Highly efficient extractive biocatalysis in the asymmetric reduction of an acyclic enone by the yeast Pichia stipitis

Article abstract of DOI:10.1016/S0957-4166(02)00798-X

A concise highly chemo- and enantioselective preparation of (S)-2-ethyl-1-phenylprop-2-en-1-ol 6 (65% yield, >99% e.e.) via extractive biocatalysis by the yeast Pichia stipitis CCT 2617-mediated reduction of 2-ethyl-1-phenylprop-2-en-1-one 5 adsorbed on Amberlite XAD-7 is reported.

Full text of DOI:10.1016/S0957-4166(02)00798-X

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 14 (2003) 43–45
Highly efficient extractive biocatalysis in the asymmetric
reduction of an acyclic enone by the yeast Pichia stipitis
Gelson J. Andrade Conceic¸a˜o, Paulo J. S. Moran and J. Augusto R. Rodrigues*
Universidade Estadual de Campinas, Instituto de Qu´ımica, CP 6154, 13083-970, Campinas-SP, Brazil
Received 23 August 2002; accepted 26 September 2002
Abstract—A concise highly chemo- and enantioselective preparation of (S)-2-ethyl-1-phenylprop-2-en-1-ol 6 (65% yield, >99%
e.e.) via extractive biocatalysis by the yeast Pichia stipitis CCT 2617-mediated reduction of 2-ethyl-1-phenylprop-2-en-1-one 5
adsorbed on Amberlite™ XAD-7 is reported. © 2003 Published by Elsevier Science Ltd.
Enantiomerically pure allylic alcohols are valuable syn-
thetic intermediates and many useful stereoselective
transformations can be performed by taking advantage
of the 1,2- or 1,3-allylic strain in such systems.1 Most
syntheses of chiral allylic alcohols rely on the resolution
of racemic alcohols by Sharpless’ asymmetric
epoxidation2 or enzyme-mediated processes.3 As such,
known examples of efficient chemo- and enantioselec-
tive reductions of a-methyleneketones 1 (Scheme 1) are
scarce.4
In a previous work, we reported our attempts to set up
stereoselective reductions of a large number of acyclic
a-methyleneketones using Baker’s yeast as biocatalyst.4a
It was found that substrates, such as 1 (Scheme 1) could
be unselectively reduced at the CꢀC double bond to
afford ketone 2, but the CꢁO bond was not reduced at
all. We felt that a screening of microorganisms coupled
to the modification of the reaction conditions would
afford alternatives to circumvent the drawbacks faced
with Baker’s yeast-mediated reduction of acyclic
enones.
Currently, much emphasis has been given to obtain
enantiomerically pure compounds in good yields under
environmentally friendly conditions.5 As a result, bio-
catalysis with either enzymes (crude or isolated) or
whole cells has blossomed over the past few years as a
viable alternative to costly or difficult chemistry in the
manufacture of chiral compounds.6 Biocatalysts display
wide substrate acceptance and can catalyse chemo-,
regio- and stereoselective transformations of organic
compounds under very mild and environmentally safe
reaction conditions.7
Of the non-conventional yeasts tested,8,9 the non-fer-
menting yeast Pichia stipitis CCT 2617 was able to
reduce (partially) the enone 5 (Scheme 2), to afford a
mixture of products. However, further assays (at ana-
lytical scale) pointed out that this enone was a very
toxic xenobiotic to the yeast at concentrations of >80
mg L−1, thus limiting the degree of conversion of the
substrate and the yield of the reaction.
Among the alternatives currently available to circum-
vent such limitations, we chose the strategy of
Scheme 1.
* Corresponding author. E-mail: jaugusto@iqm.unicamp.br
0957-4166/03/$ - see front matter © 2003 Published by Elsevier Science Ltd.
PII: S0957-4166(02)00798-X

44
G. J. A. Conceic¸a˜o et al. / Tetrahedron: Asymmetry 14 (2003) 43–45
Scheme 2.
adsorbing the substrate onto a hydrophobic polymeric
resin (mainly Amberlite™ XAD-type resins) as a mea-
sure to reduce and control the concentration of both
substrate and product in the aqueous phase.10
Acknowledgements
The authors are indebted to FAPESP and CNPq for
the financial support.
Enone 5 was prepared as previously reported, via Man-
nich reaction.8 Racemic allylic alcohol 6 for GC/MS
analysis was accessed by chemoselective reduction of 5
via Luche’s method.11 Enone 1/XAD-7 mix12 was
added to a slurry of P. stipitis CCT 261713 and the
reaction was performed for 3 days.14 This moderately
nonpolar resin was chosen since it has the desirable
property of roughly equal affinity for both product and
substrate, driving the reaction to completion while at
the same time extracting the product away from the
biocatalyst effectively.
References
1. Hoveyda, A. H.; Evans, D. A.; Fu, G. C. Chem. Rev.
1993, 93, 1307.
2. (a) For a review of practical issues in kinetic resolutions,
vide: (a) Keith, J. M.; Larrow, J. F.; Jacobsen, E. N. Adv.
Synth. Catal. 2001, 343, 5; (b) Katsuki, T.; Martin, V. S.
Org. React. 1996, 48, 1.
3. For a recent review on lipases in biocatalysis, vide: Reetz,
M. T. Curr. Opin. Chem. Biotechnol. 2002, 6, 145.
4. (a) Filho, E. P. S.; Rodrigues, J. A. R.; Moran, P. J. S.
Tetrahedron: Asymmetry 2001, 12, 847; (b) Filho, E. P. S.;
Rodrigues, J. A. R.; Moran, P. J. S. J. Mol. Catal. B:
Enzym. 2001, 15, 23; (c) Ferraboschi, P.; Reza-Elahi, S.;
Verza, E.; Santaniello, E. Tetrahedron: Asymmetry 1999,
10, 2639; (d) Sakai, T.; Matsumoto, S.; Hidaka, S.;
Imajo, N.; Tsuboi, S.; Utaka, M. Bull. Chem. Soc. Jpn.
1991, 64, 3473; (e) Utaka, M.; Onoue, S.; Takeda, A.
Chem. Lett. 1987, 971.
At the end of the reaction (complete consumption of 5),
the resin was filtered off and the beads were extracted
with ethyl acetate. Compound 6 was obtained as the
sole product with an improved isolated yield of 65%
besides excellent stereocontrol (>99% e.e., Scheme 2).15
Ozonolysis of 6 afforded acyloin 7 (60% yield).16 The
CD spectrum of 7 was closely related to that of
L-
phenylacetyl carbynol ( -PAC, 8) with known R
L
configuration.17 The absolute configuration of 7 was
assigned as R, and therefore alcohol 6 must bear S
configuration.
5. Fessner, W. D.; Jones, J. B. Curr. Opin. Chem. Biotech-
nol. 2001, 5, 103.
This hydrophobic polymer method is based on the fact
that an adsorbed hydrophobic organic compound is
partitioned into the solid organic phase and the
aqueous phase, the organic compound being largely in
the solid phase. Thus, the polymer acts as a reservoir
for the substrate, supplying by an equilibrium process
the mass of substrate consumed by the biocatalyst in
the aqueous phase. In turn, the product released by the
biocatalyst in the aqueous phase is continuously
extracted in situ by the same polymer preventing its
accumulation in the aqueous phase and cell surfaces
(which could cause cell death or enzyme inhibition).
6. For recent reviews on biocatalysis, vide inter alia: (a)
Koeller, K. M.; Wong, C. H. Nature 2001, 409, 232; (b)
Schmid, A.; Dordick, J. S.; Hauer, B.; Kiener, A.; Wub-
bolts, M.; Witholt, B. Nature 2001, 409, 258; (c) Thomas,
S. M.; DiCosimo, R.; Nagarajan, V. Trends Biotechnol.
2001, 20, 238; (d) De Conti, R.; Rodrigues, J. A. R.;
Moran. P. J. S. Quim. Nova 2001, 24, 672; (e) Patel, R. N.
Curr. Opin. Biotechnol. 2001, 12, 587; (f) Biocatalysis
from Discovery to Application; Fessner, W.-D., Ed.;
Springer: Berlin, 2000; p. 254; (g) Silverman, R. B. The
Organic Chemistry of Enzyme-Catalyzed Reactions; Aca-
demic Press: San Diego, 2000; p. 717.
7. For critical overviews, vide: (a) Walsh, C. Nature 2001,
409, 226; (b) Rozzell, J. D. Bioorg. Med. Chem. 1999, 7,
2253.
8. Yeasts evaluated: Baker’s yeast, Trichosporon cutaneum,
Kluyveromyces marxianus var. marxianus, Pichia stipitis,
P. kluyveri, Rhodotorula glutinis and Geotrichum can-
didum (yeast-like fungus).
In brief, the use of Amberlite™ XAD-7 proved to be an
efficient strategy to circunvent the inhibitory effect of
the substrate over the biocatalyst. The low concentra-
tion of the substrate in the aqueous phase drove the
reaction to completion and a single product was deliv-
ered with high chemo- and enantioselectivity.

G. J. A. Conceic¸a˜o et al. / Tetrahedron: Asymmetry 14 (2003) 43–45
45
9. For a mini-review on non-conventional yeasts, vide:
Spencer, J. F. T.; Spencer, A. L. R.; Laluce, C. Appl.
Microbiol. Biotechnol. 2002, 58, 147.
shaker (150 rpm) at 30°C until full conversion of 5 (3
days). The resin was filtered off and the beads were
extracted with ethyl acetate to afford (S)-6 in 65% yield
after purification.
10. (a) Nakamura, K.; Takenaka, K.; Fujii, M.; Ida, Y.
Tetrahedron Lett. 2002, 43, 3629; (b) Nakamura, K.;
Fujii, M.; Ida, Y. J. Chem. Soc., Perkin Trans. 1 2000,
3205; (c) D’Arrigo, P.; Fuganti, C.; Fantoni, G. P.; Servi,
S. Tetrahedron: Asymmetry 1998, 54, 15017; (d) For
large-scale biocatalysis with XAD-7, vide: Vicenzi, J. T.;
Zmijewski, M. J.; Reinhard, M. R.; Landen, B. E.; Muth,
W. L.; Marler, P. G. Enzyme Microb. Technol. 1997, 20,
494.
11. Luche, J. L. J. Am. Chem. Soc. 1978, 100, 2226.
12. Commercially available resin XAD-7 was successively
washed with distilled water and acetone and then dried
under reduced pressure. The substrate (200 mg) was
dilluted in ethyl acetate (10 mL) and added to the treated
resin (3 g). The mixture was shaken and the solvent was
evaporated to dryness at reduced pressure. The solid so
obtained was poured directly into the reaction vessel.
13. Pichia stipitis CCT 2617 stored at ‘Andre´ Tosello’
Research Foundation was collected in the brazilian rain
forest and was cultivated in YM (yeast–malt extract)
nutrient broth (1 L) for 2 days incubation at 30°C on an
orbital shaker (150 rpm) before use.
1
15. Data for (S)-6: H NMR (300 MHz, CDCl3): l 0.99 (t,
3H, J=7.3 Hz), 1.81–2.01 (m, 2H), 2.08 (s, 1H), 4.97 (s,
1H), 5.14 (s, 1H), 5.25 (m, 1H), 7.20–7.40 (m, 5H); 13C
NMR (75 MHz, CDCl3): l 12.1, 24.5, 77.4, 108.6, 126.5,
127.5, 128.2, 142.1, 152.4; IR (film) 3396, 3031, 2966,
2938, 2882, 1648, 1493, 1453, 1022, 903, 844, 763, 700
20
+
cm−1; [h]D =−40 (c 2, CHCl3); MS m/z 162 (M , 14%),
147 (4%), 133 (71%), 129 (24%), 115 (21%), 107 (42%),
105 (50%), 91 (21%), 79 (100%), 77 (92%), 55 (93%), 51
(63%), 41 (29%); HRMS m/z found: 162.1043. Calcd for
C11H14O: 162.1045.
’
1
16. Data for (R)-7: H NMR (300 MHz, CDCl3): l 1.00 (t,
3H, J=7.3 Hz), 1.26 (s, 1H), 2.36 (m, 2H), 5.10 (s, 1H),
7.25–7.40 (m, 5H); 13C NMR (75 MHz, CDCl3): l 7.6,
31.1, 79.4, 127.3, 128.6, 128.9, 138.2, 210.0; IR (film)
3460, 3088, 3064, 3033, 2980, 2940, 2880, 1715, 1598,
1494, 14521269, 1093, 1024, 756, 701 cm−1; [h]D20=−325 (c
+
’
2, CHCl3); MS m/z 164 (M , 1%), 107 (100%), 105
(13%), 91 (92%), 79 (82%), 77 (56%), 65 (1%). HRMS
m/z found: 164.0833. Calcd for C10H12O2: 164.0837.
L-PAC (R)-8: (>95% e.e.) was prepared in our laboratory
by baker’s yeast mediated reduction of the corresponding
a-diketone.
17.
14. Typical procedure: To a slurry of growing Pichia stipitis
(300 mL), 5 (200 mg) adsorbed onto XAD-7 (3 g) was
added. The resulting suspension was stirred in an orbital