Design, Synthesis, and Application of a Fluorescent Ribonucleoside Alphabet
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Design, Synthesis, and Application of a Fluorescent Ribonucleoside Alphabet


A new fluorescent ribonucleoside alphabet consisting of pyrimidines and purines, all derived from methylthieno[3,4-d]pyrimidine as the heterocyclic core, is described. Photophysical analyses reveal surprisingly large bathochromic shifts relative to previous alphabets derived from thieno[3,4-d]pyrimidine and isothiazole[4,3-d]pyrimidine. Biochemical analyses with adenosine deaminase, T7 RNA Polymerase, and guanine deaminase indicate the varying degrees of tolerance for a methyl moiety on the Hoogsteen face by different enzymes. These results indicate that the analogues can act as useful probes in the same capacity as previous alphabets and provide insight into enzyme tolerance to structural perturbations on the Hoogsteen face. Separately, the enzymatic conversion of a visibly emitting adenosine analogue, tzA, to the corresponding fluorescent inosine analogue, tzI, by adenosine deaminase (ADA) is monitored via fluorescence intensity changes. The Cohen Lab at UC San Diego has a library of over 300 compounds comprised of metal-binding pharmacophores which they have tested as novel inhibitors of a variety of metalloenzymes. The new method of monitoring ADA activity is utilized in collaboration with the Cohen Lab to screen their library of metal binding pharmacophores (MBPs) in a high throughput format as inhibitors of ADA. Several inhibitors are discovered and derivatives of the hit compounds are tested against ADA to develop a structure activity relationship. Finally, a new compound is designed and synthesized based on the findings of the structure activity relationship and found to be the most potent derivative of the original hit compound. With the new methylthieno[3,4-d]pyrimidine based alphabet in hand, the tolerance of Cytidine Deaminase (CDA) to expanded heterocycles is explored. The molecules used are mthC, thC, and tzC, three fluorescent cytidine analogues where the pyrimidine core is fused to three distinct five-membered heterocycles at the 5/6 positions. The reaction between each analogue and CDA is monitored by absorption and emission spectroscopy, revealing shorter reaction times for all three analogues than the native substrate. Pseudo-first order and Michaelis-Menten kinetic analyses provide further insight into the enzymatic deamination reactions and assist in drawing comparison to established structure activity relationships. Finally, each analogue is used to analyze two known inhibitors of CDA, zebularine and tetrahydrouridine, as a proof of concept for potential screening assays. Continuing on the success of the CDA assays, human adenosine deaminase 1 (ADA1) and 2 (ADA2) are studied. The newly synthesized adenosine analogue, mthA, as well as the two previously reported analogues thA and tzA are used to monitor ADA1 and ADA2 activity and probe their tolerance for steric hindrance on the Hoogsteen face. ADA2 is found to tolerate the missing nitrogen in thA and the perturbing methyl in mthA on the Hoogsteen face far more than ADA1. Further it is found to react faster with tzA and thA than adenosine, the native substrate. An assay is then developed in which both ADA1 and ADA2 are present, but one is “turned off” by excess selective inhibitor so that the other enzymes activity can be monitored using mthA, thA, or tzA.

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