From 2,3-Diazabicyclo[2.2.2]oct-2-ene to Fluorazophore-L, a membrane-bound fluorescent probe for antioxidants

The aim of this work was to synthesize and to establish a new fluorescent membrane probe for antioxidants by exploiting the exceptional properties of the long-lived fluorophore 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) alias Fluorazophore-P. The first step was to find an appropriate synthetic route towards a lipophilic derivative of Fluorazophore-P, namely Fluorazophore-L, that should enable an efficient and facile incorporation into model membrane systems. The water-soluble hydroxy-substituted Fluorazophore-H was chosen as a key compound and served as a versatile precursor for various members of the Fluorazophore-family, including Fluorazophore-L. For example, substantial contributions in the synthesis of fluorazophore-labeled peptides to monitor the length-dependence of end-to-end collision rates of polypeptides were done within this work: "A Fluorescence Based Method for Direct Measurement of Submicrosecond Intramolecular Contact Formation in Biopolymers: An Exploratory Study with Polypeptides", R. R. Hudgins, F. Huang, G. Gramlich, W. M. Nau, J. Am. Chem. Soc. 2002, 124, 556-564. (Appendix) In this context, the search for a mild and selective method to substitute a harsh hydrolysis step, led to a study about a photo-cleavable Fluorazophore: "A Photoactivable Fluorophore Based on Thiadiazolidinedione as Caging Group", G. Gramlich, W. M. Nau, Org. Let. 1999, 1, 603-605. (Appendix) Fluorazophore-L (Fluoazophore-L) was designed as a head-labeled palmitic acid derivative. Experiments in homogeneous solution confirmed that Fluoazophore-L preserves its photophysical properties, namely the long-lived fluorescence and the essentially diffusion-controlled reactivity towards α-tocopherol (α-Toc). Its capability to serve as a membrane probe was assessed by air/water monolayer experiments (surface pressure-area isotherms) and preliminary spectroscopic measurements. It could be shown that Fluoazophore-L partitions into monolayers of 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC) and that even pure Fluoazophore-L forms stable monolayers at the air-water interface thus presents a highly amphiphilic molecule: "A Long-Lived Amphiphilic Fluorescent Probe studied in POPC Air-Water Monolayer and Solution Bilayer Systems", G. Gramlich, J. Zhang, M. Winterhalter, W. M. Nau, Chem. Phys. Lipids 2001, 113, 1-9 (Appendix). The first assignment of Fluoazophore-L in model membranes was a study of its interaction with the water-soluble antioxidant vitamin C, thus examining interfacial reactivity. Singlet-excited Fluoazophore-L was used as a mimic for highly reactive lipid alkoxyl and peroxyl radicals. This work revealed an unexpected inversion of the pHdependent activity pattern, which could be ascribed to an interesting surface effect: "Increased Antioxidant Reactivity of Vitamin C at low pH in Model Membranes", G. Gramlich, J. Zhang, W. M. Nau, J. Am. Chem. Soc. 2002, 124, 11252-11253 (Appendix). Finally, the intrafacial reactivity of α-Toc in liposomes and micelles could be probed by means of Fluoazophore-L. In micelles and in membrane structures a more demanding quenching kinetics than in usual organic solvents arises. In the case of small micelles Poissonian statistics has to be applied while in liposomes a two dimensional diffusion rate limits the maximum reactivity. In this study, the "immobile" probe/quencher pair Fluoazophore-L/α-Toc was used for the first time and the validity of different quenching models was discussed. The resulting diffusion rate constants for α-Toc provide important benchmark values for antioxidant research: "Diffusion of α-Tocopherol in Membrane Models: Probing the Kinetics of Vitamin E Antioxidant Action by Fluorescence in Real Time", G. Gramlich, J. Zhang, W. M. Nau, J. Am. Chem. Soc. 2004, 126, 5482-5492 (Appendix). A global fitting routine was developed to allow appropriate data processing of fluorescence quenching in membrane models. This fitting procedure was also successfully employed in the simultaneous fitting of steady-state and time-resolved fluorescence quenching by host-guest complexation with cyclodextrins. "A Joint Structural, Kinetic, and Thermodynamic Investigation of Substituent Effects on Host- Guest Complexation of Bicyclic Azoalkanes by β-Cyclodextrin", X. Zhang, G. Gramlich, X. Wang, W. M. Nau, J. Am. Chem. Soc. 2002, 124, 254-263 (Appendix). For the quenching models used, it is essential to ensure that reaction between singletexcited fluorazophores and hydrogen donors as antioxidants occurs only by hydrogen transfer and upon contact of probe and quencher. To clarify this process experiments using spectroscopic methods were contributed to a detailed theoretical study of reaction pathways: "Fluorescence Quenching by Sequential Hydrogen, Electron, and Proton Transfer in the Proximity of a Conical Intersection", A. Sinicropi, R. Pogni, R. Basosi, M. A. Robb, G. Gramlich, W. M. Nau, M. Olivucci, Angew. Chem., Int. Ed. 2001, 40, 4185-4189 (Appendix). In summary, the result of this study was the design and synthesis of the new fluorescent membrane probe Fluorazophore-L that combines the unusual properties of DBO with a complete incorporation into model membranes. The properties of the new probe were assessed in monolayer and by fluorescence lifetime experiments. Its potency was proven by the interaction with natural antioxidants located in the proximity of membrane mimetic systems. These quenching experiments allowed a new insight into the processes involving antioxidants in microheterogeneous environments, especially an unusual inversion of the well-known reactivity pattern of ascorbic acid and the observation of the lateral diffusion of α-tocopherol along the surface of supramolecular assemblies

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Many students with disabilities continue to struggle to successfully make the transition from school to employment. Despite advances in employment rates for students with disabilities who have exited school, their employment rates still lag significantly behind their nondisabled peers (Blackorby & Wagner, 1996). For decades, research has shown the strong relationship between work experience during secondary school and postschool employment for youth with disabilities (Benz, Yovanoff, & Doren, 1997; Colley & Jamison, 1998; Hasazi, Gordan, & Roe, 1985). However, as the continuing disappointing postschool employment rates for youth with disabilities suggest, there remains a critical need to expand work-based learning opportunities for these youth and to integrate these experiences into secondary education. This brief highlights the benefits of work-based learning, what constitutes quality work-based learning, and selected evidence-based models of work-based learning

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