Phosphate graphene as an intrinsically osteoinductive scaffold for stem cell-driven bone regeneration.

Synthetic, resorbable scaffolds for bone regeneration have potential to transform the clinical standard of care. Here, we demonstrate that functional graphenic materials (FGMs) could serve as an osteoinductive scaffold: recruiting native cells to the site of injury and promoting differentiation into bone cells. By invoking a Lewis acid-catalyzed Arbuzov reaction, we are able to functionalize graphene oxide (GO) to produce phosphate graphenes (PGs) with unprecedented control of functional group density, mechanical properties, and counterion identity. In aqueous environments, PGs release inducerons, including Ca2+ and PO43- Calcium phosphate graphene (CaPG) intrinsically induces osteogenesis in vitro and in the presence of bone marrow stromal cells (BMSCs), can induce ectopic bone formation in vivo. Additionally, an FGM can be made by noncovalently loading GO with the growth factor recombinant human bone morphogenetic protein 2 (rhBMP-2), producing a scaffold that induces ectopic bone formation with or without BMSCs. The FGMs reported here are intrinsically inductive scaffolds with significant potential to revolutionize the regeneration of bone.

Intracellular HINT1-Assisted Hydrolysis of Nucleoside 5'-O-Selenophosphate Leads to the Release of Hydrogen Selenide That Exhibits Toxic Effects in Human Cervical Cancer Cells.

In this study, we present a new selenium derivative, 2'-deoxyguanosine-5'-O-selenophosphate (dGMPSe), synthesized by the oxathiaphospholane method and adapted here for the synthesis of nucleoside selenophosphates. Using biochemical assays (HPLC- and fluorescence-based), we investigated the enzymatic activity of HINT1 towards dGMPSe in comparison with the corresponding thiophosphate nucleoside, i.e., dGMPS. Both substrates showed similar kcat and a small difference in Km, and during the reactions the release of reducing agents such as H2Se and H2S were expected and detected. MTT viability assay and microscopic analysis showed that dGMPSe was toxic to HeLa cancer cells, and this cytotoxicity was due to the release of H2Se. The release of H2Se or H2S in the living cells after administration of dGMPSe and/or dGMPS, both without carrier and by electroporation, was observed using a fluorescence assay, as previously for NMPS. In conclusion, our comparative experiments with dGMPSe and dGMPS indicate that the HINT1 enzyme is capable of converting (d)NMPSe to (d)NMP and H2Se, both in vitro and intracellularly. Since the anticancer activity of various selenium compounds depends on the formation of hydrogen selenide, the actual inducer of cell death, we propose that selenium-containing nucleotides represent another option as novel compounds with anticancer therapeutic potential.

Green Synthesis of Iron Oxides and Phosphates via Thermal Treatment of Iron Polyphenols Synthesized by a Camellia sinensis Extract.

Iron oxide nanoparticles (FeONPs) prepared with plant extracts have been emerging as green and sustainable materials. FeONPs are usually amorphous due to the chelation of the tea polyphenols (TPs) to the iron, and the real nature of the iron compounds is not completely understood. The main goal of this study was to investigate the behavior of the green FeONPs synthesized from an Fe3+ salt and Cammelia sinensis (black tea) extract upon thermal treatment, in order to remove TPs and enable the formation of crystalline materials suitable for a thorough characterization and with the potential for diverse applications. The as-prepared FeONPs were assigned as mixed-valence Fe(III) oxyhydroxides and Fe(II)/Fe(III) ions bound to TPs. A detailed description of the phase transformation upon heating revealed the formation of the rare nano ß-Fe2O3 phase at 400 °C, followed by a transformation to α-Fe2O3 as the temperature increased. Above 600 °C, the unprecedented formation of FePO4 and Fe3PO7 was observed, produced from the reaction of Fe2O3 and free phosphate ions present in the black tea leaves, Fe3PO7 being the major phase obtained at 900 °C. Finally, the catalytic potential of the FeONPs to treat the azo dye methyl orange through a heterogeneous Fenton-like system was investigated.

Ionic liquid([C12mim][PF6])-assisted synthesis of TiO2 /Ti2O (PO4)2 nanosheets and the chemoresistive gas sensing of trimethylamine.

The architecture of PO43- modified 2D TiO2 nanosheets was constructed by ionic liquids (ILs)-assisted hydrothermal method. The nanosheet structure can be regulated by the addition of different amount of ionic liquid. Using the composite nanosheets  a chemoresistive gas sensor was prepared for trimethylamine (TMA) detection. Most reported TMA sensors need to be operated at a relatively high operating temperature, but in this paper, the as-synthesized PO43--modified 2D TiO2/Ti2O(PO4)2 nanosheet sensor has high response (S = 87.46), short response time (14.6 s), and good reproducibility to 100 ppm TMA gas, when the temperature is 170 °C. In contrast to the single-phase TiO2 sensor, the gas-sensing property of the composite one is obviously enhanced. Moreover, its response shows excellent linear relationship with TMA concentration from 0.2 to 500 ppm, and a detection limit of 0.2 ppm. The TMA detection mechanism was investigated by analyzing the changes of the surface adsorption oxygen content by XPS and gaseous products using gas chromatography after the sensor was in contact with TMA.

Enantioselective Allenoate-Claisen Rearrangement Using Chiral Phosphate Catalysts.

Herein we report the first highly enantioselective allenoate-Claisen rearrangement using doubly axially chiral phosphate sodium salts as catalysts. This synthetic method provides access to ß-amino acid derivatives with vicinal stereocenters in up to 95% ee. We also investigated the mechanism of enantioinduction by transition state (TS) computations with DFT as well as statistical modeling of the relationship between selectivity and the molecular features of both the catalyst and substrate. The mutual interactions of charge-separated regions in both the zwitterionic intermediate generated by reaction of an amine to the allenoate and the Na+-salt of the chiral phosphate leads to an orientation of the TS in the catalytic pocket that maximizes favorable noncovalent interactions. Crucial arene-arene interactions at the periphery of the catalyst lead to a differentiation of the TS diastereomers. These interactions were interrogated using DFT calculations and validated through statistical modeling of parameters describing noncovalent interactions.

Synthesis and Characterization of Thiophosphoramidate Morpholino Oligonucleotides and Chimeras.

This Article outlines the optimized chemical synthesis and preliminary biochemical characterization of a new oligonucleotide analogue called thiophosphoramidate morpholinos (TMOs). Their rational design hinges upon integrating two well-studied pharmacophores, namely, phosphorothioates (pS) and morpholinos, to create morpholino-pS hybrid oligonucleotides. Our simple synthesis strategy enables the easy incorporation of morpholino-pS moieties and therapeutically relevant sugar modifications in tandem to create novel oligonucleotide (ON) analogues that are hitherto unexplored in the oligotherapeutics arena. Exclusively TMO-modified ONs demonstrate high stability toward 3'-exonuclease. Hybridization studies show that TMO chimeras consisting of alternating TMO and DNA-pS subunits exhibit higher binding affinity toward complementary RNA relative to the canonical DNA/RNA duplex (∼10 °C). Oligonucleotides that consist entirely of TMO linkages also show higher RNA binding affinity but do not recruit ribonuclease H1 (RNase H1). Chimeric TMO analogues demonstrate high gene silencing efficacy, comparable to that of a chimeric 2'-OMe-pS/pO control, during in vitro bioassay screens designed to evaluate their potential as microRNA inhibitors of hsa-miR-15b-5p in HeLa cells.

Nonhydrolyzable Heptose Bis- and Monophosphate Analogues Modulate Pro-inflammatory TIFA-NF-κB Signaling.

d-Glycero-d-manno-heptose-1ß,7-bisphosphate (HBP) and d-glycero-d-manno-heptose-1ß-phosphate (H1P) are bacterial metabolites that were recently shown to stimulate inflammatory responses in host cells through the activation of the TIFA-dependent NF-κB pathway. To better understand structure-based activity in relation to this process, a family of nonhydrolyzable phosphonate analogues of HBP and H1P was synthesized. The inflammation modulation by which these molecules induce the TIFA-NF-κB signal axis was evaluated in vivo at a low-nanomolar concentration (6 nM) and compared to that of the natural metabolites. Our data showed that three phosphonate analogues had similar stimulatory activity to HBP, whereas two phosphonates antagonized HBP-induced TIFA-NF-κB signaling. These results open new horizons for the design of pro-inflammatory and innate immune modulators that could be used as vaccine adjuvant.

Mechanochemical Synthesis of Short DNA Fragments.

We demonstrate the first mechanochemical synthesis of DNA fragments by ball milling, enabling the synthesis of oligomers of controllable sequence and length using multi-step, one-pot reactions, without bulk solvent or the need to isolate intermediates. Mechanochemistry allowed for coupling of phosphoramidite monomers to the 5'-hydroxyl group of nucleosides, iodine/water oxidation of the resulting phosphite triester linkage, and removal of the 5'-dimethoxytrityl (DMTr) protecting group in situ in good yields (up to 60 % over three steps) to produce DNA dimers in a one-pot manner. H-Phosphonate chemistry under milling conditions enabled coupling and protection of the H-phosphonate linkage, as well as removal of the 5'-DMTr protecting group in situ, enabling a one-pot process with good yields (up to 65 % over three steps, or ca. 87 % per step). Sulfurization of the internucleotide linkage was possible using elemental sulfur (S8) or sulfur transfer reagents, yielding the target DNA phosphorothioate dimers in good yield (up to 80 % over two steps). This work opens the door to creation of solvent-free synthesis methodologies for DNA and RNA therapeutics.

d-Glycero-ß-d-Manno-Heptose 1-Phosphate and d-Glycero-ß-d-Manno-Heptose 1,7-Biphosphate Are Both Innate Immune Agonists.

d-Glycero-ß-d-manno-heptose 1,7-biphosphate (ß-HBP) is a novel microbial-associated molecular pattern that triggers inflammation and thus has the potential to act as an immune modulator in many therapeutic contexts. To better understand the structure-activity relationship of this molecule, we chemically synthesized analogs of ß-HBP and tested their ability to induce canonical TIFA-dependent inflammation in human embryonic kidney cells (HEK 293T) and colonic epithelial cells (HCT 116). Of the analogs tested, only d-glycero-ß-d-manno-heptose 1-phosphate (ß-HMP) induced TIFA-dependent NF-κB activation and cytokine production in a manner similar to ß-HBP. This finding expands the spectrum of metabolites from the Gram-negative ADP-heptose biosynthesis pathway that can function as innate immune agonists and provides a more readily available agonist of the TIFA-dependent inflammatory pathway that can be easily produced by synthetic methods.

Novel N-substituted 5-phosphate-d-arabinonamide derivatives as strong inhibitors of phosphoglucose isomerases: Synthesis, structure-activity relationship and crystallographic studies.

Phosphoglucose isomerase (PGI) is a cytosolic enzyme that catalyzes the reversible interconversion of d-glucose 6-phosphate and d-fructose 6-phosphate in glycolysis. Outside the cell, PGI is also known as autocrine motility factor (AMF), a cytokine secreted by a large variety of tumor cells that stimulates motility of cancer cells in vitro and metastases development in vivo. Human PGI and AMF are strictly identical proteins both in terms of sequence and 3D structure, and AMF activity is known to involve, at least in part, the enzymatic active site. Hence, with the purpose of finding new strong AMF-PGI inhibitors that could be potentially used as anticancer agents and/or as bioreceptors for carbohydrate-based electrochemical biosensors, we report in this study the synthesis and kinetic evaluation of several new human PGI inhibitors derived from the synthon 5-phospho-d-arabinono-1,4-lactone. Although not designed as high-energy intermediate analogue inhibitors of the enzyme catalyzed isomerization reaction, several of these N-substituted 5-phosphate-d-arabinonamide derivatives appears as new strong PGI inhibitors. For one of them, we report its crystal structure in complex with human PGI at 2.38 Å. Detailed analysis of its interactions at the active site reveals a new binding mode and shows that human PGI is relatively tolerant for modified inhibitors at the "head" C-1 part, offering promising perspectives for the future design of carbohydrate-based biosensors.

In-vitro bioactivity of silicate-phosphate glasses using agriculture biomass silica.

In the present work, silica extracted from the agricultural waste material; rice husk (RH) was utilized for the synthesis of biocompatible glass of general composition SiO2-P2O5-CaO-MgO-MoO3. In the synthesized glasses P2O5 (5%) and CaO (25%) was kept constant whereas MgO and MoO3 was varied from 10% to 20% and 0% to 5% respectively. The structural, morphological, elemental and functional properties of silica as well as the derived glasses were analyzed by X-Ray Diffraction (XRD), Field Emission Scanning Electron Microscopy (FE-SEM), Energy Dispersive X-ray spectroscopy (EDX) and Fourier Transform Infrared (FTIR) spectroscopy techniques. The effect of MoO3 on the structural and thermal properties of silicate phosphate glasses has been studied in details. The bioactivity of as-synthesized glass samples were further evaluated after immersion in Simulated Body Fluid (SBF) solution which shows bioactive properties thus enabling them to be used as scaffolds in implant materials.

Oxanosine Monophosphate Is a Covalent Inhibitor of Inosine 5'-Monophosphate Dehydrogenase.

Reactive nitrogen species (RNS) are produced during infection and inflammation, and the effects of these agents on proteins, DNA, and lipids are well recognized. In contrast, the effects of RNS damaged metabolites are less appreciated. 5-Amino-3-ß-(d-ribofuranosyl)-3 H-imidazo-[4,5- d][1,3]oxazine-7-one (oxanosine) and its nucleotides are products of guanosine nitrosation. Here we demonstrate that oxanosine monophosphate (OxMP) is a potent reversible competitive inhibitor of IMPDH. The value of Ki varies from 50 to 340 nM among IMPDHs from five different organisms. UV spectroscopy and X-ray crystallography indicate that OxMP forms a ring-opened covalent adduct with the active site Cys (E-OxMP*). Unlike the covalent intermediate of the normal catalytic reaction, E-OxMP* does not hydrolyze, but instead recyclizes to OxMP. IMPDH inhibitors block proliferation and can induce apoptosis, so the inhibition of IMPDH by OxMP presents another potential mechanism for RNS toxicity.

Cerium Phosphate-Cerium Oxide Heterogeneous Composite Nanozymes with Enhanced Peroxidase-Like Biomimetic Activity for Glucose and Hydrogen Peroxide Sensing.

In the present work, we focus on the development of CePO4-CeO2 composite nanorods with peroxidase mimetic activity for the sensitive detection of hydrogen peroxide and glucose. The Ce3+/PO43- molar ratio (CP10:1, CP5:1, CP2:1) in the hydrothermal reaction controlled the formation of pure CePO4, CePO4-CeO2 composite nanozymes with different percentages of CeO2, and its crystal structure. A higher Ce3+/PO43- molar ratio (CP10:1 or CP5:1) was required to obtain CePO4-CeO2 composite nanostructure, while a lower Ce3+/PO43- molar (CP2:1) ratio was sufficient to fabricate pure CePO4 nanorods. In the presence of hydrogen peroxide, the prepared nanozymes catalyze the oxidation of chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB). Steady state kinetic analysis based on the Michaelis-Menten model revealed that CP10:1 showed excellent affinity toward the TMB ( Km = 0.236 mM and Vmax = 8.78 × 10-8 M s-1) in comparison to the catalytic activity of CP5:1 and CP2:1 and horseradish peroxidase ( Km = 0.434 mM and Vmax = 10.0 × 10-8 M s-1). The superior peroxidase activity of CePO4-CeO2 composite nanozymes can be ascribed to the enhanced redox switching between Ce3+ ↔ Ce4+ sites from the CePO4 and CeO2 lattice, respectively. The colorimetric detection of hydrogen peroxide and glucose showed a linear response around 150 µM concentration with the limits of detection (LOD) of 2.9 and 4.1 µM, respectively.

Synthesis and characterization of pyridoxine, nicotine and nicotinamide salts of dithiophosphoric acids as antibacterial agents against resistant wound infection.

The pyridine-derived biomolecules are of considerable interest in developing medicinal compounds with various specific activities. Novel ammonium salts of pyridoxine, (S)-(-)-nicotine and nicotinamide with O,O-diorganyl dithiophosphoric acids (DTPA) were synthesized and characterized. The complexation of chiral monoterpenyl DTPA, including (S)-(-)-menthyl, (R)-(+)-menthyl, (1R)-endo-(+)-fenchyl, (1S,2S,3S,5R)-(+)-isopinocampheolyl derivatives, with pyridoxine and nicotine provided effective antibacterial compounds 3a,b,e,f, and 5a,b,d,f with MIC values against Gram-positive bacteria as low as 10 µM (6 µg/mL). Two selected pyridoxine and nicotine salts based on menthyl DTPA 3a and 5a were similarly active against antibiotic-resistant bacteria from burn wounds including MRSA. The compounds had enhanced amphiphilic and hemolytic properties and effectively altered surface characteristics and matrix-secreting ability of P. aeroginosa and S. aureus. MBC/MIC ratios of 3a and 5a suggested the bactericidal mode of their action. Furthermore, the compounds exhibited moderate cytotoxicity towards human skin fibroblasts (IC50 = 48.6 and 57.6 µM, respectively, 72 h), encouraging their further investigation as potential antimicrobials against skin and wound infections.

New phosphate derivatives of betulin as anticancer agents: Synthesis, crystal structure, and molecular docking study.

Betulin derivatives exhibit an antiproliferative activity and have been tested for many cancer cell lines. This paper describes a new series of 3-phosphate derivatives of betulin bearing different substituents at C28 position. The synthesized compounds were tested in vitro for their antiproliferative effect against human leukemia (MV-4-11 and CCRF/CEM), lung carcinoma (A549), prostate cancer (DU 145), melanoma (Hs 294T) cell lines, and murine leukemia P388. To explore the possible mechanism of anticancer activity for the most in vitro active compounds (4, 5, 7 and 8) and betulin, molecular docking was performed to the binding sites of potential anticancer targets, described for the various triterpene derivatives, including topoisomerase I and II, epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGFR), transcription factor NF-κB, anti-apoptotic protein Bcl-2 and peroxisome proliferator-activated receptor (PPARγ). According to the results of the docking, the best fit to the binding pocket of PPARγ was shown by compound 4.

Synthesis of di-docosahexaenoyl (C22:6)-bis(monoacylglycerol) phosphate in unlabelled and C-13 labelled forms for use as a biomarker of drug induced phospholipidosis.

Di-docosahexaenoyl (C22:6)-bis(monoacylglycerol) phosphate (BMP) has been identified as a promising biomarker for drug-induced phospholipidosis (DIPL). Both unlabelled and stable isotope labelled versions of BMP were desired for use as internal standards. Isopropylideneglycerol was converted to 4-methoxyphenyldiphenylmethyl-3-PMB-glycerol in three steps. Initially, the 2-postion of the glycerol was protected as a t-butyldiphenylsilyl ether, which proved to be a mistake; deprotection of the ether resulted in the decomposition of the compound. A switch to a t-butyldimethylsilyl ether protecting group resulted in an intermediate that could be deprotected to the alcohol to give the target compound after salt exchange. The same procedure was used to prepare [13 C6 ]BMP from [13 C3 ]glycerol.

Synthesis of (1,2,3-triazol-4-yl)methyl Phosphinates and (1,2,3-Triazol-4-yl)methyl Phosphates by Copper-Catalyzed Azide-Alkyne Cycloaddition.

An efficient and practical method was developed for the synthesis of new (1,2,3triazol4yl)methyl phosphinates and (1,2,3-triazol-4-yl)methyl phosphates by the copper(I)catalyzed azide-alkyne cycloaddition (CuAAC) of organic azides and prop-2-ynyl phosphinate or prop-2-ynyl phosphate. The synthesis of (1benzyl-1H-1,2,3-triazol-4-yl)methyl diphenylphosphinate was optimized with respect to the reaction parameters, such as the temperature, reaction time, and catalyst loading. The approach was applied to a range of organic azides, which confirmed the wide scope and the substituent tolerance of the process. The method elaborated represents a novel approach for the synthesis of the target compounds.

The synthesis of mono-alkyl phosphates and their derivatives: an overview of their nature, preparation and use, including synthesis under plausible prebiotic conditions.

Nucleic acids, phospholipids and other organic phosphates play central roles in biological pathways. n-Alkyl phosphates and their derivatives have been recognized as amphiphilic molecules for nearly two centuries. In the last 50 years, n-alkyl phosphate derivatives such as di-alkyl phosphates, mono-alkyl phosphatidyl ethanol amines and mono-alkyl phosphocholines have become predominant compounds with applications in different areas, from food chemistry to life science. The aim of this review is to summarize the most relevant progress made in the field of the synthesis of these molecules and to provide a concise perspective on the use of these amphiphiles as possible prebiotic membrane constituents. The first part of the review is dedicated to the analysis of the most relevant syntheses carried out in recent years with respect to those reported from the second half of the nineteenth century. The second part is dedicated to a description of the latest reports on prebiotic synthesis of mono-alkyl phosphates. In this part, the authors did not report the phosphorylation of other relevant biomolecules, such as nucleosides, which have been excellently reviewed elsewere.

Benzyl Mono-P-Fluorophosphonate and Benzyl Penta-P-Fluorophosphate Anions Are Physiologically Stable Phosphotyrosine Mimetics and Inhibitors of Protein Tyrosine Phosphatases.

α,α-Difluoro-benzyl phosphonates are currently the most popular class of phosphotyrosine mimetics. Structurally derived from the natural substrate phosphotyrosine, they constitute classical bioisosteres and have enabled the development of potent inhibitors of protein tyrosine phosphatases (PTP) and phosphotyrosine recognition sites such as SH2 domains. Being dianions bearing two negative charges, phosphonates, however, do not permeate membranes and thus are often inactive in cells and have not been a successful starting point toward therapeutics, yet. In this work, benzyl phosphonates were modified by replacing phosphorus-bound oxygen atoms with phosphorus-bound fluorine atoms. Surprisingly, mono-P-fluorophosphonates were fully stable under physiological conditions, thus enabling the investigation of their mode of action toward PTP. Three alternative scenarios were tested and mono-P-fluorophosphonates were identified as stable reversible PTP1B inhibitors, despite of the loss of one negative charge and the replacement of one oxygen atom as an H-bond donor by fluorine. In extending this replacement strategy, α,α-difluorobenzyl penta-P-fluorophosphates were synthesized and found to be novel phosphotyrosine mimetics with improved affinity to the phosphotyrosine binding site of PTP1B.

Investigation of acid phosphatase variants for the synthesis of phosphate monoesters.

The major drawback of using phosphatases for transphosphorylation reactions lies in product depletion caused by the natural hydrolytic activity of the enzymes. Variants of PhoC-Mm from Morganella morganii and NSAP-Eb from Escherichia blattae were studied for their ability to maintain a high product level in the transphosphorylation of various primary alcohols. A single amino acid exchange delivered phosphatase variant PhoC-Mm G92D, which was able to catalyze the phosphorylation of primary alcohols without any major hydrolysis of the formed phosphate esters. The mutation mostly improved the affinity of the enzyme for alcohols, while rate constants of transphosphorylation and hydrolysis were decreased, overall resulting in a superior catalytic efficiency in transphosphorylation compared to hydrolysis. The presence of residual substrate alcohol at a given concentration was crucial to suppress phosphate ester hydrolysis. The present work extends the synthetic applicability of phosphatase variants beyond the previously reported nucleosides and allows preparative-scale production of various primary phosphate esters (yields up to 42%) with high enzyme productivity (TONs up to ∼66,000). Biotechnol. Bioeng. 2017;114: 2187-2195. © 2017 Wiley Periodicals, Inc.