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Abstract

Selective chemical reactions enacted within a cellular environment can be powerful tools for elucidating biological processes or engineering novel interactions. A chemical transformation that permits the selective formation of covalent adducts among richly functionalized biopolymers within a cellular context is presented. A ligation modeled after the Staudinger reaction forms an amide bond by coupling of an azide and a specifically engineered triarylphosphine. Both reactive partners are abiotic and chemically orthogonal to native cellular components. Azides installed within cell surface glycoconjugates by metabolism of a synthetic azidosugar were reacted with a biotinylated triarylphosphine to produce stable cell-surface adducts. The tremendous selectivity of the transformation should permit its execution within a cell's interior, offering new possibilities for probing intracellular interactions.

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REFERENCES AND NOTES

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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.
10
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}.
11
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).

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Published In

Science
Volume 287 | Issue 5460
17 March 2000

Submission history

Received: 1 December 1999
Accepted: 2 February 2000
Published in print: 17 March 2000

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Authors

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Eliana Saxon
Departments of Chemistry and Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
Carolyn R. Bertozzi*
Departments of Chemistry and Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.

Notes

*
To whom correspondence should be addressed. E-mail: [email protected]

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