Resolution of non-protein amino acids via Carica papaya lipase-catalyzed enantioselective transesterification

Article abstract of DOI:10.1016/j.tetasy.2005.06.037

Carica papaya lipase-catalyzed transesterification of the 2,2,2-trifluoroethyl esters of N-benzyloxycarbonylated dl-amino acids carrying aliphatic side chains proceeded smoothly and, in almost all the cases, enantiospecifically (E = >200), affording the l-methyl esters and leaving the d-trifluoroethyl esters intact.

Full text of DOI:10.1016/j.tetasy.2005.06.037

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 16 (2005) 2569–2573
Resolution of non-protein amino acids via Carica papaya
lipase-catalyzed enantioselective transesterification
Toshifumi Miyazawa,a,* Kazuki Onishi,a Takashi Murashima,a
Takashi Yamadaa and Shau-Wei Tsaib
aDepartment of Chemistry, Faculty of Science and Engineering, Konan University, Higashinada-ku, Kobe 658-8501, Japan
bDepartment of Chemical Engineering, National Chen Kung University, Tainan 70101, Taiwan
Received 13 May 2005; accepted 27 June 2005
Abstract—Carica papaya lipase-catalyzed transesterification of the 2,2,2-trifluoroethyl esters of N-benzyloxycarbonylated DL-amino
acids carrying aliphatic side chains proceeded smoothly and, in almost all the cases, enantiospecifically (E = >200), affording the
L-methyl esters and leaving the D-trifluoroethyl esters intact.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
results have prompted us to examine CPL as a catalyst
for the resolution of N-protected amino acids as chiral
carboxylic acids. We herein report the CPL-catalyzed
highly enantioselective transesterification of N-benzyl-
oxycarbonyl (Z)-derivatives of non-protein amino acid
esters in organic solvents.
Lipases (triacylglycerol hydrolases, EC 3.1.1.3) have
been used as stereoselective catalysts for the preparation
of optically active forms of a wide variety of alcohols
and carboxylic acids.1–3 The enzymes utilized for such
a purpose are those from mammalian and microbial
sources.4–7 On the other hand, plant lipases have rarely
been employed for synthetic purposes until recently.
Some lipases from plant latexes, however, have become
available in large quantities and are now applied to
lipid conversions.8–10 Quite recently, Carica papaya
lipase (CPL), stored in the crude papain, was exploited
for the kinetic resolutions of (RS)-2-(4-chlorophen-
oxy)propanoic acid via enantioselective esterification.11
The highest enzyme activity and enantioselectivity were
attained with trimethylsilylmethanol as the acyl acceptor
in cyclohexane. CPL was more active, enantioselective,
and thermally more stable than Candida rugosa
lipase, which is known to show high enantioselectivity
toward carboxylic acids.12,13 This plant lipase was also
exploited for the enantioselective hydrolysis of (RS)-
naproxen 2,2,2-trifluoroethyl ester in water-saturated
organic solvents.14 An excellent enantioselectivity was
observed in water-saturated isooctane at 60 ꢁC. The
hydrolytic resolution of this acid with CPL was also
investigated as its 2,2,2-trifluoroethyl thioester in
water-saturated organic solvents.15 These successful
Homochiral non-protein amino acids are useful as
building blocks for the synthesis of analogues of biolog-
ically active peptides16 and as versatile chiral starting
materials or chiral auxiliaries for other synthetic pur-
poses.17 We have already reported on the resolution of
non-protein amino acids via the enantioselective hydrol-
ysis of 2-chloroethyl or 2,2,2-trifluoroethyl esters of
their N-Z derivatives catalyzed by lipases from Aspergil-
lus niger and porcine pancreas.18 We have also proposed
a transesterification procedure for their resolution med-
iated by Pseudomonas cepacia (Burkholderia cepacia)
lipase.19
The results reported herein together with those reported
earlier clearly suggest that CPL must be a very promis-
ing enzyme for the resolution of carboxylic acids via
enantioselective esterification, transesterification, or
hydrolysis.
2. Results and discussion
*
In a general experimental procedure, the 2,2,2-trifluoro-
ethyl ester of an N-Z-DL-amino acid DL-1 was allowed
Corresponding author. Tel.: +81 784314341; fax: +81 784352539;
e-mail: miyazawa@base2.ipc.konan-u.ac.jp
0957-4166/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2005.06.037

2570
T. Miyazawa et al. / Tetrahedron: Asymmetry 16 (2005) 2569–2573
H
R
R
CO2CH2CF3
CO2CH3
CO2CH2CF3
CPL
R
H
+
+
CH3OH
NHZ
NHZ
NHZ
D-1
DL
-1
L
-2
Scheme 1. CPL-catalyzed enantioselective transesterification between an N-Z-DL-amino acid 2,2,2-trifluoroethyl ester DL-1 and methanol. See Table
3 for R.
to react with an alcohol (4 M equiv) in an organic sol-
vent in the presence of CPL at a constant temperature
50
(Scheme 1). The trifluoroethyl ester was employed be-
cause of the tardiness of transesterification of other acti-
40
vated esters, such as the 2,2,2-trichloroethyl ester as well
as the conventional alkyl esters. The reaction was mon-
30
itored (conversion and enantiomeric excess values) by
HPLC on a chiral column. Initially, norleucine (2-
20
aminohexanoic acid, Nle), an isomer of the naturally
occurring leucine, was chosen as a model amino acid
10
and the effects of the reaction parameters investigated
on the enzymatic transesterification of the 2,2,2-triflu-
oroethyl ester of its N-Z-derivative DL-1d. As tempera-
ture has been recognized as one of the major potential
factors, which can affect the rate and enantioselectivity
of enzymatic reactions,20 the transesterification between
DL-1d and propanol in cyclohexane was investigated at
different temperatures (25–45 ꢁC). Lowering the temper-
ature resulted in a large decrease in the reaction rate
[temperature (ꢁC), % conversion after 24 h of incuba-
tion: 25, 11.3; 35, 21.4; 45, 43.4]. On the other hand,
temperature did not affect the enantioselectivity of the
transesterification at all: it was almost completely enan-
tioselective at each temperature (vide infra). Thus, sub-
sequent experiments were carried out at 45 ꢁC.
0
0
10
20
Time/h
30
40
50
Figure 1. Reaction profile in the CPL-catalyzed transesterification
between Z-DL-Nle-OCH2CF3 DL-1d and methanol in cyclohexane.
Symbols: s, Z-L-Nle-OCH3 L-2d; h, Z-D-Nle-OCH3 D-2d.
alcohol became longer, the conversion rate decreased
severely, with hexanol serving as the poorest nucleo-
phile, although the enantioselectivity was not affected
at all.
Recently, much attention has been focused on the sol-
vent effect on the enantioselectivity of enzymatic reac-
tions.25 Table 2 shows the effect of solvents on the
transesterification between DL-1d and methanol. Both
the reaction rate and enantioselectivity were affected lar-
gely by the solvent employed. The use of ethers, such as
isopropyl ether resulted in a marked decrease in enantio-
selectivity. The reactions were slowed down to a great
extent in solvents such as acetonitrile and chloroform,
although they proceeded in an almost completely enan-
tioselective manner. Thus, cyclohexane proved to be the
best one among the solvents examined.
Next, the effect of the alcohol nucleophile was investi-
gated on the transesterification of DL-1d in cyclohexane.
The results are summarized in Table 1. The time course
of the transesterification between DL-1d and methanol is
depicted in Figure 1. The reaction proceeded in an
almost completely enantioselective manner: the L-methyl
ester L-2d was solely formed until the reaction reached
50% conversion, while D-trifluoroethyl ester D-1d
remained nearly intact.22 The enantiomeric ratio, E,21 in
this case was evaluated to be >200.23 There was almost
no difference found between methanol and ethanol as
the reacting nucleophile with regard to the reaction rate
and enantioselectivity.24 When the chain length of the
Based on the above results, the transesterification with
methanol of the 2,2,2-trifluoroethyl esters of N-Z-DL-
Table 2. CPL-catalyzed transesterification between Z-DL-Nle 2,2,2-
trifluoroethyl ester DL-1d and methanol in different solventsa
Table 1. CPL-catalyzed transesterification of Z-DL-Nle 2,2,2-tri-
fluoroethyl ester DL-1d in cyclohexanea,b
b
Solvent
% Convn.
% eeP
E
c
Alcohol
% Convn.
% eeP
E
Cyclohexane
Isooctane
Isopropyl ether
tert-Butyl methyl ether
Chloroform
50.2
13.7
45.1
44.8
11.9
17.0
99.2
94.2
73.4
71.4
>99.8
>99.8
>200
39
12
11
>200
>200
Methanol
Ethanol
Propanol
Hexanol
50.2
50.0
43.4
20.5
99.2
>99.8
>99.8
>99.8
>200
>200
>200
>200
Acetonitrile
a Nle = 2-aminohexanoic acid.
b Conditions: 0.1 mmol of Z-DL-Nle-OCH2CF3, 0.4 mmol of an alco-
hol, and 10 mg of CPL in 0.8 ml of anhydrous cyclohexane at 45 ꢁC.
The results shown are those obtained after 24 h of incubation.
c Enantiomeric excess of the newly formed ester.
a Conditions: 0.1 mmol of DL-1d, 0.4 mmol of methanol, and 10 mg of
CPL in 0.8 ml of an anhydrous organic solvent at 45 ꢁC. The results
shown are those obtained after 24 h of incubation.
b Enantiomeric excess of the newly formed methyl ester 2d.

T. Miyazawa et al. / Tetrahedron: Asymmetry 16 (2005) 2569–2573
2571
Table 3. CPL-catalyzed transesterification between N-Z-DL-amino acid 2,2,2-trifluoroethyl esters DL-1 and methanol in cyclohexanea
b
Compound
R
% Convn.
% eeP
E
Cf. Ec
8.7
42
37
30
1a
1b
1c
1d
1e
1f
CH3
CH3CH2
CH3(CH2)2
CH3(CH2)3
(CH3)2CHCH2
CH3(CH2)4
(CH3)2CH(CH2)2
c-C6H11CH2
CH3SCH2CH2
CH3CH2SCH2CH2
55.6
50.8
46.3
50.2
43.6
46.8
48.0
26.0
50.2
47.7
78.4
95.6
>99.8
99.2
>99.8
>99.8
>99.8
>99.8
99.2
38
>200
>200
>200
>200
>200
>200
>200
>200
>200
23
1g
1h
1i
1j
>99.8
a Conditions: 0.1 mmol of DL-1, 0.4 mmol of methanol, and 10 mg of CPL in 0.8 ml of anhydrous cyclohexane at 45 ꢁC. The results shown are those
obtained after 24 h of incubation.
b Enantiomeric excess of the newly formed methyl ester 2.
c P. cepacia (B. cepacia) lipase-catalyzed transesterification between DL-1 and methanol in isopropyl ether.19
amino acids DL-1 carrying a number of aliphatic side
chains was examined in cyclohexane at 45 ꢁC. The re-
sults are summarized in Table 3, which also includes
those with some proteinogenic amino acids for the pur-
pose of comparison. The CPL-catalyzed transesterifica-
tions proceeded smoothly, especially with amino acids
bearing smaller side chains. In almost all cases exam-
ined, the reactions were almost completely enantioselec-
tive (E = >200),23 affording the L-methyl esters L-2 and
leaving the D-trifluoroethyl esters D-1 intact. One excep-
tion is the result with the alanine derivative 1a. Even in
this case, though, the E value (38) was high enough,
although it was smaller than those with other cases. In
the last column of Table 3 the E values observed in
the P. cepacia (B. cepacia) lipase-catalyzed transesterifi-
cation between DL-1 and methanol in isopropyl ether are
included.19 With this lipase, little difference was ob-
served in the solvents employed, isopropyl ether and
cyclohexane. The enantioselectivities as judged from
the E values were good to tolerable with the exception
of that obtained with alanine. The E values, however,
are smaller in one order of magnitude than those ob-
tained in the present study with CPL.
hydrolysis, taking into account that the lipase prepara-
tion is available easily and inexpensive. We have also
started an investigation on the resolution of non-protein
amino acids via the enantioselective hydrolysis of their
esters utilizing CPL.
4. Experimental
4.1. General
1H NMR spectra were obtained at 300 MHz on a Var-
ian Unity 300 spectrometer using chloroform-d as a sol-
vent with TMS as the internal standard. All organic
solvents were distilled following standard protocols
and dried over molecular sieves prior to use.
4.2. Preparation of substrates
DL-2-Aminoheptanoic acid, DL-2-amino-5-methylhexa-
noic acid, and DL-2-amino-3-cyclohexylpropanoic acid
were prepared from diethyl acetamidomalonate and
the corresponding alkyl bromide by adopting essentially
the same procedure described in the literature.29 Other
racemic amino acids were purchased from Tokyo
Chemical Industry, Wako Pure Chemical Industries,
or Aldrich Chemical Co. The amino acids were benzyl-
oxycarbonylated using benzyloxycarbonyl chloride
(Z-Cl) under the usual Schotten–Baumann conditions30
to give N-Z-DL-amino acids.18 These were purified by
recrystallization from an appropriate solvent (e.g.,
EtOAc–petroleum ether) and converted to the 2,2,2-
trifluoroethyl esters by the EDC [1-ethyl-3-(3-dimethyl-
aminopropyl)carbodiimide]-DMAP (4-dimethylamino-
pyridine) method31 using 2,2,2-trifluoroethanol. 1H
NMR data (CDCl3) of the N-Z-DL-amino acid 2,2,
2-trifluoroethyl esters thus prepared are as follows.32
In all the cases mentioned above, the preferential reac-
tion of the L-enantiomers was confirmed by comparison
with authentic samples prepared from the optically
active amino acids, if available, on HPLC or suggested
from the regularity of elution order of the enantiomers
on HPLC.26,27 This stereochemical preference is the
same as that observed in the hydrolysis of N-Z-DL-
amino acid 2-chloroethyl or 2,2,2-trifluoroethyl esters18
and the transesterification of N-Z-DL-amino acid 2,2,2-
trifluoroethyl esters19 mediated by other lipases.
3. Conclusion
The results reported herein show that CPL can act as an
enantioselective catalyst in the transesterification of N-
protected aliphatic amino acid activated esters in an
organic solvent such as cyclohexane.28 This together
with the results reported earlier clearly demonstrate that
CPL must be an enzyme of great promise for the resolu-
tion of carboxylic acids via enantioselective transesterifi-
cation as well as via enantioselective esterification or
4.2.1. Compound 1b.
2.00 (2H, m), 4.36–4.50 (2H, m), 4.56–4.69 (1H, m), 5.09
(2H, s), 5.21 (1H, br d), 7.29–7.37 (5H, m).
dH 0.97 (3H, t, J = 7.5 Hz), 1.69–
4.2.2. Compound 1c. dH 0.95 (3H, t, J = 7.3 Hz), 1.31–
1.48 (2H, m), l.61–1.90 (2H, m), 4.36–4.49 (2H, m),
4.57–4.70 (1H, m), 5.12 (2H, s), 5.17 (1H, br d,
J = 8 Hz), 7.25–7.39 (5H, m).

2572
T. Miyazawa et al. / Tetrahedron: Asymmetry 16 (2005) 2569–2573
4.2.3. Compound 1d.
d
H 0.89 (3H, t, J = 6.9 Hz), 1.25–
was collected and lyophilized to afford the crude CPL
1.40 (4H, m), 1.63–1.91 (2H, m), 4.34–4.48 (2H, m),
4.56–4.69 (1H, m), 5.11 (2H, ABq, J = 12 Hz), 5.20
(1H, br d, J = 8 Hz), 7.29–7.40 (5H, m).
(0.75 g).
4.4. HPLC analysis
Transesterification reactions were monitored by chiral
HPLC on a Chiralpak AS column (4.6 mm id · 250 mm)
or a Chiralcel OD column27 (4.6 mm id · 250 mm) (Dai-
cel Chemical Industries) using hexane–2-propanol as an
eluent. The liquid chromatograph employed was a Shi-
madzu LC-10AS instrument, equipped with a Rheodyne
7125 sample injector and a Shimadzu SPD-10A variable
wavelength UV monitor. The temperature of the column
was maintained using a thermostated bath. A Shimadzu
C-R8A data processor was used for data acquisition and
processing. The enantiomers of the methyl esters 2 of
N-Z-amino acids were separated well enough for the
accurate determination of the ee values on either of the
columns by choosing an appropriate proportion of
hexane/2-propanol for each compound. In general, the
enantiomeric separations of the corresponding 2,2,2-tri-
fluoroethyl esters 1 were inferior to those of the methyl
esters on either column. The limit of detection on the
HPLC analysis was estimated as ca. 0.1% enantiomer.
The separation of the enantiomers of N-Z-amino acid
2,2,2-trifluoroethyl esters 1 and methyl esters 2 on the
Chiralpak AS column is shown in Table 4.
4.2.4. Compound 1e. dH 0.93–0.97 (6H, m), 1.56–1.76
(3H, m), 4.35–4.50 (2H, m), 4.53–4.66 (1H, m), 5.07
(1H, br d, J = 6 Hz), 5.10 (2H, s), 5.22 (1H, br d,
J = 8 Hz), 7.36–7.40 (5H, m).
4.2.5. Compound 1f. dH 0.87 (3H, apparent t), 1.23–
1.41 (6H, m), 1.62–1.93 (2H, m), 4.34–4.48 (2H, m),
4.56–4.68 (1H, m), 5.10 (2H, ABq, J = 12 Hz), 5.16
(1H, br d, J = 8 Hz), 7.28–7.38 (5H, m).
4.2.6. Compound 1g. dH 0.80–0.93 (6H, m), 1.11–1.32
(2H, m), 1.46–1.61 (1H, m), 1.61–1.92 (2H, m), 4.35–
4.50 (2H, m), 4.57–4.70 (1H, m), 5.12 (2H, ABq,
J = 12 Hz), 5.22 (1H, br d, J = 8 Hz), 7.26–7.37 (5H,
m).
4.2.7. Compound 1h. dH 0.86–1.80 (13H, m), 4.34–4.52
(2H, m), 4.54–4.67 (1H, m), 5.07 (1H, br d, J = 6 Hz),
5.05 (1H, br d, J = 8 Hz), 5.10 (2H, ABq,
J = 13.5 Hz), 7.28–7.38 (5H, m).
4.2.8. Compound 1i. dH 1.94–2.23 (2H, m), 2.06 (3H, s),
2.53 (2H, t, J = 7.2 Hz), 4.36–4.48 (1H, m), 4.53–4.66
(2H, m), 5.10 (2H, s), 5.43 (1H, br d, J = 8 Hz), 7.28–
7.34 (5H, m).
4.5. General procedure for the CPL-catalyzed
transesterification
A solution of an N-Z-amino acid 2,2,2-trifluoroethyl
ester (0.1 mmol) and an alcohol (0.4 mmol) in an organ-
ic solvent (0.8 ml) was stirred with the CPL preparation
(10 mg) in a 1-ml screw-capped vessel in a thermostated
incubator. Monitoring the reaction and assessing the ee
value of the newly formed ester were conducted simulta-
neously by HPLC analysis on the chiral columns men-
tioned above. Aliquots (ca. 10 ll) of the reaction
mixture were withdrawn at frequent intervals, diluted
with ether (200 ll), filtered through a PTFE membrane
filter, and then injected (ca. 5 ll) onto the column. The
results obtained after 24 h of incubation are shown in
Tables 1–3.
4.2.9. Compound 1j. dH 1.22 (3H, t, J = 7.4 Hz), 1.92–
2.22 (2H, m), 2.45–2.61 (4H, m), 4.37–4.49 (1H, m),
4.53–4.66 (2H, m), 5.10 (2H, s), 5.38 (1H, br d), 7.24–
7.35 (5H, m).
4.3. Preparation of the enzyme11
The crude papain (Sigma P-3375, 5.0 g) was dissolved in
distilled water (25 ml), stirred for 30 min at 4 ꢁC, and
then centrifuged (12,000 rpm, 12 min) at the same tem-
perature. After the supernatant was removed, the above
procedure was repeated on the residue. The precipitate
Table 4. HPLC separation of the enantiomers of N-Z-amino acid 2,2,2-trifluoroethyl esters 1 and methyl esters 2a
R
2,2,2-Trifluoroethyl ester
Methyl ester
b
b
k10
ac
k10
ac
CH3
CH3CH2
CH3(CH2)2
CH3(CH2)3
(CH3)2CHCH2
CH3(CH2)4
(CH3)2CH(CH2)2
c-C6H11CH2
CH3SCH2CH2
CH3CH2SCH2CH2
1a
1b
1c
1d
1e
1f
2.50
2.00
1.77
1.43
1.43
1.17
1.20
1.57
4.07
3.27
1.16
1.00
1.13
1.12
1.23
1.06
1.19
1.15
1.05
1.07
2a
2b
2c
2d
2e
2f
3.83
3.07
3.27
2.47
2.57
2.03
2.03
3.57
7.50
5.93
1.17
1.18
1.12
1.18
1.30
1.07
1.31
1.00
1.09
1.11
1g
1h
1i
2g
2h
2i
1j
2j
a HPLC conditions: column, Chiralpak AS; mobile phase, hexane–2-propanol (95:5, v/v); flow rate 1.0 ml minꢀ1; column temperature, 30 ꢁC.
b Capacity factor: k0 = (tR ꢀ t0)/t0 where t0 = void time. The void time was estimated to be 3.0 min using 1,3,5-tri-tert-butylbenzene. The suffix 1
denotes for the faster eluting enantiomer.
c Separation factor: a ¼ k20 =k01. The suffix 2 denotes for the slower eluting enantiomer.

T. Miyazawa et al. / Tetrahedron: Asymmetry 16 (2005) 2569–2573
2573
15. Ng, I.-S.; Tsai, S.-W. Biotechnol. Bioeng. 2005, 89, 88–95.
16. Roberts, D. C.; Vellaccio, F. In Unusual Amino Acids in
Peptide Synthesis; Gross, E., Meienhofer, J., Eds.; The
Peptides; Vo1. 5; Academic Press: New York, 1983, pp
341–449.
17. Coppola, G. M.; Schuster, H. F. Asymmetric Synthesis:
Construction of Chiral Molecules Using Amino Acids; John
Wiley: New York, 1987.
Acknowledgments
This work was supported in part by a grant from the
Hirao Taro Foundation of the Konan University Asso-
ciation for Academic Research.
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