5-HT.sub.2A receptor and the natural binding partner, may increase or decrease the probability that a complex forms between the 5-HT.sub.2A receptor and the natural binding partner depending on the concentration of the compound exposed to the 5-HT.sub.2A receptor, and or may decrease the probability that a complex forms between the 5-HT.sub.2A receptor and the natural binding partner. It is further noted that the prenylated psilocybin derivatives may alter the function of a 5-HT.sub.2A receptor by acting as an agonist or antagonist of the 5-HT.sub.1A receptor, and that prenylated psilocybin derivatives according to the present disclosure may alter the function of a 5-HT.sub.2A receptor by directly interacting therewith or binding thereto, or by indirectly interacting therewith through one or more other molecular entities.
[0422] The term “5-HT.sub.2A receptor-mediated disorder”, as used herein, refers to a disorder that is characterized by abnormal 5-HT.sub.2A receptor activity. A 5-HT.sub.2A receptor-mediated disorder may be completely or partially mediated by modulating 5-HT.sub.2A receptors. In particular, a 5-HT.sub.2A receptor-mediated disorder is one in which modulation of 5-HT.sub.2A receptors results in some effect on the underlying disorder e.g., administration of a 5-HT.sub.2A receptor modulator results in some improvement in at least some of the subjects being treated.
[0423] The term “5-HT.sub.1A receptor”, as used herein, refers to a subclass of a family of receptors for the neurotransmitter and peripheral signal mediator serotonin. 5-HT.sub.1A receptors can mediate a plurality of central and peripheral physiologic functions of serotonin. Ligand activity at 5-HT.sub.1A is generally not associated with hallucination, although many hallucinogenic compounds are known to modulate 5-HT.sub.1A receptors to impart complex physiological responses (Inserra et al., 2020, Pharmacol Rev 73: 202).
[0424] The term “modulating 5-HT.sub.1A receptors”, as used herein, refers to the ability of a compound disclosed herein to alter the function of 5-HT.sub.1A receptors. A 5-HT.sub.1A receptor modulator may activate the activity of a 5-HT.sub.1A receptor, may activate or inhibit the activity of a 5-HT.sub.1A receptor depending on the concentration of the compound exposed to the 5-HT.sub.1A receptor, or may inhibit the activity of a 5-HT.sub.1A receptor. Such activation or inhibition may be contingent on the occurrence of a specific event, such as activation of a signal transduction pathway, and/or maybe manifest only in particular cell types. The term “modulating 5-HT.sub.1A receptors,” also refers to altering the function of a 5-HT.sub.1A receptor by increasing or decreasing the probability that a complex forms between a 5-HT.sub.1A receptor and a natural binding partner to form a multimer. A 5-HT.sub.1A receptor modulator may increase the probability that such a complex forms between the 5-HT.sub.1A receptor and the natural binding partner, may increase or decrease the probability that a complex forms between the 5-HT.sub.1A receptor and the natural binding partner depending on the concentration of the compound exposed to the 5-HT.sub.1A receptor, and or may decrease the probability that a complex forms between the 5-HT.sub.1A receptor and the natural binding partner. It is further noted that the prenylated psilocybin derivatives may alter the function of a 5-HT.sub.1A receptor by acting as an agonist or antagonist of the 5-HT.sub.1A receptor, and that prenylated psilocybin derivatives according to the present disclosure may alter the function of a 5-HT.sub.1A receptor by directly interacting therewith or binding thereto, or by indirectly interacting therewith through one or more other molecular entities.
[0425] The term “5-HT.sub.1A receptor-mediated disorder”, as used herein, refers to a disorder that is characterized by abnormal 5-HT.sub.1A receptor activity. A 5-HT.sub.1A receptor-mediated disorder may be completely or partially mediated by modulating 5-HT.sub.1A receptors. In particular, a 5-HT.sub.1A receptor-mediated disorder is one in which modulation of 5-HT.sub.1A receptors results in some effect on the underlying disorder e.g., administration of a 5-HT.sub.1A receptor modulator results in some improvement in at least some of the subjects being treated.
[0426] The term “reactant psilocybin derivative compound”, as used herein, refers to a psilocybin derivative compound capable of reacting in a synthetic or biosynthetic reaction to thereby form another psilocybin derivative compound, and generally includes indole structure containing reactants. The term “reactant psilocybin derivative compound” includes the term “psilocybin derivative precursor compound”.
[0427] The term “psilocybin derivative precursor compound”, as used herein, refers to a chemical compound that may serve as a precursor compound in the synthesis or biosynthesis of a multi-substituent psilocybin derivative, and includes compounds comprising an indole prototype structure, including, for example, tryptophan and tryptamine, and further includes a psilocybin derivative precursor compound having a formula (LVII):
##STR00166##
wherein at least one of R.sub.2, R.sub.4, R.sub.5, R.sub.6, or R.sub.7 is a substituent selected from (i) a halogen atom, (ii) a hydroxy group, (iii) a nitro group, (iv) a glycosyloxy group, (v) an amino group or an N-substituted amino group, (vi) a carboxyl group or a carboxylic acid derivative, (vii) an aldehyde or a ketone group, (viii) a prenyl group, and (ix) a nitrile group, and wherein each non-substituted R.sub.2, R.sub.5, R.sub.6, or R.sub.7 is a hydrogen atom and when R.sub.4 is not substituted with any of the foregoing substituents, R.sub.4 is a hydrogen atom, an O-alkyl group, an O-acyl group, or a phosphate group, and wherein R.sub.3 is a hydrogen atom or —CH.sub.2—CHNH.sub.2COOH or —CH.sub.2—CH.sub.2NH.sub.2.
[0428] The term “psilocybin biosynthetic enzyme complement”, as used herein, refers to one or more polypeptides which alone or together are capable of facilitating the chemical conversion of a psilocybin derivative precursor compound, and form a multi-substituent psilocybin derivative compound. A psilocybin biosynthetic enzyme complement can include, for example, one or more of a tryptophan synthase B polypeptide, a tryptophan decarboxylase, an N-acetyl transferase, a N-methyl transferase and a prenyl transferase.
[0429] The term “tryptophan synthase B polypeptide”, as used herein, refers to any and all enzymes comprising a sequence of amino acid residues which is (i) substantially identical to the amino acid sequences constituting any tryptophan synthase B polypeptide set forth herein, including, for example, SEQ.ID NO: 2, or (ii) encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any tryptophan synthase B polypeptide set forth herein, but for the use of synonymous codons.
[0430] The term “tryptophan decarboxylase”, as used herein, refers to any and all enzymes comprising a sequence of amino acid residues which is (i) substantially identical to the amino acid sequences constituting any tryptophan decarboxylase polypeptide set forth herein, including, for example, SEQ.ID NO: 4, SEQ.ID NO: 6 and SEQ.ID NO: 8, or (ii) encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any tryptophan decarboxylase set forth herein, but for the use of synonymous codons.
[0431] The term “N-acetyl transferase”, as used herein, refers to any and all enzymes comprising a sequence of amino acid residues which is (i) substantially identical to the amino acid sequences constituting any acetyl transferase polypeptide set forth herein, including, for example, SEQ.ID NO: 10, or (ii) encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any acetyl transferase set forth herein, but for the use of synonymous codons.
[0432] The term “N-methyl transferase”, as used herein, refers to any and all enzymes comprising a sequence of amino acid residues which is (i) substantially identical to the amino acid sequences constituting any N-methyl transferase polypeptide set forth herein, including, for example, SEQ.ID NO: 12 and SEQ.ID NO: 14, or (ii) encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any N-methyl transferase set forth herein, but for the use of synonymous codons.
[0433] The term “prenyl transferase”, as used herein, refers to any and all enzymes comprising a sequence of amino acid residues which is (i) substantially identical to the amino acid sequences constituting any prenyl transferase polypeptide set forth herein, including, for example, SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID NO: 20, and SEQ.ID NO: 22, or (ii) encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any prenyl transferase set forth herein, but for the use of synonymous codons.
[0434] The term “PsiH”, as used herein, refers to any and all enzymes comprising a sequence of amino acid residues which is (i) substantially identical to the amino acid sequences constituting any PsiH polypeptide set forth herein, including, for example, SEQ.ID NO: 24, or (ii) encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any PsiH set forth herein, but for the use of synonymous codons.
[0435] The term “CPR”, as used herein, refers to any and all enzymes comprising a sequence of amino acid residues which is (i) substantially identical to the amino acid sequences constituting any CPR polypeptide set forth herein, including, for example, SEQ.ID NO: 26, or (ii) encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any CPR set forth herein, but for the use of synonymous codons.
[0436] The term “PsiK”, as used herein, refers to any and all enzymes comprising a sequence of amino acid residues which is (i) substantially identical to the amino acid sequences constituting any PsiK set forth herein, including, for example, SEQ.ID NO: 49, or (ii) encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any PsiK set forth herein, but for the use of synonymous codons.
[0437] The terms “nucleic acid sequence encoding tryptophan synthase B polypeptide”, and “nucleic acid sequence encoding a tryptophan synthase B polypeptide”, as may be used interchangeably herein, refer to any and all nucleic acid sequences encoding a tryptophan synthase B polypeptide, including, for example, SEQ.ID NO: 1. Nucleic acid sequences encoding a tryptophan synthase B polypeptide further include any and all nucleic acid sequences which (i) encode polypeptides that are substantially identical to the tryptophan synthase B polypeptide sequences set forth herein; or (ii) hybridize to any tryptophan synthase B polypeptide nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
[0438] The terms “nucleic acid sequence encoding tryptophan decarboxylase”, and “nucleic acid sequence encoding a tryptophan decarboxylase polypeptide”, as may be used interchangeably herein, refer to any and all nucleic acid sequences encoding a tryptophan decarboxylase polypeptide, including, for example, SEQ.ID NO: 3, SEQ.ID NO: 5 and SEQ.ID NO: 7. Nucleic acid sequences encoding a tryptophan decarboxylase polypeptide further include any and all nucleic acid sequences which (i) encode polypeptides that are substantially identical to the tryptophan decarboxylase polypeptide sequences set forth herein; or (ii) hybridize to any tryptophan decarboxylase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
[0439] The terms “nucleic acid sequence encoding an N-acetyl transferase”, and “nucleic acid sequence encoding an N-acetyl transferase polypeptide”, as may be used interchangeably herein, refer to any and all nucleic acid sequences encoding an N-acetyl transferase polypeptide, including, for example, SEQ.ID NO: 9. Nucleic acid sequences encoding an N-acetyl transferase polypeptide further include any and all nucleic acid sequences which (i) encode polypeptides that are substantially identical to the N-acetyl transferase polypeptide sequences set forth herein; or (ii) hybridize to any N-acetyl transferase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under atleast moderately stringent conditions but for the use of synonymous codons.
[0440] The terms “nucleic acid sequence encoding N-methyl transferase”, and “nucleic acid sequence encoding a N-methyl transferase polypeptide”, as may be used interchangeably herein, refer to any and all nucleic acid sequences encoding a N-methyl transferase polypeptide, including, for example, SEQ.ID NO: 11 and SEQ.ID NO: 13. Nucleic acid sequences encoding a N-methyl transferase polypeptide further include any and all nucleic acid sequences which (i) encode polypeptides that are substantially identical to the N-methyl transferase polypeptide sequences set forth herein; or (ii) hybridize to any N-methyl transferase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
[0441] The terms “nucleic acid sequence encoding a prenyl transferase”, and “nucleic acid sequence encoding a prenyl transferase polypeptide”, as may be used interchangeably herein, refer to any and all nucleic acid sequences encoding a prenyl transferase polypeptide, including, for example, SEQ.ID NO: 15, SEQ.ID NO: 17, SEQ.ID NO; 19 and SEQ.ID NO: 21. Nucleic acid sequences encoding a prenyl transferase polypeptide further include any and all nucleic acid sequences which (i) encode polypeptides that are substantially identical to the prenyl transferase polypeptide sequences set forth herein; or (ii) hybridize to any prenyl transferase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
[0442] The terms “nucleic acid sequence encoding PsiH”, and “nucleic acid sequence encoding a PsiH polypeptide”, as may be used interchangeably herein, refer to any and all nucleic acid sequences encoding a PsiH, including, for example, SEQ.ID NO: 23. Nucleic acid sequences encoding a PsiH polypeptide further include any and all nucleic acid sequences which (i) encode polypeptides that are substantially identical to the PsiH polypeptide sequences set forth herein; or (ii) hybridize to any PsiH nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
[0443] The terms “nucleic acid sequence encoding CPR”, and “nucleic acid sequence encoding an CPR polypeptide”, as may be used interchangeably herein, refer to any and all nucleic acid sequences encoding a CPR, including, for example, SEQ.ID NO: 25. Nucleic acid sequences encoding a CPR polypeptide further include any and all nucleic acid sequences which (i) encode polypeptides that are substantially identical to the CPR polypeptide sequences set forth herein; or (ii) hybridize to any CPR nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
[0444] The terms “nucleic acid sequence encoding a PsiK”, and “nucleic acid sequence encoding a PsiK polypeptide”, as may be used interchangeably herein, refer to any and all nucleic acid sequences encoding PsiK, including, for example, SEQ.ID NO: 48. Nucleic acid sequences encoding a PsiK further include any and all nucleic acid sequences which (i) encode polypeptides that are substantially identical to the PsiK polypeptide sequences set forth herein; or (ii) hybridize to any PsiK nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
[0445] The terms “nucleic acid”, or “nucleic acid sequence”, as used herein, refer to a sequence of nucleoside or nucleotide monomers, consisting of naturally occurring bases, sugars and intersugar (backbone) linkages. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof. The nucleic acids of the present disclosure may be deoxyribonucleic nucleic acids (DNA) or ribonucleic acids (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine, and uracil. The nucleic acids may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil, and xanthine and hypoxanthine. A sequence of nucleotide or nucleoside monomers may be referred to as a polynucleotide sequence, nucleic acid sequence, a nucleotide sequence, or a nucleoside sequence.
[0446] The term “polypeptide”, as used herein in conjunction with a reference SEQ.ID NO, refers to any and all polypeptides comprising a sequence of amino acid residues which is (i) substantially identical to the amino acid sequence constituting the polypeptide having such reference SEQ.ID NO, or (ii) encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding the polypeptide having such reference SEQ.ID NO, but for the use of synonymous codons. A sequence of amino acid residues may be referred to as an amino acid sequence, or polypeptide sequence.
[0447] The term “nucleic acid sequence encoding a polypeptide”, as used herein in conjunction with a reference SEQ.ID NO, refers to any and all nucleic acid sequences encoding a polypeptide having such reference SEQ.ID NO. Nucleic acid sequences encoding a polypeptide, in conjunction with a reference SEQ.ID NO, further include any and all nucleic acid sequences which (i) encode polypeptides that are substantially identical to the polypeptide having such reference SEQ.ID NO; or (ii) hybridize to any nucleic acid sequences encoding polypeptides having such reference SEQ.ID NO under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
[0448] By the term “substantially identical” it is meant that two amino acid sequences preferably are at least 70% identical, and more preferably are at least 85% identical and most preferably at least 95% identical, for example 96%, 97%, 98% or 99% identical. In order to determine the percentage of identity between two amino acid sequences the amino acid sequences of such two sequences are aligned, using for example the alignment method of Needleman and Wunsch (J. Mol. Biol., 1970, 48: 443), as revised by Smith and Waterman (Adv. Appl. Math., 1981, 2: 482) so that the highest order match is obtained between the two sequences and the number of identical amino acids is determined between the two sequences. Methods to calculate the percentage identity between two amino acid sequences are generally art recognized and include, for example, those described by Carillo and Lipton (SIAM J. Applied Math., 1988, 48:1073) and those described in Computational Molecular Biology, Lesk, e.d. Oxford University Press, New York, 1988, Biocomputing: Informatics and Genomics Projects. Generally, computer programs will be employed for such calculations. Computer programs that may be used in this regard include, but are not limited to, GCG (Devereux et al., Nucleic Acids Res., 1984, 12: 387) BLASTP, BLASTN and FASTA (Altschul et al., J. Mol. Biol., 1990:215:403). A particularly preferred method for determining the percentage identity between two polypeptides involves the Clustal W algorithm (Thompson, J D, Higgines, D G and Gibson T J, 1994, Nucleic Acid Res 22(22): 4673-4680 together with the BLOSUM 62 scoring matrix (Henikoff S & Henikoff, J G, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919 using a gap opening penalty of 10 and a gap extension penalty of 0.1, so that the highest order match obtained between two sequences wherein at least 50% of the total length of one of the two sequences is involved in the alignment.
[0449] By “at least moderately stringent hybridization conditions” it is meant that conditions are selected which promote selective hybridization between two complementary nucleic acid molecules in solution. Hybridization may occur to all or a portion of a nucleic acid sequence molecule. The hybridizing portion is typically at least 15 (e.g., 20, 25, 30, 40 or 50) nucleotides in length. Those skilled in the art will recognize that the stability of a nucleic acid duplex, or hybrids, is determined by the Tm, which in sodium containing buffers is a function of the sodium ion concentration and temperature (Tm=81.5° C.–16.6 (Log10 [Na+])+0.41(% (G+C)-600/I), or similar equation). Accordingly, the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature. In order to identify molecules that are similar, but not identical, to a known nucleic acid molecule a 1% mismatch may be assumed to result in about a 1° C. decrease in Tm, for example if nucleic acid molecules are sought that have a >95% identity, the final wash temperature will be reduced by about 5° C. Based on these considerations those skilled in the art will be able to readily select appropriate hybridization conditions. In preferred embodiments, stringent hybridization conditions are selected. By way of example the following conditions may be employed to achieve stringent hybridization: hybridization at 5× sodium chloride/sodium citrate (SSC)/5×Denhardt’s solution/1.0% SDS at Tm (based on the above equation) -5° C., followed by a wash of 0.2×SSC/0.1% SDS at 60° C. Moderately stringent hybridization conditions include a washing step in 3×SSC at 42° C. It is understood however that equivalent stringencies may be achieved using alternative buffers, salts, and temperatures. Additional guidance regarding hybridization conditions may be found in: Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 1989, 6.3.1.-6.3.6 and in: Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, Vol. 3.
[0450] The term “functional variant”, as used herein in reference to polynucleotides or polypeptides, refers to polynucleotides or polypeptides capable of performing the same function as a noted reference polynucleotide or polypeptide. Thus, for example, a functional variant of the polypeptide set forth in SEQ.ID NO: 2, refers to a polypeptide capable of performing the same function as the polypeptide set forth in SEQ.ID NO: 2. Functional variants include modified a polypeptide wherein, relative to a noted reference polypeptide, the modification includes a substitution, deletion, or addition of one or more amino acids. In some embodiments, substitutions are those that result in a replacement of one amino acid with an amino acid having similar characteristics. Such substitutions include, without limitation (i) glutamic acid and aspartic acid; (i) alanine, serine, and threonine; (iii) isoleucine, leucine, and valine, (iv) asparagine and glutamine, and (v) tryptophan, tyrosine, and phenylalanine. Functional variants further include polypeptides having retained or exhibiting an enhanced psilocybin biosynthetic bioactivity.
[0451] The term “chimeric”, as used herein in the context of nucleic acids, refers to at least two linked nucleic acids which are not naturally linked. Chimeric nucleic acids include linked nucleic acids of different natural origins. For example, a nucleic acid constituting a microbial promoter linked to a nucleic acid encoding a plant polypeptide is considered chimeric. Chimeric nucleic acids also may comprise nucleic acids of the same natural origin, provided they are not naturally linked. For example a nucleic acid constituting a promoter obtained from a particular cell-type may be linked to a nucleic acid encoding a polypeptide obtained from that same cell-type, but not normally linked to the nucleic acid constituting the promoter. Chimeric nucleic acids also include nucleic acids comprising any naturally occurring nucleic acids linked to any non-naturally occurring nucleic acids.
[0452] The term “pharmaceutical formulation”, as used herein, refers to a preparation in a form which allows an active ingredient, including a psychoactive ingredient, contained therein to provide effective treatment, and which does not contain any other ingredients which cause excessive toxicity, an allergic response, irritation, or other adverse response commensurate with a reasonable risk/benefit ratio. The pharmaceutical formulation may contain other pharmaceutical ingredients such as excipients, carriers, diluents, or auxiliary agents.
[0453] The term “recreational drug formulation”, as used herein, refers to a preparation in a form which allows a psychoactive ingredient contained therein to be effective for administration as a recreational drug, and which does not contain any other ingredients which cause excessive toxicity, an allergic response, irritation, or other adverse response commensurate with a reasonable risk/benefit ratio. The recreational drug formulation may contain other ingredients such as excipients, carriers, diluents, or auxiliary agents.
[0454] The term “effective for administration as a recreational drug”, as used herein, refers to a preparation in a form which allows a subject to voluntarily induce a psychoactive effect for non-medical purposes upon administration, generally in the form of self-administration. The effect may include an altered state of consciousness, satisfaction, pleasure, euphoria, perceptual distortion, or hallucination.
[0455] The term “effective amount”, as used herein, refers to an amount of an active agent, pharmaceutical formulation, or recreational drug formulation, sufficient to induce a desired biological or therapeutic effect, including a prophylactic effect, and further including a psychoactive effect. Such effect can include an effect with respect to the signs, symptoms or causes of a disorder, or disease or any other desired alteration of a biological system. The effective amount can vary depending, for example, on the health condition, injury stage, disorder stage, or disease stage, weight, or sex of a subject being treated, timing of the administration, manner of the administration, age of the subject, and the like, all of which can be determined by those of skill in the art.
[0456] The terms “treating” and “treatment”, and the like, as used herein, are intended to mean obtaining a desirable physiological, pharmacological, or biological effect, and includes prophylactic and therapeutic treatment. The effect may result in the inhibition, attenuation, amelioration, or reversal of a sign, symptom or cause of a disorder, or disease, attributable to the disorder, or disease, which includes mental and psychiatric diseases and disorders. Clinical evidence of the prevention or treatment may vary with the disorder, or disease, the subject, and the selected treatment.
[0457] The term “pharmaceutically acceptable”, as used herein, refers to materials, including excipients, carriers, diluents, or auxiliary agents, that are compatible with other materials in a pharmaceutical or recreational drug formulation and within the scope of reasonable medical judgement suitable for use in contact with a subject without excessive toxicity, allergic response, irritation, or other adverse response commensurate with a reasonable risk/benefit ratio.
[0458] The terms “substantially pure” and “isolated”, as may be used interchangeably herein describe a compound, e.g., a psilocybin derivative, which has been separated from components that naturally accompany it. Typically, a compound is substantially pure when at least 60%, more preferably at least 75%, more preferably at least 90%, 95%, 96%, 97%, or 98%, and most preferably at least 99% of the total material (by volume, by wet or dry weight, or by mole percent or mole fraction) in a sample is the compound of interest. Purity can be measured by any appropriate method, e.g., by chromatography, gel electrophoresis or HPLC analysis.
[0459] The term “recovered”, as used herein in association with an enzyme, protein, or a chemical compound, refers to a more or less pure form of the enzyme, protein, or chemical compound.
[0460] The term “in vivo”, as used herein relation to a method of making a multi-substituent psilocybin derivative compound, refers to a method involving contacting a psilocybin derivative precursor compound with an enzyme capable of converting the psilocybin derivative precursor compound within a cell, for example, a cell or a microorganism, cultivated, for example, in a growth medium, to convert the psilocybin derivative precursor compound into a multi-substituent psilocybin derivative compound. The cell generally expresses a psilocybin biosynthetic enzyme complex, including a heterologously expressed tryptophan synthase B polypeptide, a tryptophan decarboxylase, an N-acetyl transferase, a N-methyl transferase and a prenyl transferase, for example.
[0461] The term “in vitro”, as used herein relation to a method of making a multi-substituent psilocybin derivative compound, refers to a method involving contacting a psilocybin derivative precursor compound with an enzyme capable of converting the psilocybin derivative precursor outside a cell, for example, in a microwell plate, a tube, a flask, a beaker, a tank, a reactor, or the like, to convert the psilocybin derivative precursor compound into a multi-substituent psilocybin derivative compound.
General Implementation
[0462] As hereinbefore mentioned, the present disclosure relates to psilocybin derivatives. In particular, the present disclosure provides novel multiple-substituent psilocybin derivatives. In general, the herein provided compositions exhibit functional properties which deviate from the functional properties of psilocybin. Thus, for example, the multiple-substituent psilocybin derivatives, can exhibit pharmacological properties which deviate from psilocybin. Furthermore, the multiple-substituent derivatives may psilocybin derivatives may exhibit physicochemical properties which differ from psilocybin. Thus, for example, multiple-substituent psilocybin derivatives may exhibit superior solubility in a solvent, for example, an aqueous solvent. The multiple-substituent psilocybin derivatives in this respect are useful in the formulation of pharmaceutical and recreational drug formulations. In one embodiment, the multiple-substituent psilocybin derivatives of the present disclosure can conveniently be chemically and/or biosynthetically produced. The practice of this method avoids the extraction of psilocybin from mushrooms and the performance of subsequent chemical reactions to achieve multiple-substituent derivatives. Furthermore, the growth of mushrooms can be avoided thus limiting the dependence on climate and weather, and potential legal and social challenges associated with the cultivation of mushrooms containing psychoactive compounds. The method can efficiently yield substantial quantities of multiple-substituent psilocybin derivatives.
[0463] In what follows selected embodiments are described with reference to the drawings.
[0464] Initially example multiple-substituent psilocybin derivatives will be described. Thereafter example methods of using and making the multiple-substituent psilocybin derivatives will be described.
[0465] Accordingly, in one aspect the present disclosure provides derivatives of a compound known as psilocybin of which the chemical structure is shown in
[0466] Thus, in one aspect, the present disclosure provides, in accordance with the teachings herein, in at least one embodiment, a chemical compound having a formula (I):
##STR00167##
wherein, at least two of R.sub.2, R.sub.4, R.sub.5, R.sub.6, or R.sub.7 are substituents independently selected from at least two of (i) a halogen atom, (ii) a hydroxy group, (iii) a nitro group, (iv) a glycosyloxy group, (v) an amino group or an N-substituted amino group, (vi) a carboxyl group or a carboxylic acid derivative, (vii) an aldehyde or a ketone group, (viii) a prenyl group, and (ix) a nitrile group, and wherein each non-substituted R.sub.2, R.sub.5, R.sub.6, or R.sub.7 is a hydrogen atom and when R.sub.4 is not substituted with any of the foregoing substituents, R.sub.4 is a hydrogen atom, an O-alkyl group, an O-acyl group, or a phosphate group, wherein R.sub.3a and R.sub.3b are each independently a hydrogen atom, an alkyl group, a cycloalkyl group, an acyl group, or an aryl group, and wherein and R.sub.3c is a hydrogen atom or a carboxyl group.
[0467] Thus, referring to the chemical compound having the formula (I), initially it is noted that, in an aspect hereof, at least two of R.sub.2, R.sub.4, R.sub.5, R.sub.6, or R.sub.7 are substituent entities selected from at least two of (i) a halogen atom, (ii) a hydroxy group, (iii) a nitro group, (iv) a glycosyloxy group, (v) an amino group or an N-substituted amino group, (vi) a carboxyl group or a carboxylic acid derivative, (vii) an aldehyde or a ketone group, (viii) a prenyl group, and (ix) a nitrile group. Thus, it is to be understood that, in accordance with an aspect of the present disclosure, at least two of R.sub.2, R.sub.4, R.sub.5, R.sub.6, or R.sub.7 are substituent entities. The substituent entities are each independently selected from (i) a halogen atom, (ii) a hydroxy group, (iii) a nitro group, (iv) a glycosyloxy group, (v) an amino group or an N-substituted amino group, (vi) a carboxyl group or a carboxylic acid derivative, (vii) an aldehyde or a ketone group, (viii) a prenyl group, and (ix) a nitrile group, and where two (but no more than two) substituent entities are selected, they are non-identical, and where three or more substituent entities are selected, at least two of the selected substituent entities are non-identical.
[0468] In an aspect hereof, in an embodiment, at least three of R.sub.2, R.sub.4, R.sub.5, R.sub.6, or R.sub.7 can be substituents selected from at least two of (i) a halogen atom, (ii) a hydroxy group, (iii) a nitro group, (iv) a glycosyloxy group, (v) an amino group or an N-substituted amino group, (vi) a carboxyl group or a carboxylic acid derivative, (vii) an aldehyde or a ketone group, (viii) a prenyl group, and (ix) a nitrile group.
[0469] In an aspect hereof, in an embodiment, at least three of R.sub.2, R.sub.4, R.sub.5, R.sub.6, or R.sub.7 can be substituents selected from at least three of (i) a halogen atom, (ii) a hydroxy group, (iii) a nitro group, (iv) a glycosyloxy group, (v) an amino group or an N-substituted amino group, (vi) a carboxyl group or a carboxylic acid derivative, (vii) an aldehyde or a ketone group, (viii) a prenyl group, and (ix) a nitrile group.
[0470] Continuing to refer to the chemical compound having the formula (I), in a further aspect hereof, R.sub.4 can be a phosphate group or a hydrogen atom.
[0471] Continuing to refer to the chemical compound having the formula (I), in a further aspect hereof, R.sub.3a and R.sub.3b can be a hydrogen atom, an alkyl group, an acyl group or an aryl group. Thus, R.sub.3a and R.sub.3b can each be a hydrogen atom, or R.sub.3a and R.sub.3b can each be an alkyl group, such as a methyl group, ethyl group, propyl group, or longer chain alkyl group, or R.sub.3a and R.sub.3b can be each be an acyl group, or R.sub.3a and R.sub.3b can each be an aryl group. Furthermore, one of R.sub.3a and R.sub.3b can be a hydrogen atom, and one of R.sub.3a and R.sub.3b can be an alkyl group. One of R.sub.3a and R.sub.3b can be a hydrogen atom, and one of R.sub.3a and R.sub.3b can be an acyl group. One of R.sub.3a and R.sub.3b can be a hydrogen atom, and one of R.sub.3a and R.sub.3b can be an aryl group. One of R.sub.3a and R.sub.3b can be an alkyl group, and one of R.sub.3a and R.sub.3b can be an aryl group. One of R.sub.3a and R.sub.3b can be an alkyl group, and one of R.sub.3a and R.sub.3b can be an acyl group. One of R.sub.3a and R.sub.3b can be an acyl group, and one of R.sub.3a and R.sub.3b can be an aryl group.
[0472] Continuing to refer to the chemical compound having the formula (I), in a further aspect hereof, each of the non-substituted groups R.sub.2, R.sub.5, R.sub.6, or R.sub.7 can be a hydrogen atom. Moreover, as hereinbefore noted, R.sub.4 can also be a hydrogen atom.
[0473] In accordance herewith disclosed herein, in an aspect, multiple-substituent psilocybin derivatives including two, or three substituent groups. Examples of each of these will next be discussed, by referring to selected figures. In particular, examples including multiple-substituent psilocybin derivatives including two substituent groups are discussed by referring to
[0474] Thus, referring next to
[0475] Thus, for example, referring to the chemical compound having the formula (I), in one example embodiment, two of R.sub.4, R.sub.5, R.sub.6, or R.sub.7 can be substituents, one of which can be selected from (i) a halogen atom, (ii) a prenyl group, and (iii) a nitrile group, and one of R.sub.4, R.sub.5, R.sub.6, or R.sub.7 can be a substituent selected from (i) a halogen atom, (ii) a hydroxy group, (iii) a nitro group, (iv) a glycosyloxy group, (v) an amino group or an N-substituted amino group, (vi) a carboxyl group or a carboxylic acid derivative, (vii) an aldehyde or a ketone group, (viii) a prenyl group, and (ix) a nitrile group, provided however the two substituents are non-identical, and R.sub.2 and the non-substituted R.sub.4, R.sub.5, R.sub.6 and R.sub.7 are hydrogen atoms. Thus, in such example embodiment, referring to
[0476] Thus, for example, referring to the chemical compound having the formula (I), in a further example embodiment, two of R.sub.4, R.sub.5, R.sub.6, or R.sub.7 can be substituents, one of which is a halogen atom, and one of R.sub.4, R.sub.5, R.sub.6, or R.sub.7 can be a substituent selected from (i) a hydroxy group, (ii) a nitro group, (iii) a glycosyloxy group, (iv) an amino group or an N-substituted amino group, (v) a carboxyl group or a carboxylic acid derivative, (vi) an aldehyde or a ketone group, (vii) a prenyl group, and (viii) a nitrile group, and R.sub.2 and the non-substituted R.sub.4, R.sub.5, R.sub.6 and R.sub.7 are hydrogen atoms. Thus, in such example embodiment, referring to
[0477] Thus, for example, referring to the chemical compound having the formula (I), in a further example embodiment, two of R.sub.4, R.sub.5, R.sub.6, or R.sub.7 can be substituents, one of which is a halogen atom, and one of R.sub.4, R.sub.5, R.sub.6, or R.sub.7 can be a substituent selected from (i) an amino group, (ii) a nitrile group, (iii) a nitro group, (iv) a hydroxy group, and a (vii) a prenyl group, and R.sub.2 and the non-substituted R.sub.4, R.sub.5, R.sub.6 and R.sub.7 are hydrogen atoms. Thus, in such example embodiment, referring to
[0478] Thus, for example, referring to the chemical compound having the formula (I), in a further example embodiment, two of R.sub.4, R.sub.5, R.sub.6, or R.sub.7 can be substituents, one of which is a halogen atom, and one of R.sub.4, R.sub.5, R.sub.6, or R.sub.7 can be a prenyl group, and R.sub.2 and the non-substituted R.sub.4, R.sub.5, R.sub.6 and R.sub.7 are hydrogen atoms. Thus, in such example embodiment, referring to
[0479] Thus, for example, referring to the chemical compound having the formula (I), in a further example embodiment, two of R.sub.4, R.sub.5, R.sub.6, or R.sub.7 can be substituents, one of which is a prenyl group, and one of R.sub.4, R.sub.5, R.sub.6, or R.sub.7 can be a substituent selected from (i) a hydroxy group, (ii) a nitro group, (iii) a glycosyloxy group, (iv) an amino group or an N-substituted amino group, (v) a carboxyl group or a carboxylic acid derivative, (vi) an aldehyde or a ketone group, (vii) a halogen atom, and (viii) a nitrile group, and R.sub.2 and the non-substituted R.sub.4, R.sub.5, R.sub.6 and R.sub.7 are hydrogen atoms. Thus, in such example embodiment, referring to
[0480] Thus, for example, referring to the chemical compound having the formula (I), in a further example embodiment, two of R.sub.4, R.sub.5, R.sub.6, or R.sub.7 can be substituents, one of which is a prenyl group, and one of R.sub.4, R.sub.5, R.sub.6, or R.sub.7 can be a substituent selected from (i) a carboxyl group or a carboxylic acid derivative, (ii) a halogen atom, and (iii) a hydroxy group, and R.sub.2 and the non-substituted R.sub.4, R.sub.5, R.sub.6 and R.sub.7 are hydrogen atoms. Thus, in such example embodiment, referring to
[0481] Thus, for example, referring to the chemical compound having the formula (I), in a further example embodiment, two of R.sub.4, R.sub.5, R.sub.6, or R.sub.7 can be substituents, one of which is a nitrile group, and one of R.sub.4, R.sub.5, R.sub.6, or R.sub.7 can be a substituent selected from (i) a hydroxy group, (ii) a nitro group, (iii) a glycosyloxy group, (iv) an amino group or an N-substituted amino group, (v) a carboxyl group or a carboxylic acid derivative, (vi) an aldehyde or a ketone group, (vii) a prenyl group, and (viii) a halogen atom, and R.sub.2 and the non-substituted R.sub.4, R.sub.5, R.sub.6 and R.sub.7 are hydrogen atoms. Thus, in such example embodiment, referring to
[0482] Thus, for example, referring to the chemical compound having the formula (I), in a further example embodiment, two of R.sub.4, R.sub.5, R.sub.6, or R.sub.7 can be substituents, one of which is a nitrile group, and one of R.sub.4, R.sub.5, R.sub.6, or R.sub.7 can be an amino group or an N-substituted amino group, and R.sub.2 and the non-substituted R.sub.4, R.sub.5, R.sub.6 and R.sub.7 are hydrogen atoms. Thus, in such example embodiment, referring to
[0483] Turning now to multiple-substituent psilocybin derivatives, including three substituent groups, and referring to
[0484] Further examples, wherein three of R.sub.2, R.sub.5, R.sub.6, or R.sub.7 are substituents are shown in
[0485] Further examples, wherein three of R.sub.2, R.sub.5, R.sub.6, or R.sub.7 are substituents are shown in
[0486] Further examples, wherein three of R.sub.2, R.sub.5, R.sub.6, or R.sub.7 are substituents are shown in
[0487] Further examples, wherein three of R.sub.2, R.sub.5, R.sub.6, or R.sub.7 are substituents are shown in
[0488] Further examples, wherein three of R.sub.2, R.sub.5, R.sub.6, or R.sub.7 are substituents are shown in
[0489] Further examples, wherein three of R.sub.2, R.sub.5, R.sub.6, or R.sub.7 are substituents are shown in
[0490] Yet further examples, wherein three of R.sub.2, R.sub.5, R.sub.6, or R.sub.7 are substituents are shown in
[0491] Furthermore, in each of the example embodiments shown in
[0492] It is noted that in a further aspect hereof in each of the example embodiments shown in
[0493] It is noted that in a further aspect hereof in each of the example embodiments shown in
[0494] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (IX):
##STR00168##
[0495] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (X):
##STR00169##
[0496] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XI):
##STR00170##
[0497] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XII):
##STR00171##
[0498] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XIII):
##STR00172##
[0499] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XIV):
##STR00173##
[0500] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XV):
##STR00174##
[0501] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XV):
##STR00175##
[0502] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XVII):
##STR00176##
[0503] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XVIII):
##STR00177##
[0504] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XIX):
##STR00178##
[0505] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XX):
##STR00179##
[0506] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XXI):
##STR00180##
[0507] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XXII):
##STR00181##
[0508] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XXIII):
##STR00182##
[0509] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XXIV):
##STR00183##
[0510] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XXV):
##STR00184##
[0511] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XVI):
##STR00185##
[0512] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XXVII):
##STR00186##
[0513] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XXVIII):
##STR00187##
[0514] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XXIX):
##STR00188##
[0515] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XXX):
##STR00189##
[0516] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XXXI):
##STR00190##
[0517] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XXXII):
##STR00191##
[0518] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XXXIII):
##STR00192##
[0519] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XXXIV):
##STR00193##
[0520] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XXXV):
##STR00194##
[0521] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XXXVI):
##STR00195##
[0522] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XXXVII):
##STR00196##
[0523] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XXXVIII):
##STR00197##
[0524] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XXXIX):
##STR00198##
[0525] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XL):
##STR00199##
[0526] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XLI):
##STR00200##
[0527] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XLII):
##STR00201##
[0528] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XLIII):
##STR00202##
[0529] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XLIV):
##STR00203##
[0530] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XLV):
##STR00204##
[0531] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XLVI):
##STR00205##
[0532] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XLVII):
##STR00206##
[0533] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XLVIII):
##STR00207##
[0534] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (XLIX):
##STR00208##
[0535] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (L):
##STR00209##
[0536] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (LI):
##STR00210##
[0537] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (LII):
##STR00211##
[0538] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (LIII):
##STR00212##
[0539] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (LIV):
##STR00213##
[0540] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (LV):
##STR00214##
[0541] Furthermore, in one example embodiment, a multi-substituent psilocybin derivative according to the present disclosure can be a chemical compound having a formula (LXXVI):
##STR00215##
[0542] Furthermore, it is noted that the multi-substituent psilocybin derivatives of the present disclosure include salts thereof, including pharmaceutically acceptable salts. Thus, the nitrogen atom of the ethyl-amino group extending in turn from the C.sub.3 atom may be protonated, and the positive charge may be balanced by, for example, chloride or sulfate ions, to thereby form a chloride salt or a sulfate salt. Furthermore, in compounds wherein R.sub.4 is a phosphate group, the phosphate group may be de-protonated, and the negative charge may be balanced by, for example, sodium ions or potassium ions, to thereby form a sodium salt or a potassium salt.
[0543] Furthermore, it is noted that when R.sub.4 is a phosphate group, the term prenylated psilocybin derivative also includes compounds having a formula (IV):
##STR00216##
wherein, at least two of R.sub.2, R.sub.5, R.sub.6, or R.sub.7 are substituents independently selected from at least two of (i) a halogen atom, (ii) a hydroxy group, (iii) a nitro group, (iv) a glycosyloxy group, (v) an amino group or an N-substituted amino group, (vi) a carboxyl group or a carboxylic acid derivative, (vii) an aldehyde or a ketone group, (viii) a prenyl group, and (ix) a nitrile group, and wherein each non-substituted R.sub.2, R.sub.5, R.sub.6, or R.sub.7 is a hydrogen atom, wherein R.sub.3a and R.sub.3b are each independently a hydrogen atom, an alkyl group, a cycloalkyl group, an acyl group, or an aryl group, and wherein and R.sub.3c is a hydrogen atom or a carboxyl group. When R.sub.3c is a carboxy group, further included are compounds having a formula (Iva):
##STR00217##
Further included are salts of prenylated psilocybin derivatives having a formula (IV) and (IVa), such as a sodium salt, a potassium salt, etc.
[0544] Thus, to briefly recap, the present disclosure provides multi-substituent psilocybin derivatives. The disclosure provides, in particular, a chemical compound having a formula (I):
##STR00218##
wherein, at least two of R.sub.2, R.sub.4, R.sub.5, R.sub.6, or R.sub.7 are substituents independently selected from at least two of (i) a halogen atom, (ii) a hydroxy group, (iii) a nitro group, (iv) a glycosyloxy group, (v) an amino group or an N-substituted amino group, (vi) a carboxyl group or a carboxylic acid derivative, (vii) an aldehyde or a ketone group, (viii) a prenyl group, and (ix) a nitrile group, and wherein each non-substituted R.sub.2, R.sub.5, R.sub.6, or R.sub.7 is a hydrogen atom and when R.sub.4 is not substituted with any of the foregoing substituents, R.sub.4 is a hydrogen atom, an O-alkyl group, an O-acyl group, or a phosphate group, wherein R.sub.3a and R.sub.3b are each independently a hydrogen atom, an alkyl group, a cycloalkyl group, an acyl group, or an aryl group, and wherein and R.sub.3c is a hydrogen atom or a carboxyl group.
[0545] In another embodiment, R.sub.3a and R.sub.3b are a hydrogen atom, a (C.sub.1-C.sub.20)-alkyl group, a (C.sub.6-C.sub.14)-aryl group, or a —C(═O)(C.sub.1-C.sub.20)-alkyl group. In another embodiment, R.sub.3a and R.sub.3b are a hydrogen atom, a (C.sub.1-C.sub.10)-alkyl group, a (C.sub.6-C.sub.10)-aryl group, or a —C(═O)(C.sub.1-C.sub.10)-alkyl group. In another embodiment, R.sub.3a and R.sub.3b are a hydrogen atom, a (C.sub.1-C.sub.6)-alkyl group, a phenyl group, or a—C(═O)(C.sub.1-C.sub.6)-alkyl group. In another embodiment, R.sub.3a and R.sub.3b are a hydrogen atom, a methyl group, an ethyl group, a propyl group, a phenyl group, —C(═O)—CH.sub.3, —C(═O)—CH.sub.2CH3, or —C(═O)—CH.sub.2CH.sub.2CH.sub.3.
[0546] In another embodiment, R.sub.3a and/or R.sub.3b are a (C.sub.1-C.sub.20)-cyclo-alkyl group, or a (C.sub.1-C.sub.10)-cyclo-alkyl group, a (C.sub.1-C.sub.10)-cyclo-alkyl group, or a (C.sub.1-C.sub.10)-cyclo-alkyl group. In one embodiment, R.sub.3a and/or R.sub.3b are a cyclo-propane group, a cyclo-butane group, a cyclo-pentane group, or a cyclo-hexane group.
[0547] In one embodiment, the alkyl groups (including O-alkyl) in any of the definitions of the formulas of the disclosure is C.sub.1-C.sub.20-alkyl. In another embodiment, the alkyl group is C.sub.1-C.sub.10-alkyl. In another embodiment, the alkyl group is C.sub.1-C.sub.6-alkyl. In another embodiment, the alkyl group is methyl, ethyl, propyl, butyl or pentyl.
[0548] In one embodiment, the acyl groups (including O-acyl) in any of the definitions of the formulas of the disclosure is C.sub.1-C.sub.20-acyl (or C.sub.1-C.sub.20-acyl-O-). In another embodiment, the alkyl group is C.sub.1-C.sub.10-acyl (or C.sub.1-C.sub.10-acyl-O-). In another embodiment, the alkyl group is C.sub.1-C.sub.6-acyl (or C.sub.1-C.sub.6-acyl-O-). In another embodiment, the acyl group is an O-acyl group, a methanoyl, ethanoyl, propanoyl, butanoyl or pentanoyl.
[0549] In one embodiment, the aryl groups in any of the definitions of the formulas of the disclosure is optionally substituted C.sub.6-C.sub.14-aryl. In another embodiment, the aryl group is optionally substituted C.sub.6-C.sub.10-aryl, or phenyl. In another embodiment, the aryl group is phenyl, naphthyl, tetrahydronaphthyl, phenanthrenyl, biphenylenyl, indanyl, or indenyl and the like.
[0550] The multi-substituent psilocybin derivatives of the present disclosure may be used to prepare a pharmaceutical or recreational drug formulation. Thus in one embodiment, the present disclosure further provides in another aspect, pharmaceutical and recreational drug formulations comprising multi-substituent psilocybin derivatives. Accordingly, in one aspect, the present disclosure provides in a further embodiment a pharmaceutical or recreational drug formulation comprising a chemical compound having a formula (I):
##STR00219##
wherein, at least two of R.sub.2, R.sub.4, R.sub.5, R.sub.6, or R.sub.7 are substituents independently selected from at least two of (i) a halogen atom, (ii) a hydroxy group, (iii) a nitro group, (iv) a glycosyloxy group, (v) an amino group or an N-substituted amino group, (vi) a carboxyl group or a carboxylic acid derivative, (vii) an aldehyde or a ketone group, (viii) a prenyl group, and (ix) a nitrile group, and wherein each non-substituted R.sub.2, R.sub.5, R.sub.6, or R.sub.7 is a hydrogen atom and when R.sub.4 is not substituted with any of the foregoing substituents, R.sub.4 is a hydrogen atom, an O-alkyl group, an O-acyl group, or a phosphate group, wherein R.sub.3a and R.sub.3b are each independently a hydrogen atom, an alkyl group, a cycloalkyl group, an acyl group, or an aryl group, and wherein and R.sub.3c is a hydrogen atom or a carboxyl group.
[0551] The pharmaceutical or recreational drug formulations may be prepared as liquids, tablets, capsules, microcapsules, nanocapsules, trans-dermal patches, gels, foams, oils, aerosols, nanoparticulates, powders, creams, emulsions, micellar systems, films, sprays, ovules, infusions, teas, decoctions, suppositories, etc. and include a pharmaceutically acceptable salt or solvate of the nitrilated psilocybin compound together with an excipient. The term “excipient” as used herein means any ingredient other than the chemical compound of the disclosure. As will readily be appreciated by those of skill in art, the selection of excipient may depend on factors such as the particular mode of administration, the effect of the excipient on solubility of the chemical compounds of the present disclosure and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation may be found, for example, in “Remington’s Pharmaceutical Sciences”, 22.sup.nd Edition (Pharmaceutical Press and Philadelphia College of Pharmacy at the University of the Sciences, 2012).
[0552] The dose when using the compounds of the present disclosure can vary within wide limits, and as is customary and is known to those of skill in the art, the dose can be tailored to the individual conditions in each individual case. The dose depends, for example, on the nature and severity of the illness to be treated, on the condition of the patient, on the compound employed or on whether an acute or chronic disease state is treated, or prophylaxis is conducted, on the mode of delivery of the compound, or on whether further active compounds are administered in addition to the compounds of the present disclosure. Representative doses of the present invention include, but are not limited to, about 0.001 mg to about 5000 mg, about 0.001 mg to about 2500 mg, about 0.001 mg to about 1000 mg, about 0.001 mg to about 500 mg, about 0.001 mg to about 250 mg, about 0.001 mg to about 100 mg, about 0.001 mg to about 50 mg, and about 0.001 mg to about 25 mg. Representative doses of the present disclosure include, but are not limited to, about 0.0001 to about 1,000 mg, about 10 to about 160 mg, about 10 mg, about 20 mg, about 40 mg, about 80 mg or about 160 mg. Multiple doses may be administered during the day, especially when relatively large amounts are deemed to be needed, for example 2, 3 or 4, doses. Depending on the subject and as deemed appropriate from the patient’s physician or care giver it may be necessary to deviate upward or downward from the doses described herein.
[0553] The pharmaceutical and drug formulations comprising the multi-substituent psilocybin derivatives of the present disclosure may be administered orally. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, or buccal or sublingual administration may be employed by which the compound enters the blood stream directly from the mouth. Formulations suitable for oral administration include both solid and liquid formulations.
[0554] Solid formulations include tablets, capsules (containing particulates, liquids, microcapsules, or powders), lozenges (including liquid-filled lozenges), chews, multi- and nano-particulates, gels, solid solutions, liposomal preparations, microencapsulated preparations, creams, films, ovules, suppositories, and sprays.
[0555] Liquid formulations include suspensions, solutions, syrups, and elixirs. Such formulations may be employed as fillers in soft or hard capsules and typically comprise a carrier, for example, water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifying agents and/or suspending agents. Liquid formulations may also be prepared by the reconstitution of a solid, for example, from a sachet.
[0556] Binders are generally used to impart cohesive qualities to a tablet formulation. Suitable binders include microcrystalline cellulose, gelatin, sugars, polyethylene glycol, natural and synthetic gums, polyvinylpyrrolidone, pregelatinized starch, hydroxypropyl cellulose and hydroxypropyl methylcellulose.
[0557] Tablets may also contain diluents, such as lactose (monohydrate, spray-dried monohydrate, anhydrous and the like), mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose, starch, and dibasic calcium phosphate dihydrate.
[0558] Tablets may also optionally comprise surface active agents, such as sodium lauryl sulfate and polysorbate 80. When present, surface active agents may comprise from 0.2% (w/w) to 5% (w/w) of the tablet.
[0559] Tablets may further contain lubricants such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and mixtures of magnesium stearate with sodium lauryl sulphate. Lubricants generally comprise from 0.25% (w/w) to 10% (w/w), from 0.5% (w/w) to 3% (w/w) of the tablet.
[0560] In addition to the multi-substituent psilocybin derivative, tablets may contain a disintegrant. Examples of disintegrants include sodium starch glycolate, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methyl cellulose, microcrystalline cellulose, lower alkyl-substituted hydroxypropyl cellulose, starch, pregelatinized starch and sodium alginate. Generally, the disintegrant will comprise from 1% (w/w) to 25% (w/w) or from 5% (w/w) to 20% (w/w) of the dosage form.
[0561] Other possible auxiliary ingredients include anti-oxidants, colourants, flavouring agents, preservatives, and taste-masking agents.
[0562] For tablet dosage forms, depending on the desired effective amount of the chemical compound, the chemical compound of the present disclosure may make up from 1% (w/w) to 80% (w/w) of the dosage form, more typically from 5% (w/w) to 60% (w/w) of the dosage form.
[0563] Exemplary tablets contain up to about 80% (w/w) of the chemical compound, from about 10% (w/w) to about 90% (w/w) binder, from about 0% (w/w) to about 85% (w/w) diluent, from about 2% (w/w) to about 10% (w/w) disintegrant, and from about 0.25% (w/w) to about 10% (w/w) lubricant.
[0564] The formulation of tablets is discussed in “Pharmaceutical Dosage Forms: Tablets”, Vol. 1 – Vol. 3, by CRC Press (2008).
[0565] The pharmaceutical and recreational drug formulations comprising the multi-substituent psilocybin derivatives of the present disclosure may also be administered directly into the blood stream, into muscle, or into an internal organ. Thus, the pharmaceutical and recreational drug formulations can be administered parenterally (for example, by subcutaneous, intravenous, intraarterial, intrathecal, intraventricular, intracranial, intramuscular, or intraperitoneal injection). Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (in one embodiment, to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile water.
[0566] Formulations comprising the multi-substituent psilocybin derivatives of the present disclosure for parenteral administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release. Thus the chemical compounds of the disclosure may be formulated as a solid, semi-solid, or thixotropic liquid for administration as an implanted depot providing modified release of the active compound. Examples of such formulations include drug-coated stents and poly(dl-lactic-coglycolic)acid (PGLA) microspheres.
[0567] The pharmaceutical or recreational drug formulations of the present disclosure also may be administered topically to the skin or mucosa, i.e., dermally or transdermally. Example pharmaceutical and recreational drug formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, cosmetics, oils, eye drops, dressings, foams, films, skin patches, wafers, implants, sponges, fibres, bandages and microemulsions. Liposomes may also be used. Example carriers include alcohol, water, mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol and propylene glycol. Penetration enhancers may be incorporate (see: for example, Finnin, B. and Morgan, T.M., 1999 J. Pharm. Sci, 88 (10), 955-958).
[0568] Other means of topical administration include delivery by electroporation, iontophoresis, phonophoresis, sonophoresis and microneedle or needle-free (e.g., Powderject™, Bioject™, etc.) injection.
[0569] Pharmaceutical and recreational drug formulations for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous, or organic solvents, or mixtures thereof, and powders. The liquid or solid pharmaceutical compositions can contain suitable pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical compositions are administered by the oral or nasal respiratory route for local or systemic effect. Pharmaceutical compositions in pharmaceutically acceptable solvents can be nebulized by use of inert gases. Nebulized solutions can be inhaled directly from the nebulizing device or the nebulizing device can be attached to a face mask tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder pharmaceutical compositions can be administered, e.g., orally, or nasally, from devices that deliver the formulation in an appropriate manner.
[0570] In further embodiments, in which the multi-substituent psilocybin derivative compounds of present disclosure are used as a recreational drug, the compounds may be included in compositions such as a food or food product, a beverage, a food seasoning, a personal care product, such as a cosmetic, perfume or bath oil, or oils (both for topical administration as massage oil, or to be burned or aerosolized). The chemical compounds of the present disclosure may also be included in a “vape” product, which may also include other drugs, such as nicotine, and flavorings.
[0571] Thus it will be clear the multi-substituent psilocybin derivative compounds may be used as a pharmaceutical or recreational drug. Accordingly, in another aspect the present disclosure provides, in at least one embodiment, a use of a chemical compound having a formula (I):
##STR00220##
wherein, at least two of R.sub.2, R.sub.4, R.sub.5, R.sub.6, or R.sub.7 are substituents independently selected from at least two of (i) a halogen atom, (ii) a hydroxy group, (iii) a nitro group, (iv) a glycosyloxy group, (v) an amino group or an N-substituted amino group, (vi) a carboxyl group or a carboxylic acid derivative, (vii) an aldehyde or a ketone group, (viii) a prenyl group, and (ix) a nitrile group, and wherein each non-substituted R.sub.2, R.sub.5, R.sub.6, or R.sub.7 is a hydrogen atom and when R.sub.4 is not substituted with any of the foregoing substituents, R.sub.4 is a hydrogen atom, an O-alkyl group, an O-acyl group, or a phosphate group, wherein R.sub.3a and R.sub.3b are each independently a hydrogen atom, an alkyl group, a cycloalkyl group, an acyl group, or an aryl group, and wherein and R.sub.3c is a hydrogen atom or a carboxyl group.
[0572] The pharmaceutical formulations comprising the chemical compounds of the present disclosure may be used to treat a subject, and in particular to treat a psychiatric disorder in a subject. Accordingly, the present disclosure includes in a further embodiment, a method for treating a psychiatric disorder, the method comprising administering to a subject in need thereof a pharmaceutical formulation comprising a chemical compound having a formula (I):
##STR00221##
wherein, at least two of R.sub.2, R.sub.4, R.sub.5, R.sub.6, or R.sub.7 are substituents independently selected from at least two of (i) a halogen atom, (ii) a hydroxy group, (iii) a nitro group, (iv) a glycosyloxy group, (v) an amino group or an N-substituted amino group, (vi) a carboxyl group or a carboxylic acid derivative, (vii) an aldehyde or a ketone group, (viii) a prenyl group, and (ix) a nitrile group, and wherein each non-substituted R.sub.2, R.sub.5, R.sub.6, or R.sub.7 is a hydrogen atom and when R.sub.4 is not substituted with any of the foregoing substituents, R.sub.4 is a hydrogen atom, an O-alkyl group, an O-acyl group, or a phosphate group, wherein R.sub.3a and R.sub.3b are each independently a hydrogen atom, an alkyl group, a cycloalkyl group, an acyl group, or an aryl group, and wherein and R.sub.3c is a hydrogen atom or a carboxyl group.
[0573] Psychiatric disorders that may be treated include, for example, neurodevelopmental disorders such as intellectual disability, global development delay, communication disorders, autism spectrum disorder, and attention-deficit hyperactivity disorder (ADHD); bipolar and related disorders, such as mania, and depressive episodes; anxiety disorder, such as generalized anxiety disorder (GAD), agoraphobia, social anxiety disorder, specific phobias (natural events, medical, animal, situational, for example), panic disorder, and separation anxiety disorder; stress disorders, such as acute stress disorder, adjustment disorders, post-traumatic stress disorder (PTSD), and reactive attachment disorder; dissociative disorders, such as dissociative amnesia, dissociative identity disorder, and depersonalization/derealization disorder; somatoform disorders, such as somatic symptom disorders, illness anxiety disorder, conversion disorder, and factitious disorder; eating disorders, such as anorexia nervosa, bulimia nervosa, rumination disorder, pica, and binge-eating disorder; sleep disorders, such as narcolepsy, insomnia disorder, hypersomnolence, breathing-related sleep disorders, parasomnias, and restless legs syndrome; disruptive disorders, such as kleptomania, pyromania, intermittent explosive disorder, conduct disorder, and oppositional defiant disorder; depressive disorders, such as disruptive mood dysregulation disorder, major depressive disorder, persistent depressive disorder (dysthymia), premenstrual dysphoric disorder, substance/medication-induced depressive disorder, postpartum depression, and depressive disorder caused by another medical condition, for example, psychiatric and existential distress within life-threatening cancer situations (ACS Pharmacol. Transl. Sci. 4: 553-562; J Psychiatr Res 137: 273-282); substance-related disorders, such as alcohol-related disorders, cannabis related disorders, inhalant-use related disorders, stimulant use disorders, and tobacco use disorders; neurocognitive disorders, such as delirium; schizophrenia; compulsive disorders, such as obsessive compulsive disorders (OCD), body dysmorphic disorder, hoarding disorder, trichotillomania disorder, excoriation disorder, substance/medication induced obsessive-compulsive disorder, and obsessive-compulsive disorder related to another medical condition; and personality disorders, such as antisocial personality disorder, avoidant personality disorder, borderline personality disorder, dependent personality disorder, histrionic personality disorder, narcissistic personality disorder, obsessive-compulsive personality disorder, paranoid personality disorder, schizoid personality disorder, and schizotypal personality disorder.
[0574] In an aspect, the compounds of the present disclosure may be used to be contacted with a 5-HT.sub.2A receptor to thereby modulate the 5-HT.sub.2A receptor. Such contacting includes bringing a compound of the present disclosure and 5-HT.sub.2A receptor together under in vitro conditions, for example, by introducing the compounds in a sample containing a 5-HT.sub.2A receptor, for example, a sample containing purified 5-HT.sub.2A receptors, or a sample containing cells comprising 5-HT.sub.2A receptors. In vitro conditions further include the conditions described in Example 1 hereof. Contacting further includes bringing a compound of the present disclosure and 5-HT.sub.2A receptor together under in vivo conditions. Such in vivo conditions include the administration to an animal or human subject, for example, of a pharmaceutically effective amount of the compound of the present disclosure, when the compound is formulated together with a pharmaceutically active carrier, diluent, or excipient, as hereinbefore described, to thereby treat the subject. Upon having contacted the 5-HT.sub.2A receptor, the compound may activate the 5-HT.sub.2A receptor or inhibit the 5-HT.sub.2A receptor.
[0575] Thus, in a further aspect, the condition that may be treated in accordance herewith can be any 5-HT.sub.2A receptor mediated disorder. Such disorders include, but are not limited to schizophrenia, psychotic disorder, attention deficit hyperactivity disorder, autism, and bipolar disorder.
[0576] In an aspect, the compounds of the present disclosure may be used to be contacted with a 5-HT.sub.1A receptor to thereby modulate the 5-HT.sub.1A receptor. Such contacting includes bringing a compound of the present disclosure and 5-HT.sub.1A receptor together under in vitro conditions, for example, by introducing the compounds in a sample containing a 5-HT.sub.1A receptor, for example, a sample containing purified 5-HT.sub.1A receptors, or a sample containing cells comprising 5-HT.sub.1A receptors. In vitro conditions further include the conditions described in Example 1 hereof. Contacting further includes bringing a compound of the present disclosure and 5-HT.sub.1A receptor together under in vivo conditions. Such in vivo conditions include the administration to an animal or human subject, for example, of a pharmaceutically effective amount of the compound of the present disclosure, when the compound is formulated together with a pharmaceutically active carrier, diluent, or excipient, as hereinbefore described, to thereby treat the subject. Upon having contacted the 5-HT.sub.1A receptor, the compound may activate the 5-HT.sub.1A receptor or inhibit the 5-HT.sub.1A receptor.
[0577] Thus, in a further aspect, the condition that may be treated in accordance herewith can be any 5-HT.sub.1A receptor mediated disorder. Such disorders include, but are not limited to schizophrenia, psychotic disorder, attention deficit hyperactivity disorder, autism, and bipolar disorder.
[0578] In some embodiments, upon having contacted a 5-HT.sub.1A receptor and a 5-HT.sub.2A receptor, the compound may modulate the 5-HT.sub.1A receptor, e.g., activate or inhibit the 5-HT.sub.1A receptor, however the compound may at the same time not modulate the 5-HT.sub.2A receptor.
[0579] In some embodiments, upon having contacted a 5-HT.sub.2A receptor and a 5-HT.sub.1A receptor, the compound may modulate the 5-HT.sub.2A receptor, e.g., activate or inhibit the 5-HT.sub.2A receptor, however the compound may at the same time not modulate the 5-HT.sub.1A receptor.
[0580] Turning now to methods of making the multi-substituent psilocybin derivatives of the present disclosure, it is initially noted that the multi-substituent psilocybin derivatives of the present disclosure may be prepared in any suitable manner, including by any organic chemical synthesis methods, biosynthetic methods, or a combination thereof. Next, initially example methods for chemically making the multi-substituent psilocybin derivatives of the present disclosure will be discussed. Thereafter, example biosynthetic methods for making the multi-substituent psilocybin derivatives will be discussed.
[0581] One suitable method of making the multi-substituent psilocybin derivatives of the present disclosure initially involves selecting and obtaining or preparing a reactant psilocybin derivative compound and selecting and obtaining or preparing a substituent group containing compound and, thereafter chemically or biochemically reacting the reactant psilocybin derivative compound and the substituent group containing compound to obtain a multi-substituent psilocybin derivative compound. It is noted that in embodiments hereof where the reactant psilocybin derivative compound does not already possess at least one substituent group, the non-substituent reactant psilocybin derivative compound (i.e., generally an indole structure containing reactant wherein R.sub.2, R.sub.5, R.sub.6 and R.sub.7 are each hydrogen atoms) can be reacted, generally sequentially, with at least two substituent groups containing compounds. Examples thereof are shown in
[0582] Accordingly, in one aspect, the present disclosure provides, in at least one embodiment, a method of making a psilocybin derivative or salt thereof having a formula (l):
##STR00222##
wherein, at least two of R.sub.2, R.sub.4, R.sub.5, R.sub.6, or R.sub.7 are substituents independently selected from at least two of (i) a halogen atom, (ii) a hydroxy group, (iii) a nitro group, (iv) a glycosyloxy group, (v) an amino group or an N-substituted amino group, (vi) a carboxyl group or a carboxylic acid derivative, (vii) an aldehyde or a ketone group, (viii) a prenyl group, and (ix) a nitrile group, and wherein each non-substituted R.sub.2, R.sub.5, R.sub.6, or R.sub.7 is a hydrogen atom and when R.sub.4 is not substituted with any of the foregoing substituents, R.sub.4 is a hydrogen atom, an O-alkyl group, an O-acyl group, or a phosphate group, wherein R.sub.3a and R.sub.3b are each independently a hydrogen atom, an alkyl group, a cycloalkyl group, an acyl group, or an aryl group, and R.sub.3c is a hydrogen atom or a carboxyl group, the method comprising: [0583] reacting a reactant psilocybin derivative compound having a chemical formula (ll): [0584] wherein, one of R.sub.2, R.sub.4, R.sub.5, R.sub.6, or R.sub.7 is a substituent selected from (i) a halogen atom, (ii) a hydroxy group, (iii) a nitro group, (iv) a glycosyloxy group, (v) an amino group or an N-substituted amino group, (vi) a carboxyl group or a carboxylic acid derivative, (vii) an aldehyde or a ketone group, (viii) a prenyl group, and (ix) a nitrile group, and wherein each non-substituted R.sub.2, R.sub.5, R.sub.6, or R.sub.7 is a hydrogen atom or an alcohol group and when R.sub.4 is not substituted with any of the foregoing substituents, R.sub.4 is a hydrogen atom, a hydroxy group, an O-alkyl group, O-acyl group, or a phosphate group, wherein R.sub.3a and R.sub.3b are each independently a hydrogen atom, an alkyl group, a cycloalkyl group, an acyl group, or an aryl group, and wherein and R.sub.3c is a hydrogen atom or a carboxyl group [0586] a substituent containing compound, wherein the substituent in the substituent containing compound is selected from (i) a halogen containing compound, (ii) a hydroxy group containing compound, (iii) a nitro group containing compound, (iv) a glycosyloxy group containing compound, (v) an amino group or an N-substituted amino group containing compound, (vi) a carboxyl group or a carboxylic acid derivative containing compound, (vii) an aldehyde or a ketone group containing compound, (viii) a prenyl group containing compound, and (ix) a nitrile group containing compound under reaction conditions sufficient to form the psilocybin substituent or salt thereof having chemical formula (l).
[0587] Reactant psilocybin derivative compound having formula (ll) encompasses a plurality of compounds. In general, a reactant psilocybin derivative compound having formula (ll) can be selected, by initially identifying a desired multi-substituent psilocybin derivative compound, and determining the substituent groups therein, and by thereafter selecting an appropriate reactant psilocybin derivative compound having formula (ll). Thus, for example, if it is desirable to prepare a S1.sub.4, S2.sub.6 multi-substituent psilocybin derivative, a S1.sub.4 reactant psilocybin derivative compound may be selected and reacted with an S2 substituent containing compound to form the desired S1.sub.4, S2.sub.6 multi-substituent psilocybin derivative compound, or if it is desirable to prepare a S1.sub.5, S2.sub.6 multi-substituent psilocybin derivative, a S1.sub.5 reactant psilocybin derivative compound may be selected and reacted with an S2 substituent containing compound to form the S1.sub.5, S2.sub.6 multi-substituent psilocybin derivative. Thus, furthermore it can be said that the performance of chemical reactions to make the compounds of the present disclosure, in general involves a substitution at different carbon atoms, i.e., the C.sub.2, C.sub.4, C.sub.5, C.sub.6 and/or C.sub.7 atom.
[0588] Thus, in one example embodiment, to form a multi-substituent psilocybin derivative wherein S1.sub.5 is a chlorine atom, and R.sub.2, R.sub.6, and R.sub.7 are S2.sub.2, S2.sub.6 or S2.sub.7, the reactant psilocybin derivative can be selected to be a chemical compound wherein R.sub.4 is an O-alkyl group, R.sub.2, R.sub.6, and R.sub.7 are a hydrogen atom, R.sub.5 is a chlorine atom, and R.sub.3A and R.sub.3B are a hydrogen atom, an alkyl group, an acyl group, or an aryl group, such as, for example, the reactant psilocybin derivative shown in
[0589] In one further example embodiment, to form a multi-substituent psilocybin derivative wherein S1.sub.5 is a chlorine atom, and R.sub.2, R.sub.6, and R.sub.7 are S2.sub.2, S2.sub.6 or S2.sub.7, the reactant psilocybin derivative can be selected to be a chemical compound wherein R.sub.4 is an O-acyl group, R.sub.2, R.sub.6, and R.sub.7 are a hydrogen atom, R.sub.5 is a chlorine atom, and R.sub.3A and R.sub.3B are a hydrogen atom, an alkyl group, an acyl group, or an aryl group, such as, for example, the reactant psilocybin derivative shown in
[0590] In one further example embodiment, to form a multi-substituent psilocybin derivative wherein S1.sub.5 is a chlorine atom, and R.sub.2, R.sub.6, and R.sub.7 are S2.sub.2, S2.sub.6 or S2.sub.7, the reactant psilocybin derivative can be selected to be a chemical compound wherein R.sub.4 is a hydroxyl group, R.sub.2, R.sub.6, and R.sub.7 are a hydrogen atom, R.sub.5 is a chlorine atom, and R.sub.3A and R.sub.3B are a hydrogen atom, an alkyl group, an acyl group, or an aryl group, such as, for example, the reactant psilocybin derivative shown in
[0591] In one further example embodiment, to form a multi-substituent psilocybin derivative wherein S1.sub.5 is a chlorine atom, and R.sub.2, R.sub.6, and R.sub.7 are S2.sub.2, S2.sub.6 or S2.sub.7, the reactant psilocybin derivative can be selected to be a chemical compound wherein R.sub.4 is a phosphate group, R.sub.2, R.sub.6, and R.sub.7 are a hydrogen atom, R.sub.5 is a chlorine atom, and R.sub.3A and R.sub.3B are a hydrogen atom, an alkyl group, an acyl group, or an aryl group, such as, for example, the reactant psilocybin derivative shown in
[0592] In yet one further example embodiment, to form a multi-substituent psilocybin derivative wherein S1.sub.5 is a chlorine atom, and R.sub.2, R.sub.6, and R.sub.7 are S2.sub.2, S2.sub.6 or S2.sub.7, the reactant psilocybin derivative can be selected to be a chemical compound wherein R.sub.4 is a hydrogen atom, R.sub.2, R.sub.6, and R.sub.7 are a hydrogen atom, R.sub.5 is a chlorine atom, and R.sub.3A and R.sub.3B are a hydrogen atom, an alkyl group, an acyl group, or an aryl group, such as, for example, the reactant psilocybin derivative shown in
[0593] The reactant psilocybin derivative compounds may be provided in a more or less chemically pure form, for example, in the form of a psilocybin derivative preparation having a purity of at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least 99.9%. The psilocybin derivative may be chemically synthesized, or obtained from a fine chemical manufacturer, such as, for example, Sigma-Aldrich® (St. Louis, MO, USA).
[0594] The substituent group containing compound can be any compound comprising a substituent group selected from (i) a halogen atom, (ii) a hydroxy group, (iii) a nitro group, (iv) a glycosyloxy group, (v) an amino group or an N-substituted amino group, (vi) a carboxyl group or a carboxylic acid derivative, (vii) an aldehyde or a ketone group, (viii) a prenyl group, and (ix) a nitrile group capable of reacting with the selected reactant psilocybin derivative compound.
[0595] The substituent group containing compound may be provided in a more or less chemically pure form, for example, having a purity of at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least 99.9%. The nitrile containing compound may be synthesized or purified, or can be conveniently obtained from a fine chemical manufacturer, such as, for example, Sigma-Aldrich® (St. Louis, MO, USA).
[0596] By way of an example, shown in
[0597] Thus, referring to
[0598] In one example embodiment, in an aspect, the formed first psilocybin derivative can be the compound possessing an acetyl group, such as, for example, the compound shown in
[0599] Thus, referring further to
[0600] Continuing to refer to
[0601] Continuing to refer to
[0602] Continuing to refer to
[0603] Continuing to refer to
[0604] Continuing to refer to
[0605] Referring next to
[0606] Referring further to
[0607] Continuing to refer to
[0608] Continuing to refer to
[0609] Continuing to refer to
[0610] Continuing to refer to
[0611] Continuing to refer to
[0612] Continuing to refer to
[0613] Thus, referring to
[0614] Referring further to
[0615] Referring further to
[0616] Referring further to
[0617] Referring further to
[0618] Referring further to
[0619] Referring further to
[0620] Thus, in general, a reactant psilocybin derivative is provided, and the reactant psilocybin derivative is employed to react in a chemical reaction resulting in the formation of a multiple-substituent psilocybin derivatives.
[0621] The reactions, such as the example reaction shown in
[0622] Turning next to biosynthetic methods to make the multiple-substituent compounds of the present disclosure, such methods, in general, involve the use of psilocybin biosynthetic enzyme complement to enzymatically catalyze the conversion of a psilocybin derivative precursor compound and form a multi-substituent psilocybin derivative compound. The enzymes included in the psilocybin biosynthetic enzyme complement may vary, as hereinafter will be discussed with reference to certain example enzymes and example compounds shown in
[0623] Thus, in one aspect, the present disclosure further provides a method of making a multi-substituent psilocybin derivative, the method comprising contacting a psilocybin derivative precursor compound having a formula (LVII):
##STR00224##
[0624] wherein at least one of R.sub.2, R.sub.4, R.sub.5, R.sub.6, or R.sub.7 is a substituent selected from (i) a halogen atom, (ii) a hydroxy group, (iii) a nitro group, (iv) a glycosyloxy group, (v) an amino group or an N-substituted amino group, (vi) a carboxyl group or a carboxylic acid derivative, (vii) an aldehyde or a ketone group, (viii) a prenyl group, and (ix) a nitrile group, and wherein each non-substituted R.sub.2, R.sub.5, R.sub.6, or R.sub.7 is a hydrogen atom and when R.sub.4 is not substituted with any of the foregoing substituents, R.sub.4 is a hydrogen atom, an O-alkyl group, an O-acyl group, or a phosphate group, and wherein R.sub.3 is a hydrogen atom or —CH.sub.2—CHNH.sub.2COOH or —CH.sub.2—CH.sub.2NH.sub.2, with a catalytic quantity of a psilocybin biosynthetic enzyme complement under reaction conditions permitting an enzyme catalyzed conversion of the psilocybin derivative precursor compound to form a multi-substituent psilocybin derivative compound having a formula (l):
##STR00225##
[0625] wherein, at least two of R.sub.2, R.sub.4, R.sub.5, R.sub.6, or R.sub.7 are substituents independently selected from at least two of (i) a halogen atom, (ii) a hydroxy group, (iii) a nitro group, (iv) a glycosyloxy group, (v) an amino group or an N-substituted amino group, (vi) a carboxyl group or a carboxylic acid derivative, (vii) an aldehyde or a ketone group, (viii) a prenyl group, and (ix) a nitrile group, and wherein each non-substituted R.sub.2, R.sub.5, R.sub.6, or R.sub.7 is a hydrogen atom and when R.sub.4 is not substituted with any of the foregoing substituents, R.sub.4 is a hydrogen atom, an O-alkyl group, an O-acyl group, or a phosphate group, wherein R.sub.3a and R.sub.3b are each independently a hydrogen atom, an alkyl group, a cycloalkyl group, an acyl group, or an aryl group, and wherein and R.sub.3c is a hydrogen atom or a carboxyl group.
[0626] The reaction conditions can be in vitro reaction conditions, or in vivo reaction conditions, or a combination thereof.
In Vitro Synthesis
[0627] In vitro synthesis, in general, involves initially providing the reagents, including the precursor psilocybin derivative compound and other reactants, in a more or less pure form. Thus, the reactants may be provided as a particulate in a substantially pure form, or they may be dissolved, in a more or less pure form, in a suitable solvent or diluent, such as water or a buffer. The reagents can then be combined and contacted with one another in a suitable reaction vessel, such as a tube, beaker, flask, or the like, or, at a larger scale, in a tank or reactor, generally preferably in liquid form, which may be prepared by further including a diluent, such as water or a buffer, as necessary. The combined reagents may be mixed, by, in general, gentle stirring, using a suitable stirring or mixing device, such as a laboratory size magnetic stirrer (e.g., as manufactured by Fisher Scientific®), or a handheld or industrial mixer, for example, to form a mixture. Relative quantities and absolute quantities of reagents may be selected as desired. Absolute quantities will typically depend on the scale one wishes to perform the reaction at, such as, for example, at a laboratory scale (e.g., at a less than 1 L, a less than 100 mL, a less than 10 mL, or a less than 1 mL scale), or, for example, at a commercial production scale (e.g., at a more than 100 L, a more than 1,000 L, or a more than 10,000 L scale). Relative quantities of the reagents may vary. Thus, for example, in one embodiment, stoichiometric quantities of each of a precursor psilocybin derivative and a substituent containing compound can be mixed with catalytic quantities of enzymes. If desired, off-stoichiometric quantities of reagents, for example, a molar ratio of psilocybin precursor derivative to substituent containing compound of 1 : 0.95; 1 : 0.9; 1 : 0.75; or 1 : 1.05, 1 : 1.1 or 1 : 1.25, may be selected.
[0628] As will be understood by those of skill in the art, in molar quantity terms, small quantities of enzyme suffice to conduct the reaction, since the enzyme acts as a catalytic agent, and, unlike the precursor psilocybin derivatives and the substituent containing compound, the enzyme is not consumed in the reaction. Thus, in general terms, catalytic quantities can be thought of as the at least minimal quantity of enzyme required to convert precursor psilocybin derivatives and the substituent containing compound, and form desirable quantities of multi-substituent psilocybin derivatives. Thus, for example, from 0.1 to 1,000 enzyme units (e.g., 0.1 enzyme unit, 1 enzyme unit, 10 enzyme units, 50 enzyme units, 100 enzyme units, 250 enzyme units, 500 enzyme units, or 1,000 enzyme units) may be included in a reaction mixture, wherein, as is known to those of skill in the art, 1 enzyme unit is an amount of enzyme that catalyzes 1 .Math.mole of substrate (i.e., psilocybin precursor compound) per minute. Furthermore, in vitro reaction conditions may vary and may include temperatures ranging from, for example, between about 18° C. and about 37° C., and a pH in the range of about pH 5.0 to about pH 8.5. Furthermore, other agents may be included to facilitate catalysis, for example, a diluent (e.g., water or a buffer), salts, and pH modifying agents. The in vitro reaction conditions may be adjusted and optimized, for example, by preparing a plurality of samples, each being reacted at a different operating condition, e.g., at a different temperature, a different pH, including a different quantity of enzyme, including different relative quantities of reagents, and so forth, and detecting the formed multi-substituent psilocybin derivative.
In Vivo Synthesis
[0629] In one embodiment, the psilocybin derivative precursor compound and the substituent containing compound can be contacted with the psilocybin biosynthetic enzyme complement in a host cell, wherein the host cell comprises a chimeric nucleic acid sequence comprising as operably linked components: [0630] (i) a nucleic acid sequence controlling expression in the host cell; and [0631] (ii) a nucleic acid sequence encoding psilocybin biosynthetic enzyme complement, and the host cell is grown to express the psilocybin biosynthetic enzyme complement and to produce the multi-substituent psilocybin derivative compound.
[0632] Suitable chimeric nucleic acid sequences include any nucleic acid sequence comprising a nucleic acid sequence controlling expression in the host cell operably linked to a sequence encoding psilocybin biosynthetic enzyme complement, such as, for example, one or more of tryptophan synthase subunit B polypeptide, tryptophan decarboxylase, N-acetyl transferase, N-methyl transferase, and prenyl transferase, as herein after further described.
[0633] Nucleic acid sequences capable of controlling expression of a nucleic acid sequence encoding biosynthetic enzyme complement in host cells that can be used herein include any transcriptional promoter capable of controlling expression of polypeptides in host cells. Generally, promoters obtained from bacterial cells are used when a bacterial host is selected in accordance herewith, while a fungal promoter will be used when a fungal host cell is selected, a plant promoter will be used when a plant cell is selected, and so on. Specific examples that can be used, for example for expression in yeast cells include a galactose inducible promoter, such as a Gal10/Gal 1 promoter, or for expression in Escherichia coli cells, a beta-galactosidase promoter. Further nucleic acid elements capable elements of controlling expression in a host cell include transcriptional terminators, enhancers, and the like, all of which may be included in the chimeric nucleic acid sequences of the present disclosure.
[0634] The chimeric nucleic acid sequences can be integrated into a recombinant expression vector which ensures good expression in the host cell, wherein the expression vector is suitable for expression in a host cell. The term “suitable for expression in a host cell” means that the recombinant expression vector comprises the chimeric nucleic acid sequence linked to genetic elements required to achieve expression in a cell. Genetic elements that may be included in the expression vector in this regard include a transcriptional termination region, one or more nucleic acid sequences encoding marker genes, one or more origins of replication and the like. In preferred embodiments, the expression vector further comprises genetic elements required for the integration of the vector or a portion thereof in the host cell’s genome, for example. If a plant host cell is used the T-DNA left and right border sequences which facilitate the integration into the plant’s nuclear genome.
[0635] Pursuant to the present disclosure, the expression vector may further contain a marker gene. Marker genes that may be used in accordance with the present disclosure include all genes that allow the distinction of transformed cells from non-transformed cells, including all selectable and screenable marker genes. A marker gene may be a resistance marker such as an antibiotic resistance marker against, for example, kanamycin or ampicillin, or an auxotrophic marker, for example, a leu marker (Sikorski and Hieter, 1989, Genetics 122(1): 19-27) or a ura marker (Rose and Winston, 1984, Mol. Gen. Genet. 193 (3): 557-560. Screenable markers that may be employed to identify transformants through visual inspection include β-glucuronidase (GUS) (U.S. Pat. Nos. 5,268,463 and 5,599,670) and green fluorescent protein (GFP) (Niedz et al., 1995, Plant Cell Rep., 14: 403).
[0636] A variety of host cells can be used in accordance herewith. The selected host cell may be able to naturally produce psilocybin compounds, or derivatives thereof or the cell may not be able to naturally produce psilocybin compounds or derivatives thereof. Host cells, upon the introduction of the chimeric nucleic acid sequence can be said to be able to heterologously express psilocybin biosynthetic enzyme complement.
[0637] In some embodiments, the host cell can be a microbial cell, for example, bacterial cell or a yeast cell. An example bacterial cell that can be used in accordance herewith is an Escherichia coli cell. Example yeast cells that can be in accordance herewith are a Saccharomyces cerevisiae cell or a Yarrowia lipolytica cell.
[0638] In a further embodiment, the host cell can be a plant cell or an algal cell.
[0639] A variety of techniques and methodologies to manipulate host cells to introduce nucleic acid sequences, including expression vectors comprising the chimeric nucleic acid sequences of the current disclosure, in cells and attain expression exists and are well known to the skilled artisan. These methods include, for example, cation based methods, for example, lithium ion or calcium ion based methods, electroporation, biolistics, and glass beads based methods. As will be known to those of skill in the art, depending on the host cell selected, the methodology to introduce nucleic acid material in the host cell may vary, and, furthermore, methodologies may be optimized for uptake of nucleic acid material by the host cell, for example, by comparing uptake of nucleic acid material using different conditions. Detailed guidance can be found, for example, in Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2012, Fourth Ed. It is noted that the chimeric nucleic acid is a non-naturally occurring chimeric nucleic acid sequence and can be said to be heterologous to the host cell.
[0640] One example host cell that conveniently may be used is Escherichiacoli. The preparation of the E.coli vectors may be accomplished using commonly known techniques such as restriction digestion, ligation, gel electrophoresis, DNA sequencing, the polymerase chain reaction (PCR) and other methodologies. A wide variety of cloning vectors is available to perform the necessary steps required to prepare a recombinant expression vector. Among the vectors with a replication system functional in E.coli, are vectors such as pBR322, the pUC series of vectors, the M13 mp series of vectors, pBluescript etc. Suitable promoter sequences for use in E.coli include, for example, the T7 promoter, the T5 promoter, tryptophan (trp) promoter, lactose (lac) promoter, tryptopllan/lactose (tac) promoter, lipoprotein (ipp) promoter, and λ phage PL promoter. Typically, cloning vectors contain a marker, for example, an antibiotic resistance marker, such as ampicillin or kanamycin resistance marker, allowing selection of transformed cells. Nucleic acid sequences may be introduced in these vectors, and the vectors may be introduced in E.coli by preparing competent cells, electroporation or using other well-known methodologies to a person of skill in the art. E.coli may be grown in an appropriate medium, such as Luria-Broth medium and harvested. Recombinant expression vectors may readily be recovered from cells upon harvesting and lysing of the cells.
[0641] Another example host cell that may be conveniently used is a yeast cell. Example yeast host cells that can be used are yeast cells belonging to the genus Candida, Kluyveromyces, Saccharomyces, Schizosaccharomyces, Pichia, Hansenuia, and Yarrowia. In specific example embodiments, the yeast cell can be a Saccharomyces cerevisiae cell, a Yarrowialipolytica cell, or Pichiapastoris cell.
[0642] A number of vectors exist for the expression of recombinant proteins in yeast host cells. Examples of vectors that may be used in yeast host cells include, for example, Yip type vectors, Yep type vectors, Yrp type vectors, Ycp type vectors, pGPD-2, pA0815, pGAPZ, pGAPZa, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, pPICZ, pPICZa, pPIC3K, pHWO10, pPUZZLE and 2 .Math.m plasmids. Such vectors are known to the art and are, for example, described in Cregg et al., Mol Biotechnol. (2000) 16(1): 23-52. Suitable promoter sequences for use in yeast host cells are also known and described, for example, in Mattanovich et al, Methods Mol. Biol., 2012, 824:329-58, and in Romanos et al, 1992, Yeast 8: 423-488. Examples of suitable promoters for use in yeast host cells include promoters of glycolytic enzymes, like triosephosphate isomerase (TPI), phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH or GAP) and variants thereof, lactase (LAC) and galactosidase (GAL), P.pastoris glucose-6-phosphate isomerase promoter (PPGI), the 3-phosphoglycerate kinase promoter (PPGK), the glycerol aldehyde phosphate dehydrogenase promoter (PGAP), translation elongation factor promoter (PTEF), S.cerevisiae enolase (ENO-1), S.cerevisiaegalactokinase (GAL1), S.cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP), S.cerevisiae triose phosphate isomerase (TPI), S.cerevisiae metallothionein (CUP′1), and S.cerevisiae 3-phosphoglycerate kinase (PGK), and the maltase gene promoter (MAL). Marker genes suitable for use in yeast host cells are also known to the art. Thus, antibiotic resistance markers, such as ampicillin resistance markers, can be used in yeast, as well as marker genes providing genetic functions for essential nutrients, for example, leucine (LEU2), tryptophan (TRP1 and TRP2), uracil (URA3, URA5, URA6), histidine (HIS3), and the like. Methods for introducing vectors into yeast host cells can, for example, be found in S. Kawai et al., 2010, Bioeng. Bugs 1 (6.), 395-403.
[0643] Further, guidance with respect to the preparation of expression vectors and introduction thereof into host cells, including in E. coli cells, yeast cells, and other host cells, may be found in, for example: Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2012, Fourth Ed.
[0644] Referring next to
##STR00226##
[0645] wherein, at least two of R.sub.2, R.sub.4, R.sub.5, R.sub.6, or R.sub.7 are substituents independently selected from at least two of (i) a halogen atom, (ii) a hydroxy group, (iii) a nitro group, (iv) a glycosyloxy group, (v) an amino group or an N-substituted amino group, (vi) a carboxyl group or a carboxylic acid derivative, (vii) an aldehyde or a ketone group, (viii) a prenyl group, and (ix) a nitrile group, and wherein each non-substituted R.sub.2, R.sub.5, R.sub.6, or R.sub.7 is a hydrogen atom and when R.sub.4 is not substituted with any of the foregoing substituents, R.sub.4 is a hydrogen atom, an O-alkyl group, an O-acyl group, or a phosphate group, wherein R.sub.3a and R.sub.3b are each independently a hydrogen atom, an alkyl group, a cycloalkyl group, an acyl group, or an aryl group, and wherein and R.sub.3c is a hydrogen atom or a carboxyl group.
[0646] Following the teachings set forth herein, including by referring to the examples shown in
[0647] Thus, referring further to
[0655] A first psilocybin derivative precursor compound having formula (I) can be used wherein two of R.sub.2, R.sub.4, R.sub.5, R.sub.6, or R.sub.7 are a substituent independently selected from (i) a halogen atom, (ii) a hydroxy group, (iii) a nitro group, (iv) a glycosyloxy group, (v) an amino group or an N-substituted amino group, (vi) a carboxyl group or a carboxylic acid derivative, (vii) an aldehyde or a ketone group, (viii) a prenyl group, and (ix) a nitrile group, wherein R.sub.3 is a hydrogen atom. A first multi-substituent psilocybin derivative compound having formula (I) can be formed wherein R.sub.3c is a carboxyl group. For example, a psilocybin derivative precursor compound can be a chemical compound having a formula (LVIII):
##STR00227##
wherein R.sub.4 is a hydroxy group, and wherein R.sub.6 is a chlorine atom, and a first multi-substituent psilocybin derivative compound has a formula (LV):
##STR00228##
can be formed in an in vivo or in vitro reaction catalyzed by a tryptophan synthase subunit B polypeptide.
[0656] Continuing to refer to
[0664] Continuing to refer to
##STR00229##
wherein R.sub.4 is a fluorine atom, an amino group, or a hydroxy group, wherein R.sub.6 is a fluorine atom, an amino group, a nitrile, or a bromine atom, and a first multi-substituent psilocybin derivative compound has a formula (LIX):
##STR00230##
wherein R.sub.4 is a fluorine atom, an amino group, or a hydroxy group, wherein R.sub.6 is a fluorine atom, an amino group, a nitrile, or a bromine atom can be formed. The first multi-substituent psilocybin can be decarboxylated, and a second multi-substituent psilocybin can be formed, for example, a second multi-substituent psilocybin derivative having a formula (XXII), (XXVI), (XXIX), (LII), or (LlV):
##STR00231##
##STR00232##
##STR00233##
##STR00234##
##STR00235##
[0665] A psilocybin biosynthetic enzyme complement can further, for example, comprise an N-acetyl transferase to acetylate the second psilocybin derivative having chemical formula (I) and thereby form a third multi-substituent psilocybin having chemical formula (I), wherein R.sub.3a is a hydrogen atom and R.sub.3b is an acetyl group. A N-acetyl transferase can be encoded by a nucleic acid sequence selected from: [0666] (a) SEQ.ID NO: 9; [0667] (b) a nucleic acid sequence that is substantially identical to the nucleic acid sequence of (a); [0668] (c) a nucleic acid sequence that is substantially identical to the nucleic acid sequence of (a) but for the degeneration of the genetic code; [0669] (d) a nucleic acid sequence that is complementary to the nucleic acid sequence of (a); [0670] (e) a nucleic acid sequence encoding a polypeptide having the amino acid sequence set forth in SEQ.ID NO: 10; [0671] (f) a nucleic acid sequence that encodes a functional variant of the amino acid sequence set forth in SEQ.ID NO: 10; and [0672] (g) a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a), (b), (c), (d), (e) or (f).
[0673] Thus, continuing to refer to
##STR00236##
wherein R.sub.4 is an acetamidyl group, a fluorine atom, an amino group, or a hydroxy group, wherein R.sub.6 is a fluorine atom, an amino group, a nitrile, or a bromine atom, and a first multi-substituent psilocybin derivative compound having a formula (LIX):
##STR00237##
wherein R.sub.4 is an acetamidyl group, a fluorine atom, an amino group, or a hydroxy group, wherein R.sub.6 is a fluorine atom, an amino group, a nitrile, or a bromine atom can be formed. Then the first multi-substituent psilocybin derivative can be decarboxylated and a second multi-substituent psilocybin derivative having a formula (LX):
##STR00238##
can be formed. Thereafter, the second multi-substituent psilocybin derivative can be acetylated, and a third multi-substituent psilocybin derivative can be formed, for example, a third multi-substituent psilocybin derivative having a formula (IX), (X), (XVIII), (XXI), (XXV), or (XXVIII):
##STR00239##
##STR00240##
##STR00241##
##STR00242##
##STR00243##
##STR00244##
[0674] A psilocybin biosynthetic enzyme complement, in accordance herewith can further, for example, comprise an N-methyl transferase to methylate the R.sub.3 amino group at R.sub.3 and form a fourth multi-substituent psilocybin derivative having a chemical formula (I), wherein R.sub.3a and R.sub.3b are each a methyl group, or wherein R.sub.3a is a hydrogen atom and R.sub.3b is a methyl group. A N-methyl transferase can be encoded by a nucleic acid sequence selected from: [0675] (a) SEQ.ID NO: 11 and SEQ.ID NO 13; [0676] (b) a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a); [0677] (c) a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a) but for the degeneration of the genetic code; [0678] (d) a nucleic acid sequence that is complementary to any one of the nucleic acid sequences of (a); [0679] (e) a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 12 and SEQ.ID NO 14; [0680] (f) a nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 12 and SEQ.ID NO 14; and [0681] (g) a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a), (b), (c), (d), (e) or (f).
[0682] Continuing to refer to
##STR00245##
wherein R.sub.4 is an amino group or a hydroxy group, wherein R.sub.6 is a chlorine atom, a nitrile group, or a bromine atom, and a first multi-substituent psilocybin derivative compound having a formula (LIX):
##STR00246##
wherein R.sub.4 is an amino group or a hydroxy group, wherein R.sub.6 is a chlorine atom, a nitrile group, or a bromine atom can be formed. The first multi-substituent psilocybin derivative compound can be decarboxylated to form a second multi-substituent psilocybin derivative compound, wherein the second multi-substituent psilocybin derivative has a formula (LX):
##STR00247##
The third multi-substituent psilocybin derivative compound can be methylated to form a fourth multi-substituent psilocybin derivative, for example, a fourth multi-substituent psilocybin derivative compound having a formula (XXVII), (XL), or (LIII):
##STR00248##
##STR00249##
##STR00250##
[0683] In accordance herewith, a psilocybin biosynthetic enzyme complement can, in a further example embodiment, comprise a prenyl transferase, encoded by a nucleic acid selected from: [0684] (a) SEQ.ID NO: 15, SEQ.ID NO: 17, SEQ.ID NO: 19, and SEQ.ID NO 21; [0685] (b) a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a); [0686] (c) a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a) but for the degeneration of the genetic code; [0687] (d) a nucleic acid sequence that is complementary to any one of the nucleic acid sequences of (a); [0688] (e) a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID NO: 20, and SEQ.ID NO 22; [0689] (f) a nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID NO: 20, and SEQ.ID NO 22; and [0690] (g) a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a), (b), (c), (d), (e) or (f).
[0691] Referring, in this respect, next to
##STR00251##
wherein R.sub.4 is a hydroxy group, and a first multi-substituent psilocybin derivative compound can be formed, for example, a first formed multi-substituent psilocybin derivative compound having a formula (L):
##STR00252##
[0692] Continuing to referring
[0700] A psilocybin biosynthetic enzyme complement can in a further example embodiment, comprise an N-methyl transferase to methylate the R.sub.3 amino group at R.sub.3 and form a fourth multi-substituent psilocybin derivative having a chemical formula (I), wherein R.sub.3a and R.sub.3b are each a methyl group, or wherein R.sub.3a is a hydrogen atom and R.sub.3b is a methyl group. A N-methyl transferase encoded by a nucleic acid sequence can be selected from: [0701] (a) SEQ.ID NO: 11, and SEQ.ID NO 13; [0702] (b) a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a); [0703] (c) a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a) but for the degeneration of the genetic code; [0704] (d) a nucleic acid sequence that is complementary to any one of the nucleic acid sequences of (a); [0705] (e) a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 12 and SEQ.ID NO 14; [0706] (f) a nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 12, and SEQ.ID NO 14; and [0707] (g) a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a), (b), (c), (d), (e) or (f).
[0708] Continuing to referring
##STR00253##
wherein R.sub.4 is a propionyloxy or an acetoxy group, and a first formed multi-substituent psilocybin derivative compound having a formula (LIX):
##STR00254##
wherein R.sub.4 is a propionyloxy or an acetoxy group, wherein R.sub.6 is a prenyl group is formed. The first multi-substituent psilocybin derivative compound can be decarboxylated to form a second multi-substituent psilocybin derivative having a formula:
##STR00255##
wherein R.sub.4 is a propionyloxy or an acetoxy group, wherein R.sub.6 is a prenyl group. The second multi-substituent psilocybin derivative can be methylated to for a third multi-substituent psilocybin derivative having a formula (XLI) or (XLII):
##STR00256##
##STR00257##
[0709] Referring further to
[0710] Referring next to
##STR00258##
wherein R.sub.5 is a chlorine or a fluorine atom, and a first multi-substituent psilocybin derivative compound having a formula (LXIII):
##STR00259##
wherein R.sub.5 is a chlorine or a fluorine atom, and wherein R.sub.6 is a prenyl group can be formed. The first multi-substituent psilocybin derivative can be decarboxylated to form a second multi-substituent psilocybin derivative compound having a formula (XXXVI) or (XXXVIII):
##STR00260##
##STR00261##
[0711] In one further example embodiment, a psilocybin biosynthetic enzyme complement can further comprise an N-acetyl transferase to acetylate the second psilocybin derivative having chemical formula (I) and thereby form a third multi-substituent psilocybin having chemical formula (I), wherein R.sub.3a is a hydrogen atom and R.sub.3b is an acetyl group, the N-acetyl transferase encoded by a nucleic acid sequence selected from: [0712] (a) SEQ.ID NO: 9; [0713] (b) a nucleic acid sequence that is substantially identical to the nucleic acid sequence of (a); [0714] (c) a nucleic acid sequence that is substantially identical to the nucleic acid sequence of (a) but for the degeneration of the genetic code; [0715] (d) a nucleic acid sequence that is complementary to the nucleic acid sequence of (a); [0716] (e) a nucleic acid sequence encoding a polypeptide having the amino acid sequence set forth in SEQ.ID NO: 10; [0717] (f) a nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 10; and [0718] (g) a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a), (b), (c), (d), (e) or (f).
[0719] Continuing to refer to
##STR00262##
wherein R.sub.5 is a chlorine or a fluorine atom, and a first multi-substituent psilocybin derivative compound having a formula (LXIII):
##STR00263##
wherein R.sub.5 is a chlorine or a fluorine atom, and wherein R.sub.6 is a prenyl group can be formed. The first multi-substituent psilocybin derivative compound can be decarboxylated to form a second formed multi-substituent psilocybin derivative compound having a formula (LXIV):
##STR00264##
wherein R.sub.5 is a chlorine or a fluorine atom, and wherein R.sub.6 is a prenyl group. The second multi-substituent psilocybin derivative compound can be acetylated to form a wherein third multi-substituent psilocybin derivative having a formula (XXXV) or (XXXVII):
##STR00265##
##STR00266##
[0720] Referring further to
[0721] Referring next to
[0729] A first psilocybin derivative precursor compound having formula (I) can be used wherein two of R.sub.2, R.sub.4, R.sub.5, R.sub.6, or R.sub.7 are a substituent independently selected from (i) a halogen atom, (ii) a hydroxy group, (iii) a nitro group, (iv) a glycosyloxy group, (v) an amino group or an N-substituted amino group, (vi) a carboxyl group or a carboxylic acid derivative, (vii) an aldehyde or a ketone group, (viii) a prenyl group, and (ix) a nitrile group, wherein R.sub.3 is a hydrogen atom. A first multi-substituent psilocybin derivative compound having formula (I) can be formed wherein R.sub.3c is a carboxyl group. For example, a psilocybin derivative precursor compound can be a chemical compound having a formula: (LXV):
##STR00267##
wherein R.sub.4 is a hydroxy group, and wherein R.sub.5 is a prenyl group, and a first formed multi-substituent psilocybin derivative compound having formula (LI):
##STR00268##
can be formed.
[0730] The psilocybin biosynthetic enzyme complement can further comprise a tryptophan decarboxylase to decarboxylate the R.sub.3 —CH.sub.2—CHNH.sub.2COOH group, and thereby form a second multi-substituent psilocybin derivative having formula (I) wherein an R.sub.3a and R.sub.3b each are a hydrogen atom, the tryptophan decarboxylase encoded by a nucleic acid sequence selected from: [0731] (a) SEQ.ID NO: 3, SEQ.ID NO: 5, and SEQ.ID NO: 7; [0732] (b) a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a); [0733] (c) a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a) but for the degeneration of the genetic code; [0734] (d) a nucleic acid sequence that is complementary to any one of the nucleic acid sequences of (a); [0735] (e) a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 4, SEQ.ID NO: 6, SEQ.ID NO: and SEQ.ID NO 8; [0736] (f) a nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 4, SEQ.ID NO: 6 and SEQ.ID NO 8; and [0737] (g) a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a), (b), (c), (d), (e) or (f).
[0738] Continuing to refer to
##STR00269##
wherein R.sub.4 is a fluorine atom and R.sub.5 is nitrile group, a first formed multi-substituent psilocybin derivative compound having a formula (LXIX):
##STR00270##
wherein R.sub.4 is a fluorine atom and wherein R.sub.5 is a nitrile group can be formed. A second formed multi-substituent psilocybin derivative compound can then be formed by decarboxylating the first multi-substituent derivative compound, the second multi-substituent psilocybin derivative compound having a formula (XIX):
##STR00271##
[0739] In a further embodiment, the psilocybin biosynthetic enzyme complement can further comprise an N-acetyl transferase to acetylate the second psilocybin derivative having chemical formula (I) and thereby form a third multi-substituent psilocybin having chemical formula (I), wherein R.sub.3a is a hydrogen atom and R.sub.3b is an acetyl group, the N-acetyl transferase encoded by a nucleic acid sequence selected from: [0740] (a) SEQ.ID NO: 9; [0741] (b) a nucleic acid sequence that is substantially identical to the nucleic acid sequence of (a); [0742] (c) a nucleic acid sequence that is substantially identical to the nucleic acid sequence of (a) but for the degeneration of the genetic code; [0743] (d) a nucleic acid sequence that is complementary to the nucleic acid sequence of (a); [0744] (e) a nucleic acid sequence encoding a polypeptide having the amino acid sequence set forth in SEQ.ID NO: 10; [0745] (f) a nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 10; and [0746] (g) a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a), (b), (c), (d), (e) or (f).
[0747] Continuing to refer to
##STR00272##
wherein R.sub.4 is a fluorine atom and R.sub.5 is a hydroxy group or a nitrile group, and a first formed multi-substituent psilocybin derivative compound having a formula (LXIX):
##STR00273##
wherein R.sub.4 is a fluorine atom and wherein R.sub.5 is a hydroxy group or a nitrile group can be formed. The first formed multi-substituent psilocybin derivative can be decarboxylated to form a second multi-substituent psilocybin derivative compound having a formula (LXXII):
##STR00274##
wherein R.sub.4 is a fluorine atom, and wherein R.sub.5 is a hydroxy group or a nitrile group. The second formed multi-substituent psilocybin derivative can then be acetylated to form a third multi-substituent psilocybin derivative having a formula (XVII) or (XX):
##STR00275##
##STR00276##
[0748] Referring next to
[0758] In a further embodiment, the psilocybin biosynthetic enzyme complement can further comprise a tryptophan decarboxylase to decarboxylate the R.sub.3 —CH.sub.2—CHNH.sub.2COOH group, and thereby form a second multi-substituent psilocybin derivative having formula (I) wherein an R.sub.3a and R.sub.3b each are a hydrogen atom, the tryptophan decarboxylase encoded by a nucleic acid sequence selected from: [0759] (a) SEQ.ID NO: 3, SEQ.ID NO: 5, and SEQ.ID NO: 7; [0760] (b) a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a); [0761] (c) a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a) but for the degeneration of the genetic code; [0762] (d) a nucleic acid sequence that is complementary to any one of the nucleic acid sequences of (a); [0763] (e) a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 4, SEQ.ID NO: 6, SEQ.ID NO: and SEQ.ID NO 8; [0764] (f) a nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 4, SEQ.ID NO: 6 and SEQ.ID NO 8; and [0765] (g) a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a), (b), (c), (d), (e) or (f).
[0766] Continuing to refer to
##STR00279##
wherein R.sub.4 is a fluorine atom and R.sub.7 is nitrile group, and a first multi-substituent psilocybin derivative compound having a formula (LXX):
##STR00280##
wherein R.sub.4 is a fluorine atom and wherein R.sub.7 is a nitrile group, can be formed. A second formed multi-substituent psilocybin derivative compound having a formula (XXIV):
##STR00281##
can be formed by decarboxylating the first multi-substituent psilocybin derivative compound.
[0767] In a further embodiment, the psilocybin biosynthetic enzyme complement can further comprise an N-acetyl transferase to acetylate the second psilocybin derivative having chemical formula (I) and thereby form a third multi-substituent psilocybin having chemical formula (I), wherein R.sub.3a is a hydrogen atom and R.sub.3b is an acetyl group, the N-acetyl transferase encoded by a nucleic acid sequence selected from: [0768] (a) SEQ.ID NO: 9; [0769] (b) a nucleic acid sequence that is substantially identical to the nucleic acid sequence of (a); [0770] (c) a nucleic acid sequence that is substantially identical to the nucleic acid sequence of (a) but for the degeneration of the genetic code; [0771] (d) a nucleic acid sequence that is complementary to the nucleic acid sequence of (a); [0772] (e) a nucleic acid sequence encoding a polypeptide having the amino acid sequence set forth in SEQ.ID NO: 10; [0773] (f) a nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 10; and [0774] (g) a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a), (b), (c), (d), (e) or (f).
[0775] Continuing to refer to
##STR00282##
wherein R.sub.4 is a fluorine atom or a chlorine atom and R.sub.7 is a prenyl group or a nitrile group, and a multi-substituent psilocybin derivative compound having a formula (LXX):
##STR00283##
wherein R.sub.4 is a fluorine atom or a chlorine atom and wherein R.sub.7 is a prenyl group or a nitrile group can be formed. Upon decarboxylation of the first multi-substituent psilocybin derivative compound, a second multi-substituent psilocybin derivative compound having a formula (LXXIII):
##STR00284##
wherein R.sub.4 is a fluorine atom or a chlorine atom, and wherein R.sub.7 is a prenyl group or a nitrile group, can be formed. Following acetylation, a third multi-substituent psilocybin derivative having a formula (XXIII) or (XX):
##STR00285##
##STR00286##
can be formed.
[0776] The psilocybin biosynthetic enzyme complement can further comprise an N-methyl transferase to methylate the R.sub.3 amino group at R.sub.3 and form a further multi-substituent psilocybin derivative having a chemical formula (I), wherein R.sub.3a and R.sub.3b are each a methyl group, or wherein R.sub.3a is a hydrogen atom and R.sub.3b is a methyl group, the N-methyl transferase encoded by a nucleic acid sequence selected from: [0777] (a) SEQ.ID NO: 11, and SEQ.ID NO 13; [0778] (b) a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a); [0779] (c) a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a) but for the degeneration of the genetic code; [0780] (d) a nucleic acid sequence that is complementary to any one of the nucleic acid sequences of (a); [0781] (e) a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 12 and SEQ.ID NO 14; [0782] (f) a nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 12, and SEQ.ID NO 14; and [0783] (g) a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a), (b), (c), (d), (e) or (f).
[0784] In one embodiment, for example, the psilocybin derivative precursor compound can be a chemical compound having a formula (LXVI):
##STR00287##
wherein R.sub.4 is a chlorine atom and R.sub.7 is a hydroxy group, and a first formed multi-substituent psilocybin derivative compound has a formula (LXX):
##STR00288##
wherein R.sub.4 is a hydroxy group and wherein R.sub.7 is a chlorine atom can be formed. Following decarboxylation, a second formed multi-substituent psilocybin derivative compound having a formula (LXXIII):
##STR00289##
wherein R.sub.4 is a hydroxy group, and wherein R.sub.7 is a chlorine atom can be formed Following methylation a third multi-substituent psilocybin derivative having a formula (XXXIX):
##STR00290##
can be formed.
[0785] Referring next to
[0800] Continuing to refer to
##STR00291##
wherein R.sub.5 is a fluorine atom, a chlorine atom, or a nitrile group and R.sub.6 is a fluorine atom, an amino group or a prenyl group, and a first formed multi-substituent psilocybin derivative compound has a formula (LXIII):
##STR00292##
wherein R.sub.5 is a fluorine atom, a chlorine atom, or a nitrile group and wherein R.sub.6 is a is a fluorine atom, an amino group or a prenyl group, can be formed. Following decarboxylation, a second formed multi-substituent psilocybin derivative compound having a formula (XI), (XVI), (XXXVI) or (XXXVIII):
##STR00293##
##STR00294##
##STR00295##
##STR00296##
can be formed.
[0801] The psilocybin biosynthetic enzyme complement can further comprise an N-acetyl transferase to acetylate the second psilocybin derivative having chemical formula (I) and thereby form a third multi-substituent psilocybin having chemical formula (I), wherein R.sub.3a is a hydrogen atom and R.sub.3b is an acetyl group, the N-acetyl transferase encoded by a nucleic acid sequence selected from: [0802] (a) SEQ.ID NO: 9; [0803] (b) a nucleic acid sequence that is substantially identical to the nucleic acid sequence of (a); [0804] (c) a nucleic acid sequence that is substantially identical to the nucleic acid sequence of (a) but for the degeneration of the genetic code; [0805] (d) a nucleic acid sequence that is complementary to the nucleic acid sequence of (a); [0806] (e) a nucleic acid sequence encoding a polypeptide having the amino acid sequence set forth in SEQ.ID NO: 10; [0807] (f) a nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 10; and [0808] (g) a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a), (b), (c), (d), (e) or (f).
[0809] Continuing to refer to
##STR00297##
wherein R.sub.5 is a fluorine atom or a chlorine atom and R.sub.6 is an amino group, an acetamidyl group, or a prenyl group, and a first formed multi-substituent psilocybin derivative compound having a formula (LXIII):
##STR00298##
wherein R.sub.5 is a fluorine atom or a chlorine atom and wherein R.sub.6 is an amino group, an acetamidyl group, or a prenyl group can be formed. Following decarboxylation a second multi-substituent psilocybin derivative compound having a formula (LXXIV):
##STR00299##
wherein R.sub.5 is a fluorine atom or a chlorine atom, and wherein R.sub.6 is an amino group, an acetamidyl group, or a prenyl group can be formed. Following acetylation, a third multi-substituent psilocybin derivative has a formula (XIV), (XV), (XXXV), or (XXXVII):
##STR00300##
##STR00301##
##STR00302##
##STR00303##
can be formed.
[0810] The psilocybin biosynthetic enzyme complement can further comprise an N-methyl transferase to methylate the R.sub.3 amino group at R.sub.3 and form a further multi-substituent psilocybin derivative having a chemical formula (I), wherein R.sub.3a and R.sub.3b are each a methyl group, or wherein R.sub.3a is a hydrogen atom and R.sub.3b is a methyl group, the N-methyl transferase encoded by a nucleic acid sequence selected from: [0811] (a) SEQ.ID NO: 11, and SEQ.ID NO 13; [0812] (b) a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a); [0813] (c) a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a) but for the degeneration of the genetic code; [0814] (d) a nucleic acid sequence that is complementary to any one of the nucleic acid sequences of (a); [0815] (e) a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 12 and SEQ.ID NO 14; [0816] (f) a nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 12, and SEQ.ID NO 14; and [0817] (g) a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a), (b), (c), (d), (e) or (f).
[0818] Continuing to refer to
##STR00304##
wherein R.sub.5 is a chlorine atom and R.sub.6 is a prenyl group, and a first formed multi-substituent psilocybin derivative compound has a formula (LXIII):
##STR00305##
wherein R.sub.5 is a chlorine atom and wherein R.sub.6 is a prenyl group can be formed. Following decarboxylation, a second multi-substituent psilocybin derivative compound having a formula (LXXIV):
##STR00306##
wherein R.sub.5 is a chlorine atom, and wherein R.sub.6 is a prenyl group can be formed. Following methylation, a third multi-substituent psilocybin derivative having a formula (XLIV):
##STR00307##
can be formed.
[0819] Referring next to
[0834] Continuing to refer to
##STR00308##
wherein R.sub.5 is a fluorine atom, and R.sub.7 is a nitro group or a prenyl group, a first formed multi-substituent psilocybin derivative compound having a formula (LXXI):
##STR00309##
wherein R.sub.5 is a fluorine atom, and wherein R.sub.7 is a nitro group atom or a prenyl group can be formed. Following decarboxylation, a second formed multi-substituent psilocybin derivative compound having a formula (XIII) or (XXXIII):
##STR00310##
##STR00311##
can be formed.
[0835] In one embodiment, the psilocybin biosynthetic enzyme complement can further comprise an N-acetyl transferase to acetylate the second psilocybin derivative having chemical formula (I) and thereby form a third multi-substituent psilocybin having chemical formula (I), wherein R.sub.3a is a hydrogen atom and R.sub.3b is an acetyl group, the N-acetyl transferase encoded by a nucleic acid sequence selected from: [0836] (a) SEQ.ID NO: 9; [0837] (b) a nucleic acid sequence that is substantially identical to the nucleic acid sequence of (a); [0838] (c) a nucleic acid sequence that is substantially identical to the nucleic acid sequence of (a) but for the degeneration of the genetic code; [0839] (d) a nucleic acid sequence that is complementary to the nucleic acid sequence of (a); [0840] (e) a nucleic acid sequence encoding a polypeptide having the amino acid sequence set forth in SEQ.ID NO: 10; [0841] (f) a nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 10; and [0842] (g) a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a), (b), (c), (d), (e) or (f).
[0843] Continuing to refer to
##STR00312##
wherein R.sub.5 is a fluorine atom, and R.sub.7 is a nitro group or a prenyl group, a first formed multi-substituent psilocybin derivative compound having a formula (LXXI):
##STR00313##
wherein R.sub.5 is a fluorine atom, and wherein R.sub.7 is a nitro group atom or a prenyl group can be formed. Following decarboxylation thereof a second formed multi-substituent psilocybin derivative compound has a formula (LXXV):
##STR00314##
wherein R.sub.5 is a fluorine atom, and wherein R.sub.7 is a nitro group or a prenyl group can be formed. Following acetylation thereof a third multi-substituent psilocybin derivative has a formula (XII) or (XXXII):
##STR00315##
##STR00316##
can be formed.
[0844] As hereinbefore noted, in some embodiments, a multi-substituent psilocybin derivative may be made by a employing a combination of synthetic and biosynthetic methods. Thus, for example, referring to
[0845] Thus, in one embodiment, a psilocybin biosynthetic enzyme complement can contain a prenyl transferase encoded by a nucleic acid selected from: [0846] (a) SEQ.ID NO: 15, SEQ.ID NO: 17, SEQ.ID NO: 19, and SEQ.ID NO 21; [0847] (b) a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a); [0848] (c) a nucleic acid sequence that is substantially identical to any one of the nucleic acid sequences of (a) but for the degeneration of the genetic code; [0849] (d) a nucleic acid sequence that is complementary to any one of the nucleic acid sequences of (a); [0850] (e) a nucleic acid sequence encoding a polypeptide having any one of the amino acid sequences set forth in SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID NO: 20, and SEQ.ID NO 22; [0851] (f) a nucleic acid sequence that encodes a functional variant of any one of the amino acid sequences set forth in SEQ.ID NO: 16, SEQ.ID NO: 18, SEQ.ID NO: 20, and SEQ.ID NO 22; and [0852] (g) a nucleic acid sequence that hybridizes under stringent conditions to any one of the nucleic acid sequences set forth in (a), (b), (c), (d), (e) or (f),wherein in the psilocybin derivative precursor compound having formula (LVIII), one of R.sub.2, R.sub.4, R.sub.5, R.sub.6, or R.sub.7 is a substituent selected from (i) a halogen atom, (ii) a hydroxy group, (iii) a nitro group, (iv) a glycosyloxy group, (v) an amino group or an N-substituted amino group, (vi) a carboxyl group or a carboxylic acid derivative, (vii) an aldehyde or a ketone group, (viii) a prenyl group, and (ix) a nitrile group, wherein R.sub.3 is a hydrogen atom, and wherein a first multi-substituent psilocybin derivative compound having formula (I) is formed wherein R.sub.3c is a carboxyl group or a hydrogen atom.
[0853] In one embodiment, the psilocybin derivative precursor compound can have a formula (LXXVII):
##STR00317##
and a multi-substituent psilocybin derivative compound having the formula (LXXVI):
##STR00318##
can be formed.
[0854] It will be clear to those of skill in the art that a significant variety of different psilocybin precursor compounds may be selected.
[0855] Upon production by the host cells of a multi-substituent psilocybin compound in accordance with the methods of the present disclosure, the multi-substituent psilocybin derivative compounds may be extracted from the host cell suspension, and separated from other constituents within the host cell suspension, such as media constituents and cellular debris. Separation techniques will be known to those of skill in the art and include, for example, solvent extraction (e.g., butane, chloroform, ethanol), column chromatography based techniques, high-performance liquid chromatography (HPLC), for example, and/or countercurrent separation (CCS) based systems. The recovered multi-substituent psilocybin derivative compounds may be obtained in a more or less pure form, for example, a preparation of multi-substituent derivative psilocybin compounds of at least about 60% (w/w), about 70% (w/w), about 80% (w/w), about 90% (w/w), about 95% (w/w), about 96% (w/w), about 97% (w/w), about 98% (w/w) or about 99% (w/w) purity may be obtained. Thus, in this manner, multi-substituent psilocybin derivatives in more or less pure form may be prepared.
[0856] It will now be clear from the foregoing that novel multiple-substituent psilocybin derivatives are disclosed herein, as well as methods of making multiple-substituent psilocybin derivatives. The multiple-substituent psilocybin compounds may be formulated for use as a pharmaceutical drug or recreational drug.
SUMMARY OF SEQUENCES
[0857] SEQ.ID NO: 1 sets forth a Pyrococcus furiosus nucleic acid sequence encoding tryptophan synthase subunit B polypeptide, named PfTrpB-B0A9.
[0858] SEQ.ID NO: 2 sets forth a deduced amino acid sequence of a Pyrococcus furiosus subunit B tryptophan synthase subunit B polypeptide, named PfTrpB-B0A9.
[0859] SEQ. ID NO: 3 sets forth a Bacillus atrophaeaus nucleic acid sequence encoding a tryptophan decarboxylase polypeptide, named BaTDC.
[0860] SEQ.ID NO: 4 sets forth a deduced amino acid sequence of Bacillus atrophaeaus tryptophan decarboxylase polypeptide, named BaTDC.
[0861] SEQ. ID NO: 5 sets forth a Clostridium sporidium nucleic acid sequence encoding a tryptophan decarboxylase polypeptide, named ClostSporTDC.
[0862] SEQ.ID NO: 6 sets forth a deduced amino acid sequence of Clostridium sporidium tryptophan decarboxylase polypeptide, named ClostSporTDC.
[0863] SEQ.ID NO: 7 sets forth a Psilocybe cubensis nucleic acid sequence encoding a PsiD polypeptide.
[0864] SEQ.ID NO: 8 sets forth a deduced amino acid sequence of a Psilocybe cubensis PsiD polypeptide.
[0865] SEQ. ID NO: 9 sets forth a Streptomyces griseofuscus nucleic acid sequence encoding an N-acetyl transferase, named PmsF.
[0866] SEQ.ID NO: 10 sets forth a deduced amino acid sequence of a Streptomyces griseofuscus an N-acetyl polypeptide, named PmsF.
[0867] SEQ. ID NO: 11 sets forth an Ephedra sinica nucleic acid sequence encoding an N-methyl transferase, named EsNMT.
[0868] SEQ.ID NO: 12 sets forth a deduced amino acid sequence of an Ephedra sinica an N-methyl transferase polypeptide, named EsNMT.
[0869] SEQ.ID NO: 13 sets forth a Psilocybe cubensis nucleic acid sequence encoding a PsiM polypeptide.
[0870] SEQ.ID NO: 14 sets forth a deduced amino acid sequence of a Psilocybe cubensis PsiM polypeptide.
[0871] SEQ.ID NO: 15 sets forth an Aspergillus fumigatus nucleic acid sequence encoding a tryptophan 7-prenyl transferase polypeptide, named 7DMATS.
[0872] SEQ.ID NO: 16 sets forth a deduced amino acid sequence of an Aspergillus fumigatus tryptophan 7-prenyl transferase polypeptide, named 7DMATS.
[0873] SEQ.ID NO: 17 sets forth a Streptomyces sp. RM-5-8 nucleic acid sequence encoding a 6-prenyl transferase polypeptide, named PriB.
[0874] SEQ.ID NO: 18 sets forth a deduced amino acid sequence of a Streptomyces sp. RM-5-8 6-prenyl transferase polypeptide, named PriB.
[0875] SEQ.ID NO: 19 sets forth a Streptomyces coelicolor nucleic acid sequence encoding a tryptophan 5-prenyl transferase polypeptide, named SCO7467.
[0876] SEQ.ID NO: 20 sets forth a deduced amino acid sequence of a Streptomyces coelicolor tryptophan 5-prenyl transferase polypeptide, named SCO7467.
[0877] SEQ.ID NO: 21 sets forth an Aspergillus fumigatus nucleic acid sequence encoding a tryptophan 4-prenyl transferase polypeptide, named FgaPT2.
[0878] SEQ.ID NO: 22 sets forth a deduced amino acid sequence of an Aspergillus fumigatus tryptophan 4-prenyl transferase polypeptide, named FgaPT2.
[0879] SEQ.ID NO: 23 sets forth a Psilocybe cubensis nucleic acid sequence encoding a PsiH polypeptide.
[0880] SEQ.ID NO: 24 sets forth a deduced amino acid sequence of a Psilocybe cubensis PsiH polypeptide.
[0881] SEQ.ID NO: 25 sets forth a Psilocybe cubensis nucleic acid sequence encoding a CPR polypeptide.
[0882] SEQ.ID NO: 26 sets forth a deduced amino acid sequence of a Psilocybe cubensis CPR polypeptide.
[0883] SEQ. ID NO: 27 sets forth an artificial nucleic acid useful as an integration cassette, named XII-4::TADH1-PsiH-HA-PPGK1-PTDH3-CPR-c-myc-TCYC1.
[0884] SEQ. ID NO: 28 sets forth an artificial nucleic acid useful as an integration cassette, named XII-5::TADH1-PsiK-V5-PPGK1-PTDH3-PsiM-FLAG-TCYC1.
[0885] SEQ. ID NO: 29 sets forth an artificial nucleic acid useful as an integration cassette, named pMM1-PTDH3-ClostSporTDC-His-TCYC1.
[0886] SEQ. ID NO: 30 sets forth an artificial nucleic acid useful as a promoter, named PGK1_promoter.
[0887] SEQ. ID NO: 31 sets forth an artificial nucleic acid useful as a promoter, named TDH3_promoter.
[0888] SEQ. ID NO: 32 sets forth an artificial nucleic acid useful as a promoter, named CLN1_promoter.
[0889] SEQ. ID NO: 33 sets forth an artificial nucleic acid useful as a promoter, named UGA1_promoter.
[0890] SEQ. ID NO: 34 sets forth an artificial nucleic acid useful as a vector, named pMM1.
[0891] SEQ. ID NO: 35 sets forth an artificial nucleic acid useful as a vector, named pCDM4.
[0892] SEQ. ID NO: 36 sets forth an artificial nucleic acid useful as a vector, named pET28a(+).
[0893] SEQ. ID NO: 37 sets forth an artificial nucleic acid useful as a vector, named pET23(+).
[0894] SEQ. ID NO: 38 sets forth an artificial nucleic acid encoding a polypeptide sequence useful as a tag, named HA-tag.
[0895] SEQ. ID NO: 39 sets forth an artificial polypeptide sequence useful as a tag, named HA-tag.
[0896] SEQ. ID NO: 40 sets forth an artificial nucleic acid sequence encoding a polypeptide sequence useful as a tag, named c-myc-tag.
[0897] SEQ. ID NO: 41 sets forth an artificial polypeptide sequence useful as a tag, named c-myc-tag.
[0898] SEQ. ID NO: 42 sets forth an artificial nucleic acid sequence encoding a polypeptide sequence useful as a tag, named FLAG-tag.
[0899] SEQ. ID NO: 43 sets forth an artificial polypeptide sequence useful as a tag, named FLAG-tag.
[0900] SEQ.ID NO: 44 sets forth an artificial nucleic acid sequence encoding a polypeptide sequence useful as a tag, named V5-tag.
[0901] SEQ.ID NO: 45 sets forth an artificial polypeptide sequence useful as a tag, named V5-tag.
[0902] SEQ.ID NO: 46 sets forth an artificial nucleic acid sequence encoding a polypeptide sequence useful as a tag, named His-tag.
[0903] SEQ.ID NO: 47 sets forth an artificial polypeptide sequence useful as a tag, named His-tag.
[0904] SEQ.ID NO: 48 sets forth a Psilocybe cubensis nucleic acid sequence encoding a PsiK polypeptide.
[0905] SEQ.ID NO: 49 sets forth a deduced amino acid sequence of a Psilocybe cubensis PsiK polypeptide.
[0906] SEQ.ID NO: 50 sets forth an artificial nucleic acid useful as an integration cassette, named X-3::TADH1-BaTDC-Flag-PPGK1-PTDH3-CPR-c-myc-TCYC1.
[0907] SEQ.ID NO: 51 sets forth an artificial nucleic acid useful as an integration cassette, named Xii-2::TADH 1-PPGK1-PTDH3-PriB-His-TCYC1.
[0908] SEQ.ID NO: 52 sets forth an artificial nucleic acid useful as an integration cassette, named X-3::TADH1-ClostSporTDC-Flag-PPGK1-PTDH3-CPR-c-myc-TCYC1.
[0909] SEQ.ID NO: 53 sets forth an artificial nucleic acid useful as an integration cassette, named Xii-2::TADH1-PPGK1-PTDH3-Af-7DMATS-His-TCYC1.
[0910] SEQ. ID NO: 54 sets forth an artificial nucleic acid useful as a vector, named pET26b(+).
SEQUENCES
[0911] SEQ.ID NO: 1
TABLE-US-00001 ATGTGGTTCGGTGAGTTTGGTGGACAATATGTGCCAGAGACTTTAGTGGG TCCTCTTAAGGAATTGGAAAAGGCATATAAAAGGTTCAAGGACGATGAGG AGTTCAACAGGCAACTAAACTATTATTTGAAGACATGGGCCGGTAGACCA ACGCCCTTGTATTATGCTAAGAGGTTAACTGAAAAGATTGGCGGCGCGAA AGTGTATCTGAAAAGAGAAGACCTAGTTCATGGTGGAGCACACAAGACAA ATAATGCCATTGGACAAGCACTATTGGCAAAGCTAATGGGTAAAACTAGA TTGATAGCTGAGACAGGAGCGGGTCAACATGGGGTCGCGACAGCGATGGC TGGTGCACTACTGGGGATGAAGGTAGATATTTACATGGGTGCTGAGGACG TTGAGCGTCAGAAACTAAATGTCTTCAGGATGAAGCTATTAGGTGCCAAT GTTATACCTGTAAATTCTGGCTCAAGAACACTAAAGGACGCCTTCGACGA GGCTCTTAGAGACTGGGTTGCCACTTTCGAGTATACTCATTACTTGATCG GTTCAGTGGTTGGACCACATCCATACCCAACCATCGTTAGGGACTTTCAG AGCGTGATTGGTAGAGAGGCTAAGGCACAGATCTTAGAAGCAGAGGGACA GCTACCTGACGTCATAGTTGCCTGCGTCGGCGGTGGCTCTAACGCAATGG GTATATTCTATCCATTCGTTAATGACAAGAAGGTTAAATTAGTAGGAGTC GAAGCTGGCGGAAAGGGGTTAGAGTCGGGTAAACACTCAGCAAGCTTAAA TGCAGGACAGGTAGGGGTGTCCCACGGCATGTTGTCGTATTTCTTGCAAG ACGAGGAAGGTCAGATAAAGCCAAGTCATTCAATTGCTCCAGGCCTTGAC CACCCCGGTGTTGGTCCAGAGCACGCTTACTTAAAGAAGATTCAAAGGGC CGAGTACGTCGCTGTAACAGACGAAGAGGCATTGAAAGCTTTCCATGAGC TATCCAGAACTGAGGGGATTATACCCGCCCTTGAGTCTGCCCATGCTGTG GCGTACGCCATGAAGTTAGCTAAAGAGATGTCCCGTGACGAAATCATCAT TGTAAATCTATCAGGGAGAGGAGACAAGGATTTGGACATTGTATTGAAGG CAAGCGGAAATGTTTGA
[0912] SEQ. ID NO: 2
TABLE-US-00002 MWFGEFGGQYVPETLVGPLKELEKAYKRFKDDEEFNRQLNYYLKTWAGRP TPLYYAKRLTEKIGGAKVYLKREDLVHGGAHKTNNAIGQALLAKLMGKTR LIAETGAGQHGVATAMAGALLGMKVDIYMGAEDVERQKLNVFRMKLLGAN VIPVNSGSRTLKDAFDEALRDWVATFEYTHYLIGSVVGPHPYPTIVRDFQ SVIGREAKAQILEAEGQLPDVIVACVGGGSNAMGIFYPFVNDKKVKLVGV EAGGKGLESGKHSASLNAGQVGVSHGMLSYFLQDEEGQIKPSHSIAPGLD HPGVGPEHAYLKKIQRAEYVAVTDEEALKAFHELSRTEGIIPALESAHAV AYAMKLAKEMSRDEIIIVNLSGRGDKDLDIVLKASGNV
[0913] SEQ.ID NO: 3
TABLE-US-00003 ATGATGTCTGAAAATTTGCAATTGTCAGCTGAAGAAATGAGACAATTGGG TTACCAAGCAGTTGATTTGATCATCGATCACATGAACCATTTGAAGTCTA AGCCAGTTTCAGAAACAATCGATTCTGATATCTTGAGAAATAAGTTGACT GAATCTATCCCAGAAAATGGTTCAGATCCAAAGGAATTGTTGCATTTCTT GAACAGAAACGTTTTTAATCAAATTACACATGTTGATCATCCACATTTCT TGGCTTTTGTTCCAGGTCCAAATAATTACGTTGGTGTTGTTGCAGATTTC TTGGCTTCTGGTTTTAATGTTTTTCCAACTGCATGGATTGCTGGTGCAGG TGCTGAACAAATCGAATTGACTACAATTAATTGGTTGAAATCTATGTTGG GTTTTCCAGATTCAGCTGAAGGTTTATTTGTTTCTGGTGGTTCAATGGCA AATTTGACAGCTTTGACTGTTGCAAGACAGGCTAAGTTGAACAACGATAT CGAAAATGCTGTTGTTTACTTCTCTGATCAAACACATTTCTCAGTTGATA GAGCATTGAAGGTTTTAGGTTTTAAACATCATCAAATCTGTAGAATCGAA ACAGATGAACATTTGAGAATCTCTGTTTCAGCTTTGAAGAAACAAATTAA AGAAGATAGAACTAAGGGTAAAAAGCCATTCTGTGTTATTGCAAATGCTG GTACTACAAATTGTGGTGCTGTTGATTCTTTGAACGAATTAGCAGATTTG TGTAACGATGAAGATGTTTGGTTGCATGCTGATGGTTCTTATGGTGCTCC AGCTATCTTGTCTGAAAAGGGTTCAGCTATGTTGCAAGGTATTCATAGAG CAGATTCTTTGACTTTAGATCCACATAAGTGGTTGTTCCAACCATACGAT GTTGGTTGTGTTTTGATCAGAAACTCTCAATATTTGTCAAAGACTTTTAG AATGATGCCAGAATACATCAAGGATTCAGAAACTAACGTTGAAGGTGAAA TTAATTTCGGTGAATGTGGTATCGAATTGTCAAGAAGATTCAGAGCTTTG AAGGTTTGGTTGTCTTTTAAAGTTTTCGGTGTTGCTGCTTTTAGACAAGC AATCGATCATGGTATCATGTTAGCAGAACAAGTTGAAGCATTTTTGGGTA AAGCAAAAGATTGGGAAGTTGTTACACCAGCTCAATTGGGTATCGTTACT TTTAGATACATTCCATCTGAATTGGCATCAACAGATACTATTAATGAAAT TAATAAGAAATTGGTTAAGGAAATCACACATAGAGGTTTCGCTATGTTAT CTACTACAGAATTGAAGGAAAAGGTTGTTATTAGATTGTGTTCAATTAAT CCAAGAACTACAACTGAAGAAATGTTGCAAATCATGATGAAGATTAAAGC ATTGGCTGAAGAAGTTTCTATTTCATACCCATGTGTTGCTGAATAA
[0914] SEQ.ID NO: 4
TABLE-US-00004 MMSENLQLSAEEMRQLGYQAVDLIIDHMNHLKSKPVSETIDSDILRNKLT ESIPENGSDPKELLHFLNRNVFNQITHVDHPHFLAFVPGPNNYVGVVADF LASGFNVFPTAWIAGAGAEQIELTTINWLKSMLGFPDSAEGLFVSGGSMA NLTALTVARQAKLNNDIENAVVYFSDQTHFSVDRALKVLGFKHHQICRIE TDEHLRISVSALKKQIKEDRTKGKKPFCVIANAGTTNCGAVDSLNELADL CNDEDVWLHADGSYGAPAILSEKGSAMLQGIHRADSLTLDPHKWLFQPYD VGCVLIRNSQYLSKTFRMMPEYIKDSETNVEGEINFGECGIELSRRFRAL KVWLSFKVFGVAAFRQAIDHGIMLAEQVEAFLGKAKDWEVVTPAQLGIVT FRYIPSELASTDTINEINKKLVKEITHRGFAMLSTTELKEKVVIRLCSIN PRTTTEEMLQIMMKIKALAEEVSISYPCVAE
[0915] SEQ.ID NO: 5
TABLE-US-00005 ATGAAGTTCTGGAGAAAGTACACACAACAAGAAATGGATGAAAAGATTAC TGAATCTTTGGAAAAGACTTTGAACTACGATAACACTAAGACAATCGGTA TTCCAGGTACTAAGTTGGATGATACAGTTTTCTATGATGATCATTCTTTC GTTAAGCATTCACCATACTTGAGAACTTTTATTCAAAACCCAAACCATAT CGGTTGTCATACTTATGATAAGGCTGATATCTTGTTCGGTGGTACATTCG ATATCGAAAGAGAATTAATCCAATTGTTAGCAATCGATGTTTTGAACGGT AACGATGAAGAATTTGATGGTTACGTTACTCAAGGTGGTACAGAAGCTAA CATCCAAGCAATGTGGGTTTACAGAAACTACTTCAAGAAAGAAAGAAAGG CTAAGCATGAAGAAATCGCTATCATCACTTCAGCAGATACACATTACTCT GCATACAAAGGTTCAGATTTGTTGAACATCGATATTATTAAGGTTCCAGT TGATTTTTATTCAAGAAAAATTCAAGAAAATACATTGGATTCAATTGTTA AAGAAGCTAAAGAAATTGGTAAAAAGTACTTCATCGTTATCTCTAACATG GGTACTACAATGTTTGGTTCAGTTGATGATCCAGATTTGTACGCTAACAT CTTCGATAAGTACAATTTGGAATACAAAATTCATGTTGATGGTGCATTTG GTGGTTTTATATATCCAATTGATAATAAGGAATGTAAAACTGATTTCTCT AATAAGAACGTTTCTTCAATCACATTAGATGGTCATAAGATGTTGCAAGC TCCATACGGTACTGGTATCTTCGTTTCAAGAAAGAATTTGATCCATAACA CTTTGACAAAGGAAGCAACTTACATCGAAAATTTGGATGTTACATTGTCT GGTTCAAGATCTGGTTCAAATGCTGTTGCAATTTGGATGGTTTTAGCTTC TTATGGTCCATACGGTTGGATGGAAAAGATTAATAAGTTGAGAAATAGAA CTAAATGGTTGTGTAAGCAATTGAACGATATGAGAATTAAATATTACAAA GAAGATTCAATGAATATTGTTACAATTGAAGAACAATATGTTAATAAGGA AATCGCTGAAAAGTACTTTTTAGTTCCAGAAGTTCATAACCCAACTAACA ACTGGTACAAGATCGTTGTTATGGAACATGTTGAATTGGATATCTTGAAC TCTTTGGTTTACGATTTGAGAAAGTTTAATAAGGAACATTTGAAGGCAAT GTAA
[0916] SEQ.ID NO: 6
TABLE-US-00006 MKFWRKYTQQEMDEKITESLEKTLNYDNTKTIGIPGTKLDDTVFYDDHSF VKHSPYLRTFIQNPNHIGCHTYDKADILFGGTFDIERELIQLLAIDVLNG NDEEFDGYVTQGGTEANIQAMWVYRNYFKKERKAKHEEIAIITSADTHYS AYKGSDLLNIDIIKVPVDFYSRKIQENTLDSIVKEAKEIGKKYFIVISNM GTTMFGSVDDPDLYANIFDKYNLEYKIHVDGAFGGFIYPIDNKECKTDFS NKNVSSITLDGHKMLQAPYGTGIFVSRKNLIHNTLTKEATYIENLDVTLS GSRSGSNAVAIWMVLASYGPYGWMEKINKLRNRTKWLCKQLNDMRIKYYK EDSMNIVTIEEQYVNKEIAEKYFLVPEVHNPTNNWYKIVVMEHVELDILN SLVYDLRKFNKEHLKAM
[0917] SEQ.ID NO: 7
TABLE-US-00007 ATGCAGGTGATACCCGCGTGCAACTCGGCAGCAATAAGATCACTATGTCC TACTCCCGAGTCTTTTAGAAACATGGGATGGCTCTCTGTCAGCGATGCGG TCTACAGCGAGTTCATAGGAGAGTTGGCTACCCGCGCTTCCAATCGAAAT TACTCCAACGAGTTCGGCCTCATGCAACCTATCCAGGAATTCAAGGCTTT CATTGAAAGCGACCCGGTGGTGCACCAAGAATTTATTGACATGTTCGAGG GCATTCAGGACTCTCCAAGGAATTATCAGGAACTATGTAATATGTTCAAC GATATCTTTCGCAAAGCTCCCGTCTACGGAGACCTTGGCCCTCCCGTTTA TATGATTATGGCCAAATTAATGAACACCCGAGCGGGCTTCTCTGCATTCA CGAGACAAAGGTTGAACCTTCACTTCAAAAAACTTTTCGATACCTGGGGA TTGTTCCTGTCTTCGAAAGATTCTCGAAATGTTCTTGTGGCCGACCAGTT CGACGACAGACATTGCGGCTGGTTGAACGAGCGGGCCTTGTCTGCTATGG TTAAACATTACAATGGACGCGCATTTGATGAAGTCTTCCTCTGCGATAAA AATGCCCCATACTACGGCTTCAACTCTTACGACGACTTCTTTAATCGCAG ATTTCGAAACCGAGATATCGACCGACCTGTAGTCGGTGGAGTTAACAACA CCACCCTCATTTCTGCTGCTTGCGAATCACTTTCCTACAACGTCTCTTAT GACGTCCAGTCTCTCGACACTTTAGTTTTCAAAGGAGAGACTTATTCGCT TAAGCATTTGCTGAATAATGACCCTTTCACCCCACAATTCGAGCATGGGA GTATTCTACAAGGATTCTTGAACGTCACCGCTTACCACCGATGGCACGCA CCCGTCAATGGGACAATCGTCAAAATCATCAACGTTCCAGGTACCTACTT TGCGCAAGCCCCGAGCACGATTGGCGACCCTATCCCGGATAACGATTACG ACCCACCTCCTTACCTTAAGTCTCTTGTCTACTTCTCTAATATTGCCGCA AGGCAAATTATGTTTATTGAAGCCGACAACAAGGAAATTGGCCTCATTTT CCTTGTGTTCATCGGCATGACCGAAATCTCGACATGTGAAGCCACGGTGT CCGAAGGTCAACACGTCAATCGTGGCGATGACTTGGGAATGTTCCATTTC GGTGGTTCTTCGTTCGCGCTTGGTCTGAGGAAGGATTGCAGGGCAGAGAT CGTTGAAAAGTTCACCGAACCCGGAACAGTGATCAGAATCAACGAAGTCG TCGCTGCTCTAAAGGCTTAG
[0918] SEQ.ID NO: 8
TABLE-US-00008 MQVIPACNSAAIRSLCPTPESFRNMGWLSVSDAVYSEFIGELATRASNRN YSNEFGLMQPIQEFKAFIESDPVVHQEFIDMFEGIQDSPRNYQELCNMFN DIFRKAPVYGDLGPPVYMIMAKLMNTRAGFSAFTRQRLNLHFKKLFDTWG LFLSSKDSRNVLVADQFDDRHCGWLNERALSAMVKHYNGRAFDEVFLCDK NAPYYGFNSYDDFFNRRFRNRDIDRPWGGVNNTTLISAACESLSYNVSYD VQSLDTLVFKGETYSLKHLLNNDPFTPQFEHGSILQGFLNVTAYHRWHAP VNGTIVKIINVPGTYFAQAPSTIGDPIPDNDYDPPPYLKSLVYFSNIAAR QIMFIEADNKEIGLIFLVFIGMTEISTCEATVSEGQHVNRGDDLGMFHFG GSSFALGLRKDCRAEIVEKFTEPGTVIRINEVVAALKA
[0919] SEQ.ID NO: 9
TABLE-US-00009 ATGAACACCTTCAGAACAGCCACTGCCAGAGACATACCTGATGTAGCAGC AACTCTTACGGAAGCCTTCGCAACTGATCCACCCACGCAGTGGGTGTTCC CCGACGGTACTGCCGCCGTCAGCAGGTTCTTTACACATGTTGCAGATAGG GTTCACACGGCCGGTGGTATTGTTGAGCTACTACCAGACAGAGCCGCCAT GATTGCATTGCCACCACACGTGAGGCTGCCAGGAGAAGCTGCCGACGGAA GGCAGGCGGAAATTCAGAGAAGGCTGGCAGACAGGCACCCGCTGACACCT CACTACTACCTGCTGTTTTACGGAGTTAGAACGGCACACCAGGGTTCGGG ATTGGGCGGAAGAATGCTGGCCAGATTAACTAGCAGAGCTGATAGGGACA GGGTGGGTACATATACTGAGGCATCCACCTGGCGTGGCGCTAGACTGATG CTGAGACATGGATTCCATGCTACAAGGCCACTAAGATTGCCAGATGGACC CAGCATGTTTCCACTTTGGAGAGATCCAATCCATGATCATTCTGAT
[0920] SEQ.ID NO: 10
TABLE-US-00010 MNTFRTATARDIPDVAATLTEAFATDPPTQWVFPDGTAAVSRFFTHVADR VHTAGGIVELLPDRAAMIALPPHVRLPGEAADGRQAEIQRRLADRHPLTP HYYLLFYGVRTAHQGSGLGGRMLARLTSRADRDRVGTYTEASTWRGARLM LRHGFHATRPLRLPDGPSMFPLWRDPIHDHSD
[0921] SEQ.ID NO: 11
TABLE-US-00011 ATGGGATCCATGGAAGAAGCAAAAATGGCGACCCTGGGCGGTGCGTCCTA TGCGATGATTGTGAAAACGATGATGCGCTCTCTGGAAGCAAACCTGATTC CGGATTTTGTGCTGCGTCGCCTGACGCGTATCCTGCTGGCTAGTCGCCTG AAACTGGGTTATAAGCAGACCGCTGAACTGCAACTGGCGGATCTGATGTC ATTCGTTGCGTCGCTGAAAACGATGCCGATTGCCCTGTGCACCGAAGAAG CAAAGGGTCAGCATTACGAACTGCCGACCAGCTTTTTCAAACTGGTCCTG GGCAAACATCTGAAGTATAGCTCTGCCTACTTTTCTGAACACACCCGTAC GCTGGATGAAGCGGAAGAAGCCATGCTGGCACTGTATTGCGAACGCGCCA AAATTGAAGATGGTCAGAAGATTCTGGACATCGGCTGTGGTTGGGGCAGT TTTTCCCTGTATGTGGCAGAACGTTACCCGAAATGCGAAATTACGGGCCT GTGTAACAGTTCCACCCAAAAAGCCTTCATCGAACAGCAATGCAGCGAAC GTCGCCTGTGTAATGTTACCATTTATGCAGATGACATCAGCACCTTTGAT ACGGAATCTACCTACGACCGCATTATCAGCATCGAAATGTTCGAACACAT GAAGAACTACAGTACGCTGCTGAAGAAAATTAGCAAGTGGATGAATCAGG AATGCCTGCTGTTTGTCCATTATTTCTGTCACAAAACCTTTGCGTACCAC TTCGAAGATGTGGACGAAGATGACTGGATGGCTCGTTATTTCTTTACCGG CGGCACCATGCCGGCGTCATCGCTGCTGCTGTACTTTCAGGATGACGTCT CAGTGGTTGATCATTGGCTGATTAACGGTAAACACTATGCTCAAACCTCG GAAGAATGGCTGAAGCGTATGGACCACAATCTGAGCTCTATTCTGCCGAT CTTTAACGAAACGTATGGCGAAAATGCGGCCAAAAAGTGGCTGGCATACT GGCGCACCTTTTTCATCGCAGTTGCTGAACTGTTCAAATACAACGATGGC GAAGAATGGATGGTGTCCCACTTCCTGTTCAAAAAGAAATAA
[0922] SEQ.ID NO: 12
TABLE-US-00012 MGSMEEAKMATLGGASYAMIVKTMMRSLEANLIPDFVLRRLTRILLASRL KLGYKQTAELQLADLMSFVASLKTMPIALCTEEAKGQHYELPTSFFKLVL GKHLKYSSAYFSEHTRTLDEAEEAMLALYCERAKIEDGQKILDIGCGWGS FSLYVAERYPKCEITGLCNSSTQKAFIEQQCSERRLCNVTIYADDISTFD TESTYDRIISIEMFEHMKNYSTLLKKISKWMNQECLLFVHYFCHKTFAYH FEDVDEDDWMARYFFTGGTMPASSLLLYFQDDVSVVDHWLINGKHYAQTS EEWLKRMDHNLSSILPIFNETYGENAAKKWLAYWRTFFIAVAELFKYNDG EEWMVSHFLFKKK
[0923] SEQ.ID NO: 13
TABLE-US-00013 ATGCATATCAGAAATCCTTACCGTACACCAATTGACTATCAAGCACTTTC AGAGGCCTTCCCTCCCCTCAAGCCATTTGTGTCTGTCAATGCAGATGGTA CCAGTTCTGTTGACCTCACTATCCCAGAAGCCCAGAGGGCGTTCACGGCC GCTCTTCTTCATCGTGACTTCGGGCTCACCATGACCATACCAGAAGACCG TCTGTGCCCAACAGTCCCCAATAGGTTGAACTACGTTCTGTGGATTGAAG ATATTTTCAACTACACGAACAAAACCCTCGGCCTGTCGGATGACCGTCCT ATTAAAGGCGTTGATATTGGTACAGGAGCCTCCGCAATTTATCCTATGCT TGCCTGTGCTCGGTTCAAGGCATGGTCTATGGTTGGAACAGAGGTCGAGA GGAAGTGCATTGACACGGCCCGCCTCAATGTCGTCGCGAACAATCTCCAA GACCGTCTCTCGATATTAGAGACATCCATTGATGGTCCTATTCTCGTCCC CATTTTCGAGGCGACTGAAGAATACGAATACGAGTTTACTATGTGTAACC CTCCATTCTACGACGGTGCTGCCGATATGCAGACTTCGGATGCTGCCAAA GGATTTGGATTTGGCGTGGGCGCTCCCCATTCTGGAACAGTCATCGAAAT GTCGACTGAGGGAGGTGAATCGGCTTTCGTCGCTCAGATGGTCCGTGAGA GCTTGAAGCTTCGAACACGATGCAGATGGTACACGAGTAACTTGGGAAAG CTGAAATCCTTGAAAGAAATAGTGGGGCTGCTGAAAGAACTTGAGATAAG CAACTATGCCATTAACGAATACGTTCAGGGGTCCACACGTCGTTATGCCG TTGCGTGGTCTTTCACTGATATTCAACTGCCTGAGGAGCTTTCTCGTCCC TCTAACCCCGAGCTCAGCTCTCTTTTCTAG
[0924] SEQ.ID NO: 14
TABLE-US-00014 MHIRNPYRTPIDYQALSEAFPPLKPFVSVNADGTSSVDLTIPEAQRAFTA ALLHRDFGLTMTIPEDRLCPTVPNRLNYVLWIEDIFNYTNKTLGLSDDRP IKGVDIGTGASAIYPMLACARFKAWSMVGTEVERKCIDTARLNVVANNLQ DRLSILETSIDGPILVPIFEATEEYEYEFTMCNPPFYDGAADMQTSDAAK GFGFGVGAPHSGTVIEMSTEGGESAFVAQMVRESLKLRTRCRWYTSNLGK LKSLKEIVGLLKELEISNYAINEYVQGSTRRYAVAWSFTDIQLPEELSRP SNPELSSLF
[0925] SEQ.ID NO: 15
TABLE-US-00015 ATGTCCATCGGAGCCGAGATCGATTCGCTGGTTCCTGCTCCACCGGGCCT CAACGGCACCGCTGCGGGCTATCCAGCCAAGACGCAGAAGGAGTTAAGCA ACGGAGACTTTGACGCGCACGATGGTCTTTCTCTTGCACAACTGACACCG TACGATGTCTTGACGGCTGCACTTCCGCTGCCGGCTCCGGCTTCGAGCAC AGGGTTCTGGTGGCGGGAGACGGGCCCTGTTATGAGCAAGCTTTTGGCCA AGGCGAACTACCCTCTTTACACTCATTACAAGTACCTTATGTTATACCAT ACCCATATTCTCCCATTGTTGGGACCTCGACCGCCGCTCGAGAACTCGAC GCACCCGTCGCCGAGTAACGCGCCGTGGAGGTCCTTCCTGACAGACGACT TCACTCCGCTCGAGCCGAGCTGGAACGTGAACGGGAACTCGGAAGCACAG AGCACAATCCGTCTTGGTATTGAACCTATAGGCTTTGAAGCCGGGGCTGC AGCGGACCCATTCAACCAAGCTGCCGTGACGCAGTTCATGCACTCATACG AGGCAACCGAAGTCGGTGCCACGCTGACGCTGTTCGAGCACTTCCGCAAC GACATGTTTGTTGGCCCAGAAACGTACGCTGCGTTAAGAGCGAAGATACC AGAAGGCGAGCATACCACACAGAGTTTCCTGGCGTTCGACCTGGACGCGG GTCGTGTCACCACAAAGGCGTACTTTTTCCCGATTCTCATGTCGTTGAAA ACTGGACAGAGCACAACAAAGGTGGTCTCTGATTCCATTCTGCATCTAGC GCTGAAGAGTGAGGTGTGGGGTGTGCAGACCATCGCCGCGATGTCGGTCA TGGAGGCGTGGATAGGTAGCTACGGTGGCGCGGCAAAGACGGAGATGATC AGCGTCGATTGCGTGAACGAGGCAGACTCTCGGATCAAGATATACGTGCG GATGCCACATACATCCTTGCGGAAGGTAAAAGAGGCGTACTGCTTAGGTG GGCGGTTGACAGACGAGAACACAAAGGAGGGCCTGAAGCTGCTGGACGAG CTGTGGAGGACGGTCTTCGGCATCGACGACGAGGACGCGGAGCTGCCACA GAATAGCCATCGCACCGCAGGCACAATATTCAATTTCGAGCTGAGGCCAG GGAAATGGTTCCCCGAGCCCAAGGTATACCTGCCCGTCCGACACTACTGT GAAAGTGATATGCAGATTGCTAGTCGGCTACAAACGTTCTTTGGAAGGCT CGGATGGCACAACATGGAGAAAGATTATTGCAAGCATCTGGAAGATTTGT TTCCCCATCATCCACTGTCCTCGTCAACGGGCACACACACCTTTCTCTCA TTTTCGTATAAGAAGCAGAAGGGGGTCTATATGACCATGTATTATAATCT CCGGGTGTACAGCACCTAA
[0926] SEQ.ID NO: 16
TABLE-US-00016 MSIGAEIDSLVPAPPGLNGTAAGYPAKTQKELSNGDFDAHDGLSLAQLTP YDVLTAALPLPAPASSTGFWWRETGPVMSKLLAKANYPLYTHYKYLMLYH THILPLLGPRPPLENSTHPSPSNAPWRSFLTDDFTPLEPSWNVNGNSEAQ STIRLGIEPIGFEAGAAADPFNQAAVTQFMHSYEATEVGATLTLFEHFRN DMFVGPETYAALRAKIPEGEHTTQSFLAFDLDAGRVTTKAYFFPILMSLK TGQSTTKVVSDSILHLALKSEVWGVQTIAAMSVMEAWIGSYGGAAKTEMI SVDCVNEADSRIKIYVRMPHTSLRKVKEAYCLGGRLTDENTKEGLKLLDE LWRTVFGIDDEDAELPQNSHRTAGTIFNFELRPGKWFPEPKVYLPVRHYC ESDMQIASRLQTFFGRLGWHNMEKDYCKHLEDLFPHHPLSSSTGTHTFLS FSYKKQKGVYMTMYYNLRVYST
[0927] SEQ.ID NO: 17
TABLE-US-00017 ATGGGAGGTCCGATGAGCGGTTTCCATTCGGGGGAGGCGCTGCTCGGTGA CCTCGCCACCGGTCAGCTGACCAGGCTGTGCGAGGTGGCGGGGCTGACCG AGGCCGACACGGCGGCCTACACGGGGGTGCTGATCGAAAGTCTGGGGACG TCGGCCGGACGGCCGTTGTCCCTGCCACCCCCGTCGCGGACCTTTCTCTC CGACGACCACACCCCCGTGGAGTTCTCCCTGGCCTTCCTGCCGGGACGCG CACCGCACCTGCGGGTCCTGGTGGAACCGGGCTGCTCCAGCGGCGACGAC CTGGCGGAAAACGGCCGGGCCGGTCTGCGGGCGGTCCACACCATGGCGGA CCGCTGGGGATTCTCCACCGAGCAACTCGACCGGCTGGAGGACCTGTTCT TCCCCTCCTCCCCCGAGGGCCCGCTGGCCCTGTGGTGCGCCCTGGAGCTC CGCTCCGGTGGGGTGCCGGGGGTGAAGGTCTACCTCAACCCCGCGGCGAA TGGCGCCGACCGGGCCGCCGAGACGGTACGCGAGGCGCTGGCCAGGCTGG GCCACCTGCAGGCGTTCGACGCGCTGCCCCGGGCGGACGGCTTCCCGTTC CTCGCCCTGGACCTCGGCGACTGGGACGCCCCGCGGGTGAAGATCTACCT CAAACACCTCGGCATGTCCGCCGCCGACGCGGGCTCCCTCCCCCGGATGT CGCCCGCACCGAGCCGGGAGCAGCTGGAGGAGTTCTTCCGCACCGCCGGT GACCTCCCGGCCCCGGGAGACCCGGGGCCCACCGAGGACACCGGCCGGCT CGCCGGGCGCCCCGCCCTCACCTGCCACTCCTTCACGGAGACGGCGACCG GGCGGCCCAGCGGCTACACCCTCCACGTGCCGGTCCGCGACTACGTCCGG CACGACGGCGAGGCACGGGACCGGGCGGTGGCCGTGCTGCGCGAACATGA CATGGACAGTGCGGCACTGGACCGGGCGCTGGCCGCCGTGAGCCCCCGCC CGCTGAGTGACGGGGTGGGCCTGATCGCCTATCTGGCACTGGTCCACCAG CGCGGCCGGCCGACACGGGTGACCGTCTACGTCTCCTCCGAGGCGTACGA GGTGCGGCCGCCCCGCGAGACGGTCCCCACCCGCGACCGGGCGCGGGCAC GGCTGTGA
[0928] SEQ.ID NO: 18
TABLE-US-00018 MGGPMSGFHSGEALLGDLATGQLTRLCEVAGLTEADTAAYTGVLIESLGT SAGRPLSLPPPSRTFLSDDHTPVEFSLAFLPGRAPHLRVLVEPGCSSGDD LAENGRAGLRAVHTMADRWGFSTEQLDRLEDLFFPSSPEGPLALWCALEL RSGGVPGVKVYLNPAANGADRAAETVREALARLGHLQAFDALPRADGFPF LALDLGDWDAPRVKIYLKHLGMSAADAGSLPRMSPAPSREQLEEFFRTAG DLPAPGDPGPTEDTGRLAGRPALTCHSFTETATGRPSGYTLHVPVRDYVR HDGEARDRAVAVLREHDMDSAALDRALAAVSPRPLSDGVGLIAYLALVHQ RGRPTRVTVYVSSEAYEVRPPRETVPTRDRARARL
[0929] SEQ.ID NO: 19
TABLE-US-00019 ATGAGGGCCGCGTCGACGGGCGCGGACCCGCAGGACGCATCCACGCTCGG CTCTTTCACCGGCGGCCAGTTGCGAAGACTCGGCTCGGTCGCCGGTCTGT CCCGCGCCGACGTCGAGACCTACGCACAGGTCCTGACCGACGCATTGGGC CCGGTGGCCCAGCGGCCGCTGAGCCTGGCGCCGCCCACCCGCACCTTCCT GTCGGACGACCACACCCCCGTGGAGTTCTCCCTCTCCTTCCGGCCCGGGG CGGCGCCCGCCATGCGGGTCCTCGTGGAACCGGGCTGCGGTGCGACCAGC CTGGCCGACAACGGCCGTGCCGGTCTTGAGGCGGTCCGCACGATGGCGCG GCGCTGGCACTTCACCACCGACGCCCTCGACGAACTCCTGGACCTGTTCC TGCCGCCCGCTCCGCAGGGCCCCCTCGCCCTGTGGTGCGCCCTGGAACTC AGGCCCGGGGGTGTACCGGGCGTCAAGGTCTATCTGAACCCTGCGGTGGG CGGGGAGGAACGTTCCGCCGCGACGGTGCGCGAGGCCCTGCGCCGGCTCG GGCACCACCAGGCCTTCGACAGCCTCCCCCAGGGCAGTGGATACCCGTTC CTCGCCCTGGACCTCGGGAACTGGACGGAGCCCCGGGCGAAGGTCTACCT GCGCCACGACAACCTCACGGCCGGTCGGGCCGCACGGCTGTCCCGGACGG ACTCGGGCCTCGTGCCGACCGCGGTCGAGGGTTTCTTCCGCACCGCCGCG GGTCCCGGCTCCGACGCGGGTGGGCTCGACGGGCGGCCTGCTCAGTCCTG CCACTCCTTCACCGACCCCGGCGCGGAGCGGCCGAGCGGCTTCACCCTGT ACATCCCGGTTCGTGACTACGTCCGGCATGACGGGGAGGCCCTGGCGCGG GCGTCCACCGTGCTGCACCACCACGGCATGGACGCCTCCGTGCTCCACCG CGCCCTGGCCGCCCTCACCGAGCGGCGGCCCGAGGACGGGGTGGGCCTGA TCGCCTACCTGGCCCTCGCCGGCCAACGGGACCAGCCGCCGCGGGTGACG GCCTACCTCTCCTCGGAGGCCTACACGGTCCGGCCGCCGGTCGTGGAGAC CGTCCGCCAACCGCTGTCGGTCGGCTGA
[0930] SEQ.ID NO: 20
TABLE-US-00020 MRAASTGADPQDASTLGSFTGGQLRRLGSVAGLSRADVETYAQVLTDALG PVAQRPLSLAPPTRTFLSDDHTPVEFSLSFRPGAAPAMRVLVEPGCGATS LADNGRAGLEAVRTMARRWHFTTDALDELLDLFLPPAPQGPLALWCALEL RPGGVPGVKVYLNPAVGGEERSAATVREALRRLGHHQAFDSLPQGSGYPF LALDLGNWTEPRAKVYLRHDNLTAGRAARLSRTDSGLVPTAVEGFFRTAA GPGSDAGGLDGRPAQSCHSFTDPGAERPSGFTLYIPVRDYVRHDGEALAR ASTVLHHHGMDASVLHRALAALTERRPEDGVGLIAYLALAGQRDQPPRVT AYLSSEAYTVRPPWETVRQPLSVG
[0931] SEQ.ID NO: 21
TABLE-US-00021 ATGAAGGCAGCCAATGCCTCCAGTGCGGAGGCCTATCGAGTTCTTAGTCG CGCCTTTAGATTCGATAATGAAGATCAGAAGCTGTGGTGGCACAGCACTG CCCCGATGTTTGCAAAAATGCTGGAAACTGCCAACTACACCACACCTTGT CAGTATCAATACCTCATCACCTATAAGGAGTGCGTAATTCCCAGTCTCGG ATGCTATCCGACCAACAGCGCCCCCCGCTGGTTGAGCATCCTCACTCGAT ACGGCACTCCGTTCGAATTGAGCCTAAATTGCTCTAATTCAATAGTGAGA TACACATTCGAGCCGATCAATCAACATACCGGAACAGATAAAGACCCATT CAATACGCACGCCATCTGGGAGAGCCTGCAGCACCTGCTTCCACTGGAGA AGAGCATTGATCTGGAGTGGTTCCGCCACTTCAAGCACGATCTCACCCTC AACAGTGAAGAATCTGCTTTTCTGGCTCATAATGATCGCCTCGTGGGCGG CACTATCAGGACGCAGAACAAGCTCGCGCTCGATCTGAAGGATGGCCGCT TTGCACTTAAGACGTACATATACCCGGCTCTCAAAGCTGTCGTCACCGGC AAGACAATTCATGAGTTGGTCTTTGGCTCAGTCCGCCGGCTGGCAGTGAG GGAGCCCCGAATCTTGCCCCCACTCAACATGCTGGAGGAATACATCCGAT CACGCGGTTCCAAGAGCACTGCCAGTCCCCGCCTAGTGTCCTGTGATCTG ACCAGTCCTGCCAAGTCGAGAATCAAGATCTACCTGCTGGAGCAGATGGT TTCACTAGAAGCCATGGAGGACCTGTGGACTCTGGGCGGACGGCGCCGAG ACGCTTCCACTTTAGAGGGGCTCTCTCTGGTGCGTGAGCTTTGGGATCTG ATCCAACTGTCGCCGGGATTGAAGTCCTATCCGGCGCCGTATCTGCCTCT CGGGGTTATCCCAGACGAGAGGCTGCCGCTTATGGCCAATTTCACCCTGC ACCAGAATGACCCGGTCCCAGAGCCGCAAGTATATTTCACAACCTTCGGC ATGAACGACATGGCGGTGGCGGATGCCCTGACGACGTTCTTCGAGCGCCG GGGTTGGAGTGAAATGGCCCGCACCTACGAAACTACTTTGAAGTCGTACT ACCCCCATGCGGATCATGACAAACTTAACTACCTCCACGCCTACATATCC TTCTCCTACAGGGACCGTACCCCTTATCTGAGTGTCTATCTTCAATCCTT CGAGACAGGGGACTGGGCAGTTGCAAACTTATCCGAATCAAAGGTCAAGT GTCAGGATGCGGCCTGTCAACCCACAGCTTTACCTCCAGATCTGTCAAAG ACAGGGGTATATTATTCCGGTCTCCACTGA
[0932] SEQ.ID NO: 22
TABLE-US-00022 MKAANASSAEAYRVLSRAFRFDNEDQKLWWHSTAPMFAKMLETANYTTPC QYQYLITYKECVIPSLGCYPTNSAPRWLSILTRYGTPFELSLNCSNSIVR YTFEPINQHTGTDKDPFNTHAIWESLQHLLPLEKSIDLEWFRHFKHDLTL NSEESAFLAHNDRLVGGTIRTQNKLALDLKDGRFALKTYIYPALKAVVTG KTIHELVFGSVRRLAVREPRILPPLNMLEEYIRSRGSKSTASPRLVSCDL TSPAKSRIKIYLLEQMVSLEAMEDLWTLGGRRRDASTLEGLSLVRELWDL IQLSPGLKSYPAPYLPLGVIPDERLPLMANFTLHQNDPVPEPQVYFTTFG MNDMAVADALTTFFERRGWSEMARTYETTLKSYYPHADHDKLNYLHAYIS FSYRDRTPYLSVYLQSFETGDWAVANLSESKVKCQDAACQPTALPPDLSK TGVYYSGLH
[0933] SEQ.ID NO: 23
TABLE-US-00023 ATGATCGCTGTACTATTCTCCTTCGTCATTGCAGGATGCATATACTACAT CGTTTCTCGTAGAGTGAGGCGGTCGCGCTTGCCACCAGGGCCGCCTGGCA TTCCTATTCCCTTCATTGGGAACATGTTTGATATGCCTGAAGAATCTCCA TGGTTAACATTTCTACAATGGGGACGGGATTACAGTCTGTCTTGCCGCGT TGACTTCTAATATATGAACAGCTAATATATTGTCAGACACCGATATTCTC TACGTGGATGCTGGAGGGACAGAAATGGTTATTCTTAACACGTTGGAGAC CATTACCGATCTATTAGAAAAGCGAGGGTCCATTTATTCTGGCCGGTGAG CTGATGTTGAGTTTTTTGCAATTGAATTTGTGGTCACACGTTTCCAGACT TGAGAGTACAATGGTCAACGAACTTATGGGGTGGGAGTTTGACTTAGGGT TCATCACATACGGCGACAGGTGGCGCGAAGAAAGGCGCATGTTCGCCAAG GAGTTCAGTGAGAAGGGCATCAAGCAATTTCGCCATGCTCAAGTGAAAGC TGCCCATCAGCTTGTCCAACAGCTTACCAAAACGCCAGACCGCTGGGCAC AACATATTCGCCAGTAAGTACTACTTGAGGAAAATAGCGTACGCTTCGCT GACCGGTCCGTACATCAAAGTCAGATAGCGGCAATGTCACTGGATATTGG TTATGGAATTGATCTTGCAGAAGACGACCCTTGGCTGGAAGCGACCCATT TGGCTAATGAAGGCCTCGCCATAGCATCAGTGCCGGGCAAATTTTGGGTC GATTCGTTCCCTTCTCGTGAGCATCCTTCTTCTATGTAGGAAGGGAAGGA GTCTAACAAGTGTTAGTAAAATACCTTCCTGCTTGGTTCCCAGGTGCTGT CTTCAAGCGCAAAGCGAAGGTCTGGCGAGAAGCCGCCGACCATATGGTTG ACATGCCTTATGAAACTATGAGGAAATTAGCAGTTAGTCAAATGCGTTCT CCCCGTATTTTTTCAATACTCTAACTTCAGCTCACAGCCTCAAGGATTGA CTCGTCCGTCGTATGCTTCAGCTCGTCTGCAAGCCATGGATCTCAACGGT GACCTTGAGCATCAAGAACACGTAATCAAGAACACAGCCGCAGAGGTTAA TGTCGGTAAGTCAAAAGCGTCCGTCGGCAATTCAAAATTCAGGCGCTAAA GTGGGTCTTCTCACCAAGGTGGAGGCGATACTGTAAGGATTTCTCAATCG TTAGAGTATAAGTGTTCTAATGCAGTACATACTCCACCAACCAGACTGTC TCTGCTATGTCTGCGTTCATCTTGGCCATGGTGAAGTACCCTGAGGTCCA GCGAAAGGTTCAAGCGGAGCTTGATGCTCTGACCAATAACGGCCAAATTC CTGACTATGACGAAGAAGATGACTCCTTGCCATACCTCACCGCATGTATC AAGGAGCTTTTCCGGTGGAATCAAATCGCACCCCTCGCTATACCGCACAA ATTAATGAAGGACGACGTGTACCGCGGGTATCTGATTCCCAAGAACACTC TAGTCTTCGCAAACACCTGGTGAGGCTGTCCATTCATTCCTAGTACATCC GTTGCCCCACTAATAGCATCTTGATAACAGGGCAGTATTAAACGATCCAG AAGTCTATCCAGATCCCTCTGTGTTCCGCCCAGAAAGATATCTTGGTCCT GACGGGAAGCCTGATAACACTGTACGCGACCCACGTAAAGCGGCATTTGG CTATGGACGACGAAATTGGTAAGTGCGCTTTCAGAACCCCCCCTTCCGTT GACTAGTGCCATGCGCGCATACAATATCGCTATTGATCTGATATAACTTC CCTGCGGCATTTATTTTGGCATTCCTTTAGTCCCGGAATTCATCTAGCGC AGTCGACGGTTTGGATTGCAGGGGCAACCCTCTTATCAGCGTTCAATATC GAGCGACCTGTCGATCAGAATGGGAAGCCCATTGACATACCGGCTGATTT TACTACAGGATTCTTCAGGTAGCTAATTTCCGTCTTTGTGTGCATAATAC CCCTAACGACGCACGTTTACCTTTTTGTAAAGACACCCAGTGCCTTTCCA GTGCAGGTTTGTTCCTCGAACAGAGCAAGTCTCACAGTCGGTATCCGGAC CCTGA
[0934] SEQ.ID NO: 24
TABLE-US-00024 MIAVLFSFVIAGCIYYIVSRRVRRSRLPPGPPGIPIPFIGNMFDMPEESP WLTFLQWGRDYNTDILYVDAGGTEMVILNTLETITDLLEKRGSIYSGRLE STMVNELMGWEFDLGFITYGDRWREERRMFAKEFSEKGIKQFRHAQVKAA HQLVQQLTKTPDRWAQHIRHQIAAMSLDIGYGIDLAEDDPWLEATHLANE GLAIASVPGKFWVDSFPSLKYLPAWFPGAVFKRKAKVWREAADHMVDMPY ETMRKLAPQGLTRPSYASARLQAMDLNGDLEHQEHVIKNTAAEVNVGGGD TTVSAMSAFILAMVKYPEVQRKVQAELDALTNNGQIPDYDEEDDSLPYLT ACIKELFRWNQIAPLAIPHKLMKDDVYRGYLIPKNTLVFANTWAVLNDPE VYPDPSVFRPERYLGPDGKPDNTVRDPRKAAFGYGRRNCPGIHLAQSTVW IAGATLLSAFNIERPVDQNGKPIDIPADFTTGFFRHPVPFQCRFVPRTEQ VSQSVSGP
[0935] SEQ.ID NO: 25
TABLE-US-00025 ATGGCTTCTAGTTCTTCCGATGTCTTCGTTTTGGGTCTAGGTGTTGTTTT GGCTGCCTTGTATATCTTCAGAGACCAATTATTCGCTGCTTCTAAGCCAA AGGTGGCTCCAGTTTCCACTACGAAGCCTGCCAACGGTTCCGCTAACCCA AGAGACTTCATCGCCAAGATGAAACAAGGTAAGAAGAGAATCGTAATCTT CTACGGTTCTCAAACTGGTACCGCTGAAGAATATGCTATTCGTTTGGCTA AGGAAGCTAAGCAAAAGTTCGGTCTAGCCTCCTTGGTTTGTGATCCAGAA GAATACGATTTTGAAAAGTTGGACCAATTGCCAGAAGATTCTATTGCTTT CTTCGTCGTTGCTACCTATGGTGAAGGTGAACCTACAGACAACGCTGTCC AATTGTTGCAAAACTTGCAAGATGAAAGCTTCGAATTCTCCTCTGGTGAG AGAAAGTTGTCAGGTTTGAAGTACGTTGTTTTTGGTCTGGGTAACAAGAC CTACGAACATTACAACCTCATTGGGAGAACTGTTGACGCTCAATTGGCCA AGATGGGTGCTATCAGAATCGGTGAAAGAGGTGAAGGTGATGATGACAAG TCCATGGAAGAAGACTACTTGGAATGGAAGGATGGTATGTGGGAAGCGTT TGCCACTGCTATGGGTGTTGAAGAAGGTCAAGGTGGTGACTCCGCTGATT TCGTCGTTTCCGAATTGGAATCTCACCCACCAGAAAAGGTTTACCAAGGT GAATTTTCTGCTAGAGCTTTAACCAAAACCAAGGGTATTCACGACGCTAA GAATCCTTTTGCTGCTCCAATTGCGGTTGCTAGAGAATTGTTCCAATCTG TTGTCGATAGAAACTGTGTCCACGTCGAATTCAACATTGAAGGCTCTGGT ATCACCTATCAACACGGTGACCACGTTGGTTTGTGGCCATTGAATCCAGA TGTTGAAGTCGAACGGTTGTTGTGTGTTTTAGGTTTAGCTGAAAAGAGAG ATGCTGTCATCTCCATTGAATCCTTAGACCCGGCTTTGGCTAAGGTTCCA TTCCCAGTCCCAACTACTTACGGTGCTGTGTTGAGACACTACATTGACAT CTCTGCTGTCGCCGGTAGACAAATCTTGGGTACTTTGTCCAAATTCGCTC CAACCCCAGAAGCTGAAGCTTTCTTGAGAAACTTGAACACTAACAAGGAA GAATACCACAACGTCGTCGCTAACGGTTGTTTGAAATTGGGTGAAATTTT GCAAATCGCTACCGGTAACGACATTACTGTCCCACCAACTACTGCCAACA CCACCAAATGGCCAATTCCATTCGACATCATTGTTTCTGCCATCCCAAGA TTGCAACCAAGATACTACTCTATCTCTTCTTCCCCAAAAATTCATCCAAA CACCATCCACGCTACCGTTGTTGTGCTCAAATACGAAAACGTTCCAACCG AACCAATCCCAAGAAAGTGGGTTTACGGTGTCGGTAGTAACTTCTTGTTG AATTTAAAGTACGCTGTTAACAAGGAACCAGTTCCATACATCACTCAAAA TGGCGAACAAAGAGTCGGTGTCCCGGAATACTTGATTGCTGGTCCACGTG GTTCTTACAAGACTGAATCTTTCTACAAGGCTCCAATCCATGTTAGACGT TCTACTTTCCGTTTGCCAACCAACCCAAAGTCTCCAGTCATCATGATTGG TCCAGGTACTGGTGTCGCCCCATTCAGAGGCTTCGTTCAAGAAAGAGTTG CCTTGGCCAGAAGATCCATCGAAAAGAACGGTCCTGACTCTTTGGCTGAC TGGGGTCGTATTTCCTTGTTCTACGGTTGTAGAAGATCCGACGAAGACTT CTTGTACAAGGACGAATGGCCACAATACGAAGCTGAGTTGAAGGGTAAGT TCAAGTTGCACTGTGCTTTCTCCAGACAAAACTACAAGCCAGACGGTTCT AAGATTTACGTCCAAGATTTGATCTGGGAAGACAGAGAACACATTGCCGA TGCCATCTTAAACGGTAAGGGTTACGTCTACATCTGCGGTGAAGCTAAGT CCATGTCTAAACAAGTTGAAGAAGTTCTAGCCAAGATCTTGGGCGAAGCC AAAGGTGGTTCCGGTCCAGTTGAAGGTGTTGCTGAAGTCAAGTTACTGAA GGAACGGTCCAGATTGATGTTGGATGTCTGGTCTAGG
[0936] SEQ.ID NO: 26
TABLE-US-00026 MASSSSDVFVLGLGVVLAALYIFRDQLFAASKPKVAPVSTTKPANGSANP RDFIAKMKQGKKRIVIFYGSQTGTAEEYAIRLAKEAKQKFGLASLVCDPE EYDFEKLDQLPEDSIAFFVVATYGEGEPTDNAVQLLQNLQDESFEFSSGE RKLSGLKYVVFGLGNKTYEHYNLIGRTVDAQLAKMGAIRIGERGEGDDDK SMEEDYLEWKDGMWEAFATAMGVEEGQGGDSADFVVSELESHPPEKVYQG EFSARALTKTKGIHDAKNPFAAPIAVARELFQSVVDRNCVHVEFNIEGSG ITYQHGDHVGLWPLNPDVEVERLLCVLGLAEKRDAVISIESLDPALAKVP FPVPTTYGAVLRHYIDISAVAGRQILGTLSKFAPTPEAEAFLRNLNTNKE EYHNVVANGCLKLGEILQIATGNDITVPPTTANTTKWPIPFDIIVSAIPR LQPRYYSISSSPKIHPNTIHATVVVLKYENVPTEPIPRKWVYGVGSNFLL NLKYAVNKEPVPYITQNGEQRVGVPEYLIAGPRGSYKTESFYKAPIHVRR STFRLPTNPKSPVIMIGPGTGVAPFRGFVQERVALARRSIEKNGPDSLAD WGRISLFYGCRRSDEDFLYKDEWPQYEAELKGKFKLHCAFSRQNYKPDGS KIYVQDLIWEDREHIADAILNGKGYVYICGEAKSMSKQVEEVLAKILGEA KGGSGPVEGVAEVKLLKERSRLMLDVWS
[0937] SEQ.ID NO: 27
TABLE-US-00027 GTATCCGGCTGTTCCTTCATAGCCCTTTCAATGAACGTTGCAGCCCTTTG AAGATTGGCCATTTTGTCAGGACTCGAGCCTGACAGTTGGACCAACGCAA CTTTAATTTTTTGTGAAAGAATCTTCGAAGCACTCATACTGGCGATCTTC ACGCCCTCCTGCTATTACAAAAGCTGTGTTTTTACAAGAATCAAATTAAG TTAGCAAGATATTATACAACATTATTGATAATTTCAATATCGTGTTCGTA CCTGATGACGTATCTGTGCATTGATAAGGCCCGCATGGTTTCAGAAAGCA GAGCGGAACGATTCCAAATTAGTGGCCTTGTGCTTTGCATGTCAATTGTG TTACCTTCAGCTCGTGGATTTGTTTTATCAATACACAGTCTACAGTCAAG AATTTTTTTTATCAAATTTTGCGTTCGAGCGTATAAAATAGCCGCTGTAG CTACTTAAGTTCCTGTTCAGCGATAGTTTTTTTCCATCACACGTACTATG GCAATTAAGTCCTCAGCGAGCTCGCATGGAATGCGTGCGATGAGCGACCT CATGCTATACCTGAGAAAGCAACCTGACCTACAGGAAAGAGTTACTCAAG AATAAGAATTTTCGTTTTAAAACCTAAGAGTCACTTTAAAATTTGTATAC ACTTATTTTTTTTATAACTTATTTAATAATAAAAATCATAAATCATAAGA AATTCGCTTATTTAGAAGTGTCAACAACGTATCTACCAACGGAATGCGTG CGATTCAGGCGTAGTCTGGAACGTCGTATGGGTATGGACCAGAGACGGAT TGAGAAACTTGTTCGGTTCTTGGGACGAATCTACATTGGAATGGAACTGG GTGCCTGAAGAAACCAGTGGTGAAATCAGCTGGGATGTCAATTGGCTTAC CGTTTTGGTCAACTGGTCTTTCAATGTTGAAAGCAGACAACAAAGTAGCA CCGGCAATCCAAACAGTAGATTGAGCTAGGTGAATACCTGGGCAGTTTCT TCTACCGTAACCGAAAGCAGCCTTTCTTGGGTCTCTAACAGTGTTGTCTG GTTTACCATCAGGACCCAAGTATCTTTCTGGACGGAAAACGGATGGATCT GGATAGACTTCTGGGTCATTCAAAACTGCCCAGGTGTTAGCAAAAACCAA TGTGTTCTTTGGAATCAAATAACCTCTGTAAACATCATCCTTCATCAATT TATGAGGGATGGCTAATGGAGCAATTTGGTTCCATCTGAATAATTCCTTG ATACAAGCGGTCAAATATGGGAGTGAGTCGTCTTCCTCATCGTAGTCAGG GATTTGACCGTTGTTGGTCAAAGCATCCAATTCAGCTTGAACCTTTCTTT GCACTTCTGGGTATTTAACCATGGCCAATATGAAAGCGGACATAGCAGAG ACGGTAGTGTCGCCACCACCAACGTTAACTTCAGCAGCAGTGTTCTTGAT AACATGTTCTTGGTGTTCTAAATCACCGTTCAAGTCCATAGCTTGTAATC TAGCAGAAGCGTAAGATGGTCTGGTTAGACCTTGTGGAGCCAACTTTCTC ATAGTTTCGTATGGCATGTCAACCATGTGGTCAGCGGCTTCTCTCCAGAC CTTGGCCTTACGCTTGAAGACGGCACCTGGGAACCAAGCTGGCAAGTACT TCAAAGATGGGAAGGAGTCAACCCAGAACTTACCTGGAACACTAGCAATG GCCAAACCTTCGTTGGCTAAGTGGGTAGCTTCTAACCATGGGTCATCTTC GGCTAGATCGATACCGTAACCAATGTCCAAAGACATAGCAGCGATTTGAT GTCTGATGTGTTGAGCCCACCGGTCCGGAGTCTTGGTCAATTGTTGAACC AATTGGTGAGCAGCCTTGACTTGAGCGTGTCTGAATTGCTTGATACCCTT TTCAGAGAATTCCTTAGCAAACATTCTTCTTTCTTCTCTCCAACGGTCAC CGTAAGTGATAAAACCCAGATCAAATTCCCAACCCATCAATTCATTGACC ATAGTGGATTCCAATCTACCGGAGTAGATGGAACCACGTTTTTCCAAGAG ATCCGTGATAGTTTCCAAGGTGTTTAAAATGACCATTTCGGTACCACCAG CATCTACGTACAAGATATCAGTGTTGTAGTCACGACCCCATTGCAAGAAA GTCAACCATGGAGATTCTTCTGGCATGTCGAACATGTTACCAATAAATGG GATTGGAATTCCTGGTGGACCAGGTGGCAATCTGGATCTACGAACTCTTC TGGAGACGATGTAGTAAATACAACCAGCAATGACGAAAGAGAACAAGACA GCAATCATTGTTTTATATTTGTTGTAAAAAGTAGATAATTACTTCCTTGA TGATCTGTAAAAAAGAGAAAAAGAAAGCATCTAAGAACTTGAAAAACTAC GAATTAGAAAAGACCAAATATGTATTTCTTGCATTGACCAATTTATGCAA GTTTATATATATGTAAATGTAAGTTTCACGAGGTTCTACTAAACTAAACC ACCCCCTTGGTTAGAAGAAAAGAGTGTGTGAGAACAGGCTGTTGTTGTCA CACGATTCGGACAATTCTGTTTGAAAGAGAGAGAGTAACAGTACGATCGA ACGAACTTTGCTCTGGAGATCACAGTGGGCATCATAGCATGTGGTACTAA ACCCTTTCCCGCCATTCCAGAACCTTCGATTGCTTGTTACAAAACCTGTG AGCCGTCGCTAGGACCTTGTTGTGTGACGAAATTGGAAGCTGCAATCAAT AGGAAGACAGGAAGTCGAGCGTGTCTGGGTTTTTTCAGTTTTGTTCTTTT TGCAAACAACAGTTTATTCCTGGCATCCACTAAATATAATGGAGCCCGCT TTTTAAGCTGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATATTGT TTTCTTCACCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTACA GAGAACAGGGGCACAAACAGGCAAAAAACGGGCACAACCTCAATGGAGTG ATGCAACCTGCCTGGAGTAAATGATGACACAAGGCAATTGACCCACGCAT GTATCTATCTCATTTTCTTACACCTTCTATTACCTTCTGCTCTCTCTGAT TTGGAAAAAGCTGAAAAAAAAGGTTGAAACCAGTTCCCTGAAATTATTCC CCTACTTGACTAATAAGTATATAAAGACGGTAGGTATTGATTGTAATTCT GTAAATCTATTTCTTAAACTTCTTAAATTCTACTTTTATAGTTAGTCTTT TTTTTAGTTTTAAAACACCAAGAACTTAGTTTCGAATAAACACACATAAA CAAACAAAATGGCTTCTAGTTCTTCCGATGTCTTCGTTTTGGGTCTAGGT GTTGTTTTGGCTGCCTTGTATATCTTCAGAGACCAATTATTCGCTGCTTC TAAGCCAAAGGTGGCTCCAGTTTCCACTACGAAGCCTGCCAACGGTTCCG CTAACCCAAGAGACTTCATCGCCAAGATGAAACAAGGTAAGAAGAGAATC GTAATCTTCTACGGTTCTCAAACTGGTACCGCTGAAGAATATGCTATTCG TTTGGCTAAGGAAGCTAAGCAAAAGTTCGGTCTAGCCTCCTTGGTTTGTG ATCCAGAAGAATACGATTTTGAAAAGTTGGACCAATTGCCAGAAGATTCT ATTGCTTTCTTCGTCGTTGCTACCTATGGTGAAGGTGAACCTACAGACAA CGCTGTCCAATTGTTGCAAAACTTGCAAGATGAAAGCTTCGAATTCTCCT CTGGTGAGAGAAAGTTGTCAGGTTTGAAGTACGTTGTTTTTGGTCTGGGT AACAAGACCTACGAACATTACAACCTCATTGGGAGAACTGTTGACGCTCA ATTGGCCAAGATGGGTGCTATCAGAATCGGTGAAAGAGGTGAAGGTGATG ATGACAAGTCCATGGAAGAAGACTACTTGGAATGGAAGGATGGTATGTGG GAAGCGTTTGCCACTGCTATGGGTGTTGAAGAAGGTCAAGGTGGTGACTC CGCTGATTTCGTCGTTTCCGAATTGGAATCTCACCCACCAGAAAAGGTTT ACCAAGGTGAATTTTCTGCTAGAGCTTTAACCAAAACCAAGGGTATTCAC GACGCTAAGAATCCTTTTGCTGCTCCAATTGCGGTTGCTAGAGAATTGTT CCAATCTGTTGTCGATAGAAACTGTGTCCACGTCGAATTCAACATTGAAG GCTCTGGTATCACCTATCAACACGGTGACCACGTTGGTTTGTGGCCATTG AATCCAGATGTTGAAGTCGAACGGTTGTTGTGTGTTTTAGGTTTAGCTGA AAAGAGAGATGCTGTCATCTCCATTGAATCCTTAGACCCGGCTTTGGCTA AGGTTCCATTCCCAGTCCCAACTACTTACGGTGCTGTGTTGAGACACTAC ATTGACATCTCTGCTGTCGCCGGTAGACAAATCTTGGGTACTTTGTCCAA ATTCGCTCCAACCCCAGAAGCTGAAGCTTTCTTGAGAAACTTGAACACTA ACAAGGAAGAATACCACAACGTCGTCGCTAACGGTTGTTTGAAATTGGGT GAAATTTTGCAAATCGCTACCGGTAACGACATTACTGTCCCACCAACTAC TGCCAACACCACCAAATGGCCAATTCCATTCGACATCATTGTTTCTGCCA TCCCAAGATTGCAACCAAGATACTACTCTATCTCTTCTTCCCCAAAAATT CATCCAAACACCATCCACGCTACCGTTGTTGTGCTCAAATACGAAAACGT TCCAACCGAACCAATCCCAAGAAAGTGGGTTTACGGTGTCGGTAGTAACT TCTTGTTGAATTTAAAGTACGCTGTTAACAAGGAACCAGTTCCATACATC ACTCAAAATGGCGAACAAAGAGTCGGTGTCCCGGAATACTTGATTGCTGG TCCACGTGGTTCTTACAAGACTGAATCTTTCTACAAGGCTCCAATCCATG TTAGACGTTCTACTTTCCGTTTGCCAACCAACCCAAAGTCTCCAGTCATC ATGATTGGTCCAGGTACTGGTGTCGCCCCATTCAGAGGCTTCGTTCAAGA AAGAGTTGCCTTGGCCAGAAGATCCATCGAAAAGAACGGTCCTGACTCTT TGGCTGACTGGGGTCGTATTTCCTTGTTCTACGGTTGTAGAAGATCCGAC GAAGACTTCTTGTACAAGGACGAATGGCCACAATACGAAGCTGAGTTGAA GGGTAAGTTCAAGTTGCACTGTGCTTTCTCCAGACAAAACTACAAGCCAG ACGGTTCTAAGATTTACGTCCAAGATTTGATCTGGGAAGACAGAGAACAC ATTGCCGATGCCATCTTAAACGGTAAGGGTTACGTCTACATCTGCGGTGA AGCTAAGTCCATGTCTAAACAAGTTGAAGAAGTTCTAGCCAAGATCTTGG GCGAAGCCAAAGGTGGTTCCGGTCCAGTTGAAGGTGTTGCTGAAGTCAAG TTACTGAAGGAACGGTCCAGATTGATGTTGGATGTCTGGTCTGAACAAAA GTTAATTTCTGAAGAAGATTTGGAATGAATCGCGTGCATTCATCCGCTCT AACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATT TTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTT CTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAAC CTTGCTTGAGAAGGTTTTGGGACGCTCGAAGATCGCGTCCCAATTCGCCC TATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCG TGACTGGGAAAACCCTGGCGTTACCCCTGCAGGACTAGTGCTGAGGCATT AATAGTTTACTCAATTCTTGAAGCCAATTTGTACAATTCCCCATTAGAGT CAAATAAAAGGATGCCTCACGGAGGTATGTTACCCGCGCTATTTCACATG GCTCATTGAATTAGAGGTGGAATTTGGTGTACCCTCCCCTCCTCATCTGA TGAAGTAGTGATCCGACAATTCTTAAAAGTTGTAGACATTACTTTTACCA CCAACTAAGTTGTATTTATATTGCTACCCTTATCCTTTTATATCTAACTA GCGCTCATAAGGTTGGGGCAATACTAAAACTGTGTTCTTATTCAACTCAT TAAATACGTGGCAGTACGTACCCTATTAGAAACAATAGGAAACAGCAGAG TCGGAAGAAGCCAAATGCCAGATTTGAAGTCCAAAACCTTGTCAAGCCAA TCTTTGGGAGCGGCTATTCCTCCAGAAATTGTGTACCAAATACTTACATA CCAGTTTAGGGATTTGTTAAGAAATGACCATCCAGGTACGGCAGAAA
[0938] SEQ.ID NO: 28
TABLE-US-00028 CAATCTGGCGGCTTGAGTTCTCAACATGTTTTATTTTTTACTTATATTGC TGGTAGGGTAAAAAAATATAACTCCTAGGAATAGGTTGTCTATATGTTTT TGTCTTGCTTCTATAATTGTAACAAACAAGGAAAGGGAAAATACTGGGTG TAAAAGCCATTGAGTCAAGTTAGGTCATCCCTTTTATACAAAATTTTTCA ATTTTTTTTCCAAGATTCTTGTACGATTAATTATTTTTTTTTTGCGTCCT ACAGCGTGATGAAAATTTCGCCTGCTGCAAGATGAGCGGGAACGGGCGAA ATGTGCACGCGCACAACTTACGAAACGCGGATGAGTCACTGACAGCCACC GCAGAGGTTCTGACTCCTACTGAGCTCTATTGGAGGTGGCAGAACCGGTA CCGGAGGAGGCCGCTATAACCGGTTTGAATTTATTGTCACAGTGTCACAT CAGCATTAAGTCCTCAGCGAGCTCGCATGGAATGCGTGCGATGAGCGACC TCATGCTATACCTGAGAAAGCAACCTGACCTACAGGAAAGAGTTACTCAA GAATAAGAATTTTCGTTTTAAAACCTAAGAGTCACTTTAAAATTTGTATA CACTTATTTTTTTTATAACTTATTTAATAATAAAAATCATAAATCATAAG AAATTCGCTTATTTAGAAGTGTCAACAACGTATCTACCAACGGAATGCGT GCGATTCAGGTGGAGTCCAAACCCAACAAAGGATTTGGAATTGGCTTACC GGCAGTGGAGGATTCCTTTAGCAACGTGGAAGTGATTTCACCGTTGTCGT TGTTACCTCTAGCATCGTGGAAAGCAGCAACACCCTTCTTAACAAAGTTG ATTCTTTCTTCTTCAGAACCCCATTGCATGAAGTCAGTCCACATAACAAT GTGAGCGGCGATACCAGCGGTAACCTTGGCGTAGTTGATGGAATGCTTGG AAGTACGGGCGTAAGATTGCAAGTAAGCTTGTCTCATGGTTGTACCAACT TGTTCGTCTTGGAATCTGCTAATCAAGTAACAGTCACCCAAGAAGTAACC CAAATCCAATGAAGCTGGACCGTACTTACACAATTCCCAGTCTAAGATGT AGATCTTTTGCAACTTAGATGGGTTACCTTCTTCAAGTTGCAACAAGATG TTCCCAGACCACAAGTCAGCCATGACCAAAGTTTCTTCGGAGTGCATAAC ATCGTCAACTAGATCCTTGACAACAGTTGGCAACAATGGATCATCGACGC CGTATTTAGCGGCGTTTGGGATAATAGTTTGGTACAATTGGTCAGAGGTG GTTCTACCGACAATGTTACCAGAGAAGAACTTGAATTCTGGGTCGTCTCT TCTTTCTCTACCTATGTTGTGCAATCTGGCGACGAAACCACCAATCTCGG TACCAACCAATCTAGCAATATCGGTAGCCAAAGGTGGCTTAGCAGTAACG TAGTCTAATAAGGTCTTCATTTTACCGACATCTTGCATAATCAAAGCATT GTTTTCCAAGTCATAGTTGAGACCTTCTGGAACAGAGACAATACCATCAA CACCACCCAAAACTTCTCTGTTAGCCATCATCAACTTGATAGCTTGGTAT TCGTAGACAGAACGTTCAACACCGATTTTGAAATCTTCATCAGTAGACAT GTGTGGTTGAGCGTGCTTCAAAATGATAGAAGTGTGACCTTGGTATGGAG CGTTCAATTTAATACGCCAGGTGACGTTAACGAAACCACCGGATAATCTC TTGACACCAGAAGTGTCAACATCTAAAGACAAATGCTTGGTCAAATAGGT GATTAAACCGTCTTCAGTCTTCAAGTCAAAGGCCATTGTTTTATATTTGT TGTAAAAAGTAGATAATTACTTCCTTGATGATCTGTAAAAAAGAGAAAAA GAAAGCATCTAAGAACTTGAAAAACTACGAATTAGAAAAGACCAAATATG TATTTCTTGCATTGACCAATTTATGCAAGTTTATATATATGTAAATGTAA GTTTCACGAGGTTCTACTAAACTAAACCACCCCCTTGGTTAGAAGAAAAG AGTGTGTGAGAACAGGCTGTTGTTGTCACACGATTCGGACAATTCTGTTT GAAAGAGAGAGAGTAACAGTACGATCGAACGAACTTTGCTCTGGAGATCA CAGTGGGCATCATAGCATGTGGTACTAAACCCTTTCCCGCCATTCCAGAA CCTTCGATTGCTTGTTACAAAACCTGTGAGCCGTCGCTAGGACCTTGTTG TGTGACGAAATTGGAAGCTGCAATCAATAGGAAGACAGGAAGTCGAGCGT GTCTGGGTTTTTTCAGTTTTGTTCTTTTTGCAAACAACAGTTTATTCCTG GCATCCACTAAATATAATGGAGCCCGCTTTTTAAGCTGGCATCCAGAAAA AAAAAGAATCCCAGCACCAAAATATTGTTTTCTTCACCAACCATCAGTTC ATAGGTCCATTCTCTTAGCGCAACTACAGAGAACAGGGGCACAAACAGGC AAAAAACGGGCACAACCTCAATGGAGTGATGCAACCTGCCTGGAGTAAAT GATGACACAAGGCAATTGACCCACGCATGTATCTATCTCATTTTCTTACA CCTTCTATTACCTTCTGCTCTCTCTGATTTGGAAAAAGCTGAAAAAAAAG GTTGAAACCAGTTCCCTGAAATTATTCCCCTACTTGACTAATAAGTATAT AAAGACGGTAGGTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTC TTAAATTCTACTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAAG AACTTAGTTTCGAATAAACACACATAAACAAACAAAATGCATATCAGAAA CCCATATAGAACTCCAATTGACTACCAAGCTTTGTCTGAAGCTTTCCCAC CATTGAAGCCATTTGTTTCCGTTAACGCTGATGGTACCTCCTCAGTTGAC TTGACCATTCCAGAAGCCCAAAGAGCTTTTACCGCTGCCCTTTTGCACAG AGACTTCGGCTTGACTATGACTATCCCAGAAGATCGTTTGTGTCCAACCG TTCCAAACAGATTGAACTACGTTTTGTGGATTGAAGACATTTTCAACTAC ACCAACAAGACTTTGGGTTTATCTGACGACCGTCCAATCAAGGGTGTTGA TATCGGTACCGGTGCTTCTGCCATTTACCCAATGTTGGCTTGCGCCAGAT TCAAGGCTTGGTCCATGGTTGGTACTGAAGTTGAAAGAAAGTGTATCGAC ACTGCTAGATTAAACGTTGTTGCTAACAACTTGCAAGATCGTCTATCCAT CTTGGAAACCTCTATTGACGGTCCAATTTTAGTCCCAATTTTCGAAGCTA CCGAAGAATACGAATACGAATTCACCATGTGTAACCCACCTTTCTACGAT GGTGCCGCTGACATGCAAACTAGCGATGCTGCAAAGGGTTTTGGTTTCGG TGTCGGTGCTCCACACTCTGGTACAGTCATCGAAATGTCTACTGAAGGTG GTGAATCCGCTTTCGTGGCTCAAATGGTTAGAGAATCTCTTAAGTTGAGA ACCAGATGTAGATGGTACACTTCTAACTTAGGTAAGTTGAAATCTTTGAA GGAAATTGTCGGTTTGTTGAAGGAATTAGAAATCTCTAATTACGCCATCA ACGAATATGTCCAAGGTTCCACTAGAAGATACGCTGTCGCTTGGAGTTTC ACTGATATCCAATTGCCAGAAGAATTGTCCAGACCATCTAATCCTGAATT GTCCTCTTTGTTCGACTACAAGGATGACGATGACAAATGAATCGCGTGCA TTCATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGG TCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATA TTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACAT TATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGATCGCGT ACCCAATTCGCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCG TTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCCTGCAGGACTA GTGCTGAGGCATTAATGCAACTCAGAAGTTTGACAGCAAGCAAGTTCATC ATTCGAACTAGCCTTATTGTTTTAGTTCAGTGACAGCGAACTGCCGTACT CGATGCTTTATTTCTCACGGTAGAGCGGAAGAACAGATAGGGGCAGCGTG AGAAGAGTTAGAAAGTAAATTTTTATCACGTCTGAAGTATTCTTATTCAT AGGAAATTTTGCAAGGTTTTTTAGCTCAATAACGGGCTAAGTTATATAAG GTGTTCACGCGATTTTCTTGTTATGTATACCTCTTCTCTGAGGAATGGTA CTACTGTCCTGATGTAGGCTCCTTAAATTGGTGGGCAAGAATAACTTATC GATATTTTGTATATTGGTCTTGGAGTTCACCACGTAATGCCTGTTTAAGA CCATCAGTTAACTCTAGTATTATTTGGTCTTGGCTACTGGCCGTTTGCTA TTATTCAAGTCTTTTGTGCCTTCCCGTCGGGTAAGGGAGTTATTTAGGGA TACAGAATCTAACGAAAACTAAATCTCAATGATTAACTCTATTTAATCCT TTTTTGAAAGGCAAAAGAGGTCCCTTGTTCACTTACAACGTTCTTAGCCA AATTCGCTTATCACTTACTACTTCACGATATACAGAAGTAAAAACATATA AAAAGATGTCTGTTTGTTTAGCCATCACAAAAGGTATCGCAG
[0939] SEQ.ID NO: 29
TABLE-US-00029 TGTTTTATATTTGTTGTAAAAAGTAGATAATTACTTCCTTGATGATCTGT AAAAAAGAGAAAAAGAAAGCATCTAAGAACTTGAAAAACTACGAATTAGA AAAGACCAAATATGTATTTCTTGCATTGACCAATTTATGCAAGTTTATAT ATATGTAAATGTAATTTCACGAGGTTCTACTAAACTAAACCACCCCCTTG GTTAGAAGAAAAGAGTGTGTGAGAACAGGCTGTTGTTGTCACACGATTCG GACAATTCTGTTTGAAAGAGAGAGAGTAACAGTACGATCGAACGAACTTT GCTCTGGAGATCACAGTGGGCATCATAGCATGTGGTACTAAACCCTTTCC CGCCATTCCAGAACCTTCGATTGCTTGTTACAAAACCTGTGAGCCGTCGC TAGGACCTTGTTGTGTGACGAAATTGGAAGCTGCAATCAATAGGAAGACA GGAAGTCGAGCGTGTCTGGGTTTTTTCAGTTTTGTTCTTTTTGCAAACAA CAGTTTATTCCTGGCATCCACTAAATATAATGGAGCCCGCTTTTTAAGCT GGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATATTGTTTTCTTCAC CAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTACAGAGAACAGG GGCACAAACAGGCAAAAAACGGGCACAACCTCAATGGAGTGATGCAACCT GCCTGGAGTAAATGATGACACAAGGCAATTGACCCACGCATGTATCTATC TCATTTTCTTACACCTTCTATTACCTTCTGCTCTCTCTGATTTGGAAAAA GCTGAAAAAAAAGGTTGAAACCAGTTCCCTGAAATTATTCCCCTACTTGA CTAATAAGTATATAAAGACGGTAGGTATTGATTGTAATTCTGTAAATCTA TTTCTTAAACTTCTTAAATTCTACTTTTATAGTTAGTCTTTTTTTTAGTT TTAAAACACCAAGAACTTAGTTTCGAATAAACACACATAAACAAACAAAG GATCCATGATGAAGTTCTGGAGAAAGTACACACAACAAGAAATGGATGAA AAGATTACTGAATCTTTGGAAAAGACTTTGAACTACGATAACACTAAGAC AATCGGTATTCCAGGTACTAAGTTGGATGATACAGTTTTCTATGATGATC ATTCTTTCGTTAAGCATTCACCATACTTGAGAACTTTTATTCAAAACCCA AACCATATCGGTTGTCATACTTATGATAAGGCTGATATCTTGTTCGGTGG TACATTCGATATCGAAAGAGAATTAATCCAATTGTTAGCAATCGATGTTT TGAACGGTAACGATGAAGAATTTGATGGTTACGTTACTCAAGGTGGTACA GAAGCTAACATCCAAGCAATGTGGGTTTACAGAAACTACTTCAAGAAAGA AAGAAAGGCTAAGCATGAAGAAATCGCTATCATCACTTCAGCAGATACAC ATTACTCTGCATACAAAGGTTCAGATTTGTTGAACATCGATATTATTAAG GTTCCAGTTGATTTTTATTCAAGAAAAATTCAAGAAAATACATTGGATTC AATTGTTAAAGAAGCTAAAGAAATTGGTAAAAAGTACTTCATCGTTATCT CTAACATGGGTACTACAATGTTTGGTTCAGTTGATGATCCAGATTTGTAC GCTAACATCTTCGATAAGTACAATTTGGAATACAAAATTCATGTTGATGG TGCATTTGGTGGTTTTATATATCCAATTGATAATAAGGAATGTAAAACTG ATTTCTCTAATAAGAACGTTTCTTCAATCACATTAGATGGTCATAAGATG TTGCAAGCTCCATACGGTACTGGTATCTTCGTTTCAAGAAAGAATTTGAT CCATAACACTTTGACAAAGGAAGCAACTTACATCGAAAATTTGGATGTTA CATTGTCTGGTTCAAGATCTGGTTCAAATGCTGTTGCAATTTGGATGGTT TTAGCTTCTTATGGTCCATACGGTTGGATGGAAAAGATTAATAAGTTGAG AAATGAACTAAATGGTTGTGTAAGCAATTGAACGATATGAGAATTAAATA TTACAAAGAAGATTCAATGAATATTGTTACAATTGAAGAACAATATGTTA ATAAGGAAATCGCTGAAAAGTACTTTTTAGTTCCAGAAGTTCATAACCCA ACTAACAACTGGTACAAGATCGTTGTTATGGAACATGTTGAATTGGATAT CTTGAACTCTTTGGTTTACGATTTGAGAAAGTTTAATAAGGAACATTTGA AGGCAATGCATCATCATCATCATCATTAACCGCGGCTAGCTAAGATCCGC TCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTT ATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATT TTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAA AACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGATCCAGCTGCATTAAT GAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCC GCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGC GGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGG ATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAAC GTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGAC GAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGG ACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTC CTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCG GGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGT GTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGC CCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTA AGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAG AGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACT ACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCA GTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCAC CGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAA AAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCT CAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAA AAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAA TCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATC AGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGC CTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTG GCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGAT TTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCC TGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTA GAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCT ACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTC CGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAA AAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCC GCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGT CATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGT CATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCA ATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCAT TGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGA GATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCT TTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGC CGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCT TCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGC GGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCG CACATTTCCCCGAAAAGTGCCACCTGAACGAAGCATCTGTGCTTCATTTT GTAGAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCT GAGCTGCATTTTTACAGAACAGAAATGCAACGCGAAAGCGCTATTTTACC AACGAAGAATCTGTGCTTCATTTTTGTAAAACAAAAATGCAACGCGAGAG CGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAA TGCAACGCGAGAGCGCTATTTTACCAACAAAGAATCTATACTTCTTTTTT GTTCTACAAAAATGCATCCCGAGAGCGCTATTTTTCTAACAAAGCATCTT AGATTACTTTTTTTCTCCTTTGTGCGCTCTATAATGCAGTCTCTTGATAA CTTTTTGCACTGTAGGTCCGTTAAGGTTAGAAGAAGGCTACTTTGGTGTC TATTTTCTCTTCCATAAAAAAAGCCTGACTCCACTTCCCGCGTTTACTGA TTACTAGCGAAGCTGCGGGTGCATTTTTTCAAGATAAAGGCATCCCCGAT TATATTCTATACCGATGTGGATTGCGCATACTTTGTGAACAGAAAGTGAT AGCGTTGATGATTCTTCATTGGTCAGAAAATTATGAACGGTTTCTTCTAT TTTGTCTCTATATACTACGTATAGGAAATGTTTACATTTTCGTATTGTTT TCGATTCACTCTATGAATAGTTCTTACTACAATTTTTTTGTCTAAAGAGT AATACTAGAGATAAACATAAAAAATGTAGAGGTCGAGTTTAGATGCAAGT TCAAGGAGCGAAAGGTGGATGGGTAGGTTATATAGGGATATAGCACAGAG ATATATAGCAAAGAGATACTTTTGAGCAATGTTTGTGGAAGCGGTATTCG CAATATTTTAGTAGCTCGTTACAGTCCGGTGCGTTTTTGGTTTTTTGAAA GTGCGTCTTCAGAGCGCTTTTGGTTTTCAAAAGCGCTCTGAAGTTCCTAT ACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCAAAGCGTTTCCGA AAACGAGCGCTTCCGAAAATGCAACGCGAGCTGCGCACATACAGCTCACT GTTCACGTCGCACCTATATCTGCGTGTTGCCTGTATATATATATACATGA GAAGAACGGCATAGTGCGTGTTTATGCTTAAATGCGTACTTATATGCGTC TATTTATGTAGGATGAAAGGTAGTCTAGTACCTCCTGTGATATTATCCCA TTCCATGCGGGGTATCGTATGCTTCCTTCAGCACTACCCTTTAGCTGTTC TATATGCTGCCACTCCTCAATTGGATTAGTCTCATCCTTCAATGCTATCA TTTCCTTTGATATTGGATCATACTAAGAAACCATTATTATCATGACATTA ACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGG TGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAG CTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCA GCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCA GATTGTACTGAGAGTGCACCATATTTCACACCGCATAGATCCGTCGAGTT CAAGAGAAAAAAAAAGAAAAAGCAAAAAGAAAAAAGGAAAGCGCGCCTCG TTCAGAATGACACGTATAGAATGATGCATTACCTTGTCATCTTCAGTATC ATACTGTTCGTATACATACTTACTGACATTCATAGGTATACATATATACA CATGTATATATATCGTATGCTGCAGCTTTAAATAATCGGTGTCACTACAT AAGAACACCTTTGGTGGAGGGAACATCGTTGGTACCATTGGGCGAGGTGG CTTCTCTTATGGCAACCGCAAGAGCCTTGAACGCACTCTCACTACGGTGA TGATCATTCTTGCCTCGCAGACAATCAACGTGGAGGGTAATTCTGCTAGC CTCTGCAAAGCTTTCAAGAAAATGCGGGATCATCTCGCAAGAGAGATCTC CTACTTTCTCCCTTTGCAAACCAAGTTCGACAACTGCGTACGGCCTGTTC GAAAGATCTACCACCGCTCTGGAAAGTGCCTCATCCAAAGGCGCAAATCC TGATCCAAACCTTTTTACTCCACGCACGGCCCCTAGGGCCTCTTTAAAAG CTTGACCGAGAGCAATCCCGCAGTCTTCAGTGGTGTGATGGTCGTCTATG TGTAAGTCACCAATGCACTCAACGATTAGCGACCAGCCGGAATGCTTGGC CAGAGCATGTATCATATGGTCCAGAAACCCTATACCTGTGTGGACGTTAA TCACTTGCGATTGTGTGGCCTGTTCTGCTACTGCTTCTGCCTCTTTTTCT GGGAAGATCGAGTGCTCTATCGCTAGGGGACCACCCTTTAAAGAGATCGC AATCTGAATCTTGGTTTCATTTGTAATACGCTTTACTAGGGCTTTCTGCT CTGTCATCTTTGCCTTCGTTTATCTTGCCTGCTCATTTTTTAGTATATTC TTCGAAGAAATCACATTACTTTATATAATGTATAATTCATTATGTGATAA TGCCAATCGCTAAGAAAAAAAAAGAGTCATCCGCTAGGTGGAAAAAAAAA AATGAAAATCATTACCGAGGCATAAAAAAATATAGAGTGTACTAGAGGAG GCCAAGAGTAATAGAAAAAGAAAATTGCGGGAAAGGACTGTGTTATGACT TCCCTGACTAATGCCGTGTTCAAACGATACCTGGCAGTGACTCCTAGCGC TCACCAAGCTCTTAAAACGGGAATTTATGGTGCACTCTCAGTACACGCGC CAGATCTGTTTAGCTTGCCTCGTCCCCGCCGGGTCACCCGGCCAGCGACA TGGAGGCCCAGAATACCCTCCTTGACAGTCTTGACGTGCGCAGCTCAGGG GCATGATGTGACTGTCGCCCGTACATTTAGCCCATACATCCCCATGTATA ATCATTTGCATCCATACATTTTGATGGCCGCACGGCGCGAAGCAAAAATT ACGGCTCCTCGCTGCAGACCTGCGAGCAGGGAAACGCTCCCCTCACAGAC GCGTTGAATTGTCCCCACGCCGCGCCCCTGTAGAGAAATATAAAAGGTTA GGATTTGCCACTGAGGTTCTTCTTTCATATACTTCCTTTTAAAATCTTGC TAGGATACAGTTCTCACATCACATCCGAACATAAACAACCATGGGTAAGG AAAAGACTCACGTTTCGAGGCCGCGATTAAATTCCAACATGGATGCTGAT TTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGAC AATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAAC ATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTA AACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCG TACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGCAAAACAG CATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGAT GCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTG TCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAA TGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGC TGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAGCTTTTGCCATTCTC ACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTT TTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATC GCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTT TTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATC CTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAA TCAGTACTGACAATAAAAAGATTCTTGTTTTCAAGAACTTGTCATTTGTA TAGTTTTTTTATATTGTAGTTGTTCTATTTTAATCAAATGTTAGCGTGAT TTATATTTTTTTTCGCCTCGACATCATCTGCCCAGATGCGAAGTTAAGTG CGCAGAAAGTAATATCATGCGTCAATCGTATGTGAATGCTGGTCGCTATA CTGCTGTCGATTCGATACTAACGCCGCCATCCAGTGTCGAATTCGCCATT CAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTAT TACGCCAGCTGAATTGGAGCGACCTCATGCTATACCTGAGAAAGCAACCT GACCTACAGGAAAGAGTTACTCAAGAATAAGAATTTTCGTTTTAAAACCT AAGAGTCACTTTAAAATTTGTATACACTTATTTTTTTTATAACTTATTTA ATAATAAAAATCATAAATCATAAGAAATTCGCTTATTTAGAAGTGTCAAC AACGTATCTACCAACGATTTGACCCTTTTCCATCTTTTCGTAAATTTCTG GCAAGGTAGACAAGCCGACAACCTTGATTGGAGACTTGACCAAACCTCTG GCGAAGAATTGTTAATTAAGAGCTCAGATCTTTTGCGGCCGC
[0940] SEQ.ID NO: 30
TABLE-US-00030 GTTTGCAAAAAGAACAAAACTGAAAAAACCCAGACACGCTCGACTTCCTG TCTTCCTATTGATTGCAGCTTCCAATTTCGTCACACAACAAGGTCCTAGC GACGGCTCACAGGTTTTGTAACAAGCAATCGAAGGTTCTGGAATGGCGGG AAAGGGTTTAGTACCACATGCTATGATGCCCACTGTGATCTCCAGAGCAA AGTTCGTTCGATCGTACTGTTACTCTCTCTCTTTCAAACAGAATTGTCCG AATCGTGTGACAACAACAGCCTGTTCTCACACACTCTTTTCTTCTAACCA AGGGGGTGGTTTAGTTTAGTAGAACCTCGTGAAACTTACATTTACATATA TATAAACTTGCATAAATTGGTCAATGCAAGAAATACATATTTGGTCTTTT CTAATTCGTAGTTTTTCAAGTTCTTAGATGCTTTCTTTTTCTCTTTTTTA CAGATCATCAAGGAAGTAATTATCTACTTTTTACAACAAATATAAAACA
[0941] SEQ.ID NO: 31
TABLE-US-00031 ACAGTTTATTCCTGGCATCCACTAAATATAATGGAGCCCGCTTTTTAAGC TGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATATTGTTTTCTTCA CCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTACAGAGAACAG GGGCACAAACAGGCAAAAAACGGGCACAACCTCAATGGAGTGATGCAACC TGCCTGGAGTAAATGATGACACAAGGCAATTGACCCACGCATGTATCTAT CTCATTTTCTTACACCTTCTATTACCTTCTGCTCTCTCTGATTTGGAAAA AGCTGAAAAAAAAGGTTGAAACCAGTTCCCTGAAATTATTCCCCTACTTG ACTAATAAGTATATAAAGACGGTAGGTATTGATTGTAATTCTGTAAATCT ATTTCTTAAACTTCTTAAATTCTACTTTTATAGTTAGTCTTTTTTTTAGT TTTAAAACACCAAGAACTTAGTTTCGAATAAACACACATAAACAAACAAA
[0942] SEQ.ID NO: 32
TABLE-US-00032 GCTGGATTGAGCTGAATGGTGCCAGGTCGAGGCTGGGAGGGAGACTAACT CGAAAGTGACGAAGACTCGAAAATTAAAAAAAAAGATACTGCAGAAGGCA AGATTGAGAATGGAGTAAAGGCAGCGTGGGTCCCCTGTGGAAACCGCAGT TTTCCTGCGCCAAGTGGTACCGGTGCGAGTGCAGCAATTAATCTCTCGAT ATTTTCTTAGTATCTCTTTTTATATAAGAATATATTTTGGAATTGGTAAT GCTTATCTTCAATAGTTTCTTAGTTGAATGCACACTTAAGAGCAAATTGG CCAAGGAGTTCTTCGTTCGCTTTAATTTATTTCCTGGTTATTGTCAATTT ATTCATCCCATCTCCCCAGGATAGAAGAAATTAGTGTAATTTTGCTGACA ATACATTTTAACGACGATAACAATAATAGCAATTAAATAAAATAGCACTA CCACCACTCCACTGCTCGTTAGCTATTTCTGTAAAATAAATAAAAAGATC
[0943] SEQ.ID NO: 33
TABLE-US-00033 ATTCGCGCTATCTCGATTTCTACCTATATAGTTAATCTCTGTACAAAAAC AATCTTTCCAACTATCCATTAATCATAGTATATTATCAGCGTCGGCGATT TTACCACGCTTGACAAAAGCCGCGGGCGGGATTCCTGTGGGTAGTGGCAC CGGCAGTTAATCTAATCAAAGGCGCTTGAAGGAAGAGATAGATAATAGAA CAAAGCAATCGCCGCTTTGGACGGCAAATATGTTTATCCATTGGTGCGGT GATTGGATATGATTTGTCTCCAGTAGTATAAGCAAGCGCCAGATCTGTTT ACTGTAAAATTAAGTGAGTAATCTCGCGGGATGTAATGATTTAAGGGAAT CTGGTTCAGGTTTTCACATATATTTGTATATAAGGCCATTTGTAATTTCA ATAGTTTTAGGATTTTTCCTTCTCCCAAAATACTCACTTACTGTGTTACA TTACAGAAAGAACAGACAAGAAACCGTCAATAAGAAATATAACTAAGAAC A
[0944] SEQ.ID NO: 34
TABLE-US-00034 CAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTT TTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATA AATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCC GTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCT CACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGC ACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGA GTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTG CTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGG TCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCA CAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCT GCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGAT CGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATG TAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAAC GACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAA ACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAG ACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTT CCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTC TCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCG TAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGA CAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGA CCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAAT TTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATC CCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGAT CAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGC AAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAG CTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACC AAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACT CTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCT GCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATA GTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACAC AGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGT GAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTA TCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAG GGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGA CTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAA AAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTT TTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGT ATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGA GCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAAC CGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGATCTTCGAG CGTCCCAAAACCTTCTCAAGCAAGGTTTTCAGTATAATGTTACATGCGTA CACGCGTCTGTACAGAAAAAAAAGAAAAATTTGAAATATAAATAACGTTC TTAATACTAACATAACTATAAAAAAATAAATAGGGACCTAGACTTCAGGT TGTCTAACTCCTTCCTTTTCGGTTAGAGCGGATCTTAGCTAGCCGCGGTA CCAAGCTGGTGGATCCTTTGTTTGTTTATGTGTGTTTATTCGAAACTAAG TTCTTGGTGTTTTAAAACTAAAAAAAAGACTAACTATAAAAGTAGAATTT AAGAAGTTTAAGAAATAGATTTACAGAATTACAATCAATACCTACCGTCT TTATATACTTATTAGTCAAGTAGGGGAATAATTTCAGGGAACTGGTTTCA ACCTTTTTTTTCAGCTTTTTCCAAATCAGAGAGAGCAGAAGGTAATAGAA GGTGTAAGAAAATGAGATAGATACATGCGTGGGTCAATTGCCTTGTGTCA TCATTTACTCCAGGCAGGTTGCATCACTCCATTGAGGTTGTGCCCGTTTT TTGCCTGTTTGTGCCCCTGTTCTCTGTAGTTGCGCTAAGAGAATGGACCT ATGAACTGATGGTTGGTGAAGAAAACAATATTTTGGTGCTGGGATTCTTT TTTTTTCTGGATGCCAGCTTAAAAAGCGGGCTCCATTATATTTAGTGGAT GCCAGGAATAAACTGTTGTTTGCAAAAAGAACAAAACTGAAAAAACCCAG ACACGCTCGACTTCCTGTCTTCCTATTGATTGCAGCTTCCAATTTCGTCA CACAACAAGGTCCTAGCGACGGCTCACAGGTTTTGTAACAAGCAATCGAA GGTTCTGGAATGGCGGGAAAGGGTTTAGTACCACATGCTATGATGCCCAC TGTGATCTCCAGAGCAAAGTTCGTTCGATCGTACTGTTACTCTCTCTCTT TCAAACAGAATTGTCCGAATCGTGTGACAACAACAGCCTGTTCTCACACA CTCTTTTCTTCTAACCAAGGGGGTGGTTTAGTTTAGTAGAACCTCGTGAA ACTTACATTTACATATATATAAACTTGCATAAATTGGTCAATGCAAGAAA TACATATTTGGTCTTTTCTAATTCGTAGTTTTTCAAGTTCTTAGATGCTT TCTTTTTCTCTTTTTTACAGATCATCAAGGAAGTAATTATCTACTTTTTA CAACAAATATAAAACAGCGGCCGCAAAAGATCTGAGCTCTTAATTAACAA TTCTTCGCCAGAGGTTTGGTCAAGTCTCCAATCAAGGTTGTCGGCTTGTC TACCTTGCCAGAAATTTACGAAAAGATGGAAAAGGGTCAAATCGTTGGTA GATACGTTGTTGACACTTCTAAATAAGCGAATTTCTTATGATTTATGATT TTTATTATTAAATAAGTTATAAAAAAAATAAGTGTATACAAATTTTAAAG TGACTCTTAGGTTTTAAAACGAAAATTCTTATTCTTGAGTAACTCTTTCC TGTAGGTCAGGTTGCTTTCTCAGGTATAGCATGAGGTCGCTCCAATTCAG CTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGC GCAGCCTGAATGGCGAATTCGACACTGGATGGCGGCGTTAGTATCGAATC GACAGCAGTATAGCGACCAGCATTCACATACGATTGACGCATGATATTAC TTTCTGCGCACTTAACTTCGCATCTGGGCAGATGATGTCGAGGCGAAAAA AAATATAAATCACGCTAACATTTGATTAAAATAGAACAACTACAATATAA AAAAACTATACAAATGACAAGTTCTTGAAAACAAGAATCTTTTTATTGTC AGTACTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATT CATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATG AAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTAT CGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCC CTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTG AATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCA ACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACC GTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGT TAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACT GCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATAC CTGGAATGCTGTTTTGCCGGGGATCGCAGTGGTGAGTAACCATGCATCAT CAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTC AGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACC TTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATC GATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATAC CCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAAACGTG AGTCTTTTCCTTACCCATGGTTGTTTATGTTCGGATGTGATGTGAGAACT GTATCCTAGCAAGATTTTAAAAGGAAGTATATGAAAGAAGAACCTCAGTG GCAAATCCTAACCTTTTATATTTCTCTACAGGGGCGCGGCGTGGGGACAA TTCAACGCGTCTGTGAGGGGAGCGTTTCCCTGCTCGCAGGTCTGCAGCGA GGAGCCGTAATTTTTGCTTCGCGCCGTGCGGCCATCAAAATGTATGGATG CAAATGATTATACATGGGGATGTATGGGCTAAATGTACGGGCGACAGTCA CATCATGCCCCTGAGCTGCGCACGTCAAGACTGTCAAGGAGGGTATTCTG GGCCTCCATGTCGCTGGCCGGGTGACCCGGCGGGGACGAGGCAAGCTAAA CAGATCTGGCGCGTGTACTGAGAGTGCACCATAAATTCCCGTTTTAAGAG CTTGGTGAGCGCTAGGAGTCACTGCCAGGTATCGTTTGAACACGGCATTA GTCAGGGAAGTCATAACACAGTCCTTTCCCGCAATTTTCTTTTTCTATTA CTCTTGGCCTCCTCTAGTACACTCTATATTTTTTTATGCCTCGGTAATGA TTTTCATTTTTTTTTTTCCACCTAGCGGATGACTCTTTTTTTTTCTTAGC GATTGGCATTATCACATAATGAATTATACATTATATAAAGTAATGTGATT TCTTCGAAGAATATACTAAAAAATGAGCAGGCAAGATAAACGAAGGCAAA GATGACAGAGCAGAAAGCCCTAGTAAAGCGTATTACAAATGAAACCAAGA TTCAGATTGCGATCTCTTTAAAGGGTGGTCCCCTAGCGATAGAGCACTCG ATCTTCCCAGAAAAAGAGGCAGAAGCAGTAGCAGAACAGGCCACACAATC GCAAGTGATTAACGTCCACACAGGTATAGGGTTTCTGGACCATATGATAC ATGCTCTGGCCAAGCATTCCGGCTGGTCGCTAATCGTTGAGTGCATTGGT GACTTACACATAGACGACCATCACACCACTGAAGACTGCGGGATTGCTCT CGGTCAAGCTTTTAAAGAGGCCCTAGGGGCCGTGCGTGGAGTAAAAAGGT TTGGATCAGGATTTGCGCCTTTGGATGAGGCACTTTCCAGAGCGGTGGTA GATCTTTCGAACAGGCCGTACGCAGTTGTCGAACTTGGTTTGCAAAGGGA GAAAGTAGGAGATCTCTCTTGCGAGATGATCCCGCATTTTCTTGAAAGCT TTGCAGAGGCTAGCAGAATTACCCTCCACGTTGATTGTCTGCGAGGCAAG AATGATCATCACCGTAGTGAGAGTGCGTTCAAGGCTCTTGCGGTTGCCAT AAGAGAAGCCACCTCGCCCAATGGTACCAACGATGTTCCCTCCACCAAAG GTGTTCTTATGTAGTGACACCGATTATTTAAAGCTGCAGCATACGATATA TATACATGTGTATATATGTATACCTATGAATGTCAGTAAGTATGTATACG AACAGTATGATACTGAAGATGACAAGGTAATGCATCATTCTATACGTGTC ATTCTGAACGAGGCGCGCTTTCCTTTTTTCTTTTTGCTTTTTCTTTTTTT TTCTCTTGAACTCGACGGATCTATGCGGTGTGAAATATGGTGCACTCTCA GTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCA ACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTA CAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCAC CGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTT TTATAGGTTAATGTCATGATAATAATGGTTTCTTAGTATGATCCAATATC AAAGGAAATGATAGCATTGAAGGATGAGACTAATCCAATTGAGGAGTGGC AGCATATAGAACAGCTAAAGGGTAGTGCTGAAGGAAGCATACGATACCCC GCATGGAATGGGATAATATCACAGGAGGTACTAGACTACCTTTCATCCTA CATAAATAGACGCATATAAGTACGCATTTAAGCATAAACACGCACTATGC CGTTCTTCTCATGTATATATATATACAGGCAACACGCAGATATAGGTGCG ACGTGAACAGTGAGCTGTATGTGCGCAGCTCGCGTTGCATTTTCGGAAGC GCTCGTTTTCGGAAACGCTTTGAAGTTCCTATTCCGAAGTTCCTATTCTC TAGAAAGTATAGGAACTTCAGAGCGCTTTTGAAAACCAAAAGCGCTCTGA AGACGCACTTTCAAAAAACCAAAAACGCACCGGACTGTAACGAGCTACTA AAATATTGCGAATACCGCTTCCACAAACATTGCTCAAAAGTATCTCTTTG CTATATATCTCTGTGCTATATCCCTATATAACCTACCCATCCACCTTTCG CTCCTTGAACTTGCATCTAAACTCGACCTCTACATTTTTTATGTTTATCT CTAGTATTACTCTTTAGACAAAAAAATTGTAGTAAGAACTATTCATAGAG TGAATCGAAAACAATACGAAAATGTAAACATTTCCTATACGTAGTATATA GAGACAAAATAGAAGAAACCGTTCATAATTTTCTGACCAATGAAGAATCA TCAACGCTATCACTTTCTGTTCACAAAGTATGCGCAATCCACATCGGTAT AGAATATAATCGGGGATGCCTTTATCTTGAAAAAATGCACCCGCAGCTTC GCTAGTAATCAGTAAACGCGGGAAGTGGAGTCAGGCTTTTTTTATGGAAG AGAAAATAGACACCAAAGTAGCCTTCTTCTAACCTTAACGGACCTACAGT GCAAAAAGTTATCAAGAGACTGCATTATAGAGCGCACAAAGGAGAAAAAA AGTAATCTAAGATGCTTTGTTAGAAAAATAGCGCTCTCGGGATGCATTTT TGTAGAACAAAAAAGAAGTATAGATTCTTTGTTGGTAAAATAGCGCTCTC GCGTTGCATTTCTGTTCTGTAAAAATGCAGCTCAGATTCTTTGTTTGAAA AATTAGCGCTCTCGCGTTGCATTTTTGTTTTACAAAAATGAAGCACAGAT TCTTCGTTGGTAAAATAGCGCTTTCGCGTTGCATTTCTGTTCTGTAAAAA TGCAGCTCAGATTCTTTGTTTGAAAAATTAGCGCTCTCGCGTTGCATTTT TGTTCTACAAAATGAAGCACAGATGCTTCGTT
[0945] SEQ.ID NO: 35
TABLE-US-00035 GCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCC CAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCT GGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCC ACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACT GACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGC CGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCG CGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACT GGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTG CCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTT TCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAAC GGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTG GTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCC ATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCT CTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGG CCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCC CAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAG CGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCG GCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCAC GATGCGTCCGGCGTAGCCTAGGATCGAGATCGATCTCGATCCCGCGAAAT TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTA GAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGGCAGATCTC AATTGGATATCGGCCGGCCACGCGATCGCTGACGTCGGTACCCTCGAGTC TGGTAAAGAAACCGCTGCTGCGAAATTTGAACGCCAGCACATGGACTCGT CTACTAGTCGCAGCTTAATTAACCTAAACTGCTGCCACCGCTGAGCAATA ACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGC TAGCGAAAGGAGGAGTCGACACTGCTTCCGGTAGTCAATAAACCGGTAAA CCAGCAATAGACATAAGCGGCTATTTAACGACCCTGCCCTGAACCGACGA CCGGGTCATCGTGGCCGGATCTTGCGGCCCCTCGGCTTGAACGAATTGTT AGACATTATTTGCCGACTACCTTGGTGATCTCGCCTTTCACGTAGTGGAC AAATTCTTCCAACTGATCTGCGCGCGAGGCCAAGCGATCTTCTTCTTGTC CAAGATAAGCCTGTCTAGCTTCAAGTATGACGGGCTGATACTGGGCCGGC AGGCGCTCCATTGCCCAGTCGGCAGCGACATCCTTCGGCGCGATTTTGCC GGTTACTGCGCTGTACCAAATGCGGGACAACGTAAGCACTACATTTCGCT CATCGCCAGCCCAGTCGGGCGGCGAGTTCCATAGCGTTAAGGTTTCATTT AGCGCCTCAAATAGATCCTGTTCAGGAACCGGATCAAAGAGTTCCTCCGC CGCTGGACCTACCAAGGCAACGCTATGTTCTCTTGCTTTTGTCAGCAAGA TAGCCAGATCAATGTCGATCGTGGCTGGCTCGAAGATACCTGCAAGAATG TCATTGCGCTGCCATTCTCCAAATTGCAGTTCGCGCTTAGCTGGATAACG CCACGGAATGATGTCGTCGTGCACAACAATGGTGACTTCTACAGCGCGGA GAATCTCGCTCTCTCCAGGGGAAGCCGAAGTTTCCAAAAGGTCGTTGATC AAAGCTCGCCGCGTTGTTTCATCAAGCCTTACGGTCACCGTAACCAGCAA ATCAATATCACTGTGTGGCTTCAGGCCGCCATCCACTGCGGAGCCGTACA AATGTACGGCCAGCAACGTCGGTTCGAGATGGCGCTCGATGACGCCAACT ACCTCTGATAGTTGAGTCGATACTTCGGCGATCACCGCTTCCCTCATACT CTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGA GCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGCCAGCTCA CTCGGTCGCTACGCTCCGGGCGTGAGACTGCGGCGGGCGCTGCGGACACA TACAAAGTTACCCACAGATTCCGTGGATAAGCAGGGGACTAACATGTGAG GCAAAACAGCAGGGCCGCGCCGGTGGCGTTTTTCCATAGGCTCCGCCCTC CTGCCAGAGTTCACATAAACAGACGCTTTTCCGGTGCATCTGTGGGAGCC GTGAGGCTCAACCATGAATCTGACAGTACGGGCGAAACCCGACAGGACTT AAAGATCCCCACCGTTTCCGGCGGGTCGCTCCCTCTTGCGCTCTCCTGTT CCGACCCTGCCGTTTACCGGATACCTGTTCCGCCTTTCTCCCTTACGGGA AGTGTGGCGCTTTCTCATAGCTCACACACTGGTATCTCGGCTCGGTGTAG GTCGTTCGCTCCAAGCTGGGCTGTAAGCAAGAACTCCCCGTTCAGCCCGA CTGCTGCGCCTTATCCGGTAACTGTTCACTTGAGTCCAACCCGGAAAAGC ACGGTAAAACGCCACTGGCAGCAGCCATTGGTAACTGGGAGTTCGCAGAG GATTTGTTTAGCTAAACACGCGGTTGCTCTTGAAGTGTGCGCCAAAGTCC GGCTACACTGGAAGGACAGATTTGGTTGCTGTGCTCTGCGAAAGCCAGTT ACCACGGTTAAGCAGTTCCCCAACTGACTTAACCTTCGATCAAACCACCT CCCCAGGTGGTTTTTTCGTTTACAGGGCAAAAGATTACGCGCAGAAAAAA AGGATCTCAAGAAGATCCTTTGATCTTTTCTACTGAACCGCTCTAGATTT CAGTGCAATTTATCTCTTCAAATGTAGCACCTGAAGTCAGCCCCATACGA TATAAGTTGTAATTCTCATGTTAGTCATGCCCCGCGCCCACCGGAAGGAG CTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTA ATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCC AGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCG GGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCA GTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGT TGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTT GATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGT ATCCCACTACCGAGATGTCCGCACCAACGCGCAGCCCGGACTCGGTAATG GCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGT GGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACA TGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGA GTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACT TAATGGGCCC
[0946] SEQ.ID NO: 36
TABLE-US-00036 ATCCGGATATAGTTCCTCCTTTCAGCAAAAAACCCCTCAAGACCCGTTTA GAGGCCCCAAGGGGTTATGCTAGTTATTGCTCAGCGGTGGCAGCAGCCAA CTCAGCTTCCTTTCGGGCTTTGTTAGCAGCCGGATCTCAGTGGTGGTGGT GGTGGTGCTCGAGTGCGGCCGCAAGCTTGTCGACGGAGCTCGAATTCGGA TCCGCGACCCATTTGCTGTCCACCAGTCATGCTAGCCATATGGCTGCCGC GCGGCACCAGGCCGCTGCTGTGATGATGATGATGATGGCTGCTGCCCATG GTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAGGGGAATTGT TATCCGCTCACAATTCCCCTATAGTGAGTCGTATTAATTTCGCGGGATCG AGATCTCGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCGCC ACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGA TCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGG TGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCA CCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTG CTTCCTAATGCAGGAGTCGCATAAGGGAGAGCGTCGAGATCCCGGACACC ATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGA GAGTCAATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCG CAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAG GCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGC GGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAAC AGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCG TCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAG CGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGG CGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTAT CCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGT TCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTA TTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCA TTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTC GGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAA TTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTT CAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCT GGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGT CCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACC GAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTT TCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAGG GCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGA AAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGC CGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGC AGTGAGCGCAACGCAATTAATGTAAGTTAGCTCACTCATTAGGCACCGGG ATCTCGACCGATGCCCTTGAGAGCCTTCAACCCAGTCAGCTCCTTCCGGT GGGCGCGGGGCATGACTATCGTCGCCGCACTTATGACTGTCTTCTTTATC ATGCAACTCGTAGGACAGGTGCCGGCAGCGCTCTGGGTCATTTTCGGCGA GGACCGCTTTCGCTGGAGCGCGACGATGATCGGCCTGTCGCTTGCGGTAT TCGGAATCTTGCACGCCCTCGCTCAAGCCTTCGTCACTGGTCCCGCCACC AAACGTTTCGGCGAGAAGCAGGCCATTATCGCCGGCATGGCGGCCCCACG GGTGCGCATGATCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGG GTTGCCTTACTGGTTAGCAGAATGAATCACCGATACGCGAGCGAACGTGA AGCGACTGCTGCTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGT CTTCGGTTTCCGTGTTTCGTAAAGTCTGGAAACGCGGAAGTCAGCGCCCT GCACCATTATGTTCCGGATCTGCATCGCAGGATGCTGCTGGCTACCCTGT GGAACACCTACATCTGTATTAACGAAGCGCTGGCATTGACCCTGAGTGAT TTTTCTCTGGTCCCGCCGCATCCATACCGCCAGTTGTTTACCCTCACAAC GTTCCAGTAACCGGGCATGTTCATCATCAGTAACCCGTATCGTGAGCATC CTCTCTCGTTTCATCGGTATCATTACCCCCATGAACAGAAATCCCCCTTA CACGGAGGCATCAGTGACCAAACAGGAAAAAACCGCCCTTAACATGGCCC GCTTTATCAGAAGCCAGACATTAACGCTTCTGGAGAAACTCAACGAGCTG GACGCGGATGAACAGGCAGACATCTGTGAATCGCTTCACGACCACGCTGA TGAGCTTTACCGCAGCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACC TCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGAT GCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTG TCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTG GCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATAT GCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGC GCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTG CGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGA ATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGG CCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGC CCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAA CCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCG TGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTT CTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCT CAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCC CCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCC AACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAG GATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGT GGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTG CTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAA ACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTA CGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGG TCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAA CAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGC CATATTCAACGGGAAACGTCTTGCTCTAGGCCGCGATTAAATTCCAACAT GGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAAT CAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTG TTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGAT GGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGC ATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCC GGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAA TATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTG TTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCG CAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGA GCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTT TGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGAT AACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACG AGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCC TCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGT ATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGA GTTTTTCTAAGAATTAATTCATGAGCGGATACATATTTGAATGTATTTAG AAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACC TGAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTT AAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTAT AAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAA CAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAA CCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGT TTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAG CCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGG AAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCG GTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACA GGGCGCGTCCCATTCGCCA
[0947] SEQ.ID NO: 37
TABLE-US-00037 ATCCGGATATAGTTCCTCCTTTCAGCAAAAAACCCCTCAAGACCCGTTTA GAGGCCCCAAGGGGTTATGCTAGTTATTGCTCAGCGGTGGCAGCAGCCAA CTCAGCTTCCTTTCGGGCTTTGTTAGCAGCCGGATCTCAGTGGTGGTGGT GGTGGTGCTCGAGTGCGGCCGCAAGCTTGTCGACGGAGCTCGAATTCGGA TCCTAGAGGGAAACCGTTGTGGTCTCCCTATAGTGAGTCGTATTAATTTC GCGGGATCGAGATCTCGGGCAGCGTTGGGTCCTGGCCACGGGTGCGCATG ATCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTAC TGGTTAGCAGAATGAATCACCGATACGCGAGCGAACGTGAAGCGACTGCT GCTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTC CGTGTTTCGTAAAGTCTGGAAACGCGGAAGTCAGCGCCCTGCACCATTAT GTTCCGGATCTGCATCGCAGGATGCTGCTGGCTACCCTGTGGAACACCTA CATCTGTATTAACGAAGCGCTGGCATTGACCCTGAGTGATTTTTCTCTGG TCCCGCCGCATCCATACCGCCAGTTGTTTACCCTCACAACGTTCCAGTAA CCGGGCATGTTCATCATCAGTAACCCGTATCGTGAGCATCCTCTCTCGTT TCATCGGTATCATTACCCCCATGAACAGAAATCCCCCTTACACGGAGGCA TCAGTGACCAAACAGGAAAAAACCGCCCTTAACATGGCCCGCTTTATCAG AAGCCAGACATTAACGCTTCTGGAGAAACTCAACGAGCTGGACGCGGATG AACAGGCAGACATCTGTGAATCGCTTCACGACCACGCTGATGAGCTTTAC CGCAGCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACAT GCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCA GACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCA GCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTAT GCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATATGCGGTGTGAA ATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGC TTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGG TATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGAT AACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCG TAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACG AGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGA CTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGTCTCCT GTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGG AAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGT AGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCC GACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAG ACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAG CGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTAC GGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGT TACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCG CTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAA AAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCA GTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAA GGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATC TAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAG TGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCT GACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGC CCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTT ATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTG CAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGA GTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGC AGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCG GTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAA GCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGC AGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCA TGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCA TTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAAT ACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTG GAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGA TCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTT TACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCG CAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTC CTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGG ATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCA CATTTCCCCGAAAAGTGCCACCTGAAATTGTAAACGTTAATATTTTGTTA AAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGC CGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGT TGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGAC TCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACG TGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCAC TAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAG CCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGC TAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCG CCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCA
[0948] SEQ.ID NO: 38
TABLE-US-00038 TACCCATACGACGTTCCAGACTACGCC
[0949] SEQ.ID NO: 39
TABLE-US-00039 YPYDVPDYA
[0950] SEQ.ID NO: 40
TABLE-US-00040 GAACAAAAGTTAATTTCTGAAGAAGATTTGGAA
[0951] SEQ.ID NO: 41
TABLE-US-00041 EQKLISEEDL
[0952] SEQ.ID NO: 42
TABLE-US-00042 GACTACAAGGATGACGATGACAAA
[0953] SEQ.ID NO: 43
TABLE-US-00043 DYKDDDDK
[0954] SEQ.ID NO: 44
TABLE-US-00044 GGTAAGCCAATTCCAAATCCTTTGTTGGGTTTGGACTCCACC
[0955] SEQ.ID NO: 45
TABLE-US-00045 GKPIPNPLLGLDST
[0956] SEQ.ID NO: 46
TABLE-US-00046 CATCATCATCATCATCAT
[0957] SEQ.ID NO: 47
TABLE-US-00047 HHHHHH
[0958] SEQ.ID NO: 48
TABLE-US-00048 ATGGCGTTCGATCTCAAGACTGAAGACGGCCTCATCACATATCTCACTAA ACATCTTTCTTTGGACGTCGACACGAGCGGAGTGAAGCGCCTTAGCGGAG GCTTTGTCAATGTAACCTGGCGCATTAAGCTCAATGCTCCTTATCAAGGT CATACGAGCATCATCCTGAAGCATGCTCAGCCGCACATGTCTACGGATGA GGATTTTAAGATAGGTGTAGAACGTTCGGTTTACGAATACCAGGCTATCA AGCTCATGATGGCCAATCGGGAGGTTCTGGGAGGCGTGGATGGCATAGTT TCTGTGCCAGAAGGCCTGAACTACGACTTAGAGAATAATGCATTGATCAT GCAAGATGTCGGGAAGATGAAGACCCTTTTAGATTATGTCACCGCCAAAC CGCCACTTGCGACGGATATAGCCCGCCTTGTTGGGACAGAAATTGGGGGG TTCGTTGCCAGACTCCATAACATAGGCCGCGAGAGGCGAGACGATCCTGA GTTCAAATTCTTCTCTGGAAATATTGTCGGAAGGACGACTTCAGACCAGC TGTATCAAACCATCATACCCAACGCAGCGAAATATGGCGTCGATGACCCC TTGCTGCCTACTGTGGTTAAGGACCTTGTGGACGATGTCATGCACAGCGA AGAGACCCTTGTCATGGCGGACCTGTGGAGTGGAAATATTCTTCTCCAGT TGGAGGAGGGAAACCCATCGAAGCTGCAGAAGATATATATCCTGGATTGG GAACTTTGCAAGTACGGCCCAGCGTCGTTGGACCTGGGCTATTTCTTGGG TGACTGCTATTTGATATCCCGCTTTCAAGACGAGCAGGTCGGTACGACGA TGCGGCAAGCCTACTTGCAAAGCTATGCGCGTACGAGCAAGCATTCGATC AACTACGCCAAAGTCACTGCAGGTATTGCTGCTCATATTGTGATGTGGAC CGACTTTATGCAGTGGGGGAGCGAGGAAGAAAGGATAAATTTTGTGAAAA AGGGGGTAGCTGCCTTTCACGACGCCAGGGGCAACAACGACAATGGGGAA ATTACGTCTACCTTACTGAAGGAATCATCCACTGCGTAA
[0959] SEQ.ID NO: 49
TABLE-US-00049 MAFDLKTEDGLITYLTKHLSLDVDTSGVKRLSGGFVNVTWRIKLNAPYQG HTSIILKHAQPHMSTDEDFKIGVERSVYEYQAIKLMMANREVLGGVDGIV SVPEGLNYDLENNALIMQDVGKMKTLLDYVTAKPPLATDIARLVGTEIGG FVARLHNIGRERRDDPEFKFFSGNIVGRTTSDQLYQTIIPNAAKYGVDDP LLPTVVKDLVDDVMHSEETLVMADLWSGNILLQLEEGNPSKLQKIYILDW ELCKYGPASLDLGYFLGDCYLISRFQDEQVGTTMRQAYLQSYARTSKHSI NYAKVTAGIAAHIVMWTDFMQWGSEEERINFVKKGVAAFHDARGNNDNGE ITSTLLKESSTA
[0960] SEQ.ID NO: 50
TABLE-US-00050 CGAGATCTTTGTGTTCGGTTACCCGGCTCAGATCCTAACTTCTTCTTTTG GTATGTTTATTCGTATAAGTTACTGTTGTCCACAGGCAATACTCTGCAGA AAATTAAAACGGCATTAATGCTAGGACAACCAGAATTGTTACTACTGTAT GTGCGATAGTTGATAACTGCAACATTATGCCCGGTATATTCTCAAAAAAC CCTATTACTGCATACGAAGAAATCGCAAGAGAAATCTTTCGGTTTGGAAA AGCTCACTGTGAGGTTCCTTGGAGCCAATAGTAATACAGCACAATCCAAG GAAAAATCTGGCCTATATGCAAGGAAGGAGAGATAGTCAAAAGCATTCTT TCCCCTAGAAGTTGGTGCATATATGGCATCGTTAAAACATATTACCCCCA AAATTTCTTCTCTAAACGATGTGCTTGGCCTTTGTTTTGGTTTTTGATGT CGGTCGTTTGAGGCCCCTTGCGGAAAATCGAGATCGCCGAATGGCACGCG AGGGAAGGGAAATAAGGTTTAAAGGCACTGAAACAATAGGCAAGAAGTAG GCGAGAGCCGACATACGAGACTAAATTAAGTCCTCAGCGAGCTCGCATGG AATGCGTGCGATGAGCGACCTCATGCTATACCTGAGAAAGCAACCTGACC TACAGGAAAGAGTTACTCAAGAATAAGAATTTTCGTTTTAAAACCTAAGA GTCACTTTAAAATTTGTATACACTTATTTTTTTTATAACTTATTTAATAA TAAAAATCATAAATCATAAGAAATTCGCTTATTTAGAAGTGTCAACAACG TATCTACCAACGGAATGCGTGCGATTCATTTGTCATCGTCATCCTTGTAG TCTTCAGCAACACATGGGTATGAAATAGAAACTTCTTCAGCCAATGCTTT AATCTTCATCATGATTTGCAACATTTCTTCAGTTGTAGTTCTTGGATTAA TTGAACACAATCTAATAACAACCTTTTCCTTCAATTCTGTAGTAGATAAC ATAGCGAAACCTCTATGTGTGATTTCCTTAACCAATTTCTTATTAATTTC ATTAATAGTATCTGTTGATGCCAATTCAGATGGAATGTATCTAAAAGTAA CGATACCCAATTGAGCTGGTGTAACAACTTCCCAATCTTTTGCTTTACCC AAAAATGCTTCAACTTGTTCTGCTAACATGATACCATGATCGATTGCTTG TCTAAAAGCAGCAACACCGAAAACTTTAAAAGACAACCAAACCTTCAAAG CTCTGAATCTTCTTGACAATTCGATACCACATTCACCGAAATTAATTTCA CCTTCAACGTTAGTTTCTGAATCCTTGATGTATTCTGGCATCATTCTAAA AGTCTTTGACAAATATTGAGAGTTTCTGATCAAAACACAACCAACATCGT ATGGTTGGAACAACCACTTATGTGGATCTAAAGTCAAAGAATCTGCTCTA TGAATACCTTGCAACATAGCTGAACCCTTTTCAGACAAGATAGCTGGAGC ACCATAAGAACCATCAGCATGCAACCAAACATCTTCATCGTTACACAAAT CTGCTAATTCGTTCAAAGAATCAACAGCACCACAATTTGTAGTACCAGCA TTTGCAATAACACAGAATGGCTTTTTACCCTTAGTTCTATCTTCTTTAAT TTGTTTCTTCAAAGCTGAAACAGAGATTCTCAAATGTTCATCTGTTTCGA TTCTACAGATTTGATGATGTTTAAAACCTAAAACCTTCAATGCTCTATCA ACTGAGAAATGTGTTTGATCAGAGAAGTAAACAACAGCATTTTCGATATC GTTGTTCAACTTAGCCTGTCTTGCAACAGTCAAAGCTGTCAAATTTGCCA TTGAACCACCAGAAACAAATAAACCTTCAGCTGAATCTGGAAAACCCAAC ATAGATTTCAACCAATTAATTGTAGTCAATTCGATTTGTTCAGCACCTGC ACCAGCAATCCATGCAGTTGGAAAAACATTAAAACCAGAAGCCAAGAAAT CTGCAACAACACCAACGTAATTATTTGGACCTGGAACAAAAGCCAAGAAA TGTGGATGATCAACATGTGTAATTTGATTAAAACGTTTCTGTTCAAGAAA TGCAACAATTCCTTTGGATCTGAACCATTTTCTGGGATAGATTCAGTCAA CTTATTTCTCAAGATATCAGAATCGATTGTTTCTGAAACTGGCTTAACTT CAAATGGTTCATGTGATCGATGATCAAATCAACTGCTTGGTAACCCAATT GTCTCATTTCTTCAGCTGACAATTGCAAATTTTCAGACATTGTTTTATAT TTGTTGTAAAAAGTAGATAATTACTTCCTTGATGATCTGTAAAAAAGAGA AAAAGAAAGCATCTAAGAACTTGAAAAACTACGAATTAGAAAAGACCAAA TATGTATTTCTTGCATTGACCAATTTATGCAAGTTTATATATATGTAAAT GTAAGTTTCACGAGGTTCTACTAAACTAAACCACCCCCTTGGTTAGAAGA AAAGAGTGTGTGAGAACAGGCTGTTGTTGTCACACGATTCGGACAATTCT GTTTGAAAGAGAGAGAGTAACAGTACGATCGAACGAACTTTGCTCTGGAG ATCACAGTGGGCATCATAGCATGTGGTACTAAACCCTTTCCCGCCATTCC AGAACCTTCGATTGCTTGTTACAAAACCTGTGAGCCGTCGCTAGGACCTT GTTGTGTGACGAAATTGGAAGCTGCAATCAATAGGAAGACAGGAAGTCGA GCGTGTCTGGGTTTTTTCAGTTTTGTTCTTTTTGCAAACAACAGTTTATT CCTGGCATCCACTAAATATAATGGAGCCCGCTTTTTAAGCTGGCATCCAG AAAAAAAAAGAATCCCAGCACCAAAATATTGTTTTCTTCACCAACCATCA GTTCATAGGTCCATTCTCTTAGCGCAACTACAGAGAACAGGGGCACAAAC AGGCAAAAAACGGGCACAACCTCAATGGAGTGATGCAACCTGCCTGGAGT AAATGATGACACAAGGCAATTGACCCACGCATGTATCTATCTCATTTTCT TACACCTTCTATTACCTTCTGCTCTCTCTGATTTGGAAAAAGCTGAAAAA AAAGGTTGAAACCAGTTCCCTGAAATTATTCCCCTACTTGACTAATAAGT ATATAAAGACGGTAGGTATTGATTGTAATTCTGTAAATCTATTTCTTAAA CTTCTTAAATTCTACTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACAC CAAGAACTTAGTTTCGAATAAACACACATAAACAAACAAAATGGCTTCTA GTTCTTCCGATGTCTTCGTTTTGGGTCTAGGTGTTGTTTTGGCTGCCTTG TATATCTTCAGAGACCAATTATTCGCTGCTTCTAAGCCAAAGGTGGCTCC AGTTTCCACTACGAAGCCTGCCAACGGTTCCGCTAACCCAAGAGACTTCA TCGCCAAGATGAAACAAGGTAAGAAGAGAATCGTAATCTTCTACGGTTCT CAAACTGGTACCGCTGAAGAATATGCTATTCGTTTGGCTAAGGAAGCTAA GCAAAAGTTCGGTCTAGCCTCCTTGGTTTGTGATCCAGAAGAATACGATT TTGAAAAGTTGGACCAATTGCCAGAAGATTCTATTGCTTTCTTCGTCGTT GCTACCTATGGTGAAGGTGAACCTACAGACAACGCTGTCCAATTGTTGCA AAACTTGCAAGATGAAAGCTTCGAATTCTCCTCTGGTGAGAGAAAGTTGT CAGGTTTGAAGTACGTTGTTTTTGGTCTGGGTAACAAGACCTACGAACAT TACAACCTCATTGGGAGAACTGTTGACGCTCAATTGGCCAAGATGGGTGC TATCAGAATCGGTGAAAGAGGTGAAGGTGATGATGACAAGTCCATGGAAG AAGACTACTTGGAATGGAAGGATGGTATGTGGGAAGCGTTTGCCACTGCT ATGGGTGTTGAAGAAGGTCAAGGTGGTGACTCCGCTGATTTCGTCGTTTC CGAATTGGAATCTCACCCACCAGAAAAGGTTTACCAAGGTGAATTTTCTG CTAGAGCTTTAACCAAAACCAAGGGTATTCACGACGCTAAGAATCCTTTT GCTGCTCCAATTGCGGTTGCTAGAGAATTGTTCCAATCTGTTGTCGATAG AAACTGTGTCCACGTCGAATTCAACATTGAAGGCTCTGGTATCACCTATC AACACGGTGACCACGTTGGTTTGTGGCCATTGAATCCAGATGTTGAAGTC GAACGGTTGTTGTGTGTTTTAGGTTTAGCTGAAAAGAGAGATGCTGTCAT CTCCATTGAATCCTTAGACCCGGCTTTGGCTAAGGTTCCATTCCCAGTCC CAACTACTTACGGTGCTGTGTTGAGACACTACATTGACATCTCTGCTGTC GCCGGTAGACAAATCTTGGGTACTTTGTCCAAATTCGCTCCAACCCCAGA AGCTGAAGCTTTCTTGAGAAACTTGAACACTAACAAGGAAGAATACCACA ACGTCGTCGCTAACGGTTGTTTGAAATTGGGTGAAATTTTGCAAATCGCT ACCGGTAACGACATTACTGTCCCACCAACTACTGCCAACACCACCAAATG GCCAATTCCATTCGACATCATTGTTTCTGCCATCCCAAGATTGCAACCAA GATACTACTCTATCTCTTCTTCCCCAAAAATTCATCCAAACACCATCCAC GCTACCGTTGTTGTGCTCAAATACGAAAACGTTCCAACCGAACCAATCCC AAGAAAGTGGGTTTACGGTGTCGGTAGTAACTTCTTGTTGAATTTAAAGT ACGCTGTTAACAAGGAACCAGTTCCATACATCACTCAAAATGGCGAACAA AGAGTCGGTGTCCCGGAATACTTGATTGCTGGTCCACGTGGTTCTTACAA GACTGAATCTTTCTACAAGGCTCCAATCCATGTTAGACGTTCTACTTTCC GTTTGCCAACCAACCCAAAGTCTCCAGTCATCATGATTGGTCCAGGTACT GGTGTCGCCCCATTCAGAGGCTTCGTTCAAGAAAGAGTTGCCTTGGCCAG AAGATCCATCGAAAAGAACGGTCCTGACTCTTTGGCTGACTGGGGTCGTA TTTCCTTGTTCTACGGTTGTAGAAGATCCGACGAAGACTTCTTGTACAAG GACGAATGGCCACAATACGAAGCTGAGTTGAAGGGTAAGTTCAAGTTGCA CTGTGCTTTCTCCAGACAAAACTACAAGCCAGACGGTTCTAAGATTTACG TCCAAGATTTGATCTGGGAAGACAGAGAACACATTGCCGATGCCATCTTA AACGGTAAGGGTTACGTCTACATCTGCGGTGAAGCTAAGTCCATGTCTAA ACAAGTTGAAGAAGTTCTAGCCAAGATCTTGGGCGAAGCCAAAGGTGGTT CCGGTCCAGTTGAAGGTGTTGCTGAAGTCAAGTTACTGAAGGAACGGTCC AGATTGATGTTGGATGTCTGGTCTGAACAAAAGTTAATTTCTGAAGAAGA TTTGGAATGAATCGCGTGCATTCATCCGCTCTAACCGAAAAGGAAGGAGT TAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAG TATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGA CGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTT GGGACGCTCGAAGATCGCGTCCCAATTCGCCCTATAGTGAGTCGTATTAC GCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGG CGTTACCCCTGCAGGACTAGTGCTGAGGCATTAATACCACTTTTCAATGA AACGGATATTGATATGCTAGTAAAAGGACGAGCTCAAGAGCGAAAATATA AGTAAAGAATTCGAGTGCACTTGTCTCCATGCAGCAAGATTTCATATGAG TCTTTTTTATCTTTTTACTTTTTACATTACACGATATGCACTTTATGAAA ATTTAACGAGGTTGGAAGCCGGATAATCAACCAAAATCAGGCACGAAGGC ACACTCGTATATGCATGTTGTTGAAACTCTGTTACGCTGAACTAACAATC ACACATGTAGAGGTCACCGGGAAAAGTTGCGACCCCATGGAAGGTCGATC TCTTCGTTTGGCTTTGCTTGGCTGGCGGCATTGCGCTTCTTCGCTTATAC CCGTCTCTTGACGCTCGAGCTCGTTCATTGAGATACCTTTATTCTTGCAC ATTTTCTGGCTTTTTTCGCTACTCGGGTACATGTAATCATGCACACAGAA GGTGCTGTAGGGTGAAAGTTCCTTTGTGCTGTCGTTTGTTTTTAATGCCA AACTTTCCGGTGATCAATAACCACCTC
[0961] SEQ.ID NO: 51
TABLE-US-00051 CGGCATGCAAACATCTACACAATTAGCAAGGGCAATCCATATTTTGTCTT TTCGCGCCCTGGAAAGGCCTAAGTAATGTCGTAAACGCATTCTATCTGTA CTTCAACTCTCCTCTGTGCATTGGTTTGTGCAAATCACATTTTACGATAC TGCCAGATATATGCAAAAAGAGAAAACCAAGGGACCAGAACAAAGCAAAA TTACGATATTCTTCGAATTCCTTCGTGCTTGACTAAGACAAAGGGATGGA CGTAGCGATTTTTAGCGGGCCAAGAACTGGTTCCGAAAAAGCACAGGTAC ACCGAACCCTCAGCTAAGGAGGGACAGCACCGATGCGGAAGGACAAACTT TCTTTTTGCCTATCACAGTATCTTATCGAGCTAACTATTTTCGACACACA TGAAAAAGCAGAAATATTAACGAAAAAGAAAAGAAAGACCATGTCATGTA CGGGCAATCAGAATCTGTAACAAGCGCCATTTTTTTTTCTGTATCGGGCC CTCCTTACTGCTCTCCTTCCGTGTAACGCGTTATGAAATTAAGTCCTCAG CGAGCTCGCATGGAATGCGTGCGATGAGCGACCTCATGCTATACCTGAGA AAGCAACCTGACCTACAGGAAAGAGTTACTCAAGAATAAGAATTTTCGTT TTAAAACCTAAGAGTCACTTTAAAATTTGTATACACTTATTTTTTTTATA ACTTATTTAATAATAAAAATCATAAATCATAAGAAATTCGCTTATTTAGA AGTGTCAACAACGTATCTACCAACGGAATGCGTGCGATTGTTTTATATTT GTTGTAAAAAGTAGATAATTACTTCCTTGATGATCTGTAAAAAAGAGAAA AAGAAAGCATCTAAGAACTTGAAAAACTACGAATTAGAAAAGACCAAATA TGTATTTCTTGCATTGACCAATTTATGCAAGTTTATATATATGTAAATGT AAGTTTCACGAGGTTCTACTAAACTAAACCACCCCCTTGGTTAGAAGAAA AGAGTGTGTGAGAACAGGCTGTTGTTGTCACACGATTCGGACAATTCTGT TTGAAAGAGAGAGAGTAACAGTACGATCGAACGAACTTTGCTCTGGAGAT CACAGTGGGCATCATAGCATGTGGTACTAAACCCTTTCCCGCCATTCCAG AACCTTCGATTGCTTGTTACAAAACCTGTGAGCCGTCGCTAGGACCTTGT TGTGTGACGAAATTGGAAGCTGCAATCAATAGGAAGACAGGAAGTCGAGC GTGTCTGGGTTTTTTCAGTTTTGTTCTTTTTGCAAACAACAGTTTATTCC TGGCATCCACTAAATATAATGGAGCCCGCTTTTTAAGCTGGCATCCAGAA AAAAAAAGAATCCCAGCACCAAAATATTGTTTTCTTCACCAACCATCAGT TCATAGGTCCATTCTCTTAGCGCAACTACAGAGAACAGGGGCACAAACAG GCAAAAAACGGGCACAACCTCAATGGAGTGATGCAACCTGCCTGGAGTAA ATGATGACACAAGGCAATTGACCCACGCATGTATCTATCTCATTTTCTTA CACCTTCTATTACCTTCTGCTCTCTCTGATTTGGAAAAAGCTGAAAAAAA AGGTTGAAACCAGTTCCCTGAAATTATTCCCCTACTTGACTAATAAGTAT ATAAAGACGGTAGGTATTGATTGTAATTCTGTAAATCTATTTCTTAAACT TCTTAAATTCTACTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCA AGAACTTAGTTTCGAATAAACACACATAAACAAACAAAATGGGAGGTCCG ATGAGCGGTTTCCATTCGGGGGAGGCGCTGCTCGGTGACCTCGCCACCGG TCAGCTGACCAGGCTGTGCGAGGTGGCGGGGCTGACCGAGGCCGACACGG CGGCCTACACGGGGGTGCTGATCGAAAGTCTGGGGACGTCGGCCGGACGG CCGTTGTCCCTGCCACCCCCGTCGCGGACCTTTCTCTCCGACGACCACAC CCCCGTGGAGTTCTCCCTGGCCTTCCTGCCGGGACGCGCACCGCACCTGC GGGTCCTGGTGGAACCGGGCTGCTCCAGCGGCGACGACCTGGCGGAAAAC GGCCGGGCCGGTCTGCGGGCGGTCCACACCATGGCGGACCGCTGGGGATT CTCCACCGAGCAACTCGACCGGCTGGAGGACCTGTTCTTCCCCTCCTCCC CCGAGGGCCCGCTGGCCCTGTGGTGCGCCCTGGAGCTCCGCTCCGGTGGG GTGCCGGGGGTGAAGGTCTACCTCAACCCCGCGGCGAATGGCGCCGACCG GGCCGCCGAGACGGTACGCGAGGCGCTGGCCAGGCTGGGCCACCTGCAGG CGTTCGACGCGCTGCCCCGGGCGGACGGCTTCCCGTTCCTCGCCCTGGAC CTCGGCGACTGGGACGCCCCGCGGGTGAAGATCTACCTCAAACACCTCGG CATGTCCGCCGCCGACGCGGGCTCCCTCCCCCGGATGTCGCCCGCACCGA GCCGGGAGCAGCTGGAGGAGTTCTTCCGCACCGCCGGTGACCTCCCGGCC CCGGGAGACCCGGGGCCCACCGAGGACACCGGCCGGCTCGCCGGGCGCCC CGCCCTCACCTGCCACTCCTTCACGGAGACGGCGACCGGGCGGCCCAGCG GCTACACCCTCCACGTGCCGGTCCGCGACTACGTCCGGCACGACGGCGAG GCACGGGACCGGGCGGTGGCCGTGCTGCGCGAACATGACATGGACAGTGC GGCACTGGACCGGGCGCTGGCCGCCGTGAGCCCCCGCCCGCTGAGTGACG GGGTGGGCCTGATCGCCTATCTGGCACTGGTCCACCAGCGCGGCCGGCCG ACACGGGTGACCGTCTACGTCTCCTCCGAGGCGTACGAGGTGCGGCCGCC CCGCGAGACGGTCCCCACCCGCGACCGGGCGCGGGCACGGCTGCATCATC ATCATCATCATTGAATCGCGTGCATTCATCCGCTCTAACCGAAAAGGAAG GAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATG TTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTA CAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGG TTTTGGGACGCTCGAAGATCGCGTCCCAATTCGCCCTATAGTGAGTCGTA TTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACC CTGGCGTTACCCCTGCAGGACTAGTGCTGAGGCATTAATACGACTCTCTC GAAATTTTTCTTAACGCGTCCTTGTACTGCGTCTAACGCTTTTGCCACTT GGATTTCTATTATAGGAAATAGTCTCACTTACTGGGCGACGAATTTTCGC GTTTTGATGAAGCACAGGAAGAATTTCTTTTTTTTTTGGCTTCTTCTGGT TCCGTTTTTTACGCGCACAAATCTAAAAAAAGAAATAATTATAACCTAGT CTCGAAAATTTTCATCGATCCATTCGTTCCTTTTTTTCGATTTTTTCAGA TCAAAATTCTTGTTTCTTTCTTTGTCTTAGTTTATATTAAAAGATATTTT GATTTTACTCCTGAACTATTTATTCTTTCTAAGAAGGCCAGAACACTACA GCTGTTTTAACCGACTACGAAGTTCTCCATTCTCGAACACTAGCCTTCAT TTACCAAACAGGAACTAGCGTATATCATTAGTCCTTATTCGAAAAGAGAT TGGTAGATATTTATTGTAGTTTGTGAGAAGGAGAAAATACTGTCATTGGA CTGATAGTTAGAGGACATTAACCTCTCTTACGTTCGCTCA
[0962] SEQ.ID NO: 52
TABLE-US-00052 CGAGATCTTTGTGTTCGGTTACCCGGCTCAGATCCTAACTTCTTCTTTTG GTATGTTTATTCGTATAAGTTACTGTTGTCCACAGGCAATACTCTGCAGA AAATTAAAACGGCATTAATGCTAGGACAACCAGAATTGTTACTACTGTAT GTGCGATAGTTGATAACTGCAACATTATGCCCGGTATATTCTCAAAAAAC CCTATTACTGCATACGAAGAAATCGCAAGAGAAATCTTTCGGTTTGGAAA AGCTCACTGTGAGGTTCCTTGGAGCCAATAGTAATACAGCACAATCCAAG GAAAAATCTGGCCTATATGCAAGGAAGGAGAGATAGTCAAAAGCATTCTT TCCCCTAGAAGTTGGTGCATATATGGCATCGTTAAAACATATTACCCCCA AAATTTCTTCTCTAAACGATGTGCTTGGCCTTTGTTTTGGTTTTTGATGT CGGTCGTTTGAGGCCCCTTGCGGAAAATCGAGATCGCCGAATGGCACGCG AGGGAAGGGAAATAAGGTTTAAAGGCACTGAAACAATAGGCAAGAAGTAG GCGAGAGCCGACATACGAGACTAAATTAAGTCCTCAGCGAGCTCGCATGG AATGCGTGCGATGAGCGACCTCATGCTATACCTGAGAAAGCAACCTGACC TACAGGAAAGAGTTACTCAAGAATAAGAATTTTCGTTTTAAAACCTAAGA GTCACTTTAAAATTTGTATACACTTATTTTTTTTATAACTTATTTAATAA TAAAAATCATAAATCATAAGAAATTCGCTTATTTAGAAGTGTCAACAACG TATCTACCAACGGAATGCGTGCGATTCATTTGTCATCGTCATCCTTGTAG TCCATTGCCTTCAAATGTTCCTTATTAAACTTTCTCAAATCGTAAACCAA AGAGTTCAAGATATCCAATTCAACATGTTCCATAACAACGATCTTGTACC AGTTGTTAGTTGGGTTATGAACTTCTGGAACTAAAAAGTACTTTTCAGCG ATTTCCTTATTAACATATTGTTCTTCAATTGTAACAATATTCATTGAATC TTCTTTGTAATATTTAATTCTCATATCGTTCAATTGCTTACACAACCATT TAGTTCTATTTCTCAACTTATTAATCTTTTCCATCCAACCGTATGGACCA TAAGAAGCTAAAACCATCCAAATTGCAACAGCATTTGAACCAGATCTTGA ACCAGACAATGTAACATCCAAATTTTCGATGTAAGTTGCTTCCTTTGTCA AAGTGTTATGGATCAAATTCTTTCTTGAAACGAAGATACCAGTACCGTAT GGAGCTTGCAACATCTTATGACCATCTAATGTGATTGAAGAAACGTTCTT ATTAGAGAAATCAGTTTTACATTCCTTATTATCAATTGGATATATAAAAC CACCAAATGCACCATCAACATGAATTTTGTATTCCAAATTGTACTTATCG AAGATGTTAGCGTACAAATCTGGATCATCAACTGAACCAAACATTGTAGT ACCCATGTTAGAGATAACGATGAAGTACTTTTTACCAATTTCTTTAGCTT CTTTAACAATTGAATCCAATGTATTTTCTTGAATTTTTCTTGAATAAAAA TCAACTGGAACCTTAATAATATCGATGTTCAACAAATCTGAACCTTTGTA TGCAGAGTAATGTGTATCTGCTGAAGTGATGATAGCGATTTCTTCATGCT TAGCCTTTCTTTCTTTCTTGAAGTAGTTTCTGTAAACCCACATTGCTTGG ATGTTAGCTTCTGTACCACCTTGAGTAACGTAACCATCAAATTCTTCATC GTTACCGTTCAAAACATCGATTGCTAACAATTGGATTAATTCTCTTTCGA TATCGAATGTACCACCGAACAAGATATCAGCCTTATCATAAGTATGACAA CCGATATGGTTTGGGTTTTGAATAAAAGTTCTCAAGTATGGTGAATGCTT AACGAAAGAATGATCATCATAGAAAACTGTATCATCCAACTTAGTACCTG GAATACCGATTGTCTTAGTGTTATCGTAGTTCAAAGTCTTTTCCAAAGAT TCAGTATCTTTTCATCCATTTCTTGTTGTGTGTACTTTCTCCAGAACTTC ATTGTTTTATATTTGTTGTAAAAAGTAGATAATTACTTCCTTGATGATCT GTAAAAAAGAGAAAAAGAAAGCATCTAAGAACTTGAAAAACTACGAATTA GAAAAGACCAAATATGTATTTCTTGCATTGACCAATTTATGCAAGTTTAT ATATATGTAAATGTAAGTTTCACGAGGTTCTACTAACTAAACCACCCCCT TGGTTAGAAGAAAAGAGTGTGTGAGAACAGGCTGTTGTTGTCACACGATT CGGACAATTCTGTTTGAAAGAGAGAGAGTAACAGTACGATCGAACGAACT TTGCTCTGGAGATCACAGTGGGCATCATAGCATGTGGTACTAAACCCTTT CCCGCCATTCCAGAACCTTCGATTGCTTGTTACAAAACCTGTGAGCCGTC GCTAGGACCTTGTTGTGTGACGAAATTGGAAGCTGCAATCAATAGGAAGA CAGGAAGTCGAGCGTGTCTGGGTTTTTTCAGTTTTGTTCTTTTTGCAAAC AACAGTTTATTCCTGGCATCCACTAAATATAATGGAGCCCGCTTTTTAAG CTGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATATTGTTTTCTTC ACCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTACAGAGAACA GGGGCACAAACAGGCAAAAAACGGGCACAACCTCAATGGAGTGATGCAAC CTGCCTGGAGTAAATGATGACACAAGGCAATTGACCCACGCATGTATCTA TCTCATTTTCTTACACCTTCTATTACCTTCTGCTCTCTCTGATTTGGAAA AAGCTGAAAAAAAAGGTTGAAACCAGTTCCCTGAAATTATTCCCCTACTT GACTAATAAGTATATAAAGACGGTAGGTATTGATTGTAATTCTGTAAATC TATTTCTTAAACTTCTTAAATTCTACTTTTATAGTTAGTCTTTTTTTTAG TTTTAAAACACCAAGAACTTAGTTTCGAATAAACACACATAAACAAACAA AATGGCTTCTAGTTCTTCCGATGTCTTCGTTTTGGGTCTAGGTGTTGTTT TGGCTGCCTTGTATATCTTCAGAGACCAATTATTCGCTGCTTCTAAGCCA AAGGTGGCTCCAGTTTCCACTACGAAGCCTGCCAACGGTTCCGCTAACCC AAGAGACTTCATCGCCAAGATGAAACAAGGTAAGAAGAGAATCGTAATCT TCTACGGTTCTCAAACTGGTACCGCTGAAGAATATGCTATTCGTTTGGCT AAGGAAGCTAAGCAAAAGTTCGGTCTAGCCTCCTTGGTTTGTGATCCAGA AGAATACGATTTTGAAAAGTTGGACCAATTGCCAGAAGATTCTATTGCTT TCTTCGTCGTTGCTACCTATGGTGAAGGTGAACCTACAGACAACGCTGTC CAATTGTTGCAAAACTTGCAAGATGAAAGCTTCGAATTCTCCTCTGGTGA GAGAAAGTTGTCAGGTTTGAAGTACGTTGTTTTTGGTCTGGGTAACAAGA CCTACGAACATTACAACCTCATTGGGAGAACTGTTGACGCTCAATTGGCC AAGATGGGTGCTATCAGAATCGGTGAAAGAGGTGAAGGTGATGATGACAA GTCCATGGAAGAAGACTACTTGGAATGGAAGGATGGTATGTGGGAAGCGT TTGCCACTGCTATGGGTGTTGAAGAAGGTCAGGTGGTGACTCCGCTGATT TCGTCGTTTCCGAATTGGAATCTCACCCACCAGAAAAGGTTTACCAAGGT GAATTTTCTGCTAGAGCTTTAACCAAAACCAAGGGTATTCACGACGCTAA GAATCCTTTTGCTGCTCCAATTGCGGTTGCTAGAGAATTGTTCCAATCTG TTGTCGATAGAAACTGTGTCCACGTCGAATTCAACATTGAAGGCTCTGGT ATCACCTATCAACACGGTGACCACGTTGGTTTGTGGCCATTGAATCCAGA TGTTGAAGTCGAACGGTTGTTGTGTGTTTTAGGTTTAGCTGAAAAGAGAG ATGCTGTCATCTCCATTGAATCCTTAGACCCGGCTTTGGCTAAGGTTCCA TTCCCAGTCCCAACTACTTACGGTGCTGTGTTGAGACACTACATTGACAT CTCTGCTGTCGCCGGTAGACAAATCTTGGGTACTTTGTCCAAATTCGCTC CAACCCCAGAAGCTGAAGCTTTCTTGAGAAACTTGAACACTAACAAGGAA GAATACCACAACGTCGTCGCTAACGGTTGTTTGAAATTGGGTGAAATTTT GCAAATCGCTACCGGTAACGACATTACTGTCCCACCAACTACTGCCAACA CCACCAAATGGCCAATTCCATTCGACATCATTGTTTCTGCCATCCCAAGA TTGCAACCAAGATACTACTCTATCTCTTCTTCCCCAAAAATTCATCCAAA CACCATCCACGCTACCGTTGTTGTGCTCAAATACGAAAACGTTCCAACCG AACCAATCCCAAGAAAGTGGGTTTACGGTGTCGGTAGTAACTTCTTGTTG AATTTAAAGTACGCTGTTAACAAGGAACCAGTTCCATACATCACTCAAAA TGGCGAACAAAGAGTCGGTGTCCCGGAATACTTGATTGCTGGTCCACGTG GTTCTTACAAGACTGAATCTTTCTACAAGGCTCCAATCCATGTTAGACGT TCTACTTTCCGTTTGCCAACCAACCCAAAGTCTCCAGTCATCATGATTGG TCCAGGTACTGGTGTCGCCCCATTCAGAGGCTTCGTTCAAGAAAGAGTTG CCTTGGCCAGAAGATCCATCGAAAAGAACGGTCCTGACTCTTTGGCTGAC TGGGGTCGTATTTCCTTGTTCTACGGTTGTAGAAGATCCGACGAAGACTT CTTGTACAAGGACGAATGGCCACAATACGAAGCTGAGTTGAAGGGTAAGT TCAAGTTGCACTGTGCTTTCTCCAGACAAAACTACAAGCCAGACGGTTCT AAGATTTACGTCCAAGATTTGATCTGGGAAGACAGAGAACACATTGCCGA TGCCATCTTAAACGGTAAGGGTTACGTCTACATCTGCGGTGAAGCTAAGT CCATGTCTAAACAAGTTGAAGAAGTTCTAGCCAAGATCTTGGGCGAAGCC AAAGGTGGTTCCGGTCCAGTTGAAGGTGTTGCTGAAGTCAAGTTACTGAA GGAACGGTCCAGATTGATGTTGGATGTCTGGTCTGAACAAAAGTTAATTT CTGAAGAAGATTTGGAATGAATCGCGTGCATTCATCCGCTCTAACCGAAA AGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATA GTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTT TCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTG AGAAGGTTTTGGGACGCTCGAAGATCGCGTCCCAATTCGCCCTATAGTGA GTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGG AAAACCCTGGCGTTACCCCTGCAGGACTAGTGCTGAGGCATTAATACCAC TTTTCAATGAAACGGATATTGATATGCTAGTAAAAGGACGAGCTCAAGAG CGAAAATATAAGTAAAGAATTCGAGTGCACTTGTCTCCATGCAGCAAGAT TTCATATGAGTCTTTTTTATCTTTTTACTTTTTACATTACACGATATGCA CTTTATGAAAATTTAACGAGGTTGGAAGCCGGATAATCAACCAAAATCAG GCACGAAGGCACACTCGTATATGCATGTTGTTGAAACTCTGTTACGCTGA ACTAACAATCACACATGTAGAGGTCACCGGGAAAAGTTGCGACCCCATGG AAGGTCGATCTCTTCGTTTGGCTTTGCTTGGCTGGCGGCATTGCGCTTCT TCGCTTATACCCGTCTCTTGACGCTCGAGCTCGTTCATTGAGATACCTTT ATTCTTGCACATTTTCTGGCTTTTTTCGCTACTCGGGTACATGTAATCAT GCACACAGAAGGTGCTGTAGGGTGAAAGTTCCTTTGTGCTGTCGTTTGTT TTTAATGCCAAACTTTCCGGTGATCAATAACCACCTC
[0963] SEQ.ID NO: 53
TABLE-US-00053 CGGCATGCAAACATCTACACAATTAGCAAGGGCAATCCATATTTTGTCTT TTCGCGCCCTGGAAAGGCCTAAGTAATGTCGTAAACGCATTCTATCTGTA CTTCAACTCTCCTCTGTGCATTGGTTTGTGCAAATCACATTTTACGATAC TGCCAGATATATGCAAAAAGAGAAAACCAAGGGACCAGAACAAAGCAAAA TTACGATATTCTTCGAATTCCTTCGTGCTTGACTAAGACAAAGGGATGGA CGTAGCGATTTTTAGCGGGCCAAGAACTGGTTCCGAAAAAGCACAGGTAC ACCGAACCCTCAGCTAAGGAGGGACAGCACCGATGCGGAAGGACAAACTT TCTTTTTGCCTATCACAGTATCTTATCGAGCTAACTATTTTCGACACACA TGAAAAAGCAGAAATATTAACGAAAAAGAAAAGAAAGACCATGTCATGTA CGGGCAATCAGAATCTGTAACAAGCGCCATTTTTTTTTCTGTATCGGGCC CTCCTTACTGCTCTCCTTCCGTGTAACGCGTTATGAAATTAAGTCCTCAG CGAGCTCGCATGGAATGCGTGCGATGAGCGACCTCATGCTATACCTGAGA AAGCAACCTGACCTACAGGAAAGAGTTACTCAAGAATAAGAATTTTCGTT TTAAAACCTAAGAGTCACTTTAAAATTTGTATACACTTATTTTTTTTATA ACTTATTTAATAATAAAAATCATAAATCATAAGAAATTCGCTTATTTAGA AGTGTCAACAACGTATCTACCAACGGAATGCGTGCGATTGTTTTATATTT GTTGTAAAAAGTAGATAATTACTTCCTTGATGATCTGTAAAAAAGAGAAA AAGAAAGCATCTAAGAACTTGAAAAACTACGAATTAGAAAAGACCAAATA TGTATTTCTTGCATTGACCAATTTATGCAAGTTTATATATATGTAAATGT AAGTTTCACGAGGTTCTACTAAACTAAACCACCCCCTTGGTTAGAAGAAA AGAGTGTGTGAGAACAGGCTGTTGTTGTCACACGATTCGGACAATTCTGT TTGAAAGAGAGAGAGTAACAGTACGATCGAACGAACTTTGCTCTGGAGAT CACAGTGGGCATCATAGCATGTGGTACTAAACCCTTTCCCGCCATTCCAG AACCTTCGATTGCTTGTTACAAAACCTGTGAGCCGTCGCTAGGACCTTGT TGTGTGACGAAATTGGAAGCTGCAATCAATAGGAAGACAGGAAGTCGAGC GTGTCTGGGTTTTTTCAGTTTTGTTCTTTTTGCAAACAACAGTTTATTCC TGGCATCCACTAAATATAATGGAGCCCGCTTTTTAAGCTGGCATCCAGAA AAAAAAAGAATCCCAGCACCAAAATATTGTTTTCTTCACCAACCATCAGT TCATAGGTCCATTCTCTTAGCGCAACTACAGAGAACAGGGGCACAAACAG GCAAAAAACGGGCACAACCTCAATGGAGTGATGCAACCTGCCTGGAGTAA ATGATGACACAAGGCAATTGACCCACGCATGTATCTATCTCATTTTCTTA CACCTTCTATTACCTTCTGCTCTCTCTGATTTGGAAAAAGCTGAAAAAAA AGGTTGAAACCAGTTCCCTGAAATTATTCCCCTACTTGACTAATAAGTAT ATAAAGACGGTAGGTATTGATTGTAATTCTGTAAATCTATTTCTTAAACT TCTTAAATTCTACTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCA AGAACTTAGTTTCGAATAAACACACATAAACAAACAAAATGTCCATCGGT GCTGAAATTGACTCTTTGGTTCCAGCTCCACCAGGTTTGAACGGTACCGC TGCTGGTTACCCAGCCAAGACTCAAAAGGAATTGTCTAACGGCGATTTCG ATGCTCACGATGGTCTGTCCTTGGCTCAATTGACTCCATACGATGTTTTA ACCGCTGCTTTGCCATTGCCAGCGCCAGCTTCTAGTACTGGTTTCTGGTG GAGAGAAACTGGTCCAGTTATGTCTAAGCTCTTGGCTAAAGCCAACTACC CATTGTACACCCATTACAAGTATTTAATGTTGTACCACACTCACATTTTA CCTTTGTTAGGTCCAAGACCACCTTTGGAAAATTCTACCCACCCATCTCC ATCAAATGCTCCTTGGAGATCCTTCTTGACCGATGACTTCACCCCATTAG AACCATCTTGGAACGTTAACGGTAACTCCGAAGCACAATCCACTATCAGA TTGGGTATTGAACCAATTGGTTTCGAAGCCGGTGCTGCTGCCGACCCATT CAACCAAGCTGCCGTCACCCAATTCATGCACTCCTACGAAGCTACTGAAG TTGGTGCCACTCTAACTTTGTTCGAACACTTCAGAAACGACATGTTCGTC GGTCCAGAGACTTACGCTGCCTTGAGAGCTAAGATTCCTGAAGGTGAGCA CACCACTCAATCTTTCTTGGCTTTCGACTTGGACGCCGGTCGTGTCACTA CCAAGGCTTACTTCTTCCCAATCTTGATGTCTTTGAAGACCGGTCAATCT ACGACCAAAGTTGTTTCCGATTCTATCTTGCACCTAGCTTTGAAGTCTGA AGTTTGGGGTGTCCAAACCATTGCCGCTATGTCGGTCATGGAAGCTTGGA TCGGTTCTTACGGTGGTGCTGCTAAGACCGAAATGATCTCCGTTGACTGT GTCAACGAAGCTGACTCCAGAATCAAGATCTACGTTAGAATGCCACACAC TAGCTTGAGAAAGGTCAAAGAAGCTTATTGTTTGGGTGGCCGTTTGACTG ACGAAAACACCAAGGAAGGTTTGAAATTGTTGGATGAATTGTGGAGAACT GTTTTCGGTATCGATGACGAAGATGCTGAATTACCACAAAACTCTCACAG AACTGCTGGTACTATTTTTAACTTTGAACTAAGACCAGGTAAGTGGTTCC CAGAACCAAAGGTCTACTTGCCAGTCAGACACTACTGTGAATCCGACATG CAAATTGCCTCCAGATTACAAACTTTCTTTGGTCGTTTGGGTTGGCACAA CATGGAAAAGGACTACTGCAAGCATTTGGAAGACTTATTCCCTCACCACC CATTGTCCTCCTCTACCGGTACCCACACTTTCTTGTCTTTTTCTTACAAG AAGCAAAAGGGTGTTTACATGACCATGTACTACAACTTGAGAGTTTATTC TACACACCACCATCATCATCATTGAATCGCGTGCATTCATCCGCTCTAAC CGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTT TTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTT TTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTT GCTTGAGAAGGTTTTGGGACGCTCGAAGATCGCGTCCCAATTCGCCCTAT AGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGA CTGGGAAAACCCTGGCGTTACCCCTGCAGGACTAGTGCTGAGGCATTAAT ACGACTCTCTCGAAATTTTTCTTAACGCGTCCTTGTACTGCGTCTAACGC TTTTGCCACTTGGATTTCTATTATAGGAAATAGTCTCACTTACTGGGCGA CGAATTTTCGCGTTTTGATGAAGCACAGGAAGAATTTCTTTTTTTTTTGG CTTCTTCTGGTTCCGTTTTTTACGCGCACAAATCTAAAAAAAGAAATAAT TATAACCTAGTCTCGAAAATTTTCATCGATCCATTCGTTCCTTTTTTTCG ATTTTTTCAGATCAAAATTCTTGTTTCTTTCTTTGTCTTAGTTTATATTA AAAGATATTTTGATTTTACTCCTGAACTATTTATTCTTTCTAAGAAGGCC AGAACACTACAGCTGTTTTAACCGACTACGAAGTTCTCCATTCTCGAACA CTAGCCTTCATTTACCAAACAGGAACTAGCGTATATCATTAGTCCTTATT CGAAAAGAGATTGGTAGATATTTATTGTAGTTTGTGAGAAGGAGAAAATA CTGTCATTGGACTGATAGTTAGAGGACATTAACCTCTCTTACGTTCGCTC A
[0964] SEQ.ID NO: 54
TABLE-US-00054 ATCCGGATATAGTTCCTCCTTTCAGCAAAAAACCCCTCAAGACCCGTTTA GAGGCCCCAAGGGGTTATGCTAGTTATTGCTCAGCGGTGGCAGCAGCCAA CTCAGCTTCCTTTCGGGCTTTGTTAGCAGCCGGATCTCAGTGGTGGTGGT GGTGGTGCTCGAGTGCGGCCGCAAGCTTGTCGACGGAGCTCGAATTCGGA TCCGAATTAATTCCGATATCCATGGCCATCGCCGGCTGGGCAGCGAGGAG CAGCAGACCAGCAGCAGCGGTCGGCAGCAGGTATTTCATATGTATATCTC CTTCTTAAAGTTAAACAAAATTATTTCTAGAGGGGAATTGTTATCCGCTC ACAATTCCCCTATAGTGAGTCGTATTAATTTCGCGGGATCGAGATCTCGA TCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAGGTGCG GTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCG CCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCC CCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTT GCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCTAAT GCAGGAGTCGCATAAGGGAGAGCGTCGAGATCCCGGACACCATCGAATGG CGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATT CAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATG CCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCAC GTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAA TTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGC TGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATT GTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGT GTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACA ATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGAT GACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTT ATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCC ATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCAC CAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCT GCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGA TAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACC ATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAA CGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGC GCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGC TCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCT GGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAGGGCCAGGCGG TGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACC CTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATT AATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGC AACGCAATTAATGTAAGTTAGCTCACTCATTAGGCACCGGGATCTCGACC GATGCCCTTGAGAGCCTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGG GCATGACTATCGTCGCCGCACTTATGACTGTCTTCTTTATCATGCAACTC GTAGGACAGGTGCCGGCAGCGCTCTGGGTCATTTTCGGCGAGGACCGCTT TCGCTGGAGCGCGACGATGATCGGCCTGTCGCTTGCGGTATTCGGAATCT TGCACGCCCTCGCTCAAGCCTTCGTCACTGGTCCCGCCACCAAACGTTTC GGCGAGAAGCAGGCCATTATCGCCGGCATGGCGGCCCCACGGGTGCGCAT GATCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTA CTGGTTAGCAGAATGAATCACCGATACGCGAGCGAACGTGAAGCGACTGC TGCTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTT CCGTGTTTCGTAAAGTCTGGAAACGCGGAAGTCAGCGCCCTGCACCATTA TGTTCCGGATCTGCATCGCAGGATGCTGCTGGCTACCCTGTGGAACACCT ACATCTGTATTAACGAAGCGCTGGCATTGACCCTGAGTGATTTTTCTCTG GTCCCGCCGCATCCATACCGCCAGTTGTTTACCCTCACAACGTTCCAGTA ACCGGGCATGTTCATCATCAGTAACCCGTATCGTGAGCATCCTCTCTCGT TTCATCGGTATCATTACCCCCATGAACAGAAATCCCCCTTACACGGAGGC ATCAGTGACCAAACAGGAAAAAACCGCCCTTAACATGGCCCGCTTTATCA GAAGCCAGACATTAACGCTTCTGGAGAAACTCAACGAGCTGGACGCGGAT GAACAGGCAGACATCTGTGAATCGCTTCACGACCACGCTGATGAGCTTTA CCGCAGCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACA TGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGC AGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGC AGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTA TGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATATGCGGTGTGA AATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCG CTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCG GTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGA TAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACC GTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGAC GAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGG ACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTC CTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCG GGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGT GTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGC CCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTA AGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAG AGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACT ACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCA GTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCAC CGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAA AAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCT CAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAACAATAAAAC TGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCAA CGGGAAACGTCTTGCTCTAGGCCGCGATTAAATTCCAACATGGATGCTGA TTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGA CAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAA CATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACT AAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCC GTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGGAAAACA GCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGA TGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATT GTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGA ATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGG CTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTTTGCCATTCT CACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATT TTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAAT CGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGT TTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAAT CCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTA AGAATTAATTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAA CAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAAATTGT AAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCT CATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAA GAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCC ACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATC AGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGG TCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATT TAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGA AAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTG CGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTC CCATTCGCCA
EXAMPLES
Example 1 - Biosynthesis of a First Multi-Substituent Psilocybin Derivative
[0965] E.coli strain E1 was constructed as follows. For plasmid cloning, Top10 or XL1-blue strains were used depending on antibiotic markers. Standard LB media was used for culturing. For gene expression and feeding experiments, the parent host strain employed was BL21 (DE3). First, the plasmid pET28a(+)-PfTrpB-B0A9-HIS was created by inserting an in-frame, HIS tagged (SEQ.ID NO: 46) PfTrpB-B0A9 gene (SEQ.ID NO: 1) into the NdellXhol site of pET28a(+) (SEQ.ID NO: 36). As a second step, from plasmid pCDM4 (SEQ.ID NO: 35), the plasmid pCDM4-BaTDC-HIS was created by inserting an in-frame, HIS-tagged (SEQ.ID NO: 46) BaTDC gene (SEQ.ID NO: 3) into the NdellXhol site of pCDM4. Finally, from plasmid pET23a(+) (SEQ.ID NO: 37), the plasmid pET23a(+)-PsmF-HIS was created by inserting an in-frame, HIS-tagged (SEQ.ID NO: 46) PsmF gene (SEQ.ID NO: 9) into the Nde1/Xho1 site of pET23a(+). The target plasmids pET28a(+)-PfTrpB-B0A9-HIS, pCDM4-BaTDC-HIS and pET23a(+)-PsmF-HIS were transformed into BL21 (DE3) cells as follows: pCDM4-BaTDC-HIS was transformed into BL21 (DE3) first, and transformants selected using streptomycin were transformed with pET28a(+)-PfTrpB-B0A9-HIS and pET23a(+)-PsmF-HIS together. The final E.coli strain (Ec-1) was selected with streptomycin, ampicillin, and kanamycin. Scaled-up culturing of engineered E.coli was conducted as follows: seed cultures were inoculated in AMM (Jones et al., 2015, Sci Rep. 5: 11301) medium overnight. The overnight culture was then divided into two flasks containing 500 mL each of AMM medium additionally containing 0.5% (w/v) serine, 1 M IPTG, 50 ug/L streptomycin, ampicillin, and kanamycin, and 100 mg/L indole feedstock (6-fluoro-1 H-indol-4-ylamine; www.bldpharm.com) for conversion by Ec-1. Cultures were grown for 24 h. Cultures were then centrifuged (10,000 g ×5 minutes) to remove cellular content, and culture broth containing secreted derivative was combined and stored at -80° C. until further processing. To 1.0 L of broth, 10 M NaOH solution was added until the pH reached ~7. The culture was then extracted by ethyl acetate (4×600 ml). The organic layer was combined and dried over Na.sub.2SO.sub.4, followed by concentration under reduced pressure. The residue was purified by flash chromatography on silica gel (1.fwdarw. 2% metanol in dichloromethane), to give the compound as a light yellow solid (7 mg). Following purification, high-resolution MS (HRMS), .sup.1H NMR, and selective .sup.13 C NMR were performed to assess purity, estimate total quantity, and confirm molecular structure. .sup.1H NMR (400 MHz, CD.sub.3OD): δ = 1.94 (s, 3 H), 3.00 (m, 2 H,), 3.38 (m, 2 H,), 6.10 (dd, J = 11.7, 2.2 Hz, 1 H), 6.38 (dd, J= 9.7, 2.2 Hz, 1 H), 6.84 (s, 1 H). .sup.13C NMR (100 MHz, CD.sub.3OD): δ = 21.0, 29.2, 41.9, 87.1 (d, J.sub.C,.sub.F= 26.1 Hz), 92,6 (d, J.sub.C,.sub.F= 27.8 Hz), 111.6, 112.4, 120.7, 138.0 (d, J.sub.C,.sub.F= 15.0 Hz), 141.7 (d, Jc, .sub.F = 13.2 Hz), 160.7 (d, Jc, .sub.F = 232.5 Hz), 172.1. HRMS (ESI) m/z: calcd. for C.sub.12H.sub.14FN.sub.3O [M+H].sup.+ 236.1194, found 236.1189. Purity was determined as 95% w/w. It is noted that these data confirm a chemical structure corresponding with that of example compound (IX):
##STR00319##
set forth herein.
Assessment of Cell Viability Upon Treatment of a Psilocybin Derivative
[0966] To establish suitable ligand concentrations for competitive binding assays, PrestoBlue assays were first performed. The PrestoBlue assay measures cell metabolic activity based on tetrazolium salt formation, and is a preferred method for routine cell viability assays (Terrasso et al., 2017, J Pharmacol Toxicol Methods 83: 72). Results of these assays were conducted using both control ligands (e.g., psilocybin, psilocin, DMT) and novel derivative, in part as a pre-screen for any remarkable toxic effects on cell cultures up to concentrations of 1 mM. A known cellular toxin (Triton X-100, Pyrgiotakis G. et al., 2009, Ann. Biomed. Eng. 37: 1464-1473) was included as a general marker of toxicity. Drug-induced changes in cell health within simple in vitro systems such as the HepG2 cell line are commonly adopted as first-line screening approaches in the pharmaceutical industry (Weaver et al., 2017, Expert Opin Drug Metab Toxicol 13: 767). HepG2 is a human hepatoma that is most commonly used in drug metabolism and hepatotoxicity studies (Donato et al., 2015, Methods Mol Biol 1250: 77). Herein, HepG2 cells were cultured using standard procedures using the manufacture’s protocols (ATCC, HB-8065). Briefly, cells were cultured in Eagle’s minimum essential medium supplemented with 10% fetal bovine serum and grown at 37° C. in the presence of 5% CO.sub.2. To test the various compounds with the cell line, cells were seeded in a clear 96-well culture plate at 20,000 cells per well. After allowing cells to attach and grow for 24 hours, compounds were added at 1 .Math.M, 10 .Math.M, 100 .Math.M, and 1 mM. Methanol was used as vehicle, at concentrations 0.001, 0.01, 0.1, and 1%. As a positive control for toxicity, TritonX concentrations used were 0.0001, 0.001, 0.01 and 0.1%. Cells were incubated with compounds for 48 hours before accessing cell viability with the PrestoBlue assay following the manufacture’s protocol (ThermoFisher Scientific, P50200). PrestoBlue reagent was added to cells and allowed to incubate for 1 hour before reading. Absorbance readings were performed at 570 nm with the reference at 600 nm on a SpectraMax iD3 plate reader. Non-treated cells were assigned 100% viability. Bar graphs show the mean +/- SD, n=3. Significance was determined by 2-way ANOVA followed by Dunnett’s multiple comparison test and is indicated by *** (P<0.0001), **(P<0.001), *(P<0.005). Data acquired for the derivative having chemical formula (IX) is displayed as “IX” on the x-axis of
Radioligand Receptor Binding Assays
[0967] Evaluation of drug binding is an essential step to characterization of all drug-target interactions (Fang 2012, Exp Opin Drug Discov 7:969). The binding affinity of a drug to a target is traditionally viewed as an acceptable surrogate of its in vivo efficacy (Núñez et al., 2012, Drug Disc Today 17: 10). Competition assays, also called displacement or modulation binding assays, are a common approach to measure activity of a ligand at a target receptor (Flanagan 2016, Methods Cell Biol 132: 191). In these assays, standard radioligands acting either as agonists or antagonists are ascribed to specific receptors. In the case of G protein-coupled receptor 5-HT.sub.2A, [.sup.3H]ketanserin is a well-established antagonist used routinely in competition assays to evaluate competitive activity of novel drug candidates at the 5-HT.sub.2A receptor (Maguire et al., 2012, Methods Mol Biol 897: 31). Thus, to evaluate activity of novel psilocybin derivatives at the 5-HT.sub.2A receptor, competition assays using [.sup.3H]ketanserin were employed as follows. SPA beads (RPNQ0010), [.sup.3H] ketanserin (NET1233025UC), membranes containing 5-HT.sub.2A (ES-313-M400UA), and isoplate-96 microplate (6005040) were all purchased from PerkinElmer. Radioactive binding assays were carried out using Scintillation Proximity Assay (SPA). For saturation binding assays, mixtures of 10 ug of membrane containing 5-HT.sub.2A receptor was pre-coupled to 1 mg of SPA beads at room temperature in a tube rotator for 1 hour in binding buffer (50 mM Tris-HCl pH7.4, 4 mM CaCl.sub.2, 1 mM ascorbic acid, 10 mM pargyline HCl). After pre-coupling, the beads and membrane were aliquoted in an isoplate-96 microplate with increasing amounts of [.sup.3H]ketanserin (0.1525 nM to 5 nM) and incubated for two hours at room temperature in the dark with shaking. After incubation, the samples were read on a MicroBeta 2 Microplate Counter (Perkin Elmer). Determination of non-specific binding was carried out in the presence of 20 mM of spiperone (S7395-250MG, Sigma). Equilibrium binding constants for ketanserin (K.sub.d) were determined from saturation binding curves using the ‘one-site saturation binding analysis’ method of GraphPad PRISM software (Version 9.2.0). Competition binding assays were performed using fixed (1 nM) [.sup.3H]ketanserin and different concentrations of tryptophan (3 nM to 1 mM), psilocin (30 pM to 10 mM) or unlabeled test compound (3 nM to 1 mM) similar to the saturation binding assay. K.sub.i values were calculated from the competition displacement data using the competitive binding analysis from GraphPad PRISM software. Tryptophan was included as a negative control as it has no activity at the 5-HT.sub.2A receptor. In contrast, psilocin was used as a positive control since it has established binding activity at the 5-HT.sub.2A receptor (Kim et al., 2020, Cell 182: 1574).
Cell Lines and Control Ligands Used to Assess Activity at 5-HT.SUB.1A
[0968] CHO-K1/Gα.sub.15 (GenScript, M00257) (-5-HT.sub.1A) and CHO-K⅕-HT.sub.1A/Gα.sub.15 (GenScript, M00330) (+5-HT.sub.1A) cells lines were used. Briefly, CHO-K1/Gα.sub.15 is a control cell line that constitutively expresses Gα.sub.15 which is a promiscuous G.sub.q protein. This control cell line lacks any transgene encoding 5-HT.sub.1A receptors, but still responds to forskolin; thus, cAMP response to forskolin should be the same regardless of whether or not 5-HT.sub.1A agonists are present. Conversely, CHO-K⅕-HT.sub.1A/Gα.sub.15 cells stably express 5-HT.sub.1A receptor in the CHO-K1 host background. Notably, Gα.sub.15 is a promiscuous G protein known to induce calcium flux response, present in both control and 5-HT.sub.1A cell lines. In +5-HT.sub.1A cells, Gα.sub.15 may be recruited in place of Gα.sub.i/o, which could theoretically dampen cAMP response (Rojas and Fiedler 2016, Front Cell Neurosci 10: 272). Thus, we included two known 5-HT.sub.1A agonists, psilocin (Blair etal., 2000, J Med Chem 43: 4701) and serotonin (Rojas and Fiedler 2016, Front Cell Neurosci 10: 272) as positive controls to ensure sufficient cAMP response was observed, thereby indicating measurable recruitment of Gα.sub.i/o protein to activated 5-HT.sub.1A receptors. In contrast, tryptophan is not known to activate, or modulate in any way, 5-HT.sub.1A receptors, and was thus used as a negative control. Cells were maintained in complete growth media as recommended by supplier (GenScript) which is constituted as follows: Ham’s F12 Nutrient mix (HAM’s F12, GIBCO #11765-047) with 10% fetal bovine serum (FBS) (Thermo Scientific #12483020), 200 .Math.g/ml zeocin (Thermo Scientific #R25005) and/or 100 .Math.g/ml hygromycin (Thermo Scientific #10687010). The cells were cultured in a humidified incubator with 37° C. and 5% CO.sub.2. Cells maintenance was carried out as recommended by the cell supplier. Briefly, vials with cells were removed from the liquid nitrogen and thawed quickly in 37° C. water bath. Just before the cells were completely thawed the vial’s outside was decontaminated by 70% ethanol spray. The cell suspension was then retrieved from the vial and added to warm (37° C.) complete growth media, and centrifuged at 1,000 rpm for 5 minutes. The supernatant was discarded, and the cell pellet was then resuspended in another 10 ml of complete growth media, and added to the 10 cm cell culture dish (Greiner Bio-One #664160). The media was changed every third day until the cells were about 90% confluent. The -90% confluent cells were then split 10:1 for maintenance or used for experiment.
Evaluation of 5-HT.SUB.1A Receptor Modulation
[0969] As 5-HT.sub.1A activation inhibits cAMP formation, the ability of test molecules to modulate 5-HT.sub.1A response was measured via changes in the levels of cAMP produced due to application of 4 .Math.M forskolin. Changes (should any significant change occur) in intracellular cAMP levels due to the treatment of novel molecule was evaluated using cAMP-Glo Assay kit (Promega # V1501). Briefly, +5-HT.sub.1A cells were seeded on 1-6 columns and base -5-HT.sub.1A cells were seeded on columns 7-12 of the white walled clear bottom 96-well plate (Corning, #3903). Both cells were seeded at the density of 30,000 cells/well in 100 .Math.l complete growth media and cultured 24 hrs in humidified incubator at 37° C. and 5% CO.sub.2. On the experiment day, the media of cells was replaced with serum/antibiotic free culture media. Then the cells were treated for 20 minutes with test molecules dissolved in induction medium (serum/antibiotic free culture media containing 4 .Math.M forskolin, 500 mM IBMX (isobutyl-1-methylxanthine, Sigma-Aldrich, Cat. #17018) and 100 mM (RO 20-1724, Sigma-Aldrich, Cat. #B8279)). Forskolin induced cAMP formation whereas IBMX and RO 20-1724 inhibited the degradation of cAMP. PKA was added to the lysate, mixed, and subsequently the substrate of the PKA was added. PKA was activated by cAMP, and the amount of ATP consumed due to PKA phosphorylation directly corresponded to cAMP levels in the lysate. Reduced ATP caused reduced conversion of luciferin to oxyluciferin, conferring diminished luminescence as the result 5-HT.sub.1A activation. In summary: this signal cascade permits 5-HT.sub.1A activation (positive modulation) by a test molecule to be measured in terms of decreasing % cAMP formation. Conversely, enhanced % cAMP is expected when 5-HT.sub.1A receptor is negatively modulated by a test molecule. Finally, no significant change in % cAMP - beyond that observed for negative control experiments (e.g., with tryptophan) - indicates that a test molecule does not bind 5-HT1A or that binding imparts a silent response.
Example 2 - Biosynthesis of a Second Multi-Substituent Psilocybin Derivative
[0970] Escherichiacoli strain Ec-1 was used to biosynthesize psilocybin derivative with formula (X) from derivatized indole feedstock. The construction of Ec-1 is described in Example 1. Scaled-up culturing and material storage of engineered E.coli was conducted as described in Example 1, except that 4-fluoro-1H-indol-6-ylamine (Combi-Blocks, www.combi-blocks.com) was used in place of 6-fluoro-1H-indol-4-ylamine. Scaled-up culturing and processing of engineered E.coli was conducted as described in Example 1, except that a total of 2.0 L was cultured. Two litres of E.coli culture broth was extracted by ethyl acetate (4×1.2 L). The organic layer was combined and dried over Na.sub.2SO.sub.4, followed by concentration under reduced pressure. The residue was purified by flash chromatography on silica gel (1 .fwdarw. 3% metanol in dichloromethane), to give the compound as a yellow solid (5 mg). Following purification, high-resolution MS (HRMS), .sup.1H NMR, and selective .sup.13C NMR were performed to assess purity, estimate total quantity, and confirm molecular structure. .sup.1H NMR (400 MHz, CD.sub.3OD): δ = 1.92 (s, 3H), 2.14 (s, 3H), 2.98 (t, J= 7.2, 2H), 3.47 (t, J= 7.2, 2H,), 6.82 (dd, J = 12.9, 1.6 Hz, 1H), 7.00 (s, 1H), 7.56 (d, J= 1.6 Hz, 1H). .sup.13 C NMR (100 MHz, CD.sub.3OD): δ = 21.1, 22.4, 25.9, 40.5 (d, Jc, .sub.F = 2.0 Hz), 97.5 (d, Jc, .sub.F = 24.0 Hz), 98.9 (d, Jc,.sub.F = 3.4 Hz), 110.5 (d, J.sub.C,.sub.F = 2.8 Hz), 112.6 (d, J.sub.C,.sub.F = 20.0 Hz), 122.6, 133.1 (d, Jc, .sub.F = 10.5 Hz), 139.2 (d, Jc, .sub.F = 13.6 Hz), 156.2 (d, Jc, .sub.F = 242.5 Hz), 170.0, 171.8. HRMS (ESI) m/z: calcd. for C.sub.12H.sub.16FN.sub.3O.sub.2 [M+H].sup.+ 278.1299, found 278.1298. Purity was determined as 95% w/w. It is noted that these data confirm a chemical structure corresponding with that of example compound (X):
##STR00320##
set forth herein.
Assessment of Cell Viability Upon Treatment of Psilocybin Derivative
[0971] Cell viability was assessed as described for Example 1, except the compound with formula (X) was evaluated in place of the compound with formula (IX).
Radioligand Receptor Binding Assays
[0972] Activity at 5-HT.sub.2A receptor was assessed as described for Example 1, except the compound with formula (X) was evaluated in place of the compound with formula (IX).
Cell Lines, Control Ligands, and Evaluation of 5-HT.SUB.1A Receptor Modulation
[0973] Cell lines and control ligands were as described in Example 1. Activity at 5-HT.sub.1A receptor was assessed as described for Example 1, except the compound with formula (X) was evaluated in place of the compound with formula (IX).
Example 3 - Biosynthesis of a Third Multi-Substituent Psilocybin Derivative
[0974] Escherichiacoli strain Ec-1 was used to biosynthesize psilocybin derivative with formula (XII) from derivatized indole feedstock. The construction of Ec-1 is described in Example 1. Scaled-up culturing and material storage of engineered E.coli was conducted as described in Example 1, except that 5-fluoro-7-nitro-1H-indole (www.bldpharm.com) was used in place of 6-fluoro-1H-indol-4-ylamine. Scaled-up culturing and processing of engineered E.coli was conducted as described in Example 1. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific), employing a modified version of a method described previously (Chang et al., 2015, Plant Physiol. 169: 1127-1140), with the exception that liquid chromatography was carried out using an UltiMate 3000 HPLC (Thermo Fisher Scientific) equipped with a Poroshell 120 SB-C18 column (Agilent Technologies) instead of an Accela HPLC system (Thermo Fisher Scientific) equipped with a Zorbax C18 column (Agilent Technologies). Briefly, 100 microliters of culture media were dried and resuspended in 100 microliters of DMSO. One tenth (10 microliters) of this suspension was injected at a flow rate of 0.5 mL/min and a gradient of solvent A (water with 0.1% of formic acid) and solvent B (ACN with 0.1% formic acid) as follows: 100% to 0% (v/v) solvent A over 5 min; isocratic at 0% (v/v) for 1 min; 0% to 100% (v/v) over 0.1 min; and isocratic at 100% (v/v) for 1.9 min. Total run time was 8 minutes. Heated ESI source and interface conditions were operated in positive ion mode as follows: vaporizer temperature, 400° C.; source voltage, 3 kV; sheath gas, 60 au, auxiliary gas, 20 au; capillary temperature, 380° C.; capillary voltage, 6 V; tube lens, 45 V. Instrumentation was performed as a single, HR scan event using Orbitrap detection of m/z in the range of 100-500 m/z. Ion injection time was 300 ms with scan time of 1 s. External and internal calibration procedures ensured <2 ppm error to facilitate elemental formulae predictions. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of N—[2-(5-fluoro-7-nitro-1H-indol-3-yl)ethyl]acetamide, having chemical formula (XII):
##STR00321##
eluted at 4.0 minutes (EIC, see:
TABLE-US-00055 m/z % Relative abundance Ionic species Δ ppm 207.05636 100 [M + H — C.sub.2H.sub.5NO].sup.+ 0.34 266.09362 1.1 [M + H].sup.+ 0.26 224.08303 0.8 249.06697 0.7 71.70087 0.5 66.12294 0.4 161.06346 0.3 127.24116 0.2 98.30615 0.2 192.96957 0.2
Example 4 - Biosynthesis of a Fourth Multi-Substituent Psilocybin Derivative
[0975] Escherichia coli strain Ec-1 was used to biosynthesize psilocybin derivative with formula (XIV) from derivatized indole feedstock. The construction of Ec-1 is described in Example 1. Scaled-up culturing and material storage of engineered E.coli was conducted as described in Example 1, except that 5-fluoro-1H-indol-6-ylamine (www.bldpharm.com) was used in place of 6-fluoro-1H-indol-4-ylamine. Scaled-up culturing and processing of engineered E.coli was conducted as described in Example 1. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of N-[2-(6-acetylamino-5-fluoro-1H-indol-3-yl)ethyl]acetamide, having chemical formula (XIV):
##STR00322##
eluted at 3.2 minutes (EIC, see:
TABLE-US-00056 m/z % Relative abundance Ionic species Δ ppm 219.09292 100 [M + H — C.sub.2H.sub.5NO].sup.+ 0.46 191.09787 9.0 177.08215 4.2 236.11933 2.4 234.10378 2.0 261.10332 1.4 278.12980 1.2 [M + H].sup.+ 0.47 226.11267 0.8 218.10916 0.8 260.11925 0.6
Example 5 - Biosynthesis of a Fourth Multi-Substituent Psilocybin Derivative
[0976] Escherichiacoli strain Ec-1 was used to biosynthesize psilocybin derivative with formula (XV) from derivatized indole feedstock. The construction of Ec-1 is described in Example 1. Scaled-up culturing and material storage of engineered E.coli was conducted as described in Example 1, except that culturing was performed for 14 hours instead of 24 hours, and 5-fluoro-1H-indol-6-ylamine (www.bldpharm.com) was used in place of 6-fluoro-1H-indol-4-ylamine. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of N-[2-(6-acetylamino-5-fluoro-1H-indol-3-yl)ethyl]acetamide, having chemical formula (XV):
##STR00323##
eluted at 2.5 minutes (EIC, see:
TABLE-US-00057 m/z % Relative abundance Ionic species Δ ppm 177.08158 100 [M + H — C.sub.2H.sub.5NO].sup.+ 3.78 219.09224 4.1 236.11880 3.0 [M + H].sup.+ 2.41 109.66972 0.6 157.07565 0.4 181.73305 0.2 199.48006 0.1 194.10827 0.1 220.09515 0.1 207.09179 0.1
Example 6 - Biosynthesis of a Sixth Multi-Substituent Psilocybin Derivative
[0977] Escherichia coli strain Ec-1 was used to biosynthesize psilocybin derivative with formula (XVII) from derivatized indole feedstock. The construction of Ec-1 is described in Example 1. Scaled-up culturing and material storage of engineered E.coli was conducted as described in Example 1, except that 4-fluoro-1H-indole-5-carbonitrile (www.bldpharm.com) was used in place of 6-fluoro-1H-indol-4-ylamine. Scaled-up culturing and processing of engineered E.coli was conducted as described in Example 1. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of N-[2-(5-cyano-4-fluoro-1 H-indol-3-yl)ethyl]acetamide, having chemical formula (XVII):
##STR00324##
eluted at 4.0 minutes (EIC, see:
Example 7 - Biosynthesis of a Seventh Multi-Substituent Psilocybin Derivative
[0978] Escherichiacoli strain Ec-1 was used to biosynthesize psilocybin derivative with formula (XVIII) from derivatized indole feedstock. The construction of Ec-1 is described in Example 1. Scaled-up culturing and material storage of engineered E.coli was conducted as described in Example 1, except that 6-bromo-1H-indol-4-ol (www.bldpharm.com) was used in place of 6-fluoro-1H-indol-4-ylamine. Scaled-up culturing and processing of engineered E.coli was conducted as described in Example 1. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of N-[2-(6-bromo-4-hydroxy-1H-indol-3-yl)ethyl]acetamide, having chemical formula (XVIII):
##STR00325##
eluted at 3.8 minutes (EIC, see:
TABLE-US-00058 m/z % Relative abundance Ionic species Δ ppm 237.98574 100 [M + H — C.sub.2H.sub.5NO].sup.+ 1.93 159.06737 29.5 209.99081 3.3 255.01230 1.2 279.99649 1.0 199.51968 1.0 61.09401 0.9 297.02267 0.7 [M + H].sup.+ 2.19 203.89784 0.6 177.16207 0.6
Example 8 - Biosynthesis of an Eighth Multi-Substituent Psilocybin Derivative
[0979] Escherichia coli strain Ec-1 was used to biosynthesize psilocybin derivative with formula (XXI) from derivatized indole feedstock. The construction of Ec-1 is described in Example 1. Scaled-up culturing and material storage of engineered E.coli was conducted as described in Example 1. Scaled-up culturing and processing of engineered E.coli was conducted as described in Example 1. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of N-[2-(4-acetylamino-6-fluoro-1H-indol-3-yl)ethyl]acetamide, having chemical formula (XXI):
##STR00326##
eluted at 3.2 minutes (EIC, see:
TABLE-US-00059 m/z % Relative abundance Ionic species Δ ppm 236.11928 100 [M + H — C.sub.2H.sub.2O].sup.+ 0.38 177.08202 26.0 [M + H — C.sub.2H.sub.6NO — C.sub.2H.sub.2O].sup.+ 1.30 219.09267 17.9 [M + H — C.sub.2H.sub.6NO].sup.+ 0.68 278.12986 3.0 [M + H].sup.+ 0.25 136.07571 1.1 56.17127 0.9 199.20892 0.8 88.82042 0.8 232.86285 0.8 158.02567 0.8
Example 9 - Biosynthesis of a Ninth Multi-Substituent Psilocybin Derivative
[0980] Escherichiacoli strain Ec-1 was used to biosynthesize psilocybin derivative with formula (XXIII) from derivatized indole feedstock. The construction of Ec-1 is described in Example 1. Scaled-up culturing and material storage of engineered E.coli was conducted as described in Example 1, except that 4-fluoro-1H-indole-7-carbonitrile (www.bldpharm.com) was used in place of 6-fluoro-1H-indol-4-ylamine. Scaled-up culturing and processing of engineered E.coli was conducted as described in Example 1. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of N-[2-(7-cyano-4-fluoro-1H-indol-3-yl)ethyl]acetamide, having chemical formula (XXIII):
##STR00327##
eluted at 3.9 minutes (EIC, see:
TABLE-US-00060 m/z % Relative abundance Ionic species Δ ppm 187.06606 100 [M + H — C.sub.2H.sub.3NO].sup.+ 2.89 93.13169 1.0 204.09270 0.6 229.07666 0.2 246.10356 0.2 [M + H].sup.+ 0.61 199.66049 0.2 59.34151 0.1
Example 10 - Biosynthesis of a Tenth Multi-Substituent Psilocybin Derivative
[0981] Escherichiacoli strain Ec-1 was used to biosynthesize psilocybin derivative with formula (XXV) from derivatized indole feedstock. The construction of Ec-1 is described in Example 1. Scaled-up culturing and material storage of engineered E.coli was conducted as described in Example 1, except that 4-fluoro-1H-indol-6-ylamine was used in place of 6-fluoro-1H-indol-4-ylamine. Scaled-up culturing and processing of engineered E.coli was conducted as described in Example 1. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of N-[2-(6-amino-4-fluoro-1H-indol-3-yl)ethyl]acetamide, having chemical formula (XXV):
##STR00328##
eluted at 4.1 minutes (EIC, see:
Example 11 - Biosynthesis of an Eleventh Multi-Substituent Psilocybin Derivative
[0982] Escherichiacoli strain Ec-1 was used to biosynthesize psilocybin derivative with formula (XXVIII) from derivatized indole feedstock. The construction of Ec-1 is described in Example 1. Scaled-up culturing and material storage of engineered E.coli was conducted as described in Example 1, except that 4-amino-1H-indole-6-carbonitrile (www.bldpharm.com) was used in place of 6-fluoro-1H-indol-4-ylamine. Scaled-up culturing and processing of engineered E.coli was conducted as described in Example 1. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of N-[2-(4-amino-6-cyano-1H-indol-3-yl)ethyl]acetamide, having chemical formula (XXVIII):
##STR00329##
eluted at 3.3 minutes (EIC, see:
TABLE-US-00061 m/z % Relative abundance Ionic species Empirical Formula 163.1076 100 243.1340 19.2 [M + H].sup.+ C.sub.14H.sub.15N.sub.4O 162.0760 17.3 197.1284 9.2 205.1182 9.1 144.0653 7.8 202.4956 6.9 187.1077 4.2 115.0388 2.0 186.4933 1.9
Example 12 - Biosynthesis of a Twelfth Multi-Substituent Psilocybin Derivative
[0983] Escherichiacoli strain Ec-1 was used to biosynthesize psilocybin derivative with formula (XX) from derivatized indole feedstock. The construction of Ec-1 is described in Example 1. Scaled-up culturing and material storage of engineered E.coli was conducted as described in Example 1, except that 4-fluoro-1H-indol-5-ol (www.bldpharm.com) was used in place of 6-fluoro-1H-indol-4-ylamine. Scaled-up culturing and processing of engineered E.coli was conducted as described in Example 1. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of N-[2-(4-fluoro-5-hydroxy-1H-indol-3-yl)ethyl]acetamide, having chemical formula (XX):
##STR00330##
eluted at 3.1 minutes (EIC, see:
Example 13 - Biosynthesis of a Thirteenth Multi-Substituent Psilocybin Derivative
[0984] Escherichiacoli strain Ec-2 was used to biosynthesize psilocybin derivative with formula (XIII) from derivatized indole feedstock. E.coli strain Ec-2 was constructed as follows. For plasmid cloning, Top10 or XL1-blue strains were used depending on antibiotic markers. Standard LB media was used for culturing. For gene expression and feeding experiments, the parent host strain employed was BL21 (DE3). Plasmids pET28a(+)-PfTrpB-B0A9-HIS and pCDM4-BaTDC-HIS were created as described in Example 1. The target plasmids pET28a(+)-PfTrpB-B0A9-HIS and pCDM4-BaTDC-HIS were sequentially transformed into BL21 (DE3) cells as follows: pCDM4-BaTDC-HIS was transformed into BL21 (DE3) first. Transformants selected using streptomycin were next transformed with pET28a(+)-PfTrpB-B0A9-HIS and selected with both streptomycin and kanamycin. Scaled-up culturing and material storage of engineered E.coli was conducted as described in Example 1, except that (1) only streptomycin and kanamycin were used for selection purposes; and (2) 5-fluoro-7-nitro-1H-indole (www.bldpharm.com) was used in place of 6-fluoro-1H-indol-4-ylamine. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of 2-(5-fluoro-7-nitro-1H-indol-3-yl)ethylamine, having chemical formula (XIII):
##STR00331##
eluted at 3.3 minutes (EIC, see:
Example 14 - Biosynthesis of a Fourteenth Multi-Ssubstituent Psilocybin Derivative
[0985] Escherichiacoli strain Ec-2 was used to biosynthesize psilocybin derivative with formula (XVI) from derivatized indole feedstock. The construction of Ec-2 is described in Example 13. Scaled-up culturing and material storage of engineered E.coli was conducted as described in Example 13, except that 5-fluoro-1H-indol-6-ylamine (www.bldpharm.com) was used in place of 5-fluoro-7-nitro-1H-indole. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of 3-(2-aminoethyl)-5-fluoro-1H-indol-6-amine, having chemical formula (XVI):
##STR00332##
eluted at 2.9 minutes (EIC, see:
Example 15 - Biosynthesis of a Fifteenth Multi-Substituent Psilocybin Derivative
[0986] Escherichiacoli strain Ec-2 was used to biosynthesize psilocybin derivative with formula (XIX) from derivatized indole feedstock. The construction of Ec-2 is described in Example 13. Scaled-up culturing and material storage of engineered E.coli was conducted as described in Example 13, except that 4-fluoro-1H-indole-5-carbonitrile (www.bldpharm.com) was used in place of 5-fluoro-7-nitro-1H-indole. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of 3-(2-aminoethyl)-4-fluoro-1H-indole-5-carbonitrile, having chemical formula (XIX):
##STR00333##
eluted at 3.2 minutes (EIC, see:
Example 16 - Biosynthesis of a Sixteenth Multi-Substituent Psilocybin Derivative
[0987] Escherichiacoli strain Ec-2 was used to biosynthesize psilocybin derivative with formula (XXII) from derivatized indole feedstock. The construction of Ec-2 is described in Example 13. Scaled-up culturing and material storage of engineered E.coli was conducted as described in Example 13, except that 6-fluoro-1H-indol-4-ylamine (www.bldpharm.com) was used in place of 5-fluoro-7-nitro-1H-indole. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of 3-(2-aminoethyl)-6-fluoro-1H-indol-4-amine, having chemical formula (XXII):
##STR00334##
eluted at 1.4 minutes (EIC, see:
Example 17 - Biosynthesis of a Seventeenth Multi-Substituent Psilocybin Derivative
[0988] Escherichiacoli strain Ec-2 was used to biosynthesize psilocybin derivative with formula (XXVI) from derivatized indole feedstock. The construction of Ec-2 is described in Example 13. Scaled-up culturing and material storage of engineered E.coli was conducted as described in Example 13, except that 3-(2-aminoethyl)-4-fluoro-1H-indol-6-amine (www.bldpharm.com) was used in place of 5-fluoro-7-nitro-1H-indole. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of 3-(2-aminoethyl)-4-fluoro-1H-indol-6-amine, having chemical formula (XXVI):
##STR00335##
eluted at 0.6 minutes (EIC, see:
Example 18 - Biosynthesis of an Eighteenth Multi-Substituent Psilocybin Derivative
[0989] Escherichiacoli strain Ec-2 was used to biosynthesize psilocybin derivative with formula (XXIX) from derivatized indole feedstock. The construction of Ec-2 is described in Example 13. Scaled-up culturing and material storage of engineered E.coli was conducted as described in Example 13, except that 4-amino-1H-indole-6-carbonitrile (www.bldpharm.com) was used in place of 5-fluoro-7-nitro-1H-indole. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of 4-amino-3-(2-aminoethyl)-1 H-indole-6-carbonitrile, having chemical formula (XXIX):
##STR00336##
eluted at 2.6 minutes (EIC, see:
TABLE-US-00062 m/z % Relative abundance Ionic species Δ ppm 184.08655 100 [M + H — NH.sub.3].sup.+ 2.01 97.51806 1.0 172.08669 0.7 201.11313 0.6 [M + H].sup.+ 1.69 199.55735 0.1 61.09273 0.1 143.08142 0.1 60.25552 0.1 209.32732 0.1 202.04140 0.1
Example 19 - Biosynthesis of a Nineteenth Multi-Substituent Psilocybin Derivative
[0990] Escherichiacoli strain Ec-3 was used to biosynthesize psilocybin derivative with formula (XXVII) from derivatized indole feedstock. E.coli strain Ec-2 was constructed as follows. For plasmid cloning, Top10 orXL1-blue strains were used depending on antibiotic markers. Standard LB media was used for culturing. For gene expression and feeding experiments, the parent host strain employed was BL21 (DE3). First, the plasmid pET28a(+)-EsNMT-HIS was created by inserting an in-frame, HIS tagged (SEQ.ID NO: 46) EsNMT gene (SEQ.ID NO: 11) into the NdellXhol site of pET28a(+) (SEQ.ID NO: 36). As a second step, from plasmid pCDM4 (SEQ.ID NO: 35), the plasmid pCDM4-PsiD-HIS was created by inserting an in-frame, HIS-tagged (SEQ.ID NO: 46) PsiD gene (SEQ.ID NO: 7) into the NdellXhol site of pCDM4. These target plasmids were sequentially transformed into BL21 (DE3) cells as follows: pCDM4-PsiD-HIS was transformed into BL21 (DE3) first. Transformants selected using streptomycin were next transformed with pET28a(+)-EsNMT-HIS and selected with both streptomycin and kanamycin. Scaled-up culturing and material storage of engineered E.coli was conducted as described in Example 1, except that (1) only streptomycin and kanamycin were used for selection purposes; and (2) 4-amino-1H-indole-6-carbonitrile (www.bldpharm.com) was used in place of 6-fluoro-1H-indol-4-ylamine. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of 4-amino-3-[2-(methylamino)ethyl]-1 H-indole-6-carbonitrile, having chemical formula (XXVII):
##STR00337##
eluted at 2.6 minutes (EIC, see:
Example 20 - Biosynthesis of a Twentieth Multi-Substituent Psilocybin Derivative
[0991] Yeast ( Saccharomycescerevisiae) strain Sc-1 was created through genetic engineering of a parent yeast strain, to enable bioconversion of commercially obtained, derivatized indole, tryptophan, or tryptamine feedstock to generate final product. The parent yeast ( Saccharomycescerevisiae) strain was CEN.PK with genotype Matα; ura3-52; trp1-289; leu2-3,112; his3Δ1; MAL2-8C; SUC2. The parent strain was engineered to include 7DMATS (SEQ.ID NO: 16), ClostSporTDC (SEQ.ID NO: 6), and PsmF (SEQ.ID NO: 10) which catalyzed three enzymatic steps. Engineering also included CPR (SEQ.ID NO: 26) although this enzyme was not used in the bioconversion process. 7DMATS, ClostSporTDC, and CPR were included in the strain through chromosomal homologous recombination of integration cassettes as described previously (Dastmalchi et al., 2019, Nat. Chem Biol. 15: 384-390; Chen et al., 2018, Nat. Chem Biol. 14: 738-743). Conversely, PsmF was built into a protein expression plasmid and transformed to the genomically integrated strain already harboring 7DMATS, ClostSporTDC, and CPR. 7DMATS, ClostSporTDC, and CPR were encoded by SEQ.ID NO: 15, SEQ.ID NO: 5 and SEQ.ID NO: 25, respectively, with addition of in-frame, C-terminal HIS (SEQ.ID NO: 46, SEQ.ID NO: 47), FLAG (SEQ.ID NO: 42, SEQ.ID NO: 43), and c-MYC (SEQ.ID NO: 40, SEQ.ID NO: 41) epitope tags, respectively. Integration cassettes were built using yeast promoter sequences amplified from S.cerevisiae genomic DNA as described (Dastmalchi et al., 2019; Chen et al., 2018) enabling constitutive gene expression. Amplified promoters included PGK1 (SEQ.ID NO: 30), TDH3 (SEQ.ID NO: 31), CLN1 (SEQ.ID NO: 32), and UGA1 (SEQ.ID NO: 33). Two integration cassettes were assembled: the first, (X-3)::TADH1-ClostSporTDC-Flag-PPGK1-PTDH3-CPR-c-myc-TCYC1 (SEQ.ID NO: 52), harboured tagged ClostSporTDC and CPR. The second (Xii-2)::PTDH3-7DMATS-His-TCYC1 (SEQ.ID NO: 53), harboured only tagged 7DMATS. Successive genomic integration of these cassettes was performed as described previously (Chen et al., 2018). Following stable integration of these two cassettes, the strain was further manipulated by transformation with a yeast episomal vector encoding a promiscuous N-acetyltransferase, PsmF (pMM1-pTDH3-PsmF-His-tCYC1). For construction of pMM1-pTDH3-PsmF-His-tCYC1, the gene PsmF (SEQ.ID NO: 9) fused in-frame with a HIS epitope tag (SEQ.ID NO: 46) was ligated to empty plasmid pMM1 (SEQ.ID NO: 34) using BamHI/Sacll restriction sites. For this Example, heterologous expression of a non-native or engineered TrpB gene was not necessary, as endogenous tryptophan synthase activity proved sufficient. The final engineered strain was called Sc-1. For scaled-up production of derivative product, culturing was performed as follows. Seed cultures were inoculated in SD-drop-out medium overnight. The overnight culture was then divided into two flasks containing 500 ml each of SD-drop-out medium containing 2% (w/v) glucose, 0.3%(w/v) KH.sub.2PO.sub.4, 0.05% (w/v) MgSO.sub.4.7H.sub.2O, 0.5% (w/v) (NH.sub.4).sub.2SO.sub.4 plus 500 .Math.M 4-chloro-1H-indole (www.combi-blocks.com) for conversion by Sc-1. Yeast cultures were grown for 48 h. Cultures were then centrifuged (10,000 g × 5 minutes) to remove cellular content, and culture broth containing secreted derivative product was stored at -80° C. until further processing. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of N-(2-[4-chloro-7-(3-methyl-2-butenyl)-1H-indol-3-yl]ethyl)acetamide, having chemical formula (XXXIV):
##STR00338##
eluted at 5.2 minutes (EIC, see:
TABLE-US-00063 m/z % Relative abundance Ionic species Δ ppm 246.10397 100 [M + H — NH.sub.3].sup.+ 1.74 190.04136 53.1 178.04141 2.5 232.05183 0.8 135.46806 0.7 61.09248 0.6 151.39211 0.6 134.24040 0.6 199.43795 0.6 118.45281 0.5
Example 21 - Biosynthesis of a Twenty-First Multi-Substituent Psilocybin Derivative
[0992] The same yeast strain (Sc-1) and procedures described in Example 20 were used to biosynthesize a psilocybin derivative with chemical formula (XXXIII), with the following exception: in place of 4-chloro-1H-indole, 500 .Math.M 5-fluoro-1H-indole (Combi-Blocks, www.combi-blocks.com) was supplied as feedstock for bioconversion. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of 2-[5-fluoro-7-(3-methyl-2-butenyl)-1H-indol-3-yl]ethylamine having chemical formula (XXXII):
##STR00339##
eluted at 4.9 minutes (EIC, see:
TABLE-US-00064 m/z % Relative abundance Ionic species Δ ppm 230.13354 100 [M + H — C.sub.2H.sub.6NO].sup.+ 1.78 174.07090 11.0 272.14384 1.9 233.10802 1.6 199.41144 0.8 151.39218 0.8 162.07105 0.8 216.08147 0.7 289.17040 0.7 [M + H].sup.+ 2.32 143.09824 0.7
Example 22 - Biosynthesis of a Twenty-Second Multi-Substituent Psilocybin Derivative
[0993] The same yeast strain (Sc-1) and procedures described in Example 20 were used to biosynthesize a psilocybin derivative with chemical formula (XXXIII), with the following exception: in place of 4-chloro-1H-indole, 500 .Math.M 5-fluoro-1H-indole (Combi-Blocks, www.combi-blocks.com) was supplied as feedstock for bioconversion. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of N-(2-[5-fluoro-7-(3-methyl-2-butenyl)-1H-indol-3-yl]ethyl)acetamide having chemical formula (XXXIII):
##STR00340##
eluted at 4.2 minutes (EIC, see:
Example 23 - Biosynthesis of a Twenty-Third Multi-Substituent Psilocybin Derivative
[0994] The same yeast strain (Sc-1) and procedures described in Example 20 were used to biosynthesize a psilocybin derivative with chemical formula (XLIX), with the following exception: in place of 4-chloro-1H-indole, 500 .Math.M 1H-indol-4-ol (Combi-Blocks, www.combi-blocks.com) was supplied as feedstock for bioconversion. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of 2-amino-3-[4-hydroxy-7-(3-methyl-2-butenyl)-1H-indol-3-yl]propionic acid having chemical formula (XLIX):
##STR00341##
eluted at 3.8 minutes (EIC, see:
Example 24 - Biosynthesis of a Twenty-Fourth Multi-Substituent Psilocybin Derivative
[0995] Yeast ( Saccharomycescerevisiae) strain Sc-2 was created through plasmid transformation of a parent yeast strain, to enable bioconversion of commercially obtained, derivatized indole, tryptophan, or tryptamine feedstock to generate final product. The parent yeast ( Saccharomycescerevisiae) strain was CEN.PK with genotype Matα; ura3-52; trp1-289; leu2-3,112; his3Δ1; MAL2-8C; SUC2. The parent strain was transformed with a yeast episomal vector (pMM1-pTDH3-PriB-His-tCYC1) encoding a HIS-tagged (SEQ.ID NO: 46, SEQ.ID NO: 47), promiscuous 6-prenyltransferase enzyme, PriB (SEQ.ID NO: 18). For construction of pMM1-pTDH3-PriB-His-tCYC1, the gene PriB (SEQ.ID NO: 17) fused in-frame with a HIS epitope tag (SEQ.ID NO: 46) was ligated to empty plasmid pMM1 (SEQ.ID NO: 34) using BamHI/Sacll restriction sites. For this Example, heterologous expression of a non-native or engineered TrpB gene was not necessary, as endogenous tryptophan synthase activity proved sufficient. The final engineered strain was called Sc-2. For scaled-up production of derivative product, culturing was performed as described in Example 20, with the following exception: in place of 4-chloro-1H-indole, 500 .Math.M 1H-indol-4-ol (Combi-Blocks, www.combi-blocks.com) was supplied as feedstock for bioconversion. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of 2-amino-3-[4-hydroxy-6-(3-methyl-2-butenyl)-1H-indol-3-yl]propionic acid having chemical formula (L):
##STR00342##
eluted at 3.8 minutes (EIC, see:
Example 25 - Biosynthesis of a Twenty-Fifth Multi-Substituent Psilocybin Derivative
[0996] The same yeast strain (Sc-2) and procedures described in Example 24 were used to biosynthesize a psilocybin derivative with chemical formula (XLVIII), with the following exception: in place of 1H-indol-4-ol, 5-bromo-1H-indole (Combi-Blocks, www.combi-blocks.com) was supplied as feedstock for bioconversion. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of 2-amino-3-[5-bromo-6-(3-methyl-2-butenyl)-1H-indol-3-yl]propionic acid having chemical formula (XLVIII):
##STR00343##
eluted at 4.2 minutes (EIC, see:
Example 26 - Biosynthesis of a Twenty-Sixth Multi-Substituent Psilocybin Derivative
[0997] Yeast ( Saccharomycescerevisiae) strain Sc-3 was created through plasmid transformation of a parent yeast strain, to enable bioconversion of commercially obtained, derivatized indole, tryptophan, or tryptamine feedstock to generate final product. The parent yeast ( Saccharomycescerevisiae) strain was CEN.PK with genotype Matα; ura3-52; trp1-289; leu2-3,112; his3Δ1; MAL2-8C; SUC2. The parent strain was transformed with a yeast episomal vector (pMM1-pTDH3-SCO7467-tCYC1) encoding a 5-prenyltransferase enzyme, SCO7467 (SEQ.ID NO: 20). For construction of pMM1-pTDH3-SCO7467-tCYC1, the gene SCO7467 (SEQ.ID NO: 19) was ligated to empty plasmid pMM1 (SEQ.ID NO: 34) using BamHl/Sacll restriction sites. For this Example, heterologous expression of a non-native or engineered TrpB gene was not necessary, as endogenous tryptophan synthase activity proved sufficient. The final engineered strain was called Sc-3. For scaled-up production of derivative product, culturing was performed as described in Example 20, with the following exception: in place of 4-chloro-1H-indole, 500 .Math.M 1H-Indol-4-ol (Combi-Blocks, www.combi-blocks.com) was supplied as feedstock for bioconversion. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of 2-amino-3-[4-hydroxy-5-(3-methyl-2-butenyl)-1 H-indol-3-yl]propionic acid, having chemical formula (LI):
##STR00344##
eluted at 3.8 minutes (EIC, see:
Example 27 - Biosynthesis of a Twenty-Seventh Multi-Substituent Psilocybin Derivative
[0998] Yeast ( Saccharomycescerevisiae) strain Sc-4 was created through genetic engineering of a parent yeast strain, to enable bioconversion of commercially obtained, derivatized indole, tryptophan, or tryptamine feedstock to generate final product. The parent yeast ( Saccharomycescerevisiae) strain was CEN.PK with genotype Matα; ura3-52; trp1-289; leu2-3,112; his3Δ1; MAL2-8C; SUC2. The parent strain was engineered to include PriB (SEQ.ID NO: 18), BaTDC (SEQ.ID NO: 4), and PsmF (SEQ.ID NO: 10) which catalyzed three enzymatic steps. Engineering also included CPR (SEQ.ID NO: 26) although this enzyme was not used in the bioconversion process. PriB, BaTDC, and CPR were included in the strain through chromosomal homologous recombination of integration cassettes as described previously (Dastmalchi et al., 2019, Nat. Chem Biol. 15: 384-390; Chen et al., 2018, Nat. Chem Biol. 14: 738-743). Conversely, PsmF was built into a protein expression plasmid and transformed to the genomically integrated strain already harboring PriB, BaTDC, and CPR. PriB, BaTDC, and CPR were encoded by SEQ.ID NO: 17, SEQ.ID NO: 3 and SEQ.ID NO: 25, respectively, with addition of in-frame, C-terminal HIS (SEQ.ID NO: 46, SEQ.ID NO: 47), FLAG (SEQ.ID NO: 42, SEQ.ID NO: 43), and c-MYC (SEQ.ID NO: 40, SEQ.ID NO: 41) epitope tags, respectively. Integration cassettes were built using yeast promoter sequences amplified from S.cerevisiae genomic DNA as described (Dastmalchi et al., 2019; Chen et al., 2018) enabling constitutive gene expression. Amplified promoters included PGK1 (SEQ.ID NO: 30), TDH3 (SEQ.ID NO: 31), CLN1 (SEQ.ID NO: 32), and UGA1 (SEQ.ID NO: 33). Two integration cassettes were assembled: the first, (X-3)::TADH1-BaTDC-Flag-PPGK1-PTDH3-CPR-c-myc-TCYC1 (SEQ.ID NO: 50), harboured tagged BaTDC and CPR. The second (Xii-2)::PTDH3-PriB-His-TCYC1 (SEQ.ID NO: 51), harboured only tagged PriB. Successive genomic integration of these cassettes was performed as described previously (Chen et al., 2018). Following stable integration of these two cassettes, the strain was further manipulated by transformation with a yeast episomal vector encoding a promiscuous N-acetyltransferase, PsmF (pMM1-pTDH3-PsmF-His-tCYC1). For construction of pMM1-pTDH3-PsmF-His-tCYC1, the gene PsmF (SEQ.ID NO: 9) fused in-frame with a HIS epitope tag (SEQ.ID NO: 46) was ligated to empty plasmid pMM1 (SEQ.ID NO: 34) using BamHl/Sacll restriction sites. For this Example, heterologous expression of a non-native or engineered TrpB gene was not necessary, as endogenous tryptophan synthase activity proved sufficient. The final engineered strain was called Sc-4. For scaled-up production of derivative product, culturing was performed as described in Example 20, with the following exception: in place of 4-chloro-1H-indole, 500 .Math.M 5-chloro-1H-indole (Combi-Blocks, www.combi-blocks.com) was supplied as feedstock for bioconversion. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of N-(2-[5-chloro-6-(3-methyl-2-butenyl)-1H-indol-3-yl]ethyl)acetamide, having chemical formula (XXXVII):
##STR00345##
eluted at 5.1 minutes (EIC, see:
Example 28 - Biosynthesis of a Twenty-Eighth Multi-Ssubstituent Psilocybin Derivative
[0999] The same yeast strain (Sc-4) and procedures described in Example 27 were used to biosynthesize a psilocybin derivative with chemical formula (XXXV), with the following exception: in place of 5-chloro-1H-indole, 500 .Math.M 5-fluoro-1H-indole (Combi-Blocks, www.combi-blocks.com) was supplied as feedstock for bioconversion. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of N-(2-[5-fluoro-6-(3-methyl-2-butenyl)-1H-indol-3-yl]ethyl)acetamide, having chemical formula (XXXV):
##STR00346##
eluted at 3.8 minutes (EIC, see:
Example 29 - Biosynthesis of a Twenty-Ninth Multi-Substituent Psilocybin Derivative
[1000] Yeast ( Saccharomycescerevisiae) strain Sc-5 was obtained as an intermediate in the process of assembling Sc-4. The strain Sc-5 is essentially identical to Sc-4, with the exception that Sc-5 does not harbour an additional episomal vector (pMM1-pTDH3-PsmF-His-tCYC1) encoding the promiscuous N-acetyltransferase, PsmF (SEQ.ID NO: 10). Thus, Sc-5 hosts only two enzymes through chromosomal integration - BaTDC (SEQ.ID NO: 4) and PriB (SEQ.ID NO: 18) - which participate in derivative formation. A third enzyme, CPR (SEQ.ID NO: 26) is similarly integrated but does not contribute to derivative production. Heterologous expression of a non-native or engineered TrpB gene was not necessary, as endogenous tryptophan synthase activity proved sufficient. For scaled-up production of derivative product, culturing was performed as described in Example 20, with the following exception: in place of 4-chloro-1H-indole, 500 .Math.M 5-fluoro-1H-indole (Combi-Blocks, www.combi-blocks.com) was supplied as feedstock for bioconversion. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of 2-[5-fluoro-6-(3-methyl-2-butenyl)-1H-indol-3-yl]ethylamine, having chemical formula (XXXVI):
##STR00347##
eluted at 4.3 minutes (EIC, see:
TABLE-US-00065 m/z % Relative abundance Ionic species Δ ppm 230.13358 100 [M + H — NH.sub.3].sup.+ 1.61 198.47229 0.6 89.48803 0.5 233.50280 0.5 154.92723 0.5 115.92102 0.5 132.59683 0.5 92.18462 0.5 199.41139 0.4 102.89690 0.4
Example 30 - Biosynthesis of a Thirtieth Multi-Substituent Psilocybin Derivative
[1001] The same yeast strain (Sc-5) and procedures described in Example 29 were used to biosynthesize a psilocybin derivative with chemical formula (XXXVIII), with the following exception: in place of 5-fluoro-1H-indole, 500 .Math.M 5-chloro-1H-indole (Combi-Blocks, www.combi-blocks.com) was supplied as feedstock for bioconversion. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of 2-[5-chloro-6-(3-methyl-2-butenyl)-1H-indol-3-yl]ethylamine, having chemical formula (XXXVIII):
##STR00348##
eluted at 4.4 minutes (EIC, see:
Example 31 - Biosynthesis of a Thirty-First Multi-Substituent Psilocybin Derivative
[1002] Yeast ( Saccharomycescerevisiae) strain Sc-6 was created through genetic engineering of a parent yeast strain, to enable bioconversion of commercially obtained, derivatized indole, tryptophan, or tryptamine feedstock to generate final product. The parent yeast ( Saccharomycescerevisiae) strain was CEN.PK with genotype Matα; ura3-52; trp1-289; leu2-3,112; his3Δ1; MAL2-8C; SUC2. The parent strain was engineered to include PsiH (SEQ.ID NO: 24), CPR (SEQ.ID NO: 26), ClostSporTDC (SEQ.ID NO: 6) and PsiM (SEQ.ID NO: 14) which catalyzed or supported several enzymatic steps. Engineering also included PsiK (SEQ.ID NO: 49) although this enzyme did not appear capable of contributing to the bioconversion process. PsiH, CPR, PsiM and PsiK were included in the strain through chromosomal homologous recombination of integration cassettes as described previously (Dastmalchi et al., 2019, Nat. Chem Biol. 15: 384-390; Chen et al., 2018, Nat. Chem Biol. 14: 738-743). Conversely, ClostSporTDC was built into a protein expression plasmid and transformed to the genomically integrated strain already harboring PsiH, CPR, PsiM and PsiK. PsiH, CPR, ClostSporTDC and PsiM were encoded by SEQ.ID NO: 23, SEQ.ID NO: 25, SEQ.ID NO: 5, and SEQ.ID NO: 13, respectively, with addition of in-frame, C-terminal HA (SEQ.ID NO: 38, SEQ.ID NO: 39), c-MYC (SEQ.ID NO: 40, SEQ.ID NO: 41), HIS (SEQ.ID NO: 46, SEQ.ID NO: 47) and FLAG (SEQ.ID NO: 42, SEQ.ID NO: 43) epitope tags, respectively. Integration cassettes were built using yeast promoter sequences amplified from S.cerevisiae genomic DNA as described (Dastmalchi et al., 2019; Chen et al., 2018) enabling constitutive gene expression. Amplified promoters included PGK1 (SEQ.ID NO: 30), TDH3 (SEQ.ID NO: 31), CLN1 (SEQ.ID NO: 32), and UGA1 (SEQ.ID NO: 33). Two integration cassettes were assembled: the first, XII-4::TADH1-PsiH-HA-PPGK1-PTDH3-CPR-c-myc-TCYC1 (SEQ.ID NO: 27), harboured tagged PsiH and CPR. The second, XII-5::TADH1-PsiK-V5-PPGK1-PTDH3-PsiM-FLAG-TCYC1 (SEQ.ID NO: 28), harboured tagged PsiK and PsiM. Successive genomic integration of these cassettes was performed as described previously (Chen et al., 2018). Following stable integration of these two cassettes, the strain was further manipulated by transformation with a yeast episomal vector encoding ClostSporTDC (pMM1-pTDH3-ClostSpor-His-tCYC1). For construction of pMM1-pTDH3-ClostSpor-His-tCYC1, the gene ClostSporTDC (SEQ.ID NO: 5) fused in-frame with a HIS epitope tag (SEQ.ID NO: 46) was ligated to empty plasmid pMM1 (SEQ.ID NO: 34) using BamHI/Sacll restriction sites. For this Example, heterologous expression of a non-native or engineered TrpB gene was not necessary, as endogenous tryptophan synthase activity proved sufficient. The final engineered strain was called Sc-6. Employing Sc-6, the same procedures described in Example 20 were used to biosynthesize a psilocybin derivative with chemical formula (XL), with the following exception: in place of 4-chloro-1H-indole, 500 .Math.M 6-chloro-1H-indole (Combi-Blocks, www.combi-blocks.com) was supplied as feedstock for bioconversion. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of 6-chloro-3-[2-(dimethylamino)ethyl]-1H-indol-4-ol, having chemical formula (XL):
##STR00349##
eluted at 0.38 minutes (EIC, see:
Example 32 - Biosynthesis of a Thirty-Second Multi-Substituent Psilocybin Derivative
[1003] The same yeast strain (Sc-6) and procedures described in Example 31 were used to biosynthesize a psilocybin derivative with chemical formula (XXXIX), with the following exception: in place of 4-chloro-1H-indole, 500 .Math.M 7-chloro-1H-indole (Combi-Blocks, www.combi-blocks.com) was supplied as feedstock for bioconversion. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of 7-chloro-3-[2-(dimethylamino)ethyl]-1H-indol-4-ol, having chemical formula (XXXIX):
##STR00350##
eluted at 3.1 minutes (EIC, see:
Example 33 - Biosynthesis of a Thirty-Third Multi-Substituent Psilocybin Derivative
[1004] The same yeast strain (Sc-6) and procedures described in Example 31 were used to biosynthesize a psilocybin derivative with chemical formula (LII), with the following exception: in place of 4-chloro-1 H-indole, 500 .Math.M 6-fluoro-1H-indole (Combi-Blocks, www.combi-blocks.com) was supplied as feedstock for bioconversion. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of 3-(2-aminoethyl)-6-fluoro-1H-indol-4-ol, having chemical formula (LII):
##STR00351##
eluted at 2.3 minutes (EIC, see:
Example 34 - Biosynthesis of a Thirty-Fourth Multi-Substituent Psilocybin Derivative
[1005] The same yeast strain (Sc-6) and procedures described in Example 31 were used to biosynthesize a psilocybin derivative with chemical formula (Llll), with the following exception: in place of 4-chloro-1H-indole, 500 .Math.M 6-bromo-1H-indole (Combi-Blocks, www.combi-blocks.com) was supplied as feedstock for bioconversion. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of 6-bromo-3-[2-(dimethylamino)ethyl]-1H-indol-4-ol, having chemical formula (LIII):
##STR00352##
eluted at 3.2 minutes (EIC, see:
Example 35 - Biosynthesis of a Thirty-Fifth Multi-Substituent Psilocybin Derivative
[1006] The same yeast strain (Sc-6) and procedures described in Example 31 were used to biosynthesize a psilocybin derivative with chemical formula (LlV), with the following exception: in place of 4-chloro-1H-indole, 500 .Math.M 6-bromo-1H-indole (Combi-Blocks, www.combi-blocks.com) was supplied as feedstock for bioconversion. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of 3-(2-aminoethyl)-6-bromo-1H-indol-4-ol, having chemical formula (LlV):
##STR00353##
eluted at 2.9 minutes (EIC, see:
Example 36 - Biosynthesis of a Thirty-Sixth Multi-Substituent Psilocybin Derivative
[1007] Synthesis of a psilocybin derivative was accomplished using FgaPT2 enzyme and an in vitro procedure. cDNA encoding FgaPT2 (SEQ.ID NO: 21) was synthesized and subcloned at GenScript (www.genscript.com) using Nde1 and Xho1 sites to pET26b(+) plasmid (SEQ.ID NO: 54). The final plasmid pET26b(+)-FgaPT2 encoded an in-frame, C-terminal HIS tag fusion of FgaPT2. Purified, recombinant FgaPT2 enzyme (SEQ.ID NO: 22) was raised in E.coli and isolated as follows. The plasmid pET26b(+)-FgaPT2 was transformed into Rosetta (DE3) competent E.coli cells. Transformed Rosetta (DE3) E.coli cells were grown in LB media at 30° C. for overnight and then transferred into TB (terrific broth) media to grow at 37° C. until optical density (OD.sub.600) reached 0.6-1.5. The cell culture was then transferred to a 16° C. incubator with the addition of IPTG at 0.2 mM to initiate recombinant protein expression. After 20 hours the cells were harvested by centrifugation at 5,000 × g for 6 minutes and the cell pellet was stored in -80° C. before protein extraction. For extraction and purification of FgaPT2 recombinant protein, E.coli cells were resuspended in a buffer containing 50 mM sodium phosphate (pH 7.0) and 300 mM NaCl and then sonicated for 5-10 minutes to break the cells. The cell lysate was centrifuged at 12,000 g for 30 minutes to collect the supernatant containing soluble crude protein. The supernatant was applied to cobalt resin (TALON Superflow™, Cytiva) to isolate HIS-tagged target protein. Purified protein was stored at -80° C. in a buffer containing 50 mM Tris-HCI (pH 7.0), 100 mM NaCl, and 10% glycerol. The tryptophan derivative 2-amino-3-(5-bromo-1H-indol-3-yl)propionic acid (www.sigmaaldrich.com) and DMAPP (www.sigmaaldrich.com) were used as co-substrates in the reaction. Briefly, reactions were set up as follows: 50 mM Tris-HCI (pH 8.0), 180 .Math.M DMAPP, 0.5 mM tryptophan derivative, and 300 .Math.g/mL of FgaPT2 were added together and the reaction proceeded at 37° C. for 2 hours. Equal volume of MeOH was added to quench the reaction and precipitate the protein. The sample was then centrifuged at 13,000 g for 20 minutes, allowing removal of the supernatant which contained the desired product. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of 2-amino-3-[5-bromo-4-(3-methyl-2-butenyl)-1H-indol-3-yl]propionic acid, having chemical formula (XXX):
##STR00354##
eluted at 4.2 minutes (EIC, see:
Example 37 - Biosynthesis of a Thirty-Seventh Multi-Substituent Psilocybin Derivative
[1008] Synthesis of a psilocybin derivative was accomplished using FgaPT2 enzyme and the in vitro procedure described in Example 36, with the exception that 2-amino-3-(6-fluoro-1H-indol-3-yl)propionic acid (www.sigmaaldrich.com) was used in place of 2-amino-3-(5-bromo-1H-indol-3-yl)propionic acid substrate. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of 2-amino-3-[6-fluoro-4-(3-methyl-2-butenyl)-1H-indol-3-yl]propionic acid, having chemical formula (XXXI):
##STR00355##
eluted at 4.0 minutes (EIC, see:
Example 38 - Biosynthesis of a Thirty-Eighth Multi-Substituent Psilocybin Derivative
[1009] Synthesis of a psilocybin derivative was accomplished using PriB enzyme and an in vitro procedure. cDNA encoding PriB (SEQ.ID NO: 17) was synthesized and subcloned at GenScript (www.genscript.com) using Nde1 and Xho1 sites to pET26b(+) plasmid (SEQ.ID NO: 54). The final plasmid pET26b(+)-PriB encoded an in-frame, C-terminal HIS tag fusion of PriB. Purified, recombinant PriB enzyme (SEQ.ID NO: 18) was raised in E.coli and isolated as follows. The plasmid pET26b(+)-PriB was transformed into Rosetta (DE3) competent E.coli cells. Transformed Rosetta (DE3) E.coli cells were grown in LB media at 30° C. for overnight and then transferred into TB (terrific broth) media to grow at 37° C. until optical density (OD.sub.600) reached 0.6-1.5. The cell culture was then transferred to a 16° C. incubator with the addition of IPTG at 0.5 mM to initiate recombinant protein expression. After 20 hours the cells were harvested by centrifugation at 5,000 × g for 6 minutes and the cell pellet was stored in -80° C. before protein extraction. For extraction and purification of PriB recombinant protein, E.coli cells were resuspended in a buffer containing 50 mM sodium phosphate (pH 7.0) and 300 mM NaCl and then sonicated for 5-10 minutes to break the cells. The cell lysate was centrifuged at 12,000 g for 30 minutes to collect the supernatant containing soluble crude protein. The supernatant was applied to cobalt resin (TALON Superflow™, Cytiva) to isolate HIS-tagged target protein. Purified protein was stored at -80° C. in a buffer containing 50 mM Tris-HCI (pH 7.0), 100 mM NaCl, and 10% glycerol. The co-substrates 3-[2-(dimethylamino)ethyl]-1H-indol-4-yl propionate (Indole Shop; www.theindoleshop.com) and DMAPP (www.sigmaaldrich.com) were used in the reaction. Briefly, reactions were set up as follows: 50 mM Tris-HCI (pH 8.0), 180 .Math.M DMAPP, 0.5 mM 3-[2-(dimethylamino)ethyl]-1H-indol-4-yl propionate and 392 .Math.g/mL of PriB were added together and the reaction proceeded at 37° C. for 2 hours. Equal volume of MeOH was added to quench the reaction and precipitate the protein. The sample was then centrifuged at 13,000 g for 20 minutes, allowing removal of the supernatant which contained the desired product. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of 3-[2-(dimethylamino)ethyl]-6-(3-methyl-2-butenyl)-1H-indol-4-yl propionate, having chemical formula (XLI):
##STR00356##
eluted at 4.7 minutes (EIC, see:
Example 39 - Biosynthesis of a Thirty-Ninth Multi-Substituent Psilocybin Derivative
[1010] Synthesis of a psilocybin derivative was accomplished using PriB enzyme and the in vitro procedure described in Example 38, with the exception that 3-[2-(dimethylamino)ethyl]-1H-indol-4-yl acetate (Indole Shop; www.theindoleshop.com) was used in place of 3-[2-(dimethylamino)ethyl]-1H-indol-4-yl propionate substrate. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of 3-[2-(dimethylamino)ethyl]-6-(3-methyl-2-butenyl)-1H-indol-4-yl acetate, having chemical formula (XLII):
##STR00357##
eluted at 4.4 minutes (EIC, see:
Example 40 - Biosynthesis of a Fortieth Multi-Substituent Psilocybin Derivative
[1011] Synthesis of a psilocybin derivative was accomplished using PriB enzyme and the in vitro procedure described in Example 38, with the exception that 3-[2-(diethylamino)ethyl]-1H-indol-4-yl acetate (Indole Shop; www.theindoleshop.com) was used in place of 3-[2-(dimethylamino)ethyl]-1H-indol-4-yl propionate substrate. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of 3-[2-(diethylamino)ethyl]-6-(3-methyl-2-butenyl)-1H-indol-4-yl acetate, having chemical formula (XLV):
##STR00358##
eluted at 4.6 minutes (EIC, see:
Example 41 - Synthesis of a Forty-First Multi-Substituent Psilocybin Derivative
[1012] Synthesis of a psilocybin derivative was accomplished using PriB enzyme and the in vitro procedure described in Example 38, with the exception that N,N-dimethyl[2-(5-chloro-1H-indol-3-yl)ethyl]amine (Indole Shop; www.theindoleshop.com) was used in place of 3-[2-(dimethylamino)ethyl]-1H-indol-4-yl propionate substrate. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of N,N-dimethyl(2-[5-chloro-6-(3-methyl-2-butenyl)-1H-indol-3-yl]ethyl)amine, having chemical formula (XLIV):
##STR00359##
eluted at 4.7 minutes (EIC, see:
TABLE-US-00066 m/z % Relative abundance Ionic species Δ ppm 246.10398 100 [M + H — C.sub.6H.sub.9N].sup.+ 1.71 61.09263 2.0 199.41002 1.5 181.73308 1.4 65.66295 1.2 116.30919 1.2 56.98338 1.1 80.77037 1.1 164.10424 0.9 151.38086 0.9
Example 42 - Biosynthesis of a Forty-Second Multi-Substituent Psilocybin Derivative
[1013] Synthesis of a psilocybin derivative was accomplished using (1) chemical synthesis, followed by (2) an in vitro enzymatic conversion by PriB enzyme. Chemical synthesis was conducted using the synthesis procedure shown in
[1014] To a solution of 4-Benzyloxyindole 13D-1 (1.00 mmol, 1.00 eq) in anhydrous diethyl ether (10 mL) under argon sparging at 0° C., was added oxalyl chloride (2.05 mmol, 2.05 eq) dropwise over the course of 30 minutes, and the reaction was continued at 0 – 5° C. for 3 hours. A solution of N-methylisopropylamine (5.00 mmol, 5.00 eq) in anhydrous diethyl ether (5 mL) was added dropwise over the course of 1 hour. The solution was concentrated in vacuo, and the residue was redissolved in dichloromethane (30 mL). The organic solution was washed with water (4 × 10 mL) and brine (1 × 10 mL), then dried over anhydrous Na.sub.2SO.sub.4 and concentrated under reduced pressure to yield compound 13D-2, which was used in the following step without further purification. A solution of lithium aluminum hydride in a mixture of anhydrous THF (1 M, 5.20 eq) and 1,4-dioxane (2.0 mL) was brought to 60° C. under argon. A solution of compound 13D-2 in a mixture of anhydrous THF (2.0 mL) and 1,4-dioxane (3.5 mL) was added dropwise over 30 minutes, and the reaction was brought to 70° C. for 2 hours. The reaction was further refluxed at 95° C. for 20 hours. After cooling to 0° C., excess lithium aluminum hydride was quenched through a dropwise addition of a mixture of water (0.4 mL) – THF (2.0 mL). Diethyl ether (10 mL) was added, and the reaction mixture was allowed to stir at room temperature for 30 minutes. The precipitate was removed via vacuum filtration, the filtrate was dried over anhydrous Na.sub.2SO.sub.4, and concentrated under vacuo to yield compound 3 which was used without further purification. The crude compound 13D-3 was dissolved in 95% EtOH (10 mL), and 10% palladium on activated charcoal (0.110 eq) was added. The reaction flask was evacuated then backfilled with hydrogen. After stirring at room temperature for 2 hours, the catalyst was removed by a filtration and solvent was removed under reduced pressure to yield compound 13D-4, which was purified by a reverse-phase column chromatography on C18 silica gel using a water – acetonitrile + 0.1% formic acid as the eluent. Compound 13D-4 was then used as a substrate for bioconversion by PriB enzyme, the latter which was generated and purified using the procedure described in Example 38. The in vitro conversion by PriB was conducted using the same procedure described in Example 38, with the exception that compound 13D-4 was used in place of 3-[2-(dimethylamino)ethyl]-1H-indol-4-yl propionate substrate. Analysis was carried out using high-resolution LC-HESI-LTQ-Orbitrap-XL MS (Thermo Fisher Scientific) as described in Example 3. Singly protonated product with exact m/z and expected elemental formula matching the singly protonated form of 3-(2-[(isopropyl)-N-methylamino]ethyl)-6-(3-methyl-2-butenyl)-1H-indol-4-ol, having chemical formula (LXXVI):
##STR00360##
eluted at 4.2 minutes (EIC, see: