Sterically Shielded, Stabilized Nitrile Imine for Rapid Bioorthogonal Protein Labeling in Live Cells

Abstract

In pursuit of fast bioorthogonal reactions, reactive moieties have been increasingly employed for selective labeling of biomolecules in living systems, posing a challenge in attaining reactivity without sacrificing selectivity. To address this challenge, here we report a bioinspired strategy in which molecular shape controls the selectivity of a transient, highly reactive nitrile imine dipole. By tuning the shape of structural pendants attached to the ortho position of the N-aryl ring of diaryltetrazoles-precursors of nitrile imines, we discovered a sterically shielded nitrile imine that favors the 1,3-dipolar cycloaddition over the competing nucleophilic addition. The photogenerated nitrile imine exhibits an extraordinarily long half-life of 102 s in aqueous medium, owing to its unique molecular shape that hinders the approach of a nucleophile as shown by DFT calculations. The utility of this sterically shielded nitrile imine in rapid (∼1 min) bioorthogonal labeling of glucagon receptor in live mammalian cells was demonstrated.

Figure 1. Computational studies of the steric shielding effect of the ortho N-Boc-pyrrole substituents

Computed transition states and activation energies involving nitrile imine-1 and -26 for (a) 1,3-dipolar cycloaddition with 5-methyl-spiro[2.3]hex-1-ene, and (b) nucleophilic addition with methyl thiolate, respectively. DFT calculations were performed at the ωB97x-D/6-311++G(d,p)/SMD(Water) level of theory using geometries optimized by B3LYP-D3/6-31+G(d)/SMD(Water). R = N-Boc. The distances shown are in Å, and energies are in kcal/mol.

Figure 2. Structural characterization of tetrazoles 26 and 27

(a) Chemical structure of tetrazole 26 with the proton numbering marked on the structure. (b) X-ray crystal structure of tetrazole 26 with depth cue showing co-planarity of the diphenyltetrazole (left) and perpendicular arrangement of N-Boc-pyrrole relative to the diphenyltetrazole (right). CW, clockwise. (c) Chemical structure of tetrazole 27. (d) Crystal structure of 27 with depth cue showing co-planarity between the C-phenyl and pyrrole rings, and perpendicular twisting of the tetrazole and the C-phenyl ring. The structures are viewed from the same angles as in tetrazole 26. (e) Partial ¹H-¹H ROESY spectrum of tetrazole 26 in CDCl₃ showing prominent cross-peaks between tert-butyl protons and proton-4, -5 and -7 as marked on the chemical structure.

Figure 3. Application of the sterically shielded nitrile imine to generate in situ a fluorescent sensor for probing ligand-induced QBP conformational change

(a) Scheme for in situ synthesis of a pyrazoline-based sensor from QBP-N160SphK mutant and a water-soluble tetrazole 30. The glutamine-free QBP (PDB code: 1GGG) adopts an open conformation while the glutamine-bound QBP (PDB code: 1WDN) adopts a closed conformation. (b) Fluorescence spectra of QBP-pyr-30 upon titration of glutamine (final concentration = 0.1 mM, 1 mM, 5 mM, 10 mM or 15 mM in 4:1 DPBS/acetonitrile). λ_ex = 405 nm; QBP concentration was set at 5 μM. (c) Plot of the change of fluorescence intensity at 480 nm vs. glutamine concentration. The data were fitted to one-site specific binding model with the following equation: ΔF_obs = ΔF_maxS/(K_d + S).

Figure 4. Application of the stabilized nitrile imine to bioorthogonal labeling of GCGR in live cells

(a) Scheme for bioorthogonal labeling of a GCGR-GFP mutant encoding SphK at position-372 with a Cy5-funtionalised water-soluble tetrazole 31. The C-terminal GFP is not shown for clarity. (b) Confocal micrographs of HEK 293T cells expressing GCGR-H372SphK-GFP after photoirradiation with a handheld 302-nm UV lamp for a period of 0.5, 1, or 2 min in DMEM medium containing 500 nM tetrazole-Cy5. Scale bar = 20 μm. Flow cytometry analysis of HEK 293T cells expressing (c) GCGR-H372SphK, (d) GCGR-H372SphK-GFP before labeling, and (e) GCGR-H372SphK-GFP after labeling with tetrazole 31. Cells were treated with 500 nM of tetrazole 31 and photoirradiated for 1 min. A total of 10,000 cells were analyzed in each measurement. X-axis, GFP channel; y-axis, Cy5 channel; both axes are in log scale.

Scheme 1

Strategy of employing structural pendants to stabilize the in situ generated nitrile imine and direct the reaction toward 1,3-dipolar cycloaddition over the competing nucleophilic addition.

Scheme 2

Potential products from the substituted tetrazoles under the competitive conditions. Four types of substituted tetrazoles were evaluated in this competition assay; see Table S1 in SI for structural details. PB = phosphate buffer; ACN = acetonitrile.