Beilstein J. Org. Chem. 2010, 6, 732–741.
p-Nitrophenyl 2-bromoacetate 1 (R = pNP)
into portions that were frozen in liquid nitrogen. Owing to the
A solution of pyridine (0.35 mL, 4.3 mmol) in dry DCM (5 mL) instability and crude nature of the nitrophenyl 2-S-(5′-thiophos-
was added dropwise to a stirred solution of bromoacetyl bro- phoryluridine)acetate esters in aqueous solution, only 1H NMR
mide (0.38 mL, 4.3 mmol) in dry DCM (5 mL) cooled in an ice and ES− analyses were performed. Data for p-nitrophenyl 2-S-
bath. Following the careful addition of p-nitrophenol (0.6 g, 4.3 (5′-thiophosphoryluridine)acetate 7 (R = pNP) δH (500 MHz;
mmol), the reaction mixture was stirred for 1 h. Saturated D2O) 8.11 (2 H, d, J 9.2, CHCNO2), 7.55 (1 H, d, J 8.2, 5-CH),
sodium bicarbonate solution (5 mL) was added, the layers were 7.24 (2H, d, J 9.2, CHCO), 5.72 (1H, d, J 3.9, 1′-CH), 5.55 (1H,
separated, and the organic layer was washed successively with d, J 8.2, 6-CH), 4.32–3.91 (5 H, m, 2′–5′-CH), 3.7 (2 H, d, J
water (2 × 5 mL), hydrochloric acid (0.1 M, 3 × 5 mL) and satu- 15.4, SCH2); δP (80 MHz; D2O) 19.2; m/z (ES−) 518.0 (M–H
rated sodium chloride solution (5 mL). The organic phase was for UMPS-CH2CO2-p-C6H4NO2), 379.1 (M–H for the cyclic
then dried over anhydrous magnesium sulphate and the solvent hydrolysis product, UMPSCH2CO2−).
removed under reduced pressure to give the p-nitrophenyl ester
(0.752 g, 67%); mp = 72–75 °C (dec); (Found C, 36.94; H, Hydrolysis studies on p- and m-nitrophenyl 2-S-(5′-
2.31; N, 5.17. C7H6BrNO4 requires C, 36.92; H, 2.31; N, thiophosphoryluridine)acetates 7 (R = pNP) and 7
5.38%); νmax (KBr disc)/cm−1 3110–2963 (CH), 2847 (CH2), (R = mNP)
1770 (ester CO); δH (500 MHz; CDCl3) 8.31 (2 H, d, J 9.0, Kinetic measurements were performed by mixing stock solu-
CHCNO2), 7.34 (2 H, d, J 9.3, CHCO), 4.08 (2 H, s, tion of the ester (25 μL) with buffer (1.5 mL) to give ~0.1 mM
COCH2Br); δC (125 MHz; CDCl3) 165.2 (C=O), 155.1 (CO), final concentration of ester in the cuvette. The cuvette
145.9 (CNO2), 125.6 (NO2CCH), 122.4 (CHCO), 25.3 was inserted into a thermostated (25 °C) compartment of
(CH2Br); m/z (EI) 258.9 and 260.9.
the UV–vis spectrophotometer and the increase in absorbance
of p- or m-nitrophenolate monitored at λ ~ 400 nm. The
kinetic data were fitted to the function At = A0+A∞(1–e–kobs t),
m-Nitrophenyl 2-bromoacetate 1 (R = mNP)
The procedure for p-nitrophenyl 2-bromoacetate 1 (R = pNP) and showed clean first order behaviour with observed rate
was followed except p-nitrophenol was replaced by m-nitro- constants k0.
phenol (0.6 g, 4.3 mmol). After work up, the m-nitrophenyl
ester was obtained (0.697 g, 62%); mp = 50–53 °C (dec); Aminolysis studies on p- and m-nitrophenyl 2-S-(5′-
(Found C, 36.96; H, 2.31; N, 5.39. C7H6BrNO4 requires C, thiophosphoryluridine)acetates 7 (R = pNP) and 7
36.92; H, 2.31; N, 5.38%); νmax (KBr disc)/cm−1 3116–3011 (R = mNP)
(CH), 2864 (CH2), 1777 (ester CO); δH (500 MHz; CDCl3) 8.16 D-Glucosamine solution (1.24 M, 60 μL) was mixed with buffer
(1 H, d, J 8.8, 4-CH), 8.05 (1 H, s, 4-CH ), 7.63 (1 H, t, J 8.1, (0.5 M, 1.44 mL) to generate a solution (1.5 mL) with 50 mM
5-CH), 7.53 (1 H, t, J 8.2, 6-CH), 4.10 (2 H, s, CH2Br); δC (125 final concentration of D-glucosamine in the cuvette. Stock solu-
MHz; CDCl3) 165.5 (C=O), 150.7 (CO), 149.0 (CNO2), 130.6 tion of ester was then added, and the kinetics were monitored
(5-CH), 127.8 (6-CH), 121.6 (4-CH), 117.3 (2-CH), 25.2 and analysed as described above.
(CH2Br); m/z (EI) 258.9 and 260.9.
Buffer preparation for kinetic studies
p- and m-Nitrophenyl 2-S-(5′-
thiophosphoryluridine)acetates 7 (R = pNP) and 7
(R = mNP) for kinetic studies
Buffers were prepared using CAPS (pH 10.5 and 10.17), CHES
(pH 9.81, 9.44 and 9.06), EPPS (pH 8.44 and 8.00), HEPES
(pH 7.50 and 7.10), MES (pH 6.60, 6.00 and 5.88) and acetate
The disodium salt of uridine-5′-monophosphorothioate 4 (1 eq, (pH 4.80 and 4.66) systems where pHs were adjusted by the
20 mg, 58.8 μmol) was dissolved in deionised water (0.5 mL) addition of hydrochloric acid or hydroxide solutions. Buffer
and a solution of p- or m-nitrophenyl 2-bromoacetate 1 (R = strengths of 0.05, 0.1, 0.2, 0.3 and 0.5 M were used to check for
pNP) or 1 (R = mNP) (0.8 eq, 12 mg, 46 μmol) in acetonitrile general species-promoted hydrolysis. All aminolysis studies
(0.5 mL) added. The mixture was stirred for one minute then were performed using 0.5 M buffers in the presence of 0.05 M
rapidly frozen in liquid nitrogen followed by lyophilisation to D-glucosamine.
give a light yellow solid of intermediate, p- or m-nitrophenyl
2-S-(5′-thiophosphoryluridine)acetate 7 (R = pNP) or 7 (R = Data analysis and kinetic predictions
mNP) (~80% purity by 1H NMR spectroscopy, contaminated Kinetic data were analysed using Kaleidagraph™. Kinetic
with excess uridine-5′-monophosphorothioate 4). Stock solu- predictions were based on Brønsted relationships between
tions were prepared by dissolving crude p- or m-nitrophenyl observed rate coefficients k0, kOH and kNH2 and the pKaH of the
2-S-(5′-thiophosphoryluridine)acetate 7 (R = pNP) or 7 (R = leaving group. Minimisations were performed using the Solver
mNP) (10 mg) in deionised water (3 mL), and this was divided function in Microsoft Excel™. A spreadsheet was constructed
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