Nucleotide analogues containing 2-oxa-bicyclo[2.2.1]heptane and l-α-threofuranosyl ring systems: interactions with P2Y receptors

The ribose moiety of adenine nucleotide 3′,5′-bisphosphate antagonists of the P2Y1 receptor has been successfully substituted with a rigid methanocarba ring system, leading to the conclusion that the North (N) ring conformation is preferred in receptor binding. Similarly, at P2Y2 and P2Y4 receptors, nucleotides constrained in the (N) conformation interact equipotently with the corresponding ribosides. We now have synthesized and examined as P2Y receptor ligands nucleotide analogues substituted with two novel ring systems: (1) a (N) locked-carbocyclic (cLNA) derivative containing the oxabicyclo[2.2.1]heptane ring system and (2) L-α-threofuranosyl derivatives. We have also compared potencies and preferred conformations of these nucleotides with the known anhydrohexitol-containing P2Y1 receptor antagonist MRS2283. A cLNA bisphosphate derivative MRS2584 21 displayed a Ki value of 22.5nM in binding to the human P2Y1 receptor, and antagonized the stimulation of PLC by the potent P2Y1 receptor agonist 2-methylthio-ADP (30nM) with an IC50 of 650nM. The parent cLNA nucleoside bound only weakly to an adenosine receptor (A3). Thus, this ring system afforded some P2Y receptor selectivity. A L-α-threofuranosyl bisphosphate derivative 9 displayed an IC50 of 15.3μM for inhibition of 2-methylthio-ADP-stimulated PLC activity. L-α-Threofuranosyl-UTP 13 was a P2Y receptor agonist with a preference for P2Y2 (EC50 = 9.9μM) versus P2Y4 receptors. The P2Y1 receptor binding modes, including rotational angles, were estimated using molecular modeling and receptor docking

Recognition of threosyl nucleotides by DNA and RNA polymerases

Alpha-L-threose nucleic acids (TNA) are potentially natural nucleic acids that could have acted as an evolutionary alternative to RNA. We determined whether DNA or RNA polymerases could recognize phosphorylated threosyl nucleosides. We found that for both the Vent (exo-) DNA polymerase and HIV reverse transcriptase K(m) values were increased and kcat values decreased for the incorporation of tTTP in comparison to their natural counterparts. Our results suggest that TNA may have played a role in the evolution of the DNA-RNA-protein world. Thus, TNA may be a candidate for further studies in evolutionary chemistry and biology.status: publishe

Recognition of HNA and 1,5-anhydrohexitol nucleotides by DNA metabolizing enzymes

Hexitol nucleic acids (HNA) as well as their 1,5-anhydrohexitol triphosphate building blocks were evaluated for their ability to be recognized by several DNA metabolizing enzymes. It was found that RNA polymerases can recognize the triphosphate of the adenine analogue. However, only the incorporation of a maximum of three consecutive building block analogues was possible under the applied experimental conditions. Terminal transferase was more successful succeeding in the elongation of a DNA primer with a maximum of 15 1.5-anhydrohexitol purine nucleotides. Furthermore, it was observed that the 1,5-anhydroaltritol triphosphate analogue of adenosine was a poor substrate for terminal transferase and that HNA could not act as a primer for this enzyme. Likewise, HNA did not function as a template for restriction enzymes, ligases or methylases. (C) 2002 Elsevier Science B.V. All rights reserved.status: publishe

Reverse transcriptase incorporation of 1,5-anhydrohexitol nucleotides

Several reverse transcriptases were studied for their ability to accept anhydrohexitol triphosphates, having a conformationally restricted six-membered ring, as substrate for template-directed synthesis of HNA. It was found that AMV, M-MLV, M-MLV (H(-)), RAV2 and HIV-1 reverse transcriptases were able to recognise the anhydrohexitol triphosphate as substrate and to efficiently catalyse the incorporation of one non-natural anhydrohexitol nucleotide opposite a natural complementary nucleotide. However, only the dimeric enzymes, the RAV2 and HIV-1 reverse transcriptases, seemed to be able to further extend the primer with another anhydrohexitol building block. Subsequently, several HIV-1 mutants (4xAZT, 4xAZT/L100I, L74V, M184V and K65A) were likewise analysed, resulting in selection of K65A and, in particular, M184V as the most succesful mutant HIV-1 reverse transcriptases capable of elongating a DNA primer with several 1,5-anhydrohexitol adenines in an efficient way. Results of kinetic experiments in the presence of this enzyme revealed that incorporation of one anhydrohexitol nucleotide of adenine or thymine gave an increased (for 1,5-anhydrohexitol-ATP) and a slightly decreased (for 1,5-anhydrohexitol-TTP) K(m) value in comparison to that of their natural counterparts. However, no more than four analogues could be inserted under the experimental conditions required for selective incorporation. Investigation of incorporation of the altritol anhydrohexitol nucleotide of adenine in the presence of M184V and Vent (exo(-)) DNA polymerase proved that an adjacent hydroxyl group on C3 of 1,5-anhydrohexitol-ATP has a detrimental effect on the substrate activity of the six-ring analogue. These results could be rationalised based on the X-ray structure of HIV-1 reverse transcriptase.status: publishe

Enzyme catalyzed synthesis of HNA

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Recognition of 1,5-anhydrohexitol adenine triphosphate by a DNA polymerase

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Enzymatic incorporation in DNA of 1,5-anhydrohexitol nucleotides

The ability of several DNA polymerases to catalyze the template-directed synthesis of duplex oligonucleotides containing a base pair between a nucleotide with anhydrohexitol ring and its natural complement has been investigated. All DNA polymerases were able to accept the chemically synthesized anhydrohexitol triphosphate as substrate and to catalyze the incorporation of one anhydrohexitol nucleotide. However, only family B DNA polymerases succeeded in elongating the primer after the incorporation of an anhydrohexitol nucleotide. In this family, Vent (exo(-)) DNA polymerase is the most successful one and was therefore selected for further investigation. Results revealed that at high enzyme concentrations six hATPs could be incorporated; however, a selective incorporation proved only feasible under experimental conditions where no more than two analogues could be inserted. Also the synthesis of a mixed HNA-DNA sequence was examined. Kinetic parameters for incorporation of one anhydrohexitol adenine nucleoside were similar to those of its natural analogue.status: publishe

Cleavage of DNA without loss of genetic information by incorporation of a disaccharide nucleoside

A ribose residue inserted between the 3′-OH of one nucleotide and the 5′-phosphate group of the next nucleotide, functions as a site-specific cleavage site within DNA. This extra ribose does not interrupt helix formation and it protects duplex DNA against cleavage by restriction enzymes. Cleavage can be obtained with periodate and all ribose fragments can be removed with sodium hydroxide. As a result of this, an intact natural oligodeoxynucleotide is obtained after ligation reaction, which means that site-specific cleavage and recovering of intact DNA occurs without loss of genetic information