Sinteza okoljsko občutljivih fluorescenčnih sond na osnovi nilsko rdečega za označevanje lipidnih membran

Gobec, Lucija (author), Pajk, Stane (mentor)

Tehnike na osnovi fenomena fluorescence, ki se jih uporablja na področjih biologije, farmacije in medicine, vsekakor niso nove, a še vedno tu poteka hiter tehnološki napredek. Temu mora slediti tudi razvoj fluorescenčnih sond, od katerih so odvisne praktično vse tehnike na osnovi fluorescence. Primanjkujejo sonde za selektivno označevanje membran, predvsem plazemske membrane. Slednja nas večkrat zanima že za to, da določimo meje celice, če pa so sonde okoljsko občutljive, npr. na viskoznost ali polarnost, pa lahko podajo koristne informacije o procesih v membrani. V magistrski nalogi smo uspeli od načrtovanih dveh sond pripraviti eno okoljsko občutljivo fluorescenčno sondo za označevanje plazemske membrane. Sonda je zgrajena na osnovi nilsko rdečega, ki velja za zelo okoljsko občutljiv fluorofor. V odvisnosti od polarnosti okolice se mu spremenita intenziteta fluorescence in emisijski maksimum. Da smo lahko nilsko rdeče pretvorili v membransko sondo, smo na fenoksazinski skelet nilsko rdečega najprej uvedli alkin, ki omogoča z bakrom(I) katalizirano azid-alkin cikloadicijo ali t.i. klik reakcijo s poljubnim azidom. Vzporedno smo pripravili amin, na katerega je pripeta lipofilna veriga, in azid na kratkem distančniku. Nato smo izvedli klik reakcijo med pripravljenim alkinom in azidom ter v zadnji stopnji na sondo uvedli še ion dvojček z 1,3-propansultonom. Končna spojina ima lipofilno sidro, s katerim se lahko umesti v membrano, in ion dvojček, ki predstavlja polarno glavo in naj bi preprečil ali vsaj upočasnil porazdeljevanje sonde v druge membrane celice. Pomemben del magistrske naloge je bila tudi optimizacija sinteze nilsko rdečega in identifikacija ključnih pogojev, ki vplivajo na potek reakcije. Poleg tega lahko pripravljen derivat nilsko rdečega z alkinsko skupino v prihodnosti uporabijo za pripravo novih sond. Učinkovitost pripravljene sonde bodo v prihodnosti testirali na Institutu Jožef Stefan.

• Language: Slovenian
• Keywords: fluorescenca, fluorescenčne sonde, nilsko rdeče, membranske sonde, biološke membrane
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2022
• PID: 20.500.12556/RUL-143531
• Publication date in RUL: 24.12.2022

Secondary language

• Language: English
• Title: Sinteza okoljsko občutljivih fluorescenčnih sond na osnovi nilsko rdečega za označevanje lipidnih membran

Abstract

Fluorescence-based techniques used in the fields of biology, pharmacy, and medicine are certainly not new, but there are still rapid technological advances. This must be matched by the development of fluorescent probes, upon which basically all fluorescence-based techniques depend. There is a lack of probes for selective labelling of membranes, especially plasma membranes. The latter are often of interest for determining cell boundaries, and if the probes are environmentally sensitive, e.g., to viscosity or polarity, they can provide useful information about membrane processes. In this master's thesis, we succeeded in making an environmentally sensitive fluorescence probe from the planned two probes for plasma membrane characterization. The probe is based on Nile Red, which is considered a very environmentally sensitive fluorophore. Its fluorescence intensity and emission maximum change depending on the polarity of the environment. To convert Nile Red into a membrane probe, we first added an alkyne to the phenoxazine backbone of Nile Red to enable a copper(I)-catalysed azide-alkyne cycloaddition or a so-called click reaction with any azide. At the same time, we prepared an amine to which a lipophilic chain is attached and an azide on a short spacer. The click reaction between the prepared alkyne and the azide was then carried out and, in a final step, a zwitter ion with 1,3-propanesultone was introduced onto the probe. The final compound has a lipophilic anchor that allows it to be placed in the membrane and a zwitterion that is the polar head and should prevent or at least slow down the distribution of the probe to other membranes of the cell. Another important part of the master's thesis was to optimise the synthesis of Nile Red and identify the key conditions that affect the course of the reaction. In addition, the prepared Nile Red derivative with an alkyne group can be used for the preparation of new probes in the future. The efficiency of the prepared probe will be tested in the future at the Jožef Stefan Institute.

• Keywords: fluorescence, fluorescent probes, Nile red, membrane probes, biological membranes