Resolution of racemic acids by irreversible lipase-catalyzed esterification in organic solvents

Article abstract of DOI:10.1002/1099-0690(200104)2001:8<1441::AID-EJOC1441>3.0.CO;2-D

The reversible nature of the direct esterification of acids with alcohols limits the use of this process in the biocatalytic kinetic resolution of chiral acids. A new irreversible procedure, using orthoesters, has been developed.

Full text of DOI:10.1002/1099-0690(200104)2001:8<1441::AID-EJOC1441>3.0.CO;2-D

SHORT COMMUNICATION
Resolution of Racemic Acids by Irreversible Lipase-Catalyzed Esterification in
Organic Solvents
[
a]
[a]
[a]
Raffaele Morrone, Mario Piattelli, and Giovanni Nicolosi*
Keywords: Carboxylic acids / Chiral resolution / Catalysis / Hydrolases / Orthoformates
The reversible nature of the direct esterification of acids with
alcohols limits the use of this process in the biocatalytic kin-
etic resolution of chiral acids. A new irreversible procedure,
using orthoesters, has been developed.
The pioneering work of Klibanov[1] has paved the way tion of racemic acids was based on the consideration that
for enzymatic catalysis in predominantly organic media, these orthoesters are known to be easily hydrolyzed in
and lipases (triacylglycerol acyl hydrolases, E. C. 3.1.1.3) acidic conditions according to Equation (1):[
have been used to efficiently catalyze a multitude of ester-
9]
ification, transesterification and interesterification reac-
tions. In particular, highly enantioselective direct ester-
(
1)
ification has been widely applied to the kinetic resolution
As such, orthoformates were expected to preclude accu-
of racemic alcohols and acids.[ However, the reversibility mulation of water in the reaction mixture and thus limit the
of the reaction makes it impossible to obtain the slower reverse reaction, and at the same time furnish the nucleoph-
reacting enantiomer in high optical purity simply by ex- ile for the forward reaction.
2]
tending the conversion at the expense of chemical yield. In
In this study two substrates were used as models
fact, since the same enantiomer is the fast reacting one in (Scheme 2), flurbiprofen [(R,S)-2-(2-fluoro-4-biphenyl)pro-
both the forward (esterification) and the reverse (hydrolysis) panoic acid], a member of the nonsteroidal anti-inflammat-
reaction, the optical purity of the unchanged substrate ory drugs (NSAIDs),[ and (R,S)-2-methylvaleric acid, an
drops rapidly when the conversion is extended much bey- intermediate in the synthesis of branched-chain biologically
10]
[
3]
[11]
ond 50%. In order to overcome this detrimental effect in active compounds.
the resolution of alcohols, irreversible esterification proced-
ures have been resorted to, involving the use of activated or
enol esters as acyl donors.[ In the case of the resolution
of racemic acids (Scheme 1) it is essential to decrease the
influence of the reverse process by keeping the water con-
tent to a low level.
4]
Scheme 1. Esterification of a racemic acid catalyzed by a lipase
with R-stereopreference
Scheme 2. Enzymatic esterification of (Ϯ)-flurbiprofen (A), and
Many approaches have been proposed for reducing water (Ϯ)-2-methylvaleric acid (B)
levels during esterification. They include the use of molecu-
[
5]
Trimethyl (TMOF), triethyl (TEOF), tripropyl (TPOF)
and tributyl orthoformate (TBOF) were the orthoesters ex-
amined to investigate the influence of the alkoxy groups on
the course of the reaction. Flurbiprofen was esterified in
lar sieves or the coupling of a hydrated salt and its lower
[6]
hydrated or anhydrous form, evaporation from solvent-
free reaction mixtures,[7] or sorption of water into a poly-
meric membrane followed by selective diffusion and evap-
oration into a vapor phase.[8] As these physical methods
acetonitrile in the presence of lipase from Candida antartica

(
Novozym 435 ), while for 2-methylvaleric acid a different
appeared to us to have their drawbacks or to be of limited
applicability, we considered the possibility of resorting to a
chemical method, and here we report the successful biocata-
lyzed resolution of racemic acids in the presence of ortho-
formates. The decision to use orthoformates in the resolu-
solvent (hexane), and catalyst (lipase from Candida rugosa,
CRL), were used. Each experiment was run in parallel with
a control, in which the orthoformate was replaced by the
corresponding alcohol. At the end of the incubation period
the values for conversion and enantiomeric excess (ee) were
[
a]
CNR-Istituto per lo Studio delle Sostanze Naturali di Interesse determined by chiral HPLC or GC.
Alimentare e Chimico-Farmaceutico
Via Del Santuario, 110, 95028 Valverde (CT), Italy
Fax: (internat.) ϩ39-95/721-2141
The results of the lipase-catalyzed esterification with or-
thoformates, relative to those obtained with the ‘‘normal’’
procedure with the corresponding alcohols as reagents, are
E-mail: nicolosi@issn.ct.cnr.it
Eur. J. Org. Chem. 2001, 1441Ϫ1443

WILEY-VCH Verlag GmbH, 69451 Weinheim, 2001
1434Ϫ193X/01/0408Ϫ1441 $ 17.50ϩ.50/0
1441

R. Morrone, M. Piattelli, G. Nicolosi
SHORT COMMUNICATION
shown graphically in Figure 1 and 2. Inspection of Fig-
ure 1a shows that in the normal esterification of flurbip-
rofen the ee of the unchanged substrate reaches a maximum
value of 80Ϫ85% after ca. 40 h and then begins to drop. In
contrast, when orthoformates are used the course of the
reaction is similar to an irreversible reaction (Figure 1b)
and the ee values continue to increase upon extension of the
incubation period, and, consequently, the conversion value.
With all the orthoformates tested, after 140 h of reaction
time the conversion was between 60 and 70%, with an ee
value for the unchanged acid of 95Ϫ98%.
Figure 2. Enantiomeric excess (ee) value of unchanged 2-methylva-
leric acid versus reaction time with (a) different alcohols, and
(
b) orthoformates
In summary, this study shows that orthoformates can be
used in the lipase-catalyzed esterification aimed at the kin-
etic resolution of racemic acids, circumventing the adverse
effects of the water formed in the course of the reaction.
Water not only affects negatively the position of the equilib-
rium, but also acts on the optical purity of the remaining
substrate, which cannot be raised to high levels simply by
extending the conversion. Orthoformates trap the water
Figure 1. Enantiomeric excess (ee) value of unchanged flurbiprofen through hydrolysis and therefore prevent the reverse reac-
versus reaction time with (a) different alcohols, and (b) orthoform-
tion, and at the same time provide the nucleophile for the
ates
esterification. Obviously, the method proposed here can be
used not only in the resolution of chiral acids, but also in
For 2-methylvaleric acid the trend was similar, in that the
the esterification of achiral acids to increase the yield of
normal esterification (Figure 2a) followed the expected
ester by pushing the equilibrium toward completion. In-
course for a reversible reaction, where the ee of the residual
acid decreased when conversion was extended much beyond
0%. The esterification of 2-methylvaleric acid with ortho-
creasing the yield can be of great value when the achiral
acids are expensive.
5
formates also proceeded as an irreversible reaction (Fig-
ure 2b). Of the four orthoformates tested, tributyl orthofor-
mate was found to be superior, giving ee values for the re-
maining substrate of 97 and Ͼ98% at conversions of 65 and
Experimental Section
General: 1H NMR spectra were recorded in CDCl
solution at
3
7
0%, respectively.
2
50 MHz on a Bruker AC 250 instrument. Ϫ Mass spectra were
Finally, ancillary experiments on the hydrolysis of tripro-
pyl orthoformate under different conditions demonstrated
recorded on a HewlettϪPackard GC-MS instrument, Mod. 5971A.
Ϫ Optical rotation data were measured at 25 °C on a Jasco DIP
the purely chemical nature of the reaction, without the par- 370 digital polarimeter.
Ϫ All reactions were monitored
ticipation of the enzyme.
(conversion and ee values) by HPLC using a Chirex R-NGLY &
1442
Eur. J. Org. Chem. 2001, 1441Ϫ1443

Resolution of Racemic Acids by Irreversible Lipase-Catalyzed Esterification
SHORT COMMUNICATION
DNB (250 ϫ 4.0 mm) column (0.02  ammonium acetate in meth-
anol as the eluent) or GC using a β-cyclodextrin (dimethylpenthyl-
β-cyclodextrin/OV1701 3:7) column. Ϫ Lipase from Candida rugosa
Ϫ C
10.52.
6
H
12
O
2
(116.19): calcd. C 62.04, H 10.41; found C 62.31, H
(
CRL) was purchased from Sigma. Novozym 435 (lipase from
Acknowledgments
This work was supported from CNR Target Project ‘‘Biotechno-
logy’’.
Candida antarctica, CAL) was a gift from Novo Nordisk. Ϫ Ele-
mental analyses were performed on a Carlo Erba Instrument
CHNS-O EA1108 Elemental Analyzer.
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1] [1a]
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3
CN (1 l) con-
[1b]
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taining tripropyl orthoformate (123 mmol, 26.5 mL), 0.1 mL of n-
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by shaking at 45 °C (300 rpm); the conversion and ee of unchanged
flurbiprofen were followed by chiral HPLC analysis. After 6 days
conversion had reached 60% and the reaction was stopped by fil-
tering off the enzyme. Removal of the solvent in vacuo left a residue
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Chem. Soc. 1986, 108, 2767Ϫ2768. Ϫ
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3] [3a]
that was partitioned between hexane and aq. NaHCO
mL of water). The organic phase was washed with water, dried over
Na SO and the solvent removed to afford (R)-flurbiprofen propyl
ester (6.8 g, yield 58%, ee 64%). Ϫ H NMR (CDCl
3
(3 g in 200
[
[
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4
[3b]
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3
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[3c]
J ϭ 7 Hz, 3 H), 1.54 (d, J ϭ 7 Hz, 3 H), 1.65 (m, 2 H), 3.78 (q,
J ϭ 7 Hz, 1 H), 4.06 (t, J ϭ 6 Hz, 2 H), 7.1Ϫ7.6 (m, 8 H). Ϫ
2812Ϫ2817. Ϫ
01, 711Ϫ724; Angew. Chem. Int. Ed. Engl. 1989, 28, 695Ϫ707.
C. S. Chen, C. J. Sih, Angew. Chem. 1989,
1
4] [4a]
M. Deguil-Castaing, B. De Jeso, S. Drouilard, B. Maillard,
C
18
H
2
19FO : calcd. C 75.50, H 6.69; found C 75.62, H 6.89.
[4b]
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Y. F. Wang, C. H.
[4c]
J. Hiratake,
Acidification of the aqueous phase with H
of (S)-(ϩ)-flurbiprofen (3.9 g, yield 39%, ee Ͼ98%). Ϫ C15
244.26): calcd. C 73.76, H 5.36; found C 73.90, H 5.52.
2 4
SO gave a precipitate
H13FO
2
[4d]
6
130Ϫ6133. Ϫ
Y. F. Wang, J. J. Lalonde, M. Momogan, D.
(
E. Bergbreiter, C. H. Wong, J. Am. Chem. Soc. 1988, 110,
7
200Ϫ7205.
[
[
5]
Preparation of Enantiopure (R)-(؊)-2-Methylvaleric Acid: Racemic
-methylvaleric acid (86.2 mmol, 10 g) was dissolved in hexane (500
F. Fonteyn, C. Blecker, G. Lognay, M. Marlier, M. Severin,
Biotechnol. Lett. 1994, 16, 693Ϫ696.
2
6] [6a]
P. Kuhl, P. J. Halling, Biochim. Biophys. Acta 1991, 1078,
mL) containing tributyl orthoformate (86.2 mmol, 23 mL), 0.1 mL
of n-butyl alcohol and Candida rugosa lipase (CRL) (50 g). The
mixture was incubated by shaking at 45 °C (300 rpm); the conver-
sion and ee of the butyl ester were followed by chiral GC analysis.
After 48 h conversion had reached 65% and reaction was stopped
[6b]
3
26Ϫ328. Ϫ
L. Kvittingen, B. Sjursnes, T. Anthonsen, P.
[6c]
Halling, Tetrahedron 1992, 48, 2793Ϫ2802. Ϫ
M. Zare-
v u´ cka, M. Rejzek, M. Hoskovec, A. Svato sˇ , Z. Wimmer, B.
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S. M. Kim, J. S. Rhee, J. Am. Oil Chem. Soc. 1991, 68,
[
[
[
7]
8]
9]
4
99Ϫ503.
by filtering off the enzyme. After partition with aq. NaHCO
in 200 mL of water) the hexane phase was dried over Na SO
3
(3 g
and
S. J. Kwon, K. M. Song, W. H. Hong, J. S. Rhee, Biotechnol.
Bioeng. 1995, 46, 393Ϫ395.
2
4
evaporated under vacuum to furnish (S)-2-methylvaleric butyl ester
D. Barton, W. D. Ollis, Comprehensive Organic Chemistry, Vol.
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(
9.6 g, yield 65%, ee 53%). MS data agreed with those reported in
[10]
[
12]
the literature.
20 2
Ϫ C10H O (172.27): calcd. C 69.72, H 11.70;
found C 69.98, H 11.84.
[
[
11]
12]
K. Mori, Tetrahedron 1989, 45, 3233Ϫ3298.
J. Kim Ha, R.C. Lindsay, J. Food Compos. Anal. 1989, 2,
The aqueous phase was acidified with H
2 4
SO , extracted three times
1
18Ϫ131.
with hexane and the organic phases were pooled. Removal of hex-
[13]
P. A. Levene, R. E. Marker, J. Biol. Chem. 1932, 98, 1Ϫ7.
ane under vacuum gave (R)-(Ϫ)-2-methylvaleric acid (3.5 g, yield
Received November 29, 2000
0
20
[13]
3
5%, ee Ͼ 97%). [α]2
D
ϭ Ϫ18.2 (neat); [ref. [α]
D
ϭ Ϫ18.4 (neat)].
[O00606]
Eur. J. Org. Chem. 2001, 1441Ϫ1443
1443