Fast alkene functionalization in vivo by Photoclick chemistry: HOMO lifting of nitrile imine dipoles

Abstract

Extremely fast fluorescence labeling (<1 min) of a recombinant alkene-encoded protein in living Escherichia coli cells was observed with tetrazole 1. The electron-donating methoxy substituent raises the energy of the highest occupied molecular orbital of the nitrile-imine intermediate derived from 1. This strategy greatly accelerates the functionalization of alkenes by 1,3-dipolar cycloaddition in living systems.

Figure 1

Cycloaddition rates are dependent on the nitrile imine HOMO energies. The HOMO energies of the nitrile imines were calculated using Hartree-Fock 3-21G model based on the AM1 optimized geometries (see Supporting Information for details).

Figure 2

(a) Kinetic study of the cycloaddition reactions between allyl phenyl ether and tetrazole 1 (◆) and 10 (●), respectively. A close-up view of the tetrazole 10 kinetics was rendered in the insert; (b) Kinetic study of the alkene-Z labeling by either tetrazole 1 (open bar) or 10 (filled bar). The fluorescent intensities at 4 min were treated as 100% for each reaction and used in deriving the relative intensities. The concentrations of alkene-Z and tetrazoles were 10 μM and 200 μM, respectively. Error bars represent the standard deviations from three independent measurements.

Figure 3

Selective PEGylation of alkene-Z by mPEG-tetrazole 22: (a) reaction scheme; (b) Coomassie blue staining (left panel) and in-gel fluorescence imaging (right panel, λ_ex = 365 nm) of alkene-Z upon photoirradiation for 2 min and additional incubation for 2 hr in the presence of mPEG-tetrazole 22. The experiment was repeated twice and similar results were obtained; (c) MALDI-TOF mass spectrum of the PEGylation mixture of the alkene-Z labeling by mPEG-tetrazole 22. A close-up view of the mass envelop representing mPEG-Z was rendered in the insert.

Figure 3

Selective PEGylation of alkene-Z by mPEG-tetrazole 22: (a) reaction scheme; (b) Coomassie blue staining (left panel) and in-gel fluorescence imaging (right panel, λ_ex = 365 nm) of alkene-Z upon photoirradiation for 2 min and additional incubation for 2 hr in the presence of mPEG-tetrazole 22. The experiment was repeated twice and similar results were obtained; (c) MALDI-TOF mass spectrum of the PEGylation mixture of the alkene-Z labeling by mPEG-tetrazole 22. A close-up view of the mass envelop representing mPEG-Z was rendered in the insert.

Figure 4

Fast, selective labeling of alkene-Z by tetrazole 10 in E. coli cells: DAPI channel (top row) and DIC channel (bottom row) images of bacterial cells expressing either alkene-Z (a, c) or wt-Z (b, d) after 30-sec photoirradiation in the presence of 100 μM tetrazole 10; scale bar = 5 μm.