R. Jeremy H. Davies

Absolute configurations of the arene oxide, trans-dihydrodiol and cis-dihydeodiol products resulting from metabolism of quinoline at the 5,6-bond

Abstract Trans -6-bromo-5-hydroxy-5,6,7,8-tetrahydroquinoline enantiomers have been resolved via their dibenzoyltartrate salt diastereoisomers. X-ray crystallographic analysis of the (+)-dibenzoyltartrate salt obtained from reaction of (+)-dibenzoyltartaric acid and (−)-trans-6-bromo-5-hydroxy-5,6,7,8-tetrahydro-quinoline, when allied to a stereochemical correlation sequence linking the arene oxide, trans -dihydrodiol and cis -dihydrodiol enantiomers, provides an unequivocal assignment of absolute configuration for these quinoline metabolites.

Sequence Specificity of Alkali-labile DNA Damage Photosensitized by Suprofen‡¶

Abstract On irradiation at UVB wavelengths, in aerated neutral aqueous solution, the anti-inflammatory drug suprofen (SP) photosensitizes the production of alkali-labile cleavage sites in DNA much more efficiently than direct strand breaks. It is active at submillimolar concentrations despite having no significant binding affinity for DNA. Gel sequencing studies utilizing 32P–end-labeled oligonucleotides have revealed that piperidine-sensitive lesions are formed predominantly at the positions of guanine (G) bases, with the extent of modification being UV dose- and SP concentration-dependent. Quite distinct patterns of G-specific damage are observed in single-stranded and duplex DNA molecules. The uniform attack at all G residues in single-stranded DNA, which is enhanced in D2O, is compatible with a Type-II mechanism. SP is a known generator of singlet oxygen whose participation in the reaction is supported by the effects of quenchers and scavengers. In duplex DNA, piperidine-induced cleavage occurs with high selectivity at the 5′-G of GG and (less prominently) GA doublets. This behavior is characteristic of a Type-I process involving electron transfer from DNA to photoexcited SP molecules. The ability of SP to sensitize the formation of Type-I and Type-II photo-oxidation products from 2′-deoxyguanosine attests to the feasibility of competing mechanisms in DNA.

DNA damage by low-energy ions.

Ion-beam irradiation provides a promising treatment for some types of cancer. This promise is due mainly to the selective deposition of energy into a relatively small volume (the Bragg peak), thus reducing damage to healthy tissue. Recent observations that electrons with energies below the ionization potential of DNA can cause covalent damage to the bases and backbone have led to investigations into the ability of low-energy (<1 keV x Da(-1)) ion beams to damage double-stranded DNA. It has been clearly demonstrated that these low-energy ions induce a mixture of single- and double-strand breaks to dried DNA in vacuo. These effects depend upon the number of ions incident upon the DNA, the kinetic energy of the ions and on their charge state. This DNA damage may be important, as all radiotherapies will result in the production of low-energy secondary ions as radiation passes through tissues. Currently, their effects are neglected in treatment planning, and thus more work is required to quantify and understand DNA damage by low-energy ions.

The Sunscreen Agent 2-Phenylbenzimidazole-5-Sulfonic Acid Photosensitizes the Formation of Oxidized Guanines In Cellulo after UV-A or UV-B Exposure

The sunscreen agent 2-phenylbenzimidazole-5-sulfonic acid (PBSA) is water soluble and is widely used in the cosmetic industry because it absorbs strongly at UV-B wavelengths. Previous studies have shown that PBSA, photoexcited by UV-B, oxidizes guanine bases in vitro. Because of its potential phototoxic effect, it is important to determine whether PBSA photosensitizes in cellulo the formation of oxidatively generated DNA damage on UV exposure. For this purpose, we investigated, in vitro and in cellulo, the effect of PBSA on DNA bases after UV-A or UV-B irradiation. To monitor the formation of oxidized bases and cyclobutane pyrimidine dimers (CPDs), DNA was digested either with FaPy-DNA glycosylase and endonuclease III or with T4 endonuclease V and photolyase, then analyzed by means of neutral- and glyoxal-agarose gel electrophoresis and ligation-mediated PCR. In cellulo, we found that PBSA provided good protection against CPD formation after UV-B exposure. However, PBSA also photosensitized oxidized guanines on UV-A and UV-B irradiation. Our results indicate that PBSA has the potential to function as a double-edged sword toward DNA and question its suitability for sunscreen applications.

Conversion of formycin into the fluorescent isoguanosine analogue 7-amino-3-(β-D-ribofuranosyl)-1H-pyrazolo[4,3-d]pyrimidin-5(4H)-one

On u.v. irradiation, in aqueous solution, formycin N(6)-oxide (3) undergoes photorearrangement to give 5-cyano-3-(β-D-ribofuranosyl)-4-ureido-1H-pyrazole (5) as the major product; small amounts of 7-amino-3-(β-D-ribofuranosyl)-1H-pyrazolo[4,3-d]pyrimidin-5(4H)-one (4) and formycin (1) are also formed. When treated with aqueous ammonia, compound (5) cyclizes to the isoguanosine analogue (4). The latter compound is strongly fluorescent and its c.d. spectrum resembles that of formycin. Its u.v. absorption and fluorescence characteristics are compared with those of the corresponding guanosine analogue 5-amino-3-(β-D-ribofuranosyl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (2).

Arene oxides of quinoline: epoxidation, N-oxidation and N-methylation reactions

trans- 5,6,7,8- Diepoxy-5,6,7,8-tetrahydroquinoline 5, 5,6-epoxy-5,6-dihydroquinoline 1-oxide 3, 7,8-epoxy-7,8-dihydroquinoline 1 -oxide 4 and trans- 5,6,7,8 -diepoxy - 5,6,7,8 -tetrahydroquinoline1-oxide 6 have been formed by oxidation of the corresponding arene oxides of quinoline, 5,6-epoxy5,6-dihydroquinoline 1 and 7,8-epoxy-7,8-dihydroquinoline 2. The cis-diepoxides, cis-5,6,7,8-diepoxy-5,6,7,8-tetrahydroquinoline 8 and cis-5,6,7,8-diepoxy-5,6,7,8-tetrahydroquinoline 1-oxide 9 were both obtained by a stepwise synthesis from 7,8-epoxy-7,8-dihydroquinoline 2via the bromohydrin 7. N-Methylation of 5,6-epoxy-5,6-dihydroquinoline 1 and 7,8-epoxy-7,8-dihydroquinoline 2 with methyl trifIuoromethanesulphonate yielded the corresponding N-methylquinolinium arene oxide salts 10 and 11.

Synthesis of arene oxide and trans-dihydrodiol metabolites of quinoline

Racemic samples of 5,6-epoxy-5,6-dihydroquinoline (2)(quinoline 5,6-oxide) and 7,8-epoxy-7,8-dihydroquinoline (4)(quinoline 7,8-oxide) have been synthesised by two methods from the corresponding dihydroquinoline precursors. trans-5,6-Dihydroquinoline-5,6-diol (3) and trans-7,8-dihydroquinoline-7,8-diol (5) were obtained both by multi-step synthetic routes from the corresponding dihydroquinolines and by the direct base-catalysed hydration of the corresponding arene oxides (2) and (4).

The purification of α1-antichymotrypsin from human serum using DNA-cellulose chromatography

Abstract By exploiting its capacity for binding to DNA, the protease inhibitor α 1 -antichymotrypsin has been isolated from human serum by ammonium sulfate fractionation and successive chromatography on QAE-Sephadex, DNA-cellulose, and Sephacryl S-300. This experimental procedure compares favorably with existing methods for preparing α 1 -antichymotrypsin in terms of overall yield and practical convenience. The purified α 1 -antichymotrypsin was homogeneous as judged by electrophoretic and immunoelectrophoretic criteria. From its inhibition of the fluorimetric titration of chymotrypsin with 4-methylumbelliferyl- p -trimethylammonium cinnamate it was shown to combine with chymotrypsin in a 1:1 molar ratio and thus to retain its biological activity.

The application of DNA-cellulose chromatography in the isolation of immunoglobulin M and complement component C4b-binding protein from human serum

By utilising the ability of some anionic serum proteins to bind to DNA-cellulose, at physiological pH and ionic strength, two such proteins have been purified and identified as immunoglobulin M (IgM) and complement component C4b-binding protein (C4BP). The method entails ammonium sulphate precipitation followed by QAE-Sephadex, DNA-cellulose and Sephacryl S-300 chromatographic steps. Fractionation of the anionic proteins eluting from QAE-Sephadex over a selected range of ionic strength has afforded IgM which was characterised electrophoretically and immunochemically. A mixture containing IgM complexed with C4BP has also been obtained by utilizing fractions eluted from QAE-Sephadex at higher ionic strength. C4BP can be isolated from this mixture by using immunoaffinity chromatography to remove IgM.

ALKALI-LABILE PHOTOLESIONS MAPPING TO PURINE SITES IN ULTRAVIOLET-IRRADIATED DNA

Gel sequencing experiments with the 5′‐ and 3′‐end‐labeled oligonucleotides d(A3GA4GA5GA6GA3G) and d(AT) 10 have demonstrated that dimeric adenine photoproducts and thymine‐adenine photoadducts constitute alkali‐labile lesions in UV‐irradiated DNA. On treatment with hot piperidine, DNA strand breakage occurs predominantly at the sites of 5′‐adenines in the dimeric photoproducts and of 3′‐adenines in the thymine‐adenine photoadducts. With 5′‐end‐labeled oligonucleotides of mixed sequence, major UV‐induced loci for alkaline cleavage map to purine bases flanked on their 5′‐side by two pyrimidines. This behavior does not arise from enhanced photoreactivity of purines in this sequence context as has been inferred from photofootprinting studies. Instead, as shown by 3′‐labeling and selective substitution with 5‐methylcytosine, it results from the anomalous electrophoretic mobility of 5′‐end‐labeled fragments produced by alkaline cleavage of DNA at adjacent pyrimidine (6‐4) pyrimidone photoproducts.