A Chemical Synthesis of GlcNAcβ(1→4)GlcUA-UDP to Elucidate the Catalytic Mechanism of Hyaluronic Acid Synthases (HAS)

Hironao Takaku; Hide-ki Ishida; Masaya Fujita; Toshiyuki Inazu; Hideharu Ishida; Makoto Kiso

doi:10.1055/s-2007-970766

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Synlett 2007(5): 0818-0820
DOI: 10.1055/s-2007-970766

LETTER

A Chemical Synthesis of GlcNAcβ(1→4)GlcUA-UDP to Elucidate the Catalytic Mechanism of Hyaluronic Acid Synthases (HAS)

Hironao Takaku*^a,b, Hide-ki Ishida^c,, Masaya Fujita^c, Toshiyuki Inazu^d, Hideharu Ishida^a,b, Makoto Kiso*^a,b
^a Faculty of Applied Biological Sciences, United Graduate School of Agricultural Science, Gifu University, Yanagido, Gifu-shi, Gifu 501-1193, Japan
^b CREST, Japan Science and Technology Corporation (JST), Yanagido, Gifu-shi, Gifu 501-1193, Japan
^c The Noguchi Institute, Kaga, Itabashi-ku, Tokyo 173-0003, Japan
^d Department of Applied Chemistry, School of Engineering, and Institute of Glycotechnology, Tokai University, Kitakaname, Hiratsuka-shi, Kanagawa 2591-292, Japan
Fax: +81(58)2932918; e-Mail: hironao55@yahoo.co.jp; e-Mail: kiso@cc.gifu-u.ac.jp;

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Publication History

Received 20 November 2006
Publication Date:
08 March 2007 (online)

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Abstract

A first chemical synthesis of GlcNAcβ(1→4)GlcUA-UDP is described here. This compound can be an essential tool to elucidate the catalytic mechanism of hyaluronic acid synthases (HAS) and this synthetic strategy contributes to the synthesis of various UDP-sugars which include modified GlcUA moieties.

Key words

carbohydrates - enzymes - nucleotides - oligosaccharides - synthesis

References and Notes

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1

Current address: Tokyo Chemical Industry Co., LTD., Toshima, Kita-ku, Tokyo 1140003, Japan.

15

The selected physical data of key compounds is listed. Compound 10: [α]_D +17.6° (c = 1.7, CHCl₃). ¹H NMR (500 MHz, CDCl₃): δ = 1.91 (s, 3 H, NAc), 1.94, 1.98, 2.01 (3 × s, 9 H, OAc), 3.49 (ddd, J ₅ _′,6 _′a = 2.3 Hz, J ₅ _′,6 _′b = 4.0 Hz, 1 H, H-5′), 3.57 (td, J _1,2 = 3.4 Hz, J _2,3 = 9.2 Hz, ³ J _2,P = 2.8 Hz, 1 H, H-2), 3.75 (s, 3 H, COOMe), 3.85 (t, J _2,3 = 9.2 Hz, J _3,4 = 9.2 Hz, 1 H, H-3), 3.86 (dd, J ₅ _′,6 _′a = 2.3 Hz, J ₆ _′a,6 _′b = 12.6 Hz, 1 H, H-6′a), 3.97 (t, J _3,4 = 9.2 Hz, J _4,5 = 9.7 Hz, 1 H, H-4), 3.99 (q, J ₁ _′,2 _′ = 8.6 Hz, J ₂ _′,NH = 9.2 Hz, 1 H, H-2′), 4.14 (dd, J ₅ _′,6 _′b = 4.0 Hz, J ₆ _′a,6 _′b = 12.6 Hz, 1 H, H-6′b), 4.32 (d, J _4,5 = 9.7 Hz, 1 H, H-5), 4.61 (d, 1 H, CH₂Ph), 4.64 (d, J ₁ _′,2 _′ = 8.6 Hz, 1 H, H-1′), 4.67, 4.75, 4.98 (3 × d, CH₂Ph, 3 H), 5.02-5.11 (m, 6 H, H-3′, H-4′, CH₂Ph), 5.66 (d, J _2,NH = 9.2 Hz, 1 H, NH), 5.88 (dd, J _1,2 = 3.4 Hz, ³ J _1,P = 7.4 Hz, 1 H, H-1), 7.20-7.33 (m, 20 H, CH₂Ph). ¹³C NMR (126 MHz, CDCl₃): δ = 20.5, 20.6, 20.6 (Me of OAc), 23.1 (Me of NAc), 53.0 (COOMe), 54.4 (C-2′), 61.6 (C-6′), 68.0 (C-4′), 69.2 (J _CH2Ph,P = 4.8 Hz, POCH₂Ph), 69.5 (J _CH2Ph,P = 4.8 Hz, POCH₂Ph), 71.4 (C-5), 71.9 (C-5′), 73.1 (C-3′), 73.3 (CH₂Ph), 75.3 (CH₂Ph), 77.7 (J _C-2,P = 7.2 Hz, C-2), 78.4 (C-4), 78.7 (C-3), 94.9 (J _C-1,P = 6.0 Hz, C-1), 100.0 (C-1′), 127.0, 127.0, 127.0, 127.0, 127.0, 128.0, 128.0, 128.0, 128.0, 128.0, 128.0, 135.0, 136.0, 136.0, 137.0, 139.0, 169.0 (C=O of OAc-4′), 170.0 (C=O of COOMe), 170.0 (C=O of NAc), 171.0 (C=O of OAc-6′), 171.0 (C=O of OAc-3′). ³¹P NMR (202 MHz, CDCl₃): δ = -1.83 (s, 1 P). MS (MALDI-TOF, +ve): m/z [M + Na]⁺ calcd for C₄₉H₅₆NNaO₁₈P: 1000.31; found: 1000.21. Compound 12: [α]_D +8.75° (c = 0.4, H₂O). ¹H NMR (500 MHz, D₂O): δ = 1.95 (s, 3 H, NAc), 3.36-3.37 (m, 1 H, H-5′), 3.38-3.43 (m, 2 H, H-3′, H-4′), 3.50 (td, J _1,2 = 4.0 Hz, J _2,3 = 9.2 Hz, ³ J _2,P = 2.9 Hz, 1 H, H-2), 3.59 (t, J ₁ _′,2 _′ = 8.6 Hz, 1 H, H-2′), 3.64 (t, J _2,3 = 9.2 Hz, 1 H, H-3), 3.64-3.68 (m, 2 H, H-4, H-6′a), 3.81 (d, 1 H, H-6′b), 4.01 (d, 1 H, H-5), 4.08 (ddd, J _Rib5a,Rib5b = 12.0 Hz, ³ J _Rib5a,P = 2.3 Hz, 1 H, H-5a of Rib), 4.13 (ddd, J _Rib5a,Rib5b = 12.0 Hz, ³ J _Rib5b,P = 2.3 Hz, 1 H, H-5b of Rib), 4.16-4.18 (m, 1 H, H-4 of Rib), 4.24-4.28 (m, 2 H, H-2 of Rib, H-3 of Rib), 4.42 (d, J ₁ _′,2 _′ = 8.6 Hz, 1 H, H-1′), 5.49 (dd, J _1,2 = 4.0 Hz, ³ J _1,P = 7.4 Hz, 1 H, H-1), 5.86 (d, J _Ura5,Ura6 = 8.6 Hz, 1 H, H-5 of Ura), 5.87 (d, 1 H, H-1 of Rib), 7.86 (d, J _Ura5,Ura6 = 8.6 Hz, 1 H, H-6 of Ura). ¹³C NMR (126 MHz, D₂O): δ = 23.4 (C of Me), 56.4 (C-2′), 61.6 (C-6′), 65.8 (J _C-5,P = 3.9 Hz, C-5 of Rib), 70.4 (C-3 of Rib), 70.7 (C-5′), 72.1 (C-3), 72.3 (J _C-2,P = 9.6 Hz, C-2), 74.1 (C-5), 74.9 (C-4′), 75.2 (C-2 of Rib), 77.0 (C-3′), 81.0 (C-4), 84.3 (J _C-4,P = 9.6 Hz, C-4 of Rib), 89.4 (C-1 of Rib), 96.3 (J _C-1,P = 5.8 Hz, C-1), 101.0 (C-1′), 104.0 (C-5 of Ura), 142.0 (C-6 of Ura), 153.0 (C-4 of Ura), 167.0 (C-2 of Ura), 176.0 (COOH), 176.0 (C=O of NAc). ³¹P NMR (202 MHz, D₂O): δ = -10.3 (d, J = 21.8 Hz, 1 P, P of Rib), -12.1 (d, J = 21.8 Hz, 1 P, P of GlcUA). MS (MALDI-TOF, -ve): m/z [M - H]^- calcd for C₂₃H₃₄N₃O₂₃P₂: 782.10; found: 781.96.