Nucleoside Ligands Research Articles

A Brief Overview of Telomeres and Telomerase in Aging and Cancer

Telomere is guanine-rich DNA sequence with a protein complex known as shelterin present at the chromosomal ends to protect it from destruction and is known to play a key role in aging. Telomerase is a telomeric DNA elongating enzyme, which comprises the central key components for telomeric DNA synthesis. The main objective of this review was to explore the structure and function of telomere and telomerase, and their intervention in aging, stem cells, and cancer cells. The induction of various telomeropathies and age-related diseases by Telomerase RNA component (TERC) impairment is well explained. Telomeropathies refer to bone marrow failures such as dyskeratosis congenita, aplastic anemia, etc. Telomere and telomerase have become the targets for age-related therapies, stem cell therapies, and cancer therapies. This article throws light on the telomerase-related therapies with natural compounds in regenerative medicine and cancer treatment. Telomerase inhibitors like nucleoside ligands and G4 ligands are explained in the context of anti-cancer drugs. The association of TERT promoter mutations with cancer is discussed. We conclude that understanding telomerase mechanisms in stem cells and cancer cells paves a way to translate stem cells, cancer cells, and age-related research into effective therapies. The dynamic features of telomerase and telomeres make their structural and biochemical studies difficult. Hence, further meticulous studies can be done on single-molecule approaches to facilitate feasible and effective studies of telomerase and telomere component functioning.

Purine nucleoside phosphorylase enables dual metabolic checkpoints that prevent T cell immunodeficiency and TLR7-associated autoimmunity.

Purine nucleoside phosphorylase (PNP) enables the breakdown and recycling of guanine nucleosides. PNP insufficiency in humans is paradoxically associated with both immunodeficiency and autoimmunity, but the mechanistic basis for these outcomes is incompletely understood. Here, we identify two immune lineage-dependent consequences of PNP inactivation dictated by distinct gene interactions. During T cell development, PNP inactivation is synthetically lethal with downregulation of the dNTP triphosphohydrolase SAMHD1. This interaction requires deoxycytidine kinase activity and is antagonized by microenvironmental deoxycytidine. In B lymphocytes and macrophages, PNP regulates Toll-like receptor 7 signaling by controlling the levels of its (deoxy)guanosine nucleoside ligands. Overriding this regulatory mechanism promotes germinal center formation in the absence of exogenous antigen and accelerates disease in a mouse model of autoimmunity. This work reveals that one purine metabolism gene protects against immunodeficiency and autoimmunity via independent mechanisms operating in distinct immune lineages and identifies PNP as a potentially novel metabolic immune checkpoint.

Bacterial 2'-Deoxyguanosine Riboswitch Classes as Potential Targets for Antibiotics: A Structure and Dynamics Study.

The spread of antibiotic-resistant bacteria represents a substantial health threat. Current antibiotics act on a few metabolic pathways, facilitating resistance. Consequently, novel regulatory inhibition mechanisms are necessary. Riboswitches represent promising targets for antibacterial drugs. Purine riboswitches are interesting, since they play essential roles in the genetic regulation of bacterial metabolism. Among these, class I (2′-dG-I) and class II (2′-dG-II) are two different 2′-deoxyguanosine (2′-dG) riboswitches involved in the control of deoxyguanosine metabolism. However, high affinity for nucleosides involves local or distal modifications around the ligand-binding pocket, depending on the class. Therefore, it is crucial to understand these riboswitches’ recognition mechanisms as antibiotic targets. In this work, we used a combination of computational biophysics approaches to investigate the structure, dynamics, and energy landscape of both 2′-dG classes bound to the nucleoside ligands, 2′-deoxyguanosine, and riboguanosine. Our results suggest that the stability and increased interactions in the three-way junction of 2′-dG riboswitches were associated with a higher nucleoside ligand affinity. Also, structural changes in the 2′-dG-II aptamers enable enhanced intramolecular communication. Overall, the 2′-dG-II riboswitch might be a promising drug design target due to its ability to recognize both cognate and noncognate ligands.

Discovering potential inhibitors against SARS-CoV-2 by targeting Nsp13 Helicase

The rise in the incidence of COVID-19 as a result of SARS-CoV-2 infection has threatened public health globally. Till now, there have been no proper prophylactics available to fight COVID-19, necessitating the advancement and evolution of effective curative against SARS-CoV-2. This study aimed at the nonstructural protein 13 (nsp13) helicase as a promising target for drug development against COVID-19. A unique collection of nucleoside analogs was screened against the SARS-CoV-2 helicase protein, for which a molecular docking experiment was executed to depict the selected ligand’s binding affinity with the SARS-CoV-2 helicase proteins. Simultaneously, molecular dynamic simulations were performed to examine the protein’s binding site’s conformational stability, flexibility, and interaction with the ligands. Key nucleoside ligands were selected for pharmacokinetic analysis based on their docking scores. Selected ligands (cordycepin and pritelivir) showed excellent pharmacokinetics and were well stabilized at the proteins’ binding site throughout the MD simulation. We have also performed binding free energy analysis or the binding characteristics of ligands with Nsp13 by using MM-PBSA and MM-GBSA. Free energy calculation by MM-PBSA and MM-GBSA analysis suggests that pritelivir may work as viable therapeutics for efficient drug advancement against SARS-CoV-2 Nsp13 helicase, potentially arresting the SARS-CoV-2 replication. Communicated by Ramaswamy H. Sarma

Studies on Non-Covalent Interaction of Coumarin Attached Pyrimidine and 1-Methyl Indole 1,2,3 Triazole Analogues with Intermolecular Telomeric G-Quadruplex DNA Using ESI-MS and Spectroscopy

In the present study, electrospray ionization mass spectrometry (ESI-MS) and spectroscopy have been used to evaluate the non-covalent interaction, stoichiometry, and selectivity of two synthetic coumarin-attached nucleoside and non-nucleoside 1,2,3-triazoles, namely, (1-(5-(hydroxymethyl)-4-(4-((2-oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-furan-2-yl)5-methyl pyrimidine-2,4(1H,3H)-dione (Tr1) and 4-((1-((-1-methyl-1H-indol-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (Tr2) with two different human telomeric intermolecular G-quadruplex DNA structures formed by d(T2AG3) and d(T2AG3)2 sequences. ESI-MS studies indicate that Tr1 specifically interacts with four-stranded intermolecular parallel quadruplex complex, whereas Tr2 interacts with two hairpin as well as four-stranded intermolecular parallel quadruplex complexes. UV–Visible spectroscopic studies suggest that Tr1 and Tr2 interact with G-quadruplex structure and unwind them. Job plots show that stoichiometry of ligand:quadruplex DNA is 1:1. Circular dichroism (CD) studies of G-quadruplex DNA and Tr1/Tr2 ligands manifest that they unfold DNA on interaction. Fluorescence studies demonstrate that ligand molecules intercalate between the two stacks of quadruplex DNA and non-radiative energy transfer occurs between the excited ligand molecules (donor) and quadruplex DNA (acceptor), resulting in enhancement of fluorescence emission intensity. Thus, these studies suggest that nucleoside and non-nucleoside ligands efficiently interact with d(T2AG3) and d(T2AG3)2 G-quadruplex DNA but the interaction is not alike with all kinds of quadruplex DNA, this is probably due to the variation in the pharmacophores and structure of the ligand molecules.

Phenyl 1,2,3-triazole-thymidine ligands stabilize G-quadruplex DNA, inhibit DNA synthesis and potentially reduce tumor cell proliferation over 3'-azido deoxythymidine.

Triazoles are known for their non-toxicity, higher stability and therapeutic activity. Few nucleoside (L1, L2 and L3) and non-nucleoside 1,2,3-triazoles (L4–L14) were synthesised using click chemistry and they were screened for tumor cell cytotoxicity and proliferation. Among these triazole ligands studied, nucleoside ligands exhibited higher potential than non-nucleoside ligands. The nucleoside triazole analogues, 3′-Phenyl-1,2,3- triazole-thymidine (L2) and 3′-4-Chlorophenyl-1,2,3-triazole-thymidine (L3), demonstrated higher cytotoxicity in tumor cells than in normal cells. The IC50 value for L3 was lowest (50 µM) among the ligands studied. L3 terminated cell cycle at S, G2/M phases and enhanced sub-G1 populations, manifesting induction of apoptosis in tumor cells. Confocal studies indicated that nucleoside triazole ligands (L2/L3) cause higher DNA fragmentation than other ligands. Preclinical experiments with tumor-induced mice showed greater reduction in tumor size with L3. In vitro DNA synthesis reaction with L3 exhibited higher DNA synthesis inhibition with quadruplex forming DNA (QF DNA) than non quadruplex forming DNA (NQF DNA). Tm of quadruplex DNA increased in the presence of L3, indicating its ability to enhance stability of quadruplex DNA at elevated temperature and the results indicate that it had higher affinity towards quadruplex DNA than the other forms of DNA (like dsDNA and ssDNA). From western blot experiment, it was noticed that telomerase expression levels in the tissues of tumor-induced mice were found to be reduced on L3 treatment. Microcalorimetry results emphasise that two nucleoside triazole ligands (L2/L3) interact with quadruplex DNA with significantly higher affinity (Kd≈10−7 M). Interestingly the addition of an electronegative moiety to the phenyl group of L2 enhanced its anti-proliferative activity. Though IC50 values are not significantly low with L3, the studies on series of synthetic 1,2,3-triazole ligands are useful for improving and building potential pro-apoptotic ligands.

Analysis of nucleoside-binding proteins by ligand-specific elution from dye resin: application to Mycobacterium tuberculosis aldehyde dehydrogenases

We show that Cibacron Blue F3GA dye resin chromatography can be used to identify ligands that specifically interact with proteins from Mycobacterium tuberculosis, and that the identification of these ligands can facilitate structure determination by enhancing the quality of crystals. Four native Mtb proteins of the aldehyde dehydrogenase (ALDH) family were previously shown to be specifically eluted from a Cibacron Blue F3GA dye resin with nucleosides. In this study we characterized the nucleoside-binding specificity of one of these ALDH isozymes (recombinant Mtb Rv0223c) and compared these biochemical results with co-crystallization experiments with different Rv0223c-nucleoside pairings. We found that the strongly interacting ligands (NAD and NADH) aided formation of high-quality crystals, permitting solution of the first Mtb ALDH (Rv0223c) structure. Other nucleoside ligands (AMP, FAD, adenosine, GTP and NADP) exhibited weaker binding to Rv0223c, and produced co-crystals diffracting to lower resolution. Difference electron density maps based on crystals of Rv0223c with various nucleoside ligands show most share the binding site where the natural ligand NAD binds. From the high degree of similarity of sequence and structure compared to human mitochondrial ALDH-2 (BLAST Z-score = 53.5 and RMSD = 1.5 Å), Rv0223c appears to belong to the ALDH-2 class. An altered oligomerization domain in the Rv0223c structure seems to keep this protein as monomer whereas native human ALDH-2 is a multimer.Electronic supplementary materialThe online version of this article (doi:10.1007/s10969-009-9073-z) contains supplementary material, which is available to authorized users.

Synthesis and characterization of [76Br]-labeled high-affinity A3 adenosine receptor ligands for positron emission tomography

Bromine-76-radiolabeled analogues of previously reported high-affinity A(3) adenosine receptor (A(3)AR) nucleoside ligands have been prepared as potential radiotracers for positron emission tomography. The radiosyntheses were accomplished by oxidative radiobromination on the N(6)-benzyl moiety of trimethyltin precursors. Biodistribution studies of the kinetics of uptake were conducted in awake rats. We prepared an agonist ligand {[(76)Br](1'S,2'R,3'S,4'R,5'S)-4'-{2-chloro-6-[(3-bromophenylmethyl)amino]purin-9-yl}-1'-(methylaminocarbonyl)bicyclo[3.1.0]hexane-2',3'-diol (MRS3581)} in 59% radiochemical yield with a specific activity of 19.5 GBq/micromol and an antagonist ligand {[(76)Br](1'R,2'R,3'S,4'R,5'S)-4'-(6-(3-bromobenzylamino)-2-chloro-9H-purin-9-yl)bicyclo[3.1.0]hexane-2',3'-diol (MRS5147)} in 65% radiochemical yield with a specific activity of 22 GBq/micromol. The resultant products exhibited the expected high affinity (K(i) approximately 0.6 nM) and specific binding at the human A(3)AR in vitro. Biodistribution studies in the rat showed uptake in the organs of excretion and metabolism. The antagonist MRS5147 exhibited increasing uptake in testes, an organ that contains significant quantities of A(3)AR, over a 2-h time course, which suggests the presence of a specific A(3)AR retention mechanism. We were able to compare uptake of the [(76)Br]-labeled antagonist MRS5147 to [(76)Br]agonist MRS3581. The antagonist MRS5147 shows increasing uptake in the testes, an A(3)AR-rich tissue, suggesting that this ligand may have promise as a molecular imaging agent.

Equilibrative Nucleoside Transporter Family Members from Leishmania donovani Are Electrogenic Proton Symporters

Leishmania donovani express two members of the equilibrative nucleoside transporter family; LdNT1 encoded by two closely related and linked genes, LdNT1.1 and LdNT1.2, that transport adenosine and pyrimidine nucleosides and LdNT2 that transports inosine and guanosine exclusively. LdNT1.1, LdNT1.2, and LdNT2 have been expressed in Xenopus laevis oocytes and found to be electrogenic in the presence of nucleoside ligands for which they mediate transport. Further analysis revealed that ligand uptake and transport currents through LdNT1-type transporters are proton-dependent. In addition to the flux of protons that is coupled to the transport reaction, LdNT1 transporters mediate a variable constitutive proton conductance that is blocked by substrates and dipyridamole. Surprisingly, LdNT1.1 and LdNT1.2 exhibit different electrogenic properties, despite their close sequence homology. This electrophysiological study provides the first demonstration that members of the equilibrative nucleoside transporter family can be electrogenic and establishes that these three permeases, unlike their mammalian counterparts, are probably concentrative rather than facilitative transporters.

Structural determinants of A(3) adenosine receptor activation: nucleoside ligands at the agonist/antagonist boundary.

Mutagenesis of the human A(3) adenosine receptor (AR) suggested that certain amino acid residues contributed differently to ligand binding and activation processes. Here we demonstrated that various adenosine modifications, including adenine substitution and ribose ring constraints, also contributed differentially to these processes. The ligand effects on cyclic AMP production in intact CHO cells expressing the A(3)AR and in receptor binding were compared. Notably, the simple 2-fluoro group alone or 2-chloro in combination with N(6)-substitution dramatically diminished the efficacy of adenosine derivatives, even converting agonist into antagonist. Other affinity-increasing substitutions, including N(6)-(3-iodobenzyl) 4 and the (Northern)-methanocarba 15, also reduced efficacy, except in combination with a flexible 5'-uronamide. 2-Cl-N(6)-(3-iodobenzyl) derivatives, both in the (N)-methanocarba (i.e., of the Northern conformation) and riboside series 18 and 5, respectively, were potent antagonists with little residual agonism. Ring-constrained 2',3'-epoxide derivatives in both riboside and (N)-methanocarba series 13 and 21, respectively, and a cyclized (spiral) 4',5'-uronamide derivative 14 were synthesized and found to be human A(3)AR antagonists. 14 bound potently at both human (26 nM) and rat (49 nM) A(3)ARs. A rhodopsin-based A(3)AR model, containing all domains except the C-terminal region, indicated separate structural requirements for receptor binding and activation for these adenosine analogues. Ligand docking, taking into account binding of selected derivatives at mutant A(3)ARs, featured interactions of TM3 (His95) with the adenine moiety and TMs 6 and 7 with the ribose 5'-region. The 5'-OH group of antagonist N(6)-(3-iodobenzyl)-2-chloroadenosine 5 formed a H-bond with N274 but not with S271. The 5'-substituent of nucleoside antagonists moved toward TM7 and away from TM6. The conserved Trp243 (6.48) side chain, involved in recognition of the classical (nonnucleoside) A(3)AR antagonists but not adenosine-derived ligands, displayed a characteristic movement exclusively upon docking of agonists. Thus, A(3)AR activation appeared to require flexibility at the 5'- and 3'-positions, which was diminished in (N)-methanocarba, spiro, and epoxide analogues, and was characteristic of ribose interactions at TM6 and TM7.

Formycin A and its N-methyl analogues, specific inhibitors of E. coli purine nucleoside phosphorylase (PNP): induced tautomeric shifts on binding to enzyme, and enzyme→ligand fluorescence resonance energy transfer

Steady-state and time-resolved emission spectroscopy were used to study the interaction of Escherichia coli purine nucleoside phosphorylase (PNP) with its specific inhibitors, viz. formycin B (FB), and formycin A (FA) and its N-methylated analogues, N(1)-methylformycin A (m 1FA), N(2)-methylformycin A (m 2FA) and N(6)-methylformycin A (m 6FA), in the absence and presence of phosphate (P i). Complex formation led to marked quenching of enzyme tyrosine intrinsic fluorescence, with concomitant increases in fluorescence of FA and m 6FA, independently of the presence of P i. Fluorescence of m 1FA in the complex increased only in the presence of P i, while the weak fluorescence of FB appeared unaffected, independently of P i. Analysis of the emission, excitation and absorption spectra of enzyme–ligand mixtures pointed to fluorescence resonance energy transfer (FRET) from protein tyrosine residue(s) to FA and m 6FA base moieties, as a major mechanism of protein fluorescence quenching. With the non-inhibitor m 2FA, fluorescence emission and excitation spectra were purely additive. Effects of enzyme–FA, or enzyme–m 6FA, interactions on nucleoside excitation and emission spectra revealed shifts in tautomeric equilibria of the bound ligands. With FA, which exists predominantly as the N(1)-H tautomer in solution, the proton N(1)-H is shifted to N(2), independently of the presence of P i. Complex formation with m 6FA in the absence of P i led to a shift of the amino–imino equilibrium in favor of the imino species, and increased fluorescence at 350 nm; by contrast, in the presence of P i, the equilibrium was shifted in favor of the amino species, accompanied by higher fluorescence at 430 nm, and a higher affinity for the enzyme, with a dissociation constant K d=0.5±0.1 μM, two orders of magnitude lower than that for m 6FA in the absence of P i ( K d=46±5 μM). The latter was confirmed by analysis of quenching of enzyme fluorescence according to a modified Stern-Volmer model. Fractional accessibility values ( f a) varied from 0.31 for m 1FA to 0.70 for FA, with negative cooperative binding of m 1FA and FB, and non-cooperative binding of FA and m 6FA. For all nucleoside ligands, the best model describing binding stoichiometry was one ligand per native enzyme hexamer. Fluorescence decays of PNP, FA and their mixtures were best fitted to a sum of two exponential terms, with average lifetimes (〈 τ〉) affected by their interactions. Complex formation resulted in a 2-fold increase in 〈 τ〉 of FA, and a 2-fold decrease in 〈 τ〉 of enzyme fluorescence. The amplitude of the long-lifetime component also increased, confirming the shift of the tautomeric equilibrium in favor of the N(2)-H species. The findings have been examined in relation to enzyme-nucleoside binding deduced from structural studies.

Effects of modifications in the pentose moiety and conformational changes on the binding of nucleoside ligands to uridine phosphorylase from Toxoplasma gondii

One hundred and fifty analogues of uridine, with various modifications to the uracil and pentose moieties, have been tested and compared with uridine with respect to their potency to bind to uridine phosphorylase (UrdPase, EC 2.4.2.3) from Toxoplasma gondii. The effects of the α- and β-anomers, the l- and d-enantiomers, as well as restricted syn and anti rotamers, on binding were examined. Pseudo-, lyxo-, 2,3t - ́ anhydro-2′-deoxy-, 6,5′-cyclo-, 6,3′-methano-, O 5′,6-methano- and carbocyclic uridines did not bind to the enzyme. Ribosides bound better than the corresponding xylosides, which were better than the deoxyribosides. The binding of deoxyribosides was in the following manner: 2′,3′-dideoxynucleosides > 2′,5′-dideoxynucleosides > 2′-deoxyribosides > 3′- and 5′-deoxyribosides. α-2′-Deoxyribosides bound to the enzyme, albeit less tightly than the corresponding β-anomers. The acyclo- and 2,2′-anhydrouridines bound strongly, with the 2,2′-anhydro-derivatives being the better ligands. 2,5′-Anhydrouridine bound to UrdPase less effectively than 2,2′-anhydrouridine and acyclouridine. Arabinosyluracil was at best a very poor ligand, but bound better if a benzyl group was present at the 5-position of the pyrimidine ring. This binding was enhanced further by adding a 5-benzyloxybenzyl group. A similar enhancement of the binding by increased hydrophobicity at the 5-position of the pyrimidine ring was observed with ribosides, α- and β-anomers of the 2′-deoxyribosides, acyclonucleosides, and 2,2′-anhydronucleosides. Among all the compounds tested, 5-(benzyloxybenzyl)-2,2′-anhydrouridine was identified as the best ligand of T. gondii UrdPase with an apparent K i value of 60 ± 3 nM. It is concluded that the presence of an N-glycosyl bond is a prerequisite for a nucleoside ligand to bind to T. gondii UrdPase. On the other hand, the presence of a 2′-, 3′-, or 5′-hydroxyl group, or an N-glycosyl bond in the β-configuration, enhanced but was not essential for binding. Furthermore, the potency of the binding of 2,2′-anhydrouridines (fixed high syn isomers) in contrast to the weaker binding of the 6,1′-anhydro- or 2,5′-anhydrouridines (fixed syn isomers), and the complete lack of binding of the 6,5′-cyclo, O 5′,6-methano- and 6,3′-methanouridines (fixed anti isomers) to T. gondii UrdPase indicate that the binding of ligands to this enzyme is in the syn/high syn conformation around the N-glycosyl bond. The results also indicate that the parasite but not the mammalian host UrdPase can participate in hydrogen bonding with N3 of the pyrimidine ring of nucleoside ligands. T. gondii UrdPase also has a larger hydrophobic pocket adjacent to the C5 of the pyrimidine moiety than the host enzyme, and can accommodate modifications in the pentose moiety which cannot be tolerated by the host enzyme. Most prominent among these modifications is the absence and/or lack of the ribo orientation of the 3′-hydroxyl group, which is a requirement for a ligand to bind to mammalian UrdPase. These differences between the parasite and host enzymes can be useful in designing specific inhibitors or “subversive” substrates for T. gondii UrdPase.

Structure-activity relationship for the binding of nucleoside ligands to adenosine kinase from Toxoplasma gondii

One hundred and twenty-eight purine nucleoside analogs were evaluated as ligands of Toxoplasma gondii adenosine kinase (EC 2.7.1.20) by examining their ability to inhibit this enzyme in vitro. Inhibition was quantified by determining apparent K i (app K i ) values for those compounds that inhibited this enzyme by greater than 10% at a concentration of 1 mM. Two compounds, N 6-( p-methoxybenzoyl) adenosine and 7-iodo-7-deazaadenosine (iodotubercidin), were found to bind to the enzyme (app K i = 3.9 and 1.6 μM, respectively) better than adenosine. On the basis of these data, a structure-activity relationship for the binding of ligands to T. gondii adenosine kinase was formulated using adenosine as a reference compound. It was concluded that the following structural features of purine nucleoside analogs are required or strongly preferred for binding: (1) “pyridine-type” endocyclic nitrogens at the 1- and 3-positions; (2) an exocyclic hydrogen at the 2-position; (3) 6-position exocyclic substituents in the lactim tautomeric form; (4) a “pyridine-type” endocyclic nitrogen at the 7-position or hydrophobic exocyclic substituents attached to an endocyclic carbon at the 7-position; (5) an endocyclic methine or “pyridine-type” nitrogen at the 8-position; (6) an endocyclic nitrogen at the 9-position; (7) a pentose or “pentose-like” (e.g. hydroxylated cyclopentene) moiety attached to the 9-position nitrogen; (8) hydroxyl groups at the 2′- and 3′-positions in a ribose configuration; (9) a hydroxymethyl or methyl (i.e. 5′-deoxy) group at the 5′-position; (10) a β- d-nucleoside configuration; and (11) an anti conformation around the N-glycosidic bond. In addition, there appears to be a “pocket” in the catalytic site of T. gondii adenosine kinase, adjacent to the 6-position of adenosine, that can accommodate large (preferably unsaturated or aromatic) substituents (e.g. phenyl). These findings provide the basis for the rational design of additional ligands of T. gondii adenosine kinase.

Synthesis of water-soluble (tri(hydroxymethyl)phosphine)gold(I) complexes containing a nucleoside ligand

Water-soluble (tri(hydroxymethyl)phosphine)gold(I) complexes containing a nucleoside ligand, [Au(Nuc)- {P(CH 2OH) 3}] +NO 3 − (Nuc=Guo, Ado, Cyd) have been synthesized by the reaction of AuCl(P(CH 2OH) 3 with the corresponding nucleoside in the presence of silver nitrate in ethanol at room temperature.

Flow cytometric quantitation of nucleoside transporter sites on human leukemic cells.

Quantitation of equilibrative, nitrobenzylthioinosine (NBMPR) sensitive (es) nucleoside transporters on blast cells isolated from patients with acute myeloblastic leukemia is useful in predicting intracellular accumulation of the antileukemic nucleoside drug, cytosine arabinoside. We previously reported the synthesis of a fluorescein-labeled ligand for the es nucleoside transporter, 5-(SAENTA-x2)-fluorescein. This paper reports the synthesis of 5-(SAENTA-x8)-fluorescein in which the linkage between fluorescein and nucleoside ligand has been increased from 2 atoms to 8 atoms. This new ligand had a sixfold increase in affinity (Kd 0.9 +/- 0.1 nM) as well as an 86% increase in the cell associated fluorescence output compared to its prototype 5-(SAENTA-x2)-fluorescein. The fluorescence signal arising from 5-(SAENTA-x8)-fluorescein specifically bound to freshly isolated and cultured leukemic myeloblasts was converted to molecules of equivalent soluble fluorescein (MESF) using standardized fluorescein microbeads and compared with the number of es nucleoside transporter sites assayed concurrently by [3H]NBMPR equilibrium binding analysis. A high correlation between the two assays was observed (r = 0.98), which enabled the cell-bound fluorescence output of 5-(SAENTA-x8)-fluorescein to be expressed in numbers of es nucleoside transporter sites per cell. The improved properties of 5-(SAENTA-x8)-fluorescein over those of its prototype molecule make it a suitable reagent for flow cytometric quantitation of nucleoside transporter expression on leukemic cells isolated from patient samples.

N-3 derivatives of azido- or fluoro-dideoxythymidine: Synthesis and biological activies: [formula omitted]1, [formula omitted]2, M. Lemaître 3, M. Maillard 1, D. Adams 1, C. Goulaouic 1, F. Begassat 3, A. Bugnicourt 3 C. Ferrieux 3, C. Rombi 3, E. Pacaud 3, D. Thierry 4, E. Gluckman 4, and A. Zerial 3. 1CNRS, Institut de Chimie des Substances Naturelles, 91198 Gif-sur-Yvette,

One hundred and fifty analogues of uridine, with various modifications to the uracil and pentose moieties, have been tested and compared with uridine with respect to their potency to bind to uridine phosphorylase (UrdPase, EC 2.4.2.3) from Toxoplasma gondii. The effects of the α- and β-anomers, the l- and d-enantiomers, as well as restricted syn and anti rotamers, on binding were examined. Pseudo-, lyxo-, 2,3t-́anhydro-2′-deoxy-, 6,5′-cyclo-, 6,3′-methano-, O5′,6-methano- and carbocyclic uridines did not bind to the enzyme. Ribosides bound better than the corresponding xylosides, which were better than the deoxyribosides. The binding of deoxyribosides was in the following manner: 2′,3′-dideoxynucleosides > 2′,5′-dideoxynucleosides > 2′-deoxyribosides > 3′- and 5′-deoxyribosides. α-2′-Deoxyribosides bound to the enzyme, albeit less tightly than the corresponding β-anomers. The acyclo- and 2,2′-anhydrouridines bound strongly, with the 2,2′-anhydro-derivatives being the better ligands. 2,5′-Anhydrouridine bound to UrdPase less effectively than 2,2′-anhydrouridine and acyclouridine. Arabinosyluracil was at best a very poor ligand, but bound better if a benzyl group was present at the 5-position of the pyrimidine ring. This binding was enhanced further by adding a 5-benzyloxybenzyl group. A similar enhancement of the binding by increased hydrophobicity at the 5-position of the pyrimidine ring was observed with ribosides, α- and β-anomers of the 2′-deoxyribosides, acyclonucleosides, and 2,2′-anhydronucleosides. Among all the compounds tested, 5-(benzyloxybenzyl)-2,2′-anhydrouridine was identified as the best ligand of T. gondii UrdPase with an apparent Ki value of 60 ± 3 nM. It is concluded that the presence of an N-glycosyl bond is a prerequisite for a nucleoside ligand to bind to T. gondii UrdPase. On the other hand, the presence of a 2′-, 3′-, or 5′-hydroxyl group, or an N-glycosyl bond in the β-configuration, enhanced but was not essential for binding. Furthermore, the potency of the binding of 2,2′-anhydrouridines (fixed high syn isomers) in contrast to the weaker binding of the 6,1′-anhydro- or 2,5′-anhydrouridines (fixed syn isomers), and the complete lack of binding of the 6,5′-cyclo, O5′,6-methano- and 6,3′-methanouridines (fixed anti isomers) to T. gondii UrdPase indicate that the binding of ligands to this enzyme is in the syn/high syn conformation around the N-glycosyl bond. The results also indicate that the parasite but not the mammalian host UrdPase can participate in hydrogen bonding with N3 of the pyrimidine ring of nucleoside ligands. T. gondii UrdPase also has a larger hydrophobic pocket adjacent to the C5 of the pyrimidine moiety than the host enzyme, and can accommodate modifications in the pentose moiety which cannot be tolerated by the host enzyme. Most prominent among these modifications is the absence and/or lack of the ribo orientation of the 3′-hydroxyl group, which is a requirement for a ligand to bind to mammalian UrdPase. These differences between the parasite and host enzymes can be useful in designing specific inhibitors or “subversive” substrates for T. gondii UrdPase.

NMR, CD and MCD Studies of Vanadate--Nucleoside Complexes.

Complex formation between tetraoxovanadate(V) and each of the nucleosides adenosine, guanosine, cytidine and uridine has been studied in a constant salt medium at pH 7. 13C- and 51V NMR studies show that only complexes with the formula V2L2 (V = vanadate, L = nucleoside) are formed, and their formation constants have been determined. They have 51V NMR resonances around -523 ppm relative to VOCl3 and they exhibit no CD in the spectral region of the charge-transfer transitions. MCD spectra were also measured, and all experiments are in accord with a molecular structure composed by two edge-sharing VO6 octahedra forming an O4V(mu-O)2VO4 skeleton with each of the nucleoside ligands bridging the two vanadium centres through the ribose 2',3'-oxygens, which are the oxygens outside the V2O6 plane. Admixture of imidazole-HCl buffer at pH 7 gives rise to additional complexes of 1:1 stoichiometry. They have been characterized by 51V NMR and CD, and their formation constants are reported. Vanadate(V) and the deoxynucleosides deoxyadenosine, deoxyguanosine, deoxycytidine and thymidine form very weak complexes which cannot be detected by 51V NMR or CD under conditions for which vanadate and the nucleosides form complexes.

Dissociative Ligand Exchange in Organoplatinum(II) and -gold(III) Complexes Having a Nucleoside Ligand. Dynamic NMR as a Mechanistic Probe for Ligand Exchange Process

Abstract Application of simple dynamic NMR technique to intermolecular ligand exchange reactions in dimethylchloro(guanosine)gold(III) and [ethyl(1,5-cyclooctadiene)(cytidine)platinum(II)]+ reveals that both reactions proceed by a dissociative mechanism.

Reaction of alkylcobalamins with thiols.

Carbon-13 NMR spectroscopy and phosphorus-31 NMR spectroscopy have been used to study the reaction of several alkylcobalamins with 2-mercaptoethanol. At alkaline pH, when the thiol is deprotonated, the alkyl-transfer reactions involve a nucleophilic attack of the thiolate anion on the Co-methylene carbon of the cobalamins, yielding alkyl thioethers and cob(II)alamin. In these nucleophilic displacement reactions cob(I)alamin is presumably formed as an intermediate. The higher alkylcobalamins react more slowly than methylcobalamin. The lower reactivity of ethyl- and propylcobalamin is probably the basis of the inhibition of the corrinoid-dependent methyl-transfer systems by propyl iodide. The transfer of the upper nucleoside ligand of adenosylcobalamin to 2-mercaptoethanol is a very slow process; S-adenosyl-mercaptoethanol and cob(II)alamin are the final products of the reaction. The dealkylation of (carboxymethyl)cobalamin is a much more facile reaction. At alkaline pH S-(carboxymethyl)mercaptoethanol and cob(II)alamin are produced, while at pH values below 8 the carbon-cobalt bond is cleaved reductively to acetate and cob(II)alamin. The reductive cleavage of the carbon-cobalt bond of (carboxymethyl)cobalamin by 2-mercaptoethanol is extremely fast when the cobalamin is in the "base-off" form. Because we have been unable to detect trans coordination of 2-mercaptoethanol, we favor a mechanism that involves a hydride attack on the Co-methylene carbon of (carboxymethyl)cobalamin rather than a trans attack of the thiol on the cobalt atom.

Thermolysis of Dimethylgold(III) Complexes Having a Nucleoside Ligand

Abstract Relative ease of dissociaiton of nucleosides from dimethylgold(III) complexes, estimated from the kinetics of thermolysis of dimethylgold(III) complexes having a nucleoside ligand, AuMe2(nucleoside)X, lies in the order of Cyd <Guo <Ado<<dThd.