Practical protocols for stepwise solid-phase synthesis of cysteine-containing peptides

: This study details a series of conditions that may be applied to ensure ‘safe’ incorporation of cysteine with minimal racemization during automated or manual solid‐phase peptide synthesis. Earlier studies from our laboratories [Han et al. (1997) J. Org. Chem. 62, 4307–4312] showed that several common coupling methods, including those exploiting in situ activating agents such as N‐[(dimethylamino)‐1H‐1,2,3‐triazolo[4,5‐b]pyridin‐1‐ylmethylene]‐N‐methylmethanaminium hexafluorophosphate N‐oxide (HATU), N‐[1H‐benzotriazol‐1‐yl)‐(dimethylamino)methylene]‐N‐methylmethanaminium hexafluorophosphate N‐oxide (HBTU), and (benzotriazol‐1‐yl‐N‐oxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) [all in the presence of N‐methylmorpholine (NMM) or N,N‐diisopropylethylamine (DIEA) as a tertiary amine base], give rise to unacceptable levels (i.e. 5–33%) of cysteine racemization. As demonstrated on the tripeptide model H‐Gly‐Cys‐Phe‐NH2, and on the nonapeptide dihydrooxytocin, the following methods are recommended: O‐pentafluorophenyl (O‐Pfp) ester in DMF; O‐Pfp ester/1‐hydroxybenzotriazole (HOBt) in DMF; N,N′‐diisopropylcarbodiimide (DIPCDI)/HOBt in DMF; HBTU/HOBt/2,4,6‐trimethylpyridine (TMP) in DMF (preactivation time 3.5–7.0 min in all of these cases); and HBTU/HOBt/TMP in CH2Cl2/DMF (1:1) with no preactivation. In fact, several of the aforementioned methods are now used routinely in our laboratory during the automated synthesis of analogs of the 58‐residue protein bovine pancreatic trypsin inhibitor (BPTI). In addition, several highly hindered bases such as 2,6‐dimethylpyridine (lutidine), 2,3,5,6‐tetramethylpyridine (TEMP), octahydroacridine (OHA), and 2,6‐di‐tert‐butyl‐4‐(dimethylamino)pyridine (DB[DMAP]) may be used in place of the usual DIEA or NMM to minimize cysteine racemization even with the in situ coupling protocols.