Recognition Of Peptides/Proteins By Amphiphilic Calixarenes And Their Biomedical Applications

Recently,supramolecular chemistry has made great achievements in biomedical applications,such as for biomarker detection and drug delivery,and has provided new methods for the diagnosis and treatment of many diseases.Calixarene,representing the third generation of supramolecular hosts,is one of the most widely used supramolecular hosts because of its ease of modification,excellent preorganization of skeletons,and distinctive binding properties.Amphiphilic calixarenes,which can be obtained by simple modification of hydrophilic/hydrophobic groups on the upper/lower rims,are most frequently used in biomedical applications.Amphiphilic calixarenes can selfassemble into micelles and vesicles,and can also coassemble with other amphiphiles.The recognition and assembly properties can be regulated by adjusting the hydrophilic and hydrophobic groups.Peptides and proteins are the material basis of life,important components of cells,and the main executors of biological activities.Therefore,designing and synthesizing artificial receptors that can selectively recognize peptides and proteins is of great significance for the regulation of biological processes.This thesis focuses on the recognition of peptides and proteins by amphiphilic calixarenes and their potential biomedical applications,including the transport of peptides and proteins,prevention and treatment of amyloidosis,and detoxification of toxic macromolecules.The major contents of this thesis are as follows:1.The development of calixarenes and their biomedical applications are summarized.The recognition of peptides and proteins by various artificial receptors and their applications are also outlined.2.The transport of bioactive cargo into cells is an important modality in emerging treatments.Lysine is the most frequently exposed residue among the 20 amino acids in proteins and is an important target for peptide and protein transport.Accordingly,there is high demand for lysine-compatible activators that can transport peptides and proteins.However,the ammonium group in lysine is kosmotropic,making it difficult to cross the cell membrane.Moreover,only a few artificial receptors can strongly bind to lysine.Thus,the translocation of lysine-rich peptides and proteins is a significant challenge.To overcome this challenge,amphiphilic sulfonatocalix[5]arene(sCx5-6C)was designed and synthesized according to the chemical structure of lysine.Benefiting from the dual features of lysine,compatible recognition((1.07±0.06)× 104 M-1)and embedding in membranes((2.09±0.16)× 105 M-1),sCx5-6C successfully activated the translocation of lysine-rich peptides and proteins.The transport of lysine-rich oligopeptides,polypeptides,and proteins activated by sCx5-6C was verified using artificial membranes.The transport of lysine-rich polypeptides activated by sCx5-6C was also verified using live cells.This work opens a new avenue for the research and development of novel methods in achieving peptide and protein delivery.3.Alzheimer’s disease is the most prevalent type of dementia and the most concerning form of amyloidosis.The fibrillation of β-amyloid(Aβ)monomers into mature fibrils is believed to be a significant event in Alzheimer’s disease development.Therefore,recognition of Aβ,inhibition of Aβ fibrillation,and disintegration of Aβ fibrils have strong potential in the prevention and treatment of Alzheimer’s disease.However,selective and strong binding to Aβ remains a major challenge because of the complexity of peptides and their flexible conformations.Based on the amino acid sequence of Aβ,a novel heteromultivalent recognition platform was constructed with potential as a novel anti-Aβ therapeutic agent for Alzheimer’s disease.This platform is based on the coassembly of guanidinium-modified amphiphilic calixarene(GCA),which tends to bind to negatively charged amino acids such as glutamic acid and aspartic acid,and amphiphilic cyclodextrin(CD),which favors the encapsulation of aromatic amino acids such as tyrosine and phenylalanine.These four amino acids are also significant components of the Aβ peptide.Therefore,the GCA-CD complex exhibited strong binding affinity to the Aβ peptide((3.29±0.53)× 1013 M-1).Based on the desired recognition,GCA-CD completely inhibited the fibrillation of Aβ and disintegrated Aβfibrils,significantly mitigated the cell damage induced by Aβ fibrils,and improved cognitive deficits in Alzheimer’s disease model mice.This work provides a new material for the prevention and treatment of Alzheimer’s disease.4.Parkinson’s disease is another type of amyloidosis that has received widespread attention,with an incidence of 1-2%in people over 65 years of age.The fibrillation ofα-synuclein(α-syn)monomers into mature fibrils is believed to be a significant event in Parkinson’s disease development.Similar to Alzheimer’s disease,recognizing α-syn,inhibiting its fibrillation,and disintegrating α-syn fibrils is an important approach to prevent and treat Parkinson’s disease.The above strategy of heteromultivalent recognition of Aβ and inhibition of its fibrillation was extended to α-syn,demonstrating promising results.This work provides new material for the prevention and treatment of Parkinson’s disease,and further proves that the heteromultivalent recognition strategy is universal and can be applied to other types of amyloidosis.5.Poisoning is a leading cause of admission to emergency departments and intensive care units.Supramolecular chemistry provides a new strategy for detoxification;however,the current substrates for supramolecular detoxification are mostly small molecules.Compared to small-molecule poisons,it is more challenging to design antidotes for toxic biological macromolecules because their complexity,large size,and flexible conformation,making it difficult for an artificial receptor to achieve specific and strong complexation.Based on the heteromultivalent recognition strategy described above,a complex was constructed by coassembly of carboxyl-modified amphiphilic calixarene(CCA)and CD.Melittin was then employed as a model to evaluate the therapeutic effects of CCA-CD for macromolecule poisoning.The CCACD coassembly exhibited strong binding affinity to melittin((5.14±0.23)× 108 M-1)with excellent selectivity,improved the survival rate of melittin-treated cells,reduced the hemolytic toxicity of melittin,and effectively improved the survival rate of melittinpoisoned mice.This work provides a new method for the development of toxic macromolecular antidotes.