Date of Award

Fall 2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Chemistry - (Ph.D.)

Department

Chemistry and Environmental Science

First Advisor

Lev N. Krasnoperov

Second Advisor

Arkady Mustaev

Third Advisor

Edgardo Tabion Farinas

Fourth Advisor

Tamara M. Gund

Fifth Advisor

Haidong Huang

Abstract

A variety of contemporary analytical platforms in technical and biological applications take advantage of labeling the objects of interest with fluorescent or luminescent tracers. Luminescent tracers take advantage of the unique property of some lanthanide metals to absorb and emit light. Long lifetime of lanthanide emission allows temporal gating of the signal, which avoids the short-lived background of interfering sample components. This property in combination with large Stokes shift contributes to extreme sensitivity of detection (ca. 10-13-10-14 M), which makes lanthanide-based probes suitable for large variety of challenging tasks ( e.g., intracellular detection of single DNA/RNA, or protein molecules, microbial pathogen detection in human specimens, tracing analysis, etc.). Luminescent probes include antenna fluorophore that absorbs light and transfers the excitation energy to a lanthanide metal tethered to antenna through chelation. The probe also contains crosslinking group that allows covalent labeling of the molecule of interest.

Despite great potentials of lanthanide-based tracers the wide spread of the technology is impeded by very high price of commercially available probes to their complex structure. The goal is to develop novel approaches for the synthesis of lanthanide probes with improved quantum efficiency. This work discusses development of new strategies for synthesis of antena-fluorophores, development of new methods for introduction of the crosslinking groups in the luminescent probes and elucidates the mechanisms of chemical reactions leading to principal synthetic intermediates.

New quinoline and quinolone based fluorescent compounds were synthesized, whose light emission can be conveniently tuned by simple structural modifications. Developed probes represent high-quantum yield, large Stokes shift fluorophores with amine-reactive and click-reactive groups convenient for conjugation. Some of these compounds can be used as sensitizers for lanthanide emission in design of highly sensitive luminescent probes. Obtained probes demonstrate efficient derivatization reactions allowing introduction of amine- or click-reactive crosslinking groups into the fluorophores. The reactivity of synthesized compounds is confirmed in reaction with low molecular weight nucleophiles as well as with click-reactive DNA-oligonucleotide counterparts. These reactive derivatives can be used for covalent attachment of the fluorophores to various biomolecules of interest including nucleic acids, proteins, live cells and small cellular metabolites. Synthesized compounds were characterized using N MR, steady-state and time-resolved fluorescence spectroscopy, as well UV absorption spectroscopy.

This work also discusses the development of fluorescent derivatives of the antifungal drugs, posaconazole and caspofungin, for diagnostic imaging of fungal cells. Invasive fungal infections (IFI’s) are a growing threat to human health particularly, with increase in the number of immune-compromised patient population, such as organ transplant recipients, al logeneic B MT, hematologic cancers; AIDS etc. The diagnosis of fungal infections is a complicated task and requires a combination of clinical observations, laboratory investigation, and radiological or other diagnostic imaging methods. Only 25% of I FI cases are diagnosed pre-mortem due to the current diagnostic challenges, which justifies the development of express diagnostic procedures. To address the issue fluorescent derivatives of the antifungal drugs, posaconazole and caspofungin, were synthesized. The fluorescent derivatives retained strong and highly specific binding to their cellular targets rendering the cells fluorescent. This new affinity-based approach strongly facilitates the detection of fungal pathogen thereby overcoming the current diagnostic challenges and can be used for clinical diagnostics. The power of this approach is not limited to fungal pathogens, but in fact represents a broader platform useful for detection of other classes of infections, both in the biological specimens and in the whole body using contemporary 3-D imaging approaches like fluorescence based in vivo imaging, luminescence imaging, positron emission tomography approach, and computer-assisted X-ray tomography.

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Chemistry Commons

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