S-(4-nitrobenzyl)-6-thioinosine: Tritium labelling at high specific activity.

The one-step tritiation of S-(4-nitrobenzyl)-6-thioinosine is described with characterization of the product by tritium NMR as well as mass spectrometry. The storage, stability, and repurification of [benzyl methylene-3 H]S-(4-nitrobenzyl)-6-thioinosine are also discussed.

Structures of human ENT1 in complex with adenosine reuptake inhibitors.

The human equilibrative nucleoside transporter 1 (hENT1), a member of the SLC29 family, plays crucial roles in adenosine signaling, cellular uptake of nucleoside for DNA and RNA synthesis, and nucleoside-derived anticancer and antiviral drug transport in humans. Because of its central role in adenosine signaling, it is the target of adenosine reuptake inhibitors (AdoRI), several of which are used clinically. Despite its importance in human physiology and pharmacology, the molecular basis of hENT1-mediated adenosine transport and its inhibition by AdoRIs are limited, owing to the absence of structural information on hENT1. Here, we present crystal structures of hENT1 in complex with two chemically distinct AdoRIs: dilazep and S-(4-nitrobenzyl)-6-thioinosine (NBMPR). Combined with mutagenesis study, our structural analyses elucidate two distinct inhibitory mechanisms exhibited on hENT1 and provide insight into adenosine recognition and transport. Our studies provide a platform for improved pharmacological intervention of adenosine and nucleoside analog drug transport by hENT1.

Photoreactivity of carboxylated single-walled carbon nanotubes in sunlight: reactive oxygen species production in water.

Very limited information exists on transformation processes of carbon nanotubes in the natural aquatic environment. Because the conjugated pi-bond structure of these materials is efficient in absorbing sunlight, photochemical transformations are a potential fate process with reactivity predicted to vary with their diameter, chirality, number and type of defects, functionalization, residual metal catalyst and amorphous carbon content, and with the composition of the water, including the type and composition of materials that act to disperse them into the aqueous environment. In this study, the photochemical reactions involving colloidal dispersions of carboxylated single-walled carbon nanotubes (SWNT-COOH) in sunlight were examined. Production of reactive oxygen species (ROS) during irradiation occurs and is evidence for potential further phototransformation and may be significant in assessing their overall environmental impacts. In aerated samples exposed to sunlight or to lamps that emit light only within the solar spectrum, the probe compounds, furfuryl alcohol (FFA), tetrazolium salts (NBT2+ and XTT), and p-chlorobenzoic acid (pCBA), were used to indicate production of 1O2, O2.-, and .OH, respectively. All three ROS were produced in the presence of SWNT-COOH and molecular oxygen (3O2). 1O2 production was confirmed by observing enhanced FFA decay in deuterium oxide, attenuated decay of FFA in the presence of azide ion, and the lack of decay of FFA in deoxygenated solutions. Photogeneration of O2.- and .OH was confirmed by applying superoxide dismutase (SOD) and tert-butanol assays, respectively. In air-equilibrated suspensions, the loss of 0.2 mM FFA in 10 mg/L SWNT-COOH was approximately 85% after 74 h. Production of 1O2 was not dependent on pH from 7 to 11; however photoinduced aggregation was observed at pH 3.

Testicular expression of Adora3i2 in Adora3 knockout mice reveals a role of mouse A3Ri2 and human A3Ri3 adenosine receptors in sperm.

Adenosine is a candidate modulator of sperm motility in the female reproductive tract that increases sperm flagellar beat frequency in vitro. Past work suggested that this acceleration may involve equilibrative (ENT) and concentrative (CNT) nucleoside transporters. Here we show that Slc29a1 (ENT-1) is the predominant nucleoside transporter expressed in the mouse testis. Unexpectedly, the beat of Slc29a1-null sperm still accelerates in response to 2-chloro-2'-deoxyadenosine (Cl-dAdo). Moreover, in wild-type sperm neither blockade of CNTs by removal of external Na(+), nor inhibition of ENTs with nitrobenzylthioionosine, prevents acceleration of the sperm beat by Cl-dAdo. In contrast, pertussis toxin produces strong blockade, indicating involvement of a Gα(i/o)-coupled adenosine receptor. Although agonists selective for adenosine receptors A1R, A2aR, and A2bR are ineffective, A3R-selective agonists Cl-IB-MECA and IB-MECA do accelerate the beat. Consistent with this pharmacological profile, the predominant Adora transcripts in the testis are products of the nested Adora3i1 and Adora3i2 genes. Surprisingly, Cl-IB-MECA and Cl-dAdo still accelerate the beat of Adora3i1-null sperm indicating that the remaining Adora3i2 transcript produces an A3R that functions in sperm. When cloned Adora3i2 is heterologously expressed in tsA-201 cells, Cl-dAdo decreases forskolin-evoked accumulation of cAMP, indicating that Adora3i2 specifies a functional A3Ri2 adenosine receptor that couples through Gα(i). Database mining reveals that mouse Adora3i2 is expressed primarily in testis, almost exclusively in spermatids. Expression of the orthologous ADORA3i3 transcript also is most prominent in human testis; presumably producing an A3Ri3 receptor that is functional in sperm and that may be a target for development of male-directed contraceptives.

Structure-activity relationships of carbocyclic 6-benzylthioinosine analogues as subversive substrates of Toxoplasma gondii adenosine kinase.

Carbocyclic 6-benzylthioinosine analogues were synthesized and evaluated for their binding affinity against Toxoplasma gondii adenosine kinase [EC.2.7.1.20]. Various substituents on the aromatic ring of the 6-benzylthio group resulted in increased binding affinity to the enzyme as compared to the unsubstituted compound. Carbocyclic 6-(p-methylbenzylthio)inosine 9n exhibited the most potent binding affinity. Docking simulations were performed to position compound 9n into the T. gondii adenosine kinase active site to determine the probable binding mode. Experimental investigations and theoretical calculations further support that an oxygen atom of the sugar is not critical for the ligand-binding. In agreement with its binding affinity, carbocyclic 6-(p-methylbenzylthio)inosine 9n demonstrated significant anti-toxoplasma activity (IC(50)=11.9microM) in cell culture without any apparent host-toxicity.

Photocycloaddition of the T1 excited state of thioinosine to uridine and adenosine.

Novel photoadducts were obtained by irradiation of thioinosine (6-thiopurine riboside, TI) in deaerated aqueous solution without and in the presence of uridine and adenosine. Excitation (lambda > 300 nm) of TI to its excited S2 state yields a single bimolecular photoproduct. It is a purine-pyrimidine diriboside in which the purine ring is attached to the amide nitrogen of 6-amino-4-thioxo-5-formamidopyrimidine. When TI was irradiated in the presence of an excess of adenosine, two photoproducts were isolated: diribosides of N-(4,6-diaminopirymidin-5-yl)-N-formyl-6-aminopurine and N-(4-amino-6-formylamino-pyrimidin-5-yl)-6-aminopurine, both containing a purine and a formylaminopyrimidine (Fapy) fragment. The photoreaction of TI with uridine gave two regioisomeric photoproducts identified as diribosides containing either 5- or 6-(purin-6-yl)uracil as aglycones. A multistep mechanism leading to the stable photoproducts is proposed. In the first step of the mechanism, the C=S group of the excited TI undergoes a [2 + 2] cycloaddition regioselectively to the N(7)=C(8) bond of the purine ring or adds in a non-regioselective manner to the C(5)=C(6) bond of uracil. The unstable photoproducts thus formed undergo a series of dark reactions at room temperature. The photocycloaddition reactions originate from the excited T1 state of TI. This conclusion is supported by a combination of evidence from reaction quenching studies using both steady-state quantum yield determinations and kinetics results from nanosecond laser flash photolysis. The T1 state of TI is quenched by other TI molecules in their S0 state (self-quenching) and also by uridine and adenosine, all with large rate constants (0.8-5) x 10(9) M(-1) s(-1). The quantum yields of the reactions are in general very low (phi(R) < or = 8 x 10(-3)). The sources of the inefficiency in the photocycloaddition of TI to uridine and adenosine are discussed. The photoproducts containing the Fapy residue undergo deformylation and isomerization of the ribosyl moiety (anomerization, furanose/pyranose transformation) upon heating in aqueous solution. Products of the transformations were identified.

CoMFA and CoMSIA 3D-QSAR studies on S(6)-(4-nitrobenzyl)mercaptopurine riboside (NBMPR) analogs as inhibitors of human equilibrative nucleoside transporter 1 (hENT1).

3D-QSAR (CoMFA and CoMSIA) studies were performed on human equlibrative nucleoside transporter (hENT1) inhibitors displaying K(i) values ranging from 10,000 to 0.7nM. Both CoMFA and CoMSIA analysis gave reliable models with q2 values >0.50 and r2 values >0.92. The models have been validated for their stability and robustness using group validation and bootstrapping techniques and for their predictive abilities using an external test set of nine compounds. The high predictive r2 values of the test set (0.72 for CoMFA model and 0.74 for CoMSIA model) reveals that the models can prove to be a useful tool for activity prediction of newly designed nucleoside transporter inhibitors. The CoMFA and CoMSIA contour maps identify features important for exhibiting good binding affinities at the transporter, and can thus serve as a useful guide for the design of potential equilibrative nucleoside transporter inhibitors.

Structure-activity relationships of 7-deaza-6-benzylthioinosine analogues as ligands of Toxoplasma gondii adenosine kinase.

Several 7-deaza-6-benzylthioinosine analogues with varied substituents on aromatic ring were synthesized and evaluated against Toxoplasma gondii adenosine kinase (EC.2.7.1.20). Structure-activity relationships indicated that the nitrogen atom at the 7-position does not appear to be a critical structural requirement. Molecular modeling reveals that the 7-deazapurine motif provided flexibility to the 6-benzylthio group as a result of the absence of H-bonding between N7 and Thr140. This flexibility allowed better fitting of the 6-benzylthio group into the hydrophobic pocket of the enzyme at the 6-position. In general, single substitutions at the para or meta position enhanced binding. On the other hand, single substitutions at the ortho position led to the loss of binding affinity. The most potent compounds, 7-deaza- p-cyano-6-benzylthioinosine (IC 50 = 5.3 microM) and 7-deaza- p-methoxy-6-benzylthioinosine (IC 50 = 4.6 microM), were evaluated in cell culture to delineate their selective toxicity.

Constrained NBMPR analogue synthesis, pharmacophore mapping and 3D-QSAR modeling of equilibrative nucleoside transporter 1 (ENT1) inhibitory activity.

Conformationally constrained analogue synthesis was undertaken to aid in pharmacophore mapping and 3D-QSAR analysis of nitrobenzylmercaptopurine riboside (NBMPR) congeners as equilibriative nucleoside transporter 1 (ENT1) inhibitors. In our previous study [J. Med. Chem. 2003, 46, 831-837], novel regioisomeric nitro-1,2,3,4-tetrahydroisoquinoline conformationally constrained analogues of NBMPR were synthesized and evaluated as ENT1 ligands. 7-NO(2)-1,2,3,4-Tetrahydroisoquino-2-yl purine riboside was identified as the analogue with the nitro group in the best orientation at the NBMPR binding site of ENT1. In the present study, further conformational constraining was introduced by synthesizing 5'-O,8-cyclo derivatives. The flow cytometrically determined binding affinities indicated that the additional 5'-O,8-cyclo constraining was unfavorable for binding to the ENT1 transporter. The structure-activity relationship (SAR) acquired was applied to pharmacophore mapping using the PHASE program. The best pharmacophore hypothesis obtained embodied an anti-conformation with three hydrogen-bond acceptors, one hydrophobic center, and two aromatic rings involving the 3'-OH, 4'-oxygen, the NO(2) group, the benzyl phenyl and the imidazole and pyrimidine portions of the purine ring, respectively. A PHASE 3D-QSAR model derived with this pharmacophore yielded an r(2) of 0.916 for four (4) PLS components, and an excellent external test set predictive r(2) of 0.78 for 39 compounds. This pharmacophore was used for molecular alignment in a comparative molecular field analysis (CoMFA) 3D-QSAR study that also afforded a predictive model with external test set validation predictive r(2) of 0.73. Thus, although limited, this study suggests that the bioactive conformation for NBMPR at the ENT1 transporter could be anti. The study has also suggested an ENT1 inhibitory pharmacophore, and established a predictive CoMFA 3D-QSAR model that might be useful for novel ENT1 inhibitor discovery and optimization.

Generation of superoxide from reaction of 3H-1,2-dithiole-3-thione with thiols: implications for dithiolethione chemoprotection.

3H-1,2-Dithiole-3-thione (D3T), a potent member of dithiolethiones, induces phase 2 enzymes by activating an Nrf2/Keap1-dependent signaling pathway. It was proposed that interaction between D3T and two adjacent sulfhydryl groups of Keap1 might cause dissociation of Keap1 from Nrf2, leading to Nrf2 activation. This study was undertaken to investigate the reactions between D3T and thiols, including the dithiol compound, dithiothreitol (DTT), and the monothiol, glutathione (GSH). We reported here that under physiologically relevant conditions incubation of D3T with DTT caused remarkable oxygen consumption, indicating a redox reaction between D3T and the dithiol molecule. Incubation of D3T with GSH also led to oxygen consumption, but to a less extent. Electron paramagnetic resonance (EPR) studies showed that the redox reaction between D3T and DTT generated superoxide. Superoxide was also formed from the redox reaction of D3T with GSH. These findings demonstrate that D3T reacts with thiols, particularly a dithiol, generating superoxide, which may provide a mechanistic explanation for induction of Nrf2-dependent phase 2 enzymes by D3T.

Novel C2-purine position analogs of nitrobenzylmercaptopurine riboside as human equilibrative nucleoside transporter 1 inhibitors.

Nucleoside transporter inhibitors have potential therapeutic applications as anticancer, antiviral, cardioprotective, and neuroprotective agents. S(6)-(4-nitrobenzyl)mercaptopurine riboside (NBMPR) is a prototype inhibitor of the human equilibrative nucleoside transporter (hENT1), and is a high affinity ligand with a K(d) of 0.1-1.0 nM. We have synthesized and flow cytometrically evaluated the binding affinity of a series of novel C(2)-purine position substituted analogs of NBMPR at the hENT1. The aim of this research was to understand the substituent requirements at the C(2)-purine position of NBMPR. Structure-activity relationships (SAR) indicate that increasing the steric bulk at the C(2)-purine position of NBMPR led to a decrease in binding affinity of these ligands at the hENT1. New high affinity inhibitors were identified, with the best compound, 2-fluoro-4-nitrobenzyl mercaptopurine riboside (7), exhibiting a K(i) of 2.1 nM. This information, when coupled with the information obtained from other structure-activity relationship studies should prove useful in efforts aimed at modeling the NMBPR and analogs pharmacophore of hENT1 inhibitors.

Adsorption of 6-mercaptopurine and 6-mercaptopurine-ribosideon silver colloid: a pH-dependent surface-enhanced Raman spectroscopy and density functional theory study. II. 6-mercaptopurine-riboside.

Surface-enhanced Raman spectroscopy (SERS) has been applied to characterize the interaction of 6-mercaptopurine-ribose (6MPR), an active drug used in chemotherapy of acute lymphoblastic leukemia, with a model biological substrate at therapeutic concentrations and as function of the pH value. Therefore, a detailed vibrational analysis of crystalline and solvated (6MPR) based on Density Functional Theory (DFT) calculations of the thion and thiol tautomers has been performed. 6MPR adopts the thion tautomeric form in the polycrystalline state. The SERS spectra of 6MPR and 6-mercaptopurine (6MP) recorded on silver colloid provided evidence that the ribose derivative shows different adsorption behavior compared with the free base. Under acidic conditions, the adsorption of 6MPR on the metal surface via the N7 and possibly S atoms was proposed to have a perpendicular orientation, while 6MP is probably adsorbed through the N9 and N3 atoms. Under basic conditions both molecules are adsorbed through the N1 and possibly S atoms, but 6MP has a more tilted orientation on the silver colloidal surface while 6MPR adopts a perpendicular orientation. The reorientation of the 6MPR molecule on the surface starts at pH 8 while in the case of 6MP the reorientation starts around pH 6. Under basic conditions, the presence of the anionic molecular species for both molecules is suggested. The deprotonation of 6MP is completed at pH 8 while the deprotonation of the riboside is finished at pH 10. For low drug concentrations under neutral conditions and for pH values 8 and 9, 6MPR interacts with the substrate through both N7 and N1 atoms, possibly forming two differently adsorbed species, while for 6MP only one species adsorbed via N1 was evidenced.

Inhibition of nucleoside transport proteins by C8-alkylamine-substituted purines.

4-nitrobenzylthioinosine (NBTI, 1) is a well-known inhibitor for the nucleoside transport protein ENT1. Here we report on the synthesis and the biological evaluation of compounds that are less polar than NBTI. Compound screening in our laboratory indicated that introduction of an alkylamine substituent at the C(8)-position of N(6)-cyclopentyladenosine (CPA, 2) led to an increment in affinity for the transport protein. It was investigated whether this would also apply for NBTI derivatives. Two series of C(8)-alkylamine-substituted compounds were prepared, one in which the nitro group was absent (46-58) and another in which the ribose moiety was replaced by a benzyl group (72-75). Comparison of the biological data of these compounds with 6-benzylthioinosine (4, K(i) = 53 nM) and 9-benzyl-6-(4-nitrobenzylsulfanyl)purine (59, K(i) = 135 nM) confirmed the hypothesis. The K(i) values improved upon elongation of the alkylamine chain from methylamine to n-hexylamine with an optimum for n-pentylamine (50, K(i) = 2.3 nM). Substitution with 2-methylbutylamine (52), cyclopropylamine (53), cyclopentylamine (54, 72), and cyclohexylamine (55, 73) revealed that the presence of a bulky group enhanced the affinity. The presence of tertiary amines obtained by substitution with pyrrolidine, piperidine, and morpholine gave only poor results. For both series substitution with cyclopentylamine was most effective. Compound 54 (LUF5942) proved the most active, showing a comparable affinity (K(i) = 0.64 nM) to NBTI but a significantly lower polar surface area.

Synthesis, biological activity and molecular modeling of 6-benzylthioinosine analogues as subversive substrates of Toxoplasma gondii adenosine kinase.

Toxoplasma gondii is the most common cause of secondary CNS infections in immunocompromised persons such as AIDS patients. The major route of adenosine metabolism in T. gondii is direct phosphorylation to adenosine 5'-monophosphate (AMP) catalyzed by the enzyme adenosine kinase (EC 2.7.1.20). Adenosine kinase in T. gondii is significantly more active than any other purine salvage enzyme in this parasite and has been established as a potential chemotherapeutic target for the treatment of toxoplasmosis. Subversive substrates of T. gondii,but not the human, adenosine kinase are preferentially metabolized to their monophosphorylated forms and become selectively toxic to the parasites but not their host. 6-Benzylthioinosine (BTI) was identified as an excellent subversive substrate of T. gondii adenosine kinase. Herein, we report the synthesis of new analogues of BTI as subversive substrates for T. gondii adenosine kinase. These new subversive substrates were synthesized starting from tribenzoyl protected d-ribose. To accomplish the lead optimization process, a divergent and focused combinatorial library was synthesized using a polymer-supported trityl group at the 5'-position. The combinatorial library of 20 compounds gave several compounds more active than BTI. Structure-activity relationship studies showed that substitution at the para position plays a crucial role. To investigate the reasons for this discrimination, substrates with different substituents at the para position were studied by molecular modeling using Monte Carlo Conformational Search followed by energy minimization of the enzyme-ligand complex.

Convenient synthesis of oligodeoxynucleotides containing 2'-deoxy-6-thioinosine.

A facile synthesis of oligodeoxynucleotides (ODN) containing 2'-deoxy-6-thioinosine (dI6S) based on the convertible nucleoside O6-phenyl-2'-deoxyinosine is presented. After standard solid-phase DNA synthesis and removal of the cyanoethyl protecting groups with DBU treatment with aqueous sodium hydrogen sulfide introduces the sulfur functionality, deprotects the other nucleobases and cleaves the ODN from the solid support in a one-pot reaction. In addition, the extinction coefficient of 2'-deoxy-6-thioinosine is determined by enzymatic fragmentation of the resulting ODN in the presence of adenosine deaminase.

Synthesis and flow cytometric evaluation of novel 1,2,3,4-tetrahydroisoquinoline conformationally constrained analogues of nitrobenzylmercaptopurine riboside (NBMPR) designed for probing its conformation when bound to the es nucleoside transporter.

Novel regioisomers of conformationally constrained analogues of the potent es nucleoside transporter ligand, nitrobenzylmercaptopurine riboside (NBMPR), designed for probing its bound (bioactive) conformation, were synthesized and evaluated as es transporter ligands by flow cytometry. Purine 6-position 5, 6, 7, or 8-nitro-1,2,3,4-tetrahydroisoquinolylpurine ribosides, in which the nitrobenzyl moiety in NBMPR has been locked into the nitro-1,2,3,4-tetrahydroisoquinoline system, were synthesized by reaction of the appropriate nitro-1,2,3,4-tetrahydroisoquinoline with 6-chloropurine riboside. Flow cytometry was performed using 5-(SAENTA)-X8-fluorescein as the competitive ligand. A high degree of variation in the es transporter binding capacity of the target compounds was observed, with the K(i) values ranging from 0.45 nM for the most tightly bound compound (4) to 300 nM for the least tightly bound compound (5). The K(i) of NBMPR was 0.70 nM, a little higher than that of compound 4. Compound 4 is the isomer that has the nitro group in the best orientation at the es transporter binding site compared to the other three compounds, 2, 3, and 5.

Kinetics and mechanism for platination of thione-containing nucleotides and oligonucleotides: evaluation of the salt dependence.

Reactions of cis-[PtCl(NH(3))(CyNH(2))(OH(2))](+) (Cy=cyclohexyl) with thione-containing single-stranded oligonucleotides d(T(8)XT(8)) and d(XT(16)) (X=(s6)I or (s4)U) and the mononucleotides 4-thiouridine ((s4)UMP) and 6-mercaptoinosine ((s6)IMP) have been studied in aqueous solution at pH 4.1. The reaction kinetics was followed using HPLC methodology as a function of ionic strength in the interval 5.0 mM

Topology of a human equilibrative, nitrobenzylthioinosine (NBMPR)-sensitive nucleoside transporter (hENT1) implicated in the cellular uptake of adenosine and anti-cancer drugs.

The human equilibrative nucleoside transporter hENT1, the first identified member of the ENT family of integral membrane proteins, is the primary mechanism for the cellular uptake of physiologic nucleosides, including adenosine, and many anti-cancer nucleoside drugs. We have produced recombinant hENT1 in Xenopus oocytes and used native and engineered N-glycosylation sites in combination with immunological approaches to experimentally define the membrane architecture of this prototypic nucleoside transporter. hENT1 (456 amino acid residues) is shown to contain 11 transmembrane helical segments with an amino terminus that is intracellular and a carboxyl terminus that is extracellular. Transmembrane helices are linked by short hydrophilic regions, except for a large glycosylated extracellular loop between transmembrane helices 1 and 2 and a large central cytoplasmic loop between transmembrane helices 6 and 7. Sequence analyses suggest that this membrane topology is common to all mammalian, insect, nematode, protozoan, yeast, and plant members of the ENT protein family.

Synthesis using ring closure metathesis and effect on nucleoside transport of a (N)-methanocarba S-(4-nitrobenzyl)thioinosine derivative.

[reaction: see text] A new synthetic route to ring-constrained (N)-methanocarba nucleosides and nucleotides is presented. Ring closure of a diene intermediate using Grubbs catalyst provides a new avenue for the preparation of the cyclopentenone derivative 6, which is a versatile intermediate for various carbocycles. The product was almost as potent an inhibitor of es-mediated nucleoside transport as the parent compound, inhibiting initial rates of uptake of uridine into cultured CCRF-CEM cells by 50% at approximately 30-50 nM.