Nucleobase Analogues Research Articles

Discovery of novel favipiravir derivatives with improved pharmacokinetic properties as anti-SFTSV agents

Favipiravir is a nucleobase analogue that exhibits antiviral activities against a variety of RNA viruses. Due to the lack of antivirals to combat SFTS, an emerging tickborne epidemic caused by a RNA virus belonging to the Phenuividae family within Bunyavirales, Favipiravir has been put brand new attention as optimal SFTS clinical candidate. We here disclose chemical synthesis of novel derivatives of Favipiravir. All derivatives showed favorable inhibitory effect on SFTSV replication in vitro. The 50 % effective concentration (EC50) of the most active compound H3 was 12.06 μM, better than that of Favipiravir (15.51 μM). Most importantly, compared with the clinical candidate Favipiravir, pharmacokinetic studies conducted on rats demonstrated enhanced pharmacokinetic properties for H2 and H3 including parameters of T1/2, Cmax and AUC. These combined attributes identified novel promising drug candidates for the treatment of SFTSV infection.

Interaction of Tri-Cyclic Nucleobase Analogs with Enzymes of Purine Metabolism: Xanthine Oxidase and Purine Nucleoside Phosphorylase.

Fluorescent markers play important roles in spectroscopic and microscopic research techniques and are broadly used in basic and applied sciences. We have obtained markers with fluorescent properties, two etheno derivatives of 2-aminopurine, as follows: 1,N2-etheno-2-aminopurine (1,N2-ε2APu, I) and N2,3-etheno-2-aminopurine (N2,3-ε2APu, II). In the present paper, we investigate their interaction with two key enzymes of purine metabolism, purine nucleoside phosphorylase (PNP), and xanthine oxidase (XO), using diffraction of X-rays on protein crystals, isothermal titration calorimetry, and fluorescence spectroscopy. Crystals were obtained and structures were solved for WT PNP and D204N-PNP mutant in a complex with N2,3-ε2APu (II). In the case of WT PNP-1,N2-ε2APu (I) complex, the electron density corresponding to the ligand could not be identified in the active site. Small electron density bobbles may indicate that the ligand binds to the active site of a small number of molecules. On the basis of spectroscopic studies in solution, we found that, in contrast to PNP, 1,N2-ε2APu (I) is the ligand with better affinity to XO. Enzymatic oxidation of (I) leads to a marked increase in fluorescence near 400 nm. Hence, we have developed a new method to determine XO activity in biological material, particularly suitable for milk analysis.

A nanoplatform based on allylthiopurine bio-MOF and glycosylated AIE PARP inhibitor for cancer synthetic lethal therapy.

A biological nanoplatform (Gal-ANI@ZnAP NPs) was constructed based on a prodrug-skeletal metal-organic framework (MOF) using purine nucleobase analogue prodrug 6-allylthiopurine as a bioactive ligand, and functionalized with AIE fluorescent PARP inhibitor glycoconjugate for visualization therapy and synthetic lethal cancer therapy. This nanoplatform could actively target cancer cells, selectively release drugs in response to esterase/pH, and visualize drug uptake. In vitro studies revealed that Gal-ANI@ZnAP NPs increased the synthetic lethality in cancer cells by inducing DNA repair failure with the simultaneous targeting of PARP and nucleotide metabolism, thereby exhibiting a significant cancer-killing effect. The study presents a novel strategy to construct an AIE nanoplatform using pharmaceutical molecules for drug uptake visualization and boosting synthetic lethality in cancer.

Solvent effect on the excited state photophysics of Imiquimod: A DFT/TD-DFT and spectroscopic study

Solvation plays an important role in chemistry and biology. The role of the solvent is crucial in any chemical processes such as tautomerization that controls the structure and function of biomolecules. The current study aims to explore how different solvent medium affects the tautomeric forms of an antitumor drug, Imiquimod (IMQ). We have used several spectroscopical methods, including UV–Vis absorption spectroscopy, steady-state and time-resolved fluorescence spectroscopy, and quantum mechanical (QM) calculations. Our results revealed that solvent, indeed, plays a significant role in the modulation of the photophysics of the drug. IMQ has three emission bands in protic and aprotic solvents and two bands in non-polar medium associated with different forms of the drug. Using time-resolved technique, and comparing with the predicted lifetimes from QM calculations, we succeed to assign these three forms as cation, tautomer and neutral of IMQ with 1.6 ns, 2.0 ns and 4.0 ns lifetime values, respectively. Since IMQ is a nucleobase analogue and one of the most effective medications for skin tumors; these findings about which specie of IMQ is present in a given medium, and beyond, how the medium alters the photophysics of the molecule may provide deeper insights into its structure and function.

Buffer-Dependent Photophysics of 2-Aminopurine: Insights into Fluorescence Quenching and Excited-State Interactions.

2-Aminopurine (2AP) is the most widely used fluorescent nucleobase analogue in DNA and RNA research. Its unique photophysical properties and sensitivity to environmental changes make it a useful tool for understanding nucleic acid dynamics and DNA-protein interactions. We studied the effect of ions present in commonly used buffer solutions on the excited-state photophysical properties of 2AP. Fluorescence quenching was negligible for tris(hydroxymethyl)aminomethane (TRIS), but significant for phosphate, carbonate, 3-(N-morpholino) propanesulfonic acid (MOPS), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffers. Results indicate that the two tautomers of 2AP (7H, 9H) are quenched by phosphate ions to different extents. Quenching by the H2PO4- ion is more pronounced for the 7H tautomer, while the opposite is true for the HPO42- ion. For phosphate ions, the results of the time-resolved fluorescence study cannot be explained using a simple collisional quenching mechanism. Instead, results are consistent with transient interactions between 2AP and the phosphate ions. We postulate that excited-state interactions between the 2AP tautomers and an H-bond acceptor (phosphate and carbonate) result in significant quenching of the singlet-excited state of 2AP. Such interactions manifest in biexponential fluorescence intensity decays with pre-exponential factors that vary with quencher concentration, and downward curvatures of the Stern-Volmer plots.

Conjugation of an anti-metabolite nucleobase analogue with a mitochondriotropic agent via palladium(II) against breast cancer cells

The conjugation of the uracil (a nucleobase) analogue, 6-methyl-thiouracil (MTUC), with the mitochondriotropic agent of Tri-o-Tolyl-Phosphine (TOTP) through palladium(II) leads to the formation of the metallodrug of formula [PdCl(TOTP)(MTUC)] (1). The metallodrug was characterized in solid state using Attenuated Total Reflectance-Fourier Transform Infra-Red (ATR-FTIR) spectroscopy and X-ray diffraction crystallography (XRD), while its behavior in solution was examined through Ultra Violet (UV) and 1H NMR spectroscopies. The in vitro cytotoxicity of 1 was assessed against human breast adenocarcinoma cell lines: MCF-7 (hormone-dependent (HD)) and MDA-MB-231 (hormone-independent (HI)), as well as fetal lung fibroblast (MRC-5) cells. The MCF-7 cell morphology suggests apoptotic pathway, and this was confirmed by Acridine Orange/Ethidium Bromide (AO/EB) Staining, and the loss of the permeabilization of the mitochondrial membrane. The binding affinity of 1 toward the calf thymus (CT) DNA was clarified.

Site-specific incorporation of a fluorescent nucleobase analog enhances i-motif stability and allows monitoring of i-motif folding inside cells.

The i-motif is an intriguing non-canonical DNA structure, whose role in the cell is still controversial. Development of methods to study i-motif formation under physiological conditions in living cells is necessary to study its potential biological functions. The cytosine analog 1,3-diaza-2-oxophenoxazine (tCO) is a fluorescent nucleobase able to form either hemiprotonated base pairs with cytosine residues, or neutral base pairs with guanines. We show here that when tCO is incorporated in the proximity of a G:C:G:C minor groove tetrad, it induces a strong thermal and pH stabilization, resulting in i-motifs with Tm of 39ºC at neutral pH. The structural determination by NMR methods reveals that the enhanced stability is due to a large stacking interaction between the guanines of the tetrad with the tCO nucleobase, which forms a tCO:C+ in the folded structure at unusually-high pHs, leading to an increased quenching in its fluorescence at neutral conditions. This quenching is much lower when tCO is base-paired to guanines and totally disappears when the oligonucleotide is unfolded. By taking profit of this property, we have been able to monitor i-motif folding in cells.

BODIPY-fused uracil: synthesis, photophysical properties, and applications.

Fluorescent nucleobase and nucleic acid analogs are important tools in chemical and molecular biology as fluorescent labelling of nucleobases has applications in cellular imaging and anti-tumor activity. Boron-dipyrromethene (BODIPY) dyes exhibiting high brightness and good photostability are extensively used as fluorescent labelling agents and as type II photosensitizers for photodynamic therapy. Thus, the combination of nucleobases and BODIPY to obtain new compounds with both anti-tumor activity and fluorescent imaging functions is the focus of our research. We synthesized two new nucleobase analogs 1 and 2 by fusing the BODIPY core directly with uracil which resulted in favorable photophysical properties and high emission quantum efficiencies particularly in organic solvents. Further, we explored the newly synthesized derivatives, which possessed good singlet oxygen generation efficiencies and bio-compatibility, as potential PDT agents and our results show that they exhibit in vitro anti-tumor activities.

Conformational preferences of modified nucleobases in RNA aptamers and their effect on Förster resonant energy transfer.

Förster resonant energy transfer (FRET) can be utilized in the study of tertiary structures of RNA aptamers, which bind specific fluorophoric ligands to form a fluorogenic aptamer complex. By introducing the emissive nucleobase analog 4-cyanoindole into the fluorogenic Chili RNA aptamer a FRET pair was established. The interpretation of studies aiming to investigate those tertiary structures using FRET, however, relies on prior knowledge about conformational properties of the nucleobase, which govern exciton transfer capabilities. Herein we employed classical molecular dynamics combined with Förster exciton theory to elucidate the preferred orientation relative to proximate bases and the influence on exciton transfer efficiency in multiple substitution sites. We did this by comparing the chromophoric distances emergent from MD simulations with experimental FRET data based on structural data of the native aptamer. We present the outlined methodology as a means to reliably evaluate future nucleobase analogue candidates in terms of their structural behavior and emergent exciton transfer properties as exemplified in the study of the preferred orientation of 4-cyanoindole in the Chili RNA aptamer.

The last two transmembrane helices in the APC-type FurE transporter act as an intramolecular chaperone essential for concentrative ER-exit.

FurE is a H+ symporter specific for the cellular uptake of uric acid, allantoin, uracil, and toxic nucleobase analogues in the fungus Aspergillus nidulans. Being member of the NCS1 protein family, FurE is structurally related to the APC-superfamily of transporters. APC-type transporters are characterised by a 5+5 inverted repeat fold made of ten transmembrane segments (TMS1-10) and function through the rocking-bundle mechanism. Most APC-type transporters possess two extra C-terminal TMS segments (TMS11-12), the function of which remains elusive. Here we present a systematic mutational analysis of TMS11-12 of FurE and show that two specific aromatic residues in TMS12, Trp473 and Tyr484, are essential for ER-exit and trafficking to the plasma membrane (PM). Molecular modeling shows that Trp473 and Tyr484 might be essential through dynamic interactions with residues in TMS2 (Leu91), TMS3 (Phe111), TMS10 (Val404, Asp406) and other aromatic residues in TMS12. Genetic analysis confirms the essential role of Phe111, Asp406 and TMS12 aromatic residues in FurE ER-exit. We further show that co-expression of FurE-Y484F or FurE-W473A with wild-type FurE leads to a dominant negative phenotype, compatible with the concept that FurE molecules oligomerize or partition in specific microdomains to achieve concentrative ER-exit and traffic to the PM. Importantly, truncated FurE versions lacking TMS11-12 are unable to reproduce a negative effect on the trafficking of co-expressed wild-type FurE. Overall, we show that TMS11-12 acts as an intramolecular chaperone for proper FurE folding, which seems to provide a structural code for FurE partitioning in ER-exit sites.

Enzymatic Synthesis of Aptamer-Polynucleotide Nanoparticles with High Anticancer Drug Loading for Targeted Delivery.

We report a targeted prodrug delivery platform that can deliver a cytostatic nucleobase analog with high drug loading. We chose fluorouracil (5FU), a drug used to treat various cancers, whose active metabolite 5-fluorodeoxyuridine monophosphate (5-FdUMP) is the antineoplastic agent. We use terminal deoxynucleotidyl transferase (TdT) to polymerize 5-fluorodeoxyuridine triphosphate (5-FdUTP) onto the 3'-end of an aptamer. We find that (i) addition of hydrophobic, unnatural nucleotides at the 3'-end of the 5-FdU polynucleotide by TdT leads to their spontaneous self-assembly into nuclease resistant micelles, (ii) aptamers presented on the micelle corona retain specificity for their cognate receptor on tumor cells, and (iii) the micelles deliver 5FU to tumor cells and exhibit greater cytotoxicity than the free drug. The modular design of our platform, consisting of a targeting moiety, a polynucleotide drug, and a self-assembly domain, can be adapted to encompass a range of polymerizable therapeutic nucleotides and targeting units.

Abundant extraterrestrial purine nucleobases in the Murchison meteorite: Implications for a unified mechanism for purine synthesis in carbonaceous chondrite parent bodies

Extraterrestrial nucleobases are of key interest owing to their implications for the chemical evolution of primordial molecules in the solar system and their potential contributions to prebiotic chemistry on the early Earth. Recent advances in analytical techniques capable of detecting femto-mole-order nucleobases have allowed us to identify all five exogenous nucleobases—uracil, cytosine, thymine, adenine, and guanine—in carbonaceous chondrites and to quantify uracil obtained from the carbonaceous asteroid (162173) Ryugu. In this study, we optimized the analytical method using hot-water extraction followed by 6 M hydrochloric acid (HCl) extraction from a sample of the CM2 Murchison meteorite to reassess the abundances and distributions of extraterrestrial nucleobases. The target analyses performed using high-performance liquid chromatography paired with electrospray-ionization, high-resolution mass spectrometry revealed that purine nucleobases were substantially more enriched in the subsequent 6 M HCl extract (951 ± 104 parts per billion, ppb) than in the hot-water extract (199 ± 3 ppb). The most abundant nucleobase was guanine (649 ± 103 ppb in total). The 6 M HCl extract not only contained canonical nucleobases but also included rare nucleobase analogs from the purine family, such as 8-substituted purines. Unlike purines, we preferentially detected pyrimidine nucleobases in the hot-water extract and the acid hydrolysate (185 ± 17 ppb and 297 ± 5 ppb, respectively) rather than in the 6 M HCl extract (51 ± 4 ppb). The disparate distributions of purine and pyrimidine bases in the Murchison meteorite suggests that purines are more robustly incorporated than pyrimidines into meteoritic matrices and/or insoluble macromolecular organic matter. We propose a unified formation mechanism for purine nucleobases—which involves the synthesis of 5-aminoimidazole derivatives from hydrogen cyanide and its related molecules—that can account for the molecular distributions of the extraterrestrial purine nucleobases found in the Murchison meteorite.

Screening for Novel Fluorescent Nucleobase Analogues Using Computational and Experimental Methods: 2-Amino-6-chloro-8-vinylpurine (2A6Cl8VP) as a Case Study.

Novel fluorescent nucleic acid base analogues (FBAs) with improved optical properties are needed in a variety of biological applications. 2-Amino-6-chloro-8-vinylpurine (2A6Cl8VP) is structural analogue of two existing highly fluorescent FBAs, 2-aminopurine (2AP) and 8-vinyladenine (8VA), and can therefore be expected to have similar base pairing as well as better optical properties compared to its counterparts. In order to determine the absorption and fluorescence properties of 2A6Cl8VP, as a first step, we used TD-DFT calculations and the polarizable continuum model for simulating the solvents and computationally predicted absorption and fluorescence maxima. To test the computational predictions, we also synthesized 2A6Cl8VP and measured its UV/vis absorbance, fluorescence emission, and fluorescence lifetime. The computationally predicted absorbance and fluorescence maxima of 2A6Cl8VP are in reasonable agreement to the experimental values and are significantly redshifted compared to 2AP and 8VA, allowing for its specific excitation. The fluorescence quantum yield of 2A6Cl8VP, however, is significantly lower than those of 2AP and 8VA. Overall, 2A6Cl8VP is a novel fluorescent nucleobase analogue, which can be useful in studying structural, biophysical, and biochemical applications.

Modification of N-hydroxycytidine yields a novel lead compound exhibiting activity against the Venezuelan equine encephalitis virus

Nucleoside and nucleobase analogs capable of interfering with nucleic acid synthesis have played essential roles in fighting infectious diseases. However, many of these agents are associated with important and potentially lethal off-target intracellular effects that limit their use. Based on the previous discovery of base-modified 2′-deoxyuridines, which showed high anticancer activity while exhibiting lower toxicity toward rapidly dividing normal human cells compared to antimetabolite chemotherapeutics, we hypothesized that a similar modification of the N4-hydroxycytidine (NHC) molecule would provide novel antiviral compounds with diminished side effects. This presumption is due to the substantial structural difference with natural cytidine leading to less recognizability by host cell enzymes. Among the 42 antimetabolite species that have been synthesized and screened against VEEV, one hit compound was identified. The structural features of the modifying moiety were similar to those of the anticancer lead 2′-deoxyuridine derivative reported previously, providing an opportunity to pursue further structure–activity relationship (SAR) studies directed to lead improvement, and obtain insight into the mechanism of action, which can lead to identifying drug candidates against a broad spectrum of RNA viral infections.

2-Nucleobase-substituted 4,6-Diaminotriazine Analogs: Synthesis and Anti-cancer Activity in 5-Fluorouracil-sensitive and Resistant Colorectal Cancer Cells.

Cancer continues to be the second leading cause of death worldwide, with colorectal cancer (CRC) being the third most common type. Despite significant advances in cancer therapies, the current treatment of CRC remains suboptimal. In addition, the effectiveness of available chemotherapeutic drugs such as 5-Fluorouracil (5-FU) is limited by CRC-acquired resistance. In this study, we provide innovative approaches employed in synthesizing four novel nucleobase analogs. Equally, we describe the effects of these compounds on proliferation, migration, aggregation, and adhesion of 5-FU-sensitive (HCT116) and -resistant (5-FU-R-HCT116) human CRC cells. In either cell type, our synthesized novel analogs significantly inhibited cell viability in a concentration- and time-dependent manner. This highlights the higher potency of these novel analogs. In addition, these compounds attenuated migration and adhesion of both cell types while they promoted homotypic cell-cell interaction. These changes were reflected by the downregulation of matrix metalloproteases (MMP-2 and MMP-9). Furthermore, our analogs exhibited potent anti-angiogenic activity in vivo. These novel nucleobase analogs reduced the level of secreted vascular endothelial growth factor (VEGF) and nitric oxide (NO) production in both 5-FU-sensitive and -resistant CRC cells. Taken together, our data highlight the potential chemotherapeutic properties of our novel analogs against CRC, including the 5-FU-resistant form.

Tautomeric equilibrium and spectroscopic properties of 8-azaguanine revealed by quantum chemistry methods.

8-azaguanine is a triazolopyrimidine nucleobase analog possessing potent antibacterial and antitumor activities, and it has been implicated as a lead molecule in cancer and malaria therapy. Its intrinsic fluorescence properties can be utilized for monitoring its interactions with biological polymers like proteins or nucleic acids. In order to better understand these interactions, it is important to know the tautomeric equilibrium of this compound. In this work, the tautomeric equilibrium of all natural neutral and anionic compound forms (except highly improbable imino-enol tautomers) as well as their methyl derivatives and ribosides was revealed by quantum chemistry methods. It was shown that, as expected, tautomers protonated at positions 1 and 9 dominate neutral forms both in gas phase and in aqueous solution. 8-azaguanines methylated at any position of the triazole ring are protonated at position 1. The computed vertical absorption and emission energies are in very good agreement with the experimental data. They confirm the validity of the assumption that replacing the proton with the methyl group does not significantly change the positions of absorption and fluorescence peaks.

Oxidative cyclization of allyl compounds and isocyanide: A facile entry to polysubstituted 2-cyanopyrroles

A facile TfOH-catalyzed oxidative cyclization of allyl compounds and isocyanide has been developed with the assistance of DDQ, where isocyanide is used as the crucial "N" and "CN" sources. Highly functionalized 2-cyanopyrroles are constructed efficiently through a new formal [3 + 2] mode, demonstrating diverse reactivity and synthetic utility in organic chemistry. 2-Cyanopyrrole is converted into a nucleobase analogue of Remdesivir and 5H-pyrrolo[2,1-a]isoindole through a three-step or a two-step sequence, respectively. This protocol features broad substrate scope, operational simplicity and good functional group tolerance.

Insights into the Ozonation of Antiviral Purine Derivatives by the Basic Structures’ Kinetics and Ozone Consumption Ratios

Adenine and guanine derivatives are widely applied as antiviral agents. Due to their incomplete removal in conventional wastewater treatment, these substances were detected in the aquatic environment. Ozonation as an additional treatment step is known to degrade the antiviral compounds. However, oxidation mechanisms are still poorly examined. This study focused on the basic structures of the large group of antiviral nucleobase analogues, purine, adenine, and guanine. Their reaction with ozone allows prognoses of the ozonation of structurally related micropollutants. Therefore, these structures and the two antivirals acyclovir and penciclovir are investigated regarding their kinetics and degradation. Both characteristics are the basis for further research regarding the comprehensive understanding of the purines’ ozonation. Results show differences for the basic structures but similarities between guanine derivatives. Overall, the fundamental examinations exhibit a strong pH-dependency. Investigation of kinetics and ozone consumption ratios at pH 7 indicates ozone attack at nitrogen sites for purine (k < 8 × 10–2 M–1 s–1) and adenine (k = 100 M–1 s–1) with a slight shift to a C-C double bond at a higher pH for adenine. In guanine analogues (k = 3 × 104 M–1 s–1), the attack occurs mostly at carbon. Altogether, knowledge transfer to structurally related micropollutants is possible in the absence of additional strong electron-withdrawing or electron-donating substituents.

Efficient Chemical and Enzymatic Syntheses of FAD Nucleobase Analogues and Their Analysis as Enzyme Cofactors.

Flavin adenine dinucleotide (FAD) is an essential redox cofactor in cellular metabolism. The organic synthesis of FAD typically involves coupling flavin mononucleotide (FMN) with adenosine monophosphate, however, existing synthesis routes present limitations such as multiple steps, low yields, and/or difficult-to-obtain starting materials. In this study, we report the synthesis of FAD nucleobase analogues with guanine/cytosine/uracil in place of adenine and deoxyadenosine in place of adenosine using chemical and enzymatic approaches with readily available starting materials, achieved in 1-3 steps with moderate yields (10-57 %). We find that the enzymatic route using Methanocaldococcus jannaschii FMN adenylyltransferase (MjFMNAT) is versatile and can produce these FAD analogues in high yields. Further, we demonstrate that Escherichia coli glutathione reductase is capable of binding and using these analogues as cofactors. Finally, we show that FAD nucleobase analogues can be synthesized inside a cell from cellular substrates FMN and nucleoside triphosphates by the heterologous expression of MjFMNAT. This lays the foundation for their use in studying the molecular role of FAD in cellular metabolism and as biorthogonal reagents in biotechnology and synthetic biology.

Highly Fluorescent 2‐Aminopurine Derivatives: Synthesis, Photo‐physical Characterization, and Preliminary Cytotoxicity Evaluation

AbstractNew fluorescent nucleobase analogues (FBAs) are emerging as extraordinarily useful tools for DNA labelling technologies. The highly fluorescent adenine analogue 2‐aminopurine (2AP) is still the most used within the few hundreds of newly FBAs synthesized, but its excitation in the UV region demands for high energy sources endangering living cells. New and highly fluorescent 2AP derivatives, 2‐amino‐6‐cyanopurines, were obtained using simpler but efficient synthesis method. All the new compounds exhibit advantageous photophysical properties over 2AP, showing absorption and emission bands ranging the visible region (blue‐green region), high fluorescence quantum yields and Stokes’ shifts, especially in non‐protic organic solvents. Density Functional Theory calculations (DFT) of electronic and vibrational structure were performed, allowing to predict absorption and emission spectra. In addition, these 2‐amino‐6‐cyanopurines exhibit little to no toxicity in assays using yeast cells.