Synthesis and activity of histidine-containing catalytic peptide dendrimers

Article abstract of DOI:10.1021/jo060273y

Peptide dendrimers built by iteration of the diamino acid dendron Dap-His-Ser (His = histidine, Ser = Serine, Dap = diamino propionic acid) display a strong positive dendritic effect for the catalytic hydrolysis of 8-acyloxypyrene 1,3,6-trisulfonates, which proceeds with enzyme-like kinetics in aqueous medium (Delort, E.; Darbre, T.; Reymond, J.-L. J. Am. Chem. Soc. 2004, 126, 15642-3). Thirty-two mutants of the original third generation dendrimer A3 ((Ac-His-Ser)₈(Dap-His-Ser)₄(Dap-His-Ser) ₂Dap-His-Ser-NH₂) were prepared by manual synthesis or by automated synthesis with use of a Chemspeed PSW1100 peptide synthesizer. Dendrimer catalysis was specific for 8-acyloxypyrene 1,3,6-trisulfonates, and there was no activity with other types of esters. While dendrimers with hydrophobic residues at the core and histidine residues at the surface only showed weak activity, exchanging serine residues in dendrimer A3 against alanine (A3A), β-alanine (A3B), or threonine (A3C) improved catalytic efficiency. Substrate binding was correlated with the total number of histidines per dendrimer, with an average of three histidines per substrate binding site. The catalytic rate constant k_cat depended on the placement of histidines within the dendrimers and the nature of the other amino acid residues. The fastest catalyst was the threonine mutant A3C ((Ac-His-Thr)₈(Dap-His- Thr)₄(Dap-His-Thr)₂Dap-His-Thr-NH₂), with k_cat = 1.3 min^-1, k_cat/k_uncat = 90′000, K_M = 160 μM for 8-bytyryloxypyrene 1,3,6-trisulfonate, corresponding to a rate acceleration of 18′000 per catalytic site and a 5-fold improvement over the original sequence A3.

Full text of DOI:10.1021/jo060273y

Synthesis and Activity of Histidine-Containing Catalytic Peptide
Dendrimers
†
‡
†
,†
Estelle Delort, Nhat-Quang Nguyen-Trung, Tamis Darbre, and Jean-Louis Reymond*
Department of Chemistry & Biochemistry, UniVersity of Berne, Freiestrasse 3, 3012 Berne, Switzerland,
and Chemspeed Technologies AG, Rheinstrasse 32, 4302 Augst, Switzerland
jean-louis.reymond@ioc.unibe.ch
ReceiVed February 9, 2006
Peptide dendrimers built by iteration of the diamino acid dendron Dap-His-Ser (His ) histidine, Ser )
Serine, Dap ) diamino propionic acid) display a strong positive dendritic effect for the catalytic hydrolysis
of 8-acyloxypyrene 1,3,6-trisulfonates, which proceeds with enzyme-like kinetics in aqueous medium
(
Delort, E.; Darbre, T.; Reymond, J.-L. J. Am. Chem. Soc. 2004, 126, 15642-3). Thirty-two mutants of
the original third generation dendrimer A3 ((Ac-His-Ser) (Dap-His-Ser) (Dap-His-Ser) Dap-His-Ser-NH )
8
4
2
2
were prepared by manual synthesis or by automated synthesis with use of a Chemspeed PSW1100 peptide
synthesizer. Dendrimer catalysis was specific for 8-acyloxypyrene 1,3,6-trisulfonates, and there was no
activity with other types of esters. While dendrimers with hydrophobic residues at the core and histidine
residues at the surface only showed weak activity, exchanging serine residues in dendrimer A3 against
alanine (A3A), â-alanine (A3B), or threonine (A3C) improved catalytic efficiency. Substrate binding
was correlated with the total number of histidines per dendrimer, with an average of three histidines per
substrate binding site. The catalytic rate constant kcat depended on the placement of histidines within the
dendrimers and the nature of the other amino acid residues. The fastest catalyst was the threonine mutant
-
1
A3C ((Ac-His-Thr)
90′000, K ) 160 µM for 8-bytyryloxypyrene 1,3,6-trisulfonate, corresponding to a rate acceleration
of 18′000 per catalytic site and a 5-fold improvement over the original sequence A3.
8 4 2 2
(Dap-His-Thr) (Dap-His-Thr) Dap-His-Thr-NH ), with kcat ) 1.3 min , kcat/kuncat
)
M
Introduction
vaccines, and gene delivery into cells.1,2 In the field of catalysis,
approaches to dendritic catalysts to date have capitalized on
the existence of a dendritic core providing a microenvironment,
or on multivalence and size effect accessible by attaching
multiple copies of a catalytic group at the dendrimer surface.3
Recently we applied the dendritic architecture to synthetic
peptides as a strategy to prepare artificial proteins. A dendritic
Dendrimers are tree-like molecules with various uses in
chemistry and biology, such as catalysis, drug delivery, artificial
*
Address correspondence to this author. Fax: +41 31 631 80 57.
University of Berne.
†
‡
Chemspeed Technologies AG.
10.1021/jo060273y CCC: $33.50 © 2006 American Chemical Society
4
468
J. Org. Chem. 2006, 71, 4468-4480
Published on Web 05/06/2006

Histidine-Containing Catalytic Peptide Dendrimers
peptide is topologically forced to adopt a globular shape where
intramolecular interactions between amino acids are favored over
intermolecular interactions leading to aggregation. The dendritic
architecture thus circumvents the protein folding problem
4
encountered when designing proteins from linear peptides,
which must be addressed with complex combinatorial search
5
algorithms. Dendritic peptides consisting of branched lysine
2d,e
had been investigated previously for multiple antigenic display.
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FIGURE 1. Architectures of peptide dendrimers. The variable amino
acids A are branched by using diamino acids B (b in the drawing)
such as L-2,3-diamino propionic acid (Dap). The peptide dendrimers
are synthesized by Fmoc-solid-phase peptide synthesis.
n
We prepared dendritic peptides by alternating proteinogenic
(j) Wimmer, N.; Marano, R. J.; Kearns, P. S.; Rakoczy, E. P.; Toth, I.
n
R-amino acids (A ) with a branching diamino acid (B) to form
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3
2
1
second generation dendrimers of type A 4(BA )2B-Cys-A ,
which were then dimerized by disulfide bond formation to obtain
third generation dendrimers (Figure 1). Dendrimers displaying
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hydrolysis of fluorogenic esters in aqueous media with enzyme-
like properties of substrate binding and multiple catalytic
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dimerization. The dendrimer topology was therefore modified
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purification. Peptide dendrimers were prepared with two variable
amino acid positions per branch, corresponding to a dendron
2
1
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of type B-A -A , allowing a similar number of variable amino
acid positions per dendrimer (Figure 1). In a preliminary study,
the peptide dendrimers A1-A4 were prepared with a repeating
Dap-His-Ser dendron (Dap ) L-2,3-diaminopropionic acid).
These dendrimers exhibited esterase-type catalytic activity, and
a strong positive dendritic effect was observed for the hydrolysis
1
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7
of 8-acyloxy-1,3,6-pyrene trisulfonates 1-3 (Scheme 1). Herein
we report a synthetic and mechanistic study of 32 different
dendrimer mutants in this series, which establishes the robust-
ness of the peptide dendrimer synthesis and catalytic properties
observed. An automated parallel synthesis protocol was devel-
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1
2794-12757. (i) Benson, D. E.; Haddy, A. E.; Hellinga, H. W.
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3
51, 1160-1168.
J. Org. Chem, Vol. 71, No. 12, 2006 4469

Delort et al.
8
SCHEME 1. Peptide Dendrimers A1-4 Catalyze the
Hydrolysis of 8-Acyloxypyrene 1,3,6-Trisulfonates 1-3a
(Scheme 2). Dendrimers A1-A4 were obtained by using a
tentagel-type resin with a loading of 0.25 mmol/g. The fourth
generation dendrimer A4 was obtained in only moderate yields,
suggesting an upper limit for synthesis (see Table 2 below).
Indeed an attempted synthesis of a fifth generation dendrimer
was unsuccessful.
Investigation of the catalytic properties of dendrimers A1-
A4 revealed an unusually strong positive dendritic effect for
the hydrolysis of pyrene trisulfonate esters 1-3 in aqueous
media, with both the catalytic rate constant kcat and substrate
binding 1/KM increasing with dendrimer size, resulting in a
quadratic increase in the specificity constants kcat/KM (Figure
2
).
The propotionality of the catalytic rate constant kcat to the
total number of histidine residues in dendrimers A1-A4
suggested a direct role for this residue in catalysis, which should
be visible in a pH-rate profile analysis. The pH profile for
hydrolysis of butyrate ester 3 was determined for dendrimers
A3 and A4 and the reference catalyst 4-methylimidazole (4-
MeIm), a model for the histidine side chain. The pH-rate profile
for catalysis of 4-MeIm showed a pH-dependent increase in the
range studied. On the other hand, the pH-rate profile of kcat
with the dendrimers was very different, showing a rather flat
pH dependence with a maximum activity around pH 5.5.7
The pH-rate profile of 4-MeIm was consistent with the free
base form of 4MeIm acting either as nucleophile or as general
base catalyst, as has been proposed in similar studies of histidine
9
containing artificial enzymes. The relatively flat pH-rate
profile of the peptide dendrimers, by contrast, suggested that
both protonated and free base histidine residues might act in
concert in the dendrimer-catalyzed reaction. The free base form
of histidine could act as a nucleophilic or general base catalyst
promoting nucleophilic addition to the ester carbonyl group,
while another protonated histidine side chain might help stabilize
the oxyanion intermediate (Scheme 3). Since the rate constant
kcat measures the reaction of bound substrate to bound transition
state, the stabilizing effect of the protonated histidine would be
expected in addition to a possible contribution to substrate
binding (see below). A similar bifunctional mechanism has been
suggested by other authors for similar ester hydrolysis reactions
1
0
catalyzed by imidazole containing systems.
The concerted action of histidine side chains in such a
mechanism should depend on the precise relative positioning
of at least two such side chains within the dendrimer. Such a
requirement for a precise relative positioning of catalytic groups
could imply that relatively small structural changes within the
dendrimers might lead to significant changes in catalytic
(8) (a) Lloyd-Williams, P.; Albericio, F.; Giralt, E. Chemical Approaches
to the Synthesis of Peptides and Proteins; CRC Press: Boca Raton, FL,
1997. (b) Chan, C.; White, P. D. Fmoc Solid-Phase Peptide Synthesis: a
practical approach; Oxford Press: New York, 2000.
a
Dap ) L-2,3-diaminopropionic acid. His ) L-histidine. Ser ) L-serine.
(
9) Nilsson, J.; Baltzer, L. Chem. Eur. J. 2000, 6, 2214-2220.
oped to facilitate the synthesis of analogues. The influence of
amino acid composition, dendrimer size, and amino acid side
chain ionization state on substrate binding and catalytic ef-
ficiency was determined.
(10) (a) Broo, K. S.; Brive, L., Ahlberg, P.; Baltzer, L. J. Am. Chem.
Soc. 1997, 119, 11362-11372. (b) Broo, K. S.; Nilsson, H.; Nilsson, J.;
Flodberg, A.; Baltzer, L. J. Am. Chem. Soc. 1993, 115, 4063-4068. (c)
Broo, K. S.; Nilsson, H.; Nilsson, J.; Baltzer, L. J. Am. Chem. Soc. 1998,
20, 10287-10295. (d) Baltzer, L.; Kerstin, S. B.; Nilsson, H.; Nilsson, J.
Bioorg. Med. Chem. 1999, 7, 83-91. (e) Klotz, I. M. AdV. Chem. Phys.
978, 39, 109-176. (f) Nango, M.; Klotz, I. M. J. Polym. Sci., Polym.
Chem. Ed. 1978, 16, 1265-1273. (g) Overberger, C. G.; Salomone, J. C.
Acc. Chem. Res. 1969, 2, 217-224. (h) Overberger, C. G.; Kawakami, Y.
J. Polym. Sci.: Polym. Chem. Ed. 1978, 16, 1237-1248. (i) Tomko, R.;
Overberger, C. G. J. Polym. Sci.: Polym. Chem. Ed. 1985, 23, 265-277.
j) Breslow, R.; Schmuck, C. J. Am. Chem. Soc. 1996, 118, 6601-6605.
k) Baumeister, B.; Sakai, N.; Matile, S. Org. Lett. 2001, 3, 4229-4332.
(l) Som, A.; Matile, S. Eur. J. Org. Chem. 2002, 3874-3883.
1
1
Results and Discussion
Initial Studies. Dendrimers A1-A4 built by iteration of the
diamino acid dendron Dap-His-Ser were obtained in good yields
and high purity by sequential coupling of Fmoc-protected amino
acid building block by standard solid-supported peptide synthesis
(
(
4470 J. Org. Chem., Vol. 71, No. 12, 2006

Histidine-Containing Catalytic Peptide Dendrimers
SCHEME 2. Solid Phase Synthesis of Peptide Dendrimersa
a
n
n
A ) variable amino acid. B ) Dap (2,3-diaminopropionic acid). Conditions: (a) DMF/piperidine (4:1), 2 × 10 min; (b) Fmoc-A -OH, BOP/DIEA; (c)
Fmoc)2Dap, BOP/DIEA; (d) Ac2O/DCM (1:1), 1 h; (e) TFA/TIS/H2O (94/5/1), 4 h, then preparative HPLC purification.
(
(Figure 2), was traced back to increased binding to the acyl
chain of the substrates in higher generation dendrimers. Thus,
isothermal calorimetry (ITC) measurements with dendrimers
11
A1-A4 and the nonreactive substrate 4 or the reaction product
showed that the number of binding sites per dendrimer
5
increased with generation number for both 4 and 5, with an
average of one substrate or product binding site per three
histidine residues (Table 1). A dendritic effect on binding affinity
was observed with substrate 4 following the trend in KM
observed with the reactive substrates 1-3. By contrast the
binding constant for product 5, which was 10-fold weaker than
that of the substrate, remained constant from A1 to A4. Binding
with both 4 and 5 was driven by a large and favorable enthalpy
(
∆Ha values between -7 and 11.6 kcal/mol) with favorable
contribution of ∆S that could be attributed to solvent release
12
upon binding. The strong binding between the higher genera-
tion dendrimer A4 and the longest acyl-chain substrates could
be interpreted in terms of hydrophobic interactions between the
substrate’s acyl chain and the dendrimer interior. Such hydro-
phobic interactions are also consistent with the fact that the KM
values for the more hydrophobic nonanoate ester 3 are generally
lower than those for the corresponding butyrate 2 or acetate 1
across the dendrimer series.
FIGURE 2. Dendritic effect on dendrimer-catalyzed hydrolysis of
butyrate 2 for A1 to A4. Conditions: 10-1000 µM substrate, A1 )
1
0.6 µM, A2 ) 6.4 µM, A3 ) 3.55 µM, A4 ) 1.88 µM in aqueous 5
mM citrate pH 5.5, 26 °C. nHis gives the total number of histidine
residues in the dendrimer. Similar plots were obtained on substrates 1
and 3.
(11) Dodecanoyl ester 4 gave very low conversion and competitively
inhibited catalysis for the shorter chain substrates, making this substrate an
ideal model for studying substrate binding to the dendrimers.
efficiency, suggesting that a mutational study might uncover
more active dendrimers.
The proportionality of substrate binding 1/KM to dendrimer
size, participating in the positive dendritic effect observed
(12) (a) Zechel, D. L.; Boraston, A. B.; Gloster, T.; Boraston, C. M.;
Macdonald, J. M.; Tilbrook, D. M. G.; Stick, R. V.; Davies, G. J. J. Am.
Chem. Soc. 2003, 125, 14313-14323. (b) Rekharsky, M.; Inoue Y.; Tobey,
S.; Metzger, A.; Anslyn, E. J. Am. Chem. Soc. 2002, 124, 14959-14967.
J. Org. Chem, Vol. 71, No. 12, 2006 4471

Delort et al.
TABLE 1. Calorimetric Titration of Nonreactive Substrate 4 and the Reaction Product 5 Added to A1-A4: Thermodynamic Parameters,
a
Association Constants (Ka), Number of Binding Sites (n)
NHis
n
Ka (×10 M-1)
4
∆Ha (kcal/mol)
∆Ga (kcal/mol)
∆Sa (cal/mol/K)
A1
A2
A3
A4
A1
A2
A3
A4
5
5
5
5
4
4
4
4
3
7
15
31
3
7
15
31
0.84((0.22)
1.69((0.09)
4.78((0.43)
9.43((1.5)
2.36((0.07)
1.86((0.04)
5.83((0.03)
9.88((0.04)
0.11((0.02)
0.27((0.03)
0.29((0.05)
0.28((0.07)
1.66((0.51)
10.5((3.2)
-8.36((2.46)
-7.74((0.59)
-8.89((1.07)
-10.06((1.92)
-6.91((0.33)
-12.85((0.38)
-10.85((0.11)
-11.58((0.10)
-17.47((0.45)
-19.67((0.28)
-19.91((0.43)
-19.76((0.62)
-24.20((0.77)
-28.79((0.76)
-32.98((0.53)
-33.87((0.46)
31((10)
40((3)
37((5)
32((8)
58((4)
53((4)
74((2)
74((2)
56.2((12.0)
80.8((15.0)
a
Conditions: A1 0.5 mM, A2 0.2 mM, A3 0.1 mM, A4 0.05 mM, 4 10 mM, 5 10 mM in citrate buffer pH 5.5 (5 mM), at 27 °C.
on the surface might provide interesting mimics of proteins.14
Hydrophobic core dendrimers might also allow enhanced
hydrophobic interactions with the substrates similar to those
observed by ITC with A1-A4 (Table 1).
SCHEME 3. Mechanistic Proposal for Dendrimer Catalyzed
Ester Hydrolysisa
We prepared a direct analogue series B1-B3 featuring a
phenylalanine-leucine at the core and the serine-histidine dyad
in the branches. An attempted synthesis of the fourth generation
dendrimer B4 was unsuccessful. In addition we also prepared
a diverse set of dendrimers C1-O4 incorporating negatively
charged and hydrophobic residues, which might reduce the
selectivity of the dendrimers for anionic substrates. The
syntheses were carried out with Tentagel or Rink amide resin,
and the dendrimers were isolated in pure form with satisfactory
yields. In the case of dendrimer G2, some dimerization took
place by air oxidation, and part of the dendrimer was isolated
as the disulfide bridged homodimer G22 (Table 2).
The catalytic properties of the 17 dendrimers B1-O4 were
investigated together with A1-A4. A survey of various ester
substrates showed that the substrate scope of these histidine
containing dendrimers was limited to 8-acyloxy-1,3,6-pyrene
trisulfonates. There was no significant catalysis by any of the
dendrimers for the hydrolysis of cationic substrate 7, neutral
substrates 8 and 9a-c, or the more hydrophobic fluorescein
1
5
substrates 10a-c (Figure 3).
The peptide dendrimer series B1-O4 was compared with
A1-A4 for hydrolysis of the 8-acyloxy-1,3,6-pyrene trisul-
fonates 1-4 in aqueous buffer pH 6.0. The apparent catalytic
effect in the analogue series B1-O4 was much weaker than
that with the histidine-serine series A1-A4. Dendrimer G22
formed by spontaneous oxidative dimerization of G2 during
purification also did not show strong catalysis. Dendrimers B1-
B3 bearing the leucine-phenylalanine hydrophobic core showed
acceptable catalytic properties with substrates 1-3, and were
studied in detail (see below). The generally low activity of this
dendrimer series incorporating hydrophobic core residues sug-
gested that a different strategy should be followed to improve
catalysis.
Parallel Synthesis of Analogues of Dendrimer A3. Peptide
dendrimer A3 gave the best compromise between an acceptable
chemical yield by synthesis and a strong catalytic activity. Due
to the low activity of the dendrimer analogues incorporating
hydrophobic residues discussed above, we turned our attention
to conservative replacements of serines with similarly small and
polar residues such as alanine (A3A), â-alanine (A3B), or
threonine (A3C) as a strategy to optimize catalytic activity. We
a
Reaction processes: (a) nucleophilic attack of histidine generated an
acyl-dendrimer intermediate; (b) general base catalyzed nucleophilic attack
of a water molecule
Synthesis of Hydrophobic Core Dendrimers. A series of
analogue dendrimers were prepared to investigate the effect of
amino acid substitutions on substrate binding and catalytic
efficiency. The introduction of hydrophobic residues at the
dendrimer core together with catalytic and charged amino acids
at the surface was first investigated with the aim of creating
more protein-like dendrimers. Indeed, natural protein folding
is driven by the hydrophobic collapse,13 which results in an
aggregation of apolar residues at the protein core, while polar
and charged residues are found near the surface. Similarly,
peptide dendrimers with a hydrophobic core and polar residues
(
13) (a) Gerstman, B. S.; Chapagain, P. P. J. Chem. Phys. 2005, 123,
54901/1-054901/6. (b) Zhou, R.; Huang, X.; Margulis, C. J.; Berne, B.
J. Science 2004, 305, 1605-16. (c) Tanford, C. The Hydrophobic Effect,
nd ed.; Wiley-Interscience, New York, 1980. (d) Pratt, L. R.; Pohorille,
0
(14) Clouet, A.; Darbre, T.; Reymond, J.-L. AdV. Synth. Catal. 2004,
346, 1195-1204.
(15) (a) Bensel, N.; Reymond, M. T.; Reymond, J.-L. Chem. Eur. J.
2001, 7, 4604-4612. (b) Leroy, E.; Bensel, N.; Reymond, J.-L. Bioorg.
Med. Chem. Lett. 2003, 13, 2105-2108.
2
A. Chem. ReV. 2002, 102, 2671-2691. (e) Schneider, J. P.; Kelly, J. W.
Chem. ReV. 1995, 95, 2169-2187. (f) Breslow, R.; Yang, Z.; Ching, R.;
Trojandt, G.; Odobel, F. J. Am. Chem. Soc. 1998, 120, 3536-3537.
4472 J. Org. Chem., Vol. 71, No. 12, 2006

Histidine-Containing Catalytic Peptide Dendrimers
TABLE 2. Peptide Dendrimer Series A-O of Different Generation Number Containing Histidine as the Catalytic Residue, with Yields after
Preparative HPLC, Calculated Molecular Weight, and Their Mass Obtained by Electron Spray MS (positive mode), and Their Apparent
a
Catalytic Effect Vnet/Vun
Vnet/Vun
sequence
A A .A A .A A
resin loading
(mmol/g)
m
(mg)
yield
(%)
n
n-1
4
3
2
1
no.
MW
MS (ES+)
1
2
3
c
A1
A2
A3
A4
B1
B2
B3
HS.HS
0.25
3.3
21.9
34.2
10.4
19.7
31.2
8.6
11
31
23
3.3
49
63
16
859.37
2011.85
4316.81
8926.73
895.43
859.38
2012.25
4318.13
8929.25
895.50
3.3
30
90
170
6.8
4.6
3.3
26
60
160
1.3
c
HS.HS.HS
HS.HS.HS.HS
HS.HS.HS.HS.HS
HS.LF
HS.HS.LF
HS.HS.HS.LF
0.25
38
110
180
7.0
9.8
10
c
0.25
c
0.25
c
0.18
c
0.18
2047.91
4352.87
2048.50
4353.13
13
26
0.5
0.3
c
0.18
b
C1
D2
E2
F2
G2
G22
H2
I2
HD.CS
DH.HS.CF
HD.SF.CL
0.61
4.3
11.1
11.1
4.6
3.8
1.6
13
24
14
5.9
3.9
1.6
34
29
13
30
17
45
32
3.5
881.31
2149.82
2135.85
2079.86
2493.90
4985.78
2047.89
1959.91
1895.93
1983.91
4238.67
4576.81
4512.83
9120.77
881.75
2150.00
2136.75
2080.63
2494.25
4987.88
2048.50
1960.38
1896.50
1984.63
4240.25
4578.13
4513.63
9123.75
0.3
0.4
5.5
0.2
2.8
0.0
0.0
0.7
5.7
4.4
2.6
0.0
3.2
4.5
0.4
4.2
0.3
1.9
0.0
0.2
1.1
8.2
6.5
3.6
0.0
7.3
4.5
b
0.47
3.0
0.6
1.7
0.3
0.1
0.2
3.6
3.4
1.4
0.4
1.7
2.5
b
0.61
b
HS.DF.CL
0.61
b
d
HDS.HD.CS
(HDS.HD.CS)2
HS.DF.AL
0.61
b
d
0.61
c
0.20
8.5
c
HS.AF.AL
0.18
12.7
18.2
31.3
16.1
26.7
34.6
4.6
c
J2
HA.AF.AL
HA.DF.AL
DS.SH.FL.CF
DH.HS.DF.AL
DH.HA.DF.AL
0.29
b
K2
L3
M3
N3
O4
0.43
c
0.20
c
0.20
b
0.43
b
HS.DH.HA.DF.AL
0.43
42
68
44
a
Conditions: 200 µM substrate 1, 2, 3, dendrimers 5 µM, in 20 mM Bis Tris buffer pH 6.0. Vun ) rate in buffer alone with 200 µM substrate; Vnet )
Vcat - Vun, Vcat ) apparent rate in the presence of 2.5 mol % dendrimer catalyst and 200 µM substrate. There was no catalysis with substrate 4 and any of
these dendrimers. Rink amide resin. NovaSyn TGR resin. d Isolated from the same reaction, total yield ) 5.5%.
b
c
equiv) with N-methylmorpholine as base (5 equiv).16 Cleavage
and purification of the peptides were then carried out manually.
The quality of the crude products and the isolated yields after
purification by preparative HPLC were comparable to those
obtained by manual synthesis (see the Supporting Information).
Catalytic Properties of B1-B3 and A3-Analogue Series.
The hydrophobic core series B1-B3 and 15 analogues of the
histidine-serine dendrimer A3 obtained by parallel synthesis
were characterized in detail. As for the hydrophobic core series
discussed above, catalytic activity was found to be specific for
8-acyloxy-1,3,6-pyrene trisulfonates, with no detectable catalysis
with other types of substrates (Figure 3). The dendrimers were
therefore investigated for their catalytic properties in the
hydrolysis of the fluorogenic ester substrates 1-4.
All assays were conducted in aqueous citrate buffer at pH
5.5, which are optimal conditions for this system, and the
FIGURE 3. Fluorogenic substrates tested for peptide dendrimer
catalyzed ester hydrolysis.
7
reactions were followed by fluorescence in a microtiter plate
reader. Rate enhancement was first assessed by the value of
the apparent catalytic effect Vnet/Vun measured at 200 µM
substrate and 1.8 mol % catalyst (Table 3).
also systematically exchanged histidine residues against alanine
at the inner or outer branches until complete removal to clarify
their role in substrate binding and catalysis (A3D-A3J). Finally
histidine was either used at every position (A3K) or the amino
acids were reshuffled between the different positions (A3L-
A3O) to investigate the effect of amino acid positions on
catalysis.
A parallel synthesis protocol was developed to demonstrate
the feasibility of automated dendrimer synthesis. The synthesizer
uses solutions of reagents at fixed concentrations rather than
adding solids into the reactor as during manual synthesis. This
different addition mode implies that coupling reactions require
increasing volume as the synthesis proceeds to higher genera-
tions of the dendrimer. The synthesis was carried out with
tentagel resin (0.23 mmol/g), following the synthetic route for
third generation dendrimers (Scheme 2). Coupling times were
Catalysis as judged by Vnet/Vun depended mostly on the total
number of histidine residues in the dendrimer (NHis), in
agreement with the role of histidine as a catalytic residue. Thus
the control dendrimer A3J without histidine and its analogue
A3I with only one histidine residue at the core were catalytically
inactive. On the other hand all dendrimers with 15 or more
histidines were strongly active, although structural variations
caused large differences in apparent activity. Five dendrimers
were much more active than the reference catalyst A3. These
were the A3 mutants serine f alanine A3A, serine f â-alanine
A3B, and serine f threonine A3C, dendrimer A3L with 24
histidines in the outer layers and 6 serines in the inner layers,
and its analogue A3M with alternating histidine and serine
generations. By contrast, the whole-histidine dendrimer A3K
1
4
h for the first generation, 2 h for the second generation, and
h for the third generation, using systematic double coupling.
The coupling reagent was HBTU/HOBt (0.5 M in DMF, 3
(16) Carpino, L. A.; El-Faham, A. J. Org. Chem. 1994, 59, 695-697.
J. Org. Chem, Vol. 71, No. 12, 2006 4473

Delort et al.
TABLE 3. Analogues A3A-A3O of Dendrimers A3 Prepared Par Automated Synthesis on the Chemspeed PSW1100 Peptide Synthesizera
Vnet/Vun
sequence
NHis
m (mg)
yield (%)
MW
MS (ES+)
1
2
3
A3
HS.HS.HS.HS
HA.HA.HA.HA
HâA.HâA.HâA.HâA
HT.HT.HT.HT
HS.AS.AS.AS
AS.HS.HS.HS
AS.HS.HS.HA
AS.HS.HS.AS
AS.HS.AS.AS
AS.AS.AS.HS
AS.AS.AS.AS
HH.HH.HH.HH
HH.HH.SS.SS
HH.SS.HH.SS
AH.AH.AH.AH
SH.SA.SH.SA
15
15
15
15
8
7
7
6
4
7.0
7.8
5.0
5.9
4316.81
4076.89
4076.89
4525.05
3854.66
3788.64
3772.64
3722.62
3590.57
3392.51
3326.48
5067.21
4767.05
4566.95
4076.89
3986.7
4318.13
4078.00
4079.13
4528.75
3855.88
3789.63
3773.38
3723.88
3591.5
161
160
300
260
33
77
48
67
16
0
1
36
280
250
154
90
242
370
1100
850
100
140
115
170
33
0
1.5
64
1100
760
390
190
105
140
580
280
57
81
53
37
7
1.8
0.9
17
280
560
330
74
A3A
A3B
A3C
A3D
A3E
A3F
A3G
A3H
A3I
A3J
A3K
A3L
A3M
A3N
A3O
17.2
19.1
19.0
15.8
2.0
10.3
5.8
7.9
14
2.0
23
7.4
12.9
13.3
17.3
15.0
1.9
10.2
6.2
9.8
18.3
1.0
13.3
4.7
10.4
17.6
1
0
3393.88
3327.5
30
24
20
15
10
5068.75
4768.63
4568.63
4079.13
3988.13
13.8
20.7
a
Given yields after preparative HPLC. Mass obtained by electron spray MS (positive mode). Apparent catalytic effect Vnet/Vun. Conditions: 200 µM
substrate 1, 2, 3, dendrimers 3.55 µM (1.8 mol %), in 5 mM citrate buffer pH 5.5. There was no catalysis with substrate 4 and any of these dendrimers.
TABLE 4. Michaelis-Menten Parameters on Pyrene Substrates 1, 2, and 3a
KM (mM)
kcat/kuncatb
((kcat//KM)/k2)c
dendrimer
sequence
HS.HS
HS.HS.HS
HS.HS.HS.HS
HS.HS.HS.HS.HS
NHis
1
2
3
1
2
3
1
2
3
A1
A2
A3
A4
3
7
15
31
0.840
0.110
0.041
0.035
0.450
0.140
0.063
0.029
1.60
1800
3600
6800
20000
2300
8000
17000
40000
4500
4400
6700
18000
140
140
130
3000
23000
140000
0.067
0.013
0.006
2000
10000
35000
1600
7900
38000
B1
B2
B3
HS.LF
HS.HS.LF
HS.HS.HS.LF
2
6
14
1.891
0.610
0.158
0.537
0.126
0.083
0.936
0.064
0.010
1353
4847
2990
1126
3616
5400
1322
1978
1780
45
501
1190
60
821
1872
65
1420
8198
A3A
A3B
A3C
A3D
A3E
A3F
A3G
A3H
A3I
HA.HA.HA.HA
HâA.HâA.HâA.HâA
HT.HT.HT.HT
HS.AS.AS.AS
AS.HS.HS.HS
AS.HS.HS.HA
AS.HS.HS.AS
AS.HS.AS.AS
AS.AS.AS.HS
AS.AS.AS.AS
HH.HH.HH.HH
HH.HH.SS.SS
HH.SS.HH.SS
AH.AH.AH.AH
SH.SA.SH.SA
15
15
15
8
7
7
6
4
1
0
30
24
20
15
10
0.070
0.050
0.100
0.650
0.640
0.250
0.230
0.420
d
0.060
0.070
0.160
0.240
1.100
0.200
0.310
0.740
d
0.025
0.030
0.040
0.240
0.210
0.150
0.120
d
11000
21000
22000
8000
15000
5600
7700
3000
d
23000
80000
90000
9500
52000
11000
19000
9400
d
8200
34000
16000
6700
9900
5900
3900
d
9200
28000
13000
760
1500
1400
2100
440
10500
34000
15000
1100
1400
1500
18000
360
1500
54000
20000
1300
2100
1800
1500
d
d
d
d
d
d
d
d
d
d
d
d
d
d
A3J
d
d
d
d
A3K
A3L
A3M
A3N
A3O
0.050
0.080
0.080
0.080
0.260
0.020
0.080
0.090
0.070
0.910
2600
23000
18000
11000
9800
3900
80000
10000
26000
72000
3280
18500
13800
8800
2300
4800
28000
3000
11000
2200
0.008
0.050
0.050
0.100
17000
25000
22000
7600
91000
25000
19000
3400
a
Conditions: 10-1000 µM substrate, dendrimer concentration 3.55 µM for A3 and A3A-A3O, 10.6 µM for A1, 6.4 µM for A2, 1.88 µM for A4, 16
b
-5
-1
-5
-1
-5
µM for B1, 8 µM for B2, 4 µM for B3, in aq 5 mM citrate pH 5.5, 26°C. kuncat ) 4.39 × 10 min for 1, 1.39 × 10 min for 2, and 2.20 × 10
min for 3. k2 ) 4-MeIm ) 6.96 × 10 mM min for 1, 4.86 × 10 mM min for 2, and 4.77 × 10 mM min for 3. d No activity detected.
-
1
c
-4
-1
-1
-4
-1
-1
-4
-1
-1
was much less active than A3, showing that the number of
histidines alone was not sufficient to explain the apparent
catalytic rate. By comparison the hydrophobic core dendrimers
B1-B3 with the phenylalanine-leucine dyad at positions A A
were much less catalytically active.
suggesting that this residue was responsible for both catalysis
and binding (Figure 2). To test whether this relationship held
true throughout the dendrimer series, the influence of NHis on
catalytic parameters was analyzed by multivariate statistical
analysis. Each dendrimer was described by seven variables
defined as the catalytic parameters kcat and 1/KM for substrates
1, 2, and 3, and the total number of histidine residues NHis.
Principal component analysis of this dataset showed that the
first two principal components PC1 and PC2 explained 83% of
the variability between the dendrimers (Figure 4). PC1 consisted
mostly of substrate binding (1/KM) for each of the three
substrates and NHis, and PC2 described mostly the catalytic rate
constants kcat for the three substrates.
2
1
1
7
Michaelis-Menten parameters were determined for the
different dendrimers with substrates 1-3 (Table 4). The rate
2
5
accelerations kcat/kuncat ranged from 10 to 10 , with the highest
value of kcat/kuncat ) 90 000 being observed for the histidine-
threonine dendrimer A3C with the butyrate ester 2. Substrate
binding varied between KM ) 8 µM for dendrimer A3L and
nonanoate 3, and KM ) 1100 µM for dendrimer A3E with
butyrate 2.
Kinetic Profiling and Clustering. The series A1-A4
showed a linear correlation between the total number of histidine
residues (NHis) and the catalytic parameters kcat and 1/KM,
(
17) Introduction to MultiVariate Analysis; Chatfield, C., Collins, A. J.,
Eds.; Chapman and Hall Ltd.: London, UK, 1983.
4474 J. Org. Chem., Vol. 71, No. 12, 2006

Histidine-Containing Catalytic Peptide Dendrimers
FIGURE 4. Principal component (PC) analysis of peptide dendrimers A1-4, B1-3, and A3A-A3O according to catalytic parameters kcat and
1
M
/K for substrates 1-3 and total number of histidines NHis. Main plot: PC plot according to PC1 and PC2, which explains 84% of the observed
variability. Clusters defined by hierarchical clustering are marked in circles. Upper right: Dendrogram of similarities between dendrimers (Ward’s
method with squared euclidean distances). Lower right: Loading of the principal components PC1 and PC2. The variables are normalized to an
average value of 0 and a standard deviation of 1 prior to processing.
Cluster analysis of the dendrimer series defined groups of
similar dendrimers well visible in the principal component plot
alanine (A3A) has only a minor effect on the catalytic properties
of the dendrimer.
(
Figure 4). A first group was formed by dendrimers featuring
Further Evidence for the Involvement of Histidines in
Substrate Binding. The cluster analysis above clearly pointed
to a primary role of histidine residues in substrate binding. The
pH profile data with dendrimers A3 and A4 were therefore
analyzed with respect to the pH dependence of KM. Michaelis-
Menten kinetics with butyrate 2 gave very low KM values below
pH 6.0 when measured in 20 mM Bis-tris (Bis(hydroxyethyl)-
tris(hydroxymethyl)-aminomethane) or MES (morpholinoethane
sulfonic acid) buffers, which only allowed the determination
of kcat. Substrate binding was found to be weaker in the presence
of the trianionic citrate buffer (5 mM), allowing measurable
KM values below pH 5.5 to be obtained as well. The KM values
of substrate 2 with dendrimer A3 in citrate buffer increased
linearly from 33 µM at pH 5.2 to 180 µM at pH 6.0 in that
buffer, while the catalytic rate constant showed a maximum
around pH 5.5 as in the other buffer systems (Figure 5). The
higher KM values in citrate buffer and their pH dependence can
be interpreted in terms of the citrate trianion competing against
the substrate’s sulfonate groups for binding to the dendrimer
via salt bridge interactions with the protonated histidines, and
are consistent with histidines being involved directly in substrate
modest substrate binding and catalytic properties (lower left plot
in Figure 4). These dendrimers all contained 8 or fewer histidine
residues. A second group was formed by the original sequence
A3 and several close analogues including the more weakly active
hydrophobic core analogue B3, the Ser-Ala mutants A3A and
A3N, and the permutated sequence A3M (center plot in Figure
4
). Dendrimers A3E and A3O formed a third group with
relatively good catalytic rate constants kcat but weak substrate
binding (upper left plot in Figure 4). The whole histidine
dendrimer A3K and the fourth generation His-Ser dendrimer
A4 stood out at high PC1 values by their particularly strong
substrate binding associated with a much larger number of
histidine residues (right plot in Figure 4). A4 was highly
catalytically active, while A3K was not. Finally, the three most
active dendrimers, namely the Ser-âAla mutant A3B, the Ser-
Thr mutant A3C, and the reshuffled His-Ser sequence A3L,
appeared at high PC2 values denoting high catalytic rate
constants kcat (top center plot in Figure 4).
The fact that 1/KM is found together with NHis in PC1 indicates
that the linear correlation between NHis and 1/KM is conserved
throughout the dendrimers series. By contrast NHis is only weakly
associated with PC2, which is composed of the catalytic rate
constants kcat for all three substrates (Figure 4). Indeed linear
1
8
binding interactions.
Similar binding interactions between sulfonate groups and
protonated histidines might explain the competitive inhibition
of dendrimer A3 catalyzed hydrolysis of 2 observed with pyrene
1,3,6,8-tetrasulfonate 6 (IC50(6) ) 200 µM at 200 µM 2, pH
5.5). Such interactions are presumably essential for substrate
binding and might explain the absence of catalysis with
fluorogenic substrates 8-10, which have similar or higher
chemical reactivities compared to the pyrene sulfonates 1-4,
but lack the negatively charged sulfonate groups.
2
regression analysis shows that NHis correlates with 1/KM(1) (r
2
2
)
0.669), 1/KM(2) (r ) 0.661), and 1/KM(3) (r ) 0.660), but
2
2
2
not with kcat(1) (r ) 0.290), kcat(2) (r ) 0.088), or kcat(3) (r
0.187). The correlation between substrate binding and NHis
)
suggests that a large part of the observed substrate binding can
be attributed to the presence of histidine residues. On the other
hand, the mere presence of histidine residues is clearly not
sufficient to explain the occurrence of catalysis. The difference
is striking when comparing the replacement of serine in A3 by
â-alanine (A3B) or threonine (A3C), which significantly
increases catalytic efficiency, while the simple replacement for
(18) For ion pair in water: (a) Schmuck, C.; Heil, M.; Scheiber, J.;
Baumann, K. Angew. Chem., Int. Ed. 2005, 44, 7208-7212. (b) Rensing,
S.; Schrader, T. Org. Lett. 2002, 4, 2161-2164. (c) Hossain, M. A.;
Schneider, H.-J. J. Am. Chem. Soc. 1998, 120, 11208-11209.
J. Org. Chem, Vol. 71, No. 12, 2006 4475

Delort et al.
TABLE 5. Apparent pK of Histidine Side Chainsa
compd
Me-Im
A3
A4
pKapp
pKapp with 6
4
7.8
5.7
5.0
8.0
6.8
7.3
a
Determined by titration of the trifluoroacetate salts with NaOH.
Conditions: as in Figure 7.
form for histidines at the optimum pH of 5.5 for the dendrimer
in the absence of bound substrate.
A similar titration was carried out in the presence of pyrene
1
,3,6,8-tetrasulfonate 6 as a substrate analogue to mimic the
dendrimer-substrate complex, using 1 equiv of 6 per substrate
binding site as determined by ITC (Table 1). The histidine’s
pKapp in dendrimer A3 increased by 1.1 pH units in the presence
of 6, and that of dendrimer A4 by 2.25 pH units. This
corresponds to less than 5% of histidines in the free-base form
at the optimal pH of 5.5 for both dendrimers. By comparison,
the apparent pKapp of 4-MeIm did not change significantly with
and without tetrasulfonate 6, indicating that the pKapp-shift effect
was specific to the dendrimers.
FIGURE 5. pH profile of catalytic parameters kcat and K
M
for
dendrimer A3 catalyzed hydrolysis of butyrate 2 in aqueous 5 mM
citrate buffer. Conditions: 3.55 µM A3, 10-1000 mM 2, 25 °C,
aqueous 5 mM citrate pH 4.9, 5.2, 5.5, 5.7, 5.9, 6.2, 6.7.
The titration curves in the presence of tetrasulfonate 6 indicate
50% ionization of histidines at pH 6.8, which would be expected
as the pH optimum for kcat with a bifunctional mechanism rather
than the observed pH 5.5. The pH optimum of 5.5 for kcat might
be explained in part by the fact that the number of binding sites
decreases 4-fold between pH 5.5 and pH 7.0, as evidenced by
ITC with dendrimer A3 and product 5, effectively reducing
catalytic efficiency per dendrimer (Table 6).
Ionic Strength Effects. The effects of buffer, pH, and
inhibitors discussed above clearly point to a strong electrostatic
component in binding and catalysis. On the other hand, the ITC
studies on the regular series A1-A4 and the generally lower
KM values observed with the more hydrophobic substrate/
dendrimer pairs suggest hydrophobic substrate-dendrimer
interactions. The relative strength of electrostatic versus hydro-
phobic interactions was investigated by measuring the ionic
strength dependence of catalysis. Electrostatic interactions are
expected to be weakened at high ionic strength, while those
depending on the hydrophobic effect should become stronger.19
Kinetic parameters were determined at the optimal pH of 5.5
from 0 to 500 mM KCl for dendrimer A3 and substrates 1-3
FIGURE 6. Determination of the apparent pK of histidine side chains
by titration of the dendrimer TFA salts in the absence or presence of
pyrene 1,3,6,8-tetrasulfonate 6: 4-MeIm + 1.2 equiv of TFA (b),
(Figure 7A/B). The KM values increased by approximately 10-
4
-MeIm + 1.2 equiv of TFA + 1.2 equiv of 6 (O), A3 (2), A4 (9),
fold across the KCl concentration range for all three substrates.
The weaker substrate binding at high ionic strength supports
an electrostatic component in substrate binding as inferred from
the pH effects and inhibition by the pyrene tetrasulfonate 6
discussed above. Similar ionic strength effects on binding for
substrate 2 were also observed with the hydrophobic core
dendrimer B3, the weakly active whole histidine dendrimer
A3K, and the highly efficient dendrimer A3L consisting of a
whole histidine surface around a serine core (Figure 7C/D).
These data provide further evidence that substrate binding in
the dendrimer series depends strongly on electrostatic interac-
tions between the sulfonate groups of the substrates and
protonated histidine residues on the dendrimers.
A3 + 5 equiv of 6 (4), and A4 + 10 equiv of 6 (0). Conditions: 1
mM aqueous solution of dendrimers, and 40 mM 4-methylimidazole
aqueous solution containing ca. 1.2 equiv of TFA, without or with
pyrene 1,3,6,8-tetrasulfonate 6 were titrated with aliquots of 2 mM
NaOH, 25 °C.
Ionization State of Histidine Side Chains. At any of the
pH values, the extent of histidine protonation should directly
influence binding of the trianionic substrates 1-3. Furthermore,
catalytic efficiency should depend on the percentage of free base
present in the dendrimers assuming that the free base form is
the key nucleophile or general base in catalysis (Scheme 3).
The ionization state of histidine side chains as a function of pH
was determined in peptide dendrimers A3 and A4 by direct
titration of the trifluoroacetate salts of the dendrimers, as
obtained from the preparative HPLC purification, with NaOH
The rate constant for the uncatalyzed reaction kuncat increased
approximately 2-fold for all three substrates from 0 to 500 mM
KCl, as should be expected for an ionic transition state in a
(
Figure 6). The titration curve provided apparent pK () pKapp)
values for histidine side chains (defined here as pH value for
0% ionization) of 5.7 for dendrimer A3 and 5.0 for dendrimer
A4 (Table 5). This corresponds to approximately 30% free base
(19) (a) Atkins, P. W. Physical Chemistry, 6th ed.; Oxford University
5
Press: Oxford, UK, 1998. (b) Jencks, W. P. Catalysis in Chemistry and
Enzymology; Dover Publications: New York, 1975.
4476 J. Org. Chem., Vol. 71, No. 12, 2006

Histidine-Containing Catalytic Peptide Dendrimers
TABLE 6. pH Dependence of the Calorimetric Titration of 5 Added to A3a
Ka
∆Ha
(kcal/mol)
∆Ga
(kcal/mol)
∆Sa
(cal/mol/K)
dendrimer
ligand
pH
n
(×10 M-1)
4
A3
A3
A3
A3
5
5
5
5
5.0
5.5
6.0
7.0
3.77((0.09)
4.78((0.43)
3.14((0.14)
1.34((0.93)
3.23((0.51)
0.29((0.05)
0.89((0.11)
0.21((0.05)
-9.26((0.29)
-8.89((1.07)
-6.86((0.40)
-11.00((0.67)
-25.89((0.39)
-19.91((0.43)
-22.69((0.31)
-19.10((0.65)
55((2)
37((5)
53((2)
27((4)
a
Conditions: A3 0.1 mM, 5 10 mM; buffers MES pH 5.0 (20 mM), citrate pH 5.5 (5 mM), Bis Tris pH 6.0 (20 mM), HEPES pH 7.0 (20 mM) at 27
°
C
FIGURE 7. Ionic strength effects on dendrimer-catalyzed ester hydrolysis. (A, B) Catalytic parameters for dendrimer A3 catalyzed hydrolysis of
-acyloxypyrene 1,3,6-trisulfonates 1 (acetate), 2 (butyryl), and 3 (nonanoyl) as a function of KCl concentration. (C, D) Catalytic parameters for
8
the hydrolysis of butyrate 2 with dendrimers A3, A3K, and A3L. Conditions: 10-1000 µM ester, 3.55 µM dendrimers A3, A3K, and A3L, in
aqueous 5 mM citrate pH 5.5 containing 0, 5, 10, 50, 100, 200, 300, 500 mM KCl, 26 °C.
more polar medium in the absence of interactions with a catalyst.
By contrast the rate constant kcat decreased by 2-fold with the
acetate ester substrate 1 (Figure 7A). This effect is consistent
with a specific transition state stabilizing dendrimer-substrate
interaction of electrostatic nature, for example the stabilization
of the oxyanion by a protonated histidine residue (Scheme 3).
However, the kcat values were independent of ionic strength with
the butyrate ester 2 (Figure 7A/C) and increased with the
nonanoate ester 3 (Figure 8A). This effect probably reflects
additional catalytically productive dendrimer-substrate interac-
tions of hydrophobic nature involving the substrate’s acyl chain
with these more hydrophobic substrates.
Comparison with Enzymes and Enzyme Models. Enzymes
are macromolecular catalysts acquiring activity and selectivity
from the interplay between amino acid side chains and optional
cofactors in the folded state of the protein. Hallmarks of enzyme
catalysis are selective substrate binding and catalysis in aqueous
2
0
environment. The esterase peptide dendrimers discussed here
J. Org. Chem, Vol. 71, No. 12, 2006 4477

Delort et al.
address the problem of de novo enzyme design, which strives
to construct functional protein-like structures from first prin-
ciples.
site) than our peptide dendrimers (4.5 kDal). A further approach
to artificial enzyme design consists of combining noncatalytic
proteins with catalytically active cofactors. For example, Dis-
tefano et al. reported enantioselective reductive amination
processes in a lipid-binding protein modified with a pyridox-
The key advantage of our dendrimer approach is a robust
and simple solid-phase synthesis approach. Thus, only 11
coupling steps are necessary to prepare third-generation peptide
dendrimers containing 37 amino acids, which greatly facilitates
the preparation of analogues. We have recently shown that the
synthesis is also suitable for split-and-mix synthesis and allows
active dendrimers to be discovered by screening combinatorial
libaries.21 The peptide dendrimers reported here show remark-
able catalytic efficiencies up to kcat/kuncat ) 90 000 for the Thr-
Ser mutant A3C. This corresponds to 18 000 per catalytic site
assuming three histidine residues per catalytic site as determined
by ITC, among the best enzyme models reported to date in terms
of catalytic efficiency.
2
5
amine cofactor. Ward et al. have use streptavidin combined
achiral metal complexes for enantioselective catalytic hydro-
2
6
genations and oxidations. In these systems the protein shell
is used for providing selectivity, but does not provide large rate
enhancements over the reaction of the cofactor or metal complex
alone, precluding a direct comparison with our dendrimers in
terms of efficiency.
De novo enzyme design has also been approached by using
algorithms to discover folding peptides by computational or
5
recombinant DNA methods. Thus, computational design gener-
ated an esterase active site at the surface of thioredoxin with a
single histidine residue that catalyzed the hydrolysis of nitro-
Previous enzyme models based on SPPS for synthesis used
folding linear peptides. Johnsson et al. reported a catalytic
R-helical peptide accelerating the decarboxylation of oxaloac-
4f
phenyl acetate with k /k
cat uncat
) 180 and K ) 0.17 mM. Wey
M
and Hecht have discovered a properly folded four-helix bundle
from a designed genetic library, and reported esterase activites
for nitrophenyl acetate after introduction of surface histidine
2
2
etate using three catalytic lysine residues with kcat/kuncat ) 600.
Broo et al. have reported a designed R-helix bundle displaying
multiple histidine side chains capable of hydrolyzing nitrophenyl
residues, with k /k
binding (KM ) 3 mM).
cat uncat
) 8700, but no observable substrate
4
j
esters with catalytic proficiency kcat/KM ) 230 relative to
0c
4
-methylimidazole at pH 5.1.1 SPPS has been used to prepare
combinatorial libraries of short linear peptides, and has yielded
efficient catalysts for a variety of reactions.23 These catalytic
peptides contain some side chain protected amino acids, and
show activity and selectivity in organic solvents but not in water.
Other chemical synthetic strategies to artifical enzymes include
the modification of macromolecules (e.g., cyclodextrins) and
dendrimers (PAMAM, PPI), emphasizing the preparation of
single macromolecular models without regards to enabling
evolution and functional selection. Breslow and Schmuck
reported a rate acceleration of kcat/kuncat ) 120 for the hydrolysis
of nitrophenyl phosphate catalyzed by a bis-imidazole substi-
tuted â-cyclodextrin.1 A PPI-based polyimidazole catalyst
showed only modest esterase activities with nitrophenyl
acetate.1
Conclusion
Peptide dendrimers A1-A4 composed of the repeating
dendron Dap-His-Ser catalyze the hydrolysis of 8-acyloxypyrene
1,3,6-trisulfonates 1-3 in aqueous buffer and show a strong
positive dendritic effect in catalytic proficiency. The mechanism
of the dendrimer-catalyzed ester hydrolyses is best understood
in terms of bifunctional catalysis involving a free-base histidine
acting as nucleophile or general base, together with a protonated
histidine stabilizing the oxyanion intermediate, as evidenced by
pH-rate profile data showing a maximum activity at pH 5.5.
Mutant dendrimers were prepared to examine the dependence
of catalytic activity on amino acid composition. Seventeen
dendrimers B1-O4 incorporating hydrophobic residues at the
core as well as negatively charged residues were examined to
provide more protein-like catalysts. These dendrimers were
catalytically less efficient than the parent series A1-A4. In a
second approach a series of 15 close analogues of the third
generation dendrimer A3 were prepared by parallel synthesis.
The dendrimers were investigated for their catalytic properties
for the hydrolysis of acyloxypyrene trisulfonates 1-3, which
proved to be the only reactive substrate type for this class of
dendrimers. Exchanging the serine residues in A3 against
â-alanine (A3B) or threonine (A3C), or reshuffling histidine
and serine residues between the different positions (A3L), led
to catalytically more efficient dendrimers. A3C showed the
highest rate acceleration for a third-generation dendrimer with
kcat/kuncat ) 90 000, corresponding to kcat/kuncat) 18 000 per
catalytic site, a 10-fold improvement over the original dendrimer
A3. On the other hand A3B showed the highest specific
reactivity enhancement of (kcat/KM)/k2 ) 91 000 over the
0j
0e-i
The peptide dendrimer approach may also be compared with
catalytic antibody technology.24 Esterase antibodies are among
the best reported antibodies with rate accelerations kcat/kuncat in
3
5
the range 10 -10 , comparable with our peptide dendrimers,
although the antibodies are much larger (50 kDal per catalytic
(
20) (a) Stryer, L. Biochemistry; WH Freeman and Company: New York,
999. (b) Fersht, A. Structure and Mechanism in Protein Science; WH
Freeman and Company, New York, 1999.
21) (a) Clouet, A.; Darbre, T.; Reymond, J.-L. Angew. Chem., Int. Ed.
004, 43, 4612-4615. (b) Clouet, A.; Darbre, T.; Reymond, J.-L. Biopoly-
mers (Peptide science) 2006, 84, 114-123.
22) Johnsson, K.; Allemann, R. K.; Widmer, H.; Benner, S. A. Nature
993, 365, 530-532.
23) (a) Jarvo, E. R.; Miller, S. J. Tetrahedron 2002, 58, 2481-2495.
b) Vasbinder, M. M.; Jarvo, E. R.; Miller, S. J. Angew. Chem., Int. Ed.
001, 40, 2824-2827. (c) Wennemers, H.; Conza, M.; Nold, M.; Krattiger,
1
(
2
(
1
(
(
2
P. Chem. Eur. J. 2001, 7, 3342-3347. (d) Sigman, M. S.; Jacobsen, E. N.
J. Am. Chem. Soc. 1998, 120, 4901-4902. (e) Tang, Z.; Jiang, F.; Yu,
L.-T.; Cui, X.; Gong, L.-Z.; Mi, A.-Q.; Jiang, Y.-Z.; Wu, Y.-D. J. Am.
Chem. Soc. 2003, 125, 5262-5263. (f) Tang, Z.; Jiang, F.; Cui, X.; Gong,
L.-Z.; Mi, A.-Q.; Jiang, Y.-Z.; Wu, Y.-D. Proc. Natl. Acad. Sci. U.S.A.
2
004, 101, 5755. (g) Martin, H. J.; List, B. Synlett 2003, 1901. (h) Kofoed,
J.; Nielsen, J.; Reymond, J.-L. Bioorg. Med. Chem. Lett. 2003, 13, 2445.
i) For one example in water see: Berkessel, A.; H e´ rault, D. A. Angew.
Chem., Int. Ed. 1999, 38, 102-105.
24) (a) Lerner, R. A.; Benkovic, S. J.; Schultz, P. G. Science 1991,
(25) (a) Kuang, H.; Distefano, M. D. J. Am. Chem. Soc. 1998, 120, 1072-
1073. (b) Qi, D.; Tann, C.-M.; Haring, D.; Distefano, D. Chem. ReV. 2001,
101, 3081-3111. See also: Levine, H. L.; Kaiser, E. T. J. Am. Chem. Soc.
1978, 100, 7670-7677 for a flavin modified papain system.
(26) (a) Skander, M.; Humbert, N.; Collot, J.; Gradinau, J.; Klein, G.;
Loosli, A.; Sauser, J.; Zocchi, A.; Gilardoni, F.; Ward, T. R. J. Am. Chem.
Soc. 2004, 126, 14411-14418. (b) Thomas, C. M.; Ward, T. R. Chem.
Soc. ReV. 2005, 34, 337-46.
(
(
2
52, 659-667. (b) Schultz, P. G.; Lerner, R. A. Acc. Chem. Res. 1993, 26,
3
91-395. (c) Schultz, P. G.; Lerner, R. A. Science 1995, 269, 1835-1842.
(
d) MacBeath, G.; Hilvert, D. Chem. Biol. 1996, 3, 433-445.
4478 J. Org. Chem., Vol. 71, No. 12, 2006

Histidine-Containing Catalytic Peptide Dendrimers
reference catalyst 4-methylimidazole, which amounts to 18 200-
fold higher reactivity per catalytic site. The higher catalytic
activity of dendrimer A3B, A3C, and A3L compared to A3
might reflect favorable structural changes upon mutations
resulting in a more favorable relative placement of histidines
optimizing bifunctional catalysis.
Statistical analysis of the rate data showed that the number
of histidine residues per dendrimer was correlated with its
binding affinity 1/KM to the substrates, but did not predict the
catalytic rate constants kcat, which are strongly structure de-
pendent. Analysis of pH and ionic strength effects and ITC data
indicate that substrate binding depends on electrostatic interac-
tions between the substrate’s sulfonate groups and protonated
histidine residues combined with hydrophobic intractions be-
tween the substrate’s acyl chain and the dendrimer.
synthesis was carried out in a PSW1100 peptide synthesizer
(Chemspeed AG). The resin NovaSyn TGR (0.23 mmol/g) was
acylated with each amino acid or diamino acid (0.5 M in DMF, 3
equiv) in the presence of HBTU/HOBt (0.5 M in DMF, 3 equiv)
and N-methylmorpholine (1.0 M in DMF, 6 equiv) for 1 (first
generation), 2 (second generation), and 4 h (third generation). Each
coupling was repeated twice. The Fmoc protecting group was
removed with a solution of 20% piperidine in DMF (3 × 10 min)
and the solvent was removed by filtration. After each coupling and
Fmoc-deprotection the resin was washed with DMF (3×). At the
end of the synthesis, the resin was acylated with acetic anhydride/
CH Cl (1:1) for 1 h. The resin was washed with CH Cl and dried
2
2
2
2
under reduced pressure. The resin was dried and the cleavage was
carried out with TFA/TIS/H O (94:5:1) for 4 h. The peptide was
2
precipitated with methyl tert-butyl ether then dissolved in a water/
acetonitrile mixture. All dendrimers were purified by preparative
HPLC.
Peptide dendrimers such as those reported here are very
attractive as artificial protein models. The dendrimers are well-
behaved and show excellent stability. Most importantly, the
synthesis of peptide dendrimers proceeds in excellent yields and
uses standard solid-phase synthesis protocols readily amenable
to automated synthesis. The present study illuminates a complex
structure-function relationship in catalytic peptide dendrimers
in which the nature and position of the amino acids strongly
modulate substrate binding and catalysis. Experiments toward
the discovery of enantioselective esterase peptide dendrimers
(
((Ac-His-Thr)
2 2 2 2
DapHis-Thr) DapHis-Thr) DapHis-ThrNH
(A3C): Starting with 100 mg of NovaSyn TGR resin (0.23 mmol/
g), the dendrimer A3C was obtained as a colorless foamy solid
after cleavage from the resin and preparative RP-HPLC purification
+
(
4
19.1 mg, 13.3%). MS (ES+) calcd for C187
528.05, found 4528.75.
Purification conditions and characterization of all dendrimers is
given in the Supporting Information.
. Assays and Kinetic Measurements. Assays were followed
272 75
H N O60 [M + H]
2
in individual wells of round-bottom polystyrene 96-well plates using
a fluorescence detector with preset values of the excitation and
emission wavelengths corresponding to the measured substrate. All
pipetting manipulations were done by hand with single pipets. The
measurement temperature inside the instrument was 26.5 °C. Kinetic
experiments were followed for 2-12 h. Fluorescence data were
converted to product concentration by means of a calibration curve.
2
1
by a combinatorial chemistry approach are in progress.
Experimental Part
1. Dendrimer Synthesis. Peptide dendrimers were synthesized
with the Fmoc strategy, according to standard solid-phase procedure,
as described in the Supporting Information.
2.a. Typical Measurement of Apparent Rate Enhancements.
1.a. General Procedure for Dendrimer Synthesis. Eluent A:
In a typical experiment, 20 µL of aqueous buffer were first added
water and TFA (0.1%); eluent B: acetonitrile, water and TFA (3/
in a well, followed by 2.5 µL of the dendrimer solution (typically
2/0.1%).
0.05 mM in aq buffer, final concentration in the well was 5 µM),
The synthesis of dendrimers A1-O4 has been achieved follow-
and finally 2.5 µL of substrate solution (typically 2 mM in
acetonitrile/water 1/1, final concentration in the well was 200 µM).
ing procedure A. Procedure A: The resin NovaSyn TGR (0.25
mmol/g) was acylated with each amino acid or diamino acid (3
equiv) in the presence of BOP (3 equiv) and DIEA (5 equiv) for
The rate observed under these conditions is the apparent rate Vapp
.
Vun is the rate observed with 22.5 mL of aqueous buffer and 2.5
3
3
0 min, 1 h after the first generation, 2 h after the second generation,
h after the third generation, and 5 h after the fourth generation.
µL of substrate solution (2 mM in acetonitrile/water 1/1, final
concentration in the well was 200 µM). The observed rate
After each coupling the resin was successively washed with DMF,
MeOH, and CH Cl
enhancement is defined as Vnet/Vun
.b. Michaelis-Menten Plots. Michaelis-Menten plots were
obtained from the linear double reciprocal plot of 1/Vnet versus 1/[S]
where Vnet ) Vapp - Vun and [S] is the substrate concentration)
.
2
2
(3× with each solvent), then checked for free
2
amino groups with the TNBS test. If the TNBS test indicated the
presence of free amino groups, the coupling was repeated. In case
of uncertainty in the completion of the coupling, the potential
remaining free amino groups were capped with acetic anhydride/
(
measured with dendrimers (Vapp) or without dendrimer (Vun) in
aqueous buffer. In a typical experiment, 20 µL of aqueous buffer
were first added in a well, followed by 2.5 µL of the dendrimer
solution (typically 35.5 µM in aqueous buffer, final concentration
in the well was 3.55 µM), and 10, 20, 30, 40, 60, 80, 100, 200,
2 2
CH Cl for 10 min. The Fmoc protecting groups were removed
with a solution of 20% piperidine in DMF (2 × 10 min) and the
solvent was removed by filtration. At the end of the synthesis, the
resin was acylated with acetic anhydride/CH
resin was dried and the cleavage was carried out with TFA/anisole/
,2-ethanedithiol/H O (94:5:1:1) for 4 h. The peptide was precipi-
2 2
Cl (1:1) for 1 h. The
400, 600, 800, and 1000 µM substrate in acetonitrile/water 1/1.
Initial reaction rates Vapp and Vun were obtained from the steepest
part observed in the curve that gives fluorescence versus time,
typically during the first 2000 s for Vapp and during the first 4000
s for Vun. The rate constant kuncat without catalyst was calculated
from the slope of the linear curve that gives Vun (as product
concentration per time) versus substrate concentration [S].
1
2
tated with methyl tert-butyl ether then dissolved in a water/
acetonitrile mixture. All dendrimers were purified by preparative
HPLC.
(
((Ac-His-Ser)
Starting with 200 mg of NovaSyn TGR resin (0.25 mmol/g), the
sequence (((His-Ser) BHis-Ser) BHis-Ser) BHis-SerNH was pre-
2 2 2 2
DapHis-Ser) DapHis-Ser) DapHis-SerNH (A3):
2
2
2
2
2
2.c. k (4-methylimidazole). A stock solution of 40 mM of
pared. Half of the resin was acetylated and the product cleaved
from the resin and purified by preparative HPLC to give A3 as a
4-methylimidazole was prepared, buffered in the desired aqueous
buffer. The reaction rate with 4-methylimidazole (4-MeIm) was
obtained under the same conditions as above with 40, 60, 80, 100,
200, 300, 400, 500, 800, and 1000 µM 4-MeIm, 200 µM substrate
in acetonitrile/water 1/1, and aq buffer. The second-order rate
colorless foamy solid (34.2 mg, 22.7%). MS (ES+) calcd for
+
C
172
H
242
N
75
O
60 [M + H] 4317.81, found 4318.13.
The synthesis of dendrimers A3A to A3O has been achieved by
Dr. Quang N′Guyen (Chem Speed AG) following procedure B.
Procedure B: Automated Solid-Phase Dendrimer Synthesis. The
2
constant k was calculated from linear regression of the experi-
mentally measured pseudo-first-order rate constants k′ as a function
J. Org. Chem, Vol. 71, No. 12, 2006 4479

Delort et al.
of 4-MeIm concentrations. The second-order rate constant k
2
is
mM aqueous solution of dendrimer (or 40 mM 4-methylimidazole
aqueous solution containing ca. 1.2 equiv of TFA) without or with
pyrene 1,3,6,8-tetrasulfonate 6, with respect to the number of
binding sites as determined by isothermal titration calorimetry (5
equiv of 6 was added to A3A, 10 equiv of 6 was added to A4).
The experiments were repeated and gave reproducible values within
10% error.
given by k
substrate.
2
) k′/[S], where [S] indicates the concentration of
2
.d. Inhibition with Pyrene 1,3,6,8-Tetrasulfonate. The inhibi-
tion constant K was obtained from the linear reciprocal plot of
/Vnet vs [I] measured similarly with (final concentrations) 5 µM
dendrimer and 200 µM substrate (Vapp) and 0, 10, 20, 40, 60, 80,
I
1
1
5
00, 200, 400, 600, 800, and 1000 µM pyrene 1,3,6,8-tetrasulfonate,
mM citrate pH 5.5, 26.5 °C.
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.
2.e. Ionic Strength. Ionic strength effect, i.e., initial velocities
of product formation as a function of KCl concentration, was studied
with Michaelis-Menten plots obtained as described above. Condi-
tions, if not varied as indicated: 3.55 µM dendrimer (final
concentration in the well), 10, 20, 30, 40, 60, 80, 100, 200, 400,
Supporting Information Available: Procedures for dendrimer
synthesis, HPLC, and MS data of all dendrimers synthesized,
procedures for kinetic measurements, and ITC and pH studies. This
material is available free of charge via the Internet at http://
pubs.acs.org.
6
00, 800, and 1000 µM substrate (final concentration in the well),
and 5 mM citrate buffer pH 5.5 containing KCl (respectively 0, 5,
0, 50, 100, 200, 300, and 500 mM). For each buffer, the linear
1
relationship between fluorescence and product concentration was
determined with a calibration curve.
a
2.f. pK Determination. The histidine residues of the dendrimers
were titrated by adding aliquots (20 µL) of 2 mM NaOH in a 1
JO060273Y
4480 J. Org. Chem., Vol. 71, No. 12, 2006