Structure—activity relationships for DNA photocleavage by cationic porphyrins

41

J. Photochem. Photobiol. B: Biol., 18 (1993) 41-50

Structure-activity porphyrins

relationships

for DNA photocleavage

by cationic

D. T. CrokeaptTtt, L. Perrouaultb, M. A. Sari”, J.-P. Battioni”, D. Mansuyc, C. Heleneb and T. Le Doanb “Department of Biochemishy, Royal College of Surgeons in Ireland, St. Stephens Green, Dublin 2 (Ireland) bLaboratoire de Biophysique, Museum National d’Histoire Naturelle, 43 rue Cuvier, 75231 Paris (France) ‘Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques, 45 rue des Saints Peres, 75270 Paris (France) (Received

May 7, 1992; accepted

October

8, 1992)

Abstract The influence of molecular structure and DNA binding mode on the ability of cationic porphyrins to photosensitize DNA strand break formation has been studied for a series of meso-substituted pyridinium porphyrins using electrophoretic and DNA sequencing techniques. Porphyrins substituted with pyridyl groups in which the heterocyclic nitrogen is in the para or meta position vis-d-vis the substitution point are capable of intercalative binding and are considerably more efficient DNA photosensitizers than the corresponding non-intercalating ortho compounds. Within each group of porphyrins the photosensitizer efficiency increases with the number of positive charges. Using DNA sequencing experiments, we have demonstrated that photomodification occurs primarily at the guanine and thymine bases, and that alkali-labile sites produced by photo-oxidation are as important as direct cleavage events. The kinetics of strand degradation in aerated and degassed solution suggest that type II reactions (probably mediated by singlet oxygen) occur with significantly higher yield than type I reactions and are responsible for the formation of alkali-labile sites in aerated systems. These observations seem to confirm the hypothesis that those structural features which influence the strength and mode of binding also serve to establish favourable porphyrin-DNA interactions for photosensitization.

Keywords: DNA photosensitization, relationships

porphyrins,

strand

1. Introduction The cationic porphyrins have been studied extensively in terms of their binding interactions with, and their ability to photosensitize the modification of, DNA. Despite these efforts, relatively little attention has been devoted to the structural features of the porphyrin macrocycle which influence these activities. Essentially, cationic porphyrins can bind to DNA by three distinct modes: intercalation, external binding and external binding with self-stacking (for a review, see ref. 1). A number of factors are known to influence the mode and DNA sequence selectivity of binding. The porphyrin-DNA binding interaction has been shown to be strongly dependent on ionic strength; *Also at: Laboratoire de Biophysique, Museum National d’Histoire Naturelle, 43 rue Cuvier, 75231 Paris, France. **Author to whom correspondence should be addressed.

1011-1344/93/$6.00

cleavage,

sequence

specificity,

structure-activity

increasing sodium concentration not only causes a decrease in the strength of binding, but the porphyrin molecules also shift from a predominantly intercalative binding mode in GC-rich regions (G, guanine; C, cytosine) to an external binding mode in AT-rich regions (A, adenine; T, thymine) [2]. Binding is also influenced by the complexation of a metal ion into the porphyrin macrocycle. The well-characterized meso-tetra(6 N-methylpyridinium) porphyrin is capable of intercalation both as the free base form and as a complex with Cu(I1) and Ni(II), i.e. metals without axial ligands [2-4]. In contrast, this porphyrin complexed with metals with axial ligands, e.g. Fe(III), Co(III), Zn(I1) or Mn(III), is incapable of intercalation [3]. These two classes of porphyrins (intercalators and non-intercalators) have recently been used to probe some peculiar structures in branched DNA [5].

0 1993 - Elsevier

Sequoia.

All rights reserved

42

D. T. Croke et al. / DNA photocleavage by cationic porphyrins

In addition to these factors, the binding interaction is influenced by the nature and position of substituents on the porphyrin ring system [6]. The influence of the macrocycle structure on the mode and strength of DNA binding has been extensively studied by Sari et al. [7-91 using a series of more than 30 synthetic cationic porphyrins corresponding to the general formula [meso-[N-methyl4(or 3 or 2)-pyridiniumyl],(aryl)~_,]M, where M = HZ, Cu(II) or ClFe(III) and it =2-4. By means of a variety of spectroscopic measurements, they have shown that most of the porphyrins in this series are capable of intercalative binding, including the di- and tri-cationic derivatives, provided that at least two meso-pyridiniumyl substituents in the cis position can freely rotate and come in plane with the porphyrin macrocycle. The porphyrins are also well known as photoactive molecules which can react readily with a variety of substrates including nucleic acid components and DNA [4]. The photoreactions observed with DNA are believed to be of two types: firstly, direct electron transfer processes between DNA bases (usually guanine) and porphyrin excited states, and secondly, indirect reactions mediated by reactive oxygen species (including singlet oxygen and superoxide anion) whose production from molecular oxygen is sensitized by the excited porphyrin. These reactions ultimately give rise to direct or “frank” strand breaks in the DNA and to alkalilabile sites [4, lo]. Recently, much attention has been focused on the porphyrins as potential phototherapeutic agents either alone or in combination with antisense oligonucleotides [ll, 121. There is thus a need to determine those structural features of porphyrins which influence photochemical reactivity in an effort to design porphyrin-based cleavage agents of maximal efficiency. Such knowledge would also serve to extend the use of porphyrins as probes for DNA structure and conformation [5, 131. In an effort to provide some rational basis for the selection of porphyrins for these applications, we present studies on structure-activity relationships for porphyrin-photosensitized DNA strand cleavage.

2. Materials

and methods

2.1. Materials Plasmid DNA (pBR322) was obtained from Genofit (France) and maintained as an aqueous stock solution of concentration 0.2 pg DNA ~1 -I. Porphyrin derivatives were synthesized and purified as previously described [9, 141. All other chemicals

were obtained from general laboratory suppliers. Oligonucleotides were synthesized on a Pharmacia DNA synthesizer and purified by gel electrophoresis and high performance liquid chromatography (HPLC). The complementary oligonucleotides 1 and 2 used in the base specificity studies described below were of the following sequence: 1,5’-TCC TAG CAA AGG AGG AGA CGA AGA AAA AAT GA-3’; 2, 3’-AGG ATC GTT TCC TCC TCT GCT TCT TIT T-IA CT5’. 2.2. Porphyrin-photosensitized DNA cleavage eficiency The efficiency of the porphyrins in photosensitizing DNA strand cleavage was determined by a supercoiled plasmid DNA assay system [4, 151. Irradiation mixtures (total volume, 10 ~1) were prepared in a standard irradiation buffer (100 mM NaCl, 10 mM phosphate buffer, pH 7.4) containing plasmid DNA (3.1 X 10e5 M in base pairs) and porphyrin (6.2X low8 M or 3.1 X 10e6 M) giving a molar ratio of DNA base pairs to porphyrin of 500 or 10. Samples were transferred to small glass tubes for irradiation. The glass tubes were placed in a thermostatically controlled UV cell containing ethanol for low temperature experiments. Irradiations were carried out with a mercury-xenon lamp (Oriel, 200 W) whose output was passed through an interference filter (Schott 500 FC5350) to select wavelengths in the range 418 < A < 445 nm, close to the Soret absorption maximum of the porphyrin. The intensity of the lamp output at the sample holder was found to be 75.5 W m-’ by potassium ferrioxalate actinometry [16]. Due to increased yields of photosensitization at the higher porphyrin concentration, the lamp output was reduced for these samples by the use of a neutral-density filter. Following irradiation the samples were subjected to agarose gel electrophoresis. Since high porphyrin concentrations can cause aberrant electrophoretic migration of plasmid DNA, particularly the supercoiled form, all samples with a molar ratio of DNA base pairs to porphyrin of 10 were sequentially extracted with phenol, chloroform-isoamyl alcohol and diethyl ether to remove the porphyrin. The samples were then frozen, lyophilized and resuspended in water prior to electrophoresis on 0.8% agarose gels. Samples with the lower porphyrin concentration (molar ratio of DNA base pairs to porphyrin of 500) were electrophoresed directly. Gels were electrophoresed at 5 V cm-’ for 4-5 h, stained in ethidium bromide

43

l). T. Croke et aL / DNA photocleavage by cationic porphyrins

(0.5 /xg ml-1; 90 min) and photographed on Polaroid-type .665 positive/negative Land film. The resulting negatives were analysed by densitometry (LKB Ultroscan XL densitometer) and the percentage of plasmid D N A remaining in the supercoiled form (normalized to 100% with reference to the control samples) was calculated as a function of irradiation time. A pseudo-first-order rate constant for photosensitized strand break formation (k) was then extracted from a semi-logarithmic plot of In (percentage of supercoiled D N A remaining) vs. time of irradiation (min) for each porphyrin assayed, where k is taken to be equivalent to the slope of a regression line fitted to the data. The values of k (min-1) quoted hereafter are the mean values derived from three independent estimations. It should be noted that these values are relative and are designed for comparison across the series of porphyrins studied; no attempt at absolute quantitation has been made.

2.3. Base specificity of photosensitized cleavage of DNA The base specificity of porphyrin-sensitized D N A damage was investigated using a synthetic 32 base pair, double-stranded D N A molecule created by the annealing of oligonucleotides 1 and 2 to give a D N A duplex. Prior to annealing, one or other of the oligonucleotides (as required) was radiolabelled on the 5' terminus with [.y 32p] d A T P and T4 polynucleotide kinase (Amersham, UK) to allow detection of the photosensitized reaction products by autoradiography. Irradiations were carried out at 0 °C in the standard irradiation buffer containing the 32-mer duplex (10 /xM in D N A base pairs) and porphyrin (10/zM), i.e. a 1:1 molar ratio. At this concentration it was verified that the amount of porphyrin adsorbed to glass or plastic is negligible (data not shown). Irradiation mixtures were allowed to stand on ice for 30 rain and were then transferred to small glass tubes for irradiation. The irradiation apparatus was as described above. After irradiation the samples were twice extracted with phenol and chloroform-isoamyl alcohol to remove the porphyrin, frozen and lyophilized to dryness. Where appropriate, samples were resusp e n d e d in 100 /zl of piperidine (1 M), incubated at 90 °C for 30 rain, frozen in dry-ice--ethanol and again lyophilized. The samples were finally resuspended in 4/zl of formamide and immediately loaded onto a 20% sequencing gel in parallel with Maxam and Gilbert chemical cleavage reaction products prepared from the same duplex D N A [17]. The photosensitized reaction products were visualized by autoradiography on Fuji RX or Amer-

sham Hyperfilm MP X-ray film. Following autoradiography, bands visualized on the gel were excised and their radioactivity measured by liquid scintillation counting. The yield of photoinduced cleavage of the radiolabelled D N A strands was expressed as the percentage of intact (i.e. unmodified) DNA remaining after a determined irradiation time. 3. Results

3.1. Photosensitized strand cleavage of D N A A range of nine cationic pyridinium porphyrins drawn from those described previously by Sari et al. [9] were assayed for their ability to photosensitize D N A strand break formation (Fig. 1, Table 1). The remainder of the free base porphyrins examined by Sari et al. [9] were found to be either weakly active or apparently inactive as photosensitizers and thus were not investigated further. As expected, certain porphyrins proved to be quite efficient photosensitizers of direct D N A cleavage (Fig. 2). No treatment was applied to the plasmid DNA after irradiation to reveal the photosensitized generation of alkali-labile sites. The quantitation of DNA cleavage efficiencies at high and low porphyrin concentrations showed considerable variation (as much as an order of magnitude) in the pseudo-first-order rate constant (k) for photosensitized cleavage across the range of molecules studied (Table 1). Sari et al. [9] have demonstrated a correlation between the binding affinity to calf thymus D N A (K) and the position and number of positive charges borne by the porphyrin molecule. The range of K values measured varies in the order para > meta > ortho, independent of the number of charges present on the molecule (Fig. 3(a)). It is also interesting to note that the association

c." ( X : 4

5., 3

2)

A r 1= A~ - A~ . A4r - X - N - p y r i d i n i u m y l

P4

M4

04

A r 1= P h ; A r2 - A r3 = *~r

P3

M3

O~3

Ar 1= A~ = Ph; A r3 = .~r ° X - N - pyridiniumyl

P2-cis

M2-cis

O2-cis

A r 1- A~ = Ph; A r 2 = ~ r

P2-trans

M2-trans

O2-trans

= X - N - pyr~iniurnyl

= X - N - pyridiniurnyl

Fig. 1. Nomenclature of the [meso-[N-methyl-4(or 3 or 2)-pyridiniumyl)o(aryl)4_.] cationic porphyrins, after Sari et al. [9].

D. T. Croke et al. / DNA photocleavage by cationic porphyrins

44

T A B L E 1. Summary of the binding and cleavage kinetic data for the cationic porphyrins at molar ratios of D N A base pairs to porphyrin of 500 and 10. The data presented are the apparent affinity constant for D N A binding (K), the pseudo-first-order rate constant for photosensitized strand break formation (k) and the fraction of porphyrin bound to D N A (]'Porb) under the conditions of the assay Porphyrin c

Binding mode d

K ( M - 1)

k ( m i n - l) ( + SEM) c

fPorb

P2-cis a P3 a P& M2-trans a M3 a M4 a 03 ~ P4/bz ~ M4/bz a P4b M4 b 04 b

i i i i i i e e e i i e

1.2 x 3.0× 1.0 × 1.6 × 5.1 × 1.6 × 1.6 × 5.0 × 5.1 × 1.0 × 1.6 x 1.6 x

0.007 + 0.003 0.015 -1-0.007 0.037 + 0.002 0.006 + 0.001 0.011 + 0.007 0.050 + 0.007 0.002 + 0.001 0.031 + 0.019 0.005 + 0.002 0.110 + 0.029 0.149 + 0.016 0.052 + 0.009

0.944 0.992 0.997 0.835 0.915 0.979 0.835 0.994 0.915 0.996 0.978 0.918

106 106 107 105 105 106 105 106 105 107 106 105

aDNA base pair to porphyrin ratio of 500. bDNA base pair to porphyrin ratio of 10. cp4, meso-tetrakis(N-methyl-4-pyridiniumyl)porphyrin; P3, mesotris(N-methyl-4-pyridiniumyl)phenylporphyrin; P2-cis, cis-mesobis(N-methyl-4-pyridiniumyl)diphenylporphyrin; P2-trans, transmeso-bis(N-methyi-4-pyridiniumyl)diphenylporphyrin; M4, mesotetrakis(N-methyl-3-pyridiniumyl)porphyrin; M3, meso-tris(Nmethyl-3-pyridiniumyl)phenylporphyrin; M2-cis, cis-meso-bis(Nmethyl-3-pyridiniumyl)diphenylporphyrin; M2-trans, trans-mesobis(N-methyl-3-pyridiniumyl)diphenylporphyrin; 0 4 , meso-tet rakis(N-methyl-2-pyridiniumyl)porphyrin; 03, meso-tris(Nmethyl-2-pyridiniumyl)phenylporphyrin; O2-cis, cis-meso-bis(Nmethyl-2-pyridiniumyl)diphenylporphyrin; O2-trans, trans-mesobis(N-methyl-2-pyridiniumyl)diphenylporphyrin; M4/bz and P4/ bz, the N-benzylpyridinium congeners of M4 and P4 respectively. These nomenclature indicate both the position and number of positive charges: P, para; M, meta; O, ortho. dPorphyrin binds to D N A by intercalation (i) or external binding (e). eSEM, standard error of the mean.

constants of the meta series are approximately five times greater than those of the ortho series and ten times lower than those of the para series. In an effort to assess the influence of porphyrin structure on photoreactivity, the kinetic data obtained for photosensitized D N A cleavage at the lower porphyrin concentration (molar ratio of D N A base pairs to porphyrin of 500) were analysed in analogous fashion (Fig. 3(b)). This analysis shows a correlation between the observed k values (representing relative cleavage efficiency) and the number and position of positive charges on the molecule. One possible interpretation of this finding is that the efficiency of sensitized cleavage simply reflects the amount of porphyrin bound to DNA, which will be determined by the affinity constant K. This is highly unlikely since, under the assay

1

2

3

4

5

6

7

8

Fig. 2. Plasmid D N A (pBR322) cleavage photosensitized by porphyrins. Lanes: 1, no porphyrin; 2, EcoRI-linearized plasmid DNA; 3, P4; 4, Zn-P4 (Zn congener of P4); 5, 5,10,15,20tetrasulphonatophenyl-porphyrin (TSP); 6, P2-trans; 7, P2-cis; 8, P3. Irradiation was for 6 min under the following conditions: porphyrin (P), 0.066 /xM; plasmid D N A (D), 33 ~ M in base pairs; P / D = 5 0 0 ; buffer, 100 mM NaCI in 10 mM Na phosphate buffer (pH 7.4). Plasmid D N A samples were electrophoresed directly after irradiation on 0.8% agarose gels.

conditions (see Section 2), 90% or more of the porphyrin molecules were bound to the D N A (Table 1) and all porphyrins were thus assumed to have been assayed under equivalent conditions of binding. In fact, as represented in this format, the kinetic data show the same trends as seen in the binding data. This suggests that those structural features which influence the D N A binding interaction also play a major role in determining the photochemical reactivity of these porphyrins. Only one porphyrin does not fit into this scheme: the tetra-cationic meta-substituted porphyrin (M4). This compound consistently deviates from the pattern exhibited by the other molecules in respect of the photosensitization efficiency (Table 1, Fig. 3(b)). The k value observed for M4 is approximately 35% greater than for the corresponding parasubstituted molecule (P4) even though it has an affinity constant which is significantly smaller (K=I.6X106 M -1 vs. 1.0×107 M - l ; Table 1). This deviation from the general pattern points to other less obvious factors which influence the ability of the porphyrins to photosensitize D N A damage.

3.2. Sequence specificity of DNA damage Short oligonucleotide duplexes were used to study the photochemical reactions induced by the cationic porphyrins on DNA. The use of short synthetic fragments of D N A has several advantages: (i) chemical synthesis allows reproducible production of defined sequences with considerable

D. T. Croke et al. / DNA photocleavage by cationic potphyrins

.OOl II (0)

’

2

3 Number

4

5

of charges

.l ,

.Ol _

,0°’ “:‘$“’ 4

1 Number

of charges

(b)

Fig. 3. DNA binding and strand cleavage kinetic data for the cationicporpbyrins. (a) Apparent affinity constants(K) determined by Sari et al. [9] for the cationic porphyrins as their water-soluble free bases. (b) Pseudo-first-order rate constants (k) determined in this work for the cleavage of plasmid DNA photosensitized by certain of the cationic porphyrins. The identity of the porphyrin derivatives is indicated as shown in Fig. 1 with the following additions: M4/bz and P4ibz are the N-benzylpyridinium congeners of M4 and P4 respectively.

versatility in introducing or deleting, at will, one or more nucleotides; (ii) the photochemical reaction products can be examined at the single nucleotide level by sequencing gel methodology; (iii) peculiar DNA structures arising from the supercoiling of closed circular DNA can be avoided. Regarding the stability of 32-mer duplexes in the presence of porphyrin, it can be argued that for

45

oligonucleotide duplexes of such length the melting temperature in 0.1 M NaCl is far above 0 “C, the temperature at which the experiments described herein were performed. For example, Stein et al. [18] have shown that, under similar conditions of salt concentration, the T,,, values of the duplexes dT14/dA,4 and dT,/dA, were 36 and 54 “C respectively. In the early 198Os, Fiel and Munson [19] demonstrated that cationic porphyrins stabilize DNA during thermal denaturation, as do most intercalators. The photochemical reactions sensitized by the tetra-cationic porphyrins 04, M4 and P4 (i.e. ortho, meta and para) on the radiolabelled DNA duplex were compared. As a control, the meso-substituted 5,10,15,20-tetrasulphonatophenyl-porphyrin (TSP) was used; this negatively charged molecule does not bind to DNA [lo]. The 32 base pair synthetic DNA duplex (radiolabelled on one strand only) was irradiated as described in the presence and absence of TSP and of the tetra-cationic porphyrins. The samples were treated with hot piperidine before electrophoresis (Fig. 4). The negatively charged TSP exhibited extremely weak photolytic activity. In contrast, the tetra-cationic porphyrins produced strong strand cleavage, the ortho molecule (04) being notably weaker than the para and meta derivatives. The cleavage pattern on strand 1 of the duplex (left side of autoradiogram, Fig. 4) demonstrates that the principal sites of photosensitized damage are guanine bases. Strand 2 (right side of autoradiogram, Fig. 4) also shows damage at guanine bases, but additional sites of cleavage are seen at thymine bases located in certain sequence contexts (CTC or CIT) as indicated by the asterisks in the figure. Irradiated material which is electrophoresed under neutral conditions (i.e. without piperidine treatment) consistently shows a small amount of material of higher molecular size than the starting material (data not shown); such high molecular weight bands appearing when DNA is irradiated in the presence of porphyrins can be ascribed to cross-linked DNA which can be broken down under piperidine treatment [12]. The photosensitized degradation of the radiolabelled strand 1 was monitored as a function of irradiation time (Fig. 5) before and after piperidine treatment. This allowed the relative contributions of the direct strand cleavage reaction and alkalilabile site generation by the porphyrins in DNA degradation to be estimated. The kinetics of both the direct strand cleavage reaction (Fig. 5, upper panel) and strand scission following piperidine treatment (Fig. 5, lower panel) confirm the un-

46

D. T. Croke et al. I DNA photocleavage by cationic porphyrins

1 2 3 4 5 6 7 8 9 10 11 12

O

(c-l)

20 Irradiation

0

Fig. 4. Autoradiogram of strand scissions in the 32 base pair duplex (left, strand 1; right, strand 2) after 20 min irradiation at 0 “C in the presence of: 1, 8, no porphyrin; 2, 9, P4; 3, 10, M4; 4, 11, 04; 5, 12, TSP; 6, Maxam and Gilbert [17] (G+A) reaction strand 1; 7, Maxam and Gilbert [17] (G+A) reaction strand 2. Porphyrin, 10 PM; 32-mer duplex, 10 PM in base pairs, buffer, 100 mM NaCl in 10 mM Na phosphate buffer (pH 7.4). Porphyrins were removed by solvent extraction and the samples were treated with hot piperidine prior to electrophoresis.

expected behaviour of the meta-substituted porphyrin (M4); of the three tetra-cationic isomers it is the most reactive. The degradation kinetics demonstrate that the contribution of strand breaks from direct strand cleavage is of the same order of importance as that from cleavage at alkali-labile sites. Irradiation for 60 min in the presence of porphyrin M4 can produce as much as 90% fragmentation of the DNA, including strand scission originating from alkali-labile sites. The relative efficiencies of the porphyrin isomers in DNA strand cleavage can be estimated from the irradiation times required to produce cleavage (at least once) of 50% of DNA strands, a t(50) value. The t(50) values measured for the tetra-cationic porphyrins were found to be 11, 30 and 55 min for porphyrins M4, P4 and 04 respectively (Fig. 5, lower panel). The rate of alkali-labile site generation seems to depend on the sequence and, particularly, on the number of guanine bases present in the strand (Figs. 4 and 6). The direct strand cleavage reaction

(b)

20 Irradiation

60

40 time

60

(minutes)

40

60

time

t

(minutes)

Fig. 5. Yield of strand cleavage in the 32 base pair duplex photosensitized by P4 (I), M4 (0) and 04 (A) as a function of irradiation time. Other conditions as in Fig. 4 except that the samples were either untreated (upper panel) or treated (lower panel) with hot piperidine prior to electrophoresis. The percentage of intact DNA is measured as the quantity of radiolabelled strand 1 remaining at irradiation time t.

12

E ._ ae

40-

0

0 0

20 -

0

20 Irradiation

40 time

60

6

(minutes)

Fig. 6. Kinetics of the cleavage reaction in the presence of porphyrin M4 on strand 1 of the 32 base pair duplex before (0) and after (0) treatment with hot piperidine.

is almost identical on the two strands, whereas the alkali-labile site generation reaction is more important on strand 1 compared with strand 2. The greater number of guanine bases in the se-

D. T. Croke et al. / DNA photocleavage by cationic porphyrins

quence of strand 1 may account for this disparity. The extent of photobleaching of the porphyrin under comparable irradiation conditions was studied and found to be quite small. Preliminary experiments to investigate the role of oxygen in the porphyrin-photosensitized cleavage of DNA have begun. Identical porphyrin-DNA mixtures (porphyrins P4 and M4), some of which degassed by repeated have initially been freeze-thaw under vacuum, have been irradiated in parallel. These studies indicate that the rate of DNA fragmentation is significantly greater in aerated solution and the degassed reaction yields far more adduct and cross-link formation than it does strand cleavage (data not shown). The reactivities of the Zn(II) derivatives of the three tetra-cationic porphyrins were also investigated in aerated solution. In all cases it was shown that the two photochemical reactions (direct strand cleavage and alkali-labile site generation) proceed at much higher rates in the presence of the zinc compounds compared with their free base congeners (Fig. 7). The para- and meta-substituted porphyrins (P4 and M4) were equally reactive in 100

2

60

00 0

10 Irradiation

(0)

30

20 time

40

(minutes)

100

I

ae

0

;

0 (b)

I

I

I

10

20

30

Irradiation

time

I 40

(minutes)

Fig. 7. Kinetics of the strand cleavage reaction induced by Zn(II) derivatives of P4 (I), M4 (Cl) and 04 (A) on strand 1 of the 32 base pair duplex. Upper panel, untreated samples; lower panel, piperidine-treated samples.

47

both strand cleavage and alkali-labile site generation, with the ortho isomer (04) being significantly less reactive.

4. Discussion The question which is central to this paper concerns the structural features which influence the binding interactions of the cationic porphyrins with DNA, and their ability to photosensitize covalent modification once bound. Sari et aE. [9] have demonstrated the importance of the number and relative position of positively charged pyridinium substituents (meta, para or ortho) in determining both the mode and strength of binding. Their work forms the basis of a structure-activity relationship for DNA binding which depends on two complementary factors: firstly, the greater electrostatic interaction between porphyrin and DNA with an increasing number of positive charges and, secondly, the ability of the porphyrin to intercalate between DNA base pairs. Parasubstituted compounds (PZcis, P3 and P4) freely intercalate since their pyridinium substituents are capable of unhindered rotation; as a result they exhibit the highest range of apparent affinity constants (K values). Meta substitution (MZtrans, M3 and M4) introduces an element of steric hindrance, but does not prevent intercalation. Ortho substitution (as in 03 and M4/bz) prevents intercalation because of strong hindrance to rotation of the ring substituents. The influence of freedom of substituent rotation on the mode of DNA binding is clearly demonstrated by the fact that benzyl substitution of the para and meta tetra-cationic molecules (P4/bz and M4/bz) produces a significant decrease in the apparent affinity constant when compared with the parent molecules (P4 and M4). In addition, the behaviour of the cis and trans di-cationic porphyrins has demonstrated that only half of the porphyrin ring is necessary for intercalation into DNA [9]. It is reasonable to postulate that the photosensitization of DNA cleavage may be favoured by tight binding. Our results show that a high number of positive charges determines high binding constants and also high cleavage activity (Fig. 3). Clearly, binding is not a prerequisite for cleavage mediated by compounds which sensitize the generation of diffusable radical species such as singlet oxygen [lo], but will undoubtedly play a role in direct cleavage mechanisms [20]. However, the strength of the binding interaction will not be the sole determinant of the photochemical reactivity.

48

D. T Croke et al. / DNA photocleavage by cationic porphyrins

It is possible for two molecules of equivalent binding affinity to occupy distinct chemical environments within the DNA helix and so exhibit different reactivity. Thus, in addition to the strength of binding it is expected that the mode of binding to DNA (e.g. groove binding VS. intercalation) will be an important determinant of the activity of a porphyrin as a photosensitizer. We have presented an analysis of the kinetic data obtained for photosensitized DNA cleavage analogous to that for the binding data of Sari et al. [9] in Fig. 3. Firstly, this demonstrates that para- and meta-substituted porphyrins, which are capable of intercalative binding, are considerably more efficient DNA photosensitizers than the corresponding ortho compounds, which cannot intercalate. Secondly, within each series of porphyrins, photosensitizer efficiency increases with the number of positive charges. These observations seem to confirm the hypothesis that those structural features which influence the strength and mode of binding also serve to establish favourable porphyrin-DNA interactions for photosensitization. Further, it is clear that intercalation produces a binding interaction which is far more favourable for photosensitization than is groove binding. This may suggest that proximity (in plane) to the DNA base pairs, rather than to the phosphodiester backbone in the groove, is a major prerequisite for efficient photosensitization. There is one interesting exception to this pattern, the tetra-cationic meta-substituted porphyrin (M4), which exhibits a degree of reactivity that is unexpected given the published binding data [9]. This serves to illustrate the subtle nature of the interaction between the photosensitizer and the DNA intercalation site which is involved in determining the photochemical reactivity. Clearly, the structural difference between these two compounds (meta VS. para substitution) in some way strongly favours the ability of the meta-substituted molecule to sensitize DNA damage despite the difference in DNA binding affinity. While binding parameters are undoubtedly of importance, they may not be the only parameters which contribute to the photoreactivity of the porphyrins. Intrinsic features of the porphyrins such as triplet yields and lifetimes should also be considered, but these lie outside the scope of this work. The analysis of the sequence specificity of porphyrin-sensitized DNA damage has shown that modification occurs at guanine and thymine bases, guanine being more frequently modified. The guanine base is the target of a wide range of direct (type I) and sensitized (type II) photochemical reactions of DNA. Photosensitization of DNA

mediated by singlet oxygen has been shown to cause preferential damage at guanine bases [21]. These findings reflect the fact that guanine has the lowest redox potential of the four DNA bases and is therefore most readily oxidized [22]. The sensitivity to porphyrin reaction of thymines in certain sequence contexts is not fully understood. After guanine, thymine is the next most easily oxidized base, and a range of molecules which sensitize the production of singlet oxygen have been shown to produce alkali-labile sites at guanine and thymine bases in DNA substrates [23]. In examining Fig. 4 it is clear that several of the guanine cleavage bands on strand 1 of the 32-mer duplex are stronger than others. Unfortunately, the sole use of this duplex precludes us from making definitive statements as to sequence context effects on cleavage intensity. The choice of this model duplex was dictated by subsequent work which aimed to link cationic porphyrins to an oligopyrimidine which can form a triple helical structure targeted on the duplex. This explains why strand 1 contains a run of purines while strand 2 contains a run of pyrimidines. However, it may be noted that, in general, when the sequence contains two contiguous guanine residues, the yield of alkalilabile sites is always greater for the guanine on the 5’ side than for the other. The kinetic analysis of photosensitized strand degradation and the preliminary experiments on the effect of degassing allow some mechanistic inferences to be drawn. We have demonstrated that alkali-labile sites and direct cleavage events make comparable contributions to overall strand degradation. While the direct cleavage reaction occurs equally on both strands of the duplex DNA substrate, the yield of alkali-labile sites varies with the guanine content of the strand. This suggests that it is the number of guanine bases available for attack, rather than the amount of singlet oxygen generated on irradiation, which is limiting. Therefore type II reactions, probably mediated by singlet oxygen, occur with significantly higher yield than type I reactions, which are due to electron transfer between porphyrin excited states and DNA bases. This is confirmed by the observation that strand degradation proceeds at a much reduced rate under degassed conditions. The orientation of the porphyrin sensitizer with respect to the DNA would be expected to affect type I and II reactions in different ways. Type II reactions, since they are mediated by a freely diffusable species which is relatively long lived in aqueous solution, would be less sensitive to the mode of binding of the photosensitizer. Type I reactions, in contrast, would

D. T. Croke

et al. I DNA photocleavage

be far more sensitive, since electron and energy transfer are short-range processes and will strongly depend on the geometry of the two partners within the complex. We therefore propose that the paraand meta-substituted series of porphyrins are more efficient sensitizers than the ortho series due to their ability to intercalate and thus approach their “target” more closely. This would favour type I and type II reactions with these intercalating porphyrins, in contrast with the ortho series where type I reactions should be disadvantaged by the increased distance between the excited dye molecule and the electron-donating base. Within each series (para or meta), the different efficiencies reflect the strength of the binding interaction, which is determined by the number of positive charges on the molecule. The apparently anomalous behaviour of porphyrin M4 (the meta-substituted tetra-cationic derivative and the most efficient photosensitizer) remains to be explained. It is possible that the structural difference between the meta- and para-substituted tetra-cationic porphyrins (P4 and M4) in some way leads to a more “favourable” position of the intercalated meta derivative vi+&-vis the guanine base for electron abstraction or attack by singlet oxygen. This would seem to be confirmed by molecular modelling studies which indicate that the meta isomer adopts a more stable configuration within the intercalation site than does the para isomer. Further experiments designed to investigate the role of singlet oxygen are necessary to clarify these mechanistic questions. Our studies also indicate that the porphyrin structure affects the photoproduct yield from groove-bound porphyrins. The zinc porphyrins, whether para-, meta- or ortho-substituted, are unable to intercalate due to the presence of the axially liganded zinc atom. They all bind in the minor groove and yet they exhibit differences in the efficiency with which they photosensitize DNA damage (Fig. 7). In general, zinc derivatives are more efficient in DNA cleavage compared with their free base counterparts. For example, 04-Zn is as efficient as the most reactive M4 compound (Figs. 3 and 7). The para- and meta-substituted zinc porphyrins are comparable in efficiency and are both significantly more efficient than the orthosubstituted molecule. Thus, even for external binding to DNA, the position of the porphyrin in the minor groove exercises a profound influence on the photochemical reactivity through the positioning of the sensitizer relative to the guanine base. Assuming a type II mechanism, the singlet oxygen yields of P4 and PCZn have been compared showing that ~(~0~) of PCZn is roughly 1.5 times

by cationic

porphyrins

49

that of P4 [lo]. This result can account, in part, for the differences observed between P4 and P4Zn in their DNA cleaving activity.

Acknowledgments We acknowledge the technical assistance of Mr. D. Dupre (Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques, Paris) in porphyrin synthesis and purification, the assistance of Drs. A. Tossi and M. Feeney (Trinity College, Dublin) in the degassed irradiation experiments, Dr. J. S. Sun (Laboratoire de Biophysique, Paris) for carrying out molecular modelling studies and Professor J. M. Kelly (Trinity College, Dublin) for helpful discussions during this work. D.T.C. gratefully acknowledges financial support in the form of a long-term research fellowship from the European Molecular Biology Organization (EMBO).

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