Enantioselective benzylic hydroxylation of indan and tetralin with Pseudomonas monteilii TA-5

Article abstract of DOI:10.1016/j.tetasy.2009.04.006

A set of 22 toluene- and ethylbenzene-degrading strains were screened for the enantioselective benzylic hydroxylation of indan and tetralin, and Pseudomonas monteilii TA-5 was discovered as an active and selective biocatalyst for such hydroxylations. Cells of P. monteilii TA-5 can be easily grown to a high density and demonstrated a specific hydroxylation activity of 24 U/g cdw (cell dry weight). Conditions for the hydroxylation of indan 1a and tetralin 1b with resting cells of this strain were optimized, to give the corresponding (R)-1-indanol 2a and (R)-1-tetralol 2b in 99% ee and 62-67% yields, respectively. No significant product inhibition was observed, and biohydroxylation with cell-free extracts suggested that the responsible hydroxylase is a soluble enzyme depending on either NADH or NADPH. Preparative biohydroxylation was demonstrated with resting cells as biocatalysts, affording (R)-2a in 99% ee and 65% yield, and (R)-2b in 99% ee and in 63% yield, respectively.

Full text of DOI:10.1016/j.tetasy.2009.04.006

Tetrahedron: Asymmetry 20 (2009) 1206–1211
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Enantioselective benzylic hydroxylation of indan and tetralin
with Pseudomonas monteilii TA-5
*
Felicia Lie, Yongzheng Chen, Zunsheng Wang, Zhi Li
Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117576, Singapore
a r t i c l e i n f o
a b s t r a c t
Article history:
A set of 22 toluene- and ethylbenzene-degrading strains were screened for the enantioselective benzylic
hydroxylation of indan and tetralin, and Pseudomonas monteilii TA-5 was discovered as an active and
selective biocatalyst for such hydroxylations. Cells of P. monteilii TA-5 can be easily grown to a high den-
sity and demonstrated a specific hydroxylation activity of 24 U/g cdw (cell dry weight). Conditions for the
hydroxylation of indan 1a and tetralin 1b with resting cells of this strain were optimized, to give the cor-
responding (R)-1-indanol 2a and (R)-1-tetralol 2b in 99% ee and 62–67% yields, respectively. No signifi-
cant product inhibition was observed, and biohydroxylation with cell-free extracts suggested that the
responsible hydroxylase is a soluble enzyme depending on either NADH or NADPH. Preparative biohydr-
oxylation was demonstrated with resting cells as biocatalysts, affording (R)-2a in 99% ee and 65% yield,
and (R)-2b in 99% ee and in 63% yield, respectively.
Received 19 March 2009
Accepted 15 April 2009
Available online 11 May 2009
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
an unspecified yield;7,8 Purified P450cam monooxygenase and mu-
tant were reported for the hydroxylation of indan and tetralin to
Regio- and stereoselective hydroxylations provide direct access
to chiral alcohols that are important building blocks or intermedi-
ates for the syntheses of fine chemicals, pharmaceuticals, and agro-
chemicals.1 However, these types of transformations, including the
hydroxylation on activated or non-activated carbon atoms remain
as a significant challenge in classic chemistry.2 For instance, ben-
zylic hydroxylations of benzocycloarenes were examined by using
conventional chemical methods3 as well as organocatalysts,4 but
the enantioselectivities were low. On the other hand, biohydroxy-
lations have become a useful alternative. Successful examples in-
clude selective hydroxylation non-activated atom5 as well as
benzylic hydroxylation.6–12 For example, enantioselective benzylic
hydroxylation of methyl phenylacetate with engineered cyto-
chrome P450 BM-36a and Helminthosporium sp. CIOC3316,6b
respectively, gave the corresponding (S)-methyl mandelate in
90% ee6a and 92% ee,6b respectively.
Enantioselective benzylic hydroxylations of indan and tetralin
to enantiopure 1-indanol and 1-tetralol are difficult to perform. A
number of biocatalysts have been examined for these hydroxyl-
ations,7–12 but the enantioselectivity or the productivity are not
satisfactory: Pseudomonas putida F1, P. putida F39/D, and purified
naphthalene dioxygenase from Pseudomonas sp. NCIB9816-11
were found to catalyze the hydroxylation of indan, affording 1-ind-
anol in 83% ee (R) and 96% ee (R), and 92% ee (S), respectively, with
give (R)-1-indanol in 87% ee and (R)-1-tetralol in 93–95% ee with
unspecified yields;9 Fusarium moniliforme MS31 was able to cata-
lyze the hydroxylation of indan and tetralin, but the corresponding
(R)-1-indanol and (R)-1-tetralol were obtained in only 9% ee;10 P.
putida UV4 was used to catalyze the hydroxylation of indan, to af-
ford (R)-1-indanol in 34% yield and 98% ee;11 Hydroxylation of in-
dan and tetralin with Mortierella isabellina for 2 and 4 days,
respectively, resulted in (R)-1-indanol in 64% yield and 86% ee
and (R)-1-tetralol in 38% yield and 92% ee, respectively.12 To dis-
cover more efficient and selective biocatalysts for these hydroxyl-
ations, we have been working on the screening of toluene- and
ethyl benzene-degrading microorganisms. Herein we report our
recent success on the identification of P. monteilii TA-5 as a highly
active and enantioselective biocatalyst for the desired benzylic
hydroxylation and the development of whole cells-based biohydr-
oxylation for the efficient preparation of enantiopure (R)-1-indanol
and (R)-1-tetralol.
2. Results and discussion
2.1. Screening of toluene- and ethylbenzene-degrading strains
for enantioselective benzylic hydroxylation of indan and
tetralin
In order to search for an efficient biocatalyst with high regio-
and enantioselectivity for the benzylic hydroxylations, 22 strains
were isolated from the sediment, and topsoils were collected in
* Corresponding author. Tel.: +65 6516 8416; fax: +65 6779 1936.
E-mail address: chelz@nus.edu.sg (Z. Li).
0957-4166/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2009.04.006

F. Lie et al. / Tetrahedron: Asymmetry 20 (2009) 1206–1211
1207
Table 1
Screening of efficient biocatalysts enantioselective benzylic hydroxylation of indan 1a and tetralin 1b
O
OH
n
Microorganisms
+
n
n
1a
n=1
(R)-2a n=1
3a n=1
3b
1b n=2
2b
(R)- n=2
n=2
Entry
Strains
Suba
(R)-2a Yieldb (%)
(R)-2a eeb (%)
3a Yieldb (%)
Suba
(R)-2b Yieldb (%)
(R)-2b eeb (%)
3b Yieldb (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Pseudomonas sp. TA-1
1a
1a
1a
1a
1a
1a
1a
1a
1a
1a
1a
1a
1a
1a
1a
1a
1a
1a
1a
1a
1a
1a
21
—
94
—
17
0
2
5
18
6
4
5
4
4
4
5
8
5
0
5
5
4
1b
1b
1b
1b
1b
1b
1b
1b
1b
1b
1b
1b
1b
1b
1b
1b
1b
1b
1b
1b
1b
1b
64
89
67
92
59
99
94
99
94
57
93
99
78
68
91
80
11
80
27
97
79
40
90
96
91
96
89
95
94
94
93
99
92
93
94
96
95
95
99
96
54
99
99
94
30
2
1
2
25
3
4
3
5
18
7
4
2
2
3
2
1
2
4
Pseudomonas monteilii TA-2
Pseudomonas monteilii TA-4
Pseudomonas monteilii TA-5
Pseudomonas monteilii TB-1
Pseudomonas monteilii TB-2
Pseudomonas monteilii TB-3
Pseudomonas monteilii TB-4
Pseudomonas monteilii TB-5
Pseudomonas monteilii TC-1
Rhodococcus coprophilus TC-2
Pseudomonas monteilii TC-3
Pseudomonas monteilii TC-4
Pseudomonas monteilii TC-5
Pseudomonas monteilii TD-1
Pseudomonas monteilii TD-2
Pseudomonas monteilii TD-3
Pseudomonas monteilii TD-4
Pseudomonas monteilii EBV2-3
Pseudomonas monteilii EBC-3
Pseudomonas monteilii EBV2-1
Cellulosimicrobium cellulans EB8-4
29
39
30
47
31
28
38
38
7
49
54
48
48
49
—
97
99
96
97
98
96
98
98
22
98
97
98
75
98
—
34
26
91
88
7
98
35
97
99
84
9
13
11
6
4
2
3
a
The reactions were run with 2 mM substrate in a 5-mL cell suspension (5 g cdw/L) of P. monteilii TA-5 in 50 mM KH2PO4–K2HPO4 buffer (pH 7.0) at 30 °C and 300 rpm for
45 min.
b
Determined by HPLC analysis.
Singapore through enrichment with toluene or ethylbenzene as
sole carbon sources. Eighteen toluene-degrading strains (Table 1,
entries 1–18) and four ethylbenzene-degrading strains (Table 1,
entries 19–22) were tested for biohydroxylation of 1a and 1b. As
shown in Table 1, nearly all 22 strains were able to catalyze the
hydroxylation of 1a and 1b to give the corresponding sec-alcohol
(R)-2a and (R)-2b as the major product, and the corresponding ke-
tones 3a and 3b as by products. The ee values of the obtained (R)-
2a and (R)-2b at 45 min are often higher than 90%. Strains P.
monteilii EBV2-1, P. monteilii EBC-3, and P. monteilii TA-5 were in
particular found to be able to hydroxylate 1a and 1b with consid-
erably high activity and enantioselectivity. Unfortunately, the for-
mer two strains did not work well at higher substrate
concentrations: only trace amounts of product were formed when
substrate concentration of 1a and 1b was increased from 2 mM to
5 mM. On the other hand, P. monteilii TA-5 showed higher yields
when substrate concentration was increased from 2 mM to
5 mM. This demonstrates that the P. monteilii TA-5 has a higher
substrate tolerance. Therefore, the strain P. monteilii TA-5 was se-
lected for further investigation on the enantioselective hydroxyl-
ation of indan 1a and tetralin 1b. The name of the strain TA-5
was suggested by 16S-rDNA sequence analysis by Microcheck
Inc. (Northfield, USA): this strain showed a 99.77% sequence iden-
tity with P. monteilii.
fer for the hydroxylation of 1a for 15 min to examine the specific
activity. As shown in Figure 1, cells grew fast and reached 3.4 g
cdw/L after 18 h with a hydroxylation activity of 24 U/g cdw. The
cell growth reached a late exponential phase at 21 h, and after that
time point the specific activity of the cells slightly decreased. Nev-
ertheless, the cells after 24 h still retained an activity of 18 U/g
cdw.
4
3
2
1
0
30
20
10
0
0
10
20
30
Time (h)
Figure 1. Growth curve and specific activity of P. monteilii TA-5 grown on toluene in
M9 medium. : Activity, : cell density.
2.2. Cell growth and hydroxylation activity of P. monteilii TA-5
2.3. Enatioselective benzylic hydroxylation of indan and
The cells of P. monteilii TA-5 were grown in an M9 medium with
the vapor of toluene. Samples were taken at different time points
for the determination of optical density at 450 nm. At the same
time, cells in the samples were harvested and resuspended in buf-
tetralin with resting cells of P. monteilii TA-5
The effect of substrate concentration and different pH values on
the productivity and enantioselectivity in hydroxylation of 1a and

1208
F. Lie et al. / Tetrahedron: Asymmetry 20 (2009) 1206–1211
1b with resting cells of P. monteilii TA-5 was examined. As shown
in Table 2 (entries 1–5), hydroxylation of 6–10 mM 1a at a cell den-
sity of 10 g cdw/L and pH 7.0 for 4 h gave 35–67% of (R)-2a in 97–
99% ee and 10–14 % of 3a. The best results are obtained with 8 mM
of 1a, leading to the formation of 67% of (R)-2a in 99% ee. pH of 7.0
was shown to be better than pH of 6.0 or 8.0 from entries 2, 6, and
7. On the other hand, hydroxylation of 7 mM tetralin 1b afforded
the (R)-2b with 62% yield and 99% ee (entry 8). In addition, the bio-
hydroxylations of indan or tetralin were compared with or without
glucose (2%) in the reaction medium, but no effect on the product
yield or ee was observed.
The time course of biohydroxylations of 8 mM indan 1a with
resting cells of P. monteilii TA-5 at a density of 10 g cdw/L is shown
in Figure 2. The reaction was very fast at the first 1 h; 4.6 mM (R)-
2a and 0.3 mM (S)-2a were formed after 1 h. Both (R)-2a and (S)-2a
were found to be oxidized to 3a at very slow but nearly the same
rates. There was no reduction of 3a to 1a. The ee of product (R)-
2a was increased to 99% after 4 h, and 5.4 mM (R)-2a was obtained
(entry 3). Hydroxylation of tetralin 1b afforded (R)-2b in 81% ee at
1 h, and similar to the above observation, the product ee was in-
creased to 99% at 7 h and 4.4 mM of (R)-2b was produced (entry 8).
100
80
60
40
20
0
12
10
8
6
4
2
0
10
20
30
40
50
1-indanol for pretreatment (mM)
Figure 3. Effect of the concentration of (R)-1-indanol 2a on the enantioselective
hydroxylation of 7 mM indan 1a with a resting cell (10 g cdw/L) of P. monteilii TA-5
: ee, : activity, : yield).
(
KH2PO4–K2HPO4 buffer (pH 7.0); the mixture was incubated at
30 °C and 300 rpm for 1 h; after centrifugation, the cells were
washed twice with KH2PO4–K2HPO4 buffer (50 mM, pH 7.0), and
then resuspended in KH2PO4–K2HPO4 buffer (pH 7.0) to examine
the hydroxylation of 1a. As shown in Figure 3, there was no signif-
icant influence on the final product yield when cells were pre-
treated with (R)-2a at lower than 20 mM, although the specific
activity was reduced. Thus, it was possible to use as high as
20 mM indan for the enzymatic hydroxylation without indanol
inhibition. This indicates that the stopping of the reaction after
1 h shown in Figure 2 was not caused by the product inhibition.
Increasing (R)-2a concentration to 40 mM for the pretreatment of
the cells resulted in decrease of 60% activity and 50% of final prod-
uct yields.
8
6
4
2
0
100
80
60
40
20
0
2.5. Enatioselective benzylic hydroxylation of indan and
tetralin with soluble cell-free extracts of P. monteilii TA-5
0
1
2
3
4
5
Time (h)
To obtain some information on the enzyme in P. monteilii TA-5
that is responsible for the benzylic hydroxylation, hydroxylation
with cell-free extracts of strain TA-5 was investigated. The soluble
cell-free extracts were prepared by passage of the cell suspension
(20 g cdw/L in 50 mM KH2PO4–K2HPO4 buffer at pH 7.0) through
a French press, followed by removal of the cell debris and mem-
branes by centrifugation at 16,000 rpm for 30 min. As shown in Ta-
ble 3, incubation of 1a or 1b and the equivalent NADH or NADPH
with the soluble cell-free extracts gave the same products and
same ee of (R)-2a–b as those generated with resting cells. In com-
parison, biohydroxylation of 1a–b with the soluble cell-free ex-
tracts without NADH or NADPH gave only a small amount of
Figure 2. Biohydroxylation of indan 1a (8.0 mM) with resting cells of P. monteilii
TA-5 (10 g cdw/L) in 10 mL 50 mM KH2PO4–K2HPO4 (pH 7.0). ee of 2a,
concentration of (R)-2a, N concentration of (S)-2a, concentration of 3a.
2.4. Inhibition of indanol on the hydroxylation of indan with
resting cells of P. monteilii TA-5
To investigate whether there are significant product inhibitions
for the hydroxylation of indan 1a, (R)-2a at different concentra-
tions was added to a resting cell suspension (10 g cdw/L) in
Table 2
Optimization of the reaction conditions for the hydroxylation of indan and tetralin with P. monteilii TA-5
Entry
Substratea
Concentration (mM)
Time (h)
pH
Activityb (U/g cdw)
(R)-2a
Yieldc (%)
3a Yieldc (%)
R)-2b
Yieldc (%)
3b Yieldc (%)
eec (%)
eec (%)
1
2
3
4
5
6
7
8
9
1a
1a
1a
1a
1a
1a
1a
1b
1b
1b
6
7
8
9
10
7
7
7
8
10
4
4
4
4
4
4
4.5
7
4
4
7.0
7.0
7.0
7.0
7.0
6.0
8.0
7.0
7.0
7.0
15
12
13
13
13
20
21
13
10
10
35
63
67
57
47
58
21
99
99
99
98
97
99
99
11
12
14
12
10
19
14
62
56
45
99
92
91
9
6
5
10
a
The reactions were run with a 10-mL cell suspension (10 g cdw/L) of P. monteilii TA-5 in 50 mM KH2PO4–K2HPO4 buffer (pH 7.0) at 30 °C and 300 rpm.
Activity was determined over the first 15 min.
Determined by HPLC analysis.
b
c

F. Lie et al. / Tetrahedron: Asymmetry 20 (2009) 1206–1211
1209
Table 3
Hyroxylation of indan and tetralin with soluble cell-free extracts of P. monteilii TA-5
OH
n
O
Cell-free extracts
+
n
n
(R)-2a-b
3a-b
1a-b
NAD(P)+
NAD(P)H
Entry
Substratea
Cofactor
(R)-2a
3a Yieldb (%)
(R)-2a
3b Yieldb (%)
Yieldb (%)
eeb (%)
Yieldb (%)
eeb (%)
1
2
3
4
5
6
1a
1a
1a
1b
1b
1b
—
19
44
37
97
98
99
3
9
13
NADH
NADPH
—
NADH
NADPH
2
10
5
99
98
99
0.2
2
1
a
The reactions were run with 2 mM substrate in a 2-mL solution (protein concentration 11.19 g cdw/L, 50 mM KH2PO4–K2HPO4 buffer), at 30 °C and 300 rpm for 1 h.
Determined by HPLC analysis.
b
product. These results indicated that P. monteilii TA-5 contained a
coupling constants J in hertz. The purity of product and ee were
determined with ShimadzuTM Prominence HPLC on a Daicel OB-
TM
soluble NADH- or NADPH-dependent monooxygenase for the ben-
zylic hydroxylations. Obviously, NADH is the more preferred cofac-
tor than NADPH.
H chiral column (250 ꢀ 4.6 mm, 5
lm) at 25 °C. Optical rotations
were determined in chloroform at 22 °C with a JascoTM spectropo-
larimeter. Indan (>99%), tetralin (>99%), (R)-1-indanol (>97%), (R)-
1-tetralol (>97%), (S)-1-indanol (>97%), (S)-1-tetralol (>97%), inda-
none (>99%), and tetralone (>99%) were purchased from Sigma–Al-
drich. 22 microorganisms used for screening were isolated and are
availableinour laboratory atDepartmentof ChemicalandBiomolec-
ular Engineering, National University of Singapore.
2.6. Preparation of (R)-1-indanol and (R)-1-tetralinol by
enantioselective benzylic biohydroxylation with resting cells of
P. monteilii TA-5
Preparative biotransformation of indan 1a and tetralin 1b
was carried out in a 500-mL bioreactor containing substrate 1a
(41.3 mg, 0.35 m mol) or 1b (46.2 mg, 0.35 m mol) in a 50-mL sus-
pension of resting cells (10 g cdw/L) in 50 mM of KH2PO4–K2HPO4
buffer (pH 7.0). The reactions were performed for 7 h. After work
up with centrifugation and extraction with ethylacetate (EtOAc),
the products were purified by flash chromatography on a silica gel
column (10% EtOAc: 90% n-hexane). This gave 65% yield (30.5 mg)
of (R)-2a in 99% ee and 63% yield (32.0 mg) of (R)-2b in 99% ee.
4.2. Analytical method
The ee values of bioproducts (R)-2a and (R)-2b were determined
TM
TM
with a Shimadzu Prominence HPLC on a Daicel OB-H chiral col-
umn (250 ꢀ 4.6 mm, 5 m) at 25 °C. The mobile phase used was
l
hexane/isopropanol (95:5), 1 mL/min monitored at 210 nm. Reten-
tion time: 4.1 min for 1a, 8.4 min for (R)-2a, 13.1 min for (S)-2a,
19.9 min for 3a.; 3.8 min for 1b, 7.3 min for (R)-2b, 10.9 min for
(S)-2b, 13.4 min for 3b, respectively. The absolute configuration
of the biohydroxylation products 2a and 2b was assigned by com-
parison of the retention time in HPLC analysis with those of the
standard sample of (R)-1-indanol and (R)-1-tetralol.
3. Conclusion
Twenty two toluene or ethylbenzene degrading strains were
found to catalyze the enantioselective benzylic hydroxylation of in-
dane 1a and tetralin 1b. P. monteilii TA-5 is among the best strains
with high activity, high conversion, and excellent regio- and enanti-
oselectivity. The strain grows fast, high cell density is easy to
achieve; the resting cells show a high hydroxylation activity of
24 U/gcdw. Optimalreactionconditionswiththerestingcellsasbio-
catalysts have been established and preparative biohydroxylation
has been demonstrated. Hydroxylation of indan 1a and tetralin 1b
with resting cells of P. monteilii TA-5, respectively, afforded (R)-2a
in 99% ee and 65% yield, and (R)-2b in 99% ee and 63% yield, respec-
tively. Thus, we have discovered and developed P. monteilii TA-5 as
the best catalyst known thus far for the highly active and enantiose-
lective benzylic hydroxylation of indan 1a and tetralin 1b.
4.3. Isolation of microorganisms and general culture
conditions
Thirty soil samples, five sea water samples, and five sewage
sludge samples were collected from Singapore. Luria Bertani (LB)
broth and minimal M9 medium13 supplemented with trace ele-
ment14 and different carbon (0.1% v/v) sources were used through-
out this study. Agar plates of the above-mentioned media were
TM
prepared by adding 20 g of bacto agar per 1 L of medium. All cul-
tures in liquid medium were incubated in a shaker at 30 °C and
250–300 rpm unless otherwise stated. All cultures on agar plate
were grown aerobically at room temperature with carbon source
supplied in vapor phase in desiccators unless otherwise stated.
The procedure for the isolation of the microorganisms is as follows:
enrichment cultures were prepared by adding 1 g of soil or 1 mL li-
quid of collected samples to 10 mL M9 medium and then supplying
toluene or ethylbenzene as carbon source in a tube within a 28-mL
closed glass culture bottle with a metal screw cap. After incubation
for 2 weeks at 30 °C and 250 rpm, 0.1 mL diluted enrichment cul-
4. Experimental
4.1. General
1H and 13C NMR spectra were determined at 500 (1H) and 75 (13C)
MHz, all in CDCl3, with chemical shifts in ppm relative to TMS and

1210
F. Lie et al. / Tetrahedron: Asymmetry 20 (2009) 1206–1211
ture (1000–100,000 dilution) was dispensed onto M9 agar plates.
These plates were incubated in desiccators with the vapor of tolu-
ene or ethylbenzene as the carbon source. The generated colonies
were isolated and transferred onto a new agar plate. Pure strain
was obtained by repeatedly transferring single colonies of the
strain onto a new agar plate.
was incubated at 30 °C and 300 rpm for 1 h. After centrifugation,
the cells were washed twice with 25 mL KH2PO4–K2HPO4 buffer
(50 mM, pH 7.0), then resuspended in 10 mL KH2PO4–K2HPO4 buf-
fer (pH 7.0). The substrate indan (7 mM) was added and the mix-
ture was incubated at 30 °C and 300 rpm for 7 h. The results are
shown in Figure 3.
4.4. General screening procedure of microorganisms for
enantioselective benzylic hydroxylation of indan and tetralin
4.8. Enantioselective benzylic hydroxylation of indan and
tetralin with soluble cell-free extracts of P. monteilii TA-5
At first, 1
l
L inoculated strain was transferred from M9 agar
Cells of P. monteilii TA-5 were prepared from a 21-h culture as
described above. The cells were resuspended in KH2PO4–K2HPO4
buffer (50 mM pH 7.0) to a density of 20 g cdw/L. The cell suspen-
sion was disrupted using cell disrupter at 30 kPa for 5 min. The cell
debris was removed by centrifugation at 16,099g at 4 °C for 40 min
and the supernatant was collected. The protein concentration of
the cell-free extract was 11 g/L determined by using the Bradford
protein content assay. Three parallel reactions were performed
for 1 h: 5-mL cell-free extract and 2 mM 1a; cell-free extract and
2 mM 1a and 2 mM NADH; cell free extract and 2 mM 1a and
NADPH in three different flasks. Samples were taken and analyzed
by HPLC, and the results are listed in Table 3.
plate into LB liquid medium. The culture was allowed to grow for
24 h at 30 °C and 300 rpm. One milli liter of LB seed culture was
then transferred into 50-mL M9 medium supplemented with trace
element in 125-mL conical flask, a plastic tube with length of
7.8 cm containing 0.5 mL toluene or ethylbenzene was put into
the flask and the vapor of toluene or ethylbenzene was used as car-
bon source. The cells were grown at 30 °C and 250 rpm for 2 days
and harvested by centrifugation at 8000 rpm for 10 min. The cells
of microorganism were suspended in 5 mL 50 mM KH2PO4–
K2HPO4 buffer (pH 7.0) to a density of 5 g cdw/L. Two milli molar
of indan 1a or tetralin 1b was added and the mixtures were shaken
at 30 °C and 300 rpm for 45 min. 1-mL samples were taken, cells
were removed by centrifugation, and the product was extracted
with 1 mL ethyl acetate containing 1 mM benzyl alcohol as internal
standard. The organic phase was analyzed by HPLC.
4.9. Preparation of 1-indanol (R)-2a and 1-tetralol (R)-2b by
biohydroxylation
The cells of P. monteilii TA-5 were suspended in 50 mL of 50 mM
KH2PO4–K2HPO4 buffer (pH 7.0) to a density of 10 g cdw/L, sub-
strate 1a (41.3 mg, 0.35 mmol) or 1b (46.2 mg, 0.35 mmol) was
added, and the mixture was incubated in a shaker at 30 °C and
300 rpm for 7 h. The product was extracted with EtOAc, dried over
MgSO4, and concentrated by evaporation at reduced pressure. The
crude product was purified by flash chromatography (10% EtOAc in
n-hexane, Rf = 0.2). This gave 30.5 mg of (R)-2a and 32.0 mg of (R)-
2b, corresponding to 65% and 63% yield, respectively. (R)-1-indanol
4.5. Cell growth and hydroxylation activity of P. monteilii TA-5
At first, 1 lL inoculated strain was transferred from M9 agar plate
into 5 mL LB medium in 28 mL glass bottle with screw cap. The cul-
ture was shaken at 30 °C and 300 rpm for 7 h and then added into
100 mL medium M9 liquid containing trace element in a 250-mL
shaking flask with ventilated plastic stopper to reach initial cell den-
sity of 0.1 g cdw/L. Fifteen milli liters of plastic tube with length of
9 cm containing 1 mL toluene were put into the flask, and the vapor
of toluene was used as carbon source. The culture was incubated at
30 °C and 250 rpm. After 18 h, 10-mL samples were taken after
one- or two-hour intervals, the cell density was measured by OD at
450 nm. The cells were then harvested and resuspended in buffer
for biohydroxylation of 2 mM indan 1a for 15 min to get the specific
hydroxylation activity. The cell growth reached the late exponential
stage at 21 h with highest hydroxylation activity of 24 U/g cdw. The
results are given in Figure 1.
2a: ½a 2D2
ꢁ
¼ ꢂ35:2 (c 1.05, CHCl3) {lit.15: ½a D23
¼ ꢂ36:1 (c 0.05
ꢁ
CHCl3)}; 1H NMR (CDCl3, 500 Hz): d 7.44–7.42 (d, 1H, Ph), 7.29–
7.22 (m, 3H, Ph), 5.26 (t, 1H, J = 6 Hz, CH), 3.10–3.04 (m, 1H, CH),
2.86–2.80 (m, 1H, CH), 2.53–2.46 (m, 1H, CH), 1.99–1.92 (m, 1H,
CH), 1.76 (br s, 1H, -OH); 13C NMR (CDCl3, 75 Hz): d 145.0, 143.3,
128.3, 126.7, 124.9, 124.2, 76.5, 36.0, 29.8. (R)-1-tetralol-2b:
½
a 2D2
ꢁ
¼ ꢂ34:9 (c 1.12, CHCl3) {lit.15: ½a D23
¼ ꢂ33:2 (c 0. 31 CHCl3)};
ꢁ
1H NMR (CDCl3, 500 Hz): d 7.42–7.27 (m, 1H, Ph), 7.26–7.23 (m,
2H, Ph), 7.19–7.15 (t, 1H, Ph), 4.80 (t, J = 6 Hz, 1H, CH), 2.87–
2.84 m, 1H, CH), 2.74–2.72 (m, 1H, CH), 2.04–1.75 (m, 5H, CH2,
CH2, OH); 13C NMR (CCl3, 75 Hz):138.8, 137.1, 128.9, 128.6,
127.5, 126.1, 68.1, 32.5, 29.2, 18.8.
4.6. General procedure for enantioselective benzylic
hydroxylation of indan and tetralin with resting cells of P.
monteilii TA-5
Acknowledgment
Cells of P. monteilii TA-5 were suspended in 10 mL of 50 mM
KH2PO4–K2HPO4 buffer (pH 6.0–8.0) to a density of 10 g cdw/L,
1a and 1b (6–10 mM) was added and the mixture was shaken at
300 rpm and 30 °C. The reaction was followed by HPLC analysis:
samples (0.5 mL) were taken at predetermined time points, cells
were removed by centrifugation, products were extracted with
1 mL ethyl acetate containing 1 mM benzyl alcohol as internal
standard, and organic phase was analyzed. Hydroxylation of 1a
and 1b afforded (R)-2a and (R)-2b in high ee and good yields,
shown in Table 2.
This work was supported by Science & Engineering Research
Council of A*STAR, Singapore, through a research grant (Project
No. 0621010024).
References
1. (a) Liese, A.; Seelbach, K.; Wandrey, C. Industrial Biotransformations; Wiley-
VCH: Weinheim, 2006. pp 153–254; (b) Li, Z.; Chang, D.-L. Curr. Org. Chem.
2004, 8, 1647–1658; (c) Burton, S. G. Trends Biotechnol. 2003, 12, 543–549.
2. (a) Groves, J. T.; Peter Viski’, P. J. Am. Chem. Soc. 1989, 111, 8537–8538; (b)
Battaini, G.; Monzani, E.; Perotti, A.; Para, C.; Casella, L.; Santagostini, L.;
Gullotti, M.; Dillinger, R.; Nther, C.; Tuczek, F. J. Am. Chem. Soc. 2003, 125, 4185–
4198; (c) Ishimaru, T.; Shibata, N.; Nagai, J.; Nakamura, S.; Toru, T.; Kanemasa,
S. J. Am. Chem. Soc. 2006, 128, 16488–16489; (d) Company, A.; Gmez, L.; Gell,
M.; Ribas, X.; Luis, J. M.; Que, L.; Costas, M. J. Am. Chem. Soc. 2007, 129, 15766–
15767.
4.7. Investigation of product inhibition on enantioselective
benzylic hydroxylation of idan with resting cell of P. monteilii
TA-5
3. (a) Puuuiyamurthy, T.; Iqbal, J. Tetrahedron Lett. 1994, 35, 4003–4006; (b)
Pavan, C.; Legros, J.; Bolm, C. Adv. Synth. Catal. 2005, 347, 703–705; (c) Klopstra,
M.; Hage, R.; Kellogg, R. M.; Feringa, B. L. Tetrahedron Lett. 2003, 44, 4581–4584.
At first, 10–40 mM (R)-2a was added to a resting cell suspension
(10 g cdw/L) in 10 mL KH2PO4–K2HPO4 buffer (pH 7.0), the mixture

F. Lie et al. / Tetrahedron: Asymmetry 20 (2009) 1206–1211
1211
4. (a) Tong, X.-L.; Xu, J.; Miao, H.; Gao, J. Tetrahedron Lett. 2006, 47, 1763–1766;
(b) Acocella, M. R.; Mancheño, O. G.; Bella, M.; Jrgensen, K. A. J. Org. Chem. 2004,
69, 8165–8167.
9. Mayhew, M. P.; Roitberg, A. E.; Tewari, Y.; Holden, M. J.; Vanderah, D. J.; Vilker,
V. L. New J. Chem. 2002, 26, 35–42.
10. Uzura, A.; Katsuragi, T.; Tani, Y. J. Biosci. Bioeng. 2001, 92, 381–384.
11. Bowers, N. I.; Boyd, D. R.; Sharma, N. D.; Goodrich, P. A.; Groocock, M. R.;
Blacker, A. J.; Goode, J.; Dalton, H. J. Chem. Soc., Perkin Trans. 1 1999, 1453–
1461.
12. Limberger, R. P.; Ursini, C. V.; Moranb, P. J. S.; Rodrigues, J. A. R. J. Mol. Catal. B:
Enzym. 2007, 46, 37–42.
13. Bühler, B.; Witholt, B.; Hauer, B.; Schmid, A. Appl. Environ. Microbiol. 2002, 68,
560–568.
5. (a) Chang, D.-L.; Witholt, B.; Li, Z. Org. Lett. 2000, 2, 3949–3952; (b) Chang, D.-
L.; Feiten, H. J.; Engesser, K. H.; Beilen, J. B.; Witholt, B.; Li, Z. Org. Lett. 2002, 4,
1859–1862; (c) Li, Z.; Feiten, H. J.; Chang, D.-L.; Duetz, W. A.; Beilen, J. B.;
Witholt, B. J. Org. Chem. 2001, 66, 8424–8430; (d) Chang, D.-L.; Feiten, H. J.;
Witholt, B.; Li, Z. Tetrahedron: Asymmetry 2002, 13, 2141–2147.
6. (a) Landwehr, M.; Hochrein, L.; Otey, C. R.; Kasrayan, A.; Bäckvall, J. E.; Arnold,
F. H. J. Am. Chem. Soc. 2006, 128, 6058–6059; (b) Chen, Y.-Z.; Xu, J.-G.; Xu, X.-Y.;
Xia, Y.; Lin, H.; Xia, S.-W.; Wang, L.-X. Tetrahedron: Asymmetry 2007, 18, 2537–
2540.
14. Preusting, H.; Houten, R.; Hoefs, A.; Langenberghe, E. K.; Favre-Bulle; Witholt,
O. B. Biotechnol. Bioeng. 1993, 41, 550–556.
7. Wackett, L. P.; Kwart, L. D.; Gibson, D. T. Biochemistry 1988, 27, 1360–1367.
8. Brand, J. M.; Cruden, D. L.; Zylstra, G. J.; Gibson, D. T. Appl. Environ. Microbiol.
1992, 58, 3407–3409.
15. Ma, Y.-P.; Liu, H.; Chen, L.; Cui, X.; Zhu, J.; Deng, J.-G. Org. Lett. 2003, 5, 2103–
2106.