A Bioorthogonal Chemical Reporter of Viral Infection

Neef, Anne B.; Pernot, L.; Schreier, Verena N.; Scapozza, Léonardo; Luedtke, Nathan W.

doi:10.1002/anie.201500250

Cited by 43 publications

(34 citation statements)

References 51 publications

(23 reference statements)

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“…This compound was efficiently incorporated into DNA by HeLa cells when the cells were transformed to express a low‐fidelity thymidine kinase‐1 from Herpes simplex virus‐1. In contrast, wild‐type HeLa cells exhibited little or no metabolic incorporation of AmdU into DNA, due to the relatively high fidelity of human thymidine kinases . These results are consistent with previous studies that demonstrated that the initial phosphorylation of certain nucleosides can be the limiting step for enzymatic production of nucleotide triphosphates .…”

Section: Methodssupporting

confidence: 90%

In Vivo Incorporation of Azide Groups into DNA by Using Membrane‐Permeable Nucleotide Triesters

2018

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Metabolic incorporation of bioorthogonal functional groups into cellular nucleic acids can be impeded by insufficient phosphorylation of nucleosides. Previous studies found that 5azidomethyl-2'-deoxyuridine (AmdU) was incorporated into the DNA of HeLa cells expressing a low-fidelity thymidine kinase, but not by wild-type HeLa cells. Here we report that membrane-permeable phosphotriester derivatives of AmdU can exhibit enhanced incorporation into the DNA of wild-type cells and animals. AmdU monophosphate derivatives bearing either 5'-bispivaloyloxymethyl (POM), 5'-bis-(4-acetoxybenzyl) (AB), or "Protide" protective groups were used to mask the phosphate group of AmdU prior to its entry into cells. The POM derivative "POM-AmdU" exhibited better chemical stability, greater metabolic incorporation efficiency, and lower toxicity than "AB-AmdU". Remarkably, the addition of POM-AmdU to the water of zebrafish larvae enabled the biosynthesis of azide-modified DNA throughout the body.

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Section: Methodssupporting

confidence: 90%

In Vivo Incorporation of Azide Groups into DNA by Using Membrane‐Permeable Nucleotide Triesters

2018

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“…Modified nucleosides have thus far been critical toward understanding the rate of RNA synthesis and turnover, [3] characterizing the RNA-binding proteome, [16] and even tracking viral infection. [17] As such, our azide-modified nucleosides are poised to expand the arsenal of methods used to analyze RNA in cells as well as in complex environments such as tissues and living animals.…”

mentioning

confidence: 99%

Metabolic Incorporation of Azide Functionality into Cellular RNA

Nainar¹,

Beasley²,

Fazio³

et al. 2016

ChemBioChem

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Real-time tracking of RNA expression can provide insight into the mechanisms used to generate cellular diversity, as well as help determine the underlying causes of disease. Here we present the exploration of azide-modified nucleoside analogues and their ability to be metabolically incorporated into cellular RNA. We report robust incorporation of adenosine analogues bearing azide handles at both the 2′- and N6-positions; 5-methylazidouridine was not incorporated into cellular RNA. We further demonstrate selectivity of our adenosine analogues for transcription and polyadenylation. We predict that azidonucleosides will find widespread utility in examining RNA functions inside living cells, as well as in more complex systems such as tissues and living animals.

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“…The alkyne group of EdU plays an important role as a chemical handle for reaction with an azide‐functionalized fluorophore using click chemistry, allowing detection of the DNA by high‐content imaging. A small number of nucleoside chemical probes structurally related to EdU have been synthesized and used in varying capacities as chemical probes for DNA (Neef & Luedtke, ; Neef, Pernot, Schreier, Scapozza, & Luedtke, ; Neef, Samain, & Luedtke, ). The only difference between 2ʹ‐fluoro‐arabinosyl EdU (F‐ara‐EdU) and EdU is in the 2ʹ position.…”

Section: Introductionmentioning

confidence: 99%

Synthesis of 5‐Alkynyl Substituted 2ʹ‐Arabinosyl 2ʹ‐Halogenated Uridine Nucleosides

Hilko

Bornaghi

Poulsen

2019

CP Nucleic Acid Chemistry

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This unit describes the detailed preparation of 5‐alkynyl‐2ʹ‐halogenated arabinosyl uridine nucleosides (2ʹ‐halo‐ara‐EdU) from uridine. These compounds were synthesized as prospective chemical probes for the detection of DNA synthesis in proliferating cells. Currently, this is the only synthetic methodology reported to access these compounds. The key to success of the synthetic approach was to employ a 3‐N‐nitro‐protecting group to stabilize the required 2ʹ‐triflate nucleoside precursor toward nucleophilic substitution. Several synthetic challenges were overcome to accommodate the combination of a 5‐alkyne and 3‐N‐nitro functional group, including facile introduction and removal of the N‐nitro group, and removal of the sugar acetyl groups under acidic conditions. © 2019 by John Wiley & Sons, Inc.

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A Bioorthogonal Chemical Reporter of Viral Infection

Cited by 43 publications

References 51 publications

In Vivo Incorporation of Azide Groups into DNA by Using Membrane‐Permeable Nucleotide Triesters

In Vivo Incorporation of Azide Groups into DNA by Using Membrane‐Permeable Nucleotide Triesters

Metabolic Incorporation of Azide Functionality into Cellular RNA

Synthesis of 5‐Alkynyl Substituted 2ʹ‐Arabinosyl 2ʹ‐Halogenated Uridine Nucleosides

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