31P NMR Probe Research Articles

A Novel Strategy for the Characterization of Self-Assembled Structures Using the Static Solid-State Phosphorus Nuclear Magnetic Resonance Technique.

Structural characterization of assemblies in solutions is essential for understanding the relationship between the structure and material properties. In this study, we introduce a novel approach to investigate amphiphilic self-assemblies in solutions using the phospholipid molecule 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (Lyso PC) as a 31P NMR probe. The high natural abundance and gyromagnetic ratio of 31P make it one of the most sensitive nuclei in the low-frequency region, enabling efficient detection even in dilute solutions. Lyso PC can readily co-assemble with amphiphilic molecules and ions in aqueous solutions, forming various structures, such as hexagonal, lamellar, and micellar assemblies. The characteristic line shapes of these assemblies reflect the chemical environment around the probe and provide insights into the different phase states of the assemblies. This strategy offers a simple, cost-effective, and static method for obtaining structural information about various assemblies. Our work not only introduces a sensitive probe for characterizing assemblies in a solvent environment but also inspires new ideas for the development of similar spectroscopic probes.

1,3,5-Triaza-7-Phosphaadamantane (PTA) as a 31P NMR Probe for Organometallic Transition Metal Complexes in Solution

Due to the rigid structure of 1,3,5-triaza-7-phosphaadamantane (PTA), its 31P chemical shift solely depends on non-covalent interactions in which the molecule is involved. The maximum range of change caused by the most common of these, hydrogen bonding, is only 6 ppm, because the active site is one of the PTA nitrogen atoms. In contrast, when the PTA phosphorus atom is coordinated to a metal, the range of change exceeds 100 ppm. This feature can be used to support or reject specific structural models of organometallic transition metal complexes in solution by comparing the experimental and Density Functional Theory (DFT) calculated values of this 31P chemical shift. This approach has been tested on a variety of the metals of groups 8–12 and molecular structures. General recommendations for appropriate basis sets are reported.

Highly Active Silver ion-Exchanged Silicotungstic Acid Catalysts for Selective Esterification of Glycerol with Lauric Acid

A series of highly active silver ion-exchanged silicotungstic acid (H4SiW12O40; STA) catalysts, AgxH4−xSiW12O40 (x = 1–4), were successfully synthesized and applied for the production of glycerol monolaurate (GML) via esterification of lauric acid (LA) with glycerol (GL). The physicochemical properties of these Ag-promoted STA were characterized by TGA, FE-SEM, XRD, and FT-IR, and their acidic properties were studied by a solid-state 31P NMR probe molecule method using trimethylphosphine oxide (TMPO) as the probe. Among various catalysts, the Ag2H2SiW12O40 catalyst showed extraordinary activity owing to the strong Bronsted acidity, Bronsted–Lewis acid synergy, as well as the unique Keggin structure possessed by the catalyst. A Box–Behnken design (BBD) based on response surface methodology (RSM) was exploited for the optimization of process variables. A maximum GML yield of 80.2% was achieved for the Ag2H2SiW12O40 catalyst under a GL/LA molar ratio of 4.4, catalyst amount of 4.6 wt%, reaction temperature of 429 K, and reaction time of 153 min, which was in conformity with those predicted by the mathematical model. Further recyclability and kinetic studies confirmed that the Ag2H2SiW12O40 catalyst was reusable, robust, and highly efficient for the synthesis of GML with an activation energy (Ea) of 38.14 kJ/mol. Due to the strong acidity and synergy of B/L acid sites, Ag ion-exchanged silicotungstic acid catalysts show superior activity and durability for the synthesis of glycerol monolaurate.

NMR Quantification of the Effects of Ligands and Counterions on Lewis Acid Catalysis.

The relative Lewis acidity of a variety of metal-ligand catalyst complexes is quantified using 31P NMR spectroscopy. Three 31P NMR probes, including two new bidentate binding probes, are compared on the basis of different binding modes (i.e., monodentate vs bidentate) and the relative scale of their downfield shift upon binding to Lewis acid complexes. Bidentate coordination of catalyst complexes including metal catalysts, ligands, and counterions were assessed due to their importance to asymmetric catalysis. The effect of ligands, counterions, and additives on Lewis acidity is quantified and correlated to reaction yield at an early time point as an approximation for catalytic activity/efficiency and chelation mode in two organic transformations. Binding studies were performed under catalytically relevant conditions, giving further applicability to synthesis. Insight into activation modes are revealed through this analysis.

Selective catalytic synthesis of glycerol monolaurate over silica gel-based sulfonic acid functionalized ionic liquid catalysts

A series of silica gel supported sulfonic acid functionalized ionic liquid (SAFIL/SG) catalysts have been successfully synthesized through a sol–gel method by varying the organic amine loadings. The catalysts so fabricated were characterized by various physicochemical techniques such as FT-IR, XRD, TGA, FE-SEM, and solid-state 31P NMR probe molecule method and their catalytic performances during esterification of glycerol (GL) with lauric acid (LA) were assessed. The 20% N,N-dimethyl(benzyl)ammonium propyl sulfobetaine hydrosulfate embedded silica gel catalyst (20%[DMBPSH]HSO4/SG) was found to exhibit superior performance and durability during this eco-friendly synthesis of glycerol monolaurate (GML) owing to the high surface area and strong Brønsted acidity observed for the catalyst. Moreover, an experimental design was exploited via response surface methodology (RSM) for the optimization of process variables. Accordingly, a maximum GML yield of 83.9% was achieved for the 20%[DMBPSH]HSO4/SG catalyst under the optimal conditions: amount of catalyst 2.0 wt%, GL/LA molar ratio of 4.1, reaction temperature 418 K, and reaction time 45 min, which was in conformity with those predicted by a mathematical model. Further kinetic studies confirmed that the esterification of GL with LA to GML with an activation energy of 31.51 kJ/mol.

31P NMR Spectroscopically Quantified Hydrogen‐Bonding Strength of Thioureas and Their Catalytic Activity in Diels–Alder Reactions

AbstractThe hydrogen‐bonding strength of a variety of commonly employed thiourea catalysts was quantified by using a trialkylphosphine oxide as a 31P NMR probe. Simple diarylthioureas and more complex bifunctional amine‐ and hydroxy‐substituted thiourea derivatives were examined. Their catalytic activity was determined in a Diels–Alder reaction, and the obtained pseudo‐first‐order rate constants were correlated with the 31P NMR chemical shifts. A linear correlation between both variables was observed throughout the functionalized thioureas. The 31P NMR probe correlation fared better in comparison to a pKa correlation. Accordingly, the quantification presented herein by using a 31P NMR probe offers an elegant way to estimate the catalytic activity of thiourea catalysts in hydrogen‐bond‐activated reactions such as the Diels–Alder reaction.

Solid-state NMR characterization of the acid sites in cubic mesoporous Al-MCM-48 materials using trimethylphosphine oxide as a 31P NMR probe

The acidic properties of Al-MCM-48 with Si/Al ratios ranging from 10 to 67, synthesized with Gemini surfactant as the template, have been characterized by a combination of multinuclear solid-state 1H, 23Na, 27Al, 29Si and 31P MAS (magic angle spinning) NMR and some double-resonance NMR methods using trimethylphosphine oxide (TMPO) as a probe molecule. XRD and 27Al MAS NMR results indicated that aluminum has been successfully incorporated into the framework of MCM-48 materials up to Si/Al = 17.9 by direct synthesis. 1H and 31P MAS NMR results strongly supported the generation of Brønsted acid sites in the cubic MCM-48 mesoporous material after the incorporation of aluminum, even without ion-exchange treatment. Double-resonance NMR techniques such as 31P/ 27Al TRAPDOR (Transfer Population in Double Resonance) and 29Si/ 31P REDOR (Rotational Echo in Double Resonance) NMR were performed to further correlate the TMPO probe molecule to the Brønsted acid sites in the silica framework. 31P/ 27Al TRAPDOR NMR experiments performed at different temperatures were able to establish the correlation between 31P and 27Al spins, further confirms the presence of Brønsted acid sites at 65 ppm in the 31P MAS NMR spectrum. Although the assignment of the Lewis acid sites was somehow unambiguous with 31P/ 27Al TRAPDOR NMR, the FT-IR observation of the calcined samples adsorbed with pyridine did reveal the presence of Lewis acid sites. In contrast to the pore size constraints of zeolites, 29Si/ 31P REDOR NMR results indicated that the protonated TMPO was highly mobile inside the mesoporous channels of Al-MCM-48 at the NMR time scale.

Synthesis and 31P NMR characterization of new low toxic highly sensitive pH probes designed for in vivo acidic pH studies

With the aim to provide sensitive 31P NMR probes of intra- and extracellular pH gradients that may reach cellular acidic compartments in biological systems, new α-aminophosphonates were designed to meet basic requirements such as a low pKas and a great chemical difference (Δδab) between the limiting 31P NMR chemical shifts in acidic (δa) and basic (δb) media. A series of six phosphorylated pyrrolidines and linear aminophosphonates were synthesized using aminophosphorylation reactions and were screened for cytotoxicity on cultured Müller cells. Among the compounds not being toxic under these conditions, three molecules were selected since they displayed the best in vitro (in several phosphate buffers and in a cytosol-like solution) properties as 31P NMR acidic pH markers, that is 3, 5 and 9, having the pKa values of 3.63, 5.89 and 5.66, respectively. The Δδab values of these pH markers were at least 3 times larger than that of standard 31P NMR probes, with a low sensitivity to ionic strength changes. From these data, it was proposed that 3, 5 and 9 could be used as reporting probes of subtle proton movements in acidic compartments, an area that still remains poorly investigated using non invasive 31P NMR methods.

A perifusion loop-gap resonator NMR probe for aerobic cell suspensions.

NMR studies of aerobic cell metabolism require that spectral data of high sensitivity and resolution be acquired from samples maintained under suitable conditions. The loop-gap resonator 31P NMR probe described here was designed for investigations of highly aerobic-dependent renal proximal tubules in an environment similar to those of conventional incubation flasks. The inherently higher sensitivity of the loop-gap resonator design and associated circuitry made it possible to obtain adequate spectra in short periods of time with small amounts of sample. Cellular physiological properties of perifused aerobic renal proximal tubule cell suspensions in the loop-gap resonator probe were found to be similar to equivalent tissue samples incubated in conventional flasks. Furthermore, the loop-gap perifusion probe was found to provide useful 31P NMR spectra of the kidney tubule system after acquisition times as short as 3 min.

Detection of phosphorus metabolites in human prostates with a transrectal 31P NMR probe

Detection of phosphorus metabolites in human prostates with a transrectal 31P NMR probe

NOTPME: A 31P NMR probe for measurementof divalent cations in biological systems

1, 4, 7-Triazacyclononame- N, N′,N′-tris(methylenephosphonate monoethylester) (NOTPME) has been synthesized, characterized and analyzed for use as a 31P NMR indicator of intracellular Mg 2+ and Zn 2+ ions. The 31P NMR spectrum of this chelate in the presence of metal ions shows characteristics resonances for the free chelate, Mg(NOTPME) −, Zn(NOTPME) − and Ca(NOTPME) −. The K ??? values indicate that this chelate has a 10-fold higher affinity for Mg 2+ than for Ca 2+ at physiological pH values. In the presence of Mg 2+, NOTPME is readily loaded into red blood cells. A 31P NMR spectrum of red cells taken after several washing shows resonances characteristics of entrapped NOTPME and the Mg(NOTPME) − complex, the relative areas of which report an intracellular free Mg 2+ concentration of 0.32 mM. The 31P chemical shifts of the free chelate and its metal complexes are far downfield from the typical phosphorus-containing metabolites observed in biological systems, thus making it possible to monitor intracellular cation concentration and cell energetics simultaneously.

Methyl phosphonate as a 31P-NMR probe for intracellular pH measurements in Dictyostelium amoebae

Methyl phosphonate at concentrations up to 20 mM was found to be non toxic towards the growth of Dictyostelium amoebae and the starvation-induced differentiation program. In contrast, phenyl phosphonate at the same concentrations was found to inhibit growth. Methyl phosphonate possessed good 31P-NMR characteristics for use as a pH probe in Dictyostelium. The entry of methyl phosphonate into the cytoplasm required about 150 min of incubation before an equilibrium was reached with added extracellular methyl phosphonate. A semilogarithmic plot of the efflux of methyl phosphonate out of the amoebae was linear as a function of time, and the kinetic first-order constant was K = 0.016 min −1. The kinetic parameters were consistent with an uptake of methyl phosphonate by fluid-phase pinocytosis. The pH calculated from the chemical shift of the internalized methyl phosphonate signal was found to be pH 7.4 in aerobic Dictyostelium amoebae. The intracellular methyl phosphonate environment turned more acidic in response to anaerobiosis ( ΔpH = 0.8) or in the presence of a weak acid such as propionate. These pH determinations were in agreement with the values of cytosolic pH derived from the chemical shift of Pi. Methyl phosphonate should be a useful probe for measurements in 31P-NMR studies of Dictyostelium in situations where the signal of Pi cannot be attributed with certainty.

Further investigation of the use of dimethyl methylphosphonate as a 31P-NMR probe of red cell volume

We have refined a method for measuring erythrocyte volume using the 31P-NMR spectrum of a probe molecule, dimethyl methylphosphonate. This compound, when added to an erythrocyte suspension, gives rise to two 31P-NMR resonances, and the frequency separation between them is linearly dependent on the intracellular haemoglobin concentration. If, for a given cell sample (under standard conditions), the separation of the two dimethyl methylphosphonate peaks has been measured and an independent estimation of the mean cell haemoglobin content and concentration has been obtained, then changes in the mean cell volume due to altered experimental conditions may be estimated from the peak separation measured under the new conditions. Although the peak separation was independent of extracellular pH, it did vary with (i) a range of extracellular suspension media, (ii) temperature, (iii) dimethyl methylphosphonate concentration, (iv) haemoglobin ligand state and (v) different blood donors.

Alkaline phosphatase. 31P NMR probes of the mechanism.

31P NMR signals from substrates and products of alkaline phosphatase have been adapted to measure the rates and product ratios for the hydrolysis and phosphotransferase reactions from pH 6 to 10. Below pH 8, glycerol is a poorer acceptor than H2O (glycerol phosphates:Pi = 0.5). Tris is a more effective acceptor below pH 8, showing a maximum acceptor efficiency at pH 8 (Tris phosphate:Pi = 2). Phosphotransferase efficiencies are in the order expected for the pKaS of the alcohol groups, Tris less than glycerol Cl, C3 less than glycerol C2. Tris and glycerol induce chemical shifts in 113Cd(II) present at the A site but not the B or C sites of the metal triad present at each active center of Cd(II)6 alkaline phosphatase, suggesting that the alcoxides of the acceptors coordinate the A site metal and become the nucleophiles attacking the phosphoseryl residue (E-P) in the second step of the mechanism. The interaction is through the oxygen of Tris. The transferase activity of the amino alcohol shows a bell-shaped pH dependency. Aliphatic alcohol acceptors show small increases in acceptor activity between pH 6 and 8, with 5-fold increases from pH 8 to 10 (at pH 10, glycerol phosphates:Pi = 2.5). 31P NMR inversion transfer has been used to measure the koff for Pi dissociation from the noncovalent enzyme complex (E . P). For the Zn(II)4 alkaline phosphatase koff is essentially pH independent at approximately 35 s-1. For Cd(II) or Mg(II) at the B site in place of Zn(II), koff less than or equal to 1 s-1 X Cl-ion, which appears to coordinate the A site metal ion, enhances koff, suggesting that both Cl- and HPO2-4 can coordinate the A site metal ion in a 5-coordinate intermediate. pH control of the alkaline phosphatase mechanism appears to reside in the stability of E-P and not the dissociation of E . P, compatible with the hypothesis that the activity-linked pKa is that of a H2O molecule coordinated to the A site metal, which in the hydroxide form becomes the nucleophile attacking the phosphoseryl group (E-P).

Interaction of hexane phosphonic acid diethyl ester with phospholipids in hepatic microsomes and reconstituted liposomes as studied by 31P-NMR

By use of 31P-NMR, quasi-elastic light scattering and freeze-fracture electron microscopy it is shown that hexane phosphonic acid diethyl ester (PAE) is incorporated in hepatic microsomes without any alteration of the bilayer structure at two different sites. These findings proved that PAE can be used as molecular 31P-NMR probe in microsomes to get information about lipid-protein interactions. Extensive studies on reconstituted liposomal systems which contained cytochrome P-450 and cytochrome P-450 reductase showed that both proteins influence the localization of incorporated PAE. The results indicate a specific interaction of phosphatidylethanolamine (PE) with cytochrome P-450 in microsomes.

Utilizing an intrinsic 31P NMR probe in studies of cobalamin ligation

Phosphorous 31 nuclear magnetic resonance spectroscopy has been employed to observe changes in axial ligation in hydroxycobalamin solutions. The naturally occurring phosphorous nucleus in the benzimidazole side “arm” of the corrin is shown to exhibit different chemical shifts depending upon whether the benzimidazole is coordinated to the cobalt (III) ion or whether it has been displaced. The phosphorous resonance's linewidth at half maximum peak height seems also to be an indicator of the cobalt oxidation state, exhibiting a twenty fold larger width in the coblt (II) complex. The implications of these findings for future studies on certain coenzyme B 12 dependent enzymes is discussed.

The metabolism of the phosphonium analogue of choline in vitro and in vivo, and its detection in phospholipids by 31P-NMR

1. 1. The phosphonium analogues of choline, phosphorylcholine, CDPcholine and phosphatidylcholine were synthesized chemically and characterized by 1 H- NMR and 31P-NMR; in 1,2-distearoyl- dl-glycero-3-phosphorylphosphocholine, the 31P-NMR chemical shift of phosphonium relative to phosphate was −28.2 ppm. 2. 2. A comparison was made of the rates of reaction of choline kinase, cholinephosphate cytidyltransferase, cholinephosphotransferase and phospholipase C on natural and phosphonium substrates. Enzyme reaction rates were similar for all but the cytidyltransferase, which exhibited a 3-fold preference for the normal substrate. 3. 3. Weanling rats were maintained for 6 weeks on a diet in which choline was fully replaced by phospho[1,2- 14C 2] choline mixed with a trace of [Me- 3H] choline. Incorporation of phosphocholine into liver lipids was detectable by 31P-NMR even in crude tissue homogenates. Choline-based phospholipids of liver, kidney, lung and brain were extracted, and phosphocholine incorporation calculated from 31P-NMR peak area ratios. The phosphatidylcholine analogues were separated by preparative thin-layer chromatography. Incorporation of phosphocholine ranged from 33% in lung phosphatidylcholine to 6% in kidney sphingomyelin. Variations in 14C/ 3H ratio between feed and phospholipid extracts indicated preferences for exogenous choline over phosphocholine varying from 1.3:1 in brain to 3.2:1 in liver. The results indicated that phosphocholine is a potentially useful 31P-NMR probe for the study of membrane lipids.