Trombin är ett trypsinliknande serinproteas som har stor del i kroppens reglering av blodets fluiditet och koagulation genom att det klyver faktorer som slutligen leder till att blodet kan koagulera och fibrin bildas. Syftet med det här projektet har varit att syntetisera fragment som designats med datorgrafiska metoder på Astra Zeneca och sedan analysera fragmenten med affinitetskromatografi med trypsin immobiliserad på kiselgelspartiklar. I projektet har även ett nytt koncept prövats, nämligen att analysera reaktionsblandningarna direkt med hjälp av trypsinkolonnen utan att först isolera och rena föreningarna. De designade fragmenten har syntetiserats med standardmetoder, bl.a. har N,N’dicyklohexylkarbodiimid (DCC) använts. DCC är ett kopplingsreagens som kopplar ihop aminer med karboxylsyror genom skapande av en amidbindning, peptidbindning. Ibland då lite mer komplicerade peptider skall göras, kan aminoänden eller karboxyländen skyddas med en skyddsgrupp, för att dessa inte ska reagera under reaktionssteget och för att man ska få den peptiden man är ute efter. Vätskekromatografi-masspektrometri, LC-MS har använts för identifiering av reaktionblandningarna (substanserna). Resultaten visade att två av de framställda föreningarna retarderades på trypsinkolonnen, vilket innebär att de växelverkar med trypsin och att s.k. hits, träffar erhållits. Synteserna verkar ha gått bra, då LC-MS har visat att det är rätt produkt som finns i kolven. Projektet har visat att det går att designa fragment som man syntetiserar och sen analyserar med AFC-MS. AFC-MS har dessutom visat vara en lämplig metod för screening av svagt bindande fragment. / Through different, intricate mechanisms, the body regulates the coagulation and the fluidity of the blood. This is an important part of the hemostasis if for example bleeding occurs, or to provide the body with oxygen and nutrition. Thrombin is a trypsin like serine protease that plays an important role in this process since it is cleaving factors that eventually lead to coagulation of the blood and production of fibrin. The aim of this project has been to synthesize fragments that have been designed with computer graphical methods by Astra Zeneca and then to analyze the fragments with affinity chromatography that has trypsin immobilized on silica particles. The project also introduces a new concept i.e. to analyze reaction mixtures on the trypsin column without first isolating and purifying the compounds. The design fragments are synthesized by earlier reported standard methods. N, N'-dicyclohexylcarbodiimide (DCC) was used as coupling reagents to form amide or peptide bonds between carboxylic acids and amines. In some of the reactions the amino group or the carboxylic groups of the amino acid were protected, to prevent these from interfering in the reaction and avoid to formation of unwanted substances. After the reactions the protecting groups were removed in different ways. If it is attached to the amine as a Boc-group (the most common protecting group to protect amino groups) it would be removed with trifluoracetic acid, and if the carboxyl group was protected, the protecting group would be removed with catalytical hydrogenolysis, for example by adding sodium hydroxide. To analyze if the right product has been acquired, analyzes with liquid chromatography-masspectrometry has been performed. The results showed that two of the prepared fragments were retarded on the trypsin column meaning that they interact with trypsin i.e. hits were obtained. The results showed that the two fragments that were analyzed with the trypsin column did retard a bit, which implies that they interact with trypsin. The synthesis seems to have been successful since the liquid-chromatography has shown that the right product has been made. This project has proven that it is possible to design fragments with computer graphical methods before synthesizing and analyzing with AFC-MS. AFC-MS has also been shown to be a suitable method for screening of weak binding fragments.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:lnu-6160 |
| Date | January 2010 |
| Creators | Phan, Hilda |
| Publisher | Linnéuniversitetet, Institutionen för naturvetenskap, NV |
| Source Sets | DiVA Archive at Upsalla University |
| Language | Swedish |
| Detected Language | English |
| Type | Student thesis, info:eu-repo/semantics/bachelorThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
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