Methyltetrazine as a small live-cell compatible bioorthogonal handle for imaging enzyme activities in situ

Abstract

Bioorthogonal chemistry combines well with activity-based protein profiling, as it allows for the introduction of detection tags without significantly influencing the physiochemical and biological functions of the probe. In this work, we introduced methyltetrazinylalanine (MeTz-Ala), a close mimic of phenylalanine, into a dipeptide fluoromethylketone cysteine protease inhibitor. Following covalent and irreversible inhibition, the tetrazine allows vizualisation of the captured cathepsin activity by means of inverse electron demand Diels Alder ligation in cell lysates and live cells, demonstrating that tetrazines can be used as live cell compatible, minimal bioorthogonal tags in activity-based protein profiling.

Scheme 1. The synthesis of the methyltetrazine amino acid and its incorporation in an ABP. (i) DCC, pyridine/acetone (1/1). (ii) (1) 3-Mercaptopropionic acid, hydrazine hydrate, acetonitrile. (2) NaNO₂ in AcOH/DCM (1/1). (iii) (1) Isobutylchloroformate, N-methyl morpholine. (2) Etheral diazomethane. (3) HBr/AcOH. (iv) TBAF, p-toluenesulfonic acid, THF. (v) (1) 2 M HCl/dioxane. (2) PyBoP, DIPEA, DMF. (B) The chemical structure of TAMRA-DCG-04. (C) The chemical structure of sCy5-TCO.

Fig. 1. Labeling of cysteine cathepsins by either probe 5 (A) or Z-FA-FMK (B) in Jurkat T-cell lysates. The residual cathepsin activity was labeled using TAMRA-DCG-04 (green, Cy3 settings). The unprocessed gels are shown in Fig. S1 (ESI†).

Fig. 2. Competitive labeling of Cathepsin B (A), S (B), and L (C). Live RAW 264.7 cells were incubated for 2.5 h with the indicated concentration of specific inhibitors for each cathepsin. Afterward, the cells were lysed and residual cathepsin activity was labeled with probe 5 (10 μM) and s-Cy5-TCO (2 μM). The gels were imaged in two channels, green (Cy3) and red (Cy5). The unprocessed gels are shown in Fig. S3 (ESI†).

Fig. 3. Competition labeling of cysteine cathepsins by probe 5 in Jurkat lysates. The gel was imaged using Cy3 (green)/Cy5 (red) settings. The unprocessed gels are shown in Fig. S4 (ESI†).

Fig. 4. Labeling of cysteine cathepsins by probe 5 in live Jurkat T-cells. Live cells were incubated with 2 μM of probe 5 for 2 hours. After incubation with probe 5, the cells were lysed and lysates were incubated with 2 μM of sCy5-TCO for 30 minutes. Lanes were rearranged for clarity, the unprocessed gel is shown Fig. S6 (ESI†).

Fig. 5. Imaging of probe 5 and CF©500-TCO ligation by confocal microscopy. BMDCs were incubated for 2 h with probe 5 and CF©500-TCO or CF©500-TCO. After uptake, BMDCs were fixed with 2% PFA and processed for immunoflurescence staining with LAMP-1 as a lysosomal marker (red). The nucleus was stained with Hoechst 33258 (blue) and actin was stained using Phalloidin AF555 (gray). Scale bar is 10 μm (white bar, right corner).

Fig. 6. Cellular uptake of probe 5 and ligation with CF©500-TCO. RAW 264.7 cells were incubated for 2 h with FA-FMK and/or probe 5. After uptake, RAW 264.7 cells were incubated with CF©500-TCO. (A) Flow cytometry histograms showing intensity of CF©500-TCO uptake. (B) Comparison of uptake efficiency by mean fluorescence intensity (MFI) of CF©500-TCO fluorescence. * p-value < 0.05, MFI in cells incubated with Probe 5 and CF©500-TCO (purple) compared with the rest of the experimental groups. Bars represent the mean and whiskers the SEM. Two independent experiments were performed.