Kohei Tsuji

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).

CXCL14 is a natural inhibitor of the CXCL12–CXCR4 signaling axis

CXCR4 physically interacts with CXCL14 anti bait coimmunoprecipitation by (View interaction).

The extreme N-terminal region of human apolipoprotein A-I has a strong propensity to form amyloid fibrils

The N‐terminal 1–83 residues of apolipoprotein A‐I (apoA‐I) have a strong propensity to form amyloid fibrils, in which the 46–59 segment was reported to aggregate to form amyloid‐like fibrils. In this study, we demonstrated that a fragment peptide comprising the extreme N‐terminal 1–43 residues strongly forms amyloid fibrils with a transition to β‐sheet‐rich structure, and that the G26R point mutation enhances the fibril formation of this segment. Our results suggest that in addition to the 46–59 segment, the extreme N‐terminal region plays a crucial role in the development of amyloid fibrils by the N‐terminal fragment of amyloidogenic apoA‐I variants.

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.

Application of N–C- or C–N-directed sequential native chemical ligation to the preparation of CXCL14 analogs and their biological evaluation

CXCL14 is a chemokine that exhibits chemoattractant activity for activated macrophages, immature dendric cells, natural killer cells, and epithelial tumor cells. Its potential role as a metabolic regulator has recently been disclosed. However, a complete understanding of its physiological roles remains elusive. This is partly due to the lack of appropriate CXCL14-based molecular probes to explore the biological functions of CXCL14. In this context, we have developed synthetic protocols that provide access to a wide variety of CXCL14 analogs. Two sequential native chemical ligation (NCL) protocols, which proceed in opposite directions, have been used to assemble CXCL14 analogs from peptide fragments. The first involved a conventional C-N-directed sequential NCL, and afforded wild-type CXCL14. The other used peptide thioacids in N-C-directed elongation, and yielded CXCL14 analogs with molecular diversity at the C-terminal fragment. The CXCL14 analogs prepared showed biological activity on human monocytic leukemia-derived THP-1 cells that was comparable to that of wild-type CXCL14.

Molecular Complex Composed of β-Cyclodextrin-Grafted Chitosan and pH-Sensitive Amphipathic Peptide for Enhancing Cellular Cholesterol Efflux under Acidic pH

Excess of cholesterol in peripheral cells is known to lead to atherosclerosis. In this study, a molecular complex composed of β-cyclodextrin-grafted chitosan (BCC) and cellular cholesterol efflux enhancing peptide (CEEP), synthesized by modifying pH sensitive amphipathic GALA peptide, is introduced with the eventual aim of treating atherosclerosis. BCC has a markedly enhanced ability to induce cholesterol efflux from cell membranes compared to β-cyclodextrin, and the BCC-CEEP complex exhibited a 2-fold increase in cellular cholesterol efflux compared to BCC alone under weakly acidic conditions. Isothermal titration calorimetry and fluorescence spectroscopy measurements demonstrated that the random coil structure of CEEP at neutral pH converted to the α-helical structure at acidic pH, resulting in a three-order larger binding constant to BCC (K = 3.7 × 10(7) at pH 5.5) compared to that at pH 7.4 (K = 7.9 × 10(4)). Such high-affinity binding of CEEP to BCC at acidic pH leads to the formation of 100-nm-sized aggregate with positive surface charge, which would efficiently interact with cell membranes and induce cholesterol efflux. Since the cholesterol efflux ability of HDL is thought to be impaired under acidic environments in advanced atherosclerotic lesions, the BCC-CEEP complex might serve as a novel nanomaterial for treating atherosclerosis.

Dimeric peptides of the C-terminal region of CXCL14 function as CXCL12 inhibitors

We recently reported that CXCL14 binds to CXCR4 with high affinity and inhibits CXCL12‐mediated chemotaxis. Here we found that the C‐terminal 51–77 amino acid residues of CXCL14 are responsible for CXCR4 binding. A disulfide dimer peptide of CXCL14(51–77) bound to CXCR4 with comparable affinity to full length CXCL14, and exhibited CXCL12 inhibitor activity. CXCR4 was efficiently internalized upon binding of dimeric CXCL14(51–77), thereby being reduced on the cell surface. Substitution of 5 amino acid residues in combination with the use of an oxime linker for dimerization increased the solubility and chemical stability of the dimeric CXCL14(51–77).

Development of an Anilide-Type Scaffold for the Thioester Precursor N-Sulfanylethylcoumarinyl Amide

N-Sulfanylethylcoumarinyl amide (SECmide) peptide, which was initially developed for use in the fluorescence-guided detection of promoters of N-S acyl transfer, was successfully applied to a facile and side reaction-free protocol for N-S acyl-transfer-mediated synthesis of peptide thioesters. Additionally, 4-mercaptobenzylphosphonic acid (MBPA) was proven to be a useful catalyst for the SECmide or N-sulfanylethylanilide (SEAlide)-mediated NCL reaction.

Amyloidogenic Mutation Promotes Fibril Formation of the N-terminal Apolipoprotein A-I on Lipid Membranes

Background: The N-terminal fragment of amyloidogenic apoA-I mutants deposits as fibrils by unknown mechanisms. Results: The G26R mutation partially prevents helix formation of the N-terminal fragment upon lipid binding, thereby facilitating β-transition and fibril formation. Conclusion: Membrane binding modulates fibril formation of apoA-I through partially destabilized helical conformation. Significance: The results reveal a new pathway for amyloid fibril formation by apoA-I. The N-terminal amino acid 1–83 fragment of apolipoprotein A-I (apoA-I) has a strong propensity to form amyloid fibrils at physiological neutral pH. Because apoA-I has an ability to bind to lipid membranes, we examined the effects of the lipid environment on fibril-forming properties of the N-terminal fragment of apoA-I variants. Thioflavin T fluorescence assay as well as fluorescence and transmission microscopies revealed that upon lipid binding, fibril formation by apoA-I 1–83 is strongly inhibited, whereas the G26R mutant still retains the ability to form fibrils. Such distinct effects of lipid binding on fibril formation were also observed for the amyloidogenic prone region-containing peptides, apoA-I 8–33 and 8–33/G26R. This amyloidogenic region shifts from random coil to α-helical structure upon lipid binding. The G26R mutation appears to prevent this helix transition because lower helical propensity and more solvent-exposed conformation of the G26R variant upon lipid binding were observed in the apoA-I 1–83 fragment and 8–33 peptide. With a partially α-helical conformation induced by the presence of 2,2,2-trifluoroethanol, fibril formation by apoA-I 1–83 was strongly inhibited, whereas the G26R variant can form amyloid fibrils. These findings suggest a new possible pathway for amyloid fibril formation by the N-terminal fragment of apoA-I variants: the amyloidogenic mutations partially destabilize the α-helical structure formed upon association with lipid membranes, resulting in physiologically relevant conformations that allow fibril formation.

Efficient one-pot synthesis of CXCL14 and its derivative using an N-sulfanylethylanilide peptide as a peptide thioester equivalent and their biological evaluation.

CXCL14 is a CXC-type chemokine that exhibits chemotactic activity for immature dendritic cells, activated macrophages, and activated natural killer cells. However, its specific receptor and signaling pathway remain obscure. Recently, it was reported that CXCL14 binds to CXCR4 with high affinity and inhibits CXCL12-mediated chemotaxis. Furthermore, the CXCL14 C-terminal α-helical region is important for binding to its receptor. In this context, we chemically synthesized CXCL14 and its derivative with a one-pot method using N-sulfanylethylanilide peptide as a thioester equivalent. The synthetic CXCL14 proteins possessed inhibitory activities to CXCL12-mediated chemotaxis comparable with that of recombinant CXCL14. Moreover, we proved that chemically biotinylated CXCL14 binds to CXCR4 on cells by flow cytometry analysis.