Access to Reverse Glycosyl Azides and Rare Sugar-Based Glycosyl Azides via Radical Decarboxylative Azidation: Divergent Synthesis of 4'-C-Azidonucleosides as Potential Antiviral Agents.

The radical decarboxylative azidation of structurally diverse uronic acids has been established as an efficient approach to reverse glycosyl azides and rare sugar-derived glycosyl azides under the action of Ag2CO3, 3-pyridinesulfonyl azide, and K2S2O8. The power of this method has been highlighted by the divergent synthesis of 4'-C-azidonucleosides using Vorbrüggen glycosylation of nucleobases with 4-C-azidofuranosyl acetates. The antiviral assessment of the resulting nucleosides revealed one compound as a potential inhibitor of covalently closed circular DNA.

Friedel-Crafts reactions for biomolecular chemistry.

Chemical tools and principles have become central to biological and medical research/applications by leveraging a range of classical organic chemistry reactions. Friedel-Crafts alkylation and acylation are arguably some of the most well-known and used synthetic methods for the preparation of small molecules but their use in biological and medical fields is relatively less frequent than the other reactions, possibly owing to the notion of their plausible incompatibility with biological systems. This review demonstrates advances in Friedel-Crafts alkylation and acylation reactions in a variety of biomolecular chemistry fields. With the discoveries and applications of numerous biomolecule-catalyzed or -assisted processes, these reactions have garnered considerable interest in biochemistry, enzymology, and biocatalysis. Despite the challenges of reactivity and selectivity of biomolecular reactions, the alkylation and acylation reactions demonstrated their utility for the construction and functionalization of all the four major biomolecules (i.e., nucleosides, carbohydrates/saccharides, lipids/fatty acids, and amino acids/peptides/proteins), and their diverse applications in biological, medical, and material fields are discussed. As the alkylation and acylation reactions are often fundamental educational components of organic chemistry courses, this review is intended for both experts and nonexperts by discussing their basic reaction patterns (with the depiction of each reaction mechanism in the ESI) and relevant real-world impacts in order to enrich chemical research and education. The significant growth of biomolecular Friedel-Crafts reactions described here is a testament to their broad importance and utility, and further development and investigations of the reactions will surely be the focus in the organic biomolecular chemistry fields.

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.

Recent advances in the synthesis of 4'-truncated nucleoside phosphonic acid analogues.

The synthesis of five series of 4'-truncated nucleoside phosphonic acid analogues is discussed in this review: (1) 4'-truncated furanose nucleoside phosphonic acid analogues; (2) 4'-truncated pyrrolidine nucleoside phosphonic acid analogues; (3) 4'-truncated carbocyclic nucleoside phosphonic acid analogues; (4) 4'-truncated isoxazole nucleoside phosphonic acid analogues; (5) 4'-truncated miscellaneous nucleoside phosphonic acid analogues. Five different ways are used to make the phosphonate moiety: (i) Michaelis-Arbuzov reaction of RX (X = Br, I, OTf) with trialkyl phosphate; (ii) Lewis acid catalyzed Michaelis-Arbuzov reaction of glycoside with trialkyl phosphite; (iii) nucleophilic addition of a dialkyl phosphite to a carbonyl group; (iv) direct coupling reaction with amino alkyl phosphonate; (v) de novo synthesis of phosphonated-isoxazole and 1,3-dioxolane heterocycles from phosphonated starting materials. Their biological activity results are briefly discussed.

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.

Synthesis of Nucleoside-like Molecules from a Pyrolysis Product of Cellulose and Their Computational Prediction as Potential SARS-CoV-2 RNA-Dependent RNA Polymerase Inhibitors.

(1R,5S)-1-Hydroxy-3,6-dioxa-bicyclo[3.2.1]octan-2-one, available by an efficient catalytic pyrolysis of cellulose, has been applied as a chiral building block in the synthesis of seven new nucleoside analogues, with structural modifications on the nucleobase moiety and on the carboxyl- derived unit. The inverted configuration by Mitsunobu reaction used in their synthesis was verified by 2D-NOESY correlations, supported by the optimized structure employing the DFT methods. An in silico screening of these compounds as inhibitors of SARS-CoV-2 RNA-dependent RNA polymerase has been carried out in comparison with both remdesivir, a mono-phosphoramidate prodrug recently approved for COVID-19 treatment, and its ribonucleoside metabolite GS-441524. Drug-likeness prediction and data by docking calculation indicated compound 6 [=(3S,5S)-methyl 5-(hydroxymethyl)-3-(6-(4-methylpiperazin-1-yl)-9H-purin-9-yl)tetrahydrofuran-3-carboxylate] as the best candidate. Furthermore, molecular dynamics simulation showed a stable interaction of structure 6 in RNA-dependent RNA polymerase (RdRp) complex and a lower average atomic fluctuation than GS-441524, suggesting a well accommodation in the RdRp binding pocket.

Total synthesis and chemical stability of pseudouridimycin.

We report the chemical synthesis of pseudouridimycin (1), an antimicrobial natural product that potently and selectively inhibits bacterial RNA polymerase. Chemical stability studies revealed intramolecular hydroxamate bond scission to be a major decomposition pathway for 1 in aqueous buffer. Replacement of the hydroxamate bond with a tertiary amide, as in 16, afforded a conformational isostere resistant to degradation. These studies pave the way for the design and synthesis of analogues with improved chemical stability and biological activity.

Mononucleoside phosphorodithiolates as mononucleotide prodrugs.

The synthesis and in vitro anti-HIV activity of a novel series of pronucleotides are reported. These prodrugs were characterized by a phosphorodithiolate structure, incorporating two O-pivaloyl-2-oxyethyl substituents as biolabile phosphate protections. The compounds were obtained following an original one-pot three-step procedure, involving the formation of a phosphorodithioite intermediate which is in situ oxidized. In vitro, comparative anti-HIV evaluations demonstrate that such original prodrugs are able to allow the efficient intracellular release of the corresponding 5'-mononucleotide. The pronucleotide of 2',3'-dideoxyadenosine (ddA) 3 exhibited a very potent antiretroviral effect with 50% effective concentration (EC50) values in nanomolar concentration range in various cell lines. In primary monocytes/macrophages, this derivative was 500 times more potent in inhibiting HIV replication (EC50 0.23 pM) than ddA and the selectivity index of the prodrug is fifty times higher than the one of the parent nucleoside.

Chemoselective and Diastereoselective Synthesis of C-Aryl Nucleoside Analogues by Nickel-Catalyzed Cross-Coupling of Furanosyl Acetates with Aryl Iodides.

Canonical nucleosides are vulnerable to enzymatic and chemical degradation, yet their stable mimics-C-aryl nucleosides-have demonstrated potential utility in medicinal chemistry, chemical biology, and synthetic biology, although current synthetic methods remain limited in terms of scope and selectivity. Herein, we report a cross-electrophile coupling to prepare C-aryl nucleoside analogues from readily available furanosyl acetates and aryl iodides. This nickel-catalyzed modular approach is characterized by mild reaction conditions, broad substrate scope, excellent ß-selectivity, and high functional-group compatibility. The exclusive chemoselectivity with respect to the aryl iodide enables efficient preparation of a variety of C-aryl halide furanosides suitable for various downstream transformations. The practicality of this transformation is demonstrated through the synthesis of a potent analogue of a naturally occurring NF-κB activator.

Polymerase Synthesis of DNA Containing Iodinated Pyrimidine or 7-Deazapurine Nucleobases and Their Post-synthetic Modifications through the Suzuki-Miyaura Cross-Coupling Reactions.

All four iodinated 2'-deoxyribonucleoside triphosphates (dNTPs) derived from 5-iodouracil, 5-iodocytosine, 7-iodo-7-deazaadenine and 7-iodo-7-deazaguanine were prepared and studied as substrates for KOD XL DNA polymerase. All of the nucleotides were readily incorporated by primer extension and by PCR amplification to form DNA containing iodinated nucleobases. Systematic study of the Suzuki-Miyaura cross-coupling reactions with two bulkier arylboronic acids revealed that the 5-iodopyrimidines were more reactive and gave cross-coupling products both in the terminal or internal position in single-stranded oligonucleotides (ssONs) and in the terminal position of double-stranded DNA (dsDNA), whereas the 7-iodo-7-deazapurines were less reactive and gave cross-coupling products only in the terminal position. None of the four iodinated bases reacted in an internal position of dsDNA. These findings are useful for the use of the iodinated nucleobases for post-synthetic modification of DNA with functional groups for various applications.

Direct and Catalytic C-Glycosylation of Arenes: Expeditious Synthesis of the Remdesivir Nucleoside.

Since early 2020, scientists have strived to find an effective solution to fight SARS-CoV-2, in particular by developing reliable vaccines that inhibit the spread of the disease and repurposing drugs for combatting its effects on the human body. The antiviral prodrug Remdesivir is still the most widely used therapeutic during the early stages of the infection. However, the current synthetic routes rely on the use of protecting groups, air-sensitive reagents, and cryogenic conditions, thus impeding a cost-efficient supply to patients. We have, therefore, focused on the development of a straightforward, direct addition of (hetero)arenes to unprotected sugars. Here we report a silylium-catalyzed and completely stereoselective C-glycosylation that initially yields the open-chain polyols, which can be selectively cyclized to provide either the kinetic α-furanose or the thermodynamically favored ß-anomer. The method significantly expedites the synthesis of Remdesivir precursor GS-441524 after a subsequent Mn-catalyzed C-H oxidation and deoxycyanation.

Clarifying the Use of Benzylidene Protecting Group for D-(+)-Ribono-1,4-Lactone, an Essential Building Block in the Synthesis of C-Nucleosides.

In the last two years, nucleosides analogues, a class of well-established bioactive compounds, have been the subject of renewed interest from the scientific community thanks to their antiviral activity. The COVID-19 global pandemic, indeed, spread light on the antiviral drug Remdesivir, an adenine C-nucleoside analogue. This new attention of the medical community on Remdesivir prompts the medicinal chemists to investigate once again C-nucleosides. One of the essential building blocks to synthetize these compounds is the D-(+)-ribono-1,4-lactone, but some mechanistic aspects linked to the use of different carbohydrate protecting groups remain unclear. Here, we present our investigations on the use of benzylidene as a ribonolactone protecting group useful in the synthesis of C-purine nucleosides analogues. A detailed 1D and 2D NMR structural study of the obtained compounds under different reaction conditions is presented. In addition, a molecular modeling study at the B3LYP/6-31G* level of theory with the SM8 solvation model for CHCl3 and DMSO to support the obtained results is used. This study allows for clarifying mechanistic aspects as the side reactions and structural rearrangements liked to the use of the benzylidene protecting group.

C1'-Branched acyclic nucleoside phosphonates mimicking adenosine monophosphate: Potent inhibitors of Trypanosoma brucei adenine phosphoribosyltransferase.

Some pathogens, including parasites of the genus Trypanosoma causing Human and Animal African Trypanosomiases, cannot synthesize purines de novo and they entirely rely on the purine salvage pathway (PSP) for their nucleotide generation. Thus, their PSP enzymes are considered as promising drug targets, sparsely explored so far. Recently, a significant role of acyclic nucleoside phosphonates (ANPs) as inhibitors of key enzymes of PSP, namely of 6-oxopurine phosphoribosyltransferases (PRTs), has been discovered. Herein, we designed and synthesized two series of new ANPs branched at the C1' position as mimics of adenosine monophosphate. The novel ANPs efficaciously inhibited Trypanosoma brucei adenine PRT (TbrAPRT1) activity in vitro and it was shown that the configuration on the C1' chiral centre strongly influenced their activity: the (R)-enantiomers proved to be more potent compared to the (S)-enantiomers. Two ANPs, with Ki values of 0.39 µM and 0.57 µM, represent the most potent TbrAPRT1 inhibitors reported to date and they are an important tool to further study purine metabolism in various parasites.

Antibacterial and molecular docking studies of newly synthesized nucleosides and Schiff bases derived from sulfadimidines.

A new series of nucleosides, moieties, and Schiff bases were synthesized from sulfadimidine. Infrared (IR), 1HNMR, 13C NMR, and mass spectrometry techniques and elemental analysis were employed to elucidate the synthesized compounds. The prepared analogues were purified by different chromatographic techniques (preparative TLC and column chromatography). Molecular docking studies of synthesized compounds 3a, 4b, 6a, and 6e demonstrated the binding mode involved in the active site of DNA gyrase. Finally, all synthesized compounds were tested against selected bacterial strains. The most effective synthesized compounds against S. aureus were 3a, 4d, 4b, 3b, 3c, 4c, and 6f, which exhibited inhibition zones of inhibition of 24.33 ± 1.528, 24.67 ± 0.577, 23.67 ± 0.577, 22.33 ± 1.528, 18.67 ± 1.528 and 19.33 ± 0.577, respectively. Notably, the smallest zones were observed for 4a, 6d, 6e and 6g (6.33 ± 1.528, 11.33 ± 1.528, 11.67 ± 1.528 and 14.66 ± 1.155, respectively). Finally, 6b and 6c gave negative zone values. K. pneumoniae was treated with the same compounds and the following results were obtained. The most effective compounds were 4d, 4c, 4b and 3c, which showed inhibition zones of 29.67 ± 1.528, 24.67 ± 0.577, 23.67 ± 1.155 and 19.33 ± 1.528, respectively, followed by 4a and 3d (15.33 ± 1.528 for both), while moderate results (13.67 ± 1.155 and 11.33 ± 1.528) were obtained for 6f and 6g, respectively. Finally, 6a, 6b, 6c, 3a, and 3b did not show any inhibition. The most effective compounds observed for the treatment of E. coli were 4d, 4b, 4c, 3d, 6e and 6f (inhibition zones of 26.33 ± 0.577, 21.67 ± 1.528, 21.67 ± 1.528, 19.67 ± 1.528, 17.67 ± 1.155 and 16.67 ± 1.155, respectively). Compounds 3b, 3c, 6a, 6c, and 6g gave moderate results (13.67 ± 1.528, 12.67 ± 1.528, 11.33 ± 0.577, 15.33 ± 1.528 and 12.67 ± 1.528, respectively), while 6b showed no effect. The MIC values against S. aureus ranged from 50 to 3.125 mg, while those against E. coli and K. pneumoniae ranged from 50 to 1562 mg. In vitro, the antibacterial effects were promising. Further research is required to study the in vivo antibacterial effects of these compounds and determine therapeutic doses.

A convenient synthesis of branched-chain nucleoside isothiocyanates via aza-Claisen rearrangement.

Stereocontrolled introduction of a nitrogen atom at either C-2' or C-3' positions of nucleosides derived from uridine, 4-N-benzoylcytidine and adenosine was investigated. An efficient and rapid procedure was employed for creating new chiral centers at C-2' and C-3' positions using [3,3]-sigmatropic aza-Claisen rearrangement of allyl thiocyanates under conventional and microwave conditions. Structure of isothiocyanate products was confirmed by 1-D and 2-D NMR spectral analyses including selective 1H 1-D-NOE experiments.

anti-syn Unnatural Base Pair Enables Alphabet-Expanded DNA Self-Assembly.

Self-assembly properties and diversity in higher-order structures of DNA enable programmable tools to be used to construct algorithms at the molecular level. However, the utility of DNA-based programmable tools is hampered by the low orthogonality to natural nucleic acids, especially in complex molecular systems. To address this challenge, we report here the orthogonal regulation of DNA self-assembly by using an unnatural base pair (UBP) formation. Our newly designed UBP AnN:SyN is formed in combination with anti and unusual syn glycosidic conformation with high thermal stability and selectivity. Furthermore, AnC worked as a pH-sensitive artificial nucleobase, which forms a strong base pair with cytosine under a weak acidic condition (pH 6.0). The orthogonal AnN:SyN base pair functioned as a trigger for hybridization chain reaction to provide long nicked double-stranded DNA (ca. 1000 base pairs). This work represents the first example of the orthogonal DNA self-assembly that is nonreactive to natural four-letter alphabets DNA trigger and expands the types of programmable tools that work in a complex environment.

Synthesis of acyclic nucleoside phosphonates targeting flavin-dependent thymidylate synthase in Mycobacterium tuberculosis.

Flavin-Dependent Thymidylate Synthase (FDTS) encoded by ThyX gene was discovered as a new class of thymidylate synthase involved in the de novo synthesis of dTMP named only in 30 % of human pathogenic bacteria. This target was pursed for the development of new antibacterial agents against multiresistant pathogens. We have developed a new class of ANPs based on the mimic of two natural's cofactors (dUMP and FAD) as inhibitors against Mycobacterium tuberculosis ThyX. Several synthetic efforts were performed to optimize regioselective N1-alkylation, cross-coupling metathesis and Sonogashira cross-coupling. Compound 19c showed a poor 31.8% inhibitory effect on ThyX at 200 µM.

Molecular modelling, synthesis and antimicrobial evaluation of benzimidazole nucleoside mimetics.

A series of new N-1-(ß-d-ribofuranosyl) benzimidazole derivatives has been designed using in silico methods and synthesized as probable antimicrobial agents. Further, the compounds were assessed for their antibacterial and antifungal activity. Antibacterial screening was done by employing broth micro-dilution method and compounds exhibited excellent inhibitory activity (MIC, 50-1.56 µg/mL) against different human pathogenic bacteria, viz. B. cerus, B. subtilis, S. aureus, E. coli and P. aeruginosa and drug resistant strain (DRS) of E. coli. A great synergistic effect was observed during evaluation of ∑FIC, where a combination study was performed using standard references, viz. chloramphenicol and kanamycin. The MIC data obtained from different methods of combination approach revealed 4-128 fold more potency compared to compounds tested alone. The results clearly indicated the possibility of these compounds as active ingredients of drug regimen used against MDR strains. Antifungal screening were also performed employing two different methods, viz. serial dilution method and zone inhibition method, clearly indicated that compounds were also potentially active against several species of pathogenic fungal strains, viz. A. flavus, A. niger, F. oxysporum and C. albicans. The assessment of structure activity relationship (SAR) clearly revealed that presence of less polar and more hydrophobic substituents positively favours the antibacterial activity, conversely, more polar and hydrophilic substituents favours the antifungal activities. Thus, the results positively endorsed the compounds as potent antibacterial and antifungal agents which could be developed as possible drug regimens.

Targeting SARS-CoV-2 Polymerase with New Nucleoside Analogues.

Despite the fact that COVID-19 vaccines are already available on the market, there have not been any effective FDA-approved drugs to treat this disease. There are several already known drugs that through drug repositioning have shown an inhibitory activity against SARS-CoV-2 RNA-dependent RNA polymerase. These drugs are included in the family of nucleoside analogues. In our efforts, we synthesized a group of new nucleoside analogues, which are modified at the sugar moiety that is replaced by a quinazoline entity. Different nucleobase derivatives are used in order to increase the inhibition. Five new nucleoside analogues were evaluated with in vitro assays for targeting polymerase of SARS-CoV-2.

Menaquinone Biosynthesis: The Mechanism of 5,8-Dihydroxy-2-naphthoate Synthase (MqnD).

MqnD catalyzes the conversion of cyclic dehypoxanthine futalosine (6) to 5,8-dihydroxy-2-naphthoic acid (7) and an uncharacterized product. This study describes a chemoenzymatic synthesis of 6. This synthesis achieved a 2-fold yield enhancement by using titanium(III) citrate as the reducing agent and another 5-fold yield enhancement using a fluorinated analogue of dehypoxanthine futalosine (5) that was converted to 6 by an ipso substitution mechanism. This synthetic route enabled the synthesis of 6 in sufficient quantity to identify the second reaction product and to determine that the MqnD-catalyzed reaction proceeds by a hemiacetal ring opening-tautomerization-retroaldol sequence.