Chemosynthesis and characterization of site-specific N-terminally PEGylated Alpha-momorcharin as apotential agent

Alpha-momorcharin (alpha-MC), a type I ribosome-inactivating protein (RIP) isolated from Momordica charantia seeds, has been extensively studied for its antitumor, antiviral and antifungal activities. However, as an exogenous protein, problems associated with short half-life and strong immunogenicity have limited its clinical application. Poly (ethylene glycol) (PEG), as a polyether compound, is a well established and efficient modifier to develop it as a potential agent. Nevertheless, conventional PEGylation is not site-controlled and the conjugates are often not homogenous due to the generation of multi-PEGylated derivatives. To obtain a homogenous mono-PEGylated alpha-MC, the PEGylation was carried out by coupling a 20 kDa mPEG-butyraldehyde (mPEG-ALD) with alpha-MC. The product was separated and purified by MacroCap SP chromatography. Results from SDS-PAGE and MALDI-TOF MS revealed that the PEGylated alpha-MC consisted of one molecule mPEG and alpha-MC. Edman degradation confirmed that the N-terminal residue of alpha-MC was successfully coupled with mPEG-ALD. The mono-PEGylated alpha-MC possessed an extremely similar secondary structure to native alpha-MC through spectral analyses. In addition, it also showed low immunogenicity by double immunodiffusion and preserved moderate antitumor activity to three kinds of tumor cell lines in vitro. Finally, trypsin resistance was also considerably improved.