Optimizacija ločbe in izolacije izbranega agonista NOD2 s tekočinsko kromatografijo visoke ločljivosti

Močnik Roner, Maša (author), Pajk, Stane (mentor)

Spojina SG-10 je agonist receptorja nukleotid-vezoče oligomerizacijske domene 2 (NOD2). NOD2 imajo zelo pomembno vlogo pri regulaciji prirojenega imunskega sistema, saj njihova aktivacija povzroči izločanje vnetnih citokinov in protimikrobnih peptidov. Poleg aktivacije prirojenega, ima vezava agonističnih ligandov na NOD2 vpliv tudi na pridobljeni imunski sistem, zato bi jih lahko uporabljali kot adjuvanse v cepivih. Trenutno so na trgu dovoljeni le štirje posamični adjuvansi in njihove kombinacije, zato je odkrivanje novih ključnega pomena. Pri sintezi spojine SG-10 so uporabili racemno zmes kisline, ki je bila pripeta na kiralno čist peptid z dvema kiralnima centroma. Nastali produkt, spojina SG-10, je zato zmes diastereomerov. Ker se je spojina SG-10 izkazala kot močan agonist NOD2, je pomembno, da za nadaljnje analize izoliramo oba diastereomera agonista NOD2 ter raziščemo, ali lahko s posameznim dosežemo še nižjo vrednost EC50 v primerjavi z racematom. V tej magistrski nalogi smo se ukvarjali z ločbo na sistemih tekočinske kromatografije ultravisoke ločljivosti z obrnjeno fazo ter tekočinske kromatografije visoke ločljivosti z normalno fazo. Vrhov diastereomerov na sistemu tekočinske kromatografije ultravisoke ločljivosti z obrnjeno fazo z variiranjem različnih kromatografskih pogojev (vrst stacionarnih (C18, Phenyl, Shield RP18), in mobilnih faz (različne kombinacije vode oz. 0,1 % trifluoroacetata ter vrste organskega modifikatorja (metanol in acetonitril)) nismo uspeli ločiti na bazni liniji. Bolj uspešni smo bili s sistemom tekočinske kromatografije visoke ločljivosti z normalno fazo, s pomočjo katerega smo spojino SG-10 ločili na posamična diastereomera, ju izolirali na miligramski skali, določili količino izoliranih diastereomerov in določili diastereomerni presežek. Delo je potekalo tako, da smo na analizni koloni 3-CelluCoat® najprej razvili in optimizirali metodo ločbe, nato pa smo jo prenesli na semi-preparativno kolono 5-CelluCoat® in jo optimizirali. Nato smo analita ločeno zbrali in izolirali 1,65 mg oziroma 1,76 mg posameznega diastereomera. Točne mase smo natančno določili s ponovno analizo s kolono 3-CelluCoat. Izračunali smo, da je med sintezo nastalo za približno 20 % več enega izmed diastereomerov.

• Language: Slovenian
• Keywords: agonist receptorja NOD2, NP-HPLC, RP-UHPLC, ločba diastereomerov, diastereomerni presežek
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2019
• PID: 20.500.12556/RUL-107971
• Publication date in RUL: 11.06.2019

Secondary language

• Language: English
• Title: Optimization of selected NOD2 agonist separation and isolation with high performance liquid chromatography

Abstract

The compound SG-10 is an agonist of the nucleotide-binding oligomerization domain-containing protein 2 (NOD2). NOD2 play a very important role in the regulation of the innate immune system since their activation causes secretion of pro-inflammatory cytokines and antimicrobial peptides. In addition to innate immune response activation, binding of agonistic ligands to NOD2 also affects the adaptive immune system, and could, therefore, be used as a vaccine adjuvant. Currently, only four individual adjuvants and their combinations are available on the market; consequently, the discovery of novel adjuvants is of utmost importance. During the synthesis of SG-10, a racemic mixture of acid was attached to the chirally pure peptide incorporating two stereocenters, thus the resulting product is a mixture of two diastereomers. Given the fact that SG-10 proved to be a potent NOD2 agonist, it is very important to isolate both diastereomers and ascertain whether one of them exhibits an even lower EC50 value compared to the racemate. In this Master’s thesis, we used reverse-phase ultra-high performance liquid chromatography and normal-phase high performance liquid chromatography systems to separate two analytes on a milligram scale. Firstly, reverse-phase ultra-high performance liquid chromatography system and a variety of different columns were used, such as C18 columns, phenyl columns of two different lengths and the Shield RP18 column. Despite changing the composition of the mobile phase (with the main solvent variations (water and 0.1% trifluoroacetic acid) and the variations of the type (ethanol and acetonitrile) as well as the amounts (from 5% to 95%) of the organic modifier) the peaks could not be separated at the baseline. On the other hand, the use of normal-phase high performance liquid chromatography system and the 3-CelluCoat® analytical column proved to be successful in terms of separation. We developed and optimized the separation method using the 3-CelluCoat® analytical column and then transferred it to the semi-preparative scale using 5-CelluCoat® column. The analytes were separately collected and isolated thereby obtaining 1.65 mg or 1.76 mg of each diastereomer, as determined by re-analysis with the 3-CelluCoat column. We also determined that during the synthesis of SG-10 a 20% excess of one of the diastereomers was formed.

• Keywords: NOD2 agonist, NP-HPLC, RP-UHPLC, separation of diastereomers, diastereomeric excess