Antibacterial effect of cationic porphyrazines and anionic phthalocyanine and their interaction with plasmid DNA

Accepted Manuscript Antibacterial effect of cationic porphyrazines and anionic phthalocyanine and their interaction with plasmid DNA Leila Hassani, Fatemeh Hakimian, Elham Safaei, Zahra Fazeli PII: DOI: Reference:

S0022-2860(13)00670-4 http://dx.doi.org/10.1016/j.molstruc.2013.07.054 MOLSTR 19929

To appear in:

Journal of Molecular Structure

Please cite this article as: L. Hassani, F. Hakimian, E. Safaei, Z. Fazeli, Antibacterial effect of cationic porphyrazines and anionic phthalocyanine and their interaction with plasmid DNA, Journal of Molecular Structure (2013), doi: http://dx.doi.org/10.1016/j.molstruc.2013.07.054

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Antibacterial effect of cationic porphyrazines and anionic phthalocyanine and their interaction with plasmid DNA Leila Hassani1*, Fatemeh Hakimian1, Elham Safaei2, Zahra Fazeli1 1

Department of Biological Sciences, Institute for Advanced Studies in Basic Sciences (IASBS), Zanjan 45195-1159, Iran 2

Department of Chemistry, Institute for Advanced Studies in Basic Sciences (IASBS), Zanjan 451951159, Iran

Tel.: +98 241 4153102; Fax: +98 241 4153004. E-mail address: [email protected]

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Abstract Resistance to antibiotics is a public health issue and identification of new antibacterial agents is one of the most important goals of pharmacological research. Among the novel developed antibacterial agents, porphyrin complexes and their derivatives are ideal candidates for use in medical applications. Phthalocyanines differ from porphyrins by having nitrogen atoms link the individual pyrrol units. The aza analogues of the phthalocyanines (azaPcs) such as tetramethylmetalloporphyrazines are heterocyclic Pc analogues. In this investigation, interaction of an anionic phthalocyanine (Cu(PcTs)) and two cationic tetrapyridinoporphyrazines including [Cu(2,3-tmtppa)]

4+

and [Cu(3,4-tmtppa)]4+ complexes with plasmid DNA was studied using

spectroscopic and gel electrophoresis methods. In addition, antibacterial effect of the complexes against Gram-positive (S. aureus) and Gram-negative (E.coli) bacteria was investigated using dilution test method. The results indicated that both porphyrazines have significant antibacterial properties, but Cu(PcTs) has weak antibacterial effect. Compairing the binding of the phthalocyanine and the porphyrazines to DNA demonstrated that the interaction of cationic porphyrazines is stronger than the anionic phthalocyanine remarkably. The extent of hypochromicity and red shift of absorption spectra indicated preferential intercalation of the two porphyrazine into the base pairs of DNA helix. Gel electrophoresis result implied Cu(2,3-tmtppa) and Cu(3,4-tmtppa) are able to perform cleavage of the plasmid DNA. Consequently, DNA binding and cleavage might be one of the antibacterial mechanisms of the complexes. Key words: Antibacterial, Interaction, Gel electrophoresis, Spectroflourimetery, Absorption spectroscopy

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Introduction Multiple resistances to antibiotics are a growing public health issue and identification of new antibacterial agents and generation of new strains of antibiotics to combat infections are one of the most important goals of pharmacological research [1-4]. Among the novel developed antibacterial agents, porphyrin complexes and their derivatives have attracted much attention due to their presence in natural systems which make them ideal candidates for use in medical applications [5-7]. Phthalocyanines differ from porphyrins by having nitrogen atoms link the individual pyrrol units. The aza analogues of the phthalocyanines (azaPcs) such as tetramethylmetalloporphyrazines are heterocyclic Pc analogues, which have been studied over the past decades. These complexes are phthalocyanines in which the outer aromatic benzene rings

are

replaced

with

electron-withdrawing

pyridine

rings.

The

N,N',N",N"'-

tetramethylatedquaternized forms of tetrapyridinoporphyrazines (tmtppa) are tetra-positively charged and hence water soluble. According to the literature reports, these complexes do not form aggregates in aqueous solutions [8]. The numerous biological experiments performed so far suggest that DNA is one of the most important intracellular targets of drugs and interaction of small molecules with DNA causes DNA damage, blocking the division of cell and cell death. Hence, the interaction of small molecules with DNA is an important research area for the development of new therapeutic agents [9]. Due to biological effect of porphyrines and phthalocyanines, their interaction with DNA, has been the subject of many researches and it has attracted particular great deal of attention. Even though there has been considerable research into the application of porphyrins and phthalocyanines on their anticancer and antibacterial effect and their binding to DNA, the

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tetramethylmetalloporphyrazines have received little attention. Safaei et al. demonstrated binding of some metalloporphyrazines to calf thymus DNA, but there is no report about their antibacterial effect [8, 10-11]. In

this

study,

the

antibacterial

effect

of

N,N',N",N"'-tetramethyltetra-2,3-

pyridinoporphyrazinatocopper(II), ([Cu(2,3-tmtppa)4+]) and N,N',N",N"'-tetramethyltetra-3,4pyridinoporphyrazinatocopper(II)

([Cu(3,4-tmtppa)

4+

])

as

two

cationic

tetrapyridinoporphyrazine complexes and an anionic phthalocyanine, (Cu(PcTs)) (scheme 1) towards Staphylococcus aureus and Escherichia coli growth has been studied to clarify some biological effect of copper porphyrazines and compare them with the phthalocyanine complexes. In addition, interaction of the complexes with plasmid DNA was investigated by absorption spectroscopy, ethidium bromide displacement experiments and gel electrophoresis and binding constants, binding modes, the values of the Stern–Volmer constant (KSV) and the rate constants for the quenching (kq) are determined.

Material and methods Materials Cu (PcTs) and Hinton growth medium were purchased from Merck. KH2PO4 and K2HPO4 were purchased from Fluka and Aldrich respectively. All other material was purchased from sigma.

Plasmid pEGFP-N1 was purified from XL1-blue E.coli by QIAGEN Plasmid Mega Kit. Cu(2,3tmtppa) and Cu(3,4-tmtppa) were prepared and purified by the method described previously [8]. UV-Vis absorption spectroscopy All UV-visible spectra were obtained on a Pharmacia Ultraspec 4000 at room temperature. Path length of quartz cell and scan rate was 1cm, and 100 nm.min-1. DNA concentration was determined using molar extinction coefficient values of 6600 M-1 cm-1 at 260 nm in aqueous

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solution [12]. The concentrations of Cu(2,3-tmtppa), Cu(3,4-tmtppa)

and Cu(PcTs) was

determined using molar extinction coefficient values of 1.67×105, 1.24×105 and 1.29×104 M-1cm1

[8, 13] respectively [8]. In titration experiments, 600 μl of 4.8 μM Cu(2,3-tmtppa), 5.3 μM

Cu(3,4-tmtppa) and 39 μM Cu(PcTs) was titrated with increasing amounts of 600 µM and 900 µM DNA stock solutions for both porphyrazines and the phthalocyanine respectively. Plasmid and three complexes were dissolved in 1 mM phosphate buffer, pH=7.0, containing 0.1 mM EDTA and 5 mM NaCl. The binding constant (Kb) value of the complexes to plasmid DNA was calculated by Equation 1 [14]. [DNA]/ (| εa-εf |) = [DNA]/ (| εb-εf |) + 1/ {Kb (|εb-εf |)}

(1)

Where [DNA] is the concentration of DNA in base pairs, εa correspond to Aobserved/[the complex] (Aobserved is the absorbance value of the complex titrated with DNA at the maximum of the spectra) , εf and εb are the extinction coefficient of the free complex (Afree/[ free complex]) and that of the complex in the fully bound form (Afully bounded/[ fully bound complex]), respectively. Free complex and fully bound complex is the complex in absence and saturated concentration of DNA respectively. A plot of [DNA]/ (| εa-εf |) versus [DNA] will have a slope of 1/(| εb-εf |) and an intercept equal to 1/{Kb(| εb-εf |)}. Kb is then given by the ratio of the slope to the intercept. Spectroflourimetery Fluorescence measurements were done on a Cary Eclipse fluorescence spectrophotometer (Varian, Australia) equipped with a Xenon lamp pulsed at 80 Hz. The measurements were done using a 1 cm quartz cell and a bandwidth of 5 nm for both excitation and emission monochromators. For fluorescence quenching experiments, 650 μl of ethidium bromide (EB) solution (30 μM) was transferred into the cell and 15 μl of the plasmid DNA (10 mM) was added

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to it to obtaine the molar ratio of [DNA] to EB equal to 7.7. The mixture of DNA-EB was titrated with Cu(2,3-tmtppa) and Cu(3,4-tmtppa) (16 μM) and Cu(PcTs) (600 μM) . Excitation and emission wavelengths were 460 and 665-780 nm, respectively and all the material were dissolved in 1 mM phosphate buffer, pH=7.0, containing 0.1 mM EDTA and 5 mM NaCl. To determine the quenching constant, Stern-Volmer equation (2) and its modified form (3) was applied [15]. F0/F = 1 + KSV[Q] (2) F0/F = (1 + KSV[Q]) exp(V[Q]) (3) KSV=Kq.τ0 (4) where F0 and F are the fluorescence intensities in the absence and presence of the quencher respectively. KSV is the dynamic quenching constant, [Q] is molar concentration of quencher and V is the static quenching constant. The Stern-Volmer constant is the product of the rate constant for quenching (kq) and life time of the luminescent in the absence of quencher (τ0). Curve fitting was performed with the Levenberg-Marquardt nonlinear least-squares algorithm provided by the computer program Origin (OriginPro 8.1). Gel electrophoresis For gel electrophoresis experiment, the plasmid DNA in 1 mM phosphate buffer, pH 7.00 containing 0.1 mM EDTA, was treated with the complexes. The samples were analyzed by 1% agarose gel. Electrophoresis was carried out at 65-80 V for 2 hours in 1X TAE buffer. TAE (Tris-acetate-EDTA) is a buffer solution containing a mixture of tris base, acetic acid and EDTA. 1X solution will contain 40 mM Tris, 20 mM acetic acid, and 1 mM EDTA. After electrophoresis, DNA was visualized by soaking the gel for 15 min with an aqueous solution of ethidium bromide. Bands were visualized by UV light.

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Antibacterial Susceptibility Testing Antibacterial effect of the complexes on S.aureus and E.coli was investigated using dilution test method [16]. First, one of the bacterial colonies was selected and grown in 4 ml Luria-Bertain (LB) medium at 37°C until the turbidity of 0.5 McFarland was achieved. Then, the stock solution of the complex was prepared in sterile water and poured into the sterile tube. Next, twofold serial dilutions were carried out. After that, the 0.5 McFarland standards was diluted 1000 times and 500 μl of the diluted suspension was poured into the sterilized tubes and all the tubes were incubated for 1 hour at room temperature. Finally, 500 μl sterile Mueller Hinton Broth (MHB) medium were poured into each sterile tube and all the tubes were incubated overnight (12-16 hours) at 35-37 ˚C. In each experiment, one positive control consisted of inoculums, buffer and MHB and one negative control consisted of buffer and MHB also were incubated. Minimal inhibitory concentration (MIC) was determined as the lowest concentration of the complexes displaying no visible growth in the tubes. Also, a standard loop was dipped into the tubes; a loopful of the suspension was removed, subcultured to a Mueller Hinton Agar (MHA) plate and incubated at 37˚C for 24 h. MIC was determined as the lowest concentration of the complex with no bacterial colonies.

Results and Discussion UV-Vis absorption spectroscopy The absorption spectra are the most common means to examine the interaction between metal complex and DNA. The electronic absorption spectral features of Cu(2,3-tmtppa), Cu(3,4tmtppa) and Cu(PcTs) consist of Q and B-bands. The B-band position locates in the UV-visible region at about 340 nm and it does not differ significantly among these complexes. In contrast, the Q-band of Cu(2,3-tmtppa), Cu(3,4-tmtppa) and Cu(PcTs) appears at 638, 678 and 610 nm

8

respectively [8]. Fig. 1 shows the absorption spectra of the complexes in the presence of increasing amounts of the plasmid DNA. As shown, addition of DNA to Cu(2,3-tmtppa) and Cu(3,4-tmtppa) resulted in the hypochromicity of the absorption maxima in the UV–Vis spectra. According to the literature reports, hypochromism indicates a strong interaction between the electronic states of the chromophore and that of DNA bases. Since the decrease of the strength for the electronic interaction is expected as the cube of the distance between the chromophore and the DNA bases [17-18], the observed large hypochromism in the spectra of two porphyrazines strongly suggests a close proximity of the complexes to the plasmid bases. As shown in Fig.1, the hypochromism coincidences with a large bathochromic shift of the Q-band. The red shift is also shown in table 1 quantitatively. Electrostatic perturbations, associated with changes in chromophore solvent environment results in a significant contribution to the overall spectral shift [19]. Interaction of the porphyrazine with plasmid DNA can reduce the exposure of these complexes to water molecules and therefore causes a red shift in their Q-bands. On the whole, the extent of hypochromicity and red shift is consistent with preferential intercalation of the two porphyrazine into the base pairs of DNA helix. The changes in absorbance of Cu(PcTs) upon addition of the plasmid are shown in Fig. 2. As shown, a small hyperchromicity and red shift observed upon addition of plasmid DNA to the phthalocyanine. Thus, it can be concluded that the phthalocyanine binds externally to DNA. This is supported by the values of red shift and hyperchromicity of Cu(PcTs) spectra presented in table 1. In contrast to intercalation, external binding produces a small hypochromism, or even hyperchromism, and a comparatively small bathochromic shift of 4 or 5 nm [10]. Job plot (Fig. 3) was performed to determine the binding stoichiometry of the complexes to the plasmid DNA and it was found that the stoichiometry of Cu(2,3-tmtppa) and Cu(3,4-tmtppa) to

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plasmid in base pair is 1: 6.67 and 1: 5.88 respectively. As shown, there is no large difference between the number of Cu(2,3-tmtppa) and Cu(3,4-tmtppa) molecules that bind to one base pair of plasmid DNA. As shown in Fig. 2, a little change has occurred in absorbance of Cu(PcTs) and the difference of absorbance at λmax was within the experimental error (supplementary data, Fig.S1) and it was too small to calculate the stoichiometry. The values of the equilibrium DNA binding constants, Kb, are listed in table 1. Obviously, Kb for the phthalocyanine was a small value and it was lower than that for both porphyrazine considerably indicating low affinity of this complex to DNA. It is reasonable to say that Cu(PcTs) binds to DNA weakly, because it is a negatively charged complex and therefore there is a repulsive force between this complex and the DNA backbone. As shown in table 1, maximum value of Kb was observed for Cu(3,4-tmtppa). The higher values of Kb for 3,4-isomer respect to the 2,3-isomer indicates the higher affinity of the former for DNA binding. It might be attributed to the more favorable external positioning of the cationic charges in this complex [8]. Spectroflourimetery Fluorescence is a very useful method to verify the binding mode of complexes to DNA. As the complexes is non-emissive, ethidium bromide binding study was undertaken to gain support for the extent of binding of the complexs to DNA. EB is frequently used as a sensitive probe, since its fluorescence intensity greatly enhanced in the presence of DNA, due to its strong intercalation between adjacent base pairs [20]. The emission spectra of EB bound to plasmid DNA in presence of Cu(3,4-tmtppa) and Cu(PcTs) are shown in Fig. 4. As shown, addition of the plasmid DNA to EB results in 10–11 nm blue shift with a large increase in the intensity indicating binding of EB to plasmid DNA and its strong intercalation between the adjacent DNA base pairs. Obviously, after addition of the complexes to DNA-EB, the emission intensity of EB reduces. This result

10

indicates that the complexes bound to DNA competitively and displaces the EB. The fluorescence quenching data were analyzed by Stern-Volmer equation. As shown in Fig. 5, Stern-Volmer plots for both porphyrazines exhibit a slight positive deviation from a straight line, suggesting that both static and dynamic quenching were involved. Therefore, the modified form of Stern-Volmer equation (equation 3) was applied to determine the quenching constant. The quantitative data are presented in table 2. KSV is greater than V for Cu(2,3-tmtppa) indicating that dynamic quenching plays a major role in the interaction of this complex with plasmid, but for Cu(3,4-tmtppa) the value of V is smaller than KSV and therefore, static quenching plays a major role in the interaction [15, 20]. Unlike the porphyrazines, Stern-Volmer plot for the quenching of EB by the phthalocynanine is linear (Fig. 5B), therefore Ksv was determined using linear SternVolmer equation (equation 2). As shown in Fig. 4B, a shoulder appears near 620 nm at high concentrations of Cu(PcTs). Spectral distortion may occur due to absorption of the phthalocyanine as a quencher. Optical absorption of the quencher in the excitation region decreases the elective intensity of the exciting light beam, and its absorption in the emission region decreases the measured fluorescence intensity (the inner filter effect). Since the absorption increases with the increase of the quencher concentration, this induces an apparent quenching and increases the real values of the Stern-Volmer quenching constants

obtained

from

steady

state

experiments

[21-22],

so

Ksv

for

the

phthalocynanine was obtained using linear Stern-Volmer plotted at low concentrations of the quencher (below 50 µM) when no shoulder was observed in the spectra. The value of Ksv was 1.6×104 M-1. Obviously, Ksv for the phthalocyanine is very smaller than

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that for both porphyrzines indicating binding of this complex to DNA is weak compared with the porphyrazines. Gel electrophoresis Plasmid moves on agarose gel under the influence of electrical field. The effect of binding of the complexes to plasmid DNA was also confirmed by the ability of these compounds to produce changes in the electrophoretic mobility of the supercoiled, open circular and linear forms of plasmid DNA. The changes in the migration may be due to alterations in the size, the relative charge, and/or the shape of the supercoiled form of the plasmid. In addition, the ability of the complexes to perform DNA cleavage is generally monitored by agarose gel electrophoresis [2324]. Fig. 6 shows the electrophoretic mobility pattern of plasmid incubated at increasing molar ratios of the complexes. As shown, two clear bands were observed for the plasmid (lanes 4A and 4B). The relatively fast migrated form is the intact supercoil form and the slower one is the open circular form generated from supercoiled when scission occurred on its one strand [19-20]. Lanes 1-3 and 5-7 represent the plasmid treated with different amounts of the complexes. The ratio of the complexes to the plasmid (r) varies in the ranges of 0-1. Obviously, when r equals 0.01 and 0.0033, both complexes retard the electrophoretic mobility and decrease the intensity of the open circular form of the plasmid DNA relative to the control. Electroneutralization of the negative charges of DNA backbone and/or increment of the size of the running DNA due to interaction of the complexes with the plasmid DNA can result in the observed changes. At the higher concentration of the complexes (r=0.016) a distinct difference between the effect of Cu(2,3tmtppa) and Cu(3,4-tmtppa) is obvious. The supercoiled band is disappeared remarkably due to binding of the plasmid to Cu(2,3-tmtppa), but Cu(3,4-tmtppa) hasn’t this effect suggesting the effect of Cu(2,3-tmtppa) on plasmid is more stronger than Cu(3,4-tmtppa). As shown in Fig. 6, at

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higher concentrations of both porphyrazines (r=1, 0.2 and 0.05) the plasmid band disappeared probably because of cleavage of the plasmid DNA. The result of gel electrophoresis of the plasmid in presence of Cu(PcTs) (supplementary data, Fig. S3) indicated that despite of two porphyrazines, different concentration of Cu(PcTs) has not meaningful effect on the plasmid movement. Antibacterial susceptibility testing Antibacterial effect of different concentrations of the complexes against Gram-positive (S. aureus) and Gram-negative (E.coli) bacteria was investigated using dilution test method (supplementary data). MIC values of two porphyrazines are summarized in Table 3. As shown, two porphyrazines exhibit considerable activity against both the Gram positive and the Gram negative bacteria; Cu(2,3-tmtppa) shows higher activity against S. aureus and Cu(3,4-tmtppa) has more effect on E.coli. In addition, comparing the MIC values of two porphyrazines for S. aureus indicates that Cu(2,3-tmtppa) is a more effective antibacterial complex than Cu(3,4tmtppa). MIC values were not determined for the phthalocyanine, because no clear tube and plate was observed even at the high concentrations of the complex. The result indicated that the number of the bacterial colonies in the plate supplemented with the high concentrated solutions of the phthalocyanine continued to decline (supplementary data, Fig. S5), but their growth was not inhibited completely.

Conclusion On

the

whole,

the

results

indicate

that

binding

of

two

water-soluble

tetrapyridinoporphyrazinatocopper(II) complexes had significant antibacterial properties against both the Gram positive and the Gram negative bacteria and this effect can be attributed to

13

binding of these complexes to DNA, but the phthalocyanine has not only a weak antibacterial effect but also a weak interaction with DNA. Likely, electrostatic interaction is the main reason for the observed difference between the binding of the complexes to the plasmid DNA. The phthalocyanine is a negatively charged complex and repulsion between it and phosphate group of DNA decreases the strength of the binding. Unlikely, the porphyrazines are positively charged, so attractive electrostatic interaction between them and the phosphates of the nucleic acids play important role in the binding. Grooves are the sites of location of the deepest molecular electrostatic potential in DNA [25] and therefore the electrostatic interactions play an important role in groove binding of the positively charged complexes. As also mentioned, the results suggested that the binding nature of Cu(2,3tmtppa) and Cu(3,4-tmtppa) with DNA is similar, but the value of binding constant for 3,4isomer is higher than that 2,3-isomer. This difference may be a result of the position of cation in the porphyrazines. As shown in scheme 1, the only difference between Cu(2,3-tmtppa) and Cu(3,4-tmtppa) is the position of their positive charges.

Acknowledgment: Financial support for this work was provided by Research Council of Institute for Advanced Studies in Basic Sciences.

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Abbreviations Pc

Phthalocyanine

Cu(2,3-tmtppa)

N,N',N",N"'-tetramethyltetra-2,3pyridinoporphyrazinatocopper(II)

Cu(3,4-tmtppa)

N,N',N",N"'-tetramethyltetra-3,4pyridinoporphyrazinatocopper(II)

Cu(PcTs)

Copper(II)

phthalocyanine

3,4́,4 ,4

tetrasulfonic acid, tetrasodium salt EB

Ethidium Bromide

MHB

Mueller Hinton Broth

MHA

Mueller Hinton Agar

MIC

Minimal inhibitory concentration

-

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17. K. Lang, P. Kubat, P. Lhotak, J. Mosinger and D.M. Wagnerova, Photochem. Photobiol. , 74 (2001) 558. 18. D. R. McMillin, A. H. Shelton, S. A. Bejune, P. E. Fanwick and R. K. Wall, Coordination Chemistry Reviews, 249 (2005) 1451. 19. D. J. Leggett, S. L. Kelly, L. R. Shiue, Y. T. Wu, D. Chang and K. M. Kadish, Talanta, 30 (1983) 579. 20. R. Senthil Kumar, K. Sasikala and S. Arunachalam, J. Inorg. Biochem., 102 (2008) 234. 21. J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum Press, New York, 1983. 22. I. E. Borissevitch J. Lumin. 81 (1999) 219-224. 23. S. Kashaniana, M. M. Khodaei, H. Roshanfekr, N. Shahabadi and G. Mansourid, Spectrochimica Acta, 86 (2012)531. 24. J. Toneatto, R. A. Boero, G. Lorenzatti, A. M. Cabanillas, G. A. Arguello, J. Inorg. Biochem. 104 (2010) 697. 25. A. Pullman, B. Pullman, Quart. Rev. Biophys. , 14 (1981) 289.

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Figure Legends Scheme 1: Structures of A: N,N ',N'',N'''-tetramethyltetra-2,3-pyridinoporphyrazinatocopper(II), B: N,N ',N'',N'''-tetramethyltetra-3,4-pyridinoporphyrazinatocopper(II) and C: Copper(II) phthalocyanine -3,4',4'',4'''-tetrasulfonic acid, tetrasodium salt Fig. 1: Absorption spectra of A: 4.8 μM [Cu(3,4-tmtppa)]4+, B: 5.3 μM [Cu(2,3-tmtppa)]4+ titrated with plasmid in concentration range of 0-74 μM (A), 0-103 μM (B) in 1 mM phosphate buffer containing 5 mM NaCl and 0.1 mM EDTA, pH 7.0. Fig.2: Absorption spectra of 39 μM Cu(PcTs) titrated with plasmid in concentration range of 0424 μM in 1 mM phosphate buffer containing 5 mM NaCl and 0.1 mM EDTA, pH 7.0. Fig. 3: Job plot for determining the binding stoichiometry of Cu(2,3-tmtppa) and Cu(3,4-tmtppa) to plasmid DNA. Fig. 4: Quenching effect of A: Cu(2,3-tmtppa) (0-3.5 μM) and B: Cu(PcTs) (0-209 μM ) on EBplasmid ([EB]=30 μM, [plasmid]=220 μM) fluorescence in 1 mM phosphate buffer containing 5 mM NaCl and 0.1 mM EDTA, pH 7.0 (Thick line: EB alone, thin lines: EB-plasmid titrated with the complexes). Fig: 5: Stern Volmer plots for fluorescence quenching of EB-plasmid ([EB]=30 μM and [plasmid]=220 μM) by A: Cu(2,3-tmtppa) (0-3.5 μM), Cu(3,4-tmtppa) (0-3.0 μM) and B:Cu(PcTs) (0-209 μM) in 1 mM phosphate buffer containing 5 mM NaCl and 0.1 mM EDTA, pH 7.0.

18

Fig 6: Effect of the porphyrazines on electrophoretic mobility of plasmid. In lines 1, 2, 3, 5, 6 and 7 the molar ratio of the plasmid to Cu(2,3-tmtppa) (A) and Cu(3,4-tmtppa) (B) are 1, 5, 20, 60, 100 and 300 respectively and in lines 4A and 4B the plasmid is alone ( The molar ratio of the complexes to the plasmid is 1, 0.2, 0.05, 0, 0.016, 0.01 and 0.0033 in lines 1-7). In all lines the concentration of the plasmid is 483 μM.

19

Figure Legends Scheme 1: Structures of A: N,N ',N'',N'''-tetramethyltetra-2,3-pyridinoporphyrazinatocopper(II), B: N,N ',N'',N'''-tetramethyltetra-3,4-pyridinoporphyrazinatocopper(II) and C: Copper(II) phthalocyanine -3,4',4'',4'''-tetrasulfonic acid, tetrasodium salt Fig. 1: Absorption spectra of A: 4.8 μM [Cu(3,4-tmtppa)]4+, B: 5.3 μM [Cu(2,3-tmtppa)]4+ titrated with plasmid in concentration range of 0-74 μM (A), 0-103 μM (B) in 1 mM phosphate buffer containing 5 mM NaCl and 0.1 mM EDTA, pH 7.0. Fig.2: Absorption spectra of 39 μM Cu(PcTs) titrated with plasmid in concentration range of 0424 μM in 1 mM phosphate buffer containing 5 mM NaCl and 0.1 mM EDTA, pH 7.0. Fig. 3: Job plot for determining the binding stoichiometry of Cu(2,3-tmtppa) and Cu(3,4-tmtppa) to plasmid DNA. Fig. 4: Quenching effect of A: Cu(2,3-tmtppa) (0-3.5 μM) and B: Cu(PcTs) (0-209 μM ) on EBplasmid ([EB]=30 μM, [plasmid]=220 μM) fluorescence in 1 mM phosphate buffer containing 5 mM NaCl and 0.1 mM EDTA, pH 7.0 (Thick line: EB alone, thin lines: EB-plasmid titrated with the complexes). Fig: 5: Stern Volmer plots for fluorescence quenching of EB-plasmid ([EB]=30 μM and [plasmid]=220 μM) by A: Cu(2,3-tmtppa) (0-3.5 μM), Cu(3,4-tmtppa) (0-3.0 μM) and B:Cu(PcTs) (0-209 μM) in 1 mM phosphate buffer containing 5 mM NaCl and 0.1 mM EDTA, pH 7.0. Fig 6: Effect of the porphyrazines on electrophoretic mobility of plasmid. In lines 1, 2, 3, 5, 6 and 7 the molar ratio of the plasmid to Cu(2,3-tmtppa) (A) and Cu(3,4-tmtppa) (B) are 1, 5, 20, 60, 100 and 300 respectively and in lines 4A and 4B the plasmid is alone ( The molar ratio of the

20

complexes to the plasmid is 1, 0.2, 0.05, 0, 0.016, 0.01 and 0.0033 in lines 1-7). In all lines the concentration of the plasmid is 483 μM.

21

Fig. 1

Fig. 2

22

Fig. 3

Fig. 4

23

Fig. 5

24

Fig. 6

25

Scheme Scheme 1

26

Table 1: Binding constant (Kb), binding stoichiometry (n), red shifts (nm) and hypochromicity values (%) of binding of the complexes to plasmid DNA

red-shift

hypoa and hyperb

(nm)

chromicity (%)

9.9×105

9

68.22a

Cu(3,4-tmtppa)

2.4×106

21

64.23a

Cu(PcTs)

8.4×102

4

26.48 b

complexes

Kb (M-1)

Cu(2,3-tmtppa)

Table 2: Quenching parameters of plasmid-ethidium bromide by the complexes

Complexes

KSV −1

kq×1013

τ0 (ns)

V −1

(L·mol )

(L·mol )

Cu(2,3-tmtppa)

6.3×106

3.5×106

Cu(3,4-tmtppa)

2.6×106

7×106

-1 -1

Blue-shift

(M .s )

(nm)

23

2.72

10

23

1.17

11

Table 3: MIC (µM) values of the porphyrazine complexes for S. aureus and E.coli

The complexes

S. aureus

E.coli

Cu(2,3-tmtppa)

2.97

11.87

Cu(3,4-tmtppa)

5.94

5.94