Fungal mediated kinetic resolution of racemic acetates to (R)-alcohols using Fusarium proliferatum

Article abstract of DOI:10.1016/j.tetlet.2016.08.084

Fungal mediated kinetic resolution of seven acyclic/aromatic acetates was achieved using Fusarium proliferatum to furnish (R)-alcohols in high enantiomeric excess (>95%). The kinetic resolution was established as one-pot two-step de-esterification/oxidation biocatalytic process. Further, the preparative scale synthesis of (R)-(+)-1-phenylethanol was accomplished through de-esterification/oxidation of (±)-1-phenylethyl acetate using the whole cell of F. proliferatum NCIM 1105.

Full text of DOI:10.1016/j.tetlet.2016.08.084

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Tetrahedron Letters 57 (2016) 4563–4567
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Fungal mediated kinetic resolution of racemic acetates
to (R)-alcohols using Fusarium proliferatum
Dipesh D. Jadhav a, Harshal S. Patil a, Patil S. Chaya a, Hirekodathakallu V. Thulasiram a,b,
⇑
a Chemical Biology Unit, Division of Organic Chemistry, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune 411008, India
b CSIR-Institute of Genomics and Integrative Biology, Mall Road, New Delhi 110007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Fungal mediated kinetic resolution of seven acyclic/aromatic acetates was achieved using Fusarium
proliferatum to furnish (R)-alcohols in high enantiomeric excess (>95%). The kinetic resolution was
established as one-pot two-step de-esterification/oxidation biocatalytic process. Further, the preparative
scale synthesis of (R)-(+)-1-phenylethanol was accomplished through de-esterification/oxidation of
( )-1-phenylethyl acetate using the whole cell of F. proliferatum NCIM 1105.
Received 5 August 2016
Revised 25 August 2016
Accepted 29 August 2016
Available online 30 August 2016
Ó 2016 Elsevier Ltd. All rights reserved.
Keywords:
Microorganism
Biocatalysis
Kinetic resolution
(R)-Alcohol
Acetate
Introduction
alcohols using lipases, esterases, and alcohol dehydrogenases have
been reported in the literature.9–28 For example, resolution of ( )-
Enantiomerically pure low molecular weight alcohols and their
corresponding acetates are widely useful chemicals in industry as
well as academia. They are important ingredients in the perfumery
industry due to their volatility and unique odor.1–3 Furthermore,
strategic utilization of these molecules as the chiral precursors
for the asymmetric syntheses of complex organic molecules is
well-known.4,5 For example, acyclic alcohols such as (R)-(À)-lavan-
dulol (1a) exist naturally as one of the major components of the
lavender essential oil and is used in perfumery and cosmetic indus-
try. (R)-(À)-Lavandulyl propionate has been investigated as the sex
pheromone in mealy bug, whereas its (S)-enantiomer was found to
be inactive (Fig. 1).6 Both the enantiomers of 2-hexyl acetate (2)
and 2-heptyl acetate (3) along with their corresponding alcohols
are used in flavor and fragrance industry. On the other hand, (R)-
(À)-2-hexanol (2a) and (S)-(+)-2-hexanol (2b) were used in the
preparation of key intermediates in the total synthesis of anti-viral
glycolipid cycloviracin B1.7 (R)-(À)-2-Heptanol (3a) was utilized in
resolving the racemic mixture of a key intermediate in the synthe-
sis of 2,3,4,5-tetrahydro-1H-1-benzdiazepine derivatives, known
to be a strong vasopressin V2 receptor agonist.8
lavandulol has been achieved previously with various lipases such
as Candida antarctica lipase B (CAL B),9 Hog pancreas lipase,29 Porcine
pancreas lipase,26 and Yarrowia lipolytica lipases.30 However,
enzyme catalyzed resolution process is associated with several
disadvantages such as higher cost, low substrate concentration,
instability and cofactor dependency of the enzymes in several
occasions. On the other hand, whole cell biocatalysis offers an inex-
pensive choice in which enzymes are stable within cellular environ-
ment and the microbial cells themselves act as the source of
cofactors for the biocatalyst mediated conversion.31–33 The present
study describes one-pot two-step de-esterification followed by
selective (S)-enantiomer oxidation of seven acyclic and aromatic
acetates using Fusarium proliferatum (National Collection of Indus-
trial Microorganisms/NCIM, catalog no. 1105). (R)-Alcohols pro-
duced using whole cells of Fusarium proliferatum showed >95% of
ee. Substrate concentration and incubation time were optimized
and the efficiency of resolution process [enantiomeric excess (ee)]
was evaluated for each of these substrates. Furthermore, prepara-
tive scale resolution was successfully achieved on ( )-1-pheny-
lethyl acetate (6) to isolate (R)-(+)-1-phenylethanol (6a).
Resolution of the racemic mixture of these alcohols/acetates is
highly challenging and available chromatographic techniques are
ineffective to achieve the desired resolution, especially in prepara-
tive scale separation. Enzyme mediated kinetic resolution of such
Results and discussion
The fungal system, Fusarium proliferatum (NCIM, Catalog no.
1105) was screened for its ability to convert racemic acetates into
corresponding (R)-alcohols in efficient manner. ( )-Lavandulyl
⇑
Corresponding author. Tel.: +91 2025902478; fax: +91 2025902636.
E-mail address: hv.thulasiram@ncl.res.in (H.V. Thulasiram).
http://dx.doi.org/10.1016/j.tetlet.2016.08.084
0040-4039/Ó 2016 Elsevier Ltd. All rights reserved.

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acetate (1), an acyclic racemic ester was used as the model sub-
strate for the screening of fungal mediated kinetic resolution of
esters. Fermentation procedures were carried out as reported ear-
lier.31,34 Among various fungal systems screened for hydrolytic
kinetic resolution of (1), Fusarium proliferatum was able to convert
it to (R)-(À)-lavandulol (1a) in an efficient manner with very high
ee. Biocatalytic resolution was validated with two independent
control experiments; substrate control (with substrate and
without organism), and organism control (with organism and
without substrate). Both the control experiments did not show
any evidence for the formation of (R)-(À)-lavandulol (1a) as ana-
lyzed by GC-FID and GC–MS (see ESI).34 In addition, the resolution
achieved through the resting cells of F. proliferatum confirmed that
the enzyme seems to be constitutive to the whole-cell used. Sub-
strate concentration 0.6 g LÀ1 was found to be optimum to achieve
the highest ee. Use of higher concentration of substrate led to the
lower ee and conversion rate. After 3 days of incubation, ( )-lavan-
dulyl acetate (1) was converted to (R)-(À)-lavandulol (1a) with
95.3% conversion and 99.6% ee. Time-course experiments (Fig. 2A)
indicated that F. proliferatum was able to transform almost 95% of
(1) in to (R)-lavandulol (1a) and (S)-lavandulol (1b) (R/S 45.9:49.4)
after 12 h incubation. With prolonged incubation up to 3 days, the
relative abundance of (R)-lavandulol (1a) increased to 99.8% and
Figure 1. Kinetic resolution of ( )-lavandulyl acetate to (R)-(À)-lavandulol by
F. proliferatum.
Figure 2. Time-course experiment of kinetic resolution using F. proliferatum in graphical representation: (A) Whole cells incubated with ( )-lavandulyl acetate at 0.6 g LÀ1
concentration; (B–I) resting cells incubated with ( )-lavandulyl acetate (B), ( )-2-hexyl acetate (C), ( )-2-heptyl acetate (D), ( )-3-hexyl acetate (E), ( )-1-octen-3-yl acetate
(F), ( )-1-phenylethyl acetate (G), ( )-3-methyl-1-phenylethyl acetate (H), ( )-1-phenylpropyl acetate (I) respectively with 0.1 g LÀ1 substrate concentration.

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D. D. Jadhav et al. / Tetrahedron Letters 57 (2016) 4563–4567
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Table 1
Hydrolytic kinetic resolution achieved on various racemic acyclic/aromatic acetates through resting cells of F. proliferatum
Entry
Substratea
Time (h)
Product
Configuration and % ee
1
24
R, 96.8
R, 99.9
2
6
3
4
5
6
R, 99.9
R, 62.5
R, 99.9
12
8
6
7
12
48
R, 97.5
0
8
9
36
24
R, 99.9
R, 98.2
a
Substrate concentration: 0.1 g LÀ1 and substrate conversion: 100% for each entry.
the percentage of (S)-lavandulol (1b) steeply diminished to 0.18%
in the fermentation broth. Continuing the incubation up to 5 days
didn’t alter the relative abundance of the individual enantiomer in
the reaction mixture. Resting cell experiments were carried out as
reported earlier31 by incubating well washed F. proliferatum with
0.1 g LÀ1 concentration of ( )-lavandulyl acetate (1). Time-course
experiment with the resting cells revealed that after 6 h of incuba-
tion, the abundance of (R)-lavandulol (1a) starts to increase in the
fermentation broth, further reaching up to 94.4% and 96.8% ee at
24 h of incubation with quantitative consumption of the acetate
(Fig. 2B). To investigate the substrate scope of the whole cell bio-
catalyst four racemic acyclic esters [( )-2-hexyl acetate (2), ( )-2-
heptyl acetate (3), ( )-3-hexyl acetate (4), and ( )-1-octen-3-yl
acetate (5)] and four aromatic esters [( )-1-phenylethyl acetate
(6), ( )-2-methyl-1-phenylethyl acetate (7), ( )-3-methyl-1-phe-
nylethyl acetate (8) and ( )-1-phenylpropyl acetate (9)] were cho-
sen as the substrates (Table 1). The kinetic resolution of these
substrates was assessed by incubating it with resting cells of F. pro-
liferatum and carrying out time course study (Fig. 2C–I). Resting
cell experiments were carried out with acyclic esters [(2), (3), (4),
and (5)] at substrate concentration of 0.1 g LÀ1 for different time
interval of incubation. Esters (2) and (3) yielded enantiomerically
pure (R)-alcohols (2a) and (3a) with 100% conversion and 99.9%
ee respectively after 6 h of incubation (Fig. 2C and D).
experiments indicated that the fungal systems converted ( )-1-
phenylethyl acetate (6) and ( )-3-methyl-1-phenylethyl acetate
(8) to (R)-(+)-1-phenyl ethanol (6a) and (R)-(+)-3-methyl-1-phenyl
ethanol (8a) in quantitative yields with 100% conversion and 99.9%
ee at the end of incubation periods 12 h and 36 h respectively
(Fig. 2G and H).There was no enantioselectivity observed with aro-
matic ester (7) with whole cells of F. proliferatum, which may be
due to presence of 2C-methyl group on phenyl ring of the com-
pound (7). On the other hand, the fungal system efficiently con-
verted the acetate ( )-1-phenylpropyl acetate (9) to (R)-(+)-1-
phenylpropanol (9a) with 100% conversion and 98.2% ee after
24 h of incubation in the resting cell experiment (Fig. 2I).
Time-course study experiments have indicated that during
initial period of incubation racemic acetates were converted into
individual enantiomeric alcohols at the same rate and accumula-
tion of (R)-alcohols with decrease in (S)-alcohols levels in the assay
mixture was observed with continued incubation (Fig. 2). Careful
analysis of the GC and GC–MS chromatograms (see ESI)34 of the
extracted fermentation broth revealed the presence of correspond-
ing ketones in case of all seven substrates [(2), (3), (4), (5), (6), (8),
and (9)] during later stage of incubation (Fig. 3B). These results
indicate that there is a selective oxidation of (S)-alcohol (see ESI)
to the corresponding ketone with faster rate in comparison to
(R)-alcohol, leading to kinetic resolution with high ee (Fig. 3A). To
further substantiate this pathway of hydrolytic kinetic resolution,
racemic mixture of alcohols was incubated with F. proliferatum
which resulted in decrease in the relative abundance of (S)-alcohol
in the reaction mixture along with the formation of corresponding
prochiral ketone. Similar case studies were performed using
individual (R) and (S)-alcohol with F. proliferatum, which showed
While esters (4) and (5) were converted into corresponding (R)-
alcohols (4a) and (5a) with 100% conversion and 62.5%, 99.9% ee at
the end of 12 h and 8 h of incubation period respectively
(Fig. 2E and F). Similar experiments were performed with the race-
mic aromatic esters [(6), (7), (8), and (9)] using resting cells of F.
proliferatum at the concentration of 0.1 g LÀ1. Time course study

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D. D. Jadhav et al. / Tetrahedron Letters 57 (2016) 4563–4567
Thus, the fungal system, F. proliferatum can be used for large-
scale production of corresponding (R)-alcohols through kinetic res-
olution of acyclic and aromatic acetates with fine tuning of the fer-
mentation conditions.
Conclusion
In conclusion, an efficient one-pot two step de-esterification/
oxidation biocatalytic technique was developed for the kinetic res-
olution of acyclic and aromatic acetates by using the whole-cells of
Fusarium proliferatum. The fungal system was able to carry out the
kinetic resolution of four racemic acyclic esters [( )-lavandulyl
acetate (1), ( )-2-hexyl acetate (2), ( )-2-heptyl acetate (3) and
( )-1-octen-3-yl acetate (5)] and three aromatic esters [( )-1-phe-
nylethyl acetate (6), ( )-3-methyl-1-phenylethyl acetate (8) and
( )-1-phenylpropyl acetate (9)] into corresponding (R)-alcohols in
an efficient manner with high ee. Enantioselective hydrolysis of
( )-1-phenylethyl acetate (6) to (R)-(+)-1-phenylethanol (6a) was
successfully scaled up to preparative scale, which indicated great
potential of the developed process to be applied in large scale
preparation of enantiopure (R)-alcohols.
Acknowledgement
D.D.J. and H.S.P. acknowledges ICMR and CSIR, New Delhi, India
for their research fellowship. We thank Mr. G. Nagesh Reddy for
helping in synthesis. This work is supported by CSIR-New Delhi,
India sponsored network project (CSC0106 and CSC0130).
Figure 3. (A) Schematic representation of de-esterification/oxidation one-pot two-
step kinetic resolution of acyclic/aromatic acetates by F. proliferatum; (B) GC-FID
chromatograms of (i) extracted reaction mixture of ( )-2-hexyl acetate with resting
cell of F. proliferatum after 2 h of incubation period, (ii) standard 2-hexanone (iii)
standard (S)-(+)-2-hexanol, (iv) standard (R)-(À)-2-hexanol, (v) standard (S)-(+)-2-
hexyl acetate and (vi) standard (R)-(À)-2-hexyl acetate.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2016.08.
084.
References and notes
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that the microorganism could transform (6) in to (R)-(+)-1-pheny-
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Increase in the substrate concentration decreased the level of
metabolite formation as well as enantiomeric excess (ee). Time
course experiments indicate that F. proliferatum could transform
100% of (6) into (6a) at the end of 3 days of incubation period.
The fermentation volume was scaled up to 1.0 L with substrate
concentration 0.4 g LÀ1 34
Purification of the alcohol fraction from
.
1.0 L fermentation medium containing 0.4 g racemic acetate (6)
resulted in the isolation of 0.14 g pure (R)-(+)-1-phenylethanol
(6a) with a yield of 47% and 99.9% ee. Purified (R)-(+)-1-pheny-
lethanol (6a) was further characterized by analytical techniques
such as NMR (1H, 13C) and optical rotation. (R)-1-Phenylethanol:
colorless liquid; [a]
25 = +36.2. 1H NMR (CDCl3, 200 MHz) d: 7.25
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