Thymine Derivatives Research Articles

Unlocking precision in aptamer engineering: a case study of the thrombin binding aptamer illustrates why modification size, quantity, and position matter.

The thrombin binding aptamer (TBA) is a prototypical platform used to understand the impact of chemically-modified nucleotides on aptamer stability and target affinity. To provide structural insight into the experimentally-observed effects of modification size, location, and number on aptamer performance, long time-scale molecular dynamics (MD) simulations were performed on multiple binding orientations of TBA-thrombin complexes that contain a large, flexible tryptophan thymine derivative (T-W) or a truncated analogue (T-K). Depending on modification position, T-W alters aptamer-target binding orientations, fine-tunes aptamer-target interactions, strengthens networks of nucleic acid-protein contacts, and/or induces target conformational changes to enhance binding. The proximity and 5'-to-3' directionality of nucleic acid structural motifs also play integral roles in the behavior of the modifications. Modification size can differentially influence target binding by promoting more than one aptamer-target binding pose. Multiple modifications can synergistically strengthen aptamer-target binding by generating novel nucleic acid-protein structural motifs that are unobtainable for single modifications. By studying a diverse set of modified aptamers, our work uncovers design principles that must be considered in the future development of aptamers containing chemically-modified nucleotides for applications in medicine and biotechnology, highlighting the value of computational studies in nucleic acids research.

Synthesis of Chiral Acyclic Pyrimidine Nucleoside Analogues from DHAP-Dependent Aldolases.

Dihydroxyacetone phosphate (DHAP)-dependent aldolases catalyze the aldol addition of DHAP to a variety of aldehydes and generate compounds with two stereocenters. This reaction is useful to synthesize chiral acyclic nucleosides, which constitute a well-known class of antiviral drugs currently used. In such compounds, the chirality of the aliphatic chain, which mimics the open pentose residue, is crucial for activity. In this work, three DHAP-dependent aldolases: fructose-1,6-biphosphate aldolase from rabbit muscle, rhanmulose-1-phosphate aldolase from Thermotoga maritima, and fuculose-1-phosphate aldolase from Escherichia coli, were used as biocatalysts. Aldehyde derivatives of thymine and cytosine were used as acceptor substrates, generating new acyclic nucleoside analogues containing two new stereocenters with conversion yields between 70% and 90%. Moreover, structural analyses by molecular docking were carried out to gain insights into the diasteromeric excess observed.

Molecular aggregation by hydrogen bonding in cold-crystallization behavior of mixed nucleobases analyzed by temperature-controlled infrared spectroscopy.

The cold-crystallization behaviors of dodecyl-substituted nucleobases (adenine, uracil, and thymine) were analyzed. The dodecyl derivative from uracil alone did not exhibit cold crystallization; however, a mixture of adenine and uracil derivatives at a molar ratio of 1 : 1 exhibited cold crystallization. These results are similar to the thermal behavior of dodecyl derivatives of adenine and thymine alone and in mixtures reported in a previous study. Temperature-controlled infrared spectroscopy was used to observe the molecular assembly states of the liquid, supercooled state, and cold-crystallized compounds. Hydrogen-bonded molecular pairs in the high-temperature liquid state, multiple hydrogen-bonded networks in the supercooled state, and reverse Hoogsteen-type complementary hydrogen bonds in cold-crystallized compounds were observed using infrared spectroscopy. The heterogeneity of the system, due to multiple types of hydrogen bonding, retarded the crystallization rate, resulting in supercooling and cold crystallization. Infrared spectroscopy, which can be used to measure the aggregation state of molecules, including the liquid and supercooled states, is an effective analytical method for clarifying the process of cold crystallization.

Investigation of the interaction of thymine drugs with Be12O12 and Ca12O12 nanocages: A quantum chemical study

Based on the DFT in a Wb97xd/6-311+G* level of theory, the interaction of thymine derivatives with Be12O12 and Ca12O12 nanocages was investigated. It was found that adsorption energies of thymine molecules on the Be12/Ca12-O12 surface was around −43.16, −60.06 and −29.62, −50.71, −45.95, −30.27 kcal/mol, for thymine (TH1), 1-amino thymine (TH2) and thymine glycol (TH3), respectively and this result supported the drug’s adsorption. Additionally, according to the FMOs and MEP studies, a charge transfer from TH’s to nanocages. Additionally, both molecular orbitals demonstrate that the LUMO and HOMO are primarily found on the BeO’s surface.

Arabinofuranosyl Thymine Derivatives-Potential Candidates against Cowpox Virus: A Computational Screening Study.

Cowpox is caused by a DNA virus known as the cowpox virus (CPXV) belonging to the Orthopoxvirus genus in the family Poxviridae. Cowpox is a zoonotic disease with the broadest host range among the known poxviruses. The natural reservoir hosts of CPXV are wild rodents. Recently, the cases of orthopoxviral infections have been increasing worldwide, and cowpox is considered the most common orthopoxviral infection in Europe. Cowpox is often a self-limiting disease, although cidofovir or anti-vaccinia gammaglobulin can be used in severe and disseminated cases of human cowpox. In this computational study, a molecular docking analysis of thymine- and arabinofuranosyl-thymine-related structures (1-21) on two cowpox-encoded proteins was performed with respect to the cidofovir standard and a 3D ligand-based pharmacophore model was generated. Three chemical structures (PubChem IDs: 123370001, 154137224, and 90413364) were identified as potential candidates for anti-cowpox agents. Further studies combining in vitro and in silico molecular dynamics simulations to test the stability of these promising compounds could effectively improve the future design of cowpox virus inhibitors, as molecular docking studies are not sufficient to consider a ligand a potential drug.

Structure-Based design of [(2-Hydroxyethoxy)methyl]-6-(phenylthio)-thymine derivatives as nonnucleoside HIV-1 reverse transcriptase Inhibitors: From HEPTs to Sulfinyl-substituted HEPTs

The [(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) analogs were reported to be a kind of promising lead compounds as nonnucleoside HIV-1 reverse transcriptase inhibitors. In this work, a series of novel sulfinyl-substituted analogs were designed by structure-based design strategy with the purpose of improving the activity of HEPT, followed by evaluating their anti-HIV-1 activity in MT-4 cells. Most of the final compounds had moderate to strong activity against wild-type HIV-1 strain (IIIB) with EC50 values in the range of 0.21–1.91 μM, which were around 4 ∼ 32-fold better than the reference compound HEPT. Some of them showed higher sensitivity toward clinically relevant mutant L100I and E138K viruses than NVP. Selected compounds were further evaluated for their activity against wild-type reverse transcriptase (RT), and most of them exhibited nanomolar activity, suggesting a good correlation with the cell-based activity. The compounds 11h, 11l, and 11ab displayed the best anti-HIV-1 activity against wild-type HIV-1 strain (EC50 = 0.280, 0.209, and 0.290 μM) and nanomolar activity against mutant strains (L100I and E138K), superior to HEPT and NVP. Molecular modeling studies were also performed to elucidate the biological activity, providing a structural insight for follow-up research on HEPT optimization.

Structure-Based Design of [(2-Hydroxyethoxy)Methyl]-6-(Phenylthio)-Thymine Derivatives as Nonnucleoside Hiv-1 Reverse Transcriptase Inhibitors: From HEPTs to Sulfinyl-Substituted HEPTs

Structure-Based Design of [(2-Hydroxyethoxy)Methyl]-6-(Phenylthio)-Thymine Derivatives as Nonnucleoside Hiv-1 Reverse Transcriptase Inhibitors: From HEPTs to Sulfinyl-Substituted HEPTs

Design of a protein-targeted DNA aptamer using atomistic simulation

The concentrations of specific macromolecular species can be quantified using diagnostic tools that rely on molecular recognition by nucleic acid aptamers. One such approach involves the formation of osmium tetroxide 2,2'-bipyridine protein adducts, followed by electrochemical detection of analytes that bind specifically to electrode-tethered aptamers. In conjunction with a 27-mer DNA aptamer that binds specifically to exosite II on human alpha thrombin, this technique permits, in theory, a highly sensitive diagnostic tool for the quantification of serum thrombin levels. However, thrombin's aptamer binding site is lined by two tryptophan residues and the conjugation of bulky osmium groups to these residues weakens aptamer binding by an estimated 4 to 12 kcal/mol, undermining detection sensitivity. Therefore, we have rationally modified this DNA aptamer to strengthen its thrombin binding in the presence of conjugated osmium. Specifically, aptamers carrying long hydrophobic thymine derivatives in place of guanine 21 have binding affinities for osmium-conjugated thrombin that are enhanced by 10 to 15 kcal/mol, suggesting that these modified aptamers may be effective in a highly sensitive electrochemical sensor for the quantification of low concentrations of thrombin. Our approach of using molecular simulation to subtly re-engineer a DNA aptamer may be generally applicable for the optimization of other macromolecular binding interfaces. Communicated by Ramaswamy H. Sarma

Unexpected Reaction Products of Uracil and Its Methyl Derivatives with Acetic Anhydride and Methylene Chloride.

New acetyl derivatives of uracil, 6-methyluracil, and thymine were obtained in the course of an unconventional synthesis in methylene chloride. It was shown that products with the acetyloxymethyl fragment are formed according to a mechanism different from that for products with the acetyloxyethyl group. In particular, for uracil it was proven that the reaction with Ac2O, TEA, and CH2Cl2 leads to 1-acetyloxymethyluracil, where the N1 substituent is composed of the -CH2- fragment that originated from CH2Cl2 and the 1-acetyloxy moiety from Ac2O. The reaction of uracil with Ac2O, TEA, CH2Cl2, and DMAP leads to an acetyloxyethyl derivative in which the -CH2-CH2- fragment originates from TEA and the 1-acetyloxy moiety from Ac2O. A possible mechanism for the formation of new compounds was suggested and supported by the density functional theory/B3LYP quantum mechanical calculations. New compounds (39 in total, including seven deuterated) were fully characterized by nuclear magnetic resonance and high-resolution mass spectrometry techniques.

Hydrogen‐Bonding Interactions of Methylated Adenine Derivatives

AbstractHydrogen bonding between nucleobases is a crucial noncovalent interaction for the life on Earth. Apart from Watson‐Crick binding, Hoogsteen pairing has been found in many structures of nucleic acids. Methylation of nucleic acids (NAs) is a post‐replication or post‐transcription mechanism that can modulate the structure and function of a NA without changing its sequence. Methylation of adenine at the N6 position to form m6A is one of the most important and common epigenetic markers. This paper describes an investigation of intermolecular H‐bonding interactions of methylated derivatives of adenine with its complementary partner, thymine. Adenine derivatives with (di)methylamino groups in positions 2 or 6 have been prepared and their interactions with a thymine derivative have been studied by NMR spectroscopy and DFT calculations. It has been found that Hoogsteen pairing is preferred for adenine derivatives, which offer two hydrogen‐bond sites on both Watson‐Crick and Hoogsteen sides of the molecule. Methylation of the N6 position leads to further stabilization of the Hoogsteen pair.

Diversity-oriented functionalization of 2-pyridones and uracils

Heterocycles 2-pyridone and uracil are privileged pharmacophores. Diversity-oriented synthesis of their derivatives is in urgent need in medicinal chemistry. Herein, we report a palladium/norbornene cooperative catalysis enabled dual-functionalization of iodinated 2-pyridones and uracils. The success of this research depends on the use of two unique norbornene derivatives as the mediator. Readily available alkyl halides/tosylates and aryl bromides are utilized as ortho-alkylating and -arylating reagents, respectively. Widely accessible ipso-terminating reagents, including H/DCO2Na, boronic acid/ester, terminal alkene and alkyne are compatible with this protocol. Thus, a large number of valuable 2-pyridone derivatives, including deuterium/CD3-labeled 2-pyridones, bicyclic 2-pyridones, 2-pyridone-fenofibrate conjugate, axially chiral 2-pyridone (97% ee), as well as uracil and thymine derivatives, can be quickly prepared in a predictable manner (79 examples reported), which will be very useful in new drug discovery.

Supramolecular Functionalizable Linear-Dendritic Block Copolymers for the Preparation of Nanocarriers by Microfluidics.

Hybrid linear–dendritic block copolymers (LDBCs) having dendrons with a precise number of peripheral groups that are able to supramolecular bind functional moieties are challenging materials as versatile polymeric platforms for the preparation of functional polymeric nanocarriers. PEG2k-b-dxDAP LDBCs that are based on polyethylene glycol (PEG) as hydrophilic blocks and dendrons derived from bis-MPA having 2,6-diacylaminopyridine (DAP) units have been efficiently synthesized by the click coupling of preformed blocks, as was demonstrated by spectroscopic techniques and mass spectrometry. Self-assembly ability was first checked by nanoprecipitation. A reproducible and fast synthesis of aggregates was accomplished by microfluidics optimizing the total flow rate and phase ratio to achieve spherical micelles and/or vesicles depending on dendron generation and experimental parameters. The morphology and size of the self-assemblies were studied by TEM, Cryogenic Transmission Electron Microscopy (cryo-TEM), and Dynamic Light Scattering (DLS). The cytotoxicity of aggregates synthesized by microfluidics and the influence on apoptosis and cell cycle evaluation was studied on four cell lines. The self-assemblies are not cytotoxic at doses below 0.4 mg mL−1. Supramolecular functionalization using thymine derivatives was explored for reversibly cross-linking the hydrophobic blocks. The results open new possibilities for their use as drug nanocarriers with a dynamic cross-linking to improve nanocarrier stability but without hindering disassembly to release molecular cargoes.

In silico modeling of some HEPT analogues as anti -HIV agents using QSAR and molecular docking studies

Quantitative structure–activity relationship (QSAR), Density functional theory and molecular docking studies have been performed on a series of 49 1-[2-Hydroxyethoxy) methyl]-6-(phenylthio)-thymine (HEPT) derivatives as anti-HIV agents. A genetic algorithm multiple linear regression (GA-MLR) analysis has revealed that the five variable model comprises of 2D autocorrelation, 2D atom pairs and constitutional descriptors of these compounds are the governing factors for their biological activity. Using this GA-MLR model some new compounds have been proposed that have higher potency than the existing ones. The molecular docking study has shown that these new compounds can form hydrogen bonds with the receptor and have effective steric interactions.

Enhanced duplex- and triplex-forming ability and enzymatic resistance of oligodeoxynucleotides modified by a tricyclic thymine derivative.

We designed and synthesized an artificial nucleic acid, [3-(1,2-dihydro-2-oxobenzo[b][1,8]naphthyridine)]-2'-deoxy-D-ribofuranose (OBN), with a tricyclic structure in a nucleobase as a thymidine analog. Oligodeoxynucleotides (ODNs) containing consecutive OBN displayed improved duplex-forming ability with complementary single-stranded (ss) RNA and triplex-forming ability with double-stranded DNA in comparison with ODNs composed of natural thymidine. OBN-modified ODNs also displayed enhanced enzymatic resistance compared with ODNs with natural thymidine and phosphorothioate modification, respectively, due to the structural steric hindrance of the nucleobase. The fluorescence spectra of OBN-modified ODNs showed sufficient fluorescence intensity with ssDNA and ssRNA, which is an advantageous feature for fluorescence imaging techniques of nucleic acids with longer emission wavelengths than bicyclic thymine (bT).

Triphenylphosphonium conjugates of 1,2,3-triazolyl nucleoside analogues. Synthesis and cytotoxicity evaluation

A series of triphenylphosphonium (TPP) conjugates of 1,2,3-triazolyl analogues of several pyrimidine nucleosides was synthesized and evaluated for the in vitro cytotoxicity against human cancer cell lines M-HeLa, MCF-7, PANC-1, PC-3, DU145, SKOV-3, A275, and normal human cell line WI-38. In these TPP-conjugates triphenylphosphonium cation was attached via a tetramethylene chain to the N-3 atom of the heterocycle moiety (uracil, thymine, quinazoline-2,4-dione), which was coupled with the D-ribofuranosyl-1,2,3-triazol-4-yl fragment via methylene or tetramethylene linker. It was shown for the first time that the conjugation of 1,2,3-triazolyl derivatives of uridine, its analogues featuring quinazoline-2,4-dione fragment as well as uracil and thymine derivatives, having propargyl or a 1,2,3-triazolyl substituent at the N-1 atom, with a TPP-butyl cation endowed some of them with cytotoxic activity against human cancer cells. Among all human cancer cell lines used, DU-145 and A375 cells were the most sensitive to these TPP conjugates. At the same time, all tested compounds did not inhibit growth of normal cells WI-38. Propargyl containig TPP-conjugates of uracil 4f, 4j, and thymine 5f showed the highest cytotoxicity with IC50 values in the low micromolar concentration range. The present findings suggest that TPP-conjugates of uracil and thymine derivatives would be promising for further development as an anticancer agent.

Catalysis for Fine Chemicals

2′-Methoxyacetophenone (2M) presents improved UVA absorption as compared with other acetophenone derivatives. On the basis of transient infrared spectroscopy it has been previously claimed that 2M is an interesting photosensitiser for cyclobutane pyrimidine dimers (CPDs) formation. In the present paper, a complete UV–Vis transient absorption spectroscopic characterisation of this compound is provided, including triplet-triplet spectra, triplet lifetimes and rate constants for quenching of 2M by a dimeric thymine derivative. Furthermore, generation of singlet oxygen has been proven by time-resolved near IR phosphorescence measurements. Overall, the obtained results confirm the potential of 2M as a DNA photosensitiser, not only for CPDs formation, but also for oxidative damage.

Frequency and hydrogen bonding of nucleobase homopairs in small molecule crystals.

We used the high resolution and accuracy of the Cambridge Structural Database (CSD) to provide detailed information regarding base pairing interactions of selected nucleobases. We searched for base pairs in which nucleobases interact with each other through two or more hydrogen bonds and form more or less planar structures. The investigated compounds were either free forms or derivatives of adenine, guanine, hypoxanthine, thymine, uracil and cytosine. We divided our findings into categories including types of pairs, protonation patterns and whether they are formed by free bases or substituted ones. We found base pair types that are exclusive to small molecule crystal structures, some that can be found only in RNA containing crystal structures and many that are native to both environments. With a few exceptions, nucleobase protonation generally followed a standard pattern governed by pKa values. The lengths of hydrogen bonds did not depend on whether the nucleobases forming a base pair were charged or not. The reasons why particular nucleobases formed base pairs in a certain way varied significantly.

A Click Synthesis, Molecular Docking, Cytotoxicity on Breast Cancer (MDA-MB 231) and Anti-HIV Activities of New 1,4-Disubstituted-1,2,3-Triazole Thymine Derivatives

A new series of 1,4-disubstituted-1,2,3-triazolethymine derivatives (VIa–e) were synthesized and characterized by spectroscopic studies. The in vitro cytotoxic activities of selected compounds against human cancer cell line (MDA-MB 231) were evaluated by MTT assay. 4-Azido-N-substituted-benzenesulfonamides (Vc–e) and 4,4'-(4,4'-((5-methyl-2,4-dioxopyrimidine-1,3(2H,4H)-diyl)bis(methylene))-bis(1H-1,2,3-triazole-4,1-diyl))-bis(N-(4-methyl pyrimidin-2-yl)benzenesulfonamide) (VIc) displayed a significant cytotoxic activity with IC50 values of 1.61, 1.41, 1.61 and 1.81 μM, respectively. Molecular docking study of 4‑azido-N-(4,6-dimethylpyrimidin-2-yl)benzenesulfonamide (Vd) and 4,4'-(4,4'-((5-methyl-2,4-dioxopyrimidine-1,3(2H,4H)-diyl)bis(methylene))-bis(1H-1,2,3-triazole-4,1-diyl))-bis (N-(4-methyl pyrimidin-2-yl)benzenesulfonamide) (VIc) showed hydrogen bonding with the amino acid residues of the receptors 1X7R and 1A53, respectively. These derivatives are useful as starting points for further study of new anticancer drugs and to confirm the potential of triazole-sulfonamide analogues as lead compounds in anticancer drug discovery. In addition, 1,4-disubstituted-1,2,3-triazolethymine derivatives (VIa–e) were evaluated in vitro for antiviral activity against the replication of HIV-1 and HIV-2 in MT-4 cells. The results showed that 1,4-disubstituted-1,2,3-triazolethymine derivatives (VIc–e) possess a potent activity against HIV-1 replication with IC50 values of 11.42, ≥15.25,and 14.36 μM, SI > 4, ≤6, >9, respectively.

Regioselectivity in the adiabatic photocleavage of DNA-based oxetanes.

Direct absorption of UVB light by DNA may induce formation of cyclobutane pyrimidine dimers and pyrimidine-pyrimidone (6-4) photoproducts. The latter arise from the rearrangement of unstable oxetane intermediates, which have also been proposed to be the electron acceptor species in the photoenzymatic repair of this type of DNA damage. In the present work, direct photolysis of oxetanes composed of substituted uracil (Ura) or thymine (Thy) derivatives and benzophenone (BP) have been investigated by means of transient absorption spectroscopy from the femtosecond to the microsecond time-scales. The results showed that photoinduced oxetane cleavage takes place through an adiabatic process leading to the triplet excited BP and the ground state nucleobase. This process was markedly affected by the oxetane regiochemistry (head-to-head, HH, vs. head-to-tail, HT) and by the nucleobase substitution; it was nearly quantitative for all investigated HH-oxetanes while it became strongly influenced by the substitution at positions 1 and 5 for the HT-isomers. The obtained results clearly confirm the generality of the adiabatic photoinduced cleavage of BP/Ura or Thy oxetanes, as well as its dependence on the regiochemistry, supporting the involvement of triplet exciplexes. As a matter of fact, when formation of this species was favored by keeping together the Thy and BP units after splitting by means of a linear linker, a transient absorption at ∼400 nm, ascribed to the exciplex, was detected.

Electron and radical cation of sulfur-substituted thymine derivatives produced near photoionization threshold can alter and distort double-helix DNA structure

Results from DFT calculations show that the aqueous adiabatic ionization energies for thymine, 2-thiothymine, 2,4-dithiothymine, and 4-thiothymine are 4.95, 4.99 eV, 4.94 eV and 4.81 eV, respectively. The calculated absorption spectra for the radical cations of thymine and 4-thiothymine exhibit significant red-shift and show characteristics of noncanonical structures. The calculated large aqueous adiabatic electron affinity for 2,4-dithiothymine (AEA 2.67 eV) suggests that it can be a potent radio chemotherapeutic agent in cancer treatment.