Suzana M Andrade
email: suzana.andrade@ist.utl.pt

Raquel Teixeira

Sílvia M B Costa

Abílio J F N Sobral

SELF-AGGREGATION OF FREE BASE PORPHYRINS IN AQUEOUS SOLUTION AND IN DMPC VESICLES

Keywords: Porphyrins, DMPC vesicles, J-aggregates, Fluorescence, FLIM

Free base porphyrin (PPhe), derivatized with aminosulfonyl groups linked to the aromatic amino acid phenylalanine substituents in meso-positions, were mixed with DMPC vesicles. The resulting interaction was studied by absorption, steady state and transient state fluorescence, at different pHs.

At pH=2 to pH=9, the aforementioned porphyrin predominates as an aggregated species, with a co-facial arrangement of the molecules taking into account the blue-shift of the Soret band (414 nm for the monomer and 401 nm for the aggregate). Upon interaction with DMPC vesicles, the competing hydrophobic interactions with the bilayer destabilize the aggregated species in favor of monomer incorporation. Fluorescence lifetimes also show that the long component assigned to the monomer contributes only 30% to the overall decay in solution (e.g. pH=7.0) whereas in DMPC vesicles this contribution increases up to 85 % independent of the solution pH, which confirms a location of the probe in an environment "protected" from free water.

The picture changes completely in the case of TSPP, an anionic porphyrin which does not incorporate in DMPC vesicles. Remarkably, at pH=2.5 all the experimental findings point to the self-assembling of the porphyrin units in J-aggregates induced at the surface of the DMPC vesicle. In fact, upon removal of the aqueous solvent, we could define by fluorescence lifetime imaging microscopy (FLIM) regions where the fluorescence lifetime is that characteristic of the Jaggregate (<τ F > 0.11 ns).

A C C E P T E D M A N U S C R I P T INTRODUCTION

Porphyrins, as prosthetic groups of enzymes and carrier proteins are important to perform several functions. For example, porphyrins participate as electron transporters in oxidationreduction processes of cells, in oxygen transport and storage.

Porphyrins and their analogues are known to self-aggregate which is facilitated by a flat, wide, and electron-rich surface, creating van der Waals, π-π stacking, charge-transfer interactions.

Porphyrin aggregates also play specific roles in nature namely in photosynthetic plants and in organisms and have potential uses as nonlinear optical materials. In fact, much of the development in molecular electronics has been derived from attempts to mimic the highly efficient electron and energy transfer processes which take place in the light-harvesting complexes and reaction centers of photosynthetic organisms. [START_REF] Gust | Mimicking photosynthetic solar energy transduction[END_REF][START_REF] Van Amerongen | Photosynthetic excitons[END_REF] A variety of factors can influence the extent of self-association including the nature of the porphyrin itself (metal ion, number and nature of peripheral groups) as well as external changes as ionic strength, pH, solvent nature, temperature, among others.

A special interest has lied upon strongly coupled aggregates with delocalized excitonic states due to their potential applications as nonlinear optical materials [START_REF] Yamaguchi | Macroscopic spinning chirality memorized in spin-coated films of spatially designed dendritic zinc porphyrin J-aggregates[END_REF][START_REF] Misawa | Ultrafast exciton and excited-exciton dynamics in J-aggregates of three-level porphyrin molecules[END_REF]. According to Kasha's exciton theory, J-aggregates are formed when the transition dipole moments of the monomer molecules are aligned parallel to the line that joins the molecular centers in the aggregate ("head-to-tail"). By contrast, in the case of H-aggregates, the transition dipole moments of the monomer molecules are perpendicular to the line of centers ("face-to-face") [START_REF] Mcrae | Enhancement of phosphorescence ability upon aggregation of dye molecules[END_REF]. It is generally accepted that a red-shift in the electronic absorption spectra relatively to that of the monomer is proof of J-aggregate whereas a blue shift is proof of H-aggregates. Nevertheless, these two types are ideal cases which provide indications regarding the monomers' self-assembling in the aggregate.

A C C E P T E D M A N U S C R I P T

Although in literature J-aggregates are especially reported relatively to molecules belonging to the carbocyanine family [START_REF] Higgins | Excitonic transitions in J-aggregates probed by near-field scanning optical microscopy[END_REF][START_REF] Tatikolov | Effects of normal and reverse micellar environment on the spectral properties, isomerization and aggregation of a hydrophilic cyanine dye[END_REF][START_REF] Spitz | Polarized absorption and anomalous temperature dependence of fluorescence depolarization in cylindrical J-aggregates[END_REF], a significant body of investigation in this matter has been devoted

to the water-soluble ionic meso-tetra(p-sulfonatophenyl)porphyrin, TSPP. [START_REF] Ohno | J-aggregate formation of a water-soluble porphyrin in acidic aqueous-media[END_REF][START_REF] Maiti | Fluorescence dynamics of noncovalently linked porphyrin dimers and aggregates[END_REF][START_REF] Akins | Absorption and Raman-scattering by aggregated mesotetrakis(p-sulfonatophenyl)porphine[END_REF] The latter was reported to form, under suitable conditions of pH (below 1) and ionic strength, homoassociated arrays stabilized through a hydrogen bond network acting between the anionic sulfonate groups and the charged protonated nitrogen atoms. [START_REF] Ribó | Aggregation in water solutions of tetrasodium diprotonated meso-tetrakis(4-sulfonatophenyl)porphyrin[END_REF][START_REF] Rubires | Self-assembly in water of the sodium salts of meso-sulfonatophenyl substituted porphyrins[END_REF] The coherence length, i.e. the number of molecules collectively excited, has been reported to be 5 -7, although evidence for a much larger physical size has been given from light scattering techniques [START_REF] Mallamace | Experimental evidence for self-similar structures in the aggregation of porphyrins in aqueous solutions[END_REF][START_REF] Collings | Resonance light scattering and its application in determining the size, shape, and aggregation number for supramolecular assemblies of chromophores[END_REF][16].

This process of aggregation has been reported to be more efficient when the porphyrin interacted with templates such as: proteins [START_REF] Borissevitch | Fluorescence and optical absorption study of interaction of two water soluble porphyrins with bovine serum albumin. The role of albumin and porphyrin aggregation[END_REF][START_REF] Andrade | Spectroscopic studies on thr interaction of a water soluble porphyrin with two drug carrier proteins[END_REF][START_REF] Andrade | Aggregation kinetics of meso-tetrakis(4-sulfonatophenyl) porphine in the presence of proteins: Temperature and ionic strength effects[END_REF], surfactants [START_REF] Maiti | J-and H-aggregates of porphyrin-surfactant complexes: time-resolved fluorescence and other spectroscopic studies[END_REF], microemulsions [START_REF] Castriciano | Aggregation behavior of tetrakis (4-sulfonatophenyl) porphyrin in AOT/water/decane microemulsions[END_REF][START_REF] Andrade | Spectroscopic studies of water-soluble porphyrins with protein encapsulated in bis(2-ethylhexyl)sulfosuccinate (AOT) reverse micelles: aggregation versus complexation[END_REF], and dendrimers [START_REF] Paulo | Non-covalent dendrimer-porphyrin interactions: the intermediacy of H-aggregates?[END_REF] among others. Moreover, in some of these systems both the size and the type of aggregates could be tuned and in fact, H-aggregates were also detected.

Only more recently, the evidence of J-aggregation was reported for another water-soluble porphyrin meso-tetra(p-carboxyphenyl)porphyrin, TCPP, in very acidic conditions which was counterion dependent. [START_REF] Choi | Counterion-dependent excitonic spectra of tetra(p-carboxyphenyl)porphyrin aggregates in acidic aqueous solution[END_REF] The present work encompasses a new class of sulfonamide meso-tetraphenylporphyrin derivatives linked to amino acid phenylalanine groups -PPhe, scheme 1. Besides the terminal carboxylic groups similar to TCPP, Phe contributes to increase the dye hydrophobicity which were shown determinant for incorporation in liposomes. The sulfonamide group, known to be present in many drugs with bactericide and chemotherapeutic applications [START_REF] Winum | Carbonic anhydrase inhibitors: Synthesis and inhibition of cytosolic membrane-associated carbonic anhydrase isozymes I, II, and IX with sulfonamides incorporating hydrazino moieties[END_REF], may contribute to molecular association/dissociation through intra-/intermolecular hydrogen bonding. In fact, the existence of small aggregates was reported for PPhe in AOT RM, dependent on the amount of water present in the system [START_REF] Andrade | Self-association of free base porphyrins with aminoacid substituents in AOT reverse micelles[END_REF].

We have investigated and report herein, the influence of the microenvironment provided by DMPC bilayer vesicles in the self-aggregation pattern of the aforementioned porphyrins, TSPP and PPhe. These vesicles can be considered as a close model of biological membranes which

A C C E P T E D M A N U S C R I P T
offer the advantage of being thermodynamically stable and easily reproducible. The importance of understanding the interactions of these self-organized nanoscale molecular assemblies with porphyrins are stressed by their potential application in molecular photonics and artificial photosynthesis.

We will report spectroscopic evidence for a complex pattern of PPhe aggregation in water at different pH that simplifies in the presence of DMPC vesicles where essentially monomers are incorporated. By contrast, TSPP exists as a monomer in water which does not incorporate in DMPC bilayers. In the zwitterionic form, TSPP aggregates at the vesicle surface which is well supported by fluorescence lifetime imaging.

Experimental

Materials

The porphyrin meso-tetra (phenylalanine-4-amino sulfonyl-phenyl) porphyrin (PPhe) was synthesized and purified as described elsewhere [START_REF] Sobral | New sulfonamide and sulfonic ester porphyrins as sensitizers for photodynamic therapy[END_REF]. TSPP was obtained from Fluka ≥98% purity (catalogue no. 88074). 1,2-Dimirystoyl-sn-glycero-3-phosphocholine (DMPC) >99% purity was obtained from Avanti Polar Lipids (Alabaster, AL, USA). Buffer solutions were made up with bidistilled water, following the recommended procedures. The buffer citrate-(sodium)phosphate was employed for pH=2.5 to 7.0, and the more acidic pH or more alkaline pH were obtained by adding a few drops of highly concentrated solutions of HCl and NaOH, respectively. The buffer concentration was 25 mM. All solvents were spectroscopic grade.

Vesicle preparation

Phospholipid vesicles were prepared at concentrations between 0.01 and 0.8 mM. The appropriate amount of phospholipid was dissolved in a chloroform/methanol 2:1 v/v mixture (~100 µL). The solvent was evaporated under a nitrogen stream to obtain a thin lipid film.

Residual solvent was removed under vacuum overnight. The flask containing the lipid film was

A C C E P T E D M A N U S C R I P T
placed in a warm water-bath (with the temperature above the phospholipids main phase transition, T m =24 ºC at pH=6 and T m =27.5 ºC at pH=2.5) and the film was hydrated with the citrate-phosphate buffer of desired pH under mild agitation. To obtain the vesicles the hydrated phospholipid dispersions were extruded using a LiposoFast Basic mini-extruder (Avestin, Ottawa, Canada) by means of 15x through 0.4 µm pore diameter polycarbonate filters (Whatman, Clifton, NJ, USA) while maintaining the temperature above the main phospholipid phase transition. The liposomes were then stored at 4ºC and incubated at working temperature for at least 1 h before usage. Stock PPhe solutions were prepared in acetone, the desired volume of this solution was evaporated and mixed with 5 mL of buffer solutions. The final porphyrin concentrations used in this work were in the range 0.1-1.10 -6 M. The size distribution of the vesicles was obtained using a multi-angle Brookhaven Instruments (BI-200SM

goniometer and a BI-2030AT autocorrelator) with a He-Ne laser (632.8 nm, 35 mW) from Spectra Physics (model 127) under controlled temperature, T=32±1 ºC. A relatively monodisperse population (polydispersity =0.17) was obtained at both pH=2.5 and pH=7, with an average hydrodynamic diameter, D H =340 ± 8 nm at pH=7 (similar to that in water) and D H =145 ± 2 nm at pH=2.5 with an observation angle of 90º. The value obtained at pH=2.5 may not be associated to smaller vesicles but rather to changes in shape since a dependence on the observation angle was detected [START_REF] Han | Spontaneous fragmentation of dimyristoylphosphatidylcholine vesicles into a discoidal form at low pH[END_REF].

Photophysical studies

UV-Vis absorption spectra of the samples were measured using a Jasco V -560 spectrophotometer (Easton, MD, USA) and, when necessary, corrections for turbidity were carried out. [START_REF] Castanho | Separating the turbidity spectra of vesicles from the absorption spectra of membrane probes and other chromophores[END_REF] CD spectra were obtained with a Jasco spectropolarimeter J-720 with spectral averaging and baseline correction. The studies have been performed at a constant TSPP concentration of 2 µM in the visible range and varied DMPC concentration All cells used were

A C C E P T E D M A N U S C R I P T

quartz with 1 cm pathlengths and temperature was maintained at 32.0±0.2 ºC in a thermostated chamber by using a circulating water bath.

Steady-state fluorescence measurements were performed with a SPEX® Fluorolog spectrofluorimeter with a FL3-11 configuration. The instrumental response at each wavelength was corrected by means of a correction function provided by the manufacturer.

Fluorescence decays were obtained with a time-correlated single-photon counting (TC-SPC) technique using commercial equipment M icrotime 200 from Picoquant GmbH described elsewhere. [START_REF] Paulo | Porphyrin-dendrimer assemblies studied by electronic absorption spectra and time-resolved fluorescence[END_REF] Briefly, the measurements are performed in front-face geometry and the backscattered light is attenuated through the use of appropriate filters. Excitation was achieved using a pulsed laser diode head at 405 nm or 638 nm, with varied repetition rate (10, 20 or 40 MHz). The maximum output power is 0.4 mW and a minimum pulse width of 54 ps is obtained. A band-pass filter with a transmission in the range 600-800 nm (for 405 nm excitation) or a 695/55 (for 638 nm excitation) was used to eliminate backscattered light in the photomultiplier tube from Picoquant (model PMA-182). Data acquisition can be recorded with 4096 channels per range with a time increment smaller than 40 ps. Data analysis was performed by a deconvolution method using a non-linear least-squares fitting program, based on the Marquardt algorithm. The goodness of the fit was evaluated by the usual statistical criteria and by visual inspection of the distribution of weighted residuals and the autocorrelation function. Fluorescence Lifetime Imaging was performed in the same setup and a more detailed description may be found elsewhere [START_REF] Togashi | Lipophilic porphyrin microparticles induced by AOT reverse micelles -A fluorescence lifetime imaging study[END_REF].

Results

-Self-aggregation properties of PPhe induced by pH changes.

PPhe solubility in aqueous solutions is highly dependent on the solution pH which is related to changes in the porphyrin ionization state and the porphyrin tendency to self-aggregate. At

A C C E P T E D M A N U S C R I P T

pH=0.8, the absorption spectra show only two Q-bands (Table 1) characteristic of the porphyrin with their pyrrole nitrogens protonated, which is also reflected in a single fluorescence band centered at ~674 nm. However, the absorption Soret band is quite broad (λ max ~442 nm) and different from the excitation spectra obtained by collecting emission at 670 nm (or at 640 nm).

The latter show a narrow (fwhm~950 cm -1 ) Soret band centered at 435 nm, common to the protonated free-base porphyrins. This puts into evidence the existence of non-fluorescent aggregates which absorb essentially in the red-portion of Soret band. The contribution of such aggregates decreases as the solution pH is raised until no contribution from the diprotonated porphyrin is detected (i.e until pH=5). The emission decays (Table 2) show a biexponential behavior with a long lifetime τ F ~3.3 ns as the major contribution similar to that obtained for the diprotonated form of TSPP and a shorter one (τ F ~0.6 ns) with increasing contribution upon pH raise in the range 0.8-4, as we will discuss below.

At pH=2-4 there are some solubility difficulties which are reflected in a drastic decrease in the absorbance and fluorescence intensity. The splitting of the Q-band absorption and emission also observed hints for the presence of a neutral form. Moreover, excitation spectra obtained at 640 nm or 670 nm no longer coincide and are much broader (fwhm~2500 cm -1 ). The former shows more of species absorbing with maximum around 420 nm which indicates that neutral forms are already present contributing to solubility decrease. A blue-shifted absorption is also denoted around 405 nm which is associated to red-emission (~670 nm). The formation of aggregates and the consequent light scattering from the solutions on increasing pH in this range prevent a quantitative analysis of titration curves and thus the estimation of the pKa for the protonation of the nitrogen atoms.

By raising the temperature, at pH=3, Fig. 1A, the aggregation is disfavored, and indeed, the species absorbing more to the blue is destabilized and fluorescence emission is enhanced.

A C C E P T E D M A N U S C R I P T

The contribution of the short lifetime, τ F ~0.6 ns, is promoted at these pHs together with an increase in the long component lifetime, Table 2. Moreover, in the pH range of 4 to 5, the fluorescence decays would be more accurately described by a distribution model than with the discrete exponential one where both pre-exponential as well as lifetime components are changing.

Upon pH increase from pH=5 up to pH=9, we observe gradual changes which follow a similar

pattern. An increase in solubility is detected by absorbance and fluorescence intensity increase more pronounced at pH=5-6 which probably coincides with the changes in peripheral carboxylic groups protonation. As in the case of the diacid species, the determination of the pK a for this process was prevented by the light scattering of the solutions. For TCPP, a meso-substituted carboxyphenyl porphyrin, these changes occur in the 5-7 pH region (the highest pK a value reported is at pH=6.6 [START_REF] Clarke | Aqueous complexation equilibria of mesotetrakis(4-carboxyphenyl)porphyrin with viologens: Evidence for 1:1 and 1:2 complexes and induced porphyrin dimerization[END_REF]). Therefore, a tetra-anionic species will tend to be more soluble than a neutral one, the latter prevalent at pH=2 -4.

A decomposition of the Soret band by a Gaussian function shows two main contributions: a band centered at 403 nm which prevails in absorption and in excitation (with λ em =670 nm) and another at 415 nm more important in excitation (with λ em =640 nm). By raising the temperature in this pH region, pH=7 Fig. 1B, we see that the latter is promoted. Moreover, we see an increase in fluorescence intensity and a blue shift (from 667 to 654 nm) and could detect an isoemissive point (not shown). These data clearly suggests a deaggregation process where the aggregated species has a lower fluorescence efficiency than the monomer in which it is converting.

The overall spectroscopic features are consistent with the existence of dimers with a H-type geometry according to excitonic coupling model [START_REF] Mcrae | Enhancement of phosphorescence ability upon aggregation of dye molecules[END_REF]. As we may see from [START_REF] Schneckenburger | Timeresolved in vivo fluorescence of photosensitizing porphyrins[END_REF][START_REF] Yamashita | Picosecond timeresolved fluorescence spectroscopy of hematoporphyrin derivatives[END_REF]. In the case of bis-glycosamide derivatives of protoporphyrin IX a model was assumed in which a face-to-face dimer of porphyrins (with a lateral offset) is stacked to another one stabilized through a network of hydrogen bonds connecting the lateral glycosamide groups [START_REF] Fuhrhop | Chiral micellar porphyrin fibers with 2-aminoglycosamide head groups[END_REF]. Another model is proposed for protoporphyrin IX where protonated carboxylic groups are hydrogen bonded to the inner NH [START_REF] Scolaro | Aggregation behavior of protoporphyrin IX in aqueous solutions: Clear evidence of vesicle formation[END_REF] In the case of PPhe we could envisage that possibility for PPhe aggregates at pH below 6. At higher pH, there must be an additional role for the sulfonamide group in minimizing repulsions between the negatively charged groups of neighbor molecules and adding favorable p -p interactions together with possible H-bonding sites.

Pasternack et al. [START_REF] Pasternack | Resonance light-scattering -a new technique for studying chromophore aggregation[END_REF], showed that scattered light could be measured in a conventional spectrofluorimeter and could be related to the extent of electronic coupling between neighbour chromophores in the aggregate and respective size and geometry. The RLS of PPhe resulted in low Rayleigh scattering from the solution and a well due to photon absorption in almost the entire pH region under study (data not shown). Only at pH=0.8 -1, a more intense signal to the red of absorption (~465 nm) was found without any specific resonant peak. According to literature, in the case of TCPP a resonant peak could only be found in aqueous HNO 3 at pH=0.9 [START_REF] Choi | Counterion-dependent excitonic spectra of tetra(p-carboxyphenyl)porphyrin aggregates in acidic aqueous solution[END_REF]; whereas for TSPP J-aggregates, an even sharper and intense resonant peak red-shifted (~499 nm) from that in absorption is observed. According to RLS theory, this means that the structural arrangement of PPhe has a weak electronic coupling, involving only a dimer. For instance, in the case of Protoporphyrin IX no aggregated species could be detected at pH below 1 [START_REF] Scolaro | Aggregation behavior of protoporphyrin IX in aqueous solutions: Clear evidence of vesicle formation[END_REF]. The presence of aromatic groups at the meso-positions is considered to be necessary for the formation of porphyrin J -aggregates. However, the presence of sulphonamide and phenylalanine groups in PPhe may introduce some steric hindrance and prevent such highly

A C C E P T E D M A N U S C R I P T
organized molecular arrangement. Furthermore, the zwitterionic nature of TSPP monomers was suggested to contribute with additional Coulombic interactions to stabilize an arrangement with a larger number of interacting chromophores in close association. By opposition, in PPhe the carboxylic groups are uncharged in very acidic conditions, as in the case of TCPP, where a distribution of physical aggregate sizes was reported [START_REF] Choi | Counterion-dependent excitonic spectra of tetra(p-carboxyphenyl)porphyrin aggregates in acidic aqueous solution[END_REF].

-PPhe interaction with DMPC vesicles.

The affinity of PPhe towards DMPC vesicles was investigated. For this purpose we followed both This suggests that the same PPhe species associates with the lipidic environment of the vesicle which is "protected" from differences in the pH of the aqueous surroundings. At the interface of a phospholipid layer, water is perturbed (the network of H -bonds is modified and thus the properties of interfacial water -density, mobility, lifetime of H bond -are different from those of free water) by up to 1 nm away from it. [START_REF] Milhaud | New insights into water-phospholipid model membrane interactions[END_REF] At the same [DMPC], the ratio monomer/aggregate is always higher for the lowest [PPhe],

pointing for the preferential association of monomeric species with DMPC, which can be rationalized from the following equilibria:

[ ] [ ] DMPC K DMPC K A A A L L L W + + = 1
Eq. ( 1)

As the total absorbance of the partitioning molecule as well as its absorbance in aqueous solution, A W are known, the limit absorption in the lipid environment, A L , and the binding constant can be estimated from an A vs.

[DMPC] plot by a non-linear least square analysis of the data. An analogous equation can be drawn [START_REF] Huang | Partition coefficients of fluorescent probes with phospholipids membranes[END_REF] to calculate the binding constant from the plot of fluorescence intensity, I F , data vs.

[DMPC].

The values obtained at the two pHs are not significantly different: K L = (3.2±0.5)x10 4 M -1 at pH=3

and K L = (2.0±0.5)x10 4 M -1 at pH=7.

Data from literature show that a neutral chlorin with ring substitutions in meso positions has a quite higher binding constant (K L ˜ 114 mM -1 ), [START_REF] Kepczynski | Do liposome-binding constants of porphyrins correlate with their measured and predicted partitioning between octanol and water?[END_REF] which must be not only due to differences in the buffer and lipid nature but also due to the fact that this porphyrin does not aggregate in aqueous solutions in contrast with PPhe. The same authors reported that the cationic meso-

A C C E P T E D M A N U S C R I P T

tetra(N-methyl-4-pyridyl) porphyrin (TMpyP) incorporates into lipid vesicles ( K L ˜ 6 mM -1 ) whereas the anionic meso-tetra(4-carboxyphenyl) porphyrin (at pH=7.3) does not.

Comparatively, protoporphyrin IX with a quite different structure with two propionic acids attached on the same side of the ring deprotonated at pH=7.3 presents a much higher binding constant (K L ˜ 110 mM -1 ).

These results suggest that several factors contribute to porphyrin incorporation in membranes.

The amphiphilic character of the dye (as in protoporphyrin) takes advantage of the nature of the lipid membrane allowing for the hydrophobic core of the porphyrins to be buried in contact with the lipid alkyl chain while the charged substituent groups interact with the charged polar head of the lipid. Previous studies using TCPP with four negatively charged phenylcarboxylic groups symmetrically placed showed no binding to lipid vesicles [START_REF] Kepczynski | Do liposome-binding constants of porphyrins correlate with their measured and predicted partitioning between octanol and water?[END_REF]. In the case of PPhe incorporation can be facilitated by the presence of the aminosulfonyl groups and the hydrophobic nature of the phenylalanine groups, in an environment which leads to no charge separation i.e. increases pK a of carboxylic groups and therefore these remain uncharged.

-TSPP interaction

A C C E P T E D M A N U S C R I P T

pH=2.5: At pH=2.5, besides the four negative peripheral sulfonate groups also the two nitrogens in the inner tetrapyrrole are charged. Data from Brault et al. [START_REF] Brault | Spectrofluorimetric study of porphyrin incorporation into membrane models -evidence for pH effects[END_REF] showed that protonation of one inner nitrogen was sufficient to prevent incorporation of HP into lipid vesicles. From this, we could anticipate that TSPP with symmetrical negative groups and protonated inner nitrogens would not incorporate into DMPC bilayers. Although incorporation does not seem to occur, data obtained in this system shows markedly differences in TSPP spectra upon addition of DMPC. with KCl [START_REF] Maiti | Fluorescence dynamics of noncovalently linked porphyrin dimers and aggregates[END_REF][START_REF] Akins | Absorption and Raman-scattering by aggregated mesotetrakis(p-sulfonatophenyl)porphine[END_REF] or varying protein concentration [START_REF] Andrade | Spectroscopic studies on thr interaction of a water soluble porphyrin with two drug carrier proteins[END_REF].

Noteworthy is the fact that at higher [DMPC] the absorbance of these two new bands decreases and there is a blue shift in the monomer Soret band from 435 to around 422 nm, similar to that already reported for TSPP in AOT RM at high water contents (e.g.:[water]/[AOT]=ω 0 =50 [START_REF] Andrade | Spectroscopic studies of water-soluble porphyrins with protein encapsulated in bis(2-ethylhexyl)sulfosuccinate (AOT) reverse micelles: aggregation versus complexation[END_REF]).

The origin of the absorption band at 422 nm has been the subject of some controversy in literature. The band has been assigned to an H-aggregate of TSPP based on Raman data.

However, the calculated value for the face-to-face packing in this type of aggregate is much larger than this experimental one at 422 nm. Moreover, Scolaro et al. [START_REF] Castriciano | Aggregation behavior of tetrakis (4-sulfonatophenyl) porphyrin in AOT/water/decane microemulsions[END_REF] obtained essentially the same predominant lifetime upon TSPP excitation at 424 nm or 490 nm. Thus, it seems more plausible to attribute both bands to the splitting of TSPP B-band as a consequence of symmetry lowering of the porphyrin macrocycle in the aggregates [START_REF] Andrade | Spectroscopic studies of water-soluble porphyrins with protein encapsulated in bis(2-ethylhexyl)sulfosuccinate (AOT) reverse micelles: aggregation versus complexation[END_REF]. Additional evidence may be obtained from CD measurements.

values from the corrected bandwidth of the aggregate. This number represents the number of adjacent molecules that are collectively excited. The values obtained are shown in the inset of fig. 4C and give lower values as compared to those obtained in AOT RM but similar to those reported in CTAB micelles. The possibility of defect states in the molecular arrangement was raised to justify these lower values of N [START_REF] Maiti | J-and H-aggregates of porphyrin-surfactant complexes: time-resolved fluorescence and other spectroscopic studies[END_REF]. Perhaps the inner aqueous pool of AOT RM is a more controlled environment less prone to dynamical changes in the template as it is the case of the surfaces of micelles or vesicles.

D. Steady-state and time-resolved fluorescence:

The fluorescence obtained upon excitation at 425 nm shows a single band centered around 675 nm, which is quenched upon increase of

[DMPC] until it reaches a plateau at [DMPC]=0.25 mM, Fig. 4C. From previous data we determined a fluorescence quantum yield of 0.14 for TSPP protonated monomer in aqueous solution [START_REF] Andrade | Spectroscopic studies on thr interaction of a water soluble porphyrin with two drug carrier proteins[END_REF] whereas the same value for J-aggregates is significantly lower, 10 -3 [START_REF] Miura | Femtosecond fluorescence spectroscopy and near-field spectroscopy of water-soluble tetra(4-sulfonatophenyl)porphyrin and its J-aggregate[END_REF]. In the presence of DMPC, global fluorescence quantum yield of TSPP obtained upon excitation at 425 nm decreases drastically, inset Fig. 4C, as expected, reflecting the formation of J-aggregates at the expenses of the monomer.

When the 490 nm absorption band exists, fluorescence spectra depend on excitation wavelength. Upon excitation at such wavelength the band peak at 675 nm is replaced by a new maximum around 720 nm which corresponds to the fluorescence of J-aggregates and therefore has low intensity but increases concomitantly with addition of more DMPC since more aggregates are formed.

Time resolved measurements were performed upon excitation at 638 nm and collected in the emission range of 667 -722 nm. In the absence of lipid vesicles, the fluorescence decay is monoexponential with a fluorescence lifetime τ F = 3.83±0.02 ns, which agrees well with data published in literature [START_REF] Andrade | Spectroscopic studies on thr interaction of a water soluble porphyrin with two drug carrier proteins[END_REF][START_REF] Castriciano | Aggregation behavior of tetrakis (4-sulfonatophenyl) porphyrin in AOT/water/decane microemulsions[END_REF][START_REF] Miura | Femtosecond fluorescence spectroscopy and near-field spectroscopy of water-soluble tetra(4-sulfonatophenyl)porphyrin and its J-aggregate[END_REF]. In the presence of DMPC, the decays become bi-exponential and, on average, τ F decreases until reaching a plateau at [DMPC]>0.25 mM. A global analysis

A C C E P T E D M A N U S C R I P T

fitting of the data shows that the contribution of the longer component, assigned to TSPP protonated monomer, decreases as [DMPC] increases, by opposition to the shorter one which can be associated to the aggregated species of TSPP. In fact, upon excitation at 490 nm (Jaggregate absorption) and collecting emission in the whole spectrum, we obtained a maximal contribution of 90% (with [DMPC]=0.5 mM) from a sub-picosecond component τ 1 = 0.07±0.04 ns and 10% of a longer one (τ 2 = 3.1±0.2 ns) due to some overlap from the monomer emission. This seems in agreement with literature data, where a value of 0.1±0.03 ns was obtained for TSPP Jaggregate in CTAB micelles [START_REF] Maiti | J-and H-aggregates of porphyrin-surfactant complexes: time-resolved fluorescence and other spectroscopic studies[END_REF]. In water at pH=1, a tri-exponential decay with τ 1 = 0.055 ns (75%), τ 2 = 0.36 ns (15%) and τ 3 = 3.4 ns (10%), was reported.

E. FLIM:

Fluorescence lifetime imaging spectroscopy (FLIM) allows the lifetimes of one or more fluorophores to be spatially resolved and can be used to provide information about the state of the fluorescent species and their microenvironment, since the fluorescence lifetime is sensitive to environmental conditions such as pH, viscosity, polarity and interactions with other molecules as well as aggregation state.

A spin-coated sample of aqueous TSPP shows a rather uniform picture with a single exponential decay with τ F =4.95 ns, Fig. 5A due to TSPP monomers adsorbed to the glass coverslip. An heterogeneous image 3x3 µm in XY direction (0.02 µm/pixel), is obtained from a spin-coated sample of TSPP in the presence of [DMPC]=0.25 mM, Fig. 5B. In the latter we may see approximately round spots with average sizes in the range of 800-1000 nm (optical resolution ~320 nm), which are much larger than those obtained in solution by DLS (without TSPP). PC vesicles were reported to flatten considerably upon adsorption to the glass (width-to-height-ratio ˜5) [START_REF] Schonherr | Vesicle adsorption and lipid bilayer formation on glass studied by atomic force microscopy[END_REF]. Besides, PC vesicles were also shown to fuse in glass substrate governed by pH and ionic strength [START_REF] Cremer | Formation and spreading of lipid bilayers on planar glass supports[END_REF], thus contributing to larger adsorbed round structures. Fluorescence decays obtained within such spots show that the main contribution (A=85%) is associated to a short fluorescence lifetime, τ F =0.112 ns, which may safely be attributed to the J -aggregates

A C C E P T E D M A N U S C R I P T

fluorescence resulting from interaction of TSPP with the DMPC vesicles. Under analogous excitation and emission conditions, we were not able to detect the short lifetime for TSPP in DMPC solution due to the fact that fluorescence quantum yield from the prevailing monomers in solution (φ F ~0.14 [START_REF] Andrade | Spectroscopic studies on thr interaction of a water soluble porphyrin with two drug carrier proteins[END_REF]) is much more efficient than that of the J-aggregate (φ F ~0.001 [START_REF] Miura | Femtosecond fluorescence spectroscopy and near-field spectroscopy of water-soluble tetra(4-sulfonatophenyl)porphyrin and its J-aggregate[END_REF]).

The longer lifetime is similar to that obtained in the absence of DMPC and assigned to the monomer adsorbed. An intermediate lifetime is probably associated to some less ordered aggregates induced upon film formation on the glass.

Conclusions

We have presented s pectroscopic as well as photophysical data for two water-soluble porphyrins regarding their possible association with DMPC vesicles.

PPhe, a free base porphyrin with aminosulfonyl groups linked to an aromatic amino acid, phenylalanine, presented a complex aggregation pattern in aqueous solution strongly dependent on the pH. Under very acidic media, the predominant diprotonated PPhe coexists with some nonspecific nonfluorescent aggregates. However, upon pH increase and taking into account the spectroscopic data, a small dimension aggregate, a dimer in a co-facial arrangement predominates due to π-π interactions between porphyrin macrocycles and possible intramolecular H-bonds which involve the sulfonamide groups. Nevertheless, this porphyrin associates to the DMPC bilayer as a noncharged monomer regardless the pH of the aqueous solution. The combination of these studies with those in cells is important to envisage the potential application of PPhe as photosensitizer.

On the other hand, TSPP a monomer in solution does not incorporate within DMPC vesicles at any of the two prototropic forms studied. However, TSPP J-aggregates are induced at the vesicle surface under acidic conditions. In the case of the latter, protonation of the macrocycle together with peripheral anionic substituents are unfavorable conditions for binding to DMPC but

A C C E P T E D M A N U S C R I P T

  the absorption and fluorescence signals of PPhe in the presence of increasing concentrations of DMPC at pH 3, Fig. 2 and pH 7, Fig. 3. The latter was performed at [PPhe]=0.1 µM and 1 µM to evaluate how the presence of aggregates in solution affect the association process. The results obtained in the presence of DMPC show a similar pattern and at the highest [DMPC] studied the spectra are almost superimposable at both pHs. Likewise, in organic solvents like DMSO or dioxane, an increase in absorbance together with a significant red shift of Soret band maximum is observed. The fluorescence also reflects a similar trend: a slight red shift is detected in the fluorescence maxima. Fluorescence lifetimes obtained for PPhe in the presence of high [DMPC] are also independent of the solution pH.

  of DMPC vesicles, the absorbance signal at 422 nm, the emission at 650 nm or fluorescence excitation at 424 nm, increase with increasing DMPC concentrations until reaching a plateau value. This trend can be rationalized by means of the model in which the porphyrin is assumed to distribute between the lipid phase (L) and the aqueous phase (W), with a partition coefficient variation of the absorbance A with [DMPC] is given by:

  with DMPC vesicles. -pH=7: At physiological conditions TSPP is a tetra-anion and from what was just discussed in the previous section, it is not expected to incorporate in the DMPC bilayer. In fact, apart from an increase in the solution turbidity due to the increase in vesicle concentration, absorption spectra remain identical in the range of[DMPC] used (up to 0.5 mM). In the same way, fluorescence spectra obtained upon excitation at 425 nm show some quenching only at high [DMPC] (above 0.25 mM). The fluorescence excitation spectra are similar to absorption a t all studied concentrations confirming the absence of incorporation of TSPP in DMPC vesicles. TCPP and TSPP have the four negatively charged groups in a symmetrical placing around the π-system, thus preventing their incorporation.

A.

  Absorption spectra: The absorbance in the Soret band region, which has a maximum at 435 nm characteristic of protonated inner nitrogens, decreases together with the appearance of a new band at around 489 nm which increases following the concomitant increase in [DMPC] until up to 0.25 mM, Fig. 4A. Similarly, in the Q-band region the absorbance at 643 nm decreases and a new band appears around 705 nm that increases at the expenses of the other until [DMPC]=0.25 mM. These two new bands were already assigned to J-aggregates of TSPP. Two isosbestic points relatively well defined are found at 448 and 662 nm which indicates the interconversion of two species. Similar values were obtained when varying the ionic strength

A

  C C E P T E D M A N U S C R I P T adequate for forming highly ordered aggregates. The ability of these vesicles to induce such aggregates opens up the possibility for their application in nonlinear optics. Besides, further exciton characterization of porphyrin self-assembly may serve for a better understanding of aggregates in photosynthetic organisms. A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T

Figure 1 -Figure 2 -

 12 Figure 1 -Normalized fluorescence excitation spectra of PPhe (λ em = 640 nm) in aqueous solution and in the presence of DMPC vesicles at (A) pH=3 and at (B) pH=7: 1-[PPhe]=1 µM and T=32 ºC; 2-[PPhe]=0.1 µM and T=32 ºC; 3 -[PPhe]=0.1 µM and T=62 ºC; 4 -[PPhe]=0.1 µM, [DMPC]=0.25 mM and T=32 ºC;

Figure 3 -

 3 Figure 3 -(A) Normalized fluorescence excitation and (B) emission spectra of PPhe in the

Figure 4 -Figure 5 -Scheme 1 29 Figure 1 -Figure 2 Figure 3 32 Figure 4 -Figure 5 -

 451291233245 Figure 4 -Spectroscopic data of TSPP in aqueous solution at pH=2.5 in the presence of

table 2

 2 solvents like DMSO; and a short one (τ F ~0.7 ns) which prevails in water where aggregation is more important. Since the close proximity of chromophores in dye aggregates can promote nonradiative decay, thus contributing to fluorescence quenching, the fastest component detected in water may be attributed to PPhe oligomers

	, at pH=6 the

F ~11-12 ns) which can be safely assigned to the monomeric species and an intermediate one (τ F ~3 ns) attributable to a dimeric form, are similar to those obtained in A C C E P T E D M A N U S C R I P T

Table 1 -

 1 Absorption peaks, Soret bandwidth and fluorescence emission maxima of PPhe in different media, ([DMPC]=0.25 mM; [HSA]=25 µM; λexc=415 nm; T=32.0 ºC).

	pH	Soret	Absorption maxima/nm Q-bands		fwhm /cm -1	Emission max. /nm
	0.8	442	---	---	598	648.5	2860	675	---
	2.0-4.0	419.5 (405)	516.5	551	n.d.	n.d.	2890	666.5	715
	5.0-9.0	401 (415)	517	553	591	644	1880	669 (655)	716
	DMPC pH=3.0	418	517	548	587	n.d.	850	649.5	715
	DMPC pH=7.0	418.5	516.5	547.5	589	n.d.	904	651	716
	HSA pH=3.0	420.5	516.5	544	593.5	n.d.	885	650.5	716.5
	HSA pH=6-9	419	516	551	590	n.d.	930	648.5	715
	DMSO	419	514	548	588	642	825	650.5	716

Table 2 -

 2 Fluorescence lifetimes of PPhe in different media. λ exc =405 nm; T=25ºC.

medium A 1 (%) τ 1 /ns A 2 (%) τ 2 /ns A 3 (%) τ 3 /ns χ 2

	Buffer pH=1.0 a	68	3.53	---	---	32	0.61	1.09
	Buffer pH=3.0 a	15	4.73	---	---	85	0.61	1.09
	Buffer pH=4.0 a	5.5	5.42	23	1.00	71	0.55	1.09
	Buffer pH=7.0 a	30	11.76	12	2.66	41	0.70	1.09
	DMPC pH=3.0 b	85	11.90	9	4.32	6	0.82	1.07
	DMPC pH=7.0 b	79	11.86	9	4.40	12	0.81	1.05
	DMSO a	92	11.09	8	2.85	---	---	1.10
	HSA pH=7.0 b	99	12.10	1	3.61	---	---	1.10

a [PPhe]=1 µM; b [PPhe]=0.1 µM

Acknowledgments

. This work was supported by Project POCI/QUI/57387/2004 and 3º Quadro Comunitário de Apoio (FEDER). The authors thank Professor J.M.G. Martinho for the use of DLS equipment and Prof. João Costa Pessoa for the use of the spectropolarimeter. S.M. Andrade thanks FCT for SFRH/BPD/24367/2005 grant and R. Teixeira thanks for a grant from Pluriannual Project/CQE.

In fact, Maiti et al. [START_REF] Maiti | J-and H-aggregates of porphyrins with surfactants: fluorescence, stopped flow and electron microscopy studies[END_REF] showed that the macroscopic appearance of a linear arrangement of Jaggregates induced by K + was a fiber-like structure whereas a helical arrangement was proposed for J-aggregates induced by CTAB molecules.

C. Spectroscopic aggregate size: Although the physical size and the number of porphyrin units involved in the aggregate may be large, exciton theory allows for the calculation of the spectroscopic aggregation number, N, from the spectral width of the absorption band of the Jaggregate, this being proportional to N -1/2 . By using a previously described procedure [START_REF] Andrade | Spectroscopic studies of water-soluble porphyrins with protein encapsulated in bis(2-ethylhexyl)sulfosuccinate (AOT) reverse micelles: aggregation versus complexation[END_REF]43] RLS data were used to withdraw the scattering contribution from the absorption spectra, which in the red portion of the aggregate absorption is rather significant and hence, calculate the N A C C E P T E D M A N U S C R I P T


S Sánchez-Carrasco

J G Delcros
email: delcros@univ-rennes1.fr

A A Moya-García

F Sánchez-Jiménez

F J Ramírez
email: ramirez@uma.es

Study by Optical Spectroscopy and Molecular Dynamics of the Interaction of Acridine-Spermine Conjugate with DNA Authors

Keywords: Acridine, spermine, DNA, Spectroscopy, Molecular Dynamics

We report a spectroscopic and theoretical study of the interaction between doublestranded oligonucleotides containing either adenine-thymine or guanine-cytosine alternating sequences and N1-(Acridin-9-ylcarbonyl)-1,5,9,14,18-pentazaoctadecane, or ASC, which is formed by the covalent bonding of spermine and 9-amidoacridine moieties via a trimethylene chain. Solutions containing the oligonucleotides and the conjugate at different molar ratios were studied using complementary spectroscopic techniques, including electronic absorption, fluorescence emission, circular dichroism, and Raman spectroscopy. The spectroscopical properties of ASC at both the vibrational and the electronic levels were described by means of ab initio quantum-chemical calculations on 9-amidoacridine, used as a model compound.

Molecular dynamics calculations, based on the QM/MM methodology, were also performed using previously docked structures of two oligonucleotide-ASC complexes containing the A-T and the G-C sequence. Our data, taken all together, allowed us to demonstrate that conjugation of spermine to acridine modulates and gives additional properties to the interaction of the latter with DNA. As the ASC molecule has a high affinity by the polyamine transport system, these results are promising for their application in the development of new anti-tumor drugs.

A C C E P T E D M A N U S C R I P T

Introduction

Acridine is an heteroaromatic molecule able to intercalate into DNA [START_REF] Albert | The Acridines[END_REF]. This structural property confers antitumour capabilities to acridine and its derivatives [START_REF] Demeunynck | Interest of acridine derivatives in the anticancer chemotherapy[END_REF], since DNA is the main target in anticancer drug design [START_REF] Neidle | Chemical approaches to the discovery and development of cancer therapies[END_REF][START_REF] Graves | Intercalative binding of small molecules to nucleic acids[END_REF]. Intercalation of a planar system between DNA base pairs can induce structural alterations in such a way that cell proliferation is blocked [START_REF] Mcdonald | Inhibition of topoisomerase II catalytic activity by pyridoacridine alkaloids from a Cystodites sp. ascidian: A mechanism for the apparent intercalator-induced inhibition of topoisomerase II[END_REF][START_REF] Dias | Targeting DNA with novel diphenylcarbazole[END_REF]. A usual mechanism to explain this fact is related to the DNA-binding enzymes which have essential roles in reactions directly involved in cell proliferation, such as topoisomerases and telomerases [START_REF] Larsen | Catalytic topoisomerase II inhibitors in cancer therapy[END_REF][START_REF] Marco | Molecular determinants of topoisomerase I poisoning by Lamellarins: comparison eith Camptothecin and structure-activity relationships[END_REF]. The DNA-intercalator complexes prevent, in most cases, DNA recognition by these enzymes, thus disrupting the whole process.

It is known that one of the main problems of anti-tumour therapies is the secondary effects caused by the action of cytotoxic drugs on healthy cells. Most of the promising drugs preserve cytotoxic activity, although they have tumour cells as their main target. In the case of intercalators, this selectivity can be acquired by attaching an aliphatic side chain [START_REF] Graves | Intercalative binding of small molecules to nucleic acids[END_REF][START_REF] Andrew | Structure-activity studies of anthracycline-DNA complexes[END_REF]. These chemical substituents can also provide both cell-specificities and new contributions to DNA binding, as they can establish additional interactions with intra-cellular reactive sites. As a consequence, changes can appear in the thermodynamic parameters of binding and in useful biochemical properties, such as uptake systems or sequence selectivity. This strategy is being currently tested by functionalizing the acridine molecule [START_REF] Demeunynck | Interest of acridine derivatives in the anticancer chemotherapy[END_REF]. Acridine based anti-tumour drugs have been developed and studied [START_REF] Delisha | Acridine-modified, clamp-forming antisense oligonucleotides synergize with cisplatin to inhibit c-Myc expression and B16-F0 tumor progression[END_REF][START_REF] Baguley | Mechanisms of action of DNA intercalating acridine-based drugs: how important are contributions from electron transfer and oxidative stress?[END_REF], as amsacrine or asulacrine, which present excellent activity against some cases of leukaemia [START_REF] Sklarin | A phase II trial of CI-921 in advanced malignancies[END_REF]. Acridine derivatives recently synthesized have been proven to stabilize G-quadruplex structures in telomeres, inhibiting telomerase action [START_REF] Harrison | Trisubstituted acridine derivatives as potent and selective telomerase inhibitors[END_REF].

The biogenic polyamines are organic polycations essential for cell proliferation [START_REF] Cohen | Molecular effects on internal celular polymers: transfer RNA and DNA[END_REF].

Tumour cells uptake biogenic polyamines in greater amounts than do healthy cells [START_REF] Thomas | Polyamines in cell growth and cell death: molecular mechanisms and therapeutic application[END_REF][START_REF] Bachrach | Polyamines and cancer: minireview article[END_REF],

thus making them suitable candidates for the improving the anti-cancer action of intercalators A C C E P T E D M A N U S C R I P T like acridine. Three important features have been recently proven for a series of acridinepolyamine covalent conjugates [START_REF] Blagbrough | Asymmetric intercalation of N1-(acridin-9-ylcarbonyl)spermine at homopurine sites of duplex DNA[END_REF][START_REF] Phanstiel | The effect of polyamine homologation on the transport and cytotoxicity properties of polyamine-(DNA-Intercalator) conjugates[END_REF][START_REF] Wang | The influence of polyamine architecture on the transport and topoisomerase II inhibitory properties of polyamine DNA-intercalator conjugates[END_REF][START_REF] Carrington | A novel solid-phase reductive alkylation route to acridine and dansyl polyamine conjugates[END_REF][START_REF] Seiler | Polyamine transport in mammalian cells. An update[END_REF]: i) they present higher affinities for DNA than acridine; ii) they preserve the topoisomerase II inhibitory activity of acridine and, iii) they are recognized by the polyamine transport system. Acridine-polyamine conjugates are, therefore, promising chemical systems for the development of more efficient anti-tumour drugs.

Nevertheless, this task requires a deeper knowledge of the structure and binding properties of these molecules [START_REF] Neidle | Chemical approaches to the discovery and development of cancer therapies[END_REF], which enhances the role of physico-chemical tools in developing anticancer drugs and in understanding their structure-activity relationships.

In this work, we used electronic (absorption, fluorescence, circular dichroism) and vibrational (Raman) spectroscopies to investigate the interaction with DNA of a conjugate molecule formed by acridine and spermine bound by means of a trimethylene-amide group (hereafter ASC), Fig. 1. The affinity of spermine to the DNA major groove has been evidenced by different experimental and theoretical studies [START_REF] Ruiz-Chica | Fourier transform Raman study of the structural specificities on the interaction between DNA and biogenic polyamines[END_REF][START_REF] Feuerstein | Molecular mechanics of the interactions of spermine with DNA: DNA bending as a result of ligand binding[END_REF][START_REF] Drew | Structure of a B-DNA dodecamer. III. Geometry of hydration[END_REF]. Here, we will discuss the influence of spermine conjugation on the intercalation of acridine into DNA chains. For the DNA models, we have chosen the 16-mer oligonucleotides ds(dG-dC) [START_REF] Marco | Molecular determinants of topoisomerase I poisoning by Lamellarins: comparison eith Camptothecin and structure-activity relationships[END_REF] and ds(dA-dT) [START_REF] Marco | Molecular determinants of topoisomerase I poisoning by Lamellarins: comparison eith Camptothecin and structure-activity relationships[END_REF] .

Comparison between results from these two sequences allowed us to investigate the sequence selectivity in the DNA binding of this compound, as observed for other acridine derivatives with applied interest [START_REF] Adams | Crystal structures of acridine complexes with nucleic acids[END_REF]. Ultraviolet-visible (UV-vis) absorption, emision fluorescence, circular dichroism (CD) and Fourier transform (FT) Raman spectra of solutions containing these oligonucleotides and ASC, at different molar ratios, were recorded. Thermal denaturation experiments in the absence and in the presence of the conjugate were performed on genomic DNA and poly(dA-dT).

In order to support the experimental results, density functional theory (DFT) calculations, using ab initio methodology at the 6-31G** level and simulating the solutesolvent electrostatic interactions by means of the polarizable continuum model (PCM), were performed on the molecule 9-(N-methylamide)-acridine, as a model compound. These

A C C E P T E D M A N U S C R I P T

calculations provided us a reliable assignment of the electronic and vibrational transitions of the acridine-spermine conjugate in aqueous solution. Molecular dynamics (MD) calculations of the interaction between the conjugate and both oligonucleotide sequences were also performed on the basis of a hybrid quantum mechanics/molecular mechanics (QM/MM) approach. This methodology allows the conjugate to always be described as a quantum system, while the oligonucleotides are treated with a classical MM force field. The optimized conjugate-oligonucleotide structures for both sequences were validated by the experimental data.

Experimental

Materials. Details about the synthesis of N1-(Acridin-9-ylcarbonyl)-1,5,9,14,18pentazaoctadecane, hereafter referred to as the acridine-spermine conjugate or ASC, are given elsewhere [START_REF] Delcros | Effect of spermine conjugation on the cytotoxicity and cellular transport of acridine[END_REF]. The single-stranded 16-mer oligonucleotides ss(dG-dC) 8 and ss(dA-dT) 8 , calf-thymus genomic DNA and poly-ds(dA-dT) were synthesized by Sigma Chemical Co.

Double-stranded oligonucleotides, ds(dG-dC) 8 and ds(dA-dT) 8 , were obtained and tested as previously reported [START_REF] Ruiz-Chica | Raman study of the interaction between polyamines and a GC oligonucleotide[END_REF][START_REF] Ruiz-Chica | Raman spectroscopy study of the interaction between biogenic polyamines and an alternating AT oligodeoxyribonucleotide[END_REF]. To keep conditions close to the physiological environment, 10 mM TRIS buffer, 100 mM sodium chloride, was always used as the solvent. appreciable change in the total volume; a new spectrum was recorded after each addition. The entire protocol was repeated at least twice for each oligonucleotide, in order to confirm the results. All spectra were recorded at least 10 minutes after oligonucleotide addition, although the complex was always formed within a few seconds. To improve the separation of peaks and shoulders in overlapping absorption bands, a Fourier self deconvolution procedure was performed using a standard Gaussian line-broadening function (deconvolution factor 276; suppression factor 0.2). These parameters allowed suitable resolution of the acridine absorptions in the visible region between 300 and 450 nm.

Thermal denaturing studies were performed in a variable temperature cell Specac P/N 2100 equipped with a Copper-Constanton thermocouple for temperature monitoring purposes.

Data were always acquired after thermal equilibrium was reached, which was automatically checked by the acquisition software. Thermal analysis for poly-ds(dG-dC) was not achieved because it does not melt within the range of available temperatures. The melting temperature (T m ) was determined over the A 260 versus the temperature curves, using the first derivative method. Fluorescence spectra were measured in a Photon Technology International (PTI) spectrofluorimeter. Two kind of experiments were performed for each oligonucleotide.

Firstly, the influence of conjugate addition on oligonucleotide-bound ethidium fluorescence was studied. Secondly and separately, the behaviour of the fluorescence emission of ASC in the presence of the oligonucleotides at different molar ratios was studied. In this case, successive fluorescence spectra were recorded following the same titration procedure described previously for the absorption spectra. As previously seen in the corresponding

A C C E P T E D M A N U S C R I P T

excitation fluorescence spectra, maximal quantum yields were achieved using radiation at 471 nm for ethidium and at 360 nm for ASC. Electronic CD spectra were recorded in a JASCO J-810 spectropolarimeter. Standard quartz cells of 1 or 10 mm path length were used for conjugate concentrations higher or lower than 100 µM, respectively, in order to avoid the detector's overload of the detector. Buffer corrections were applied immediately after recording the spectra. The spectra were expressed in terms of molar ellipticity of the oligonucleotide, which was calculated from the equation [Θ] = Θ /cl [START_REF] Snatzke | Circular Dichroism: an introduction[END_REF]. In this equation Θ is the ellipticity, c is the oligonucleotide molar concentration, and l is the cell pathlength in cm.

FT-Raman spectra of the oligonucleotides, ASC and their complexes were recorded at room temperature (20 o C) by means of a Bruker Equinox 55 spectrometer supplied with a Raman module. Spectra were obtained at a resolution better than 2 cm -1 , using excitation radiation wavenumber at 1064 nm from a Nd-YAG laser working at 500 mW. Samples were analysed using a sapphire cell suitable for low volumes of liquid samples. The Raman spectra were recorded using the general procedure previously reported elsewhere [START_REF] Ruiz-Chica | Raman study of the interaction between polyamines and a GC oligonucleotide[END_REF][START_REF] Ruiz-Chica | Raman spectroscopy study of the interaction between biogenic polyamines and an alternating AT oligodeoxyribonucleotide[END_REF]. To discuss the Raman peaks, the following selection criteria were adopted: i) peaks must appear with the same wavenumber and intensity in the spectra of independent samples; ii) peaks must exhibit an intensity clearly higher than the experimental noise level; and iii) peaks should be previously reported and assigned in the literature. Spectral treatment was performed using the Bruker OPUS © spectroscopic software. All the Raman spectra were normalized to the sapphire band at 750 cm -1 .

Theoretical calculations. The GAUSSIAN'03 package of programs [START_REF] Frisch | Gaussian 03, Revision B.0[END_REF] was used for DFT quantum chemical calculations. We used the hybrid functional B3PW91 [START_REF] Becke | Density-functional exchange-energy approximation with correct asymptotic behaviour[END_REF][START_REF] Perdew | Accurate and simple analytic representation of the electron-gas correlation energy[END_REF], which has been successfully proven to accurately reproduce structural and spectroscopic features of biological molecules [START_REF] Tortonda | Aminoacid Zwitterions in solution: geometric, energetic, and vibrational analysis using density functional theory-continuum model calculations[END_REF][START_REF] Ramírez | Structural and vibrational study of the taumerism of histamine free-base in solution[END_REF][START_REF] Ruiz-Chica | On the interpretation of Raman spectra of 1-aminooxy-spermine/DNA complexes[END_REF]. To simulate an aqueous environment, a PCM was employed A C C E P T E D M A N U S C R I P T [START_REF] Miertus | Electrostatic interaction of a solute with a continuum. A direct utilization of ab initio molecular potencials for the prevision of solvent effects[END_REF][START_REF] Miertus | Approximate evaluations of the electrostatic free energy and internal energy changes in solution[END_REF].This model places the solute molecule into a size-adapted cavity formed from interlocking atom-centered van der Waals spheres, while the solvent is assimilated into a continuum characterized by its dielectric constant (78.7 for water).

Spectroscopic features were computed on the optimized molecular structure, which was obtained by allowing all the geometric parameters to vary independently. Electronic excitation energies were obtained by using time-dependent (TD) formalism [START_REF] Gross | Time-dependent density-functional theory[END_REF][START_REF] Casida | Time-dependent density functional response theory for molecules[END_REF].

Quadratic force constants were calculated from the analytical second derivatives of the molecular energy with respect to the cartesian coordinates, in the geometry of minimal energy. Raman intensities were calculated by numerical differentiation of dipole derivatives with respect to the electric field. The theoretical Raman spectrum was obtained from the calculated vibrational wavenumbers and the Raman intensities. Every Raman band was represented by a Gaussian function of 20 cm -1 half-height width.

We used the split-valence 6-31G** basis set [START_REF] Clark | Efficient diffuse function-augmented basis sets for anion calculations III. The 3-21+G basis set for the elements Li-F[END_REF][START_REF] Hariharan | The influence of polarization functions on molecular orbital hydrogenation energies[END_REF], which includes six s-type and three p-type polarization functions, for structural optimization, contour surfaces and timedependent calculations. Force field, vibrational wavenumbers, Raman intensities and normal coordinates were computed using the 3-21G* basis set, with only s-type polarization functions, over the 6-31G** optimized geometry. The use of polarization functions is necessary in order to correctly describe the solute-solvent interactions, because they allow for a suitable charge distribution adapted to the presence of an external electric field, such as a polar environment.

Docking calculations were performed using the GRAMM (Global Range Molecular Matching) v1.03 program [START_REF] Vakser | Protein docking for low-resolution structures[END_REF]. This is a fast Fourier transform based method that works from the atomic coordinates of the two interacting systems, without requiring any additional information about the binding sites. The structure of the complex is achieved after a systematic six-dimensional grid search through the relative molecular rotations and translations. Atomic coordinates of the ASC and the oligonucleotides were obtained in

A C C E P T E D M A N U S C R I P T different ways.
The ASC geometry of minimal energy was obtained using the GAUSSIAN'03 package at the same level of calculation previously described, although not including solvent effects. Atomic coordinates for the oligonucleotides were obtained from standard structural parameters for nucleic acids using the HyperChem program [43].

The two better-scored intercalated ASC-oligonucleotide structures, one for each sequence, were used as starting points of a deeper theoretical study based on QM/MM hybrid methodology. Once the initial interaction was defined, the complete system was placed in a 68 x 47 x 42 Å side box of water molecules, from which molecules overlapping with ASC or the oligonucleotides were removed. The simulation box also contained sodium ions to compensate for the negative charges of the ASC-oligonucleotide complex, resulting in a electrically neutral system hydrated with 1284 water molecules. The full system was described using a hybrid QM/MM Hamiltonian. The ASC molecule was the quantum subsystem, being treated using the semiempirical Hamiltonian AM1 [START_REF] Dewar | Development and use of quantum mechanical molecular models. 76. AM1: a new general purpose quantum mechanical molecular model[END_REF]; the oligonucleotides, water molecules and sodium ions were the classical sub-system, being treated at a MM level.

To describe the oligonucleotide sequences, we used the CHARMM force field as developed by MacKerell et al. [START_REF] Foloppe | All-atom empirical force field for nucleic acids: 1. parameter optimization based on small molecule and condensed phase macromolecular target data[END_REF][START_REF] Mackerell | All-atom empirical force field for nucleic acids: 2) Application to molecular dynamics simulations of DNA and RNA in solution[END_REF], whereas we used a flexible TIP3P potential for water molecules [START_REF] Jorgensen | Comparison of simple potential functions for simulating liquid water[END_REF]. Sodium ions and van der Waals parameters of the ASC molecule were also taken from the CHARMM force field [START_REF] Brooks | CHARMM: A program for macromolecular energy, minimization and dynamics calculations[END_REF].

Once the material model was defined, we were ready to study the behaviour of the two ASC-oligonucoleotide models. A detailed theoretical protocol has been included as Supplementary Material. All calculations were made with the CHARM31 program [START_REF] Brooks | CHARMM: A program for macromolecular energy, minimization and dynamics calculations[END_REF]. We employed periodic boundary conditions and a switched cut-off radius of 10 Å for all types of interactions. The bond lengths of all hydrogen atoms of the classical sub-system were kept constrained to their equilibrium lengths using the SHAKE algorithm [START_REF] Ryckaert | Numerical integration of the Cartesian equation of motion of a system with constraints: molecular dynamics of N-alkanes[END_REF]. MD simulations were carried out using the leap-frog [START_REF] Swope | A computer simulation method for the calculation of equilibrium constants for the formation of physical clusters of molecules: Application to small water clusters[END_REF] integration algorithm with a time step of 1 fs.

A C C E P T E D M A N U S C R I P T

Results and Discussion

Results obtained from the spectral measurements

The spectrum of ASC in the absence of oligonucleotides is shown in Fig. 2. It exhibits two band systems: a very strong absorption in the UV region, centred at 251 nm (which has a second component observed as a shoulder at a higher energy), and a more complex system in the visible region with a maximum at 360 nm. Fourier self-deconvolution enabled us to obtain the main components of this broad band, which were measured at 345, 375 and 388 nm. This medium-intensity multiplet presents the advantage of being free of the oligonucleotide absorptions which appear at lower wavelengths. This region, therefore, is suitable for monitoring the conjugate-oligonucleotide interaction. In order to provide a supported assignment of these bands, we carried out a theoretical prediction of the electronic excitation energies. Taking into account that the electronic absorption in the visible region should correspond to π→π* and n→π* transitions [START_REF] Fornasiero | Analysis of the visible-near-ultraviolet spectrum of 9aminoacrdine using dichroic spectra in stretched polymer films[END_REF][START_REF] Van Holde | Principles of Physical Biochemistry[END_REF], TD calculations were carried out on the 9-(N-methyl-amide)-acridine molecule. The results concerning the calculated energies of the electronic transitions and their assignments in terms of the frontier molecular orbitals are available as Supplementary Material. The calculated wavelengths fit well to the experimental values, and the assignments indicate that the band system between 300 and 400 nm observed in the ASC electronic absorption spectrum involves a partial charge transfer from the amide group to the acridine ring.

We have displayed in Fig. 3 (Panel 1) the effect of the oligonucleotide addition in the electronic spectrum of ASC between 300 and 400 nm. A table containing the relevant wavelength changes has been included as Supplementary Material. Both figures show strong hypochromism and a red-shift of the absorption maxima of ASC when increasing the oligonucleotide concentration. The titration with ds(dA-dT) 8 also shows an isosbestic point at

A C C E P T E D M A N U S C R I P T

393 nm, which was not observed for ds(dG-dC) 8 . On the other hand, 73% and 65% of the final hypochromism of the 360 nm band were measured in the first titration step for ds(dA-dT) 8 and ds(dG-dC) [START_REF] Marco | Molecular determinants of topoisomerase I poisoning by Lamellarins: comparison eith Camptothecin and structure-activity relationships[END_REF] , respectively, that is to say, at an ASC:oligonucleotide molar ratio of The Raman spectra of aqueous solutions containing the oligonucleotides alone and in the presence of either 3 and 10 mM of ASC are shown in Fig. 7. Wavenumbers for the more relevant bands and their assignments are summarized in Tables 1 and2, which correspond to ds(dA-dT) 8 and ds(dG-dC) 8 , respectively. Under our experimental settings, the solutions of ds(dA-dT) 8 were transparent and presented a relatively high fluidity. The assignments of the oligonucleotide Raman bands are based on vibrational studies reported for the polymer, and they have been widely discussed elsewhere [START_REF] Ruiz-Chica | Raman study of the interaction between polyamines and a GC oligonucleotide[END_REF][START_REF] Ruiz-Chica | Raman spectroscopy study of the interaction between biogenic polyamines and an alternating AT oligodeoxyribonucleotide[END_REF]. The assignments of the ASC bands have been based on a force field and the same normal coordinate calculations for the molecule as the electronic structure calculations, using DFT methodology at the 6-31G** level. A set of the obtained theoretical vibrational data has been included as Supplementary Material.

A C C E P T E D M A N U S C R I P T

Analysis and discussion of the spectral results.

The aforementioned spectral data support the following conclusions:

1.

The acridine moieties of ASC can intercalate between adjacent base pairs of oligonucleotide sequences. The first evidence of this conclusion is found in the hypochromism and red-shift observed by UV-vis absorption spectroscopy, Fig. 3. ASC is also able to displace ethidium from its intercalation sites in oligonucleotides, as shown in the Fig. 5. The observed splitting of the phosphodioxy stretching band in the Raman spectra of both ds(dA-dT) 8 and ds(dG-dC) 8 are also a consequence of the structural re-organization of the double helix upon acridine intercalation.

2.

One 16-mer double-stranded chain can host more that one ASC molecule by intercalation. This fact is evidenced by comparing the intensity reduction of the ASC electronic absorption band in the initial steps of the titrations, that is to say, when an excess of conjugate is present. Thus, we measured 73% and 65% of the final hypochromism for ds(dA-dT) 8 and ds(dG-dC) 8 , respectively, in the first oligonucleotide addition, which corresponds to an oligonucleotide:ASC molar ratio of 1:3. A similar behaviour was observed in the fluorescence spectra showed in the Fig. 5, thus supporting this assertion.

3.

The isosbestic point at 393 nm observed in Fig. 3 indicates the existence of a conformational equilibrium between bonded and free ASC molecules during the titration with ds(dA-dT) 8 . The thermal denaturing curves, Fig. 4, exhibit structured profiles for both poly-ds(dA-dT) and calf-thymus DNA. These profiles are explained as the result of at least two melting processes [START_REF] Crothers | Statistical Thermodynamics of Nucleic Acid Melting Transitions with Coupled Binding Equilibria[END_REF][START_REF] Mcghee | Theoretical Calculations of the Helix-Coil Transition of DNA in the Presence of Large, Cooperatively Binding Ligands[END_REF]. The first of them would be a partial opening of the helix near the occupied sites, accompanied by a redistribution of the bound ligands to allow the helix to melt in larger loops. In a second process, the ligands are removed from the helix before complete melting. However, it has been theoretically

A C C E P T E D M A N U S C R I P T

demonstrated that multiphasic melting profiles is not an evidence cooperative binding [START_REF] Mcghee | Theoretical Calculations of the Helix-Coil Transition of DNA in the Presence of Large, Cooperatively Binding Ligands[END_REF]. On the other hand, the observed increases of the melting points indicates stabilization of the duplexes, which is an usual characteristic of groove interaction.

4.

Evidence of the interaction of the spermine tails with the double-helix grooves was ii) The UV-absorption titrations of ASC exhibit differences with respect of the base sequence (hypochromism, behaviour of the main oligonucleotide absorption band, presence of isosbestic points).

iii) ASC is able to remove more efficiently ethidium from ds(dG-dC) 8 than from ds(dA-dT) 8 .

iv) The quenching of the fluorescence of ASC when adding ds(dA-dT) 8 is noticeably different to that observed for ds(dG-dC) 8 .

v) The CD spectra of ds(dG-dC) 8 in the presence of ASC exhibit a biphasic signal which has been assigned to the acridine moieties placed in a chiral environment (the intercalation sites). This phenomenon was not observed for ds(dA-dT) 8 .

A C C E P T E D M A N U S C R I P T

The Raman spectra, Fig. 7, allowed us to extend the structural details known so far on the ASC-oligonucleotide complexes. The measured wavenumber shifts for the ds(dA-dT) 8

Raman bands at 1248 and 729 cm -1 could be due to the interaction of the spermine side chain with reactive sites of the bases of the oligonucleotide grooves. This hypothesis is also supported by the most intense ASC Raman band, measured at 1418 cm -1 , which has been assigned to a methylene bending vibration of the side chain, ν(CH 2 ), scissoring. It appeared at 1416 cm -1 in the Raman spectrum of the ds(dA-dT) 8 solution, 3 mM in ASC. The ds(dA-dT) 8

band at 1577 cm -1 was measured at 1580 cm -1 at 10 mM ASC. This band involved relevant contributions from both the C(6)-NH 2 bending vibration (major groove) and aromatic stretching modes in which the N(3) atom (minor groove) presents a significant vibrational amplitude [START_REF] Dhaouadi | Vibrational motions of bases of nucleic acids as revealed by neutron inelastic scattering and resonance Raman Spectroscopy. 1. Adenine and its deuterated species[END_REF]. Interaction with adenine was also supported by the ds(dA-dT) 8 band at 1341 cm -1 , which has been assigned to a stretching vibration of the purine moieties [START_REF] Movileanu | Temperature dependence of the Raman spectrum of DNA. Part I-Raman signaturas of premelting and melting transitions of Poly(dA-dT)-Poly(dA-dT)[END_REF][START_REF] Movileanu | Temperature dependence of the Raman spectrum of DNA. II Raman signatures of premelting and melting transitions of poly(dA)-Poly(dT) and comparison with poly (dA-dT)•poly(dA-dT)[END_REF]. It also shifted upwards by adding 10 mM ASC, similar to the 1577 cm -1 band. Upshifting of a stretching vibration is evidence of stronger bonds, which are often accompanied by downshifting of the related bending modes [START_REF] Mayo | Course notes on the interpretation of infrared and Raman spectra[END_REF]. Here, this fact was observed for the purine bending vibration at 729 cm -1 , which is considered to be an adenine marker band [START_REF] Movileanu | Temperature dependence of the Raman spectrum of DNA. Part I-Raman signaturas of premelting and melting transitions of Poly(dA-dT)-Poly(dA-dT)[END_REF][START_REF] Katahira | Local and overall conformations of DNA double helices with the A-T Base pairs[END_REF] being measured at 727 cm -1 in the presence of 10 mM ASC.

Interaction of the spermine tail of ASC with the nucleotide bases of ds(dG-dC) 8 was supported by the measured shifts in the oligonucleotide bands at 1317 cm -1 (guanine), 1219 cm -1 (cytosine) and 1177 cm -1 (guanine, cytosine) [START_REF] Ruiz-Chica | Raman study of the interaction between polyamines and a GC oligonucleotide[END_REF][START_REF] Benevides | Characterization of DNA structures by Raman spectroscopy: high-salt and low-salt forms of double helical poly(dG-dC) in H2O and D 2 O solutions and application to B, Z and A-DNA[END_REF]. Similar to the ds(dA-dT) 8 solutions, the δ(CH 2 ) band at 1418 cm -1 shifted to 1416 cm -1 , while the acridine vibrations measured at 1308 and 1221 cm -1 downshifted by 5 and 4 cm -1 , respectively. On the other hand, the amide III band at 1379 cm -1 exhibited the opposite behaviour of that observed with ds(dA-dT) 8 , which indicates that the amide group of the intercalated ASC molecules interacts differently in these two sequences. present in the solid phase, while monovalent cations remain partially in solution. This prediction agrees with the absence of a significant ASC Raman signal at 10 mM ASC upon oligonucleotide precipitation. However, this phenomenon was not observed for the ds(dA-dT) 8 sequence, supporting that cation-induced precipitation of DNA and other nucleotide chains cannot be exclusively explained on the basis of charge neutralization, but a significant degree of structural specificity is involved [START_REF] Raspaud | Precipitation of DNA by Polyamines: A Polyelectrolyte Behavior[END_REF][START_REF] Thomas | Ionic and structural effects on the thermal helix-coil transition of DNA complexed with natural and synthetic polyamines[END_REF][START_REF] Saminathan | Ionic and structural specificity effects of natural and synthetic polyamines on the aggregation and resolubilization of single-, double-, and triple-stranded DNA[END_REF][START_REF] Saminathan | Polyamine structural effects on the induction and stabilization of liquid crystalline DNA: potential applications to DNA packaging, gene therapy and polyamine therapeutics[END_REF][START_REF] Geall | Efficient calf thymus DNA condensation upon binding with novel bile acid polyamine amides[END_REF].

Molecular dynamics calculations.

As aforementioned, we performed MD calculations on the basis of a hybrid QM/MM approach in order to find interactional theoretical models in agreement with previous experimental results. Since there is a large conformational flexibility of oligonucleotides, MD simulations are often dependent on the starting structure [START_REF] Becker | Dynamics Methods[END_REF]. In order to build suitable ASC-

A C C E P T E D M A N U S C R I P T

oligonucleotide starting models, the Protein Data Bank (PDB) [START_REF] Berman | The Protein Data Bank[END_REF] provided us with geometric parameters of a drug-intercalated 12-mer double-stranded oligonucleotide. Then, we removed the drug and we substituted the nucleic bases one-to-one to build the two alternating purine-pyrimidine double-stranded sequences, ds(dA-dT) 6 and ds(dG-dC) 6 , which maintaining the original inter-base hole able to host a planar molecule. The ASC structure was optimized using DFT/6-31G** gas-phase methodology.

High-resolution rigid-body docking calculations were performed for the two ASColigonucleotide systems. Up to 1000 matches, using a grid step of 1.7 Å, was scored for each sequence. After removing the matches based on end-chain interactions, the best scored structures for both sequences presented the acridine ring intercalated into the double helix, as can be seen in Fig. 8. On the other hand, the calculated interactional energy for the best scored complex with ds(dG-dC) 6 was higher (in absolute value) than for the one with ds(dA-dT) 6 . This result is compatible to a deeper interaction of ASC with the G-C sequence, which agrees with the Raman data. However, the lack of torsional flexibility enables interaction of the tail with the reactive sites of the grooves.

The stability of the two best scored ASC-oligonucleotide complexes obtained by docking calculations was checked using QM/MM methodology. In both cases, the MD simulations were successful, thus achieving relaxed final structures. They are shown in Fig. 8.

As it can be observed, the two final complexes exhibit a fully intercalated acridine ring. Three main common facts can be pointed out: i) conformational changes of the bases in the hole, ii) appreciable out-of-plane deviations of the acridine moieties, and iii) adaptation of the spermine tail to the oligonucleotide groove to achieve additional interatomic contacts. The highest deviations measured for the dihedral angles within the acridine ring were 24 and 21 deg. for the complexes with ds(dA-dT) 6 and ds(dG-dC) 6 , respectively.

The optimized complexes ASC-ds(dA-dT) [START_REF] Dias | Targeting DNA with novel diphenylcarbazole[END_REF] shows the acridine ring deeply intercalated into the double-helix. As a consequence, it is able to interact with the two

A C C E P T E D M A N U S C R I P T

adenine-thymine base pairs of the hole. The Watson-Crick hydrogen bonding is broken in the hole, which allows the amide group of ASC to interact with the adenine-N1 atom. The H ... N1 calculated distance is 1.98 Å, which is within the range of a medium-intensity hydrogen bond [START_REF] Taylor | Comparison of X-ray and neutron diffraction results for the N-H … O=C hydrogen bond[END_REF]. In addition, the oxygen atom of the amide group has captured one acidic hydrogen atom of the second NH 2 + group of spermine. These interactions agree with the observed Raman shift of 5 cm -1 for the amide III band of ASC at 1379 cm -1 . The C(6)-NH 2 moieties of the two adenine bases of the hole have rotated their amino groups by about 90 deg with respect to their starting positions, which is well correlated with the observed behaviour for the oligonucleotide Raman band at 1577 cm -1 . As can be easily observed in the Fig. 8, the spermine side chain is adapted to the groove shape, which allows it to establish weak atomic contacts with the phosphate groups and the adenine bases.

The structural specificities of the complex with ds(dG-dC) 6 are different than those observed for ds(dA-dT) 6 . Thus, the π-stacking interaction of the acridine moiety is only clearly established with a purine ring within the hole. This theoretical prediction could explain the observed trends in the fluorescence spectra (Fig. 5). If we accept that the intensity reduction upon intercalation was assigned to a charge-transfer phenomenon between the bases and the ligand, the greater π-stacking predicted for acridine with the bases of the adeninethymine oligonucleotide will give rise to a more efficient charge-transfer. As a consequence, quenching of the ASC fluorescence will be more intense, as observed in the Fig. 5. On the other hand, the amide group of ASC interacts through the hydrogen atom with a cytosine-N3 atom (1.90 Å), which has also lost its hydrogen bond with the corresponding guanine base. In this case, no hydrogen migration was predicted, which can be well correlated with the different response shown by the amide band at 1379 cm -1 of ASC, depending on the oligonucleotide sequence. The side chain deviation from the linear structure was greater in the complex with ds(dG-dC) 8 . As a consequence, stronger hydrogen bonds are established, which involve a guanine-O6 atom (1.66 Å) and a phosphate moiety (1.64 Å). This result is relevant because a strong interaction of the polycationic tail is required to induce oligonucleotide condensation at high ASC concentrations, as demonstrated by Raman spectroscopy.

Conclusions

In summary, our results present a coherent set of experimental and theoretical data aimed at finding new insights about the interaction of DNA with intercalator drugs. In this study we have obtained new insights on preferential binding sites of the acridine-spermine conjugate. Our results indicate that the spermine tail modulates the interaction of acridine with oligonucleotide chains. This interaction appears as base sequence dependent, which was not previously reported for acridine. Our findings support the possibilities of these new molecules to be applied to anti-tumor pharmacology and biotechnology, since they add several specific characteristics provided by the polyamine moiety to the previously described interesting properties of acridine derivatives. Thus, we can postulate a potential application as stabilizers of GC-containing sequences, maybe blocking their ability to be substrates (or ligands) of essential DNA-modifying enzymes (proteins). Further efforts, coming from biophysics and molecular biology, will be necessary to complete the characterization of the potential usefulness of these compounds.

  Final pH was adjusted to the physiological value (7.5) by using aqueous hydrochloric acid. Stock solutions of 120 mM (in phosphate) oligonucleotides were stored at 4 o C until ready for use in the experiments. Solutions for different experiments were prepared by dilution of these stock solutions up to the suitable concentration. Spectral measurements. UV-vis absorption spectra were recorded in an Agilent 8453 UV-visible spectrophotometer supplied with a diode array detector. The oligonucleotideconjugate interaction was studied by observing the behavior of the conjugate absorption bands in the visible region, where they are free from oligonucleotide absorption, while A C C E P T E D M A N U S C R I P T increasing the oligonucleotide concentration, either ds(dG-dC) 8 or ds(dA-dT) 8 . The following general procedure was used: an initial spectrum of a 2 µM ASC solution was recorded; then, 5 1-µl aliquots of an oligonucleotide solution of 40 mM (in phosphate) were sequentially added to increase the concentration until reaching a final titration point of 120 µM without an

3: 1 .Fig. 5 (

 15 Fig. 5 (Panel 1) shows lowering of the fluorescence emission band of oligonucleotide-

  found in the very intense bands measured in the CD spectra of the oligonucleotides at ASC concentrations of 250 µM. These intensity increase are distinctive of cholesteric liquid-crystal structures named Ψ-DNA[START_REF] Fornasiero | Circular dichroism spectra and the interaction between acridine dyes and deoxyribonucleic acid[END_REF][START_REF] Zacharias | Condensed form of (dG-dC)n´(dG-dC), as an intermediate between the B-and Z-Type conformations induced by sodium acetate[END_REF][START_REF] Phillips | Circular differential scattering and circular differential absorption of DNA-protein condensates and of dyes bound to DNA-protein condensates[END_REF][START_REF] Livolant | The highly concentrated liquidcrystalline phase of DNA is columnar hexagonal[END_REF][START_REF] Leforestier | Liquid crystalline ordering of nucleosome core particles under macromolecular crowding conditions: evidence for a discotic columnar hexagonal phase[END_REF]. They are highly ordered structures which give rise to long-range interactions between the base pairs and, as a consequence, strong increases of the oligonucleotide CD bands[START_REF] Gray | A new approach to the study of secuence-dependent propierties of polynucleotides[END_REF][START_REF] Ramírez | Effects of aminooxy analogues of biogenic polyamines on aggregation and stability of calf thymus DNA[END_REF].5.The interactions of ASC with ds(dA-dT) 8 and ds(dG-dC) 8 exhibited different spectroscopic features, which can indicate base selectivity. The main features which support this point are the following:i) The melting curves of poly-ds(dA-dT) and DNA behave differently with the presence of ASC.

Fig. 7 (

 7 Panel 2) shows that a significant intensity reduction A C C E P T E D M A N U S C R I P T of the Raman intensities of both ds(dG-dC) 8 and ASC is observed when increasing the ASC concentration to 10 mM. This fact indicates that these molecules are no longer in solution, but a solid phase has appeared as a consequence of the interaction between these two polyelectrolytes. It has been demonstrated that DNA collapse is observed in concentrated DNA solutions (millimolar) regardless of the DNA length [68-70]. Raspaud et al. [68] found an empirical relationship to calculate the minimal counterion concentration required to induce DNA collapse, C ion = [C DNA ] 0,77±0.03 , which was in good agreement with the theoretical predictions. At 60 mM oligonucleotide (in phosphate) this equation predicts a minimal cation concentration between 26.5 and 20.7 mM. Taking into account that ASC is a polycation with a molecular charge near +4 at our experimental settings, this threshold was clearly surpassed at 10 mM ASC concentration (equivalent to almost 40 mM in positive charges), while at 3 mM, the cation concentration (near 12 mM) is still insufficient. The same authors predicted that, upon cation-induced precipitation of DNA or oligonucleotides, polycations are all

Figure 1 .

 1 Figure legends

Figure 2 .

 2 Figure 2. Electronic absorption spectra of ASC (solid lines) and deconvoluted spectra (dotted

Figure 3 .

 3 Figure 3. Electronic absorption spectra in the visible region of solutions 2 µM ASC and increased concentrations of the oligonucleotides up to 120 µM. The oligonucleotide concentration increment in each step was 20 µM. Panel 1: ds(dA-dT) 8 . Panel 2: ds(dG-dC) 8 .

Figure 4 .

 4 Figure 4. Thermal denaturing curves from the electronic absorbance at 260 nm (solid lines) of poly-ds(dA-dT) 8 and ct DNA, both at a concentration 60 µM in phosphate, alone (circles) and in the presence of 3µM ASC (squares). Temperature increment was 2 or 0.5 o C for

Figure 5 .

 5 Figure 5. Panel 1: fluorescence emission spectra of oligonucleotide-intercalated ethidium (2 µM of ethidium and 60 µM, in phosphate, of the oligonucleotides) in the presence of 0, 4 and 8 µM of ASC. Panel 2: fluorescence emission spectra solutions 2 µM ASC in the presence of increased concentrations of the oligonucleotides up to 80 µM. The oligonucleotide concentration increment in each step was 20 µM.

Figure 6 .

 6 Figure 6. Electronic CD spectra of the oligonucleotides (60 µM in phosphate) in the presence of different concentrations of ASC: 10 µM (circles), 50 µM (diamonds), 200 µM (squares) and 500 µM (triangles). Solid lines are the spectra in absence of ASC.

Figure 7 .

 7 Figure 7. Raman spectra of solutions (A) 60 mM oligonuleotide, in phosphate; (B) 60 mM

Figure 8 .

 8 Figure 8. Comparison between the docked (left) and MD relaxed (right) structures of ASC

Figure 8

 8 Figure 1

Table 1 .

 1 Wavenumbers and assignments for relevant bands measured in the Raman spectra of solutions 60 mM ds(dA-dT) 8 , in phosphate, and different concentrations of

	ASC.
	ds(dA-dT) 8	ds(dA-dT) 8 3 mM ASC	ds(dA-dT) 8 10 mM ASC	ASC	Assignments a
			1594	1596	acridine, (arom)
	1577	1577	1580		adenine (C6-NH 2 , N3)
			1538	1541	acridine, (arom)
	1510	1510	1509		adenine
		1495	1495	1495	acridine, (C-H) + (arom)
	1461	1459	1458		deoxyribose
		1416	1415	1418	(CH 2 )
			1384	1379	amide III + ( arom)
	1375	1375			thymine (CH 3 ),
	1341	1342	1344		adenine
			1306	1308	acridine, (C-H)
	1304	1301	1301		adenine, thymine
			1280	1282	(arom) + (C-H)
	1248	1251			adenine (C6-NH 2 ), thymine
	1092	1092	1092		phosphodioxy
		1021	1018	1021	acridine, (C-H)
	840	840/832	839		phosphodiester,deoxyribose
	793	793	793		phosphodiester, thymine
	729	727	727		adenine (N3)

a Symbols used: (stretching), (bending), (out-of-plane bending).

Table 2 .

 2 Wavenumbers and assignments for relevant bands measured in the Raman spectra of solutions containing 60 mM ds(dG-dC) 8 , in phosphate, alone and in the

	presence of 3 mM ASC.
	ds(dG-dC) 8 ds(dG-dC) 8 -ASC	ASC	Assignments a
	1577	1578		guanine (N3)
	1488	1488		guanine (N7)
		1416	1418	(CH 2 )
		1375	1379	amide III + ( arom)
	1362	1364		deoxyribose
	1317	1314		guanine
		1303	1308	acridine, (C-H)
	1291	1294		cytosine
		1218	1221	acridine, (C-H)
	1177	1173		cytosine, guanine
		1092		phosphodioxy
	896	896		phosphodiester, deoxyribose
	828	828/816		phosphodiester
	784	784		phosphodiester, cytosine

a Symbols used: (stretching), (bending).

supplying the acridine-spermine conjugate. We would also like to thank COST-922 (ESF) program and PAI (research groups FQM-159 and CVI-267) for financial support.

Acknowledgements

The Spanish Ministry of Science and Technology, grants CTQ2006-00686/BQU and SAF2005-01812, has supported this work. The authors greatly thank Prof. I.S. Blagbrough for

A C C E P T E D

M A N U S C R I P T