Insight into the SEA amide thioester equilibrium. Application to the synthesis of thioesters at neutral pH

Abstract

The bis(2-sulfanylethyl)amide (SEA) N,S-acyl shift thioester surrogate has found a variety of useful applications in the field of protein total synthesis. Here we present novel insights into the SEA amide/thioester equilibrium in water which is an essential step in any reaction involving the thioester surrogate properties of the SEA group. We also show that the SEA amide thioester equilibrium can be efficiently displaced at neutral pH for accessing peptide alkylthioesters, i.e. the key components of the native chemical ligation (NCL) reaction.

Insights into the Mechanism and Catalysis of Peptide Thioester Synthesis by Alkylselenols Provide a New Tool for Chemical Protein Synthesis

“…We also noticed an increase of the pH of the reaction mixture, a phenomenon that was not observed with the other selenols. Since previous studies established that the SEA/thioester ratio at equilibrium increases with pH [45,47], the pH drift observed in these experiments might explain in part the diminished conversion observed with catalyst 14. Taken together, the lower conversion and rates observed with catalyst 14 preclude its use as an interesting alternative to 8a for the production of peptide thioesters from SEA peptides.…”

“…The design of catalyst 8a has been inspired by the observation that the simpler diselenol 8b (R = H, Figure 1D) was able to catalyze the SEA/thiol exchange reaction, albeit with byproduct formation due to the presence in 8b of a nucleophilic secondary amine, hence the introduction of a blocking alkyl group on the nitrogen. The diverse kinetic data collected so far support that the catalysis of the SEA/thiol exchange reaction by selenols proceeds as shown in Figure 2 [45,47]. According to this mechanism, the SEA peptide 9 undergoes a spontaneous N,S-acyl shift to produce the transient SEA thioester 10.…”

“…One popular mode of use of N,S-acyl shift systems is the production of alkylthioesters by exchanging the N-(2-mercaptoethyl)amine unit by a thiol used in excess, i.e., R SH in Figure 1B. Such exchange reactions are equilibrated, the equilibrium being displaced toward the thioester form at acidic pH probably due to the masking of the departing N-(2-mercaptoethyl)amine by protonation [45]. Detailed mechanistic studies performed on the bis(2-sulfanylethyl)amido (SEA) system established that in such conditions the thiolthioester exchanges are rate limiting.…”

Accelerated microfluidic native chemical ligation at difficult amino acids toward cyclic peptides

“…This allowed us to simplify the preliminary attempts by avoiding the reduction of disulfide 3 into dithiol 4 . The equilibrium between SEA on amides 4 and SEAE peptides 5 considerably favors the amide at neutral pH to a point where SEAE peptides 5 are barely detectable by HPLC 39 . In contrast, the equilibrium favors the SEAE peptide 5 at pH 1 by ≥90% so that the activation was examined at this pH.…”

Insights into the Mechanism and Catalysis of Peptide Thioester Synthesis by Alkylselenols Provide a New Tool for Chemical Protein Synthesis

“…R'SH in Figure 1B. Such exchange reactions are equilibrated, the equilibrium being displaced toward the thioester form at acidic pH probably due to the masking of the departing N- (2-mercaptoethyl)amine by protonation [40]. Detailed mechanistic studies performed on the bis(2-sulfanylethyl)amido (SEA) system established that in such conditions the thiol-thioester exchanges are rate limiting.…”