Engineering of a single conserved amino acid residue of herpes simplex virus type 1 thymidine kinase allows a predominant shift from pyrimidine to purine nucleoside phosphorylation.
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Studies of herpes simplex virus type 1 (HSV-1) thymidine (dThd) kinase (TK) crystal structures show that purine and pyrimidine bases occupy distinct positions in the active site but approximately the same geometric plane. The presence of a bulky side chain, such as tyrosine at position 167, would not be sterically favorable for pyrimidine or pyrimidine nucleoside analogue binding, whereas purine nucleoside analogues would be less affected because they are located further away from the phenylalanine side chain. Site-directed mutagenesis of the conserved Ala-167 and Ala-168 residues in HSV-1 TK resulted in a wide variety of differential affinities and catalytic activities in the presence of the natural substrate dThd and the purine nucleoside analogue drug ganciclovir (GCV), depending on the nature of the amino acid mutation. A168H- and A167F-mutated HSV-1 TK enzymes turned out to have a virtually complete knock-out of dThd kinase activity (at least approximately 4-5 orders of magnitude lower) presumably due to a steric clash between the mutated amino acid and the dThd ring. In contrast, a full preservation of the GCV (and other purine nucleoside analogues) kinase activity was achieved for A168H TK. The enzyme mutants also markedly lost their binding capacity for dThd and showed a substantially diminished feedback inhibition by thymidine 5'-triphosphate. The side chain size at position 168 seems to play a less important role regarding GCV or dThd selectivity than at position 167. Instead, the nitrogen-containing side chains from A168H and A168K seem necessary for efficient ligand discrimination. This explains why A168H-mutated HSV-1 TK fully preserves its GCV kinase activity (Vmax/Km 4-fold higher than wild-type HSV-1 TK), although still showing a severely compromised dThd kinase activity (Vmax/Km 3-4 orders of magnitude lower than wild-type HSV-1 TK). (+info)
Carbocyclic pyrimidine nucleosides as inhibitors of S-adenosylhomocysteine hydrolase.
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The design, synthesis, and unexpected inhibitory activity against S-adenosyl-homocysteine (SAH) hydrolase (SAHase, EC 3.3.1.1) for a series of truncated carbocyclic pyrimidine nucleoside analogues is presented. Of the four nucleosides obtained, 10 was found to be active with a Ki value of 5.0 microM against SAHase. (+info)
Regulation by thymidine monophosphate and other nucleotides of thymidine kinase activity in extracts from primary rabbit kidney cells infected by HSV types 1 and 2.
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In an attempt to differentiate between thymidine kinase (EC.2.4.1.21) induced by herpesvirus hominis type I (TK I) and type 2 (TK2), the different susceptibilities to the modifying effects of some thymidine analogues proved to be useful criteria: (I)2'-deoxythymidine-5'-triphosphate (dThd-5'-PPP) inhibits TK 2 at a concentration of 0-125 mM by 90%, whereas TK I is inhibited at 4-03 mM by 50%. (2) 2'-deoxythymidine-5'-monophosphate (dThd-5'-P) competitively inhibits TK 2 at all concentrations tested. On the other hand, the direction of its effect on TK I is concentration dependent: at 500 mum it stimulates and at 8 mM inhibits TK I activity. During enzyme kinetic studies, TK I displays substrate inhibition which is reversed by dThd-5'-P. This result explains the stimulating effect of dThd-5'-P at 500 muM. This phenomenon suggests the existence on the enzyme molecule of a second binding site for dThd which mediates substrate inhibition and which can be occupied also by dThd-5'-P. After polyacrylamide gel electrophoresis of TK I, the stimulation by dThd-5'-P disappears, suggesting the separation of the second binding site from the catalytic centre. (+info)
Examination of protecting groups in the synthesis of nucleosides by intramolecular glycosylation.
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Influence of carbohydrate protecting groups on intramolecular glycosylation was examined, and it was found that the use of 4-methoxybenzyl group as the protection for the 4-O position as well as t-butyldimethylsilyl group at the 3-O position was necessary to obtain desired beta-nucleosides in good yields. (+info)
Effects of 5-substituted pyrimidine nucleoside bases of WNA on stability of triplex DNA.
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Triplex-forming oligonucleotides (TFOs) are potential DNA-targeting molecules, but their recognizable duplexes are limited to homopurine:homopyrimidine sequences by interruption of pyrimidine bases in the purine strand. Despite numerous studies, this problem has not been generally solved. We have recently demonstrated that the new nucleoside analogues, WNA-betaT and WNA-betaC exhibit selective stabilization of the triplexes at a TA and a CG interrupting site, respectively. However, subsequent investigations have shown that there are limitations in recognizable sequences by these analogs. In this study, we performed further systematic investigation on WNA analogs by synthesizing a variety of WNA analogs having 5-substituted cytosine and uracil, and found that WNA-betaFU exhibit high CG-selectivity. (+info)
DNA recognition and recognition control of alpha-peptide ribonucleic acids by external factors.
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Peptide ribonucleic acid (PRNA) is one of artificial nucleic acids, which enables us to control its nucleobase orientation and recognition behavior by external factors through the synergetic effects of reversible borate ester formation and intramolecular hydrogen bond formation. In this study, a series of novel alpha-PRNA oligomers, possessing alternative alpha-PRNA/basic amino acid sequences, were newly designed, synthesized, and evaluated as the second-generation PRNA. As expected, these alpha-PRNAs indeed formed highly stable sequence-specific complexes with the complementary DNAs, for which both the conventional hydrogen-bonding interactions between the complementary nucleobase pairs and the electrostatic interactions between the basic amino acid's ammonium cation and the DNA's phosphate anion on the backbone are jointly responsible. (+info)
Design, synthesis, and anticancer activity of fluorocyclopentenyl-pyrimidines.
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Novel pyrimidine nucleosides with fluorocyclopentene ring were synthesized from D-ribose via stereoselective Grignard reaction and electrophilic vinyl fluorination as key steps. Among compounds tested, cytosine derivative 15a was found to show high growth inhibition against a broad range of human tumor cell lines. (+info)
Synthesis of 2'- or 3'-O-heteroaryl substituted nucleic acid derivatives and their biological properties.
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The successful preparation of 3'-O-heteroaryl substituted thymidine derivatives were developed by the reaction of 5'-O-(tert-butyldimethylsilyl)thymidine with heteroaromatic halides in the presence of sodium hydride. In a similar manner, 2' or 3'-O-heteroaryl substituted uridine derivatives were obtained by the use of 5'-O-(tert-butyldimethylsilyl)uridine. All the arylations proceeded selectively at the site of the hydroxyl group and reactions of heteroaromatic halides with the nucleobase residues were not observed. Desilylation of the nucleic acid derivatives synthesized afforded 2'- or 3'-O-heteroaryl substituted thymidine or uridine derivatives in good yields. The anti-BVDV assay of the synthesized 2'- or 3'-O-heteroarylsubstituted thymidine and uridine derivatives were studied. (+info)