Facile Synthesis of β(1→6)-D-Galactosylated Oligosaccharides Employing the β(1→6)-D-GalT-II from the Albumen Gland of the Snails Helix Pomatia

Abstract

Investigations of a β(1→6)-d-galactosyltransferase found in the galactan-synthesising albumen glands of the snail Helix pomatia show formation of a β(1→6)-d-Gal linkage to a terminal d-galactopyranosyl unit β(1→3)-linked to a subterminal d-Gal, which was already the site of β(1→6)-d-Gal branching.

chemoenzymatic oligosaccharide synthesis - β(1→6)-d-galactosyltransferase - Helix pomatia - H antigen

References

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15

The Helix pomatia albumen glands (1-2 g) was homogenised in Tris/HCl buffer (50 mM, pH = 7.6, 5 mL) in a Potter-Elvehjem homogeniser, then centrifuged at 4000 rpm for 45 min at 4 °C. The supernatant was removed from the pellet and discarded. This process was repeated five times with 30 min centrifugation at 4000 rpm, whereupon the final albumen gland sediment (approx 2000 µL) was rehomogenised and used immediately. Albumen gland sediment, which was not used immediately was stored at -70 °C for later use. The amount of 2.81 µmol of acceptor is treated with 300 µL of albumen gland sediment. One snail can provide one albumen gland, which has an average weight of approximately 2.0 g. A solution of the trisaccharide 2 (5 mg, 9.65 µmol), UDP-d-Gal (11.8 mg, 19.3 µmol, as 100 µg/µL aq solution), 30 µL of calf intestine alkaline phosphatase (185 mU/µL), 30 µL MnCl₂ (380 mM), 20 µL NaN₃ (10 mg/mL) and 1000 µL of H. pomatia albumen gland sediment (pH = 7.6) was incubated at 28 °C for 36 h. The reaction was terminated by centrifugation (5 min, 10000 rpm). The supernatant was removed and the pellet washed (3 × 500 µL). The supernatant and washings were combined, lyophilised and treated again with UDP-d-Gal (11.8 mg, 19.3 µmol, as 100 µg/µL aqueous solution), 30 µL of calf intestine alkaline phosphatase (185 mU/µL), 30 µL MnCl₂ (380 mM), 20 µL NaN₃ (10 mg/mL) and 1000 µL of fresh H. pomatia albumen gland sediment (pH = 7.6), with reincubation at 28 °C for 36 h. The reaction was terminated by centrifugation (5 min, 10000 rpm), the supernatant removed, and the pellet washed (3 × 500 µL). The combined washings and supernatant were lyophilised, and peracetylated by adding pyridine (2 mL) and Ac₂O (2 mL) and heating to 80 °C for 2 h. Pyridine and Ac₂O were removed from the reaction mixture by evaporation under N₂ flow. The resulting residue was dissolved in CHCl₃ (3 mL) and washed with bidistilled H₂O (2 × 3 mL). The organic layer was dried and evaporated, yielding a yellow oil which was purified by preparative TLC (CHCl₃:acetone 18:3), giving tetrasaccharide 5 as a clear glass (5.8 mg, 50% yield).

16

Methyl 2,4-di-O-acetyl-3-O-[2,3,4-tri-O-acetyl-6-O-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)-β-d-galactopyranosyl]-6-O-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)-β-d-galactopyranoside (5): ¹H NMR (500 MHz; CDCl₃): δ = 5.33 (m, 1 H, H-4′′), 5.31 (m, 1 H, H-4′′′), 5.27 (m, 1 H, H-4), 5.25 (m, 1 H, H-4′), 5.12 (dd, 1 H, H-2′′′), 5.11 (dd, 1 H, H-2), 5.08 (dd, 1 H, H-2′′), 4.99 (dd, 1 H, H-2′), 4.94 (dd, 1 H, H-3′′), 4.91 (dd, 1 H, H-3′′′), 4.83 (dd, 1 H, H-3′), 4.50 (d, 1 H, H-1′′′), 4.48 (d, 1 H, H-1′), 4.39 (d, 1 H, H-1′′), 4.22 (d, 1 H, H-1), 4.10-4.05 (m, 4 H, H-6a′′, H-6b′′, H-6a′′′, H-6b′′′), 3.88-3.82 (m, 5 H, H-5′′, H-5′′′, H-3, H-5, H-6a), 3.79-3.70 (m, 2 H, H-5′, H-6a′), 3.63 (dd, 1 H, H-6b′), 3.57 (dd, 1 H, H-6b), 3.43 (s, 3 H, OCH₃), 2.10, 2.08, 2.07, 2.06, 2.04, 2.00, 1.98, 1.97, 1.95, 1.94, 1.91, 1.91, 1.89 (13 × s, each 3 H, CH₃COO) ppm. Coupling constants: J _1,2 = 8.2, J _2,3 = 10.4, J _3,4 = 3.2, J _4,5 = 0-1.0, J _5,6b = 7.3, J _6a,6b = 10.1, J _{1 ′ ,2} = 7.9, J _{2 ′ ,3 ′} = 10.4, J _{3 ′ ,4 ′} = 3.5, J _{4 ′ ,5 ′} = 0-1.0, J _{5 ′ ,6b ′} = 7.3, J _{6a ′ ,6b ′} = 9.5, J _{1 ′′ ,2 ′′} = 7.9, J _{2 ′′ ,3 ′′} = 10.7, J _{3 ′′ ,4 ′′} =& nbsp;3.5, J _{4 ′′ ,5 ′′} = 0-1.0, J _{1 ′′′ ,2 ′′′} = 7.9, J _{2 ′′′ ,3 ′′′} = 10.4, J _{3 ′′′ ,4 ′′′} = 3.2, J _{4 ′′′ ,5 ′′′} = 0-1.0 Hz. ¹³C NMR (100.6 MHz, CDCl₃): δ = 102.21 (C-1), 101.54, 101.52 (C-1′, C-1′′′), 101.11 (C-1′′), 75.22 and 74.39 (C-3), 72.72 (C-5′), 71.51 (C-2) ′ 74.39 and 75.22, 71.32, 71.30, 71.26, 71.24, 71.20 (C-5′′, C-5′′′, C-5, C-3′, C-3′′, C-3′′′), 70.47 (C-4), 69.25, 69.23 (C-2′′, C-2′′′), 68.89 (C-2′), 68.87 (C-6), 67.67 (C-4′), 67.42 (C-6′), 67.36, 67.36 (C-4′′, C-4′′′), 61.76, 61.70 (C-6′′, C-6′′′), 57.31 (OCH₃) ppm.