PMC

Spectra of ODF-HaloTag ligands. A) Absorption spectra, and B) normalized fluorescence emission spectra. Conditions: 2.0 μM ODF-HaloTag ligand in PBS (ex. 344 nm).

Fluorescence emission spectra ODF ligands alone (A) and of protein-ODF conjugates (B). ODF-HaloTag ligands are 1.0 μM in PBS; protein-ODF conjugates prepared from ODF 1.0 μM, HaloTag protein 2.5 μM, PBS, 30 min, 37 °C. Ex 344 nm.

Structures in this study. A) Monomers used for making chloroalkyl-ODF HaloTag ligands. B) Structure of a typical ODF-HaloTag ligand with the sequence of 5′-htS₂EY (haloalkyl group marked in blue). C) Illustration of conjugate between the engineered dehalogenase enzyme and an ODF ligand (5′-htS₂EY).

Multispectral labeling of GST-HaloTag fusion protein (lane 2-9) and HaloTag protein (lane 10-17) with ODF-HaloTag ligands. A) fluorescence image (Ex 365 nm), and B) Coomassie blue staining: lane 1: marker, lane 2 & 10: htS₂YYYY, lane 3 & 11: htS₂EY, lane 4 & 12: htS2YKY, lane 5 & 13: htS₂FYF, lane 6 & 14: htS₂YZY, lane 7 & 15: htS₂EYF, lane 8 & 16: htS₂EYKa, lane 9 & 17: htS₂EYKb.

Live HeLa cell imaging showing two-color, single-excitation protein labeling. Cytoplasmic α-tubulin was first expressed and labeled with cyan ODF HaloTag ligand htS₂EY, and cell surface protein was then expressed and (48 h later) labeled with red htS₂YKY. Laser confocal imaging was carried out in phenol-free growth media (excitation 405 nm). A) Multiple cell view. B) Closeup of single cell.

Imaging α-tubulin in live HeLa cells after labeling cytoplasmic tubulin-Halotag fusion protein with three different chloroalkyl-ODF ligands (A-C) (excitation 405 nm; bar denotes ca. 5 microns). (D,E) SDS PAGE gel of cell extracts showing labeling of 88.5 kD fusion protein (D: fluorescence scan; E: Coomassie blue staining). Lane 1: size marker, lane 2: control HeLa cells, lane 3: HeLa cells expressing α-tubulin fusion protein.

Imaging cell surface protein in live HeLa cells with ODF-HaloTag ligands (A-C) by confocal microscopy (excitation 405 nm; bar denotes ca. 20 microns). (D,E) SDS PAGE gel of cell extracts showing labeling of 66.8 kD cell surface protein (D: fluorescence scan; E: Coomassie blue staining). Lane 1: size marker, lane 2: control HeLa cells, lane 3: HeLa cells expressing cell surface protein.

Reagents and conditions: i) Boc₂O, anhyd. EtOH, 0 °C to rt., 2 h, 99%. ii) NaH, 6-chloro-1-iodohexane, THF/DMF, 0 °C to rt, o/n, 69%. iii) TFA, CH₂Cl₂, 0 °C to rt, 2 h, 81%. iv) DMT-Cl, Et₃N, CH₂Cl₂, rt, 6 h, 67 %. v) Methyl acrylate, NaH, THF, 0 °C, 1 h, 75 %. vi) LiOH, MeOH/H₂O, rt, 2h, 93 %. vii) N-hydroxysuccinimide, DCC, CH₂Cl₂, 0 °C to rt, o/n, 95%. viii) DIPEA, CH₂Cl₂, rt, o/n, 82%. ix) AcOH, H₂O, rt, 2h, 68%. ×) DIPEA, 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, CH₂Cl₂, 0 °C, 45 min, 98%.

Reagents and conditions: i) MeI, KOH, KI, DMSO, rt, 24h, 97%. ii) nBuLi, DMF, THF, -78 °C, 1+2h, 90%. iii) diphenylamine, Cs₂CO₃, Pd(OAc)₂, t-Bu₃P, toluene, reflux, 24 h, 67%. iv) 2-Amino-5-bromobenzenethiol 5, TsOH, toluene, reflux, 2 d, 80%. v) Cy₂NMe, 3′-O-TBDPS-1,2-dehydro-2-deoxy-d-ribofuranose, Pd(t-Bu₃P)₂, Bu₄NBr, dioxane, 90 C, 36 h. vi) TBAF, AcOH, THF, 0 °C, 1 h, 25% (3 steps). viii) Na(OAc)₃BH, HOAc, CH₃CN, THF, -10 °C, 1 h. viii) DMT-Cl, pyridine, DIPEA, rt, 3 h, 82%.