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Yoshiaki Nakahara

Publications and source records attributed to Yoshiaki Nakahara.

18 recordsLinked to original sources
PubMed2006

Chemoenzymatic synthesis of a MUC1 glycopeptide carrying non-natural sialyl TF-beta O-glycan.

A MUC1 type of glycopeptide was synthesized by the 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS) protocol using benzyl and benzylidene-protected beta-D-Gal-(1-->3)-beta-D-GalNAc-Ser/Thr (TF-beta: a stereoisomer of the Thomsen-Friedenreich antigen). The synthetic glycopeptide was released from the resin with reagent K, and the resulting benzylated glycopeptide was deprotected under conditions of low-acidity trifluoromethanesulfonic acid (TfOH). The glycopeptide carrying duplicate non-natural O-glycans was dominant in the products, but was accompanied by a considerable amount of the glycopeptide missing one of the O-glycans. In contrast, the native alpha-glycoside was relatively stable under the acidic debenzylation conditions as shown by a parallel experiment with the glycopeptide involving alpha-D-GalNAc-Ser/Thr linkage. Enzymatic glycosylation with CMP-NeuAc was successful with both natural and non-natural O-glycans of the synthetic glycopeptide.

Antigens, Neoplasm↗
PubMed2006

Efficient microwave-assisted tandem N- to S-acyl transfer and thioester exchange for the preparation of a glycosylated peptide thioester.

A peptide carrying a mercaptomethylated proline derivative at the C-terminus was prepared by solid-phase peptide synthesis (SPPS) and converted to the thioester of 3-mercaptopropionic acid (MPA) by aqueous MPA under microwave irradiation conditions. This post-SPPS thioesterification reaction was successfully applied to the synthesis of a glycopeptide thioester composed of 25 amino acid (AA) residues, which was then used for the preparation of a 61-AA glycopeptide by the thioester condensation method.

3-Mercaptopropionic Acid↗
PubMed2006

Structural characterization of glycopeptides by N-terminal protein ladder sequencing.

High-sensitivity and high-throughput mass spectrometry (MS) has become an important tool for characterizing glycopeptides. Here, we analyzed synthetic O-linked glycopeptides using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. First, we applied MALDI-quadrupole ion trap (QIT)-TOF MS, which enables collision-induced dissociation-MSn analysis for fine structural characterization. Subsequent MS/MS of sodium adduct ions selected as precursor ions yielded detailed information about the site of oligosaccharide attachment as well as the carbohydrate and amino acid sequences; however, these MS/MS spectra were very complex. To obtain easily interpretable and simple spectra, we used N-terminal protein ladder sequencing coupled with MALDI-TOF MS. From the extremely simple resulting spectra, we were able to determine the glycosylation sites, amino acid sequences, and oligosaccharide molecular weights of the glycopeptides.

Amino Acid Sequence↗
PubMed2006

Synthesis of a glycosylated peptide thioester by the Boc strategy and its application to segment condensation.

The synthesis of a chitobiosylated peptide thioester by the t-butoxycarbonyl (Boc) strategy is demonstrated. Boc-Asn carrying benzyl-protected chitobiose was introduced during application of the Boc mode solid-phase method. HF treatment of the resulting protected peptide resin gave the desired chitobiosylated peptide thioester. This thioester was used to prepare the peptide sequence derived from extracellular matrix metalloproteinase inducers (emmprin) (34-94), (34-118) and (22-118) by the thioester segment condensation method. The conformation of these glycopeptides is characterized by circular dichroism (CD) spectral measurement.

Amino Acid Sequence↗
PubMed2005

Chemical synthesis of 23 kDa glycoprotein by repetitive segment condensation: a synthesis of MUC2 basal motif carrying multiple O-GalNAc moieties.

Peptide thioester corresponding to a MUC2 tandem repeat unit, which retains seven GalNAc moieties, was prepared by the Fmoc method followed by the low TfOH treatment to remove benzyl groups at the carbohydrate portions. The glycosylated peptide thioester was then consecutively joined by the activation of a thioester group by silver ions to obtain a MUC2 tandem repeat model composed of 141 amino acids with 42 GalNAc moieties.

Acetylgalactosamine↗
PubMed2005

Combinatorial synthesis of MUC1 glycopeptides: polymer blotting facilitates chemical and enzymatic synthesis of highly complicated mucin glycopeptides.

The chemoselective polymer blotting method allows for rapid and efficient synthesis of glycopeptides based on a "catch and release" strategy between solid-phase and water-soluble polymer supports. We have developed a heterobifunctional linker sensitive to glutamic acid specific protease (BLase). The general procedure consists of five steps, namely (i) the solid-phase synthesis of glycopeptide containing BLase sensitive linker, (ii) subsequent deprotections and the release of the glycopeptide from the resin, (iii) chemoselective blotting of the glycopeptide intermediates in the presence of water-soluble polymers with oxylamino functional groups, (iv) sugar elongations using glycosyltransferases, and (v) the release of target glycopeptides from the polymer platform by selective BLase promoted hydrolysis. The combined use of the solid-phase chemical syntheses of peptides and the enzymatic syntheses of carbohydrates on water-soluble polymers would greatly contribute to the production of complicated glycopeptide libraries, thereby enhancing applicative research. We report here a high-throughput synthetic system for the various types of MUC1 glycopeptides exhibiting a variety of sugar moieties. It is our belief that this concept will become part of the entrenched repertoire for the synthesis of biologically important glycopeptides on the basis of glycosyltransferase reactions in automated and combinatorial syntheses.

Carbohydrate Sequence↗
PubMed2005

Solid-phase synthesis of core 8 O-glycan-linked MUC5AC glycopeptide.

The benzyl-protected disaccharide building blocks of core 8 O-glycan (15a/15b) for glycopeptide were stereoselectively synthesized by two glycosidation reactions with the glycosyl fluoride method. The building blocks were utilized in the solid-phase synthesis of a glycopeptide carrying two O-glycans with the consensus sequence of the tandem-repeat domain of MUC5AC. The synthetic glycopeptide was detached from the resin with reagent K, and subsequent debenzylation under conditions of low-acidity TfOH afforded glycopeptide 2. The synthetic sample will be used as a suitable standard in studies of the physicochemical or immunochemical characterization of mucin glycoforms.

Carbohydrate Sequence↗
PubMed2004

Synthesis and conformational characterization of the epidermal growth factor-like domain of blood coagulation factor IX carrying xylosyl-glucose.

Solid-phase synthesis of glycopeptide generally requires the protection of both peptide side chains and hydroxyl groups of the carbohydrate portion. However, if the mild coupling conditions are used, the protection of the carbohydrate portion can be omitted. In this paper, we demonstrated it by the synthesis of Fmoc-serine carrying unmasked xylosyl glucose followed by the solid-phase synthesis of epidermal growth factor (EGF)-like domain of factor IX (45-87) using the unit. The product was well characterized by enzymatic digestion, amino acid analysis and mass spectrometry. The secondary structure of the product as well as glucosylated and non-glycosylated EGF-like domain was characterized by circular dichroism (CD) spectroscopy.

Amino Acid Sequence↗
PubMed2003

Application of Fmoc-amino acid carrying an unmasked carbohydrate to the synthesis of the epidermal growth factor-like domain of bovine blood coagulation factor IX.

The epidermal growth factor (EGF)-like domain of the bovine blood coagulation factor IX (45-87) carrying glucose at Ser(53) was synthesized by a solid-phase method using 9-fluorenylmethoxycarbonyl (Fmoc)-amino acids. The introduction of Ser(53) was carried out using the benzotriazolyl ester of Fmoc-serine carrying an unmasked glucose. The remaining sequence was also introduced using the benzotriazolyl ester. HPLC analysis of the crude peptide shows that acylation of the free hydroxyl group of the glucose was not significant, demonstrating that the amino acid carrying an unmasked carbohydrate is a useful building block for solid-phase synthesis.

Amino Acid Sequence↗
PubMed2003

Synthesis of an unnatural N-glycan-linked dolichyl pyrophosphate precursor.

An unnatural alpha-D-mannopyranose-linked chitobiosyl dolichyl pyrophosphate, a stereoisomer of the N-glycan biosynthesis intermediate, was synthesized. The protected trisaccharide, alpha-D-Man-(1-->4)-beta-D-GlcNAc-(1-->4)-D-GlcNAc, carrying a 4-methylbenzoyl group was prepared for the convenience of a TLC analysis. 1-O-Phosphorylation, condensation with dolichyl phosphate, and subsequent deprotection afforded the title compound.

Carbohydrate Conformation↗
PubMed2002

Hypothermia augments polymorphonuclear leukocyte degranulation and interleukin-8 production from human umbilical vein endothelial cells and increases lipopolysaccharide-induced polymorphonuclear leukocyte-endothelial cell interaction when followed by normothermia.

OBJECTIVE: To determine if hypothermia followed by normothermia (rewarmed to 37 degrees C) changes the inflammatory response of polymorphonuclear leukocytes (PMNs) and human umbilical vein endothelial cells (HUVEC). DESIGN: Prospective, controlled, in vitro study. SETTING: University laboratory. PARTICIPANTS: PMNs from 4 healthy volunteers and HUVEC. MEASUREMENTS AND MAIN RESULTS: PMNs and HUVEC were incubated for 3 hours at 20 degrees C, 30 degrees C, or 37 degrees C followed by 37 degrees C again. PMN degranulation, cytokine production from HUVEC, and PMN-HUVEC interaction were compared among the 3 experimental temperatures. Interleukin (IL)-8-induced PMN degranulation measured by myeloperoxidase concentrations was significantly higher in the 20 degrees C and 30 degrees C groups than the 37 degrees C groups. Bacterial lipopolysaccharide (LPS)-induced IL-8 production from HUVEC, measured by enzyme-linked immunosorbent assay, was significantly higher in the 20 degrees C group than the other 2 groups; however, tumor necrosis factor-alpha was not detectable in any of the groups. LPS-induced cell injury measured by cellular (51)Cr release was significantly higher in the 20 degrees C and 30 degrees C groups than in the 37 degrees C groups. This injury was significantly inhibited by IL-8 antibody. CONCLUSION: Hypothermic (20 degrees C and 30 degrees C) incubation followed by rewarming augmented IL-8-induced PMN degranulation and LPS-induced IL-8 production from HUVEC and LPS-induced PMN-endothelial interaction. IL-8 plays an important role in this increased injury. This increased inflammatory response may support the positive outcomes of normothermic CPB.

Cardiopulmonary Bypass↗
PubMed2002

Pulmonary colonization by Chrysosporium zonatum associated with allergic inflammation in an immunocompetent subject.

We report a case of noninvasive pulmonary disease due to Chrysosporium zonatum in an immunocompetent male. The fungus colonized an existing tuberculous cavity and was isolated from transbronchial lavage fluid and from a percutaneous aspiration specimen. The disease was accompanied by the unusual feature of an allergic reaction. The fungus ball was successfully treated by intracavitary administration of amphotericin B. C. zonatum is the anamorph of the heterothallic ascomycete Uncinocarpus orissi, and the identity of the case isolate was verified by formation of ascospores in mating tests with reference isolates.

Aged↗
PubMed2002

Synthesis of core-class 2 O-linked glycopeptides: a benzyl-protected tetrasaccharyl serine and its derivative carrying a hydrophobic cholestanyl group.

A core-class 2 tetrasaccharide-linked serine was synthesized in a convergent manner. The coupling reaction of disaccharide glycosyl donor 3 and acceptor 4 stereoselectively afforded tetrasaccharide 15, which was converted to glycosyl fluoride 20. Glycosylation of Fmoc serine allyl ester 5 with 20 produced alpha- and beta-glycosides in 40% and 33% yields, respectively. Alpha-Isomer 21 was converted into 1, a useful building block for the solid-phase synthesis of glycopeptides. On the other hand, 21 was N-deprotected and condensed with hydrophobic cholestanol through a succinyl spacer. The same compound was alternatively synthesized by coupling 20 and 28. Functional group manipulation and hydrogenation afforded core 2 tetrasaccharide-cholestanol conjugate 2.

Glycopeptides↗
PubMed2002

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 x Ser x OAll and Fmoc x Thr x 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.

Esters↗
PubMed2001

Solid-Phase Capture-Release Strategy Applied to Oligosaccharide Synthesis on a Soluble Polymer Support This work was supported in part by the Mizutani Foundation for Glycoscience (grant to Y.I.), the RIKEN Presidential Fund (grant to S.M.), and the Ministry of Education, Culture, Sports, Science and Technology (Grant-in-Aid for Scientific Research to S.M.). We thank Dr. Teiji Chihara and his staff for the elemental analyses, Ms. Yasuko Tanaka for her contribution at the exploratory stage of this work, and Ms. Akemi Takahashi for her technical assistance.

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Journal Article↗