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

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

2 Copyright Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2007 Supporting Information for Head-to-Tail Cyclized Cystine-Knot Peptides via a Combined Recombinant and Chemical Route of Synthesis Olga Avrutina, Hans-Ulrich Schmoldt, Dusica Gabrijelcic-Geiger, Alexander Wentzel, Holm Frauendorf, Christian P. Sommerhoff, Ulf Diederichsen and Harald Kolmar Reagents and solvents: All chemicals were of highest grade available and used as supplied. High performance liquid chromatography: HPLC was performed on a Pharmacia Äcta basic system (pump type P-900, variable wavelength detector, type UV-900) by using the following columns: Analytical HPLC: Preparative HPLC: YMC J sphere ODS H-80, RP C-18 ( mm, 4 µm, 8 nm) Phenomenex Synergi 4u Hydro-RP 80 Å ( mm, 4 µm, 8 nm) YMC J sphere ODS H-80, RP C-18 ( mm, 4 µm, 8 nm) Phenomenex Synergi 4u Hydro-RP 80 Å ( mm, 4 µm, 8 nm) - 1 -

3 HPLC runs were performed with the following eluents: A (0.1 % aq. TFA), B1 (90 % aq. acetonitrile, 0.1 % TFA), and B2 (100 % acetonitrile, 0.1 % TFA). Two gradients were used. Gradient 1: a) 10 % B2 (6 min), b) 10? 26 % B2 (6 min); c) 26? 30 % B2 (12 min); d) 30? 44 % B2 (6 min). Gradient 2: 10? 40 % B1 (30 min). For analysis and purification HPLC grade acetonitrile and millipore water were used. Flow rates were 1 ml/min for analytical (column mm), 5 ml/min for semi-preparative (column mm), and 10 ml/min for preparative purifications (column mm). UV detection was performed at 215 nm for fully deprotected peptides and at 280 nm for the hydrazone-containing peptides to detect hydrazone formation. 1. McoEeTI SDG homoserine lactone (4) O H-SDGGVCPKILKKCRRDSDCLAGCVCGPNGFCGV NH O C 139 H 224 N 44 O 45 S 6 [ ] Barnase -McoEeTI fusion protein was dissolved in 0.2 M HCl/8 M urea and 0.6 µl of 5 M BrCN solution was added. After overnight incubation the sample was diluted 1:10 with eluent A containing 5 % acetonitrile and subjected to a XK26 column (Amersham Biosciences) containing Amberchrom CG-300M (Tosoh Bioscience, 100 ml bed volume). After washing with 0.1% Vol. aqueous TFA, the cleaved microprotein was separated from the barnase'-carrier using a gradient from 5 to 50% eluent B1 within 30 min. McoEeTI containing fractions were combined and lyophilized. Additional purification was performed by the preparative HPLC to give 6 mg of 4. Analytical data: HPLC (Figure S1): R t = (gradient 1); MS (ESI): m/z: [M + 4 H] 4+, [M + H] + ; HRMS (ESI): calcd for C 139 H 224 N 44 O 45 S 6 : , , found [M + 4 H] 4+, [M 0 ]

4 Figure S1. Analytical HPLC of 4 at 215 nm. Column - Phenomenex Synergi 4u Hydro-RP 80 Å ( mm, 4 µm, 8 nm). 2. McoEeTI SDG hydrazide (6) H-SDGGVCPKILKKCRRDSDCLAGCVCGPNGFCGVHse-NHNH 2 C 139 H 228 N 46 O 45 S 6 [3456.2] Hydrazine hydrate (7 µl, 0.14 mmol) was added to McoEeTI SDG homoserine lactone 4 (4.7 mg, 1.4 µmol) solution in water (2 ml). The mixture was stirred for 1 h at room temperature. Reaction was monitored by analytical HPLC (Figure S2). The reaction mixture was lyophilized to remove the excess of hydrazine. The sample was redissolved in 10 % eluent B2 and purified by a preparative HPLC to yield 2.1 mg (44.3%) of 6. Peak at min corresponds to a peptide with C-terminal Hse. (MS: calcd ; found m/z [M + 4 H] 4+, [M + 3 H] 3+, [M + 3 H + TFA] 3+ ). Analytical data: HPLC (Figure S3): R t = (gradient 1); MS (ESI): m/z: [M + 4 H] 4+, [M + 3 H] 3+, [M + 3 H + TFA]

5 Figure S2. Monitoring of hydrazinolysis of 4 at 215 and 280 nm. Reaction progress after 1 h. Column - Phenomenex Synergi 4u Hydro-RP 80 Å ( mm, 4 µm, 8 nm). Figure S3. Analytical HPLC of 6 at 215 and 280 nm in phosphate buffer. Column - Phenomenex Synergi 4u Hydro-RP 80 Å ( mm, 4 µm, 8 nm)

6 3. cmcoeeti SDG (8) O DGGVCPKILKKCRRDSDCLAGCVCGPNGFCGVHse N NH C136H218N44O43S6 [3407.2] McoEeTI SDG hydrazide 6 (1.6 mg, 0.46 µmol) was dissolved in 1 ml 10 mm phosphate buffer ph 7.0. NaIO 4 (1 mg, 4.6 µmol) was added as a solution in phosphate buffer (1 ml). After 5 min at room temperature the reaction was terminated by HPLC injection. Monitoring was performed at 280 nm to detect macrocycle formation (Figure S4). Pure yield of 8 was 1 mg (63.7%). Analytical data: HPLC (Figure S5): R t = (gradient 2); MS (ESI): m/z: [M + 3 H] 3+, [M + 3 H + TFA] 3+, [M + 2 H] 2+. Figure S4. Monitoring of sodium periodate oxidation-cyclization of 6 at 280 nm in phosphate buffer. Reaction progress after 5 min. Column - Phenomenex Synergi 4u Hydro-RP 80 Å ( mm, 4 µm, 8 nm)

7 Figure S5. Analytical HPLC of 8 at 215 and 280 nm. Gradient 2. Column - YMC J sphere ODS H-80, RP C-18 ( mm, 4 µm, 8 nm). 4. McoEeTI KKV homoserine lactone (3) O H-SGVCPKILKKCRRDSDCLAGCVCGPNGFCGAKKV NH O C 148 H 246 N 46 O 42 S 6 [ ] McoEeTI KKV homoserine lactone was synthesized according to the method described in section 1. Additional purification was made by the preparative HPLC to give 8 mg of 3. Analytical data: HPLC (Figure S6): R t = min (gradient 2); MS (ESI): m/z: [M + 4 H] 4+, [M + H] + ; HRMS (ESI): calcd for C 148 H 246 N 46 O 42 S 6 : , ; found [M + 4 H] 4+, [M 0 ]

8 Figure S6. Analytical HPLC of 3 at 215 nm. Gradient 2. Column - YMC J sphere ODS H-80, RP C-18 ( mm, 4 µm, 8 nm). 5. McoEeTI KKV hydrazide (5) H-SGVCPKILKKCRRDSDCLAGCVCGPNGFCGAKKVHse-NHNH 2 C 148 H 250 N 48 O 42 S 6 [ ] Hydrazine hydrate (10 µl, 0.2 mmol) was added to McoEeTI KKV homoserine lactone 3 (7 mg, 2 µmol) solution in 2 ml 10 mm phosphate buffer (ph 6). The mixture was stirred for 1 h at room temperature. Reaction was controlled by analytical HPLC (Figure S7). After the starting lactone was completely consumed, the reaction mixture was lyophilized to remove the excess of hydrazi ne. The dry residue after lyophilization was re-dissolved in water-acetonitrile mixture and subjected to a preparative HPLC affording 4.5 mg (64.3%) of 5. Analytical data: HPLC (Figure S8) : R t = (gradient 2); MS (ESI): m/z: [M + 4 H] 4+, [M + 3 H] 3+, [M + H]

9 Figure S7. Monitoring of hydrazinolysis of 3 at 215 nm in water. Reaction progress after 1 h. Gradient 2. Column - YMC J sphere ODS H-80, RP C-18 ( mm, 8 nm). Figure S8. Analytical HPLC of 5 at 215 nm in phosphate buffer. Gradient 2. Column - YMC J sphere ODS H-80, RP C-18 ( mm, 4 µm, 8 nm)

10 6. McoEeTI KKV aldehyde (7) OHCCO-GVCPKILKKCRRDSDCLAGCVCGPNGFCGAKKVHse-NHNH 2 C 147 H 245 N 47 O 42 S 6 [ ] McoEeTI KKV hydrazide 5 (4 mg, 1.1 µmol) was dissolved in phosphate buffer 1 ml 10 mm phosphate buffer ph 7.0. NaIO 4 (2.4 mg, 11 µmol) was added as a solution in phosphate buffer (1 ml). After 5 min at room temperature the reaction was terminated by HPLC injection (Figure S9). Yield of 7 was 2.5 mg (62.8%). Analytical data: HPLC: R t = min (gradient 2); MS (ESI): m/z: [M + H] +. Figure S9. Monitoring of sodium periodate oxidation of 5 at 215 nm in phosphate buffer. Reaction progress after 5 min. Gradient 2. Column - YMC J sphere ODS H-80, RP C-18 ( mm, 8 nm)

11 7. cmcoeeti KKV (9) O GVCPKILKKCRRDSDCLAGCVCGPNGFCGAKKVHse N NH C 147 H 243 N 47 O 41 S 6 [ ] cmcoeeti KKV aldehyde 7 (2 mg, 0.56 µmol) was dissolved in 1 ml of 0.25 M sodium acetate buffer (ph = 5) and incubated at room temperature. The reaction progress was monitored by analytical HPLC at 215 and 280 nm (Figure S10). After 72 h the reaction mixture was subjected to preparative HPLC to yield 0.5 mg (25 %) of cmcoeeti KKV hydrazone (9). Analytical data: HPLC (Figure S11): R t = min (gradient 2); MS (ESI): m/z: [M + 5 H] 5+, [M + 4 H] 4+, [M + 3 H] 3+, [M + H] +. Figure S10. Monitoring of macrocyclization of 7 at 215 and 280 nm in sodium acetate buffer. Reaction progress after 72 h. Gradient 2. Column - YMC J sphere ODS H-80, RP C-18 ( mm, 8 nm)

12 Figure S11. Analytical HPLC of 9 at 215 nm. Gradient 2. Column - YMC J sphere ODS H-80, RP C-18 ( mm, 4 µm, 8 nm)