Highly chemo- and regio-selective hydroxylations of o- and m-substituted toluenes to benzyl alcohols with Cellulosimicrobium cellulans EB-8-4

Article abstract of DOI:10.1016/j.tet.2010.06.039

Highly chemo- and regio-selective benzylic hydroxylations of o- and m-substituted toluenes were achieved with the easily available and easy-to-handle resting cells of Cellulosimicrobium cellulans EB-8-4 as biocatalysts, giving the corresponding benzyl alcohols as single product. Benzyl alcohols were obtained in 78-94% yield, demonstrating the first green, clean, and simple method for the preparation of benzyl alcohols via hydroxylations. Biotransformation of 4-methylbenzyl chloride with the same strain gave 4-methylbenzyl alcohol in 67-81% yield, suggesting a novel dehalogenation activity of the cells and providing a novel, green, and efficient method for the preparation of 4-methylbenzyl alcohol as well as the application potential in biodegradation of chlorine-containing aromatics.

Full text of DOI:10.1016/j.tet.2010.06.039

Tetrahedron 66 (2010) 6919e6923
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Highly chemo- and regio-selective hydroxylations of o- and m-substituted
toluenes to benzyl alcohols with Cellulosimicrobium cellulans EB-8-4
a
b
a
a
a,
*
Shiyao Dai , Jinchuan Wu , Zunsheng Wang , Yongzheng Chen , Zhi Li
a
Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117576
Institute of Chemical and Engineering Sciences, 1 Pesek Road, Jurong Island, Singapore 627833
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 April 2010
Received in revised form 26 May 2010
Accepted 14 June 2010
Available online 18 June 2010
Highly chemo- and regio-selective benzylic hydroxylations of o- and m-substituted toluenes were ach-
ieved with the easily available and easy-to-handle resting cells of Cellulosimicrobium cellulans EB-8-4 as
biocatalysts, giving the corresponding benzyl alcohols as single product. Benzyl alcohols were obtained
in 78e94% yield, demonstrating the first green, clean, and simple method for the preparation of benzyl
alcohols via hydroxylations. Biotransformation of 4-methylbenzyl chloride with the same strain gave
4
-methylbenzyl alcohol in 67e81% yield, suggesting a novel dehalogenation activity of the cells and
Keywords:
providing a novel, green, and efficient method for the preparation of 4-methylbenzyl alcohol as well as
the application potential in biodegradation of chlorine-containing aromatics.
Ó 2010 Elsevier Ltd. All rights reserved.
Biotransformations
Alkyl toluenes
Enzyme catalysis
Hydroxylations
Benzyl alcohols
1. Introduction
2-ethylphenyl methanol, 2-propylphenyl methanol, is difficult,
which involves the reduction of their corresponding aldehydes or
1
1e13
14,15
Chemo-, regio-, and stereoselective hydroxylations are impor-
tant reactions for the preparation of alcohols. For instance, selective
benzylic hydroxylation could give rise to the direct synthesis of
benzyl alcohols that are useful fine chemicals and pharmaceutical
intermediates.1 However, chemical benzylic hydroxylation suffers
from low chemo- and regioselectivity and often results in a mixture
of different products. As easily available industry feedstocks,
xylenes are typical substrates for the preparation of benzyl alcohols
and thus have been examined for the benzylic hydroxylations. Mn-
complexes-catalyzed oxidation of o-xylene 1 gave 55% benzylic
acids
or the reductive cleavage of phthalans.
Enzyme-catalyzed hydroxylation is a green alternative for selec-
tive benzylic hydroxylation.
been explored for the hydroxylation of xylenes,
are not satisfactory: Chloroperoxidase (CPO) catalyzed the oxidation
of o-, m-, and p-xylene to give abundant tolualdehydes with only
0.2e0.4 mM methybenzyl alcohols. Cytochrome P450-catalyzed
hydroxylation of m- and p-xylene gave 80% alcohols with 20% alde-
hyde as by-product; xylene monooxygenase-catalyzed hydroxyl-
ation of m- and p-xylene
from xylene to its alcohol and then to the aldehyde. A cloned
p-cymene monooxygenase and a cytochrome P450 monooxygenase
were also reported as potential enzymes for the selective bio-
hydroxylation of m- and p-xylenes, but with no information on the
selectivity and product purity.
16e18
Different enzymatic systems have
1
9e25
but the results
e4
19
2
0
21,22
was known to be a multiple oxidation
5
23
selectivity with the alcohol and aldehyde in 1:2. Mo-peroxo com-
plex-catalyzed oxidation of o-xylene 1 afforded a mixture of the
mono- and diol in 2:1 with 23% yield, and oxidation of p-xylene with
the same system yielded 27% of the same product mixture in the
same ratio.6 Similar results were reported by N-hydroxyph-
thalimide-catalyzed oxidation of o-, m-, and p-xylene, with 5.2e28%
of the corresponding methylbenzyl alcohols and simultaneously
24,25
Recently, we discovered Cellulo-
simicrobium cellulans EB-8-4 for the regio- and stereo-selective al-
lylic hydroxylation of
2
6
D
-limonene to (þ)-trans-carveol. Since the
7,8
produced aldehydes and acids. Therefore, the current industrial
preparation of these methylbenzyl alcohols has to be achieved by
other methods, such as the non-green one, which involves chlori-
strain was isolated by growing on ethyl benzene, it may contain the
enzyme that catalyzes the benzylic hydroxylation. Thus, we have
investigated the hydroxylation of xylenes 1,2 and other substituted
toluenes 3e6 and 13e15 with this strain. Here, we report the highly
chemo- and regio-selective benzylic hydroxylations and the green
and efficient syntheses of the corresponding benzyl alcohols. In ad-
dition, the novel enzymatic activity of dehalogenation of chlorine-
containing aromatic substrate 13e15 with this strain is also reported.
9
,10
nation and hydrolysis with base at high temperature.
Moreover,
the preparation of other substituted benzyl alcohols, such as
*
Corresponding author. Tel.: þ65 6516 8416; fax: þ65 6779 1936; e-mail address:
chelz@nus.edu.sg (Z. Li).
0
040-4020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.06.039

6920
S. Dai et al. / Tetrahedron 66 (2010) 6919e6923
2
. Results
The reaction courses of hydroxylation of 1 and 2 are shown in
Figure 2, the activity calculated over the first 1 h reaction reached
3.3 U/g cdw (U¼mmol/min; cdw¼cell dry weight) and 8.7 U/g cdw
for the biohydroxylation of 1 to 7 and 2 to 8, respectively. The bio-
hydroxylation of 1 to 7 was still active from 6e10 h and gave product
concentration of 9.8 mM in 82% yield. On the other hand, the bio-
hydroxylation of 2 to 8 was much faster and mostly completed at
6 h. 11.3 mM product in 94% yield was obtained. As a result, no
further reaction was observed from 6e10 h, possibly due to the low
substrate concentration in the reaction system. As shown in Table 1,
the increase of substrate concentration from 12 to 24 mM in the
biohydroxylation of 2 to 8 gave an improvement in product con-
centration to 15.6 mM with 65% yield. However, a similar increase of
substrate concentration did not result in the increase of product
concentration in the biohydroxylation of 1 to 7. There was no re-
action for the biotransformation of p-xylene for 10 h at different
substrate concentrations under the same reaction conditions. This
2
3
.1. Biohydroxylation of xylenes 1e2 and other alkyl toluenes
e6 to benzyl alcohols 7e12
To explore the chemo- and regio-selective benzylic bio-
hydroxylation, o-xylene 1, m-xylene 2, and p-xylene were chosen as
model substrates (Scheme 1). The hydroxylation was carried out by
using the resting cells of C. cellulans EB-8-4 as an easy-to-handle
catalyst, which was prepared on a large scale by simple growing
procedures. With the cell density of 10 g cdw/L, different substrate
concentrations of 12 mM, 24 mM and 50 mM were examined for the
ꢀ
biohydroxylation of 1 and 2 at 30 C and 200 rpm. The reactions were
followed by taking samples at different time points for GC analysis.
Calibration curves for 1, 2, 7, and 8 were established with 1 mM
n-hexadecane as internal standard to quantify the substrate and
product concentrations. Highly regio-selective biohydrxoylations of
1
7
and 2 were achieved. As shown in Figure 1, only the product peak of
at 12.6 minwas detected in theGC spectra of the sample taken from
1
1
2
0
8
6
4
2
0
the biohydroxylation of 1 at 2 h. When the reaction was further
conducted to 8 h, no by-product such as benzylic aldehyde or ben-
zylic acid was detected from the GC spectrum as we ll. Similarly,
a clean reaction was also found in the biohydroxylation of 2 to 8.
Cellulosimicrobium
o-xylene 1
-methylbenzylalcohol 7
m-xylene 2
-methylbenzylalcohol 8
cellulans EB-8-4
OH
R
R
2
3
1
-6
7-12
7 R= o-CH
1
R= o-CH3
3
2
3
4
5
6
R= m-CH3
8 R= m-CH
3
R= o-CH CH
9 R= o-CH CH
2 3
2
2
2
2
3
3
2
2
R= m-CH CH
10 R= m-CH CH
2 3
0
2
4
6
8
10
R= o-CH CH CH
11 R= o-CH CH CH
2 2 3
3
3
Time (h)
R= m-CH CH CH
12 R= m-CH CH CH
2 2 3
Scheme 1. Biohydroxylation of o- and m-alkyl toluenes 1e6 to their corresponding
benzyl alcohols 7e12.
Figure 2. Reaction course of biohydroxylation of 12 mM xylenes 1e2 in 10 mL cell
suspension (10 g cwd/L) in K-buffer (pH¼7.0) at 30 ꢀC and 200 rpm.
ꢀ
Figure 1. GC spectra of samples taken from biohydroxylation of o-xylene (12 mM) in 10 mL cell suspension (10 g cdw/L) in K-buffer (pH¼7.0) at 30 C and 200 rpm (i) at 2 h;
(
ii) at 8 h.

S. Dai et al. / Tetrahedron 66 (2010) 6919e6923
6921
Table 1
increase theproductconcentration. Anincreaseofconcentrationof11
from 8.5 to 10.1 mM was achieved for the hydroxylation of substrate 5
by increasing the substrate concentration from 10 mM to 20 mM. The
biohydroxylations of 4 to 10 and 6 to 12 were relatively less active, but
the concentration of product 10 still reached 5.2 mM in 52% yield,
whiletheconcentrationofproduct12 reachedonly 1.6 mM. Theeffect
of substrate structure on the biohydroxylation rate is also shown in
Table 1. The comparison between the biohydroxylations of 3 and 4
showed that m-substitution resulted in a much slower reaction rate
than that of o-substitution. This trend was much clear in the com-
parison between the reactions of 5 and 6. Under the same reaction
conditions, hydroxylation of 5 with o-substituted propyl group pro-
duced 8.5 mM product, which is about five times higher than the
product concentration obtained from biohydroxylation of 6 with m-
substituted propyl group. However, both the biohydroxylation of 1
and 2 showed high reaction rate and high product concentration,
although they have o- and m-substitution in their structures, re-
spectively. A possible reason is that the methyl group at a different
position may have an effect on the binding of substrate 1 or 2 to the
catalytic pocket of the enzyme, but the freedom is still enough for
them to reach the catalytic sites. So, the length of side chain may be an
important factor that caused the difference in reaction rate, especially
for m-substituted compounds. For the o-substituted compounds,
substrate 5 showed comparable product concentration as substrate 3
and 1. Therefore, the side-chain-length effect was less significant for
o-substituted compounds in our study.
Chemo- and regio-selective biohydroxylation of o- and m-alkyl toluenes 1e6 to the
corresponding benzyl alcohols 7e12
Entrya
Substrate
Concn. Time
Product
Concn.
(mM)
Yield
(%)
(mM)
(h)
1
2
3
4
5
1
1
2
2
3
12
24
12
24
10
10
10
6
7
7
8
8
9
9.8
6.2
81
26
78
65
94
OH
OH
11.3
15.6
9.4
10
5
6
7
8
3
3
3
15
20
40
9
9
9
OH
9
9
9
11.6
8.8
9.2
77
44
23
9
4
10
7
10
5.2
52
1
1
1
0
1
2
4
4
4
15
20
40
10
7
9
10
10
10
6.6
4.7
5.5
44
23
14
OH
OH
1
1
3
4
5
5
10
20
9
9
11
8.5
85
51
11 10.1
1
1
5
6
6
6
10
20
9
9
12
12
1.6
3.0
16
4.3
OH
a
Biohydroxylation of o- and m-alkyl toluenes 1e6 (10e40 mM) was carried out in
1
0 mL cell suspension (10 g cwd/L) in K-buffer (pH¼7.0) at 30 ꢀC and 200 rpm.
may due to the substrate specificity of the hydroxylation enzyme in
the cells.
The mono-hydroxylation selectivity was recognized in the bio-
hydroxylation of 1 and 2 with C. cellulans EB-8-4 cells. Benzyl al-
cohols 7 and 8 were produced as the only products, respectively,
without the formation of any diol. The highly selective hydroxyl-
ation of the benzylic methyl group was also demonstrated in the
hydroxylation of 3, 4, 5, and 6. The biotransformation of 1,3-
diethylbenzene with C. cellulans EB-8-4 cells was also examined,
but no reaction occurred within 8 h.
To further study the regioselectivity of the benzylic bio-
hydroxylation with resting cells of C. cellulans EB-8-4 and extend
the substrate spectra for producing more useful benzyl alcohols,
another five alkyl-toluenes were investigated for the bio-
hydroxylation (Scheme 1). The high regioselectivity was also
demonstrated for the biohydroxylation of 2-ethyltoluene 3, 3-eth-
yltoluene 4, 2-propyltoluene 5, and 3-propyltoluene 6. Single
product of 2-ethylphenyl methanol 9, 3-ethylphenyl methanol 10,
2
-propylphenyl methanol 11, or 2-propylphenyl methanol 12 was
2
.2. Biohydroxylation of methylbenzyl chlorides 13e15 to
detected from GC analysis, respectively. The results are summarized
in Table 1 (entries 5e16). The reaction courses of hydroxylation of 3,
benzylic alcohols
4
, and 5 are shown in Figure 3. The activity over the first 1 h re-
To explore the possibility of biohydroxylation of chloride-
substituted aromatic compounds by C. cellulans EB-8-4, 4-methyl-
benzyl chloride 13, 2-methylbenzylchloride 14 and 3-methylbenzyl
chloride 15 were investigated. It was surprising to find that a differ-
ent type of reaction happened in the biotransformation: 4-methyl-
benzyl alcohol 16 was formed. This demonstrated that the C. cellulans
EB-8-4 cell was able to catalyze a dehalogenation reaction. As shown
in Table 2, the biotransformation of 30 mM 13 gave 24.3 mM 16 in
action was calculated as 6.2 U/g cdw, 5.4 U/g cdw, and 5.7 U/g cdw
for the biohydroxylation of 3 to 9, 4 to 10, and 5 to 11, respectively.
After 10 h biohydroxylation, 9.4 mM 9 (94% yield), 5.2 mM 10 (52%
yield), and 8.5 mM 11 (85% yield) were obtained, respectively.
As shown in Table 1, concentration of the product 9 in the bio-
hydroxylation of 3 was increased to 11.6 mM (77% yield) by using
15 mM substrate. Further increase of substrate concentration did not
8
3
1% yield, while the biotransformation of 50 mM 13 produced
3.4 mM 16 in 67% yield. The C. cellulans EB-8-4 cell was also found to
1
0
8
6
4
2
0
survive at 100 mM 13 to produce 30.7 mM 16 without significant
inhibition effect. Although the detailed mechanism of the dehalo-
genation reaction is not clear from this study, a high productivity for
the biotransformation of 13 to 16 with 33.4 mM product, 67% yield,
and good substrate resistance of 100 mM were successfully dem-
onstrated. This provides a novel, green, and efficient method for the
preparation of benzyl alcohol 16, and it could also be a useful method
for biodegradation of chlorine-containing aromatics.
o-ethyltoluene 3
o-ethylbenzylalcohol 9
m-ethyltoluene 4
The dehalogenation reaction was further confirmed by the
biotransformation of substrates 14 and 15. As shown in Table 2
m-ethylphenylmethanol 10
o-propyltoluene 5
o-propylphenylmethanol 11
(entry 4), a mixture of 3.0 mM 17, 0.37 mM 7, and 0.84 mM 18
were formed at 5 h. The products were isolated and characterized
1
by GCeMS and H NMR spectroscopy. Compound 7 from the mix-
ture was produced by the dehalogenation reaction, while 17 was
synthesized from the direct hydroxylation of the methyl group in
0
2
4
6
8
Time (h)
14. When product concentrations between 1 h and 5 h (entry 4)
were compared, the concentration of 18 increased from 0.06 mM to
0.84 mM. A control experiment by using 7 as substrate for
Figure 3. Reaction course of biohydroxylation of 10 mM alkyl toluenes 3e5 in 10 mL
cell suspension (10 g cwd/L) in K-buffer (pH¼7.0) at 30 ꢀC and 200 rpm.

6922
S. Dai et al. / Tetrahedron 66 (2010) 6919e6923
Table 2
tolulene 4 (99%), 2-methylbenzylchloride 14 (99%), 3-methylbenzyl
chloride 15 (98%), and 4-methyl-benzylchloride 13 (98%) were
purchased from SigmaeAldrich. 2-Propyltoluene 5 (98.5%) and
3-propyltoluene 6 (99.7%) were obtained from TCI (Japan). 1,3-
diethylbenzene (>99%) and ethyl benzene were ordered from Fluka.
n-Hexadecane (>99%) was obtained from Merck. Ethyl acetate was
bought from Fisher.
Chemo- and regio-selective biohydroxylation of methylbenzyl chlorides 13e15 to
the corresponding benzyl alcohols
Entrya
Substrate
Concn.
mM)
Time
(h)
Product
Concn.
(mM)
Yield
(%)
(
Cl
OH
1
13
30
9
16
24.3
81
2
3
13
13
50
100
9
9
16
16
33.4
30.7
67
31
Analytical methods: Gas chromatography (GC) analysis was
performed on an Agilent 7890A system with a flame ionization
Cl
Cl
4
14
5
1
17
2.75
55
OH
detector on a HP-5 capillary column (30.0 mꢁ322
m
mꢁ0.25
mm).
Helium was used as the carrier gas at a flow rate of 1.0 mL/min. Inlet
OH
ꢀ
and detector temperature were 280 and 300 C, respectively. 1 mM
1
7
0.24
4.8
n-hexadecane was used as internal standard. The temperature was
ꢀ
ꢀ
ꢀ
programmed to increase from 60 C to 100 C at a rate of 5 C/min,
OH
ꢀ
ꢀ
ꢀ
ꢀ
increase from 100 C to 116 C at a rate of 2 C/min, hold at 116 C
for 2 min, increase from 116 C to 280 C at a rate of 30 C/min, and
hold at 280 C for 3 min. Retention time: 6.2 min for o-xylene 1,
.8 min for m-xylene 2, 7.9 min for 2-ethyltoluene 3, 7.5 min for
-ethyltoluene 4, 10.2 min for 2-propyltoluene 5, 9.7 min for
1
18
0.06
1.2
OH
ꢀ
ꢀ
ꢀ
5
5
5
17
7
18
3.01
0.37
0.84
60
7.4
17
ꢀ
5
3
Cl
Cl
5
15
5
1
19
1.02
20
OH
3-propyltoluene 6, 11.8 min for 2-methylbenzylchloride 14,
1.7 min for 3-methylbenzyl chloride 15, 11.9 min for 4-methyl-
benzyl chloride 13, 12.6 min for 2-methylbenzyl alcohol 7, 12.3 min
for 3-methylbenzyl alcohol 8, 12.2 min for 4-methylbenzyl alcohol
OH
1
1
8
0.17
3.4
OH
16, 15.7 min for 2-ethylphenyl-methanol 9, 15.8 min for 3-ethyl-
1
20
<0.01
0
phenyl methanol 10, 19.2 min for 2-propylphenyl methanol 11,
19.3 min for 3-propylphenyl methanol 12, 20.2 min for 2-(chlor-
omethyl)-phenyl methanol 17, 20.6 min for 3-(chloromethyl)-
phenyl methanol 19, and 22.3 min for n-hexadecane.
OH
5
5
5
19
8
20
1.97
0.47
0.19
39
9.4
3.8
a
Biohydroxylation of methylbenzyl chlorides 13e15 (5e100 mM) was performed
in 10 mL cell suspension (10 g cwd/L) in K-buffer (pH¼7.0) at 30 ꢀC and 200 rpm.
GCeMS analysis was performed with an Agilent 7890A GC sys-
tem coupled with a 5975C mass selective detector (EI) on the same
HP5 column as described above. Helium was used as the carrier gas
biotransformation was carried out, and the result showed that <1%
8 was produced after 6 h. Thus, it could be deducted that com-
at a flow rate of 1.0 mL/min. Inlet and detector temperature were
1
ꢀ
2
80 and 300 C, respectively. The temperature program was the
pound 18 was formed from 17 via dehalogenation catalyzed by the
resting cells. The activities for the production of 7, 17, and 18 were
calculated over the first 1 h reaction as 0.4 U/g cdw, 4.6 U/g cdw,
and 0.1 U/g cdw, respectively. The ratio of 7, 17, and 18 in the
product mixture changed slightly during biotransformation. A
similar phenomenon was observed in the biotransformation of 15
with the resting cells of C. cellulans EB-8-4, and product 19, 8, and
0 were obtained in a different ratio and lower yield. It is not clear
whether the dehalogenation was catalyzed by the same enzyme for
the hydroxylation or other enzymes in C. cellulans EB-8-4 cells.
same as the GC analysis mentioned above. The temperature was
ꢀ
ꢀ
ꢀ
increased from 60 C to 100 C at a rate of 5 C/min, increase from
ꢀ ꢀ ꢀ ꢀ
1
00 C to 116 C at a rate of 2 C/min, hold at 116 C for 2 min,
ꢀ
ꢀ
ꢀ
increase from 116 C to 280 C at a rate of 30 C/min, and hold at
ꢀ
2
80 C for 3 min.
1H NMR (500 MHz) spectra were recorded in CDCl
at room
3
temperature with a Bruker AMX 500 NMR instrument. Chemical
shifts were referred to TMS at 0 ppm.
2
Optical density of cell cultures was measured at 450 nm (OD450
)
with a Hitachi U-1900 spectrophotometer. A cell density of 1 g cdw/
L corresponds to OD450 of 0.26.
3
. Conclusion
Growth of C. cellulans EB-8-4: C. cellulans EB-8-4 was cultured
2
6
according to pervious report. The strain was grown in 5 mL LB
Highly chemo-, and regio-selective biohydroxylation of o- and
ꢀ
medium at 30 C and 250 rpm for 14e16 h, and 3 mL LB culture
m-alkyl toluenes 1e6 was achieved by using the easily available and
easy-to-handle resting cells of C. cellulans EB-8-4 as biocatalyst.
This synthetic route via biohydroxylation gave 100% regiose-
lectivity, no by-product, and good to excellent yields, being much
better than any other known oxidation methods for the preparation
of pure benzyl alcohols without any aldehyde or acid by-products.
A dehalogenation activity was also discovered in C. cellulans EB-8-4
cells, which provides with a novel, green, and efficient method for
the preparation of 4-methylbenzyl alcohol 16 in 67e81% yield by
biotransformation of 4-methylbenzyl chloride 13 with this strain.
The novel enzymatic dehydrogenation could be useful for the
biodegradation of chlorine-containing aromatics.
were then transformed into 100 mL M9 medium in a 250-mL
shaking flask with a tube containing 1 mL ethyl benzene. The cul-
ꢀ
ture was incubated at 30 C and 250 rpm. Cells were harvested at
early stationary phase at 24 h with a cell density of 2.9 g cdw/L.
General procedures for biohydroxylation of alkyl toluenes 1e6
and methylbenzyl chlorides 13e15 to the corresponding benzyl
alcohols with resting cells of C. cellulans EB-8-4: Cells of C. cellulans
EB-8-4 was resuspended to 10 g cdw/L in 10 mL K-buffer (pH¼7.0)
containing 100 mM glucose in a 125-mL conical flask with glass
stopper sealed with parafilm. Alkylbenzene 1e6 (10e40 mM) or
methylbenzyl chlorides 13e15 (5e100 mM) were added to the cell
ꢀ
suspension and the biotransformation was performed at 30 C and
200 rpm. At regular time intervals, a 0.2 mL sample was taken and
4
4
. Experimental section
.1. General
mixed with 0.4 mL ethyl acetate containing 1 mM n-hexadecane.
After extraction, centrifugation and separation, the organic phase
was analyzed by GC.
Product identification: Benzyl alcohols 9, 10, 11 and 12 produced
from biohydroxylation of substrate 3, 4, 5, and 6 were isolated by
column chromatography on silica gel with ethyl acetate: n-hexane
Chemicals: o-xylene 1 (anhydrous, 97%), m-xylene 2 (anhydrous,
9%), p-xylene (anhydrous, 99%), 2-ethyl tolulene 3 (99%), 3-ethyl
9

S. Dai et al. / Tetrahedron 66 (2010) 6919e6923
6923
(
1:4) as eluent. Benzyl alcohols 19 from substrate 15 were isolated
by column chromatography on silica gel with ethyl acetate:
n-hexane (1:1) as eluent. The R value for all alcohols was in the
range of 0.2e0.3. The isolated benzyl alcohols had a purity of >99%
2. Ullmann’s Encyclopaedia of Industrial Chemistry, 6th ed.; Wiley-VCH: Weinheim,
002; Vol. 5; p 83.
2
3
4
. Kim, J. W.; He, J.; Yamaguchi, K.; Mizuno, N. Chem. Lett. 2009, 38, 920e921.
. Sun, H.; Su, F.; Ni, J.; Cao, Y.; He, H.; Fan, K. Angew. Chem., Int. Ed. 2009, 48,
4390e4393.
f
1
5. Bennur, T. H.; Sabne, S.; Deshpande, S. S.; Srinivas, D.; Sivasanker, S. J. Mol. Catal.
A 2002, 185, 71e80.
(
GC). Their H NMR and Mass (from GCeMS) data were given as
1
1 1
follows: 9 : H NMR (CDCl
3
, 500 MHz):
OH), 2.705e2.750 (m, 2H, CH
d
¼7.221e7.286 (m, 4H, Ph),
6. Das, S.; Bhowmick, T.; Punniyamurthy, T.; Dey, D.; Nath, J.; Chaudhuri, M. K.
4
3
5
2
(
(
.732 (s, 2H, CH
H, CH
), m/z¼136 (M ). Compound 10 : H NMR (CDCl
00 MHz): OH),
¼7.133e7.299 (m, 4H, Ph), 4.679 (s, 2H, CH
.658e2.688 (m, 2H, CH Me), 1.235e1.261 (m, 3H, CH
), m/z¼136
¼7.222e7.383
OH), 2.647e2.678 (m, 2H, CH CH Me),
Me), 0.976e1.005 (m, 3H, CH
), m/z¼150
¼7.111e7.291
OH), 2.586e2.617 (m, 2H, CH CH Me),
), m/z¼150
2
2
Me),1.236e1.266 (m,
Tetrahedron Lett. 2003, 44, 4915e4917.
7. Figiel, P. J.; Sobczak, J. M. New J. Chem. 2007, 31, 1668e1673.
þ
13
1
3
3
,
8
9
. Nishiwaki, Y.; Sakaguchi, S.; Ishii, Y. J. Org. Chem. 2002, 67, 5663e5668.
. Kiyotak, O. Jpn. Kokai Tokkyo Koho 2,007,186,440, Jul. 2007.
d
2
2
3
10. Du, J.; Chen, Z.; Zou, X. YouJi HuaXue 2004, 24, 1122e1124.
11. Hayes, R.; Li, K. D.; Leeming, P.; Wallace, T. W.; Williams, R. C. Tetrahedron 1999,
þ
14
1
M ). Compound 11 : H NMR (CDCl
m, 4H, Ph), 4.728 (s, 2H, CH
.587e1.688 (m, 2H, CH
3
, 500 MHz): d
5
5, 12907e12928.
2. Wagner, P. J.; Meador, M. A.; Zhou, B.; Park, B. S. J. Am. Chem. Soc. 1991, 113,
9630e9639.
2
2
2
1
1
2
3
þ
27 1
13. Lee, J. P.; Ke, Z.; Ramirez, M. A.; Gunnoe, T. B.; Cundari, T. R.; Boyle, P. D.;
(
(
M ). Compound 12 : H NMR (CDCl
m, 4H, Ph), 4.669 (s, 2H, CH
3
, 500 MHz): d
Petersen, J. L. Organometallics 2009, 28, 1758e1775.
2
2
2
14. Azzena, U.; Demartis, S.; Fiori, M. G.; Melloni, G.; Pisano, L. Tetrahedron Lett.
1
(
(
.621e1.696 (m, 2H, CH
2
Me), 0.938e0.994 (m, 3H, CH
3
1995, 36, 8123e8126.
15. Azzena, U.; Demartis, S.; Melloni, G. J. Org. Chem. 1996, 61, 4913e4919.
þ
28
þ
29
1
M ). Compound 17 : m/z¼156 (M ). Compound 19
:
H NMR
1
1
6. Holland, H. L.; Weber, H. K. Curr. Opin. Biotechnol. 2000, 11, 547e553.
7. Li, Z.; Beilen, J. B.; Duetz, W. A.; Schmid, A.; Raadt, A.; Griengl, H.; Witholt, B.
Curr. Opin. Chem. Biol. 2002, 6, 136e144.
8. Burton, S. G. Trends Biotechnol. 2003, 21, 543e549.
9. Park, J.; Clark, D. S. Biotechnol. Bioeng. 2006, 94, 189e192.
0. Maruyama, T.; Iida, H.; Kakidani, H. J. Mol. Catal. B 2003, 21, 211e219.
CDCl
3
, 500 MHz):
d
¼7.261e7.407 (m, 4H, Ph), 4.708 (s, 2H, CH
2
Cl),
þ
4
.596 (s, 2H, CH
2
OH), m/z¼156 (M ).
1
1
2
Acknowledgements
21. Buehler, B.; Schmid, A.; Hauer, B.; Witholt, B. J. Biol. Chem. 2000, 275,
This work was financially supported by Science and Engineering
Research Council of A*STAR, Singapore, through a research grant
project No. 0621010024).
10085e10092.
2
2. Buehler, B.; Witholt, B.; Hauer, B.; Schmid, A. Appl. Environ. Microbiol. 2002, 68,
560e568.
(
2
2
2
2
3. Nakano, T.; Kawabata, S.; Sugihara, T.; Agatsuma, N.; Kakuda, H.; Mori, Y. Bull.
Chem. Soc. Jpn. 2003, 76, 2353e2360.
4. Nishio, T.; Patel, A.; Wang, Y.; Lau, P. C. K. Appl. Microbiol. Biotechnol. 2001, 55,
Supplementary data
321e325.
5. Liu, L.; Schimid, R. D.; Urlacher, V. B. Appl. Microbiol. Biotechnol. 2006, 72,
876e882.
6. Wang, Z.; Lie, F.; Lim, E.; Li, K.; Li, Z. Adv. Synth. Catal. 2009, 351, 1849e1856.
The Supplementary data associated with this article can be
found in the online version at doi:10.1016/j.tet.2010.06.039.
2
7. Kazuhiko, M.; Masayuki, H.; Keiichiro, N.; Toshio, F. Pestic. Biochem. Physiol.
1
989, 35, 300e314.
References and notes
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