Nickel-Catalyzed Difluoroalkylation of (Hetero)Arylborons with Unactivated 1-Bromo-1,1-difluoroalkanes.

A nickel-catalyzed cross-coupling between (hetero)arylborons and unactivated 1-bromo-1,1-difluoroalkanes has been developed. The use of two ligands (a bidentate bipyridine-based ligand, 4,4'-ditBu-bpy, and a monodentate pyridine-based ligand, DMAP) offers a highly efficient nickel-based catalytic system to prepare difluoroalkylated arenes which have important applications in medicinal chemistry.

Building a nanostructure with reversible motions using photonic energy.

Recently, the specific hybridization of DNA molecules has been used to construct self-assembled devices, such as the mechanical device to mimic cellular protein motors in nature. Here, we present a new light-powered DNA mechanical device based on the photoisomerization of azobenzene moieties and toehold-mediated strand displacement. This autonomous and controllable device is capable of moving toward either end of the track, simply by switching the wavelength of light irradiation, either UV (365 nm) or visible (>450 nm). This light-controlled strategy can easily solve one main technical challenge for stepwise walking devices: the selection of routes in multipath systems. The principle employed in this study, photoisomerization-induced toehold length switching, could be further useful in the design of other mechanical devices, with the ultimate goal of rivaling molecular motors for cargo transport and macroscopic movement.

Photon-regulated DNA-enzymatic nanostructures by molecular assembly.

Future smart nanostructures will have to rely on molecular assembly for unique or advanced desired functions. For example, the evolved ribosome in nature is one example of functional self-assembly of nucleic acids and proteins employed in nature to perform specific tasks. Artificial self-assembled nanodevices have also been developed to mimic key biofunctions, and various nucleic acid- and protein-based functional nanoassemblies have been reported. However, functionally regulating these nanostructures is still a major challenge. Here we report a general approach to fine-tune the catalytic function of DNA-enzymatic nanosized assemblies by taking advantage of the trans-cis isomerization of azobenzene molecules. To the best of our knowledge, this is the first study to precisely modulate the structures and functions of an enzymatic assembly based on light-induced DNA scaffold switching. Via photocontrolled DNA conformational switching, the proximity of multiple enzyme catalytic centers can be adjusted, as well as the catalytic efficiency of cofactor-mediated DNAzymes. We expect that this approach will lead to the advancement of DNA-enzymatic functional nanostructures in future biomedical and analytical applications.

3-Methyl-3-deazaadenine, a stable isostere of N3-methyl-adenine, is efficiently bypassed by replication in vivo and by transcription in vitro.

The goal of the present work was to determine the impact of N3-methyladenine (3-mA), an important lesion generated by many environmental agents and anticancer drugs, on in vivo DNA replication and in vitro RNA transcription. Due to 3-mA chemical instability, the stable isostere 3-methyl-3-deazaadenine (3-m-c(3)A) was site specifically positioned into an oligodeoxynucleotide. The oligomer was, then incorporated into a vector system that is rapidly converted to ssDNA inside yeast cells and requires DNA replication opposite the lesion for plasmid clonal selection. For control purposes, an adenine or a stable apurinic/apyrimidinic (AP)-lesion was placed at the same site. The presence of each lesion in the oligonucleotide was confirmed by MALDI-TOF analysis. Plasmids were then transfected into yeast cells. While the AP-site dramatically reduced plasmid replication in all strains, the 3-m-c(3)A had a slight effect in the rad30 background which significantly increased only in a rev3rad30 background. Considering TLS events opposite 3-m-c(3)A, the lack of Polη was associated with a substantial increase in AT>GC transitions (p=0.0011), while in the absence of Polζ only events derived from an error free bypass were detected. The 3-m-c(3)A also did not affect in vitro transcription, while the AP-site was a strong block to T7 RNA progression when located in the transcribed strand. We conclude that, in these experimental systems, 3-m-c(3)A is efficiently bypassed by replication in vivo and by transcription in vitro.

Effect of n3-methyladenine and an isosteric stable analogue on DNA polymerization.

N3-methyladenine (3-mA) is a cytotoxic lesion formed by the reaction of DNA with many methylating agents, including antineoplastic drugs, environmental agents and endogenously generated compounds. The toxicity of 3-mA has been attributed to its ability to block DNA polymerization. Using Me-lex, a compound that selectively and efficiently reacts with DNA to afford 3-mA, we have observed in yeast a mutational hotspot at the 5'-terminus of an A(4) tract. In order to explore the potential role of sequence-dependent DNA polymerase bypass of 3-mA, we developed an in vitro system to prepare 3-mA modified substrates using Me-lex. We detail the effects of 3-mA, its stable isostere analogue, 3-methyl-3-deazaadenine, 3-deazaadenine and an THF abasic site on DNA polymerization within an A(4) sequence. The methyl group on 3-mA and 3-methyl-3-deazaadenine has a pronounced inhibitory effect on DNA polymerization. There was no sequence selectivity for the bypass of any of the lesions, except for the abasic site, which was most efficiently by-passed when it was on the 5'-terminus of the A(4) tract. The results indicate that the weak mutational pattern induced by Me-lex may result form the depurination of 3-mA to an abasic site that is bypassed in a sequence dependent context.

Thermodynamic characterization of DNA with 3-deazaadenine and 3-methyl-3-deazaadenine substitutions: the effect of placing a hydrophobic group in the minor groove of DNA.

In many high-resolution structures of DNA there are ordered waters associated with the floor of the minor groove and extending outward in several layers. It is thought that this hydration structure, along with cations, reduces the Coulombic repulsion of the interstrand phosphates. In previous studies, the replacement of the 3-N atom of adenine with a C-H to afford 3-deazaadenine was shown to decrease the thermodynamic stability of DNA via a reduction in the enthalpic term. Using spectroscopic and calorimetric methods, we report herein a rigorous examination of the thermodynamics of DNA with 3-deazaadenine modifications, and report for the first time how the presence of a minor groove methyl group, i.e., 3-methyl-3-deazaadeine, affects DNA stability, hydration, and cation binding. The methylation of adenine at the N3-position to yield N3-methyladenine represents an important reaction in the toxicity of many anticancer compounds. This minor groove lesion is unstable and cannot be readily studied in terms of its effect on DNA stability or structure. Our studies show that 3-methyl-3-deazaadenine, an isostere of N3-methyladenine, significantly destabilizes DNA (DeltaDeltaG > 4 kcal x mol(-1)) due to a significant drop in the enthalpy (DeltaH) term, which is associated with a lower hydration of the duplex relative to the unfolded state.

Introduction of cationic charge into DNA near the major groove edge of a guanine x cytosine base pair: characterization of oligodeoxynucleotides substituted with 7-aminomethyl-7-deaza-2'-deoxyguanosine.

The replacement of the 7-N atom on guanine (G) with a C-H to give 7-deazaguanine (c(7)G) alters the electronic properties of the heterocyclic base and eliminates a potential major groove cation binding site, which affects the organization of salts and water in the major groove. This has a destabilizing effect on DNA. We report herein the characterization of DNA oligomers containing 7-(aminomethyl)-7-deazaguanine (1) residues using a variety of spectroscopic and thermodynamic approaches. 1 is an intramolecular model for the major groove binding of cations and basic amino acid residues to G. In contrast to c(7)G, the tethering of a cation in the major groove using 1 affords DNA that is as, or more, stable than the corresponding unmodified DNA. The stabilization is associated with the folding enthalpy and hydration.

A facile synthetic approach to 7-deazaguanine nucleosides via a Boc protection strategy.

An efficient route to the preparation of 5-substituted 2-amino-7-((2R,4R,5R)-tetrahydro-4-hydroxy-5-(hydroxymethyl)furan-2-yl)-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one compounds has been developed by the condensation of omega-substituted aldehydes with 2,6-diaminopyrimidin-4(3H)-one, followed by Boc protection to afford the corresponding N(2),N(2),N(7)-tris-Boc-O(4)-t-Bu-5-substituted 2-amino-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one, which is amenable to direct condensation with 1-chloro-2-deoxy-3,5-di-O-p-toluoyl-alpha-D-erythro-pentofuranose. This route affords an efficient synthesis to 2-amino-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one, 2-amino-5-alkyl-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one, and guanine nucleosides.

Difluoromethylenated polyhydroxylated pyrrolidines: facile synthesis, crystal structure and biological evaluation.

In our ongoing program aimed at the design, synthesis and biological evaluation of novel gem-difluoromethylenated glycosidase inhibitors, the gem-difluoromethylenated polyhydroxylated pyrrolidines as analogues of 2,5-dideoxy-2,5-imino-D-mannitol (DMDP) were designed and prepared. The crystal structure of gem-difluoromethylenated polyhydroxylated pyrrolidine 17 contains an N-H…F intermolecular hydrogen bond. The biological assessment of gem-difluoromethylenated polyhydroxylated pyrrolidines showed that the modification by the gem-difluoromethylene group decreased the inhibitory activities of DMDP.

Synthesis and biological evaluation of glycosidase inhibitors: gem-difluoromethylenated nojirimycin analogues.

In our ongoing program aimed at the design, synthesis, and biological evaluation of novel gem-difluoromethylenated glycosidase inhibitors, gem-4,4-difluoromethylenated iminosugars (5-9) were synthesized. The biological evaluation of these synthetic iminosugars showed that the gem-difluoromethylenyl group generally reduced the inhibition of glycosidases. However, this was not the case at pH 5.0, where the gem-difluoromethylenated iminosugar 6 was a stronger inhibitor than comparable iminosugars 1 and 36, suggesting that the influence of this group is mainly through its effect on the amine. It is proposed that the unprotonated iminosugar is the species preferably bound by beta-glucosidase, due to the lower pK(a) value of iminosugar 6 than of 1 or 36, leaving iminosugars 1 and 36 mostly protonated at pH 5.0, while iminosugar 6 is not. Iminosugar 6 also displayed good and selective inhibition of beta-glucosidase at pH 6.8.

Highly stereocontrolled synthesis of gem-difluoromethylenated azasugars: D- and L-1,4,6-trideoxy-4,4-difluoronojirimycin.

[reaction: see text]. D-1,4,6-trideoxy-4,4-difluoronojirimycin and L-1,4,6-trideoxy-4,4-difluoronojirimycin, a novel series of gem-4,4-difluoromethylenated azasugars, were synthesized from CF3CH2OH in 10 steps. A key step was the highly diastereoselective construction of the piperidine ring via reductive amination.