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

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.

carbohydrates - enzymes - nucleotides - oligosaccharides - synthesis

References and Notes

1

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

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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.