New phosphorylating reagents for deoxyribonucleosides and oligonucleotides

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New phosphorylating reagents 1 and 2 were prepared in three steps from 4-hydroxybenzaldehyde. They showed good efficiency in the solid phase synthesis of 5′-phosphate monoester nucleosides. End-phosphate DNA sequence synthesis demonstrated the efficiency of the new reagents (1 and 2) according to the general procedure of automated DNA synthesis. The oxidation of P(III) to P(V) and the removal of benzyl protecting groups were achieved in a single step by treatment with a 0.02 M I2/pyridine/H2O solution. Due to this one-pot treatment, it is possible to use the phosphorylating reagents (1 and 2) for the synthesis of base-sensitive ODNs. The reagents 1 and 2 are unique among phosphorylating reagents.

A versatile reagent for the synthesis of 5′-phosphorylated, 5′-thiophosphorylated or 5′-phosphoramidate-conjugated oligonucleotides

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We report the synthesis of a new phosphorylating reagent that is easily accessible and allows not only the chemical synthesis of 5′-phosphorylated and 5′-thiophosphorylated oligonucleotides but also the 5′-conjugation through a phosphoramidate linkage. 5′-Amino-linker and 5′-alkyne oligonucleotides were obtained and the latter was conjugated by a 1,3-dipolar cycloaddition (click chemistry) with a galactosylated azide derivative to afford 5′-galactosyl oligonucleotide with high efficiency.

Novel reagents for terminal phosphorylation and thiophosphorylation of synthetic oligonucleotides

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Two novel phosphoramidite building blocks and a solid support that allow an efficient solid-phase phosphorylation or thiophosphorylation of synthetic oligonucleotides were developed. The utility of these synthetic tools was demonstrated in the preparation of 5′- or 3′-thiophosphorylated oligonucleotides, which were subsequently labeled at the termini with fluorescent reporters.

Chemical ligation of oligonucleotides using an electrophilic phosphorothioester

We developed a new approach for chemical ligation of oligonucleotides using the electrophilic phosphorothioester (EPT) group. A nucleophilic phosphorothioate group on oligonucleotides was converted into the EPT group by treatment with Sanger’s reagent (1-fluoro-2,4-dinitrobenzene). EPT oligonucleotides can be isolated, stored frozen, and used for the ligation reaction. The reaction of the EPT oligonucleotide and an amino-modified oligonucleotide took place without any extra reagents at pH 7.0–8.0 at room temperature, and resulted in a ligation product with a phosphoramidate bond with a 39–85% yield. This method has potential uses in biotechnology and chemical biology. (Nucleic Acids Res. 2017 Jul 7; 45(12): 7042–7048.)

Fragment-based solid-phase assembly of oligonucleotide conjugates with peptide and polyethylene glycol ligands

Ligand conjugation to oligonucleotides is an attractive strategy for enhancing the therapeutic potential of antisense and siRNA agents by inferring properties such as improved cellular uptake or better pharmacokinetic properties. Disulfide linkages enable dissociation of ligands and oligonucleotides in reducing environments found in endosomal compartments after cellular uptake. Solution-phase fragment coupling procedures for producing oligonucleotide conjugates are often tedious, produce moderate yields and reaction byproducts are frequently difficult to remove. We have developed an improved method for solid-phase coupling of ligands to oligonucleotides via disulfides directly after solid-phase synthesis. A 2′-thiol introduced using a modified nucleotide building block was orthogonally deprotected on the controlled pore glass solid support with N-butylphosphine. Oligolysine peptides and a short monodisperse ethylene glycol chain were successfully coupled to the deprotected thiol. Cleavage from the resin and full removal of oligonucleotide protection groups were achieved using methanolic ammonia. After standard desalting, and without further purification, homogenous conjugates were obtained as demonstrated by HPLC, gel electrophoresis, and mass spectrometry. The attachment of both amphiphilic and cationic ligands proves the versatility of the conjugation procedure. An antisense oligonucleotide conjugate with hexalysine showed pronounced gene silencing in a cell culture tumor model in the absence of a transfection reagent and the corresponding ethylene glycol conjugate resulted in down regulation of the target gene to nearly 50% after naked application.(European Journal of Medicinal Chemistry.,Volume 121, 4 October 2016, Pages 132-142.)

Solid-phase synthesis of 5′-triantennary N-acetylgalactosamine conjugated antisense oligonucleotides using phosphoramidite chemistry

A convenient solid-phase synthetic method was developed for assembling a triantennary N-acetylgalactosamine (GalNAc) cluster on the 5′-end of antisense oligonucleotide using phosphoramidite chemistry. Conjugation of the 5′-triantennary GalNAc cluster improved potency of the 14 mer ASO 7-fold in mice and more than 50 fold in hepatocytes. The synthetic approach described in this Letter simplifies the synthesis of 5′-triantennary GalNAc cluster conjugated ASOs and helps understand the structure–activity relationship for targeting hepatocytes with oligonucleotide therapeutics.(Bioorganic & Medicinal Chemistry Letters.,Volume 25, Issue 19, 1 October 2015, Pages 4127-4130.)

MMT, Npeoc-protected spermine, a valuable synthon for the solid phase synthesis of oligonucleotide oligospermine conjugates via guanidine linkers

Solid phase spermine oligomerization via guanidine linkers was achieved using activated thiourea coupling reaction with primary amino group. Disymmetric spermine synthon was efficiently synthesised in eight steps from spermine. MMT group was used as coupling monitor and resulting oligomeric spermines were conjugated to oligonucleotides. (Bioorganic & Medicinal Chemistry., Volume 19, Issue 6, 15 March 2011, Pages 1972-1977.)