1. 研究目的与意义
CONTENT: 1. Solid phase chemical synthesis of CGYRHGKANC-NH22. develop and validate the analytical methods for the quantification of CGYRHGKANC-NH23. Do researches on plasma stability of CGYRHGKANC-NH2SIGNIFICANCE:To learn the synthesis of peptides, find analytical methods for the quantification and detection of CGYRHGKANC-NH2.
2. 文献综述
Researches on the quantification, stability, and delivery of bioactive peptidesXu Zhuo(Nanjing University of Chinese Medicine, Nanjing, China)Bioactive peptides, generally mass less than 6000 Da, are beneficial to life activities of living organisms or have physiological effect [1]. The synthesis of polypeptides is mainly divided into two ways: chemical synthesis and biosynthesis. Chemical synthesis is further divided into liquid phase synthesis and solid phase synthesis. Solid phase synthesis methods can be divided into tert-butyloxycarbonyl (Boc) and 9-fluorenylmethyloxycarbonyl (Fmoc) methods. The biosynthesis methods of polypeptides mainly include fermentation and enzymatic hydrolysis [2-4]. Bioactive peptides have a variety of human metabolic and physiological functions, and have achieved good efficacy in clinical applications. For example, Oren et al [5] isolated a 33-peptide antibacterial peptide from leopard pupa, which has stronger antibacterial activity than melittin. Its antibacterial mechanism is to dissolve the cell wall of bacteria. Since the first discovery of cyclic peptide antitumor active components from sea rabbits in 1976, 18 anticancer active peptides Dolastatin 1-18 have been traced. Clinical trials were conducted in the United States and Dolastatin18 are mainly used for the treatment of solid tumors such as small cell lung cancer, ovarian cancer, melanoma and prostate cancer. Dolastatin 15 and Dolastatin 10 have been artificially synthesized and are undergoing clinical trials in the U.S., which are mainly used for small cell lung cancer, ovarian cancer, and melanoma treatment of solid tumors such as prostate cancer [6]. Bioactive peptides are the most popular research topics in the international food industry as well as the functional factors with glorious development prospects. However, bioactive peptides still have many problems to be solved from basic research to widespread clinical applications. This review will focus on the quantification, stability and delivery of bioactive peptides in order to provide a theoretical basis for the clinical application of bioactive peptides.1 Quantification of bioactive peptidesQuantitative bioactive peptide is the first step for bioactive peptides in the clinical application. Accurate quantification of peptides is the prerequisite. Ji et al [7] proposed the strategy of quantitative analysis of relatively low molecular mass peptides in biological samples based on HPLC-MS for the first time. Huang T et al [8] proposed an accurate measurement method to detect the purity of small molecules of impurities. As a new method of qNMR, the proton of the separated peptide can be measured accurately and the application of qNMR from small molecular compound to peptide can be expanded. In recent years, the quantitative analysis of peptides by on-line SPE LC-MS/MS with high analytical flux has been proved to be a remarkable technique [9], which can avoid other impurities in the sample preparation process, reduce sample loss and provide better extraction efficiency and reproducibility [10]. Online SPE is a solid-phase extraction process that can do on-line extraction, washing, elution, separation, detection and rebalance of the sample column. The key of this method is to optimize the conditions of mass spectrometry, sample preprocessing and chromatographic analysis, and establish the quantitative analysis method of target peptides [11]. Based on the established method, the working curve can be drawn, and the precision, accuracy, recovery and specificity of the method can be evaluated, and the stability and dilution linearity of bioactive peptides will be explored.2 Stability of bioactive peptidesDue to the complex amino acid composition of bioactive polypeptides, they are easy to be affected by the environment in the process of synthesis, processing and storage. At the same time, if the peptide were ingested into the body, it would also be inactivated by the digestive system. Therefore, the stability of bioactive polypeptides is particularly important, which can be mainly divided into in vivo and in vitro. The former generally need to investigate the effects of temperature, pH, metal ions and drying methods on the peptides. While the latter usually depends on multimode human gastric juice, intestinal juice, plasma environment. The stability of bioactive peptide will be evaluated by HPLC to determine the half-life or activity retention of the bioactive peptide in different environments. Zhu CZ et al [12] investigated the stability of the antioxidant peptide extracted from Jinhua ham from NaCl, temperature, pH, light intensity and simulated gastrointestinal digestion. And they found the suitable conditions for maintaining antioxidant activity. In addition to studying the factors affecting the stability of bioactive peptides, it is particularly important to explore ways to improve and enhance the stability of bioactive peptides. Pan R et al [13] found that peptides combined with diethylene glycol can improve serum stability. Chan LYs [14] result of the study showed that the stability of Gomesin could be improved by natural chemical linkage. Wang J et al [15] found that the stability of the peptide varies widely depending on the various amino acid. In human gastric juice, smaller peptides show good stability. 3 The delivery of bioactive peptidesPeptide drugs have come into our world since insulin was firstly discovered and used in the last century. Extragastrointestinal administrations are still the main drug delivery methods of peptides to relieve patients suffering, such as subcutaneous, intravenous injection. Other drug delivery routes, such as pulmonary administration, transdermal administration, cavity administration and so on, also have short bioactive peptides half-Life, resulting in frequent use of drugs, which will bring inconvenience to patients. Deputy F [16] found that inhalant administration has the advantage of large alveolar area and rapid absorption, but lung proteases will degrade peptides, leading to low bioavailability. Lochhead JJ [17] found that the delivery of macromolecular mass biological agents into the nose was a potentially useful strategy for treating various diseases and disorders of CNS, because nasal administration can bypass the blood-brain barrier and enter the central nervous system directly. However, the metabolism enzyme and small surface area of nasal mucosa can also reduce the bioavailability. Namjoshi S et al [18] conjugated the tetrapeptide AAPV to Laas by solid phase synthesis and determined Peptide stability, skin distribution and permeability, elastase activity and surface activity. This study showed that the Laa conjugation method can be used to enhance the penetration of peptide drugs of medium size and may be used to treat skin diseases. Lee YH et al [19] found that the oral absorption properties of the model peptide drug sCT can be modulated by changing the intestinal pH. sCT is a substrate for the pancreatic serine protease trypsin, which has maximal activity at pH 5 to 6. Therefore, in order to widely apply peptides in clicics, the expansion and rational use of drug delivery methods is also a subject that requires in-depth researches. There are now two approaches available to address the short half-lives and low bioavailability of peptide drugs, including chemical modification and colloidal delivery systems [20, 21]. The new method can not only prolong the half-life, enhance the curative effects, but also be convenient to take medicine, reduce the cost, and benefit the popularization of peptide drugs.4 ProspectsIn conclusion, the widespread clinical application of bioactive peptides is still an urgent and arduous task. The problem begins with the synthesis, purification and quantification of bioactive peptides, followed by a series of processes involving stability, including storage, transport, processing, and, finally, the delivery which leading directly to bioavailability. In this case, each step is a key step to push the bioactive peptide into clinical. Nowadays, bioactive peptides have been used clinically and have the advantages of good efficacy and small side effects. The synthetic methods of biologically active peptides have been relatively mature, and its purification and quantification also have some research basis. The stability studies are the most critical steps before the wide use in clinical practice. With the development of modern science and technology, the researches on the quantification, stability, and delivery of bioactive peptides will continue to develop, and an extensive clinical application of bioactive peptides will be realized as soon as possible.References:[1] Lule VK1, Garg S, Pophaly SD, et al. Potential health benefits of lunasin: a multifaceted soy-derived bioactive peptide[J]. J Food Sci., 2015, 80(3): R485-94.[2] Sistiabudi Ｒizaldi, Ivanisevic Albena. Patterning of polypeptides on a collagen-terminated tissue surface［J］．Journal of Physics and Chemistry C，2007，111( 31) : 11676-11681．[3] Sun Jing, Chen Xuesi, Deng Chao, et al. Direct formation of giant vesicles from synthetic polypeptides[J]. Langmuir, 2007, 23( 16): 8308-8315．[4] Giovannoni J, Didierjean C, Durand P, et al. Synthesis and structure of symmetrical bicyclic hexapeptides bridged by metathesis reaction [J]. Organic Letters, 2004, 6 ( 20 ): 3449-3452．[5] Oren Z, ShaiY. Selectivelysis of bacteria but not mammalian cells by diaster eomers of melittin: structure-function study[J]. Biochemistry, 1997, 36(7): 1826-1835.[6] Chirmer A, Gadkari R, ReevesCD, et al. Metagenomic analysis re-veals diverse polyketide synthase gene clusters in microorganisms associ-ated with marine sponge Discodermia dissolute[J]. Appl Environ Micro-biol, 2005, 71(8): 4840-4849.[7] Ji QC, Rodila R, Gage EM, et al. Strategy of plasma protein quantitation by selective reaction monitoring of an intact protein[J]. Anal Chem, 2003, 75(24): 7008-7014. [8] Ting Huang, Wei Zhang,Xinhua Dai,Xiaoguang Zhang,Can Quan,Hongmei Li,Yi Yang. Precise measurement for the purity of amino acid and peptide using quantitative nuclear magnetic resonance[J]. Talanta,2014,125.[9] Dai SJ, Song HF, Dou GF, et al. Quantification of sifuvirtide in monkey plasma by an on-line solid-phase extraction procedure combined with liquid chromatography/electrospray ionization tandem mass spectrometry[J]. Rapid Communications in Mass Spectrometry, 2005, 19(10): 1273-1282. [10] Liu A, Tweed J, Wujcik CE, et al. Investigation of an on-line two-dimensional chromatographic approach for peptide analysis in plasma by LC-MS-MS[J]. J Chromatogr B, 2009, 877(20-21): 1873-81 [11] Neubauer W, Konig A, Bolek R, et al. Determination of the antifungal agent posaconazole in human serum by HPLC with parallel column-switching technique[J]. J Chromatogr B, 2009, 877(24): 2493-2498. [12] Chao-Zhi Zhu, Wan-Gang Zhang, Zhuang-Li Kang, Guang-Hong Zhou, Xing-Lian Xu. Stability of an antioxidant peptide extracted from Jinhua ham[J]. Meat Science,2014,96(2).[13] Pan R, Xu W, Jafari M, et al. DEGylation Enhanced the Stability of Peptide-siRNA Complexes in Serum. J Nanosci Nanotechnol. 2015, 15(12): 9982-90.[14] Chan LY, Zhang VM, Huang YH, et al. Cyclization of the antimicrobial peptide gomesin with native chemical ligation: influences on stability and bioactivity[J]. Chembiochem. 2013, 14(5): 617-24.[15] Wang J, Yadav V, Smart AL, et al. Toward oral delivery of biopharmaceuticals: an assessment of the gastrointestinal stability of 17 peptide drugs[J]. Mol Pharm, 2015,12(3):966-73[16] Depreter F, Pilcer G, Amighi K. Inhaled proteins: challenges and perspectives. Int. J. Pharm. 2013, 447(1-2): 251280.[17] Lochhead JJ, Thorne RG. Intranasal delivery of biologics to the central nervous system. Adv. Drug Deliv. Rev. 2012, 64(7): 614628.[18] Namjoshi S， Toth I， Blanchfield JT, et al. Enhanced transdermal peptide delivery and stability by lipid conjugation: epidermal permeation, stereoselectivity and mechanistic insights. Pharm Res. 2014, 31(12):3304-12.[19] Lee YH ， Perry BA， Labruno S， et al. Impact of regional intestinal pH modulation on absorption of peptide drugs: oral absorption studies of salmon calcitonin in beagle dogs. Pharm Res. 1999, 16(8): 1233-9.[20] Pisal DS, Kosloski MP, Balu-Iyer SV. Delivery of therapeutic proteins. J. Pharm. Sci. 2010, 99(6): 25572575.[21] Kumar TR, Soppimath K, Nachaegari SK. Novel delivery technologies for protein and peptide therapeutics. Curr. Pharm. Biotechnol. 2006, 7(4): 261276.
3. 设计方案和技术路线
Research Programmes:First, solid-phase synthesis method was used to repeat the addition of precisely-named amino acids required for synthesis from the oxime end (carboxyl group) to the oxime end (amino group). In order to prevent the occurrence of side reactions, the side faces of the amino acids participating in the reaction are protected by Fmoc, and the C-terminal is released and activated before the reaction. The resin was added to immobilize the polypeptide - CGYRHGKANC-NH2, which was lyophilized for use. Coupled with mass spectrometry to determine the molecular weight of the polypeptide, plus known sequence information, tentatively verified whether the sequence of the polypeptide meets the requirements. Finally, through the relevant antibacterial experiments, the function of the peptide was identified and screened.Technical Route:Peptide synthesis-Purification of the peptide solution-1. Develop analytical methods for the quantification2.Do researches on plasma stability of CGYRHGKANC-NH2-validate its analytical methods for the quantification
4. 工作计划
2022.03.03~03.08 Read articles regarding to the experiment and do the presentation completed2022.03.09~03.23 Do the experient completed2022.03.24~04.04 Read relative articles and write the initiating report and the review paper completed2022.04.05~04.15 Finish the experiment To be completed2022.04.16~05.15 Analyze the experimental data and write the research paper To be completed
5. 难点与创新点
The target peptide is an antibacterial peptide precursor. This work will finally synthetise a new peptide and provide new analytical methods for the quantification of the novel peptides.
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