Supporting Information: Reinvestigation of the Branimycin Stereochemistry

Transcription

1 Supporting Information: Reinvestigation of the Branimycin Stereochemistry at position 17-C Ana Čikoš*, Nicolas Triballeau, Paul A. ubbard, Dinko Žiher, Pieter F. W. Stouten #, Julien G. P.-. Doyon #, Tiny Deschrijver #, Johan Wouters, Renaud. M. Lépine, Laurent Sanière Fidelta Ltd, Prilaz baruna Filipovića 29, Zagreb, Croatia Galapagos SASU, Parc Biocitech, 102 Avenue Gaston Roussel, Romainville, France BioFocus DPI Ltd, Chesterford Research Park, Saffron Walden, CB10 1XL, United Kingdom #Galapagos NV, Industriepark Mechelen Noord, Generaal De Wittelaan L11 A3, 2800 Mechelen, Belgium Université de Namur ASBL, Rue de Bruxelles 61, 5000 Namur, Belgium Corresponding author: SI1

2 Contents SI1. Experimental... 3 SI1.1. Crystallization... 3 SI1.2. X-ray Crystallography... 3 SI1.3. NMR Spectroscopy... 3 SI1.4. Molecular Modeling... 3 SI2. Characterization Data... 4 SI2.1. X-ray... 4 SI2.2. Molecular modelling... 6 SI2.3. NMR Chemical Shifts... 7 SI2.4. NMR Coupling Constants... 8 SI2.5. NMR Interactions through Space... 9 SI2.6. Comparison of NMR Chemical Shifts SI2.7. NMR Spectra SI2

3 SI1. Experimental SI1.1. Crystallization A brownish batch of branimycin was optained from Saccharothrix xinjiangensis G60/1571 according to the previously described procedure (Dudfield, P et al., W A1, 2015). 500 mg was dissolved in 2 ml sbuac. A precipitate was formed and filtered off (~300 mg). The filtrate was left to evaporate and during which crystals were formed. SI1.2. X-ray Crystallography A crystal measuring x x mm was mounted on the goniometer head of an xforddiffraction Gemini R Ultra diffractometer equipped with a Ruby CCD detector. Diffraction intensities were collected at room temperature using Mo Kα radiation. Data collection, cell parameters refinement and data reduction were performed with CrysAlisPro (xford Diffraction, CrysAlisPro Software System). The structure was solved by direct methods and refined on F 2 using the Shelx-2014 program (Sheldrick, G. M. (2008) Acta Cryst. A64, ). Non-hydrogen atoms were refined anisotropically. ydrogen atoms were allowed to ride on the carrier atom from their calculated positions. Molecular overlay with X-ray structure of nodusmicin (CSD ref code NDMSCN) was performed in Discovery Studio 4.1 (Accelrys, San Diego CA, USA). Four matching atoms located in the most rigid part of the structures (4-C, 5-C, 6-C and the bridge oxygen in branymicin with 5-C, 6-C, 7-C and the bridge oxygen in nodusmicin) were selected to define the tethers needed to superimpose the two structures. SI1.3. NMR Spectroscopy The complete 1 and 13 C assignments, as well as the conformational analysis in solution were made on basis of one- and two-dimensional NMR spectra ( 1, DEPTq, CSY, RESY, NESY, edited SQC and MBC). All spectra were recorded on Bruker Avance III 600 and Bruker Avance AV400 spectrometer equipped with room temperature 5 mm diameter SEI and BB probes, respectively. The spectra were acquired using standard Bruker pulse sequences on samples dissolved in CDCl 3 and DMS-d 6 at 25 C in 3 mm NMR tubes. Conformational analysis and configuration at position 17-C were investigated using the combination of coupling constants (from 1 NMR spectrum) and re/ne interactions (from RESY and NESY spectra) in both DMS-d 6 and CDCl 3 at 25 C. SI1.4. Molecular Modeling Starting conformation of branimycin was generated from IsisDraw structure in Maestro 9.2 (Maestro, version 9.2, Schrödinger, LLC, New York, NY, 2011.). and minimized by MacroModel (MacroModel, version 9.9, Schrödinger, LLC, New York, NY, 2011.) using PLS 2005 force field and ε r (DMS) = Constraints (100 kcal mol-1) based on vicinal coupling constants (marked grey in Table SI6) and re signals (marked grey in Table SI7) were applied for torsion angles and distances, respectively, during Conformational Search procedure (PLS 2005, ε r (DMS) = 47.2). Generated conformations were minimized without constrains afterwards. ur choice of method was based on the size of branimycin, SI3

4 availability of NMR data (constraints) and our previous experience with conformations of large macrocyclic molecules were we found that the conformational search procedure based on molecular mechanic calculation (PLS force field) yields best results in time effective manner. After it was obtained, X-ray structure was used as a starting conformation of (R)-17-C branimycin and minimized with weighted heavy atoms for comparison with previously observed NMR data and literature (S)-17-C branimycin. For additional comparison (S)-17-C branimycin was generated by inverting 17-C configuration in crystal structure of branimycin. Most probable conformers of both epimers were generated by conformational search procedure applied only on 16-C substituent with fixed ring atoms followed by whole molecule minimization. All calculations were performed using the PLS 2005 force field. Software used for calculations and visualizations were MacroModel 9.9 and Maestro 9.3, implemented in Schrodinger Suite package SI2. Characterization Data SI2.1. X-ray Coordinates of branimycin and structure factors from the X-ray diffraction experiment have been deposited at the Cambridge Crystallographic Data Center under the entry CCDC Table SI1. Crystal data Formula C Formula Weight Crystal System Monoclinic Space group P2 1, (No. 4) a, b, c [Å] (4), (2), (4) α, β, γ [ ] 90, (4), 90 V [Å 3 ] (8) Z 2 D(calc) [g/cm] 1.26 µ(mok α ) [mm -1 ] F(000) 520 Crystal Size [mm] 0.19 x 0.32 x 0.35 Table SI2. Data collection Temperature [K] 293 Radiation [Å] MoK α θ Min-Max [ ] 3.4, 25.0 Dataset -12: 12 ; -14: 14 ; -13: 13 Tot., Uniq. Data, R(int) 9212, 4475, bserved data [I > 2.0 sigma(i)] 4349 SI4

5 Table SI3. Structure refinement Nref, Npar 4475, 324 R, wr 2, S , , 1.04 w = 1/[σ 2 (Fo 2 )+(0.0383P) P] where P=(Fo 2 +2Fc 2 )/3 Max. and Av. Shift/Error 0.00, 0.00 Flack x 0.0(2) Min. and Max. Resd. Dens. [e/å 3 ] -0.14, 0.16 Table SI4. Selected -bond parameters A (3) _ A (3) _ A (3) _ A (2) _565 [ ] = 1-x,-1/2+y,-z [ ] = x,1+y,z [ ] = -x,1/2+y,-1-z Figure SI1. The crystal packing of branimycin viewed down the crystallographic c axis. ydrogen bonds are shown as doted blue lines. For clarity, only polar hydrogens are shown. SI5

6 nodusmicin branimycin Figure SI2. Molecular overlay of our branimycin X-ray structure (green carbon) with that of nodusmicin (orange carbon). The four atoms used to perform the alignment are depicted with spheres. SI2.2. Molecular modelling Table SI5. Energies and geometrical parameters for calculated conformations and their comparison to X-ray structure of branimycin (R)-17-C conformer (S)-17-C conformer X-ray E(DMS) / kj mol d(17- =C1) / Å d(17- -C1) / Å SI6

7 2'Me SI ' NMR Chemical Shifts Me 15 15Me ' 102 9'Me Me Figure SI3. Structure and numbering of branimycin 1 NMR (600 Mz, DMS-d 6 ): δ = 1.12 (d, J = 6.8 z, 3, 15-Me), 1.56 (s, 3, 13-Me), (dddd, J = 11.6, 9.3, 4.2 z, 1, 9-), 2.21 (d, J = 2.4 z, 1, 11-), 2.56 (d, J = 7.0 z, 1, 6- ), 2.71 (sxt, J = 7.0 z, 1, 15-), 2.81 (ddd, J = 10.8, 9.6, 5.2 z, 1, 2-), 2.85 (dt, J = 9.6, 2.3 z, 1, 3-), 3.16 (s, 3, 9'-Me), 3.18 (s, 3, 2'-Me), 3.20 (dd, J = 10.1, 7.2 z, 1, 18-), 3.22 (s, 3, 18-Me), 3.36 (dd, J = 10.1, 2.6 z, 1, 18-), 3.36 (dd, J = 9.1, 5.2 z, 1, 2'-), 3.36 (dd, J = 8.7 z, 1, 9'-), 3.41 (dd, J = 8.7, 4.2 z, 1, 9'-), 3.44 (dd, J = 10.6, 9.1 z, 1, 2'-), 3.46 (ddd, J = 11.6, 3.8, 2.9 z, 1, 10-), 3.71 (q, J = 4.8 z, 1, 8-), 3.77 (dddd, J = 10.0, 7.2, 6.6, 2.6 z, 1, 17-), 3.91 (d, J = 4.9 z, 1, 7-), 4.66 (d, J = 5.1 z, 1, 8-), 4.70 (d, J = 3.8 z, 1, 10-), 4.80 (dd, J = 10.0, 6.5 z, 1, 16-), 5.06 (d, J = 6.6 z, 1, 17-), 5.35 (dd, J = 9.8, 2.6 z, 1, 4-), 5.48 (dd, J = 8.0, 1.0 z, 1, 14-), 6.00 (ddd, J = 9.5, 7.1, 2.1 z, 1, 5-) ppm 13 C NMR (151 Mz, DMS-d 6 ): δ = 15.3 (15-Me), 16.6 (13-Me), 31.6 (15-C), 37.9 (6-C), 39.8 (9-C), 45.5 (2-C), 48.7 (11-C), 51.4 (3-C), 57.9 (9'-Me), 58.2 (2'-Me), 58.4 (18-Me), 67.1 (8-C), 67.5 (17- C), 69.7 (10-C), 70.3 (9'-C), 73.0 (2'-C), 75.6 (18-C), 78.8 (16-C), 84.1 (7-C), 88.1 (12-C), (4-C), (5-C), (13-C), (14-C), (1-C) ppm 1 NMR (600 Mz, CLRFRM-d): δ = 1.27 (d, J = 7.0 z, 3, 15-Me), 1.69 (s, 3, 13-Me), 2.17 (d, J = 5.5 z, 1, 10-), 2.30 (dddd, J = 11.1, 5.3, 4.8 z, 1, 9-), 2.48 (d, J = 2.6 z, 1, 11-), 2.55 (d, J = 4.2 z, 1, 17-), 2.72 (d, J = 7.0 z, 1, 6-), 2.88 (sxt, J = 7.0 z, 1, 15- ), (m, 1, 2-), 3.03 (d, J = 2.8 z, 1, 8-), 3.05 (dd, J = 9.5, 2.6 z, 1, 3-), 3.32 (s, 3, 2'-Me), 3.36 (s, 3, 9'-Me), 3.40 (s, 3, 18-Me), 3.41 (dd, J = 9.7, 7.3 z, 1, 18-), 3.48 (dd, J = 8.6, 4.8 z, 1, 2'-), (m, 1, 2'-), 3.57 (dd, J = 9.7, 2.9 z, 1, 18-), 3.64 (dd, J = 9.5, 5.3 z, 1, 9'-), 3.77 (dd, J = 9.4, 4.4 z, 1, 9'-), 4.02 (ddd, J = 11.1, 4.9, 2.8 z, 1, 10-), (m, 1, 17-), (m, 1, 8-), 4.10 (d, J = 5.0 z, 1, 7-), 5.04 (dd, J = 9.9, 6.8 z, 1, 16-), 5.39 (dd, J = 9.8, 1.7 z, 1, 4-), 5.68 (d, J = 8.1 z, 1, 14- ), 6.05 (ddd, J = 8.8, 7.0, 2.0 z, 1, 5-) ppm SI7

8 13 C NMR (151 Mz, CLRFRM-d): δ = 15.5 (15-Me), 17.0 (13-Me), 32.5 (15-C), 38.6 (6-C), 40.5 (9-C), 46.2 (2-C), 48.3 (11-C), 51.8 (3-C), 59.3 (2'-Me), 59.3 (18-Me), 59.4 (9'-Me), 68.2 (17- C), 72.0 (8-C, 10-C), 73.4 (9'-C), 73.8 (2'-C), 74.7 (18-C), 79.4 (16-C), 84.1 (7-C), 88.8 (12-C), (4-C), (5-C), (13-C), (14-C), (1-C) ppm SI2.4. NMR Coupling Constants Table SI6. Vicinal and geminal coupling constants for branimycin at 25 C Atom 1 Atom 2 3 J 1,2 / z in J Constraint / 1,2 / z in DMS-d 6 CDCl 3 2 2'a overlap 2 2'b < 1 < < 1 < 'a 'b Me 1.0 < Me a b J 1,2 2 J 1,2 Atom 1 Atom 2 2'a 2'b 'a 9'b a 18b grey used as constraints for molecular modeling SI8

9 SI2.5. NMR Interactions through Space Table SI7. re correlations for branimycin from RESY spectrum at 25 C in DMS-d 6 2 2'a 2'b 2'Me 'a 9'b 9'Me Me Me a 18b 18Me 2 w w s 2'a m 2'b s 2'Me 3 m w m 4 m s m w 5 w s m w 6 s s m s m 7 w s s s 8 s s s 8 m s s w 9 s m 9'a m w 9'b w s 9'Me m 10 s s s 10 w s s s 11 m m s s 13Me s s s s 14 m s w s 15 s w s w 15 Me s s w 16 s s s w s 17 s s m w w 17 w m s m 18a w s 18b s m w 18Me w s - strong, m - medium, w weak, grey used as constraints SI9

10 Table SI8. ne correlations for branimycin from NESY spectrum at 25 C in CDCl 3 2 2'a 2'b 2'Me 'a 9'b 9'Me Me Me a 18b 18Me 2 m m s 2'a m s m 2'b m s s 2'Me 3 m w m s 4 m s m 5 w s m w 6 s s m s m 7 w s s s 8 s s s 8 m s s w w m 9 s m m m 9'a m s m w m 9'b w m s s m 9'Me m m w 10 s w s s m 10 w s s s 11 m w w m m s s 13Me m s s w s s 14 m s w s m 15 s w s s 15 Me s s w s m 16 s s s w s 17 m s s m w 17 w m s w m 18a w s w s 18b s m m s 18Me m m s - strong, m - medium, w - weak SI10

11 SI2.6. Comparison of NMR Chemical Shifts Table SI9. Chemical shift comparison of branimycins with different origin a) dissolved crystal (600 Mz), b) synthetic* (600 Mz), c) natural* (600 Mz) and d) original isolated # (200 Mz), all at 25 C in CDCl 3 Proton a) Crystal b) Synthetic c) Natural d) Isolated Carbon a) Crystal b) Synthetic c) Natural d) Isolated 15-Me 1.27 (d, J = 7.0 z) 1.27 (d, J= 7.0 z) 1.27 (d, J= 7.0 z) 1.27 (d, J=7.2 z) 15-Me Me 1.69 (s) 1.69 (d, J= 0.9 z) 1.69 (d, J= 0.8 z) 1.70 (s) 13-Me (d, J = 5.5 z) 2.09 (d, J= 5.5 z) 2.09 (d, J= 5.5 z) n/a 15-C (dq, J = 11.1, 5.3, 4.8 z) (m) (m) 2.34 (m) 6-C (d, J = 2.6 z) 2.48 (d, J= 2.5 z) 2.48 (d, J= 2.7 z) 2.47 (d, J=2.3 z) 9-C (d, J = 4.2 z) 2.49 (d, J= 4.3 z) 2.50 (d, J= 4.0 z) n/a 2-C (d, J = 7.0 z) 2.72 (d, J= 7.2 z) 2.72 (d, J= 7.0 z) 2.72 (d, J=7.4 z) 11-C (sxt, J = 7.0 z) (m) (m) 2.87 (ddq, J=7.4 z) 3-C (m) (m) (m) 3.04 (m) 2 -Me (d, J = 2.8 z) 2.99 (d, J= 2.6 z) 3.00 (d, J= 2.1 z) n/a 18-Me (dd, J = 9.5, 2.6 z) (m) (m) 3.04 (m) 9 -Me Me 3.32 (s) 3.32 (s) 3.32 (s) 3.27 (s) 17-C Me 3.36 (s) 3.36 (s) 3.36 (s) 3.31 (s) 8-C Me 3.40 (s) 3.40 (s) 3.40 (s) 3.36 (s) 10-C b 3.41 (dd, J = 9.7, 7.3 z) (m) (m) 9 -C b 3.48 (dd, J = 8.6, 4.8 z) 3.48 (dd, J= 8.5, 4.8 z) 3.48 (dd, J= 8.4, 4.6 z) 2 -C a (m) (m) 18-C (m) (m) 18- a 3.57 (m, J = 9.7, 2.9 z) 16-C b 3.64 (dd, J = 9.5, 5.3 z) 3.64 (dd, J= 9.6, 5.2 z) 3.64 (dd, J= 9.5, 5.1 z) 7-C a 3.77 (dd, J = 9.4, 4.4 z) 3.78 (dd, J= 9.6, 4.6 z) 3.78 (dd, J= 9.7, 4.7 z) 3.78 (dd, J=10.0, 4.7 z) 12-C (ddd, J = 11.1, 4.9, 4.02 (ddd, J= 11.1, 5.3, z) z) 4-C (m) (m) (m) 5-C (m) (m) 13-C (d, J = 5.0 z) 14-C (dd, J = 9.9, 6.8 z) 5.04 (dd, J= 10.0, 6.7 z) 5.04 (dd, J= 9.9, 6.8 z) 5.03 (dd, J=9.8, 6.4 z) 1-C (dd, J = 9.8, 1.7 z) 5.39 (dd, J= 9.7, 2.2 z) 5.38 (dd, J= 9.9, 2.3 z) 5.37 (d, J=9.8 z) (d, J = 8.1 z) 5.68 (dd, J= 8.0, 1.2 z) 5.68 (d, J= 8.1 z) S.67 (d, J=7.8 z) *Enev, V. S.; Felzmann, W.; Gromov, A.; Marchart, S.; Mulzer, J. Chem. Eur. J. 2012, 18 (31), and Stefan Marchart thesis Total Synthesis of the Antibiotic (+) Branimycin, University of Vienna, #Michael Speitling thesis Isolierung und Strukturaufklärung von Branimycin, Brom-alterochromid A/B und weiteren Stoffwechselprodukten, University of Göttingen, 1998 SI11

12 SI2.7. NMR Spectra BRANIMYCIN_ _36_DMS_.ESP 2'Me ' Me Me 8 9' 102 9'Me 9' 18 9' 18 water 10 9'Me Date 08 Dec :48:48 Solvent DMS-d6 Pulse Sequence zg Temperature (degree C) Acquisition Time (sec) Number of Transients 64 Points Count Sweep Width (z) Spectrum ffset (z) riginal Points Count Nucleus 1 Frequency (Mz) Me Me 2'Me Me Me DMS Chemical Shift (ppm) Figure SI4. Proton spectrum of branimycin in DMS-d 6 at 25 C (14.10 T) SI12

13 BRANIMYCIN_ _36_DMS_C.ESP 'Me 2'Me Me Me ' 2'Me Me105 2' Me 15 15Me ' 102 9'Me 2' Date 08 Dec :50:56 Solvent DMS-d6 Pulse Sequence deptqgpsp.2 Temperature (degree C) Acquisition Time (sec) Number of Transients 5000 Points Count Sweep Width (z) Spectrum ffset (z) riginal Points Count 8192 Nucleus 13C Frequency (Mz) Chemical Shift (ppm) Figure SI5. Carbon spectrum of branimycin in DMS-d 6 at 25 C (14.10 T) SI13

14 BRANIMYCIN_CRYSTAL_ _13_CDCL3_.ESP 2'Me ' Me 15 15Me ' 102 9'Me 2'<'> 18<'> 2'<''> 18<''> 2'Me 9'Me Date 28 Jul :16:16 Solvent CLRFRM-d Pulse Sequence zg30 Temperature (degree C) Acquisition Time (sec) Number of Transients 64 Points Count Sweep Width (z) Spectrum ffset (z) riginal Points Count Nucleus 1 Frequency (Mz) Me Me 13Me Me Chemical Shift (ppm) Figure SI6. Proton spectrum of branimycin in CDCl 3 at 25 C (14.10 T) SI14

15 2'Me ' Me 15 15Me ' 102 9'Me Date 29 Jul :13:36 Solvent CLRFRM-d Pulse Sequence deptqgpsp.2 Temperature (degree C) Acquisition Time (sec) Number of Transients 1000 Points Count Sweep Width (z) Spectrum ffset (z) riginal Points Count 8192 Nucleus 13C Frequency (Mz) BRANIMYCIN_CRYSTAL_ _13_CDCL3_C.ESP Me ' 18 9' , 'Me Me Me 18Me Chemical Shift (ppm) Figure SI7. Carbon spectrum of branimycin in CDCl 3 at 25 C (14.10 T) SI15

16 Figure SI8. RESY spectrum of branimycin in DMS-d 6 at 25 C (14.10 T) SI16

17 Figure SI9. NESY spectrum of branimycin in CDCl 3 at 25 C (14.10 T) SI17

18 Figure SI10. Comparison of proton spectra of branimycin: a) authentic sample from Laatsch group, b) amorphous and c) crystal, all in CDCl 3 at 25 C (14.10 T) SI18

19 Figure SI11. Comparison of carbon spectra of branimycin: a) authentic sample from Laatsch group, b) amorphous and c) crystal, all in CDCl 3 at 25 C (14.10 T) SI19

20 Figure SI12. Comparison of SQC spectra of branimycin: authentic sample from Laatsch group (black/red signals), amorphous (blue/pink signals) and crystal (green/orange) in CDCl 3 at 25 C (14.10 T), signals shifted in F1 domain to avoid overlap SI20

21 Figure SI13. CSY spectrum of branimycin in DMS-d 6 at 25 C (14.10 T) SI21

22 Figure SI14. Edited SQC spectrum of branimycin in DMS-d 6 at 25 C (14.10 T) SI22

23 Figure SI15. MBC spectrum of branimycin in DMS-d 6 at 25 C (14.10 T) SI23

24 Figure SI16. CSY spectrum of branimycin in CDCl 3 at 25 C (14.10 T) SI24

25 Figure SI17. Edited SQC spectrum of branimycin in CDCl 3 at 25 C (14.10 T) SI25

26 Figure SI18. MBC spectrum of branimycin in CDCl 3 at 25 C (14.10 T) SI26