Synthesis Of Nucleosides Research Articles

Glycolysis addiction compensating for a defective pentose phosphate pathway confers gemcitabine sensitivity in SETD2-deficient pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy driven by genetic mutations and/or epigenetic dysregulation. Gemcitabine chemotherapy is the first-line regimen for pancreatic cancer but has limited efficacy. Our previous study revealed the role of SETD2-H3K36me3 loss in the initiation and metastasis of PDAC, but little is known about its role in tumor metabolism. Here, we found that SETD2-deficient PDAC enhanced glycolysis addiction via upregulation of glucose transporter 1 (GLUT1) to meet its large demand for glucose in progression. Moreover, SETD2 deficiency impaired nucleoside synthesis by directly downregulating the transcriptional level of transketolase (TKT) in the pentose phosphate pathway. The metabolic changes confer SETD2-deficient PDAC cells with increased sensitivity to gemcitabine under glycolysis restriction conditions. Collectively, our study provides mechanistic insights into how SETD2 deficiency reprograms glycolytic metabolism to compensate for insufficient nucleoside synthesis, suggesting that glycolysis restriction combined with gemcitabine might be a potential therapeutic strategy for PDAC patients with SETD2 deficiency.

Synthesis and Biological Assessment of New Nucleosides of Pyrido[2,3-d]pyrimidine Derivatives Bearing Ribose and 2-Deoxyribose Moieties

Herein, it is unprecedentedly reported the synthesis of some new β-D-ribo and β-D-2-dexoyribofuranosyl nucleosides of pyrido[2,3-d]pyrimidines derivatives where the sugar moiety is linked to pyrido[2,3-d]pyrimidines via S or N glycosidic bond. Therefore, pyrido[2,3-d]pyrimidine derivatives 3 and 6 were utilized as scaffolds to be incorporated with ribose and 2-deoxyribose moieties. The glycosylation reactions were carried out under optimized conditions to enhance the regioselectivity and stereoselectivity during the glycosylation coupling. Various strategies have been applied to afford the desired N-nucleosides 16 and 19. Other approaches were utilized to afford the S-nucleosides 9, 13, 21, and 24. The structural elucidations of the novel nucleosides formed are reported and ascertained by using IR, 1H NMR, 13C NMR spectra and microanalysis. All the synthesized compounds were screened against a panel of microorganism strains for antibacterial and antifungal activities as well as antitumor evaluation.

Impact of loss or regain of Equilibrative Nucleoside Transporter (ENT) Subtype‐1 on ENT Subtype‐2 pharmacology in a Novel CRISPR‐Cas9 HEK293‐ENT1KO Cell Model

BackgroundEquilibrative nucleoside transporters (ENT) mediate the transmembrane flux of endogenous nucleosides and nucleoside‐analog drugs that are used to treat cancers and viral infections. Of the two major ENT subtypes (ENT1, ENT2), ENT1 has been studied the most due to its relative predominance in tissues, and the availability of the highly selective ENT1 inhibitor, nitrobenzylthioinosine (NBMPR). In contrast, there is much less information on how drugs interact with ENT2 or factors that affect ENT2 expression. This is because ENT2 is always expressed concomitantly at lower levels with ENT1, and there is no selective ENT2 inhibitor available. Given the emerging evidence suggesting that ENT2 has distinct physiological roles from ENT1, it is critical that its functional characteristics and modes of regulation are better defined in its native environment. To address these knowledge gaps, we removed ENT1 from Human Embryonic Kidney cells (HEK293) via CRISPR‐Cas 9, creating a cell line that has ENT2 as the only functional ENT in the cells (HEK293‐ENT1KO).ObjectivesTo confirm that any changes observed in the HEK293‐ENT1KO cells were due to the removal of ENT1, we have introduced recombinant ENT1 back into these cells and assessed the function and expression of the endogenous ENT2.MethodsStable transfection of MYC‐ENT1 in pcDNA3.1 was conducted in HEK293‐ENT1KO cell line using calcium phosphate precipitation method and stable clones selected with G418(Geneticin).Transporter function was assessed through measurement of the initial rates of [3H]2‐chloroadenosine uptake (2.5–300µM). Kivalues for inhibitors were defined using the IC50 derived from concentration‐response curve analyses and the Km of [3H]2‐chloroadenosine for ENT2 determined in this model. The presence or absence of ENT1 was determined based on the binding of [3H]NBMPR. Protein levels were assessed by immunoblotting using ENT‐specific antibodies and changes in expression of metabolic genes involved in de novo synthesis of nucleosides were examined by qPCR. Data are expressed as mean±SEM.ResultsMYC‐ENT1‐HEK293‐ENT1KO had a similar level of ENT2 uptake (Km‐32µM±9.7, Vmax‐0.6pmol/µl/s±0.1) as wild‐type (WT) HEK293 (Km‐19µM±1.2, Vmax‐0.7pmol/µl/s±0.3) and HEK293‐ENT1KO (Km‐25µM±17, Vmax‐0.6pmol/µl/s±0.1). In contrast, kinetic profile of the MYC‐ENT1 transfected model showed significant higher ENT1‐mediated uptake (Km‐97µM±68, Vmax‐11pmol/µl/s±9.1) than that observed in the WT‐HEK293 (Km‐90µM±27, Vmax‐3.6pmol/µl/s±2.4). Also, the number of [3H]NBMPR binding sites were also found to be higher in the transfected model. The complete regain of ENT1 in MYC‐ENT1‐HEK293‐ ENT1KO cell model was further confirmed in protein and gene expression studies. However, the presence of recombinant ENT1 did not change the endogenous ENT2 protein expression. Loss of endogenous ENT1 showed no changes in gene expression levels of other transporters or metabolic enzymes except NT5E (5′‐Nucleotidase Ecto) which reduced significantly.ConclusionOur data suggest that neither removing endogenous ENT1 from HEK293 cells nor introducing recombinant ENT1 in the HEK293‐ENT1KO model impacts the expression or function of ENT2.

Constructing Cell-Free Expression Systems for Low-Cost Access.

Cell-free systems for gene expression have gained attention as platforms for the facile study of genetic circuits and as highly effective tools for teaching. Despite recent progress, the technology remains inaccessible for many in low- and middle-income countries due to the expensive reagents required for its manufacturing, as well as specialized equipment required for distribution and storage. To address these challenges, we deconstructed processes required for cell-free mixture preparation and developed a set of alternative low-cost strategies for easy production and sharing of extracts. First, we explored the stability of cell-free reactions dried through a low-cost device based on silica beads, as an alternative to commercial automated freeze dryers. Second, we report the positive effect of lactose as an additive for increasing protein synthesis in maltodextrin-based cell-free reactions using either circular or linear DNA templates. The modifications were used to produce active amounts of two high-value reagents: the isothermal polymerase Bst and the restriction enzyme BsaI. Third, we demonstrated the endogenous regeneration of nucleoside triphosphates and synthesis of pyruvate in cell-free systems (CFSs) based on phosphoenol pyruvate (PEP) and maltodextrin (MDX). We exploited this novel finding to demonstrate the use of a cell-free mixture completely free of any exogenous nucleotide triphosphates (NTPs) to generate high yields of sfGFP expression. Together, these modifications can produce desiccated extracts that are 203–424-fold cheaper than commercial versions. These improvements will facilitate wider use of CFS for research and education purposes.

Investigations to mechanism and applications of the glycosylation protocol employing 8-methyltosylaminoethynyl-1-naphthyl (MTEAN) glycoside donors

Based on the capability of silyl ethers for efficient glycosylation, the new MTEAN glycosylation protocol utilizing 8-methyltosylaminoethynyl-1-naphthyl (MTEAN) glycosides as donors were extended to one-pot synthesis of nucleosides, late-stage modification of bioactive molecules, and stereoselective construction of 1,2-cis-glucosidic linkages using silyl ethers of the acceptors. Moreover, the reaction mechanism was systematically investigated by control reactions and side product characterizations, leading to the determination of a TfOH-catalyzed ynamide functionality-initiated process, which was further corroborated by identifications of the departure form of leaving group and the real catalyst.

Efficient Synthesis and Antimicrobial Evaluation of Acyclic Pyrimidine Nucleosides and Their Sulfanyl Analogs

Efficient Synthesis and Antimicrobial Evaluation of Acyclic Pyrimidine Nucleosides and Their Sulfanyl Analogs

Synthesis, Characterization and Molecular Docking of New Nucleosides and Schiff Bases Derived from Ampyrone as Antiviral Agents to Contain the COVID-19 Virus

A new series of nucleoside derivatives was prepared from the reaction of 4-aminoantipyrene with different sugar moieties. In addition, ampyrone’s reaction with different aromatic aldehydes gave the corresponding Schiff base derivatives, which were also synthesized. Both molecular docking and in vitro antiviral activities at different concentrations of different synthesized compounds against SARS-CoV-2 were screened. All newly synthesized compounds were characterized on the basis of IR, 1H NMR and 13C NMR spectral data and physical data. The compounds were screened for potential cytotoxic activities. The molecular docking analysis showed that compounds 6b, 6e, 6c, 6f and 6d exhibited relatively higher binding energies (−8.1, −8.1, −8.3, −8.4 and −8.7 kcal/mol, respectively) compared to all the other compounds. However, the different compounds did not show any promising in vitro antiviral activities against SARS-CoV-2.

Azide-Modified Nucleosides as Versatile Tools for Bioorthogonal Labeling and Functionalization.

Azide-modified nucleosides are important building blocks for RNA and DNA functionalization by click chemistry based on azide-alkyne cycloaddition. This has put demand on synthetic chemistry to develop approaches for the preparation of azide-modified nucleoside derivatives. We review here the available methods for the synthesis of various nucleosides decorated with azido groups at the sugar residue or nucleobase, their incorporation into oligonucleotides and cellular RNAs, and their application in azide-alkyne cycloadditions for labelling and functionalization.

Plasma metabolomics to predict chemotherapy (CTX) response in advanced pancreatic cancer (PC) patients (pts) on enteral feeding for cachexia.

600 Background: We evaluated the potential of plasma metabolites as predictors of response to CTX in a prospective cohort of pts who received enteral feeding for cachexia and advanced PC. Methods: The PANCAX-1 (NCT02400398) prospective trial enrolled 31 cachectic advanced PC pts to receive jejunal tube peptide-based diet for 12 weeks (wks) who were planned for palliative CTX. Out of 16 evaluable pts, 62.5% receiving enteral feeding met the primary endpoint of weight stability at 12 wks. As part of an exploratory analysis of the PANCAX-1 trial, serial blood samples were collected at 3 predefined timepoints over 12 wks of enteral feeding. Up to 219 plasma metabolites were analyzed by mass spectrometry and high-performance liquid chromatography. Analytes were compared by relative area under the curve (AUC) and differences evaluated by two-sample t-tests. The mean AUC was used in pts with metabolites measured from > 1 timepoint of collection. Pts were stratified by stable disease (SD), partial response (PR), or progressive disease (PD) as best overall response to standard CTX. Results: Of 31 pts with advanced PC prospectively enrolled for enteral feeding, there were 55 blood samples collected from 28 pts available for plasma metabolomics. 20/28 (71%) pts received first-line CTX, the majority of whom (90%) received gemcitabine-based CTX. There were 2 PRs (7%) and 10 with SD (36%) as best response to CTX. Overall, there were statistically significant differences in levels of intermediates involved in multiple metabolic pathways including glycolysis, the tricarboxylic acid (TCA) cycle, fatty acid synthesis, and nucleoside synthesis in pts with PR/SD vs. PD to CTX (all p < 0.05). When stratified by CTX regimen, PD to 5-fluorouracil-based CTX (e.g., FOLFIRINOX) was associated with decreased levels of essential amino acids (AAs, L-leucine, L-methionine, L-tryptophan) and non-essential AAs (L-arginine, L-serine, L-tyrosine, all p < 0.05). For gemcitabine-based CTX (e.g., gemcitabine/nab-paclitaxel), PD was associated with increased levels of intermediates of glycolysis (pyruvate), TCA cycle (L-glutamate), nucleoside synthesis (xanthine), and bile acid metabolism (taurocholic acid, all p < 0.05). Conclusions: We are the first to demonstrate the feasibility of plasma metabolomics in a prospective cohort of advanced PC pts on enteral feeding as their primary source of nutrition. Metabolic signatures unique to FOLFIRINOX or gemcitabine/nab-paclitaxel may be predictive of response and warrant further study.

Immobilized Palladium Nanoparticles on Phosphanamine-grafted Cellulose for Arylation of Uracil

Background: The synthesis of 5-arylation uracil nucleosides is an imperative challenge, especially for the method of Suzuki reaction using N-unprotected uracil as materials, which holds the potential to enhance the yield. Objective: The objective of the study was to find a more efficient catalyst to increase the yield of aryluracils and aryluridines. Methods: We first constructed the phosphanamine-grafted cellulose (PAGC) from cellulose material. Then, we prepared the nanocatalyst PAGC/Pd(0) through heating and reducing the mixture of PAGC and Pa(OAc)2. Results: When using this nanocatalyst to catalyze the Suzuki reaction of 5-iodouracil or 5-iodouridine and aryl heterocyclic boronic acids, the arylation yields have been significantly improved. Conclusion: This means that the resultant nanocatalyst exhibits a remarkable catalytic efficacy for Suzuki arylation of 5-iodouracil and 5-iodouridine.

A Plausible Prebiotic One-Pot Synthesis of Orotate and Pyruvate Suggestive of Common Protometabolic Pathways.

A reaction between two prebiotically plausible building blocks, hydantoin and glyoxylate, generates both the nucleobase orotate, a precursor of biological pyrimidines, and pyruvate, a core metabolite in the citric acid cycle and amino acid biosynthesis. The reaction proceeds in water to provide significant yields of the two widely divergent chemical motifs. Additionally, the reaction of thiohydantoin and glyoxylate produces thioorotate in high yield under neutral aqueous conditions. The use of an open‐chain thiohydantoin derivative also enables the potential pre‐positioning of a nucleosidic bond prior to the synthesis of an orotate nucleoside. The observation that diverse building blocks of modern metabolism can be produced in a single reaction pot, from common reactants under mild conditions, supports the plausibility of orthogonal chemistries operating at the origins of chemical evolution.

A Plausible Prebiotic One‐Pot Synthesis of Orotate and Pyruvate Suggestive of Common Protometabolic Pathways

AbstractA reaction between two prebiotically plausible building blocks, hydantoin and glyoxylate, generates both the nucleobase orotate, a precursor of biological pyrimidines, and pyruvate, a core metabolite in the citric acid cycle and amino acid biosynthesis. The reaction proceeds in water to provide significant yields of the two widely divergent chemical motifs. Additionally, the reaction of thiohydantoin and glyoxylate produces thioorotate in high yield under neutral aqueous conditions. The use of an open‐chain thiohydantoin derivative also enables the potential pre‐positioning of a nucleosidic bond prior to the synthesis of an orotate nucleoside. The observation that diverse building blocks of modern metabolism can be produced in a single reaction pot, from common reactants under mild conditions, supports the plausibility of orthogonal chemistries operating at the origins of chemical evolution.

Total Synthesis of Pseudouridimycin.

Pseudouridimycin (1), a potent antibiotic against both Gram-positive and Gram-negative bacteria including multi-drug-resistant strains with a new mode of action isolated from Streptomyces sp., was synthesized by a convergent strategy from 5'-amino-pseudouridine 5 and N-hydroxy-dipeptide 26 in 23% total yield. The key intermediate 26 was synthesized by hydroxylaminolysis of the nitrone derived from glutamine and subsequent glycylation with glycine chloride. The synthetic method provides an efficient and practical way for the synthesis of N-hydroxylated peptidyl nucleoside.

Synthesis of the New Nucleoside 5'-Alpha-Iminophosphates Using Staudinger Reaction

Synthesis of the New Nucleoside 5'-Alpha-Iminophosphates Using Staudinger Reaction

Diversity-Oriented Synthesis of 2-Substituted Purine Nucleosides from Available Nucleosides via the Late-Stage Nitration/Derivatization

Diversity-Oriented Synthesis of 2-Substituted Purine Nucleosides from Available Nucleosides via the Late-Stage Nitration/Derivatization

Electrochemical enantioselective dihydroxylation reaction of N-alkenyl nucleobases for the construction of chiral acyclic nucleosides

A simple and sustainable KI-mediated electrochemical enantioselective dihydroxylation reaction for the synthesis of chiral acyclic nucleosides was developed for the first time with only a 0.1 mol% Os catalyst.

Design, synthesis, and greener pasture biological assessment of a novel nucleoside: 1-(α-D-ribofuranosyl)-6,7-difluoro-2-methyl-4-quinazolinone as an inhibitor of COVID-19 and Alzheimer's disease.

Synthesis of a new fluorinated nucleoside of 6,7-difluoro-2-methyl-4-quinazolinone was described. 2-Amino-4,5-difluorobenzoic acid 1 reacts with (CH3CO)2O followed by ammonia to form (1H)-6,7-difluoro-2-methyl-4-quinazolinone 3a. Ribosylation of a silylated 4 with l-O-acety1-2,3-5-tri-O-benzoyl-α-D-ribofuranose 5 forms a protected nucleoside 6 then unprotected from 6 to give a free nucleoside 7. Greener pasture biological docking of the cystine protease of COVID-19 [Mpro, code 7BQY, PDB] by novel nucleoside and fluoroquinazoline compounds is presented. LIGPLOT (2D) representations calculated for the same ligands are shown. A superposition of remdesivir approved medicine, N3 inhibitor, and our ligands docked together into the binding protein of 7BQY is also given for a fair comparison. The binding affinities of remdesivir, N3 inhibitor, the nucleoside 7, and fluoroquinazoline 3a, 3b compounds with 7BQY calculated under the same conditions are -7.7, -7.4, -7.6, -6.1, and -6.1 kcal mol-1, respectively. The high values were due to the existence of many hydrophobic interactions and hydrogen bonds between the ligands and the active amino acid residues of the receptor, indicating a promising candidate as a COVID-19 inhibitor. Pro Tox -II server showed that compound 7 has a similar feature to the approved antiviral drug remdesivir for COVID-19. Additionally, a fascinating molecular modeling investigation showed that our nucleoside demonstrated good binding inhibition of AChE enzyme towards advancing an efficient medication against Alzheimer's disease. Finally, DFT has been conducted to illustrate the MD results in terms of the molecular descriptor-based structural activity relationship calculated from FMOs.

A Study on Synthesis and Upscaling of 2'-O-AECM-5-methyl Pyrimidine Phosphoramidites for Oligonucleotide Synthesis.

2′-O-(N-(Aminoethyl)carbamoyl)methyl-modified 5-methyluridine (AECM-MeU) and 5-methylcytidine (AECM-MeC) phosphoramidites are reported for the first time and prepared in multigram quantities. The syntheses of AECM-MeU and AECM-MeC nucleosides are designed for larger scales (approx. 20 g up until phosphoramidite preparation steps) using low-cost reagents and minimizing chromatographic purifications. Several steps were screened for best conditions, focusing on the most crucial steps such as N3 and/or 2′-OH alkylations, which were improved for larger scale synthesis using phase transfer catalysis (PTC). Moreover, the need of chromatographic purifications was substantially reduced by employing one-pot synthesis and improved work-up strategies.

The Synthesis of Nucleoside 2',3'-Cyclic Monophosphate Analogs with Regio- and Stereospecificity.

This article presents a new method for the rapid, stereoselective and regioselective synthesis of nucleoside 2',3'-O,O-phosphorothioate and 2',3'-O,O-phosphoroselenoate molecules. The method avoids the use of protection groups, chiral reagents, and chiral metal catalysts, as well as complicated chiral separations. This synthetic method has been applied successfully to all of the natural nucleosides. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Preparation of 2-chloro-1,3,2-dithiophospholane (6) Basic Protocol 2: Preparation of 2-cyanoethoxy-thio-dithiophospholane (8) Basic Protocol 3: Preparation of 2-cyanoethoxy-seleno-dithiophospholane (9) Basic Protocol 4: Preparation of uridine-2',3'-O,O-phosphorothioate (Sp, exo; 1A) Basic Protocol 5: Preparation of uridine-2',3'-O,O-phosphoroselenoate (Sp, exo; 11) Basic Protocol 6: Preparation of adenosine-2',3'-O,O-phosphorothioate (Sp, exo; 12) Basic Protocol 7: Preparation of thymidine-2',3'-O,O-phosphorothioate (Sp, exo; 13) Basic Protocol 8: Preparation of cytosine-2',3'-O,O-phosphorothioate (Sp, exo; 14) Basic Protocol 9: Preparation of guanosine-2',3'-O,O-phosphorothioate (Sp, exo; 15).

Engineered Ribosyl-1-Kinase Enables Concise Synthesis of Molnupiravir, an Antiviral for COVID-19.

Molnupiravir (MK-4482) is an investigational antiviral agent that is under development for the treatment of COVID-19. Given the potential high demand and urgency for this compound, it was critical to develop a short and sustainable synthesis from simple raw materials that would minimize the time needed to manufacture and supply molnupiravir. The route reported here is enabled through the invention of a novel biocatalytic cascade featuring an engineered ribosyl-1-kinase and uridine phosphorylase. These engineered enzymes were deployed with a pyruvate-oxidase-enabled phosphate recycling strategy. Compared to the initial route, this synthesis of molnupiravir is 70% shorter and approximately 7-fold higher yielding. Looking forward, the biocatalytic approach to molnupiravir outlined here is anticipated to have broad applications for streamlining the synthesis of nucleosides in general.