Synthesis and characterization of bay region halohydrins derived from Benzo[a]pyrene diol epoxide and their role as intermediates in halide-catalyzed cis adduct formation.

The bay region epoxide of benzo[a]pyrene (anti-BPDE) alkylates DNA to form adducts with >98% trans stereochemistry. Halide ions catalyze this reaction; however, this pathway is characterized by the formation of adducts with altered cis stereochemistry. Bay region halohydrins are possible intermediates in these reactions, but are too unstable to be isolated from aqueous solutions. However, we successfully synthesized halohydrins in tetrahydrofuran (THF) by treatment of anti-BPDE with the corresponding lithium halide salt in the presence of acetic acid. Absorbance and CD spectroscopy clearly indicated the formation of chloro-, bromo-, and iodohydrins. The structure and stereochemistry of the chlorohydrin was established by NMR. Chloride addition is exclusively at the benzylic position of the epoxide, and the stereochemistry of the C-9 and -10 positions is trans. The long-wavelength absorbance band in the chloro-, bromo-, and iodohydrin is red-shifted 7, 13, and 22 nm, respectively, relative to the hydrolysis product of anti-BPDE. The ellipticity of the same absorbance band in CD spectra of enantiomerically pure halohydrins is opposite in sign compared to that of the corresponding anti-BPDE enantiomer. The relative stability of these derivatives is chlorohydrin > bromohydrin > iodohydrin. The chloro- and bromohydrins were isolated, but the iodohydrin decomposed during this manipulation. The addition of 500 mM chloride decreased the hydrolysis rate of the chlorohydrin 4-fold in 50% THF/buffer. Direct evidence for the transient formation of the iodohydrin in aqueous buffer/acetone mixtures was obtained by absorbance spectroscopy. At 1 M chloride, bromide, and iodide, alkylation of deoxyadenosine by anti-BPDE in aqueous buffer yields 85, 91, and 92% cis adducts, respectively. In the absence of halide, alkylation of deoxyadenosine in buffer by anti-BPDE, the chlorohydrin, and the bromohydrin yields 32, 65, and 83% cis adducts, respectively.

High-resolution polyacrylamide gel electrophoresis of carbohydrates derivatized with a visible dye.

A technique for carbohydrate analysis that is both inexpensive and easily performed is currently unavailable. In this communication we address the problem and have outlined a method for labeling saccharides with a visible dye, 4-amino-1,1'-azobenzene-3, 4'-disulfonic acid, which has an absorption maximum of 489 nm and an extinction coefficient of 37,615, to facilitate visible detection at low levels. The visible dye was coupled by a reductive amination to different sugars. The sugar-dye adducts were then separated by electrophoresis on alkaline polyacrylamide gels. The gels were scanned with a densitometer, and visible sugar-dye adducts were qualitatively analyzed by identifying them according to their mobilities. The sugar-dye adducts were quantified by determining their densitometric volume. The kinetics of the reductive amination reactions, performed at 37 degrees C, were different for each of three saccharides tested. The rate constants for glucose and fucose were 1.31 times greater and 1.8 times greater, respectively, than that of maltotriose. The reductive amination reactions were essentially complete after approximately 16 h under the given experimental conditions. A linear dose-response relationship was observed between the amount of sugar (monosaccharide, trisaccharide, or heptasaccharide) in the reductive amination reaction. The quantity of saccharide-dye adduct that could be visually detected for glucose, maltotriose, and maltoheptaose, was 25, 25, and 50 nmol, respectively. Sugar-dye adducts were separated from one another by varying the acrylamide concentration in the polyacrylamide gels. Sugar-dye adducts of monosaccharides, disaccharides, trisaccharides, and heptasaccharides were separated on alkaline 30% polyacrylamide gels with mobilities of 0.778, 0.667, 0.639, and 0.375. Adducts of glucose, fucose, galactose, and mannose were separated with mobilities of 0.844, 0.833, 0.820, and 0.810, respectively, on a 30 to 40% gradient polyacrylamide gel. Adducts of glucose and glucose derivatives were separated on a 35% polyacrylamide gel. This technique provides an inexpensive and easily performed method of carbohydrate analysis to laboratories that do not have the highly trained personnel nor the expensive equipment needed for other methods of carbohydrate analysis. The method is most applicable to research problems where sensitivity (20 pmol) is not a problem. The simplicity of the method also makes it easily incorporated into teaching laboratories.

Benzo[a]pyrene diol epoxide-DNA cis adduct formation through a trans chlorohydrin intermediate.

Alkylation of DNA by 7r,8t-dihydroxy,9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) forms mainly trans adducts (with respect to the C-9/10 positions). We recently described a halide-catalyzed pathway that preferentially generates cis adducts and now report that the trans chlorohydrin of anti-BPDE (trans-BPDCH) is an intermediate in the chloride-catalyzed reaction. trans-BPDCH was synthesized, and both it and anti-BPDE were reacted with deoxyadenosine as a model DNA nucleophile. The stereochemistry and yields of deoxyadenosine adducts were determined as a function of chloride concentration. In the absence of salt, the fraction of cis adducts obtained from anti-BPDE and trans-BPDCH are 0.33 and 0.67, respectively. Adding sodium chloride increases the fraction of cis adducts (and consequently decreases the fraction of trans adducts), with the midpoint of the increase for both substrates at approximately 35-40 mM chloride. The chloride-dependent curves for BPDE and BPDCH converge at 1 M chloride, where the fraction of cis adducts is 0.88. Chloride also increases the total yield of cis adducts with either substrate, whereas the yield of trans adducts from the chlorohydrin is not significantly changed. These results support a mechanism by which chloride ion undergoes nucleophilic addition to the benzylic C-10 position of anti-BPDE. This generates a trans halohydrin that alkylates DNA with inversion of configuration to form a cis adduct. This pathway may have biological significance because chlorohydrins could form in serum or in cells with relatively high intracellular concentrations of chloride.