Native chemical ligation (NCL) has shown great utility in protein chemistry and has yielded impressive success in the preparation of a wide variety of proteins.[1] This methodology requires peptide thioesters that serve as chemoselective acylating agents for N-terminal cysteinyl peptides to afford ligated peptides through a sequence of reactions consisting of S-S and S-N acyl transfers. The susceptibility of the thioester moiety to basic reagents has necessitated the preparation of the key intermediate by Boc-based solid-phase peptide synthesis (Boc-SPPS) without requiring a nucleophile-mediated deprotection procedure.[2] However, the preferred use of Fmoc-based SPPS with piperidine treatment demands the development of a synthetic methodology using peptide thioesters that are compatible with Fmoc chemistry.In this context, many research groups, including ours, have explored an Fmoc-based synthetic protocol for thioesters. [3][4][5] Among the reported studies, N-S acyl-transfer-mediated procedures have great potential in Fmoc chemistry.[5] We have also developed an N-sulfanylethylaniline linker that can be used for the acyl-transfer-mediated synthesis of peptide thioesters.[5g]Standard Fmoc-SPPS on the sulfanylethylaniline linker followed by N-S acyl transfer under acidic conditions (4 m HCl in DMF) efficiently yielded peptide thioesters (Scheme 1). On the basis of these experimental results, we attempted to utilize an N-terminal cysteinyl N-sulfanylethylanilide (SEAlide) peptide as the middle fragment(s) for sequential NCL, which features the use of more than one thioester fragment.[6] Here, involvement of the SEAlide peptide in the first NCL with a peptide thioester would seem to selectively afford the corresponding ligated SEAlide peptide, which can be used in the second NCL step after conversion of the anilide moiety to the thioester under acidic conditions. The first NCL doubtlessly proceeded; however, contrary to our expectations, a not insignificant amount of cyclic material resulting from the unanticipated intramolecular NCL of the cysteinyl SEAlide peptide was observed (Scheme 2). This unexpected result indicated that the SEAlide moiety could work as a thioester in the presence of an N-terminal cysteinyl residue even under neutral NCL conditions to afford the corresponding NCL product. [7] In this study, we first examined the feasibility of the SEAlide peptide as a crypto-thioester peptide, and discuss the utility of the cryptic thioester in a kinetically controlled NCL. [8] Initial evaluation of the utility of the SEAlide peptide under NCL conditions was attempted through model coupling reactions between peptide 1 and 2 (Table 1). After preliminary experiments, we first fixed the control coupling conditions (1 mm each peptide in 6 m guanidine·HCl (Gn·HCl)-0.2 m sodium phosphate in the presence of 100 mm (4-carboxymethyl)thiophenol (MPAA) [9,10] and 40 mm tris(2-carboxyethyl)phosphine (TCEP), pH 7.3, 37 8C). Under standard conditions, the attempted NCL between 1 a and 2 was almost complete in 48 h and ...

N-->S acyl-transfer-mediated synthesis of peptide thioesters utilizing an N-aminoacyl-N-sulfanylethylaminobenzoic acid derivative has been examined. The developed synthetic methodology for peptide thioesters is compatible with Fmoc solid-phase peptide synthesis (SPPS).

No abstract

The ability to temporally and spatially control the function of peptides/proteins by a stimulus has received increased attention due to its potential in various fields such as chemical biology and drug delivery. Recently, photo-induced processing (peptide bond cleavage) or conformational change has been successfully applied to convert inactive (or active) peptides and proteins into their corresponding active (or inactive) forms at a desired time and location. [1a-i] Increasing the diversity of the trigger that is involved in a processing reaction might facilitate stimulus-responsive processing to become a general method for controlling peptide functions. Therefore, we attempted to design an amino acid derivative that induces a processing reaction as a response to a wide variety of stimuli. Inspired by the trimethyl lock system, [2,3] we designed stimulus-responsive peptide 1, which can release a functional peptide after the stimulus-induced removal of a phenolic protective group (PG) and subsequent processing reaction (Scheme 1). Peptide 1 features the nucleophilic involvement of a regenerated phenolic hydroxyl group to an adjacent peptide bond to release functional peptides. In this investigation, we chose a photo-removable o-nitrobenzyl (o-NB) and a phosphat-A C H T U N G T R E N N U N G ase-removable phosphate group for phenolic protection to afford stimulus-responsive model peptides.Scheme 2 shows the synthesis for photo-responsive model peptide 8. Aldehyde 2[2] was a-aminated with di-tert-butyl azodicarboxylate in the presence of pyrrolidine. After reduction of the aldehyde group with sodium borohydride, the resulting alcohol was protected with a tert-butyldimethylsilyl (TBS) group to give silyl ether 3. Trifluoroacetylation of the terminal nitrogen in 3 with trifluoroacetic anhydride (TFAA) and subsequent NÀN bond cleavage by sammarium(II) iodide in the presence of hexamethylphosphoramide (HMPA) and tert-butanol gave amino alcohol 4. The benzyl group on 4 was removed by hydrogenolysis and the generated phenolic hydroxyl group was protected with an o-nitrobenzyl group to afford o-NB ether 5. After removing the TBS group of 5 under acidic conditions, a two-step oxidation was performed to give carboxylic acid 6. The tert-butyloxycarbonyl (Boc) group on 6 was removed with hydrogen chloride in ethyl acetate to yield an amine, which was reprotected with a fluorenylmethoxycarbonyl (Fmoc) group to give Fmoc-protected photo-responsive processing device 7. The total yield of Fmoc derivative 7 was 11 % over 12 steps from aldehyde 2. Finally, the incorporation of amino acid 7 into the peptide by standard Fmoc solid-phase peptide synthesis (SPPS) afforded photo-responsive model peptide 8 as a diastereomeric mixture.To examine the photoreactivity of model peptide 8, we performed the photo-processing reaction that is outlined in Scheme 1. Design of a stimulus-responsive peptide.Scheme 2. Reagents and conditions. a) di-tert-butyl azodicarboxylate, pyrrolidine, CH 2 Cl 2 , 85 %; b) NaBH 4 , MeOH, 100

Disulfide bond formation performed in solution with the tert-butylthio (StBu) group was accomplished using a free peptide having only the sulfhydryl groups of Cys protected with the aid of postsynthetic S-tritylation. This facilitated removal of the StBu group with subsequent disulfide formation without any difficulty. This strategy using the StBu group in combination with the widely used acetamidomethyl (Acm), 4-methylbenzyl/4-methoxybenzyl (Meb/Mob), and trityl (Trt) groups enables reliable and regioselective synthesis of multicystine peptides.