The investigation of the bicyclo[3.1.0]hexane template as a platform for the construction of novel conformationally locked carbocyclic nucleosides continues to yield many interesting compounds. We use these modified nucleosides with the intent of: (1) determining the conformational preferences of enzymes involved in the biochemistry of nucleosides, nucleotides, and oligonucleotides; and (2) developing selected compounds as possible antitumor/antiviral drugs based on our understanding of their biochemical mechanism of action. These efforts have resulted in the discovery of N-methanocarbathymidine (N-MCT), a compound active against herpes viruses 1 and 2, as well as human herpesvirus 8 associated with Kaposi sarcoma. This year we contracted a semi-prep synthesis of N-MCT (65 g) to use for future toxicological and pharmacological studies in vivo. Last years discovery of D-carba-T (carbocyclic thymidine) as a successfully kinased nucleoside that functions as a kinetic delayed chain terminator and is effective against HIV-infected cells led to a full investigation of its mechanism of action. D-carba-TTP is efficiently incorporated by HIV-RT; however, the next deoxynucleotide triphosphate is added slowly to D-carba-TMP at the primer terminus. With a DNA template, some pausing was detectable, suggesting that D-carba-dTPP is incorporated to some extent. However, with the DNA template, D-carba-dTTP did not appear to compete effectively with the normal dTTP, since most of the products are full length. With the RNA template, there was more pausing than with the DNA template, suggesting that D-carba-dTPP is better able to compete with TTP with an RNA template. Since D-carba-T is able to inhibit viral replication in cell culture, these data suggest that most of the inhibitory activity occurs during first strand synthesis. In addition, D-carba-T was able to block the replication of multi-drug resistant HIV-1 vectors in cultured cells (J. Med. Chem. to be submitted). Conformationally locked North and South versions of puromycin analogues built on a bicyclo[3.1.0]hexane pseudosugar template were synthesized. The final assembly of the products was accomplished by the Staudinger-Vilarrasa coupling of the corresponding North and South 3-azidopurine carbanucleosides with the Fmoc-protected 1-hydroxybenzotriazole ester of 4-methoxy-L-tyrosine (manuscript submitted to J. Org. Chem.). The synthesis of oligonucleotides carrying novel nucleosides that are locked toward one specific ring puckering is a field of increasing interest. Oligonucleotides carrying adenine and guanine bicyclo[3.1.0]hexane pseudosugars have been prepared. The substitution of one adenine or guanine with either North- or South-bicyclo[3.1.0]hexane pseudosugar derivatives have little effect on the global stability of a duplex DNA structure. Three derivatives of the thrombin binding aptamer carrying one modified bicyclo[3.1.0]hexane guanine residue were studied. The replacement of one guanine residue of the thrombin binding aptamer with a bicyclic guanine pseudosugar does not change the antiparallel quadruplex structure of the aptamer but it has a strong influence in the stability of the quadruplex. The substitution of a syn-guanine with equivalent syn-guanine pseudosugar locked in the South-conformation maintains the stability. Changing a guanine that is in the anti orientation for a guanine pseudosugar locked in a syn conformation induces a strong destabilization of the quadruplex (manuscript in preparation). For more than a decade our laboratory has been studying the properties of locked carbocyclic nucleosides built on a bicyclo[3.1.0]hexane template (Marquez, V. E. The properties of Locked Methanocarba Nucleosides in Biochemistry, Biotechnology and Medicine, Chapter 12. In: Modified Nucleosides in Biochemistry, Biotechnology and Medicine, P. Herdewijn Ed. Wiley-VCH, 2008, pp 307-341). One of the drawbacks of using such a template is the loss of the O4 oxygen and its role in the anomeric effect. To reintroduce an oxygen in an equivalent position adjacent to the glycosyl C-N bond, we have synthesized two oxobicyclo[3.1.0]hexane nucleosides in an attempt to reinstate the anomeric effect. Even more importantly, these two structures freeze the orientation of the oxygens lone pair orbitals in relation to the C-N antibonding orbital to either augment (antiperiplanar) or diminish (gauche) the strength of the anomeric effect. This allowed us to study the consequences of delocalizing the oxygens lone pair into the antibonding C-N bond orbital, a phenomenon also known as the anomeric effect. Using Gaussian 98 and NBO5.0 the second order perturbation interaction between the oxygen lone pair with the highest p orbital component and the adjacent C-N bond was studied for both molecules. The geometries of the molecules were optimized at the B3LYP/6-31G* level of theory. The strongest lone pair-antibonding orbital interaction was observed for the compound with the antiperiplanar orientation and expectedly the bond length was greater. This orbital interaction increases even more in the protonated species and lengthens the C-N bond by 0.034 . To help correlate these results with experimental data we measured the acid stability of these molecules at pH 2 (37 C). The compound for which the anomeric effect was significantly reduced (gauche) was quite stable and had a half-life of 52.8 min, while for the antiperiplanar disposed compound with a strong anomeric effect, the C-N bond was cleaved with a half-life of 6.46 min (Nucleic Acids Symposium Series No. 52, 2008, 543-544). The first synthesis of the ribo-like South bicyclo[3.1.0]hexane nucleoside in enantiomerically pure form was achieved during this period. This was accomplished through the use of several functional group transformations on a sensitive bicyclo[3.1.0]hexane system. D-Ribose was transformed into a methanocarba alcohol followed by conversion of the OH group to a nitrile with inversion of configuration. The nitrile group was subsequently reduced in two stages to the 5'-hydroxymethyl group. An ester group was then appended to a tertiary carbon (C1), which was transformed to an amino group as a nucleobase precursor (J. Org. Chem. in press).
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