Supporting Information. Copyright Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2008

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1 Supporting Information Copyright Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2008

2 Copyright Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2008 Supporting Information for Efficient N-Terminal Glycoconjugation of Proteins by the N-End Rule Lars Merkel, Henning S. G. Beckmann, Valentin Wittmann,* and Nediljko Budisa* Figure S1. Top: Deconvoluted ESI-MS spectra of Aha-ψ-b*. Bottom: Theoretical mass distribution of Aha-ψ-b*. The almost identically isotopic pattern indicates a high rate of Met? Aha substitution.

3 Figure S2. Coomassie stained 20 % gel after SDS-PAGE. (1) Marker (Fermantas Page- Ruler Prestained Protein Ladder SM0671), (2) ψ-b*, () Aha-ψ-b*, (4) Aha(GlcNAc)-ψ-b*, (5) Aha(ChiAc 2 )-ψ-b*. Slight shifts in migration height of glycoproteins are detected. Figure S. Thermal unfolding of ψ-b*, Aha-ψ-b*, Aha(GlcNAc)-ψ-b* and Aha(ChiAc 2 )-ψ-b*. The unfolding was monitored at 220 nm. The protein solutions were heated from 4 C to 95 C with a rate of 0 C h 1 in 110-QS Hellma quartz cells with an optical path length of 0.1 cm. T m values are in the range of 68 C (ψ-b*) and 64 C (Aha-ψ-b* and glycoconjugates of Aha-ψ-b*).

4 Figure S4. CD spectra (left) and fluorescence spectra (right) of ψ-b*, Aha-ψ-b*, and Aha- (GlcNAc)-ψ-b*. Fluorescence emission maxima were measured in the range nm upon excitation at 280 nm. The concentrations of the samples were 0.5 µm. CD spectroscopy was performed at 20 C. Protein samples (0.2 mg ml 1 ) diluted in 1 x PBS buffer were measured using quartz Hellma 110-QS cells with 0.1 cm optical path length. Further measurement parameters: speed 50 nm min 1, bandwidth 1 nm and response time 1 s. At least three scans were accumulated in the range nm. Table S1. Analytical data of ψ-b*, Aha-ψ-b* and glycoconjugated Aha-ψ-b* variants. Protein T m / C H / J mol 1 λ max / nm M calcd / Da M found / Da ψ-b* ± 0.18 Aha-ψ-b* ± 0.0 Aha(GlcNAc)-ψ-b* ± 0.05 Aha(ChiAc 2 )-ψ-b* 6.86 ± ± ± ± ±

5 Experimental Section General Methods: Analytical thin layer chromatography (TLC) was performed on Merck Silica Gel 60 F254 aluminum sheets. Compound spots were visualized by quenching of fluorescence and/or by charring after treatment with cerium reagent (5 g molybdatophosphoric acid, 2.5 g ceric sulphate tetrahydrate, 25 ml sulfuric acid and 225 ml water), ethanolic ninhydrin ( % w/v), and ethanolic sulfuric acid (15 % v/v), respectively. Flash column chromatography was performed on Macherey-Nagel Silica Gel 60 ( mm; mesh ASTM). 1 H and 1 C NMR spectra were recorded at room temperature on a Bruker Avance DRX 600 spectrometer. 1 H chemical shifts are referenced to residual protic solvent (CD D, δ H =.1; [D 6 ]- DMS, δ H = 2.50). 1 C chemical shifts are referenced to the solvent signal (CD D, δ C = 49.1; [D 6 ]DMS, δ C = 9.4). ESI-IT mass spectra were recorded on a Bruker Daltonics Esquire 000 plus instrument. MALDI-TF mass spectra were recorded on a Bruker Biflex III spectrometer in positive, linear mode with a delayed extraction MALDI source and a pulsed nitrogen laser (7 nm). Combustion elemental analyses were performed on an elementar vario EL analyzer. High resolution mass spectrometry of protein samples was performed on a micro- TF-LC mass spectrometer (Bruker Daltonics, Bremen, Germany). Samples were pre-separated by HPLC on a C4 column (Symmetry, Waters, Eschborn, Germany) using a gradient from 90 % A (0.05 % TFA in water) to 90 % B (0.05 % TFA in acetonitrile) within 20 min at a flow rate of 250 µl min 1. The absorbance was detected at 210 nm. Far-UV CD spectroscopy of protein samples was carried out at 20 C on a JASC J- 715 dichrograph. Protein samples (0.2 mg ml 1 ) diluted in 1X PBS buffer were measured using quartz Hellma 110-QS cells with 0.1 cm optical path length. Further measurement parameters: speed 50 nm min 1, bandwidth 1 nm, response time 1 s. At least three scans were accumulated in the range nm. Melting curves of ψ-b* and related variants were measured at a concentration of 0.2 mg ml -1 by monitoring the changes in residual ellipticity (i.e. unfolding) at 220 nm. The protein solutions were heated from 4 C to 95 C with a rate of 0 C h 1 in 110- QS Hellma quartz cells with an optical path length of 0.1 cm. These experiments were performed on a JASC J-715 dichrograph equipped with a Peltier-type FDCD

6 attachment (model PFD-50S/50L). At 95 C and after cooling back to 4 C CD spectra were measured again to analyze denaturation and renaturation. Fluorescence spectra of protein samples were measured on a luminescence spectrometer LS 50 B (PerkinElmer Life Sciences; Boston MA) with an excitations/emissions slit of 5 nm. The concentrations of the samples were 0.5 µm. The proteins were excited at 280 nm and the fluorescence was measured in a range of 00 to 500 nm at 20 C. L-Azidohomoalanine (Aha): Aha was synthesized as described before. [1] Propargyl 2-acetamido-,6-di--acetyl-2-deoxy-4--(2-acetamido-,4,6-tri-acetyl-2-deoxy-b-D-glucopyranosyl)-b-D-glucopyranoside (4) 4' ' 6' 4 1' 6 1 HNAc H BF Et 2 4' 6' ' 4 1' '' 2'' '' Chitobiose octaacetate [2] (2.1 g,.15 mmol) and propargyl alcohol (.75 ml, 6.0 mmol) were dissolved in 1,2-dichloroethane (20 ml) and trimethylsilyl triflate (625 µl,.46 mmol) was added. After stirring for 22 h at 50 C, the reaction mixture was neutralized with triethylamine and evaporated. After flash chromatography (CHCl /MeH 15:1) pure 2 was isolated in % yield. 1 H NMR ([D 6 ]DMS, 600 MHz): δ = 7.99 (d, J = 9.1 Hz, 1 H; NH ), 7.89 (d, J = 9.2 Hz, 1 H; NH), 5.11 (ψt, J = 9.9 Hz, 1 H; H- ), 4.97 (ψt, J = 9.5 Hz, 1 H; H-), 4.81 (ψt, J = 9.7 Hz, 1 H; H-4 ), 4.65 (d, J = 8.5 Hz, 1 H; H-1 ), 4.62 (d, J = 8.4 Hz, 1 H; H-1), (m, H; H-6a, H-6a, H-1a ), 4.17 (d, J = 16. Hz, 1 H; H-1b ), 4.07 (dd, J = 6.2 and 11.9 Hz, 1 H; H-6b),.89 (d, J = 11.9 Hz, 1 H; H-6b ),.8.79 (m, 1 H; H-5 ),.72 (ψt, J = 9.4 Hz, 1 H; H-4), (m, 1 H; H-2), (m, 2 H; H-2, H-5), (m, 1 H; H- ), 2.06 (s, H; C()CH ), 2.00 (s, H; C()CH ), 1.94 (s, H; C()CH ), 1.89 (s, H; C()CH ), 1.75 (s, H; C()CH ), 1.74 ppm (s, H; C()CH ); 1 C NMR ([D 6 ]DMS, MHz): δ = 170.0, 169.9, 169.5, 169., 169.1, (each C=), (C-1 ), 98.2 (C-1), 79.1 (C- ), 77.6 (C-2 ), 75.8 (C-4), 7. (C-), 72.2 (C- ), 71.9 (C-5), 70.4 (C-5 ), 68.1 (C-4 ), 62.2 (C-6), 61.5 (C-6 ), 55. (C-1 ), 5.6 (C-2 ), 5.1 (C-2), 22.6, 22.5, 20.6, 20., 20., 20.2 ppm (each CH ); MS (MALDI+): m/z

7 67.2 [M + H] +, [M + Na] +, [M + K] + ; elemental analysis calcd (%): C 51.78, H 5.99, N 4.16; found: C 51.40, H 6.01, N Propargyl 2-acetamido-2-deoxy-4--(2-acetamido-2-deoxy-b-D-glucopyranosyl)- b-d-glucopyranoside (2) 4' 6' ' 4 1' '' 2'' '' NaMe CH 2 Cl 2 MeH 4' H H ' 6' H 4 1' H 2 6 H 1 1'' 2'' '' Propargyl glycoside 4 (128 mg, 0.19 mmol) was dissolved in dry CH 2 Cl 2 /MeH (1:1, 2 ml) and a solution of NaMe in methanol (0.5 M, 8 µl, mmol) was added. After 17 h, the precipitate was dissolved by addition of water (0.5 ml) and the mixture was neutralized with ion exchange resin (DWEX 50 WX8, H + form), filtrated and evaporated to give 2 in quantitative yield. 1 H NMR ([D 6 ]DMS, 600 MHz): δ = 7.9 (d, 1 H, J = 9.2 Hz; NH ), 7.81 (d, 1 H, J = 8.6 Hz; NH), 4.41 (d, 1 H, J = 8.1 Hz; H-1), 4.4 (d, 1 H, J = 8.5 Hz; H-1 ), 4. (dd, 1H, J = 2.4 and 15.8 Hz; H-1a ), 4.2 (dd, 1 H, J = 2. and 15.8 Hz; H-1b ),.71 (d, 1 H, J = 10.2 Hz; H-6a ),.61 (d, 1 H, J = 11.8 Hz; H-6a),.5.2 (m, 8 H; H-2, H-2, H-, H-, H-4, H-6b, H-6b and ), (m, 2 H; H-5 and H-5 ),.02 (ψt, 1 H, J = 9.2 Hz; H-4 ), 1.81 (s, H; C()CH ), 1.79 ppm (s, H; C()CH ); 1 C NMR ([D 6 ]DMS, MHz): δ = (C=), (C=), (C-1 ), 98.8 (C-1), 81.2 (C-4), 79.6 (C- ), 77. (C-2 ), 76.9 (C-5 ), 75.0 (C-5), 7.9 (C- ), 72. (C-), 70.6 (C-4 ), 60.9 (C-5 ), 59.9 (C-5), 55. (C-2 ), 54.7 (C-1 ), 54.2 (C-2), 2.0 (CH ), 22.9 ppm (CH ); MS (ESI, MeH): m/z [M H] Propargyl 2-acetamido-2-deoxy-b-D-glucopyranoside (1) [] H H 1 H 1' 2' ' The title compound was synthesized from 2-acetamido-1,,4,6-tetra--acetyl-2-deoxy-α-D-glucopyranose as described for 2. 1 H NMR (CD D, 600 MHz): δ = 4.59 (d, 1 H, J = 8.44 Hz; H-1), 4.7 (dd, 1 H, J = 2.5 and 16.0 Hz; H-1a ), 4.4 (dd, 1 H, J = 2.5 and 16.0 Hz; H-1b ),.87 (dd, 1 H, J = 1.9 and 12.0 Hz; H-6a), (m, 2

8 H; H-2 and H-6b),.47 (dd, 1 H, J = 8.6 and 10.2 Hz; H-),.1.25 (m, 2 H; H-4 and H-5), 2.84 (t, 1H, J = 2.4 Hz; H- ), 1.97 ppm (s, H; C()CH ); 1 C NMR (CD D, MHz): δ = 17.9 (C=), (C-1), 80.1 (C- ), 78.1 (C-5), 76.2 (C- 2 ), 76.0 (C-), 72.1 (C-4), 62.8 (C-6), 57.1 (C-2), 56.5 (C-1 ), 2.0 ppm (CH ); MS (ESI+, MeH): m/z = [M + H] +, [M + Na] +, [M + K] + Aha incorporation and purification of y-b* and Aha-y-b*: The plasmid pqe80ψb* for the expression of ψ-b* and Aha-ψ-b* was transformed into the Met-auxotrophic Escherichia coli strain B84(DE) (F - ompt hsds D (r - D m - D ) gal dcm mete (DE)) (Novagen Merck Chemicals Ltd., Nottingham, UK) following standard protocols. For the expression of Met? Aha substituted ψ-b* and Aha-ψ-b* in inclusion bodies, we followed previously described protocols. [4] Briefly, cells were grown to mid-log phase (D ) in synthetic medium containing mm of Met. Met-starved cells were induced for protein expression (IPTG) with concomitant addition of either 50 mg L 1 Met or 100 mg L 1 of Aha. The inclusion body isolation and refolding protocol was identical for both variants. Cells were first harvested and lysed in lysis buffer (50 mm Tris-HCl ph 8, 1 mm PMSF) and lysozyme was added. After 0 min at 0 C the suspension was sonicated and subsequently spun down at g, 4 C for 40 min. The pellet with inclusion bodies was resuspended and homogenized in urea buffer (7.5 M urea, 50 mm Tris-HCl ph 8). Insoluble material was removed by centrifugation at g, 4 C for 0 min. The supernatant was dialyzed three times against 50 mm Tris-HCl ph 8 and 0.1 mm NaCl at 4 C. After another spin as described above, the supernatant containing the desired renatured ψ-b* variant and residual cellular proteins was loaded onto HiTrap Q-sepharose (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), washed with 50 mm Tris-HCl ph 8.0 and eluted with a gradient of 0.1 M NaCl in 50 mm Tris-HCl ph 8.0 to 1 M NaCl in 50 mm Tris-HCl ph 8.0. References [1] L. Merkel, Y. Cheburkin, B. Wiltschi, N. Budisa, ChemBioChem 2007, 8, [2] M. Spinola, R. W. Jeanloz, J. Biol. Chem. 1970, 245, [] B. Vauzeilles, B. Dausse, S. Palmier, J.-M. Beau, Tetrahedron Lett. 2001, 42, ; S. I. van Kasteren, H. B. Kramer, H. H. Jensen, S. J. Campbell, J. Kirkpatrick, N. J. ldham, D. C. Anthony, B. G. Davis, Nature 2007, 446, [4] A. J. Link, D. A. Tirrell, Methods 2005, 6,