Occurrence and Minimization of Cysteine Racemization during Stepwise Solid-Phase Peptide Synthesis1,2

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Abstract
Contrary to the conventional wisdom of the peptide synthesis field, N,S-protected derivatives of cysteine can undergo substantial levels of racemization with widely-used reagents and protocols for stepwise incorporation. A systematic study of this problem has been carried out as a function of coupling conditions and β-thiol protecting groups, i.e., S-acetamidomethyl (Acm), S-triphenylmethyl (trityl or Trt), S-2,4,6-trimethoxybenzyl (Tmob), and S-9H-xanthen-9-yl (Xan), taking advantage of a convenient and quantitative model system assay involving HPLC resolution of H-Gly-l-Cys-Phe-NH2 from H-Gly-d-Cys-Phe-NH2. For example, standard protocols for couplings mediated by phosphonium and aminium salts, e.g., (benzotriazolyloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HBTU), N-[[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-yl]methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU), and (7-azabenzotriazol-1-yloxy)tris(pyrrolidino)phosphonium hexafluorophosphate (PyAOP), typically involve 5-min preactivation times and are conducted in the presence of suitable additives such as 1-hydroxybenzotriazole (HOBt) or 7-aza-1-hydroxybenzotriazole (HOAt) plus a tertiary amine base such as N,N-diisopropylethylamine (DIEA) or N-methylmorpholine (NMM). Under such conditions, the levels of racemization in the model peptide, expressed as the ratio of d:l peptide formed, were in the entirely unacceptable range of 5−33%. However, these levels were in general reduced by a factor of 6- or 7-fold by avoiding the preactivation step. Additional strategies to reduce racemization involved change to a weaker base, with 2,4,6-trimethylpyridine (TMP, collidine) being substantially better than DIEA or NMM; 2-fold reduction in the amount of base; and change in solvent from neat N,N-dimethylformamide (DMF) to the less polar CH2Cl2−DMF (1:1). Coupling methods for the safe incorporation of cysteine with minimal racemization (2Cl2−DMF (1:1), DIPCDI/HOBt (or HOAt) (4:4) with 5-min preactivation, and preformed pentafluorophenyl (Pfp) esters in CH2Cl2−DMF (1:1).

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