Sankha Pattanayak

Improved Protocol for the Synthesis of Flexibly Protected Morpholino Monomers from Unprotected Ribonucleosides

An inexpensive and much improved protocol has been developed for the synthesis of protected morpholino monomers from unprotected ribonucleosides in high overall yield, using oxidative glycol cleavage and reductive amination strategy. Unlike the previous methods, the present strategy allows installing the exocyclic amine protections at a later stage, and thus avoids the use of expensive, or commercially unavailable, exocyclic amine-protected ribonucleosides as starting materials. To demonstrate the flexibility of the present method in choosing protecting groups, the monomers have been protected with several such groups of different deblocking properties at the exocyclic amine position.

A cationic morpholino antisense oligomer conjugate: synthesis, cellular uptake and inhibition of Gli1 in the hedgehog signalling pathway

A guanidinium-linked morpholino pentamer is synthesized and evaluated for cellular penetration properties. A regular morpholino and the pentamer conjugate can exhibit antisense effects when designed against Gli1-transcription factors of the Hh signalling pathway, which is a potential target for cancer therapy. Evidence was found from luciferase assays and western blot analysis.

Piperazic acid derivatives inhibit Gli1 in Hedgehog signaling pathway

Piperazic acid, a non-proteinogenic amino acid, found in complex secondary metabolites and peptide natural substances, has shown down regulation of Gli1 expression in Hedgehog signaling pathway in cell based assays. Further structure activity relationship study indicated that amide derivatives of piperazic acid are more potent than piperazic acid itself, with little to no toxicity. However, other cellular components involved in the pathway were not affected. To the best of our knowledge, this is the first report on the inhibitory property of piperazic acid in this pathway. Hence, this molecule could serve as a useful tool for studying Hedgehog signaling.

Hedgehog Antagonist Pyrimidine–Indole Hybrid Molecule Inhibits Ciliogenesis through Microtubule Destabilisation

One of the crucial regulators of embryonic patterning and tissue development is the Hedgehog‐glioma (Hh‐Gli) signalling pathway; its uncontrolled activation has been implicated in different types of cancer in adult tissues. Primary cilium is one of the important factors required for the activation of Hh signalling, as it brings the critical components together for key protein–protein interactions required for Hh pathway regulation. Most of the synthetic and natural small molecule modulators of the pathway primarily antagonise Smoothened (Smo) or other effectors like Hh ligand or Gli. Here, we report a previously described Hh antagonist, with a pyrimidine–indole hybrid (PIH) core structure, as an inhibitor of ciliogenesis. The compound is unique in its mode of action, as it shows perturbation of microtubule dynamics in both cell‐based assays and in vivo systems (zebrafish embryos). Further studies revealed that the probable targets are α‐tubulin and its acetylated form, found in the cytoplasm and primary cilia. PIH also showed axonal defasiculation in developing zebrafish embryos. We thus propose that PIH antagonises Hh signalling by repressing cilia biogenesis and disassembling α‐tubulin from its stabilised form.

Synthesis of Morpholino Monomers, Chlorophosphoramidate Monomers, and Solid‐Phase Synthesis of Short Morpholino Oligomers

Phosphorodiamidate morpholino oligomers (PMOs) are a highly capable class of synthetic antisense oligonucleotides that are used to study gene functions in in vitro and in vivo models. This unit describes the synthesis of exocyclic‐amine‐protected 7′‐hydroxy and 7′‐chlorophosphoramidate‐activated morpholino monomers of A, T, G, and C, together with their incorporation into short PMO oligomers by solid‐phase synthesis. Starting from ribonucleosides, the exocyclic‐amine‐protected 7′‐hydroxy monomers are prepared following a modified Summerton protocol, which consists of a periodate cleavage/Schiff base formation/reduction cycle. The exocyclic amine protections are installed at a later stage (except G) to avoid the use of costly exocyclic‐amine‐protected counterparts that give control over protecting group manipulation. The 7′‐hydroxy monomers with N‐Trit/N‐MMTr are then converted to the 7′‐chlorophosphoramidate morpholino monomers in one step employing a combination of lithium bromide and DBU. These chlorophosphoramidate monomers are finally assembled by solid‐support synthesis to obtain the short PMO oligomers.