A peptide dendrimer enzyme model with a single catalytic site at the core

Article abstract of DOI:10.1021/ja074115f

Catalytic esterase peptide dendrimers with a core active site were discovered by functional screening of a 65 536-member combinatorial library of third-generation peptide dendrimers using fluorogenic 1-acyloxypyrene-3,6,8- trisulfonates as substrates. In the best catalyst, RMG3, ((AcTyrThr) ₈(DapTrpGly)₄-(DapArgSerGly)₂DapHisSerNH ₂), ester hydrolysis is catalyzed by a single catalytic histidine residue at the dendrimer core. A pair of arginine residues in the first-generation branch assists substrate binding. The catalytic proficiency of dendrimer RMG3 (k_cat/K_M = 860 M^-1 min ^-1 at pH 6.9) per catalytic site is comparable to that of the multivalent esterase dendrimer A3 ((AcHisSer)₈(DapHisSer) ₄(DapHisSer)₂DapHisSerNH₂) which has fifteen histidines and five catalytic sites (Delort, E. et al. J. Am. Chem. Soc. 2004, 126, 15642-15643). Remarkably, catalysis in the single site dendrimer RMG3 is enhanced by the outer dendritic branches consisting of aromatic amino acids. These interactions take place in a relatively compact conformation similar to a molten globule protein as demonstrated by diffusion NMR. In another dendrimer, HG3 ((AcllePro)₈(DaplleThr)₄(DapHisAla) ₂DapHisLeuNH₂) by contrast, catalysis by a core of three histidine residues is unaffected by the outer dendritic layers. Dendrimer HG3 or its core HG1 exhibit comparable activity to the first-generation dendrimer A1 ((AcHisSer)₂DapHisSerNH₂). The compactness of dendrimer HG3 in solution is close to that a denatured peptide. These experiments document the first esterase peptide dendrimer enzyme models with a single catalytic site and suggest a possible relationship between packing and catalysis in these systems.

Full text of DOI:10.1021/ja074115f

Published on Web 10/09/2007
A Peptide Dendrimer Enzyme Model with a Single Catalytic
Site at the Core
Sacha Javor, Estelle Delort, Tamis Darbre,* and Jean-Louis Reymond*
Contribution from the Department of Chemistry and Biochemistry, UniVersity of Berne,
Freiestrasse 3, 3012 Berne, Switzerland
Received June 23, 2007; E-mail: jean-louis.reymond@ioc.unibe.ch
Abstract: Catalytic esterase peptide dendrimers with a core active site were discovered by functional
screening of a 65 536-member combinatorial library of third-generation peptide dendrimers using fluorogenic
1
(
-acyloxypyrene-3,6,8-trisulfonates as substrates. In the best catalyst, RMG3, ((AcTyrThr)
DapArgSerGly) DapHisSerNH ), ester hydrolysis is catalyzed by a single catalytic histidine residue at the
dendrimer core. A pair of arginine residues in the first-generation branch assists substrate binding. The
8 4
(DapTrpGly) -
2
2
) 860 M- min at pH 6.9) per catalytic site is comparable
1
-1
catalytic proficiency of dendrimer RMG3 (kcat/K
to that of the multivalent esterase dendrimer A3 ((AcHisSer)
which has fifteen histidines and five catalytic sites (Delort, E. et al. J. Am. Chem. Soc. 2004, 126, 15642-
5643). Remarkably, catalysis in the single site dendrimer RMG3 is enhanced by the outer dendritic branches
M
8
(DapHisSer) (DapHisSer) DapHisSerNH
4
2
2
)
1
consisting of aromatic amino acids. These interactions take place in a relatively compact conformation
similar to a molten globule protein as demonstrated by diffusion NMR. In another dendrimer, HG3
(
(AcIlePro)
residues is unaffected by the outer dendritic layers. Dendrimer HG3 or its core HG1 exhibit comparable
activity to the first-generation dendrimer A1 ((AcHisSer) DapHisSerNH ). The compactness of dendrimer
8 4 2 2
(DapIleThr) (DapHisAla) DapHisLeuNH ) by contrast, catalysis by a core of three histidine
2
2
HG3 in solution is close to that a denatured peptide. These experiments document the first esterase peptide
dendrimer enzyme models with a single catalytic site and suggest a possible relationship between packing
and catalysis in these systems.
7
Introduction
include cofactor engineering, the installation of cofactors or
8
catalytic metal complexes into noncatalytic proteins, and the
search for promiscuous enzyme activities by active site or
The catalytic function of enzymes arises through the relative
positioning of functional groups in space to create a catalytic
site. Designing enzymes de noVo is one of the most challenging
tasks in macromolecular chemistry. Most approaches to create
functional analogues of enzymes are based on modifying the
enzymes themselves or other proteins, for example by selection
for transition state analogue binding, by directed evolution,
or by computational protein design. Artificial enzymes may
also be formed by design or functional selection from folded
linear peptides or noncatalytic proteins. Additional strategies
9
substrate modifications.
The dendrimer approach to artificial enzymes follows a
different strategy, belonging to the general theme of synthetic
enzyme models, and proposes to organize building blocks into
a globular macromolecule by means of topology rather than by
folding. Most experiments toward catalytic dendrimers ad-
1
10
2
3
4
11
dressed the multivalent display of catalytic groups at the end
of the dendritic branches, which was found in several cases
5
6
1
12
to enhance catalysis by cooperative multivalency effects despite
steric crowding, following earlier reports in polymers with
multiple catalytic and binding groups investigated as hydrolase
mimics. In the context of catalysis in aqueous medium, we
recently reported strong enhancement of the catalytic potency
of histidine and N-terminal proline residues by multivalent
(
1) Toscano, M. D.; Woycechowsky, K. J.; Hilvert, D. Angew. Chem., Int.
Ed. 2007, 46, 3212-3236.
(
2) (a) Lerner, R. A.; Benkovic, S. J.; Schultz, P. G. Science 1991, 252, 659-
6
67. (b) Schultz, P. G.; Lerner, R. A. Acc. Chem. Res. 1993, 26, 391-395.
13
(
c) Schultz, P. G.; Lerner, R. A. Science 1995, 269, 1835-1842. (d)
MacBeath, G.; Hilvert, D. Chem. Biol. 1996, 3, 433-445. (e) Thomas, N.
R. Nat. Prod. Rep. 1996, 13, 479-511. (f) Stevenson, J. D.; Thomas, N.
R. Nat. Prod. Rep. 2000, 17, 535-537. (g) Keinan, E., Ed. Catalytic
Antibodies; Wiley-VCH: Weinheim, 2005.
(
3) (a) Reetz, M. T. Angew. Chem., Int. Ed. 2001, 40, 284-310. (b) Bloom, J.
D.; Meyer, M. M.; Meinhold, P.; Otey, C. R.; MacMillan, D.; Arnold, F.
H. Curr. Opin. Struct. Biol. 2005, 15, 447-452.
(7) (a) van de Velde, F.; Arends, I. W.; Sheldon, R. A. J. Inorg. Biochem.
2000, 80, 81-89. (b) Wahler, D.; Reymond, J.-L. Can. J. Chem. 2002, 80,
665-670.
(
4) (a) Bolon, D. N.; Voigt, C. A.; Mayo, S. L. Curr. Opin. Chem. Biol. 2002,
6, 125-129. (b) Kuhlman, B.; Baker, D. Curr. Opin. Struct. Biol. 2004,
14, 89-95. (c) Dwyer, M. A.; Hellinga, H. W. Curr. Opin. Struct. Biol.
2004, 14, 495-504. (d) Moffet, D. A.; Hecht, M. H. Chem. ReV. 2001,
101, 3191-3203.
(8) Thomas, C. M.; Ward, T. R. Chem. Soc. ReV. 2005, 34, 337-346.
(9) Hult, K.; Berglund, P. Curr. Opin. Biotechnol. 2003, 14, 395-400.
(10) (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. U.S.A.,
1997, 94, 11156-11158.
(
5) Berkessel, A. Curr. Opin. Chem. Biol. 2003, 7, 409-419.
(
6) (a) Qi, D.; Tann, C.-M.; Haring, D.; Distefano, M. D. Chem. ReV. 2001,
1
01, 3081-3112. (b) Andersson, L. K.; Caspersson, M.; Baltzer, L. Chem.
Eur. J. 2002, 8, 3687-3689.
1
3238 J. AM. CHEM. SOC. 2007, 129, 13238-13246
9
10.1021/ja074115f CCC: $37.00 © 2007 American Chemical Society

Peptide Dendrimer Enzyme Model
A R T I C L E S
Figure 1. Esterase peptide dendrimer RMG3 catalyzed hydrolysis of pyrene trisulfonate esters (green arrows indicate a possible general base mechanism;
red arrow indicates an alternative nucleophilic catalysis). The yellow circle highlights the reactive core with substrate.
complex or functional group placed at the dendritic core by
grafting dendritic branches around it, in the spirit of creating
dendritic enzyme mimics with a single catalytic site.1
2a-d
It is
indeed the hallmark of natural enzymes that the bulk of the
protein is able to enhance the efficiency of a few catalytic
residues placed at the active site not by providing additional
catalytic functionality but rather through indirect effects such
as directed substrate binding, preorganization of catalytic
residues, and polarization effects stabilizing the transition state.18
(
11) (a) Newkome, G. R.; Moorefield, C. N.; V o¨ gtle, F. Dendritic Molecules:
Concepts, Synthesis, Applications; VCH: Weinheim, 2001. (b) Tomalia,
D. A.; Dvornic, P. R. Nature 1994, 372, 617-618. (c) Smith, D. K.;
Diederich, F. Top. Curr. Chem. 2000, 210, 183-227. (d) Helms, B.; Fr e´ chet,
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.; Fr e´ chet, J.
M. J. Prog. Polym. Sci. 2005, 30, 385-402. Grayson, S. M.; Fr e´ chet, J.
M. J. Chem. ReV. 2001, 101, 3819-3868. (g) Lee, C. C.; Mackay, J. A.;
Fr e´ chet, J. M. J.; Szoka, F. C. Nat. Biotechnol. 2005, 23, 1517-1526.
12) Reviews on catalysis with dendrimers: (a) Oosterom, G. E.; Reek, J. N.;
Kamer, P. C.; van Leeuwen, P. W. Angew. 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. Catal. 2005, 347, 329-338. (e) Helms, B.; Fr e´ chet,
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.; Majoral, 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.
Figure 2. Combinatorial library of 65 536 peptide dendrimers. The
branching points consist of (S)-2,3-diaminopropanoic acid (Dap). All
N-terminal positions are acetylated. The solid support in the library is
tentagel with an ꢀ-aminohexanoylglycine spacer.
(
13) For early examples of catalytic polymers with imidazole groups: (a)
14
Overberger, C. G.; Salamone, J. C. Acc. Chem. Res. 1969, 2, 217-224.
display within peptide dendrimers for the catalysis of ester
(
b) Overberger, C. G.; Mitra, S. Pure Appl. Chem. 1979, 51, 1391-1404.
1
5,16
17
hydrolysis
and aldol reactions, respectively.
For other examples and reviews: (c) Suh, J. Acc. Chem. Res. 2003, 36,
5
62-570. (d) Benaglia, M. Puglisi.; Cozzi, F. Chem. ReV. 2003, 103, 3401-
429. (e) Bosman, A. W.; Vestberg, R.; Heumann, A.; Fr e´ chet, J. M. J.;
Other studies of catalytic dendrimers have addressed the
possible enhancement of the catalytic properties of a metal
3
Hawker, C. J. J. Am. Chem. Soc. 2003, 123, 5908-5917.
J. AM. CHEM. SOC.
9
VOL. 129, NO. 43, 2007 13239

A R T I C L E S
Javor et al.
Figure 3. pH-profile of catalytic parameter for dendrimer RG3 (b) and HG3 (2) catalyzed hydrolysis of butyrate 1b. A: Catalytic rate constant kcat. B:
Michaelis constant KM. Conditions: 5.0 µM dendrimer, 29-1000 µM 1b, 10 mM Bis-Tris buffer, 34 °C.
Table 1. Peptide Dendrimer Sequences Identified by Amino Acid Analyses of Active Beads from Selection Assay on Substrate 1ba
no.
intensityb
X8
X7
X6
X5
X4
X3
X2
X1
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
++
++
++
++
+
I
I
I
I
I
I
Y
Y
Y
Y
I
I
I
Y
I
Y
F
P
T
T
F
T
T
T
P
F
T
F
T
G
P
T
W
I
I
W
W
Y
I
G
T
P
H
H
C
R
H
R
R
R
H
R
H
H
H
R
R
R
A
A
S
A
A
S
S
L
S
D
A
L
A
S
R
C
R
C
R
C
C
C
H
R
H
H
C
C
V
H
A
S
L
S
A
L
S
L
L
L
A
L
L
S
S
S
P
G
G
T
F
G
G
T
G
T
T
F
+
++
+
I
++
++
++
+
Y
Y
Y
Y
Y
Y
I
0
1
2
3
4
5
++
+
+
L
S
6 (RG3)
+++
W
G
a
Conditions: 45 mg batch of on-bead library swollen in 500 µL Bis-Tris pH 6.0 (20 mM) for 40 min, then mixed with 500 µL of substrate 1b (80 µM
in Bis-Tris pH 6.0) for 8 min, then spread on a silica plate, UV irradiation 365 nm. b + weak fluorescence, ++ strong fluorescence, +++ very strong
fluorescence.
To date there are only very few examples where addition of
dendritic branches to a catalytic core resulted in significant
increases in rates or selectivity. These include catalysis of
transamination by a dendritic pyridoxal cofactor,19 the catalysis
been shown to catalyze the dehydrohalogenation of 2-iodo-2-
2
2
methylheptane with high turnover. In each case the rate
enhancement effect was attributed to the effect of the nanoen-
vironment on substrate binding and/or transition state stabiliza-
tion.
20
of singlet oxygen cycloaddition by a dendritic benzophenone,
and catalysis of transesterification by a dendritic 4-pyrrolidi-
Herein we report the first catalytic peptide dendrimers with
a single catalytic site at the core as an example of an ester
hydrolysis reaction. These dendrimers were discovered by
activity screening of combinatorial dendrimer libraries using
our recently reported method for the synthesis and decoding of
nopyridine.21 Unimolecular dendritic reverse micelles have also
(
14) (a) Crespo, L.; Sanclimens, G.; Pons, M.; Giralt, E.; Royo, M.; Albericio,
F. Chem. ReV. 2005, 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.
2
3
such libraries. We have used this approach previously to
(
15) (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-
2
3
17
discover multivalent esterase and aldolase dendrimers, as
24
well as multivalent lectin binding glycodendrimers. The library
used here to discover single site catalytic dendrimers features
catalytic amino acids at the core and noncatalytic residues in
the outer shells to provide a favorable protein-like environment.
1
226. (c) Douat-Casassus, C.; Darbre, T.; Reymond, 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.
(16) (a) Delort, E.; Darbre, T.; Reymond, J.-L. J. Am. Chem. Soc. 2004, 126,
1
5642-15643. (b) Delort, E.; Nguyen-Trung, N.-Q.; Darbre, T.; Reymond,
J.-L. J. Org. Chem. 2006, 71, 4468-4480.
(
17) Kofoed, J.; Darbre, T.; Reymond, J.-L. Org. Biomol. Chem. 2006, 3268-
(22) Piotti, M. E.; Rivera, F., Jr.; Bond, R.; Hawker, C. J.; Fr e´ chet, J. M. J. J.
Am. Chem. Soc. 1999, 121, 9471-9472.
3
281.
(18) (a) Warshel, A.; Florian, J. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 5950-
(23) (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.
5
955. (b) Bruice, T. C. Chem. ReV. 2006, 106, 3119-3139. (c) Benkovic,
S. J.; Hammes-Schiffer, S. Science 2003, 301, 1196-1202.
(
(
(
19) Liu, L.; Breslow, R. J. Am. Chem. Soc. 2003, 125, 12110-12111.
20) Hecht, S.; Fr e´ chet, J. M. J. J. Am. Chem. Soc. 2001, 123, 6959-6960.
21) Liang, C. O.; Helms, B.; Craig, J.; Frechet, J. M. J. Chem. Commun. 2003,
(24) (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.; J a¨ ger, K.-E.; Darbre, T.; Reymond, J.-L. New J. Chem.
2007, 31, 1291-1299.
2
524-2525
13240 J. AM. CHEM. SOC.
9
VOL. 129, NO. 43, 2007

Peptide Dendrimer Enzyme Model
A R T I C L E S
Figure 4. Space filling model of a minimized energy conformation of dendrimer RG3 (left) and RMG3 (right). The pyrene trisulfonate substrate 1b was
placed manually on the surface. The arginine and histidine residues are shown in stick model and the dendrimer in molecular surface representation colored
by atom type (green: carbon; white: hydrogen; red: oxygen; blue: nitrogen). The image was generated from coordinates produced by GROMACS using
2
9
PyMol. Energy-minimized conformations were computed at pH 7 in water (SPC model) as explicit solvent by gradual cooling from 800 to 300 K over 180
ps. Similar relative arrangement of histidine and arginine side chains, compatible with the proposed mechanism, were observed in the energy-minimized
conformations at 300 K generated from 20 different starting conformations sampled at 800 K.
Figure 5. Esterase dendrimers and analogues. The branching points consist of (S)-2,3-diaminopropanoic acid (Dap). All sequences are acetylated at the
N-termini and have their C-terminus as amide (CONH2).
The dendrimers catalyze the hydrolysis of 1-acyloxypyrene-
,6,8-trisulfonates 1a/b in aqueous buffer. In the best catalyst
per catalytic site in our previously reported multivalent esterase
1
6a
3
dendrimer system.
Dendrimer RMG3 adopts a globular
identified, RMG3 catalysis by the dendritic core is enhanced
by the outer layers of aromatic amino acids (Figure 1). The
reactivity enhancement effect on the catalytic histidine residue
in this single-site system is comparable to the effect observed
conformation in solution with packing similar to a molten
globule protein as demonstrated by diffusion NMR measure-
ments. A second catalytic dendrimer, HG3, featuring a hydro-
phobic aliphatic outer layer shows no catalysis enhancement
J. AM. CHEM. SOC.
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VOL. 129, NO. 43, 2007 13241

A R T I C L E S
Javor et al.
X1
Table 2. Peptide Dendrimer Sequences Identified by Amino Acid Analysis of Active Beads from Selection Assay on Substrate 1da
no.
intensityb
X8
X7
X6
X5
X4
X3
X2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
+++
+++
++
+
Y
W
Y
Y
I
Y
Y
I
T
F
P
T
P
F
F
T
P
F
T
P
T
T
P
I
I
I
I
I
I
I
Y
I
I
I
W
W
I
T
T
T
T
T
T
T
T
T
T
G
T
T
T
T
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
A
A
A
A
A
S
H
H
V
V
H
H
V
H
H
R
H
H
H
H
H
A
L
S
S
S
L
S
L
L
S
L
S
L
S
A
+
+
+
S
++
++
+
A
A
S
A
A
A
A
A
(HG3)
0
I
Y
Y
I
I
Y
I
1
2
3
4
5
+
+++
++
+
+
I
a
Conditions: Same as in Table 1 using substrate 1d. b + weak fluorescence, ++ strong fluorescence, +++ very strong fluorescence.
Table 3. Synthesis of Core Active Site Esterase Dendrimers and Analogues
no.
sequence
yield (%)
m (mg)
MS calcd
MS obsda
R-G0
R-G1
R-G2
R-G3
RMG0
RM-G1
RM-G2
RM-G3
H-G0
H-G0′
H-G1′
H-G1
H-G2
H-G3
HM-G1
HM-G2
HM-G3
AcRSAHS
(AcRS)2BHS
(AcWG)4(BRS)2BHS
(AcYT)8(BWG)4(BRS)2BHS
AcRSGAHS
2.2
27.5
5.4
2.9
8.5
3.3
598.3
898.5
2127.0
4752.1
655.3
1012.5
2241.0
4866.1
589.3
476.2
788.4
854.4
1967.1
4160.4
968.5
2081.1
4274.5
598.6
897.4
2126.8
4754.4
655.4
1012.8
2241.5
4868.7
589.4
476.4
788.6
854.6
1967.4
4162.2
968.6
2081.4
4277.4
1.7
5.5
50.6
51.9
5.2
70.4
40.4
7.7
(AcRSG)2BHS
(AcWG)4(BRSG)2BHS
(AcYT)8(BWG)4(BRSG)2BHS
AcHAAHL
AcHAAH
(AcHA)2BAL
7.3
24.8
78.8
67.3
73.3
40.4
10.2
46.7
40.1
35.5
28.6
61.4
60.9
45.9
58.7
7.7
16.0
53.2
25.5
9.5
(AcHA)2BHL
(AcIT)4(BHA)2BHL
(AcIP)8(BIT)4(BHA)2BHL
(AcHAG)2BHL
(AcIT)4(BHAG)2BHL
(AcIP)8(BIT)4(BHAG)2BHL
a
+
(
ES+) [M + H] peak. For the complete ion interpretation, see Supporting Information. B ) branching unit (S)-2,3-diaminopropanoic acid (Dap).
Amino acids indicated with one-letter code.
by the outer layers, and a less compact conformation closer to
a denatured peptide.
hydrophobic residues were distributed evenly. The library was
prepared on a 400 mg batch of tentagel resin and capped by
acetylation after removal of the last Fmoc protecting group. The
quality of the library was checked by submitting randomly
picked beads to amino acid analysis, which gave the expected
average distribution of amino acids at variable positions.
Results and Discussion
To search for a core-active site peptide dendrimer enzyme
model, we focused our attention on the hydrolysis of 1-acy-
loxypyrene-3,6,8-trisulfonates 1a-d, which is a favorable model
reaction for the study of aqueous esterolysis. These fluorogenic
pyrene substrates render direct on-bead screening possible and
facilitate kinetic studies. Furthermore, the reaction is catalyzed
by 4-methylimidazole and therefore should require only a single
histidine side chain for catalysis. Nevertheless, dendrimers with
a single catalytic histidine at their core, such as (Ac-Ala-Ser)8-
The peptide dendrimer library was first screened with the
fluorogenic butyrate ester 1b by soaking the beads with an
aqueous buffered solution of the substrate and plating out on a
silica gel plate. Strongly green fluorescent beads appeared after
∼30 min incubation on the plates and were picked for
sequencing. Sequence assignment from amino acid analysis was
successful in approximately 80% of the analyzed beads (Table
1). The sequences displayed at least one histidine or arginine
(
Dap-Ala-Ser)4(Dap-Ala-Ser)2Dap-His-Ser-NH2, showed no de-
16b
2
4
tectable activity for this reaction, prompting an alternative
exploratory approach based on combinatorial chemistry to
identify favorable sequences.
residue at position X and X , combined with predominantly
6
8
aromatic residues at the outer positions X -X . Dendrimer
sequences with one or two arginines but lacking the critical
catalytic histidine residue probably showed positive in the assay
due to product binding. On the other hand, sequences with at
least one histidine residue presumably indicated catalytically
active peptide dendrimers. The library was also screened against
the undecanoyl ester 1d to probe the possibility of lipase-like
activity in the dendrimers. In this case, sequencing of fluorescent
beads in the assay gave a consensus for dendrimers bearing three
Library Design, Synthesis, and Screening. A 65 536-
member combinatorial library of third-generation peptide den-
drimers was prepared following our combinatorial approach to
23
peptide dendrimers. Variable amino acids at the core positions
1
-4
X
included nucleophilic (His, Cys) and cationic (Arg)
residues for binding and catalysis (Figure 2). Aromatic residues
5-8
(
Tyr, Phe, Trp) were placed in the outer positions X to assist
1
3
substrate binding. Small and polar, negatively charged, and
histidine residues at positions X and X , combined with
13242 J. AM. CHEM. SOC.
9
VOL. 129, NO. 43, 2007

Peptide Dendrimer Enzyme Model
A R T I C L E S
Table 4. Kinetic Parameters for Ester Hydrolysisa
cat (min-1)
k
cat/kuncat
K
(µ
M)
k
cat/K
(M- min-1)
1
100
×
k
M
M
citrate 5.5
Bis-Tris 6.9
citrate 5.5
1b
Bis-Tris 6.9
1a 1b
citrate 5.5
1a 1b
Bis-Tris 6.9
1a 1b
citrate 5.5
Bis-Tris 6.9
1a 1b
compd
1a
1b
1a
1b
1a
1a
1b
R-G0
R-G1
R-G2
R-G3
RM-G0
RM-G1
RM-G2
RM-G3
H-G0
H-G0
H-G1
H-G1
H-G2
1.1
5.8
1.2
0.47
1.3
1.1
0.83
0.29
1.6
1.3
1.7
8.1
3.0
1.6
2.0
1.3
1.6
0.8
4.6
0.84
6.7
2.1
4.2
2.4
1.9
2.0
1.7
-
-
3.8
8.6
2.6
2.5
2.7
2.5
2.9
-
440
2400
510
190
550
460
340
120
660
540
680
3300
1200
670
840
540
650
600
3300
570
150
550
500
430
60
970
720
960
4600
960
930
1100
700
910
2200
17000
26
210
66
130
75
60
63
54
130
60
120
270
81
77
83
-
580
410
83
100
1250
300
69
130
450
530
550
405
160
240
300
59
720
560
110
74
910
250
96
2200
220
60
60
290
86
61
77
-
-
790
490
150
300
380
150
390
-
18
140
150
46
11
37
120
22
35
25
30
200
180
66
69
220
85
12
81
70
27
8.3
28
62
34
22
16
20
150
160
63
50
200
68
3.8
1.2
210
70
470
53
150
260
860
4.3
2.5
28
170
99
-
-
150
120
160
140
210
180
39
-
110
32
310
350
700
82
220
330
220
4.5
2.6
48
170
180
82
0.79
0.20
0.76
0.69
0.60
0.08
1.4
1.0
1.3
6.4
1.3
1.3
1.5
1.0
1.3
3.1
24
1.9
1.5
2.1
1.8
2.7
2.3
0.5
0.036
3.2
22
4.7
3.2
4.1
6.0
3.3
1.5
390
120
104
27
25
600
620
660
440
82
210
300
50
1200
140
1200
1300
470
330
750
760
320
360
63
2.7
250
1700
360
240
310
460
250
120
2000
H-G3
98
54
79
100
42
HM-G1
HM-G2
HM-G3
71
170
74
77
90
180
190
450
63
b
A1
69
3800
b
A3
26
4100
a
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 ×
-
5
-1
-4
-1
-5
-1
-4
-1 b
1
0
min , kuncat (1a, pH 6.9) ) 3.2 × 10 min , and kuncat (1b, pH 5.5) ) 1.4 × 10 min ; kuncat (1b, pH 6.9) ) 1.3 × 10 min
. Data for A1 and
A3 from ref 16. Dendrimer A1: (AcHisSer)2(DapHisSerNH2), Dendrimer A3: (AcHisSer)8(DapHisSer)4(DapHisSer)2DapHisSerNH2. The parameters are
derived from Lineweaver-Burk plots with eight data points with r > 0.95. The corresponding plots are given in the SI. The error on kcat/KM is approximately
10-20% based on triplicate measurements.
2
(
Table 5. Compaction Factors C of Proteins and Denatured Peptides and Peptide Dendrimers As Determined by Diffusion NMR
peptide
no. of residues
radius (nm)
C
R-G3
RM-G3
H-G3
37
39
37
39
58
129
104
153
32
1.44
1.52
1.56
1.62
1.58
2.05
2.01
2.53
1.55
3.46
0.76
0.65
0.45
0.40
0.95
0.93
0.86
0.74
0.15
0.04
HM-G3
b
bovine pancreatic trypsin inhibitor
hen lysozymeb
b
horse cytochrome c (NaCl-induced molten globule)
sperm whale apomyoglobin pH 4 (molten globule)
residues 2-38 from D3 of fibronectin binding protein
hen lysozyme
b
b,c
b,c
129
a
b
The compaction factors were calculated from the hydrodynamic radii as described in ref 31. All data are for ca.1 mM aqueous solution (D2O). Data
c
from ref 31. Determined under strong denaturing conditions.
6
8
predominantly hydrophobic residues at positions X -X and a
Both dendrimers catalyzed the hydrolysis of 1a/b with
saturation kinetics and multiple turnover. The pH-rate profile
of RG3 in the accessible range pH 5.5-7.5 showed a pH-
independent KM and a pH-dependent increase in kcat (Figure
5
threonine residue at position X . All sequences of active beads
displayed at least one histidine residue, as expected from the
requirement for esterolytic activity with the substrates.
3
). This pH-dependence is consistent with a guanidinium-
Synthesis and Characterization of Library Hits. Dendrim-
ers (AcTyrThr)8(DapTrpGly)4 (DapArgSer)2DapHisSer (RG3)
2
5
sulfonate salt bridge for substrate binding and the histidine
side-chain as a free base triggering catalysis. Similar arginine-
sulfonate interactions have been proposed in the binding of
2
4
with a single histidine at X and two arginines at X , and
AcIlePro)8(DapIleThr)4(DapHisAla)2DapHisLeu (HG3) with
(
2
6
2
4
coenzyme M to hydrogenases and the binding of the hapten
three histidines at X -X , were resynthesized as representative
members of the screening results with substrates 1b and 1d,
respectively. The syntheses were carried out on Rink-amide
resin. The dendrimers were deprotected, cleaved from the resin,
purified by preparative HPLC, and investigated as soluble
dendrimers in aqueous buffer. The dendrimers catalyzed the
hydrolysis of substrates 1a-d; however, the activity was very
low with the long chain ester substrates 1c and 1d. The low
activity of dendrimer HG3 with 1d contrasts with the activity
observed during screening and might be caused by the much
lower dendrimer concentration in this assay compared to the
situation on the beads. The dendrimers were therefore further
studied using the more reactive substrates 1a and 1b.
2
7
naphthalene-1,5-disulfonate to an antibody. In the case of
HG3, the pH-rate profile showed that both KM and kcat
increased with pH. This suggests that the three histidines control
both binding and catalysis in this dendrimer.
For both dendrimers the mechanism of esterolysis probably
involves binding of the sulfonate groups of the substrate to the
pair of arginine or protonated histidine residues at position X ,
positioning the reacting acyl group in the substrate close to a
histidine side chain at position X which catalyzes hydrolysis
4
2
(25) Schug, K. A.; Lindner, W. Chem. ReV. 2005, 105, 67-113.
(26) Clark, D. D.; Boyd, J. M.; Ensign, S. A. Biochemistry 2004, 43, 6763-
6771.
(27) Hotta, K.; Wilson, I. A.; Hilvert, D. Biochemistry 2002, 41, 772-779.
J. AM. CHEM. SOC.
9
VOL. 129, NO. 43, 2007 13243

A R T I C L E S
Javor et al.
were observed with the third-generation dendrimer RG3 and
its analogue RMG3 at pH 6.9. Under these conditions the single
catalytic histidine at the core is mostly present as free base while
the pair of arginine residues are positively charged and probably
assist in substrate binding. The catalytic proficiency however
decreased in the lower generation dendrimers RG2/RG1 and
RMG2/RMG1 which also contain these three residues in the
same relative arrangement and was even lower in the linear core
analogues RG0 and RMG0. The positive effect of the outer
dendritic layers on catalytic proficiency kcat/KM was particularly
strong with the butyrate substrate 1b in the series RG0fRG3,
in which the third-generation dendrimer RG3 was 390-fold more
active than its linear core analogue RG0. In both series the outer
dendritic layers enhanced catalysis by an increase in substrate
binding (lower KM) at approximately constant kcat.
The positive effect of the outer dendritic layers on catalysis
in the R-series dendrimers at pH 6.9 was not observed at pH
5
.5, under which conditions the best catalysis was observed at
the level of the first-generation dendrimers RG1 and RMG1.
Although addition of the outer denritic layers caused an increase
in substrate binding comparable to that observed at pH 6.9, the
catalytic rate constant kcat decreased strongly, in particular upon
addition of the third layer to form RG3 and RMG3. The outer
dendritic layers in the R-series dendrimers consist of a 4-fold
tryptophan-glycine dyad at G2 and an 8-fold tyrosine-threonine
dyad at G3. The increase in substrate binding observed upon
addition of these layers might be triggered by enhanced
hydrophobic substrate-dendrimer interactions. The hydrophobic
nature of the amino acids in G2 and G3 might also explain the
low kcat values observed at pH 5.5, considering that the ester
hydrolysis reaction is favored in a highly polar medium. Indeed
several previous attempts to combine aromatic residues with
histidines in multivalent peptide dendrimer esterases did not
Figure 6. Michaelis-Menten plot for dendrimers RG1, RG2, RG3 at pH
6
.9 with substrate 1b. Conditions as in Table 4.
of the acyl group as a nucleophile or general base (Figure 1).
Molecular dynamics simulations using GROMACS2 for den-
drimer RG3 and its analogue RMG3 (see below) suggest that
although the dendrimers adopt multiple conformations in
aqueous solution, the pair of arginine residues and the catalytic
histidine should be found mostly at the dendrimer surface in a
relative orientation compatible with recognition and esterolysis
of the pyrene trisulfonate substrates according to the proposed
mechanism (Figure 4).
8
Synthesis of Dendrimer Analogues. The role of the dendritic
structure on catalysis by RG3 and HG3 was investigated by
gradual stripping of the outer branches to form the second-
generation dendrimers RG2 and HG2, the dendritic catalytic
core RG1, HG1, and HG1′ (an analogue of HG1 with an
1
5b
produce active catalysts. The positive effect of the aromatic
layers G2 and G3 on catalytic proficiency at pH 6.9 is therefore
particularly remarkable and might reflect the onset of a
catalytically favorable conformation upon neutralization of the
histidine side-chain at the higher pH value.
2
HisfAla exchange at position X ), and finally the linear core
peptides RG0, HG0, and HG0′ (an analogue of HG0 with
1
deletion of Leu at position X ) (Figure 5). The spacing between
2
the core histidine residue at position X , presumably responsible
Effects of the Dendritic Layers in the H-Series Dendrim-
ers. The exceptional nature of the positive dendritic effect
observed by addition of the noncatalytic outer layers on the
catalytic core in the R-series was further highlighted by the
catalytic behavior of the H-series dendrimers featuring a three-
histidine catalytic core surrounded by outer layers of hydro-
phobic amino acids and proline (G2 ) 4 × Ile-Thr, G3 ) 8 ×
Ile-Pro). In this series the best catalysts were the first-generation
dendrimers HG1/HMG1 at pH 5.5 and the second-generation
dendrimers HG2/HMG2 at pH 6.9. Addition of the third
dendritic layer either had a negligible effect (substrate 1b at
pH 6.9) or reduced catalytic proficiency (1a at pH 6.9 and 1a/b
at pH 5.5) by influencing both the catalytic rate constant (lower
kcat) and substrate binding (higher KM).
for nucleophilic catalysis, and the pair of arginine or histidine
residues at position X that probably participate in substrate
4
binding to the sulfonate groups, might also be expected to
influence catalysis. To investigate this point, we prepared two
related series RMG0fRMG3 and HMG0fHMG3 with an
additional glycine residue after the first branching. All den-
drimers and peptides were prepared by solid-phase peptide
synthesis and obtained in satisfactory purity after preparative
HPLC purification (Table 3).
Positive Dendritic Effect in Catalysis by the R-Series Core
Active Site Dendrimers. Michaelis-Menten parameters were
determined for all dendrimers and lower generation analogues
for substrates 1a and 1b in aqueous buffers at pH 5.5 and pH
6
.9 (Table 4, Figure 6 and 7). The catalytic effects observed in
Single-Site versus Multivalent Esterase Dendrimers. The
most active core active site dendrimer catalysts as measured
by the catalytic proficiency kcat/KM were the third-generation
dendrimers RG3 with substrate 1a and RMG3 with substrate
terms of rate acceleration kcat/kuncat and catalytic proficiency kcat/
KM were generally higher at the higher pH value, in agreement
with the pH-rate profile analysis. The strongest catalytic effects
1
M
b. The catalytic proficiency RMG3 at pH 6.9 (kcat/KM ) 820
(
28) (a) Berendsen, H. J. C.; van der Spoel, D.; van Drunen, R. Comput. Phys.
Commun. 1995, 91, 43-56. (b) Lindahl, E.; Hess, B.; van der Spoel, D. J.
Mol. Model. 2001, 7, 306-317.
-1
-1
min , Table 4) is 5-fold lower than our previously reported
multivalent esterase dendrimer A3 ((AcHisSer)8(DapHisSer)4-
(DapHisSer)2DapHisSerNH2), which displayed 15 catalytic
(29) DeLano, W. L. The PyMOL Molecular Graphics System (2002) DeLano
Scientific, San Carlos, CA. http://www.pymol.org.
13244 J. AM. CHEM. SOC.
9
VOL. 129, NO. 43, 2007

Peptide Dendrimer Enzyme Model
A R T I C L E S
Figure 7. Catalytic proficiency kcat/KM of peptide dendrimers with acetate ester substrate 1a and butyrate ester substrate 1b at pH 5.5 and pH 6.9. Data from
-
1
-1
-1
-1
Table 4. The second-order rate constant for catalysis by 4-methylimidazole is k2 (1a) ) 11 M min and k2 (1b) ) 8.6 M min (pH 6.9).
dendrimers show higher rate acceleration over background at
this lower pH value.
Diffusion-NMR. The very different behavior of the R- and
RM-series versus the H- and HM-series dendrimers is in part
due to the difference between their catalytic cores. On the other
hand, the very different nature of their outer layers reflecting
different selections during the combinatorial experiment might
also cause a different structural behavior. To test this hypothesis,
the dendrimers were investigated by diffusion NMR spectros-
3
0
copy. This analysis allows one to determine the average
hydrodynamic radius of macromolecules in solution and has
31
been used to characterize native and denatured proteins as well
3
2
as various dendrimers. The dendrimers were investigated as
-
1
5
mg mL solutions in D2O (>1 mM). All dendrimers were
fully soluble under these conditions, and the diffusion NMR
data showed that all species were monomeric in solution.33 The
increase in hydrodynamic radius as a function of molecular
weight for the series RG1fRG3 and RMG1fRMG3 was
comparable to that reported for PAMAM dendrimers. By
contrast, the hydrodynamic radius increased much faster with
MW in the series HG1fHG3 and HMG1fHMG3 (Figure
Figure 8. Hydrodynamic radii (Rh) measured by diffusion NMR spec-
troscopy as a function of dendrimer molecular weight. (2) RG1-3; (4)
RMG1-3; (9) HG1-3; (0) HMG1-3. The Rh were determined from
-
1
the diffusion coefficients D at 5 mg mL peptide dendrimer in D2O at
00 K. The estimated error on Rh is (2.6%; see Experimental Section and
SI for details. (×) PAMAM dendrimers measured in MeOH (data from ref
2a).
3
8
). With 37-39 residues, the third-generation dendrimers can
also be compared to proteins, whose hydrodynamic radius as
3
(
(
(
30) Cohen, Y.; Avram, L.; Frish, L. Angew. Chem., Int. Ed. 2005, 44, 520-
554.
histidines and acquired a high catalytic efficiency by cooper-
ativity effects between these residues forming five active sites.
31) Wilkins, D. K.; Grimshaw, S. B.; Receveur, V.; Dobson, C. M.; Jones, J.
A.; Smith, L. J. Biochemistry 1999, 38, 16424-16431.
16
32) (a) Newkome, G. R.; Young, J. K.; Baker, G. R.; Potter, 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.
Considering that the single site dendrimer RMG3 features only
a single histidine residue, its catalytic activity is thus comparable
to that of dendrimer A3 when counted per catalytic site. On
the other hand, lowering of the pH from 6.9 to 5.5 reduced the
catalytic proficiency of RMG3 by protonation of the catalytic
histidine, which lowers kcat at constant KM but had almost no
influence on the catalytic proficiency of the multivalent histidine
dendrimer A3, its core analogue A1, or dendrimers in the
H-series, which all feature three histidine residues in a similar
relative arrangement. As a consequence, these polyhistidine
(
c) Ihre, H.; Hult, A.; S o¨ derlind, E. J. Am. Chem. Soc. 1996, 118, 6388-
6
395. (d) Hecht, S.; Vladimirov, N.; Fr e´ chet, J. M. J. J. Am. Chem. Soc.
2
001, 123, 18-25. (e) Jeong, S. W.; O’Brien, D. F.; Or a¨ dd, G.; Lindblom,
G. Langmuir 2002, 18, 1073-1076. (f) Fritzinger, 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.
(
33) Addition of hydroxypyrene-trisulfonates to solutions of RG3 and RMG3
lead to precipitation precluding the determination of hydrodynamic radii
for the dendrimer-substrate complexes. Precipitation was also observed
upon attempted microcalorimetric measurements with these dendrimers.
J. AM. CHEM. SOC.
9
VOL. 129, NO. 43, 2007 13245

A R T I C L E S
Javor et al.
determined by NMR is larger in the denatured state than in the
native state. Analysis in terms of the compaction factor C, as
defined by Wilkins et al.,31 shows that RG3 and RMG3 have
a compaction factor typical for a molten globule state of proteins,
while HG3 and HMG3 have a lower compaction factor
suggesting a more disordered conformation (Table 5). The
relatively tight packing of the RG3/RMG3 dendrimers might
be related to the catalytically productive effects of the outer
dendritic layers on catalysis observed at neutral pH.
with 40 µL of buffer and 40 µL of substrate in a Costar flat-bottom
polystyrene 96-well-plate (150 µL). The formation of 2 was followed
by fluorescence emission 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 45 000-55 000 for
the 100 µM 2 well). The calibration curve (40 µL of 2, 40 µL of buffer,
and 40 µL of H
and 40 µL of H
2
O) and the blank (40 µL of substrate, 40 µL of buffer,
O) were recorded for every experiment in the same
2
time. The temperature inside the instrument was adjusted to 34.0 °C.
Kinetic experiments were followed for typically 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 calculated from the steepest linear part observed in the curve
that gives fluorescence Versus time, typically between 500 and 2000 s,
corresponding to less than 10% conversion.
Measurement of Apparent Rate Enhancements and Kinetic
M
Parameters kcat and K . Vcat is the apparent rate in the presence
dendrimer catalyst; Vun is the rate in buffer alone. The observed rate
enhancement is defined as Vnet/Vun with Vnet ) Vcat - Vun. Michaelis-
Conclusion
In summary, the first core active site esterase peptide
dendrimers were discovered by activity screening of a combi-
natorial library of dendrimers designed to present catalytic
residues at the core combined with hydrophobic and aromatic
residues in the outer layers, thus overcoming difficulties
encountered previously in our attempts to combine histidine
residues with hydrophobic and/or aromatic residues to obtain
dendritic esterase models.15b-c Catalytically productive interac-
tions between the catalytic core and the outer dendritic layers
containing aromatic amino acids occur in dendrimers RG3 and
RMG3, such that these third-generation dendrimers are more
active than the corresponding catalytic cores RG1/RMG1 and
the second-generation dendrimers RG2/RMG2. By contrast, no
such productive interactions occur in dendrimers HG3 and
HMG3 where the outer dendritic layers feature hydrophobic
amino acids and proline, such that their catalytic activity is
already fully present in the first-generation core dendrimers
HG1/HMG1. The different influence of the outer dendritic
layers in R- versus H-series dendrimers might be related to the
different degree of compaction of the dendrimers in the two
series as suggested by the hydrodynamic radii determined by
diffusion NMR. The possible relationship between packing and
function in peptide dendrimers provides an attractive hypothesis
for further investigations of dendrimers as models for proteins.
Menten parameters kcat (rate constant) and K
determined from the linear double reciprocal plot 1/Vnet versus 1/[S]
where [S] is the substrate concentration). The rate constant kuncat without
catalyst was calculated from the slope of the linear curve that gives
un (as product concentration per time) Versus substrate concentration
[S].
M
(Michaelis constant) were
(
V
2
k (4-Methylimidazole). The solutions of 4-methylimidazole were
prepared by serial dilution from a stock solution (3.0 mM) adjusted to
the desired pH value using HCl 1 M. The reaction rate with
4
-methylimidazole (4-MeIm) was obtained under the same conditions
as described above. The final concentrations in the plate were 0, 87.9,
32, 198, 296, 444, and 667 µM 4-MeIm, 200 µM of substrate, and 10
mM of buffer. The second-order rate constants k were calculated from
1
2
linear regression of the experimentally measured pseudo first-order rate
constants k′ as a function of 4-MeIm concentrations. The second-order
rate constants k2 is given by k2 ) k′/[S], where [S] indicates the
concentration of substrate.
Diffusion NMR Measurements. The standard PGSE diffusion NMR
experiments were performed by the NMR service of the Department
of Chemistry and Biochemistry of the University of Bern. The
measurements were carried using a Bruker DRX400 or DRX500 with
Experimental Section
2
dilute solutions (typically 5 mg/mL) in D O at 300 K. The gradient
Dendrimer Synthesis. Peptide dendrimers were synthesized by the
Fmoc strategy according to standard solid-phase procedure and purified
by preparative reverse-phase HPLC. Details of the synthetic procedure
for library and single dendrimers and all characterizations are described
in Supporting Information.
Assays and Kinetic Measurements. Kinetic measurements were
carried out using a CytoFluor Series 4000 multiwell plate reader from
PerSeptive Biosystems. Dendrimers were used as 10 µM, 15 µM, or
with a maximum strength of 50 × 10 T cm-1 was calibrated using
-4
the HOD proton signal in 99.997% D O. The diffusion time ∆ was 50
2
ms and the gradient duration δ was 7 ms. The diffusion coefficient D
was derived from peak integrals or intensities using the Simfit software
from Bruker. The hydrodynamic radii were calculated from the diffusion
coefficient D using the Stokes-Einstein equation30 with η ) 1.089
mPa for D O at 300 K. The compaction factor was calculated as
2
originally proposed.31 See the Supporting Information table for values
and errors.
3
0 µM freshly prepared solutions in milliQ water. Solutions were
prepared by dissolving the dry TFA salts of dendrimers. Substrate
solutions for the Michaelis-Menten kinetics were prepared by serial
dilution by a factor 2/3 (7×) of a freshly prepared 3.0 mM solution of
substrate in milliQ water (final concentration on the plate 60-1000
Acknowledgment. This work was financially supported by
the University of Berne, the Swiss National Science Foundation,
and the European Marie-Curie Training Network IBAAC.
µM). Low K
substrate concentrations, prepared by serial dilution by factors 2/3 and
/2. Eight solutions of 8-hydroxypyrene-1,3,6-trisulfonic acid sodium
M
determinations were carried using 30-200 µM final
Supporting Information Available: Synthetic procedures and
1
1
HPLC, MS (ES+), and H NMR analysis of all dendrimers and
salt 2 ranging from 0 µM to 100 µM in buffer were used for the
calibration 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 pipet, 40 µL of dendrimer was mixed
peptides synthesized, Michaelis-Menten plots for each data
point, and diffusion coefficients for the dendrimers. This material
is available free of charge via the Internet at http://pubs.acs.org.
JA074115F
13246 J. AM. CHEM. SOC.
9
VOL. 129, NO. 43, 2007