N. Berezina et al. / Tetrahedron: Asymmetry 13 (2002) 1953–1955
1955
able to perform biotransformations at rather high pH.12
Thus, for analytical purposes, an experiment was con-
ducted using 2.8 g (dry weight) of cells13 suspended in 1
L of a pH 8.5 glycine/NaOH buffer (50 mM) contain-
ing 1% of glycerol. 300 mg (1.5 mM) of substrate 1
(solubilized in 10 mL ethanol) were added.† The pH
was regulated at a value of 9 by automated addition of
0.1N NaOH. The reaction was followed by withdraw-
ing aliquots which were analyzed for yield and ee. The
results shown in Fig. 3 indicate that the substrate
disappeared within a period of about 27 h, whereas the
corresponding lactone was formed with an analytical
yield of about 75% and an ee of about 98%. This high
analytical yield clearly indicates that a dynamic kinetic
resolution process did take place as expected. A similar
bioconversion, conducted for preparative purposes and
using 5.3 g (dry weight) of cells, was stopped after 24 h
by addition of HCl until acidic pH. The medium was
continuously extracted with dichloromethane contain-
ing Amberlite IR 120 resin. Purification by flash chro-
matography afforded a 85% isolated yield of nearly
enantiopure (R)-6-benzyloxymethyl-tetrahydropyran-2-
one 2 (275 mg, 96% ee, [h]2D5=−8 (c 1.1, CHCl3)). This
confirmed the above proposed dynamic kinetic
resolution.
concentration as we have previously described in the
case of bicyclo[3.2.0]hept-2-en-6-one.13
Acknowledgements
This work was funded by the European Community
Fourth Framework Program (project BIO4-CT98-
0267). One of us (N.B.) expresses her gratitude to the
EC for financial support. We are very grateful to
Professor J. Ward (UCL, London, UK) for having
provided us with the recombinant E. coli strain.
References
1. (a) For reviews about chemical asymmetric Baeyer–Vil-
liger oxidation, see: Strukul, G. Angew. Chem., Int. Ed.
1998, 37, 1199–1209; (b) Watanabe, A.; Uchida, T.; Ito,
K.; Katsuki, T. Tetrahedron Lett. 2002, 43, 4481–4485
and references cited therein.
2. For reviews about biocatalysed asymmetric Baeyer–Vil-
liger oxidation see for instance: (a) Willetts, A. Trends
Biotechnol. 1997, 15, 515–62; (b) Roberts, S. M.; Wan, P.
W. H. J. Mol. Catal. B: Enz. 1998, 4, 111–136; (c)
Alphand, V.; Furstoss, R. Baeyer–Villiger oxidations. In
Enzyme Catalysis in Organic Synthesis: a Comprehensive
Handbook, Drauz, K.; Waldmann, H., Eds.; VCH: Wein-
heim, 1995; Vol. 2, pp. 745–772.
3. For reviews about dynamic resolution, see: (a) Strauss,
U. T.; Felfer, U.; Faber, K. Tetrahedron: Asymmetry
1999, 10, 107–117; (b) Kitamura, K.; Tokunaga, M.;
Noyori, R. Tetrahedron 1993, 49, 1853–1860; (c) Azerad,
R.; Buisson, D. Curr. Opin. Biotechnol. 2000, 11, 565–
571.
3. Conclusion
A dynamic kinetic resolution process was successfully
applied, for the first time, to a Baeyer–Villiger micro-
biological oxidation using a recombinant E. coli strain
overexpressing the well known cyclohexanone
monooxygenase enzyme. Nearly enantiopure (R)-6-ben-
zyloxymethyl-tetrahydropyran-2-one 2 (96% ee) was
thus obtained with a 85% overall yield from racemic
2-benzyloxymethylcyclopentanone 1. The application of
this dynamic kinetic resolution process to other sub-
strates is in progress in our laboratory. We also are
currently studying the possibility of increasing the pro-
ductivity of this process, i.e. to increase the substrate
4. Schulze, B.; Wubbolts, M. G. Curr. Opin. Biotechnol.
1999, 10–6, 609–6615.
5. Taylor, R. J. K.; Wiggins, K.; Robinson, D. H. Synthesis
1990, 589–590.
6. Borjesson, L.; Csoregh, I.; Welch, C. J. J. Org. Chem.
1995, 60, 2989–2999.
7. Alphand, V.; Furstoss, R.; Pedragosa-Moreau, S.;
Roberts, S. M.; Willetts, A. J. J. Chem. Soc., Perkin
Trans. 1 1996, 1867–1872.
8. Chen, C. S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J. J.
Am. Chem. Soc. 1982, 104, 7294–7299.
9. Sharpless, K. B.; Amberg, W.; Bennani, Y. L.; Crispino,
G. A.; Hartung, J.; Jeong, K. S.; Kwong, H. L.;
Morikawa, K.; Wang, Z. M.; Xu, D.; Zhang, X. L. J.
Org. Chem. 1992, 57, 2768–2771.
10. Liu, Z. Y.; Ji, J. X.; Li, B. G. J. Chem. Soc., Perkin
Trans. 1 2000, 3519–3521.
11. (a) Doig, S. D.; O’Sullivan, L. M.; Patel, S.; Ward, J. M.;
Woodley, J. M. Enzyme Microb. Technol. 2001, 28, 265–
274; (b) Zambianchi, F.; Pasta, P.; Carrea, G.; Colonna,
S.; Gaggero, N.; Woodley, J. M. Biotechnol. Bioeng.
2002, 78, 489–496.
12. Doig, S. D.; Simpson, H. D.; Alphand, V.; Furstoss, R.;
Woodley, J. M. Enzyme Microb. Technol. in press.
13. Simpson, H. D.; Alphand, V.; Furstoss, R. J. Mol. Cat.
B: Enz. 2001, 16, 101–108.
Figure 3. Biotransformation using E. coli TOP10(pQR239) at
pH 9: ꢀ ketone yield, ꢁ ketone ee, ꢂ lactone yield, ꢃ
lactone ee.
† The biotransformation was carried out in a 2 L Setric fermentor
under the following conditions: 400 rpm, 0.27 vvm aeration at 30°C.