The synthesis, photophysics, and biochemical utility of a fluorescent NAD⁺ analogue based on an isothiazolo[4,3-d]pyrimidine core (N^tzAD⁺) are described. Enzymatic reactions, photophysically monitored in real time, show N^tzAD⁺ and N^tzADH to be substrates for yeast alcohol dehydrogenase and lactate dehydrogenase, respectively, with reaction rates comparable to that of the native cofactors. A drop in fluorescence is seen as N^tzAD⁺ is converted to N^tzADH, reflecting a complementary photophysical behavior to that of the native NAD⁺/NADH. N^tzAD⁺ and N^tzADH serve as substrates for NADase, which selectively cleaves the nicotinamide's glycosidic bond yielding ^tzADP-ribose. N^tzAD⁺ also serves as a substrate for ribosyl transferases, including human adenosine ribosyl transferase 5 (ART5) and Cholera toxin subunit A (CTA), which hydrolyze the nicotinamide and transfer ^tzADP-ribose to an arginine analogue, respectively. These reactions can be monitored by fluorescence spectroscopy, in stark contrast to the corresponding processes with the nonemissive NAD⁺.

A series of emissive ribonucleoside purine mimics, all comprised of an isothiazolo[4,3-d]pyrimidine core, was prepared using a divergent pathway involving a key Thorpe-Ziegler cyclization. In addition to an adenosine and a guanosine mimic, analogues of the noncanonical xanthosine, isoguanosine, and 2-aminoadenosine were also synthesized and found to be emissive. Isothiazolo 2-aminoadenosine, an adenosine surrogate, was found to be particularly emissive and effectively deaminated by adenosine deaminase. Competitive studies with adenosine deaminase with each analogue in combination with native adenosine showed preference for the native substrate while still deaminating the isothiazolo analogues.

An evolved fluorescent ribonucleoside alphabet comprising isomorphic purine ((tz)A, (tz)G) and pyrimidine ((tz)U, (tz)C) analogues, all derived from isothiazolo[4,3-d]pyrimidine as a common heterocyclic core, is described. Structural and biochemical analyses illustrate that the nucleosides, particularly the C-nucleosidic purine analogues, are faithful isomorphic and isofunctional surrogates of their natural counterparts and show improved features when compared to an RNA alphabet derived from thieno[3,4-d]-pyrimidine. The restoration of the nitrogen in a position equivalent to the purines' N7 leads to "isofunctional" behavior, as illustrated by the ability of adenosine deaminase to deaminate (tz)A as effectively as adenosine, the native substrate.

The enzymatic conversion of isothiazolo[4,3-d]pyrimidine-based adenosine (tz A) and 2-aminoadenosine (tz 2-AA) analogues to the corresponding isothiazolo[4,3-d]pyrimidine-based inosine (tz I) and guanosine (tz G) derivatives is evaluated and compared to the conversion of native adenosine to inosine. Henri-Michaelis-Menten analyses provides the foundation for a high-throughput screening assay, and the efficacy of the assay is showcased by fluorescence-based analysis of tz A conversion to tz I in the presence of known and newly synthesized inhibitors.

An enzyme-mediated approach for the assembly of singly modified RNA constructs in which specific G residues are replaced with ^th G, an emissive isomorphic G surrogate, is reported. Transcription in the presence of ^th G and native nucleoside triphosphates enforces initiation with the unnatural analogue, yielding 5'-end modified transcripts that can be mono-phosphorylated and ligated to provide longer site-specifically modified RNA constructs. The scope of this unprecedented enzymatic approach to non-canonical purine-containing RNAs is explored via the assembly of several altered hammerhead (HH) ribozymes and a singly modified HH substrate. By strategically modifying key positions, a mechanistic insight into the ribozyme-mediated cleavage is gained. Additionally, the emissive features of the modified nucleoside and its responsiveness to environmental changes can be used to monitor cleavage in real time by steady state fluorescence spectroscopy.

A series of enzymatic transformations, which generate visibly emissive isofunctional cofactors based on an isothiazolo[4,3-d]pyrimidine analogue of adenosine (tz A), was developed. Nicotinamide adenylyl transferase condenses nicotinamide mononucleotide and tz ATP to yield Ntz AD+ , which can be enzymatically phosphorylated by NAD+ kinase and ATP or tz ATP to the corresponding Ntz ADP+ . The latter can be engaged in NADP-specific coupled enzymatic transformations involving conversion to Ntz ADPH by glucose-6-phosphate dehydrogenase and reoxidation to Ntz ADP+ by glutathione reductase. The Ntz ADP+ /Ntz ADPH cycle can be monitored in real time by fluorescence spectroscopy.

Guanine deaminase (GDA) deaminates guanine to xanthine. Despite its significance, the study of human GDA remains limited compared to other metabolic deaminases. As a result, its substrate and inhibitor repertoire are limited, and effective real-time activity, inhibitory, and discovery assays are missing. Herein, we explore two emissive heterocyclic cores, based on thieno[3,4-d]pyrimidine (^thN) and isothiazole[4,3-d]pyrimidine (^tzN), as surrogate GDA substrates. We demonstrate that, unlike the thieno analog, ^thG_N, the isothiazolo guanine surrogate, ^tzG_N, does undergo effective enzymatic deamination by GDA and yields the spectroscopically distinct xanthine analog, ^tzX_N. Further, we showcase the potential of this fluorescent nucleobase surrogate to provide a visible spectral window for a real-time study of GDA and its inhibition.

Bacterial cyclic dinucleotides (CDNs) play important roles in regulating biofilm formation, motility and virulence. In eukaryotic cells, theses bacterial CDNs are recognized as pathogen-associated molecular patterns (PAMPs) and trigger an innate immune response. We report the photophysical analyses of a novel group of enzymatically synthesized emissive CDN analogues comprised of two families of isomorphic ribonucleotides. The highly favorable photophysical features of the CDN analogues, when compared to their non-emissive natural counterparts, are used to monitor in real time the dinucleotide cyclase-mediated synthesis and phosphodiesterase (PDE)-mediated hydrolysis of homodimeric and mixed CDNs, providing effective means to probe the activities of two classes of bacterial enzymes and insight into their biomolecular recognition and catalytic features.

Adenosine to inosine RNA editing catalyzed by ADAR enzymes is common in humans, and altered editing is associated with disease. Experiments using substrate RNAs with adenosine analogues at editing sites are useful for defining features of the ADAR reaction mechanism. The reactivity of ADAR2 was evaluated with RNA containing the emissive adenosine analogue thieno[3,4-d]-6-aminopyrimidine ((th)A). This nucleoside was incorporated into a mimic of the glutamate receptor B (GluR B) mRNA R/G editing site. We found that (th)A is recognized by AMV reverse transcriptase as A, and is deaminated rapidly by human ADAR2 to give (th)I. Importantly, ADAR reaction progress can be monitored by following the deamination-induced change in fluorescence of the (th)A-modified RNA. The observed high (th)A reactivity adds to our understanding of the structural features that are necessary for an efficient hADAR2 reaction. Furthermore, the new fluorescent assay is expected to accelerate mechanistic studies of ADARs.

Cyclic dinucleotides (CDNs) trigger the innate immune response in eukaryotic cells through the stimulator of interferon genes (STING) signaling pathway. To decipher this complex cellular process, a better correlation between structure and downstream function is required. Herein, we report the design and immunostimulatory effect of a novel group of c-di-GMP analogues. By employing an "atomic mutagenesis" strategy, changing one atom at a time, a class of gradually modified CDNs was prepared. These c-di-GMP analogues induce type-I interferon (IFN) production, with some being more potent than c-di-GMP, their native archetype. This study demonstrates that CDN analogues bearing modified nucleobases are able to tune the innate immune response in eukaryotic cells.