Solid Phase Synthesis Of Growth Hormone Releasing Peptide

Recombinant Human Somatropin (rhGH) has a very broad applicationprospects in clinical.The curative effect of rhGH is positive, while formingantibody, insulin resistance, hypothyroidism, expensive, long-term injectionproblem have also caused public attention. Therefore, the researchers activelyseek some drugs which were more simple and effective. In1982, researcherssynthetized a kind of natural biological active peptide on the basis ofMet-Enkephalin. This peptide was composed of6amino acids, which containstwo kinds of D-amino acids, called growth hormone releasingpeptide-6(GHRP-6). In the following years, research group synthesis ofGHRP-1and GHRP-2, their effect of stimulating the secretion of growthhormone (GH)was stronger than GHRP-6. In addition to the strong promotionof GH release, the effect of GHRP on cardiovascular function, feeding andadipocyte differentiation, regulation of gastric acid secretion andgastrointestinal movement have attracted people’s attention. Especially,GHRP’s effect of regulating and improving on cardiac function become aresearch hotspot in recent years. GHRP have no species specificity. It hasmulti-aspect functions on animals in promoting GH release, stimulatingappetite, promoting animal growth, affecting the animal productionperformancing and improving the level of animal immune. The developmentof GHRP is of great significance to livestock production.At present, many domestic and international scholars carried on a greatdeal of research on the mechanism of action and physiological function withGHRP. It has been reported Boc solid phase synthesis method for synthesis ofGHRP-6in China in the1990s. On this basis, it has been more and moreimportant to explore the synthetic methods of GHRP, optimize the synthesisprocess and reduce the cost of synthesis. GHRP determined by a large numberstructure modification which contain a variety of aromatic amino acids and itssequence of D-amino acid which plays an important role in maintaining biological activity can inhibit protease recognition and hydrolysis effectively.This subject adopts Fmoc solid phase synthesis method, uses Rink Amide-AMresin as carrier, and DIC/HOBt as coupling reagents. Protection group of therough peptides were cleaved by Several different reagent combination. Therough peptide was purified on preparative RP-HPLC, and freeze drying to getthe final products. Pure products identified by RP-HPLC, ESI-MS.Objective:Fmoc solid phase peptide synthesis method is used to synthesis ofGHRP-6,GHRP-2and GHRP-1. Optimized synthesis conditions, establishproduct identified method. With fewer synthetic cost get high purity of targetproduct.Methods:1Solid phase synthesis of GHRPUse Fmoc solid phase peptide synthesis methods, choose RinkAmide-AM resin as solid carrier. Choose coupling reagents which can producelow rate of racemization. Control reagent mole ratio and reaction time. Judgethe connection degree of amino acids by ninhydrin reaction. To explore theinfluence of substitutability of Rink Amide-AM resin on synthesis.2Purification of GHRPOptimize the dissociation conditions and selection simple and low costmethod. Centrifugal collection product after peptide was precipitatated by iceether. The rough peptide was purified on preparative RP-HPLC, explore theappropriate purification conditions, get high purity of the peptide.3Identification of GHRPThe study established the identified method. Using RP-HPLC method todetermine the purity of product. GHRP-6,GHRP-1and GHRP-2identifiedby RP-HPLC, ESI-MS.Results:1Using Rink Amide-AM resin as carrier, DIC/HOBt as couplingreagents synthesis of GHRP-6, GHRP-2and GHRP-1. Reagent mole ratio of1:4. The reaction time of150min. It should avoid high temperature reaction when coupling the amino acid such as Phe, His and shorten reaction time ataround120min. The appropriate substitutability of Fmoc-Lys(Boc)-RinkAmide-Am resin is0.45mmol/g.2Three kinds of cleavage cocktails can cleaving the protection groups ofGHRP-6, the purity of products are similar. Among themTFA/m-Cresol(99:1,V/V) is simple and the cost is low. Two kinds of cleavagecocktails of GHRP-2and GHRP-1had the similar cleaving effect. Thereaction time of0.5h in the ice bath, then30℃for1h.3GHRP-6, GHRP-2and GHRP-1identified by RP-HPLC, ESI-MS.GHRP-6and GHRP-2after the purification of preparative HPLC the puritywas increased to more than95%. The purity of GHRP-1was greater than85%.Conclusions:GHRP-6,GHRP-2and GHRP-1were synthesized by Fmoc solid phasepeptide synthesis. Three peptides were included in D-amino acids. We couldreduce side reaction in synthesis process by choosing low rate racemizationcoupling reagents and controling of reaction time. Optimized dissociationconditions. The rough peptide was purified on preparative RP-HPLC. Targetproduct purity can reach more than95%. The molecular weight of GHRP-6，GHRP-2and GHRP-1were determined by ESI-MS. Using reversed phaseHPLC to identify the purity.