KINETIC RESOLUTION OF CHIRAL ALKOHOLS, ACIDS,
AND ESTERS IN ENZYME MEMBRANE REACTORS
Józef Ceynowa, Marta Rauchfleisz
Nicholas Copernicus University, Faculty of Chemistry,
Gagarin St. 7, 87-100 Toruń, Poland
ABSTRACT
Enzyme membrane reactors were used for kinetic resolution of chiral
alcohols (trans-2-methyl-1-cyclohexanol, menthol), carboxylic acids (2-(2fluoro-4-biphenyl)propanoic acid, (4-isobutylphenyl)propanoic acid) and esters
(trans-2-methyl-1-cyclohexyl acetate, trans-2-methyl-1-cyclohexyl benzoate).
The enzyme membranes for the reactors were prepared by chemical
immobilisation of the chosen lipase within asymmetric polyamide capillary
membranes. In all kinds of processes the highest catalytic activity and
enantioselectivity is exhibited by lipase from Pseudomonas sp. The lipase
manifests a catalytic action preferentially to (R) – chiral centres. The influence
of various parameters on the processes has been estimated, and the processes
have been characterised by enantioselectivities, enantiomeric excess of
products and substrates as well as the constants of Michaelis – Menten
equation.
Keywords: Kinetic resolution, enzyme membrane reactor, immobilised lipase,
enantioselective hydrolysis, esterification and transesterification.
INTRODUCTION
Biological properties of organic compounds depend on their enantiomeric purity as the
particulate enantiomers can display various biological activities: from distinguishable smell
and flavour to the opposite influence on living organisms. Thus, compounds such as
pharmaceuticals, pesticides, pheromones, and some food should be produced from pure
enantiomers [1].
An application of enzymes in organic synthesis is one of the most useful and practical
methods for the preparation of compounds with high optical purity. Enzymes act in aqueous
and organic solvents at mild conditions, with high catalytic activity. Especially advantageous
is the application of the enzymes immobilised within microfiltration membranes, which
placed in a membrane module, constitutes the main part of the enzyme membrane reactor.
The processes in such a reactor can conveniently be performed in a continuous mode of
operation. It is environmentally safe and does not require sophisticated equipment [2].
One of the effective ways to produce enantiomerically pure chemicals relies on the
kinetic resolution of racemates of substances such as chiral acids, alcohols and esters. It can
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be accomplished in the processes of hydrolysis, esterifications, and transesterifications under
the stereospecific catalytic action of lipases.
In this work, polyamide membrane with immobilised lipase has been applied for
kinetic resolution of trans-2-methyl-1-cyclohexanol and menthol, 2-(2-fluoro-4biphenyl)propanoic acid and (4-isobutylphenyl)propanoic acid, as well as trans-2-methyl-1cyclohexyl acetate and trans-2-methyl-1-cyclohexyl benzoate. The hydrolyses were
performed in biphasic, and esterifications / transesterifications - in one phase systems (with
the reactants dissolved in the proper organic solvent).
EXPERIMENTAL
One of the three lipases: from Pseudomonas sp. (Type XIII, 2160 U/mg), from
Candida rugosa (Type VII, 1500 U/mg) or from Porcine pancreas (Type VI-S, 205 U/mg)
was immobilised in an asymmetric capillary polyamide membrane (nominal molecular weight
cut-off of 50 kDa, i.d. 0.6 mm, o.d. 1.2 mm). The reactors consisted of five capillaries
(200 mm in length) encased in a glass tube (Fig.1).
The procedure of immobilisation: i) acidolise of a part of amide bounds with 1.8 M
HCl (3 h); ii) modification by 1,4-butadiene (0.1 M, 3 h); iii) activation by glutaraldehyde
(5 % in phosphate buffer pH 7.2, 3 h); and iv) immobilisation of lipase from phosphate buffer
( 0.5 M, pH 7.2). The load density of the bonded lipase was determined according to Sigma
Diagnostics procedure based on the Tietz and Fiereck method. The catalytic activity of the
lipase immobilised in the polyamide membrane equals 27% of its activity in the native form.
The enzyme membranes exhibit constant activity for more than 500 h of continuous
operation. All the reactions were carried out at 310 K. The temperature has been estimated as
optimal in the additional experiments.
Esterification and transesterification were performed in one-phase system (solutions in
n-heptane) in cross-flow circulation of the reaction mixture at both sides of the membrane and
its partial permeation through the membrane. Hydrolyses were carried out in a two-phase
reactor with ester solution in n-heptane and phosphate buffer (pH 7.2, 0.5 M) circulating
countercurrently on the definite side of the enzyme membrane. The progress of the reaction
and the enantiomeric resolution were followed by high performance liquid chromatography
using a chiral column Chiralcel OD-H (I.D. 0.46 cm, I.L. 25 cm) with a precolumn Chiralcel
OD (I.D. 0.46 cm, I.L. 5 cm), Shimadzu SPD-10A (VP) UV-VIS detector and Shimadzu
LC-10AD (VP) pump. Optical rotation was measured with Polamat A Carl Zeiss Jena
polarimeter.
Enantioselectivity of each process, represented by enantiomeric ratio, (E) was
calculated according to the following formula [3]:
c  cS
(k / K M ) R ln[( 1  )(1  e.e.)]
E  cat

, where: e.e.  R
, kcat – catalytic rate constant,
(k cat / K M )S ln[( 1  )(1  e.e.)]
c R  cS
KM – Michaelis constant,  - extent of conversion, e.e. – enantiomeric excess, cR, cS –
concentration of (R) and (S) enantiomers, respectively.
RESULTS
Lipases catalyse preferentially the processes with (R)-enantiomer [4], but the
enantioselectivity of kinetic resolution in the very reaction depends on the kind of
immobilised lipase. This was checked in the proper kinetic resolution process. The results are
presented in Tab. 1.
As it can be seen, lipase from Pseudomonas sp. exhibits highest enantioselectivity in
all the processes (esterification, hydrolysis and transesterification). The best enantioselectivity
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values were observed in the esterification of 2-propanoic acids. Esters react much slower and
with the lowest enantioselectivities.
Table .1. Enantioselestivities of lipases
Source of lipase
Racemic substrate
Pseudomonas species
Candida cylindracea
Porcine pancreas
Pseudomonas species
Candida cylindracea
Porcine pancreas
Pseudomonas species
Candida cylindracea
Porcine pancreas
Pseudomonas species
Candida cylindracea
Porcine pancreas
Pseudomonas species
Candida cylindracea
Porcine pancreas
Pseudomonas species
Candida cylindracea
Porcine pancreas
trans-2-methyl-1-cyclohexanol *)
*)
menthol *)
trans-2-methyl-1-cyclohexyl acetate **)
trans-2-methyl-1-cyclohexyl benzoate **)
2-(2-fluoro-4-biphenyl)propanoic acid ***)
(4-isobutylphenyl)propanoic acid ***)
Enantioselectivity, E
37
21
<5
41
9
10
35
17
<5
36
17
<5
40
33
11
40
31
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– transesterification with vinyl acetate, **) – hydrolysis, ***) – esterification with ethanol.
One of the most important factors in the balance between stabilisation and inactivation
of the enzyme catalytic centre, due to the organic phase, is the solvent polarity [5]. It is
decisive in one-phase processes of esterification and transesterification. The best solvent was
selected in transesterification of ()-trans-2-methyl-1-cyclohexanol with vinyl acetate from
among ten solvents, differing in hydrofobicity (measured by log P). The highest catalytic
activity of the lipase was observed for n-hexane and n-heptane – the most hydrophobic
solvents. The lipase was completely inactivated by solvents with logP <2. n-Hexane has been
accepted as the most adequate solvent as it was selected also as a component of the mobile
phase for HPLC.
The kinetic parameters of the processes were calculated from the Michaelis – Menten
equation. They are listed in Tab.2.
Table 2. Enantiomeric excess (e.e.) and kinetic parameters (V max, KM) of enzymatic reactions catalysed
by immobilised Pseudomonas sp. lipase in membrane bioreactor.
Reaction
transesterification
esterification
hydrolysis
Chiral substrate
trans-2-methyl-1-cyclohexanol
menthol
2-(2-fluoro-4-biphenyl)propanoic acid
(4-isobutylphenyl)propanoic acid
trans-2-methyl-1-cyclohexyl acetate
trans-2-methyl-1-cyclohexyl benzoate
Vmax [mol/h mg]
3.90 x 10-4
8.79 x 10-5
5.34 x 10-6
5.79 x 10-6
3.28 x 10-4
1.16 x 10-4
KM [M]
1.70 x 10-2
9.93 x 10-3
7.55 x 10-3
6.23 x 10-3
1.01 x 10-2
1.35 x 10-2
Another problem concerning the above mentioned processes is the content of water
that should be added to organic solvent; It is necessary to maintain the proper structure of the
enzyme catalytic centre. It was found that both in esterification and transesterification, the
optimum constants of the Michaelis – Menten equation (KM and Vmax) can be obtained at 1%
(v/v) concentration of water.
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a)
product
substrate
b)
product
substrate
The studied reactions were carried out
at various chiral substrate concentrations and
the constant molar ratio of chiral / achiral
substrates. It was proved that the optical
purity of the chiral products and/or substrates
is related to the degree of conversion and
does not depend on the initial concentration
of substrates. The processes are typically
reversible, so it was important to estimate the
optimum molar excess of the achiral
substrate. In transesterification, it equals ~10,
and in estrification - in the range 2 - 5.
The courses of the three kinds of
processes are presented in Fig.1 a,b,c as
dependences of the enentiomeric excess of
the proper substrate and product versus the
process conversion. Plots for the other
substrate are similar.
c)
product
substrate
Figure 1.
Dependences of e.e. versus conversion for: a)
transesterification
of
trans-2-methyl-1cyclohexanol, b) esterification of 2-(2-fluoro-4biphenyl)propanoic acid, c) hydrolysis of trans-2methyl-1-cyclohexyl acetate.
CONCLUSION
The enzyme membrane reactor could be used for producing substances with pure
enantiomeric blocks needed for further syntheses of enantiomerically pure compounds. The
enantiomeric excess could be as high as 95 % and more. It is convenient to perform the
processes in reactors with lipase immobilised chemically within the polyamide membrane.
The catalytic activity of lipase from Pseudomonas sp. immobilised in the membranes was
more stable when compared to the activity of its native form. The membranes allowed at least
500 h operation of the reactors at constant activity. The reaction rate and optical purity depend
dramatically on some factors such as the type of solvent, the amount of water phase (in
esterification and transesterification) and chiral/achiral substrate molar ratio.
The processes can be easily carried out, and the strategy is worth applying for
resolution of many other chiral alcohols, carboxylic acids and esters, too.
LITERATURE: [1] S.M. Roberts, J. Chem. Soc., Perkin Trans. 1 (1999) 1-21, [2] K. Sakaki, L. Giorno,
E. Drioli, J. Membr. Sci. 184 (2001) 27-38, J. Ceynowa, I. Koter, Acta Biotechnol. 17 (1997) 253-263, [3]
C.S. Chen, C.J. Shin, Angew. Chem. 28 (1989) 695-707, [4] V.S. Parmar, K.S. Bisht, A. Singh, Proc. Indian.
Acad. Scai. 18 (1996) 575-583, [5] C.C. Akoh, L.N. Yee, J. Mol. Catal. B: Enzymatic 4 (1998) 149-153.
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