Inverse electron demand Diels-Alder reactions between s-tetrazines and strained dienophiles have numerous applications in fluorescent labeling of biomolecules.H erein, we investigate the effect of the dienophile on the fluorescence enhancement obtained upon reaction with at etrazinequenched fluorophore and study the possible mechanisms of fluorescence quenching by both the tetrazine and its reaction products.T he dihydropyridazine obtained from reaction with astrained cyclooctene shows aresidual fluorescence quenching effect, greater than that exerted by the pyridazine arising from reaction with the analogous alkyne.L inear and ultrabroadband two-dimensional electronic spectroscopye xperiments reveal that resonance energy transfer is the mechanism responsible for the fluorescence quenching effect of tetrazines, whereas am echanism involving more intimate electronic coupling,l ikely photoinduced electron transfer,i sr esponsible for the quenchinge ffect of the dihydropyridazine.T hese studies uncover parameters that can be tuned to maximize fluorogenic efficiency in bioconjugation reactions and reveal that strained alkynes are better reaction partners for achieving maximum contrast ratio.

Fluorescent indicators based on β-keto-acid bidentate coordination motifs display superior metal selectivity profiles compared to current o-aminophenol-N,N,O-triacetic acid (APTRA) based chelators for the study of biological magnesium. These low denticity chelators, however, may allow for the formation of ternary complexes with Mg2+ and common ligands present in the cellular milieu. In this work, absorption, fluorescence, and NMR spectroscopy were employed to study the interaction of turn-on and ratiometric fluorescent indicators based on 4-oxo-4H-quinolizine-3-carboxylic acid with Mg2+ and ATP, the most abundant chelator of biological magnesium, thus revealing the formation of ternary complexes under conditions relevant to fluorescence imaging. The formation of ternary species elicits comparable or greater optical changes than those attributed to the formation of binary complexes alone. Dissociation of the fluorescent indicators from both ternary and binary species have apparent equilibrium constants in the low millimolar range at pH 7 and 25 °C. These results suggest that these bidentate sensors are incapable of distinguishing between free Mg2+ and MgATP based on ratio or intensity-based steady-state fluorescence measurements, thus posing challenges in the interpretation of results from fluorescence imaging of magnesium in nucleotide-rich biological samples.

The design and optimization of fluorescent labels and fluorogenic probes rely heavily on their ability to distinguish among multiple competing fluorescence quenching mechanisms. Cresyl violet, a member of the 1,4-oxazine family of dyes, has generally been regarded as an exemplary fluorescent probe; however, recent ultrafast experiments revealed an excited-state decay kinetic of 1.2 ps, suggesting the presence of a transient photochemical state. Here, we present ultrabroadband two-dimensional electronic spectroscopy (2D ES) measurements of cresyl violet in the presence of the fluorescence quenching agent 3,6-di­(2-hydroxyethyl)-1,2,4,5-tetrazine. The broad spectral bandwidth allows for the evaluation of multiple fluorescence quenching mechanisms such as exciton formation, photoinduced electron transfer, resonance energy transfer, and excited-state proton transfer. The 2D electronic spectra in the presence and absence of the quencher suggest that excited-state proton transfer drives the system’s excited-state dynamics and leads to a cresyl violet tautomer involved in fluorescence quenching. The invocation of the tautomeric form of cresyl violet neatly resolves longstanding inconsistencies in the photophysics of oxazine dyes more generally. Although still under development, the application of ultrabroadband 2D ES to a molecular system represents a compelling demonstration of the technique’s future role in the study of photochemical reaction mechanisms.

Inverse electron demand Diels-Alder reactions between s-tetrazines and strained dienophiles have numerous applications in fluorescent labeling of biomolecules.H erein, we investigate the effect of the dienophile on the fluorescence enhancement obtained upon reaction with at etrazine-quenched fluorophore and study the possible mechanisms of fluorescence quenching by both the tetrazine and its reaction products.T he dihydropyridazine obtained from reaction with astrained cyclooctene shows aresidual fluorescence quenching effect, greater than that exerted by the pyridazine arising from reaction with the analogous alkyne.L inear and ultrabroadband two-dimensional electronic spectroscopye xperiments reveal that resonance energy transfer is the mechanism responsible for the fluorescence quenching effect of tetrazines, whereas am echanism involving more intimate electronic coupling,l ikely photoinduced electron transfer,i sr esponsible for the quenching effect of the dihydropyridazine.T hese studies uncover parameters that can be tuned to maximizef luorogenic efficiency in bioconjugation reactions and reveal that strained alkynes are better reaction partners for achieving maximum contrast ratio.

Introduction Lipopolysaccharide (LPS) is the outer membrane component of Gram-negative bacteria. LPS-binding protein (LBP) is an acute-phase reactant that mediates immune responses triggered by LPS and has been used as a blood marker for LPS. LBP has recently been indicated to be associated with Parkinson’s disease (PD) in small-scale retrospective case–control studies. We aimed to investigate the association between LBP blood levels with PD risk in a nested case–control study within a large European prospective cohort. Methods A total of 352 incident PD cases (55% males) were identified and one control per case was selected, matched by age at recruitment, sex and study center. LBP levels in plasma collected at recruitment, which was on average 7.8 years before diagnosis of the cases, were analyzed by enzyme linked immunosorbent assay. Odds ratios (ORs) were estimated for one unit increase of the natural log of LBP levels and PD incidence by conditional logistic regression. Results Plasma LBP levels were higher in prospective PD cases compared to controls (median (interquartile range) 26.9 (18.1–41.0) vs. 24.7 (16.6–38.4) µg/ml). The OR for PD incidence per one unit increase of log LBP was elevated (1.46, 95% CI 0.98–2.19). This association was more pronounced among women (OR 2.68, 95% CI 1.40–5.13) and overweight/obese subjects (OR 1.54, 95% CI 1.09–2.18). Conclusion The findings suggest that higher plasma LBP levels may be associated with an increased risk of PD and may thus pinpoint to a potential role of endotoxemia in the pathogenesis of PD, particularly in women and overweight/obese individuals.