Spectroscopic and Electrochemical Investigations into the Interactions of Metal Ions with a Ferrocenoyl-Histidine Peptide Conjugate

Article abstract of DOI:10.1002/ejic.201000658

A new ferrocene-peptide conjugate Fc-[His(DNP)-Gly-OMe]₂ (2) was synthesized and characterized spectroscopically. The conjugate displays the familiar 1,2a-P helical Herrick conformation in solution and exhibits a reversible one-electron oxidati

Full text of DOI:10.1002/ejic.201000658

FULL PAPER
DOI: 10.1002/ejic.201000658
Spectroscopic and Electrochemical Investigations into the Interactions of Metal
Ions with a Ferrocenoyl–Histidine Peptide Conjugate
Lan-Ya Cheng,[a,b] Yi-Tao Long,*[b] He Tian,[b] and Heinz-Bernhard Kraatz*[a]
Keywords: Cyclic voltammetry / NMR spectroscopy / N ligands / Metal recognition / Chiral induction
A
new ferrocene–peptide conjugate Fc-[His(DNP)-Gly- 180 mV) Ͼ Na+ (–75 mV). The formation of a 1:1 metal com-
OMe]2 (2) was synthesized and characterized spectroscopi- plex was postulated on the basis of CV and NMR spectro-
cally. The conjugate displays the familiar 1,2Ј-P helical Her- scopic titration experiments. The complexes themselves were
rick conformation in solution and exhibits a reversible one- characterized by electrospray mass spectrometry. NMR spec-
electron oxidation at 380 mV versus Fc/Fc+. The interactions troscopic studies show that the His imidazole is the site of
of the metal ions Na+, Mg2+, Fe2+, Cu2+, Zn2+, and Cd2+ with metal coordination. As is confirmed by CD spectroscopy,
conjugate 2 were probed by using a range of techniques, in- metal coordination itself results in conformational changes of
cluding cyclic voltammetry, and NMR and CD spectrosco- the ferrocene core that are dependent on the particular metal
pies. Electrochemical studies showed that the system exhib- ion and its coordination preferences. Whereas P-helicity of
its a rare cathodic potential shift upon the addition of metal the conjugate is maintained during the interactions with
ions, which followed the order Cu2+ (–436 mV) Ͼ Fe2+ (– Mg2+, Zn2+, and Cd2+, M-helicity is adopted for Fe2+ and
284 mV) Ͼ Zn2+ (–270 mV) Ͼ Cd2+ (–238 mV) Ͼ Mg2+ (– Cu2+
.
Whereas Fc–peptide conjugates have been explored to
Introduction
probe cell uptake and targeting in medicinal applications,[3]
and used as part of biosensor platforms,[4] their coordina-
tion chemistry with metal ions and their effects on the
structure of the conjugate and the stereochemistry of the
Fc core remains largely unexplored. Metzler-Nolte and co-
workers recently reported the structure of an Fc–cysteine
derivative in which the two Cys sulfur atoms coordinate to
an Fe–carbonyl fragment.[5] Hirao et al. reported a Pd2+
complex of an Fc–peptide–pyridine conjugate in which a
Pd2+ ion is coordinated to the two pyridine ligands.[6] Re-
sults on a cyclic Fc–His derivative show that alkali metals
can coordinate presumably to the amide carbonyl groups,
giving rise to significant changes in the redox potential of
the Fc core.[7] A similar coordination behavior was ob-
served for the interaction of alkali metal ions with cyclic
Fc–peptides in which the metal ions coordinate to the
amide C=O oxygen atom.[8]
Peptides can readily interact with metal ions through the
peptide backbone, involving the O or N site of the amide
group.[9] While such interactions are pH-dependent and
generally show poor selectivity to metal ions, peptide-in-
volving functional side groups, such as His, are expected to
enhance the metal selectivity. The study of these com-
pounds is of growing interest in an attempt to understand
the role of metal ions in peptide interactions and aggrega-
tions.[10] In addition, they are being exploited in sensor sur-
faces for Cu recognition. For example, the tripeptide Gly–
Gly–His readily binds to Cu2+ ions at ultralow concentra-
tions through the imidazole group.[11]
The ferrocenyl (Fc) group has proven itself as a useful
scaffold for the design of well-defined peptide structural
motifs, including β-sheets, γ-turns, and other more complex
foldamer structures.[1] The separation between the two cy-
clopentadienyl rings is ideal to support hydrogen-bonding
interactions between the peptide substituents on the two Cp
rings that help to stabilize structural motifs. The H-bonding
interactions, however, are controlled to some degree by the
particular amino acid. Most bis(peptide)-substituted Fc
conjugates display two H-bonding interactions involving
the conformation that locks the central Fc core into a spe-
cific axial conformation.[2] Generally, for most -amino
acids, a 1,2Ј-P-helical arrangement is preferred in solution
and in the solid state. The CD spectra of such conjugates
exhibit a characteristic signal with a positive Cotton effect
in the Fc-based absorption region from λ = 440–480 nm.
The corresponding -amino acids give rise to a 1,5Ј-M-
helical conformation with the corresponding negative CD
signal in this region.[2b,2d,2e,2n]
[a] Department of Chemistry, The University of Western Ontario,
London, Ontario N6A 5B7, Canada
Fax: +1-519-661-3022 x81561
E-mail: hkraatz@uwo.ca
[b] Department of Chemistry, East China University of Science
and Technology,
Shanghai 200237, China
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejic.201000658.
View this journal online at
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L.-Y. Cheng, Y.-T. Long, H. Tian, H.-B. Kraatz
FULL PAPER
Here, we focus on the use of a histidine-containing Fc the NMR spectroscopic properties of conjugate 2 were in-
conjugate and probe its interactions with a range of metal vestigated at various temperatures ranging from 290 to
ions. Histidine plays a critical role in many metalloproteins 320 K and at concentrations ranging from 1 to 20 m. The
and provides a coordination site for metals such as Fe, Cu, H-bonding interactions are temperature-sensitive and, at
and Zn. We will provide evidence that in a bis(peptide)–Fc higher temperatures, H-bonding interactions are weakened.
conjugate, metal coordination to the imidazole of the His This is readily detected by NMR spectroscopy. If H-bond-
moiety influences the stereochemistry of the central core ing interactions are present, an increase in the temperature
and also gives rise to significant changes in the redox prop- will weaken these interactions resulting in an upfield shift
erties of the Fc group.
of the resonances assigned to the amide protons. At low
concentrations (below the self-association concentration of
the peptide conjugate), an upfield shift indicates the pres-
ence of intramolecular H-bonding. A study of the concen-
tration dependence of the chemical shift will allow an evalu-
ation which of the amide protons are involved in intermo-
lecular H-bonding interactions. Downfield shifts of the
amide proton with increasing concentration indicate the in-
volvement of intermolecular H-bonding interactions. Plots
showing the effects of varying the temperature and the con-
centrations on the chemical shifts of NHa and NHb are
shown in Figure 1. Several findings are noteworthy. At a
concentration of 1 m, the chemical shift of the His NHa
proton is affected more by changes in the temperature com-
pared with that of the Gly NHb proton. The ∆δ/T values of
the amide protons are –6 ppb/K for NHa and –3.2 ppb/K
for NHb. Temperature coefficients larger than 4 ppb/K indi-
cate the involvement of the amide protons in H-bond-
ing.[2b,12]
Results and Discussion
Preparation and Characterization of Fc[CO-His(DNP)-Gly-
OMe]2 (2)
A brief overview of the synthetic strategy leading to
peptide conjugate 2 is shown in Scheme 1, essentially fol-
lowing well-documented coupling strategies, which involve
1,nЈ-ferrocenedicarboxylic acid [Fc(COOH)2], O-benzotri-
azole-N,N,NЈ,NЈ-tetramethyluronium hexafluorophosphate
(HBTU)/NЈ-[3-(dimethylamino)propyl]-N-ethylcarbodiimide
(EDC) as coupling reagent, and the deprotected dipeptide
Boc-His(DNP)-Gly-OMe (1; DNP = 2,4-dinitrophenol).[2]
Figure 1. Effects of temperature and concentration on the chemical
shift of the amide protons of compound 2 in CD3CN solution. (A)
VT NMR plots of the resonances for NHa and NHb at 293.15,
298.15, 303.15, 308.15, 313.15, and 318.15 K ([conjugate 2] = 1 m,
CD3CN). (B) VC NMR plots of the resonances of NHa and NHb
at 1, 2, 5, 10, and 20 m concentrations in CD3CN.
Scheme 1. (A) Synthesis of the ferrocene conjugate Fc[CO-
His(DNP)-Gly-OMe]2 (2) by cross-coupling with the dipeptide
Boc-His(DNP)-Gly-OMe under suitable conditions (DNP = 2,4-
dinitrophenol). (a) HOBT, HBTU, TEA, H-Gly-OMe; (b) TFA/
CH2Cl2 (1:1); (c) Fc(COOH)2, HOBT, EDC. (B) “Herrick” motif
showing the 1,2Ј-arrangement of the two peptide substituents and
the interstrand H-bonding interactions between the NHa and His
CO on opposite strands.
Although both amide signals exhibit concentration de-
pendence, the effect of concentration on the chemical shift
of NHb is stronger. Increasing the concentration will favor
intermolecular interactions, therefore suggesting that the
The Fc–peptide conjugate 2 was purified by column Gly NHb moiety is involved in intermolecular H-bonding
chromatography and obtained as a yellow–orange solid in interactions. These spectroscopic results are compatible
40% yield. Time-of-flight mass spectrometry measurements with the Fc-proximal NHa being involved in intramolecular
show a strong [M + H]+ peak at m/z = 1023.1920. Com- cross-strand H-bonding interactions, presumably giving rise
pound 2 has a good solubility in most organic solvents and to “Herrick”-type H-bonding, which is characteristic for
was characterized spectroscopically in CD3CN solution. At many bis(peptide)-substituted 1,nЈ-Fc conjugates. This
room temperature, compound 2 displays two signals as- leaves the distal Gly NHb moiety free to engage in intermo-
signed to the two amide protons (10 m) at δ = 8.23 (NHa) lecular interactions. The “Herrick” pattern locks the Fc
and 7.62 ppm (NHb). The position of these two amide sig- core into a 1,2Ј-P-helical conformation.[2d] The conforma-
nals is indicative of hydrogen-bonding interactions.[2d,2e] To tion of compound 2 was studied by CD spectroscopy in
evaluate if intra- or intermolecular interactions are respon- acetonitrile solution at 1 m. The CD plot of compound 2
sible for the observed chemical shifts of the amide protons, in the range of 300–600 nm is shown in Figure S25 in the
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Interactions of Metal Ions with a Ferrocenoyl–Histidine Peptide Conjugate
Supporting Information. The system exhibits a CD band in served for all metal ions. The cathodic shift decreases in the
the Fc region with a positive Cotton effect and a maximum order Cu2+ (–403 mV)
Ͼ Ͼ
Zn2+ (–268 mV) Fe2+
absorbance at λ = 480 nm, indicating a P-helical conforma- (–254 mV) Ͼ Cd2+ (–242 mV) Ͼ Mg2+ (–137 mV) Ͼ Na+
tion around the Fc group. The properties of the Fc-centered (–40 mV), indicating the Fc component with the various
CD signal are similar to those of a wide range of bis(pep- metals are more easily oxidized in the order Cu2+ Ͼ Zn2+
tide)-substituted Fc derivatives derived from -amino ac- Ͼ Fe2+ Ͼ Cd2+ Ͼ Mg2+ Ͼ Na+. For the most part, cation
ids,[2d,2e] but differences are noticeable in the band at λ = coordination to an Fc host is expected to result in an anodic
350 nm showing a negative CD signal. This band is ration- shift of the redox potential, making the Fc group more dif-
alized largely by transitions involving the Cp and the Fe ficult to oxidize, whereas anions can cause cathodic
and is significantly more intense compared with other Fc– shifts.[13] There are few examples showing similar cathodic
peptide conjugates, presumably due to contributions from shifts. Beer and co-workers reported an Fc–crown-ether
the DNP-protecting group on the imidazole ring of His conjugate with a thioether linker, which showed an anodic
moiety, which proved to be useful in this context.[2p]
potential shift when Na+ ions were added, but displayed a
The redox properties of conjugate 2 were tested in aceto- surprising cathodic potential shift when K+ ions were
nitrile solution with cyclic voltammetry (CV) by using tetra- added.[13b] This was ascribed to the formation of a 1:1 K+
butylammonium perchlorate (TBAP) as the supporting complex. Another example of a cathodic shift is observed
electrolyte. Figure S26 shows a typical voltammogram of for the alkali metal complex of a cyclic 1,1Ј-FcCO–His con-
conjugate 2 at a scan rate of 100 mV. The system displays a jugate (FcCO-His-OMe), in which cyclization is achieved
single one-electron oxidation with a halfwave potential through the ε-N atom of the imidazole.[7] Metal binding
(E1/2) of 380(5) mV (vs. Fc/Fc+), a peak separation (∆E) of occurs presumably through the oxygen atom of the Fc-C=O
89(5) mV at 100 mV, and a linear relationship between the moiety, which translates into the slight conformational
peak potential ipa and the square-root of the scan rate. In changes in the Fc core. Another interesting example is a
addition, the peak current ratio ipa/ipc is close to unity Pd2+ complex of Fc[CO--Ala--Pro-NHPy]2, which has
(Table S1).
the metal ion complexed through the C-terminal pyridine
groups. Upon coordination, the Fc group displays a cath-
odic shift of about 20 mV versus the free Fc–peptide.[6]
Interactions with Metal Ions
The behavior of conjugate 2 in the presence of metal ions
was probed by CV. Since the conjugate contains His resi-
dues it can be expected that metal ions can coordinate to
the imidazole groups of the two proximal His residues
(Scheme 2).[9a] Metal coordination to the imidazole groups
is expected to influence the redox properties of the Fc group
and, in addition, should further be detectable by a shift in
the resonance of the imidazole protons in the 1H NMR
spectrum.
Scheme 2. Proposed interaction of conjugate 2 with transition
metal ions involving the two His residues.
Figure 2. The cyclic voltammograms of conjugate 2 in acetonitrile
before (_____) and after (-----) the addition of metal perchlorate salts.
The addition of metal salts caused cathodic shifts ([conjugate 2] =
1 m, acetonitrile, 1  TBAP was added as supporting electrolyte;
glassy carbon working electrode, Pt wire counter electrode, Ag/
AgCl reference electrode; Fc/Fc+ was used as an external refer-
First, the effects on the electrochemical properties were
investigated. Figure 2 shows CV plots of conjugate 2 in the
presence of 2 equiv. of the metal ions Na+, Mg2+, Zn2+
,
Cd2+, Fe2+, and Cu2+. A shift to lower potential was ob- ence).
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L.-Y. Cheng, Y.-T. Long, H. Tian, H.-B. Kraatz
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A titration experiment in which metal ions were added with 1:1 stoichiometry. This would presumably involve N-
to a solution of conjugate 2 was carried out, and the cath- coordination of the imidazole group. This is supported by
odic shift as a function of added metal ions was measured. our NMR spectroscopic titration experiments.
The cathodic shift decreases in the order Cu2+ (–436 mV)
Ͼ Fe2+ (–284 mV) Ͼ Zn2+ (–270 mV) Ͼ Cd2+ (–238 mV)
Ͼ Mg2+ (–180 mV) Ͼ Na+ (–75 mV) when 4 equiv. of the
metal ions were added. The collective result of this study
is shown in Figure 3. The results clearly demonstrate that
cathodic shifts increase significantly upon the addition of
1 equiv. of metal ion. The addition of excess metal ions does
not affect the redox potential significantly, and further ad-
dition of metal ions shows negligible changes. This suggests
that the Fc conjugate 2 binds to a single metal ion, which
gives rise to a cathodic shift. The effect of introducing water
to the solution was also considered, but the shifts caused
by water are significantly smaller than the effects of the
added metal ions (see Figures S27 and S28 in the Support-
ing Information).
Figure 4. 1H NMR spectroscopic titration experiments of Cd2+
.
Cd2+ was added at 0, 0.2, 0.4, 0.6, 1.0, 1.2, 1.4, and 2.0 equiv.
shown from top to bottom ([conjugate 2] = 1 m, CD3CN; the
asterisk indicates residual CHCl3).
Figure 3. Effects of metal ion addition on the half-wave potential
of compound 2 (1 m in acetonitrile). The following metal ions
were added as their perchlorate salts: o = Na+; ᭹ = Zn2+; ᭿ =
Cd2+; ᭢ = Mg2+; ∆ = Fe2+; ◂ = Cu2+
.
A series of 1H NMR spectroscopic titrations was carried
out, in which the chemical shifts of conjugate 2 were moni-
tored as a function of added metal ion. The 1H NMR spec-
tra for the titrations of Cd2+ to a solution of conjugate 2 in
CD3CN are shown in Figure 4. It is clearly seen that the
signals of the imidazole protons δ2 and ε1 are significantly
affected by the addition of the Cd2+ ions. The amide proton
is also affected by the addition of metal ions. Similar results
were observed on the addition of the other transition metal
ions to a solution of conjugate 2, indicating that metal co-
Figure 5. Changes in the chemical shifts of the two His protons ε1
and δ2 as a function of added Cd2+ to a 1 m solution of conjugate
2 in CD3CN. ᭹ = ε1 of His, o = δ2 of His, ᭿ = NHa. Note that
after the addition of 1 equiv. of Cd2+, no additional chemical shift
changes are observed.
There are some additional interesting changes noticeable
1
ordination involves the imidazole group of His. A plot of in the H NMR spectra shown in Figure 5, presumably re-
the chemical shift change (∆δ) versus the equivalents of lated to the structural rigidity of the metal complex. Before
Cd2+ added to the solution is shown in Figure 5. Essen- metal addition, the two diastereotopic β-protons are ob-
tially, the results are comparable with our CV titration ex- served as a single multiplet, which upon addition of Cd2+
periments and show that the chemical shift is affected until splits into two separate multiplets; this indicates enhanced
1 equiv. of metal ion is added. Addition of excess metal ion conformational rigidity upon metal coordination to the His
does not cause any significant changes of the chemical imidazole. This is a reasonable assumption, since side-chain
shifts. It can be assumed that transition metals prefer to rotation will not be restricted for a metal complex. Ad-
1
bind to conjugate 2 involving the formation of a chelate ditional H NMR spectroscopic titrations can be found in
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Interactions of Metal Ions with a Ferrocenoyl–Histidine Peptide Conjugate
Figures S12–S17 in the Supporting Information. The ad-
dition of Na+ ions to 2 did not cause any visible change
during the titration, which is comparable with the electro-
chemical results. In the case of Fe2+ and Cu2+, the para-
magnetic nature of the hexaqua ion essentially causes line
broadening.
Our spectroscopic and electrochemical studies suggest
the formation of a 1:1 complex between the conjugate 2 and
metal ions, such as Zn2+, Cd2+, Fe2+, Mg2+, and Cu2+. Next
we carried out additional experiments focused on evaluat-
ing the stoichiometry of the complex in more detail. For
this purpose, we carried out studies by electrospray time-
of-flight mass spectrometry at the ratio of 4:1 of metal ions/
conjugate 2. A typical mass spectrum of the solution of
2·Cd2+ displays a peak at m/z = 1133.2 assigned to [M +
Cd – H]+ and 1233.2 assigned to [M + Cd + 2 CH3CN +
H2O – H]+. In both cases, the mass spectrum shows an
isotope envelope indicative of a Cd complex. The [M +
Cd – H]+ cluster was analyzed further by high-resolution
MS, and the experimental result, together with a theoretical
isotope pattern of the molecular ion [M + Cd – H]+ at m/z
= 1133.0789, is shown in Figure 6. Similar MS results were
obtained for Zn2+, Fe2+, and Cu2+ and are summarized in
Table 1 (see also the Supporting Information).
As we know the stoichiometry of our Fc–peptide metal
complexes, it is of interest to evaluate if metal coordination
will cause any structural changes in the Fc conjugate. For
this purpose, we used CD spectroscopy. The 1,nЈ-bis(pep-
tide)–Fc conjugates display a characteristic CD signal in the
Fc region of λ = 400–500 nm that is linked to the axial
chirality of the Fc group and its substitution
pattern.[2b,2d,2e] Figure 7 shows the CD spectra of the Fc–
peptide conjugate 2 in the presence of various metal ions in
1 m acetonitrile solution. The spectrum of conjugate 2 is
characterized by a negative CD signal at λ = 350 nm, a
negative shoulder at λ ≈ 420 nm, and a positive signal at λ
= 490 nm, indicative of a P-helical arrangement of the Fc
core. This is in line with the results from the 1H NMR spec-
troscopic studies of conjugate 2, which indicated that the
proximal amide protons are involved in intramolecular H-
bonding interactions.
Figure 6. Partial ES-Tof-MS results showing the experimental and
theoretical isotopic pattern of the Cd2+ complex of conjugate 2,
assigned to [M + Cd – H]+. Top: calculated molecular isotopic
distribution for [M + Cd – H]+; bottom: observed partial spectrum.
Table 1. Results of the high-resolution electrospray mass spectrom-
etry analysis of acetonitrile solutions of ferrocene conjugate 2 in
the presence of Fe2+, Zn2+, Cu2+, and Cd2+
.
Complex compostion
Mass
Calcd. mass
[M+Fe – H]+
[M+Zn – H]+
[M+Cu]+
1077.1110
1085.1067
1085.1165
1133.0789
1077.1149
1085.1091
1085.1174
1133.0789
As expected, the addition of Na+ ions does not cause
any significant changes in the CD spectrum of compound
2, whereas the addition of all other metal ions caused sig-
nificant changes. Two changes are particularly noteworthy.
[M+Cd – H]+
Firstly, for all metal ions the signal at λ = 330 nm has a conformational changes in the structure of the Fc core and
positive Cotton effect and experiences a hypsochromic shift, a change to an M-helical arrangement is indicated by the
and its intensity is affected by the specific metal ion. Sec- CD signal with a negative Cotton effect at λ ≈ 470 nm for
ondly, the Fc-based signal remains positive on addition of both complexes. Presumably, coordination of the two imid-
Mg2+, Zn2+, and Cd2+ ions, indicating that the helical chi- azole ligands to the metal ions is responsible for the changes
rality of the Fc core retains P-helicity. This would suggest in the Fc conformation. The disposition of the two imid-
that metal coordination to the two imidazoles of the conju- azoles around the metal ion is expected to be 90° due to
gate does not alter the conformation around the Fc core. It the preference of the Fe2+ and Cu2+ ions for octahedral to
should be noted that all three metal ions can accommodate distorted octahedral (or square-pyramidal to square-
a tetrahedral coordination environment, which presumably planar) environments. This in turn might influence the
does not cause any significant steric stress on the Fc core structure of the Fc core and distort the Cp rings away from
upon coordination in a bidentate fashion. In contrast, the a 1,2Ј-P helical conformation towards a more open M-heli-
addition of Fe2+ and Cu2+ ions appears to cause significant cal structure.
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L.-Y. Cheng, Y.-T. Long, H. Tian, H.-B. Kraatz
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sized from ferrocene according to literature procedures.[14,15] Boc-
His(DNP)-OH·IPA (IPA
=
2-propanol), H-Gly-OMe·HCl,
HOBT·H2O, and EDC·HCl were obtained from Advanced Chem-
Tech. Cd(ClO4)2·6H2O, Zn(ClO4)2·6H2O, Cu(ClO4)2·6H2O, and
Fe(ClO4)2·xH2O were bought from Aldrich; Mg(ClO4)2·6H2O and
NaClO4 were bought from Alfa–Aesar. All chemicals were used
without further purification. All 1H NMR spectroscopic experi-
ments were carried out with a Varian Nova 400M NMR spectrom-
eter at 25 °C, except the VT NMR experiments. All 1H NMR spec-
troscopic titrations (except the VC NMR spectroscopic experiment)
and CV titration experiments were carried out at an Fc conjugate
concentration of 1 m in acetonitrile. The metal concentration was
changed by the addition of aliquots of metal salt solutions in
[D]acetonitrile by using microliter syringes. Mass spectrometry was
carried out with a Finnigan MAT 8200 mass spectrometer. A
JASCO J-810 CD spectrometer was used to evaluate the solution
conformation of the Fc–peptide conjugate 2; 1 mm cuvettes were
used for these studies and a solution of conjugate 2 in freshly dis-
tilled acetonitrile (1 m concentration). A 0.1  metal perchlorate
solution was prepared in dry, distilled acetonitrile. The metal salt
solution was added in 0.2 µL aliquots. and the resulting concentra-
tions were adjusted.
Synthesis of Boc-His(DNP)-Gly-OMe (1): Boc-His(DNP)-OH·IPA
(1.44 g, 3 mmol) was dissolved in distilled CH2Cl2 (50 mL). The
solution was cooled in an ice bath, and solid HOBT (1.2 eqiuv.,
0.49 mg, 3.6 mmol) and HBTU (1.36 g, 3.6 mmol) were added im-
mediately, followed by the addition of triethylamine (TEA;
0.75 mL). After stirring for 0.5 h, H-Gly-OMe·HCl (0.38 g,
3 mmol) in distilled CH2Cl2 (30 mL), together with TEA (0.75 mL),
was added to the stirred, activated mixture of Boc-His(DNP)-OH
after 10 min. The ice bath was removed and the stirring continued
at r.t. overnight. The reaction solution was then dried in vacuo and
purified on a silica column by using a solvent gradient (CH2Cl2 to
2% methanol/CH2Cl2). The desired peptide was obtained as a yel-
Figure 7. CD spectra of the six metal complexes prepared with
2 equiv. of metal ions with compound 2 (dashed line), compared
with compound 2 (1 m; solid line) in acetonitrile.
Conclusion
1
low powder (1.26 g, 85.4%). Rf = 0.24 (CH2Cl2/MeOH, 91:9). H
A novel disubstituted ferrocene–peptide histidine conju-
gate has been successfully synthesized, and its coordination
chemistry with a range of metal ions was studied. Our stud-
NMR (CDCl3): δ = 8.84 (d, J = 2.4 Hz, 1 H, H-DNP), 8.57 (m, 1
H, H-DNP), 7.78 (d, J = 8.8 Hz, 1 H, H-DNP), 7.62 (s, 1 H, Hε1),
7.00 (br. s, 1 H, NHa), 6.96 (s, 1 H, Hδ2), 6.18 (br. s, 1 H, NHb),
ies show that the metal ions coordinate to the His imid- 4.54 (br. s, 1 H, CHα), 3.99 (m, 2 H, CH2-ester), 3.70 (s, 3 H,
COOCH3), 3.12 (m, 2 H, CH2-imidazole), 1.47 (s, 9 H, H-Boc)
ppm. 13C NMR (CDCl3): δ = 171.9, 170.4, 147.1, 144.6, 140.3,
136.7, 135.4, 129.8, 128.4, 121.5, 118.1, 80.4, 54.5, 52.4, 47.4, 41.3,
30.3, 28.5, 8.92 ppm. TOF MS (ES+): found for C20H24N6O9
492.1625.
azoles. Furthermore, metal coordination to the imidazoles
influences the helicity of the central Fc group. For Mg2+
,
Zn2+, and Cd2+, the Fc maintains P-helicity, whereas for
Fe2+ and Cu2+ the Fc group is clearly M-helical. Changes
in the Fc helicity are thought to be the result of differences
in the coordination environments between metals preferring
a tetrahedral to an octahedral (square-planar) geometry.
Presumably, a tetrahedral geometry is more amenable to a
P-helicity.
Synthesis of Fc[CO-His(DNP)-Gly-OMe]2 (2): Boc-His-Gly-OMe
(0.98 g, 2 mmol) was dissolved in distilled CH2Cl2 (20 mL). TFA
(20 mL) was added slowly, and the solution was stirred at r.t. for
20 min and then dried under vacuum. The residue was then dis-
solved in CH2Cl2 (40 mL), and TEA was added to the solution (ca.
2 mL). After 5 min, the clear solution was again concentrated to
dryness and divided into portions. Fc(COOH)2 (0.14 g, 0.5 mmol)
was dissolved in distilled CH2Cl2 (30 mL). Solid HOBT·H2O
(0.18 g, 1.2 mmol) and EDC·HCl (0.21 g, 1.1 mmol) were added,
The metal complexes of our Fc–His conjugate also gives
rise to cathodic potential shifts of the Fc signal. This dem-
onstrates the 1,nЈ-disubstituted Fc–histidine conjugates are
a good scaffolds to recognize the metal ions through elec-
trochemical observation, and different chiral changes, affec- and the reaction mixture was stirred overnight. The reaction solu-
tion was then cooled in an ice bath, and the deprotected peptide
(0.49 g,1.2 mmol) dissolved in CH2Cl2 was added, followed by the
addition of TEA (0.6 mL). Stirring was continued at r.t. for 4 d.
The reaction solution was then dried under vacuum and purified
chromatographically by using a gradient eluent from chloroform to
chloroform/methanol (93:7). The desired product was obtained as
a brown powder (0.20 g, 39.1%.). 1H NMR (CDCl3, 298 K, c =
10 m): δ = 8.74 (m, 2 H, H-DNP), 8.50 (m, 2 H, H-DNP), 8.47
(d, J = 7.4 Hz, 2 H, NHa), 7.72 (m, 2 H, H-DNP), 7.63 (s, 2 H,
ted by different metal chelation, will show different shifts in
potential.
Experimental Section
General: All syntheses were carried out in air unless otherwise indi-
cated. CH2Cl2 (BDH; ACS grade), used for synthesis, was dried
with CaH2 and distilled. Ferrocenedicarboxylic acids were synthe-
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Eur. J. Inorg. Chem. 2010, 5231–5238

Interactions of Metal Ions with a Ferrocenoyl–Histidine Peptide Conjugate
Hε1), 7.45 (t, J = 6.2 Hz, 2 H, NHb), 7.04 (s, 2 H, Hδ2), 4.93 (m, 2
H, CHα), 4.78 (m, 2 H, H-Fc), 4.73 (m, 2 H, H-Fc), 4.43 (m, 2 H,
H-Fc), 4.27 (m, 2 H, H-Fc), 3.99 (m, 4 H, CH2-ester), 3.58 (s, 6 H,
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[2]
COOCH3), 3.09 (d, J = 7.3 Hz, 4 H, CH2-imidazole) ppm. 13C
NMR (CDCl3, 298 K, c = 10 m): δ = 173.5, 170.7, 170.4, 147.0,
140.7, 136.7, 135.4, 129.6, 128.4, 121.4, 118.2, 77.2, 76.4, 72.1, 71.6,
1
70.6, 70.4, 53.6, 52.4, 41.7, 29.7 ppm. H NMR (CD3CN, 298 K,
C = 1 m): δ = 8.77 (s, 1 H, H-DNP), 8.76 (s, 1 H, H-DNP), 8.545
(m, 2 H, H-DNP), 8.53 (d, J = 7.8 Hz, 2 H, NHa), 7.80 (m, 2 H,
H-DNP), 7.74 (s, 2 H, Hε1), 7.55 (t, J = 5.7 Hz, 2 H, NHb), 7.16
(s, 2 H, Hδ2), 4.81 (m, 2 H, CHα), 4.73 (m, 2 H, H-Fc), 4.68 (m, 2
H, H-Fc), 4.43 (m, 2 H, H-Fc), 4.34 (m, 2 H, H-Fc), 3.95 (m, 4 H,
CH2-ester), 3.55 (s, 6 H, COOCH3), 3.07 (m, 4 H, CH2-imidazole)
ppm. 13C NMR (CD3CN, 298 K, c = 1 m): δ = 174.1, 170.5,
169.9, 168.6, 166.5, 147.1, 130.0, 128.8, 121.5, 110.4, 76.8, 71.8,
71.5, 71.0, 69.9, 53.5, 52.0, 41.4, 37.0, 32.8 ppm. TOF MS (ES+):
calcd. for C42H38FeN12O16 [M + H]+ 1023.1956; found 1023.1920.
C42H38FeN12O16 (1022.67): calcd. C 49.33, H 3.75, N 16.44; found
C 49.60, H 3.87, N 16.66.
Electrochemical Studies: All electrochemical experiments described
here make use of a glassy carbon working electrode (diameter
3 mm), Pt wire as counter electrode, and Ag wire as pseudo-refer-
ence electrode. The glassy carbon electrode was polished with
0.05 µm Al2O3, sonicated in MilliQ water for 1 min to fully remove
any absorbed Al2O3, rinsed in MilliQ water, and dried under N2.
[3]
The Pt wire was sonicated and rinsed with ethanol and MilliQ
water, dried under N2, and flamed by using a propane torch until
glowing red. The Ag wire was sonicated and rinsed with ethanol
and MilliQ water, and polished with sand paper. All electrochemi-
cal data were reported relative to the Fc/Fc+ redox couple. Acetoni-
[4]
trile was dried with CaH2 and freshly distilled prior to use. TBAP
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gate 2 was studied at concentrations of 1 m in the supporting
electrolyte solution. Since most of the metal salts contain water, it
was decided to probe the effect of water since it is known that the
[5]
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Supporting Information (see footnote on the first page of this arti-
1
[8]
cle): CV, CD spectroscopy, H, g-cosy, VC, and VT NMR spectra
1
of conjugate 2, CV titration plots and H NMR titration spectra
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Acknowledgments
The authors thank Natural Sciences and Engineering Research
Council of Canada and the China Scholarship Council for finan-
cial support in the form of a scholarship to L.-Y. C. We acknow-
ledge the financial support of the Shanghai Shuguang Project
(07SG36) and funding from the Ministry of Health (Grant
2009ZX10004-301). We also want to acknowledge discussions with
Dr. Sanela Martic.
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www.eurjic.org
5237

L.-Y. Cheng, Y.-T. Long, H. Tian, H.-B. Kraatz
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Received: June 15, 2010
Published Online: October 11, 2010
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Eur. J. Inorg. Chem. 2010, 5231–5238