Discovery of hydrolytic catalysts in a peptidocalixarene library by binding assay with a transition state analogue for the hydrolysis

Article abstract of DOI:10.1039/b913907a

Hydrolytic catalysts were found in a peptidocalixarene library by binding assay with a transition state analogue for the hydrolysis. The rate of the reaction can be specifically enhanced up to 50-fold in the presence of the discovered catalyst. The Royal

Full text of DOI:10.1039/b913907a

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Discovery of hydrolytic catalysts in a peptidocalixarene library by
binding assay with a transition state analogue for the hydrolysisw
Hideaki Hioki,* Ryosuke Nishimoto, Kota Kawaguchi, Miwa Kubo, Kenichi Harada and
Yoshiyasu Fukuyama
Received (in Cambridge, UK) 13th July 2009, Accepted 5th October 2009
First published as an Advance Article on the web 15th October 2009
DOI: 10.1039/b913907a
Hydrolytic catalysts were found in a peptidocalixarene library
by binding assay with a transition state analogue for the
hydrolysis. The rate of the reaction can be specifically enhanced
up to 50-fold in the presence of the discovered catalyst.
The development of artificial enzymes that specifically catalyze
the reaction with a target molecule is a challenging research
area in the field of supramolecular chemistry.1 Catalytic
antibodies are established examples of tailor-made enzymes.2
Scheme 1 Discovery of catalysts for the hydrolysis of 1.
This approach has limited application, however, due to the
instability of the antibodies under various reaction conditions
(i.e., solvent, temperature), because antibodies are high
molecular-weight proteins. In addition, special equipment is
required to prepare the catalytic antibodies. On the other
hand, calixarenes are widely used as a platform for functional
molecules, such as artificial receptors, sensors and catalysts.3
Their potential function depends on the modification of the
core calixarene. We previously synthesized calixarene-based
peptide libraries to rapidly access desired functional molecules
such as chemical sensors for specific oligopeptides.4,5 We
expected that catalysts for specific substrates would be easily
found among the library in the same manner as the desired
catalytic antibodies, because the library members can be
postulated to function as antibodies.6,7
as a reporter group to allow visualization at the time of
the binding assay with previously synthesized resin-bound
peptidocalixarene library 5, which has 1000 members.9
The transition state analogue
4 was synthesized from
Fmoc-tyrosine in six steps (Fig. 1 and 2).
We incubated 3.5 Â 10À3 mol LÀ1 of 4 with ca. 5 mg
(ca. 3000 beads) of the library 5 in pH 6.86 phosphate buffer
for 15 h to screen the library 5 for binding with 4. The
screening was performed by a post-labeling color assay utilizing
the Trinder reaction.10 Almost 5% of the beads were stained
light purple as shown in Fig. 3. Thirty relatively dark colored
beads were isolated and decoded to identify their amino acid
sequences. The results are summarized in Table 1. All 30 beads
had at least one basic amino acid residue (Arg, His, Lys). In
particular, 20 of the 30 beads had His in AA3. These findings
suggested that, as expected, the binding nature in water is
dominated by electrostatic interactions.
First, we chose hydrolysis of phenol ester 1 as a study model
because it was the first successful system to generate a catalytic
antibody with hydrolytic activity.8 Phosphoric ester 4 was
designed to mimic the tetrahedral geometry of the transition
state for the hydrolysis of 1. The aniline group in 4 was loaded
Four peptidocalixarenes 5a–d, shown in Fig. 4, were selected
and synthesized on beads in large amounts to estimate their
hydrolytic activity. We mixed 1.25 Â 10À3 mol LÀ1 of 1 with
0.2 equiv. peptidocalixarene 5a–d in pH 6.86 phosphate buffer
Fig. 1 Transition state analogue for the reaction shown in Scheme 1.
Faculty of Pharmaceutical Sciences, Tokushima Bunri University,
Yamashiro-cho, Tokushima 770-8514, Japan.
E-mail: hioki@ph.bunri-u.ac.jp; Fax: +81 88 655 3051;
Tel: +81 88 602 8437
w Electronic supplementary information (ESI) available: Experimental
details for the synthesis of substrates 1, 4, 6, 7, 8 and 9. Determination
of kinetic parameters for the hydrolysis of 1, 7 and 9. See DOI:
10.1039/b913907a
Fig. 2 Peptidocalixarene library 5.
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Fig. 3 Screening of the library 5 for binding with 4 (3.5 Â 10À3 mol LÀ1
)
by a post-labeling color assay utilizing the Trinder reaction.9
Fig. 5 Concentration–time profile of the hydrolysis of 1 (1.25 Â
10À3 mol LÀ1) with 5a–e (0.2 equiv.). Solvent: aqueous phosphate
buffer, pH 6.86, Temperature: 30 1C. The concentration of product 2
was determined by quantitative HPLC analysis.
Table 1 Peptide sequences of the library members in 5 binding to a
transition state analogue 4a
Entry
AA1
AA2
AA3
Frequencyb
1
2
3
4
5
6
7
His
Pro
x1
Arg
Arg
Arg
Lys
Lys
Lys
His
His
His
His
His
His
His
2
1
4
1
1
4
7
10
30
Typical kinetic behavior based on the Michaelis–Menten
principle was observed. The kinetic parameters, such as the
Michaelis constant (Km) and rate enhancement (kcat/kuncat),
for the hydrolysis were calculated using a non-linear least-
squares regression method (Table 2). An approximately
50-fold rate enhancement was observed in the presence of
the peptidocalix[4]arene catalyst 5a. If the transition state of
the hydrolysis of 1 was stabilized by binding with 5a, the
reaction should be inhibited by the addition of the transition
state analogue 6. As expected, the reaction rate was clearly
decreased. Lineweaver–Burk plots in the presence or absence
of 6 are shown in Fig. 6. The two lines intersected in the left (– +)
quadrant, indicating mixed-type inhibition, suggesting that the
inhibitor 6 (Fig. 7) binds not only the active site for the
hydrolysis, but also other sites of the peptidocalix[4]arene.
c
Tyr
Ser
d
x2e
e
x3
x4
Others
8
Total
a
b
Exact sequences of all the 30 peptides are shown in ESI.w Number
of beads having the indicated sequences. Any amino acid except His
c
d
e
and Pro. Any amino acid except Tyr and Ser. x3 and x4 are
combinations of any amino acids except for combinations appearing
in entries 1–6.
Table 2 Michaelis constant (Km) and rate enhancement (kcat/kuncat
for the hydrolysis of 1, 7 and 9 catalyzed by peptidocalix[4]arene 5a
)
Substrate
Km/mol LÀ1
kcat/kuncat
1
7
9
1.59 Â 10À3
1.86 Â 10À3
4.02 Â 10À3
53.7
42.8
14.9
Fig. 4 Selected candidates to catalyze the hydrolysis of 1.
at 30 1C. The reaction was quantitatively monitored by HPLC.
The concentration–time profile of the product 3 is shown in
Fig. 5. The peptidocalixarenes clearly promoted the hydrolysis
compared to the negative control 5e or no beads. Despite
their binding with transition state analogue 4, their catalytic
activities were significantly different from each other (Fig. 5);
5a and 5b, possessing Arg in AA2, had a higher catalytic
activity than 5c and 5d with Lys in AA2. These results
indicated that the combining site of the peptidocalixarenes
binding with 4 is not necessarily the phosphoric ester function.
The same phenomenon was reported in the case of catalytic
antibodies coupled with a phage-display method.11
Fig. 6 Inhibition of hydrolysis of 1 by transition state analogue 6.
Lineweaver–Burk plots in the absence (blue) and the presence (red) of 6.
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Notes and references
1 For reviews on artificial enzymes, see: (a) R. Breslow and
S. D. Dong, Chem. Rev., 1998, 98, 1997–2012; (b) R. Breslow,
Acc. Chem. Res., 1995, 28, 146–153; (c) Y. Murakami, J. Kikuchi,
Y. Hisaeda and O. Hayashida, Chem. Rev., 1996, 96, 721–758;
(d) A. J. Kirby, Angew. Chem., Int. Ed. Engl., 1996, 35, 706–724;
(e) G. J. Rowlands, Tetrahedron, 2001, 57, 1865–1882.
2 (a) Catalytic Antibodies, ed. E. Keinan, Wiley-VCH Verlag GmbH
& Co. KGaA, Weinheim, Germany, 2005; (b) P. G. Schultz, Acc.
Chem. Res., 1989, 22, 287–294.
3 (a) C. D. Gutsche, in Calixarenes Revisited, Monograph in
Supramolecular Chemistry, ed. J. F. Stoddart, Royal Society of
Chemistry, Cambridge, 1998; (b) Calixarenes in Action,
ed. L. Mandolini and R. Ungaro, Imperial College Press, London,
2000.
4 (a) H. Hioki, Y. Ohnishi, M. Kubo, E. Nashimoto, Y. Kinoshita,
M. Samejima and M. Kodama, Tetrahedron Lett., 2004, 45,
561–564; (b) H. Hioki, M. Kubo, H. Yoshida, M. Bando,
Y. Ohnishi and M. Kodama, Tetrahedron Lett., 2002, 43,
7949–7952.
5 Recent research works on peptidocalixarenes by other groups, see:
(a) L. Baldini, F. Sansone, F. Scaravelli, C. A. Massera, Casnati
and R. Ungaro, Tetrahedron Lett., 2009, 50, 3450–3453;
(b) A. V. Yakovenko, V. I. Boyko, V. I. Kalchenko, L. Baldini,
A. Casnati, F. Sansone and R. Ungaro, J. Org. Chem., 2007, 72,
3223–3231; (c) S. Francese, A. Cozzolino, I. Caputo, C. Esposito,
M. Martino, C. Gaeta, F. Troisi and P. Neri, Tetrahedron Lett.,
2005, 46, 1611–1615.
Fig. 7 Structures of transition state analogue 6 without a reporter
group and reference substrates 7, 8 and 9 for hydrolysis catalyzed by 5a.
This finding implies that all four peptide arms in 5a are not
involved in binding the substrate. The inhibition constant (Ki)
was estimated to be 1.8 Â 10À3 mol LÀ1, which was calculated
using a nonlinear least-squares regression method. The value
was comparable to that of the Michaelis constant (Km).
Finally, substrate specificity was investigated using reference
compounds 7, 8 and 9. Compound 8 was poorly soluble under
the same reaction conditions as those applied for 1. Km and
k
cat/kuncat for the hydrolysis of 7 and 9 are shown in Table 2.
6 For recent reviews on peptide catalysts, see: (a) E. A. C. Davie,
S. M. Mennen, Y. Xu and S. J. Miller, Chem. Rev., 2007, 107,
5759–5812; (b) J. D. Revell and H. Wennemers, Curr. Opin. Chem.
Biol., 2007, 11, 269–278.
7 For selected reports on the discovery of catalysts using a combi-
natorial approach. see: (a) C. Schmuck and J. Dudaczek, Org.
Lett., 2007, 9, 5389–5392; (b) P. Krattiger, C. McCarthy, A. Pfaltz
and H. Wennemers, Angew. Chem., Int. Ed., 2003, 42, 1722–1724;
(c) A. Berkessel, Curr. Opin. Chem. Biol., 2003, 7, 409–419;
(d) G. T. Copeland and S. J. Miller, J. Am. Chem. Soc., 1999,
121, 4306–4307.
8 (a) A. Tramontano, K. D. Janda and R. A. Lerner, Science, 1986,
234, 1566–1570; (b) S. J. Pollack, J. W. Jacobs and P. G. Schultz,
Science, 1986, 234, 1570–1573.
9 M. Kubo, E. Nashimoto, T. Tokiyo, Y. Morisaki, M. Kodama
and H. Hioki, Tetrahedron Lett., 2006, 47, 1927–1931.
10 M. Kubo, R. Nishimoto, M. Doi, M. Kodama and H. Hioki,
Chem. Commun., 2006, 3390–3392.
The Km was slightly increased and rate enhancement was
slightly decreased for monoester 7. On the other hand, large
differences in both values were observed for diester 9, suggesting
that catalyst 5a discriminates between 1 and 7 or 9 and that the
carboxylic acid in the substrates 1 and 7 is important for
binding with 5a.
In conclusion, hydrolytic catalysts for 1 were found
in peptidocalixarene library 5 using a binding assay with
transition state analogue 4 for the hydrolysis of 1. The rate
of the hydrolysis of 1 can be enhanced up to 50-fold in the
presence of the discovered catalyst 5a. This technique might be
useful for creating tailor-made substrate specific catalysts.
Design and synthesis of a large-sized peptidocalixarene library
is in progress to find highly active and specific catalysts for
various reactions.
11 I. Fujii, F. Tanaka, H. Miyashita, R. Tanimura and K. Kinoshita,
J. Am. Chem. Soc., 1995, 117, 6199–6209.
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7196 | Chem. Commun., 2009, 7194–7196