The amine‐donor substrate specificity of tissue‐type transglutaminase has been studied in a series of recombinant αA‐crystallin mutants. These mutant proteins have been provided with a potential substrate lysine residue, flanked by different amino acid residues, in the C‐terminal extended arm of αA‐crystallin. A biotinylated amine‐acceptor hexapeptide was used as a probe for labelling the amine‐donor sites. Wild‐type bovine αA‐crystallin does not function as an amine‐donor substrate for tissue‐type transglutaminase. Yet, upon introduction of a lysine residue at the C‐terminal or penultimate position, all mutant αA‐crystallins act as amine‐donor substrates, although to different extents. This shows that accessibility is the primary requirement for a lysine residue to function as an amine‐donor substrate for transglutaminase and that the enzyme has a broad tolerance towards the neighbouring residues. However, the nature of the flanking amino acid residues does clearly affect the reactivity of the substrate lysine residue. Notably, we found that a proline or glycine residue in front of the substrate lysine has a strong adverse effect on the substrate reactivity as compared to a preceding leucine, serine, alanine or arginine residue.

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Abstract. N-Acetyl-S-(alky1sulfanyl)cysteine benzyl esters were synthesized as models for Nacylated S-(alkylsulfanyl)cysteine residues linked via an ester bond to a solid phase. The S-protections involved were: the ethylsulfanyl, the well-established tert-butylsulfanyl and the newly developed tritylsulfanyl group. We investigated the chemical behaviour of the disulfides in reagents commonly applied in solid-phase peptide synthesis using the Fmoc strategy (Fmoc SPPS). It was found that the tritylsulfanyl group as a thiol protection is comparable with the tert-butylsulfanyl group in these respects. It is stable in trifluoroacetic acid and is rapidly reduced by thiols and phosphines. For all three cysteine esters rapid racemization was observed in piperidine (25%) in DMF, the amides being chirally stable. The demonstrated chiral instability of cysteine esters has consequences for the solid-phase synthesis of peptides using the Fmoc protocol.

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