Solid Phase Synthesis Of Pramlintide

According to a recent World Health Organization statistics shows thereare more than360million diabetes patients in the world, the number of Chinais the highest. At present the sort of glucose-lowering drugs is more,hypoglycemic mechanism is not the same, but still can’t meet the need ofclinical application, developing a new drug still has a long way to go. March2005, the FDA approved Pramlintide injection used in type1and type2diabetes adult, and has made good clinical effect. Pramlintide can auxiliarytreat for type1and type2diabetes, mainly for the diabetic oralglucose-lowering drugs not working. It urges the human body form a kind ofinternal environment, make oral glucose-lowering drugs working. In addition,have Research Report, Pramlintide also has the function of regulate appetite，the United States Amylin pharmaceuticals companies are using Pramlintidedevelopment medicine for reducing weight. A study reported HuangYa Donget al. used genetic method for a structure change of people amyloidpeptide-pramlintide. Recently, there are many studies about the research ofpramlintide solid phase synthesis at home and abroad, mainly concentrated inthe segment synthesis. Overall, pramlintide injection bring hope of diagnosisto sufferers special diabetes, but price of pramlintide is expensive and hard tomeet the needs of the domestic market. Committed to the R&D of the activepharmaceutical pramlintide is of great significance.Pramlintide contains37amino acids and a pair of disulfide bridge.Pharmacological studies indicate that1-22amino acid, disulfide bridge, amideof terminal position are important for molecular biology function. Pramlintidewas synthesized by Fmoc solid phase peptide synthesis methods, using RinkAmide-AM resin as solid carrier, and HBTU-HOBt-DIEA as couplingreagents. Protection groups of the crude peptide were cleaved by a mixture ofTFA-Thioanisole-phenol-H₂O-EDT-TIS, disulfide bridge was formed by air,DMSO or H₂O₂oxidation. The crude pramlintide was purified on preparative RP-HPLC, and identified by RP-HPLC, ESI-MS, MALDI-TOF-MS oranalysis of amino acid composition. The purity of the crude peptide was about50.0%. After the purification of preparative HPLC the purity was increased tomore than95.0%.At present, the pramlintide solid phase synthesis method and the R&D ofthe active pharmaceutical pramlintide is one of the difficult research projects.To develop the solid phase synthesis method for reduceing the cost is essential.Objective:This study established the peptide synthesis process and identifiedmethod. By the synthesis study of pramlintide, it reduced the cost ofpramlintide, it can provide reference for the industrial production.Methods:1Detection methods of solid phase peptide synthesis:Fmoc Spectrophotometry is used to determine contents ofmulti-component amino acid, quantitative calculating the amino acidconnection efficiency. By the color reaction of amino acid and ninhydrin, wetest the connection of the amino acids. Using RP-HPLC and ESI-MS checkthe synthesis process.2Solid phase synthesis of Pramlintide:Laboratory substitution degree coefficient identified by the referencestandard Fmoc-Gly-Wang-Resin. Substitutability of Rink Amide-AM-Resincould control by condensation reagent, the molar ratio and the reaction time.Manual synthetic and instrumentation synthetic process performed all at thesame time, explores the effect of substitutability, the molar ratio and thereaction time.To develop the splitting formula, calculation yield and purity of crudepramlintide. Disulfide bridge was formed by three kinds of oxidation method.The crude pramlintide was purified on preparative RP-HPLC by the optimizecondition. Pramlintide identified by RP-HPLC, ESI-MS, MALDI-TOF-MS oranalysis of amino acid composition. Results:1Laboratory substitution degree coefficient is6L·（mmol·cm）-1by FmocSpectrophotometry, pramlintide was optimized synthesized by0.25mmol·g-1substitution of Fmoc-Tyr（tBu）-Rink Amide-Am-Resin.2The condensation reagent of HBTU-HOBt-DIEA, the molar ratio of5:1,the reaction time of150min were meet the synthesized needs well. Protectiongroups of the crude peptide were cleaved by a mixture ofTFA-Thioanisole-phenol-H₂O-EDT-TIS, disulfide bridge was formed by air,DMSO or H₂O₂oxidation and H₂O₂was best, The crude pramlintide waspurified on preparative RP-HPLC.3The pramlintide has been identified by RP-HPLC, ESI-MS,MALDI-TOF-MS or analysis of amino acid composition. The purity of thecrude peptide was about50.0%. After the purification of preparative HPLC thepurity was increased to more than95.0%.Conclusions:1Manual synthetic and instrumentation synthetic performed well all atthe same time.2Protection groups of the crude peptide were cleaved by a mixture ofoptimized splitting formula.3Disulfide bridge was formed by simple techniques and H₂O₂oxidationis the best.4The crude pramlintide was purified on preparative RP-HPLC. Thepurity of the crude peptide was about50.0%. After the purification ofpreparative HPLC the purity was increased to more than95.0%.5Pramlintide identified by RP-HPLC, ESI-MS, MALDI-TOF-MS oranalysis of amino acid composition.6The synthetic method of pramlintide is brief with higher purity andyield, and it can provide reference for the industrial production.