| Diabetes is a complex chronic metabolic disease characterized by hyperglycemia,which is widely distributed in the world.The hyperglycemia symptoms of diabetes can lead to the metabolic disorder of sugar and lipid in patients,and then cause a series of chronic complications.With the continuous progress of human civilization and the continuous improvement of living standards,the prevalence of diabetes in human beings is increasing year by year.Among them,the number of type 2 diabetes has increased significantly,accounting for more than 90%of the total diabetic population.Type 2 diabetes is characterized by insulin resistance,i.e.decreased glucose metabolism of cells regulated by insulin or decreased insulin secretion caused by impaired function of islet ? cells,or both.This disease is more and more threatening to human life and health.At present,there are many kinds of therapeuticmethods and drugs for the treatment of type 2 diabetes,but it is still a great challenge to find a safe and effective drug to treat type 2 diabetes.Therefore,it is an urgent task to find a safer and more effective drug for the treatment of type 2 diabetesOn December 5,2017,the US FDA approved the listing of a new drug semaglutide(also known as ozempic)for the treatment of type 2 diabetes,which was developed by Novo Nordisk,Denmark.Semaglutide is a GLP-1 receptor agonist and can reduce the blood glucose level of patients by stimulating the secretion of insulin,as well as the blood pressure and weight of patients.This drug has a half-life of up to 165 hours in human body,and is the only GLP-1 receptor agonist that can be administered once a week.It is also extremely safe and has a very low risk of causing pancreatitis or hypoglycemia due to repeated administration.In view of its above advantages,semaglutide is becoming the first-line drug for the treatment of type 2 diabetes in Europe,America and other countries.Semaglutide is a 31 peptide with a long fat side chain,which was prepared by replacing eighth glutamate residue(Glu)of the GLP-1 receptor agonist liraglutide with the 2-aminoisobutyrate residue(Aib)and palmitoyl glutamate on amino group at the position 6 of the 27th lysine(Lys)residue with a long fatty chain composed of two 8-amino-3,6-dioxooctanoic acid(AEEA),glutamic acid(Glu)and octadecanoic acid.As a polypeptide drug,somalutide is synthesized by solid-phase peptide synthetic method,which is similar to other polypeptide drugs.Considering of the shortcomongs of solid-phase synthesis and the characteristics of semaglutide structure(it contains a long fat side chain made from expensive raw materials),we tried to apply a new hydrophobic-supports-assisted liquid-phase peptide synthetic method to semaglutide synthesis and preliminarily optimize the process conditions on this basis.The principle of this new method is similar to that of the traditional solid-phase method.It is also to couple the amino acids at the C-terminal of polypeptide with the supports,then get the desired fragments by one-by-one coupling of the amino acids,and finally obtain the products by acid cracking.However,this method is different from the solid-phase synthesis method in nature:the the supports used in this method is highly soluble in non-polar solvents,but insoluble in polar solvents;Coupling and deprotection reactions are homogeneous reactions that can be carried out in solution,which can be easily and intuitively monitored by TLC method,effectively reducing the generation of impurities;When the reaction is completed,the product can be crystallized by adding the poor solvent into the reaction solution,and the separation and purification steps are as easy to handle as the solid-phase synthesis method;because of the homogeneous reaction,the ratio of amino acids and catalysts is more reasonable,which does not need to be excessive as the solid-phase synthesis method,thereby greatly reducing the preparation cost.According to the characteristics of this method and the structural properties of somaluptide,we divided semaglutide into six the following fragments:Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH(fragment 1)?Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-OH(fragment 2),Fmoc-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH(fragment 3),Fmoc-Gln(Trt)-Ala-Ala-Lys[-AEEA-AEEA-?-Glu(?-OtBu)-Oct(OtBu)]-Glu(OtBu)-Phe-OH(fragment 4),Fmoc-Ile-Ala-Trp(Boc)-Leu-OH(fragment 5)and Fmoc-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH(fragment 6).We selected 2,4-di(docosanoxy)benzyl alcohol as the soluble hydrophobic support,and coupled the carboxyl group of amino acids to the alcohol hydroxyl group of the supports through esterification reaction,and then coupled the amino acids successively through Fmoc protecting method to obtain six Fmoc full-protected fragments:Tag-fragment 1-6.Then,the soppurts of tag fragments 1-5 were removed,and according to the sequence from fragment 5 to 1(i.e.the sequence from C-terminal to N-terminal of somaluptide main chain),these fragments were coupled to Tag-fragment 6 to obtain the full-protected Tag-somaluptide.Finally,somaluptide was prepared by deprotection of all protecting groups and preparative HPLC purification.In the study of somaluptide synthesis,Alloc-Lys(Fmoc)-OH was selected as the raw material of fragment coupling lysine residue because of the long fat side chain attached to the amino group of lysine residue in fragment 4 structure.When coupling to this amino acid residue,the Fmoc protecting group was first removed and the side chain was synthesized by successively coupling three kinds of raw materials needed for the side chain to the lysine residue.Then the Alloc protecting group was deprotected and the remaining amino acid residues were coupled to the main chain to obtain fragment 4.This was also the first application of Alloc protecting group method in the hydrophobic-supports-assisted liquid-phase peptide synthetic method.Then,the synthetic process of semaglutide was further optimized.We established a simple synthetic route with low cost,excellent yield and high purity byinvestigating the effects of various factors such as catalysts,reaction temperature,ratio of the reactants and post-treatment methods on the reaction..The main findings of this study weres as follows:?In the reaction of coupling the C-terminal amino acids of the fragment to the supports,when the ratio of supports,amino acid and DIC is 1:1.2:1.5,four kinds of high purity Fmoc protected Tag-fragments:Fmoc-Gly-O-Tag,Fmoc-Phe-O-Tag,Fmoc-Asp(OtBu)-O-Tagand Fmoc-Leu-O-Tag were synthesized at 40?,which greatly reduced the difficulty of subsequent separation and purification;?In the coupling reaction of C-terminal amino acid and supports,it is very difficult to filter because of the fine and viscous particles of coupling products.Before filtration,diatomite was added to the crystal suspension of the poor solvent to fully mix,and then the filtration was carried out again,which solved the technical difficulties in filtration.Then,a good solvent(such as tetrahydrofuran)was used to extract filter cake(the mixture particles of diatomite and product)to achieve the purpose of high efficiency separation and purification;? In the reaction of removing the supports from Tag-fragment 1,a decrease in the amount of trifluoroacetic acid could reduce the loss of triphenylmethyl protecting group on histidine residue,which increased the yield from 73.28%to 93.02%and the purity of the product from 81.95%to 92.86%;?In the synthesis of fragment 4,we applied Alloc chemistry to this hydrophobic-supports-assisted liquid-phase peptide synthetic method by using tetra(triphenylphosphine)palladium and phenylsilane catalyst system,which improved the purity of fragment 4 to more than 98%;?In the reaction of removing Fmoc protecting group,when the pH is adjusted to 6,the difficulty of crystallization of Tag-fragment 6 could be effectively overcome,so that the yield and purity of this fragment reached 76.73%and 96.73%,respectively;?In the synthesis of the fully protected Tag-somaluptide by coupling each fragment in turn,we screened and optimized the reaction system with reasonable ratio of the reactants using DMT-MM as coupling agent,which significantly improved the total yield and purity of the crude somaluptide after deprotection of the supports,reaching 51.82%and 62.25%respectively.In contrast,the total yield and purity of somaluptide synthesized by the solid-phase peptide synthetic method were 39.22%and 53.51%respectively.Therefore,this greatly improved the synthetic efficiency of somaluptide.In conclusion,compared with the solid-phase peptide synthetic method,the hydrophobic-supports-assisted liquid-phase peptide synthetic method for somaluptide has more reasonable ratio of the reactants,more exenllent yield and higher purity product,which can be directly reflected in the cost reduction.In addition,the technology process of somaluptide established by the hydrophobic-supports-assisted liquid-phase peptide synthetic method in this paper has the advantages of simple operation,mild reaction conditions and potential industrial application value. |
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