Chemoenzymatic Synthesis And Application Of Amphiphilic Oligo (Phenylalanine-Lysine)

Deep Eutectic Solvents（DESs）have attracted extensive attention due to their low volatility,biodegradability,low toxicity,and low cost,and are considered as potential alternative solvents for traditional organic solvents and ionic liquids.Chemoenzymatic synthesis of peptides is an emerging efficient and green method for peptide synthesis.It has the advantages of mild reaction conditions,high reaction selectivity,and non-racemic products.The limitation of reaction medium has always been one of the challenges of peptide synthesis,because hydrophobic and hydrophilic substrates usually need to be combined at the same time.Using water as the reaction medium will shift the reaction equilibrium to the side reaction of hydrolysis,rather than the synthesis of peptides.Therefore,non-aqueous medium has unique advantages in the synthesis of peptides.DESs have good biocompatibility,degradability and nonvolatilization,and are easy to prepare.It is an excellent medium for enzyme reaction and has been widely studied in the field of biocatalysis,especially enzyme catalysis.In this paper,the enzymatic synthesis of polypeptides in DES and its mechanism were studied.Then,an amphiphilic phenylalanine-lysine random copolymer was synthesized in choline chloride aqueous solution and it was used to stabilize squalane/water high internal phase emulsion.The main results are as follows:（1）Seven kinds of DESs were selected and synthesized according to the relevant papers.The reaction of papain catalyzed synthesis of oligo-L-phenylalanine（OPhe）was carried out using these DESs as solvents.Among them,the C-2U synthesized by choline chloride and urea at a molar ratio of 1:2 was selected as the best reaction solvent.The optimum reaction time,temperature,water content,the amount of papain and p H range in C-2U was determined to be 3 h,45°C,20%,50 mg and 7.0-8.5,respectively.And the yield was up to 73%.Through the analysis of the results of ¹H NMR spectrum and mass spectrum,it was determined that the degree of polymerization of the product was mainly 7-8.（2）The effects of various components used as additives in the synthesis of DES on the yield of OPhe catalyzed by papain were studied.It was found that 20%（w/v）choline chloride had a promoting effect.GROMACS was used for molecular dynamics simulation and Gaussian was used for DFT calculation.It was found that papain could maintain structural stability in aqueous solution of choline chloride,and choline chloride formed hydrogen bond interaction with amino acid ester substrate,which reduced the energy barrier and promoted the reaction.The existence of this hydrogen bonding was verified by FT-IR spectroscopy.（3）Three random copolymers of phenylalanine and lysine called O-Phe_xLys_y-OMe which have different feed ratios were synthesized in 20%（w/v）choline chloride aqueous solution with papain as catalyst.And the reaction conditions were optimized.The optimum reaction time,temperature,total reaction substrate concentration and immobilized papain amount were 1-2 h,40°C,0.3-0.5 mol/L and 10-12 mg,respectively.The highest yields of O-Phe₁Lys₄-OMe,O-Phe₁Lys₁-OMe and O-Phe₄Lys₁-OMe were 29.9%,42.9%and 58.3%,respectively.The terminal methyl ester of O-Phe_xLys_y-OMe was hydrolyzed and the hydrolysate was named O-Phe_xLys_y-OH.（4）The results of interfacial tension test showed that the three O-Phe_xLys_y-OH emulsifiers could effectively reduce the interfacial tension of squalene and water.O-Phe₁Lys₄-OH,O-Phe₁Lys₁-OH and O-Phe₄Lys₁-OH were used to stabilize the high internal phase emulsion of squalene and water.The required emulsifier concentrations were as low as0.5,0.5 and 3 g/L,respectively.The oil phase volume fraction was up to 80%,and the stability was various at different p H.The CLSM pictures confirmed that the three emulsifier-stabilized high internal phase emulsions were O/W Pickering emulsions.