Synthesis of o-Nitrobenzyl Amine/Alcohol Derivatives for Solid-phase Organic Synthesis and Cell Membrane Protein Isolation

Abstract

Photochemistry is one of the unique and useful subdiscipline of chemistry. Photoreactive molecules, the core compounds in photochemistry, are undergone chemical reactions by irradiation of light, while remaining stable under various conditions such as acidic or basic reaction conditions. With such characteristic features, photoreactive molecules have been used as a linker for solid-phase organic synthesis or a photolabile protecting groups. Furthermore, the light with a wavelength above 315 nm does not give severe damages to biomolecules, and thus, the related photochemistry has been applied to chemical biology field. In this thesis, o-nitrobenzyl amine/alcohol derivatives which can absorb UVA light (365 nm) well are synthesized for solid-phase organic synthesis and isolation cell membrane proteins. In the first part, a novel photocleavable linker which contains o-nitrobenzyl amine moiety was effectively synthesized in six steps with 33 % of synthetic yield, without any complex purification steps. Synthesized photocleavable linker could absorb UVA light with wavelength of 330 to 370 nm well, and showed similar or better photocleavage kinetics compared with established photolinkers. Based on these results, synthesized photocleavable linker was successfully applied to solid-phase organic synthesis. Leu-enkephalin amide (H-YGGFL-NH2) was synthesized by using photolinker-coupled polymer supports, with high purity. Acyl-phenylhydrazone of peptide C-terminus was oxidized by photo-oxidation of photocleavable linker, and peptide acid and ester were synthesized by addition of nucleophiles to acyl-phenyldiazene. Glycopeptides-immobilized polymer supports which can be applied to bioassays were prepared by imine formation reaction between glycans with peptides which coupled with polymer supports via photocleavable linker, and the glycopeptides were analyzed by mass spectroscopy analysis after photocleavage. The synthesized peptides on the photocleavable linker coupled polymer supports were analyzed by laser desorption-ionization mass spectroscopy method without using any additional cleavage steps and matrices. In the second part of thesis, isolation method of the cell membrane protein by using o-nitrobenzyl alcohol moiety containing linkers is described. For the isolation of cell membrane protein, linkers which consisted of amine catchable part, photoreactive part, hydrophilic spacer, and tethering part for immobilization were synthesized. As functional group for tethering, azide which can undergo the copper assisted azide-alkyne cycloaddition reaction and biotin which has high affinity with streptavidin were selected. The reactivity of synthesized molecules toward amine and alkyne was confirmed by a model reaction with amino acids and 4-pentynoic acid in solution phase. Copper assisted azide-alkyne reaction underwent between azide labeled lysine with 4-pentynoic acid in solution-phase

however, it did not undergo between azide-labeled bovine serum albumin with 4-pentynoic coupled polymer supports. Instead of azide-containing molecule, biotin-containing molecule was used for the isolation of proteins. Bovine serum albumin was labeled with biotin-containing molecule, and this labeled protein was bound with streptavidin which immobilized on the beads. And also, cell membrane proteins of Escherichia coli which were labeled with biotin-containing molecule were bound with streptavidin-coated beads. Bound proteins were released from the beads by irradiation of UVA light. Isolated proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and compared with the results of authentic proteins.