Načrtovanje in sinteza fluorescentno označenega zaviralca imunoproteasoma

Zupan, Petra (author), Obreza, Aleš (mentor), Sosič, Izidor (comentor)

Jedro sistema ubikvitin – proteasom predstavlja 26S proteasom, multiproteazni kompleks, ki je odgovoren za proteolitično razgradnjo z ubikvitinom označenih proteinov. Številni ključni celični procesi so odvisni od njegove aktivnosti in posledično igra pomembno vlogo pri različnih patoloških stanjih, kot so rakave, avtoimune in nevrodegenerativne bolezni. Imunoproteasom je različica proteasoma, ki se normalno nahaja v celicah imunskega sistema, ob vnetju pa različni vnetni citokini inducirajo njegov nastanek tudi v celicah nehematopoetskega izvora. Bortezomib in karfilzomib sta že registrirana zaviralca proteasoma za zdravljenje multiplega mieloma, vendar gre zaradi neselektivnega delovanja in posledično povzročenih resnih neželenih učinkov razvoj v smer iskanja selektivnih zaviralcev imunoproteasoma. Pomembno orodje pri razumevanju njegove funkcije in iskanju novih selektivnih zaviralcev tega encima predstavlja uporaba na aktivnosti temelječih fluorescentnih sond. Te spojine se selektivno vežejo na želeno tarčo in omogočajo njeno vizualizacijo znotraj celice. V okviru magistrske naloge smo sintetizirali prekurzorje fluorescentno označenega zaviralca β5i podenote imunoproteasoma. Slednja je poglavitna pri nastajanju vnetnih citokinov in ima kot farmakološka tarča največji potencial. Za omenjeno sondo je značilno, da ima reaktivno elektrofilno glavo, ki kovalentno ireverzibilno reagira z nukleofilnim delom treonina v aktivnem mestu β5i. V ta namen smo načrtovali pripravo spojine z epoksidnim obročem, za katerega je značilno, da z aktivnim mestom reagira na specifičen način s tvorbo morfolinskega obroča, s čimer je dosežena večja selektivnost. Sintezni načrt smo začeli z uspešno pretvorbo L-fenilalanina v Weinrebov amid in preko Grignardove reakcije v α, β-nenasičen keton, naslednja stopnja epoksidacije pa ni bila uspešna. V drugem delu smo uspešno pripravili končno spojino iz L-lizina, D-alanina in 3-metilinden-2-karboksilne kisline. Sonda zaradi neuspele sinteze spojine z epoksidnim obročem še ni sintetizirana, saj le-ta predstavlja manjkajoči člen v verigi sintez, ki vodijo do končne spojine. Ker so bile direktne epoksidacije na konjugiranem sistemu neuspešne, bi bilo v prihodnosti reakcijo epoksidacije smiselno poskušati le z reducirano obliko alilnega alkohola in svežim Dess-Martinovim reagentom zaradi njegove izjemne občutljivosti na atmosfersko vlago in svetlobo.

• Language: Slovenian
• Keywords: zaviralec proteasoma ubikvitin-proteasom epoksidni obroč fluorescentna sonda neželeni učinki zdravil
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FFA - Faculty of Pharmacy
• Place of publishing: Ljubljana
• Publisher: [P. Zupan]
• Year: 2015
• Number of pages: IV, 54 f.
• PID: 20.500.12556/RUL-121067
• UDC: 615.2:616-097(043.3)
• COBISS.SI-ID: 3982449
• Publication date in RUL: 29.09.2020

Secondary language

• Language: English
• Title: Design and synthesis of fluorescent-labelled inhibitor of immunoproteasome

Abstract

The core of the ubiquitin – proteasome system represents 26S proteasome, a multiprotease complex that is responsible for proteolytic degradation of proteins with ubiquitin tag. Numerous cellular processes rely on proteasomal activity and so it is reasonable that proteasome plays an important role in pathological states such as cancer, autoimmune and neurodegenerative diseases. Immunoproteasome is type of proteasome that is normally expressed in cells of haematopoetic origin, however during inflammation cytokines induce its formation also in cells of non-haematopoetic origin. Bortezomib and carfilzomib are approved proteasome inhibitors for multiple myeloma treatment but their nonselective action towards constitutive proteasome and consequentially serious unwanted side effects led to the development of immunoproteasome selective inhibitors. Activity-based fluorescent probes represent an important tool for increasing our understanding of the function of immunoproteasome subunits and development of new selective inhibitors. These molecules selectively bind to catalytically active immunoproteasome subunit and enable its visualization in cells. In our work we synthesized precursors for fluorescent-labelled inhibitor of β5i subunit. This subunit has major role in cytokine production and therefore has the biggest potential as pharmacological target. Activity-based fluorescent probes use electrophilic warhead that covalently irreversibly react with nucleophilic threonine in β5i active site. We planned to synthesize a molecule with epoxide ring that reacts in a unique way by forming morpholine adduct with active site which results in better selectivity. Our synthesis plan was started with L-phenylalanine, which was successfully converted to Weinreb amide and next with Grignard reagent into α, β-unsaturated ketone but the epoxidation reaction in the final step failed. In the second part we successfully prepared a final compound from L-lysine, D-alanine and 3-methylindene-2-carboxylic acid. The activity-based fluorescent probe is not yet prepared because of failed epoxidation reaction and so the compound with epoxide ring represents the missing piece. Due to unsuccessful direct epoxidation reactions we suggest trying with reduced form of allylic alcohols and fresh prepared Dess-Martin reagent because of its high sensitivity when exposed to atmospheric moist and light.

• Keywords: immunoproteasome inhibitor epoxide ring activity-based fluorescent probe