tert-Butyldithiomethyl (DTM), a novel hydroxyl protecting group, cleavable under reductive conditions, was developed and applied for the protection of 2'-OH during solid-phase RNA synthesis. This function is compatible with all standard protecting groups used in oligonucleotide synthesis, and allows for fast and high-yield synthesis of RNA. Oligonucleotides containing the 2'-O-DTM groups can be easily deprotected under the mildest possible aqueous and homogeneous conditions. The preserved 5'-O-DMTr function can be used for high-throughput cartridge RNA purification.
Using the intramolecular 5-exo-5-hexenyl radical as a key cyclization step, we previously reported an unambiguous synthesis of carba-LNA thymine (cLNA-T), which we subsequently incorporated in antisense oligonucleotides (AON) and investigated their biochemical properties [J. Am. Chem. Soc.2007, 129 (26), 8362-8379]. These cLNA-T incorporated oligos showed specific RNA affinity of +3.5-5 °C/modification for AON:RNA heteroduplexes, which is comparable to what is found for those of LNAs (Locked Nucleic Acids). These modified oligos however showed significantly enhanced nuclease stability (ca. 100 times more) in the blood serum compared to those of the LNA modified counterparts without compromising any RNase H recruitment capability. We herein report the synthesis of 5-methylcytosine-1-yl ((Me)C), 9-adeninyl (A), and 9-guaninyl (G) derivatives of cLNA and their oligonucleotides and report their biochemical properties as potential RNA-directed inhibitors. In a series of isosequential carba-LNA modified AONs, we herein show that all the cLNA modified AONs are found to be RNA-selective, but the magnitude of RNA-selectivity of 7'-R-Me-cLNA-G (cLNA-G) (ΔT(m) = 2.9 °C/modification) and intractable isomeric mixtures of 7'-(S/R)-Me-cLNA-T (cLNA-T, ΔT(m) = 2.2 °C/modification) was found to be better than diastereomeric mixtures of 7'-(S/R)-Me-cLNA-(Me)C with trace of cENA-(Me)C (cLNA-(Me)C, ΔT(m) = 1.8 °C/modification) and 7'-R-Me-cLNA-A (cLNA-A, ΔT(m) = 0.9 °C/modification). cLNA-(Me)C modified AONs however exhibited the best nuclease stability, which is 4-, 7-, and 20-fold better, respectively, than cLNA-T, cLNA-A, and cLNA-G modified counterparts, which in turn was more than 100 times stable than that of the native. When the modification sites are appropriately chosen in the AONs, the cLNA-A, -G, and -(Me)C modified sites in the AON:RNA hybrids can be easily recognized by RNase H, and the RNA strand of the hybrid is degraded in a specific manner, which is important for the design of oligos for therapeutic purposes. The cLNA-(Me)C modified AON/RNA, however, has been found to be degraded 4 times faster than cLNA-A and G modified counterparts. By appropriately choosing the carba-LNA modification sites in AON strands, the digestion of AON:RNA can be either totally repressed or be limited to cleavage at specific sites or at a single site only (similar to that of catalytic RNAzyme or DNAzyme). Considering all physico- and biochemical aspects of cLNA modified oligos, the work suggests that the cLNA modified antisense oligos have the potential of being a promising therapeutic candidate due to their (i) higher nucleobase-specific RNA affinity and RNA selectivity, (ii) greatly improved nuclease stability, and (iii) efficient RNase H recruitment capability, which can induce target RNA cleavage in a very specific manner at multiple or at a single site, in a designed manner.
Base-catalyzed reaction of 2,6-dibenzylidenecyclohexanone and alkylguanidines gave 2-alkylamino-8-benzylidene-4-phenyl-3,4,5,6,7,8-hexahydroquinazolines IVa-c, which were oxidized to 2-alkylamino-8-benzylidene-4-phenyl-5,6,7,8-tetrahydroquinazolines Va-c. The acylamino (VIa-c, VIIa-d, IXa,b) and diacylamino derivatives (VIIIa,b) of the 2-amino-8-benzylidene-4-pheny]-3,4,5,6,7,8-hexahydroquinazoline (II), 2-amino-8-benzylidene-4-phenyl-5,6,7,8-tetrahydroquinazoline (III) and compounds Va-c have also been prepared. These compounds having the E-configuration were converted into the Z isomers XIIa-e by photoisomerization. The structures were confirmed by spectroscopic methods (IR, 1H NMR, 13C NMR).
Completely homogeneous polyacrylamide-based gels were used for capillary electrochromatography (CEC) of drug enantiomers. Like continuous beds (also called continuous polymer rods, silica rods, monoliths) they do not require frits to support the bed because it is covalently linked to the capillary wall. A long lifetime is an important feature of the beds. The gel matrices can be prepared in any laboratory and for specific interactions they can be derivatized with appropriate ligands. The application range is, therefore, broad. For chiral electrochromatography, negatively and positively charged polyacrylamide gels copolymerized with 2-hydroxy-3-allyloxy-propyl-beta-cyclodextrin (allyl-beta-CD) were prepared. The latter monomer was synthesized from beta-CD and allylglycidyl ether by a very simple one-step procedure. Eight acidic, neutral and basic drug compounds were resolved into their enantiomers, most of them with baseline separation. Interestingly, the resolution is independent of the electroendosmotic velocity, i.e., rapid analyses will not give low resolution. Upon increasing this velocity, the plate height for the fast enantiomer did not change (or decreased slightly), whereas that for the slow enantiomer increased. Only the last term in the van Deemter equation contributed significantly to the total plate height. The composition of the gel was chosen such that the "pores" became large enough to guarantee a satisfactory electroendosmotic flow (EOF). This open gel structure explains why acetone diffused as in free solution, i.e., independently of the presence of the gel matrix. This finding also indicates that the separation of small molecules in polyacrylamide gels cannot be explained by "molecular-sieving", but rather by some type of adsorption ("aromatic adsorption"?).
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