Conjugates of 1,4,7-triazacyclononanes, dinuclear metal complexes of such conjugates, and methods of use for both 1,4,7-triazacyclononanes and conjugate

Abstract

Conjugates of 1,3-bis(1,4,7-triazacyclonon-1-yl)-2-hydroxypropanes with a variety of conjugating members are used in the formation of dinuclear metal complexes which bind to phosphate esters. By virtue of their conjugated forms, the complexes are incorporated into chromatographic media, affinity binding reagents, and dyes, which make the complexes useful in a wide range of assays, separations, and purifications. In addition, dinuclear metal complexes of 1,3-bis(1,4,7-triazacyclonon-1-yl)-2-hydroxypropanes that are not so conjugated are used in the detection of phosphate esters of biological species by either MALDI-TOF mass spectrometry or by dye displacement.

Claims

1. A method for the preparation of a separation medium for use in electrophoresis of phosphorylated species, said method comprising polymerizing a monomer mixture comprising acrylamide, a crosslinker, an initiator, and a compound having the formula ##STR00014## in which: one of R.sup.1 through R.sup.6 is -L-R.sup.7 in which L is a member selected from the group consisting of CH.sub.2C(O)NH(C.sub.1-C.sub.4 alkyl), CH.sub.2C(O)NH(C.sub.1-C.sub.4 alkyl)NH, and (C.sub.1-C.sub.4 alkyl)NH, and R.sup.7 is an acrylamide group; the remainder of R.sup.1 through R.sup.6 are independently selected from the group consisting of H and C.sub.1-C.sub.6 alkyl; and M is a divalent metal.

2. The method of claim 1 wherein M is a member selected from the group consisting of Ca, Zn, Cr, Mn, Fe, Co, Ni, and Cu.

3. The method of claim 1 wherein M is a member selected from the group consisting of Zn and Mn.

4. The method of claim 1 wherein R.sup.5 is -L-R.sup.7, and R.sup.1, R.sup.2, R.sup.3, R.sup.4, and R.sup.6 are independently selected from the group consisting of H and C.sub.1-C.sub.6 alkyl.

5. The method of claim 1 wherein R.sup.5 is -L-R.sup.7, and R.sup.1, R.sup.2, R.sup.3, R.sup.4, and R.sup.6 are H.

6. The method of claim 1 wherein R.sup.2 is -L-R.sup.7, and R.sup.1, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are independently selected from the group consisting of H and C.sub.1-C.sub.6 alkyl.

7. The method of claim 1 wherein R.sup.2 is -L-R.sup.7, and R.sup.1, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are H.

8. The method of claim 1 wherein R.sup.6 is -L-R.sup.7, and R.sup.1, R.sup.2, R.sup.3, R.sup.4, and R.sup.5 are independently selected from the group consisting of H and C.sub.1-C.sub.6 alkyl.

9. The method of claim 1 wherein R.sup.6 is -L-R.sup.7, and R.sup.1, R.sup.2, R.sup.3, R.sup.4, and R.sup.5 are H.

10. The method of claim 1 wherein L is a member selected from the group consisting of CH.sub.2C(O)NHCH.sub.2CH.sub.2, CH.sub.2C(O)NHCH.sub.2CH.sub.2NH, and C.sub.2H.sub.5NH.

11. The method of claim 1 wherein R.sup.5 is CH.sub.2C(O)NHCH.sub.2CH.sub.2NHC(O)CHCH.sub.2, and R.sup.1, R.sup.2, R.sup.3, R.sup.4, and R.sup.6 are H.

12. The method of claim 1 wherein R.sup.5 is CH.sub.2CH.sub.2NHC(O)CHCH.sub.2, and R.sup.1, R.sup.2, R.sup.3, R.sup.4, and R.sup.6 are H.

13. The method of claim 1 wherein R.sup.2 is CH.sub.2CH.sub.2NHC(O)CHCH.sub.2, and R.sup.1, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are H.

14. The method of claim 1 wherein R.sup.6 is CH.sub.2CH.sub.2NHC(O)CHCH.sub.2, and R.sup.1, R.sup.2, R.sup.3, R.sup.4, and R.sup.5 are H.

Description

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

(1) The ligands, which are substituted or unsubstituted 1,3-bis(1,4,7-triazacyclonon-1-yl)-2-hydroxypropane, are represented by generic Formula (I):

(2) ##STR00001##

(3) In Formula (I), the groups R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are the same or different (some the same and others different) and are either H or a lower alkyl group. The expression lower alkyl is defined as an alkyl group of six carbon atoms or less. Preferred groups for R.sup.1 through R.sup.6 are either H, C.sub.1-C.sub.3 alkyl, or combinations selected from H and C.sub.1-C.sub.3 alkyl, and the most preferred is H. The term substituted in this context denotes that one or more of R.sup.1, R.sup.2, R.sup.3, R.sup.4, and R.sup.6 is an alkyl group rather than an H atom.

(4) Ligands that are described herein as not being conjugated are those in which R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6 are the same or different and each is either H or a lower alkyl group. The conjugates of the ligands are those compounds of Formula (I) in which a conjugating member occupies any of the positions indicated by R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.6, through a linking group joining the conjugating member to the remainder of the structure. Any such R groups that do not represent the conjugating member are either H or a lower alkyl group, as indicated above. For the conjugates, one or more of R.sup.1 through R.sup.6 is thus -L-R.sup.7, where L represents the linking group and R.sup.7 is the conjugating member, and the remaining group(s) of R.sup.1 through R.sup.6, which are either the same or different (some the same and others different), are each either H or C.sub.1-C.sub.6 alkyl. For the functionalized ligands, one or more of R.sup.1 through R.sup.6 is -L, where L represents the unreacted linking group that is reactive with the conjugating member. In the functionalized ligands as in the conjugates, the remaining group(s) of R.sup.1 through R.sup.6, which are either the same or different (some the same and others different), are each either H or C.sub.1-C.sub.6 alkyl. In preferred functionalized ligands or conjugates, only one of R.sup.1 through R.sup.6 is -L or -L-R.sup.7, respectively, and in further preferred ligands or conjugates, only R.sup.5, only R.sup.6, or only R.sup.2 is -L or -L-R.sup.7. Of the R.sup.1 through R.sup.6 groups that are not replaced by -L or -L-R.sup.7, such groups are preferably either H, C.sub.1-C.sub.3 alkyl, or combinations selected from H and C.sub.1-C.sub.3 alkyl, and the most preferred is H. The conjugating member R.sup.7 is either an acrylamide group, a fluorescent dye, an affinity-type binding member, or a solid chromatographic support. The linking group L or L is any conventional linker with two binding sites, one of which forms a covalent bond with the conjugating member R.sup.7 and the other forms a covalent bond with the adjacent carbon atom on the remainder of Formula (I). The linking group generally has from 3 to 30 atoms selected from C, N, O, P, and S, in addition to hydrogen atoms filling available valences, and is either cyclic, acyclic, aromatic or a combination of cyclic, acyclic, and aromatic moieties. Examples of linking groups are amine alkyl linkages, alkenyl linkages, amide or amide-containing linkages, ester or ester-containing linkages, and ether or ether-containing linkages. Preferred linking groups for L are (C.sub.1-C.sub.4 alkyl)-NH.sub.2, (C.sub.1-C.sub.4 alkyl)-C(O)NH.sub.2, (C.sub.1-C.sub.4 alkyl)-NHCO.sub.2H, (C.sub.1-C.sub.4 alkyl)-C(O)NH(C.sub.1-C.sub.4 alkyl)-NH.sub.2, and (C.sub.1-C.sub.4 alkyl)-NHC(O)(C.sub.1-C.sub.4)NH.sub.2, and particularly preferred are CH.sub.2CH.sub.2NH, CH.sub.2C(O)NH.sub.2, CH.sub.2CH.sub.2NHCO.sub.2H, CH.sub.2C(O)NHCH.sub.2CH.sub.2NH.sub.2, and CH.sub.2CH.sub.2NHC(O)CH.sub.2CH.sub.2NH.sub.2. Preferred groups for L are (C.sub.1-C.sub.4 alkyl)-C(O)NH(C.sub.1-C.sub.4 alkyl)-, (C.sub.1-C.sub.4 alkyl)-NHC(O) (C.sub.1-C.sub.4 alkyl)-NH, and (C.sub.1-C.sub.4 alkyl)-NH, and particularly preferred are CH.sub.2C(O)NHCH.sub.2CH.sub.2, CH.sub.2CH.sub.2NHC(O) CH.sub.2CH.sub.2NH, and CH.sub.2CH.sub.2NH.

(5) When R.sup.7 is an acrylamide group, -L-R.sup.7 is preferably CH.sub.2C(O)NH(CH.sub.2).sub.nNHC(O)CHCH.sub.2 or (CH.sub.2).sub.2NHC(O)(CH.sub.2)NHC(O)CHCH.sub.2 where n is 1 to 6, and the most preferred are CH.sub.2C(O)NH(CH.sub.2).sub.2NHC(O)CHCH.sub.2 and (CH.sub.2).sub.2NHC(O)(CH.sub.2).sub.2NHC(O)CHCH.sub.2. When this particular -L-R.sup.7 replaces R.sup.5 and the remaining R's are all H, the conjugate has the formula

(6) ##STR00002##

(7) Functionalized polyacrylamide gels can be formed from conjugates and dinuclear metal complexes in accordance with this invention where the conjugating member is acrylamide. The functional group in these gels is the dimetal complex of the acrylamide-containing conjugate, and the resulting gels can be used for electrophoretic separations of phosphate monoesters of proteins, peptides, or nucleic acids from the non-phosphorylated forms of these species. Phosphate monoesters of proteins, peptides, or nucleic acids within the mixture will migrate more slowly during electrophoresis due to interaction between the phosphate esters and the dimetal complex functional groups on the gels. Phosphorylated components of the mixture can thereby be distinguished from their unphosphorylated counterparts. The functionalized gel is formed by including the dimetal complex in the monomer mixture, which will generally also include non-functionalized acrylamide, a crosslinker, and an initiator. Suitable crosslinkers and initiators are well known in the art. Examples of crosslinkers are bisacrylamide and ethylene diacrylate, and examples of initiators are riboflavin, ammonium persulphate, and tetramethylethylenediamine (TEMED). The conditions for polymerization of the monomers and the concentrations of the various components of the monomer mixture are the same as those used in procedures of the prior art for the formation of the polyacrylamide gels.

(8) Dinuclear metal complexes of the ligands of Formula (I) for use in the practice of this invention are those having Formula (II):

(9) ##STR00003##
In Formula (II), R.sup.1 through R.sup.6 are as defined above, and M is a divalent metal. A preferred group of divalent metals is Ca, Zn, Cr, Mn, Fe, Co, Ni, and Cu (all in divalent form). Particularly preferred divalent metals are Zn (II) and Mn(II).

(10) When used in the various methods of the present invention, the dinuclear metal complexes of Formula (II) form phosphate association complexes of Formula (III):

(11) ##STR00004##

(12) In Formula (III), X is the residue of a peptide, protein, nucleic acid, or in general any species to which the complexes of this invention are to be applied for any of the various purposes stated herein.

(13) When R.sup.7 is a fluorescent dye, the dye can be any of the variety of fluorescent dyes known for use in the labeling of biological species. Examples are: fluorescein and fluorescein derivatives such as fluorescein isothiocyanate, rhodamine derivatives such as tetramethyl rhodamine, rhodamine B, rhodamine 6G, sulforhodamine B, rhodamine 101 (Texas Red), and rhodamine 110, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) and derivatives thereof, pyrenes and pyrene derivatives such as 8-methoxypyrene-1,3,6-trisulfonic acid, pyridyloxazole derivatives, dapoxyl derivatives, umbelliferone, 1-anilino-8-naphthalenesulfonic acid, 3,6-disulfonate-4-amino-naphthalimide, tri-, penta-, and heptamethine cyanine dyes, and luminescent transition metal complexes such as tris(2,2-bipyridine)ruthenium(II) or cyclometalated complexes of Ir(III).

(14) When R.sup.7 is tetramethyl rhodamine, for example, -L-R.sup.7 is preferably

(15) ##STR00005##
where the asterisk denotes the site of attachment to the 1,3-bis(1,4,7-triazacyclonon-1-yl)-2-hydroxypropane. When this linker and dye replace R.sup.5 and R.sup.1 through R.sup.4 are all H, the resulting conjugate has the formula

(16) ##STR00006##

(17) Dinuclear metal complexes in accordance with this invention that are formed from conjugates containing fluorescent dyes can be used for the staining of gels or blots to identify and quantify phosphate esters, such as phosphoproteins, for example, in samples of biological fluids and in assay media in general.

(18) When R.sup.7 is an affinity-type binding member, examples of such binding members are biotin, avidin, streptavidin, antibodies, and antibody fragments. When R.sup.7 is biotin, -L-R.sup.7 is preferably

(19) ##STR00007##
where the asterisk denotes the site of attachment to the 1,3-bis(1,4,7-triazacyclonon-1-yl)-2-hydroxypropane. When this functional group replaces R.sup.5 and R.sup.1 through R.sup.4 are all H, the resulting conjugate has the formula

(20) ##STR00008##

(21) Dinuclear metal complexes in accordance with this invention in which R.sup.6 is an affinity binding member are useful in the detection of phosphoproteins and other phosphate esters bound to or otherwise immobilized on a solid support. The ability of the affinity binding member to bind with specificity to a second affinity binding member to form an affinity binding pair allows the attachment of a reporter group to the complexes through the binding pair, either before or after the complexes have associated with the phosphate esters. This allows detection by any type of reporter group that can be conjugated to an affinity binding member. Affinity binding pairs are well known; examples are biotin and avidin, biotin and streptavidin, and any of various antibodies or antibody fragments and antigens. Examples or reporter groups are radioactive labels, chemiluminescent labels, and enzymes. Examples of enzymes are horseradish peroxidase, chloramphenicol acetyl transferase, -galactosidase, alkaline phosphatase, and luciferase. To illustrate the use of dinuclear metal complexes containing an affinity binding member, the solid support, such as for example, a blotting membrane, on which the phosphate esters have been captured is first contacted with the complex. If, for example, the conjugating member on the complex is biotin, the support is contacted with a streptavidin-enzyme conjugate. This is followed by incubation of the support with an enzyme substrate and detection of the change, most often a color change, in the substrate, mediated by the enzyme. Optimal conditions for the contact and incubation are readily apparent to those of skill in the art.

(22) When R.sup.7 is a solid chromatographic support, the term solid is used herein to include semi-solids such as gels, flexible solids such as membranes and films, and rigid solids such as those used in the formation of incompressible beads, granules, and column or tube walls. Examples of such solids are polyacrylamide, crosslinked silicon polymers, silica gel, agarose, polyvinyl alcohols, cellulose and nitrocellulose. A chromatographic support bearing the dinuclear metal complexes of this invention can be used to extract phosphorylated compounds (phosphate esters) from fluids such as biological fluids, assay media, or any fluids containing phosphorylated compounds, for purposes of purification or enrichment.

(23) Conjugates in accordance with this invention can be prepared by conventional procedures, beginning with 1,4,7-triazacyclononane derivatives with one or more of the secondary amine groups protected either with a suitable protecting group or by formation of a 1,4,7-triazacyclononane tricyclic orthoamide. Appropriate functional groups can be selectively placed in one or more of the unprotected R.sup.1 through R.sup.4 positions. An appropriate functional group can also be placed in the R.sup.5 position by selection of an appropriate synthetic intermediate that will join the two 1,4,7-triazacyclononane groups by connecting one ring nitrogen from each macrocycle with a three-carbon chain. The choice of functional group will vary with the choice of linking group and with the means by which the linking group is attached, such attachment being achievable by any of the wide range of linking reactions known in the art. Prominent example of functional groups are carboxylic acids or esters of carboxylic acids that can react with an amine functionality appended to the conjugating member, or an amine group that can react with an active carboxylic acid ester or acid chloride, both resulting in the formation of an amide linkage. Reaction of a carboxylic acid-functionalized ligand with an amine-bearing conjugation partner, as known in the art, is readily achieved either directly or in the presence of an activated acid such as an acid chloride, acid anhydride, or succinimidyl ester. Reaction of an amine-functionalized ligand with an activated ester, or acid chloride-bearing conjugation partner, as known in the art, is readily achieved.

(24) The preparation of functionalized ligands is illustrated by a ligand in which a methyl ester occupies the R.sup.5 position, with all other R-groups being H. An example of such a preparation is one that begins with 1,4,7-triazacyclononane and reacts with dimethylformamide dimethyl acetal to form 1,4,7-triazacyclononane tricyclic orthoamide, which is then reacted with methyl 3,5-dibromolevulinate to form methyl 3,5-bis(4-formyl-1,4,7-triazonan-1-yl)-4-oxopentanoate. The latter is then reacted with sodium borohydride to form methyl 3,5-bis(4-formyl-1,4,7-triazonan-1-yl)-4-hydroxypentanoate, which is then reacted with hydrochloric acid to produce methyl 4-hydroxy-3,5-di(1,4,7-triazonan-1-yl)pentanoate. This sequence of reactions is shown below:

(25) ##STR00009##

(26) Variations of this procedure to place the methyl ester group at other locations on the molecule and to obtain the functionalized ligand with additional substitutions will be apparent to those skilled in organic chemical syntheses. One example is shown below, which places the amine group and starts with 1,4,7-triazacyclononane that is protected at two N atoms with butoxycarbonyl groups is reacted with 1,3-dibromo-2-propanol to form di-tert-butyl 7-(3-bromo-2-hydroxypropyl)-1,4,7-triazonane-1,4-dicarboxylate (Step 1). Separately, unprotected 1,4,7-triazacyclononane is reacted with N,N-dimethylformamide dimethyl acetal to form the corresponding tricyclic structure, which is then reacted with tert-butyl (2-bromoethyl)carbamate and hydrolyzed, yielding tert-butyl (2-(4-formyl-1,4,7-triazanon-1-yl)-3-)1,4,7-triazonan-1-yl)ethyl carbamate. The two products are then reacted to form 1-(4-(2-aminoethyl)-1,4,7-triazonan-1-yl)-3-(1,4,7-triazonan-2-yl)propan-2-ol following deprotection (Step 3).

(27) ##STR00010## ##STR00011##

(28) A further example is shown below, starting with the hydration of N-(3-butynyl)phthalamide to N-(3-oxobutyl) phthalamide. This is followed by reaction with bromine, then with a Wittig reagent, then with peroxyacetic acid, then with 1,4,7-triazacyclononane protected at two N atoms, followed by deprotection to form 2-(1,4,7-trazonan-1-yl)methyl)-4-1-(1,4,7-triazonan-1-yl)butan-2-ol.

(29) ##STR00012## ##STR00013##

(30) Complexation of either the ligands or the conjugates for use in the present invention can be performed on the ligand, on the functionalized ligand prior to conjugation, or on the conjugate itself. In either case, complexation can be achieved by combining an appropriate salt of the metal, for example Zn(NO.sub.3).sub.2 where a Zn complex is to be formed, with the hydrobromide or hydrochloride salt of the ligand at an appropriate molar ratio, and adjusting the pH to 6.5-7.0.

(31) In the claims appended hereto, the term a or an is intended to mean one or more. The term comprise and variations thereof such as comprises and comprising, when preceding the recitation of a step or an element, are intended to mean that the addition of further steps or elements is optional and not excluded. All patents, patent applications, and other published reference materials cited in this specification are hereby incorporated herein by reference in their entirety. Any discrepancy between any reference material cited herein or any prior art in general and an explicit teaching of this specification is intended to be resolved in favor of the teaching in this specification. This includes any discrepancy between an art-understood definition of a word or phrase and a definition explicitly provided in this specification of the same word or phrase.