Sterically shielded tetrazoles for a fluorogenic photoclick reaction: tuning cycloaddition rate and product fluorescence

Abstract

A panel of sterically shielded tetrazoles with different N-aryl groups were synthesized and subsequently evaluated in the photoinduced tetrazole-alkene cycloaddition reaction. It was found that increase in the HOMO energy of the corresponding nitrile imines leads to a faster cycloaddition reaction along with a red shift in the fluorescence emission of the pyrazoline cycloadduct.

Figure 1

Plot of logk₂ of the cycloaddition reaction vs. HOMO energy of the nitrile imines in the photoclick reaction with Sph. For substituted phenyl rings, only para-substituents are shown.

Figure 2

Characterization of the fluorescent properties of the in situ formed pyrazolines. (a) Fluorescent images of the pyrazolines from the reactions of 10 μM tetrazoles 1–9 with 250 μM Sph in 1:1 PB/ACN. The cuvettes were illuminated with a 365 nm UV light. (b) Fluorescence spectra of the pyrazolines. λ_ex = 360 nm for tetrazoles 2–4 and 405 nm for tetrazoles 5–9; see Figure S3 in Electronic Supplementary Information for selection of appropriate λ_ex. For pyrazolines containing N-phenyl groups, only para-substituents are shown. (c) Solvent-dependent fluorescence spectra of the in situ generated pyrazoline 6. λ_ex = 405 nm.

Figure 3

Fluorogenic labelling of sfGFP-Q204SphK by the sterically shielded tetrazoles. (a) Reaction scheme: a mixture of 10 μM sfGFP mutant encoding SphK at position-204 and 200 μM tetrazole in PBS/ACN (1:1) was photoirradiated with a handheld 302-nm UV lamp for 30 sec. (b) SDS-PAGE/in-gel fluorescence analysis of the reaction mixtures. Top panel, Coomassie blue stained gel; bottom panel, in-gel fluorescence by illuminating the gel with a 365-nm UV light. The non-fluorescent sfGFP variant containing the T65G/Y66G mutations was used in this experiment. (c) Fluorescence spectra of the reaction mixtures. The emission maximum of sfGFP-Q204Pyr is marked on top of each spectrum. λ_ex = 405 nm.

Scheme 1

Synthesis of the sterically shielded tetrazoles 2–9.

Chart 1

Structures of sterically shielded tetrazoles 1–9.