Synthesis and Fluorescence Imaging of π-Extended Aromatic Ring Fused Rhodamines

Author:Wang Qing

Supervisor:zhao lian cheng zeng xian shun


Degree Year:2018





Rhodamine dyes have been broadly used as platforms to design fluorescent probes because of their advantages such as high molar extinction coefficients,excellent quantum yields,good photobleaching and biocompatibility.However,most rhodamine dyes have their absorption and emission maxima in the visible region(500-600 nm),which renders them unsuitable for in vivo imaging.Therefore,the structures of rhodamines need to be modified to extend their absorption and emission wavelengths to deep-red and NIR regions to make them ideal biomarkers or fluorophores for probe design.Based on the systematical summary of present literatures,in this paper,a series of fluorescent rhodamine dyes have been designed and synthesized using the method of fusing aromatic rings to the core xanthene skeleton of rhodamine to extend theπ-conjugation system.The spectral properties of these dyes and their applications in biological imaging and probe design are systematically studied.Two fluorescent rhodamine dyes fused with fluorescein were designed by sequential Friedel–Crafts acylation reactions.With the addition of external stimuli such as acid,alkali or metal ions in methanol solution,the two spirolactone rings of each dyes exhibited selectively"open-off"character,which can be used to regulate the spectral properties and solutions color changes of the dyes.The two spirolactone rings of both dyes S1 and S2 were completely opened in neutral and alkaline aqueous solutions,and the maximum emission wavelengths for both dyes were above 610 nm.The obvious red-shift of the maximum emission wavelengths of both dyes compared with the template rhodamine proved the feasibility of our design.Using the changes in the UV/Vis absorption spectra under the influence of H+and OH-,two half-subtractors that can operate subtraction operation were achieved.Finally,based on the ring opening character in aqueous phase,both S1 and S2 can localize at mitochondria selectively.Using intramolecular SNArH reaction,a rhodamine dye fused with benzoxazine(RO1)has been synthesized successfully.The SNArH reaction presented herein can be performed under mild reaction temperatures without any expensive transition metal catalyst,extra strong bases or oxidant.To evaluate the influences of some factors,such as electronic electron-donating group,electron-withdrawing group,as well as further increasing the conjugation system on the feasibility of this SNArH reaction and spectral properties of dyes,we synthesized another four rhodamine dyes RO2-RO5.RO4 was synthesized by replacing the benzene of benzoxazine in RO1 with pyridine;RO3 and RO3 contain an electron-donating methoxyl group;RO5 is a quinoline-fused rhodamine with six fused rings.The successful synthesis of dyes RO1-RO5 proved the feasibility of this SNArH strategy.The maximum emission wavelengths of these five dyes are all above 650 nm,showed obvious red-shift compared with that of the template rhodamine.Using RO1 as a template,four probes(two probes for Hg2+and two probes for Cu2+,respectively)were synthesized by modifying the spirolactone of RO1 All these probes can recognize target ions with high selectivity and sensitivity.Linking the nitrogen atom on oxazine ring of RO1 with morpholine moiety,another dye(RO1-MPL)that can target lysosomes of cells was synthesized successfully.Based on the successful design and synthesis of dyes RO1-RO5 using the SNArH strategy,two fluorescent rhodamine dyes(RS1 and RS2)were synthesized by introducing aromatic thiazine ring into xanthene dyes.Utilizing the response of sulfur atoms to hypochlorite,RS1 and RS2 can be used as fluorescent probes for hypochlorite.Due to the introduction of sulfur atoms,the emission wavelengths of RS1 and RS2exhibit further red-shift(to above 700 nm).After reacted with hypochlorite,the fluorescence emission wavelength of the probe showed hypsochromic shift(to590nm),which makes RS1 and RS2 can be used as ratiometric fluorescence probes for hypochlorite.The probes can target lysosomes with good photostability,which enable them to ratiometrically image hypochlorite in intracellular lysosomes.Based on the mechanism of oxidating 2-aminophenol to o-quinoneimide by ClO-,the intermediate 4X that obtained in the process of synthesizing near-infrared fluorescent dyes can be used for detecting ClO-with high selectivity.At the same times,4X can be used to image ClO-in cells.Inspired by probe 4X,the oxygen atom on2-aminophenol was replaced by nitrogen and a new probe ROPD with o-phenylenediamine group as the reaction site was successfully synthesized.Probe ROPD exhibits high selectivity and sensitivity towards NO with fast response and can be used for the detection of endogenous and exogenous NO in three different cell lines.