Chemical synthesis of oligonucleotides. 3: Synthesis and characterization of N, O-protected ribophosphoesters for applications in RNA synthesis

The chemical synthesis of RNA, in contrast to that of DNA, poses problems due to fi) additional requirement of 2'-hydroxyl protection of the ribose moieties, and (ii) high lability of inter-ribophosphate bonds. Herein we report the synthesis and characterisation of N, O-protected ribophosphoesters 1 which are key monomeric derivatives in phosphotriester methodology for RNA synthesis. Both the isomeric 2' and 3'-O-phosphates have been obtained and characterised. The utility of the t-butyl dimethylsilyl group for 2'-hydroxyl protection in the phosphotriester method is demonstrated by the synthesis of r(AUAU),r(UAUA) and r(CACA)

Regioselective 2'/3'-O-allylation of pyrimidine ribonucleosides using phase transfer catalysis

A short and convenient procedure for regiospecific O-allylation of uridine is reported by employing dibutyltin oxide as a mild base in conjunction with a phase transfer catalyst tetrabutylammonium bromide. The resulting isomeric 2'/3'-O-allyl uridines were separated after conversion into their corresponding 5'-O-DMT derivatives. The 2'-O-allyluridine 3 was then transformed into 2'-O-allylcytidine 7 and both were individually converted into the corresponding β-cyanoethyl phosphoramidite monomers (9 and 10) and a phosphodiester monomer 11, required for oligonucleotide assembly. The utility of 11 is demonstrated by synthesis and characterization of a 2'-O-allyl ribodinucleotide UpU

Regioselective allylations of pyrimidine ribonucleosides using Pd(0) catalyst

A novel procedure for regiospecific O-allylation of pyrimidine ribonucleosides is reported by using Pd(PPh<SUB>3</SUB>)<SUB>4</SUB>-allyl ethyl carbonate reagent to synthesize 2'-O-allyluridine and 2'-O-allylcytidine. 3-N-allylation of the pyrimidine ring is prevented by protection of uridine imide function by 4-O-(2,5-dimethylphenyl) group which can be transformed to 4-oxo function of uridine or exocyclic amino function of cytidine

Influence of internucleotide phosphate linkage on relative base stacking in 3'-5' and 2'-5' RNA: a circular dichroic spectroscopic study of RNA hexamer AACCUU

The variations in base stacking interactions of two isomeric RNA hexamers, 3'-5'r(AACCUU) and 2'-5'r'(AACCUU), have been studied using temperature dependent CD spectroscopy. Both RNA hexamers, in single strand form, exhibited a right handed helical sense. Van't Hoff analysis of the CD spectral results, derived from a two state model, gave a higher enthalpy of stacking for 3'-5' RNA than for 2'-5'RNA. The results suggest that 3'-5' linkage in RNA facilitates formation of better helical stacks in relation to an isomeric 2'-5' linkage

Spectroscopic and enzymatic characterization of 2'-5' and 3'-5' RNA hexamers AACCUU synthesised by phosphotriester approach in solution using 2'-t-butyldimethylsilyl protection

Comparative <SUP>1</SUP>H,<SUP> 31</SUP>P NMR and CD spectroscopic results and enzymatic cleavage of single stranded RNA hexamers which have identical base sequence (AACCUU) but are regioisomeric at the internucleotide phosphate linkages (2'-t', 3'-5' and their covalent hybrid) are presented. The <SUP>31</SUP>P NMR results revealed significant differences in local phosphate backbone conformation among these isomers, with 2'-5' isomer exhibiting maximum heterogeneity as compared to the 3'-5' isomer, the analogous DNA hexamer and the covalent hybrid hexamer. In contrast to this, there are no appreciable differences in the overall base-base stacking as seen in the CD spectra of 2'-5' and 3'-5' isomers. All RNA hexamers were synthesised by solution phase phosphotriester chemistry with t-butyldimethylsilyl (TBDMS) as 2'-O-protecting group. The fully protected oligorobomers were deprotected in two steps: (i) saturated anhydrous MeOH---NH<SUB>3</SUB> for phosphate and amino deprotections and (ii) TBAF for removal of 2'-O-TBDMS group. Use of sat. MeOH---NH<SUB>3</SUB> (instead of aq. NH<SUB>3</SUB>) prevents 2'-3' internucleotide phosphate migrations, chain fragmentations and 5'-terminal modifications by neighbouring group participation (NGP). The retention of isomeric integrity and absence of 5'-terminal modification in the final products was established by digestion with several nucleases. <SUP>1</SUP>H, <SUP>31</SUP>P NMR and CD spectroscopic studios of titled RNA compounds and an isomeric covalent hybrid indicates conformational differences among these, arising due to different nature of phosphodiester linkages. The compounds, synthesised by solution phase phosphotriester chemistry and 2'-TBDMS protecting group, were shown to be isomerically pure by their specificities towards digestion with the enzyme pancreatic ribonuclease

NP001 regulation of macrophage activation markers in ALS: A phase I clinical and biomarker study

This is a phase I, placebo-controlled, single ascending dose safety and tolerability study of NP001 in patients with ALS. NP001 is a novel regulator of inflammatory macrophages and monocytes. As ALS progression is thought to be related to neuroinflammation, an additional objective of the study was to assess the effects of NP001 administration on monocyte activation markers. Thirty-two ALS patients were enrolled and received either placebo (eight) or one of four (six at each dose) ascending single i.v. doses (0.2, 0.8, 1.6 and 3.2 mg/kg NP001). Patients were monitored for safety, and blood monocyte immune activation markers CD16 and HLA-DR were assessed pre- and 24 h post-dosing. Changes from baseline were calculated. Results showed that NP001 was generally safe and well tolerated. Importantly, a single dose of NP001 caused a dose-dependent reduction in expression of monocyte CD16, a marker of monocyte activation/inflammation. Additionally, monocyte HLA-DR expression was also decreased in those patients with elevated values at baseline. In conclusion, these data indicate that NP001 has an acute effect on inflammatory monocytes in ALS patient blood. The potential for modulation of inflammation in the context of ALS disease progression will require further study with long-term follow-up

NP001 regulation of macrophage activation markers in ALS: A phase I clinical and biomarker study

This is a phase I, placebo-controlled, single ascending dose safety and tolerability study of NP001 in patients with ALS. NP001 is a novel regulator of inflammatory macrophages and monocytes. As ALS progression is thought to be related to neuroinflammation, an additional objective of the study was to assess the effects of NP001 administration on monocyte activation markers. Thirty-two ALS patients were enrolled and received either placebo (eight) or one of four (six at each dose) ascending single i.v. doses (0.2, 0.8, 1.6 and 3.2 mg/kg NP001). Patients were monitored for safety, and blood monocyte immune activation markers CD16 and HLA-DR were assessed pre- and 24 h post-dosing. Changes from baseline were calculated. Results showed that NP001 was generally safe and well tolerated. Importantly, a single dose of NP001 caused a dose-dependent reduction in expression of monocyte CD16, a marker of monocyte activation/inflammation. Additionally, monocyte HLA-DR expression was also decreased in those patients with elevated values at baseline. In conclusion, these data indicate that NP001 has an acute effect on inflammatory monocytes in ALS patient blood. The potential for modulation of inflammation in the context of ALS disease progression will require further study with long-term follow-up