Polymer-protein conjugation via photo-induced inverse electron demand Diels-Alder (photo-IEDDA) reaction

Abstract

Click chemistry and bioorthogonal reactions have drawn attention in recent years which brought Noble Prize in 2022. This can be explained by the fact that these reactions have fast kinetics and not interferring other chemical reactions in vivo. There are several bioorthogonal click reactions, one of which is inverse electron-demand Diels-Alder cycloaddition (IEDDA). IEDDA is drawing attention due to its fast kinetics and is being utilized in different areas such as drug delivery, protein modification, cell imaging, etc. This reaction has fast kinetics and can be performed without any catalysts. Initiation of this reaction can be controlled by light; so that it is named photo-induced inverse electron-demand Diels-Alder reacytion (photo-IEDDA) which enables spatial and temporal control. In this study, polymer-protein conjugates were synthesized by photo-IEDDA. Briefly, 6-(6-pyridin-2-yl)-1,4-dihydro-1,2,4,5-tetrazin-3-yl)-pyridin-3-amine (PPA-dHTz) was synthesized by the reaction between 2-pyridine-carbonitrile and 5-amino-2-pyridine-carbonitrile in the presence of sulphur. PPA-dHTz was modified with succinic anhydride to obtain carboxylic acid end-functional PPA-dHTz. The resultant precursor was attached to 5 kDa poly(ethylene glycol) (PEG) using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 4-dimethylaminopyridine (DMAP) to obtain PEG-COO-PPA-dHTz. Also, end groups of a poly(beta-amino ester) (PβAE) were modified with PPA-dHTz using EDC and N-hydroxysuccinimide (NHS) in a different experiment. Chemical structures of both polymers were confirmed by 1H-NMR spectroscopy. On the other hand, trans-5-hydroxy-1-cyclooctene (TCO) was obtained by converting cis-isomer to trans-isomer via 254 nm irradiation. Trans isomer of TCO had polar and apolar isomers which polar isomer was used in the next experiments. The polar TCO was modified with succinimidyl ester and TCO-NHS obtained. TCO-NHS was attached to Concanavalin A protein. The PPA-dHTz end-functionalized PβAE and TCO functionalized Concanavalin A were conjugated to via photo-IEDDA with 680 nm laser irradiation in the presence of photosensitizer, Methylene Blue. The preleminary results were obtained with low yield; hence, the studies are in progress to achieve a improve the yields. 1H-NMR, UV-vis and MALDI-TOF mass spectroscopies were used to characterize above mentioned precursors and resultants.