High-Resolution capillary electrophoresis separation of large RNA under non-aqueous conditions.

Large RNAs including messenger RNAs (mRNAs) are promising candidates for development of new drug products and vaccines. Development of high resolution methods for direct analysis of large RNAs, especially for purity in general and size or length in particular, is critical to support new drug development and manufacture. However, resolution based on size or length for large RNAs is limited even by capillary electrophoresis (CE), which is one of the most efficient separation methods for nucleic acids in general. This paper presents a capillary gel electrophoresis (CGE) method for separating large RNA molecules by size or length under strongly denaturing, non-aqueous conditions. We believe that our work constitutes the first time that a gel suitable for CGE prepared with high molecular weight polymers and using only formamide as solvent has been successfully employed to analyze large RNAs on the basis of their size or length with high resolution. With an eye toward application for mRNAs in particular, separation conditions in this work were optimized for RNAs approximately 2000 nucleotides (nt) in length. As compared to a standard CGE method using an aqueous gel, resolution for commercially-available RNA ladder components at 1500 and 2000 nt is increased approximately 6-fold. The impacts of polymer type, molecular weight of the polymer, and polymer concentration on the separation were studied and optimized. Analysis of the results presented here also provides guidance for optimization of separation conditions for RNAs with different sizes as needed for particular applications in the future.

Exploration of long-acting implant formulations of hepatitis B drug entecavir.

Alternative formulations of entecavir, a once daily oral hepatitis B antiretroviral, may improve treatment adherence by patients. We explored the use of biocompatible polymers to control entecavir dissolution in two formats suitable for subcutaneous implantation. Hot melt extrudates were prepared by extruding entecavir-polymer blends at specified weight ratios. Dip-coated tablets were prepared by compressing entecavir in a multi-tip tooling. Tablets were dip-coated in solutions of polymer and dried. In rodents, entecavir-poly(caprolactone) extrudates demonstrated >180 days of continuous drug release, although below the estimated efficacious target input rate. Drug pharmacokinetic profiles were tunable by varying the polymer employed and implant format. The rank order trends of drug input rates observed in vitro were observed in vivo in the detected plasma concentrations of entecavir. In all dose groups entecavir was not tolerated locally at the site of administration where adverse event severity correlated with drug input rate. These polymer-based implantable formats have applicability to long-acting formulations of high solubility compounds beyond entecavir.

Rapid HATU-mediated solution phase siRNA conjugation.

Conditions for facile solution-phase amide conjugation of amine-modified siRNA with a diverse set of carboxylic acid partners using the coupling reagent HATU are described. These conditions eliminate the need for isolated activated esters and allow for rapid access to conjugates with a wide range of lipophilicity and functionality in good yield.

Metal-ligand charge-transfer-promoted photoelectronic Bergman cyclization of copper metalloenediynes: photochemical DNA cleavage via C-4' H-atom abstraction.

Metal-to-ligand charge-transfer (MLCT) photolyses (lambda > or = 395 nm) of copper complexes of cis-1,8-bis(pyridin-3-oxy)oct-4-ene-2,6-diyne (bpod, 1), [Cu(bpod)(2)]PF(6) (2), and [Cu(bpod)(2)](NO(3))(2) (3) yield Bergman cyclization of the bound ligands. In contrast, the uncomplexed ligand 1 and Zn(bpod)(2)(CH(3)COO)(2) compound (4) are photochemically inert under the same conditions. In the case of 4, sensitized photochemical generation of the lowest energy (3)pi-pi state, which is localized on the enediyne unit, leads to production of the trans-bpod ligand bound to the Zn(II) cation by photoisomerization. Electrochemical studies show that 1, both the uncomplexed and complexed, exhibits two irreversible waves between E(p) values of -1.75 and -1.93 V (vs SCE), corresponding to reductions of the alkyne units. Irreversible, ligand-based one-electron oxidation waves are also observed at +1.94 and +2.15 V (vs SCE) for 1 and 3. Copper-centered oxidation of 2 and reduction of 3 occur at E(1/2) = +0.15 and +0.38 V, respectively. Combined with the observed Cu(I)-to-pyridine(pi) MLCT and pyridine(pi)-to-Cu(II) ligand-to-metal charge transfer (LMCT) absorption centered near approximately 315 nm, the results suggest a mechanism for photo-Bergman cyclization that is derived from energy transfer to the enediyne unit upon charge-transfer excitation. The intermediates produced upon photolysis degrade both pUC19 bacterial plasmid DNA, as well as a 25-base-pair, double-stranded oligonucleotide. Detailed analyses of the cleavage reactions reveal 5'-phosphate and 3'-phosphoglycolate termini that are derived from H-atom abstraction from the 4'-position of the deoxyribose ring rather than redox-induced base oxidation.

Electroreductive intramolecular cyclization of a bromo propargyloxy ester catalyzed by nickel(i) tetramethylcyclam electrogenerated at carbon cathodes in dimethylformamide.

Cyclic voltammetry and controlled-potential electrolysis have been employed to investigate and characterize the reductive intramolecular cyclization of ethyl 2-bromo-3-(3',4'-dimethoxyphenyl)-3-(propargyloxy)propanoate (1) promoted by (1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane)nickel(I), [Ni(tmc)](+), electrogenerated at glassy carbon cathodes in dimethylformamide containing tetraalkylammonium salts. Cyclic voltammograms for reduction of [Ni(tmc)](2+) in the presence of 1 reveal that [Ni(tmc)](+) catalytically reduces 1 at potentials more positive than those required for direct reduction of 1. During controlled-potential electrolyses of solutions containing [Ni(tmc)](2+) and 1, catalytic reduction of the latter proceeds via one-electron cleavage of the carbon-bromine bond to form a radical intermediate that undergoes cyclization to afford 2-(3',4'-dimethoxyphenyl)-3-(ethoxycarbonyl)-4-methylenetetrahydrofuran (2). In the presence of a base (either electrogenerated or deliberately added as potassium tert-butoxide), 2 rearranges to give 2-(3',4'-dimethoxyphenyl)-3-(ethoxycarbonyl)-4-methyl-2,5-dihydrofuran (3). A mechanistic scheme is proposed to explain the results obtained by means of cyclic voltammetry and controlled-potential electrolysis.