Mechanistic Evaluation of Bioorthogonal Decaging with trans-Cyclooctene: The Effect of Fluorine Substituents on Aryl Azide Reactivity and Decaging from the 1,2,3-Triazoline

Abstract

Bioorthogonal prodrug activation/decaging strategies need to be selective, rapid and release the drug from the masking group upon activation. The rates of the 1,3-dipolar cycloaddition between a trans-cyclooctene (TCO) and a series of fluorine-substituted azido-PABC self-immolative spacers caging two model drugs, and subsequent release from the 1,2,3-triazoline are reported. As the number of fluorine substituents on the PABC linker increases from one to four, the rate of cycloaddition increases by almost one order of magnitude. Using a combination of fluorescence, H/F NMR, and computational experiments, we have been able to determine how substituents on the PABC ring can influence the degradation rates and also the product distribution of the 1,2,3-triazoline. We have also been able to determine how these substituents influence the rate of imine hydrolysis and 1,6-self-immolation decaging rates of the generated anilines. The NMR and computational studies demonstrate that fluorine substituents on the aromatic ring lower the transition state energy required for converting the triazoline to the imine or aziridine intermediates via extrusion of diatomic nitrogen, and that in the case of a tetrafluoro substituted aromatic ring, it is the imine hydrolysis and 1,6-self-immolation that is rate-limiting. This knowledge further enhances the understanding of factors which influence the stability of triazolines, and enables potential applications of fluorinated aromatics, in particular, perfluorinated aromatics, in synthetic chemistry and sustained-release drug delivery systems.

Alkene–Azide 1,3‐Dipolar Cycloaddition as a Trigger for Ultrashort Peptide Hydrogel Dissolution

“…Next we investigated the dissolution of hydrogel 1 using TCO as the bioorthogonal trigger (Scheme ). The same mechanism as for prodrug activation was proposed for hydrogel dissolution. In the first step, 1,3‐dipolar cycloaddition between TCO and azide 1 is expected to result in the formation of 1,2,3‐triazoline 6 , which is relatively unstable under aqueous conditions and converted to the imine 7 via extrusion of diatomic nitrogen (expected to result in visual bubbles within the gel).…”

“…Therefore, to estimate the rate of the 1,3‐dipolar cycloaddition for hydrogel 1 , we monitored the reaction of azido‐dipeptide 1 and TCO in solution phase (MeCN:PBS; 1:1). Using HPLC (Figure S9 and S10), the disappearance of 1 ( R T =7.9 min) was measured under pseudo first‐order conditions (at 254 nm), and the second‐order rate constant was determined as 0.018 m −1 s −1 , a rate comparable to our previously reported bioorthogonal prodrug activation . In the absence of HPLC data on the gel‐sol transition itself, we performed a time‐dependent analysis of the 1,3‐dipolar cycloaddition using IR spectroscopy.…”

“…Using HPLC ( Figure S9 and S10), the disappearance of 1 (R T = 7.9 min) was measured under pseudo first-order conditions (at 254 nm), and the second-order rate constant was determined as 0.018 m À1 s À1 ,arate comparable to our previously reported bioorthogonal prodrug activation. [24,25] In the absence of HPLC data on the gel-sol transition itself, we performed at ime-dependentanalysisoft he 1,3-dipolarc ycloaddition using IR spectroscopy. The IR results demonstrated that after 1h our as mall amount of azide wasy et to react with TCO (presence of azide peak evident), but by 4h ours all of the azide in the gel had been consumed ( Figure S11).…”

Dissociative Bioorthogonal Reactions

“…The cycloaddition step occurs at a rate of 0.137 m −1 s −1 (mouse serum/PBS (1:1), T =37 °C); however, degradation of the triazoline and imine hydrolysis might both be rate limiting. In an effort to accelerate the reaction, fluorine substituents were introduced onto the p ‐azidobenzyl ring . A structure–reactivity analysis revealed that the introduction of fluorine substituents increased the rate of the cycloaddition step, but decelerated the 1,6‐elimination step from PABC.…”

“…In an effort to accelerate the reaction, fluorine substituents were introduced onto the p-azidobenzyl ring. [75] A Scheme9.Reactionmechanism between vinyl ether derivatives and tetrazines. Following cycloaddition and the expulsion of nitrogen gas, aromatization of the pyridazines tructure affords efficient release of the leavinggroup.…”