Supporting Information. for. Angew. Chem. Int. Ed Wiley-VCH 2004

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1 Supporting Information for Angew. Chem. Int. Ed Wiley-VCH Weinheim, Germany

2 Isolation, Crystal and Solution Structure, and Biosynthesis of Tubulysins - Powerful Inhibitors of Tubulin Polymerisation from Myxobacteria** Heinrich Steinmetz, Nicole Glaser, Eberhardt Herdtweck, Florenz Sasse, Hans Reichenbach, and Gerhard Höfle * [*] Prof. Dr. G. Höfle, Ing. H. Steinmetz, Dr. N. Glaser, Dr. F. Sasse Bereich Naturstoffe, Gesellschaft für Biotechnologische Forschung mbh Mascheroder Weg 1, Braunschweig gho@gbf.de Dr. E. Herdtweck Institut für Anorganische Chemie Technische Universität München General Experimental Procedures. Optical rotations were determined on a Perkin-Elmer 241 instrument. UV spectra were recorded on a Shimadzu UV-2102 PC scanning spectrometer. IR spectra were measured with a Nicolet 20DXB FT-IR spectrometer. NMR spectra were recorded in DMSO-d 6 on a Bruker DMX-600 spectrometer. For structural analogs only a selection of significant NMR data are given. EI mass spectra were obtained on a Finnigan MAT 95 spectrometer, high resolution data were aquired using peak matching (M/DM = 10000). ESI mass spectra were obtained on a QTOF 2 mass spectrometer (Micromass, Manchester). Pure compounds were characterized by analytical HPLC on Nucleosil C 18 (column 125 x 2 mm, 5µm, flow 0.3 ml/min), acetonitrile/10 mm ammonium acetate buffer, ph 5.5, gradient 30:70 to 95:5 in 20 min, diode array and ESI-MS detection. Preparative HPLC on Nucleosil (column 250 x 21 mm, 7 µm, flow 16 ml/min),solvent: acetonitril/ 50mM ammonium acetate buffer, ph 6.5, isocratic 35:65, detection: UV absorption at 227 nm. Sephadex LH20 column, h = 80 cm, = 18 cm, solvent: methanol, flow: 40 ml/min, detection: UV absorption at 254 nm. Analytical TLC (TLC aluminium 1

3 sheets silica gel Si 60 F 254 (Merck), solvent: dichloromethan/acetone/methanol 70:20:10, detection: UV absorption at 254 nm, dark blue (1-3, 7, 9), faint blue (4-6, 8) spots after spraying with Cer (IV) ammonium sulfate reagent and heating to 120 C. Tubulysin B (2): colorless amorphous solid; t R 12.6 min; R f 0.39; [α] 22 D 11.3 (c 5.0, MeOH); UV (MeOH) λ max nm (lgε) 205 (4.43), 225 (4.23), 250 (3.91), 280 (3.26); IR (KBr) ν max 3421, 2964, 2935, 2878, 1742, 1667, 1550, 1517, 1235 cm -1 ; 1 H NMR (DMSO) identical to tubulysin A, except for Tuv: δ 2.36 (1H, m, H-6b), 2.18 (1H, m, H-2 b), 2.15 (1H, m, H-2 a), 2.08 (1H, m, H-6a), 1.50 (1H, m, H-3 b), 1.48 (1H, m, H-3 a), 0.82 (3H, t, 7.0 Hz, H-4 ); 13 C NMR (DMSO) identical to tubulysin A, except for Tuv: 35.5 (t, C-2 ), 17.6 (t, C-3 ), 10.7 (q, C-4 ); DCI-MS m / z [M+H + ] 830 (40), 742 (41), 504 (8), 421 (17), 239 (60) 89 (100); HRDCI- MS m / z (M+H + ) (calcd. for C 42 H 64 N 5 O 10 S, ). Tubulysin C (3): colorless amorphous solid; t R 11.6 min; R f 0.37; [α] 22 D 12.2 (c 1.04, MeOH); UV (MeOH) λ max nm (lgε) 225 (4.17), 250 (3.92); IR (KBr) ν max 3388, 2963, 2935, 2875, 1743, 1667, 1544, 1226 cm -1 ; 1 H NMR (DMSO) identical to tubulysin A, except for Tuv: δ 2.35 (1H, m, H-6b), 2.29 (1H, m, H-2 b) 2.27 (1H, m, H-2 a), 2.16 (1H, m, H-6a); 13 C NMR (DMSO) identical to tubulysin A, except for Tuv: δ (s, C-1 ), 28.0 (t, C-2 ), 8.6 (q, C-3 ); DCI-MS m / z [M+H + ] 816 (23), 742 (24), 239 (100), 225 (20); HRDCI-MS m / z (M+H + ) (calcd. for C 41 H 62 N 5 O 10 S, ). Tubulysin D (4): colorless amorphous solid; t R 17.5 min; R f 0.52; [α] 22 D 12.8 (c 4, MeOH); UV (MeOH) λ max nm (lgε) 204 (4.57), 232 (4.13); IR (KBr) ν max 3394, 2963, 2936, 2875, 1741, 1669, 1541 cm -1 ; 1 H NMR (DMSO) identical to tubulysin A (1), except for Tup: δ 7.81 d, 9.0 Hz, 4-NH; 7.2 m, 7-H 2, 8-H 2, 9-H; 13 C NMR (DMSO) identical to tubulysin A, except for Tup: δ (C-6); (C-7); (C-8); (C-9); ESI-MS m / z [M+H + ] 828 (100); EI-MS m / z [M + ] 827 (3); 736 (100); 725 (6); HREI-MS m / z (M-C 7 H 7 ) (calcd. for C 36 H 58 N 5 O 9 S, ). Tubulysin E (5): colorless amorphous solid; t R 16.4 min; R f 0.50; [α] 22 D 12.6 (c 5, MeOH); UV (MeOH) identical to tubulysin D; 1 H NMR (DMSO) identical to tubulysin D (4), except for Tuv: δ 2.27 (m, 2 -H 2 ); 1.60 (q, t, J = 7 Hz, 3 -H 2 ); 0.90 (t, J = 7 Hz, 4 -H 3 ); EI-MS m / z [M+H + ] 814; HRDCI-MS m / z (M+H + ) (calcd for C 42 H 64 N 5 O 9 S, ). 2

4 Tubulysin F (6): colorless amorphous solid; t R 15.3 min; R f 0.47; UV (MeOH) identical to tubulysin D; 1 H NMR (CD 3 OD) Tuv: δ 2.32 (m, 2 -H 2 ), 1.08 (t, J = 7Hz, 3 -H 3 ); ESI-MS m / z [M+H + ] 800 (100); HRDCI-MS m / z (M+H + ) (calcd. for C 42 H 64 N 5 O 9 S, ). Tubulysin G (7): colorless amorphous solid; t R 13.0 min; R f 0.35; [α] 22 D 20.7 (c 7, MeOH); UV (MeOH) λ max (lgε) 204 (4.51), 223 (4.41), 250 sh (3.89), 275 (3.12), 285 nm (2.94); IR (KBr) ν max 3384, 2964, 2935, 1664, 1545, 1226 cm -1 ; 1 H NMR (DMSO) identical to tubulysin A, except for Tuv: δ 6.32 (1H, d, J = 12.1 Hz, H-11b), 5.73 (1H, dd, H-5), 5.55 (1H, s, H-2 ), 5.22 (1H, d, J = 12.0 Hz, H-11a), 4.40 (1H, m, H-7), 2.37 (1H, m, H-6b), 2.10 (1H, m, H-6a), 2.05 (3H, s, H-4 ), 1.84 (3H, s, H-5 ), 1.80 (1H, m, H-8), 0.97 (3H, d, H-9), 0.67 (3H, d, H- 10); 13 C NMR (DMSO) identical to tubulysin A, except for Tuv: δ (s, C-1 ), (s, C-3 ), (d, C-2 ), 68.1 (t, C-11), 27.0 (q, C-4 ), 19.9 (q, C-5 ); DCI-MS m / z [M+H + ] 842 (10), 742 (9), 256 (4), 239 (100), 135 (24), 118 (30); HRDCI-MS m / z (M+H + ) (calcd. for C 43 H 64 N 5 O 10 S, ). Tubulysin H (8): colorless amorphous solid; t R 13.9 min.; R f 0.47; UV (MeOH) identical to tubulysin D (4); 1 H NMR (CD 3 OD) Tuv: δ 2.03 (s, 2 -H 3 ); ESI-MS m / z [M+H + ] 786 (100); HRDCI-MS m / z (M+H + ) (calcd. for C 40 H 60 N 5 O 9 S, ). Tubulysin I (9): colorless amorphous solid; t R 10.4 min.; R f 0.23; [α] 22 D 6.7 (c 0.74 MeOH); UV (MeOH) λ max nm (lgε) 226 (4.07), 249 (3.91); IR (KBr) ν max 3388, 2964, 2936, 1745, 1665, 1229 cm -1 ; 1 H NMR (DMSO) identical to tubulysin A, except for Tuv: δ 6.19 (1H, d, J = 11.8 Hz, H-11b), 5.28 (1H, d, J = 12.1 Hz, H-11a), 2.10 (3H, s, H-5OAc), 2.0 (3H, s, 2 - H 3 ); 13 C NMR (DMSO) identical to tubulysin A, except for Tuv: δ (s, C-1 ), 20.5 (C- 2 ); DCI-MS m / z [M+H + ] 802 (20), 742 (14), 256 (4), 239 (100), 94 (4); HRDCI-MS m / z (M+H + ) (calcd. for C 40 H 60 N 5 O 10 S, ). 13 C-Labeling of tubulysin A (1) and B (2): Two 10-L cultures of strain Ar 315 were grown for 4 6 days in presence of 100 ml Amberlite XAD-16, 1.0 g sodium [ 13 C 2 ] acetate (96% 13 C) and 0.2 g L-[ 13 CH 3 ]methionine (96 % 13 C) were added to individual cultures and cultivation continued for 16 h. The adsorber resin was collected on a sieve, washed with water, transferred to an open column and eluted with 0.6 L of acetone. After evaporation of the acetone the aqueous phase was extracted with ethyl acetate to give 0.56 g and 0.55 g crude extracts, respectively. Chromatography on Sephadex LH-20 and silica gel RP-18 as described 3

5 above yielded acetate-labeled tubulysin A (1a) (13 mg) and tubulysin B (2a) (7 mg) as well as methionin-labeled tubulysin A (1b) (4 mg) and tubulysin B (2b) (3 mg). Table C NMR data for enriched carbon atoms of tubulysin 1a and 1b (DMSO, 80 C, 150 MHz). Compound Atom (ppm) Multipl. Rel. intensitiy [1] J cc (Hz) 1a Tuv C d C d OAc d OAc 20.7 d Tut C d C d b Mep C s 6.5 [2] - Tuv C s 6.0 [2] - Tut C s [1] Non-labeled carbons = 1; [2] line broadening. Crystal structure analysis of tubulysin A (1): C 43 H 65 N 5 O 10 S, 5(H 5 O), M r = , colorless plate ( mm 3 ), tetragonal, P (No.: 96), a = b = (1), c = (13) Å, V = (2) Å 3, Z = 8, d calc = gcm -3, F 000 = 4032, µ = mm -1. Preliminary examination and data collection were carried out on a kappa-ccd device (NONIUS MACH3) at the window of a rotating anode (NONIUS FR591) with graphite monochromated Mo-K α radiation (λ = Å). Data collection was performed at T = 173 K within the Θ range of 2.43 < Θ < A total of 8759 reflections were integrated and corrected for Lorentz and polarization effects. A correction for absorption effects and/or decay was applied during the scaling procedure. After merging (R int = ), 4442 [3669: I o >2σ(I o )] independent reflections remained and all were used to refine 562 parameters. The structure was solved by a combination of direct methods and difference-fourier syntheses. All non-hydrogen atoms (excluding C28, C29, and C30) were refined with anisotropic displacement parameters. All hydrogen atoms were calculated in ideal positions (riding model). The hydrogen atoms of the water molecules were not included. Full-matrix leastsquares refinements were carried out by minimizing Σw(F 2 o -F 2 c ) 2 and converged with R1 = 4

6 [I o >2σ(I o )], wr2 = [all data], GOF = 1.117, and shift/error < The final difference-fourier map shows no striking features ( e min/max = +0.78/-0.34 eå -3 ). The absolute configuration was proved by retro-synthesis, the trend is given correctly by Flack s parameter ε = 0.2(3). Crystallographic data (excluding structure factors) for the structure reported in this paper have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC (1). Copies of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: (+44) ; deposit@ccdc.cam.ac.uk). b) Data Collection Software for Nonius kappa- CCD devices, Delft (The Netherlands), 2001; c) Z. Otwinowski, W. Minor, Methods in Enzymology 1997, 276, 307ff; d) A. Altomare, G. Cascarano, C. Giacovazzo, A. Guagliardi, M. C. Burla, G. Polidori, M. Camalli, SIR92, J. Appl. Crystallogr. 1994, 27, ; e) International Tables for Crystallography, Vol. C, Tables , , and (Hrsg.: A. J. C. Wilson), Kluwer Academic Publishers, Dordrecht (The Netherlands), 1992; f) A. L. Spek, PLATON, Utrecht University, Utrecht (The Netherlands), 2001; g) G. M. Sheldrick, SHELXL-97, Universität Göttingen, Göttingen (Germany), Figure 1. Stereo-view of the crystal structure of tubulysin A (1) (ORTEP plot, hydrogen atoms added). 5

7 Uptake of tubulysin A and D by mouse fibroblast cells: A 10 ml culture of cell line L929 in DME medium containing 10 % FCS at 37 C and 10 % CO 2 was treated with 0.5 µg each of tubulysin A and D. After 18 h the cell mass was harvested by centrifugation and extracted repeatedly with ethanol. The supernatant was extracted with ethyl acetate. Both extracts were analysed by HPLC/ESI-MS (ion current for M+H + ) to contain 45 ng of tubulysin A and 125 ng of tubulysin D in the cell extract, and 335 ng and 250 ng in the supernatant corresponding to a recovery of 75 %. At an estimated volume of the cell mass of 50 µl/10 ml culture the intracellular concentrations of tubulysin A and tubulysin D are 25 and 100 fold higher than in the culture medium. 6