Computationally guided discovery of a reactive, hydrophilic trans-5-oxocene dienophile for bioorthogonal labeling

The use of organic chemistry principles and prediction techniques has enabled the development of new bioorthogonal reactions.

Splitting patterns are designated as follows: s, singlet; d, doublet; t, triplet; q, quartet; quin, quintet; sext, sextet; m, multiplet. When an APT pulse sequence was used to distinguish 13 C NMR multiplicities, quaternary and methylene carbons appear 'up' (C or CH2) and methane and methyl carbons appear 'down' (CH or CH3). Mass spectrometry was carried out on a Waters GCT Premier and was prepared by dissolving sodium dihydrogen phosphate (NaH2PO4, 76.7 mg) and disodium hydrogen phosphate (Na2HPO4, 203.5 mg) in 20 mL D2O to make a 0.1 M solution. The pD value was confirmed on a Fisher Scientific AB15 Plus pH meter. The pH readings were converted to pD by adding 0.4 units. [1] All other solvents and chemical reagents were purchased from commercial suppliers and used without further purification. All non-aqueous reactions were carried out in flame-dried glassware under an inert atmosphere of nitrogen. Brine refers to a saturated solution of sodium chloride in water.

S2. Photoisomerization apparatus
Photoisomerizations were carried out using a modified procedure previously developed in our lab. [2] Reactions were done in a quartz flask (Southern New England Ultraviolet Company) using a Southern New England Ultraviolet Company Rayonet® reactor model RPR-100 or RPR-200, equipped with 8 lowpressure mercury lamps (2537 Å). Biotage® SNAP cartridges (Biotage part No.

S3. Silver nitrate-impregnated silica gel
Flash silica gel (90 g, Silicycle cat # R12030B, 60 Å) was suspended in 100 mL of water in a 2 L round bottom flask. The flask was covered with aluminum foil and a silver nitrate (10 g) solution in water (10 mL) was added. The resulting mixture was thoroughly mixed. Water was evaporated under reduced pressure on the rotavap (bath temperature ~ 65 °C) using a bump trap with a coarse fritted disk. To remove the remaining traces of water, toluene (2 x 200 mL) was added and subsequently evaporated by rotary evaporation. The silver nitrate impregnated silica was then dried under vacuum overnight at room temperature.
The mixture was warmed to room temperature and stirred for 2 h, cooled again to 0 °C, diluted with DCM (30 mL) and water (50 mL) was added slowly. The phases were separated and the aqueous phase extracted with DCM (3 x 30 mL).
The combined organics were washed with brine (50 mL), dried over sodium sulfate and concentrated under reduced pressure. The crude material was purified by silica gel chromatography (5-20% toluene in hexane) to yield butenyl ether 8 as a colorless oil (1.15 g, 78%). 1

sfGFP150Tet-v.2.0 (11)
Prepared by previously developed method: see reference [3] THF, reflux found to be 14.7 mM and the aqueous phase was found to be 4.5 mM. Log(P) was then calculated as 0.51.

S8. Partition Coefficient (LogP) Determination
As a comparison, the partition of equatorial 5-hydroxy-transcyclooctene was measured using the same procedure. The log(P) was calculated as 1.1

Stability of oxoTCO 3 in methanol-d4
A solution of oxoTCO 3 (3.5 mg, 25 μmol, 2.5:1 d.r.) in methanol-d4 (750 μL) was monitored by quantitative proton NMR on a 600 MHz instrument to observe the decomposition of the trans-isomer. t-Butyl methyl ether (3.0 μL, 25 μmol) was used as an internal standard. After 14 days there was no measureable decomposition of oxoTCO 3 ( Fig. S9-a). A waterfall plot of the periodic 1 H NMR spectra is provided.

In Vivo Mass Spec Analysis
A 50 mL culture of E. coli overexpressing GFP-TetV2.0 11 was resuspended in 15 mL PBS buffer and washed three times. Excess oxoTCO 3 was added to a 500 μL cell suspension at a final concentration of 400 μM and allowed to react at room S28 temperature for one hour. The 500 μL cell suspension was spun down and washed 3 times with PBS. The cell pellet was resuspended in lysis buffer (50 mM Tris, NaCl 150 mM, sodium deoxycholate 0.5%, Triton X-100 1%, pH 7.5) and rocked for 30 minutes at room temperature. The cell solution was spun down, the supernatant was purified by nickel affinity chromatography. The eluted fractions were applied to a 10 mL Amicon centrifugal filter (10,000 MWCO, EMD Millipore) and underwent three rounds of buffer exchange with 50 mM KPi, pH 7.5, 0.1mM EDTA. The cell lysis solution was analyzed by LC-ESI-MS (Fig. S12-c).