Lipase-catalyzed synthesis of carboxylic amides: Nitrogen nucleophiles as acyl acceptor

Article abstract of DOI:10.1007/s007060070086

The lipase-catalyzed aminolysis of carboxylic esters is a fairly general reaction that has been performed with a wide range of esters and amines, generally in anhydrous organic media to avoid undesirable hydrolysis of the ester. Alternatively, carboxylic amides can be synthesized by lipase mediated condensation of carboxylic acids and amines if an excess of either reactant is avoided. Chiral carboxylic esters have been resolved by lipase-catalyzed aminolysis. In the majority of these resolutions, Candida antarctica lipase B has been employed as the catalyst. A range of chiral amines has been resolved by lipase mediated acylation, using mainly the lipases from C. antarctica (B type) and Pseudomonas species. The enantiorecognition was frequently found to depend critically on the acylating agent and the reaction medium.

Full text of DOI:10.1007/s007060070086

Monatshefte fuÈr Chemie 131, 549±569 (2000)
Invited Review
Lipase-Catalyzed Synthesis of Carboxylic
Amides: Nitrogen Nucleophiles as Acyl Acceptor
Ã
Fred van Rantwijk , Michiel A. P. J. Hacking, and Roger A. Sheldon
Laboratory of Organic Chemistry and Catalysis, Delft University of Technology, NL-2628 BL Delft,
The Netherlands
Summary. The lipase-catalyzed aminolysis of carboxylic esters is a fairly general reaction that has
been performed with a wide range of esters and amines, generally in anhydrous organic media to
avoid undesirable hydrolysis of the ester. Alternatively, carboxylic amides can be synthesized by
lipase mediated condensation of carboxylic acids and amines if an excess of either reactant is
avoided.
Chiral carboxylic esters have been resolved by lipase-catalyzed aminolysis. In the majority of
these resolutions, Candida antarctica lipase B has been employed as the catalyst. A range of chiral
amines has been resolved by lipase mediated acylation, using mainly the lipases from C. antarctica
(B type) and Pseudomonas species. The enantiorecognition was frequently found to depend critically
on the acylating agent and the reaction medium.
Keywords. Lipase; Candida antarctica lipase; Amine; Carboxylic amide; Enantioselectivity.
Introduction
There is currently a marked trend in the ®ne chemicals industry towards the
reduction, or preferably elimination, of waste by widespread substitution of classical
syntheses employing stoichiometric reagents with cleaner catalytic alternatives [1].
Enzymes occupy a special position in the catalysis ®eld because they operate at
moderate temperature and mild reaction conditions, which is advantageous with
sensitive reactants and products. Moreover, enzymes are inherently enantioselec-
tive. In consequence, enzyme catalysis is destined to play a major role in the
transition from stoichiometric to catalytic transformations, particularly if it can be
made to transcend the limitations of natural reactions in aqueous medium by
replacement of the natural reactant by a non-natural one. Lipases, the lipid
hydrolyzing catalysts of nature, are eminently suited for such an approach because
of their stability and the simplicity of their catalytic machinery (Fig. 1). These
characteristics have made them outstanding catalysts for synthetic biotransforma-
tions that involve the carboxyl group, such as esteri®cation, transesteri®cation,
Ã
Corresponding author

550
F. van Rantwijk et al.
perhydrolysis, and aminolysis in which the natural nucleophile (water) is replaced
by alcohols, hydroperoxides, or amines [2, 3]. The principle of lipase-catalyzed
aminolysis, resulting in the synthesis of carboxylic amides, has been demonstrated
for the ®rst time in 1984/85 [4, 5], and a multitude of publications describing its
application has been published since.
This review describes the use of nitrogen nucleophiles in the acyl accepting
role as a synthetic route to carboxylic amides as well as the resolution of chiral
carboxylic acids, esters, and amides by lipase catalyzed aminolysis.
Mechanistic Considerations
Lipases, the lipid splitting catalysts of nature (EC 3.1.1.3), operate by essentially the
same mechanism as that of the serine proteases [6]. The active site (see Fig. 1)
contains a serine residue that is activated by histidine and aspartate residues; these
together form the catalytic triad. The reactant ester forms a tetrahedral acyl-enzyme
intermediate by reaction with the OH group of the catalytic serine residue; the
resulting excess of negative charge that develops on the carbonyl oxygen atom is
stabilized by the oxyanion hole. Next, the tetrahedral intermediate collapses to the
serinate ester with elimination of the alcohol. Subsequent reaction of the
acylenzyme intermediate with the nucleophile ± the acyl acceptor ± affords the
product. In the case of hydrolysis, which is the natural reaction of lipases, the
nucleophile is water. However, almost any nucleophile can, in principle, react with
the acyl-enzyme intermediate [2].
Ester aminolysis can be regarded as irreversible, because the transformation is
strongly exothermic under normal reaction conditions [7]. Amide hydrolysis, which
is, inter alia, not readily mediated by lipases, can also be excluded due to the almost
universally employed anhydrous reaction conditions (see below). Lipase mediated
aminolysis of a carboxylic amide, i.e. transamidation, has been reported only once
and involved a highly activated N-2,2,2-tri¯uoroethyl amide as donor [8].
Fig. 1. Reaction mechanism of lipase catalysis; the numbering is for Candida antarctica lipase,
- - - - - denotes a hydrogen bond; step (iii) is the microscopic reversal of steps (i) and (ii)

Lipase-Catalyzed Synthesis of Amides
551
Although lipases have not been designed for enantioselectivity, they are chiral
and, hence, inherently enantioselective towards all components of the reaction: the
acyl group, the leaving group, and the nucleophile. The resolution of chiral amines
is one of the major practical applications of lipase-catalyzed aminolysis.
The Use of Lipases in Aminolysis
Lipases1 are ubiquitous in Nature. Initially the very impure [9] porcine pancreas
lipase (PPL) ± easily obtained from slaughter waste ± and Candida rugosac2 lipase
(CrL), a rather unstable enzyme actually consisting of a mixture of isozymes, were
widely used. The recombinant microbial lipases that are now widely available
generally do not contain contaminating activity and are much more stable. Some of
these have been developed for application in synthetic biotransformations, but most
of them were originally intended to be used in laundry formulations or the
upgrading of food triglycerides.
Water, which is the natural reaction medium of enzymes, is not a good solvent
for aminolysis, because hydrolysis is bound to predominate. In consequence,
organic solvents are used almost universally. Even trace quantities of water in the
reaction mixture will lead to undesirable hydrolysis of the acyl donor; apart from
the resulting loss in yield, the liberated acid may effect deactivation of the lipase.
Hence, aminolysis is preferably carried out in a strictly anhydrous medium; in
some cases, activated zeolites have been added to the reaction mixture to ensure
anhydrous reaction conditions. Lipases, in particular those from microbial sources
that are commonly used nowadays, are exceptionally rugged enzymes and tolerate
anhydrous organic solvents rather well. In particular, Candida antarctica lipase B
(CaLB) [10, 11], which actually seems to prefer anhydrous conditions [12], has
been widely used in consequence. We note that some lipases, such as that from
Thermomyces lanuginosus3 (TIL), that require some water to catalyse esteri®ca-
tion, still act as an ammoniolysis catalyst in dry medium [13]. Presumably,
ammonia acts as a water replacement.
We have found that ammoniolysis catalyzed by Novozym 435 (CaLB on
Lewatit E) is sometimes accompanied by unexpected hydrolysis of the reactant in
spite of rigorous exclusion of moisture. Apparently, the acrylic carrier strongly
adsorbs traces of water that are ¯ushed out by ammonia. This could be easily
remedied by using the hydrophobic Accurel EP 100 (a macroporous polypropy-
lene) as lipase carrier [7, 14].
The ef®cient use of enzymes in non-aqueous media necessitates their
immobilization on a suitable carrier material [15], presumably because the use
of `free' protein dispersions renders the major part of the enzyme molecules
inaccessible to the reaction medium. Nevertheless, suspensions of lyophilisate are
still often used. Adsorption at the non-polar surface of organic polymers has
evolved into a dominant technique for the immobilization of lipases. Lipases
generally adsorb readily at a non-polar surface, because in their natural function
1
A review of the structure, mechanism, and application of lipases is given in Ref. [3]
2
Candida rugosa was previously known as Candida cylindracea
3
Previously classi®ed as Humicola lanuginosa

552
F. van Rantwijk et al.
they adsorb at the water-lipid interface; a number of lipases are even interfacially
activated. It should be kept in mind, however, that adsorption is a reversible process
in aqueous medium [15]. Moreover, a major part of the activity is lost [15], which
is probably caused by the partial disruption of the native structure by the
hydrophobic interaction of lipase and carrier [16]. We have shown, nevertheless,
that in ammoniolysis lipases adsorbed on macroporous polypropylene (Accurel
EP100) are more ef®cient than a free suspension of an equal amount of lyophilisate
[15].
Alternatively, lipases can be covalently bound to a solid carrier such as Eupergit
C. The activated carrier is expensive, however, and its protein binding capacity is
low. The encapsulation of lipases in hydrophobic sol-gel materials which has
recently been developed [17] shows considerable potential. The very stable lipase
cross-linked crystals [18] (CLCs) such as ChiroCLEC-CR and -PC from CrL and
Burkholderia cepacia4 lipase (BcL), respectively, have the advantage that the
catalyst material is 100% protein. It has also become clear recently that CLCs of
CrL maintain their activity under (aminolysis) conditions that deactivate the native
lipase [19].
A number of lipases are commercially available as adsorbed, covalently bound,
encapsulated, or cross-linked immobilisates. Moreover, adsorption of lipases on
Accurel EP 100 is simple and straightforward [20, 21]. Finally, the modi®cation of
lipases to render them soluble in organic media deserves to be mentioned.
Although a solubilized lipase has been employed in the ®rst published example of
lipase-catalyzed aminolysis [4], this methodology has not found wide application.
Synthesis of Carboxylic Amides
The lipase-catalyzed aminolysis of carboxylic esters has ®rst been described in
1984 by Inada et al. [4] in a paper that also constituted the ®rst use of a lipase in
organic solvent, closely followed by a publication by Zaks and Klibanov [5].
Amines
Aminolysis seems to be a fairly general reaction that has been reported for a wide
range of alkylamines, including allyl- and benzylamine [22]. Aniline derivatives
are weak bases and ± similar to phenols ± weak nucleophiles. Only a few examples
of lipase-catalyzed anilide synthesis, which required massive amounts of CrL, have
been published [23,24]. Hydroxyalkylamines are preferentially acylated at the
nitrogen atom [25,26], as would be expected on the basis of nucleophilicity as well
as bond strength. The competition between the acylation of a secondary amine and
a primary alcohol function was less one-sided [27, 28], and the actual outcome of
the acylation of N-methylglucamine (Fig. 2) depended on the ionization state and,
hence, the nucleophilicity of the nitrogen atom [28].
Although ammonia is the simplest amine, its use as acyl acceptor ±
ammoniolysis ± was reported only fairly recently by us [29] as well as others
[22]. Many lipases catalyzed this reaction, although some do not maintain their full
4
Burkholderia cepacia was previously classi®ed as Pseudomonas capacia

Lipase-Catalyzed Synthesis of Amides
553
Fig. 2. O-vs. N-acylation of N-methylglucamine mediated by Rhizomucor miehei lipase (RmL)
Fig. 3. Ammoniolysis of olive oil
activity at high ammonia concentrations [30]. CaLB (Novozym 435) is particularly
stable and ef®cient in ammoniolysis as well as in aminolysis is general. It has been
used, for example, in the transformation of triolein (olive oil) into oleamide (see
Fig. 3) [31]. A potential advantage of the enzymatic procedure is that it affords a
product of higher purity which obviates the need for puri®cation via distillation.
Hydroxylamine and hydrazine, which are also weak bases but, due to the ꢀ-effect,
strong nucleophiles, readily act as acyl acceptor. Accordingly, the hydroxyl-
aminolysis of fatty acid esters, catalyzed by Rhizomucor miehei lipase (RmL),
afforded the corresponding hydroxamic acids (N-hydroxyamides) [32]. Hydrazino-
lysis was, remarkably, ®rst reported for a number of ± deactivated ± acylhydrazines
[33] andonlylateron alsoforhydrazine proper[34], usingmassive amountsof Amano
P lipase in both cases.
The hydroxylaminolyis and hydrazinolysis of ethyl octanoate have been
extensively investigated in our group [20]. Both nucleophiles require the presence
of at least one equivalent of water to prevent irreversible deactivation of the
biocatalyst. Hence, hydroxylaminolysis is accompanied by partial hydrolysis of the
acyl donor, whereas in hydrazinolysis the hydrolytic side reaction was almost absent.
Because the acid resulting from hydrolysis also was converted [32a], although at a
lower rate (see below), a nearly quantitative conversion into the hydroxamic acid was
eventually achieved.
Acyl donors: carboxylic esters
A wide range of esters, including ꢀ,ꢁ-alkenic [35, 36] and ꢁ-keto esters [22] have
been used as acyl donor. Ethyl propiolate, which gave Michael adducts with

554
F. van Rantwijk et al.
Fig. 4. Time course of the hydroxylaminolysis and competing hydrolysis of ethyl octanoate
catalyzed by TIL [20]; *: ethyl octanoate, ~: octanoic acid, !: octanohydroxamic acid; reaction
conditions: 688 mg (4 mmol) ethyl octanoate, 0.5 M NH2OH, 1.75 M H2O, 10 mg immobilized TIL
on Accurel EP 100 in 10 cm3 tert-butyl alcohol, 40ꢀC
Fig. 5. Aminolysis of diethyl (S)- and (R)-N-benzyloxycarbonylglutamate
alkylamines [23], reacted with aniline derivatives and ammonia [35] to give the
corresponding amides in high yield. The selective transformation of diesters to the
monoamide has been observed in hydrophilic solvents [36±39], whereas formation
of the succinimide was observed upon aminolysis of diethyl succinate in hexane
[38]. An interesting effect of the absolute con®guration of the reactant on the
regioselectivity is presented by diethyl N-benzyloxycarbonylglutamate (Fig. 5) [37].
Lactams were readily formed by the intramolecular aminolysis of 4- and 5-
amino-alkanoic esters, but 6-aminoalkanoic esters reacted only sluggishly to give
the "-lactams [40].
Dialkyl carbonates, the close structural relatives of carboxylic esters, have not
often been subjected to lipase-catalyzed transformations. We have found that only
CaLB converted carbonates from dibutyl carbonate upwards at a useful rate [41].
Reaction with alkyl amines or ammonia afforded the corresponding carbamate
without any further reaction to the ureum derivative (Fig. 6).

Lipase-Catalyzed Synthesis of Amides
555
Fig. 6. Aminolysis of dialkyl carbonates
Peptide synthesis
Lipase-catalyzed peptide synthesis offers a number of potential advantages com-
pared with competing methodologies. In contrast to chemical synthesis, circuitous
protection/deprotection strategies are not required. Compared with proteases, which
are restricted to L-amino acids, lipases offer a much more relaxed substrate
speci®city; moreover, product hydrolysis is virtually absent. These advantages were
recognized quite early, and a number of lipase-mediated dipeptide syntheses have
been published from 1987 onwards [42±45]. In order to impart the desired
selectivity, the amino function in the acyl donor was protected with an acyl group.
Out of a large number of lipases tested, only PPL mediated the synthesis at a useful
rate [45]. PPL was speci®c for donors with L-con®guration [44], whereas the
enantiomeric preference towards the acceptor was generally slight [42, 45] (Table
1). These results should be interpreted with some caution, because crude PPL is
known to contain protease and carboxylesterase contaminants which, in principle,
could be the origin of the observed activity [43]. CrL, which was much less active
than PPL, preferentially converted donors with D-con®guration [44].
Table 1. PPL mediated dipeptide synthesis
Donor
Acceptor
Solvent
Product
Time Yield Ref.
(h) (%)
Ac-N-L-Phe-OEtCl L-Leu-NH2 Toluene
Ac-N-L-Phe-OEtCl D-Leu-NH2 Toluene
N-Ac-L-Phe-L-Leu-NH2
72 83
[42]
[42]
[43]
[43]
[45]
[45]
N-Ac-L-Phe-D-Leu-NH2 > 72 76
Z-L-Phe-L-Ala-OBus
Z-L-Phe-D-Ala-OBus
Z-L-Phe-OMe
Z-L-Phe-OMe
Bz-N-L-Tyr-OEt
Bz-N-L-Tyr-OEt
L-Ala-OBus Et2O
D-Ala-OBus Et2O
16 85
16 60
L-Thr-OMe 3-Me-pentanol Bz-N-L-Tyr-L-Thr-OMe
D-Thr-OMe 3-Me-pentanol Bz-N-L-Tyr-D-Thr-OMe
7
7
92
73
Acyl donors: carboxylic acids
Because carboxylic acids and amines tend to form unreactive salts, it was generally
assumed that their condensation to the corresponding amides would not be feasible.
This opinion has persisted until recently in spite of a number of contrary results
that have appeared in the literature. Tuccio et al. found that a number of lipases
catalyzed the condensation of alkylamines and fatty acids ± ranging from acetic to
palmitic acid ± in anhydrous hexane [46]. The condensation of taurine and oleic
acid (Fig. 7) was mediated by Rml as well as CaLB [47], and it has been shown

556
F. van Rantwijk et al.
Fig. 7. Condensation of taurine and oleic acid
that for optimum yield the acid and amine reactants should be present in equal
amounts to maximize the concentration of both reactants in solution. The synthesis
of N-oleyl-N-methylglucamine from oleic acid and N-methylglucamine has been
investigated by the same group [28]. Quite recently, Litjens et al. showed that the
lipase-catalyzed reaction of butyric acid and ammonia is quite feasible [48]; these
authors have also established the parameters of the equilibria involved [49].
The lipase-catalyzed condensation of carboxylic acids with hydroxylamine and
hydrazine [20, 32a, 50] as well as their derivatives [20] presents a special case
because of the stability of the resulting amide bond. These reactions proceed readily
to essentially quantitative conversions (Table 2); the condensation of octanoic acid
and hydroxylamine has even be performed in water [20]. The advantage of starting
from the acid instead of the ester is twofold: it may save a reaction step, and there is
virtually no uncatalyzed background reaction, which is important in kinetic
resolutions (see later).
Even more surprisingly, many lipases also mediated the condensation of a
carboxylic acid with an acyl hydrazide to the N,N0-diacylhydrazine (Fig. 8) [51].
Table 2. Acylation of hydroxylamine and hydrazine derivatives
Donor
Ethyl octanoate
Octanoic acid
Initial rate
Initial rate
Yielda
(%)
1
1
1
1
Nucleophile
(mmol Á g Á min
}
(mmol Á g Á min
)
H2NOH
910
n.d.
200
n.d.
540
52
160
< 5
87
99
66
98
65
91
95
96
H2NOPh
H2NOCH2Ph
HN(CH2Ph)OH
H2NNH2
< 5
70
H2NNHPh
H2NNHCH2Ph
70
200
100
a
Reaction conditions: 57.6 mg (0.4 mmol) octanoic acid, 1.0 mmol nucleophile, and 50 mg Novozym
435 in 1.0 cm3 tert-butyl alcohol, 20 h, 40ꢀC

Lipase-Catalyzed Synthesis of Amides
557
Fig. 8. Time course of the reaction of hydrazine and octanoic acid in isooctane [51]; lipase: A: TIL,
B: PaL; ^: octanoic acid, !: octanohydrazine, ~: N,N0-dioctanoylhydrazine; reaction conditions:
1 mmol hydrazine hydrate, 2 mmol octanoic acid, 50 mg immobilized lipase in 5 cm3 isooctane,
40ꢀC; 100% yield of N,N0-dioctanoylhydrazine equals 1 mmol
TIL was conspicious for its high activity in this reaction. Some lipases, such as
Pseudomonas lipoprotein lipase, P. alcaligenes lipase (PaL), and C. antarctica
lipase A acylated octanohydrazide faster than hydrazine itself when both
nucleophiles were present in the reaction mixture.
Enantioselective Aminolysis
Although lipases have not been designed for enantioselectivity, it was recognized
quite early [52] that they are chiral and, hence, inherently enantioselective. This
feature has been exploited in aminolysis with the purpose of resolving chiral acyl
donors as well as amines. An inherent advantage of aminolysis as a resolution
strategy is that, due to the stability of the amide bond, the formation of a carboxylic
amide is virtually irreversible [7], which is a prerequisite for an ef®cient resolution
[53].
It is common practice to discuss a kinetic resolution in terms of its enantiomeric
ratio E [54] which is equal to the ratio of the pseudo-®rst-order rate constants of the
enantiomers according to Eq. 1. We note that formally this practice is not correct for
lipase-catalyzed resolutions, because lipases do not obey minimal Michaelis-
Menten kinetics and, hence, slight deviations of the predicted behaviour are to be
expected [55].
E ˆ ꢁkcat=Km†R=ꢁKcat=Km†
ꢁ1†
S
Resolution of the acyl donor
Because of the ping-pong mechanism of lipases the conversion of the acyl donor
involves a number of steps (see Fig. 1), each of which is subject to ± potentially
con¯icting ± enantiomer discriminating effects. The resulting enantiomeric

558
F. van Rantwijk et al.
Fig. 9. Effect of the acceptor on the CaLB-catalyzed resolution of ibuprofen
preference of the transformation is the sum of all these enantiodiscriminatory
processes. Hence, the outcome may depend on the reaction conditions and, in
particular, on the nature of the acyl acceptor. The CrL mediated aminolysis of methyl
2-chlorobutanoate, for example, was moderately (S)-selective (E ˆ 9)5 [8, 56, 57]. In
contrast, the esteri®cation of 2-chloropropionic acid, mediated by the same catalyst,
was highly (R)-speci®c [58, 59], whereas the hydrolysis of methyl 2-chlorobutanoate
was only slightly biased in favour of the (R)-enantiomer (E ˆ 2) [57, 60]. The N-alkyl-
azetidine-2-carboxylates (1, Fig. 12) present a comparable case: their resolution could
not be accomplished by enzymatic hydrolysis [61], but ammoniolysis catalyzed by
CaLB gave the (R)-ester and the (S)-amide with high selectivity [62]. A ®nal example
is the CaLB mediated ammoniolysis of the chloroethyl ester of ibuprofen, which was
much more enantioselective than its hydrolysis or alcoholysis (Fig. 9) [29, 30].
In contrast with CaL, CrL is known to convert the physiologically active (S)-
ibuprofen preferentially [63]. This feature was exploited in the synthesis of (S)-
ibuproxam, a prodrug, by condensation of racemic ibuprofen with hydroxylamine
in aqueous medium (Fig. 10). Crosslinked crystals of CrL (CLEC-CR) were used
as the catalyst, because the native enzyme was deactivated under the reaction
conditions [19].
Acyl donor resolution would become much more interesting if it could be
combined with racemization of the slow-reacting enantiomer to achieve a 100%
Fig. 10. CLEC-CR mediated synthesis of (S)-ibuproxam
5
E-factors, when not provided by the authors, were calculated from the conversion (c) and the
enantiomeric purity of the product (eep) using the equation derived by Chen et al. [54]:
E ˆ lnꢁ1 c Á ꢁ1 ‡ eep††=lnꢁ1 c Á ꢁ1 eep††

Lipase-Catalyzed Synthesis of Amides
559
Fig. 11. Ammoniolytic synthesis of (R)-phenylglycine amide by dynamic kinetic resolution
Fig. 12. Enantiorecognition of chiral acyl groups by CaLB
yield of enantiomerically pure product. This has been demonstrated for the CaLB
mediated synthesis of (R)-phenylglycine amide from its racemic ester [64]. By
combining the ammoniolysis with in situ-racemization of the slow-reacting (S)-
ester via a Schiff base, a dynamic kinetic resolution was developed [65]. Initially
the ee of the amide was rather low, but it could be improved signi®cantly by
lowering the reaction temperature (Fig. 11) [14].
The enantiomeric preference of CaLB in the aminolysis of ꢀ-substituted acyl
donors is summarized in Fig. 12. The preferentially formed enantiomers of 1 [62],
ibuprofen amides (2) [19, 29, 30], phenylglycine amide (3) [64], and derivatives of
glutamic acid (4) [37] seem to correspond with a common steric model with the
substituents arranged as indicated in Fig. 12. Only the selective (E ˆ 25) formation
of (S)-N-pentylpyroglutamic amide (5) [66] does not easily ®t into the pattern.

560
F. van Rantwijk et al.
Fig. 13. BcL mediated resolution of benzyl ꢀ-hydroxycarboxylates
A number of ꢀ-hydroxycarboxylic acid benzyl esters were transformed into
their benzylamides in the presence of BcL [67]. The (S)-amides were preferentially
formed (Fig. 13), whereas the CaLB mediated aminolysis of ꢀ-aminocarboxylic
acid esters was (R)-speci®c (Fig. 12).
Chiral 3-substituted acyl groups are hardly less ef®ciently discriminated by CaLB
than 2-subsituted ones, in spite of the increasing distance from the reactive centre. The
ethyl ester of 3-aminobutyric acid [68] was converted into benzamide 6 with moderate
enantioselectivity, but the corresponding 3-hydroxy compound (7) was formed with
almost absolute enantioselectivity [69, 70] (Fig. 14); the ammoniolysis of ethyl 4-
chloro-3-hydroxybutyrate likewise was highly selective [71]. In contrast, BcL acted
with negligible enantioselectivity in the benzylaminolysis of ethyl 3-hydroxybutyrate
[69].
Prochiral diesters, such as dimethyl 3-hydroxyglutarate, are interesting reactants
because their monoaminolysis could, in principle, afford a 100% yield of enantiopure
product. Indeed it was found [72] that the pro-R ester group in dimethyl 3-hydroxy-
glutarate was preferentially converted to the monoamides 9 with complete selectivity
by CaLB mediated aminolysis (Fig. 14). The enantioselective aminolysis of chiral
vinyl carbonates to the corresponding carbamates (10), which is not really an acyl
resolution, was also quite ef®cient [73].
It has been explained above that the enantiorecognition of a chiral acyl group
involves many steps. Hence, the structure of the amine would be expected to
contribute to the overall effect, and intuitively one would expect enantiorecognition
to become more ef®cient with sterically demanding amines. Systematic studies in
this ®eld have been few; the available data, which involve ꢁ-amino- and ꢁ-
hydroxyesters, are compiled in Table 3. Based on this very restricted data set,
benzylamine would seem to be the resolving agent of choice.
Fig. 14. Enantiomeric preferences of CaLB in the aminolysis of ꢁ-substituted acyl donors

Lipase-Catalyzed Synthesis of Amides
561
Table 3. Effect of the amine on the enantiorecognition of CaLBa
Product
R1
CH3
CH2CH3 CH2CH2Cl CH2CH3 n-C6H13 C6H5
R2 ˆ H
4
14
68
44b
R2 ˆ n-Bu
R2 ˆ Benzyl
R2 ˆ n-Octyl
Reference
17
27
52
43
> 100
20
90
700
160
73
24
200
10
63
183
22
> 100
30
> 100
70
70
70
73
73
a
Enantiomeric ratios as taken from the references or calculated from conversion and ee; bdata taken
from Ref. [71]
Solvents often exert dramatic effects on enzymatic resolutions [74], and an
inversion of the enantiopreference has even been observed in certain cases. The
effect of the solvent on the CaLB mediated condensation of ibuprofen and
hydroxylamine to (R)-ibuproxam (2, R ˆOH) was only modest, however [19]. E
ranged from a value of 4 in water and some moderately hydrophilic solvents via 8
in isooctane or tert-butyl alcohol to 13 in dioxane. The enantiomer discrimination
in the CaLB-catalyzed ammoniolysis of phenylglycine methyl ester (Fig. 11) was
likewise little affected by the nature of the solvent [14].
Resolution of chiral amines
Acylation studies of secondary alcohols have shown that the nucleophile subsites
of all lipases have the same steric preference. A model that has originally been
constructed for CrL and BcL [75] was later found to be valid for many other lipases
± although not always to the same extent [76] ± including CaLB [77]. An overview
of the aliphatic and arylaliphatic amines that have been resolved by lipase-
catalyzed acylation is given in Fig. 15. The con®guration of the preferentially
converted enantiomer corresponds with the steric model mentioned above.
The magnitude of the enantiomeric preference, i.e. E, depends, often critically,
on the nature of the acyl reagent and the solvent. Systematic studies in this ®eld are
almost non-existent, but we have compiled the available data, which mainly
involve CaLB, in Table 4. From these it becomes clear that almost any secondary
alkylamine can be resolved once the right reaction conditions have been found. In
general, a bulky acyl group, which restricts the conformational freedom of the
nucleophile, would be expected to assist the enantiodiscrimination. Indeed 2-
aminobutane (11a) was much more ef®ciently resolved by methyl methacrylate
than by methyl acrylate [23b]. However, dimethyl succinate also worked rather
well in dioxane; in hexane the enantioselectivity was poor [38]. This latter pattern,
with the best results being obtained in mildly hydrophilic media, will often be
repeated although it is by no means universal.
2-Aminopentane (11b) has been resolved, also using CaLB, under completely
different conditions: by ethyl acetate in a solvent-free medium [78]. A comparable

562
F. van Rantwijk et al.
Fig. 15. Preferentially acylated enantiomers of aliphatic and arylaliphatic amines6
result was obtained with Pseudomonas aeruginosa lipase (PaL) in diethyl ether
[79]. The bulky dibenzyl carbonate was an unexpectedly inef®cient resolving agent
[41], perhaps because hexane is not the optimum solvent considering the solvent
effect in the acylation of 11a [38].
In the resolution of 2-aminoheptane (11c) via acylation by dimethyl succinate
the solvent effect was the opposite of that observed earlier: a good enantiomeric
ratio (E ˆ 68) was obtained in hexane, whereas reaction in dioxane resulted in only
half that value [38]. Another attempt to resolve 11c in dioxane using the activated
reagent octyl vinyl carbonate also failed [80]; 11a was not resolved by this system
either. Finally, ethyl octanoate, an acyl donor that has not been mentioned so far,
resolved 2-aminooctane (11d) almost quantitatively [81] in a solventless and,
hence, hydrophobic reaction medium. However, dibenzyl carbonate in hexane
again was quite inef®cient [41].
The branched and, therefore, more sterically restricted 2-amino-3,3-dimethyl-
butane (11e) was acylated with low enantioselectivity by neat ethyl acetate [82],
whereas this reaction system had given excellent results with 11b [78]. Even more
surprisingly, the E factor of 11e improved by more than an order of magnitude
when the acyl donor was isopropyl acetate. Reaction in 1,2-dimethoxyethane
(DME), which is a more hydrophilic medium, resulted in a further improvement by
a factor of seven6.
1-Cyclohexylethylamine (12) was acylated with a moderate E of 17 by
dibenzyl carbonate in toluene [83]. The more hindered and less ¯exible amine 13
behaved similarly to 11e upon acylation by acetic acid esters in the presence of
CaLB: the enantiodiscrimination was modest when the ethyl ester was used as
donor but, quite remarkably, absolute with the isopropyl ester [82]; BcL was much
less selective, although the same trend became apparent.6 It should be noted that 13
has two chiral centres; the acylation yields [82] indicate that only one (presumable
that attached to the nitrogen atom) was resolved.
6
The absolute con®gurations of the acylation products of 11e and 13 were not stated by the authors
[82]; by analogy with comparable resolutions, the (R)-enantiomer should be expected to be the fast
reacting one

Lipase-Catalyzed Synthesis of Amides
563
Table 4. Resolution of chiral amines 11±17
Amine
Lipase
Donor
Solvent
T/ꢀC
E
Ref.
11a
CaLB
CaLB
CaLB
CaLB
CaLB
CaLB
CaLB
PaL
Me acrylate
THF
30
30
30
30
8
55
35
6
[23b]
[23b]
[38]
[38]
[80]
[78]
[41]
[79]
[38]
[38]
[80]
[81]
[41]
[82]
[82]
[82]
[82]
[83]
[82]
[82]
[82]
[82]
[78]
[81]
[38]
[38]
[80]
[80]
[80]
[83]
[83]
[41]
[84]
[78]
[82]
[82]
[82]
[82]
[82]
[83]
[41]
[41]
Me methacrylate
Dimethyl succinate
Dimethyl succinate
Octyl vinyl carbonate
Ethyl acetate
THF
Dioxane
Hexane
Dioxane
7
11b
11c
±
> 200
4
Dibenzyl carbonate
Ethyl acetate
Hexane
r.t.
r.t.
30
30
30
39
r.t.
28
r.t.
28
r.t.
r.t.
28
28
r.t.
r.t.
Diethyl ether
> 100
37
CaLB
CaLB
CaLB
CaLB
Dimethyl succinate
Dimethyl succinate
Octyl vinyl carbonate
Ethyl octanoate
Dioxane
Hexane
68
Dioxane
3
11d
11e
±
> 100
5
Dibenzyl carbonate
Ethyl acetate
Hexane
CaLB
CaLB
CaLB
CaLB
CaLB
CaLB
CaLB
BcL
±
8
Ethyl acetate
DME
29
i-Propyl acetate
i-Propyl acetate
Dibenzyl carbonate
Ethyl acetate
±
109
> 700
17
DME
12
13
Toluene
±
14
i-Propyl acetate
Ethyl acetate
±
450
2
±
BcL
i-Propyl acetate
Ethyl acetate
±
28
14
CaLB
CaLB
CaLB
CaLB
CaLB
CaLB
CaLB
CaLB
CaLB
CaLB
BpL
±
110
> 200
95
Ethyl octanoate
±
39
30
30
Dimethyl succinate
Dimethyl succinate
Octyl vinyl carbonate
Octyl vinyl carbonate
Octyl vinyl carbonate
Dibenzyl carbonate
Dibenzyl carbonate
Dibenzyl carbonate
Ethyl methoxyacetate
Ethyl acetate
Dioxane
Hexane
Dioxane
Di-i-propyl ether
Hexane
Toluence
Hexane
Hexane
MTBE
±
6
10
200
200
> 300
20
r.t.
r.t.
r.t.
50
114
24
15
CaLB
CaLB
CaLB
CaLB
BcL
Ethyl acetate
±
28
28
r.t.
r.t.
r.t.
r.t.
r.t.
r.t.
15
i-Propyl acetate
i-Propyl acetate
Ethyl acetate
±
100
650
3
DME
±
BcL
i-Propyl acetate
Dibut-3-enyl carbonate
Dibenzyl carbonate
Dibenzyl carbonate
±
18
16
17
CaLB
CaLB
CaLB
Toluene
Hexane
Hexane
> 200
72
19
1-Phenylethylamine (14), which has attracted a major share of the efforts in this
®eld, has successfully been resolved under a wide range of reaction conditions. Neat
ethyl acetate [78] or octanoate [81] gave a high selectivity; a comparable result was
achieved with dimethyl succinate in dioxane, but in hexane the enantioselectivity

564
F. van Rantwijk et al.
was low [38] as observed with 11a. Octyl vinyl carbonate showed the opposite
solvent effect: 14 was resolved with absolute selectivity in diisopropyl ether or
hexane, but not in dioxane [80]. Dibenzyl carbonate acted with excellent selectivity
in toluene [83], but in hexane [41, 83] the result was much more modest, seemingly
con®rming that hexane is not the optimum medium for this acyl donor.
Finally, the acyl donor methoxyacetic acid ethyl ester deserves to be
mentioned. This compound was developed by BASF as a mildly activated and
easily recyclable reagent for the enzymatic resolution of chiral alcohols and
amines. 14 was ef®ciently resolved by Bukholderia plantarii lipase (BpL) mediated
methoxyacetylation in tert-butyl methyl ether (TBME) [84].
The enantiodiscrimination of 1-(1-naphthyl)ethylamine (15) in the CaLB
mediated acylation by neat ethyl acetate was only modest [78, 82], but E could be
improved dramatically to over 600 by using isopropyl acetate in DME [82] similar
to 11e and 13.
Finally, the bicyclic amine 1-aminoindane (16) was ef®ciently resolved [41, 83],
but the closely related 1-aminotetraline (17) reacted with a moderate E of 19 [41].
A dramatic effect of the leaving group (ethyl vs. isopropyl) has manifested itself in
the acylation of 11e, 13, and 15 [82]. The obvious conclusion is that the liberated
alcohol in¯uences the enantiorecognition of the amine, presumably because it
binds in the active site of the lipase. It is pertinent to note that the interaction with
the inihibition of lipases by hydroxylic reaction media is well documented [7, 85].
It has already been noted that in situ-racemization of the slowly reacting
enantiomer has the potential advantage that the fast reacting enantiomer can be
isolated in 100% yield. This has been accomplished with 14 by combining its
CaLB mediated acylation with palladium-catalyzed reacemization of the unreacted
amine in one pot (Fig. 16) [86].
A few resolutions of more bulky ꢀ-phenyl substituted amines have been carried
out. The CaLB mediated resolution of a range of 1-phenyl-2-propynylamines (18)
has been accomplished with absolute enantioselectivity in neat ethyl acetate [87].
Apparently the acetylene moiety is readily accommodated in the part of the
nucleophile subsite where the M-group binds in the resolutions of 11±15. 1-Amino-
1-phenylacetonitrile (19), the nitrile analogue of 18, reacted with 2,2,2-tri¯uoro-
ethyl acetate in diisopropyl ether with moderate (S)-selectivity (E ˆ 12±15) in the
presence of PaL or Chromobacterium viscosum lipase (CvL), but BcL converted
(R)-19 slightly faster [88]. The CaLB mediated reaction of 19 and phenylacetic
Fig. 16. Dynamic kinetic resolution of 1-phenylethylamine (14)

Lipase-Catalyzed Synthesis of Amides
565
Fig. 17. Enantiopreference of CaLB in the acylation of 18 and 19
Fig. 18. CaLB mediated enantioselective acylation of functionalized amines 20±24
acid methyl ester was highly (S)-selective [89], which may be explained as a
bene®cial effect of steric congestion in the active site.
A few functionalized amines, such as ꢁ-hydroxyamines and amino acid esters
have been resolved by enantioselective acylation (Fig. 18), and the question arises
whether or not the same steric model applies. The CaLB mediated acylation of trans-
1-amino-2-hydroxycyclopentane with dimethyl glutarate to 20 was only modestly
enantioselective [90]. The enantiomers of its C6-homologue were more readily
discriminated (21a) when the reaction was carried out in dioxane, but no resolution
was possible in toluene. This enantiopreference would seem to be consistent with the
steric model, with the hydroxyl group binding as the L substituent. Surprisingly, the
enantiopreference was inverted with dibenzyl carbonate as the acyl donor [83, 90];
21b was formed with E ˆ 16 in either dioxane or toluene. The acylation of amino
acid esters was (R)-selective, as would be expected on the basis of the peptide
synthesis experiments. Proline tert-butyl ester (22) was inef®ciently resolved using
dibenzyl carbonate [83] but, surprisingly, the acylation of the methyl esters of
phenylalanine (23) and ꢁ-aminobutyric acid (24) with methoxyacetic acid ethyl ester
resulted in a nearly quantitative (E ˆ 200) resolution [91]. The reaction of ꢁ-
aminobutyric acid ethyl ester with ethyl acetate was somewhat less enantioselective
[68].

566
F. van Rantwijk et al.
Fig. 19. Combined resolutions
Combined resolutions
Considering that lipases can resolve the acyl donor as well as the acyl acceptor, it
might be attractive to accomplish both resolutions in one reaction. Such a one-step
resolution of a chiral vinyl carbonate and chiral amines, mediated by CaLB, was
only moderately successful, because the products, such as e.g. 25 (see Fig. 19),
were formed in a moderate diasteromeric and/or enantiomeric excess [92]. In
contrast, 26 and some related amides were obtained as the almost pure (3S,10R)
stereoisomers [93].
Conclusion
The lipase-catalyzed aminolysis of carboxylic esters is a fairly general methodology
for the synthesis of carboxylic amides under mild conditions. The condensation of
carboxylic acids and the amines provides an alternative route to carboxylic amides.
Lipase-catalyzed aminolysis is a useful method to resolve chiral carboxylic esters as
well as chiral amines. The enantiorecognition of chiral amines often depends
critically on the acylating agent and the reaction medium.
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Received December 20, 1999. Accepted January 1, 2000