Synthesis, biophysical properties and biological activity of second generation antisense oligonucleotides containing chiral phosphorothioate linkages

Abstract

Bicyclic oxazaphospholidine monomers were used to prepare a series of phosphorothioate (PS)-modified gapmer antisense oligonucleotides (ASOs) with control of the chirality of each of the PS linkages within the 10-base gap. The stereoselectivity was determined to be 98% for each coupling. The objective of this work was to study how PS chirality influences biophysical and biological properties of the ASO including binding affinity (Tm), nuclease stability, activity in vitro and in vivo, RNase H activation and cleavage patterns (both human and E. coli) in a gapmer context. Compounds that had nine or more Sp-linkages in the gap were found to be poorly active in vitro, while compounds with uniform Rp-gaps exhibited activity very similar to that of the stereo-random parent ASOs. Conversely, when tested in vivo, the full Rp-gap compound was found to be quickly metabolized resulting in low activity. A total of 31 ASOs were prepared with control of the PS chirally of each linkage within the gap in an attempt to identify favorable Rp/Sp positions. We conclude that a mix of Rp and Sp is required to achieve a balance between good activity and nuclease stability.

Quantitative Analysis of Imetelstat in Plasma with LC–MS/MS Using Solid-Phase or Hybridization Extraction

“…These chemical modifications include: sulfurization of the phosphodiester bond to avert degradation by enlindogenous exonucleases, addition of methoxy or methoxyethyl groups to sugar moieties [7,8], locked and unlocked nucleic acids, and alterations in the internucleotide linkage (e.g., amide linkage) resulting in peptide nucleic acids [1]. Sulfurization results in phosphorothioate analogs that are more hydrophobic, exhibit more complex secondary structures with higher protein binding and greater accumulation in organs than phosphodiester analogs [9][10][11].Imetelstat is a novel, first-in-class telomerase inhibitor ODN [12], being investigated for the treatment of hematologic myeloid malignancies [13,14] and certain solid tumors [12,15,16]. Imetelstat is a 13-mer oligonucleotide N3 -P5 thio-phosphoramidate (NPS) with a covalently linked C16 (palmitoyl) lipid moiety at the 5 -end (Figure 1; Supplementary Table 1).…”

“…These chemical modifications include: sulfurization of the phosphodiester bond to avert degradation by enlindogenous exonucleases, addition of methoxy or methoxyethyl groups to sugar moieties [7,8], locked and unlocked nucleic acids, and alterations in the internucleotide linkage (e.g., amide linkage) resulting in peptide nucleic acids [1]. Sulfurization results in phosphorothioate analogs that are more hydrophobic, exhibit more complex secondary structures with higher protein binding and greater accumulation in organs than phosphodiester analogs [9][10][11].…”

Chromatographic approaches for the characterization and quality control of therapeutic oligonucleotide impurities

“…Consequently, PS n ‐mer oligonucleotides may be present as a mixture of 2 ( n − 1) diastereomers (where n is the length of the oligonucleotide). This can cause an increase in sample complexity to a level beyond the current available technologies, especially if there is a need to completely separate and resolve this mixture using conventional analytical techniques such as chromatography and capillary electrophoresis (Gilar, Belenky, & Cohen, ; Nikcevic et al, ; Wan et al, ).…”

“…However, this method did not show any significant advantage for therapeutic applications over the conventional method producing diastereomeric mixtures. To date, this approach has been limited owing to the added complexity and cost related to the synthesis and analysis (Wan et al, ).…”

Phosphate-modified analogues of m 7 GTP and m 7 Gppppm 7 G—Synthesis and biochemical properties

“…A greater excess of a P 1 ,P 2 -di(1-imidazolyl) derivative results in the formation of nucleoside pentaphosphates as side products. After the optimisation, the desired products, GTP (20) or Gp BH3 pp (21), were isolated in 65% and 44% yields, respectively. If compound 11 was used in the reaction with Gp BH3 (18) instead of 10, the reaction proceeded significantly faster, requiring less than 5 h for completion, and affording Gp BH3 pCH 2 p (22) in a slightly lower isolated yield (39%).…”