STUDY OF PHOTOCHEMISTRY AND THERMAL CHEMISTRY OF NEWLY DESIGNED ORGANIC COMPOUNDS AND A METAL COMPLEX TOWARDS INTERACTIONS WITH PROTEINS

Abstract

In this study, the ability of new chemical reagents, a pyrene derivative, fluorescein derivatives, and a metal complex, to initiate the cleavage of proteins at specific sites was investigated. The selective cleavage of bovine serum albumin (BSA) by PMA-IB was successful by irradiating the mixture of BSA and PMA-IB at 346 nm, in the presence of electron acceptor, cobalt (III) hexamine trichloride (CoHA). The cleavage of BSA resulted in two new fragments with molecular weights of approximately 45 and 22 kDa. The spectroscopic method and molecular docking indicated that PMA-IB was bound to BSA with a high binding affinity and the location of PMA-IB was located within the subdomain IIIB of BSA. Fluorescein sodium salt (F-1), 5(6)-carboxyfluorescein diacetate (F-2) and biotin-4-Fluorescein (F-3) were also selected as fluorescent probes for initiation of the protein cleavage reaction. All three probes cleaved lysozyme and avidin after exposure the light at 490 - 494 nm, in the presence of CoHA. N-terminal amino acid sequencing of peptide fragments indicated the cleavage site of lysozyme between Trp108 and Val109 for all three probes, while the cleavage of avidin by F-1 and F-2 was detected between Trp70 – Lys71. However, the cleavage site of avidin by F-3 could not be determined due to small cleavage yields. In addition, the spectroscopic method and molecular docking studies clarified the binding location and binding affinity of F-1, F-2 and F-3 on lysozyme and avidin, corresponding with the results obtained from the protein photocleavage experiments. In case of metal complex, MoO(O2)2(asparagine)(H2O) successfully cleaved pepsin by activating it with light (320 and 340 nm) and heat (37 °C ), without the requirement of a reducing agent and H2O2. The cleavage of pepsin resulted in at least two fragments with molecular weights of 22 and 9 kDa in photoreaction, and resulted in at least three fragments with molecular weights of 30, 26 and 22 kDa for a thermal reaction. These synthesized chemical reagents can serve as potential artificial peptidases for applications in biological fields, and can be applied in the design of novel therapeutic agents in the future.

-