Real-time bioluminescence imaging of glycans on live cells

Abstract

Cell-surface glycans are attractive targets for molecule imaging due to their reflection of cellular processes associated with development and disease progression. In this paper, we describe the design, synthesis, and biological application of a new phosphine probe for real-time imaging of cell-surface glycans using bioluminescence. To accomplish this goal, we took advantage of the bioorthogonal chemical reporter technique. This strategy uses a two-step labeling procedure in which an unnatural sugar analogue containing a functional handle is (1) incorporated into sugar-bearing proteins via the cell's own biosynthetic machinery and then (2) detected with an exogenously added probe. We designed phosphine-luciferin reagent 1 to activate bioluminescence in response to Staudinger ligation with azide-labeled glycans. We chose to use a phosphine probe because, despite their slow reaction kinetics, they remain the best-performing reagents for tagging azidosugars in mice. Given the sensitivity and negligible background provided by bioluminescence imaging (BLI), we reasoned that 1 might be able to overcome some of the limitations encountered with fluorescent phosphine probes. In this work, we synthesized the first phosphine-luciferin probe for use in real-time BLI and demonstrated that azide-labeled cell-surface glycans can be imaged with 1 using concentrations as low as single digit nanomolar and times as little as 5 min, a feat that cannot be matched by any previous fluorescent phosphine probes. Even though we have only demonstrated its use in visualizing glycans, it can be envisioned that this probe could also be used for bioluminescence imaging of any azide-containing biomolecule, such as proteins and lipids, since azides have been previously incorporated into these molecules. The phosphine-luciferin probe is therefore poised for many applications in real-time imaging in cells and whole animals. These studies are currently in progress in our laboratory.

Figure 1

Use of 1 for bioluminescence imaging of cell-surface azidosugars. Compound 1 releases firefly luciferin upon Staudinger ligation with azides. After luciferin diffuses into cells, luciferase-catalyzed conversion of luciferin to oxyluciferin is accompanied by production of a photon of light, which is detected using a CCD camera.

Scheme 1. Synthesis of Phosphine−Luciferin Conjugate 1

Figure 2

Labeling of cell-surface azidosugars with phosphine−luciferin 1. LNCaP-luc cells, a prostate cancer cell line stably transfected with luciferase, were incubated for 2 days in the presence of various concentrations of Ac₄ManNAz, Ac₄GalNAz, Ac₄GlcNAz, or media. The cells were washed three times with 200 μL of PBS and then treated with 1 (100 μM) for 120 min. The total bioluminescence was obtained by calculating the area under the curves represented by those in Figure S2 (SI). Error bars represent the standard deviation of the mean for three replicate experiments.

Figure 3

Sialidase treatment reduces labeling seen with 1. LNCaP-luc cells were incubated for 2 days in the presence of 50 μM Ac₄ManNAz, Ac₄GalNAz, Ac₄GlcNAz, or media. Cells were treated with Arthrobacter ureafaciens sialidase (patterned bars) or left untreated (black bars). The cells were then treated with 1 (100 μM) for 1 h, and the total bioluminescence was quantified. Error bars represent the standard deviation of the mean for three replicate experiments.

Figure 4

Sensitivity of labeling of SiaNAz with 1. LNCaP-luc cells were incubated for 2 days in the presence of 50 μM Ac₄ManNAz (◼) or media (●). The cells were washed three times with PBS and then treated with 1 for 30 min. For each concentration, the total bioluminescence was quantified over 30 min. Error bars represent the standard deviation of the mean for three replicate experiments.