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

Pattanayak, Sankha; Paul, Subhojit; Nandi, Bappaditya; Sinha, Surajit

doi:10.1080/15257770.2012.724491

Cited by 24 publications

(24 citation statements)

References 51 publications

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“…Accordingly, deprotonations were carried out with sodium hydride and subsequent quenching was done with N,N ‐dimethylphosphoramic dichloride, 5 a to obtain chlorophosphoramidate activated monomer 6 as a 1:1 diastereomeric mixture due to the formation of a new chiral phosphorus centre, as confirmed by 31 P NMR. To achieve transporter G3 ( 1 ), the 7′‐azido morpholino monomer unit 7 was used as the starting monomer, obtained from N 4 ‐benzoyl‐7′‐hydroxy‐ N ‐trityl morpholinocytidine in 84 % yield over three steps (See Supporting Information Scheme S1). Free morpholine amine of 7 was then coupled with activated monomer 6 to get dimer 8 as 1:1 diastereomeric mixture of almost same polarity in 73 % total yield.…”

Section: Resultsmentioning

confidence: 99%

Cationic Cytosine Morpholino‐Based Transporters: Synthesis and Regulation of Intracellular Localization

et al. 2017

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Cationic guanidinium and phosphonium functionalized cytosine morpholino tetramer (G3) and trimer (P3) have been reported for the first time. They show efficient cellular uptake properties in full growth medium, quantified by fluorescence activated cell sorting (FACS) and visualized by fluorescence microscopy. An interesting feature of these transporters is their localization in the nucleolus or the rest of nucleus, determined by the nature of cationic moieties attached with cytosine base. Co-staining with MitoTracker Deep Red depicts their ability to penetrate mitochondrial membrane. Fluorescence imaging displays their uniform distribution throughout zebrafish larvae with no significant toxicity. FACS analysis at 4 8 C indicated that transfection of G3 transporter is energy dependent whereas P3 is energy independent. When covalently conjugated, G3 is capable of transporting a 25-mer phosphorodiamidate morpholino oligomer (PMO) into the cells. It exhibits antisense effect against Gli1 gene in hedgehog pathway, confirmed in preliminary b-glactosidase assay performed in Ptch1 -/cells. Further antisense effect was confirmed when G3-conjugated zebrafish Gli1-PMO was injected into embryo to obtain Gli1dependent phenotypes. To the best of our knowledge, herein, we first report that molecular transporters with a minimum number of three cationic groups are capable of efficiently passing through plasma, nuclear and mitochondrial membranes and show distribution in zebrafish larvae.[a] Dr. Figure 1. Chemical structures of (a) DNA, (b) PMO, transporters (c) G3 (1) and (d) P3 (2).

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Section: Resultsmentioning

confidence: 99%

Cationic Cytosine Morpholino‐Based Transporters: Synthesis and Regulation of Intracellular Localization

et al. 2017

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“…Reduction with sodium cyanoborohydride in the presence of glacial acetic acid provided crude morpholine 78 . 44,45 N -Tritylation furnished 79 in 43% yield over four synthetic steps. 44,46 Desilylation followed by sulfamoylation of 80 in DME–MeCN afforded sulfamate 81 that was biotinylated using our standard protocol.…”

Section: Resultsmentioning

confidence: 99%

“…44,45 N -Tritylation furnished 79 in 43% yield over four synthetic steps. 44,46 Desilylation followed by sulfamoylation of 80 in DME–MeCN afforded sulfamate 81 that was biotinylated using our standard protocol. The synthesis was completed by ammonolysis to remove the N 6 -benzoyl group and by subsequent treatment with 80% aqueous acetic acid to deprotect the N -trityl group to give 82 in 36% yield over three steps from 81 .…”

Section: Resultsmentioning

confidence: 99%

Targeting Mycobacterium tuberculosis Biotin Protein Ligase (MtBPL) with Nucleoside-Based Bisubstrate Adenylation Inhibitors

et al. 2015

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Mycobacterium tuberculosis (Mtb) responsible for both latent and symptomatic tuberculosis (TB) remains the second leading cause of mortality among infectious diseases worldwide. Mycobacterial biotin protein ligase (MtBPL) is an essential enzyme in Mtb and regulates lipid metabolism through the post-translational biotinylation of acyl coenzyme A carboxylases. We report the synthesis and evaluation of a systematic series of potent nucleoside-based inhibitors of MtBPL that contain modifications to the ribofuranosyl ring of the nucleoside. All compounds were characterized by isothermal titration calorimetry (ITC) and shown to bind potently with KD's below 2 nM. Additionally, we obtained high-resolution co-crystal structures for a majority of the compounds. Despite fairly uniform biochemical potency, the whole-cell Mtb activity varied greatly with minimum inhibitory concentrations (MIC) ranging from 0.78 to >100 μM. Cellular accumulation studies showed a nearly 10-fold enhanced accumulation of a C-2′-α analog over the corresponding C-2′-β analog, consistent with their differential whole-cell activity.

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“…Morpholinos are prepared on a solid‐support using an iterative two‐step process that consists of detritylation and successive coupling of the chlorophosphoramidate‐activated monomers (Summerton and Weller, ). In this unit, the synthesis of the 7′‐hydroxyl morpholino monomers, starting from unprotected ribonucleosides of adenosine, guanosine, 5‐methyluridine, and cytidine (see and Alternate Protocols 1 to 3) using a periodate cleavage/Schiff base formation/reduction cycle is described (Pattanayak et al, ). These hydroxyl monomers are then converted to chlorophosphoramidate‐activated monomers using a combination of lithium bromide and DBU (Pattanayak and Sinha, ).…”

Section: Introductionmentioning

confidence: 99%

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

Bhadra

Pattanayak

Sinha

2015

CP Nucleic Acid Chemistry

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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.

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

Cited by 24 publications

References 51 publications

Cationic Cytosine Morpholino‐Based Transporters: Synthesis and Regulation of Intracellular Localization

Cationic Cytosine Morpholino‐Based Transporters: Synthesis and Regulation of Intracellular Localization

Targeting Mycobacterium tuberculosis Biotin Protein Ligase (MtBPL) with Nucleoside-Based Bisubstrate Adenylation Inhibitors

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

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