Metal-based turn-on fluorescent probes for nitric oxide sensing

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

Sensitivity of Ru(bpy)_2dppz^(2+) Luminescence to DNA Defects

The luminescent characteristics of Ru(bpy)_2dppz^(2+) (dppz = dipyrido[3,2-a:2′,3′-c]phenazine), a DNA light switch, were investigated in the presence of oligonucleotides containing single base mismatches or an abasic site. In water, the ruthenium luminescence is quenched, but, bound to well matched duplex DNA, the Ru complex luminesces. Here we show that with DNAs containing a defect, rac-, Δ-, and Λ-Ru(bpy)_2dppz^(2+) exhibit significant luminescent enhancements above that with well matched DNA. In the presence of a single base mismatch, large luminescent enhancements are evident for the Δ-Ru isomer; the Λ-isomer shows particularly high luminescence bound to an oligonucleotide containing an abasic site. Similar increases are not evident with two common DNA-binding organic fluorophores, ethidium bromide and TO-PRO-3. Titrations with hairpin oligonucleotides containing a variable mismatch site show correlation between the level of luminescent enhancement and the thermodynamic destabilization associated with the mismatch. This correlation is reminiscent of that found earlier for a bulky rhodium complex that binds mismatched DNA sites through metalloinsertion, where the complex binds the DNA from the minor groove side, ejecting the mismatched bases into the major groove. Differential quenching studies with minor and major groove quenchers and time-resolved emission studies support this metalloinsertion mode for the dppz complex at the defect site. Certainly these data underscore the utility of Ru(bpy)_2dppz^(2+) as a sensitive luminescent reporter of DNA and its defects

The central role of the d-block metals in the periodic table. Editorial for a themed issue of the journal

No abstract

Myricetin: A Naturally Occurring Regulator of Metal-Induced Amyloid-β Aggregation and Neurotoxicity

No AbstractPeer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/84385/1/1198_ftp.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/84385/2/cbic_201000790_sm_miscellaneous_information.pd

The Characteristics of Action Potentials in Primo Vessels and the Effects of Acetylcholine Injection to the Action Potentials

In a previous study, we found that Primo vessels generate different action potentials in smooth muscles, but this study compared the pulse shape to distinguish the two tissues. Thus, a more sophisticated extracellular experiment was performed in this study using an acetylcholine injection; we then observed changes in the amplitude, FWHM (full width at half maximum), and period to explore Primo vessel function. A third type of pulse was recorded for Primo vessels. We observed fast depolarizing and repolarizing phases for this pulse. Further, its FWHM was 30 ms between smooth muscles and neurons. Acetylcholine affected only the period. The amplitude and FWHM were consistent after injection. Primo-vessels generated action potentials at twice the frequency after injection. From the results, we speculate that Primo-vessels perform a role in transferring signals in a different manner, which may be relevant for acupuncture treatment

Recent Development of Bifunctional Small Molecules to Study Metal-Amyloid-β Species in Alzheimer's Disease

Alzheimer's disease (AD) is a multifactorial neurodegenerative disease related to the deposition of aggregated amyloid-β (Aβ) peptides in the brain. It has been proposed that metal ion dyshomeostasis and miscompartmentalization contribute to AD progression, especially as metal ions (e.g., Cu(II) and Zn(II)) found in Aβ plaques of the diseased brain can bind to Aβ and be linked to aggregation and neurotoxicity. The role of metal ions in AD pathogenesis, however, is uncertain. To accelerate understanding in this area and contribute to therapeutic development, recent efforts to devise suitable chemical reagents that can target metal ions associated with Aβ have been made using rational structure-based design that combines two functions (metal chelation and Aβ interaction) in the same molecule. This paper presents bifunctional compounds developed by two different design strategies (linkage or incorporation) and discusses progress in their applications as chemical tools and/or potential therapeutics

A lab-on-a-disc platform enables serial monitoring of individual CTCs associated with tumor progression during EGFR-targeted therapy for patients with NSCLC

Rationale: Unlike traditional biopsy, liquid biopsy, which is a largely non-invasive diagnostic and monitoring tool, can be performed more frequently to better track tumors and mutations over time and to validate the efficiency of a cancer treatment. Circulating tumor cells (CTCs) are considered promising liquid biopsy biomarkers; however, their use in clinical settings is limited by high costs and a low throughput of standard platforms for CTC enumeration and analysis. In this study, we used a label-free, high-throughput method for CTC isolation directly from whole blood of patients using a standalone, clinical setting-friendly platform. Methods: A CTC-based liquid biopsy approach was used to examine the efficacy of therapy and emergent drug resistance via longitudinal monitoring of CTC counts, DNA mutations, and single-cell-level gene expression in a prospective cohort of 40 patients with epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer. Results: The change ratio of the CTC counts was associated with tumor response, detected by CT scan, while the baseline CTC counts did not show association with progression-free survival or overall survival. We achieved a 100% concordance rate for the detection of EGFR mutation, including emergence of T790M, between tumor tissue and CTCs. More importantly, our data revealed the importance of the analysis of the epithelial/mesenchymal signature of individual pretreatment CTCs to predict drug responsiveness in patients. Conclusion: The fluid-assisted separation technology disc platform enables serial monitoring of CTC counts, DNA mutations, as well as unbiased molecular characterization of individual CTCs associated with tumor progression during targeted therapy

KITENIN increases invasion and migration of mouse squamous cancer cells and promotes pulmonary metastasis in a mouse squamous tumor model

AbstractKAI1 C-terminal interacting tetraspanin (KITENIN) is reported to promote metastasis in mouse colon cancer models. We investigated the role of KITENIN on the progression of squamous cell carcinoma (SCC). In a preliminary clinical study using resected tissues from head and neck SCC patients, KITENIN was highly expressed in tumors and metastatic lymph nodes, while KAI1 was more increased in adjacent mucosa than in tumor. KITENIN-transfected mouse squamous cancer (SCC VII/KITENIN) cells showed significantly higher invasion, migration, and proliferation than empty vector-transfected cells. In syngeneic mouse squamous tumor models, more increased tumor volume and enhanced lung metastasis were found in SCC VII/KITENIN cells-injected mice. Thus, KITENIN increases invasion and migration of squamous cancer cells and thereby promotes distant metastasis in mouse squamous tumor models

Purification and characterization of angiotensin-1 converting enzyme (ACE)-inhibitory peptide from the jellyfish, Nemopilema nomurai

The Nemopilema nomurai hydrolysate was produced by the reaction of papain, and an angiotensin-Ι converting enzyme (ACE)-inhibitory peptide was purified by using the molecular cut-offs membrane filter, the gel filtration chromatography with Sephadex LH-20 and the reverse phase chromatographic method using C18 and C12 columns. Purification yield of the active peptide was estimated to be 0.2 ± 0.1%, starting from the lyophilized jellyfish. The infrared (IR), proton nuclear magnetic resonance spectroscopy (1H NMR), carbon nuclear magnetic resonance (13C NMR) and mass spectrometry (MS) spectrometer analyses elucidated that the structure of the purified peptide is tyrosine-isoleucine (Tyr-Ile). The inhibitory concentration at 50% (IC50) and Ki values were calculated to be 2.0 ± 0.3 μg/ml and 3.3 ± 0.3 μM, respectively, which acts as a competitive inhibitor to ACE.Keywords: Angiotensin-Ι converting enzyme, Jellyfish, Nemopilema nomurai, Papain hydrolysate, Tyrosine-IsoleucineAfrican Journal of Biotechnology Vol. 12(15), pp. 1888-189