ABSTRACT
We have investigated the cleavage rates of various protecting groups for the exocyclic amine of cytosine, adenine, and guanine bases. Specifically, deprotection of N-benzoyl (Bz), N-acetyl (Ac), N-isobutyryl (iBu), N-phenoxyacetyl (PAC) and N-tert-butylphenoxyacetyl (tBPAC) groups from 2'-deoxyribonucleosides was effected under various cleavage conditions and the rates of cleavage (half-lives) were determined. Aqueous methylamine cleaves all of the examined protecting groups from the exocyclic amine the fastest among the six methods used. Ethanolic ammonia showed the highest selectivity between standard protecting groups (Ac, Bz, iBu) and fast-deprotecting groups (PAC, tBPAC). Under ammonia conditions, it was possible to cleave PAC and tBPAC rapidly and selectively in 2h, while still retaining the large majority of the acetyl, benzoyl and isobutyryl groups. The results of this study allowed us to perform mild and complete deprotection of an oligoribonucleotide while still attached to the support with a light labile linker. This procedure simplifies and speeds up post-synthesis processing of the RNA chain and offers a new route to the synthesis of sensitive oligonucleotide derivatives on solid supports.
Subject(s)
Nucleosides/chemical synthesis , Oligonucleotides/chemical synthesis , RNA/chemical synthesis , Adenine/chemistry , Chromatography, Thin Layer , Cytosine/chemistry , Guanine/chemistry , Nucleosides/chemistry , Oligonucleotides/chemistry , RNA/chemistry , Uracil/chemistryABSTRACT
Cystic fibrosis is a genetic disease caused by mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. In vitro experiments have demonstrated that 4-methyl-2-(5-phenyl-1H-pyrazol-3-yl)phenol (VRT-532, 1) is able to partially restore the function of mutant CFTR proteins. To help elucidate the nature of the interactions between 1 and mutant CFTR, molecular probes based on the structure of 1 have been prepared. These include a photoreactive aryl azide derivative 11 and a fluorescent dansyl sulfonamide 15. Additionally, a method for hydrogen isotope exchange on 1 has been developed, which could be used for the incorporation of radioactive tritium. Using iodide efflux assays, the probe molecules have been demonstrated to modulate the activity of mutant CFTR in the same manner as 1. These probe molecules enable a number of biochemical experiments aimed at understanding how 1 rescues the function of mutant CFTR. This understanding can in turn aid in the design and development of more efficacious compounds which may serve as therapeutic agents in the treatment of cystic fibrosis.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Fluorescent Dyes/chemical synthesis , Molecular Probes/chemical synthesis , Photoaffinity Labels/chemical synthesis , Animals , Azides/chemical synthesis , Azides/chemistry , Azides/pharmacology , Cell Line , Cresols/pharmacology , Cricetinae , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Dansyl Compounds/chemical synthesis , Dansyl Compounds/chemistry , Dansyl Compounds/pharmacology , Fluorescent Dyes/chemistry , Fluorescent Dyes/pharmacology , Isotope Labeling , Molecular Probes/chemistry , Molecular Probes/pharmacology , Mutation , Photoaffinity Labels/chemistry , Photoaffinity Labels/pharmacology , Pyrazoles/chemical synthesis , Pyrazoles/chemistry , Pyrazoles/pharmacology , Structure-Activity Relationship , TritiumABSTRACT
We report on the synthesis and properties of oligonucleotides containing 2'-O-(levulinic acid) and 2'-O-(amino acid) acetalesters. Given that esters serve as promoieties in several therapeutic prodrugs, we believe that these derivatives will have potential use as nucleic acid prodrugs. In addition, we report on the synthesis of a novel solid support with a photolabile linker that not only allows for the synthesis of oligonucleotides containing various 2'-O-acetalesters, but can be generally adopted to the synthesis of base-sensitive oligoribonucleotides. The release of oligonucleotides from this support is faster than with conventional linkers.