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Hydrolysis of dissolved organic phosphorus by marine planktonic microorganisms is a key process in the P cycle, particularly in P -depleted, oligotrophic environments. The present study assessed spatiotemporal variations in phosphomonoesterase (PME) and phosphodiesterase (PDE) activities using concentration kinetics in the eastern Mediterranean Sea in two contrasting situations: the end of winter (including a small bloom period) and autumn. The distribution and regulation of the maximum hydrolysis rate (Vm) and half-saturation constant (Km) of both ectoenzymes were assessed in relation to the vertical structure of the epipelagic layers. PME reached its maximum activities (Vm) after the addition of 1 µM MUF-P (4-methylumbelliferyl phosphate), whereas, for PDE, it was necessary to add up to 50 µM bis(4-methylumbelliferyl)phosphate (bis-MUF-P) to reach saturation state. On average, the Km of PDE was 33 ± 25 times higher than that of PME. The Vm of PME and Vm of PDE were linearly correlated. Conversely to the Km values, Vm values were on the same order of magnitude for both ectoenzymes, with their ratio (Vm PME : Vm PDE) ranging between 0.2 and 6.3. Dissolved organic phosphorus (DOP) and the phosphomonoesterase hydrolysable fraction of DOP explained most of the lack of variability in Vm PME and Vm PDE. On the contrary, Vm of both phosphohydrolase enzymes was inversely correlated to the concentration of dissolved inorganic phosphorus. The particular characteristics of concentration kinetics obtained for PDE (saturation at 50 µM , high Km , high turnover times) are discussed with respect to the possible unequal distribution of PDE and PME among the size continuum of organic material and accessibility of phosphodiesters. [ABSTRACT FROM AUTHOR]

PNAzymes are a group of artificial enzymes which show promising results in selective and efficient cleavage of RNA targets. In the present study, we introduce a series of metal chelating groups based on N,N-bis(2-picolyl) groups (parent, 6-methyl and 6-amino substituted) as the active sites of novel PNAzymes. An improved synthetic route for the 6-amino analogues is described. The catalytic activity of the chelating groups for cleaving phosphodiesters were assessed with the model substrate 2-hydroxypropyl p-nitrophenyl phosphate (HPNPP), confirming that the zinc complexes have the reactivity order of parent < 2-methyl < 2-amino. The three ligands were conjugated to a PNA oligomer to form three PNAzymes which showed the same order of reactivity and some sensitivity to the size of the RNA bulge designed into the catalyst–substrate complex. This work demonstrates that the kinetic activity observed for the model substrate HPNPP could be translated onto the PNAzymes, but that more reactive Zn complexes are required for such PNAzymes to be viable therapeutic agents. [ABSTRACT FROM AUTHOR]

Dissolved organic phosphorus hydrolysis by marine planktonic microorganisms is a key process in the P cycle, particularly in P-depleted, oligotrophic environments. The present study assessed spatio-temporal variations of phosphomonoesterase (PME) and phosphodiesterase (PDE) activities using concentration kinetics in the eastern Mediterranean Sea during 2 contrasted situations: the end of winter (including a small bloom period), and autumn. The distribution and regulation of the maximum hydrolysis rates (Vm) and half-saturation constants (Km) of both ectoenzymes were assessed in relation to the vertical structure of the epipelagic layers. PME reached their maximum activities (Vm) after 1 µM MUF-P addition whereas, for PDE, it was necessary to add up to 50 µM bis-MUF-P to reach saturation state. On average, the Km of PDE was 33 ± 25- times higher than that of the PME. Vm of PME and Vm of PDE were linearly correlated. Conversely to the Km values, Vm were on the same order of magnitude for both ectoenzymes, their ratio (Vm PME:Vm PDE) ranging between 0.2 and 6.3). Dissolved organic phosphorus (DOP) and the phosphomonoesterease hydrolysable fraction of DOP explained mostly no variability of Vm PME nor Vm PDE. On the contrary, Vm of both phosphohydrolase enzymes was inversely correlated to DIP concentration. The particular characteristics of concentration kinetics obtained for PDE (saturation at 50 µM, high Km, high turnovertimes) are discussed with respect to possible inequal distribution of PDE and PME among organic material size continuum, and accessibility to phosphodiesters. [ABSTRACT FROM AUTHOR]

Dissolved organic phosphorus hydrolysis by marine planktonic microorganisms is a key process in the P cycle, particularly in P-depleted, oligotrophic environments. The present study assessed spatio-temporal variations of phosphomonoesterase (PME) and phosphodiesterase (PDE) activities using concentration kinetics in the eastern Mediterranean Sea during 2 contrasted situations: the end of winter (including a small bloom period), and autumn. The distribution and regulation of the maximum hydrolysis rates (Vm) and half-saturation constants (Km) of both ectoenzymes were assessed in relation to the vertical structure of the epipelagic layers. PME reached their maximum activities (Vm) after 1 μM MUF-P addition whereas, for PDE, it was necessary to add up to 50 μM bis-MUF-P to reach saturation state. On average, the Km of PDE was 33 ± 25-times higher than that of the PME. Vm of PME and Vm of PDE were linearly correlated. Conversely to the Km values, Vm were on the same order of magnitude for both ectoenzymes, their ratio (Vm PME:Vm PDE) ranging between 0.2 and 6.3). Dissolved organic phosphorus (DOP) and the phosphomonoesterease hydrolysable fraction of DOP explained mostly no variability of Vm PME nor Vm PDE. On the contrary, Vm of both phosphohydrolase enzymes was inversely correlated to DIP concentration. The particular characteristics of concentration kinetics obtained for PDE (saturation at 50 μM, high Km, high turnovertimes) are discussed with respect to possible inequal distribution of PDE and PME among organic material size continuum, and accessibility to phosphodiesters. [ABSTRACT FROM AUTHOR]

In solid-phase oligonucleotide (ON) synthesis, especially for 3′-modified ONs, a universal linker attached to a solid support is widely used. In this study, benzo-fused 7-oxabicyclo[2.2.1]heptane-2,3-diol derivatives are designed, synthesized, and evaluated as universal linkers. The designed linkers show reactivity comparable to that of a conventional universal linker for releasing the desired ONs. Additionally, these materials exhibit a more robust structure under basic conditions, as generally used in ON synthesis, and hydrophobic properties relative to the conventional universal linker. Notably, when diphenyl-substituted (terphenyl) and phenanthrene-type (PT) linkers are used, cyclic phosphodiesters derived from linker units as byproducts, which are produced by release of ONs from the linker units, are detected in the HPLC chromatograms. The PT linker is applicable to various ON syntheses using controlled pore glass (CPG) and polystyrene (PS) resins. These results demonstrate that the PT linker can serve as an alternative to conventional universal linkers. [ABSTRACT FROM AUTHOR]

Many cells adapt to hyperosmolal conditions by upregulation of organic osmolytes to maintain cell function and integrity. Glycerophosphocholine (GPC), a recognized osmolyte in renal medullary cells, is the major phosphodiester (PDE) in human skeletal muscle, wherefore we hypothesized muscular GPC to be associated with surrogate parameters of fluid status and osmolality in healthy humans. The objective of this study was to investigate the relationship of muscular GPC with surrogate parameters of body fluid status and osmolality. We analyzed data of 30 healthy volunteers who underwent noninvasive 31P-magnetic resonance spectroscopy of either calf (n = 17) or thigh (n = 13) muscle. Therefore, we conducted correlation analyses between phosphor metabolites, and blood values depicting body fluid status and osmolality. Relevant parameters were further implemented in a multivariable regression model to evaluate if GPC concentrations can depict variations in fluid and electrolyte balance. Uric acid (0.437, P = 0.018) and urea (0.387, P = 0.035) were significantly correlated with GPC, which in case of uric acid was independent of sex. Considering sex, following multivariable regression reported GPC as suitable parameter to predict uric acid (R² = 0.462, adjusted R² = 0.421; P < 0.001). Our data indicate a connection between muscular GPC concentrations and uric acid, which is a marker of body fluid status, in healthy human subjects, suggesting that skeletal muscle might regulate GPC content in adaptation to changes in fluid status. [ABSTRACT FROM AUTHOR]

(1) Background: the theoretical modelling of reactions occurring in liquid phase is a research line of primary importance both in theoretical–computational chemistry and in the context of organic and biological chemistry. Here we present the modelling of the kinetics of the hydroxide-promoted hydrolysis of phosphoric diesters. (2) Method: the theoretical–computational procedure involves a hybrid quantum/classical approach based on the perturbed matrix method (PMM) in conjunction with molecular mechanics. (3) Results: the presented study reproduces the experimental data both in the rate constants and in the mechanistic aspects (C–O bond vs. O–P bond reactivity). The study suggests that the basic hydrolysis of phosphodiesters occurs through a concerted A N D N mechanism, with no formation of penta-coordinated species as reaction intermediates. (4) Conclusions: the presented approach, despite the approximations, is potentially applicable to a large number of bimolecular transformations in solution and therefore leads the way to a fast and general method to predict the rate constants and reactivities/selectivities in complex environments. [ABSTRACT FROM AUTHOR]

In the Pseudomonas aeruginosa type strain PA14, 40 genes are known to encode for diguanylate cyclases (DGCs) and/or phosphodiester ases (PDEs), whic h modulate the intr acellular pool of the nucleotide second messenger c-di-GMP. While in general, high levels of c-di-GMP drive the switch from highly motile phenotypes to war ds a sessile lifestyle, the different c-di-GMP modulating enzymes are r esponsib le for smaller and in parts nonoverlapping phenotypes. In this study, we sought to utilize previously recorded P. aeruginosa gene expression datasets on 414 clinical isolates to uncover tr anscriptional changes as a result of a high expression of genes encoding DGCs. This approach might provide a unique opportunity to bypass the problem that for many c-di-GMP modulating enzymes it is not known under which conditions their expression is activated. However, while we demonstrate that the selection of subgroups of clinical isolates with high versus lowexpression of sigma factor encoding genes served the identification of their downstream regulons, we wer e una b le to confirm the predicted DGC regulons, because the high c-di-GMP associated phenotypes were r apidly lost in the clinical isolates,. Further studies are needed to determine the specific mechanisms underlying the loss of cyclase activity upon pr olonged cultivation of clinical P. aeruginosa isolates. [ABSTRACT FROM AUTHOR]

Among natural and synthetic polymers, main-chain phosphorus-containing polyacids (PCPAs) (polyphosphodiesters), stand in a unique position at the intersection of chemistry, physics, biology and medicine. The structural similarity of polyphosphodiesters PCPAs to natural nucleic and teichoic acids, their biocompatibility, mimicking to biomolecules providing the 'stealth effect', high bone mineral affinity of polyphosphodiesters resulting in biomineralization at physiological conditions, and adjustable hydrolytic stability of polyphosphodiesters are the basis for various biomedical, industrial and household applications of this type of polymers. In the present review, we discuss the synthesis, properties and actual applications of polyphosphodiesters. [ABSTRACT FROM AUTHOR]

The cleavage of RNA phosphodiester bonds by RNase A and hammerhead ribozyme at neutral pH fundamentally differs from the spontaneous reactions of these bonds under the same conditions. While the predominant spontaneous reaction is isomerization of the 3',5'‐phosphodiester linkages to their 2',5'‐counterparts, this reaction has never been reported to compete with the enzymatic cleavage reaction, not even as a minor side reaction. Comparative kinetic measurements with structurally modified di‐nucleoside monophosphates and oligomeric phosphodiesters have played an important role in clarification of mechanistic details of the buffer‐independent and buffer‐catalyzed reactions. More recently, heavy atom isotope effects and theoretical calculations have refined the picture. The primary aim of all these studies has been to form a solid basis for mechanistic analyses of the action of more complicated catalytic machineries. In other words, to contribute to conception of a plausible unified picture of RNA cleavage by biocatalysts, such as RNAse A, hammerhead ribozyme and DNAzymes. In addition, structurally modified trinucleoside monophosphates as transition state models for Group I and II introns have clarified some features of the action of large ribozymes. [ABSTRACT FROM AUTHOR]

A method to estimate phosphorus (31P) transversal relaxation times (T2s) of coupled spin systems is demonstrated. Additionally, intracellular and extracellular pH and relaxation‐corrected metabolite concentrations are reported. Echo time (TE) series of 31P metabolite spectra were acquired using stimulated echo acquisition mode (STEAM) localization. Spectra were fitted using LCModel with accurately modeled Versatile Simulation, Pulses and Analysis (VeSPA) basis sets accounting for J‐evolution of the coupled spin systems. T2s were estimated by fitting a single exponential two‐parameter model across the TE series. Fitted inorganic phosphate frequencies were used to calculate pH, and estimated relaxation times were used to determine the relaxation‐corrected brain metabolite concentrations on an assumption of 3 mM γ‐ATP. The method was demonstrated in healthy human brain at a field strength of 9.4 T. T2 times of ATP and nicotinamide adenine dinucleotide (NAD) were shortest between 8 and 20 ms, followed by T2s of inorganic phosphate between 25 and 50 ms, and phosphocreatine with a T2 of 100 ms. Phosphomonoesters and phosphodiesters had the longest T2s of about 130 ms. The measured T2s are comparable with literature values and fit in a decreasing trend with increasing field strengths. Calculated pHs and metabolite concentrations are also comparable with literature values. [ABSTRACT FROM AUTHOR]

In addition to classical steroids, which have cholesterol as a precursor, there are steroids with 7-dehydrocholesterol as a precursor. This review describes the identification of these steroids, their biosynthesis, and some aspects of their function. There are three classes of these compounds, distinguished by the number of their carbon atoms, 23, 24, and 25. Each class has a spiral steroid and is a phosphodiester. Up until these investigations, no spiral steroids or steroid phosphodiesters were known. There are at least 13 compounds, of which six have been purified to near homogeneity; each one has been characterized by its mass and proposed composition, and they function by regulating the NaK-ATPase. Based on the tissues in which they have been detected, each class of compound seems to regulate a different isoform of the NaK-ATPase. This is an important site of endocrine regulation. [ABSTRACT FROM AUTHOR]

Electric forces play a key role in the interaction of negatively charged phosphate groups with the dipolar water molecules of an aqueous environment. Both fluctuation amplitudes and effective spatial range of the electric fields that fluctuate on a multitude of time scales have remained highly controversial. We employ the dimethylphosphate anion (DMP) as a fundamental building block of the phosphodiester backbone in DNA to model electric fields at the phosphate-water interface. DMP is considered to be solvated in bulk water and the fluctuating electric forces exerted on the (PO2)- moiety are calculated by combining the ab initio based effective fragment potential approach that accounts for electric fields due to static multipoles and polarization contributions due to induced dipoles, with molecular dynamics. We demonstrate that the total time-averaged electric field generated by water molecules arises to a large extent from the first water layer. The second layer contributes some 18% with noticeable contributions from induction. We further show that the solvent electric field experienced by the phosphate group is the dominant contribution to the pronounced solvatochromism of the asymmetric (PO2)- stretch vibration. Accounting for a field expansion up to quadrupoles and polarization due to induced dipoles allows us to simulate solvent induced frequency shifts and lineshapes in almost quantitative agreement to experiment. Our theoretical model strongly supports the picture of short-range electric forces that arise locally from the first and second hydration shell. [ABSTRACT FROM AUTHOR]

Enantioselective iodocycloetherifications can be conducted using sterically highly demanding BINOL‐based phosphoric acid diesters as catalyst. To achieve highly enantioselective reactions, cocatalysis by triphenylphosphine selenide is necessary. With cocatalysis, good to excellent enantioselectivities can be achieved for a broad range of substrates using catalyst and cocatalyst loadings as low as 1 mol %. The triphenylphosphine selenide cocatalyst itself does not strongly influence diastereoselectivity, but leads to higher reactivity and, in combination with a BINOL‐based phosphoric acid diester, to good enantioselectivity. [ABSTRACT FROM AUTHOR]

Phosphodiesters are important structural motifs observed in a diverse field of molecular science. It is, thus, important to develop a simple and robust way to synthesize them from corresponding alcohols. Here we report a single-step asymmetric phosphodiester synthesis from alcohols with phosphoenolpyruvate phosphodiesters as phosphoryl donors. This transformation allows for the use of various functionalized alcohols as substrates and would be useful for diverse fields including biology and medicine. [ABSTRACT FROM AUTHOR]

The cleavage of uridine 3'‐phosphodiesters bearing alcohols with pKa ranging from 7.14 to 14.5 catalyzed by AuNPs functionalized with 1,4,7‐triazacyclononane‐Zn(II) complexes has been studied to unravel the source of catalysis by these nanosystems (nanozymes). The results have been compared with those obtained with two Zn(II) dinuclear catalysts for which the mechanism is fairly understood. Binding to the Zn(II) ions by the substrate and the uracil of uridine was observed. The latter leads to inhibition of the process and formation of less productive binding complexes than in the absence of the nucleobase. The nanozyme operates with these substrates mostly via a nucleophilic mechanism with little stabilization of the pentacoordinated phosphorane and moderate assistance in leaving group departure. This is attributed to a decrease of binding strength of the substrate to the catalytic site in reaching the transition state due to an unfavorable binding mode with the uracil. The nanozyme favors substrates with better leaving groups than the less acidic ones. [ABSTRACT FROM AUTHOR]

The kinetics for the cleavage of the phosphotriester PDE6D inhibitors 1 (Deltaflexin‐2) and 3 (Deltaflexin‐1) and their derivatives 2, 4 and 5 is presented under various conditions in aqueous solutions. The conversion of the phosphotriesters (1–5) into the phosphodiesters 1**–5** was detected to take place as a major degradation process. In the absence of enzyme, the 4‐acetylthio‐2,2‐dimethyl‐3‐oxobutyl protected compounds (1, 4 and 5) are one order of magnitude more stable than the 4‐acetylthio‐2‐ethoxycarbonyl‐3‐oxo‐2‐methylbutyl protected ones (2 and 3). In cell culture (DMEM) containing fetal bovine serum and l‐glutamine, an intermediary formation of S−S‐dimer competed with the removal of the protecting group after deacetylation of the starting material. In addition, the susceptibility of the compounds to amine nucleophiles as well as their stability under acidic and basic condition were determined in non‐aqueous solutions. [ABSTRACT FROM AUTHOR]

The labile fraction of dissolved organic phosphorus (DOP) – predominantly consisting of phosphoric esters – is an important microbial P source in the subtropical oligotrophic ocean. However, unlike phosphate, knowledge for labile DOP is still limited due to the scarcity of broad and intensive observations. In this study, we examined the concentrations and size-fractionated hydrolysis rates of labile phosphoric monoesters and diesters along a >10,000 km longitudinal transect in the North Pacific (23°N; upper 200-m layer). Depth-integrated monoesters decreased westward with a maximum difference of fivefold. Vertical profiles of monoesters in the eastern and western basins showed decreasing and increasing trends with depth, respectively. The monoester-depleted shallow layer of the western basin was associated with phosphate depletion and monoesterase activity was predominant in the large size fraction (>0.8 μm), suggesting that monoesters are significant P sources particularly for large microbes. In contrast, diester concentrations were generally lower than monoester concentrations and showed no obvious horizontal or vertical variation in the study area. Despite the unclear distribution pattern of diesters, diesterase activity in the particulate fraction (>0.2 μm) increased in the phosphate-depleted shallow layer of the western basin, suggesting that the targeted diesters in the assay were also important microbial P sources. Diesterase activities in the dissolved fraction (<0.2 μm) were not correlated with ambient phosphate concentrations; however, cell-free diesterase likely played a key role in P cycling, as dissolved diesterase activities were substantially higher than those in the particulate fraction. The horizontal and vertical variability of labile monoesters in the subtropical North Pacific were therefore predominantly regulated by P stress in particularly large microbes, whereas the distributions of labile diesters and diesterase activities were generally independent of microbial P stress, indicating a more complex regulation of diesters to that of monoesters. [ABSTRACT FROM AUTHOR]

While most restriction enzymes catalyze the hydrolysis of phosphodiester bonds at specific nucleotide sequences in DNA, restriction enzymes of the HALFPIPE superfamily cleave N-glycosidic bonds, similar to DNA glycosylases. Apurinic/apyrimidinic (AP) sites generated by HALFPIPE superfamily proteins are cleaved by their inherent AP lyase activities, other AP endonuclease activities or heat-promoted β-elimination. Although the HALFPIPE superfamily protein R.PabI, obtained from a hyperthermophilic archaea, Pyrococcus abyssi, shows weak AP lyase activity, HALFPIPE superfamily proteins in mesophiles, such as R.CcoLI from Campylobacter coli and R. HpyAXII from Helicobacter pylori, show significant AP lyase activities. To identify the structural basis for the AP lyase activity of R.CcoLI, we determined the structure of R.CcoLI by X-ray crystallography. The structure of R.CcoLI, obtained at 2.35-Å resolution, shows that a conserved lysine residue (Lys71), which is stabilized by a characteristic β-sheet structure of R.CcoLI, protrudes into the active site. The results of mutational assays indicate that Lys71 is important for the AP lyase activity of R.CcoLI. Our results help to elucidate the mechanism by which HALFPIPE superfamily proteins from mesophiles efficiently introduce double-strand breaks to specific sites on double-stranded DNA. [ABSTRACT FROM AUTHOR]

A mechanistic understanding of dissolved organic phosphorus (DOP) utilization, and its role in the marine P cycle, requires knowledge of DOP molecular composition. In this study, a recently developed approach coupling electrodialysis and reverse osmosis with solution 31P‐NMR analysis was used to examine DOP composition within a tidally dominated salt‐marsh estuary (North Inlet, South Carolina) over seasonal and tidal time frames. The isolation technique allowed for near complete recovery of the DOP pool (90% ± 13%; n = 12) with six broad compound classes quantified: phosphonates, phosphomonoesters, phosphodiesters, pyrophosphate, di‐ and tri‐phosphate nucleotides (nucleoPα), and polyphosphate. Our results indicate that phosphomonoesters (ca. 61%) and phosphodiesters (ca. 31%) comprise the majority of the DOP pool, with relatively small contributions from pyrophosphates (ca. 4%), phosphonates (ca. 2%), nucleoPα (ca. 1%), and polyphosphates (ca. 1%). The study found no significant differences in DOP composition or concentration between tidal stages, despite significant tidal changes in dissolved organic nitrogen (DON):DOP stoichiometry. Significant seasonal variation was observed, with higher concentrations of phosphonates, nucleoPα, and monophosphates and lower phosphomonoester concentrations in Fall relative to all other seasons. We hypothesize that these seasonal variations reflect the balance between specific compound class seasonal production, lability, and local P demands associated with marine vs. terrestrial sources. Our results indicate that DOP composition exists at a dynamic equilibrium that is strongly conserved across diverse marine environments. [ABSTRACT FROM AUTHOR]

Phosphodiester hydrogels offer a wide range of fascinating properties. Not only do they exhibit excellent hemocompatibility and cellular compatibility, they also show a remarkable resistance to protein adsorption, thereby limiting the foreign body response. In this work, phosphodiester‐crosslinked hydrogels are produced by a simple free‐radical polymerization of a phosphotriester crosslinker. In a second step, this material is transformed to the phosphodiester, by heating it up to 60 °C in phosphate‐buffered saline. Compared to earlier methods, there is no need for acids, bases, or oxidizing agents to achieve this final conversion to the phosphodiester. This method thus reduces the risk to damage or degrade any sensitive biomolecules that might be of interest to tissue engineers, such as various growth factors or other proteins. The phosphotriester crosslinker is readily synthesized out of common laboratory chemicals in multigram quantities with good yield and easy workup and purification. [ABSTRACT FROM AUTHOR]

Organic phosphorus (P) accumulates in soil during pedogenesis, yet information on the composition and transformation of organic P during the early stages of soil development remains scarce. We studied the top 5 cm of mineral soil immediately beneath the organic horizon from six sites (0, 35, 45, 57, 85 and 125 years) along the Hailuogou glacier foreland chronosequence, on the southeastern edge of the Tibetan Plateau. Phosphorus compounds in the soils were determined by NaOH–EDTA extraction and solution phosphorus-31 nuclear magnetic resonance (31P NMR) spectroscopy. Extractable P was dominated by phosphomonoesters (up to 51.5%) and orthophosphate (37.9–44.6%) throughout the chronosequence. The phosphomonoesters were mainly hydrolysis products of RNA and phospholipids and followed a unimodal pattern with soil age, with maximum concentration at the 57-year-old site. myo -Inositol hexakisphosphate was not detected, although scyllo -inositol hexakisphosphate accounted for 4.7–9.3% of the extracted P and d - chiro - and neo -inositol hexakisphosphates occurred small amounts in a few soils (1.1–3.3% of the extracted P). DNA accounted for 4.0–8.3% of extracted P and increased continuously along the chronosequence, associated with increased inputs of plant and microbial residues and stronger sorption to soil surfaces during the rapid decline in soil pH. Pyrophosphate, an inorganic polyphosphate, occurred in small concentrations (up to 5.0%) that fluctuated with soil age. We conclude that organic P compounds accumulate rapidly in the top mineral soil during the early stages of pedogenesis, predominantly as relatively labile compounds from plant and microbial residues. • Phosphomonoesters dominated organic P and showed a unimodal trend during 125-years of pedogenesis. • Phosphomonoesters were mainly the hydrolysis products of RNA and phospholipids. • myo -Inositol hexakisphosphate were not detected in these young soils. • The concentrations of DNA increased through time. • The composition of soil P o reflects an accumulation of plant and microbial residues. [ABSTRACT FROM AUTHOR]

Thin molecular films of the short single strand of DNA, GCAT, were bombarded under vacuum by electrons with energies between 4 and 15 eV. Ex vacuo analysis by high-pressure liquid chromatography of the samples exposed to the electron beam revealed the formation of a multitude of products. Among these, 12 fragments of GCAT were identified by comparison with reference compounds and their yields were measured as a function of electron energy. For all energies, scission of the backbone gave nonmodified fragments containing a terminal phosphate, with negligible amounts of fragments without the phosphate group. This indicates that phosphodiester bond cleavage by 4–15 eV electrons involves cleavage of the C–O bond rather than the P–O bond. The yield functions exhibit maxima at 6 and 10–12 eV, which are interpreted as due to the formation of transient anions leading to fragmentation. Below 15 eV, these resonances dominate bond dissociation processes. All four nonmodified bases are released from the tetramer, by cleavage of the N-glycosidic bond, which occurs principally via the formation of core-excited resonances located around 6 and 10 eV. The formation of the other nonmodified products leading to cleavage of the phosphodiester bond is suggested to occur principally via two different mechanisms: (1) the formation of a core-excited resonance on the phosphate unit followed by dissociation of the transient anion and (2) dissociation of the CO bond of the phosphate group formed by resonance electron transfer from the bases. In each case, phosphodiester bond cleavage leads chiefly to the formation of stable phosphate anions and sugar radicals with minimal amounts of alkoxyl anions and phosphoryl radicals. [ABSTRACT FROM AUTHOR]

Keywords: Amino Acids; Genetic Copying; Prebiotic Chemistry; RNA; Template Effect EN Amino Acids Genetic Copying Prebiotic Chemistry RNA Template Effect 1 1 1 10/05/23 20231009 NES 231009 B A new synergy b between fundamental classes of biomolecules is reported by Clemens Richert et al. in their Research Article (e202307591). Prolinyl nucleotides, such as the one shown in the upper right-hand corner of the image, act as building blocks in the enzyme-free genetic copying of RNA. Amino Acids, Genetic Copying, Prebiotic Chemistry, RNA, Template Effect. [Extracted from the article]

• Deamination as a C-polysaccharide degradation method to improve vaccine production. • C-polysaccharide removed from pneumococcal polysaccharides except serotype 1. • Proposed deamination mechanisms for C-polysaccharide and serotype 1 polysaccharide. Pneumococcal cell wall polysaccharide (C–PS), a contaminant in pneumococcal capsular polysaccharide (Pn-PS) vaccines is degraded by mild deamination of the 4-amino-2-acetamido-2,4,6-tri-deoxy-galactose (AAT) in C–PS, which was carried out by addition of 5% aqueous sodium nitrite to a solution of polysaccharide in 5% aqueous acetic acid. Glycosidic linkage and functional groups such as O-acetates, phosphodiesters, and pyruvates were preserved under the conditions. The small fragments from degraded C–PS were removed by ultrafiltration or dialysis to provide essentially C–PS free Pn-PS. Because of the presence of AAT in its structure the deamination is not suitable for the purification of type 1 Pn-PS. Meanwhile, the mass and NMR spectroscopic analysis on the deamination products suggests that both type 1 Pn-PS and C–PS degraded following a major pathway of 5,4-hydride shift, cleavage of AAT O5-C1 bond, C1 hemiacetal formation, and its hydrolysis to release neighboring GalA- in type 1 Pn-PS and GalNAc(6-O-PCho)- in C–PS. [ABSTRACT FROM AUTHOR]

Small self-cleaving ribozymes catalyze site-specific cleavage of their own phosphodiester backbone with implications for viral genome replication, pre-mRNA processing, and alternative splicing. We report on the 2.1-Å crystal structure of the hatchet ribozyme product, which adopts a compact pseudosymmetric dimeric scaffold, with each monomer stabilized by long-range interactions involving highly conserved nucleotides brought into close proximity of the scissile phosphate. Strikingly, the catalytic pocket contains a cavity capable of accommodating both the modeled scissile phosphate and its flanking 5' nucleoside. The resulting modeled precatalytic conformation incorporates a splayed-apart alignment at the scissile phosphate, thereby providing structure-based insights into the in-line cleavage mechanism. We identify a guanine lining the catalytic pocket positioned to contribute to cleavage chemistry. The functional relevance of structure-based insights into hatchet ribozyme catalysis is strongly supported by cleavage assays monitoring the impact of selected nucleobase and atom-specific mutations on ribozyme activity. [ABSTRACT FROM AUTHOR]

Ribonuclease H (RNase H) specifically hydrolyzes the 5'-phosphodiester bonds of the RNA of RNA/DNA hybrid. Both types 1 and 2 RNases H act on the RNA strand of the hybrid, while only type 2 acts on the single ribonucleotide embedded in DNA duplex. In this study, to explore the role of mammalian type 2 RNase H (RNase H2) in cells, we constructed the RNase H2 knockout NIH3T3 cells (KO cells) by CRISPR/Cas9 system. KO cells hydrolyzed RNA strands in RNA/DNA hybrid, but not single ribonucleotides in DNA duplex, while wild-type NIH3T3 cells (WT cells) hydrolyzed both. Genomic DNA in the KO cells was more heavily hydrolyzed than in the WT cells by the alkaline or RNase H2 treatment, suggesting that the KO cells contained more ribonucleotides in genomic DNA than the WT cells. The growth rate of the KO cells was 60% of that of the WT cells. Expression of interferon-stimulated genes (ISGs) in the KO cells was not markedly elevated compared with the WT cells. These results suggest that in NIH3T3 cells, RNase H2 is crucial for suppressing the accumulation of ribonucleotides in genomic DNA but not for the expression of ISGs. [ABSTRACT FROM AUTHOR]

Abstract In this contribution, a new highly sensitive and selective sensor of the determination of tyrosine has been proposed based on the downturn effect of light scattering (LS) using phosphodiesters quaternary ammonium nanoparticles (PQANPs). Phosphodiesters quaternary ammonium (PQA), one of Gemini zwitterionic surfactants, self-aggregated into the micelle named as PQANPs, which generated strong LS signal in aqueous solution under the optimum condition. Interestingly, the powerful LS intensity of PQANPs with the maximum peak located at 391 nm significantly decreased after introducing trace amount of tyrosine. The decreased value of the LS intensity of the PQA–tyrosine system (Δ I LS) was in proportion to tyrosine concentration in the ranges from 5.5 × 10−8 mol/L to 4.68 × 10−6 mol/L, with a detection limit of 1.38 × 10−8 mol/L. Based on this decreased LS situation, the novel approach of the determination of tyrosine was first developed. The reaction mechanism for the interaction between PQANPs and tyrosine was also investigated. Moreover, the proposed LS assay was applied to the detection of tyrosine concentration in human serum and urine samples with satisfactory results. Graphical Abstract Unlabelled Image Highlights • Establish of a new approach of detecting tyrosine based on the downturn effect of light scattering using PQANPs. • The present light scattering (LS) assay exhibits label-free, facile fabrication, high sensitivity and selectivity advantages. • The proposed LS approach was successfully applied to the detection of tyrosine in real samples with satisfactory results. [ABSTRACT FROM AUTHOR]

Here we describe a DNA analog in which the mesyl (methanesulfonyl) phosphoramidate group is substituted for the natural phosphodiester group at each internucleotidic position. The oligomers show significant advantages over the often-used DNA phosphorothioates in RNA-binding affinity, nuclease stability, and specificity of their antisense action, which involves activation of cellular RNase H enzyme for hybridization-directed RNA cleavage. Biological activity of the oligonucleotide analog was demonstrated with respect to pro-oncogenic miR-21. A 22-nt anti-miR-21 mesyl phosphoramidate oligodeoxynucleotide specifically decreased the miR-21 level in melanoma B16 cells, induced apoptosis, reduced proliferation, and impeded migration of tumor cells, showing superiority over isosequential phosphorothioate oligodeoxynucleotide in the specificity of its biological effect. Lower overall toxicity compared with phosphorothioate and more efficient activation of RNase H are the key advantages of mesyl phosphoramidate oligonucleotides, which may represent a promising group of antisense therapeutic agents. [ABSTRACT FROM AUTHOR]

A combination of chromatographic and spectroscopic (Q-ToF/MS; FTIR) studies were conducted to further understand hydrolytic processes in reactions of phosphate mono- and phosphodiesters with model Cobalt(III)-tetraamine metallohydrolases of the type [CoL(OH)(OH2)]2+; L = (en)2, (tn)2; en = 1,2-diaminoethane, tn = 1,3-diaminopropane. High resolution mass spectroscopic (HRMS) analysis of the reaction fragments indicates that the monoester substrates undergo initial coordination to the metal center followed by an intramolecular hydroxyl attack on phosphorus, a process that is concerted with liberation of the bound alcoholic leaving group. The CoIII(tn)2 complex displays twice as more reactivity towards both mono- and phosphodiesters compared to the (en)2 analog under similar conditions, a behavior that is discussed in terms of the structural differences in the coordinated amine ligands. Plausible mechanisms for Co(III)-promoted hydrolysis of both the mono- and phosphodiesters are proposed. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract FDA approval of CpG oligodeoxynucleotide (CpG ODN) adjuvants for a human hepatitis B virus vaccine has been delayed until late 2017 because of concerns regarding the severe side effects, which may be attributed to the high dosage and systemic diffusion of this proinflammatory material. Considering that PLGA could provide shelter to resist nucleases in tissue and that cationic lipids could confine anionic oligonucleotides in the nanoparticles via electrostatic attraction to avoid systemic diffusion, we encapsulated a natural phosphodiester or the expensive phosphorothioate CpG ODNs in our previously reported hyaluronic acid-modified cationic lipid-PLGA hybrid nanoparticles and evaluated vaccine efficacy in a TC-1-grafted mouse model. Our results showed that together with Poly I:C, CpG ODN could promote the maturation of bone marrow-derived dendritic cells and the cross-presentation of exogenous antigens in vitro. For the coencapsulation with Poly I:C, in vivo studies showed that adjuvant effects on the vaccine efficacy of tumor depression, immune cell activation, and memory T-cell elevation of phosphodiester CpG ODNs were comparable to those of the phosphorothioate CpG ODNs at a low concentration (5 µg/dose). In conclusion, the combination of oligonucleotide adjuvants and synthetic particulate systems not only potentiated the immunogenicity of these nanoparticles but also made these adjuvants safer and more economical, which may be helpful for their wide application. [ABSTRACT FROM AUTHOR]

Heart failure (HF) is one of the most common causes of morbidity and mortality worldwide. Although many patients suffering from HF die from sudden cardiac death caused by arrhythmias, the mechanism linking HF remodeling to an increased arrhythmogenic propensity remains incomplete. HF is typically characterized by a progressive loss of transverse tubule (T-tubule) domains, which leads to an altered distribution of L-type calcium channels (LTCCs). Microdomain degradation also causes the disruption of the β2 adrenergic receptor (β2AR) and phosphodiesterase (PDE) signaling localization, normally confined to the dyadic space. The goal of this study was to analyze how these subcellular changes affect the function of LTCCs and lead to the emergence of ventricular cell-level triggers of arrhythmias. To accomplish this, we developed a novel computational model of a human ventricular HF myocyte in which LTCCs were divided into six different populations, based on their location and signaling environment they experience. To do so, we included T-tubular microdomain remodeling which led to a subset of LTCCs to be redistributed from the T-tubular to the surface membrane and allowed for different levels of phosphorylation of LTCCs by PKA, based on the presence of β2ARs and PDEs. The model was used to study the behavior of the LTCC current (ICaL) under basal and sympathetic stimulation and its effect on cellular action potential. Our results showed that channels redistributed from the T-tubular membrane to the bulk of the sarcolemma displayed an altered function in their new, non-native signaling domain. Incomplete calcium dependent inactivation, which resulted in a longer-lasting and larger-in-magnitude LTCC current, was observed when we decoupled LTCCs from ryanodine receptors and removed them from the dyadic space. The magnitude of the LTCC current, especially in the surface sarcolemma, was also increased via phosphorylation by the redistributed β2ARs and PDEs. These changes in LTCC current led to the development of early afterdepolarizations. Thus, our study shows that altered LTCC function is a potential cause for the emergence of cell-level triggers of arrhythmia, and that β2ARs and PDEs present useful therapeutic targets for treatment of HF and prevention of sudden cardiac death. [ABSTRACT FROM AUTHOR]

Deoxyribozymes are synthetic enzymes made of DNA that can catalyze the cleavage or formation of phosphodiester bonds and are useful tools for RNA biochemistry. Herein, we report new RNA‐cleaving deoxyribozymes to interrogate the methylation status of target RNAs, thereby providing an alternative method for the biochemical validation of RNA methylation sites containing N6‐methyladenosine, which is the most wide‐spread and extensively investigated natural RNA modification. The developed deoxyribozymes are sensitive to the presence of N6‐methyladenosine in RNA near the cleavage site. One class of these DNA enzymes shows faster cleavage of methylated RNA, while others are strongly inhibited by the modified nucleotide. The general applicability of the new deoxyribozymes is demonstrated for several examples of natural RNA sequences, including a lncRNA and a set of C/D box snoRNAs, which have been suggested to contain m6A as a regulatory element that influences RNA folding and protein binding. [ABSTRACT FROM AUTHOR]

Since 2011, Borrelia miyamotoi disease (BMD) has been reported in five countries in the northern hemisphere. The causative agent of BMD is transmitted by Ixodes ticks, which are also vectors of Lyme disease borreliae. In this study, we examined 459 cases of clinically suspected Lyme disease (LD group), and found twelve cases that were seropositive for the glycerophosphodiester phosphodiesterase (GlpQ) antigen derived from B . miyamotoi . The retrospective surveillance revealed that the seroprevalence of anti-GlpQ in the LD group was significantly higher than in a healthy cohort. Seropositive cases were observed from spring through autumn when ticks are active, and the cases were geographically widespread, being found in Hokkaido-Tohoku, Kanto, Chubu, Kinki, and Kyushu-Okinawa regions. Seropositive cases for GlpQ were most frequent in the Chubu region (6.3%) where B . miyamotoi has been found in Ixodes ticks. Out of the twelve cases that were found in the LD group, three cases exhibited concomitant seropositivity to Lyme disease borreliae by western blot assay. This is the first report of serological surveillance for BMD in Japan, and we conclude that BMD occurs nationwide. [ABSTRACT FROM AUTHOR]

Phosphodiesters of glucose-2-phosphate (G2P) are found only in few natural compounds such as agrocinopine D and agrocin 84. Agrocinopine D is a G2P phosphodiester produced by plants infected by Agrobacterium fabrum C58 and recognized by the bacterial periplasmic binding protein AccA for being transported into the bacteria before cleavage by the phosphodiesterase AccF, releasing G2P, which promotes virulence by binding the repressor protein AccR. The G2P amide agrocin 84 is a natural antibiotic produced by the non-pathogenic Agrobacterium radiobacter K84 strain used as a biocontrol agent by competing with Agrobacterium fabrum C58. G2P esters are also found in irregular glycogen structures. The rare glucopyranosyl-2-phophoryl moiety found in agrocin 84 is the key structural signature enabling its action as a natural antibiotic. Likewise, G2P and G2P esters can also dupe the Agrobacterium agrocinopine catabolism cascade. Such observations illustrate the importance of G2P esters on which we have recently focused our interest. After a brief review of the reported phosphorylation coupling methods and the choice of carbohydrate building blocks used in G2P chemistry, a flexible access to glucose-2-phosphate esters using the phosphoramidite route is proposed.

Lysophosphatidyl glucoside (LPGlc) is a structurally unique glycolipid that acts as a guidance cue for extending axons during central nervous system development by activating the class A G protein coupled receptor (GPR) 55 of spinal cord sensory axons. GPR55 not only plays an important role during development, but is also implicated in many disease states, rendering molecules that target GPR55 of widespread interest. In this study, we developed synthetic access to a novel class of LPGlc analogues featuring a squaryl diamide group as surrogate for the phosphodiester. We report the facile synthesis of a series of LPGlc analogues, their GPR dependent biological activity and a systematic analysis of the structure–activity relationship in regards to GPR55 modulation. The lead compound featuring identical configuration at all stereocenters compared to natural LPGlc exhibits an activity to repel axons of dorsal root ganglion (DGR) nociceptive neurons. [ABSTRACT FROM AUTHOR]

Background and Purpose: To investigate accumulative aging effects on neurometabolism in human brain and to collect a reference dataset.Methods: Fifty-four healthy volunteers aged evenly between 22 and 73 years were studied using whole-brain 1 H-MR spectroscopic imaging in combination with 31 P-MRS at 3T. Global metabolite concentrations of brain N-acetylaspartate (NAA), total choline (tCho), and total creatine (tCr), as well as phosphocreatine (PCr), adenosine-5'-triphosphate (ATP), phosphomonoesters (PME), phosphodiesters (PDE), and inorganic phosphate (Pi) were determined. Fractional volumes of brain gray matter (FVGM), white matter (FVWM), and total tissue (FVTB, GM+WM) were also estimated.Results: With age, NAA, ATP, and PME, as well as FVTB and FVGM decreased and tCho and FVWM increased linearly. Positive correlations were found between FVGM and global concentrations of NAA, ATP, PME, and Pi.Conclusion: Age-related accumulative metabolic changes in aging human brain correlated with reduced neuronal metabolic activity and density, reflected by decreased NAA, reduced mitochondrial activity by decreased ATP, and reduced membrane synthesis by decreased PME. These changes are associated with age-related decrease of neuronal volume. Global NAA and ATP might be used as surrogate biomarker for monitoring aging in human brain. [ABSTRACT FROM AUTHOR]

Purpose: To determine the phosphorus‐31 T2s of phosphomonoesters, phosphodiesters, and inorganic phosphate in the healthy human brain at 7T. Methods: A 3D chemical shift imaging multi‐echo sequence with composite block pulses for refocusing was used to measure one free induction decay (FID) and seven full echoes with an echo spacing of 45 ms on the brain of nine healthy volunteers (age range 22–45 years; average age 27 ± 8 years). Spectral fitting was used to determine the change in metabolic signal amplitude with echo time. Results: The average apparent T2s with their standard deviation were 202 ± 6 ms, 129 ± 6 ms, 86 ± 2 ms, 214 ± 10 ms, and 213 ± 11 ms for phosphoethanolamine, phosphocholine, inorganic phosphate, glycerophosphoethanolamine, and glycerophosphocholine, respectively. Conclusion: The determined apparent T2 for phosphoethanolamine, glycerophosphocholine, and glycerophosphoethanolamine is approximately 200 ms. The lower apparent T2 value for phosphocholine is attributed to the overlap of this resonance with the 3‐phosphorous resonance of 2,3‐diphosphoglycerate from blood, with an apparent shorter T2. Omitting the FID signal and the first echo of phosphocholine leads to a T2 of 182 ± 7 ms, whereas a biexponential analysis leads to 203 ± 4 ms. These values are more in line with phosphoethanolamine and the phosphodiesters. The short T2 of inorganic phosphate is subscribed to the fast reversible exchange with γ‐adenosine triphosphate, which is mediated by glyceraldehyde‐3‐phosphate dehydrogenase and phosphoglycerate kinase within the glycolytic pathway. Magn Reson Med 80:29–35, 2018. © 2017 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. [ABSTRACT FROM AUTHOR]

Understanding the atmospheric fate of organophosphorus plasticizers is important for their environmental risk assessment. However, limited information is available at present. In this study, density functional theory (DFT) calculations were performed to investigate the transformation mechanism and kinetics of tri-p-cresyl phosphate (TpCP) initiated by OH. Results show that the initial reactions are dominated by H-abstraction and OH addition to form TpCP-radical, TpCP–OH adducts and aryl phosphodiester. The H-abstraction pathways are more favorable than the OH addition pathways. The TpCP-radical and TpCP–OH adducts can further react with O 2 in the atmosphere to finally form benzaldehyde phosphate, hydroxylated TpCP and bicyclic radicals. Based on the transition state theory, the calculated rate constant ( k OH ) of TpCP with OH at T  = 298 K is 1.9 × 10 −12 cm 3 molecule −1 s −1 with an atmospheric lifetime of 4.2 days, which demonstrates that gaseous TpCP is atmospherically persistent. This study provides a comprehensive investigation of the OH-initiated oxidation of TpCP, which is useful for understanding its mechanism of transformation and evaluating the risk in atmospheric environment. [ABSTRACT FROM AUTHOR]

New silibinin phosphodiester glyco-conjugates were synthesized by efficient phosphoramidite chemistry and were fully characterized by 2D-NMR. A wide-ranging study focused on the determination of their pKa and E ° values as well as on their radical scavenging activities by different assays (DPPH, ABTS + and HRSA) was conducted. The new glyco-conjugates are more water-soluble than silibinin, and their radical scavenging activities are higher than those of silibinin. The conjugation therefore improves both the water solubilities and antioxidant activities of the flavonolignan moieties. The serum stability was evaluated under physiological conditions, and the glyco-conjugates degraded with half-lives of 40–70 h, making them useful in pro-drug approaches. We started by treating androgen-dependent prostate cancer (PCa) LNCaP cells and then expanded our studies to androgen-independent PCa PC3 and DU145 cells. In most cases, the new derivatives significantly reduced both total and live cell numbers, albeit at different levels. Anti-HIV activities were evaluated and the glucosamine-phosphate silibinin derivative showed higher activity (IC 50  = 73 μM) than silibinin. [ABSTRACT FROM AUTHOR]

Based on molecular dynamics simulations and functional studies, a conformational mechanism is posited for forward translocation by RNA polymerase (RNAP). In a simulation of a ternary elongation complex, the clamp and downstream cleft were observed to close. Hinges within the bridge helix and trigger loop supported generation of translocation force against the RNA-DNA hybrid resulting in opening of the furthest upstream i¡8 RNA-DNA bp, establishing conditions for RNAP sliding. The β flap tip helix and the most N-terminal β' Zn finger engage the RNA, indicating a path of RNA threading out of the exit channel. Because the β flap tip connects to the RNAP active site through the β subunit double-Ψ-β-barrel and the associated sandwich barrel hybrid motif (also called the flap domain), the RNAP active site is coupled to the RNA exit channel and to the translocation of RNA-DNA. Using an exonuclease III assay to monitor translocation of RNAP elongation complexes, we show that K+ and Mg2+ and also an RNA 3'-OH or a 3'-H2 affect RNAP sliding. Because RNAP grip to template suggests a sticky translocation mechanism, and because grip is enhanced by increasing K+ and Mg2+ concentration, biochemical assays are consistent with a conformational change that drives forward translocation as observed in simulations. Mutational analysis of the bridge helix indicates that 778-GARKGL-783 (Escherichia coli numbering) is a homeostatic hinge that undergoes multiple bends to compensate for complex conformational dynamics during phosphodiester bond formation and translocation. [ABSTRACT FROM AUTHOR]

Nanopore analysis, which is, currently, chiefly used for DNA sequencing, is also an appealing technique for characterizing abiotic polymers. As a first step toward this goal, nanopore detection of non-natural monodispersed poly(phosphodiester)s as candidate backbone structures is reported herein. Two model homopolymers containing phosphopropyl repeat units (i.e., 56 or 104 r.u.) and a short thymidine nucleotide sequence are analyzed in the present work. They are tested in two different biological nanopores, α-hemolysin from Staphylococcus aureus, and aerolysin from Aeromonas hydrophila. These recordings are performed in aqueous medium at different KCl concentrations and various driving voltages. The data show a complex interaction with evidence for voltage dependence and threading, and underline the influence of the molecular structure and orientation of the precision poly(phosphodiester)s on the observed residual current signal as well as on the translocation dynamics. In particular, they suggest a dominant entropic contribution due to the high flexibility of the phosphodiester homopolymer. [ABSTRACT FROM AUTHOR]

A photoregulated phosphoramidite iterative process is studied for the synthesis of non-natural, digitally encoded oligo(phosphodiester)s. The oligomers are prepared using two reactive phosphoramidite monomers containing a 2-(2-nitrophenyl)propoxycarbonyl (NPPOC) protected OH group. The stepwise synthesis is performed on an OH-functional soluble polystyrene support, which allows recycling by precipitation in a nonsolvent. Repeating cycles involving phosphoramidite coupling, oxidation of phosphite to phosphate, and NPPOC deprotection by light irradiation at λ = 365 nm are performed in order to prepare oligomers with different lengths and sequences. Synthesis is conducted on a micromolar scale and good recycling yields are obtained in all cases. The use of a soluble polymer support allows an in-depth characterization of the NPPOC photo-deprotection step by 1H NMR, UV spectroscopy, and size exclusion chromatography, and thus identification of optimal synthesis conditions. After cleavage from the support, the oligo(phosphodiester)s are characterized by tandem mass spectrometry, which confirms preparation of uniform sequence-coded oligomers. [ABSTRACT FROM AUTHOR]

Apurinic/apyrimidinic (AP) sites are among the most frequent DNA lesions. The first step in the AP site repair involves the magnesium-dependent enzyme AP endonuclease 1 (APE1) that catalyzes hydrolytic cleavage of the DNA phosphodiester bond at the 5′ side of the AP site, thereby generating a single-strand DNA break flanked by the 3′-OH and 5′-deoxyribose phosphate (dRP) groups. Increased APE1 activity in cancer cells might correlate with tumor chemoresistance to DNA-damaging treatment. It has been previously shown that the multifunctional oncoprotein Y-box-binding protein 1 (YB-1) interacts with APE1 and inhibits APE1-catalyzed hydrolysis of AP sites in single-stranded DNAs. In this work, we demonstrated that YB-1 stabilizes the APE1 complex with double-stranded DNAs containing the AP sites and stimulates cleavage of these AP sites at low magnesium concentrations. [ABSTRACT FROM AUTHOR]

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Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2′, 3′ -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2′-5′ and 3′-5′ phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (KD = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors. [ABSTRACT FROM AUTHOR]

Cytosine-phosphate-guanine (CpG) DNA is known to increase the potency of vaccines. Here, in vitro and in vivo stimulation of toll-like receptor 9 by CpG DNA incorporated into polypod-like DNA nanostructures was evaluated by measuring the levels of tumor necrosis factor alpha released from macrophage-like RAW 264.7 cells and plasma interleukin (IL)-12p40 in vivo following intravenous injection into mice. Phosphodiester CpG1668 was selected as the CpG DNA, and tripodna and hexapodna, which were CpG1668-containing tripod and hexapod-like DNA nanostructures, respectively, were designed. CpG-tripodna and CpG-hexapodna induced tumor necrosis factor alpha release from RAW 264.7 cells about 10- and ∼30-fold higher than single-stranded CpG1668 (CpG-SS). Moreover, in all cases examined, plasma IL-12p40 concentrations increased after intravenous injection into mice, with peak levels depending on the samples and the doses. The area under the plasma concentration-time curves indicated that the CpG-hexapodna was approximately 20-fold more efficient in inducing IL-12p40 production than CpG-SS. The efficiency of CpG-tripodna and CpG-hexapodna to increase the potency of CpG-SS in vivo was comparable to that observed in cultured RAW 264.7 cells. These results provide experimental evidence that in vitro studies can be used to estimate the in vivo immunostimulatory activity of CpG DNA incorporated into DNA nanostructures. [ABSTRACT FROM AUTHOR]

Internucleoside amide linkages are excellent mimics of phosphodiesters in RNA and may be used to optimize the properties of short interfering RNAs. Herein we report a remarkably straightforward, efficient and step economic synthesis of C3'-homologated uridine and adenosine amino acids starting from nucleosides in six steps (31% overall yield) and eight steps (16% overall yield), respectively. The key enabling step is a one-pot multifunctional group transformation including a stereoselective hydrogenation, termed "Global Hydrogenation". [ABSTRACT FROM AUTHOR]