Revealing the mechanism of action of a first-in-class covalent inhibitor of KRASG12C (ON) and other functional properties of oncogenic KRAS by 31P NMR.

Individual oncogenic KRAS mutants confer distinct differences in biochemical properties and signaling for reasons that are not well understood. KRAS activity is closely coupled to protein dynamics and is regulated through two interconverting conformations: state 1 (inactive, effector binding deficient) and state 2 (active, effector binding enabled). Here, we use 31P NMR to delineate the differences in state 1 and state 2 populations present in WT and common KRAS oncogenic mutants (G12C, G12D, G12V, G13D, and Q61L) bound to its natural substrate GTP or a commonly used nonhydrolyzable analog GppNHp (guanosine-5'-[(ß,γ)-imido] triphosphate). Our results show that GppNHp-bound proteins exhibit significant state 1 population, whereas GTP-bound KRAS is primarily (90% or more) in state 2 conformation. This observation suggests that the predominance of state 1 shown here and in other studies is related to GppNHp and is most likely nonexistent in cells. We characterize the impact of this differential conformational equilibrium of oncogenic KRAS on RAF1 kinase effector RAS-binding domain and intrinsic hydrolysis. Through a KRAS G12C drug discovery, we have identified a novel small-molecule inhibitor, BBO-8956, which is effective against both GDP- and GTP-bound KRAS G12C. We show that binding of this inhibitor significantly perturbs state 1-state 2 equilibrium and induces an inactive state 1 conformation in GTP-bound KRAS G12C. In the presence of BBO-8956, RAF1-RAS-binding domain is unable to induce a signaling competent state 2 conformation within the ternary complex, demonstrating the mechanism of action for this novel and active-conformation inhibitor.

Role of isotropic lipid phase in the fusion of photosystem II membranes.

It has been thoroughly documented, by using 31P-NMR spectroscopy, that plant thylakoid membranes (TMs), in addition to the bilayer (or lamellar, L) phase, contain at least two isotropic (I) lipid phases and an inverted hexagonal (HII) phase. However, our knowledge concerning the structural and functional roles of the non-bilayer phases is still rudimentary. The objective of the present study is to elucidate the origin of I phases which have been hypothesized to arise, in part, from the fusion of TMs (Garab et al. 2022 Progr Lipid Res 101,163). We take advantage of the selectivity of wheat germ lipase (WGL) in eliminating the I phases of TMs (Dlouhý et al. 2022 Cells 11: 2681), and the tendency of the so-called BBY particles, stacked photosystem II (PSII) enriched membrane pairs of 300-500 nm in diameter, to form large laterally fused sheets (Dunahay et al. 1984 BBA 764: 179). Our 31P-NMR spectroscopy data show that BBY membranes contain L and I phases. Similar to TMs, WGL selectively eliminated the I phases, which at the same time exerted no effect on the molecular organization and functional activity of PSII membranes. As revealed by sucrose-density centrifugation, magnetic linear dichroism spectroscopy and scanning electron microscopy, WGL disassembled the large laterally fused sheets. These data provide direct experimental evidence on the involvement of I phase(s) in the fusion of stacked PSII membrane pairs, and strongly suggest the role of non-bilayer lipids in the self-assembly of the TM system.

Combined nuclear magnetic resonance methods in quality control of lubricants in green energy production.

NMR methods were applied for lubricant analysis. Different factors influence the real aging of lubricants on diverse length scales and are captured by NMR. Chemical conversion of additives is addressed by NMR spectroscopy. High-field NMR experiments allow the identification and quantification of chemical components and are transferred to benchtop devices. Molecular dynamics and contaminations like fuel or abrasion are addressed via NMR relaxation and diffusion. Quality parameters were extracted via suitable data analysis of NMR raw data, which allow the detection of aging and indicate changes in the oil composition. At the same time, the methodology is optimized to the conditions in quality control. The feasibility is shown the example of a series of lubricants from applications in regenerative energy production, namely, wind turbine oils and biogas motor oils.

Quantitative 31P-NMR for the Purity Determination of the Organophosphorus Compound Brigatinib and Its Method Validation.

The spectrum of 31P-NMR is fundamentally simpler than that of 1H-NMR; consequently identifying the target signal(s) for quantitation is simpler using quantitative 31P-NMR (31P-qNMR) than using quantitative 1H-NMR (1H-qNMR), which has been already established as an absolute determination method. We have previously reported a 31P-qNMR method for the absolute determination of cyclophosphamide hydrate and sofosbuvir as water-soluble and water-insoluble organophosphorus compounds, respectively. This study introduces the purity determination of brigatinib (BR), an organophosphorus compound with limited water solubility, using 31P-qNMR at multiple laboratories. Phosphonoacetic acid (PAA) and 1,4-BTMSB-d4 were selected as the reference standards (RSs) for 31P-qNMR and 1H-qNMR, respectively. The qNMR solvents were chosen based on the solubilities of BR and the RSs for qNMR. CD3OH was selected as the solvent for 31P-qNMR measurements to prevent the influence of deuterium exchange caused by the presence of exchangeable intramolecular protons of BR and PAA on the quantitative values, while CD3OD was the solvent of choice for the 1H-qNMR measurements to prevent the influence of water signals and the exchangeable intramolecular protons of BR and PAA. The mean purity of BR determined by 31P-qNMR was 97.94 ± 0.69%, which was in agreement with that determined by 1H-qNMR (97.26 ± 0.71%), thus indicating the feasibility of purity determination of BR by 31P-qNMR. Therefore, the findings of this study may provide an effective method that is simpler than conventional 1H-qNMR for the determination of organophosphorus compounds.

Phosphorus speciation in different sewage sludges and their biochars and its implications for movement of labile phosphate in two soils.

It is essential to understand the P dynamics of recycled biomaterials, like biochar derived from sewage sludge, especially with potential application as fertilizers. The objective of this study was to understand how pyrolysis affects the speciation of P in sewage sludge and thereby the effect on labile P pools and mobility of P in soil. The P speciation and lability of two sewage sludges (one biologically treated and one iron-precipitated) and their biochars (pyrolyzed at 400 °C and 600 °C) were determined by liquid state 31P nuclear magnetic resonance spectroscopy, X-ray absorption near edge spectroscopy, and sequential chemical extraction. These biomaterials were applied in a concentrated band to two soils, and P lability was studied in the adjacent soil at varying distances. Speciation techniques showed P was more closely associated with Ca and Fe for the iron-precipitated sludge and its biochars than the biologically treated sludge and its biochars. Instead, the P in the biologically treated biochars was found to be largely (40% or more) in polymeric forms (pyro- or poly-phosphates). The relationship between the speciation and the mobility of P in soil (as assessed by incubating biomaterials in a one-dimensional reaction system) was more evident when incubating the sewage sludges than the respective biochars. Particularly, the biologically treated sludge had a high proportion of labile P (56% water-extractable P), as determined by sequential extraction, and upon incubation, it was also the only material where water-extractable P remained significantly above the control soil level up to 3 mm from the biomaterial layer. After pyrolysis, this lability decreased significantly (up to a 25-fold decrease in water-extractable P), and this was reflected in the immobility of P in the biochars during incubation in the two soils. Differences in speciation between biochars were not reflected in the incubation experiment, as the differences in P release and mobility were not significant.

Biochar application regulates organic phosphorus fractions and the release of available phosphorus in farmland soil.

BACKGROUND: The relationship between phosphorus (P) related enzymatic activity and organic P turnover remains unclear, particularly in the context of biochar application. Field experiments were conducted on Phaeozem and Luvisol soil types to investigate the effects of biochar application rates - 0 t ha-1 (CK), 22.5 t ha-1 (D1), 67.5 t ha-1 (D2), and 112.5 t ha-1 (D3) - on soil organic fractions using 31P nuclear magnetic resonance (NMR) spectroscopy and relevant phosphatase activity. RESULTS: The application of biochar increased the soil organic carbon (SOC), pyrophosphate (pyro), and orthophosphate (ortho) content, as well as the acid phosphomonoesterase (AcP), alkaline phosphomonoesterase (AlP), inorganic pyrophosphatase (IPP), and phosphodiesterase (PD) activities. Biochar application also increased soil organic P (OPa), the sum of inorganic P forms (IP), ortho, monoesters, and myo-IHP contents, the pH value, AlP and PD activities in Phaeozem, but it significantly reduced diesters, polyphosphate (poly) contents, and IPP and AcP activities compared to those in Luvisol. Acid phosphomonoesterase and PD activities also showed an opposite trend in Luvisol. The structural equation model showed that the potential mechanism of organic P turnover in response to biochar application differed depending on the soil types, potentially influenced by P availability. CONCLUSION: Overall, the findings of this study enhance the comprehension of the variation of P fractions and their availability in the context of biochar application for agricultural production in northeastern China. © 2024 Society of Chemical Industry.

Variations of sediment organic phosphorus and organic carbon during the outbreak and decline of algal blooms in Lake Taihu, China.

In this study, sediment organic phosphorus (OP) and organic carbon (OC) in Lake Taihu, China, as well as their relationships, were analyzed during the outbreak and decline of algal blooms (ABs) over a five-month field study. The results showed synchronous temporal changes in the sediment OP and OC contents with the development of ABs. In addition, there was a significant positive correlation between the sediment OP and OC (p < 0.01), suggesting simultaneous deposition and consumption during the ABs outbreak. The sediment OP and OC contents decreased significantly at the early and last stages of the ABs outbreak and increased at the peak of the ABs outbreak and during the ABs decline. These temporal variation patterns suggest that the sediment OC and OP contents did not consistently increase during the ABs outbreak, even though algae are an important source of organic matter in sediments. The depletion or enrichment of OC and OP in sediments may also depend on the scale of the ABs outbreak. The obtained results revealed significant differences in the sediment OC and OP contents between the months (p < 0.05). In addition, OP in the sediments was dominated by orthophosphate diester (phospholipids and DNA-P) and orthophosphate monoester during the ABs outbreak and decline, respectively. The active OC contents and proportions in the sediments in the ABs outbreak were significantly lower than those observed in the ABs decline period, demonstrating the significant impacts of the ABs outbreak and decline on the sediment OC and OP in Lake Taihu.

New Biocompatible ß-Phosphorylated Linear Nitrones Targeting Mitochondria: Protective Effect in Apoptotic Cells.

The mitochondrion, an essential organelle involved in cellular respiration, energy production, and cell death, is the main cellular source of reactive oxygen species (ROS), including superoxide. Mitochondrial diseases resulting from uncontrolled/excess ROS generation are an emerging public health concern and there is current interest in specific mitochondriotropic probes to get information on in-situ ROS production. As such, nitrones vectorized by the triphenylphosphonium (TPP) cation have recently drawn attention despite reported cytotoxicity. Herein, we describe the synthesis of 13 low-toxic derivatives of N-benzylidene-1-diethoxyphosphoryl-1-methylethylamine N-oxide (PPN) alkyl chain-grafted to a pyridinium, triethylammonium or berberinium lipophilic cation. These nitrones showed in-vitro superoxide quenching activity and EPR/spin-trapping efficiency towards biologically relevant free radicals, including superoxide and hydroxyl radicals. Their mitochondrial penetration was confirmed by 31 P NMR spectroscopy, and their anti-apoptotic properties were assessed in Schwann cells treated with hydrogen peroxide. Two pyridinium-substituted PPNs were identified as potentially better alternatives to TPP nitrones conjugates for studying mitochondrial oxidative damage.

Perfusion system for studying dynamic metabolomics in rat brain slices exposed to oxygen-glucose deprivation using proton and phosphorus nuclear magnetic resonance.

The aim of the current study was to establish a controlled and reproducible model to study metabolic changes during oxygen-glucose deprivation (OGD) in rat brain using a nuclear magnetic resonance (NMR)-compatible perfusion system. Rat brains were cut into 400-µm thick slices and perfused with artificial cerebrospinal fluid (aCSF) in a 10-mm NMR tube inside a 600-MHz NMR spectrometer. Four experimental conditions were tested: (1) continuous perfusion with aCSF with glucose and normoxia, and (2) 30-, (3) 60-, or (4) 120-min periods of OGD followed by reperfusion of aCSF containing glucose and normoxia. The energetic state of perfused brain slices was measured using phosphorus (31 P) NMR and metabolite changes were measured using proton (1 H) NMR. aCSF samples were collected every 30 min and analyzed using 1 H NMR. The sample temperature was maintained at 36.7 ± 0.1°C and was checked periodically throughout the experiments. Brain slice histology was compared before and after OGD in the perfusion system using hematoxylin-eosin-saffron staining. NMR data clearly distinguished three severity groups (mild, moderate, and severe) after 30, 60, and 120 min of OGD, respectively, compared with the control group. 31 P NMR spectra obtained from controls showed that phosphocreatine levels were stable for 5 h inside the perfusion system. Control 1 H NMR spectra showed that lactate, N-acetylaspartic acid, glutamate, γ-aminobutyric acid, and creatine metabolite levels were stable over time, with lactate levels having a tendency to gradually increase due to the recirculation of the aCSF in the perfusion system. A controlled and reproducible perfusion system was established to study the energetic and metabolic changes in rat brain slices during and after OGD of varying severity.

C- and N-Phosphorylated Enamines-An Avenue to Heterocycles: NMR Spectroscopy.

The review presents extensive data (from the works of the author and literature) on the structure of C- and N-chlorophosphorylated enamines and the related heterocycles obtained by multipulse multinuclear 1H, 13C, and 31P NMR spectroscopy. The use of phosphorus pentachloride as a phosphorylating agent for functional enamines enables the synthesis of various C- and N-phosphorylated products that are heterocyclized to form various promising nitrogen- and phosphorus-containing heterocyclic systems. 31P NMR spectroscopy is the most convenient, reliable and unambiguous method for the study and identification of organophosphorus compounds with different coordination numbers of the phosphorus atom, as well as for the determination of their Z- and E-isomeric forms. An alteration of the coordination number of the phosphorus atom in the phosphorylated compounds from 3 to 6 leads to a drastic screening of the 31P nucleus from about +200 to -300 ppm. The unique structural features of nitrogen-phosphorus-containing heterocyclic compounds are discussed.

Effects of different chemical agents on changes in sediment phosphorus composition and the response of sediment microbial community.

Controlling the release of sediment phosphorus (P) using chemical agents is a promising method for controlling internal P in eutrophic lakes. However, mineral P formation and changes in the organic P composition after sediment amendment with P-inactivation agents remain poorly understood. Furthermore, little is known about the changes in the sediment microbial community composition after remediation. Here, various ratios of poly aluminum chloride (PAC) and lanthanum-modified bentonite (LMB) were added to nutrient-rich sediments and incubated. Sequential P extraction, solution/solid-state 31P nuclear magnetic resonance (NMR), and microbial analyses were periodically performed on the inactivated sediments. The results indicate that PAC and LMB effectively reduced sediment iron-bound P and organic P, respectively, markedly increasing the content of aluminum- and calcium-bound P in the sediment, respectively. Solid-state 31P NMR results confirmed the formation of rhabdophane (LaPO4. nH2O) in the LMB-amended sediment. Solution 31P NMR results showed that PAC preferentially reduced the organic P fractions of pyrophosphate, whereas LMB efficiently reduced the organic P fractions of orthophosphate, monoesters, and diesters in the sediment. Compared with the control sediment, PAC addition can cause short-term negative effects on sediment microbes at high doses, whereas LMB addition can increase bacterial diversity or richness in the sediment. These results provide a deeper understanding of the differences between PAC and LMB in internal sediment P control.

Strategies for the Preparation of Phosphorus Janus Dendrimers and Their Properties.

Dendrimers, being highly branched monodispersed macromolecules, predominantly exhibit identical terminal functionalities within their structural framework. Nonetheless, there are instances where the presence of two distinct surface functionalities becomes advantageous for the fulfilment of specific properties. To achieve this objective, one approach involves implementing Janus dendrimers, consisting of two dendrimeric wedges terminated by dissimilar functionalities. The prevalent method for creating these structures involves the synthesis of dendrons that possess a core functionality that complements that of a second dendron, facilitating their coupling to generate the desired dendrimers. In this comprehensive review, various techniques employed in the fabrication of phosphorus-based Janus dendrimers are elucidated, displaying the different coupling methodologies employed between the two units. The advantages of phosphorus dendrimers over classic dendrimers will be shown, as the presence of at least one phosphorus atom in each generation allows for the easy monitoring of reactions and the confirmation of purity through a simple technique such as 31P NMR, as these structures typically exhibit easily interpretable patterns.

Organophosphorus Azoles Incorporating a Tetra-, Penta-, and Hexacoordinated Phosphorus Atom: NMR Spectroscopy and Quantum Chemistry.

The review presents extensive data (from the author's work and the literature) on the stereochemical structure of functionalized organophosphorus azoles (pyrroles, pyrazoles, imidazoles and benzazoles) and related compounds, using multinuclear 1H, 13C, 31P NMR spectroscopy and quantum chemistry. 31P NMR spectroscopy, combined with high-level quantum-chemical calculations, is the most convenient and reliable approach to studying tetra-, penta-, and hexacoordinated phosphorus atoms of phosphorylated N-vinylazoles and evaluating their Z/E isomerization.

Hydrophyte Debris Induced Sedimentary Phosphorus Release in Tuojiang Rivers, China.

Hydrophyte debris decomposition may contribute to phosphorus (P) release from the sediments in riverine systems, but the transport and transformation of organic phosphorus during this process has not been studied well. Here, a ubiquitous hydrophyte in southern China (Alternanthera philoxeroides, A. philoxeroides) was selected to identify the processes and mechanisms of sedimentary P release in late autumn or early spring by laboratory incubation. The results showed that the physio-chemical interactions changed quickly during the beginning of the incubation, where the redox potential and dissolved oxygen at the water-sediment interface decreased rapidly, reaching reducing (299 mV) and anoxic (0.23 mg∙L-1) conditions, respectively. Soluble reactive P, dissolved total P and total P concentrations in overlying water all increased with time from 0.011, 0.025 and 0.169 mg∙L-1 to 0.100, 0.100 and 0.342 mg∙L-1 on average, respectively. Furthermore, the decomposition of A. philoxeroides induced sedimentary organic P release to overlying water, including phosphate monoester (Mono-P), and orthophosphate diesters (Diesters-P). The proportions of Mono-P and Diesters-P were higher at 3 to 9 days than at 11 to 34 days, being 29.4% and 23.3 for Mono-P, 6.3% and 5.7% for Diesters-P, respectively. Orthophosphate (Ortho-P) increased from 63.6 to 69.7% during these timeframes, which indicated the transformations of both Mono-P and Diester-P to bio-available orthophosphate (Ortho-P), causing the rising P concentration in the overlying water. Our results revealed that hydrophyte debris decomposition in river systems might lead to autochthonous P contribution even without external P import from the watershed, accelerating the trophic state of receiving waterbodies.

Characteristics and distribution of phosphorus in surface sediments of a shallow lake.

Phosphorus (P) in sediments plays an important role in shallow lake ecosystems and has a major effect on the lake environment. The mobility and bioavailability of P primarily depend on the contents of different P forms, which in turn depend on the sedimentary environment. Here, sediment samples from Baiyangdian (BYD) lake were collected and measured by the Standards, Measurements, and Testing procedure and Phosphorus-31 nuclear magnetic resonance spectroscopy (31P NMR) to characterize different P forms and their relationships with sediment physicochemical properties. The P content in the sediments varied in different areas and had characteristics indicative of exogenous river input. Inorganic P (334-916 mg/kg) was the dominant form of P. The 31P NMR results demonstrated that orthophosphate monoesters (16-110 mg/kg), which may be a source of P when redox conditions change, was the dominant form of organic P (20-305 mg/kg). The distribution of P forms in each region varied greatly because of the effects of anthropogenic activities, and the regions affected by exogenous river input had a higher content of P and a higher risk of P release. Principal component analysis indicated that P bound to Fe, Al, and Mn oxides and hydroxides (NaOH-P) and organic P were mainly derived from industrial and agricultural pollution, respectively. Redundancy analysis indicated that increases in pH lead to the release of NaOH-P. Organic matter plays an important role in the organic P biogeochemical cycle, as it acts as a sink and source of organic P.

The Ad-MD method to calculate NMR shift including effects due to conformational dynamics: The 31 P NMR shift in DNA.

A method for averaging of NMR parameters by molecular dynamics (MD) has been derived from the method of statistical averaging in MD snapshots, benchmarked and applied to structurally dynamic interpretation of the 31 P NMR shift (δ31P ) in DNA phosphates. The method employs adiabatic dependence of an NMR parameter on selected geometric parameter(s) that is weighted by MD-calculated probability distribution(s) for the geometric parameter(s) (Ad-MD method). The usage of Ad-MD for polymers is computationally convenient when one pre-calculated structural dependence of an NMR parameter is employed for all chemically equivalent units differing only in dynamic behavior. The Ad-MD method is benchmarked against the statistical averaging method for δ31P in the model phosphates featuring distinctively different structures and dynamic behavior. The applicability of Ad-MD is illustrated by calculating 31 P NMR spectra in the Dickerson-Drew DNA dodecamer. δ31P was calculated with the B3LYP/IGLO-III/PCM(water) and the probability distributions for the torsion angles adjacent to the phosphorus atoms in the DNA phosphates were calculated using the OL15 force field.

Phosphatic metabolism in dark- and light-adapted rat retinas.

This study defines retinal phosphatic metabolites and their adjustment to illumination in rat retinas under conditions that preserve retinal function. Metabolic data are measured using high-performance liquid chromatography (HPLC) and 31P nuclear magnetic resonance (31P NMR) spectroscopy after 10 min of light exposure in vivo compared with retinas from dark-adapted rats. Multiple high-energy and low-energy phosphatic metabolites of intermediary metabolism were quantified. The concentration of the high-energy phosphate adenosine triphosphate (ATP) remained unchanged from dark- to light-adaptation. Under the same conditions the concentrations of the high-energy phosphates guanosine triphosphate (GTP) and creatine phosphate increased, whereas the inorganic phosphate decreased. Comparing dark-adapted controls with retinas light-adapted either in vitro or in vivo, the evidence is consistent with a light-dependent increase in GTP and a decrease in cyclic guanosine monophosphate. Although cyclic adenosine monophosphate (cAMP) levels were lower in retinas light-adapted in vivo than in the dark-adapted controls, this did not seem to be an effect of light, as cAMP levels decreased similarly after 10 min incubation in dark or light in parallel with recovery of ATP/adenosine diphosphate ratios. This study: (1) reports on retinal metabolic changes with adjustment in illumination, (2) provides baseline measurements of retinal phosphatic metabolites in whole retinas, and (3) reports on the validity of chromatographic and spectroscopic methods used for studying retinal metabolism establishing a high correlation among measurements made using HPLC and 31P NMR.

Elucidating structure and dynamics of crystalline α-zirconium phosphates intercalated with water and methanol by multinuclear solid-state MAS NMR: A comprehensive NMR approach.

Solid-state NMR experiments on 2 H, 31 P, 13 C, and 1 H nuclei, including 31 P T1 , 1 H T1 , and 1 H T1ρ measurements, as well as on the kinetics of proton-phosphorus cross-polarization have been performed to characterize the crystalline and amorphous α-zirconium phosphates, which were intercalated with D2 O and/or CD3 OD. The 13 C{1 H} CP MAS NMR experiment performed for compound 1-CD3 OD (Zr (HPO4 )2. 0.2CD3 OD) with carbon cross-polarization via protons of phosphate groups has provided a prove that the methanol was intercalated into the interlayer spaces of this compound. The variable-temperature 2 H solid-echo MAS NMR spectra of intercalated compounds demonstrated that the methanol molecules, in contrast to the mobile water, were immobile, keeping, however, free CD3 rotations around the C3 -axis. It has been demonstrated that the intercalated species, D2 O and CD3 OD, do not affect the high-frequency motions of the phosphate groups. By utilizing local structural models that satisfy the constraints of the experimental data, it has been suggested that the immobile methanol molecules are located in the cavity between two neighboring layers of the zirconium phosphates. Thus, the present work illustrates the reliable criteria in a comprehensive NMR approach to structural and dynamic studies of such systems.

Quantitative 31P-NMR for Purity Determination of Sofosbuvir and Method Validation.

Quantitative 1H-NMR (1H-qNMR) is useful for determining the absolute purity of organic molecules; however, it is sometimes difficult to identify the target signal(s) for quantitation because of their overlap and complexity. Therefore, we focused on the 31P nucleus because of the simplicity of its signals and previously reported 31P-qNMR in D2O. Here we report 31P-qNMR of an organophosphorus compound, sofosbuvir (SOF), which is soluble in organic solvents. Phosphonoacetic acid (PAA) and 1,4-bis(trimethylsilyl)benzene-d4 (1,4-BTMSB-d4) were used as reference standards for 31P-qNMR and 1H-qNMR, respectively, in methanol-d4. The purity of SOF determined by 31P-qNMR was 100.63 ± 0.95%, whereas that determined by 1H-qNMR was 99.07 ± 0.50%. The average half bandwidths of the 31P signal of PAA and SOF were 3.38 ± 2.39 and 2.22 ± 0.19 Hz, respectively, suggesting that the T2 relaxation time of the PAA signal was shorter than that of SOF and varied among test laboratories. This difference most likely arose from the instability in the chemical shift due to the deuterium exchange of the acidic protons of PAA, which decreased the integrated intensity of the PAA signal. Next, an aprotic solvent, dimethyl sulfoxide-d6 (DMSO-d6), was used as the dissolving solvent with PAA and sodium 4,4-dimethyl-4-silapentanesulfonate-d6 (DSS-d6) as reference standards for 31P-qNMR and 1H-qNMR, respectively. SOF purities determined by 31P-qNMR and 1H-qNMR were 99.10 ± 0.30 and 99.44 ± 0.29%, respectively. SOF purities determined by 31P-qNMR agreed with the established 1H-qNMR values, suggesting that an aprotic solvent is preferable for 31P-qNMR because it is unnecessary to consider the effect of deuterium exchange.

5'-Chalcogen-Substituted Nucleoside Pyrophosphate and Phosphate Monoester Analogues: Preparation and Hydrolysis Studies.

Novel sulfur and selenium substituted 5',5'-linked dinucleoside pyrophate analogues were prepared in a vibration ball mill from the corresponding persilylated monophosphate. The chemical hydrolysis of pyrophosphorochalcogenolate-linked dimers was studied over a wide pH-range. The effect of the chalcogeno-substitution on the reactivity of dinucleoside pyrophosphates was surprisingly modest, and the chemical stability is promising considering the potential therapeutic or diagnostic applications. The chemical stability of the precursor phosphorochalcogenolate monoesters was also investigated. Hydrolytic desilylation of these materials was effected in aqueous buffer at pH 3, 7 or 11 and resulted in phosphorus-chalcogen bond scission which was monitored using 31P NMR. The rate of dephosphorylation was dependent upon both the nature of the chalcogen and the pH. The integrity of the P-S bond in the corresponding phosphorothiolate was maintained at high pH but rapidly degraded at pH 3. In contrast, P-Se bond cleavage of the phosphoroselenolate monoester was rapid and the rate increased with alkalinity. The results obtained in kinetic experiments provide insight on the reactivity of the novel pyrophosphates studied as well as of other types of thiosubstituted biological phosphates. At the same time, these results also provide evidence for possible formation of unexpectedly reactive intermediates as the chalcogen-substituted analogues are metabolised.