Rapid and efficient bioorthogonal ligation reaction and boron-containing heterocycles useful in conjunction therewith

1. A process comprising:

(a) providing:

(1) a composition having a boron atom bonded to a sp 2 hybridized carbon, conjugated with a cis-carbonyl, and linked to a first moiety, the boron atom having at least one labile substituent, the composition being configured to spontaneously react with an α-effect amine in an aqueous solvent at −5° C., and, to undergo a replacement of the carbonyl oxygen by formation of a carbon-nitrogen bond in the aqueous solvent; and

(2) an α-effect amine, linked to a second moiety, the α-effect amine being configured to spontaneously react with the composition in an aqueous solvent at −5° C., and to undergo replacement of the carbonyl oxygen by the formation of the carbon-nitrogen bond in the aqueous solvent; and

(b) contacting the composition with the α-effect amine, in the aqueous solvent at a temperature below about 55° C., to spontaneously form an adduct having an interacting boron of the composition and a nitrogen of the α-effect amine, linking the first moiety and the second moiety,

wherein at least one of the first moiety and the second moiety is selected from the group consisting of a probe, a protein, an antibody, a peptide, an amino acid, a nucleic acid, a linker, a drug, a radiolabel, a fluorophore, a sugar, a carbohydrate, a support, a surface, a bead, and a nanoparticle,

wherein the composition is:

wherein:

X 1 , X 2 are groups that can hydrolyze from the boron in the aqueous solvent to yield boronic acid; and

R 1 , R 2 , and R 3 are selected from the group consisting of hydrogen, organic ligands, and heterorganic ligands, and at least one of R 1 , R 2 , and R 3 are selected from the group consisting of hydrogen, organic ligands, and heterorganic ligands.

2. The process according to claim 1 , wherein the contacting is performed at temperatures between about −5° C. to 55° C., and at a pH between 2 and 8, and wherein the composition, the α-effect amine, and the aqueous solvent are biorthogonal and the composition is provided at a concentration of less than or equal to 5 mM.

3. The process according to claim 1 , wherein the composition comprises a cis-carbonyl-substituted arylboronic acid or ester.

4. The process according to claim 1 , wherein:

R 1 , R 2 , and R 3 are independently selected from the group consisting of H, CH 3 , C 1 -C 6 alkyl, C 1 -C 6 alkyl which incorporates one further heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur, C 2 -C 6 alkanoyl, CH 2 Ar, CH 2 CH 2 Ar, an aromatic ring, and an aromatic ring substituted with a substituent selected from the group consisting of a fluorescent group, a sugar, and a polyethylene glycol chain, and

Ar is selected from the group consisting of a phenyl, a substituted phenyl ring, a naphtyl, a heteroaromatic ring, and a fused ring comprising at least one ring heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur.

5. The process according to claim 1 , wherein the α-effect amine is selected from the group consisting of:

H 2 N—X 3 R 4 ,

wherein:

X 3 is O or N; and

R 4 is an alkyl, aryl or a heteroatom containing group.

6. The process according to claim 5 , wherein R 4 is selected from the group consisting of: H, CH 3 , CH 2 CH 3 , CH 2 Ph, p-COOH Ph, o-NH 2 Ph, o-OH Ph, COH, COCH 3 , COCH 2 Ph, COPh, CO-coumarin, and CONH 2 .

7. The process according to claim 1 , wherein the α-effect amine is selected from the group consisting of an alpha hydrazide of tyrosine, phenylalanine, alanine, beta-alanine, glycine, dimethylglycine, and CBz-serine.

8. The process according to claim 1 , wherein the α-effect amine is selected from the group consisting of: a hydrazine; a semicarbazide, a thiosemicarbazide; a hydrazide, a thiohydrazide, a hydroxylamine, an O-alkylhydroxylamine, and an O-arylhydroxylamine.

9. The process according to claim 1 , wherein:

(a) the α-effect amine is selected from the group consisting of:

H 2 N—X 3 R 4 ,

wherein:

X 3 is O or N; and

R 4 is an alkyl, aryl or a heteroatom containing group; and

(c) the adduct comprises a composition selected from the group consisting of:

and a further product thereof formed through at least one of dehydration, intramolecular reaction, interaction with the solvent, and interaction with a reactive heteroatom in the solvent.

10. The process according to claim 9 , wherein the further product comprises a dehydration product selected from the group consisting of:

wherein X 4 is selected from the group consisting of alkyl, aryl, heteroalkyl, heteroaryl, hydroxyl, and water.

11. The process according to claim 1 , wherein the spontaneously formed adduct is selected from the group consisting of a hydrazono arylboronic acid, an imino arylboronic acid, a 3,4-borazaisoquinoline and a 1,2-dihydrobenzo [d][1,2,3]diazaborinin-1-uide.

12. The process according to claim 1 , wherein the adduct is selected from the group consisting of:

wherein:

R 2 is H or CH 3 ,

R 3 and R 6 are independently selected from the group consisting of alkyl or OR, wherein R is selected from the group consisting of alkyl, heteroalkyl, heteroaryl, alkylamine, alkylthiol, alkylbromide, arylbromide, C 2 -C 6 alkanoyl, CH 2 Ar or CH 2 CH 2 Ar,

in which a heteroatom of the heteroalkyl and heteroaryl is selected from the group consisting of nitrogen, oxygen, and sulfur,

the Ar group of CH 2 Ar or CH 2 CH 2 Ar is selected from the group consisting of a phenyl, a substituted phenyl ring, a naphtyl, a heteroaromatic ring, and a fused ring comprising at least one ring heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur, a 4to 7 member ring optionally incorporating one or more heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur, an aromatic ring optionally substituted with a fluorescent group, a sugar, and a polyethylene glycol chain; and

R 5 is selected from the group consisting of H, CH 3 , CH 2 CH 3 , CH 2 Ph, Ph, substituted Ph, and NH 2 .

13. The process according to claim 1 , wherein the composition comprises a cis-carbonyl substituted arylboronic acid selected from the group consisting of:

an ortho formyl phenylboronic acid or ester derivative;

an ortho ketone phenylboronic acid or ester derivative;

an ortho aldehyde phenylboronic acid ester derivative of an amino acid;

a ketone phenylboronic acid or ester derivative of an amino acid;

an ortho aldehyde phenylboronic acid derivatized with an orthogonal reactive functional group; and

a ketone phenylboronic acid derivatized with an orthogonal reactive functional group.

14. The process according to claim 1 , wherein the aqueous solvent has a pH of about 7, and the spontaneous formation of the adduct is substantially complete within a period of less than about 10 minutes at a temperature of about 25° C., and the composition is present at a concentration of less than or equal to 5 mM.

15. An adduct composition, formed by a process comprising:

providing:

(a) a composition having a boron atom bonded to a sp 2 hybridized carbon, conjugated with a cis-carbonyl, and linked to a first moiety, the boron atom having at least one labile substituent, the composition being configured to spontaneously react in an aqueous solvent at −5° C. with an α-effect amine to undergo replacement of the carbonyl oxygen by formation of a carbon-nitrogen bond in the aqueous solvent,

wherein the composition is:

wherein:

X 1 , X 2 are groups that can hydrolyze from the boron in the aqueous solvent to yield boronic acid; and

R 1 , R 2 , and R 3 are selected from the group consisting of hydrogen, organic ligands, and heterorganic ligands, and at least one of R 1 , R 2 , and R 3 are selected from the group consisting of hydrogen, organic ligands, and heterorganic ligands; and

(b) an α-effect amine, linked to a second moiety, the α-effect amine being configured to spontaneously react in an aqueous solvent at −5° C. with the composition, to undergo the replacement of the carbonyl oxygen and the formation of the carbon-nitrogen bond in the aqueous solvent; and

contacting the composition with the α-effect amine, in the aqueous solvent at a temperature below about 55° C., to spontaneously form an adduct having an interacting boron of the composition and nitrogen of the α-effect amine, linking the first moiety and the second moiety, wherein at least one of the first moiety and the second moiety is selected from the group consisting of a probe, a protein, an antibody, a peptide, an amino acid, a nucleic acid, a linker, a drug, a radiolabel, a fluorophore, a sugar, a carbohydrate, a support, a surface, a bead, and a nanoparticle.

16. The adduct composition according to claim 15 , wherein the composition is:

the α-affect amine is:

and

the spontaneously formed adduct is selected from the group consisting of:

wherein:

X 1 and X 2 are groups that can hydrolyze from the boron to yield boronic acid;

R 2 is selected from the group consisting of H and CH 3 ;

R 3 and R 6 are independently selected from the group consisting of H, OH, O-alkyl, O-heteroalkyl, O-heteroaryl, O-alkylbromide, O-arylbromide, O-alkylamine, O-alkylamide, O-alkylthiol, O-alkylthioester, O-C 2 -C 6 alkanoyl, O-CH 2 Ar, and —CH 2 CH 2 Ar, alkyl, aryl, heteroalkyl, heteroaryl, alkylamine, alkylamide and alkylbromide; and

R 5 is selected from the group consisting of H, —CH 3 , —CH 2 CH 3 , —CH 2 Ph, Ph, substituted-Ph, and —NH 2 in which a heteroatom of the heteroalkyl and heteroaryl is selected from the group consisting of nitrogen, oxygen, and sulfur, and

the Ar group of CH 2 Ar or CH 2 CH 2 Ar is selected from the group consisting of a phenyl, a substituted phenyl ring, a naphtyl, a heteroaromatic ring or fused ring comprising at least one ring heteroatom selected from nitrogen, oxygen and sulfur, a 4 to 7 ring optionally incorporating one or more heteroatoms selected from oxygen, nitrogen or sulfur, an aromatic ring optionally substituted with a fluorescent group, sugars or polyethylene glycol chain.

17. A kit, comprising:

(a) a composition linked to a first moiety, having a boron atom bonded to a sp 2 hybridized carbon, the boron having at least one labile substituent, conjugated with a cis-carbonyl,

wherein the composition is:

wherein:

X 1 , X 2 are groups that can hydrolyze from the boron in the aqueous solvent to yield boronic acid; and

R 1 , R 2 , and R 3 are selected from the group consisting of hydrogen, organic ligands, and heterorganic ligands, and at least one of R 1 , R 2 , and R 3 are selected from the group consisting of hydrogen, organic ligands, and heterorganic ligands; and

(b) an α-effect amine linked to a second moiety,

wherein at least one of the first moiety is selected from the group consisting of a probe, a protein, an antibody, a peptide, an amino acid, a nucleic acid, a linker, a drug, a radiolabel, a fluorophore, a sugar, a carbohydrate, a support, a surface, a bead, and a nanoparticle,

the composition and the α-effect amine each being respectively configured to spontaneously react with each other in an aqueous solvent at a temperature below about 55° C. to undergo replacement of the carbonyl oxygen cis-conjugated to the boron, by formation of a carbon-nitrogen bond with the amine nitrogen of the α-effect amine in the aqueous solvent to form an adduct.

18. The kit according to claim 17 , wherein:

the α-effect amine is selected from the group consisting of:

H 2 N—X 3 R 4 ,

wherein:

X 3 is O or N; and

R 4 is an alkyl, aryl or a heteroatom containing group;

wherein the composition and α-effect amine are configured to spontaneously form at least one adduct at pH 7, 25° C., in the aqueous solvent selected from the group consisting of:

and optionally at least one of:

a further product formed from the adduct through dehydration selected from the group consisting of:

wherein X 4 is selected from the group consisting of alkyl, aryl, heteroalkyl, heteroaryl, hydroxyl and water,

a further product formed from the at least one adduct through interaction with the solvent;

a further product formed from the at least one adduct through interaction with a reactive heteroatom in the solvent or the adduct.

19. A compound, comprising an anhydrous or hydrated, compound selected from the group consisting of:

wherein X═O or NH.