Fmoc Solid-phase Synthesis Of Novel Casomorphin And Study On Its Property

Opioids, a kind of neuromimune peptides, can be divided into Endogenous Opioid Peptides and Exogenous Opioid Peptides. They work in the central nervous system and peripheral organs. Lots of opioid peptides had been discovered comes from casein. Hidden or inactive in the amino-acid sequence of casein, they can be released or activated via specific, enzyme-mediated proteolysis. In this paper, the synthesized casomorphin is from the 144-158 sequence of Alpha-S1–casein: YFYPELFRQFYQLDA, its molecular weight is 2252.48.Because of its short sequence, this novel casomorphin can be synthetized conveniently by Fmoc solid-phase peptide synthesis method (SPPS method). This casomorphin was synthesized successfully from C-terminal to N-terminal according to its amino acids sequence with raw material of Fmoc- amino acids and Wang resin as the vector. The optimized technology of this opioid synthesis was as follows: using DCM as the main coupling solvent, TBTU/Hobt/DIEA as the coupling reagent, the synthesis reaction time 3h, and TFA/H2O/thioanisole/Phenol (Volune ratio of 85: 5: 5: 5) as resin cleavage condition, the cleavage reaction time 2h, 24℃as the cleavage temperature. Then the white power of novel casomorphin was precipitated using absolute ether. And then the crude peptide was frozen-dried. The yield of crude product was 57.7%. Sephadex G-15 was used to separate and purify the novel opioid, then collected peaks were analyzed by HPLC-MS. From the chromatogram map, the purity of this peptide was 43%, and the mass spectrogram told the opioids molecular weight was 2253.4. In order to test activities of the opioid, the hot plate test and the writhing method were used. Results showed that this synthesis opioid not only has the active of center analgesia, but also has active of peripheral analgesia. As well as it had obvious anti-fatigue effects since it could prolonged the swimming time of mice. From the t-test results, it was also found that the bio-activities of synthesized casomorphin was corresponded with the ones of nature casomorphin.