G Model
PRBI-10178; No. of Pages10
ARTICLE IN PRESS
F. Boraty n´ ski et al. / Process Biochemistry xxx (2014) xxx–xxx
3
of LiAlH . The mixture was then acidified with 0.1 M HCl and the
products were extracted with chloroform. Then the extract was
2.3.8. cis-exo-3-Oxabicyclo[2.2.1]dec-7-en-2-one (± )-(2d)
4
1
Yield 16.5%. H NMR (500 MHz, CDCl ) ı: 1.46 (d, J = 9.7 Hz, 1H,
3
washed with saturated NaCl and dried over anhydrous MgSO . The
one of CH -10), 1.54 (td, J = 7.7, 1.6 Hz, 1H, one of CH -10), 2.54 (t,
4
2
2
crude products were purified by column chromatography (silica
gel, methylene chloride–methanol, 95:5). The yields and spectral
data of diols 3c–e and lactones 2a–e obtained are given below. The
spectral data of diols 3a and 3b have been describe previously [13].
J = 8.3 Hz, 1H, CH-5), 2.63 (d, J = 8.5 Hz, 1H, CH-1), 2.88 (m, 1H, CH-
6), 3.26 (m, 1H, CH-9), 3.97 (dd, J = 9.8, 3.5 Hz, 1H, one of CH -4),
2
4.46 (t, J = 9.1, 1H, one of CH -4), 6.15–6.24 (m, 2H, CH-7, CH-8);
2
1
3
C NMR (151 MHz, CDCl ) ı: 41.92 (CH-5), 43.29 (CH -10), 46.41
3
2
(
1
1
CH-9), 47.93 (CH-1), 48.16 (CH-6), 71.85 (CH -4), 137.55 (CH-7),
37.73 (CH-8), 177.70 (C O); IR (film, cm ): 1756 (m); GC–EIMS:
50 (M+1).
2
−1
2.3.1. cis-1,2-Bis(hydroxymethyl)cyclohexane (3a)
Yield 53.6%.
2
.3.9. cis-endo-2,3-bis(hydroxymethyl)bicyclo[2.2.2]oct-5-ene)
2
.3.2. cis-3-Oxabicyclo[4.3.0]nonan-2-one (± )-(2a)
(
3e)
1
Yield 28.6%. H NMR (500 MHz, CDCl ) ı: 0.80–0.98 (m, 1 H, one
3
Yield 78.3%. 1H NMR (500 MHz, CDCl ) ı: 1.15–1.29 (m, 2H,
3
of CH -6), 1.05–1.30 (m, 5H, CH -8, CH -7, one of CH -6), 1.34 (d,
1
2
2
2
2
CH -8), 1.49–1.62 (m, 2H, CH -7), 2.17–2.28 (m, 2H, CH-1, CH-
2
J = 11.2, 3.2 Hz, 2H, CH -OH), 3.62 (t, J = 11.2 Hz, 2H, CH -OH), 6.12
2
2
H, J = 10.2 Hz, one of CH -9), 1.45–1.95 (m, 1H, one of CH -9), 2.10
2
2
), 2.40–2.46 (m, 2H, CH-1, CH-4), 2.78 (s, 2H, 2xOH), 3.52 (dd,
(
dd, 1H, J = 23.3, 11.0 Hz, H-1), 2.35–2.70 (m, 1H, H-5), 3.92 (d, 1H,
2
2
J = 8.8 Hz, one of CH -4), 4.16 (dd, 1H, J = 8.8, 5.0 Hz, one of CH -4);
2
2
13
(dd, J = 4.6, 3.2 Hz, 2H, CH-5, CH-6); C NMR (151 MHz, CDCl ) ı:
13
3
C NMR (151 MHz, CDCl ) ı: 22.40 (CH -6), 22.80 (CH -8), 23.30
3
2
2
2
(
5.44 (CH -8, CH -7), 34.47 (CH-1, CH-4), 45.61 (CH-2, CH-3), 65.33
2 2
(
CH -7), 27.10 (CH -9), 35.30 (CH-5), 39.40 (CH-1), 71.70 (CH -4),
2
2
2
−1
CH -OH), 132.99 (CH-5, CH-6); IR (film, cm ): 3020 (s); GC–EIMS:
2
−
1
1
78.60 (C O); IR (film, cm ): 1766 (s); GC–EIMS: 140 (M+1).
1
2
1
68 (M+1).
2
.3.3. cis-4,5-Bis(hydroxymethyl)cyclohexene (3b)
Yield 73.1%.
.3.10. cis-endo-3-Oxabicyclo[2.2.2]undec-7-en-2-one (± )-(2e)
1
Yield 3.8%. H NMR (500 MHz, CDCl ) ı: 1.09–1.36 (m, 2H, CH -
3
2
1), 1.38–1.64 (m, 2H, CH -10), 2.67 (m, 2H, CH-9, CH-6), 2.74 (dd,
2
2
.3.4. cis-3-Oxabicyclo[4.3.0]non-7-en-2-one (± )-(2b)
J = 10.2, 3.2 Hz, 1H, CH-5), 3.06 (m, 1H, CH-1), 3.82 (dd, J = 9.3, 3.9 Hz,
1H, one of CH2-4), 4.32 (t, J = 8.9 Hz, 1H, one of CH2-4), 6.23–6.34
Yield 7.5%. 1H NMR (500 MHz, CDCl ) ı: 1.77–2.05 (m, 1H, one
3
1
3
of CH -6), 1.90–2.90 (m, 3H, one of CH -6, H-5, one of CH -9),
2
of CH -4), 4.30 (dd, 1H, J = 8.8, 5.1 Hz, 1H, one of CH -4), 5.60–5.70
(m, 2H, CH-7, CH-8); C NMR (151 MHz, CDCl3) ı: 23.38 (CH2-
10), 23.43 (CH2-11), 31.78 (CH-1), 33.37 (CH-6), 38.02 (CH-9), 44.77
(CH-5), 72.37 (CH2-4), 132.62 (CH-7), 134.31 (CH-8), 179.29 (C O);
2
2
2
.74–2.80 (m, 2H, one of CH -9, H-1), 4.00 (dd, 1H, J = 8.8, 2.0 Hz, one
2
2
2
13
−1
(m, 2H, H-8, H-7); C NMR (151 MHz, CDCl ) ı: 21.91 (CH -6),
IR (film, cm ): 1757 (m); GC–EIMS: 164 (M+1).
3
2
2
4.60 (CH -9), 31.89 (CH-1), 37.19 (CH-5), 72.70 (CH -4), 124.77
2
2
−
1
(
CH-7), 125.05 (CH-8), 179.09 (C O); IR (film, cm ): 1771 (s);
2.4. Growth conditions
GC–EIMS: 138 (M+1).
The composition of culture media (g/1 L H O) consist of 20 g
2
glucose, 10 g peptone, 2 g casein hydrolysate, 2 g yeast extract, 6 g
NaCl.
2
(
.3.5. cis-endo-2,3-Bis(hydroxymethyl)bicyclo[2.2.1]hept-5-ene
3c)
Yield 55.8%. H NMR (500 MHz, CDCl ) ı: 1.34–1.42 (m, 2H, CH -
1
3
2
2.5. Microorganisms
7
2
), 2.52 (m, 2H, CH-2, CH-3), 2.78 (m, 2H, CH-1, CH-4), 3.27 (s, 2H,
xOH), 3.38 (t, J = 10.8 Hz, 2H, CH -OH), 3.63 (dd, J = 10.9, 3.3 Hz, 2H,
2
1
3
The following bacteria strains were used for screening: Bacillus
CH -OH), 6.02 (t, J = 1.8 Hz, 2H, CH-5, CH-6); C NMR (151 MHz,
2
pumilus PCM 1852, Bacillus megaterium PCM 1855, Dietzia sp. DSM
44016, Gordonia sp. DSM 44456, Streptomyces griseus PCM 2325,
Micrococcus sp. DSM 30771, Rhodococcus sp. DSM 364, Rhodococ-
cus erythropolis DSM 44534. The microorganisms came from the
German Collection of Microorganisms and Cell Cultures (DSMZ)
in Braunschweig and the Polish Collection of Microorganisms
CDCl ) ı: 45.11 (CH-2, CH-3), 46.54 (CH-1, CH-4), 49.92 (CH -7),
6
GC–EIMS: 154 (M+1).
3
2
−
3.58 (CH -OH), 134.80 (CH-5, CH-6); IR (film, cm ): 3020 (s);
2
1
2
.3.6. cis-endo-3-Oxabicyclo[2.2.1]dec-7-en-2-one (± )-(2c)
Yield 25.5%. 1H NMR (500 MHz, CDCl ) ı: 1.44 (d, J = 8.7 Hz, 1H,
3
(
PCM) Institute of Immunology and Experimental Therapy, Polish
one of CH -10), 1.62 (td, J = 8.6, 1.5 Hz, 1H, one of CH -10), 3.07
2
2
◦
Academy of Sciences in Wroclaw. They were maintained at 4 C on
Sabouraud agar slants containing peptone (10 g), glucose (40 g) and
agar (15 g) dissolved in water (1 L) at pH 5.5.
(m, 1H, CH-6), 3.09 (m, 1H, CH-5), 3.22 (dd, J = 9.3, 4.7 Hz, 1H, CH-
1
), 3.31 (m, 1H, CH-9), 3.77 (dd, J = 9.7, 3.1 Hz, 1H one of CH -4),
2
4
.26 (t, J = 8.6 Hz, 1H, one of CH -4), 6.23–6.31 (m, 2H, CH-7, CH-8);
2
1
3
C NMR (151 MHz, CDCl ) ı: 40.28 (CH -5), 45.76 (CH-6), 46.12
3
2
2.6. Biotransformations of meso diols 3a–e
(
1
CH-9), 47.56 (CH-1), 51.81 (CH-10), 70.25 (CH -4), 134.36 (CH-7),
36.91 (CH-8), 178.00 (C O); IR (film, cm ): 1758 (m); GC–EIMS:
2
−
1
2.6.1. Rapid screening procedure in microtiter plate (all in MTP
1
50 (M+1).
technique)
2
4-Deepwell polypropylene microtiter plate containing 4 mL
2
.3.7. cis-exo-2,3-Bis(hydroxymethyl)bicyclo[2.2.1]hept-5-ene)
of sterile medium was inoculated by 0.1 mL of overnight precul-
tured bacterial strain. To prevent cross-contamination between
suspended cultures in adjacent wells during shaking, the microtiter
plate was covered with a sandwich cover, then clamped accord-
ing the manufacturer guideline [16]. Subsequently, the MTP was
(
3d)
Yield 50.6%. 1H NMR (500 MHz, CDCl ) ı: 1.19 (d, J = 8.0 Hz, 1H,
3
one of CH -7), 1.29 (d, J = 8.4 Hz, 1H, one of CH -7), 1.74 (m, 2H, CH-
2
4
CDCl ) ı: 43.21 (CH-2, CH-3), 43.69 (CH -7), 45.59 (CH-1, CH-4),
6
2
2
, CH-3), 2.47 (s, 2H, CH-1, CH-4), 3.66 (d, J = 3.8 Hz, 4H, CH -OH),
2
1
3
.56 (s, 2H, 2xOH), 6.12 (m, 2H, CH-5, CH-6); C NMR (151 MHz,
◦
incubated at 25 C on a rotary shaker (180 rpm, shaking diameter
3
2
50 mm). After 1–2 days of cultivation, 1 mg of substrate 3a–e in
0.05 mL of acetone was added to the grown cultures. For the time-
course analysis at appropriate intervals (4 and 7 days), 2 mL of the
−
2
1
Please cite this article in press as: Boraty n´ ski F, et al. Microbial alcohol dehydrogenase screening for enantiop-
ure lactone synthesis: Down-stream process from microtiter plate to bench bioreactor. Process Biochem (2014),