Rosemarie F. Hartman

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 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.

DETECTION OF THE EXCITED SINGLET STATE OF A DEPROTONATED, REDUCED FLAVIN

Abstract— The excited singlet state of a deprotonated, reduced flavin [1, 5‐dihydro‐N(3)‐carboxymethyllumiflavin] in aqueous solution at pH 8 has been detected by laser flash photolysis. The broad absorption band maximized at ∼ 490 nm (ε= 9.9 × 103M‐1 cm‐1). The lifetime of the transient was found to be 100 ± 15 ps. The lifetime was not affected by the presence of pyrimidine dimers, which would be monomerized under these conditions. A longer‐lived transient, tentatively identified as the solvated electron, was also detected. The neutral reduced flavin did not give a detectable transient.

Detection of radical ion intermediates in flavin-photosensitized pyrimidine dimer splitting.

Photosensitized splitting of cis‐syn‐ and trans‐syn‐l,3‐dimethyluracil dimers by 2′,3′,4′,5′‐tetraacetylri‐boflavin in acetonitrile containing a trace of perchloric acid was studied by laser flash photolysis. Protonation of the flavin prior to excitation resulted in excited singlet and triplet states that abstracted an electron from the dimers and yielded the protonated flavin radical (F1H2′+), which was detected by absorption spectroscopy. Electron abstraction by the excited singlet state predominated over abstraction by the triplet state. Approximately one‐third to one‐half of the excited states quenched by the trans‐syn dimer yielded F1H2+, the balance presumably undergoing back electron transfer within the geminate radical ion pair generated by the initial electron transfer. A covalently linked dimer‐flavin exhibited very inefficient flavin radical ion formation, consistent with the known low efficiency of dimer splitting in this system. These results constitute the first identification of a flavin radical ion intermediate in photosensitized pyrimidine dimer splitting.

Emollient, humectant, and fluorescent α,β-unsaturated thiol esters for long-acting skin applications

We describe compounds in which an emollient or a humectant bears an alpha,beta-unsaturated thiol ester capable of reacting with nucleophilic amino acids in stratum corneum proteins. These compounds should serve as long-lasting moisturizers for skin. The emollient derivatized was octadecyl propanoate, and the humectant was poly(ethylene glycol). These hydrophobic and hydrophilic compounds, as well as a fluorescent, dansyl-containing thiol ester, were found to react within minutes with the thiol N-acetylcysteamine upon addition of a catalytic amount of an organic base in chloroform. The structures of the products resulting from conjugate addition to the unsaturated thiol esters were determined by NMR spectroscopy. In the case of the alpha,beta,gamma,delta-unsaturated (sorboyl) thiol ester, both the 1,4-addition product and the beta,gamma-unsaturated-1,6-addition product formed, followed by diadduct. An in vivo test of the fluorescent alpha,beta-unsaturated thiol ester showed that this compound persisted on skin for 3 weeks vs. 6 days for the non-bonding control compound.