Supporting Information. Copyright Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2007

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1 Supporting Information Copyright Wiley-VC Verlag Gmb & Co. KGaA, Weinheim, 2007

2 Caged substrates for protein labeling and immobilization Sambashiva Banala, Anke Arnold, Kai Johnsson* École Polytechnique Féderale de Lausanne, Institute of Chemical Sciences and Engineering, C Lausanne, Switzerland. General Standard chemicals were purchased from Fluka, Aldrich, Acros or Roche. Flash column chromatography was performed on silica gel 60 (40-63 µm, Fluka). Mass spectra were recorded by electrospray ionization on a Finnigan MAT ACPI/ESI SSQ 710C spectrometer. MR spectra were recorded on a Bruker ARX-400 Mz spectrometer. Reverse phase PLC was accomplished on a waters 2790 separations module with a binary gradient from water/tfa and acetonitrile and the use of absorption detection at 280 nm and 350 nm. Synthesis of BG- 3 (1) To a solution of 14-azido-3,6,9,12-tetraoxatetradecan-1-oic acid [1] (3.0 g, 10.8 mmol) in anhydrous DMF (10 ml) under 2 atmosphere were added PyBP (8.6 g, 16.5 mmol, 1.5 eq) and Et 3 (4.5 ml, 32.4 mmol, 3 eq). After 30 min of stirring at room temperature, 6 -(4- aminomethyl-benzyl)guanine [2] (2.9 g, 10.8 mmol) was added and reaction was continued for overnight. Solvent was removed under reduced pressure, crude product was diluted with 100 ml of ethyl acetate and washed with water and brine. rganic layer was dried over MgS 4 concentrated in vacuo and the product was purified by flash column chromatography (Me/C 2 Cl 2 5/95). Yield: 4.0 g ( 7.55 mmol, 70%). 1 MR (400 Mz, DMS-d 6 ): δ (s, 1), 8.25 (t, J = 6.1 z, 1), 7.80 (s, 1), 7.45 (d, J = 7.9 z, 2), 7.29 (d, J = 7.9 z, 2), 6.29 (s, 2), 5.45 (s, 2), 4.32 (d, J = 6.1 z, 2), 3.95 (s, 2), (m, 14), 3.37 (t, J = 5.1 z, 2). 13 C MR (100.6 Mz, DMS-d 6 ): δ 41.5, 50.0, 66.6, 69.3, 69.6, 69.69, 69.74, 69.8, 69.78, 69.80, 69.82, 70.0, 70.4, 79.2, 127.3, 128.6, 135.3, 139.4, 159.7,

3 MS(ESI) m/z: [M+] + calcd for C , ; found, (1-bromoethyl)-2-nitrobenzene (2). Br 2 1-ethyl-2-nitrobenzene (10.0 g, 66.2 mmol) and BS (12.9 g, 72.8 mmol, 1.1 eq.) were dissolved in 35 ml of CCl mg of AIB were added and reaction was heated to 85 C for 6 h. Insoluble by-products were filtered off and the filtrate was concentrated in vacuo. Crude product was diluted with 200 ml of ethyl acetate and washed with water and brine. Solvent was removed under reduced pressure and purified by flash column chromatography. Yield: 13.6 g (59.5 mmol, 90%) as a brown liquid. R f = 0.47(AcEt/ n-hexane 1/10). 1 MR (400 Mz, CDCl 3 ): δ 7.88 (d, J = 8.0 z, 1), 7.82 (d, J = 8.0 z, 1), 7.64 (t, J = 8.0 z, 1), 7.43 (t, J = 8.0 z, 1), 5.80 (q, J = 6.7 z, 1), 2.08 (d, J = 6.7 z, 3). 13 C MR (100.6 Mz, CDCl 3 ): δ 147.6, 137.8, 133.5, 129.9, 129, 124.4, 41.9, PE-BG- 3 and 9 PE-BG- 3 (3 and 4 respectively) PE-BG PEE-BG- 3 4 To a solution of BG- 3 1 (200 mg, mmol) in anhydrous DMF (1.5 ml) under 2 atm was added K 2 C 3 (164 mg, 1.18 mmol, 3 eq.), after 5 min of stirring 1-(1-bromoethyl)-2-nitrobenzene 2 (104 mg, mmol, 1.2 eq) was added. The reaction was continued for overnight at room temperature. Solvent was removed under reduced pressure and crude product was diluted with 50 ml of water and extracted with ethyl acetate (2x60 ml). rganic layer was washed with brine, dried over MgS 4 and concentrated in vacuo. Product was purified by flash column chromatography using a stepwise gradient of methanol in dichloromethane (1/50 to 1/10). Yield : 2

4 7 derivative 50 mg (28%, R f = 0.47), 9 derivative 130 mg (72%, R f = 0.52). Me/C 2 Cl 2 1/10 ). verall yield : 70%. (R f values in 7 deriivative (3) 1 MR (400 Mz, CDCl 3 ) : δ 8.16 (s, 1, -8), 7.89 (dd, J = 8.0, 1.3 z, 1), (m, 2), 7.37 (dt, J = 8.0, 1.3 z, 1), 7.18 (d, J = 8.0 z, 2), 7.07 (d, J = 8.0 z, 2), 6.80 (dd, J = 8.0, 1.3 z, 1), 6.42 (q, J = 7.0 z, 1), 5.30 (d, J = 12.2 z, 1), 5.18 (d, J = 12.2 z, 1), 4.82 (s, 2), 4.46 (d, J = 6.1 z, 2), 4.06 (s, 2), (m, 2), (m, 4), (m, 12), 3.34 (t, J = 5.1 z, 2), 2.0 (d, J = 7.0 z, 2). 13 C MR (100.6 Mz, CDCl 3 ) δ 170.2, 163.7, 159.3, 157.2, 147.4, 142.3, 138.3, 137.8, 134.8, 134.3, 128.8, 128.7, 127.8, 126.7, 125.2, 107.1, 71.1, 70.7, 70.1, 70.51, 70.50, 70.3, 70.0, 68.0, 67.5, 53.5, 50.7, 42.5, MS(ESI) m/z: [M+] + calcd for C , ; found, derivative (4) 1 MR (400 Mz, CDCl 3 ) : δ 7.95 (dd, J = 8.0, 1 z, 1), 7.84 (s, 1, -8), 7.51 (dt, J = 8.0, 1 z, 1), (m, 4), 7.27 (d, J = 8.0 z, 1), 7.21 (d, J = 8.0 z, 1), 6.31 (q, J = 7.0 z, 1), 5.51 (d, J = 12.8 z, 1), 5.47 (d, J = 12.8 z, 1), 4.78 (s, 2), 4.46 (d, J = 6.1 z, 2), 4.04 (s, 2), 3.74 (t, J = 6.1 z, 1), (m, 2), (m, 12), 3.33 (t, J = 5.1 z, 2), 2.01 (d, J = 7.0 z, 2). 13 C MR (100.6 Mz, CDCl 3 ) δ 170.2, 160.9, 159.2, 154.1, 148.5, 138.2, 137.2, 136.5, 135.7, 133.9, 128.9, 128.7, 127.9, 127.7, 125.1, 116.3, 115.9, 71.2, 70.3, 70.7, 70.6, 70.5, 70.3, 70.1, 67.8, 50.7, 40.6, 42.6, MS(ESI) m/z: [M+] + calcd for C , ; found, Differentiation between 7 and 9 substituted guanine derivatives Two model compounds (M 1, M 2) were synthesized for differentiating between the two isomers by MR studies. Synthetic procedure was same as above PE-BG M 1 9 PE-BG M 2 3

5 7 PE-BG M 1 1 MR (400 Mz, CDCl 3 ): δ 8.18 (s, 1, -8), 7.89 (d, J = 8.0 z, 1), 7.45 (t, J = 8.0 z, 1), 7.35 (t, J = 8.0 z, 1), (m, 3), 7.11 (d, J = 8.0 z, 2), 6.80 (d, J = 8.0 z,1), 6.44 (q, J = 6.7 z, 1), 5.32 (d, J = 12.2 z, 1, -C 2 -Ph), 5.22 (d, J = 12.2 z, 1, -C 2 -Ph), 4.96 (s,2, 2 -guanine), 2.01 (d, J = 6.7 z, 3). 9 PE-BG M 2 1 MR (400 Mz, CDCl 3 ): δ 7.95 (d, J = 8.0 z, 1), 7.85 (s, 1, -8), (m, 3), 7.40 (t, J = 8.0 z, 1), (m, 3), 7.22 (d, J = 8.0 z, 1), 6.32 (q, J = 6.7 z, 1), 5.52 (d, J = 12.2 z, 1, -C 2 -Ph), 5.48 (d, J = 12.2 z, 1, -C 2 -Ph), 4.78 (s, 2, 2 -guanine), 2.02 (d, J = 6.7 z, 3). Characterization 1. The E spectrum of 7 caged derivative shows cross peaks between benzylic protons and proton of nitro phenyl derivative. These cross peaks are not observed in 9 caged derivative characteristic E 2 2. The chemical shift values of 1-8, which for the 7 isomer appears (δ 8.18) downfield of that 9 isomer (δ 7.85). [3] 1D and ESY spectrums of both compounds are attached at end of the experimental part. General procedure to reduce azides to amines: The benzylguanine azide derivatives (0.2 mmol) were dissolved in a solvent mixture consisting of TF (1 ml) and water (50 µl). To this solution triphenylphospine (0.6 mmol) was added and stirred for overnight at room temperature. Solvents were removed in vacuo and the product was purified by flash column chromatography (gradient methanol/dichloromethane from 1:20 to 1:5, 1% TEA) to obtain pure amines in 65-80% yields. 4

6 7 PE-BG- 2 (5) : 1 MR (400 Mz, CDCl 3 ): δ 8.19 (s, 1), 7.90 (dd, J = 8.0, 1.6 z, 1), 7.67 (t, J = 6.4 z, 1), 7.47 (dt, J = 7.7, 1.6 z, 1), 7.39 (dt, J = 7.7, 1.6 z, 1), 7.19 (d, J = 8.0 z, 2), 7.06 (d, J = 8.0 z, 2), 6.83 (dd, J = 8.0, 1.6 z, 1), 6.40 (q, J = 7.0 z, 1), 5.30 (d, J = 12.2 z, 1), 5.19 (d, J = 12.2 z, 1), 5.07 (s, 2), 4.45 (d, J = 6.1 z, 2), 4.08 (s, 2), (m, 4), (m, 10), 2.90 (t, J = 5.4, 1), 2.02 (d, J = 7.0 z, 3). RMS (ESI) m/z: [M+] + calcd for C , ; found, PE-BG- 2 (6) : 1 MR (400 Mz, CDCl 3 ): δ 7.98 (d, J = 8.0, 1.6 z, 1), 7.86 (s, 1), 7.59 (t, J = 6.1 z, 1), 7.53 (dt, J = 7.7, 1.3 z, 1), (m, 3), (m, 3), 7.26 (dd, J = 7.7, 1.3 z, 1), 6.33 (q, J = 7.0 z, 1), 5.53 (d, J = 12.5 z, 1), 5.49 (d, J = 12.5 z, 1), 4.84 (br s, 2), 4.49 (d, J = 6.1 z, 2), 4.06 (s, 2), (m, 4), (m, 8), 3.46 (t, J = 5.4 z, 2), 2.82 (t, J = 5.4 z, 2), 2.04 (d, J = 7.0 z, 3), 2.02 (br s, 2). MALDI (TF): [M+] + calcd for C , ; found, General procedure to attach fluorescein to amine group: The corresponding benzylguanine amine derivative (6 mg, 9.2 µmol) were dissolved in anhydrous DMF (0.5 ml). Then 5(6)- carboxyfluorescein -hydroxysuccinimide ester (3 mg, 6.3 µmol) was added followed by triethylamine (1.9 µl, 13.8 µmol). Reaction was continued overnight at room temperature. Product was purified by PLC with a linear gradient of solvent 2 (0.1% TFA)/C 3 C (0.08%TFA) 80/20 to 20/80. The solution was adjusted to p 7 and the solvents were removed in vacuo. Yield : 65-80%. 7 PE-BG-FL (7): RMS (ESI) m/z: [M+] + calcd for C , , found, PE-BG-FL (8): RMS (ESI) m/z: [M+] + calcd for C , , found, PE-BG-Dig (9) and 9 PE-BG-Dig (10). Digoxigenin-3--methyl carbonyl-ε-aminocaproic acid -hydroxysuccinimide ester (5 mg, 7.6 μmol) were dissolved in 0.5 ml anhydrous DMF. 7 or 9 PE-BG-PEG- 2 (10 mg, 15.3 μmol) and Et 3 (30 μl, 21.4 μmol) were added and stirred for overnight at room temperature. The reaction was quenched by adding 0.1 ml of water. Product was purified by PLC with a linear gradient of solvent 2 (0.1% TFA)/ C 3 C (0.08%TFA) 95/5 to 30/70. Solvents were removed in vacuo. Yield: mg (45-65%). 5

7 7 PE-BG-Dig (9): MS(ESI) m/z: [M] + calcd for C , ; found, PE-BG-Dig (10): MS(ESI) m/z : [M] + calcd for C , ; found, Caged CoDi (11) PE-BG-PEG. 3 3 (9.6 mg, 14.3 µmol) and BG-alkyne 15 (5 mg, 14.3 µmol) were suspended in a solvent mixture consisting of 2-propanol (50 µl) and water (400 µl) and sonicated for 5 min. To this added 2.85 µmol (20 mol%) of sodium ascorbate (11.2 μlfrom a stock of 50 mg/ml in water) and 1.43 µmol (10 mol%) of CuS (14.2 µl from a stock of 25 mg/ml in water) were added. Reaction was continued for 1 d at room temperature. Product was purified by PLC with a linear gradient of solvent 2 (0.1% TFA)/C 3 C (0.08%TFA) 80/20 to 20/80. The solution was adjusted to p 7 and the solvents were removed in vacuo to yield 11.7 mg (11.4 µmol, 80%) of product. 1 MR (400 Mz, DMS-d 6 ) δ 8.54 (s, 1), 8.49 (t, J = 6.0 z, 1), 8.30 (t, J = 6.0 z, 1), 7.96 (d, J = 7.9 z, 1), 7.74 (s, 1), 7.62 (t, J = 7.9 z, 1), 7.51 (t, J = 7.9 z, 1), 7.42 (d, J = 7.9 z, 3), 7.20 (d, J = 7.9 z, 2), 7.11 (d, J = 7.9 z, 2), 7.05 (m, 2), 6.70 (br s, 2), 6.25 (br s, 1), 6.18 (q, J = 7.0 z, 1), 6.14 (s, 2), 5.45 (s, 2), 5.25 (d, J = 12.1 z, 1), 5.13 (d, J = 12.1 z, 1), 4.41 (t, J = 5.1 z, 2), 3.94 (s, 2), 3.72 (t, J = 5.1 z, 2), 3.5 (m, 12), 3.31 (s, 1), 2.85 (t, J = 7.6 z, 2), 1.95 (d, J = 7.0 z, 3). 13 C MR (100.6 Mz, DMS-d 6 ) : δ 172.7, 172.1, 170.3, , 160.4, 159.9, 159.5, 159.2, 158.9, 156.8, 148.2, 146.7, 144.5, 141.6, 139.8, 138.2, 135.6, 135.1, 129.8, 129.7, 128.9, 128.0, 127.9, 125.6, 123.1, 122.5, 119.5, 116.6, 113.6, 106.4, 71.1, 70.7, 70.5, 70.3, 70.2, 69.6, 67.3, 66.9, 53.5, 52.3, 50.0, 49.4, 42.7, 42.2, 35.6, 29.8, 23.2, 22.1, MS(ESI) m/z: [M+] + calcd for C , ; found

8 BG derivatives R 2 BG--R BG-FL 12 (R = Fluorescein) BG-Dig 13 (R = Digoxigenin) BG (R = ) Synthetic procedures for the preparation of these compounds were the same as for caged derivatives. BG-FL (12) : RMS (ESI) m/z: [M+] + calcd for C , ; found, BG- 2 (14) : RMS(ESI) m/z : [M+] + calcd for C , ; found, MR (400 Mz, DMS-d6) : δ 8.33 (t, J = 5.7 z, 1), 7.83 (s, 1), 7.46 (d, J = 7.6 z, 2), 7.29 (d, J = 7.6 z, 2), 6.28 (br s, 2), 5.46 (s, 2), 5.32 (s, 2), 4.32 (d, J = 6.0 z, 2), 3.96 (s, 2), (m, 14), 2.81 (t, J = 5.4, 2). BG-alkyne (15). 2 To a solution of 4-pentynoic acid (0.42 g, 4.3 mmol) and Et 3 (1.5 ml, 10.8 mmol) in anhydrous DMF (10 ml) under nitrogen atmosphere was added PyBP (3.37 g, 6.5 mmol). After 30 min of stirring BG- 2 (1.0 g, 3.9 mmol) was added and stirred for 3 h. Solvent was removed under reduced pressure, crude product is dissolved in C 2 Cl 2 (50 ml) and washed with aq. ac 3 (p = 10) and brine solution. rganic phase is dried over MgS 4, concentrated in vacuo and the product was purified by flash column chromatography using a stepwise gradient of methanol in 7

9 dichloromethane (1/50 to 1/10). Yield: 0.8 g (2.28 mmol, 62%). R f : 0.51 (methanol/dichloromethane 15/85). 1 MR (400 Mz, DMS-d 6 ): δ 8.41 ( t, J = 5.7 z, 1 ), 7.81 (d, J = 8.3 z, 1, 8), 7.56 (d, J = 8.3 z, 1), 7.45 (d, J = 7.9 z, 2), 7.29 (d, J = 7.9 z, 2), 6.29 (s, 2), 5.45 (s, 2), 4.29 (d, J = 5.7 z, 2), 2.79 (t, J = 2.5 z, 1), 2.39 (m, 2), 2.35 (m, 2). 13 C MR (100.6 Mz, DMS-d 6 ): δ 170.3, 159.7, 142.9, 132.3, 135.3, 128.6, 127.3, 124.9, 123.5, 118.6, 83.8, 71.4, 66.6, 41.9, 34.2, MS(ESI) m/z: [M+] + calcd for C , ; found, Photolysis study of compounds 3 and µm stock solutions of 3 and 4 were prepared in water (with 1% DMS as a co solvent) and samples were irradiated with a spectral line of nm (major peak is 365 nm from 100W g lamp). Photo conversion was analyzed by PLC. Both samples showed 90% conversion after 20 sec and complete conversion after 40 sec of irradiation. Product formation in the illuminated samples was also confirmed by mass spectrometry and MR analysis (in which case uncaging was performed in CD 3 D). Light-induced dimerization of SAP-ACP with caged CoDi (11): To follow the kinetics of dimerization of SAP-ACP by caged CoDi 11, the fusion protein was first labeled with Cy3 via ACP using previously published procedures. [4] Briefly, SAP-ACP (1 µm) was incubated for 30 min with 2 µm of CoA-Cy3, 1.5 µm of phospopantetheine transferase AcpS and 10 mm of MgCl 2 in epes buffer (100mM p 7.2) containing 1 mm DTT. Then, caged CoDi 11 was added with and without prior illumination (365 nm, 1min) to a final concentration of 0.70 µm. Aliquots were taken at different time points and the reaction was quenched by addition of SDS sample buffer and heating the sample to 95 C for 5 min. Samples were analyzed by SDS-PAGE and laserbased scanning (Pharos SX; BioRad). Preparation of BG-covered glass slides and protein immobilization: The coupling of BG to PEGMA-covered glass slides was performed as described previously. [5] Briefly, PEGMA polymer brushes on glass slides were activated by application of a freshly prepared mixture of p- nitrophenyl chloroformate (PC, 282 mg, 1.4 mmol) and triethylamine (140 mg, 1.4 mmol) in anhydrous TF for one hour at room temperature. Excess PC was removed from the surfaces through extensive washing with anhydrous TF and the slides were subsequently dried under a flow of nitrogen. ext, 100 mm of BG (positive control), benzyl amine (negative control) and 7 PE-BG- 2 5 were spotted on the PC-activated PEGMA slides by microarray robot (Qarray-mini, Genetix, UK) at ambient temperature. A humidity-controlled chamber was used to 8

10 maintain humidity at 90%. After spotting, the chips were incubated for an hour. The slides were immersed in ethanolamine (1M) for 50 min then washed extensively with deionized water for times. A part of the chip was covered with a mask (black paper, one side coated with aluminum), and the chip was illuminated for 20 min (365 nm, 100 W g lamp). The chip was then incubated with 5 µm of Cy5-labeled SAP-ACP for 1 h (Cy5- labeling of SAP-ACP was achieved by pre-incubation of SAP-ACP (5 µm) with CoA-Cy5 (10 µm), AcpS (1 µm), and MgCl 2 (1 mm) for 30 min). Subsequently, the chip was washed three times (20 min each) with PBS buffer. Finally, the chip was dried and analyzed with a fluorescence laser array scanner (Genepix 4000B) at excitation wavelength 635 nm. References [1] A. W. Schwabacher, J. W. Lane, K. Leigh, C. W. Johnson, J. rg. Chem 1998, 63, [2] A. Keppler, M. Kindermann, S. Gendreizig,. Pick,. Vogel, K. Johnsson, Methods 2004, 32, 437. [3] W. McComas, L. Chen, K. K, Tetrahedron Lett 2000, 41, [4] G. Lemercier, S. Gendreizig, M. Kindermann, K. Johnsson, Angew. Chem. Int. Ed. 2007, 46, [5] S. Tugulu, A. Arnold, I. Sielaff, K. Johnsson,. A. Klok, Biomacromolecules 2005, 6,

11 7 PE-BG M (t) 7.89 (d) 7.45 (t) 6.80 (d) , 5.22 (d) 2 characteristic E 10

12 9 PE-BG M , 5.48(d) (d) (t) 7.95 (d) 7.40 (t) 11