1. The problems that the research wants to solve:Cyclotides usually are ribosomally produced from processing of dedicated genes and thus can be extracted from plants. Also, recent technologies in the fields of engineered proteins and molecular biology enables scientists to acquire cyclotides inside some bacterial cells by using standard heterologous expression systems. However, these ways all have their own disadvantages involving needing harsh conditions and long time as well as obtaining diverse cyclotides difficultly.Solid phase peptide synthesis ( SPPS ) is an efficient method for synthesizing peptides, which can provide diverse peptides quickly under relatively mild conditions. In this work, we intend to develop a novel method by using SPPS to solve problems of synthesizing natural cyclotide MCoTI-I. Natural cyclotide MCoTI-I is one of novel trypsin inhibitor subfamily of cyclotides which share a head-to-tail cyclized backbone stabilized by three disulfide bonds forming a cyclic cysteine knot ( CCK ) motif. Due to its unqiue structure, cyclotides from this family are not toxic to mammalian cells, do not interact with membranes, and can be easily engineered to introduce novel biological functions. Therefore, our work is able to offer an effective chemical method for synthesis of cyclotide MCoTI-I which can lay a solid foundation for further applications such as imaging probes and therapeutics.2. Research Method and TechnologyThis work mainly depends on the novel technique of solid phase peptide synthesis ( SPPS ) by using a machine. After that, it relies on traditional technique of organic synthesis to proceed with cleavage, cyclization and folding.Specifically, the linear peptide precursor will be synthesized by peptide synthesizer at first. Then, the linear peptide precursor will be manually cleaved from the resin and deprotected the protecting groups through three steps involving alkylation, thiolysis and acidolysis. Next, linear peptide precursor will be cyclized and folded in a single one-pot reaction using GSH. After this step, the peptide will be purified by C18-solid-phase extraction cartridges and semi-preparative HPLC. Then it will be characterized by analytical HPLC and ESMS.3. Work Schedule:2.09-2.16 Take GLS and BBP exams2.19-3.03 Read papers, organize reading materials and write opening report3.05-3.23 Learn the experiment3.26-5.04 Peptide synthesis5.02-5.22 Write dissertation and attend graduation defense4. Research ReviewCyclotides are a very special family of circular proteins that were discovered and extracted from plants. These peptides typically constructed of 28 to 37 amino acid residues, share a unique head-to-tail circular knotted topology of three disulfide bridges, with one disulfide penetrating through a macrocycle formed by the two other disulfides and interconnecting peptide backbones,forming what is called a cyclic cystine knot motif ( CCK ).[1] The interlocking structure of the CCK motif makes cyclotides very compact and provide a highly rigid structure.[2] Due to this unique structure, cyclotides have shown high resistance to thermal or chemical denaturation and enzymatic decomposition. Natural cyclotides also display a series of exciting biological activities involving antimicrobial, protease inhibitory, insecticidal, anti-HIV, cytotoxic, and hormone-like activities.[3] Except for their bioactivities, compared with other proteins, they are very stable and able to cross the membrane easily, which indicated that some of them can have satisfying orally bioactivities. For example, the first discovered cyclotide kalata B1 and its derivatives such as some engineered cyclotides based on kalata B1 are orally effective.[4] Therefore, from the perspective of therapeutics, cyclotides have displayed strong potential to be developed as therapeutics targeting intracellular protein interactions both in vitro and in vivo. Furthermore, some cyclotides appear to be promising leads or frameworks for the development of novel peptide-based diagnostics like imaging probes and research tool[5].This article provides a brief review for cyclotides.The first cyclotide is called kalata B1 discovered by a Norwegian doctor, Lorents Gran in 1960s. He noticed that the natives in Africa drunk a kind of traditional medicinal herbal tea by boiling the plant Oldenlandia affinis to accelerate the childbirth. He then took samples and extracted the active compound, the cyclotide kalata B1. This tea needs to be boiled at 100℃, which can be viewed as another evidence about cyclotides stability. However, given the limitations of protein-based chemistry technique, it was not until 1995 that the CCK motif of kalata B1 was determined.[6] At nearly same time, different research scholars found several macrocyclic peptides that have similar size, sequence and structure of the Rubiaceae and Violaceae families, which led to the definition of cyclotides. Now, cyclotides have been also isolated from the other plant families including Cucurbitaceae, Fabaceae, Solanaceae, and Apocynaceae families.Cyclotides are classified into three subfamilies known as the Mbius, bracelet, and trypsin inhibitor cyclotide subfamilies.[7] Although all subfamilies have the same cyclic cystine knot motif, the specific compositionofthe loops is a little different. The Mbius subfamily of cyclotides including kalata B1, contain a cis-proline residue at loop 5 resulting in a slight twist of the backbone. However, bracelet cyclotides do not have the same structure. Bracelet cyclotides which are more diverse are a little bit larger than Mbius cyclotides. They are also much more numerous than Mbius cyclotides because bracelet cyclotides have constituted two-thirds of the known sequenced cyclotides. Despite of their dominance in number, bracelet cyclotides are more difficult to fold in vitro than either Mbius or trypsin inhibitor cyclotides, hence making them more difficult to synthesize chemically by using standard peptide synthesis protocols. Consequently, this type of cyclotide has been utilized less in biotechnological applications.The third subfamily of cyclotides is the trypsin inhibitor cyclotides which are very important in our research. This subfamily is the smallest one, which consists of the trypsin inhibitor cyclotides MCoTI-I and MCoTI-II. These cyclotides have been extracted from seeds of the plant Momocordica cochinchinesis of the Cucurbitaceae (squash) family.[8] These cyclotides are potent trypsin inhibitors. Although these cyclotides naturally contain the cystine-knot topology and they are backbone cyclized, they do not share a considerable amount of sequence homology with the other families of cyclotides. Instead, these two cyclotides show homology with other trypsin inhibitors which are not determined as cyclotides. Peptides from this family are linear variants of cyclotides and have a cystine-knot motif but lack the residues necessary to backbone cyclize. For this reason, they are also sometimes referred ascyclic knottins. Cyclotides from this family possess a longer sequence in loop 1, making the cystine knot slightly less rigid than in cyclotides from the other two subfamilies.As many studies have showed, cyclotides have many amazing biological activities. Originally, deduced from occurring cyclotides in plants and insects, it is high chance that cyclotides in these creatures play a role as host-defense agents.[9] They have shown that they can inhibit the growth and development of nematodes and trematodes and of mollusks efficiently.[10,11] Cyclotides seem to interact with cellular membranes and disrupt their normal functions. For example, the midgut membranes of Lepidopteran species are seriously disrupted after ingesting cyclotides.[12] However, the antimicrobial activity of cyclotides that are tested in vitro seems to occur only under non-physiological conditions involving the use of low ionic strength buffers, which significantly limits its potential for the design of antimicrobial therapeutics.In addition, the anti-HIV activity of cyclotides attracts a lot of scientists to dig into. From the reported cyclotides with anti-HIV activity that are used for identifying novel natural antiviral compounds to several new cyclotides from Mbius subfamilies, so far, it has been studied extensively.[13,14] While the exact molecular mechanism of action is not fully understood, some studies suggest that cyclotides may exert their anti-HIV activity by disrupting the binding or fusion of the virus to the cellular membrane. The inability of cyclotides to inhibit HIV reverse transcriptase activity seems to suggest that the antiviral activity may occur before entry of the virus into the host cell.[15]Several cyclotides have been reported that they have selective cytotoxicity against several cancer cell lines including primary cancer cell lines compared to normal cells, which means they possess anticancer activity, to some extent. For example, the cytotoxic activity of cyclotide vingo 5 from Viola ignobilis has been shown to be apoptosis-dependent when tested in HeLa cells.[16] Besides, three cyclotides extracted from Hedyotis diffusa, a Chinese medicinal plant from the Rubiaceae family, have been shown that they could induce apoptosis and inhibit proliferation and migration of several prostate cancer cell lines. However, these cyclotides still require efforts to optimize them to be developed as actual and effective therapeutics because of their not high therapeutic index.The first cyclotides were discovered and isolated from plants. Many studies involving cyclotides produce them through biosynthesis and the isolation of them from natural sources. However, it is easier for medium-sized peptides containing between 28-37 residues to readily synthesize the linear precursors of cyclotides using chemical methods. It can be accomplished by using solid-phase peptide synthesis (SPPS).[17] Both 9-Fluorenyloxycarbonyl (Fmoc)- and tert-butyloxycarbonyl (Boc)- based solid-phase peptide synthesis approaches have been successfully used. These two strategies can provide suitable linear peptide precursors for backbone cyclization using method of native chemical ligation (NCL).[18] However, the tendency has been towards Fmoc-based SPPS because of the more hazardous reagents used in Boc-based SPPS. In this work, the synthesis of cyclotide MCoTI-I will be proceeded with Fmoc-based SPPS, too. Fmoc- based SPPS has to use a sulfonamide safety-catch linker.[19] To produce the C-terminal thioester for NCL, the 4-sulfamylbutyryl AM resin will be used in this work. This approach requires the coupling of the C-terminal residue before synthesis. This type of resin will lead to a completed synthesis where the protected peptide is attached to the resin through that safety-catch linker. Once the resin is synthesized it has to be cleaved from sulfonamide resin by several steps. The first thing is activation of sulfonamide resin by alkylating the N-acyl-sulfonamide to make it more reactive towards S- nucleophiles. Sodium thiophenolate and ethyl thioglycolate then will be used for thiolysis.The sodium thiophenolate was used as a catalysis to quickly produce the initial thioester after the resin is cleaved. However, it is then replaced by the thioester of ethyl thioglycolate to form a more stable thioester. Following this reaction, the cleaved peptide is then fully deprotected by acidolytic treatment with trifluoroacetic acid ( TFA ). After that, the linear precursor contains the N-terminal cysteine and the C-terminal thioester.[20] After the linear thioester is prepared, it is necessary to then cyclize the backbone of the peptide and then fold it to form three disulfide bonds constituting the CCK motif. Cyclization and the oxidative folding of the CCK motif can then be treated in a single one-pot reaction under thermodynamic control conditions.[21] In this reaction, glutathione plays a role as thiol to promote cyclization of the thioester as well as to provide an appropriate redox buffer to aid in the oxidative folding, which is helpful for disulfides to be broken and reformed in the correct positions which are usually the most stable folded confirmation.[22] To obtain a satisfying yield and purity, diluting the concentration of linear thioester to centration range about 25mu;M-50mu;M is a must for avoiding the polymerization. Also, keeping PH range about 7.2-7.4 can treat this reaction in a timely manner and degassing the buffer before the reaction is able to keep the reduced glutathione and provide the suitable redox buffer for oxidative folding. Usually, the cyclization and folding need 24-92h. During the period, the reaction mixture will be monitored by HPLC. When the reaction is completed, the reaction mixture will be added acetic acid to stop the folding. The cyclotides are first purified using C18-solid-phase extraction cartridges to remove the buffer components and to concentrate the cyclotides. The folded cyclotides are finally purified by semi-preparative HPLC and characterized by analytical HPLC and electro-spray mass spectrometry (ESMS). The peptides are then lyophilized and stored at -20C until needed.Cyclotides now have been exclusively studied for diverse applications and imaging probes is a good example. The development and application of effective molecular diagnostic tool is a key determinant for early detection and successful treatment of plenty of aggressive diseases such as cancer and HIV. The advances in instrument have dramatically increased the need for novel imaging agents. Although there have not been a successful report of using cyclotides as imaging probes, there is a promising future for trypsin inhibitor cyclotides as imaging probes due to the similarity in sequence and structure between trypsin inhibitor cyclotides and the linear squash trypsin inhibitors. For example, G protein-coupled receptor CXCR4 which is overexpressed in cancer cells and associated with a propensity for metastasis and poor prognosis has been announced as a biomarker. MCoTI-I now has showed that it is able to antagonize CXCR4 with low nanomolar affinity.[23] Therefore, the development of MCoTI-I based cyclotides as imaging agents should be feasible. Above all, given their unique structure and bioactivity, cyclotides have become well-studied biomolecules. These unique polypeptide characteristics enable them to provide promising leads or frameworks to peptide drug design. Furthermore, the relatively small size of cyclotides makes them readily chemically synthesize, allowing introduction of chemical modifications such as unnatural amino acids and PEGylation to improve their pharmacological properties. It is anticipated that there will be more studies about oral bioavailability which is the main challenge of cyclotides. Although no one cyclotide has been into human clinical experiment, some records from animal models suggest that it might happen in a foreseeable future.1． Daly, N.L., K.J. Rosengren, and D.J. Craik, Discovery, structure and biological activities of cyclotides. Adv Drug Deliv Rev, 2009. 61(11): p. 918-30.2． S.S. Puttamadappa, et al., Backbone dynamics of cyclotide MCoTI-I free and complexed with trypsin. Angew Chem Int Ed Engl, 2010. 49(39): p. 7030-4.3． Aaron G. Poth, Michelle L. Colgrave, Russell E. Lyons, Norelle L. Daly and David J. Craik, Discovery of an unusual biosynthetic origin for circular proteins in legumes. PNAS 2011 June, 108 (25) 10127-10132.4． C. T. Wong, D. K. Rowlands, C.H.Wong, T. W. Lo, G.K.Nguyen, H. Y. Li, J. P. Tam, Orally Active Peptidic Bradykinin B1 Receptor Antagonists Engineered from a Cyclotide Scaffold for Inflammatory Pain Treatment. Angew. Chem. Int. Ed. 2012, 51,56205624.5． S. T. Henriques, D. J. Craik, Cyclotides as templates in drug design. Drug Discovery Today 2010, 15,5764.6． O. Saether, D.J.Craik, I. D. Campbell, K. Sletten, J. Juul, D. G. Norman, Elucidation of the Primary and Three-Dimensional Structure of the Uterotonic Polypeptide Kalata B1. Biochemistry 1995, 34,41474158.7． A. G. Poth, M. L. Colgrave, R. E. Lyons, N. L. Daly, D.J.Craik, Discovery of an unusual biosynthetic origin for circular proteins in legumes. Proc. Natl. Acad. Sci. USA 2011, 108,1012710132.8． A. Heitz, J. F. Hernandez, J. Gagnon, T. T. Hong, T. T. Pham, T. M. Nguyen, D. Le-Nguyen, L. Chiche, Solution Structure of the Squash Trypsin Inhibitor MCoTI-II. A New Family for Cyclic Knottins. Biochemistry 2001, 40,79737983.9． C. Jennings, J. West, C.Waine, D. Craik, M. Anderson, Biosynthesis and insecticidal properties of plant cyclotides: The cyclic knotted proteins from Oldenlandia affinis. Proc. Natl. Acad. Sci. USA 2001, 98,1061410619.10． M.L.Colgrave, A. C. Kotze, D.C.Ireland, C. K. Wang, D. J. Craik, The Anthelmintic Activity of the Cyclotides: Natural Variants with Enhanced Activity. ChemBioChem 2008,9,19391945.11． M. R. Plan, I. Saska, A. G. Cagauan, D. J. Craik, Backbone Cyclised Peptides from Plants Show Molluscicidal Activity against the Rice Pest Pomacea canaliculata (Golden Apple Snail). J. Agric. Food Chem. 2008, 56,52375241.12． B. L. Barbeta, A. T. Marshall, A. D. Gillon, D.J.Craik, M. A. Anderson, Plant cyclotides disrupt epithelial cells in the midgut of lepidopteran larvae. Proc. Natl. Acad. Sci. USA 2008, 105,12211225.13． K. R. Gustafson, T. C. McKee, H. R. Bokesch, Effects of cyclotides against cutaneous infections caused by Staphylococcus aureus. Curr. Protein Pept. Sci. 2004, 5,331340.14． K. R. Gustafson, L. K. Walton, R. C. Sowder,Jr.,D.G.Johnson, L.K.Pannell, J. H. Cardellina, Jr., M. R. Boyd, Anti-HIV Cyclotides from the Chinese Medicinal Herb Viola yedoensis. J. Nat. Prod. 2000, 63,176178.15． K. R. Gustafson, R. C. Sowder, L.E.Louis, E. Henderson, I.C.Parsons, Y. Kashman, J. H. Cardellina, J. B. McMahon, R. W. Buckheit, L.K.Pannell, M. R. Boyd, Circulins A and B. Novel human immunodeficiency virus (HIV)-inhibitory macrocyclic peptides from the tropical tree Chassalia parvifolia. J. Am. Chem. Soc. 1994, 116,93379338.16． M.A. Esmaeili, N. Abagheri-Mahabadi, H.Hashempour, M.Farhadpour, C. W. Gruber, A. Ghassempour, The alpine violet, Viola biflora, is a rich source of cyclotides with potent cytotoxicity , Fitoterapia 2016, 109,162168.17． Marglin, A. and R.B. Merrifield, Chemical synthesis of peptides and proteins. Annu Rev Biochem, 1970. 39: p. 841-66.18． Aboye, T.L., et al., Efficient one-pot cyclization/folding of Rhesus theta-defensin-1 (RTD1). Bioorg Med Chem Lett, 2012. 22(8): p. 2823-6.19． Camarero, J.A. and A.R. Mitchell, Synthesis of proteins by native chemical ligation using Fmoc-based chemistry. Protein Pept Lett, 2005. 12(8): p. 723-8.20． Camarero, J.A.P., J; Muir, TW, Chemical Synthesis of a Circular Protein Domain: Evidence for Folding-Assisted Cyclization. Angew Chem Int Ed Engl, 1998. 37(3): p. 347-9.21． Kimura, R.H., A.T. Tran, and J.A. Camarero, Biosynthesis of the cyclotide Kalata B1 by using protein splicing. Angew Chem Int Ed Engl, 2006. 45(6): p. 973-6.22． Leta Aboye, T., et al., Ultra-stable peptide scaffolds for protein engineering-synthesis and folding of the circular cystine knotted cyclotide cycloviolacin O2. Chembiochem,2008. 9(1): p. 103-13.23． Teshome L. Aboye1, Helen Ha1, Subhabrata Majumber3, Frauke Christ4, Zeger Debyser4, Alexander Shekhtman3, Nouri Neamati1, and Julio A. Camarero1,2,*. Design of a novel cyclotide-based CXCR4 antagonist with antihuman immunodeficiency virus (HIV)-1 activity. J Med Chem. 2012 December 13; 55(23): 1072910734.
以上是毕业论文文献综述，课题毕业论文、任务书、外文翻译、程序设计、图纸设计等资料可联系客服协助查找。
