A convenient incorporation of conformationally constrained 5,5‐dimethylproline into the ribonuclease A 89–124 sequence by condensation of synthetic peptide fragments

Abstract

The presence of l-5,5-dimethylproline (dmP) within an amino acid sequence results in the formation of an X-dmP peptide bond predominantly locked in a cis conformation. However, the common use of this unnatural amino acid has been hampered by the difficulty of the economical incorporation of the dmP residue into longer peptide segments due to the steric hindrance imposed by the dimethyl moieties. Here, we describe synthesis of the C-terminal 36-residue peptide, corresponding to the 89-124 sequence of bovine pancreatic ribonuclease A (RNase A), in which dmP is incorporated as a substitute for Pro93. The peptide was assembled by condensation of protected 5- and 31-residue peptide fragments, which were synthesized by solid-phase peptide methodology using fluorenylmethyloxycarbonyl chemistry. We focused on optimizing the synthesis of the Fmoc-Ser(tBu)-Ser(tBu)-Lys(Boc)-Tyr(tBu)-dmP-OH pentapeptide (residues 89-93) with efficient acylation of the sterically hindered dmP residue. In a comparative study, the reagent O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate was found to be superior to bromo-tris-pyrrolidino-phosphonium hexafluorophosphate and tetramethylfluoroformamidinium hexafluorophosphate for the synthesis of the -Tyr(tBu)-dmP- peptide bond in solution as well as on a resin.

A structural and mechanistic study of π-clamp-mediated cysteine perfluoroarylation

“…To understand if the conformation of the Cys-Pro amide bond perturbs the reaction we used mutation studies. Proline analogues containing certain functional groups can constrain the Cys-Pro conformation; analogues α-methylproline (αMePro) 16 , 17 and 5,5-dimethylproline (5,5-dmP) 18 , 19 strongly promote the trans and cis conformations, respectively. Three peptides incorporating proline (the π-clamp, peptide 1A ), α-methylproline (peptide 1B ) and 5,5-dimethylproline (peptide 1C ) at residue 3 were synthesized.…”