Sulphydryl reactive conjugates

A sulphydryl reactive derivative of CyS then conjugated to cysteine 40 (introduced to facilitate ligation)... 

From the numerous conjugation methods available (1), only a few are described in this chapter. One example concerns the application of the homobifxmctional cross-linker glutaraldehyde. Heterobifunctional cross-linking is illustrated by coupling of thiol-containing peptides or carriers to sulphydryl-reactive carriers or peptides, respectively. 

Another approach towards conjugation is the use of cross-linkers containing two different reactive groups. Compared with homobifunctional cross-linkers, heterobifunctional linkers offer the possibility to prepare better-defined conjugates, since peptide-peptide and carrier-carrier conjugation can be prevented. Convenient procedures are based on couplings of thiols to sulphydryl-reactive groups. 

Cysteine-containing peptides can be conjugated to sulphydryl-reactive proteins, provided that the cysteine is not essential for the biological activity. If the peptide is devoid of Cys, this amino acid can be added N- or C-terminally during synthesis. Depending on the linker attached to the solid phase, C-terminal incorporation is not recommended to avoid possible racemization of Cys (17). 

Instead of using Cys-containing peptides it is convenient to use S-acetylmercaptoacetyl (SAMA) peptides for conjugation purposes. Unlike Cys, SAMA is not sensitive towards (air) oxidation during purification of the peptide. SAMA can be introduced into deprotected peptides in solution by reaction with N-succinimidyl S-acetylmercaptoacetate (18, 24). Selective introduction of SAMA can be achieved at the end of solid phase synthesis by N-terminal modification (Chapter 6) of the side-chain protected and resin-bound peptide with pentafluorophenyl 5-acetylmercaptoacetate (SAMA-OPfp) and 1-hydroxybenzotriazoIe (25). The thiol group of SAMA-peptides can be liberated by reaction with hydroxylamine. This S-deacetylation can be performed in the conjugation reaction mixture (25,26), i.e. in the presence of the sulphydryl-reactive protein Protocol 2). 

The reactive nitrenium or carbonium ions postulated to be produced will react with nucleophilic groups in nucleic acids, proteins and sulphydryl compounds such as glutathione and methionine. The arylation of DNA by acetylaminofluorene has been demonstrated in vivo and in vitro. The involvement of sulphate conjugation brings other factors into play. Depletion of body sulphate reduces and supplementation with organic sulphate increases the carcinogenicity of acetylaminofluorene. The production of covalent adducts between acetylaminofluorene and cellular macromolecules in vivo can be shown to be correspondingly decreased and increased by manipulation of body sulphate levels. 

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