Redox-active bis-cysteinyl peptides. I. Synthesis of cyclic cystinyl peptides by conventional methods in solution and...

Cyclic mono-cystinyl active-site fragments of thioredoxin and thioredoxin reductase were synthesized as N-acetyl and C-amide octapeptides by conventional methods of peptide synthesis in solution and on solid supports. Using a side-chain protection based on acid-labile tert-butanol-derived groups and on the S-tert-butylthio unsymmetric disulfide for the thiol functions, in combination with N alpha-Z- or N alpha-Nps derivatives in the chain elongation steps, the synthesis in solution was carried out in straightforward manner yielding the fully protected octapeptides as well characterized compounds. Upon deprotection with trifluoroacetic acid and reduction of the unsymmetrical disulfides with tri-butylphosphine, the resulting bis-cysteinyl-octapeptides were oxidized in dimethylformamide with azodicarboxylic acid di-tert-butyl ester to produce the desired cyclic compounds in good overall yields. For the synthesis on solid supports a similar acid-labile side-chain protection was applied in combination with the N alpha 9-flourenylmethyoxycarbonyl derivatives in the chain elongation steps. Thereby acylations were performed with the related amino acid N-carboxyanhydrides (UNCAs) or by the O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium-tetraf luoroborate/1- hydroxybenzotriazole (TBTU/HOBt) procedure. The solid phase synthesis of the two octapeptides led to unexpected difficulties in terms of recovery of peptidic material from the resins in the final acidolytic cleavage step as well as of racemization at the level of the cysteine residues by the TBTU/HOBt coupling method. Racemization was efficiently suppressed by employing the related pentafluorophenyl ester and this method led to crude octapeptide products of a degree of purity comparable to those obtained by the synthesis in solution. However, the recovery of the peptides from the resin, i.e., irreversible reattachment of cleaved peptidic material via alkylation of various side-chain functions, could not be avoided even using the most efficient scavengers or their cocktails.