Chiral separation and isolation of enantiomers of selected InhA inhibitors

Tuberkuloza je kronična nalezljiva bolezen. Povzroča jo bakterija Mycobacterium tuberculosis. S tuberkulozo, večinoma z latentno obliko, je okuženega približno ? svetovnega prebivalstva od katerih je letno 10 milijonov novih primerov. Težava je predvsem v bakterijski rezistenci, kar vodi do velikega zdravstvenega problema. Izoniazid je učinkovina 1. izbora pri tuberkulozi, ki ima v visokih koncentracijah baktericidno delovanje. Učinkuje tako, da zavira biosintezo encima 2-trans-enoil ACP reduktaze (InhA), in s tem prepreči sintezo mikolnih kislin v bakterijski celični steni ter smrt bakterij. Odpornost sevov M. tuberculosis izhaja iz mutacije gena katG, ki pretvori izoniazid v aktivno učinkovino. V magistrski nalogi smo s HPLC sistemom opremljenim s kolono s kiralnim selektorjem razvili metodo za kiralno ločbo racematov izbranih zaviralcev InhA. Encimi kot tarče učinkovin imajo sposobnost kiralnega prepoznavanja. S pomočjo kiralne kromatografije smo želeli ločiti zmes racematov izbranih zaviralcev InhA (v našem primeru spojine PZZ-24 in PAS-17) z namenom pridobitve optično čistih enantiomerov. Ti imajo namreč pogosto različne biološke učinke, zato pa je smiselno posamezna enantiomera ločiti in testirati vsakega posebej. Metodo smo naprej razvijali na analitski koloni s tehniko normalnofazne kromatografije in z uporabo mobilnih faz, sestavljenih iz zmesi različnih topil. Rezultate smo ovrednotili in izbrali metodo z najbolj ustrezno ločbo. To smo prenesli na semi-preparativno kolono, kjer smo jo nadalje optimizirali. Spojine PZZ-24 nismo uspeli ločiti, ločba se je sicer nakazovala, a nismo uspeli ločiti vrhov na bazni liniji, kljub uporabi različnih kombinacij mobilnih faz. Nasprotno smo spojino PAS-17 uspeli zadovoljivo ločiti na analitski kakor tudi na semi-preparativni koloni. Na koncu smo zbrali frakcije s posameznim enantiomerom in jih uparili ter raztopili v DMSO. S pomočjo reverznofazne kromatografije smo pripravili umeritveno krivuljo standardom spojine PAS-17 in nato določili koncentracijo spojine PAS-17 v DMSO raztopini. S tehtanjem izoliranih enantiomerov bi zaradi malih mas (okoli 4 mg) namreč naredili večjo napako. Zaradi male količine izoliranega enantiomera meritev optične sučnosti nismo izvedli. Smo pa izračunali enantiomerni presežek izoliranim frakcijam posameznega enantiomeraTuberculosis is a chronic infectious disease. It is caused by Mycobacterium tuberculosis. It is estimated that one quarter of the world\u27s population is infected with tuberculosis (mostly in latent form), with 10 million new cases annually. The problem is mainly in bacterial resistance, which leads to a major health problem. Isoniazid is the active substance of the first choice for the treatment of tuberculosis infection and has bactericidal activity in high concentrations. It is effective in inhibiting the biosynthesis of 2-trans-enoyl ACP reductase enzyme (InhA), thereby preventing the synthesis of mycolic acids, a key component of the bacterial cell wall. The resistance of M. tuberculosis strains towards isoniazid derives from the mutation of the katG gene, which converts isoniazid into the active form. In the master\u27s thesis we developed a method for the chiral separation of racemates of selected InhA inhibitors using HPLC system equipped with a column with chiral selector. Enzymes are able to distinguish between enantiomers, therefore identification of more active enantiomer or eutomer is important for drug development. By chiral chromatography, we wanted to separate the mixture of enantiomers of certain InhA inhibitors (in our case compounds PZZ-24 and PAS-17) so the inhibitory optically pure enantiomers could be tested. Namely, the chiral active enantiomers often have different biological effects, therefore it is desirable to detect the affinity, efficacy, purity of each enantiomer separately. The method was first developed on the analytical column, we used normal phase chromatography and tested several mobile phases. We evaluated the results and selected the method with the most appropriate separation and transferred it to the semi-preparative column where we further optimized it. Separation of compound PZZ-24 was found to be inadequate and we therefore concentrated on compound PAS-17 where separation was excellent. Finally, the best method was transferred from analytical to semi-preparative column and optimized again. Following optimization we started collecting resolved signals and evaporated the mobile phase of collected fractions. We further conducted analysis of each enantiomer - we determined the enantiomeric excess, however we were not able to determine optical rotation, because of too small quantities of separated enantiomers. Collected samples were dissolved in DMSO, this solutions served for determination of IC50 values. We determined the exact concentrations of each enantiomer in DMSO solutions with reverse phase chromatography. Weighing of isolated enantiomer would produce much greater error