31P NMR probes of chemical dynamics: paramagnetic relaxation enhancement of the (1)H and (31)P NMR resonances of methyl phosphite and methylethyl phosphate anions by selected metal complexes.

Methyl phosphite ((CH(3)O)P(H)(O)(2)(-); MeOPH) and methylethyl phosphate ((CH(3)O)P(OCH(2)CH(3))(O)(2)(-); MEP) are two members of a class of anionic ligands whose (31)P T(2) relaxation rates are remarkably sensitive to paramagnetic metal ions. The temperature dependence of the (31)P NMR line broadenings caused by the Mn(H(2)O)(6)(2+) ion and a water-soluble manganese(III) porphyrin (Mn(III)TMPyP(5+)) indicates that the extent of paramagnetic relaxation enhancement is a measure of the rate at which the anionic probes come into physical contact with the paramagnetic center (i.e., enter the inner coordination shell); that is, piDeltanu(par) = k(assn)[M], where Deltanu(par) is the difference between the line widths of the resonance in paramagnetic and diamagnetic solutions, and k(assn) is the second-order rate constant for association of the phosphorus ligand with the metal, M. Comparison of the (31)P T(1) and T(2) relaxation enhancements shows that rapid T(2) relaxation by the metal ion is caused by scalar interaction with the electronic spin. Relaxation of the phosphorus-bound proton of MeOPH ((1)H-P) by Mn(III)TMPyP(5+) displayed intermediate exchange kinetics over much of the observable temperature range. The field strength dependence of (1)H-P T(2) enhancement and the independence of the (31)P T(2) support these assertions. As in the case of the (31)P T(2), the (1)H-P T(2) relaxation enhancement results from scalar interaction with the electronic spin. The scalar coupling interpretation of the NMR data is supported by a pulsed EPR study of the interactions of Mn(H(2)O)(6)(2+) with the P-deuterated analogue of methyl phosphite, CH(3)OP((2)H)(O)(2)(-). The electron to (31)P and (2)H nuclear scalar coupling constants were found to be 4.6 and 0.10 MHz, respectively. In contrast, the effects of paramagnetic ions on the methoxy and ethoxy (1)H resonances of MeOPH and MEP are weak, and the evidence suggests that relaxation of these nuclei occurs by a dipolar mechanism. The wide variation in the relaxation sensitivities of the (1)H and (31)P nuclei of MeOPH and MEP permits us to study how differences in the strengths of the interactions between an observed nucleus and a paramagnetic center affect NMR T(2) relaxations. We propose that these anion ligand probes may be used to study ligand-exchange reactivities of manganese complexes without requiring variable temperature studies. The (31)P T(2) is determined by chemical association kinetics when the following condition is met: (T(2M,P)/T(2M,H))(Deltanu(P)/Deltanu(HP) - 1) < 0.2 where T(2M,P) and T(2M,H) are the transverse relaxation times of the (31)P and (1)H nuclei when the probe is bound to the metal, and Deltanu(P) and Deltanu(HP) are the paramagnetic line broadenings of the (31)P and (1)H-P nuclei, respectively. We assert that the ratio T(2M,P)/T(2M,H) can be estimated for a general metal complex using the results of EPR and NMR experiments.

Accumulation of a polyisoprene-linked amino sugar in polymyxin-resistant Salmonella typhimurium and Escherichia coli: structural characterization and transfer to lipid A in the periplasm.

Polymyxin-resistant mutants of Escherichia coli and Salmonella typhimurium accumulate a novel minor lipid that can donate 4-amino-4-deoxy-l-arabinose units (l-Ara4N) to lipid A. We now report the purification of this lipid from a pss(-) pmrA(C) mutant of E. coli and assign its structure as undecaprenyl phosphate-alpha-l-Ara4N. Approximately 0.2 mg of homogeneous material was isolated from an 8-liter culture by solvent extraction, followed by chromatography on DEAE-cellulose, C18 reverse phase resin, and silicic acid. Matrix-assisted laser desorption ionization/time of flight mass spectrometry in the negative mode yielded a single species [M - H](-) at m/z 977.5, consistent with undecaprenyl phosphate-alpha-l-Ara4N (M(r) = 978.41). (31)P NMR spectroscopy showed a single phosphorus atom at -0.44 ppm characteristic of a phosphodiester linkage. Selective inverse decoupling difference spectroscopy demonstrated that the undecaprenyl phosphate group is attached to the anomeric carbon of the l-Ara4N unit. One- and two-dimensional (1)H NMR studies confirmed the presence of a polyisoprene chain and a sugar moiety with chemical shifts and coupling constants expected for an equatorially substituted arabinopyranoside. Heteronuclear multiple-quantum coherence spectroscopy analysis demonstrated that a nitrogen atom is attached to C-4 of the sugar residue. The purified donor supports in vitro conversion of lipid IV(A) to lipid II(A), which is substituted with a single l-Ara4N moiety. The identification of undecaprenyl phosphate-alpha-l-Ara4N implies that l-Ara4N transfer to lipid A occurs in the periplasm of polymyxin-resistant strains, and establishes a new enzymatic pathway by which Gram-negative bacteria acquire antibiotic resistance.

Phosphorus 31 magnetic resonance spectroscopy of perifused human placental villi under varying oxygen concentrations.

OBJECTIVE: Initial phosphorus magnetic resonance spectroscopy observations on the oxygen metabolism of placental villi from normal term pregnancies are described. STUDY DESIGN: Villi were suspended in medium and perifused within a custom-designed 30 mm nuclear magnetic resonance probe in a superconducting vertical nuclear magnetic resonance magnet where pH, temperature, and oxygenation were monitored. RESULTS: Phosphorus resonances were observed from adenosine triphosphate, phosphomonoesters. inorganic phosphate, and phosphodiesters. No phosphocreatine signal was observed. The placental villus tissue responded to an increase in oxygen concentration of the perifusate with a rise in the adenosine triphosphate level and a concomitant decline in the inorganic phosphate and the phosphomonoester signals. CONCLUSION: The changes observed reflect continuing dynamic glycolysis and oxidative phosphorylation. The absence of a phosphocreatine peak suggests that aerobic pathways not driven by creatine kinase are important for placental metabolism. Our system demonstrates dynamic oxygen metabolism in perifused viable placental villus tissue by means of magnetic resonance spectroscopy.

Comparative analysis of normal and growth-retarded placentas with phosphorus nuclear magnetic resonance spectroscopy.

OBJECTIVE: Phosphorus 31 magnetic resonance spectroscopy studies were carried out on placentas from normal vaginal and elective cesarean deliveries without antenatal complications and from pregnancies complicated by intrauterine growth retardation of unknown cause to determine differences. STUDY DESIGN: Perchloric acid extraction was performed on frozen tissue, and quantitative analysis was carried out for well-resolved resonances representing adenosine triphosphate, sugar phosphate, inorganic phosphorus, diphosphoglycerate, glycerophosphorylethanolamine, and glycerophosphorylcholine. RESULTS: Adenosine triphosphate levels were highest in the growth-retarded group. There were significantly higher levels of sugar phosphate, diphosphoglycerate, and glycerophosphorylcholine in the placentas of the growth-retarded pregnancies compared with those from normal placentas. CONCLUSION: These differences may represent a response to hypoxia and an increase in the amount of blood in the placenta. The results demonstrate the utility of nuclear magnetic resonance spectroscopy for studying the pathology of abnormal placentas to gain a better understanding of the pathology and represent early steps toward in vivo spectroscopic studies of the placenta.

Phosphorus 31 magnetic resonance spectroscopy of human placenta and quantitation with perchloric acid extracts.

Phosphorus 31 magnetic resonance spectroscopic studies of fresh placental tissue are reported that indicate resonances for adenosine triphosphate, inorganic phosphate, sugar phosphates-phosphomonoesters, and phosphodiesters. Perchloric acid extract methods were used to further characterize and quantitate phosphorous metabolites in term human placentas by phosphorus 31 magnetic resonance spectroscopy. The perchloric acid extracts give enhanced resolution of phosphorus signals and allow identification of other phosphorus metabolites including small amounts of phosphocreatine. Emphasis was placed on quantitating adenosine triphosphate levels in the acid extracts with the use of the external reference standard hexachlorocyclotriphosphazene in a coaxial capillary system. Adenosine triphosphate levels measured in this way ranged from 0.404 to 0.709 mumol per gram wet weight. Comparison with an internal standard method with phosphocreatine is also reported. Contribution to the measured high-energy phosphate pool from blood in the highly vascularized tissue was found to be relatively large and could range from 30% to 50% of the total adenosine triphosphate measured.