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

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 -9 H -xanthen-9-yl (Xan), taking advantage of a convenient and quantitative model system assay involving HPLC resolution of H-Gly- l -Cys-Phe-NH 2 from H-Gly- d -Cys-Phe-NH 2 . For example, standard protocols for couplings mediated by phosphonium and aminium salts, e.g., (benzotriazolyloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), N -[(1 H -benzotriazol-1-yl)(dimethylamino)methylene]- N -methylmethanaminium hexafluorophosphate N -oxide (HBTU), N -[[(dimethylamino)-1 H -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 CH 2 Cl 2 −DMF (1:1). Coupling methods for the safe incorporation of cysteine with minimal racemization (<1% per step) in 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis include BOP (or HBTU or HATU)/HOBt (or HOAt)/TMP (4:4:4) without preactivation in CH 2 Cl 2 −DMF (1:1), DIPCDI/HOBt (or HOAt) (4:4) with 5-min preactivation, and preformed pentafluorophenyl (Pfp) esters in CH 2 Cl 2 −DMF (1:1).