Synthesis Of Coumarin-Based Cationic Photosensitizers For Antibacterial Photodynamic Therapy

In recent years,the rapid growth of serious dieases such as skin diseases,inflammation,and meningitis caused by pathogenic microorganisms infections,and the emergence of“superbacteria”and“superfungi”due to antibiotics abuse has weakened the efficacy of most traditional antibacterial drugs against pathogenic microorganisms infections.Therefore,it is thus urgent to develop new methods to distinguish,detect and kill pathogenic bacteria with rapid,sensitive,efficient properties and low side effects for overcoming human resistance to pathogenic microorganisms.Owing to its noninvasive character,accurate drug tracking and spatial-temporal control,fluorescence imaging-guided photodynamic therapy（PDT）has exhibited significant advantages,and has surpassed the traditional detection and treatment methods with operational steps and susceptibility to drug resistance.Not much research has been done on type-Ⅲ,and this paper focused on the application of type-?and type-Ⅱ.type-?PDT has garnered more promising applications in combating pathogenic microorganism infections in hypoxic microenvironment in comparison comparision to the efficacy of type-ⅡPDT largely depends on the O₂level.Since PDT employs photosensitizer absorption of specific wavelengths of lights that can undergo energy transfer or electron transfer pathways to generate reactive oxygen species（ROSs）.Unfortunately,some conventional photo-sensitizers preferentially undergo the type-Ⅱ pathway which have limited their antimicrobial applications in hypoxic conditions.Therefore,the development of high-performance type-?photosensitizers is essential for PDT therapy.In this paper,we designed and synthesized a series of cationic organic conjugated molecules based on coumarin derivatives by adjusting the electron donor unit on coumarin derivatives and increasing the donor-π-acceptor（D-π-A）structure to achieve selective fluorescence imaging and type-Ⅰ/ⅡPDT mechanism against pathogenic microorganisms such as S.aureus.The main research is as follows:1.A series of conjugated organic molecules with a D-π-A structure were designed and synthesized,named ICM-n（n=1,2）,for the study of the detection and killing mechanism of pathogenic microorganisms.This series of molecules consists of three main parts:diphenyl isoquinoline unit（A）,coumarin derivatives（D）,and benzene ring（π-bridge）.The diphenyl-isoquinoline unit has a highly distorted molecular structure and strong electron pulling ability.The bridged benzene ring is the connection between the diphenyl isoquinoline moiety and coumarin,which can effectively extend the conjugated and twisted structure.Coumarin derivatives as donor units have large conjugation systems,easily modified molecular structures and good membrane-active.Cyclic amino and dibutylamino groups were introduced as electron donors to the 7-substituted groups of donor coumarin derivatives to form ICM-1 and ICM-2.In addition,its cationic pyridinium structure endows ICM-n（n=1,2）to further promote electrostatic binding with surface of negative pathogenic microorganisms.ICM-n（n=1,2）showed stronger hydroxyl radicals（·OH）generation than the commercially available photosensitizer Chlorin e6（Ce6）owing to the effective intramolecular charges effect（ICT）and enhanced intersystem crossing（ISC）efficiency,accompanied by a certain singlet oxygen（¹O₂）production.Further experiments demonstrated that 2μM ICM-n（n=1,2）up to100%inhibition rate is achieved for S.aureus（Gram-positive bacteria）under white light irradiation（15 m W/cm²）.Moreover,ICM-1 could image S.aureus（Gram-positive bacteria）and C albicans（fungi）,not stain E.coli（Gram-negative bacteria）;ICM-2 could image three pathogenic microorganisms,which showed the alkyl chains may increase the hydrophobic effect and thus promote the ability of ICM-2 to bind to the outer membrane of Gram-negative bacteria.In summary,the organic conjugated molecules ICM-1 and ICM-2 based on coumarin derivatives can achieve type-Ⅰ/Ⅱ PDT antibacterial treatment and differentiation detection of pathogenic microorganisms.2.Based on the above work,we prepared and synthesized binary（D-π-A）-linker-（D-π-A）photosensitizers ICM-3 and ICM-4,which use flexible alkyl chain connecting two identical momo-D-π-A units to form a binary（D-π-A）-linker-（D-π-A）photosensitizers.The momo-D-π-A structure consists of donor coumarin derivative,bridging benzene ring（π）,and acceptor diphenyl isoquinoline unit.ICM-3 showed stronger·OH and ¹O₂production compared to ICM-2 with the monomeric D-π-A structure,notably,the dimerization could significantly enhance·OH production.Given that ICM-3 with butylamino group as 7-substituent on coumarin derivatives,ICM-4 with different alkyl side chain lengths（ethylamino group）on the substituent was designed in order to further enhance the ROS production.ICM-4demonstrated predominate·OH and ¹O₂generation and certain dark antimicrobial ability,further light irradiation leads to better bacteria inactivation properties.Therefore,increasing the D-π-A structure as well as controlling the length of alkyl side chains on the donor could be an effective strategy to enhance ROS production.Given that ICM-3 and ICM-4 have higher·OH and ¹O₂production capacity than the commercially available photosensitizers Ce6 and methylene blue（MB）,we further determined the killing ability against S.aureus by plate counting method.We further determined that 2μM ICM-3 with up to 97%inhibition rate and0.25μM ICM-4 with up to 95%inhibition rate are achieved for S.aureus under an ultralow dose white light irradiation（5 m W/cm²）.Moreover,cationic ICM-3 and ICM-4 can be combined with different pathogenic microorganisms to achieve broad-spectrum fluorescence imaging capability.In summary,the coumarin-based cationic photosensitizer ICM-n（n=1-4）can efficiently generate ROS via type-Ⅰ/Ⅱ PDT.The result indicated structural modifications with an electron donor,alkyl side chain and dimerization could achieve reasonable regulation of energy levels of these compounds,intramolecular charge transfer efficiency,and intersystem crossing.Such these strategies provide a feasible design strategy for the development of new efficient photosensitizers.