Leishmania beta-1,2-mannan is assembled on a mannose-cyclic phosphate primer -- Sernee et al. 103 (25): 9458 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Sernee et al. 10.1073/pnas.0603539103.

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Fig. 6. GDP-Man is not the direct precursor for Man-a-1,4-cyclic phosphate. Cell lysates derived from L. mexicana Dgmp promastigotes were incubated with GDP-[³H]Man (1 mM) for 1 h, and the polar products were analyzed by HPAEC. [³H]Man was incorporated into phosphomannose oligomers (peaks Y and Z) but not into Man-cyclic phosphate (peak X). The elution positions of Man-1-P, Man-6-P, and GDP-Man and peaks X, Y, and Z are indicated.

Supporting Text

Synthesis and Analysis of Mannose Phosphates

General Methods. Petroleum spirits refers to a mixed fraction boiling at 40-60°C. TLC was performed with Merck Silica Gel 60 F₂₅₄ by using mixtures of petroleum spirits-ethyl acetate unless otherwise stated. Detection was effected by either charring in a mixture of 5% sulfuric acid-MeOH and/or by visualization in UV light. NMR spectra were obtained on Unity 300- or Inova 400- or 500-MHz instruments (Melbourne, Australia). Flash chromatography was performed according to the method of Still et al. (1) with Merck Silica Gel 60 by using adjusted mixtures of ethyl acetate-petroleum spirits unless otherwise stated. Dichloromethane was dried over CaH₂. Solvents were evaporated under reduced pressure by using a rotary evaporator. Optical rotations were obtained by using a JASCO DIP-1000 polarimeter. [a]_D values are given in 10^-1·cm^2·g^-1. The melting point was obtained using an electrothermal melting point apparatus and is uncorrected. Elemental analyses were performed by CMAS (Belmont, Victoria, Australia). High-resolution mass spectra were performed by Sally Duck (Monash University, Victoria, Australia).

1,2,3,6-Tetra-O-acetyl-a-D-mannopyranose.
A mixture of D-mannose (5.03 g, 22.5 mmol), acetic anhydride (15.0 ml, 159 mmol), and sodium acetate (1.50 g, 18.3 mmol) was stirred at room temperature for 48 h, then filtered and concentrated in vacuo. The residue was dissolved in chloroform (100 ml), washed with water (3 × 100 ml) and saturated NaHCO₃ (2 × 100 ml), dried (MgSO₄), and concentrated in vacuo. Flash chromatography (50–60% ethyl acetate in petroleum spirits) gave an oil, which consisted of a mixture of a- and b-anomers. This oil was further purified by flash column chromatography (15–25% diethyl ether in dichloromethane) to give 1,2,3,6-tetra-O-acetyl-a-D-mannopyranose as a colorless oil (1.36 g, 14%), [a]_D²³ +29.8° (c 3.17, CHCl₃), (ref. 2) [a]_D²⁷ +27.3° (c 0.2, CHCl₃). ¹H NMR (400 MHz, CDCl₃): d 2.01 (s, 3H, CH₃); 2.07 (s, 3H, CH₃); 2.08 (s, 3H, CH₃); 2.10 (s, 3H, CH₃); 3.05 (bs, 1H, OH); 3.88–3.79 (m, 2H, H4,5); 4.23 (dd, J_6,6 12.4, J_5,6 1.4 Hz, 1H, H6); 4.41 (dd, J_6,6 12.4, J_5,6 4.0 Hz, 1H, H6); 5.18–5.12 (m, 2H, H2,3); 5.98 (d, J_1,2 1.6 Hz, 1H, H1). ¹³C NMR (100 MHz, CDCl₃): d 20.52, 20.62 (×2), 20.69 (4C, CH₃CO); 62.87, 64.76, 68.34, 70.81, 72.74 (5C, C2,3,4,5,6); 90.67 (1C, C1); 168.21, 169.56, 170.45, 171.53 (4C, C = O). The ¹H NMR and ¹³C NMR spectra were consistent with that of the a-anomer reported by Crout and colleagues (2). Dibenzyl 1,2,3,6-Tetra-O-acetyl-a-D-mannopyranos-4-yl Phosphate.
Dibenzyl N,N-diisopropylphosphoramidite (620 ml, 1.88 mmol) was added to a suspension of 1H-tetrazole (264 mg, 3.76 mmol) in dichloromethane (5 ml), and the mixture was stirred under N₂ for 15 min. A solution of 1,2,3,6-tetra-O-acetyl-a-D-mannopyranose (262 mg, 752 mmol) in dichloromethane (5 ml) was added, and the resultant mixture was stirred for 1.5 h. The reaction mixture was cooled to 0°C, m-CPBA (556 mg, 2.26 mmol) was added, and the resultant mixture was stirred for 1 h. The reaction mixture was diluted with dichloromethane (20 ml), washed with aqueous 10% Na₂S₂O₃ (2 × 30 ml), 1 M HCl (2 × 30 ml), saturated NaHCO₃ (2 × 30 ml), and brine (3 × 30 ml), dried (MgSO₄) and concentrated in vacuo. Flash chromatography (14–25% diethyl ether in dichloromethane) gave dibenzyl 1,2,3,6-tetra-O-acetyl-a-D-mannopyranos-4-yl phosphate as a pale yellow oil (406 mg, 89%), [a]_D²⁴ +33° (c 0.71, CHCl₃). ¹H NMR (300 MHz, CDCl₃): d 1.85 (s, 3H, CH₃); 2.06 (s, 3H, CH₃); 2.15 (s, 3H, CH₃); 2.17 (s, 3H, CH₃); 4.02 (m, 1H, H5); 4.28 (dd, J_5,6 4.7, J_6,6 12.4 Hz, 1H, H6); 4.36 (dd, J_5,6 2.2, J_6,6 12.4 Hz, 1H, H6); 4.80 (ddd, J_3,4 9.6, J_4,5 9.6, J_4,P 9.6 Hz, 1H, H4); 4.87 (m, 4H, CH₂Ph); 5.23 (dd, J_1,2 1.5, J_2,3 3.8 Hz, 1H, H2); 5.41 (dd, J_2,3 3.8, J_3,4 9.6 Hz, 1H, H3); 6.06 (d, J_1,2 1.5 Hz, 1H, H1); 7.36–7.21 (m, 10, Ph). ¹³C NMR (100 MHz, CDCl₃): d 20.51, 20.67, 20.73, 20.83 (4C, CH₃); 62.07, 68.50, 68.82 (d, J_C,P 3.0 Hz), 69.53 (d, J_C,P 6.0 Hz), 69.68 (d, J_C,P 5.6 Hz), 70.88 (d, J_C,P 6.0 Hz), 70.98 (d, J_C,P 6.0 Hz) (7C, C2,3,4,5,6,CH₂Ph); 90.33 (1C, C1); 127.83–135.33 (4C, Ph); 168.00, 169.62, 170.06, 170.56 (4C, C = O). ³¹P NMR (161.8 MHz, CDCl₃): d –2.36 (P). Anal. calc. for C₂₈H₃₃O₁₃P: C, 55.26; H, 5.47. Found: C, 55.18; H, 5.39. D
-Mannopyranose-4-phosphate. A mixture of palladium hydroxide on carbon (20%, 100 mg) and 1,2,3,6-tetra-O-acetyl-a-D-mannopyranos-4-yl phosphate (381 mg, 625 mmol) in methanol (10 ml) was stirred under H₂ at atmospheric pressure for 2 h. The reaction mixture was then filtered through Celite, and the residue was treated with triethylamine (174 ml, 1.25 mmol) and concentrated in vacuo to yield crude bis(triethylammonium) 1,2,3,6-tetra-O-acetyl-a-D-mannopyranos-4-yl phosphate as a thick, colorless oil. Without purification, the crude product was dissolved in MeOH:H₂O:Et₃N (7:2:1, 20 ml), and the solution was stirred for 18 h and then concentrated in vacuo. The resulting material was purified by flash chromatography (EtOAc:MeOH:H₂O at 3:2:1 then 2:2:1) and then converted to the cyclohexylammonium salt by way of the intermediate pyridinium salt. Thus, the product after flash chromatography was passaged through cation exchange resin (Dowex 50W X8-400, pyridinium form, 2 × 5 cm). Fractions containing the product were pooled and concentrated in vacuo. The residue was treated with cyclohexylamine (300 ml, 2.62 mmol) and coevaporated with toluene (3 × 5 ml). The residue was dissolved in H₂O/MeOH and precipitated with acetone, and the residue was collected by centrifugation to yield cyclohexylammonium D-mannopyranose-4-phosphate as a yellow solid (82 mg, 28%). For characterization purposes, the phosphate was converted to the pyridinium salt. Thus, the cyclohexylammonium salt was passaged through cation exchange resin (Dowex 50W X8-400, H⁺ form, 2 × 5 cm), and fractions containing the product were pooled and concentrated in vacuo. The residue was treated with pyridine (3 × 5 ml) and concentrated in vacuo to yield bis(pyridinium) D-mannopyranose-4-phosphate, [a]_D²⁴ +36° (c 0.58, MeOH). ¹H NMR (400 MHz, D₂O): d 3.59–3.77 (m, 4H, H2,5,6); 3.81 (dd, J_2,3 3.2, J_3,4 9.2 Hz, 1H, H3); 4.11 (ddd, J_3,4 9.2, J_4,5 9.2, J_4,P 9.2 Hz, 1H, H4); 4.72 (s, 1H, H1b); 4.99 (s,1H, H1a); 7.72 (dd, J 7.0, J 7.0 Hz, 4H, PyH3); 8.23 (t, J 8.0 Hz, 2H, PyH4); 8.53 (bs, 4H, PyH2). ¹³C NMR (100 MHz, D₂O): d 61.46 (a), 61.50 (b), 70.77 (d, J_C,P 1.5 Hz, a), 71.17 (a), 71.57 (b), 71.74 (d, J_C,P 6.1 Hz, b), 72.01 (d, J_C,P 5.3 Hz, a), 72.17 (d, J_C,P 6.8 Hz, a), 73.54 (d, J_C,P 1.5 Hz, b), 76.05 (d, J_C,P 6.1 Hz, b) (5C, C2,3,4,5,6); 94.40 (1C, C1, a and b); 127.24, 143.99, 145.05 (3C, Py). ³¹P NMR (161.8 MHz, D₂O): d 3.10 (P). HRMS (ESI^-, m/z) calc. for C₆H₁₃O₉P [M-H]^- 259.0219, found: 259.0216. Diphenyl 2,3,4,6-Tetra-O-acetyl-b-D-mannopyranosyl Phosphate.
A solution of diphenylchlorophosphate (3.09 ml, 14.9 mmol) in dichloromethane (15 ml) was added to a solution of 2,3,4,6-tetra-O-acetyl-D-mannopyranose (2.20 g, 6.32 mmol) in dichloromethane (37 ml) containing 4-dimethylaminopyridine (1.75 g, 14.4 mmol) over a period of 90 min. After stirring for a further 30 min, the reaction mixture was diluted with dichloromethane (50 ml) and washed successively with ice-cold water (2 × 100 ml), ice-cold 0.5 M HCl (2 × 100 ml), and saturated NaHCO₃ (2 × 100 ml). The organic layer was dried (MgSO₄), concentrated in vacuo, and purified by flash chromatography (40–50% ethyl acetate in petroleum spirits) to give diphenyl 2,3,4,6-tetra-O-acetyl-b-D-mannopyranosyl phosphate as a pale yellow oil (3.04 g, 83%), [a]_D²⁴ –38.7° (c 1.40, CHCl₃), (ref. 3) [a]_D²⁵ –6.7° (c 0.84, CHCl₃). ¹H NMR (400 MHz, CDCl₃): d 1.99 (s, 3H, CH₃); 2.04 (s, 3H, CH₃); 2.05 (s, 3H, CH₃); 2.10 (s, 3H, CH₃); 3.78 (m, 1H, H5); 4.10 (dd, J_5,6 1.4, J_6,6 12.4, Hz, 1H, H6); 4.27 (dd, J_5,6 5.4, J_6,6 12.2 Hz, 1H, H6); 5.06 (dd, J_2,3 3.2, J_3,4 10.0 Hz, 1H, H3); 5.25 (dd, J_3,4 9.6, J_4,5 9.6 Hz, 1H, H4); 5.48 (d, J_2,3 3.6 Hz, 1H, H2); 5.60 (dd, J_1,P 7.2, J_1,2 1.2 Hz, 1H, H1); 7.15–7.36 (m, 10H, Ph); ¹³C NMR (100 MHz, CDCl₃): d 20.47, 20.58, 20.66 (×2) (4C, CH₃); 62.03, 65.37, 70.13, 73.14 (4C, C3,4,5,6); 68.04 (d, J_C,P 9.1 Hz, 1C, C2); 94.76 (d, J 4.6 Hz, 1C, C1); 120.21–150.39 (4C, Ph); 169.68, 169.77, 170.50 (4C, C = O). Cyclohexylammonium b-D-mannopyranosyl phosphate.
A solution of diphenyl 2,3,4,6-tetra-O-acetyl-b-D-mannopyranosyl phosphate (306 mg, 527 mmol) in EtOAc:EtOH (1:1, 8 ml) containing PtO₂ (8 mg) was shaken under H₂ in a Parr apparatus at 380 kPa for 18 h. The reaction mixture was filtered through Celite, and the filtrate was treated with triethylamine (148 ml, 1.06 mmol) and then concentrated in vacuo to yield crude bis(triethylammonium) 2,3,4,6-tetra-O-acetyl-b-D-mannopyranosyl phosphate. Without purification, this crude material was dissolved in MeOH:H₂O:Et₃N (7:2:1, 16 ml) and stirred for 20 h. The reaction mixture was concentrated in vacuo, and the resulting product was converted to the pyridinium salt by passage through a cation exchange column (Dowex 50W X8-400, pyridinium form, 2 × 5 cm). Fractions containing the product were pooled and concentrated in vacuo. Half of this material was treated with cyclohexylamine (300 ml) and evaporated to dryness by coevaporation with toluene (3 × 5 ml). The residue was dissolved in H₂O, and acetone was added to afford a precipitate that was collected to yield cyclohexylammonium b-D-mannopyranosyl phosphate (70 mg, 74%) as a white solid, mp 141-142°C, [a]_D²⁴ –11° (c 0.42, methanol). ¹H NMR (400 MHz, D₂O): d 0.91–1.82 (m, 9H, cyclohexyl); 2.95 (m, 1H, CHNH₂); 3.23 (m, 1H, H5); 3.35 (dd, J_3,4 9.6, J_4,5 9.6 Hz, 1H, H4); 3.49–3.54 (m, 2H, H3,5); 3.73 (dd, J_6,6 12.0, J_5,6 2.2 Hz, 1H, H6); 3.82 (d, J_2,3 3.2 Hz, 1H, H2); 4.96 (d, J_1,P 8.8 Hz, 1H, H1). ¹³C NMR (100 MHz, D₂O): d 24.51, 24.99, 31.03, 51.07 (4C, cyclohexyl); 61.82, 67.22, 71.65 (d, J_C,P 6.8 Hz), 73.23, 77.49 (C2,3,4,5,6); 95.89 (d, J_1,P 4.5 Hz, 1C, C1). ³¹P NMR (161.8 MHz, D₂O): d 0.53 (P). Anal. calc. for C₁₂H₃₀NO₁₁P.2H₂O: C, 36.46; H, 7.65; N, 3.54. Found: C, 36.22; H, 7.58; N, 3.20. Mannose-b-1,2-cyclic phosphate.
Dicyclohexylcarbodiimide (DCC) (58 mg, 281 mmol) was added to a solution of b-D-mannopyranosyl phosphate, pyridinium salt (12 mg, 28.7 mmol), and triethylamine (23 ml, 0.16 mmol) in MeOH (4 ml), and the resultant solution was stirred under N₂ for 20 h. Additional DCC (38 mg, 184 mmol) was added, and the mixture was stirred for a further 6 h. The reaction was diluted with water (4 ml) and then washed with ether (3 × 10 ml). The aqueous layer was concentrated in vacuo to yield mannose-b-1,2-cyclic phosphate as a glassy solid (7.0 mg, 87%). ¹H NMR (400 MHz, D₂O): d 3.19–3.23 (m, 1H, H5); 3.53–3.69 (m, 4H, H3,4,6,6); 4.42 (s, 1H, H2); 5.34 (d, J_1,P 25.2 Hz, 1H, H1). ¹³C NMR (100 MHz, D₂O): d 60.99 (1C, C6); 66.36 (1C, C4); 71.70 (d, J_3,P 10.0 Hz, 1C, C3); 75.50 (1C, C5); 78.96 (d, J_2,P 3.0 Hz, 1C, C2); 96.99 (1C, C1). ³¹P NMR (161.8 MHz, CDCl₃): d 19.17 (P). The ¹H and ¹³C NMR spectra were in accordance with that reported in ref. 4. Man-2-P was generated from synthetic mannose-b-1,2-cyclic phosphate by mild acid hydrolysis (5 mM TFA, 100°C, 10 min). Dibenzyl 1,2,4,6-Tetra-O-acetyl-a-D-mannopyranos-3-yl Phosphate.
Dibenzyl N,N-diisopropylphosphoramidite (316 ml, 960 mmol) was added to a suspension of 1H-tetrazole (135 mg, 1.92 mmol) in dichloromethane (3 ml), and the mixture was stirred under N₂ for 15 min. A solution of 1,2,4,6-tetra-O-acetyl-a-D-mannopyranose (5) (134 mg, 385 mmol) in dichloromethane (3 ml) was added, and the resultant mixture was stirred for 1.5 h. The reaction mixture was cooled to 0°C, m-CPBA (285 mg, 1.15 mmol) was added, and the resultant mixture was stirred for 1 h. The reaction mixture was diluted with dichloromethane (10 ml), washed with aqueous 10% Na₂S₂O₃ (2 × 20 ml), 1 M HCl (2 × 20 ml), saturated NaHCO₃ (2 × 20 ml), and brine (3 × 20 ml), dried (MgSO₄), and concentrated in vacuo. Flash chromatography (14–25% diethyl ether in dichloromethane) gave dibenzyl 1,2,4,6-tetra-O-acetyl-a-D-mannopyranos-3-yl phosphate as a pale yellow oil (214 mg, 92%), [a]_D²⁴ +14.5° (c 1.19, CHCl₃). ¹H NMR (400 MHz, CDCl₃): d 1.90 (s, 3H, CH₃); 2.08 (s, 3H, CH₃); 2.10 (s, 3H, CH₃); 2.12 (s, 3H, CH₃); 3.96–4.00 (m, 1H, H5); 4.08 (dd, J_5,6 2.4, J_6,6 12.4 Hz, 1H, H6); 4.26 (dd, J_5,6 4.8, J_6,6 12.4 Hz, 1H, H6); 4.90 (ddd, J_2,3 3.6, J_3,4 9.6, J_3,P 9.6 Hz, 1H, H3); 4.94–5.12 (m, 4H, CH₂Ph); 5.33 (dd, J_1,2 2.0, J_2,3 3.6 Hz, 1H, H2); 5.40 (dd, J_3,4 10.0, J_4,5 10.0 Hz, 1H, H4); 6.09 (d, J_1,2 2.0 Hz, 1H, H1); 7.28–7.37 (m, 10, Ph). ¹³C NMR (100 MHz, CDCl₃): d 20.44, 20.55 (×2), 20.67 (4C, CH₃); 61.87, 66.02 (d, J_C,P 4.5 Hz), 69.03 (d, J_C,P 2.3 Hz), 69.30 (d, J_C,P 6.1 Hz), 69.58 (d, J_C,P 6.0 Hz), 70.30, 73.15 (d, J_C,P 5.3 Hz) (7C, C2,3,4,5,6, CH₂Ph); 90.22 (1C, C1); 127.70–135.37 (4C, Ph); 167.77, 169.42, 169.61, 170.42 (4C, C = O). ³¹P NMR (161.8 MHz, CDCl₃): d –2.17 (P). Anal. calc. for C₂₈H₃₃O₁₃P: C, 55.26; H, 5.47. Found: C, 55.31; H, 5.39. Bis(pyridinium) D-mannopyranose-3-phosphate.
A mixture of 10% palladium on carbon (22.5 mg) and 1,2,4,6-tetra-O-acetyl-a-D-mannopyranos-3-yl phosphate (181 mg, 297 mmol) in EtOAc:MeOH (1:1, 20 ml) was stirred under H₂ at atmospheric pressure for 2 h. The reaction mixture was then filtered through Celite, and the residue was treated with triethylamine (83 ml, 595 mmol) and concentrated in vacuo to yield crude bis(triethylammonium) 1,2,4,6-tetra-O-acetyl-a-D-mannopyranos-3-yl phosphate as a thick colorless oil. Without purification, the crude product was dissolved in MeOH:H₂O:Et₃N (7:2:1, 10 ml), and the mixture was stirred for 48 h, concentrated in vacuo, and then converted to the cyclohexylammonium salt by way of the intermediate pyridinium salt. Thus, the crude product was passaged through cation exchange (Dowex 50W X8-400, H⁺ form, 2 × 5 cm). Fractions containing the product were pooled and concentrated in vacuo. The residue was treated with cyclohexylamine (300 ml, 2.62 mmol) and coevaporated with toluene (3 × 5 ml). The residue was dissolved in H₂O/MeOH and precipitated with acetone, and the residue was collected by centrifugation to yield cyclohexylammonium D-mannopyranose-3-phosphate as a white solid (71.4 mg, 68%). For characterization purposes, the phosphate was converted to the pyridinium salt by passage through cation exchange resin (Dowex 50W X8-400, H⁺ form, 2 × 5 cm). Fractions containing the product were pooled and concentrated in vacuo. The residue was treated with pyridine (3 × 5 ml) and concentrated in vacuo to yield bis(pyridinium) D-mannopyranose-3-phosphate. [a]_D²⁴ +23° (c 0.80, MeOH). ¹H NMR (500 MHz, D₂O) d 3.51–3.94 (m, 5H, H2,4,5,6); 4.13 (ddd, J_2,3 3.5, J_3,P 9.0, J_3,4 9.0 Hz, 1H, H3); 4.74 (s, 1H, H1b); 4.99 (s, 1H, H1a); 7.67 (dd, J 7.5, J 7.5 Hz, 4H, PyH3); 8.17 (t, J 8.0 Hz, 2H, PyH4); 8.50 (bs, 4H, PyH2). ¹³C NMR (125.6 MHz, CDCl₃): d 61.66 (a), 61.70 (b), 66.60 (d, J_C,P 5.5 Hz, b), 66.83 (d, J_C,P 5.5 Hz, a), 70.66 (d, J_C,P 2.3 Hz, a), 71.12 (d, J_C,P 2.3 Hz, b), 73.07 (a), 75.74 (d, J_C,P 5.5 Hz, a), 76.52 (b), 78.02 (d, J_C,P 5.5 Hz, b) (5C, C2,3,4,5,6); 94.15 (1C, C1b); 94.51 (1C, C1a); 127.08, 144.43, 144.47 (3C, Py). ³¹P NMR (161.8 MHz, CDCl₃): d 2.50 (P). HRMS (ESI^-, m/z) calc. for C₆H₁₃O₉P [M-H]^- 259.0219, found: 259.0224.

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