Modification Of Recombinant Hirudin And Loxenatide With Fatty Acid

Protein and peptide drugs have several advantages such as high activity,high specificity and clear biological function.However,their shortcomings such as short circulation half-life in vivo and strong immunogenicity have limited their clinical application.Therefore,the development of long-acting protein and peptide drugs using chemical modification has become a trend in recent years.PEGylation is one of the most widely used methods.However,PEGylation also exists some problems in safe medication.To overcome these problems,new agents such as fatty acid are developing to replace the PEG.Recently,the reported studies on the modification of protein and peptide drugs with fatty acid are still limited.These studies focus on the the modification of theε-amino group of lysine residues in proteins and peptides using saturated fatty acids.Since more than one lysine residue exists in proteins and peptides,it is difficult to achieve site-specific modification,thereby resulting in the heterogeneity of the product.Therefore,the development of site-specific modification strategy has become a trend in recent years.Additionally,unsaturated fatty acids can play a more important role in human body than saturated fatty acids.In this study,recombinant hirudin（HV2）and loxenatide were chosen as model proteins and peptides to be modified with fatty acids.The main contents were as follows:Firstly,a saturated fatty acid benzotriazole（namely,palmitic acid benzotriazole（PAL-BTA））and an unsaturated fatty acid benzotriazole（namely,oleic acid benzotriazole（OA-BTA））were synthesized using acyl chloride method and NN’-dicyclohexylcarbodie（DCC）condensation method,and used to modify the HV2.Compared with the acyl chloride method,DCC condensation method achieved higher yields of PAL-BTA and OA-BTA（45%and 40%,respectively）.The condtions for the synthesis of PAL-BTA and OA-BTA modified HV2 were optimized,and three mono-fatty acid modified HV2 products and a few di-fatty acid modified HV2 products were achieved,in which the yields of mono-fatty acid modified HV2 products for PAL-BTA and OA-BTA were 8.89%and 10.91%,respectively.Secondly,a saturated fatty acid N-hydroxysuccinimide（namely,palmitic acid N-hydroxysuccinimide（PAL-NHS））and two unsaturated fatty acid N-hydroxysuccinimide（namely,oleic acid N-hydroxysuccinimide（OA-NHS）and linoleic acid N-hydroxysuccinimide（LOA-NHS））were synthesized using DCC condensation method,and the yields of PAL-NHS,OA-NHS and LOA-NHS were 40%,37%and 34%,respectively.The three fatty acid N-hydroxysuccinimide were used to modify HV2.The yields of the modified products decreased with the increaseing unsaturated degree of fatty acids.The condtions for the synthesis of PAL-NHS modified HV2 were optimized,and three mono-fatty acid modified HV2 products were achieved at a ratio of 0.8:1:2.2.The isomers of the three mono-fatty acid modified HV2products were futher purified using preparative liquid chromatography,which retained respectively 35.71%,3.79%and 88.74%in vitro anticoagulant activity of unmodified HV2.Finally,a palmitic acid-polyethylene glycol-maleimide（PAL-PEG-MAL）was synthesized using amino-PEG-maleimide（MAL-PEG-NH₂）,and used for site-specific modification of the thiol group of loxenatide.Effects of the volume ratio of H₂O to DMSO and pH were investigated,and the optimized condtion was pure DMSO solution under alkaline pH.In summary,fatty acid reagents for the modification of amine or thiol groups of protein and peptide drugs were synthesized and used to modify recombinant hirudin or loxenatide.This study provided the basis for the modification of protein and peptide drugs with fatty acids,which can also provide new strategies for the development of long-acting protein drugs.