1. 研究目的与意义

OBJECTIVE: Synthesis of a peptide that reorganized from nature peptide and test of its antimicrobial activity against staphylococcus aureus, escherichia coli and candida albicansSIGNIFICANCE: Antimicrobial peptides(AMPs) are an indispensable part of the innate immune system for its broad spectrum of activity against pathogenic bacteria and fungi, and often, their killing activities extend to enveloped viruses, parasites and sometimes even to cancerous cells. Unfortunately, the number of new antibiotics in the pipeline of the big pharmaceutical companies has been declining because they have shifted their attention towards more lucrative areas of drug development even though the emergence of multidrug-resistant super drug bacteria has created an urgent need for the development of novel classes of antimicrobials.Given the situation, our group keens on optimizing the peptides segregated from natural animals like frogs, snakes and scorpions to heighten its resistance to digestive enzymes, fighting capacity to bacteria and etc. This research is a part of exploration about a synthetic peptide transformed from discovered natural peptide and its antimicrobial activity.

2. 文献综述

Antimicrobial peptides prevention of bacteria biofilm Liu NairongSchool of pharmacy, Nanjing University of Chinese MedicineABSTRAT: The ability on drug resistance of some pathogenic bacteria improved because of the existence of biofilms making it an intractable problem in clinical and creating an urgent need for the development of novel class of antimicrobials. Antimicrobial peptides (AMPs) are an indispensable part of the innate immune system for its broad spectrum of activity against pathogenic bacteria and fungi, and often, their killing activities extend to enveloped viruses, parasites and sometimes even to cancerous cells. For now, many modes of their action have been presented and one of them is the interaction with biofilm of the bacteria.KEY WORDS: biofilm, AMPs, mechanism, interactionBIOFILMA biofilm comprises any syntrophic consortium of microorganisms in which cells stick to each other and often also to a surface. These adherent cells become embedded within a slimy extracellular matrix that is composed of extracellular polymeric substances (EPS). The cells within the biofilm produce the EPS components, which are typically a polymeric conglomeration of extracellular polysaccharides, proteins, lipids and DNA[1][2].The discovery of biofilms challenged the thought on bacteria that they are single and free swimming microbial cell. And the existence of biofilms greatly enhanced the tolerance of some bacteria to the sever environments for that they can avoid being washed away by blood by simply sticking to the surface of organization and prevent the permeation of antibiotic drugs with the help of EPS. Even though the bacteria itself might be sensitive to certain drugs, the tolerance of bacteria in biofilm is orders of magnitude higher than free swimming bacteria.The biofilm can exist generally at two circumstance[3]. One of them is at the surface ofthe hosts organization like epithelial cell, mucosal surface, teeth and etc, which may cause urinary tract infections, middle-ear infections, formation of dental plaque, gingivitis and other infections. The other comes from their residence at the surface of non-living things like medical apparatus and instruments, which may result in disease like bacterial vaginosis, coating contact lenses, catheter infections and so on. In this case, the pathogenic bacteria might escape from the biofilm and flow with blood causing some other infections.As mentioned before, EPS is an important weapon to help the battle with antibiotics, for it can reduce or prevent the permeation of antibiotic drugs into the bacteria. Also the growth rate of bacteria in the biofilm may slow down even stop making it stronger to antibiotics. Whats more, quorum sensing (QS) system of the bacteria might be stimulated to induce and regulate the genetic expression[4]. The structure of biofilm makes it drug-fast and its mechanism may be one or multiple. Whichever mode it works, biofilm is hard to deal with clinically. AMPs, however, have been proved being a prospective way to fight with pathogenic bacteria and biofilm.ANTIMICROBIAL PEPTIDESSince the initial discovery of cecropins in the 1980 by Swedish scientists from insects[5], AMPs have been heralded as a promising alternative to todays antibiotics. APMs typically have a broad spectrum of activity against pathogenic bacteria and fungi, and often, their killing activities extend to enveloped virus, parasites and sometimes even to cancerous cells[6].Generally, two physical features are common for AMPs: a cationic charge and a significant proportion of hydrophobic residues. The former property promotes selectivity for negatively charged microbial cytoplasmic membranes over zwitterionic mammalian membranes whereas the latter facilitates interactions with the fatty acyl chain[6].Numerous researches have verified that AMPs can inhibit the formation of biofilm. LL-37 is an important antimicrobial peptide in human body and has been proved to have biofilm inhibitory ability[7]. According to Shis research, LL-37 can refrain Acinetobacter baumanniis biofilm even far away from minimun inhibitory concentration[8]. A Chinese team found that LL-37 has great antimicrobial activity to biofilm ofStaphylococcus aureu, Pseudomonas aeruginosa, Acinetobacter baumannii which prevents its adherence and gathering and prohibits dispersion of mature biofilm[9]. Anunthawas team researched that amphoteric cationic peptides KT2 and RT2 can enter and kill the bacteria whether it is free or covered by biofilm[10]. In addition to the peptides mentioned, many other peptides have also been certified having interaction with biofilm like BMAP-27 from cow, SMAP-29 from sheep and NA-CATH from Naja atra[4]. A huge number of peptides have been or being discovered which can interact with biofilm and this mechanism is just a small part of the research of AMPs action mode. The territory of AMPs still has immense space for us to explore.REFERENCES[1]Vert, Michel; Doi, Yoshiharu; Hellwich, Karl-Heinz; Hess, Michael; Hodge, Philip; Kubisa, Przemyslaw; Rinaudo, Marguerite; Schu, Franois. Terminology for biorelated polymers and applications (IUPAC Recommendations 2012) . Pure and Applied Chemistry. 2012, 84 (2): 377410.[2]Lpez, Daniel; Vlamakis, Hera; Kolter, Roberto. "Biofilms". Cold Spring Harbor Perspectives in Biology. 2010,2 (7): a000398.[3]Research on microbial biofilms.NIH, National Heart, Lung, and Blood Institute. 2002-12-20.[4]李艳. 抗菌多肽对生物被膜的作用研究进展. 临床医药文献杂志[J]. 2018, 5(31).[5]MERRIFIELD R B, VIZIOLI L D, BOMAN H G. Synthesis of the antibacterial peptide cecropin A (1-33)[J]. Biochemistry, 1982, 21(20): 5020-5031.[6]Leonard T. Nguyen, Evan F. Haney and Hans J. Vogel. The expanding scope of antimicrobial peptide structures and their modes of action[J]. Cell.Trends in Biotechnology, 2011, 29(9).[7]STREMPEL N, STREHMEL J, OVERHAGE J. Potential application of antimicrobial peptides in the treatment of bacterial biofilm infections[J]. CURR Pharm DES, 2015,21(1).[8]SHI, GAO Y, LU z, ect. Effect of antibacterial peptide LL-37 on the integrity of Acinctobacter baumannii biofilm[J]. NAN FANG YI KE DA XUE BAO,2014,34(3):426-429.[9]回丽媛, 李军, 朱明星, 等. 抗菌肽17BIOHE2对金黄色葡萄球菌生物被膜的抑制作用[J]. 微生物学通报, 2017(04):872-880.[10]ANUNTHAWAN T, FUENTE-NUNEZ C L, HANCOCK R W, et. Cationic amphipathic peptides KT2 and RT2 are taken up into bacterial cells and kill planktonic and biofilm bacteria[J]. Biochimica ET Biophysica ACTA, 2015, 1848(6):1352-1358.

3. 设计方案和技术路线

STEP 1: SOLID PHASE PEPTIDE SYNTHESISRink amide MBHA resin protected by Fmoc is used as the holder of the synthesizing peptide which has amino tail to protect its structure from disorganization during the progress. 20%Dimethylformamide(DMF) in piperidine is chosen to deprotect the Rink amide MBHA resin while the terminal amino acid is activated by N-Methylmorpholine(NMM) thus they can combine by peptide bond with the catalyzing of HBTU. And so on, each amino acid selected is able to bond on the construction backwards forming a peptide with resin and side chain protector. Residual DMF will be wash by Dichloromethane(DCM) which has high volatility during the synthesizing progress. Then comes the cleavage reaction, 94%Trifluoroacetic acid(TFA), 2%water, 2%Thioanisole(TIS) and 2% 1,2-Ethanedithiol(EDT) are used as the cleavage solution to remove the resin and side chain protector and the free peptide is synthesized. Wash the final product with DCM and then extrat with diethyl ether after concentration.After repeating wash with Et2O, mobile phase of HPLC ought to be added into the peptide to make sure the environment is the same as the structural trial. At last the peptide need to be frozen to dry to get the proper crystal form.STEP 2: STRUCTURAL IDENTIFICATIONHPLC and LC-MS are used to inspect if the anticipatory peptide is successfully synthesized.STEP 3: ANTIMICROBIAL ACTIVITY ASSAYThree bacteria, staphylococcus aureus, escherichia coli and candida albicans, which respectively belong to gram-positive bacteria, gram-negative bacterium and fungus, are chosen to test the antimicrobial activity of the synthesized peptide with minimal inhibitory concentration(MIC) as the index.

4. 工作计划

● 04/03/2022-08/03/2022: Study relevant knowledge and read related documents● 11/03/2022-15/03/2022: Prepare for the synthesis and write the initiating report and review paper● 19/03/2022-22/03/2022: Get the final product● 25/03/2022-28/03/2022: Test construction of the synthesized peptide ● 08/04/2022-12/04/2022: Run the antimicrobial activity assay● 15/04/2022-19/04/2022:Analyze the data and meet the tutor● 22/04/2022-17/05/2022:Write the reporting paper and get prepared for the graduation oral examination

5. 难点与创新点

Today, Fmoc SPPS is the method of choice for peptide synthesis. Very‐high‐quality Fmoc building blocks are available at low cost because of the economies of scale arising from current multiton production of therapeutic peptides by Fmoc SPPS. Many modified derivatives are commercially available as Fmoc building blocks, making synthetic access to a broad range of peptide derivatives straightforward. The number of synthetic peptides entering clinical trials has grown continuously over the last decade, and recent advances in the Fmoc SPPS technology are a response to the growing demand from medicinal chemistry and pharmacology.