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 (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⁺.

Figure 1

Comparing the photophysical behavior of native NAD⁺ and N^tzAD⁺ in reactions involving alcohol dehydrogenase.

Figure 2

(a) Enzymatic cycle for NAD⁺ consumption and regeneration with ADH and LDH. (b) ADH-mediated oxidation of ethanol to acetaldehyde using N^tzAD⁺ followed by UV and fluorescence spectroscopies (λ_ex = 330 nm). (c) As in b, showing N^tzAD⁺ (red) to N^tzADH (orange) conversion, followed by HPLC (monitored at 330 nm). (d) ADH-mediated oxidation of ethanol to acetaldehyde followed by LDH-mediated reduction of pyruvic acid to lactic acid with N^tzAD⁺ (red/orange) and NAD⁺ (gray) followed by real-time emission at 410 nm (λ_ex = 330 nm) and 465 nm (λ_ex = 335 nm), respectively. Dashed lines represent weighted curve fits.

Figure 3

(a) Enzymatic cycle for N^tzAD⁺ consumption by ADH and NADase. (b) UV–vis and emission (λ_ex = 330 nm) spectra of N^tzAD⁺ at time 0 (red), after oxidizing ethanol to acetaldehyde with ADH (orange) and subsequent treatment with NADase (blue). (c) Real-time emission intensity at 410 nm (λ_ex = 330 nm) of the enzymatic oxidation of ethanol to acetaldehyde by ADH with N^tzAD⁺ (orange, bottom time scale) followed by cleavage with NADase (blue, top time scale). Inset: Cleavage of N^tzAD⁺ with NADase (blue) followed by real-time emission at 410 nm (λ_ex = 330 nm).

Figure 4

(a) Treatment of N^tzAD⁺ with ART5 and CTA to yield ADPR and ADPR-agmatine, respectively. (b) Steady state emission spectra following treatment of N^tzAD⁺ with ART5 at 0 (blue) and 18 min (red), as well as NAD⁺ at 0 (green) and 18 min (orange), λ_ex = 335 nm. Inset: Fluorescence based kinetics of aforementioned reaction (λ_em = 410 nm, λ_ex = 335 nm, blue solid), and normalized HPLC-monitored product formation from reactions with N^tzAD⁺ (blue, dashed) and native NAD (red, dashed). (c) Steady state emission spectra (λ_ex = 335 nm) following treatment of N^tzAD⁺ with CTA; reaction sampled at 0 (blue), 20 (green), 50 (orange), and 90 min (pink). Inset: Reactions with CTA following normalized emission intensity at 410 nm (λ_ex = 335 nm, blue, solid), normalized HPLC-monitored product formation from reactions with N^tzAD⁺ (blue, dashed) and native NAD⁺ (red, dashed).

Scheme 1. Synthesis of N^tzAD

^aReagents and conditions: (a) POCl₃, proton sponge, trimethyl phosphate, 4 °C, 2 h, 50%. (b) i. β-Nicotinamide mononucleotide, CDI, Et₃N, DMF, rt, 6 h; ii. ^tzAMP, DMF, rt, 4 days, 20%.