Synthesis Of Fluorescent Lipid-like Vesicles Based On Michael Addition Reaction

To explore the form of primitive life,construction of primitive cells with specific structural functions is quite vital.At present,structures commonly used in the construction of primitive cells include vesicle,condensed droplet and oil droplet.Among these structures,vesicle is most similar to the existing cell structure for its interface composed of amphiphilic molecule.The main component of modern cell’s membrane is phospholipid.However,structure of phospholipid is relatively complex,and its synthesis in vivo includes multi-step reaction with several enzymes.To simulate the formation of phospholipid from the primeval environments,it is necessary to produce amphiphilic molecules with long hydrophobic chains from the homogeneous aqueous phase,which was very difficult in the past.We use dibromomaleimide(DBM)and mercaptan to complete this process.DBM-mercaptan click reaction is a novel bridging method.In addition to the advantages of high efficiency and high conversion,compared with the traditional maleimide-mercaptan click reaction,it retains the original double bond of maleimide group,and therefore,its product has significant yellow fluorescence.Besides,product of this click reaction could have two hydrophobic chains similar to phospholipids.In this paper,efficient click reaction of DBM is used to generate droplet-shaped protocell with more than5μm diameter and significant yellow fluorescence.These droplets are photosensitive.They respond significantly to 450-460 nm blue light,changing rapidly from droplets to vesicles.This paper explores the formation process of the droplet precursors and giant vesicles.We characterize the kinetics characteristics of in situ reaction in many aspects,and explore its potential as a hydrophobic vehicle for drug delivery and others.Main research and results of this paper are as below:(1)Using thiol ester with carboxylic acid head group and DBM molecule as reactants,the click reaction of DBM and mercaptan is used to generate a large number of liquid droplets with yellow fluorescence in the water phase.These droplets could into vesicles with a diameter of microns under the illumination of about 450 nm wavelength.The concentration and p H conditions of the reaction were controlled in the experiment.It takes 6-7 hours for the reaction to be complete and a large number of droplets to be formed.(2)The kinetic process of the reaction was studied by various means.It is confirmed that the click reaction of DBM in the aqueous phase is a two-steps process,that is,firstly the single hydrophobic chain substitution product is mainly formed,hours later it converts into substitution product with double hydrophobic chains.These two products are both colored and DTM has fluorescence.Therefore,this process can be monitored by measuring the absorption curve and fluorescence emission at any time in the reaction process.Besides,this process was also quantitatively confirmed by HPLC.(3)The lipase with certain hydrophobicity is loaded by the in-situ generated aggregate,and the subsequent catalytic reaction is carried out.In the process of in-situ loading,lipase can,on the one hand,catalyze the formation of the aggregate itself by catalyzing the hydrolysis of thiol ester,and on the other hand,it can be loaded on the surface of the generated vesicles in situ along with the reaction process.At this time,lipase still has catalytic activity,and can catalyze the hydrolysis of substrates such as p NPA and 4-methylumbelliferone butyrate.Because the product DTM has significant fluorescence characteristics,it can also be used as a donor or acceptor of fluorescence resonance energy transfer to characterize the changes in the closeness of intermolecular arrangement in the environment.The further modification of the in-situ formed membrane can be achieved by adding a small amount of membrane stability factor at the initial stage of the reaction.