Design and synthesis of Cu-free click chemistry reagents. (A) The copper-catalyzed azide–alkyne cycloaddition. (B) The Cu-free click reaction of azides and DIFOs. (C) (i) Sodium hydride, allyl bromide, 52%. (ii) Pyridinium chlorochromate, 91%. (iii) Lithium hexamethyldisilazide (LHMDS), chlorotriethylsilane (TESCl), 91%. (iv) Selectfluor, 95%. (v–vii). Cat. LHMDS, 95%. (viii) Potassium hexamethyldisilazide (KHMDS), TESCl, 97%. (ix) Selectfluor, 74%. (x) Cat. RuCl₃, NaIO₄, 96%. (xi) KHMDS, N-phenylbis(trifluoromethanesulfonamide), 46%. (xii) Lithium diisopropylamide, 11%. (D) Derivatives of DIFO and a linear alkyne (alk) containing Alexa Fluor 488, Alexa Fluor 568, or biotin.

Time-lapse imaging of glycan trafficking using an Alexa Fluor 488 derivative of DIFO. (A–H) CHO cells were incubated with 100 μM Ac₄ManNAz (A–D) or 100 μM Ac₄ManNAc as a negative control (E–H) for 3 days and subsequently labeled with 100 μM DIFO-488 at 37°C for 1 min. (I–N) Time-lapse imaging of a single cell from the previous experiment over 1 h at 25°C (I–M, Ac₄ManNAz; N, Ac₄ManNAc).

Comparison of Cu-free click chemistry with existing bioorthogonal ligations. (A) Reactions of 10 ng of azidohomoalanine-labeled DHFR with 25 μM DIFO-488 or alk-488 were allowed to proceed for the time indicated. Reactions with alk-488 were performed as described in ref. 26. A negative control reaction (−) using 10 ng of azide-free DHFR was allowed to proceed for 60 min. (B) Schematic for metabolic labeling and detection of cell-surface glycans using Ac₄ManNAz and DIFO-based reagents. (C and D) Flow cytometry plots of labeling experiment described in B using Jurkat cells. (C) Cells were labeled for 1 h with 100 μM biotinylated derivatives of a phosphine (Staudinger ligation) (20), a nonfluorinated cyclooctyne (strain-promoted cycloaddition) (14), and DIFO (Cu-free click chemistry). In all cases, control cells (incubated in azido sugar-free medium but carried through an identical labeling procedure) displayed mean fluorescence intensity (MFI, arbitrary units) values < 20. (D) Cells were labeled for 1 h with 10 nM–100 μM DIFO-biotin. Error bars represent the standard deviation of three replicate experiments. Solid line, + Ac₄ManNAz; dashed line, − Ac₄ManNAz.

Multicolor, dynamic imaging of glycan trafficking using Alexa Fluor derivatives of DIFO. CHO cells were grown for 2 days in 100 μM Ac₄ManNAz (A–E) or 100 μM Ac₄ManNAc (data not shown) and labeled for 1 h at 37°C with 10 μM DIFO-488 (t = 0 h, A and B). The cells were returned to medium supplemented with the appropriate sugar for 23 h and then labeled for 1 h at 37°C with 10 μM DIFO-568 (t = 24 h, C–E). Labeling in the Golgi apparatus and endosomes (filled arrowheads) and lysosomes (open arrowheads) was confirmed in colocalization experiments with known markers (SI Fig. 9).