Efficient Chemical Protein Synthesis using Fmoc‐Masked N‐Terminal Cysteine in Peptide Thioester Segments

Abstract

We report an operationally simple method to facilitate chemical protein synthesis by fully convergent and one‐pot native chemical ligations utilizing the fluorenylmethyloxycarbonyl (Fmoc) moiety as an N‐masking group of the N‐terminal cysteine of the middle peptide thioester segment(s). The Fmoc group is stable to the harsh oxidative conditions frequently used to generate peptide thioesters from peptide hydrazide or o‐aminoanilide. The ready availability of Fmoc‐Cys(Trt)‐OH, which is routinely used in Fmoc solid‐phase peptide synthesis, where the Fmoc group is pre‐installed on cysteine residue, minimizes additional steps required for the temporary protection of the N‐terminal cysteinyl peptides. The Fmoc group is readily removed after ligation by short exposure (<7 min) to 20 % piperidine at pH 11 in aqueous conditions at room temperature. Subsequent native chemical ligation reactions can be performed in presence of piperidine in the same solution at pH 7.

One‐Pot Chemical Protein Synthesis Utilizing Fmoc‐Masked Selenazolidine to Address the Redox Functionality of Human Selenoprotein F**

“…The presence of 20 % Pip in the reaction was compatible with the following NCL reaction conditions, as Pip is fully protonated at neutral pH, preventing any side reaction with the thioester [24] . Without further purification step, SELENOF(89‐95)‐COSR was dissolved in phosphate buffer at pH 7 containing 0.05 M TCEP, 0.1 M sodium ascorbate and 0.1 mM MPAA and was added directly for the second NCL reaction.…”

“…Next, without a purification step, the pH was adjusted to 5.5 and the deselenization of SELENOF(96–165)(U96FmocSez/A141U) was performed under anaerobic conditions using 50 equiv TCEP at 37 °C. After 13 h (Figures 2c and S8), the reaction mixture was treated with 20 % Pip in phosphate buffer containing 0.1 M sodium ascorbate at pH 10 [22,24] . Without NH 2 OMe, [17] Cu ions [18] or any additive, [30] we were delighted to find that these conditions provided the Fmoc deprotection and Sez opening in one step within 5 h (Figures 2b, c and S9).…”

“…Previously, different protecting groups were developed to increase the stability of Thz during hydrazide oxidation to azides, [21] among these were tert ‐butyldisulfanylethyloxycarbonyl (Tbeoc), [21] 9‐fluorenylmethoxy‐carbonyl (Fmoc) [22] and trifluoroacetyl (Tfa) groups [23] . It is worth noting that Fmoc‐Cys was recently used to mask the N‐terminal Cys for one‐pot CPS [24] . Following ligation, Fmoc is removed by 20 % piperidine in aqueous solution [24] .…”

“…It is worth noting that Fmoc‐Cys was recently used to mask the N‐terminal Cys for one‐pot CPS [24] . Following ligation, Fmoc is removed by 20 % piperidine in aqueous solution [24] . Hence, we decided to test Fmoc as a protecting group of Sez during CPS, which prevented the undesired deselenization at position 96 in the redox motif, and provided milligram quantities of the Trx‐like domain of SELENOF.…”

Repetitive Thiazolidine Deprotection Using a Thioester‐Compatible Aldehyde Scavenger for One‐Pot Multiple Peptide Ligation**

“…Among various one‐pot peptide ligation strategies, [15–24] the use of protecting groups at the N‐terminal amine or thiol of Cys on internal segments is the simplest approach reported by many research groups. For example, acetamidomethyl (Acm) group, [23] trifluoroacetamidomethyl (Tfacm) [25] and 7‐(piperazin‐1‐yl)‐2‐(methyl)quinolinyl (PPZQ) [26] for thiol group, 4‐(dimethylamino)phenacyloxycarbonyl (Mapoc), [27] p ‐borobenzyloxycarbonyl group (Dobz), [20] allyloxycarbonyl (Alloc), [28–30] and 9‐fluorenylmethyloxycarboyl (Fmoc) for amine group [31] were successfully used as N‐terminal cysteinyl protecting groups in one‐pot peptide ligation.…”