Following the lipase catalyzed enantioselective hydrolysis of amino acid esters with capillary electrophoresis using contactless conductivity detection

Article abstract of DOI:10.1002/chir.20746

The progress of the enzymatic hydrolysis of racemic mixtures of the enantiomers of the methyl esters of serine and threonine was monitored. This was possible in a reaction vessel of 1.5 mL by direct sampling of volumes in the nanoliter-range directly into an electrophoresis capillary. Contactless conductivity detection was used for quantification as the analytes are not accessible by UV-detection in capillary electrophoresis. Porcine pancreatic lipase and wheat germ lipase both showed a preference for the L-enantiomers of both amino acid esters. The selectivity of the porcine lipase between the two L-esters of the two amino acids was also studied and it was found that the production of L-threonine had priority over L-serine.

Full text of DOI:10.1002/chir.20746

CHIRALITY 22:331–335 (2010)
Following the Lipase Catalyzed Enantioselective Hydrolysis of
Amino Acid Esters with Capillary Electrophoresis Using
Contactless Conductivity Detection
AIPING SCHUCHERT-SHI, AND PETER C. HAUSER*
Department of Chemistry, University of Basel, 4056 Basel, Switzerland
ABSTRACT
The progress of the enzymatic hydrolysis of racemic mixtures of the
enantiomers of the methyl esters of serine and threonine was monitored. This was pos-
sible in a reaction vessel of 1.5 mL by direct sampling of volumes in the nanoliter-range
directly into an electrophoresis capillary. Contactless conductivity detection was used
for quantification as the analytes are not accessible by UV-detection in capillary electro-
phoresis. Porcine pancreatic lipase and wheat germ lipase both showed a preference for
the L-enantiomers of both amino acid esters. The selectivity of the porcine lipase
between the two L-esters of the two amino acids was also studied and it was found that
the production of L-threonine had priority over L-serine. Chirality 22:331–335,
2
010. VV C 2009 Wiley-Liss, Inc.
KEY WORDS: enzymatic reactions; enantiomeric separations; ester hydrolysis; lipase
kinetic resolution; amino acids; capillary electrophoresis; conductivity
detection
INTRODUCTION
coupled contactless conductivity detection.’’ Its fundamen-
5
–8
tals have been studied extensively and at least two com-
mercial devices are now available and can be retrofitted to
existing CE-instrumentation. Recent reviews on applica-
Capillary electrophoresis (CE) is an attractive method
for the separation of enantiomers as in contrast to HPLC it
is not necessary to use a chiral column. Chiral reagents
can be dissolved in the relatively low volumes of the sepa-
ration buffer and do not have to be covalently bound to the
packing material of separation columns. Optimization is
possible by adjusting concentration, pH or ratios of differ-
ent dissolved reagents without having to resort to modifi-
cations of a stationary phase. This application has there-
fore become one of the major uses of CE. For a recent
review, see for example Ref 1. A further advantage of CE
is the fact that only small sample volumes are required as
the separation is carried out in narrow capillaries.
A shortcoming of CE has long been the limitation of
commercial instrumentation to UV-absorbance or fluores-
cence detection. Because of the short optical pathlengths
available, this has meant that weakly absorbing species,
such as most amino acids, could only be detected follow-
ing chemical derivatization to render them accessible by
optical means. Often studies of amino acids by CE have
been restricted to those few species which have an aro-
matic moiety and are thus easily detectable by UV-absorb-
ance. See for example, the recent review by Lindner and
4
9–11
tions of C D are also available.
Several research groups have demonstrated the deter-
mination of amino acids at low pH in cationic form using
1
2–16
17
contactless conductivity detection.
Gong et al. dem-
onstrated that the determination of the enantiomers of
4
non-UV-absorbing amino acids is possible by CE-C D
using a combination of the noncharged additives hydroxy-
propyl-b-cyclodextrin and (1)-(18-crown-6)-2,3,11,12-tetra-
carboxylic acid (18C6H ) as the chiral separators. It has
4
furthermore been shown that capillary electrophoresis-
capacitively coupled contactless conductivity detection
4
(
CE-C D) may be used for the monitoring of enzymatic
reactions when the species involved are not detectable by
optical means. The nonionic species glucose, ethanol,
ethyl acetate, and ethyl butyrate were made accessible for
analysis by CE via charged products or byproducts
obtained in enzymatic conversions using hexokinase,
2
Contract grant sponsor: Swiss National Science Foundation;
Contract grant number: 200020-105176/1.
Contract grant sponsor: Marie Heim-V o¨ gtlin Scholarship; Contract grant
number: PMCD2-110198/1.
coworkers. This shortcoming can, however, be overcome
by using conductivity detection, which is universal for all
ionic species.
*
Correspondence to: Peter C. Hauser, Department of Chemistry, Univer-
Conductivity detection has been used for electromigra-
sity of Basel, Spitalstrasse 51, 4056, Switzerland.
tion separation techniques for a long time, but has played E-mail: peter.hauser@unibas.ch
Received for publication 11 February 2009; Accepted 29 April 2009
DOI: 10.1002/chir.20746
Published online 18 June 2009 in Wiley InterScience
a marginal role until the introduction of the contactless
3
,4
approach relying on a pair of external tubular electrodes.
4
The method has been termed C D for ‘‘capacitively (www.interscience.wiley.com).
VV C 2009 Wiley-Liss, Inc.

3
32
SCHUCHERT-SHI AND HAUSER
glucose oxidase, alcohol dehydrogenase, and esterase,
1
8
respectively. Recently, urea was also determined by CE-
4
C D in clinical samples via enzymatic conversion to ammo-
1
9
nium ion with urease.
4
The application of CE-C D to the monitoring of the
lipase-catalyzed hydrolysis of amino acid esters is
reported herein. Many workers have investigated the use
of lipases for enantioselective synthesis. In particular
2
0–22
Reetz et al.
have studied the optimization of the
enzyme structures in an approach named ‘‘directed evolu-
tion.’’ This reaction has also been investigated as a useful
path to the resolution of racemates of the amino acids,
2
3
and work in this area was reviewed by Miyazawa. More
2
4
recently, Malhotra and Zhao have researched the enan-
tioseparation of the esters of a-amino acids by lipase in an
ionic liquid.
Fig. 1. Electropherograms of the concurrent separation of the enan-
tiomers of (1) DL-SME and DL-serine, and (2) DL-TME and DL-threonine.
Buffer: 2 M acetic acid/5 mM 18C6H
4
. Capillary: 50 lm i.d., total length
5
0 cm, and effective length 45 cm. Injection: hydrodynamic, 15 cm height
elevation for 10 s. Separation voltage: 115 kV.
MATERIALS AND METHODS
Chemicals and Procedures
MacLab/4e analog-to-digital converter system (AD Instru-
ments, Castle Hill, Australia).
All chemicals were of reagent grade and deionized water
(
Millipore, Bedford, MA) was used throughout. All chemi-
cals were purchased either from Sigma, Fluka, Aldrich
Buchs, Switzerland), or Acros Organics (Geel, Belgium).
RESULTS AND DISCUSSION
Separation and Detection of the Enantiomers of Serine
and Threonine
(
The details of the suppliers are as follows: DL-serine methyl
ester hydrochloride (Sigma), L-serine methyl ester (SME)
hydrochloride (Aldrich), DL-threonine methyl ester (TME)
hydrochloride (Sigma), L-threonine methyl ester hydro-
chloride (Fluka), DL-serine (Fluka), L-serine (Sigma), DL-
threonine (Fluka), and L-threonine (Sigma). Acetic acid
was purchased from Fluka. HPLC-grade acetonitrile was
obtained from Acros. Sodium bicarbonate was purchased
from Sigma. (1)-(18-(Crown-6)-2,3,11,12-tetracarboxylic
acid was bought from Fluka. The enzymes lipase from por-
cine pancreas (Type II) and lipase from wheat germ
To monitor the hydrolysis, it is necessary to be able to
separate and detect both enantiomers of the amino acids.
DL-serine methyl ester and DL-threonine methyl ester were
chosen as model compounds. These compounds do not
contain benzyl groups and it would thus not be possible to
detect them optically in CE. The choices were made partly
also owing to the commercial availability of the esters. An
electrolyte solution consisting of 2 M acetic acid to render
the analytes in the protonated positively charged form and
5
6
mM of the chiral crown ether 18C6H [(1)-(18-(crown-
4
(
WGL) (Type I) were also purchased from Sigma. All stock
)-2,3,11,12-tetracarboxylic acid] was used as selector.
solutions were prepared in deionized water. Fused-silica
capillaries of 50 lm ID and 375 lm OD were purchased
from Polymicro Technologies (Phoenix, AZ) and were pre-
conditioned with 1 mM sodium hydroxide solution and
then flushed with water followed by 1 mM hydrochloride
solution and again by water. Before use, they were flushed
with the running buffer. All capillaries had a total length of
This was a variation of a buffer found suitable by Gong
17
et al. for the separation of the enantiomers of amino acids.
As illustrated by electropherogram 1 of Figure 1, which
is the result of the injection of a mixture of 5 mM DL-SME
and 5 mM DL-serine, the enantiomers of serine can be sep-
arated from the substrate, and the two enantiomers are
well resolved. Furthermore, it is possible to also separate
the enantiomers of the methyl esters using the electrolyte
solution containing the chiral reagent. For the enantiom-
5
0 cm and 45 cm effective length. Sample injection was
carried out manually directly from the reaction vessels in a
hydrodynamic manner at 10 cm elevation for 10 s. The
separation voltage was 115 kV unless stated otherwise.
ers of serine a high resolution, R , of 3.7 was obtained (the
s
separation factor, a, is 1.2). The enantiomeric separation
of DL-SME is also good with a resolution of 3.8 (a 5 1.3).
The result of the separation of a mixture of DL-TME and
Instrumentation
Separations were carried out on an electrophoretic DL-threonine is shown in electropherogram 2 of Figure 1.
instrument made in-house which is based on a high-volt- The separation is generally not as good as that of the ser-
age power supply from Spellman (model CZE 2000, Pul- ine species, but baseline resolution was still obtained for
borough, UK). It comprises a Perspex box which is fitted all compounds. The R values were calculated as 2.0 and
s
with a microswitch to interrupt the power supply for safety 1.3 for the threonine and TME enantiomers respectively
reasons when opening. The contactless conductivity detec- (a 5 1.1, 1.2). A calibration curve for L-serine was
tor was also constructed in-house, and details can be found acquired for the concentration range from 0.5 to 25 mM.
2
5,26
27
elsewhere.
The cell design detailed in Zhang et al.
The following regression equation was obtained: peak
was used. Data acquisition was carried out with a Macin- area (mV s) 5 20.0616 1 0.9451 3 concentration (correla-
tosh personal computer (Apple, Cupertino, CA) using a tion coefficient, r 5 0.9999). A calibration curve for L-threo-
Chirality DOI 10.1002/chir

ENZYMATIC HYDROLYSIS OF THE AMINO ACID ESTERS
333
Fig. 2. Plot of the ee (n) of DL-serine and the yield (l) of L-serine
resulting from the hydrolysis of DL-SME to DL-serine by PPL versus reac-
tion time. Conditions for the reaction are given in the text. Separation as
for Figure 1.
Fig. 4. Plot of the yield of L-serine produced from the hydrolysis of DL-
SME by WGL (n) and PPL (l) versus reaction time. Conditions for the
reaction are given in the text. Separation as for Figure 1.
nine in the range from 0.5 to 10 mM resulted in the follow-
ing regression equation: peak area (mV s) 5 0.012 1 values are given in Figure 2. Clearly, at the beginning of
0
.8054 3 concentration (r 5 0.9993).
the hydrolysis, a high selectivity for L-serine was obtained,
which then decreased with time as the yield increased.
Hydrolysis of the Methyl Esters of DL-Serine with
Porcine Lipase
Hydrolysis of the Methyl Esters of DL-Threonine with
Porcine Lipase
The enzyme catalyzed hydrolysis requires a pH-value
Five milligrams of a racemic mixture of DL-TME hydro-
around 7.4. The reaction could thus not be carried out in chloride and 2.5 mg of the PPL were mixed in the same
the electrolyte solution used for separation, which has to fashion as described earlier for DL-SME and the mixture
have a low pH-value to render the analytes in the proto- was incubated again at 378C. The reaction mixture was an-
nated cationic form. Into a 1.5 mL microvial, total volume 1 alyzed immediately after mixing, and then after 15 min, 2
mL of a NaHCO aqueous buffer solution (0.2 M, pH 7.8) h, 5 h, and 21 h. The results are shown in Figure 3. The
3
was placed, 10 mg of a racemic mixture of DL-SMT hydro- pattern is similar to that obtained with serine, with the
chloride was added as solid, and the enzyme porcine pan- most pronounced enantiomeric excess obtained early in
creas lipase (PPL) (2.5 mg) was added dissolved in 0.5 mL the reaction. However, the selectivity of this reaction was
of 15% acetonitrile in water. After mixing, the vial was generally less pronounced than for the hydrolysis of the
placed in a water bath of 378C and the reaction was moni- serine ester.
tored for 2 days. Analysis of the reaction mixture was
performed immediately after mixing, and after 20 min, 2 h,
Comparison Between Porcine Lipase and Wheat
Germ Lipase
1
9 h, and 43 h. The separation was carried out in the acetic
acid electrolyte containing the chiral crown ether as
Next, a comparative study of the selectivity of two differ-
described earlier. The concentrations of D- and L-serine ent lipases was carried out using the hydrolysis of DL-
were then determined from the calibration curve. Plots of SMT. A total of 2.5 mg of porcine lipase and of the same
enantioselectivity expressed as the enantiomeric excess amount of wheat germ lipase were added respectively to
(ee) value and the yield for L-serine (fraction of the L-SMT two batches of 10 mg of the racemic mixture of the sub-
converted to L-serine) against time calculated from these strate. The same protocol as outlined earlier was used. As
Fig. 3. Plot of the ee (n) of DL-threonine and the yield (l) of L-threo-
nine resulting from the hydrolysis of DL-TME to DL-threonine by PPL ver-
sus reaction time. Conditions for the reaction are given in the text. Separa-
tion as for Figure 1.
Fig. 5. Plot of the ee of DL-serine produced from the hydrolysis of DL-
SME by WGL (n) and PPL (l) versus reaction time. Conditions for the
reaction are given in the text. Separation as for Figure 1.
Chirality DOI 10.1002/chir

3
34
SCHUCHERT-SHI AND HAUSER
observe the progress of the conversion, which would not
have been possible by HPLC.
LITERATURE CITED
1
2
. G u¨ bitz G, Schmid MG. Chiral separation by capillary electromigration
techniques. J Chromatogr A 2008;1204:140–156.
. Preinerstorfer B, L ¨a mmerhofer M, Lindner W. Advances in enantiose-
lective separation techniques using electromigration capillary techni-
ques. Electrophoresis 2009;30:100–132.
3
4
. Fracassi da Silva JA, do Lago CL. An oscillometric detector for capil-
lary electrophoresis. Anal Chem 1998;70:4339–4343.
. Zemann AJ, Schnell E, Volgger D, Bonn GK. Contactless conductivity
detection for capillary electrophoresis. Anal Chem 1998;70:563–567.
Fig. 6. Plot of the yields of L-threonine and L-serine resulting from the
competitive hydrolysis between L-TME (n) and L-SME (l) by PPL versus
reaction time. Conditions for the reaction are given in the text. Separation
as for Figure 1.
5. Brito-Neto JGA, Fracassi da Silva JA, Blanes L, do Lago CL. Under-
standing capacitively coupled contactless conductivity detection in
capillary and microchip electrophoresis. I. Fundamentals. Electroanal-
ysis 2005;17:1198–1206.
6
. Brito-Neto JGA, Fracassi da Silva JA, Blanes L, do Lago CL. Under-
standing capacitively coupled contactless conductivity detection in
capillary and microchip electrophoresis. II. Peak shape, stray capaci-
tance, noise, and actual electronics. Electroanalysis 2005;17:1207–
in the previous experiments, the most pronounced enan-
tiomeric selectivity was obtained at early times, the analy-
sis of the reaction mixture was carried out at short inter-
vals of 15 min for the first hour of the reaction. In Figure
1
214.
7
. Kub a´ nˇ P, Hauser PC. Fundamental aspects of contactless conductivity
detection for capillary electrophoresis. I. Frequency behavior and cell
geometry. Electrophoresis 2004;25:3387–3397.
4
, the yield of the reaction is shown in terms of production
of L-serine. It is evident that for the conditions used, the
rate of reaction is slightly higher for the porcine lipase
than for the wheat germ lipase. The results for the enantio-
meric excess given in Figure 5 indicate that while the
wheat germ lipase also prefers the L-serine ester, a higher
selectivity for the L-form is found for the porcine lipase. No
attempt was made to correct the data for differences in ac-
tivity, but it appears that the difference is partially due to
the initially higher rate of reaction for the wheat germ
lipase (see Fig. 4).
8. Kub a´ nˇ P, Hauser PC. Fundamental aspects of contactless conductivity
detection for capillary electrophoresis. II. Signal-to-noise ratio and
stray capacitance. Electrophoresis 2004;25:3398–3405.
9
. Kub a´ nˇ P, Hauser PC. A review of the recent achievements in capaci-
tively coupled contactless conductivity detection. Anal Chim Acta
2
008;607:15–29.
1
0. Kub a´ nˇ P, Hauser PC. Ten years of axial capacitively coupled contact-
less conductivity detection for CZE—a review. Electrophoresis
2009;30:176–188.
ˇ
ˇ
11. Sol ´ı nov a´ V, Ka sˇ icka V. J Sep Sci 2006;29:1743–1762.
1
2. Coufal P, Zuska J, van de Goor T, Smith V, Ga sˇ B. Separation of
twenty underivatized essential amino acids by capillary zone electro-
phoresis with contactless conductivity detection. Electrophoresis
2003;24:671–677.
The Selectivity Between the Methyl Esters of L-Serine
and L-Threonine
1
1
3. Tanyanyiwa J, Schweizer K, Hauser PC. High-voltage contactless con-
ductivity detection of underivatized amino acids in capillary electro-
phoresis. Electrophoresis 2003;24:2119–2124.
In the final experiment, L-SME and L-TME were selected
to react together to investigate the preference of the por-
cine lipase for the two substrates. Five milligrams of the L-
SME and 5 mg of the L-TME were mixed with 2.5 mg of
the PPL, using the protocol used thus far. The progress of
4. Samcov a´ E, T u˚ ma P. Determination of proteinogenic amino acids in
human plasma by capillary electrophoresis with contactless conductiv-
ity detection. Electroanalysis 2006;18:152–157.
hydrolysis was monitored over the period of 24 h and the 15. T u˚ ma P, Samcov a´ E, And eˇ lov a´ K. Determination of free amino acids
and related compounds in amniotic fluid by capillary electrophoresis
with contactless conductivity detection. J Chromatogr B 2006;839:12–
results in terms of the yield for the two amino acids are
given in Figure 6. Clearly, in the competitive situation the
1
8.
rate of reaction is higher for the threonine ester, the com-
pound for which the discrimination between the two enan-
tiomers is less pronounced. As the structure of the two
amino acids is identical except for the methyl group in the
b-position, the presence of the latter must be responsible
for the differences observed.
1
6. Zuskov a´ I, Novotn a´ A, V cˇ el a´ kov a´ K, Ga sˇ B. Determination of limiting
mobilities and dissociation constants of 21 amino acids by capillary
zone electrophoresis at very low pH. J Chromatogr B 2006;841:129–
1
34.
1
1
7. Gong XY, Kub a´ nˇ P, Tanyanyiwa J, Hauser PC. Separation of enantiom-
ers in capillary electrophoresis with contactless conductivity detection.
J Chromatogr A 2005;1082:230–234.
8. Schuchert-Shi A, Kub a´ nˇ P, Hauser PC. Monitoring of enzymatic reac-
tions using conventional and on-chip capillary electrophoresis with
contactless conductivity detection. Electrophoresis 2007;26:4690–
CONCLUSIONS
4
696.
4
It has been demonstrated that the CE-C D method is 19. Schuchert-Shi A, Hauser PC. Monitoring the enzymatic conversion of
urea to ammonium by conventional or microchip capillary electropho-
resis with contactless conductivity detection. Anal Biochem
well suited for the monitoring of an enantioselective reac-
tion involving compounds which are not readily detectable
2
008;376:262–267.
by UV-absorption measurement. The fact that only low vol-
umes are consumed for each analysis enabled the
repeated sampling from a very small reaction volume to
Chirality DOI 10.1002/chir
2
0. Reetz MT, Zonta A, Schimossek K, Liebeton K, Jaeger K-E. Creation
of enantioselective biocatalysts for organic chemistry by in vitro evolu-
tion. Angew Chem Int Ed Engl 1997;36:2830–2832.

ENZYMATIC HYDROLYSIS OF THE AMINO ACID ESTERS
335
2
2
2
2
1. Reetz MT, Jaeger K-E. Enantioselective enzymes for organic synthesis
created by directed evolution. Chem Eur J 2000;6:407–412.
25. Tanyanyiwa J, Galliker B, Schwarz MA, Hauser PC. Improved capaci-
tively coupled conductivity detector for capillary electrophoresis. Ana-
lyst 2002;127:214–218.
2. Reetz MT. Application of directed evolution in the development of
enantioselective enzymes. Pure Appl Chem 2000;72:1615–1622.
26. Tanyanyiwa J, Hauser PC. High voltage contactless conductivity
detection of metal ions in capillary electrophoresis. Electrophoresis
2002;23:3781–3786.
3. Miyazawa T. Enzymatic resolution of amino acids via ester hydrolysis.
Amino Acids 1999;16:191–213.
4. Malhotra SV, Zhao H. Enantioseparation of the esters of a-N-ace-
tyl amino acids by lipase in ionic liquid. Chirality 2005;17:S240–
S242.
27. Zhang L, Khaloo SS, Kub a´ nˇ P, Hauser PC. Analysis of electroplating
baths by capillary electrophoresis with high voltage contactless con-
ductivity detection. Meas Sci Technol 2006;17:3317–3322.
Chirality DOI 10.1002/chir