An Effective Sialylation Method Using N-Troc- and N-Fmoc-protected β-Thiophenyl Sialosides and Application to the One-pot Two-step Synthesis of 2,6-Sialyl-T Antigen

Masaatsu Adachi; Hiroshi Tanaka; Takashi Takahashi

doi:10.1055/s-2004-817759

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Synlett 2004(4): 609-614
DOI: 10.1055/s-2004-817759

LETTER

An Effective Sialylation Method Using N-Troc- and N-Fmoc-protected β-Thiophenyl Sialosides and Application to the One-pot Two-step Synthesis of 2,6-Sialyl-T Antigen

Masaatsu Adachi, Hiroshi Tanaka, Takashi Takahashi*
Department of Applied Chemistry, Tokyo Institute of Technology, Ookayama, Meguro, Tokyo 152-8552, Japan
Fax: +81(3)57342884; e-Mail: thiroshi@apc.titech.ac.jp;

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Publication History

Received 4 December 2003
Publication Date:
10 February 2004 (online)

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Abstract

We describe an efficient sialylation method using β-thiosialosides with various N-protecting groups. Modification of the C-5 amino group of β-thiosialosides into N-Fmoc and N-Troc derivatives enhanced their reactivity in glycosidation. In addition, a minimum amount of MS-3 Å was effective to improve the yield of α-linked sialoside. Branched type one-pot glycosylation initiating glycosidation of the N-Troc-protected β-thiophenyl sialoside at a primary alcohol provided the protected 2,6-sialyl-T antigen in good yield, which was converted to the fully deprotected glycosyl amino acid.

Key words

sialic acids - one-pot glycosylation - glycopeptides - glycosyl amino acids - thioglycosides

References

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10

We have reported a preliminary result of the sialylation in 83rd Spring Meeting (Tokyo) in 2002 of Japan Chemical Society (3D1-12).

19

Selected ¹H NMR assignment of disaccharide 4e-g: For 4e-α-isomer: H-3eq (δ = 2.70 ppm), H-4 (δ = 4.90 ppm), J _7,8 = 9.2 Hz, Δδ{H-9′-H-9} = 0.31 ppm. For 4e-β-isomer: H-3eq (δ = 2.54 ppm), H-4 (δ = 5.21 ppm), J _7,8 = 8.7 Hz, Δδ{H-9′-H-9} = 0.93 ppm. For 4f-α-isomer: H-3eq (δ = 2.72 ppm), H-4 (δ = 4.98 ppm), Δδ{H-9′-H-9} = 0.27 ppm. For 4f-β-isomer: H-3eq (δ = 2.54 ppm), H-4 (δ = 5.25 ppm), J _7,8 = 4.8 Hz, Δδ{H-9′-H-9} = 0.91 ppm. For 4g-α-isomer: H-3eq (δ = 2.70 ppm), H-4 (δ = 4.99 ppm), J _7,8 = 8.8 Hz, Δδ{H-9′-H-9} = 0.30 ppm. For 4g-β-isomer: H-3eq (δ = 2.45 ppm), H-4 (δ = 5.21 ppm), Δδ{H-9′-H-9} = 0.91 ppm.

30

The α/β ratio was determined by HPLC analysis based on refractive index detection (Eluent: hexane/2-propanol = 90:10, 3.0 mL/min; Retention time: α-isomer 10.7 min, β-isomer 11.7 min). The anomeric configuration of disaccharide 8 was determined by ¹H NMR measurement of the isolated isomers according to empirical rule.

31

Analytical data of 8: For 8-α: [α]_D ²¹ +43.4 (c 1.00, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ = 7.30-7.60 (m, 15 H, aromatic), 5.89 (d, 1 H, Ser-NH, J = 8.3 Hz), 5.40 (ddd, 1 H, Neu-H-8), 5.36 (dd, 1 H, Neu-H-7, J _6,7 = 1.4 Hz, J _7,8 = 8.8 Hz), 5.35 (d, 1 H, J _gem = 12.2 Hz), 5.14 (d, 1 H, J _gem = 12.6 Hz), 5.11 (d × 2, 2 H), 5.00 (br dd, 1 H, Neu-H-4, J _3ax,4 = 4.9 Hz, J _3eq,4 = 10.8 Hz), 4.94 (d, 1 H, Neu-Troc-NH, J = 10.3 Hz), 4.90 (d, 1 H, J _gem = 12.2 Hz), 4.79 (d, 1 H, J _gem = 11.7 Hz), 4.78 (d, 1 H, GalNAc-H-1, J _1,2 = 3.4 Hz), 4.65 (d, 1 H, J _gem = 11.2 Hz), 4.60 (m, 1 H, Ser-α), 4.36 (d, 1 H, J _gem = 12.2 Hz), 4.24 (d, 1 H, Neu-H-9′, J _8,9’ = 1.9 Hz, J _gem = 12.7 Hz), 4.16 (br d, 1 H, Neu-H-6, J = 10.8 Hz), 4.06 (dd, 1 H, Neu-H-9, J _8,9 = 4.9 Hz, J _gem = 12.7 Hz), 3.91-4.01 (m, 3 H, GalNAc-H-6′, Ser-β), 3.79-3.85 (m, 3 H, GalNAc-H-3, H-4, H-6), 3.65-3.69 (m, 4 H, Neu-H-5, OMe), 3.52 (br dd, 1 H, GalNAc-H-5), 3.36 (dd, 1 H, GalNAc-H-2, J _1,2 = 3.0 Hz, J _2,3 = 10.3 Hz), 2.66 (dd, 1 H, Neu-H-3eq, J _3eq,4 = 4.9 Hz, J _gem = 13.2 Hz), 2.25 (d, 1 H, GalNAc-H-3, OH, J = 8.8 Hz), 1.97, 2.00, 2.09, 2.11 (4 s, 12 H, Ac), 1.92 (dd, 1 H, Neu-H-3ax, J _3ax,4 = 10.8 Hz). ¹³C NMR (100 MHz, CDCl₃): δ = 172.0, 170.7, 170.5, 169.8, 166.6, 156.0, 154.5, 138.0, 136.0, 134.9, 128.6, 128.6, 128.5, 128.4, 128.1, 127.9, 127.8, 98.7, 98.2, 95.5, 77.9, 77.2, 75.4, 74.4, 72.3, 71.8, 70.7, 69.2, 68.4, 68.2, 68.1, 67.9, 67.2, 63.3, 62.7, 60.4, 53.8, 52.7, 50.9, 37.0, 20.9, 20.8, 20.7, 20.6. IR (KBr): 3347, 2534, 2109, 1745, 1523, 1369, 1218, 1038, 738, 738, 698 cm^-1. Anal. Calcd for C₅₂H₆₀Cl₃N₅O₂₂: C, 41.47; H, 4.98; N, 5.77. Found: C, 51.03; H, 5.02; N, 5.47. For 8-β: [α]_D ²² +37.5 (c 1.00, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ = 7.30-7.39 (m, 15 H, aromatic), 6.36 (d, 1 H, Ser-NH, J = 8.8 Hz), 6.22 (d, 1 H, Neu-Troc-NH, J = 10.3 Hz), 5.54 (ddd, 1 H, Neu-H-4, J _3ax,4 = 12.2 Hz, J _3eq,4 = 4.4 Hz), 5.47 (br s, 1 H, Neu-H-7), 5.37 (d, 1 H, J _gem = 13.9 Hz), 5.34 (m, 1 H, Neu-H-8), 5.12 (d, 1 H, J _gem = 13.9 Hz), 5.11 (d × 2, 2 H), 4.96 (br d, 1 H, Neu-H-9′, J = 11.2 Hz), 4.89 (d, 1 H, GalNAc-H-1, J _1,2 = 2.9 Hz), 4.76 (m, 1 H, Ser-α), 4.75 (d, 1 H, J _gem = 12.2 Hz), 4.69 (d × 2, 2 H), 4.53 (d, 1 H, J _gem = 11.7 Hz), 4.10 (m, 1 H, Ser-β′), 4.03 (dd, 1 H, Neu-H-9, J _8,9 = 8.8 Hz, J _gem = 12.7 Hz), 3.94 (m, 2 H, Neu-H-6, GalNAc-H-5), 3.62 (m, 9 H, Neu-H-5, OMe, GalNAc-H-3, H-4, H-6′, H-6, Ser-β), 3.36 (dd, 1 H, GalNAc-H-2, J _1,2 = 2.9 Hz, J _2,3 = 10.8 Hz), 2.51 (dd, 1 H, Neu-H-3eq, J _3eq,4 = 4.4 Hz, J _gem = 13.9 Hz), 1.96 (d, 1 H, GalNAc-H-3-OH), 1.91, 1.97 × 2, 2.01 (3 s, 12 H, Ac), 1.83 (dd, 1 H, Neu-H-3ax, J _3ax,4 = 12.2 Hz). ¹³C NMR (100 MHz, CDCl₃): δ = 170.7, 170.3, 170.0, 169.8, 169.7, 167.7, 156.0, 154.0, 138.0, 156.0, 138.0, 136.1, 135.1, 128.5, 128.5, 128.1, 128.0, 128.0, 127.8, 99.3, 98.4, 95.3, 76.6, 75.1, 74.4, 71.9, 69.6, 69.1, 68.3, 68.0, 67.9, 67.5, 67.3, 67.0, 63.0, 62.2, 60.8, 54.4, 52.8, 51.4, 37.7, 21.0, 20.8, 20.7, 20.6. IR (KBr): 3368, 2955, 2110, 1744, 1530, 1370, 1223, 1036, 944, 736, 696 cm^-1.

32

The α/β ratio was determined by HPLC analysis based on refractive index detection (Eluent: hexane/2-propanol = 95:5, 3.0 mL/min; Retention time: α-isomer 12.9 min, β-isomer 16.1 min).

33

Selected analytical data of 10: For α-sialoside 10 (α-isomer): ¹H NMR (400 MHz, CDCl₃): δ = 5.61 (dd, 1 H, Gal-H-2, J _1,2 = 3.4 Hz, J _2,3 = 10.3 Hz), 5.55 (d, 1 H, Gal-H-1, J _1,2 = 3.4 Hz). For α-sialoside 10 (β-isomer): [α]_D ²¹ +23.8 (c 1.36, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ = 8.05 (d, 2 H, aromatic), 7.13-7.57 (m, 33 H, aromatic), 5.74 (dd, 1 H, Gal-H-2, J _1,2 = 7.3 Hz, J _2,3 = 8.2 Hz), 5.74 (d, 1 H, Ser-NH, J = 6.8 Hz), 5.39 (m, 1 H, Neu-H-8), 5.35 (dd, 1 H, Neu-H-7, J _6,7 < 1 Hz, J _7,8 = 8.3 Hz), 5.09-5.17 (m, 4 H), 5.05 (d, 1 H, J = 11.2 Hz), 4.97 (ddd, 1 H, Neu-H-4, J _3ax,4 = 12.2 Hz, J _3aq,4 = 4.9 Hz, J _4,5 = 9.6 Hz), 4.94 (d, 1 H, J = 11.7 Hz), 4.90 (d, 1 H, J = 12.7 Hz), 4.87 (d, 1 H, J = 10.8 Hz), 4.77 (d, 1 H, Gal-H-1, J _1,2 = 7.8 Hz), 4.69 (d, 1 H, GalNAc-H-1, J _1,2 = 3.4 Hz), 4.67 (d, 1 H, J = 12.2 Hz), 4.61 (d, 1 H, J = 11.2 Hz), 4.58 (d, 1 H, J = 11.7 Hz), 4.56 (m, 1 H, Ser-α), 4.53 (d, 1 H, J = 12.7 Hz), 4.46 (d, 1 H, J = 12.2 Hz), 4.45 (d, 1 H, J = 12.2 Hz), 4.29 (br dd, 1 H, Neu-H-9′, J _gem = 11.2 Hz), 4.14 (dd, 1 H, Neu-H-9, J _8,9 = 4.9 Hz, J _gem = 11.2 Hz), 4.13 (dd, 1 H, Neu-H-6, J _5,6 = 9.8 Hz, J _6,7 < 1 Hz), 4.05 (br dd, 1 H, Gal-H-4), 3.89-3.99 (m, 4 H, Ser-β, Gal-H-3, H-6′), 3.67-3.69 (m, 6 H, GalNAc-H-4, H-6, Gal-H-3, H-5, H-6, H-6′), 3.59-3.64 (m, 4 H, Neu-H-5, OMe), 3.50 (dd, 1 H, GalNAc-H-2, J _1,2 = 3.0 Hz, J _2,3 = 11.2 Hz), 3.20 (ddd, 1 H, GalNAc-H-5, J = 4.9 Hz, J = 9.3 Hz), 2.56 (dd, 1 H, Neu-H-3eq, J _3eq,4 = 9.3 Hz, J _gem = 12.7 Hz), 1.97, 1.99, 2.07, 2.11 (4 s, 12 H, Ac), 1.84 (dd, 1 H, Neu-H-3ax, J _3ax,4 = 12.2 Hz). ¹³C NMR (100 MHz, CDCl₃): δ = 170.6, 170.3, 170.1, 169.8, 169.6, 169.6, 167.7, 165.3, 156.0, 154.0, 138.6, 138.5, 137.7, 137.6, 136.2, 135.1, 132.8, 132.8, 130.2, 129.8, 128.7, 128.6, 128.5, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6, 127.4, 127.3, 102.6, 99.3, 98.4, 95.4, 79.7, 77.2, 76.8, 75.3, 74.5, 74.4, 74.3, 73.6, 73.4, 72.6, 72.1, 72.0, 71.8, 69.8, 68.7, 68.6, 68.4, 68.3, 67.5, 67.1, 63.8, 62.1, 59.4, 54.5, 52.7, 51.5, 37.7, 21.0, 20.7, 20.7. IR (KBr): 3347, 2952, 2109, 1745, 1520, 1454, 1218, 1040, 737, 698 cm^-1.

36

Analytical data for 1a: [α]_D ²⁶ +67.0 (c 0.16, H₂O). ¹H NMR (400 MHz, D₂O, 303 K): δ = 4.88 (d, 1 H, GalNAc-H-1, J _1,2 = 3.9 Hz), 4.43 (d, 1 H, Gal-H-1, J _1,2 = 7.8 Hz), 4.31 (dd, 1 H, GalNAc-H-2, J _1,2 = 3.4 Hz, J _2,3 = 11.2 Hz), 4.22 (br d, GalNAc-H-4, J = 2.9 Hz), 4.10 (dd, 1 H, Ser-β′, J _α, _β _′ = 2.4 Hz, J _gem = 10.7 Hz), 4.04 (dd, 1 H, GalNAc-H-3, J _2,3 = 11.2 Hz, J _3,4 = 2.9 Hz), 4.01 (m, 1 H, GalNAc-H-5), 3.97 (dd, 1 H, Ser-α, J _α, _β _′ = 2.4 Hz, J _α, _β = 4.9 Hz), 3.90 (dd, 1 H, Ser-β, J _α, _β = 4.9 Hz, J _gem = 10.7 Hz), 3.61-3.88 (12 H, Neu5Ac-H-4,5,6,7,8,9′,9, GalNAc-H-6′,6, Gal-H-5,6′), 3.58 (dd, 1 H, Gal-H-3, J _2,3 = 10.3 Hz, J _3,4 = 3.9 Hz), 3.55 (dd, 1 H, Gal-H-6, J _5,6 = 1.0 Hz, J _gem = 10.3 Hz), 3.48 (dd, 1 H, Gal-H-2, J _1,2 = 7.8 Hz, J _2,3 = 10.3 Hz), 2.70 (dd, 1 H, Neu5Ac-H-3eq, J _3eq,4 = 4.9 Hz, J _gem = 12.7 Hz), 2.00, 2.01 (2 s, 6 H, Ac), 1.65 (dd, 1 H, Neu5Ac-H-3ax, J _3ax,4 = 12.2 Hz). ¹³C NMR (100 MHz, D₂O, acetone-d ₆): δ = 175.8, 175.4, 173.9, 172.0, 105.5, 100.9, 99.1, 77.4, 75.8, 73.5, 73.4, 72.4, 71.4, 70.3, 69.4, 69.3, 69.7, 68.9, 67.3, 64.6, 63.5, 61.8, 54.8, 52.6, 49.2, 40.9, 22.9 × 2. IR (KBr): 3330, 1641, 1572, 1393, 1121, 1055, 930, 669 cm^-1. MS (ESI-TOF): Calcd for C₂₈H₄₇N₃O₂₁Na [M + Na]⁺ 784.3; found: 784.3.