A strong positive dendritic effect in a peptide dendrimer-catalyzed ester hydrolysis reaction

Article abstract of DOI:10.1021/ja044652p

The contribution of the dendritic structure in catalysis of ester hydrolysis was investigated with a systematic peptide dendrimer series of increasing generation number (G1-G4) containing a catalytic consensus sequence His-Ser in all branches. A strong positive dendritic effect was observed with up to 100-fold increased histidine reactivity between G1 and G4. Kinetic studies and isothermal calorimetric titration experiments showed that the strong positive dendritic effect resulted from cooperativity between binding and catalysis. Copyright

Full text of DOI:10.1021/ja044652p

Published on Web 11/11/2004
A Strong Positive Dendritic Effect in a Peptide Dendrimer-Catalyzed Ester
Hydrolysis Reaction
Estelle Delort, Tamis Darbre, and Jean-Louis Reymond*
Department of Chemistry and Biochemistry, UniVersity of Berne, Freiestrasse 3, 3012 Berne, Switzerland
Received September 3, 2004; E-mail: jean-louis.reymond@ioc.unibe.ch
Scheme 1. Peptide Dendrimer G1-G4-Catalyzed Ester
Hydrolysis
Dendrimers are treelike molecules with various uses in chemistry
and biology such as catalysis, drug delivery, artificial vaccines, and
gene delivery into cells.1 Most applications in catalysis consist of
attaching a catalytic module to the dendrimer surface to afford an
easily separable macromolecular catalyst.2 This multiple display
at the dendrimer surface, however, can induce a decrease in catalytic
efficiency per catalyst unit due to steric crowding.3 A strong positive
dendritic effect was observed in the oxidation of bromide by H2O2
catalyzed by dendritic phenylselenides.4 Herein we report the first
strong positive dendritic effect for a peptide dendrimer-catalyzed
ester hydrolysis reaction and show that the effect originates from
cooperativity between substrate binding and catalysis.
During our investigation of peptide dendrimers as enzyme
models, we reported that peptide dendrimers displaying multiple
histidine residues at their surface catalyze ester hydrolysis reactions.5
In particular, dendrimers with surface His-Ser catalyzed the hydrol-
ysis of pyrene trisulfonate esters 1-3.6 Hydrolysis seems to occur
by nucleophilic catalysis by the histidine side-chain without par-
ticipation of serine.6a To determine the contribution of the dendrimer
structure in catalysis, we have investigated a systematic peptide
dendrimer series of increasing generation number containing a
catalytic consensus sequence His-Ser in all branches (Scheme 1).
Synthesis was carried out by Fmoc strategy on NovaSyn Tentagel
resin (0.25 mmol/g), affording the peptide dendrimers in good yields
(G1 15%, G2 44%, G3 32%, G4 4.6%). Hydrolysis of pyrene
trisulfonate esters 1-4 (0.01-1.0 mM) catalyzed by dendrimers
G1-G4 (G1 10.6 µM, G2 6.4 µM, G3 3.5 µM, G4 1.9 µM) or
4-methyl-imidazole (0.04-1 mM) was followed by fluorescence
(λexc ) 460 nm, λem ) 530 nm) in a 96 well microtiter-plate setup.
All dendrimers were catalytically active with multiple turnover and
displayed enzymelike Michaelis-Menten kinetics. The pH rate
profile with substrate 2 indicated an optimal activity in the range
pH 5-6 (Figure 1).
Kinetic parameters showed that catalysis increased with increas-
ing generation number (Table 1). The catalytic rate constants kcat
and the substrate binding constants 1/KM with substrates 1-3 were
directly proportional to the total number of histidines per dendrimer,
resulting in a quadratic increase in catalytic efficiency kcat/KM as a
function of dendrimer size (Table 1, Figure 2). In the best case of
dendrimer G4 and nonanoyl ester 3, the dendrimer was 140 000-
fold more efficient than 4-methyl-imidazole as a reference catalyst,
amounting to a 4500-fold acceleration per histidine side-chain. The
positive dendritic effect in catalysis can be measured by the increase
in histidine reactivity (kcat/KM)/k2/His between G1 and G4, which
amounts to 24-fold for substrate 1, 25-fold for 2, and 100-fold
for 3.
increase from G1 to G4, following the trend in KM observed with
the reactive substrates 1-3 (Table 1). By contrast, product 5 binding
remained constant across the series G1-G4. Therefore, the positive
dendritic effect on substrate binding 1/KM is best interpreted in terms
of hydrophobic interactions between the substrate’s acyl chains and
the dendrimers.
The experiments above show that the esterolytic activity of
peptide dendrimers involves a strong positive dendritic effect
resulting from cooperative binding and catalysis (Figure 2). Catalytic
efficiency increases with generation despite steric crowding of
catalytic groups at the periphery, which must occur in higher
generation dendrimers. The effect of increasing dendrimer size
Binding of the dendrimers to the slow reacting substrate 47 or
product 5 was investigated by isothermal titration calorimetry (ITC)
(Table 2). Binding was strongly exothermic in both cases. Substrate
4 binding was 15-300 times stronger than product binding and
increased in higher generation number with an overall 50-fold
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J. AM. CHEM. SOC. 2004, 126, 15642-15643
10.1021/ja044652p CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
Table 2. Calorimetric Titration of 4 and 5 Added to G1-G4a
Figure 1. pH rate profile for substrate 2 (4-Me-Im ) 4-methyl-imidazole).
Table 1. Michaelis-Menten Parameters for Dendrimers G1-G4a
1
n
Ka
(×
104 M-
)
G1
G2
G3
G4
G1
G2
G3
G4
4
4
4
4
5
5
5
5
2.36
1.86
5.83
9.88
0.84
1.69
4.78
9.43
1.66 ((0.51)
10.5 ((3.2)
56.2 ((12.0)
80.8 ((15.0)
0.11 ((0.02)
0.27 ((0.03)
0.29 ((0.05)
0.28 ((0.07)
G1
G2
G3
G4
1
2
3
KM (µM)
840
0.080
1800
140
46
450
0.031
2200
140
48
110
0.16
3600
2000
280
140
0.11
8000
1600
230
67
41
0.30
6800
10 500
700
63
0.24
17 000
7900
530
13
0.15
6700
23 000
1600
35
0.86
20 000
35 000
1100
29
k
k
cat (min-1
)
b
cat/kuncat
c
(kcat/KM)/k2
(kcat/KM)/k2/His
K
M (µM)
cat (min-1
)
0.55
a Association constants (Ka) and number of binding sites (n); raw data
for titration of 4 added to G4 and enthalpogram corrected for the heat of
dilution. Conditions: G1 0.5 mM, G2 0.2 mM, G3 0.1 mM, G4 0.05 mM,
4 10 mM, 5 10 mM in citrate buffer pH 5.5 (5 mM), at 27 °C.
k
k
b
cat/kuncat
39 000
38 000
1200
5.8
0.39
18 000
c
(kcat/KM)/k2
(kcat/KM)/k2/His
K
M (µM)
1600
k
k
cat (min-1
)
0.099
0.096
Acknowledgment. This work was financially supported by the
University of Berne, the Swiss National Science Foundation, the
COST program D25, and the European Marie Curie Traning
Network IBAAC.
b
cat/kuncat
4500
130
44
4400
3000
420
c
(kcat/KM)/k2
(kcat/KM)/k2/His
140 000
4500
b
a Conditions: 5 mM aq citrate pH 5.5, 27 °C. kuncat(min-1) ) 4.4 ×
10-5 (1), 1.4 × 10-5 (2), 2.2 × 10-5 (3). c k2(4-Me-Im) (mM-1 min-1) )
7.0 × 10-4 (1), 4.9 × 10-4 (2), 4.8 × 10-4 (3).
Supporting Information Available: Synthetic procedures and data
for all dendrimers and details of kinetic and ITC measurements (PDF).
This material is available free of charge via the Internet at http://
pubs.acs.org.
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Figure 2. Dendritic effect on dendrimer-catalyzed hydrolysis of
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(7) Dodecanoyl ester 4 was a poor substrate (see Figure S1, Supporting
Information) and competitively inhibited catalysis for the shorter chain
substrates, making this substrate an ideal model for studying substrate
binding to the dendrimers.
rather induces catalytically productive interactions such as (1)
modulation of the histidine side-chain’s pKa, as evidenced from
the very different pH rate profile of kcat for the dendrimers vs
4-methyl-imidazole (Figure 1) and (2) creation of a hydrophobic
microenvironment allowing substrate binding by the acyl chains.
Fine-tuning of this dendritic effect toward selective substrate
recognition and turnover might enable the preparation of more
efficient and selective dendritic catalysts.
JA044652P
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