Fluorogenic protein labeling using a genetically encoded unstrained alkene† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc03635j Click here for additional data file.

A new fluorogenic bioorthogonal reaction between styrene (an unstrained alkene) and a tetrazine was developed.

Five equivalents of styrene (229 µL, 2 mmol) were added into the solution. The resulting pink mixture was stirred overnight to form yellow solid, which was collected by vacuum filtration as desired product. To improve the final yield, the filtrate was extracted by diethyl ether (3 × 50 mL). Combined organic layers were dried over Na 2 SO 4 . The solvent was removed by vacuum evaporation.
The residue was further purified by flash chromatography with ethyl acetate/hexane (10% to 25%) to afford a yellow solid. PDHP (111 mg) was obtained in 89% yield by combining two portions of yellow solid. 1  Kinetic measurements.
The reactions were carried out in indicated solvent under pseudo first-order conditions with 10-

Computational methodology
All the quantum mechanical (QM)/FixSol 5 calculations were performed by using the Quantum chemistry Polarizable force field program (QuanPol) 6  Cells were imaged on an Olympus FV500 inverted (Olympus IX-81) confocal microscope.
PDHP channel was excited by DAPI excitation wavelength (405 nm) and imaged using GFP emission filter (510 nm).  12 2 0.036 (in PBS buffer) 12 3 0.0042 (in 1:1 methanol/water) 13 4 0.048 (in 1:1 methanol/water) 13 5 0.078 (in 1:3 methanol/water; this work) Note: These rate constants were measured in different solvents using different methods. The comparison is not absolute.             Figure S15. Stability study of PDHP in DMSO-d 6 . The solution was stored in NMR tube at room temperature (21 o C) in DMSO/D 2 O (4:1). The 1 H NMR data was collected at indicated time points. PDHP stayed to be the major component after 26 hours. Note: We were unable to conduct the NMR stability study of PDHP in higher percentage of water due to the compound's limited solubility. In fact, PDHP crystals were formed during the incubation. More crystals were observed with longer incubation time. This was reflected in the decrease of the 1 H NMR signal.

III. Supplemental tables
After 26 h, the crystals were collected and redissolved in DMSO/D 2 O (4:1) at 37 o C. Based on 1H NMR, the crystals were still PDHP. Following labeling reactions, protein samples were denatured by heating, analyzed by SDS-PAGE, and imaged by GE Typhoon imager (Excitation/Emission filter: LPB (510LP)). The fluorescence intensity of each labeled protein band was measured by ImageJ. The fluorescence intensity was normalized to the amount of protein.