Poly(dG–dC)₂ and poly(dA–dT)₂ have marked influences on the rate of insertion of copper(II) into cationic porphyrins reflecting the interaction mode of the porphyrin with the nucleic acid

Ellinas, Panayiotis A., , \u2787

Gibbs, E. J.

Mah, Stanley C., , \u2787

Pasternack, Robert F.

Santucci, R.

Schaertel, Stephanie A., , \u2786

Works

Swarthmore CollegeWorksChemistry & Biochemistry Faculty Works Chemistry & Biochemistry12-1-1987Influence Of DNA On The Rate Of PorphyrinMetallationRobert F. PasternackSwarthmore College, rpaster1@swarthmore.eduE. J. GibbsR. SantucciStephanie A. Schaertel , '86Panayiotis A. Ellinas , '87See next page for additional authorsFollow this and additional works at: http://works.swarthmore.edu/fac-chemistryPart of the Inorganic Chemistry CommonsThis Article is brought to you for free and open access by the Chemistry & Biochemistry at Works. It has been accepted for inclusion in Chemistry &Biochemistry Faculty Works by an authorized administrator of Works. For more information, please contact myworks@swarthmore.edu.Recommended CitationRobert F. Pasternack; E. J. Gibbs; R. Santucci; Stephanie A. Schaertel , '86; Panayiotis A. Ellinas , '87; and Stanley C. Mah , '87. (1987)."Influence Of DNA On The Rate Of Porphyrin Metallation". Journal Of The Chemical Society: Chemical Communications. Issue 23.1771-1774.http://works.swarthmore.edu/fac-chemistry/123AuthorsRobert F. Pasternack; E. J. Gibbs; R. Santucci; Stephanie A. Schaertel , '86; Panayiotis A. Ellinas , '87; andStanley C. Mah , '87This article is available at Works: http://works.swarthmore.edu/fac-chemistry/123J. CHEM. SOC. ,  CHEM. COMMUN., 1987 1771 Influence of DNA on the Rate of Porphyrin Metallation Robert F. Pasternack,*a Esther J. Gibbs,b Roberto Santucci,c Stephanie Schaertep Panayiotis Ellinas,a and Stanley C. Maha a Department of Chemistry, Swarthmore College, Swarthmore, Pennsylvania 19801, U.S.A. b Department of Chemistry, Goucher College, Towson, Maryland 21204, U.S.A. c lstituto di Chimica Biologica, Facolta di Roma 'La Sapienza,' 00185 Rome, Italy Poly(dG-dC)2 and poly(dA-dT)p have marked influences on the rate of insertion of copper(it) into cationic porphyrins reflecting the interaction mode of the porphyrin with the nucleic acid. Nucleic acids in solution with their negatively charged phosphate backbone, tend to concentrate cations of added electrolytes in their immediate environment. 1 Therefore, it might be anticipated that reactions between cationic reactants will be catalysed by the presence of DNA;2 similar catalyses at polymeric surfaces have been demonstrated for poly- vinylsulphonate, for example .3 However, cations can interact with nucleic acids in a variety of ways involving not only couiombic attractions but also complexation of purinelpyrimi- dine bases, 'external' (e.g. groove binding) and 'internal' (i.e. intercalation) processes in which the purine/pyrimidine bases also play a role.4 The influence of added DNA on the rate of a reaction involving cations may be expected to reflect the manner in which one or the other of the reactants interacts with the nucleic acid. Conversely, it might prove possible to gain further insight into the nature of a cation-nucleic acid interaction by determining the effect of DNA on the rate of some standard chemical process. We investigated the interactions of cationic water soluble porphyrins and metalloporphyrins with nucleic acidss--9 and find that H2TMpyP4+ and its metal derivatives not possessing axial iigands are capable of intercalating into poly(dG-dC);! but not p~ly(dA-dT)~. t  At the low levels of drug load utilized for the kinetic studies reported here there is a correlation between mode of porphyrin binding to non-aggregated nucleic acids and the profile of the circular dichroism (c.d.) spectrum induced in the Soret region; i.e. negative bands reflect intercalation, positive bands, external (groove) binding, and conservative spectra suggest external bonding with porphyrin aggregation .5.9,10 For H2TMpyP-24+ at these same low drug loads, no c.d. spectrum is induced with poly(dG-dC)2 but a positive band is produced with p~ly(dA-dT)~.  Recent studies of H2TAP4+ with a natural DNA have shown that this porphyrin does not interclate.1" Based upon c.d. measure- ments, it appears that, under the conditions of these experi- ments, H2TAP4+ is capable of groove binding to either p~ly(dG-dC)~ or poly( dA-dT)2, perhaps with some aggrega- tion. We have considered the rate of insertion of copper(r1) into each of these porphyrins both with and without added p~ly(dG-dC)~ or p~ly(dA-dT)~.  To conduct experiments in a pH range in which the polynucleotide duplex is stable requires the addition of some potential ligand to copper(r1) to prevent the precipitation of the metal hydroxide. We have used both ammonia and imidazole for this purpose (see Table 1). Previous work has shown that hexaquacopper(I1) interacts extensively with DNA giving rise to structural and conforma- t P~ ly (dG-dC)~  and poly(dA-dT), obtained from P-L Biochemicals are synthetic deoxyribonucleic acid duplexes containing alternating guanine and cytosine bases or adenine and thymine bases, respect- ively. The cationic porphyrins supplied by Midcentury Chemical Company are tetrakis(4-N-methylpyridy1)porphyrin (H2TMpyP4+), tetrakis-(2-N-rnethylpyridyl)porphyrin ( H , T M P ~ P - ~ ~ + ) ,  and tetra- kis(4-N,N,N-trimethylanilinium)porphyrin (H2TAP4+). See Figure 1. All porphyrins were used in the chloride form. Me. Me H2TMpyP4+ Me \Me +/ N Me / > Me Me / N Me \ N +  /Me /+\ Me Me H,TAP4+ /. -\Me Me tional changes of the polymer.11 These processes are likely to be less important when the metal ion is complexed to other ligands, and in the present study, more than 85% of the copper ion in solution is bound to two or more nitrogenous bases (ammonia or imidazole). Indeed, results obtained with H2TMpyP-24+ (vide infra) imply that if such copper(1r)-DNA Published on 01 January 1987. Downloaded by Swarthmore College on 28/04/2015 16:42:20. View Article Online / Journal Homepage / Table of Contents for this issue1772 J. CHEM. SOC., CHEM. COMMUN., 1987 Table 1. Experimental rate constants for the insertion of copper(i1) into cationic porphyrins. ( [Cu2+] 380 ELM; [poly(dG-dC),] 100 VM; [Porphyrin C14], 10 VM; 25 "C; pH 8.) A. H2TMpyP4+ (a) in presence of NH4CI and NaCl [ NH,+]/M [ N ~ + ] / M  0.05 0.10 0.05 0 (b) in presence of imidazole (Imid) and NaCl [Imid] x 1 0 3 / ~  5.0 5.0 5.0 5.0 B. H ~ T M P Y P - ~ ~ +  (a) in presence of NH4C1 and NaCl [ N H 4 + ] / ~  0.05 0.05 (b) in presence of imidazole and NaCl [Imid] X l O 3 / ~  5.0 5.0 5.0 5.0 C. H2TAP4+ (a) in presence of NH4C1 and NaCl [NH,+)/M 0.05 0.05 (b) in presence of imidazole and NaCl [Imid] x ~ O Y M  5.0 5.0 5.0 5.0 [ Na + ]/M 0.15 0.05 0.01 0.001 [ N ~ + ] / M  0.10 0 [N~+] /M 0.15 0.05 0.01 0.001 [ N ~ + ] / M  0.10 0 [N~+] /M 0.15 0.05 0.01 0.001 poly( dG-dC)za N Y N Y poly( dG-dC)z N Y N Y N Y N Y poly( dG-dC)z N Y N Y poly(dG-dC), N Y N Y N Y N Y poly( dG-dC)z N Y N Y poly( dG-dC)Z N Y N Y N Y N Y a Y = yes i .e. p~ly(dG-dC)~ added; N = no p~ly(dG-dC)~. b p poly(dG-dC),) under otherwise identical conditions. kls-1 2.5 X 10-2 2.2 x 10-2 very slow 1.8 x 10-4 kls- 1 1.4 x 10-2 9.1 X 10-3 very slow very slow 6.2 x 10-3 very slow 1.9 x 10-4 8.1 x 10-3 kls- 1 2.5 X 10-2 2.3 X 3.0 x 10-2 2.1 x 10-2 kls-1 1.8 X 10-2 1.8 x 10-2 1.6 X 10-2 1.3 x 10-2 1.5 X 10-2 1.0 x 10-2 1.6 x 10-2 1.6 x 10-2 kls- 1 9.6 X 10-3 9.9 x 10-4 7.6 x 10-3 1.4 x 10-4 kiss I 1.6 X 10-2 1.2 x 10-2 1.2 x 10-2 5.4 x 10-3 2.5 x 10-4 4.7 x 10-4 9.2 x 10-3 1.1 x 10-3 Pb 0.0072 4 0 - 3  P 0.014 4 0 - 3  4 0 - 3  4 0 - 3  P 0.92 0.70 P 1 .o 0.81 0.67 1 .o P 0.10 0.018 P 0.34 0.021 0.039 0.12 = (k in presence of poly(dG-dC),)l(k in absence of interactions exist for these species they have little effect on the minimize ambiguities in the interpretation of the results, kinetics of metallation. Furthermore, the spectral properties experiments were conducted for each porphyrin at identical of the free base porphyrin-DNA complexes are unaffected by pH, ionic strengths, temperature, and concentrations of the addition of such copper-nitrogenous base complexes. To copper(Ir), nitrogenous ligand, porphyrin, and DNA. The Published on 01 January 1987. Downloaded by Swarthmore College on 28/04/2015 16:42:20. View Article OnlineJ .  CHEM. SOC., CHEM. COMMUN., 1987 1773 reactions can be represented by equation (1). k CuL,2+ + H2P4+ --+ CuP4+ + nL + 2H+ (1) The wavelengths of observation were: for H2TMpyP4+, 518 nm (no DNA) and 532 nm (with DNA); for H2TMpyP-24+, 510 nm both with and without DNA; and for H2TAP4+, 514 nm (no DNA) and 518 nm (with DNA). The results of these experiments are shown in Tables 1 and 2. Rate constants are reported to within +20% based upon multiple runs of the same experiment. As can be seen from Table 1, similar kinetic patterns are obtained whether ammonia or imidazole is used to bind the copper ion. Furthermore, under identical experimental condi- tions in the absence of DNA, the copper insertion rates are remarkably similar for all three porphyrins although there are marked differences in basicities for these species12 ranging from a pK, <1 for H4TMpyP-26+ to pK, -4 for H4TAP6+. As may also be seen from Table 1, the presence of p~ly(dG-dC)~ markedly inhibits the metallation of H2TMpyP4+ presumably because the porphyrin intercalates into this synthetic DNA and is effectively removed from the copper(I1)-containing solution. To account for these results we suggest the model shown in equation 2 where 'out' and 'in' refer to noninter- calated and intercalated porphyrin , respectively. H ~ P ~ + ~ ~ ~  'w CUL,2+ +& t CuP4+ + nL + 2H+ (2) HP+~, < We define an apparent equilibrium constant, K ,  in equation (2), since the intercalation process is known to be much more rapid than the metallation.6 (3) K ,  as defined here, is not a true constant13 but depends upon r (where r E [H2Pi,]/[poly(dG-dC)2]o), so that we can at best provide an estimate of K from these kinetic results. The [p~ly(dG-dC)~] term is the concentration of binding sites available at equilibrium taking into account near-neighbour exclusion and any other co-operativity effects.5.9.13 Because our experiments were conducted with a large excess of poly(dG-dC)2 to porphyrin, we can estimate this term using equations provided by McGhee and von Hippel.13 Comparing equations (2) and (3) to equation (1) leads to equation (4). (4) Since the rate of the reaction for H2TMpyP4+ goes virtually to zero when the ionic strength is lowered to a point at which the porphyrin is almost totally intercalated,g we estimate kin = 0 dm3 mol-1 s-1. Furthermore, we approximate k,,, with the rate constant obtained in the absence of polynucleotide. This leads to the ratio p, as defined in equation (5). From the k p = = (1 + K[poly(dG-dC)2])-1 ( 5 )  KOUt results of Table 1A we obtain K -106dm3 mol-1 at p = 0.15 M, in good agreement with the value of 8 X 105 dm3 mol-1 determined earlier from titration experiments under similar conditions.5 Table 2. Comparison of polynucleotide effects on copper(I1) insertion into cationic porphyrins. ([Cuz+] 380 p . ~ ;  [polynucleotide] 100 p . ~ ;  [Porphyrin C14], 10 p . ~ ;  25 "C; pH 8.) p.lM PGC PAT HZTMpyP'+ 0.15 0.013 0.017 0.05 4 0 - 3  0.015 0.01 4 0 - 3  0.016 0.001 <lo-3 0.032 H2TMp~p-24~ 0.15 1 .o 0.43 0.05 0.81 0.069 0.01 0.67 0.058 0.001 1 .o 0.094 H*TAP"+ 0.15 0.33 0.002 0.028 0.05 0.021 0.003 0.01 0.039 <10-3 0.001 0.13 0.01 8 This determination was conducted at [HzTAP4+] = 1 p . ~  using fluoresence detection. All other conditions were the same as in other experiments. We contrast these results with those obtained for HzTMpyP-24+ (Table 1B). This latter porphyrin does not intercalate into p ~ l y ( d G - d C ) ~  and, under the conditions of our experiments, the presence of the polymer has very little effect on the metallation kinetics. Whether this result implies that H2TMpyP-24+ is not concentrated at the surface of p~ly(dG-dC)~ or that two off-setting effects are at work here (concentration vs. partitioning of ions and/or protection of the porphine core), is not clear from these experiments. However, it is evident that poly(dG-dC);! has a very different influence on the metal insertion kinetics for H2TMpyP-24+ than for H2TMpyP4+. By way of comparison, we note that at p = With H2TAP4+, although the effect is not nearly as dramatic as for H2TMpyP4+, the influence of poly(dG-dC)2 is to inhibit metallation. While this porphyrin does not interca- late into p ~ l y ( d G - d C ) ~ ,  its interaction is apparently profound enough to partially protect the porphine core from metalla- tion. These results are consistent with the induced c.d. spectrum in the Soret region which yields a small signal, primarily positive but with some conservative character implying a degree of porphyrin aggregation. None of the porphyrins tested intercalates into poly (dA-dT)2 but they all provide c.d. signals in the Soret region. The profile is a simple positive band for each of H2TMpyP-24+ and H2TMpyP4+, but is more complicated for H2TAP4+. At low drug load, a split positive band is obtained for H2TAP4+ but as the porphyrin : nucleic acid ratio (r,) is increased, a conservative spectrum is obtained suggesting porphyrin aggregation at the nucleic acid surface. Table 2 shows a comparison of the metallation rates for p~ly(dG-dC)~ and p~ly(dA-dT)~.  The effect of the poly- nucleotide is still the most modest for H2TMpyP-24+ but, for this porphyrin, the effect of poly(dA-dT)2 is much more profound than that of p ~ l y ( d G - d C ) ~ .  This is true also for the other non-intercalating porphyrin, H2TAP4+. Generally, groove binding is more favourable in A-T regions of DNA than G-C regions for both electronic and steric reasons. The degree of aggregation of the porphyrin may also be a factor in determining these rates. In the absence of poly(dA-dT)2, the copper(I1) insertion rate constant obtained via absorption ([H2TAP4+] = 10 p~), agreed with one obtained via fluor- Oa15M, PH2TMpyP -100. Published on 01 January 1987. Downloaded by Swarthmore College on 28/04/2015 16:42:20. View Article Online1774 J. CHEM. SOC., CHEM. COMMUN., 1987 escence ([H2TAP4+] = 1 p ~ )  to within 5%.  But in the presence of the DNA, the insertion of copper(1r) into H2TAP4+ is an order of magnitude faster at low drug load (ro-l -100) where aggregation is less important than at ro-1 = 10. For H2TMpyP4+ alone, the only one of these porphyrins which can intercalate into nucleic acids is poly(dG-dC)*, a more effective inhibitor of metallation than poly(dA-dT);?, particularly at low ionic strength. Based upon the evidence obtained thus far, protection against metallation appears to follow the order: intercalation > external binding with aggregation > external binding, no aggregation > electro- static interactions only. We gratefully acknowledge financial support for this work by the National Institutes of Health (GM 34676) and the National Science Foundation (CHE-8613592). We also thank Professor Maurizio Brunori for providing space to one of us (R. F. P.) and for most useful conversations. Received, 26th May, 1987; Corn. 712 References 1 G. S. Manning, Q. Rev. Biophys., 1978, 11, 179; G. S.  Manning, 2 J. K. Barton, C. V. Kunar, and N. J .  Turro, J.  A m .  Chern. Soc., J .  Phys. Chem., 1981, 85, 870. 1986, 108, 6391. 3 H. Morawetz, Acc. Chem. Res., 1970,3,354; H. Morawetz and B. Vogel, J .  Am. Chem. Soc., 1969,91, 563. 4 D. F. Wilson and R. L. Jones in ‘Intercalation Chemistry,’ Academic Press, New York, 1982, 445. 5 R. F. Pasternack, E. J .  Gibbs, and J. J .  Villafranca, Biochemistry, 1983, 22, 2406. 6 R. F. Pasternack, E. J .  Gibbs, and J. J .  Villafranca, Biochemistry, 1983, 22, 5409. 7 R. F. Pasternack, D. Sidney, P. A.  Hunt, E. A. Snowden, and E. J .  Gibbs, Nucl. Acids Res., 1986, 14, 3927. 8 R. F. Pasternack, P. Garrity, B. Ehrlich, C. B. Davis, E. J. Gibbs, G. Orloff, A. Giartosio, and C. Turano, Nucl. Acids Res., 1986, 14, 5919. 9 E. J. Gibbs, M. C. Maurer, J. H. Zhang, W. M. Reiff, D. Hill, M. Malicka-Blaszkiewicz, S. C. Mah, and R. F. Pasternack, J .  Znorg. Biochem., in the press. 10 M. J. Carvlin, N .  Datta-Gupta, and R. Fiel, Biochem. Biophys. Res. Commun., 1982, 108, 66. 11 V. A. Sorokin, Y. P. Blagoi, V. A. Valeev, S. V. Kosnilova, G. 0. Gladchenko, I. D. Reva, and V. I.  Sokhan, J .  Znorg. Biochem., 1987, 30, 87, and references therein. 12 M. Krishnamurthy, Znd. J .  Chem. B ,  1977,15,964; P. Hambright, T. Gore, and M. Burton, Znorg. Chem., 1976, 15, 2314; R. F. Pasternack, P. R. Huber, P. Boyd, G. Engasser, L. Francesconi, E. Gibbs, P. Fasella, G. C. Venturo, and L. de C. Hinds, J .  A m .  Chem. SOC. ,  1972, 94,4511. 13 J. D. McGhee and P. H. von Hippel, J .  Mol. Bid . ,  1974,86,469. Published on 01 January 1987. Downloaded by Swarthmore College on 28/04/2015 16:42:20. View Article Online

Influence Of DNA On The Rate Of Porphyrin Metallation

Swarthmore College 
Works 
Chemistry & Biochemistry Faculty Works Chemistry & Biochemistry 
12-1-1987 
Influence Of DNA On The Rate Of Porphyrin Metallation 
Robert F. Pasternack 
Swarthmore College, rpaster1@swarthmore.edu 
E. J. Gibbs 
R. Santucci 
Stephanie A. Schaertel , '86 
Panayiotis A. Ellinas , '87 
See next page for additional authors 
Follow this and additional works at: https://works.swarthmore.edu/fac-chemistry 
 Part of the Inorganic Chemistry Commons 
Let us know how access to these works benefits you 
 
Recommended Citation 
Robert F. Pasternack; E. J. Gibbs; R. Santucci; Stephanie A. Schaertel , '86; Panayiotis A. Ellinas , '87; and 
Stanley C. Mah , '87. (1987). "Influence Of DNA On The Rate Of Porphyrin Metallation". Journal Of The 
Chemical Society: Chemical Communications. Issue 23. 1771-1774. DOI: 10.1039/c39870001771 
https://works.swarthmore.edu/fac-chemistry/123 
This work is brought to you for free by Swarthmore College Libraries' Works. It has been accepted for inclusion in 
Chemistry & Biochemistry Faculty Works by an authorized administrator of Works. For more information, please 
contact myworks@swarthmore.edu. 
Authors 
Robert F. Pasternack; E. J. Gibbs; R. Santucci; Stephanie A. Schaertel , '86; Panayiotis A. Ellinas , '87; and 
Stanley C. Mah , '87 
This article is available at Works: https://works.swarthmore.edu/fac-chemistry/123 
J. CHEM. SOC. ,  CHEM. COMMUN., 1987 1771 
Influence of DNA on the Rate of Porphyrin Metallation 
Robert F. Pasternack,*a Esther J. Gibbs,b Roberto Santucci,c Stephanie Schaertep Panayiotis Ellinas,a and 
Stanley C. Maha 
a Department of Chemistry, Swarthmore College, Swarthmore, Pennsylvania 19801, U.S.A. 
b Department of Chemistry, Goucher College, Towson, Maryland 21204, U.S.A. 
c lstituto di Chimica Biologica, Facolta di Roma 'La Sapienza,' 00185 Rome, Italy 
Poly(dG-dC)2 and poly(dA-dT)p have marked influences on the rate of insertion of copper(it) into cationic porphyrins 
reflecting the interaction mode of the porphyrin with the nucleic acid. 
Nucleic acids in solution with their negatively charged 
phosphate backbone, tend to concentrate cations of added 
electrolytes in their immediate environment. 1 Therefore, it 
might be anticipated that reactions between cationic reactants 
will be catalysed by the presence of DNA;2 similar catalyses at 
polymeric surfaces have been demonstrated for poly- 
vinylsulphonate, for example .3 However, cations can interact 
with nucleic acids in a variety of ways involving not only 
couiombic attractions but also complexation of purinelpyrimi- 
dine bases, 'external' (e.g. groove binding) and 'internal' (i.e. 
intercalation) processes in which the purine/pyrimidine bases 
also play a role.4 The influence of added DNA on the rate of a 
reaction involving cations may be expected to reflect the 
manner in which one or the other of the reactants interacts 
with the nucleic acid. Conversely, it might prove possible to 
gain further insight into the nature of a cation-nucleic acid 
interaction by determining the effect of DNA on the rate of 
some standard chemical process. 
We investigated the interactions of cationic water soluble 
porphyrins and metalloporphyrins with nucleic acidss--9 and 
find that H2TMpyP4+ and its metal derivatives not possessing 
axial iigands are capable of intercalating into poly(dG-dC);! 
but not p~ly(dA-dT)~. t  At the low levels of drug load utilized 
for the kinetic studies reported here there is a correlation 
between mode of porphyrin binding to non-aggregated nucleic 
acids and the profile of the circular dichroism (c.d.) spectrum 
induced in the Soret region; i.e. negative bands reflect 
intercalation, positive bands, external (groove) binding, and 
conservative spectra suggest external bonding with porphyrin 
aggregation .5.9,10 For H2TMpyP-24+ at these same low drug 
loads, no c.d. spectrum is induced with poly(dG-dC)2 but a 
positive band is produced with p~ly(dA-dT)~.  Recent studies 
of H2TAP4+ with a natural DNA have shown that this 
porphyrin does not interclate.1" Based upon c.d. measure- 
ments, it appears that, under the conditions of these experi- 
ments, H2TAP4+ is capable of groove binding to either 
p~ly(dG-dC)~ or poly( dA-dT)2, perhaps with some aggrega- 
tion. 
We have considered the rate of insertion of copper(r1) into 
each of these porphyrins both with and without added 
p~ly(dG-dC)~ or p~ly(dA-dT)~.  To conduct experiments in a 
pH range in which the polynucleotide duplex is stable requires 
the addition of some potential ligand to copper(r1) to prevent 
the precipitation of the metal hydroxide. We have used both 
ammonia and imidazole for this purpose (see Table 1). 
Previous work has shown that hexaquacopper(I1) interacts 
extensively with DNA giving rise to structural and conforma- 
t P~ ly (dG-dC)~  and poly(dA-dT), obtained from P-L Biochemicals 
are synthetic deoxyribonucleic acid duplexes containing alternating 
guanine and cytosine bases or adenine and thymine bases, respect- 
ively. The cationic porphyrins supplied by Midcentury Chemical 
Company are tetrakis(4-N-methylpyridy1)porphyrin (H2TMpyP4+), 
tetrakis-(2-N-rnethylpyridyl)porphyrin ( H , T M P ~ P - ~ ~ + ) ,  and tetra- 
kis(4-N,N,N-trimethylanilinium)porphyrin (H2TAP4+). See Figure 1. 
All porphyrins were used in the chloride form. 
Me. Me 
H2TMpyP4+ 
Me 
\Me 
+/ 
N 
Me 
/ 
> 
Me 
Me 
/ 
N 
Me 
\ 
N +  /Me 
/+\ 
Me Me 
H,TAP4+ 
/. -\Me 
Me 
tional changes of the polymer.11 These processes are likely to 
be less important when the metal ion is complexed to other 
ligands, and in the present study, more than 85% of the copper 
ion in solution is bound to two or more nitrogenous bases 
(ammonia or imidazole). Indeed, results obtained with 
H2TMpyP-24+ (vide infra) imply that if such copper(1r)-DNA 
Pu
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View Article Online / Journal Homepage / Table of Contents for this issue
1772 J. CHEM. SOC., CHEM. COMMUN., 1987 
Table 1. Experimental rate constants for the insertion of copper(i1) into cationic porphyrins. ( [Cu2+] 380 ELM; [poly(dG-dC),] 
100 VM; [Porphyrin C14], 10 VM; 25 "C; pH 8.) 
A. H2TMpyP4+ 
(a) in presence of NH4CI and NaCl 
[ NH,+]/M [ N ~ + ] / M  
0.05 0.10 
0.05 0 
(b) in presence of imidazole (Imid) and NaCl 
[Imid] x 1 0 3 / ~  
5.0 
5.0 
5.0 
5.0 
B. H ~ T M P Y P - ~ ~ +  
(a) in presence of NH4C1 and NaCl 
[ N H 4 + ] / ~  
0.05 
0.05 
(b) in presence of imidazole and NaCl 
[Imid] X l O 3 / ~  
5.0 
5.0 
5.0 
5.0 
C. H2TAP4+ 
(a) in presence of NH4C1 and NaCl 
[NH,+)/M 
0.05 
0.05 
(b) in presence of imidazole and NaCl 
[Imid] x ~ O Y M  
5.0 
5.0 
5.0 
5.0 
[ Na + ]/M 
0.15 
0.05 
0.01 
0.001 
[ N ~ + ] / M  
0.10 
0 
[N~+] /M 
0.15 
0.05 
0.01 
0.001 
[ N ~ + ] / M  
0.10 
0 
[N~+] /M 
0.15 
0.05 
0.01 
0.001 
poly( dG-dC)za 
N 
Y 
N 
Y 
poly( dG-dC)z 
N 
Y 
N 
Y 
N 
Y 
N 
Y 
poly( dG-dC)z 
N 
Y 
N 
Y 
poly(dG-dC), 
N 
Y 
N 
Y 
N 
Y 
N 
Y 
poly( dG-dC)z 
N 
Y 
N 
Y 
poly( dG-dC)Z 
N 
Y 
N 
Y 
N 
Y 
N 
Y 
a Y = yes i .e. p~ly(dG-dC)~ added; N = no p~ly(dG-dC)~. b p 
poly(dG-dC),) under otherwise identical conditions. 
kls-1 
2.5 X 10-2 
2.2 x 10-2 
very slow 
1.8 x 10-4 
kls- 1 
1.4 x 10-2 
9.1 X 10-3 
very slow 
very slow 
6.2 x 10-3 
very slow 
1.9 x 10-4 
8.1 x 10-3 
kls- 1 
2.5 X 10-2 
2.3 X 
3.0 x 10-2 
2.1 x 10-2 
kls-1 
1.8 X 10-2 
1.8 x 10-2 
1.6 X 10-2 
1.3 x 10-2 
1.5 X 10-2 
1.0 x 10-2 
1.6 x 10-2 
1.6 x 10-2 
kls- 1 
9.6 X 10-3 
9.9 x 10-4 
7.6 x 10-3 
1.4 x 10-4 
kiss I 
1.6 X 10-2 
1.2 x 10-2 
1.2 x 10-2 
5.4 x 10-3 
2.5 x 10-4 
4.7 x 10-4 
9.2 x 10-3 
1.1 x 10-3 
Pb 
0.0072 
4 0 - 3  
P 
0.014 
4 0 - 3  
4 0 - 3  
4 0 - 3  
P 
0.92 
0.70 
P 
1 .o 
0.81 
0.67 
1 .o 
P 
0.10 
0.018 
P 
0.34 
0.021 
0.039 
0.12 
= (k in presence of poly(dG-dC),)l(k in absence of 
interactions exist for these species they have little effect on the minimize ambiguities in the interpretation of the results, 
kinetics of metallation. Furthermore, the spectral properties experiments were conducted for each porphyrin at identical 
of the free base porphyrin-DNA complexes are unaffected by pH, ionic strengths, temperature, and concentrations of 
the addition of such copper-nitrogenous base complexes. To copper(Ir), nitrogenous ligand, porphyrin, and DNA. The 
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J .  CHEM. SOC., CHEM. COMMUN., 1987 1773 
reactions can be represented by equation (1). 
k CuL,2+ + H2P4+ --+ CuP4+ + nL + 2H+ (1) 
The wavelengths of observation were: for H2TMpyP4+, 518 
nm (no DNA) and 532 nm (with DNA); for H2TMpyP-24+, 
510 nm both with and without DNA; and for H2TAP4+, 514 
nm (no DNA) and 518 nm (with DNA). The results of these 
experiments are shown in Tables 1 and 2. Rate constants are 
reported to within +20% based upon multiple runs of the 
same experiment. 
As can be seen from Table 1, similar kinetic patterns are 
obtained whether ammonia or imidazole is used to bind the 
copper ion. Furthermore, under identical experimental condi- 
tions in the absence of DNA, the copper insertion rates are 
remarkably similar for all three porphyrins although there are 
marked differences in basicities for these species12 ranging 
from a pK, <1 for H4TMpyP-26+ to pK, -4 for H4TAP6+. As 
may also be seen from Table 1, the presence of p~ly(dG-dC)~ 
markedly inhibits the metallation of H2TMpyP4+ presumably 
because the porphyrin intercalates into this synthetic DNA 
and is effectively removed from the copper(I1)-containing 
solution. To account for these results we suggest the model 
shown in equation 2 where 'out' and 'in' refer to noninter- 
calated and intercalated porphyrin , respectively. 
H ~ P ~ + ~ ~ ~  'w 
CUL,2+ +& t CuP4+ + nL + 2H+ (2) 
HP+~, < 
We define an apparent equilibrium constant, K ,  in equation 
(2), since the intercalation process is known to be much more 
rapid than the metallation.6 
(3) 
K ,  as defined here, is not a true constant13 but depends 
upon r (where r E [H2Pi,]/[poly(dG-dC)2]o), so that we can at 
best provide an estimate of K from these kinetic results. The 
[p~ly(dG-dC)~] term is the concentration of binding sites 
available at equilibrium taking into account near-neighbour 
exclusion and any other co-operativity effects.5.9.13 Because 
our experiments were conducted with a large excess of 
poly(dG-dC)2 to porphyrin, we can estimate this term using 
equations provided by McGhee and von Hippel.13 Comparing 
equations (2) and (3) to equation (1) leads to equation (4). 
(4) 
Since the rate of the reaction for H2TMpyP4+ goes virtually to 
zero when the ionic strength is lowered to a point at which the 
porphyrin is almost totally intercalated,g we estimate kin = 0 
dm3 mol-1 s-1. Furthermore, we approximate k,,, with the 
rate constant obtained in the absence of polynucleotide. This 
leads to the ratio p, as defined in equation (5). From the 
k 
p = = (1 + K[poly(dG-dC)2])-1 ( 5 )  
KOUt 
results of Table 1A we obtain K -106dm3 mol-1 at p = 0.15 M, 
in good agreement with the value of 8 X 105 dm3 mol-1 
determined earlier from titration experiments under similar 
conditions.5 
Table 2. Comparison of polynucleotide effects on copper(I1) insertion 
into cationic porphyrins. ([Cuz+] 380 p . ~ ;  [polynucleotide] 100 p . ~ ;  
[Porphyrin C14], 10 p . ~ ;  25 "C; pH 8.) 
p.lM PGC PAT 
HZTMpyP'+ 0.15 0.013 0.017 
0.05 4 0 - 3  0.015 
0.01 4 0 - 3  0.016 
0.001 <lo-3 0.032 
H2TMp~p-24~ 0.15 1 .o 0.43 
0.05 0.81 0.069 
0.01 0.67 0.058 
0.001 1 .o 0.094 
H*TAP"+ 0.15 0.33 0.002 
0.028 
0.05 0.021 0.003 
0.01 0.039 <10-3 
0.001 0.13 0.01 
8 This determination was conducted at [HzTAP4+] = 1 p . ~  using 
fluoresence detection. All other conditions were the same as in other 
experiments. 
We contrast these results with those obtained for 
HzTMpyP-24+ (Table 1B). This latter porphyrin does not 
intercalate into p ~ l y ( d G - d C ) ~  and, under the conditions of 
our experiments, the presence of the polymer has very little 
effect on the metallation kinetics. Whether this result implies 
that H2TMpyP-24+ is not concentrated at the surface of 
p~ly(dG-dC)~ or that two off-setting effects are at work here 
(concentration vs. partitioning of ions and/or protection of the 
porphine core), is not clear from these experiments. However, 
it is evident that poly(dG-dC);! has a very different influence 
on the metal insertion kinetics for H2TMpyP-24+ than for 
H2TMpyP4+. By way of comparison, we note that at p = 
With H2TAP4+, although the effect is not nearly as 
dramatic as for H2TMpyP4+, the influence of poly(dG-dC)2 is 
to inhibit metallation. While this porphyrin does not interca- 
late into p ~ l y ( d G - d C ) ~ ,  its interaction is apparently profound 
enough to partially protect the porphine core from metalla- 
tion. These results are consistent with the induced c.d. 
spectrum in the Soret region which yields a small signal, 
primarily positive but with some conservative character 
implying a degree of porphyrin aggregation. 
None of the porphyrins tested intercalates into poly 
(dA-dT)2 but they all provide c.d. signals in the Soret region. 
The profile is a simple positive band for each of H2TMpyP-24+ 
and H2TMpyP4+, but is more complicated for H2TAP4+. At 
low drug load, a split positive band is obtained for H2TAP4+ 
but as the porphyrin : nucleic acid ratio (r,) is increased, a 
conservative spectrum is obtained suggesting porphyrin 
aggregation at the nucleic acid surface. 
Table 2 shows a comparison of the metallation rates for 
p~ly(dG-dC)~ and p~ly(dA-dT)~.  The effect of the poly- 
nucleotide is still the most modest for H2TMpyP-24+ but, for 
this porphyrin, the effect of poly(dA-dT)2 is much more 
profound than that of p ~ l y ( d G - d C ) ~ .  This is true also for the 
other non-intercalating porphyrin, H2TAP4+. Generally, 
groove binding is more favourable in A-T regions of DNA 
than G-C regions for both electronic and steric reasons. The 
degree of aggregation of the porphyrin may also be a factor in 
determining these rates. In the absence of poly(dA-dT)2, the 
copper(I1) insertion rate constant obtained via absorption 
([H2TAP4+] = 10 p~), agreed with one obtained via fluor- 
Oa15M, PH2TMpyP -100. 
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1774 J. CHEM. SOC., CHEM. COMMUN., 1987 
escence ([H2TAP4+] = 1 p ~ )  to within 5%.  But in the 
presence of the DNA, the insertion of copper(1r) into 
H2TAP4+ is an order of magnitude faster at low drug load 
(ro-l -100) where aggregation is less important than at ro-1 = 
10. For H2TMpyP4+ alone, the only one of these porphyrins 
which can intercalate into nucleic acids is poly(dG-dC)*, a 
more effective inhibitor of metallation than poly(dA-dT);?, 
particularly at low ionic strength. Based upon the evidence 
obtained thus far, protection against metallation appears to 
follow the order: intercalation > external binding with 
aggregation > external binding, no aggregation > electro- 
static interactions only. 
We gratefully acknowledge financial support for this work 
by the National Institutes of Health (GM 34676) and the 
National Science Foundation (CHE-8613592). We also thank 
Professor Maurizio Brunori for providing space to one of us 
(R. F. P.) and for most useful conversations. 
Received, 26th May, 1987; Corn. 712 
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http://works.swarthmore.edu/cgi/viewcontent.cgi?article=1122&context=fac-chemistry

Influence Of DNA On The Rate Of Porphyrin Metallation

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