UnyLinker: An Efficient and Scaleable Synthesis of Oligonucleotides Utilizing a Universal Linker Molecule: A Novel Approach To Enhance the Purity of Drugs

A novel universal linker (UnyLinker) molecule which has a conformationally rigid and chemically stable bridge head ring oxygen atom carrying a conventional 4,4′-dimethoxytrityl (DMT) and succinyl groups locked in a syn orientation has been developed to carry out oligonucleotide synthesis efficiently and smoothly. The geometry of the vicinal syn oxygen functionalized group allows fast and clean cleavage under standard aqueous ammonia deprotection conditions to afford high-quality oligonucleotides. No base modification is observed, based on the ion-pair HPLC−UV−MS (IP-HPLC−UV−MS) method with detection limit of <0.1%. A class of impurities formed by branching from the exocyclic amino group of nucleosides loaded onto a solid support has been eliminated by the use of this method. Examples demonstrating the versatile nature of this molecule are shown by syntheses of different chemistries such as 2′-deoxy, 2′-O-methyl, 2′-O-methoxyethyl, Lock nucleic acids (LNA), 2′-α-fluoro nucleic acids (FANA), conjugates such as 5′-phosphate monoester and biotin, and phosphate diester and phosphorothioate backbone modifications. This molecule was loaded onto several commercial solid supports and used in both gas-sparged and packed-bed automated DNA/RNA synthesizers. Large-scale syntheses (up to 700 mmol) of multiple phosphorothioate first- and second-generation antisense drugs on GE-Amersham’s OligoProcess synthesizer are demonstrated further, showing that this chemistry could be used for efficient synthesis of multiple oligonucleotide drugs using a single raw material, thereby eliminating a difficult to characterize nucleoside-loaded polymer matrix used as a starting material. A mechanism for deprotection and cleavage of the linker molecule to liberate the free oligonucleotide is proposed. Characterization of the cyclic byproduct formed during release of the oligonucleotide is presented. The exo-syn configuration of the dihydroxy structure of the UnyLinker molecule is conclusively established by X-ray crystallography studies. A novel method to remove the last traces of osmium used during the synthesis of the UnyLinker molecule to reach undetectable levels (<1 ppm) is also described.