Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2006. / Vita. / Includes bibliographical references. / Chapter 1. Metal-Based Turn-On Fluorescent Probes for Sensing Nitric Oxide. Nitric oxide, a reactive free radical, regulates a variety of biological processes. The absence of tools to detect NO directly, rapidly, specifically and selectively motivated us to develop metal-based fluorescent probes to visualize the presence of NO. We have prepared and investigated Co(II), Fe(II), Ru(II), Rh(II), and Cu(II) complexes as turn-on fluorescent NO sensors. Our exploration has provided insight into how the interaction of transition metal centers with nitric oxide can be utilized for NO sensing. Chapter 2. Fluorescence-Based Nitric Oxide Detection by Ruthenium Porphyrin Fluorophore Complexes. The ruthenium(II) porphyrin fluorophore complexes [Ru(TPP)(CO)(Ds-R)] (TPP = tetraphenylporphinato dianion; Ds = dansyl; R = imidazole (im), 1, or thiomorpholine (tm), 2) were synthesized and investigated for their ability to detect nitric oxide (NO) based on fluorescence. The X-ray crystal structures of 1 and 2 were determined. The Ds-im or Ds-tm ligand coordinates to an axial site of the ruthenium(II) center through a nitrogen or sulfur atom, respectively. Both exhibit quenched fluorescence when excited at 368 or 345 nm. / (cont.) Displacement of the metal-coordinated fluorophore by NO restores fluorescence within minutes. These observations demonstrate fluorescence-based NO detection using ruthenium porphyrin fluorophore conjugates. Chapter 3. Nitric Oxide-Induced Fluorescence Enhancement by Displacement of Dansylated Ligands from Cobalt. The cobalt complexes, [Co(Ds-AMP)2] (1) and [Co(Ds-AQ)2] (2), where Ds-AMP and Ds-AQ are the conjugate bases of dansyl aminomethylpyridine, Ds-HAMP, and dansyl aminoquinoline, Ds-HAQ, respectively, were synthesized in two steps as fluorescence-based nitric oxide (NO) sensors and characterized by X-ray crystallography. The fluorescence of both complexes was significantly quenched in CH3CN or CH3OH compared to that of the free Ds-HAMP or Ds-HAQ ligands. Addition of NO to a CH3CN solution of 1 or 2 enhanced the integrated fluorescence emission by factors of 2.1(_-+0.3) or 3.6(+0.4) within 35 or 20 min, respectively. Introduction of NO to methanolic solutions similarly increased the fluorescence by 1.4(±0.1) for 1 or 6.5(±1.4) for 2 within 1 h. / (cont.) These studies demonstrate that 1 and 2 can monitor the presence of NO with turn-on emission, and that their fluorescence responses are more rapid than those of previously reported cobalt systems in coordinating solvents such as CH3CN and CH3OH. H NMR and IR spectroscopic data revealed the formation of a {Co(NO)2'0 cobalt dinitrosyl adduct with concomitant dissociation of one ligand from the cobalt center as the metal-containing product of the NO reactions, indicating NO-induced ligand release to be the cause of the fluorescence Chapter 4. Fluorescent Nitric Oxide Detection by Copper Complexes Bearing Anthracenyl and Dansyl Fluorophore Ligands. Anthrancenyl and dansyl fluorophore ligands (AnCH2pipCS2K (1), Ds-Hen (2), Ds-HAMP (3), Ds-HAQ (4), and Ds-HAPP (5)) were prepared for copper(II). Five copper complexes, [Cu(AnCH2pipCS2)2] (6), [Cu(Ds-en)2] (7), [Cu(Ds-AMP)2] (8), [Cu(Ds-AQ)2] (9), and [Cu(Ds-APP)(OTf)] (10), were synthesized for fluorescent nitric oxide (NO) detection and were characterized by X-ray crystallography. A decrease in fluorescence of free ligands (1- 5) coordinated to the Cu(II) center was observed in all Cu(II) complexes (6-10). / (cont.) The fluorescence of fluorophore ligands in Cu(II) complexes was restored in the presence of NO in a CH3OH/CH2C12 solvent. Furthermore, compounds 7, 8, and 10, exhibited fluorescence response to NO in aqueous pH 7.0 or 9.0 buffered solutions. Fluorescence enhancement of these Cu(II) complexes occurs by NO-induced reduction from Cu(II) to Cu(I), as demonstrated spectroscopically. The present work suggest that a copper(II) complex would be effective as a fluorescent probe for sensing NO in both organic and aqueous settings. Chapter 5. Direct Nitric Oxide Detection In Aqueous Solution by Copper(II) Fluorescein Complexes. Fluorescein-based ligands (FL., n = 1 - 5) for Cu(II) were synthesized and their photophysical properties were determined. Introduction of nitric oxide (NO) to a pH 7.0 buffered solution of Cu(FLn) (1 M CuCl2 and 1 uM F Ln) induces an increase in fluorescence at 37 °C. The fluorescence response of Cu(FL.) is direct and specific, which is a significant improvement of commercially available small molecule-based probes that are capable only of indirect NO detection. NO-triggered fluorescence increase of Cu(FLn) occurs by reduction of Cu(II) to Cu(I) with concomitant dissociation of the N-nitrosated fluorophore ligand from copper. / (cont.) Spectroscopic and product analyses of the reaction of the copper fluorescein complex with NO suggest that the N-nitrosated fluorescein ligand (FLn-NO) is the species for fluorescence turn-on. Density functional theory (DFT) calculations of FL5 versus FL-NO reveal how N-nitrosation of the fluorophore ligand causes the fluorescence increase. The investigation of copper-based probes described in the present work is the basis for developing a metal complex for fluorescent NO detection. Chapter 6. Visualization of Nitric Oxide in Living Cells by a Copper-Based Fluorescent Probe. Nitric oxide (NO) is a highly reactive gaseous free radical that serves as a messenger for cellular signaling. To visualize NO in living cells, a turn-on fluorescent probe was designed and synthesized for use in combination with microscopy. Unlike existing fluorescent sensors, the construct, a Cu(II) complex of a fluorescein modified with an appended metal-chelating ligand (FL), directly and immediately images NO rather than a derivative reactive nitrogen species (RNS). Nitric oxide produced by both constitutive (cNOS) and inducible (iNOS) NO synthases in live neurons and macrophages is detected by the Cu(II)-based imaging agent in a concentration- and time-dependent manner. / (cont.) The sensitivity to nM levels of NO and the spatiotemporal information provided by this complex demonstrate its value for numerous biological applications. Appendix. Fluorescent Detection of Nitric Oxide by a Rhodium Fluorophore Embedded in a Silastic Polymer Using Two-Photon Microscopy. A Silastic membrane embedded with a dirhodium fluorophore conjugate, [Rh2(M-O2CPr)4(Ds-pip)] (Ds-pip = dansyl-piperzine), was prepared. Nitric oxide (NO) in aqueous media replaces the Ds-pip bound to the dirhodium core in the solid state, inducing the fluorescence increase observed by two-photon spectroscopy. This observation is the first effort for NO detection using two-photon microscopy and represents an initial step toward fiber-optic-based NO sensing in aqueous media using this dirhodium-containing polymer. / by Mi Hee Lim. / Ph.D.
| Identifer | oai:union.ndltd.org:MIT/oai:dspace.mit.edu:1721.1/36267 |
| Date | January 2006 |
| Creators | Lim, Mi Hee |
| Contributors | Stephen J. Lippard., Massachusetts Institute of Technology. Dept. of Chemistry., Massachusetts Institute of Technology. Dept. of Chemistry. |
| Publisher | Massachusetts Institute of Technology |
| Source Sets | M.I.T. Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 274 p., application/pdf |
| Rights | M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission., http://dspace.mit.edu/handle/1721.1/7582 |
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