Synthesis of selenium-alkylated selenocysteine derivatives and their use in native chemical ligation, expressed protein ligation and the synthesis of dehydropeptides

Selenocysteine (Sec) has long been recognized as the 21st genetically encoded amino acid. However, until recently no good method for the incorporation of Sec into peptides and proteins existed. The machinery that codes for and inserts this unique amino acid into proteins in vivo is complex, and in some ways, not well understood. The complexity of the decoding apparatus has caused limited success in attempts to overexpress selenoproteins. This is unfortunate, since Sec mutation could be an effective tool to investigate the role of cysteine thiols in enzyme catalysis.; Several amino acid derivatives were synthesized that could be unmasked after peptide synthesis to give Sec. Specifically, Fmoc-p-methoxybenzylselenocysteine, Fmoc-acetamidomethylselenocysteine and Fmoc-2,4-dimethoxybenzylselenocysteine were synthesized in moderate to high yields. All three amino acids could be deprotected in good yield to give selenopeptides. Peptides synthesized with an N-terminal Sec were found to undergo native chemical ligation with C-terminal peptide thioesters. In this way, several mutants of the C-terminal peptide of E. coli ribonucleotide reductase were synthesized. Also, N-terminal Sec peptides underwent expressed protein ligation with C-terminal protein thioesters generated with an intein system. The electron transport protein, azurin, was reengineered to contain a Sec residue in its metal binding site. C112U azurin bound copper and exhibited the features of the type I copper binding site. The mutant azurin contained a Cu-Se bond that was less covalent than the Cu-S bond in WT as indicated by various spectroscopies. In addition, L. leichmannii ribonucleoside triphosphate reductase was mutated to contain Sec near its C-terminus. In the future, it may be possible to use this mutant to trap an enzyme intermediate of ribonucleotide reduction based on the thermodynamic stability of S-Se bonds.; Finally, an improved, scalable synthesis of Fmoc-phenylselenocysteine is reported. The amino acid was coupled to a model peptide on the solid phase. It was demonstrated that the selenide could be oxidatively eliminated to give a dehydropeptide while still bound to the solid resin. In addition, the peptide acted as a Michael acceptor for a thiol nucleophile. These solid phase reactions were monitored by magic angle spinning NMR spectroscopy.