Recombinant human growth hormone (rhGH) has a very broad application prospect in clinical practice. At the same time of affirming the efficacy of rhGH, the emergence of antibody formation, insulin resistance, hypothyroidism, expensive, long-term injection inconvenience and other problems have also caused people‘s concern. Therefore, researchers are actively looking for simpler and more effective drugs that promote the secretion of pituitary growth hormone. In 1982, the researchers synthesized the first non-natural bio active peptide that stimulated the growth hormone secretion based on the structure of the Enkephalin analog. The peptide consists of six amino acids, including two D-amino acids. It is called Growth hormone releasing peptide-6 (GHRP-6). In the following years, the research group synthesized GHRP-1 of heptad peptide and GHRP-2 of hexapeptide on the basis of GHRP-6. These two active peptides stimulated the secretion of growth hormone more strongly than GHRP-6. In addition to the strong promotion of growth hormone release, GHRP has also attracted attention to the effects of GHRP on cardiovascular function, feeding, adipocyte differentiation, gastric acid secretion and gastrointestinal motility, especially GHRP on the heart. The regulation and improvement of various functions has become a hot topic in recent years. GHRP has no species specificity and has many functions such as promoting growth hormone release, stimulating animal appetite, promoting animal growth, affecting animal performance and improving animal immunity. The research and development of GHRP is also very important for animal husbandry production. Significance.
At present, a large number of theoretical and practical studies have been carried out on the mechanism of action and physiological effects of GHRP at home and abroad. In the 1990s, Boc solid phase synthesis strategy for the synthesis of GHRP-6 was reported in China. Recently, research has focused on GHRP pharmacology research. On this basis, exploring the synthesis method of GHRP, optimizing the synthesis process, and reducing the synthesis cost have become more and more important topics. GHRP is modified by a large number of structures and contains a variety of aromatic amino acids. The D-type amino acids in the sequence can effectively inhibit the recognition and hydrolysis of proteases , let the utilization of various routes of administration higher than other peptide drugs. It plays an important role in maintaining the biological activity of the polypeptide . This topic choose Fmoc solid phase synthesis strategy. Rink Amide-AM resin was used as carrier to select carbodiimide type condensation reagent DIC. The HOBt reagent is added to the activation to reduce the racemization reaction, the peptide reaction was carried out by stepwise condensation. The crude peptide was cleaved from the resin by several different cutting reagents to compare the cutting effect. The crude peptide was separated and purified by semi-preparative high performance liquid chromatography, and then freeze-dried to obtain a final product. The obtained pure peptide was subjected to purity analysis by RP-HPLC, and structural analysis was carried out by ESI-MS.
The three short peptides of GHRP-6, GHRP-1 and GHRP-2 were synthesized by Fmoc solid phase peptide synthesis method, the synthesis conditions, cutting conditions and purification methods affecting the product results are explored. Establish the products of detection method. Reduce the synthesis cost and obtain the target product with higher purity.
1 GHRP solid phase synthesis
GHRP-6, GHRP-1 and GHRP-2 were synthesized by Fmoc solid phase synthesis. Using a suitable resin as a carrier, selecting a condensation reagent capable of producing the lower rate of rotation. Control the molar ratio of the feed and the reaction time, standardize operation, reduce the by-products produced during the peptide reaction; the degree of amino acid linkage in the process of peptide extraction was judged by the color change of the ninhydrin detection reaction; explore the effect of resin substitution on the synthesis reaction.
2 GHRP cutting, purification
According to different protection groups of amino acids, different cutting reagent formulas are selected for parallel experiments; the cutting conditions are optimized, and the cutting method with simple and less side reactions is selected; the polypeptide is cleaved by ice diethyl ether precipitation, and the product is collected by centrifugation to obtain a crude peptide; Preparative RP-HPLC separation and purification; simple treatment of the purified product, followed by lyophilization to obtain the final product.
3 Structural analysis and identification of GHRP
The retention time of the synthesized GHRP-6, GHRP-1 and GHRP-2 was determined by RP-HPLC, and the purity analysis was performed. The HPLC chromatograms of the cutting effects of different cutting reagents were compared, and the HPLC spectra of the peptides before and after purification were compared. Comparisons were made; the molecular weight was confirmed by ESI-MS analysis.
1 GHRP is the structure of peptide amide, Rink Amide-AM resin is selected as the carrier; DIC/HOBt is selected as the condensation system, the price is low, and the combination of the two can effectively reduce the racemization rate, and the side reaction can be controlled to a very low range. The molar ratio of the reaction feed can be fully reacted at 1:4; the reaction time is generally 150 min. When linking Phe, His, etc., which are prone to racemic side reactions, avoid high temperature reaction environment and shorten the reaction time by about 120 min; The degree of synthesis is better at 0.45 mmol/g.
2 The three cleavage reagents of GHRP-6 have similar effects on the removal of protecting groups, and the purity of the obtained products is similar. The ratio of the cutting solution TFA: m-cresol (V/V99:1) is the easiest and the least cost. The two cutting reagents used in GHRP-1 and GHRP-2 also have good effects. The two cutting reagents used in GHRP-1 and GHRP-2 also have good effects. When the cutting experiment was carried out, the reaction liquid was first reacted in an ice bath for 0.5 h, and the product obtained by the cracking condition at 30 ° C for 1 h had less by-products and a higher yield.
3 The synthetic hexapeptide GHRP-6, GHRP-2 and heptapeptide GHRP-1 were detected and determined molecular weight by RP-HPLC and by ESI-MS. After, separated and purified by semi-preparative RP-HPLC, the purity of GHRP-6 and GHRP-2 is greater than 95%, and the purity of GHRP-1 is greater than 85%.
In this study, a short peptide GHRP-6 and GHRP-2 consisting of six amino acids and a short peptide GHRP-1 consisting of seven amino acids were synthesized by Fmoc solid phase peptide synthesis method. All three peptides contained D-type unnatural amino acids. In the synthesis, by selecting a condensation reagent having a small racemic ratio, the reaction time is controlled to reduce side reactions; The cutting effect of several different cutting reagents on the peptide resin was compared, and the cutting conditions were optimized; The purity of target product is over 95%, after separated and purified by semi-preparative RP-HPLC; The obtained product was determined the retention time by RP-HPLC, and confirmed the molecular ion peak by ESI-MS, which was consistent with the theoretical molecular weight. The synthesis process of this study is simple in operation, low in synthesis cost, and can achieve high purity requirements, and can provide reference experience for other units to synthesize such products.
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Solid phase synthesis of Growth hormone releasing peptide-6