Date of Award

1-1-2022

Language

English

Document Type

Master's Thesis

Degree Name

Master of Science (MS)

College/School/Department

Department of Chemistry

Content Description

1 online resource (xi, 56 pages) : illustrations (some color)

Dissertation/Thesis Chair

Maksim Royzen

Committee Members

Alexander Shekhtman

Keywords

Bio-orthogonal Chemistry, Click Chemistry, Fluorescent Labelling of RNA, TCO, Tubercidin Analogue, Uridine Analogue, Fluorescent labeling, RNA

Subject Categories

Chemistry

Abstract

This thesis describes a bio-orthogonal chemistry-based approach for new methods of fluorescence labelling of RNA in live HeLa cells via an IEDDA reaction of TCO with tetrazine. The key step of the approach is synthesis of two nucleobase analogues which were modified with TCO and a masked 5′-phosphate group using aryl phosphonamidite chemistry. Synthesis of tubercidin and uridine analogues were considered in this investigation for incorporation into RNA in live HeLa cells. Synthetic approach was successful at installing the handle containing TCO to tubercidin and uridine to make compounds 12 and 21 respectively (the synthesis of both of which are for the first time reported here). However, the last step of synthesis of both analogues, adding the masked phosphate group, was not successful. So, in both cases, synthetic processes need to be further optimized or new pathways need to be examined. Incorporation of a previously synthesized adenosine analogue (compound 23) into ribosomal RNA of live HeLa cells was studied using UHPLC/MS/MS. The results did not show a trend and was not compatible with previous studies and needs further investigation. HeLa cells were treated with 5-Ethynyl uridine (EU) and 5-bromo uridine (BrU), RNAs of the treated cells were extracted, hydrolyzed and prepared for UHPLC/MS/MS studies and stored for future studies.

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