<p>The purpose of this study was to evaluate enantioselective analytical methods by separation of the enantiomers of four drugs (citalopram, zopiclone, tramadol and methylphenidate) and their metabolites. The analyses were performed with HPLC-UV with columns whose stationary phases were based on macrocyclic glycopeptides (Chirobiotic V, V2 and T) and polysaccharides (Lux Cellulose-1, Cellulose-2 and Amylose-2).</p><p>The Chirobiotic V column showed high selectivity for citalopram and its metabolites. High resolution was obtained using a mobile phase consisting of methanol, acetic acid and ammonia. High selectivity for the enantiomers of zopiclone and its metabolites were obtained on the Cellulose-2 column using a mobile phase consisting of acetonitrile and ammonium acetate buffer.</p><p>The enantiomers of tramadol were separated with the Amylose-2 column. However, changes in the pressure arose, probably caused by the additive NH₄HCO₃. When the analysis was repeated at a later occasion, reproducible results were not obtained. With the Cellulose-1 column, lower selectivity was obtained, resulting in unacceptably long analysis time.</p><p>Only a few analyses of methylphenidate were performed and the results indicated that the glycopeptide columns had higher selectivity for this compound than the polysaccharides.</p>

The purpose of this study was to evaluate enantioselective analytical methods by separation of the enantiomers of four drugs (citalopram, zopiclone, tramadol and methylphenidate) and their metabolites. The analyses were performed with HPLC-UV with columns whose stationary phases were based on macrocyclic glycopeptides (Chirobiotic V, V2 and T) and polysaccharides (Lux Cellulose-1, Cellulose-2 and Amylose-2). The Chirobiotic V column showed high selectivity for citalopram and its metabolites. High resolution was obtained using a mobile phase consisting of methanol, acetic acid and ammonia. High selectivity for the enantiomers of zopiclone and its metabolites were obtained on the Cellulose-2 column using a mobile phase consisting of acetonitrile and ammonium acetate buffer. The enantiomers of tramadol were separated with the Amylose-2 column. However, changes in the pressure arose, probably caused by the additive NH4HCO3. When the analysis was repeated at a later occasion, reproducible results were not obtained. With the Cellulose-1 column, lower selectivity was obtained, resulting in unacceptably long analysis time. Only a few analyses of methylphenidate were performed and the results indicated that the glycopeptide columns had higher selectivity for this compound than the polysaccharides.

Användningen av biokatalysatorer, särskilt enzymer, inom kemikalie- och läkemedelsindustrin erbjuder betydande fördelar jämfört med de traditionella kemo-katalytiska metoderna som historiskt har dominerat industrin. En viktig klass av enzymer, transaminaser, spelar en central roll i tillverkningen av kirala aminer, som utgör grundläggande byggstenar i dessa industriella sektorer. Denna studie är inriktad på ett specifikt amin transaminas från Silicibacter pomeroyi. Tidigare har detta enzym visat förmåga att katalysera en mängd olika reaktioner för produktion av kirala aminer, men för att realisera dess fulla potential inom industriella tillämpningar krävs förbättrad stabilitet vid högre temperaturer. I motsats till de vanligt förekommande metoderna för proteinteknik, såsom rationell design och riktad evolution, används i denna studie släktsekvensrekonstruktion för att skapa mer temperaturtåliga varianter av detta enzym. Tidigare användning av denna metod har visat sig kunna generera proteiner med högre temperaturtålighet. Genom denna metod, där förfäder till detta enzym återskapas utifrån bevarade sekvenser, förväntas generering av varianter som kan bibehålla sin funktion vid högre temperaturer under en längre tid. Genom att utforska denna alternativa strategi för proteinteknik strävar studien efter att ge mer robusta biokatalysatorer för industriella tillämpningar. Utfallet från denna studie visade att två förfäder hade ökad termostabilitet. Detta visade sig dels genom analys av T5015 som påvisade en 3.9 och 6 C° förbättring för respektive förfader. Vidare påvisade t1/2 mätningar att dessa enzymer kunde utstå 2.06 till 3.72 gånger så lång tid vid 55 C° innan de inaktiverades. De påvisade dock lägre specifik aktivitet vid rumstemperatur, där en bidragande faktor till detta var att enbart en liten fraktion av förfäderna är korrekt veckade. Detta visar att det är nödvändigt med fortsatta förbättringar och fortsatta studier kring substratacceptans och stabilitet i olika lösningsmedel. Sammanfattningsvis påvisar resultaten att släktsekvensrekonstruktion är en proteinteknik som fungerar för att skapa proteiner med ökad termostabilitet och bör ses som ett mer självklart alternativ till riktad evolution och rationell design. / The utilisation of biocatalysts, particularly enzymes, in chemical and pharmaceutical industries presents significant advantages over the traditional chemocatalytic methods that historically dominated the industry. A crucial class of enzymes, transaminases, play a central role in the production of chiral amines, fundamental building blocks in these industrial sectors. This study focuses on a specific amine transaminase from Silicibacter pomeroyi. While this enzyme has previously demonstrated the ability to catalyse a variety of reactions for chiral amine production, realising its full potential in industrial applications requires enhanced stability at higher temperatures. In contrast to commonly employed protein engineering methods such as rational design and directed evolution, this study utilises ancestral sequence reconstruction to generate more temperature-resistant variants of this enzyme. Previous applications of this method have shown promising results in generating proteins with increased thermal stability. Through this approach, wherein ancestors of this enzyme are recreated from extant sequences, it is expected that variants capable of maintaining function at higher temperatures will be produced. By exploring this alternative strategy for protein engineering, the study aims to provide more robust biocatalysts for industrial applications. The outcome of this study is that two ancestors exhibited increased thermostability. This was evidenced by the analysis of T5015, which showed an improvement of 3.9 and 6 °C for each respective ancestor. Furthermore, t1/2 measurements indicated that they remained active for 2.06 to 3.72 fold longer at 55 °C before becoming inactive. However, they exhibited lower specific activity at room temperature, partially due to only a small fraction of the ancestral protein samples being properly folded. This suggests further improvements and continued investigations into substrate acceptance and stability in different solvents are required. In conclusion, this study demonstrates that ancestral sequence reconstruction is a protein engineering technique effective in enhancing protein thermostability and should be considered a more viable alternative to directed evolution and rational design.