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

• US 9,758,533 B2
• Filed: 04/23/2015
• Issued: 09/12/2017
• Est. Priority Date: 04/23/2014
• Status: Active Grant

• Current Assignee: The Research Foundation for The State University of New York (State University of New York)
• Original Assignee: The Research Foundation for The State University of New York (State University of New York)
• Inventors: Tuttle, Susan Bane, Dilek, Ozlem, Mukherjee, Kamalika
• Primary Examiner(s): Balasubramanian, Venkataraman
• Application Number:
  US14/694,744
• Publication Number:
  US 20150329568A1
• Time in Patent Office: 873 Days
• Field of Search: 544229, 544 69
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 31/69 Boron compounds
• CPC Class Codes: C01B 35/10 Compounds containing boron and oxygen C01B35/06 takes precedence
• CPC Class Codes: C01B 35/146 Compounds containing boron and nitrogen, e.g. borazoles ammonium tetrafluoborates C01B35/063; ammonium borates C01B35/12
• CPC Class Codes: C07F 5/02 Boron compounds
• CPC Class Codes: C07F 5/025 Boronic and borinic acid compounds
• CPC Class Codes: C07H 21/00 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 Class Codes: C07K 14/765 Serum albumin, e.g. HSA
• CPC Class Codes: C07K 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 16/40 against enzymes

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 sp² 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.

Claims

1. A process comprising:

(a) providing;

(1) a composition having a boron atom bonded to a sp² 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;

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:

R₁, R₂, and R₃ are independently selected from the group consisting of H, CH₃, C₁-C₆ alkyl, C₁-C₆ alkyl which incorporates one further heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur, C₂-C₆ alkanoyl, CH₂Ar, CH₂CH₂Ar, 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;

H₂N—

X₃R₄,

wherein;

X₃ is O or N; and

R₄ is an alkyl, aryl or a heteroatom containing group.

6. The process according to claim 5, wherein R₄ is selected from the group consisting of:

H, CH₃, CH₂CH₃, CH₂Ph, p-COOH Ph, o-NH₂ Ph, o-OH Ph, COH, COCH₃, COCH₂Ph, COPh, CO-coumarin, and CONH₂.

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;

H₂N—

X₃R₄,

wherein;

X₃ is O or N; and

R₄ is an alkyl, aryl or a heteroatom containing group; and

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

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

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:

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 sp² 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;

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

17. A kit, comprising:

(a) a composition linked to a first moiety, having a boron atom bonded to a sp² hybridized carbon, the boron having at least one labile substituent, conjugated with a cis-carbonyl,

wherein the composition is;

18. The kit according to claim 17, wherein:

the α

-effect amine is selected from the group consisting of;

H₂N—

X₃R₄,

wherein;

X₃ is O or N; and

R₄ 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;

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