Enhancing protease enantioselectivity by ionic liquids based on chiral- or ω-amino acids

Article abstract of DOI:10.1016/j.tetasy.2006.05.022

Ionic liquids (ILs) carrying anions of chiral- or ω-amino acids were prepared. The enzymatic hydrolysis of phenylalanine methyl ester was studied in aqueous solutions of these ILs. These ILs were found capable of stabilizing the protease activity and enantioselectivity at low concentrations. Interestingly, higher ees and yields of l-phenylalanine were generally observed in ILs based on d-amino acids rather than in those derived from l-isomers. The reason could be that d-amino acids are more kosmotropic than l-isomers. Meanwhile, the IL-D₂O solution was able to further enhance the enzymatic resolution, when comparing with that in an IL-H₂O system.

Full text of DOI:10.1016/j.tetasy.2006.05.022

Tetrahedron: Asymmetry 17 (2006) 1549–1553
Enhancing protease enantioselectivity by ionic liquids
based on chiral- or x-amino acids
Hua Zhao,* Lee Jackson, Zhiyan Song and Olarongbe Olubajo
Chemistry Program, Savannah State University, Savannah, GA 31404, USA
Received 21 April 2006; accepted 29 May 2006
Abstract—Ionic liquids (ILs) carrying anions of chiral- or x-amino acids were prepared. The enzymatic hydrolysis of phenylalanine
methyl ester was studied in aqueous solutions of these ILs. These ILs were found capable of stabilizing the protease activity and enantio-
selectivity at low concentrations. Interestingly, higher ees and yields of L-phenylalanine were generally observed in ILs based on D-amino
acids rather than in those derived from L-isomers. The reason could be that D-amino acids are more kosmotropic than L-isomers. Mean-
while, the IL–D2O solution was able to further enhance the enzymatic resolution, when comparing with that in an IL–H2O system.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
There are several pieces of experimental evidence to
strengthen this statement: (1) the effect of two amino acids
(glycine and b-alanine) on the esterase activity of bovine
carbonic anhydrase was observed close to that of kosmo-
tropic acetate anion;19 (2) the effect of amino acid salts
on the pig heart mitochondrial dehydrogenase (phm-
MDH) against temperature induced changes is in the order
of stabilization of NaGlutamate, NaAspartate > NaGlyci-
nate > lysineÆHCl > arginineÆHCl;20 (3) glycine, alanine
and proline showed nonperturbing or favorable effects on
the enzyme-substrate and enzyme-cofactor complex forma-
tion, catalytic velocity and protein structural stability;14
and (4) Nc-acetyldiaminobutyrate (NADA) displayed a
greater ability in protecting rabbit muscle lactate dehydro-
genase against thermal inactivation than ectoine or potas-
sium diaminobutyrate.15 Furthermore, Wiggins suggested
that D-amino acids are kosmotropes while L-isomers are
chaotropes.21 Meanwhile, as reviewed by Baudequin
et al.,22 various chiral ILs have showed very promising
applications in asymmetric synthesis. Therefore, it would
be interesting to examine the enzyme enantioselectivity in
chiral ILs based on D- or L-amino acids (AA), in the form
of [EMIM+][D-AAÀ] or [EMIM+][L-AAÀ] (EMIM+ is 1-
ethyl-3-methylimidazolium). This is the first objective of
this study. Fortunately, the preparation of these ILs based
on racemic amino acids ([EMIM+][DL-AAÀ]) has already
been reported by Fukumoto et al.23
Ions have a profound impact on the protein stabilization
and enzyme activity.1–5 Ion interactions with protein mole-
cules can be specific (following the ion kosmotropicity, so
called Hofmeister series6,7), or nonspecific (simply depend-
ing on the ionic strength).1 In particular, the ion-specific
interaction has been an attractive focus for many scientists
who routinely work with proteins and enzymes. Such an
interaction was recently adopted by our group to help ex-
plain the effect of ionic liquids (ILs) as new ionic solvents
on the enzyme stability8,9 and enantioselectivity.10 Our
studies suggested that in aqueous solutions, kosmotropic
anions and chaotropic cations of ILs stabilize the enzyme
activity and enantioselectivity since hydrophilic ILs dissoci-
ate into individual cations and anions in water. It is also
important to note that the enzymatic catalysis in hydro-
phobic ILs containing low water contents seems to follow
different mechanisms.5,11–13
Encouraged by these findings, our group has been search-
ing for new ILs containing kosmotropic anions and chao-
tropic cations. It has come to our attention that most
amino acids (a-, b-, or c-) are actually so called compensa-
tory (or compatible) solutes of proteins,14–17 even though
not all amino acids and short peptides stabilize the globular
proteins (e.g., arginine is known as a protein denaturant).18
It is very important to emphasize that some x-amino
acids are also known as compensatory solutes with a
*
Corresponding author. Tel.: +1 912 353 5290; fax: +1 912 691 6839;
e-mail: zhaoh@savstate.edu
0957-4166/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2006.05.022

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H. Zhao et al. / Tetrahedron: Asymmetry 17 (2006) 1549–1553
structure-enforcing effect.24–26 It was observed that c-ami-
no acids are more structure-making than their a-isomers
based on the more negative differential entropies of dilu-
tion in aqueous solutions of c-amino acids than those of
a- and b-ones.27 The x-amino acids, such as 4-aminobuta-
noic acid (4-ABA), 5-aminopentanoic acid (5-APA), 6-
aminohexanoic acid (6-AHA), have high viscosity B-values
and negative values of dB/dT, suggesting that these amino
acids are strong kosmotropes.28 Therefore, several ionic
liquids containing x-amino acids as anions ([EMIM+][x-
AAÀ]) were also prepared and used as media for the enzy-
matic resolution of phenylalanine. The last objective of this
study is to examine the protease behavior in IL–heavy
water (D2O) solutions, because D2O has showed a higher
ability in stabilizing proteins against denaturation than or-
dinary water (H2O).29,30 Cioni and Strambini31 observed a
higher rigidity of most protein structures in D2O than in
H2O, especially at higher temperatures. Based on the in-
verse relationship between the structural flexibility and sta-
bility to thermal denaturation,32 they suggested that the
folded state is more stable in D2O than in H2O.
100
80
60
40
20
0
ee
yield
0
0.5
1
1.5
2
Concentration (M) of [EMIM][5-APA]
Figure 1. Effect of IL concentration on the protease enantioselectivity
(40 min reaction time and 30 ꢁC).
2. Results and discussion
ee
yield
[EMIM][OTs]
[EMIM][6-AHA]
[EMIM][5-APA]
[EMIM][4-ABA]
[EMIM][D-Pro]
[EMIM][L-Pro]
[EMIM][D-Lys]
[EMIM][L-Lys]
[EMIM][D-Glu]
[EMIM][L-Glu]
[EMIM][D-Ala]
[EMIM][L-Ala]
[EMIM][Gly]
2.1. Effect of IL concentration
89.0
77.6
The enzymatic hydrolysis of phenylalanine methyl ester
was conducted as a model reaction for examining protease
activity and enantioselectivity. At first, we investigated the
concentration effect of a promising IL ([EMIM][5-APA])ꢀ
based on an x-amino acid (5-aminopentanoic acid). The
[EMIM]+ cation was chosen because it is a chaotrope,33
and stabilizes the enzyme.8–10 As seen in Figure 1, a high
enantiomeric excess (ee, 92.2%) and yield (97.2%) of L-
phenylalanine were obtained in a low IL concentration
(0.5 M), suggesting high enzyme activity in this medium.
These values are very comparable with those in pure water
(94.2% ee and 94.6% yield). However, with the increase of
IL concentration, both ee and yield decreased, suggesting a
lower enzyme activity and enantioselectivity. This observa-
tion is consistent with previous reports from our group8,10
as well as others34–37 indicating that high concentrations of
hydrophilic ILs destabilize the enzyme.
92.2
90.4
92.9
85.5
86.4
87.2
91.4
90.6
92.1
90.9
88.6
94.2
H2O
2.2. Effect of amino acid anions on the enzymatic
resolution
0.0
20.0 40.0 60.0 80.0 100.0
ee or yield (%)
We carried out the same enzymatic resolution in 0.5 M of
other ILs containing anions of various chiral- or x-amino
acids (Fig. 2). The chiral amino acids proline, lysine, ala-
nine, and glutamic acid were chosen because the former
two (proline and lysine) are known as important compen-
satory solutes in nature with structure-making abil-
ity,14,26,38–40 alanine showed favorable effects on the
enzyme–substrate and enzyme–co-factor complex forma-
tion,14 and sodium glutamate salt stabilized pig heart mito-
chondrial dehydrogenase (phm-MDH) against temperature
induced changes.20 Glycine was considered for the purpose
of comparing the kosmotropicity of different amino acids.
Figure 2. Comparison of the enzymatic resolution of phenylalanine
methyl ester in 0.5 M ILs and water (40 min reaction time and 30 ꢁC).
In general, a moderate to high enzyme activity and enantio-
selectivity were observed in most amino acid based ILs,
suggesting that they are compatible solutes. Meanwhile,
slightly higher ees were seen in D-amino acid based ILs
than in those based on L-isomers (except very comparable
ees in D- and L-lysine based ILsꢁ); higher yields were also
ꢁ The optimum resolution was actually achieved at 20 min of reaction time
in both 0.5 M [EMIM][L-Lys] (94.1% ee and 74.6% yield) and 0.5 M
[EMIM][D-Lys] (90.9% ee and 98.9% yield).
ꢀ The abbreviations of amino acids and ionic liquids are in Section 4.

H. Zhao et al. / Tetrahedron: Asymmetry 17 (2006) 1549–1553
1551
observed in the same trend with the exception of D/L-gluta-
mate. A possible explanation is the following: since
kosmotropic anions strongly stabilize the enzyme,5 our
observation is consistent with Wiggins’ hypothesis that D-
amino acids are usually more kosmotropic than their L-iso-
mers.21 On the other hand, relatively low ees were found in
solutions of [EMIM][OTs] and [EMIM][Gly]. The reason
for the former IL is that OTsÀ was suspected to be a chao-
trope based on our previous enzyme-stability studies.8,9
The reason for the later IL could be explained by the kos-
motropicity of amino acids. As discussed in detail by our
recent review on this subject,28 the viscosity B-coefficients
of amino acids at 25 ꢁC are 0.143 (glycine), 0.252 (alanine),
0.268 (proline), 0.29 (glutamic acid), 0.312 (4-ABA), 0.383
(5-APA), and 0.489 (6-AHA). Since a higher B-value sug-
gests a higher kosmotropicity, glycine is least kosmotropic
than other amino acids.
(being similar to Fig. 1). These data confirmed that
[D-Glu]À is more kosmotropic than [L-Glu]À.
2.3. Effect of heavy water (D2O) on the enzyme
enantioselectivity
We next carried out the enzymatic reaction in D2O solu-
tions instead of ordinary H2O solutions. As illustrated in
Figure 4, ees obtained in pure H2O and pure D2O are
not distinguishable, while a lower yield was found in
D2O. This observation suggests that the enzyme enantiose-
lectivity is about the same in H2O and D2O, while a lower
enzyme activity is observed in D2O. The lower reaction rate
in D2O might be due to higher enzyme rigidity in D2O than
in H2O,31 and/or due to the enzyme being partially deuter-
ated from the H/D exchange causing a slower reaction rate.
However, when an IL (0.5 M [EMIM][5-APA]) was added,
a higher ee (97.5%) was observed in D2O solution than in
H2O solution (91.0%) although the yield is still lower in
D2O solution.
On the other hand, the ions of amino acids are more
kosmotropic than zwitterionic amino acids (AA) in a
decreasing order of anion > cation > AA;28 for example,
B-coefficienÀts of glycine ions at 25 ꢁC are in the order of
0.242 (Gly ) > 0.160 (Gly+) > 0.143 (Gly).28 Therefore,
amino acid anions are stronger enzyme stabilizers than
zwitterionic amino acids. In the case of x-amino acids,
higher ees were observed in [5-APA]À than in [4-ABA]À
due to higher kosmotropicity of the former anion. How-
ever, a lower ee was found in [6-AHA]À which should be
the most kosmotropic anion in the series. This contradic-
tory result might be due to the fact that an optimal stabil-
ization of enzyme can usually be achieved through a
balance between kosmotropicÀanions and chaotropic cat-
ions.2,41,42 The anion [6-AHA] could be too kosmotropic,
breaking such a balance and causing a lower enzyme
activity.
97.5
94.8
94.4
100.0
80.0
60.0
40.0
20.0
0.0
91.0
79.1
74.4
58.6
54.5
Figure 3 further demonstrates that with the increasing
concentration of [EMIM][L-Glu] and [EMIM][D-Glu], the
difference of ees in 1.0 M ILs (25.0% Dee) was more
pronounced than that in 0.5 M ILs (0.8% Dee), although
lower ee and yield were observed at a higher concentration
Reaction medium
Figure 4. Effect of D2O on the enzymatic resolution (20 min reaction time,
[EMIM][5-APA] in 0.5 M, and 30 ꢁC).
100.0
We suspected that the cation and/or anion hydration might
be responsible for such a difference. As briefly discussed by
Jenkins and Marcus,43 negative B-coefficients of ions are
more negative in D2O than in H2O, while there is little
difference in the positive B-values in D2O and H2O. The
B-coefficients of both [EMIM]+ and [5-APA]À in H2O
are expected to be quite positive,28,33 therefore, the hydra-
tion of these two ions in D2O and H2O should not be
considerably different based upon their B-coefficients.
However, the comparison made by Jenkins and Marcus
was limited to several ions (including tetraalkylammonium
cations). The hydration of large hydrophobic cations (such
as [EMIM]+) and anions (such as [5-APA]À) in D2O is not
well understood.
ee
80.0
60.0
40.0
20.0
0.0
yield
L-Glu (0.5 M) D-Glu (0.5 M) L-Glu (1.0 M) D-Glu (1.0 M)
Concentration of [EMIM][D/L-Glu]
Meanwhile, the hydrogen at the C2 position of the
imidazolium cation is rather acidic (pKa = 21–23) and is
subject to deprotonation under basic conditions.44–46 Since
Figure 3. Effect of concentration of chiral ILs on the enzymatic resolution
(40 min reaction time and 30 ꢁC).

1552
H. Zhao et al. / Tetrahedron: Asymmetry 17 (2006) 1549–1553
our enzymatic reactions were conducted in a NaHCO3 buf-
fer, the H/D exchange of the imidazolium ring is likely to
occur through carbene intermediates, especially on the C2
position.46 The H/D exchange might also occur for the an-
ion (e.g., H/D exchange of hydrogen atoms on the amine
group).47,48 These partially deuterated cations or anions
have different properties (such as hydration behavior) in
the solution, which could be responsible for the difference
of enantioselectivity in D2O and H2O. However, the exact
mechanism is not clear.
into the [EMIM][OH] (1 equiv) solution. The mixture was
stirred for 12 h (with gentle heat or microwave heating
for some amino acids), followed by the removal of water
under vacuum through a rotary evaporator at 60 ꢁC. A
mixed solution of acetonitrile and methanol (9:1, v/v)
was added into the crude product. The mixture was then
stirred vigorously to dissolve the IL. The excess amino acid
(precipitate) was filtered off, and the solvents were removed
from the filtrate through a rotary evaporator. The IL prod-
uct was dried at 90 ꢁC for 24 h. All product yields are great-
er than 80%. The IL purity was ensured by measurements
1
of IR, H NMR and HPLC. The thermal properties and
ionic conductivity of most of these ILs have been reported
3. Conclusion
in a recent paper.23
Generally, moderate to high enzyme enantioselectivities
were observed in ILs based on chiral- or x-amino acids.
ILs based on D-amino acids are more favorable than those
based on L-amino acids in terms of improving ee and yield.
It seems that an enhanced enzymatic resolution could be
achieved in the IL–D2O solution rather than in the
IL–H2O solution.
4.3. Enzymatic resolution
DL-Phenylalanine methyl ester (10 mg) was dissolved in
1.0 mL solvent consisting of an IL and 0.2 M NaHCO3
buffer. Immediately, 0.5 mg of enzyme was added to the
reaction mixture at time zero. The reaction was shaken
and maintained at 30 1 ꢁC. Samples were withdrawn
from the reaction mixture periodically and analyzed by
the HPLC. All experiments were run in duplicates. The
averaged values are reported.
4. Experimental
4.1. Materials
4.4. HPLC analysis
1-Ethyl-3-methylimidazolium bromide ([EMIM]Br), Bacil-
lus licheniformis protease (subtilisin Carlsberg), DL-phenyl-
alanine methyl ester hydrochloride, Amberliteꢂ IRA-400
Cl resin, glycine (Gly), D- and L-alanine (Ala), D- and L-ly-
sine (Lys), D- and L-glutamic acid (Glu), D- and L-proline
(Pro), 4-aminobutanoic acid (4-ABA), 5-aminopentanoic
acid (5-APA), 6-aminohexanoic acid (6-AHA), and other
reagents were purchased from Sigma–Aldrich.
The samples were analyzed by a LC-10AT Schimadzu
HPLC equipped with a SPD-10A UV–vis dual wavelength
detector, and
a
Crownpak CR(+) chiral column
(150 mm · 4.0 mm, particle size 5 lm). The mobile phase
was 1.0 mL/min HPLC-grade water containing 0.1 M
HClO4. The detection wavelength is 210 nm (254 nm for
reactions in pure water). The eluting sequence of four
isomers is in the increasing retention time of D-acid < L-
acid < D-ester < L-ester.
4.2. IL preparations
[EMIM][OH] was prepared according to a literature meth-
od23 by using the anion exchange resin (Amberliteꢂ IRA-
400 Cl). A brief description of the procedures is the follow-
ing: about 100 mL of the resin packed in a glass column
was thoroughly washed with distilled water until no yellow
color was observed in the eluting water and no precipitate
could be detected by 0.1 M AgNO3 solution. Next, the ClÀ
ions on the resin were exchanged by OHÀ ions through
slow washing of the column with about 100 mL 3.0 M
NaOH solution until no white precipitate (AgCl) could
be detected by 0.1 M AgNO3 solution; 20 g [EMIM]Br dis-
solved in 100 mL water was made to slowly drip through
the column, and the eluting solution was monitored by
0.1 M AgNO3 to ensure the absence of BrÀ. If necessary,
the resulting solution was run through the column (OHÀ
form) for the second time. Water was then removed from
the resulting [EMIM][OH] solution through a rotary evap-
orator under vacuum at 60 ꢁC.
4.5. Calculations of ee and L-yield
The ee of L-phenylalanine was calculated from the HPLC
integration area as (L area À D area)/(L area + D area) ·
100%. The yield of L-acid (maximum is 100% for a com-
plete conversion of L-ester) was calculated from comparing
the current area of L-acid with that of complete conver-
sion of the L-ester. The L-acid area for the complete conver-
sion of the L-ester was measured by HPLC individually for
each sample, typically at 2–3 h of reaction time.
Acknowledgements
This project was supported by a CASTME Research
Program under the Title III Grant from the Department
of Education.
The method for preparing the chiral- or x-amino acid
based ILs is based on a literature method (for preparing
ILs based on racemic amino acids):23 an aqueous solution
of D-, L-, or x-amino acid (1.2 equiv) was added dropwise
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