Metabolic Glycan Imaging by Isonitrile–Tetrazine Click Chemistry

Seeing the sugar coating: N-Acetyl-glucosamine and mannosamine derivatives tagged with an isonitrile group are metabolically incorporated into cell-surface glycans and can be detected with a fluorescent tetrazine. This bioorthogonal isonitrile-tetrazine ligation is also orthogonal to the commonly used azide-cyclooctyne ligation, and so will allow simultaneous detection of the incorporation of two different sugars.


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General Information 1 H-NMR spectra were recorded with an internal deuterium lock for the residual protons in CDCl 3 (δ 7.26) at ambient probe temperature on a Bruker DRX400 instrument. Data are presented as follows: chemical shift (in ppm), integration, multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, qu=quintet, m=multiplet, br=broad, app=apparent), coupling constants in Hz and interpretation. Coupling constants are reported as they appear in the spectra. Assignments were determined either on the basis of unambiguous chemical shift data or coupling patterns, COSY experiments or by analogy to fully interpreted spectra for related compounds. 13 C-NMR spectra were recorded with an internal deuterium lock for CDCl 3 (δ 77.0) at ambient probe temperature. Chemical shifts are given in ppm. Infrared spectra were recorded on a Perkin Elmer Spectrum One (FT-IR) spectrophotometer. Wavelengths of maximum absorbance (ν max ) are quoted in wavenumbers (cm −1

Stability of isonitriles in the presence of biologically relevant functional groups
Stock solutions of glutathione, n-pentyl isocyanide and t-butyl isocyanide were prepared in CD 3 CN/D 2 O (1:1) at 20 mM. The glutathione solution was basified to pH 7.4. The following mixtures were prepared: (a) 1:1 molar equivalent of glutathione/pentyl isocyanide at 10 mM (700 µL total) (b) 1:1 molar equivalent of glutathione/t-butyl isocyanide at 10 mM (700 µL total) (c) Individual controls (glutathione and isonitriles) diluted to 10 mM. The NMR tubes (a) and (b) were incubated at 37 o C for 24 h. 1 H NMR spectra were run at 2 and 24 h.

3-Formamidopropanoic acid (A)
3-Aminopropionic acid (10 g, 112 mmol) and formic acid (8.78 g, 191 mmol) were heated at reflux in DMF (51 ml) for 2 hr. The solvent was evaporated to give fine white crystals which were suspended in ether, filtered, washed and dried to give the title compound (13.14 g, 112 mmol, 100%

Tetra-O-acetyl-N-(2-isocyano2-methylpropanoyl)-D-galactosamine (Ac 4 GalN-t-Iso).
This was synthesised from Ac 4 GalNH 3 Cl by the above method to give the title compound as a colourless oil (15 mg, 0.034 mmol, 26%  The solvent was evaporated under reduced pressure and the residue was dissolved in pyridine (1 ml), cooled to 0 o C and treated with acetic anhydride (1.12 ml). The mixture was stirred for 1 hr then warmed to RT and stirred overnight. The mixture was evaporated and then co-evaporated with water (3x) and toluene (3x) then rapidly eluted through a column of silica gel (approx 20 ml) with 2% MeOH/DCM to remove EDC impurities. Fractions were evaporated, dissolved in DCM (1 ml), cooled to -40 o C and then treated successively with Et 3 N (63 µl, 0.45 mmol) and phosphorus oxychloride (13.4 µl, 0.14 mmol). After 1 hr the reaction was quenched with saturated aq. NaHCO 3 solution and extracted with EtOAc. The combined extracts were washed with water, saturated aq. NaHCO 3 , and brine, dried over MgSO 4 and evaporated. The product was purified by silica column chromatography, eluting with 2% MeOH/DCM, to yield the title compound as a colourless oil (8.9 mg, 0.02 mmol, 6.3%).  Cell surface isocyanoglycan labeling and quantification with fluorescence microscopy imaging LL2 cells were seeded onto 4-well coverslip chambers (Lab-Tek ™ borosylicate, Nunc, Roskilde, Denmark) at 2×10 4 /cm 2 and allowed to adhere to the plate surface for 6 hr. After adhesion, cells were pulsed with either isonitrile sugar or vehicle for 24 hr. Cells were then washed 3 times in ice cold FACS buffer before being incubated (30 min, 37 o C) in situ in 200 µL of FACS buffer containing 300 µM Tz-Biotin, followed by 2 washes as above and a subsequent incubation (15 min, 37 o C) with 50 µg/ml NeutrAvidin-Dylight680 and 300 nM DAPI (Invitrogen). After being washed as above, cells were fixed in PBS containing 4% formalin (RT, 15 min, 37 o C) and washed again twice in cold PBS. The chambers were scanned on an iCys Research Imaging Cytometer (CompuCyte, Westwood, MA, U.S.A.) using 405 nm and 633 nm lasers. A 60x objective was used with 0.5 µm X-step size, giving a field size of 500 µm x 132 µm. A total of 300 fields were scanned for each chamber well. Watershed filters were also included in the protocol to ensure separate contouring on closely spaced cells. Images were analysed using primary and peripheral contours to sample cytoplasmic and membrane staining per cell respectively. The primary contour was set on the blue (DAPI) channel with an integration contour of 8 pixels to include cytoplasmic staining. The peripheral contour was set to a width of 8 pixels measuring from the outer edge of the integration contour. A sub contour was set with a threshold on the long red channel to measure total Alexa Fluor 647 staining per cell.

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Flow cytometry data Figure S1. -Flow cytometry dot plots of labelling experiment described in Fig. 1. Each dot represents 1 of 10,000 events recorded. Y-axis measures NAD autofluorescence and X-axis measures Sytox Green, a cell death marker. Scales are logarithmic spanning 5 log units. Cells circled in pink were considered viable and are the only ones used to calculate the DyLight680 mean fluorescence intensity (MFI). The percentage of viable cells in the total population is shown in pink on each plot.