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R. Kourist et al. / Tetrahedron: Asymmetry 19 (2008) 1839–1843
70%). 1H NMR: d = 1.4 (s, 9H, C(CH3)3), 2.3 (s, 3H, CH3), 5.9 (s, 1H,
NH), 7.4 (m, 3H, H-Ar), 7.5 (m, 2H, H-Ar).13C NMR: d = 21.4, 28.3,
52.3, 83.6 (q, J = 28.3), 122.7 (q, J = 285.5), 127.3, 128.4, 129.6,
131.3, 162.8, 167.9. MS (EI): 318 (M+), 274, 260, 242, 219, 156,
77, 57, 43. Anal. Calcd for C15H18F3NO3: C, 56.78; H, 5.72; N,
4.41. Found: C, 57.07; H, 55.74; N, 4.33. HRMS: 298.101
[MÀCH3COOH+Na+].
(s, 1H, OH), 6.2 (s, 1H, NH), 7.4 (m, 3H, H-Ar), 7.7 (m, 2H, H-Ar).
13C NMR: d = 28.4, 52.9, 77.8 (q, J = 28.5), 123.9 (q, J = 286.1),
128.8, 128.9, 129.6, 134.8, 166.0. MS (EI): 232 (MÀ43), 176, 156,
105, 84, 77, 57. HRMS: 298.121 [M+Na+].
4.4. High-throughput screening
The test kit for the determination of released acetic acid was
from R-Biopharm GmbH (Darmstadt, Germany) and applied
according to the manufacturer’s protocol as described previously.29
4.2.4. (R,S)-2-(tert-Butylcarbamoyl)-1,1,1-trifluorobut-2-yl
butyrate, 4a
Compound 4a was obtained after 7 d in dichloromethane as a
white powder (1064 mg, 3.6 mmol, 92%, mp 72 °C). 1H NMR:
d = 0.9 (t, J = 7.52, 3H, CH3), 1.0 (t, 3H, CH3), 1.4 (s, 9H, (CH3)3),
1.7 (m, 2H, CH2), 2.5 (t, 2H, CH2), 2.6 (m, 2H, CH2), 5.8 (s. 1H,
NH). 13C NMR: d = 7.5, 22.4, 13.5, 18.3, 28.3, 36.6, 51.9, 83.8, (q,
J = 27.8), 123.4 (q, J = 285.6), 163.2, 170.0. MS (EI): 297 (M+), 279,
254, 242, 172, 100, 71, 57. Anal. Calcd for C13H22F3NO3: C, 52.52;
H, 7.46; N, 4.71; O, 16.14. Found: C, 52.52; H, 7.46; N, 4.71. HRMS:
320.144 [M+Na+].
Enzyme solution (20
of racemic 6a were added to a mixture (150
l
L) and substrate solution (20
lL; 7.5 mg/mL)
lL) of the test kit com-
ponents. The increase of NADH was monitored at 340 nm by using
the Fluostar Galaxy or Fluostar Optima (BMG, Offenburg, Ger-
many). Mixtures of test kit components with buffer or cell lysate
from induced Escherichia coli, that contained pUC18 without an
esterase gene, served as controls.
4.5. General procedure for esterase-catalyzed small-scale
resolutions
4.2.5. (R,S)-2-(tert-Butylcarbamoyl)-1,1,1-trifluorobut-2-yl
benzoate, 5a
To a stirred solution of the acetate (25 mM) in a phosphate buf-
fer (100 mM, pH 7.5) and the appropriate amount of co-solvent,
esterase solution was added to a total volume of 2 mL. The reaction
mixture was stirred in a thermoshaker (Eppendorf, Hamburg, Ger-
many) at the appropriate temperature. Samples were taken after
1 h, 4 h and 24 h. The reaction mixture was extracted twice with
Compound 5a was obtained after 2 d in dichloromethane and
column chromatography (n-pentane/ethyl acetate) as colourless
oil (845 mg, 2.8 mmol, 93%, mp 102 °C). 1H NMR: d = 0.9 (t,
J = 7.45, 3H, CH3), 2.5 (m, 2H, CH2), 5.9 (s, 1H, NH), 7.3 (m, 2H, H-
Ar), 7.6 (m, 1H, H-Ar), 8.1 (m, 1H, H-Ar). 13C-NMR: d = 7.6, 22.6,
28.3, 52.0, 84.1 (q, J = 27.9), 123.6 (q, J = 283.2), 129.3, 129.8,
130.1, 134.0, 163.2, 163.3. MS (EI): 332 (M+), 316, 259, 232, 105,
77, 57. Anal. Calcd for C15H17F3NO3: C, 58.00; H, 6.08; N, 4.23.
Found: C, 58.45; H, 6.03; N, 4.215. HRMS: 354.129 [M + Na+].
400 lL dichloromethane, the combined organic layers were dried
over anhydrous sodium sulfate and the organic solvent was re-
moved under nitrogen. For the determination of the enantiomeric
purity of the remaining substrate a it was hydrolyzed to the corre-
sponding alcohol b. Enantioselectivity and conversion were calcu-
lated according to Chen et al.25
4.3. General procedure for the synthesis of racemic N-tert-
butyl-2-hydroxyamides (serving as standards)
4.6. Esterase-catalyzed preparative-scale resolution of 2a
General procedure for the preparation of hydroxyamides: 2 mmol
of tertiary ester of protected a,a-dialkyl-a-hydroxycarboxylic acid
To a solution of 2a (120 mg, 0.55 mmol) in phosphate buffer
(100 mM, pH 7.5) and 5% (v/v) dimethyl formamide, esterase solu-
tion was added to a total volume of 40 mL. The solution was stirred
for 4 h at 4 °C, and the product was extracted three times with
methyl tert-butyl ether. After drying over Na2SO4 the solvent was
removed by distillation under reduced pressure, and the product
was purified by column chromatography (n-pentane/ethyl
acetate).
were dissolved in an aqueous solution of NaOH (0.1 N, 50 mL) with
DMSO (10%, v/v) as co-solvent and stirred for 1 h. The product was
extracted three times with dichloromethane. After drying over
Na2SO4, the solvent was removed by distillation under reduced
pressure.
4.3.1. (R,S)-N-tert-Butyl-2-hydroxy-2-(trifluoromethyl)-
butanamide, 1b
Compound 1b was obtained after as yellowish powder (69 mg,
0.3 mmol, 83%). 1H NMR: d = 0.92 (t, J = 7.41, 3H, CH3), 1.4 (s, 9H,
(CH3)3), 1.9 (m, 2H, CH2), 4.5 (s, 1H, OH), 6.1 (s,1H, NH). 13C
NMR: d = 6.33, 25.61, 28.35, 52.18, 77.15 (t, J = 28.0), 126.2 (t,
J = 285.4), 166.0. MS (EI): 212 (M+À15). HRMS: 228.119 [M+H+].
GC analysis: 5.3 min (+), 8.0 min (À) at a column temperature of
75 °C.
4.6.1. (À)-2-(tert-Butylcarbamoyl)-1,1,1-trifluorobut-2-yl
acetate, 1a
Compound (À)-1a was obtained after column chromatography
(n-pentane/ethyl acetate) as a white powder (30 mg, 0.11 mmol,
30%). Enantiomeric excess was determined after hydrolysis to 1b
as 47%ee.
4.3.2. (R,S)-N-tert-Butyl-2-hydroxy-2-methyl butanamide, 2b
Compound 2b was obtained after as white powder 72 mg,
0.4 mmol, 90%, mp 71 °C. 1H NMR: d = 0.9 (t, J = 7.44, 3H, CH3),
1.4 (s, 3H, CH3), 1.3 (s, 9H, (CH3)3), 1.7 (m, 2H, CH2), 2.4 (2H,
CH2), 2.9 (s, 1H, OH), 6.5 (s, 1H, NH). 13C NMR: d = 7.8, 26.6, 33.4,
27.7, 50.7, 75.9, 174.8. MS (EI): 144 (M+À29), 130, 101, 73, 58,
41. HRMS: 174.150 [M+H+]. GC analysis: 41.2 min (À), 44.0 min
(+) at a column temperature of 90 °C.
4.6.2. (+)-N-tert-Butyl-2-hydroxy-2-(trifluoromethyl)-
butanamide, 1b
Compound (+)-1b was obtained after column chromatography
(n-pentane/ethyl acetate) as a white powder (25 mg, 0.12 mmol,
32%, 87%ee).
4.6.3. (+)-1-(tert-Butylamino)-2-methyl-1-oxobutyl-2-yl
acetate, 2a
Compound (+)-2a was obtained after column chromatography
(n-pentane/ethyl acetate) as a white powder (46 mg, 0.22 mmol,
38%). Enantiomeric excess was determined after hydrolysis to 2b
as 93%ee.
4.3.3. (R,S)-N-tert-Butyl-3,3,3-trifluoro-2-hydroxy-2-
phenylpropanamide, 3b
Compound 3b was obtained after as white powder (61 mg,
0.26 mmol, 67%, mp 115 °C). 1H NMR: d = 1.31 (s, 9H, (CH3)3), 5.3