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CHAPTER 18
USE OF LIPASES IN ORGANIC SYNTHESIS
VICENTE GOTOR-FERNÁNDEZ AND VICENTE GOTOR
∗
Departamento de Química Orgánica e Inorgánica, Instituto Universitario de Biotecnología,
Universidad de Oviedo, Oviedo, Spain
∗
vgs@fq.uniovi.es
1. INTRODUCTION
The use of enzymes for organic synthesis has become an interesting area for organic
and bio-organic chemists. Since many enzymes have been demonstrated to possess
activity against non-natural substrates in organic media they have become widely
used to carry out synthetic transformations. Hydrolases are the most frequently
used enzymes due to their broad substrate spectrum and considerable stability.
Additionally, many of them are commercially available and they work under mild
reaction conditions and without the necessity for cofactors. Among the hydro-
lases, lipases (EC 3.1.1.3) are considered the most popular and useful enzymes for
asymmetric synthesis (Wiktelius, 2005). Applications for lipases include kinetic
resolution of racemic alcohols, acids, esters or amines (Ghanem and Aboul-Enein,
2004), as well as the desymmetrization of prochiral compounds (García-Urdiales
et al., 2005). They are also successfully employed in regioselective esterification
or transesterification of polyfunctional compounds, for instance in the chemoen-
zymatic synthesis of nucleoside derivatives (Ferrero and Gotor, 2000). Recently,
non-conventional processes, such as aldol reactions or Michael addition have been
achieved using lipases (Bornscheuer and Kazlauskas, 2004).
2. LIPASES IN ORGANIC SOLVENTS
One of the more serious drawbacks for the use of enzymes in aqueous organic
synthesis is the poor water solubility of organic compounds with more of four carbon
atoms. Water is also a poor solvent for most applications in industrial chemistry,
since many organic compounds are unstable in aqueous solution. Furthermore,
301
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© 2007 Springer.
302 GOTOR-FERNÁNDEZ AND GOTOR
its removal is more tedious and expensive than that of organic solvents the
boiling points of which are lower. The use of organic solvents presents several
advantages including: (a) easier recovery of products with high yields; (b) the
possibility of using non-polar substrates; (c) organic solvents avoid many side
reactions, (d) in many cases lipases are thermodynamically more active than
in water; (e) shifting the thermodynamic equilibrium to favour synthesis over
hydrolysis.
Biocatalysis in non-aqueous media has been widely used for the resolution of
alcohols, acids or lactones by enzymatic transesterification reactions using different
lipases (Klibanov, 2001). Moreover, other processes such as the enzymatic acylation
of amines or ammonia have shown themselves to be of great utility for the resolution
of amines and the preparation of chiral amides.
The main difference between the enzymatic acylation of alcohols and amines in
organic solvents is the use of the corresponding acyl donor since activated esters
which are useful in the enzymatic acylation of alcohols, such as halogen-ethyl or
methyl esters, oxime ester, anhydrides and especially vinyl esters cannot be used
with amines because they normally react in the absence of a biocatalyst. Thus non-
activated esters must be used to carry out enzymatic aminolysis or ammonolysis
reactions.
Although the enzymatic acylation of alcohols or amines is the process that has
been most exhaustively studied, enzymatic alkoxycarbonylation for the activation
and protection of hydroxyl or amino groups is a process of great utility. In this type
of processes benzyl or allyl carbonates together with oxime or vinyl carbonates are
the most efficient alkoxycarbonylating reagents (Takayama et al., 1999).
3. LIPASES IN IONIC LIQUIDS
The use of ionic liquids has recently emerged in organic synthesis and in some cases
can be of great utility in biocatalysis (Jain et al., 2005). It constitutes an alternative
for carrying out processes that present serious difficulties in organic solvents or
water (Irimescu and Kato, 2004). These solvents can be used in three different ways
in an enzymic system: (a) as a cosolvent in the aqueous phase, (b) as a pure solvent,
and (c) as a two-phase system together with other solvents. Another possibility in
biocatalysis is to carry out reactions under supercritical conditions. Although some
progress has been achieved in this field, a major inconvenience is the need for
sophisticated equipment. Nevertheless, this ‘Green Chemistry’ alternative could be
of particular interest in the future.
One of the first examples of the use of ionic liquids was the synthesis of
octanamide (Madeira Lau et al., 2000) by the reaction of octanoic acid and
ammonia using Candida antarctica lipase B (CAL-B) as the biocatalyst (Scheme 1).
Enzymatic aminolysis of carboxylic acids is more difficult to accomplish than that
of esters because of the tendency of the reactants to form unreactive salts, however
using carboxylic acids in the presence of ionic liquids the process takes place with
high yields.
USE OF LIPASES IN ORGANIC SYNTHESIS 303
Scheme 1. Enzymatic preparation of octanamide in ionic liquids
4. HYDROLYSIS OF ESTERS AND CARBONATES
Nowadays enzymatic transesterification is a process that is widely used for the
preparation of chiral compounds. However, enzymatic hydrolysis, the natural
reaction of lipases, is also very useful for the resolution of racemic mixtures or the
desymmetrization of prochiral compounds. Hydrolysis and transesterification can
be complementary processes for the resolution of secondary alcohols (Scheme 2).
Both reactions fit Kazlauskas’ rule (Kazlauskas et al., 1991) and the R-isomer
reacts in both processes faster than its S-counterpart.
There are a number of enzymatic hydrolysis reactions that are of great utility
for the synthesis of pharmaceuticals or their intermediates (Gotor, 2002). For
example, -adrenergic blocking agents such as propanolol, have been synthesized
by chemoenzymatic methods, where the key step is an enzymatic hydrolysis. The
main reason for preparing these amino alcohols in optically pure form is that
the activity of these pharmaceuticals resides in the S-enantiomer. In addition,
the preparation of single enantiomers of arylpropionic acids (non-steroidal anti-
inflammatory agents) and optically pure 1,4-dihydropyridine derivatives (calcium
antagonists) are important issues in the pharmaceutical industry.
The enzymatic desymmetrization of the prochiral compound diethyl 3-(3
,
4
- dichlorophenyl)-glutarate (Homann et al., 2001), an intermediate in the synthesis
of neurokinin receptor antagonists, has been successfully developed and scaled up
by enzymatic hydrolysis with CAL-B, obtaining the acid-ester enantiopure with
80% yield (Scheme 3).
Hydrolysis and transesterification desymmetrization processes are in several
cases complementary reactions of utility in obtaining both enantiomers of a
compound with very high enantioselectivity. The synthesis of R- and S-1-amino-
2,2-difluorocyclopropanecarboxylic acids via lipase-catalysed desymmetrization of
Scheme 2. Symmetry in enzymatic hydrolysis and transesterification
304 GOTOR-FERNÁNDEZ AND GOTOR
Scheme 3. Hydrolytic desymmetrization of 3-substituted glutarates
Scheme 4. Symmetry of hydrolysis and acylation in enzymatic desymmetrization
prochiral diols and diacetates has been reported (Kirihara et al., 2003). In both
cases the products were isolated with very high yield and enantiomeric excesses
(Scheme 4).
Although enzymatic ester hydrolysis has been widely applied in enzymatic
resolution or desymmetrization processes, the enzymatic hydrolysis of carbonates
has been scarcely reported. A practical example that we have carried out is the
chemoenzymatic synthesis of S-zoplicone, a hypnotic that has a pharmaceutical
profile of high efficacy and low toxicity. This was achieved by the hydrolysis of
carbonates using lipases as biocatalysts (Fernández-Solares et al., 2002).
5. ESTERIFICATION AND TRANSESTERIFICATION PROCESSES
Enzymatic transesterification processes are more widely used than esterification
reactions in resolution or desymmetrization processes, and the acylation of alcohols
using lipases is currently the most frequently used process in biocatalysis (Faber,
2004). Whilst several agents can be employed, the most efficient are vinyl
esters, especially vinyl acetate which in many cases is used as both solvent and
acyl donor. There are many examples of the resolution of primary and secondary
alcohols. Although the reaction with tertiary alcohols is more difficult some
examples have been reported (Krishna et al., 2002).
Here we choose a few representative examples of acylation of alcohols where
enzymatic transesterification is the key step in the synthesis of chiral pharmaceu-
ticals. Fluoxetine, tomoxetine and nisoxetine, three antidepressants, were synthe-
sised from racemic 3-chloro-1-phenylpropan-1-ol (Liu et al., 2000) by enzymatic
USE OF LIPASES IN ORGANIC SYNTHESIS 305
Scheme 5. Enzymatic transesterification of pharmaceutical intermediates
transesterification in hexane using vinyl butanoate (VB) as the acyl donor and
CAL-B as the best biocatalyst (Scheme 5). It is also possible to obtain these pharma-
ceuticals by resolution of 3-hydroxy-3-phenylpropanenitrile as the starting material
using Pseudomonas cepacia lipase (PSL) and vinyl acetate (VA) as the acyl donor
(Kamal et al., 2002).
Kinetic resolution (KR) is a useful method to obtain enantiomerically pure
compounds but suffers the drawback that the maximum yield is 50% of the starting
material. This limitation can be overcome via dynamic kinetic resolution (DKR),
in which the slower reacting enantiomer racemises during the process (Scheme 6).
This procedure can theoretically lead to a single product enantiomer with 100%
yield.
DKR has appeared in asymmetric catalysis in the last decade as a common
methodology involving a lipase as the biocatalyst and a metal-organic complex as
the chemical catalyst (Pàmies and Bäckvall, 2003). This strategy has been widely
used for the resolution of alcohols by enzymatic hydrolysis or transesterification
processes. For instance, the synthesis of S-propanolol by DKR of the corres-
ponding azidoalcohol using a combination of a ruthenium complex and CAL-B in
toluene at 80
C and p-chlorophenyl acetate (PCPA) as the acyl donor (Scheme 7)
has been described. After 1 day the R-acetate was produced with > 99% ee and
94% conversion (86% isolated yield). The enzyme was recycled and used again for
another DKR without any loss of activity (Pàmies and Bäckvall, 2001).
Scheme 6. Schematic representation of a DKR process
306 GOTOR-FERNÁNDEZ AND GOTOR
Scheme 7. Chemoenzymatic synthesis of S-propanolol by DKR
In some cases the use of vinyl or alkyl esters as acyl donors has the drawback
of the need to separate the ester (product) from the alcohol (substrate). A practical
strategy to avoid this problem is the use of cyclic anhydrides (Bouzemi et al., 2004).
In this case an acid is obtained as the product which can be readily separated
from the unreacted alcohol by a simple aqueous base-organic solvent liquid-liquid
extraction (Scheme 8).
This strategy has been applied to the resolution of N -substituted trans-
4-(4
-fluorophenyl)-3-hydroxymethylpiperidines as key intermediates in the
synthesis of −-paroxetine which is a potent and selective inhibitor of
5-hydroxytryptamine reuptake and is used in the treatment of a variety of human
diseases such as depression (De Gonzalo et al., 2003). The best results were obtained
with a combination of CAL-B, glutaric anhydride and toluene as the solvent. In
addition, the reuse of the immobilized lipase afforded the same enantioselectivity
in the second and third cycles with just a moderate loss of enzyme activity in the
fourth and fifth cycles.
Lipases are also valuable tools for the resolution of biaryl derivatives with axial
chirality (Sanfilippo et al., 2003). Thus, Pseudomonas cepacia lipase (PSL) has been
used for the transesterification of 2 2
-dihydroxy-6 6
-dimethoxy-11
-biphenyl in
a reaction using vinyl acetate as the acyl donor and tert-butyl methyl ether as
Scheme 8. Enzymatic resolution of alcohols using anhydrides as acyl reagents
USE OF LIPASES IN ORGANIC SYNTHESIS 307
the solvent. Product of configuration R was obtained with >98% ee while the
S-substrate was recovered with >96% ee (Scheme 9). These compounds are of
great interest as ligands in asymmetric organic synthesis.
As commented above, in enzymatic desymmetrization processes it is possible
to achieve an enantiopure compound with a maximum yield of 100%. For this
reason these reactions constitute a very interesting alternative to KRs. One of the
families of substrates to which more attention has been being paid is that containing
the propane-1,3-diol moiety because this group is present or can easily lead to
many molecules that play important roles in medicinal chemistry and/or asymmetric
synthesis (Neri and Williams, 2003).
The first enzymatic desymmetrization of prochiral phosphine oxides has recently
been published (Kielbasinski et al., 2003). In this case the chiral centre is the
phosphorus atom. The prochiral compound bis(hydroxymethyl)phenyl-phosphine
oxide was desymmetrized using either lipase-catalysed acetylation with vinyl acetate
as acyl donor in an organic solvent, or enzymatic hydrolysis of the corresponding
diacetate in phosphate buffer and solvent (Scheme 10). The monoacetate was
obtained in 79% ee and 76% yield.
Chiral 1,2 and 1,3-amino alcohols have proven to be a functionally active class
of compounds of wide application in medical chemistry. The N -acylation to obtain
the corresponding amide gives very poor yields because migration of the acyl group
normally takes place. For this reason, to achieve a good bioresolution with amino
alcohols it is necessary to protect the amino or hydroxyl group. For instance, the
four isomers of 1,2-aminoindanols can be resolved using carbamate derivatives. Of
special relevance is the enzymatic transesterification of cis-1S2R-1-aminoindan
-2-ol (Luna et al., 1999), a key component of indinavir which is a potent inhibitor
of the protease of the human immunodeficiency virus (HIV). Optically active
trans-2-(N N -dialkylamino)-cyclohexanols have been easily prepared in a two-
step sequence: ring opening of cyclohexene oxide and subsequent resolution of
the resulting racemic amino alcohol by transesterification catalysed by PSL-C
Scheme 9. Enzymatic resolution of a compound with axial chirality
Scheme 10. Enzymatic desymmetrization of prochiral phosphine oxide