Synthesis of 4-thiouridines with prodrug functionalization for RNA metabolic labeling

Moreno, Sarah; Brunner, Melanie; Delazer, Isabel; Rieder, Dietmar; Lusser, Alexandra; Micura, Ronald

doi:10.1039/d2cb00001f

Cited by 4 publications

(3 citation statements)

References 76 publications

(108 reference statements)

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“…This functionalization was achieved using mild phosphor( III )amidite chemistry catalyzed by 5-(benzylthio)-1 H -tetrazole. The required reagents 4-[({[bis(propan-2-yl)amino](9 H -fluoren-9-ylmethoxy)phosphanyl}oxy)methyl]phenyl acetate I , and bis-( S -pivaloyl-2-thioethyl)- N , N -diisopropylaminophosphoramidite II , , respectively, were synthesized as described earlier . Selective oxidation of the phosphite P(III) triester intermediates to the phosphate triester P(V) compounds 3a and 3b was accomplished using tert -butyl hydroperoxide at low temperature and short reaction times, immediately followed by 1,4-dithiothreitol (DTT) treatment to reduce S–S dimers and eliminate the slight excess of oxidizing reagent.…”

Section: Resultsmentioning

confidence: 99%

“…Prodrug 5 was obtained by a fast reaction sequence starting from precursor 2 , applying the reagent bis-(4-acetyloxybenzyl)- N , N -diisopropylaminophosphoramidite III , ,− followed by the oxidation procedure described above (Scheme ). Filtration on silica gel immediately followed by deprotection of the 2′- O and 3′- O TBS groups gave prodrug 5 in high purity and sufficient yields.…”

Section: Resultsmentioning

confidence: 99%

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6-Thioguanosine Monophosphate Prodrugs Display Enhanced Performance against Thiopurine-Resistant Leukemia and Breast Cancer Cells

et al. 2022

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Thiopurines are in widespread clinical use for the treatment of immunological disorders and certain cancers. However, treatment failure due to resistance or adverse drug reactions are common, asking for new therapeutic strategies. We investigated the potential of 6-thioguanosine monophosphate (6sGMP) prodrugs to overcome resistance to 6-thioguanine. We successfully developed synthetic routes toward diverse 6sGMP prodrugs, tested their proliferation inhibitory potential in different cell lines, and examined their mode of action. Our results show that 4-acetyloxybenzyl- and cycloSaligenyl-derivatized 6sGMP prodrugs are effective antiproliferative compounds in cells that are resistant to thiopurines. We find that resistance is related to the expression of salvage pathway enzyme HGPRT. Using TUC-seq DUAL, we demonstrate the intracellular conversion of 6sGMP prodrugs into bioactive 6sGTPs. Thus, our study offers a promising strategy for thiopurine therapy by using 6sGMP prodrugs, and it suggests TUC-seq DUAL as a simple and fast method to measure the success of thiopurine therapy.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

6-Thioguanosine Monophosphate Prodrugs Display Enhanced Performance against Thiopurine-Resistant Leukemia and Breast Cancer Cells

et al. 2022

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“…for biotinylation and RNA pull-down, covalent attachment to fluorophores, or photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP). [28][29][30][31] Moreover, metabolic labeling-based RNA sequencing methods (e.g. Thiouridine-to-Cytidine Conversion Sequencing, TUC-seq) have emerged which became indispensable tools for analysing cellular RNA dynamics.…”

Section: Papermentioning

confidence: 99%

Robust synthesis of 2′-azido modified RNA from 2′-amino precursors by diazotransfer reaction

et al. 2022

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Time-resolved single-cell RNA-seq using metabolic RNA labelling

Erhard

Lusser

Toussaint

et al. 2022

Nat Rev Methods Primers

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Single-cell RNA sequencing offers snapshots of whole transcriptomes but obscures the temporal RNA dynamics. Here we present single-cell metabolically labeled new RNA tagging sequencing (scNT-seq), a method for massively parallel analysis of newly transcribed and pre-existing mRNAs from the same cell. This droplet microfluidics-based method enables high-throughput chemical conversion on barcoded beads, efficiently marking newly transcribed mRNAs with T-to-C substitutions. Using scNT-seq, we jointly profiled new and old transcriptomes in 55,000 single cells. These data revealed time-resolved transcription factor activities and cell-state trajectories at the single-cell level in response to neuronal activation. We further determined rates of RNA biogenesis and decay to uncover RNA regulatory strategies during stepwise conversion between pluripotent and rare totipotent two-cell embryo (2C)-like stem cell states. Finally, integrating scNT-seq with genetic perturbation identifies DNA methylcytosine dioxygenase as an epigenetic barrier into the 2C-like cell state. Time-resolved single-cell transcriptomic analysis thus opens new lines of inquiry regarding cell-type-specific RNA regulatory mechanisms.

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Synthesis of 4-thiouridines with prodrug functionalization for RNA metabolic labeling

Abstract: Synthetic access to 4-thiouridine (4sU) derivatives with monophosphate prodrug patterns creates additional possibilities for metabolic labeling of RNA for different applications.

Cited by 4 publications

References 76 publications

6-Thioguanosine Monophosphate Prodrugs Display Enhanced Performance against Thiopurine-Resistant Leukemia and Breast Cancer Cells

6-Thioguanosine Monophosphate Prodrugs Display Enhanced Performance against Thiopurine-Resistant Leukemia and Breast Cancer Cells

Robust synthesis of 2′-azido modified RNA from 2′-amino precursors by diazotransfer reaction

Time-resolved single-cell RNA-seq using metabolic RNA labelling

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