Resolution of racemic 1-arylethyl acetates by Pseudomonas fluorescens in the presence of a surfactant

Article abstract of DOI:10.1016/j.tetlet.2003.10.045

The Pseudomonas fluorescens RRLJ 134 strain efficiently resolved (R/S)-1-arylethyl acetates into S-(-)-1-arylethyl acetates and R-(+)-1-arylethanol in the presence of the surfactant Tween-8. The surfactant reverses the enantioselectivity compared to the surfactant free medium.

Full text of DOI:10.1016/j.tetlet.2003.10.045

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 9099–9102
Resolution of racemic 1-arylethyl acetates by Pseudomonas
fluorescens in the presence of a surfactant
a
a
a
b
b
a,
U. Bora, C. J. Saikia, A. Chetia, A. K. Mishra, B. S. D. Kumar and R. C. Boruah *
a
Medicinal Chemistry Division, Regional Research Laboratory, Jorhat 785006, India
Soil Microbiology Division, Regional Research Laboratory, Jorhat 785006, India
b
Received 2 September 2003; revised 29 September 2003; accepted 10 October 2003
Abstract—The Pseudomonas fluorescens RRLJ 134 strain efficiently resolved (R/S)-1-arylethyl acetates into S-(−)-1-arylethyl
acetates and R-(+)-1-arylethanol in the presence of the surfactant Tween-80. The surfactant reverses the enantioselectivity
compared to the surfactant free medium.
©
2003 Elsevier Ltd. All rights reserved.
The preparation of enantiomerically pure substrates
using bio-catalysis has received considerable attention
in recent years because of significant demand for opti-
strategies in bio-organic transformations. To improve
stereoselectivity, different methods have been developed
by modification of the whole-cell reaction conditions
1
4
cally pure compounds. Indeed, the bio-catalytic resolu-
such as treatment of microbial cells with additives,
5
tion of racemates is now recognised as a valuable tool
addition of an inhibitor to specific enzymes and the use
2
6
for the synthesis of optically active compounds. Two
of hydrophobic polymers. Although these methods are
approaches have been used for bio-catalytic resolu-
tions—one involves the use of isolated enzymes and the
other involves the use of microbial whole-cells. In com-
parison to enzymatic reactions, the whole-cell reactions
usually afford poor enantioselectivity as several
enzymes in the cell can participate in the reaction with
one enzyme affording the desired enantiomer while
effective, many reactions are unsuitable for these types
of stereochemical control and consequently new strate-
gies to increase the stereoselectivity of the microbial
whole-cell reactions are still desirable.
There are several examples available for enzyme or
7
whole-cell catalysed resolution of arylpropionic acids.
3
another enzyme may produce its antipode. However,
Further, the complete reversal of enantioselectivity of
an enzyme catalysed reaction by directed evolution
using error prone PCR and DNA shuffling has recently
because of the cost effectiveness, availability, scope for
discovery of newer lipase activities and ease of han-
dling, the whole-cell reactions continue to be preferred
8
gained importance. Also, the effect of the surfactant
*
0
Corresponding author. Tel.: +91 376 2370327; fax: +91 376 2370011; e-mail: rc boruah@yahoo.co.in
–
040-4039/$ - see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2003.10.045

9
100
U. Bora et al. / Tetrahedron Letters 44 (2003) 9099–9102
a
Table 1. The optical resolution of (R/S)-1–4 by P. fluorescens RRLJ 134
Entry
Acetate
Surfactant (%)
Conv. (%)b
Substrate
Yieldc
Product
Yieldc
Ed
Ee (%)
Ee (%)
1
2
3
4
5
6
7
8
1
1
2
2
3
3
4
4
Nil
0.01
Nil
0.01
Nil
0.01
Nil
60
53
59
55
56
56
55
60
(R)-1
(S)-1
(R)-2
(S)-2
(R)-3
(S)-3
(R)-4
(S)-4
38
33
42
34
41
45
35
41
30
95
22
99
23
28
20
25
(S)-5
(R)-5
(S)-6
(R)-6
(S)-7
(R)-7
(S)-8
(R)-8
33
38
38
30
28
34
27
38
20
80
15
80
18
22
16
20
1.9
43
1.6
49
1.7
2
2.2
1.9
0.01
a
Reaction time=36 h.
b
c
Conversions were deduced from the ees of the substrate (ee ) and the product (ee ): c=ee /(ee +ee ).
Calculated taking into account the percentage of conversion.
Enantiomeric ratio calculated according to Sih et al.
s
p
s
s
p
d
16
2
5
Tween-80 on enantioselective hydrolysis of propionic
R-(+)-5, [h] =+52 (c 1.4, CHCl ) were obtained with
14
D
3
esters by crude or purified lipases as well as whole-cell
reversal of enantioselectivity (Table 1, entry 2). The
enantiomeric ratio E, which reflects the relative reaction
rate of the two enantiomers, turned out to be 43 in
comparison to the surfactant-free medium (E=1.9).
Similarly (R/S)-2 underwent enantioselective deracem-
ization in the presence of Tween-80 to afford S-(−)-2 and
R-(+)-6 in high enantiomeric excess. The enantiomeric
ratio being E=49 in comparison to the surfactant-free
medium where E=1.6 (Table 1, entries 3–4). However,
the hydrolytic resolution of both (R/S)-3 and (R/S)-4
with surfactant or surfactant-free conditions afforded the
products in poor enantioselectivities (Table 1, entries
9
performance is documented. To the best of our knowl-
edge such a study on the kinetic resolution of an
1-arylethyl acetate is rare.
The fluorescent Pseudomonads are well known bacterial
1
0
biocontrol agents and are involved in various bio-trans-
1
1
formation reactions. Recently we have demonstrated
that the whole-cell-catalysed reaction of P. fluorescens
RRLJ 134 can be successfully applied to the hydrolysis
1
2
of acetates into symmetrical alcohols. Herein, we report
the use of P. fluorescens RRLJ 134 in the hydrolytic
kinetic resolution of (R/S)-1-arylethyl acetates with a
slight preference for the S-alcohol. We further demon-
strate that the enantioselectivity of the bio-catalyst can
be completely reversed and significantly enhanced by
using the surfactant Tween-80.
5
–8).
It was further observed that the enzyme activity was
influenced by surfactant concentration. When the
hydrolyses of (R/S)-(1–2) were carried at a concentration
of 10 mg/100 ml (0.01%) of the nutrient broth, the
The kinetic resolution of (R/S)-1-phenylethyl acetate 1
13
with P. fluorescens RRLJ 134 in succinate media at pH
2
5
7
1
1
.0 afforded 1-phenylethyl acetate R-(+)-1, [h] =+25 (c
D
25
D
.5, CHCl ) and 1-phenylethanol S-(−)-5, [h] =−14 (c
3
.4, CHCl ) in poor enantioselectivity (Table 1, entry 1).
3
The result suggested that the resolution of (R/S)-1 by P.
fluorescens was conducted by more than one enzyme,
wherein one enzyme might hydrolyse the substrate to
S-(−)-5, while another afforded the antipode on
hydrolytic resolution.
The enhancing effect of the surfactant Tween-80 on
lipase performance as well as whole-cell bacteria in the9
enantioselective hydrolysis of arylpropionic esters
prompted us to test the effect of the surfactant on the
resolution reaction of (R/S)-1 using whole-cell P. fluores-
cens RRLJ 134. We assumed that the relative activities
of the enzymes that conduct hydrolytic resolution might
be enhanced in the presence of the surfactant in compari-
son to the surfactant-free medium.
Thus, when the whole-cell P. fluorescens RRLJ 134 was
incubated at 30°C with (R/S)-1 in the presence of the
surfactant Tween-80 (10 mg/100 ml of nutrient broth),
Figure 1. The effect of the surfactant Tween-80 on the enan-
tioselective resolution of (R/S)-1–2 by P. fluorescens RRLJ 134.
25
the acetate S-(−)-1, [h] =−86 (c 0.5, CHCl ) and alcohol
D
3

U. Bora et al. / Tetrahedron Letters 44 (2003) 9099–9102
9101
enantiomeric excesses (ee) of S-(−)-1–2 and R-(+)-5–6
2000, 11, 4263; (f) Dehli, J. R.; Gotor, V. Tetrahedron:
Asymmetry 2001, 12, 1485; (g) Yadav, J. S.; Reddy, P. T.;
Nanda, S.; Rao, A. B. Tetrahedron: Asymmetry 2001, 12,
3381; (h) Mukherjee, S.; Prasad, A. K.; Parmar, V. S.;
Howarth, O. W. Bioorg. Med. Chem. Lett. 2001, 11,
2117; (i) Poonam; Prasad, A. K.; Azim, A.; Kumar, R.;
Jain, S. C.; Parmar, V. S.; Olsen, C. E.; Errington, W.
Tetrahedron 2001, 57, 7395.
15
were significantly enhanced (95–99%, and 80%). How-
ever, changing the concentration of the surfactant to 5
mg/100 ml or 20 mg/100 ml (0.005 or 0.02%) led to a
decrease in enantiomeric excesses (ee) of S-(−)-1–2 and
R-(+)-5–6 (Fig. 1).
For the determination of the absolute configuration of
the resolved products, products S-(−)-1–4 were
hydrolysed into S-(−)-5–8 with methanolic KOH at
room temperature. The S-configurations of the alcohols
2. (a) Fantin, G.; Fogagnolo, M.; Guerrini, A.; Medici, A.;
Pedrini, P.; Fontana, S. Tetrahedron: Asymmetry 2001,
12, 2709; (b) Bakker, M.; Spruijt, A. S.; Rantwijk, F. V.;
Sheldon, R. A. Tetrahedron: Asymmetry 2000, 11, 1801;
(c) Liu, H. L.; Hoff, B. H.; Anthonsen, T. J. Chem. Soc.,
Perkin Trans. 1 2000, 1767; (d) Nakamura, K.; Fujii, M.;
Ida, Y. Tetrahedron: Asymmetry 2001, 12, 3147; (e)
Kamal, A.; Khanna, G. B. R. Tetrahedron: Asymmetry
2001, 12, 405; (f) Yadav, J. S.; Nanda, S. Tetrahedron:
Asymmetry 2001, 12, 3223; (g) Nair, M. S.; Joly, S.
Tetrahedron: Asymmetry 2000, 12, 2049; (h) Singh, S.;
Kumar, S.; Chimni, S. S. Tetrahedron: Asymmetry 2001,
12, 2457; (i) Lo, L. C.; Shie, J. J.; Chou, T. C. J. Org.
Chem. 2002, 67, 282; (j) Idogaki, H.; Kasai, N.; Takeuchi,
M.; Hatada, M.; Suzuki, T. Tetrahedron: Asymmetry
2001, 12, 369.
5
–8 were assigned by comparisons of their specific
17
rotations with literature data.
Thus we have found that the surfactant Tween-80 can
reverse the enantioselectivity and enhance the reactivity
of the P. fluorescens lipase that catalyses the resolution
of (R/S)-1–2 to S-(−)-1–2 and R-(+)-5–6. The reversal
of enantioselectivities could be due to an inhibitory
influence of the surfactant Tween-80 on S-selective
enzymes in preference to R-selective enzymes during
hydrolysis. Further, the enantioselectivity is dependent
on the surfactant concentration as well as the nature of
the aryl substituents. An electron donating substituent
on the aryl moiety favours enantioselectivity whereas
an electron withdrawing group reduces the degree of
resolution. The poor enantioselective resolution of sub-
strates (R/S)-3–4 could probably be due to the unfa-
vourable electron deficient situation at the binding site
of the enzyme-substrate due to the electron withdraw-
ing group at the p-position of the aryl substrates.
3. Nakamura, K.; Yamanaka, R. Tetrahedron: Asymmetry
2002, 13, 2529.
4. (a) Ghanem, A.; Schurig, V. Tetrahedron: Asymmetry
2001, 12, 2761; (b) Nakamura, K.; Takenaka, K. Tetra-
hedron: Asymmetry 2002, 13, 415.
5. Nakamura, K.; Kawai, Y.; Nakajima, N.; Ohno, A. J.
Org. Chem. 1991, 56, 4778.
6
. (a) Nakamura, K.; Inoue, Y.; Matsuda, T.; Misawa, I. J.
Chem. Soc., Perkin Trans. 1 1999, 2397; (b) Nakamura,
K.; Kondo, S.; Nakajima, N.; Ohno, A. Tetrahedron
1995, 51, 687.
7. (a) Kumar, I.; Manju, K.; Jolly, R. S. Tetrahedron:
Asymmetry 2001, 12, 1431; (b) Kato, D.; Mitsuda, S.;
Ohta, H. Org. Lett. 2002, 4, 371; (c) Menzal, S.; Waibel,
R.; Brune, K.; Geisslinger, G. Biochem. Pharmacol. 1994,
48, 1056; (d) Sheih, W.-R.; Chen, C.-S. J. Biol. Chem.
1993, 268, 3487; (e) Rhys-Williams, W.; Thomason, M.
J.; Hung, Y.-F.; Hanolon, G. W. Pharm. Sci. 1996, 2,
In conclusion, we have reported the first example of the
kinetic resolution of racemates with reversal of enan-
tioselectivity using a microorganism in the presence of a
surfactant in comparison with the surfactant-free
medium. Furthermore, a p-substituent on the aryl
group influences the enantioselectivities of the products
thus providing an example for the electronic effect of
18
the substituent on the aromatic ring towards the
binding site of the enzymes.
537.
Acknowledgements
8. (a) Zha, D.; Wilensek, S.; Hermes, M.; Jaeger, K. E.;
Reetz, M. T. Chem. Commun. 2001, 2664; (b) Reetz, M.
T.; Wilensek, S.; Zha, D.; Jaeger, K. E. Angew. Chem.,
Int. Ed. 2001, 40, 3589.
9. (a) Xu, S.; Cao, G.; Xu, Q.; Wang, W.; Zho, J.
Weishengwu Xuebao 1995, 35, 190; Chem. Abstr. 1995,
123, 79240t; (b) Liu, Y.-Y.; Xu, J. H.; Hu, Y. J. Mol.
Cat. B: Enzym. 2000, 10, 523.
We thank Dr. P. G. Rao, Director of Regional
Research laboratory, Jorhat for providing laboratory
facilities to carry out this work. Also the support of the
Department of Science and Technology (DST), New
Delhi is gratefully acknowledged.
1
0. (a) Exploitation of Microorganism; Jones, D. J., Ed.;
Chapman & Hall: London, 1993; p. 49; (b) Kumar, B. S.
D.; Berggren, I.; Martensson, A. M. Plant and Soil 2001,
References
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. (a) Sehgal, A.; Kelly, R. M. J. Am. Chem. Soc. 2002, 124,
11. (a) Torimura, M.; Yoshida, H.; Kano, K.; Ikeda, T.;
Nagasawa, T.; Ueda, T. Chem. Lett. 1998, 295; (b)
Soeda, Y.; Toshima, K.; Matsumura, S. Chem. Lett.
2001, 76; (c) Ghanem, A.; Schurig, V. Tetrahedron:
Asymmetry 2001, 12, 2761.
12. Bora, U.; Longchar, M.; Chetia, A.; Kumar, B. S. D.;
Boruah, R. C.; Sandhu, J. S. Tetrahedron Lett. 2002, 43,
2269.
8
190 and references cited therein; (b) Park, S.;
Kazlauckas, R. J. J. Org. Chem. 2001, 66, 8395; (c) Perez,
H. I.; Luna, H.; Manjarrez, N.; Solis, A. Tetrahedron:
Asymmetry 2001, 12, 1709; (d) Gefflaut, T.; Martin, C.;
Delor, S.; Besse, P.; Veschanmbre, H.; Bolte, J. J. Org.
Chem. 2001, 66, 2296; (e) Perez, H. I.; Luna, H.; Manjar-
rez, N.; Solis, A.; Nunez, M. A. Tetrahedron: Asymmetry

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102
U. Bora et al. / Tetrahedron Letters 44 (2003) 9099–9102
13. (a) King, E. O.; Wood, M. K.; Raney, D. E. J. Lab. Clin.
chromatography to afford S-(−)-5, yield 0.03 g (80%),
Med. 1954, 44, 301; (b) Meyer, J. M.; Abdallah, M. A. J.
Gen. Microbiol. 1978, 107, 319; (c) Plant Pathologist’s
Book, 1968, Commonwealth Mycological Institute, Sur-
rey, KEW, UK, p. 267.
R =0.35 (hexane/ethyl acetate=90/10); IR (KBr) 3490
f
−
1 1
cm ; H NMR (300 MHz, CDCl ) l 6.70–7.00 (m, 5H,
3
aromatic protons), 4.40 (q, 1H, J=6.5 Hz, methine pro-
ton), 3.50 (bs, 1H, hydroxyl proton), 1.18 (d, 3H, J=7.0
+
1
4. Typical experimental procedure: To 100 ml of succinate
media in a 250 ml Erlenmeyer flask were added (R/S)-1-
phenylethyl acetate 1 (0.046 g, 0.278 mM) in methanol (2
ml), Tween-80 [polyoxyethylene(20) sorbitan monooleate,
Hz, methyl protons); MS (ESI) m/z 122 (M ); ee 94%;
2
5
17b
27
[
h] =−60.2 (c 1.4, CHCl ); [lit.,
[h] =−66.5 (c 1.4,
D
3
D
CHCl )].
3
S-(−)-2, yield 34%, R =0.65 (hexane/ethyl acetate=90/
f
1
0 mg] and P. fluorescens RRLJ 134 strain (0.5 ml) grown
−1
+
1
(
0); IR (KBr) 1730 cm ; MS (ESI) m/z 178 (M ), 136
in King’s B medium. The reaction was incubated (30°C)
at 120 rpm rotation for 36 h with shaking. The resulting
mixture was extracted with ethyl acetate, washed with
water, dried over anhydrous sodium sulfate. Removal of
the solvent and separation of the products by preparative
silica gel TLC afforded S-(−)-1-phenylethyl acetate S-(−)-
+
25
D 3
M −42); ee 99%; [h] =−49 (c 0.5, CHCl ). R-(+)-6:
yield 30%, R =0.35 (hexane/ethyl acetate=90/10); IR
f
−1
+
(
[
KBr) 3495 cm ; MS (ESI) m/z 136 (M ); ee 77%;
25
D D
17c
27
h] =+32 (neat); [lit., [h] =+56 (neat)].
S-(−)-3, yield 45%, R =0.70 (hexane/ethyl acetate=90/
f
−1
+
1
2
0
0); IR (KBr) 1730 cm ; MS (ESI) m/z 198 (M ); ee
8%; [h] =−15 (c 0.5, CHCl ). R-(+)-7: yield 34%, R =
.30 (hexane/ethyl acetate=90/10); IR (KBr) 3495 cm ;
MS (ESI) m/z 156 (M ); ee 22%; [h] =+12 (c 0.5,
1
and R-(+)-1-phenylethanol R-(+)-5. S-(−)-1, yield 33%,
25
D
3
f
−1
R =0.6 (hexane/ethyl acetate=90/10); IR (KBr) 1730
f
−
1
1
cm ; H NMR (300 MHz, CDCl ) l 6.8–7.1 (m, 5H,
+
25
D
3
aromatic protons), 5.58 (q, 1H, J=7.0 Hz, methine pro-
ton), 1.75 (s, 3H, methyl protons), 1.28 (d, 3H, J=7.0
CHCl3).
S-(−)-4, yield 41%, R =0.60 (hexane/ethyl acetate=90/
+
+
f
Hz, methyl protons); MS (ESI) m/z 164 (M ), 122 (M −
−
1
1
(
0); IR (KBr) 1730, 1535, 1348 cm ; MS (ESI) m/z 209
25
4
3
3
5
2); ee 95%; [h] =−86 (c 0.5, CHCl ). R-(+)-5: yield
8%, R =0.35 (hexane/ethyl acetate=90/10); IR (KBr)
490 cm ; H NMR (300 MHz, CDCl ) l 6.70–7.00 (m,
H, aromatic protons), 4.40 (q, 1H, J=6.5 Hz, methine
D 3
+
25
D 3
M ); ee 25%; [h] =−15 (c 0.5, CHCl ). R-(+)-8: yield
f
−
1
1
38%, R =0.30 (hexane/ethyl acetate=90/10); IR (KBr)
f
3
−1
+
25
3
495 cm ; MS (ESI) m/z 167 (M ); ee 20%; [h] =+20 (c
D
0
.5, CHCl3).
proton), 3.55 (bs, 1H, hydroxyl proton), 1.18 (d, 3H,
+
15. Enantiomeric excesses (ees) were determined by HPLC
analysis with a chiral cell OD column.
6. Chen, C. S.; Fugimoto, Y.; Girdaukas, G.; Sih, C. J. J.
Am. Chem. Soc. 1982, 104, 7294.
7. (a) Ley, S. V.; Parra, M.; Redgrave, A. J.; Sternfeld, F.
Tetrahedron 1990, 46, 4995; (b) Kamal, A.; Sandbhor,
M.; Ramana, K. V. Tetrahedron: Asymmetry 2002, 13,
J=7.0 Hz, methyl protons); MS (ESI) m/z 122 (M ); ee
2
5
17a
25
8
0%; [h] =+52 (c 1.4, CHCl ) [lit.,
[h] =+41.5
D
3
D
1
(neat)].
Hydrolysis of S-(−)-1 to 1-phenylethanol S-(−)-5
To a solution of S-(−)-1 (0.05 g, 0.3 mM) in methanol (2
ml) was added a methanolic solution of KOH (0.3 ml,
1
1
0%) dropwise and the reaction mixture was stirred for 2
815; (c) Dictionary of Organic Compounds, 6th Ed.; Cado-
h at room temperature. It was then poured into water (50
ml), extracted with dichloromethane (2×5 ml) and dried
over sodium sulfate. Removal of the solvent afforded a
liquid product, which was purified by silica gel column
gan, J. I. G.; Ley, S. V.; Patterson, G., Eds.; Chapman &
Hall: London, 1996; Vol. 5.
18. Kato, D.; Mitsuda, S.; Ohta, H. Org. Lett. 2002, 4, 371.