General procedure. NMR spectra were recorded on Bruker DRX-500, AMX-500 or AMX-300 instruments. IR spectra were recorded on a Perkin-Elmer 1600 series FT-IR spectrometer. High-resolution mass spectra (HRMS) were recorded on a VG ZAB-ZSE mass spectrometer using MALDI (matrix-assisted laser-desorption ionization) or ESI (electrospray ionization).3α,7α,12α,24-Tetrahydroxycholane (3): [1] To a suspension of LiAlH 4 (3.85 g, 100 mmol) in dry THF (100 mL) at 0°C was added dropwise a solution of cholic acid (15.03 g, 35 mmol) in dry THF (200 mL) with vigorous stirring under nitrogen atmosphere. The reaction mixture was then heated to reflux with stirring for overnight. Upon completion, the reaction was carefully quenched with saturated aqueous NH 4 Cl solution at RT. Then the mixture was acidified with 1N HCl to pH 1~2. The precipitate was collected via filtration and washed with water and acetone to give the product 2 (12.1 g, 88%) as a white solid.7α,12α-Dihydroxycholane (2): To a solution of tetrahydroxycholane 3 (17.4 g, 44.2 mmol) and triethylamine (11.8 g, 116.6 mmol) in dry THF (300 mL) was added dropwise a solution of methanesulfonyl chloride (11.1 g, 97 .2 mmol) in dry THF (100 mL) at 0°C. Then the reaction mixture was slowly warmed up to RT. After 30 minutes, the reaction was quenched with saturated aqueous NH 4 Cl solution. The organic solvents were removed under vacuum and the left aqueous solution was extracted with ethyl acetate. The combined organic portions were washed with brine and then dried over anhydrous Na 2 SO 4 . The filtered solution was concentrated under vacuum to give the product 7α,12α-Dihydro-3α,24-dimethylsulfonate-cholane (25.6 g, 95%) which was directly dissolved in dry THF for the next step.A solution of LiAlH 4 (6.0 g, 158 mmol) in dry THF (300 mL) was added dropwise to the above obtained THF solution of 7α,12α-Dihydro-3α,24-dimethylsulfonate-cholane at 0°C. The reaction mixture was heated to reflux with stirring for overnight. Then the reaction was quenched with saturated aqueous NH 4 Cl solution at RT. The organic solvents were evaporated under vacuum and the left aqueous solution was acidified with 1N HCl to pH 1. The white precipitate formed was collected via filtration, washed with water and acetone to give the crude product, and then crystallized in dichloromethane and methanol to afford the pure compound 7α,12α-dihydrocholane (12.8 g, 80% over two steps 7α, 12α-Di-(O-β-D-maltosyl)-cholane (1):A mixture of compound 2 (210 mg, 0.58 mmol), 1-thio-ethyl-hepta-o-benzoyl-β-D-maltose (2.1 g, 2.08 mmol) and 4 Ǻ molecule sieves (800 mg) in dry CH 2 Cl 2 (50 mL) was stirred at RT for 30 minutes. The reaction mixture was then cooled to -15 °C, to which was added crystallized N-iodosuccinimide (500 mg, 2.22 mmol) and silver trifluorosulfonate (100 mg, 0.39 mmol). The reaction mixture was slowly warmed up to RT with stirring. The reaction was monitored by TLC. Upon completion, the reaction was quenched with triethylamine. The mixture was filtered and the filtrate was concentrated under vac...

Development of Vibrio cholerae lipopolysaccharide (LPS) as a cholera vaccine immunogen is justified by the correlation of vibriocidal anti-LPS response with immunity. Two V. cholerae O1 LPS serotypes, Inaba and Ogawa, are associated with endemic and pandemic cholera. Both serotypes induce protective antibody following infection or vaccination. Structurally, the LPSs that define the serotypes are identical except for the terminal perosamine moiety, which has a methoxyl group at position 2 in Ogawa but a hydroxyl group in Inaba. The terminal sugar of the Ogawa LPS is a protective B-cell epitope. We chemically synthesized the terminal hexasaccharides of V. cholerae serotype Ogawa, which comprises in part the O-specific polysaccharide component of the native LPS, and coupled the oligosaccharide at different molar ratios to bovine serum albumin (BSA). Our initial studies with Ogawa immunogens showed that the conjugates induced protective antibody. We hypothesized that antibodies specific for the terminal sugar of Inaba LPS would also be protective. Neoglycoconjugates were prepared from synthetic Inaba oligosaccharides (disaccharide, tetrasaccharide, and hexasaccharide) and BSA at different levels of substitution. BALB/c mice responded to the Inaba carbohydrate (CHO)-BSA conjugates with levels of serum antibodies of comparable magnitude to those of mice immunized with Ogawa CHO-BSA conjugates, but the Inaba-specific antibodies (immunoglobulin M [IgM] and IgG1) were neither vibriocidal nor protective in the infant mouse cholera model. We hypothesize that the anti-Inaba antibodies induced by the Inaba CHO-BSA conjugates have enough affinity to be screened via enzyme-linked immunosorbent assay but not enough to be protective in vivo.

The rate limiting step in biophysical characterization of membrane proteins is often the availability of suitable amounts of protein material. It was therefore of interest to demonstrate that microcoil nuclear magnetic resonance (NMR) technology can be used to screen microscale quantities of membrane proteins for proper folding in samples destined for structural studies. Micoscale NMR was then used to screen a series of newly designed zwitterionic phosphocholine detergents for their ability to reconstitute membrane proteins, using the previously well characterized beta-barrel E. coli outer membrane protein OmpX as a test case. Fold screening was thus achieved with microgram amounts of uniformly (2)H, (15)N-labeld OmpX and affordable amounts of the detergents, and prescreening with SDS-gel electrophoresis ensured efficient selection of the targets for NMR studies. A systematic approach to optimize the phosphocholine motif for membrane protein refolding led to the identification of two new detergents, 138-Fos and 179-Fos, that yield 2D [ (15)N, (1)H]-TROSY correlation NMR spectra of natively folded reconstituted OmpX.

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