chen si accept cg 1408821869 3 - nature · the column was then washed with 500 ml dichloromethane...

S1

Supplementary Information

Diels–Alder reaction-triggered bioorthogonal protein decaging in living cells

Jie Li1,3, Shang Jia1,3, Peng R. Chen1,2★

1Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China, 2Peking-Tsinghua Center for Life Sciences, Beijing 100871, China. 3These authors contributed equally to this work. ★e-mail: [email protected]

Ø Supplementary Note

Ø Supplementary Results

Ø References

Nature Chemical Biology: doi:10.1038/nchembio.1656

S2

Supplementary Note 1

Synthesis of (Z)-cyclooct-2-en-1-ol

We synthesized these compounds according to the reference2,3. A mixture of (Z)-cyclooctene (30.0 mL, 231 mmol), N-Bromosuccinimide (30.0 g, 168.6 mmol) and azobisisobutyronitrile (AIBN, 23 mg，0.14 mmol) in carbon tetrachloride (120 mL) was purged with N2 and was stirred under reflux for 2 h. The reaction was cooled at 0 °C and the precipitate were removed by filtration. The solvent was rotary vaporized to give (Z)-3-bromocyclooctene as light yellow oil. The product was subsequently dissolved in a mixture of acetone (240 mL) and water (120 mL). NaHCO3 (30 g, 360 mmol) was added to the solution and the mixture was stirred under reflux for 1 h. The mixture was filtered and the filtrate was extracted with diethyl ether (3×120 mL). The solvent in the ether layer was rotary vaporized to give (Z)-cyclooct-2-enol as a light yellow oil (19.59 g, 67%). 1H NMR (CDCl3, 300 MHz): δ (ppm): 5.46-5.70 (m, 2H), 4.66 (m, 1H), 2.2-2.0 (m, 2H), 2.0-1.8 (m, 1H), 1.79 (s, 1H), 1.7-1.3 (m, 7H). MS (ESI) m/z calculated for [M+Na]+: 149.09, found: 149.29. Synthesis of (E)-cyclooct-2-enol

We synthesized these compounds according to the reference2,3. (Z)-cyclooct-2-enol (8.5 g，67 mmol) and methyl benzoate (9.2 g，67 mmol) was dissolved in a mixture of 400 mL diethyl ether and 800 mL petroleum ether. The solution was irradiated for 24 h while it was constantly led through a column containing silica (117 g) treated with AgNO3 (13.19g, 77.6 mmol). The column was placed in the dark during the reaction. The column was then washed with 500 mL dichloromethane and dried with air. The silica was collected, stirred in a mixture of aqueous ammonia (500 mL) and dichloromethane (500 mL). The organic layer was separated, and the aqueous layer was extracted with dichloromethane (750 mL). The organic layer was combined, dried over Na2SO4, filtered and vaporized to give brown thick oil. The crude product was purified by column chromatography using petroleum ether and ethyl acetate (100:1 to 75:1) to give axial isomer of (E)-cyclooct-2-enol (first fraction, 1.84 g, 22%) as colorless thick oil and equatorial isomer of (E)-cyclooct-2-enol (second fraction, 1.94 g, 23 %) as light yellow thick oil.

Axial isomer: 1H NMR (CDCl3, 300 MHz) δ (ppm): 6.00 (m, 1H), 5.57 (dd, J1=16.8 Hz, J2=2.4 Hz, 1H), 4.62 (s, 1H), 2.49 (m, 1H), 2.1-1.7 (m, 4H), 1.7-1.3 (m, 4H), 1.2-1.0 (m, 1H)，0.9-0.7 (m, 1H). MS (ESI) m/z calculated for [M+Na]+: 149.09, found: 149.37. Equatorial isomer: 1H NMR (CDCl3, 300 mHz) δ (ppm): 5.7-5.4 (m，2H), 4.25 (m, 1H), 2.40 (m, 1H), 2.12 (m, 1H), 2.3-1.7 (m, 4H), 1.6-1.3 (m, 3H), 1.0-0.6 (m，2H). MS (ESI) m/z calculated for [M+Na]+:


S3

149.09, found: 149.29. Synthesis of Nα-Fmoc-Nε-(((E)-cyclooct-2-enyl)oxycarbonyl)-ʟ-lysine (1)

Axial isomer of (E)-cyclooct-2-enol (1.0 g，7.9 mmol) and pyridine (0.94 g, 12 mmol) was dissolved in dichloromethane and cooled at 0 °C. 4-nitrophenyl chloroformate (1.92 g, 9.5 mmol) was added in multiple portions and the reaction was allowed to reach room temperature. The mixture was stirred in the dark for 3 h and was poured into water (25 mL). The aqueous layer was extracted with diethyl ether (3×60 mL). The ether layer was combined, washed with aqueous acetic acid (pH=3, 3×50 mL), saturated aqueous NaHCO3 (3×50 mL) and saturated aqueous NaCl (50 mL). The organic layer was dried over Na2SO4, filtered and the solvent was removed to give (E)-cyclooct-2-enyl (4-nitrophenyl) carbonate as a white solid. Nα-Fmoc-L-lysine hydrochloride (4.8 g, 12 mmol) and DIPEA (2.8 mL, 16 mmol) was dissolved in DMF (30 mL) and was cooled at 0 °C. (E)-cyclooct-2-enyl (4-nitrophenyl) carbonate was added. The mixture was stirred at room temperature in the dark overnight. DMF was vaporized under reduced pressure and the resulting mixture was purified by column chromatography using 5:1 petroleum ether and ethyl acetate followed by 20:1 dichloromethane and methanol to obtain the axial isomer of Nα-Fmoc-Nε-(((E)-cyclooct-2-enyl)oxycarbonyl)-ʟ-lysine (1a) as a colorless foamy thick oil (4.5 g, 100%). 1H NMR (CDCl3, 300 MHz) δ (ppm): 11.05 (br, 1H), 8.04 (s, 2H), 7.76 (d, J=7.2 Hz, 2H), 7.61 (d, 2H), 7.4-7.2 (m, 4H), 5.9-5.7 (m, 1H), 5.7-5.6 (m, 1H), 5.6-5.4 (m, 1H), 5.34 (s, 1H), 4.38 (d, 2H), 4.22 (t, J=6.9 Hz, 1H), 3.7-3.6 (m, 2H), 2.7-3.2 (m, 6H), 2.44 (m, 1H), 2.1-1.3 (m, 9H). 13C NMR (CD3OD, 125 MHz) δ (ppm): 175.81, 158.57, 158.34, 145.33, 145.14, 142.52, 132.91, 132.50, 128.76, 128.15, 128.13, 126.27, 126.25, 120.90, 74.94, 67.89, 55.25, 54.82, 49.51, 49.34, 49.17, 49.00, 48.83, 48.66, 48.49, 48.40, 41.63, 41.37, 36.99, 36.90, 36.72, 32.35, 31.64, 30.42, 30.04, 25.14, 24.16. HRMS (ESI) m/z calculated for [M-H]-: 519.24951, found 519.25027. The equatorial isomer of Nα-Fmoc-Nε-(((E)-cyclooct-2-enyl)oxycarbonyl)-ʟ-lysine (1e) was synthesized from equatorial isomer of (E)-cyclooct-2-enol in a similar fashion as a light yellow foamy thick oil. 1H NMR (CDCl3, 300 MHz) δ (ppm): 11.03 (br, 1H), 8.02 (s, 2H), 7.40 (d, J=7.5 Hz, 2H), 7.58 (s, 2H), 7.4-7.2 (m, 4H), 5.8-5.3 (m, 2H), 5.04 (s, 1H), 4.82 (s, 1H), 4.34 (s, 2H), 4.20 (t, 1H), 3.7-3.5 (m, 2H), 3.1-2.8 (m, 6H), 2.34 (s, 1H), 2.16 (s, 1H), 1.9-1.2 (m, 8H). 13C NMR (CD3OD, 125 MHz) δ (ppm): 175.84, 158.67, 158.57, 145.43, 145.28, 142.60, 133.79, 133.42, 128.97, 128.37, 128.36, 126.48, 121.13, 79.27, 68.07, 55.39, 55.11, 49.76, 49.59, 49.42, 49.25, 48.91, 48.74, 48.51, 43.89, 42.29, 41.50, 37.17, 36.90, 36.70, 36.42, 32.52, 31.90, 30.82, 30.57, 30.14, 28.54, 24.28. HRMS (ESI) m/z calculated for [M-H]-: 519.24951, found 519.24990. Synthesis of Nε-(((E)-cyclooct-2-enyl)oxycarbonyl)-ʟ-lysine (TCOK, 10)


S4

Axial isomer of Nα-Fmoc-Nε-(((E)-cyclooct-2-enyl)oxycarbonyl)-ʟ-lysine (4.1g, 79 mmol) was dissolved in dichloromethane (100 mL) and 24 mL piperidine was added. The solution was stirred for 1 h in the dark. The solvent and piperidine was removed under reduced pressure to give a yellow solid. The crude product was dissolved in methanol and reprecipitated by adding diethyl ether. The precipitate was filtered, washed with diethyl ether and dried to give axial isomer of Nε-(((E)-cyclooct-2-enyl)oxycarbonyl)-ʟ-lysine (10a) as a white powder. 1H NMR (CD3OD, 500 MHz) δ (ppm): 5.84 (t, J=14 Hz, 1H), 5.55 (d, J=16 Hz, 1H), 5.22 (s, 1H), 3.53 (m, 1H), 2.44 (m, 1H), 2.08-1.93 (m, 2H), 1.93-1.84 (m, 2H), 1.84-1.77 (m, 4H), 1.73-1.67 (m, 4H), 1.58-1.41 (m, 4H), 1.16 (m, 1H), 0.87 (m, 1H). 13C NMR (CD3OD, 125 MHz) δ (ppm): 174.50, 158.53, 132.97, 132.64, 75.08, 56.17, 45.77, 41.69, 41.31, 37.07, 36.78, 32.02, 30.66, 30.12, 25.23, 23.80, 23.61, 23.11. HRMS (ESI) m/z calculated for [M+H]+: 299.19653, found 299.19626. The equatorial isomer of Nε-(((E)-cyclooct-2-enyl)oxycarbonyl)-ʟ-lysine (10e) was synthesized from equatorial isomer of Nα-Fmoc-Nε-(((E)-cyclooct-2-enyl)oxycarbonyl)-ʟ-lysine in a similar fashion as a white power. 1H NMR (d6-DMSO, 500 MHz) δ (ppm): 7.02 (s, 1H), 5.70 (t, J=13, 1H), 5.51 (m, 1H), 4.97 (m, 1H), 3.18 (m, 1H), 2.34 (m, 1H), 2.10 (m, 1H), 1.92 (m, 1H), 1.78 (m, 1H), 1.73-1.58 (m, 9H), 1.44-1.20 (m, 4H), 0.89-0.74 (m, 2H). 13C NMR (d6-DMSO, 125 MHz) δ (ppm): 170.79, 155.68, 132.28, 132.21, 77.36, 53.89, 43.50, 40.92, 35.01, 34.80, 30.60, 29.13, 28.46, 26.82, 22.33, 22.14, 21.76. HRMS (ESI) m/z calculated for [M+H]+: 299.19653, found 299.19641. Synthesis of 1,2,4,5-tetrazine

We synthesized these compounds according to the reference1. Formamidine acetate (12 g, 0.115 mol) was dissolved in methanol (20 mL) and cooled at 0 °C. Hydrazine hydrate (12.0 g, 0.253 mmol) was added to the solution with stirring, and acetic acid (40 mL) was added dropwise. The mixture was stirred at room temperature for 0.5 h and cooled to 10 °C. NaNO2 (17 g, 0.25 mmol) was added and the mixture was stirred for 1 h at 10 °C and 1h at room temperature. NaHCO3 (25 g) was added and the mixture was stirred for another 1 h, filtered to give a red solution. The solution was extracted with dichloromethane (3×40 mL). The combined organic layer was dried over Na2SO4 and carefully concentrated. The resulting solution was purified by sublimation under reduced pressure to give 1,2,4,5-tetrazine as a red crystal (70 mg, 1.5%). 1H NMR (CDCl3, 300 MHz) δ (ppm): 10.43 (s, 2H). Synthesis of 3,6-dimethyl-1,2,4,5-tetrazine


S5

We synthesized these compounds according to the reference1. Hydrazine hydrate was added to a suspension of acetamidine hydrochloride (4.0 g, 42.0 mmol) in ethanol (20 mL) under an atmosphere of nitrogen. The mixture was stirred for 18 h to give a pink suspension. The precipitate was removed by filtration and the solution was diluted with water (60 mL). The solution was stirred vigorously in an erlenmeyer under an atmosphere of air. The solution was extracted with dichloromethane (30 mL), and the aqueous layer was subsequently stirred and extracted until it no longer turned red. The solvent in combined organic layer was mostly rotary vaporized, and was purified by column chromatography using diethyl ether as eluent to give 3,6-dimethyl-1,2,4,5-tetrazine as a dark purple crystal (532 mg, 23%). 1H NMR (CDCl3，300 MHz) δ (ppm): 3.03(s).


S6

Supplementary Results

Supplementary Table 1. Comparison of reactivity of 1a and 1e towards tetrazines 2–5 as measured by UPLC analysis. 1a/1e (100 µM) were treated with each of these tetrazines (5 equiv) in 20% acetonitrile/water solution for 3 h at 37 oC followed by UPLC-PDA/MS analysis (PDA = photo diode array).

Supplementary Figure 1. Potential mechanisms for inv-DA mediated elimination reaction. The amine containing elimination product S6 is generated from the reaction of the TCO carbamate caged S1 with tetrazines through the intermediates S3 and S4. Not all possible tautomer conversions and stereoisomers are shown.

Supplementary Figure 2. LC spectra of the reactions between 1a/1e and tetrazine 3. The reactions were analyzed by UPLC-PDA/MS (PDA = photo diode array) after 3 h. Shown are the LC spectra of PDA channel at 210 nm.


S7

Supplementary Figure 3. Immunoblotting analysis confirming incorporation of TCOK-a (10a) and TCOK-e (10e) into the GFP-N149-TAG template protein with a C-term His-tag. The yield of GFP containing either of the TCOK (10) isomers was essentially at the same level, indicating that the mutant MmPylRS (Y306A/Y384F, TCOKRS) recognizes these two isomers with a similar efficiency.

Supplementary Figure 4. ESI-MS analysis of GFP-N149-TCOK-e and its decaging products. TCOK-e was site-specifically incorporated at residue Asn 149 on GFP in order to generate GFP-N149-TCOK-e. The deconvoluted mass spectra of GFP-N149-TCOK (black) and their reaction products (red) upon addition of 3 (Me2Tet) are shown. GFP-N149-TCOK-e, expected mass 27,889 Da, found mass 27,889 Da (major), and 27,758 Da (minor). GFP-N149-K (the elimination product), expected mass 27,737 Da, found mass 27,737 Da (major), 27,760 Da (27,737 Da with Na+), and 27,606 Da (minor). The minor peak corresponds to the same protein (the major peak) with the N-terminal Met (–132 Da) posttranslationally cleaved.


S8

Supplementary Figure 5. ESI-MS spectra of the invDA-mediated elimination reaction on protein GFP-N149-TCOK-a/-e at indicated time points. The MS spectra indicated that both GFP-N149-TCOK-e (a) and GFP-N149-TCOK-a (b) were successfully decaged via inv-DA reaction. (Representative MS peaks in the figure were labeled in (b): GFP-N149-K/Met: 27606 Da, Peak 1; GFP-N149-K/Met+Na+: 27628 Da, Peak 2; GFP-N149-K: 27737 Da, Peak 3; GFP-N149-K+Na+: 27759 Da, Peak 4; GFP-N149-TCOK: 27889 Da, Peak 5; GFP-N149-TCOK+Na+: 27911Da, Peak 6; GFP-N149-TCOK+Me2Tet-N2: 27971 Da, Peak 7; GFP-N149-TCOK+Me2Tet-N2+Na+: 27993, Peak 8) (c) Equation for calculating the elimination yields on protein. The elimination yields on protein GFP-N149-TCOK in the main-text and Supplementary Fig. 7 were calculated from MS spectra (a,b) according to this equation.


S9

Supplementary Figure 6. Yield of elimination product GFP-N149-K from GFP-N149-TCOK-a (red) or GFP-N149-TCOK-e (black) at indicated time points. The reactions were quenched by TCOK-a (10a) followed by the LC-MS analysis. Error bars represent ± s.d. from three independent experiments.

Supplementary Figure 7. Incorporation of TCOK-a/-e into GFP within mammalian cells. The transfected HEK293T cells were incubated at 37 °C with (+) and without (-) 1 mM TCOK-a/-e (10a/e) in the cell culture media for 24 h before being visualized by a fluorescence microscope (a) and subsequently analyzed by immunoblotting assay (b). Scale bars, 100 µm.


S10

Supplementary Figure 8. The invDA-mediated elimination reaction on GFP inside living cells. The inv-DA mediated elimination reaction inside living cells was monitored through LC-MS-MS analysis of the four target peptides after trypsin digestion. The target peptides sequences and reaction sites are listed in Fig. 2d. LC spectra of GFP-Y40-TCOK-a (a) and GFP-Y40-TCOK-e (b) at different time points (labeled in purple) in the reaction are shown. The reaction yield of the final product GFP-Y40-K is labeled in each spectrum, which was calculated from LC-MS spectra (a,b) according to the equation(c).


S11


S12

Supplementary Figure 9. The MS-MS spectra of the identified target peptides shown in Fig.2b and Supplementary Fig. 8.


S13

Supplementary Figure 10. Cytotoxicity measurement of the tetrazine molecule Me2Tet. HEK293T cells were treated by tetrazine molecule (Me2Tet) at different concentrations for 2 h before being analyzed by MTS assay. Error bars represent ±1 s.d. from three independent experiments.

Supplementary Figure 11. InvDA-mediated activation of caged firefly luciferase (fLuc) in living cells. Schematic representation of generation and activation of chemical-caged fLuc (C-fLuc) through the invDA mediated elimination. The chemical-caged mutant K529TCOK-a of fLuc, generated by incorporating TCOK-a (10a) at residue K529, no longer adenylates luciferin with Mg-ATP, resulting in abandoned enzymatic activity, which thus accounts for the interruption of the H-bonds formed between ε-amine ion of K529 with luciferin and ATP. Addition of Me2Tet would regenerate free ε-amine group of K529 and restore fLuc’s activity in catalyzing the conversion of D-luciferin to the luminescent oxyluciferin in the presence of Mg-ATP.


S14

Supplementary Figure 12. InvDA-mediated activation of caged firefly luciferase (fLuc) in living cells. HEK293T cells expressing C-fLuc supplemented with or without 1 mM TCOK-a (10a) were mixed with 100 µM Me2Tet and analyzed by the chemiluminescent channel of ChemiDoc (BL) with bright field (BF) images taken as a control. Immunoblotting analysis was used to compare the amount of luciferase protein carrying a C-terminal Histag (anti-His) being used as well as the equal loading (anti-β-tubulin). Scale bars (white line), 1 mm.

Supplementary Figure 13. Me2Tet has no influence on the enzymatic activity of fLuc. HEK293T cells expressing fLuc-WT were mixed with or without 100 µM Me2Tet and analyzed by the chemiluminescent channel of ChemiDoc (BL) with bright field (BF) images taken as a control. Immunoblotting analysis was used to compare the amount of luciferase protein carrying a C-terminal Histag (anti-His) being used as well as the equal loading (anti-β-tubulin). Scale bars (white line), 1 mm.


S15

Supplementary Figure 14. Quantitative measurement of invDA-mediated activation of caged firefly luciferase (fLuc) in living cells. HEK293T cells expressing C-fLuc supplemented with or without 1 mM TCOK-a (10a) were treated with Me2Tet in different concentrations and analyzed with the microplate reader at different time points. Immunoblotting analysis was used to show the comparable amount of luciferase protein carrying a C-terminal Histag (anti-His) being used and equal loading (anti-β-tubulin). Error bars represent ± s.d. from three independent experiments.

Supplementary Figure 15. Uncut gel images in Fig. 3a. (luciferase protein carrying a C-terminal Histag ,anti-His)


S16

Supplementary Figure 16. 1H-NMR spectrum of TCOK-a (10a).

Supplementary Figure 17. 13C-NMR spectrum of TCOK-a (10a).


S17

Supplementary Figure 18. HRMS spectrum of TCOK-a (10a).

Supplementary Figure 19. 1H-NMR spectrum of TCOK-e (10e).


S18

Supplementary Figure 20. 13C-NMR spectrum of TCOK-e (10e).

Supplementary Figure 21. HRMS spectrum of TCOK-e (10e).

References

1. Sauer, J. et al. 1,2,4,5-Tetrazine: Synthesis and Reactivity in [4+2] Cycloadditions. Eur. J. Org. Chem. 1998, 2885-2896 (1998).

2. Royzen, M., Yap, G.P.A. & Fox, J.M. A Photochemical Synthesis of Functionalized trans-Cyclooctenes Driven by Metal Complexation. J. Am. Chem. Soc. 130, 3760-3761 (2008).

3. Nikić, I. et al. Minimal Tags for Rapid Dual-Color Live-Cell Labeling and Super-Resolution Microscopy. Angew. Chem. Int. Ed. 53, 2245-2249 (2014).


chen si accept cg 1408821869 3 - nature · the column was then washed with 500 ml dichloromethane...

Documents