Title:
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Novel dinucleotides, precursors to fluorescent cyclic nucleotides
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While the function of NAD+ as energy transducer has been well established, it is
also now known to play an important role in cellular processes such as gene regulation
and cell maintenance. In addition; NAD is a precursor to biologically active metabolites
such as ADPR, ADP_ribosyl-derivatives and cADPR. cADPR is a Ca
2 -modulating second messenger formed by the cyclisation of NAD+ by ADP-ribosyl cyclases. While
three different enzymes have been identified as cyclases, the mechanism and site of
activity of such enzymes has not yet been fully established. The aim of this work is thus
to synthesise base-modified NAD+ analogues that can reach the cyclase active site, and
be enzymatically converted to cyclic products that fluoresce at wavelengths not harmful
to cells. Such analogues may facilitate establishing the location and mechanism of the
cyclase and also the metabolic pathway of cADPR.
Nine novel n~cleosides were synthesised by glycosylation of the selected
heterocycles with a protected ribose using Vorbriiggen conditions. Subsequent
phosphorylation using PM chemistry developed by Yoshikawa afforded the nucleotide
analogues.
Fluorescence studies of the nucleosides were then carried out to provide
preliminary information as to whether any of the analogues would fluoresce, and at
which excitation and emission wavelengths the fluorescence would occur.
This synthetic route was designed to introduce the relatively unstable
nicotinamide moiety at the final step. However, coupling to nicotinamide
mononucleotide to form the cyclic precursors proved unfruitful, despite subjection to
numerous different reaction conditions.
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