Synthesis of the Phosphono Analogue of the Dimeric Subunit of Neisseria ­meningitidis Type A Capsular Polysaccharide

Abstract

The development of a glycoconjugate vaccine against N. meningitidis type A bacterium is greatly hampered by the chemical lability of the phosphodiester bridges joining the N-acetyl ­mannosamine repeating units of its capsular polysaccharide. We ­describe the first synthesis of the phosphonodisaccharide α-d-ManpNAc-[1→CH₂-P(O)(O^-)→6]-β-d-ManpNAc-(1→O) (CH₂)₃NH₂ as a stable analogue of the corresponding phosphate-bridged disaccharide. The key phosphonoester linkage is obtained by condensation of monosaccharide building blocks under Mitsunobu conditions. Moreover, the protected precursor of the target compound is suitably designed to allow further elongation and synthesis of higher oligomers.

carbohydrates - Mitsunobu reaction - phosphonates - protecting groups - glycoconjugate vaccines

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Spectroscopic and analytical data of compound 11: [α]_D ²⁵ +20.7 (c 1.0, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ = 7.37-7.19 (m, 15 H, Ph), 5.87 (d, 1 H, J = 9.3 Hz, NH), 4.65, 4.56 (each d, each 1 H, J _gem = 11.7 Hz, CH ₂ Ph), 4.55, 4.51 (each d, each 1 H, J _gem = 11.9 Hz, CH ₂ Ph), 4.46, 4.41 (each d, each 1 H, J _gem = 11.5 Hz, CH ₂ Ph), 4.36 (dt, 1 H, J _2,1 = 3.5 Hz = J _2,3, H-2), 4.16 (m, 1 H, H-1), 4.00 (dd, 1 H, J _5,6a = _5.1 = J _5,4, J _5,6b = 9.7 Hz, H-5), 3.82 (dd, 1 H, J _6a,6b = 10.0, H-6a), 3.78-3.69 (m, 3 H, H-3, H-4, H-6b), 3.74 (d, 3 H, J _Me,P = 11.3 Hz, OMe), 3.71 (d, 3 H, J _Me,P = 11.3 Hz, OMe), 2.13 (dt, 1 H, J _7a,1 = 9.3 Hz, J _7a,7b = 15.7 Hz = J _7a,P, H-7a), 2.04 (dt, 1 H, J _7b,1 = 4.0 Hz, J _7b,P = 15.7 Hz, H-7b), 1.87 (s, 3 H, COCH ₃). ¹³C NMR (100.6 MHz, CDCl₃): δ = 170.37 (CO), 138.39, 138.27, 137.74 (C ipso), 129.19-128.15 (Ph), 76.49 (C-3), 74.06 (C-5), 73.81, 73.35, 72.31 (3 CH ₂ Ph), 72.31 (C-4), 68.79 (C-1), 68.69 (C-6), 53.26 (d, J _OMe,P = 5.9 Hz, OMe), 52.60 (d, J _OMe,P = 5.9 Hz, OMe), 45.28 (d, J _2,P = 14.8 Hz, C-2), 28.06 (d, J _7,P = 142.0 Hz, C-7), 23.71 (COCH₃). ³¹P NMR (162 MHz, CDCl₃): δ = 32.48. HRMS (MALDI): m/z calcd for C₃₂H₄₀NO₈PNa: 620.2389 [M + Na⁺]; found: 620.2386; m/z calcd for C₃₂H₄₀NO₈PK: 636.2129 [M + K⁺]; found: 636.2132.

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<A NAME="RG07005ST-24">24</A>

A fraction containing a single phosphorous diastereoisomer of 16 was isolated during column chromatography and fully characterized: [α]_D ²⁵ +8.1 (c 0.25, CHCl₃). ¹H NMR (500 MHz, CDCl₃): δ = 8.25 (br s, 1 H, NH), 7.46-7.25 (m, 25 H, Ph), 5.76 (br d, 1 H, NH), 5.28 (br s, 1 H, NH), 5.11 (s, 2 H, CH ₂ Ph Z), 4.95, 4.60 (each d, each 1 H, J _gem = 10.7 Hz, CH ₂ Ph), 4.89 (1 H, overlapping signal, H-2), 4.89, 4.51 (each d, each 1 H, J _gem = 10.9 Hz, CH ₂ Ph), 4.66, 4.59 (each d, each 1 H, J _gem = 11.8 Hz, CH ₂ Ph), 4.49, 4.42 (each d, each 1 H, J _gem = 11.9 Hz, CH ₂ Ph), 4.45 (br s, 1 H, H-1), 4.37-4.29 (m, 2 H, H-2′, H-6a), 4.22 (br dd, 1 H, J _6a,6b = 12.0 Hz, H-6b), 4.14 (m, 1 H, H-1′), 4.04 (dd, 1 H, J _{3 ′ ,4 ′} = 9.7 Hz, J _{3 ′ ,2 ′} = 5.4 Hz, H-3′), 3.92-3.77 (m, 6 H, H-4, H-7a, H-4′, H-5′, H-6′a, H-6′), 3.75 (d, 3 H, J _Me,P = 10.9 Hz, OMe), 3.69 (dd, 1 H, J _3,4 = 8.3 Hz, J _3,2 = 3.7 Hz, H-3), 3.58 (m, 1 H, H-7b), 3.38 (br d, 1 H, J _5,4 = 9.5 Hz, H-5), 3.27 (m, 2 H, H-9a, H-9b), 2.23-2.06 (m, 2 H, H-7′a, H-7′b), 1.84 (br s, 6 H, COCH ₃ ), 1.78 (m, 2 H, H-8a, H-8b). ¹³C NMR (125.7 MHz, CDCl₃): δ = 171.62 (CO), 169.82 (CO), 156.52 (CO Z), 138.21 (2 × C), 137.92, 137.61, 137.11 (C ipso), 128.73-127.80 (Ph), 99.57 (C-1), 79.88 (C-3), 75.92, 73.01, 71.81 (C-4, C-4′, C-5′), 75.24, 73.35, 72.80, 71.06 (4 CH₂Ph), 74.51 (C-5), 73.75 (C-3′), 68.24 (C-1′), 68.21 (C-6′), 67.49 (C-7), 66.79 (d, J _6,P = 38.5 Hz, C-6), 66.54 (CH₂Ph Z), 53.87 (d, J _OMe,P = 6.8 Hz, OMe), 49.94 (C-2), 48.68 (d, J _{2 ′ ,P} = 12.8, C-2′), 38.46 (C-9), 30.81 (C-7′), 29.64 (C-8), 23.98 (COCH₃), 23.31 (COCH₃). ³¹P NMR (202.5 MHz, CDCl₃): δ = 29.71. HRMS (ESI): m/z calcd for C₅₇H₇₀N₃O₁₅PNa: 1090.4436 [M + Na⁺]; found: 1090.4410.

<A NAME="RG07005ST-25">25</A>

Spectroscopic and analytical data of compound 17: [α]_D ²⁵ -40.5 (c 1.0, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ = 7.78 (br s, 1 H, NH), 7.34-7.20 (m, 30 H, Ph), 5.92 (d, 1 H, J = 8.9 Hz, NH), 5.14 (s, 2 H, CH ₂ Ph Z), 4.98, 4.62 (each d, each 1 H, J _gem = 10.8 Hz, CH ₂ Ph), 4.98, 4.52 (each d, each 1 H, J _gem = 10.8 Hz, CH ₂ Ph), 4.80 (br dd, 1 H, H-2), 4.74, 4.50 (each d, each 1 H, J _gem = 11.3 Hz, CH ₂ Ph), 4.63-4.52 (m, 3 H, H-1, H-2′, H-6a), 4.54, 4.47 (each d, each 1 H, J _gem = 11.4 Hz, CH ₂ Ph), 4.42, 4.32 (each d, each 1 H, J _gem = 12.1 Hz, CH ₂ Ph), 4.26 (m, 1 H, H-1′), 4.18 (br dd, 1 H, J _6a,6b = 11.4 Hz, H-6b), 3.98-3.87 (m, 3 H, H-4, H-5′, H-7a), 3.81-3.79 (m, 3 H, H-3′, H-4′, H-6′a), 3.73 (dd, 1 H, J _{6 ′ b,6 ′ a} = 9.7 Hz, J _{6 ′ b,5} = 2.9 Hz, H-6′b), 3.68 (dd, 1 H, J _3,2 = 3.5 Hz, J _3,4 = 9.4 Hz, H-3), 3.53 (br s, 1 H, H-7b), 3.39 (d, 1 H, J _5,4 = 9.5 Hz, H-5), 3,34 (m, 1 H, H-9a), 3.10 (m, 1 H, H-9b), 2.27 (dt, 1 H, J _{7 ′ a,1 ′} = 10.2 Hz, J _{7 ′ a,7 ′ b} = 16.8 Hz = J _{7 ′ a,P}, H-7′a), 2.07 (1 H, overlapping signal, H-7′b), 2.07 (s, 3 H, COCH ₃ ), 1.85 (s, 3 H, COCH ₃ ), 1.73 (m, 2 H, H-8a, H-8b). ¹³C NMR (100.6 MHz, CDCl₃): δ = 171.79 (CO), 169.88 (CO), 159.46 (CO Z), 138.34 (2 × C), 138.11, 138.02, 137.87 (2, C ipso), 128.58-127.66 (Ph), 97.27 (C-1), 80.12 (C-3), 76.82 (C-3′), 75.38, 73.86, 73.30, 71.37, 70.74 (5 CH₂Ph), 73.86 (C-5), 73.10 (C-4′), 72.74 (C-4, C-5′), 71.05 (C-1′), 68.40 (C-6′), 67.62 (CH₂Ph Z), 65.13 (d, J _6,P = 37.8 Hz, C-6), 62.09 (C-7), 49.47 (C-2), 48.90 (d, J _{2 ′ ,P} = 15.3, C-2′), 37.40 (C-9), 28.76 (d, J _{7 ′ ,P} = 147.6 Hz, C-7′), 27.21 (C-8), 23.42 (COCH₃), 22.94 (COCH₃). ³¹P NMR (202.5 MHz, CDCl₃): δ = 24.83. HRMS (MALDI): m/z calcd for C₆₃H₇₃N₃O₁₅PNa: 1166.4677 [M + H⁺]; found: 1167.4669; m/z calcd for C₆₃H₇₃N₃O₁₅PNa₂: 1188.4575 [M + H + Na⁺]; found: 1189.4552.

<A NAME="RG07005ST-26">26</A>

Spectroscopic and analytical data of compound 1: [α]_D ²⁵ -17.8 (c 1.0, H₂O). ¹H NMR (400 MHz, D₂O): δ = 4.94 (d, 1 H, J _1,2 = 1.7 Hz, H-1), 4.69 (1 H, overlapped by HDO signal, H-2), 4.51 (br dd, 1 H, H-2′), 4.38-4.23 (m, 3 H, H-1′, H-6a, H-6b), 4.17-4.08 (m, 2 H, H-3′, H-7a), 4.02-3.93 (m, 4 H, H-3, H-7b, H-6′a, H-6′b), 3.81-3.73 (m, 3 H, H-4, H-4′, H-5′), 3.66 (m, 1 H, H-5), 3.26 (br t, 2 H, J = 7.5 Hz, H-9a, H-9b), 2.40-2.27 (m, 2 H, H-7′a, H-7′), 2.22 (s, 3 H, COCH ₃ ), 2.20 (s, 3 H, COCH ₃ ), 2.11 (m, 2 H, H-8a, H-8b). ¹³C NMR (100.6 MHz, D₂O): δ = 175.73 (CO), 174.60 (CO), 99.73 (C-1), 75.63 (C-5), 74.44, 67.70, 66.90 (C-4, C-4′, C-5′), 72.84 (C-1′), 71.86 (C-3), 69.57 (C-3′), 67.97 (C-7), 63.38 (C-6), 60.77 (C-6′), 53.27 (C-2), 53.05 (d, J _{2 ′ ,P} = 9.7 Hz, C-2′), 38.02 (C-9), 27.80 (d, J _{7 ′ ,P} = 134.6 Hz, C-7′), 26.85 (C-8), 22.31 (2 COCH₃). ³¹P NMR (162 MHz, D₂O): δ = 22.95. HRMS (ESI): m/z calcd for C₂₀H₃₈N₃O₁₃PNa: 582.2034 [M + H⁺]; found: 582.2035; m/z calcd for C₂₀H₃₇N₃O₁₃PNa₂: 604.1853 [M + Na⁺]; found: 604.1857.