Molecular engineering strategies for expanding the capabilities of fluorescent zinc (II) sensors

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2004.Vita.Includes bibliographical references.(cont.) affords the two fluorophores, such that excitation of the coumarin at 445 nm and measurement of the emission at 488 nm affords information of the amount of sensor present, while excitation of the fluorescein at 505 nm and measurement of the emission at 535 nm indicates the amount of sensor in the zinc(II)-bound form. This system has been characterized and applied to the study of exogenous zinc(II) fluxes in HeLa cells. Chapter 4: Unimolecular Two-Fluorophore Ratiometric Zn²⁺ Sensing Systems. Dichlorofluorescein compounds covalently bound to zinc(II)-insensitive reporter fluorophores via a rigid cyclohexyl linker have been prepared and characterized. Based on favorable photophysical properties, a Zinpyr-1 species covalently bound to coumarin 343 has been prepared and shown to afford a ratiometric response to excess zinc(II). Chapter 5: ZPI Synthons for Functionalization of Biological Targets. Installation of a functional group prior to Mannich reaction is impractical in many cases. This chapter describes the preparation of reactive ZP1 synthons for direct functionalization of biological targets containing an amine or azide, and reports applications to the synthesis of ZPI conjugates. Appendix 1: Synthetic Approaches to Other Isomerically Pure Functionalizable Fluorophores. Crystallization approaches have been applied to separate fluorescein 5- and 6-sulfonic acid, and subsequent generation of the sulfonyl chlorides is discussed. A dibromofluoran approach to isomerically pure rhodamine carboxylates is based on a similar separation. Basic hydrolysis of the previously described 2',7'-dichlorofluorescein ...Chapter 1: The Development and Use of Fluorescent Sensors in the Imaging of Physiological Zinc(II): A Review This chapter presents an overview of fluorescent techniques used to image chelatable zinc(II) in vivo. Many intensity-based sensors take advantage of photoinduced electron transfer quenching pathways. Peptide- and protein-based sensors offer excellent selectivity but are poorly suited to intracellular applications. Recently, ratiometric sensors in which the zinc(II) binding event interrupts or alters conjugation within the fluorophore have been described. Chapter 2: Carboxylate-Functionalized Zinpyr-1 Sensors: Synthesis, Characterization, and In Vivo Staining Patterns. A class of Zinpyr-1 sensors containing a carboxylic acid or ester at the 5- or 6-position of the fluorescein has been prepared. These sensors offer decreased background fluorescence and enhanced fluorescence response compared to the parent Zinpyr-1. The acid-functionalized sensors bear a negative charge at physiological pH, rendering them cell-impermeable. The esterified sensors are cell-permeable, but are hydrolyzed in vivo by intracellular esterases, affording a clear delineation of zinc(II)-containing damaged neurons in mechanically-injured or seizure-induced rats, rather than the punctate staining pattern obtained with Zinpyr-1. Chapter 3: Esterase-Dependent Two-Fluorophore Ratiometric Sensing of Zinc(II). This chapter describes a new approach to ratiometric sensing in which a zinc(II)-sensitive fluorescein fluorophore based on Zinpyr-1 is functionalized with a zinc(lI)-insensitive coumarin fluorophore via a flexible ester linker. The flexible linker enables intramolecular quenching of the two fluorophores. Esterase hydrolysis of the linkerby Carolyn C. Woodroofe.Ph.D

Click beetle luciferase mutant and near infrared naphthyl-luciferins for improved bioluminescence imaging

The sensitivity of bioluminescence imaging in animals is primarily dependent on the amount of photons emitted by the luciferase enzyme at wavelengths greater than 620 nm where tissue penetration is high. This area of work has been dominated by firefly luciferase and its substrate, D-luciferin, due to the system's peak emission (~ 600 nm), high signal to noise ratio, and generally favorable biodistribution of D-luciferin in mice. Here we report on the development of a codon optimized mutant of click beetle red luciferase that produces substantially more light output than firefly luciferase when the two enzymes are compared in transplanted cells within the skin of black fur mice or in deep brain. The mutant enzyme utilizes two new naphthyl-luciferin substrates to produce near infrared emission (730 nm and 743 nm). The stable luminescence signal and near infrared emission enable unprecedented sensitivity and accuracy for performing deep tissue multispectral tomography in mice

Fluorine-18 Labeled Urea-Based Ligands Targeting Prostate-Specific Membrane Antigen (PSMA) with Increased Tumor and Decreased Renal Uptake

High expression of prostate-specific membrane antigen (PSMA) in prostate cancers prompted the development of the PSMA-targeted PET-imaging agent [18F]DCFPyL, which was recently approved by the FDA. Fluorine-18-labeled Lys–Urea–Glu-based oxime derivatives of [18F]DCFPyL were prepared for the comparison of their in vitro and in vivo properties to potentially improve kidney clearance and tumor targeting. The oxime radiotracers were produced by condensation of an aminooxy functionalized PSMA-inhibitor Lys–Urea–Glu scaffold with fluorine-18-labeled aldehydes. The radiochemical yields were between 15–42% (decay uncorrected) in 50–60 min. In vitro saturation and competition binding assays with human prostate cancer cells transfected with PSMA, PC3(+), indicated similar high nM binding affinities to PSMA for all radiotracers. In vivo biodistribution studies with positive control PC3(+) tumor xenografts showed that the kidneys had the highest uptake followed by tumors at 60 min. The PC3(+) tumor uptake was blocked with non-radioactive DCFPyL, and PC3(−) tumor xenograft (negative control) tumor uptake was negligible indicating that PSMA targeting was preserved. The most lipophilic tracer, [18F]2a, displayed comparable tumor-targeting to [18F]DCFPyL and a desirable alteration in pharmacokinetics and metabolism, resulting in significantly lower kidney uptake with a shift towards hepatobiliary clearance and increased liver uptake

Mass Spectrometry Compatible Surfactant for Optimized In-Gel Protein Digestion

Identification of proteins resolved by SDS-PAGE depends on robust in-gel protein digestion and efficient peptide extraction, requirements that are often difficult to achieve. A lengthy and laborious procedure is an additional challenge of protein identification in gel. We show here that with the use of the mass spectrometry compatible surfactant sodium 3-((1-(furan-2-yl)­undecyloxy)­carbonylamino)­propane-1-sulfonate, the challenges of in-gel protein digestion are effectively addressed. Peptide quantitation based on stable isotope labeling showed that the surfactant induced 1.5–2 fold increase in peptide recovery. Consequently, protein sequence coverage was increased by 20–30%, on average, and the number of identified proteins saw a substantial boost. The surfactant also accelerated the digestion process. Maximal in-gel digestion was achieved in as little as one hour, depending on incubation temperature, and peptides were readily recovered from gel eliminating the need for postdigestion extraction. This study shows that the surfactant provides an efficient means of improving protein identification in gel and streamlining the in-gel digestion procedure requiring no extra handling steps or special equipment

Click beetle luciferase mutant and near infrared naphthyl-luciferins for improved bioluminescence imaging

textabstractThe sensitivity of bioluminescence imaging in animals is primarily dependent on the amount of photons emitted by the luciferase enzyme at wavelengths greater than 620 nm where tissue penetration is high. This area of work has been dominated by firefly luciferase and its substrate, D-luciferin, due to the system's peak emission (~ 600 nm), high signal to noise ratio, and generally favorable biodistribution of D-luciferin in mice. Here we report on the development of a codon optimized mutant of click beetle red luciferase that produces substantially more light output than firefly luciferase when the two enzymes are compared in transplanted cells within the skin of black fur mice or in deep brain. The mutant enzyme utilizes two new naphthyl-luciferin substrates to produce near infrared emission (730 nm and 743 nm). The stable luminescence signal and near infrared emission enable unprecedented sensitivity and accuracy for performing deep tissue multispectral tomography in mice

Novel Heterocyclic Analogues of Firefly Luciferin

Five novel firefly luciferin analogues in which the benzothiazole ring system of the natural substrate was replaced with benzimidazole, benzofuran, benzothiophene, benzoxazole, and indole were synthesized. The fluorescence, bioluminescence, and kinetic properties of the compounds were evaluated with recombinant <i>Photinus pyralis</i> wild type luciferase. With the exception of indole, all of the substrates containing heterocycle substitutions produced readily measurable flashes of light with luciferase. Compared to that of luciferin, the intensities ranged from 0.3 to 4.4% in reactions with varying pH optima and times to reach maximal intensity. The heteroatom changes influenced both the fluorescence and bioluminescence emission spectra, which displayed maxima of 479–528 and 518–574 nm, respectively. While there were some interesting trends in the spectroscopic and bioluminescence properties of this group of structurally similar substrate analogues, the most significant findings were associated with the benzothiophene-containing compound. This synthetic substrate produced slow decay glow kinetics that increased the total light-based specific activity of luciferase more than 4-fold versus the luciferin value. Moreover, over the pH range of 6.2–9.4, the emission maximum is 523 nm, an unusual 37 nm blue shift compared to that of the natural substrate. The extraordinary bioluminescence properties of the benzothiophene luciferin should translate into greater sensitivity for analyte detection in a wide variety of luciferase-based applications

NanoBRETA Novel BRET Platform for the Analysis of Protein–Protein Interactions

Dynamic interactions between proteins comprise a key mechanism for temporal control of cellular function and thus hold promise for development of novel drug therapies. It remains technically challenging, however, to quantitatively characterize these interactions within the biologically relevant context of living cells. Although, bioluminescence resonance energy transfer (BRET) has often been used for this purpose, its general applicability has been hindered by limited sensitivity and dynamic range. We have addressed this by combining an extremely bright luciferase (Nanoluc) with a means for tagging intracellular proteins with a long-wavelength fluorophore (HaloTag). The small size (19 kDa), high emission intensity, and relatively narrow spectrum (460 nm peak intensity) make Nanoluc luciferase well suited as an energy donor. By selecting an efficient red-emitting fluorophore (635 nm peak intensity) for attachment onto the HaloTag, an overall spectral separation exceeding 175 nm was achieved. This combination of greater light intensity with improved spectral resolution results in substantially increased detection sensitivity and dynamic range over current BRET technologies. Enhanced performance is demonstrated using several established model systems, as well as the ability to image BRET in individual cells. The capabilities are further exhibited in a novel assay developed for analyzing the interactions of bromodomain proteins with chromatin in living cells