Synthesis of chondroitin sulfate CC and DD tetrasaccharides and interactions with 2H6 and LY111

https://doi.org/10.1016/j.bmc.2018.01.011Get rights and content

Abstract

We synthesized the biotinylated chondroitin sulfate tetrasaccharides CS-CC [-3)βGalNAc6S(1–4)βGlcA(1-]2 and CS-DD [-3)βGalNAc6S(1–4)βGlcA2S(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.

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)βGalNAc6S(1–4)βGlcA2S(1-] and CS-E [-3)βGalNAc4S6S(1–4)βGlcA(1-] promote neuronal growth, whereas CS-A [-3)βGalNAc4S(1–4)βGlcA(1-] and CS-C [-3)βGalNAc6S(1–4)βGlcA(1-] do not.1, 2, 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 disaccharide 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 oligosaccharides by adopting stepwise glycan elongation.4, 5, 6, 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 oligosaccharides 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 disaccharide had the GlcA-GalNAc-type sequence.9, 11 In spite of the significance 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)βGalNAc6S(1–4)βGlcA(1-]2 (1) and CS-DD [-3)βGalNAc6S(1–4)βGlcA2S(1-]2 (2), as dimers bound with a biotin linker (Fig. 1). These tetrasaccharides were equipped with biotin at the reducing end via a hydrophilic linker for use as biological probes. CS-CC had sulfate groups at two O-6s of GalNAc, while CS-DD had additional sulfate groups at two O-2s of GlcA. We enabled the efficient synthesis of CS-CC and -DD tetrasaccharides. We also analyzed the interactions among 1 and 2 and the antibodies 2H6 and LY111. These antibodies are known to bind with CS-A, while facile recognition sites (sulfation patterns of CS) have not yet been examined in detail. In the present study, the DD-type tetrasaccharide (2) was shown for the first time to bind with both antibodies.

Section snippets

Results and discussion

Prior to the synthesis of tetrasaccharides, we examined the synthetic strategy for the synthesis of the disaccharide analogue, D-type biotinylated CS disaccharide (10) (Scheme 1). The azide group of the suitably protected disaccharide (3)12 was reduced to amine with zinc under acidic conditions, and subsequent acetylation afforded the corresponding acetamide (4) in 96% yield in 2 steps. The allyl group of 4 was removed with the iridium complex to give the hemiacetal 5, which transformed to

Experimental

General methods: Optical rotations were measured at 22 ± 3°C with the HORIBA automatic polarimeter SEPA-500. Melting points were measured with Büchi B-545 and were uncorrected. 1H and 13C NMR assignments were confirmed by two-dimensional HH COSY and 1H and 13C HSQC techniques using the Bruker AVANCE II 600 MHz spectrometer with Me4Si and tert-BuOH as internal standards (0 and 1.23 ppm) in CDCl3 and D2O, respectively. As an example of signal assignments, H-13 stands for a proton at C-1 of sugar

Acknowledgments

This study was supported by a Grant-in-Aid for Scientific Research in Innovative Areas (23110003) from MEXT, Japan. The authors thank Mrs. Mayumi Ikenari and Mrs. Mizuki Yokono (Research Center for Bioscience and Technology, Division of Instrumental Analysis, Tottori University) for performing ESI-HRMS.

References (15)

  • A.M. Clement et al.

    J Biol Chem

    (1998)
  • M. Hikino et al.

    J Biol Chem

    (2003)
  • F. Bélot et al.

    Carbohydr Res

    (2000)
  • J. Tamura et al.

    Bioorg Med Chem Lett

    (2012)
  • K. Matsushita et al.

    Carbohydr Res

    (2015)
  • N. Sugiura et al.

    J Biol Chem

    (2012)
  • H. Wang et al.

    J Cell Sci

    (2008)
There are more references available in the full text version of this article.

Cited by (10)

  • Spatiotemporal distribution of chondroitin sulfate proteoglycans after optic nerve injury in rodents

    2020, Experimental Eye Research
    Citation Excerpt :

    To assess the time course and spatial distribution of CSPG and 4S GAG deposition after injury, optic nerve crush or dorsal column crush surgery was performed on adult mice and rats (Fig. 1), and tissue was collected 1, 3, 7, 14, and 21 dpc. The composition of GAGs in the lesion area was assessed using immunohistochemistry with either CS-56, an antibody that reacts with GAG chains of various sulfation patterns (Sugiura et al., 2012), or 2H6, an antibody that reacts predominantly with 4S (Yamamoto et al., 1995), and to a lesser degree, with 6S (Sugiura et al., 2012) and 2,6S (Matsushita et al., 2018). The intensity of GAG immunoreactivity was increased at the lesion during the scar-forming phase at 7 dpc in all conditions (Fig. 2a–d).

  • Chondroitin sulfate-D promotes neurite outgrowth by acting as an extracellular ligand for neuronal integrin αVβ3

    2019, Biochimica et Biophysica Acta - General Subjects
    Citation Excerpt :

    The sugars CS-C from shark cartilage (average molecular mass 43 kDa), CS-D from shark cartilage (average molecular mass 30 kDa), and CS-E from squid cartilage (average molecular mass 70 kDa), standard unsaturated CS disaccharides, and the enzyme Proteus vulgaris chondroitinase ABC (ChABC, EC 4.2.2.4) were purchased from Seikagaku Corp. (Tokyo, Japan). The CS-C disaccharide GalNAc(6-O-sulfate)-GlcA-O-propyl (CS-C-di) [29], CS-D disaccharide [(GalNAc(6-O-sulfate)-GlcA(2-O-sulfate)-O-propyl) (CS-D-di) [29], and biotinylated CS tetrasaccharides, CC– and DD-type tetrasaccharides linked with a biotin linker [30], denoted as CS-C-tetra and CS-D-tetra, respectively, were previously chemically synthesized. A monoclonal antibody 473HD (rat IgM) was purchased from Merck (Tokyo, Japan).

View all citing articles on Scopus
e

Present address: Fujimoto Pharmaceutical Corporation, Nishi-otsuka 1-3-40, Matsubara, Osaka 580-8503, Japan.

View full text