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G. A. Petkova, V. Král / Bioorg. Med. Chem. 18 (2010) 6651–6656
115, 116, 117, 122 and 123) or 100 mM Tris–HCl (pH 7.0—for KRED
102, 106, 121, 124 and 127) with 0.05 mM NADH. Ketone 1a
10 mM was added as liquid, dissolved in 5% (v/v) 2-propanol. The
reaction was initialized by the addition of 1 mg mLꢁ1 enzyme.
Then, 0.1 mL of the reaction mixture was periodically withdrawn
and the reaction progress monitored by HPLC. Variable reaction
times were required to reach moderate or good conversion. Once
the reduction process reached a completion the reaction was ter-
minated by extraction with 2 ꢀ 5 mL CHCl3. Then the solvent was
evaporated and the sample resuspended in methanol for a chiral
HPLC analysis.
Acknowledgment
This work was supported by grants of the Ministry of Education
of the Czech Republic MSM6036137307, LC06077 and LC512, by
the grant P301/10/1426 awarded by the Grant Agency of the Czech
Republic, and by grant KAN200200651 awarded by the Grant
Agency of the Academy of Sciences of the Czech Republic.
Reference and notes
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Angew. Chem., Int. Ed. 2007, 46, 3091.
4.3.3. Enzymatic reduction of ketone 1a in the presence of
3. Keinan, E.; Hafeli, E. K.; Seth, K. K.; Lamed, R. J. Am. Chem. Soc. 1986, 108, 162.
4. Truppo, M. D.; Kim, J.; Brower, M.; Madin, A.; Sturr, M. G.; Moore, J. C. J. Mol.
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Asymmetry 2006, 17, 554.
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various concentrations of a-, b- or c-CDs
The reaction process followed the same protocol as Sec-
tion 4.3.2. In the experiments the molar ratios of 1a to CDs were:
for
for
a
-CD—1/1,5; 1/3,1 and 1/5,1; for b-CD—1/1,3; 1/2,6 and 1/4,4;
c-CD—1/1,1; 1/2,3; 1/3,2; 1/3,8 and 1/4,6. 10 mM ketone dis-
solved in 5% (v/v) 2-propanol was added to solution of desired con-
centration CDs and left under stirring for 10 min. Afterward, NADH
and 1 mg mLꢁ1 KRED 115 or 122 were added.
11. Eckstein, M.; Daubmann, T.; Kragl, U. Biocatal. Biotransform. 2004, 22, 89.
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4.3.4. Circular dichroism
The experiments were performed with KRED 115 and 122 in
100 mM phosphate buffer (pH 6.8) in a 2 mL volume and arranged
as series of independent measurements. In a general procedure, to
b- or c-CD solution was added 20 lg enzyme. After 10 min incuba-
tion time, the ECD spectra were recorded. In the experiments
amount of CDs to enzyme was: 1:1; 30:1, 60:1; 80:1; 100:1 and
130:1 (w/w) (0.02; 0.6; 1.2; 1.6; 2 and 2.6 mg, respectively).
4.3.5. Fluorescence spectroscopy
The samples were prepared and the experiments arranged as
Section 4.3.4. Duplicate fluorescence intensity measurements were
carried out.
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4.3.61H NMR measurements
1H NMR spectra of 1-(p-methoxyphenyl)-propan-2-one were
recorded in the presence and absence of c-CD. The stock 1a and
c
-CD solutions were freshly prepared by dissolving 1a in D2O.
The concentration of the 1a was 2.03 ꢀ 10ꢁ3 M and that of
c-CD
was varied from 0.223 to 4.8 ꢀ 10ꢁ2 M.
4.3.6.1 1-(p-Methoxyphenyl)-propan-2-ol rac-1b.
13C NMR: 22.7; 45.0; 56.0; 69.3; 114.2; 116.2; 130.5; 158.5. 1H
NMR: 1.2 (3H, CH3, d, J = 6.3 Hz); 2.6 (2H, CH2, m); 3.0 (1H, OH, s);
3.8 (1H, OCH3, s); 3.9–4.1 (1H, CH, m); 6.9 (2H, Ar2-6-H, d, J = 9 Hz);
7.14 (2H, Ar3–5-H, d, J = 8.7 Hz), Refs. 36–38.
37. Douzelli, F.; Fuganti, C.; Mendozza, M.; Pedrocchi-Fantoni, G.; Servi, S.; Zucchi,
G. Tetrahedron: Asymmetry 1996, 7, 3129.
38. Erdelyi, B.; Szabo, A.; Seres, G.; Birincsik, L.; Ivanics, J.; Szatzker, G.; Poppe, L.
Tetrahedron: Asymmetry 2006, 17, 268.
4.3.6.2. (R)-1-(p-Methoxyphenyl)-propan-2-ol.
Yield: 45%;
ee = 92% (HPLC); ½a D20
ꢃ
ꢁ21.0 (c = 0.5 in CHCl3), Refs. 36–38.