Structure-activity relationship studies in single-site esterase peptide dendrimers

Article abstract of DOI:10.1560/IJC.49.1.129

We recently reported on peptide dendrimers with a single catalytic site at the dendrimer core catalyzing the hydrolysis of acetoxy- and butyryloxy-pyrene trisulfonate 1a/b in aqueous buffer with Michaelis-Menten kinetics. Substrate binding is mediated by

Full text of DOI:10.1560/IJC.49.1.129

Israel Journal of Chemistry
Vol. 49
DOI: 10.1560/IJC.49.1.129
2009 pp. 129–136
Structure–Activity Relationship Studies in Single-Site
Esterase Peptide Dendrimers
Sacha Javor and Jean-Louis Reymond*
Department of Chemistry and Biochemistry, University of Berne, Freiestrasse 3, CH-3012, Berne, Switzerland
(Received 6 August 2008 and in revised form 4 November 2008)
Abstract. We recently reported on peptide dendrimers with a single catalytic site
at the dendrimer core catalyzing the hydrolysis of acetoxy- and butyryloxy-pyrene
trisulfonate 1a/b in aqueous buffer with Michaelis–Menten kinetics. Substrate bind-
ing is mediated by a pair of protonated arginine or histidine residues in the first gen-
eration branch, and esterolysis is performed by the imidazole side-chain of a histi-
dine residue in the core acting as a general base or nucleophile. Herein we report on a
structure–activity relationship study searching for an optimal combination between
amino acid sequence and catalytic machinery. Installation of histidine residues onto
the aromatic dendrimer framework “R” leads to 10-fold higher rate acceleration up
to kcat/kuncat = 1.5 * 103 at pH 5.5 with dendrimers RG3H (AcYT)8(BWG)4(BHS)2BHS
and RMG3H (AcYT)8(BWG)4(BHSG)2BHS (one-letter codes for L-amino acids;
Ac = acetyl, B = L-2,3-diaminopropionic acid branching point, C-terminus is amide
–CONH2). These dendrimers reach the compactness of a native folded protein.
IntRoductIon
Peptide dendrimers can be easily modified by varia-
tions of the amino acid building blocks, which allows
one to assess structure–activity relationships (SAR) that
may not be accessible in other synthetic dendrimers less
amenable to modifications. In the case of multivalent
esterase dendrimers featuring multiple histidine resi-
dues as catalytic and binding groups,10 such SAR studies
showed that histidine multivalency primarily influenced
substrate binding, whereas the catalytic rate constant
kcat was more strongly influenced by the nature of other
amino acids in the dendrimers and the exact placement
of histidine residues within the structure.11 Catalysis
also depended on dendrimer size, with the occurrence of
a positive dendritic effect on substrate binding and ca-
talysis, as evidenced by the investigation of dendrimers
at various generation numbers.
Due to their globular shape, dendrimers1 are attractive
synthetic models for proteins, in particular as catalysts.2
In recent years we have shown that peptide dendrimers3
assembled by solid-phase peptide synthesis using pro-
teinogenic amino acid building blocks and branching
diamino acids may exhibit a variety of protein-like
functions, such as catalysis, cofactor and protein bind-
ing, and drug delivery.3c Peptide dendrimers are particu-
larly attractive as enzyme models4 since they are formed
from the same building blocks as natural enzymes and
only differ in their topology. We have recently identi-
fied peptide dendrimers with a variety of protein-like
functions by screening combinatorial libraries of den-
drimers.5 These included peptide dendrimers with ester-
ase-like6 and aldolase-like catalysis,7 cofactor-binding
dendrimers,8 and multivalent glycodendrimers for lectin
recognition.9
*Author to whom correspondence should be addressed.
E-mail: jean-louis.reymond@ioc.unibe.ch

130
Scheme 1. Esterase peptide dendrimer catalyzed hydrolysis of acyloxypyrene trisulfonates 1a/b.
ExpERIMEntAl
We recently reported a series of dendrimer esterase
models with a single catalytic site at the dendrimer
core.12 These dendrimers catalyzed the hydrolysis of ac-
yloxypyrene trisulfonates 1a/1b in aqueous buffer with
Michaelis–Menten kinetics. Substrate binding was me-
diated by a pair of protonated arginine or histidine resi-
dues in the first generation branch, and esterolysis was
performed by the imidazole side-chain of a histidine
residue in the dendrimer core acting as a general base or
nucleophile (Scheme 1). An initial series of dendrimer
analogues addressed the role in catalysis of the outer,
non-catalytic layers of the dendrimers and of the relative
placement of catalytic residues within the core. Herein
we report further SAR studies concerning the search for
an optimal combination between dendrimer sequence
and catalytic machinery. We show that installation of
histidine residues onto the aromatic dendrimer frame-
work “R” leads to a 10-fold gain in rate acceleration.
The combination showing enhanced activity is also the
most compact as measured by hydrodynamic radius.
Peptide Dendrimer Synthesis
Peptide dendrimers were synthesized by the Fmoc strategy
using a Chemspeed PSW 1100 automatic synthesizer. Prior
to every reaction the resin was swelled in CH2Cl2. The resin
NovaSyn TGR was acylated with each amino acid or diamino
acid (3.0 equiv) using procedures BOP (3.0 equiv) and DIEA
(5.0 equiv) in a mixture of NMP/DCM (3:1) for 2 h. To ensure
the completion, all the acylation steps were repeated twice.
The Fmoc protecting groups were removed with a solution of
20% piperidine in DMF (2 × 20 min). At the end of the synthe-
sis, the resin was acylated with acetic anhydride/CH2Cl2 (1:1)
for 20 min. The cleavage was carried out with TFA/TIS/H2O
(94:5:1) for 6 h. The peptide was precipitated with methyl tert-
butyl ether, and then dissolved in a water/acetonitrile mixture.
All dendrimers were purified by preparative HPLC and ob-
tained as TFA salts after lyophilization.
(((Ac-Tyr-Thr)2Dap-Trp-Gly)2Dap-His-Ser)2Dap-His-Ser-
NH2 (RG3H): Starting with 250 mg of NovaSyn TGR resin
(0.23 mmol/g), the dendrimer RG3H was obtained as a color-
less foamy solid using the automated synthesis after cleavage
Israel Journal of Chemistry
49
2009

131
from the resin and preparative RP-HPLC purification (1.9 mg, gives fluorescence vs. time, typically between 500 and 2000 s,
0.7%). MS (ES+) calcd for C220H278N55O64 [M+H]+: 4714.0. corresponding to less than 10% conversion. Michaelis–Men-
Found: 4716.8; [M+Na]+: 4736.0. Found: 4738.4.
ten parameters kcat (rate constant) and KM (Michaelis constant)
(((Ac-Tyr-Thr)2Dap-Trp-Gly)2Dap-His-Ser-Gly)2Dap- were determined from the linear double reciprocal plot 1/Vnet
His-Ser-NH2 (RMG3H): Starting with 250 mg of NovaSyn vs. 1/[S] (Lineweaver–Burk plot).
TGR resin (0.23 mmol/g), the dendrimer RMG3H was ob-
Diffusion-NMR
The diffusion constants of the dendrimers were obtained
by H NMR using a stimulated echo (STE) pulse sequence.
tained as a colorless foamy solid using the automated synthe-
sis after cleavage from the resin and preparative RP-HPLC pu-
rification (1.0 mg, 0.3%). MS (ES+) calcd for C224H284N57O66
[M+H]+: 4828.1. Found: 4830.4; [M+Na]+: 4850.1. Found:
4852.0; [M+K]+: 4867.1. Found: 4871.4.
(((Ac-Ile-Pro)2Dap-Ile-Thr)2Dap-Arg-Ala)2Dap-His-
Leu-NH2 (HG3R): Starting with 250 mg of NovaSyn TGR
resin (0.23 mmol/g), the dendrimer HG3R was obtained as
a colorless foamy solid using the automated synthesis using
procedure A after cleavage from the resin and preparative
RP-HPLC purification (18.4 mg, 7.0%). MS (ES+) calcd for
C195H330N53O49 [M+H]+: 4198.5. Found: 4200.0.
(((Ac-Ile-Pro)2Dap-Ile-Thr)2Dap-Arg-Ala-Gly)2Dap-His-
Leu-NH2 (HMG3R): Starting with 250 mg of NovaSyn TGR
resin (0.23 mmol/g), the dendrimer HMG3R was obtained as
a colorless foamy solid using the automated synthesis after
cleavage from the resin and preparative RP-HPLC purification
(7.1 mg, 2.7%). MS (ES+) calcd for C199H336N55O51 [M+H]+:
4312.5. Found: 4312.8
1
The measurements were carried out using a Bruker DRX400
or DRX500 with dilute solutions (typically 5 mg·mL−1) in
D2O at 300 K. The gradient with a maximum strength of 50
× 10−4 T·cm−1 was calibrated using the HOD proton signal in
99.997% D2O. The diffusion time Δ was 50 ms and the gradi-
ent duration δ was 7 ms. The diffusion coefficient D was de-
rived from peak integrals or intensities using the Simfit soft-
ware from Bruker. The hydrodynamic radii were calculated
from the diffusion coefficient D using the Stokes–Einstein
equation with η = 1.089 mPa for D2O at 300 K. The compac-
tion factor was calculated as originally proposed.13
REsults And dIscussIon
In searching for core active site esterase activity in a
combinatorial library of peptide dendrimers using a
solid-supported assay, we had identified two types of
amino acid sequences leading to activity.12 In the first
case, RG3, the core active site catalytic histidine was
assisted by a pair of arginine residues in the 1st genera-
tion branch for substrate binding, and this core was sur-
rounded by 2nd- and 3rd-generation layers containing
the aromatic residues tryptophan and tyrosine. In the
Kinetic Assays
Kinetic measurements were carried out using a CytoFluor
Series 4000 multi-well plate reader from PerSeptive Bio-
systems. Dendrimers were used as 10 µM or 15 µM freshly
prepared solutions in milliQ water. Solutions were prepared
by dissolving the dry TFA salts of dendrimers. Substrate solu-
tions for the Michaelis–Menten kinetics were prepared by se-
rial dilution by a factor 2/3 (7´) of a freshly prepared 3.0 mM second case, HG3, the catalytic core displayed three
solution of substrate in milliQ water (final concentration on
the plate 60–1000 µM). Low KM determinations were carried
using 30–440 µM final substrate concentrations. Eight solu-
tions of 8-hydroxypyrene-1,3,6-trisulfonic acid sodium salt 2
ranging from 0 µM to 100 µM in buffer were used for the cali-
bration curve. Bis-Tris 30 mM or citrate 15 mM was used as
buffer and the pH was adjusted to the desired value with HCl
1.0 M and NaOH 1.0 M using a Metrohm 692 pH/ion meter.
In a typical experiment, using a multichannel pipette, 40 µL
of dendrimer was mixed with 40 µL of buffer and 40 µL of
substrate in a Costar flat-bottom polystyrene 96-well-plate 150
histidine residues presumably sharing substrate binding
and catalytic functions, and was surrounded by outer
layers of aliphatic hydrophobic amino acids.
Dendrimers RG3 and HG3 both proved catalyti-
cally active, but they differed in the pH profile of sub-
strate binding. The KM value was pH-independent in the
case of RG3, as expected from a salt-bridge interaction
between the protonated arginine side chains and the
sulfonate group on substrate 1a/b. On the other hand,
KM increased with increasing pH in the case of HG3,
µL. The formation of 2 was followed by fluorescence emission reflecting the neutralization of the protonated histidines
using absorbance filter 450/50 and emission filter 530/25. The
gain was adjusted using the signal of the calibration curve
prior to every experiment (typically a signal 45000–55000 for
the 100 µM 2 well). The calibration curve (40 µL 2, 40 µL
buffer, and 40 µL H2O) and the blank (40 µL substrate, 40 µL
buffer, and 40 µL H2O) were recorded for every experiment
at the same time. The temperature inside the instrument was
adjusted to 34.0 °C. Kinetic experiments typically were fol-
lowed for 180 min. The data points were measured every 90 s.
Fluorescence data were converted to product concentration by
means of the calibration curve. Initial reaction rates were cal-
involved in a similar salt-bridge, resulting in weaker sub-
strate binding. Both dendrimers exhibited a comparable
increase in the catalytic rate constant upon increasing
pH. Overall, the strong substrate binding of RG3 at neu-
tral pH led to an increase in catalytic proficiency of this
dendrimer compared to acidic conditions, while HG3
did not show increased activity at that pH. Most interest-
ingly, the catalytic proficiency of RG3 depended on the
presence of the outer layer of aromatic amino acids, and
was thus much more active than its lower generation an-
culated from the steepest linear part observed in the curve that alogues RG0➔RG2. By contrast, HG3 showed catalytic
Javor and Reymond / SAR in Esterase Peptide Dendrimers

132
Fig. 1. Catalytic proficiencies of peptide dendrimers as a function of generation number, for the hydrolysis of substrate 1b. Con-
ditions: 3.3, 5.0, or 10 µM dendrimer, 30–1000 µM substrate 10 mM aq. Bis-Tris buffer pH 6.9, 34 °C. Data from ref 12.
parameters that were very similar to its lower generation effect of the non-catalytic aromatic amino acids was
analogues HG0➔HG2, and its outer layers of hydropho- indeed correct.
bic amino acids seemed not to play any significant role in
catalysis. The kinetic parameters with the butyrate ester G1 to the positive dendritic effect, we set out to investi-
1b are representative of these trends (Fig. 1). gate the kinetic parameters of all possible exchange mu-
To probe the contributions of the cationic residues at
The existing SAR data indicated that the positive tants in the form of RG3H, RMG3H for the arginine-to-
dendritic effect on catalysis was determined by the histidine exchanges in RG3 and RMG3, and HG3R and
nature of the G2 and G3 layers. However, it is also HMG3R for the histidine-to-arginine exchanges in HG3
conceivable that the positive effect observed in the R and HMG3, respectively. The four dendrimers were pre-
series on catalysis at pH 7 might depend on the pres- pared by solid-phase synthesis on tentagel TGR resin as
ence of arginine residues for strong substrate binding. previously described using Fmoc chemistry (Table 1).
In this case one would expect to see enhanced catalysis
All four dendrimers were catalytically active for the
for the HG3 and HMG3 dendrimers by exchanging the hydrolysis of acyloxypyrene trisulfonates 1a and 1b.
pair of histidine residues at G1 for a pair of arginine The kinetic parameters for the hydrolysis of these two
residues. On the other hand, combining the “R” series substrates were determined in aqueous buffer at pH 5.5
framework of RG3 and RMG3 for substrate binding and pH 6.9 (Fig. 2). The kinetic parameters of the four
with histidine residues at G1 for efficient catalysis analogues showed remarkable differences from the
might lead to enhanced catalytic activity if the assigned parent dendrimers obtained by activity screening. As
Table 1. Synthesis of mutant single-site esterase dendrimers
no.
sequence
yield (%)
m (mg)
MS calcd
MS obs.a
RG3H
(AcYT)8(BWG)4(BHS)2BHS
(AcYT)8(BWG)4(BHSG)2BHS
(AcIP)8(BIT)4(BRA)2BHL
(AcIP)8(BIT)4(BRAG)2BHL
0.7
0.3
7.0
2.7
1.9
1.0
18.4
7.1
4714.0
4828.1
4198.5
4312.5
4716.8
4830.4
4200.0
4312.8
RMG3H
HG3R
HMG3R
a(ES+) [M+H]+ peak. For the complete ion interpretation see the Supporting Information. B = branching unit (S)-2,3-diamino-
propanoic acid (Dap). Amino acids indicated with one letter code. All dendrimers were pure by analytical HPLC and gave the
expected signals by 1H NMR spectroscopy.
Israel Journal of Chemistry
49
2009

133
previously observed, the pH profile of substrate binding
was primarily determined by the nature of the cationic
residues at G1. Thus, the KM values for both substrates
approximately doubled between pH 5.5 and pH 6.9 for
analogues RG3H and RMG3H, reflecting weaker sub-
strate binding at higher pH due to neutralization of the
positive charge in the histidines, as observed with the
histidine-containing dendrimers HG3 and HMG3. On
the other hand the KM values of the arginine analogues
HG3R and HMG3R decreased by approximately 50%
between pH 5.5 and pH 6.9, similar to the trend ob-
served with RG3 and RMG3.
A
The most remarkable effect in the original series
concerned an increase by one order of magnitude in kcat
between pH 5.5 and pH 6.9 for the arginine-contain-
ing dendrimers RG3 and RMG3, which was assigned
to the neutralization of the core nucleophilic histidine.
However, none of the analogues showed a similar ef-
fect. Thus, the kcat values, at most, doubled between pH
5.5 and pH 6.9 in HG3R/HMG3R dendrimers, as well
as in the RG3H/RMG3H dendrimers, which is even
less than what was observed in the histidine-containing
dendrimers HG3/HMG3. Thus, exchanging the binding
residues from one dendrimer type to the other gave ana-
logues with decreased pH-dependence of catalysis.
Analysis of the catalytic proficiency kcat/KM, which
reflects the overall catalyst performance, showed that
none of the analogues surpassed the original dendrimers
RG3 and RMG3, which have the highest catalytic pro-
ficiency in the series at pH 6.9 for the butyrate substrate
1b. On the other hand, the introduction of histidine
residues in this dendrimer type in the form of RG3H
and RMG3H resulted in dendrimers retaining an ex-
cellent catalytic proficiency at pH 5.5, with values one
B
Fig. 2. Michaelis–Menten plot for dendrimers RG3H,
RMG3H, HG3R, HMG3R at A: pH 5.5 and B: pH 6.9, with
substrate 1b. Conditions as in Table 2.
Table 2. Kinetic parameters for ester hydrolysis. Conditions: 3.3, 5.0, or 10 µM catalyst, 30–1000 µM substrate 1a or 1b, 10 mM
aq. Bis-Tris buffer pH 6.9, or 5 mM citrate buffer pH 5.5, 34 °C. The formation of 2 was followed by fluorescence at λex
=
450 nm, λem = 530 nm. Under these conditions, the background rate is kuncat(1a, pH 5.5) = 2.4 × 10–5 min–1, kuncat(1a, pH 6.9) =
3.2 × 10–4 min–1, and kuncat(1b, pH 5.5) = 1.4 × 10–5 min–1; kuncat(1b, pH 6.9) = 1.3 × 10–4 min-1. The parameters are derived from
Lineweaver–Burk plots with 8 data points with r2 > 0.95. The error on kcat/KM is approximately ± 10–20% based on triplicate
measurements
100 ´ kcat (min–1)
citrate bis-tris
5.5 6.9
kcat/kuncat
citrate
5.5
KM (µM)
kcat/KM (M–1 min–1)
bis-tris
6.9
citrate
bis-tris
citrate
bis-tris
5.5
6.9
5.5
6.9
cpd
RG3
RG3H
RMG3
RMG3H
HG3
HG3R
HMG3
HMG3R
1a
1b
1a 1b
4.2 1.5
6.1 3.1
1.7 2.3
5.7 2.9
2.5 3.2
1.5 1.1
2.9 3.3
1.1 0.88
1a
1b
1a 1b
130 120
190 240
54 180
180 220
77 240
47 85
90 250
34 68
1a
1b
1a 1b
60 32
199 125
77 27
193 127
300 330
31 41
390 320
27 41
1a 1b
46 27
360 284 305 244
22 34 220 860
363 254 298 228
66 63
139 84
85 68
131 63
1a 1b
700 470
0.47 0.20
3.7 2.1
0.29 0.08
3.7 2.2
1.6 1.3
0.68 0.68
1.6 1.3
0.52 0.51
190 150
1540 1500
120 60
1540 1570
670 930
280 490
650 910
220 360
100 74
103 75
130 25
101 87
240 210
48
180 190
40 81
82 98
472 272
74 100
409 217
81
Javor and Reymond / SAR in Esterase Peptide Dendrimers

134
Table 3. Compaction factors C of proteins and denatured peptides and peptide dendrimers as
determined by diffusion NMR. The compaction factors were calculated from the hydrodynamic
radii as described in ref 14. All data are for ca. 1 mM aqueous solution (D2O)
peptide
RG3a
RG3H
RMG3a
RMG3H
HG3a
HG3R
HMG3a
HMG3R
no. of residues
radius (nm)
C
37
37
39
39
37
37
39
39
1.44
1.35
1.52
1.34
1.56
1.50
1.62
1.54
1.58
2.05
2.01
2.53
1.55
3.46
0.76
1.00
0.65
1.09
0.45
0.60
0.40
0.58
0.95
0.93
0.86
0.74
0.15
0.04
bovine pancreatic trypsin inhibitorb
hen lysozymeb
horse cytochrome c (NaCl-induced molten globule)b
sperm whale apomyoglobin pH 4 (molten globule)b
residues 2–38 from D3 of fibronectin-binding proteinb,c
hen lysozymeb,c
58
129
104
153
32
129
aData from ref 12. bData from ref 14. cDetermined under strong denaturing conditions.
order of magnitude higher than those of the parent den- (Table 3).13 This analysis has been used to character-
drimers RG3 and RMG3 and approximately twofold ize native and denatured proteins14 as well as various
higher than the other histidine-only dendrimers HG3 dendrimers.15 As in our previous study, the diffusion
and HMG3.
NMR data showed that all species were monomeric
The new dendrimer analogues RG3H and RMG3H in solution. The data indicated that dendrimers RG3H
featuring three histidine residues at the core attached and RMG3H have a significantly lower hydrodynamic
to the “R” framework with aromatic amino acids in the radius compared to their parent dendrimers RG3 and
outer layers showed the highest rate accelerations kcat/ RMG3, leading to a higher compaction factor, which
kuncat in the series, giving values of up to kcat/kuncat= 1500 is of comparable value to a fully folded protein, such
at pH 5.5, which is an order of magnitude higher than as bovine pancreatic inhibitor or hen lysozyme. How-
the parent “R” series dendrimers and 2–3-fold higher ever dendrimers HG3R and HMG3R were also more
than the “H” series dendrimers. The effect can be traced compact than the parent dendrimers HG3 and HMG3
back to the combination of efficient substrate binding as despite being catalytically less active, implying that cat-
in the “R” series dendrimers with a high catalytic rate alytic efficiency is not simply related to compactness.
constant at pH 5.5 as in the “H” series dendrimers. The
relatively modest rate accelerations kcat/kuncat observed
at pH 6.9 despite higher catalytic rate constants kcat are
conclusIon
caused by the fact that the uncatalyzed hydrolysis of the The series of peptide dendrimers described above cata-
substrate in buffer (kuncat) is approximately 5-fold faster lyzing the hydrolysis of acyloxypyrene trisulfonates
at the higher pH value.
in aqueous buffer using a single catalytic site at the
The enhanced catalytic efficiency of RG3H and dendrimer core were originally discovered by activity
RMG3H in terms of both rate acceleration kcat/kuncat and screening of a combinatorial library, which returned two
catalytic proficiency kcat/KM at pH 5.5 is remarkable. possible sequence motives for catalysis. The first den-
In our initial study the higher catalytic proficiency of drimer type combined a core catalytic histidine residue
RG3 and RMG3 was correlated to their higher com- at the core with a pair of cationic arginine residues for
paction factor, and we hypothesized that reaching a binding at G1 and two outer branches of aromatic amino
more protein-like dense packing might be beneficial for acids at G2 at G3. The second dendrimer type displayed
catalysis. To investigate this point, the hydrodynamic three histidine residues at the core and G1 for both
radii of the four dendrimers RG3H, RMG3H, HG3R, substrate binding and catalysis, combined with outer
and HMG3R were determined by diffusion NMR, and branches of mostly hydrophobic amino acids at G2 and
the values were used to compute the compaction factor G3. A strong positive dendritic effect on catalytic profi-
Israel Journal of Chemistry
49
2009

135
Catal. 2005, 347, 329–338. (e) Helms, B.; Fréchet,
J.M.J. Adv. Synth. Catal. 2006, 348, 1125–1148. Recent
reports: (f) Wang, Z.-J; Deng, G.-J.; Yong, L.; He, Y.-
M.; Tang, W.-J.; Fan, Q.-H. Org. Lett. 2007, 9, 1243–
1246. (g) Ouali, A.; Laurent, R.; Caminade, A.M.; Ma-
joral, J.-P.; Taillefer, M. J. Am. Chem. Soc. 2006, 128,
15990–15991. (h) van de Coevering, R.; Alfers, A.P.;
Meeldiijk, J.D.; Martinez-Viviente, E.; Pregosin, P.S.;
Klein Gebbink, R.J.M.; van Koten, G. J. Am. Chem.
Soc. 2006, 128, 12700–12713. (i)Abu-Reziq, R.;Alper,
H.; Wang, D.; Post, M.L. J. Am. Chem. Soc. 2006, 128,
5279–5282.
ciency was identified in the first dendrimer type, the “R”
series, and suggested that the outer layers of aromatic
residues contributed positively to catalysis.
In the present study, we exchanged the catalytic
machinery in these dendrimers by replacing the pair
of arginine residues useful for binding with a pair of
histidine residues, so as to obtain the same combina-
tion as that found in the second, “H” series dendrimers.
The exchange produced two new dendrimers, RG3H
and RMG3H, which display higher catalytic activity
at pH 5.5, with rate accelerations up to kcat/kuncat = 1500.
These new dendrimers also possess a lower hydrody-
namic radius as determined by diffusion NMR and a
higher compaction factor, which is similar to a folded
protein. By contrast, introducing arginine residues at
G1 in the “H” series dendrimers to form HG3R and
HMG3R gave generally less active dendrimers com-
pared to HG3 and HMG3.
While the catalytic efficiency of our single-site es-
terase dendrimers for hydrolyzing acyloxypyrene tri-
sulfonates is primarily determined by the nature of the
catalytic residues in the active site, their combination
with aromatic amino acids in the outer shells seems in-
strumental in obtaining enhanced catalytic efficiencies.
Future effort will address core active site dendrimers
with enhanced enzyme-like catalytic activities through
efficient high-throughput screening of new libraries.
The exploration of peptide dendrimers as synthetic en-
zyme models provides an uprecedented opportunity to
explore fundamental aspects of enzyme design.
(3) (a) Crespo, L.; Sanclimens, G.; Pons, M.; Giralt, E.;
Royo, M.; Albericio, F. Chem. Rev. 2005, 105, 1663–
1681. (b) Sadler, K.; Tam, J.P. Rev. Mol. Biotechnol.
2002, 90, 195–229. (c) Darbre, T.; Reymond, J.-L. Acc.
Chem. Res. 2006, 39, 925–934.
(4) (a) Kirby, A.J. Angew. Chem., Int. Ed. 1996, 35, 706–
724. (b) Woggon, W.–D. Acc. Chem. Res. 2005, 38,
127–136. (c) Motherwell, W.B.; Bingham, M.J.; Six,
Y. Tetrahedron 2001, 57, 4663–4686. (d) Breslow, R.;
Zhang, X.; Huang, Y. J. Am. Chem. Soc. 1997, 119,
4535–4536. (e) Breslow, R.; Huang, Y.; Zhang, X.;
Yang, J. Proc. Natl. Acad. Sci. USA 1997, 94, 11156–
11158.
(5) (a) Clouet, A.; Darbre, T.; Reymond, J.-L. Angew.
Chem., Int. Ed. 2004, 43, 4612-4615. (b) Clouet, A.;
Darbre, T.; Reymond, J.-L. Biopolymers 2006, 84, 114-
123.
(6) (a) Esposito, A.; Delort, E.; Lagnoux, D.; Djojo, F.;
Reymond, J.-L. Angew. Chem., Int. Ed. 2003, 42, 1381–
1383. (b) Lagnoux, D.; Delort, E.; Douat-Casassus, C.;
Esposito, A.; Reymond, J.-L. Chem. Eur. J. 2004, 10,
1215–1226. (c) Douat-Casassus, C.; Darbre, T.; Rey-
mond, J.-L. J. Am. Chem. Soc. 2004, 126, 7817–7826.
(d) Clouet, A.; Darbre, T.; Reymond, J.-L. Adv. Synth.
Catal. 2004, 346, 1195–1204.
Acknowledgments. This work was supported financially by the
University of Berne, the Swiss National Science Foundation,
and the Marie Curie Training Network IBAAC.
(7) Kofoed, J.; Darbre, T.; Reymond, J.-L. Org. Biomol.
Chem. 2006, 3268–3281.
REfEREncEs And notEs
(1) (a) Newkome, G.R.; Moorefield, C.N.; Vögtle, F. Den-
dritic Molecules: Concepts, Synthesis, Applications;
VCH: Weinheim, 2001. (b) Tomalia, D.A.; Dvornic,
P.R. Nature 1994, 372, 617–618. (c) Smith, D.K.; Die-
derich, F. Top. Curr. Chem. 2000, 210, 183–227. (d)
Helms, B.; Fréchet, J.M.J. Adv. Synth. Catal. 2006, 348,
1125–1148. (e) Smith, D.K.; Diederich, F. Chem. Eur.
J. 1998, 4, 1353–1361. (f) Liang, C.; Fréchet, J.M.J.
Prog. Polym. Sci. 2005, 30, 385–402. Grayson, S.M.;
Fréchet, J.M.J. Chem. Rev. 2001, 101, 3819–3868. (g)
Lee, C.C.; Mackay, J.A.; Fréchet, J.M.J.; Szoka, F.C.
Nat. Biotechnol. 2005, 23, 1517–1526.
(8) Sommer, P.; Uhlich, N.; Reymond, J.-L.; Darbre, T.
ChemBiochem 2008, 9, 689–693.
(9) (a) Kolomiets, E.; Johansson, E.M.V.; Renaudet, O.;
Darbre, T.; Reymond, J.-L. Org. Lett. 2007, 9, 1465–
1468. (b) Johansson, E.M.V.; Kolomiets, E.; Rosenau,
F.; Jäger, K.-E.; Darbre, T.; Reymond, J.-L. New J.
Chem. 2007, 31, 1291–1299. (c) Johansson, E.M.V.;
Crusz, S.A.; Kolomiets, E.; Buts, L.; Kadam, R.U.;
Cacciarini, M.; Bartels, K.-M.; Diggle, S.P.; Cámara,
M.; Williams, P.; Loris, R.; Nativi, C.; Rosenau, F.;
Jaeger, K.-E.; Darbre, T.; Reymond, J.-L. Chem. Biol.
2008, 15, 1249–1257.
(2) Reviews on catalysis with dendrimers: (a) Oosterom,
G.E.; Reek, J.N.; Kamer, P.C.; van Leeuwen, P.W. An-
gew. Chem., Int. Ed. 2001, 40, 1828–1849. (b) Twyman,
L.J.; King, A.S.; Martin, I.K. Chem. Soc. Rev. 2002,
31, 69–82. (c) Kofoed, J.; Reymond, J.-L. Curr. Opin.
Chem. Biol. 2005, 9, 656–664. (d) Astruc, D.; Heuze,
K.; Gatard, S.; Mery, D.; Nlate, S.; Plault, L. Adv. Synth.
(10) For early examples of catalytic polymers with imidaz-
ole groups: (a) Overberger, C.G.; Salamone, J.C. Acc.
Chem. Res. 1969, 2, 217–224. (b) Overberger, C.G.;
Mitra, S. Pure Appl. Chem. 1979, 51, 1391–1404. For
other examples and reviews: (c) Suh, J. Acc. Chem.
Res. 2003, 36, 562–570. (d) Benaglia, M.; Puglisi,
A.; Cozzi, F. Chem. Rev. 2003, 103, 3401–3429. (e)
Javor and Reymond / SAR in Esterase Peptide Dendrimers

136
Bosman, A.W.; Vestberg, R.; Heumann, A.; Fréchet,
J.M.J.; Hawker, C.J. J. Am. Chem. Soc. 2003, 123,
5908–5917.
R.L.; Audoly, L.; Cooper, D.; Weis, C.D.; Morris, K.;
Johnson, C.S.,Jr. Macromolecules 1993, 26, 2394–
2396. (b) Young, J.K.; Baker, G.R.; Newkome, G.R.;
Morris, K.F.; Johnson, C.S.,Jr. Macromolecules 1994,
27, 3464–3471. (c) Ihre, H.; Hult, A.; Söderlind, E. J.
Am. Chem. Soc. 1996, 118, 6388–6395. (d) Hecht, S.;
Vladimirov, N.; Fréchet, J.M.J. J. Am. Chem. Soc. 2001,
123, 18–25. (e) Jeong, S.W.; O’Brien, D.F.; Orädd, G.;
Lindblom, G. Langmuir 2002, 18, 1073–1076. (f) Frit-
zinger, B.; Scheler, U. Macromol. Chem. Phys. 2005,
206, 1288–1291. (g) Wong, S.; Appelhans, D.; Voit,
B.; Scheler, U. Macromolecules 2001, 34, 678–680.
(h) Ong, W.; Grindstaff, J.; Sobransingh, D.; Toba, R.;
Quintela, J.M.; Peinador, C.; Kaifer, A.E. J. Am. Chem.
Soc. 2005, 127, 3353–3361.
(11) (a) Delort, E.; Darbre, T.; Reymond, J.-L. J. Am. Chem.
Soc. 2004, 126, 15642–15643. (b) Delort, E.; Nguyen-
Trung, N.-Q.; Darbre, T.; Reymond, J.-L. J. Org. Chem.
2006, 71, 4468–4480.
(12) Javor, S.; Delort, E.; Darbre, T.; Reymond, J.-L. J. Am.
Chem. Soc. 2007, 129, 13238–13246.
(13) Cohen, Y.; Avram, L.; Frish, L. Angew. Chem., Int. Ed.
2005, 44, 520–554.
(14) Wilkins, D.K.; Grimshaw, S.B.; Receveur, V.; Dobson,
C.M.; Jones, J.A.; Smith, L.J. Biochemistry 1999, 38,
16424–16431.
(15) (a) Newkome, G.R.; Young, J.K.; Baker, G.R.; Potter,
Israel Journal of Chemistry
49
2009