Enhanced Sialylating Activity of O-Chloroacetylated 2-Thioethyl Sialosides

Abstract

It has been shown that O-chloroacetyl protecting groups enhance significantly sialylating activity of 2-thioethyl sialosides in model reactions of α(2-8)-sialylation. Ethyl 4,7,8,9-tetra-O-chloroacetyl-3,5-dideoxy-2-thio-5-trifluoroacetamido-d-glycero-α-d-galacto-non-2-ulopyranoside that combines electron-withdrawing O-chloroacetyl and N-trifluoroacetyl protecting groups displayed the best reactivity and enabled the most efficient synthesis of the Neu5Acα(2-8)Neu5Ac dimer. The GD₃ tetrasaccharide was synthesized in stepwise manner using this sialyl donor in the key (2-8)-coupling, albeit the yield was noticeably lower than in the model sialylation of monosaccharide acceptors.

chloroacetyl protecting group - 2-thioethyl sialoside - glycosylation - reactivity - GD₃ oligosaccharide

References

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Analytical data for 7b: mp 145-146 °C (EtOAc-hexane); [α]_D +24 (c 1, CHCl₃). ¹H NMR (500 MHz, CDCl₃): δ = 1.21 (t, 3 H, J = 7.5 Hz, SCH₂ CH ₃ ), 2.03 (t, 1 H, J _3ax,3eq = 12.9 Hz, J _3ax,4 = 12.3 Hz, H-3ax), 2.55 (dq, 1 H, J _gem = 12.3 Hz, SCH ₂ CH₃), 2.75 (dq, 1 H, SCH ₂ CH₃), 2.85 (dd, 1 H, J _3eq,4 = 4.8 Hz, H-3eq), 3.83 (s, 3 H, CH₃O), 3.99, 4.02 (2 d, 2 H, J _gem = 15.0 Hz, ClCH₂), 4.07 (m, 4 H, H-5, H-6, ClCH₂), 4.17, 4.20 (2 d, 2 H, J _gem = 14.6 Hz, ClCH₂), 4.20, 4.32 (2 d, 2 H, J _gem = 15.2 Hz, ClCH₂), 4.25 (dd, 1 H, J _{9,9 ′} = 12.7 Hz, J _9,8 = 5.3 Hz, H-9), 4.58 (dd, 1 H, J _{9 ′ ,8} = 2.5 Hz, H-9′), 5.08 (m, 1 H, J _4,5 = 10.0 Hz, H-4), 5.38 (d, 1 H, J _7,8 = 8.0 Hz, H-7), 5.50 (m, 1 H, H-8), 6.91 (d, 1 H, J _NH,5 = 9.9 Hz, NH).

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General Procedure for Model Sialylation.
A mixture of sialyl donor (0.33 mmol), sialyl acceptor (0.165 mmol) and powdered molecular sieves 3 Å (1.5 g) in MeCN (4 mL) was stirred at r.t. for 2 h, then cooled to -40 °C. Then, NIS (149 mg, 0.66 mmol) and TfOH (6 µL, 0.07 mmol) were added and the resulting mixture was stirred at -40 °C for 1.5 h. A drop of pyridine was added, the mixture was diluted with CHCl₃ and filtered through Celite. The solids were washed with CHCl₃, combined filtrates were washed with 1 M Na₂S₂O₃ solution, H₂O, and concentrated. The residue was dissolved in a mixture of MeOH (3 mL) and HOAc (1 mL) and thiourea (20 mg) was added. After being stirred for 7-8 min, the mixture was diluted with CHCl₃, thoroughly washed with H₂O, and the solvent was evaporated. The residue was subjected to gel permeation chromatography on a column with Bio Beads S-X3 (Bio-Rad) in toluene. Disaccharide fractions were pooled and concentrated to give a mixture of 18 and 19. Pure anomers were isolated by preparative HPLC (LiChrosorb Si-60 column, K-2401 RI detector, both from Knauer) using toluene-acetone mixtures as eluents.

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<A NAME="RG03405ST-24">24</A>

Selected ¹H NMR assignments for anomeric (2-8)-disialosides 18 and 19.
Compound 18a: δ = 4.86 (H-4′), 3.95 (H-6′), J _{7 ′ ,8 ′} = 8.3 Hz, 4.52 (H-8), Δδ{H-9_a′-H-9_b′} = 0.28.
Compound 19a: δ = 5.31 (H-4′), 4.55 (H-6′), J _{7 ′ ,8 ′} = 4.3 Hz, 4.06 (H-8), Δδ{H-9_a′-H-9_b′} = 0.51.
Compound 18b: δ = 4.88 (H-4′), 3.97 (H-6′), J _{7 ′ ,8 ′} = 8.7 Hz, 4.56 (H-8), Δδ{H-9_a′-H-9_b′} = 0.37.
Compound 19b: δ = 5.37 (H-4′), 4.72 (H-6′), J _{7,8 ′} = 2.8 Hz, 4.07 (H-8), Δδ{H-9_a′-H-9_b′} = 0.60.
Compound 18c: δ = 5.03 (H-4′), 4.11 (H-6′), J _{7 ′ ,8 ′} = 8.0 Hz, 4.53 (H-8), Δδ{H-9_a′-H-9_b′} = 0.25.
Compound 19c: δ = 5.42 (H-4′), 4.76 (H-6′), J _{7 ′ ,8 ′} = 4.6 Hz, 4.03 (H-8), Δδ{H-9_a′-H-9_b′} = 0.45.
Compound 18d: δ = 5.06 (H-4′), 4.15 (H-6′), J _{7 ′ ,8 ′} = 8.6 Hz, 4.58 (H-8), Δδ{H-9_a′-H-9_b′} = 0.36.
Compound 19d: δ = 5.47 (H-4′), 4.89 (H-6′), J _{7 ′ ,8 ′} = 2.8 Hz, 4.02 (H-8), Δδ{H-9_a′-H-9_b′} = 0.63.
Compound 18e: δ = 5.09 (H-4′), 4.13 (H-6′), J _{7 ′ ,8 ′} = 8.3 Hz, 4.40 (H-8), Δδ{H-9_a′-H-9_b′} = 0.32.
Compound 19e: δ = 5.47 (H-4′), 4.82 (H-6′), J _{7 ′ ,8 ′} = 2.8 Hz, 4.03 (H-8), Δδ{H-9_a′-H-9_b′} = 0.60.

<A NAME="RG03405ST-25">25</A> Castro-Palomino JC. Simon B. Speer O. Leist M. Schmidt RR. Chem.-Eur. J.  2001,  7:  2178 

<A NAME="RG03405ST-26">26</A>

Analytical data for 21a: [α]_D +39 (c 1, CHCl₃). ¹H NMR (500 MHz, CDCl₃): δ = 1.22 (t, 3 H, J = 7.5 Hz, SCH₂ CH ₃ ), 1.78 (s 3 H, CH₃CO), 2.11 (t, 1 H, J _3ax,3eq = 12.7 Hz, J _3ax,4 = 12.2 Hz, H-3ax), 2.58 (dq, 1 H, J _gem = 12.3 Hz, SCH ₂ CH₃), 2.80 (dq, 1 H, SCH ₂ CH₃), 2.94 (dd, 1 H, J _3eq,4 = 4.6 Hz, H-3eq), 3.56 (dd, 1 H, J _{9,9 ′} = 11.0 Hz, J _9,8 = 4.6 Hz, H-9), 3.77 (dd, 1 H, J _{9 ′ ,8} = 3.5 Hz, H-9′), 3.86 (s, 3 H, CH₃O), 3.96 (dd, 1 H, J _6,7 = 1.4 Hz, H-6), 4.06, 4.23 (2 d, 2 H, J _gem = 14.7 Hz, ClCH₂), 4.22, 4.38 (2 d, 2 H, J _gem = 15.0 Hz, ClCH₂), 4.29 (q, 1 H, J _5,6 = 10.7 Hz, H-5), 4.49, 4.53 (2 d, 2 H, J _gem = 11.8 Hz, PhCH ₂ ), 5.07 (m, 1 H, J _4,5 = 11.0 Hz, H-4), 5.41 (d, 1 H, J _NH,5 = 9.9 Hz, NH), 5.49 (m, 1 H, H-8), 5.55 (dd, 1 H, J _7,8 = 8.8 Hz, H-7), 7.17-7.98 (m, 10 H, arom.).

<A NAME="RG03405ST-27">27</A> Cheshev PE. Khatuntseva EA. Tsvetkov YE. Shashkov AS. Nifantiev NE. Russ. J. Bioorg. Chem.  2004,  30:  60 

<A NAME="RG03405ST-28">28</A>

Analytical data for 23a: [α]_D +11 (c 1, CHCl₃). ¹H NMR (500 MHz, CDCl₃): δ = 1.78 (s, 3 H, CH₃CO), 2.14 (t, 1 H, J _3ax,3eq = 12.6 Hz, J _3ax,4 = 11.4 Hz, H-3ax NeuAc), 2.73 (dd, 1 H, J _3eq,4 = 4.5 Hz, H-3eq NeuAc), 3.73, 4.02 (2 d, 2 H, J _gem = 14.9 Hz, ClCH₂), 3.81 (s, 3 H, CH₃O), 4.15, 4.29 (2 d, 2 H, J _gem = 15.0 Hz, ClCH₂), 4.40 (d, 1 H, J _1,2 = 8.0 Hz, H-1 Glc), 4.65 (d, 1 H, J _1,2 = 7.8 Hz, H-1 Gal), 5.04 (m, 1 H, J _4,5 = 10.3 Hz, H-4 NeuAc), 5.35 (d, 1 H, J _NH,5 = 10.1 Hz, NH), 5.51 (dd, 1 H, J _7,6 = 1.7 Hz, J _7,8 = 9.0 Hz, H-7 NeuAc), 5.57 (m, 1 H, H-8 NeuAc).

<A NAME="RG03405ST-29">29</A>

Analytical data for 25a: [α]_D -15 (c 1, CHCl₃). ¹H NMR (500 MHz, CDCl₃): δ = 1.92 (s, 3 H, CH₃CO), 2.18 (t, 1 H, J _3ax,3eq = 13.2 Hz, J _3ax,4 = 11.6 Hz, H-3ax NeuTFA), 2.21 (t, 1 H, J _3ax,3eq = 13.1 Hz, J _3ax,4 = 11.2 Hz, H-3ax NeuAc), 2.61 (dd, 1 H, J _3eq,4 = 4.8 Hz, H-3eq NeuAc), 2.77 (br d, 1 H, 4-OH Gal), 2.82 (dd, 1 H, J _3eq,4 = 4.7 Hz, H-3eq NeuTFA), 3.79 (s, 3 H, CH₃O), 3.85 (s, 5 H, CH₃O, ClCH₂), 4.05 (s, 2 H, ClCH₂), 4.12 (dd, 1 H, J _6,5 = 10.5 Hz, J _6,7 = 1.6 Hz, H-6 NeuTFA), 4.13, 4.16 (2 d, 2 H, J _gem = 15.0 Hz, ClCH₂), 4.17, 4.28 (2 d, 2 H, J _gem = 15.1 Hz, ClCH₂), 4.19 (dd, 1 H, J _{9,9 ′} = 12.4 Hz, J _9,8 = 6.0 Hz, H-9 NeuTFA), 4.28 (m, 1 H, H-8 NeuAc), 4.40 (dd, 1 H, J _1,2 = 7.8 Hz, H-1 Glc), 4.45 (dd, 1 H, J _1,2 = 8.2 Hz, H-1 Gal), 4.55 (dd, 1 H, J _{9 ′ ,8} = 2.6 Hz, H-9′ NeuTFA), 5.05 (ddd, 1 H, J _4,5 = 10.4 Hz, H-4 NeuTFA), 5.27 (dd, 1H, J _7,8 = 8.8 Hz, H-7 NeuTFA), 5.32 (ddd, 1 H, J _4,5 = 10.5 Hz, H-4 NeuAc), 5.52 (m, 1 H, H-8 NeuTFA), 6.00 (d, 1 H, J _NH,5 = 8.7 Hz, NH NeuAc), 6.40 (d, 1 H, J _NH,5 = 9.4 Hz, NH NeuTFA).

<A NAME="RG03405ST-30">30</A>

Analytical data for 26: [α]_D +5 (c 0.5, H₂O). ¹H NMR (500 MHz, D₂O, relative to internal acetone, δ_H = 2.225); terminal Neu5Ac: δ = 2.03 (CH₃CO), 2.78 (dd, J _3eq,4 = 4.1 Hz, J _3eq,3ax = 12.2 Hz, H-3eq), 1.74 (t, H-3ax), 3.67 (H-4), 3.83 (H-5), 3.61 (H-6), 3.58 (H-7), 3.88 (H-8), 3.65 (H-9), 3.87 (H-9′); internal Neu5Ac: δ = 2.06 (CH₃CO), 2.68 (dd, J _3eq,4 = 3.9 Hz, J _3eq,3ax = 12.0 Hz, H-3eq), 1.74 (t, H-3ax), 3.61 (H-4), 3.82 (H-5), 3.71 (H-6), 3.86 (H-7), 4.11 (H-8), 3.75 (H-9), 4.17 (dd, J _{9 ′ ,8} = 2.8 Hz, J _{9 ′ ,9} = 12.1 Hz, H-9′); Gal: δ = 4.52 (d, J _1,2 = 7.6 Hz, H-1), 3.57 (dd, J _2,3 = 9.7 Hz, H-2), 4.08 (dd, J _3,4 = 2.3 Hz, H-3), 3.96 (d, H-4), 3.72 (H-5), 3.73 (H-6), 3.75 (H-6′); Glc: δ = 4.54 (d, J _1,2 = 8.2 Hz, H-1), 3.38 (t, J _2,3 = 8.5 Hz, H-2), 3.66 (H-3), 3.68 (H-4), 3.62 (H-5), 3.86 (H-6), 4.00 (dd, J _{6 ′ ,6} = 12.4 Hz, J _{6 ′ ,5} = 1.8 Hz, H-6′), 3.27 (t, J = 4.9 Hz, OCH₂CH ₂ NH₂), 3.88, 4.12 (OCH ₂ CH₂NH₂). ¹³C NMR (125 MHz, D₂O, relative to internal acetone, δ_C = 31.45); terminal Neu5Ac: δ = 176.2 (C-1), 41.7 (C-3), 69.7 (C-4), 53.0 (C-5), 74.0 (C-6), 69.4 (C-7), 72.2 (C-8), 63.9 (C-9), 23.3 (CH₃CO), 174.4 (CH₃ CO); internal Neu5Ac: δ = 176.2 (C-1), 41.0 (C-3), 69.0 (C-4), 53.5 (C-5), 75.1 (C-6), 70.5 (C-7), 79.3 (C-8), 62.7 (C-9), 23.5 (CH₃CO), 174.4 (CH₃ CO); Gal: δ = 104.0 (C-1), 70.5 (C-2), 76.7 (C-3), 68.7 (C-4), 76.5 (C-5), 62.3 (C-6); Glc: δ = 103.2 (C-1), 73.9 (C-2), 75.3 (C-3), 79.2 (C-4), 76.0 (C-5), 61.1 (C-6); 67.0 (OCH₂CH₂NH₂), 40.7 (OCH₂ CH₂NH₂).

<A NAME="RG03405ST-31">31</A> Zou W. Borelli S. Gilbert M. Liu T. Pon RA. Jennings HJ. J. Biol. Chem.  2004,  279:  25390