Synthesis of peptide substrates for mammalian thioredoxin reductase.
Mammalian thioredoxin reductase (TR) catalyzes the reduction of the redox-active disulfide bond of thioredoxin (Trx) and is similar in structure and mechanism to glutathione reductase except for a C-terminal 16-amino acid extension containing a rare vicinal selenylsulfide bond. This vicinal selenylsulfide bond is essentially a substrate for the enzyme's N-terminal redox center. Here we report the synthesis of peptide substrates for the truncated enzyme missing the C-terminal redox center. We developed a procedure for the synthesis of peptides containing cyclic vicinal disulfide/selenylsulfide bonds as well as their corresponding acyclic heterodimers. Vicinal disulfide bonds form eight-membered ring structures and are difficult to synthesize owing to their propensity to dimerize during oxidation. Our procedure makes use of two key improvements for on-resin disulfide bond formation presented previously by Galande and coworkers (Galande AK, Weissleder R, Tung C-H. An effective method of on-resin disulfide bond formation in peptides. J. Comb. Chem. 2005; 7: 174-177). First, the addition of an amine base to the deprotection solution allows the complete removal of the StBu group, allowing it to be replaced with a 5-Npys group. The second enhancement is the direct use of a Cys(Mob) or Sec(Mob) derivative as the nucleophilic partner instead of utilizing a naked sulfhydryl or selenol. These improvements result in the formation of a vicinal disulfide (or selenylsulfide) bond in high purity and yield. A direct comparison with the Galande procedure is presented. We also present a novel strategy for the formation of an acyclic, interchain selenylsulfide-linked peptide (linking H-PTVTGC-OH and H-UG-OH). Cysteine analogs of the cyclic and acyclic peptides were also synthesized. The results show that the ring structure contributes a factor of 52 to the rate, but the presence of selenium in the peptide is more important to catalysis than the presence of the ring.
Flemer
Stevenson
S
Department of Biochemistry, Given Laboratory, Burlington, VT 05405, USA.
Lacey
Brian M
BM
Hondal
Robert J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004220:Disulfides / Q000737:chemistry; D004579:Electron Transport; D066298:In Vitro Techniques; D051379:Mice; D015394:Molecular Structure; D009842:Oligopeptides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010456:Peptides, Cyclic / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004220:Disulfides; D009842:Oligopeptides; D010446:Peptide Fragments; D010456:Peptides, Cyclic; D011994:Recombinant Proteins; D013880:Thioredoxin-Disulfide Reductase