Study On The Property Regulation And Imaging Application Of Anthocyanidin-based Fluorescent Dyes

Fluorescence imaging technology has attracted more and more attention because of its advantages such as simplicity,high spatio-temporal resolution,and no damage detection.Among them,the two-photon fluorescence imaging technology has the advantages of low autofluorescence,weak photodamage to biological samples,and deeper tissue penetration,etc.So it is widely applied in cell,tissue and in vivo imaging research.In order to meet the imaging and detection needs of various biomolecules,researchers have developed a large number of functional fluorescent dyes.However,due to the shortcomings of short emission wavelength,small Stokes shift,and the lack of suitable optically tunable group,most of the two-photon fluorescent dyes have faced the problems such as low signal-to-background ratio and low sensitivity in the development of fluorescent probes for biological imaging appl ications.Therefore,long-wavelength two-photon dyes with tunable optical properties can play an important role in the development of high-performance“activated”probes and their applications in biological imaging analysis.Regarding the above problems,t his paper chooses anthocyanidins(2-phenylbenzopyran salts)with good water solubility as the dye core,and through the combination of molecular engineering and density functional theory(DFT)calculations,a series of the long-wavelength two-photon fluorescent dyes with optically tunable hydroxyl group are designed and synthesized.Meanwhile,we constructed a series of fluorescent probes,which realize highly sensitive detection of nitroreductase(NTR)and pH in biological environment.The specific research contents are as follows:Firstly,in response to the common problems such as the short emission wavelength of two-photon fluorescent dyes and the lack of suitable optical tunable groups,a multi-functional long-wavelength fluorophore LDOH-4 with an optically tunable hydroxyl group,which is based on the anthocyanin dye ACF4 developed by our research group,was successfully developed by regulating molecular planarity and the twisted intramolecular charge transfer effect with a protected and deprotected hydroxyl group approach via“step by step”modifying strategy.LDOH-4 has good optical properties,such as suitable p K_a value(5.13),suitable fluorescence quantum yield(0.55),long absorption and emission wavelengt h(?_ab/?_{e m}=574/633 nm),and large two-photon absorption cross section(89 GM).In addition,its fluorescence intensity can be regulated by hydroxyl protection and deprotection reactions,indicating that LDOH-4 has a good application prospect in the design and application of“activated”fluorescent probes.Secondly,on the basis of the above work,we choosed LDOH-4 as the two-photon fluorescent report unit and p-nitrobenzyl as the recognition and departure group,and developed a nitroreductase-activated two-photon fluorescent probe LDO-NTR with a protected and deprotected hydr00000oxyl group approach.Because the hydroxyl group in the probe molecule is protected,the p-?conjugation effect between oxygen atom and anthocyanin mother nucleus is weakened,while the TICT effect of dimethylamino and the photoinduced electron transfer(PET)effect between nitro group and dye are intensified,which lead to extremely low background fluorescence of the probe.Hence,LDO-NTR can sensitively recognize NTR with more than 310-fold response signal enhancement in vitro(the detection limit is 0.79 ng/m L).In addition,LDO-NTR can not only detect the activity of NTR in hypoxic cells and tumor tissues,but also visualize the activity of NTR in hypoxic tumor mice.Thirdly,the pH value in mitochondria is weakly alkalin e(?8.0),and the weak change of pH can affect its physiological function,but there is a lack of highly sensitive fluorescence probes to study the changes of pH in mitochondria in various physiological activities.To solve this problem,on the basis of t he dye LDOH-4developed previously,we further optimized its properties by introducing benzothiazole groups in the ortho position of hydroxyl groups and regulat ed its molecular planarity,and constructed four new types of pH long-wavelength fluorescent probes.Compared with the other three probes,NIR-OH-1 has excellent optical properties,such as emission wavelength close to near infrared region(?_{e m}=656 nm),suitable p K_a(7.77,consistent with mitochondrial pH gradient),and highly sensitive pH response(acid-base response range is approximately 1 pH unit,and the multiple of fluorescent response is 12.5 times),indicating that the probe is suitable for monitoring weak changes of pH in mitochondria.The results of cell imaging experiments showed that NIR-OH-1 could selectively light up cell mitochondria and highly sensitively monitor the degree of mitochondrial acidification induced by carbonyl cyanide m-chlorophenylhydrazone(CCCP)in He La cells.It is suggested that NIR-OH-1 has great application potential in monitoring the changes of mitochondrial pH in various physiological and pathological activities.