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

Abstract

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-NH(2), 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 CH(2)Cl(2)/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.

Optimized Fmoc solid‐phase synthesis of the cysteine‐rich peptide linaclotide

“…In all cases, linear peptides were manually synthesized by 9-fluorenylmethoxycarbonyl (Fmoc) chemistry using 1-[bis(dimethylamino)methylene]-6-chloro-1H-benzotriazolium hexafluorophosphate 3-oxide (HCTU) and diisopropylethylamine (DIEA) in N,N-dimethylformamide (DMF) for 1 h at 258C to incorporate all amino acids other than Cys. To prevent racemization, 15,16 Cys was coupled using N,N 0 -diisopro- Random Strategy: Disulfide-rich peptides have commonly been synthesized by regioselective methods to assure the correct pairing of Cys. [17][18][19] Nevertheless, some examples can be found in nature, such as cyclotides 20 (*30 residues), which behave like miniproteins and fall into the right conformation without assistance.…”

Structure–activity relationships of bifunctional cyclic disulfide peptides based on overlapping pharmacophores at opioid and cholecystokinin receptors

“…The Δδ/ΔT (-ppb/K) values for 1, as well as comparative data for DPDPE and nonsulfated Tyr CCK-8, are summarized in Table 7. The large temperature dependence for Gly 3 , Trp 4 and Phe 7 indicates that the amide protons in these peptides are exposed to solvent. These amide protons may not have a role in stabilizing a turn.…”

“…), and HOBt (3 eq.) in a solution of DMF/DCM (1:1) with no preactivation, and the base used was 2,4,6-trimethylpyridine (TMP, 6 eq collidine) [4]. The cleavage from the resin was achieved in the 95% TFA in the presence of thiol and silane based scavengers for 1.5 hours.…”

Amino Acid-Protecting Groups

“…324 -Racemization: Cys is highly prone to racemize during the anchoring to the solid support or during the couplings. 329 330 The extent of the racemization also depends on the S-protecting groups (S t Bu > Trt > Acm > MeBn > t Bu) 331,332,333,334,335 and coupling methods used (favoured if pre-activation in the presence of base is performed and in the coupling methods involving the use of base). Epimerization of the Cys linked to a hydroxyl resin can even take place during the synthesis as a result of the repetitive base treatments to remove the Fmoc group, 2-chlorotrityl resin being the least prone to this process.…”