Changes In 31P NMR Spectra Research Articles

The investigation of stereogenic properties of cyclotriphosphazene derivatives with two different chiral centres

Hexachlorocyclotriphosphazene N 3P 3Cl 6 and gem-disubstituted cyclotriphosphazene derivatives N 3P 3Cl 4X 2 (X = Ph, PhS, PhNH) were reacted with N-methyl-1,3-propanediamine and 3-amino-1-propanol to give compounds ( 9a– 12a, 9b– 12b) which exist as cis and trans geometric isomers and are two different racemic isomers, respectively to describe the stereogenic properties of a series of chiral cyclotriphosphazene compounds with two different centres of chirality. The geometric isomers were separated by column chromatography on silica gel and analysed by elemental analysis, mass spectrometry, and 31P and 1H NMR spectroscopies, and also the geometric forms ( cis or trans) of 9b, 10a, 11a, 11b and 12a have been determined by the X-ray crystallography. The enantiomers of all racemic compounds have been analysed by the changes in 31P NMR spectra on addition of a Chiral Solvating Agent (CSA), (R)-(+)-2,2,2-trifluoro-1-(9′-anthryl)ethanol. On the other hand, the racemic forms of chiral cyclotriphosphazene derivatives have been confirmed by contribution of chiral HPLC methods which have been developed for this study.

Amphotericin B covalent dimers forming sterol-dependent ion-permeable membrane channels.

Polyenemacrolides such as amphotericin B (AmB) were thought to assemble together and form an ion channel across plasma membranes. Their antimicrobial activity has been accounted for by this assemblage, whose stability and activity are dependent on sterol constituents of lipid bilayer membranes. The structure of this channel-like assemblage formed in biomembranes has been a target of extensive investigations for a long time. For the first step to this goal, we prepared several AmB dimers with various linkers and tested for their channel-forming activity. Among these, AmB dimers that bore an aminoalkyl-dicarboxylate tether covalently linked between amino groups of AmB showed potent hemolytic activity. Furthermore, K+ influx actions monitored by measuring the pH of the liposome lumen by 31P NMR revealed that the dimers formed the molecular assemblage similar to that of AmB in phospholipid membrane. Judging from changes in 31P NMR spectra, the dimers appeared to induce "all-or-none"-type ion flux across the liposome membrane in the presence of ergosterol, which suggested that the ion channel formed by ergosterol/dimer is similar to that of AmB. With these data in hand, we are now trying to elucidate the structure of the ion-channel complex by making the labeled conjugates of AmB for NMR measurements.

31P NMR spectroscopic studies on purified, native and cloned, expressed forms of NADPH-cytochrome P450 reductase.

31P NMR spectroscopy has been utilized in conjunction with site-directed mutagenesis and phospholipid analysis to determine structural aspects of the prosthetic flavins, FAD and FMN, of NADPH-cytochrome P450 reductase. Comparisons are made among detergent-solubilized and protease (steapsin)-solubilized preparations of porcine liver reductases, showing unequivocally that the 31P NMR signals at approximately 0.0 ppm in the detergent-solubilized, hydrophobic form are attributable to phospholipids. By extraction and TLC analysis, the phospholipid contents of detergent-solubilized rat liver reductase, both tissue-purified and Escherichia coli-expressed, have been determined to reflect the membranes from which the enzyme was extracted. In addition, the cloned, wild-type NADPH-cytochrome P450 reductase exhibits an additional pair of signals downfield of the normal FAD pyrophosphate resonances reported by Otvos et al. [(1986) Biochemistry 25, 7220-7228], but these signals are not observed with tissue-purified or mutant enzyme preparations. The Tyr140----Asp140 mutant, which exhibits only 20% of wild-type activity, displays no gross changes in 31P NMR spectra. However, the Tyr178----Asp178 mutant, which has no catalytic activity and does not bind FMN, exhibits no FMN 31P NMR signal and a normal, but low intensity, pair of signals for FAD. The latter experiments, taking advantage of mutations in residues putatively on either side of the FMN isoalloxazine ring, suggest subtle to severe changes in the binding of the flavin prosthetic groups and, perhaps, cooperative interactions of flavin binding to NADPH-cytochrome P450 reductase.

31P NMR spectroscopic study of bioenergetic changes in radiation-induced fibrosarcoma-1 after radiation therapy.

The effects of localized gamma-irradiation on the in vivo 31P NMR spectra of RIF-1 tumors grown subcutaneously in C3H/HeN mice have been examined before and during the week after treatment. Increases in the ratio of phosphocreatine (PCr) to inorganic phosphate (Pi) and in tumor pH, and decreases in the ratio of Pi to the beta phosphorus resonance of the nucleotide triphosphates (beta NTP) were observed in irradiated tumors. The time course of changes in the 31P spectrum following treatment was the opposite of the pattern during untreated growth, and the magnitude and duration of the changes increased with increasing radiation dose, decreasing clonogenic cell survival and increasing growth delay. To examine the possibility that nontherapeutic systemic effects of the tumor irradiation were responsible for the changes observed, a number of animals bearing two tumors were examined. One tumor on each mouse was selectively irradiated. Changes in tumor volume, Pi/beta NTP, PCr/Pi, the ratio of phosphomonoesters to beta NTP, and tumor pH were all significantly different in the treated compared to the untreated tumor on each animal, indicating that these changes in 31P NMR spectra were a response to radiation therapy and not a systemic response to radiation toxicity.

Phosphate metabolites in single barnacle muscle fibers investigated by phosphorus-31 nuclear magnetic resonance

1. 1. A study has been made of phosphate-containing metabolites in single barnacle muscle fibers using phosphorus-31 nuclear magnetic resonance. 2. 2. Spectra from single fibers (∼50 mg in wet weight) show major resonances from sugar phosphates, inorganic phosphate, arginine phosphate and the α, β and γ phosphorus atoms of ATP. 3. 3. The approximate “free” concentration of each metabolite was determined by integration of the spectrum, using a sample of 1 M-methylene diphosphonic acid as a reference. A notable feature of the results obtained is that the concentrations of SP&P i in freshly dissected fibers are low. 4. 4. Time-dependent changes in 31P-NMR spectra indicate that ArP declines fairly slowly, while SP and P i rises. The half-life of ArP at 26°C turns out to be about 8 hr. ATP remains relatively constant for the first 8 hr but disappears following the disappearance of ArP. As the intensity of the P i resonance increases with time, it broadens and moves upfield, suggesting internal acidosis. 5. 5. These results demonstrate that 31P-NMR can provide useful information about metabolism and its regulation in single barnacle muscle fibers.