Phosphoramidate Group Research Articles

Structure-activity relationship study of mesyl and busyl phosphoramidate antisense oligonucleotides for unaided and PSMA-mediated uptake into prostate cancer cells.

Phosphorothioate (PS) group is a key component of a majority of FDA approved oligonucleotide drugs that increase stability to nucleases whilst maintaining interactions with many proteins, including RNase H in the case of antisense oligonucleotides (ASOs). At the same time, uniform PS modification increases nonspecific protein binding that can trigger toxicity and pro-inflammatory effects, so discovery and characterization of alternative phosphate mimics for RNA therapeutics is an actual task. Here we evaluated the effects of the introduction of several N-alkane sulfonyl phosphoramidate groups such as mesyl (methanesulfonyl) or busyl (1-butanesulfonyl) phosphoramidates into gapmer ASOs on the efficiency and pattern of RNase H cleavage, cellular uptake in vitro, and intracellular localization. Using Malat1 lncRNA as a target, we have identified patterns of mesyl or busyl modifications in the ASOs for optimal knockdown in vitro. Combination of the PSMA ligand-mediated delivery with optimized mesyl and busyl ASOs resulted in the efficient target depletion in the prostate cancer cells. Our study demonstrated that other N-alkanesulfonyl phosphoramidate groups apart from a known mesyl phosphoramidate can serve as an essential component of mixed backbone gapmer ASOs to reduce drawbacks of uniformly PS-modified gapmers, and deserve further investigation in RNA therapeutics.

New Zwitterionic Oligonucleotides: Preparation and Complementary Binding

New zwitter-ionic oligonucleotide derivatives containing 1,2,3,4-tetrahydroisoquinoline-7-sulfonyl phosphoramidate group are described. Automated synthesis of these compounds was carried out according to the β-cyanoethyl phosphoramidite scheme using the Staudinger reaction between 2-trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-7-sulfonyl azide and phosphite triester within an oligonucleotide grafted to a polymer support. 1,2,3,4-Tetrahydroisoquinoline-7-sulfonyl phosphoramidate group (THIQ) proved to be stable under the conditions of standard oligonucleotide synthesis, including the removal of protective groups and cleavage of the oligonucleotide from the polymer support by treatment with a mixture of concentrated aqueous solutions of ammonia and methylamine (1 : 1) at 55oC. Oligonucleotides modified by one to five THIQ groups in various positions were obtained. The zwitter-ionic character of the obtained derivatives was reflected in their different mobility under conditions of denaturing PAGE. The thermal stability of the duplexes of oligodeoxynucleotides containing THIQ groups with complementary DNA and RNA differed only slightly from that of natural DNA:DNA and DNA:RNA duplexes. The results obtained suggest the possible use of oligonucleotides modified with zwitterionic THIQ groups as antisense therapeutic agents.

New Zwitter-Ionic Oligonucleotides: Preparation and Complementary Binding

New zwitter-ionic oligonucleotide derivatives containing 1,2,3,4-tetrahydroisoquinoline-7-sulfonyl phosphoramidate group are described. Automated synthesis of these compounds was carried out according to the β-cyanoethyl phosphoramidite scheme via the Staudinger reaction between 2-trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-7-sulfonyl azide and phosphite triester within oligonucleotide grafted to polymer support. 1,2,3,4-Tetrahydroisoquinoline-7-sulfonyl phosphoramidate group (THIQ) was stable under the conditions of standard oligonucleotide synthesis, including the removal of protective groups and cleavage of the oligonucleotide from the polymer support by treatment with a mixture of concentrated aqueous solutions of ammonia and methylamine (1 : 1) at 55°C. Oligonucleotides modified by one to five THIQ groups in various positions were obtained. The zwitter-ionic character of the obtained derivatives was reflected in their varying mobility under conditions of denaturing PAGE. The thermal stability of the duplexes of oligodeoxynucleotides containing THIQ groups with complementary DNA and RNA only slightly differed from that of natural DNA:DNA and DNA:RNA duplexes. The results reported suggest that oligonucleotides modified with zwitter-ionic THIQ groups as antisense therapeutic agents.

Post-synthetic modification of graphitic carbon nitride with PCl3 and POCl3 for enhanced photocatalytic degradation of organic compounds

Graphitic carbon nitride (g-C 3 N 4 ) was synthesised from melamine at 550 °C in the air for a period of 4 h. As such prepared g-C 3 N 4 was dispersed in PCl 3 and POCl 3 with and without pyridine at an ambient temperature for us to dope g-C 3 N 4 with phosphorus. The bulk structural properties of g-C 3 N 4 examined by X-ray diffraction (XRD) and Fourier transformed infrared (FTIR) spectroscopy were not changed. On the contrary, a surface modification in terms of pore size distribution studied using physisorption of nitrogen and electron microscopy was observed. Using PCl 3 (in the presence of pyridine), nitrogen vacancies were filled with phosphorus and phosphoramidate groups were formed (with and without pyridine). When POCl 3 was used nitrogen vacancies were removed and the surface structure was rearranged, but no phosphorus was doped in g-C 3 N 4 . The band gap energies varied from 2.69 to 2.73 eV and specific surface areas varied from 8 to 11 m 2 g −1 . The g-C 3 N 4 surface structure rearrangement was associated with altered electronic properties which led to higher photocatalytic activity observed by the degradation of Ofloxacin, Amoxicillin and Rhodamine B (RhB) under LED irradiation of 420 nm. A degradation efficiency decreased in the order: Ofloxacin > RhB > Amoxicillin. Superoxide radicals were found to be able to react with all the organic compounds, but holes could react only with Ofloxacin and RhB. All the modified materials were more active than the pristine g-C 3 N 4 and the best photocatalyst was prepared through the reaction with PCl 3 in the presence of pyridine. • g-C 3 N 4 was synthetized from melamine at 550 °C in the air for 4 h. • Then, g-C 3 N 4 was modified with PCl 3 and POCl 3 . • Using PCl 3 , the surface of g-C 3 N 4 was doped with phosphorus. • In the case of POCl 3 , g-C 3 N 4 was undoped but its surface structure was changed. • These modifications led to the enhanced photocatalytic activity of g-C 3 N 4 .

Mesyl Phosphoramidate Oligonucleotides as Potential Splice-Switching Agents: Impact of Backbone Structure on Activity and Intracellular Localization.

A series of 2'-deoxy and novel 2'-O-methyl and 2'-O-(2-methoxyethyl) (2'-MOE) oligonucleotides with internucleotide methanesulfonyl (mesyl, μ) or 1-butanesulfonyl (busyl, β) phosphoramidate groups has been synthesized for evaluation as potential splice-switching oligonucleotides. Evaluation of their splice-switching activity in spinal muscular atrophy patient-derived fibroblasts revealed no significant difference in splice-switching efficacy between 2'-MOE mesyl oligonucleotide and the corresponding phosphorothioate (nusinersen). Yet, a survival study with model neonatal mice has shown the antisense 2'-MOE mesyl oligonucleotide to be inferior to nusinersen at the highest dose of 40 mg/kg. A reason for their lower activity in vivo as ascertained by cellular uptake study by fluorescent confocal microscopy in HEK293 cell line could possibly be ascribed to compromised endosomal release and/or nuclear uptake of the 2'-OMe or 2'-MOE μ- and β-oligonucleotides compared to their phosphorothioate analog.

Novel Lipid-Oligonucleotide Conjugates Containing Long-Chain Sulfonyl Phosphoramidate Groups: Synthesis and Biological Properties

New lipid conjugates of DNA and RNA incorporating one to four [(4-dodecylphenyl)sulfonyl]phosphoramidate or (hexadecylsulfonyl)phosphoramidate groups at internucleotidic positions near the 3′ or 5′-end were synthesized and characterized. Low cytotoxicity of the conjugates and their ability to be taken up into cells without transfection agents were demonstrated. Lipid-conjugated siRNAs targeting repulsive guidance molecules a (RGMa) have shown a comparable gene silencing activity in PK-59 cells to unmodified control siRNA when delivered into the cells via Lipofectamine mediated transfection.

The Importance of Phosphates for DNA G-Quadruplex Formation: Evaluation of Zwitterionic G-Rich Oligodeoxynucleotides.

A quaternary ammonium butylsulfonyl phosphoramidate group (N+) was designed to replace all the phosphates in a G-rich oligodeoxynucleotide d(TG4 T), resulting in a formally charge-neutral zwitterionic N+TG4 T sequence. We evaluated the effects of N+phosphate modifications on the structural, thermodynamic and kinetic properties of the parallel G-quadruplexes (G4) formed by TG4 T and compared them to the properties of the recently published phosphoryl guanidine d(TG4 T) (PG-TG4 T). Using size-exclusion chromatography, we established that, unlike PG-TG4 T, which exists as a mixture of complexes of different molecularity in solution, N+TG4 T forms an individual tetramolecular complex. In contrast to PG modifications that destabilized G4s, the presence of N+ modifications increased thermal stability relative to unmodified [d(TG4 T)]4 . The initial stage of assembly of N+TG4 T proceeded faster in the presence of Na+ than K+ ions and, similarly to PG-TG4 T, was independent of the salt concentration. However, after complex formation exceeded 75 %, N+TG4 T in solution with Na+ showed slower association than with K+ . N+TG4 T could also form G4s in solution with Li+ ions at a very low strand concentration (10 μM); something that has never been reported for the native d(TG4 T). Charge-neutral PG-G4s can invade preformed native G4s, whereas no invasion was observed between N+and native G4s, possibly due to the increased thermal stability of [N+TG4 T]4 . The N+ modification makes d(TG4 T) fully resistant to enzymatic digestion, which could be useful for intracellular application of N+-modified DNA or RNA.

Phosphoramidates as Transient Precursors of Nitrogen‐Centered Radical Under Visible‐Light Irradiation: Application to the Synthesis of Phthalazine Derivatives

AbstractPhosphoramidates are for the first time presented as efficient N‐Centered Radical (NCR) precursors under visible‐light irradiation. More precisely among this class of phosphorus‐derived compounds, we studied the radical reactivity of phosphonohydrazones, under mild reaction conditions, which allowed the synthesis of a wide and diversified library of the scarcely reported phthalazine scaffold. Mechanistic investigations confirmed the formation of a NCR from these brand‐new phosphonohydrazones (derivatived from phosphoramidates), which were further engaged in an intramolecular 6‐exo‐dig cyclization to provide phthalazines. Compared to other pre‐activated moieties, the phosphoramidate group is self‐immolative, thus enhancing its attractiveness for the C−N bond formation.magnified image

Biosynthesis of GDP-d-glycero-α-d-manno-heptose for the Capsular Polysaccharide of Campylobacter jejuni.

The capsular polysaccharide (CPS) structure of Campylobacter jejuni contributes to its robust fitness. Many strains contain heptose moieties in their CPS units. The precursor heptose is GDP-d-glycero-α-d-manno-heptose; modifications to the stereochemistry at C3-C6 as well as additions of methyl and phosphoramidate groups lend to the hypervariability of the C. jejuni CPS structures. Synthesis of GDP-d-glycero-α-d-manno-heptose has been described previously, but using enzymes from Aneurinibacillus thermoaerophilus DSM 10155. Here we describe the complete synthesis of GDP-d-glycero-α-d-manno-heptose using enzymes from C. jejuni NTCC 11168: Cj1152 and Cj1423-Cj1425. Our results yield kinetic parameters for these enzymes and outline a successful strategy for milligram-gram scale synthesis of GDP-d-glycero-α-d-manno-heptose. This achievement is critical for the characterization of other carbohydrate tailoring enzymes, which are expected to utilize GDP-d-glycero-α-d-manno-heptose for the biosynthesis of more complex carbohydrates in the CPS of C. jejuni.

Selective Liquid‐Liquid Extraction of Uranium by Phosphoramidate‐Group Bearing Ionic Liquid

AbstractExtraction of pure uranium (U) from its ore or spent nuclear fuel and separation of U for trace impurities characterization of nuclear fuels are the processes resulting into the accumulation of large amounts of low boiling and volatile organic waste. The limited reusability and degradation (chemical and radiation induced) of these waste solutions turning out to be an environmental threat. The authors have addressed this issue by developing an eco‐friendly, simple and selective liquid‐liquid extraction (LLE) procedure for U employing a phosphoramidate group bearing task specific ionic liquid (TSIL). Optimization of various LLE parameters resulted in high distribution ratio (D) value of 174±5 for U when extracted from 5 mol L−1 HNO3 medium. A detailed study on the infrared and extended X‐ray absorption fine structure spectroscopy of U loaded ionic liquid phase reveals the extraction mechanism. Such high DU value resulted in high separation factors for U with respect to the elements which are commonly present in the ore, processed nuclear fuel and spent nuclear fuel. The structural integrity and metal ion extractability of the TSIL is found to remain unaffected when irradiated up to an absorbed dose of 400 kilogray (kGy). Sodium carbonate solution is found to back‐extract ≥ 95% of U from the ionic liquid phase and regenerate it for further usage.

On the synthesis, optical and computational studies of novel BODIPY-based phosphoramidate fluorescent dyes

New boro-dipyrromethene (BODIPY) fluorophores substituted with phosphoramidate groups have been synthesized using environmentally friendly methodologies, and their photophysical properties were evaluated experimentally and characterized in detail with respect to DFT and TD-DFT using B3LYP/6-31+G(d) level of theory. These fluorophores may be used as promising candidates for biological probes and optical materials.

Mesyl phosphoramidate antisense oligonucleotides as an alternative to phosphorothioates with improved biochemical and biological properties

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.

Phosphoramidate‐functionalized Merrifield resin: synthesis and application in actinide separation

AbstractA novel phosphoramidate‐functionalized polymeric resin was synthesized by anchoring diethyl (3‐(1H‐imidazol‐1‐yl)propyl)phosphoramidate group with chloromethylated polystyrene divinylbenzene resin. The grafted resin was characterized using elemental analysis, Fourier transform infrared, solid‐state 31P NMR energy‐dispersive X‐ray spectroscopies, and thermogravimetric analysis. The surface morphology of the functionalized resin was investigated using scanning electron microscopy and the surface area was measured with Brunauer–Emmett–Teller analysis. Sorption affinities of the functionalized resin towards actinides such as Th(IV) and U(VI) were determined under various concentrations of nitric acid solution (0.01–6 mol L−1). Desorption of the actinides from the loaded resin was performed using a solution of 0.01 mol L−1 nitric acid. Reusability of the grafted resin was studied over various successive cycles and the stability of the functional groups on the synthesized resin was monitored using various analytical techniques. © 2017 Society of Chemical Industry

New oligodeoxynucleotide derivatives containing N-(methanesulfonyl)-phosphoramidate (mesyl phosphoramidate) internucleotide group

Novel oligonucleotide derivatives containing N-(methanesulfonyl)-phosphoramidate (mesyl phosphoramidate) group have been described. Solid-phase synthesis of these compounds using an automated DNA synthesizer has been performed for the first time, including the Staudinger reaction between methanesulfonyl azide (mesyl azide) and 3′,5′-dinucleoside 2-cyanoethyl phosphite within an oligonucleotide immobilized on the polymer support, which is a product of phosphoramidite coupling. The mesyl phosphoramidate group is stable to the conditions of oligonucleotide synthesis, in particular, during acidic detritylation and subsequent removal of protecting groups and cleavage of an oligonucleotide from the polymer support by concentrated aqueous ammonia or methylamine at 55°C. It has been shown that the stability of complementary duplexes of oligodeoxynucleotides containing the mesyl phosphoramidate group with a single-stranded DNA is not inferior to the stability of native DNA:DNA duplex. Furthermore, mesyl phosphoramidate oligonucleotides are able to form a complementary duplex with RNA, which is only slightly less stable than the equivalent DNA:RNA duplex. This raises the possibility of their application as potential antisense therapeutic agents.

Selective Micellar Extraction of Ultratrace Levels of Uranium in Aqueous Samples by Task Specific Ionic Liquid Followed by Its Detection Employing Total Reflection X-ray Fluorescence Spectrometry.

A task specific ionic liquid (TSIL) bearing phosphoramidate group, viz., N-propyl(diphenylphosphoramidate)trimethylammonium bis(trifluoromethanesulfonyl)imide, was synthesized and characterized by 1H NMR, 13C NMR, 31P NMR, and IR spectroscopies, elemental (C H N S) analysis, and electrospray ionization mass spectrometry (ESI-MS). Using this TSIL a cloud point extraction (CPE) or micelle mediated extraction procedure was developed for preconcentration of uranium (U) in environmental aqueous samples. Total reflection X-ray fluorescence spectrometry was utilized to determine the concentration of U in the preconcentrated samples. In order to understand the mechanism of the CPE procedure, complexation study of the TSIL with U was carried out by isothermal calorimetric titration, liquid-liquid extraction, 31P NMR and IR spectroscopies, and ESI-MS. The developed analytical technique resulted in quantitative extraction efficiency of 99.0 ± 0.5% and a preconcentration factor of 99 for U. The linear dynamic range and method detection limit of the procedure were found to be 0.1-1000 ng mL-1 and 0.02 ng mL-1, respectively. The CPE procedure was found to tolerate a higher concentration of commonly available interfering cations and anions, especially the lanthanides. The developed analytical method was validated by determining the concentration of U in a certified reference material, viz., NIST SRM 1640a natural water, which was found to be in good agreement at a 95% confidence limit with the certified value. The method was successfully applied to the U determination in three natural water samples with ≤4% relative standard deviation (1σ).

New oligodeoxyribonucleotide derivatives bearing internucleotide N-tosyl phosphoramidate groups: Synthesis and complementary binding to DNA and RNA

N-Sulfonyl phosphoramidate derivatives of oligodeoxyribonucleotides containing N-tosyl phosphoramidate groups are first reported. The synthesis is based on Staudinger reaction between tosyl azide and 3′,5′-dinucleoside β-cyanoethyl phosphite comprising the immobilized oligonucleotide, which is obtained by the phosphoramidite coupling during the solid-phase oligonucleotide synthesis. The N-tosyl phosphoramidate group was stable under conditions of the oligonucleotide synthesis, in particular, upon acidic detritylation followed by the removal of protective groups and cleavage from the polymer support by the treatment with concentrated aqueous ammonia at 55°C. The stability of DNA and RNA duplexes of the model oligonucleotides containing N-tosyl phosphoramidate groups was only slightly lower than that of native DNA:DNA and DNA:RNA duplexes, respectively.

ChemInform Abstract: Direct Synthesis of Methyl Phosphoramidates in Carbohydrates

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ChemInform Abstract: Synthesis, Crystal Structures, and in silico Toxicity Prediction of Thienopyridine Phosphoramidates.

Abstract New thieno[2,3-b]pyridine phosphoramidates compounds were synthesized and characterized by infrared; 1H, 13C, and 31P NMR spectroscopy; and high-resolution mass spectrometry. The products were obtained in good yields (64–82%) under mild conditions by nucleophilic aromatic substitution reaction of aminoalkylphosphoramidates over 4-chlorothieno[2,3-b]pyridine-5-carbonitrile. The crystal structures of two compounds were solved by x-ray diffraction and showed a network of intermolecular interactions involving phosphoramidate groups. Druglike properties and toxicity of the new compounds were studied with the help of the software Molinspiration, Osiris, and Toxtree, and were compared with the standard drugs amphotericin B, miltefosine, benznidazole, and nifurtimox. [Supplementary materials are available for this article. Go to the publisher's online edition of Synthetic Communications® for the following free supplemental resource(s): Full experimental and spectral details.]

Synthesis and characterization of new 1H-pyrazolo[3,4-b]pyridine phosphoramidate derivatives

Twelve new 1 H-pyrazolo[3,4-b]pyridine phosphoramidate derivatives were synthesized under mild conditions by nucleophilic aromatic substitution reaction of aminoalkylphosphoramidates over 4-Cl substituted pyrazolo[3,4-b]pyridine in good yields. The new compounds were characterized by IR, 1 H, 13 C and 31 P NMR spectroscopy and HRMS. The crystal structure of one compound was solved by X-ray diffraction and showed a network of intermolecular interactions involving phosphoramidate groups.

Synthesis, Crystal Structures, and in Silico Toxicity Prediction of Thienopyridine Phosphoramidates

New thieno[2,3-b]pyridine phosphoramidates compounds were synthesized and characterized by infrared; 1H, 13C, and 31P NMR spectroscopy; and high-resolution mass spectrometry. The products were obtained in good yields (64–82%) under mild conditions by nucleophilic aromatic substitution reaction of aminoalkylphosphoramidates over 4-chlorothieno[2,3-b]pyridine-5-carbonitrile. The crystal structures of two compounds were solved by x-ray diffraction and showed a network of intermolecular interactions involving phosphoramidate groups. Druglike properties and toxicity of the new compounds were studied with the help of the software Molinspiration, Osiris, and Toxtree, and were compared with the standard drugs amphotericin B, miltefosine, benznidazole, and nifurtimox. [Supplementary materials are available for this article. Go to the publisher's online edition of Synthetic Communications® for the following free supplemental resource(s): Full experimental and spectral details.]