J. Phys. Chem. B 2009, 113, 5369–5375
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UV-Vis Spectra of the Anticancer Campothecin Family Drugs in Aqueous Solution: Specific Spectroscopic Signatures Unraveled by a Combined Computational and Experimental Study Nico Sanna,*,† Giovanni Chillemi,† Lorenzo Gontrani,†,‡ Andrea Grandi,† Giordano Mancini,†,§ Silvia Castelli,| Giuseppe Zagotto,⊥ Costantino Zazza,† Vincenzo Barone,*,# and Alessandro Desideri*,| CASPUR, Consortium for Supercomputing in Research, Via dei Tizii 6, 00185 Roma, Italy; Dipartimento di Scienze Chimiche, UniVersita` di Cagliari, Cittadella UniVersitaria di Monserrato, 09042 Monserrato (Cagliari), Italy; Istituto CNR-IMIP sezione di Bari, c/o Dipartimento di Chimica, UniVersita` di Bari, Via Orabona 4, Bari, Italy; Dipartimento di Biologia and Centro di Bioinformatica e Biostatistica, UniVersita` di Roma “Tor Vergata”, Via della Ricerca Scientifica, 00133 Roma, Italy; Dipartimento di Scienze Farmaceutiche, UniVersita` di PadoVa, Via Marzolo 5, 35131 PadoVa, Italy; and Istituto CNR-IPCF, Area di Ricerca, Via Moruzzi 1, 56124 Pisa, Italy, and Scuola Normale Superiore, piazza dei CaValieri 7, 56126 Pisa, Italy ReceiVed: NoVember 6, 2008; ReVised Manuscript ReceiVed: February 20, 2009
The ultraviolet-visible absorption spectrum of camptothecin (CPT) has been been recorded in aqueous solution at pH 5.3, where the equilibrium among the different CPT forms is shifted toward the lactonic one. Timedependent density functional theory (TD-DFT) computations lead to a remarkable reproduction of the experimental spectrum only upon addition of explicit water molecules in interaction with specific moieties of the camptothecin molecule. Molecular dynamics (MD) simulations enforcing boundary periodic conditions for CPT embedded with 865 water molecules, with a force field derived from DFT computations, show that the experimental spectrum is due to the contributions of CPT molecules with different solvation patterns. A similar solvent effect is observed for several CPT derivatives, including the clinically relevant SN-38 and topotecan drugs. The quantitative agreement between TD-DFT/MD computations and experimental data allow us to identify specific spectroscopic signatures diagnostic of the drug environment and to develop procedures that can be used to monitor the drug-DNA/protein interaction. 1. Introduction Camptothecin (CPT), a natural cytotoxic alkaloid isolated from the Camptotheca acuminata,1 and its derivatives have been the subject, in the last 20 years or so, of a huge production of scientific publications and industrial patents2 aimed at identifying their chemical properties and find applications in medicinal chemistry. The interest in CPT is due to the fact that it has as unique target topoisomerase I (Top1), a monomeric enzyme that catalyzes the relaxation of supercoiled DNA.3 Eukaryotic Top1 relaxes DNA superhelical tension by introducing a transient single-strand break in one strand of duplex DNA and forming a covalent phosphotyrosyl bond with the 3′-end of the broken DNA strand. CPT binds reversibly to the covalent DNA-enzyme intermediate and stabilizes the cleavable complex that collides with the progression of the replication fork, producing lethal double strand DNA break and cell death.3 The X-ray structures of Top1 and DNA in ternary complex with CPT or CPT derivatives have been resolved and have permitted us to understand some peculiarities of the Top1 drug interaction.4 However, the fine details of this interaction have not yet been * To whom correspondence should be addressed. E-mail:
[email protected] (N.S.);
[email protected] (A.D.);
[email protected] (V.B.). † CASPUR, Consortium for Supercomputing in Research. ‡ Universita` di Cagliari. § Universita` di Bari. | Universita` di Roma “Tor Vergata”. ⊥ Universita` di Padova. # Istituto CNR-IPCF, and Scuola Normale Superiore.
fully elucidated and a deeper understanding of the spectroscopic features of CPT may provide a useful tool to this aim, especially if specific spectroscopic signatures could be determined for differenttautomericformsand/orsolute/solute-solventinteractions. The UV-vis spectra of drugs of the CPT family have been reported for different solvents and at different concentrations;5,6 however, their interpretation in terms of structural and electronic characteristics is still lacking and requires an integrated approach combining spectroscopic determinations and state of the art quantum mechanical (QM) calculations. Making use of this approach, we have recently interpreted the UV-vis spectrum of topotecan (TPT) in terms of an equilibrium between different forms that is modulated by the microenvironment embedding the drug.6 However, that work still left open the assignment of one of the most intense bands. In order to evaluate environmental (e.g., solvent and/or DNA) effects on the spectroscopic features, classical molecular dynamics (MD) simulations can be performed using a reliable force field built from purposely tailored QM calculations. Next, TDDFT computations can be performed on a representative number of frames in which distant parts of the solvent are replaced by a polarizable continuum. In the past few years, we have made such MD simulations to study the binary Top1-DNA complex and gain information on protein-DNA interactions, on the protein domain concerted motions, on the role of water in protein-DNA recognition and in the catalytic reaction, and on the open state of Top1 in the absence of DNA.7-10 In the present work, we have calculated the absorption spectrum of CPT in
10.1021/jp809801y CCC: $40.75 2009 American Chemical Society Published on Web 03/30/2009
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aqueous solution by means of TD-DFT computations on suitable clusters obtained either by direct energy minimization or extracted by a MD simulation. We show that the most intense absorption bands are sensitive to the solute-solvent interactions and provide a distinctive signature of specific interactions. These findings can lead to the development of detection protocols based on specific spectroscopic signatures, and make it possible to monitor modifications of the chemical environment during the binding of the drug to its molecular target. 2. Materials and Methods 2.1. Experimental Methods. Camptothecin was purchased from Sigma; SN-38 was a kind gift of Professor L. Merlini, University of Milano; Topotecan was a kind gift of Dr. C. Pisano from Sigma Tau; CPT-20-O-methylthiomethyl ether (20-mod CPT) was synthesized as described by Zhao et al.,11 but by a simplified procedure. In detail, camptothecin (0.30 g, 0.86 mmol), DMSO (18 mL), and acetic anhydride (6 mL) were stirred at room temperature for 2 days. The reaction mixture was centrifuged and the liquid phase discarded. The solid residue was washed with water and then with diethyl ether to give the desired pure product, as verified by NMR and mass spectroscopy. For UV-vis absorption spectra the compounds were dissolved in DMSO to form stock solutions and then diluted in sodium acetate buffer 10 mM (pH 5.0) to a final concentration of 5 µM. The UV-vis spectra were recorded with a Cary 5 spectrophotometer. The following extinction coefficients (M-1 cm-1) have been used to calculate the drug concentrations: CPT ε369 ) 22 866; 20-mod CPT ε367 ) 17 380; SN-38 ε378 ) 20 423; TPT ε381 ) 25 580. 2.2. Theoretical Calculations. All computations have been performed with the Gaussian03 package12 using increasing levels of sophistication for both the methods and the basis set. Starting from X-ray crystallographic data4 of the camptothecin ternary complex with the DNA-Top1 system, we extracted the pentacyclic structure of the lactonic form of CPT, which was then fully optimized at the HF/3-21G level both in vacuo and in aqueous solution (see section 1 of Supporting Information for further details), including in the latter case solvent effects by means of the CPCM variant of the polarizable continuum model (PCM).13 Correlation effects were next taken into account via the density functional theory (DFT) using the B3LYP14 hybrid functional with the 6-31G* basis set, and full geometry optimizations were carried out on the in vacuo and solvated geometries of the lactone CPT. The UV-vis spectra of CPT were then calculated performing single-point computations on the structures previously optimized. To this end, we resorted to the TD-DFT approach15 at the B3LYP/6-31G* level, coupled to a PCM treatment taking into account nonequilibrium solvent effects.16,17 Although in principle the TD-DFT approach has inherent problems with extended π systems, the results reported in the following suggest that valence excitations of lactone CPT are well represented by a TD-DFT approach employing hybrid functionals, as already shown on related systems.18 The results in a recent paper by Jaquemin et al.19 inspired us to investigate the performance of the TD-DFT+PCM methods using various DFT functionals, and in the case of the studied molecules, the B3LYP/6-31G* method gave the best results. The MD calculations were carried out with the Amber suite of programs.20 Generation of a force field for the CPT molecule was accomplished by optimizing restricted electrostatic potential (RESP) charges21 for all atoms and using PARM94/GAFF22 atom types with optimized ab initio distances as equilibrium values. A 2.6 ns trajectory in an NVT ensemble was obtained for the drug kept fixed in the optimized ab initio structure, while
Figure 1. (A) UV-vis experimental absorption spectrum of 1.2 µM CPT at pH 5.0 in acetate buffer and in the 225-500 nm region. The three arrows indicate the most intense band maxima at 369, 354, and 334 nm. (B) Molecular structure of the CPT molecule in the lactonic form with standard atom numbering.
sampling only the motion of the surrounding 865 water molecules. After an equilibration time of 1 ns, 160 frames were extracted every 100 ps from the last 1.6 ns of simulation, and treated at the TD-DFT//PCM(B3LYP/6-31G*) level, to calculate the UV-vis spectra of the different CPT + nH2O structures. The radial distribution functions were calculated using the g_rdf program of the GROMACS package; the original functions were plotted as running averages over 10 data points (figures with the nonaveraged data are presented in section 5 of Supporting Information). The spatial distribution functions were calculated using the g_sdf program from the GROMACS package.23 3. Results and Discussion The UV-vis spectrum in acetate buffer of CPT at pH 5.0 is shown in Figure 1A. At this pH the equilibrium of camptothecin and its derivatives is shifted toward the closed E ring lactonic form. Therefore, all the calculations were carried out on this form (Figure 1B), which hereafter will be referred to as CPT. The calculated maxima of the UV-vis spectral bands in the 300-400 nm region are compared in Table 1 with the experimental values. The low-energy π f π* bands involve a limited number of Kohn-Sham orbitals that give rise to two transitions, having a HOMO f LUMO (H f L) and a HOMO-1 f LUMO (H-1 f L) character, respectively (see section 2 in the Supporting Information for further details). The λ(HfL) reproduces quite well the experimental λ1 band, centered at 369 nm, although the computed maximum is marginally shifted with respect to its experimental counterpart. The low-intensity experimental λ3 band, centered at 334 nm, is also quite well reproduced by the λ(H-1fL), but the TD-DFT/PCM spectrum of the isolated CPT never showed the λ2 band, centered at 354 nm. Additional calculations were therefore carried out adding explicit solvent molecules to the sites of the CPT molecule mainly involved in the H f L electronic transition. In particular, the effects of one
Spectroscopic Signature of Camptothecin Family Drugs TABLE 1: Experimental Absorption Band Maxima of CPT in Aqueous Solution at pH 5.0 and Calculated TD-DFT// PCM(B3LYP/6-31G*) Vertical Transitions (in nm) with Oscillator Strength (OS) in Parenthesesa λ1 experimentalb PCM PCM + 1w N/B PCM + 1w O/D PCM + 1w N/B + 1w O/D PCM + 2w O/D PCM + 2w O/E PCM + 1w N/B + 2w O/D PCM + 1w N/B + 2w O/E PCM + 1w N/B + 2w O/D + 2w O/E type of transitionc
369 (s) 367 (0.47) 374 (0.45)
λ2 354 (s) 360 (0.53)
366 (0.50) 353 (0.59) 366 (0.49) 360 (0.54) 372 (0.46)
334 (w) 329 (0.04) 332 (0.04) 332 (0.04) 334 (0.04) 336 (0.03) 331 (0.04) 337 (0.04)
TABLE 2: TD-PCM(B3LYP/6-31G*) Vertical Transitions (in nm, OS in Parentheses) with 1 (1w) or 2 (2w) Water Molecules in Proximity of the Carbonyl Group of Ring Da type of transitionb 1w O/D 2w O/D
QM/PCM charge/PCM QM/PCM charge/PCM
λ(HfL)
λ(H-1fL)
360 (0.53) 361 (0.52) 353 (0.59) 354 (0.57)
332 (0.04) 332 (0.03) 336 (0.03) 334 (0.03)
a Calculations were carried out with explicit (QM/PCM) or point charge (charge/PCM) water molecules. b H ) HOMO and L ) LUMO.
333 (0.04) 359 (0.56)
λ(HfL)
λ3
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λ(HfL)
339 (0.04) λ(H-1fL)
a
Calculations are carried out with polarizable continuum method (PCM) and in presence of 1 (1w) or 2 (2w) explicit water molecules in proximity of the indicated chemical group (atom/ring). b s ) strong and w ) weak. c H ) HOMO and L ) LUMO.
Figure 3. Experimental UV-vis spectrum of CPT in water (solid line) superimposed on the fit (dashed line) obtained by summing two Lorentzian curves. The fit maxima are located at 369 nm (short dotdashed line) and 355 nm (long dot-dashed line) nm. The two Lorentzians have a half-width half-maximum (hwhm) of 9 nm (short dot-dashed line) and 24 nm (long dot-dashed line) and the fit has a correlation coefficient of 0.991, rms 0.02%.
Figure 2. Shape of the ΨHOMO-(-ΨLUMO) Kohn-Sham orbitals involved in the π f π* transition. Red and blue represent positive and negative values, respectively.
or two solvent molecules on the CPT chromophores, in close proximity to the N site of the B ring and to the CO sites of rings D and E (see Figure 1A) have been investigated. Modeling of the direct solute-solvent interaction was suggested by the shape of the ΨHOMO-(-ΨLUMO) Kohn-Sham orbitals involved in the π f π* transition (Figure 2). In fact, the large value of the electronic density, found along the N atom (ring B) and CO groups (rings D and E) chromophores, suggests these molecular sites to be the most sensitive ones to a non covalent perturbation by water molecules. The presence of explicit solvent molecules on ring E does not affect the λ(HfL) and λ(H-1fL) band maxima in a significant way. On ring B the presence of one water molecule causes a red shift of the λ(HfL) peak from 367 to 374 nm, while an even stronger effect is observed adding two water molecules to the ring D carbonyl (Table 1). In this case, the presence of one or two water molecules around the CO group of D ring caused a blue shift of the calculated λ(HfL) transition from 367 nm to 360 and 353 nm, respectively (Table 1). These values are very close to the 354 nm experimentally measured maximum of the λ2 band (in particular, the second one almost reproduces the experimental value), suggesting that this band is due to specific solute-solvent interactions, i.e., the formation of one or more H-bonds between the D ring CO site with one or, more probably,
two water molecules. The blue shift induced by the presence of two water molecules close to the CO group of ring D remains also upon addition of water molecules in the proximity of ring B and E (Table 1). A similar result has been reported in a recent QM/MM study24 where a comprehensive analysis of the water solvation around the CO in the acetone molecule suggested the presence of up to three water molecules close to this chromophore. In the same study, a more general analysis of the hydrogen bond interaction energies concluded that on average two water molecules are hydrogen-bonded to the acetone carbonyl oxygen. The strength of the hydrogen bond25 between CPT and water molecules calculated at the TD-DFT (charge/PCM) level of theory, and which give rise to a similar blue shift, amount to 0.152 eV (14.7 kJ/mol) for the CPT-(H2O)2 and to 0.084 eV (8.1 kJ/mol) for the CPT-H2O with respect to the bare CPT molecule in solution. These H-bond energies are about half the values found in the corresponding models of acetone interaction with water,24 thus suggesting the presence of a more complex pattern of water solvation around the CPT chromophores that is not fully described by the static QM models (see the MD discussion below). The absorption band shift induced by the solvent is mainly an electrostatic effect. In fact, TD-DFT calculations including a background charge distribution made up of point charges placed at the water atom positions give almost the same results as the corresponding full QM models (Table 2). It is important to notice that the λ(H-1fL) band maximum is only slightly affected by the presence of the explicit water molecules (Table 1). These results indicate that the two bands,
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Figure 4. (A) Radial distribution function g(r) of water oxygen atoms and the corresponding number of coordinated water molecules N(r) calculated for the carbonyl oxygen atom on ring D of CPT. Frames with a λ(HfL) vertical transition above or below 362 nm are shown in red and blue; solid and dot-dashed lines are used for g(r) and N(r), respectively. (B) Spatial distribution functions of water oxygen atoms around ring D for frames characterized by λ(HfL) transition above (red solid surface) and below (blue mesh surface) 362 nm.
centered at high wavelengths, are due to the same π f π* electronic transition whose maximum mainly depends on the CPT solvent environment, surrounding the CO group of ring D, and to a lower extent the other chromophores. We therefore propose that the CPT UV-vis spectrum in aqueous solution is due to the statistical distribution of CPT molecule populations having different water molecule environments. To further investigate this hypothesis, we have simulated the hydration of CPT with classical MD. The ability of the MD simulation to explore several solvation microstates, coupled with the calculation of the vertical transitions associated with each individual microstate, can improve our understanding of the solvation effects on the position of the λ1 and λ2 peaks. We have calculated the UV spectrum associated with each of the 160 configurations extracted from the classical trajectory (picking a frame every 100 ps to ensure their uncorrelation). Each frame includes all the 865 water molecules, considered as point charges, because this approximation produces results similar to those obtained with a full QM treatment (Table 2). The inclusion of all the water molecules requires a considerable computational effort, but prevents an arbitrary a priori choice of the positions of the water molecules that may have an effect on the λ values of the vertical transitions. The experimental spectrum in the 340-380 nm range is shown in Figure 3. The vertical transitions, calculated for 160 extracted frames at the TD-DFT (charge/PCM) level of theory, are centered around 330 and 360 nm (data not shown), corresponding to the λ(H-1fL) and λ(HfL) transitions, respectively. The transition at lower wavelength corresponds to the experimental weak band at 334 nm; the range of calculated values goes from 322 to 335 nm, but more than 90% of them are in the range 330 ( 2 nm, indicating that this band is only slightly influenced by the solvent environment. The calculated λ(HfL)
Sanna et al.
Figure 5. Radial distribution function g(r) of water oxygen atoms and the corresponding number of coordinated water molecules N(r) calculated on rings B and E. Frames with a λ(HfL) vertical transition above or below 362 nm are shown in red and blue; solid and dotdashed lines are used for g(r) and N(r), respectively. (A) Radial distribution functions and corresponding running integrals calculated around the nitrogen atom of ring B. (B) Radial distribution functions and corresponding running integrals calculated around the carbonyl oxygen atom of ring E.
Figure 6. 2D Molecular structure of the three CPT derivatives investigated.
transition is mainly localized in the 351-383 nm range, covering the values of the experimental λ1 and λ2 bands centered at 354 and 369 nm, respectively. The large majority of band positions (>90%) falls in the range 350-370 nm, in excellent agreement with the experimental spectrum and with the results obtained by the ab initio optimized models (Table 1).
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Figure 7. UV-vis experimental spectra of 5 µM CPT derivatives at pH 5.3 in acetate buffer 10 mM, in the 300-400 nm region.
TABLE 3: Experimental UV-Vis Band Maxima of 20-mod CPT, SN-38 and TPT in Water Solution at pH 5.0 and TD-PCM(B3LYP/6-31G*) Vertical Transitions (in nm) with OS in Parenthesesa λ1 20-mod CPT SN-38
TPT
experimentalb PCM PCM + 2w O/D experimentalb PCM PCM + 2w O/D PCM + 2w O/D + 3w OH/A experimentalb PCM PCM + 2w O/D + 3w OH/A type of transitionc
367 (s) 366 (0.48) 378 (s) 376 (0.59) 374 (0.58) 382 (s) 382 (0.44) λ(HfL)
λ2 351 (s) 353 (0.58) 362 (s) 367 (0.63) 368 (s) 367 (0.58) λ(HfL)
λ3
λ4
332 (w) 330 (0.03) 335 (0.03) 325 (w) 340 (0.04) 339 (0.10) 335 (0.08) 328 (w) 325 (0.12) 325 (0.05) λ(H-1fL)
310 (w) 315 (0.10) 314 (0.12) 310 (0.12) 314 (w) 312 (0.11) 306 (0.13) λ(HfL+1)
a
Calculations are carried out with polarizable continuous method (PCM) and in the presence of 1 (1w), 2 (2w), or 3 (3w) explicit water molecules in proximity of the indicated chemical groups (atom/ring). b s ) strong and w ) weak. c H ) HOMO and L ) LUMO.
In Figure 3 is also reported the fit of the experimental spectrum that was obtained by summing two Lorentzian curves. The mean value of the maxima for the two fitted curves is at 362 nm, and this value has been taken as a threshold to investigate if it is possible to single out specific solvent distribution patterns around the CPT molecule, diagnostic for the vertical transition above or below this threshold. The value of this wavelength has then been used as a cutoff to divide the 160 frames, extracted from the MD simulation, into two groups, thus obtaining 59 frames with λ(HfL) falling above and 101 frames with λ(HfL) below 362 nm. Following this classification, the distribution of water molecules around the 3 CPT chromophores (i.e., the carbonyl groups on rings D and E and the nitrogen atoms on ring B) has been investigated for each frame. Figure 4A shows the radial distribution function, g(r), of the water oxygen atoms around the carbonyl oxygen on ring D and the corresponding number of coordinated water molecules (running integrals), N(r), obtained by integration of the g(r). Both the frames with λ(HfL) below (Figure 4A, blue line) and above (Figure 4A, red line) 362 nm show the presence of two coordinated water molecules within a distance of 3 Å. However, the frames with absorption below 362 nm have an oxygen/water-oxygen/CPT g(r) peak centered at shorter distances. The different distribution of the solvent in the two conformation families can be appreciated in Figure 4B, which shows the isodensity surfaces (spatial distribution functions26) of the water oxygen atoms. The frames below 362 nm, in fact,
show the presence of two water “clouds” at short distance from the carbonyl oxygen whereas only one close water is seen for the frames above 362 nm. On the other hand, the same kind of analyses carried out on the other two CPT chromophores of ring B and E reported in Figure 5 does not show any appreciable difference in the g(r), independently if they are above or below the 362 nm threshold, confirming that the value of the λ(HfL) absorption band is mainly dependent on the chemical environment around ring D. The results obtained for the CPT molecule have encouraged us to apply the same experimental and computational procedures to some CPT derivatives in their lactonic forms. In detail, we have focused our attention on the 20-mod CPT, having a different substitutions on the asymmetric carbon of ring E, to SN-38 and to TPT, having different chemical entities on ring A/B, as depicted in Figure 6. For all the selected CPT derivatives we recorded the UV-vis spectrum (Figure 7) and carried out the QM part of the computational procedure already described. The experimental spectrum of the 20-mod CPT derivative is close to that of CPT, being characterized in the 300-400 region by two strong λ1 and λ2 bands and by a weak one (λ3) at lower wavelength (Figure 7 and Table 3). The TD-DFT/PCM calculations identify the λ(HfL) and λ(H-1fL) transitions that well reproduce the λ1 and λ3 experimental bands, but again the λ2 maximum is never reproduced (Table 3). Also in this case the presence of two water molecules in proximity of the CO site of
5374 J. Phys. Chem. B, Vol. 113, No. 16, 2009 the D ring shifts the λ(HfL) maximum toward the λ2 experimental value (Table 3). These results indicate that chemical modifications of the asymmetric carbon of ring E do not perturb the electronic distributions of the CPT-derived molecule, giving rise to very similar absorption spectra. In the same way, other chemical substitutions on this ring, like OCH3, OC2H5, NH2, Br, and Cl, give rise to identical calculated vertical transitions and to the same solvent effects for water molecules in proximity of the CO group of ring D (see section 3 of Supporting Information for further details). The experimental spectrum of SN-38 displays two strong λ1 and λ2 bands at high wavelengths (378 and 362 nm, respectively) and two weak λ3 and λ4 bands at low wavelengths (325 and 310 nm, respectively), shown in Figure 5 and Table 3. The TDDFT/PCM calculations on SN-38 without explicit water molecules clearly identify the λ(HfL) and λ(HfL+1) bands that well reproduce the λ1 and λ4 experimental bands, and with less accuracy the λ(H-1fL) band, that likely corresponds to the λ3 UV-vis signal, but again the λ2 band is never reproduced. Addition of two water molecules in proximity of the CO site of the D ring is not sufficient to blue shift the λ(HfL) transition toward the λ2 experimental maximum, as observed in CPT calculations, with PCM modeling without explicit water molecules (Table 3). The only significant difference is observed at the level of the λ(H-1fL) transition that doubles its oscillator strength value (Table 3), thus suggesting a more complicated pattern of solute-solvent interaction, perhaps including other chromophores not present in the CPT molecule. In fact, the addition of three further water molecules around the OH group of ring A induces a shift of the λ(HfL) transition from 376 to 367 nm, closer to the experimental λ2 maximum centered at 362 nm. Moreover, the calculated λ(HfL+1) vertical transition and the λ4 experimental maximum now have an identical value, and the λ(H-1fL) calculated transition shifts toward lower wavelength values (335 nm) relatively close to the experimental λ3 maximum (325 nm). An identical approach has been applied to the TPT molecule. The experimental spectrum is quite close to that observed for the SN-38 drug, being characterized by the presence of two strong λ1 and λ2 bands at 382 and 367 nm and two weak λ3 and λ4 bands at 328 and 314 nm, respectively. The TD-DFT/PCM calculations identify the λ(HfL), the λ(H-1fL), and the λ(HfL+1) transitions that well reproduce the λ1, the λ3, and the λ4 experimental absorptions, respectively, but again the λ2 band is not reproduced at all. Addition of two and three water molecules in proximity of the CO group of the D ring and of the OH group of the A ring, respectively, leads to a very close agreement between the experimental and calculated transitions, providing a further and, in our opinion, fully convincing demonstration of the importance of the non covalent interactions in the separation of the electronic levels of this class of drug molecules. 4. Conclusions The UV-vis spectroscopic signatures of the natural alkaloid CPT, and three derivates (20-mod CPT, SN38, and TPT), have been systematically investigated using a combined computational procedure and results have been compared to experimental spectrum in water. We successfully provide, for the first time, a full interpretation of the absorption spectrum of CPT and of its derivatives in aqueous solution, and show that the overall spectral shapes result from the contribution of different drug populations having defined water distribution around them. The finding of a specific structure-spectroscopic feature correlations
Sanna et al. can pave the route to experiments able to monitor drug binding to DNA or to the DNA-topoisomerase I binary complex, by following the variation of the spectroscopic bands. This may permit the identification of the drug structural environments, such as the specific protein-DNA anchor sites. Furthermore, our finding represents a significant step ahead in the understanding of the behavior of CPT derivatives in aqueous solution since the λ2 experimental absorption band has been successfully reproduced by considering a direct solute-solvent interaction mechanism that is found also in clinically relevant derivatives like SN-38. Acknowledgment. The authors thank the CASPUR consortium for the computing facilities used in this work. A.D. acknowledges support by the AIRC project “Characterization of human topoisomerase I mutants resistant to camptothecin and its derivatives”. S.C. acknowledges a fellowship from AIRC/ FIRC. L.G. acknowledges support by MIUR under project PONCyberSar. C.Z. acknowledges support by the FILAS-Lazio regional agency under the project CAMPTOFAR. Finally, we thank J. Z. Pedersen for the critical reading of the manuscript. Supporting Information Available: Additional calculated data report: (1) Structural parameters; (2) TD-DFT Kohn-Sham (KS) orbitals; (3) UV-vis data; (4) QM minimum energy molecular geometries of the CPT-nH2O models investigated; (5) Nonaveraged radial distribution functions. Complete refs 12 and 20. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Wall, M. E.; Wani, M. C.; Cook, C. E.; Palmer, K. H.; McPhail, A. T.; Sim, G. A. J. Am. Chem. Soc. 1966, 88, 3888–3890. (2) (a) Bailly, C. Crit. ReV. Oncol. Hematol. 2003, 45, 91–108. (b) Nagourney, R. A.; Sommers, B. L.; Harper, S. M.; Radecki, S.; Evans, S. S. Br. J. Cancer 2003, 89, 1789–1795. (c) Garcia-Carbonero, R.; Supka, J. G. Clin. Cancer Res. 2002, 8, 641–661. (d) Accessing the European Patent website http://ep.espacenet.com/ using “camptothecin” as a simple search keyword has given, at present time, 1746 hits. (3) (a) Pommier, Y.; Pourquier, P.; Fan, Y.; Strumberg, D. Biochim. Biophys. Acta 1998, 1400, 83–106. (b) Pommier, Y.; Redon, C.; Rao, V. A.; Seiler, J. A.; Sordet, O.; Takemura, H.; Antony, S.; Meng, L. H.; Liao, Z. Y.; Kohlhagen, G.; Zhang, H. L.; Kohn, K. W. Mutat. Res. 2003, 532, 173–203. (c) Leppard, J. B.; Champoux, J. J. Chromosoma 2005, 114, 75– 85. (4) (a) Staker, B. L.; Hjerrild, K.; Feese, M. D.; Behnke, C. A.; Burgin, A. B.; Stewart, L. Proc. Natl. Acad. Sci. 2002, 99, 15387–15392. (b) Staker, B. L.; Feese, M. D.; Cushman, M.; Pommier, Y.; Zembower, D.; Stewart, L.; Burgin, A. B. J. Med. Chem. 2005, 48, 2336–2345. (c) Chrencik, J. E.; Staker, B. L.; Burgin, A. B.; Pourquier, P.; Pommier, Y.; Stewart, L.; Redinbo, M. R. J. Mol. Biol. 2004, 339, 773–784. (5) (a) Posokhov, Y.; Biner, H.; Icli, S. J. Photochem. Photobiol. A: Chem. 2003, 158, 13–20. (b) Dey, J.; Warner, I. M. J. Photochem. Photobiol. A: Chem. 1996, 101, 21–27. (c) Nabiev, I.; Fleury, F.; Kudelina, I.; Pommier, Y.; Charton, F.; Riou, J. F.; Alix, A. J. P.; Manfait, M. Biochem. Pharm. 1998, 55, 1163–1174. (d) Thomas, C. J.; Rahier, N. J.; Hecht, S. M. Bioorg. Med. Chem. 2004, 12, 1585–1604. (e) Du, W. Tetrahedron 2003, 59, 8649– 8687. (6) Sanna, N.; Chillemi, G.; Grandi, A.; Castelli, S.; Desideri, A.; Barone, V. J. Am. Chem. Soc. 2005, 127 (44), 15429–15436. (7) Chillemi, G.; Castrignano`, T.; Desideri, A. Biophys. J. 2001, 81, 490–500. (8) Chillemi, G.; Fiorani, P.; Benedetti, P.; Desideri, A. Nucleic Acids Res. 2003, 31, 1525–1535. (9) Chillemi, G.; Redinbo, M.; Bruselles, A.; Desideri, A. Biophys. J. 2004, 87, 4087–4097. (10) Chillemi, G.; Bruselles, A.; Fiorani, P.; Bueno, S.; Desideri, A. Nucleic Acids Res. 2007, 35 (9), 3032–8. (11) Zhao, H.; Lee, C.; Sai, P.; Choe, Y. H.; Boro, M.; Pendri, A.; Guan, G.; Greenwald, R. B. J. Org. Chem. 2000, 65, 4601–4606. (12) Frisch, M. J.; et al. Gaussian 03 ReV. C2; Gaussian, Inc.: Wallingford, CT, 2004. (13) (a) Cossi, M.; Rega, N.; Scalmani, G.; Barone, V. J. Chem. Phys. 2002, 117, 43–54. (b) Cossi, M.; Rega, N.; Scalmani, G.; Barone, V.
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