Design And Biological Activity Of Trivalent Iron Porphyrin-Peptide Peroxidase Mimics

Reactive oxygen species such as hydrogen peroxide have been associated with many non-infectious chronic diseases such as diabetes,atherosclerosis,arthritis and cancer.Peroxidase is of great significance to the prevention and treatment of many diseases caused by free radical damage.Therefore,people try to use the method of mimicking enzymes to prepare mimics of peroxidase with biological activity.Both the heme group itself or after incorporating linear peptides have been shown to scavenge peroxides and show a protective effect in the Caenorhabditis elegans model.According to the structure of microperoxidase MP-9 in the preliminary research of our laboratory,a peroxidase mimic enzyme deuterohemin tripeptide(Dh HP-3,Dh-?-Ala-His-Glu)was designed.It has high peroxidase-like activity and catalase-like activity in vitro,can effectively scavenge free radicals,and has the functions of prolonging nematode lifespan,anti-oxidation and anti-aging in the body.However,peptides must resist the degradation of gastrointestinal proteases and be completely absorbed through the intestinal barrier in order to exert their physiological effects in the human system.Considering the poor stability of Dh-?-Ala-His-Glu in the gastrointestinal tract,replacing key residues with unnatural amino acids may be an effective strategy to improve peptide stability.2-Aminobutyric acid(Abu)and Phenylglycine(Phg)are two unnatural amino acids.They are introduced into the peptide sequence to replace?-Ala to achieve the purpose of improving the stability of the peptide while ensuring its biological activity.Three deuterohemin tripeptides(Dh-?-Ala-His-Glu,Dh-Abu-His-Glu,and Dh-Phg-His-Glu)were synthesized by solid-phase peptide synthesis.After HPLC analysis,MALDI-TOF-MS detection,separation and purification,the deuterohemin peptide compounds with a purity of more than 90%and stable physical and chemical properties can be obtained.The secondary structures determined by CD were?-helix,random coil,and?-sheet.This article confirms that the three deuterohemin tripeptides have dual enzyme-like activities,and the activities are similar.Through the DPPH and ABTS free radical scavenging methods,it is proved that the deuterohemin tripeptides have anti-oxidation effects,and the three deuterohemin peptide compounds have different mechanisms of anti-oxidation ability.Combined with HPLC and MS analysis and detection,in the gastrointestinal tract simulation fluid where peptides are very easy to degrade,the substitution of unnatural amino acids can effectively improve the stability of the peptide.Using wild-type Caenorhabditis elegans N₂,it was found that the deuterohemin tripeptide can prolong the life of the nematode.Its anti-aging effect cannot be achieved by affecting the growth and metabolism of bacteria,but by improving the resistance to stress without affecting the nematode growth and reproduction.In summary,the deuterohemin tripeptide designed,synthesized and prepared in this paper not only has peroxidase-like activity,catalase-like activity,and antioxidant effects,but also can improve the stability of the gastrointestinal tract.At the same time,they play an anti-aging effect by improving the ability of nematodes to resist oxidative stress induced by H₂O₂,and have high safety.