title: Advanced aqueous-phase phosphoramidation reactions for effectively synthesizing peptide-oligonucleotide conjugates trafficked into a human cell line.
abstract: Peptide-oligonucleotide conjugates (POCs) have held promise as effective therapeutic agents in treating microbial infections and human genetic diseases including cancers. In clinical applications, POCs are especially useful to circumvent cellular delivery and specificity problems of oligonucleotides. We previously reported that nucleic acid phosphoramidation reactions performed in aqueous solutions have the potential for facile POC synthesis. Here, we carried out further studies to significantly improve aqueous-phase two-step phosphoramidation reaction yield. Optimized reactions were employed to effectively synthesize POCs for delivery into human A549 cells. We achieved optimization of aqueous-phase two-step phosphoramidation reaction and improved reaction yield by (1) determining appropriate co-solutes and co-solute concentrations to acquire higher reaction yields, (2) exploring a different nucleophilicity of imidazole and its derivatives to stabilize essential nucleic acid phosphorimidazolide intermediates prior to POC formation, and (3) enhancing POC synthesis by increasing reactant nucleophilicity. The advanced two-step phosphoramidation reaction was exploited to effectively conjugate a well-studied cell penetrating peptide, the Tat(48-57) peptide, with oligonucleotides, bridged by either no linkers or a disulfide-containing linker, to have the corresponding POC yields of 47-75%. Phosphoramidation-synthesized POCs showed no cytotoxicity to human A549 cells at studied POC concentrations after 24 h inoculation and were successfully trafficked into the human A549 cell line as demonstrated by flow cytometry, fluorescent microscopy, and confocal laser scanning microscopy study. The current report provides insight into aqueous-phase phosphoramidation reactions, the knowledge of which was used to develop effective strategies for synthesizing POCs with crucial applications including therapeutic agents for medicine.
authors.affiliation: Department of Medicinal and Applied Chemistry, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, 80708, Taiwan. tzupinw@cc.kmu.edu.tw
authors.forename: Tzu-Pin
authors.initials: TP
authors.lastname: Wang
authors.forename: Ni Chien
authors.initials: NC
authors.lastname: Ko
authors.forename: Yu-Chih
authors.initials: YC
authors.lastname: Su
authors.forename: Eng-Chi
authors.initials: EC
authors.lastname: Wang
authors.forename: Scott
authors.initials: S
authors.lastname: Severance
authors.forename: Chi-Ching
authors.initials: CC
authors.lastname: Hwang
authors.forename: Ying Ting
authors.initials: YT
authors.lastname: Shih
authors.forename: Min Hui
authors.initials: MH
authors.lastname: Wu
authors.forename: Yen-Hsu
authors.initials: YH
authors.lastname: Chen
mesh_terms: D000577:Amides / Q000737:chemistry*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D057846:Cell-Penetrating Peptides / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D004220:Disulfides / Q000737:chemistry; D005434:Flow Cytometry; D006801:Humans; D007093:Imidazoles / Q000737:chemistry*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009696:Nucleic Acids / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry*; D010756:Phosphoric Acids / Q000737:chemistry*; D021381:Protein Transport; D012996:Solutions; D014867:Water; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology
chemical_list: D000577:Amides; D057846:Cell-Penetrating Peptides; D004220:Disulfides; D007093:Imidazoles; D009696:Nucleic Acids; D009841:Oligonucleotides; D010756:Phosphoric Acids; D012996:Solutions; D054322:tat Gene Products, Human Immunodeficiency Virus; D014867:Water; C011067:phosphoramidic acid