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

Abstract

A reaction method comprising combining a carbonyl-substituted arylboronic acid or ester and an -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 boron compound having a boron atom bonded to a sp.sup.2 hybridized carbon conjugated with a cis-carbonyl, the boron having at least one labile substituent, with an -effect amine, in a solvent for a time sufficient to form an adduct, which may proceed to further products.

Claims

1. A process comprising: (a) providing: (1) a boron compound having a boron atom covalently bonded to an sp.sup.2 hybridized carbon conjugated with a cis-carbonyl, selected from the group consisting of: ##STR00014## wherein: X.sub.1, X.sub.2 are independently selected from groups that can hydrolyze from the boron to yield boronic acid; and R.sub.1, R.sub.2, and R.sub.3 are independently selected from the group consisting of hydrogen, organic ligands, and heterorganic ligands and optionally joined in one or more rings, wherein X.sub.1, X.sub.2, R.sub.1, R.sub.2, and R.sub.3 exclude -effect amines, and wherein the boron compound is adapted to selectively and spontaneously react with an -effect amine at in a liquid solvent, a temperature of less than 55 C., and at a pH above 2, to at least form an adduct by formation of a covalent bond between the boron compound and the -effect amine; and (2) an -effect amine comprising an amino acid, and having an amine group bonded to an oxygen, nitrogen, or sulfur; and (b) contacting the boron compound with the -effect amine in the liquid solvent at the temperature of less than 55 C. and at the pH above 2, to spontaneously form the adduct.

2. The process according to claim 1, wherein the solvent comprises an aqueous medium, the contacting being performed at temperatures between about 5 C. to 55 C., and at a pH between 2 and 8, and wherein the boron compound, the -effect amine, and the solvent are bioorthogonal.

3. The process according to claim 1, wherein the -effect amine comprises an amine group bonded to an oxygen which is reactive with the boron compound.

4. The process according to claim 1, wherein the -effect amine comprises an amine group bonded to a nitrogen which is reactive with the boron compound.

5. The process according to claim 1, wherein the boron compound comprises a carbonyl-substituted arylboronic acid or ester.

6. The process according to claim 1, wherein the adduct undergoes a molecular rearrangement after forming an initial covalent bond between the cis-carbonyl carbon and the -effect amine.

7. The process according to claim 1, wherein: R.sub.2 is selected from the group consisting of: H, CH.sub.3; and R.sub.3 is selected from the group consisting of OH, O-alkyl, O-alkylbromide, O-alkylamine, O-alkylamide, O-alkylthiol, O-alkylthioester, alkylamine, alkylamide and alkylbromide.

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

9. The process according to claim 1, wherein the -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.

10. The process according to claim 1, wherein the boron compound comprises a 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 a reactive functional group which is orthogonal with respect to the boron compound and the -effect amine; and a ketone phenylboronic acid derivatized with a reactive functional group which is orthogonal with respect to the boron compound and the -effect amine.

11. The process according to claim 1, wherein the liquid solvent is an aqueous medium having a pH of about 6-8, and the spontaneous formation of the adduct is substantially complete within a period of less than about 10 minutes at a temperature of about 0-42 C.

12. The process according to claim 1, wherein the adduct comprises a boron compound selected from the group consisting of: ##STR00015## and a further product thereof formed through at least one of dehydration, interaction with the solvent, and interaction with a reactive heteroatom in the solvent, wherein: X.sub.3 is O or N; and R.sub.4 is an alkyl, aryl, or heteroatom containing group.

13. The process according to claim 12, wherein the further product comprises a dehydration product selected from the group consisting of: ##STR00016## wherein X.sub.3 is N, and X.sub.4 is selected from the group consisting of alkyl, aryl, heteroalkyl, heteroaryl, hydroxyl, and water.

14. The process according to claim 12, wherein R.sub.4 is selected from the group consisting of: H, CH.sub.3, CH.sub.2CH.sub.3, CH.sub.2Ph, p-COOH Ph, o-NH.sub.2Ph, o-OH Ph, COH, COCH.sub.3, COCH.sub.2Ph, COPh, CO-coumarin, and CONH.sub.2.

15. A boron compound comprising a boron atom bonded to a sp.sup.2 hybridized carbon conjugated with a cis-carbonyl and a biomolecule, selected from the group consisting of: ##STR00017## wherein: X.sub.1, X.sub.2 are independently selected from groups that can hydrolyze from the boron to yield boronic acid; and R.sub.1, R.sub.2, and R.sub.3 are independently selected from the group consisting of hydrogen, organic ligands, and heterorganic ligands and optionally joined in one or more rings, at least one of R.sub.1, R.sub.2, and R.sub.3 being the biomolecule; wherein X.sub.1, X.sub.2, R.sub.1, R.sub.2, and R.sub.3 exclude -effect amines, the boron compound being selectively and spontaneously reactive to form at least a covalently bonded adduct by formation of a covalent bond between the boron compound and an -effect amine comprising an amine group bonded to an oxygen, nitrogen, or sulfur, and non-reactive to form a covalently bonded adduct with a non -effect amine, in an aqueous solution at pH 7 at 25 C., wherein the biomolecule comprises at least one of an amino acid, a peptide, a nucleotide, a carbohydrate, and a drug.

16. The boron compound according to claim 15, wherein the adduct is selected from the group consisting of: (i) ##STR00018## (ii) a further product formed from (I) formed through dehydration: ##STR00019## wherein X.sub.3 is N, and X.sub.4 is selected from the group consisting of alkyl, aryl, heteroalkyl, heteroaryl, hydroxyl and water, (iii) a further product formed from (I) through interaction with the solvent; and (iv) a further product formed from (I) through interaction with a reactive heteroatom in the solvent.

17. The boron compound according to claim 15, wherein the adduct is selected from the group consisting of: ##STR00020## wherein: R.sub.2 is H or CH.sub.3; R.sub.3 and R.sub.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.sub.2-C.sub.6 alkanoyl, CH.sub.2Ar or CH.sub.2CH.sub.2Ar, wherein R.sub.3 further comprises the biomolecule, 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.sub.2Ar or CH.sub.2CH.sub.2Ar 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 4 to 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.sub.5 is selected from the group consisting of H, CH.sub.3, CH.sub.2CH.sub.3, CH.sub.2Ph, Ph, substituted Ph, and NH.sub.2.

18. The boron compound according to claim 15, wherein the boron compound comprises a 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 a reactive functional group which is orthogonal with respect to the boron compound and the -effect amine; and a ketone phenylboronic acid derivatized with a reactive functional group which is orthogonal with respect to the boron compound and the -effect amine.

19. The boron compound according to claim 15, provided as a kit in combination with the -effect amine.

20. A process comprising: combining: (1) a biorthogonal compound having a boron atom covalently bonded to an sp.sup.2 hybridized carbon conjugated with a cis-carbonyl, selected from the group consisting of: ##STR00021## wherein: X.sub.1, X.sub.2 are independently selected from groups that can hydrolyze from the boron to yield boronic acid; and R.sub.1, R.sub.2, and R.sub.3 are independently selected from the group consisting of hydrogen, organic ligands, and heterorganic ligands and optionally joined in one or more rings, wherein X.sub.1, X.sub.2, R.sub.1, R.sub.2, and R.sub.3 exclude -effect amines, and wherein the boron compound is adapted to selectively and spontaneously react with an -effect amine at in a liquid solvent, a temperature of less than 55 C., and at a pH above 2, to at least form a covalently bonded adduct by formation of a covalent bond between the boron compound and the -effect amine; and (2) an -effect amine comprising an amino acid, having an amine group bonded to an oxygen, nitrogen, or sulfur, in the liquid solvent at the temperature of less than 55 C. and at the pH above 2, to spontaneously form the covalently bonded adduct between the biorthogonal compound and the -effect amine to essential completion within 10 minutes, wherein at least one of the biorthogonal compound and the -effect amine comprises at least one of an amino acid, a peptide, a nucleotide, a carbohydrate, a drug, a dye, a fluorophore, a radionuclide, and an isotopically enriched compound.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The Invention will now be described with reference to the drawings, in which:

(2) FIG. 1 shows the reaction of aromatic boronic acid or ester with -effect amines at neutral pH.

(3) FIG. 2 shows p-boronophenylalanine.

(4) FIG. 3 shows reaction pathways of SAL of 2-formylphenylboronic acid (fPBA) with PhNHNH.sub.2.

(5) FIGS. 4A-4D show reaction kinetics of SAL and PhNHNH.sub.2, compared with PBA and PhNHN.sub.2

(6) FIG. 5 shows a reaction of an aromatic aldehyde or boronic acid with a hydrazide.

(7) FIGS. 6A-6D show reaction kinetics of F-Tyr and CHzide, compared with fPBA and CHzide.

(8) FIG. 7 shows the 2-boronic acid benzophenone with a hydrazide.

(9) FIGS. 8A and 8B show reaction kinetics of 2-acetylphenylboronic acid with coumarin hydrazide.

(10) FIG. 9 shows the reaction of fPBA with phenylhydrazide and phenylhydrazine in neutral buffer.

(11) FIG. 10 shows reaction kinetics of phenylhydrazide and benzyl hydrazine with fPBA.

(12) FIGS. 11 and 12 shows products of fPBA with O-methylhydroxylamine, hydroxylamine and hydrazine hydrate, and their respective NMR spectra.

(13) FIG. 13 shows a reaction of Tyrosine hydrazide tubulin and fPBA.

(14) FIG. 14 shows a change in UV absorbance spectra over time.

(15) FIG. 15 shows linking of a protein with an fPBA linked to a substance of interest.

(16) FIGS. 16, 17, 18, 19 and 20 show fPBA or esters thereof having various second functional groups.

(17) FIG. 21 shows a prior art Drug-Antibody Conjugates, Trastuzumab emtansine, for HER2-positive metastatic breast cancer linked through an MCC linker to maytansine.

(18) FIGS. 22 and 23 show Taxol linked through a spacer modified to terminate in a formylboronic acid fPBA for coupling to monoclonal antibodies.

(19) FIGS. 24A and 24B show antibodies modified with a non-natural amino acid containing a hydrazide or a fPBA functionality.

(20) FIGS. 25A and 25B show a coupling of Antibody-hydrazide with fPBA-derivatized taxol and a coupling of Antibody-2fPBA with hydrazide-derivatized taxol.

(21) FIGS. 26A and 26B show biomolecule immobilization with hydrazide-functionalized surface and biomolecule immobilization with fPBA-functionalized surface, bead, resin, or other material

(22) FIG. 27 shows hydrazide-oligonucleotide immobilization on fPBA-functionalized surface, bead, resin, or other material.

(23) FIGS. 28 and 29 show a prior art and a new .sup.18F PET radionuclide, respectively.

(24) FIG. 30 shows a schematic depiction of metabolic labeling.

(25) FIGS. 31A and 31B show semicarbazide and azide derivatized sugars.

(26) FIG. 32 shows a generic reaction according to the present technology.

(27) FIGS. 33A, 33B and 33C show alternate products of the reaction.

(28) FIGS. 34A, 34B, 34C and 34D show alternate products of the reaction.

(29) FIGS. 35A and 35B show a cyclized boronic acid reaction product in anhydrous and hydrated form.

(30) FIG. 36 shows a tetracyclic product according to the present technology.

(31) FIGS. 37 and 38 show antibody linkage reactions according to the present technology.

(32) FIGS. 39A-39D and 40A-40C show carbonyl-substituted arylboronic acid moieties according to the present technology.

(33) FIG. 41 shows reaction of a boron atom bonded to a sp.sup.2 hybridized carbon, the boron having at least one labile substituent, conjugated with a cis-carbonyl, with a second composition having an -effect amine, in an aqueous medium, which may proceed to form further products.

(34) FIGS. 42A and 42B show linkage of hydrazine- and hydrazide-containing protein (bovine serum albumen) with coumarin-fPBA.

(35) FIG. 42C shows a UV fluorescence plot of the reaction.

(36) FIG. 43 shows a hydrazine-protein labeled with coumarin-fPBA imaged under long wavelength UV (left panel) and Coomassie blue dyed electrophoresis gel (right panel).

(37) FIG. 44 shows a coupling reaction of a thiol linked boronic acid according to the present technology.

(38) FIGS. 45 and 46 show prior art azide-alkyne cycloaddition coupling reactions.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(39) Procedure for forming a hydrazono or imino arylboronic acid, a 3,4-borazaisoquinoline or a related boron-containing heterocycle such as a 1,2-dihydrobenzo[d][1,2,3]diazaborinin-1-uide under biocompatible conditions.

(40) General: Each component (o-carbonyl-substituted phenylboronic acid, alpha-effect amine-containing molecule) is dissolved in water or in buffer, normally pH 7, at room temperature. The solutions are mixed such that the molar ratio of the components in the mixture is 1:1. The concentration of each reagent is normally 30 M to 5 mM. Normally a single product is formed. Stereoisomers can be formed when the product contains a chirality center. The structure of the product is identified by .sup.1H, .sup.13C and .sup.11B NMR. Reactions are followed by absorption difference spectroscopy.

Example 1

(41) Demonstration of reaction kinetics. Salicylaldehyde (Sal) represents a typical aromatic aldehyde used in a coupling reaction. Ortho-formylphenylboronic acid (2f-PBA) exemplifies the effect of the boronic acid at the ortho position. The -effect amine is an aromatic hydrazine, e.g., phenylhydrazine. The reaction is shown in FIG. 3. Therefore the boronic acid substituted fPBA produces a stable cyclic product, while the product Sal with phenylhydrazine does not cyclize.

Example 2

(42) 3-Formyltyrosine represents a typical aromatic aldehyde used in a coupling reaction. fPBA exemplifies the effect of the boronic acid at the ortho position. The -effect amine is an aromatic hydrazide, e.g., 7-diethylamino coumarin 3-carbohydrazide (CHzide), as shown in FIG. 5. Hydrazides react with aldehydes only very slowly at neutral pH. The corresponding reaction with a boronic acid ortho- to the aldehyde is complete in minutes. The 3-formyl-4-boronophenylalanine derivative would be expected to engage in similar reactions, with similar kinetics, to fPBA, in this reaction, and therefore may be used to derivatize peptides for compatibility with the present click reaction.

Example 3

(43) 2-Acetylphenylboronic acid exemplifies a ketone-substituted phenylboronic acid. Acetophenone exemplifies an aromatic ketone. See FIG. 7. No reaction was observed with acetophenone under the general conditions noted above. However, the reaction of 2-acetylphenylboronic acid with 7-(diethylamino) coumarin-3-carbohydrazide does lead to an adduct. Note that this reaction is faster than the reaction in example 2. This is the opposite of the normal trend for hydrazone-forming reactions.

Example 4

(44) Comparison of Hydrazide vs. Hydrazine. A test was conducted to compare the reaction of fPBA with phenylhydrazide and benzylhydrazine in pH 7 buffer. The hydrazide reagent reacts faster. See FIG. 9.

Example 5

(45) Reaction of fPBA with -effect amines in pH 7 buffer. Each component (o-carbonyl-substituted phenylboronic acid, alpha-effect amine-containing molecule) is dissolved in 0.1 M phosphate buffer, pH 7, at room temperature. The concentration of each reagent is normally 1-2 mM. The solutions are mixed such that the molar ratio of the components in the mixture is 1:1. Proton NMR spectra are collected within 10 minutes after mixing. Shown in FIG. 12 are spectra of the results from mixing fPBA with O-methylhydroxylamine, hydroxylamine and hydrazine hydrate. See FIG. 11.

Example 6

(46) Coupling to Proteins. Hydrazine- and hydrazide-containing bovine serum albumen (BSA) were used as a model to demonstrate the coupling reaction between fPBA and alpha-effect amines on proteins. BSA containing phenylhydrazine functionalities was prepared by allowing the hydroxysuccimidyl ester of 4-(2-(propan-2-ylidene)hydrazinyl)benzoic acid to react with the protein. Hydrazine-BSA or unmodified BSA (final concentration 15 M) was allowed to react with coumarin-2fPBA at room temperature for 5 min. See FIGS. 42A, and 43. The samples were then prepared for SDS PAGE analysis. The gel was visualized using long wave UV light and was then stained for protein, as shown in FIG. 43, in which the left panel is imaged under long wavelength UV (negative of the image is shown for clarity), and the right panel shows a Coomassie stained gel.

(47) BSA with hydrazide functionalities was prepared by allowing oxidized BSA to react with adipic dihydrazide. BSA hydrazide (Zide-BSA, final concentration 10 M) or oxidized BSA (O-BSA, final concentration 10 M) was allowed to react with 20 M C-2fPBA for 30 min in 10 mM sodium phosphate buffer (pH 7) prior to rapid gel filtration. The samples were excited at 340 nm, and emission spectra were collected. Each spectrum was normalized by protein concentration estimated by BCA assay. See FIGS. 42B and 42C.

Example 7

(48) Bifunctional linkers. Molecules that possess a second reactive functional groups may be used to link the boronic acid or boronic ester conjugated to a carbonyl-containing moiety to the desired partner, as shown in FIG. 16. A boronic acid or boronic ester conjugated to a carbonyl-containing moiety. The ring may be substituted. A reactive functional group (R.sub.3) is appended to the ring or part of a substituent attached to the ring. See FIG. 1, left structure.

Example 8

(49) C-terminal protein labeling. Tyrosine hydrazide (Y-zide) is covalently bonded to the carboxyl terminus of alpha-tubulin using the enzyme tubulin tyrosine ligase as described in Mukherjee and Bane. (Mukherjee, K., and Bane, S. L. Site-specific fluorescent labeling of tubulin, In Microtubules, In Vitro 2nd ed., pp 1-12 (2013)). Y-zide-tubulin is equilibrated in PME buffer (0.1 M PIPES, 1 mM MgSO.sub.4, 2 mM EGTA, pH 6.9) using rapid gel filtration. To the Y-zide-tubulin solution is added fPBA in 10 mM phosphate buffer, pH 7. The final concentration of Y-zide-tubulin and of fPBA is 93 M. The reaction progress is monitored by absorption spectroscopy. The appearance of a shoulder at 310 nm is indicative of product formation. See FIG. 13.

(50) C-terminal hydrazide-containing proteins may be synthesized as described by Thom et al (Jennifer Thom; David Anderson; Joanne McGregor; Graham Cotton; Bioconjugate Chem. 2011, 22, 1017-1020). The probe or substance of interest (fluorophore, nanoparticle, protein, carbohydrate, surface, etc.) is covalently bonded to the reactive functional group on the probe (such as amine, thiol, azide, alkyne) using standard methods. The hydrazide-containing protein is allowed to react with the probe at neutral pH at microM to millimolar concentration and 1:1 stoichiometry at room temperature for 5-60 min. Progress of the reaction may be monitored by a change in the absorption spectrum. See FIG. 15.

Example 9

(51) In addition to the method shown in Example 6, internal amino acid protein labeling may be performed using unnatural amino acid mutagenesis. Unnatural amino acid mutagenesis is a known method for adding reactive functional groups to proteins. This has been done with boronophenylalanine. (Liu, C. C., and Schultz, P. G. Adding New Chemistries to the Genetic Code, In Annual Review of Biochemistry, Vol 79 (Kornberg, R. D., Raetz, C. R. H., Rothman, J. E., and Thorner, J. W., Eds.), pp 413-444 (2010), U.S. Pat. No. 8,637,306, US 20090148887; WO 2009/059056 entitled A Genetically Encoded Boronate Amino Acid, filed Oct. 30, 2008; U.S. Pat. Nos. 8,637,306; 8,632,970; 8,609,383; US 20110312027; US 20100297693; WO 2013/084198; Miyaura and Suzuki (1995) Palladium-Catalyzed Cross-Coupling Reactions of Organoboron Compounds, Chemical Reviews 95: 2457 and Suzuki (1999) Recent advances in the cross-coupling reactions of organoboron derivatives with organic electrophiles, 1995-1998, Journal of Organometallic Chemistry 576:147.) Therefore, the present technology provides that a protein labeled with an ortho-carbonyl substituted boronophenylalanine can be coupled using a click chemistry reaction to a hydrazide molecule as discussed herein for labeling C-termini. Alternatively, protein with an alpha-effect amine-containing unnatural amino acid could be prepared using unnatural amino acid mutagenesis or synthetic chemistry techniques for coupling to an ortho-carbonyl-substituted phenylboronic acid.

Example 10

(52) Drug-Antibody Conjugates are provided. Procedures for coupling fluorophores to proteins are generally applicable to coupling drugs to targeting proteins such as antibodies. One example of an antibody-drug complex is Trastuzumab emtansine, which has been used for HER2-positive metastatic breast cancer. Maytansine can be attached to the antibody through an MCC linker, as shown below. See, Elkins K et al. Mol Cancer Ther 2012; 11:2222-2232. See FIG. 20.

(53) Likewise, Taxol may be linked through a spacer modified to terminate in a formylboronic acid fPBA or a hydrazide for coupling to targeting proteins such as antibodies. A Taxol derivative with an amine-terminated spacer is known. Altering the spacer to terminate in an alpha-effect amine or an ortho carbonyl-phenylboronic acid allows for the drug to be attached to the appropriately modified antibody. See FIGS. 21 and 22.

(54) In order to add a hydrazide or ortho-carbonyl phenylboronic acid functionality to an antibody, a standard coupling procedure that uses maleimide reactive groups and Cys residues in the protein can be employed (Shen, B.-Q.; Xu, K.; Liu, L.; Raab, H.; Bhakta, S.; Kenrick, M.; Parsons-Reponte, K. L.; Tien, J.; Yu, S.-F.; Mai, E.; Li, D.; Tibbitts, J.; Baudys, J.; Saadi, O. M.; Scales, S. J.; McDonald, P. J.; Hass, P. E.; Eigenbrot, C.; Trung, N.; Solis, W. A.; Fuji, R. N.; Flagella, K. M.; Patel, D.; Spencer, S. D.; Khawlil, L. A.; Ebens, A.; Wong, W. L.; Vandlen, R.; Kaur, S.; Sliwkowski, M. X.; Scheller, R. H.; Polakis, P.; Junutula, J. R., Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates. Nat. Biotechnol. 2012, 30 (2), 184-189.) A bifunctional reagent that possesses a maleimide and either a hydrazide or a formylboronic acid can be used to add the linker, as shown in FIG. 23.

(55) Alternatively, the antibody may be expressed with unnatural amino acids, as is known with different reactive amino acids. (Axup, J. Y., Bajjuri, K. M., Ritland, M., Hutchins, B. M., Kim, C. H., Kazane, S. A., Halder, R., Forsyth, J. S., Santidrian, A. F., Stafin, K., Lu, Y., Tran, H., Seller, A. J., Biroc, S. L., Szydlik, A., Pinkstaff, J. K., Tian, F., Sinha, S. C., Felding-Habermann, B., Smider, V. V., and Schultz, P. G. (2012) Synthesis of site-specific antibody-drug conjugates using unnatural amino acids, Proc. Natl. Acad. Sci. USA 109, 16101-16106; Tian, F.; Lu, Y.; Manibusan, A.; Sellers, A.; Tran, H.; Sun, Y.; Phuong, T.; Barnett, R.; Hehli, B.; Song, F.; DeGuzman, M. J.; Ensari, S.; Pinkstaff, J. K.; Sullivan, L. M.; Biroc, S. L.; Cho, H.; Schultz, P. G.; DiJoseph, J.; Dougher, M.; Ma, D.; Dushin, R.; Leal, M.; Tchistiakova, L.; Feyfant, E.; Gerber, H.-P.; Sapra, P., A general approach to site-specific antibody drug conjugates. Proc. Natl. Acad. Sci. USA 2014, 111 (5), 1766-1771.). See FIGS. 24 and 25.

(56) A key advantage of this technology is that the drug may be coupled to the antibody quickly, at neutral pH and without excess reagent. Integrating a non-natural amino acid reduces post-production steps for the antibody, and allows the antibody to be labeled after addition to a biological system; the standard coupling procedure is not selective, and will add hydrazine functionality to all exposed cysteine residues.

Example 11

(57) Nanoparticle-biomolecule conjugates. Gold nanoparticles (1-100 nm), nanospheres and nanorods have applications in photothermal therapy and optical and contrast imaging techniques. (Algar, W. R., Prasuhn, D. E., Stewart, M. H., Jennings, T. L., Blanco-Canosa, J. B., Dawson, P. E., and Medintz, I. L. (2011) The Controlled Display of Biomolecules on Nanoparticles: A Challenge Suited to Bioorthogonal Chemistry, Bioconj. Chem. 22, 825-858.) Targeting nanoparticles to particular in vivo locations requires conjugation to a biological moiety. The gold nanostructure must first be coated with an appropriate linkers, which is generally accomplished through bifunctional thiol ligands, as shown in FIG. 45.

Example 12

(58) Biomolecules may be attached to a surface according to the present technology. 96 well plates with hydrazide functional groups are commercially available (for example, Corning Carbo-BIND 96 well plates). Biomolecules that have been covalently modified with the reactive ortho-carbonyl phenylboronic acid can then be attached to these plates by adding the desired molecule to the well. If the modified protein is an antibody, then such plates may be used for ELISA (Brillhart, K. L., and Ngo, T. T. (1991) Use Of Microwell Plates Carrying Hydrazide Groups To Enhance Antibody Immobilization In Enzyme Immunoassays, J. Immunol. Methods 144, 19-25.). Other commercially available products include hydrazide-modified magnetic beads (BcMag Hydrazide-modified Magnetic Beads; Bioclone Inc), hydrazide containing resins (Affi-Gel Hz hydrazide gel, Bio-Rad), glass slides, membranes, plates, and nanoparticles (Biosynthesis, Inc.) See also Applying Genomic and Proteomic Microarray Technology in Drug Discovery, Second edition. (Robert S. Matson, ed. CRC Press, 2013). See FIG. 26A.

(59) A bifunctional ortho-carbonyl phenylboronic acid linker can also be attached to appropriately functionalized solid supports, surfaces or beads. The production is within the ordinary skill in the art, for example, immobilized phenylboronic acids are commercially available (Pierce Boronic Acid Resin, Affi-Gel Boronate Affinity Gel), Such surfaces would be then be available for linking to substances containing alpha-effect amines. See FIG. 26B.

Example 13

(60) Nucleic acid conjugates are provided. Hydrazides can be appended easily to nucleic acids. (See, for example, Ghosh, S. S., Kao, P. M., and Kwoh, D. Y. (1989) Synthesis Of 5-Oligonucleotide Hydrazide Derivatives And Their Use In Preparation Of Enzyme Nucleic-Acid Hybridization Probes, Analytical Biochemistry 178, 43-51; Raddatz, S., Mueller-Ibeler, J., Kluge, J., Wass, L., Burdinski, G., Havens, J. R., Onofrey, T. J., Wang, D., and Schweitzer, M. (2002) Hydrazide oligonucleotides: new chemical modification for chip array attachment and conjugation, Nucleic Acids Res. 30, 4793-4802; Zatsepin, T. S., Stetsenko, D. A., Gait, M. J., and Oretskaya, T. S. (2005) Use of carbonyl group addition-elimination reactions for synthesis of nucleic acid conjugates, Bioconj. Chem. 16, 471-489.) These hydrazide modified nucleic acids are then attached to a probe, nanoparticle, surface, etc. using the boronic acid-based linker. See FIG. 27.

Example 14

(61) Targeting PET Probes. A problem for preparing radiolabeled conjugates for positron emission tomography (PET) use is the short half-life of .sup.18F, a commonly used emitter. The longer the time required to prepare the conjugate, the less isotope will be available for patient imaging. The present technology can shorten the time to prepare the conjugate, and the reaction is stoichiometric, and the reagents and product are bioorthogonal (to the limits of .sup.18F radiopharmaceutical pharmacology), so a purification step is not necessary. Click-Chemistry Reactions in Radiopharmaceutical Chemistry: Fast & Easy Introduction of Radiolabels into Biomolecules for In Vivo Imaging. Current Medicinal Chemistry, 2010, 17, 1092-1116. Therefore, 4-.sup.18F-fluoro-N-(prop-2-ylyl)-benzyamide (FIG. 28) is replaced with 4-.sup.18F-fluoro-benzoyl hydrazine (FIG. 29).

Example 15 Carbohydrate Labeling

(62) Laughlin et al. were able to image glycans in developing zebrafish by the use of click chemistry. Cancer Biother Radiopharm. 2009 June; 24(3): 289-302. In this study, embryonic zebrafish were incubated with an azide-peracetylated N-azidoacetylgalactosamine derivative (Ac.sub.4-GalNAz), which was then reacted with a difluorinated cyclooctyne attached to a dye. See FIG. 30. A semicarbazide derivative of galactosamine is used in place of Ac.sub.4-GalNAz, as shown in FIGS. 31A and 31B.

(63) All patents and publications mentioned in this specification are expressly incorporated herein by reference in their entirety, and may be pertinent to various issues.

(64) It is to be understood that while a certain form of the invention is illustrated, it is not to be limited to the specific form or arrangement herein described and shown. It will be apparent to those skilled in the art that various changes may be made without departing from the scope of the invention and the invention is not to be considered limited to what is shown and described in the specification and any drawings/figures included herein. The embodiments, methods, procedures and techniques described herein are presently representative of the preferred embodiments, are intended to be exemplary and are not intended as limitations on the scope. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention and are defined by the scope of the appended claims.