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in higher generation dendrimers. The positive dendritic effect on catalysis lead to a
remarkable rate acceleration of k
cat
/k
uncat
ΒΌ
39,000 with the fourth-generation den-
drimer
G4
(AcHisSer)
16
(DapHisSer)
8
(DapHisSer)
4
(DapHisSer)
2
DapHisSerNH
2
and substrate
. This corresponds to a 1200-fold increase in reactivity per histidine
residue when compared to 4-methylimidazole as reference monovalent catalyst.
Isothermal titration calorimetry showed that the esterase dendrimer
1
and its
lower generation analogs bind one substrate molecule for three histidine residues.
The ester substrate
G4
binds the dendrimer approximately 10-fold stronger than the
hydroxypyrene trisulfonate reaction product
1
by hydrophobic interactions between
the acyl group and the dendrimer interior, which avoids product inhibition and allows
multiple turnover to take place. Acid-base titration showed a significant spread in the
pK
a
values of histidine side chains toward lower values within the dendrimer [28].
This suggests that the positive dendritic effect is caused in part by allowing the
coexistence of protonated and free base forms of the histidine side chains in the same
dendrimer. The protonated histidine favors electrostatic binding of the anionic
substrate
2
, while the free base form is necessary to promote nucleophilic or general
base catalyzed cleavage of the ester.
A similar positive dendritic effect by multivalent display of catalytic groups was
observed in aldolase peptide dendrimers with N-terminal proline residues, for exam-
ple,
1
AlaVal)
2
DapTyrLeuNH
2
(Figure 15.5),
which were investigated for catalysis of the coupling of acetone and cyclohexanone
to 4-nitrobenzaldehyde and dihydroxyacetone to 4-bromobenzaldehyde to form the
corresponding aldols [29]. The catalytic sequences were identified by on-bead
screening of a combinatorial library using either staining with a dye-labeled 1,3-
diketone to mark enamine-reactive amino groups, or the fluorogenic substrate dihy-
droxyacetone coumarin ether to detect enolization catalysis [30]. Iteration of the
(ProLys)
2
Dap and (ProThr)
2
Dap dendrons identified in active sequences to form
dendrimers of first, second, and third generation showed that the activity perN-terminal
proline for acetone aldolization was highest for the tetravalent second-generation
dendrimers
L2K7
(ProLys)
8
(DapSerArg)
4
(Dap
b
(ProLys)
4
(Dap-
ProLys)
2
DapProLysNH
2
. Trapping of the enamine intermediate with acetone by
reduction gave no reaction of lysine side chains and alkylation of 50% of the N-
terminal prolines, suggesting that the multivalency effect was caused by cooperativity
between pairs of proline residues, with one residue forming the enamine and the other
acting as general acid-base catalyst in the aldol reaction.
R1G2
(ProThr)
4
(DapProThr)
2
DapProThrNH
2
and
R2G2
15.3.2 Hydrophobic and Electrostatic Effects
Hydrophobic and electrostatic effects controlled dendrimer binding to the acylox-
ypyrene trisulfonates
discussed
above (Figure 15.4). These factors also determined the properties of three series of
peptide dendrimers designed to mimic enzymes with a single active site. These
dendrimers are equipped with catalytic residues at the dendrimer core, while amino
acids in the outer dendrimer branches modulate global properties such as charges and
hydrophobicity.
1-3
in the multivalent esterase dendrimers such as
G4
