Chapter
1
Introduction
The aim if this thesis is to merge spectroscopic and theoretical investigations to shed light in the complex mechanism of binding of a typical molecular fluorescent rotor (Auramine) to deoxyribonucleic acid (DNA).
1. Fluorescent molecular rotors
In the present context, the term fluorescent molecular rotor is used to describe a family of fluorescent molecules featuring intramolecular charge transfer (ICT) and a twisting motion in the relaxation process of the excited states [1]. This group of fluorophores is often also known as twisted intramolecular charge transfer (TICT) complexes. After photoexcitation, the molecular rotor can go back to the ground state from the locally excited (LE) state or from a twisted conformation either radiatively or nonradiatively.[2] It has also to be noted that the twisted-state de-excitation rate is highly dependent on the local environment, namely on the microviscosity and polarity of the solvent; this feature has been intensively investigated for its importance in the tuning of the radiative and nonradiative properties of the system.
The common feature of an ICT dye is the presence of an electron donor group, that usually provides n-electrons, and an acceptor groups connected via a π-conjugation system, often called the bridge, (see Fig 1).
D π A
Figure 1. General structure of a molecular rotor, highlighting the electron donating subunit (green), the electron accepting subunit (red), and the spacer, or bridge, unit (blue).
This π-conjugation allows interaction between the donor and acceptor groups so that the non-bonding electrons of the donor can be delocalized to the unoccupied molecular orbitals, mainly localized on the acceptor group. In this configuration, the response to photoexcitation consists in an intramolecular charge transfer from the donor to the acceptor unit. Usually, the three subunits (the donor, the bridge and the acceptor) assume a planar or near-planar configuration in the ground state. In the excited state the promotion of an electron to an anti-bonding orbital causes an intramolecular twisting motion of the sub-groups relative to each other [3]. This non-planar (twisted) geometry usually has a lower excited-state energy and a higher ground-state energy than the planar one. As a consequence, the relaxation from the twisted state causes fluorescence emission either to be red-shifted or to totally disappear, depending on the system [2, 4, 5]. As shown in Fig. 2, the twisted configuration generally has a lower excited state energy and higher ground state energy than the planar structure, but the vertical LE is seprated from the TICT by an energy barrier. [6].
The dihedral angle which defines the excited state torsional motion is not the only coordinate that has to be taken into account when dealing with the twisting: many other coordinates are in fact involved, such as the bond lengths of the π subunit (usually resumed in the BLA –Bond Length Alternation– value) [7, 8], the set of coordinates relative to a change of hybridization of the electron donor atom [3], and the set of coordinates relative to environment (either solvent or organized environment) reorientation [9]. For example, for a set of molecules with a nitrogen acting as an electron donor, Zachariasse and co-workers observed that the twisting requires the nitrogen atom in the donor group to be able to undergo
1 Fluorescent molecular rotors 3
Figure 2. Ground-state and excited-state energies in the planar and twisted configurations of a TICT molecule. By absorption the systems reaches the LE state in the planar configuration; from the LE state, the molecule can return to the ground state emitting a photon either directly or after intramolecular rotation. From the twisted state, the molecule can return to the ground state or back to the LE state: the probability of the return to LE is usually very small, but it can increase in nonpolar solvents [2].
a change from a pyramidal conformation to a planar conformation going from the ground state to the excited charge transfer state, and that placing the amino group within a heterocyclic ring, thus delaying the planarization of the nitrogen, causes an overall increase in the energy barrier between LE and TICT states [10–13]. Also the atoms of the acceptor groups have to change their hybridization in the excited state (for example the central iminic nitrogen in Auramine is planar in the ground state and pyramidal in the excited state) and any structural modification that delays this conformational change should result in an increase of the energy barrier between the two excited state geometries.
Without any doubt, the most important feature of molecular fluorescent rotors is the sensitivity of their optical characteristics to the environment, that makes them useful in a very wide range of applications. The main goal of the first studies (see for example the work on Auramine by Oster and Nishijima [14]) was the understanding of the dependence of fluorescence quantum yield from the viscosity
of the solvent. However, finding the fluorescence quantum yield dependence from the viscosity for a certain rotor is not an easy task, as it is different for each molecule, because the viscosity probed by the rotor is not always equal to the macroscopic (shear) viscosity. In fact specific interactions with some components of an inhomogeneous environment (from a nanoscale point of view) comes into play. For example, if a positively charged rotor binds preferentially (or exclusively) polyanions, the addition of other kind of a polycation polymer will not cause any effect on the observed optical properties, although shear viscosity is increased. Moreover, in some cases, relaxation rates depend strongly on the polarity of the solvent, and interpretation of data is difficult because it requires an adequate and reliable kinetic model. Furthermore, for some systems the dependence of quantum yield from solvent polarity and viscosity cannot be decoupled. Finally, specific interactions with the solvent (e.g. hydrogen bonds) can increase or decrease the energy barriers between the excited states, adding another variable to this already complex picture.
Fluorescent Molecular Rotors have recently received popularity thanks to their easy applicability as non-mechanical viscosity sensors. The very high sensitiv-ity towards viscossensitiv-ity changes allowed the development of optical methods based on fluorescent molecular rotors having a precision comparable to the ones based on commercial mechanical rheometers [15] with shorter measurement times [16]. Moreover, FMRs have an immense variety of biological and biomedical applica-tions. For instance, they can be used for protein characterization and local mi-croviscosity imaging [17–20]. Accordingly, there is high interest in enlightening the detailed mechanism of molecular rotors binding to biosubstrates and on the analysis of the photophysical changes upon binding.
2 Interaction of small molecules with DNA 5
2. Interaction of small molecules with DNA
Numerous small organic molecules are known to bind to DNA, and there is great structural variety within this family. Many molecules that interact with DNA are of pharmaceutical interest, since they can have a direct influence on the functionality of DNA and then on the processes that depend on it. Cancer and genetic diseases may be caused by mutations in the DNA sequence or by base-pair mismatches that sometimes happen during replication. Most of cancer chemotherapy, indeed, is based on molecules that interact with DNA [21, 22]. The study of the interaction of DNA with small molecules is thus of very large interest, since it can shed light on the mechanism of actions of drugs and pave the way for the development of new compounds of therapeutic or diagnostic interest. A future goal in this field will certainly be the design of molecules that can bind selectively to some sequences or to specific structural elements, as natural DNA-binding proteins are able to do.
Given the high structural variability of DNA binders, it is not surprising that these molecules can interact with DNA in several different ways:
(1) By forming covalent bonds;
(2) By establishing electrostatic interactions with the negatively charged sugar-phosphate backbone (these ligands usually bear a positive charge);
(3) By establishing non-covalent interactions with the major or minor grove (in this process hydrophobic interactions may have a fundamental role); (4) By intercalation between the base pairs;
(5) By combining two (or possibly more) of the abovementioned mechanisms. We will analyse each case in detail.
2.1. Molecules that form covalent bonds with DNA. Cancer chemother-apy started with molecules that can irreversibly react with DNA [23], generally by alkylation by a SN mechanism. This action is also the reason of the car-cinogenic activity of strong alkylating agents, and especially of powerful methy-lating reagents such as N -methyl-N -nitrosourea, dimethylnitrosoamine, dimethyl
sulphate, methyl methane sulphonate [24] and halogenomethanes [25]. The sites most susceptible to alkylation are the purine bases, and more in detail the N7 and O6 positions of guanine and the N3 of adenine. Nitrogen mustard (Fig. 3), initially developed as a chemical warfare agent and then entered into the clinical practice [26–28], is a typical representative of this class. Cyclophosphamide (Fig. 3) is an alkylating agent currently used in cancer chemotherapy that acts by cross-linking DNA strands [29]. As for inorganic molecules, the metal centres contained in transition metal complexes can covalently bind DNA and this is the basis of their cellular activity. This is the binding mode of cis-platinum, the first anticancer agent discovered [30], and the progenitor of many other metal complexes that are studied until possible clinical trial, for improved efficacy.
Cl N CH3 Cl Cl N P Cl HN O O Nitrogen mustard Cyclophosphamide
Figure 3. Molecular structure of two alkylating agents: Nitrogen Mustard and Cyclophosphamide.
2.2. Molecules that establish electrostatic interactions with DNA. DNA is a polyanion due to the negative charge of the sugar-phosphate backbone. Normally, electrostatic interactions are not the only forces that make a molecule bind to DNA, but generally the complex formed between cationic molecules and the nucleic acid itself is also stabilized by these interactions. Actually, naturally oc-curring DNA-binding proteins (histones) are generally rich in amino-acid residues featuring positive charge at physiological pH (Arg, Lys) [31]. The ionic force of the solution has a strong influence on the affinity of a ligand for DNA if electro-static interactions are important. At high salt concentrations, the charges are more screened and the apparent affinity constants decrease. A quantitative description of this process has been proposed by Record and co-workers [32, 33].
2 Interaction of small molecules with DNA 7
2.3. Groove-binders. In principle, molecules can bind either on the major groove or on the minor groove of double stand DNA. The major groove, as the name implies, is much larger than the minor groove (about 12 ˚A and 6 ˚A respectively). Most DNA-binding proteins and large molecules such as oligonucleotides interact with the major groove [34–36], while there are few small molecules that display this interaction mode [37, 38].
Major groove binders: It is especially interesting to develop major-groove binders, since they can in principle displace or tune the affinity of DNA-binding proteins. Some examples of synthetic molecules of this class are the so-called peptide nucleic acids (Fig. 4).
H N N O H N N O H N O B O B B = nitrogenous base
Peptide nucleic acid
Figure 4. General molecular structure of a peptide nucleic acid.
They are inhibitor of the transcription process and they act by compet-ing with the bindcompet-ing of transcription factors and of DNA polymerase [39]. Since they can be used as general recognition agents, these compounds can be useful in many biochemical procedures, for example in alternative to (strept)avidin and biotin for pretargeting studies [40].
Minor groove binders: Minor groove binders are generally low-molecular weight compounds. The common features of most of them are a positive charge, structural flexibility, curvature, and the possibility to establish hydrogen bonds [41–43]. A crescent-like shape, with a radius of curvature complementary to the one of the minor groove, is usually considered to be prerequisite for minor groove binding [44], but some molecules that violate this empirical rule have been found [45] and a family of them were recently
synthesised [46]. The structures of some typical synthetic minor groove binders are summarized in Fig. 5. Some naturally occurring antibiotics belong to this class too [47].
H2N NH HN O NH N O NH NH2 NH N O N H Netropsin N H HN NH2 NH NH2 DAPI HO N H N N H N N N Hoechst 33258
Figure 5. Molecular structures of some typical synthetic minor groove-binders: Netropsin, DAPI, and Hoechst 33258.
Most minor grove binders display some selectivity for AT-rich regions [48] that is probably due to multiple reasons [38, 49]:
• because an AT base pair has only two hydrogen bonds, it can propeller twist more than a GC pair with three such bonds, shifting the positions of C1’ atoms [50] and producing a narrower groove that is hospitable to a planar molecule;
• AT-containing regions have a more negative electrostatic potential than CG-containing ones (groove binders are generally cationic);
2 Interaction of small molecules with DNA 9
• the amine group of guanine, which points towards the minor groove itself, may disfavour the binding of small molecules because of steric hindrance. Sequence-selectivity, if any, is due to the formation of specific hydrogen bond networks with the nitrogenous bases. The structural modifications induced by groove binders are in general less important than the ones due to intercalation.
2.4. Intercalators. Intercalation has been the first binding manner that has been proposed [51–53] and has been studied extensively. It is characterized by the insertion of a planar aromatic moiety between two consecutive base pairs in double strand DNA. The stabilization of intercalation complexes is due to a combi-nation of contributions of Van Der Waals, hydrophobic, stacking and electrostatic interactions [54].
Medhi et al. [55] assessed that the electrostatic interaction with the base pairs plays a dominant role in the intercalation of positively charged chromophores, and that a degree of electrostatic potential complementarity between the drug and the two DNA base pair is probably a prerequisite for intercalation of charged chromophores. They also stated that, among all thermodynamic contributions, electrostatic component is likely to be the decisive factor for the base pair selectiv-ity of intercalators, since all other energetic contributions do not vary very much with the base pairs sequence. Generally it is necessary that the double helix un-winds partially and the concerned base pair unstack for the intercalation to take place.
These considerations, however, do not lead to any possible criterion for se-quence selectivity, since interaction takes place only with two base pairs. This binding mode generally brings about more distortion of the tridimensional struc-ture of double strand DNA than groove binding, disrupting severely much of the molecular machinery that interacts with DNA [56].
Among the classical intercalators there are synthetic compounds such as the dyes Ethidium bromide and acridine Orange, commonly used in molecular biol-ogy to stain DNA [57–59], the antimalarial drug Quinacrine [60, 61], and natural
compounds such as the alkaloid Berberine, (Fig. 6) used in the cure of gastric and colon cancer [62, 63]. N H2N NH2 Ethidium bromide N HN N OMe Cl Quinacrine N O O O O Berberine N N N Acridine orange
Figure 6. Molecular structures of three well-known intercalators: Ethidium Bromide, Quinacrine, Berberine, and Acirdine Oragne.
It has been generally noticed that compounds containing not fused aromatic rings with terminal basic functions bind to DNA in one of the grooves while fused ring aromatic cations bind most strongly to DNA in an intercalation complex [64]. Denny and co-workers [65] have assessed that, in order to intercalate in the DNA, a small molecule must have a “minimal intercalating structure” consisting in a planar moiety containing several condensed aromatic rings (at least two) in which the chromophore size and cross-sectional width are sufficiently small to fit between the base pairs [66].
However, some intercalators without this structural pattern have also been found. An example is Thalidomide (Fig. 7), whose teratogenicity mechanism in-volves its intercalation as the first step [67]. An extensive discussion with several other examples of atypical intercalators is given by Snyder [68] (Fig. 7).
2 Interaction of small molecules with DNA 11 N N Cl Chlorpheniramine N N S N S 1-S N N S N S N M-30 N O O NH O O Thalidomide
Figure 7. Molecular structures of four atypical intercalators.
2.5. Agents that bind to DNA through multiple modes. Threading intercalators are characterized by the same aromatic structure typical of ordinary intercalators, in addition to flexible side chains that can interact with the major or minor groove at the same time as the planar moiety is inserted between two pair of bases. Naturally occurring anthracyclines (bacterial metabolites that find use as anti-tumour agents) are representative of this class. The structures of Daunorubicin and Doxorubicin, two compounds of this kind, are depicted in Fig. 8.
O O O OH OH O OH O O OH NH2 Daunorubicin O O O OH OH O OH O O NH2 OH Doxorubicin OH H H
The complexation of anthracyclines to DNA, overall, benefits from intercala-tion, partial groove-binding, and electrostatic interaction [69]. Synthetic molecules that have been designed to bind with DNA by two mechanisms at the same time are called combilexins. They combine the potent disruption of the DNA confor-mation induced by intercalators with the potential specificity of groove binders. They can also have long residence time, thus interfering strongly with the activity of DNA-processing enzymes (e.g. they could inhibit topoisomerase [70, 71]).
3. Auramine
Auramine (Fig. 9), bis(4-(dimethylamino)phenyl)methanimine (-ium), is a diphenyl-methane imine (iminium) dye. Throughout this entire work, with this name we will indicate its protonated form.
N N H H N Cl Auramine O
Figure 9. Molecular structure of Auramine
The best known and commercialized form of Auramine is its hydrochloride salt, Auramine O (other salts, like Auramine perchlorate [72], have been synthesized and characterized as well). It is a brilliant yellow dye with high colour strength [73] and is commercialized mainly as dyestuff [74]. It has at least 40 other trade names [74] and roughly 1000 tons per year were sold in 2000 [73], surprisingly not so much considering that in 1933 about 500 tons per year were produced only in the USA [75].
Auramine is a synthetic molecule and it was patented in 1884 [76]. It was produced from Michler’s ketone and ammonium chloride in the presence of zinc chloride. A second process, patented in 1889 (although patented in the name of Adolph Feer, it is credited to Traugott Sandmeyer [77]), is very similar to the
3 Auramine 13
methods used nowadays (Fig. 10). This industrial process starts from bis(4-dimethylaminophenyl)-methane, which is heated to 200 with ammonium chlo-ride, sulfur, and sodium chloride. 4,4’-Bis(dimethylaminophenyl) thiobenzophe-none is formed as an intermediate and is readily converted into the imine Auramine O with ammonia (a large number of German patents by BASF dating from the end of the XIX century are reported in the Ullmann’s Encyclopedia of Industrial Chemistry [73]) N N H H N Cl N N S8 NH4Cl NaCl 200 °C N N S NH3 / NH4Cl Auramine O
Figure 10. Scheme of industrial synthesis of Auramine
Auramine has many industrial applications, including colour filters [78, 79], plasma displays [80, 81], semiconductor devices [82], inks [83], highlighters [84], image-recording materials [85, 86], printing plates [87], toners [88], adhesives [89], fuels [90], pesticides [91], and textiles [92] and wood [93] dyeing. It has been also used to colour smoke in military applications [94] and in firework displays [95].
It is know that Auramine is toxic for humans and animals: it has acute oral toxicity [96], it is cytotoxic [97], genotoxic [98, 99], mutagen [100], and it is known to damage DNA [99, 101]. Its carcinogenicity has been already proven for rats an mice [102–104], but not yet for humans. Studies on workers involved in its manufacture [105, 106] demonstrated that Auramine production causes cancer of the urinary bladder, probably due to intermediates, while there is inadequate evidence in humans for the carcinogenicity of Auramine itself [107, 108]. It has also been used in the past in some countries of Latin America as a food colourant
[109] and same traces have been found in food samples from India [110, 111] and China [112].
Auramine has many biological applications in treatment of cancers [113], car-diovascular diseases [114], pain associated with diabetes [115], mechanical allody-nia [116], metabolic syndrome [117]. It can also be used in fungal inhibitors [118] and in the treatment of protozoan infection in fish [119]. Finally, it can be used in targeted drug delivery [120].
The main biological use of Auramine has always been in fluorescence mi-croscopy as staining agent for bacteria, in particular in the detection of Mycobac-terium tuberculosis (an extensive recent review with references is provided by Steingart et al. [121]). Nowadays staining with Auramine, alone or in combina-tion with other dyes such as Acridine Orange [122] or Rhodamine B (to give the Truant stain [123, 124]), is a standard procedure for identification of many kinds of microorganisms and its range of use is destined to spread, especially in poor countries, due to its easy applicability and low cost (for an example see [125]).
3.1. Photophysical properties and interaction with the environment. Auramine is composed of a protonated imine group conjugated to two electron-rich nitrogen atoms through an extended polimethine bridge. Thus, it is a typical molecular fluorescent rotor in the framework of what discussed in at the beginning of the chapter. Like the major part of florescent rotors, its main spectroscopic feature is the extreme sensitivity of its fluorescence spectrum (both intensities and position of the peaks) on the environment. As a matter of fact, most of the studies (from the 1950s to now) on this molecule have been focused on elucidating its photophysical behaviour in different environments (vacuo, solvents, and organized environments).
Fluorescence of Auramine has been studied in a great variety of solvents (in-cluding iso-propanol, iso-butanol, glicerol [126], the first 10 n-alcohols [127], ethy-lene glycol, acetonitrile, various mixtures of ethyethy-lene glycol–water [128], methanol– water [129], and water–sucrose [130]) and other kinds of environment (microcrys-talline cellulose [131], montmorillonite clays [132], silica solid matrix [133], Aerosol
3 Auramine 15
OT (dioctyl sodium sulfosuccinate) regular [134] and reverse [135] micelles , AOT– water–heptane microemulsions [136], cyclodestrin nanocavities [137], composite films [138] and water-soluble calixarenes [139]). It has a very low fluorescence quantum yield in non-viscous solvents, particularly in polar solvents (Chen [140] reports Φf = 4 · 10−5 in water), that raises slightly in apolar solvents and becomes appreciable in viscous solvents and in organized environments.
Auramine has been proposed as fluorescent probe for polyelectrolites [141–144] and for different biological substrates like small [145] and globular proteins [146], and membranes [130]. Some studies have been published recently on the possible use as fluorescent marker for the detection of amyloid fibrils of Auramine instead of Thioflavine T [147, 148], indicating a preference for the first with respect to the latter [148, 149], due to the comparison of an additional red-shifted emission band exclusively in the fibrillar medium.
3.2. Interaction of Auramine with DNA. As already cited above, Au-ramine can be used in biological applications, it is cytotoxic, causes cancer and damages DNA. This bio-activity, together with some similarity with the interca-lating dye acridine orange, suggests possible intercalation of Auramine between DNA base pairs. Moreover, Auramine belongs to the family of cyanine dyes which do bind DNA and can also sometimes be molecular rotors.
From a structural point of view, cyanines are characterized by the presence of a nitrogen atom in the electron-donor group and in the electron-acceptor group as well, joined by a polymethine chain. Cyanine dyes are generally divided in three types, depending on the location of such nitrogen atoms (see Fig. 11). Cyanines in which only one nitrogen atom is within a heterocycle (pyrrole, imidazole, thiazole, pyridine, quinoline, indole, benzothiazoleare etc.) are called hemicyanines, while cyanines in which both nitrogen atoms are within a heterocycle are called closed chain cyanines. Finally, streptocyaninces or open chain cyanines do not have any nitrogen within a cycle.
Figure 11. General structural formulae of cyanines: closed chain cyanines (left), hemicyanines (center) and streptocyanines (right).
One of the main reasons cyanines are so intensively studied is their biomedical and biological use as molecular probes [150, 151]. Most of fluorescent dyes com-monly used nowadays for DNA visualization assays are cyanines [41, 152], because compounds belonging to this family show strong absorption in the visible range of the spectrum, high affinity for nucleic acid double strands, and sharp increase of flu-orescence emission when interacting with DNA [153–157]. Cyanine dyes are widely used in DNA sequencing, fluorescence microscopy, and conformational studies via fluorescence energy transfer [158–160], DNA analysis in polymerization chain re-actions [161–163], agarose gel and capillary electrophoresis staining [164–166] and flow cytometry [167].
As for DNA binding, cyanine dyes usually have characteristics of both inter-calators (aromatic rings, usually condensed) and minor groove binders (positive charge, curvature, flexibility and hydrogen bond donor and acceptor groups), so it is fairly natural that both binding modes are commonly observed. Even subtle changes in either the structure of the dye or sequence of the DNA may cause a variation from one binding mode to the other. Moreover, some works have shown that cyanines can also assemble into supramolecular aggregates by using DNA as a template (for some examples see [41] and references therein).
Among their works on the interactions between cyanine dyes and nucleic acids, Yarmoluk and co-workers have extensively studied Cyan 2 (also called CCyan 2) and some other molecule belonging to this family (Fig. 12). Cyan 2 has been proven to bind DNA principally by intercalation, Biver and co-workers [168] found m0φ = 1.1 according to the Manning–Record theory [33, 169]).
3 Auramine 17 N S N S Cyan 2 N N S Cyan 40 S N N Thiazole orange
Figure 12. Molecular structure of Cyan2, Cyan40, and Thiazole orange.
In the same work, the “half-intercalation” model was preferred to describe the binding mode of the relatively similar compound Cyan 40 (Fig. 12). According to this model one heterocycle is intercalated and the remaining stays in the groove. Thiazole orange (TO, Fig. 12) is another typical cyanine dye that was found to bind to double stranded DNA with the 2:1 ratio stoichiometry, characteristic of intercalation [170]. Lately, many works confirmed, experimentally and computa-tionally, this guess [171].
Cyanine dyes can also exhibit non-intercalative binding modes [172–174], that usually appear at higher dye:DNA ratios. Increasing the link between the two aro-matic rings in the dye can lead to even more complex binding behaviour: length-ening of the bridge confers to cyanine dyes a structure more similar to classical minor groove binders and this is probably the main contribution to the complexity of DNA binding by TO-PRO-3 [174]. It is also to be noted that, in aqueous solu-tion, fluorescence quantum yield of these dyes is very low, due to fast nonradiative decay of the excited state through torsional motion in the methine bridge joining the two rings (see for example [172]). However, restriction of this conformational mobility upon DNA-binding (especially intercalation) can cause fluorescence en-hancements greater than 1000-fold for the compounds of this class [150, 172].
Despite all the studies of optical properties of Auramine in so many kinds of different environments, the literature regarding the topic of the present work, namely interaction of Auramine with DNA, is surprisingly poor and controversial. Auramine began to be massively used in laboratory practice during the 1930s (brief historical reviews in English are provided by Lempert [175] and Matthaei [176]), when fluorescence microscopy began to replace conventional staining in the detecting acid-fast bacilli (at that time mainly Mycobacterium Tubercolosis). However, fluorescence enhancement of Auramine in presence of these bacteria was found to be due to its binding to mycolic acids [177], long fatty acids characteristic of Mycobacteria (C88 in Mycobacterium Tubercolosis), discovered and isolated in that period [178], and the possibility of an interaction of Auramine with DNA was neglected for long time.
The first Auramine–DNA interaction study was reported by Oster in 1951 [179]. He measured the equilibrium constant of the binding reaction of Auramine with DNAs of different molecular weights and RNA, obtaining ∆G = −10.8 kcal mol and ∆H = −7.0 kcal
mol at 25. He also observed a strongly positive change in entropy for the binding process (−T ∆S = −3.8 kcal
mol at 25) [141]. Usually compounds having such a high favourable enthalpic contribution to the free binding energy are considered to be intercalators [180], but intercalation is characterized by negative or negligible entropy, and a dye binding to DNA that benefits from both considerably favourable enthalpic and entropic contributions is, at least, very uncommon [56].
In 1971 Hadwiger and Schwochau [181] studied the specificity of 38 potential DNA-intercalating compounds in the control of Phenylalanine Ammonia Lyase (PAL) and pisatin levels in pea tissue. It was proposed in precedent studies [182, 183] that increased PAL and pisatin synthesis occurs in pea tissue as a result of gene activation due to the conformational change of double-stranded DNA. Being intercalation the main non-covalent interaction capable of modify the ds-DNA conformation (this is not rigorously true, for example Bleomycin binds the minor groove and induces double strand breaks [184], but this is a very rare exception),
3 Auramine 19
the authors assessed that positivity on both these tests (and this is the case of Auramine) is synonymous of intercalation.
Auramine has been assumed not to be able to intercalate DNA due to structural limitations by M¨uller and Gautier in 1975 [185]. They measured the binding constants of several compounds including Auramine and found 5.8 · 10−2 < Keq < 2.0·10−3, that is 3.7 kcal
mol < ∆G
< 4.5 kcal
mol at 25 in cacodylate buffer, depending on the kind of DNA. They found some selectivity for AT-rich regions and hence assessed that, as they expected, Auramine is a minor groove binder.
In 1980 Ridler and Jennings [186] studied the orientation relative to the axis of DNA of the aromatic moieties of 11 dyes known to interact with it. For 8 of these they obtained the expected result (6 of these are well-known intercalators and 2 are well-known minor groove binders), and for the remaining 3 (whose binding mode was to be determined) intercalation was assessed to be the main binding mode, as they saw that these dyes bind DNA with their aromatic rings perpendicular to the axis of DNA.
In 1990 Stockert and co-workers [187] studied the fluorescence enhancement of a set of non-rigid DNA-binding compounds (including Auramine) upon interaction with complex cellular structures containing DNA such as chromatin of interphase nuclei, meiotic and polytene chromosomes, spermatozoa heads and kinetoplasts of Trypanosoma cruzi epimastigotes. Regarding the mode of binding of these molecules, the interesting result is that all of them showed preference or AT-rich regions. The authors thus suggested that there is a high possibility that minor groove could be the binding site of these molecules. The same group analysed a series of compounds that bind chromatin DNA, which only some of them are able to selectively label pericentromeric heterochromatin (C-bands) in metaphase chromosomes [188]. This is a AT-rich region of DNA, and Auramine was expected to show selectivity for it. However a second condition was required according to the authors, namely hydrogen bonding capability. In the case of Auramine, they concluded that although it has AT selectivity, the lack of H-bond capability prevents it from biding to this particular region. The same authors [189] published
a study on the development and validation of a simple method for predicting intercalation or groove binding of dyes with double stranded DNA based on a quantitative assessment of the aspect (width to length) ratio of the dyes. Although Auramine is counted by them as a groove binder, with this brutal method they predicted properties that are sort of intermediate between the two binding modes. In 2002 Snyder and Arnone [190] studied the interaction between topoisomerase II and 14 dyes that are well-known or suspected intercalators. According to this work, Auramine is a topoisomerase II poison acting through DNA intercalation. This statement is supported by some evidences:
(1) a red-shift in the absorption spectra is detected (λmax= 448nm bound to DNA while it was 427 without DNA);
(2) Ethidium Bromide displacement assay [191] suggests that these two com-pounds compete for the same site;
(3) positivity in the Bleomycin amplification assay [192] is intended by the authors as a clear evidence of intercalation, since intercalated compounds are supposed to stabilize double strand breaks caused by the groove binder Bleomycin, thus enhancing its effect [193], while groove binders should not do it (they should also displace Bleomycin);
(4) the clastogenicity observed in V79 cells treated with Auramine is strongly antagonized by catalytic topoisomerase II inhibitors, namely Chloroquine [194], Ethidium Bromide [57], 9-aminoacridine [195], and Tacrine [196], all well-known intercalators.
The authors noticed that Auramine has never been tested for topoisomerase II inhibition because of its unfused bicyclic structure, that makes it not an obvious intercalating agent (it lacks the “minimal intercalating structure” [66] discussed above).
Chapter
2
Free Auramine
To understand the changes in the photo-physical behaviour of Auramine in dif-ferent environmental conditions, we analysed the electronic and structural prop-erties of both ground and excited states of the isolated and solvated dye using a TD-DFT approach combined with a PCM description of the solvent. Starting from these structures we finally computed the excitation and emission energies and compared with the measured spectra.
1. Ground State properties
Like for conjugated push-pull systems, also for Auramine it is possible to draw two limit resonance structures, as depicted in Fig. 1.
In order to quantify how the environment affects the relative contribution of the two resonance structures, we analysed the two most important geometrical parameters, namely the Bond Length Alternation (BLA) and the C=N iminic bond length.
BLA is defined as the average of the difference in the length between adjacent carbon–carbon bonds in a polymethine ((CH)n) chain. A BLA = 0 indicates a perfectly delocalized structure, while BLA < 0 indicates a partially quinoid structure.
Figure 1. The two limit resonance structures: benzenoid (left) and quinoid (right)
As here the solvent explored is water and Auramine in protic solvents is likely to interact through hydrogen bonds with the solvent molecules within the first solvation shells, we have also considered two clusters formed by Auramine and one (or two) water molecule(s) H-bonded to the protonated imine group (see Fig. 2).
The choice of the H-bonding sites in Auramine has been the CNH+2 group act-ing as electron acceptor, rather than the NMe2 groups acting as electron donors, becasue the lone pairs on the amine nitrogens are not available due to the high delocalization caused by the overall +1 charge of the molecule and the steric hin-drance of the methyl substituents. To also include the bulk effects of the outer shells of solvent, the cluster has been embedded in a PCM solvent.
BLA and C=N bond length in vacuo, continuum solvent, and continuum sol-vent + 1 and + 2 explicit water molecules are listed in the Table 1.
The inclusion of solvent effects enhances the weight of the benzenoid structure, and the value of BLA becomes more negative. This behaviour is further increased when explicit water molecules are also taken into account: the water oxygens stabilize the partial positive charge on the imine moiety donating electron density through H-bond, while no H-bond stabilization can occour for the dimethylaminic nitrogens (see Fig. 3).
1 Ground State properties 23
Figure 2. Geometry of the clusters of Auramine with one (left) and two (right) hydrogen bonded waters optimized at the DFT/6-311+G(d,p) level: front view (upper panels) and side view (lower panels). Environment BLA (˚A) C=N (˚A) vacuo -0.0445 1.331 water (PCM) -0.0407 1.321 water (PCM + 1 explicit) -0.0388 1.316 water (PCM + 2 explicit) -0.0369 1.312
Table 1. BLA and C=N bond length, in the four different envi-ronments, as computed at the M06-2X/6-311+G(d,p) level.
Another important structural parameter for Auramine is represented by the two torsional angles between the two para-dimethylaminophenyl groups (see Fig. 4). It is in fact known that the changes in fluorescence quantum yield are related to intramolecular torsions. In fact, as evidenced in the first work by Oster and Nishijima [14] and then in experimental and computational works by other groups
Figure 3. Oxygen lone pairs can interact with the protonated imine moiety (left) but cannot interact with the + charged dimethyl-aminic nitrogens (right)
[127, 197–204]), being a molecular rotor is the most interesting characteristic of Auramine. A detailed analysis of the dependence of fluorescence on the rotation coordinates will be given later on.
Figure 4. The two main rotational degrees of freedom of Auramine
A convenient way to study the electronic distribution is to divide the whole molecule in moieties and use the M¨ulliken charges formalism [205–207]. This partition will be used throughout the following and the next analysis.
2 Absorption 25
From Table 2 we can see that about one half of the formal + charge is localized on the imine moiety, while the other half is split into the two rings.
Environment Imine moiety Ring
vacuo 0.418 0.289
water (PCM) 0.486 0.257
Table 2. M¨ulliken charges of Auramine in vacuo and in water (IEF-PCM), as computed at the M06-2X/6-311+G(d,p) level.
2. Absorption
2.1. Measured spectra. In Fig 5 we report the absorption spectra of Au-ramine obtained in a buffered water solution ([NaCl] = 0.1M, [NaCac] = 0.01M 1, pH = 7.0, T = 25 ), at different concentrations (Cd) ranging from 5 · 10−6M to 5 · 10−5M.
This buffered solution has been used in all the experiments (unless otherwise specified), also in the presence of DNA, because it has been found that cacody-late buffer does not affect the interaction between Auramine and DNA, and it is commonly used in staining too (see for example [208–220]).
The spectrum has a broad symmetric band in the violet region, at 432 nm or 2.87 eV, that gives rise to the intense yellow colour perceived by the human eyes, and a smaller band in the near UV region, 371 nm or 3.34 eV.
In order to check if the dye has some tendency to self-aggregation, the depen-dence of the ratio of the intensities of the two peaks from the concentration has
1Cac=(CH3)2AsO -2
Figure 5. Absorption spectra of several Auramine water solutions (5 · 10−6M < Cd < 5 · 10−5M, [NaCl] = 0.1M, [NaCac] = 0.01M, pH = 7.0, T = 25 )
been analysed (Fig. 6). This method has already been used in some works of this group [221–224].
From this analysis we can say that the chromophore does not self-aggregate, or, at least, that if self-aggregation is happening, then it does not affect absorption spectra.
As cyanine dyes have a very high tendency to self-aggregate [41, 168, 225– 231], a common procedure (for instance see [171]) is also to study if the dye begin to self-aggregate varying temperature or salt concentration ([NaCl] up to 3M). In every case we obtained the same results and we can assess that no particular ionic strength or temperature condition (in the range of these measures) can cause significant self-aggregation.
2.2. Calculated spectra. To characterize the nature of the excitation we have analyzed the molecular orbitals mostly involved in the transition and the difference in the the electron density between ground and excited state. These data are plotted in Fig. 7.
2 Absorption 27
Figure 6. Concentration dependence of Auramine absorption peaks intensities (top) and their ratio (bottom).
From these plots we can clearly see that the electronic transition has a strong charge-transfer character: electrons are moving from the dimethylamine groups to the imine moiety. Moreover we can see that the double imine bond loses electron density while the C and N atoms gain density.
Excitation energies of Auramine, in the four different environments speci-fied above, have been computed with four different DFT functionals and the 6-311+G(d,p) basis set. Solvent effect has been taken into account with the PCM-cLR (corrected linear response) formalism. The results are listed in the table below together with the experimental value (in eV).
Environment B3LYP PBE0 M06-2X CAM-B3LYP EXP
vacuo 2.93 3.01 3.21 3.26
-water (PCM) 2.97 3.06 3.31 3.35 2.87
water (PCM + 1 explicit) 3.02 3.12 3.38 3.43 2.87
water (PCM + 2 explicit) 3.06 3.15 3.44 3.50 2.87
Table 3. Excitation energies (in eV) of Auramine in the four envi-ronments computed at the TD-DFT/6-311+G(d,p)/cLR-PCM level.
Although the observed off-sets (for some functionals of the order of 0.40-0.45 eV) are slightly larger than the TD-DFT systematic error [171, 232–236], these results are quite satisfactory considering that cyanines are one of the most difficult classes of chromophores to treat with TD-DFT [233–249].
As pointed out by many authors, part of the difficulties involving the sim-ulation of spectra of cyanine dyes can be attributed to the fact that calculated vertical excitation energies are not directly comparable with the energies corre-sponding to the experimental absorption band maxima, but the overestimation persists when comparing TD-DFT with accurate theoretical vertical excitation energies [233, 234, 237, 238]. The discrepancy can be partly ascribed to an insuffi-cient description of double-excitations at TD-DFT level [237, 238, 241, 243, 250], and partly imputed to nuclear relaxations upon excitation, which makes vertical transitions not corresponding to maximum absorption wavelengths [244, 250, 251].
2 Absorption 29
Figure 7. Molecular orbitals involved in the first electronic tran-sition: HOMO (upper panel) and LUMO (central panel), with their phases. Lower panel: Electron density difference between ground and first excited state. The regions coloured in light blue lost den-sity in the transition while the ones coloured in dark blue gained density
We also note that errors are larger for functionals including a larger contribu-tion of the HF exact exchange to exchange funccontribu-tional. As the poor performance of TD-DFT for the cyanines was attributed to the strong multideterminantal nature of the states of these dyes [237, 246], probably this correlation is originated by the fact that HF exact exchange is more accurate when it is possible to accurately describe ground and excited states with only one determinant, and then a big HF contribution to the exchange functional will stress once more the monodeterminan-tal character of the DFT method. However, as discussed many times in literature by Jacquemin and co-workers, Minnesota functional M06-2X still represents the best choice among DFT functional to study electronic excitations of this class of compounds [233, 234, 239].
3. Emission
Excited state dynamics of Auramine in solution have been studied for the past 20 years [127, 129, 134, 197–202, 252] and also very recently [148, 204].
After excitation, Auramine undergoes a relaxation process involving the reori-entation of the dimethylanilino ring. According to Palit and co-workers [200] , the intramolecular torsion around the dihedral angle φ should be a barrierless process. However, Rafiq and Sen [201] reported that the magnitude of the energy barrier between the locally excited state (LE), with geometry similar to the ground state and the supposed equilibrium excited state with φ ≈ 90° varies significantly shift-ing solvent from methanol to chloroform (these solvents have the same viscosity, but different polarity). It is likely that highly polar solvents catalyse the formation of an intermediate charge-transfer state (evidenced experimentally by Singh et al. [200] but not described computationally) eliminating the energy barrier in the re-laxation to the TICT non-emissive state. Meech and co-workers [134] proposed that in bulk water, since the solvation is very fast, it promotes a facile barrierless formation of the (CT) state and hence relaxation of Auramine is decided primarily by aqueous solvation and not by the solvent viscosity. More precisely, all the au-thors agree that the relevant reaction coordinates in the relaxation process are not
3 Emission 31
only the torsions around the φ and ψ dihedral angles. The solvation coordinate (that depends at least on polarity and viscosity, and probably on the hydrogen-bonding capability of the solvent too) and the set of coordinates relative to the pyramidalization of the nitrogens must be considered as well.
The optimization of first excited state was carried out with 4 different DFT functionals (namely B3LYP, CAM-B3LYP, M06-2X, and PBE0) and the 6-311+G(d,p) basis set, using the ground state geometry as starting point.
The optimization carried out with B3LYP and PBE0 converged to a twisted geometry indicated in literature as non-emissive state. This state is called in literature Twisted Internal Charge Transfer (TICT) State [201] or Transition State II (TSII) [200]. With CAM-B3LYP or M06-2X, the optimization converged to a geometry similar to the ground state, indicated in literature as emissive state. From now on our analysis will be focused only on this emissive state and the results obtained with the B3LYP and PBE0 functionals discarded.
As we can see from Fig. 8 and Table 4, the excitation causes two main changes in the geometry: a partial planarization of the whole molecule and an increase of the central C-N bond length, Both these changes are consistent with the nature of the transition.
Figure 8. Auramine geometry in its first excited state computed at the TD-M06-2X/6-311+G(d,p) level in water (PCM)
φ (deg.) ψ (deg.) C=N (˚A) Ground State -31.6 -31.6 1.321 Excited State -29.3 -24.8 1.384
Table 4. Dihedral angles φ and ψ amplitudes, and imine C=N bond length of the ground and excited state structures optimized at the M06-2X/6-311+G(d,p)/ level, in water (PCM).
As we can see from table 5, the migration of the electron density from the aromatic rings to the protonated iminic EWG group causes an increase and an inversion of the polarity of the molecule.
Iminium moiety Ring1 Ring2
Ground State 0.486 0.257 0.257
Excited State 0.006 0.481 0.513
Table 5. M¨ulliken charges of the excited state of Auramine in wa-ter (IEF-PCM), as computed at the M06-2X/6-311+G(d,p) level.
This plot shows a clear relation between the torsional angle φ and the emission energies as well as the oscillatory strengths. The emission energy of the lowest state drops rapidly with increasing angles over 60°. An opposite trend is instead found for the second state for which both the emission energy and the oscillator strength increase for angles larger than 60°. This behaviour can be explained in terms of a loss of conjugation between the aromatic rings.
3 Emission 33
Figure 9. Emission energies of Auramine as a function of the dihe-dral angle φ for the first two singlet excited states (Exc1 and Exc2) of Auramine in water; their oscillator strengths are also shown as dashed lines. For each value of φ, all the other geometrical parame-ters have been optimized (TD-M06-2X/6-311+G(d)/PCM)
Chapter
3
Auramine bound to DNA
1. The structure
Because of the large size of the system, we studied the intercalation process using a simplified model which limits the interaction between Auramine and the double-stranded DNA (dsDNA) to the nearest neighborhoods. Only the four ad-jacent nucleic acid bases and the sugar/phosphate connecting groups have been considered. The representativeness of this model has been already verified in other works of this group [171, 232].
As experimental structures of the DNA–Auramine complex are not available in literature, we used the structure of a DNA–Proflavine complex, obtain by X-ray crystallography [253], as a starting point to generate a reasonable guess for the DNA–Auramine complex.
The ground and excited state geometry optimizations were performed with the TD-DFT method using the M06-2X functional. This functional was chosen for its ability to describe the most important non-covalent interactions in nucleic acids such as stacking effects and T-shaped interactions [254], and also for its ability to correctly model cyanine compounds, as discussed above [233, 234]. As usual, 6-311+G(d,p) basis set was employed in the optimization, and IEF-PCM was used to take into account solvent effects. Optimization has been attempted also with PBE0 functional combined with Grimme’s D3 empirical functional for dispersion
[255], as suggested by Rutledge and Wetmore [254], but the optimization did not converge to any stable state.
1.1. Optimized ground state geometry. The optimized structure is de-picted in Fig. 1.
Figure 1. Optimized structure of the model Auramine-DNA com-puted at the M06-2X/6-311+G(d,p) level: front view (upper panel) and top view (lower panel).
Overall, the ground state equilibrium geometry in DNA is quite similar to the one in water, with some small differences. As we can see from Table 1, interac-tion with DNA causes a partial planarizainterac-tion of the molecule, and a modest shift towards the more benzenoid structure.
An interesting question at this point could be if the DNA stabilizes a more or a less polar form with respect to water.
Table 2 shows that interaction with DNA causes the whole molecule to have a total charge of +0.70 a.u. instead of +1.00 a.u. of the free chromophore. This observation can be explained by donation of electronic density by the DNA bases to the Auramine via stacking interaction. The two dimethylaniline rings, thus,
2 Absorption 37
Environment BLA (˚A) C=N (˚A) φ (deg.) ψ (deg.)
vacuo -0.0445 1.331 -31.7 -31.6
water (PCM) -0.0407 1.321 -31.6 -31.6
DNA -0.0392 1.320 -23.9 -27.7
Table 1. BLA and C=N bond length, in the three different envi-ronments, as computed at the M06-2X/6-311+G(d,p) level.
are less charged and the charge is mainly localized on the imine. This moiety is not shielded by the DNA, so it is exposed to water, which stabilises the charge by electrostatic interactions; the PCM solvation model adequately accounts for this effect.
Environment Imine moiety Ring1 Ring2 Total
Vacuo 0.418 0.289 0.289 1.00
Water 0.486 0.257 0.257 1.00
DNA 0.431 (62%) 0.110 (16%) 0.154 (22%) 0.70 Table 2. M¨ulliken charges (in a.u.) of the ground state of Au-ramine in vacuo, water (IEF-PCM), and DNA as computed at the M06-2X/6-311+G(d,p) level.
2. Absorption
In Fig. 2 are shown the spectra of several buffered solutions of Auramine and DNA at different concentrations, corrected for dilution. As the DNA concentration increases, the spectra progressively decrease in intensity of the absorption band
of Auramine (hypochromic effect), going from 27100 cm-1/M of free Auramine to 21100 cm-1/M of completely bound Auramine. and a simultaneous shift of the maximum towards the red (bathochromic effect), going from 432 nm (2.87 eV) of the free Auramine to 444 nm (2.79 eV) of the completely bound Auramine.
Figure 2. Absorption spectra of several Auramine-DNA buffered water solutions (7.5·10−6M < Cd< 1.1·10−5M, 0 < Cp < 6.7·10−4M, [NaCac] = 0.01M, pH = 7.0, T = 25)
Both these changes in the absorption spectra are very modest compared to the ones in the emission spectrum, hence only emission has been chosen to perform accurate thermodynamic studies of the interaction between Auramine and DNA.
The excitation energy of the complex Auramine-DNA has been computed at the TD-M06-2X/6-311+G(d,p) level, with the corrected linear response PCM taking into account the effect of the water surrounding the system.
3 Emission 39
The obtained value of 3.22 eV for the vertical excitation, although not corre-sponding to the experimental measured maximum of absorption, correctly repro-duces the decrease in energy associated with the binding, with the surprisingly precise shift of 0.09 nm (see Tab. 3).
Environment Comp. Energy (eV) Exp Energy (eV) f
Water 3.30 2.87 1.11
DNA 3.21 2.79 0.75
Shift 0.09 0.08
Table 3. Computed excitation energies and relative oscillator strengths of Auramine in water (IEF-PCM), and bound to the DNA, as computed at the TD-M06-2X/6-311+G(d,p) level.
Both bathchromism and hypochromism are usually associated with intercala-tion of an aromatic moiety into the DNA double strands [256] , and the correct reproduction of both these effect by our computational model makes it actually a very reliable tool for prediction of structural and electronic properties of the DNA–Auramine complex.
3. Emission
The first excited state of the complex Auramine–DNA has been optimized the M06-2X/6-311+G(d,p) level with the IEF-PCM including the electrostatic effect of the water environment. Only Auramine coordinates have been allowed to optimize while DNA ones have been kept frozen during the geometry optimization.
As we can see from Table 4, the excitation process of the complex Auramine– DNA causes only minimum structural changes compared to the excitation in water. This result is partially expected because binding with DNA restricts the main de-grees of freedom of the molecule, namely the two rotation around the φ and ψ dihedral angles. Consequently, also the other main geometrical parameter, the central C=N bond length, (that is closely dependent on the torsions) is not signif-icantly affected by the excitation.
Water DNA
C=N (˚A) φ (deg.) ψ (deg.) C=N (˚A) φ (deg.) ψ (deg.)
Ground State 1.321 -31.6 -31.6 1.320 -23.9 -27.7
Excited State 1.384 -29.3 -24.8 1.328 -24.0 -27.4
Table 4. Computed excitation energies and relative oscillator strengths of Auramine in water (IEF-PCM), and bound to the DNA, as computed at the TD-M06-2X/6-311+G(d,p) level.
As shown in Table 5, the excitation causes only a very modest change in the electronic strucutre of the complex Auramine-DNA, if compared with the difference between excited and ground state of Auramine in water.
Environment Imine moiety Ring1 Ring2 Total
Water – Ground State 0.486 0.257 0.257 1.00
Water – Excited State 0.006 0.481 0.513 1.00
DNA – Ground State 0.431 (62%) 0.110 (16%) 0.154 (22%) 0.70 DNA – Excited State 0.426 (67%) 0.089 (14%) 0.120 (19%) 0.63
Table 5. M¨ulliken charges of the ground and excited states of Au-ramine free and bound to DNA in water (IEF-PCM), as computed at the M06-2X/6-311+G(d,p) level.
Most likely DNA protects Auramine from the interaction with the solvent, con-sidered crucial by many authors ([134, 201] and references therein) in the relaxation dynamic of the excited state
4 Fluorescence titrations 41
These results are also consistent with the modest difference in the main geo-metrical parameters and with the idea that geometry and electronic distribution are strongly correlated.
In order to avoid complications due Raman bands of the solvents, all the flu-orescence measurements were not obtained by excitation at the absorption max-imum (between 432 and 444 nm), but at 400 nm. The vibrational Raman band associated to OH and CH stretching (between 2900 and 3400 cm−1) would appear beetwen 494 and 523 nm if excitation was performed at the maximum of absorp-tion, and it would thus be superimposed to the fluorescence peak of interest. To double-check that there is no particular difference we performed two fluorescence spectra in the same conditions, but the excitation wavelength. Fig. 3 shows two emission spectra of an Auramine–DNA buffered water solution obtained with an excitation wavelength of 400 nm and 430 nm.
4. Fluorescence titrations
Fluorometric titration of the system Auramine–DNA have been performed fol-lowing the fluorescence intensity variation after the addition of increasing quanti-ties of nucleic acid.
The quantitative analysis of the equilibrium of the binding reaction1: P + D −−*)−− PD
was carried out by plotting ∆FC
D = F −F0
CD against CP in the so-called binding
isotherm 2.
The overall fluorescence is given by the sum of two contributions 3
:
F = φD[D] + φPD[PD]
1P indicates a binding site of the Polymer (DNA), D a Dye molecule, and PD a Dye molecule bound to the site of the Polymer
2F0 is the initial fluorescence without any DNA addition, F is the fluorescence read at a certain DNA concentration CP and CD is the Auramine concentration.
3φD and φPD are the fluorescence coefficients of the free and bound dye, respectively, and ∆φ is their difference
Figure 3. Emission spectra of an Auramine-DNA buffered water solution (Cd = 1.6 · 10−6M, Cp = 2.8 · 10−3M, [NaCac] = 0.01M, [NaCl] = 0.1M, pH = 7.0, T = 25 ) obtained at two different excitation wavelengths (in red λexc = 400 nm and in blue λexc= 430 nm)
Following the treatment of Benesi and Hildebrand [257], we obtain the Benesi– Hildebrand equation (for the derivation see the section in the methods chapter):
CD· CP ∆F + ∆F (∆φ)2 = 1 ∆φ(CD+ CP) + 1 Keq · 1 ∆φ
This is a linear equation of the form y = mx + q and the value of the left-hand-side is plotted against the value of (CD+ CP). The values of ∆F come directly from the measures, and the value of ∆φ can be found by an iterative method. A linear least-square fit gives the value of the equilibrium constant as the reciprocal of the intercept multiplied by a coefficient.
5 Thermodynamic study 43
Figure 4. Top: binding isotherm of a titration of the equilibrium of the binding reaction (T = 25, pH = 7.0, I = 0.01). Bottom: plot of the same data using the Benesi–Hildebrand equation. The intercept is 1.02 · 10−11M2, so Keq = 3.01 · 103M
5. Thermodynamic study
5.1. Titrations at different temperatures: Chaires’ Plot. In order to obtain a dependence of the apparent equilibrium constant for the complexation of Auramine with DNA from the temperature, we performed a series of titrations
at different temperature keeping fixed all the other conditions, as shown in Fig. 5, More in detail, we plotted the natural logarithm of the equilibrium constant against the inverse of the absolute temperature and fitted the Van ’t Hoff equation between the two quantities:
ln K = −∆H R · 1 T + ∆S R
Figure 5. Van ’t Hoff plot for the binding reaction between Au-ramine and DNA. The natural logarithm of the equilibrium constant K is reported as a function of 1/T . Least squares linear regression is shown as a solid line (R2 = 0.9846).
Chaires plotted in a graph ∆H (T = 25) against −T ∆S (T = 25) us-ing the standard enthalpy and entropy values for 26 drug-DNA bindus-ing reactions [56], and found an empirical correlation between the position of the point in the graph and the type of interaction involved in the binding reaction. He found that the points obtained with thermodynamic data of intercalation reactions lie on one
5 Thermodynamic study 45
curve, while the points obtained with thermodynamic data of groove-binding re-actions lie on another curve. These two curves are in two different region of the Chaires’ plot.
According to Chaires’ plot (see Fig. 6) a binding reaction with the enthalpy and entropy we have found (∆H = −6.65 kcal
mol and −T ∆S
= 2.26 kcal
mol at 25) should be an intercalation.
Figure 6. Chaires’ plot [56] with experimentally found thermody-namic data for Auramine (in red)
5.2. Titrations at different ionic strengths: Record’s Equation. The binding constant of the Auramine-DNA system depends on salt concentration as shown in Fig. 7. The trend of ln K as a function of ln I is linear, in agreement with the Manning and Record equation [33].
The intercept of the plot yields ln K0, which has been defined as the binding constant in the absence of electrostatic effects [33, 169]. The slope, whose value
Figure 7. The natural logarithm of the equilibrium constant K is reported as a function of the natural logarithm of the ionic strength I. Least squares linear regression is shown as a solid line (R2 = 0.8931)
is 0.89, corresponds to m0ψ, where m0 is the number of phosphodiesteric residues occupied by one dye molecule and ψ is the extent of DNA charge shielded by counter-ions. Being ψ = 0.88 [258], it follows that m0 = 1.01. The value of ψ is made by a shielding contribution ψs and by a contribution from ion condensation ψc. Being for DNA ψ ≈ ψc, it turns out that m0represents the number of condensed sodium ions displaced by one dye molecule. The obtained value is in the range of the results reported in the literature for similar compounds [259] (for example Biver et al. found 0.99 for Thioflavine T [260]). As we obtained the value m0 = Z (molecular charge), all the +1 charge of Auramine interacts with DNA. A possible interpretation can be that all the molecular charge distribution is able to interact with the DNA and this means that a very deep insertion is happening.
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