Summary of Serge L. Beaucage’s Research Program

1. Summary of Serge L. Beaucage’s Research Program Thermosensitivethiophosphate protecting groups in the development of thermolyticphosphorothioate DNA oligonucleotide prodrugs In 2005 we demonstrated that the co-administration of a CpG-containing phosphorothioate DNA prodrug with thermosentive 2-(N-formyl-N-methylamino)ethyl groups for thiophosphate protection and a conventional phosphorothioate DNA sequence in mice, resulted in a significantly prolonged immunoprotection of the animal against specific viral infections. In 2010,thermolytic thiophosphate-protecting groups derived from 2-(N-formyl-N-methyl)aminoethanol and 4-(methylthio)butan-1-ol have been developed. The conversion of DNA prodrug models to bioactive drugs occurred with half-times ranging from 20 hrs to 265 hrs at 37°C. This work may lead to the development of improved thermolyticCpG-containing DNA prodrug formulations, which could potentially induce a strong and sustained immune response against infectious diseases in animal models over extended periods of time.

2. Solid-phase synthesis and thermolytic release of DNA sequences conjuga-ted with functional groups at either the 3’-terminus or at both 5’- and 3’-termini • The availability of the 5’- and/or 3’-terminal hydroxyl groups for conjugation with various reporter or functional groups is particularly useful in the synthesis oligonucleotide conjugates. In 2008 we engineered a solid support functionalized with dinucleoside phosphate/thiophosphatetriesters through a thermosensitivelinker. • This support allows one to: (i) conjugate any functional groups at the 3’-terminus of the dinucleotide; (ii) grow a DNA chain through the 5’-terminus; (iii) conjugate the 5’-end of the DNA sequence with any functional groups; (iv) deprotect the DNA sequence while still being linked to the support; and (v) thermolytically release the DNA sequence from the support,free of deprotection side products. • Biotinylationof a propargylated cyclic (3’-5’) diguanylic acid and of its mono-6-thioated analog under “click” conditions • The synthesis of biotinylated c-di-GMP conjugates has led the identification and characterization of proteins binding to this ligand. UV-crosslinking and competition studies have shown that c-diGMP physically associate with a limited set of murine or human macrophage proteins, which directs activation of a potent inflammatory response.

3. Designing and implementing protecting groups for the 2’-hydroxy function of ribonucleosides in the chemical synthesis of RNA (Dr. JacekCieślak) • With the advent of RNA interference, as a means to silence gene expression, oligonucleotides consisting of less than 25 nucleotides are now being investigated as potential drugs for various biomedical indications. • RNA interference has spurred a renewed interest in the development of rapid and efficient methods for solid-phase RNA synthesisto enable the production of RNA sequences in sufficient quantity and purity for pharmaceutical applications. • Given that the most formidable challenge in the chemical synthesis of RNA oligonucleotides is designing a 2’-hydroxyl protecting group that would provide ribonucleosidephosphoramidites with rapid coupling kinetics and high coupling efficiencies, the 4-(N-dichloroacetyl-N-methylamino) benzyloxymethyl group was reported for 2’-hydroxyl protection of ribonucleosides for this purpose in 2007 and 2008. • Although the 4-(N-dichloroacetyl-N-methylamino)benzyloxymethyl group is suitable for 2’-hydroxyl protection of ribonucleosides, its synthesis is not cost-effective. Furthermore, two synthetic approaches were required for the preparation of the ribonucleosidephosphoramidites needed for RNA synthesis.

4. Designing and implementing protecting groups for the 2’-hydroxy function of ribonucleosides in the chemical synthesis of RNA (Cont’d) • In 2012, we reported the synthesis of 2’-O-aminooxymethyl ribonucleo-sides, which have been useful in the preparation of permanent or reversible ribonucleoside 2’-conjugates. The synthetic approach allows 2’-functionalization of all four ribonucleosides in a cost-effective process. • The reversibility of 2’-O-aminooxymethyl ribonucleoside conjugates provides new opportunities for the discovery and implementation of novel 2’-hydroxy protecting groups for ribonucleosides in the synthesis of native and/or modified RNA sequences for RNA interference applications. • 2’-O-(2-Cyano-2,2-dimethylethanimine-N-oxymethyl)ribonucleosides have been prepared from 2’-O-aminooxymethyl ribonucleosides and have been successfully used as phosphoramidite derivatives in the solid-phase synthesis of RNA sequences. • The fluoride-assisted removal of 2’-O-(2-cyano-2,2-dimethylethani-mine-N-oxymethyl) groups from RNA sequences is however chain length-dependent. This limitation has been alleviated through the development of novel 2’-O-iminooxymethyl conjugates, which are found to be easily cleaved from RNA sequences through an intramolecular process.

5. Assessment of the internalization of positively charged thermolyticphosphorothioate DNA prodrugs in cell cultures • The potential nucleic acid-based drugs for therapeutic applications is difficult to evaluate given that the polyanionic nature of these biopolymers has been and still is a major limitation to their cellular internalization. • We discovered that the incorporation of four positively charged 3-(N,N-dimethylamino)-prop-1-yl phosphorothioatetriester functions into a 5’-fluoresceinated neutral DNA prodrug sequence resulted in a ~30-fold, 40-fold and 4-fold increased uptake of this modified DNA sequence in Vero, GC-2 and HeLacells,respectively. The positively charged DNA sequence did not show cytotoxicity in Vero cells even at a concentration of 50 µM. • Confocal microscopy showed that the fluorescence of the positively charged DNA prodrug sequence appeared within the nucleus of Vero and GC-2 cells, thereby suggesting that the positively charged phosphoro-thioatetriester functions of the neutral DNA sequence contributed to the successful escape of the modified DNA prodrug sequence from endosomal vesicles in either Vero or GC-2 cells.

6. Synthesis of ultra long DNA sequences • The published synthesis and assembly of a 582,970-base pair Mycoplasma genitaliumgenome demonstrated the power of DNA synthesis in the context of potential gene therapy applications. • Our approach to the synthesis of ultra long (~1 Kb) DNA sequences consists of using a non-porous solid support functionalized with a linker, which will be used to anchor the growing DNA chains through their 3’-termini. • Deoxyribonucleosidephosphoramidites functionalized with a 5’-fluorescent protecting group will be used to monitor the efficiency of each chain elongation step and various techniques will be employed to minimize the entropy of the growing DNA chains. Regulatory significance A research program on the development of novel methods aimed at improving the chemical synthesis of DNA and RNA oligonucleotides for therapeutic, diagnostic and genetic engineering applications should lead to: (i) a more rigorous evaluation of the novel strategies employed for manufacturing synthetic oligonucleotides of the highest purity, (ii) a better understanding of the parameters linked to the delivery and bioactivity of oligonucleotides, and (iii) the development of guidelines and regulations to ensure safety and efficacy of synthetic oligonucleotides for the diagnosis and treatment of cancer and infectious diseases and of gene therapy protocols.