Structure-Dependent Complexation of Fe3+ by Anthracyclines. 1. The

May 22, 2013 - ABSTRACT: We have investigated the stability of doxor- ubicin and daunorubicin complexes with Fe3+ in aqueous solution. Doxorubicin and...
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Structure-Dependent Complexation of Fe3+ by Anthracyclines. 1. The Importance of Pendent Hydroxyl Functionality Krzysztof Nawara,†,‡ John L. McCracken,‡ Paweł Krysiński,† and G. J. Blanchard*,‡ †

Department of Chemistry, University of Warsaw, Pasteura 1, Warsaw 02-093, Poland Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, United States

‡

ABSTRACT: We have investigated the stability of doxorubicin and daunorubicin complexes with Fe3+ in aqueous solution. Doxorubicin and daunorubicin are anthracycline chemotherapeutic agents that differ structurally by the presence of a hydroxymethylketone functionality in doxorubicin versus a methyl ketone moiety in daunorubicin. We demonstrate that the presence of the hydroxyl group in doxorubicin enhances its 1:1 complexation with Fe3+ relative to that seen for daunorubicin. We utilize UV−visible absorbance, circular dichroism, fluorescence and EPR spectroscopies, molecular dynamics, and semiempirical calculations to understand how the presence of an additional hydroxyl group affects the interactions of anthracyclines with Fe3+. Our data indicate that the binding mode of iron in the complex is different for doxorubicin and daunorubicin.

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dynamics, and semiempirical calculations, we find different binding modes for Fe3+:doxorubicin and Fe3+:daunorubicin complexes. The difference in the chemical structures of these complexes underscores the importance of chemical functionalities that are not central to the chromophore in determining molecular interactions.

INTRODUCTION The anthracyclines doxorubicin (adriamycin) and daunorubicin (cerubidine) are potent anticancer agents. These two compounds are structurally similar, differing only in the presence of one hydroxyl group (Figure 1). Both compounds are known to produce severe side effects, including myelosuppression, neutropenia, leukopenia, hand−foot syndrome, and drug-induced heart failure.1 It is well established that doxorubicin is twice as cardiotoxic as daunorubicin.2,3 At the present time, the toxicity of anthracyclines is believed to be caused by two factors: the formation of reactive oxygen species (ROS)4,5 and the disturbance of iron homeostasis.6,7 There are several known routes for the anthracycline-mediated formation of ROS. It was found initially that doxorubicin could be reduced enzymatically to the corresponding semiquinone, with that species reoxidizing spontaneously to form doxorubicin and hydrogen peroxide.5,8 We have found recently that a similar reaction sequence proceeds in the absence of enzyme, utilizing UV light to effect the photoreduction of doxorubicin,9 and the mechanistic details of this process are understood.10 Anthracyclines are also known to disturb iron homeostasis in the cell. This process does not generate ROS but is strictly related to the cellular iron pool and iron chelation by anthracyclines and may lead to apoptosis.7 The aim of this work is to investigate how specific structural features of anthracyclines affect their propensity for complex formation with iron(III). In this paper we investigate the effect of the pendent hydroxyl group on complexation. The companion paper discusses effects of methoxy and daunosamine functionalities.11 We examine differences in the kinetics behavior of these complexes. Using UV−visible absorbance, circular dichroism, fluorescence and EPR spectroscopies, molecular © XXXX American Chemical Society

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MATERIALS AND METHODS Chemicals. Doxorubicin hydrochloride (>99% purity) and daunorubicin hydrochloride (>99% purity) were purchased from Selleck Chemicals and were used without further purification. Ferrous sulfate heptahydrate was purchased from J. T. Baker Chemicals. HEPES buffer (>99.5% purity) and sodium hydroxide were obtained from Sigma-Aldrich. Sodium acetate (>99% purity) was purchased from Columbus Chemical Industry Inc. Hydrogen peroxide (15% aqueous solution) was obtained from Rocky Mountain Reagents. All aqueous solutions were prepared with Milli-Q water. The concentrations of doxorubicin and daunorubicin were determined spectrophotometrically using ε480 = 1.15 × 104 M−1 cm−1.12 The concentration of freshly prepared ferrous sulfate solution was determined indirectly after oxidation with hydrogen peroxide. The absorbance of Fe3+ was measured at 340 nm.13 Absorbance Measurements. Absorbance measurements were performed using a Cary model 300 double beam UV− visible absorption spectrometer with a spectral resolution of 1 nm. The full spectrum kinetics measurements (350−700 nm) were collected every 55 s. The solutions were made with 1:1 Received: March 7, 2013 Revised: May 20, 2013

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Figure 1. Structures of (a) doxorubicin and (b) daunorubicin.

detected simultaneously using two microchannel plate photomultiplier tube detectors (MCP-PMT, Hamamatsu R3809U50), each mounted on a subtractive double monochromator (Spectral Products CM-112). The detection electronics (Becker & Hickl SPC-132) resolve the parallel and perpendicular polarized transients separately yielding ∼30 ps response functions for each detection channel. Data acquisition, detector bias, and collection wavelength are all controlled using an in-house written LabVIEW (National Instruments) program on a PC. 100 μM solutions of doxorubicin and daunorubicin were measured in HEPES buffer. The light intensity was controlled by neutral density filters to avoid anthracycline photodegradation.10 Electron Paramagnetic Resonance Measurements. EPR spectra were collected using a Bruker ESP-300E spectrometer operating at X-band. The spectrometer was equipped with a 4102ST cavity and an Oxford ESR-900 liquid helium cryostat for these measurements. Samples were prepared in the same stoichiometry as for absorbance measurements but with 1 mM anthracycline. The iron− anthracycline complex containing solutions were frozen in N2(l) at specified time intervals after preparation to monitor their kinetics stability. Semiempirical Calculations. Semiempirical calculations of doxorubicin and daunorubicin with Fe3+ were performed using Hyperchem 8.0 with PM3 parametrization. The initial doxorubicin and daunorubicin structures were optimized using molecular mechanics, followed by geometry optimization at the semiempirical level. Then a Fe3+ ion was placed in the vicinity of the anthracycline but at a distance no closer than 4 Å to avoid any biased initial orientation (stoichiometry 1:1). These systems were subsequently subjected to geometry optimization using PM3 parametrization with conjugate gradient minimization. Several distinct initial geometries were evaluated to obtain different stable Fe3+:anthracycline conformers. Molecular Dynamics. Molecular dynamics calculations were performed using the Desmond Molecular Dynamics System (version 2.4., D. E. Shaw Research, New York, NY, 2010) and Maestro-Desmond Interoperability Tools (version 2.4, Schrödinger, New York, NY, 2010).14 The dynamics of the doxorubicin and daunorubicin molecules were studied in a cubic box of total volume ∼245 000 Å3. We employed the TIP3P15 explicit water model and the OPLS2005 all atom force field.16 The system was allowed to relax according to the default procedure. Isothermal−isobaric (NPT) ensemble simulation was carried out for 10 ns with both recording interval and trajectory interval set to 1.2 ps.

iron/anthracycline stoichiometry. Initial concentrations of reactants in a cuvette were 80 μM anthracycline (doxorubicin or daunorubicin), 0.1 M HEPES (pH 7.0), and 80 μM FeSO4. The Fe2+ solution was added to the buffered anthracycline solution and stirred for ∼15 s before kinetics measurements were initiated. Circular Dichroism Measurements. Circular dichroism measurements were performed using a JASCO J-810 spectropolarimeter. All spectra were measured in millidegrees of rotation at a scanning speed of 100 nm/min using a 1 cm path length UV-quartz cell. Solution concentrations were the same as for the absorbance measurements. Fluorescence Measurements. Fluorescence measurements were performed using SPEX Fluorolog 3 fluorescence spectrometer equipped with a 450 W Xe arc lamp and single monochromators for both excitation and emission. Solution concentrations were the same as for absorbance measurements. Kinetics measurements were performed in the front face mode with excitation at 530 nm (1.5 nm bandpass) and emission at 590 nm (2 nm bandpass) showing maximum values for the free anthraquinones. To avoid changes associated with variations in lamp intensity over time, the collected fluorescence intensity signal was divided by the lamp intensity signal that was recorded simultaneously during the measurement. Both signals were collected at 20 s intervals. Time-Correlated Single-Photon-Counting (TCSPC) Measurements. Fluorescence lifetime data were acquired using a time-correlated single-photon-counting (TCPSC) instrument that has been described previously,10 and we recap its salient features here. The source laser is a CW passively mode-locked diode-pumped Nd:YVO4 laser (Spectra Physics Vanguard) that produces 2.5 W average power at 355 nm and at 532 nm, at 80 MHz repetition rate, with 13 ps fwhm pulses at both wavelengths. The Nd:YVO4 laser pumps a cavitydumped dye laser (Coherent 702-2), which operates in the range 430−850 nm producing 5 ps pulses. The repetition rate of the dye laser is adjustable between 80 MHz and 80 kHz through cavity-dumping electronics (Gooch & Housego). The laser output is polarized linearly with a polarization extinction ratio of approximately 100:1. The excitation pulse at 483 nm from the dye laser is divided with one portion directed to a reference photodiode (Becker & Hickl PHD-400-N) and the other portion sent to the sample. Emission at 590 nm is collected using a 40× reflecting microscope objective (Ealing). The collected emission is separated into polarization components parallel (0°) and perpendicular (90°) to the vertically polarized excitation pulse using a polarizing cube beam splitter (Newport, extinction ratio of ≥500:1). The parallel and perpendicular polarized signal components are B

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Figure 2. Contour plots of time-resolved absorption spectra for (a) doxorubicin and (b) daunorubicin. In the panels above the contour plots, horizontal (time) line scans of the data are presented for the 480 nm (free) and 600 nm (complexed) bands as a function of time. The panels to the right of the contour plots are wavelength line scans at selected times after initial mixing.

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and are characterized by the same molar absorptivity.12 In addition, the circular dichroism spectra of these two compounds are likewise identical (vide infra). The fluorescence lifetime of doxorubicin (τfl = 1026 ± 5 ps) and daunorubicin (τfl = 1033 ± 3 ps) are the same to within the experimental uncertainty. These data indicate that there is no observable intramolecular interaction of the doxorubicin pendent hydroxymethyl ketone moiety with the chromophore ring system. We consider next the role of the doxorubicin hydroxymethyl ketone group in iron(III) complexation. A potentially complicating issue associated with formation of the

RESULTS AND DISCUSSION The primary goal of this paper is to understand the consequences of subtle anthracycline structural differences on their complexation with Fe3+. In order to understand the differences between Fe3+:doxorubicin and Fe3+:daunorubicin complexes, we have utilized several spectroscopic and computational techniques. The data acquired reveal a difference in the structures of the two complexes. We start with an examination of the spectroscopic properties of doxorubicin and daunorubicin. These two chromophores exhibit absorption spectra that are indistinguishable (Figure 2) C

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Fe3+:doxorubicin complex is the generation of insoluble ferric hydroxide when Fe3+ salts are introduced directly to solutions at physiological pH.17 To circumvent this issue, we employed a method developed by Myers and co-workers,18 where ferrous (Fe2+) ions are added to the solution. At pH 7.0, Fe2+ is oxidized rapidly to Fe3+,19 which is known to form complexes with doxorubicin and daunorubicin. This method does not prevent the formation of ferric hydroxide but allows for competition between the formation of Fe(OH)3 and the Fe3+:anthracycline complex. To compare the spectroscopic properties of Fe3+:doxorubicin and Fe3+:daunorubicin complexes, the same anthracycline concentration in aqueous HEPES buffer (pH 7.0) was mixed in a 1:1 ratio with an aliquot of ferrous sulfate. The complexes of Fe3+:doxorubicin and Fe3+:daunorubicin have several common properties. A previous report on the Fe3+:doxorubicin complex has shown that the stoichiometry of the complex can vary with the solution stoichiometry and that a stable 1:1 complex will form.18 Absorbance data of both complexes exhibit the disappearance of the free anthracycline band centered at 480 nm and the appearance of a red-shifted band centered around ∼600 nm. This band is associated with the interaction of Fe3+ with the anthracycline, most likely with the quinone and hydroquinone groups on the anthracycline chromophore ring system.20 Examination of the absorbance data for these complexes (Figures 2) shows that the band intensity at ∼600 nm changes in inverse proportion to the 480 nm band. There is an isosbestic point at 520 nm for daunorubicin (Figure 2b), showing that free daunorubicin exists in equilibrium with the iron complex. The spectral profile of the doxorubicin/Fe3+ system appears to also exhibit an isosbestic point, but the Fe3+ concentration dependence of this band does not have the same linear dependence seen for the daunorubicin/Fe3+ system. The Fe3+:doxorubicin complex absorption band is ∼15% lower in intensity than the Fe3+:daunorubicin band, suggesting a difference in the details of metal ion binding. We assert that the difference between these complexes is reflective of the role that the hydroxymethyl ketone group on the 9-position of the doxorubicin “A” ring plays in iron complexation. It is instructive to examine the time evolution of the absorbance spectra of these two complexes. These data are presented in Figures 2, where the contour plots and their line traces show substantially different time-evolution behavior for doxorubicin (Figure 2a) and daunorubicin (Figure 2b). It is important to address the relative stability of these complexes. For doxorubicin (Figure 2a) the maximum at 480 nm, corresponding to the free chromophore, decreases very rapidly upon addition of Fe2+ (which is rapidly oxidized to Fe3+ by O2 solution (vide infra)) and then achieves a plateau. This behavior is accompanied by the transient appearance of a band in the 580−610 nm range, as noted above, and it too reaches a plateau intensity following the initial change. The data for daunorubicin (Figure 2b) exhibit somewhat different spectral behavior. For daunorubicin, the maximum at 480 nm likewise decreases almost instantaneously upon the addition of Fe2+, but following the initial decrease, the signal recovers in intensity, and the ∼600 nm band that is characteristic of the Fe3+ complex decreases in intensity correspondingly. This phenomenon can also be seen in fluorescence kinetics measurements (Figure 3), which monitor time-dependent changes in free anthracycline concentration because iron− anthracycline complexes emit with very low quantum efficiency. For daunorubicin, the kinetics profile shows significant recovery

Figure 3. Emission kinetics data of daunorubicin and doxorubicin, acquired at 590 nm.

of the free chromophore, while doxorubicin exhibits only very small changes in concentration over time following its initial decrease. Daunorubicin forms a complex with Fe3+ that disappears slowly, leading to recovery of free daunorubicin. The Fe3+:doxorubicin complex does not diminish in concentration over the same time period. The fluorescence emission intensity minima for doxorubicin and daunorubicin reach the same level (Figure 3); thus, the increase in absorption band at 600 nm by ∼15% for daunorubicin must be related to different molar absorptivity coefficients for the Fe3+:doxorubicin and Fe3+:daunorubicin complexes. This finding indicates that the details of complexation between Fe3+ and the two anthracyclines must be significantly different. Both iron complexes coexist with free Fe3+ in solution (in vanishingly small steady state concentrations) and with Fe(OH)3. Our time-dependent spectroscopic data (Figures 2 and 3) reflect differences in the kinetics of formation and dissociation for the Fe3+:anthracycline complex and for Fe(OH)3, and the functional form of the absorbance data indicate that the binding constant for the Fe3+:doxorubicin complex is larger than the binding constant of the Fe3+:daunorubicin complex. We show in Figure 4 the expected changes in absorbance bands for the free chromophore and the Fe3+:anthracycline complex as a function of the relative values of the rate constants for the complex and Fe(OH)3. These calculated results are for the Fe3+:anthracycline 1:1 complex because the formation of this complex is a precursor to any other complexes with different stoichiometry.21 It is important to note that the final ratio of Fe3+:anthracycline to Fe(OH)3 depends on the equilibrium constants for each process (Ksp ≈ 2.3 × 10−37 for Fe(OH)3);22 the differences in time-dependent species concentrations (Figures 2 and 3) are determined by the relative magnitudes of the dissociation rate constant for the complex and the formation rate constant for Fe(OH)3. While the time profiles reported in these calculations are not intended to quantitatively reproduce the experimental absorbance (Figures 2) and emission (Figure 3) results, the differences in the functional forms of these profiles underscore the point that our experimental data can be understood in terms of differences in the equilibrium constants for the two Fe3+:anthracycline complexes. On the basis of the observations that both doxorubicin and daunorubicin interact with Fe3+ to form complexes ranging up to 3:1 stoichiometry18,21 and that the equilibrium constants for the complexes with the two anthracyclines are different D

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of this functionality in daunorubicin implies that at least some Fe3+:anthracycline complex formation involves the chromophore ring functionalities. We expect that the involvement of the pendent hydroxyl functionality affects the spectroscopic properties of doxorubicin to a relatively limited extent, but interactions between Fe3+ and the diol/dione moieties will have a more pronounced spectroscopic effect. In an effort to determine directly whether or not the Fe3+:doxorubicin and Fe3+:daunorubicin complexes are structurally distinct, we have acquired circular dichroism (CD) data for the two anthracyclines and for the Fe3+:anthracyclines with 1:1 stoichiometry (Figure 5). The data in Figure 5a for the Fe3+:anthracyclines

Figure 5. (a, top) Circular dichroism spectra of Fe3+:daunorubicin (- - -) and Fe3+:doxorubicin (−). Spectra were acquired for 1:1 Fe3+/ anthracycline stoichiometry. (b, bottom) Circular dichroism spectra of daunorubicin (- - -) and doxorubicin (−) with no Fe3+ present in solution.

demonstrate distinct differences, while the data for the uncomplexed anthracyclines (Figure 5b) show their structures to be essentially the same. The daunorubicin complex possesses a highly chiral band at 650 nm, in contrast to the doxorubicin complex. Additionally, the complexation of Fe3+ by doroxubicin produces substantial changes in the chromophore aromaticity. The CD spectrum of Fe3+:doxorubicin differs substantially from the CD spectrum of doxorubicin (Figure 5), while the Fe3+:daunorubicin CD spectrum is qualitatively similar to the CD spectrum of daunorubicin, with an additional band arising from the chromophore interaction with iron. These findings further support the assertion that the hydroxymethyl ketone functionality must be involved in iron binding. A central issue in this work is the interaction between Fe3+ and the anthracyclines. To this point we have focused on information obtained through examination of the anthracycline. It is also useful to examine the Fe3+. The specific information of importance here is the oxidation state of the iron and its spin state. We have used electron paramagnetic resonance (EPR) to address these issues. EPR spectra of Fe3+ complexes with doxorubicin and daunorubicin, frozen 60 min after mixing, are shown in Figure 6a and Figure 6b, respectively. Both samples show a single resonance at g = 4.3 indicative of high-spin Fe3+

Figure 4. Calculated time evolution of the populations of selected species for two reactions that compete for Fe3+. The calculations are not intended for the extraction of quantitative rate constant data but are for the purpose of demonstrating that such competition gives rise to species distributions consistent with those found in our experimental data. Relative rate constants are indicated in the figure panels.

(Figures 2−4), two pieces of information can be inferred. The first is that the doxorubicin pendent hydroxyl functionality must play some role in determining K e q for the Fe3+:doxorubicin complex, and the second is that the absence E

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reference electrode). As has been reported in the literature,24 within this potential window, upon scanning toward negative potentials, we should observe the redox behavior of the anthracycline (2e−/2H+) process centered at approximately −0.7 V and a small peak, centered at approximately −0.5 V, related to the Fe3+:anthracycline complex formed. The experiments cited were performed at pH 9, but under our experimental conditions at pH 7 (data not shown), we could not observe the electrochemical response in the region of the Fe3+/2+ redox couple to be able to evaluate the anthracyclinedependent differences in either electron transfer kinetics or redox potential. We believe that the positions of doxorubicin and daunorubicin redox signals, being pH dependent, largely overlap the signal from iron(III)−anthracycline complexes, and this is why we could not resolve the signal associated with the complexes that had formed. We note, however, that the reduction of the anthracyclines releases iron from the complex, which can be seen clearly in subsequent cycles. We have utilized computational methods in an attempt to gain some insight into the structural differences of the Fe3+:anthracycline complexes. We have focused both experimentally and computationally on the 1:1 complex for two reasons. Not only has the 1:1 complex been shown to be stable,18 it is also the least likely complex to exhibit potentially complicating steric issues and it must be the initial complex formed for systems with higher anthracycline-to-iron stoichiometry. The calculations we present below first deal with the ability of the doxorubicin hydroxymethyl ketone functionality to orient in a manner that will facilitate interactions with metal ions. To evaluate potential binding modes for the Fe3+:anthracycline complexes, we performed molecular dynamics simulations of doxorubicin and daunorubicin in aqueous solution. The focus of the simulation was the C9 pendent hydroxymethyl ketone functionality (Figure 1). Fluctuations of the torsional angle between the ketone group and the C9hydroxyl group indicate that for doxorubicin and daunorubicin there is substantially free rotation of the pendent ketone moiety. We found no significant difference in behavior of doxorubicin and daunorubicin. Both molecules can adopt syn and anti conformations, while syn geometry is favored energetically (Figure 7a,b). The mobility of the doxorubicin terminal hydroxymethyl ketone group is, in contrast, more restricted (Figure 7c). During 10 ns dynamics, we found only the syn conformation present, reflective of the stabilizing effect of the ketone moiety on the orientation of the α-hydroxyl group. Thus, for doxorubicin, the hydroxymethyl ketone functionality exhibits predominantly one structure, which is capable of bidentate coordination to a metal ion, and this side group exhibits sufficient rotational freedom to facilitate metal ion coordination with the anthracycline ring functionalities (vide infra). For daunorubicin, any metal ion interactions with the C9 ketone functionality must be monodentate. It is important to note that while the molecular dynamics OPLS force field is well parametrized to model the behavior of organic molecules,16 this approach cannot be used to determine anthracycline interactions with ferric ions. We have used semiempirical calculations with PM3 parametrization to examine Fe3+ interactions with anthracyclines. The aim of these calculations is to evaluate the effect of the doxorubicin hydroxymethyl ketone group on Fe3+ binding. We have focused on the 1:1 complex because achieving an understanding of its structure will provide insight into the differences in Fe3+-binding behavior between doxorubicin and

Figure 6. EPR spectra collected in the g = 4 region for solutions of 1 mM FeSO4 in 0.1 M HEPES buffer, pH 7, with (a) 1 mM doxorubicin and (b) 1 mM daunorubicin. The EPR spectra for traces a and b were obtained from solutions that were frozen 60 min after mixing. The spectrum shown in trace c is for a blank solution of FeSO4 in HEPES buffer. The spectra shown as traces d, e, and f are for Fe3+:daunorubicin samples frozen after incubation times of (d) 7 min, (e) 30 min, and (f) 50 min. Conditions common to all six measurements were the following: microwave frequency 9.475 GHz, microwave power 200 μW, field modulation amplitude 1.6 mT, time constant 80 ms, video gain 5 × 104, and sample temperature 5 K. The spectra are displayed on the same vertical scale but offset from one another for display purposes.

in a rhombic coordination environment (E/D = 1/3).23 The corresponding spectrum of a control sample of 1 mM Fe3+ in HEPES buffer (Figure 6c) shows no EPR response, indicating that all of the Fe has precipitated as Fe(OH)3 under the conditions of our measurement. Taken together, these results indicate that the EPR signals shown in Figure 6 can be attribut ed to Fe 3 + :doxorubicin (Figure 6a) and Fe3+:daunorubicin (Figure 6b) complexes. The stability of the Fe3+:daunorubicin complex was also studied by EPR. Spectra for Fe3+:daunorubicin samples frozen 7, 30, and 50 min after mixing are shown in spectra d, e, and f of Figure 6, respectively. While the amplitude of the Fe3+:daunorubicin EPR signal recorded 7 min after mixing (Figure 6d) is about 40% lower than that of the Fe3+:doxorubicin sample (Figure 6a), the daunorubicin complex shows a broader line width. As a result, the integrated intensities of these two signals, indicative of the amount of each complex present, are similar. The time course shows that the strength of the EPR signal from the Fe3+:daunorubicin complex decreases steadily over time, leading to the nearly 5-fold difference in amplitude observed between the two complexes 60 min after mixing. These findings are consistent with the fluorescence data for daunorubicin shown in Figure 3. Given that the Fe3+:doxorubicin and Fe3+:daunorubicin complexes exhibit structural and kinetics differences, evaluating the Fe3+:anthracycline binding electrochemically could provide additional insight. We have attempted to acquire cyclic voltammetry data for the Fe3+:anthracycline complexes within the potential window −0.8 to +0.4 V (vs Ag,AgCl|1 M KClaq F

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Figure 8. Interaction of the Fe3+ ion (green) with C9-chain of (a) doxorubicin and (b) daunorubicin. Interactions of the Fe3+ ion (green) with quinone−hydroquinone anthracycline functionalities: (c) doxorubicin; (d) daunorubicin.

Figure 7. Variation in torsional angles between ring attached hydroxyl group with C9-chain ketone group: (a) daunorubicin; (b) doxorubicin. (c) Variation in torsional angle between terminal hydroxyl group and ketone group (doxorubicin).

the process of forming a stable complex, with Fe3+ interacting with two quinone oxygens, an α-hydroxy terminal group and the ketone of the C9 side group (Figure 8c). This cagelike structure appears to be stabilized by cation−π interactions between iron and the hydroquinone ring system. The energy for this conformer is much lower (E = −7180 kcal/mol), and the torsional angle between the terminal α-hydroxyl and ketone groups remains in the syn conformation, which was also predicted as the most stable geometry from molecular dynamics simulation. This binding mode explains how the pendent hydroxyl group can coordinate with iron and at the same time mediate changes in the conjugated ring system of doxorubicin. This calculated result is consistent with CD data for the

daunorubicin that are responsible for our experimental data. One stable doxorubicin conformer involves Fe3+ being chelated by the C9-ketone oxygen and the two available α-hydroxyl groups (E = −7006 kcal/mol, Figure 8a). The energy of the analogous conformer for daunorubicin is E = −6302 kcal/mol (Figure 8b). The energy for initial geometries was Edoxorubicin = −5994 kcal/mol and Edaunorubicin = −5904 kcal/mol. These were not, however, the most stable conformers. The addition of Fe3+ had a structurally perturbative effect on the anthracyclines. Geometry optimization (energy minimization) calculations performed from an initial state, where Fe3+ is located in the vicinity of the quinone−hydroquinone group (∼4 Å distance), revealed that doxorubicin can alter its geometry significantly in G

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doxorubicin complex (Figure 5b) that show pronounced changes in the spectral region associated with the conjugated ring system. It is not possible for daunorubicin to adopt an analogous stable geometry (Figure 8d) because of the absence of the hydroxymethyl ketone moiety, making the interaction with Fe3+ weaker. Daunorubicin prefers its fully conjugated conformation, where Fe3+ interacts with the hydroquinone group oxygen and the C9 ketone functionality (E = −6244 kcal/mol). These calculations are thus consistent with the difference in stability of the Fe3+:anthracycline complexes which we observe experimentally.

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CONCLUSIONS Doxorubicin and daunorubicin differ structurally only by one hydroxyl group. Our data and calculations indicate that the Fe3+:daunorubicin complex decomposes quickly in water solution (pH 7.0) whereas the Fe3+:doxorubicin complex is stable under the same conditions. This difference in stability results from different coordination of the Fe3+ ion by the two anthracyclines. Circular dichroism data show different chirality for the two Fe3+:anthracyclines as well as substantial changes in the aromatic system upon complexation for doxorubicin. Semiempirical calculations showing a direct effect of the doxorubicin hydroxymethyl ketone group of doxorubicin on the complex structure are consistent with the experimental data. The presence of the additional hydroxyl group in doxorubicin does not alter the spectroscopic properties of the aromatic chromophore system for free anthracyclines, but it does play an important role in the coordination of Fe3+. This structuredependent difference in iron binding may play a role in understanding the disparity in biological properties observed for doxorubicin and daunorubicin.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +011 517 355 9715, extension 224. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Science Foundation Grant CHE 0808677 and the ACS Petroleum Research Fund Grant 52692-ND6. K.N. gratefully acknowledges support of the Foundation of Polish Science MPD Program cofinanced by the European Regional Development Fund. We are grateful to Professor Babak Borhan and Carmin Burrell (Michigan State University) for their assistance with the acquisition of the circular dichroism data.

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REFERENCES

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(24) Cakir, S.; Bicer, E.; Coskun, E.; Cakir, O. Electrochemical Monitoring of the Interaction of Doxorubicin with Nicotinamide and Fe(III) Ions under Aerobic and Anaerobic Conditions. Bioelectrochemistry 2003, 60, 11−19.

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dx.doi.org/10.1021/jp402349e | J. Phys. Chem. B XXXX, XXX, XXX−XXX