Multifunctional Linkers Of Photoaffinity Labeling:Design,Synthesis And Bioorthogonal Reactions

Photoaffinity labeling has been a valuable biochemical tool for studying the interactions of protein-protein,protein-peptide and DNA-DNA,and finding the binding target proteins or imaging the location of target proteins in organisms.A classical photoaffinity labeling probe is usually composed of a biologically active component,a photoaffinity crosslinking group,and a tracing group bearing the element of radioisotope or stable isotope,or a fluorescent group.Thus the probe can be used as a reporter to trace the biological reaction process and find their binding biomolecule target.Although these methods have been widely used,some limitations,including long synthetic route,poor general utility,low resolution and radioactivity,etc.,result in the low versatility and efficiency of the probes.Therefore,design and synthesis of multifunctional linkers,which bear high efficient photo-crosslinking group and specificity-linkable groups with less impact on the probe activity,will be of great importance to the fast construction of versatile and multifunctional probes.Ideal multifunctional linkers should contain three function moieties,namely a photoaffinity group and two reactive groups.The two reactive groups are respectively used for connecting with active molecule and reporter.Photoaffinity group can be photolyzed under mild condition to covalently bind with protein with high efficiency.One functional group that is used to connect with a bioactive molecule should be able to connect with the molecule under mild condition;and the another that is used to connect with one reporter should undergo an efficient and fast bioorthogonal reaction with reporter molecule under physiological condition.Based on the relevant reports,we design and synthesize a series of multifunctional linkers,whose structures are confirmed by NMR,13C NMR,19F NMR,LC-MS,MS,IR and Elemental analysis.We also investigate the photolysis reaction and bioorthogonal reaction of the as synthezied linkers.Main works are listed as follows:1.The development of photoaffinity labeling probes and their applications such as target localization,imaging,isolation and identification are outlined.On this base,the research topic of design and synthesis multifunctional linkers are put forward,with the intention to develop the method for fast construction of versatile multifunctional molecular probes in the future.2.According to the relevant literatures and the availability of chemical reagents,two aromatic diazirine compounds(2-7 and 2-16)and two aliphatic diazirine compounds(3-6 and 3-8)were synthesized,and their molecular structures confirmed.Among these compounds,compounds 2-16,3-6,and 3-8 are new and not reported in literature.3.Taking use of UV-vis spectroscopy,1H NMR and 19F NMR,the photolysis processes were monitored,yielding their maximum absorption wavelength ?max,molar absorption coefficient ?,and photolysis kinetics constant k.Photolysis processes under UV light irradiation are consistent with the mechanism reported in literature,which means the compounds produce active intermediate carbene and diazo by-products.For the diazirine with a-CF3 attachment,it can effectively supress the generation of by-products.Compared to the photolysis processes of aliphatic diazirine compounds,aromatic diazirine compounds have faster rates of photolysis.4.Given the advantages of Diels-Alder cycloaddition due to the fast reaction,mild reaction condition and metal catalyst-free process,functional cyclopropylene and tetrazine compounds were designed and synthesized as functional groups for the binding with reporter molecules.The bioorthogonal reactions in vitro were studied using UV-vis spectroscopy and fluorescence spectroscopy,and the suitable reaction ratio 1:10 with a secondary reaction kinetics constant k=3.07±0.002 M-1s-1 was found.The connection of tetrazine with FITC induces the quenching the fluorescenc with a fluorescence intensity drop down to 0.01 times,and following the click reaction with a cyclopropylene derivative,the fluorescence intensity recover 40%of the original,meaning a 34-fold increase in the fluorescence intensity.The reaction possesses the merits of fast reaction and turn off/on effect,which are valuable in biological imaging since the background interference can be greatly reduced in practical applications.5.In order to achieve connection reaction under mild conditions and reduce the non-specific adsorption of linker molecules,four linker molecules,namely a-amino or hydroxyl-?-azido or progargyl polyether connectors,werer synthesized,which can be used as the bifunctional linkers in connection of different probe components.