Synthesis of 2,3-dideoxy-2-fluoro-2,3-endo-methylene- and 2,3-Dideoxy-2-fluoro-3-C-hydroxymethyl-2,3-endo-methylene-pentofuranoses and their use in the preparation of conformationally locked bicyclic nucleosides.

Construction of protected 2,3-dideoxy-2-fluoro-2,3-endo-methylene-pentofuranoses from d-glyceraldehyde and 2,3-dideoxy-2-fluoro-3-C-hydroxymethyl-2,3-endo-methylene-pentofuranoses from d-isoascorbic acid, via Simmons-Smith-type stereoselective cyclopropanations on the respective fluoroallyl alcohols, is described. Synthesis of the corresponding conformationally locked sugar modified uridine and guanosine nucleosides was achieved via Vorbrüggen or Mitsunobu methodologies. Stereochemical confirmation of the novel nucleosides was performed on the basis of 2D NOESY NMR experiments. Analysis of 2',3'-dideoxy-2'-fluoro-3'-C-hydroxymethyl-2',3'-endo-methylene-uridine by X-ray crystallography yielded the principal conformational parameters and indicated that the furanoid ring adopted an (o)E/(o)T1, East pucker. The uridine and guanosine nucleosides were found to be inactive in the hepatitis C virus (HCV) cell-based replicon assay, which was corroborated on examination of the corresponding nucleoside triphosphates against the HCV NS5B polymerase.

Stereoselective cyclopropanation in the synthesis of 3'-deoxy-3'-C-hydroxymethyl-2',3'-methylene-uridine.

The synthesis of the novel 2',3'-cyclopropane nucleoside 3'-deoxy-3'-C-hydroxymethyl-2',3'-methylene-uridine is described. Stereoselective construction of the cyclopropane ring was achieved via Simmons-Smith cyclopropanation of a benzyl protected silyl enol ether, which was itself derived from 1,2-O-isopropylidene-α-D-xylofuranose.

A new modular approach to nanoassembly: stable and addressable DNA nanoconstructs via orthogonal click chemistries.

Thermodynamic instability is a problem when assembling and purifying complex DNA nanostructures formed by hybridization alone. To address this issue, we have used photochemical fixation and orthogonal copper-free, ring-strain-promoted, click chemistry for the synthesis of dimeric, trimeric, and oligomeric modular DNA scaffolds from cyclic, double-stranded, 80-mer DNA nanoconstructs. This particular combination of orthogonal click reactions was more effective for nanoassembly than others explored. The complex nanostructures are stable to heat and denaturation agents and can therefore be purified and characterized. They are addressable in a sequence-specific manner by triplex formation, and they can be reversibly and selectively deconstructed. Nanostructures utilizing this orthogonal, chemical fixation methodology can be used as building blocks for nanomachines and functional DNA nanoarchitectures.

Fast and efficient DNA crosslinking and multiple orthogonal labelling by copper-free click chemistry.

Two new dibenzocyclooctyne-thymidine monomers were incorporated into oligonucleotides and crosslinked to azide-labelled complementary strands across the DNA grooves. Equivalent reactions were successful using a (bicyclo[6.1.0]nonyne) alkyne. Oligonucleotides containing internal cyclooctyne and amino groups were simultaneously reacted with azides and NHS esters of different fluorescent dyes to produce functional genetic probes.

2'-Aminoethoxy-2-amino-3-methylpyridine in triplex-forming oligonucleotides: high affinity, selectivity and resistance to enzymatic degradation.

The phosphoramidite monomer of the C-nucleoside 2'-aminoethoxy-2-amino-3-methylpyridine (AE-MAP) has been synthesized for the first time and incorporated into triplex-forming oligonucleotides (TFOs). Ultraviolet melting and DNase I footprinting studies show that AE-MAP is a potent triplex-stabilizing monomer that is selective for GC base pairs. TFOs containing AE-MAP bind with high affinity to duplexes but only weakly to single stranded DNA. In addition, AE-MAP confers high nuclease resistance on oligonucleotides. TFOs containing AE-MAP have potential for gene knock-out and gene expression studies.

Fast copper-free click DNA ligation by the ring-strain promoted alkyne-azide cycloaddition reaction.

Templated DNA strand ligation by the ring-strain promoted alkyne-azide [3+2] cycloaddition reaction is very fast; with dibenzocyclooctyne, the reaction is essentially complete in 1 min. It is inhibited by the presence of a single mismatched base pair suggesting applications in genetic analysis.