Emissive Synthetic Cofactors: An Isomorphic, Isofunctional, and Responsive NAD⁺ Analogue

Abstract

The synthesis, photophysics, and biochemical utility of a fluorescent NAD+ analogue based on an isothiazolo[4,3-d]pyrimidine core (NtzAD+) are described. Enzymatic reactions, photophysically monitored in real time, show NtzAD+ and NtzADH 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 NtzAD+ is converted to NtzADH, reflecting a complementary photo-physical behavior to that of the native NAD+/NADH. NtzAD+ and NtzADH serve as substrates for NADase, which selectively cleaves the nicotinamide’s glycosidic bond yielding tzADP-ribose. NtzAD+ 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+.

Emissive Synthetic Cofactors: A Highly Responsive NAD+Analogue Reveals Biomolecular Recognition Features

“…Although the thieno[3,4‐ d ]pyrimidine family of nucleosides displays outstanding emission features, the isothiazolo[4,3 ‐d ]pyrimidine‐based analogues, albeit somewhat less emissive, provide enhanced isomorphicity and isofunctionality . Having access to both purine analogues, offers, however, a unique opportunity to assess biomolecular recognition features using sensitive fluorescence‐based tools.…”

Emissive Synthetic Cofactors: Enzymatic Interconversions of tzA Analogues of ATP, NAD+, NADH, NADP+, and NADPH

“…[13] Since the native NAD + , and NADP + are nonemissive, developing and implementing isomorphic and isofunctional fluorescent surrogates will allow one to monitor their biochemical transformation in real time using common fluorescence spectrometers. [13b] As the synthesis of such cofactors is frequently challenging, their adoption by the community tends to lag behind. We have therefore sought to advance enzymatic pathways to their preparation, which could be employed in any laboratory using available enzymes [14] and monitored by conventional steady-state fluorescence measurements.…”

“…[13b] Thus, over 3 hours, an increase in absorbance at 333 nm and a concomitant decrease of the emission intensity at 410 nm (λ ex : 330 nm) are observed (Figure 2b). The overall reaction half-time, derived from the variation of emission spectra over time (Figure 2c), are 3.3 and 3.6 × 10 2 s for the native and the tz A -based analogue nucleosides, respectively, assuming pseudo-first-order kinetics.…”

“…Conveniently, this N tz ADP + / N tz ADPH couple showed a photophysical behavior complementary to the photophysical changes seen for the native NADP + and NADPH. [13b] …”

Emissive Synthetic Cofactors: Enzymatic Interconversions of tzA Analogues of ATP, NAD+, NADH, NADP+, and NADPH

“…[9][10][11] While the incorporation of as ingle modified nucleoside into an oligonucleotide could be structurally and functionally benign, as detrimental effects might be masked, the adequate performance of nucleosides and nucleotides as cofactors and secondary messengers represents ad emanding test for their isomorphism and isofunctionality. [13] Since the native NAD + ,a nd NADP + are nonemissive,d eveloping and implementing isomorphic and isofunctional fluorescent surrogates will allow one to monitor their biochemical transformation in real time using common fluorescence spectrometers. [13] Since the native NAD + ,a nd NADP + are nonemissive,d eveloping and implementing isomorphic and isofunctional fluorescent surrogates will allow one to monitor their biochemical transformation in real time using common fluorescence spectrometers.…”