Remarkable activation of an enzyme by (R)-pyrrolidine-substituted imidazolium alkyl PEG sulfate

Article abstract of DOI:10.1002/adsc.200800382

The chiral pyrrolidine-substituted imidazolium cetyl-PEG10-sulfate (D-ProMe) derived from D-proline worked as an excellent activating agent of Burkholderia cepacia lipase; it is particularly interesting that the D-isomer of the imidazolium salt worked better than the L-isomer. This suggests that the imidazolium cation group directly interacts with the enzyme protein and causes preferable modification of the reactivity.

Full text of DOI:10.1002/adsc.200800382

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DOI: 10.1002/adsc.200800382
Remarkable Activation of an Enzyme by (R)-Pyrrolidine-
Substituted Imidazolium Alkyl PEG Sulfate
Yoshikazu Abe,a Takuya Hirakawa,a Shino Nakajima,a Nagisa Okano,a
Shuichi Hayase,a Motoi Kawatsura,a Yoshihiko Hirose,b and Toshiyuki Itoha,*
a
Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, Tottori 680-8552, Japan
Fax : (+81)-857-31-5259; e-mail: titoh@chem.tottori-u.ac.jp
Amano Enzyme, Ltd., Kagamihara, Gifu 509-0108, Japan
b
Received: June 17, 2008; Published online: August 13, 2008
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/adsc.200800382.
Abstract: The chiral pyrrolidine-substituted imida-
zolium cetyl-PEG10-sulfate (d-ProMe) derived
from d-proline worked as an excellent activating
agent of Burkholderia cepacia lipase; it is particu-
larly interesting that the d-isomer of the imidazoli-
um salt worked better than the l-isomer. This sug-
gests that the imidazolium cation group directly in-
teracts with the enzyme protein and causes prefera-
ble modification of the reactivity.
lipase PS from Burkholderia cepacia contains ca. 20
wt% of glycine as essential stabilizer during the prep-
aration of the lipase protein by the lyophilization pro-
cess.[8,11] We were fascinated by this glycine-mediated
stabilization of lipase protein and investigated the
role of glycine; it was found to work only as a stabiliz-
er of the enzyme and had no influence on the reactiv-
ity of lipase PS.[8b] Since it was anticipated that a
chiral amino acid may provide for a modification of
the enantioselectivity of an enzymatic reaction, we
next prepared amino acid-coated PS (i.e., with l-Phe
and l-Pro) and investigated their properties for the
activation of lipase PS [Eq. (1)].[12]
Keywords: acceleration; enhanced enantioselectivi-
ty; enzyme catalysis; lipase; transesterification
Lipases are one of the most widely used enzymes ap-
plicable for various substrates,[1] however, the enantio-
selectivity is significantly dependent on both sub-
strates and reaction media.[1] Therefore, the develop-
ment of a strategy to improve the reaction perfor-
mance of lipases is desirable.[1,2] Several methods have
been reported for activation of lipases in a non-aque-
ous medium: lipid coating-mediated activation,[3] en-
As can be seen in Table 1, however, coating of
trapment of lipases in hydrophobic sol-gel materials,[4] lipase PS protein by l-Phe (entry 2) or l-Pro (entry 3)
molecular bioimprinting,[5] salt-mediated activa- neither accelerated the reaction nor enhanced the
tion,[6,7,8] and use of some surfactants such as polyoxy- enantioselectivity. On the contrary, a slight reduction
ethylene alkyl ether.[9] Recently we reported an effi- of enantioselectivity was recorded when the reaction
cient activation protocol for lipases by coating them was carried out using l-Pro-PS and the E value[13]
with [1-butyl-2,3-dimethylimidazolium] [cetyl-PEG10- dropped to 91 (entry 3) from >200 which was ob-
sulfate] (IL1).[8] Although the activation effect was tained for the GF-PS-catalyzed reaction (entry 1).
dependent on the substrate, a more than 1000-fold ac- Coating treatment by glycine rather reduced the
celeration was accomplished for some substrates with enantioselctivity of the enzymatic reaction (entry 4)
excellent enantioselectivity when Burkholderia cepa- and a similar reaction rate to those of l-Phe-coating
cia lipase (lipase PS) or Candida rugosa lipase was enzyme or l-Pro-coating enzyme was obtained. IL1
coated with IL1.[8,10]
coating, on the other hand, caused a great accelera-
Amino acids have been used as a stabilizer of an tion of the enzymatic reaction for GF-PS (entry 5). To
enzyme during the purification process; commercial our delight, we discovered a very interesting coopera-
1954
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2008, 350, 1954 – 1958

Remarkable Activation of an Enzyme
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Table 1. Transesterification of 1-phenylethanol (1) by glycine-free lipase PS (GF-PS) or amino acid coated lipase PS.
Entry
Lipase
Time [h]
Yield of
Yield of
% Conversion[b]
Relative rate
(% conv/h)
E[b]
(R)-2 (eeP %)[a]
(S)-1 (eeS %)[a]
ACHTREUNG
1
2
3
4
5
6
7
GF-PS
48
48
60
26
20
21
4
5 (>99)
10 (>99)
14 (97)
44 (33)
53 (23)
58 (23)
72 (15)
35 (64)
38 (63)
35 (62)
25
19
19
15
39
39
38
0.52
0.40
0.32
0.58
2.0
>200
>200
91
l-Phe-PS
l-Pro-PS
Gly-PS
IL1-PS
l-Phe-IL1-PS
9 (87)
17
22 (>99)
25 (>99)
22 (>99)
>200
>200
>200
1.9
9.5
l-Pro-IL1-PS
[a]
Deternined by HPLC, Chiralcel OD-H, i-PrOH: hexane=9:1.
[b]
Calculated from eeP and eeS: E=ln[(1Àc)(1+eeP)]/ln
A
U
E
following formula: c=eeP/
AHCTREUNG
tive activation of the enzyme for IL1 with an amino types of chiral pyrrolidine-substituted imidazolium
acid. A great acceleration was obtained for l-Pro- cetyl-PEG10-sulfates as listed in Figure 1.[15] Although
IL1-PS (entry 7), which was prepared by treatment of glycine-free lipase PS was employed for the initial ex-
glycine-free PS with 100 mol equiv. of l-Pro and IL1, periments described in Table 1, commercial lipase PS
and the reaction conversion reached 38% after only a was this time used as a source of lipase protein, which
4-h reaction time (9.5% conversion/h); on the contra- contained 20 wt% of glycine as stabilizing agent, be-
ry, it reached only 19% conversion in a 60-h reaction cause glycine had no modification effect on the enan-
time for the l-Pro-PS-catalyzed reaction (entry 4: tioselectivity.
0.32% conversion/h). It was thus found that the com-
Figure 2 summarizes the results of transesterifica-
bination of IL1 and amino acid, particularly l-Pro tion of (Æ)-1-phenylethanol (1) using commercial
with IL1, was essential for realizing an effective acti- lipase PS and four types of 2nd generation imidazoli-
vation of the lipase.
um salt-coated lipase PS using two types of acyl
We previously reported that MALDI-TOF mass ex- donor [Eq. (2)].
periments suggested that IL1 binds with the enzyme
protein, therefore it was assumed that the modified
activity of the lipase might be due to flexibility or
conformation change of the lipase protein upon IL1
binding.[8b] Furthermore, it was established that the
imidazolium cation affected the enantioselectivity of
the lipase; 1-butyl-2,3-dimethyimidazolium salt-
coated lipase PS gave a better enantioselectivity than
a 1-butyl-3-methylimidazolium salt-coated enzyme.
Based on these results, we attempted to refine the
design of the activating agent for lipase by modifica-
tion of the imidazolium group. Our idea is that the in-
troduction of an appropriate chiral functional group
on the imidazolium group may create a more efficient
activation of an enzyme (Figure 1). We hypothesized
that a chiral pyrrolidine moiety-substituted imidazoli-
um cetyl-PEG10-sulfate may have the desired ability
to control the stereochemistry of the enzymatic reac-
tion. Recently Cheng and co-workers reported the
synthesis of the imidazolium ionic liquid derived from
l-proline and used it as an efficient asymmetric orga-
nocatalyst.[14] Following their method, we prepared a
pyrrolidine-substituted imidazolium bromide and con-
verted it to cetyl-PEG10-sulfate species by a metal
exchange reaction.[15] Since it was anticipated that the
stereochemistry of the proline moiety to which the
Figure 1. Design of 2nd generation ionic liquid type activat-
imidazolium ring was attached may influence the
enantioselectivity of the enzyme, we prepared four ing agents for enzyme.
Adv. Synth. Catal. 2008, 350, 1954 – 1958
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
1955

Yoshikazu Abe et al.
COMMUNICATIONS
previously reported,[8] the functional group on the 2-
position of the imidazolium ring had a certain impact
on the modified property. Both l- and d-ProMe gave
better results than those of corresponding l- and d-
ProH salts. The results seem to suggest that the cat-
ionic part of the imidazolium salt bound with the
lipase protein and affected its reaction profile; this
may explain why our ionic liquid coating activation of
We conducted the reaction using 1.5 equiv. of vinyl enzyme depends on the substrate. It was thus estab-
acetate or 2,2,2-trifluoroethyl acetate as acylating re- lished that d-ProMe worked an excellent activator of
agent. The results were very satisfactory. The reaction lipase PS. No significant diffference in the reaction
rate of the commercial lipase PS-catalyzed reaction rate between acyl agents was obtained, while better
was faster than that of the GF-PS reaction (column enantioselectivity was recorded when the esterifica-
1), although the enantioselectivity of commercial tion of (Æ)-1 was carried out in the presence of 2,2,2-
lipase PS was inferior to that of GF-PS reaction (see trifluoroethyl acetate as acyl donor (column 7). A re-
Table 1, entry 1). As we previously reported,[8b] coat- markable acceleration was again obtained for the d-
ing of lipase PS protein by IL1 (1st generation activat- ProMe-PS-catalyzed reaction even when 2,2,2-tri-
ing agent) caused a great acceleration and enhanced fluoroethyl acetate was employed as acyl donor
tghe enantioselectivity (column 2). As expected, it (column 8), although a slightly reduced activation was
was established that the chiral pyrrolidine-substituted observed compared to that of vinyl acetate-mediated
imidazolium salt worked as an excellent activator of reaction using the same enzyme (column 6). There-
lipase PS. In particular, the (R)-pyrrolidine-substitut- fore, we assume that the origin of the activation prop-
ed salt (d-ProMe), which was derived from unnatural erty might be unaffected by the acylation agent. We
d-proline, was found to be the best agent: an extraor- further confirmed that it was possible to use d-
dinary acceleration was accomplished with perfect ProMe-PS more than 10 times while maintaining per-
enantioselectivity for the d-ProMe-PS-catalyzed reac- fect reactivity when the reaction was carried out in an
tion and a 58-times faster reaction (vs. lipase PS) was ionic liquid solvent system.[16]
recorded (column 6). To the best of our knowledge,
To gain more detailed information of how the pres-
this is a record for the most rapid transesterification ent d-ProMe caused activation of lipase, the kinetic
of 1-phenylethanol by a lipase PS. l-ProMe-PS also parameters of lipase PS-catalyzed transesterification
showed a significant acceleration (column 5), but it of (R)-3 or (S)-3 were measured [Eq. (3), Eq. (4), and
was obviously inferior to that of d-ProMe-PS. As we Table 2].[17]
[a]
Figure 2. Activation of lipase PS by the coating of the 2nd generation imidazolium salt. The reaction was carried out using
vinyl acetate as acyl donor. [b] 2,2,2-Trifluoroethyl acetate was used as acylating reagent.
1956
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ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2008, 350, 1954 – 1958

Remarkable Activation of an Enzyme
COMMUNICATIONS
ed activation of an enzyme will make it even more
valuable.
Experimental Section
Preparation of (D)-ProMe-Supported Lipase PS (d-
ProMe-PS) byLyophilization
The imidazolium cetyl-PEG10 sulfate (IL1) coating
significantly accelerated the reaction using two enan-
tiomers in a similar magnitude (ca. 10–20-fold). A
particularly great acceleration (17-fold vs. PS) was ob-
tained for the d-ProMe-PS-catalyzed reaction of (R)-
3 (entry 4); but, unexpectedly, a slightly greater accel-
eration was obtained for the (S)-isomer (21-fold vs.
PS). The most interesting result of kinetic parameter
determination of the d-ProMe-PS-catalyzed reaction
was found in the modified Km values between the en-
antiomers: the Km value of (S)-isomer was reduced
compared with that of PS when d-ProMe-PS was
used as catalyst (entry 8), while the value was in-
creased for (R)-3 (entry 4). This clearly suggests that
the cationic part of the ionic liquid might bind with
the lipase protein, thus causing a conformational
change of the enzyme and contributing to the expan-
sion of the difference in Km between enantiomers.
In summary, we succeeded in developing a very ef-
fective 2nd generation activation agent for lipase pro-
tein: a novel chiral imidazolium cetyl-PEG10-sulfate
(d-ProMe) derived from d-proline worked as an ex-
cellent activator of lipase PS and was ca. 20-fold
stronger than the IL1. It is particularly noteworthy
that the (R)-isomer of the imidazolium salt worked
better than the (S)-isomer. This suggests that the imi-
dazolium cation group directly interacts with the
enzyme protein and causes a modification of the reac-
tivity. Since we have discovered that even activation
of an oxide-reductase can be realized by the proline-
substituted imidazolium salt coating,[18] we are expect-
ing that introduction of an appropriate amino acid or
oligopeptide moiety on the imidazolium ring may
makes it possible to design more efficient activating
agents for various types of enzymes. Further investi-
gation of the scope and limitation of this salt-mediat-
Lipase PS (Amano) (1.0 g, since this contained 20 wt% of
glycine and 1.0 wt% of lipase protein, therefore amount of
lipase protein was estimated as 3.1”10À4 mmol) was dis-
solved in 8.0 mL of 0.1M potassium phosphate buffer
(pH 7.2) and the mixture was centrifuged at 3,500 rpm for
5 min (3 times). 2.0 mL of 0.1M potassium phosphate buffer
(pH 7.2) solution of d-ProMe (30 mg; ca. 3.1”10À2 mmol
because the estimated molecular weight of the salt was
984.37) was mixed with the resulting supernatant and dried
by lyophilization to give d-ProMe-PS (219 mg) as a hygro-
scopic white powder. The lipase protein of this enzyme
powder was therefore estimated as 4.2 wt%. l-ProH-PS, l-
ProMe-PS, and d-ProH-PS were prepared by the same pro-
cedure.
d-ProMe-PS-Catalyzed Acylation of (Æ)-1-
Phenylethanol (1) in i-Pr2O
To a solution of (Æ)-1a (50 mg, 0.41 mmol) and vinyl acetate
(52.9 mg, 0.62 mmol) in i-Pr2O (2.0 mL) was added d-
ProMe-PS (6.0 mg, corresponding to 0.25 mg of enzyme)
and the mixture was stirred at 358C. The reaction course
was monitored by capillary GC-analysis and silica gel TLC.
The reaction mixture was filtered through a sintered glass
filter with a celite pad to remove the lipase, and the filtrate
was concentrated under vacuum. Preprative silica gel TLC
afforded (R)-2 (25.0 mg, 0.15 mmol) in 37% yield and (S)-1
(19.5 mg, 0.16 mmol) in 39% yield. The enantioselectivity
was determined by HPLC analysis using a chiral column
(Chiralcel OB, hexane: 2-propanol=9: 1). (R)-2: [a]2D5: +102
(c 1.0, CHCl3), >99% ee; (S)-1: [a]2D5: À54.0 (c 1.0, CHCl3),
97% ee.
Supporting Information
Detailed experimental procedures, and NMR spectra data
for new compounds are avairable in the Supporting Infor-
mation.
Table 2. Results of kinetic experiments of IL-coated PS-catalyzed transesterification of 3-hydroxypentanenitrile (3).
[a]
Entry
Substrate
Enzyme
Vmax
Km [M]
Kcat [minÀ1]
Kcat/Km
1
2
3
4
5
6
7
8
(R)-3
(R)-3
(R)-3
(R)-3
(S)-3
(S)-3
(S)-3
(S)-3
PS
IL1-PS
l-ProMe-PS
d-ProMe-PS
PS
IL1-PS
l-ProMe-PS
d-ProMe-PS
7.2”10À2
4.4”10À1
9.1”10À1
1.2
0.23
0.30
0.69
0.51
0.19
0.24
0.31
0.15
0.29
1.8
3.6
1.3
6.0
5.3
9.7
0.12
1.4
1.4
3.3
5.0
5.8”10À3
8.7”10À2
1.1”10À1
1.2”10À1
0.023
0.35
0.44
0.49
[a]
M, minÀ1, mg (enzyme)À1.
Adv. Synth. Catal. 2008, 350, 1954 – 1958
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
1957

Yoshikazu Abe et al.
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Acknowledgements
Abe, S.-H. Han, S. Wada, S. Hayase, M. Kawatsura, S.
Takai, M. Morimoto, Y. Hirose, Chem. Eur. J. 2006, 12,
9228–9237.
The present work was supported by a Grant-in-Aid for Scien-
tific Research in a Priority Area, “Science of Ionic Liquids”,
from the Ministry of Educatio, Culture, Sports, Science and
Technology of Japan.
[9] For examples, see: a) S. Park, R. J. Kazlauskas, J. Org.
Chem. 2001, 66, 8395–8401; b) T. Maruyama, S. Naga-
sawa, M. Goto, Biotechnol. Lett. 2002, 24, 1341–1345;
c) U. Bora, C. J. Saikia, A. Chetia, A. K. Mishra,
B. S. D. Kumar, R. C. Boruah, Tetrahedron Lett. 2003,
44, 9099–9102.
[10] IL1-PS is commercially available from Tokyo Chemical
Industry Co., Ltd.; Tel.: (+81)-3-5640-8857, Fax: (+
81)-3-5640-8868.
[11] Commercial lipase PS (Amano) was supported by a
Celite with 20 wt% of glycine. Protein content of PS is
stated to be 1.0 wt% by Amano Enzyme Ltd.; the
amino acid sequence of this lipase is known, so the mo-
lecular weight can be given precisely as 32 Kda.
[12] Glycine-free PS was prepared from the Celite-free
lipase PS which was provided by Amano Enzyme and
the details are reported in the Supporting Information.
[13] C. S. Chen, Y. Fujimoto, G. Girdauskas, C. J. Sih, J.
Am. Chem. Soc. 1982, 102, 7294–7298.
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1958
asc.wiley-vch.de
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2008, 350, 1954 – 1958