The synthesis and characterization of the extended thieno[3,4-b]pyrazine analogues acenaphtho[1,2-b]thieno[3,4-e]pyrazine (3a), 3,4-dibromoacenaphtho[1,2-b]thieno[3,4-e]pyrazine (3b), 3-octylacenaphtho[1,2-b]thieno[3,4-e]pyrazine (3c), dibenzo[f,h]thieno[3,4-b]quinoxaline (4), and thieno[3',4':5,6]pyrazino[2,3-f][1,10]phenanthroline (5) are reported. Comparison of structural, electrochemical, and photophysical properties to those of simple thieno[3,4-b]pyrazines are provided in order to provide structure-function relationships within this series of compounds.

Aldehyde dehydrogenase 2 (ALDH2) catalyzes oxidation of toxic aldehydes to carboxylic acids. Physiologic levels of Mg2+ ions influence ALDH2 activity in part by increasing NADH binding affinity. Traditional fluorescence measurements monitor the blue shift of the NADH fluorescence spectrum to study ALDH2-NADH interactions. By using time-resolved fluorescence spectroscopy, we have resolved the fluorescent lifetimes (τ) of free NADH (τ = 0.4 ns) and bound NADH (τ = 6.0 ns). We used this technique to investigate the effects of Mg2+ on the ALDH2-NADH binding characteristics and enzyme catalysis. From the resolved free and bound NADH fluorescence signatures, the KD for NADH with ALDH2 ranged from 468 µM to 12 µM for Mg2+ ion concentrations of 20 µM to 6000 µM, respectively. The rate constant for dissociation of the enzyme-NADH complex ranged from 0.4 s−1 (6000 µM Mg2+) to 8.3 s−1 (0 µM Mg2+) as determined by addition of excess NAD+ to prevent re-association of NADH and resolving the real-time NADH fluorescence signal. The apparent NADH association/re-association rate constants were approximately 0.04 µM−1s−1 over the entire Mg2+ ion concentration range and demonstrate that Mg 2+ ions slow the release of NADH from the enzyme rather than promoting its re-association. We applied NADH fluorescence lifetime analysis to the study of NADH binding during enzyme catalysis. Our fluorescence lifetime analysis confirmed complex behavior of the enzyme activity as a function of Mg2+ concentration. Importantly, we observed no pre-steady state burst of NADH formation. Furthermore, we observed distinct fluorescence signatures from multiple ALDH2-NADH complexes corresponding to free NADH, enzyme-bound NADH, and, potentially, an abortive NADH-enzyme-propanal complex (τ = 11.2 ns).

Aldehyde dehydrogenase 1 (ALDH1A1) catalyzes the oxidation of toxic aldehydes to carboxylic acids. Physiologic levels of Mg2+ ions decrease ALDH1 activity in part by increasing NADH binding affinity to the enzyme. By using time-resolved fluorescence spectroscopy, we have resolved the fluorescent lifetimes (τ) of free NADH in solution (τ = 0.4 ns) and two enzymebound NADH states (τ = 2.0 ns and τ = 7.7 ns). We used this technique to investigate the effects of Mg2+ ions on the ALDH1A1-NADH binding characteristics and enzyme catalysis. From the resolved free and bound NADH fluorescence signatures, the KD values for both NADH conformations in ALDH1A1 ranged from about 24 µM to 1 µM for Mg2+ ion concentrations of 0 µM to 6000 µM, respectively. The rate constants for dissociation of the enzyme-NADH complex ranged from 0.03 s−1 (6000 µM Mg2+) to 0.30 s−1 (0 µM Mg2+) as determined by addition of excess NAD+ to prevent re-association of NADH and resolving the real-time NADH fluorescence signal. During the initial reaction of enzyme with NAD+ and butyraldehyde, there was an immediate rise in the NADH fluorescence, due to the formation of bound NADH complexes, with a constant steady-state rate of production of free NADH. As the Mg2+ ion concentration was increased, there was a consistent decrease of the enzyme catalytic turnover from 0.31 s−1 (0 µM Mg2+) to 0.050 s−1 (6000 µM Mg2+) and a distinct shift in steady-state conformational population from one that favors the ALDH1-NADH complex with the shorter fluorescence lifetime (33% excess) in the absence of magnesium ion to one that favors the ALDH1-NADH complex with the longer fluorescence lifetime (13% excess) at 6000 µM Mg2+. This shift in conformational population at higher Mg2+ ion concentrations and to lower enzyme activity may be due to longer residence time of the NADH in the ALDH1 pocket. The results from monitoring enzyme catalysis in the absence of magnesium suggests that the ALDH1-NADH complex with the shorter fluorescence lifetime is the form initially produced, and the complex with the longer fluorescence lifetime is produced through isomerization.

Structural Effects on the Electronic Properties of Extended Fused-Ring Thieno[3,4-b]pyrazine Analogues. -Based on electrochemical and spectroscopic studies, clear relationships between chemical structures of fused thienopyrazines and their electronic properties are found. -(NIETFELD, J. P.; SCHWIDERSKI, R. L.; GONNELLA, T. P.; RASMUSSEN*, S. C.; J. Org. Chem. 76 (2011) 15, 6383-6388, http://dx.

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