Stereoselective benzylic hydroxylation of ethylbenzene and propylbenzene using the mycelia of Aspergillus flavus MTCC-1783 and MTCC-1884

Article abstract of DOI:10.1139/v2012-034

The aim of this study was to provide syntheses of optically pure (R)-1-phenylethanol and (R)-1-phenylpropanol from ethylbenzene and propylbenzene, respectively, using the fungal mycelia of new fungal species, namely Aspergillus flavus MTCC-1783 and Aspergillus flavus MTCC-1884, as catalysts. The mycelia of A. flavus MTCC-1783 and A. flavus MTCC-1884 were prepared by growing the fungal strains in liquid culture medium containing ethylmethylketone as the sole carbon source. The mycelia were suspended in potassium phosphate buffer pH 7.0. The suspensions of mycelia were used for the transformations of ethylbenzene and propylbenzene. Ethylbenzene and propylbenzene were converted to (R)-1-phenylethanol and (R)-1-phenylpropanol, in 100% and 99% ee, respectively. The mycelia of A. flavus MTCC-1783 and A. flavus MTCC-1884 can be used for the preparation of (R)-1-phenylethanol and (R)-1-phenylpropanol in 100% and 99% ee, respectively, from ethylbenzene and propylbenzene, respectively. The studies report convenient methods for the syntheses of optically pure isomers, (R)-1-phenylethanol and (R)-1-phenylpropanol, which are important chiral building blocks in the preparations of fine chemicals and pharmaceuticals. The reactions are ecofriendly, occur at 30 °C, and the time required was 24 h.

Full text of DOI:10.1139/v2012-034

597
Stereoselective benzylic hydroxylation of
ethylbenzene and propylbenzene using the
mycelia of Aspergillus flavus MTCC-1783 and
MTCC-1884
Saroj Yadav, R.S.S. Yadav, and K.D.S. Yadav
Abstract: The aim of this study was to provide syntheses of optically pure (R)-1-phenylethanol and (R)-1-phenylpropanol
from ethylbenzene and propylbenzene, respectively, using the fungal mycelia of new fungal species, namely Aspergillus fla-
vus MTCC-1783 and Aspergillus flavus MTCC-1884, as catalysts. The mycelia of A. flavus MTCC- 1783 and A. flavus
MTCC-1884 were prepared by growing the fungal strains in liquid culture medium containing ethylmethylketone as the sole
carbon source. The mycelia were suspended in potassium phosphate buffer pH 7.0. The suspensions of mycelia were used
for the transformations of ethylbenzene and propylbenzene. Ethylbenzene and propylbenzene were converted to (R)-1-
phenylethanol and (R)-1-phenylpropanol, in 100% and 99% ee, respectively. The mycelia of A. flavus MTCC-1783 and A.
flavus MTCC-1884 can be used for the preparation of (R)-1-phenylethanol and (R)-1-phenylpropanol in 100% and 99% ee,
respectively, from ethylbenzene and propylbenzene, respectively. The studies report convenient methods for the syntheses of
optically pure isomers, (R)-1-phenylethanol and (R)-1-phenylpropanol, which are important chiral building blocks in the
preparations of fine chemicals and pharmaceuticals. The reactions are ecofriendly, occur at 30 °C, and the time required was
24 h.
Key words: Aspergillus flavus, benzylic hydroxylation, biocatalyst, (R)-1-phenylethanol, (R)-1-phenylpropanol.
Résumé : On a réalisé la synthèse des (R)-1-phényléthanol et (R)-1-phénylpropanol, à partir respectivement de l’éthylben-
zène et du propylbenzène, en présence de mycélium fongiques provenant de nouvelles espèces fongiques, soit l’Aspergillus
flavus MTCC-1783 et l’Aspergillus flavus MTCC-1884, utilisées comme catalyseurs. Les mycélium d’A. flavus MTCC-
1783 et d’A. flavus MTCC-1884 ont été préparés en procédant à la culture des souches fongiques dans des milieux de
culture liquide ne contenant que de l’éthylméthylcétone comme source de carbone. Les mycélium ont été suspendus dans un
tampon de sulfate de potassium, de pH 7,0. On a ensuite utilisé les suspensions de mycélium pour les transformations de
l’éthylbenzène et du propylbenzène. L’éthylbenzène et le propylbenzène ont été convertis en (R)-1-phényléthanol et en (R)-
(1)-phénylpropanol avec des excès énantiomères respectivement de 100 % et 99 %. En conclusion, les mycélium d’A. flavus
MTCC-1783 et d’A. flavus MTCC-1884 peuvent être utilisés dans la préparation de (R)-1-phényléthanol et (R)-(1)-phényl-
propanol avec des excès énantiomères respectivement de 100 % et 99 %, à partir respectivement l’éthylbenzène et du propyl-
benzène. Les études décrites dans ce travail rapportent la mise au point de méthodes commodes de synthèses d’isomères
optiquement purs de (R)-1-phényléthanol et de (R)-(1)-phénylpropanol qui sont des synthons chiraux importants dans les
préparations de produits chimiques fins et pharmaceutiques. Les réactions sont écologiques; elles se produisent à 30 °C et
les temps requis sont de l’ordre de 24 heures.
Mots‐clés : Aspergillus flavus, hydroxylation benzylique, biocatalyseur, (R)-1-phényléthanol, (R)-(1)-phénylpropanol.
[Traduit par la Rédaction]
Other applications include use as a salvatochromic dye,9 use
Introduction
as an opthalic preservative, and use as an inhibitor of choles-
terol intestinal absorption.10 b-Aminoalcohols have been used
for the syntheses of 1-phenylethanol-2-[(2-phenyl-1-alkyl-
ethyl)amino]ethanol derivatives, a new important class of
antidiabetic agents.11 A large genus of mushroom flies has
been reported to be attracted to 1-phenylethanol in field
tests.12 Thus, secondary alcohols act as pheromones also.
However, the chemical methods for their syntheses are not
Chiral alcohols are important intermediates and are struc-
tural elements in the syntheses of biologically active com-
pounds and natural products.1–5 The optically active 1-
phenylethanol is used as a chiral building block and synthetic
intermediate in fine chemicals and in pharmaceutical indus-
tries.6–8 (R)-1-Phenylethanol is widely used as a fragrance in
the cosmetic industry because it has a mild floral odour.
Received 2 December 2011. Accepted 20 April 2012. Published at www.nrcresearchpress.com/cjc on 3 July 2012.
S. Yadav, R.S.S. Yadav, and K.D.S. Yadav. Department of Chemistry, D.D.U. Gorakhpur University, Gorakhpur-273 009, India.
Corresponding author: Saroj Yadav (e-mail: s_saroj123@rediffmail.com).
Can. J. Chem. 90: 597–599 (2012)
doi:10.1139/V2012-034
Published by NRC Research Press

598
Can. J. Chem. Vol. 90, 2012
convenient and ees are generally low.13 On the other hand,
biocatalytic methods are convenient and enantiomeric ex-
cesses are high.14
Bennett’s agar medium,17 which consisted of 1.5% (w/v) glu-
cose, 0.5% peptone, 0.2% yeast extract, 0.2% Ehlrich’s beef
extract, and 1.5% agar in tap water.
Several biocatalytic methods to synthesize enantiomeric
pure secondary alcohols have been developed during the re-
cent past owing to the increasing demand for these valuable
compounds.5 Stereoselective reduction of ketones14 and enan-
tioselective oxidation of racemic secondary alcohols have
been studied for the preparations of pure enantiomeric secon-
dary alcohols. Although the hydroxylation of nonactivated
centres in hydrocarbons is one of the most useful biotransfor-
mations, so far it has been used mainly for the hydroxylation
of steroids and terpeniods.3,13 The biotransformation of ethyl-
benzene to 1-phenylethanol has rarely been studied.15–20 The
conversion of ethylbenzene and a number of its para-substituted
derivatives to the corresponding optically active 1-phen-
ylethanols with enantiomeric excesses varying between 5%
and 40% using the fungus Mortierella isabellina have been
reported.15 The fungi Cunninghamella echinulata var. ele-
gans and Heminthosporium were also capable of performing
some of these transformations.15 The hydroxylation of ethyl-
benzene almost exclusively at the secondary carbon atom
giving 1-phenylethanols in the ratio 2:1 of the R and S iso-
mers using cytochrome P450camp has been reported.16 Sev-
enteen fungi and two yeast species that could hydroxylate
ethylbenzene and propylbenzene to 1-phenylethanol and 1-
phenylpropanol, respectively, have been reported.17 One po-
tent strain, Fusarium moniliforme, oxidizes ethylbenzene
and propylbenzene to the corresponding benzylic alcohols
with enantiomeric excesses of 98% in the (R) (+) form.17
The involvement of cytochrome P450 in this transformation
has been demonstrated.18 Szaleniec et al.19 reported the oxi-
dation of ethylbenzene to (S)-(–)-1-phenylethanol by the de-
nitrifying bacterium Azoarcus species strain EbN1. A nobel
Mo–Fe–S enzyme, anaerobic ethylbenzene dehydrogenase,
has been isolated and characterized.20 Keeping in mind the
potential for the biotransformation of ethylbenzene to opti-
cally pure isomers of 1-phenylethanol, we initiated a search
for the fungal strains that could carry out these useful trans-
formations. In this communication, we report two fungal
strains, namely Aspergillus flavus MTCC-1783 and Asper-
gillus flavus MTCC-1884, which transform ethylbenzene to
(R)-1-phenylethanol and propylbenzene to (R)-1-phenylpro-
panol in 100% and 99% ee, respectively.
Preparation of mycelia
The microorganisms were cultivated in 100 mL of basal
medium I (BM I) containing 1 mL of unsterilized ethylmeth-
ylketone (v/v) in 250 mL Erlenmeyer flasks at 30 °C on a
rotary shaker at 150 rpm for 3 days. BM I contained 10 g of
NaNO3, 2 g of NH4Cl, 2 g of KH2PO4, 3 g of K2HPO4, 2 g
of NaCl, 0.2 g of MgSO4·7H2O, 0.5 g of yeast extract, and
2 mL of metal solution having pH 7.0 in 1 L of deionized
water. The metals solution consisted of 400 mg of
MnCl2·2H2O, 350 mg of FeCl2·4H2O, 200 mg of ZnCl2,
20 mg of CoCl2, 20 mg of CuCl2·H2O, 10 mg of
Na2MoO4·2H2O, 10 mg of Na2B4O7·10H2O, and 2 mL of
concentrated HCl in 100 mL of deionized water. The mycelia
were collected by filtration on ordinary filter papers, washed
twice with 30 mL of 25 mmol/L potassium phosphate buffer
(KPB, pH 7.0), and were used fresh.
Biotransformation reactions
The biotransformation reactions were performed using the
reported method.17 Wet mycelia (0.1 g) were suspended in
2 mL of 25 mmol/L KPB in test tubes (17 mm in diameter
and 150 mm in height), and 200 µmol of ethylbenzene
(21 µL) was added. The test tubes were closed with stoppers
and incubated at 30 °C in a reciprocal shaker at 200 rpm.
After 24 h, the reaction solutions were acidified by the addi-
tion of 0.2 mL of 6 N HCl. The products formed in the reac-
tion solution were extracted thrice using 2.0 mL of n-hexane
each time. Biotransformations of propylbenzene and methyl-
benzene were also studied using the above method. The ex-
tracts were analysed for 1-phenylethanol, 1-phenylpropanol,
and benzylalcohol by using HPLC as mentioned in the fol-
lowing. In one typical scale-up experiment, 7.5 g of the my-
celia of A. flavus MTCC-1884 suspended in 100 mL of
25 mmol/L KPB and 1.5 mL of ethylbenzene were reacted
for 24 h at 25 °C and then extracted with 30 mL of n-hexane;
20 µL of the extract was subjected to HPLC analysis. In an-
other experiment, 14 g of the mycelia of A. flavus MTCC-
1783 suspended in 100 mL of KPB and 3.0 mL of ethylben-
zene at 25 °C was reacted for 24 h, then extracted with
30 mL of n-hexane, and then 20 µL of the extract was sub-
jected to HPLC analysis. Similar experiments were per-
formed with propylbenzene.
Materials and methods
Chemicals
HPLC analysis
Methylbenzene, ethylbenzene, propylbenzene, ethylmethyl-
ketone, racemic ( )-1-phenylethanol, (R)-1-phenylethanol,
racemic ( )-1-phenylpropanol, and (R)-1-phenylpropanol
were purchased from Sigma-Aldrich Chemicals Private Lim-
ited, New Delhi, India. All other chemicals were purchased
either from S.D. Fine-chem Ltd., Mumbai, India, or from
Qualigens Chemicals, Mumbai, India, and were used without
further purification.
HPLC analyses were done using a Waters HPLC (model
600E) and a Spherisorb C18 5 UV (4.5 mm × 250 mm) col-
umn. The eluent phase was a methanol–water mixture in a
1:1 (v/v) ratio at 1 mL/min. The n-hexane extracts of the
products (20 µL) were injected and the detections were made
using a Waters UV detector (model 2487) at 254 nm.
Spectroscopic analysis
The identifications of the biotransformation products were
1
Fungal strains
done by IR and H and 13C NMR spectroscopic techniques.
Both fungal strains were procured from the Microbial Type
Culture Collection Centre and Gene Bank, Institute of Micro-
bial Technology, Chandigarh, India, and were maintained on
1H NMR
The 1H NMR spectra of the biotransformation products
Published by NRC Research Press

Yadav et al.
599
were recorded using a Jeol AL 300 MHz spectrometer in
CDCl3 using TMS as the internal standard reference at the
Department of Chemistry, Banaras Hindu University, Vara-
nasi, India.
the journal Web site at http://nrcresearchpress.com/doi/suppl/
10.1139/v2012-034.
Acknowledgements
The financial support of CST, U.P., Lucknow, India,
through project No. CST/SERPD/ D-2895/2008 is thankfully
acknowledged. The academic support of Professor V.K. Ya-
dav, Department of Chemistry, Indian Institute of Technol-
ogy, Kanpur, India, in the determination of enantiomeric
excess, and Professor R. Gurunath, of the same department,
in GC–MS analysis is thankfully acknowledged.
13C NMR
The 13C NMR spectra of the biotransformation products
were recorded on a Jeol-ECX 500 Hz spectrophotometer in
CDCl3 using TMS as the internal standard reference at the
Department of Chemistry, Indian Institute of Technology,
Kanpur, India. The chemical shift values were reported in
ppm.
References
IR
(1) Theil, F. Chem. Rev. 1995, 95 (6), 2203. doi:10.1021/
cr00038a017.
(2) Nakamura, K.; Matsuda, T. J. Org. Chem. 1998, 63 (24), 8957.
The IR spectra of the biotransformation products were re-
corded on a Bruker IR-VERTEX Flash 70 spectrometer in
the range of 400–4000 cm–1 at the Department of Chemistry,
Indian Institute of Technology, Kanpur, India.
doi:10.1021/jo9812779.
(3) Borgas, K. B.; de Souza Borges, W.; Durán-Patrón, R.; Pupo,
M. T.; Bonato, P. S.; Collado, I. G. Tetrahedron Asymmetry
2009, 20, 385. doi:10.1016/j.tetasy.2009.02.009.
(4) Ou, L.; Xu, Y.; Ludwig, D.; Pan, J.; Xu, J. H. J. Org. Process
Res. Dev. 2008, 12 (2), 192. doi:10.1021/op700253t.
(5) Bisogno, F. R.; Lavandera, I.; Kroutil, W.; Gotor, V. J. Org.
Chem. 2009, 74 (4), 1730. doi:10.1021/jo802350f.
(6) Suginaka, K.; Hayashi, Y.; Yamamoto, Y. Tetrahedron
Asymmetry 1996, 7 (4), 1153. doi:10.1016/0957-4166(96)
00121-8.
(7) Ivanov, A. E.; Schneider, M. P. J. Mol. Catal. B: Enzym. 1997,
3 (6), 303. doi:10.1016/S1381-1177(97)00012-X.
(8) de los Ríos, A. P.; Hernández-Fernández, F. J.; Tomás-Alonso,
F.; Gómez, D.; Víllora, G. Can. J. Chem. Eng. 2010, 88, 442.
doi:10.1002/cjce.20285.
GC–MS
Products were analysed by GC–MS using an HP GC–MS
model No. 5975C-inert MSDS with triple axis detector using
helium gas (5 mL/min) as the mobile phase at the Depart-
ment of Chemistry, Indian Institute of Technology, Kanpur,
India.
Determination of ee
The ees were determined using a Chiralcel OD column
(4.6 mm × 250 mm) manufactured by Daicel Chiral Technol-
ogies Pvt. Ltd. (Japan) using a 90:10 (v/v) mixture of n-hexane
and isopropylalcohol as the eluent phase at a flow rate of
0.5 mL/min at the Department of Chemistry, Indian Insti-
tute of Technology, Kanpur, India.
(9) Berglund, P. Biomol. Eng. 2001, 18 (1), 13. doi:10.1016/
S1389-0344(01)00081-8.
(10) Chua, L. S.; Sarmidi, M. R. J. Mol. Catal. B: Enzym. 2004, 28
(2–3), 111. doi:10.1016/j.molcatb.2004.02.004.
(11) Lohse, O.; Spöndlin, C. Org. Process Res. Dev. 1997, 1 (3),
247. doi:10.1021/op9600264.
(12) Kamm, J. A.; Buttery, R. G.; Robinson, W. H. J. NewYork
Entomol. Soc. 1987, 95, 19.
(13) Faber, K. Biotransformation Inorganic Chemistry; 5th ed.,
Springer-Verlag: Berlin, 2004.
(14) Wu, X.; Xiao, J. J. Chem. Commun. (Camb.) 2007, 24 (24),
Results and discusion
The results of the identification of the biotransformation
products, (R)-1-phenylethanol and (R)-1-phenylpropanol, us-
ing the suspensions of fungal mycelia of the strains A. flavus
MTCC-1783 and A. flavus MTCC-1884 are given in the
Supplementary data. The fungal mycelia of A. flavus
MTCC-1783 converted ethylbenzene to (R)-1-phenylethanol
in 100% enantiomeric excess and propylbenzene to (R)-1-
phenylpropanol in 99% enantiomeric excess. The reaction
times were 24 h and no ethylbenzene and propylbenzene
were detected at the end of transformation reactions showing
that ethylbenzene and propylbenzene were fully transformed
to their corresponding biotransformation products. Methyl-
benzene was also transformed to benzylalcohol by these two
fungal strains showing that hydroxylations occur at benzylic
carbon centre. In typical scale up experiments as mentioned
in the materials and methods section (Biotransformation reac-
tions) 1.5 mL of ethylbenzene and 3.0 mL of propylbenzene
were fully converted to (R)-1-phenylethanol and (R)-1-phen-
ylpropanol, respectively, showing the feasibility of the
method on laboratory scale.
2449. doi:10.1039/b618340a.
(15) Holland, H. L.; Bergen, E. J.; Chenchaiah, P. C.; Khan, S. H.;
Munoz, B.; Ninniss, R. W.; Richards, D. Can. J. Chem. 1987,
65 (3), 502. doi:10.1139/v87-087.
(16) Filipovic, D.; Paulsen, M. D.; Loida, P. J.; Sligar, S. G.;
Ornstein, R. L. Biochem. Biophys. Res. Commun. 1992, 189
(1), 488. doi:10.1016/0006-291X(92)91584-D.
(17) Uzura, A.; Katsuragi, T.; Tani, Y. J. Biosci. Bioeng. 2001, 91,
217.
(18) Uzura, A.; Katsuragi, T.; Tani, Y. J. Biosci. Bioeng. 2001, 91,
580.
(19) Szaleniec, M.; Hagel, C.; Menke, M.; Nowak, P.; Witko, M.;
Heider, J. J. Biochemistry 2007, 46 (25), 7637. doi:10.1021/
bi700633c.
(20) Johnson, H. A.; Pelletier, D. A.; Spormann, A. M. J. Bacteriol.
2001, 183 (15), 4536. doi:10.1128/JB.183.15.4536-4542.2001.
Supplementary data
Supplementary data are available with the article through
Published by NRC Research Press