Taku Yoshiya

Isopeptide method: development of S-acyl isopeptide method for the synthesis of difficult sequence-containing peptides

A novel strategy for a more efficient synthesis of difficult sequence‐containing peptides, the S‐acyl isopeptide method, was developed and successfully applied. A model pentapeptide Ac–Val–Val–Cys–Val–Val–NH2 was synthesized via its water‐soluble S‐acyl isopeptide using an S‐acyl isodipeptide unit, Boc–Cys(Fmoc–Val)–OH. An S‐acyl isopeptide possessing excellent water solubility could be readily and quantitatively converted to the native peptide via an SN intramolecular acyl migration reaction at pH 7.4. Thus, the S‐acyl isopeptide method provides a useful tool in peptide chemistry. Copyright

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.

Epimerization-free synthesis of cyclic peptide by use of the O-acyl isopeptide method.

A head‐to‐tail cyclization of a protected linear hexapeptide with a C‐terminal O‐acyl isopeptide proceeded to give a cyclic O‐acyl isopeptide without epimerization. The cyclic O‐acyl isopeptide possessed different secondary structures compared with the native cyclic peptide. The isopeptide was then efficiently converted to the desired cyclic peptide via an O‐to‐N acyl migration reaction using a silica gel‐anchored base. Copyright

Click Peptide concept: o-acyl isopeptide of islet amyloid polypeptide as a nonaggregative precursor molecule.

The O‐acyl isopeptide (1) of islet amyloid polypeptide (IAPP), which contains an ester moiety at both Ala8‐Thr9 and Ser19‐Ser20, was prepared by sequential segment condensation based on the O‐acyl isopeptide method. Isopeptide 1 possessed nonaggregative properties, retaining its random coil structure under the acidic conditions; this suggests that the insertion of the O‐acyl isopeptide structures in IAPP suppressed aggregation of the molecule. As a result of the rapid O‐to‐N acyl shift of 1 under neutral pH, in situ‐formed IAPP adopted a random‐coil structure at the start of the experiment, and then underwent conformational change to α‐helix/β‐sheet mixed structures as well as aggregation. The click peptide strategy with the nonaggregative precursor molecule 1 could be a useful experimental tool to identify the functions of IAPP, by overcoming the handling difficulties that arise from IAPPs intense and uncontrollable self‐assembling nature.

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.

S-acyl isopeptide method: Use of allyl-type protective group for improved preparation of thioester-containing S-acyl isopeptides by Fmoc-based SPPS†

We have studied the “S‐acyl isopeptide method” for the synthesis of peptides containing difficult sequences. The S‐acyl isopeptide, which contains a β‐thioester instead of the native N‐acyl bond at a Cys residue, can be converted into the target peptide via an S‐to‐N intramolecular acyl migration reaction. However, the synthesis of the S‐acyl isopeptide structure by Fmoc‐based SPPS is hampered by repetitive base treatments; decomposition of the thioester and the epimerization of the thioesterified residue are commonly observed. Here, we adopted allyloxycarbonyl (Aloc) protective group to avoid the problem. Catalytic amount of Pd in the presence of scavengers such as PhSiH3 and dimedone selectively removed the Aloc group with neither decomposition of the thioester structure nor epimerization at the thioesterified residue. A model pentapeptide and amylin(1‐12) with difficult sequences were efficiently synthesized by the improved S‐acyl isopeptides method. Finally, the isolated S‐acyl isopeptides were quantitatively converted into the desired peptides via the S‐to‐N intramolecular acyl migration reaction. The S‐acyl isopeptide method will be a useful method to prepare the difficult sequence‐containing peptides with Cys residue.

Synthesis and conformation of an analog of the helix-loop-helix domain of the Id1 protein containing the O-acyl iso-prolyl-seryl switch motif

Synthetic peptides reproducing the helix‐loop‐helix (HLH) domains of the Id proteins fold into highly stable helix bundles upon self‐association. Recently, we have shown that the replacement of the dipeptide Val‐Ser at the loop–helix‐2 junction with the corresponding O‐acyl iso‐dipeptide leads to a completely unfolded state that only refolds after intramolecular O → N acyl migration. Herein, we report on an Id HLH analog based on the substitution of the Pro‐Ser motif at the helix‐1–loop junction with the corresponding O‐acyl iso‐dipeptide. This analog has been successfully synthesized by solid‐phase Fmoc chemistry upon suppression of DKP formation. No secondary structure could be detected for the O‐acyl iso‐peptide before its conversion into the native form by O → N acyl shift. These results show that the loop–helix junctions are determinant for the folded/unfolded state of the Id HLH domain. Further, despite the high risk of DKP formation, peptides containing O‐acyl iso‐Pro‐Ser/Thr units are synthetically accessible by Fmoc chemistry. Copyright

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