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

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.

oligosaccharides - carbohydrates - glycosylation - conjugate vaccine - cholera

References and Notes

1 Manning PA. Stroeher UH. Morona R. In Vibrio cholerae and Cholera: Molecular to Global Perspectives   Wachsmuth IK. Blake PA. Olsvik O. American Society for Microbiology; Washington DC: 1994.  p.77-94  

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3 Szu SC, Kossaczka Z, and Robbins JB. inventors; US 7 527 797  B1. 

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5 Kováč P. In Protein-Carbohydrate Interactions in Infectious Diseases   Bewley C. Royal Society of Chemistry; London: 2006.  p.175-220  

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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.

16 Hou S.-j. Kováč P. Carbohydr. Res.  2011,  346:  1394 

17 Moreau V. Norrild JC. Driguez H. Carbohydr. Res.  1997,  300:  271 

18

86%, [α] _d +11 (c 1, CHCl₃). ^¹H NMR: δ_H-1 ^I = 5.05 ppm,
δ_H-1 ^II = 4.48 ppm, δ_C-1 ^I = 98.41 ppm, δ_C-1 ^II = 103.47 ppm, absence of singlet for the benzylidene proton present in the spectrum of 2 at δ = 5.45; free OH-4^II was evidenced by upfield shift of the signal for C-4^II 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.

19 Sherman AA. Yudina ON. Mironov YV. Sukhova EV. Shashkov AS. Menshov VM. Nifantiev NE. Carbohydr. Res.  2001,  336:  13 

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-glucopyranoside^¹8 (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 NH^I at 7.22 and NH^II 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-4^IV), 5.19 (dd, J _1,2 = 7.9 Hz, J _2,3 = 10.2 Hz, 1 H, H-2^IV), 5.16 (d, J _1,2 = 8.6 Hz, 1 H, H-1^III), 5.00 (d, J _1,2 = 6.6 Hz, 1 H, H-1^I), 4.92 (d, J = 10.6 Hz, 1 H, PhCH), 4.88-4.84 (m, 2 H, H-3^IV 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-1^IV 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-1^II), 4.39-4.35 (m, 4 H, H-3^I, H-3^III, and PhCH₂), 4.23 (dd, J = 4.9 Hz, 10.5 Hz, 1 H, H-6^III _a), 4.11 (dd, J = 8.0 Hz, 11.2 Hz, 1 H, H-6^IV _a), 4.02 (dd, J = 5.9 Hz, 11.2 Hz, 1 H, H-6^IV _b), 3.98 (d, J = 2.9 Hz, 1 H, H-4^II), 3.92 (m, 1 H, H-1′_a), 3.79 (s, 3 H, OCH₃), 3.87-3.71 (m, 4 H, including H-6^I _a at 3.76, H-5^I and H-5^IV at 3.73, H-6^III _b at 3.72), 3.70-3.66 (m, 2 H, including H-4^I at 3.69 and H-4^III at 3.67), 3.65 (t, 2.87, 1 H, H-6^I _b), 3.64-3.61 (m, 2 H, including H-6^II _a,b at 3.63 and H-2^II at 3.62), 3.60 (m, 1 H, H-2^III), 3.59-3.54 (m, 9 H, H-2′, H-3′, H-4′, H-5′, H-2^I at 3.55), 3.46-3.42 (m, 2 H, H-3^II and H-5^II), 3.40 (m, 1 H, H-5^III), 3.35 (m, 1 H, H-6′). ^¹³C NMR (150 MHz, CDCl₃): δ = 103.4 (C-1^II), 101.0 (PhCH), 100.1 (C-1^IV), 99.8 (C-1^III), 98.6 (C-1^I), 92.5 (CCl₃), 92.1 (CCl₃), 80.6 (C-3^II), 80.1 (C-2^II), 79.1 (C-4^III), 77.2 (C-4^I), 77.1 (C-3^I), 76.2 (C-3^III), 75.5 (CH₂), 74.2 (CH₂), 73.9 (C-5^I), 73.4 (CH₂), 73.2 (C-5^II), 73.0 (CH₂), 70.9 (2 CH₂), 70.7 (C-5^IV), 70.5 (CH₂), 70.4 (CH₂), 70.3 (CH₂), 69.9 (C-6^II), 68.9 (C-2^IV), 68.8 (CH₂), 68.5 (C-6^III), 68.4 (CH₂), 66.7 (C-4^IV), 66.2 (C-5^III), 60.8 (C-6^IV), 58.6 (C-2^III), 57.4 (C-2^I), 55.2 (OCH₃), 50.6 (CH₂), 33.1 (C-6^I), 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.

22 David S. Thieffry A. Veyrieres A. J. Chem. Soc., Perkin Trans. 1  1981,  1796 

23 Kováč P. Taylor RB. Glaudemans CPJ. Carbohydr. Res.  1985,  142:  158 

24

72%; [α]_D -5.2 (c 1, CHCl₃). ^¹H NMR: δ_H-1 ^I = 4.98 ppm,
δ_H-1 ^II = 4.48 ppm, δ_H-1 ^III = 5.03 ppm, δ_H-1 ^IV = 4.28 ppm; ^¹³C NMR: δ_C-1 ^I = 98.67 ppm, δ_C-1 ^II = 103.40 ppm, δ_C-1 ^III = 100.72 ppm, δ_C-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: δ_H-1 ^I = 5.02 ppm,
δ_H-1 ^II = 4.43 ppm, δ_H-1 ^III = 5.07 ppm, δ_H-1 ^IV = 4.38 ppm; ^¹³C NMR: δ_C-1 ^I = 98.43 ppm, δ_C-1 ^II = 103.52 ppm, δ_C-1 ^III = 100.58 ppm, δ_C-1 ^IV = 102.26 ppm; ^³¹P NMR: δ_P = -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: δ_P -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.

27 Oikawa Y. Yoshioka T. Yonemitsu O. Tetrahedron Lett.  1982,  23:  885 

28

79%; [α]_D -11.2 (c 0.3, CHCl₃). ^¹H NMR: δ_H-1 ^I = 5.06 ppm, δ_H-1 ^II = 4.67 ppm, δ_H-1 ^III = 5.40 ppm, δ_H-1 ^IV = 4.41 ppm; ^¹³C NMR: δ_C-1 ^I = 99.68 ppm, δ_C-1 ^II = 104.27 ppm, δ_C-1 ^III = 101.24 ppm, δ_C-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: δ_P = -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.

29 Epp JB. Widlanski TS. J. Org. Chem.  1999,  64:  293 

30 Walvoort MTC. Sail D. van der Marell GA. Codee JD. In Carbohydrate Chemistry: Proven Synthetic Methods   Kováč P. CRC/Taylor and Francis; Boca Raton: 2011. 

31

72%; ^¹H NMR: δ_H-1 ^I = 5.03 ppm, δ_H-1 ^II = 4.49 ppm, δ_H-1 ^III = 5.12 ppm, δ_H-1 ^IV = 4.39 ppm; ^¹³C NMR: δ_C-1 ^I = 98.51 ppm, δ_C-1 ^II = 103.08 ppm, δ_C-1 ^III = 100.08 ppm, δ_C-1 ^IV = 102.33; absence of signal for C-6^II at δ = 61.33 ppm present in the spectrum of 8; ^³¹P NMR: δ_P = -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.

32 Sakagami M. Hamana H. Tetrahedron Lett.  2000,  41:  5547 

33

90%; ^¹H NMR: δ_H-1 ^I = 5.78 ppm, δ_H-1 ^II = 4.33 ppm, δ_H-1 ^III = 5.19 ppm, δ_H-1 ^IV = 4.51 ppm; ^¹³C NMR: δ_C-1 ^I = 98.70 ppm, δ_C-1 ^II = 104.39 ppm, δ_C-1 ^III = 99.46 ppm, δ_C-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 δ_P = -10.02 ppm. TOF-HRMS: m/z calcd for C₇₈H₉₀BrCl₉N₆O₂₅P [M + NH₄]⁺: 1935.2037; found: 1935.1966.

34 Ruttens B. Kováč P. Synthesis  2004,  2505 

35 Lemieux RU. Hendriks KB. Stick RV. James K. J. Am. Chem. Soc.  1975,  97:  4056 

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 presumably^¹4 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-1^I-IV), 5.68, 5.35 (H-1^V,VI, J _1,2 = 3.0 and 3.1 Hz, respectively]; ^¹³C NMR: 103.60, 102.82, 99.62, 98.72 (C-1^I-IV), 98.39, 98.25 (C-1^V,VI). ^³¹P NMR: δ_P = -10.31 ppm; 2 doublets at δ = 1.82 and 1.60 (J _5,6 = ca. 6.4 Hz, H-6^V,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-1^V,VI), 4.82, 4.67, 4.65, 4.47 (H-1^I-IV); ^¹³C NMR: δ = 103.05, 102.72, 101.24, 101.09 (C-1^I-IV), 99.57, 98.04 (C-1^V,VI) ppm. ^³¹P NMR: δ_P = -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).