Enantioselective ester hydrolysis catalyzed by β-cyclodextrin conjugated with β-hairpin peptides

Article abstract of DOI:10.1016/j.bmcl.2003.11.046

Designed cyclodextrin-peptide conjugates, which have one or two β-hairpin peptides, have been synthesized as catalysts for ester hydrolysis. One or two β-hairpin peptides were located at the primary hydroxyl group side of β-cyclodextrin so as to arrange t

Full text of DOI:10.1016/j.bmcl.2003.11.046

Bioorganic & Medicinal Chemistry Letters 14 (2004) 723–726
Enantioselective ester hydrolysis catalyzed by ꢀ-cyclodextrin
conjugated with ꢀ-hairpin peptides
Hiroshi Tsutsumi, Hiroshi Ikeda, Hisakazu Mihara* and Akihiko Ueno*
Department of Bioengineering, Graduate School of Bioscience and Biotechnology,
Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan
Received 7 October 2003;accepted 13 November 2003
This work is dedicated to the late professor Akihiko Ueno (deceased March 23, 2003)
Abstract—Designed cyclodextrin–peptide conjugates, which have one or two b-hairpin peptides, have been synthesized as catalysts
for ester hydrolysis. One or two b-hairpin peptides were located at the primary hydroxyl group side of b-cyclodextrin so as to
arrange two histidine residues that act as a general acid and a general base catalysts and provide the substrate recognition subsite.
Kinetic studies revealed that the two-b-hairpin peptide was more effective than that of the one-b-hairpin peptide for substrate
recognition.
# 2003 Elsevier Ltd. All rights reserved.
Much effort has been devoted to demonstrate the con-
struction methods of catalysts for the desirable reac-
tions.1 Enzymes generate their high catalytic activities
and substrate selectivities by arranging multiple func-
tional groups masterly in their catalytic centers. Then, a
lot of entries as trials for artificial enzymes have been
reported by chemical approaches.2 One of the essential
factors for constructing artificial enzymes is arranging
the multiple functional groups at appropriate positions
for a selective substrate binding and an effective cataly-
tic reaction. In the design of artificial enzymes, con-
jugation of cyclodextrin (CD) and polypeptide could be
a promising technique for providing binding sites and
scaffolds. In the modified CD derivatives, CD acts as a
substrate binding site because of its remarkable ability
to bind a guest molecule.3 In the de novo designed pep-
tides, their well-defined secondary structures are useful
as scaffolds on which multiple functional moieties are
placed at their appropriate positions.4 Previously, we
bCD cavity might not be enough for selective substrate
binding. The multi-points binding is important for
selective binding of substrates. In addition to the bind-
ing site by a CD cavity, another substrate binding/
recognition site is also necessary. Because it is difficult
to construct substrate binding site using single a-helix
peptide, three- or four-helix bundle structures are uti-
lized for the construction of substrate binding site.6
Here, we selected an antiparallel b-sheet peptide as a
scaffold to arrange functional groups for catalytic reac-
tion and substrate binding. A b-sheet peptide can be
designed in a smaller size so that the side chains are
located vertically for its backbone. Although the prin-
ciples behind b-sheet formation are less well under-
stood, many trials to construct the stable b-sheet
structure have been reported.7 A b-hairpin structure is
the smallest b-sheet structure unit in which two anti-
parallel b-strands are connected by a b-turn. The
sequence of BH8 peptide (-ITVNGKTY-) is one of the
b-hairpin peptides that afford a stable b-sheet structure
in aqueous solution.8 In this peptide, the side chains of
Ile, Val and Tyr are arranged at the same side and form
a hydrophobic face when this peptide takes b-hairpin
conformation. Hence, two b-hairpin peptide chains
were arranged at the primary hydroxyl group side of
bCD so as to form a hydrophobic site as an additional
recognition site. In this study, an ester hydrolysis was
chosen as a model catalytic reaction. Two histidine
reported that
a cyclodextrin-peptide (CD-peptide)
hybrid having a bCD, an imidazole and a carboxylate
group on an a-helix peptide hydrolyzed ester substrates
effectively with the cooperative work between the imi-
dazole and the carboxylate groups.5 In this case, how-
ever, the remarkable substrate selectivity was not
observed because the simple substrate binding site of the
* Corresponding author. Tel.: +81-45-924-5756;fax: +81-45-924-
5833;e-mail: hmihara@bio.titech.ac.jp
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.11.046

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H. Tsutsumi et al. / Bioorg. Med. Chem. Lett. 14 (2004) 723–726
residues were arranged so as to work as a general acid
and a general base catalysts for the ester hydrolysis (Fig.
1). In b(AD)-AH, the b-hairpin peptide has one histi-
dine residue at the C-terminal side and two peptide
chains were linked to A and D positions of bCD, and
totally two histidine residues were arranged around a
bCD cavity. In order to confirm the effect of two-b-
hairpin peptide, b-HH that has one-b-hairpin peptide
was designed. In b-HH, two histidine residues were
arranged at both N-terminal and C-terminal sides in a
single chain (Fig. 1).
Both peptides were synthesized by stepwise elongation
of Fmoc-amino acids on a Rink amide resin.9 The side
chains of histidine, asparagine and cystein were pro-
tected by trityl groups, and those of glutamic acid,
threonine and tyrosine were protected by tert-butyl
groups. The side chain of Lys was protected by tert-
butyloxycarbonyl group. Synthesized peptides were
cleaved from resin, and at this stage, all protecting
groups were removed. Crude peptides were purified with
reversed phase HPLC, and purified peptides were reac-
ted with 6-mono(N-bromoacetylamino)-bCD or 6A,6D-
bis(N-bromoacetylamino)-bCD10,11 in the Tris–HCl
buffer solution (pH 8.5) to give the bCDs conjugated
with peptides. Products were purified with reversed-
phase HPLC and identified by MALDI-TOFMS
(b(AD)-AH m/z 4360.9 [(M+Na)+], calcd 4361.0 and
b-HH m/z 2803.2 [(M+H)+], calcd 2804.0).
Figure 2. Circular dichroism spectra of b(AD)-AH and b-HH in
5.0Â10À2 M, pH 6.5 phosphate buffer solution containing 0 or 30%
TFE at 25 ꢀC. b(AD)-AH (——) and b-HH (- - - - -) in 0 vol% TFE
and b(AD)-AH (– – – –) and b-HH (— — — —) in 30 vol% TFE buffer
solution (pH 6.5), respectively.
the presence of 30% 2,2,2-trifluoroethanol (TFE), both
b-HH and b(AD)-AH showed almost the same spectra
with a negative minimum point at 216 nm. In the NMR
analyses of b(AD)-AH and b-HH in water, the long
range NOE signals between Tyr10(CdH) and
Ile3(CdH3), Tyr10(CeH) and Val5(CbH) were observed
for only b(AD)-AH (data not shown). These results
suggest that the peptide chains of b(AD)-AH interact
with each other and stabilize their secondary structures
but the structure of b-HH is more flexible. Since these
peptides can form an amphiphilic structure as designed,
the hydrophobic interaction between the two peptides
might be effective.
Circular dichroism spectra of b(AD)-AH and b-HH are
shown in Figure 2. In the circular dichroism studies,
there was no pH dependence (from 5.5 to 8.0) and no
concentration effect (from 5.0Â10À6 M to 1.0Â10À4 M)
for both catalysts. b(AD)-AH had a negative maximum
point at 216 nm, whereas b-HH showed a smaller peak
around 216 nm. b(AD)-AH showed a similar spectrum
pattern compared with BH8 in aqueous solution.8a In
Figure. 1. Illustration of b(AD)-AH and b-HH and their amino acid sequences.

H. Tsutsumi et al. / Bioorg. Med. Chem. Lett. 14 (2004) 723–726
725
In the kinetic studies of ester hydrolysis, Boc-l-alanine
p-nitrophenyl ester (l-AlaONp) and Boc-d-alanine
p-nitrophenyl ester (d-AlaONp) were chosen as sub-
strates. The hydrolysis was performed in various pHs of
phosphate buffer solution and monitored as increasing
UV–vis absorbance of the product, p-nitrophenolate
(320 or 400 nm). In all measurements, the concentration
of each catalyst was fixed at 2.0Â10À5 M. The substrate
concentration was varied from 1.0Â10À4 to 5.0Â10À4 M
(l- and d-AlaONp). Excess conditions of the substrates
were kept in each measurement. The pH dependence in
initial rates of hydrolysis was measured under the con-
ditions of the fixed substrate concentration of 3.0Â10À4
M. Both catalysts had a maximum point around pH 6.5
in the hydrolysis of l-AlaONp and d-AlaONp. Since
the secondary structure of the catalysts do not sig-
nificantly depend on pH, the existence of a maximum
point must arise from two imidazole groups. Two
imidazole groups might exist as a protonated form and
a free form, which could work as a general acid and a
general base catalysts in this pH.12 Then, kinetic ana-
lyses for the ester hydrolysis by b(AD)-AH and b-HH
were performed at pH 6.5 and obtained kinetic para-
meters are summarized in Table 1. In the absence of any
catalyst, the rate constants of the hydrolyses for
d-AlaONp and l-AlaONp (kun) were 1.86Â10À5 sÀ1
(1.02Â10À4 M) to b-HH (1.63Â10À4 M) for l-AlaONp.
On the other hand, the Km value of b(AD)-AH for
d-AlaONp (7.66Â10À4 M) was slightly inferior to that
of b-HH (6.75Â10À4 M). As a result, b(AD)-AH
showed higher selectivity for l-AlaONp (7.5 fold) than
b-HH (4.1 fold). This result suggests that the higher
substrate selectivity of b(AD)-AH for l-AlaONp might
be due to the hydrophobic site constructed with Ile and
Tyr residues of two peptide chains. The hydrophobic
site constructed with two b-hairpin peptides would serve
favorably for the l-AlaONp binding. Thus, the two-b-
hairpin peptide-bCD conjugate, b(AD)-AH, gained the
higher catalytic activity for l-AlaONp than d-AlaONp
(6.3 times higher kcat/Km value).
In this study, we have demonstrated that the b-hairpin
peptide is useful as a new scaffold to arrange the func-
tional groups and construct substrate binding subsite in
the peptide-CD conjugates. The usage of two b-hairpin
peptides is effective for the selective substrate recogni-
tion and catalysis. Although the reported peptide
sequence is used as a model case in this study, design
and selection of new b-hairpin scaffold suitable for cat-
alysis will lead to the higher reaction and recognition
abilities with smaller artificial molecules.
and 1.89Â10À5
s
À1, respectively. In the presence of cat-
alysts, b(AD)-AH showed higher kcat values than b-HH
for both substrates. Because the secondary structure of
b(AD)-AH appeared to be more stable than that of b-HH,
two histidine residues of b(AD)-AH were fixed at their
appropriate positions so as to hydrolyze the substrates
efficiently, resulting that b(AD)-AH showed the higher
kcat values as compared with b-HH. However, b-HH
also showed the maximum point for ester hydrolysis
around pH 6.5, two histidine residues of b-hairpin pep-
tide might be arranged near the bCD cavity and act
cooperatively on the catalytic center. The Km values of
b(AD)-AH for l-AlaONp and d-AlaONp were
1.02Â10À4 M and 7.66Â10À4 M. b(AD)-AH showed 7.5
times superior Km value for l-AlaONp. It is noted that
there was no significant difference in Km values between
l-AlaONp and d-AlaONp by the reported bis-imida-
zole modified bCDs (for example, l-AlaONp: 8.0Â10À4
M and d-AlaONp: 6.0Â10À4 M).12b In addition,
b(AD)-AH showed a superior Km value to bis-imidazole
modified bCDs for l-AlaONp, while b(AD)-AH
showed almost the same value for d-AlaONp. These
results suggest that the selectivity of b(AD)-AH for
l-AlaONp is derived from the b-hairpin peptide scaf-
fold. b(AD)-AH showed the slightly superior Km value
Acknowledgements
We are grateful to Nihon Shokuhin Kako Co., Ltd. for
a generous supply of cyclodextrins. This work was
partly supported by
a Grant-in-Aid for Science
Research from the Ministry of Education, Culture,
Sports, Science and Techniology, and the COE21 pro-
gram in Tokyo Institute of Technology.
References and notes
1. (a) Stevenson, J. D.;Thomas, N. R. Nat. Prod. Rep. 2000,
17, 535. (b) Matsuo, T.;Hayashi, T.;Hisaeda, Y. J. Am.
Chem. Soc. 2002, 124, 11234.
2. Motherwell, W. B.;Bingham, M. J.;Six, Y. Tetrahedron
2001, 57, 4663.
3. (a) For the review of cyclodextrin as enzyme mimics:
Breslow, R.;Dong, S. Chem. Rev. 1998, 98, 1997. (b)
Komiyama, M.;Shigekawa, H. Comprehensive Supramo-
lecular Chemistry, Vol. 3. Cyclodextrins;Szejtli, J.;Osa,
T., Ed.;Pergamon: Oxford, New York, Tokyo, 1996, p
401.
4. (a) For the review of de novo design of peptides:
DeGrado, W. F.;Summa, C. M.;Pavone, V.;Nastri, F.;
Lombardi, A. Ann. Rev. Biochem. 1999, 68, 779. (b)
Schneider, J. P.;Kelly, J. W. Chem. Rev. 1995, 95, 2164.
5. (a) Tsutsumi, H.;Hamasaki, K.;Mihara, H.;Ueno, A.
Bioorg. Med. Chem. Lett. 2000, 10, 741. (b) Tsutsumi, H.;
Hamasaki, K.;Mihara, H.;Ueno, A. J. Chem. Soc., Per-
kin Trans. 2 2000, 1813.
Table 1. Kinetic parameters for the hydrolysis of Boc-l-alanine p-
nitrophenyl ester (l-AlaONp) and Boc-d-alanine p-nitrophenyl ester
(d-AlaONp) catalyzed by b(AD)-AH and b-HH at 25 ꢀC in pH 6.5
phosphate buffer solution (5.0Â10À2 M)
6. (a) Obataya, I.;Sakamoto, S.;Ueno, A.;Mihara, H. Bio-
polymers 2001, 59, 65. (b) Tomizaki, K.;Tsunekawa, Y.;
Akisada, H.;Mihara, H.;Nishiso, N. J. Chem. Soc., Per-
kin Trans. 2 2000, 813.
7. (a) Nesloney, C. L.;Kelly, J. W. Bioorg. Med. Chem.
1996, 4, 739. (b) Ramirez-Alvarado, M.;Kortemme, T.;
Blanco, F. J.;Serrano, L. Bioorg. Med. Chem. 1999, 7, 93.
Catalyst
Substrate
kcat
[10À4 sÀ1
km
[10À4 M]
kcat/Km
[MÀ1 s1]
]
b(AD)-AH
b-HH
l-AlaONp
d-AlaONp
l-AlaONp
d-AlaONp
12.1
14.4
8.89
10.0
1.02
7.66
1.63
6.75
11.9
1.88
5.45
1.48

726
H. Tsutsumi et al. / Bioorg. Med. Chem. Lett. 14 (2004) 723–726
8. (a) Ramirez-Alvarado, M.;Blanco, F. J.;Serrano, L. Nat.
Struct. Biol. 1996, 3, 604. (b) Ramirez-Alvarado, M.;
tration and to filtrate was added the corresponding mono
or 6A,6D-bis(6-deoxy-6-amino)-bCDs and stirred for 2 h.
The reaction mixture was poured into excess acetone and
formed precipitates were collected by filtration. The pre-
cipitates were washed by acetone several times and dried
in reduced pressure condition.
Blanco, F. J.;Niemann, H.;Serrano, L.
1997, 273, 898.
J. Mol. Biol.
9. Chan, W. C.;White, P. D. In Fmoc Solid Phase Peptide
Synthesis: A Practical Approach;Chan, W. C., White, P.
D., Eds.;Oxford University Press;New York, 2000;p 41.
10. Mono(6-deoxy-6-amino)-bCD and 6A,6D-bis(6-deoxy-6-
amino)-bCD were synthesized as previously described.11
N-Bromoacetylamino-CD derivatives were synthesized as
follows: Bromoacetic acid and dicyclohexylcarbodiimide
were dissolved in DMF and solution was stirred at ice
bath. After 1 h, dicyclohexylurea was removed by fil-
11. (a) Hamasaki, K.;Ikeda, H.;Nakamura, A.;Ueno, A.;
Toda, F.;Suzuki, I.;Osa, T. J. Am. Chem. Soc. 1993, 115,
5035. (b) Fujita, K.;Matsunaga, A.;Imoto, T.
hedron Lett. 1984, 25, 5533.
Tetra-
12. (a) Broo, K. S.;Nilsson, H.;Nillson, J.;Flodberg, A.;
Baltzer, L. J. Am. Chem. Soc. 1998, 120, 4063. (b) Lee,
W.-S.;Ueno, A. Chem. Lett. 2000, 258.