Primary Photophysics of Nicotinamide Chromophores in Their Oxidized and Reduced Forms

Abstract

Nicotinamide adenine dinucleotide (NADH) is an important enzyme cofactor with emissive properties that allow it to be used in fluorescence microscopies to study cell metabolism. Its oxidized form NAD + , on the other hand, is considered to produce negligible fluorescence. In this contribution, we describe the photophysics of the isolated nicotinamidic system in both its reduced and oxidized states. This was achieved through the study of model molecules that do not carry the adenine nucleotide since its absorbance would overlap with the absorption spectrum of the nicotinamidic chromophores. We studied three model molecules: nicotinamide (niacinamide, an oxidized form without nitrogen substitution), the oxidized chromophore 1-benzyl-3-carbamoyl-pyridinium bromide (NBzOx), and its reduced form 1-benzyl-1,4-dihydronicotinamide (NBz). For a full understanding of the dynamics, we performed both femtosecond-resolved emission and transient absorption experiments. The oxidized systems, nicotinamide and NBzOx, have similar photophysics, where the originally excited bright state decays on an ultrafast timescale of less than 400 fs. The depopulation of this state is followed by excited-state positive absorption signals, which evolve in two timescales: the first one is from 1 to a few picoseconds and is followed by a second decaying component of 480 ps for nicotinamide in water and of 80−90 ps for nicotinamide in methanol and NBzOx in aqueous solution. The long decay times are assigned as the S 1 lifetimes populated from the original higher-lying bright singlet, where this state is nonemissive but can be detected by transient absorption. While for NBzOx in aqueous solution and for nicotinamide in methanol, the S 1 signal decays to the solvent-only level, for the aqueous solutions of nicotinamide, a small transient absorption signal remains after the 480 ps decay. This residual signal was assigned to a small population of triplet states formed during the slower S 1 decay for nicotinamide in water. The experimental results were complemented by XMS-CASPT2 calculations, which reveal that in the oxidized forms, the rapid evolution of the initial π−π* state is due to a direct crossing with lower-energy dark n−π* singlet states. This coincides with the experimental observation of long-lived nonemissive states (80 to 480 ps depending on the system). On the other hand, the reduced model compound NBz has a long-lived emissive π−π* S 1 state, which decays with a 510 ps time constant, similarly to the parent compound NADH. This is consistent with the XMS-CASPT2 calculations, which show that for the reduced chromophore, the dark states lie at higher energies than the bright π−π* S 1 state.

Sub-100-fs energy transfer in coenzyme NADH is a coherent process assisted by a charge-transfer state

“…The high conformational flexibility afforded by the phosphate bridge linking the two monomers leads to DNA-like stacked (folded) or unstacked (unfolded) conformers whose ratio depends on solvent polarity, pH, and temperature 29 – 33 . While in alcoholic environments, like propylene glycol or methanol, there is a high propensity of unstacked conformations, a considerable percentage of stacked conformers is observed in water 34 – 37 .…”