Live-Cell Imaging of Sterculic Acid-a Naturally Occurring 1,2-Cyclopropene Fatty Acid-by Bioorthogonal Reaction with Turn-On Tetrazine-Fluorophore Conjugates

Abstract

In the field of lipid research, bioorthogonal chemistry has made the study of lipid uptake and processing in living systems possible, whilst minimising biological properties arising from detectable pendant groups. To allow the study of unsaturated free fatty acids in live cells, we here report the use of sterculic acid, a 1,2-cyclopropene-containing oleic acid analogue, as a bioorthogonal probe. We show that this lipid can be readily taken up by dendritic cells without toxic side effects, and that it can subsequently be visualised using an inverse electron-demand Diels-Alder reaction with quenched tetrazine-fluorophore conjugates. In addition, the lipid can be used to identify changes in protein oleoylation after immune cell activation. Finally, this reaction can be integrated into a multiplexed bioorthogonal reaction workflow by combining it with two sequential copper-catalysed Huisgen ligation reactions. This allows for the study of multiple biomolecules in the cell simultaneously by multimodal confocal imaging.

Keywords: Click Chemistry; Cyclopropene; Inverse Electron-Demand Diels-Alder Reaction; Lipids; Sterculic Acid.

Figure 1

Schematic of the approach to label dendritic cells with sterculic acid (1), followed by an IEDDA reaction with tetrazine‐fluorophore conjugates to allow for live‐cell confocal imaging, due to fluorophore unquenching upon reaction.

Scheme 1

The synthesis of the tetrazine‐fluorophore conjugates 6–11 and 19–20. A) Synthesis of BODIPY‐FL and AF488‐tetrazines through late stage fluorophore introduction: a) Boc₂O, NaOH, H₂O. b) (1) NH₂NH₂, Zn(OTf)₂, acetonitrile/2‐cyanopyridine or formamidine acetate. (2) NaNO₂ in DCM/AcOH (1 : 1, v:v). c) (1) 4 m HCl, dioxane/DCM (1 : 1, v/v) (2) AF488‐NHS or BODIPY‐FL NHS, DIPEA, DCM. B) Synthesis of BODIPY‐Tetrazines 19–20, through fluorophore formation on a tetrazine scaffold: d) DMP, DCM e) (1) 2,4‐Dimethylpyrrole, TFA. (2) DDQ. (3) TEA, BF₃⋅OEt_2.

Figure 2

Average turn‐on ratio of tetrazine‐fluorophore conjugates 6–11 and 19–20 upon reaction with 1 in PBS at 25 °C. All conditions were measured in triplicate, and standard deviations are indicated.

Figure 3

Confocal imaging of DC2.4 cells incubated with 1 (+StA, 50 μM) or without the probe (‐StA). A) Live‐cell imaging of labelled cells visualised with 19 (5 μM). B) Fixed‐cell imaging of labelled cells visualised with 8 (5 μM). The samples were washed after metabolic incorporation of 1 and after ligation with the fluorophore‐tetrazines, and were imaged at >4 distinct locations in the same well. DNA was counterstained with Hoechst 33342 (blue) for reference. Scale bars represent 10 μm.

Figure 4

Confocal imaging of triple‐bioorthogonally labelled DC2.4 cells incubated with A) 5‐ethynyl‐2′deoxyuridine (EdU, 10 μM, yellow) for 20 h, followed by 1 (StA, 50 μM, green) and azido palmitic acid (azPA, 100 μM, red) simultaneously for 1 h. The probes were visualised with AZDye™ 555‐azide, compound 7, and AZDye™ 647‐alkyne (all 5 μM), respectively. B) The cells were incubated without probes and treated in the same triple‐click manner as described above to show the background signal. CCHL reactions were performed using ascorbate as a reducing agent and THPTA as a ligand. The samples were washed between each metabolic incorporation and between each respective bioorthogonal reaction, and were imaged at 3 distinct locations in the same well. DNA was counterstained with Hoechst 33342 (blue) for reference. All scale bars represent 10 μm.

Figure 5

Proteomic analysis of proteins modified with 1 in DC2.4 cells by chemical proteomics. DC2.4 cells were stimulated with or without LPS (100 ng mL⁻¹) for 24 h and treated with 1 (10 μM) for 20 h. Left: Volcano plot of proteins identified in pulldown experiment with 1 (10 μM) and biotin‐PEG4‐tetrazine (200 μM). Proteins with a ratio >2.5 and p‐value <0.05 are considered specifically enriched and are highlighted in black. Right: Difference in sterculic acid‐labeling of proteins between LPS‐ or vehicle‐treated DC2.4 cells. LFQ‐values of specifically sterculic acid‐enriched proteins were compared and proteins with significantly higher LFQ abundance between the two conditions are marked in red and blue for LPS‐ and vehicle‐treated conditions, respectively.