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with AcOEt and washed successively with H2O and 1m aq. NaOH. Evaporation of the org. phase and FC
(hexane/AcOEt 3 :2; Rf 0.2) gave (R)-5 (298 mg, 67%). Colorless crystalline solid. M.p. 888. [a]2D5 4 (c
1.35, CHCl3). IR (CHCl3): 3434, 2934, 2254, 1738, 1614, 912, 730. 1H-NMR (300 MHz, CDCl3): 7.62 (d, J 9.4,
1 H); 7.36 (d, J 8.5, 1 H); 6.84 (m, 2 H); 6.24 (d, J 9.4, 1 H); 3.99 (m, 2 H); 3.70 (m, 2 H); 2.24 (m, 1 H);
1.08 (d, J 6.9, 3 H). 13C-NMR (75 MHz, CDCl3): 162.9; 161.9; 156.5; 144.1; 129.4; 113.7; 113.5; 113.2; 102.1;
71.6; 65.7; 36.2; 14.3. Anal. calc. for C13H14O4 (234.25): C 66.65, H 6.02; found: C 66.65, H 6.01.
A similar procedure starting with (E)-3-bromo-2-methylpropan-1-ol (0.3 g, 1.96 mmol) gave (S)-5 (287 mg,
65%) as colorless crystalline solid.
(R)- and (S)-7-(3-Acetoxy-2-methylpropoxy)-2H-1-benzopyran-2-one (6). Acetylation of 5 as above gave
6 in quantitative yield as colorless solids. Anal. data for (R)-6: M.p. 518. [a]D25
2 (c 0.5, CHCl3). IR
1
(CHCl3): 2972, 2254, 1738, 1614, 1230, 1124, 910, 732, 650. H-NMR (300 MHz, CDCl3): 7.62 (d, J 10, 1 H);
7.36 (d, J 8.5, 1 H); 6.84 (m, 2 H); 6.24 (d, J 8.5, 1 H); 4.14 (m, 2 H); 3.95 (m, 2 H); 2.34 (m, 1 H); 2.08
(s, 3 H); 1.12 (d, J 15, 3 H). 13C-NMR (75 MHz, CDCl3): 171.7; 162.7; 161.8; 156.5; 144.1; 129.4; 113.8; 113.6;
113.2; 101.9; 70.6; 66.4; 33.5; 21.5; 14.5. Anal. calc. for C15H16O5 (276.29): C 65.21, H 5.84; found: C 65.21,
H 5.86.
Fluorescence Measurements. Substrates were diluted from a 10 mm stock soln. in 50% aq. MeCN. All
buffers and solns. were prepared using MilliQ-deionized H2O. Enzymes were diluted from 1 mg/ml stock solns.
in PBS (PBS 10 mm aq. phosphate, 160 mm NaCl, pH 7.4). BSA was diluted from a 40 mg/ml stock soln. in
either 20 mm borate (pH 8.8) (assays with 4) or 20 mm Bis-tris ( 2,2-bis(hydroxymethyl)-2,2',2''-nitrilo-
triethanol {[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]imino}bis[ethanol] (pH 7.2) (assays with 6). Reactions
were initiated by addition of lipase or esterase to a soln. containing substrate, HLDH, and NAD .
The 200-ml assays were followed in individual wells of round-bottom polypropylene 96-well plates (Costar)
with a Cytofluor-II fluorescence-plate reader (Perseptive Biosystems, filters lex 360 Æ 20 nm, lem 460 Æ 20 nm).
For each assay, fluorescence was converted to umbelliferone (3) concentration according to a calibration curve
with pure 3 in the same buffers containing BSA. The reference maximum rate with alcohol substrates was taken
from the linear portion of the curves (data points 0 to 5000 s). For assays with acetates, the linear portions of the
curves (data points 15000 to 30000 s) were used to derive the reaction rates.
Preparative assays: Solid enzyme (1 mg) weighed into a 1.5-ml Eppendorf tube was dissolved in 20 mm aq.
borate (pH 8.8; 200 ml). Then, a soln. of racemic acetate 4 (50 ml; 50 mg/ml in MeCN) was added and the
resulting suspension agitated on a plate stirrer at r.t. To follow the reaction, 2-ml aliquots were taken and diluted
with hexane/i-PrOH 1:1. Then, this soln. (20 ml) was injected on a Chiralpak AS column (Daicel, 0.45 Â 22 cm,
flow 1.0 ml ´ min 1, hexane/i-PrOH 1 : 2): tR 10.9 ((R)-1), 23.8 ((S)-1), 12.1 ((R)-4), 13.1 ((S)-4) min. The
integral of the peaks recorded by UVat 325 nm was used to calculate the conversion and ee of released alcohol.
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Â
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Received November 20, 1998