Rapid Cu-free click chemistry with readily synthesized biarylazacyclooctynones

Abstract

Bioorthogonal chemical reactions, those that do not interact or interfere with biology, have allowed for exploration of numerous biological processes that were previously difficult to study. The reaction of azides with strained alkynes, such as cyclooctynes, readily forms a triazole product without the need for a toxic catalyst. Here we describe a biarylazacyclooctynone (BARAC) that has exceptional reaction kinetics and whose synthesis is designed to be both modular and scalable. We employed BARAC for live cell fluorescence imaging of azide-labeled glycans. The high signal-to-background ratio obtained using nanomolar concentrations of BARAC obviated the need for washing steps. Thus, BARAC is a promising reagent for in vivo imaging.

Figure 1

Bioorthogonal reaction of cyclooctyne probes with azide-labeled biomolecules allows their interrogation in cell-based systems. (A) Cells are treated with azide-functionalized metabolic substrates. The azides are then detected with a cyclooctyne-functionalized probe. (B) Cyclooctynes designed for fast Cu-free click chemistry (1−3) and reactivity studies (4). The R-group denotes the location for linkage to a probe moiety.

Scheme 1. Retrosynthesis of BARAC (1)

Scheme 2. Synthesis of BARAC (15)

Figure 2

BARAC-probe conjugates label live cells with superior sensitivity compared to DIFO and DIBO reagents. (A) Structures of BARAC-biotin (16) and BARAC-Fluor (17). (B−C) Flow cytometry plots of live cell labeling with BARAC-biotin. Jurkat cells were incubated with (+Az) or without (−Az) 25 μM Ac₄ManNAz for 3 days. The cells were labeled with 1 μM cyclooctyne-biotin for various times and then treated with FITC-avidin. Cyclooctyne-biotin probes used were DIBO-biotin, BARAC-biotin, or DIFO-biotin. The degree of labeling was quantified by flow cytometry. The level of fluorescence is reported in mean fluorescence intensity (MFI, arbitrary unit). Error bars represent the standard deviation of three replicate experiments. (B) Comparison of the efficiencies of labeling of different cyclooctyne reagents after 1 h. (C) Time-dependent labeling of cyclooctyne-biotin probes. MFI reported as difference between signal of cells +Az and signal of cells −Az.

Figure 3

Imaging of azide-labeled glycans on live cells using BARAC-Fluor (17). (A−P) CHO cells were incubated with (A−D, I−L) or without (E−H, M−P) 50 μM Ac₄ManNAz for 3 days. (A−H) The cells were subsequently labeled with 5 μM BARAC-Fluor and Hoechst-33342 for 5 min and then washed and imaged. (I−P) The cells were subsequently labeled with 250 nM BARAC-Fluor for 30 min and Hoechst-33342 and then imaged without washing. Channels shown are differential interference contrast bright-field (A, E, I, M), the blue DAPI channel (B, F, J, N), the green FITC channel (C, G, K, O), and the DAPI/FITC channels merged (D, H, L, P).