Faculty Bibliography

The new adduct 3-(2-carboxyethyl)cytosine (3-CEC) was isolated following in vitro reaction of the carcinogen beta-propiolactone (BPL) with calf thymus DNA. The structure of 3-CEC was confirmed by synthesis from BPL and dCyd. Reaction of BPL with cCyd (pH 7.0-7.5, 37 degrees C) gave 3-(2-carboxyethyl)deoxycytidine (3-CEdCyd) (9% yield) and 3,N4-bis(2-carboxyethyl)deoxycytidine (3,N4-BCEdCyd) (0.6% yield). 3-CEdCyd and 3,N4-BCEdCyd were hydrolyzed (1.5 N HCl, 100 degrees C, 2 h) to 3-CEC and 3,N4-bis(2-carboxyethyl)cytosine (3,N4-BCEC), respectively. The structure of 3-CEC was assigned on the basis of UV and NMR spectra and the electron impact (EI) mass spectra of 3-CEC and a tri-trimethylsilyl (TMS) derivative of 3 CEC as well as deuterated (d27) tri-TMS derivative of 3-CEC. The structure of 3,N4-BCEC was assigned on the basis of UV spectra and the EI mass spectra of a tri-TMS derivative. Ei and isobutane chemical ionization mass spectra of 3-methylcytosine (3-MeCyt) and a di-TMs derivative of 3-MeCyt were obtained and were helpful in deducing the structures of 3-CEC and 3,N4-BCEC. This is the first report of the alkylation by BPL of an exocyclic atom on a base in DNA. Compound 3,N4-BCEC was not detected in BPL-reacted calf thymus DNA. The relative amounts of 1-(2-carboxyethyl)-adenine (1-CEA), 7-(2-carboxyethyl)guanine (7-CEG), 3-(2-carboxyethyl)-thymine (3-CET) and 3-CEC isolated from BPL-reacted DNA following perchloric acid hydrolysis were 0.23, 1.00, 0.39 and 0.41 respectively, when the alkylation reaction was conducted in phosphate buffer at 0-5 degrees C and pH 7.5 and 0.10, 1.00, 0.29 and 0.28 respectively when the reaction was conducted in H2O at 37b degrees C and pH 7.0-7.5.

Previous work has shown that unsaturated fatty acid components of model membrane phospholipids in vitro, damaged via a free radical mechanism, are protected by the presence of cholesterol in these membranes. The participation of these membrane lipids in the pathogenesis of traumatic injury to brain was studied in vivo using the Klatzo method of cryogenic injury in rats. Increased edema 4 hr after cryogenic injury was noted on the lesioned side. Total cerebral cholesterol was decreased significantly in the lesioned hemispheres 10 hr following injury. In lesioned animals pretreated and post-treated with methylprednisolone, there were no significant differences in the cholesterol levels. Arachidonic acid isolated from total membrane phospholipids was significantly reduced on the injured side 24 hr after injury, but not before. Other fatty acids were not significantly affected. Methylprednisolone treatment prevented the decrease in arachidonic acid. Animals that had received a cold injury had significant decreases in ascorbic acid levels after 4 hr on the lesioned side of the brain. This decrease was significantly ameliorated by corticosteroid administration. These results support the hypothesis that the protective effect of corticosteroids in cryogenic cerebral trauma may be due to antioxidant protection of major cell membrane lipid components such as cholesterol and phospholipids

3-(2-Carboxyethyl)thymine (3-CET) was synthesized from beta-propiolactone (BPL) and dThd 5'P at pH 9.0--9.5 via the intermediate 3-(2-carboxyethyl)-thymidine-5'-monophosphoric acid (3-CEdThd5'P). 3-CEdThd5'P was converted to 3-CET by hydrolysis in 1.5 N HCl at 100 degrees C for 2 h. The structure of 3-CET was assigned on the basis of UV spectra, electron impact (EI) and isobutane chemical ionization mass spectra and the EI mass spectrum of a trimethylsilyl derivative of 3-CET. BPL was reacted in vitro with calf thymus DNA at pH 7.5. 100 A units of BPL-reacted DNA yielded, following perchloric acid hydrolysis and preparative paper chromatography, 3 A units of 3-CET. Reaction of BPL with the phosphodiester thymidylyl-(3'-5')-thymidine gave 3-(2-carboxyethyl)thymidylyl-(3'-5')-3-(2-carboxyethyl)-thymidine (approximately 3%). Phosphotriester formation was not detected.

The new adduct N6-(2-carboxyethyl)adenine (N6-CEA) was prepared from 1-(2-carboxyethyl)adenine (1-CEA) by base catalyzed (Dimroth) rearrangement of 1-CEA. The structure of N6-CEA was assigned on the basis of UV spectra and electron impact and isobutane chemical ionization mass spectra. When the carcinogen beta-propiolactone was reacted in vitro with calf thymus DNA, 1-CEA but not N6-CEA was detected on paper chromatograms following acid hydrolysis of the DNA. When BPL-reacted single-stranded DNA was incubated at pH 11.7 (37 degrees C, 18 h) prior to acid hydrolysis, it was found that 1-CEA was completely converted to N6-CEA in DNA by Dimroth rearrangement, whereas no conversion occurred at pH 7.5. The extent of Dimroth rearrangement at various pHs and temperatures was determined for 1-CEA, 1-methyladenine (1-MeA), 1-(2-carboxyethyl)-deoxyadenosine-5'-monophosphoric acid (1-CEdAdo5'P) and the phosphodiester 5'-O-(2-carboxyethyl)phosphono-1-(2-carboxyethyl)deoxyadenosine (1-CE-Ado-5'-P-CE).