Zinc-Binding Oligonucleotide Backbone Modifications for Targeting a DNA-Processing Metalloenzyme.

A series of chemically-modified oligonucleotides for targeting the DNA repair nuclease SNM1A have been designed and synthesised. Each oligonucleotide contains a modified internucleotide linkage designed to both mimic the native phosphodiester backbone and chelate to the catalytic zinc ion(s) in the SNM1A active site. Dinucleoside phosphoramidites containing urea, squaramide, sulfanylacetamide, and sulfinylacetamide linkages were prepared and employed successfully in solid-phase oligonucleotide synthesis. All the modified oligonucleotides were found to interact with SNM1A in a gel electrophoresis-based assay, demonstrating the first examples of inhibition of DNA damage repair enzymes for many of these groups in oligonucleotides. One strand containing a sulfinylacetamide-linkage was found to have the strongest interaction with SNM1A and was further tested in a real-time fluorescence assay. This allowed an IC50 value of 231 nM to be determined, significantly lower than previously reported substrate-mimics targeting this enzyme. It is expected that these modified oligonucleotides will serve as a scaffold for the future development of fluorescent or biotin-labelled probes for the in vivo study of DNA repair processes.

Oligonucleotides Containing C5-Propynyl Modified Arabinonucleic Acids: Synthesis, Biophysical and Antisense Properties.

The introduction of chemical modifications on the nucleic acid scaffold has allowed for the progress of antisense oligonucleotides (ASOs) in the clinic for the treatment of a variety of disorders. In contribution to the repertoire of gene-silencing nucleic acid modifications, herein we report the synthesis and incorporation of C5-propynyl arabinouridine (araUP ) and arabinocytidine (araCP ) into mixed-base ASOs containing a pyrimidine core. Substitution of the core with araUP and araCP resulted in stabilization of the duplex formed with RNA but not with DNA. Similar results were obtained with ASOs bearing phosphorothioate linkages or methoxyethyl (MOE) wings in a gapmer design. All modified ASOs were compatible with E. coli RNase H mediated degradation of target RNA. Substitution of DNA for araUP and araCP in the central portion of a gapmer with MOE wings demonstrated improved nuclease resistance. These results suggest C5-modified arabinonucleic acids may serve as a potential chemical modification for therapeutic ASOs.

Prebiotic Nucleoside Synthesis: The Selectivity of Simplicity.

Ever since the discovery of nucleic acids 150 years ago,[1] major achievements have been made in understanding and decrypting the fascinating scientific questions of the genetic code.[2] However, the most fundamental question about the origin and the evolution of the genetic code remains a mystery. How did nature manage to build up such intriguingly complex molecules able to encode structure and function from simple building blocks? What conditions were required? How could the precursors survive the unhostile environment of early Earth? Over the past decades, promising synthetic concepts were proposed providing clarity in the field of prebiotic nucleic acid research. In this Minireview, we show the current status and various approaches to answer these fascinating questions.

Preparation of 5'-deoxy-5'-amino-5'-C-methyl adenosine derivatives and their activity against DOT1L.

From a readily available 5-C-Me ribofuranoside, we have realized a reliable route to valuable 5'-deoxy-5'-amino-5'-C-methyl adenosine derivatives at gram scale with confirmed stereochemistry. These adenosine derivatives are useful starting materials for the preparation of 5'-deoxy-5'-amino-5'-C-methyl adenosine derivatives with higher complexity. From one of the new adenosine derivatives, some 5'-deoxy-5'-amino-5'-C-methyl adenosine DOT1L inhibitors were prepared in several steps. Data from DOT1L assay indicated that additional 5'-C-Me group improved the enzyme inhibitory activity.

Enzymatic synthesis and RNA interference of nucleosides incorporating stable isotopes into a base moiety.

Thymidine phosphorylase was used to catalyze the conversion of thymidine (or methyluridine) and uracil incorporating stable isotopes to deoxyuridine (or uridine) with the uracil base incorporating the stable isotope. These base-exchange reactions proceeded with high conversion rates (75-96%), and the isolated yields were also good (64-87%). The masses of all synthetic compounds incorporating stable isotopes were identical to the theoretical molecular weights via EIMS. (13)C NMR spectra showed spin-spin coupling between (13)C and (15)N in the synthetic compounds, and the signals were split, further proving incorporation of the isotopes into the compounds. The RNA interference effects of this siRNA with uridine incorporating stable isotopes were also investigated. A 25mer siRNA had a strong knockdown effect on the MARCKS protein. The insertion position and number of uridine moieties incorporating stable isotopes introduced into the siRNA had no influence on the silencing of the target protein. This incorporation of stable isotopes into RNA and DNA has the potential to function as a chemically benign tracer in cells.

Improved Metal-Free Approach for the Synthesis of Protected Thiol Containing Thymidine Nucleoside Phosphoramidite and Its Application for the Synthesis of Ligatable Oligonucleotide Conjugates.

Oligonucleotide conjugates are versatile scaffolds that can be applied in DNA-based screening platforms and ligand display or as therapeutics. Several different chemical approaches are available for functionalizing oligonucleotides, which are often carried out on the 5' or 3' end. Modifying oligonucleotides in the middle of the sequence opens the possibility to ligate the conjugates and create DNA strands bearing multiple different ligands. Our goal was to establish a complete workflow that can be applied for such purposes from monomer synthesis to templated ligation. To achieve this, a monomer is required with an orthogonal functional group that can be incorporated internally into the oligonucleotide sequence. This is followed by conjugation with different molecules and ligation with the help of a complementary template. Here, we show the synthesis and the application of a thiol-modified thymidine nucleoside phosphoramidite to prepare ligatable oligonucleotide conjugates. The conjugations were performed both in solution and on solid phase, resulting in conjugates that can be assembled into multivalent oligonucleotides decorated with tissue-targeting peptides using templated ligation.

Synthesis of oxygen-linked 8-phenoxyl-deoxyguanosine nucleoside analogues.

Nucleobase adducts, which form in vivo by the nucleophilic attack of nucleobases on exogenous electrophilic species, can impact conformation and biological influences of the adducted nucleoside. Contemporary studies aim to address the occurrence and relevance of O-linked 8-phenoxy-purine adducts; however, preparative techniques for synthesizing these nucleosides were not previously described. Reported herein is a relatively facile synthesis of O-linked 8-dG phenol adducts with a wide variety of electron-donating, electron-withdrawing, and sterically demanding phenols.

Practical and reliable synthesis of 2',3',5',5″-tetradeuterated uridine derivatives.

Deuterated drugs are valuable in the fields of drug discovery and medicinal chemistry. 2',3',5',5″-tetradeuterated uridine derivatives were synthesised from 2,3,5,5'-selectively tetradeuterated ribose using Sajiki's H-D exchanged Ru/C-H2-D2O-NaOH system and silyl-Hilbert-Johnson methods. The total deuterium content of the tetradeuterated uridines was over 92% using either basic or acidic reaction conditions. These derivatives would be expected as building blocks for the synthesis of deuterium-substituted nucleic acid probes for tracking the pharmacokinetics of nucleic acid drugs.

Chemical ribosylation of 5-substituted 1,2,4-triazole-3-carboxylates.

The chemical ribosylation pathways of 5-substituted-1,2,4-triazole-3-carboxylates are discussed. For the products of the chemical synthesis of the 3(5)-alkyl- or 3(5)-aryl-substituted ribavirin analogues the anomer configuration and isomer composition were determined.

Gemcitabine analogues with 4-N-alkyl chain modified with fluoromethyl ketone group.

Gemcitabine analogues with a lipophilic 4-N-alkyl chain bearing a terminal ß-keto sulfonate moiety suitable for fluorination compatible with 18F-radiolabeling have been explored. Displacement of p-toluenesulfonylamino in protected 4-N-tosylgemcitabine with 1-amino-10-undecene gave 4-N-(10-undecenyl)-3',5'-di-O-benzoyl-2'-deoxy-2',2'-difluorocytidine. Oxidation of the terminal double bond in the latter with OsO4/NMO afforded 4-N-(10,11-dihydroxyundecanyl) derivative. Regioselective sulfonation of primary hydroxyl followed by oxidation of secondary hydroxyl with Collin's reagent yielded desired ß-keto sulfonate analogues 8 or 9. Subsequent displacement of the mesylate or tosylate group with KF in the presence of Kryptofix 2.2.2. or 18-crown-6 ether followed by deprotection with NH3/MeOH gave 4-N-(11-fluoro-10-oxoundecanyl)-2'-deoxy-2',2'-difluorocytidine 11.

Exploring the Emergence of RNA Nucleosides and Nucleotides on the Early Earth.

Understanding how life began is one of the most fascinating problems to solve. By approaching this enigma from a chemistry perspective, the goal is to define what series of chemical reactions could lead to the synthesis of nucleotides, amino acids, lipids, and other cellular components from simple feedstocks under prebiotically plausible conditions. It is well established that evolution of life involved RNA which plays central roles in both inheritance and catalysis. In this review, we present historically important and recently published articles aimed at understanding the emergence of RNA nucleosides and nucleotides on the early Earth.

Tricyclic nitrogen base 1,N6-ethenoadenine and its ribosides as substrates for purine-nucleoside phosphorylases: Spectroscopic and kinetic studies.

The title compound is an excellent substrate for E. coli PNP, as well as for its D204N mutant. The main product of the synthetic reaction is N9-riboside, but some amount of N7-riboside is also present. Surprisingly, 1,N6-ethenoadenine is also ribosylated by both wild-type and mutated (N243D) forms of calf PNP, which catalyze the synthesis of a different riboside, tentatively identified as N6-ß-D-ribosyl-1,N6-ethenoadenine. All ribosides are susceptible to phosphorolysis by the E. coli PNP (wild type). All the ribosides are fluorescent and can be utilized as analytical probes.

Synthesis of ß-Nicotinamide Riboside Using an Efficient Two-Step Methodology.

A two-step chemical method for the synthesis of ß-nicotinamide riboside (NR) is described. NR has achieved wide use as an NAD+ precursor (vitamin B3) and can significantly increase central metabolite NAD+ concentrations in mammalian cells. ß-NR can be prepared with an efficient two-step procedure. The synthesis is initiated via coupling of commercially available 1,2,3,5-tetra-O-acetyl-ß-D-ribofuranose with ethyl nicotinate in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf). 1 H NMR showed that the product was formed with complete stereoselectivity to produce only the ß-isomer in high yield (>90% versus starting sugar). The clean stereochemical result suggests that the coupling proceeds via a cationic cis-1,2-acyloxonium-sugar intermediate, which controls addition by nucleophiles to generate predominantly ß-stereochemistry. The subsequent deprotection of esters in methanolic ammonia generates the desired product in 85% overall yield versus sugar. © 2017 by John Wiley & Sons, Inc.

Dual addressing of thymidine synthesis pathways for effective targeting of proliferating melanoma.

Here, we examined the potential of blocking the thymidine de novo synthesis pathways for sensitizing melanoma cells to the nucleoside salvage pathway targeting endogenous DNA irradiation. Expression of key nucleotide synthesis and proliferation enzymes thymidylate synthase (TS) and thymidine kinase 1 (TK1) was evaluated in differentiated (MITFhigh [microphthalmia-associated transcription factor] IGR1) and invasive (MITFmedium IGR37) melanoma cells. For inhibition of de novo pathways cells were incubated either with an irreversible TS inhibitor 5-fluoro-2'-deoxyuridine (FdUrd) or with a competitive dihydrofolate-reductase (DHFR) inhibitor methotrexate (MTX). Salvage pathway was addressed by irradiation-emitting thymidine analog [123/125 I]-5-iodo-4'-thio-2'-deoxyuridine (123/125 I-ITdU). The in vivo targeting efficiency was visualized by single-photon emission computed tomography. Pretreatment with FdUrd strongly increased the cellular uptake and the DNA incorporation of 125 I-ITdU into the mitotically active IGR37 cells. This effect was less pronounced in the differentiated IGR1 cells. In vivo, inhibition of TS led to a high and preferential accumulation of 123 I-ITdU in tumor tissue. This preclinical study presents profound rationale for development of therapeutic approach by highly efficient and selective radioactive targeting one of the crucial salvage pathways in melanomas.

Regioselective Mitsunobu Reaction of Partially Protected Uridine.

Mitsunobu reaction of partially acylated uridine proceeds with high regioselectivity for intramolecular SN2 anhydro linkage closuring. Under the reaction conditions, an isomeric mixture of diacyl uridine derivatives with either free 2'- or 3'-hydroxyl group was transformed into a single cyclonucleosidic product, 2,2'-anhydro-3',5'-di-O-acyluridine. This paper presents a possible mechanism of the reactions, the explanation of observed phenomenon based on semiempirical and density functional theory (DFT) calculations and possible utility of this synthetic pathway.

Efficient Synthesis of Cladribine via the Metal-Free Deoxygenation.

The efficient synthesis of cladribine via the metal-free deoxygenation was developed. Using (Bu4N)2S2O8/HCO2Na instead of Bu3SnH/AIBN as deoxygenation system, cladribine could be obtained with good yield and even on tens of grams scales. The intermediates and product could be purified by simple work-up process and chromatography was avoided, which showed the good future for industrial applications.

Pseudoknot interaction-mediated activation of type I hammerhead ribozyme: a new class of gene-therapeutic agents.

Recently discovered hammerhead ribozymes that are activated through pseudoknot interactions (Watson-Crick base pairs between loops) are attractive candidates as gene-therapeutic agents because sequences of gene-therapeutic ribozymes can be designed simply based on the sequence complementarity against target RNAs. Herein, we examined if the newly found pseudoknot-type hammerhead ribozyme with type I topology is activated through the pseudoknot interactions. Substitutions of pseudoknot sequences into fully mismatched ones significantly reduced the activity of type I pseudoknot-type hammerhead ribozyme, while those with full-matched pseudoknot sequences were highly active. The results indicated that the pseudoknot interactions activated type I pseudoknot-type hammerhead ribozyme, making them suitable as gene-therapeutic agents.

2,8-Dihydroxyadenine urolithiasis: a not so rare inborn error of purine metabolism.

Adenine phosphoribosyltransferase (APRT) deficiency is a rare inherited metabolic disorder that leads to the formation and hyperexcretion of 2,8-dihydroxyadenine (DHA) into urine. The low solubility of DHA results in precipitation and formation of urinary crystals and kidney stones. The disease can be present as recurrent urolithiasis or nephropathy secondary to crystal precipitation into renal parenchyma (DHA nephropathy). The diagnostic tools available, including stone analysis, crystalluria, and APRT activity in red blood cells, make the diagnosis easy to confirm when APRT deficiency is suspected. However, the lack of recognition of this metabolic disorder frequently resulted in a delay in diagnosis and treatment with grave consequences. The early recognition and treatment of APRT deficiency are of crucial importance to prevent irreversible loss of renal function. This review summarizes the genetic and metabolic mechanisms underlying DHA stones formation and chronic kidney disease, along with the issues of diagnosis and management of APRT deficiency. Moreover, we report the mutations in the APRT gene responsible for APRT deficiency in 51 French patients (43 families) including 22 pediatric cases (18 families) among the 64 patients identified in the biochemistry laboratories of Necker Hospital, Paris (1978-2013).