Section Page Experimental methods and materials S1-S3 Supplemental references S3 Figures S4-S7 Tables S8-S13 HRMS and NMR spectra S14-S58
Experimental methods and materials.
General. Unless otherwise stated, all general chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO), ENZO Life Sciences (Farmingdale, NY) or Selleckchem (Houston, TX) and were reagent grade or better. E. coli BL21(DE3) competent cells were purchased from Invitrogen (Carlsbad, CA). The pET28a E. coli expression vector was purchased from Novagen (Madison, WI). PD-10 columns and Ni- NTA superflow columns were purchased from GE Healthcare (Piscataway, NJ). Plate-based assays were conducted using a FLUOstar Omega plate reader (BMG LABTECH GmbH, Offenburg, Germany) at λ = 410 nm with a bandwidth of 2 nm and the path length was corrected to a depth corresponding to 20 µL liquid volume per well. NMR spectra were obtained on Varian Unity Inova 400 instrument (Palo Alto, CA) at the NMR facility of the College of Pharmacy at University of Kentucky using 1 13 “DMSO-d6 (D, 99.9%)” from Cambridge Isotopes (Cambridge Isotope Laboratories, MA). H and C chemical shifts were referenced to internal solvent resonances. Multiplicities are indicated by s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), quin (quintet), m (multiplet), and br (broad). Chemical shifts are reported in parts per million (ppm) and coupling constants J are given in Hz. Routine 13C NMR spectra were fully decoupled by broad-broad WALTZ decoupling. All NMR spectra were recorded at ambient temperature. NMR assignments were performed with the aid of gCOSY, HSQC, and HMBC experiments. Analytical TLC was performed on silica gel glass TLC plates (EMD Chemical Inc., Billerica, MA). Visualization was accomplished with UV light (254 nm) followed by staining with dilute H2SO4 (5% in EtOH) solution. HPLC was accomplished using an Agilent 1260 system (Santa Clara, CA, USA) equipped with a DAD detector (Method A) or a Varian ProStar (Palo Alto, CA, USA) system equipped with a PDA detector (Method B). Method A (HPLC): Analytical reverse-phase (RP) HPLC was conducted with a Luna C18 (5 μm, 4.6 mm × 250 mm; Phenomenex, Torrance, California, USA) column (5% B to 95% B, 25 min; -1 95% B, 2 min; 95% B to 5% B, 0.1 min; 5% B, 5 min; A = ddH2O, 0.1% TFA; B = CH3CN; 1.0 mL min ; A254). Method B (HPLC): Preparative RP-HPLC was conducted with a Discovery® BIO Wide Pore C18 (10 µm, 21.2 250 mm; Supelco, Bellefonte, Pennsylvania, USA) column (5% B to 55% B, 25 min; 55%
B to 100% B, 2 min; 100% B, 5 min; 100% to 5% B, 1 min; 5% B, 4 min; A = ddH2O, 0.1% TFA; B = CH3CN; 5.0 mL min-1 ; A254). HPLC peak areas were integrated using Star Chromatography Workstation Software (Agilent 1260 system, Agilent Technologies, Santa Clara, CA, USA). For pure compounds, high resolution electrospray ionization (ESI) mass spectra (HRMS) were recorded on an AB Sciex Triple TOF 5600 (Redwood City, CA, USA) instrument coupled with an Eksigent Ekspert micro LC 200 system with source temperature of 150 °C, ion spray voltage floating (ISVF) of 5000 V in positive mode. Samples were infused at 20 µL min-1 and spectra collected for 3 min at a resolution greater than 31000. In negative mode ISVF of -4000 V was used. C17 lysophosphatidyl choline with a mass of 510.3554 and C17 lysophosphatidic acid with a mass of 423.2517 were used as internal references to calibrate the spectra in positive and negative modes, respectively. For crude reaction mixtures, high-resolution electrospray ionization mass spectra were recorded on a Thermo Scientific (Rockford, IL, USA) Q Exactive Orbitrap mass spectrometer via direct infusion at 3 μL min-1. Full-scan mass spectra were recorded in positive (3.8 kV) and negative (3.8 kV) ion modes (capillary temperature: 225 °C; nominal resolution: 140,000).
S1
Protein production, purification and storage. Heterologous protein (OleD Loki) production from the strain pET28a/oleD[P67T/I112P/T113M/S132F/A242I] (OleD Loki)/E. coli, protein purification and protein handling/storage followed previously reported protocols.[1] OleD plate-based glycosylation assays. Using a select set of representative hydroxamates (Figure 1), reactions were conducted in a final volume of 30 µL in a flat bottom 384-well microtiter plate and contained 0.25 µM of pure enzyme, 0.1 mM UDP, 2.0 mM PNP-Glc, 25 mM Tris-HCl (pH 8.0), 5 mM MgCl2, and 1 mM of putative acceptor, unless otherwise noted. Two separate control reactions were conducted in parallel, a known substrate (4-methylumbelliferone) positive control and vehicle (DMSO) negative control. Reactions were allowed to proceed at 30°C for 2 hrs with continuous monitoring (ΔA410). For positives, reactions were subsequently quenched with an equal volume of MeOH, centrifuged (18,000 x g for 20 min) and the recovered supernatant analyzed by analytical reverse-phase HPLC (method A; Figure S1) and HRMS (Table S1). Product and starting material HPLC peak integration were used to determine percent conversion as previously described.[2] Scale-up and purification of representative glycosides. Reactions contained 1 mM acceptor [abexinostat (1), dacinostat (2), givinostat (3), panobinostat (4) or tubastatin A (5)], 2 mM ClNP-β-D-glc, 0.5 mM UDP and 0.25 μM OleD Loki in a total volume of 30 mL 25 mM Tris-HCl, 5 mM MgCl2, pH 8.0 at 30 °C. Reaction progress was monitored via HPLC (method A) and, upon completion (~24 hour), enzyme was removed via forced dialysis (30 kDa Amicon Ultra centrifugal filter, EMD Chemical Inc, Billerica, MA; 6,500 x g, 45 min, 4 °C). Solid-phase capture of reactants and products was accomplished via the addition of 15% weight/volume XAD (Amberlite, XAD16N, Sigma Aldrich, St. Louis, MO) followed by gentle stirring for 4 hours. The resin was filtered, washed (200 mL ddH2O, x 3) and products and reactants eluted via the addition of MeOH and gentle stirring overnight. The eluent was recovered via filtration and the resin washed with additional MeOH (100 mL, x 3). The combined eluent was concentrated in vacuo and the desired products subsequently resuspended in 1 mL of MeOH/DMSO and purified by preparative HPLC (method B). For each, final fractions containing pure glycoside products were subsequently combined, frozen (- 80°C) and lyophilized to dryness. pH degradation assays. Assays were conducted in a volume of 1 mL and contained a final concentration of 1 mM of either gavinostat-β-D-glucoside (3a) or panobinostat-β-D-glucoside (4a) glycoside (DMSO) in pH adjusted buffer as follows: pH 5 (50 mM NaOAc), pH 7.5 (50 mM NaPO4) or pH 10 (50 mM NH4HCO3) at room temperature. Aliquots of 20 µl were taken at fixed timepoints (0 min, 30 min, 90 min, 3 hrs, 5 hrs, 8 hrs, 24 hrs and 720 hrs) and added to equal volume of acetonitrile and centrifuged at 18,000 x g for 2 min. Supernatant was analyzed via analytical reverse-phase HPLC (method A) for hydrolysis of the sugar. Samples were also subjected to concentrated H2SO4 (16 N) at equal volume DMSO (containing glycoside) and acid. Samples were left at room temp for 5 min before being neutralized with 5 M NH4HCO3 (pH 10) at a 1:10 ratio acid:base. Samples were centrifuged at 18,000 x g for 2 min and analyzed via analytical reverse-phase HPLC (method A) or via TLC using 5% MeOH/CHCl3. Enzymatic degradation assays. Reactions were conducted in parallel in a final volume of 30 µL in a flat bottom 384-well microtiter plate with 1 µM of Saccharomyces cerevisiae α-glucosidase (Sigma Aldrich G5003) or almond β-glucosidase (Sigma Aldrich 49290), 25 mM Tris-HCl (pH 8.0) and 1 mM of putative substrate. Two separate control reactions (4-methylumbelliferone α- and β-glucosides) were conducted for each glucosidase. Reactions were allowed to proceed at 30°C and aliquots were taken at fixed timepoints (0 min, 5 min, 10 min, 30 min, 60 min, 2 hr, 4 hr, 72 hr), quenched with MeOH, centrifuged (18,000 x g for 20 min) and the recovered supernatant analyzed by analytical reverse-phase HPLC (method A, Figure S3). Product and starting material HPLC peak integration were used to determine percent conversion as previously described.[2]
S2
Axolotl embryo tail regeneration assay. The tail regeneration assay was conducted as previously described.[3, 4] The use of pre-feeding stage axolotls does not require a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at University of Kentucky. However, embryos used in this study were treated according to the same ethical standards that apply to feeding axolotls, which are cared for using standard axolotl husbandry methods approved under IACUC protocol 2017-2580. Assays employed Mexican axolotl (Ambystoma mexicanum) embryos (RRID:AGSC_100E) obtained from the Ambystoma Genetic Stock Center (RRID:SCR_006372).
Cytotoxicity assays. A resazurin-based cytotoxicity assay, in triplicate, was used to assess the cytotoxicity of chemicals against the human lung non-small cell carcinoma cell line A549 and colorectal carcinoma cell line HCT 116, as previously described.[4, 5]
Computational studies. The X-ray structure of oleandomycin glycosyltransferase (OleD) in complex with erythromycin A (PDB: 2IYF) was selected to conduct molecular docking. All missing atoms in the X-ray crystal structure (chain B) were added using the H++ server.[6] Each ligand (abexinostat, MC1568, or tubacin) was docked to the substrate-binding site of OleD using the AutoDock Vina software.[7] Docked binding modes were subsequently manually inspected, and the most favorable binding structures were energy-minimized. Briefly, the AMBER14SB force field[8] and the second generation of the general AMBER force field (gaff2) were used for the protein and ligands, respectively. The nonbonded cutoff for the interatomic interactions was set to 10 Å. The binding structures were energy-minimized using the Sander module of AmberTools18[9] with a hybrid protocol of the steepest descent minimization (8000 steps) and then conjugate gradient minimization to meet the convergence criterion (the root-mean-square of the energy gradient was smaller than 1.0 × 10–4 kcal/mol·Å). The energy-minimized protein-ligand complex structures were used for binding mode analysis.
Supplemental references.
[1] R. W. Gantt, P. Peltier-Pain, S. Singh, M. Zhou, J. S. Thorson, Proc. Natl. Acad. Sci. USA 2013, 110, 7648-7653. [2] R. R. Hughes, K. A. Shaaban, J. Zhang, H. Cao, G. N. Phillips, J. S. Thorson, Chembiochem, 2017,18, 363-36. [3] L. V. Ponomareva, A. Athippozhy, S. R. Voss, J. S. Thorson, Comp. Biochem. Physiol. C Toxicol. Pharmacol. 2015, 178, 128-135. [4] X. Wang, Y. Zhang, L. V. Ponomareva, Q. Qiu, R. Woodcock, S. I. Elshahawi, X. Chen, Z. Zhou, B. E. Hatcher, J. C. Hower, C. G. Zhan, S. Parkin, M. K. Kharel, S. R. Voss, K. A. Shaaban, J. S. Thorson, Angew. Chem. Int. Ed. 2017, 56, 2994-2998. [5] M. Abbas, S. I. Elshahawi, X. Wang, L. V. Ponomareva, I. Sajid, K. A. Shaaban, J. S. Thorson, J. Nat. Prod. 2018, 81, 2560−2566. [6] J. C. Gordon, J.B. Myers, T. Folta, V. Shoja, L.S. Heath, A. Onufriev, Nucl. Acids Res. 33, 2005, W368- W371. [7] O. Trott, A. J. Olson, J. Comput. Chem. 2010, 31, 455-461. [8] J.A. Maier, C. Martinez, K. Kasavajhala, L. Wickstrom, K. E. Hauser, C. Simmerling, J. Chem. Theory Comput. 2015, 11, 3696-3713. [9] D.A. Case, I.Y. Ben-Shalom, S.R. Brozell, D.S. Cerutti, T.E. Cheatham, III, V.W.D. Cruzeiro, T.A. Darden, R.E. Duke, D. Ghoreishi, M.K. Gilson, H. Gohlke, A.W. Goetz, D. Greene, R Harris, N. Homeyer, S. Izadi, A. Kovalenko, T. Kurtzman, T.S. Lee, S. LeGrand, P. Li, C. Lin, J. Liu, T. Luchko, R. Luo, D.J. Mermelstein, K.M. Merz, Y. Miao, G. Monard, C. Nguyen, H. Nguyen, I. Omelyan, A. Onufriev, F. Pan, R. Qi, D.R. Roe, A. Roitberg, C. Sagui, S. Schott-Verdugo, J. Shen, C.L. Simmerling, J. Smith, R. Salomon-Ferrer, J. Swails, R.C. Walker, J. Wang, H. Wei, R.M. Wolf, X. Wu, L. Xiao, D.M. York and P.A. Kollman, AMBER 2018, University of California, San Francisco.
S3
^ * * ^ A) B) C) ^ D) * * ^
^
^ * E) F) G) * H) *
^ * ^ ^
^ ^ I) ^ J) K) ^ L) * * * *
M) * N) O) * P) ^ ^ * *
^ ^
Figure S1. Analytical HPLC (method A) for crude reactions containing hydroxamate acceptors. A) abexinostat, B) CUDC-101, C) CUDC-907, D) dacinostat. E) droxinostat, F) givinostat, G) M344, H) MC1568, I) nexturastat A, J) panobinostat, K) PCI-34051, L) quisinostat, M) roclinostat, N) scriptaid, O) tepoxalin and P) tubastatin A. Crude reactions (red); aglycon standard (blue); * denotes aglycon; ^ denotes glycoside product; peaks near 13.5 and 9 min represent ClNP and ClNP- β-D-Glc , respectively. For corresponding trichostatin A analytical HPLC analyses see previously reported work.[2]
S4
p H 2 d epg Hra d2 adteiognra d a tio n p H 5 d epgHra 5d adteiognra d a tio n
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Figure S3. Enzymatic stability assay. A) almond β-glucosidase (positive control 4-methylumbelliferone β-glucoside, green) and B) Saccharomyces cerevisiae α-glucosidase (positive control 4- methylumbelliferone -glucoside, green); 3a, orange; and 4a, blue. See Supplemental Methods for assay conditions.
S5
Figure S4. 1H-1H COSY (▬) and key HMBC (→) correlations of β-D-glucosyl-abexinostat (1a), β-D- glucosyl-dacinostat (2a), β-D-glucosyl-givinostat (3a), β-D-glucosyl-panobinostat (4a) and β-D-glucosyl- tubastatin A (5a).
S6
Figure S5. Single dose axolotl tail regeneration assay of representative HDACi-glucosides 2a and 4a and their parental comparators at 10 μM concentration. Control 0.1% DMSO showed no effect on regeneration (see Supplementary Methods). For the chemical structures of test agents, see Figures 2 and 3. Treatment with 4 led to the loss of one embryo due to toxicity.
S7
Table S1. (+)HR-ESI-MS and HPLC % conversions for crude reactions (Figure 1)
Hydroxamate Substrate % conversion, HPLC Expected Mass + [H] Observed Mass abexinostat quantitative 560.224 560.224 CUDC-101 60 597.256 597.255 CUDU-907 96 671.224 671.223 dacinostat 93 542.250 542.248 droxinostat 43 406.126 406.125 givinostat quantitative 584.260 584.260 M344 quantitative 470.250 470.248 MC1658 9 477.167 477.165 nexturastat A 90 504.234 504.233 panobinostat 80 512.239 512.239 PCI-34051 quantitative 459.176 459.176 quisinostat 84 557.272 557.271 roclinostat 20 596.272 596.271 scriptaid 50 489.187 489.186 tepoxlalin 31 548.180 548.181 trichostatin A 74 465.223 465.223 tubastatin A 86 498.224 498.223
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Table S2. 13C (100 MHz) and 1H (400 MHz) NMR spectroscopic data of abexinostat (1) and
[a] β-D-glucosyl-abexinostat (1a) in DMSO-d6.
Position abexinostat (1) β-D-glucosyl-abexinostat (1a)
δC, type δH (mult, J in [Hz]) δC, type δH (mult, J in [Hz])
2 145.8, C 146.6, C 3 114.1, C 114.9, C 3a 126.9, C 127.4, C 4 121.6, CH 7.86 (d, 7.8) 121.6, CH 8.02 (d, 7.5) 5 123.3, CH 7.33 (t, 7.5) 124.3, CH 7.47 (dt, 7.1, 0.9) 6 128.4, CH 7.46 (t, 7.8) 128.0, CH 7.58 (ddd, 8.5, 7.2, 1.3) 7 111.7, CH 7.64 (d, 8.3) 112.0, CH 7.72 (d, 8.4) 7a 152.9, C 152.9, C 8 159.2, C 159.1, C
9 51.2, CH2 3.82 (s) 49.2, CH2 4.75 (s)
9-N(CH3)2 44.3, CH3 2.19 (s) 42.7, CH3 2.84 (s)
10 38.4, CH2 3.69 (q, 5.3) 38.3, CH2 3.72 (q, 5.8) 10-NH 10.06 (t, 5.1) 9.35 (t, 5.6) 11 66.3, CH2 4.20 (t, 5.4) 65.9, CH2 4.25 (t, 5.7) 12 160.6, C 161.2, C 13/17 128.7, CH 7.73 (d, 8.8) 114.3, CH 7.04 (d, 8.9) 14/16 114.1, CH 7.03 (d, 8.8) 129.4, CH 7.81 (d, 8.9) 15 125.1, C 123.6, C 18 163.9, C 165.7, C 18-NH 11.06 (s) 12.03 (s) 18-NHOH 8.89 (s) 1′ 106.6, CH 4.59 (d, 8.0) 2′ 71.9, CH 3.12 (t, 8.4) 3′ 76.0, CH 3.23 (m) 4′ 69.7, CH 3.07 (t, 9.1) 5′ 77.5, CH 3.23 (m)
6′ 61.0, CH2 3.72 (m), 3.50 (m) 2′/3′/4′/6′-OH 5.38 (brs, 1H), 5.06 (brs, 2H), 4.50 (brs, 1H)
[a] Assignments supported by gCOSY, gHSQC and gHMBC experiments (Figures S6-8, S10-14).
S9
Table S3. 13C (100 MHz) and 1H (400 MHz) NMR spectroscopic data of dacinostat (2) and β- [a] D-glucosyl-dacinostat (2a) in DMSO-d6.
Position dacinostat (2) β-D-glucosyl-dacinostat (2a)
δC, type δH (mult, J in [Hz]) δC, type δH (mult, J in [Hz])
1 10.72 (brs) 10.97 (s) 2 122.5, CH 7.09 (d, 2.1) 123.3, CH 7.20 (brd, 2.3) 3 111.3, C 108.9, C 3a 127.2, C 126.6, C 4 118.1, CH 7.36 (d, 7.5) 118.1, CH 7.46 (d, 7.9) 5 118.2, CH 6.90 (td, 7.0, 1.1) 118.5, CH 6.97 (t, 7.5) 6 120.8, CH 7.02 (td, 7.0, 1.1) 121.2, CH 7.08 (t, 7.6) 7 112.5, CH 7.30 (d, 8.1) 111.6, CH 7.35 (d, 8.1) 7a 136.2, C 136.2, C
8 22.6, CH2 2.84 (dd, 9.8, 5.6) 19.4, CH2 3.17 (brm)
9 54.9*, CH2 2.73 (dd, 9.5, 5.5) 52.8*, CH2 3.41-3.20 (brm)
11 55.9*, CH2 2.63 (t, 6.5) 53.7*, CH2 3.41-3.20 (brm)
12 58.2, CH2 3.51 (t, 6.3) 55.3, CH2 3.81 (brm) 12-OH 4.37 (brs) 5.03 (brs)
13 59.4, CH2 3.71 (s) 56.2, CH2 4.67-4.35 (brs) 14 141.8, C 135.6, C 15/19 129.1, CH 7.34 (d, 8.0) 131.8, CH 7.62-7.56 (brm) 16/18 127.3, CH 7.49 (d, 8.1) 128.1, CH 7.62-7.56 (brm) 17 133.3, C 131.8, C 20 138.2, CH 7.44 (d, 15.8) 139.8, CH 7.60 (brd, 15.5) 21 118.4, CH 6.44 (d, 15.8) 119.0, CH 6.60 (d, 16.0) 22 162.9, C 164.2, C 22-NH 10.72 (brs) 11.73 (s) 22-NHOH 10.72 (brs) 1′ 106.0, CH 4.67-4.35 (brm) 2′ 71.5, CH 3.11 (m) 3′ 75.9, CH 3.24 (m) 4′ 69.7, CH 3.08 (m) 5′ 77.6, CH 3.24 (m)
6′ 61.0, CH2 3.72 (brd, 11.6), 3.49 (dd, 10.8m 5.6) 2′/3′/4′/6′-OH 5.46-5.02 (brs, 3H), 4.46 (brs, 1H)
[a] Assignments supported by gCOSY, gHSQC and gHMBC experiments (Figures S15-17, S19-23). * Overlapped signals/signals may be exchanged
S10
Table S4. 13C (100 MHz) and 1H (400 MHz) NMR spectroscopic data of givinostat (3) and β- [a] D-glucosyl-givinostat (3a) in DMSO-d6.
Position givinostat (3) β-D-glucosyl-givinostat (3a)
δC, type δH (mult, J in [Hz]) δC, type δH (mult, J in [Hz])
1 126.5, CH 8.01 (brs) 126.5, CH 8.02 (brs) 2 135.3, C 135.4, C 3 126.6, CH 7.63 (dd, 8.5, 1.6) 126.7, CH 7.65 (m) 4 128.4, CH* 7.97 (d, 8.6) 128.4, CH 8.02 (m) 4a 132.7, C 132.8, C 5 130.5, CH 8.15 (brs) 130.6, CH 8.10 (brs) 6 128.4, C* 128.1, C 7 128.3, CH* 8.02 (d, 8.4) 128.2, CH 8.02 (m) 8 128.5, CH* 7.83 (brd, 8.5) 128.6, CH 7.65 (m)
8a 132.1, C 132.1, C
9 54.8, CH2 4.43 (brs) 55.0, CH2 4.47 (brs)
9-N(CH2CH3)2 45.8, CH2 3.06 (brm) 46.1, CH2 3.12 (m)
9-N(CH2CH3)2 8.4, CH3 1.26 (t, 7.2) 8.4, CH3 1.25 (t, 6.3) 10 65.9, CH2 5.36 (s) 65.9, CH2 5.37 (s) 11 153.2, C 153.2, C 12 141.7, C 142.5, C 12-NH 10.11 (brs) 10.2 (brs) 13/17 117.4, CH 7.54 (d, 8.8) 117.4, CH 7.57 (brd, 7.4) 14/16 127.8, CH 7.70 (d, 8.8) 128.5, CH 7.78 (brd, 7.4) 15 126.6, C 124.9, C 18 163.9, C 165.7, C 18-NH 11.10 (s) 12.02 (s) 18-NHOH 10.66 (brs) 1′ 106.7, CH 4.60 (brd, 6.6) 2′ 71.9, CH 3.12 (m) 3′ 76.0, CH 3.23 (m) 4′ 69.7, CH 3.08 (m) 5′ 77.5, CH 3.23 (m)
6′ 61.0, CH2 3.72 (brd, 11.6), 3.49 (m) 2′/3′/4′/6′-OH 5.37 (brs, 1H), 5.02 (brs, 2H), 4.46 (brs, 1H)
[a] Assignments supported by gCOSY, gHSQC and gHMBC experiments (Figures S24-26, S28-32). * Overlapped signals
S11
Table S5. 13C (100 MHz) and 1H (400 MHz) NMR spectroscopic data of panobinostat (4) and [a] β-D-glucosyl-panobinostat (4a) in DMSO-d6.
Position panobinostat (4) β-D-glucosyl-panobinostat (4a)
δC, type δH (mult, J in [Hz]) δC, type δH (mult, J in [Hz])
1 10.66 (s) 10.87 (s) 2 131.7, C 132.7, C 2-CH3 11.3, CH3 2.30 (s) 11.1, CH3 2.32 (s) 3 108.2, C 104.9, C 3a 127.3, C 127.7, C 4 117.3, CH 7.36 (brd, 7.3) 117.1, CH 7.41 (d, 7.6) 5 118.0, CH 6.88 (t, 7.4) 118.4, CH 6.95 (td, 7.4, 1.2) 6 119.8, CH 7.95 (t, 7.5) 120.3, CH 7.00 (td, 7.0, 1.2) 7 110.3, CH 7.20 (d, 7.7) 110.6, CH 7.25 (d, 7.9) 7a 135.1, C 135.2, C
8 24.5, CH2 2.67 (t, 7.4) 20.7, CH2 3.01 (brm)
9 49.7, CH2 2.79 (t, 7.3) 47.1, CH2 3.07 (brm) 10 9.00 (brs) 11 52.5, CH2 3.74 (s) 49.6, CH2 4.24 (brs)
12 133.1, C 133.7, C 13/17 128.3, CH 7.48 (brd, 7.9) 130.4, CH 7.55 (brd, 8.1) 14/16 127.3, CH 7.34 (brd, 7.3) 128.1, CH 7.41 (brd, 7.7) 15 138.1, C 135.1, C 18 142.6, CH 7.42 (d, 15.8) 139.9, CH 7.59 (brd, 16.2) 19 118.3, CH 6.43 (d, 15.8) 118.7, CH 6.58 (d, 16.1) 20 162.8, C 164.2, C 22-NH 11.72 (s) 1′ 105.9, CH 4.60-4.40 (brm) 2′ 71.6, CH 3.13-3.03 (m) 3′ 75.9, CH 3.24 (m) 4′ 69.7, CH 3.13-3.03 (m) 4′-OH 5.03 (d, 5.4)
5′ 77.6, CH 3.24 (m)
6′ 61.0, CH2 3.72 (brdd, 11.8, 5.0), 3.48 (m) 2′/3′/6′-OH 5.32 (brs, 1H), 5.10 (brs, 1H), 4.51 (brs, 1H)
[a] Assignments supported by gCOSY, gHSQC and gHMBC experiments (Figures S33-35, S37-41).
S12
Table S6. 13C (100 MHz) and 1H (400 MHz) NMR spectroscopic data of tubastatin A (5) and β- [a] D-glucosyl-tubastatin A (5a) in DMSO-d6.
Position tubastatin A (5) β-D-glucosyl-tubastatin A (5a)
δC, type δH (mult, J in [Hz]) δC, type δH (mult, J in [Hz]) 2 133.8, C 131.2, C 3 107.9, C 102.4, C 3a 125.2, C 124.4, C 4 117.4, CH 7.38 (brd, 7.2) 117.8, CH 7.49 (d, 8.5) 5 118.8, CH 6.98 (td, 7.4, 1.1) 119.8, CH 7.09 (t, 7.4) 6 120.6, CH 7.03 (m) 122.0, CH 7.16 (t, 8.7) 7 109.5, CH 7.35 (brd, 7.9) 110.1, CH 7.47 (d, 9.2) 7a 136.2, C 136.6, C
8 22.5, CH2 2.79-2.66 (brm) 19.7, CH2 3.08 (brm)
9 51.3, CH2 2.79-2.66 (brm) 50.2, CH2 3.76 (brm), 3.51 (brm)
10-CH3 45.5, CH3 2.42 (s) 41.9, CH3 3.00 (s)
11 52.0, CH2 3.55 (brs) 50.6, CH2 4.66 (brs), 3.34 (brs)
12 45.4, CH2 5.37 (s) 45.6, CH2 5.48 (brs)
13 141.6, C 142.0, C 14/18 126.3, CH 7.04 (d, 8.3) 126.6, CH 7.14 (d, 8.1) 15/17 127.2, CH 7.65 (d, 8.3) 127.8, CH 7.75 (d, 8.0) 16 131.8, C 130.5, C 19 163.8, C 165.4, C 19-NH 10.23 (brs)[b] 12.08 (brs) 19-NHOH 10.23 (brs)[b] 106.5, CH 4.58 (d, 8.0) 71.9, CH 3.10 (m) 76.0, CH 3.28-3.16 (m) 69.7, CH 3.07 (m) 77.5, CH 3.28-3.16 (m)
61.0, CH2 3.70 (m), 3.47 (m) 5.30 (brs, 1H), 5.10 (brs, 1H), 4.50-4.30 (brs, 2H)
[a] Assignments supported by gCOSY, gHSQC and gHMBC experiments (Figures S42-44, S46-50). [b] Broad signals presumably due to the equilibrium,
S13
Spectroscopic Data
1 Figure S6. H NMR spectrum (DMSO-d6, 400 MHz) of abexinostat (1).
S14
13 Figure S7. C NMR spectrum (DMSO-d6, 100 MHz) of abexinostat (1).
S15
1 1 Figure S8. H- H COSY spectrum (DMSO-d6, 400 MHz) of abexinostat (1).
S16
[M+H]+
Figure S9. (+)-HRESI-MS of β-D-glucosyl-abexinostat (1a).
S17
1 Figure S10. H NMR spectrum (DMSO-d6, 400 MHz) of β-D-glucosyl-abexinostat (1a).
S18
13 Figure S11. C NMR spectrum (DMSO-d6, 100 MHz) of β-D-glucosyl-abexinostat (1a).
S19
1 1 Figure S12. H- H COSY spectrum (DMSO-d6, 400 MHz) of β-D-glucosyl-abexinostat (1a).
S20
Figure S13. HSQC spectrum (DMSO-d6, 400 MHz) of β-D-glucosyl-abexinostat (1a).
S21
Figure S14. HMBC spectrum (DMSO-d6, 400 MHz) of β-D-glucosyl-abexinostat (1a).
S22
1 Figure S15. H NMR spectrum (DMSO-d6, 400 MHz) of dacinostat (2).
S23
13 Figure S16. C NMR spectrum (DMSO-d6, 100 MHz) of dacinostat (2).
S24
1 1 Figure S17. H- H COSY spectrum (DMSO-d6, 400 MHz) of dacinostat (2).
S25
[M+H]+
Figure S18. (+)-HRESI-MS of β-D-glucosyl-dacinostat (2a).
S26
1 Figure S19. H NMR spectrum (DMSO-d6, 400 MHz) of β-D-glucosyl-dacinostat (2a).
S27
13 Figure S20. C NMR spectrum (DMSO-d6, 100 MHz) of β-D-glucosyl-dacinostat (2a).
S28
1 1 Figure S21. H- H COSY spectrum (DMSO-d6, 400 MHz) of β-D-glucosyl-dacinostat (2a).
S29
Figure S22. HSQC spectrum (DMSO-d6, 400 MHz) of β-D-glucosyl-dacinostat (2a).
S30
Figure S23. HMBC spectrum (DMSO-d6, 400 MHz) of β-D-glucosyl-dacinostat (2a).
S31
1 Figure S24. H NMR spectrum (DMSO-d6, 400 MHz) of givinostat (3).
S32
13 Figure S25. C NMR spectrum (DMSO-d6, 100 MHz) of givinostat (3).
S33
1 1 Figure S26. H- H COSY spectrum (DMSO-d6, 400 MHz) of givinostat (3).
S34
[M+H]+
Figure S27. (+)-HRESI-MS of β-D-glucosyl-givinostat (3a).
S35
1 Figure S28. H NMR spectrum (DMSO-d6, 400 MHz) of β-D-glucosyl-givinostat (3a).
S36
13 Figure S29. C NMR spectrum (DMSO-d6, 100 MHz) of β-D-glucosyl-givinostat (3a).
S37
1 1 Figure S30. H- H COSY spectrum (DMSO-d6, 400 MHz) of β-D-glucosyl-givinostat (3a).
S38
Figure S31. HSQC spectrum (DMSO-d6, 400 MHz) of β-D-glucosyl-givinostat (3a).
S39
Figure S32. HMBC spectrum (DMSO-d6, 400 MHz) of β-D-glucosyl-givinostat (3a).
S40
1 Figure S33. H NMR spectrum (DMSO-d6, 400 MHz) of panobinostat (4).
S41
13 Figure S34. C NMR spectrum (DMSO-d6, 100 MHz) of panobinostat (4).
S42
1 1 Figure S35. H- H COSY spectrum (DMSO-d6, 400 MHz) of panobinostat (4).
S43
[M+H]+
Figure S36. (+)-HRESI-MS of β-D-glucosyl-panobinostat (4a).
S44
1 Figure S37. H NMR spectrum (DMSO-d6, 400 MHz) of β-D-glucosyl-panobinostat (4a).
S45
13 Figure S38. C NMR spectrum (DMSO-d6, 100 MHz) of β-D-glucosyl-panobinostat (4a).
S46
1 1 Figure S39. H- H COSY spectrum (DMSO-d6, 400 MHz) of β-D-glucosyl-panobinostat (4a).
S47
Figure S40. HSQC spectrum (DMSO-d6, 400 MHz) of β-D-glucosyl-panobinostat (4a).
S48
Figure S41. HMBC spectrum (DMSO-d6, 400 MHz) of β-D-glucosyl-panobinostat (4a).
S49
1 Figure S42. H NMR spectrum (DMSO-d6, 400 MHz) of tubastatin A (5).
S50
13 Figure S43. C NMR spectrum (DMSO-d6, 100 MHz) of tubastatin A (5).
S51
1 1 Figure S44. H- H COSY spectrum (DMSO-d6, 400 MHz) of tubastatin A (5).
S52
[M+H]+
Figure S45. (+)-HRESI-MS of β-D-glucosyl-tubastatin A (5a).
S53
1 Figure S46. H NMR spectrum (DMSO-d6, 400 MHz) of β-D-glucosyl-tubastatin A (5a).
S54
13 Figure S47. C NMR spectrum (DMSO-d6, 100 MHz) of β-D-glucosyl-tubastatin A (5a).
S55
1 1 Figure S48. H- H COSY spectrum (DMSO-d6, 400 MHz) of β-D-glucosyl-tubastatin A (5a).
S56
Figure S49. HSQC spectrum (DMSO-d6, 400 MHz) of β-D-glucosyl-tubastatin A (5a).
S57
Figure S50. HMBC spectrum (DMSO-d6, 400 MHz) of β-D-glucosyl-tubastatin A (5a).
S58