Copper-free click chemistry for dynamic in vivo imaging

Supporting Information

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Fig. 5. Structural determination of monofluoroketone 10b. (a) Decomposition reaction of 10b to yield bicyclic acetal 11. (b) ORTEP diagram of 11 at 50% probability.

Fig. 6. Analytical HPLC trace of purified DIFO-488. Method: 0% to 50% B from 0-40 min, 50% to 100% B from 40-50 min. Solvent A: water + 0.1% trifluoroacetic acid; solvent B: acetonitrile + 0.1% trifluoroacetic acid.

Fig. 7. Analytical HPLC trace of purified DIFO-FLAG. Method: 0% to 50% B from 0-40 min, 50% to 100% B from 40-50 min. Solvent A: water + 0.1% trifluoroacetic acid; solvent B: acetonitrile + 0.1% trifluoroacetic acid.

Fig. 8. Proton NMR spectrum of purified DIFO-biotin. Solvent: CD₃OD, 500 MHz.

Fig. 9. Kinetic data for the reaction of 1 with benzyl azide. Substrates were dissolved separately in CD₃CN and mixed in a 1:1 ratio at a final concentration of 18 mM. Percentage conversion was monitored by ¹H-NMR. The second-order rate constant for the reaction was determined by plotting 1/[benzyl azide] versus time and analysis by linear regression. The second-order rate constant corresponds to the determined slope. Error bars represent standard deviations from three replicate experiments.

Fig. 10. Silver stain of DHFR labeling experiments. Azido-DHFR (all lanes except right-most) or DHFR (-), ~10 ng per reaction, was labeled with DIFO-488 (Upper) or alk-488 and appropriate reagents (Cu-catalyzed) (Lower). Reactions were allowed to progress for the number of minutes indicated (60 min for -) and then quenched with 2-azidoethanol and urea. After gel electrophoresis and fluorescence measurement (Fig. 2A), the gels were stained for protein content by using silver stain (Bio-Rad).

Fig. 11. Labeling of live CHO cells with DIFO-Alexa Fluor conjugates. CHO cells were incubated for 2 days in media containing 100 mM Ac₄ManNAz (a-c, g-i, m-u, and y-dd) or Ac₄ManNAc (d-f, j-l, and v-x). (a-f) The cells were labeled at 4°C for 1 h with 100 mM DIFO-488 (a and d), DIFO-568 (b and e), or DIFO-647 (c and f), and then Hoechst 33342 dye (dark blue). (g-l) The cells were labeled at 37°C for 1 h with 10 mM DIFO-488 (g and j), DIFO-568 (h and k), or DIFO-647 (i and l), and then Hoechst 33342 dye (dark blue). (m-dd) The cells were labeled at 37°C for 1 h with 10 mM DIFO-488 (m-aa) or 10 mM alk-488, 1 mM Tris-carboxyethylphosphine, 100 mM TBTA, and 1 mM CuSO₄ (bb-dd), then Hoechst 33342 dye, and BODIPY ceramide TR (m-o), Alexa Fluor 647-conjugated human transferrin (p-r), LysoTracker Red (s-v), or 0.33 mg/ml propidium iodide (v-dd).

Fig. 12. In vivo labeling of glycans with DIFO. Mice were injected with Ac₄ManNAz (300 mg·kg^-1 in 70% DMSO, +) or vehicle (70% DMSO, -) once daily for 7 days. On the 8th day, DIFO-FLAG (0.16 mmol·kg^-1 in 70% DMSO) was injected. After 3 h, the animals were killed, and the splenocytes were isolated, treated with FITC-anti-FLAG antibody, and analyzed by flow cytometry. Each diamond represents a sample of splenocytes from one mouse, and the dashed line represents the average MFI value for the population of identically treated mice.

Scheme 1. Synthesis of DIFO (1), part I. 1,5-cyclooctanediol is monosubstituted with an allyl group (7), and the remaining alcohol is oxidized to the corresponding ketone (8). A fluorine atom is installed using Selectfluor from the silyl enol ether (9), generating two diastereomeric silyl enol ethers (10a and 10b). As monofluoroketone 10b will be carried on, 10a can be epimerized to a separable mixture of 10a and 10b. NaH, sodium hydride; PCC, pyridinium chlorochromate; LHMDS, lithium hexamethyldisilazide; Et₃SiCl, chlorotriethylsilane.

Scheme 2. Synthesis of DIFO (1), part II. From monofluoroketone 10b, fluoro silyl enol ether 12 is formed under kinetic enolization conditions, followed by fluorination to yield difluoroketone 13. Oxidation of the allyl group to the corresponding carboxylic acid (14), followed by generation (15) and elimination (1) of a vinyl triflate, yielded DIFO (1). KHMDS, potassium hexamethyldisilazide; Tf₂NPh, N-phenylbis(trifluoromethanesulfonamide); LDA, lithium diisopropylamide.

Scheme 3. Synthesis of derivatives of DIFO containing fluorophores (DIFO-488, DIFO-568, and DIFO-647), biotin (DIFO-biotin), and the FLAG peptide (DIFO-FLAG). For the Alexa Fluor and biotin derivatives, DIFO was converted to the pentafluorophenyl ester (DIFO-PFP), which then was coupled to the appropriate amino derivative. In the case of DIFO-FLAG, DIFO-PFP was coupled to an amino-maleimide linker (17) to generate DIFO-maleimide (5f), which was coupled to a fully deprotected, soluble cysteine-terminated FLAG peptide. PFP-TFA, pentafluorophenyltrifluoroacetate; DIEA, N,N-diisopropylethylamine; DMF, dimethylformamide.

SI Materials and Methods

All chemical reagents were of analytical grade, obtained from commercial suppliers, and used without further purification unless otherwise noted. With the exception of reactions performed in aqueous media, all reaction vessels were flame-dried before use. Reactions were performed in an N₂ atmosphere, except in the case of reactions performed in aqueous media, and liquid reagents were added with a syringe unless otherwise noted. Tetrahydrofuran (THF) was distilled under N₂ from Na/benzophenone immediately before use, and CH₂Cl₂ was distilled from CaH₂ immediately before use. Flash chromatography was carried out with Merck 60 230-400 mesh silica gel according to the procedure described by Still (1). Reactions and chromatography fractions were analyzed with Analtech 250-mm silica gel G plates and visualized by staining with ceric ammonium molybdate, anisaldehyde, or by absorbance of UV light at 245 nm. Organic extracts were dried over MgSO₄, and solvents were removed with a rotary evaporator at reduced pressure (20 torr), unless otherwise noted. Unless otherwise noted, ¹H-, ¹³C-, and ¹⁹F-NMR spectra were obtained with 300 MHz or 400 MHz Bruker spectrometers. Chemical shifts are reported in d ppm referenced to the solvent peak for ¹H and ¹³C and relative to CFCl₃ for ¹⁹F. Coupling constants (J) are reported in Hz. Low- and high-resolution fast-atom bombardment (FAB), and electron impact (EI) mass spectra were obtained at the University of California Berkeley Mass Spectrometry Facility, and FT-ICR mass spectra were obtained at the Howard Hughes Medical Institute Mass Spectrometry Facility at University of California Berkeley. Reversed-phase HPLC was performed by using a Rainin Dynamax SD-200 HPLC system with 210-nm detection on a Microsorb C18 analytical or preparative column.

Dulbecco's PBS, fluorescein isothiocyanate (FITC)-anti-FLAG, and BSA were purchased from Sigma. RPMI medium 1640 was obtained from Invitrogen Life Technologies, Inc., and FBS was purchased from HyClone Laboratory. FITC-conjugated mouse IgG₁ isotype control was obtained from BD PharMingen. Flow cytometry analysis was performed on a BD FACSCalibur flow cytometer using a 488-nm argon laser. At least 10⁴ cells were analyzed for each sample. Cell viability was ascertained by gating the samples on the basis of forward scatter (to sort by size) and side scatter (to sort by granularity). The average fluorescence intensity was calculated from each of three replicate experiments to obtain a representative value in arbitrary units. For all flow cytometry experiments, data points were collected in triplicate and are representative of at least three separate experiments.

Synthetic Procedures

The synthesis of DIFO (1) was carried out as outlined in SI Schemes 1 and 2. Various derivatives of DIFO containing fluorophores (DIFO-488, DIFO-568, and DIFO-647), biotin (DIFO-biotin), and the FLAG peptide (DIFO-FLAG) were synthesized as outlined in SI Scheme 3.

Monoallylated Cyclooctanediol (7). To a solution of cis-1,5-cyclooctanediol (50.0 g, 347 mmol) in DMF (800 ml) at 0°C was added NaH (60% dispersion in mineral oil, 15.3 g, 381 mmol). The suspension was stirred for 1 h at 0°C, and allyl bromide (29.3 ml, 347 mmol) then was added to the reaction vessel by using a syringe pump over 1 h at 0°C. The reaction was warmed to room temperature overnight while stirring, and the reaction mixture then was quenched with water (800 ml). The aqueous layer was extracted with ether (5 ´ 400 ml), and the combined organic fractions were washed with water (3 ´500 ml) and brine (1 ´ 300 ml) and then dried over MgSO₄. Chromatography of the crude product (8:1 to 2:1 hexanes/EtOAc) yielded a clear oil (33.2 g, 52%, R_f = 0.30 in 2:1 hexanes/EtOAc). ¹H-NMR (CDCl₃, 400 MHz): d 1.48 (m, 2H), 1.62 (m, 4H), 1.86 (m, 6H), 3.40 (t, 1H, J = 8.8 Hz), 3.80 (t, 1H, J = 9.0 Hz), 3.95 (d, 2H, J = 5.6 Hz), 5.15 (d, 1H, J = 10.0 Hz), 5.26 (dd, 1H, J = 1.2, 17.2 Hz), and 5.90 (m, 1H). ¹³C-NMR (CDCl₃, 100 MHz): d 20.6, 32.7, 36.3, 69.1, 71.6, 78.6, 116.4, and 135.3. FAB-HRMS: Calculated for C₁₁H₂₁O₂⁺ [M+H]⁺: 185.1542; found: 185.1544.

Allylated Cyclooctanone (8). To a stirring solution of alcohol 7 (33.2 g, 180 mmol) in CH₂Cl₂ (650 ml) at room temperature was added pyridinium chlorochromate (54.4 g, 252 mmol) over 2 h, 6.8 g every 15 min. After an additional 30 min of stirring, the reaction mixture was concentrated on a rotary evaporator, and the product was purified directly by column chromatography (8:1 to 3:1 hexanes/EtOAc) to yield a clear oil (30.0 g, 91%, R_f = 0.30 in 5:1 hexanes/EtOAc). ¹H-NMR (CDCl₃, 400 MHz): d 1.63-1.90 (m, 6H), 2.04 (m, 2H), 2.28 (m, 2H), 2.55 (m, 2H), 3.16 (tt, 1H, J = 2.8, 8.6 Hz), 3.89 (d, 1H, J = 5.2 Hz), 5.13 (d, 2H, J = 10.4 Hz), 5.22 (dd, 1H, J = 1.2, 17.2 Hz), 5.85 (ddd, 1H, J = 5.6, 10.4, 22.2 Hz). ¹³C-NMR (CDCl₃, 100 MHz): d 22.9, 33.7, 42.3, 69.5, 77.6, 116.7, 135.0, and 216.5. FAB-HRMS: Calculated for C₁₁H₁₉O₂⁺ [M+H]⁺: 183.1385; found: 183.1380.

Silyl Enol Ether (9). To a stirring solution of lithium hexamethyldisilazide (LHMDS, 181 ml, 181 mmol, 1.00 M solution in THF) in THF (750 ml) at -78°C was added a solution of ketone 8 (30.0 g, 165 mmol) in THF (40 ml) over 1 h using a syringe pump. After an additional 20 min of stirring at -78°C, chlorotriethylsilane (31.8 ml, 189 mmol) was added. The solution was stirred at -78°C for 10 min, removed from the cold bath, warmed to room temperature with a water bath, and stirred for 1 h. The reaction mixture was concentrated on a rotary evaporator, and the crude product was purified directly by column chromatography (100% hexanes to 25:1 hexanes/EtOAc) to yield a clear oil (44.2 g, 91%, R_f = 0.70 in 9:1 hexanes/EtOAc). ¹H-NMR (CDCl₃, 400 MHz): d 0.67 (q, 6H, J = 8.0 Hz), 0.98 (t, 9H, J = 8.0 Hz), 1.52 (m, 2H), 1.70-2.01 (m, 5H), 2.13 (m, 2H), 2.26 (m, 1H), 3.40 (m, 1H), 3.95 (m, 2H), 4.76 (dd, 1H, J = 7.2, 9.2 Hz), 5.14 (dd, 1H, J = 1.6, 10.4 Hz), 5.26 (dd, 1H, J = 1.8, 15.4 Hz), and 5.91 (ddd, 1H, J = 5.4, 10.6, 22.6 Hz). ¹³C-NMR (CDCl₃, 100 MHz): d 5.0, 6.7, 22.4, 24.5, 31.8, 33.7, 36.2, 69.2, 79.9, 104.3, 116.3, 135.5, and 152.7. Calculated for C₁₇H₃₃O₂Si⁺ [M+H]⁺: 297.2250; found: 297.2246.

Monofluoroketones (10a and 10b). To a stirring solution of Selectfluor (63.4 g, 179 mmol) in DMF (150 ml) at 0°C was added a solution of silyl enol ether 9 (44.2 g, 149 mmol) DMF (180 ml) via an addition funnel over 30 min. The reaction was allowed to slowly warm to room temperature while stirring over 30 min, and then it was quenched with water (350 ml). The aqueous layer was extracted with ether (4 ´ 350 ml), and the combined organic extracts were washed with water (3 ´300 ml) and brine (1 ´ 200 ml). The crude product was purified by column chromatography (10:1 to 5:1 hexanes/EtOAc) to yield two diastereomers, both clear oils [10a (cis), 19.5 g, 65%, R_f = 0.40 in 9:1 hexanes/EtOAc and 10b (trans), 9.10 g, 30%, R_f = 0.20 in 9:1 hexanes/EtOAc]. Relative stereochemistry was assigned upon determination of the x-ray crystal structure of 11, a decomposition product of 10b (SI Fig. 5).

10a (trans): ¹H-NMR (CDCl₃, 400 MHz): 1.59 (m, 1H), 1.69 (m, 2H), 1.90 (m, 1H), 2.02 (m, 1H), 2.16-2.42 (m, 3H), 2.88 (m, 1H), 3.26 (m, 1H), 3.91 (m, 2H), 4.92 (ddd, 1H, J = 2.6, 6.4, 50.4 Hz), 5.16 (dd, 1H, J = 1.6, 10.4 Hz), 5.25 (dd, 1H, J = 1.6, 17.2 Hz), and 5.91 (ddd, 1H, J = 5.2, 10.4, 22.4 Hz). ¹³C-NMR (CDCl₃, 100 MHz): d 20.1 (d, J = 5 Hz), 27.2 (d, J = 3 Hz), 30.7 (d, J = 21 Hz), 33.4, 40.2, 69.4, 77.5, 95.8 (d, J = 185 Hz), and 116.8, 134.8, 213.5 (d, J = 24 Hz). ¹⁹F-NMR (CDCl₃, 376 MHz): d -189.1 (app t, J = 43 Hz) FAB-HRMS: Calculated for C₁₁H₁₈FO₂⁺ [M+H]⁺: 201.1291; found: 201.1291.

10b (trans): ¹H-NMR (CDCl₃, 400 MHz): 1.62-1.91 (m, 4H), 1.93-2.11 (m, 3H), 2.27-2.55 (m, 3H), 3.35 (m, 1H), 3.92 (m, 2H), 4.99 (ddd, 1H, J = 3.4, 6.8, 48.0 Hz), 5.15 (dd, 1H, J = 1.6, 10.4 Hz), 5.24 (dd, 1H, J = 1.8, 17.2 Hz), and 5.91 (ddd, 1H, J = 5.6, 10.8, 22.8 Hz). ¹³C-NMR (CDCl₃, 100 MHz): d 22.2, 26.6 (d, J = 22 Hz), 27.0 (d, J = 4 Hz), 31.7, 38.5, 69.5, 76.0, 93.9 (d, J = 184 Hz), and 116.8, 134.9, 210.3 (d, J = 17 Hz). ¹⁹F-NMR (CDCl₃, 376 MHz): d -188.0 (app t, J = 41 Hz) FAB-HRMS: Calculated for C₁₁H₁₈FO₂⁺ [M+H]⁺: 201.1291; found: 201.1286.

Monofluoroketone (10b, trans). To a stirring solution of compound 10a (26.5 g, 132 mmol) in THF (300 ml) at 0°C was added KHMDS (2.64 ml, 1.32 mmol, 0.500 M solution in toluene). After 1 h, the reaction mixture was concentrated on a rotary evaporator and the crude product was purified by column chromatography (10:1 to 5:1 hexanes:EtOAc) to yield two diastereomers, both clear oils (10a, 14.4 g, 54%, and 10b, 11.8 g, 45%).

Fluorinated Silyl Enol Ether (12). To a stirring solution of potassium hexamethyldisilazide (KHMDS, 225 ml, 113 mmol, 0.500 M solution in toluene) in THF (800 ml) at -78°C was added a solution of ketone 10b (18.8 g, 93.9 mmol) in THF (40 ml) dropwise, using a syringe pump, over 2 h. After an additional 30 min of stirring at -78°C, chlorotriethylsilane (20.5 ml, 122 mmol) was added. The solution was stirred at -78°C for 30 min, removed from the cold bath, and stirred for 1 h at room temperature. The reaction mixture was concentrated on a rotary evaporator, and the crude product was purified directly by column chromatography (100% hexanes to 25:1 hexanes/EtOAc) to yield a clear oil (28.7 g, 97% as a 5:1 mixture of desired:undesired regioisomers, R_f = 0.70 in 9:1 hexanes/EtOAc). ¹H-NMR (CDCl₃, 400 MHz): d 0.67 (q, 6H, J = 8.0 Hz), 0.97 (t, 9H, J = 8.0 Hz), 1.50-1.91 (m, 5H), 2.02-2.13 (m, 2H), 2.14-2.30 (m, 2H), 2.27-2.55 (m, 1H), 3.43 (m, 1H), 3.96 (m, 2H), 5.15 (dd, 1H, J = 1.6, 10.4 Hz), 5.27 (dd, 1H, J = 1.6, 17.2 Hz), and 5.91 (ddd, 1H, J = 5.2, 10.4, 22.4 Hz). ¹³C-NMR (CDCl₃, 100 MHz): d 5.2, 6.7, 24.5 (d, J = 3 Hz), 24.5 (d, J = 27 Hz), 31.2, 32.9, 33.5 (d, J = 2 Hz), 69.3, 79.0, 104.3, 116.4, 128.6 (d, J = 81 Hz), and 135.2, 144.2 (d, J = 239 Hz). ¹⁹F-NMR (CDCl₃, 376 MHz): d -129.5 (dd, J = 21, 28 Hz).

Allylated Difluoroketone (13). To a stirring solution of Selectfluor (35.8 g, 101 mmol) in DMF (100 ml) was slowly added a solution of silyl enol ether 12 (24.4 g, 77.7 mmol) in DMF (180 ml) over 30 min using an addition funnel, at 0°C. The reaction mixture was allowed to warm to room temperature, and, after 2 h of additional stirring, the reaction mixture was quenched with water (100 ml). The aqueous layer was extracted with ether (4 ´ 200 ml), and the combined organic extracts were washed with water (3 ´200 ml) and brine (1 ´ 100 ml) and then dried over MgSO₄. The crude product was purified by column chromatography (11:1 to 6:1 hexanes/EtOAc) to yield a clear oil (12.5 g, 74%, R_f = 0.60 in 4:1 hexanes/EtOAc). ¹H-NMR (CDCl₃, 300 MHz): 1.65-1.93 (m, 5H), 2.04-2.20 (m, 2H), 2.27-2.47 (m, 1H), 2.48-2.61 (m, 1H), 2.75-2.85 (m, 1H), 3.35 (m, 1H), 3.92 (dt, 2H, J = 1.4, 5.7 Hz), 5.17 (dd, 1H, J = 1.4, 10.4 Hz), 5.25 (dd, 1H, J = 1.5, 17.4 Hz), and 5.91 (ddd, 1H, J = 5.6, 10.8, 22.8 Hz). ¹³C-NMR (CDCl₃, 100 MHz): d 21.0, 25.9 (t, J = 6 Hz), 31.0 (t, J = 25 Hz), 31.4, 38.0, 69.4, 75.9, 116.9, 118.5 (t, J = 250 Hz), and 134.7, 203.4 (t, J = 28 Hz). ¹⁹F-NMR (CDCl₃, 376 MHz): d -105.0 (app q, J = 244 Hz) FAB-HRMS: Calculated for C₁₁H₁₇F₂O₂⁺ [M+H]⁺: 219.1197; found: 219.1223.

Difluoroketone Carboxylic Acid (14). To a stirring solution of alkene 13 (4.90 g, 22.5 mmol) in CCl₄ (45 ml), CH₃CN (45 ml), and H₂O (68 ml) at 0°C in a three-necked flask equipped with an overhead stirrer was added NaIO₄ (19.3 g, 90.0 mmol) and RuCl₃·H₂O (117 mg, 0.563 mmol). After 10 min of vigorous stirring at 0°C, the reaction was allowed to warm to room temperature. After an additional 2.5 h of vigorous stirring of the suspension at room temperature, the reaction mixture was concentrated on a rotary evaporator and diluted with 1 M HCl (200 ml) and brine (200 ml). The aqueous layer was extracted with CH₂Cl₂ (7 ´ 200 ml) and dried extensively over MgSO₄. The crude product was purified by column chromatography (4:1 to 1:1 hexanes/EtOAc, with 1% acetic acid) to yield a clear oil, which turned to a white solid upon storage at -20°C overnight (5.10 g, 96%, R_f = 0.30 in 1:1 hexanes/EtOAc with 1% acetic acid). ¹H-NMR (CD₃CN, 400 MHz): 1.62-1.97 (m, 5H), 1.98-2.20 (m, 2H), 2.27-2.42 (m, 1H), 2.53 (m, 1H), 2.72 (m, 1H), 3.35 (tt, 1H, J = 3.6, 7.6 Hz), and 3.99 (s, 2H). ¹³C-NMR (CD₃CN, 100 MHz): d 22.1, 26.4 (t, J = 6 Hz), 31.0 (t, J = 25 Hz), 31.5, 38.4, 66.2, 78.6, 119.8 (t, J = 248 Hz), and 172.2, 204.2 (t, J = 28 Hz). ¹⁹F-NMR (CD₃CN, 376 MHz): d -105.9 (app t, J = 15 Hz) FAB-HRMS: Calculated for C₁₀H₁₅F₂O₄⁺ [M+H]⁺: 237.0938; found: 237.0934.

Vinyl Triflate (15). To a solution of KHMDS (106 ml, 52.9 mmol, 0.500 M solution in toluene) in THF (700 ml) was added a solution of ketone 14 (6.10 g, 25.8 mmol) in THF (20 ml) at -78°C dropwise over 20 min. After an additional 1 h of stirring at -78°C, a solution of N-phenyl-bis(trifluoromethylsulfonamide) (Tf₂NPh, 18.9 g, 52.9 mmol) in THF (40 ml) was added and the reaction was allowed to warm to room temperature. After 30 min, the reaction mixture was concentrated on a rotary evaporator, and the crude product was diluted with CH₂Cl₂ (200 ml), 1 M HCl (200 ml), and brine (200 ml). The aqueous layer was extracted with CH₂Cl₂ (5 ´ 150 ml) and dried extensively with MgSO₄. The crude product was purified by column chromatography (4:1 to 1:1 hexanes/EtOAc with 1% acetic acid) to yield the desired product (4.50 g, 47%, R_f = 0.35 in 1:1 hexanes/EtOAc with 1% acetic acid) as a colorless oil. ¹H-NMR (CD₃CN, 400 MHz): d 1.74 (m, 1H), 1.92 (m, 3H), 2.21-2.43 (m, 2H), 2.58-2.74 (m, 2H), 3.59 (tt, 1H, J = 4.0, 7.8 Hz), 4.04 (d, 2H, J = 1.6 Hz), and 6.35 (td, 1H, J = 2.8, 9.6 Hz). ¹³C-NMR (CD₃CN, 100 MHz): d 19.8, 26.4 (t, J = 2 Hz), 31.7, 32.1 (t, J = 25 Hz), 66.4, 77.6, 119.8 (t, J = 237 Hz), 130.6 (t, J = 5 Hz), and 143.0 (t, J = 29 Hz), 172.2. ¹⁹F-NMR (CD₃CN, 376 MHz): d -74.4 (s, 3F), -89.2 (dd, 2F, J = 30, 273 Hz), and -92.6 (dd, 2F, J = 30, 273 Hz). FAB-HRMS: Calculated for C₁₁H₁₃F₅O₆SLi⁺ [M+H]⁺: 375.0513; found: 375.0509.

DIFO (1). To a solution of diisopropylamine (10.1 ml, 71.3 mmol) in THF (86 ml) at -78°C was added nBuLi (23.8 ml, 59.4 mmol, from a 2.5 M solution in hexanes) dropwise and stirred at -78°C for 45 min. In a separate flask, a solution of vinyl triflate 15 (7.31 g, 19.8 mmol) in THF (400 ml) was prepared and kept at -15°C by using a MeOH/ice bath. LDA was added to this solution dropwise, via syringe pump, at the rate of 1.0 equivalent (~40 ml of the solution prepared above) per 30 min, with vigorous stirring, until 2.5 equivalents of LDA was added. Note that the color of the solution turned from clear to yellow to amber. The reaction mixture was quenched by the addition of 10 ml of saturated ammonium chloride solution, and then the solvent was removed by rotary evaporation. To the residue was added CH₂Cl₂ (100 ml), 1 M HCl (50 ml), and brine (50 ml). The aqueous layer was extracted with CH₂Cl₂ (5 ´ 40 ml) and washed extensively with MgSO₄. The crude product was purified by column chromatography (4:1 to 1:1 hexanes/EtOAc with 1% acetic acid) to yield the desired product (466 mg, 11%, R_f = 0.35 in 1:1 hexanes/EtOAc with 1% acetic acid) as a colorless oil. ¹H-NMR (CD₃CN, 400 MHz): d 1.94 (m, 1H), 2.07 (m, 1H), 2.11-2.33 (m, 3H), 2.41 (m, 1H), 2.52 (m, 2H), 3.55 (app t, J = 7.2 Hz, 1H), and 4.05 (s, 2H). ¹³C-NMR (CD₃CN, 100 MHz): d 17.6, 34.4 (d, J = 6 Hz), 39.2, 43.2 (t, J = 28 Hz), 66.5, 84.4, 85.2 (dd, J = 48, 43 Hz), 113.9 (t, J = 11 Hz), 120.5 (t, J = 233 Hz), and 172.2. ¹⁹F-NMR (CD₃CN, 376 MHz): d -86.6 (dddt, J = 259.0, 26.0, 12.8, 6.0 Hz, 1F), -88.6 (dm, J = 260.9 Hz, 1F). FAB-HRMS: Calculated for C₁₀H₁₂F₂O₃Li⁺ [M+H] ⁺: 225.0915; found: 225.0912.

General Procedure for Synthesis of Conjugates of DIFO (5a-f). The pentafluorophenyl ester of DIFO (16) was prepared as follows and used immediately. DIFO (1, 1.0 equivalent) was dissolved in CH₂Cl₂ (final concentration of 0.1-0.2 M) and N,N-diisopropylethylamine (2.0 equivalents) was added. The solution was cooled to 0°C and pentafluorophenyltrifluoroacetate (1.05 equivalents) was added dropwise. After 1 h, the solvent and residual pentafluorophenol was removed on a rotary evaporator. The crude product was either used immediately or quickly purified by column chromatography (R_f = 0.3 in 9:1 hexanes:ethyl acetate) and then used immediately. ¹H-NMR (CD₃CN, 400 MHz): d 1.99 (m, 1H), 2.09 (m, 1H), 2.15-2.35 (m, 3H), 2.43 (m, 1H), 2.54 (m, 2H), 3.63 (app t, J = 7.2 Hz, 1H), and 4.53 (s, 2H). ¹⁹F-NMR (CD₃CN, 376 MHz): d -86.6 (dddt, J = 259.0, 26.0, 12.8, 6.0 Hz, 1F), -88.6 (dm, J = 260.9 Hz, 1F), -153.6 (d, J = 19 Hz, 2F), -159.2 (t, 21 Hz, 1F), and -163.6 (dd, J = 17, 4 Hz, 2F).

Synthesis of Alexa Fluor Derivatives (5a, DIFO-488; 5d, DIFO-568; 5e, DIFO-647; 6a, Alk-488). To a solution of the appropriate Alexa Fluor cadaverine (1.0 equivalent) in DMF (final concentration of 0.2 M) was added a solution of the pentafluorophenyl ester of DIFO (16) or 4-pentynoic acid (2.0 equivalents) and then N,N-diisopropylethylamine (5.0 equivalents). The solution was stirred at room temperature overnight in the dark, and then the solvent was removed on a rotary evaporator. The residue was dissolved in water or 9:1 water:acetonitrile, purified by reversed-phase HPLC using water and acetonitrile, and lyophilized to a fine powder.

DIFO-488, FT-ICR-MS: Calculated for C₃₆H₃₆F₂N₄O₁₂S₂⁺ [M⁺]: 818.1739; found: 818.1725.

DIFO-568, FT-ICR-MS: Calculated for C₄₈H₅₂F₂N₄O₁₂S₂⁺ [M⁺]: 978.2986; found: 978.2971.

DIFO-647, FT-ICR-MS: Found: 1142.3509.

Alk-488, FT-ICR-MS: Calculated for C₃₁H₃₀N₄O₁₁S₂⁺ [M⁺]: 698.1352; found: 698.1318.

DIFO-biotin (5b). To a solution of biotin-PEG-amine (2) (15 mg, 0.034 mmol) in DMF (0.5 ml) was added a solution of pentafluorophenyl ester 16 (13 mg, 0.034 mmol) in DMF (1.0 ml) and then N,N-diisopropylethylamine (9.0 ml, 0.051 mmol). The solution was stirred overnight at room temperature, the DMF was removed on a rotary evaporator, and the residue was purified by silica gel chromatography (100% CH₂Cl₂ to 9:1 CH₂Cl₂:MeOH) to yield 19 mg (87%) of a clear oil (R_f in 9:1 CH₂Cl₂:MeOH = 0.40). ¹H-NMR (MeOD, 500 MHz): d 1.43 (m, 2H), 1.55-1.70 (m, 3H), 1.71-1.83 (m, 5H), 1.95 (m, 1H), 2.07-2.35 (m, 4H), 2.18 (t, J = 8.0 Hz, 2 H), 2.43 (m, 1H), 2.52 (m, 2H), 2.70 (d, J = 12.5 Hz, 1H), 2.92 (dd, J = 12.5, 5.0 Hz, 1H), 3.20 (m, 1H), 3.26 (t, J = 6.8 Hz, 2H), 3.34 (t, J = 6.8 Hz, 2H), 3.53 (m, 5H), 3.59 (m, 4H), 3.64 (m, 4H), 3.95 (d, J = 3.5 Hz, 2H), 4.30 (dd, J = 7.5, 4.5 Hz, 1H), and 4.49 (dd, J = 7.5, 5.0 Hz, 1H). ¹³C-NMR (MeOD, 125 MHz): d 18.2, 27.4, 30.0, 30.3, 30.9, 35.3 (d, J = 6 Hz), 37.4, 38.3 (d, J = 4 Hz), 40.3, 41.6, 44.2 (t, J = 28 Hz), 57.5, 62.1, 63.9, 69.4, 70.4, 70.8, 71.8, 72.0, 72.1, 85.2, 86.4 (dd, J = 48, 42 Hz), 113.9 (t, J = 11 Hz), 120.5 (t, J = 234 Hz), and 166.6, 172.9, 176.5. ¹⁹F-NMR (MeOD, 376 MHz): d -88.0 (dddt, J = 266.5, 26.3, 12.4, 6.0 Hz, 1F), and -90.0 (dm, J = 259.4 Hz, 1F). FAB-HRMS: Calculated for C₃₀H₄₈F₂N₄O₇SLi⁺ [M+H]⁺: 653.3372; found: 653.3379.

Maleimide-amine (17). A dry 500-ml round-bottom flask was charged with maleimide (3.12 g, 32.2 mmol) and triphenylphosphine (8.29 g, 31.6 mmol) and then THF (150 ml). N-(tert-butoxycarbonyl)ethanolamine (5.00 ml, 29.3 mmol) and diisopropylazidodicarboxylate (6.80 ml, 35.1 mmol) were added in succession. The flask was stirred under a nitrogen atmosphere overnight, the reaction mixture was concentrated on a rotary evaporator, and the crude product was filtered through a plug of silica gel by using a 2:1 mixture of hexanes:ethyl acetate as the eluent. The crude product was dissolved in 100 ml of a 60:35:5 mixture of CH₂Cl₂:trifluoroacetic acid:water and stirred at room temperature for 2 h. The reaction mixture was diluted with CH₂Cl₂ (50 ml) and water (50 ml), transferred to a separatory funnel, and the organic layer was extracted with water (3 ´ 25 ml). The combined aqueous layers were washed with CH₂Cl₂ (3 ´ 50 ml) and concentrated on a rotary evaporator to yield the desired product (7.51 g, 96%) as a yellow oil. ¹H-NMR (DMSO-d₆, 400 MHz): d 1.76 (app quintet, 2H, J = 7.2 Hz), 2.76 (m, 2H), 3.45 (t, 2H, J = 6.8 Hz), 7.02 (s, 2H), and 7.79 (br s, 3H). ¹³C-NMR (DMSO-d₆, 100 MHz): d 26.7, 34.6, 37.0, 115.6 (q, J = 290 Hz), 134.7, and 158.9 (q, J = 36 Hz), 171.3. ¹⁹F-NMR (DMSO-d₆, 376 MHz): d -73.9.

DIFO-Maleimide (5f). To a solution of maleimide-amine 17 (74 mg, 0.27 mmol) in CH₂Cl₂ (1.5 ml) was added N,N-diisopropylethylamine (160 ml, 0.91 mmol). The solution was cooled to 0°C, and then a solution of pentafluorophenyl ester 16 (97 mg, 0.23 mmol) in CH₂Cl₂ (1 ml) was added dropwise. The reaction mixture was stirred for 2 h, the solvent was removed on a rotary evaporator, and the residue was purified by silica gel chromatography (2:1 to 1:3 hexanes:ethyl acetate) to yield 62 mg (76%) of a clear oil (R_f = 0.20 in 1:1 hexanes:ethyl acetate). ¹H-NMR (CDCl₃, 500 MHz): d 1.81 (app q, J = 6.5 Hz, 2H), 2.05-2.31 (m, 5H), 2.48 (m, 2H), 2.60 (m, 1H), 3.26 (t, J = 6.5 Hz, 2H), 3.55 (t, J = 6.8 Hz, 1H), 3.60 (t, J = 6.5 Hz, 2H), 3.95 (dd, J = 20.5, 15.0 Hz, 2H), 6.74 (s, 2H), and 7.12 (br s, 1H). ¹⁹F-NMR (CD₃CN, 376 MHz): d -86.6 (dddt, J = 266.5, 26.3, 12.4, 6.0 Hz, 1F), -88.5 (dm, J = 259.4 Hz, 1F).

DIFO-FLAG (5c). Cysteine-modified FLAG peptide (FLAG-C, H₂N-DYKDDDDKC-CO₂H) was synthesized by using established automated protocols on a Perkin-Elmer ABI 431 A peptide synthesizer using fluorenylmethoxycarbonyl (Fmoc)-Cys(Trt)-Wang resin (Novabiochem). The peptide was cleaved by using a solution of trifluoroacetic acid:triisopropylsilane:water (95:2.5:2.5), precipitated with ether, and the crude product was dried and used without further purification. To a solution of FLAG-C (214 mg, 0.192 mmol) in 1 ml of water was added a solution of DIFO-maleimide (5f, 62 mg, 0.175 mmol) in 1 ml of DMF at 0°C. The reaction mixture was allowed to warm to room temperature and stir overnight. The solvents were removed on a rotary evaporator, and the residue was purified by reversed-phase HPLC (5% to 40% acetonitrile in water over 60 min, product eluting at 30-35 min) and lyophilized to yield 125 mg (49%) of a white solid. MALDI-TOF: Calculated [M+H]⁺: 1470.5540; found: 1470.5526.

Kinetic Evaluation of the [3 + 2] Cycloaddition of DIFO and Benzyl Azide

Stock solutions of DIFO (1, 20 mM) and benzyl azide (200 mM) were made in CD₃CN. An NMR tube was charged with 450 ml of the solution of 1, and, immediately before lowering into the NMR magnet, 50 ml of the benzyl azide solution, and the reaction was monitored over time by using ¹H-NMR spectroscopy. The kinetic data were derived by monitoring the change in integration of resonances corresponding to the benzylic protons in benzyl azide (d~4.4 ppm) compared to the corresponding resonances of the triazole products (d ~5.5 to 5.7 ppm).

The second-order rate constant for the reaction was determined by plotting 1/[benzyl azide] versus time, followed by subsequent analysis by linear regression (SI Fig. 9). The second-order rate constant (k, M^-1·s^-1) corresponds to the determined slope. Error bars represent standard deviations from three replicate experiments.

Mice and Animal Protocol

B6D2F1 mice were obtained from The Jackson Laboratory. All animals were housed and monitored at the Northwest Animal Facility (Berkeley, CA), and experiments were performed according to guidelines established by the Animal Care and Use Committee at the University of California, Berkeley (protocols R234-0503B, -0505B, -0606B, and -0607B).

General Protocol for Compound Administration

For in vivo metabolic labeling experiments, mice were administered daily doses of Ac₄ManNAz (300 mg/kg in ~150 ml of 70% DMSO, from a stock solution of 50 mg/ml) i.p. for 7 days as previously described (3). Sixteen to 24 h after the final azido sugar bolus, mice were injected i.p. with DIFO-FLAG (5c, 0.16 mmol kg^-1 in 70% DMSO) or vehicle (70% DMSO). After 3 h, the mice were killed, and organs were harvested.

Detection of Splenocyte Cell Surface FLAG Epitopes After in Vivo Ligation

To determine the extent of ligation of azides with DIFO-FLAG in vivo, splenocytes from B6D2F1 mice treated with the appropriate combination of Ac₄ManNAz, DIFO-FLAG, or vehicle (70% DMSO), as described above, were isolated and probed for the presence of cell-surface FLAG epitopes by using a flow cytometry assay. Briefly, splenocytes from one spleen were suspended in RPMI medium 1640 and distributed among wells of a 96-well V-bottom tissue culture plate (three wells per treatment, ~5 ´ 10⁵ cells per well). The cells were pelleted, rinsed with labeling buffer (PBS, pH 7.4 containing 1% FBS), and incubated with a FITC-conjugated anti-FLAG antibody (1:900 dilution) in labeling buffer for 30 min on ice. All cells were analyzed by flow cytometry.

1. Still WC, Kahn M, Mitra A (1978) J Org Chem 43:2923-2925.

2. Vocadlo DJ, Hang HC, Kim EJ, Hanover JA, Bertozzi CR (2003) Proc Natl Acad Sci USA 100:9116-9121.

3. Prescher JA, Dube DH, Bertozzi CR (2004) Nature 430:873-877.