Phosphoryl Of Non-Natural Amino Acids Of Fatty Acid Chain And Study On Property

The nature chooses phosphorus as the control center of life activity. The introduction of the phosphoryl can activate the natural alpha amino acids’s carboxyl and change the chemical and physical property of the natural alpha amino acids and endow them with a lot of important and special characteristics. Good surface activity could make the phosphorus acylation of amino acid form stable LB membrane. The property of the self-assembled polymerization into peptide could provide the theoretical basis for imitation biochemical research. A wide range of biological activity has important application prospects in the food. At present, there is no report of the research about the non-natural fatty acid chains of amino acids of phosphorus acylation and its characters. So,the study on the behavior of the new type of green phosphorus acylation molecular self-assemble into vesicle and the interaction with trypsin has important significance in the food processing technology, original cell model, clinical medicine and other fields.This topic has selected four non-nature amino acid L-chain fatty acids as the substrates and synthesized the non-natural phosphorus acylation fatty acid chains of amino acids independently, and characterized their structures with Nuclear Magnetic Resonance Spectroscopy (1H NMR) and Electrospray Mass Spectrometry (ESI-MS), High Performance Liquid Chromatography (HPLC).We have investigated the critical aggregation concentration (CAC) of non-natural phosphorus acylation fatty acid chains of amino acids, and the influence of temperature on CAC by Dynamic Light Scattering Instrument (DLS) and the Isothermal Titer Heat Meter (ITC). It’s property of self-assembling into peptide of non-natural fatty acid chains of amino acids was studied by Fourier Transform Infrared Spectroscopy (FT-IR) and have studied the behavior of self-assembling into vesicles by non-natural fatty acid chain phosphorus acylation successfully. At the same time, since the trypsin has extensive application in the food technology, therefore, I have discussed the mechanism and effect of the fluorescence quenching of the non-natural fatty acid chain to trypsin by the Three-Dimensional Fluorescence Spectrum (FL7000). The article contains the follow aspects:(1)The design and synthesis of new type amino acid molecules with non natural phosphorus acylation fatty acid chain This project designed four different side chain length fatty acid chains of non-natural amino acids (L-2-amino valeric acid, heptanoic acid, octanoic acid, nonanoc acid) and selected the four natural fatty acid chain L amino acid as the substrate, used phosphorous acid diisopropyl ester carbon tetrachloride in water diagnosis step reaction generated four crude product, conducted repeated experiments to explore the different conditions of column chromatography, The four products were isolated and purified. NMR spectrometer (1H NMR)、 Electrospray Ionization Mass Spectrometry (ESI-MS)、High Performance Liquid Chromatograph (HPLC) charactered the structure of the product. The results showed that the purity of the product can reach more than90%and the correctness of the target product, the purity could achieve the requirement for the experiment. At the same time, The experimental results also proved that carbon tetrachloride in water method could apply to the phosphorus acylation of natural fatty acid chains of amino acids, it has important reference significance for more non-natural amino acid phosphorylation.(2) Study on physical and chemical properties of the new type amino acid molecules with non-natural phosphorus acylation fatty acid chainSince a long chain of phosphorus acylation amino acids amphiphilic molecules has good surface activity, therefore, using Isothermal Titer Heat Meter (ITC) Conductivity Method (DLS) to study the CAC of phosphorus acylation of valeric acid, heptanoic acid, octanoic acid, nonanoc acid. Phosphorus acylation valeric acid, heptanoic acid can be dissolved at25℃; phosphorylation octanoic acid, nonanoc acid can be dissolved at50℃. The results show that the CAC of N-phosphorus acylation valeric acid and heptanoic acid is0.0806mmol/L、0.0125mmol/L respectively at25℃. The CAC of N-phosphorylation octanoic acid and nonanoc acid is0.2029mmol/L、0.2397mmol/L respectively at50℃. At the same time, I have used DLS to investigate the temperature’s effect on CAC of phosphorus acylation of amino acid. The results show that CAC is decreasing with the increase of temperature. At the same time, using FT-IR to study the property of forming peptide for four unnatural phosphorus acylation and the fatty acid chains of amino acids and it suggests that the non-natural phosphorus acylation fatty acid chains of amino acids can also form into peptide in the absence of catalyst and condensing agent under the moderate condition, and with the increase of amino acid side chain length, it’s self-polymerization time of forming into peptides gradually reduced. Using the property of forming peptides in isopropyl alcohol solvent to prepare vesicles (50℃,10r/min, pH=2-3), Transmission Electron Microscopy (TEM) was used to characterize the morphology and observed that there was a stable single vesicle aggregates. It indicated that the non-natural phosphorus acylation fatty acid chains of amino acids could self-assemble into vesicles. The study provides an important basis for building self replication of vesicles, autocatalytic, self-assembly.(3) Study on the fluorescence quenching effect between N-phosphoryl amino acids of non-natural of fatty acids chain and trypsinTrypsin could chang the property of food protein, so it could improve the function of food. At present, trypsin has been used in food field widely, In recent years, the research about inhibition to trypsin of N-phosphoryl amino acids of non-natural of fatty acids chain hasn’t been reported. Trypsin has the fluorescence. Fluorescence method was used to study the interactions between N-phosphoryl non-natural amino acids and trypsin solution in detail.The results indicated that L-2-amino valeric acid, heptanoic acid, octanoic acid and nonanoc acid can’t decrease the fluorescence intensity of trypsin solution, but N-phosphoryl amino valeric acid, heptanoic acid, octanoic acid and nonanoc acid can quench the fluorescence of trypsin solution.At the same time, N-phosphoryl amino acid of different concentration, different length have different quenching effects on trypsin.With the increasing of carbon chain length of N-phosphoryl amino acid and the increasing of N-phosphoryl heptanoic acid’s concentration, the intensity of trypsin decreased fast.What’s more, the fluorescence of trypsin could be quenched completely by N-phosphoryl amino heptanoic acid. It showed that N-phosphoryl amino acids could change the interactions with trypsin. The phosphoryl of non-amino acids could increase the weak interaction between N-phosphoryl amino acids of non-natural of fatty acids chain and trypsin and improve the affinity of them. The study could promote the progress of inhibition to trypsin of N-phosphoryl amino acids of non-natural of fatty acids chain.The subject has designed and synthesized four N-phosphoryl amino acids of non-natural of fatty acids chain by myself. By taking advantage of important and special property of N-phosphoryl amino acid, I have studied the physical behavior of self-assemble to vesicle of N-phosphoryl amino acids of non-natural of fatty acids chain and the quenching effect to the trypsin successfully. The study could promote the the investigation of chemical property and biological activity of N-phosphoryl amino acids of non-natural of fatty acids chain.It could provide powerful reference and foundation of establishing the system of self-assembling,self-catalyzing,self-recycling.On the other hand, it is conductive to investigate the application of N-phosphoryl amino acids of non-natural of fatty acids chain in food technology,medicine control and release, biological membrane simulation and other related fields.