N-terminus FITC labeling of peptides on solid support: the truth behind the spacer

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Fluorescein isothiocyanate (FITC) is an amine reactive derivative of fluorescein dye that has wide ranging application in biochemistry. It has been extensively used to label peptides and proteins. However, its use in solid phase peptide synthesis is restricted. Indeed, in acidic conditions required for linker cleavage, N-ter FITC-labeled peptides undergo a cyclization leading to the formation of a fluorescein with subsequent removal of the last amino acid. This can be avoided when a spacer such as amino hexanoic acid is used or if non-acidic cleavage is operated to release targeted peptide from the resin. (Tetrahedron Letters. Volume 50, Issue 3, 21 January 2009, Pages 260-263.)

GalNAc-siRNA Conjugates: Leading the Way for Delivery of RNAi Therapeutics

Short-interfering RNA (siRNA)-induced RNAi responses have great potential to treat a wide variety of human diseases from cancer to pandemic viral outbreaks to Parkinson’s Disease. However, before siRNAs can become drugs, they must overcome a billion years of evolutionary defenses designed to keep invading RNAs on the outside cells from getting to the inside of cells. Not surprisingly, significant effort has been placed in developing a wide array of delivery technologies. Foremost of these has been the development of N-acetylgalactosamine (GalNAc) siRNA conjugates for delivery to liver. Tris-GalNAc binds to the Asialoglycoprotein receptor that is highly expressed on hepatocytes resulting in rapid endocytosis. While the exact mechanism of escape across the endosomal lipid bilayer membrane remains unknown, sufficient amounts of siRNAs enter the cytoplasm to induce robust, target selective RNAi responses in vivo. Multiple GalNAc-siRNA conjugate clinical trials, including two phase III trials, are currently underway by three biotech companies to treat a wide variety of diseases. GalNAc-siRNA conjugates are a simple solution to the siRNA delivery problem for liver hepatocytes and have shown the RNAi (and antisense oligonucleotide) field the path forward for targeting other tissue types.

Despite RNAi’s promising therapeutic features, due to its size (∼14,000 Da) and 40 negatively charged phosphates, siRNA RNAi therapeutics cannot enter cells on their own and require a delivery agent. Moreover, naked siRNAs are rapidly degraded in blood by RNAses, cleared by the kidneys, absorbed by liver scavenger receptors, and can activate the innate immune TLR3/7/8, RIG-I, and MDA-5 systems. All these factors contribute to a poor drug profile for siRNA, which must be addressed before the therapeutic potential of siRNAs can be realized. (https://www.liebertpub.com/doi/10.1089/nat.2018.0736)

An efficient deprotection method for 5′-[O,O-bis(pivaloyloxymethyl)]-(E)-vinylphosphonate containing oligonucleotides

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5′-(E)-Vinylphosphonate (VP) is an effective bioisostere of the natural 5′-monophosphate in small interfering RNAs (siRNAs). Solid-phase synthesis of VP-siRNAs requires the use of appropriately protected VP-phosphoramidites in combination with optimal oligonucleotide deprotection conditions. Addition of 3% (v) neat diethylamine to the standard aqueous ammonia deprotection conditions allows clean and rapid one-step deprotection of 5′-[O,O-bis(pivaloyloxymethyl)] (POM)-protected VP oligonucleotides, minimizing side reactions and impurities, which broadly enhances the scope of VP oligonucleotide synthesis. (Tetrahedron.Volume 74, Issue 42, 18 October 2018, Pages 6182-618.)

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.