Reactivity and Chemical Synthesis of L-Pyrrolysine— the 22nd Genetically Encoded Amino Acid Original Research Article

L-pyrrolysine, the 22nd genetically encoded amino acid, was previously deduced to be (4R, 5R)-4-substituted-pyrroline-5-carboxylate attached to the ϵ-nitrogen of lysine based on the crystal structure of the M. barkeri monomethylamine methyltransferase (MtmB). To confirm L-pyrrolysineʹs identity, structures of MtmB have been determined following treatment with hydroxylamine, N-methylhydroxylamine, or dithionite. Analysis of these structures has provided additional support for the presence of the pyrroline ring and, together with previous mass spectroscopy data, has led us to assign the C4-substituent to a methyl group. Based on this assignment, synthetic L-pyrrolysine was prepared by chemical methods. Detailed study of this chemically synthesized L-pyrrolysine has allowed us to characterize its physical properties, to study its chemical stability, and to elucidate the role of its C4 substituent. Future applications of this synthetic L-pyrrolysine include its in vivo incorporation into recombinant proteins.