Evaluation of acid‐labile S‐protecting groups to prevent Cys racemization in Fmoc solid‐phase peptide synthesis

Abstract

Phosphonium and uronium salt-based reagents enable efficient and effective coupling reactions and are indispensable in peptide chemistry, especially in machine-assisted SPPS. However, after the activating and coupling steps with these reagents in the presence of tertiary amines, Fmoc derivatives of Cys are known to be considerably racemized during their incorporation. To avoid this side reaction, a coupling method mediated by phosphonium/uronium reagents with a weaker base, such as 2,4,6-trimethylpyridine, than the ordinarily used DIEA or that by carbodiimide has been recommended. However, these methods are appreciably inferior to the standard protocol applied for SPPS, that is, a 1 min preactivation procedure of coupling with phosphonium or uronium reagents/DIEA in DMF, in terms of coupling efficiency, and also the former method cannot reduce racemization of Cys(Trt) to an acceptable level (<1.0%) even when the preactivation procedure is omitted. Here, the 4,4′-dimethoxydiphenylmethyl and 4-methoxybenzyloxymethyl groups were demonstrated to be acid-labile S-protecting groups that can suppress racemization of Cys to an acceptable level (<1.0%) when the respective Fmoc derivatives are incorporated via the standard SPPS protocol of phosphonium or uronium reagents with the aid of DIEA in DMF. Furthermore, these protecting groups significantly reduced the rate of racemization compared to the Trt group even in the case of microwave-assisted SPPS performed at a high temperature. © 2013 The Authors. European Peptide Society published by John Wiley & Sons, Ltd.

Disulfide Formation Strategies in Peptide Synthesis

“…[48] The highest level of racemization was observed during coupling with uronium activation in the presence of base with Trt-protected Cys (8 %, as described above), whereas the protecting groups MBom (2), Dpm (3), and Ddm (6) caused 0.4, 1.2, and 0.8 % racemization, respectively. In a recent paper by Hibino et al, the acid-labile Cys protecting groups MBom (2), Dpm (3), Ddm (6), and Trt were evaluated for their propensity to reduce Cys racemization.…”

“…The protecting groups Dpm, 4MeO-2MeBn, and 2,6diMeOBn were found to be appropriate for this purpose. [48] The peptide was prepared by uronium activation, and the degree of racemization was determined. In contrast, Ddm was readily removed with 10 % TFA and could be applied as a racemization-suppressing replacement for the Trt protecting group.…”

“…[48,77] Quan-Scheme 5. However, microwave-assisted peptide synthesis with Cys-containing peptides can be hindered by increased Cys racemization.…”

Trends to Acid-Labile Cys Protecting Groups: Thp as an Efficient and Non-Aromatic Cys Protecting Group for Fmoc Chemistry

“…Although trityl-, diphenylmethyl-, and benzyl-like scaffolds are the most used for the protection of Cys, in the recent years the studies with S,O-acetal protecting groups has presented extremely low racemization levels [25]. While benzyloxymethyl (Bom) is used for Boc chemistry, 4-methoxybenzyloxymethyl (MBom) and the recently published tetrahydropyranyl (Thp) are compatible with the Fmoc/tBu approach.…”

Peptides 2015, Proceedings of the 24th American Peptide Symposium

“… Obviously, Dts cannot be used to protect the N α -imino group of proline, but fortunately we were able to optimize open-chain carbamoyl disulfide protection for this residue [24].  We can "invert" the fundamental mechanism of thiolytic deprotection to develop protecting groups for the sulfhydryl side-chain of cysteine, and for "directed" syntheses of inter-and intramolecular disulfides [25,26].  The fact that Dts derivatives are "masked" isocyanates [21] can be very important, given all the difficulties and complications of working directly with isocyanates.…”