Synthesis and Esterolytic Activity of Catalytic Peptide Dendrimers

Article abstract of DOI:10.1002/chem.200305578

Peptide dendrimers were prepared by solid-phase peptide synthesis. Monomeric dendrimers were first obtained by assembly of a hexa-peptide sequence containing alternate standard a-amino acids with diamino acids as branching units. The monomeric dendrimers were then dimerized by disulfide-bridge formation at the core cysteine. The synthetic strategy is compatible with functional amino acids and different diamino acid branching units. Peptide dendrimers composed of the catalytic triad amino acids histidine, aspartate, and serine catalyzed the hydrolysis of N-methylquinolinium salts when the histidine residues were placed at the outermost position. The dendrimer-catalyzed hydrolysis of 7-isobutyryl-N-methylquinolinium followed saturation kinetics with a rate constant of catalysis/rate constant without catalysis (k_cat/k_uncat) value of 3350 and a rate constant of catalysis/Michaelis constant (k_cat/K_M) value 350-fold larger than the second-order rate constant of the 4-methylimidazole-catalyzed reaction; this corresponds to a 40-fold rate enhancement per histidine side chain. Catalysis can be attributed to the presence of histidine residues at the surface of the dendrimers.

Full text of DOI:10.1002/chem.200305578

FULL PAPER
Synthesis and Esterolytic Activity of Catalytic Peptide Dendrimers
David Lagnoux,[a] Estelle Delort,[a] Cÿline Douat-Casassus,[a] Annamaria Esposito,[b] and
Jean-Louis Reymond*[a]
Abstract: Peptide dendrimers were
prepared by solid-phase peptide syn-
thesis. Monomeric dendrimers were
first obtained by assembly of a hexa-
peptide sequence containing alternate
standard a-amino acids with diamino
acids as branching units. The monomer-
ic dendrimers were then dimerized by
disulfide-bridge formation at the core
cysteine. The synthetic strategy is com-
patible with functional amino acids and
different diamino acid branching units.
Peptide dendrimers composed of the
catalytic triad amino acids histidine, as-
partate, and serine catalyzed the hy-
drolysis of N-methylquinolinium salts
when the histidine residues were
placed at the outermost position. The
dendrimer-catalyzed hydrolysis of 7-
constant of catalysis/rate constant with-
out catalysis (kcat/kuncat) value of 3350
and a rate constant of catalysis/Michae-
lis constant (kcat/KM) value 350-fold
larger than the second-order rate con-
stant of the 4-methylimidazole-cata-
lyzed reaction; this corresponds to a
40-fold rate enhancement per histidine
side chain. Catalysis can be attributed
to the presence of histidine residues at
the surface of the dendrimers.
isobutyryl-N-methylquinolinium
fol-
lowed saturation kinetics with a rate
Keywords: dendrimers
models ester hydrolysis
peptides ¥ solid-phase synthesis
¥
enzyme
¥
¥
Introduction
The analysis of protein structures over the years has given
sufficient insights to permit the rational design and synthesis
of single synthetic peptides that fold into predetermined
topologies, such as four-helix bundles.[2] Nevertheless the ap-
proach is limited. The vast majority of artificial proteins are
produced by semiempirical approaches which, in one way or
another, circumvent the need for complete rational design
by applying experimental or theoretical search algorithms
inspired by natural selection and evolution. These methods
are based on defining boundaries for active structures and
randomly generating candidates within these boundaries;
the candidates are then selected on the basis of their proper-
ties. The strategy concerns computational predictions of the
three-dimensional structure of proteins,[3] catalytic antibod-
ies,[4] and the directed evolution of enzymes.[5]
Our approach to de novo enzyme design is based on pep-
tide dendrimers (Scheme 1). Dendrimers are ramified struc-
tures that adopt globular or disk-shaped structures as a con-
sequence of topology rather than folding.[6] Catalytic den-
drimers are known that are based on ether linkages with in-
corporation of catalytically active subunits such as metal
complexes and cofactors, either at the surface or at the core
of the dendrimer, which results in systems reproducing the
catalytic properties of these cofactors.[7] The dendrimer is
used either to provide a particular microenvironment or to
increase molecular size and facilitate catalyst separation and
recovery. We reasoned that dendrimeric architectures ap-
plied to a peptide sequence would provide an efficient strat-
Enzyme catalysis is one of the most fascinating properties
encountered in proteins. Enzyme catalysis is characterized
by the emergence of selective molecular recognition, transi-
tion-state stabilization, and regulation processes in the
folded protein which are not encountered at the level of the
amino acid building blocks.[1] These processes appear as a
consequence of the interplay between amino acids within
the ordered three-dimensional structure produced by the
folding process. Rational de novo enzyme design, which is
one of the main goals of supramolecular chemistry and bio-
chemistry, would require a complete understanding of both
folding and catalysis. The key difficulty resides in choosing
the right amino acid sequence and length among the combi-
natorial infinity of possibilities to fold into a stable function-
al structure.
[a] D. Lagnoux, E. Delort, Dr. C. Douat-Casassus, Prof. J.-L. Reymond
Department of Chemistry and Biochemistry
University of Bern
Freiestrasse 3, 3012 Bern (Switzerland)
Fax : (+41)31-631-80-57
E-mail: reymond@ioc.unibe.ch
[b] Dr. A. Esposito
Ecogreen s.r.L.
Via Bachelet 13, 07036 Sennori-SS (Italy)
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
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Chem. Eur. J. 2004, 10, 1215 1226
DOI: 10.1002/chem.200305578
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

FULL PAPER
egy to circumvent the protein-folding problem. Such an ap-
proach would lead to protein-like structures where catalysis
or molecular-recognition phenomena might appear by con-
structive interactions between amino acids as in natural pro-
teins. Herein we report a versatile synthetic strategy for
functionalized peptide dendrimers. More than one hundred
different peptide dendrimers were prepared based on solid-
phase peptide synthesis. The catalytic properties of estero-
lytic dendrimers obtained by using catalytic triad amino
acids are discussed.[8]
Results
Strategy: Dendrimers are usually synthesized either by a di-
vergent or a convergent strategy. In the divergent strategy
sequential assembly starts from the dendrimer core and ex-
tends to the outside by addition of branching units and
spacers. This approach is applied in polymerization or oligo-
merization protocols to produce statistical dendrimer popu-
lations with average molecular sizes. The convergent strat-
egy proceeds in the opposite direction by condensing preas-
sembled dendrons to a common core to form a new, larger
dendron and by repeating this cycle iteratively. The ap-
proach is equivalent to the convergent strategy in natural
product synthesis and is usually applied for the synthesis of
dendrimers consisting of a single molecular species with de-
fined molecular weight.
Peptides are most efficiently prepared by solid-phase pep-
tide synthesis (SPPS), whereby N-protected amino acid
building blocks are condensed sequentially to the free
amino terminus of a growing peptide chain attached to the
solid support.[9] The key advantage is the possibility of using
a large excess of N-protected activated amino acid in solu-
tion to maximize the coupling efficiency to the amino termi-
nus anchored on the solid support. SPPS can furthermore be
automated and all common building blocks and reagents are
commercially available. We envisioned a divergent synthesis
of peptide dendrimers based on SPPS whereby standard a-
amino acid building blocks would alternate with tailored di-
amino acids as branching units (Scheme 1). The exploration
of synthetic peptide dendrimers would only be interesting if
a certain level of structural diversity could be achieved. Di-
versity would be realized by using different a-amino acids at
the variable positions between the branching diamino acids.
We also took the opportunity to multiply structural diversity
by chain dimerization through disulfide bridges. Disulfide
coupling is an efficient synthetic protocol to assemble frag-
ments of peptides or proteins and enables the formation of
both homo- and heterodimers.[10] This design applied to
second-generation dimeric dendrimers could provide as
many as 1.7î109 different sequences from 19 proteinogenic
a-amino acids and 3 different branching diamino acids. Our
first attempt at preparing functional peptide dendrimers was
aimed at finding peptide dendrimers that could catalyze an
ester hydrolysis reaction. Following the well-known model
of esterolytic enzymes, peptide dendrimers containing com-
binations of the catalytic triad amino acids aspartate, histi-
dine, and serine were prepared.
Scheme 1. General structure of peptide dendrimers. An =a-amino acid,
B=branching diamino acid, Fmoc=9-fluorenylmethoxycarbonyl.
Design and synthesis of building blocks: Ordered polylysine
dendrimers have been prepared on solid support as antigen-
display units.[11] However, we wanted to use shorter, more
symmetrical branching units than lysine to obtain symmetri-
cally growing dendrimers. We therefore chose diamino acids
1 3 as branching units. The branching diamino acid 1 was
prepared in three steps from 1,3-diamino-2-propanol
(Scheme 2). O-alkylation of the Boc-protected derivative 6
with ethyl bromoacetate gave 7. Saponification of the ethyl
ester with LiOH in THF, followed by treatment with 3n
HCl, gave the free diamino acid, which was reprotected with
Fmoc-Cl in 1,4-dioxane in the presence of sodium hydrogen
carbonate to give the bis-Fmoc derivative 1. The synthesis
of branching diamino acid 2 started with double alkylation
of dimethyl malonate with benzyl bromoacetate (NaH,
THF, 98%). Reductive debenzylation gave diacid 9, which
was converted into the bis(acyl) azide via the acyl chloride.
A thermal Curtius rearrangement in refluxing anhydrous
toluene in the presence of 9-fluorenemethanol gave carba-
mate 10. Finally, saponification and decarboxylation of the
remaining diester function with AcOH and 5n HCl under
reflux conditions gave the desired branching unit 2. The
commercially available Fmoc-protected (S)-2,3-diaminopro-
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Chem. Eur. J. 2004, 10, 1215 1226

Catalytic Peptide Dendrimers
1215 1226
ble combinations of aspartate, histidine, and serine at posi-
tions A1, A2, and A3. Each monomeric dendrimer was puri-
fied by preparative reversed-phase (RP) HPLC. The puri-
fied monomeric dendrimers were then dimerized with aldri-
thiol,[12] a reaction that allowed the preparation of all 21
possible combinations of disulfide-bridged dimeric dendrim-
ers. The same protocol was used to prepare equivalent den-
drimer families based on branching diamino acids 2 and 3
(Scheme 1). Somewhat to our surprise, the 2,3-diaminopro-
panoic acid branching unit 3 yielded to the same protocol
and gave similar yields of isolated products in spite of it
being the smallest branching unit.
Screening for esterolytic activity: The 60 dimeric dendrimers
obtained were assayed for hydrolysis of fluorogenic and
chromogenic ester substrates. We used a series of 7-hydroxy-
coumarin esters and acyloxymethyl ethers from our labora-
tory,[13] as well the commercially available esterase sub-
strates 8-acetoxypyrene-1,3,6-trisulfonate, 7-acetoxycoumar-
in-3-carboxylic acid, and 7-acetoxy-1-methylquinolinium
iodide (14). The dendrimers were assayed for activity in
aqueous buffer (pH 7.0 or 6.0) in the presence of the differ-
ent substrates in a microtiter-plate setup. The reactions were
followed over several hours and the apparent rates of hy-
drolysis were compared to the rates in the absence of den-
drimer. The rates of hydrolysis of 7-acetoxy-1-methylquino-
linium iodide (14) were accelerated by some of the dendrim-
ers at pH 6.0 (Scheme 3, Table 1). Calibration with the prod-
uct 18 in the presence of the dendrimers showed that the
effect observed was not due to a dendrimer-induced fluores-
cence enhancement. Product formation was confirmed by
HPLC analysis of the reaction mixture. The catalytic den-
Scheme 2. Synthesis of dendrimer building blocks. Conditions:
a) (Boc)2O, Et3N, CH2Cl2/MeOH, 2 h, 258C, 98%; b) BrCH2CO2Et,
NaH, THF, 258C, 5 h, 54%; c) LiOH, THF/H2O, 25 8C, 15 h; then 3n aq.
HCl, 608C, 3 h; then, Fmoc-Cl, NaHCO3, H2O/dioxane, 258C, 15 h, 66%;
d) H2 (4 bars), Pd/C, EtOH, 14 h, 91%; e) (COCl)2, CH2Cl2, cat. DMF,
258C, 7 h; then TMSN3, CH2Cl2, 258C, 20 h; then 9-fluorenemethanol,
toluene, reflux, 18 h, 43%; f) AcOH, 2n HCl, reflux, 15 h, 65%; g) 40%
aq. CH2O, NaBH3CN, DMF, 258C, 5 h, 83%; g) (COCl)2, CH2Cl2, cat.
DMF, 258C, 2 h; then KOtBu (3 equiv), CH2Cl2, 08C, 3 h, 67 %; h) KOH
(1 equiv), tBuOH/CH2Cl2, 408C, 12 h; then HCl, 37%. Boc=tert-butoxy-
carbonyl,
DMF=N,N-dimethylformamide,
THF=tetrahydrofuran,
TMS=trimethylsilyl.
panoic acid 3 was used as a third branching unit. In addition
to the linkers, we also prepared the monoprotected 5-dime-
thylamino-isophthalic acid 4 from 5-amino-isophthalic acid
11, to be used as capping group. Addition of this acid as the
last building block at the dendrimer surface would provide
homogeneous properties such as aqueous solubility at neu-
tral pH values, by means of the negatively charged carboxy-
late, and easy traceability by HPLC due to the aminobenza-
mide chromophore.
SPPS of peptide dendrimers: The SPPS protocol for den-
drimer synthesis was developed with diamino acid 1, which
was expected to be the least problematic in terms of steric
crowding. We adopted an Fmoc strategy on polystyrene
resin functionalized with Rink amide (0.47 mmolgꢀ1) and
used 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumte-
trafluoroborate (TBTU) or benzotriazol-1-yloxytris(dime-
thylamino)phosphonium hexafluorophosphate (BOP) as the
coupling reagent. This protocol was used to prepare the six
monomeric dendrimers, A1 F1, resulting from the six possi-
Scheme 3. Acyloxy-N-methylquinolinium derivatives assayed for hydroly-
sis by catalytic peptide dendrimers. Esters 14 17 are accepted as sub-
strates to give the fluorescent product 18. Ester 19 21 are not accepted
as substrates.
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J.-L. Reymond et al.
FULL PAPER
Table 1. Initial screening results for the hydrolysis of quinolinium ester 14 by catalytic peptide dendrimers.
tyryl ester 16. The dendrimers
did not show any significant
degree of enantioselectivity
with the enantiomeric substrate
pair 17 (Table 2, Figure 1, and
Figure 2).
[a]
The apparent rate enhancement (Vnet/Vuncat
)
is reported for monomeric dendrimers and all combinations of
dimers.
Monomer
A1
B1
C1
D1
E1
F1
A1
B1
C1
D1
E1
F1
0.5
0.0
0.6
0.4
2.1
1.7
0.2
0.7
0.3
0.2
3.1
2.1
0.5
0.6
0.5
1.9
0.7
0.4
0.7
1.6
1.8
0.4
1.4
1.0
Dendrimer variations: Since the
esterolytic activity of our cata-
lytic peptide dendrimers was
strongly correlated with the
presence of histidine residues at
the outermost position in the
sequence, we set out to prepare
a new dendrimer family incor-
porating histidine as the catalyt-
ic amino acid together with a
broader selection of other
amino acids. A new family of
22 dendrimers incorporating
histidine together with the
amino acids leucine, arginine,
tryptophan, or phenylalanine,
as well as aspartate and serine,
was designed (Table 3). All the
dendrimers were prepared with
9.2
12.0
6.4
Monomer
A2
B2
C2
D2
E2
F2
A2
B2
C2
D2
E2
F2
0.2
0.1
0.4
0.4
2.0
1.5
0.0
0.5
0.4
0.5
1.8
1.5
0.2
1.1
0.4
1.9
1.5
0.4
0.4
1.4
0.4
1.0
1.1
2.8
1.9
2.8
Monomer
A3
B3
C3
D3
E3
F3
A3
B3
C3
D3
E3
F3
2.2
0.8
2.3
1.8
12.2
9.7
0.3
0.2
0.4
2.1
1.4
0.2
0.4
4
0.9
0.4
3.2
1.9
7.6
3.7
8.4
5.8
0.9
5.6
4.6
[a] With 5 mm dendrimer and 200 mm substrate. For other conditions and methods, see the Experimental Sec-
tion. Vuncat is the hydrolysis rate of 14 in buffer without catalysis. Vnet =VappꢀVuncat, where Vapp is the apparent
hydrolysis rate of 14 in the presence of dendrimer.
drimers retained activity upon resynthesis from the amino
acid constituents, a result showing that the observed effects
were not due to impurities in the samples. The dendrimers
appeared unchanged upon HPLC and MS analysis after the
reactions; this shows that they were not permanently acylat-
ed during the reaction. Catalysis was further confirmed by
the observation of multiple turnovers.
diamino acid 1 as the branching unit and were obtained in
satisfactory yields. Aldrithiol-mediated dimerization was
then carried out for all combinations. Disulfide-bond forma-
tion was much slower with hydrophobic dendrimers, with re-
action times extending up to 24 h for the more hydrophobic
dendrimers. Homo- and heterodimers were nevertheless ob-
tained in good yields, with the exception of the HWS HWS
and DHF DHL dendrimers, which were not formed even
after repeated attempts under various conditions. In fact,
the aldrithiol-activated HWS dendrimer did not undergo
any reaction and the corresponding heterodimers were ob-
tained by using the HWS dendrimer as a free thiol nucleo-
phile in combination with another thiol-activated dendrimer.
Kinetic studies: A pH-profile study under screening condi-
tions (5 mm dendrimer, 200 mm substrate) showed a maxi-
mum of activity at pH 6.0, in agreement with a possible role
of the histidine side chain as a catalytic side chain. All fur-
ther measurements were therefore carried out at that pH
value. Catalysis was proportional to the dendrimer concen-
tration up to 10 mm dendrimer. However catalysis did not in-
crease above that concentration, a fact suggesting the forma-
tion of catalytically inactive aggregates.
Catalysis with a range of additional quinoline-derived sub-
strates was also examined. The substrates were prepared by
esterification of hydroxyquinoline, followed by quaterniza-
tion with dimethylsulfate.[14] The hexanoyl 15, isobutyryl 16,
and (R)- and (S)-a-methylphenacetyl derivatives 17 were
also accepted as substrates. By contrast, the more sterically
hindered pivalate 19 and benzoate 20, as well as the isomer-
ic 6-acetoxy-1-methylquinolinium iodide (21), were not hy-
drolyzed by the dendrimers. The kinetics of hydrolysis with
the reactive substrates 14 17 and the most active dendrimers
were characterized in detail and followed the Michaelis
Menten model, with Michaelis constant (KM; dissociation
constant of the substrate dendrimer complex) values in the
range of 0.1 1 mm and rate constant of catalysis/rate con-
stant without catalysis (kcat/kuncat) values in the range of 102
103. The best substrate across all dendrimers was the isobu-
Figure 1. Time-course graph for the hydrolysis of 16 to form 18 in the ab-
sence (*) or presence of dendrimers E1 F1 (*), E2 E2 (&), or E3 E3
(~), with 200 mm substrate and 5 mm catalyst.
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Chem. Eur. J. 2004, 10, 1215 1226

Catalytic Peptide Dendrimers
1215 1226
Table 2. Michaelis Menten parameters for the six most active dendrimers on substrates 14 17.[a]
We also prepared the all-histi-
dine monomeric dendrimer
(HHH) derived with branching
unit 1. The hydrophobic den-
drimer LRH was coupled with
this all-histidine monomeric
dendrimer, as well as with a se-
lection of dendrimers from the
aspartate/histidine/serine family
(Table 3).
14
15
16
(S)-17
(R)-17
E[b]
kuncat [minꢀ1
]
3.6î10ꢀ4
6.7î10ꢀ3
2.4î10ꢀ4
5.6î10ꢀ3
1.2î10ꢀ4
4.3î10ꢀ3
2.2î10ꢀ4
5.0î10ꢀ3
2.2î10ꢀ4
4.8î10ꢀ3
4-methylimidazole
k2[c] [mmꢀ1 minꢀ1
]
E1 E1
KM [mm]
0.20
0.31
860
230
9.2
0.21
0.26
1050
220
0.11
0.28
2333
592
0.17
0.21
944
244
17.8
0.22
0.20
891
192
15.5
kcat [minꢀ1
kcat/kuncat
]
[c]
kcat/KM:k2
Vnet/Vuncat
1.27
[d]
12.5
23.3
E1 F1
E2 E2
E2 F2
E3 E3
E3 F3
KM [mm]
0.14
0.12
340
130
12.0
0.12
0.33
1380
360
0.23
0.30
2455
280
0.12
0.14
637
229
13.0
0.15
0.12
487
148
10.4
The new series was screened
for catalysis with the substrates
kcat [minꢀ1
kcat/kuncat
]
[c]
kcat/KM:k2
Vnet/Vuncat
1.55 used for the catalytic triad den-
drimer series described above.
There was no significant cata-
lytic effect in any of the den-
drimers tested, including LRH
HHH, which contained the all-
histidine monomeric dendrimer.
An all-histidine monomeric
dendrimer was also prepared
with diamino acid 3, 3(HHH),
and converted into the corre-
sponding all-histidine homo-
dimer 3(HHH HHH). The all-
histidine homodimer showed
esterolytic activity with sub-
strates 14, 15, 16, (S)-17, and
(R)-17, with apparent rates of
[d]
18.8
26.5
KM [mm]
0.25
0.10
268
57
0.07
0.09
380
226
3.7
0.69
0.25
2010
83
kcat [minꢀ1
kcat/kuncat
]
[c]
kcat/KM:k2
Vnet/Vuncat
[d]
2.8
11.1
KM [mm]
0.27
0.06
173
30
0.24
0.08
322
57
0.48
0.15
1214
72
kcat [minꢀ1
kcat/kuncat
]
[c]
kcat/KM:k2
Vnet/Vuncat
[d]
1.9
2.3
6.3
KM [mm]
0.29
0.14
380
70
0.32
0.17
690
90
0.264
0.41
3350
360
0.23
0.18
816
154
7.8
0.16
0.18
828
243
11.8
kcat [minꢀ1
kcat/kuncat
]
[c]
kcat/KM:k2
Vnet/Vuncat
[d]
4.1
6.2
31.8
hydrolysis (Vnet/Vuncat) of 6.5,
KM [mm]
0.23
0.08
220
50
0.14
0.08
330
100
4.9
0.634
0.25
2011
90
0.16
0.11
519
142
6.9
0.08
0.10
463
278
6.4
kcat [minꢀ1
kcat/kuncat
]
6.7, 6.2, 5.3, and 6.1, respective-
ly.
[c]
kcat/KM:k2
Vnet/Vuncat
[d]
2.7
16.2
[a] With 5 mm dendrimer and 40 700 mm substrate. For other conditions and methods, see the Experimental
Section. The kinetic constants given are derived from the linear double-reciprocal plots of 1/Vnet against 1/[S]
(Figure 1), with r2 >0.95. [b] E=(kcat/KM((S)-17))/(kcat/KM((R)-17)). [c] k2 is the second-order rate constant for
the reaction of the substrate with 4-methylimidazole. The ratio of kcat/KM to k2 gives a quantitative comparison
of dendrimer catalysis under the reaction conditions. [d] Vnet/Vuncat was observed with 5 mm dendrimer and
200 mm substrate (see legend of Table 1).
Discussion
The chemical synthesis of large
molecules is intrinsically com-
plex and synthetic availability is
a key issue in supramolecular
chemistry. In the context of
enzyme models, synthesis plan-
ning should also incorporate diversity to allow for variations
in binding and catalysis. The preparation of catalytic peptide
dendrimers demonstrated here is advantageous in both re-
spects. By using a carefully optimized peptide-coupling pro-
tocol we obtained yields of up to 30% after HPLC purifica-
tion for the monomeric dendrimers, which is in the range ex-
pected for linear peptide sequences of similar length.
Branching and resin loading did not affect synthesis signifi-
cantly. The fact that synthesis succeeded with the standard
Rink-amide resin at the usual loading of 0.47 mmolgꢀ1 is
noteworthy, as are the high yields obtained with the com-
mercially available branching unit 2,3-diaminopropanoic
acid 3. Dendrimer synthesis was exemplified with a broad
range of amino acids, including acidic, basic, polar, hydro-
phobic, and aromatic amino acids. Diversity was efficiently
increased by the dimerization strategy through disulfide
bridges, which enabled us to access a sizeable structural
Figure 2. Double reciprocal plot for hydrolysis of ester 16 catalyzed by
peptide dendrimers E1 F1 (*) and E3 F3 (~).
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J.-L. Reymond et al.
FULL PAPER
Table 3. Hydrophobic dendrimers with branching unit 1. Amino acids A1, A2, and A3 correspond to one mon-
quantitative comparison of den-
drimer catalysis along these
lines is given by the ratio of
second-order rate constants for
the reaction of the substrates
with the dendrimers (kcat/KM)
and with 4-methyl-imidazole
(k2) under the reaction condi-
tions (see Table 2). The best
dendrimers accelerate hydroly-
sis by approximately 350-fold
over the rate with 4-methylimi-
dazole. Since these dendrimers
display a total of eight histi-
dines at their surface, this cor-
responds to a 40-fold rate en-
hancement per histidine side
chain. Similar acceleration fac-
tors have been reported by
Baltzer and co-workers for the
hydrolysis of nitrophenyl esters
by using designed helical pepti-
des containing six histidines.[18]
Catalytic efficiency in den-
drimers having the same amino
acid sequence was strongly in-
omeric dendrimer and A1’, A2’, and A3’ correspond to the second monomeric dendrimer.
Entry
A3
A2
A1
A1’
A2’
A3’
Yield[a] [%]
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
D
D
H
L
L
L
L
L
L
L
H
F
L
H
S
11
20
12
14
26
27
40
74
54
54
45
30
47
39
55
63
60
47
54
30
39
35
W
R
R
R
R
R
R
R
R
R
W
W
W
W
H
H
H
H
F
H
H
H
H
H
H
H
H
H
S
S
S
S
L
L
L
L
H
H
S
L
W
H
F
S
H
S
H
R
H
S
H
S
S
H
S
H
D
D
H
D
D
H
L
D
H
D
D
H
D
D
D
H
D
H
D
S
H
H
H
L
D
S
H
D
S
L
L
H
H
H
H
D
D
D
D
D
D
H
L
D
H
H
S
F
F
[a] Entries 1 4 give yields for the monomer after HPLC purification. Entries 5 22 give yields of dimerization
after HPLC purification.
family from only a small number of monomeric dendrimer
building blocks. Semipreparative HPLC on milligram quan-
tities of dendrimers provided a convenient method to obtain
each dendrimer in pure form. All the dendrimers prepared
were very well behaved in terms of chemical stability. In
particular, the heterodimers did not show any disproportio-
nation, a fact indicating that disulfide-exchange reactions
were slow. The low reactivity of the disulfide group was also
evident from the long dimerization times of up to 24 h ob-
served in some of the dendrimers. These results suggest that
larger scale preparation of any dendrimer is possible and
that the procedure can be automated.
While only proteinogenic l-amino acids were used here,
structural diversity in the dendrimers could be further ex-
tended to the enantiomeric d-amino acids and to nonnatural
amino acid side chains. Amino acids bearing catalytic side
chains[15] and fluorescent groups[16] would be relevant for de-
veloping other catalytic or biosensor peptide dendrimers.
Dendrimers based on b- or g-amino acids[17] could also be
considered. Combinatorial split-and-mix protocols were not
addressed in this study since the dependence of coupling ef-
ficiency on sequence and on the nature of the diamino acid
branching unit was not known.
The choice of an ester hydrolysis reaction was guided by a
number of precedents in the history of enzyme models. Tar-
geting ester hydrolysis naturally led to the choice of the cat-
alytic triad amino acids aspartate, histidine, and serine as
the variable amino acids. All substrates tested were catalyti-
cally hydrolyzed by 4-methylimidazole, which is the simplest
model for the histidine side chain. The fact that catalytic ac-
tivity only appeared when histidine was positioned at the
outermost position in the dendrimer suggests that nucleo-
philic catalysis by the imidazole side chain is involved. A
fluenced by the linking diamino acid used. The best results
were observed for the homodimer E3 E3 with the diamino
acid linker 3 on the isobutyryl ester 16. Dendrimers pre-
pared with the bis(b-alanine) linker 2 showed almost no ac-
tivity with the acetate ester 14 and the hexanoate 15 and
only showed sizable activity with the isobutyryl substrate 16.
By contrast, dendrimers made from linkers 1 and 3 were
quite active across all the different quinolinium esters 14
17.
Catalytic activity was also observed with the monomeric
dendrimers derived from the shorter diamino acids 2 and 3.
The catalytic efficiency in these monomeric dendrimers was
slightly higher than that of the dimeric dendrimers when re-
ferred to the number of catalytic histidine residues. By con-
trast, with the longer branching unit 1 there was almost no
catalysis in the monomeric dendrimers. These observations
suggest that a productive interplay between several histidine
side chains only occurs under certain conditions. In the case
of the longer linker 1, dimerization is probably required to
enforce steric crowding of the side chains and the monomer-
ic dendrimers are too flexible to acquire strong activity. In
the dendrimers made with the shorter linkers 2 and 3, the
amino acids are close enough for productive interaction in
the monomeric dendrimers and dimerization does not en-
hance catalysis.
The observation of histidine-only catalysis was challenged
with the preparation of a dendrimer family containing histi-
dine and noncatalytic amino acids. To our surprise, there
was no significant catalytic effect within this series with any
of the substrates tested, in particular with the dendrimers
displaying surface histidine side chains as in the catalytic
series above. This was all the more puzzling since these den-
drimers were similar to those of the histidine/aspartate/
1220
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2004, 10, 1215 1226

Catalytic Peptide Dendrimers
1215 1226
with NaOH pellets and then to pH 8 with aq. sat. NaHCO3 (10 mL). A
solution of Fmoc-Cl (1.30 g, 5.02 mmol) in dioxane (15 mL) was added at
08C and the mixture was stirred overnight at room temperature. After
addition of water (until the mixture became cloudy), the solution was
washed twice with diethyl ether. The aqueous phase was acidified to
pH 2 with aq. conc. HCl and extracted with EtOAc. The organic phase
was evaporated and the residue purified by flash column chromatography
(hexane/EtOAc/AcOH (3.8:6:0.2)) to give 1 (895 mg; 66%) as a colorless
solid; m.p. 2058C; 1H NMR (300 MHz, [D6]DMSO): d=7.66 (m, 4H),
7.40 (m, 4H), 7.31 (m, 8H), 4.23 (m, 6H), 4.10 (s, 2H), 3.36 (m, 1H),
3.12 (m, 4H) ppm; 13C NMR (75 MHz, DMSO-d6): d=162.07, 145.87,
133.48, 130.32, 117.24, 116.70, 114.80, 109.72, 67.02, 55.87, 55.14, 36.33,
31.38 ppm; FABMS: 593 [M+]; HRMS: calcd. for C35H32N2O7: 593.2288;
found: 593.2287.
serine series. Most strikingly, heterodimers containing one
of the ™catalytic∫ halves combined with a more hydrophobic
half were also not active (LRH HHH). These results indi-
cate that other factors beyond histidine proximity at the sur-
face control catalytic activity. The more hydrophobic den-
drimers might adopt a compact, noncatalytic structure due
to hydrophobic packing of the side chains.
Conclusion
Peptide dendrimers were prepared by solid-phase synthesis,
alternating standard a-amino acids with diamino acid
branching units, followed by disulfide dimerization of core
cysteine residues. 103 different dendrimers were obtained
from diamino acids 1 3 as branching units and a variety of
acidic, basic, hydrophobic, aromatic, and hydrophilic amino
acids. Dendrimers containing catalytic triad amino acids
with the histidine at the outermost position catalyzed the
hydrolysis of N-methylquinolinium esters 14 17 with
enzyme-like kinetic behavior, thereby demonstrating that
the dendrimers can display functionality. Catalysis was
strongly influenced by the diamino acid branching unit and
the actual amino acid sequence. Further experimentation
will be required to discover catalytic dendrimers with practi-
cal catalytic potencies or selective molecular recognition
properties. The general strategy for dendrimer assembly de-
lineated here enables the assembly of billions of different
structures. A combinatorial synthetic protocol[19] combined
with activity screening can be considered to explore much
larger numbers of dendrimers.
N-(3-(9-Fluorenylmethyloxycarbonylamino)-2-carboxyprop-1-yl)carbamic
acid 9-fluorenylmethyl ester (2): A solution of 10 (2.89 g, 4.55 mmol) in
AcOH (30 mL) and aq. 2n HCl (5 mL) was stirred under reflux condi-
tions for 15 h. The solution was cooled and toluene was added. The pre-
cipitate was filtered to gave 2 (1.67 g, 2.98 mmol, 65%) as a white solid;
m.p. 2018C; IR (KBr): n˜ =3326 (m), 2946 (s), 1699 (s), 1538 (m), 1450
1
(m), 1258 (w), 1450 (m), 1258 (w) cmꢀ1; H NMR (300 MHz, CDCl3): d=
7.35 (d, J=7.35 Hz, 4H), 7.57 (d, J=7.71 Hz, 4H), 7.27 (m, J=7.35,
11.0 Hz, 8H), 5.74 (t, J=6.27 Hz, 2H), 4.35 (d, J=7.0 Hz, 4H), 4.18 (t,
J=7.0 Hz, 2H), 3.61 (m, 2H), 3.29 (m, 2H), 2.74 (t, J=5.5 Hz, 1H) ppm;
13C NMR (75 MHz, CDCl3): d=175.22, 157.49, 144.45, 14.86, 128.27,
127.65, 125.171, 120.53, 67.41, 47.87, 45.87, 39.51, 21.49 ppm; HRMS:
calcd for C34H30N2O6: 563.2182; found: 563.2181.
5-Dimethylamino-isophthalic acid mono-tert-butyl ester (4): A solution
of 13 (5.5 g, 17.11 mmol) in tert-butanol (55 mL) and dry CH2Cl2 (11 mL)
at room temperature was treated with 20n KOH (855 mL, 1 equiv) and
heated at 408C for 12 h. After removal the solvent, the crude product
was dissolved in CH2Cl2 and extracted with water (3î). The water layer
was filtered over celite and then acidified with 1n HCl to pH 4 5 to give
4
(1.66 g, 37%) as a
white precipitate; m.p. 175 1838C; 1H NMR
(300 MHz, DMSO): d=7.72 (s, 1H), 7.43 (s, 1H), 7.39 (s, 1H), 2.98 (s,
6H), 1.55 (s, 9H) ppm; 13C NMR (75 MHz, DMSO): d=167.2, 164.9,
150.2, 132.3, 131.8, 117.0, 116.2, 115.8, 80.9, 39.9, 27.4 ppm; MS (EI): 265
[M+]; HRMS (ESI): calcd for C14H20NO4: 266.1392; found: 266.1398.
N-(3-(tert-Butoxycarbonylamino)-2-hydroxyprop-1-yl)carbamic acid tert-
butyl ester (6): A solution of di-tert-butyl dicarbonate (4.80 g, 22 mmol)
in CH2Cl2 (4 mL) was added slowly to a stirred solution of 1,3-diamino-
propanol (5; 901 mg, 10.0 mmol) and triethylamine (164 mL, 1.18 mmol)
in CH2Cl2/MeOH (1:5, 10 mL). The reaction was complete in 2 h (as
monitored by TLC). The solution was concentrated and the oily residue
was purified by flash column chromatography (hexane/EtOAc (1:4)) to
give the Boc-protected intermediate 6[20] as a colorless solid (2.83 g,
98%); 1H NMR (300 MHz, CDCl3) d=3.70 (m, 1H), 3.20 (m, 4H), 1.27
(s, 18H) ppm; 13C NMR (75 MHz, CDCl3) d=157.27, 80.07, 71.20, 43.61,
28.37 ppm.
Experimental Section
General: All reagents were either purchased from Aldrich or Fluka or
synthesized following literature procedures. Amino acids and their deriv-
atives were purchased from Senn Chemicals or Novabiochem (Switzer-
land). Rink amide resin and TGR resin were purchased from Novabio-
chem. All solvents used were analytical grade. Chromatography (flash)
was performed with Merck silica gel 60 (0.040ꢁ0.063 mm). Analytical
HPLC was performed in a Waters (996 photodiode array detector) chro-
matography system with a Vydac 218TP54 column (RP-C18, 300 ä pore
size, 0.4î22 cm, flow rate of 1.5 mLminꢀ1) or a chromolith column (0.4î
5 cm, flow rate of 5 mLminꢀ1). Preparative HPLC was performed with
HPLC-grade acetonitrile and MilliQ deionized water in a Waters prepak
cartridge (500 g; RP-C18, 300 ä pore size, 20 mm) installed on a Waters
Prep LC4000 system from Millipore (flow rate of 100 mLminꢀ1, gradient
of 0.5% CH3CN per min). Fluorescence measurements were carried out
with a spectraMAX fluorescence detector. NMR spectra were recorded
on a Bruker AC-300 instrument (1H, 300 MHz; 13C, 75 MHz). Infrared
spectroscopy was performed with a Perkin Elmer 1600 series FTIR appa-
ratus. Frequencies (n) are given in cmꢀ1. Optical rotations were measured
with a Perkin Elmer 241 digital polarimeter with a 1-dm cell. Melting
points were determined on a B¸chi 510 apparatus and are not corrected.
TLC was performed with fluorescent F254 glass plates. MS was provided
by Dr. Thomas Schneeberger (University of Bern, Switzerland).
N-(3-(tert-Butoxycarbonylamino)-2-(ethoxycarbonylmethyloxy)prop-1-
yl)carbamic acid tert-butyl ester (7): Ethyl bromoacetate (1.18 mL,
10.7 mmol) was added to a stirred solution of 6 (1.23 g, 4.23 mmol) in dry
THF (2 mL) at room temperature. Sodium hydride (0.47 g, 4.5 equiv)
was added slowly over 1 h. After an additional 5 h the reaction mixture
was filtered over celite and evaporated. The residue was purified by flash
column chromatography (hexane/EtOAc (8:2)) to give 7 (858 mg, 54%)
1
as a colorless oil; H NMR (300 MHz, CDCl3): d=4.21 (q, J=7 Hz, 2H),
4.15 (s, 2H), 3.45 (m, 1H), 3.48 3.06 (m, 4H), 1.42 (s, 18H), 1.27 (t, J=
7 Hz, 3H) ppm; 13C NMR (75 MHz, CDCl3) d=171.09, 156.37, 79.38,
78.91, 67.16, 61.13, 40.56, 28.35, 14.10 ppm; MS (FAB): 377 [M+H]+
HRMS: calcd for C17H32N2O7 +Na: 399.2113; found: 399.2107.
;
Dibenzyl-3,3-bis(methoxycarbonylmethyl)glutarate (8): A solution of di-
methyl malonate (13.0 mL, 0.112 mol) in dry THF (170 mL) was added
dropwise to a stirred suspension of NaH (10.66 g, 0.235 mol, 2.1 equiv;
55 65% oil dispersion) in dry THF (170 mL) under N2. The solution was
stirred at room temperature for 2 h and then a solution of benzyl 2-bro-
moacetate (35.2 mL, 0.224 mol, 2 equiv) in dry THF (160 mL) was added
dropwise. The reaction mixture was stirred at room temperature for 4 h
and then poured into aq. sat. NH4Cl. After extraction with AcOEt (2î),
the combined organic phases were dried with Na2SO4. Removal of the
N-(3-(9-Fluorenylmethyloxycarbonylamino)-2-(carboxymethyloxy)prop-
1-yl)carbamic acid 9-fluorenylmethyl ester (1):
A solution of LiOH
(164 mg, 6.85 mmol) in water (10 mL) was added to a stirred solution of
7 (858 mg, 2.28 mmol) in 10 mL THF. The mixture was stirred overnight.
The solvent was evaporated and the residue was lyophilized to give a
white solid. 3n HCl (10 mL) was added to the solid and the mixture was
stirred at 608C for 3 h. The pH value of the solution was adjusted to 5
1221
Chem. Eur. J. 2004, 10, 1215 1226
www.chemeurj.org
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

J.-L. Reymond et al.
FULL PAPER
solvents in vacuum gave 8 (47.9 g, 98%) as a yellow oil; IR (CHCl3): n˜ =
(3 mL). Dimethylsulfate (0.47 mL, 5.00 mmol, 7 equiv) was added and
the solution was stirred for 12 h at 258C. After solvent evaporation, the
residue was purified by preparative RP HPLC (C-18, acetonitrile/water
gradient containing 0.1% trifluoroacetic acid (TFA)). Lyophilization of
the product-containing fractions gave 15 (71.6 mg, 0.245 mmol, 64%) as
an orange oil; 1H NMR (300 MHz, D2O): d=9.14 (d, J=5.84 Hz, 1H),
9.04 (d, J=8.29 Hz, 1H), 8.34 (d, J=9.04 Hz, 1H), 8.10 (d, J=1.88 Hz,
1H), 7.93 (q, J=8.47, 6.02 Hz, 1H), 7.72 (dd, J=8.86, 1.89 Hz, 1H), 4.51
(s, 3H), 2.70 (t, J=7.35 Hz, 2H), 1.71 (q, J=7.35 Hz, 2H), 1.32 (m, 4H),
0.83 (t, J=7.35 Hz, 3H) ppm; 13C NMR (75 MHz, D2O): d=117.24,
157.93, 152.33, 149.85, 142.21, 134.93, 130.37, 128.12, 123.88, 113.22, 47.81,
36.24, 32.94, 26.19, 24.13, 15.63 ppm; MS (EI): 258 [M+]; HRMS: calcd
for C16H20NO2: 258.149290; found: 258.149404.
3034 (s), 2955 (s), 1741 (s), 1170 (w), 1227 (s) cmꢀ1 1H NMR (300 MHz,
;
CD3OD): d=7.33 (s, 10H), 5.10 (s, 4H), 3.66 (s, 6H), 3.22 (s, 4H) ppm;
13C NMR (75 MHz, CD3OD): d=170.88, 170.19, 136.14, 129.0, 128.96,
67.37, 53.81, 38.44, 30.38 ppm; MS (EI): 428 [M+]; HRMS: calcd. for
C23H24O8: 429.147118; found: 428.147130.
3,3-Bis(methoxycarbonylmethyl)glutaric acid (9): A solution of 8 (48.6 g,
0.113 mol) in absolute EtOH (60 mL) was hydrogenated with H2 (4 bars)
over 10% Pd/C (4 g) in a Parr shaker for 14 h. The catalyst was removed
by filtration through celite and the filtrate was concentrated. The solid
residue was dissolved in a small amount of AcOEt and precipitated by
addition of hexane. After filtration, 9 (25.6 g, 91%) was obtained as a
white solid; m.p. 1268C; IR (KBr): n˜ =3418 (br), 2924 (s), 2854 (m), 1731
(s), 1395 (s), 1220 (s) cmꢀ1
;
1H NMR (300 MHz, CD3OD): d=3.71 (s,
7-Isobutyryloxy-1-methylquinolinium trifluoracetate (16): The same pro-
cedure as for 15 was applied with 7-hydroxyquinoline (18; 50.0 mg,
0.34 mmol) in THF (2 mL), isobutyryl chloride (55 mL, 0.52 mmol,
1.5 equiv), and DIEA (118 mL, 0.69 mmol, 2 equiv), followed by dime-
thylsulfate (0.225 mL, 2.4 mmol, 7 equiv) to give, after lyophilization, 16
6H), 3.13 (s, 4H) ppm; 13C NMR (75 MHz, CD3OD): d=173.77, 171.38,
54.59, 53.46, 38.51 ppm; MS (FAB): 249 [M+H]; HRMS: calcd for
C9H12O8: 249.0610; found: 249.0599.
N-(3-(9-Fluorenylmethyloxycarbonylamino)-2,2-bis(methoxycarbonyl)-
prop-1-yl)carbamic acid 9-fluorenylmethyl ester (10): A solution of 9
(8.15 g, 0.033 mol) in dry CH2Cl2 was treated at 258C with oxalyl chloride
(8.50 mL, 0.098 mol, 3 equiv) and DMF (1 drop) and the mixture was stir-
red for 7 h. Evaporation to dryness gave the bis-acyl chloride as an
orange oil (8.66 g, 0.030 mol), which was dissolved in dry CH2Cl2 and
treated with azidotrimethylsilane (14.90 mL, 0.113 mol, 3.7 equiv). After
stirring for 20 h at 258C, evaporation to dryness gave the corresponding
1
(71.6 mg, 0.245 mmol, 72%) as an orange oil; H NMR (300 MHz, D2O):
d=9.14 (d, J=5.84 Hz, 1H), 9.04 (d, J=8.39 Hz, 1H), 8.34 (d, J=
9.04 Hz, 1H), 8.10 (d, J=1.88 Hz, 1H), 7.93 (q, J=8.39, 5.84 Hz, 1H),
7.73 (dd, J=9.04, 1.88 Hz, 1H), 4.51 (s, 3H), 2.95 (m, 1H), 1.28 (d, J=
6.97 Hz, 6H) ppm; 13C NMR (75 MHz, D2O): d=180.42, 158.10, 152.32,
149.85, 134.92, 130.37, 128.11, 126.59, 123.88, 113.18, 47.83, 36.53,
20.41 ppm; MS (EI): 230 [M+]; HRMS: calcd for C14H16NO2:
230.117570; found: 230.118104.
bis-acyl azide as an orange oil.
A solution of acyl azide (5.53 g,
18.50 mmol) and 9-fluorenemethanol (15.80 g, 80.51 mmol, 4.3 equiv) in
dry toluene (120 mL) was stirred under reflux conditions for 18 h. After
removal of the solvent, the crude product was purified by flash column
chromatography (hexane/AcOEt (8:2)) to give 10 (5.51 g, 8.68 mmol,
43%) as a colorless solid; m.p. 758C; IR (CHCl3): n˜ =3447 (m), 3014 (s),
(S)-7-(a-methylphenylacetoxy)-1-methylquinolinium trifluoracetate ((S)-
17): A solution of 7-hydroxyquinoline (18; 66.6 mg, 0.46 mmol) in THF
(4 mL) with (S)-(+)-2-phenylpropionic acid (75.8 mg, 0.51 mmol,
1.1 equiv), DCC (114.0 mg, 0.55 mmol, 1.2 equiv), and 4-dimethylamino-
pyridine (6.1 mg, 0.05 mmol, 0.1 equiv) was stirred for 15 h at 258C. The
solvent was evaporated and the residue was dissolved in THF (3 mL) and
treated with dimethylsulfate (0.3 mL, 3.20 mmol, 7 equiv). After 12 h at
258C, the solvent was evaporated and the residue was purified by prepa-
rative RP HPLC (C18, water/acetonitrile gradient containing 0.1%
TFA). Lyophilization of the product-containing fractions gave (S)-17
(71.6 mg, 0.245 mmol, 53%) as orange oil; 1H NMR (400 MHz, D2O):
d=9.07 (d, J=5.77 Hz, 1H), 8.96 (d, J=8.43 Hz, 1H), 8.21 (d, J=
8.93 Hz, 1H), 7.91 (d, J=1.83 Hz, 1H), 7.88 (q, J=8.43, 5.77 Hz, 1H),
7.54 (dd, J=8.93, 1.83 Hz, 1H), 7.40 (m, 4H), 7.31 (m, 1H), 4.40 (s, 3H),
4.15 (t, J=6.91 Hz, 1H), 1.55 (d, J=6.91 Hz, 3H) ppm; 13C NMR
(100 MHz, DMSO): d=172.23, 155.14, 150.72, 146.81, 132.14, 128.88,
127.68, 127.45, 125.54, 121.78, 111.29, 45.47, 44.67, 18.65 ppm; [a]2D0 =ꢀ70
(c=1m, MeOH); MS (EI): 292 [M+]; HRMS: calcd for C19H18NO2:
292.133880; found: 292.133754.
2955(s), 1732 (s), 1516 (m), 1451(s), 1229 (s) cmꢀ1 1H NMR (300 MHz,
;
CDCl3): d=7.78 (d, J=7.4 Hz, 4H), 7.60 (d, J=7.4 Hz, 4H), 7.32 (m, J=
6.6, 10.7 Hz, 8H), 5.58 (t, 2H), 4.44 (d, J=6.99 Hz, 4H), 4.25 (q, J=
6.99 Hz, 2H), 3.76 (d, 4H), 3.74 (s, 6H) ppm; 13C NMR (75 MHz,
CDCl3): d=169.91, 157.60, 144.49, 142.00, 128.42, 127.78, 125.77, 120.67,
67.79, 59.67, 53.73, 47.88, 42.11 ppm; MS (FAB): 635 [M+]; HRMS: calcd
for C37H34N2O8: 635.2393; found: 635.2362.
5-Dimethylamino-isophthalic acid (12): Formaldehyde (ꢂ37wt% in
water, 24 mL, 10 equiv) was added to a solution of 5-amino-isophthalic
acid (11; 5.50 g, 30.4 mmol) in DMF at 258C for 20 minutes. The solution
was cooled to 08C and NaBH3CN (5.70 g, 90.7 mmol, 3 equiv) was slowly
added. The solution was stirred for 5 h at 258C. After evaporation of the
solvent, the residue was dissolved in water and precipitated by addition
of 1n HCl. The white precipitate was filtrated and washed with water
until the pH value of the filtrate was neutral, to yield 12 (5.28 g, 83%) as
a colorless solid; IR (DMSO): n˜ =3424 (s), 2151 (w), 1653 (m), 1437 (w),
(R)-(7-a-methylphenylacetoxy)-1-methylquinolinium
trifluoracetate
((R)-17): The same procedure as above was applied with 18 (67 mg,
0.46 mmol) and (R)-(+)-2-phenylpropionic acid (76.0 mg, 0.51 mmol) to
give (R)-17 (84.6 mg, 63%) as an orange oil; [a]2D0 =+70 (c=1m,
MeOH).
1430 (w), 1420 (w), 1405 (w), 1347 (w), 1317 (w), 1229 (w) cmꢀ1
;
1H NMR (300 MHz, DMSO): d=7.78 (s, 1H), 7.43 (s, 2H), 2.97 (s,
6H) ppm; 13C NMR (75 MHz, DMSO): d=167.3, 150.2, 131.8, 117.5,
116.2, 40.0 ppm; MS (EI): 209 [M+]; HRMS (ESI): calcd for C10H12NO4:
210.0766; found: 210.0767.
7-(2,2-Dimethyl-propionyloxy)-1-methylquinolinium trifluoracetate (19):
The same procedure as for 15 was applied with 7-hydroxyquinoline (18;
50.0 mg, 0.34 mmol) in THF (2 mL), pivaloylchloride (64 mL, 0.52 mmol,
1.5 equiv), and DIEA (118 mL, 0.69 mmol, 2 equiv), followed by dime-
thylsulfate (0.23 mL, 2.4 mmol, 7 equiv) to give, after lyophilization, 19
5-Dimethylamino-isophthalic acid di-tert-butyl ester (13): A solution of
12 (4.0 g, 19.1 mmol) in dry CH2Cl2 (80 mL) was treated with oxalyl chlo-
ride (4.9 mL, 57.1 mmol, 3 equiv) and a few drops of DMF and the mix-
ture was stirred at 258C for 2 h. Evaporation to dryness gave a crude
yellow acyl chloride, which was dissolved in dry CH2Cl2 (80 mL) and
cooled to 08C. KOtBu (6.4 g, 57.3 mmol, 3 equiv) was slowly added at
08C. The solution was stirred for 3 h and then evaporated to dryness.
Aqueous work-up (AcOEt/water) and purification by flash column chro-
matography (CH2Cl2) gave 13 (4.14 g, 67%) as a yellow solid; m.p. 149
1518C; 1H NMR (300 MHz, CDCl3): d=7.67 (s, 1H), 7.27 (s, 2H), 2.79
(s, 6H), 1.37 (s, 18H) ppm; 13C NMR (75 MHz, CDCl3): d=165.9, 150.3,
132.7, 118.2, 116.7, 81.2, 40.5, 28.1 ppm; MS (EI): 321 [M+]; HRMS
(ESI): calcd for C18H28NO4: 322.2018; found: 322.2029.
1
(41.5 mg, 0.167 mmol, 50%) as an orange oil; H NMR (300 MHz, D2O):
d=9.14 (d, J=5.86 Hz, 1H), 9.05 (d, J=8.47 Hz, 1H), 8.35 (d, J=
9.03 Hz, 1H), 8.09 (d, J=1.98 Hz, 1H), 7.94 (q, J=8.47, 5.86 Hz, 1H),
7.74 (dd, J=9.03, 1.98 Hz, 1H), 4.52 (s, 3H), 1.35 (s, 9H) ppm; 13C NMR
(75 MHz, D2O): d=181.93, 158.40, 152.33, 149.87, 142.24, 134.92, 130.38,
128.12, 123.89, 113.14, 47.85, 41.61, 28.62 ppm; MS (EI): 244 [M+];
HRMS: calcd for C15H18NO2: 244.133710; found: 244.133754.
7-Benzoyloxy-1-methylquinolinium trifluoracetate (20): The same proce-
dure as for 15 was applied with 7-hydroxyquinoline (18; 51 mg,
0.35 mmol) in THF (2 mL), benzoyl chloride (165 mL, 0.52 mmol,
1.5 equiv), and DIEA (118 mL, 0.69 mmol, 2 equiv), followed by dime-
thylsulfate (0.23 mL, 2.4 mmol, 7 equiv) to give, after lyophilization, 20
7-Hexanoyloxy-1-methylquinolinium trifluoroacetate (15): Hexanoyl
chloride (145 mL, 1.04 mmol, 1.5 equiv) and N,N-diisopropylethylamine
(DIEA; 250 mL, 1.44 mmol, 2 equiv) were added to a solution of 7-hy-
droxyquinoline (18; 103 mg, 0.71 mmol) in THF (4 mL). After 20 h at
258C, the solvent was evaporated and the residue was dissolved in THF
1
(64.0 mg, 0.242 mmol, 69%) as an orange oil; H NMR (300 MHz, D2O):
d=9.18 (d, J=5.84 Hz, 1H), 9.08 (d, J=8.40 Hz, 1H), 8.40 (d, J=
9.04 Hz, 1H), 8.27 (d, J=1.79 Hz, 1H), 8.17 (d, J=7.16 Hz, 2H), 7.97
1222
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Chem. Eur. J. 2004, 10, 1215 1226

Catalytic Peptide Dendrimers
1215 1226
(dd, J=8.40, 5.84 Hz, 1H), 7.90 (dd, J=9.04, 1.79 Hz, 1H), 7.71 (t, J=
7.54 Hz, 1H), 7.55 (t, J=7.53 Hz, 2H), 4.54 (s, 3H) ppm; 13C NMR
(75 MHz, D2O): d=168.83, 158.21, 152.41, 149.93, 142.22, 137.44, 135.05,
132.74, 131.49, 130.44, 130.25, 128.15, 124.00, 113.34, 47.86 ppm;
MS (EI): 264 [M+]; HRMS: calcd for C17H14NO2: 264.102450; found:
264.102454.
17.25 min (A/B=80/20!50/50 in 30 min); MS (ES+): calcd for
C92H117N27O30S: 2111.83; found: 2112.38.
Dendrimer A2: From Rink amide resin (200 mg, 0.61 mmolgꢀ1), the se-
quence ((Cap-Ser)2B2Asp)2B2CysHisNH2 was obtained as
a colorless
foamy solid after preparative HPLC purification (29.8 mg, 12.9%); RP
HPLC: tR =14.0 min (A/B=80/20!50/50 in 30 min); MS (ES+): calcd
for C81H105N21O31S: 1899.70; found: 1900.25.
6-Acetoxy-1-methylquinolinium trifluoracetate (21): Acetyl chloride
(53.0 mL, 0.75 mmol, 2.0 equiv) and DIEA (0.13 mL, 0.76 mmol, 2 equiv)
were added to a solution of 7-hydroxyquinoline (18; 54.2 mg, 0.37 mmol)
in THF (0.5 mL). The solution was stirred overnight at room tempera-
ture. The solvent was removed under vacuum and the residue was taken
up into THF (0.5 mL). Dimethyl sulfate (0.25 mL, 2.6 mmol, 7 equiv) was
added and the solution was stirred for 20 h at room temperature. The sol-
vent was removed under vacuum and the residue was purified by prepa-
rative HPLC. After lyophilization, 21 (32.1 mg, 0.16 mmol, 43%) was
Dendrimer B2: From Rink amide resin (200 mg, 0.47 mmolgꢀ1), the se-
quence ((Cap-Asp)2B2Ser)2B2CysHisNH2 was obtained as
a colorless
foamy solid after preparative HPLC purification (8.2 mg, 4.5%); RP
HPLC: tR =15.6 min (A/B=70/30!40/60 in 30 min); MS (ES+): calcd
for C83H105N21O33S: 1955.69; found: 1956.25.
Dendrimer C2: From Rink amide resin (150 mg, 0.61 mmolgꢀ1), the se-
quence ((Cap-Asp)2B2His)2B2CysSerNH2 was obtained as
a colorless
foamy solid after preparative HPLC purification (14.7 mg, 8.0%); RP
HPLC: tR =15.6 min (A/B=80/20!50/50 in 30 min); MS (ES+): calcd
for C86H107N23O32S: 2005.72; found: 2006.39.
Dendrimer D2: From TGR resin (400 mg, 0.20 mmolgꢀ1), the sequence
((Cap-Ser)2B2His)2B2CysAspNH2 was obtained as a colorless foamy solid
after preparative HPLC purification (22.9 mg, 14.9%); RP HPLC: tR =
24.8 min (A/B=90/10!50/50 in 40 min); MS (ES+): calcd for
C83H107N23O23S: 1921.73; found: 1922.13.
obtained as
a
white solid; 1H NMR (300 MHz, D2O): d=9.15 (d,
J=5.74 Hz, 1H), 9.00 (d, J=8.43 Hz, 1H), 8.41 (d, J=9.42 Hz, 1H),
8.04 (d, J=2.45 Hz, 1H), 7.97 (q, J=8.43, 5.77 Hz, 2H), 3.08
(s, 3H), 2.38 (d, J=6.91 Hz, 3H) ppm; 13C NMR (75 MHz, DMSO):
d=174.70, 152.77, 151.85, 149.71, 133.28, 133.03, 124.82, 123.84, 122.93,
48.09, 22.93 ppm; HRMS: calcd for C12H12NO2: 202.0868; found:
202.0862.
Dendrimer synthesis: The dendrimer synthesis was carried out by using
standard Fmoc SPPS on Rink amide resin (0.47 mmolgꢀ1). The resin was
acylated with each amino acid or diamino acid (3 equiv) in the presence
of TBTU or BOP (3 equiv) and DIEA (5 equiv). After 30 min, the resin
was successively washed with DMF, MeOH, and CH2Cl2 (2î with each
solvent), then checked for free amino groups with the 2,4,6-trinitroben-
zenesulfonic acid test. The Fmoc protecting group was removed with a
solution of 20% piperidine in DMF for 2î10 min and the solution was
then removed by filtration. The resin was washed with DMF, MeOH, and
CH2Cl2 (2î with each solvent). At the end of the synthesis, the resin was
acylated with three equivalents of the capping group in the presence of
BOP (3 equiv) and DIEA (5 equiv) for 2 h. The resin was dried and the
cleavage was carried out with TFA/1,2-ethanediothiol/H2O/triisopropylsi-
lane (94.5:2.5:2.5:1) for 4 h. The peptide was precipitated with methyl
tert-butyl ether then dissolved in a water/acetonitrile mixture. All den-
Dendrimer E2: From Rink amide resin (150 mg, 0.61 mmolgꢀ1), the se-
quence ((Cap-His)2B2Asp)2B2CysSerNH2 was obtained as
a colorless
foamy solid after preparative HPLC purification (9.7 mg, 5.2%); RP
HPLC: tR =22.4 min (A/B=80/20!50/50 in 30 min); MS (ES+): calcd
for C90H111N27O26S: 2049.78; found: 2050.86.
Dendrimer F2: From TGR resin (400 mg, 0.20 mmolgꢀ1), the sequence
((Cap-His)2B2Ser)2B2CysAspNH2 was obtained as a colorless foamy solid
after preparative HPLC purification (21.8 mg, 13.5%); RP HPLC: tR =
17.2 min (A/B=80/20!50/50 in 30 min); MS (ES+): calcd for
C89H111N27O27S: 2021.79; found: 2022.25.
Dendrimer A3: From Rink amide resin (150 mg, 0.61 mmolgꢀ1), the se-
quence ((Cap-Ser)2B3Asp)2B3CysHisNH2 was obtained as
a colorless
foamy solid after preparative HPLC purification (9.7 mg, 5.5%); RP
HPLC: tR =34 min (l=214 nm, A/B=90/10!60/40 in 60 min); MS
(ES+): calcd for C78H99N21O31S: 1857.65; found: 1858.50.
drimers were purified by preparative HPLC (flow rate of 100 mLminꢀ1
;
Dendrimer B3: From Rink amide resin (100 mg, 0.47 mmolgꢀ1), the se-
eluent A=water and 0.1% TFA; eluent B=acetonitrile, water, and TFA
(3:2:0.1%); column: Waters prepak cartridge 500 g RP-C18, 20 mm, pore
size: 300 ä, detection: l=214 nm).
quence ((Cap-Asp)2B3Ser)2B3CysHisNH2 was obtained as
a colorless
foamy solid after preparative HPLC purification (21.4 mg, 24%); RP
HPLC: tR =34 min (l=214 nm, A/B=97/3!50/50 in 47 min); MS (ES+):
calcd for C80H100N21O33S: 1914.64; found: 1914.38.
Dendrimer A1: From Rink amide resin (200 mg, 0.061 mmolgꢀ1), the se-
quence ((Cap-Ser)2B1Asp)2B1CysHisNH2 was obtained as
a colorless
Dendrimer C3: From Rink amide resin (150 mg, 0.61 mmolgꢀ1), the se-
foamy solid after preparative HPLC purification (27 mg, 16%); RP
HPLC: tR =18.56 min (A/B=80/20!50/50 in 30 min); MS (ES+): calcd
for C84H111N21O34S: 1989.73; found: 1990.00.
quence ((Cap-Asp)2B3His)2B3CysSerNH2 was obtained as
a colorless
foamy solid after preparative HPLC purification (7.0 mg, 4%); RP
HPLC: tR =23 min (l=214 nm, A/B=90/10!70/30 in 20 min, then !60/
40 in 40 min); MS (ES+): calcd for C83H102N23O32S: 1964.87; found:
1964.13.
Dendrimer B1: From Rink amide resin (200 mg, 0.061 mmolgꢀ1), the se-
quence ((Cap-Asp)2B1Ser)2B1CysHisNH2 was obtained as
a colorless
foamy solid after preparative HPLC purification (25 mg, 15%); RP
HPLC: tR =16.96 min (A/B=80/20!50/50 in 30 min); MS (ES+): calcd
for C86H111N21O36S: 2045.72; found: 2046.00.
Dendrimer D3: From Rink amide resin (150 mg, 0.61 mmolgꢀ1),
the sequence ((Cap-Ser)2B3His)2B3CysAspNH2 was obtained as
a
Dendrimer C1: From Rink amide resin (200 mg, 0.061 mmolgꢀ1), the se-
colorless foamy solid after preparative HPLC purification (50.4 mg,
29.3%); RP HPLC: tR =27 min (l=214 nm, A/B=90/10!50/50
in 40 min); MS (ES+): calcd for C80H102N23O29S: 1880.69; found:
1880.13.
quence ((Cap-Asp)2B1His)2B1CysSerNH2 was obtained as
a colorless
foamy solid after preparative HPLC purification (23 mg, 12%); RP
HPLC: tR =19.55 min (A/B=80/20!50/50 in 30 min); MS (ES+): calcd
for C89H113N23O35S: 2095.76; found: 2096.13.
Dendrimer E3: From Rink amide resin (150 mg, 0.61 mmolgꢀ1), the se-
Dendrimer D1: From Rink amide resin (400 mg, 0.061 mmolgꢀ1), the se-
quence ((Cap-His)2B3Asp)2B3CysSerNH2 was obtained as
a colorless
quence ((Cap-Ser)2B1His)2B1CysAspNH2 was obtained as
a
colorless
foamy solid after preparative HPLC purification (17.1 mg, 9.3%); RP
HPLC: tR =17 min (l=214 nm, A/B=90/10!70/30 in 20 min, then !60/
40 in 40 min); MS (ES+): calcd for C87H105N27O28S: 2008.73; found:
2008.73.
foamy solid after preparative HPLC purification (34 mg, 16%); RP
HPLC: tR =17.48 min (A/B=80/20!50/50 in 30 min); MS (ES+): calcd
for C86H113N23O32S: 2011.78; found: 2012.13.
Dendrimer E1: From Tentagel resin (300 mg, 0.2 mmolgꢀ1), the sequence
((Cap-His)2B1Asp)2B1CysSerNH2 was obtained as a colorless foamy solid
after preparative HPLC purification (18 mg, 12%); RP HPLC: tR =
17.96 min (A/B=80/20!50/50 in 30 min); MS (ES+): calcd for
C93H117N27O31S: 2139.83; found: 2140.38.
Dendrimer F3: From Rink amide resin (150 mg, 0.61 mmolgꢀ1), the se-
quence ((Cap-His)2B3Ser)2B3CysAspNH2 was obtained as
a colorless
foamy solid after preparative HPLC purification (47.2 mg, 26%); RP
HPLC: tR =34 min (l=214 nm, A/B=90/10!60/40 in 60 min); MS
(ES+): calcd for C86H105N27O27S: 1981.99; found: 1980.38.
Dendrimer F1: From Tentagel resin (400 mg, 0.2 mmolgꢀ1), the sequence
((Cap-His)2B1Ser)2B1CysAspNH2 was obtained as a colorless foamy solid
after preparative HPLC purification (44 mg, 18%); RP HPLC: tR =
Dendrimer DFH: From Novasyn TGR resin (400 mg, 0.02 mmolgꢀ1), the
sequence ((Cap-Asp)2B2Phe)2B2CysHisNH2 was obtained as a colorless
foamy solid after preparative HPLC purification (24.8 mg, 20%); RP
1223
Chem. Eur. J. 2004, 10, 1215 1226
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¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

J.-L. Reymond et al.
FULL PAPER
HPLC: tR =28.4 min (A/B=80/20!40/60 in 40 min); MS (ES+): calcd
Dendrimer A1 F1: Yield: 1.1 mg (0.23 mmol), 49%; RP HPLC: tR =
14.50 min (A/B=70/30!50/50 in 20 min); MS (ES+): calcd for
for C98H119N21O34S: 2165.79; found: 2166.63.
Dendrimer HWS: From Rink amide resin (200 mg, 0.47 mmolgꢀ1), the
sequence ((Cap-His)2B2Trp)2B2CysSerNH2 was obtained as a colorless
foamy solid after preparative HPLC purification (26 mg, 12%); RP
HPLC: tR =24.6 min (A/B=80/20!40/60 in 40 min); MS (ES+): calcd
for C99H123N21O34S: 2281.92; found: 2282.88.
Dendrimer LRH: From Novasyn TGR resin (400 mg, 0.02 mmolgꢀ1), the
sequence ((Cap-Leu)2B2Arg)2B2CysHisNH2 was obtained as a colorless
foamy solid after preparative HPLC purification (23.3 mg, 14%); RP
HPLC: tR =24.6 min (A/B=60/40!20/80 in 40 min); MS (ES+): calcd
for C99H123N21O34S: 2176.09; found: 2177.13.
Dendrimer DHL: From Rink amide resin (200 mg, 0.47 mmolgꢀ1), the
sequence ((Cap-Asp)2B2His)2B2CysLeuNH2 was obtained as a colorless
foamy solid after preparative HPLC purification (21 mg, 11%); RP
HPLC: tR =22.1 min (A/B=80/20!40/60 in 40 min); MS (ES+): calcd
for C93H121N23O33S: 2121.16; found: 2122.25
Dendrimer HHH: From Novasyn TGR resin (100 mg, 0.02 mmolgꢀ1), the
sequence ((Cap-His)2B2His)2B2CysHisNH2 was obtained as a colorless
foamy solid after preparative HPLC purification; MS (ES+): calcd for
C
176H228N48O64S2: 4099.56; found: 4101.25.
Dendrimer B1 C1: Yield: 0.6 mg (0.13 mmol), 29%; RP HPLC: tR =
15.80 min (A/B=70/30!50/50 in 20 min); MS (ES+): calcd for
C
175H224N44O71S2: 4139.49; found: 4140.63.
Dendrimer B1 D1: Yield: 0.9 mg (0.20 mmol), 43%; RP HPLC: tR =
15.43 min (A/B=70/30!50/50 in 20 min); MS (ES+): calcd for
C
172H224N44O68S2: 4055.5; found: 4056.63.
Dendrimer B1 E1: Yield: 0.8 mg (0.17 mmol), 36%; RP HPLC: tR =
13.68 min (A/B=70/30!50/50 in 20 min); MS (ES+): calcd for
C
179H228N48O67S2: 4183.55; found: 4185.00.
Dendrimer B1 F1: Yield: 0.4 mg (0.08 mmol), 18%; RP HPLC: tR =
14.25 min (A/B=70/30!50/50 in 20 min); MS (ES+): calcd for
C
178H228N48O66S2: 4155.55; found: 4157.25.
Dendrimer C1 D1: Yield: 0.5 mg (0.11 mmol), 25%; RP HPLC: tR =
15.32 min (A/B=70/30!50/50 in 20 min); MS (ES+): calcd for
C
175H226N46O67S2: 4105.54; found: 4106.63.
Dendrimer C1 E1: Yield: 0.7 mg (0.08 mmol), 19%; RP HPLC: tR =
13.82 min (A/B=70/30!50/50 in 20 min); MS (ES+): calcd for
C
100H123N33O26S: 2233.90; found 2235.26.
Dendrimer dimerization: Homodimers: The monomeric dendrimer X
(1 mg) was dissolved in water (25 mL) and solution of aldrithiol
C
182H228N50O66S2: 4233.59; found: 4234.50.
Dendrimer C1 F1: Yield: 1.4 mg (0.29 mmol), 29%; RP HPLC: tR =
14.26 min (A/B=70/30!50/50 in 20 min); MS (ES+): calcd for
a
(0.0454m in MeOH, 0.45 equiv) was added. The pH value was adjusted
to 8 with 20 mm aq. NH4HCO3 and the solution was stirred for 30 min at
258C then acidified with TFA (1 drop). The homodimer X X was puri-
fied by semipreparative RP HPLC (column: Vydac 218TP510, C18, 1.0î
25 cm, flow rate of 4 mLminꢀ1; eluents A and B as above, gradient of elu-
ents as indicated in the characterization; detection by UV, l=220 nm).
Heterodimers: Dendrimer X (5 mg) was dissolved in water (125 mL) and
aldrithiol (0454m in MeOH, 25 equiv) was added. The methanol was
evaporated and the excess of aldrithiol was removed by extraction with
dichloromethane. The activated dendrimer solution was divided in five
parts. A solution of dendrimer Y (1 mg in 25 mL of H2O, 1 equiv) was
added to each part and the final solution was adjusted to pH 8 by addi-
tion of 20 mm aq. NH4HCO3. The solution was stirred for 30 minutes and
then acidified with TFA (1 drop). The heterodimer was purified by semi-
preparative RP HPLC as above.
C
181H230N50O62S2: 4205.59; found: 4207.25.
Dendrimer D1 E1: Yield: 0.4 mg (0.08 mmol), 18%; RP HPLC: tR =
12.50 min (A/B=70/30!50/50 in 20 min); MS (ES+): calcd for
C
179H230N50O63S2: 4150.38; found: 4149.61.
Dendrimer D1 F1: Yield: 1.1 mg (0.23 mmol), 51%; RP HPLC: tR =
15.28 min (A/B=70/30!50/50 in 20 min); MS (ES+): calcd for
C
178H228N50O62S2: 4121.61; found: 4122.63.
Dendrimer E1 F1: Yield: 1.0 mg (0.19 mmol), 50%; RP HPLC: tR =
13.00 min (A/B=70/30!50/50 in 20 min); MS (ES+): calcd for
C
185H234N54O61S2: 4249.66; found: 4251.88.
Dendrimer A2 A2: Yield: 0.6 mg (0.149 mmol), 60%; semipreparative
RP HPLC: tR =21.6 min (A/B=80/20!A/B=50/50 in 30 min); MS
(ES+): calcd for C162H208N42O62S2: 3797.39; found: 3798.75.
Dendrimer B2 B2: Yield: 0.5 mg (0.121 mmol), 50%; semipreparative RP
HPLC: tR =26.7 min (A/B=80/20!50/50 in 30 min); MS (ES+): calcd
for C166H208N42O66S2: 3909.37; found: 3910.75.
Dendrimer A1 A1: Yield: 0.8 mg (0.19 mmol), 80%; RP HPLC: tR =
13.95 min (A/B=70/30!50/50 in 20 min); MS (ES+): calcd for
C
168H222N42O68S2: 3977.46; found: 3978.75.
Dendrimer C2 C2: Yield: 0.45 mg (0.10 mmol), 45%; semipreparative RP
HPLC: tR =27.1 min (A/B=80/20!50/50 in 30 min); MS (ES+): calcd
for C172H212N46O64S2: 4009.42; found: 3910.75.
Dendrimer B1 B1: Yield: 0.4 mg (0.09 mmol), 40%; RP HPLC: tR =
16.70 min (A/B=70/30!50/50 in 20 min); MS (ES+): calcd for
C
172H222N42O72S2: 4089.44; found: 4090.75.
Dendrimer D2 D2: Yield: 0.45 mg (0.09 mmol), 45%; semipreparative
RP HPLC: tR =25.3 min (A/B=80/20!50/50 in 30 min); MS (ES+):
calcd for C166H212N46O58S2: 3841.45; found: 3842.88.
Dendrimer C1 C1: Yield: 0.8 mg (0.17 mmol), 80%; RP HPLC: tR =
15.85 min (A/B=70/30!50/50 in 20 min); MS (ES+): calcd for
C
178H226N46O70S2: 4189.52; found: 4191.00.
Dendrimer E2 E2: Yield: 0.7 mg (0.14 mmol), 70%; semipreparative RP
HPLC: tR =22.5 min (A/B=80/20!50/50 in 30 min); MS (ES+): calcd
for C180H220N54O56S2: 4097.55; found: 4099.13.
Dendrimer D1 D1: Yield: 0.5 mg (0.11 mmol), 50%; RP HPLC: tR =
14.88 min (A/B=70/30!50/50 in 20 min); MS (ES+): calcd for
C172H226N46O64S2: 4021.56; found: 4022.63.
Dendrimer F2 F2: Yield: 0.9 mg (0.18 mmol), 45%; semipreparative RP
HPLC: tR =22.6 min (A/B=80/20!50/50 in 30 min); MS (ES+): calcd
for C178H220N54O54S2: 4041.56; found: 4043.13.
Dendrimer E1 E1: Yield: 0.9 mg (0.17 mmol), 90%; RP HPLC: tR =
10.30 min (A/B=70/30!50/50 in 20 min); MS (ES+): calcd for
C
186H234N54O62S2: 4277.66; found: 4279.13.
Dendrimer A2 B2: Yield: 0.5 mg (0.12 mmol), 25%; semipreparative RP
HPLC: tR =22.6 min (A/B=80/20!50/50 in 30 min); MS (ES+): calcd
for C164H208N42O64S2: 3853.38; found: 3854.88.
Dendrimer F1 F1: Yield: 0.5 mg (1.0 mmol), 50%; RP HPLC: tR =
13.90 min (A/B=70/30!50/50 in 20 min); MS (ES+): calcd for
C
184H234N54O60S2: 4221.66; found: 4223.63.
Dendrimer A2 C2: Yield: 0.5 mg (0.12 mmol), 25%; semipreparative RP
HPLC: tR =23.9 min (A/B=80/20!50/50 in 30 min); MS (ES+): calcd
for C167H210N44O63S2: 3903.40; found: 3904.63.
Dendrimer A1 B1: Yield: 1.5 mg (0.35 mmol), 75%; RP HPLC: tR =
14.65 min (A/B=70/30!50/50 in 20 min); MS (ES+): calcd for
C170H222N42O70S2: 4033.45; found: 4034.88.
Dendrimer A2 D2: Yield: 1.3 mg (0.31 mmol), 65%; semipreparative RP
HPLC: tR =24.7 min (A/B=80/20!50/50 in 30 min); MS (ES+): calcd
for C164H210N44O60S2: 3819.42; found: 3820.63.
Dendrimer A1 C1: Yield: 0.9 mg (0.20 mmol), 45%; RP HPLC: tR =
14.90 min (A/B=70/30!50/50 in 20 min); MS (ES+): calcd for
C173H224N44O69S2: 4083.49; found: 4085.25.
Dendrimer A2 E2: Yield: 0.8 mg (0.160 mmol), 40%; semipreparative
RP HPLC: tR =21.6 min (A/B=80/20!50/50 in 30 min); MS (ES+):
calcd for C180H220N54O56S2: 4097.55; found: 3948.63.
Dendrimer A1 D1: Yield: 0.5 mg (0.11 mmol), 25%; RP HPLC: tR =
14.05 min (A/B=70/30!50/50 in 20 min); MS (ES+): calcd for
C
170H224N44O66S2: 3999.51; found: 4001.13.
Dendrimer A2 F2: Yield: 1.1 mg (0.245 mmol), 55%; semipreparative RP
HPLC: tR =21.8 min (A/B=80/20!50/50 in 30 min); MS (ES+): calcd
for C170H214N48O58S2: 3919.47; found: 3920.75.
Dendrimer A1 E1: Yield: 0.9 mg (0.19 mmol), 40%; RP HPLC: tR =
11.75 min (A/B=70/30!50/50 in 20 min); MS (ES+): calcd for
C
177H228N48O65S2: 4127.56; found: 4129.00.
1224
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Catalytic Peptide Dendrimers
1215 1226
Dendrimer B2 C2: Yield: 1.1 mg (0.256 mmol), 55%; semipreparative RP
HPLC: tR =24.7 min (A/B=80/20!50/50 in 30 min); MS (ES+): calcd
for C169H210N44O65S2: 3959.39; found: 3961.74.
Dendrimer C3 D3: Yield: 0.2 mg, 10%; semipreparative RP HPLC: tR =
22.00 min (l=225 nm, A/B=80/20!40/60 in 40 min); MS (ES+): calcd
for C163H201N46O61S2: 3842.34; found: 3842.88.
Dendrimer B2 D2: Yield: 0.5 mg (0.120 mmol), 25%; semipreparative
RP HPLC: tR =25.0 min (A/B=80/20!50/50 in 30 min); MS (ES+):
calcd for C166H210N44O62S2: 3875.21; found: 3876.75.
Dendrimer C3 E3: Yield: 0.7 mg, 39%; semipreparative RP HPLC: tR =
8.00 min (l=225 nm, A/B=70/30!40/60 in 30 min); MS (ES+): calcd
for C170H205N50O60S2: 3970.39; found: 3970.50.
Dendrimer B2 E2: Yield: 0.8 mg (0.175 mmol), 40%; semipreparative RP
HPLC: tR =22.6 min (A/B=80/20!50/50 in 30 min); MS (ES+): calcd
for C173H214N48O61S2: 4003.46; found: 4005.13.
Dendrimer C3 F3: Yield: 1.4 mg, 70%; semipreparative RP HPLC: tR =
17.80 min (l=225 nm, A/B=80/20!40/60 in 40 min); MS (ES+): calcd
for C169H205N50O59S2: 3942.39; found: 3942.88.
Dendrimer B2 F2: Yield: 0.6 mg (0.132 mmol), 30%; semipreparative RP
HPLC: tR =24.0 min (A/B=80/20!50/50 in 30 min); MS (ES+): calcd
for C172H214N48O60S2: 3975.46; found: 3976.75.
Dendrimer D3 E3: Yield: 0.8 mg, 39%; semipreparative RP HPLC: tR =
17.80 min (l=225 nm, A/B=80/20!40/60 in 40 min); MS (ES+): calcd
for C167H205N50O57S2: 3886.40; found: 3886.88.
Dendrimer C2 D2: Yield: 0.7 mg (0.160 mmol), 35%; semipreparative
RP HPLC: tR =24.0 min (A/B=80/20!50/50 in 30 min); MS (ES+):
calcd for C169H212N46O61S2: 3925.43; found: 3927.54.
Dendrimer D3 F3: Yield: 1.0 mg, 48%; semipreparative RP HPLC: tR =
18.80 min (l=225 nm, A/B=90/10!40/60 in 40 min); MS (ES+): calcd
for C166H205N50O56S2: 3858.41; found: 3858.88.
Dendrimer C2 E2: Yield: 1.0 mg (0.199 mmol), 50%; semipreparative RP
HPLC: tR =22.7 min (A/B=80/20!50/50 in 30 min); MS (ES+): calcd
for C176H216N50O60S2: 4053.48; found: 4055.00.
Dendrimer E3 F3: Yield: 2.3 mg, 100%; semipreparative RP HPLC:
tR =6.50 min (l=225 nm, A/B=70/30!40/60 in 30 min); MS (ES+):
calcd for C173H209N54O55S2: 3986.46; found: 3986.88.
Dendrimer D2 E2: Yield: 1.8 mg (0.387 mmol), 90%; semipreparative
RP HPLC: tR =21.6 min (A/B=80/20!50/50 in 30 min); MS (ES+):
calcd for C173H216N50O57S2: 3969.50; found: 3970.63.
Dendrimer DHL DHL: Yield: 0.6 mg (0.107 mmol), 60%; semiprepara-
tive RP HPLC: tR =17.9 min (A/B=70/30!30/70 in 40 min); MS (+
ESI): calcd for C184H236N46O68S2: 4241.59; found: 4243.38.
Dendrimer D2 F2: Yield: 1.3 mg (0.281 mmol), 65%; semipreparative RP
HPLC: tR =21.6 min (A/B=80/20!50/50 in 30 min); MS (ES+): calcd
for C172H216N50O56S2: 3941.50; found: 3943.38.
Dendrimer LRH LRH: Yield: 0.6 mg (0.105 mmol), 60%; semiprepara-
tive RP HPLC: tR =27.7 min (A/B=60/40!20/80 in 40 min); MS (+
ESI): calcd for C200H296N54O52S2: 4350.16; found: 4352.00.
Dendrimer E2 F2: Yield: 1.4 mg (0.281 mmol), 70%; semipreparative RP
HPLC: tR =20.4 min (A/B=80/20!50/50 in 30 min); MS (ES+): calcd
for C179H220N54O55S2: 4069.55; found: 4071.00.
Dendrimer DFH DFH: Yield: 0.7 mg (0.127 mmol), 70%; semiprepara-
tive RP HPLC: tR =27.7 min (A/B=70/30!30/70 in 40 min); MS (+
ESI): calcd for C197H240N42O68S2: 4345.61; found: 4352.00.
Dendrimer A3 A3: Yield: 0.4 mg, 40%; semipreparative RP HPLC: tR =
19.80 min (l=225 nm, A/B=80/20!40/60 in 40 min); MS (ES+): calcd
for C156H197N42O62S2: 3714.29; found: 3714.63.
Dendrimer LRH HWS: Yield: 0.4 mg (0.088 mmol), 26%; semiprepara-
tive RP HPLC: tR =21.1 min (A/B=60/40!20/80 in 40 min); MS (+
ESI): calcd for C207H274N56O53S2: 4455.99; found: 4458.00.
Dendrimer B3 B3: Yield: 0.4 mg, 50%; semipreparative RP HPLC: tR =
21.75 min (l=225 nm, A/B=80/20!50/50 in 30 min); MS (ES+): calcd
for C166H197N42O66S2: 3826.27; found: 3726.63.
Dendrimer LRH DHL: Yield: 0.4 mg (0.093 mmol), 27%; semiprepara-
tive RP HPLC: tR =25.1 min (A/B=60/40!20/80 in 40 min); MS (+
ESI): calcd for C192H266N50O60S2: 4295.87; found: 4297.50.
Dendrimer C3 C3: Yield: 0.4 mg, 40%; semipreparative RP HPLC: tR =
21.25 min (l=225 nm, A/B=80/20!40/60 in 40 min); MS (ES+): calcd
for C166H201N46O64S2: 3926.33; found: 3926.38.
Dendrimer LRH DFH: Yield: 0.6 mg (0.138 mmol), 31%; semiprepara-
tive RP HPLC: tR =16.3 min (A/B=50/50!10/90 in 40 min); MS (+
ESI): calcd for C198H266N48O60S2: 4339.87; found: 4341.50.
Dendrimer D3 D3: Yield: 1.3 mg, 100%; semipreparative RP HPLC:
tR =21.00 min (l=225 nm, A/B=80/20!40/60 in 40 min); MS (ES+):
calcd for C160H201N46O58S2: 3758.36; found: 3758.75.
Dendrimer LRH HSD: Yield: 1.1 mg (0.257 mmol), 53%; semiprepara-
tive RP HPLC: tR =35.6 min (A/B=70/30!30/70 in 40 min); MS (+
ESI): calcd for C192H264N54O56S2: 4285.89; found: 4287.50.
Dendrimer E3 E3: Yield: 0.5 mg, 50%; semipreparative RP HPLC: tR =
17.00 min (l=225 nm, A/B=80/20!50/50 in 30 min); MS (ES+): calcd
for C174H209N42O60S2: 4014.45; found: 4014.75.
Dendrimer LRH DHS: Yield: 0.8 mg (0.188 mmol), 41%; semiprepara-
tive RP HPLC: tR =16.5 min (A/B=50/50!10/90 in 40 min); MS (+
ESI): calcd for C189H260N50O61S2: 4269.82; found: 4271.38.
Dendrimer F3 F3: Yield: 0.6 mg, 60%; semipreparative RP HPLC: tR =
16.20 min (l=225 nm, A/B=80/20!50/50 in 30 min); MS (ESI): calcd
for C172H209N54O54S2: 3958.46; found: 3959.00.
Dendrimer LRH DSH: Yield: 0.8 mg (0.190 mmol), 42%; semiprepara-
tive RP HPLC: tR =19.2 min (A/B=50/50!10/90 in 40 min); MS (+
ESI): calcd for C186H258N48O62S2: 4219.80; found: 4221.00.
Dendrimer A3 B3: Yield: 0.1 mg, 10%; semipreparative RP HPLC: tR =
42.80 min (l=225 nm, A/B=80/20!70/30 in 20 min, then !40/60 in
40 min); MS (ES+): calcd for C158H197N42O64S2: 3770.28; found: 3771.13.
Dendrimer LRH HHH: Yield: 0.4 mg (0.091 mmol), 31%; semiprepara-
tive RP HPLC: tR =22.7 min (A/B=60/40!20/80 in 40 min); MS (+
ESI): calcd for C200H270N60O52S2: 4407.98; found: 4409.99.
Dendrimer A3 D3: Yield: 0.1 mg, 9%; semipreparative RP HPLC: tR =
30.25 min (l=225 nm, A/B=90/10!50/50 in 40 min); MS: calcd for
Dendrimer HWS DHL: Yield: 0.6 mg (0.136 mmol), 33%; semiprepara-
tive RP HPLC: tR =21.6 min (A/B=70/30!30/70 in 40 min); MS (+
ESI): calcd for C199H244N52O61S2: 4401.72; found: 4403.13.
C
158H199N44O60S2: 3736.32; found: 3736.63.
Dendrimer A3 E3: Yield: 0.9 mg, 51%; semipreparative RP HPLC: tR =
18.00 min (l=225 nm, A/B=80/20!40/60 in 40 min); MS (ES+): calcd
for C165H203N48O59S2: 3864.37; found: 3865.00).
Dendrimer HWS DHS: Yield: 0.7 mg (0.160 mmol), 39%; semiprepara-
tive RP HPLC: tR =10.7 min (A/B=60/40!20/80 in 40 min); MS (+
ESI): calcd for C196H238N52O62S2: 4375.67; found: 4378.61.
Dendrimer A3 F3: Yield: 1 mg, 58%; semipreparative RP HPLC: tR =
18.25 min (l=225 nm, A/B=80/20!40/60 in 40 min); MS (ES+): calcd
for C164H203N48O58S2: 3836.38; found: 3836.75.
Dendrimer HWS DSH: Yield: 0.8 mg (0.185 mmol), 45%; semiprepara-
tive RP HPLC: tR =11.4 (A/B=60/40!20/80 in 40 min); MS (+ESI):
calcd for C193H236N50O63S2: 4325.64; found: 4328.59.
Dendrimer B3 C3: Yield: 0.2 mg, 20%; semipreparative RP HPLC: tR =
39.00 min (l=225 nm, A/B=90/10!70/30 in 20 min, then !A/B=40/60
Dendrimer HWS HSD: Yield: 0.5 mg (0.114 mmol), 26%; semiprepara-
tive RP HPLC: tR =11.8 min (A/B=60/40!20/80 in 40 min); MS (+
ESI): calcd for C199H242N56O57S2: 4391.74, found: 4393.00.
in 60 min); MS (ES+): calcd for
C163H199N44O66S2: 3876.30; found:
3777.13.
Dendrimer DHL HSD: Yield: 0.9 mg (0.213 mmol), 43%; semiprepara-
tive RP HPLC: tR =6.7 min (A/B=60/40!20/80 in 40 min); MS (+ESI):
calcd for C184H234N50O64S2: 4231.62; found: 4233.55.
Dendrimer B3 D3: Yield: 0.9 mg, 74%; semipreparative RP HPLC: tR =
21.25 min (l=225 nm, A/B=80/20!40/60 in 40 min); MS (ES+): calcd
for C160H199N44O62S2: 3792.31; found: 3793.00.
Dendrimer DHL DHS: Yield: 0.7 mg (0.166 mmol), 35%; semiprepara-
tive RP HPLC: tR =8.5 min (A/B=60/40!A/B=20/80 in 40 min); MS
(+ESI): calcd for C181H230N46O69S2: 4215.55; found: 4217.31.
Dendrimer B3 F3: Yield: 1.3 mg, 74%; semipreparative RP HPLC: tR =
18.50 min (l=225 nm, A/B=80/20!50/50 in 30 min); MS (ES+): calcd
for C166H203N48O60S2: 3892.37; found: 3893.25.
1225
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¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

J.-L. Reymond et al.
FULL PAPER
Dendrimer DHL DSH: Yield: 0.8 mg (0.192 mmol), 42%; semiprepara-
tive RP HPLC: tR =18.4 min (A/B=70/30!30/70 in 40 min); MS (+
ESI): calcd for C178H228N44O70S2: 4165.52; found: 4166.38.
91, 10747 10751; c) W. P. C. Stemmer, Nature 1994, 370, 389 391;
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Reetz, K.-E. Jaeger, Chem. Eur. J. 2000, 6, 407 412; f) S. V. Taylor,
P. Kast, D. Hilvert, Angew. Chem. 2001, 113, 3408 3436; Angew.
Chem. Int. Ed. 2001, 40, 3310 3335.
Dendrimer DFH HSD: Yield: 0.6 mg (0.140 mmol), 28%; semiprepara-
tive RP HPLC: tR =10.5 min (A/B=50/50!10/90 in 40 min); MS (+
ESI): calcd for C191H238N48O64S2: 4275.61; found: 4277.25.
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Assays and kinetic measurements: Dendrimers were kept as 1m stock
solutions in acetonitrile/water (1:1) and preserved at 48C in Eppendorf
vials. Dendrimers were freshly diluted to 0.05 mm in 20 mm aq. bis(2-hy-
droxyethyl)amino-tris(hydroxymethyl)methane (BisTris, pH 6.0) before
each measurement. The BisTris buffer (pH 6.0) was prepared with MilliQ
deionized water and the pH value was adjusted with aq. NaOH and aq.
HCl solutions. Assays were followed in individual wells of round-bot-
tomed polystyrene 96-well plates (Costar) by using a SPECTRAMax
fluorescence detector with preset values of the excitation and emission
wavelengths corresponding to the measured substrate. All pipetting ma-
nipulations were done by hand with single pipettes (Pipetman form,
Gilson). The measurement temperature inside the instrument was 258C.
Kinetic experiments were followed for 2 h. Fluorescence data were con-
verted into product concentrations by means of a calibration curve. Ini-
tial reaction rates were calculated from the steepest part observed during
the first 2000 s of each curve. In a typical experiment, 20 mm aq. BisTris
(20 mL; pH 6.0) was added to a well first, followed by dendrimer solution
(2.5 mL, 0.05 mm in aq. BisTris (pH 6.0), final concentration in the well=
5 mm) and then substrate solution (2.5 mL, 2 mm in acetonitrile/water
(1:1), final concentration in the well=200 mm). The rate observed under
these conditions is the apparent rate Vapp. Vuncat is the rate observed with
20 mm aq. BisTris (22.5 mL; pH 6.0) and substrate solution (2.5 mL, 2 mm
in acetonitrile/water (1:1), final concentration in the well=200 mm). The
observed rate enhancement Vnet/Vuncat is defined as (Vapp/Vuncat)ꢀ1. Mi-
chaelis Menten parameters were obtained from the linear double recip-
rocal plot of 1/Vnet against 1/[S], measured similarly with (final concentra-
tions) of 5 mm dendrimer (Vapp) or no dendrimer (Vuncat), 40, 60, 80, 100,
200, 300, 400, 500, 600, or 700 mm substrate, and 20 mm BisTris at 258C.
The reaction rate with 4-methylimidazole was obtained under the same
conditions with 40, 60, 80, 100, 200, 300, 400, and 500 mm 4-methylimida-
zole and 200 mm substrate.
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Received: September 26, 2003 [F5578]
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