Metabolic incorporation of unnatural sialic acids into Haemophilus ducreyi lipooligosaccharides -- Data Supplement - Supporting Information -- Proceedings of the National Academy of Sciences

Supporting information for Goon et al. (2003) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0437851100

Spectroscopic Data for Synthetic Mannosamine and Sialic Acid Compounds.
¹H and ¹³C NMR spectra were obtained on a Bruker DRX-500 spectrometer. High resolution fast atom bombardment (FAB⁺) analysis of synthetic compounds were obtained by the Mass Spectrometry Facility at the University of California, Berkeley. N-Propanoylmannosamine.
¹H NMR (500 MHz, D₂O): d 1.04-1.14 (m, 6H), 2.25 (app q, 2H, J = 7.69), 2.30 (app q, 2H, J = 7.69), 3.34 (1H, ddd, J = 2.26, 5.00, 9.89), 3.45 (app t, 1H, J = 9.85), 3.56 (app t, 1H, J = 9.55), 3.72-3.83 (m, 6H), 3.99 (dd, 1H, J = 4.71, 9.83), 4.26 (dd, 1H, J = 1.49, 4.69), 4.39 (dd, 1H, J = 1.52, 4.43), 4.96 (d, 1H, J = 1.63), 5.04 (d, 1H, J = 1.51); ¹³C NMR (125 MHz): d 9.4, 16.7, 28.9, 53.0, 53.8, 57.3, 60.3, 66.4, 66.6, 68.7, 71.9, 76.2, 92.9, 93.1, 178.6, 179.5; HRMS (FAB⁺) calculated for C₉H₁₉NO₆ (M+H)⁺ 236.1134, found 236.1134. N-Butanoylmannosamine.
¹H NMR (500 MHz, D₂O): d 0.89 (t, 3H, J = 7.44), 0.92 (t, 3H, J = 7.42), 1.60 (sxt, 4H, J = 7.46), 2.27 (t, 2H, J = 7.38), 2.31 (t, 2H, J = 7.43), 3.39 (ddd, 1H, J = 2.13, 4.91, 9.81), 3.50 (t, 1H, J = 9.84), 3.60-3.68 (m, 2H), 3.77-3.89 (m, 6H), 4.04 (dd, 1H, J = 4.70, 9.81), 4.32 (app d, 1H, J = 4.64), 4.44 (app d, 1H, J = 4.21), 5.01 (d, 1H, J = 0.98), 5.10 (app s, 1H); ¹³C NMR (125 MHz): d 12.7, 12.9, 16.8, 18.9, 37.4, 37.7, 53.1, 53.9, 57.4, 60.4, 66.5, 66.7, 68.7, 72.0, 76.3, 92.9, 93.2, 177.8, 178.7; HRMS (FAB⁺) calculated for C₁₀H₂₀NO₆ (M+H)⁺ 250.1291, found 250.1291. N-Pentanoylmannosamine.
¹H NMR (500 MHz, D₂O): d 0.84 (t, 3H, J = 7.37), 0.85 (t, 3H, J = 7.36), 1.28 (m, 4H), 1.53 (m, 4H), 2.27 (app t, 2H, J = 7.49), 2.31 (app t, 2H, J = 7.59), 3.36 (ddd, 1H, J = 2.23, 4.86, 9.86), 3.47 (app t, 1H, J = 9.84), 3.58 (app t, 1H, J = 9.38), 3.74-3.86 (m, 6H), 4.01 (dd, 1H, J = 4.71, 9.82), 4.28 (dd, 1H, J = 1.34, 4.63), 4.40 (dd, 1H, J = 1.34, 4.36), 4.98 (d, 1H, J = 1.56), 5.06 (d, 1H, J = 1.33); ¹³C NMR (125 MHz): d 12.9, 13.0, 21.5, 21.6, 27.5, 35.3, 35.5, 53.0, 53.8, 60.3, 66.4, 66.6, 68.7, 71.9, 76.2, 92.9, 93.1, 178.0, 178.8; HRMS (FAB⁺) calculated for C₁₁H₂₂NO₆ (M+H)⁺ 264.1447, found 264.1447. N-Hexanoylmannosamine.
¹H NMR (500 MHz, D₂O): d 0.80-0.83 (m, 6H), 1.21-1.28 (m, 8H), 1.51-1.57 (m, 4H), 2.25 (app t, 2H, J = 7.45), 2.29 (app t, 2H, J = 7.57), 3.35 (ddd, 1H, J = 2.26, 4.90, 9.86), 3.46 (app t, 1H, J = 9.84), 3.55-3.61 (m, 2H), 3.73-3.84 (m, 5H), 3.99 (dd, 1H, J = 4.73, 9.80) 4.26 (dd, 1H, J = 1.49, 4.67), 4.39 (dd, 1H, J = 1.50, 4.41), 4.96 (d, 1H, J = 1.63), 5.04 (d, 1H, 1.48); ¹³C NMR (125 MHz): d 13.1, 13.2, 16.7, 21.6, 25.0, 30.4, 30.5, 35.5, 35.7, 53.0, 53.8, 57.3, 60.3, 66.4, 66.6, 68.7, 71.9, 76.2, 92.9, 93.1, 177.9, 178.8; HRMS (FAB⁺) calculated for C₁₂H₂₄NO₆ (M+H)⁺ 278.1604, found 278.1604. N-Heptanoylmannosamine.
¹H NMR (500 MHz, D₂O): d 0.80-0.83 (m, 6H ), 1.24 (m, 12H), 1.54 (m, 4H), 2.26 (app t, 2H, J = 7.47) 2.30 (app t, 2H, J = 7.57), 3.35 (ddd, 1H, J = 2.06, 4.78, 9.76), 3.47 (app t, 1H, J = 9.81), 3.58 (app t, 1H, J = 9.41), 3.74-3.85 (m, 6H), 4.00 (dd, 1H, J = 4.68), 4.27 (app d, 1H, J = 4.60), 4.39 (app d, 1H, J = 3.41), 4.97 (app s, 1H), 5.05 (app s, 1H); ¹³C NMR (125 MHz): d 13.3, 21.8, 25.3, 27.9, 28.0, 30.7, 35.6, 35.8, 53.0, 53.8, 60.3, 66.4, 66.7, 68.7, 71.9, 72.0, 76.2, 92.9, 93.1, 177.9, 178.7; HRMS (FAB⁺) calculated for C₁₂H₂₆NO₆ (M+H)⁺ 292.1760, found 292.1760. N-Octanoylmannosamine.
¹H NMR (500 MHz, D₂O): d 0.80 (m, 6H), 1.22 (m, 16H), 1.54 (m, 4H), 2.25 (app t, 2H, J = 7.47), 2.29 (app t, 2H, J = 7.59), 3.35 (ddd, 1H, J = 2.24, 4.95, 9.85), 3.46 (app t, 1H, J = 9.83), 3.57 (app t, 1H, J = 9.50), 3.73-3.84 (m, 6H), 4.00 (dd, 1H, J = 4.71, 9.80), 4.27 (dd, 1H, J = 1.48, 4.67), 4.39 (dd, 1H, J = 1.50, 4.41), 4.97 (d, 1H, J = 1.59), 5.04 (d, 1H, J = 1.45); ¹³C NMR (125 MHz): d 8.15, 13.3, 18.1, 21.9, 25.3, 27.4, 28.13, 28.22, 28.3, 30.9, 35.6, 35.8, 46.6, 53.0, 53.8, 60.3, 60.4, 66.4, 66.7, 68.7, 71.9, 72.0, 76.3, 92.9, 93.1, 177.8, 178.8; HRMS (FAB⁺) calculated for C₁₄H₂₈NO₆ (M+H)⁺ 306.1917, found 306.1917. N-Propanoyl sialic acid.
¹H NMR (500 MHz, D₂O): d 1.05 (t, 3H, J = 7.67), 1.81 (dd, 1H, J = 11.7, 12.9), 2.22-2.26 (m, 3H), 3.46 (app d, 1H, J = 9.22), 3.54 (dd, 1H, J = 6.33, 11.9), 3.67 (ddd, 1H, J = 2.68, 6.32, 9.05), 3.76 (dd, 1H, J = 2.63, 11.9), 3.86 (app t, 1H, J = 10.27), 3.98-4.03 (m, 2H); ¹³C NMR (125 MHz): d 9.5, 29.2, 38.8, 51.8, 63.1, 66.5, 68.2, 70.0, 70.4, 95.2, 173.2, 178.7; HRMS (FAB⁺) calculated for C₁₂H₂₂NO₉ (M+H)⁺, found 324.1295. N-Butanoyl sialic acid.
¹H NMR (500 MHz, D₂O): d 0.85 (t, 3H, J = 7.37), 1.55 (sxt, 2H, J = 7.37), 1.81 (app t, 1H, J = 12.31), 2.19-2.26 (m, 3H), 3.47 (app d, 1H, J = 9.21), 3.53 (dd, 1H, J = 6.33, 11.9), 3.68 (ddd, 1H, J = 2.65, 6.33, 9.01), 3.75 (dd, 1H, J = 2.56, 11.9), 3.87 (app t, 1H, J = 10.3), 3.97-4.02 (m, 2H); ¹³C NMR (125 MHz): d 12.8, 18.9, 37.8, 38.9, 51.8, 63.1, 66.5, 68.2, 70.0, 70.4, 95.2, 173.2, 177.8; HRMS (FAB⁺) calculated for C₁₃H₂₄NO₉ (M+H)⁺, found 338.1451. N-Pentanoyl sialic acid.
¹H NMR (500 MHz, D₂O): d 0.84 (t, 3H, J = 7.36), 1.27 (sxt, 2H, J = 7.39), 1.50-1.56 (m, 2H), 1.82 (app t, 1H, J = 12.2), 2.23-2.27 (m, 3H), 3.48 (app d, 1H, J = 9.22), 3.55 (dd, 1H, J = 6.28, 11.9), 3.69 (ddd, 1H, J = 2.41, 6.32, 8.9), 3.77 (dd, 1H, J = 2.46, 11.9), 3.88 (app t, 1H, J = 10.24), 3.98-4.03 (m, 2H); ¹³C NMR (125 MHz): d 12.9, 21.5, 27.5, 35.6, 35.7, 39.0, 51.9, 63.1, 66.6, 68.3, 70.1, 70.4, 95.3, 173.6, 178.1; HRMS (FAB⁺) calculated for C₁₄H₂₆NO₉ (M+H)⁺, found 352.1608. N-Hexanoyl sialic acid.
¹H NMR (500 MHz, D₂O): d 0.80 (t, 3H, J = 6.90), 1.20-1.27 (m, 4H), 1.55 (qnt, 2H, J = 7.06), 1.81 (dd, 1H, J = 11.7, 12.9), 2.22-2.27 (m, 3H), 3.48 (app d, 1H, J = 9.19), 3.54 (dd, 1H, J = 6.36, 11.9), 3.69 (ddd, 1H, J = 2.66, 6.33, 9.05), 3.77 (dd, 1H, J = 2.61, 11.9), 3.86 (app t, 1H, J = 10.3), 3.97-4.03 (m, 2H); ¹³C NMR (125 MHz): d 13.1, 21.6, 25.0, 30.5, 35.9, 38.9, 51.8, 63.1, 66.4, 68.3, 70.1, 70.4, 95.2, 173.2, 178.1; HRMS (FAB⁺) calculated for C₁₅H₂₈NO₉ (M+H)⁺, found 366.1764. N-Heptanoyl sialic acid.
¹H NMR (500 MHz, D₂O): d 0.77 (t, 3H, J = 6.78), 1.20 (m, 6H), 1.50-4.53 (m, 2H), 1.79 (dd, 1H, J = 11.7, 12.8), 2.20-2.24 (m, 3H), 3.45 (app d, 1H, J = 6.44, 11.9), 3.66 (ddd, 1H, J = 2.61, 6.42, 9.06), 3.76 (dd, 1H, J = 2.57, 11.9), 3.84 (app t, 1H, J = 10.3), 3.95-4.00 (m, 2H); ¹³C NMR (125 MHz): d 13.3, 21.8, 25.0, 25.3, 27.9, 30.6, 35.9, 38.9, 51.8, 63.1, 66.4, 68.3, 70.0, 70.4, 95.2, 173.1, 178.1; HRMS (FAB⁺) calculatedd for C₁₆H₃₀NO₉ (M+H)⁺, found 380.1921. N-Octanoyl sialic acid.
¹H NMR (500 MHz, D₂O): d 0.80 (app t, 3H, J = 6.90), 1.22-1.25 (m, 6H), 1.52-1.57 (m, 2H), 1.83 (app t, 1H, J = 12.3), 2.23-2.27 (m, 2H), 3.47 (d, 1H, J = 9.44), 3.60 (dd, 1H, J = 6.54, 11.8), 3.70 (ddd, 1H, J = 2.56, 6.39, 9.11), 3.78 (dd, 1H, J = 2.58, 11.9), 3.88 (app t, 1H, J = 10.3), 3.98-4.03 (m, 2H); ¹³C NMR (125 MHz): d 13.27, 21.86, 25.34, 28.02, 28.14, 30.89, 35.90, 3895, 51.84, 63.19, 66.54, 68.36, 70.09, 70.35, 95.32, 173.53, 178.07; HRMS (FAB⁺) calculated for C₁₇H₃₁NO₉ (M+H)⁺ 394.2078, found 394.2071. Quantification Studies of Neuraminic Acid using Mass Spectrometric Methods and SDS/PAGE Analysis.
Methods.
The following bacterial strains were used in this separate quantification study, H. ducreyi strains 35000HP-RSM202, (a neuA mutant) which lacked the sialic acid-containing glycoform and strain 35000HP-RSM203(pLST), (a complemented lst mutant) which overexpressed the lst gene and therefore yielded a high level of sialic acid-containing glycoforms (1). LOS and O-deacylated LOS were prepared as described in the article. The LOS was analyzed using a 16% acrylamide resolving gel with a 4% stacking gel (14 cm ´ 16 cm ´ 0.75 mm) as described (1). Before loading on the gel the LOS samples (80 ng) were not boiled in case this might release some of the NeuAc. After silver staining, gels were scanned with an Amersham Pharmacia LKB UltroScan XL densitometer and individual components were quantified by peak height. Matrix-assisted laser desorption ionization MS (MALDI) and electrospray ionization (ESI) MS were similar to the ones described in the article. Samples were analyzed on a Voyager-DE time-of-flight (TOF) mass spectrometer (Applied Biosystems) and on a Mariner orthogonal-TOF ESI mass spectrometer (Applied Biosystems). Both instruments were operated in the negative ion mode. Decomposition of LOS glycoforms during mass spectrometric analysis was minimized.

1. Bozue, J. A., Wang, J., Tullius, M. V., Gibson, B. W. & Munson, J. R. S. (1999) J. Biol. Chem. 274, 4106-4114.