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

Abstract

Apart from its vital function as a redox cofactor, nicotinamide adenine dinucleotide (NAD+) has emerged as a crucial substrate for NAD+‐consuming enzymes, including poly(ADP‐ribosyl)transferase 1 (PARP1) and CD38/CD157. Their association with severe diseases, such as cancer, Alzheimer's disease, and depressions, necessitates the development of new analytical tools based on traceable NAD+ surrogates. Here, the synthesis, photophysics and biochemical utilization of an emissive, thieno[3,4‐d]pyrimidine‐based NAD+ surrogate, termed NthAD+, are described. Its preparation was accomplished by enzymatic conversion of synthetic thATP by nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1). The new NAD+ analogue possesses useful photophysical features including redshifted absorption and emission maxima as well as a relatively high quantum yield. Serving as a versatile substrate, NthAD+ was reduced by alcohol dehydrogenase (ADH) to NthADH and afforded thADP‐ribose (thADPr) upon hydrolysis by NAD+‐nucleosidase (NADase). Furthermore, NthAD+ was engaged in cholera toxin A (CTA)‐catalyzed mono(thADP‐ribosyl)ation, but was found incapable in promoting PARP1‐mediated poly(thADP‐ribosyl)ation. Due to its high photophysical responsiveness, NthAD+ is suited for spectroscopic real‐time monitoring. Intriguingly, and as an N7‐lacking NAD+ surrogate, the thieno‐based cofactor showed reduced compatibility (i.e., functional similarity compared to native NAD+) relative to its isothiazolo‐based analogue. The distinct tolerance, displayed by diverse NAD+ producing and consuming enzymes, suggests unique biological recognition features and dependency on the purine N7 moiety, which is found to be of importance, if not essential, for PARP1‐mediated reactions.

Functionalised Cofactor Mimics for Interactome Discovery and Beyond

“…Various biotin‐, [82] alkyne‐, [78] azide‐ [84] and photoaffinity‐functionalised probes [86, 87] . Various fluorophore probes [83, 100, 104–108] …”

“…[100] However, fluorophores can also be nested in the structure of the cofactor rather than appended via a linker. Classic examples of this include the numerous fluorescent adenosine analogues developed and applied to generate fluorescent mimics of ATP, [93,101] SAM, [102,103] and NAD [104][105][106][107][108] (4.2-4.4; reviewed elsewhere [81] ). The most successful analogues maintain similar size and H-bonding pattern to adenosine to minimise functional perturbation and thereby enable the real-time monitoring of the respective cofactor-mediated transformations.…”

Fundamental photophysics of isomorphic and expanded fluorescent nucleoside analogues

“…Therefore, the N-tz ADP + /N-tz ADP cycle could be followed in real time by fluorescence spectroscopy. Finally, N-th AD + has been compared to N-tz AD + as emissive cofactors in a number of enzymatic reactions that play central roles in the cell 206 . N-th AD + was found to be substantially more responsive than N-tz AD + and thus appeared as a more suited probe for real-time applications.…”

Funktionalisierte Cofaktor‐Analoga für die Erforschung von Interaktomen und darüber hinaus

“…[86,87] Verschiedene Fluorophor-Sonden. [83,100,[104][105][106][107][108] NCB10 (Herz-Kreislauf-Erkrankungen, S-Adenosyl-L-homocysteinhydrolase) [141] Pyridoxalphosphat (PLP) Alkin-, Azid-und elektrophile Sonden, [21,25,35] fluoreszierende Chemosensor-Analoga [111][112][113] S-Adenosyl-L-methionin (SAM)Verschiedene Alkin-, [75] Azid- [72] und Photoaffinitäts-funktionalisierte Sonden [76] . Verschiedene Fluorophor-Sonden.…”