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

Patent metadata

Field	Value
Publication number	US-9758533-B2
Application number	US-201514694744-A
Country	US
Kind code	B2
Filing date	Apr 23, 2015
Priority date	Apr 23, 2014
Publication date	Sep 12, 2017
Grant date	Sep 12, 2017

Abstract

A reaction method comprising combining a carbonyl-substituted arylboronic acid or ester and an α-effect amine in aqueous solution at a temperature between about −5 C to 55 C, and a pH between 2 and 8 to produce an adduct. A process is also provided comprising: contacting a composition having a boron atom bonded to a sp 2 hybridized carbon, the boron having at least one labile substituent, conjugated with a cis-carbonyl, with an α-effect amine, in an aqueous medium for a time sufficient to form an adduct, which may proceed to further products.

First claim

What is claimed is:

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

Assignees

• Univ New York State Res Found

Inventors

• Tuttle Susan Bane
• Dilek Ozlem
• Mukherjee Kamalika

Classifications

• C01B35/146

  Compounds containing boron and nitrogen, e.g. borazoles (ammonium tetrafluoborates C01B35/063; ammonium borates C01B35/12) · CPC title

• C07H21/00

  Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids · CPC title

• C07F5/025 Primary

  Boronic and borinic acid compounds · CPC title

• C01B35/10

  Compounds containing boron and oxygen (C01B35/06 takes precedence) · CPC title

• C07F5/02

  Boron compounds · CPC title