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This observation, together with the fact that 1 equivalent of DTT is not consumed in the reaction, indicated that intramolecular amide formation by nucleophilic attack of the AX side chain amino group to its electrophilic -lactam carbonyl, under these conditions, occurs only in the presence of DTT

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Therefore, AX reactivity and conjugation to carrier molecules have been the subject of thorough investigation ( Initial assays preincubating proteins with reducing agents before treatment with AX showed a clear decrease in AX-protein adduct formation in the presence of DTT, whereas the effect of GSH was less consistent ( Figure 1 )

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