Efficient enzymatic kinetic resolution of 2-heptylamine with a highly active acyl donor

Article abstract of DOI:10.1016/j.catcom.2010.04.015

Novozyme435 facilitated kinetic resolution of 2-heptylamine was here presented. Methyl methoxyacetate was used as acyl donor. A survey of influencing factors including hydrogen bonding effect, solvent effect, steric effect, temperature and the amount of acyl donor were investigated in detail. At the optimum conditions, the enantiomeric separation was successfully obtained within 8 h at 20 °C, and gave high conversion and optical purity of (R)-2-heptylamine, 48.9% and over 99% respectively. The immobilized lipase B was found to be suitable for the enantiomeric separation of aliphatic amines with good recyclability.

Full text of DOI:10.1016/j.catcom.2010.04.015

Catalysis Communications 11 (2010) 987–991
Contents lists available at ScienceDirect
Catalysis Communications
journal homepage: www.elsevier.com/locate/catcom
Efficient enzymatic kinetic resolution of 2-heptylamine with a highly active
acyl donor
⁎
Jian-Hua Sun, Rong-Ji Dai, Wei-Wei Meng, Yu-lin Deng
School of Life Science, Beijing Institute of Technology, Beijing 100081, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 March 2010
Received in revised form 15 April 2010
Accepted 20 April 2010
Available online 28 April 2010
Novozyme435 facilitated kinetic resolution of 2-heptylamine was here presented. Methyl methoxyacetate
was used as acyl donor. A survey of influencing factors including hydrogen bonding effect, solvent effect,
steric effect, temperature and the amount of acyl donor were investigated in detail. At the optimum
conditions, the enantiomeric separation was successfully obtained within 8 h at 20 °C, and gave high
conversion and optical purity of (R)-2-heptylamine, 48.9% and over 99% respectively. The immobilized lipase
B was found to be suitable for the enantiomeric separation of aliphatic amines with good recyclability.
© 2010 Elsevier B.V. All rights reserved.
Keywords:
Enzymatic kinetic resolution
Novozyme435
2-Heptylamine
Methyl methoxyacetate
1. Introduction
2. Experimental
Over the last years, single-enantiomer drugs became more and
more important in the pharmaceutical industry. Optically active
aliphatic amines are a type of compounds for the enantioselective
synthesis of various pharmaceuticals [1]. Among these amines, 2-
heptylamine (2-HA) is usually used to prepare neurological disease
drugs [2]. Enantiomeric forms of these drugs always differ in potency,
toxicity and behavior in biological systems. As a result, it is essential to
study the resolution methods of 2-HA.
2.1. General
2, 3, 4, 6-Tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate
(GITC) was synthesized according to the method introduced by
Deng and Wan [19]. 2-HA 99%, 2-pentylamine 99%, 4-methyl-2-
pentylamine 99%, 2-hexylamine 99%, 3-heptyl amine 99%, 2-
nonylamine 99%, α-phenethylamine 98%, methyl methoxyacetate
99%, and methyl dimethoxyacetate 99% were all purchased from J&K
Lipase B from Candida antarctica (CALB) has been demonstrated to
be a very useful catalyst for the resolution of chiral amines. In order to
improve the activity of CALB, a variety of new reaction systems [3–6]
and new immobilization methods [7–11] have been reported. Other
reports focused their attentions on seeking new acyl donors with high
activity, such as ethyl caprylate [12], propylene acetate [13], dibenzyl
carbonate [14], capric acid [15], ethyl acetate [16,17] and isopropenyl
acetate [18]. However these acyl donors are not suitable for large-
scale application in industry because of their low activity. The
produced esters or amides were usually difficult to be transferred
into corresponding alcohols or amines. In this paper, we reported a
highly active acyl donor, methyl methoxyacetate (MMA), which could
be supported commercially. Improved condition for enzymatic kinetic
resolutions of 2-HA and other aliphatic amines was also provided.
Typical reaction was shown in Scheme 1.
Chemical Ltd. Novozyme435 (immobilized lipase B from C.
antarctica, 4500 LU/g) was purchased from Novozymes (Denmark).
All other chemicals and reagents were obtained commercially and
were of analytical grade. All solvents were dried by 4 Å molecular
sieves before being used.
2.2. Lipase-catalyzed acylation of amine
Acyl donor (2 mmol) and amine (2.5 mmol) were dissolved in
10 ml ethyl ether. Novozyme435 (200 mg) was then added. The
mixture was incubated at set temperature for a certain time. The
reaction was stopped by removal of the enzyme. The unreacted
amine was isolated by extraction with hydrochloric acid solution
(1.2 N, 10 ml). The organic fraction was concentrated under vacuum
and pure amide was obtained. The pH of aqueous solution was
adjusted to over 10 with sodium hydroxide solution (1 N). Ethyl
ether (15 ml) was introduced and the phases were separated. The
unreacted amine was obtained by concentration of organic fraction
under vacuum.
⁎
Corresponding author. Tel.: +86 10 68914607.
E-mail address: deng@bit.edu.cn (Yu-lin Deng).
1566-7367/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.catcom.2010.04.015

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J.-H. Sun et al. / Catalysis Communications 11 (2010) 987–991
Scheme 1. Enzymatic kinetic acylation of amines.
solution (50 μl) and amine solution (50 μl) were mixed together with
2.3. Hydrolysis of amide
scroll instrument for 10min. The mixture was placed for 30 min at
room temperature.
A mixture of (R)-amide (2.5 mmol), triethanolamine (200 mg)
and 50% sodium hydroxide solution (400 mg) were heated at 120 °C
for 6h. The reaction was quenched by adding 10 ml H2O. The mixture
was then cooled to room temperature. Ethyl ether (30 ml) was
introduced and the phases were separated. The organic extract was
washed with 10 ml H2O. The R-amine was obtained as a colorless
liquid by distilling the organic fraction under vacuum.
HPLC separation was achieved on a Diamonsil C18 column using an
isocratic flow (1 ml/min). The mobile phase consisted of a mixture of
TEAA buffer (0.1 M, pH 5.5) and methanol. The ratio of buffer and
methanol was 38:62. Twenty microliters sample was injected into
HPLC system every time. Wavelength of UV detector was 254 nm.
3. Results and discussion
2.4. Analysis of the samples
The mechanism of enzymatic kinetic resolution of alcohol has been
described by Cygler et al. [20] and Castro and Gago [21]. Cygler and his
research group [20] also identified how the enantiomers of menthol, a
typical secondary alcohol, bind to lipase B in the transition state using
X-ray crystallography. According to the report, mechanism of amine
resolution could be summarized by Scheme 2. The overall progress
included two defined steps: 1) formation of acyl-enzyme and 2)
amination of the acyl-enzyme.
The optical purity and yield measurement were performed on a
HPLC system (Shimadzu) by pre-column derivation. Typical deriva-
tion of GITC and amine was carried out as followed: a certain amount
of GITC was dissolved with acetonitrile to give 2 mol/L solution. Fatty
amine (1 mmol) was diluted with a mixed solvent of acetonitrile and
three steaming water (VCH3CN:VH2O =50:50), which also contained
1 g/L triethylamine. The final volume of amine solution was 5 ml. GITC
Scheme 2. Enzymatic kinetic acylation mechanism of amines.

J.-H. Sun et al. / Catalysis Communications 11 (2010) 987–991
989
Hydrogen bonding was supposed to play more important role in
the transformation than inductive effect [22]. To confirm this
hypothesis, two amines (2-HA and α-phenethylamine) were
employed as substrates, and two esters (methyl methoxyacetate
and ethyl chloroacetate) were used as acyl donors. A detailed
comparison of the effect of acyl donor on conversion of the enzymatic
resolution had been described in Fig. 1. Just as shown in Fig. 1, when
methyl methoxyacetate was performed as acyl donor, the acylation of
2-HA was faster than the reaction with ethyl chloroacetate as acyl
donor. According to previous paper [23], rate of the reaction with
methyl methoxyacetate as acyl donor should be slower if the
inductive effect of β-position on acyl donor largely affected the
acylation. The results in the experiments disagreed with the inductive
effect, even though Cl atom is more electronegative than methoxyl
group, and a possible explanation is that Cl only forms weaker
hydrogen comparing to oxygen [24]. Similar results were observed
when α-phenethylamine was employed as substrate, which was
shown in Fig. 1. On the base of these results, the rate increase might be
attributed to the hydrogen bonding between acyl donor and enzyme.
Methyl methoxyacetate was an ideal acyl donor because it could form
hydrogen bonding easily with enzyme.
Polarity of organic solvent was major criteria in solvent selection
due to its close relationship with the formation of hydrogen bonding.
As shown in Fig. 2, an increasing conversion and eeS with increasing
hydrophobicity were observed when ethyl ether, THF and isopropanol
were used as solvents. It would appear likely that the differences were
due mainly to their effect on the formation of hydrogen bonding. In
more hydrophobic solvent, hydrogen bonding between substrate and
Novozyme435 could form more easily. In particular, the conversion
and eeS of 2-HA in n-heptane or n-hexane are lower than those in
ethyl ether, isopropyl ether or methyl tert-butyl ether, which might
be related to the solvent swelling of polyacrylic resin particles where
the enzyme is immobilized. The carrier of the enzyme is polyacrylic
resin particle, which has certain hydrophilicity. Considering the more
conformability the more consistencies, the polyacrylic resin ball is
swelling more easily in middle hydrophobic solvents than hydropho-
bic solvent. Accordingly, the immobilized enzyme cannot be dispersed
well in the reaction system using hydrophobic solvent. The same
solvent effect was also reported by literature [18]. The results of our
experiments suggested that the resolution should be operated in
Fig. 2. Effect of solvent on the enzymatic acylation conversion and eeS of 2-HA. Reaction
conditions: methyl methoxyacetate, 4 mmol; 2-HA: 5 mmol; Novozyme435: 200 mg;
solvent (10 ml): 1 n-heptane, 2 n-hexane, 3 isopropyl ether, 4 methyl tert-butyl ether, 5
ethyl ether, 6 THF, and 7 isopropanol; a slightly elevated temperature 20 °C was used
during the reactions.
middle hydrophobicity solvents, such as ethyl ether, isopropyl ether
and methyl tert-butyl ether.
Steric effect was also studied by the comparison between the
reactions with methyl methoxyacetate and methyl dimethoxyacetate
as acyl donors. The reactions were carried out in ethyl ether system at
20 °C. Methyl dimethoxyacetate was supposed to form hydrogen
bonding with enzyme more easily than methyl methoxyacetate
because it had two methoxyl groups. However, it could be clearly
seen from Fig. 3 that the conversion of 2-HA declined largely when
methyl dimethoxyacetate was used as acyl donor. The result might be
attributed to another influencing factor (steric effect). Two methoxyl
groups on methyl dimethoxyacetate configuration made the combi-
nation of enzyme and acyl donor unstable, which led to lower
conversion. These results showed that straight chain acyl donors were
desirable.
Fig. 1. Effect of acyl donor on the enzymatic acylation conversion of 2-HA and α-
phenethylamine. Reaction conditions: (●) methyl methoxyacetate: 4 mmol, 2-HA:
5 mmol (■) ethyl chloroacetate:4 mmol, 2-HA: 5 mmol ( ) methyl methoxyacetate:
Fig. 3. Effect of acyl donor on the enzymatic acylation conversion of 2-HA. Reaction
conditions: (■) methyl methoxyacetate, 4 mmol (●) methyl dimethoxyacetate,
4 mmol; 2-HA: 5 mmol; Novozyme435: 200 mg; 10 ml ethyl ether was used as solvent.
A slightly elevated temperature 20 °C was used during the reaction.
a
4 mmol, α-phenethylamine: 5 mmol ( ) ethyl chloroacetate: 4 mmol, α-phenethy-
Y
lamine: 5 mmol; Novozyme435, 200 mg; 10 ml ethylether was used as solvent. A
slightly elevated temperature 20 °C was used during the reactions.

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J.-H. Sun et al. / Catalysis Communications 11 (2010) 987–991
Table 1
Enzyme catalyzed resolution of aliphatic aminesa.
Entry Amine
Time (h) eeS% eeP% Eb
Conversion%
1
2
3
4
5
6
7
2-Hexylamine
8
8
8
2
2
2
8
96.9 N99 N1000 49.5
97.7 N99 N1000 49.7
95.1 N99 N1000 48.9
96.3 N99 N1000 49.3
96.4 N99 N1000 49.3
4-Methyl-2-pentylamine
2-Heptylamine
3-Heptylamine
2-Pentylamine
2-Heptylamine
2-Nonylamine
80.5 N99 N611
44.8
n.d.
–
–
–
a
All reactions were conducted in dry ether using 5 mmol amine, 4 mmol methyl
methoxyacetate and 200 mg. Novozym 435 at 20 °C for 8 h. Conversion and ee were
determined by HPLC.
b
E=ln[(1−C)(1−eeS)]/ln[(1−C)(1+eeS)] [25].
inhibition to the activity of Novozyme435 obviously. In order to
reduce the cost, Novozyme435-catalyzed resolution would better be
carried out at the molar ratio of acyl donor and 2-HA, 4:5.
A remaining task of the overall process was the hydrolysis of the R-
configured amide. Based on the above results, 2-HA was firstly
separated to give R-configured amide. Hydrolysis of (R)-amide to (R)-
amine was operated in NaOH-triethanolamine system. Optical purity
of (R)-2-HA was determined on HPLC with the method of pre-column
derivation. The final optical purity was 99.6% ee, and conversion of
(R)-2-HA was 48.9%, which was calculated according to literature
[25].
To test the recyclability, Novozyme435 was repeatedly recovered
from the reaction mixture, washed with ethyl ether, and reused. After
just eight cycles at 20 °C, the conversion of 2-HA was still over 48%.
These experiments showed that this immobilized CALB showed a
good stability in our conditions. The slight decline was mainly due to
the leakage of the enzyme from polyacrylic resin balls on which the
enzyme was non-covalently absorbed. Accordingly, wild stirring,
which might shatter the polyacrylic resin ball, should be avoided. In
the experiments, the reaction system was incubated by rotating the
reactor.
By the enzymatic procedure, various racemic aliphatic amines
(Table 1, Entry 1–5) were successfully isolated into optically pure
amines in high convention and high enantiomeric excess, over 48.9%
and 99% respectively. It was observed that for hydrophilic amines,
such as 2-pentylamine (Table 1, Entry 5), 3-heptylamine (Table 1,
Entry 4), the enantiomers were obtained within 2h, but at the same
conditions, the conversion and eeS of the amines with longer carbon
chain, such as 2-HA (Table 1, Entry 6) reduced to 44.8% and 80.5%.
When 2-nonylamine (Table 1, Entry 7) was used as a substrate, the
enantiomeric separation did not proceed, even though the reaction
time was extended to 8h. These results suggested that increase of
hydrophilic nature in amines might increase the reaction activity, and
finally lead to better enantiomeric separation. Comparing previous
studies [12–18], an ideal acyl donor was used for the enantiomeric
resolution of aliphatic amines, and thus high conversion and optical
purity of (R)-amines were achieved.
Fig. 4. Effect of reaction temperature on the enzymatic acylation conversion and eeS of
2-HA. Reaction conditions, varying temperature from 10 °C to 30 °C; Methyl
methoxyacetate, 4 mmol; 2-HA: 5 mmol; Novozyme435: 200 mg; 10 ml ethyl ether
was used as solvent.
The choice of temperature was of great importance for the
asymmetric acylation of 2-HA. A survey of temperatures below
30 °C were investigated because the reactions were operated in ethyl
ether system. As shown in Fig. 4, the conversion and eeS of 2-HA
increased when temperature was increased from 10 °C to 20 °C. The
highest conversion and eeS came close to 50% and 95%. But when
temperature was increased sequentially to 30 °C, the conversion and
eeS declined to 46% and 85%. The results showed that asymmetric
acylation of 2-HA should be carried out at 20 °C.
The effect of amount of acyl donor on the asymmetric acylation
was investigated to confirm acyl donor inhibition. The results were
shown in Fig. 5. When the amount of methyl methoxyacetate was
lower than 4 mmol, the conversion and eeS increased with the
increased amount of acyl donor. When the amount of acyl donor was
increased from 4 mmol to 30 mmol, the conversion and eeS did not
show significant change. The results indicate that acyl donor has no
4. Conclusions
Novozyme435-catalyzed asymmetric acylation of 2-HA was
successfully carried out in ethyl ether system. Methyl methoxyacetate
was found to be an ideal acyl donor for this enzymatic resolution, and
high conversion and optical purity of enantiomers of amines were
obtained. It somehow suggested that hydrogen bonding and steric
effect might play important roles in the reaction. Finally, the enzyme
and the solvent could be reused in the next run, which significantly
reduced the cost of the process. In summary, a highly efficient
protocol for asymmetric resolution of 2-HA and its homologous
compounds was developed.
Fig. 5. Effect of amount of acyl donor on the enzymatic acylation conversion and eeS of
2-HA. Reaction conditions, varying the amount of methyl methoxyacetate from
2.5 mmol to 30 mmol; 2-HA: 5 mmol; Novozyme435: 200 mg; 10 ml ethyl ether was
used as solvent. A slightly elevated temperature 20 °C was used during the reaction.

J.-H. Sun et al. / Catalysis Communications 11 (2010) 987–991
991
[9] K. Mondal, P. Mehta, B.R. Mehta, D. Varandani, M.N. Gupta, Biochim. Biophys. Acta
1764 (2006) 1080–1086.
Acknowledgments
[10] B. Krajewska, Enzyme Microb.Technol. 35 (2004) 126–139.
[11] M. Matsumoto, K. Ohashi, Biochem. Eng. J. 14 (2003) 75–77.
[12] N. Öhrner, C. Orrenius, A. Mattson, T. Norin, K. Hult, Enzyme Microb.Technol. 19
(1996) 328–331.
[13] D. Xu, Z. Li, S. Ma, Tetrahedron Lett. 44 (2003) 6343–6346.
[14] C.E. Hoben, L. Kanupp, J.E. Bäckvall, Tetrahedron Lett. 49 (2008) 977–979.
[15] A.T. Gavilán, J. Escalante, I. Regla, A.L. Munguía, E. Castillo, Tetrahedron Asymmetr.
18 (2007) 2621–2624.
The research of our group is supported financially by the grants
2008BAK03A02 and 2007CB914100 projects from the Ministry of
Science and Technology of China. The authors are also thankful for the
financial support of Beijing Institute of Technology GenYuan Phar-
maceutical Development Center Co., Ltd.
[16] S.A. Santamaría, V.G. Fernández, V. Gotor, Eur. J. Org. Chem. (2009) 2533–2538.
[17] J. Paetzold, J.E. Bäckvall, J. Am. Chem. Soc 127 (2005) 17620–17621.
[18] I.I. Gill, J. Das, R.N. Patel, Tetrahedron Asymmetr. 18 (2007) 1330–1337.
[19] X.J. Deng, Q.H. Wan, Fine Chemicals 22 (2005) 307–310.
[20] M. Cygler, P. Grochulski, R.J. Kazlauskas, J.D. Schrag, F. Bouthillier, B. Rubin, A.N.
Serreqi, A.K. Guptai, J. Am. Chem. Soc. 116 (1994) 180–186.
[21] M.S. Castro, J.V.S. Gago, Tetrahedron 54 (1998) 2877–2892.
[22] H. Ismail, R.M. Lau, F. Rantwijk, R.A. Sheldon, Adv. Synth. Catal. 350 (2008)
1511–1516.
[23] F. Balkenhohl, K. Ditrich, B. Hauer, W. Ladner, J. Prakt. Chem. 339 (1997) 381–384.
[24] M.B. Smith, J. March, March's Advanced Organic Chemistry, Reactions, Mechan-
isms, and Structure, fifth editionJohn Wiley & Sons, Inc, USA, 2001 p101.
[25] D.H.Yu.Z. Wang, P. Chen, L. Jin, Y.M. Cheng, J.G. Zhou, S.G. Cao, J. Mol. Catal. B:
Enzym. 48 (2007) 51–57.
References
[1] H. Thomas, M. Roger, H.M. Brunne, R. Felix, Angew. Chem. Int. Ed. 38 (1999)
643–647.
[2] P.H. Yu, B.A. Davis, A.A. Boulton, J. Med. Chem. 35 (1992) 3705–3713.
[3] J.K. Lee, M.J. Kim, J. Org. Chem. 67 (2002) 6845–6847.
[4] R. Irimescu, K. Kato, Tetrahedron Lett. 45 (2004) 523–525.
[5] R. Irimescu, K. Kato, J. Mol. Catal. B: Enzym. 30 (2004) 189–194.
[6] K.W. Kim, B. Song, M.Y. Choi, M. Kim, Org. Lett. 3 (2001) 1507–1509.
[7] D.S. Rodrigues, A.A. Mendes, W.S. Adriano, J. Mol. Catal. B: Enzym. 51 (2008)
100–109.
[8] B. Chen, J. Hu, E.M. Miller, W. Xie, M. Cai, R.A. Gross, Biomacromolecules 9 (2008)
463–471.