Anti-ACQ Cyanine Dyes Activated By Click-chemistry Via Large Steric Hindrance And Their Application In Bioimaging

Near-infrared(NIR)fluorescence imaging technology has been widely concerned and researched due to its advantages of high sensitivity,non-radiation and low cost.Small organic fluorescence dyes,especially Cyanines have been enabled great progress in bioimaging area.Some existing NIR probes exhibit outstanding imaging performance,the currently FDA-approved clinical NIR probe still limited for,indocyanine green(ICG)for the sake of biocompatibility.Owing to their low toxicity and feasible structures,the majority of NIR probes reported so far are based on cyanine derivatives(such as ICG,Cy5.5 and IRDye800CW),cyanines have adjustable wavelength from 400~1100 nm as well as facile functionalization and have been widely exploited in biomedical,clinical imaging and phototherapy applications.However,traditional fluorescence dyes are still suffering from aggregation-caused quenching(ACQ)owing to ?-? stacking,resulting in low imaging sensitivity and limiting their applications.In this work,we developed an anti-ACQ and large stokes shift NIR cyanine dye with conformational activation for in vivo biorthogonal imaging.Furthermore,investigating the imaging performance in vitro and in vivo,the antiACQ probe markedly achieves high target-to-background ratios.The paper's content is outlined as:1.Among the various biocompatible requirements,good water-solubility of fluorophores is foremost factor for using in living systems and clinically promising.Common strategy to enhance water-solubility for cyanines relies on introducing sulfonic acid groups(e.g.,ICG),but which are usually accompanied by increased negative charges,leading to poor cell membrane permeability and short half-life periods.To endow the cyanine with good water solubility,herein,short hydrophilic polyethylene glycol(PEG)chains were introduced to the heterocyclic rings and resulted in PEGylated CyP7 dyes,which exhibited good solubility in aqueous solution.Then click module(Tz)were substituted chloro-atoms of substrate cyanine CyP7 via the nucleophilic substitution reaction and resulted in CyP7 T dye.All synthetic process conditions were mild and the products were easy to separate and purify.CyP7 T demonstrated large stokes shifts about 110 nm which was larger than CyP7 of 29 nm.Furthermore,CyP7 T showed concentration-dependent fluorescence enhancement after click-reacting with TCO,namely anti-ACQ effect.2.To comprehend the large stokes shifts and anti-ACQ mechanism of the cyanines,theoretical calculations of optimized configurations,orbitals and energies were carried out by density functional theory DFT at B3LYP/6-31G* level.The HOMO of CyP7 was located on the whole structure,and the LUMO was mostly located on the heptamethine chains,indicating a strong intramolecular charge transfer(ICT)from HOMO to LUMO.However,nitrogen with a lone pair electron on the meso-position of polymethine chain(soft segment)strongly broke the planarity of nitrogen-cyanine,resulting in twisted intramolecular charge transfer(TICT),which able to produce large Stokes shifts.A transformation from E-isomer to Z-isomer was generated when CyP7 T reacted with TCO via click-chemistry,whereas Zisomer has weaker conjugated system than E-isomer,which result in large steric hindrance and inhibit molecular aggregation.The substrate dye CyP7 with good planarity has a strong intramolecular charge transfer(ICT)from HOMO to LUMO.Further,nitrogen-cyanine CyP7 T were out-of-plane resulting in twisted intramolecular charge transfer(TICT),which were able to produce large Stokes shifts.A conformational transformation from E-isomer to Z-isomer was generated for CyP7 TT by biorthogonal reaction,which result in large steric hindrance and anti-ACQ effect.3.The conformational activation of anti-ACQ NIR cyanines was also investigated in vitro and in vivo level.Compared with ICG,PEGylated cyanines exhibited good cell membrane permeability.PEGylation enables CyP7 to accelerate metabolism in liver and increase the accumulation in tumor in vivo.The anti-ACQ probe markedly achieved target-tobackground(TBR)ratios as high as about 5.7 after 24 h tail intravenous injection.The proposed method shows a new path for improving imaging sensitivity by click-activated anti-ACQ not only for cyanines but also can be expanded for other organic fluorophores.