Enhanced activity of α-chymotrypsin in organic media using designed molecular staples

Article abstract of DOI:10.1016/j.tet.2005.04.063

We report the enhancement of α-chymotrypsin activity in organic solvents using modified peptides bearing two crown ethers. The transesterification of N-acetyl-l-phenylalanine ethyl ester with 1-propanol was used as model reaction. Co-lyophilization of cro

Full text of DOI:10.1016/j.tet.2005.04.063

Tetrahedron 61 (2005) 6824–6828
Enhanced activity of a-chymotrypsin in organic media using
designed molecular staples
*
´ ˆ ´
Melanie Tremblay, Simon Cote and Normand Voyer
´ ´ ´ ´ ´
Centre de recherche sur la fonction, la structure, et l’ingenierie des proteines, Departement de chimie, Faculte des Sciences et de Genie,
´
Universite, Laval, Que., Canada G1K 7P4
Received 24 March 2005; revised 28 April 2005; accepted 28 April 2005
Abstract—We report the enhancement of a-chymotrypsin activity in organic solvents using modified peptides bearing two crown ethers.
The transesterification of N-acetyl-L-phenylalanine ethyl ester with 1-propanol was used as model reaction. Co-lyophilization of crown ether
modified peptides with a-chymotrypsin prior to use resulted in an increase of enzyme catalytic activity in non-aqueous media. The efficiency
of enzyme activation is dependent on the amino acid sequence of peptidic additives and on the positions of the amino acids bearing the crown
ligand.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
therefore highly desirable. Towards that goal, Reinhoudt
et al. have studied the use of crown ethers.13 They observed
an improvement of chymotrypsin activity up to 500 fold in
organic media by co-lyophilization of the biocatalyst with
250 equiv of 18-crown-6. Recent studies by this group, on
the mechanism of crown-ether-induced activation of
enzymes in non-aqueous media revealed the possibility of
a conformational stabilization in organic solvent by crown
ethers.18 On the other hand, Griebenow et al.20,21 have
demonstrated by infrared spectroscopy that no relationship
could be established between secondary structure and
activity in various subtilisin-crown ether preparations. The
crown ether enhancing effect has also been associated with a
molecular imprinting effect. Indeed, it has been proposed
that the crown ether is not able to stabilize the overall active
3D structure of the enzyme, but rather helps preserve the
active site structure.19
Enzymes are continuously gaining importance as ‘green’
bio catalysts in organic processes and useful tools in organic
synthesis due to their exquisite enantioselectivity and
chemoselectivity.1–4 Furthermore, enzymes often simplify
the synthetic protocol by reducing the overall number of
steps required. Thus, enzymes are competitive and
economic alternatives for small and large scale production
of enantiopure pharmaceutical intermediates and other
useful chiral synthons.5
Unfortunately, activity of enzymes decreases in non-
aqueous media, in which starting materials are soluble in.
To overcome such problems, considerable developments
have been made in the past to understand enzyme behavior
and reactivity in non-aqueous media.6,7 Several procedures
involving additives have been devised to improve efficiency
of enzymes in organic media.8–12 Among these procedures,
the use of additives such as crown ether,13 sorbitol,14
sugars,14 polyethylene glycol,15 cyclodextrins,16 and salts,17
represents an attractive method as it is both straightforward
and economical.
Towards more efficient crown systems, Shinkai et al.22 have
developed multi-crown ether compounds, ‘crowned’
arborols. They have tested the efficiency of these arborols
to solubilize myoglobin. Their results showed that smaller
‘crowned’ arborols gave better results for dissolution of
myoglobin in DMF. The authors proposed that the larger
multiple crown systems were less efficient probably due to
their lower ability to cover the myoglobin surface
accurately.
However, all of these additives are non-specific and their
efficiency is unpredictable. Tailor-made devices specifically
designed to enhanced stability, activity and solubility of
many different enzymes in non-aqueous media, are
On the basis of our groundwork on functional peptidic
devices,23 we sought to exploit bis-crown ether modified
peptides for the structural stabilization and solubilization of
enzymes in organic media.
Keywords: Peptide nanostructure; Enzyme stabilization; Crown ether;
Organic solvent.
*
Corresponding author. Tel.: C1 418 6562131x4946; fax: C1 418
6567916; e-mail: normand.voyer@chm.ulaval.ca
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.04.063

M. Tremblay et al. / Tetrahedron 61 (2005) 6824–6828
6825
Herein, we report our results on the ability of bis-crown
peptides 1–3 to act as ‘molecular staples’ for protein
surfaces and their effect on the catalytic activity of
a-chymotrypsin in an organic environment.
Scheme 1. Transesterification reaction of N-acetyl-L-phenylalanine ethyl
ester catalyzed by a-chymotrypsin used as model system in the present
studies.
behavior, and ion binding ability of peptides 1–3. These
compounds were shown to complex CsC ions selectively by
forming ‘sandwich’ type complexes with one CsC by the
cooperative action of the two 18-crown-6 rings.24 Further-
more, they were also shown to bind selectively and
efficiently diammonium compounds.25 The alanine based
heptapeptide frameworks were chosen for their ability to
adopt several different conformations, hence to facilitate the
cooperative complexation of two charged groups at variable
distances on the surface of a-chymotrypsin. The crown
ether residue was synthesized from L-DOPA and the
peptides were prepared by solid phase peptide synthesis
using the oxime resin.24
Macrocyclic ligands of the 18-crown-6 family are known to
complex NaC, KC, and R-NHC3 ions. Hence, we envisioned
that bis-crown peptides like 1–3 could potentially bind
tightly at the surface of globular proteins through coopera-
tive complexation of two adjacent charged groups from
exposed Lys, Arg, Asp, and Glu residues (Fig. 1).
2. Results and discussion
The hydrophobic nature of the peptidic framework and the
neutralization of charged groups at the surface should
enhanced solubility in low polarity solvents. Likewise, the
‘linkage’ of distant functional groups by bis-crown ether
devices should also stabilize the active 3D structures of the
enzyme. In other words, peptidic devices 1–3 could possibly
acts as flexible but rigidifying ‘staples’ that allow
stabilization of enzyme active conformation without
significant lost of mobility. To demonstrate the proof of
concept, we investigated the model reaction consisting
of transesterification of N-Ac-L-Phe ethyl ester with
n-propanol catalyzed by a-chymotrypsin in cyclohexane
(Scheme 1). This transformation was used previously by
several groups for enzyme activity studies.13
a-Chymotrypsin was coated with variable amount of 1, 2 or
3 by adding the bis-crown additive to an aqueous solution
(pH 7.8) of the biocatalyst, and then lyophilizing to dryness.
As control, we also prepared the enzyme coated with twice
as much of 18-crown-6 by the same procedure.
To affect the transesterification, coated enzymes were
suspended at room temperature in cyclohexane in the
presence of N-acetyl-L-phenylalanine ethyl ester and
n-propanol. The rate of transesterification was followed by
high performance liquid chromatography (HPLC) measur-
ing the appearance of N-Ac-L-Phe-O-n-Pr. Initial rates (Vo)
were calculated from conversion !10%. In order to define
the most efficient conditions, experiments with a-chymo-
trypsin coated with 5, 10, 25, and 50 equiv of bis-crown
peptides 1–3, as compared to enzyme, were performed.
Results are shown in Table 1.
We have already described the synthesis, conformational
It has to be noted that almost no activity was observed with
a-chymotrypsin alone under the reaction conditions used.
For a-chymotrypsin coated with 5 equiv of each additive, an
increase of enzymatic activity was observed. However, no
differences could be noted between biocatalyst stabilized
with bis-crown ether additives 1, 2, 3, and 18-Crown-6.
When a-chymotrypsin coated with 10 equiv of peptide
additives were used, the initial rate increased by 40 fold
compared to a-chymotrypsin without additives, but only
two-fold compared to 18-Crown-6. The conversion of the
substrate after 30 min is more than 30% for the systems with
peptide 1 and with 18-Crown-6, and 15% for both 2 and 3
(Table 1). Interestingly, in this case a-chymotrypsin coated
with bis-crown peptide 1 is a little more active then
a-chymotrypsin coated with peptide 2 and 3 and 18-Crown-
6. This difference could be due to the better enzyme surface
Figure 1. Proposed complexation mode of molecular staples on the surface
of a globular protein.

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M. Tremblay et al. / Tetrahedron 61 (2005) 6824–6828
Table 1. Effect of peptides 1–3 on the activity of a-chymotrypsin in the transesterification of N-acetyl-L-phenylalanine ethyl ester in cyclohexane/1 M 1-PrOHa
Vo (Cadditive)
Conversion (%)
30 min
Conversion (%)
2 h
(10K5 M minK1
)
Mole additive
Vo (Cadditive)
Additive
(mole a-chymotrypsin)
Vo
None
0
5
5
12
125
95
90
65
1
10
7
7
5
!5
!10
!10
!10
!10
30–35
15–20
15–20
25–30
80
!10
20–25
15–20
15–20
15–20
60–70
35–40
20–25
55–60
O95
Peptide 1
Peptide 2
Peptide 3
18-Crown-6
Peptide 1
Peptide 2
Peptide 3
18-Crown-6
Peptide 1
Peptide 2
Peptide 3
18-Crown-6
18-Crown-6
Peptide 1
Peptide 2
Peptide 3
18-Crown-6
5
10
10
10
10
20
25
25
25
50
50
50
50
50
100
515
420
200
285
2600
755
450
420
3.3b
3175
3695
1255
10
41
33
16
23
208
60
36
34
41b
254
295
100
!1
30–35
20–25
40
60
40–50
60–70
70–80
70–80
60–70
!5
O 95
O95
70–80
!5
a Conditions: 2.5 mM substrate, 1 mg mLK1 enzyme powder (not corrected for buffer salts weight), 22 8C.
b Results obtained by Reinhoudt and co-workers under the same conditions.13
complexation of bis-crown peptide 1 that has the two crown
ether residues separated by only one alanine.
thus obtained, decreased dramatically. This behavior has
also been reported when using more then 250 equiv of
18-Crown-6 with a-chymotrypsin.18 The important lost of
activity is probably due to enzyme denaturation during
co-lyophilization.
The most pronounced effect was observed with 25 equiv of
1 (Table 1). In only 30 min, the conversion of the substrate
reached 80% and completion after 1 h with a-chymotrypsin
coated with this additive compared to 40% and even less
when using 18-Crown-6, peptides 2 or 3. An improvement
of chymotrypsin activity up to 200 fold is observed with
peptide 1, compared to 30 fold for 18-Crown-6. So, the
peptide chain allows a six-fold increase of enzyme activity.
In order to make this procedure more convenient for
synthetic chemists, reactions were performed using con-
ventional glassware and magnetic stirring comparatively to
mechanical stirring frequently used with biocatalytic
processes. Interestingly, no significant changes were
observed in the results between reactions carried out with
magnetic or mechanical stirring for reaction times of 2 h or
less. However, for reactions requiring longer period of time
(O2 h) to attain a reasonable conversion level, it is
preferable to use mechanical stirring. Along the same
lines, it is important to note that very high conversion of the
substrate can be achieved with only 10 equiv of peptidic
additive 1 after 24 h (O95% with magnetic stirring and
O99% with mechanical stirring).
Although, not optimized crown peptide additives compared
favorably then with commonly used additives (Table 2).
These new additives could eventually be used with other
enzyme or mixed with other additives like KCl to increase
enzyme activation.
Using a-chymotrypsin coated with 50 equiv of peptide 1
lead to a modest improvement of rate compared to reaction
with 25 equiv of 1. Improvement was more important with
50 equiv of peptide 2 and 3 but without a good
reproducibility. This is probably due to difficulties of
additive dissolution in phosphate buffer with a larger
amount of peptide 2 or 3. Indeed in all other cases,
experiments were highly reproducible in duplicate runs.
When 100 equiv of 18-Crown-6 were colyophilized with
a-chymotrypsin, the rate enhancement of the biocatalyst
These results show the efficiency of crown ether peptide to
enhance the a-chymotrypsin activity in cyclohexane.
However, more work is required to understand the
mechanism of enzyme activation by peptide 1–3. CD and
IR studies could not be investigated due to the chiral nature
of additives 1–3. Hence, it is not possible to study their
effect on structural stability of chymotrypsin as their own
Table 2. Rate enhancement a-chymotrypsin colyophilized with different additives
Entry
Additives
Enhancement
Reference
1
2
3
4
5
6
Crown peptide 1 (25 equiv)
18-C-6 (50 equiv)
KCl 94% w/wa
208
41
51
2–40
264
19
13
17
16
14
14
Various cyclodextrinsb
Substrate analogc
Sorbitolc
a For transesterification reaction of N-Ac-L-Phe-OEt in anhydrous hexane.
b For transesterification reaction of N-Ac-L-Tyr-OMe in various organic solvent/water (97:3).
c For transesterification reaction of N-Ac-L-Phe-OEtCl in anhydrous carbon tetrachloride.

M. Tremblay et al. / Tetrahedron 61 (2005) 6824–6828
6827
spectrum interferes with the one of the enzyme. The
different enhancing ability between 1–3 demonstrates that
the peptidic framework plays a functional role in the
stabilization of the enzyme structure. Therefore, it is fair to
say that bis-crown peptides with other amino acid sequences
could be engineered to stabilize numerous enzymes of
industrial and synthetic interest.
enhanced activity that catalyzes efficiently a model
transesterification reaction. The best results were obtained
with a-chymotrypsin coated with 25 equiv of bis-crown
peptide 1 in cyclohexane with complete conversion of the
substrate after less than an hour, a tremendous improvement
over the control reaction using uncoated a-chymotrypsin.
Important differences of efficiency between additives 1–3
point out the functional role of the peptidic framework to
allow the two binding groups (crown ether) to cooperatively
complex and bridge charged groups at the biocatalyst
surface. Therefore, it is possible to envision that this
stabilization strategy could be applied to numerous enzymes
of interest and that efficiency of peptidic devices could be
improved rapidly by parallel solid phase synthesis.
Although, ‘tailor-made’ nanoscale additives are presently
more expensives then polyethylene glycol, their potential
applicability to a wide variety of enzymes not responding to
actual additives make them attractive and a valuable
alternative. Improvement of 1–3 through parallel synthesis
and their use with other biocatalysts are currently underway
in our laboratories.
To evaluate the influence of bis-crown peptide 1 on the
enantioselectivity of a-chymotrypsin, the transesterification
reaction was studied with N-Ac-D-Phe-OEt and N-Ac-D,L-
Phe-OEt as substrates using a-chymotrypsin coated with
10 equiv of 1 as catalyst. After 2 h, almost no conversion
was observed for the experiment with the D substrate,
whereas around 40% conversion was measured when using
the racemic substrate. With the latter reaction, we have
determined the enantiomeric excess of product and remain-
ing substrate (eep and ees) to calculate the enantiomeric ratio
(E) of the bis-crown peptide enzyme system.26 An E value
O200 was obtained; no D-enantiomer could be detected by
chiral HPLC even after four days. These results clearly
demonstrate that the enantioselectivity of the enzyme is not
altered significantly by the presence of crown peptide.
4. Experimental
The activity of a-chymotrypsin stabilized with 25 equiv of
bis-crown peptide 1 was also studied at different tempera-
ture in cyclohexane (Table 3). In comparison with room
temperature (22 8C), the activity decreased slightly at 30 8C
and significantly at 40 and 50 8C. Therefore, for practical
purpose the actual synthetic procedure should be carried out
at room temperature.
Synthesis, purification, and characterization of bis-crown
ether peptides 1–3 was done according to the reported
procedures.23 a-Chymotrypsin (E.C. 3.4.21.1), type II, from
bovine pancreas and 18-Crown-6 were obtained from
Aldrich (Milwaukee, WI, USA) and used without further
purification. Distilled cyclohexane over molecular sieves
was used. n-Propanol was purified by a benzene azeotropic
distillation.
Table 3. Effect of increasing temperature on catalytic activity of a-
chymotrypsin coated with 25 equiv of bis-crown device 1 in the
transesterification of N-acetyl-L-phenylalanine ethyl ester in cyclohexane/
1 M 1-PrOHa
4.1. Coating of a-chymotrypsin
T (8C)
Vo (Cadditive)
Conversion
(%) 30 min
Conversion
(%) 2 h
(10K5M minK1
)
a-Chymotrypsin (10 mg, 4E-4 mmol) and the appropriate
amount of bis-crown ether peptides or 18-Crown-6 were
dissolved in 50 mM sodium phosphate buffer, pH 7.8
(2 mL). The solution was shaken manually (for the solution
with 50 equiv of bis-crown ether peptide, a short sonication
gave a better dissolution but it was still incomplete). The
samples were lyophilized to a white powder after freezing in
liquid nitrogen.
22
30
40
50
2600
2210
770
45
80
50
25
!10
O95
65
30–35
10–15
a Conditions: 2.5 mM substrate, 1 mg mLK1 enzyme powder.
Because peptides 1–3 incorporate derivatives of phenyl-
alanine, their stability to degradation in presence of
a-chymotrypsin was verified. Studies were done in phosphate
buffer with bis-crown peptide 3 and compared to its
phenylalanine analog 4 that gives no enhancement of catalytic
activity in cyclohexane. HPLC analysis demonstrated a rapid
degradation of peptide 4, but complete stability of peptide 3
towards hydrolytic degradation by a-chymotrypsin. The
crown ether ring attached to the phenyl group therefore
prohibits the accessibility to the active site of a-chymotrypsin,
leading to enzymatic degradation stability.
4.2. Catalytic activity
Reactions were performed on a 1.5 mL scale with magnetic
stirring. In every reaction, 1.5 mg of a-chymotrypsin, free
or coated, was used (quantities were adjusted to always have
1.5 mg of a-chymotrypsin content. For example, 3.5 mg of
the solid obtained from co-lyophilization of 25 equiv of
peptide 1 and a-chymotrypsin were used). The biocatalyst
was added to a cyclohexane solution containing N-Ac-L-
Phe-OEt (2.5 mM) and 1-propanol (1 M) at 22 8C. The
transesterification reaction was immediately followed by
high-performance liquid chromatography (HPLC) monitor-
ing the appearance of the reaction product (tretZ5.2 min).
HPLC analyses were performed on an Agilent 1050 HPLC
system using an analytical C18 reverse phase column
(Vydac, Hesperia, CA, USA). Column was eluted isocratic
at a flow rate of 1 mL/min with 45% acetonitrile in water
3. Conclusions
We have reported the use of peptides bearing two crown
ethers as tailor-made additives for the stabilization of the
structure and for increasing activity of a-chymotrypsin in
organic solvents. Co-lyophilization of a-chymotrypsin with
different amount of 1–3 lead to coated biocatalysts with

6828
M. Tremblay et al. / Tetrahedron 61 (2005) 6824–6828
12. Kragl, U.; Eckstein, M.; Kaftzik, N. Curr. Opin. Biotechnol.
2002, 13, 565–571.
(with 0.1% TFA). 10 mL of reaction mixtures were injected
at regular intervals.
13. Engbersen, J. F. J.; Broos, J.; Verboom, W.; Reinhoudt, D. N.
Pure Appl. Chem. 1996, 68, 2171–2178.
Chiral HPLC was performed with a Hypersil Phenylglycine
column (250!4.6 mm, 5 mm) using hexane with 1%
ethanol as eluent (1.5 mL minK1).
14. Dabulis, K.; Klibanov, A. M. Biotechnol. Bioeng. 1993, 41,
566–571.
15. Murakami, Y.; Hirata, A. Biotechnol. Tech. 1999, 13,
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16. Hasegawa, M.; Yamamoto, S.; Kobayashi, M.; Kise, H.
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Acknowledgements
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J. Am. Chem. Soc. 1994, 116, 2647–2648.
The financial support of this work by NSERC of Canada,
´
FQRNT of Quebec, and Universite Laval is gratefully
acknowledged.
18. Van Unen, D.-J.; Engbersen, J. F. J.; Reinhoudt, D. N.
Biotechnol. Bioeng. 1998, 59, 553–556.
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