Primary Photophysics of Nicotinamide Chromophores in Their Oxidized and Reduced Forms.

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 S1 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 S1 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 S1 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* S1 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* S1 state.

Sensitization of Nd3+ Luminescence by Simultaneous Two-Photon Excitation through a Coordinating Polymethinic Antenna.

We have designed and synthesized two new cyaninic Nd3+ complexes where the lanthanide emission can be induced from simultaneous two-photon absorption followed by energy migration. These complexes correspond to a molecular design that uses an antenna ligand formed by the functionalization of a heptamethine dye with 5-ol-phenanthroline or 4-phenyl-terpyridine derivatives. These complexes employ the important nonlinear optical properties of symmetric polymethines to sensitize the lanthanide ion. We verified that simultaneous biphotonic excitation indirectly induces the 4F3/2 → 4I11/2 Nd3+ emission using femtosecond laser pulses tuned below the first electronic transition of the antenna. The simultaneous two-photon excitation events initially form the nonlinear-active second excited singlet of the polymethine antenna, which rapidly evolves into its first excited singlet. This state in turn induces the formation of the emissive Nd3+ states through energy transfer. The role of the first excited singlet of the antenna as the donor state in this process was verified through time resolution of the antenna's fluorescence. These measurements also provided the rates for antenna-lanthanide energy transfer, which indicate that the phenanthroline-type ligand is approximately five times more efficient for energy transfer than the phenyl-terpyridine derivative due to their relative donor-acceptor distances. The simultaneous two-photon excitation of this polymethine antenna allows for high spatial localization of the Nd3+excitation events.

Ultrafast Fluorescence Signals from ß-Dihydronicotinamide Adenine Dinucleotide: Resonant Energy Transfer in the Folded and Unfolded Forms.

ß-Dihydronicotinamide adenine dinucleotide (NADH) plays a critical role in biological redox processes. Inside the cell, NADH can be in a folded or an unfolded conformation, depending on the chemical environment that surrounds it. It is known that selective excitation of adenine in NADH can induce energy transfer events from this nucleotide to the reduced nicotinamide chromophore. From the anticipated time scales, this process must compete with adenine's internal conversion channel, which is known to occur in the sub-picosecond time scale. In this work, we studied the dynamics of the excited states of both chromophores in NADH through the time resolution of the spontaneous emission from both nucleotides. Through these experiments, we extend the knowledge about the competition between the different photophysical channels both in the folded and unfolded states. The study involved the folded and unfolded states of NADH by experiments in water and methanol solutions. Our femtosecond fluorescence results were complemented by the first nuclear magnetic resonance through space magnetization transfer measurements on NADH, which establish the solvent-dependent folded versus unfolded states. We determined the dynamics of the excited states by direct excitation of dihydronicotinamide at 380 nm and adenine at 266 nm. From this, we were able to measure for the folded state, a time constant of 90 fs for energy transfer. Additionally, we determined that even in what is referred to as an unfolded state in methanol, non-negligible excitation transfer events do take place. Spontaneous emission anisotropy measurements were used in order to confirm the presence of a minor energy transfer channel in the methanol solutions where the unfolded state dominates.