Ferrocenedicarboxylic Acid: Structural, Electrochemical, and Metal Ion

Article pubs.acs.org/Organometallics

Bis-amino Acid Derivatives of 1,1′-Ferrocenedicarboxylic Acid: Structural, Electrochemical, and Metal Ion Binding Studies Bimalendu Adhikari,†,‡ Alan J. Lough,‡ Bryan Barker,† Afzal Shah,†,⊥ Cuili Xiang,† and Heinz-Bernhard Kraatz*,†,‡ †

Department of Physical and Environmental Sciences, University of Toronto, 1265 Military Trail, Toronto, M1C 1 A4 Canada Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario M5S 3H6 Canada ⊥ Department of Chemistry, Quaid-i-Azam University, 45320 Islamabad, Pakistan ‡

S Supporting Information *

ABSTRACT: We report on the structural and electrochemical investigation of 1,1′-ferrocenedicarboxylic acid derivatives of tryptophan (Fc[CO-Trp-OMe]2 - Fc-conjugate 1), threonine (Fc[CO-Thr-OMe]2 − Fc-conjugate 2), aspartic acid (Fc[COAsp-OMe]2 - Fc-conjugate 3) and glutamic acid (Fc[COGluOMe]2 - Fc-conjugate 4) and their hydrolyzed analogues 1a−4a respectively (Scheme 1). CD and NMR spectroscopy established 1,2′-“Herrick conformation” in solution, having intramolecular interstrand hydrogen bonds for all Fc-conjugates. However, in solid state, Fc[CO-Trp-OMe]2 exists in “Herrick conformation” whereas Fc[CO-Thr-OMe]2 is present in anti conformation. In solution, the involvement of indole NH of Trp and alcoholic proton of Thr in intermolecular hydrogen bonding has been explored by temperature- and concentration-dependent NMR studies. The half-wave potentials (E1/2) of ferroceneconjugates follow the sequence 1 < 2 < 4 < 3 which is explained by the contribution of amino acid side chain functionalities toward the stability of ferrocenium ion. The CV of the Fc-conjugate 1/1a (having Trp moiety) displays two redox processes, one of which is assigned to the Fc group, and the other being related to the indole group. The oxidation peak potential of indole was found to depend strongly on the pH of the medium. The values of diffusion coefficient (D) and electron transfer rate constant (ksh) for all Fc-conjugates were determined from their corresponding cyclic voltammograms. In addition, metal ion interactions were studied with hydrolyzed Fc-conjugates 2a−4a using CV and DPV. Upon binding to metal ions, the electrochemical changes associated with the hydrolyzed Fc-conjugates correlated to the charge density of the binding metal ion.

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INTRODUCTION The bioorganometallic chemistry of ferrocene has been of great interest in recent years due to its versatility and reversible redox behavior.1,2 Amino acid and peptide conjugates of ferrocene (Fc) have received considerable attention due to their unique structural properties1,3,4 and potential bioanalytical applications.2 A number of Fc bioconjugates of amino acids,5−20 peptides,3,21−38 nucleic acids,39−41 peptide−nucleic acids,42,43 and carbohydrates44−46 have been studied for applications in peptide foldamers,26 as enzyme mimics,2,13,32 in electrochemical detection,1,2 and in biomedical applications.1 The separation (3.3 Å) between the two cyclopentadienyl rings in ferrocene is ideal to maintain hydrogen-bonding interactions between the amino acid/peptide substituents on the two cyclopentadienyl (Cp) rings that help to stabilize structural motifs. Most bis-amino acid derivatives of 1,1′-ferrocenedicarboxylic acid display a “Herrick conformation”, which is a 1,2′conformation stabilized by two intramolecular hydrogen bonds (Scheme 2a).3,5,10 However, the H-bonding interactions are controlled to some degree by the particular amino acid. For © XXXX American Chemical Society

example, a different conformation with one hydrogen bond has been observed for the phenylalanine derivative in the solid state which is called “van Staveren conformation”.3,7 Finally, an “open/Xu conformation” is feasible if no hydrogen bonds between substituents on the two different Cp rings form (Scheme 2a), and this is generally observed for proline derivatives due to the absence of amide functionality.3,23 However, most of the studies are limited to nonfunctional side chain-containing amino acids. It is interesting to investigate the structural and electrochemical properties of functional side chain-containing amino acid derivatives of 1,1′-ferrocenedicarboxylic acid as those functional groups can affect their properties significantly. We previously reported bis(histidine) derivatives of 1,1′-ferrocenedicarboxylic acid and the interaction with metal ions.11,16 Metzler-Nolte and co-workers reported the Special Issue: Organometallic Electrochemistry Received: January 28, 2014

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dx.doi.org/10.1021/om500032p | Organometallics XXXX, XXX, XXX−XXX

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Scheme 2. (a) Schematic Representation of P-1,2′-“Herrick”, P-1,3′-“Xu” and Anti Conformation Obtained from Bisamino Acid Derivatives of 1,1′-Ferrocenedicarboxylic Acid. Here, Conformations Are Described in Terms of the Dihedral Angles (ω) between the Two Ring-Bound Substituents of Ferrocene; in Ideal Cases, the Dihedral Angles (ω) are 72°, 144°, and 180° for P-1,2′-“Herrick”, P1,3′-“Xu” and Anti Conformation, Respectively; It Is Noted That Two Cyclopentadienyl Rings Are Staggered for Anti Conformation whereas Eclipsed for P-1,2′-“Herrick” and P1,3′-“Xu” Conformations; (b) ω Is the Dihedral Angle between the Two Ring-Bound Substituents: C(ipso)− Cp(centroid)−Cp(centroid)−C(ipso); Tilt Angle, θ, Is the Dihedral Angle between the Two Cp Rings; β Is the Dihedral Angle between the Cp Ring and the −COR (β C O) Substituent

Scheme 1. Chemical structures of Fc-conjugates 1−4 and their corresponding hydrolyzed Fc-conjugates 1a−4a used in this study

to the amino acids.11,13 Previously, researchers used different ferrocene scaffolds for electrochemical sensing of metal ions using both solution and surface electrochemistry.9,11,13,16,47−52 Beer and co-workers have made a significant contribution on ferrocene conjugate-based electrochemical sensing of cation and anions.51−55 In this study, we report the synthesis, characterization, structural and electrochemical properties of 1,1′-ferrocenedicarboxylic acid derivatives of tryptophan (Fc[CO-Trp-OMe]2 Fc-conjugate 1), threonine (Fc[CO-Thr-OMe]2 - Fc-conjugate 2), aspartic acid (Fc[CO-Asp-OMe]2 - Fc-conjugate 3) and glutamic acid (Fc[CO-GluOMe]2 - Fc-conjugate 4) and their hydrolyzed products, 1a−4a, respectively (Scheme 1). All of these Fc-conjugates show “Herrick conformation” in solution. Interestingly, Fc[CO-Thr-OMe]2 exhibits anti conformation in solid state. Moreover, metal−ion interactions were studied with hydrolyzed Fc-conjugates 1a−4a.

structure of an Fc-cysteine derivative in which the two Cys sulfur atoms coordinated to an Fe-carbonyl fragment.13 In metalloproteins, metal ions are often coordinated to a variety of nucleophilic donor groups that exist on the side chains of the amino acids. Amino acids that contain polar or electrically charged side chains are prime candidates for metal coordination. For example, human carbonic anhydrase is only fully functional due to its zinc core, which is directly coordinated to the polar imidazole groups on histidine and indirectly coordinated to threonine. The study of metal interactions and the amino acids involved in such interactions can be conducted through linking of the L-amino acids to a ferrocene scaffold and monitoring the change in the electrochemical behavior of that scaffold as metal ions are coordinated

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RESULTS AND DISCUSSION The L-amino acid derivatives of 1,1′-ferrocenedicarboxylic acid of tryptophan (Fc-conjugate 1), threonine (Fc-conjugate 2), aspartic acid (Fc-conjugate 3) and glutamic acid (Fc-conjugate 4) were synthesized by using HOBt/EDC methodology in solution. Fc-conjugates 1−4 were hydrolyzed into their corresponding free acids (Fc-conjugates 1a−4a). These conjugates are depicted in Scheme 1. B


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Figure 1. Variable-temperature 1H NMR studies of Fc-conjugates in CDCl3 at a concentration 10 mM in the temperature range of 10−50 °C: (a) Stacked spectra for Fc-conjugate 1 and (b) stacked spectra for Fc-conjugate 2. (c) Plots of indole NH (square symbol) and amide NH (circle symbol) obtained from Fc-conjugate 1. (d) Table for chemical shift (δ) at 10 °C and temperature coefficient (Δδ/T) of amide protons for Fcconjugates 1−4.

Figure 2. Variable-concentration 1H NMR studies of Fc-conjugates in CDCl3 at room temperature in the concentrations ranging from 10 to1.25 mM. (a) Fc-conjugate 1 and (b) Fc-conjugate 2.

NMR Spectroscopy. 1H NMR spectroscopy is a useful tool to identify the H-bonding interaction and conformation of ferrocene peptides in solution.3,10,25 The chemical shift of an amide NH proton changes with temperature, and the relationship between the change and the hydrogen-bonding environment of the amide NH was delineated by individual studies of Gellman and Kelly.56,57 In this study, the variable

temperature (VT-NMR) experiments were carried out with Fcconjugates 1−4 in CDCl3 in the temperature range of 10−50 °C to investigate the hydrogen-bonding interactions (Figure 1). We noticed temperature-dependent variations in the chemical shifts (Figure 1a). At 10 °C, the indole NH protons and amide NH appeared at 8.68 and 7.68 ppm, respectively. With an increase in temperature, the peak at 8.68 ppm gradually shifted C


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Figure 3. (a) CD spectra of different Fc-conjugates in chloroform at constant concentration (1.25 mM) and temperature (22 °C). Solid black, red, green and blue lines represent Fc-conjugates 1, 2, 3, and 4, respectively. (b) Comparison between two enantiomers of Fc-conjugate 1: Fc[CO-L-TrpOMe]2 () and Fc[CO-D-Trp-OMe]2 (- - -).

upfield and appeared at 8.16 ppm at 50 °C. The temperature coefficient of indole NH and amide NH protons were found to be −14.1 ppb K−1 and −5.3 ppb K−1 respectively (Figure 1c,d). On the other hand, the lower temperature coefficient of the amide NH and the presence of amide resonances above 7 ppm in non-hydrogen-bonding solvents (in this case, CDCl3) presumably suggests the presence of intramolecular hydrogen bonds.3,56 It was observed that one βH of the Cp rings showed significant temperature dependence. It appeared at 3.9 ppm at 10 °C and gradually shifted downfield with an increase in temperature, finally appearing at 4.3 ppm at 50 °C. This occurrence is presumably due to the shielding effect, as this βH is in the vicinity of the indole ring at lower temperatures where molecules are intermolecularly associated (vide inf ra in crystallographic analysis). Next, VT-NMR experiments of Fcconjugate 2 showed that the temperature coefficient of the amide NH and alcoholic OH groups were −4.4 ppb K−1 and −2.2 ppb K−1, respectively (Figure 1b,d). The amide NH groups appeared at 7.44 ppm (10 °C) in non-hydrogenbonding solvents (in this case, CDCl3), which most likely arose from the presence of a “Herrick conformation” in solution (Figure 1d). Similarly, Fc conjugates 3 and 4 exhibited a chemical shift higher than 7 ppm and little temperature dependence for their amide protons (Figure 1d and Figure S1 in the Supporting Information [SI]), which may be an indication of “Herrick conformation” in these solutions as well.3,7,25 The variable-concentration 1H NMR (VC-NMR) studies were carried out with Fc-conjugates 1−4 in CDCl3 at room temperature in the concentrations ranging from 10 to 1.25 mM. For Fc-conjugate 1, the indole NH and amide NH protons appeared at 8.44 and 7.67 ppm, respectively, at a concentration of 10 mM (Figure 2). With a decrease in concentration, the peak at 8.44 ppm gradually shifted upfield, and the peak at 7.67 ppm remained almost constant. This may suggest that these protons are involved in inter- and intramolecular hydrogen bonds. However, more studies will be necessary to ensure that the insight provided by Gellman and Kelly conclusions54,55 on amide NH also hold true for the indole NH of Trp and the alcoholic OH of Thr. In case of Fc-conjugate 2, the hydroxyl group showed a change in chemical shift with concentration, presumably indicating intermolecular interaction. To the best of our knowledge, the involvement of Trp and Thr in

intermolecular hydrogen bonding through side-chain functionalities has not been previously explored in detail. Although there are some reports about the conformational analysis of tryptophan and threonine residues containing peptides,58,59 none makes claims about hydrogen bonding through indole NH and side-chain alcoholic protons. These hydrogen-bonding interactions in NMR studies match well with the crystallographic data obtained, where the ferrocenyl CO of one molecule is intermolecularly hydrogen bonded with the indole NH of another molecule at the supramolecular level (vide inf ra). For all Fc-conjugates 1−4, amide protons showed a chemical shift higher than 7.4 ppm, and they were almost independent over a range of concentrations (Figure 2 and Figure S2 in the SI), suggesting the potential formation of “Herrick conformation” in solution. Circular Dichroism (CD) Spectroscopy. CD is useful for the elucidation of conformation and metallocene chirality.3 The CD studies of Fc-conjugates 1−4 in chloroform showed a strong positive band at λ = 475−485 nm (Figure 3a). Bands between 300 and 600 nm are characteristic of metal-centered dd transitions, and bands below 300 nm originate from the chiral amino acids. The strong positive Cotton effect indicates the formation of ferrocene core-based chiral P-helical axial chirality as the result of intramolecular H-bonding interactions.3 The CD spectrum for the enantiomer of Fc-conjugate 1, Fc[CO-DTrp-OMe]2, exhibits a strong negative band around 500 nm, and this CD signal is the mirror image of the CD signal obtained from the Fc-conjugate 1 Fc[CO-L-Trp-OMe]2 (Figure 3b). This indicates the formation of an M-helical chirality of the ferrocene core for Fc[CO−D-Trp-OMe]2. We have also performed CD studies for all hydrolyzed Fc-conjugates 1a− 4a in 1:1 acetonitrile−water, and they showed positive Cotton effect around 480 nm, suggesting the preservation of the Phelical ferrocene core (Figure S3 in the SI). Crystallographic Analysis. Single crystals suitable for Xray analysis were obtained for two Fc-conjugates, 1 and 2, in this study. Fc-conjugate 1 crystallizes in the orthorhombic space group P212121 and was obtained by a slow evaporation of a mixture of toluene and methanol. Slow evaporation of a toluene/dichloromethane solvent mixture was successful for Fcconjugate 2 crystals having monoclinic space group P21. X-ray crystallography of Fc-conjugate 1 revealed the presence of two crystallographically independent molecules that displayed D


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Figure 4. ORTEP diagrams showing hydrogen-bonding interactions of two independent molecules A (a) and B (b) as obtained from single-crystal X-ray diffraction studies of Fc-conjugate 1 (d[N(1A)···O(6A)] = 3.06 Å and d[N(2A)···O(4A)] = 3.02 Å for molecule A). Thermal ellipsoids are depicted at 30% probability. (c) Conformation of Fc core showing dihedral angle (ω) between the two ring-bound substituents. The value of ω is 69° for molecule A, which suggests P-1,2′ “Herrick conformation”. (d) Intermolecular hydrogen bonding interactions (d[N(3A)···O(2A)] = 2.81 Å and d[N(4A)···O(1A)#1] = 2.96 Å for molecule A) at supramolecular level. For clarity, only intra- and intermolecular hydrogen bonds involving the asymmetric unit are shown. H atoms bonded to C atoms are not shown, and the H-bond acceptor O atoms are labeled.

significant differences in dihedral angles, β, thus reflecting differences in conformation (see Table S1 in SI, Scheme 2b). Two different solvent molecules (toluene and methanol) were also found within the asymmetric unit of the structure (Figure 4). The ORTEP diagrams for two crystallographically independent molecules are depicted in Figure 4a,b. Each molecule displays the amino acid substituents in the 1 and 2′ positions and has two intramolecular interstrand hydrogen bonds between the methyl ester carbonyl oxygen of Trp of one strand and the amide NH on the Trp of the other strand (d[N(1A)···O(6A)] = 3.06 Å and d[N(2A)···O(4A)] = 3.02 Å for molecule A). Consequently, a 10-membered H-bonded ring is formed, and this structural element is classified as a β-turn. This overall pattern is known as the “Herrick conformation” (Figure 4a,b and Scheme 2a).3 This compares well with an

earlier report.12b In addition to the intramolecular hydrogen bonds, there are also intermolecular hydrogen-bond interactions at the supramolecular level (Figure 4c). A ferrocenyl CO of one strand is intermolecularly hydrogen bonded with the indole NH of the Trp of the adjacent molecule (d[N(3A)··· O(2A)] = 2.81 Å and d[N(4A)···O(1A)#1] = 2.96 Å for molecule A). The ferrocenyl CO of the other strand is hydrogen bonded with methanol (d[O(1S)···O(1A)] = 2.90 Å) (Figure S4 in SI). A Cp βH of ferrocene interacts with the πsystem of a neighboring fused five-membered ring of an indole system [d(ring centroid and βH) = 2.85, 2.73, and 2.86 Å]. These hydrogen-bonding interactions in the solid state match well with our findings about the hydrogen-bonding patterns of this conjugate in solution from NMR studies (vide supra). The solid-state structure for the enantiomer of Fc-conjugate 1, E


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Figure 5. ORTEP diagram of the asymmetric unit of Fc-conjugate 2. Thermal ellipsoids are depicted at 30% probability. (b) Conformation of Fc core showing dihedral angle (ω) between the two ring-bound substituents. The value of ω is 169.4°, which suggests almost anti conformation. (c) Intermolecular hydrogen-bonding interactions at supramolecular level. For clarity, only intra- and intermolecular hydrogen bonds involving the asymmetric unit are shown. H atoms bonded to C atoms are not shown, and the H-bond acceptor O atoms are labeled.

noted that Fc−conjugate 2 forms “Herrick conformation” in solution, whereas it shows anti conformation in the solid state. It can be hypothesized that packing forces are responsible for a different conformation in the crystal of Fc-conjugate 2, but in solution, the more stable “Herrick conformation” is obtained.7 A summary of crystallographic data for Fc-conjugates 1 and 2 are given in the SI (Table S2). Moreover, a number of structural parameters, such as the tilt and other specific angles are presented in Scheme 2b and Table S1 (in the SI). Electrochemical Studies. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) of Fc-conjugates 1−4 were carried out in acetonitrile and dichloromethane solutions with tetrabutyl ammonium perchlorate (TBAP) as the supporting electrolyte. Cyclic voltammograms of all Fcconjugates (1−4) exhibited reversible one-electron redox processes of ferrocene moiety, as judged by the peak separation (ΔE) between cathodic and anodic peaks, which were in the range of 60−65 mV (Figure 6). All halfwave electrode potentials (E1/2) of Fc-conjugates 1−4 occurred in the range of 722 mV−800 mV in two different solvents. The CV and DPV of Fc-conjugate 1 and 2 are shown in Figure 6a−d and other Fc-conjugates can be found in the SI (Figure S6). The increase of the oxidation potential upon substitution of the Cp

Fc[CO-D-Trp-OMe]2, exhibits features similar to those of Fc[CO-L-Trp-OMe]2 and can be found in the SI (Figure S5). Here, we compare the structural properties of Fc-conjugate 1 with other pseudopeptide, dicarboxylic acid derivatives of amino acids.60,61 A 1,1-cyclopropane dicarboxylic acid derivative of an amino acid (Aib) adopts a folded structure with the formation of an intramolecular hydrogen-bonded nine-membered ring conformation.60 In another study, a malonic acid derivative of valine shows a turn-like molecular conformation with involvement of a six-membered intramolecular hydrogen bond.61 Figure 5 shows the crystal structure of Fc−conjugate 2, which indicates a zigzag hydrogen bonding arrangement and does not contain any intramolecular hydrogen bonds. Interestingly, it displays a ω value equal to 169.4°, which can be termed as anti confomation (Figure 5a and Scheme 2b). One molecule is intermolecularly hydrogen bonded with four adjacently located molecules (Figure 5c). One ferrocenyl CO of one molecule is simultaneously hydrogen bonded with amide NH and alcoholic OH of Thr of another molecule (d[N(2)··· O(1)#2] = 2.89 Å and d[N(1)···O(5)#1] = 2.84 Å for amide NH; d[O(2)···O(5)#1] = 2.79 Å and d[O(6)···O(1)#2] = 2.79 Å for alcoholic OH), resulting in infinite chains. It should be F


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Figure 6. Electrochemical properties of Fc-conjugates 1−4. CVs of (a) Fc-conjugate 1 and (b) Fc-conjugate 2 in acetonitrile () and in dichloromethane (- - -); DPVs of (c) Fc-conjugate 1 and (d) Fc-conjugate 2 in acetonitrile (solid line) and in dichloromethane (- - -). (e) Summary of electrochemical results: half-wave potentials [E1/2, mV], peak separation [ΔEp, mV], diffusion coefficients of reduced and oxidized species [DR and DO, cm2/s] and heterogeneous electron transfer rate constant [ksh, cm/s] of Fc-conjugates 1−4 obtained by corresponding CV/DPV measurements. E1/2 and ΔEp are given with a maximum uncertainty of ±4 mV. The maximum error associated with DR/DO/ksh is 5%. Concentration of Fcconjugates was 0.5 mM with 0.5 M TBAP as a supporting electrolyte. Glassy carbon, Pt wire, and Ag/AgCl were used as working, counter, and reference electrodes, respectively.

reaction is followed by a chemical reaction leading to the formation of an electro-inactive species (Figure S7a, SI).62 It is noted that the redox chemistry of all Fc-conjugates is influenced by the solvent. For all Fc-conjugates, the halfwave potential shows lower values in acetonitrile than in dichloromethane (Figure 6e). This change in E1/2 is related to the different strengths of interaction of each redox state with the solvent molecules.21 E1/2 of different Fc-conjugates exhibits the following order: 1 < 2 < 4 < 3 in both solvents. This suggests

ring by amide groups can be explained by the withdrawal of electron density from the ferrocene moiety by these substituents.7 Interestingly, the CV and DPV of the Fcconjugate 1, having the Trp moiety show two redox processes, one corresponds to the Fc group, and the other at higher potential is related to the indole group. The indolic redox process is irreversible and disappears upon successive scanning due to a probable EC mechanism in which an electron transfer G


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Figure 7. Electrochemical comparisons between ester-protected Fc-conjugates 1-4 () and hydrolyzed Fc-conjugates 1a−4a (- - -) in 1:1 acetonitrile−water. CVs of (a) Fc-conjugates 1/1a and (b) Fc-conjugates 2/2a. (c) Summary of electrochemical results: half-wave potentials [E1/2, mV], peak separation [ΔE, mV], diffusion coefficients of reduced and oxidized species [DR and DO, cm2/s] and heterogeneous electron transfer rate constant [ksh, cm/s] of all Fc-conjugates obtained from their corresponding CV/DPV measurements. E1/2 and ΔEp are given with a maximum uncertainty of ±6 mV. The maximum error associated with DR/DO/ksh is 5%. Concentration of Fc-conjugates was 0.5 mM with 0.5 M LiClO4 as supporting electrolyte. Glassy carbon, Pt wire, and Ag/AgCl were used as working, counter, and reference electrodes, respectively.

The D values of all Fc-conjugates 1−4 are higher in magnitude in acetonitrile than dichloromethane, presumably due to the lower viscosity of acetonitrile compared to dichloromethane.64 The diffusion coefficients of reduced species (DR) are greater in magnitude than those of oxidized species (D O). Using the Nicholson equation k sh = Ψ(πD0αν)1/2,65 comments can be made about the reversible nature of the electron transfer (ET) process. For a fully reversible ET process, the heterogeneous electron transfer rate constant (ksh) value is greater than 0.020 cm/s. The values of ksh of all Fc-conjugates 1−4 were determined, which fall in the range of 2.77−14.02 × 10−2 cm/s, suggesting the Fc-conjugates follow reversible redox behavior (see section 1 of the SI for details). The electrochemical investigations of all Fc-conjugates were carried out in 1:1 acetonitrile−water system to examine the change in redox behavior upon the removal of the ester

that oxidation of the Fc moiety is easiest for Fc-conjugate 1 and the most difficult for conjugate 3. The lower E1/2 of Fcconjugates 1 and 2 can be rationalized by considering the ability of the indole and hydroxyl group to stabilize the ferrocenium moiety in 1 and 2, respectively. The higher electron-donating ability of the indole group results in a more cathodic shift of the redox potential of the Trp derivative 1 compared to that of the Thr derivative 2. The E1/2 values of all Fc-conjugates 1−4 with respect to Fc/Fc+ reference electrodes can be seen in the SI (Table S3). The diffusion coefficients (D) of all Fc-conjugates 1−4 are listed in Figure 6e and were determined from their corresponding CVs using the Randles−Sevcik equation (ipa = 2.69 × 105n3/2ACD1/2ν1/2; where ipa is the anodic peak current, A the surface area of the electrode, C the bulk concentration of the analyte, n number of electrons, D the diffusion coefficient and ν the scan rate).63 H


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Figure 8. pH-dependent cyclic voltammograms: (a) Fc-conjugates 1a, (b) Fc-conjugates 4a in (unbuffered) pH solution (1:1 acetonitrile-pH solution) using 0.5 M NaNO3 supporting electrolyte and 0.1 M HNO3/NaOH was used to alter the pH. Black, red, and green lines represent the pH values 4, 7, and 10, respectively.

Figure 9. (a) Cyclic voltammograms and (b) differential pulse voltammograms of Fc-conjugate 4a (in acetonitrile−water system at 0.5 mM concentrations) before (black line) and after addition of one equivalent of different metal ions: Na+ (red), Ca2+ (green), Ba2+ (blue), and Al3+ (cyan). (c) Summary of electrochemical data: half-wave potentials [E1/2, mV] and peak separation [ΔE, mV] (given to the nearest 5 mV) of Fc-conjugates 2a−4a obtained from CV. LiClO4 (0.5M) was added as supporting electrolyte. Glassy carbon, Pt wire, and Ag/AgCl were used as working, counter, and reference electrodes, respectively.

protecting groups (Figure 7c). The diffusion coefficients of reduced species (DR) are an order of magnitude greater than those of oxidized species (DO). For comparison, we determined the diffusion coefficients of 1,1′-ferrocenedicarboxylate ester (5), 1,1′-ferrocenedicarboxylic acid (5a), and ferrocene (Fc) (Figure 7 and Figure S9 in the SI). The difference between DR and DO may be related to the stabilization of ferrocenium state by the residues attached to ferrocene. The pH-dependent electrochemical studies of all Fcconjugates were performed in the pH range 4−10 (Figure 8, Figures S10, S11 in the SI). The oxidation peak of ferrocene of all hydrolyzed conjugates 1a−4a significantly shifted to lower potential with increase in pH of the medium. This can presumably be related to the deprotonation of the carboxylic acid group at pH > 4. Additionally, the reversible redox response of ferrocene switches to quasi-reversible upon

protecting groups. The results showed a measurable effect on the E1/2, presumably due to the changes in the electronic environment of the overall molecule by the removal of the ester protecting groups from Fc-conjugates 1−4 (Figure 7). The electrochemical properties have been summarized in Figure 7c. The largest change in E1/2 was observed for the threonine conjugate 2. In contrast, a minimum change in E1/2 was observed for Fc-conjugate 1, which has a more hydrophobic tryptophan moiety. Furthermore, it can be mentioned that Fcconjugate 1a also shows an additional irreversible oxidation peak at a potential of 955 mV due to presence of electroactive indole moiety (Figure S7b−d, SI). Electrochemical data for others Fc-conjugates can be found in the SI (Figure S8). The diffusion coefficients (D) and heterogeneous electron transfer rate constant (ksh) of all Fc-conjugates were determined in 1:1 acetonitrile−water system before and after removal of ester I


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Ca]2+ respectively suggesting the formation of a 1:1 complex (Table S4 in SI). Similar mass spectrometric results were obtained for other Fc-conjugates (Table S4 in SI). Tentative models have been proposed for the complexation of different Fc-conjugates with metal ions (Figure S13, SI). CD spectroscopy was used to evaluate if metal coordination would cause any structural changes in the Fc conjugates. Figure 10a shows the CD spectra of the Fc-conjugates in the presence

increasing the pH from acidic to neutral/alkaline conditions. Interestingly, a pH-dependent irreversible oxidation peak of indole was observed for Fc-conjugate 1a (Figure 8a). The pHdependency of the redox event of indole is already established.62 Another oxidation peak of indole appears around 1150 mV at pH 10, indicating a further oxidation of indole.62 Interactions with Metal Ions. The behavior of Fcconjugates 2a−4a in the presence of metal ions was probed by electrochemical techniques. The Fc-conjugates are expected to coordinate metal ions due to the presence of polar functional groups on the two attached amino acids that exist in Herrick conformation. Since metal coordination most likely influences the redox properties of the Fc group, binding can be evaluated by monitoring the halfwave potentials. The interactions of an assortment of mono-, di-, and trivalent metal ions including: Na+, Ca2+, Ba2+, and Al3+ were studied. Figure 9 shows CV plots of conjugate 4a in the presence of one equivalent of the metal ions: Na+, Ca2+, Ba2+, and Al3+. For all Fc-conjugates, cyclic voltammograms showed prominent shifts in oxidation potential (Figure 9a,b and Figure S12 in the SI). DPVs showed almost symmetrical peaks, which suggested that the redox properties of the Fc-conjugates were reversible. Additionally, in DPV, the peak intensity of the Fc-conjugate increased upon metal ion binding. This is presumably due to the increase in overall charge density of the Fc-conjugate upon complexation with metal ions. The cation coordination to an Fc host is expected to result in an anodic shift of the redox potential, making the Fc group more difficult to oxidize. The collective result of this study is shown in Figure 9c. Overall, for all Fc-conjugates, the anodic shift decreases in the order of the charge: Al3+ > Ca2+/Ba2+ > Na+. The change in halfwave potential for all conjugates was the most significant upon aluminum binding, with the most significant change being 27 mV for Fc-conjuate 2a. This may be because aluminum ion has much higher charge density than others. With regard to the divalent interactions, Ca2+ showed stronger binding than Ba2+. This may arise because the calcium ion has higher charge density compared to barium ion. Therefore, the calcium ion’s ability to shift electron density toward itself is much stronger than the barium ion’s. This leads to the observed changes in the redox behavior of this scaffold. Beer and co-workers studied metal ion interactions with 1,1′-ferrocene-bis(methylene aza18-crown-6) ligand, and they interpreted results by developing an “intramolecular through-space electrostatic interaction model”.52 According to this model, the mean potential shift difference (between free ligand and complex) is proportional to the bound cation’s effective charge (Qcation). Our results also show that the halfwave potential shift difference is proportional to the charge density of the cations, which follow the order: Al3+ > Ca2+ > Ba2+ > Na+. With comparison to the Fc-conjugate 3a (Fc-[CO-Asp-OH]2), the Fc-conjugate 4a (Fc-[CO-GluOH]2) showed larger shifts in halfwave potential upon binding to Ca2+. The presence of an additional methylene group in the side chains of Glu might make it more flexible than Asp, thus optimizing coordination and maximizing the potential for the metal cations to withdraw electron density from the scaffold. ESI-TOF mass spectrometric studies were carried out to determine the stoichiometry of the complex formation by Fcconjugates with metal ions. Even in excess amounts of metal ions, only the 1:1 species were observed. A typical mass spectrum of the solution of Fc conjugate 2 with Ca2+ displays a peak at m/z = 515.0361 and 258.0219 with theoretical values of 515.168 and 258.084, assigned to [M + Ca − H]+ and [M +

Figure 10. (a) CD spectra of Fc-conjugate 2a (in 1:1 acetonitrile− water at 2 mM concentrations) before (black line) and after addition of one equivalent of different metal ions: Na+ (red), Ca2+ (green), Ba2+ (blue), and Al3+ (cyan). (b) 1H NMR spectra of Fc-conjugate 2a with the continuous variation of Al3+ for Job plot. Total concentration of ([2a] + [Al3+]) for each sample is 5 mM in DMSO-D6.

of different metal ions. For all Fc-conjugates, the addition of Na+, Ca2+, and Ba2+ ions did not cause any significant changes in the CD spectrum. In contrast, the addition of Al3+ ions caused significant changes in the CD spectrum for Fc-conjugate 2a. This suggests changes in the structure of the Fc core and a change from a P- to M-helical arrangement, which is evident from the observed negative Cotton effect at λ ≈ 500 nm. The strong binding of Al3+ to the Fc-conjugate 2a was further investigated through 1H NMR titration experiments (Figure 10b). A significant chemical shift of βH of Thr presumably indicates the binding site is close to βH and a Job plot (Table S5 and Figure S14 in the SI) shows that the stoichiometry of the complex is 2:1 (2a:Al3+).

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CONCLUSION In this study, we report the synthesis, characterization, conformational and electrochemical analysis of functional side J


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(30 mL) was added to the reaction mixture. The organic layer was washed with saturated aqueous sodium carbonate (2 × 30 mL), 10% citric acid solution (2 × 30 mL), and brine (2 × 30 mL), respectively. The washed organic solution was dried over anhydrous sodium sulfate and evaporated in vacuum. Then the crude product was purified by flash chromatography (SiO2, 230−400 mesh) using DCM:MeOH gradient. Usually, at the beginning, the eluent was DCM only, and the MeOH content of the eluent was increased gradually, depending on the separation. Isolated yields remain 40−60% for Fc-conjugates 1−4. The Rf values can be found in the Table S6 in the SI. Fcconjugates 1a−4a were obtained by hydrolysis of methyl esters of corresponding Fc-conjugates 1−4. Procedures for the Synthesis of Fc-Conjugates 1a−4a by the Hydrolysis of Fc-Conjugates 1−4. Hydrolysis of FcConjugates 1. One mmol (674 mg) of Fc-conjugate 1 was dissolved in 20 mL MeOH. Two mL of 2 (M) NaOH was added into the reaction mixture and was stirred for a day. Then, methanol was removed under vacuum, and the residue was taken in 20 mL of water and washed with diethyl ether. The pH of the aqueous layer was then adjusted to 2−3 using HCl; the aqueous layer was extracted with ethyl acetate. The extracts were pooled, dried over anhydrous sodium sulfate, and evaporated in vacuum. Then the product was purified by flash chromatography using DCM:MeOH gradient. Yield 80% (517 mg). Hydrolysis of Fc-Conjugates 2. One mmol (504 mg) of Fcconjugate 2 was dissolved in 20 mL acetone and it was stirring. Four mmol of NaOH was dissolved in 5 mL of a 4:1 MeOH:H2O mixture. This mixture was added into the stirring solution of Fc-conjugates 2 and reaction mixture was allowed to stir for 2 days at room temperature. After this time, 2 mL water was added, and the acetone was removed under reduced pressure. Then, the aqueous layer was washed twice with DCM. The remaining aqueous layer was acidified over ice with HCl to a pH 2−3. It was dried, and product purified was purified by flash chromatography using DCM:MeOH gradient. Yield 50% (238 mg). Hydrolysis of Fc-Conjugates 3/4. Thirty-two mmol (8 equiv per ester protecting group) of NaOH was dissolved in 16 mL of a 1:1 MeOH:H2O mixture. While this mixture was stirring, 1 mmol (560 mg/588 mg) of Fc-conjugates 3/4 was added. The resulting reaction mixture was covered from light and allowed to stir for 2 days at room temperature. After this time, the reaction mixture was washed twice with DCM and ethyl acetate. The aqueous layer was collected, and the MeOH from this aqueous layer was removed under reduced pressure. The remaining aqueous layer was acidified over ice with HCl to a pH 2−3. Following acidification, the aqueous layer was extracted several times with ethyl acetate. The combined ethyl acetate was collectively dried over sodium sulfate and evaporated in vacuum. Yield 70% (353 mg) and 50% (266 mg) for Fc-conjugates 3 and 4, respectively. All the compounds were fully characterized by ESI-TOF mass spectrometry, 1D proton, 1D carbon, 2D COSY, and 2D gHMBCAD NMR spectroscopy. Methanol was used as solvent for all ESI-TOF mass spectrometric studies. Characterization of Synthetic Fc-Conjugates 1−4. Fcconjugates 1-Fc[CO-L-Trp-OMe]2: 1H NMR (500 MHz, CDCl3, 10 mM, 22 °C): δ 8.472 (d, 2H, J = 5; indole NH of Trp), δ 7.715−7.680 (m, 4H; H of fused 6-membered ring of indole-H8, see spectra with chemical structure in the SI and amide NH of Trp), δ 7.442−7.426 (d, 2H; H of fused 6-

chain-containing amino acid conjugates of 1,1′-ferrocenedicarboxylic acid. NMR spectroscopy and CD studies in solution demonstrate the presence of 1,2′-“Herrick conformation” comprising intramolecular interstrand hydrogen bonds for all Fc-conjugates. Interestingly, Fc-conjugate 1 crystallizes into two crystallographically independent molecules which differ significantly in dihedral angles (β); however, they both exist in “Herrick conformation” and connect to one another by intermolecular hydrogen bonds between ferrocenyl CO and the indole NH of the Trp. In contrast, the crystal structure of Fc-conjugate 2 indicates the existence of anti conformation since it lacks intramolecular hydrogen bonds. Rather, a zigzag hydrogen-bonding arrangement arises due to the intermolecular hydrogen bonding between each conjugate and four adjacently located molecules. More precisely, the ferrocenyl CO of one molecule is simultaneously hydrogen bonded with the amide NH and alcoholic OH of a Thr on a neighboring molecule, resulting in infinite chains. The “open/ Xu conformation” is usually observed for proline derivatives due to the absence of amide functionality; however, our finding of anti conformation is unusual for Fc-conjugate 2 containing amide groups. It should be noted that Fc-conjugate 2 forms “Herrick conformation” in solution, whereas it shows anti conformation in the solid state. Packing forces are probably responsible for a different conformation in the solid state of Fcconjugate 2, but in solution the more stable “Herrick conformation” is obtained. The study of all Fc-conjugates through CV and DPV was a quintessential part of this study. The half-wave potential considerably varied among different Fc-conjugates, suggesting that the side-chain functionalities of corresponding amino acids significantly influence electrode potentials. The half-wave potentials of different Fc-conjugates also showed dependency on the solvent, removal of the ester group, and pH of the medium. Interestingly, Fc-conjugate 1 exhibited an additional irreversible oxidation peak at higher potential which is related to the indole group. The indolic redox process is irreversible and disappears upon successive scanning due to a probable EC mechanism in which an electron transfer reaction is followed by a chemical reaction leading to the formation of an electroinactive species. Finally, metal ion interactions were studied with hydrolyzed Fc-conjugates 2a−4a using the electrochemical properties of ferrocene. It was found that electrochemical behavior was influenced by the charge density of the binding metal ion, with Al3+ showing the most striking change, particularly for Fc-conjugate 2a. Further investigation into Al3+ binding to Fc-conjugate 2a via CD suggested that Al3+ ions cause changes in the conformation of the ferrocene.

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EXPERIMENTAL SECTION General Synthesis of Ferrocene Bioconjugates. 1,1′Ferrocenedicarboxylic acid was synthesized according to a modified literature procedure.20 Amino acid derivatives of 1,1′ferrocenedicarboxylic acid were synthesized by using the standard EDC/HOBt method. Fc[COOH]2 (2 mmol) was first dissolved in dry CH2Cl2 (70 mL) with stirring, with HOBt (8 mmol) and EDC (8 mmol) being added consecutively into the reaction mixture. The reaction mixture was stirred for 15 min followed by the addition of a methyl-ester protected amino acid (H-AA-OMe; AA stands for amino acid), which was isolated from 8 mmol of the corresponding methyl ester hydrochloride by neutralization with TEA. The reaction was stirred overnight at room temperature. After this period, DCM K


Organometallics

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bending of amide II); mp 189 °C; Elemental analysis calcd (%) for C24H28FeN2O10: C 51.44, H 5.04, N 5.00; found C 51.39, H 5.02, N 4.98. Fc-conjugates 4−Fc[CO-L-Glu-(OMe)2]2: 1H NMR (500 MHz, CDCl3, 10 mM, 22 °C): δ 7.80 (d, 2H, J = 8.5; NH), δ 7.29 (s; CDCl3 solvent residual peak), δ 4.93−4.92 (2H; aromatic αH of ferrocene), δ 4.88−4.84 (2H; α CH of Glu), δ 4.77−4.76 (2H; aromatic αH of ferrocene), δ 4.59−4.58 (2H; aromatic βH of ferrocene), δ 4.40−4.39 (2H; aromatic βH of ferrocene), δ 3.84 (s, 6H; OCH3), δ 3.69 (s, 6H; OCH3), δ 2.57−2.45 (m, 4H; γ CH2 of Glu), δ 2.30−2.24 (m, 2H; β CH2 of Glu), 1.95−1.88 (m, 2H; β CH2 of Glu), δ 1.60 (br; HDO present in solvent). 13C NMR (CDCl3, 22 °C): δ 175.5 (2C of COOMe, side chain of Glu), δ 172.9 (2C of COOMe, main chain of Glu), δ 170.5 (2C of CONH), δ 77.2−77.0 (C of CDCl3), δ 75.6 (2C, ipso C of ferrocene), δ 72.0 (2C, α C of ferrocene), δ 71.5 (2C, α C of ferrocene), δ 70.3 (2C, β C of ferrocene), δ 70.2 (2C, β C of ferrocene), 52.9 (2C, α C of Glu), δ 51.8 (2C, C of OCH3; main chain), δ 51.5 (2C, C of OCH3; side chain), δ 30.7 (2C, γ C of Asp), δ 26.2 (2C, β C of Asp). MS: m/z 589.14 [M + H]+, 611.13 [M + Na]+, 627.10 [M + K]+; FTIR (cm−1): 3296 (N−H stretch of amide), 1731 (CO stretch of ester), 1633 (CO stretch of amide I), 1537 (N−H bending of amide II); mp 108 °C; Elemental analysis calcd (%) for C26H32FeN2O10: C 53.07, H 5.48, N 4.76; found C 53.25, H 5.49, N 4.74. Characterization of Fc-conjugates 1a−4a. Fc[CO-L-TrpOH]2: 1H NMR (500 MHz, DMSO-d6, 22 °C) δ (ppm): δ 12.85 (br, 2H; COOH), δ 10.87 (d, J = 2.5; 2H; indole NH of Trp), δ 8.06 (d, J = 8.5; 2H; amide NH of Trp), δ 7.64 (d, J = 7.5; 2H; H of fused 6-membered ring of indole-H8, see spectra with chemical structure in the SI), δ 7.33 (d, J = 8.0; 2H; H of fused 6-membered ring of indole-H5), δ 7.27 (d, J = 2.5; 2H; H of fused 5-member ring of indole-H2), δ 7.06 (t, J = 7.0; 2H; H of fused 6-membered ring of indole-H7), δ 7.00 (t, J = 8.0; 2H; H of fused 6-membered ring of indole-H6), δ 4.69−4.65 (m, 2H; α CH of Trp), 4.64−4.63 (m, 2H; aromatic αH of ferrocene), 4.54−4.53 (m, 2H; aromatic αH of ferrocene), 4.08−4.07 (m, 2H; aromatic βH of ferrocene), 4.05−4.04 (m, 2H; aromatic βH of ferrocene), δ 3.30 (d, 2H; β CH2 of Trp), δ 3.17−3.12 (m, 2H; β CH2 of Trp). 13C NMR (125.653 MHz, DMDO-d6, 22 °C) δ (ppm): δ 174.7 (2C of COOH), δ 169.5 (2C of CONH), δ 136.6 (2C, aromatic indole ring-C9), δ 127.4 (2C, aromatic indole ring-C4), δ 124.2 (2C, aromatic indole ring-C2), δ 121.4 (2C, aromatic indole ring-C6), δ 118.9 (2C, aromatic indole ring-C7), δ 118.5 (2C, aromatic indole ring-C5), δ 111.9 (2C, aromatic indole ring-C8), δ 110.8 (2C, aromatic indole ring-C3), δ 77.1 (2C, ipso C of ferrocene), δ 72.3 (2C, α C of ferrocene), δ 71.3 (2C, α C of ferrocene), δ 71.1 (2C, β C of ferrocene), δ 69.2 (2C, β C of ferrocene), δ 53.6 (2C, α C of Trp), δ 40.5−40.1 (C of DMSO-d6), δ 27.0 (2C, C of β CH2). ESI-MS: m/z 669.1440 [M + Na]+. FTIR (cm−1): 3327 (N−H stretch of amide), 1712 (CO stretch of COOH), 1600 (CO stretch of amide I), 1538 (N−H bending of amide II); mp 173 °C; Elemental analysis calcd (%) for C34H30FeN4O6: C 63.17, H 4.68, N 8.67; found C 62.99, H 4.70, N 8.64. Fc[CO-L-Thr-OH]2: 1H NMR (500 MHz, DMSO-d6, 22 °C) δ (ppm): δ 8.26 (br, 2H; NH), δ 5.03 (s, 2H; aromatic αH of ferrocene), δ 4.78 (s, 2H; aromatic αH of ferrocene), δ 4.42− 4.38 (m, 4H; aromatic βH of ferrocene and α CH of Thr), δ 4.29 (s, 2H; aromatic βH of ferrocene), δ 4.01−3.99 (m, 2H; β CH of Thr), δ 3.34 (br; HDO for solvent), δ 2.51 (br; solvent),

membered ring of indole-H5), δ 7.289−7.250 (d, 2H; H of fused 5-member ring of indole-H2), δ 7.251−7.212 (m, 2H; H of fused 6-membered ring of indole-H7), δ 7.198−7.161 (m, 2H; H of fused 6-membered ring of indole-H7), δ 5.089 (br, 2H; α CH of Trp), 4.689−4.679 (m, 2H; aromatic αH of ferrocene), 4.638 (2H; aromatic αH of ferrocene), 4.187−4.173 (m, 2H; aromatic βH of ferrocene), 4.157−4.143 (m, 2H; aromatic βH of ferrocene), δ 3.832 (s, 6H; OCH3), δ 3.490− 3.457 (m, 2H; β CH2 of Trp), δ 3.199−3.150 (m, 2H; β CH2 of Trp). 13C NMR (CDCl3, 22 °C): δ 175.6 (2C of COOMe), δ 170.6 (2C of CONH), δ 136.3 (2C, aromatic indole ring-C9), δ 127.0 (2C, aromatic indole ring-C4), δ 122.6(2C, aromatic indole ring-C2), δ 122.2 (2C, aromatic indole ring-C6), δ 119.6 (2C, aromatic indole ring-C7), δ 118.2 (2C, aromatic indole ring-C5), δ 111.4 (2C, aromatic indole ring-C8), δ 110.9 (2C, aromatic indole ring-C3), δ 77.5−76.7 (C of CDCl3), δ 75.1 (C, ipso C of ferrocene), δ 74.1 (C, ipso C of ferrocene), δ 71.8 (2C, α C of ferrocene), δ 71.3 (2C, α C of ferrocene), δ 70.1 (2C, β C of ferrocene), δ 70.0 (2C, β C of ferrocene), δ 53.4 (2C, α C of Trp), δ 52.7 (2C, C of OCH3), δ 26.9 (2C, C of β CH2). MS: m/z 675.18 [M + H]+, 697.17 [M + Na]+, 713.14 [M+K]+; FTIR (cm−1): 3239 (N−H stretch of amide), 1723 (CO stretch of ester), 1628 (CO stretch of amide I), 1538 (N−H bending of amide II); mp 125 °C; Elemental analysis calcd (%) for C36H34FeN4O6: C 64.10, H 5.08, N 8.31; found C 63.98, H 5.10, N 8.30. Fc-conjugates 2−Fc[CO-L-Thr-OMe]2: 1H NMR (500 MHz, CDCl3, 10 mM, 22 °C): δ 7.40 (d, J = 9, 2H; NH), δ 7.29 (s; CDCl3; solvent), δ 4.93−4.91 (m, 4H; aromatic αH of ferrocene), δ 4.89−4.87 (dd, 2H; α CH of Thr), δ 4.57−4.56 (m, 2H; aromatic βH of ferrocene), δ 4.49−4.48 (m, 2H; aromatic βH of ferrocene), δ 4.47−4.45 (m, 2H; β CH of Thr), δ 3.87 (s, 6H; OCH3), δ 2.69 (br, 2H; β OH of Thr), δ 1.63 (br; HDO from solvent), δ 1.33 (d, J=6; 6H; γCH3 of Thr). 13C NMR (CDCl3, 22 °C): δ 173.7 (2C of COOMe), δ 170.9 (2C of CONH), δ 77.2−76.1(C of CDCl3), δ 75.9 (2C, ipso C of ferrocene), δ 71.8 (4C, α C of ferrocene), δ 70.7 (2C, β C of ferrocene), δ 70.6 (2C, β C of ferrocene), δ 67.9 (2C, β C of Thr), δ 58.0 (2C, α C of Thr), δ 53.1 (2C, C of OCH3), δ 20.5 (2C, γ C of Thr). MS: m/z 505.1297 [M + H]+, 527.1101 [M + Na]+. FTIR (cm−1): 3403 (O−H stretch), 3295 (N−H stretch of amide), 1741 (CO stretch of ester), 1609 (CO stretch of amide I), 1542 (N−H bending of amide II); mp 184 °C; Elemental analysis calcd (%) for C22H28FeN2O8: C 52.40, H 5.60, N 5.55; found C 52.25, H 5.59, N 5.58. Fc-conjugates 3−Fc[CO-L-Asp-(OMe)2]2: 1H NMR (500 MHz, CDCl3, 10 mM, 22 °C): δ 7.58 (d, 2H, J = 8.5; NH), δ 7.29 (s; CDCl3 solvent residual peak), δ 5.24−5.20 (m, 2H; α CH of Asp), δ 4.87−4.84 (m, 4H; aromatic αH of ferrocene), δ 4.54−4.53 (2H; aromatic βH of ferrocene), δ 4.44−4.43 (2H; aromatic βH of ferrocene), δ 3.86 (s, 6H; OCH3 of α ester), δ 3.73 (s, 6H; OCH3 of side chain β ester), δ 3.06−2.95 (m, 4H; β CH2 of Asp), δ 1.62(br; HDO present in solvent). 13C NMR (CDCl3, 22 °C): δ 172.9 (2C of main chain COOMe), δ 171.0 (2C of side chain COOMe and 2C of CONH), δ 77.2−77.0 (C of CDCl3), δ 75.9 (2C, ipso C of ferrocene), δ 71.9 (2C, α C of ferrocene), δ 71.8 (2C, α C of ferrocene), δ 70.5 (2C, β C of ferrocene), δ 70.4 (2C, β C of ferrocene), 52.9 (2C, C of OCH3, main chain of Asp), δ 52.0 (2C, C of OCH3, side chain of Asp), δ 48.7 (2C, α C of Asp), δ 36.0 (2C, β C of Asp). MS: m/z 561.11 [M + H]+, 583.10 [M + Na]+, 599.07 [M + K]+; FTIR (cm−1): 3298 (N−H stretch of amide), 1736 (CO stretch of ester), 1634 (CO stretch of amide I), 1536 (N−H L


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δ 1.06 (d, J = 6.2; 6H; γCH3 of Thr). MS: m/z 499.0791 [M + Na]+. FTIR (cm−1): 3325 (N−H stretch of amide), 1715 (C O stretch of COOH), 1601 (CO stretch of amide I), 1525 (N−H bending of amide II). Fc[CO-L-Asp-(OH)2]2: 1H NMR (500 MHz, DMSO-d6, 22 °C) δ (ppm): δ 12.55 (br, 4H; COOH), δ 8.11 (d, J = 8, 2H; NH), δ δ 4.84−4.82 (m, 4H; aromatic αH of ferrocene), δ 4.71−4.65 (m, 2H; α CH of Asp), δ 4.40−4.35 (4H; aromatic βH of ferrocene), 3.35 (br; HDO for solvent), δ 2.87−2.81 (m, 2H; β CH2 of Asp), δ 2.72−2.67 (m, 2H; β CH2 of Asp), δ 2.51 (solvent). 13C NMR (125.653 MHz, DMDO-d6, 22 °C) δ (ppm): δ 173.3 (2C of main chain COOH), δ 172.4 (2C of side chain COOH), δ 168.9 (2C of CONH), δ 77.0 (2C, ipso C of ferrocene), δ 72.7 (4C, α C of ferrocene), δ 70.1 (4C, β C of ferrocene), δ 49.4 (2C, α C of Asp), δ 40.5−40.1 (C of DMSO-d6), δ 36.4 (2C, β C of Asp). MS: m/z 527.0358 [M + Na]+. FTIR (cm−1): 3298 (N−H stretch of amide), 1703 (C O stretch of COOH), 1602 (CO stretch of amide I), 1538 (N−H bending of amide II); mp 141 °C; Elemental analysis calcd (%) for C20H20FeN2O10: C 47.64, H 4.00, N 5.56; found C 47.45, H 4.02, N 5.53. Fc[CO-L-Glu-(OH)2]2: 1H NMR (500 MHz, DMSO-d6, 22 °C) δ (ppm): δ 12.42 (br, 4H; COOH), δ 8.0 (d, 2H; NH), δ 4.89 (2H; aromatic αH of ferrocene), δ 4.84 (2H; aromatic αH of ferrocene), δ 4.44−4.43 (2H; aromatic βH of ferrocene), δ 4.39−4.38 (2H; aromatic βH of ferrocene), δ 4.38−4.37 (2H; α CH of Glu), 3.35 (br; HDO for solvent), δ 2.51 (solvent), δ 2.39−2.36 (m, 4H; γ CH2 of Glu), δ 2.12−2.06 (m, 2H; β CH2 of Glu), 1.93−1.87 (m, 2H; β CH2 of Glu. 13C NMR (125.653 MHz, DMDO-d6, 22 °C) δ (ppm): δ 174.2 (2C of side chain COOH), δ 173.2 (2C of main chain COOH), δ 169.5 (2C of CONH), δ 77.1 (2C, ipso C of ferrocene), δ 72.6 (4C, α C of ferrocene), δ 69.7 (4C, β C of ferrocene), 56.2 (2C, α C of Glu), δ 40.5−40.1 (C of DMSO-d6), δ 30.0 (2C, γ C of Asp), δ 27.5 (2C, β C of Asp). MS: m/z 555.0616 [M + Na]+. FTIR (cm−1): 3327 (N−H stretch of amide), 1708 (CO stretch of COOH), 1604 (CO stretch of amide I), 1538 (N−H bending of amide II); Elemental analysis calcd (%) for C22H24FeN2O10: C 49.64, H 4.54, N 5.26; found C 49.43, H 4.56, N 5.24. NMR Study. All synthetic Fc-conjugates were fully characterized using NMR pectroscopy. 1D 1H and 2D COSY spectra were recorded on a Bruker Advanced 500 spectrometer (operating at 500 MHz for 1H). The spectrometer was equipped with a four channel (1H, 13C, 19F, D) direct broad band observed probe. The 1D 13C spectra were acquired at 25 °C on an Agilent DD2 500 spectrometer (Agilent, Walnut Creek, CA) operating at 499.664 MHz for 1H and 125.653 for 13 C. The spectrometer was equipped with an XSens C13sensitive Cold Probe. Two-dimensional gHMBCAD spectra were acquired at 50 °C on an Agilent DD2 spectrometer operating at 699.805 MHz for 1H and 175.981 for C13 (Agilent, Walnut Creek, CA). The spectrometer was equipped with a 5 mm HFCN Cold Probe. CDCl3 and DMSO-d6 were used as NMR solvents for Fcconjugates 1−4 and Fc-conjugates 1a−4a respectively. Variable-temperature 1H NMR (VT-NMR) studies were used to investigate hydrogen-bonding interactions for Fc-conjugates 1−4 by dissolving them in CDCl3 at concentrations of 10 mM in the temperature range of 10−50 °C. Variable-concentration 1 H NMR (VC-NMR) measurements were performed to evaluate the conformation of ferrocene and to investigate the involvement of hydrogen bonding in Fc-conjugates 1−4, which

were in CDCl3 at room temperature and in the concentrations range of 10−1.25 mM. For characterization of Fc-conjugates, all 13 C NMR studies were performed at a concentration of 10 mM. Circular Dichroism (CD) Study. CD spectroscopy was used for determining the conformation of all Fc-bioconjugates and interaction of metal ions. CD spectra were recorded between 200 and 600 nm by a JASCO J-810 spectrometer using a 1 mm path length cell at room temperature. Experiments were carried out in chloroform at concentrations of 1.25 mM for Fc-conjugates 1−4 and in acetonitrile−water mixture (1:1) at concentrations of 2 mM for Fc-conjugates 1a−4a. Crystallographic Analysis. Crystallographic data were collected on a Bruker Kappa APEX-DUO diffractometer using a Copper ImuS tube with multilayer optics or monochromated (Triumph) Mo Kα radiation which were measured using a combination of ϕ scans and ω scans. The data were processed using APEX2 and SAINT (Bruker, 2007). Absorption corrections were carried out using SADABS (Bruker, 2007). The structure was solved and refined using SHELXTL (Sheldrick, 2008) for full-matrix least-squares refinement that was based on F2. All H atoms were included in calculated positions and allowed to refine in riding-motion approximation with U ∼ iso ∼ tied to the carrier atom. CCDC1009307 (Fc-conjugate 1Fc[CO-L-Trp-OMe]2), CCDC1009308 (Fc[CO-D-Trp-OMe]2) and CCDC-1009309 (Fcconjugate 2Fc[CO-L-Thr-OMe]2) contain supplementary crystallographic data for this paper. Electrochemical Studies. The electrochemical experiments were carried out at room temperature using a CHI660B electrochemical workstation (CH instruments Inc.). All electrochemical experiments described in this study make use of a glassy carbon working electrode (diameter 3 mm), Pt wire as counter electrode, and Ag/AgCl (saturated with 3 M KCl) reference electrode. The glassy carbon electrode was polished with 0.05 μm Al2O3, sonicated successively in Milli-Q water, ethanol, and Milli-Q water again to fully remove any absorbed Al2O3. The electrodes were thoroughly rinsed in Milli-Q water after each sonication. For all electrochemical cyclic voltammetric experiments, 100 mV/s scan rate was used. The experimental conditions for DPV were amplitude of 50 mV, incremental potential of 4 mV and pulse period of 0.5 s. For Fc conjugates 1−4, a 0.5 mM acetonitrile (or dichloromethane) solution containing 0.5 M tetrabutylammonium perchlorate (TBAP) was used for cyclic voltammetry (CV) and differential pulse voltammetry (DPV). For Fc conjugates 1a−4a, solutions of CH3CN−H2O (1:1 mixture) at a concentration of 0.5 mM were used that contains 0.5 M LiClO4 as supporting electrolyte. A mixture of CH3CN−H2O was used because all of the Fc conjugates 1a−4a are neither soluble in absolute water nor in absolute acetonitrile. E1/2 values are given to the nearest ±2, ±4, ±2, and ±3 mV for Fcconjugates 1−4, respectively. E1/2 values are given to the nearest ±6, ±4, ±3, and ±3 mV for Fc-conjugates 1a−4a, respectively. The oxidation peak of indole is provided with an uncertainty of ±6 for Fc-conjugates 2/2a. The procedure for the determination of error can be found in the SI (Figure S15). The pH-dependent electrochemical studies were performed in both buffered and unbuffered pH solution. In this study, phosphate buffer solutions in the pH range 4−10 were used which also acted as a supporting electrolyte. In case of unbuffered pH solution, 0.5 M NaNO3 was used as a supporting electrolyte and 0.1 M HNO3 and 0.1 M NaOH were used to control the pH of the medium. The metal M


Organometallics

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perchlorate salts were added in 1 equiv portions to the analytic solution.

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ASSOCIATED CONTENT

S Supporting Information *

Additional figures and tables, characterization, and spectra of synthetic Fc-bioconjugates. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by NSERC, Higher Education Commission of Pakistan and the University of Toronto Scarborough. We would like to thank Tony Adamo and Darcy Burns for their assistance in data collection.

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REFERENCES

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