Modifikované nukleotidy a DNA nesoucí reaktivní skupiny pro bioconjugace a cross-linkování s aminokyselinami

Spampinato, Ambra

This PhD thesis is divided in four parts describing the development and the synthesis of nucleotides and DNA-reactive probes bearing different reactive groups for bio-imaging, bioconjugation and cross-linking with aminoacids and peptides. The first part regarding the design and synthesis of a series of 2'-deoxyribonucleoside triphosphates (dNTPs) and DNA-reactive probes bearing 2- or 4- linked trans-cyclooctene (TCO) or bicyclononyne (BCN) tethered through a shorter propargylcarbamate or longer triethyleneglycol-based spacer for in vitro and in cellulo labeling with various tetrazines by IEDDA reaction. The general strategy was based on the functionalization of the position 5 of pyrimidines via acqueous Sonogashira coupling with propargylamino linker and then addition of reactive building block bearing TCO or BCN by nucleophilic substitution to synthetize modified 2'-deoxycitidine monophosphate (dCMP) and triphosphates (dCTPs). The following step was enzymatic incorporation of such functionalized dCTPs into DNA via primer extension (PEX). The 4- linked TCO monophosphate derivative was used, as model compound, for reaction with 3,6- dipyridyl tetrazine to prove the formation of the conjugate by NMR analysis. In parallel, the reactivity of all functionalized triphosphates with excess of various...

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