Hironobu Hojo

Preparation of Peptide Thioesters using Fmoc-Solid-Phase Peptide Synthesis and its Application to the Construction of a Template-Assembled Synthetic Protein (TASP)

Preparation of peptide thioesters was conducted through peptide chain construction with Fmoc-solid-phase peptide synthesis on a 2-chlorotrityl resin followed by coupling with HS-(CH2)2-COOEt and deprotection with 95% aqueous CF3COOH. The peptide thioester corresponding to a transmembrane segment of the calcium channel (S4 in repeat IV) thus obtained was introduced onto a peptide template to give an artificial four-helix-bundle protein.

The first synthesis of peptide thioester carrying N-linked core pentasaccharide through modified Fmoc thioester preparation: synthesis of an N-glycosylated Ig domain of emmprin

Peptide thioester carrying N-linked core pentasaccharide was prepared by the Fmoc solid-phase method with a combination of the benzyl-protection strategy at the carbohydrate portion. The obtained peptide thioester was successfully used for the synthesis of the first Ig domain of emmprin composed of 61 amino acid residues.

Enhanced cell adhesion on RGDS-carrying keratin film

Abstract Keratin, which was extracted from wool as a reduced form, was subjected to modification with cell adhesion peptide, Arg-Gly-Asp-Ser (RGDS), at free cysteine residues. L929 mouse fibroblast cells were cultured on micro cover glasses coated with various amounts of keratin or RGDS-carrying keratin. One hour after cell inoculation, more cells adhered on RGDS-carrying keratin-coated surface than on a plain glass or untreated keratin-coated glass surfaces. The cell number adhered on RGDS-carrying keratin-coated surface increased with an increasing amount of coated proteins, while that on keratin-coated glass was unaffected by the amount of coated proteins. Significant number of cells with spindle-shape morphology was observed on RGDS-carrying keratin-coated surface, while the cells on a plain glass or keratin-coated glass surfaces had round morphology. Upon cultivation for 24 h, the cells on RGDS-carrying keratin-coated glass proliferated well to reach confluence, while cells on keratin-coated or untreated glasses remained sparse. Thus, RGDS-carrying keratin was found to be a good substrate for mammalian cells because of their high cell adhesive property. And keratin was demonstrated to be potential biomaterials applicable to versatile purposes by introducing modifications to abundantly present cysteine residues.

Functional and possible physical association of scavenger receptor with cytoplasmic tyrosine kinase Lyn in monocytic THP-1-derived macrophages

Acetyl LDL (modified low‐density lipoprotein), which is thought to be taken up through scavenger receptor A (SR‐A), rapidly induced the appearance of phosphotyrosine proteins in monocytic THP‐1‐derived macrophages in vitro. The two alternative forms of Lyn (p53 and p56) were found to be tyrosine‐phosphorylated within 30 s after the stimulation with acetyl LDL. The catalytic activity of Lyn measured by an in vitro kinase assay had also increased in acetyl LDL‐stimulated THP‐1‐derived macrophages. Furthermore, Lyn could be co‐immunoprecipitated with SR‐A from the cell lysate. These observations suggest a functional and possible physical association of SR‐A with Lyn in THP‐1‐derived macrophages, and also imply a possible involvement of Lyn in SR‐A signal transduction.

Preparation of core 2 type tetrasaccharide carrying decapeptide by benzyl protection-based solid-phase synthesis strategy

Abstract β- d -Gal-(1→4)-β- d -GlcNAc-[β- d -Gal-(1→3)]-α- d -GalNAc-(1→3)- l -Ser/Thr building blocks for solid-phase synthesis of glycopeptide were stereoselectively synthesized in a benzyl-protected form. The key glycosylation reaction to form β- d -GlcNAc linkage was established by the use of protected N -trichloroacetyl- d -lactosaminyl fluoride. Usefulness of the building block was demonstrated by solid-phase synthesis of a segment of human leukosialin. Benzyl protecting group was efficiently removed by ‘low acidity TfOH’ conditions.

Three-dimensional structure of the S4-S5 segment of the Shaker potassium channel.

The propagation of action potentials during neuronal signal transduction in phospholipid membranes is mediated by ion channels, a diverse group of membrane proteins. The S4-S5 linker peptide (S4-S5), that connects the S4 and S5 transmembrane segments of voltage-gated potassium channels is an important region of the Shaker ion-channel protein. Despite its importance, very little is known about its structure. Here we provide evidence for an amphipathic alpha-helical conformation of a synthetic S4-S5 peptide of the voltage-gated Drosophila melanogaster Shaker potassium channel in water/trifluoroethanol and in aqueous phospholipid micelles. The three-dimensional solution structures of the S4-S5 peptide were obtained by high-resolution nuclear magnetic resonance spectroscopy and distance-geometry/simulated-annealing calculations. The detailed structural features are discussed with respect to model studies and available mutagenesis data on the mechanism and selectivity of the potassium channel.

Studies directed toward the synthesis of protein-bound GPI anchor

Abstract Mannobioside-linked phosphoethanolamine 10 , a prototype model of the GPI anchor, was synthesized via glycosidation of the monosaccharide donor and acceptor, and subsequent phosphorylation. In order to test the reactivity of the amino group involved in 10 against the activated amino acid esters, 10 was reacted with N -protected amino acid pentafluorophenyl esters in the presence of HOBt. The reactions gave the aminoacylated products in moderate yields. When Fmoc-Ser-OPfp 12 and Fmoc-Cys(SBu t )-OPfp 14 were reacted with 10 , byproducts 19 , 20 and 21 derived from N - and O -acylation were produced. In contrast, reactions of 10 and N -protected amino acid thioesters were promoted with AgNO 3 , HOSu, and DIEA to afford the coupling products without the undesired O -acylation. Peptidylation of 10 with the synthesized oligopeptide thioesters 24 and 27 was also successful under the segment coupling conditions of the peptide thioester method as well as those of the native chemical ligation.

Silyl Linker-based Approach to the Solid-phase Synthesis of Fmoc Glycopeptide Thioesters

An efficient solid-phase synthesis of Fmoc (glyco)peptide thioesters is described. Fmoc·Ser·OAll and Fmoc·Thr·OAll bound to resin with a silyl ether linker were deallylated by Pd(0) catalysis and condensed with thiophenol, benzyl mercaptane, and ethyl 3-mercaptopropionate by activation with DCC/HOBt. The thioesters were released from the resin either by treatment with CsF-AcOH or by acidic hydrolysis. The effectiveness of this silyl linker strategy is further demonstrated by the synthesis of more complex (glyco)peptide thioesters 25, 26 and 27 involving N→C and C→N peptide elongation.

Synthesis and structural characterization of triple-helical peptides which mimic the ligand binding site of the human macrophage scavenger receptor

Abstract A synthetic method for triple-helical peptides was developed. Peptides with and without glutamic acid α-thioester at their N-termini are prepared by the solid-phase method. These peptides are crosslinked at their N-termini one by one to generate trimeric peptides using the activation of the thioester group by silver ions. This method was applied to the synthesis of model peptides, which mimic the binding site of modified low density lipoprotein (LDL) in the human scavenger receptor (SR). These models possessed different spacers, which connect the peptide chains and the crosslinking site. CD and DSC analysis of the peptides revealed that spacer length has a critical effect on the stability of the triple helix.

SYNTHESIS AND LIPOSOME-FORMATION OF A THERMOSTABLE LIPID BEARING CELL ADHESION PEPTIDE SEQUENCE

Abstract A peptide containing the cell adhesion sequence, Arg-Gly-Asp-Ser, was attached to 2,3-phytanoxypropaneamine (NH 2 -DPhy), an archaebacterial lipid model. The peptide moiety contained a thioester group at its C -terminus and was synthesized by the solid-phase method. The thioester group was activated with silver ions to yield a peptide bond between the peptide and the archaebacterial lipid. The resulting peptide-lipid conjugate formed stable liposomes upon sonication.