Development of a novel, coumarin-europium sensor for reactive oxidizing species

The synthetic framework has been developed for two fluorescent probes of Reactive Oxidizing Species (RoxS) based on a coumarin-europium fluorescence pair. The coumarin moiety's selectivity for hydrogen peroxide coupled with 620 nm shifted emission of the europium will make these novel sensors biologically useful imaging tools. The two probe compounds, L1 and L2, are based on known Europium Chelators and were easily synthesized from small molecule building blocks in good yield.

DEVELOPMENT OF A NOVEL, COUMARIN-EUROPIUM SENSOR FOR REACTIVE OXIDIZING SPECIES by Sara Sanders A Senior Honors Thesis Submitted to the Faculty of The University of Utah In Partial Fulfillment of the Requirements for the Honors Degree in Bachelor of Science In Chemistry Approved: ____________________ Amy M. Barrios Supervisor ____________________ Henry S. White Chair, Department of Chemistry ____________________ Jennifer Heemstra Department Honors Advisor ____________________ Dr. Sylvia D. Torti Dean, Honors College May 2012   ii     ABSTRACT The synthetic framework has been developed for two fluorescent probes of Reactive Oxidizing Species (RoxS) based on a coumarin-europium fluorescence pair. The coumarin moiety's selectivity for hydrogen peroxide coupled with 620 nm shifted emission of the europium will make these novel sensors biologically useful imaging tools. The two probe compounds, L1 and L2, are based on known Europium Chelators and were easily synthesized from small molecule building blocks in good yield.     iii     TABLE OF CONTENTS ABSTRACT ii INTRODUCTION 1 SYNTHESIS AND CHARACTERIZATION 5 SYNTHESIS OF COUMARIN BORONATE ESTER 5 SYNTHESIS OF L1 AND L2 7 CONJUGATION OF THE BORONATE ESTER AND EUROPIUM CHELATE 9 CONCLUSIONS 10 FUTURE DIRECTIONS 11 EXPERIMENTAL 12 REFERENCES 17     1     INTRODUCTION Cells generate reactive oxidizing species (ROxS) in response to cellular stimuli such as inflammation, an immune response to microbial invasion, and as second messengers in cellular signal transduction pathways. In signal transduction, hydrogen peroxide can chemoselectively oxidize certain amino acid residues, but real time tracking of these ROxS in vivo is difficult.1 Additionally, there is growing evidence that oxidative stress plays a role in a number of diseases, including cardiovascular disease, heart disease, and diabetes.11-13 Understanding the cellular functions of hydrogen peroxide in these and other processes is the first step to uncovering scientifically relevant information that could potentially the new animal models of disease and new drug targets and leads. Selectively targeting a molecule as transient as hydrogen peroxide is a difficult task, so care was taken in choosing the probe's features. A fluorescent probe was chosen, as they are typically faster and more sensitive at low concentrations than other methods, particularly indirect reporting or colorimetric assays.14,15 Fluorescent probes may also be used in tissues non-invasively, which allows for real-time tracking of ROxS. ROxS in cell culture and even organs have successfully been imaged under laboratory generated oxidative stress.9 Figure 1 illustrates a literature-reported example of such imaging. In this study, a general oxidizing sensing fluorescent probe 4,5-diaminofluorescein, DAF, was loaded into lung venular capillaries at left atrial pressure of 5 or 15 cmH2O. At the lower vascular pressure, the probe gave little signal, but when the pressure was increased, a significant increase in fluorescence was observed. This result indicated that upon increased vascular pressure, oxidative stress also increases and oxidizing species are   2     released into the capillary. The quantified signal was depicted as a colored intensity gradient where background fluorescence was set as zero and colored black. Figure 1. Intensity of DAF fluorescent probe in a mouse capillary upon increased capillary pressure. Non-fluorescent areas in the image were shaded black, with increasing probe intensities shaded blue, green, yellow and the most intense probe turnover in red.9 Another feature necessary for our probe was the anticipated emission in the red region of the electromagnetic spectrum, around 620 nm. Coumarin alone has an emission in the blue region of the spectrum, around 460 nm. This blue emission is undesirable because shorter wavelengths have more difficulty permeating tissue, which limits the scope of blue-emitting probes to cell culture experiments. Other probes, including fluorescein based probes, such as DAF, have emission in the green region of the spectrum, around 520 nm.10 While the permeability of the green emission is better than blue, cellular auto-fluorescence is also green. If activated probe and background are the same color, the detection limit is necessarily much higher, and measurements at very small concentrations are difficult to obtain. In order to make a red probe, Europium will be chelated via an organic ligand attached to a coumarin to form a Fluorescence Resonance Energy Transfer (FRET) pair. Europium (Eu), a lanthanide, is a unique     3     fluorescent reagent that has been used in many biomedical-imaging applications.2 Coumarin has been successfully used as a Europium sensitizing ligand in the literature.316 The final goal was to create a hydrogen peroxide selective probe. To achieve this, a boronate ester or acid was introduced to mask the coumarin sensitizer prior to oxidation. The coumarin-europium probe is expected to exhibit little fluorescence prior to exposure to H2O2, and significant fluorescence after oxidation. The probe is also expected to show a preference for H2O2 detection based on previous studies of similar coumarins and their selectivity for H2O2 at low concentrations.4 One example of a coumarin boronate ester probe from previous work in our lab is shown in Figure 2. In this graph, the boronated coumarin shown in (a) was reacted with several different oxidants. The xaxis is time and the y-axis is the concentration of free, fluorescent coumarin produced. This data shows the definite selectivity of the boronate ester protected coumarin for hydrogen peroxide, especially at low concentrations.4 Figure 2. a) Reaction of boronated coumarin with hydrogen peroxide to produce free coumarin. b) Graphical depiction of data from a fluorescence assay showing the selectivity of the boronated coumarin for hydrogen peroxide.     4     This novel coumarin and europium-based sensor for ROxS overcomes many of the problems with current fluorescent sensors, including emission wavelength, and one compound, hydrogen peroxide, specificity. For this project we designed a probe that would (1) show an increased fluorescence (2) with emission in the red, and (3) be selective for H2O2. Figure 3 depicts the general probe design and the organic synthesis is outlined in Schemes 1-5. O O OH N NR2 DCC, HOBt R O O R O O L1 or L2 Figure 3. General Probe Design. The coumarin boronate ester probe is coupled through peptide-like coupling conditions to make one of two probes. L1 uses a secondary amine, and L2 uses a primary amine.     5     SYNTHESIS AND CHARACTERIZATION All compounds synthesized in Schemes 1-5 were monitored using Thin Layer Chromatography with UV, KMnO4, or Bromocresol Green for visualization, and characterized by NMR (Mercury 400). SYNTHESIS OF COUMARIN BORONATE ESTER Scheme 1: Synthesis of Coumarin Compounds 1-4 2-(7-hydroxy-2-oxo-2H-chromen-4-yl) acetic acid4 (Compound 1) was synthesized in concentrated sulfuric acid using resorcinol powder and 3-oxopentanedioic acid. The reaction stirred overnight and was recrystallized to afford 1. After formation of the acid coumarin, the hydroxyl group of the carboxylic acid was methyl protected for the duration of the synthesis and removed quantitatively in the end. Protection of the carboxylic acid proceeded with SOCl2 in methanol to yield the methyl esterified Compound 2. To attach the triflate group, 2 was combined with DMAP, 2,6-lutidine, and triflic anhydride. This, after purification, yielded a light yellow crystalline solid that was     6     verified to be the desired coumarin triflate, Compound 3.4 Next, 3 was combined with bis(pinacolato)diboron, Pd(dppf)2Cl2-CH2Cl2, and KOAc in dioxane under inert conditions. This reaction was purified to yield the boronated coumarin, which acts as the H2O2 selective functional group as Compound 4 in good yield.4 After formation of 4, the ester may be removed to form the boronic acid functional group (Compound 5) using sodium periodate and ammonium acetate.4 The solid reagents were dissolved in water in one round-bottom flask while the coumarin was dissolved in acetone in a separate flask. Both flasks were allowed to stir for one hour before adding the coumarin slowly to the sodium periodate and ammonium acetate mixture. After a simple separation the organics were dried and yielded pure Compound 5. Finally, after addition of either of the boron containing groups, hydrolysis of the ester with KOH in a water and methanol mixture yielded the final coumarins ready to couple with the chelating ligands (Compounds 6.1 and 6.2, Scheme 2). O O a) O O NaIO4, NH4OAc Acetone O B O O HO O B Compound 4 HO O O Compound 5 65.6% O b) O O OH KOH, H2O, MeOH N2 (g) R O O R= Boronate Ester, Compound 4 R= Boronic Acid, Compound 5 R O O R= Boronate Ester, Compound 6.1 R= Boronic Acid, Compound 6.2 Scheme 2. a) Boronate ester to acid, b) hydrolysis to remove protecting group     7     SYNTHESIS OF L1 AND L2 Two chelating ligands were used in the synthesis. First, a synthetically constructed diethyltriamine-based compound, L1, was synthesized, and second, a commercially available, known europium chelator, L2, was used. Scheme 3. Synthetic ligand, L1, synthesis To the diethylene triamine framework, protecting groups were added to construct the chelating ligand for Eu. To protect the terminal nitrogens, N,N-bis(2-aminoethyl) aminovaleric acid (Compound 7) was synthesized. Phthalic anhydride was added to a solution of diethyenetriamine in glacial acetic acid and then refluxed.6 While this procedure did yield compound 7, another method provided a faster, more effective route to its synthesis. N,N-bis(2-aminoethyl)aminovaleric acid, also known as 1,5-     8     Bis(phthalimido)-3-azapentane and compound 4, was made instead by adding phthalic anhydride and diethylenetriamine to chloroform and then refluxed.8 Another protecting group was then added to the central nitrogen to yield N1,N3Bis(phthalimido)-N2-benzoxycarbonyl-diethylenetriamine (Compound 8). Compound 7, DMAP, diisopropylethylamine (DIPEA), and Benzylchloroformate were mixed in anhydrous DCM under nitrogen gas. terminal nitrogens were then Once the central nitrogen was protected, the deprotected to form N2-Benzoxycarbonyl- diethylenetriamine (Compound 9).7 Hydrazine hydrate was added to Compound 8 in acetonitrile, which, when filtered, yielded yellow oil in a quantitative reaction where the protecting group crashed out of solution. Compound 9 was then dissolved in DMF with KHCO3 (to basify the solution) and stirred at room temperature after addition of tert-butyl bromoacetate, which added the desired chelating groups to the terminal nitrogens. After purification by column chromatography, which was when the bulk of the percent yield was lost, N2benzoxycarbonyl-diethylenetriamine-tetra-t-butylacetate (Compound 10) was obtained as another yellow oil.7 One last deprotection was required before coupling with coumarin. To deprotect the central nitrogen, Compound 11 was dissolved in MeOH and the protecting group cleaved with H2 (g) and Pd/C catalyst. Purification on an alumina plug yielded the desired product. The synthesis of the chelating agent was fairly inexpensive, since it stemmed from small, common molecules, which is a plus when considering using these molecules for larger-scale imaging projects.     9     CONJUGATION OF THE BORONATE ESTER AND EUROPIUM CHELATE A coupling of the synthetic amine (Compound 11) and Compound 6.1, the boronate ester coumarin, was then attempted. (Scheme 4) The formation of Compound 12 was difficult to monitor on TLC, and crude NMR data failed to provide useful information. This could be due to the fact that the coupling reaction took place under conditions that are normally reserved for primary amines: DCC and HOBt in DMF. In the future, a stronger coupling reagent or a short linker will be added to connect the two pieces, but in the short term, the commercially available amine has been the primary focus. Scheme 4. Coupling of synthetic amine, L1, with boronate ester coumarin While difficulties arose with the secondary synthetic amine, the commercially available amine coupled smoothly to form Compound 13. It reacted with the boronate acid coumarin, Compound 6.2, in a solution of DCC, HOBt, MeCN and DMF. The reaction stirred for approximately 24 hours and was purified by column chromatography to yield the desired product. The compound stuck on the baseline on TLC and silica and could be washed out of the column with increasing percentages of methanol in the solvent system. NMR indicated that the product was synthesized successfully. The purification may be simpler in the future, since the boronate ester coupling product is     10     expected to travel on silica rather than sit on the baseline. Having the compound on the baseline would normally be a good thing, but in this case, the boronate acid coumarin starting material also sits on the baseline and is difficult to separate. Scheme 5. Coupling of commercially available amine with coumarin CONCLUSIONS In summary, synthesis of a coumarin-europium sensor for hydrogen peroxide has been successfully completed. The probe includes features that give this probe advantages over others in the literature. These features include (1) fluorescence upon oxidation, (2) emission in the red, around 620 nm, and (3) selectivity for H2O2 using a boronate ester moiety. After further research, this probe shows promise as a biological imaging tool for cell culture and animal models.     11     FUTURE DIRECTIONS Chelation and Characterization Once the final coupling steps have been completed (the commercial amine with 6.1, and the synthetic amine with both 6.1 and 6.2), preparations will begin for europium chelation. First, the two coupling compounds will be fully chemically characterized. Then, instead of wasting the synthesized compounds, the europium chelation will be optimized using the commercially available europium chelator. This complex will also be fully chemically characterized. The boronated probe complex will next be reacted with different oxidants to track selectivity with the hypothesis that the probe will be selective for hydrogen peroxide. When the probe is excited with blue light, but before an oxidant is introduced to the compound, no fluorescence emission is expected. Once an oxidant reacts with the boronate ester moiety, an emission in the red should be produced, around 650 nm. Potential oxidants for this study include hydrogen peroxide, potassium superoxide, sodium nitroprusside, S-nitrosocysteine, and sodium hypochlorite. From these oxidant reactions the selectivity, sensitivity and kinetics of the probe will be documented. In Vivo Studies Finally, the fully characterized probe will be validated in cell culture with cellular assays and then with a mouse heart-lung model. Working with collaborators in the Medical School, the probe will finally be used to image the production of ROxS in lung endothelial cells. This probe has utility as a biological imaging tool, which could ultimately be applied to animal models for disease study and then eventually drug development.     12     EXPERIMENTAL Compound 1: Concentrated sulfuric acid cooled was to -5ºC, and under brisk stirring, finely ground resorcinol powder was mixed in, followed by the addition of 3oxopentanedioic acid. The reaction mixture stirred overnight as monitored by TLC. At completion, the reaction mixture was then poured into ice water and stirred for 1 hour. The white precipitate was collected by filtration and washed with ice water until filtrate neutral. The precipitate was dried under reduced pressure to afford an off-white precipitate. This solid was recrystallized in reagent alcohol (ethanol) yielding a white precipitate.4 Yield: 66.6%, 1H-NMR (DMSO, 400 MHz): δ = 3.824 (s, 2H), 6.223 (s, 1H), 6.729 (s, 1H), 6.789-6.811 (dd, 1H), 7.517-7.539 (d, 1H) ppm. Compound 2: Compound 1 was added to a dried round-bottomed flask, evacuated, and filled with nitrogen gas. The compound was dissolved in MeOH and SOCl2 was added dropwise via syringe. The reaction was stirred for 24 hours during which the desired compound crashed out of solution as a white solid. The solid was washed with MeOH until the filtrate ran colorless, and the solid was characterized as Compound 2 by NMR.4 Yield: 77.4%, 1H-NMR (MeOD:CDCl3 1:1, 400 MHz): δ = 3.753 (s, 3H), 3.825 (s, 2H), 6.207 (s, 1H), 6.789 (s, 1H), 6.823 (d, 1H), 7.481 (d, 1H) ppm. Compound 3: Compound 2 and DMAP were dissolved in DCM at 0ºC, and then 2,6lutidine was added dropwise, followed by the dropwise addition of triflic anhydride. The reaction mixture stirred for 2 hours, and was then quenched with ice and 0.5 N HCl. Washing of the water layer with DCM, washing of the DCM extracts with HCl, and NaHCO3, drying over Na2SO4, and concentration by rotary evaporation yielded a brown oil, which was purified on a silica column in 3:1 Hex: EtOAc. The desired spot was     13     collected and washed with hexanes which yielded a light yellow crystalline solid.4 Yield: 96.9%, 1H-NMR (CDCl3, 400 MHz): δ = 3.757 (s, 3H), 3.789 (s, 2H), 6.461 (s, 1H), 7.235 (d, 1H), 7.307 (s, 1H), 7.695 (d, 1H) ppm. Compound 4: Compound 3 was combined with bis(pinacolato)diboron, Pd(dppf)2Cl2CH2Cl2, and KOAc in dioxane under inert conditions. The mixture was heated to 85ºC and stirred for 20 hours. Once cooled to room temperature, the reaction mixture was filtered through celite, and the dioxane solvent removed in vacuo. Column chromatography was used to purify the resulting orange-brown oil.4 Yield: 85.7%, 1HNMR (CDCl3, 400 MHz): δ = 1.356 (s, 12H), 3.723 (s, 3H), 3.932 (s, 2H), 6.422 (s, 1H), 7.506 (d, 1H), 7.550 (d, 1H), 7.681 (d, 1H) ppm. Compound 5: The methyl boronic acid coumarin was synthesized from the methyl boronate ester coumarin. Compound 4 was dissolved in acetone in a round-bottom flask, and sodium periodate and ammonium acetate were dissolved in water in a separate flask. After approximately one hour of stirring, the coumarin/acetone mixture was added dropwise to the NaIO4 and NH4OAc flask and stirred overnight. Next, the acetone was removed by rotary evaporation. The remaining liquid was transferred to a separatory funnel and extracted with water and ethyl acetate. The organics were dried and yielded a white powder with one spot on TLC.4 1H-NMR (MeOD, 400 MHz): δ = 3.727 (s, 3H), 3.968 (s, 2H), 6.469 (s, 1H), 7.506-7.664 (m, 3H) ppm. Compound 6.1, 2: The procedure for demethylation for both the boronic acid coumarin and boronate ester coumarin was the same. The respective coumarin was added to a flask along with excess KOH. The flask was evacuated and flushed with nitrogen gas for an inert atmosphere. Methanol was added via syringe (due to solubility) and the mixture     14     stirred for 2 days. The organics were dried and then separated with EtOAc and 0.5 N Hcl. Organics were collected and dried yielding the desired compound.4 1H-NMR (CDCl3, 400 MHz): Compound 6.1: δ = 1.356 (s, 12H), 3.823 (s, 2H), 6.460 (s, 1H), 7.552 (d, 1H), 7.683 (d, 1H), 7.758 (d, 1H) ppm. Compound 6.2: δ = 3.833 (s, 2H), 6.452 (s, 1H), 7.531 (d, 1H), 7.671 (d, 1H), 7.756 (d, 1H) ppm. Compound 7: Phthalic anhydride was added to CHCl3, followed by the dropwise addition of diethylenetriamine. This mixture was refluxed for 5 hours. The solvent was then removed under reduced pressure to yield a solid material. This solid was dissolved in CH2Cl2 and the insoluble material filtered off leaving the desired product in the filtrate. This filtrate was dried under vacuum again, and once washed with ethanol, compound 7 was obtained as verified by NMR spectroscopy.8 Yield: 43%, 1H-NMR (CDCl3, 400 MHz): δ = 7.64-7.80 (m, 8H), 3.77 (t, 4H), 2.96 (t, 4H). Compound 8: Compound 7, 4-Dimethylaminopyridine diisopropylethylamine (DIPEA) were added to an oven dried flask. (DMAP) and The flask was evacuated and filled with nitrogen gas. Anhydrous DCM was added, and the resulting slurry was cooled to 0ºC. Benzylchloroformate was dissolved in anhydrous DCM in a separate round bottom flask, also under nitrogen gas. The solvated benzylchloroformate was added dropwise to the slurry over a period of 15 minutes. The solution stirred briskly for 48 hours, after which the mixture was washed 3x with acidic water (a pH of 4), 3x with saturated bicarbonate, and 3x with ultrapure water. The organic fraction was dried over sodium sulfate and dried in vacuo to produce a solid, which was then washed with     15     anhydrous ether to remove the excess benzochloroformate. This wash yielded compound 8 in the form of a white solid.7 Yield: 72%, 1H-NMR (CDCl3, 400 MHz): δ = 7.68-7.80 (m, 8H), 7.23 (m, 3H), 7.17 (d, 2H), 4.81 (s, 2H), 3.89 (dt, 4H), 3.59 (t, 4H) ppm. (7) Compound 9: Compound 8 was slurried in acetonitrile, followed by the addition of hydrazine hydrate via syringe. The solution stirred for approximately 24 hours, and a precipitate formed. Thin layer chromatography was used to determine reaction completion. Upon completion, the precipitate was gravity filtered from the solution and thoroughly washed with acetonitrile. The acetonitrile washes and the initial solution were combined and dried in vacuo yielding compound 9 in the form of a yellow oil.7 Yield: 95%, 1H-NMR (CDCl3, 400 MHz): δ = 1.36 (bs, 4H), 2.85 (s, 4H), 3.357 (s, 4H), 5.14 (s/d, 2H), 7.36 (m, 5H) ppm. Compound 10: Compound 9 was dissolved in DMF and KHCO3, stirring at room temperature. Tert-butyl bromoacetate was added dropwise to the stirring solution. The solution stirred for 48 hours at room temperature, and then heated to 60ºC for another 2 hours. Then, diethyl ether and saturated sodium bicarbonate were added and the solution stirred for 5 more minutes. The ether layer was retained and washed 3x with saturated sodium bicarbonate, followed by dH2O. Drying over anhydrous sodium sulfate and evaporation under vacuum yielded the crude product as yellow oil. The crude product was dry loaded onto a silica column, and the product-containing fractions were again dried under vacuum.7 Yield: 33%, 1H-NMR (CDCl3, 400 MHz): δ = 1.44 (s, 36H), 2.86 (s, 4H), 3.39 (s, 8H), 3.47(s, 4H), 7.36 (m, 5H) ppm. Compound 11: Compound 10 was dissolved in MeOH, and Pd/C catalyst was added. Several liters of H2 (g) were bubbled through the room temperature solution with stirring     16     overnight. After 24-36 hours the reaction was determined complete by TLC, and the catalyst was removed by filtering through celite, and then dried in vacuum yielding Diethylenetriamine-tetra-t-butylacetate (Compound 11).7 Yield: 65%, 1H-NMR (CDCl3, 400 MHz): δ = 1.45 (s, 36H), 3.05 (t, 4H), 3.24 (t, 4H), 3.518 (s, 8H) ppm. Compound 12: HOBt and Compound 6.1 were combined in a round-bottom flask and dissolved in DCM. DCC was added to this mixture and stirred for 20 minutes. The synthetic amine, tert-butyl 2,2’,2’’,2’’’-(2,2’-azanediylbis(ethane-2,1- diyl)bis(azanetriyl)) tetraacetate, was dissolved in DMF and added dropwise to the coumarin solution, and stirred overnight. The reaction was monitored by TLC, but showed either no reaction or minimal reaction. No NMR data available. Compound 13: Compound 6.2 was mixed with HOBt and DCC and dissolved in acetonitrile. The reaction mixture was cooled to 0°C and stirred for 30 minutes. The Commercially available amine, di-tert-butyl 4-amino-(3-tert-butoxy-3-oxopropyl) heptanedioate, was dissolved in DMF and added dropwise to the flask. The reaction mixture was stirred for 3 hours, and stored in the fridge for one week. An alumina column was run using MeCN and increasing percentages of MeOH with a final MeOH wash to pull any last compound off the alumina. A number of impure fractions resulted, however one happened to contain pure Compound 13. An NMR was obtained, but further studies and optimizations are required before an accurate percent yield may be reported. 1H-NMR (MeOD, 400 MHz): δ = 1.426 (s, 27H), 1.943 (t, 6H), 2.195 (t, 6H), 3.125 (s, 2H), 6.294 (s, 1H), 7.503 (d, 1H), 7.578 (m, 2H) ppm.         17     REFERENCES           1. Miller EW, Tulyathan O, Isacoff EY, Chang CJ “Molecular Imaging of Hydrogen Peroxide Produced for Cell Signaling” Nature Chem. Biol. 2007 2(5), 263-267. 2. Munzel T, et. al. “Is oxidative stress a therapeutic target in cardiovascular disease?” Eur. Heart J. 2010. 31(22), 2741-2748. 3. Giugliano D, et. al. “Diabetes Mellitus, Hypertension, and Cardiovascular Disease: Which Role for Oxidative Stress?” Metabolism. 1995. 44(3), 363-368. 4. Singal PK, Khaper N, Palace V, Kumar D. “The role of oxidative stress in the genesis of heart disease.” Cardiovascular Research. 1998. 40, 426 –432. 5. Xiaoqiang C, et al. “Fluorescent and luminescent probes for detection of reactive oxygen and nitrogen species” Chem. Soc. Rev., 2011, 40, 4783–4804. 6. Coutlee F, et. al. “Comparison of Colorimetric, Fluorescent, and Enzymatic Amplification Substrate Systems in an Enzyme Immunoassay for Detection of DNA-RNA Hybrids”, Journal of Clinical Microbiology, 1989, 1002-1007. 7. Kuebler W, Uhlig, U, et. al. “Stretch Activates Nitric Oxide Production in Pulmonary Vascular Endothelial Cells In Situ.” Am. J. Respir. Crit. Care Med. 2003. 168, 1391-1398. 8. Sun WC, et. al. “Synthesis of Fluorinated Fluoresceins” J. Org. Chem. 1997. 62, 6469-6475. 9. Richardson FS “Terbium (III) and Europium (III) Ions as Luminescent Probes and Stains for Biomolecular Systems” Chem. Rev. 1982. 82, 541-552. 10. Feau C, Klein E, Dosche C, Kerth P, Lebau L, “Synthesis and Characterization of Coumarin-based Europium Complexes and Luminescence Measurements in Aqueous Media” Org. Biomol. Chem. 2009. 7, 5259-5270. 11. Pershagen E, et. al. “Luminescent Lanthanide Complexes with Analyte-Triggered Antenna Formation” JACS. 2011. dx.doi.org/10.1021/ja3004045. 12. Hubbard, C. et.al “Courmarin-based Fluorescent Probes for the Visualization of Oxidants in T Cells” manuscript in progress. 13. Benerjee SR, Babich JW, Zubieta J “Bifunctional chelaes with aliphatic amine donors for labeling of biomolecules with the {Tc(CO)3}+ and {Re(CO)3}+ cores: the crystal and molecular structure of [Re(CO)3{(H2NCH2CH2)2 N(CH2)4CO2Me}]Br” Inorg. Chem. Comm. 2004. 7, 481-84. 14. Teramae S, Osako T, Nagatomo S, Kitagawa T, Fukuzumi S, Itoh S “Dinuclear copper-dioxygen intermediates supported by polyamine ligands” J. Inorg. Biochem. 2004. 98(5), 746-757. 15. Bryson J, Chu WJ, Lee JH, Reineke T “A -Cyclodextrin “Click Cluster” Decorated with Seven Paramagnetic Chelates Containing Two Water Exchange Sites” Bioconjugate Chem. 2008. 19(8), 1505-09.