Chemical Physics 275 (2002) 93–108 www.elsevier.com/locate/chemphys

Camptothecins–guanine interactions: mechanism of charge transfer reaction upon photoactivation K. Steenkeste a, E. Guiot b, F. Tfibel a, P. Pernot c, F. M
erola c, P. Georges b, M.P. Fontaine-Aupart a,* a

Laboratoire de Photophysique Mol
eculaire, UPR 3361 CNRS, Universit
e Paris-Sud, Bat 213, 91405 Orsay Cedex, France b Laboratoire Charles Fabry de l’Institut d’Optique, UMR 8501, 91405 Orsay Cedex, France c Laboratoire de Chimie Physique, UMR 8000, Universit
e Paris-Sud, 91405 Orsay Cedex, France Received 17 May 2001; in final form 30 July 2001

Abstract The potent activity exhibited by the antitumoral camptothecin (CPT) and its analog irinotecan (CPT-11) is known to be related to a close contact between the drug and the nucleic acid base guanine. This specificity of interaction between these two chromophores was examined by following changes in the photophysical properties of the drug using steadystate as well as time-resolved absorption and fluorescence methods. The observed effects on absorption, fluorescence emission and singlet excited state lifetimes give evidence for the occurrence of a stacking complex formation restricted to the quinoline part of CPT or CPT-11 and the guanine base but also with the adenine base. The triplet excited state properties of the drugs have been also characterized in absence and in presence of guanosine monophosphate and reveal the occurrence of an electron transfer from the guanine base to the drug. Support for this conclusion was obtained from the studies of a set of biological targets of various oxido-reduction potentials, adenosine monophosphate, cytidine, cytosine, tryptophan, tyrosine and phenylalanine. This finding gives an interpretation of the CPT-induced guanine photolesions previously reported in the literature. These data taken together are discussed in connection with the drug activity. The stacking complex CPT/guanine is necessary but not sufficient to explain the role of the chirality and of the lactone structure in the function of the drug. A stereospecific interaction with the enzyme topoisomerase I seems necessary to stabilize the stacking complex. The first experiments using time-resolved fluorescence by two-photon excitation confirms that CPT does not bind to the isolated enzyme. Ó 2002 Elsevier Science B.V. All rights reserved. Keywords: Camptothecins; Fluorescence spectroscopy; Triplet state; Two-photon excitation

*

Corresponding author. Tel.: +1-33-1-69-15-73-64; fax: +1-69-15-67-77. E-mail address: [email protected] (M.P. Fontaine-Aupart). Abbreviations: CPT, camptothecin; GMP, guanosine monophosphate; AMP, adenosine monophosphate; CMP, cytidine; Trp, tryptophan; Tyr, tyrosine; Phe, phenylalanine; TopoI, topoisomerase I; DMSO, dimethylsulfoxide; OPE, one-photon excitation; TPE, two-photon excitation. 0301-0104/02/$ - see front matter Ó 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 1 - 0 1 0 4 ( 0 1 ) 0 0 5 2 9 - 8

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1. Introduction The current interest in studying the antitumoral drug camptothecin (CPT) [1] and its analogues such as irinotecan (CPT-11) successfully used in colon cancer and leukemia [2–4] arises from the recognition that these compounds belong to a restricted class of molecules which act as inhibitors of human DNA topoisomerase I (TopoI) [2,5–7]. This enzyme mediates the relaxation of supercoiled DNA; it binds to DNA and then nicks it on one strand via the formation of a covalent link between the 30 -phosphate of the cleaved strand and the phenolic hydroxyl group of a tyrosine (Tyr 723 in human TopoI). In a second step, TopoI catalyzes the religation of the 50 -hydroxyl group of the broken DNA strand after one turn of the helix. It is now accepted that CPTs molecules activity is correlated to a stabilization of the covalent TopoI– DNA complex (ternary complex) thus preventing DNA religation by the enzyme [7–11] and ultimately leading to DNA fragmentation and cell death [12]. Several structural features of CPT are essential for its activity. One requirement is the planarity of

the five rings of the polycyclic compound (Scheme 1a). Modifications at the C-9 and C-10 positions of the A-ring and the C-7 position of the B-ring very often enhance the potency of the drug in both in vitro and in vivo studies [4,10,12]. Besides, C-11, C-12 of the A-ring and C, D, E rings of CPT cannot be substituted without severely affecting or inhibiting its biological function [4,10,12]. In particular, the configuration of the C-20 chiral center is very important, the 20(S) hydroxyl enantiomer is active while the 20(R) CPT form, which does not exist in nature, was found to be inactive [10]. The lactone moiety of the E-ring is also of importance. The intact hydroxy-lactone ring is a structural requirement for the biological activity of CPT; the ring opened carboxylate form of CPT is not only inactive [10] but also toxic [13,14]. The way of formation of the ternary complex TopoI–DNA–CPT prerequisite for the drug activity remains a controversial subject. The results of structure–activity studies [7] and fluorescence intensity measurements [15] preclude binding of CPT to TopoI alone. Moreover, equilibrium dialysis studies [7] provided no evidence of drug binding to DNA. However, in experimental

Scheme 1. Structures of the S enantiomer of CPT (a) and of CPT-11 (b).

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conditions, such as the DNA/drug ratio is higher than 103 , it has been shown that the active lactone forms of CPTs molecules are stabilized through interactions (intercalation, surface binding) with DNA [3,15] and sometimes with a base nature specificity [15]. On the basis of these results, it has been proposed that the DNA–drug association could be the initial event for the subsequent drug– DNA–TopoI complex formation [3]. On the other hand, when intercalation has been demonstrated (DNA/drug ratio > 1000), this mode of complexation has been found to have no specificity for the nature of base sequence and to be in rapid equilibrium with an external backbone binding mode, revealing a very low affinity of the drug for DNA [16]. Whatever the ternary complex formation process, it has been revealed that CPT is selectively active at TopoI cleavage site bearing a guanine base at the +1 position on the scissile strand, immediately downstream of the cleavage site [17,18] suggesting specific interactions between the drug and the base. Furthermore, the activity of the drug is enhanced upon photoactivation specially in anaerobic conditions [19]. The present paper is focussed on the study of the molecular recognition of guanosine monophosphate (GMP) by CPT in its ground and excited (singlet and triplet) states by means of steady-state and time-resolved absorption and fluorescence spectroscopy. The involvement of a stacking process and/or a charge transfer process has been examined by extending the study to a series formed by the complexes of CPT with different nucleotides adenosine monophosphate (AMP), cytidine (CMP) and cytosine. In order to further discuss the results obtained, the features involved in complex formation have also been examined in the case of CPT/amino acid complexes (CPT–tryptophan, CPT–tyrosine, CPT–phenylalanine). We provide evidence for the occurrence of different photoprocesses in the singlet and triplet excited states of CPT in the presence of the biological targets. The effects identified in both kinetics and spectra are discussed in connection with a possible molecular interaction of the drug in the molecular assembly CPT–DNA–TopoI. The interaction between CPT and TopoI which seems to

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be a prerequisite for the drug activity may be too weak to be detected using fluorescence intensity [15] and dialysis [7] measurements but should be revealed by means of fluorescence decay analysis which is a more sensitive method. Dynamic fluorescence spectroscopy under two-photon excitation (TPE) has thus been used in order to investigate a possible CPT lactone ring–TopoI interaction.

2. Material and methods 2.1. Material S-CPT (CPT), GMP, AMP, CMP, cytosine, and D enantiomers of tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe) were obtained from Sigma and used as received. S isomer of CPT-11 was a generous gift of Rhone-Poulenc-Rorer. TopoI was purchased from Life Technologies. At pH 4.0, CPT and CPT-11 were first dissolved in dimethylsulfoxide (DMSO) solution (Prolabo, Normapur) and then diluted in 20 mM acetate buffer. The final percentage of DMSO can be neglected. In basic media (pH 8.0), the drugs were directly dissolved in 20 mM tris-HCl buffer. At pH 7.0, the lactone form of CPT was obtained by dissolving the drug in 25 mM potassium phosphate buffer, 50% (v/v) glycerol (glycerol being necessary for the experiments with TopoI) and used just after the dilution. According to the procedure described by Chourpa et al. [4], the carboxylate form of the drug for studies at pH 7.0, was generated by preparing the solute in phosphate buffer pH 8.0, 50% (v/v) glycerol, followed by a readjustment of the pH of the CPT solution to a value of 7.0. The two-photon fluorescence excitation measurements of the carboxylate form of CPT were performed immediately after. The interaction studies of the drugs with the nucleotides or the amino acids were performed by keeping the concentration of CPT and CPT-11 constant while varying the biological target concentration (dilution effects were taken into account). The ratio of nucleotide or amino acid to the drug concentrations was defined as r. The concentrations of drugs, nucleotides and amino acids were determined spectrophotometrically L

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using the following molar absorption coefficients: e370 ¼ 19000M1 cm1 for CPT [20], e355 ¼ 25000 M1 cm1 for S-CPT11 [21], e255 ¼ 14000 M1 cm1 for GMP [22], e260 ¼ 15000 M1 cm1 for AMP [22], e275 ¼ 9000 M1 cm1 for CMP [22], e266 ¼ 6000 M1 cm1 for cytosine [23], e280 ¼ 5600 M1 cm1 for Trp [23] e275 ¼ 1400 M1 cm1 for Tyr [23] and e257 ¼ 200 M1 cm1 for Phe [23]. It must be noted that it was necessary to study the highly water soluble nucleotides (GMP, AMP) rather than the free bases (guanine, adenine) since high concentrations were required to obtain significant complexation of the drug with the nucleic acid bases. The samples were deaerated by bubbling argon through the solution prior to the experiments. When necessary, the solvated electron was eliminated by saturation of the solution with N2 O and 0.05 M tert-butyl alcohol which scavenged the free OH: radicals formed during the electron N2 O reaction. The oxygenated samples were obtained by maintaining 1 atm O2 upon the solutions. For the detection of TopoI-CPT complex formation, each sample (11 ll total volume) contained 10 ll (2 lM) of TopoI (stocked in 30 mM potassium phosphate pH 7.0, 5 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA, 50% (v/v) glycerol, 0.1% (w/v) Triton X-100) and 1 ll (2.2–22 lM) of CPT at pH 7.0 (hereafter, 1–10 enzyme/drug molar ratio). 2.2. Techniques Stationary absorption spectra were recorded on a Cary 210 spectrophotometer (Varian); fluorescence emission was measured on a Perkin Elmer MPF-3L spectrofluorimeter using an emission and excitation bandwidth of 3 nm and a cell with an optical path length of 1cm. The absorbance of the samples at the excitation wavelength was maintained less than 0.1 making inner filter effects negligible. Rayleigh and Raman scattering from the buffer were subtracted from the fluorescence of the samples. Excitation spectra were corrected from the fluctuations of the Xe-lamp intensity in the range 275–380 nm. The time-correlated single photon counting method [24] was employed to determine the lifetimes of the fluorescent singlet excited state of free

CPT or complexed to the different nucleotides, amino acids and TopoI. The time-resolved fluorescence measurements by one-photon excitation (OPE) used to study free CPT at both pH 4.0 and 8.0, were performed using the synchrotron radiation from the Orsay storage ring (super ACO, LURE) working at a frequency of 8.33 MHz, 650 ps pulse width. After excitation at 370 nm, the emission signal at 440 nm (maximum fluorescence wavelength) was collected as described previously [25]. The instrumental function response (100 ps) was recorded by detecting the light scattered by a solution of Ludox (DuPont Co.) at the emission wavelength. For each sample, approximately 10 million counts (giving approximately 4  104 counts in the peak channel) were stored in the total fluorescence decays. The latter were analyzed by the quantified maximum entropy method (MEM) [26]. The time resolution obtained after pulse convolution is 100 ps. The TPE experimental setup consisting of a Ti:sapphire laser system (MIRA 900, Coherent Inc., Santa Clara, CA) pumped in the green by a cw diode-pumped solid state laser (VERDI, Coherent Inc., Santa Clara, CA) (excitation pulse width 100fs, repetition rate 76 MHz, excitation wavelength 760 nm) was described previously [27]. For the present experiments, the repetition rate of 3.8 MHz was achieved with a pulse picker model 9200 from Coherent. The average output power was 10 mW. The laser beam enters through a Zeiss Axiovert 135 microscope and is focused on the sample with a Zeiss oil immersion objective (63x, N:A: ¼ 1:4). The radius of the focussed beam was 0.5 lm. Fluorescence photons are collected by the same objective, separated from the excitation radiation by a dichro€ic mirror and detected by means of a microchannel plate photomultiplier (Hamamatsu R3809U-52), connected to an 1 GHz amplifier and timing discriminator (gain 0–150 mV) (EGG Ortec 9327) and a picosecond time analyzer (PTA) (EGG Ortec 9308). The time resolution of the experimental setup was 55 ps. Approximately 5 million counts (giving approximately 1:5  104 counts in the peak channel) were stored for each fluorescence decay. The intensity decays were analyzed in terms of a multiexponential model. The parameter values given in Table 2 (Ai , population

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of molecules fluorescing at a lifetime si ) were determined by non-linear least squares by minimizing the goodness-of-fit parameter v2 . The excitation source of the nanosecond photolysis system was a YAG laser (Quantel, YG441) of 3 ns full width at half maximum with third harmonic (355 nm) generation [28]. The 355 nm beam was directed onto one side of a 10 mm square silica cell containing the sample. The transient transmission variations were monitored at right angles to the excitation in a cross beam arrangement using a xenon flash lamp, a monochromator, a photomultiplier (HTV R928, response time 1 ns) and a digitized oscilloscope (Tektronix 2440) controlled by a microcomputer. The fluence of the incident laser pulse in the sample was obtained by calibration of the joulemeter using anthracene in deaerated cyclohexane as a triplet actinometer, with UT ¼ 0:7 and eT ¼ 67 500 M1 cm1 at 422 nm [23]. This actinometer was also used for the determination of the triplet quantum yield of CPT. The transient absorption data can be expressed as a sum of contributions from the spectrally active species: X DAðk; tÞ ¼ Ei ðkÞ Di ðtÞ;

97

The steady-state absorption and fluorescence properties of CPT depend on its protonation state [4,20,30]. As can be seen in the Fig. 1a, the absorption spectrum of the drug in its lactone form (pH 4.0) is characterized by a strong band centered at 370 nm with a shoulder at 355 nm. This lowest singlet state transition is of p–p character [30]. At pH 8.0 (carboxylate form of CPT), the vibrational

where Ei (k) is the absorption spectrum of species i, andDi (t) the corresponding kinetic evolution functions. The kinetic parameters are determined by non-linear iterative method minimizing the sum of weighted-least-squares [29].

3. Results 3.1. CPT–GMP interaction 3.1.1. Steady-state absorption properties According to the previous reports [4,30] which describe the hydrolysis and lactonization reactions of CPT as a function of pH, it could be assumed that at pH 6 5:0 CPT is present in its lactone form (active form) and at pH P 8:0 mainly in the carboxylate form. Thus, all the experiments described hereafter have been carried out both at pH 4.0 and 8.0.

Fig. 1. (a) Absorption spectra at pH 4.0 (acetate buffer) of free CPT (10 lM) (––) and of CPT in the presence of GMP (- ), AMP (- - -) or L -Tryptophan ( ) for r ¼ 600. (b) Absorption spectra at pH 8.0 (tris buffer) of the same complexes for r ¼ 1000.

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structure is blurred and a red shift of the maximum from 370 to 371 nm is observed (Fig. 1b). The position of these bands reflects the extended conjugation of the quinoline ring with the adjacent C and D rings of the drug [4,20]. Fig. 1a and b show the changes in the electronic absorption spectrum of CPT in its lactone or carboxylate form induced by the addition of GMP. An hypochromic effect of 11.2% and 4.3% at pH 4.0 and pH 8.0 respectively is observed as well as the presence of an isosbestic point at 380 nm which support the formation of a CPT–GMP complex in the ground state. By increasing the nucleotide concentrations from 8.5 to 10.5mM, no further absorption change is observed, a result revealing that molecular association was complete at these concentrations. Such complexes may result from stacking interaction between the two chromophores. However, GMP is also known to be easily oxidized. Thus, the involvement of stacking effect and/or oxidation process in the ground state complex formation was investigated by studying the absorption properties of CPT in the presence of different nucleotides and amino acids with different aromatic structures and various oxidation potentials. The oxidation potentials of these compounds at the two studied pH (pH 4.0 and 8.0) are collected in Table 1 [31–33]. Although the oxidation

potential of Phe has not been reported, its value can be expected to be higher than that of cytosine. This assumption is based on the absence of triplet quenching of drugs by Phe via a charge transfer mechanism by comparison with the other nucleic acid bases and amino acids investigated in this study [34]. The addition of equal amounts of AMP or Trp to CPT in its lactone and carboxylate forms results in similar but less intense modification of the absorption spectrum of the drug to that observed in the presence of GMP (Fig. 1a and b). Whatever the pH, no absorption spectral changes were observed when CPT was in the presence of the DNA base cytosine and its corresponding nucleic acid CMP, or in the presence of the amino acids Tyr and Phe. 3.1.2. Steady-state fluorescence properties The fluorescence properties of CPT are also pH dependent [4,20,30]. Its emission maximum is centered at 438 nm at pH 4.0 and 447 nm at pH 8.0. We have verified that the emission spectra of CPT are independent of the excitation wavelength and that the excitation spectra are superimposed on the absorption spectra, which supports the existence of only one form of the drug in the ground state. The changes in fluorescence spectrum induced upon addition of CPT to various amounts of GMP together with the fluorescence spectrum of the free

Table 1 Oxidation potentials of the biological targets and steady-state absorption and fluorescence features of free or complexed CPT as a function of pH (d½CPT ¼ 10 lM, r ¼ 600 at pH 4.0 and r ¼ 1000 at pH 8.0) pH 4.0 E0oxy

vs NHE

(V) CPT CPT–GMP CPT–AMP CPT–CMP CPT–Cytosine CPT–Tr CPT–Phe CPT–Tyr

1.31c 1.60c >1.86c 1.15d 1.10d

pH 8.0 Absorption hypochromicitya (%)

Fluorescence quenchingb (%)

11.2 5.0 0 0 7.6 0 0

35 35 0 0 35 0 0

E0oxy vs NHE (V) 1.12c 1.47c >1.35c 0.95d 0.88d

Absorption hypochromicitya (%)

Fluorescence quenchingb (%)

4.3 3.5 0 0 3.8 0 0

32 32 0 0 55 0 0

a The hypochromicity of the absorption spectra is calculated as follows: (A0  AÞ=A, A and A0 corresponding to the absorbance of complexed and free CPT respectively, measured at 370 nm. b The fluorescence quenching is calculated as follows: ðF0  F Þ=F0 , F and F0 corresponding to the area under the fluorescence spectrum of complexed and free CPT respectively upon excitation at 382 nm. c See Ref. [31,33]. d See Ref. [32].

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drug at pH 8.0 are shown in Fig. 2a. The fluorescence is quenched without spectral change up to a maximum extent of 35%. The ratio (F0 =F ) of the fluorescence intensities of CPT in the absence and in the presence of GMP varies linearly with the GMP concentration up to a concentration of 8.5 mM (Fig. 2a, inset) also allowing the determination of the quenching constant K from the slope of this

Fig. 2. (a) Fluorescence spectra obtained at pH 8.0 of CPT (3.2 lM) with increasing concentration of GMP (r ranging from 0 to 3200) upon excitation at 370 nm. Inset: Ratio of the fluorescence intensity for free CPT (F0 ) to the fluorescence intensity for the complexes CPT–GMP (F ), as a function of GMP concentration. (b) Fluorescence spectra measured at pH 4.0 of free CPT (10 lM) (solid line) and of CPT bound to GMP, AMP or Trp (dashed line) for r ¼ 600 upon excitation at 382 nm.

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linear plot, K ¼ 60 M1 . Since this quenching is of static nature (see below), K corresponds to the binding constant of the drug to the nucleotide. Similar fluorescence quenching effects were obtained at pH 4.0 and summarized in Table 1. The fluorescence emission spectra of CPT at pH 4.0 as a function of biological target nature (nucleotides and amino acids) are also recorded and displayed in Fig. 2b. The results substantiate the observation obtained from absorption spectroscopy. Similar maximum amounts of fluorescence quenching (35%) are observed when the drug is in the presence of GMP, AMP and Trp while Tyr, Phe, CMP and cytosine do not affect the fluorescence of the drug. These results are not influenced by the protonation state of CPT (pH 8.0) as revealed by the data of Table 1. 3.1.3. Time-resolved fluorescence spectroscopy The photophysical properties of CPT singlet excited state complexed with GMP have been investigated and the results compared with those obtained under the same experimental conditions for the free drug in its lactone and carboxylate states. At both pH 4.0 and 8.0, the fluorescence decays of CPT can be described by two exponential components (Table 2). The nanosecond lifetimes are in good agreement with previous reports [20] but an additional picosecond component has also been observed whatever the pH. Presumably this is due to the presence of CPT dimers. Indeed, it has been reported that the dimerization constant for such molecules was 2  104 M1 [21] and thus for a drug concentration of 6  106 M as used in this study, we can calculate that 15% of dimers are formed in solution which corresponds to the relative amplitude of the fast component in the fluorescence decay (Table 2). The fluorescence decays of CPT both at pH 4.0 and 8.0 are not affected by the presence of GMP, showing that no new fluorescent complexes are formed. The same behavior has been observed in the presence of AMP, CMP and the amino acids Trp, Tyr, Phe. 3.1.4. Transient absorption spectroscopy In order to determine whether CPT could induce specific reaction in the triplet excited state

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Table 2 Summarized fluorescence decay parameters of free and complexed CPT in its lactone and carboxylate form Lactone forma CPT

OPE

CPT–GMP

TPE OPE

CPT–Tryptophan

OPE

CPT–TopoI

TPE

Carboxylate forma

sb (ns)

Ac (%)

v2

sb (ns)

Ac (%)

v2

0:13 0:01 3:1 0:2 3:6 0:2 0:16 0:01 3:1 0:2 0:26 0:01 3:2 0:2 3:6 0:2

13.7 86.3 82.0 16.6 83.4 11.6 88.4 89.0

1.13

0:21 0:02 4:6 0:2 4:1 0:2 0:20 0:02 4:6 0:1 0:23 0:01 4:4 0:1 4:1 0:2

11.9 88.1 78.0 14.9 85.1 9.2 90.8 78.0

1.02

1.21 1.07 1.04 1.18

1.29 1.10 2.10 1.29

a

The OPE experiments were realized in acetate buffer solutions pH 4.0 (lactone form) or in TRIS buffer solutions pH 8.0 (carboxylate form), whereas the TPE measurements were carried out in phosphate buffer solutions pH 7.0 containing 50% (v/v) glycerol according to the method described in the experimental section. b s is the fluorescence lifetime. c A is the population of molecules fluorescing with a lifetime s.

with the different biological targets, laser flash photolysis experiments have been carried out. A prerequisite for this investigation was the detailed study of the triplet state of the free drug. 3.1.4.1. CPT in solution. Fig. 3a shows the transient spectra obtained from 355 nm laser excitation of a N2 saturated solution of CPT at pH 4.0, recorded at different delay times after the end of the laser pulse. At the end of the laser pulse, the difference spectrum is characterized by a positive broad band centered at 400 nm and a negative band due to ground state depletion around 370 nm. The dependence of the transient signal at 400 nm on the laser energy is linear up to an incident fluence of 15.3 mJ/cm2 , as expected for a monophotonic process (Fig. 3b). Furthermore, this transient is quenched by oxygen (1 atm) with a rate constant evaluated to 1:3  109 M1 s1 demonstrating that it can be attributed to the triplet state of CPT. Molecules in their triplet state may be quenched by molecules in the ground state thus influencing the triplet state lifetime. This was checked by varying CPT concentration in deaerated solutions. The results show that the absorption decay is affected by the concentration of the drug. The Stern–Volmer relationship allows the determination of the intrinsic rate constant of decay of the CPT triplet ki ¼ 4:1  104 s1 ( 10%) and the rate constant of

the quenching by ground-state molecules kq ¼ 7:1  108 M1 s1 ( 10%). This kinetic analysis also reveals that at the end of CPT triplet deactivation, the absorption of the ground state of the drug is recovered. Thus, the residual absorption (Fig. 3a) observed at 40 ls (upper limit of our experimental analysis system) can be assigned to CPT radical cation (CPT þ ) absorption. Indeed, preliminary experiments carried out in both argon and N2 O saturated solutions reveal the occurrence of a biphotonic ionization of the drug with an apparent yield of 20% (data not shown) leading to the formation of CPT þ ; it is well known that in deaerated conditions, such radicals disappear by dismutation on a much longer time scale [35]. The shape of the triplet absorption spectrum of CPT in its carboxylate form (pH8.0) is similar to that obtained at pH 4.0 with only a red shift of the maximum from 400 to 420 nm (Fig. 3c). This spectral effect was the same as that observed in the ground state suggesting the lack of significant change in pKa between the ground and the triplet excited state of the drug. The rate constants of triplet decay are the same at both pH. The triplet quantum yield UT of CPT has been measured in acetate buffer, assuming that no photoionization process occurred at the low energies used in this experiment. Using the method described previously [36], we obtained from the slopes of the curves of Fig. 3b:

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UT CPT eCPT ¼ 0:023UT anthracene eanthracene : T T The determination of the molar absorption coefficient of the CPT triplet has been undertaken by simulating complete conversion of the CPT ground state to the triplet state. This has been achieved by adding the transient absorption measurements to a fraction að0 < a < 1Þ of the ground state absorption spectrum of CPT. Different values of a have been tried and the resulting spectrum of the triplet has been compared with that of the ground state of the molecule. Under the assumption that the triplet spectrum is different from that of the ground state between 320 and 600 nm, acceptable values of a lie in the range 0.26–0.33. Under these conditions, the eCPT maximum value T at 400 nm has been estimated to be 14 700 3000 M1 cm1 (Fig. 3c), leading to a CPT triplet quantum yield equal to 0:07 0:02, a plausible value considering the fluorescence quantum yield reported in literature (Uf ¼ 0:64) [20].

Fig. 3. (a) Transient absorption spectra of free CPT (44 lM) in N2 O saturated buffered solution pH 4.0 at the end of the laser pulse (j) and 40 ls after the end of the laser pulse (d). (b) Transient absorption at 422 nm due to the triplet population on laser excitation of anthracene 13 lM in cyclohexane plotted against the laser energy (d), transient absorption at 400 nm due to the triplet population of CPT (44 lM) in buffered solution pH 4.0 (j). (c) Calculated absorption spectrum of CPT triplet, as described in text, in buffered solution pH 4.0 (j) and pH 8.0 (d). The ground state absorption spectra are also reported for comparison: pH 4.0 (––), pH 8.0 ( ).

3.1.4.2. Campthotecin-GMP complexes. Excitation at 355 nm of CPT in the presence of GMP under N2 O saturation conditions leads to absorbance changes in the spectral range 300–600 nm, whatever the pH (Fig. 4a). This spectrum closely matches both in shape and intensity the triplet absorption spectrum of the free molecule obtained using the same concentration (Fig. 3a). Thus, it can be stated that both the triplet absorption coefficient and the triplet quantum yield of CPT as well as the biphotonic ionization process are not affected by complexation with GMP. The evolution of the T1 –Tn absorption of CPT in the presence of GMP under N2 O-saturation conditions differs from that of the free drug and is characterized by the formation of a new transient. At pH 4.0, a build-up of the absorbance for the wavelengths between 450–525 nm can be observed on a few microseconds associated to an absorbance decrease for the wavelengths shorter than 450 nm and higher than 525 nm (Fig. 4a). When the drug is in its carboxylate form (pH 8.0), the build-up of the absorbance extends over a wider spectral range (400–600 nm). According to the model analysis (see above), the kinetics of CPT triplet state decay in the presence

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Fig. 4. ((a) Transient absorption spectra of CPT (44 lM) in the presence of GMP (r ¼ 230) in N2 O saturated buffered solution pH 4.0: (j) absorbance change at the end of the laser pulse, ( ) evolution of the absorbance change 2 ls after the end of the laser pulse and (N) absorbance change remaining 40 ls after the end of the laser pulse. (b) Absorption spectra of the B species (see in the text) obtained from the difference between the spectra measured at 40 ls after the end of the laser pulse for free CPT (Fig. 3a) and for CPT/nucleotides or CPT/amino acids complexes: (j) CPT/ Tryptophan complexes (r ¼ 230), ( ) CPT/GMP complexes (r ¼ 230), (M) CPT/Tyrosine (r ¼ 96), (.) CPT/AMP (r ¼ 230). For each complex, the CPT concentration was maintained to 44 lM.





of GMP at pH 4.0 and pH 8.0 are well fitted by the sum of two exponentials according to the same following scheme:

Part of the triplet (about 50% at both pH 4.0 and 8.0) deactivates to the ground state by firstorder kinetics with the same rate constant as for the free drug. The remaining population leads within about 100 ns to the formation of a new species B which stays stable during the 40 ls of the observation. According to this kinetic analysis, the absorption lasting over the limit of observation in the absence of GMP (Fig. 3a) was subtracted from that measured in the presence of GMP (Fig. 4a), the resulting absorption therefore being attributed to the B species (Fig. 4b). When the laser flash experiments are performed with an oxygen saturated solution, the formation of the transient species B is not observed due to the more rapid quenching of the triplet state by O2 . The deactivation of the triplet state of CPT in the presence of the different biological targets having decreasing redox potentials (Table 1) has also been performed. A spectro-kinetic evolution similar to that described in the presence of GMP, is observed with AMP, Trp and Tyr. In the presence of CMP, cytosine and Phe, the CPT triplet state deactivates like that of the free drug at both pH. 3.2. Two-photon excitation emission of CPT in the presence of TopoI The TPE of the lactone form of CPT and of its analogue topotecan has been demonstrated in previous reports [16], but according to our knowledge, this is the first determination of the CPT fluorescence lifetimes, both in its lactone and carboxylate forms at physiological pH. Upon TPE, the fluorescence decay profile of CPT in its two forms shows a monoexponential behavior (Fig. 5a). Deconvolution of the decay traces re-

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sulted effectively in a nearly single nanosecond lifetime, the same as that measured upon OPE at pH 4.0 and 8.0 respectively (Table 2). Small amplitude picosecond components ( 450 nm, the absorbance measured in the spectral range 450–700 nm can be attributed to the absorbance of the radical anion of CPT also formed upon reaction (1). This radical absorbance explains the higher absorbance measured between 425 and 550 nm compared to that of the biological radical cation alone (GMP þ , AMP þ , Trp þ ). The thermodynamic driving force for an electron transfer process (DG) can be estimated from the Rehm–Weller equation [48]: DG° ¼ e½E°ðD þ =DÞ  EðCPT=CPT  ÞDE00 þ w; where E°(D þ /D) corresponds to the oxidation potential of the donor, E°(CPT/CPT  ) to the reduction potential of CPT, DE00 to the electronic excitation energy for CPT (3.20 eV) and w < 0:1eV (in highly polar solvents) to the coulombic energy term characterizing the interaction of the two radical ion pairs. Thus, considering that AMP can be oxidized (DG° < 0) and not cytosine (DG° > 0), E°(CPT/CPT  ) belongs to the interval [1:60 V; 1:34 V]. The same electron transfer reaction has been also observed with the analogue CPT-11.

4.3. Biological relevance Our absorption and fluorescence spectroscopic measurements carried out with a DNA/drug ratio