Papers - Fujimoto Yukari

The key entity of a DCAR agonist, phosphatidylinositol mannoside Ac ₁PIM ₁: Its synthesis and immunomodulatory function

Arai Y., Torigoe S., Matsumaru T., Yamasaki S., Fujimoto Y.

Organic and Biomolecular Chemistry 18 ( 19 ) 3659 - 3663 2020.05

Accepted,  ISSN  14770520

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© The Royal Society of Chemistry 2020. Ac1PIM1is a potential biosynthetic intermediate for phosphatidylinositol mannosides (PIMs) fromMycobacterium tuberculosis. We achieved the first synthesis of Ac1PIM1by utilizing an allyl-type protecting group strategy and regioselective phosphorylation of inositol. A very potent agonist of an innate immune receptor DCAR, which is better than previously known agonists, is demonstrated.

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Design and Discovery of Covalent α-GalCer Derivatives as Potent CD1d Ligands

Kishi J., Inuki S., Kashiwabara E., Suzuki T., Dohmae N., Fujimoto Y.

ACS Chemical Biology 15 ( 2 ) 353 - 359 2020.02

Accepted,  ISSN  15548929

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© 2020 American Chemical Society. CD1d is a nonpolymorphic antigen-presenting protein responsible for the regulation of natural killer T (NKT) cell activation. α-Galactosyl ceramide (α-GalCer, KRN7000) is the representative CD1d ligand that can bind to the CD1d protein. The resulting complex is recognized by the T cell receptors of the NKT cell, inducing various immune responses. Previous structure-activity relationship studies of α-GalCer have revealed that the ability of NKT cells to induce cytokines depends on the ligand structure, and in particular, ligands that form more stable complexes with CD1d display potent activity. We focused on the Cys residue of the large hydrophobic pockets of CD1d (A′ pocket) and developed α-GalCer derivatives containing groups that can form covalent bonds. The assay results revealed that these ligands displayed higher levels of cytokine production and Th2 cell-type cytokine polarization response. Furthermore, the LC-MS/MS analysis indicated that the chloroacetylamide-containing ligand was covalently bound to Cys12 of CD1d, which suggests that the enhanced activities result from the formation of a stable CD1d-ligand complex. To our knowledge, this is the first ligand that allows covalent bond formation to CD1d under physiological conditions.

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Potent Th2 Cytokine-Bias of Natural Killer T Cell by CD1d Glycolipid Ligands Based on “Anchoring Effect” of Polar Groups in Their Lipid Component.

Inuki, S., Kashiwabara, E., Hirata, N., Kishi, J., Nabika, E., Fujimoto, Y.

Angew. Chem. Int. Ed. 57 ( 31 ) 9655 - 9659 2018

Research paper (scientific journal), Joint Work, Accepted,  ISSN  14337851

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© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Th2-biasing CD1d ligands are attractive potential candidates for adjuvants and therapeutic drugs. However, the number of potent ligands is limited, and their biasing mechanism remain unclear. Herein, a series of novel Th2-biasing CD1d glycolipid ligands, based on modification of their lipid part, have been identified. These have shown high binding affinities and efficient Th2 cytokine production. Importantly, the truncated acyl chain containing variants still retain their binding affinities and agonistic activities, which can be associated with an “anchoring effect,” that is, formation of a buried hydrogen bond between a polar group on the acyl chain and the CD1d lipid-binding pocket. The analysis indicated that the appearance rates of ligand–CD1d complexes on the cell surface were involved in Th2-biasing responses. The designed ligands, having the anchor in the shorter lipid part, are one of the most potent Th2-biasing ligands among the known ligands.

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Syntheses and Immunological Evaluation of Self-Adjuvanting Clustered N-Acetyl and N-Propionyl Sialyl-Tn Combined with A T-helper Cell Epitope as Antitumor Vaccine Candidates.

Chang, T. C., Manabe, Y., Fujimoto, Y., Ohshima, S., Kametani, Y., Kabayama, K., Nimura, Y., Lin, C. C., Fukase, K.

Angew. Chem. Int. Ed. (Angewandte Chemie - International Edition)  57 ( 27 ) 8219 - 8224 2018

Research paper (scientific journal), Joint Work,  ISSN  14337851

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© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Sialyl-Tn (STn) is a tumor-associated carbohydrate antigen (TACA) rarely observed on healthy tissues. We synthesized two fully synthetic N-acetyl and N-propionyl STn trimer (triSTn) vaccines possessing a T-helper epitope and a TLR2 agonist, since the clustered STn antigens are highly expressed on many cancer cells. Immunization of both vaccines in mice induced the anti-triSTn IgG antibodies, which recognized triSTn-expressing cell lines PANC-1 and HepG2. The N-propionyl triSTn vaccine induced the triSTn-specific IgGs, while IgGs induced by the N-acetyl triSTn vaccine were less specific. These results illustrated that N-propionyl triSTn is a valuable unnatural TACA for anticancer vaccines.

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Isolated Polar Amino Acid Residues Modulate Lipid Binding in the Large Hydrophobic Cavity of CD1d

Inuki, S., Aiba, T., Hirata, N., Ichihara, O., Yoshidome, D., Kita, S., Maenaka, K., Fukase, K., Fujimoto, Y.

ACS Chem. Biol. 11   3132 - 3139 2016.09

Research paper (scientific journal), Accepted

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Trehalose diesters containing a polar functional group-modified lipid moiety: Synthesis and evaluation of Mincle-mediated signaling activity.

Matsumaru, T., Sueyoshi, K., Okubo, K., Fujii, S., Sakuratani, K., Saito, R., Ueki, K., Yamasaki, S., Fujimoto, Y.

Bioorg. Med. Chem. 75   117045 2022.12

Research paper (scientific journal), Accepted

Precise immunological evaluation rationalizes the design of a self-adjuvanting vaccine composed of glycan antigen, TLR1/2 ligand, and T-helper cell epitope.

Chang, T. C., Manabe, Y., Ito, K., Yamamoto, R., Kabayama, K., Ohshima, S., Kametani, Y., Fujimoto, Y., Lin, C. C., Fukase, K.

RSC Advances (The Royal Society of Chemistry)  12   18985 - 18993 2022.06

Research paper (scientific journal), Accepted

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β-mannosyloxymannitol glycolipids and their analogues: synthesis and Mincle-mediated signaling activity.

Matsumaru, T., Sakuratani, K., Yanaka, S., Kato, K., Yamasaki, S., Fujimoto, Y. Fungal

European journal of Organic Chemistry    e202200109 2022.04

Research paper (scientific journal), Corresponding author, Accepted

Alkyne-Tagged Dopamines as Versatile Analog Probes for Dopamin-ergic System Analysis.

Nuriya, M., Ashikari, Y., Shou, J., Iino, T., Asai, T., Karasawa, K., Nakamura, K., Ozeki, Y., Fujimoto, Y.

Analytical Chemistry (ACS)  93 ( 27 ) 9345 - 9355 2021.07

Research paper (scientific journal), Accepted

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Lipopolysaccharide derived from the lymphoid-resident commensal bacteria Alcaligenes faecalis functions as an effective nasal adjuvant to augment IgA antibody and Th17 cell responses.

Wang, Y., Hosomi, K., Shimoyama, A., Yoshii, K., Nagatake, T., Fujimoto, Y., Kiyono, H., Fukase, K., Kunisawa, J.

Frontiers in immunology 12   699349 2021.07

Research paper (scientific journal), Accepted

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Lipopolysaccharide from Gut-Associated Lymphoid-Tissue-Resident Alcaligenes faecalis: Complete Structure Determination and Chemical Synthesis of Its Lipid A.

Shimoyama A, Di Lorenzo F, Yamaura H, Mizote K, Palmigiano A, Pither MD, Speciale I, Uto T, Masui S, Sturiale L, Garozzo D, Hosomi K, Shibata N, Kabayama K, Fujimoto Y, Silipo A, Kunisawa J, Kiyono H, Molinaro A, Fukase K.

Angew Chem Int Ed Engl 60 ( 18 ) 10023 - 10031 2021.01

Research paper (scientific journal), Accepted

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Polar functional group-containing glycolipid CD1d ligands modulate cytokine-biasing responses and prevent experimental colitis

Inuki S., Hirata N., Kashiwabara E., Kishi J., Aiba T., Teratani T., Nakamura W., Kojima Y., Maruyama T., Kanai T., Fujimoto Y.

Scientific Reports (Scientific Reports)  10 ( 1 )  2020.12

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The MHC class I-like molecule CD1d is a nonpolymorphic antigen-presenting glycoprotein, and its ligands include glycolipids, such as α-GalCer. The complexes between CD1d and ligands activate natural killer T cells by T cell receptor recognition, leading to the secretion of various cytokines (IFN-γ, IL-4, IL-17A, etc.). Herein, we report structure–activity relationship studies of α-GalCer derivatives containing various functional groups in their lipid acyl chains. Several derivatives have been identified as potent CD1d ligands displaying higher cytokine induction levels and/or unique cytokine polarization. The studies also indicated that flexibility of the lipid moiety can affect the binding affinity, the total cytokine production level and/or cytokine biasing. Based on our immunological evaluation and investigation of physicochemical properties, we chose bisamide- and Bz amide-containing derivatives 2 and 3, and evaluated their in vivo efficacy in a DSS-induced model of ulcerative colitis. The derivative 3 that exhibits Th2- and Th17-biasing responses, demonstrated significant protective effects against intestinal inflammation in the DSS-induced model, after a single intraperitoneal injection.

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Efficient preparation of human and mouse CD1d proteins using silkworm baculovirus expression system

Kusaka H., Kita S., Tadokoro T., Yoshida K., Kasai Y., Niiyama H., Fujimoto Y., Hanashima S., Murata M., Sugiyama S., Ose T., Kuroki K., Maenaka K.

Protein Expression and Purification (Protein Expression and Purification)  172 2020.08

ISSN  10465928

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© 2020 Elsevier Inc. CD1d is a major histocompatibility complex (MHC) class I-like glycoprotein and binds to glycolipid antigens that are recognized by natural killer T (NKT) cells. To date, our understanding of the structural basis for glycolipid binding and receptor recognition of CD1d is still limited. Here, we established a preparation method for the ectodomain of human and mouse CD1d using a silkworm-baculovirus expression system. The co-expression of human and mouse CD1d and β2-microglobulin (β2m) in the silkworm-baculovirus system was successful, but the yield of human CD1d was low. A construct of human CD1d fused with β2m via a flexible GS linker as a single polypeptide was prepared to improve protein yield. The production of this single-chained complex was higher (50 μg/larva) than that of the co-expression complex. Furthermore, differential scanning calorimetry revealed that the linker made the CD1d complex more stable and homogenous. These results suggest that the silkworm-baculovirus expression system is useful for structural and biophysical studies of CD1d in several aspects including low cost, easy handling, biohazard-free, rapid, and high yielding.

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Synthetic Studies on FNC-RED and Its Analogues Containing an All syn-Cyclopentanetetrol Moiety.

Inuki, S., Sato, K., Zui, N., Yamaguchi, R., Matsumaru, T., Fujimoto, Y.

J. Org. Chem. (Journal of Organic Chemistry)  84 ( 19 ) 12680 - 12685 2019.09

Research paper (scientific journal), Accepted,  ISSN  00223263

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© 2019 American Chemical Society. FNC-RED exhibits innate immune receptor Toll-like receptor 4 (TLR4)/myeloid differentiation factor-2 (MD2) stimulatory activity. We have developed a divergent synthetic route to FNC-RED derivatives containing various alkyl side chains. Key features of the synthetic study include stepwise palladium catalyzed cross-coupling reactions and the construction of an all syn-cyclopentanetetrol moiety.

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Total synthesis of naturally occurring chiral cyclopropane fatty acids and related compounds

Inuki S., Fujimoto Y.

Tetrahedron Letters (Tetrahedron Letters)  60 ( 16 ) 1083 - 1090 2019.04

ISSN  00404039

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© 2019 Elsevier Ltd Cyclopropane fatty acids (CFAs) and related compounds containing CFAs commonly occur in various natural sources, including plants or microorganisms. Some CFAs exhibit intriguing biological activities such as antifungal or immunosuppressive activities. Due to their biological importance and structural features, a number of synthetic studies have been conducted to date. These total syntheses have helped to elucidate their structural determinants or biological activities. Herein the total syntheses of naturally occurring CFAs and related compounds are summarized.

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Structure-activity relationship studies of Bz amide-containing α-GalCer derivatives as natural killer T cell modulators

Kishi J., Inuki S., Hirata N., Kashiwabara E., Yoshidome D., Ichihara O., Fujimoto Y.

Bioorganic and Medicinal Chemistry Letters (Bioorganic and Medicinal Chemistry Letters)  29 ( 8 ) 970 - 973 2019.04

ISSN  0960894X

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© 2019 Elsevier Ltd CD1d is a non-polymorphic antigen-presenting glycoprotein that recognizes glycolipids as ligands. Ligands bind to the hydrophobic grooves of CD1d, and the resulting ligand-CD1d complexes activate natural killer T (NKT) cells by means of T cell receptor recognition, leading to the secretion of various cytokines. However, details of the ligand recognition mechanism of a large hydrophobic ligand binding pocket and the relationship between cytokine induction and ligand structure are unclear. We report the synthesis of α-GalCer derivatives containing a Bz amide group having various substituting groups in the ceramide moiety, and the analysis of the structure-activity relationships. The assays reveal that the Bz amide-containing CD1d ligands function as NKT cell modulators displaying Th2 cytokine biasing responses. Furthermore, molecular dynamics simulation studies suggest that the phenyl groups can interact with the aromatic amino acid residues in the lipid binding pocket of CD1d.

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Glycans in infection and immunity

Suzuki T., Okamatsu M., Sakoda Y., Kinoshita T., Katayama T., Kiyono H., Goto Y., Takegawa K., Yokoyama N., Fujimoto Y., Angata T., Ohtani K., Wakamiya N., Arase H., Nishihara S., Suda Y.

Glycoscience: Basic Science to Applications: Insights from the Japan Consortium for Glycobiology and Glycotechnology (JCGG) (Glycoscience: Basic Science to Applications: Insights from the Japan Consortium for Glycobiology and Glycotechnology (JCGG))     227 - 257 2019.01

ISSN  9789811358555

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Synthesis of glycerolipids containing simple linear acyl chains or aromatic rings and evaluation of their Mincle signaling activity

Matsumaru T., Ikeno R., Shuchi Y., Iwamatsu T., Tadokoro T., Yamasaki S., Fujimoto Y., Furukawa A., Maenaka K.

Chemical Communications (Chemical Communications)  55 ( 5 ) 711 - 714 2019

ISSN  13597345

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© The Royal Society of Chemistry. Mincle, expressed in activated phagocytes, recognizes the lipid ligand to activate the innate immune system. We have synthesized glycerol derivatives possessing simple alkyl chains or aromatic rings and elucidated their structure-activity relationships using a Mincle-mediated signaling assay. The activity depends on the length of the simple acyl chains of the glycerol derivatives.

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Synthesis of Cyclopropane Fatty Acids by C(sp3)–C(sp3) Cross-coupling Reaction and Formal Synthesis of α-Mycolic Acid.

Iwasaki, T., Terahigashi, S., Wang, Y., Tanaka, A., Zhao, H., Fujimoto, Y., Fukase, K., Kambe. N.

Adv. Synth. Catal. (Advanced Synthesis and Catalysis)  in press ( 19 ) 3810 - 3817 2018

Research paper (scientific journal), Joint Work,  ISSN  16154150

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© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim An iterative Ni-catalyzed C(sp3)−C(sp3) cross-coupling reaction of a novel cis-cyclopropane containing bifunctional building blocks with alkyl halides and alkyl Grignard reagents enabled the introduction of a cyclopropane ring into the desired position(s) of saturated carbon frameworks, providing a straightforward synthetic route to cyclopropane fatty acids. The present method creates a direct route for the construction of saturated carbon frameworks, and can avoid the tedious multistep operations based on unsaturated functional group manipulations that are often employed in conventional synthetic routes. This method could be applicable to the synthesis of trans-cyclopropane fatty acids and enantioenriched cyclopropane fatty acids. Formal synthesis of α-mycolic acid was achieved by the C(sp3)−C(sp3) cross-coupling reaction of cyclopropane-containing bifunctional building blocks. (Figure presented.).

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Lymphoid tissue-resident Alcaligenes LPS induces IgA production without excessive inflammatory responses via weak TLR4 agonist activity.

Shibata, N., Kunisawa, J., Hosomi, K., Fujimoto, Y., Mizote, K., Kitayama, N., Shimoyama, A., Mimuro, H., Sato, S., Kishishita, N., Ishii, K. J., Fukase, K., Kiyono, H.

Mucosal Immunol. (Mucosal Immunology)  11 ( 3 ) 693 - 702 2018

Research paper (scientific journal), Joint Work, Accepted,  ISSN  19330219

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© The Author(s) 2018. Alcaligenes are opportunistic commensal bacteria that reside in gut-associated lymphoid tissues such as Peyer's patches (PPs); however, how they create and maintain their homeostatic environment, without inducing an excessive inflammatory response remained unclear. We show here that Alcaligenes-derived lipopolysaccharide (Alcaligenes LPS) acts as a weak agonist of toll-like receptor 4 and promotes IL-6 production from dendritic cells, which consequently enhances IgA production. The inflammatory activity of Alcaligenes LPS was weaker than that of Escherichia coli-derived LPS and therefore no excessive inflammation was induced by Alcaligenes LPS in vitro or in vivo. Alcaligenes LPS also showed adjuvanticity, inducing antigen-specific immune responses without excessive inflammation. These findings reveal the presence of commensal bacteria-mediated homeostatic inflammatory conditions within PPs that produce optimal IgA induction without causing pathogenic inflammation and suggest that Alcaligenes LPS could be a safe and potent adjuvant.

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