Synthesis And Application Of Cyclodextrin Derivatives For The Adapter Of Nanopore

Small organic molecules(such as ATP,glutathione,et al.)play a vital role in many life processes like energy supply,response to oxidative stress in cells.Recognizing and detecting these molecules can provide important information for revealing the mechanics of life process.Nanopore technique is a simple and label-free single-molecule analysis method.Based on ?-hemolysin nanopore and?-cyclodextrin,several kinds of organic molecules have been recognized and detected,including nucleotides,amantadine,ampicillin,et al.However,many organic molecules still can not be recognized by existing nanopore systems and it is a necessity to develop a novel platform to enhance the nanopore performance.Based on this,the thesis functionalized the C-6 sites of natural?-cyclodextrin through a series of organic reaction.The functional ?-cyclodextrin derivatives are utilized as an adapter in ?-hemolysin to recognize small organic molecules and revealing information of enzymatic kinetics,order of chemical reaction,as well as chemical reaction of single molecule.This work includes:1.Design and synthesis of artificial receptor for selectively recognizing ATP,ADP and AMPBased on natural ?-cyclodextrin,all the C-6 sites are modified as the following order:per-6-iodine-?-cyclodextrin,per-6-azido-?-cyclodextrin,per-6-amino-?-cyclodextrin,then the product per-6-quatemary ammonium-?-cyclodextrin ?(p-QABCD I).Its structure resembles that of mitochondrial ADP/ATP carrier and p-QABCDI is used as an adapter in nanopore.ATP,ADP and AMP create distinct current signature and the formation equilibrium constant varies obviously.After molecular docking simulation by DOCK 6 software,three different interaction modes are demonstrated:ATP,ADP and AMP attract the functional group in p-QABCD ? and cause varied constriction degree resulting in different residential current.With the different number of salt bridges and hydrogen bonds,ATP,ADP and AMP could bind with p-QABCD ? for different time.Consequently,this platform has accomplished the recognition and detection of essential molecules in biology.Based on the recognition ability of p-QABCD I,the kinetic process of the ATP hydrolysis by calf intestinal alkaline phosphatase(CIALP)was monitored continuously in real time and in situ.The concentration of ATP,ADP and AMP can be obtained in the process.This nanopore-based platform may provide a competitive and label-free method to evaluate the enzyme activity as well as to assess inhibitors of ATPase or CIALP.2.Design and synthesis of per-6-quatemary ammonium-?-cyclodextrin ?(p-QABCD ?)for selective detecting glutathione oxidizedThe determination of glutathione reduced(GSH)or oxidized(GSSG)in bulk solution has been reported previously.However,it is still critically important to set up a simple and label-free method to recognize GSSG and GSH selectively and dynamically,especially at single-molecule level.Here we report a novel nanopore method to recognize GSSG based on a newly synthesized per-6-quaternary ammonium-?-cyclodextrin ?(p-QABCD ?),which is used as both the molecular adaptor of protein nanopore and the recognizing element of GSSG.Distinct current signature has been observed upon GSSG binding in a mutant protein nanopore(M113R)7 equipped with p-QABCD?,while there is no signal for GSH.Thus,GSSG in the mixture can be selectively detected in the concentration range of6.00-90.0 ?M.Furthermore,the conversion between GSH and GSSG both in bulk solution and in nanochannel can be continuously monitored in real time and in situ.This label-free method provides a possibility to investigate enzymatic activity as well as its activators or inhibitors related to the transformation between GSH and GSSG.