spectroscopic and physicochemical methods for

DEVELOPMENT OF PHYSICOCHEMICAL AND SPECTROSCOPIC METHODS FOR STUDYING THE INTERACTION OF CATIONIC PORPHYRIN WITH DNA (PART I : DETERMINATION OF BINDING MODE)

Daryono H. Tjahjono,*1) Slamet Ibrahim,1) Amir Musadad,1) Mudasir2) and Hidenari Inoue3) 1

Pharmaceutical Analysis and Medicinal Chemistry Research Group, Department of Pharmacy, Institut Teknologi Bandung, Jalan Ganesha 10 Bandung 40132, Indonesia E-mail : [email protected] 2 Laboratory of Analytical Chemistry, Department of Chemistry, Universitas Gajah Mada, Sekip Utara P.O. Box Bls. 21, Yogyakarta 55281, Indonesia 3 Laboratory of Coordination Chemistry, Department of Applied Chemistry, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522 Japan ABSTRACT A combination of viscometric, melting temperature, visible absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) methods have been developed and applied to study the interaction of cationic porphyrin with DNA. This method could be used to judge the binding mode of porphyrin-DNA interaction and predict in detail the position and orientation of porphyrin molecule relative to DNA. H2-1 was bound edge-on at the 5'TA3' steps of the minor groove of ctDNA, while its isomer H2-2 was intercalated into the 5'GC3' step of ctDNA. Cu-2 was intercalated into the 5'GC3' steps but Zn-2 only outside bound at the 5'TA3' step of the major groove of ctDNA. H2-3 was intercalated into AT and GC sites of ctDNA, but the base step had not been clarified yet. Key word : Cationic porphyrin, DNA, interaction, physicochemical spectroscopic.

ABSTRAK Suatu kombinasi metode viskometri, suhu pelelehan, spetrofotometri sinar tampak, dikroisme sirkular dan dikroisme sirkular magnetik telah dikembangkan dan diterapkan untuk mempelajari interaksi antara kationik porfirin dengan DNA. Metode ini dapat digunakan untuk menentukan mode pengikatan dari interaksi porfirin dengan DNA dan dapat memperkirakan posisi dan orientasi molekul porfirin terhadap DNA. H2-1 terikat edge-on pada lekukan minor ctDNA dengan pusat porfirin terletak pada urutan basa 5'TA3', sedangkan isomernya H2-2 terinterkalasi ke dalam urutan basa 5'GC3' dari ctDNA. Cu-2 terinterkalasi ke dalam urutan basa 5'GC3' dari ctDNA tetapi Zn-2 hanya terikat diluar pada lekukan major ctDNA dengan pusat porfirin terletak pada urutan basa 5'TA3'. H2-3 dapat terinterkalasi pada daerah AT maupun GC dari ctDNA tetapi urutan basanya belum dapat dijelaskan. Kata kunci : Porfirin kationik, DNA, interaksi, fisikokimia spektroskopi

78 – Acta Pharmaceutica Indonesia

INTRODUCTION Many experimental techniques have been applied to study the interactions of porphyrins with DNA. A variety of spectroscopic methods, e.g. UV-visible absorption and circular dichroism (CD) [1-3], fluorescence [4], Raman [5], NMR [5-6], ESR [7], and magnetic circular dichroism (MCD) [8] spectroscopics have been used to evaluate the binding interaction of porphyrin to DNA. Moreover, a few instrumental analytical techniques applicable to an aqueous buffer solution of DNA have been particularly developed to investigate porphyrin-DNA interactions. On the other hand, a number of physicochemical methods, e.g. gel electrophoresis [9], melting temperature measurements [10] and hydrodynamic methods such as viscosity and sedimentation measurements [11], have also been used for determining the binding modes of porphyrin to DNA. However, no single technique can be used to determine the binding mode. Therefore a judicious combination of some techniques to provide a strong basis for characterizing the interactions of porphyrin with DNA is a necessary. For the purpose of preliminary study of molecule-DNA interaction, a spectrophotometric technique so-called spectrophotometric titration of a certain molecule is usually performed in a buffer solution by DNA. This rather conventional analytical method provides a lot of information about the interactions of compound with DNA. The tendency and magnitude of the peak shift and hypo-or hyperchrominisity of a band is useful for assessing the binding mode and binding constants [1-3]. The circular dichroism (CD) spectra are indispensable not only for structural analysis of DNA but also for in vitro studies of small molecule-DNA interaction. This instrumental technique has been developed mainly for structural analysis of molecule-DNA complexes. In addition, this technique also makes a unique contribution to the stereo analysis, because so-called induced circular dichroism is observed upon coupling of electric dipole transition moments between base pair of DNA and molecule [12-13]. In many cases, achiral molecules give raises a magnetic circular dichroism (MCD) in the certain band region. A change in the MCD spectra of molecules upon their interaction with DNA brings about variable information on molecule-DNA interaction [8]. In the MCD spectra, the directly measurable MCD/optical parameter ratio is used for assessing the binding mode, because it is proportional to the excited-state angular momentum, (Lj) [14]. When a DNA is heated in solution, the absorbance at about 260 nm increases. For double-stranded (double-helix) DNA, there is an S-shaped increase in absorbance as single strands are produced. The absorbance versus temperature curve is called melting curve, and the midpoint of transition is called “melting temperature” (Tm). The Tm of DNA is sensitive to its double helix stability and the binding of

Vol. XXVIII, No. 4, Desember, 2003 - 79

compound to DNA alters the Tm depending on the strength of interactions [10]. Therefore, it can be used as an indicator of binding properties of molecules to DNA and their binding strength. Binding of small molecules to DNA can alter not only the helical twist, but also the contour of length and stiffness (both bending and torsional), and can induce systematic bend into the DNA double helix. A hydrodynamic method such as viscometric measurements in which the solution viscosity of DNA is sensitive to the changes in the effective length of DNA molecules is one of the most critical tests for inferring the binding mode of DNA in solution, i.e. intercalation or other binding modes. Generally an increase in the relative viscosity is ascribed to a length increase of the DNA double helix due to intercalation. On the other hand, a decrease in the relative viscosity due to the reduction of the effective length of DNA molecules results from the bending of the DNA double helix caused by electrostatic interaction between the anion site of phosphate of DNA and the cation site of molecule [15]. In recent years, studies on the interaction of cationic porphyrin with DNA have received much attention because of the importance in DNA-probing and photodynamic therapy of cancer. In the present work we have developed the application of visible absorption, CD and MCD spectroscopy as well as melting temperature and viscosity measurements to studies of porphyrin-DNA interactions. As model of cationic porphyrins, we have chosen meso-tetrakis(1,3dimethylimidazolium-2-yl)porphyrin (H2ImP, H2-1), meso-tetrakis(1,2-dimethylpyrazolium-4-yl)porphyrin (H2PzP, H2-2) and its copper and zinc complexes (Cu-2, Zn-2), and 5,15-bis (1,3-dimethylimidazolium-2-yl)porphyrin (H2BImP, H2-3). These cationic porphyrins have been recently synthesized and characterized in our laboratory [16]. The structure of these water-soluble cationic porphyrins is shown in Fig. 1. This paper will focus on the determination of binding modes of the interaction between cationic porphyins and calf thymus DNA (ctDNA) by the above mentioned spectroscopic and physicochemical methods in detail more than general intercalation and outside binding modes only.

EXPERIMENTAL Materials and Instruments NaH2PO4, Na2HPO4, EDTA and NaCl were purchased from Kanto Chemical Co. Ltd. Calf thymus DNA (ctDNA) was purchased from Sigma Co. Ltd. and used as received. Other chemicals were used as received without further purification and all solvents were of reagent grade. Cationic porphyrins H2-1, H2-2, Cu-2, Zn-2, and H2-3 were synthesized and characterized according to the previous method [16].


UV-visible absorption spectra were recorded in solutions at 25 °C (unless otherwise stated) on a JASCO V-570 spectrophotometer equipped with a JASCO ETC-505T temperature controller using 10 mm quartz cells. CD and MCD spectra were recorded on a JASCO J-720 WI spectropolarimeter using 10 mm quartz cells. The viscosity of buffer solution of DNA was measured by an Ubbelohde viscometer. 1H NMR spectra were recorded at 300 MHz on a JNM-LA300 spectrometer. Elemental analyses were performed at the Central Laboratory of the Faculty of Science and Technology, Keio University and the Shonan Bunseki Center. R2

N

N M

R1 N

R1 N

R2 Me

Me

N 1 : R1 = R2 =

N M = H2

R2 = H

3 : R1 =

N

M = H2

N

Me

Me

2 : R1 = R2 = Cu-2 : M = Cu(II) Zn-2 :

N N

Me

M = H2 Me

M = Zn(II)

Fig. 1 : Stucture of cationic porphyrin 1. Measurements of Viscosity The viscosity of DNA solutions was measured at 30 ± 1°C in a temperaturecontrolled circulating water-bath using an Ubbelohde viscometer. Typically, 10.0 mL of phosphate buffer was transferred to the viscometer to obtain the reading of flow time. For the determination of solution viscosity, 10.0 mL of 45 M DNA in phosphate buffer was taken to the viscometer and a flow time reading was obtained. An appropriate amount of porphyrin in a buffered solution was then added to the viscometer to give a certain R value (=[porphyrin]/[DNA in base pair]) while keeping the DNA concentration constant, and the flow time was read. The flow time of samples was measured after the thermal equilibrium of the viscometer


was achieved (ca. 60 min). Each point measured was the average of at least five readings with a relative standard deviation (RSD) of less than 1 %. The data obtained were presented as (/o) versus R, where  is the reduced specific viscosity of DNA in the presence of porphyrin and o is the reduced specific viscosity of DNA alone [11,15]. 2. Measurement of Melting Temperature (Tm) The melting curves of both free DNA and porphyrin-DNA complex in phosphate buffer were obtained by measuring the hyperchromicity of DNA at 260 nm as a function of temperature. The melting temperatures were measured in 45 M DNA in phosphate buffer at pH 6.8 ( = 0.2 M NaCl). The temperature was scanned from 25 to 95 °C at a speed of 2 °C per min. The melting temperature (Tm) was taken as the mid-point of the hyperchromic transition. All measurements were repeated three times and the data presented are the average values with a RSD of lower than 5%. 3. Spectral Measurements All experiments, unless specifically indicated, were performed at 25 °C in a phosphate buffer (pH 6.8). The buffer solution consists of 6 mM Na2HPO4, 2 mM NaH2PO4, 1 mM EDTA and a sufficient amount of NaCl to give a final ionic strength of  = 0.2 M. A stock solution of ctDNA was prepared and stored in phosphate buffer. An extinction coefficient of 260 = 1.31 × 104 M–1 cm–1 was used to determine the concentration in base pairs of ctDNA [17]. Porphyrin concentrations were determined spectrophotometrically with 407 = 1.57 × 105 M–1 cm–1 for H2-1 [18], 412 = 2.50 × 105 M–1 cm–1 for H2-2 [18], 412 = 2.74 × 105 M–1 cm–1 for Cu-2, 419 = 3.4 × 105 M–1 cm–1 for Zn-2 and 394 = 1.31 × 105 M–1 cm–1 for H2-3 [16]. In the absence of DNA, cationic porphyrins studied neither dimirize nor aggregate under the present experimental conditions (phosphate buffer, pH 6.8,  = 0.2 M). Visible absorption spectra were recorded on a JASCO V-570 spectrophotometer at a spectral bandpass of 0.2 nm with 0.05 nm spectral resolution. Wavelength calibration was carried out using a holmium oxide-glass standard. Induced CD spectra were obtained on a JASCO J-720 WI spectropolarimeter, in which the calibration of wavelength and intensity was carried out using a 0.060% (w/v) aqueous solution of l-ammonium-camphore-10-sulfonate (Aldrich). CD spectra were recorded with the following instrument parameter settings: 0.7 nm bandwidth, 2.0 s response time, 0.5 nm step resolution and 20 nm min–1 scan speed between 500 and 350 nm. The spectra were obtained from 4 time scans. MCD spectra were also measured on a JASCO J-720 WI spectropolarimeter equipped with a JASCO water-cooled electromagnet and magnetic shielding of the detector. Wavelength


and intensity were ascertained by 1 mM aq. solution of K3[Fe(CN)6] and (+)-[Co(en)3]Cl3. Measurements of MCD spectra were made in an applied magnetic field of H0 = 1.1 T and with the instrument parameter setting which is the same as CD spectral measurements. Total MCD spectra, [CD + MCD], were recorded for free and DNA-bound porphyrin solutions. Each MCD recording was preceded by a CD recording (H0 = 0.0 T) of the sample in order to obtain the pure, net MCD spectra, i.e. net MCD = [CD + MCD] – [CD]. Typically, the porphyrin was titrated by stepwise addition of the concentrated stock solution of DNA directly to 2.5 mL of starting volume of porphyrin solution. Visible absorption, induced CD and MCD spectra were measured subsequently after the sample solutions (porphyrin-DNA mixture) were allowed to equilibrate prior to recording of data, i.e. 15 min at each addition of DNA. The excited state angular momentum (Lj) was calculated from the ratio of []p-t/max according to the literature method [14].

RESULTS AND DISCUSSION The porphyrins studied here are structurally different from each other. The meso-substituents of H2-1 have a large steric hindrance due to the presence of two methyl groups at 1N and 3N of the imidazolium rings. Therefore, these imidazolium rings can not rotate freely to porphyrin macrocycle due to a high rotational barrier of the C-C bond joining the porphyrin Cmeso and imidazole-C2, caused by steric hindrance between the methyl group of imidazole and the porphyrin -hydrogens. Consequently, these meso-substituents will make a position of nearly perpendicular against its porphyrin macrocycle. Porphyrin H2-2 has methyl groups at position 1N and 2N of pyrazolium rings so that it can rotate freely and may make a planar position against its porphyrin macrocycle. Insertion of metal ion into porphyrin H2-2 can change its planarity which depend on the kind of metal. Complex Cu-2 has the same planarity with that of free base H2-2 because the copper(II) ion ligate only four inner nitrogen of porphyrin while Zn-2 may ligate one molecule of H2O as fifth ligand. Porphyrin H2-3 has only two meso-substituents. The  electronic system effective for the DNA binding is widely extended in H2-3 compared to that of H2-1 with four imidazolium rings at the meso-position. Therefore, the absence of two meso-substituents at the trans-position may facilitate easily the intercalation of the porphyrin into the DNA base pairs. 1.

Physicochemical Evidence for Binding Mode of Cationic Porphyrin-DNA Interaction In the absence of X-ray and NMR structural data, hydrodynamic methods such as viscosity and sedimentation measurements are arguably the most critical test of the


classical intercalation model and provide the most definitive means of inferring the binding mode of porphyrins to DNA in solution [15]. For this reason, the binding modes of cationic porphyrins to polynucleotides were investigated by viscometric measurements. As shown in Fig. 2, the viscosity of the buffer solution of ctDNA was increased with the concentration of H2-2, Cu-2 and H2-3. The increasing tendency in the relative viscosity is ascribed to a length increase of the DNA helix due to the intercalation [19]. Thus, ctDNA is intercalated by H2-2, Cu-2 and H2-3. In contrast, the viscosity of the buffer solution of ctDNA was decreased with increasing concentrations of H2-1 and Zn-2. The decrease of the viscosity due to shortening of the effective length of DNA is attributed to the bending and/or kinking of the DNA helix. This is typical of the outside binding of porphyrin [20], and thus ctDNA is outside bound by H2-1 and Zn-2. The above viscometric consequency is consistent with the following conclusion from the visible, induced CD and MCD spectra. 1.4

2 Cu-2 3

/0

1.2

1 Zn-2

1.0 0.8 0.6 0.00

0.02

0.04

0.06

0.08

0.10

R (= [Por]/[DNA])

Fig. 2

Plots of the relative viscosity of ctDNA versus R values of porphyrins in phosphate buffer (pH 6.8,  = 0.2 M).

The stability of the DNA double helix influences the melting temperature (Tm) of DNA, while the binding of compounds to DNA alters the Tm depending on the strength of interactions. Upon heating DNA in a buffer solution, firstly the A-T base pairs having two hydrogen bonds are dissociated, secondly the G-C base pairs with three hydrogen bonds are cleaved, and finally the DNA double helix turns into two single strands. It is known that ctDNA is consisted of 42% of G-C base pairs and 58% of A-T base pairs [21]. Thus, measurements of the Tm of ctDNA in porphyrin solutions can explore how strong the interaction between porphyrin and DNA is. To investigate this characteristic, the changes in the Tm upon the increasing addition of H2-1, H2-2, Cu-2, Zn-2 and H2-3 to the phosphate buffer solution of


ctDNA were measured in the range of R ([Porphyrin]/[DNA in base pair]) = 0  0.1. Plots of the increase in melting temperature (Tm) versus R values are shown in Fig. 3. The Tm of ctDNA is always increased with the addition of H2-1, H2-2, Cu-2, Zn-2 or H2-3 to the ctDNA buffer solution. The above results have shown that all the porphyrins studied interact with ctDNA to stabilize the duplex structure. Moreover, the insertion of the metal ions into H2-2 leads to change in Tm, suggesting that the interaction between porphyrin and ctDNA is influenced by insertion of metal ion the free-metal porphyrin. It is also noteworthy that the Tm of Zn-2 is larger than that of Cu-2 and thus Zn-2 stabilizes the double helix more than Cu-2. This remarkable feature probably not only comes from the difference in the binding modes as suggested by the viscosity data and as described later in detail, but also come from that in the binding strength as will be published elsewhere (part II: affinity and thermodynamic parameters).

Zn-2 2 Cu-2 1 3

6

o

T m / C

8

4 2 0 0

0.02

0.04

0.06

0.08

0.1

R (= [Por] / [DNA])

Fig. 3

Plots of the increase in ctDNA melting temperature (Tm) versus R values of porphyrins in phosphate buffer (pH 6.8,  = 0.2 M).

2. Spectroscopic Evidence for Binding Mode of Cationic Porphyrin-DNA Interaction In the interpretation of induced CD due to porphyrin-DNA interaction, there is a convention that a negative induced signal indicates intercalation, while a positive signal suggests an outside (groove) binding mode [1]. However, there is no detail explanation about the relationship between sign and magnitude of the signal and base step of binding site. In order to systematically study how the induced CD of a ligand depends on its position and orientation relative to DNA, it is convenient to determine reference geometry for ligand-DNA complex. The standard of electric dipole transition moments (edtm, e) of the ligand is set to be parallel with the


groove, and the standard position to be halfway between two successive base pairs at a distance of 7 Å from the helix axis[12-13]. Moreover, in the interpretation of induced CD and MCD spectra, the porphyrin molecule is placed in the xy-plane and the inner hydrogen-hydrogen axis of the porphyrin is set to the x-axis while the other inner N-N axis is to the y-axis. For the metallopyrazoliumylporphyrin, each of the inner N-N axes is set to the x- and y-axis. The two e of the porphyrin are in the direction of each axis. On the basis of Weiss model [22], ex is the most perturbed direction, while ey is the least perturbed direction. The CD results calculated by the CD matrix method [23-24], which is abbreviated as CD-mm and supported by other approaches [12-13], were used to interpret the CD spectra and to asses the binding mode of the porphyrin to DNA. The parameters , ,  and  define the position and orientation of the porphyrin molecule relative to DNA. The X axis of the DNA coordinate system extends from the minor groove and constitutes a true C2 axis of the B-DNA helix. The parameter  is the angle of edtm of porphyrin with respects to the DNA helix axis (Z);  is the azimuthal angle between the X axis and the line connecting the edtm of porphyrin;  is the azimuthal angle between the line from the helix axis to the position of the edtm of porphyrin and the projection of the edtm of porphyrin onto a plane perpendicular to the helix axis and containing the same line;  is the angle from the X axis to the edtm of porphyrin in the X-Y plane of intercalation pocket; X, Y and Z axes are defined as a right-hand Cartesian coordinate system (for detail, see ref. 23-24). In addition, the MCD results were used to confirm the binding mode by comparing the excited state angular momenta (Lj), which are proportional to []p-t/max of free and DNA-bound porphyrins, i.e. the energy differences in the - and *-MOs (1a1u 3a2u 4eg) of porphyrins [14]. The Soret band of H2-1 is virtually unaffected by the addition of ctDNA because the bathochromic shift is 1 nm and the hypochromicity is only 2 %. The induced CD spectra showed a single positive band at 408 nm. On the basis of these visible and induced CD spectral changes it suggest that H2-1 is outside bound to ctDNA. Furthermore, the CD-mm suggests that H2-1 is bound edge-on at the 5'TA3' steps of the minor groove. Upon interaction of H2-1 with ctDNA, the MCD spectra were not changed significantly as shown by very small changes (ca 0.5%) in the excited state angular momentum (Lj) of H2-1 (Fig. 4). Such changes also support the outside binding mode. The (+) induced CD at 408 nm is aligned with the high energy (+) MCD band. Thus, it suggests that the (+) induced CD band is associated with the least perturbed edtm ey of H2-1. Therefore, outside binding edge-on of H2-1 at the 5'TA3' steps of the minor groove is positioned by 90º/0º/0º and 45º/0º/90º for // of ex and ey, respectively. In the presence of ctDNA, the Soret band of H2-2 showed a large bathocromic shift


(14 nm) and a substantial hypochromicity (45%) which finally became a new single peak at 425 nm at R-values of 0.04 and lowers. At these R-values, the induced CD spectra of H2-2 were composed of only a single negative band at 426 nm (Fig. 5). These visible spectral changes and induced CD spectral pattern suggest that H2-2 intercalate into ctDNA. The application of the CD-mm to the observed induced CD spectra has revealed that H2-2 is intercalated into the 5'GC3' step of ctDNA because the CD-mm correctly predicts weak (+) and strong (–) bands for  = 0º and  = 90º, respectively. The MCD spectrum of H2-2 showed a (+) pseudo A-term with a positive peak at 407 nm and a negative peak at 435 nm. In the presence of ctDNA, the MCD spectrum of H2-2 shifts to the longer wavelength (13 nm) and the intensity (/H) decreases, but the sign is retained as the (+) pseudo A-term. This MCD change has revealed that the excited state angular momentum (Lj), i.e. []p-t/max, is reduced ca. 23% from 2.56 to 1.98 (deg cm2 dmol–1 T–1/M–1 cm–1), indicating that the perturbation to the localized * MOs (1a1u 3a2u 4eg) is strong due to intercalation of H2-2. In addition, the (–) induced CD band is well aligned with the crossover of the MCD spectra, suggesting that the porphyrin plane is located at the center of base pairs.

Abs

0.3

0.2

0.1

0 350

400

450

500

400

450

500

10

-1

4

 / M cm

6

-1

8

2 0 -2 350


-1 -1

 / H (M cm T )

45 30

-1

15 0 -15 -30 -45 350

400

450

500

Wavelength / nm

Fig. 4

Visible, CD and MCD spectra of free (solid line) and ctDNA bound (dashed line) H2-1 in phosphate buffer (pH 6.8,  = 0.2 M) at R (= [porphyrin]/[DNA]) = 0.02.

Abs

0.10

0.05

0.00 350

400

450

500

400

450

500

400

450

500

0

-1

 / M cm

-1

3

-3 -6 -9 -12 350

-1

-1

-1

 / H (M cm T )

120 60 0 -60 -120 350

Wavelength / nm


Fig. 5

Visible, CD and MCD spectra of free (solid line) and ctDNA-bound (dashed line) H2-2 in phosphate buffer (pH 6.8,  = 0.2 M) at R (= [porphyrin]/[DNA]) = 0.02.

All the spectroscopic data measured for the DNA binding of Cu-2 and Zn-2 and H2-3 are listed together with some assignments in Table 1. The binding of Cu-2 to ctDNA caused 9% of hypochromicity and 6 nm of red shift of the Soret band. At an R-value of 0.02, the induced CD showed a strong negative band at 421 nm and a weak positive band at 436 nm. The MCD of free Cu-2 showed a strong (+) A-term with its positive and negative band at 406 nm and 419 nm, respectively. Upon interaction of Cu-2 with ctDNA, the MCD is shifted 8 nm to the longer wavelength and accompanied by a decrease in /H, and the positive and negative bands appeared at 413 and 427 nm, respectively. The spectral pattern of the induced CD suggests that Cu-2 is intercalated into the DNA base pairs. A decrease in the excited state angular momentum also supports this binding mode. The (–) induced CD band at 421 nm is aligned with the higher energy (+) MCD band at 413 nm. Thus, it is concluded that the (–) induced CD band is associated with the least perturbed edtm ey of Cu-2. The CD-mm correctly predicts a weak (+) and a strong (–) band for  = 0º and  = 90º, respectively, for intercalation into the 5'GC3' steps. When Cu-2 intercalates into the 5'GC3' steps of ctDNA, the ex is nearly perpendicular to the base pair and ey is parallel to the base pair. Upon interaction of Zn-2 with ctDNA, the Soret band showed 5 nm of red shift and 13% of hyperchromicity. The induced CD of Zn-2 appeared as a single positive band at 416 nm accompanied by a weak negative band at a longer wavelength at 440 nm. The MCD of Zn-2 was observed as a (+)-A term with a strong positive and a negative band at 414 and 426 nm, respectively. Upon interaction of Zn-2 with ctDNA, the MCD was shifted about 4 nm to the longer wavelength and accompanied by an increase in /H. These visible and induced CD spectral changes indicate that Zn-2 is outside bund to DNA. An increase in Lj (ca 1%) of Zn-2 upon binding to ctDNA also supports the outside binding mode. Furthermore, the application of the CD-mm has revealed that Zn-2 is bound face-on at the 5'TA3' step of the major groove. In addition, the (+) induced CD band at 416 nm is well aligned with the (+) MCD band at 418 nm, suggesting that this band is due to the least perturbed edtm ey. Thus, the position of Zn-2 at the 5'TA3' step of the major groove of ctDNA is predicted to be 45º/180º/90º and 45º/180º/270º for // of ex and ey, respectively.


In the presence of ctDNA, the Soret band of H2-3 showed large red shift (12 nm) and substantial hypochromicity (34%) and finally changed to a new single peak at 406 nm at R values of 0.02 and lower. At these R values the induced CD spectra of H2-3 appeared as a weak conservative signal in a pattern similar to that of meso-tetakis(4-N-methyl-pyridiniumyl)porphyrin (H2TMPyP) [25]. First, this CD-spectral feature suggests that H2-3 is intercalated into CTDNA although the intercalation is probably accompanied by outside binding. The appearance of this conservative spectrum also suggests the possibility of exciton coupling of the edtm of porphyrin due to inter-porphyrin self-association. However, such an assumption can be ruled out, because this induced CD was observed at R = 0.02 where the porphyrin load is very low. The application of the CD-mm to our induced CD leads first to an edge-on binding of H2-3 to AT sites, especially to the 5'AT3' step.


However, the substantial hypochromicity and large red shift of the Soret band do not support this interpretation, because only small hypochromicity and small red shift of the Soret band are usually observed for the outside binding [25]. Therefore, an arguable binding mode is intercalation, i.e. intercalation of H2-3 into both AT and GC sites. This probability of the binding of H2-3 to two different sites is also supported by the appearance of the two induced CD bands which are separated by ~ 1482 cm–1 (26 nm). Upon interaction with ctDNA, the MCD of H2-3 shifted to the longer wavelength and accompanied by a large decrease in /H and Lj. This feature also supports an intercalation binding mode. In addition, the (+) induced CD band at 406 nm is well aligned with the lower energy (–) MCD band at 406 nm. Therefore, it is concluded that the (+) induced CD band is concerned with the most perturbed direction that coincides with the edtm ex of H2-3. The above results show that the physicochemical methods can only determined whether a molecule is intercalated into or outside bound to DNA base pairs, and visible absorption, induced CD and MCD spectroscopic can complete the data to dissect in detail the binding mode of porphyrin-DNA interaction (illustrations for the binding of cationic porphyrin to DNA see ref. 26).

CONCLUSIONS A combination of physicochemical and spectroscopic methods can be applied to characterize the binding mode of the interaction between porphyrin and DNA. H 2-1 was outside bound at the minor groove of ctDNA, while its isomer H 2-2 was intercalated into ctDNA base pair. Insertion of copper(II) ion into H2-2 did not change the binding mode significantly. On the other hand, metallation of H 2-2 with zinc(II) ion changed the binding mode from intercalation to outside binding mode. The absence of two meso-substituents at the trans-position has promoted H2-3 to


intercalate easily into the DNA base pairs although its parent tetracationic H2-1 was blocked for intercalation.

ACKKNOWLEDGEMENTS This work was supported in part by a Grant-in-Aid for Scientific Research (No. 13554025) from the Ministry of Education, Science, Sports, and Culture of the Japanese Government. The first author (D.H.T.) acknowledges financial support from Keio Leading-Edge Laboratory Program, Keio University, Japan and The Hitachi Scholarship Foundation, Tokyo, Japan through Research Fellowship Program. REFERENCES

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5.

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26.

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Table 1 Spectral data of cationic porphyrins upon interaction with ctDNAa Free porphyrin Method Visible

H2-1

H2-2 Cu-2 Zn-2 H2-3 H2-1 H2-2

Cu-2

Zn-2

H2-3

406

412

412

419

394

407

425

418

424

406

1.57

2.50

2.74

3.40

1.32

1.53

1.58

2.49

3.84

0.87

%H

2.55

36.5

9.04

–12.8

34.1

o, nm

408

427

421; 436

416; 440

406; 432

o, nm

max, cm M × 10 –1

CD

ctDNA-bound porphyrin

Parameters

–1

5

, cm–1M–1 MCD

+7.87 –4.81 –13.3; +1.91 +14.0; –1.46

+1.31; –0.78

crossover, nm

414

414

412

420

398

415

427

420

426

413

p, nm

404

407

406

414

390

405

420

413

418

404


p/H, cm–1M–1T–1 t, nm

+43.3 +96.3 +98.9 +174 +37.9 +42.3 +41.1 425

421

419

426

406

426

435

+94.7

+197

+18.9

427

430

423

t/H, cm M T 

–36.1 –97.4 –89.5 –153 –33.6 –34.9 –54.5

–74.3

–176

–18.0

[]p-t/max, deg cm2

1.67

2.24

3.21

1.40

–1

–1 –1

–1 –1

–1

2.56

2.27

3.17

1.79

1.66

1.98

–1

dmol T /cm M a

in phosphate buffer (pH 6.8,  = 0.2 M) at R (= [porphyrin]/[DNA]) = 0.02; %H is hypochromicity (%); p and t are peak and trough, respectively; [] =  × 3300.14

Table 1 Spectral data of cationic porphyrins upon interaction with ctDNAa Free porphyrin Method Visible

ctDNA-bound porphyrin

Parameters H2-1 H2-2 Cu-2 Zn-2 H2-3 H2-1 H2-2

Cu-2

Zn-2

H2-3

o, nm

406

412

412

419

394

407

425

418

424

406

max, cm–1M–1 × 105

1.57

2.50

2.74

3.40

1.32

1.53

1.58

2.49

3.84

0.87

2.55

36.5

9.04

–12.8

34.1

408

427

421; 436

416; 440

406; 432

+14.0; –1.46

+1.31; –0.78

%H CD

o, nm , cm M –1

MCD

–1

+7.87 –4.81 –13.3; +1.91

crossover, nm

414

414

412

420

398

415

427

420

426

413

p, nm

404

407

406

414

390

405

420

413

418

404

+94.7

+197

+18.9

427

430

423

–36.1 –97.4 –89.5 –153 –33.6 –34.9 –54.5

–74.3

–176

–18.0

1.67

2.24

3.21

1.40

p/H, cm–1M–1T–1 t, nm

425

t/H, cm–1M–1T–1 [] /max, deg cm p-t

–1 –1

+43.3 +96.3 +98.9 +174 +37.9 +42.3 +41.1

2

–1

421

2.56

419

2.27

426

3.17

406

1.79

426

1.66

435

1.98

–1

dmol T /cm M a

in phosphate buffer (pH 6.8,  = 0.2 M) at R (= [porphyrin]/[DNA]) = 0.02; %H is hypochromicity (%); p and t are peak and trough, respectively; [] =  × 3300.14
