Synlett 2011(20): 3013-3017  
DOI: 10.1055/s-0031-1289876
LETTER
© Georg Thieme Verlag Stuttgart ˙ New York

Towards the First Chemical Synthesis of the Hexasaccharide O-Antigen of Vibrio cholerae O139

Shu-jie Hou, Deepak Sail, Pavol Kováč*
NIDDK, LBC, National Institutes of Health, Bethesda, MD 20892-0815, USA
Fax: +1(301)4963569; e-Mail: kpn@helix.nih.gov;
Further Information

Publication History

Received 22 July 2011
Publication Date:
11 November 2011 (online)

Abstract

The hexasaccharide O-antigen of Vibrio cholerae O139 was synthesized in its protected form from thioglycosides and glycosyl bromides as glycosyl donors by a stepwise and blockwise approach. The synthesis was designed to permit a global, one-step deprotection (H2, Pd/C). It allows the transformation of 15 functionalities in one operation, namely the removal of ten benzyl protecting groups, the removal of a trichloroethyl phosphate protecting group, the conversion of two N-trichloroacetyl into N-acetyl groups, a bromomethyl into a methyl group, and the conversion of an azido into an amino group.

    References and Notes

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15

¹H NMR, ¹³C NMR, and ³¹P NMR spectra we taken at 22 ˚C for solutions in CDCl3 (2-9), C6D6 (10 and 11), and D2O (1) at 600 MHz, 150 MHz, and 162 MHz, respectively. The chemical shifts are reported relative to the characteristic solvent peaks. When reporting chemical shifts, sugar residues are serially numbered, beginning with the one bearing the aglycon, and are identified by a Roman numeral superscript. Signals associated with colitose linked to glucosamine and galactose are identified by superscripts V and VI, respectively. The molecular formulas for all new compounds were confirmed by HRMS. Expected conversions during chemical transformations were confidently confirmed in this way. Purity of products were verified by TLC, NMR, and, in most cases, by combustion analysis. However, some compounds tenaciously retained traces of solvents, despite exhaustive drying, and analytical figures for carbon could not be obtained within 0.4%. Structures of these compounds follow unequivocally from the mode of synthesis, NMR data and m/z values.

18

86%, [α] d +11 (c 1, CHCl3). ¹H NMR: δH-1 I = 5.05 ppm,
δH-1 II = 4.48 ppm, δC-1 I = 98.41 ppm, δC-1 II = 103.47 ppm, absence of singlet for the benzylidene proton present in the spectrum of 2 at δ = 5.45; free OH-4II was evidenced by upfield shift of the signal for C-4II from δ = 77.4 ppm in 2 ¹6 to δ = 66.46 ppm. TOF-HRMS: m/z calcd for C49H62BrCl3N5O13 [M + NH4]+: 1112.2593; found: 1112.2622. Anal. Calcd for C49H58BrCl3N4O13: C, 53.64; H, 5.33; N, 3.11. Found: C, 53.66; H, 5.44; N, 5.08.

20

NIS (1.0 g, 4.5mmol), followed by AgOTf (386 mg, 1.5mmol) was added to a stirred mixture of 3 (3.3 g, 3.0 mmol), ethyl 4,6-O-benzylidene-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)-1-thio-2-trichloroacetamido-β-d-glucopyranoside¹8 (4.7 g, 6.0 mmol), 4 Å MS (6.0 g), and CH2Cl2 (50 mL), which had been stirred under N2 for 30 min at r.t. After 1.5 h, the mixture was neutralized with Et3N (0.5 mL), filtered, the filtrate was concentrated, and chromatography (2:1 → 1:1, hexane-EtOAc) afforded 4 (4.3 g, 79%); mp 75-77 ˚C (acetone-hexane); [α]D -16.2 (c 1, CHCl3). ¹H NMR (600 MHz, CDCl3): δ = 7.57-6.86 (m, 26 H, arom. protons, including NHI at 7.22 and NHII at 6.96), 5.56 (s, 1 H, PhCH), 5.31 (dd, J 3,4 = 3.4 Hz, J 4,5 = 1.0 Hz, 1 H, H-4IV), 5.19 (dd, J 1,2 = 7.9 Hz, J 2,3 = 10.2 Hz, 1 H, H-2IV), 5.16 (d, J 1,2 = 8.6 Hz, 1 H, H-1III), 5.00 (d, J 1,2 = 6.6 Hz, 1 H, H-1I), 4.92 (d, J = 10.6 Hz, 1 H, PhCH), 4.88-4.84 (m, 2 H, H-3IV and PhCH), 4.76-4.74 (2 d, J = 11.9, 10.5 Hz, 2 PhCH), 4.64-4.62 (m, 2 H, including H-1IV at 4.62, J 1,2 = 8.0 Hz and PhCH at 4.64, J = 11.9 Hz), 4.57 (d, J = 10.5 Hz, 1 H, PhCH), 4.47 (d, J 1,2 = 7.9 Hz, 1 H, H-1II), 4.39-4.35 (m, 4 H, H-3I, H-3III, and PhCH2), 4.23 (dd, J = 4.9 Hz, 10.5 Hz, 1 H, H-6III a), 4.11 (dd, J = 8.0 Hz, 11.2 Hz, 1 H, H-6IV a), 4.02 (dd, J = 5.9 Hz, 11.2 Hz, 1 H, H-6IV b), 3.98 (d, J = 2.9 Hz, 1 H, H-4II), 3.92 (m, 1 H, H-1′a), 3.79 (s, 3 H, OCH3), 3.87-3.71 (m, 4 H, including H-6I a at 3.76, H-5I and H-5IV at 3.73, H-6III b at 3.72), 3.70-3.66 (m, 2 H, including H-4I at 3.69 and H-4III at 3.67), 3.65 (t, 2.87, 1 H, H-6I b), 3.64-3.61 (m, 2 H, including H-6II a,b at 3.63 and H-2II at 3.62), 3.60 (m, 1 H, H-2III), 3.59-3.54 (m, 9 H, H-2′, H-3′, H-4′, H-5′, H-2I at 3.55), 3.46-3.42 (m, 2 H, H-3II and H-5II), 3.40 (m, 1 H, H-5III), 3.35 (m, 1 H, H-6′). ¹³C NMR (150 MHz, CDCl3): δ = 103.4 (C-1II), 101.0 (PhCH), 100.1 (C-1IV), 99.8 (C-1III), 98.6 (C-1I), 92.5 (CCl3), 92.1 (CCl3), 80.6 (C-3II), 80.1 (C-2II), 79.1 (C-4III), 77.2 (C-4I), 77.1 (C-3I), 76.2 (C-3III), 75.5 (CH2), 74.2 (CH2), 73.9 (C-5I), 73.4 (CH2), 73.2 (C-5II), 73.0 (CH2), 70.9 (2 CH2), 70.7 (C-5IV), 70.5 (CH2), 70.4 (CH2), 70.3 (CH2), 69.9 (C-6II), 68.9 (C-2IV), 68.8 (CH2), 68.5 (C-6III), 68.4 (CH2), 66.7 (C-4IV), 66.2 (C-5III), 60.8 (C-6IV), 58.6 (C-2III), 57.4 (C-2I), 55.2 (OCH3), 50.6 (CH2), 33.1 (C-6I), 20.8 (COCH3), 20.7 (COCH3), 20.6 (COCH3), 20.5 (COCH3). TOF-HRMS: m/z calcd for C78H94BrCl6N6O27 [M + NH4]+: 1835.3481; found: 1835.3502. Anal. Calcd for C49H58BrCl3N4O13: C, 51.41; H, 4.98; N, 3.84. Found: C, 51.30; H, 5.02; N, 3.99.

21

TOF-HRMS: m/z calcd for C70H82BrCl6N5O23K [M + K]+: 1688.2352; found: 1688.2347.

24

72%; [α]D -5.2 (c 1, CHCl3). ¹H NMR: δH-1 I = 4.98 ppm,
δH-1 II = 4.48 ppm, δH-1 III = 5.03 ppm, δH-1 IV = 4.28 ppm; ¹³C NMR: δC-1 I = 98.67 ppm, δC-1 II = 103.40 ppm, δC-1 III = 100.72 ppm, δC-1 IV = 102.25 ppm. TOF-HRMS: m/z calcd for C77H92BrCl6N6O23 [M + NH4]+: 1757.3528; found: 1757.3555. Anal. Calcd for C77H88BrCl6N5O23: C, 53.02; H, 5.09; N, 4.02. Found: C, 53.24; H, 5.02; N, 3.90.

25

49%; [α]D -12.6 (c 1, CHCl3). ¹H NMR: δH-1 I = 5.02 ppm,
δH-1 II = 4.43 ppm, δH-1 III = 5.07 ppm, δH-1 IV = 4.38 ppm; ¹³C NMR: δC-1 I = 98.43 ppm, δC-1 II = 103.52 ppm, δC-1 III = 100.58 ppm, δC-1 IV = 102.26 ppm; ³¹P NMR: δP = -10.65 ppm. TOF-HRMS: m/z calcd for C79H92BrCl9N6O25P [M + NH4]+: 1949.2230; found: 1949.2305. Anal. Calcd for C79H88BrCl9N5O25P: C, 48.97; H, 4.58; N, 3.61. Found: C, 48.75; H, 4.57; N, 3.67.

26

40%; [α]D -0.7 (c 1, CHCl3). ³¹P NMR: δP -8.88 ppm. Anal. Calcd for C79H88BrCl9N5O25P: C, 48.97; H, 4.58; N, 3.61. Found: C, 48.85; H, 4.58; N, 3.61.

28

79%; [α]D -11.2 (c 0.3, CHCl3). ¹H NMR: δH-1 I = 5.06 ppm, δH-1 II = 4.67 ppm, δH-1 III = 5.40 ppm, δH-1 IV = 4.41 ppm; ¹³C NMR: δC-1 I = 99.68 ppm, δC-1 II = 104.27 ppm, δC-1 III = 101.24 ppm, δC-1 IV = 103.86 ppm; absence of OMe signal in the ¹H spectrum, which was present in the ¹H spectrum of 7A at δ = 3.78 ppm; ³¹P NMR: δP = -10.22. TOF-MS: m/z = 1835.2
[M + NH4]+, 1856.1 [M + K]+. Anal. Calcd for C71H80BrCl9N5O24P: C, 46.92; H, 4.44; N, 3.85. Found: C, 46.71; H, 4.48; N, 3.87.

31

72%; ¹H NMR: δH-1 I = 5.03 ppm, δH-1 II = 4.49 ppm, δH-1 III = 5.12 ppm, δH-1 IV = 4.39 ppm; ¹³C NMR: δC-1 I = 98.51 ppm, δC-1 II = 103.08 ppm, δC-1 III = 100.08 ppm, δC-1 IV = 102.33; absence of signal for C-6II at δ = 61.33 ppm present in the spectrum of 8; ³¹P NMR: δP = -10.46 ppm. TOF-HRMS:
m/z calcd for C78H88BrCl9N6O25P [M + NH4]+: 1933.1917; found: 1933.1948. Anal. Calcd for C78H84BrCl9N5O25P: C, 48.76; H, 4.41; N, 3.64. Found: C, 48.60; H, 4.40; N 3.66.

33

90%; ¹H NMR: δH-1 I = 5.78 ppm, δH-1 II = 4.33 ppm, δH-1 III = 5.19 ppm, δH-1 IV = 4.51 ppm; ¹³C NMR: δC-1 I = 98.70 ppm, δC-1 II = 104.39 ppm, δC-1 III = 99.46 ppm, δC-1 IV = 103.69 ppm; absence of the singlet for the PhCH proton present in the spectrum of 9 at δ = 5.47 ppm; ³¹P NMR δP = -10.02 ppm. TOF-HRMS: m/z calcd for C78H90BrCl9N6O25P [M + NH4]+: 1935.2037; found: 1935.1966.

36

The reaction was performed as described,¹4 except 10 equiv of the bromide was used. After 2 d, when TLC (hexane-acetone = 3:2) showed that all glycosyl acceptor was consumed, the mixture was worked up and chromatographed (hexane-acetone = 3:2). The slowest moving zone contained a mixture (MS, NMR) of a pentasaccharide {TOF-MS: m/z 2249.5 [M + Na]+} having presumably¹4 the benzylated colitose linked to the terminal galactosyl residue and 11. The mixture was re-chromatographed (toluene-EtOAc = 5:1) to afford pure 11 {43%; ¹H NMR: δ = 5.60, 5.12, 4.85, 4.46 (Η-1I-IV), 5.68, 5.35 (H-1V,VI, J 1,2 = 3.0 and 3.1 Hz, respectively]; ¹³C NMR: 103.60, 102.82, 99.62, 98.72 (C-1I-IV), 98.39, 98.25 (C-1V,VI). ³¹P NMR: δP = -10.31 ppm; 2 doublets at δ = 1.82 and 1.60 (J 5,6 = ca. 6.4 Hz, H-6V,VI) ppm. TOF-HRMS: m/z calcd for C118H134BrCl9N6O31P [M + NH4]+: 2555.5211; found: 2555.5317. Anal. Calcd for C118H130BrCl9N5O31P: C, 55.70; H, 5.15; N, 2.75. Found: C, 55.42; H, 5.39; N. 2.67}.

37

¹H NMR: δ = 5.13, J 1,2 = 3.5 Hz; δ = 4.92, J 1,2 = 3.3 Hz (H-1V,VI), 4.82, 4.67, 4.65, 4.47 (H-1I-IV); ¹³C NMR: δ = 103.05, 102.72, 101.24, 101.09 (C-1I-IV), 99.57, 98.04 (C-1V,VI) ppm. ³¹P NMR: δP = -3.72 ppm. TOF-HRMS: m/z calcd for C46H79N3O31P [M + H]+: 1200.4435; found: 1200.4447.

38

Normal phase silica gel, Varian, SuperFlash Si35 columns, using Biotage Isolera Flash Chromatograph; slowest moving product, MeOH-25% NH4OH (10:1).