RESUMO
Rhodamine spirolactams (RSLs) have recently emerged as popular fluorescent pH probes due to their fluorescence turn-on capability and ease of functionalization at the spirolactam nitrogen. Design of RSLs is often driven by biological targeting or compatibility concerns, rather than the pH sensitivity of the probe, and the relationship between RSL structure and pKa is not well understood. To elucidate the relationship between pKa values and the properties of substituents attached to the spirolactam nitrogen, a series of 19 aniline-derived RSLs is presented. RSLs derived from di-ortho-substituted anilines exhibit pKa tunability across the moderately acidic region (ca. pHâ 4-6). Evaluation of pKa data using the Fujita-Nishioka model for ortho substituent effects reveals that both steric and electronic substituent properties influence RSL pH responsiveness, with pKa values increasing as substituent size and electron withdrawing character increase. These trends are attributed to changes in the RSL structure induced by large substituents, and to electronic influences on the protonated spirocyclic reaction intermediate. To demonstrate the practical applicability of these probes in completely aqueous environments, RSL-doped conjugated polymer nanoparticles that exhibit a ratiometric fluorescence response to changing pH levels are presented.
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The ability to modulate protein function through minimal perturbations to amino acid structure represents an ideal mechanism to engineer optimized proteins. Due to the novel spectroscopic properties of green fluorescent protein, it has found widespread application as a reporter protein throughout the fields of biology and chemistry. Using site-specific amino acid mutagenesis, we have incorporated various fluorotyrosine residues directly into the fluorophore of the protein, altering the fluorescence and shifting the pKa of the phenolic proton associated with the fluorophore. Relative to wild type GFP, the fluorescence spectrum of the protein is altered with each additional fluorine atom, and the mutant GFPs have the potential to be employed as pH sensors due to the altered electronic properties of the fluorine atoms.
Assuntos
Aminoácidos/química , Fluorescência , Proteínas de Fluorescência Verde/química , Técnicas Biossensoriais , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Halogenação , Modelos Moleculares , Conformação Molecular , Mutação , Espectrometria de Fluorescência/métodosRESUMO
The emission of conjugated polymer nanoparticles (CPNs or Pdots) is often tailored for specific uses by functionalizing CPNs with dyes that act as fluorescence resonance energy transfer (FRET) acceptors. A number of dye functionalization methods for CPNs have been developed, ranging from simple noncovalent doping to covalent attachment. We seek to develop guidelines for when noncovalent doping is acceptable and when covalent attachment is necessary to achieve the desired result. We present results of CPNs functionalized with photochromic spirooxazines by four different methods: simple doping, doping with an amphiphilic coating polymer, covalent functionalization prior to CPN formation, and covalent functionalization after CPN formation. The different CPNs are evaluated in terms of their fluorescence photomodulation properties to determine how the preparation method affects the CPN-dye photophysical interactions. Doping preparations yield the most efficient quenching of CPN emission due to shorter donor-acceptor distances in these CPNs compared to those with covalently tethered dyes. Aging studies reveal that the photochromic dyes in doped samples degrade over time to a far greater extent than those in covalently functionalized samples. These results suggest that dye-doped CPNs are appropriate for short-term experiments where highly efficient FRET is desired while covalent dye functionalization is a better choice for experiments executed over an extended time frame.
RESUMO
Fluorescent turn-on probes based on a rhodamine spirolactam (RSL) structure have recently become a popular means of detecting pH, metal ions, and other analytes of interest. RSLs are colorless and non-fluorescent until the target analyte induces opening of the spirocyclic ring system, revealing the fully conjugated and highly fluorescent rhodamine dye. Among RSLs opened by acid, we have observed wide variation in the kinetics of the fluorescence turn-on process such that some probes would not be usable in situations where a rapid reading is desired or the pH fluctuates temporally. Herein we present a systematic investigation of the fluorescence turn-on kinetics of RSLs to probe the hypothesis that the reaction rates are influenced by the electronic properties of the spirolactam ring system. A series of 8 aniline-derived RSLs with para substituents ranging from electron-donating to electron-withdrawing was prepared from rhodamine B. The fluorescence turn-on rates are observed to increase by a factor of four as the substituent is tuned from methoxy to nitro. This effect is explained in terms of the destabilization of the reaction intermediate by the substituent. As the reaction rates increase across the series, a concomitant increase in fluorescence intensity is also observed. This result is attributed to an increase in the concentration of the fluorescent form of the dye and is consistent with the expected equilibrium properties of this system. These findings are applied to the design of a faster-reacting and more intensely fluorescent RSL pH probe.
Assuntos
Corantes Fluorescentes/química , Rodaminas/química , Concentração de Íons de Hidrogênio , Cinética , Estrutura MolecularRESUMO
Conjugated polymer nanoparticles (CPNs or Pdots) have become increasingly popular fluorophores for multimodal applications that combine imaging with phototherapeutic effects. Reports of CPNs in photodynamic therapy applications typically focus on their ability to generate singlet oxygen. Alternatively, CPN excited states can interact with oxygen to form superoxide radical anion and a CPN-based hole polaron, both of which can have deleterious effects on fluorescence properties. Here, we demonstrate that CPNs prepared from the common conjugated polymer poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-co-(1,4-benzo-{2,1',3}-thiadiazole)] (PFBT, also known as F8BT) generate superoxide upon irradiation. We use the same CPNs to detect superoxide by doping them with a superoxide-responsive hydrocyanine dye developed by Murthy and co-workers. Superoxide induces off-to-on fluorescence switching by converting quenching hydrocyanine dyes to fluorescent cyanine dyes that act as fluorescence resonance energy transfer (FRET) acceptors for PFBT chromophores. Amplified FRET from the multichromophoric CPNs yields fluorescence signal intensities that are nearly 50 times greater than when the dye is excited directly or over 100 times greater when signal readout is from the CPN channel. The dye loading level governs the maximum amount of superoxide that induces a change in fluorescence properties and also influences the rate of superoxide generation by furnishing competitive excited state deactivation pathways. These results suggest that CPNs can be used to deliver superoxide in applications in which it is desirable and provide a caution for fluorescence-based CPN applications in which superoxide can damage fluorophores.
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We present dye-doped polymer nanoparticles that are able to detect mercury in aqueous solution at parts per billion levels via fluorescence resonance energy transfer (FRET). The nanoparticles are prepared by reprecipitation of highly fluorescent conjugated polymers in water and are stable in aqueous suspension. They are doped with rhodamine spirolactam dyes that are nonfluorescent until they encounter mercury ions, which promote an irreversible reaction that converts the dyes to fluorescent rhodamines. The rhodamine dyes act as FRET acceptors for the fluorescent nanoparticles, and the ratio of nanoparticle-to-rhodamine fluorescence intensities functions as a ratiometric fluorescence chemodosimeter for mercury. The light harvesting capability of the conjugated polymer nanoparticles enhances the fluorescence intensity of the rhodamine dyes by a factor of 10, enabling sensitive detection of mercury ions in water at levels as low as 0.7 parts per billion.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Mercúrio/análise , Nanopartículas/química , Polímeros/química , Água/análise , Íons/química , Rodaminas/química , Poluentes Químicos da Água/análiseRESUMO
Conjugated polymer nanoparticles (CPNs or Pdots) are used to sensitize the photorelease reaction of a BODIPY photoremovable protecting group. Sensitization yields effective values of ελΦpr - the product of the extinction coefficient at the irradiation wavelength and the photorelease quantum yield - that are more than 60-fold greater than those measured upon direct excitation.
RESUMO
We present fluorescence studies of quenching behavior in photoaddressable azobenzene-substituted derivatives of the fluorescent conjugated polymer poly(p-phenylenevinylene) (PPV). The azobenzene side chains partially quench the PPV fluorescence, and we have shown previously that the quenching efficiency is greater when the azobenzene side chains are cis than when they are trans. This effect provides a photoaddressable means of modulating the fluorescence intensity of PPV derivatives. To optimize the efficiency of photoinduced intensity modulation, it is important to understand the molecular nature of quenching by both trans- and cis-azobenzene side chains. Here we investigate the photophysical origins of quenching by the two isomers using steady-state and time-resolved fluorescence spectroscopy. We present results from the azobenzene-modified PPV derivative poly(2-methoxy-5-((10-(4-(phenylazo)phenoxy)decyl)oxy)-1,4-phenylenevinylene) (MPA-10-PPV) and two new related polymers, a copolymer lacking half of the azobenzene side chains and an analogue of MPA-10-PPV with a tert-butyl-substituted azobenzene. These studies reveal that steric interactions influence the extent of PPV emission quenching by trans-azobenzene but do not affect the efficient quenching by cis-azobenzene. The difference in dynamic quenching efficiencies between trans- and cis-azobenzene isomers is consistent with fluorescence resonance energy transfer. These results show that it is possible to control the efficiency of photoswitchable fluorescence modulation through specific structural variations designed to encourage or block quenching by trans-azobenzene. This is a promising approach to providing useful general guidelines for designing photomodulated PPV derivatives.
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Conjugated polymer nanoparticles doped with a reverse photochromic dye exhibit highly quenched fluorescence that can be reversibly activated by controlling the form of the photochrome with visible light.
Assuntos
Luz , Nanopartículas/química , Polímeros/química , FluorescênciaRESUMO
Reverse transcription of the HIV-1 genome is a complex multi-step process. HIV-1 nucleocapsid protein (NC) is a nucleic acid chaperone protein that has been shown to greatly facilitate the nucleic acid rearrangements that precede the minus-strand transfer step in reverse transcription. NC destabilizes the highly structured transactivation response region (TAR) present in the R region of the RNA genome, as well as a complementary hairpin structure ("TAR DNA") at the 3'-end of the newly synthesized minus-strand strong-stop DNA ((-) SSDNA). Melting of the latter structure inhibits a self-priming (SP) reaction that competes with the strand transfer reaction. In an in vitro minus-strand transfer system consisting of a (-) SSDNA mimic and a TAR-containing acceptor RNA molecule, we find that when both nucleic acids are present, NC facilitates formation of the transfer product and the SP reaction is greatly reduced. In contrast, in the absence of the acceptor RNA, NC has only a small inhibitory effect on the SP reaction. To further investigate NC-mediated inhibition of SP, we developed a FRET-based assay that allows us to directly monitor conformational changes in the TAR DNA structure upon NC binding. Although the majority ( approximately 71%) of the TAR DNA molecules assume a folded hairpin conformation in the absence of NC, two minor "semi-folded" and "unfolded" populations are also observed. Upon NC binding to the TAR DNA alone, we observe a modest shift in the population towards the less-folded states. In the presence of the RNA acceptor molecule, NC binding to TAR DNA results in a shift of the majority of molecules to the unfolded state. These measurements help to explain why acceptor RNA is required for significant inhibition of the SP reaction by NC, and support the hypothesis that NC-mediated annealing of nucleic acids is a concerted process wherein the unwinding step occurs in synchrony with hybridization.
Assuntos
Repetição Terminal Longa de HIV/genética , HIV-1/genética , HIV-1/metabolismo , Conformação de Ácido Nucleico , Proteínas do Nucleocapsídeo/metabolismo , RNA Viral/química , RNA Viral/metabolismo , Transcrição Gênica/genética , DNA Viral/biossíntese , DNA Viral/química , DNA Viral/genética , DNA Viral/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Transferência Ressonante de Energia de Fluorescência , RNA Viral/genética , Ativação TranscricionalRESUMO
Main chain polymeric radicals from several acrylic polymers, produced by laser flash photolysis at 248 nm in liquid solution, have been studied using direct detection time-resolved electron paramagnetic resonance (TREPR) spectroscopy at 9.5 GHz. Highly isotactic poly(methyl methacrylate) (i-PMMA) shows a sharp, well-resolved spectrum at about 95 degrees C. Using synthetic methodology to disrupt the tacticity of i-PMMA, we observed different fast-motion hyperfine coupling constants for the main chain radicals. By raising the temperature of observation, we returned the coupling constants to the same value as those in the highly isotactic sample. This result is related qualitatively to the degree of stiffness of the polymer chains as a function of tacticity. The concept is tested further by comparison to two other acrylic polymers with bulky side chains: poly(fluorooctyl methacrylate) (PFOMA) and poly(adamantyl methacrylate) (PAMA), whose main chain radicals show significant line broadening even at 110 degrees C. Solvent effects on both spectral appearance (the alternating line-width effect) and kinetic decays (attributed to T1 relaxation) are also presented and discussed in terms of main chain conformational motion.
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X-band time-resolved electron paramagnetic resonance (TREPR) spectra of three flexible biradicals of varying chain length and structure were obtained in liquid and supercritical carbon dioxide (CO2) solutions and compared to conventional solvents. For C16 acyl-alkyl biradical 1a, an average spin exchange interaction between the radical centers, J(avg), was obtained by spectral simulation using a simple model for spin-correlated radical pairs (SCRPs) and a small amount of T2 relaxation from a previously established J modulation mechanism. A large solvent effect on J(avg) was observed for the first time, varying by almost 1 order of magnitude from CO2 (J(avg) = -120 +/- 10 MHz) to heavy mineral oil (-11 +/- 3 MHz) for 1a. For C15 bis(alkyl) biradical 1b, spectra obtained under supercritical conditions are only slightly different from those detected in liquid CO2 but differ from spectra taken in benzene. For C10 acyl-alkyl biradical 2a, more emissive spin polarization due to S-T- mixing is observed in CO2 than in benzene. These results are discussed in terms of solvent properties such as dielectric constant, viscosity, and specific interactions. Both chain dynamics and changes to the equilibrium distribution of end-to-end distances can alter J(avg) and the observed ratio of S-T0 to S-T- mixing; however, faster chain dynamics is concluded to be the most likely cause of the observed effects in these systems.
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We report fluorescence studies of phototriggered changes in spectral position and shape for two azobenzene-functionalized poly(p-phenylenevinylene) derivatives, poly(2-methoxy-5-(4-phenylazophenyl-4'-(1,10-dioxydecyl))-1,4-phenylenevinylene) (MPA-10-PPV) and poly(2-hexyloxy-5-(4-phenylazophenyl-4'-(1,10-dioxydecyl))-1,4-phenylenevinylene) (HPA-10-PPV). Upon trans --> cis azobenzene photoisomerization, small (ca. 1 nm) blue shifts in spectral position are observed for MPA-10-PPV in 100% toluene, a good solvent for this polymer. These shifts are reversed upon visible irradiation and can be cycled many times. To probe the dependence of these shifts on initial polymer conformation, a dichloromethane-methanol cosolvent study was performed in which the solvent quality was decreased incrementally to induce a reduction in polymer coil dimensions. Unirradiated dichloromethane solutions of both MPA-10-PPV and HPA-10-PPV showed a red shift and reduction in quantum yield with increasing methanol concentration as expected based on literature results for other poly(p-phenylenevinylene) derivatives. These changes have been attributed to a dramatic conformational collapse by others and occur for these azo polymers over the 30-60% (v/v) methanol range. While little or no light-induced spectral shifting was observed at low (
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Reverse transcription of the HIV-1 RNA genome involves several complex nucleic acid rearrangement steps that are catalyzed by the HIV-1 nucleocapsid protein (NC), including for example, the annealing of the transactivation response (TAR) region of the viral RNA to the complementary region (TAR DNA) in minus-strand strong-stop DNA. We report herein single-molecule fluorescence resonance energy transfer measurements on single immobilized TAR DNA hairpins and hairpin mutants complexed with NC (i.e., TAR DNA/NC). Using this approach we have explored the conformational distribution and dynamics of the hairpins in the presence and absence of NC protein. The data demonstrate that NC shifts the equilibrium secondary structure of TAR DNA hairpins from a fully "closed" conformation to essentially one specific "partially open" conformation. In this specific conformation, the two terminal stems are "open" or unwound and the other stems are closed. This partially open conformation is arguably a key TAR DNA intermediate in the NC-induced annealing mechanism of TAR DNA.