Construction of affinity sorbents utilizing glutathione analogs

Solution-phase N-fluorenylmethoxycarbonyl (Fmoc) mediated peptide synthesis has been adapted to the synthesis of glutathione (gamma-glutamyl-cysteinyl-glycine) analogs. A protecting group strategy has been devised in which all of the masking groups are removed with mild base. This allows for the synthesis of acid-sensitive materials and lessens concerns about the alkylation at sulfur by carbocations known to be present in the trifluoroacetic acid mixtures usually employed for deprotection of peptides made by the Fmoc methodology. A series of structurally varied glutathione analogs were prepared by modifying the peptide in two ways. The first involved C-terminal substitution for glycine by one of several different amino acids. The second involved substitution of one of five alkyl or aryl groups onto the cysteine sulfhydryl. The complete set of all combinations would yield 48 reagents, of which 25 have actually been synthesized. Following confirmation of the structures by FAB mass spectrometry, the peptides were immobilize by conjugation to epoxyfunctionalized Sepharose at pH 11-12. The amount and identity of immobilized peptide was assayed by amino acid analysis of acid-hydrolyzed resin. One of the tripeptides was purified by ion-exchange and preparative HPLC.