Substrates of DNA polymerases with planar conformation of sugar: model of substrate transition state?

Abstract

Several years ago, we published an hypothesis concerning conformation of the glycone moiety of different substrates in active centers of several DNA metabolizing enzymes (Nucleosides & Nucleotides 1993, 12, 649–670). This hypothesis prompted us to further study the subtle conformational changes on substrates of DNA polymerases. Data collected in our, as well as other laboratories, have been analyzed, and models of active centers of different DNA polymerases are discussed below. Based on the model of substrate requirements, we now can divide DNA polymerases into two distinguished classes.     

Modified nucleosides and nucleotides inhibiting HIV replication: analysis of the situation and potential prospects]

The results of an eight year search of blockaders of human immunodeficiency virus (HIV) among groups of modified nucleosides and nucleotides are reviewed. The molecular mechanism of action of these compounds is based on the inhibition of DNA polymerases activity. Attempts of systematic analysis of structure--anti-HIV activity relationship for modified substrates of DNA biosynthesis are made. In this analysis attention is focused on the evaluation of general properties of enzymes of the phosphorylation cascade and DNA polymerases. Such properties are analyzed on the basis of empiric rules which summarize electronic, steric and conformational properties of substrate analogs. The ability of phosphorylating nucleosides to block HIV reproduction are analyzed. The interdependence of structure of several most known inhibitors with their action on nucleic acids components metabolism as well as the structure--therapeutic properties relationship are discussed.     

Some Examples of the Use of Trifluoromethane Sulfonic Anhydride in Nucleic Acid Chemistry

Abstract

Trifluoromethane sulfonic anhydride has been used for the inversion of configuration at the 3′-position of 2′-deoxypurine nucleosides, for the modification of the base moiety of purine and pyrimidine nucleosides, for nucleophilic substitution in the sugar moiety, for the synthesis of O²,3′-cyclothymidine and for sugar-base condensation reactions. Reaction can be carried out under very mild conditions. The conditions for these reactions are quite different so that a good selectivity can be obtained when different reactive groups are present.     

Substrate/Inhibitor Properties of Human Deoxycytidine Kinase (dCK) and Thymidine Kinases (Tk1 And Tk2) Towards the Sugar Moiety of Nucleosides,Including O′-Alkyl Analogues

Abstract

Nucleoside analogues with modified sugar moieties have been examined for their substrate/inhibitor specificities towards highly purified deoxycytidine kinase (dCK) and thymidine kinases (tetrameric high-affinity form of TK1, and TK2) from human leukemic spleen. In particular, the analogues included the mono-and di-O′-methyl derivatives of dC, dU and dA, syntheses of which are described. In general, purine nucleosides with modified sugar rings were feebler substrates than the corresponding cytosine analogues. Sugar-modified analogues of dU were also relatively poor substrates of TK1 and TK2, but were reasonably good inhibitors, with generally lower K_i values vs TK2 than TK1. An excellent discriminator between TK1 and TK2 was 3′-hexanoylamino-2′,3′-dideoxythymidine, with a K_i of ～600 μM for TK1 and ～0.1 μM for TK2. 3′-OMe-dC was a superior inhibitor of dCK to its 5′-O-methyl congener, consistent with possible participation of the oxygen of the (3′)-OH or (3′)-OMe as proton acceptor in hydrogen bonding with the enzyme. Surprisingly α-dT was a good substrate of both TK1 and TK2, with K_i values of 120 and 30 μM for TK1 and TK2, respectively; and a 3′-branched α-L-deoxycytidine analogue proved to be as good a substrate as its α-D-counterpart. Several 5 ′-substituted analogues of dC were     

Configurational and Conformational Analysis of Regular and Modified Nucleosides by 1D-NOE Difference Spectroscopy

Abstract

1D-Nuclear Overhauser enhancement (NOE) spectroscopy on regular and modified nucleosides provides qualitative and semiquantitative information about configurational and conformational parameters. The formula scheme shows selected nucleosides which were studied with this technique.     

Synthesis and Anti-HIV Activity of β-D-3′-Azido-2′,3′-unsaturated Nucleosides and β-D-3′-Azido-3′-deoxyribofuranosylnucleosides

Since the discovery of 3′-azido-3′-deoxythymidine (AZT) and 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) as potent and selective inhibitors of the replication of human immunodeficiency virus (HIV), there has been a growing interest for the synthesis of 2′,3′-didehydro-2′,3′-dideoxynucleosides with electron withdrawing groups on the sugar moiety. Here we described an efficient method for the synthesis of such nucleoside analogs bearing structural features of both AZT and d4T. The key intermediate, 3-azido-1,2-bis-O-acetyl-5-O-benzoyl-3-deoxy-D-ribofuranose, 5 was synthesized from commercially available D-xylose in five steps, from which a series of pyrimidine and purine nucleosides were synthesized in high yields. The resultant protected nucleosides were converted to target nucleosides using appropriate chemical modifications. The final nucleosides were evaluated as potential anti-HIV agents.     

Molecular Conformation of 2′-Deoxy-2′-methylidene-cytidine: A Potent Antineoplastic Nucleoside

Abstract

2′-Deoxy-2′-methylidenecytidine (DMDC), a potent inhibitor of the growth of tumor cells, was crystallized with two different forms. One is dihydrated (DMDC·2H₂O) and the other is its hydrochloride salt (DMDC·HCLl). Both crystal and molecular structures have been determined by the X-ray diffraction method. In both forms the glycosidic and sugar conformations are anti and C(4′)-exo, respectively, whereas the conformation about the exocyclic bond is trans for DMDC·2H₂O and gauche ⁺ for DMDC·HCl. Proton nuclear magnetic resonance data of DMDC indicate a preference for the anti C(4′)-exo conformation found in the solid state. These molecular conformations were compared with the related pyrimidine nucleosides. When the cytosine bases are brought into coincidence, DMDC displays the exocyclic C(4′)-C(5′) bond located on the very close position to those of pyrimidine nucleosides with typical overall conformations. On the other hand, the hydroxyl O(3′)-H groups are separated by ca. 3 Å in the cases of DMDC and other pyrimidine nucleosides which have the C(2′)-endo sugar conformation. This result may be useful for the implication about the mechanism of the biological activity of DMDC.     

Synthesis of Branched-Chain and Bicyclic Thiosugar Nucleosides

Abstract

The synthesis of strategically protected nucleosides bearing β-mercaptoethyl chains at the α-C-3′ position from 1,2-di-O-acetyl-2′-S-acetyl-5-t?butyldiphenylsilyl-3-deoxy-3-C-(2′-mercaptoethyl)-α-D-ribofuranose 1 is described. It was found that treatment of the 5-O-methanesulfonyl sugar 19 or nucleoside 5 with either benzylmercaptan or methoxide resulted in rapid cleavage of the thiolester followed by intramolecular cyclization. This was used to prepare the novel trans?fused oxathiahydrindane nucleosides 7 and 27 as well as the cAMP analogue 29.     

The Synthesis of Novel Transition State Analogues Related to Imidazole and Purine Nucleotides Involved in the De novo Biosynthesis of Purines

Abstract

5-Amino-4-sulphonamidoimidazole ribofuranosyl nucleosides and a related imidazothiadiazine dioxide nucleoside have been synthesized as potential inhibitors of enzymes involved in the de novo biosynthesis of purine nucleotides.     

Synthesis and Biological Activity of 5-Fluoro-2-thiocytosine Nucleosides

Abstract

Two pathways are described for the synthesis of the 2′-deoxynucleosides of 2-thiocytosine and 5-fluoro-2-thiocytosine: (a) by nucleoside condensation, (b) by amination of the corresponding nucleosides of 2,4-dithiouracil. Biological activities vs two cell systems are described. The nucleosides are moderate to weak substrates of deoxycytidine kinase and, partly as a result of this, reasonable good inhibitors of the enzyme     

THE SYNTHESIS OF ANTI-FIXED 3-METHYL-3- DEAZA-2′-DEOXYADENOSINE AND OTHER 3H-IMIDAZO[4,5-c]PYRIDINE ANALOGS

ABSTRACT

Rotation of a heterocyclic base around a glycosidic bond allows the formation of syn and anti conformations in nucleosides. The syn conformation has been observed primarily in purine-purine mismatches in DNA duplexes. Such mismatches give rise to false positive oligonucleotide hybridization in DNA-based diagnostics. Here we describe the synthesis of an analog of 2′-deoxyadenosine that retains its Watson-Crick functional groups, but cannot form the syn conformation. In this analog, the N3 atom of 2′-deoxyadenosine is replaced by a C-CH₃ group to give 7-methyl-1-β-D-deoxyribofuranosyl-1H-imidazo[4,5-c]pyridin-4-ylamine or 3-methyl-3-deaza-2′-deoxyadenosine (3mddA). This modification sterically prevents the syn conformation and 3mddA becomes an anti-fixed nucleoside analog of 2′-deoxyadenosine. The synthesis and conformational analysis of 3mddA and several analogs with an 3H-imidazo[4,5-c]pyridine skeleton are described, as well as their potential applications.     

Conformational Studies of 3′-Spironucleosides (TSAO-T and Analogues) by NMR Spectroscopy

Abstract

The conformational properties in solution of the prototype compound TSAO-T (1) and its two analogues 2 and 3 have been determined by H and C NMR techniques. The three compounds showed a sugar ring conformation rare among HIV-inhibitory nucleosides, probably due to the presence, at the 3′-position of the spiro moiety.     

Enzymatic Degradation of GlycosaminogIycans

Abstract

Glycosarninoglycans (GAGs) play an intricate role in the extracellular matrix (ECM), not only as soluble components and polyelectrolytes, but also by specific interactions with growth factors and other transient components of the ECM. Modifications of GAG chains, such as isomerization, sulfation, and acetylation, generate the chemical specificity of GAGs. GAGS can be depolymerized enzymatically either by eliminative cleavage with lyases (EC 4.2.2.-) or by hydrolytic cleavage with hydrolases (EC 3.2.1.-). Often, these enzymes are specific for residues in the polysaccharide chain with certain modifications. As such, the enzymes can serve as tools for studying the physiological effect of residue modifications and as models at the molecular level of protein-GAG recognition. This review examines the structure of the substrates, the properties of enzymatic degradation, and the enzyme substrate-interactions at a molecular level. The primary structure of several GAGS is organized macro-scopicallyby segregation into alternating blocks of specific sulfation patterns and microscopicallyby formation of oligosaccharide sequences with specific binding functions. Among GAGs, considerable dermatan sulfate, heparin and heparan sulfate show conformational flexibility in solution. They elicit sequence-specific interactions with enzymes that degrade them, as well as with other proteins, however, the effect of conformational flexibility on protein-GAG interactions is not clear. Recent findings have established empirical rules of substrate specificity and elucidated molecular mechanisms of enzyme-substrate interactions for enzymes that degrade GAGs. Here we propose that local formation of polysaccharide secondary structure is determined by the immediate sequence environment within the GAG polymer, and that this secondary structure, in turn, governs the binding and catalytic interactions between proteins and GAGs.     

Conformational Analysis of Nucleosides Constructed on a Bicyclo[3.1.0]hexane Template. Structure-Antiviral Activity Analysis for the Northern and Southern Hemispheres of the Pseudorotational Cycle

Abstract

A conformational analysis of carbocyclic nucleosides built on a rigid bicyclo[3.1.0]hexane template (1–4, Northern and 5–8 Southern) showed that the Northern conformation prefers an anti glycosyl torsion angle whereas the Southern conformation favors the syn range. Antiviral activity was mostly associated with the Northern conformers.     

SYNTHESIS AND IN VITRO ACTIVITY OF D- AND L-ENANTIOMERS OF 5-(TRIFLUOROMETHYL)URACIL NUCLEOSIDE DERIVATIVES

Recently, β-L-nucleoside analogues have emerged as a new class of sugar modified nucleosides with potential antiviral and/or antitumoral activity. As a part of our ongoing research on this topic, we decided to synthesize 5-CF₃-β-L-dUrd (7), the hitherto unknown L-enantiomer of Trifluridine, an antiherpetic drug approved by FDA but only used in topical applications due to concomitant cytotoxicity. 5-CF₃-β-L-dUrd (7) as well as some other related L-nucleoside derivatives were stereospecifically prepared and tested in vitro against viral (HSV-1 and HSV-2) and human thymidine kinases (TK).     

The Synthesis of Deuterionucleosides

Abstract

The synthesis of deuterionucleosides for site-specific incorporation into oligo-DNA or -RNA is herein reviewed for NMR or biological studies. The review covers the following aspects: (i) deuteration of the aglycone; (ii) single-site chemical deuteration of the sugar residues; (iii) multiple-site chemical deuteration of the sugar residues; (iv) enzymatic synthesis of deuterated nucleosides or nucleotides; and (v) synthesis of labelled nucleosides with multiple isotopes.