Highly selective enzymatic kinetic resolution of primary amines at 80°C: A comparative study of carboxylic acids and their ethyl esters as acyl donors

Article abstract of DOI:10.1021/jo071069t

(Chemical Equation Presented) Optimization of the kinetic resolution of 2-amino-4-phenyl-butane was achieved at 80°C using CAL-B-catalyzed aminolysis of carboxylic acids and their ethyl esters. The reactions carried out with long chain esters and the corresponding acids as acyl donors proceeded with remarkably high enantioselectivity. The use of carboxylic acids as acylating agents led to a marked acceleration of the reaction rate compared to their ester counterparts. Laurie acid led to enantiomeric excesses superior to 99.5% for both the remaining amine and the corresponding lauramide at 50% conversion (reached in 3 h). These optimized conditions were applied to the resolution of a series of aliphatic and benzylic amines.

Full text of DOI:10.1021/jo071069t

Highly Selective Enzymatic Kinetic Resolution of Primary Amines at
80 °C: A Comparative Study of Carboxylic Acids and Their Ethyl
Esters as Acyl Donors
Malek Nechab,† Nadia Azzi,† Nicolas Vanthuyne,† Miche`le Bertrand,*,‡
Ste´phane Gastaldi,*,‡ and Ge´rard Gil*,†
Laboratoire de Ste´re´ochimie Dynamique et Chiralite´, UMR 6180, Chirotechnologies: Catalyse et
Biocatalyse UniVersite´ Paul Ce´zanne, Aix-Marseille III, Faculte´ des Sciences St Je´roˆme, AVenue Escadrille
Normandie-Niemen, 13397 Marseille Cedex 20, France, and Laboratoire de Chimie Mole´culaire
Organique, UMR 6517, Chimie, Biologie, et Radicaux Libres, Boite 562, UniVersite´ Paul Ce´zanne,
Aix-Marseille III, Faculte´ des Sciences St Je´roˆme, AVenue Escadrille Normandie-Niemen,
13397 Marseille Cedex 20, France
michele.bertrand@uniV-cezanne.fr; stephane.gastaldi@uniV.u-3mrs.fr; gerard.gil@uniV-cezanne.fr
ReceiVed May 24, 2007
Optimization of the kinetic resolution of 2-amino-4-phenyl-butane was achieved at 80 °C using CAL-
B-catalyzed aminolysis of carboxylic acids and their ethyl esters. The reactions carried out with long
chain esters and the corresponding acids as acyl donors proceeded with remarkably high enantioselectivity.
The use of carboxylic acids as acylating agents led to a marked acceleration of the reaction rate compared
to their ester counterparts. Lauric acid led to enantiomeric excesses superior to 99.5% for both the remaining
amine and the corresponding lauramide at 50% conversion (reached in 3 h). These optimized conditions
were applied to the resolution of a series of aliphatic and benzylic amines.
Introduction
asymmetric synthesis of agrochemicals and pharmaceuticals
products.1 Biotransformations are becoming more and more
important in industrial processes.2 Dynamic kinetic resolution
(DKR),3-5 and deracemization3,6 involving biocatalysts offer
alternatives to purely chemical asymmetric catalysis which
generally requires expensive chiral catalysts.7
Optically pure amines are building blocks of major impor-
tance to industrial chemistry, since they are used for the
† Laboratoire de Ste´re´ochimie Dynamique et Chiralite´, UMR 6180, Chiro-
technologies: Catalyse et Biocatalyse, Universite´ Paul Ce´zanne.
We have recently reported that the combination of enzymatic
resolution with thiyl radical-mediated racemization led to
optically pure aliphatic amides from the corresponding racemic
aliphatic amines in good yield and high enantiomeric excess.5
‡ Laboratoire de Chimie Mole´culaire Organique, UMR 6517, Chimie,
Biologie, et Radicaux Libres, Boite 562, Universite´ Paul Ce´zanne.
(1) (a) Lawrence, S. A. In Amines: Synthesis, Properties and Applica-
tions; Cambridge University Press: Cambridge, U.K., 2004. (b) Breuer,
M.; Ditrich, K.; Habicher, T.; Hauer, B.; Keâeler, M.; Stu¨rmer, R.; Zelinski,
T. Angew. Chem., Int. Ed. 2004, 43, 788-824. (c) Hieber, G.; Ditrich, K.
Chim. Oggi 2001, 19, 16-20.
(4) For DKR see: (a) Reetz, M. T.; Schimossek, K. I. Chimia 1996, 50,
668-669. (b) Choi, Y. K.; Kim, M. J.; Ahn, Y.; Kim, M.-J. Org. Lett.
2001, 3, 4099-4101. (c) Kim, M.-J.; Kim, W.-H.; Han, K.; Choi, Y. K.;
Park, J. Org. Lett. 2007, 9, 1157-1159. (d) Paetzold, J.; Ba¨ckvall, J.-E. J.
Am. Chem. Soc. 2005, 127, 17620-17621. (e) Parvulescu, A.; De Vos, D.;
Jacobs, P. Chem. Commun. 2005, 5307-5309. (f) Parvulescu, A. N.; Jacobs,
P. A.; De Vos, D. E. Chem. Eur. J. 2007, 3, 2034-2043. (g) Stirling, M.;
Blacker, J.; Page, M. I. Tetrahedron Lett. 2007, 48, 1247-1250. (h)
Crawford, J. B.; Skerlj. R. T.; Bridger, G. J. J. Org. Chem. 2007, 72, 669-
671.
(2) (a) Jaegger, K.-E.; Reetz, M. T. Tibtech. 1998, 16, 396-403. (b)
Koeller, K. M.; Wong, C.-H. Nature 2001, 409, 232-240.
(3) For concepts and general reviews see: (a) Faber, K.; Chem. Eur. J.
2001, 7, 5004-5010. (b) Pe´lissier, H. Tetrahedron 2003, 59, 8291-8327.
(c) Ward, R. S. Tetrahedron: Asymmetry 1995, 6, 1475-1490. (d) Pa`mies,
O.; Ba¨ckvall, J.-E. Chem. ReV. 2003, 103, 3247-3261. (e) Pa`mies, O.;
Ba¨ckvall, J.-E. Curr. Opin. Biotechnol. 2003, 14, 407-413. (f) Kim, M.-
J.; Ahn, Y.; Park, J. Curr. Opin. Biotechnol. 2002, 13, 578-587. (f) Strauss,
U. T.; Felber, U.; Faber, K. Tetrahedron: Asymmetry 1999, 10, 107-117.
10.1021/jo071069t CCC: $37.00 © 2007 American Chemical Society
Published on Web 08/03/2007
6918
J. Org. Chem. 2007, 72, 6918-6923

Enzymatic Kinetic Resolution of Primary Amines
The racemization process involves reversible H-abstraction at
the chiral center in position R relative to the NH2 group.8 The
rate constant for the limiting step, that is, bimolecular hydrogen
abstraction by alkylthiyl radical from aliphatic amines was
estimated to be 104 M-1 s-1 at 80 °C.8 The thermally initiated
racemization carried out in the presence of AIBN proceeds
through a chain mechanism. To be efficient, the reaction has to
be performed at a temperature close to 80 °C. Thereby, the
performance of a highly selective enzymatic resolution at 80 °C
was of paramount importance to optimize the DKR process.
We report in this article the detailed study of kinetic resolution
optimization that enabled the performance of the above-cited
dynamic kinetic resolutions of aliphatic amines.5 The screening
of selected acyl donors was achieved with the purpose to
improve the kinetic resolution of primary aliphatic amines using
the immobilized CAL-B catalyst (Novozym 435 from Novo
Nordisk A/S, Denmark) at 80 °C. 2-Amino-4-phenyl-butane (1)
was chosen as model for this study, the best conditions were
then applied to a series of aliphatic and benzylic amines.
aminolysis have been performed by Gotor,10 Sheldon,11 and
other groups.12-14 Improving the lipase catalytic activity and
selectivity can be made by changing the solvent,14 or the acyl
donor,15 or both. The lipase-catalyzed resolution of racemic
amines is reputedly successful in organic solvent of low
polarity.14 Since the final goal behind this study was to carry
out DKR reactions under optimal conditions,5 the enzymatic
resolution was optimized in heptane (one of the best solvents
for the radical-mediated racemization) despite the rather poor
results reported for CAL-B-mediated acylations in hexane16 or
cyclohexane.14c,15c
The selection of appropriate acyl donors is crucial for the
kinetic resolution of amines.2b,15,16 Although they are highly
reactive irreversible acyl donors, enol ethers are not suitable
for this purpose, since they release carbonyl compounds that
may react with the aminogroup to form imines. Furthermore,
since amines are good nucleophiles, highly reactive acyl donors
might lead to amides through spontaneous nonenzymatic
nucleophilic displacement by the racemic substrate, which would
lower the enantiomeric excess of the acylated product. Carbon-
ates have been used as acyl donors for the resolution of amines,
since they lead to carbamates from which amines are easily
recovered. A preliminary trial was carried out in the presence
of 1 equiv of diallylcarbonate. At 80 °C, 64% conversion was
reached in 7.5 h, and the reaction led to rather low enantiomeric
excesses for both the remaining amine (S)-1 (46% eeS, 39%
yield) and the corresponding (R)-carbamate (26% eeR, 71%
yield). Even though at 80 °C spontaneous aminolysis was shown
not to interfere with the enzymatic reaction, this family of acyl
donors was discarded, and further assays were limited to ethyl
esters and corresponding acids.
Results and Discussion
The thermostable lipase B from Candida antartica (CAL-B)
has proven to be the most effective biocatalyst for aminolysis
reactions in organic solvents.9 Numerous studies of enzymatic
(5) Gastaldi, S.; Escoubet, S.; Vanthuyne, N.; Gil, G.; Bertrand, M. P.
Org. Lett. 2007, 9, 837-839.
(6) For deracemization, see: Soda, K.; Oikawa, T.; Yokoigawa, K. J.
Mol. Catal. B: Enzym. 2001, 11, 149-153. (b) Turner, N. J. Curr. Opin.
Biotechnol. 2003, 14, 401-406. (c) Gruber, C. C.; Lavandera, I.; Faber,
K.; Kroutil, W. AdV. Synth. Catal. 2006, 348, 1789-1805. (d) Dunsmore,
C. J.; Carr, R.; Fleming, T.; Turner, N. J. J. Am. Chem. Soc. 2006, 128,
2224-2225. (e) Turner, N. J. Curr. Opin. Chem. Biol. 2004, 8, 114-119.
(7) For examples, see: (a) Uematsu, N.; Fujii, A.; Hashigushi, S.; Ikariya,
T.; Noyori, R. J. Am. Chem. Soc. 1996, 118, 4916-4917. (b) Mao, J.; Baker,
D. Org. Lett. 1999, 1, 841-843. (c) Kobayashi, S.; Kobayashi, S.; Ishitani,
H. Chem. ReV. 1999, 99, 1069-1094. (d) Hansen, M. C.; Buchwald, S.
Org. Lett. 2000, 2, 713-715. (e) Trifonova, A.; Diesen, J. S.; Chapman,
C. J.; Andersson, P. G. S. Org. Lett. 2004, 6, 3825-3827. (f) Bra¨se, S.;
Baumann, T.; Dahmen, S.; Vogt, H. Chem. Comm. 2007, 1881-1890.
(8) (a) Escoubet, S.; Gastaldi, S.; Vanthuyne, N.; Gil, G.; Siri, D.;
Bertrand, M. P. J. Org. Chem. 2006, 71, 7288-7292. (b) Escoubet, S.;
Gastaldi, S.; Vanthuyne, N.; Gil, G.; Siri, D.; Bertrand, M. P. Eur. J. Org.
Chem. 2006, 3242-3250.
Comparative results obtained with racemic amine (1) are
given in Table 1.
We first used simple esters, the most common one being ethyl
acetate (entry 1). At 80 °C, ethyl acetate led to 50% conversion
in 7 h, but the enantiomeric excesses did not exceed 91% for
(R)-amide 1a and 92% for the remaining (S)-amine 1.17 The
enantioselectivity was the lowest in the series.
(12) (a) Skupinska, K. A.; McEachern, E. J.; Baird, I. R.; Skerlj. R. T.;
Bridger, G. J. J. Org. Chem. 2003, 68, 3546-3551. (b) Davis, B. A.; Durden,
D. A. Synth. Commun. 2001, 31, 569-578. (c) O¨ hrner, N.; Orrenius, C.;
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348, 797-812. (b) Gotor-Ferna´ndez, V.; Gotor, V. Curr. Org. Chem. 2006,
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dron: Asymmetry 2005, 16, 3070-3076. (e) Gonza´lez-Sab´ın, J.; Gotor,
V.; Rebolledo, F. Tetrahedron: Asymmetry 2004, 15, 481-488. (f)
Gonza´lez-Sab´ın, J.; Gotor, V.; Rebolledo, F. Tetrahedron: Asymmetry 2002,
13, 1315-1320. (g) Garc´ıa-Urdiales, E.; Rebolledo, F.; Gotor, V. Tetra-
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H. A.; Schoevaart, R.; van Rantwijk, F.; Sheldon, R. A. Enzyme Microb.
Technol. 2007, 40, 1095-1099. (b) Youshko, M. I.; van Rantwijk, F.;
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M. A. P. J.; van Rantwijk, F.; Sheldon, R. A. J. Mol. Catal. B: Enzym.
2000, 9, 201-208. (d) van Langen, L. M.; Oosthoek, N. H. P.; Guranda,
D. T.; van Rantwijk, F.; Sˇvedas, V. K.; Sheldon, R. A. Tetrahedron:
Asymmetry 2000, 11, 4593-4600. (e) Zoete, M. C.; Kock-van Dalen, A.
C.; van Rantwijk, F.; Sheldon, R. A. J. Mol. Catal. B: Enzym. 1996, 1,
109-113.
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2005, 100, 662-666.
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2254. (b) Klibanov, A. M. Nature 2001, 409, 241-246. (c) Kitaguchi, H.;
Fitzpatrick, P. A.; Huber, J. E.; Klibanov, A. M. J. Am. Chem. Soc. 1989,
111, 3094-3095. (d) Re´jasse, B.; BessonEn, T.; Legoy, M.-D.; Lamare, S.
Org. Biomol. Chem. 2006, 4, 3703-3707. (e) Lozano, P.; de Diego, T.;
Carrie´, D.; Vaultier, M.; Iborra, J. L. Chem. Commun. 2002, 692-693.
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127-128. (c) Goswami, A.; Guo, Z.; Parker, W. L.; Patel, R. N.
Tetrahedron: Asymmetry 2005, 16, 1715-1719. (d) Cammenberg, M.; Hult,
K.; Park, S. ChemBioChem 2006, 7, 1745-1749. (e) Ottoson, K.; Hult, J.
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Cai, X.-Q.; Wu, Q.; Lin, X.-F. J. Mol. Catal. B: Enzym. 2007, 44, 1-7.
(16) Puertas, S.; Brieva, R.; Rebolledo, F.; Gotor, V. Tetrahedron 1995,
51, 1495-1502.
(17) The absolute configuration of the remaining amine and the amides
were assigned by analogy with literature data (ref 10f), in agreement with
Kazlauskas’ empirical rule (ref 22).
(18) Chen, C. S.; Fujimoto, Y.; Girdaukas, C. J.; Sih, C. J. J. Am. Chem.
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J. Org. Chem, Vol. 72, No. 18, 2007 6919

Nechab et al.
TABLE 1. Influence of the Acyl Donor on the Kinetic Resolution of Amine (1) with CAL-B at 80 °C in Heptane
(S)-1
(R)-amide
time
(h)
T
(°C)
yield %
(NMR)c
yield %e
(NMR)c
entry
acyl donora
ees (%)b
amide
eeR (%)d
c (%)f
Eg
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
MeCO2Et
MeOCH2CO2Et
C3H7CO2Et
7
1.5
9
6
6
5
4
3
3
80
80
80
80
80
80
80
80
80
80
70
60
50
80
80
80
92.0
>99.5
97.6
>99.5
>99.5
>99.5
99.0
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
(43)
(40)
(47)
(47)
(47)
(40)
(41)
(48)
(39)
(39)
(41)
(50)
(49)
(50)
(48)
(49)
1a
1b
1c
1d
1e
1f
1c
1d
1e
1e
1e
1e
1e
1e
1e
1f
91.0
77.4
95.5
98.0
97.0
99.2
97.1
98.6
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
23 (46)
39 (53)
- (47)
42 (48)
50 (42)
- (46)
- (44)
48 (49)
- (45)
- (46)
- (44)
- (46)
- (52)
- (50)
- (48)
49 (50)
50.3
56.4
50.5
50.5
50.8
50.2
50.5
51.5
50.0
50.0
50.0
50.0
50.0
50.0
50.0
50.2
69
100
>200
.500
.500
.500
.200
.500
.500
.500
.500
.500
.500
.500
.500
.500
C7H15CO2Et
C11H23CO2Etk
C15H31CO2Etk
C3H7CO2H
C7H15CO2H
C11H23CO2H
C11H23CO2Hh
C11H23CO2H
C11H23CO2H
C11H23CO2H
C11H23CO2Hi
C11H23CO2Hj
C15H31CO2H
3
4.5
5.3
10
5
6
3
a General conditions unless otherwise stated: 1 (1 mmol); acyl donor (1 mmol); CAL-B, Novozym 435 (200 mg); heptane (10 mL), 80 °C. b Determined
by chiral GC after derivatization in trifluoroacetamide. c Pentamethylbenzene was used as internal standard. d Determined by chiral HPLC. e Isolated yield.
f Calculated according to c ) eeamine/(eeamine+ eeamide). g Enantioselectivity factors were calculated according to E ) ln[(1 - c)(1 - eeamine)]/ln[(1 - c)(1 +
eeamine)].18 Owing to sensitivity to experimental errors, E values calculated in the range 200-300 are reported as >200, values in the range 300-500 are
k
given .200, and values in the range 900-1000 are given .500. h Lauric acid (0.6 equiv). i CAL-B (100 mg). j CAL-B (50 mg). C11H23CO2Et (ethyl
laurate), C15H31CO2Et (ethyl palmitate).
The time necessary to reach 50% conversion increased, going
from 7 h for ethyl acetate to 9 h for ethyl butyrate. Further
increase of the acyl group chain length resulted in the accelera-
tion of the reaction, since the same conversion was reached in
6 h for ethyl octanoate and ethyl laurate and in 5 h for ethyl
palmitate. Simultaneously, the enantioselectivity reached re-
markably high values, the rate constant for the conversion of
the (R)-enantiomer being more than 2 orders of magnitude
higher than the rate constant for the conversion of the (S)-
enantiomer.
A similar effect of the acyl group chain-length has been
reported for the enzymatic resolution of sec-butylamine; the
highest enantioselectivities were obtained with ethyl esters of
long chain fatty acids, although the rate of conversion at 25 °C
decreased, while the carbon chain length of the acid moiety
increased.15c It must be noted that the influence of the number
of carbons in the acyl moiety on CAL-B-catalyzed aminolysis
of esters should by no means be considered as general trend
for lipase catalysis. Contradictory reports about the influence
of the chain length of the acylating agent are known for
transesterifications catalyzed by lipase PS.19 In the enzymatic
FIGURE 1. Plot of amine 1 enantiomeric excess versus time for the
acylation of alcohols using Pseudomonas sp. lipase and acid
anhydrides as acyl donors, increasing the chain length resulted
in the acceleration of the conversion rate, but with concomitant
lowering of the enantioselectivity.20 The lipase’s affinity toward
long-chain acyl groups might be responsible for the accelaration,
and according to CAL-B structure, the acyl moiety lies in a
large hydrophobic pocket.10c,21,22
aminolysis of ethyl esters.
Increasing the acyl moiety chain length improved the enan-
tioselectivity. The enantiomeric excess of the amide varied from
95.5% for ethyl butyrate (1c) to 97 and 98% for ethyl laurate
(1e) and ethyl octanoate (1d), respectively, and became superior
to 99% for ethyl palmitate (1f) for conversions close to 50%
(entries 3-6). Concomitantly, the enantiomeric excess of the
remaining amine varied from 97.6% for ethyl butyrate to
>99.5% for ethyl octanoate, ethyl laurate, and ethyl palmitate.
Figure 1 shows the plot for the variation of the amine
enantiomeric excess versus time for the various esters.
(19) Ema, T.; Maeno, S.; Takaya, Y.; Sakai, T.; Utaka, M. Tetrahe-
dron: Asymmetry 1996, 7, 625-628.
(20) Miyazawa, T.; Kaito, E.; Yukawa, T.; Murashima, T.; Yamada, T.
J. Mol. Catal. B: Enzym. 2005, 37, 63-67.
6920 J. Org. Chem., Vol. 72, No. 18, 2007

Enzymatic Kinetic Resolution of Primary Amines
Because of the electron-withdrawing effect of the methoxy-
group, ethyl 2-methoxyacetate deserves a separate comment
(Table 1, entry 2). The R-methoxy group activates the reactivity
at the carbonyl group. Ethyl 2-methoxyacetate was more re-
active than ethyl acetate and all the other esters in the series.
This ester has been used in other lipase-mediated resolution of
amines.10e,15d,23 It is reputed to be the optimum acyl donor
for industrial amine resolution processes.1b,c Park has sug-
gested that hydrogen bonding between the â-oxygen atom in
methoxyacetate and the proton of the amine in the transition
state might be a key factor in the rate enhancement.15d In
our case, the rate of the enzymatic reaction was very fast, and
56.4% conversion was reached in only 1.5 h. Within less
than 1 h, the amide enantiomeric excess reached a maximum
and started decreasing. The enantioselectivity was high
(E ) 100), but it was far lower than those that were registered
with long chain esters (the low enantiomeric excess of amide
1b reflects the partial acylation of (S)-1 due to the 56.4%
conversion).
As briefly mentioned previously, a leaving group that is too
good is not appropriate for enzymatic acylation of good
nucleophiles like amines. The influence of the nature of the
leaving group on the resolution step itself is not clear-cut.
According to the mechanism generally accepted for lipases,21,24
the leaving group is not present in the enantioselectivity
determining step. We have focused our attention on the use of
carboxylic acids as acylating agents.25 The latter have been
scarcely used as acyl donors in enzymatic aminolysis because
one would predict that the formation of an unreactive amine
salt would prevent the formation of the acyl-enzyme intermedi-
ate due to the low reactivity of the carboxylate. However, the
formation of the salt is an equilibrium, and the small amount
of free acid available to the enzyme is sufficient for the
aminolysis to proceed, making prior activation of the acid
unnecessary.
FIGURE 2. Plot of amine 1 enantiomeric excess versus time for
carboxylic acids acyl donors.
The data given in Table 1 (entries 7-16) demonstrate that
with an acid/amine ratio of 1,26 contrary to the prediction of a
slower rate of conversion, 50% conversion was obtained in
reaction times ranging from 3 to 6 h depending on the acid. It
can be noted that lowering the amount of acid to 0.6 equiv
(entries 9 and 10) had nearly no effect on the reaction rate.
At 80 °C, the time needed to reach 50% conversion was
roughly half the time needed when using the corresponding ethyl
ester (entries 3 and 7; 4 and 8; 5 and 9; 6 and 16).27 It has been
suggested that the negative effect produced on the reaction rate
resulting from the formation of the amine salt might be
compensated by the higher specificity of the enzyme for the
acid than for the ester.25c The incidence of the chain length on
the reaction rate paralleled that observed with ethyl esters as
acyl donors (Figures 1 and 2).28
Amides 1e and 1f were prepared in very high enantiomeric
excesses. The enantioselectivity was not significantly affected
by the nature of the leaving group. This is consistent with the
mechanism, since the selectivity is determined in the acyl-
enzyme complex cleavage step.
Acetic acid and 2-methoxyacetic acid are exceptions. The
former did not give any conversion. In the case of the latter, in
5 h, the conversion reached 43.9%; the enantiomeric excess of
the remaining amine and the amide were 76% and 97%,
respectively. The reaction was shown not to be reversible.
The influence of the temperature on the reaction rate was
examined (entries 9 and 11-13). Lowering the temperature from
80 to 50 °C resulted in slowing down the rate of the enzymatic
The enzymatic reaction in organic solvent was slow when
using oleic acid and lauramine to prepare the corresponding
amide (60% conversion in 12 days at room temperature, 80%
in 15 days).25a It was further demonstrated that the amides
derived from 1-phenylethylamine and dodecanoic acid, and other
aliphatic acids, could be synthesized in high enantiomeric excess,
under reduced pressure in non-solvent system, and in ionic
liquids at temperature ranging from 30 to 55 °C.25b The rate of
amonolysis of octanoic acid in ionic liquids was found
comparable or even faster than the ones observed in organic
solvents.25d
(26) The rate of CAL-B-catalyzed amidation of oleic acid with N-
methylglucamine in organic solvents was reported to decrease at acid/amine
ratios higher than 1, see: Maugard, T.; Remaud-Simeon, M.; Monsan, P.
Biochim. Biophys. Acta 1998, 16, 181-204.
(27) Upon request of a referee, attempts to perform the reaction in
acetonitrile (HPLC grade, <0.01% (v/v) H2O), a non-hydrogen bonding
solvent, were made. This solvent was selected for its polar character (ꢀ )
32), quite different from heptane (ꢀ ) 1.92). It led to very poor results, no
conversion after 24 h in the presence of lauric acid, and amine ee was only
21% after 6 h in the presence of ethyl laurate. In this case, owing to the
slow rate of conversion, ethyl methoxy acetate would be a better acylating
agent than ethyl laurate (87% amine ee, 98% amide ee; 47% conversion (E
) 270, after 24 h at 80°C).
(28) The rate of enzymatic acylation of chiral alcohols with fatty acids
is also influenced by the number of carbons in the acyl chain. The acyl
binding site of Chirazyme L2 (CAL-B) can accomodate acyl groups up to
the carbon number 16, see: (a) Suan, C.; Sarmidi, M. R. J. Mol. Catal. B:
Enzym. 2004, 28, 111-119. For related observations in the acylation of
flavonoids, see: (b) Ardhaoui, M.; Falcimaigne, A.; Ognier, S.; Engasser,
J. M.; Moussou, P.; Pauly, G.; Ghoul, M. J. Biotechnol. 2004, 110, 265-
271.
(21) (a) Bornscheuer, U. T.; Kazlauskas, R. J. In Hydrolases in Organic
Synthesis. Regio- and StereoselectiVe Biotransformations, 2nd ed.; Wiley-
VCH: Weinheim, Germany, 2006. (b) Mezzetti, A.; Keith, C.; Kazlauskas,
R. J. Tetrahedron: Asymmetry 2003, 14, 3917-3924.
(22) Kazlauskas, R. J. Curr. Opin. Chem. Biol. 2000, 4, 81-88.
(23) Balkenhohl, F.; Ditrich, K.; Hauer, B.; Ladner, W. J. Prakt. Chem.
1997, 339, 381-384.
(24) Kraut, J. Ann. ReV. Biochem. 1977, 46, 331-358.
(25) (a) Montet, D.; Pina, M.; Graille, J.; Renard, G.; Grimaud, J. Fat.
Sci. Technol. 1989, 91, 14-18. (b) Irimescu, R.; Kato, K. Tetrahedron Lett.
2004, 45, 523-525. (c) Irimescu, R.; Saito, T.; Kato, K. J. Am. Oil Chem.
Soc. 2003, 80, 659-663. (d) Madeira Lau, R.; van Rantwijk, F.; Sheldon,
K. R.; Sheldon, R. A. Org. Lett. 2000, 2, 4189-4191. (e) Litjens, M. J. J.;
Straathof, A. J. J.; Jongejan, J. A.; Heijnen, J. J. Tetrahedron 1999, 55,
12411-12418. (f) Prasad, A. K.; Husain, M.; Singh, B. K.; Gupta, R. K.;
Manchanda, V. K.; Olsen, C. E.; Parmar, V. S. Tetrahedron Lett. 2005, 46,
4511-4514. (g) Tufvesson, P.; Annerling, A.; Hatti-kaul, R.; Adlereutz,
D. Biotechnol. Bioeng. 2006, 97, 447-453. (h) Slotema, W. F.; Sandoval,
G.; Guieysse, D.; Straathof, A. J. J.; Mary, A. Biotechnol. Bioeng. 2003,
82, 664-669.
J. Org. Chem, Vol. 72, No. 18, 2007 6921

Nechab et al.
TABLE 2. Influence of Enzyme Recycling on the Aminolysis of
Lauric Acid with Amine 1 in 5h at 80 °C
(S)-1
yield (%)c
(R)-1e
yield (%)c
cyclea
eeb
eed
1 (fresh)
>99.5
>99.5
99.4
97.9
92.6
46
42
46
43
43
99.2
99.3
99.2
99.1
99.0
42
41
42
41
41
2
3
4
5
a General conditions: 1 (1 mmol); lauric acid (1 mmol); CAL-B,
Novozym 435 (100 mg); heptane (10 mL), 80 °C. The resin was filtered
off, rinsed with ether, and then dried under vacuum for 2 h before being
recycled. b Determined by chiral GC after derivatization in trifluoroaceta-
mide. c NMR yield with pentamethylbenzene as internal standard. d Deter-
mined by chiral HPLC.
FIGURE 3. Structures of amines 2-14.31
reaction roughly by a 2.7 factor. The initial rate was 2.15 µmol
h-1 mg-1 at 80 °C, while it was 0.79 µmol h-1 mg-1 at 50 °C.
The enantioselectivity ratio did not change significantly due to
its high range.
Dividing the amount of CAL-B relative to the amine by 4
resulted in slowing down the reaction rate approximately by a
factor of 2 (entries 9, 14, and 15).
residual activity was 95-70% after 6-9 h of reaction. Thereby,
it can be concluded that most of the loss of activity originated
from thermal degradation.
The optimal conditions determined for the resolution of amine
1 with lauric acid as the acyl donor were then applied to amines
2-14 (Figure 3). The data are collected in Table 3.
The small difference in the steric bulk of ethyl group
compared to methyl group explains the low factor of discrimi-
nation between the two enantiomers of amine 2. The enanti-
oselectivity factor was 4 times higher than that reported by
Gotor.10g
The higher reactivity, associated to high enantiomeric ratios,
that was observed with carboxylic acids might be due to the
release of a water molecule in the stereospecificity pocket. The
role of water on the enzyme activity is fundamental.14a Enzyme
activity correlates to water activity, and less water is necessary
in hydrophobic solvents. It has recently been reported that the
presence of a water molecule enhances the enantioselectivity
of CAL-B with respect to the acylation of 2-pentanol. Molecular
modeling suggested that the binding of a water molecule in the
active site could obstruct the binding of the slowly reacting
enantiomer.29
It has also been pointed out by several authors that the
reactions of amines are often slower than the reactions of
alcohols in spite of their higher nucleophilicity.10c,12c,25g In
redesigning the commonly accepted serine-mediated mechanism
for the aminolysis of esters, Gotor10c has suggested that
unassisted attack of the amine onto the carbonyl group was
plausible and that a water molecule might be necessary to
facilitate the proton transfer from the resulting ammonium group
to the histidine residue. This is consistent with the rationale
proposed by Hult,12c according to which the proton transfer from
the acyl acceptor to the histidine residue would be slower in
the case of an amine than in the case of an alcohol.
Attention was paid to the recycling of the enzyme. The NMR
yields and enantiomeric excesses determined after four consecu-
tive recycling cycles of the enzyme are given in Table 2.
The thermal stability of the enzyme was evaluated in the
absence of substrate and acyl donor in heptane at 80 °C and
200 rpm. No change in lipase activity was recorded after 5 h at
80 °C; a 72% residual activity was registered after heating for
22 h. The stability of Novozym 435 was also examined by
determining its residual activity after incubation under the
reaction conditions.30 Under the aminolysis conditions, 100-
95% residual activity remained after 2-5 h of reaction. The
Aliphatic amines structurally close to 1, that is, 3, 4, 5, 6,
and 7, were very efficiently resolved at 80 °C. The time
necessary to reach 50% conversion ranged from 6 to 9.5 h. For
all these amines, the enantioselectivity factor was far greater
than 500. Ramification with a methyl group at a remote position
(5) slightly slowed down the reaction, more than the trisubsti-
tuted-double bond in 6 (entries 4 and 5). The terminal diethy-
lamino group in 7 slowed down the reaction some more,
possibly because the additional basic function contributes to
decrease the amount of free acid in the reaction medium.
It must be noted that the isolated yield of the remaining (S)-
amine was low in several instances. Amine volatility resulted
in loss during the isolation. This is the case for amine 2 which
is the most volatile in the series and for which the yield of the
recovered substrate was not determined.32
The enzymatic reaction was much slower with amine 8, since
14 h were needed to reach 50% conversion, presumably because
of the steric bulk of the cyclohexyl substituent. Surprizingly,
the acylation was slower for amine 9, bearing a phenyl group
in â-position relative to the amino group, than for amine 10
bearing a much hindered aryloxy group. Although lower, the E
factor remained very high.
Benzylic amines 11-14 gave E factors as high as aliphatic
amines with the exception of amine 12. It must be noted that
the enzymatic reaction was extremely slow, even at 80 °C for
amines 12 and 13, whereas the times necessary to reach 50%
(31) For comparative data for the resolution of amine 1, see refs 10f,
12b,d; for amine 2, see refs 10g, 12c, 13b, 15c; for amine 3, see refs 10g,
13b; for amine 4, see refs 12c, 13b,c; for amine 8, see ref 15b; for amine
9, see ref 13g; for amine 10, see ref 10f; for amine 11, see refs 10g, 12a,d,
13b,c,g, 14c, 15b,d; for amine 12, see refs 10g, 13a,e,g, 14c; for amine 13,
see refs 12c, 13a,e; for amine 14, see refs 12b,c,d, 15b.
(29) Le´onard, V.; Fransson, L.; Lamare, S.; Hult, K.; Graber, M.
ChemBioChem 2007, 8, 662-667.
(30) The activity was measured using a standard protocol, i.e., by
incubating 10-20 mg of lipase in 10 mL of heptane containing 0.1 M lauric
acid, 0.1 M 1-propanol, and 0.05 M n-hexadecane as internal standard for
quantitative GC analysis, at 40°C and 200 rpm for 2-10 min.
(32) Boiling points: 2 (63°C), 2 (142°C), 5 (154°C), heptane (102°C).
6922 J. Org. Chem., Vol. 72, No. 18, 2007

Enzymatic Kinetic Resolution of Primary Amines
TABLE 3. Kinetic Resolution of Amines 2-14 with CALB at 80 °C in Heptane with Lauric Acid as the Acyl Donora
(S)-remaining amine
(R)-amide
yield % isolated
yield % isolated
(NMR)c
entry
amine
time (h)
ees (%)b
(NMR)c
amide
eeR (%)d
C (%)e
Ef
16
1
2
2
3
6
8
95.0
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
98.0
>99.5
>99.5
97.9
>99.5
>99.5
Nd
2e
3e
4e
5e
6e
7e
8e
9e
10e
11e
12e
13e
14e
65.0
96.9
>99.5
>98.0
99.5
99.8
99.0
98.0
>98.0
99.0
94.0
96.7
55
59.4
50.8
50
50.5
50.1
50
50.2
50
50.5
50.2
51
10 (41)
41 (43)
17 (41)
28
45 (49)
45 (41)
38 (43)
42
44 (44)
49.5
46
39 (41)
46
48
.500
.500
.500
.500
.500
.500
.200
.500
.500
>100
.500
.500
3
4
6
4
5
6
7
5
6
7
8
8.5
7.5
9.5
14
9
31 (43)
48
8
9
27g
9
10
11
12
13
14
7
4
25
26
2
27 (44)
45
42
34
44
10
11
12
13
42
47
50.8
50
>99.5
a General conditions unless otherwise stated: substrate (1 mmol); lauric acid (1 mmol); CAL-B, Novozym 435 (100 mg); heptane (10 mL), 80 °C.
b Determined by chiral GC after derivatization in trifluoroacetamide. c Pentamethylbenzene was used as internal standard. d Determined by chiral HPLC.
e Calculated according to c ) eeamine/(eeamine + eeamide). f Calculated according to E ) ln[(1 - c)(1 - eeamine)]/ln[(1 - c)(1 + eeamine)].18 Owing to sensitivity
to experimental errors, E values calculated in the range 100-200 are reported as >100, values calculated in the range 200-300 are reported as >200, values
in the range 300-500 are given .200, and values in the range 900-1000 are given .500. g Remaining 9 was isolated in 38% yield when starting from
5mmol of substrate.
conversion were the shortest in all the series for amine 11 and
particularly for amine 14.
to 99.5% for both the remaining amine and the corresponding
lauramide at 50% conversion (reached in 3 h). Excellent results
were similarly recorded for a series of aliphatic and benzylic
amines. Finally, it must be recalled that this optimization of
enzymatic resolution with CAL-B at 80 °C found its justification
in the performance of DKR experiments associating enzymatic
resolution with CAL-B and thiyl radical-mediated racemization
at 80 °C that were successful with both ethyl laurate and lauric
acid as acyl donors.5,34
Very low selectivity factors (5 and 17, respectively) were
reported for the CAL-B-mediated resolutions of amines 12 and
13 performed at 60 °C in isopropyl ether, in the presence of
4-5 equiv of ethyl acetate.12a A plausible correlation was made
with the pKa of the amines (pKa ) 9.5 for 12 and pKa ) 9.7 for
13). Since a higher pKa value might result in a higher reactivity
of both enantiomers of the amine, one might expect a decrease
in selectivity. In our case, there was no significant effect on
the selectivity, but the pKa value should exert two opposite
influences. A more basic amine should slow down the rate of
conversion by decreasing the amount of free acid in the medium,
and at the same time, it should increase the rate of nucleophilic
attack on the acyl-enzyme complex. It is thereby very difficult
to evaluate the relative contribution of steric hindrance and
basicity upon the rate of the enzymatic conversion and its
enantioselectivity.
Experimental Section
General Procedure for Kinetic Resolutions. A solution of
amine (1 mmol), acyl donor (1 mmol), and C. antartica lipase B
(CAL-B, Novozym 435) (200 mg) in heptane (10 mL) was heated
for 1.5 to 26 h at 80 °C (with a magnetic agitation at 250 rpm).
The reaction was monitored by GC. When the analysis showed
the completion of the reaction, the mixture was cooled down to
room temperature, and the resin was filtered through a glass sinter
and rinsed with Et2O. The filtrate was concentrated in Vacuo. The
residue was dissolved in CDCl3, and pentamethylbenzene (0.25
mmol) was added as an NMR internal standard to afford the crude
yields for amine and amide. The crude was dissolved in CH2Cl2,
and K2CO3 was added. The solution was filtered and concentrated
in Vacuo. The residue was purified by column chromatography on
basic alumina (neat Et2O to Et2O/MeOH 90:10) to give amide as
a white solid and amine as a colorless oil.
1-Aminoindane (14) and, to a lesser extent, 1-phenethyl amine
(11) reacted like amine 1 much faster than the other amines in
the series.33 This cannot be ascribed to its basicity (pKa ) 9.53).
The aromatic substituents might be favorable in this case for
reactivity. As suggested by Sheldon,9c nonsteric interactions also
contribute to the enantiorecognition.
Conclusion
Acknowledgment. A gift of Novozym 435 from de Novo
Nordisk (Denmark) is gratefully acknowledged. The authors
thank the National Agency for Research for funding (ANR, no.
Blan06-3_142460).
The kinetic resolution of 2-amino-4-phenyl-butane was
optimized at 80 °C using CAL-B-catalyzed aminolysis of
carboxylic acids and their ethyl esters. The reactions carried
out with long chain esters and the corresponding acids as acyl
donors all proceeded with remarkably high enantioselectivity.
The use of carboxylic acids as acylating agents led to a marked
acceleration of the reaction rate compared to their ethyl ester
counterparts. Lauric acid led to enantiomeric excesses superior
Supporting Information Available: Experimental procedures
and NMR spectra for 1a-f, 2e, 3e, 4e, 5e, 6, 6e, 7e, 8e, 9, 9e, 10e,
11e, 12e, 13e, and 14e. This material is available free of charge
via the Internet at http//pubs.acs.org.
JO071069T
(33) (a) For comparative resolution of these amines with aminoacylase
I from Aspergillus melleus in the presence of 2-methoxyacetate, see ref
12b. (b) For their resolution with penicillin acylase from Alcaligenes faecalis
in aqueous solvent in the presence of phenylacetamide, see ref 12d.
(34) In the DKR experiments reported in ref 5, the use of lauric acid as
acylating agent led to transform rac-1 into (R)-1e (71% yield and ee >
99%); the use of ethyl laurate led to the same amide in 70% isolated yield
and 99% ee.
J. Org. Chem, Vol. 72, No. 18, 2007 6923