Polaina J., MacCabe A.P. (ed.). Industrial Enzymes. Structure, Function and Applications

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308 GOTOR-FERNÁNDEZ AND GOTOR

Scheme 11. Enzymatic resolution of 1,2-amino alcohols

(González-Sabin et al., 2004). In most cases conversions reached values closed to

50%, isolating substrate and product in enantiopure form (Scheme 11). The utility

of these -amino alcohols as chiral ligands has also been investigated.

6. ENZYMATIC AMINOLYSIS AND AMMONOLYSIS REACTIONS

Traditional synthetic methods to obtain optically pure amines have used chiral

catalysts for the reduction of amine precursors. However the preparation of enantio-

enriched amines via lipase-catalysed enantioselective acylation can be accomplished

using mild conditions, non-toxic reagents and easy experimental procedures, and it

is also possible to recycle the biocatalyst. As a result, the use of enzymatic methods

for the preparation of chiral nitrogenated compounds has rapidly gained prominence

in Green Chemistry and, of course, in large-scale industrial applications. In addition,

lipases are the most efficient hydrolases for catalysing the acylation of amines and

ammonia because they have very low amidase activity.

In the last few years several reviews have been published showing the utility

of lipases in ammonolysis and aminolysis reactions for the synthesis of nitrogen-

containing organic compounds (Gotor, 1999; van Rantwijk and Sheldon, 2004;

Alfonso and Gotor, 2004). In this section we describe a few representative examples

of enzymatic aminolytic and ammonolytic processes using lipases in organic

solvents. The best biocatalyst for these enzymatic reactions is generally CAL-B,

although in some cases CAL-A or PSL-C can also be used depending of the structure

of the amine.

Scheme 12 shows the general strategy to obtain enantiopure amines (González-

Sabín et al., 2002). Normally ethyl acetate is used for the acylation of amines,

in many cases as both acyl donor and solvent. Other acylating agents such alkyl

methoxy acetates are also of utility, however vinyl esters, the best reagents for the

resolution of alcohols, are not adequate for the resolution of primary amines due to

their high reactivity. Although there are many examples of KR of primary amines

(Alfonso and Gotor, 2004), few examples of the preparation of enantiomerically

pure secondary amines by enzymatic acylation have been reported.

In some cases sequential biocatalytic resolutions by ‘one-pot’ double enzymatic

reactions are of great utility because with moderate enantioselectivity in both

processes it is possible to achieve a high ee of substrate and product. An example

of this is the resolution of trans-cyclohexane-1,2-diamine and trans-cyclopentane-

1,2-diamine (Alfonso et al., 1996; Luna et al., 2002).

USE OF LIPASES IN ORGANIC SYNTHESIS 309

Scheme 12. KR of primary amines by enzymatic acylation

Scheme 13. DKR of an ester by aminolysis

Examples of DKR via enzymatic aminolysis reactions are scarce in the liter-

ature. Nevertheless, the resolution of racemic ethyl 2-chloropropionate amines using

Candida cylindracea lipase (CCL) has been carried out in this way catalysed by

encapsulated CCL in the presence of triphenylphosphonium chloride immobilized

on Merrifield resin (Scheme 13). This process yielded the S-enantiomer with high

yield and ee (Badji

c et al., 2001).

An elegant example of the DKR of amines has been described using ketoximes

as the starting material (Choi et al., 2001). The coupling of Pd-catalysed reduction

of ketoximes and the subsequent Pd and CAL-B catalysed DKR of the resulting

racemic amine afforded acetamides with very high chemical and optical yields. In

this process an additive such as N -ethyldiisopropylamine was required to suppress

the reductive deamination of the amine (Scheme 14). This procedure improves the

results of the first DKR reported (Reetz and Schimossek, 1996). By this means

the concentration of amine is low and the formation of by-products via reductive

amination is less favoured.

The resolution of secondary amines is a process that presents more difficulty than

the resolution of primary amines. Cyclic secondary amines are structurally easier to

resolve than acyclic compounds and examples of the resolution of pyrrolidine and

310 GOTOR-FERNÁNDEZ AND GOTOR

Scheme 14. DKR of racemic amines from ketoximes

piperidine derivatives have been described. The enzymatic acylation of pipecolic

acid derivatives has been catalysed by CAL-A with good results (Liljeblad et al.,

2002). This enzyme seems to have a larger pocket in the active site than CAL-B and

accepts bulkier substrates. This reaction has recently been applied to a pyrrolidine

ring and the enzyme was very efficient in catalysing the acylation of the secondary

amino group with very high enantioselectivity (Scheme 15).

The resolution of secondary amines via enzyme-catalysed acylation is not

frequently used. In the case of piperidine shown in Scheme 16, the molecule exists

as a pair of enantiomers due to atropisomerism about the exocyclic double bond.

The use of the lipase Toyobo LIP-300 and trifluoroethyl isobutyrate as the acylating

agent resulted in isobutyrylation of the +-enantiomer which was used as the

starting material for the synthesis of a product of physiological interest (Morgan

et al., 2000).

The first example of desymmetrization by enzymatic aminolysis and ammonolysis

reactions was described several years ago (Puertas et al., 1996). The aminolysis

of dimethyl 3-hydroxyglutarate with amines and ammonia in the presence

of CAL-B led exclusively to the corresponding monoamide of configuration

S (Scheme 17). The ammonia-derived enantiopure monoamide has been

Scheme 15. Example of the resolution of secondary amines

Scheme 16. KR of atropisomers with lipases

USE OF LIPASES IN ORGANIC SYNTHESIS 311

Scheme 17. Synthesis of R-GABOB by ammonolytic desymmetrization

Scheme 18. Resolution of a secondary amine by enzymatic alkoxycarbonylation

used to prepare the biologically interesting -amino acid R-3-hydroxy-4-

aminobutanoic acid [R-GABOB]. This desymmetrization process has also been

applied to other 3-substituted glutarates (López-García et al., 2003a), and some

of these enantiopure monoamides have been used to prepare -amino acids

(López-García et al., 2003b).

Recently enzymatic alkoxycarbonylation has been applied to resolve the

secondary amine 1-methyl tetrahydroisoquinoline (Scheme 18) using substituted

phenyl allylcarbonates as acylating reagents and Candida rugosa lipase (CRL)

as biocatalyst (Breen, 2004). The best solvent was found to be toluene and the

S-amine was recovered with 46% yield and 99% ee whilst the R-carbonate was

obtained at 47% yield and 98% ee. To achieve these results the amount of water

used in the process is critical.

7. NON-CONVENTIONAL REACTIONS OF LIPASES

In many cases, enzymes are able to catalyse more than one reaction. The challenge

is to use mechanistic reasoning to discover these new processes. Among the lipases

CAL-B is the biocatalyst that has shown the greatest promiscuity (Kazlauskas,

2005). This author defines ‘catalytic promiscuity’ as the ability of a single active

site to catalyse more than one chemical reaction. It has been reported that this lipase

can catalyse aldol condensations and Michael additions.

Aldol condensation of hexanal in cyclohexane is catalysed by CAL-B (Branneby

et al., 2003). The reaction is not enantioselective and the authors hypothesized that

the formation of a carbon-carbon bond did not require the active site serine. Indeed

replacement with alanine increased the aldol addition approximately two-fold. The

calculated transition-state structure for enolate formation is shown on the right in

Scheme 19.

Two recent articles have noted the potential of CAL-B to catalyse Michael-

type additions. This lipase catalyses the addition of thiols or secondary amines to

312 GOTOR-FERNÁNDEZ AND GOTOR

Scheme 19. Aldol condensation of hexanal catalysed by lipases

Scheme 20. CAL-B catalyses Michael additions

 -unsaturated aldehydes (Carlqvist et al., 2004). Quantum-modelling suggests

that the oxyanion hole in CAL-B activates the aldehyde for addition, the

histidine residue acts as a base, while serine is not involved in this process

(Branneby et al., 2004).

The reaction of acrylonitrile with secondary amines in the presence of CAL-B led

to the production of the corresponding Michael adduct faster than in the absence of

biocatalyst (Scheme 20) and a tentative mechanism for this new process has been

proposed (Torre et al., 2004).

A new strategy for the enzymatic synthesis of pyrimidine derivatives containing

a sugar branch has been developed combining enzymatic Michael addition and

acylation processes. The first step in the reaction between pyrimidines and vinyl

3-propionyloxy propionate was catalysed by Amano lipase M from Mucor javanicus

in DMSO, while the regioselective acylation of D-glucose and D-mannose with

the Michael adducts was catalysed by alkaline protease from Bacillus subtilis in

pyridine (Xu et al., 2005).

8. CONCLUDING REMARKS

The use of lipases has become a conventional process in organic synthesis, not

only for the preparation of optically pure compounds but also for regioselective

and chemoselective processes. Their utility in carrying out selective transformations

under mild reaction conditions make them attractive catalysts for performing certain

transformations that are difficult to achieve by chemical procedures. Nowadays

many companies use lipases for the preparation of chemicals instead of using

chemical catalysis because the use of these biocatalysts has enormous advan-

tages including the economy of the process, the environmental friendliness of the

USE OF LIPASES IN ORGANIC SYNTHESIS 313

catalysts and their recyclability. In addition, genetic engineering techniques can be

expected to play a major role in future research providing new biocatalytic pathways

ultimately leading to the generation of a great variety of new products.

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CHAPTER 19

USE OF LIPASES FOR THE PRODUCTION

OF BIODIESEL

ANDREA SALIS

∗

, MAURA MONDUZZI AND VINCENZO SOLINAS

Dipartimento di Scienze Chimiche, Università di Cagliari - CSGI, Monserrato (CA), Italia

∗

asalis@unica.it

1. INTRODUCTION

1.1. Why Biodiesel?

Biodiesel is composed of a mixture of fatty acid alkyl esters. It is a natural substitute

for petroleum-derived diesel fuel and has similar or better specifications (density,

viscosity, cetane number, flash point, etc.). Biodiesel is industrially obtained by

transesterification of vegetable oils or animal fats with short chain alcohols. When

methanol is used, the resulting biodiesel is a mixture of fatty acid methyl esters

(FAME). Other alcohols such as ethanol, iso-propanol or longer linear or branched

chains can also be used. Whilst methanol is the cheapest alcohol, the other alcohols

yield products with better performances (Knothe, 2005a). Since biodiesel comes

from renewable sources it is CO

-neutral, biodegradable and conserves fossil fuels.

Compared to traditional diesel fuel its combustion leads to a substantial reduction

in polluting emissions. Finally, biodiesel is less dangerous to handle than diesel

fuel because of its higher flash point (120



C compared to 61



C).

Interest in alternative energy sources is justified by high petroleum prices and

increasing environmental concerns. Countries committed to the Kyoto protocol

must decrease CO

and the other greenhouse gas emissions by 8% by 2012 –

with respect to those measured in 1990. In this context the European Union

has decided to increase the use of biofuels from 1.7 % in 2003 to 5.75 %

of total diesel fuel consumption by 2010. This means that production must

triplicate in the next few years, and justifies the major interest being given to this

biofuel.

317

J. Polaina and A.P. MacCabe (eds.), Industrial Enzymes, 317–339.

318 SALIS ET AL.

1.2. Disadvantages of Presently used Processes for Biodiesel Production

Fig. 1a shows the industrially used reaction for biodiesel production by homoge-

neous alkaline catalysis. The basic catalyst, i.e. sodium hydroxide, reacts with

methanol to yield sodium methoxide (Fig. 1b) that then reacts with the triglyceride to

produce the FAME. In order to increase the reaction rate, temperatures greater than

60–70



C are needed, demanding high energy consumption. The reaction products

are a mixture of esters and several by-products. The main by-product is glycerol

that, after purification, can be used for cosmetic and pharmaceutical purposes. Other

by-products are di- and mono-glycerides arising from the partial alcoholysis of

triglycerides. These by-products must be separated and pumped back to the reactor.

Free fatty acids, water and unreacted alkaline catalyst are also present. Thus compli-

cated purification processes are needed in order to obtain a pure biodiesel achieving

the standard requirements.

When the acidity of a feedstock is high the reaction between the free fatty acids

and the basic catalyst produces soap (Fig. 1c). Since soap is a surfactant it forms

emulsions and makes the separation between FAME and glycerol difficult. Thus, in

CHO

C OCH

CHOH

3 CH

Catalyst

(a)

OH + NaOH CH

–

+ H

(b)

RCOOH + NaOH RCOO

–

Fatty acid

Soap

(c)

(d)

RCOOH + CH

RCOOCH

+ H

FAME

Fatty acid

OCH

Methanol

Figure 1. (a) Methanolysis of a generic triglyceride for the production of Fatty Acid Methyl Esters

(FAME). (b) Formation of the methoxide. (c) Unwanted reaction of soap formation. (d) Acid catalysed

esterification of free fatty acids