Synthesis of chondroitin sulfate CC and DD tetrasaccharides and interactions with 2H6 and LY111 Kenya Matsushita a,e , Tomomi Nakata b , Naoko Takeda-Okuda c , Satomi Nadanaka d , Hiroshi Kitagawa d , Jun-ichi Tamura a,b,c,⇑ a Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, Koyamacho-Minami 4-101, Tottori 680-8552, Japan b Department of Regional Environment, Faculty of Regional Sciences, Tottori University, Koyamacho-Minami 4-101, Tottori 680-8551, Japan c Department of Life and Environmental Agricultural Sciences, Faculty of Agriculture, Tottori University, Koyamacho-Minami 4-101, Tottori 680-8553, Japan d Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan article info Article history: Received 9 December 2017 Revised 17 January 2018 Accepted 18 January 2018 Available online 3 February 2018 Keywords: Chondroitin sulfate Glycosylation Oligosaccharide synthesis 2H6 LY111 abstract We synthesized the biotinylated chondroitin sulfate tetrasaccharides CS-CC [-3)bGalNAc6S(1–4)bGlcA(1- ] 2 and CS-DD [-3)bGalNAc6S(1–4)bGlcA2S(1-] 2 which possess sulfate groups at O-6 of GalNAc and an additional sulfate group at O-2 of GlcA, respectively. We also analyzed interactions among CS-CC and CS-DD and the antibodies 2H6 and LY111, both of which are known to bind with CS-A, while CS-DD was shown for the first time to bind with both antibodies. Ó 2018 Elsevier Ltd. All rights reserved. 1. Introduction Chondroitin sulfate (CS) is a linear polysaccharide consisting of D-glucuronic acid (GlcA) and N-acetyl-D-galactosamine (GalNAc), the hydroxyl groups of which are often regiospecifically sulfated. CS exhibits various bioactivities that depend on sulfation patterns. Highly sulfated CS-D [-3)bGalNAc6S(1–4)bGlcA2S(1-] and CS-E [-3) bGalNAc4S6S(1–4)bGlcA(1-] promote neuronal growth, whereas CS-A [-3)bGalNAc4S(1–4)bGlcA(1-] and CS-C [-3)bGalNAc6S(1–4) bGlcA(1-] do not. 1–3 However, these findings were obtained using naturally occurring CS enriched with these disaccharide units. Characteristic bioactivities are often caused by the localized fine structures of glycans. Chemically synthesized oligosaccharides are structurally defined and are ideal tools for identifying the real functional domain of the glycan exhibiting biological activity. We focused on small differences between the two types of disaccha- ride units, particularly the CS-C and -D units possessing sulfate groups at O-6 of GalNAc and an additional sulfate group at O-2 of GlcA, respectively. To the best of our knowledge, some research groups including our team have been synthesizing CS-C- and -D-type oligosaccha- rides by adopting stepwise glycan elongation. 4–7 Most of these oligosaccharides were not designed for conjugation procedures. Our group has synthesized biotinylated CS oligosaccharides and elucidated the degree of the interaction between glycans and midkine. 8 Our glycans have the GalNAc-GlcA-type sequence, while Jacquinet’s group also obtained some biotinylated CS oligosaccha- rides of the GlcA-GalNAc-type sequence up to the tetrasaccharides, CS-AA, -CC, -OA, and -OC, in addition to the octasaccharide of CS-O. 9,10 In that study the disaccharide unit was obtained by the acid hydrolysis of natural CS (polysaccharide); thus, the disaccha- ride had the GlcA-GalNAc-type sequence. 9,11 In spite of the signif- icance of its biological role, a biotinylated CS-DD tetrasaccharide has yet to be synthesized for biological use. In the present study, we synthesized the CS tetrasaccharides, CS-CC [-3)bGalNAc6S(1–4)bGlcA(1-] 2 (1) and CS-DD [-3)bGal- NAc6S(1–4)bGlcA2S(1-] 2 (2), as dimers bound with a biotin linker https://doi.org/10.1016/j.bmc.2018.01.011 0968-0896/Ó 2018 Elsevier Ltd. All rights reserved. Abbreviations: CS, chondroitin sulfate; GlcA, D-glucuronic acid; GalNAc, N- acetyl-D-galactosamine; MS, molecular sieves; TMSOTf, trimethylsilyl triflate; NAP, (2-naphtyl)methyl; DDQ, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone; MBz, 4- methylbenzoyl; Z, benzyloxycarbonyl. ⇑ Corresponding author at: Department of Life and Environmental Agricultural Sciences, Faculty of Agriculture, Tottori University, Koyamacho-Minami 4-101, Tottori 680-8553, Japan. E-mail address: jtamura@muses.tottori-u.ac.jp (J.-i. Tamura). e Present address: Fujimoto Pharmaceutical Corporation, Nishi-otsuka 1-3-40, Matsubara, Osaka 580-8503, Japan. Bioorganic & Medicinal Chemistry 26 (2018) 1016–1025 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc