Cell Surface Engineering by a Modified Staudinger Reaction

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Synthesis of N-azidoacetylmannosamine (3) and acetylated 3. A solution of mannosamine hydrochloride (250 mg, 1.16 mmol) and sodium methoxide (1.16 ml of a 1 M methanolic solution) in dry MeOH (10 ml) was stirred for 1 hour at room temperature, after which chloroacetic anhydride (991 mg, 5.80 mmol) was added. The resulting solution was stirred overnight at room temperature under an atmosphere of N₂ and then quenched with H₂O (5 ml) for 1 hour. The solution was neutralized with saturated NaHCO₃ and concentrated, and the residue was filtered through a plug of silica gel eluting with 5:1 CHCl₃/MeOH. The crude product obtained was dissolved in dimethylformamide (10 ml) and NaN₃ (78 mg, 1.39 mmol) was added. After heating at reflux overnight, the solution was cooled and concentrated. Purification by silica gel chromatography eluting with a gradient of 50:1 to 6:1 CHCl₃/MeOH afforded 179 mg of compound 3 (59% over two steps). The compound was peracetylated before incubation with cells as follows. A solution of 3 (25 mg, 0.095 mmol), acetic anhydride (1.0 ml, 11 mmol), and a catalytic amount of 4-dimethylaminopyridine in pyridine (2 ml) was cooled to 0°C. The mixture was stirred overnight, warmed to room temperature, then diluted with CH₂Cl₂ (100 ml) and washed with 1 N HCl (3 × 50 ml), saturated NaHCO₃ (1 × 50 ml), water (1 × 50 ml), and saturated NaCl (1 × 50 ml). The combined organic layers were dried over Na₂SO₄ and concentrated. The crude product was purified by silica gel chromatography eluting with a gradient of 1:10 to 1:2 EtOAc/hexanes to afford 39 mg (95%) of acetylated 3.

Synthesis of intermediate phosphine 4. A solution of NaNO₂ (180 mg, 2.64 mmol) in 1 ml of H₂O was added dropwise to a solution of 1-methyl-2-aminoterephthalate (500 mg, 2.56 mmol) in 5 ml of cold concentrated HCl. The mixture was stirred for 30 min at room temperature and then filtered through glass wool into a solution of KI (4.30 g, 25.0 mmol) in 7 ml of H₂O. The dark red solution was stirred for 1 hour and then diluted with CH₂Cl₂ (100 ml) and washed with saturated Na₂SO₃ (2 × 10 ml). The organic layer was washed with water (2 × 20 ml) and saturated NaCl (1 × 20 ml). The combined aqueous layers were back extracted with CH₂Cl₂ (20 ml). The combined organic layers were dried over Na₂SO₄ and concentrated. The crude product was dissolved in a minimum amount of MeOH and H₂O was added until the solution appeared slightly cloudy. Cooling to 4°C and subsequent filtration afforded 449 mg (57%) of a yellow solid. To a flame-dried flask was added this product (300 mg, 1.00 mmol), dry MeOH (3 ml), triethylamine (0.3 ml, 2 mmol), and palladium acetate (2.2 mg, 0.010 mmol). While stirring under an atmosphere of Ar, diphenylphosphine (0.17 ml, 1.0 mmol) was added to the flask by means of a syringe. The resulting solution was heated at reflux overnight, and then allowed to cool to room temperature and concentrated. The residue was dissolved in 250 ml of a 1:1 mixture of CH₂Cl₂/H₂O and the layers were separated. The organic layer was washed with 1 M HCl (1 × 10 ml) and concentrated. The crude product was dissolved in a minimum amount of methanol and an equal amount of H₂O was added. The solution was cooled to 4°C for 2 hours and the resulting solid was collected by filtration. The pure product, compound 4, was isolated in 69% yield (245 mg). This compound can be coupled with amines by using standard procedures {such as EDC [1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride] or DCC (1,3-dicyclohexylcarbodiimide) coupling reactions}.

Acetylated monosaccharides are metabolized 200-fold more efficiently than the free sugars owing to improved cellular uptake, which is followed by deacetylation by cytosolic esterases [C. L. Jacobs and C. R. Bertozzi, unpublished results;

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We acknowledge generous funding from the W. M. Keck Foundation, Glaxo Wellcome, and the Burroughs Wellcome Fund. E.S. was supported by a Howard Hughes Medical Institute Predoctoral Fellowship. This research was supported by the Office of Naval Research, grant N00014-98-1-0605 and order N00014-98-F-0402 through the U.S. Department of Energy under contract DE-AC03-76SF00098, and by the National Institutes of Health (GM58867-01).