ABSTRACT
Short oligonucleoside boranophosphates containing all four nucleosides were synthesized on solid support using base-unprotected nucleoside H-phosphonate monomers. This strategy avoided irreversible base modifications during the boronation procedure. Structures of the boranophosphate oligomers were confirmed by 1H, 31P, 10B NMR and MS analysis as well as by enzymatic hydrolysis.
Subject(s)
Boron Compounds/chemical synthesis , Oligonucleotides/chemical synthesis , Organophosphonates/chemistry , Chromatography, High Pressure Liquid , Magnetic Resonance Spectroscopy , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
A new method for synthesis of N-alkylated nucleosides was developed. Exceptionally mild and selective conversion of N-acyl to the corresponding N-alkyl nucleosides was achieved by reduction with borane-amine complexes. The borane-amine complexes were also used as efficient scavengers of a 4,4'-dimethoxytrityl (DMT) cation. Neutralization of the cation eliminated the boranophosphate group degradation during acidic DMT deprotection and allowed milder acidic conditions for the deprotection.
Subject(s)
Amines/chemistry , Boranes/chemistry , Nucleosides/chemical synthesis , Alkylation , Reducing Agents/chemistry , Trityl Compounds/chemistryABSTRACT
Antisense oligonucleotides are potentially a powerful tool for the therapeutic manipulation of genes associated with cancer. However, pharmacological applications of oligonucleotides have been hindered by the inability to effectively deliver these compounds to their sites of action within cells. In this study, we have prepared peptide-oligonucleotide conjugates with the intent of improving intracellular delivery. The phosphorothioate oligonucleotide component of the conjugates was complementary to a site flanking the AUG of the message for P-glycoprotein, a membrane ATPase associated with multidrug resistance in tumor cells. Two types of peptide-antisense oligonucleotide conjugates, but not mismatched control conjugates, provided substantial inhibition of cell surface expression of P-glycoprotein. Surprisingly, the peptide-oligonucleotide conjugates were more potent in the presence of serum than when used under serum-free conditions; this is in striking contrast to most other approaches for intracellular delivery of nucleic acids. Effective inhibition of P-glycoprotein expression was attained with submicromolar concentrations of antisense conjugates under serum-replete conditions. The combination of relatively modest molecular size and good efficacy in the presence of serum proteins suggests that peptide-antisense oligonucleotide conjugates may have significant promise for in vivo therapeutic applications.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/biosynthesis , Gene Expression/drug effects , Oligonucleotides, Antisense/pharmacology , 3T3 Cells , ATP Binding Cassette Transporter, Subfamily B, Member 1/antagonists & inhibitors , Amino Acid Sequence , Animals , Dose-Response Relationship, Drug , Gene Products, tat/chemistry , Humans , Mice , Molecular Sequence Data , Oligonucleotides, Antisense/chemistry , Peptides/chemistry , Peptides/pharmacology , TransfectionABSTRACT
Borane-amine complexes provide an unusually fast and selective reduction of a deoxynucleoside N-acyl group to a corresponding N-alkyl group. Three different nucleosides (dG, dA, and dC) each having one of three N-protecting groups (benzoyl, isobutyryl, or acetyl) were used to prepare N-alkylated nucleosides in good yields under mild conditions. Deoxyribose O-acyl protecting groups remain intact at the conditions of N-acyl group reduction.
