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Development and implementation of isomorphic and isofunctional fluorescent nucleosides

  • Author(s): Rovira, Alexander Raymond
  • Advisor(s): Tor, Yitzhak
  • et al.

Given that the molecules composing RNA and DNA are naturally nonemissive, we make use of fluorescent nucleoside analogues engaged in the same biological processes as their native counterparts to probe the mechanisms involved in such biological processes. I developed a synthesis to create a family of isothiazolo-pyrimidine nucleoside analogues. A necessity for these analogues was proposed out of the loss of key hydrogen-bond acceptor, nitrogen-7, found in the native purine scaffolds, missing in a previously synthesized family of fluorescent nucleoside analogues composed of a thiopheno-pyrimidine framework. Ultimately I pursued a route involving a Thorpe-Ziegler reaction to construct the isothiazole ring on an alkylated ribofuranose unit and synthesized each canonical RNA purine-based nucleoside analogue, adenosine (tzA) and guanosine (tzG). Cytosine (tzC), and uridine (tzU) analogues were synthesized as well using traditional nucleoside synthetic methods. Each final product was extensively studied to determine the photophysical properties, and found to be emissive as well as responsive to changes in pH and polarity. I compared the activity of our analogues in enzymatic systems through the use of adenosine deaminase (ADA), an enzyme found in many purine metabolic pathways. The deamination of our fluorescent analogue, tzA, can be followed by both emission and absorption. When subjecting tzA to adenosine deaminase, I found effective deamination to isothiazolo-inosine (tzI) at the same rate as the native adenosine, and significantly faster than our previously synthesized thiophene-based adenosine analogue. The initial publication of these fluorescent analogues has given way to several ongoing collaborations and is finding utility in the fluorescent nucleoside community.

The set of aforementioned synthesized RNA analogues was expanded to include several “non-canonical” RNA nucleosides such as xanthosine (tzX), isoguanosine (tzisoG), and diaminopurine (tz2-AA). Alongside these newly introduced nucleosides, my endeavors to synthesize this set of molecules with a unique heterocyclic scaffold were discussed in great detail, as non-traditional synthetic methods were required to synthesize each nucleoside. We conducted extensive studies on the photophysical properties of each molecule, identifying pKa values in both the ground and excited states, and identifying tautomeric species existing in the excited state of several molecules. I further explored the adenosine deaminase assay previously developed in our lab by looking at the effects of several of my synthesized molecules as direct inhibitors of this enzymatic process. These studies gave interesting results as the synthesized nucleoside analogues seemed to react with adenosine deaminase faster than their native counterparts.

To further exemplify the power and utility of these fluorescent probes, we sought to develop fluorescent nucleoside-containing secondary metabolites in an effort to better understand relevant biological processes. Nicotinamide adenine dinucleotide (NAD+) is a coenzyme found in all living cells. In addition to its metabolic roles, it also serves as a substrate to enzymes involved in posttranslational modifications of proteins (e.g., ribosylation), thus rendering these enzymes important targets for drug discovery. An NAD+ analogue, which contains a fluorescent adenosine surrogate, has the potential to find broad utility in sensitive fluorescent assays. I incorporated our fluorescent adenosine analogue, tzA, into a fully functional NAD+ surrogate through chemical synthesis. This molecule, named NtzAD+, was found to be emissive and function exactly as the naturally occurring NAD+ in several systems that I employed, including alcohol dehydrogenase, an enzyme which oxidizes ethanol to acetaldehyde through the conversion of NAD+ to NADH, followed by treatment with lactate dehydrogenase, an enzyme which reduces pyruvic acid to lactic acid through the conversion of NADH to NAD+, and arginine-specific mono ADP ribosyl transferases, enzymes that play a crucial role in post-translational modifications. To our knowledge, this is the first fluorescent NAD+ analogue known to exhibit changes between oxidized and reduced forms, lending itself to have great potential for biophysical assays.

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