Anthony N. Scozzari

Applications of green chemistry in the manufacture of oligonucleotide drugs

We have modified the current phosphoramidite-based, solid-phase synthesis of antisense oligonucleotides to accommodate principles of green chemistry. In this article, we summarize key accomplishments that reduce or eliminate the use or generation of toxic materials, solvents, and reagents. Also discussed are methodologies that allow reuse of valuable materials such as amidites, solid-support, and protecting groups, thus improving the atom economy and cost-efficiency of oligonucleotide manufacture. Approaches to accident prevention and the use of safer reagents during oligonucleotide synthesis are also covered.

Chemical Synthesis and Purification of Phosphorothioate Antisense Oligonucleotides

Over a dozen antisense oligonucleotide drugs are undergoing human clinical trials for the treatment of viral infections, cancers, and a range of inflammatory disorders (Table 1). One of these was recently the first antisense oligonucleotide to demonstrate clinical safety and efficacy in pivotal Phase III clinical trials, in this case for the treatment of cytomegalovirus retinitis (Sanghvi et al., 1998). A dozen more antisense oligonucleotides have demonstrated pre-clinical efficacy (Crooke, 1998) and are under consideration for clinical development. In addition, use of antisense gene expression modulation to produce well-defined pharmacological effects is now a routine procedure (Akhtar et al., 1997). Automation of synthesis and ready access to required raw materials are two key reasons for the tumultuous growth in this area of research and development. Methods that allow preparation of a large number of pure oligonucleotides at reasonable cost expedite not only antisense drug discovery, but also open the door to manufacture of these drugs for market and the ultimate goal of delivery to patients. This chapter focuses on advances made in oligonucleotide process chemistry, the introduction and use of new reagents, and purification and analysis of antisense oligonucleotides.

Polysulfide Reagent in Solid-Phase Synthesis of Phosphorothioate Oligonucleotides: Greater than 99.8% Sulfurization Efficiency

A solution of sulfur (0.1 M) and sodium sulfide (0.01 M) in 3-picoline, referred to as polysulfide reagent, rapidly converts trialkyl and triaryl phosphite triesters to the corresponding phosphorothioate derivatives. Greater than 99.8% average stepwise sulfurization efficiency is obtained in the solid-phase synthesis of DNA and RNA phosphorothioate oligonucleotides via the phosphoramidite approach.

Solution Stability and Degradation Pathway of Deoxyribonucleoside Phosphoramidites in Acetonitrile

The impuritiy profiles of acetonitrile solutions of the four standard O‐cyanoethyl‐N,N‐diisopropyl‐phosphoramidites of 5′‐O‐dimethoxytrityl (DMT) protected deoxyribonucleosides (dGib, dAbz, dCbz, T) were analyzed by HPLC‐MS. The solution stability of the phosphoramidites decreases in the order T, dC>dA>dG. After five weeks storage under inert gas atmosphere the amidite purity was reduced by 2% (T, dC), 6% (dA), and 39% (dG), respectively. The main degradation pathways involve hydrolysis, elimination of acrylonitrile and autocatalytic acrylonitrile‐induced formation of cyanoethyl phosphonoamidates. Consequently, the rate of degradation is reduced by reducing the water concentration in solution with molecular sieves and by lowering the amidite concentration. Acid‐catalyzed hydrolysis could also be reduced by addition of small amounts of base.

Synthesis of antisense oligonucleotides. using environmentally friendly and safe deprotection procedures

It is demonstrated that mixed-sequence phosphorothioate oligodeoxyribonucleotides can be synthesized on scales up to 80 mmol without using chlorinated solvents like dichloromethane, while preserving both high yield and purity of the product. A solution of dichloroacetic acid in toluene cleanly and efficiently removes 4,4′-dimethoxytrityl groups from the 5′-terminus of the growing oligonucleotide chain during synthesis on solid support. Ammonium hydroxide treatment at room temperature at atmospheric pressure furnishes deprotected oligonucleotides reducing the risk that pressurized reaction glass vessels pose. To ensure facile separation of polymer beads (Primer HL 30) and oligonucleotide solution, minimum agitation of the reaction mixture is applied.