Protein bioconjugation reactions

Singh, P. (1998) Terminal groups in starburst dendrimers Activation and reactions with proteins. Bioconjugate Chem. 9, 54-63. 

These considerations form the basis for the numerous techniques that are now available for the chemical modification of proteins. The sections that follow will examine these techniques and the reactive principles by which they function. A section describing reactions that display orthogonal reactivity to native protein functional groups has also been included because of the growing importance of these reactions as tools to label proteins in complex mixtures. Because it is not practical to summarize all protein bioconjugation methods here, this information instead is intended to serve as an introduction to the concepts that drive the development of these reactions. Several additional reviews and books on protein modification have been listed in the Further Reading section. 

Fig. 10.3-1 A survey of molecules and materials that are commonly attached to proteins through bioconjugation reactions.

Fig. 10.3-2 Common strategies for protein bioconjugation, targeting lysine, cysteine, aspartic acid, and glutamic acid residues. In most situations, only cysteine modification reactions are site selective.

The above studies emphasize the ability of diazonium-coupling reactions to modify proteins with extremely high efficiency, but one of the limitations of this method is the lack of selectivity that can be obtained when there are multiple tyrosines on the surface of a single protein. This has not been problematic for the viral capsids shown above, as only one tyrosine is accessible on each monomer, but many applications demand higher levels of selectivity than allowed by these coupling reactions. To address this need, and to increase the substrate scope for bioconjugation reactions in general, a versatile Mannich-type reaction has been developed for tyrosine modification, Fig. 10.3-5 [25]. In this reaction, aldehydes and anilines are mixed to form... 

The labeling of a single biomolecule in a complex protein mixture presents a particularly difficult challenge, as no bioconjugation reaction targeting a... 

Several reports have been made using this reaction as a protein bioconjugation technique. In the first report, 60 azide functional groups attached to the surface of the cowpea mosaic virus (CPMV) using NHS ester or iodoacetamide reactions served as attachment sites for fluorescent alkyne derivatives [92]. The optimal conditions for the reaction were 21 pM protein (based on azide-functionalized capsid monomers), 1 mM CuS04, 2 mM TCEP, and 2 mM 58 in pH 8 buffer at 4°C for 16 h. As an alternative, small amounts of copper wire were also effective in generating and maintaining the Cu(I) species. The reaction was also successful when the alkynes were attached to the viral capsid and azides were attached to the dye. 

As the field of bioconjugation continues to evolve at a rapid pace, there are a number of challenges that are likely to be addressed. The first of these involves improvements in the overall reliability of existing protein modification reactions. A lesson from the field of natural product synthesis... 

Given this structural diversity, the continued development of new reactions is also crucial. Even in cases where a modification strategy is already in place for a particular functional group, alternative reactions can allow expansions in substrate scope, alterations in modification selectivity, synthetic convenience, and perhaps even greater biocompatibility. Just as a well-trained synthetic chemist must know a dozen methods for the oxidation of an alcohol to an aldehyde, protein bioconjugation will be approached with much more success if many techniques are available to address the situation at hand. 

In proteins, cysteine residues are not always readily accessible, since they are often involved in disulfide bridges within the complex three-dimensional biomacromolecular structures. Therefore, only a small number of cysteine residues can be used for bioconjugation reactions. The ligation of polymer bound dibromomaleimides takes advantage of this circumstance, as it allows for the functionalization of disulfide moieties. Haddleton and coworkers demonstrated the applicability of this reaction for bioconjugation of salmon calcitonin (sCT). ... 

This reaction has been used to make rapid and reliable covalent connections to micromolar concentrations of protein in a bioconjugation reaction in water, as well as construct a dendrimer with exceptionally high yields. ... 

F. Meng, B.N. Manjula, P.K. Smith, S.A. Acharya, PEGylation of human serum albumin reaction of PEG-phenyhsothiocyanate with proteins. Bioconjugate Chem., 19 (7) 1352-1360, 2008. 

The aqueous Diels-Alder reaction has also been used for bioconjugate studies. A Diels-Alder reaction of diene oligonucleotides with maleimide dieneophiles was used to prepare oligonucleotide conjugates in aqueous media under mild conditions (Eq. 12.39).102 A Diels-Alder-type cycloaddition of an electronically matched pair of saccharide-linked conjugated dienes and a dienophile-equipped protein was the... 

Metz B, Kersten GFA, Baart GJ, et al. Identification of formaldehyde-induced modifications in proteins reactions with insulin. Bioconjug. Chem. 2006 17 815-822. 

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