Protonation of phosphoramide mustard and other phosphoramides.

The chemistry of the bifunctional alkylating agent phosphoramide mustard and model phosphoramides was probed by multinuclear NMR spectroscopy as a function of pH. Between pH 1 and 11, both the 31P and 15N resonances for phosphoramide mustard displayed a single monobasic titration curve with a pKa of 4.9. The protonation below pH 4.9 correlates with the loss in reactivity of the mustard. The 17O NMR spectrum of 17O-enriched phosphoramide mustard shows little change with pH. The data on the mustard was compared to 15N and 31P NMR data on 15N-enriched phosphoramidic acid, phosphorodiamidic acid, and phosphoric triamide. Contrary to the conclusions of previous studies, our combined 31P, 15N, and 17O NMR results are more consistent with N-protonation of phosphoramide mustard rather than an O-protonation. Theoretical calculations on the phosphoramidic acid, phosphorodiamidic acid, and phosphoric triamide show O-protonation to be more stable in the gas phase. For the latter two compounds, the calculations suggest that N-protonation may be the most stable protonated form in the aqueous phase. These findings influence our understanding of the structure-activity relationships of phosphoramide mustards.

Shift reagent enhanced concurrent 23Na and 1H magnetic resonance spectroscopic studies of transcellular sodium distribution in the dog brain in vivo.

The intracellular to extracellular sodium distribution is one of the primary determinants of action potentials necessary for the electrical function of organs such as brain, heart and skeletal muscle. The ability of shift reagent enhanced 23Na MRS to directly measure the intracellular and extracellular sodium distribution in brain is controversial and centers on the relative contributions of bulk magnetic susceptibility and hyperfine interactions to the observed chemical shifts. In this study, infusion of dysprosium (III) triethylenetetraminehexacetate (Dy(TTHA)-3), resulted in a 23Na MRS spectrum of dog brain with two well resolved peaks at 9 and 0.4 ppm. The 9 ppm peak corresponded to the resonance seen in aspirated blood. After disruption of the blood brain barrier, the single peak at 0.4 ppm split into two peaks at 3 and 0 ppm. The ability of Dy(TTHA)-3 enhanced 23Na MRS to follow global changes in brain sodium distribution was tested during cardiac arrest. The expected rapid Na influx into the intracellular space produced a marked decrease in the 3 ppm signal and a parallel increase in the 0 ppm peak. This is consistent with the assignment of the 3 ppm peak as interstitial sodium and the 0 ppm peak as intracellular sodium.

Two-dimensional nMR study of bleomycin and its zinc(II) complex: reassignment of 13C resonances.

Two-dimensional NMR experiments--one bond 1H-13C correlation spectroscopy and heteronuclear multiple bond correlation spectroscopy, both performed in the reverse detection mode--have been employed to unambiguously assign all of the 13C resonances of the antibiotic bleomycin and its zinc(II) complex. Previous 1H resonance assignments of bleomycin (Chen et al. (1977) Biochemistry 16, 2731-2738) were confirmed on the basis of homonuclear Hartmann-Hahn and homonuclear COSY experiments. The 13C assignments differ substantially from those previously obtained by other investigators (Naganawa et al., (1977) J. Antibiot. 30, 388-396; Dabrowiak et al., (1978) Biochemistry 17, 4090-4096) but are in agreement with those reported by Akkerman et al. (1988) (Magn. Reson. Chem. 26, 793-802). The more recent study employed similar two-dimensional correlation experiments (performed in the direct detection mode) in conjunction with attached proton tests. Their study often required model compound data to identify carbonyls adjacent to aliphatic moieties. Previous 13C NMR studies of the structure, pH titration, and molecular dynamics of bleomycin and its zinc complex have been reinterpreted in terms of the revised assignments.

Computational analysis of structural and energetic consequences of DNA methylation.

The conformational and energetic consequences of cytosine methylation in eukaryotes related to transcription or formation of chromatin structure are not well understood. Structures of methylated and unmethylated DNA sequences from biologically relevant sources were studied by theoretical methods under different ionic conditions and demonstrate that cytosine methylation produces a localized pattern of steric, hydrophobic, energetic, conformational and electrostatic alterations in DNA. These findings suggest how this modification may influence protein-DNA interactions and support current hypotheses. The results reveal previously unrecognized potential effects of cytosine methylation which could critically affect normal and neoplastic cellular processes by altering transcriptional events, histone binding, chromosomal stability and cellular differentiation.

Dynamic monitoring of corneal carbohydrate metabolism using high-resolution deuterium NMR spectroscopy.

Glucose metabolism in rabbit corneas was monitored with deuterium (D or 2H) NMR spectroscopy. The corneas were incubated in 5.5 mM deuterated glucose (glucose-6, 6-D2). A 2.5 micrograms change in lactate and a 4.1 micrograms change in glucose could be detected by the NMR method. The mean rates of glucose utilization and lactate production in intact rabbit corneas were 248- and 151 micrograms h-1, respectively. The lactate production/glucose utilization ratio of 0.60, i.e. 60% of total glucose is metabolized to lactate, confirms that glycolysis is the principal pathway for glucose catabolism. Further, based on enrichment of the HDO signal (which refers to the naturally abundant deuterium signal arising from deuterons in water), glucose oxidation through Krebs cycle and its associated pathways is estimated to be 90 micrograms h-1 or 36% of total consumption. The significant advantages of deuterium NMR spectroscopy over other NMR techniques (e.g. 13C spectroscopy) are: (1) shorter acquisition times because of the short relaxation times of deuterated metabolites; (2) the HDO signal can be used as the internal reference; and (3) significant reduction in cost and high availability of 2H-labeled compounds. Deuterium NMR spectroscopy is therefore a reliable and effective means with which the corneal glycolytic activity prior to transplantation can be readily assessed.

The study of diabetic cataractogenesis in the intact rabbit lens by deuterium NMR spectroscopy.

The polyol pathway has been implicated in the process of diabetic cataractogenesis. We report the use of deuterium (2H) spectroscopy for dynamically monitoring the polyol and glycolytic pathways in the single intact rabbit lens. Using 2H labeled C-1 D-glucose, the formation of sorbitol from glucose and the metabolism of sorbitol to fructose was dynamically monitored at 5.5 mM and 35.5 mM glucose concentrations. The accumulation of sorbitol at 35.5 mM glucose concentration was prevented by the inhibition of aldose reductase using an inhibitor (Sorbinil). 2H spectra were obtained in short acquisition times because of the short T1's of deuterated metabolites. A further advantage of 2H spectroscopy is that the natural abundance resonance of water (HDO) can be used as an internal reference standard. These findings confirm previous studies and demonstrate for the first time by NMR spectroscopy activity in the polyol pathway at low glucose concentrations.