Development of Kilogram-Scale Convergent Liquid-Phase Synthesis of Oligonucleotides.

Oligonucleotide drugs show promise to treat diseases afflicting millions of people. To address the need to manufacture large quantities of oligonucleotide therapeutics, the novel convergent liquid-phase synthesis has been developed for an 18-mer oligonucleotide drug candidate. Fragments containing tetra- and pentamers were synthesized and assembled into the 18-mer without column chromatography, which had a similar impurity profile to material made by standard solid-phase oligonucleotide synthesis. Two of the fragments have been synthesized at ∼3 kg/batch sizes and four additional tetra- and pentamer fragments were synthesized at >300-g scale, and a 34-mer was assembled from the fragments. Critical impurities are controlled in the fragment syntheses to provide oligonucleotides of purities suitable for clinical use after applying standard full-length product purification process. Impurity control in the assembly steps demonstrated the potential to eliminate chromatography of full-length oligonucleotides, which should enhance scalability and reduce the environmental impact of the process. The convergent assembly and telescoping of reactions made the long synthesis (>60 reactions) practical by reducing production time, material loss, and chances for impurity generation.

Solid-Phase Synthesis of Phosphorothioate Oligonucleotides Using Sulfurization Byproducts for in Situ Capping.

Oligonucleotides containing phosphorothioate (PS) linkages have recently demonstrated significant clinical utility. PS oligonucleotides are manufactured via a solid-phase chain elongation process in which a four-reaction cycle consisting of detritylation, coupling, sulfurization, and failure sequence capping with Ac2O is repeated. In the capping step, uncoupled sequences are acetylated at the 5'-OH to stop the chain growth and control the levels of deletion, or ( n-1), impurities. Herein, we report that the byproducts of commonly used sulfurization reagents react with the 5'-OH and cap the failure sequences. The standard Ac2O capping step can therefore be eliminated, and this 3-reaction cycle process affords a higher yield and higher or comparable overall purity compared to the conventional 4-reaction synthesis. This improvement results in reducing the number of reactions from ∼80 to ∼60 for the synthesis of a typical length 20-mer oligonucleotide. For every kilogram of an oligonucleotide intermediate synthesized, > 500 L of reagents and organic solvents is saved, and the E-factor is decreased to <1500 from ∼2000.

Cell penetrating agents based on a polyproline helix scaffold.

Cell penetrating agents were designed and synthesized that introduce cationic and hydrophobic moieties along the backbone of a polyproline helix (PPII) in an amphiphilic manner. The CD profile has the features that are expected for a PPII helix, demonstrating that the addition of these groups had little effect on the backbone structure. Dramatic increases in uptake were found with MCF-7 cells when up to six guanidinium groups were positioned on the polyproline helix, whereas only modest increases in cellular uptake were observed with the amine-containing polyproline compounds as compared to their flexible counterparts. Amphiphilicity played a key role in the enhanced cell translocation, as scrambled versions of the designed agents, with hydrophobic and cationic groups on all faces of the helix, were only as effective as their flexible peptide counterparts. Interestingly, the most potent agent, P11LRR, demonstrated almost an order of magnitude more efficient cellular uptake as compared to that of the well-studied Tat peptide, with minimal cytotoxicity.