Study On The Self-assembly Behaviors Of Phenylalanine Derivatives And Their Applications

Biomaterials possess broad application prospects in material science,biology and medicine.Peptides and protein molecules can be assembled into biological nanomaterials with different morphologies through hydrophobic interaction,π-πstacking,and electrostatic interaction.By imitating the assembly processes of biopeptides or proteins in nature,many bionically or biologically inspired nanomaterials have been designed and synthesized.Among them,hydrophobic phenylalanine plays an important role in the self-assembly process of peptides or proteins.For example,the KLVFF fragment serves as the core recognition sequence for the self-assembly of amyloid-beta（Aβ）,and Aβaggregates with different shapes can be formed through the hydrophobic interaction between phenylalanine,which is one of the important causes of Alzheimer’s disease.Therefore,the Aβaggregation inhibitors so far reported are mainly aimed at inhibiting the assembly of KLVFF fragments.In addition,through hydrophobic interaction andπ-πstacking interaction,phenylalanine can be self-assembled into the nanomaterials with various shapes,such as nanofibers,nanotubes,hydrogels,nanospheres,etc.These nanomaterials have shown good application performance in drug delivery,biosensing,optical imaging,etc.By modifying phenylalanine with other small molecules,a large variety of new functional nanomaterials can be designed.In this thesis,the self-assembly behaviors of several phenylalanine derivatives are studied,and their applications in inhibition of Aβaggregation and analyte analysis are explored.In addition,based on the interaction between streptavidin and biotin,two kinds of electrochemical sensors are designed.The specific research contents are as follows.1.The self-assembly behavior of natural neurokinin containing phenylalanine and its mechanism of inhibiting Cu²⁺activity were studied.It was found that neurokinin B（NKB,DMHDFFVGLM-NH₂）could be self-assembled into oligomers and fibers.Cu²⁺can interact with NKB with the formation of a complex,thus inhibiting the transition of NKB from oligomer to fiber.In addition,Cu²⁺can destroy the fibers to oligomers.The redox behavior and catalytic activity of NKB-Cu²⁺complex was investigated by electrochemistry,UV-vis and fluorescence spectra.It was found that NKB could effectively inhibit the catalytic activity of Cu²⁺,but the NKB-Cu²⁺complex possessed great cytotoxicity.Based on the above results,the self-assembly behaviors of neurokinin A and neuromedin C（NKA and NKC,respectively）and their effects on inhibiting Cu²⁺catalytic activity and Aβaggregation were studied.It was found that NKA could be self-assembled into nanoplates with the thickness of about 6 nm.NKA and NKC can inhibit Cu²⁺activity to varying degrees.These studies provide important information for understanding the role of amyloid peptide and copper ion in neurodegenerative diseases,and expand the applications of natural small molecules including FF dipeptides in the self-assembled nanomaterials.2.The FF dipeptides labeled with pyrroloquinoline quinone（PQQ）and4-（10,15,20-triphenyl-5-porphyrin）benzoic acid（CATPP）were designed and synthesized,and two kinds of nanomaterials were prepared.The effects of the above two nanomaterials on inhibiting Aβaggregation,and the fluorescence quenching ability were studied.The PQQ-FF nanoparticles could effectively inhibit the aggregation of Aβ,and CATPP-FF nanoparticles could not only effectively inhibit the aggregation of Aβ,but also destroy Aβfibrils under 808 nm laser irradiation.In addition,both organic self-assembled materials showed good fluorescence quenching ability,which could effectively quench the fluorescence of fluorophore-labeled peptides or DNA probes.This work is of great significance for exploiting new multifunctional biological nanomaterials.3.The biotinylated phenylalanine（biotin-F）was designed and synthesized,and the self-assembly behavior of biotin-F was studied.It was found that biotin-F could be self-assembled into stable nanoparticles（biotin-FNP）in weak alkaline condition.In the presence of streptavidin,the formation of the network structure from the nanoparticles was achieved.Based on the interaction between streptavidin and biotin and the in-situ self-assembly on the electrode surface,an electrochemical sensor for detecting caspase-3 activity was designed.The biotinylated polypeptide modified on the electrode could be used to capture the tetrameric streptavidin in the solution,and the biotin-F nanoparticles in the solution could be further captured by streptavidin.The process occurred repeatedly,and finally the streptavidin-biotin-FNP network structure was formed on the electrode surface,resulting in the increase of impedance signal.Once the polypeptide on the electrode surface was cleaved by protease（such as caspase-3）,the biotin group on the polypeptide was detached from the electrode surface,thus hindering the capture of streptavidin and the assembly of biotin-F nanoparticles on the electrode surface.In this case,the impedance signal did not increase.This method possesses good sensitivity for the detection of caspase-3,and has been successfully used for the determination of caspase-3 activity in apoptotic cells.4.Based on the interaction between streptavidin and biotin,a sensing strategy of transforming homogeneous analysis into surface electrochemical analysis was proposed,and the feasibility of the method was verified by using ligase Sortase A（Srt A）as a model analyte.Bio-LPETGG and GGGK-bio were used as the substrates,and Srt A could catalyze the reaction of these two substrates with the formation of Bio-LPETGGK-Bio,thus inducing the aggregation of streptavidin-modified gold nanoparticles.As a result,the colorimetric assay of Srt A was achieved.Based on this principle,the enzyme-catalyzed product（bio-LPETGGGK-bio）could be captured by the gold electrode modified with streptavidin,thus inducing the in-situ self-assembly of streptavidin on the electrode surface.The formation of（SA-bio-LPETGGGK-bio）n assembly could significantly inhibit the electron transfer of[Fe（CN）₆]^3-/4-.In this method,the enzymatic reaction was carried out in solution,and the electrochemical detection was performed on the electrode surface.Therefore,the advantages of homogeneous reaction and surface analysis were combined,providing a new idea for the design of other novel biosensors.