Seth D. Rose

PHOTOSENSITIZATION OF PYRIMIDINE DIMER SPLITTING BY A COVALENTLY BOUND INDOLE

Abstract— Photosensitized pyrimidine dimer splitting characterizes the enzymatic process of DNA repair by the DNA photolyases. Possible pathways for the enzymatic reaction include photoinduced electron transfer to or from the dimer. To study the mechanistic photochemistry of splitting by a sensitizer representative of excited state electron donors, a compound in which an indole is covalently linked to a pyrimidine dimer has been synthesized. This compound allowed the quantitative measurement of the quantum efficiency of dimer splitting to be made without uncertainties resulting from lack of extensive preassociation of the unlinked dimer and sensitizer free in solution. Irradiation of the compound with light at wavelengths absorbed only by the indolyl group (approximately 280 nm) resulted in splitting of the attached dimer. The quantum yield of splitting of the linked system dissolved in N20‐saturated aqueous solution was found to be 0.04 ± 0.01. The fluorescence typical of indoles was almost totally quenched by the attached dimer. A splitting mechanism in which an electron is efficiently transferred intramolecularly from photoexcited indole to ground state dimer has been formulated. The surprisingly low quantum yield of splitting has been attributed to inefficient splitting of the resulting dimer radical anion. Insights gained from this study have important mechanistic implications for the analogous reaction effected by the DNA photolyases.

SOLVENT DEPENDENCE OF PYRIMIDINE DIMER SPLITTING IN A COVALENTLY LINKED DIMER-INDOLE SYSTEM

Abstract— –Cyclobutadipyrimidines (pyrimidine dimers) undergo splitting that is photosensitized by indole derivatives. We have prepared a compound in which a two‐carbon linker connects a dimer to an indolyl group. Indolyl fluorescence quenching indicated that the two portions of the molecule interact in the excited state. Intramolecular photosensitization of dimer splitting was remarkably solvent dependent, ranging from φspl= 0.06 in water to a high value of φspl= 0.41 in the least polar solvent mixture examined, l,4‐dioxane‐isopentane(5: 95). A derivative with a 5‐methoxy substituent on the indolyl ring behaved similarly. These results have been interpreted in terms of electron transfer from the excited indolyl group to the dimer, which would produce a charge‐separated species. The dimer anion within such a species could split or undergo back electron transfer. The possibility that back electron transfer is in the Marcus inverted region can be used to rationalize the observed solvent dependence of splitting. In the inverted region, the high driving force of a charge recombination exceeds the reorganization energy of the solvent, which is less for solvents of low polarity than those of high polarity. If this theory is applicable to the hypothetical charge‐separated species, a slower back electron transfer, and consequently higher splitting efficiencies, would be expected in solvents of lower polarity. Photolyases may have evolved in which a low polarity active site retards back transfer of an electron and thereby contributes to the efficiency of the enzymatic dimer splitting.

TRANSIENT INTERMEDIATES IN INTRAMOLECULARLY PHOTOSENSITIZED PYRIMIDINE DIMER SPLITTING BY INDOLE DERIVATIVES

Abstract— Intramolecularly photosensitized pyrimidine dimer splitting can serve as a model for some aspects of the monomerization of dimers in the enzyme‐substrate complex composed of a photolyase and UV‐damaged DNA. We studied compounds in which a pyrimidine dimer was covalently linked either to indole or to 5‐methoxyindole. Laser flash photolysis studies revealed that the normally observed photoejection of electrons from the indole or the 5‐methoxyindole to solvent was diminished by an order of magnitude for indoles with dimer attached (dimer‐indole and dimer‐methoxyindole). The fluorescence lifetime of dimer‐indole in aqueous methanol was 0.85 ns, whereas that of the corresponding indole without attached dimer (tryptophol) was 9.7 ns. Similar results were obtained for the dimer‐methoxyindole (0.53 ns) and 5‐methoxytryptophol (4.6 ns). The quantum yield of dimer splitting for the dimer‐methoxyindole (φ287K7 = 0.08) was only slightly greater than the value found earlier for the dimer bearing the unsubstituted indole (4>2K7= 0.04). Transient absorption spectroscopy also revealed lower yields of indole radical cations following laser flash photolysis of dimer‐indole compared to the indole without attached dimer. Dimer‐methoxyindole behaved similarly. These results are interpreted in terms of an enhanced rate of radiationless relaxation of the indole and methoxyindole excited singlet states in dimer‐indoles. The possible quenching of the indole and methoxyindole excited states via electron abstraction by the covalently linked dimer is discussed.

PHOTO‐CIDNP DETECTION OF PYRIMIDINE DIMER RADICAL CATIONS IN ANTHRAQUINONESULFONATE‐ SENSITIZED SPLITTING

Abstract— –Anthraquinone‐2‐sulfonate (AQS) photosensitizes pyrimidine dimer splitting. Electron abstraction from the dimer is thought to induce dimer splitting, but direct evidence for the existence and intermediacy of dimer radical cations has been lacking. By employing photochemically induced dynamic nuclear polarization, we have found emission signals in the NMR spectra of dimers upon photolysis of dimers in the presence of anthraquinone‐2‐sulfonate. The two dimers employed were ci?, syn‐thymine dimer in which the JV(l)‐positions were linked by a three‐carbon bridge and the N(3), N(3′)‐dimethyle derivative of that compound. The anthraquinone‐2‐sulfonate sensitized photochemically induced dynamic nuclear polarization spectrum of the methylated derivative exhibited an emission signal from the dimer‐C(6) hydrogens. This result implied the existence of a dimer radical cation (mD+) formed by electron abstraction by excited anthraquinone‐2‐sulfonate and nuclear spin sorting within a solvent caged radical ion pair [mD+ AQS‐]. Product pyrimidine photochemically induced dynamic nuclear polarization signals were also seen [enhanced absorption by C(6)‐hydrogens and emission by C(5)‐methyl groups]. Nuclear spin polarization in the product resulted from spin sorting in one or more of its precursors, including mD+. The results support the conclusion that dimer radical cations not only exist but are intermediates in the photosensitized splitting of pyrimidine dimers by anthraquinonesulfonate.

THERMAL REQUIREMENTS OF PHOTOSENSITIZED PYRIMIDINE DIMER SPLITTING

Abstract— A cis, syn‐pyrimidine dimer (derived from thymine and orotate) covalently linked to 5‐methoxyindole has been studied as a mechanistic model of photosensitized pyrimidine dimer splitting. In this dimer‐indole, photoinitiated electron transfer to the dimer causes splitting in a manner that parallels the mechanism by which the DNA photolyases are thought to act. Dissolved in EPA (diethyl ether‐isopentane‐ethyl alcohol, 5: 5: 1, by vol) at room temperature, the dimer‐indole exhibited indole fluorescence quenching and underwent splitting upon irradiation at 300 nm. In an EPA glass at 77 K, however, no splitting was detectable. To distinguish the effects of temperature and immobilization, photolysis experiments were performed on PMM [poly(methyl methacrylate)] films containing dimer‐indole. In PMM at room temperature, dimer‐indole underwent splitting when irradiated at 300 nm, which indicated that immobilization per se was not responsible for the failure of dimer‐indole to split at low temperature. Furthermore, no splitting was observed when dimer‐indole was irradiated in PMM at 77 K. These results imply that a step following photoinitiated, intramolecular electron transfer from indole to dimer has an insurmountable activation barrier at 77 K. The mechanistic implications for the photolyases are considered.

Pyrimidine dimer splitting in covalently linked dimer—arylamine systems

Cyclobutadipyrimidines (pyrimidine dimers) undergo photosplitting which is sensitized by electron donors. We prepared a series of compounds in which a dimer is directly linked to an arylamine, which acts as sensitizer for dimer splitting. Two diastereomers of the dimer-arylamine exhibited very different splitting efficiencies. Also studied were N-methyl, ring methoxy, as well as deuterated derivatives of the sensitizer. These dimer-arylamines had an absorption band with lambda max approximately 300 nm. In each case intramolecular photosensitization of dimer splitting was highly dependent on the solvent, ranging in one instance from phi spl = 0.02 in water to a high value of 0.31 in the least polar solvent mixture examined (1,4-dioxane: isopentane, 1:99). A mechanism is proposed which involves photoinduced electron transfer from arylamine to dimer and splitting of the dimer radical anion. The dependence of splitting on the solvent was rationalized on the basis of retardation of back electron transfer due to Marcus inverted behavior of the charge-separated species. Photolyases might achieve their high efficiency of dimer splitting in part by employing a hydrophobic active site to slow back electron transfer in a similar manner.

PHOTO-CIDNP STUDY OF PYRIMIDINE DIMER SPLITTING I: REACTIONS INVOLVING PYRIMIDINE RADICAL CATION INTERMEDIATES

The light‐induced splitting of pyrimidine dimers was studied using the electron acceptor anthraquinone‐2‐sulfonate (AQS) as a photosensitizer. To this end, photochemically induced dynamic nuclear polarization (photo‐CIDNP) experiments were performed on a series of pyrimidine monomers and dimers. The CIDNP spectra demonstrate the existence of both the dimer radical cation, which is formed by electron transfer from the dimer to the photoexcited sensitizer AQS*, and its dissociation product, the monomer radical cation. In spectra of 1,1′‐trimethylene bridged cis,syn pyrimidine dimers, polarization is observed that originates from a spin‐sorting process in the dimer radical pair. This points to a relatively long lifetime of the dimer radical cation involved, which is presumably due to stabilization by the trimethylene bridge. Polarization originating from a dimer radical pair is detected in the spectrum of trans,anti (1,3‐dimethyluracil) dimer as well. The spectra of the bridged pyrimidines also demonstrate the reversibility of the dissociation of dimer radical cation into monomer radical cation, which is concluded from the observation of polarization in the dimer as a result of spin sorting in the monomer radical pair.

Energy and electron transfer processes in flavoprotein-mediated DNA repair

Abstract The flavin photoenzyme, DNA photolyasesm utilizes a pMhotom to repair the main damage to DNA (pyrimidine dimers) caused by UV light. DNA photolyases are monomeric flavoproteins that bind to the cis-syn-pyrimidine dimer in DNA in a light-independent step, forming a stable enzyme_substrate complex. This complex absorbs a photon, leading to electron transfer to the pyrimidine dimer, producing a unstable cyclobutane radical anion and thereby initiating bond cleavage in the dimer. The mechanism and energetics involved in the action of this remarkable enzyme are reviewed.

FLAVIN‐SENSITIZED PHOTOCHEMICALLY INDUCED DYNAMIC NUCLEAR POLARIZATION DETECTION OF PYRIMIDINE DIMER RADICALS

A photochemically induced dynamic nuclear polarization (photo‐CIDNP) study of carb‐oxymethyllumiflavin‐sensitized splitting of pyrimidine dimers has been carried out. In aqueous solution at high pH, an emission signal (δ 3.9 ppm) was observed from the dimer C(6)‐ and C(6′)‐protons of an N(l),N(1′)‐trimethylene‐bridged thymine dimer (1). The dimer photo‐CIDNP signal was seen only above pD 11.6 and was most intense at pD 12.9. Also observed were weak enhanced absorption signals from the product of splitting, trimethylenebis(thymine) (8 1.7 and 7.2 ppm). In contrast, cis, syn‐thymine dimer (3) gave no photo‐CIDNP signals from the dimer. An enhanced absorption at 1.8 ppm. however, due to the product of splitting (thymine) was observed. It was found that dimer 1 and, to a lesser extent, dimer 3 quenched flavin fluorescence. An N(3),N(3′)‐dimethylated derivative of 1, however, failed to quench flavin fluorescence. Comparison of the pD profile of the dimer photo‐CIDNP signal to the pKa values for thymine dimer suggested that principally the dideprotonated dimer undergoes electron abstraction by the excited flavin.

Cell Cycle Arrest and Apoptosis Induction by an Anticancer Chalcone Epoxide

Safe and effective chemotherapeutic agents for the treatment of pancreatic cancer remain elusive. We found that chalcone epoxides (1,3‐diaryl‐2,3‐epoxypropanones) inhibited growth in two pancreatic cancer cell lines, BxPC‐3 and MIA PaCa‐2. Three compounds were active, with GI50 values of 5.6 to 15.8 µM. Compound 4a, 1,3‐bis‐(3,4,5‐trimethoxyphenyl)‐2,3‐epoxypropanone, had an average GI50 of 14.1 µM in the NCI 60‐cell‐line panel. To investigate the mode of action, cell cycle analyses of BxPC‐3 cells were carried out. Treatment of cells with 50 µM 4a resulted in dramatic accumulation at G2/M (61% after 12 h for 4a vs. 15% for untreated cells). The cells rapidly entered apoptosis. After 12 h, 26% of cells treated with 50 µM 4a had entered apoptosis vs. 4% for cells treated with 100 µM etoposide and 2% for untreated cells. Compound 4a interfered with paclitaxel enhancement of tubulin polymerization, suggesting microtubules as the site of action. Reaction of thiol nucleophiles with 4a under basic conditions resulted in epoxide ring‐opening and retroaldol fragmentation, yielding alkylated thiol. MALDI mass spectrometry showed that retroaldol reaction occurred upon treatment of β‐tubulin with 4a. The site of alkylation was identified as Cys354. Chalcone epoxides warrant further study as potential agents for treatment of cancer.