Peptide dendrimer enzyme models for ester hydrolysis and aldolization prepared by convergent thioether ligation

Article abstract of DOI:10.1039/c1ob05877k

Peptide dendrimers with multiple histidines or N-terminal prolines efficiently catalyze ester hydrolysis or aldol reactions in aqueous medium. Part of the catalytic proficiency of these dendritic enzyme models stems from multivalency effects observed in G2, G3 and G4 dendrimers displaying multiple catalytic groups in their branches. To study multivalency in higher generation systems, G4, G5 and G6 peptide dendrimers were prepared by a convergent assembly. Thus, peptide dendrimers bearing four or eight chloroacetyl groups at their N-termini underwent multiple thioether ligation with G2 and G3 peptide dendrimers with a cysteine residue at their focal point, to give G4, G5 and G6 dendrimers containing up to 341 amino acids, including multiple histidines or N-terminal prolines. While the efficiency of the esterase catalysts was comparable to that of their lower generation analogs, a remarkable reactivity increase was observed in G5 and G6 aldolase dendrimers.

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PAPER
Peptide dendrimer enzyme models for ester hydrolysis and aldolization
prepared by convergent thioether ligation†
Nicolas A. Uhlich, Tamis Darbre and Jean-Louis Reymond*
Received 2nd June 2011, Accepted 20th July 2011
DOI: 10.1039/c1ob05877k
Peptide dendrimers with multiple histidines or N-terminal prolines efficiently catalyze ester hydrolysis
or aldol reactions in aqueous medium. Part of the catalytic proficiency of these dendritic enzyme
models stems from multivalency effects observed in G2, G3 and G4 dendrimers displaying multiple
catalytic groups in their branches. To study multivalency in higher generation systems, G4, G5 and G6
peptide dendrimers were prepared by a convergent assembly. Thus, peptide dendrimers bearing four or
eight chloroacetyl groups at their N-termini underwent multiple thioether ligation with G2 and G3
peptide dendrimers with a cysteine residue at their focal point, to give G4, G5 and G6 dendrimers
containing up to 341 amino acids, including multiple histidines or N-terminal prolines. While the
efficiency of the esterase catalysts was comparable to that of their lower generation analogs, a
remarkable reactivity increase was observed in G5 and G6 aldolase dendrimers.
terminal proline residues for aldol reactions. However, the limits
Introduction
of the divergent synthesis by SPPS precluded the systematic
Dendrimers are regularly branched synthetic macromolecules
investigation of multivalency effects in higher generation systems.
assembled from a variety of dendron building blocks, which dis-
Herein, we report on the properties of G4, G5 and G6
play useful properties for technical and biomedical applications.1
catalytic peptide dendrimers obtained by a one-step “dendrimers-
Dendrimers have been compared to proteins because of their
on-dendrimer” ligation of peptide dendrimer building blocks
roughly globular shape enforced by the dendritic topology. Peptide
themselves prepared by standard SPPS (Fig. 1). Thus, G2 or
dendrimers2 assembled from proteinogenic amino acids and
G3 peptide dendrimer “cores” functionalized with chloroacetyl
branching diamino acids are particularly well suited to study
groups on their four or eight N-termini underwent 4-fold or 8-
dendritic analogs of proteins since they are composed of the
fold thioether ligation with peptide dendrimer “arms” with a
same building blocks as natural proteins and only differ through
cysteine residue at their focal point, to form G4, G5 and G6
their dendritic rather than linear topology. Peptide dendrimers can
peptide dendrimers such as G5P5 (Fig. 2). This approach is
inspired by convergent approaches to dendrimers.12 The resulting
dendrimers display remarkable catalytic properties in aqueous
media for their respective reactions, namely the hydrolysis of
acetoxypyrene trisulfonate 1 and isobutyryl fluorescein 3 to give
the fluorescent hydroxypyrene trisulfonate 2 and fluorescein 4, and
the aldol addition of acetone to 4-nitrobenzaldehyde 5 to give aldol
6 (Scheme 1).
be obtained by solid phase peptide synthesis (SPPS) alternating
standard a-amino acids with branching diamino acids.3 This
method is efficient up to G3 dendrimers with eight end-groups,
and lends itself to combinatorial synthesis,4 enabling the screening
of relatively large libraries for function using either on-bead or off-
bead assays for binding5 or catalysis.6 Upon resynthesis of library
hits, the resulting dendrimers exhibit the expected functions, and
are also surprisingly resistant towards proteolysis.7
Screening peptide dendrimer libraries for various catalytic
reactions allowed us to identify peptide dendrimer enzyme models8
for ester hydrolysis9,10 and aldolization.11 In both reaction types
significant rate enhancements were observed by multivalency
effects, in particular by cooperativity between multiple histidine
residues for ester hydrolysis reactions, and between multiple N-
Results and discussion
Ligation strategy
SPPS of peptide dendrimers gives access to peptide dendrimers up
to G3 in excellent yields and purities, and is compatible with an
extremely large functional diversity as given by the available amino
acid building blocks, including various hydrophilic, hydrophobic,
aromatic, basic and acidic functional groups. However SPPS is
reliable only up to G3 dendrimers with eight end-groups and up
to 40–50 amino acids. To access higher generation peptide den-
drimers, we set out to investigate a “dendrimers-on-dendrimer”
Department of Chemistry and Biochemistry, University of Berne, Freiestrasse
3, 3012 Berne, Switzerland. E-mail: jean-louis.reymond@ioc.unibe.ch
† Electronic supplementary information (ESI) available: HPLC/MS spec-
tra and amino acid analysis data on all dendrimer building blocks
and ligation products and additional experimental details. See DOI:
10.1039/c1ob05877k
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with a purified peptide dendrimer “arm” bearing the correspond-
ing reactive group at its focal point.
Attempts to implement this strategy to prepare higher gener-
ation multivalent enzyme models using either click chemistry13
or native chemical ligation14 did not yield the desired products,
probably due to steric hindrance of the reactive focal points or
interference of the multiple catalytic groups, in particular histidine
residues. We then turned our attention to the thioether ligation
between a cysteine thiol and a haloacetyl group, which has been
used previously to couple unprotected linear peptide fragments
to form polymers, synthetic proteins and cyclic peptides.15 The
reaction is compatible with unprotected side chains and is reported
to proceed in good yields in either buffered water (pH 5.5–8.5) with
optional potassium iodide catalyst,15f or in DMF with diisopropyl
ethylamine (DIEA) as base.15j The resulting thioether bond is
stable towards hydrolysis and presents a minimal departure from
a pure peptide-type structure. The thioether ligation has been
used to conjugate the amino termini of PAMAM dendrimers with
linear peptides.16 As discussed below, we found this reaction to be
well suited for a one-step “dendrimers-on-dendrimer” convergent
assembly to access higher generation aldolase and esterase peptide
dendrimer enzyme models.
Synthesis of esterase peptide dendrimers by thioether ligation
The thioether ligation was first used to prepare higher genera-
tion analogs of previously reported esterase peptide dendrimers
consisting of the repeating (His-Ser)2Dap dendron.9 The corre-
sponding dendrimer cores G2C1 and G3C1 with four and eight
chloroacetyl groups, respectively, at the N-termini as well as the
two G2 dendrimer arms G2M1 and G2M2 with a cysteine residue
at their focal point were prepared by SPPS and obtained in good
yields as pure products after purification by preparative HPLC
(Table 1).
Fig. 1 Multivalent thioether ligation of G4, G5 and G6 enzyme models.
The thioether ligation required 1.5 equivalents of cysteine per
chloroacetyl group under inert atmosphere and slightly basic
conditions, ensuring that the starting materials and products were
all soluble. In each case the reaction proceeded to an endpoint
where the product composition did not evolve even upon addition
of excess dendrimer arm. The ligation products were purified
by preparative RP-HPLC, which separated minor amounts of
incompletely ligated core and unreacted arm present as disulfide
bridged dimers.
The ligation reaction was initially tested by coupling the
chloroacetylated cores G2C1 and G3C1 with the polyan-
ionic arm B1G2 (AcGlu-Ala)4(Lys-Amb-Tyr)2Dap-Cys-AspNH2,
whose cysteine thiol was known to be accessible for reaction
from its high reactivity with cobalamin (Scheme 2).17 The reaction
proceeded well in aqueous phosphate buffer pH 8.5 and yielded
G4E and G5E in 28% and 30%, respectively, after preparative
HPLC purification. The chloroacetylated core G2C1 reacted
under the same conditions with the polyhistidine “arms” G2M1
and G2M2 to give the polyhistidine G4 dendrimers G4H1 and
G4H2. However, reaction of the G3 core G3C1 with G2M1 and
G2M2 led to precipitation and incomplete conversion under a
variety of conditions, including aqueous, aqueous–organic, or
completely organic conditions. A G5 polyhistidine dendrimer was
obtained in the form of dendrimer G5H by ligation of core G3C2,
containing the (Pro-Ser)2Dap dendrons (see below), with arm
Scheme 1 Reactions catalyzed by multivalent esterase and aldolase
enzyme models.
strategy by which a purified peptide dendrimer “core” would
undergo multiple ligation at its suitably functionalized N-termini
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Fig. 2 Structural formula of peptide dendrimer G5P5.
G2M2 in 1 : 1 DMF/water with diisopropylethylamine (DIEA)
as base in the presence of potassium iodide as catalyst (Table 2).
dendrimers with multiple N-terminal prolines as catalytic groups,
in particular peptide dendrimers with repetitive (Pro-Thr)2Dap or
(Pro-Lys)2Dap dendrons.11 We chose to assemble higher gener-
ation aldolase dendrimers based on N-chloroacetylated G2 and
G3 dendrimer “cores” G2C2 and G3C2 composed of the related
(Pro-Ser)2Dap dendron, and the G3 core G3C3 assembled from
the sterically less congested (Pro-Ser)2Lys dendron. Six different
Synthesis of aldolase peptide dendrimers by thioether ligation
Our previous studies with aldolase peptide dendrimers showed
that efficient catalysis could be obtained with G2 and G3
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Table 1 Building blocks for the synthesis of esterase dendrimers by thioether ligation
Yieldc
MW (Da)d
Found
IDa
Sequenceb
%
mg
Calc.
G2C1
G3C1
G2M1
G2M2
(ClAcHS)4(BHS)2BHSNH2
(ClAcHS)8(BHS)4(BHS)2BHSNH2
(AcHS)4(BHS)2BCNH2
44
15
40
42
157
116
123
148
2150.0
4594.0
1891.0
2116.0
2150.8
4594.8
1891.9
2116.1
(AcHS)4(BHS)2BHSCNH2
a Synthesized on Tenta Gel S RAMꢀR resin, loading 0.24 mmol g-1. b B = Dap = (S)-2,3-diaminopropionic acid branching. c Yields were calculated for the
TFA salts of the products. d ESI+.
Table 2 Esterase peptide dendrimers and analogs prepared by thioether ligation
Yield
%
MW (Da)c
Found
d
ID
Sequencea
C.b
mg
Calc.
NAA
+
7
Rh (nm)e
G4E
G5E
G4H1
G4H2
G5H
(AcEA)16(KXY)8(BC(D)xHS)4(BHS)2BHSNH2
(AcEA)32(KXY)16(BC(D)xHS)8(BHS)4(BHS)2BHSNH2
(AcHS)16(BHS)8(BCxHS)4(BHS)2BHSNH2
I
I
I
I
28
30
12
8
8.23
13.3
4.02
2.24
13.7
10562.0
21417.0
9572.0
10471.0
20634.0
10561.9
21416.1
9572.5
10470.4
20633.6
85
173
81
89
181
1.98 0.08
2.35 0.02
2.63 0.06
2.92 0.09
2.51 0.07
15
31
35
56
(AcHS)16(BHS)8(BHSCxHS)4(BHS)2BHSNH2
(AcHS)32(BHS)16(BHSCxPS)8(BPS)4(BPS)2BPSNH2
II
50
a B = Dap = (S)-2,3-diaminopropionic acid branching; K = lysine branching; X = Amb = 4-aminomethylbenzoic acid; x = thioether linkage. b Conditions:
I: 0.2 M phosphate pH 8.5; II: DMF/H2O, 55 equiv. DIEA, 20 equiv. KI. c ESI+ for all compounds except G4E/G5E (ESI-). d + charges calculated for
pH 2. e From PGSE diffusion NMR in D2O, 303 K, pH 2 (as formic acid salts).
Table 3 Peptide dendrimer building blocks for the synthesis of G4–G6 aldolase dendrimers
Yieldc
%
MW (Da)d
Found
IDa
Sequenceb
mg
Calc.
G2C2
G3C2
G3C3
G2M3
G2M4
G2M5
G2M6
G2M7
G3M1
(ClAcPS)4(BPS)2BPSNH2
(ClAcPS)8(BPS)4(BPS)2BPSNH2
(ClAcPS)8(KPS)4(KPS)2KPSNH2
(PS)4(BPS)2BPSCNH2
46
25
18
47
49
55
59
45
36
104
120
150
119
111
109
196
150
149
1869.6
3993.9
4287.9
1668.0
1863.0
1651.0
1955.0
2307.5
3485.0
1870.6
3994.4
4289.0
1667.8
1863.9
1651.7
1955.5
2308.4
3485.7
(PD)4(BPD)2BPDCNH2
(PD)4(BPD)2BCNH2
(PK)4(BPK)2BPKCNH2
(pPE)4(BPD)2BPDCNH2
(PS)8(BPS)4(BPS)2BPSCNH2
a Synthesized on Tenta Gel S RAMꢀR resin, loading 0.24 mmol g-1. b B = Dap = (S)-2,3-diaminopropionic acid branching; K = lysine branching. c Yields
were calculated for the TFA salts of the products. d ESI+.
dendrimer “arms” with a cysteine as the focal point were prepared
featuring various types of potentially catalytic di- or tri-peptides
at their N-termini, including Pro-Ser (G2M3, G3M1), Pro-Asp
(G2M4), G2M5), Pro-Lys (G2M6), and D-Pro-Pro-Glu (G2M7)
which has been shown to be a particularly active organocatalyst
for various aldol-type reactions (Table 3).18
The thioether ligation was then used to couple various combi-
nations of cores and arms to prepare G4, G5 and G6 aldolase
peptide dendrimers (Table 4). The reactions proceeded well under
a variety of conditions, including water/DMF as used for G5H,
or water with NaHCO3 as base and potassium iodide catalyst with
or without acetonitrile as cosolvent, to yield the ligation products
after preparative RP-HPLC.
starting materials, the disulfide bridged dimer of the arm, and
the intermediates of partial ligation. The complete (4-fold or 8-
fold) ligation product was in each case the major product detected
by MS, either as the only peak, or together with minor peaks
corresponding to the incomplete (3-fold, 7-fold or 6-fold) ligation
products (Fig. 3A and the ESI†). These peaks could be assigned to
an unreacted chloroacetyl (G5E and G4H2) or iodoacetyl group
(G5P3, G5P4, G5P6, G5P7), or a hydroxyacetyl group (G5H and
G5P5). The two G6 dendrimers also gave sharp UPLC peaks;
however, their MS spectra could not be deconvoluted.
Characterization by SDS-PAGE showed relatively sharp bands
for the dendrimers. In several cases such as G5P3 a second
weaker band was separated, which probably corresponded to the
incomplete ligation product observed by MS. Surprisingly, the
highly cationic G5P4 did not stain at all with Coomassie blue or
silver staining, and G4P and G4E only stained very weakly (Fig.
3B). The migration behavior of the dendrimers was very different
from that of linear peptides and was strongly sequence dependent.
Characterization of the peptide dendrimers
The HPLC-purified ligation products gave sharp peaks by an-
alytical UPLC under conditions which clearly separated the
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Scheme 2 Thioether ligation between the dendrimer core G2C1 and the dendrimer arm B1G2.
Such anomalous behavior has also been reported with peptide
dendrimers obtained by native chemical ligation of linear peptides
and proteins to PPI dendrimers.14b
Standard PGSE diffusion NMR experiments were used to
determine the hydrodynamic radii of the dendrimers.10,17,19 The
analysis indicated that the dendrimers were homogeneous, with
hydrodynamic radii (Rh) ranging from 1.98 to 5.51 nm de-
pending on molecular weight and amino acid sequence (Tables
2 and 4). A linear relationship between hydrodynamic radius
and molecular weight was observed for the homologous series
G4P→G5P2→G6P1 (Fig. 4A). A comparable increase in hydro-
dynamic radius as a function of MW occurred in the three peptide
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Table 4 Synthesis of aldolase peptide dendrimers and analogs by thioether ligation
Yield
MW (Da)c
Found
b
d
ID
Sequencea
C.
%
mg
Calc.
NAA
+
Rh (nm)e
G4P
(PS)16(BPS)8(BPSCxPS)4(BPS)2BPSNH2
(PD)32(BPD)16(BCxPS)8(BPS)4(BPS)2BPSNH2
II
III
II
IV
IV
V
II
II
II
II
55
57
40
62
77
11
66
56
45
51
2.05
10.3
1.48
14.7
16.4
2.77
11.8
15.6
17.7
18.2
8393.0
16916.0
17043.0
18620.0
19353.0
17645.0
18907.0
22459.0
n.d.f
8395.9
89
16
32
32
32
88
47
32
32
64
64
2.09 0.07
2.76 0.05
3.42 0.04
3.12 0.04
4.74 0.15
3.64 0.10
3.07 0.05
3.21 0.08
5.51 0.07
4.88 0.18
G5P1
G5P2
G5P3
G5P4
G5P5
G5P6
G5P7
G6P1
G6P2
16916.1
17045.1
18619.7
19352.4
17645.5
18908.2
22464.8
31588.4
31883.0
165
181
181
181
181
181
213
341
341
(PS)32(BPS)16(BPSCxPS)8(BPS)4(BPS)2BPSNH2
(PD)32(BPD)16(BPDCxPS)8(BPS)4(BPS)2BPSNH2
(PK)32(BPK)16(BPKCxPS)8(BPS)4(BPS)2BPSNH2
(PS)32(BPS)16(BPSCxHS)8(BHS)4(BHS)2BHSNH2
(PD)32(BPD)16(BPDCxPS)8(KPS)4(KPS)2KPSNH2
(pPE)32(BPD)16(BPDCxPS)8(KPS)4(KPS)2KPSNH2
(PS)64(BPS)32(BPS)16(BPSCxPS)8(BPS)4(BPS)2BPSNH2
(PS)64(BPS)32(BPS)16(BPSCxPS)8(KPS)4(KPS)2KPSNH2
n.d.f
a B = Dap = (S)-2,3-diaminopropionic acid branching; K = lysine branching; x = thioether linkage. b Conditions: II: DMF/H2O, 55 equiv. DIEA, 20 equiv.
KI; III: 0.1 M aq. phosphate pH 8.5, 20 equiv. KI; IV: 0.5 M NaHCO3 pH 8.0, 20 equiv. KI; V: 0.5 M NaHCO3 pH 8.0/CH3CN, 20 equiv. KI. c ESI+
for all compounds. d + charges calculated for pH 2. e From PGSE diffusion NMR in D2O, 303 K, pH 2 (as formic acid salts). f n.d. = not detected.
Fig. 3 Analysis of ligation products. A. Direct MS (ESI+) of a sample of the HPLC-purified ligated dendrimer G5P3 C757H1105N221O316S8 (8-fold ligation
product, main peak) found/calc. 18620.0/18619.7 [M + 6H]+. 7-Fold ligation product with unreacted iodoacetyl group C682H996IN199O284S7 (peak height
26% of main peak) found/calc. 16876.0/16877.7 [M]+; G5P4 C869H1497N277O204S8 (8-fold ligation product, main peak) found/calc. 19353.0/19352.4 [M
+ 6H]+. 7-Fold ligation product with one hydroxyacetyl group C780H1340N248O187S7 (peak height 10% of main peak) found/calc. 17411.0/17411.0 [M +
2H]+. B. SDS-PAGE of thioether ligation products.
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(kcat/kuncat ~ 104, KM ~ 50 mM, Table 5). The catalytic proficiency
per histidine residue was however independent of dendrimer size,
and was in the range of that previously reported for dendrimer
A2 (dendrimer A2 structure: (AcHis-Ser)4(Dap-His-Ser)2Dap-His-
SerNH2) under the same conditions ((kcat/KM)/k2)/NHis = 280
with NHis = 7),9 with no significant additional positive effect
from the increased multivalency. Considering that the histidine-
containing dendrimer arms G2M1 and G2M2 used to prepare the
ligated dendrimers correspond to dendrimer A2 appended with
an additional cysteine at the focal point, the data suggest that
the extended tripeptide or pentapeptide branch at the level of
the thioether bond results in reduced steric congestion preventing
additive multivalency.
Hydrolysis of the hydrophobic fluorescein isobutyryl ester 3
was also catalyzed by the G4 and G5 esterase dendrimers.
We recently reported the use of substrate 3 to study peptide
dendrimer enzyme models, and showed that the substrate reacts
faster with more hydrophobic dendrimers than substrate 1.20
Ester 3 was converted with efficiencies per histidine residue in
the range of 2 < ((kcat/KM)/k2)/NHis < 20 for various histidine
containing catalysts including linear polyhistidine peptides,21,22
and G3 peptide dendrimers. The highest values were observed
with G3 peptide dendrimers H7 (His)8(Lys-Phe)4(Lys-Pro)2Lys-
aProNH2) and AcH3 (AcHis)8(Lys-Leu)4(Lys-Val)2Lys-LysOH,
an effect attributed to the presence of hydrophobic residues in
the sequence.20 The present G4 and G5 peptide dendrimers gave
a comparable value of ((kcat/KM)/k2)/NHis ~10; however, with
the important difference that pre-equilibrium substrate binding
in the range of KM ~ 50 mM was observed. By contrast, the
lower generation peptide dendrimers such as H7 and AcH3 gave
no detectable substrate binding. This apparent substrate binding
in the G4 and G5 esterase dendrimers might indicate more
potent hydrophobic interactions with the substrate created by a
microenvironment effect possible only in these larger dendrimers.
Fig. 4 Hydrodynamic radii Rh of the ligation products in water pH 2 as
a function of A. molecular weight, and B. the number of positive charges
calculated at pH 2.
dendrimers with a Lys-branched core G5P6→G5P7→G6P2.
These dendrimers were more compact than their analogs with
a Dap-branched core probably due to a tighter packing enabled by
the longer but more flexible Lys branching unit. The hydrodynamic
radius also correlated with the number of positive charges (at pH
2 corresponding to the pH of dissolved formic acid salts), sug-
gesting that electrostatic repulsion between dendrimer branches
enforced an extended conformation as previously observed with
polyanionic peptide dendrimers (Fig. 4B).17
Overall, the MS, SDS-PAGE and NMR data on the isolated
products were consistent with the products resulting from a
4-fold or 8-fold ligation of the cysteine-containing dendrimer
“arms” onto the chloroacetylated dendrimer “cores”. The ligation
products were also analyzed by quantitative amino acid analysis,
in which a weighted sample is hydrolyzed by complete acidic hy-
drolysis, and the released amino acids are quantified by analytical
HPLC as phenylthiocarbamoyl derivatives. The observed amino
acid composition was in all cases consistent with the calculated
composition. The quality of the samples obtained was judged
sufficient to study the effect of multivalency in catalysis in these
higher generation peptide dendrimer enzyme models.
Catalytic properties of the aldolase enzyme models
The G4, G5 and G6 dendrimers with multiple N-terminal prolines
were investigated for catalysis of the aldol addition of acetone to 4-
nitrobenzaldehyde 5 to form aldol 6 (Scheme 1). The reaction was
conducted in 50% aqueous acetone at neutral pH using 100 mM 4-
nitrobenzaldehyde and 0.64 mM (0.64 mol%) N-terminal proline
added either as free L-proline or as G4, G5 or G6 dendrimer
(Fig. 5A). The reaction was efficiently catalyzed by several of
the dendrimers, in particular G6P1, G6P2 and G5P5. Catalysis
with the best catalyst G6P1 corresponded to a 150-fold reactivity
enhancement per N-terminal proline (over proline itself), which
compared favorably to the only 4.3-fold increase in MW per N-
terminal proline (Fig. 5B). This relatively strong dendritic effect
with the G5 and G6 dendrimers was surprising since previously
studied peptide dendrimer aldolases showed only a 4-fold activity
increase per N-terminal proline with G2 dendrimers with no
further increase at G3.11 Catalysis with G6P1 was highest at
neutral pH, which might indicate bifunctional catalysis involving
a free-base and a protonated proline with depressed pKa due to
multivalency (Fig. S1, ESI†).
Catalytic properties of the esterase enzyme models
Dendrimers G4H1, G4H2 and G5H, G5P5 with multiple His-
Ser branches catalyzed the ester hydrolysis of 8-acetoxypyrene-
1,3,6-trisulfonate 1 in aqueous buffer with enzyme-like kinetics
The enantioselectivity of the aldol reaction was determined with
1 mol% N-terminal proline under aqueous conditions, as well
as under organic conditions (DMSO/acetone) under which the
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Table 5 Catalytic parameters for ester hydrolysis of substrates 1 and 3
G4H1
31
G4H2
35
G5H
56
G5P5
15
NHis
Substrate 1
Substrate 3
KM (mM)
42
3
50
1
72
3
48
1
kcat (min-1)
0.32 0.0067
7600 582
250
8740
7580
0.32 0.0034
6330 170
180
8590
6310
180
39
0.034 0.004
880 88
25
0.43 0.0081
6050 355
110
11800
6040
110
29
0.037 0.002
1290 70
23
1540
570
0.33 0.0079
6790 155
450
8900
6770
450
50
0.017 0.0007
340 19
23
690
150
k
k
k
cat/KM (min-1 M-1)
cat/KM/NHis (min-1 M-1)
cat/kuncat
(kcat/KM)/k2
(kcat/KM)/k2/NHis
KM (mM)
250
65 10
0.047 0.004
730 85
24
1940
320
7
kcat (min-1)
k
k
k
cat/KM (min-1 M-1)
cat/KM/NHis (min-1 M-1)
cat/kuncat
1390
390
11.1
(kcat/KM)/k2
(kcat/KM)/k2/NHis
10.3
10.1
10.0
Conditions: 5 mM aq. citric acid-sodium citrate pH 5.5, with 11% acetonitrile for substrate 3; 1.39–1.86 mM peptide dendrimers; 120 mL assays in
microtiterplate wells were followed by fluorescence (lexc = 450 25 nm, lem = 530 12 nm) at 34 ◦C; all kinetics result from three independent
measurements. KM = reciprocal substrate binding; kuncat (1) = 3.9 ¥ 10-5 min-1; kuncat(3) = 2.5 ¥ 10-5 min-1; k2(1) = 1.0 M-1min-1; k2 (3) = 2.26 M-1min-1 (2nd
order rate constant with 4-methyl imidazole); NHis = number of histidine residues in the dendrimer.
Conclusion
Esterase and aldolase peptide dendrimers were prepared in a
single step by a multivalent dendrimers-on-dendrimer ligation
strategy from G2 and G3 peptide dendrimer building blocks,
which were themselves prepared readily by SPPS from standard
building blocks. The G4 and G5 esterase peptide dendrimers thus
obtained showed catalytic proficiencies per histidine residue which
were comparable to previously reported G3 dendrimers. On the
other hand, G5 and G6 aldolase peptide dendrimers exhibited
remarkable aldolase reactivity not present in lower generation
analogs, although without significant enantioselectivity. The al-
dolase reactivity was also largely sequence dependent.
The simple one-step ligation approach presented to assemble
higher generation peptide dendrimers gives access to products
with MW above 20 kDa, a size range corresponding to proteins
and which is not accessible by direct SPPS. This convergent
synthesis gave products with satisfactory yields and purities,
although minor amounts of incomplete ligation products were
also observed. The thioether ligation has the advantage of being
compatible with unprotected amino acid side chains. In addition,
the reaction does not require any unusual building blocks apart
from the chloroacetyl group, which is simply added by acylation
of the N-termini at the last step of the dendrimer “core” SPPS.
This multivalent dendrimers-on-dendrimer strategy should be
generally useful to explore the structure and function of other
types of peptide dendrimers.
Fig. 5 Dendrimer-catalyzed aldol reaction. A. Time-course of product
Experimental section
formation with 100 mM 4-nitrobenzaldehyde and 0.64 mol% N-terminal
proline in acetone/aq. HEPES pH 7.0 (1/1) at 25 ◦C. Followed by
SPPS
RP-HPLC. B. Initial rates of the dendrimer-catalyzed aldol reaction with
100 mM 4-nitrobenzaldehyde acetone/aq. HEPES pH 7.0 (1/1) as a
function of the dendrimers’ MW.
Dendrimers were synthesized by adding 500–800 mg of Tenta
R
Gel S RAMꢀ resin (loading: 0.24 or 0.26 mmol g-1) in a 10 mL
reference catalyst L-proline is active at 30 mol% catalyst and gives
significant enantioselectivity (76% ee).23 The dendrimers showed
only low levels of enantioselectivity in both cases (9–41% ee, Table
S1, ESI†).
polypropylene syringe fitted with a polyethylene frit, a Teflon
stopcock and stopper. The resin was swollen in DCM for 15 min.
After removal of the DCM the first amino acid was coupled.
Stirring of the reaction mixture at any given step described below
7078 | Org. Biomol. Chem., 2011, 9, 7071–7084
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was performed by attaching the closed syringe to a rotating axis.
The following coupling conditions were used:
1.405 min (A/D 100/0 to 0/100 in 5 min, l = 214 nm). MS (ESI+):
C73H109Cl4N21O28 found/calc. 1869.6/1870.6 [M]+.
Coupling of the Fmoc-protected amino acids
(ClAc-His-Ser)8(Dap-His-Ser)4(Dap-His-Ser)2Dap-His-Ser-
NH2(G3C1)
3.0 equivalents of Fmoc-protected amino acid and 3.0 equivalents
of PyBOP in 6 mL of NMP were added to the resin. 5.0
equivalents of DIEA were added and the reaction was stirred
for 30 min (reaction times were prolonged for the amino acids
after 1 branching unit (60 min), after 2 branching units (90 min)
and 3 branching units (120 min); for branching units twice the
reaction time was used). The resin was then washed (3¥ each)
with NMP, MeOH and DCM. The effectiveness of the coupling
was monitored by the trinitrobenzensulfonic acid (TNBS)24 or
chloranil test.
G3C1 was obtained as a foamy yellowish solid after preparative
RP-HPLC (116.2 mg, 18.4 mmol, 15%). Analytical RP-HPLC:
tR = 9.766 min (A/D 100/0 to 50/50 in 10 min, l = 214 nm). MS
(ESI+): C172H233Cl8N75O60 found/calc. 4594.0/4594.8 [M]+.
(ClAc-Pro-Ser)8(Dap-Pro-Ser)4(Dap-Pro-Ser)2Dap-Pro-Ser-NH2
(G3C2)
G3C2 was obtained as a foamy yellowish solid after preparative
RP-HPLC (119.9 mg, 30.0 mmol, 25%). Analytical RP-UPLC: tR =
1.515 min (A/D 100/0 to 0/100 in 5 min, l = 214 nm). MS (ESI+):
C157H233Cl8N45O60 found/calc. 3993.9/3994.4 [M]+; 4015.4/4017.4
[M + Na]+; 4034.3/4033.5 [M + K]+.
Cleavage of the Fmoc protecting group
The Fmoc protecting group was removed with 6 mL of a solution
of DMF/piperidine (1/4, v/v) for 10 min. After filtration the
procedure was repeated and finally the resin washed (3¥ each)
with NMP, MeOH and DCM.
(ClAc-Pro-Ser)8(Lys-Pro-Ser)4(Lys-Pro-Ser)2Lys-Pro-Ser-NH2
(G3C3)
N-Acetylation/N-chloroacetylation
G3C3 was obtained as a foamy colourless solid after preparative
RP-HPLC (149.7 mg, 34.9 mmol, 18%). Analytical RP-UPLC: tR =
1.510 min (A/D 100/0 to 0/100 in 5 min, l = 214 nm). MS (ESI+):
C178H275Cl8N45O60 found/calc. 4287.9/4289.0 [M]+; 4310.0/4312.0
[M + Na]+; 4326.7/4328.1 [M + K]+; 4348.8/4351.1 [M + Na +
K]+; 4364.1/4367.2 [M + 2K]+.
When necessary the resin was acetylated/chloroacetylated with a
solution of acetic acid anhydride/DCM (1/1, v/v) or a solution of
chloroacetic acid anhydride (10.0 equivalents)/DCM for 30 min.
After filtration the resin was washed (3¥ each) with NMP, MeOH
and DCM.
TFA cleavage
(Ac-His-Ser)4(Dap-His-Ser)2Dap-Cys-NH2 (G2M1)
The cleavage was carried out using
a
TFA/H2O/TIS
G2M1 was obtained as a foamy colourless solid after preparative
RP-HPLC (123.0 mg, 47.8 mmol, 40%). Analytical RP-HPLC:
tR = 7.682 min (A/D 100/0 to 50/50 in 10 min, l = 214 nm). MS
(ESI+): C74H106N32O26S found/calc. 1891.0/1891.9 [M]+.
(95/2.5/2.5 v/v/v) solution (if the dendrimer contained Cys a
TFA/H2O/TIS/EDT (94/1/2.5/2.5 v/v/v) solution was used
instead) for 4.5 h.
Purification
(Ac-His-Ser)4(Dap-His-Ser)2Dap-His-Ser-Cys-NH2 (G2M2)
After TFA cleavage the dendrimers were separated from the resin
by filtration and precipitated with ice-cold MTBE. The MTBE
was removed by centrifugation and the crude product dried under
vacuum. It was then dissolved in a water/acetonitrile mixture,
purified by preparative RP-HPLC and lyophilized. Yields were
calculated for the TFA salts of the products. For characterization
by analytical RP-HPLC the following elution solutions were used:
A = mQ-deionized H2O with 0.1% TFA; D = mQ-deionized
H2O/HPLC-grade CH3CN (40 : 60) with 0.1% TFA.
G2M2 was obtained as a foamy colourless solid after preparative
RP-HPLC (147.9 mg, 50.7 mmol, 42%). Analytical RP-HPLC:
tR = 7.569 min (A/D 100/0 to 50/50 in 10 min, l = 214 nm). MS
(ESI+): C83H118N36O29S found/calc. 2116.0/2116.1 [M]+.
(Pro-Ser)4(Dap-Pro-Ser)2Dap-Pro-Ser-Cys-NH2(G2M3)
G2M3 was obtained as a foamy colourless solid after preparative
RP-HPLC (118.8 mg, 56.0 mmol, 47%). Analytical RP-HPLC:
tR = 9.142 min (A/D 100/0 to 50/50 in 10 min, l = 214 nm).
MS (ESI+): C68H110N22O25S found/calc. 1668.0/1667.8 [M]+;
1709.0/1708.8 [M + 2H + K]+.
(ClAc-His-Ser)4(Dap-His-Ser)2Dap-His-Ser-NH2 (G2C1)
G2C1 was obtained as a foamy colourless solid after preparative
RP-HPLC (156.7 mg, 53.1 mmol, 44%). Analytical RP-HPLC:
tR = 9.071 min (A/D 100/0 to 50/50 in 10 min, l = 214 nm).
MS (ESI+): C80H109Cl4N35O28 found/calc. 2150.0/2150.8 [M]+;
2189.0/2189.9 [M + K]+.
(Pro-Asp)4(Dap-Pro-Asp)2Dap-Pro-Asp-Cys-NH2(G2M4)
G2M4 was obtained as a foamy colourless solid after preparative
RP-HPLC (110.5 mg, 59.3 mmol, 49%). Analytical RP-HPLC:
tR = 9.258 min (A/D 100/0 to 50/50 in 10 min, l = 214 nm).
MS (ESI+): C75H110N22O32S found/calc. 1863.0/1863.9 [M]+;
1902.0/1903.0 [M + K]+.
(ClAc-Pro-Ser)4(Dap-Pro-Ser)2Dap-Pro-Ser-NH2(G2C2)
G2C2 was obtained as a foamy colourless solid after preparative
RP-HPLC (104.0 mg, 55.6 mmol, 46%). Analytical RP-UPLC: tR =
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(Pro-Asp)4(Dap-Pro-Asp)2Dap-Cys-NH2(G2M5)
(Ac-Glu-Ala)16(Lys-Amb-Tyr)8(Dap-Cys(Asp)-x-His-Ser)4(Dap-
His-Ser)2Dap-His-Ser-NH2 (G4E)
G2M5 was obtained as a foamy colourless solid after prepar-
ative RP-HPLC (109.0 mg, 66.0 mmol, 55%). Analytical RP-
HPLC: tR = 8.330 min (A/D 100/0 to 50/50 in 10 min, l =
214 nm). MS (ESI+): C66H98N20O28S found/calc. 1651.0/1651.7
[M]+; 1690.0/1690.8 [M + K]+.
From starting materials G2C1 and B1G2 using the general
procedure described above (solvent: 0.2 M phosphate buffer pH
8.5), G4E was obtained as a colourless solid after preparative
RP-HPLC (8.23 mg). Amino acid analysis: protein content 52.5%
(4.32 mg, 0.40 mmol, yield 28%); found/calc. (%) Glu 17.6/18.8,
Ala 17.3/18.8, Dap 11.8/8.2, Tyr 10.0/9.4, Amb 9.5/9.4, Lys
9.2/9.4, His 7.8/8.2, Ser 7.1/8.2, Asp 4.8/4.7, CM-Cys 4.8/4.7.
Analytical RP-UPLC: tR = 1.467 min (A/D 100/0 to 0/100 in
5 min, l = 214 nm). MS (ESI-): C464H629N119O160S4 found/calc.
10562.0/10561.9 [M]-; 10624.0/10624.0 [M + Na + K]-.
(Pro-Lys)4(Dap-Pro-Lys)2Dap-Pro-Lys-Cys-NH2 (G2M6)
G2M6 was obtained as a foamy colourless solid after prepar-
ative RP-HPLC (195.9 mg, 71.2 mmol, 59%). Analytical RP-
HPLC: tR = 9.523 min (A/D 100/0 to 50/50 in 10 min, l =
214 nm). MS (ESI+): C89H159N29O18S found/calc. 1955.0/1955.5
[M]+; 1996.0/1994.6 [M + K]+; 2069.0/2069.5 [M + TFA]+;
2183.0/2183.5 [M + 2TFA]+; 2297.0/2297.5 [M + 3TFA]+;
2411.0/2411.5 [M + 4TFA]+; 2525.0/2525.5 [M + 5TFA]+.
(Ac-Glu-Ala)32(Lys-Amb-Tyr)16(Dap-Cys(Asp)-x-His-Ser)8(Dap-
His-Ser)4(Dap-His-Ser)2Dap-His-Ser-NH2 (G5E)
From starting materials G3C1 and B1G2 using the general
procedure described above (solvent: 0.2 M phosphate buffer
pH 8.5), G5E was obtained as a foamy colourless solid after
preparative RP-HPLC (13.3 mg). Amino acid analysis: protein
content 32.3% (4.29 mg, 0.19 mmol, yield 30%); found/calc. (%)
Ala 19.2/18.5, Glu 15.2/18.5, Dap 13.1/8.7, Tyr 9.8/9.2, His
9.7/8.7, Lys 9.2/9.2, Amb 9.0/9.2, Ser 8.7/8.7, CM-Cys 4.2/4.6,
Asp 1.9/4.6. Analytical RP-UPLC: tR = 1.480 min (A/D 100/0
to 0/100 in 5 min, l = 214 nm). MS (ESI-): C940H1273N243O324S8
(8-fold ligation product, main peak) found/calc. 21417.0/21416.1
[M - H]-. C844H1143ClN222O291S7 (7-fold ligation product with one
unreacted chloroacetyl group, peak height 33% of main peak)
found/calc. 19311.0/19313.3 [M - H]-.
(DPro-Pro-Glu)4(Dap-Pro-Asp)2Dap-Pro-Asp-Cys-NH2 (G2M7)
G2M7 was obtained as a foamy colourless solid after preparative
RP-HPLC (149.9 mg, 54.2 mmol, 45%). Analytical RP-UPLC: tR =
1.267 min (A/D 100/0 to 0/100 in 5 min, l = 214 nm). MS (ESI+):
C99H146N26O36S found/calc. 2307.5/2308.4 [M]+.
(Pro-Ser)8(Dap-Pro-Ser)4(Dap-Pro-Ser)2Dap-Pro-Ser-Cys-NH2
(G3M1)
G3M1 was obtained as a foamy colourless solid after preparative
RP-HPLC (149.0 mg, 42.8 mmol, 36%). Analytical RP-HPLC: tR =
8.995 min (A/D100/0 to 50/50 in 10 min, l = 214 nm). MS (ESI+):
C144H230N46O53S found/calc. 3485.0/3485.7 [M]+; 3527.0/3526.8
[M + 2H + K]+; 3568.0/3567.9 [M + 4H + 2K]+; 3610.0/3610.0
[M + 7H + 3K]+; 3650.0/3650.2 [M + 8H + 4K]+.
(Ac-His-Ser)16(Dap-His-Ser)8(Dap-Cys-x-His-Ser)4(Dap-His-
Ser)2Dap-His-Ser-NH2 (G4H1)
From starting materials G2C1 and G2M1 using the general
procedure described above (solvent: 0.2 M phosphate buffer
pH 8.5), G4H1 was obtained as a foamy colourless solid after
preparative RP-HPLC (4.02 mg). Amino acid analysis: protein
content 46.6% (1.87 mg, 0.17 mmol, yield 12%); found/calc. (%)
His 47.8/38.3, Ser 31.5/38.3, Dap 16.3/18.5, CM-Cys 4.4/4.9.
Analytical RP-UPLC: tR = 1.228 min (A/D 100/0 to 0/100 in
5 min, l = 214 nm). MS (ESI+): C376H529N163O132S4 found/calc.
9572.0/9572.5 [M]+.
Thioether ligation
In a typical experiment a solution of core dendrimer (sequence
containing chloroacetyl end groups, ~3 mg, 1.0 equiv.) and KI
(20 equiv.) in DMF/H2O (1/1 v/v) (300 mL) was prepared in a
5 mL pointed glass flask. The mixture was degassed (Ar) during
10 min. In a 5 mL round bottomed glass flask arm dendrimer
(Cys containing sequence, 1.5 equiv. per chloroacetyl end group
in core sequence) was prepared (without solvent) and the flask
degassed (Ar) during 10 min. The core dendrimer solution was
transferred to the round bottomed glass flask containing the arm
dendrimer with a gas tight syringe. DIEA (55 equiv.) was added
and the solution stirred at rt. The reaction was followed by RP-
UPLC (1.0 mL reaction mixture taken with a gas tight 10 mL glass
syringe + 200 mL of A). After completion (usually overnight for 4th
generation and 18 to 22 h for 5th and 6th generation sequences),
the reaction was quenched by adding 3.5 mL of A, filtered and
directly injected to preparative RP-HPLC. The purified product
was lyophilized (3¥) with formic acid 1%. Yields were calculated
based on amino acid analysis.
(Ac-His-Ser)16(Dap-His-Ser)8(Dap-His-Ser-Cys-x-His-Ser)4(Dap-
His-Ser)2Dap-His-Ser-NH2 (G4H2)
From starting materials G2C1 and G2M2 using the general
procedure described above (solvent: 0.2 M phosphate buffer
pH 8.5), G4H2 was obtained as foamy colourless solid after
preparative RP-HPLC (2.24 mg). Amino acid analysis: protein
content 59.7% (1.34 mg, 0.11 mmol, yield 8%); found/calc. (%)
His 48.8/39.3, Ser 32.1/39.3, Dap 14.7/16.9, CM-Cys 4.3/4.5.
Analytical RP-UPLC: tR = 1.230 min (A/D 100/0 to 0/100
in 5 min, l = 214 nm). MS (ESI+): C412H577N179O144S4 (4-fold
ligation product, main peak) found/calc. 10471.0/10470.4 [M +
H]+; 10511.0/10510.5 [M + 2H + K]+. C329H460ClN143O115S3 (3-
fold ligation product with one unreacted chloroacetyl group, peak
height 9% of main peak) found/calc. 8392.0/8390.7 [M + H]+.
Several variants of the reaction were performed in the following
solvents instead of DMF/H2O (1/1 v/v): 0.1 M or 0.2 M
phosphate buffer pH 8.5, 0.5 M NaHCO3 buffer pH 8.0, 0.5 M
NaHCO3 buffer pH 8.0/CH3CN (2/1 v/v). The reactions were
performed with or without addition of KI.
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(Ac-His-Ser)32(Dap-His-Ser)16(Dap-His-Ser-Cys-x-Pro-
Ser)8(Dap-Pro-Ser)4(Dap-Pro-Ser)2Dap-Pro-Ser-NH2 (G5H)
preparative RP-HPLC (14.7 mg). Amino acid analysis: protein
content 63.6% (9.33 mg, 0.46 mmol, yield 62%); found/calc. (%)
Pro 40.4/39.2, Asp 31.9/30.9, Dap 17.1/17.1, Ser 6.6/8.3, CM-
Cys 4.1/4.4. Analytical RP-UPLC: tR = 1.245 min (A/D 100/0 to
0/100 in 5 min, l = 214 nm). MS (LC-MS+): C757H1105N221O316S8
(8-fold ligation product, main peak) found/calc. 18620.0/18619.7
[M + 6H]+. C682H996IN199O284S7 (7-fold ligation product with
one unreacted iodoactyl group, peak height 26% of main peak)
found/calc. 16876.0/16877.7 [M]+.
From starting materials G3C2 and G2M2 using the general pro-
cedure described above (solvent: DMF/H2O (1/1 v/v), 20 equiv.
KI), G5H was obtained as foamy colourless solid after preparative
RP-HPLC (13.7 mg). Amino acid analysis: protein content 64.1%
(8.75 mg, 0.38 mmol, yield 50%); found/calc. (%) His 37.2/30.9,
Ser 31.8/39.2, Dap 15.6/17.1, Pro 10.9/8.3, CM-Cys 4.5/4.4.
Analytical RP-UPLC: tR = 1.243 min (A/D 100/0 to 0/100
in 5 min, l = 214 nm). MS (LC-MS+): C821H1169N333O292S8 (8-
fold ligation product, main peak) found/calc. 20634.0/20633.6
[M + 2H]+. C738H1053N297O264S7 (7-fold ligation product with one
hydroxyacetyl group, peak height 23% of main peak) found/calc.
18535.0/18534.5 [M]+.
(Pro-Lys)32(Dap-Pro-Lys)16(Dap-Pro-Lys-Cys-x-Pro-Ser)8(Dap-
Pro-Ser)4(Dap-Pro-Ser)2Dap-Pro-Ser-NH2 (G5P4)
From starting materials G3C2 and G2M6 using the general
procedure described above (solvent: 0.5 M NaHCO3 buffer pH 8.0,
20 equiv. KI), G5P4 was obtained as crystalline colourless solid af-
ter preparative RP-HPLC (16.4 mg). Amino acid analysis: protein
content 82.3% (13.5 mg, 0.58 mmol, yield 77%); found/calc. (%)
Pro 40.8/39.2, Lys 31.5/30.9, Dap 17.8/17.1, Ser 6.4/8.3, CM-
Cys 3.5/4.4. Analytical RP-UPLC: tR = 1.255 min (A/D 100/0 to
0/100 in 5 min, l = 214 nm). MS (LC-MS+): C869H1497N277O204S8
(8-fold ligation product, main peak) found/calc. 19353.0/19352.4
[M + 6H]+. C780H1340N248O187S7 (7-fold ligation product with one
hydroxyacetyl group, peak height 10% of main peak) found/calc.
17411.0/17411.0 [M + 2H]+.
(Pro-Ser)16(Dap-Pro-Ser)8(Dap-Pro-Ser-Cys-x-Pro-Ser)4(Dap-
Pro-Ser)2Dap-Pro-Ser-NH2 (G4P)
From starting materials G2C2 and G2M3 using the general
procedure described above (solvent: DMF/H2O (1/1 v/v),
20 equiv. KI), G4P was obtained as a foamy colourless solid after
preparative RP-HPLC (2.05 mg). Amino acid analysis: protein
content 65.8% (1.35 mg, 0.15 mmol, yield 55%); found/calc. (%)
Pro 44.0/39.3, Ser 33.8/39.3, Dap 16.9/16.9, CM-Cys 5.4/4.5.
Analytical RP-HPLC: tR = 9.156 min (A/D 100/0 to 50/50
in 10 min, l = 214 nm). MS (LC-MS+): C345H545N109O128S4
found/calc. 8393.0/8395.9 [M]+.
(Pro-Ser)32(Dap-Pro-Ser)16(Dap-Pro-Ser-Cys-x-His-Ser)8(Dap-
His-Ser)4(Dap-His-Ser)2Dap-His-Ser-NH2 (G5P5)
(Pro-Asp)32(Dap-Pro-Asp)16(Dap-Cys-x-Pro-Ser)8(Dap-Pro-
Ser)4(Dap-Pro-Ser)2Dap-Pro-Ser-NH2 (G5P1)
From starting materials G3C1 and G2M3 using the general
procedure described above (solvent: 0.5 M NaHCO3 buffer pH
8.0/CH3CN (2/1 v/v), 20 equiv. KI), G5P5 was obtained as
a foamy colourless solid after preparative RP-HPLC (2.77 mg).
Amino acid analysis: protein content 52.2% (1.45 mg, 0.07 mmol,
yield 11%); found/calc. (%) Pro 37.5/30.9, Ser 31.2/39.2, His
10.7/8.3, Dap 16.0/17.1, CM-Cys 4.6/4.4. Analytical RP-UPLC:
tR = 1.144 min (A/D 100/0 to 0/100 in 5 min, l = 214 nm).
MS (ESI+): C716H1105N251O260S8 (8-fold ligation product, main
peak) found/calc. 17645.0/17645.5 [M]+. C648H997N229O236S7 (7-
fold ligation product with one hydroxyacetyl group, peak height
19% of main peak) found/calc. 15995.0/15995.7 [M]+.
From starting materials G3C2 and G2M5 using the general pro-
cedure described above (solvent: 0.1 M phosphate buffer pH 8.5,
20 equiv. KI), G5P1 was obtained as a foamy colourless solid after
preparative RP-HPLC (10.3 mg). Amino acid analysis: protein
content 76.5% (7.86 mg, 0.43 mmol, yield 57%); found/calc. (%)
Pro 39.4/38.2, Asp 30.7/29.1, Dap 19.0/18.8, Ser 6.3/9.1, CM-
Cys 4.7/4.8. Analytical RP-UPLC: tR = 1.245 min (A/D 100/0
to 0/100 in 5 min, l = 214 nm). MS (ESI+): C685H1009N205O284S8
found/calc. 16916.0/16916.1 [M]+.
(Pro-Ser)32(Dap-Pro-Ser)16(Dap-Pro-Ser-Cys-x-Pro-Ser)8(Dap-
Pro-Ser)4(Dap-Pro-Ser)2Dap-Pro-Ser-NH2 (G5P2)
(Pro-Asp)32(Dap-Pro-Asp)16(Dap-Pro-Asp-Cys-x-Pro-Ser)8(Lys-
Pro-Ser)4(Lys-Pro-Ser)2Lys-Pro-Ser-NH2 (G5P6)
From starting materials G3C2 and G2M3 using the general proce-
dure described above (solvent: DMF/H2O (1/1 v/v), 20 equiv.
KI), G5P2 was obtained as a foamy colourless solid after
preparative RP-HPLC (1.48 mg). Amino acid analysis: protein
content 62.4% (0.92 mg, 0.05 mmol, yield 40%); found/calc. (%)
Pro 44.1/39.2, Ser 33.3/39.2, Dap 17.2/17.1, CM-Cys 5.4/4.4.
Analytical RP-HPLC: tR = 9.132 min (A/D 100/0 to 50/50 in
10 min, l = 214 nm). MS (ESI+): C701H1105N221O260S8 found/calc.
17043.0/17045.1 [M]+.
From starting materials G3C3 and G2M4 using the general proce-
dure described above (solvent: DMF/H2O (1/1 v/v), 20 equiv.
KI), G5P6 was obtained as a foamy colourless solid after
preparative RP-HPLC (11.8 mg). Amino acid analysis: protein
content 80.0% (9.41 mg, 0.46 mmol, yield 66%); found/calc. (%)
Pro 39.6/39.2, Asp 31.7/30.9, Dap 12.3/13.3, Ser 7.5/8.3, CM-
Cys 4.6/4.4, Lys 4.3/3.9. Analytical RP-UPLC: tR = 1.214 min
(A/D 100/0 to 0/100 in 5 min, l = 214 nm). MS (LC-
MS+): C778H1147N221O316S8 (8-fold ligation product, main peak)
found/calc. 18907.0/18908.2 [M]+. C703H1038IN199O284S7 (7-fold
ligation product with one unreacted iodoacetyl group, peak
height 28% of main peak) found/calc. 17165.0/17172.3 [M]+.
C628H929I2N177O252S6 (6-fold ligation product with two unreacted
iodoacetyl groups, peak height 11% of main peak) found/calc.
15467.0/15436.3 [M]+.
(Pro-Asp)32(Dap-Pro-Asp)16(Dap-Pro-Asp-Cys-x-Pro-Ser)8(Dap-
Pro-Ser)4(Dap-Pro-Ser)2Dap-Pro-Ser-NH2 (G5P3)
From starting materials G3C2 and G2M4 using the general
procedure described above (solvent: 0.5 M NaHCO3 buffer pH 8.0,
20 equiv. KI), G5P3 was obtained as a foamy colourless solid after
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(DPro-Pro-Glu)32(Dap-Pro-Asp)16(Dap-Pro-Asp-Cys-x-Pro-
Ser)8(Lys-Pro-Ser)4(Lys-Pro-Ser)2Lys-Pro-Ser-NH2 (G5P7)
mQ-deionized H2O. The actual peptide content of the solutions
was determined by amino acid analysis which lead to the following
concentrations in the deep well plate: 5.37 mM (G4H2), 4.17 mM
(G4H1), 4.53 (G5P5), 5.58 mM (G5H). 8-Acetoxypyrene-1,3,6-
trisulfonic acid trisodium salt (1) or isobutyryl fluorescein (3)
were used as substrates. Dilutions by 2/3 with mQ-deionized H2O
(for 1) or mQ-deionized H2O/CH3CN (2/1, v/v) (for 3) gave
eight substrate concentrations ranging from 0.18 to 3 mM (for
1) or 0.018 to 0.3 mM (for 3). 8-Hydroxypyrene-1,3,6-trisulfonic
acid trisodium salt (HPTS) and fluorescein (fluo) were used as
fluorescent standards. Dilutions of stock solutions of HPTS or
fluorescein in mQ-deionized H2O or mQ-deionized H2O/CH3CN
(2/1, v/v), respectively, gave eight fluorescent standard concentra-
tions ranging from 0 to 0.3 mM (HPTS) or 0 to 0.03 mM (fluo).
Citric acid–sodium citrate 15 mM at pH 5.5 was used as buffer
and the pH was adjusted to the desired value with HCl 1.0 M or
NaOH 1.0 M using a Metrohm 692 pH/ion meter.
40 mL of buffer were mixed with 40 mL of dendrimer solution and
40 mL of substrate solution in a 96-well half area flat bottom plate
(190 mL). Thus all initial concentrations of the prepared solutions
were divided by 3 to give eight final substrate concentrations
ranging from 58.5 to 1000 mM in buffer (for 1) or 5.85 to 100 mM
in CH3CN/buffer (11 : 89) (for 3) for each Michaelis–Menten plot.
The eight final fluorescent standard concentrations ranged from
0 to 100 mM in buffer (HPTS) or 0 to 10 mM in CH3CN/buffer
(11 : 89) (fluo) and were used for the calibration curve.
The formation of HPTS or fluorescein was followed by flu-
orescence emission using an absorbance filter 450/50 and an
emission filter 530/25. The calibration curve and the background
(40 mL buffer, 40 mL mQ-deionized H2O and 40 mL substrate
solution) were recorded for every experiment in parallel. Prior
to every experiment the gain was adjusted to a signal of ~50 000
for the well with the maximum concentration of HPTS (for 1)
or fluores◦cein (for 3). The temperature inside the instrument was
set to 34 C. Kinetic experiments were followed for 3 h and the
data points were measured every 90 s. Fluorescence data were
converted into product concentration by means of the calibration
curve. Initial reaction rates were calculated from the steepest linear
part observed in the curve “fluorescence vs. time”. All values result
from three independent measurements.
From starting materials G3C3 and G2M7 using the general pro-
cedure described above (solvent: DMF/H2O (1/1 v/v), 20 equiv.
KI), G5P7 was obtained as foamy colourless solid after preparative
RP-HPLC (15.6 mg). Amino acid analysis: protein content
60.7% (9.46 mg, 0.40 mmol, yield 56%); found/calc. (%) Pro
51.7/48.4, Glu 15.3/15.0, Asp 11.6/11.3, Dap 10.6/11.3, Ser
6.6/7.0, CM-Cys 4.2/3.8, Lys 3.7/3.3. Analytical RP-UPLC:
tR = 1.304 min (A/D 100/0 to 0/100 in 5 min, l = 214 nm).
MS (LC-MS+): C970H1435N253O348S8 (8-fold ligation product, main
peak) found/calc. 22459.0/22464.8 [M]+. C871H1290IN227O312S7 (7-
fold ligation product with one unreacted iodoacetyl group, peak
height 12% of main peak) found/calc. 20270.0/20271.9 [M]+.
(Pro-Ser)64(Dap-Pro-Ser)32(Dap-Pro-Ser)16(Dap-Pro-Ser-Cys-x-
Pro-Ser)8(Dap-Pro-Ser)4(Dap-Pro-Ser)2Dap-Pro-Ser-NH2
(G6P1)
From starting materials G3C2 and G3M1 using the general proce-
dure described above (solvent: DMF/H2O (1/1 v/v), 20 equiv.
KI), G6P1 was obtained as a foamy colourless solid after
preparative RP-HPLC (17.7 mg). Amino acid analysis: protein
content 65.8% (11.6 mg, 0.34 mmol, yield 45%); found/calc. (%)
Pro 44.2/39.6, Ser 30.3/39.6, Dap 22.9/18.5, CM-Cys 2.6/2.3.
Analytical RP-UPLC: tR = 1.189 min (A/D 100/0 to 0/100 in
5 min, l = 214 nm).
(Pro-Ser)64(Dap-Pro-Ser)32(Dap-Pro-Ser)16(Dap-Pro-Ser-Cys-x-
Pro-Ser)8(Lys-Pro-Ser)4(Lys-Pro-Ser)2Lys-Pro-Ser-NH2 (G6P2)
From starting materials G3C3 and G3M1 using the general proce-
dure described above (solvent: DMF/H2O (1/1 v/v), 20 equiv.
KI), G6P2 was obtained as a foamy colourless solid after
preparative RP-HPLC (18.2 mg). Amino acid analysis: protein
content 68.6% (12.5 mg, 0.36 mmol, yield 51%); found/calc. (%)
Pro 44.2/39.6, Ser 32.9/39.6, Dap 17.4/16.4, CM-Cys 3.0/2.3,
Lys 2.6/2.1. Analytical RP-UPLC: tR = 1.202 min (A/D 100/0 to
0/100 in 5 min, l = 214 nm).
PGSE diffusion NMR
Kinetic parameters kcat and KM
Standard PGSE diffusion NMR experiments were performed
using a Bruker DRX500 with dilute solutions of dendrimer
(1–5 mM) in D2O (pH 2–3, at 303 K). The gradient with a
maximum strength of 50 ¥ 10-4 T cm-1 was calibrated using
the HOD proton signal in D2O (99.997%). The diffusion time
D was 110 ms and the gradient duration d was 4 ms. Data
analysis was performed by using the Bruker Simfit software and
the diffusion coefficient D [m2 s-1] was derived from peak integrals
or intensities. The hydrodynamic radii were calculated from the
diffusion coefficient D using the Stokes–Einstein equation Rh =
kT/6phD with Boltzmann constant k = 1.380 ¥ 10-23 J K-1,
temperature T in K and viscosity h = 1.089 mPa s for D2O at
303 K.
Vcat is the apparent rate in the presence of dendrimer catalyst;
V
uncat is the rate in buffer alone. The observed rate enhancement is
defined as Vnet/Vuncat with Vnet = Vcat - Vuncat. Michaelis–Menten
parameters kcat (rate constant) and KM (Michaelis–Menten con-
stant) were determined by fitting the data to the Michaelis–Menten
model. The rate constant kuncat without dendrimer catalyst was
calculated from the slope of the linear curve that gives Vuncat (as
product concentration per time) vs. substrate concentration [S].
The following values were obtained: kuncat (for 1, pH 5.5) = 3.891 ¥
10-5 min-1, kuncat (for 3, pH 5.5) = 2.512 ¥ 10-5 min-1.
Second order rate constant k2 of reference catalyst
Kinetics of ester hydrolysis
4-methylimidazole
All necessary solutions were prepared in a 96-deep well plate
(2000 mL). Peptide dendrimer solutions were freshly prepared in
The solutions of 4-methylimidazole (4-MeIm) were prepared by
serial dilution (2/3) from stock solutions (3 mM for the use with
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8-acetoxypyrene-1,3,6-trisulfonic acid trisodium salt (1) or
0.3 mM for the use with isobutyryl fluorescein (3)) adjusted to the
desired pH value using HCl 1 M. The reaction rate with 4-MeIm
was obtained under the same conditions as described above. The
final concentrations in the 96-well half area flat bottom plate were
0, 88, 132, 198, 296, 444, 667 and 1000 mM of 4-MeIm (for 1)
or 0, 8.78, 13.2, 19.8, 29.6, 44.4, 66.7 and 100 mM of 4-MeIm
(for 3); 200 mM (for 1) or 20 mM (for 3) of substrate and 5 mM
(citrate buffer pH 5.5) of buffer. The second order rate constants
k2 were calculated from linear regression of the experimentally
measured pseudo first order rate constants k2¢ as a function of 4-
MeIm concentrations. The second order rate constants k2 is given
by k2 = k2¢/[S]. The following values were obtained: k2 (for 1, pH
5.5) = 1.00 min-1 M-1, k2 (for 3, pH 5.5) = 2.26 min-1 M-1.
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A freshly prepared solution of 4-nitrobenzaldehyde in acetone
(200 mM, 50 mL) was mixed with a solution of dendrimer catalyst
in buffer (50 mL) in a 1 mL glass vial and stirred for 14 to
24 h at rt by means of a miniature stirring ball. The initial
reaction concentrations of dendrimers were determined using
amino acid analysis: 0.04 mM (= 1/25 mol%) for the 4th generation
compound, 0.02 mM (= 1/50 mol%) for 5th generation com-
pounds or 0.01 mM (1/100 mol%) for 6th generation compounds
corresponding to 0.64 mol% N-terminal Pro. Aq. bicine buffer
0.1 M at pH 8.5, aq. HEPES buffer 0.1 M at pH 7.0 or aq.
citric acid–sodium citrate buffer 0.1 M at pH 6.0 or 5.0 were
used as buffers and the pH was adjusted to the desired value
with HCl 1.0 M or NaOH 1.0 M using a Metrohm 692 pH/ion
meter. A variant of the reaction consisted in replacing the solvent
system by DMSO/acetone (4/1 v/v), buffered with x equiv. of
N-methylmorpholine (NMM, where x = number of formic acid–
amine salts for the dendrimer tested). An aliquot (1 mL) of the
reaction mixture was taken every 30 min or 1 h, quenched with
A (mQ-deionized H2O with 0.1% TFA, 200 mL) and injected to
analytical RP-UPLC. A gradient of A/D 100/0 to 0/100 in 5 min
was used and the reaction followed at l = 254 nm. Conversion was
calculated using the area under the peaks. The initial reaction rate
was deduced from the initial slope (first 5 h) of the curves “aldol
product 6 [mM] vs. time [h]”. L-Pro (0.64 mM) was used as refer-
ence catalyst and the background (4-nitrobenzaldehyde (100 mM)
in buffer or DMSO/acetone (4/1 v/v)) was recorded for every
experiment in parallel. The enantiomeric excess was determined
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Acknowledgements
This work was supported financially by the Swiss National Science
Foundation, the Marie-Curie Training Network IBAAC, and the
University of Berne.
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