HaloTag protein-mediated specific labeling of living cells with quantum dots.

General methods and chemicals Unless otherwise stated, all reagents and solvents were obtained from commercial sources and used without purification. Water was deionized by passing through a Milli-Q water filtration system. Analytical TLC was performed with 0.25 mm silica gel 60F plates with fluorescent indicator (254 nm). Plates were visualized by ultraviolet light. 1 H NMR spectra were measured on a Varian INOVA 400 magnetic resonance spectrometer. Data for 1 H NMR spectra are reported as follows: chemical shifts are reported as δ in units of parts per million (ppm) relative to chloroform-d (δ 7.26, s) and Methanol-d 4 (δ 4.87, s); multiplicities are reported as follows: s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), m (multiplet), or br (broadened); coupling constants are reported as a J value in Hertz (Hz); the number of protons (n) for a given resonance is indicated nH, and is based on spectral integration values. EI-MS spectrometric analyses were performed at the Mass Spectrometry Facility of Stanford University, California. Synthesis of biotinylated HaloLigands 1 and 2 HaloTag ligands 1 and 2 are prepared according to Scheme 2 . 3 : To a solution of 2-(2-aminoethoxy)ethanol (1.05 g, 10 mmol) in anhydrous EtOH (20 mL) at 0 °C was added Boc 2 O (2.2 g, 10 mmol). After stirring at room temperature for 2 h, the reaction mixture was diluted with CH 2 Cl 2 , washed with brine and the organic layer was dried over Na 2 SO 4 . The solvent was removed to get the residue 3 (2.05 g, 100%), which was used for the next step without further purification. 1 HNMR: (400 MHz, CDCl 3 ) δ 5.07 (br, 1 H), 3.74(m, 2 H), 3.60 (m, 4 H), 3.33 (m, 2 H), 2.69 (m, 1 H), 1.45 (s, 9 H). 4 : Under argon, to a solution of 3 (1.0 g, 5.0 mmol) in anhydrous DMF (5 mL) at 0 °C was added 80% of NaH (192 mg, 6.0 mmol). After stirring at 0 °C for 30 min, 1-chloro-6- iodohexane (1.5 g, 6.1 mmol) was added to the reaction mixture. After 4h the reaction was quenched with 1N of HCl, and extracted with CH 2 Cl 2 . The organic layer was washed with brine and dried over Na 2 SO 4 and then evaporated. The residue was purified by flash column on silica gel to afford 0.9 g (56%) of 4 as colorless oil. 1 HNMR: (400 MHz, CDCl 3 ) δ 5.07 (br, 1 H), 3.64−3.50 (m, 8 H), 3.47(t, J = 6.8 Hz, 3 H), 3.36−3.28 (m, 2 H), 1.82−1.74 (m, 2 H), 1.66−1.57 (m, 2 H), 1.50−1.34 (m, 4 H), 1.45 (s, 9 H). EI-MS: m/z 324.2 [M+1] + ; calc 323.2. 5 : Compound 4 (650 mg, 2.0 mmol) was dissolved in 4 mL of CH 2 Cl 2 and treated with TFA (1.0 mL) at room temperature for 2h. The solvent was removed and the residue was treated with anhydrous K 2 CO 3 (550 mg, 4.0 mmol) in MeOH (5 mL). The mixture was filtered and the filtrate was concentrated to give amine 5 (406 mg, 91%) as colorless oil. 1 HNMR: (400 MHz, CDCl 3 ) δ 3.95−3.40 (m, 8 H), 3.10−2.80 (m, 2 H), 1.82−1.74 (m, 2 H), 1.65−1.56 (m, 2 H), 1.50−1.32 (m, 4 H). EI-MS: m/z 223.8 [M+1] + ; calc 223.1. 8 : To a mixture of 6 (200 mg, 0.5 mmol), TBTU (160 mg, 0.5 mmol) and HOBt (80 mg, 0.6 mmol) in 3.0 mL of anhydrous CH 2 Cl 2 was added DIPEA ( 175 μL, 1.0 mmol) at room temperature. The reaction mixture was stirred for 5 min and then 5 (130 mg, 0.6 mmol) was added. After stirring for 1h, the reaction mixture was quenched with NH 4 Cl and extracted with EtOAc. The organic layer was washed with brine, dried over Na 2 SO 4 and evaporated. The residue was purified by flash column on silica gel to get 250 mg (83 %) of 8 as colorless oil. 1 HNMR: (400 MHz, CDCl 3 ) δ 7.76 (d, J = 7.6 Hz, 1 H), 7.61 (d, J = 7.2 Hz, 1 H), 7.42−7.36 (m, 2 H), 7.34−7.27 (m, 2 H), 6.63 (br, 1 H), 5.78 (br, 1 H), 4.43 (d, J = 6.8 Hz, 2 H), 4.24−4.21 (m, 2 H), 3.73 (t, J = 6.4 Hz, 2 H), 3.66−3.40 (m, 20 H), 2.45 (t, J = 6.4 Hz, 2 H), 1.80−1.72 (m, 2 H), 1.64−1.54 (m, 2 H), 1.48−1.30 (m, 4 H). EI-MS: m/z 605.3 [M+1] + ; calc 604.3. 9 : To a mixture of compound 6 (130 mg, 0.25 mmol), TBTU (80 mg, 0.25 mmol) and HOBt (40 mg, 0.3 mmol) in 1.5 mL of anhydrous CH 2 Cl 2 was added DIPEA ( 100 μL, 0.6 mmol) at room temperature. The reaction mixture was stirred for 5 min and then 5 (70 mg, 0.31 mmol) was added. After stirring for 1h, the reaction mixture was quenched with NH 4 Cl and extracted with EtOAc. The organic layer was washed with brine, dried over Na 2 SO 4 and evaporated. The residue was purified by flash column on silica gel to afford 140 mg (76 %) of 9 as colorless oil. 1 HNMR: (400 MHz, CDCl 3 ) δ 7.76 (d, J = 7.2 Hz, 1 H), 7.61 (d, J = 7.2 Hz, 1 H), 7.41−7.37 (m, 2 H), 7.34−7.29 (m, 2 H), 6.64 (br, 1 H), 5.56 (br, 1 H), 4.40 (m, 2 H), 4.24−4.22 (m, 2 H), 3.72 (t, J = 6.0 Hz, 2 H), 3.66−3.40 (m, 34 H), 2.45 (t, J = 6.0 Hz, 2 H), 1.80−1.72 (m, 2 H), 1.62−1.54 (m, 2 H), 1.50−1.30 (m, 4 H). EI-MS: m/z 767.5 [M+1] + ; calc 766.4. 1 : To a solution of 8 (61 mg, 0.10 mmol) in 1.5 mL of anhydrous CH 2 Cl 2 was added piperidine (300 L) at room temperature. After stirring for 1h, the solvent was removed under reduced pressure and the residue was dried in vacuum overnight. The resultant amine was dissolved in DMF. To this solution was added DIPEA (100 L) and Biotin-NHS (50 mg, 0.15 mmol) at room temperature. The reaction mixture was diluted with EtOAc, washed with brine, dried over Na 2 SO 4 and evaporated. The residue was purified by flash column on silica gel to get 22 mg (36%) of 1 . 1 HNMR: (400 MHz, CD 3 OD) δ 4.51−4.48 (m, 1 H), 4.32−4.28 (m, 1 H), 3.74 (t, J = 6.0 Hz, 1 H), 3.62−3.52 (m, 14 H), 3.49 (t, J = 6.0 Hz, 1 H), 3.40−3.28 (m, 6 H), 3.24−3.18 (m, 1 H), 2.96−2.90 (m, 1 H), 2.74−2.68 (m, 1 H), 2.46 (m, J = 6.0 Hz, 1 H), 2.22 (m, J = 7.2 Hz, 1 H), 1.80−1.52 (m, 8 H), 1.50−1.26 (m, 6H). EI-MS: m/z 609.4 [M+1] + ; calc 608.3. 2 : To a solution of 9 (78 mg, 0.10 mmol) in 1.5 mL of anhydrous CH 2 Cl 2 was added piperidine (300 μL) at room temperature. After stirring for 1h, the solvent was removed under reduced pressure and the residue was dried in vacuum overnight. The resultant amine was dissolved in DMF. To this solution was added DIPEA (100 μL) and Biotin-NHS (50 mg, 0.15 mmol) at room temperature. The reaction mixture was diluted with EtOAc, washed with brine, dried over Na 2 SO 4 and evaporated. The residue was purified by flash column on silica gel to get 21 mg (27%) of 2 . 1 HNMR: (400 MHz, CD 3 OD) δ 4.51−4.46 (m, 1 H), 4.32− 4.28 ( m, 1 H), 3.72 (t, J = 6.4 Hz, 1 H), 3.66−3.52 (m, 32 H), 3.49 (t, J = 6.4 Hz, 1 H), 3.38−3.34 (m, 4 H), 3.32−3.29 (m, 2 H), 3.24−3.18 (m, 1 H), 2.96−2.90 (m, 1 H), 2.71 (d, J = 12.8 Hz, 1 H), 2.45 (t, J = 6.4 Hz, 2 H), 2.22 (t, J = 7.2 Hz, 2 H), 1.80−1.54 (m, 8 H), 1.52−1.36 (m, 6H). EI-MS: m/z 785.4 [M+1] + ; calc 784.4.

Plasmid constructs

All mammalian expression vectors were constructed by standard cloning procedures. PCR amplification was done using Pfu ultra high-fidelity DNA polymerase (Stratagene) to avoid any undesired mutations. The gene encoded for the HaloTag protein without the stop codon was obtained by PCR from the HaloTag pHT2 vector (Promega) with a BglII site straddling the initial methionine codon and a PstI site attached to the 3' end. The PCR product was digested and ligated with a BglII/PstI digested pDisplay plasmid (Invitrogen, Carlsbad, CA) to afford the plasmid pmHT. that is included an N-terminal signal peptide, a c-terminal PDGFR transmembrane domain and a stop codon. The CMV-null plasmid was made from the pDsRed2-N1 vector (Clontech) after removal of the DsRed cDNA as a control vector. The pEGFP-C1 plasmid (Clontech) was used as the transfection marker. Cell lines, cell culture, and transfection protocols COS7 cells (monkey kidney cell line) were grown in Dulbecco’s Modified Eagle medium supplemented with 10% fetal bovine serum at 37°C and 5% CO 2 . Cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Two-step labeling of living cell COS7 cells were transfected with 0.6 μg of the pmHT or cmv-null plasmid along with 0.2 μg of pEGFP-C1 plasmid by lipofectamine 2000 (Invitrogen). 24 h after transfection, cells were reseeded into a 35-mm glass bottom culture dish (MatTek, Ashland, MA). 24 h after reseeding cells were rinsed twice with Hanks' balanced salt solution (HBSS, Gibco). Cells were incubated with 50 μM of the biotinylated HaloTag ligand for 30 min at 37 °C, 5% CO 2 , and then labeled with the 5 nM of streptavidin-QD655 conjugate (Invitrogen) in HBSS for 30 min at room temperature. The cells were washed 5 times with HBSS before imaging on an Axiovert 200M fluorescence microscope (Zeiss) using a 40x oil-immersion lens. QD655 (420/440 excitation, 660/40 emission, 470dcxr), and EGFP (480/30 excitation, 535/40 emission, 505dclp) images were collected and analyzed using MetaMorph Image analysis software version 5.0 (Molecular Devices). Direct labeling of living cell with QDs The biotinylated HaloTag ligand was mixed with the streptavidin coated QDs (sQD) at a ratio of 50:1 in 10 mM phosphate buffer (pH=7.4) for 30 min in room temperature, and free ligand was removed with three washes using 10K NanoSep filter (Pall) by centrifugation at 7000 rpm for 3 min at 4 °C. Final QD complex was collected with HBSS buffer (pH=7.4) for the determination of the concentration. Cells were prepared using previous protocol for labeling. After rinsing twice with HBSS, transfected cells were incubated with 20 nM of QD/HaloTag ligand conjugates for 30 minutes in 37°C, 5% CO 2 . The incubation medium was removed and the cells were rinsed 5 times with HBSS before imaging.

General methods and chemicals Unless otherwise stated, all reagents and solvents were obtained from commercial sources and used without purification. Water was deionized by passing through a Milli-Q water filtration system. Analytical TLC was performed with 0.25 mm silica gel 60F plates with fluorescent indicator (254 nm). Plates were visualized by ultraviolet light. 1 H NMR spectra were measured on a Varian INOVA 400 magnetic resonance spectrometer. Data for 1 H NMR spectra are reported as follows: chemical shifts are reported as δ in units of parts per million (ppm) relative to chloroform-d (δ 7.26, s) and Methanol-d 4 (δ 4.87, s); multiplicities are reported as follows: s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), m (multiplet), or br (broadened); coupling constants are reported as a J value in Hertz (Hz); the number of protons (n) for a given resonance is indicated nH, and is based on spectral integration values. EI-MS spectrometric analyses were performed at the Mass Spectrometry Facility of Stanford University, California.

Cell lines, cell culture, and transfection protocols COS7 cells (monkey kidney cell line) were grown in Dulbecco’s Modified Eagle medium supplemented with 10% fetal bovine serum at 37°C and 5% CO 2 . Cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol.

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