Cell-Trappable Quinoline-Derivatized Fluoresceins for Selective and Reversible Biological Zn(II) Detection

The synthesis and spectroscopic characterization of two new, cell-trappable fluorescent probes for Zn(II) are presented. These probes, 2-(4,5-bis(((6-(2-ethoxy-2-oxoethoxy)quinolin-8-yl)amino)methyl)-6-hydroxy-3-oxo-3H-8 xanthen-9-yl)benzoic acid (QZ2E) and 2,2′-((8,8′-(((9-(2-carboxyphenyl)-6-hydroxy-3-oxo-3H-xanthene-4,5-diyl)bis(methylene))bis(azanediyl))bis(quinoline-8,6-diyl))bis(oxy))diacetic acid (QZ2A), are poorly emissive in the off-state but exhibit dramatic increases in fluorescence upon Zn(II) binding (120 ± 10-fold for QZ2E, 30 ± 7-fold for QZ2A). This binding is selective for Zn(II) over other biologically relevant metal cations, toxic heavy metals, and most first-row transition metals and is of appropriate affinity (K[subscript d1](QZ2E) = 150 ± 100 μM, K[subscript d2](QZ2E) = 3.5 ± 0.1 mM, K[subscript d1](QZ2A) = 220 ± 30 μM, K[subscript d2](QZ2A) = 160 ± 80 μM, K[subscript d3](QZ2A) = 9 ± 6 μM) to reversibly bind Zn(II) at physiological levels. In live cells, QZ2E localizes to the Gogli apparatus where it can detect Zn(II). It is cell-membrane-permeable until cleavage of its ester groups by intracellular esterases produces QZ2A, a negatively charged acid form that cannot cross the cell membrane.National Science Foundation (U.S.) (CHE-0907905

Detecting and Understanding the Roles of Nitric Oxide in biology

We are pursuing a dual strategy for investigating the chemistry of nitric oxide as a biological signaling agent. In one approach, metal-based fluorescent sensors for the detection of NO in living cells are evaluated, and a sensor based on a copper fluorescein complex has proved to be a valuable lead compound. Sensors of this class permit identification of NO from both inducible and constitutive forms of nitric oxide synthase and facilitate investigation of different NO functions in response to external stimuli. In the other approach, we employ synthetic model complexes of iron−sulfur clusters to probe their reactivity toward nitric oxide as biomimics of the active sites of iron−sulfur proteins. Our studies reveal that NO disassembles the Fe−S clusters to form dinitrosyl iron complexes.National Science Foundation (U.S.) (CHE-0907905)National Institute of General Medical Sciences (U.S.) (Grant F32 GM082031-03

Seminaphthofluorescein-Based Fluorescent Probes for Imaging Nitric Oxide in Live Cells

Fluorescent turn-on probes for nitric oxide based on seminaphthofluorescein scaffolds were prepared and spectroscopically characterized. The Cu(II) complexes of these fluorescent probes react with NO under anaerobic conditions to yield a 20–45-fold increase in integrated emission. The seminaphthofluorescein-based probes emit at longer wavelengths than the parent FL1 and FL2 fluorescein-based generations of NO probes, maintaining emission maxima between 550 and 625 nm. The emission profiles depend on the excitation wavelength; maximum fluorescence turn-on is achieved at excitations between 535 and 575 nm. The probes are highly selective for NO over other biologically relevant reactive nitrogen and oxygen species including NO3–, NO2–, HNO, ONOO–, NO2, OCl–, and H2O2. The seminaphthofluorescein-based probes can be used to visualize endogenously produced NO in live cells, as demonstrated using Raw 264.7 macrophages.National Science Foundation (U.S.) (CHE-0611944)National Institutes of Health (U.S.) (K99GM092970

Mechanism of Nitric Oxide Reactivity and Fluorescence Enhancement of the NO-Specific Probe CuFL1

The mechanism of the reaction of CuFL1 (FL1 = 2-{2-chloro-6-hydroxy-5-[(2-methylquinolin-8-ylamino)methyl]-3-oxo-3H-xanthen-9-yl}benzoic acid) with nitric oxide (NO) to form the N-nitrosated product FL1-NO in buffered aqueous solutions was investigated. The reaction is first-order in [CuFL1], [NO], and [OH−]. The observed rate saturation at high base concentrations is consistent with a mechanism in which the protonation state of the secondary amine of the ligand is important for reactivity. This information provides a rationale for designing faster-reacting probes by lowering the pKa of the secondary amine. Activation parameters for the reaction of CuFL1 with NO indicate an associative mechanism (ΔS‡ = −120 ± 10 J/mol·K) with a modest thermal barrier (ΔH = 41 ± 2 kJ/mol; Ea = 43 ± 2 kJ/mol). Variable-pH electron paramagnetic resonance experiments reveal that, as the secondary amine of CuFL1 is deprotonated, electron density shifts to yield a new spin-active species having electron density localized on the deprotonated amine nitrogen atom. This result suggests that FL1-NO formation occurs when NO attacks the deprotonated secondary amine of the coordinated ligand, followed by inner-sphere electron transfer to Cu(II) to form Cu(I) and release of FL1-NO from the metal.National Science Foundation (U.S.) (grant CHE-0907905)National Institutes of Health (U.S.) (Postdoctoral Fellowship (5 F32 GM085930))National Institutes of Health (U.S.) (Postdoctoral Fellowship (1 K99 GM092970

Cell-Trappable Fluorescent Probes for Nitric Oxide Visualization in Living Cells

Two new cell-trappable fluorescent probes for nitric oxide (NO) are reported based on either incorporation of hydrolyzable esters or conjugation to aminodextran polymers. Both probes are highly selective for NO over other reactive oxygen and nitrogen species (RONS). The efficacy of these probes for the fluorescence imaging of nitric oxide produced endogenously in Raw 264.7 cells is demonstrated.National Science Foundation (U.S.) (grant CHE-0907905)National Institutes of Health (U.S.) (Postdoctoral Fellowship (5 F32 GM085930)

Nitroreductase-Activatable Morpholino Oligonucleotides for <i>in Vivo</i> Gene Silencing

Phosphorodiamidate morpholino oligonucleotides are widely used to interrogate gene function in whole organisms, and light-activatable derivatives can reveal spatial and temporal differences in gene activity. We describe here a new class of caged morpholino oligonucleotides that can be activated by the bacterial nitroreductase NfsB. We characterize the activation kinetics of these reagents <i>in vitro</i> and demonstrate their efficacy in zebrafish embryos that express NfsB either ubiquitously or in defined cell populations. In combination with transgenic organisms, such enzyme-actuated antisense tools will enable gene silencing in specific cell types, including tissues that are not amenable to optical targeting

Optochemical Dissection of T‑box Gene-Dependent Medial Floor Plate Development

In addition to their cell-autonomous roles in mesoderm development, the zebrafish T-box transcription factors <i>no tail a</i> (<i>ntla</i>) and <i>spadetail</i> (<i>spt</i>/<i>tbx16</i>) are required for medial floor plate (MFP) formation. Posterior MFP cells are completely absent in zebrafish embryos lacking both Ntla and Spt function, and genetic mosaic analyses have shown that the two T-box genes promote MFP development in a non-cell-autonomous manner. On the basis of these observations, it has been proposed that Ntla/Spt-dependent mesoderm-derived signals are required for the induction of posterior but not anterior MFP cells. To investigate the mechanisms by which Ntla and Spt regulate MFP development, we have used photoactivatable caged morpholinos (cMOs) to silence these T-box genes with spatiotemporal control. We find that posterior MFP formation requires Ntla or Spt activity during early gastrulation, specifically in lateral margin-derived cells that converge toward the midline during epiboly and somitogenesis. Nodal signaling-dependent MFP specification is maintained in the absence of Ntla and Spt function; however, midline cells in <i>ntla</i>;<i>spt</i> morphants exhibit aberrant morphogenetic movements, resulting in their anterior mislocalization. Our findings indicate that Ntla and Spt do not differentially regulate MFP induction along the anterior–posterior axis; rather, the T-box genes act redundantly within margin-derived cells to promote the posterior extension of MFP progenitors