•A new trinuclear Cu(II) complex was synthesized.•The complex photocleaves plasmid DNA with high efficiency at neutral pH.•Hydroxyl radicals and Cu(I) are generated during the reaction.•Singlet oxygen does not contribute to DNA photocleavage.•The complex associates with DNA through external interactions.This paper describes the synthesis of a trinuclear Cu(II) complex (4) containing a central 1,4,5,8,9,12-hexaazatriphenylene-hexacarboxylate (hat) core (3). Low, micromolar concentrations of the negatively charged parent ligand 3 and the neutral trinuclear complex 4 were found to photocleave negatively charged pUC19 plasmid DNA with high efficiency at neutral pH (350 nm, 50 min, 22 °C). The interactions of complex 4 with double-helical DNA were studied in detail. Scavenger and colorimetric assays pointed to the formation of Cu(I), superoxide anion radicals, hydrogen peroxide, and hydroxyl radicals during photocleavage reactions. UV–visible absorption, circular dichroism, DNA thermal denaturation, and fluorescence data suggested that the Cu(II) complex contacts double-stranded DNA in an external fashion. The persistent association of ligand 3 and complex 4 with Na(I) and/or other cations in aqueous solution might facilitate electrostatic DNA interactions.A tri-nuclear Cu(II) complex containing a central, negatively charged 1,4,5,8,9,12-hexaazatriphenylene-hexacarboxylate core photocleaves plasmid DNA in 98% to 99% yield at low micromolar concentrations of complex (350 nm, 22 °C, pH 7.0). The cleavage process involves the generation of Cu(I) and hydroxyl radicals.Download high-res image (115KB)Download full-size image

Photosensitizing agents that oxidatively cleave DNA are important to photodynamic therapy (PDT), an emerging treatment option for patients diagnosed with age-related macular degeneration, pre-cancerous conditions such as Barrett's esophagus, and localized cancers that include inoperable neoplasms. Excitation with low energy irradiation activates the PDT agents, causing spatially targeted, oxidative damage to DNA and other macromolecules in diseased cells. While most routinely used PDT photosensitizers rely on visible light sources that emit at wavelengths ≤689 nm, excitation that extends from the far-red into the near-infrared wavelength range is desirable. Due to low absorption by biogenic chromophores, light in this region has a greater penetration depth through tissue when compared to visible irradiation that is higher in energy. In the present review, we describe the development of new, long wavelength DNA photo-oxidizing agents. We have attempted to correlate the structural elements of the photosensitizers with their reported light-induced nuclease activities (λex ≥ 690 nm). Reaction pathways that lead to DNA cleavage are discussed, including 3O2 dependent Type I electron transfer and Type II energy transfer processes and anaerobic Type I hydrogen atom abstraction from deoxyribose. The summary discussion contained in this review is intended to contribute to the discovery of new phototherapeutic agents that are activated with far-red to near-infrared light.

Niemann-Pick disease and drug-induced phospholipidosis are lysosomal storage disorders in which there is an excessive accumulation of sphingomyelin in cellular lysosomes. Here we have explored the possibility of developing metal-based therapeutic agents to reverse phospholipid build-up through phosphate ester bond hydrolysis at lysosomal pH (~4.8). Towards this end, we have utilized a malachite green/molybdate-based colorimetric assay to quantitate the inorganic phosphate released upon the hydrolysis of sphingomyelin by twelve d- and f-block metal ion salts. In reactions conducted at 60 °C, the yields produced by the cerium(IV) complex Ce(NH4)2(NO3)6 were superior. An Amplex® Red-based colorimetric assay and mass spectrometry were then employed to detect choline. The data consistently showed that Ce(IV) hydrolyzed sphingomyelin more efficiently at lysosomal pH: i.e., yields of choline and phosphate were 54 ± 4 and 22 ± 5 % at pH ~ 4.8, compared to 8 ± 1 and 5 ± 2 % at pH ~ 7.2. Hydrolysis at 60 °C could be significantly increased by converting sphingomyelin vesicles to mixed lipid vesicles and mixed micelles of Triton X-100. We then utilized cerium(IV) to cleave sphingomyelin at 37 °C (no Triton X-100). Although choline and phosphate levels were relatively low, hydrolysis continued to be considerably more efficient at lysosomal pH. A side by side comparison to phosphatidylcholine was then made. While the yields of choline and phosphate produced by phosphatylcholine were higher, the ratio of pH ~ 4.8 hydrolysis to pH ~ 7.2 hydrolysis was usually more favorable for sphingomyelin (37 and 60 °C).

This paper describes the synthesis of a new 9-aminomethylanthracene dye N-substituted with a pyridinylpolyamine side chain (4). The effects of NaCl and KCl on anthracene/DNA interactions were then studied, with the goal of simulating the conditions of high ionic strength that a DNA photosensitizer might encounter in the cell nucleus (∼150 mM of NaCl and 260 mM of KCl). As exemplified by methylene blue (5), the expected effect of increasing ionic strength is to decrease DNA binding and photocleavage yields. In contrast, the addition of 150 mM of NaCl in combination with 260 mM of KCl to photocleavage reactions containing micromolar concentrations of 4 triggers the conversion of supercoiled, nicked, and linear forms of pUC19 plasmid into a highly degraded band of DNA fragments (350 nm hν, pH 7.0). Circular dichroism spectra point to a correlation between salt-induced unwinding of the DNA helix and the increase in DNA photocleavage yields. The results of circular dichroism, UV–vis absorption, fluorescence emission, thermal denaturation, and photocleavage inhibition experiments suggest that the combination of salts causes a change in the DNA binding mode of 4 from intercalation to an external interaction. This in turn leads to an increase in the anthracene-sensitized production of DNA-damaging reactive oxygen species.

Niemann–Pick disease and drug-induced phospholipidosis are examples of lysosomal storage disorders in which serious respiratory infections are brought on by high levels of the phospholipid phosphatidylcholine in the acidic lamellar bodies and lysosomes of pulmonary cells. One approach to developing an effective therapeutic agent could involve the use of a metal to preferentially hydrolyze phospholipid phosphate ester bonds at mildly acidic, lysosomal pH values (~ pH 4.8). Towards this end, here we have investigated phosphatidylcholine hydrolysis by twelve metal ion salts at 60 °C. Using a malachite green/molybdate-based colorimetric assay to detect inorganic phosphate released upon metal-assisted phosphate ester bond hydrolysis, Ce(IV) was shown to possess outstanding reactivity in comparison to the eleven other metals. We then utilized cerium(IV) to hydrolyze phosphatidylcholine at normal, core body temperature (37 °C). The malachite green/molybdate assay was used to quantitate free phosphate and an Amplex® Red-based colorimetric assay and matrix-assisted laser desorption ionization time-of-flight mass spectrometry were employed to detect choline. Ce(IV) hydrolyzed phosphatidylcholine more efficiently at lysosomal pH: i.e., at a Triton X-100:phosphatidylcholine molar mixing ratio of 1.57, yields of choline and phosphate were 51 ± 4% and 40 ± 4% at ~ pH 4.8, compared to 28 ± 4% and 27 ± 5% at ~ pH 7.2.Phosphatidylcholine was screened against twelve commercially available metal ion salts. Ce(NH4)2(NO3)6, ZrCl4, and HfCl4 produced higher levels of phosphate ester bond hydrolysis at lysosomal pH (~ 4.8) compared to near neutral, cytoplasmic pH (~ 7.2). The cerium(IV) salt was the most reactive, displaying considerable activity at 60 °C and 37 °C.

We report the synthesis of photonuclease 3 consisting of two acridine rings joined by a 2,6-bis(aminomethyl)pyridine copper-binding linker. In reactions containing micromolar concentrations of 3, irradiation at 419 nm produces efficient, copper(II)-dependent cleavage of plasmid DNA in the presence of the high concentrations of salt that exist in the cell nucleus (150 mM NaCl and 260 mM KCl). The DNA interactions of 3 are compared to an analogous bis-acridine (4) containing a more flexible 2,6-bis{[(methoxycarbonylamino)-ethyl]methylaminomethyl}pyridine unit.Copper(II) chloride enhances DNA photocleavage by bis-acridine 3 in the presence of physiological concentrations of salt.

We report the synthesis and characterization of N,N′-bis[(7-dimethylamino)phenothiazin-5-ium-3-yl]-4,4′-ethylenedipiperidine diiodide (3), consisting of two photosensitizing phenothiazinium rings attached to a central ethylenedipiperidine linker. At all time points (10, 30, 60 min) and all wavelengths (676, 700, 710 nm) tested, photocleavage of pUC19 plasmid DNA (22 °C and pH 7.0) was markedly enhanced by 1 μM of 3 in comparison to 1 μM of the parent phenothiazine methylene blue (MB). At concentrations of phenothiazine ranging from 5 to 0.5 μM, the photocleavage levels produced by compound 3 were consistently higher than the cleavage produced using approximately twice the amount of MB (e.g., 710 nm irradiation of 5 μM of 3 and 10 μM of MB cleaved the plasmid DNA in 93% and 71% yields, respectively). Scavenger assays provided evidence for the involvement of singlet oxygen and, to a lesser extent, hydroxyl radicals in DNA damage. Analysis of photocleavage products at nucleotide resolution revealed that direct strand breaks and alkaline-labile lesions occurred predominantly at guanine bases. While compound 3 and MB were both shown to stabilize duplex DNA, the ΔTm values of calf thymus (CT) and C. perfringens DNAs were approximately three fold higher in the presence of compound 3. Finally, viscometric data indicated that CT DNA interacts with compound 3 and MB by a combination of groove binding and monofunctional intercalation, and with compound 3 by a third, bisintercalative binding mode.

Zirconium(IV) hydrolyzes the 30-mer oligopeptide oxidized bovine insulin chain B after 4 h to 8 h of treatment at ∼pH 7.0 and 60 °C: MALDI-TOF and HPLC-ESI-MS and MS/MS data show significant levels of cleavage at Gly8-Ser9, Gly20-Glu21, Ser9-His10, Cys(SO3H)7-Gly8, and Cys(SO3H)19-Gly20 amide bonds within the oligopeptide.

The present study has compared the effects of a total of 17 ligands on Zr(IV)-assisted hydrolysis of the dipeptide Gly–Gly (60 °C, pH 6.8–7.4, t = 4 h and t = 10 h). The macrocyclic azacrown ether ligands 1,4,10,13-tetraoxa-7,16-diazacyclooctadecane and 1,4,10-trioxa-7,13-diazacyclopentadecane produced the overall highest amounts of hydrolysis, followed by the open-chain ligand 2-(2-aminoethoxy)-ethanol. While it was not necessary to have a ring structure to enhance Zr(IV) reactivity, the structural feature “ROCH2CH2OCH2CH2NR” appeared to contribute to increased levels of peptide cleavage.Seventeen ligands were compared to observe their effects on Zr(IV)-assisted hydrolysis of Gly–Gly (60 °C, pH 6.8–7.4, t = 4 and 10 h). The diazacrown ethers 1,4,10,13-tetraoxa-7,16-diazacyclooctadecane and 1,4,10-trioxa-7,13-diazacyclopentadecane and the open-chain ligand 2-(2-aminoethoxy)-ethanol produced the most cleavage, indicating that the structural feature “ROCH2CH2OCH2CH2NR” contributes to peptide hydrolysis.

We report the syntheses and characterization of a series of compounds based on 1,10-phenanthroline covalently tethered, at the 2 and 9 positions, to either two benzene, naphthalene, acridine or anthracene chromophores. The acridine and anthracene derivatives are shown to efficiently cleave pUC19 plasmid DNA upon irradiation with ultraviolet light (pH = 7.0, 22 °C, 350 nm). Furthermore, photocleavage levels are markedly increased by the addition of Cu2+ to the DNA photolysis reactions. Interestingly, when the concentrations of the anthracene compounds are lowered from 35 µM to 0.25 µM, the reverse trend is observed. DNA photocleavage is markedly reduced in the presence of copper(II).

Rapid, microplate-based fluorogenic screening of a positional scanning combinatorial library accurately predicts the sequence specificity of metal-assisted peptide hydrolysis.

Synthesis of two (2,2′-bipyridine)platinum(II) complexes tethered to one or two acridine chromophores is reported. These acridine complexes efficiently unwind and photocleave supercoiled plasmid DNA under physiological conditions of temperature and pH.Synthesis and DNA interaction of two acridine (2,2′-bipryidine)platinum(II) complexes are reported.

We report the synthesis and characterization of N,N′-bis[(7-dimethylamino)phenothiazin-5-ium-3-yl]-4,4′-ethylenedipiperidine diiodide (3), consisting of two photosensitizing phenothiazinium rings attached to a central ethylenedipiperidine linker. At all time points (10, 30, 60 min) and all wavelengths (676, 700, 710 nm) tested, photocleavage of pUC19 plasmid DNA (22 °C and pH 7.0) was markedly enhanced by 1 μM of 3 in comparison to 1 μM of the parent phenothiazine methylene blue (MB). At concentrations of phenothiazine ranging from 5 to 0.5 μM, the photocleavage levels produced by compound 3 were consistently higher than the cleavage produced using approximately twice the amount of MB (e.g., 710 nm irradiation of 5 μM of 3 and 10 μM of MB cleaved the plasmid DNA in 93% and 71% yields, respectively). Scavenger assays provided evidence for the involvement of singlet oxygen and, to a lesser extent, hydroxyl radicals in DNA damage. Analysis of photocleavage products at nucleotide resolution revealed that direct strand breaks and alkaline-labile lesions occurred predominantly at guanine bases. While compound 3 and MB were both shown to stabilize duplex DNA, the ΔTm values of calf thymus (CT) and C. perfringens DNAs were approximately three fold higher in the presence of compound 3. Finally, viscometric data indicated that CT DNA interacts with compound 3 and MB by a combination of groove binding and monofunctional intercalation, and with compound 3 by a third, bisintercalative binding mode.