Design And Synthesis Of Novel Xanthene Dyes And Fluorescent Probes For Bioimaging

Recently, fluorescence imaging is one of the most powerful techniques formonitoring biomolecules in living systems, while fluorescent probe with excellentperformance is the foundation for imaging. Xanthene dyes (including rhodamine andfluoresceine) is one of the most robust platform for fluorescent probe developmentbecause of their excellent photophysical properties. However, there are still someproblems in constructing fluorescent probes based on xanthene dyes, which constrainthe full potential of their applications. First, the vast majority of rhodamine-basedfluorescent probes response to an analyte of interest with optical signal changes onlyin fluorescent intensity. A main limitation of intensity-based probes is that variationsin probe concentration, probe environment, and excitation intensity may beproblematic for utilization in quantitative measurements. Although partial success hasbeen obtained in design of first generation rhodamine-based FRET probes viamodulation of acceptor molar absorptivity, the first generation rhodamine-basedFRET ratiometric fluorescent probes are associated with several drawbacks (e.g.complicated synthetic problems associated with the existing strategies; the existingstrategies is not suitable for analytes which may induce the cleavage of the FRETdyad), further improvements in terms of versatility, sensitivity, and syntheticaccessibility are required. Second, most of xanthene–based fluorescent probes exciteonly in the visible region, which limit their applications on fluorescence imaging forchemical biology and clinical diagnosis due to photodamage to biological samples,limited tissue penetration and interference from background auto-fluorescence inliving systems. Third, xanthene dyes have the fatal disadvantage of very small Stokesshifts (typically less than25nm), which can lead to serious self-quenching andfluorescence detection error due to excitation backscattering effects.Therefore, in this thesis, we are focusing on the integration of organic chemistry,analytical chemistry, materials chemistry, and biology, to conquer the abovebottle-necks in the development of fluorescent probes for bioimaging based onxanthene dyes. For this purpose, we have carried out the following works:(1) To address the issues associated with the first generation rhodamine-basedFRET probes, we have proposed a strategy for the design of second generationrhodamine-based FRET probes. To demonstrate the usefulness of our novel strategy, we have employed it to create FRET imaging probe for diverse targets including Cu2+,NO, and HOCl. When compared to the existing approaches, our strategy has thefollowing advantages: a) The central theme of the strategy is to assemble the FRETplatform prior to the interaction site. Thus, the complicated synthetic problemsassociated with the existing strategies may be alleviated; b) our strategy renders theFRET platforms with an intact2-position carboxyl group, which can be readilyfunctionalized to afford diverse array of interaction sites of interest. Thus, ourstrategy should be robust and simple to be employed; c) our FRET strategy is suitablefor target analytes which could induce the cleavage of the FRET dyad. Furthermore,all of these probe have two well-resolved emission before and after interact with theanalytes, which is highly favorable for the accuracy in determination of thefluorescence ratios.(2) Based on the above work, we present the rational design, synthesis, spectralproperties, and living cell imaging studies of FP-H2O2-NO, the firstsingle-fluorescent-molecule, that can respond to H2O2, NO, and H2O2/NO with threedifferent sets of fluorescence signals. FP-H2O2-NO senses H2O2, NO, and H2O2/NOwith a fluorescence signal pattern of blue-black-black, black-black-red, andblack-red-red, respectively. Significantly, we have further demonstrated for the firsttime that FP-H2O2-NO, a single fluorescent probe, is capable of simultaneouslymonitoring endogenously produced NO and H2O2in living macrophages cells inmulticolor imaging. We envision that FP-H2O2-NO will be a unique molecular tool toinvestigate the interplaying roles of H2O2and NO in the complex interaction networksof the signal transduction and oxidative pathways.(3) Fluorescent probes with absorption and emission in the near infrared (NIR)region are favorable for biological imaging applications in living animals, as NIRlight leads to minimum photo-damage, deep tissue penetration, and minimumbackground auto-fluorescence interference. We have introduced a new strategy todesign NIR functional dyes with the carboxylic-acid-controlled fluorescence on-offswitching mechanism by the spirocyclization. Based on the design strategy, we havedeveloped a series of Changsha NIR fluorophores, a unique new class of NIRfunctional fluorescent dyes. Significantly, the new CS1-6NIR dyes are superior tothe traditional rhodamine dyes with both absorption and emission in the NIR regionwhile retaining the rhodamine-like fluorescence on-off switching mechanism. Inaddition, we have performed quantum chemical calculations with the B3LYPexchange functional employing6-31G*basis sets to shed light on the structure-optical properties of the new CS NIR dyes. Furthermore, we furtherconstructed the novel NIR fluorescent turn-on probe NIR-HOCl, which is capable ofimaging endogenously produced HClO in the living animals.(4) We also describe the development of a new class of NIR fluorescent dyes,namely Huda NIR fluorophores, which are superior to the traditional fluorescein withboth absorption and emission in the NIR region while retaining an optically tunablehydroxyl group. We have performed quantum chemical calculations with the B3LYPexchange functional employing6-31G(d) basis sets to provide insights into thestructure-optical properties of the novel class of NIR fluorescent dyes. The uniqueoptical properties of the new type of fluorescent dyes can be exploited as a newstrategy for development of NIR fluorescent probes with both absorption andemission in the NIR region. Employing this innovative strategy, two different types ofNIR fluorescent probes, NIR-H2O2and NIR-Thiol, for H2O2and thiols, respectively,were constructed. These novel probes respond to H2O2or thiols with a large turn-onNIR fluorescence signal upon excitation in the NIR region. Furthermore, NIR-H2O2and NIR-Thiol are capable of imaging endogenously produced H2O2and thiols,respectively, not only in living cells, but also in living mice, demonstrating the valueof the new NIR fluorescent probe design strategy.(5) We present the rational design, synthesis, spectral properties, and living tissueimaging studies of GCTPOC, a unique family of GFP-chromophore conformationallyrestricted analogues with tunable two-photon action cross-sections. GCTPOC havelarge two-photon excitation action cross-section in the NIR region, especially, thetwo-photon excitation action cross-section of GCTPOC containing an opticallytunable hydroxyl group can be easily controlled by hydroxyl group. Based on thetwo-photon platform, we further constructed the novel two-photon fluorescent thiolprobe. We have further demonstrated that the two-photon fluorescence turn-on probecould stain endogenous thiols in tissue slices, indicating that GCTPOC can beemployed as new two-photon fluorescent platforms for rational design of two-photonfluorescent probes for bioimaging.(6) Finally, we have developed a series of rhodamine derivaties and analogueswith larger stokes shift: a) A new class of coumarin-rhodamine through-bond energytransfer (TBET) cassettes with minimal spectral overlap between the donor emissionand the acceptor absorption show desirable features of very large pseudo-Stokes shifts(up to230nm) and huge emission shifts (up to170nm). The utility of the novelTBET platform for TBET-based probe development was demonstrated by a new ratiometric fluorescent pH probe. b) A novel class of analogues of Changsha (CS) NIRdyes with large Stokes shifts were designed, synthesized and evaluated. Using one ofthe new dyes as a platform, we further developed the new NIR fluorsecent probe,which is suitable for imaging endogenously produced HClO in RAW264.7macrophage cells.Conclusively, we are focusing on the design and synthesis novel xanthene dyes tosolve the bottle-necks in fluorescent probes based on xanthene dyes, and then developa series of fluorescent probes for bioimaging.