Remote control of bipyridine-metal coordination within a peptide dendrimer

Article abstract of DOI:10.1039/b912291e

The metal coordinating ability of a bipyridine ligand at the core of a peptide dendrimer was found to be controlled by the nature of amino acids placed at the dendrimer periphery, with coordination being promoted by anionic residues and inhibited by catio

Full text of DOI:10.1039/b912291e

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Remote control of bipyridine–metal coordination within a
peptide dendrimerw
Nicolas A. Uhlich, Peter Sommer, Claudia Buhr, Stefan Schurch, Jean-Louis Reymond* and
¨ ¨
Tamis Darbre*
Received (in Cambridge, UK) 23rd June 2009, Accepted 14th August 2009
First published as an Advance Article on the web 2nd September 2009
DOI: 10.1039/b912291e
The metal coordinating ability of a bipyridine ligand at the core
of a peptide dendrimer was found to be controlled by the nature
of amino acids placed at the dendrimer periphery, with
coordination being promoted by anionic residues and inhibited
by cationic residues; heterotrimers with mixed charges were
preferentially formed.
We asked the question whether the remote control of peptide
dendrimer core properties by peripheral residues might also
allow tuning of strong metal–ligand interactions as in the case
of the bidentate bipyridine.9 To test this hypothesis we prepared
a
16 384-membered second generation peptide dendrimer
combinatorial library10 incorporating a bipyridine ligand in the
form of amino acid 1 surrounded by dendritic branches displaying
anionic, cationic, aromatic and hydrophobic amino acids. Starting
with the commercially available 5,50-dimethyl-2,20-bipyridine,
amino acid 1 was prepared in a 20% yield in an efficient synthesis
with only one purification step.11 The amino group in 1 was
coupled to the branching unit (S)-2,3-diaminopropanoic acid
under symmetrical anhydride conditions.12 The metal coordinating
abilities of the dendritic bipyridine library was tested using a
colorimetric on-bead assay for Fe2+-binding (Fig. 1).
The binding affinities of nucleophilic ligands towards metal ions
generally depend on the structure of the coordinating groups
and can be varied by the addition of electron-donating or
electron-withdrawing groups, or by changing the steric bulk
close to the coordinating atoms. Herein we report that the metal
coordinating ability of a ligand can also be controlled indirectly
by remote substituents when the ligand is placed within a
peptide dendrimer. Thus, the formation of an Fe2+–bipyridine
complex is shown to depend on the nature of amino acid
residues at the dendrimer periphery placed up to ten bonds
away from the coordinating atoms. Such indirect control
of metal coordination illustrates the potential of dendritic
macromolecules to create new chemical functions. In nature,
protein folding can be metal directed as in the case of finger
proteins which are completely unfolded in the absence of a
metal ion.1 Few cases have been reported where amino acid
composition of peptides exert control over metal ion ligation.2
Dendrimers are regularly branched, synthetic, tree-like
macromolecules suitable for a variety of macromolecular
functions, usually obtained by exploiting multivalency at the
dendrimer surface and microenvironment effects at the
dendrimer core.3 We recently showed that peptide dendrimers
prepared by inserting a branching diamino acid at every
second, third or fourth position in a linear peptide sequence,
provide useful functional4 and structural5 synthetic analogs
of proteins and glycoproteins.6 In one particular study of
‘‘core-active-site’’ peptide dendrimers,7 we found that amino
acid residues placed in the outermost dendrimer branches
strongly influence the properties of functional groups at
the dendrimer core, such as substrate binding and catalytic
turnover in esterolytic peptide dendrimers, and the binding to
cobalamin mediated by cysteine or histidine coordination to
cobalt in models for vitamin-B12 transport proteins.8
Under selective assay conditions the polymer beads were
differently stained, indicating that binding to Fe2+ was
strongly dependent on the dendrimer sequence.13 Analysis of
the deeply red-stained beads showed a strong consensus for
anionic residues in the outermost dendrimer layers. By contrast,
unstained beads had predominantly cationic residues in their
sequence. Three dendrimers from the on-bead assay (E1–E3)
and two additional ones with intermediate charges (G1, G2)
were prepared by solid-phase peptide synthesis (SPPS) and
purified by preparative reversed phase HPLC to test their
metal-binding properties in solution (Table 1).14
UV-Vis titrations of G1 and E1 with Fe2+ showed the
formation of the magenta Fe2+–bipyridine complexes (lmax
=
541, 325 nm)15 with clear isosbestic points, indicating the
presence of a single species with a 3 : 1 ligand-to-metal ratio
with the very high binding constants typical of 2,20-bipyridine
ligands16,17 (Ka of 1.1 ꢀ 1015 Mꢁ3 for Fe(G1)3 and 3.1 ꢀ
1014 Mꢁ3 for Fe(E1)3) (Fig. 2). The Fe(E1)3 complex was
isolated by reversed phase HPLC and gave the expected mass
by +ESI-MS (Fig. S3w). Dendrimers G1 and G2 with four
positive and two negative residues in the peripheral layers
similarly formed complexes as observed for E1, confirming the
strong Fe2+-complexation ability of the bipyridine amino acid
within the peptide dendrimer architecture. In contrast, no Fe2+
-
complexation could be detected either colorimetrically or by MS
with dendrimers E2 and E3, bearing 8–10 positive charges and no
negative charge, as observed for similar cationic dendrimers in the
on-bead assay. Libraries without bipyridine did not give red beads
with iron(II) and binding in solution could not be detected in spite
of the presence of glutamate residues in the dendrimers.
Department of Chemistry and Biochemistry, University of Berne,
Freiestrasse 3, CH-3012 Berne, Switzerland.
E-mail: darbre@ioc.unibe.ch, jean-louis.reymond@ioc.unibe.ch;
Fax: +41 31 631 80 57; Tel: +41 31 631 43 25
w Electronic supplementary information (ESI) available: Experimental
procedures and supplementary spectra. See DOI: 10.1039/b912291e
Complexation equilibria were further investigated by LC-MS
in the case of G2, E1 and E2, in particular to probe the possibility
ꢂc
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Chem. Commun., 2009, 6237–6239 | 6237

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Fig. 1 Top: solid phase synthesis with coupling conditions (a) 1,
PyBOP, DIEA, DMF, 2 h; (b) piperidine/DMF 1/4 v/v, 2 ꢀ 10 min;
(c) (Fmoc–Dap)2O, DIC, DCM, 18 h. For A1, A2, A3 to A6: same
conditions as (a) and (b), An = natural amino acids, ꢃ = branching
(S)-2,3-diaminopropanoic acid (Dap). Table: amino acids used in each
of the 6 variable positions An of the peptide dendrimer combinatorial
library E which was synthesized on TG resin (0.30 mmol gꢁ1).
N-termini were acetylated. A linker (6-aminohexanoylglycine) was
attached between A1 and the resin. Bottom right: on-bead screening
for Fe2+-binding on library E (incubation with 30 mM Fe2+ for 5 min),
pH 6.0 (MES buffer 20 mM). Amb = 4-aminomethyl benzoic acid.
Fig. 2 Top: UV-Vis titration of dendrimer G1 (conc. = 80 mM) with
Fe2+. Insert top right: molar ratio plot at 541 nm indicating a 3 : 1
complex of dendrimer G1 relative to Fe2+ and binding constant Ka =
1.1 ꢀ 1015
M
ꢁ3; pH 6.5 (HEPES buffer 20 mM), 25 1C. Bottom:
UV-Vis spectra from 400 nm to 700 nm of solutions containing Fe2+
and one of the dendrimers G1, G2 or E1–E3; dendrimers (50 mM),
Fe2+ (17 mM), pH 6.5 (HEPES 20 mM), 25 1C.
of mixed complexes (Table 2). Mixed complexation of G2 and
E1, which are both able to bind Fe2+ alone, lead to heterotrimers
Fe(G2)2E1 (6829 Da) and FeG2(E1)2 (6747 Da) as the main
products (entry 4, Table 2, Fig. S4w). The cationic bipyridine
dendrimer E2, which did not form any detectable complexes
either alone (entry 3, Table 2) or as a mixed complex with G2
(entries 6 and 7, Table 2), formed the heterotrimer Fe(E1)2E2
(7007 Da) in the presence of the anionic dendrimer E1 (entries 8
and 9, Table 2, Fig. 3). The heterotrimer Fe(E1)2E2 is the main
product (peaks 2–5 in Fig. 3 corresponding to diastereoisomers
of Fe(E1)2E2)) with Fe(E1)3 being formed as a minor product
(peak 6, Fig. 3). The uncomplexed dimer E1E2 (4750 Da) was
also detected in this experiment, presumably formed by multiple
carboxylate–arginine interactions.
The experiments above show that Fe2+-complexation
of a bipyridine ligand placed at the core of a peptide dendrimer
is controlled by the nature of amino acid residues in the
dendrimer branches. Thus, the strong Fe2+-complexing
ability of the bipyridine amino acid in dendrimer E1
(AcET)4(BEV)2BBypX0DNH2 is entirely blocked within the
related dendrimer E2 (AcRK)4(BHV)2BBpyXYNH2. The effect
is probably caused by the overall charge of the dendrimers,
Heterotrimers combining positive and negative charges are
preferentially formed indicating that electrostatic interactions
Table 1 Sequences, yields and +ESI-MS data of peptide dendrimers G1, G2, E1–E3. B = branching unit (S)-2,3-diaminopropanoic acid (Dap),
Ac = acetyl, X = 4-aminomethyl benzoic acid, X0 = b-alanine
+ESI-MS
Yielda/mg (%)
Found
Calc.
Compound
Sequence
Charge
E1
E2
E3
G1
G2
(AcET)4(BEV)2BBypX0DNH2
(AcRK)4(BHV)2BBpyXYNH2
(AcRK)4(BFK)2BBpyX0YNH2
(AcKA)4(BEP)2BGBpyGLNH2
(AcKS)4(BYD)2BGBpyGLNH2
7ꢁ
8+
33.9 (28)
21.7 (11)
52.8 (27)
15.4 (13)
40.7 (31)
2203
2547
2563
2117
2285
2203
2545
2561
2116
2284
10+
4+/2ꢁ
4+/2ꢁ
a
Yields were calculated for the TFA-salts after RP-HPLC purification.
ꢂc
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Table 2 Peptide dendrimer trimers from combinations of G2, E1
and E2 with Fe2+. The complexes were identified by LC-MS analysis.
The composition of the different Fe2+-dendrimer solutions were stable
over time (15 days)
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1
2
3
3G2 + Fe2+
3 E1 + Fe2+
3 E2 + Fe2+
Fe(G2)3
Fe(E1)3
FeG2
a
4b
5c
6
2G2 + 1E1 + Fe2+ Fe(G2)2E1 Fe(G2)2 FeG2E1 FeG2
1G2 + 2E1 + Fe2+ FeG2(E1)2 Fe(E1)2 FeG2E1
2G2 + 1E2 + Fe2+ Fe(G2)3
1G2 + 2E2 + Fe2+ Fe(G2)3
2E1 + 1E2 + Fe2+ Fe(E1)2E2
1E1 + 2E2 + Fe2+ Fe(E1)2E2
FeG2
7
8cd
9d
FeE1E2 FeE1
FeE1E2 FeE1
a
b
No complex formed; Fe(G2)3 and FeG2(E1)2 were formed as
c
d
minor products; Fe(E1)3 was formed as a minor product; the mass
corresponding to E1E2 was also observed.
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F. N. Castellano, Angew. Chem., Int. Ed., 2000, 39, 4301–4305;
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11 Fmoc-protected bipyridine derived amino acid building block 1
was prepared as follows:
Fig. 3 LC-MS analysis of a mixture of E1 and E2 in the presence of
Fe2+ (entry 8, Table 2). Top: absorption at 541 nm. Bottom: total ion
current chromatogram (m/z 400–1800). Fe(E1)2E2 diastereoisomers:
peaks 2–5, 7007 Da; Fe(E1)3: peak 6, 6665 Da; E1: peak 7, 2203 Da
and E2: peak 1, 2546 Da.
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the beads. We assume that a mixture of complexes is formed.
14 For examples of combinatorial libraries of metal ligands see:
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are involved in trimer formation and stabilization. In addition,
the amino acid composition can induce different conformations
in the dendritic structure, exposing or hiding the bipyridine
group. Such electrostatic tuning of metal complexation might
be generally useful to design dendritic sensing devices based on
relaying information from the branches to the core.
This work was financially supported by the Swiss National
Science Foundation and the Marie-Curie Training Network
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ꢂc
This journal is The Royal Society of Chemistry 2009
Chem. Commun., 2009, 6237–6239 | 6239