Enantioselectivity of alcohol-treated Candida rugosa lipase in the kinetic resolution of racemic methyl 2-aryloxypropionates in water and aqueous organic media

Article abstract of DOI:10.1016/S0957-4166(99)00500-5

Racemic methyl 2-aryloxypropionates (±)-1 were subjected to hydrolysis in water and in a series of two-phase aqueous organic media in the presence of Candida rugosa lipase (CRL). The biocatalytic material used was the enzyme of commercial CRL purified by treatment with different alcohols. The purification of CRL and the reaction medium play an important role in the enantioselection of racemates (±)-1. While it is not possible to use the same protocol for all substrates, by combining the different ways of purifying the enzyme with the various reaction media, it is possible to achieve high enantioselectivities of racemic esters. (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0957-4166(99)00500-5

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 10 (1999) 4599–4605
Enantioselectivity of alcohol-treated Candida rugosa lipase in the
kinetic resolution of racemic methyl 2-aryloxypropionates in
water and aqueous organic media
Antonio Cipiciani,∗ Francesca Bellezza, Francesco Fringuelli and Massimiliano Stillitano
Dipartimento di Chimica, Università di Perugia, Via Elce di Sotto 8, 06100 Perugia, Italy
Received 5 November 1999; accepted 10 November 1999
Abstract
Racemic methyl 2-aryloxypropionates (±)-1 were subjected to hydrolysis in water and in a series of two-phase
aqueous organic media in the presence of Candida rugosa lipase (CRL). The biocatalytic material used was the
enzyme of commercial CRL purified by treatment with different alcohols. The purification of CRL and the reaction
medium play an important role in the enantioselection of racemates (±)-1. While it is not possible to use the same
protocol for all substrates, by combining the different ways of purifying the enzyme with the various reaction
media, it is possible to achieve high enantioselectivities of racemic esters. © 1999 Elsevier Science Ltd. All rights
reserved.
1. Introduction
Lipases are enzymes widely used in organic synthesis because of their ability to regio-, diastereo-
and enantioselectively catalyze hydrolysis and esterification in water as well as in aqueous organic
media.1 The enzyme is sometimes more active in aqueous organic media than in water and this can be
explained by hypothesizing an oil–water interfacial activation associated with a conformational change
of the protein molecule.2 The activity of lipases is also increased by using a purified or a semipurified
native material.
Several protocols have been proposed to obtain a biocatalyst with different degrees of purification,
with the most common ones being a multi-step purification,3–7 dialysis,8 direct treatment of crude lipase
with deoxycolate followed by ethanol–ethyl ether,7 and the iso-propanol treatment.9,10
Candida rugosa lipase (CRL) is an extracellular protein. Structural analysis reveals that the catalytic
triad is not exposed to the reaction medium, and the polypeptide lid, which covers the active site of the
∗
Corresponding author. E-mail: cipan@unipg.it
0957-4166/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(99)00500-5
tetasy 3135

4600
A. Cipiciani et al. / Tetrahedron: Asymmetry 10 (1999) 4599–4605
native enzyme, is displaced before the substrate approaches the active site.11 Scattered data indicate that
the method of CRL purification and the reaction medium12 strongly influence the activity and selectivity
of lipase but systematic studies in this area are rare.10,13
Optically active 2-aryloxypropionic acids are a family of compounds of great biological interest14
which, in principle, should be easily obtained by lipase kinetic resolution of parent esters. The literature
data show that crude CRL in water is poorly enantioselective towards this class of compounds and with
the exception of our recently reported hydrolysis of racemic methyl 2-(2,4-dichlorophenoxy)propionate
by treated CRL,10 no investigation in an aqueous organic medium has been reported.
As a continuation of the research, we report a systematic study on the ability of CRL, purified
by treatment with different alcohols, in the kinetic resolution of racemic methyl aryloxypropionates
(±)-1 in both water and aqueous organic media. The hydrolysis of esters was chosen rather than the
transesterification reaction because the acid reaction product can be easily separated by washing with
alkali and no reagent other than the substrate is required.
2. Results and discussion
Firstly we studied the hydrolyses of esters (±)-1 (Fig. 1) in water by CRL purified by treatment with
simple aliphatic alcohols (methanol, ethanol, n-propanol and iso-propanol).
Fig. 1.
The results of the reactions carried out at room temperature and at pH=7.2 are illustrated in Table 1.
The acids (+)-(R)-2 and the esters (−)-(S)-3 were isolated in all the experiments. It is known that an
enantiomeric ratio value of E≥20 indicates a high level of enantioselection.15 On this basis, the MeOH-
or n-PrOH-purified CRL in water enantioselectively hydrolyzes only the (+)-(R) enantiomer of (±)-1d
yielding enantiomerically pure forms of (+)-2d and (−)-3d. Treatments with EtOH and i-PrOH allow
CRL to also recognize the (+)-(R) esters of (±)-1b and (±)-1e and to obtain (+)-2b and (−)-3e with high
enantiomeric excesses.
The effect of the reaction medium was evaluated by carrying out the hydrolyses at room temperature
and pH=7.2 with iso-propanol-treated CRL in homogeneous and heterogeneous 10:1 v/v aqueous organic
media. The 10:1 v/v water:organic solvent ratio was chosen based on previous experiences10 which
indicated that beyond 10% of the amount of organic fraction, the E value does not change, while the
rate of hydrolysis decreases. The organic solvents (acetone, diethyl ether, benzene and cyclohexane)
were chosen in order to cover a wide range of hydrophobicity values expressed by log P (from −0.23 to

A. Cipiciani et al. / Tetrahedron: Asymmetry 10 (1999) 4599–4605
4601
Table 1
Hydrolysis of (±)-methyl 2-aryloxypropionates 1 in water at room temperature and pH 7.2 by CRL
treated with the simplest aliphatic alcohols
3.20). The results are illustrated in Table 2. The isolated compounds were again the acids (+)-(R)-2 and
the esters (−)-(S)-3. The aqueous–organic medium influences the enantioselectivity, as well as the rate
of hydrolyses. The water–benzene medium gives the best results: the reactions are slower than in water
but the acids (+)-2b and (+)-2e are isolated in optically pure form while the acids (+)-2a and (+)-2d are
enantiomerically pure after only one recrystallization.
In water–acetone and water–diethyl ether media the CRL purified by i-PrOH only recognizes the (+)-
(R) enantiomer of racemic 1a, 1b and 1d. The water–cyclohexane medium deactivates the enzyme.
On the basis of these results, the water–benzene medium was also used to test the enantioselectivity
of the methanol-, ethanol- and n-propanol-purified CRL. The results are illustrated in Table 3 along with
those of iso-propanol-treated CRL. The water–benzene medium always improves the enantioselectivity
of the enzyme with respect to water only (Tables 1 and 3). This is particularly important for the hydrolysis
of (±)-1c because only by using this protocol is it possible to obtain enantiomerically pure (+)-2c.
The ee of products obtained in the hydrolyses with E≥20, using different protocols are summarized
in Table 4. The table shows that none of the protocols is effective for all five esters (±)-1, rather it is

4602
A. Cipiciani et al. / Tetrahedron: Asymmetry 10 (1999) 4599–4605
Table 2
Hydrolysis of (±)-methyl 2-aryloxypropionates 1 by iso-propanol purified CRL in different aqueous
organic media at room temperature and pH=7.2
necessary to combine the methods of CRL purification and the reaction medium in order to achieve the
best results.
Enantiomerically pure acids (+)-2b and (+)-2d can be easily obtained by using several procedures
(seven for the former and nine for the latter: entries 1, 2, 5, 8–11 and 1–5, 7, 9–11, respectively) while
(+)-2c can only be prepared by using the protocol of entry 9. The (+)-2 and (−)-3 are simultaneously
obtained, with a high enantiomeric excess, only from (±)-1d (entries 1, 3 and 7) and from (±)-1e (entry
5). All acids (+)-2 and the esters (−)-3d and (−)-3e are obtained enantiomerically pure in good yield
by using the suitable protocol, while no procedure yields (−)-3a, (−)-3b and (−)-3c with high ee value
and satisfactory yield. The procedure that uses i-PrOH-purified CRL and water–cyclohexane as reaction
medium is ineffective for all substrates.
In conclusion, the CRL purification and the reaction medium play an important role in the enantioselec-
tion of racemates (±)-1 but it is not possible to use the same protocol for all substrates. The purification
eliminates non-essential material or that which hinders the reaction, while the reaction medium influences

A. Cipiciani et al. / Tetrahedron: Asymmetry 10 (1999) 4599–4605
4603
Table 3
Enantioselection of (±)-methyl 2-aryloxypropionates 1 in water–benzenea at room temperature by
CRL treated with the simplest aliphatic alcohols
the conformational state of the enzyme (i.e., open or closed form) which then determines the geometry
of the active site, allowing one compound to be recognized more easily than another.
3. Experimental
3.1. General
Lipase from Candida rugosa (crude CRL E.C. 3.1.1.13 type VII) was purchased from Sigma Chemical
Co. Racemic 2-aryloxypropionic acids were obtained from Aldrich Chemical Co. All the organic solvents
were of reagent grade and used without further purification. The ees of acids (+)-2 were determined
on corresponding methyl esters obtained by treatment with CH2N2. [Eu(hfc)3] was used as chiral shift
reagent to determine enantiomeric excess recording the 1H NMR spectra in CDCl3 solution on a Bruker
AC 200 MHz spectrometer. The ees were determined after the usual work-up of the reaction mixture

4604
A. Cipiciani et al. / Tetrahedron: Asymmetry 10 (1999) 4599–4605
Table 4
Enantioselection of (±)-methyl 2-aryloxypropionates 1 by CRL under different conditions
before the purification of the reaction products. Racemic esters 1 were prepared in 90% yield by treating
commercial (±)-2 with anhydrous methanol in the presence of catalytic amounts of p-toluenesulfonic
acid at reflux for 3 h. The products were separated and purified by usual chemical work-up and spectral
analyses were consistent with those previously described.15 Purification of crude lipase from Candida
rugosa by treatment with different alcohols was carried out according to the general procedure reported
below. The specific activity of purified lipase was determined as previously described.10 The hydrolysis
of (±)-1d with iso-propanol-treated CRL in water–ethyl ether is reported as the typical procedure.
3.2. Alcohol-treated CRL
Commercial material (10 g, Sigma Chemical Co.) was mixed in phosphate buffer (50 mL, 50 mM, pH
7.2, 4°C) by stirring for 30 min. Alcohol (Table 1) (50 mL) was added dropwise over 40 min at 4°C.
This heterogeneous medium was stirred at 4°C for 46 h. The mixture was centrifuged (3000 rpm, 30 min,
4°C) to remove the precipitate. The supernatant was dialyzed against phosphate buffer solution (1×2 L,
50 mM, pH 7.2) and then against deionized distilled water (1×2 L). The protein solution was stored at
4°C and used within one week. Protein concentrations were obtained by spectroscopic determination at
288 nm using bovine serum albumin (Sigma Chemical Co.) as standard (3 mg/mL).
3.3. Hydrolysis of methyl 2-(4-chlorophenoxy)propionate (±)-1d with iso-propanol-treated CRL in
water–ethyl ether
Ethyl ether (1.2 mL) was added to a phosphate buffer (20 mM, pH 7.2) solution of iso-propanol-treated
CRL (12 mL, 260 units, with p-NPA assay) and the mixture stirred for 15 min at room temperature in
a closed vessel. The resulting enzymatic system was added to the substrate (214 mg) and the mixture

A. Cipiciani et al. / Tetrahedron: Asymmetry 10 (1999) 4599–4605
4605
stirred and maintained at pH 7.2 by automatic titration with NaOH (0.2N) using a Mettler DK pH-Stat.
When the hydrolysis reached 50% conversion (23 h), a saturated solution of NaCl (15 mL) was added to
the reaction mixture, the pH was adjusted to 2 using 6N HCl and extracted with ether (3×30 mL). The
combined ether extracts were treated with saturated aqueous NaHCO3 (3×40 mL) and the layers were
separated. The ether layer was dried with Na2SO4 and concentrated in vacuo giving the ester (−)-3d: 104
25
25
mg (90% yield), ee 94%, [α] −38.6, lit.17 [α] −41.1 (c=50, EtOH). The combined aqueous layers
D
D
were acidified to pH 2, extracted with ether (3×30 mL), dried with Na2SO4 and concentrated in vacuo
giving the acid (+)-2d: 84 mg (85% yield), ee 92%, [α] +36.6, lit.18 [α]25 +39.8 (c=30, EtOH). After
25
D
D
one recrystallization from hexane:ethyl acetate (10:1), ee 99%.
Acknowledgements
The Ministero dell’Università e della Ricerca Scientifica e Tecnologica (MURST) Consiglio Nazionale
delle ricerche (CNR, Rome) and Università degli Studi di Perugia are thanked for financial support.
References
1. Klibanov, A. M. Acc. Chem. Res. 1990, 23, 114. Santaniello, E.; Ferraboschi, P.; Grisenti, P.; Manzocchi, A. Chem. Rev.
1992, 92, 1071. Wong, C. H.; Whitesides, G. M. Enzymes in Synthetic Organic Chemistry; Pergamon: New York, 1994.
2. Holmquist, M.; Martinelle, M.; Berglund, P.; Clausen, I. G.; Patkar, S.; Svendsen, A.; Hult, K. J. Prot. Chem. 1993, 12, 749.
3. Tomizuka, N.; Ota, Y.; Yamada, K. Biol. Chem. 1966, 30, 576, 1090.
4. Veeragavan, K.; Gibbs, B. F. Biotechnol. Lett. 1989, 11, 345.
5. Shaw, J. F.; Chang, C. H.; Wang, Y. J. Biotechnol. Lett. 1989, 11, 779.
6. Brahimi-Horn, M. C.; Guglielmino, M. L.; Elling, L.; Sparrow, L. G. Biochim. Biophys. Acta 1990, 1042, 51.
7. Wu, S. H.; Guo, Z. H.; Sih, C. J. J. Am. Chem. Soc. 1990, 112, 1990.
8. Hernaiz, M. J.; Sànchez-Montero, J. M.; Sinisterra, J. V. Tetrahedron 1994, 50, 10749.
9. Colton, I. J.; Sharmin, N. A.;. Kazlauskas, R. J. J. Org. Chem. 1995, 60, 212.
10. Cipiciani, A.; Cittadini, M.; Fringuelli, F. Tetrahedron 1998, 54, 7890.
11. Grochulski, P.; Li, Y.; Schrag, J. D.; Bouthillier, F.; Smith, P.; Harrison, D.; Rubin, B.; Cygler, M. J. Biol. Chem. 1993, 268,
12843. Grochulski, P.; Li, Y.; Schrag, J. D.; Cygler, M. Protein Sci. 1994, 3, 82.
12. Rua, M. L.; Diaz-Maurino, T.; Fernandez, V. M.; Otero, C.; Ballesteros, A. Biochim. Biophys. Acta 1993, 1156, 181.
13. Cipiciani, A.; Fringuelli, F.; Mancini, V.; Piermatti, O.; Scappini, A. M.; Ruzziconi, R. Tetrahedron 1997, 53, 11853.
14. The Pesticide Manual; Worthing, C. R., Ed.; 6th ed.; BCPC Publication: London, 1979; p. 329. Secor, J.; Cséke, C.; Owen,
W. J. In Biocatalysis in Agricultural Biotechnology; Whitaker, J. R.; Sonnet, P. E., Eds.; American Chemical Society:
Washington, 1989; p. 265. ConteCamerino, D.; Mambrini, M.; De Luca, A.; Tricarico, D.; Bryant, S. H.; Tortorella, V.;
Bettoni, G. Pflugers Arch. Eur. J. Physiol. 1988, 413, 105. Fredga, A.; Weidler, A. M.; Gronwall, C. Ark. Kemi 1960, 17,
265.
15. Chen, C. S.; Wu, S. H.; Girdauskas, G.; Sih, C. J. J. Am. Chem. Soc. 1987, 109, 2812.
16. Chen, C. S.; Fujimoto, Y.; Girdauskas, G.; Sih, C. J. J. Am. Chem. Soc. 1982, 104, 7294.
17. Kirchner, G.; Scollar, M. P.; Klibanov, A. M. J. Am. Chem. Soc. 1985, 107, 7072.
18. Witiak, D. T.; Ho, T. C.-L.; Hakney, R. E.; Connor, W. E. J. Med. Chem. 1968, 11, 1086.