Facile synthesis of amino-functionalized magnetic materials for efficient enrichment of anionic metabolites from biological samples.

The analysis of biological samples is often affected by the background matrix. Proper sample preparation is a critical step in the analytical procedure for complex samples. In this study, a simple and efficient enrichment strategy based on Amino-functionalized Polymer-Magnetic MicroParticles (NH2-PMMPs) with coral-like porous structures was developed to enable the detection of 320 anionic metabolites, providing detailed coverage of phosphorylation metabolism. Among them, 102 polar phosphate metabolites including nucleotides, cyclic nucleotides, sugar nucleotides, phosphate sugars, and phosphates, were enriched and identified from serum, tissues, and cells. Furthermore, the detection of 34 previously unknown polar phosphate metabolites in serum samples demonstrates the advantages of this efficient enrichment method for mass spectrometric analysis. The limit of detections (LODs) were between 0.02 and 4 nmol/L for most anionic metabolites and its high sensitivity enabled the detection of 36 polar anion metabolites from 10 cell equivalent samples. This study has provided a promising tool for the efficient enrichment and analysis of anionic metabolites in biological samples with high sensitivity and broad coverage, facilitating the knowledge of the phosphorylation processes of life.

Nucleotide Analogues Bearing a C2' or C3'-Stereogenic All-Carbon Quaternary Center as SARS-CoV-2 RdRp Inhibitors.

The design of novel nucleoside triphosphate (NTP) analogues bearing an all-carbon quaternary center at C2' or C3' is described. The construction of this all-carbon stereogenic center involves the use of an intramoleculer photoredox-catalyzed reaction. The nucleoside analogues (NA) hydroxyl functional group at C2' was generated by diastereoselective epoxidation. In addition, highly enantioselective and diastereoselective Mukaiyama aldol reactions, diastereoselective N-glycosylations and regioselective triphosphorylation reactions were employed to synthesize the novel NTPs. Two of these compounds are inhibitors of the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2, the causal virus of COVID-19.

2',3'-Cyclic GMP-AMP Dinucleotides for STING-Mediated Immune Modulation: Principles, Immunotherapeutic Potential, and Synthesis.

The cGAS-STING pathway discovered ten years ago is an important component of the innate immune system. Activation of cGAS-STING triggers downstream signalling, such as TBK1-IRF3, NF-κB and autophagy, which in turn leads to antipathogen responses, durable antitumour immunity or autoimmune diseases. 2',3'-Cyclic GMP-AMP dinucleotides (2',3'-cGAMP), the key second messengers produced by cGAS, play a pivotal role in cGAS-STING signalling by binding and activating STING. Thus, 2',3'-cGAMP has immunotherapeutic potential, which in turn has stimulated research on the design and synthesis of 2',3'-cGAMP analogues for clinical applications over the past ten years. This review presents the discovery, metabolism, and function of 2',3'-cGAMP in the cGAS-STING innate immune signalling axis. The enzymatic and chemical syntheses of 2',3'-cGAMP analogues as STING-targeting therapeutics are also summarized.

Simple and Easy Synthesis of γ-Amido-dNTPs in Water and Their Polymerase Reaction Properties.

γ-Amido-modified 2'-deoxynucleoside triphosphates (dNTPs) and nucleoside triphosphates (NTPs) are becoming increasingly important as biological tools. We herein describe the simple and easy synthesis of γ-amido-dNTPs and -NTPs from commercially available corresponding dNTPs and NTPs in a one-pot reaction using water-soluble carbodiimide and ammonia solution. We examined the effects of synthesized γ-amido-dNTPs on the DNA polymerase reaction. The results obtained showed the incorporation of these derivatives into the DNA primer while maintaining nucleobase selectivity; however, their incorporation efficiency by DNA polymerase was lower than that of dNTP. This is the first study to demonstrate the successful synthesis of four sets of γ-amido-dNTPs and clarify their properties.

Non-Canonical Helical Structure of Nucleic Acids Containing Base-Modified Nucleotides.

Chemically modified nucleobases are thought to be important for therapeutic purposes as well as diagnosing genetic diseases and have been widely involved in research fields such as molecular biology and biochemical studies. Many artificially modified nucleobases, such as methyl, halogen, and aryl modifications of purines at the C8 position and pyrimidines at the C5 position, are widely studied for their biological functions. DNA containing these modified nucleobases can form non-canonical helical structures such as Z-DNA, G-quadruplex, i-motif, and triplex. This review summarizes the synthesis of chemically modified nucleotides: (i) methylation, bromination, and arylation of purine at the C8 position and (ii) methylation, bromination, and arylation of pyrimidine at the C5 position. Additionally, we introduce the non-canonical structures of nucleic acids containing these modifications.

Synthesis of a novel cyclopropyl phosphonate nucleotide as a phosphate mimic.

The inherent in vivo instability of oligonucleotides presents one of many challenges in the development of RNAi-based therapeutics. Chemical modification to the 5'-terminus serves as an existing paradigm which can make phosphorylated antisense strands less prone to degradation by endogenous enzymes. It has been recently shown that installation of 5'-cyclopropyl phosphonate on the terminus of an oligonucleotide results in greater knockdown of a targeted protein when compared to its unmodified phosphate derivative. In this paper we report the synthesis of a 5'-modified uridine.

Syntheses of Morpholine-Based Nucleotide Analogs for Hepatic siRNA Targeting and Stabilization.

A morpholine-based nucleotide analog was developed as a building block for hepatic siRNA targeting and stabilization. Attachment of an asialoglycoprotein-binding GalNAc ligand at the morpholine nitrogen was realized with different linkers. The obtained morpholino GalNAc scaffolds were coupled to the sense strand of a transthyretin-targeting siRNA and tested for their knockdown potency in vitro and in vivo. A clear structure-activity relationship was developed with regard to the linker type and length as well as the attachment site of the morpholino GalNAc moieties at the siRNA sense strand. Further, simple alkylation of the morpholine nitrogen led to a nucleotide analog, which increased siRNA stability, when used as a double 3'-overhang at the sense strand sequence. Combination of the best morpholino GalNAc building blocks as targeting nucleotides with an optimized stabilizing alkyl-substituted morpholine as 3'-overhangs resulted in siRNAs without any phosphorothioate stabilization in the sense strand and clearly improved the duration of action in vivo.

4-Substituted-1,2,3-triazolo nucleotide analogues as CD73 inhibitors, their synthesis, in vitro screening, kinetic and in silico studies.

Three series of nucleotide analogues were synthesized and evaluated as potential CD73 inhibitors. Nucleobase replacement consisted in connecting the appropriate aromatic or purine residues through a triazole moiety that is generated from 1,3-dipolar cycloaddition. The first series is related to 4-substituted-1,2,3-triazolo-ß-hydroxyphosphonate ribonucleosides. Additional analogues were also obtained, in which the phosphonate group was replaced by a bisphosphonate pattern (P-C-P-C, series 2) or the ribose moiety was removed leading to acyclic derivatives (series 3). The ß-hydroxyphosphonylphosphonate ribonucleosides (series 2) were found to be potent inhibitors of CD73 using both purified recombinant protein and cell-based assays. Two compounds (2a and 2b) that contained a bis(trifluoromethyl)phenyl or a naphthyl substituents proved to be the most potent inhibitors, with IC50 values of 4.8 ± 0.8 µM and 0.86 ± 0.2 µM, compared to the standard AOPCP (IC50 value of 3.8 ± 0.9 µM), and were able to reverse the adenosine-mediated immune suppression on human T cells. This series of compounds illustrates a new type of CD73 inhibitors.

Synthesis of α,δ-Disubstituted Tetraphosphates and Terminally-Functionalized Nucleoside Pentaphosphates.

The anion [P4O11]2-, employed as its bis(triphenylphosphine)iminium (PPN) salt, is shown herein to be a versatile reagent for nucleophile tetraphosphorylation. Treatment under anhydrous conditions with an alkylamine base and a nucleophile (HNuc1), such as an alcohol (neopentanol, cyclohexanol, 4-methylumbelliferone, and Boc-Tyr-OMe), an amine (propargylamine, diethylamine, morpholine, 3,5-dimethylaniline, and isopropylamine), dihydrogen phosphate, phenylphosphonate, azide ion, or methylidene triphenylphosphorane, results in nucleophile substituted tetrametaphosphates ([P4O11Nuc1]3-) as mixed PPN and alkylammonium salts in 59% to 99% yield. Treatment of the resulting functionalized tetrametaphosphates with a second nucleophile (HNuc2), such as hydroxide, a phenol (4-methylumbelliferone), an amine (propargylamine and ethanolamine), fluoride, or a nucleoside monophosphate (uridine monophosphate, deoxyadenosine monophosphate, and adenosine monophosphate), results in ring opening to linear tetraphosphates bearing one nucleophile on each end ([Nuc1(PO3)3PO2Nuc2]4-). When necessary, these linear tetraphosphates are purified by reverse phase or anion exchange HPLC, yielding triethylammonium or ammonium salts in 32% to 92% yield from [PPN]2[P4O11]. Phosphorylation of methylidene triphenylphosphorane as Nuc1 yields a new tetrametaphosphate-based ylide ([Ph3PCHP4O11]3-, 94% yield). Wittig olefination of 2',3'-O-isopropylidene-5'-deoxy-5'-uridylaldehyde using this ylide results in a 3'-deoxy-3',4'-didehydronucleotide derivative, isolated as the triethylammonium salt in 54% yield.

Synthesis of selected unnatural sugar nucleotides for biotechnological applications.

Sugar nucleotides are the principal building blocks for the synthesis of most complex carbohydrates and are crucial intermediates in carbohydrate metabolism. Uridine diphosphate (UDP) monosaccharides are among the most common sugar nucleotide donors and are transferred to glycosyl acceptors by glycosyltransferases or synthases in glycan biosynthetic pathways. These natural nucleotide donors have great biological importance, however, the synthesis and application of unnatural sugar nucleotides that are not available from in vivo biosynthesis are not well explored. In this review, we summarize the progress in the preparation of unnatural sugar nucleotides, in particular, the widely studied UDP-GlcNAc/GalNAc analogs. We focus on the "two-block" synthetic pathway that is initiated from monosaccharides, in which the first block is the synthesis of sugar-1-phosphate and the second block is the diphosphate bond formation. The biotechnological applications of these unnatural sugar nucleotides showing their physiological and pharmacological potential are discussed.

Prebiotic Syntheses of Noncanonical Nucleosides and Nucleotides.

The origin of nucleotides is a major question in origins-of-life research. Given the central importance of RNA in biology and the influential RNA World hypothesis, a great deal of this research has focused on finding possible prebiotic syntheses of the four canonical nucleotides of coding RNA. However, the use of nucleotides in other roles across the tree of life might be evidence that nucleotides have been used in noncoding roles for even longer than RNA has been used as a genetic polymer. Likewise, it is possible that early life utilized nucleotides other than the extant nucleotides as the monomers of informational polymers. Therefore, finding plausible prebiotic syntheses of potentially ancestral noncanonical nucleotides may be of great importance for understanding the origins and early evolution of life. Experimental investigations into abiotic noncanonical nucleotide synthesis reveal that many noncanonical nucleotides and related glycosides are formed much more easily than the canonical nucleotides. An analysis of the mechanisms by which nucleosides and nucleotides form in the solution phase or in drying-heating reactions from pre-existing sugars and heterocycles suggests that a wide variety of noncanonical nucleotides and related glycosides would have been present on the prebiotic Earth, if any such molecules were present.

Rapid Synthesis of Nucleoside Triphosphates and Analogues.

Nucleoside triphosphates (NTPs) are essential biomolecules involved in almost all biological processes, and their study is therefore critical to understanding cellular biology. Here, we describe a chemical synthesis suitable for obtaining both natural and highly modified NTPs, which can, for example, be used as surrogates to probe biological processes. The approach includes the preparation of a reagent that enables the facile introduction and modification of three phosphate units: cyclic pyrophosphoryl P-amidite (c-PyPA), derived from pyrophosphate (PV ) and a reactive phosphoramidite (PIII ). By using non-hydrolyzable analogues of pyrophosphate, the reagent can be readily modified to obtain a family of non-hydrolyzable analogues containing CH2 , CF2 , CCl2 , and NH that are stable in solution for several weeks if stored appropriately. They enable the synthesis of NTPs by reaction with nucleosides to give deoxycyclotriphosphate esters that are then oxidized to cyclotriphosphate (cyclo-TP) esters. The use of different oxidizing agents provides an opportunity for modification at P-α. Furthermore, terminal modifications at P-γ can be introduced by linearization of the cyclo-TP ester with various nucleophiles. © 2020 The Authors. Basic Protocol 1: Synthesis of cyclic pyrophosphoryl P-amidite (c-PyPA) and derivatives (c-PyNH PA, c-PyCH2 PA, c-PyCCl2 PA, c-PyCF2 PA) Basic Protocol 2: Synthesis of 3'-azidothymidine 5'-γ-P-propargylamido triphosphates and analogues Basic Protocol 3: Synthesis of 2'-deoxythymidine 5'-γ-P-propargylamido triphosphate (15) Basic Protocol 4: Synthesis of adenosine 5'-γ-P-amido triphosphate (19) and adenosine 5'-γ-P-propargylamido triphosphate (20) Basic Protocol 5: Synthesis of d4T 5'-γ-propargylamido ß,γ-(difluoromethylene)triphosphate Support Protocol: Synthesis of diisopropylphosphoramidous dichloride.

Synthesis and Biochemical Evaluation of Noncyclic Nucleotide Exchange Proteins Directly Activated by cAMP 1 (EPAC1) Regulators.

Exchange proteins directly activated by cAMP (EPAC) play a central role in various biological functions, and activation of the EPAC1 protein has shown potential benefits for the treatment of various human diseases. Herein, we report the synthesis and biochemical evaluation of a series of noncyclic nucleotide EPAC1 activators. Several potent EPAC1 binders were identified including 25g, 25q, 25n, 25u, 25e, and 25f, which promote EPAC1 guanine nucleotide exchange factor activity in vitro. These agonists can also activate EPAC1 protein in cells, where they exhibit excellent selectivity toward EPAC over protein kinase A and G protein-coupled receptors. Moreover, 25e, 25f, 25n, and 25u exhibited improved selectivity toward activation of EPAC1 over EPAC2 in cells. Of these, 25u was found to robustly inhibit IL-6-activated signal transducer and activator of transcription 3 (STAT3) and subsequent induction of the pro-inflammatory vascular cell adhesion molecule 1 (VCAM1) cell-adhesion protein. These novel EPAC1 activators may therefore act as useful pharmacological tools for elucidation of EPAC function and promising drug leads for the treatment of relevant human diseases.

Highly regioselective methylation of inosine nucleotide: an efficient synthesis of 7-methylinosine nucleotide.

A facile, straightforward, reliable, and an efficient chemical synthesis of inosine nucleotides such as 7-methylinosine 5'-O-monophosphate, 7-methylinosine 5'-O-diphosphate, and 7-methylinosine 5'-O-triphosphate, starting from the corresponding inosine nucleotide is delineated. The present methylation reaction of inosine nucleotide utilizes dimethyl sulfate as a methylating agent and water as a solvent at room temperature. It is noteworthy that the present methylation reaction proceeds smoothly under aqueous conditions that is highly regioselective to afford exclusive 7-methylinosine nucleotide in good yields with high purity (>99.5%).

Radiosynthesis of [18F]-Labelled Pro-Nucleotides (ProTides).

Phosphoramidate pro-nucleotides (ProTides) have revolutionized the field of anti-viral and anti-cancer nucleoside therapy, overcoming the major limitations of nucleoside therapies and achieving clinical and commercial success. Despite the translation of ProTide technology into the clinic, there remain unresolved in vivo pharmacokinetic and pharmacodynamic questions. Positron Emission Tomography (PET) imaging using [18F]-labelled model ProTides could directly address key mechanistic questions and predict response to ProTide therapy. Here we report the first radiochemical synthesis of [18F]ProTides as novel probes for PET imaging. As a proof of concept, two chemically distinct radiolabelled ProTides have been synthesized as models of 3'- and 2'-fluorinated ProTides following different radiosynthetic approaches. The 3'-[18F]FLT ProTide was obtained via a late stage [18F]fluorination in radiochemical yields (RCY) of 15-30% (n = 5, decay-corrected from end of bombardment (EoB)), with high radiochemical purities (97%) and molar activities of 56 GBq/µmol (total synthesis time of 130 min.). The 2'-[18F]FIAU ProTide was obtained via an early stage [18F]fluorination approach with an RCY of 1-5% (n = 7, decay-corrected from EoB), with high radiochemical purities (98%) and molar activities of 53 GBq/µmol (total synthesis time of 240 min).

Thiophene-linked tetramethylbodipy-labeled nucleotide for viscosity-sensitive oligonucleotide probes of hybridization and protein-DNA interactions.

Cytosine 2'-deoxyribonucleoside dCTBdp and its triphosphate (dCTBdpTP) bearing tetramethylated thiophene-bodipy fluorophore attached at position 5 were designed and synthesized. The green fluorescent nucleoside dCTBdp showed a perfect dependence of fluorescence lifetime on the viscosity. The modified triphosphate dCTBdpTP was substrate to several DNA polymerases and was used for in vitro enzymatic synthesis of labeled oligonucleotides (ONs) or DNA by primer extension. The labeled single-stranded ONs showed a significant decrease in mean fluorescence lifetime when hybridized to the complementary strand of DNA or RNA and were also sensitive to mismatches. The labeled dsDNA sensed protein binding (p53), which resulted in the increase of its fluorescence lifetime. The triphosphate dCTBdpTP was transported to live cells where its interactions could be detected by FLIM but it did not show incorporation to genomic DNA in cellulo.

Chemistry of Abiotic Nucleotide Synthesis.

The chemistry of abiotic nucleotide synthesis of RNA and DNA in the context of their prebiotic origins on early earth is a continuing challenge. How did (or how can) the nucleotides form and assemble from the small molecule inventories and under conditions that prevailed on early earth 3.5-4 billion years ago? This review provides a background and up-to-date progress that will allow the reader to judge where the field stands currently and what remains to be achieved. We start with a brief primer on the biological synthesis of nucleotides, followed by an extensive focus on the prebiotic formation of the components of nucleotides-either via the synthesis of ribose and the canonical nucleobases and then joining them together or by building both the conjoined sugar and nucleobase, part-by-part-toward the ultimate goal of forming RNA and DNA by polymerization. The review will emphasize that there are-and will continue to be-many more questions than answers from the synthetic, mechanistic, and analytical perspectives. We wrap up the review with a cautionary note in this context about coming to conclusions as to whether the problem of chemistry of prebiotic nucleotide synthesis has been solved.

P(V) Reagents for the Scalable Synthesis of Natural and Modified Nucleoside Triphosphates.

Natural and modified nucleoside triphosphates impact nearly every major aspect of healthcare research from DNA sequencing to drug discovery. However, a scalable synthetic route to these molecules has long been hindered by the need for purification by high performance liquid chromatography (HPLC). Here, we describe a fundamentally different approach that uses a novel P(V) pyrene pyrophosphate reagent to generate derivatives that are purified by silica gel chromatography and converted to the desired compounds on scales vastly exceeding those achievable by HPLC. The power of this approach is demonstrated through the synthesis of a broad range of natural and unnatural nucleoside triphosphates (dNTPs and xNTPs) using protocols that are efficient, inexpensive, and operationally straightforward.

Targeting Huntingtin Expression in Patients with Huntington's Disease.

BACKGROUND: Huntington's disease is an autosomal-dominant neurodegenerative disease caused by CAG trinucleotide repeat expansion in HTT, resulting in a mutant huntingtin protein. IONIS-HTTRx (hereafter, HTTRx) is an antisense oligonucleotide designed to inhibit HTT messenger RNA and thereby reduce concentrations of mutant huntingtin. METHODS: We conducted a randomized, double-blind, multiple-ascending-dose, phase 1-2a trial involving adults with early Huntington's disease. Patients were randomly assigned in a 3:1 ratio to receive HTTRx or placebo as a bolus intrathecal administration every 4 weeks for four doses. Dose selection was guided by a preclinical model in mice and nonhuman primates that related dose level to reduction in the concentration of huntingtin. The primary end point was safety. The secondary end point was HTTRx pharmacokinetics in cerebrospinal fluid (CSF). Prespecified exploratory end points included the concentration of mutant huntingtin in CSF. RESULTS: Of the 46 patients who were enrolled in the trial, 34 were randomly assigned to receive HTTRx (at ascending dose levels of 10 to 120 mg) and 12 were randomly assigned to receive placebo. Each patient received all four doses and completed the trial. Adverse events, all of grade 1 or 2, were reported in 98% of the patients. No serious adverse events were seen in HTTRx-treated patients. There were no clinically relevant adverse changes in laboratory variables. Predose (trough) concentrations of HTTRx in CSF showed dose dependence up to doses of 60 mg. HTTRx treatment resulted in a dose-dependent reduction in the concentration of mutant huntingtin in CSF (mean percentage change from baseline, 10% in the placebo group and -20%, -25%, -28%, -42%, and -38% in the HTTRx 10-mg, 30-mg, 60-mg, 90-mg, and 120-mg dose groups, respectively). CONCLUSIONS: Intrathecal administration of HTTRx to patients with early Huntington's disease was not accompanied by serious adverse events. We observed dose-dependent reductions in concentrations of mutant huntingtin. (Funded by Ionis Pharmaceuticals and F. Hoffmann-La Roche; ClinicalTrials.gov number, NCT02519036.).

[Hachimoji DNA extends coding possibilities]. / Extension du domaine du codage : l'ADN hachimoji - Chroniques génomiques.

The synthesis of four new nucleotide analogues that can form hydrogen bonds within the DNA double helix and can be incorporated without distortion of the structure extends the possibilities of synthetic biology. Although functional use of these analogues remains in the future, they already have interesting applications and represent an important step forward.