DESIGN, SYNTHESIS AND APPLICATIONS OF FLUORESCENT AND ELECTROCHEMICAL PROBES

“Seeing is believing” the proverb well suits for fluorescent imaging probes. Since we can selectively and sensitively visualize small biomolecules, organelles such as lysosomes, neutral molecules, metal ions, anions through cellular imaging, fluorescent probes can help shed light on the physiological and pathophysiological path ways. Since these biomolecules are produced in low concentrations in the biochemical pathways, general analytical techniques either fail to detect or are not sensitive enough to differentiate the relative concentrations. During my Ph.D. study, I exploited synthetic organic techniques to design and synthesize fluorescent probes with desirable properties such as high water solubility, high sensitivity and with varying fluorescent quantum yields. I synthesized a highly water soluble BOIDPY-based turn-on fluorescent probe for endogenous nitric oxide. I also synthesized a series of cell membrane permeable near infrared (NIR) pH activatable fluorescent probes for lysosomal pH sensing. Fluorescent dyes are molecular tools for designing fluorescent bio imaging probes. This prompted me to design and synthesize a hybrid fluorescent dye with a functionalizable chlorine atom and tested the chlorine re-activity for fluorescent probe design. Carbohydrate and protein interactions are key for many biological processes, such as viral and bacterial infections, cell recognition and adhesion, and immune response. Among several analytical techniques aimed to study these interactions, electrochemical bio sensing is more efficient due to its low cost, ease of operation, and possibility for miniaturization. During my Ph.D., I synthesized mannose bearing aniline molecule which is successfully tested as electrochemical bio sensor. A Ferrocene-mannose conjugate with an anchoring group is synthesized, which can be used as a potential electrochemical biosensor

Nitro-group functionalization of dopamine and its contribution to the viscoelastic properties of catechol-containing nanocomposite hydrogels

Linear polyacrylamide (PAAm) is modified with dopamine or nitrodopamine (PAAm-D and PAAm-ND, respectively) to evaluate the effect of nitro-group modification on the interfacial binding properties of polymer-bound catechol. Nanocomposite hydrogels are prepared by mixing PAAm-based polymers with Laponite and the viscoelastic properties of these materials are determined using oscillatory rheometry. The incorporation of a small amount of catechol (≈0.1 wt% in swollen hydrogel) drastically increases the shear moduli by 1–2 orders of magnitude over those of the catechol-free control. Additionally, PAAm-ND exhibits higher shear moduli values than PAAm-D across the whole pH range tested (pH 3.0–9.0). Based on the calculated effective crosslinking density, effective functionality, and molecular weight between crosslinks, nitro-group functionalization of dopamine results in a polymer network with increased crosslinking density and crosslinking points with higher functionality. Nitro-functionalization enhances the interfacial binding property of dopamine and increases its resistant to oxidation, which results in nanocomposite hydrogels with enhanced stiffness and a viscous dissipation property

Deep-red emissive conjugated poly(2,6-BODIPY-ethynylene)s bearing alkyl side chains

Novel deep-red emissive poly(2,6-BODIPY-ethynylene)s bearing dodecyl side chains (polymers A, B, and C) have been prepared by palladium-catalyzed Sonogashira polymerization of 2,6-diiodo-functionalized BODIPY monomers with 2,6-diethynyl-functionalized BODIPY monomers. These polymers emit in the deep-red region with emission maxima at up to 690 nm, and exhibit significant red shifts (up to 166 and 179 nm) of both absorption and emission maxima compared with their parent BODIPY dyes due to significant extension of π-conjugation. These polymers possess good thermal stability with decomposition temperature between 270 and 360°C. The polymers exhibit a little larger Stokes shifts and shorter lifetime than their corresponding BODIPY dyes. The solid state thin films of polymers A, B, and C emit in near-infrared region between 723 and 743 nm, and show significantly red shifts (up to 57 nm) in absorption and emission maxima relative to their polymer solution

Synthesis and optical properties of red and deep-red emissive polymeric and copolymeric BODIPY dyes

Deep-red emissive polymeric BODIPY dyes (polymers A and B), poly(2,6-BODIPY-ethynylene)s, were prepared by palladium-catalyzed Sonogashira polymerization of 2,6-diiodo-functionalized BODIPY monomers with 2,6-diethynyl-functionalized BODIPY monomers. Poly(2,6-BODIPY-ethynylene)s emit in the deep-red region with emission spectral maxima at around 680 nm and exhibit significant red shifts (up to 163 and 172 nm) of both absorption and emission maxima compared with their initial BODIPY dyes due to significant extension of π-conjugation. Red emissive copolymeric BODIPY dyes (polymers C, D, and E) were also prepared by palladium-catalyzed Sonogashira polymerization of a diethynyl-functionalized BODIPY monomer with 9,9-bis(6′-(hexylthio)hexyl)-2,7-diiodo-9H-fluorene, 1,4-diiodo-2,5-didecyloxybenzene, and 2,5-diiodo-3-decylthiophene, respectively. Incorporation of different band gap monomer units into poly(2,6-BODIPY-ethynylene)s resulted in copolymers with a range of emission wavelengths from 641 to 664 nm. The fluorescence lifetimes of these polymers (polymers A−D) are from 2.8 to 3.8 ns except the copolymer with thiophene moieties (polymer E), which displays a much shorter lifetime of 0.23 ns with low fluorescence quantum yield due to efficient intersystem crossing induced by the heavy atom effect of sulfur

Highly water-soluble BODIPY-based fluorescent probe for sensitive and selective detection of nitric oxide in living cells

A highly water-soluble BODIPY dye bearing electron-rich o-diaminophenyl groups at 2,6-positions was prepared as a highly sensitive and selective fluorescent probe for detection of nitric oxide (NO) in living cells. The fluorescent probe displays an extremely weak fluorescence with fluorescence quantum yield of 0.001 in 10 mM phosphate buffer (pH 7.0) in the absence of NO as two electron-rich o-diaminophenyl groups at 2,6-positions significantly quench the fluorescence of the BODIPY dye via photoinduced electron transfer mechanism. The presence of NO in cells enhances the dye fluorescence dramatically. The fluorescent probe demonstrates excellent water solubility, membrane permeability, and compatibility with living cells for sensitive detection of NO. © 2013 American Chemical Society

Near-infrared fluorescent probe for sensitive detection of Pb(II) ions in living cells

A new near-infrared fluorescent probe (NIR-PbP) for sensitive detection of Pb(II) ions in solution and living cells has been rationally designed and synthesized. The NIR-PbP is inherently non-fluorescent and gains fluorescence in the presence Pb(II) ions. The ion detection is based on Pb(II)-induced unmasking the fluorophore through the opening of the spyrocycle, with more than 500-fold fluorescence enhancement for sub-micromolar Pb(II) concentration. The NIR-PbP has high sensitivity, good photo-stability, low detection limit, and reversible response to Pb(II) ions

Highly Water-Soluble BODIPY-Based Fluorescent Probe for Sensitive and Selective Detection of Nitric Oxide in Living Cells

A highly water-soluble BODIPY dye bearing electron-rich <i>o</i>-diaminophenyl groups at 2,6-positions was prepared as a highly sensitive and selective fluorescent probe for detection of nitric oxide (NO) in living cells. The fluorescent probe displays an extremely weak fluorescence with fluorescence quantum yield of 0.001 in 10 mM phosphate buffer (pH 7.0) in the absence of NO as two electron-rich <i>o</i>-diaminophenyl groups at 2,6-positions significantly quench the fluorescence of the BODIPY dye via photoinduced electron transfer mechanism. The presence of NO in cells enhances the dye fluorescence dramatically. The fluorescent probe demonstrates excellent water solubility, membrane permeability, and compatibility with living cells for sensitive detection of NO

Thiophene bridged aldehydes (TBAs) image ALDH activity in cells via modulation of intramolecular charge transfer.

Aldehyde dehydrogenases (ALDHs) catalyze the oxidation of an aldehyde to a carboxylic acid and are implicated in the etiology of numerous diseases. However, despite their importance, imaging ALDH activity in cells is challenging due to a lack of fluorescent imaging probes. In this report, we present a new family of fluorescent probes composed of an oligothiophene flanked by an aldehyde and an electron donor, termed thiophene-bridged aldehydes (TBAs), which can image ALDH activity in cells. The TBAs image ALDH activity via a fluorescence sensing mechanism based on the modulation of intramolecular charge transfer (ICT) and this enables the TBAs and their ALDH-mediated oxidized products, thiophene-bridged carboxylates (TBCs), to have distinguishable fluorescence spectra. Herein, we show that the TBAs can image ALDH activity in cells via fluorescence microscopy, flow cytometry, and in a plate reader. Using TBA we were able to develop a cell-based high throughput assay for ALDH inhibitors, for the first time, and screened a large, 1460-entry electrophile library against A549 cells. We identified α,β-substituted acrylamides as potent electrophile fragments that can inhibit ALDH activity in cells. These inhibitors sensitized drug-resistant glioblastoma cells to the FDA approved anti-cancer drug, temozolomide. The TBAs have the potential to make the analysis of ALDH activity in cells routinely possible given their ability to spectrally distinguish between an aldehyde and a carboxylic acid