Anion effects in Cu-crosslinked palindromic artificial tripeptides with pendant Bpy ligands

Article abstract of DOI:10.1002/ejic.201100567

An artificial peptide with three pendant bipyridine (bpy) ligands on a palindromic backbone was designed and used to make multimetallic assemblies. The tripeptide is synthesized by addition of ligand-substituted aminoethylglycine monomers to both ends of a bpy-substituted diacid. The tripeptide was treated with Cu^II acetate, tetrafluoroborate, and nitrate salts, and the chelation stoichiometry was confirmed in spectrophotometric titrations. NMR spectroscopy, mass spectrometry, and analytical HPLC separations confirm the identity and purity of the product, which is a tripeptide duplex with three Cu^II coordinative crosslinks. UV/Vis absorbance and EPR spectroscopy were used to assess the geometry of the metal complexes and examine the effects of coordinative anions on the coordination geometry about the Cu centers. We find that the distorted square-planar geometry in the Cu duplex shifts to a tetrahedral geometry upon addition of I^- and that counteranions can be used to tune the interaction between metal complexes in multimetallic assemblies.

Full text of DOI:10.1002/ejic.201100567

FULL PAPER
DOI: 10.1002/ejic.201100567
Anion Effects in Cu-Crosslinked Palindromic Artificial Tripeptides with
Pendant Bpy Ligands
[a]
[a]
[a]
Joy A. Gallagher, Lauren A. Levine, and Mary Elizabeth Williams*
Keywords: Self-assembly / Molecular recognition / Copper / Anion effects
An artificial peptide with three pendant bipyridine (bpy) li-
gands on a palindromic backbone was designed and used to
make multimetallic assemblies. The tripeptide is synthesized
and purity of the product, which is a tripeptide duplex with
three Cu coordinative crosslinks. UV/Vis absorbance and
EPR spectroscopy were used to assess the geometry of the
II
by addition of ligand-substituted aminoethylglycine mono- metal complexes and examine the effects of coordinative
mers to both ends of a bpy-substituted diacid. The tripeptide
was treated with Cu acetate, tetrafluoroborate, and nitrate
salts, and the chelation stoichiometry was confirmed in spec-
trophotometric titrations. NMR spectroscopy, mass spectrom-
etry, and analytical HPLC separations confirm the identity
anions on the coordination geometry about the Cu centers.
We find that the distorted square-planar geometry in the Cu
duplex shifts to a tetrahedral geometry upon addition of I
and that counteranions can be used to tune the interaction
between metal complexes in multimetallic assemblies.
II
–
Introduction
initial work implemented solid-phase peptide synthesis to
create oligopeptide strands with N and C termini.[
2a,2b,3a]
Inorganic biomimetic structures have been designed by
using biological macromolecules for inspiration.[1] Inor-
ganic analogs have the potential for encoding tunable mag-
netic, electronic, chemical, and physical properties and ex-
tending these beyond the capabilities of natural systems. To
create large, functional architectures, directed self-assembly
is a particularly attractive and viable route. Nature employs
molecular recognition of a finite number of discrete, modu-
lar units in the form of amino acids and nucleobases, which
encode structure and function in a bottom-up manner for
all biological process. We aim to develop structural and
functional inorganic analogs of deoxyribonucleic acid
To eliminate the formation of parallel and antiparallel iso-
mer duplexes and to control the alignment of the strands
in metal-linked oligopeptide duplexes, we developed a syn-
[
5]
thetic strategy to prepare symmetric peptides. The palin-
dromic motif eliminates the formation of parallel and anti-
parallel isomers, which are possible as a result of alternating
termini (Figure 1).
(DNA), which use metals to crosslink artificial oligopep-
tides that assemble into multimetallic architectures for func-
tional materials.
Our group has employed an aminoethylglycine backbone
with pendant heterocyclic ligands that bind metal ions to
create single-stranded,[ duplex, and hairpin structures.
By using solely chelating ligands, our systems assemble via
metal coordinative bonds rather than hydrogen-bonding or
π–π interactions of nucleic acid–base pairs. Molecular re-
cognition between oligopeptide strands is based on metal
2]
[3]
[4]
Figure 1. (A) Schematic of a metal-induced duplex formation of a
monofunctional, self-complementary tripeptide with N and C ter-
coordinative saturation: in the presence of a tetracoordinate mini. (B) Schematic of metal-coordination-induced parallel duplex
formation with palindromic tripeptides.
metal, the complementarity of artificial bases will follow a
bidentate–bidentate [2ϫ2] or tridentate–monodentate
In this study, the palindromic motif has been utilized to
[
3ϫ1] pattern, analogous to that in adenine–thiamine [A–
form metal-linked duplexes after the synthesis and charac-
terization of the bpy–tripeptide. We then use these as model
systems to study the role and impact of the counteranion
on the geometries of the metal complex crosslinks, since this
is a determining factor for the interactions between metal
complexes within multimetallic assemblies.
T] and guanine–cytosine [G–C] base pairs in DNA. Our
[
a] Department of Chemistry, The Pennsylvania State University,
1
04 Chemistry Building, University Park, PA 16802, USA
E-mail: mbw@chem.psu.edu
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejic.201100567.
4168
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Anion Effects in Cu-Crosslinked Palindromic Artificial Tripeptides
Results and Discussion
with two Fmoc-deprotected H N-bpy(aeg)-OtBu mono-
2
mers 1d to yield tripeptide 3 in a facile one-pot synthesis.
Synthesis and Characterization of Artificial Tripeptides
In contrast to the ligand-substituted artificial oligopeptides
[
2,3]
we have previously made,
tripeptide 3 is prepared by this
Our group has previously reported the synthesis of li-
gand-substituted artificial oligopeptides by solution-phase
techniques, which sequentially add monomers to the grow-
route from the monomers in a single step in 54.1% yield, a
significant increase in comparison with our previously re-
[
2a]
ported solid-phase peptide synthesis (24.8%)
first solution-phase tripeptide (1.19%).
and our
[
3b]
ing chain terminus.
In this paper, we present the synthe-
[
3b]
sis and characterization of a new symmetric bipyridine tri-
peptide by using a strategy akin to divergent dendrimer syn-
[
6]
thesis. Our initial report using the divergent synthetic Synthesis and Characterization of Duplexes
strategy employed a 9-fluorenylmethoxycarbonyl (Fmoc)
Bulk-scale preparations of the Cu-containing peptide
complex were performed by reacting three molar equiva-
lents of copper(II) nitrate with two molar equivalents of the
tripeptide, and the product was isolated by precipitation
with sodium tetrafluoroborate or ammonium hexafluoro-
phosphate. Alternatively, the product of the reaction with
copper(II) acetate was isolated by concentration under re-
duced pressure, followed by recrystallization from methanol
protecting group; however, the product and metal com-
plexes had limited solubility.[
3b]
To increase solubility and
overall yield, we have instead used the approach shown in
Scheme 1, in which a bpy-substituted iminodiacetic acid
backbone protected with ethyl esters (compound 2) is used
as the central unit. After deprotection to give terminal acid
2d, this is reacted by using standard coupling techniques
1
and diethyl ether. The purity was confirmed by using H
NMR spectroscopy (see Supporting Information). Al-
though bound Cu2 ions induce a paramagnetic shift of the
ligand proton resonances, integration of aliphatic peaks and
comparison to the amide proton peak resonances confirms
that all of the tripeptide in solution has bound Cu2 metal
ions. HPLC was further used to assess the purity of the
Cu-containing products. As we have done previously,[ the
analytical chromatograms of these complexes and of the
unmetallated tripeptide were obtained by using a mobile
phase of 85:15 acetonitrile with 0.1% TFA and water. The
elution time of the duplexes increases with molecular
weight of the counteranion: PF (146 g/mol) ϾBF (86 g/
+
+
7]
6
4
mol) ϾCH CO (59 g/mol), suggesting these are tightly as-
3
2
sociated with the metallated oligopeptide structures. Vapor
pressure osmometry experiments provide molecular weights
that are equivalent to product species with two tripeptide
strands with three bound Cu2 ions, with some experimen-
tal variance that arises from residual solvent present in the
Cu complexes. Together, the NMR spectra, HPLC, and
vapor pressure osmometry data confirm that the predomi-
+
Scheme 1. Artificial tripeptide 3 synthesis. (i) 20% piperidine in
2+
nant product of the reaction of tripeptide 3 with Cu un-
ACN, 30 min; (ii) NaOH in THF overnight, 0 °C; reduce to pH to
6+
1; (iii) 2:1 mol ratio of 1d/2d with EDC, HOBt, DIPEA.
der these conditions is [Cu 3 ] .
3 2
Figure 2. Molecular ion peaks observed by positive-ion electrospray mass spectrometry, plotted together with the calculated mass (above)
5
+
4+
3+
2+
3 2 6 3 2 6 2 3 2 6 3 3 2 6 4
and isotopic splitting patterns (below) for (A) [Cu 3 PF ] , (B) [Cu 3 (PF ) ] , (C) [Cu 3 (PF ) ] , and (D) [Cu 3 (PF ) ] .
Eur. J. Inorg. Chem. 2011, 4168–4174
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4169

J. A. Gallagher, L. A. Levine, M. E. Williams
FULL PAPER
High-resolution electrospray mass spectrometry was used photometric titrations. Figure 3 contains representative dif-
to identify the product of the reaction and compare its iso- ference spectra in the UV and visible regions as Cu(BF4)2
tope pattern to the calculated isotope pattern. For example is incrementally added to a solution containing the tripep-
in Figure 2, multiple molecular ion peaks of the PF salt of tide. In Figure 3A, addition of metal ion causes a shift in
6
2
+
[
Cu 3 ] are observed and correspond to species with the wavelength of the peak absorbance at 670 to 722 nm,
charge +2 to +5. Rather than differences in the oxidation which is attributed to the d–d transition of [Cu(bpy) ] .
3 2
2+ [8]
2
state of the metal ions, these molecular ions differ in charge The titration curve in Figure 3B is a plot of the change in
because of the number of anions associated with the +6- absorbance at 718 nm, which increases and levels off at a
charged molecular ion in the gas phase. Comparisons of stoichiometric Cu/peptide ratio of 1.5:1 or 3:2. This stoichi-
these molecular ion peaks with those in theoretical spectra ometry is consistent with the molecular ions observed in
2
+
confirmed the identity of the species {[Cu 3 ](PF ) } , the gas phase mass spectrometry experiments. At this point,
3
2
6 4
3
+
4+
5+
{
[Cu 3 ](PF ) } , {[Cu 3 ](PF ) } , and{[Cu 3 ](PF ) } . the extinction coefficient of the species formed is calculated
3 2 6 3 3 2 6 2 3 2 6 1
Analogously, multiple molecular ion peaks are observed for to be 100 m–1 cm at 718 nm, which is consistent with the
–1
6
+
2+
the BF salt of the [Cu 3 ] complex. However, despite value for [Cu(bpy)] . Alternatively, titration of the tripep-
4
3 2
exhaustive efforts we have been unable to observe molecular tide with Cu(OAc) in the Figure 3B is monitored at 920 nm
2
ion peaks for the analogous acetate complex, which may be and similarly contains an inflection at Cu/peptide ≈ 1.5:1.
–1
due to differences in the charge of this complex.
The extinction at this point is 60 m–1 cm at 920 nm; the
differences in extinction of the acetate and tetrafluorobor-
ate complexes are consistent with known differences for
Spectrophotometric Titrations
[
Cu(bpy) ](BF ) at 660 nm and [Cu(bpy) ](OAc)2 at
2 4 2 2
[
8b]
Our molecular design was based on the hypothesis that 925 nm.
Cu2+ ions would form coordinative crosslinks between the
In contrast to the visible absorbance spectra, four
pendant ligands on the tripeptides. To further investigate isosbestic points are observed in the UV difference spectra
the stoichiometry of the reaction, the reaction of tripeptide in Figure 3C. These are the result of a drastic redshift in
II
3
with each of the Cu salts was monitored during spectro- the π–π* transition for the bpy ligand from 278 to 302 nm
Figure 3. Spectrophotometric titration of the reaction of Cu2+ with the tripeptide in spectroscopic grade methanol: (A) 15 mm
Cu(BF titrated in 50 μL increments into 3.16 mm tripeptide 3. (B) Plot of the change in the absorbance at 712 nm vs. molar ratio of
added Cu to peptide for Cu(BF
circles). (C) Difference spectra acquired during the spectrophotometric titration of the reaction of Cu with 2.5 mL of 14.5 μL tripeptide
in methanol. Cu2+ is added in 50 μL increments of 100 μm Cu(BF
. (D) Plots of the change in the absorbance as a function of the
4 2
)
2+
4 2 2
) titration monitored at 718 nm (full circles) and Cu(OAc) titration monitored at 920 nm (empty
2
+
3
4 2
)
2+
relative molar ratio of added Cu to 3; Cu(BF
4
)
2
titration monitored at 310 nm (black circles) and 277 nm (red circles); Cu(OAc)
2
titration monitored at 310 nm (black triangles) and 277 nm (red triangles).
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Eur. J. Inorg. Chem. 2011, 4168–4174

Anion Effects in Cu-Crosslinked Palindromic Artificial Tripeptides
upon metal ion binding[9] as well as a slight increase fol- anions associate to the duplex, presumably in the Cu axial
II
lowed by a decrease in intensity of the peak at 357 and positions by displacement of more weakly associated
3
94 nm, which we hypothesize is due to charge transfer be- anions, thereby affecting the coordination environment. In-
[10]
tween the bipyridine ligands.
Titration curves (Fig- creased extinction in the electronic absorbance spectra fur-
ure 3D) at 274 and 310 nm each contain a stoichiometric ther suggests a distortion of the square-planar coordination
+
2
point at a Cu /tripeptide mol ratio of 1.5:1, again consis- geometry. Table 1 summarizes the spectral changes caused
tent with the formation of a trimetallic species with three by the addition of salt to each Cu tripeptide complex. The
2
+
[
Cu(bpy)2] complexes forming crosslinks between two tri- degree of spectral shifts and changes in extinction are in-
peptide strands, similar to the titration curve at 712 nm. dicative of the affinity for the Cu complexes and changes in
Spectrophotometric titration data of Cu(NO3)2 and the molecular geometry.
Cu(CH CO ) are found in the Supporting Information. At
3
2 2
the equivalence point, the extinction coefficients at 310 nm
–
1
–1
are approximately 17,000 and 11,000 m cm for the OAc
and BF titrations, respectively, which compare favorably
4
with the values measured for both the duplex structures and
literature reports for [Cu(bpy)]2+ at 310 nm.[9a]
Role of the Counteranion
2
+
The coordination geometry of [Cu(bpy) ] has been
2
shown to be impacted by the counteranions.[
8,11]
For exam-
ple, [Cu(bpy) ](BF ) is known to have either a distorted
2
4 2
tetrahedral geometry, where the BF does not interact with
4
[
11]
the Cu center,
or an elongated rhombic octahedral one,
in which the BF anions are loosely coordinated in the
–
4
[
8,12]
+
axial position.
has been classified as cis-distorted octahedral,
Cu(bpy) ]I is known to adopt a distorted trigonal bipy-
The [Cu(bpy) NO ] stereochemistry
2 3
[
13]
and
[
2
2
[
14]
ramidal geometry.
Although it is known that anion ef-
fects alone cannot dictate the geometry around the Cu cen-
ter[ and that steric effects can play a strong role, it is clear
from the anion-dependent geometries of these small mole-
cule analogs that this may have an impact in our trimetallic
structures. We therefore investigated the role of the counter-
ion on the Cu coordination geometry by monitoring
changes in the electronic absorbance spectra at the d–d Cu
transition and the electron paramagnetic resonance (EPR)
spectra, since these could be used to tune structure and elec-
tronic communication in the Cu-linked tripeptide duplexes.
To rapidly assay for impacts of the anion on the visible
absorbance in this region, titrations with a series of sodium
15]
Figure 4. Change in absorbance of solutions containing (top)
.53 m [Cu ](BF or (bottom) 0.52 m [Cu ](OAc) duplexes as
a function of increasing mol of salt, as indicated, in spectroscopic
grade methanol.
0
3
3
2
4
)
6
3
3
2
6
3 2 4 6
Table 1. Absorbance data for salt titrated into [Cu 3 ](BF ) and
–
–
–
–
–
–
salts (I , Br , Cl , NO , CH CO , and BF ) were per-
3
3
2
4
[Cu 3 ](OAc) recorded in air at room temperature in spectroscopic
3
2
6
formed in a 96-well plate and measured by using a micro- grade MeOH.
plate reader. Spectral changes of the d–d transition of the
Salt
[Cu
3
3
2
](BF
4
)
6
3 2 6
[Cu 3 ](OAc)
λmax (nm)
II
6+
Cu centers for the [Cu 3 ] are compared to what is
[a]
ε (m–1 cm–1)[b]
[a]
Δε (m–1 cm–1)[b]
3
2
added λmax (nm)
2
+
known for the small molecule [Cu(bpy)2] analogs; for ex-
7
770
15
680
761
741
710
680
680
680
ample, changes in absorbance at 770 nm were chosen for
NaI
259
76
45
14
27
30
101
48
35
24
25
25
both BF and OAc salts of the Cu complex, because of the NaBr 745
4
significant spectral increase and slight redshift in the λmax NaCl 738
at the d–d transition region upon addition of NaI. Figure 4
compares the change in absorbance per molar equivalent of
added salt for each of the series of sodium salts. These data
reveal that the most extensive spectral shifts occur upon
addition of NaI, which causes increased extinction and a
redshift of the peak absorbance for both the OAc and BF4
NaOAc 703
NaNO
NaBF
3
715
715
4
[
a] Peak absorbance after addition of six molar equivalents of salt
to complex. [b] Change in extinction at λmax
.
Redshift and intensity enhancement of the d–d bands are
complexes. This increase in absorbance levels off at a NaI/ recognized indicators of the transformation from square-
Cu 3 ] ratio of approximately 6:1, suggesting that six I– planar to tetrahedral stereochemistry, since the symmetry
6
+
[
3 2
Eur. J. Inorg. Chem. 2011, 4168–4174
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J. A. Gallagher, L. A. Levine, M. E. Williams
FULL PAPER
of the ligand field is decreased allowing for easier d–d tran- indicates that the metal centers are at a distance greater
sitions as electric dipole transitions.[ The electronic ab- than 6 Å. At this distance, it is unlikely that acetate brid-
16]
[19]
[
20]
sorption spectral changes predict that the intensity of a d– ges two Cu centers.
d transition will increase as the symmetry of the ligand field Since the addition of NaI caused the most dramatic
decreases. Since the d–d transitions become allowed as elec- changes in the electronic absorbance spectra, we examined
–
tric dipole transitions, I has the largest spectral impact fol- the EPR spectra of the complexes with six molar equiva-
–
–
lowed by Br . Whereas addition of OAc to the lents of added NaI. In Figure 5 (dashed lines), added NaI
Cu 3 ](BF ) complex induces spectral shifts, the opposite results in increases in g , g , and A . The g /A value
[
3
2
4 6
ʈ
Ќ
ʈ
ʈ
ʈ
is not true. Using the data in Figure 4 and Table 2, we can (120 cm) for [Cu 3 ](BF ) with NaI suggests that the soft
3
2
4 6
–
compare the degree of spectral shift and change in ab- anion I associates with Cu in the axial positions. The lack
sorbance; it appears that the trend in the extent of anion of hyperfine interactions for [Cu 3 ](OAc) with NaI sug-
3
2
6
association with the trimetallic duplex is: I ϾBr– gests that the addition of I prevents nuclear interactions of
–
–
–
–
–
–
ϾCH CO ϾCl ≈ NO ≈ BF .
the Cu centers and deforms these toward distorted-tetrahe-
3
2
3
4
[
21]
dral geometry.
Insertion of anions in the trimetallic
Table 2. Measured g tensor and hyperfine coupling constants from structure and distortion of geometry is likely to cause an
EPR spectra.
Complex
increase in the metal–metal distances. Because the aeg
backbone is flexible, the tripeptide is able to accommodate
the I anions. However, the dynamic motions of the mole-
cule have also made crystallographic-quality crystals elus-
ive, so that in our ongoing efforts we use the high-energy
beam of the synchrotron to determine the metal–metal dis-
tances in solution.
(10–4 cm–1)
A
ʈ
g
ʈ
g
Ќ
–
[Cu
[Cu
[Cu
[Cu
3
3
3
3
3
2
3
2
3
2
3
2
](BF
](BF
](OAc)
](OAc)
4
)
)
6
2.183
+ 6NaI 2.198
2.228
2.098
2.094
2.124
2.107
161
182
4
6
6
6
+ 6NaI 2.203
The observed influences of the counterion in the ab-
sorbance spectra led us to characterize the molecular geo-
metries with use of EPR spectroscopy. Figure 5 contains
the EPR spectra of solutions containing [Cu 3 ](BF ) and
Conclusions
Using the symmetric bipyridine tripeptide as a platform,
we have investigated the formation of [Cu(bpy)2]2
crosslinked tripeptide duplexes and the role of the
counteranion on the resulting trimetallic structure. We find
that association with anions in the axial positions has ob-
servable impact on the absorbance and EPR spectra, which
is indicative of impacts on the complex geometry. Because
coupling between adjacent metal ions is important for elec-
tron transfers in the structures and for long-range photoin-
duced charge separation in chromophore-linked assemblies
3
2
4 6
+
[
Cu 3 ](OAc) ; analysis of these provides the g tensor values
3 2 6
and hyperfine couplings listed in Table 2. The tensor values
in the range g Ͼ2.1Ͼ g Ͼ2.0 and hyperfine coupling
ʈ
Ќ
–4
–
1
II
constant A Ͼ130 (cm / 10 ) suggest that the Cu geome-
try is tetragonal. The g /A quotient value of 135 cm for
ʈ
[
17]
ʈ
ʈ
[
Cu 3 ](BF ) is at the upper limit of the range assigned
3 2 4 6
to square-planar geometry, suggesting a distortion toward
tetrahedral geometry.[18] Values of the g tensor for
[
Cu 3 ](OAc) are also in the range g Ͼ2.1Ͼ g Ͼ2.0,
3 2 6 ʈ
Ќ
[
4a,4c]
and molecular materials,
portant and felxible role that axial ligation can play in tun-
ing the functionality of multimetallic structures.
these studies reveal the im-
suggesting a tetragonal coordination sphere; however, the
lack of hyperfine interaction for the [Cu 3 ](OAc) duplex
3
2
6
Experimental Section
Instrumentation and Analysis: UV/Vis absorbance spectra were ob-
tained with a double-beam spectrophotometer (Varian, Cary 500)
by using a 1-cm quartz cuvette. The salt titrations were performed
with a Molecular Devices SpectraMax M2 microplate reader. Mass
spectrometric analysis was performed with a Waters LCT Premier
time-of-flight (TOF) mass spectrometer. Samples were introduced
into the mass spectrometer by using direct infusion via a syringe
pump built into the instrument. The mass spectrometer was set
to scan the range m/z = 100–2500 in the positive-ion mode, with
electrospray ionization (ESI). The drying gas temperature was set
to 80 °C, and the capillary voltage was 2200 V. X-band electron
paramagnetic resonance (EPR) spectra were acquired with a
9
1
.5 GHz Bruker ESP-300 spectrometer with a liquid He cryostat at
1
2.9 K with 5 mW power and 10.2 G modulation amplitude. H,
Figure 5. EPR spectra of 0.53 mm [Cu
3
3
2
](BF
4
)
6
(solid line) along
13
C, HMQC, HMBC, and COSY NMR spectra were collected with
with the same solution containing 6 molar equivalents of NaI
dashed line). The inset contains the EPR spectra of 0.52 mm
Cu ](OAc) (solid line) in methanol together with this solution
containing 6 molar equivalents of NaI (dashed line).
a 400 MHz spectrophotometer (Bruker).
(
[
3
3
2
6
Chemicals: The syntheses of 4Ј-methyl-2,2Ј-bipyridine-4-acetic
acid,[
22]
tert-butyl N-[2-(N-9-fluorenylmethoxycarbonyl)amino-
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Anion Effects in Cu-Crosslinked Palindromic Artificial Tripeptides
ethyl]glycinate hydrochloride (Fmoc-aeg-OtBu·HCl),[23] and tert-
butyl {N-[2-(N-9-fluorenylmethyoxycarbonylamino)ethyl]-N-[2-(4Ј-
(t, J = 5 Hz, 1 H), 7.0–7.22 (dd, J = 5, 61 Hz, 2 H), 8.04–8.19 (d,
J = 34 Hz, 2 H), 8.36–8.52 (dd, J = 5, 35 Hz, 2 H) ppm.
methyl-2,2Ј-bipyridyl)acetyl]amino}acetate
[Fmoc-aeg(Bpy)-
tert-Butyl-(aeg)(bpy)-(diamide)(bpy)-(aeg)(bpy)-tert-butyl (3): In an
ice bath, 2d (0.50 g, 1.5 mmol), EDC (0.70 g, 3.7 mmol), and HOBt
[
4b]
OtBu]
(1) were performed as reported. N-hydroxybenzotriazole
(
HOBT) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hy-
drochloride (EDC) were purchased from Advanced ChemTech. O-
Benzotriazole-N,N,NЈ,NЈ-tetramethyluronium hexafluorophos-
(
0.50 g, 3.7 mmol) were added to dry dichloromethane (75 mL).
The suspension was stirred for 15 min and DIPEA (1.5 mL,
.9 mmol) was added. The yellow solution was allowed to stir for
8
phate (HBTU) was purchased from NovaBiochem. All solvents
were used as received without further purification unless otherwise
noted.
an additional 90 min and 1d (2.2 g, 5.7 mmol) was added. The solu-
tion was allowed to react for four days, and the volume was then
reduced under vacuum. The product was purified by column
chromatography using a 10% methanol/90% dichloromethane mo-
bile phase. Like fractions were combined to yield 0.85 g of yellow
Syntheses
Diethyl Iminodiacetate: By using a modified literature method,[24]
iminodiacetic acid (10.33 g, 77.7 mmol) was added to hydrochloric
acid (100 mL of 2.5 m in ethanol). This solution was heated at re-
flux for 24 h and then allowed to cool to room temperature. Water
+
+
foam (54.1%). HRMS (ESI ): calcd. for [M + H] 1076.5358;
found 1076.5358. C59 ·CH OH·CH Cl : calcd. C 61.69, H
.39, N 12.93; found C 61.43, H 6.36, N 12.92. See Supporting
H
69
N
11
O
9
3
2
2
6
1
13
Information for H and C NMR spectral assignments.
(ca. 200 mL) was added, and the solution was neutralized with so-
dium hydrogen carbonate. The solution was extracted with dichlo-
romethane (3ϫ75 mL), and the combined organic solutions were
dried with sodium sulfate. The solvent was removed under vacuum
Cu [3] (BF and Cu [3] (PF : In a 50 mL round-bottomed flask,
3
2
4
)
6
3
2
6 6
)
3
(141 mg, 0.13 mmol) and copper(II) nitrate hexahydrate (45.9 mg,
0.19 mmol) were added to methanol (ca. 10 mL) and heated at re-
+
to yield 3.62 g of colorless oil (38.4%). MS (ESI ): calcd. for [M +
4
flux overnight. To produce the BF salt, the solution was cooled to
+
1
3
H] 190.1; found 190.3. H NMR (400 MHz, CDCl ): δ = 1.13 (t,
room temperature, and the solvent was evaporated under vacuum.
The remaining mixture was redissolved in a 1:4 mixture of meth-
anol/water, and a saturated aqueous solution of ammonium tetra-
J = 7 Hz, 6 H), 2.10 (s, 1 H), 3.35 (s, 4 H), 4.10 (q, J = 7 Hz, 4 H)
ppm.
Ethyl [N-2-(4Ј-Methyl-2,2Ј-bipyridyl)acetyl]iminodiacetate (EtO- fluoroborohyrate was slowly added. The product was filtered and
Bpy-OEt) (2): 4Ј-methyl-2,2Ј-bipyridine-4-acetic acid (3.40 g,
washed with methanol and diethyl ether. The olive-green powder
was dried under vacuum overnight to yield 75.6 mg (20.1%). H
1
1
1
4.0 mmol), HBTU (5.29 g, 14.0 mmol), and HOBT (1.89 g,
4.0 mmol) were added to dry dichloromethane (150 mL) stirred in
NMR (400 MHz, [D ]ACN, Figure S5): δ = 4.67–2.57 (br. m, 44
3
+
an ice bath. The suspension was stirred for 15 min, DIPEA
4.6 mL, 27.9 mmol) was added, and the yellow solution was stirred
for an additional 45 min. To this was added diethyl iminodiacetate
1.32 g, 7.0 mmol) in dry dichloromethane (50 mL), and the solu-
H), 1.57–1.23 (br. s, 36 H) ppm. MS (ESI ): calcd. for {[Cu (3) ]-
628.9; found 631.0; calcd. for {[Cu (3) ](BF ) -
3 2 4 4
CH CN} 1357.9; found 1359.4. To produce the PF salt, a satu-
3
2
+
4
(
(BF ) }
4
2
+
2
3
6
(
rated solution of hexafluorophosphate was added to precipitate the
copper duplex, and the product was isolated and purified as before.
tion was allowed to reach room temperature. After stirring for 24 h,
the solvent was removed under vacuum, and the product was iso-
lated and purified by silica column chromatography with a mobile
phase of methanol (5%) in dichloromethane. Like fractions were
combined, and the solvent was evaporated to yield 1.29 g of yellow
+
For [Cu (3) ](PF ) : MS (ESI ) (Figure 3): calcd. for {[Cu (3) ]-
3
2
6 x
3
2
+3
+
2
(PF ) }
6
4
3 2 6 3
1461.4; found 1461.2; calcd. for {[Cu (3) ](PF ) }
925.9; found 925.6; calcd. for {[Cu (3) ](PF ) } 658.2; found
+
4
3
2
6 2
+
5
1
658.2; calcd. for {[Cu (3) ](PF )} 497.6; found 497.6. H NMR
3
2
6
+
+
oil (46.3%). MS (ESI ): calcd. for [M + H] 400.4; found 400.5.
(400 MHz, [D ]ACN, Figure S7): δ = 4.74–2.43 (br. m, 44 H), 1.54–
3
1
H NMR (400 MHz, CDCl
s, 3 H), 3.45 (s, 4 H), 3.7–3.8 (s, 2 H), 4.15 (m, 4 H), 8.60–7.18 (d,
J = 64 Hz, 2 H), 7.89–8.16 (d, J = 36 Hz, 2 H), 8.3–8.5 (d, J =
1 Hz, 2 H) ppm.
3
): δ = 1.2 (m, 6 H), 2.10 (s, 1 H), 2.40
1.31 (br. s, 36 H) ppm.
(
Cu
(126 mg, 0.117 mmol) and copper acetate (34.3 mg, 0.176 mmol)
were stirred in methanol overnight. The solution was recrystallized
from methanol and diethyl ether, washed with H O, collected, and
dried overnight to yield an emerald green powder, yield 151 mg
3 2 3 2
[3] (CH CO )6: In a 50 mL round-bottomed flask, compound
3
4
Ethyl [N-2-(4Ј-Methyl-2,2Ј-bipyridyl)acetyl]iminodiacetic Acid (2d):
2
[25]
According to an adapted synthesis,
compound 2 (1.29 g,
3.2 mmol) was dissolved in tetrahydrofuran (13 mL), and sodium
1
(
47.8%). H NMR (400 MHz, [D
3
]ACN, Figure S6): δ = 4.74–2.43
hydroxide (8 mL, 2 m) was added dropwise and allowed to stir over-
night. Water (25 mL) was added and extracted with dichlorometh-
ane (2ϫ50 mL). The aqueous layer was retained and cooled by
stirring over ice. The pH was lowered to 7 with HCl (2 m), and the
solvent was removed under vacuum to yield 1.05 g of bright yellow
(
br. m, 44 H), 1.54–1.31 (br. s, 36 H) ppm.
Spectrophotometric Titrations: Titrations were performed by using
spectroscopic grade methanol solutions of known concentrations
of tripeptide and Cu(NO
measurements of visible wavelength absorbance, approximately
15 mm Cu was titrated into approximately 3 mm tripeptide; and
for absorbance measurements in the ultraviolet region, approxi-
mately 100 μm Cu was titrated into approximately 15 μm tripep-
tide solution. Background subtractions were performed by using
the double beam of the spectrometer.
3 2 4 2 3 2
) , Cu(BF ) , and Cu(CH COO) . For
+
+
solid (94.7%). MS (ESI ): calcd. for [M + H] 344.3; found 344.2.
2
+
1
H NMR (400 MHz, MeOD): δ = 2.50 (s, 3 H), 3.27 (s, 4 H), 3.86
(s, 2 H), 7.19–7.47 (dd, J = 4, 69 Hz, 2 H), 7.92–8.26 (d, J = 43 Hz,
2
+
2
H), 8.3–8.75 (dd, J = 25, 5 Hz, 2 H) ppm.
tert-Butyl {N-[2-Aminoethyl]-N-[2-(4Ј-methyl-2,2Ј-bipyridyl)-acetyl]-
amino} Acetate (1d): This synthesis was adapted from a published
[
26]
procedure.
idine (140 mL, 20%) in acetonitrile. The solution was stirred for
0 min and extracted with hexanes (3ϫ75 mL). The acetonitrile
layer was reduced under vacuum to yield 1.65 g of yellow oil
To 1 (2.64 g, 4.4 mmol) was added dropwise piper-
Spectrophotometric Titrations with Addition of Salts: Titrations
were performed by using spectroscopic grade methanol solutions of
2
+
3
approximately 0.5 mm Cu duplexes (BF
approximately 10 mm salts (NaI, NaBr, NaCl, NaNO
NaCH CO , and NaBF ) were added incrementally, and the pro-
cess was monitored with a BD Falcon 96 well-plate and well-plate
reader in the visible region.
4 3 2
and CH CO ), to which
3
,
+
+
1
(
98.6%). MS (ESI ): calcd. for [M + H] 385.5; found 385.2. H
NMR (400 MHz, CDCl ): δ = 1.33 (s, 9 H), 2.33 (s, 3 H), 2.70 (t,
J = 5 Hz, 2 H), 3.10–3.35 (q, J = 8 Hz, 2 H), 3.60 (m, 2 H), 6.85
3
2
4
3
Eur. J. Inorg. Chem. 2011, 4168–4174
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjic.org
4173

J. A. Gallagher, L. A. Levine, M. E. Williams
FULL PAPER
Supporting Information (see footnote on the first page of this arti-
cle): NMR spectroscopic data, spectrophotometric titrations of
Cu(NO ) and Cu(CH O ) into 3.
3 2 3 2 2
Youm, L. A. Levine, J. R. Miller, C. P. Myers, M. E. Williams,
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4174
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Eur. J. Inorg. Chem. 2011, 4168–4174