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Glycolipid antigens are presented by CD1d on antigen-presenting cells to T cell receptors (TCRs) of natural killer T (NKT) cells, leading to immune responses via cytokine induction. Although various lipid antigens have been found, there are only a limited number of glycolipid antigens having selective cytokine induction. In this study, we identified the glycolipids (α-GalCer nitro-type) that exhibit highly selective induction of Th2 and Th17 type cytokines, with very high binding affinity to CD1d, by introducing nature-inspired nitro-modified fatty acyl groups. The natural nitroalkene moiety of fatty acyl groups in the glycolipids effectively enhances the affinity to CD1d through presumed hydrogen-bonding and NO2–π interactions, leading to the distinctive function in cytokine induction and selectivity.

Introduction: Grass pollen triggers nearly 30% of bronchial allergic asthma cases. While most Q8 research focuses on pollen allergens, pollen lipids may also influence allergic reactions. Previous studies demonstrated that Timothy grass (TG, Phleum pratense) lipids, such as phytoprostanes, can activate immune cells, promoting pro-allergic responses. However, the role of water-insoluble pollen glycolipids in allergic airway inflammation remains unclear. Thus, this study aimed to isolate and characterize glycolipids from TG pollen and evaluate their bioactivity in allergic airway inflammation. Methods: Lipids were extracted from the water-insoluble pollen fraction, separated by silica gel, and fractionated by HPLC. GC-MS, HR ESI-MS, and NMR confirmed the presence of di-galactosyldiacylglycerol (DGDG). The biological activity of fractions containing DGDG (DGDG-3 and DGDG-4) and synthetic DGDG variants was tested in vitro in murine and human cell systems and in vivo in mice. Results: Fraction 4 induced strong proliferation of murine NKT cells and upregulated CD69 expression in human NKT cells. Synthetic DGDG variants (DGDG-1, DGDG-2, and DGDG-3) with defined acylation profiles stimulated robust NKT-cell proliferation, with DGDG-2 and DGDG-3 increasing IL-13 production, one of the key Th2 cytokines. In vivo, only these variants caused lung inflammation marked by eosinophil infiltration but did not increase airway resistance. Discussion: This study reveals for the first time the structure-dependent role of DGDG of TG pollen grains in immune cell recognition in the context of allergic inflammation. Our data may pave the way for therapies targeting lipid components in combination with protein allergens.

Newly developed synthetic methods for monoglycosylceramides enabled representative brain glycolipid synthesis. Immunomodulatory activities of the glycolipids were examined via CD1d and Mincle. CD1d strongly bound endogenous GlcCer with longer unsaturated fatty acids (e.g., C24:1), partly inhibiting other antigens and stabilizing CD1d's immunological role in presenting lipid antigens to NKT cells.

© 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.

© 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.

© 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.

© 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.

The dopaminergic system is essential for the function of the brain in health and disease. Therefore, detailed studies focused on unraveling the mechanisms involved in dopaminergic signaling are required. However, the lack of probes that mimic dopamine in living tissues, owing to the neurotransmitter’s small size, has hampered analysis of the dopaminergic system. The current study aimed to overcome this limitation by developing alkyne-tagged dopamine compounds (ATDAs) that have a minimally invasive and uniquely identifiable alkyne group as a tag. ATDAs were established as chemically and functionally similar to dopamine and readily detectable by methods such as specific click chemistry and Raman scattering. The ATDAs developed here were verified as analogue probes that mimic dopamine in neurons and brain tissues, allowing the detailed characterization of dopamine dynamics. Therefore, ATDAs can act as safe and versatile tools with wide applicability in detailed studies of the dopaminergic system. Furthermore, our results suggest that the alkyne-tagging approach can also be applied to other small-sized neurotransmitters to facilitate characterization of their dynamics in the brain.

Mincle, a C-type lectin receptor (CLR), senses certain lipid conjugates, leading to the activation of the innate immune system. The lipid conjugate ligands include glyco-, glycero-, and mannitol lipids. Recently, a fungal β-mannosyloxymannitol glycolipid demonstrating a novel architecture, “44-2,” was isolated from the fungus Malassezia and displayed potent Mincle-mediated signaling activity. For this study, we synthesized the diastereomeric pair of mannosyloxymannitol glycolipids and their analogues and analyzed the structure-activity relationships of Mincle-mediated signaling. The results suggest that the dimannosyl-fatty acid moiety in the mannosyloxymannitol glycolipid-type structure plays a major role in the molecular recognition of Mincle.

Peptidoglycan (PGN) is a component of bacterial cell walls; its fragments are recognized by the cytoplasmic receptors Nod1 and Nod2, thereby promoting the production of inflammatory cytokines and antibodies. To further elucidate these biological defense mechanisms, a large and stable supply of the PGN fragments via chemical synthesis is essential. However, the synthesis and purification of long PGN fragments are quite challenging due to their low solubility. In this study, we efficiently synthesized PGN fragments via solid-phase oligosaccharide synthesis (SPOS). Using the JandaJel™ Wang resin (JJ-Wang), an octasaccharide glycan chain of PGN was constructed by repeating glycosylation reactions to elongate β-1,4-linked disaccharide units composed of MurNAc and GlcNAc. To enhance reactivity, glycosylation was performed in a mixed solvent comprising C4F9OEt/CH2Cl2/THF with the intention of promoting substrate concentration onto the solid support through the fluorophobic effect, affording the PGN octasaccharide in a 19% overall yield (10 steps). Subsequently, after deprotection of the O-Fmoc, N-Troc, and ethyl ester groups, N- and O-acetylation proceeded smoothly, owing to the high swelling property of JJ-Wang. Peptide condensation with L-Ala-D-isoGln(OBn) and carboxylic acids was also achieved. Finally, cleavage of the PGN fragment from the resin with TFA afforded the desired octasaccharide with dipeptides in a 2.3% overall yield (15 steps).