Polymerizable compound, compound, and method for producing boranophosphate oligomer
11542292 · 2023-01-03
Assignee
Inventors
Cpc classification
Y02P20/55
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
C07H1/00
CHEMISTRY; METALLURGY
C07H19/073
CHEMISTRY; METALLURGY
C07F9/6558
CHEMISTRY; METALLURGY
C12N15/113
CHEMISTRY; METALLURGY
C07H23/00
CHEMISTRY; METALLURGY
C07H21/00
CHEMISTRY; METALLURGY
C07F9/6584
CHEMISTRY; METALLURGY
International classification
C07H19/073
CHEMISTRY; METALLURGY
C07H19/173
CHEMISTRY; METALLURGY
C07H21/00
CHEMISTRY; METALLURGY
C07F9/6584
CHEMISTRY; METALLURGY
C07F9/6558
CHEMISTRY; METALLURGY
C12N15/113
CHEMISTRY; METALLURGY
Abstract
Provided a polymerizable compound represented by the following Formula A-1 or Formula A-2: In Formula A-1 or Formula A-2, R.sup.1 represents an electron-donating group; n represents an integer from 1 to 5; R.sup.2 represents a hydrogen atom, a halogen atom, or —OR.sup.O, wherein R.sup.O represents a hydrogen atom, an alkyl group, or a protecting group of a hydroxy group; R.sup.3 represents a hydrogen atom or a protecting group of a hydroxy group; and X represents a structure represented by any one of Formula B-1 to Formula B-5.
Claims
1. A polymerizable compound represented by the following Formula A-1 or Formula A-2: ##STR00029## ##STR00030## wherein, in Formula A-1 or Formula A-2, R.sup.1 represents an electron-donating group selected from the group consisting of an alkoxy group, —NR.sup.N.sub.2, a hydroxyl group, an aryl group, and an alkyl group, wherein each R.sup.N independently represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms; n represents an integer from 1 to 5; R.sup.2 represents a hydrogen atom, a halogen atom, or —OR.sup.O, wherein R.sup.O represents a hydrogen atom, an alkyl group, or a protecting group of a hydroxy group; R.sup.3 represents a hydrogen atom or a protecting group of a hydroxy group; and X represents a structure represented by any one of Formula B-1 to Formula B-5, and wherein, in Formula B-1 to Formula B-5, R.sup.T represents a hydrogen atom, an alkyl group, an alkenyl group, or an alkynyl group; each of R.sup.pC, R.sup.pA, and R.sup.pG represents a protecting group that is removed under an acidic condition; R.sup.pC2 represents a hydrogen atom or an alkyl group; R.sup.pG2 represents a protecting group; R.sup.pG3 represents a protecting group that is removed under an acidic condition, or a hydrogen atom; and represents a binding site to a carbon atom at a 1′-position of an adjacent pentose sugar.
2. A compound comprising a structural unit represented by the following Formula T-1 and a structural unit represented by the following Formula D-1 or Formula D-2: ##STR00031## ##STR00032## wherein, in Formula T-1, R.sup.2 represents a hydrogen atom, a halogen atom, or —OR.sup.O, wherein R.sup.O represents a hydrogen atom, an alkyl group, or a protecting group of a hydroxy group; Z represents a structure represented by any one of Formula B-6 to Formula B-9; and each of * and ** represents a binding site with another structure, wherein, in Formula D-1 or Formula D-2, R.sup.1 represents an electron-donating group selected from the group consisting of an alkoxy group, —NR.sup.N.sub.2, a hydroxyl group, an aryl group, and an alkyl group, wherein each R.sup.N independently represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms; n represents an integer from 1 to 5; R.sup.2 represents a hydrogen atom, a halogen atom, or —OR.sup.O, wherein R.sup.O represents a hydrogen atom, an alkyl group, or a protecting group of a hydroxy group; R.sup.3 represents a hydrogen atom or a protecting group of a hydroxy group; X represents a structure represented by any one of Formula B-1 to Formula B-5; TfO represents a triflate anion; and ● represents a binding site with another structure, wherein, in Formula B-1 to Formula B-5, R.sup.T represents a hydrogen atom, an alkyl group, an alkenyl group, or an alkynyl group; each of R.sup.pC, R.sup.pA, and R.sup.pG represents a protecting group that is removed under an acidic condition; R.sup.pC2 represents a hydrogen atom or an alkyl group; R.sup.pG2 represents a protecting group; R.sup.pG3 represents a protecting group that is removed under an acidic condition, or a hydrogen atom; and represents a binding site to another structure, and wherein, in Formula B-6 to Formula B-9, R.sup.T represents a hydrogen atom, an alkyl group, an alkenyl group, or an alkynyl group; each of R.sup.C, R.sup.A, and R.sup.G represents a hydrogen atom; R.sup.C2 represents a hydrogen atom or an alkyl group; and
represents a binding site to a carbon atom at a 1′-position of an adjacent pentose sugar.
3. The compound according to claim 2, further comprising one or both structural units represented by the following Formula C-1 or Formula C-2: ##STR00033## wherein, in Formula C-1 or Formula C-2, R.sup.2 represents a hydrogen atom, a halogen atom, or —OR.sup.O, wherein R.sup.O represents a hydrogen atom, an alkyl group, or a protecting group of a hydroxy group; Z represents a structure represented by any one of Formula B-6 to Formula B-9; and each of ** and ● represents a binding site with another structure, and wherein, in Formula B-6 to Formula B-9, R.sup.T represents a hydrogen atom, an alkyl group, an alkenyl group, or an alkynyl group; each of R.sup.C, R.sup.A, and R.sup.G represents a hydrogen atom; R.sup.C2 represents a hydrogen atom or an alkyl group; and represents a binding site to a carbon atom at a 1′-position of an adjacent pentose sugar.
4. A method of producing a boranophosphate oligomer, comprising condensing the polymerizable compound according to claim 1.
Description
BRIEF DESCRIPTION OF DRAWINGS
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
DESCRIPTION OF EMBODIMENTS
(10) Hereinafter, the present disclosure will be described in detail.
(11) In addition, the description “xx to yy” herein represents a numerical value including xx and yy.
(12) In addition, in the present disclosure, “% by mass” and “% by weight” are synonymous and “parts by mass” and “part by weight” are synonymous.
(13) In addition, in the present disclosure, a combination of two or more preferred embodiments is a more preferred embodiment.
(14) In the present disclosure, regarding a representation of a group in a compound represented by a formula, when “substituted or unsubstituted” is not described and the group can further have a substituent, not only an unsubstituted group but also a substituted group is included, unless otherwise specified. For example, in the formula, when it is described that “R represents an alkyl group”, it means that “R represents an unsubstituted alkyl group or a substituted alkyl group”.
(15) The word “step” herein is included in the term, not only in the case where the step is an independent step, but also even in the case where the step cannot be clearly distinguished from other steps, when an intended purpose of the step is achieved.
(16) Hereinafter, the present disclosure will be described in detail.
(17) (Polymerizable Compound)
(18) The polymerizable compound according to the present disclosure is a compound represented by the following Formula A-1 or Formula A-2.
(19) It is preferred that the polymerizable compound according to the present disclosure is a polymerizable compound for forming a boranophosphate oligomer.
(20) ##STR00006## ##STR00007##
(21) In Formula A-1 or Formula A-2, R.sup.1 represents an electron-donating group; n represents an integer of 1 to 5; R.sup.2 represents a hydrogen atom, a halogen atom, or —OR.sup.O; R.sup.O represents a hydrogen atom, an alkyl group, or a protecting group of a hydroxy group; R.sup.3 represents a hydrogen atom or a protecting group of a hydroxy group; and X represents a structure represented by any one of Formula B-1 to Formula B-5.
(22) In Formula B-1 to Formula B-5, R.sup.T represents a hydrogen atom, an alkyl group, an alkenyl group, or an alkynyl group; each of R.sup.pC, R.sup.pA, and R.sup.pG represents a protecting group that is removed under acidic conditions; R.sup.pC2 represents an alkyl group; R.sup.pG2 represents a protecting group; R.sup.pG3 represents a protecting group that is removed under an acidic condition or a hydrogen atom; and a wavy line () represents a binding site to another structure.
(23) In Formula A-1 or Formula A-2, R.sup.1 represents an electron-donating group, and preferably an alkoxy group, —NR.sup.N.sub.2, a hydroxy group, an aryl group, or an alkyl group, more preferably an alkoxy group, still more preferably an alkoxy group having 1 to 4 carbon atoms, and particularly preferably a methoxy group.
(24) R.sup.N each independently represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms.
(25) In Formula A-1 or Formula A-2, n represents an integer of 1 to 5, preferably an integer of 1 to 3, and more preferably 1.
(26) When n is 2 or more, a plurality of R.sup.1 may be identical or different from each other.
(27) In Formula A-1 or Formula A-2, R.sup.2 represents a hydrogen atom, a halogen atom, or —OR.sup.O.
(28) Examples of the halogen atom of R.sup.2 include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom, and a fluorine atom is preferred.
(29) R.sup.O represents a hydrogen atom, an alkyl group, or a protecting group of a hydroxy group, and as a protecting group of a hydroxy group, for example, it is possible to use a protecting group conventionally known as a protecting group used for protecting the hydroxy group at 2-position of a ribose structure commonly used in the synthesis of RNA or its derivative, reference can be also made to protecting groups described in publications such as Green, et al., Protective Groups in Organic Synthesis, 3rd Edition, 1999, John Wiley & Sons, Inc., and examples thereof include an acetyl group, a phenoxyacetyl group, a pivaloyl group, a benzyl group, a 4-methoxybenzyl group, a benzoyl group, a triphenylmethyl group, a 4,4′-dimethoxytrityl (DMTr) group, a 4-methoxytrityl (MMTr) group, a 9-phenylxanthenyl group, a trimethylsilyl group, a cyanomethoxymethyl group, a 2-(cyanoethoxy)ethyl group, and a cyanoethoxymethyl group, and preferably a 4,4′-dimethoxytrityl (DMTr) group.
(30) R.sup.3 represents a hydrogen atom or a protecting group of a hydroxy group, and as a protecting group of a hydroxy group, reference can be made to protecting groups described in publications such as Green, et al., Protective Groups in Organic Synthesis, 3rd Edition, 1999, John Wiley & Sons, Inc., and examples thereof include an acetyl group, a phenoxyacetyl group, a pivaloyl group, a benzyl group, a 4-methoxybenzyl group, a benzoyl group, a triphenylmethyl group, a 4,4′-dimethoxytrityl (DMTr) group, a 4-methoxytrityl (MMTr) group, a 9-phenylxanthenyl group, a trimethylsilyl group, a cyanomethoxymethyl group, a 2-(cyanoethoxy)ethyl group, and a cyanoethoxymethyl group, and preferably a 4,4′-dimethoxytrityl (DMTr) group.
(31) In Formula A-1 or Formula A-2, X represents a structure represented by any one of Formula B-1 to Formula B-5, Formula B-1 corresponds to a thymine structure or uracil structure, Formula B-2 corresponds to a cytosine structure, Formula B-3 corresponds to an adenine structure, and Formula B-4 and Formula B-5 correspond to a guanine structure, respectively.
(32) In addition, the thymine structure, the uracil structure, the cytosine structure, the adenine structure, and the guanine structure include each structure having a substituent.
(33) In Formula B-1 to Formula B-5, it is preferred that R.sup.T represents a hydrogen atom, an alkyl group, an alkenyl group, or an alkynyl group, and a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an alkenyl group having 2 to 4 carbon atoms, or an alkynyl group having 2 to 4 carbon atoms is preferred.
(34) Each of R.sup.pC, R.sup.pA, and R.sup.pG represent a protecting group that is removed under acidic conditions, reference can be made to protecting groups described in publications such as Green, et al., Protective Groups in Organic Synthesis, 3rd Edition, 1999, John Wiley & Sons, Inc., for example, a tert-butoxycarbonyl group, a benzyloxycarbonyl group, a 4,4′-trimethoxytrityl (TMTr) group, a 4,4′-dimethoxytrityl (DMTr) group, a 4-methoxytrityl (MMTr) group, or 4-methoxybenzyloxycarbonyl (MCBz) group is preferred, and from a viewpoint of reducing steric hindrance and improving reactivity of a monomer, 4-methoxybenzyloxycarbonyl (MCBz) group is more preferred.
(35) R.sup.pC2 represents a hydrogen atom or an alkyl group, preferably an alkyl group having 1 to 4 carbon atoms, and more preferably a methyl group.
(36) R.sup.pG2 represents a protecting group, and as the protecting group, for example, it is possible to use a known protecting group of a hydroxy group without limitation, reference can be also made to protecting groups described in publications such as Green, et al., Protective Groups in Organic Synthesis, 3rd Edition, 1999, John Wiley & Sons, Inc., and examples thereof include an acetyl group, a phenoxyacetyl group, a pivaloyl group, a benzyl group, a 4-methoxybenzyl group, a benzoyl group, a triphenylmethyl group, a 4,4′-dimethoxytrityl (DMTr) group, a 4-methoxytrityl (MMTr) group, a 9-phenylxanthenyl group, a trimethylsilyl group, a trimethylsilylethyl group, a cyanomethoxymethyl group, a 2-(cyanoethoxy)ethyl group, and a cyanoethoxymethyl group, and preferably a trimethylsilylethyl group.
(37) In addition, as R.sup.pG2, a protecting group that is removed under an acidic condition is preferred.
(38) R.sup.pG3 represents a protecting group or a hydrogen atom that is removed under acidic conditions in R.sup.pG described above, and examples of the protecting group include the same protecting group as those removed under an acidic condition, and the same applies to a preferred embodiment thereof.
(39) <Method of Producing a Polymerizable Compound>
(40) Hereinafter, an example of the method of producing a polymerizable compound according to the present disclosure will be described. However, the present disclosure is not limited thereto.
(41) The polymerizable compound according to the present disclosure can be synthesized, for example, according to the following Scheme 1.
(42) In the following Scheme 1, Et.sub.3N is triethylamine.
(43) In the following Scheme 1, (Rp)or(Sp)-20a-d is a compound represented by Formula A-1 or Formula A-2.
(44) ##STR00008##
(45) In Scheme 1, R.sup.1, n, R.sup.2, R.sup.3, and X have the same meaning as R.sup.1, n, R.sup.2, R.sup.3 and X in Formula A-1 or Formula A-2, respectively, and the same applies to a preferred embodiment thereof.
(46) Compound 9a-d in Scheme 1 can be synthesized by using for example, thymidine, uridine, cytidine, adenosine, or guanosine as a raw material.
(47) Compound (4S,5R)-18 in Scheme 1 can be synthesized, for example, according to the following Scheme 2.
(48) In the following Scheme 2, Me is a methyl group, MMTrCl is 4-methoxytrityl chloride, Et.sub.3N is triethylamine, SO.sub.3—Py is a pyridine-sulfur trioxide complex, DCA is dichloroacetic acid, and PCl.sub.3 is phosphorus trichloride.
(49) In addition, though the following Scheme 2 uses L-proline as a starting material, compound (4R,5S)-18 can be synthesized by performing similar synthesis using D-proline as a starting material.
(50) ##STR00009## ##STR00010##
(Method of Producing Boranophosphate Oligomer)
(51) A method of producing a boranophosphate oligomer according to the present disclosure is a method of producing a boranophosphate oligomer, the method including a step of condensing a polymerizable compound according to the present disclosure (hereinafter, also referred to as “condensation step”).
(52) Hereinafter, an example of the method of producing a boranophosphate oligomer according to the present disclosure will be described. However, the present disclosure is not limited thereto.
(53) <Condensation Step>
(54) A condensation step in the present disclosure follows, for example, the following Scheme 3.
(55) ##STR00011##
(56) In Scheme 3, R.sup.1, n, R.sup.2, R.sup.3, and X have the same meaning as R.sup.1, n, R.sup.2, R.sup.3, and X in Formula A-1 or Formula A-2, respectively, and the same applies to a preferred embodiment thereof.
(57) In addition, in Scheme 3, Z represents a structure represented by any one of Formula B-6 to Formula B-9 described later.
(58) In Scheme 3, deprotection of R.sup.3 is performed in Compound 27 loaded on a solid support. The deprotection of R.sup.3 is performed, for example, under acidic conditions.
(59) Subsequently, an oligomer chain is extended by repeating a condensation reaction and deprotection of an asymmetric auxiliary group, a protecting group of a base, and R.sup.3, using the polymerizable compound and an activator. Deprotection of the asymmetric auxiliary group and the protecting group of a base is performed under an acidic condition. By using a protecting group that is removed under acidic conditions as R.sup.3, it is possible to perform deprotection of R.sup.3 simultaneously with the deprotection of the asymmetric auxiliary group and the protecting group of a base.
(60) In Scheme 3, though the polymerizable compound 20a-d and compound 21 (CMPT, N-(cyanomethyl)pyrrolidinium triflate) which is an activator are used, the activator is not limited thereto and can be changed.
(61) After H-phosphonate oligomer 23 is formed, boronation is performed, thereby boranophosphate oligomer 24a-d is obtained.
(62) In Scheme 3, though a synthesis method by a solid phase method is shown, it is possible to perform synthesis by a liquid phase method by the same scheme.
(63) From a viewpoint of stereoselectivity and reaction rate, it is preferred to perform synthesis by a solid phase method.
(64) In Scheme 3, details of an extension reaction of an oligomer chain are shown in the following Scheme 4.
(65) ##STR00012##
(66) In Scheme 4, R.sup.1, n, R.sup.2, R.sup.3, and X each independently have the same meaning as R.sup.1, n, R.sup.2, R.sup.3, and X in Formula A-1 or Formula A-2, and the same applies to a preferred embodiment thereof.
(67) n represents an integer of 0 to 100, preferably an integer of 1 to 100, more preferably an integer of 9 to 100, and still more preferably an integer of 11 to 100.
(68) In Scheme 4, TfO (OTf) represents a triflate anion, and Z represents a structure represented by any one of Formula B-6 to Formula B-9 described later.
(69) In Scheme 4, (Rp) or (Sp)-20a-d which is the polymerizable compound according to the present disclosure is bonded to a hydroxy group at 5′-position of a sugar structure at the end of H-phosphonate substituted nucleotide in the presence of an activator 21 and forms an intermediate 28. Thereafter, an asymmetric auxiliary group from the intermediate 28, a protecting group of a base, and R.sup.3 are deprotected to form an oligomer 29. Further, (Rp) or (Sp)-20a-d is bonded to a hydroxy group at 5′-position of a sugar structure at the end of the oligomer 29. This is repeated to extend the oligomer chain.
(70) In Scheme 4, though for convenience, all configurations in which a phosphorus atom contained in the intermediate 28 or the oligomer 29 is an asymmetric point is described as an S configuration (Sp isomer), a compound represented by Formula A-1 and a compound represented by Formula A-2 are properly used as a monomer, whereby an H-phosphonate structure of a S configuration (Sp isomer) and an H-phosphonate structure of a R configuration (Rp isomer) can be introduced at any position.
(71) That is, the intermediate 28 is a compound including a structural unit represented by the following Formula T-1 and a structural unit represented by the following Formula D-1 or D-2.
(72) ##STR00013## ##STR00014##
(73) In Formula T-1, R.sup.2 represents a hydrogen atom, a halogen atom, or —OR.sup.O; R.sup.O represents a hydrogen atom, an alkyl group, or a protecting group of a hydroxy group; Z represents a structure represented by any one of Formula B-6 to Formula B-9; and each of * and ** represents a binding site with another structure.
(74) In Formula D-1 or Formula D-2, R.sup.1 represents an electron-donating group; n represents an integer of 1 to 5; R.sup.2 represents a hydrogen atom, a halogen atom, or —OR.sup.O; R.sup.O represents a hydrogen atom, an alkyl group, or a protecting group of a hydroxy group; R.sup.3 represents a hydrogen atom or a protecting group of a hydroxy group; X represents a structure represented by any one of Formula B-1 to Formula B-5; TfO represents a triflate anion; and a black circle (.circle-solid.) represents a binding site with another structure.
(75) In Formula B-1 to Formula B-5, R.sup.T represents a hydrogen atom, an alkyl group, an alkenyl group, or an alkynyl group; each of R.sup.pC, R.sup.pA, and R.sup.pG represents a protecting group that is removed under acidic conditions; R.sup.pC2 represents a hydrogen atom or an alkyl group; R.sup.pG2 represents a protecting group; R.sup.pG3 represents a protecting group that is removed under an acidic condition or a hydrogen atom; and a wavy line () represents a binding site to another structure.
(76) In Formula B-6 to Formula B-9, R.sup.T represents a hydrogen atom, an alkyl group, an alkenyl group, or an alkynyl group; R.sup.C, R.sup.A, and R.sup.G represent a hydrogen atom; R.sup.C2 represents a hydrogen atom or an alkyl group; and a wavy line represents a binding site to another structure.
(77) In Formula T-1, R.sup.2 has the same meaning as R.sup.2 in Formula A-1 or Formula A-2, and the same applies to a preferred embodiment thereof.
(78) In Formula T-1, * represents a binding site to another structure, and when the synthesis is performed by solid phase synthesis, it is preferred that * represents a binding site to a support.
(79) In Formula T-1, Z is preferably a structure represented by any one of Formula B-6 to Formula B-9, and more preferably any one of a thymine structure, a uracil structure, a cytosine structure, an adenine structure, or a guanine structure.
(80) In Formula D-1 and Formula D-2, R.sup.1, n, R.sup.2, R.sup.3, and X have the same meaning as R.sup.1, n, R.sup.2, R.sup.3, and X in Formula A-1 or Formula A-2, and the same applies to a preferred embodiment thereof.
(81) Formula B-1 to Formula B-5 have the same meaning as Formula B-1 to Formula B-5 in Formula A-1 or Formula A-2 described above, and the same applies to a preferred embodiment thereof.
(82) According to a method of producing a boranophosphate oligomer according to the present disclosure, both boranophosphate DNA and boranophosphate RNA can be produced, depending on the selection of a polymerizable compound used in the condensation step.
(83) In the present disclosure, boranophosphate DNA refers to DNA in which one of non-bridging oxygen atoms in a phosphodiester structure of natural DNA is substituted by a borano group (—BH.sub.3).
(84) In the present disclosure, boranophosphate RNA refers to RNA in which one of non-bridging oxygen atoms in a phosphodiester structure of natural RNA is substituted by a borano group (—BH.sub.3).
(85) In any one of the DNA and RNA, it is possible to select the polymerizable compound according to the embodiment so that a base sequence is complementary to a base sequence of a target nucleic acid and perform synthesis.
(86) In addition, the compound may further include any one or both of structural units represented by the following Formula C-1 or Formula C-2.
(87) The structural unit represented by the following Formula C-1 or Formula C-2 is a structural unit which is increased by one by one cycle of a condensation cycle described in Scheme 4 described above, and may exist in plural.
(88) The total number of the structural units represented by Formula C-1 or Formula C-2 included in the compound is preferably 9 to 100, and more preferably 11 to 50.
(89) ##STR00015##
(90) In Formula C-1 or Formula C-2, R.sup.2 represents a hydrogen atom or —OR.sup.O; R.sup.O represents a hydrogen atom, an alkyl group, or a protecting group of a hydroxy group; Z represents a structure represented by any one of Formula B-6 to Formula B-9; and ** and a black circle (.circle-solid.) represent a binding site with another structure.
(91) In Formula B-6 to Formula B-9, R.sup.T represents a hydrogen atom, an alkyl group, an alkenyl group, or an alkynyl group; R.sup.C, R.sup.A, and R.sup.G represent a hydrogen atom; R.sup.C2 represents a hydrogen atom or an alkyl group; and a wavy line () represents a binding site to another structure.
(92) In Formula C-1 and Formula C-2, Z and R.sup.2 have the same meaning as Z and R.sup.2 in Formula T-1, respectively, and the same applies to a preferred embodiment thereof.
(93) When a structure represented by Formula C-1 or Formula C-2 exists in plural, a plurality of Z and R.sup.2 in Formula C-1 or Formula C-2 may be the same or different.
(94) Formula B-6 to Formula B-9 have the same meaning as Formula B-6 to Formula B-9 in Formula T-1 described above, and the same applies to a preferred embodiment thereof.
(95) In addition, the compound may further include a structural unit derived from at least one selected from the group consisting of the polymerizable compound represented by the following Formula E-1 to Formula E-4.
(96) In the condensation step of the present disclosure, at least one polymerizable compound selected from the group consisting of the polymerizable compound represented by Formula E-1 to Formula E-4 may be further used as other polymerizable compounds.
(97) That is, the boranophosphate oligomer produced by the method of producing a boranophosphate oligomer according to the present disclosure may be boranophosphate LNA.
(98) In the present disclosure, LNA refers to an RNA derivative having a structure in which an oxygen atom at 2′-position and a carbon atom at 4′-position of a ribose ring are crosslinked via methylene, and boranophosphate LNA refers to LNA in which one of non-bridging oxygen atoms in a phosphodiester structure of LNA is substituted by a borano group (—BH.sub.3).
(99) ##STR00016##
(100) In Formula E-1 to Formula E-4, R.sup.1, n, R.sup.3, and X have the same meaning as R.sup.1, n, R.sup.3, and X in Formula A-1 or Formula A-2, and the same applies to a preferred embodiment thereof.
(101) In Formula E-3 and Formula E-4, R.sup.4 represents a protecting group of a hydroxy group, and any of those exemplified as the protecting group of a hydroxy group in R.sup.3 described above can be used without limitation.
(102) R.sup.5 represents a hydrogen atom, a hydroxy group having a protecting group, a halogen atom, or an alkoxy group.
(103) When the polymerizable compound represented by Formula E-1 or Formula E-2 is used in the method of synthesizing of a boranophosphate oligomer according to the present disclosure, it is possible to perform synthesis by the same method as the case where the polymerizable compound represented by Formula A-1 or Formula A-2 is used.
(104) In addition, when a polymerizable compound represented by Formula E-3 or Formula E-4 is used in the method of synthesizing a boranophosphate oligomer according to the present disclosure, it is possible to perform synthesis by a known method of synthesizing DNA or RNA, using a known condensation reagent.
(105) By using the polymerizable compound represented by Formula E-3 or Formula E-4, it is possible to synthesize a boranophosphate oligomer partially including a phosphodiester structure.
(106) <Purification Step>
(107) The method of producing a boranophosphate oligomer according to the present disclosure may further include a step of producing a boranophosphate oligomer (purification step).
(108) In a purification step, the boranophosphate oligomer is purified by a known purification method such as reverse phase high performance liquid chromatography (reverse phase HPLC), ion exchange HPLC, column chromatography, or recrystallization.
(109) In the method of producing a boranophosphate oligomer according to the present disclosure is preferably a method of producing a boranophosphate oligomer of a 10-mer to a 100-mer, more preferably a method of producing a boranophosphate oligomer of a 10-mer to a 50-mer, and still more preferably a method of producing a boranophosphate oligomer of a 12-mer to a 50-mer.
(110) According to the method of producing a boranophosphate oligomer according to the present disclosure, it is possible to produce a boranophosphate oligomer in which the Sp isomer and the Rp isomer described above are freely combined.
(111) For example, by forming only both ends of the boranophosphate oligomer as an Rp isomer and the other structure as a Sp isomer, it is possible to produce a boranophosphate oligomer having improved enzyme resistance.
(112) The boranophosphate oligomer obtained by the method of producing a boranophosphate oligomer according to the present disclosure can be used as an antisense molecule having excellent duplex formation ability to a target nucleic acid, by designing the boranophosphate oligomer to be complementary to a base sequence of a target nucleic acid.
(113) For example, when the target nucleic acid corresponds to a partial sequence of a disease-associated gene, the boranophosphate oligomer is preferably used in a medical use such as an antisense drug having high translation inhibition ability.
(114) Furthermore, application to boron neutron capture therapy (BNCT) can be also considered.
EXAMPLES
(115) Hereinafter, the present disclosure will be described in detail by way of the Examples, but the present disclosure is not limited thereto.
(116) In addition, in the following description, “%” is based on mass, unless otherwise specified.
(117) Details of analysis equipment used in the Examples are as follows.
(118) .sup.1H-nuclear magnetic resonance spectrum (.sup.1H-NMR): JNM-LA 400 (400 MHz)
(119) .sup.31P-nuclear magnetic resonance spectrum (.sup.31P-NMR): JNM-LA 400 (161.8 MHz)
(120) In addition, in .sup.1H-NMR, tetramethylsilane (TMS) is used as an internal standard, and in .sup.31P-NMR, 85% H.sub.3PO.sub.4 is used as an external standard.
(121) ESI-MS: Varian 910-MS
(122) Details of terms used in the Examples are as follows.
(123) MMTr=4-methoxytrityl
(124) DMTr=4,4′-dimethoxytrityl
(125) TBS=tert-butyldimethylsilyl
(126) TMS=trimethylsilyl
(127) TFA=trifluoroacetic acid
(128) DCA=dichloroacetic acid
(129) MCbz=4-methoxybenzyloxycarbonyl
(130) Tse=trimethylsilylethyl
(131) CDI=1,1′-carbonyldiimidazole
(132) HMDS=hexamethyldisilazane
(133) DEAD=diethylazodicarboxylate
(134) CMPT=N-(cyanomethyl)pyrrolidinium tri fluoromethanesulfonate
(135) DMAc=N,N-dimethylacetamide
(136) BSA=N,O-bis(trimethylsilyl)acetamide
(137) Th (T)=thymine
(138) Cy (C)=cytosine
(139) Ad (A)=adenine
(140) Gu (G)=guanine
EXAMPLES AND COMPARATIVE EXAMPLES
(141) <Synthesis of Polymerizable Compound>
(142) [Synthesis of (2S)-5]L-proline (1) (11.51 g, 100 mmol) was dissolved in MeOH (100 mL) and cooled to 0° C. To the solution, SOCl.sub.2 (14.42 mL, 200 mmol) was slowly added with a dropping funnel. The reaction mixture was stirred at room temperature for 3 hours, and the solvent was removed under reduced pressure to obtain (2S)-2 as a crude product. (2S)-2 was used for the next step, without further purification.
(143) The crude product (2S)-2 was repeatedly azeotropically dried with toluene and CHCl.sub.3, and dissolved in CH.sub.2Cl.sub.2 (250 mL), and Et.sub.3N (55.75 mL, 400 mmol) and MMTrCl (40.14 g, 130 mmol) were added thereto. The reaction mixture was stirred at room temperature for 17 hours, and a saturated aqueous NH.sub.4Cl solution-concentrated aqueous NH.sub.3 solution (2:1, v/v) (150 mL) was added thereto. The organic layer was separated and washed with a saturated aqueous NaHCO.sub.3 solution (3×100 mL), and the collected washing liquid was extracted with CH.sub.2Cl.sub.2 (100 mL). The collected organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure to obtain (2S)-3 as a crude product. (2S)-3 was used for the next step, without further purification.
(144) The crude product (2S)-3 was repeatedly azeotropically dried with pyridine, toluene, and CHCl.sub.3, then dissolved in THF (100 mL). The solution was slowly added to a THF solution (100 mL) of LiAlH.sub.4 (4.93 g, 130 mmol) at 0° C. with a dropping funnel. The reaction mixture was stirred at room temperature for 12 hours and cooled to 0° C., and H.sub.2O (5 mL), a 15% aqueous NaOH solution (5 mL), and H.sub.2O (15 mL) were sequentially slowly added with a dropping funnel. The reaction mixture was stirred at room temperature for 30 minutes, anhydrous MgSO.sub.4 was added thereto, and the mixture was stirred for another 30 minutes. The suspension was filtered through Celite and washed with AcOEt (500 mL). The solution was concentrated under reduced pressure and to the residue CH.sub.2Cl.sub.2 (300 ml) was added. Washing was performed with a saturated aqueous NaHCO.sub.3 solution (3×100 mL), and the collected washing liquid was extracted with CH.sub.2Cl.sub.2 (100 mL). The collected organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure to give (2S)-4 as a crude product. (2S)-4 was used for the next step, without further purification.
(145) The crude product (2S)-4 was dissolved in CH.sub.2Cl.sub.2 (400 mL), and Et.sub.3N (83.18 mL, 600 mmol) was added thereto. To the solution, a DMSO solution (100 mL) of a pyridine-sulfur trioxide complex (47.75 g, 300 mmol) was added at 0° C. The reaction mixture was stirred at room temperature for 3 hours, and a saturated aqueous NaHCO.sub.3 solution (100 mL) was added thereto. The organic layer was separated and washed with saturated aqueous NaHCO.sub.3 solutions (3×100 mL), and the collected washing liquid was extracted with CH.sub.2Cl.sub.2 (100 mL). The collected organic layer was concentrated under reduced pressure, Et.sub.2O (300 mL) was added to the residue, and washing was performed with a saturated aqueous NaCl solution (5×100 mL). The collected organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography [neutral silica gel, hexane-AcOEt (6:1, v/v), pyridine 1%] to recover a fraction containing (2S)-5. The solvent was removed under reduced pressure to give (2S)-5 as a light yellow foamed material.
(146) .sup.1H NMR (400 MHz, CDCl.sub.3) δ9.84 (s, 1H), 7.55-7.43 (m, 6H), 7.28-7.14 (m, 6H), 6.82-6.78 (m, 2H), 3.78 (s, 3H), 3.29-3.23 (m, 1H), 2.93-2.87 (m, 1H), 1.64-1.55 (m, 2H), 1.47-1.37 (m, 1H), 1.19-1.10 (m, 1H), 0.90-0.77 (m, 1H). FAB-HRMS: Calcd. for [M+Na].sup.+; 394.1783. Found; 394.1788.
Synthesis of (2R)-5
(147) (2R)-5 was synthesized in the same manner as (2S)-5, using D-proline (1) as a starting material.
(148) .sup.1H NMR (300 MHz, CDCl.sub.3) δ9.83 (s, 1H), 7.56-7.43 (m, 6H), 7.28-7.12 (m, 6H), 6.84-6.78 (m, 2H), 3.77 (s, 3H), 3.30-3.21 (m, 1H), 2.94-2.85 (m, 1H), 1.65-1.55 (m, 2H), 1.49-1.37 (m, 1H), 1.20-1.08 (m, 1H), 0.90-0.75 (m, 1H). FAB-HRMS: Calcd. for [M+Na].sup.+; 394.1783. Found; 394.1784.
Synthesis of (αR,2S)-7
(149) (2S)-5 (26.00 g, 70 mmol) was repeatedly azeotropically dried with pyridine, toluene, and CHCl.sub.3, then dissolved in Et.sub.2O (300 mL) and cooled to −78° C. To the solution, a 0.5 M 4-methoxyphenyl magnesium bromide/THF solution (420 mL, 210 mmol) was slowly added with a dropping funnel. The reaction mixture was stirred at room temperature for 18 hours, and a saturated aqueous NH.sub.4Cl solution-concentrated aqueous NH.sub.3 solution (2:1, v/v) (300 mL) was added thereto at 0° C. The suspension was filtered through Celite and washed with Et.sub.2O (300 mL). The organic layer was separated and washed with saturated aqueous NaHCO.sub.3 solutions (3×100 mL), and the collected washing liquid was extracted with Et.sub.2O (100 mL). The collected organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure to give (αR,2S)-6 as a crude product. (αR,2S)-6 was used for the next step, without further purification.
(150) To the crude product (αR,2S)-6, a 3% DCA/CH.sub.2Cl.sub.2 solution (300 mL) was added. The reaction mixture was stirred at room temperature for 10 minutes, H.sub.2O (300 mL) was added thereto, the aqueous layer was separated, washed with CH.sub.2Cl.sub.2 (5×100 mL), and the collected washing liquid was extracted with water (100 mL). To the collected aqueous layer, a 5 M aqueous NaOH solution was added until the pH reached 11. CH.sub.2Cl.sub.2 (300 mL) was added thereto, the organic layer was separated and washed with a saturated aqueous NaHCO.sub.3 solution (3×100 mL), and the collected washing liquid was extracted with CH.sub.2Cl.sub.2 (100 mL). The collected organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure to give (αRS,2S)-7.
(151) To (αRS,2S)-7 (5.18 g, 25 mmol), trans-cinnamic acid (3.70 g, 25 mmol) was added and recrystallization was performed using EtOH. To the precipitated crystal, a 2M aqueous KOH solution (100 mL) and CH.sub.2Cl.sub.2 (100 mL) was added, and the organic layer was separated. Washing was performed with saturated aqueous NaHCO.sub.3 solutions (3×100 mL), and the collected washing liquid was extracted with CH.sub.2Cl.sub.2 (100 mL). The collected organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure to give (αR,2S)-7 as a light yellow oily material.
(152) .sup.1H NMR (400 MHz, CDCl.sub.3) δ7.30-7.27 (m, 2H), 6.89-6.86 (m, 2H), 4.68 (d, J=4.0 Hz, 1H), 3.80 (s, 3H), 3.42-3.37 (m, 1H), 3.05-2.99 (m, 1H), 2.96-2.91 (m, 1H), 2.43 (br, 2H), 1.79-1.62 (m, 3H), 1.55-1.47 (m, 1H). FAB-HRMS: Calcd. for [M+H].sup.+; 208.1338. Found; 208.1337.
Synthesis of (αS,2R)-7
(153) (αS,2R)-7 was synthesized in the same manner as (αR,2S)-7, using (2R)-5 as a starting material.
(154) .sup.1H NMR (400 MHz, CDCl.sub.3) 57.30-7.27 (m, 2H), 6.89-6.86 (m, 2H), 4.66 (d, J=4.0 Hz, 1H), 3.80 (s, 3H), 3.40-3.35 (m, 1H), 3.03-2.98 (m, 1H), 2.94-2.88 (m, 1H), 2.54 (br, 2H), 1.79-1.61 (m, 3H), 1.55-1.46 (m, 1H). FAB-HRMS: Calcd. for [M+H].sup.+; 208.1338. Found; 208.1338.
(155) ##STR00017## ##STR00018##
Synthesis of 5′-O-(DMTr)thymidine [9a]
(156) Synthesis was performed according to the method described in the literature, using thymidine (8) as a starting material. .sup.1H NMR spectrum was consistent with the literature value.
(157) ##STR00019##
Synthesis of 3′,5′-O-bis(TBS)-2′-deoxycytidine [14]
(158) Synthesis was performed according to the method described in the literature, using 2′-deoxycytidine (13) as a starting material. .sup.1H NMR spectrum was consistent with the literature value.
Synthesis of 3′,5′-O-bis(TBS)-N.SUP.4.-MCbz-2′-deoxycytidine [15]
(159) 3′,5′-O-bis(TBS)-2′-deoxycytidine (14) (4.55 g, 10 mmol) was repeatedly azeotropically dried with pyridine, toluene, and CHCl.sub.3, then dissolved in 1,2-dichloroethane (100 mL), CDI (2.60 g, 16 mmol) was added, and heated to reflux for 20 hours. 4-methoxybenzylalcohol (2.00 mL, 16 mmol) was added thereto, and the mixture was heated to reflux for further 18 hours. A saturated aqueous NaHCO.sub.3 solution (100 mL) was added thereto, and the organic layer was separated. Washing was performed with saturated aqueous NaHCO.sub.3 solutions (3×100 mL), and the collected washing liquid was extracted with CH.sub.2Cl.sub.2 (100 mL). The collected organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography [neutral silica gel, hexane-AcOEt (1:1, v/v)] to recover a fraction containing 15. The solvent was removed under reduced pressure to give 15 (4.64 g, 7.49 mmol, 75%) as a colorless foamed material.
(160) .sup.1H NMR (300 MHz, CDCl.sub.3) δ8.38 (d, 1H), 7.46 (br, 1H), 7.34 (d, 2H), 7.18 (d, 1H), 6.93 (d, 2H), 6.25 (t, 1H), 5.15 (s, 2H), 4.41 (q, 1H), 3.98-3.94 (m, 2H), 3.82-3.76 (m, 4H), 2.56-2.47 (m, 1H), 2.17-2.08 (m, 1H), 0.93-0.88 (m, 18H), 0.12-0.05 (m, 12H). FAB-HRMS: Calcd. for [M+H].sup.+; 620.3187. Found; 620.3187.
Synthesis of N.SUP.4.-MCbz-5′-O-DMTr-2′-deoxycytidine [9b]
(161) 3′,5′-O-bis(TBS)-N.sup.4-MCbz-2′-deoxycytidine (15) (4.34 g, 7 mmol) was dissolved in THF (35 mL), 1 M TBAF/THF (35 mL) was added thereto, and stirring was performed at room temperature for 1 hour. A saturated aqueous NaHCO.sub.3 solution (100 mL) and AcOEt (300 mL) were added, and the organic layer was separated. Washing was performed with saturated aqueous NaHCO.sub.3 solutions (3×100 mL), and the collected washing liquid was extracted with AcOEt (100 mL). The collected organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. To the residue, pyridine (70 mL) and DMTrCl (2.37 g, 7 mmol) were added, and stirred at room temperature for 14 hours. MeOH (30 mL), CHCl.sub.3 (300 mL), and a saturated aqueous NaHCO.sub.3 solution (100 mL) were added, and the organic layer was separated. Washing was performed with saturated aqueous NaHCO.sub.3 solutions (3×100 mL), and the collected washing liquid was extracted with CHCl.sub.3 (100 mL). The collected organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography [neutral silica gel, CH.sub.2Cl.sub.2-MeOH-pyridine (99:0.5:0.5 to 94.5:5:0.5, v/v)] to recover a fraction containing 9b. The solvent was removed under reduced pressure to give 9b (3.47 g, 5 mmol, 71%) as a colorless foamed material.
(162) .sup.1H NMR (300 MHz, CDCl.sub.3) (8.25 (d, 1H), 7.44-7.39 (m, 3H), 7.34-7.22 (m, 9H), 7.02 (d, 1H), 6.92-6.84 (m, 6H), 6.25 (t, 1H), 5.13 (s, 2H), 4.47 (br, 1H), 4.09 (q, 1H), 3.80 (s, 9H), 3.55-3.39 (m, 2H), 2.71-2.62 (m, 1H), 2.30-2.22 (m, 1H). FAB-HRMS: Calcd. for [M+H].sup.+; 694.2765. Found; 694.2763.
(163) ##STR00020##
Synthesis of 3′,5′-O-bis(TBS)-2′-deoxyadenosine [11]
(164) Synthesis was performed according to the method described in the literature, using 2′-deoxyadenosine (10) as a starting material. .sup.1H NMR spectrum was consistent with the literature value.
Synthesis of 3′,5′-O-bis(TBS)-N.SUP.6.-MCbz-2′-deoxyadenosine [12]
(165) 3′,5′-O-bis(TBS)-2′-deoxyadenosine (11) (7.19 g, 15 mmol) was repeatedly azeotropically dried with pyridine, toluene, and CHCl.sub.3 to form a 1,2-dichloroethane solution (150 mL), CDI (3.89 g, 24 mmol) was added, and heated to reflux for 17 hours. 4-methoxybenzylalcohol (3.00 mL, 24 mmol) was added thereto, and heated to reflux for further 20 hours. A saturated aqueous NaHCO.sub.3 solution (100 mL) was added thereto, and the organic layer was separated. Washing was performed with saturated aqueous NaHCO.sub.3 solutions (3×100 mL), and the collected washing liquid was extracted with CH.sub.2Cl.sub.2 (100 mL). The collected organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography [neutral silica gel, hexane-AcOEt (2:1, v/v)] to recover a fraction containing 12. The solvent was removed under reduced pressure to give 12 (7.26 g, 11 mmol, 75%) as a colorless foamed material.
(166) .sup.1H NMR (400 MHz, CDCl.sub.3) δ8.75 (s, 1H), 8.41 (s, 1H), 8.26 (s, 1H), 8.38 (d, 2H), 6.90 (d, 2H), 6.49 (t, 1H), 5.23 (s, 2H), 4.63-4.59 (m, 1H), 4.04 (q, 1H), 3.89-3.75 (m, 5H), 2.67-2.61 (m, 1H), 2.48-2.42 (m, 1H), 0.92-0.87 (m, 18H), 0.10-0.04 (m, 12H). FAB-HRMS: Calcd. for [M+H].sup.+; 644.3300. Found; 644.3298.
Synthesis of N.SUP.6.-MCbz-5′-O-DMTr-2′-deoxyadenosine [9c]
(167) 3′,5′-O-bis(TBS)-N.sup.6-MCbz-2′-deoxyadenosine (12) (7.26 g, 11 mmol) was dissolved into THF (55 mL), 1 M TBAF/THF (55 mL) was added thereto, and stirring was performed at room temperature for 1 hour. A saturated aqueous NaHCO.sub.3 solution (100 mL) and AcOEt (300 mL) were added, and the organic layer was separated. Washing was performed with saturated aqueous NaHCO.sub.3 solutions (3×100 mL), and the collected washing liquid was extracted with AcOEt (100 mL). The collected organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. To the residue, pyridine (110 mL) and DMTrCl (4.48 g, 13.2 mmol) were added, and stirred at room temperature for 18 hours. MeOH (50 mL), CHCl.sub.3 (300 mL), and a saturated aqueous NaHCO.sub.3 solution (100 mL) were added, and the organic layer was separated. Washing was performed with a saturated aqueous NaHCO.sub.3 solution (3×100 mL), and the collected washing liquid was extracted with CHCl.sub.3 (100 mL). The collected organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography [neutral silica gel, CH.sub.2Cl.sub.2-MeOH-pyridine (98:1.5:0.5 to 96.5:3:0.5, v/v)] to recover a fraction containing 9c. The solvent was removed under reduced pressure to give 9c (5.08 g, 7 mmol, 64%) as a colorless foamed material.
(168) .sup.1H NMR (300 MHz, CDCl.sub.3) δ8.68 (s, 1H), 8.16 (br, 1H), 8.06 (s, 1H), 7.40-7.36 (m, 2H), 7.31-7.16 (m, 9H), 6.91-6.77 (m, 6H), 6.46 (t, 1H), 5.23 (s, 2H), 4.70 (br, 1H), 4.16 (q, 1H), 3.77 (s, 9H), 3.41-3.39 (m, 2H), 2.91-2.82 (m, 1H), 2.59-2.51 (m, 1H), 1.65 (br, 1H). FAB-HRMS: Calcd. for [M+H].sup.+; 718.2877. Found; 718.2880.
(169) ##STR00021##
Synthesis of O.SUP.6.-Tse-5′-O-DMTr-2′-deoxyguanosine [9d]
(170) Synthesis was performed according to the method described in the literature, using 2′-deoxyguanosine (1) as a starting material. .sup.1H NMR spectrum was consistent with the literature value.
(171) ##STR00022##
Synthesis of 3′,5′-bis-O-benzoyl-N.SUP.2.-MCbz-deoxyguanosine [6]
(172) 3′,5′-bis-O-benzoyldeoxyguanosine (4) (0.52 g, 1.09 mmol) was dissolved in THF (13.0 mL), DIPEA (0.95 mL, 5.5 mmol) and TMSCI (0.15 mL, 1.0 mmol) were added thereto, and stirring was performed at room temperature for 1 hour. Triphosgene (0.1 g, 0.35 mmol) was added and stirred at 0° C. for 1 hour. 4-methoxybenzylalcohol (0.17 g, 1.2 mmol) was added at room temperature, and heated to reflux overnight. Washing was performed with saturated saline solutions (3×10 mL), and the collected washing liquid was extracted with CH.sub.2Cl.sub.2 (5 mL). The collected organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. The residue was separated and purified by silica column chromatography [neutral silica gel, CH.sub.2Cl.sub.2-MeOH-pyridine (99:0.5:0.5 to 94.5:5:0.5, v/v)] to recover a fraction containing 6. The solvent was removed under reduced pressure to give 6 (0.09 g, 0.40 mmol, 37%) as a yellow foamed material.
(173) .sup.1H NMR (300 MHz, CDCl.sub.3) δ 11.31 (s, 1H), 8.35 (s, 1H), 8.06-8.04 (d, J=7.5 Hz, 2H), 7.96-7.93 (d, J=7.2 Hz, 2H), 7.72 (s, 1H), 7.64-7.59 (m, 1H), 7.55-7.45 (m, 9H), 7.39-7.34 (m, 4H), 6.94-6.92 (m, 2H), 6.30-6.26 (t, J=7.2, 6.6 Hz, 1H), 5.80-5.78 (m, 1H), 5.23 (s, 2H), 4.94-4.90 (m, 1H), 4.75-4.69 (m, 1H), 4.69-4.67 (m, 1H), 3.83 (s, 1H), 3.17-3.07 (m, 1H), 2.70-2.65 (m, 1H).
(174) ##STR00023##
Synthesis of 2-chloro-1,3,2-oxazaphospholidine [(4S,5R)-18]
(175) (αR,2S)-7 (1.07 g, 5.16 mmol) was repeatedly azeotropically dried with toluene, then dissolved in toluene (3 mL), and N-methylmorpholine (1.13 mL, 10.32 mmol) was added thereto. The mixed solution was slowly added to a solution of phosphorus trichloride (0.45 mL, 5.16 mmol) in toluene (2.5 mL) at 0° C. with a syringe. The reaction mixture was stirred at room temperature for 2 hours, and the resulting salt was filtered off at −78° C. under an Ar atmosphere and concentrated under reduced pressure under an Ar atmosphere to give 2-chloro-1,3,2-oxazaphospholidine (4S,5R)-18 (1.28 g, 4.71 mmol). Light yellow oil. (4S,5R)-18 was used for the reaction, without further purification.
Synthesis of [(4R,5S)-18]
(176) Synthesis was performed in the same manner as (4S,5R)-18, using (αS,2R)-7 as a starting material.
(177) ##STR00024##
Synthesis of oxazaphospholidine monomer [(Rp)-20a]
(178) 5′-O-(DMTr)thymidine (9a) (0.86 g, 1.58 mmol) was repeatedly azeotropically dried with pyridine, toluene and THF, then dissolved in THF (8 mL), and Et.sub.3N (1.52 mL, 11 mmol) was added thereto. The mixed solution was cooled to −78° C. and a solution of 0.5 M (4S,5R)-18 in THF (9.5 mL, 4.75 mmol) was slowly added thereto with a syringe. The reaction solution was stirred at room temperature for 2 hours, and CHCl.sub.3 (300 mL) and a saturated aqueous NaHCO.sub.3 solution (100 mL) was added thereto. The organic layer was separated and washed with saturated aqueous NaHCO.sub.3 solutions (2×100 mL), and the collected washing liquid was extracted with CHCl.sub.3 (100 mL). The collected organic phase was dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography [NH-silica gel, toluene-AcOEt (7:3, v/v), Et.sub.3N 0.1%], a fraction containing (Rp)-20a was collected, and the solvent was removed under reduced pressure to give (Rp)-20a (0.53 g, 0.68 mmol, 43%) as a colorless foamed material.
(179) .sup.1H NMR (400 MHz, CDCl.sub.3) δ7.60 (s, 1H), 7.41-7.22 (m, 11H), 6.89-6.79 (m, 6H), 6.43 (t, 1H), 5.68 (d, 1H), 4.94-4.89 (m, 1H), 4.13 (q, 1H), 3.80-3.77 (m, 10H), 3.60-3.53 (m, 1H), 3.49-3.36 (m, 2H), 3.20-3.13 (m, 1H), 2.60-2.55 (m, 1H), 2.40-2.33 (m, 1H), 1.66-1.58 (m, 2H), 1.42 (s, 3H), 1.21-1.14 (m, 1H), 1.00-0.91 (m, 1H). .sup.31P NMR (161 MHz, CDCl.sub.3) b 155.65. FAB-HRMS: Calcd. for [M+Na].sup.+; 802.2869. Found; 802.2866.
(180) The crude products of (Rp)-20b-d and (Sp)-20a-d were all obtained by the same method as described above. Only the conditions for silica gel column chromatography are shown for these compounds.
Synthesis of oxazaphospholidine monomer [(Rp)-20b]
(181) The crude product of (Rp)-20b was obtained from 9b (1.02 g, 1.47 mmol) and (4S,5R)-18 (0.80 g, 2.94 mmol), by the same method as (Rp)-20a. Purification was performed by silica gel column chromatography [NH-silica gel, toluene-AcOEt (7:3, v/v), Et.sub.3N 0.1%] to give (Rp)-20b (0.65 g, 0.70 mmol, 47%) as a colorless foamed material.
(182) .sup.1H NMR (400 MHz, CDCl.sub.3) δ8.25 (d, 1H), 7.39-7.13 (m, 13H), 6.91-6.79 (m, 8H), 6.26 (t, 1H), 5.68 (d, 1H), 5.13 (s, 2H), 4.87-4.81 (m, 1H), 4.16 (q, 1H), 3.83-3.76 (m, 14H), 3.60-3.52 (m, 1H), 3.47 (d, 2H), 3.22-3.13 (m, 1H), 2.82-2.75 (m, 1H), 2.38-2.32 (m, 1H), 1.67-1.57 (m, 2H), 1.22-1.15 (m, 1H), 1.01-0.94 (m, 1H). .sup.31P NMR (161 MHz, CDCl.sub.3) b 156.72. FAB-HRMS: Calcd. for [M+H].sup.+; 929.3527. Found; 929.3528.
Synthesis of oxazaphospholidine monomer [(Rp)-20c]
(183) The crude product of (Rp)-20c was obtained from 9c (1.05 g, 1.47 mmol) and (4S,5R)-18 (0.80 g, 2.94 mmol), by the same method as (Rp)-20a. Purification was performed by silica gel column chromatography [NH-silica gel, toluene-AcOEt (8:2, v/v), Et.sub.3N 0.1%] to give (Rp)-20c (0.62 g, 0.65 mmol, 44%) as a colorless foamed material.
(184) .sup.1H NMR (400 MHz, CDCl.sub.3) δ8.67 (s, 1H), 8.30 (br, 1H), 8.06 (s, 1H), 7.38-7.35 (m, 4H), 7.29-7.14 (m, 9H), 6.91-6.86 (m, 4H), 6.75-6.70 (m, 4H), 6.47 (t, 1H), 5.77 (d, 1H), 5.22 (s, 2H), 5.09-5.03 (m, 1H), 4.29 (q, 1H), 3.88-3.84 (m, 1H), 3.81 (s, 6H), 3.74 (s, 6H), 3.62-3.54 (m, 1H), 3.41-3.33 (m, 2H), 3.20-3.11 (m, 1H), 2.99-2.92 (m, 1H), 2.71-2.65 (m, 1H), 1.68-1.59 (m, 2H), 1.26-1.16 (m, 1H), 1.03-0.94 (m, 1H). .sup.31P NMR (161 MHz, CDCl.sub.3) b 154.97. FAB-HRMS: Calcd. for [M+Na].sup.+; 975.3458. Found; 975.3459.
Synthesis of oxazaphospholidine monomer [(Rp)-20d]
(185) The crude product of (Rp)-20d was obtained from 9d (0.67 g, 1.00 mmol) and (4S,5R)-18 (1.07 g, 3.95 mmol), by the same method as (Rp)-20a. Purification was performed by silica gel column chromatography [NH-silica gel, toluene-AcOEt (9:1, v/v), Et.sub.3N 0.1%] to give (Rp)-20d (0.34 g, 0.38 mmol, 38%) as a colorless foamed material.
(186) .sup.1H NMR (400 MHz, CDCl.sub.3) δ7.69 (s, 1H), 7.43-7.13 (m, 11H), 6.92-6.74 (m, 6H), 6.33 (t, 1H), 5.75 (d, 1H), 5.04 (m, 1H), 4.65-4.53 (m, 4H), 4.26 (q, 1H), 3.92-3.74 (m, 10H), 3.64-3.53 (m, 1H), 3.41-3.30 (m, 2H), 3.20-3.13 (m, 1H), 2.57-2.55 (m, 1H), 2.24-2.19 (m, 1H), 1.68-1.55 (m, 2H), 1.26-1.12 (m, 3H), 1.03-0.96 (m, 1H), 0.08 (s, 9H). .sup.31P NMR (161 MHz, CDCl.sub.3) b 155.04. FAB-HRMS: Calcd. for [M+H].sup.+; 905.3823. Found; 905.3826.
(187) [Oxazaphospholidine monomer [(Sp)-20a]]
(188) The crude product of (Sp)-20a was obtained from 9a (0.85 g, 1.57 mmol) and (4R,5S)-18 (1.28 g, 4.71 mmol), by the same method as (Rp)-20a. Purification was performed by silica gel column chromatography [NH-silica gel, toluene-AcOEt (7:3, v/v), Et.sub.3N 0.1%] to give (Sp)-20a (0.82 g, 1.05 mmol, 67%) as a colorless foamed material.
(189) .sup.1H NMR (400 MHz, CDCl.sub.3) δ8.02 (br, 1H), 7.64 (s, 1H), 7.42-7.16 (m, 11H), 6.90-6.82 (m, 6H), 6.41 (t, 1H), 5.63 (d, 1H), 4.94-4.89 (m, 1H), 4.18 (q, 1H), 3.86-3.76 (m, 10H), 3.56-3.48 (m, 1H), 3.39-3.34 (m, 2H), 3.20-3.13 (m, 1H), 2.50-2.43 (m, 1H), 2.39-2.31 (m, 1H), 1.64-1.59 (m, 2H), 1.40 (s, 3H), 1.25-1.19 (m, 1H), 1.06-0.90 (m, 1H). .sup.31P NMR (161 MHz, CDCl.sub.3) b 156.12. FAB-HRMS: Calcd. for [M+Na].sup.+; 802.2869. Found; 802.2874.
Synthesis of oxazaphospholidine monomer [(Sp)-20b]
(190) The crude product of (Sp)-20b was obtained from 9b (1.01 g, 1.46 mmol) and (4R,5S)-18 (1.00 g, 3.67 mmol), by the same method as (Rp)-20a. Purification was performed by silica gel column chromatography [NH-silica gel, toluene-AcOEt (7:3, v/v), Et.sub.3N 0.1%] to give (Sp)-20b (0.55 g, 0.59 mmol, 40%) as a colorless foamed material.
(191) .sup.1H NMR (400 MHz, CDCl.sub.3) δ8.36 (d, 1H), 7.43-7.16 (m, 13H), 6.94-6.84 (m, 8H), 6.24 (t, 1H), 5.68 (d, 1H), 5.13 (s, 2H), 4.90-4.83 (m, 1H), 4.18 (q, 1H), 3.87-3.76 (m, 14H), 3.57-3.44 (m, 3H), 3.21-3.13 (m, 1H), 2.69-2.63 (m, 1H), 2.41-2.32 (m, 1H), 1.69-1.60 (m, 2H), 1.25-1.17 (m, 1H), 1.04-0.95 (m, 1H). .sup.31P NMR (161 MHz, CDCl.sub.3) b 155.97. FAB-HRMS: Calcd. for [M+Na].sup.+; 951.3346. Found; 951.3349.
Synthesis of oxazaphospholidine monomer [(Sp)-20c]
(192) The crude product of (Sp)-20c was obtained from 9c (1.05 g, 1.46 mmol) and (4R,5S)-18 (1.00 g, 3.67 mmol), by the same method as (Rp)-20a. Purification was performed by silica gel column chromatography [NH-silica gel, toluene-AcOEt (8:2, v/v), Et.sub.3N 0.1%] to give (Sp)-20c (0.67 g, 0.70 mmol, 48%) as a colorless foamed material.
(193) .sup.1H NMR (400 MHz, CDCl.sub.3) δ8.67 (s, 1H), 8.29 (br, 1H), 8.11 (s, 1H), 7.41-7.35 (m, 4H), 7.30-7.12 (m, 9H), 6.91-6.74 (m, 8H), 6.46 (t, 1H), 5.72 (d, 1H), 5.22 (s, 2H), 5.06-5.00 (m, 1H), 4.34 (q, 1H), 3.91-3.85 (m, 1H), 3.80 (s, 6H), 3.76 (s, 6H), 3.62-3.52 (m, 1H), 3.48-3.34 (m, 2H), 3.22-3.13 (m, 1H), 2.94-2.88 (m, 1H), 2.63-2.58 (m, 1H), 1.69-1.58 (m, 2H), 1.26-1.19 (m, 1H), 1.06-0.98 (m, 1H). .sup.31P NMR (161 MHz, CDCl.sub.3) b 155.36. FAB-HRMS: Calcd. for [M+H].sup.+; 953.3639. Found; 953.3643.
Synthesis of oxazaphospholidine monomer [(Sp)-20d]
(194) The crude product of (Sp)-20d was obtained from 9d (0.67 g, 1.00 mmol) and (4R,5S)-18 (1.07 g, 3.95 mmol), by the same method as (Rp)-20a. Purification was performed by silica gel column chromatography [NH-silica gel, toluene-AcOEt (9:1, v/v), Et.sub.3N 0.1%] to give (Sp)-20d (0.34 g, 0.38 mmol, 38%) as a colorless foamed material.
(195) .sup.1H NMR (400 MHz, CDCl.sub.3) δ7.73 (s, 1H), 7.44-7.13 (m, 11H), 6.88-6.78 (m, 6H), 6.30 (t, 1H), 5.71 (d, 1H), 5.01 (m, 1H), 4.61-4.53 (m, 4H), 4.30 (q, 1H), 3.90-3.77 (m, 10H), 3.62-3.52 (m, 1H), 3.46-3.42 (m, 1H), 3.33-3.30 (m, 1H), 3.22-3.13 (m, 1H), 2.52-2.48 (m, 1H), 2.25-2.20 (m, 1H), 1.66-1.59 (m, 2H), 1.26-1.19 (m, 3H), 1.07-0.98 (m, 1H), 0.08 (s, 9H). .sup.31P NMR (161 MHz, CDCl.sub.3) b 155.36. FAB-HRMS: Calcd. for [M+H].sup.+; 905.3823. Found; 905.3825.
(196) ##STR00025##
(197) 9a to 9d (base protected by a protecting group), Compound 18 used for synthesis of each monomer, a yield of each monomer, and a mass ratio of an Rp isomer and a Sp isomer contained in the obtained monomer are described in Table 1.
(198) In Table 1, Th represents thymine, Cy.sup.MCbz represents cytosine of which the amino group is protected by an MCbz group, Ad.sup.MCbz represents adenine of which the amino group is protected by an MCbz group, and Gu.sup.Tse represents guanine of which the amino group is protected by a trimethylsilyl group, respectively.
(199) TABLE-US-00001 TABLE 1 9a to 9d Used 20 Entry compound Included base 18 Yield (%) Rp:Sp.sup.b 1 9a Th (4S,5R) 43 >99:1 2 9b Cy.sup.MCbz (4S,5R) 47 >99:1 3 9c Ad.sup.MCbz (4S,5R) 44 >99:1 4 9d Gu.sup.Tse (4S,5R) 37 >99:1 5 9a Th (4R,5S) 67 >1:99 6 9b Cy.sup.MCbz (4R,5S) 40 >1:99 7 9c Ad.sup.MCbz (4R,5S) 48 >1:99 8 9d Gu.sup.Tse (4R,5S) 38 >1:99 .sup.aIn entries 4 to 8, a processing condition by Et.sub.3N described above was at −78° C. .sup.b31P NMR was used for measurement.
<Synthesis 1 of Boranophosphate DNA>
(200) Manual solid phase synthesis of boranophosphate DNA (PB-DNA) (24a-d,25,26) was performed using a controlled-pore glass (CPG) as a solid support and a glass filter (10 mm×50 mm) having a cock at the bottom as a reaction vessel.
(201) First, 5′-O-(DMTr)thymidine (27) supported on CPG via succinyl linker was treated with 1% TFA/CH.sub.2Cl.sub.2 (4×5 s) to remove a 5′-O-DMTr group, and the following steps (i) to (v) were performed to synthesize H-phosphonate DNA (23). (i) washing (CH.sub.2Cl.sub.2, CH.sub.3CN) (ii) coupling (0.2M monomer 20a-d and 1.0M CMPT(21) in CH.sub.3CN;5 min), (iii) washing (CH.sub.3CN, CH.sub.2Cl.sub.2) (iv) 1% TFA/CH.sub.2Cl.sub.2-Et.sub.3SiH (1:1,v/v)(4×1 min) (v) washing (CH.sub.2Cl.sub.2, CH.sub.3CN)
(202) Subsequently, DMAc (0.8 mL), BSA (0.1 mL), and BH.sub.3.SMe2 (0.1 mL) were added to the synthesized 23 at room temperature. After 15 minutes, the reaction solution was removed and CPG was washed with DMAc and CH.sub.3CN. CPG was treated with concentrated aqueous NH.sub.3 solution-EtOH (3:1, v/v) at 25° C. for 3 hours or 55° C. for 12 hours, CPG was filtered off, and the filtrate was concentrated under reduced pressure. The residue was analyzed and identified by RP-HPLC and ESI-MS.
(203) ##STR00026##
(204) A sequence of the obtained boranophosphate DNA (PB-DNA), reaction conditions for release of the oligomer from a solid support (temperature and retention time under a condition of conc. NH.sub.3-EtOH (3:1, v/v)), a yield, and a stereochemical purity of a dimer are listed in Table 2, respectively. In addition, in Table 2, description of Rp or Sp indicates whether the absolute configuration of a boranophosphate structure in the oligomer is Sp or Rp. The description of all-(Rp) indicates that the absolute configuration of a boranophosphate structure in the oligomer is all Rp, and the description of all-(Sp) indicates that the absolute configuration of a boranophosphate structure in the oligomer is all Sp, respectively.
(205) TABLE-US-00002 TABLE 2 Conditions for release of the oligomer from solid Yield Stereochemical Entry Sequence of PB-DNA.sup.a support (%).sup.b purity.sup.b 1 (Rp)-24a (Rp)-T.sub.BT 25° C., 3 h 91 98:2 2 (Rp)-24b (Rp)-dC.sub.BT 25° C., 3 h 91 >99:1 3 (Rp)-24c (Rp)-dA.sub.BT 25° C., 3 h — >99:1 4 (Rp)-24c (Rp)-dA.sub.BT 55° C., 12 h 88 >99:1 5 (Rp)-24d (Rp)-dG.sub.BT 55° C., 12 h 77 >99:1 6 (Sp)-24a (Sp)-T.sub.BT 25° C., 3 h 88 >99:1 7 (Sp)-24b (Sp)-dC.sub.BT 25° C., 3 h 93 98:2 8 (Sp)-24c (Sp)-dA.sub.BT 25° C., 3 h — >99:1 9 (Sp)-24c (Sp)-dA.sub.BT 55° C., 12 h 87 >99:1 10 (Sp)-24d (Sp)-dG.sub.BT 55° C., 12 h 77 97:3 11 25 all-(Rp)- 55° C., 12 h .sup. 14.sup.c — d(C.sub.BA.sub.BG.sub.BT) 12 26 all-(Sp)- 55° C., 12 h .sup. 23.sup.c — d(C.sub.BA.sub.BG.sub.BT) .sup.a“B” represents being bonded by a boranophosphate structure. .sup.bCalculated by reverse phase HPLC. The HPLC charts are shown in FIGS. 1 to 7. .sup.cIsolated yield
(206)
(207) The measurement conditions of the reverse phase HPLC were as follows.
(208) Gradient cycle: After a linear gradient of acetonitrile from 0% to 10% for 30 minutes in a 0.1 M triethylammonium buffer solution (pH 7.0), acetonitrile was held at 10% for 30 minutes.
(209) Measurement temperature: 30° C.
(210) Flow rate: 0.5 mL/min
(211) Column: μBondasphere 5 μm C18 column (100 Å, 3.9 mm×150 mm) (Waters)
(212)
(213) The measurement conditions of the reverse phase HPLC were as follows.
(214) Gradient cycle: After a linear gradient of acetonitrile from 0% to 10% for 30 minutes in a 0.1 M triethylammonium buffer solution (pH 7.0), acetonitrile was held at 10% for 20 minutes. Measurement temperature: 30° C. Flow rate: 0.5 mL/min Column: μBondasphere 5 μm C18 column (100 Å, 3.9 mm×150 mm) (Waters)
(215)
(216) The measurement conditions of the reverse phase HPLC were as follows.
(217) Gradient cycle: A linear gradient of acetonitrile from 0% to 30% for 60 minutes in a 0.1 M triethylammonium buffer solution (pH 7.0). Measurement temperature: 30° C. Flow rate: 0.5 mL/min Column: μBondasphere 5 μm C18 column (100 Å, 3.9 mm×150 mm) (Waters)
(218)
(219) The measurement conditions of the reverse phase HPLC were as follows. Gradient cycle: A linear gradient of acetonitrile from 0% to 30% for 60 minutes in a 0.1 M triethylammonium buffer solution (pH 7.0). Measurement temperature: 30° C. Flow rate: 0.5 mL/min Column: μBondasphere 5 μm C18 column (100 Å, 3.9 mm×150 mm) (Waters)
(220)
(221) The measurement conditions of the reverse phase HPLC were as follows. Gradient cycle: A linear gradient of acetonitrile from 0% to 30% for 60 minutes in a 0.1 M triethylammonium buffer solution (pH 7.0). Measurement temperature: 30° C. Flow rate: 0.5 mL/min Column: μBondasphere 5 μm C18 column (100 Å, 3.9 mm×150 mm) (Waters)
(222)
(223) The measurement conditions of the reverse phase HPLC were as follows. Gradient cycle: A linear gradient of acetonitrile from 0% to 20% for 60 minutes in a 0.1 M triethylammonium buffer solution (pH 7.0). Measurement temperature: 30° C. Flow rate: 0.5 mL/min Column: μBondasphere 5 μm C18 column (100 Å, 3.9 mm×150 mm) (Waters)
(224)
(225) The measurement conditions of the reverse phase HPLC were as follows. Gradient cycle: A linear gradient of acetonitrile from 0% to 20% for 60 minutes in a 0.1 M triethylammonium buffer solution (pH 7.0). Measurement temperature: 30° C. Flow rate: 0.5 mL/min Column: μBondasphere 5 μm C18 column (100 Å, 3.9 mm×150 mm) (Waters)
(226) The sequence of the obtained boranophosphate DNA (PB-DNA), the chemical formula [—H.sup.+], the theoretical value of a molecular weight (MS), and the measured value by ESI-MS are shown in Table 3, respectively. In addition, in Table 3, description of Rp or Sp indicates whether the absolute configuration of a boranophosphate structure in the oligomer is Sp or Rp. The description of all-(Rp) indicates that the absolute configuration of a boranophosphate structure in the oligomer is all Rp, and the description of all-(Sp) indicates that the absolute configuration of a boranophosphate structure in the oligomer is all Sp, respectively.
(227) TABLE-US-00003 TABLE 3 Chemical Formula Theoretical Measured PB-DNA.sup.a [—H.sup.+] value value (Rp)-24a (Rp)-T.sub.BT C.sub.20H.sub.29BN.sub.4O.sub.11P.sup.− 542.1705 542.1695 (Rp)-24b (Rp)-dC.sub.BT C.sub.19H.sub.28BN.sub.5O.sub.10P.sup.− 527.1709 527.1708 (Rp)-24c (Rp)-dA.sub.BT C.sub.20H.sub.28BN.sub.7O.sub.9P.sup.− 551.1821 551.1826 (Rp)-24d (Rp)-dG.sub.BT C.sub.20H.sub.28BN.sub.7O.sub.10P.sup.− 567.1770 567.1761 (Sp)-24a (Sp)-T.sub.BT C.sub.20H.sub.29BN.sub.4O.sub.11P.sup.− 542.1705 542.1700 (Sp)-24b (Sp)-dC.sub.BT C.sub.19H.sub.28BN.sub.5O.sub.10P.sup.− 527.1709 527.1710 (Sp)-24c (Sp)-dA.sub.BT C.sub.20H.sub.28BN.sub.7O.sub.9P.sup.− 551.1821 551.1821 (Sp)-24d (Sp)-dG.sub.BT C.sub.20H.sub.28BN.sub.7O.sub.10P.sup.− 567.1770 567.1763 25 all-(Rp)- C.sub.39H.sub.57B.sub.3N.sub.15O.sub.19P.sub.3.sup.− 1166.3549 1166.3547 d(C.sub.BA.sub.BG.sub.BT) 26 all-(Sp)- C.sub.39H.sub.57B.sub.3N.sub.15O.sub.19P.sub.3.sup.− 1166.3549 1166.3542 d(C.sub.BA.sub.BG.sub.BT) .sup.a“B” represents being bonded by a boranophosphate structure.
<Synthesis 2 of boranophosphate DNA>
(228) According to the following scheme, boranophosphate DNA (PB-DNA) [27] was synthesized. The sequence was all-(Sp)-d(G.sub.BT.sub.B(A.sub.BC.sub.BT.sub.B).sub.3T). A controlled-pore glass (CPG) as a solid support and a glass filter (10 mm×50 mm) having a cock at the bottom of a reaction vessel were used. The isolated yield was 1.5%.
(229) Other detailed reaction conditions are shown in the following Table 4.
(230) ##STR00027##
(231) TABLE-US-00004 TABLE 4 step manipulation reagents and solvents time 1 detritylation 1% TFA in DCM 15 s 2 wash (1) DCM (2) CH.sub.3CN — 3 drying — 5 min 4 condensation monomer units (0.2M), 5 min CMPT (1.0M)/CH.sub.3CN 5 wash CH.sub.3CN — 6 drying — 5 min 7 wash (1) DCM (2) CH.sub.3CN — 8 detritylation and 1% TFA in DCM-Et.sub.3SiH (1:1, v/v) 5 min deprotection repeat steps 2-8 in order to synthesize the objective sequence 9 wash (1) DCM (2) CH.sub.3CN — 10 boronation BH.sub.3•SMe.sub.2-BSA-DMAc 15 min (1:1:8, v/v/v) 11 wash (1) DMAc (2) CH.sub.3CN — 12 release the oligomer sat.NH.sub.3aq-EtOH (3:1, v/v, 50° C.) 12 h from the CPG
(232) In Table 4, monomer units represent Compound 20a-d.
(233)
(234) The measurement conditions of the reverse phase HPLC were as follows.
(235) Gradient cycle: A linear gradient of acetonitrile from 0% to 40% for 60 minutes in a 0.1 M triethylammonium buffer solution (pH 7.0). Measurement temperature: 30° C. Flow rate: 0.5 mL/min Column: μBondasphere 5 μm C18 column (100 Å, 3.9 mm×150 mm) (Waters)
<Synthesis 3 of boranophosphate DNA (Comparative Example)>
(236) According to the following scheme, boranophosphate DNA (PB-DNA) [28, 29] was synthesized. The sequence was all-(Sp)-d(C.sub.BA.sub.BG.sub.BT.sub.B).sub.2(CBABGB)T [28] or all-(Rp)-d(C.sub.BA.sub.BG.sub.BT.sub.B).sub.2(CBABGB)T [29]. A controlled-pore glass (CPG) as a solid support and a glass filter (10 mm×50 mm) having a cock at the bottom of reaction vessel were used.
(237) Other detailed reaction conditions are shown in the following Table 5.
(238) ##STR00028##
(239) TABLE-US-00005 TABLE 5 step manipulation reagents and solvents time 1 detritylation 1% TFA in DCM 15 s 2 wash (1) DCM (2) CH.sub.3CN — 3 drying — 10 min 4 condensation monomer units (0.2M), 5 min CMDMT (0.5M)/CH.sub.3CN-NMP (4:1, v/v) 5 wash CH.sub.3CN — 6 drying — 5 min 7 capping capping amidite (0.5M), 5 min CMPT (1.0M)/CH.sub.3CN 8 wash (1) DCM (2) CH.sub.3CN — 9 detritylation and 1% TFA in DCM-Et.sub.3SiH (1:1, v/v) 15 s deprotection repeat steps 2-9 in order to synthesize the objective sequence 10 wash (1) DCM (2) CH.sub.3CN — 11 boronation BH.sub.3•SMe.sub.2-BSA-DMAc 15 min (1:1:8, v/v/v) 12 wash (1) DMAc (2) CH.sub.3CN (3) CH.sub.3OH — 13 release the oligomer sat.NH.sub.3 in CH.sub.2OH 12 h from the HCP resin (50° C., 0.1 mM)
(240)
(241) The measurement conditions of the reverse phase HPLC were as follows.
(242) Gradient cycle: A linear gradient of acetonitrile from 0% to 40% for 60 minutes in a 0.1 M triethylammonium buffer solution (pH 7.0). Measurement temperature: 30° C. Flow rate: 0.5 mL/min Column: μBondasphere 5 μm C18 column (100 Å, 3.9 mm×150 mm) (Waters)
(243) As can be seen from