Shugo Tsuda

N-->S acyl-transfer-mediated synthesis of peptide thioesters using anilide derivatives.

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).

N-Sulfanylethylanilide Peptide as a Crypto-Thioester Peptide

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. 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. 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. Among the reported studies, N–S acyl-transfer-mediated procedures have great potential in Fmoc chemistry. We have also developed an N-sulfanylethylaniline linker that can be used for the acyl-transfer-mediated synthesis of peptide thioesters. 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. 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).

Synthesis of a stimulus-responsive processing device and its application to a nucleocytoplasmic shuttle Peptide.

Stimulus-responsive processing (peptide bond cleavage) devices were developed. The processing reaction was triggered by stimulus-induced removal of a PG and the processing products were obtained in good purity. A photo-responsive processing device was successfully applied to develop a nucleocytoplasmic shuttle peptide. (F: fluorophore, NES: nuclear export signal. NLS: nuclear localization signal. PG: stimulusresponsive protective group)

Synthetic procedure for N-Fmoc amino acyl-N-sulfanylethylaniline linker as crypto-peptide thioester precursor with application to native chemical ligation.

N-sulfanylethylanilide (SEAlide) peptides 1, obtainable using Fmoc-based solid-phase peptide synthesis (Fmoc SPPS), function as crypto-thioesters in native chemical ligation (NCL), yielding a wide variety of peptides/proteins. Their acylating potential with N-terminal cysteinyl peptides 2 can be tuned by the presence or absence of phosphate salts, leading to one-pot/multifragment ligation, operating under kinetically controlled conditions. SEAlide peptides have already been shown to be promising for use in protein synthesis; however, a widely applicable method for the synthesis of N-Fmoc amino acyl-N-sulfanylethylaniline linkers 4, required for the preparation of SEAlide peptides, is unavailable. The present study addresses the development of efficient condensation protocols of 20 naturally occurring amino acid derivatives to the N-sulfanylethylaniline linker 5. N-Fmoc amino acyl aniline linkers 4 of practical use in NCL chemistry, except in the case of the proline- or aspartic acid-containing linker, were successfully synthesized by coupling of POCl(3)- or SOCl(2)-activated Fmoc amino acid derivatives with sodium anilide species 6, without accompanying racemization and loss of side-chain protection. Furthermore, SEAlide peptides 7 possessing various C-terminal amino acids (Gly, His, Phe, Ala, Asn, Ser, Glu, and Val) were shown to be of practical use in NCL chemistry.

Synthesis of Cysteine-Rich Peptides by Native Chemical Ligation without Use of Exogenous Thiols

Native chemical ligation (NCL) performed without resorting to the use of thiol additives was demonstrated to be an efficient and effective procedure for synthesizing Cys-rich peptides. This method using tris(2-carboxyethyl)phosphine (TCEP) as a reducing agent facilitates the ligation reaction even at the Thr-Cys or Ile-Cys site and enables one-pot synthesis of Cys-rich peptides throughout NCL and oxidative folding.

A fluorogenic probe for γ-glutamyl cyclotransferase: application of an enzyme-triggered O-to-N acyl migration-type reaction.

Light it up: human chromosome 7 ORF 24, a tumor-related protein, has been identified as a γ-glutamyl cyclotransferase (GGCT) in the glutathione homeostasis cycle. The singular substrate preference of the enzyme has hampered chemical probe development, and no fluorogenic probe has been reported. Here we report the first fluorogenic dipeptide probe, LISA-4, which should contribute toward further understanding of GGCT.

Regioselective formation of multiple disulfide bonds with the aid of postsynthetic S-tritylation.

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.

Imidazole‐Aided Native Chemical Ligation: Imidazole as a One‐Pot Desulfurization‐Amenable Non‐Thiol‐Type Alternative to 4‐Mercaptophenylacetic Acid

Various bioactive proteins have been synthesized by native chemical ligation (NCL) and its combination with subsequent desulfurization (e.g., conversion from Cys to Ala). In NCL, excess 4-mercaptophenylacetic acid (MPAA) is generally added to facilitate the reaction. However, co-elution of MPAA with the ligation product during preparative high-performance liquid chromatography sometimes reduces its usefulness. In addition, contamination of MPAA disturbs subsequent desulfurization. Here, we report for the first time that imidazole can be adopted as an alternative to MPAA in NCL using a peptide-alkylthioester. The efficiency of the imidazole-aided NCL (Im-NCL) is similar to that of traditional MPAA-aided NCL. As model cases, we successfully synthesized adiponectin(19-107) and [Ser(PO3 H2 )65 ]-ubiquitin using Im-NCL with a one-pot desulfurization.

Development of a sufficiently reactive thioalkylester involving the side-chain thiol of cysteine applicable for kinetically controlled ligation.

Nα‐Trifluoroacetyl‐Cys‐Leu‐NH2 (TfaC‐Leu‐NH2) was incorporated into thioesters through its side‐chain thiol group to develop a more reactive peptide‐thioester than the commonly used peptide‐3‐mercaptopropionic acid (MPA)‐thioester. The TfaC‐thioester could be readily synthesized by solid‐phase peptide synthesis (SPPS) with Boc chemistry using in situ neutralization protocols in sufficient yield without any side reaction associated with the use of TfaC. This thioester proved to display a much higher reactivity in the thiol‐free native chemical ligation (NCL) reaction than the MPA‐thioester and to be comparable to the thioarylester, such as the 4‐mercaptophenylacetic acid (MPAA)‐thioester, in terms of the ligation rate. We were able to demonstrate the usefulness of the TfaC‐thioester by using it to synthesize neuromedin S via a one‐pot sequential NCL approach followed by desulfurization.

Design of fluorogenic probes and fluorescent-tagged inhibitors for γ-glutamyl cyclotransferase

A tumor‐related protein, human chromosome 7 ORF 24 (C7orf24), is involved in regulation of the glutathione homeostasis cycle as a γ‐glutamyl cyclotransferase (GGCT). The singular substrate preference of this enzyme had long hampered its chemical probe development. That is, substrate of GGCT is definitely ‘γ‐Glu‐Xaa’, where Xaa is an L‐α‐amino acid. Based on the structure of substrates and GGCT fluorogenic probes, LISA‐4/101, we succesfully developed a fluorescent‐tagged inhibitor, gKFA. These chemical tools will assist cancer‐related researches in the future. Copyright