Proflavine poly

Planar dyes such as proflavine can bind to DNA by intercalation between base pairs or by stacking along the groove of the helix. The former process is favored when the nucleic acid is in excess (Nuc/D 4) and in high salt solution. We are interested in the intercalation process and hence our NMR studies have focussed on an investigation of the proflavine poly(dA-dT) complex as a function of the Nuc/D ratio in 1 M NaCl in an attempt to probe the structure and dynamics of mutagen-nucleic acid interactions in solution (25). 

Hydrogen Bonding The thymidine H-3 Watson-Crick proton can be readily detected in the proflavine poly(dA-dT) complex,... 

Melting Transition Typical 360 MHz proton NMR spectra of proflavine poly(dA-dT) complexes, Nuc/D = 24 and Nuc/D = 8, in 1 M NaCl solution at temperatures below the midpoint for the dissociation of the complex are presented in Figures 17A and B respectively. The stronger base and sugar resonances can be readily resolved from the weaker proflavine resonances (designated by asterisks) in the presence of excess nucleic acid (Figure 17) so that the resonances of the synthetic DNA and the mutagen can be monitored independently of each other. 

The dissociation of the proflavine poly(dA-dT) complex can be followed by monitoring the temperature dependent chemical shift or the line width as demonstrated by shift data on the thymidine CH3-5 resonance (Figure 18A) and width data on the adenosine H-8 resonance (Figure 18B). The proton resonances shift as average peaks during the dissociation of the complex, indicative of fast exchange ( dissociation 10 sec l at the transition midpoint) between the complex and its dissociated components on the NMR time scale. 

Figure 16. The temperature dependence of the thymidine H-3 resonance in poly(dA-dT) (O) and the proflavine poly(dA-dT) complex Nuc/D = 8(0) in /M NaCl, lOmM cacodylate, ImM EDTA, HtO at pH 6.53, and pH 7.1...

Figure 17. The 360-MHz proton NMR spectra of the proflavine poly(dA-dT) complex in 1M NaCl, lOmM cacodvlate, JOmM EDTA, zHzO, pH 7. The top spectrum represents the Nuc/D = 24 complex at 78.5°C ( ut of the proflavine resonances in the complex is 80°C), while the bottom spectrum represents the Nuc/D = 8 complex at 8l.4°C ft,/, of proflavine resonances in complex is 84.3°C). The proflavine resonances are designated by asterisks.

Figure 19. The temperature dependence of the nucleic acid (O) and proflavine (0) chemical shifts between 5.5 and 8.6 ppm for poly(dA-dT) and the Nuc/D = 24 and 8 proflavine poly(dA-dT) complexes in /M NaCl, lOmWl cacodylate, lOmM EDTA, 2 HO between 50° and 100°C. The poly(dA-dT) concentration was fixed at I2.6mM in phosphates and the proflavine concentration was varied to make the different Nuc/D ratio complexes.

Experimental and Calculated Upfield Proflavine Complexation Shifts on Formation of the Proflavine Poly(dA-dT) Complex... 

Summary The proton resonances of the nucleic acid and the mutagen are well resolved in the proflavine poly(dA-dT) complex and can be monitored independently of each other. The resonances shift as average peaks during the thermal dissociation of the complex with stabilization of the duplex by bound mutagen. 

The dye exhibits different binding affinities for pyrimidine(3 -5 )purine and purine(3 -5 )pyrimidine sites and this is readily demonstrated by the observation of resolved resonances in the spectrum of the proflavine poly(dA-dT) complex in solution. 

Figure 24. The proton noise decoupled 145.7-MHz 31P NMR spectra of (A) poly-(dA-dT) in 1M NaCl, lOmM cacodylate, lOmM EDTA, H20, pH 6.2 at 65°C and (B) the proflavine poly(dA-dT) complex, Nuc/D = 10, in 1M NaCl, lOmNi cacodylate, lOmM EDTA, 2H20 at 65°C. The scale is upfleld from standard trimethylphosphate.

Photocatalytically active polymer having acridine units in the backbone was synthesized by the polycondensation of 3,6-diaminoacridine (proflavine) as a diamine and m-dibromobenzene (Scheme 13) [68]. Mullen and coworkers synthesized a series of novel poly(imino ketone)s via Pd-catalyzed polycondensation of aromatic dichloro or dibromo ketones with various aromatic diamines (Scheme 14) [69]. The Mw values were in the range of 19500-474 500. The FT-IR spectra of the obtained polymer revealed that in the solid state intermolecular and intramolecular hydrogen bonding (N-H 0=C)... 

We first describe the NMR parameters for the duplex to strand transition of the synthetic DNA poly(dA-dT) (18) with occasional reference to poly(dA-dU) (24) and poly(dA- brdU) and the corresponding synthetic RNA poly(A-U) (24). This is followed by a comparison of the NMR parameters of the synthetic DNA in the presence of 1 M Na ion and 1 M tetramethylammonium ion in an attempt to investigate the effect of counterion on the conformation and stability of DNA. We next outline structural and dynamical aspects of the complexes of poly(dA-dT) with the mutagen proflavine (25) and the anti-tumor agent daunomycin (26) which intercalate between base pairs and the peptide antibiotic netropsin (27) which binds in the groove of DNA. 

Nucleic Acid Base Resonances The chemical shifts of the nonexchangeable protons in poly(dA-dT), the Nuc/D = 24 complex and the Nuc/D = 8 complex in 1 M NaCl solution are plotted as a function of temperature in Figure 19. The nucleic acid nonexchangeable proton chemical shifts in the duplex state are either unperturbed (adenosine H-8, H-2, and thymidine CH3-5) or shift slightly upfield (thymidine H-6) on complex formation (Figure 19). By contrast, the thymidine H-3 exchangeable proton located in the center of the duplex resonates 0.35 ppm to higher field in the Nuc/D = 8 proflavine complex compared to its position in the... 

It should be noted that the H-3 shifts on proflavine complex formation (Figure 20) parallel those reported for ethidium bromide complex formation with poly(dA-dT) (11). 

We observe structural changes in the glycosidic torsion angle(s) (monitored at the H-l protons) and the sugar ring parameters (monitored at the H-3 protons) of poly(dA-dT) on proflavine complex formation. 

We have attempted to investigate the daunomycin complex with poly(dA-dT) in order to set constraints on possible overlap geometries in the intercalation complex (26) using methods described in the previous section on the proflavine complex (25). There are nonexchangeable proton markers on ring D and exchangeable proton markers on ring B of the planar portion of the... 

In the center of the duplex shifts upfield by 0.15 ppm (Table VI, Figure 25) and the thymidine CH3-5 which Is directed towards the major groove shifts upfield by 0.1 ppm (Figure 29). It should be noted that such an upfield shift of the thymidine CH3-5 group was not observed in the intercalation complexes of ethidium (11), proflavine (25), terpyridylplatinum II (11) and nitroaniline dication with poly(dA-dT). The results require that at least one thymidine CH3-5 group project onto the periphery of the anthra-cycline ring system at the intercalation site. 

Overlap Geometry at the Intercalation Site We shall attempt to utilize the nucleic acid base and anthracycline ring proton com-plexation shifts to deduce which anthracycline aromatic ring(s) overlap with nearest neighbor base pairs in the daunomycin poly-(dA-dT) intercalation complex. It should be noted that the nonplanarity of ring A in the antibiotic requires that the aromatic portion of the anthracycline chromophore cannot intercalate with its long axis colinear to the direction of the Watson-Crick hydrogen bonds at the intercalation site as was demonstrated for proflavine-nucleic acid complexes. 

The purine(3 -5 Ipyrimidine and the pyrimidine(3 -5 )purine phosphodiester linkages are partially resolved in the proflavine and daunomycin intercalation complexes with poly(dA-dT) with the phosphodiester at the intercalation site shifting to low field. This suggests that these intercalating agents exhibit a sequence specificity in their complexes with alternating purine-pyrimidine polynucleotides. 

Poly dA-dT Proflavine-complexes dye laser 430 nm Nj-laser 337 nm ionization Proflavine 2-step damage Fluorescence- decrease 82,83)... 

The authors found a different damage probability for the dye proflavine bound either to poly AT or to poly GC. The damage was measured by the reduction of the fluorescence when the complexes were excited at 430 nm alone. 

The quantum yield for production by electron transfer from proflavin as sensitizer to a Pt-charged Ti02 colloid using P-CD and poly(vinylalcohol) as stabilizers were compared. The system using P-CD was found to be three times more efficient, because inclusion of proflavin in the cavity increases the number of dye-semiconductor interactive sites (Scheme 35) [343],... 

It has been shown that DNA intercalators (ethidium bromide and proflavine) form complexes with single-stranded DNA and homopolynucleotides [3]. It was of interest, therefore, to determine the effect of intercalators on the degradation of the homopolymer (ADP-ribose)jj by the poly (ADP-ribose) glycohydrolase. 

In the absence of DNA-histone complexes, ethacridine, proflavine, tilorone R10,556 DA, ellipticine and daunomycin inhibit the poly(ADP-ribose) glycohydrolase activity... 

Effect of DNA and DNA-Histone Complexes on the Inhibition of Poly(ADP-Ribose) Glycohydrolase by Intercalators. The inhibition of glycohydrolase activity by intercalators (proflavine, ellipticine, tilorone R10,556 DA) is relieved by the addition of 100 Mg mr calf thymus DNA (data not shown). This is probably due to the well-known binding of the intercalators to the added DNA. However, addition of DNA-histone complexes (100 jug nil" of each) slightly increases the inhibition of the glycohydrolase activity by those intercalators which are inhibitory (Table 1). These include ethacridine, tilorone R10,556 DA, eUipticine, daunomycin and proflavine. 

The kinetics of stacking have been investigated for three systems in aqueous and mixed aqueous solutions. The three systems, thionine, proflavin, and polyriboadenylie acid (poly A), represent strong, moderate, and weak stacking, respectively. 

---