Supplementary information
DMN-Tre labelling for detection and High Content Screening of compounds against intracellular Mycobacteria
Henok A. Sahile1, Celine Rens1, Tirosh Shapira1, Raymond J. Andersen3, Yossef Av-Gay 1, 2*
1Division of Infectious Diseases, Departments of Medicine and 2Microbiology and Immunology, Life Sciences Institute, University of British Columbia, 2350 Health Sciences Mall, V6T 1Z3, Vancouver, British Columbia, Canada. 3Department of Earth, Ocean and Atmospheric Sciences, Faculty of Science, University of British Columbia, 2036 Main Mall, Vancouver, BC, V6T 1Z1, Canada.
Contents
Synthesis of DMN-Tre...S2 General experimental methods...S2 Synthetic protocol...S3 1HNMR spectra of DMN-Tre...S8 Table S1: List of Mtb mutants resistant to the corresponding anti-Mtb compounds...S9 Figures S2-S6: DMN-Tre labelling of Mtb mutants in vitro...S10 Figure S7: Effect of DMN-Tre on the viability of THP-1...S14 Figure S8: Intracellular labelling of different mycobacterial species with DMN-Tre...S15 References...S16
Synthesis of DMN-Tre
General experimental methods
All chemical reagents obtained from commercial suppliers were used without further purification. Merck Type 5554 silica gel plates and Whatman MKC18F plates were used for analytical thin layer chromatography. Purification of intermediate compounds were performed by column chromatography using Silica gel 60 (0.063–0.200 mm, Merk) for the stationary phase. 1H NMR spectra were recorded on a Bruker AV-600 spectrometer with a 5 mm CPTCI cryoprobe. Low and high resolution ESI-QIT-MS were recorded on a Bruker-Hewlett Packard 1100 Esquire–LC system mass spectrometer. Reversed-phase HPLC purifications were performed on a Waters 1525 Binary HPLC Pump attached to a Waters 2998 Photodiode Array Detector using a C18 column (InertSustain, 5 µm, 25 x 1 cm). Solvent A consisted of water; solvent B consisted of acetonitrile. The method involved isocratic elution, with a flow rate of 2.0 mL/min (30% A, 70% B). All solvents used for HPLC were Fisher HPLC grade.
Synthetic protocol 2, 3, 4, 6, 2’, 3’, 4’, 6’-Octakis-O-(trimethylsilyl)- α,α-trehalose (1) O O O TMSO OTMS TMSO TMSO TMSO OTMS OTMS OTMS
Compound 1 was synthesized from trehalose following the method of Toubiana et al1. Trimethylsilyl chloride (7.6 mL, 12 mmol) was added drop wise over a period of 1 hour to a 0 °C-cooled and stirred solution of trehalose (1.71 g, 5 mmol) in pyridine (30 mL). The reaction mixture was then stirred for additional 18 hours at room temperature and then partitioned between ethyl acetate (100 mL) and cold water (100 mL). The organic layer was separated, washed twice with cold water (50 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The pyridine was then co-evaporated twice with toluene to obtain 1 as a white solid. The isolated yield was 5.1 g(90%), 1H NMR (400 MHz, CDCl3) δ 4.91 (d, J = 3.1 Hz, 2H), 3.88 (t, J = 8.8 Hz, 2H), 3.80-3.77 (m, 2H), 3.71-3.64 (m, 4H), 3.42 (t, J = 9.3 Hz, 2H), 3.39 (dd, J = 3.1, 9.3 Hz, 2H), 0.14 (s, 18H), 0.13 (s, 18H), 0.11 (s, 18H), 0.09 (s, 18H).
2, 3, 4, 2’, 3’, 4’, 6’-Octakis-O-(trimethylsilyl)- α,α-trehalose-6-diol (2) O O O HO OTMS TMSO TMSO TMSO OTMS OTMS OTMS
Compound 2 was synthesized from 1 following the method of Narouz et al2. Briefly, compound 1 (3.0 g, 3.3 mmol) was kept in pre-cooled 0.2 % methanolic K2CO3 (40 mL) and stirred for 20 minutes while the reaction mixture was maintained at 0 °C. After quenching the reaction by addition of acetic acid (4 mL), the mixture was partitioned between chloroform (100 mL) and water (100 mL) and the aqueous layer was washed twice with cold chloroform (50 mL). The combined chloroform extract was dried over anhydrous Na2SO4, filtered and concentrated in vacuo to get a colourless semisolid mass that was purified on column chromatography using silica gel as the stationary phase and ethyl acetate/hexane (1:13) as the mobile phase. The yield was 1.1 g (40 %), mp 76-79, ESI-MS m/z calculated for C33H78O11Si7:846.39; found: 845.5[M-H]-.
6-Azido-6-deoxy-α,α-trehaloses (5) O O O N3 OH HO HO HO OH OH OH
Compound 2 (0.8 g, 0.9 mmol) was charged into a dry round-bottom flask equipped with magnetic stirring bar. The flask was sealed with a rubber septum and subjected to high vacuum for 2–3 h to ensure anhydrous conditions. Anhydrous CH2Cl2 (10 mL/g) and pyridine (4 mL) were added to the flask and the reaction mixture was cooled to 0 °C while being stirred. Triflic anhydride (2 eq, 302 µL, 1.8 mmol) was added slowly while the reaction mixture was continually maintained at 0 °C. After 2 h stirring at room temperature, the solvent was concentrated in vacuo and kept under strong vacuum overnight to ensure complete removal of traces of pyridine. The crude mass collected in the above step (compound 3) was dissolved in anhydrous DMF (6 mL) and kept at 0 °C; NaN3 (5 eq, 0.29 g, 4.5 mmol) was then added. The reaction mixture was warmed up to room temperature and stirred overnight, then poured into water and extracted with ethyl acetate (3 x 40 mL). The combined organic layer was washed with water, dried over anhydrous Na2SO4, filtered and evaporated. The crude mass (containing compound 4) was incubated with a cleaving cocktail made of trifluoroacetic acid/tetrahydrofuran/water (8:17:33, 10 mL) for 1 h at room temperature with stirring until TLC analysis showed the hydrolysis was complete. Purification by chromatography on silica, followed by eluting with chloroform/methanol/hexane (10:4:1) afforded compound 5 as an amorphous solid. The isolated yield was 109 mg (33 %), mp 193-195 °C, 1H NMR (D
2O); δ 5.15, 5.14 (2d, 2H, J = 4.5, 4.0 Hz); 3.93-3.89 (m, 1H); 3.81–3.37 (m, 11H); ESI-MS m/z: calculated for C12H21N3O10, 367.31; found 366.3 [M - H+]- .
6-Amino-6-deoxy-α,α-trehalose (6) O O O H2N OH HO HO HO OH OH OH
Compound 5 (90 mg, 0.25 mmol), methanol (40 mL) and a catalytic amount of Pd/C (10 %) were added to a dry round-bottom flask equipped with a magnetic stirring bar. The flask was then sealed with a rubber septum and purged with argon gas to displace the air from the flask. The mixture was stirred under hydrogen gas for 1 hour until the hydrogenation was complete (as monitored by TLC). Finally, the catalyst was filtered and the solvent was evaporated to obtain compound 6 as a white solid mass. Yield = 75 mg (90 %), ESI-MS m/z calculated for C12H23NO10:341.13; found: 342.24[M+H]+.
Synthesis of 4-N, N-dimethylaminonaphthalic anhydride(7)
O O
O
N
Compound 7 was synthesized following the method of Kollár et al.3. 4-Bromo-1,8-naphthalic anhydride (1.4 g, 5 mmol) was dissolved in 3-methyl-1-butanol (35 ml) and the solution was heated to 132 °C while being stirred. Following this, 3-dimethylaminopropionitrile (20 mmol) was added and the reaction was allowed to continue for 12 h. After the completion of the reaction, as monitored by TLC, the formed crystal was filtered and washed with cool isohexane to yield an orange crystal. Yield = 0.94 g (78 %), mp 205-07 °C, literature3 mp 208-210 °C; ESI-MS m/z calcd for C14H11NO3:241.07; found: 264.29 [M+Na]+.
Synthesis of DMN-Tre (8) O O O N OH HO HO OH OH OH N O O HO
DMN-Tre was synthesized from 6 and 7 following the method of Kamariza et al.4. 6-Amino-6-deoxy-α,α-trehalose (6, 35 mg, 0.1 mmol) and 4-N,N-dimethylaminonaphthalic anhydride (24 mg, 0.1 mmol) (7) were charged to a flask along with 5 mL of absolute ethanol. Sodium bicarbonate (25 mg) was added to the mixture and formed a yellow suspension that was heated to 85 °C under argon atmosphere and stirred for 8 h until it turned to a red-orange solution. The obtained red-orange solution was concentrated by rotary evaporation and the suspension was dissolved in 10 mL of H2O, filtered through a plug of cotton, and re-concentrated. The remaining orange residue was first purified by column chromatography on silica gel using chloroform/methanol/hexane (10:4:1) then purified by reversed-phased HPLC (70 % MeCN in H2O). Yield of DMN-Tre (8) = 20 mg (35 %), RP-HPLC (UV) purity: 99 %, RT 27.3 min, HRMS m/z: calculated for C26H32N2O12 [M+Na]+ 587.1853, found 587.1850. 1H NMR (400 MHz, D2O) δ 8.21 (d, J = 8.5 Hz, 1H), 8.13 (d, J = 7.3 Hz, 1H), 7.95 (d, J = 8.4 Hz, 1H), 7.46 (t, J = 7.9 Hz, 1H), 6.90 (d, J = 8.6 Hz, 1H), 4.97 (d, J = 3.8 Hz, 1H), 4.53 (d, J = 3.7 Hz, 1H), 4.23 (d, J = 6.2 Hz, 2H), 4.03 (dt, J = 12.3, 6.4 Hz, 1H), 3.79 (t, J = 9.4 Hz, 1H), 3.73 – 3.61 (m, 4H), 3.56 (dd, J = 12.1, 5.4 Hz, 1H), 3.44 (t, J = 9.5 Hz, 1H), 3.18 (t, J = 9.5 Hz, 1H), 3.07 (m, 1H), 3.05 (s, 6H).
1HNMR spectra of DMN-Tre
Figure S1: 1HNMR spectra of DMN-Tre. a) measured 1H NMR spectrum of DMN-Tre
(400 MHz, D2O), b) 1H NMR spectrum of DMN-Tre previously reported in the literature4 (600 MHz, D2O).
Table S1: List of Mtb mutants resistant to the corresponding anti-Mtb compounds
Identified anti-Mtb compound (Lab ID)
Mtb mutant strain grown Mtb 66-1 YA-66 Mtb 66-2 Mtb 66-3 Mtb 31-1 Mtb 31-2 YA-31 Mtb 31-3 Mtb 25-2 Mtb 25-3 YA-25 Mtb 25-4 Mtb 15-3 YA-15 Mtb 15-4 YA-5 Mtb 5-1 Mtb 1-1 YA-1 Mtb 1-2
Figures S2-S6: DMN-Tre labelling of Mtb mutants in vitro
Figure S2: In vitro labelling with DMN-Tre (75 µM) of three Mtb mutants grown in the
presence of the identified anti-Mtb hit compound, YA-66. Images were collected in the differential interference contrast (DIC), green fluorescent protein (GFP, DMN fluorescence), or red fluorescent protein (RFP, dTomato fluorescence) channels using the Evos FL auto cell imaging system fluorescence microscope (Thermo Fisher). Magnification 20X. “Overlay” represents merged images from all channels. Scale bars, 50 μm.
Figure S3: In vitro labelling with DMN-Tre (75 µM) of three Mtb mutants grown in the presence of the identified anti-Mtb hit compound, YA-31. Scale bars, 50 μm.
Figure S4: In vitro labelling with DMN-Tre (75 µM) of three Mtb mutants grown in the presence of the identified anti-Mtb hit compound, YA-25. Scale bars, 50 μm.
Figure S5: In vitro labelling with DMN-Tre (75 µM) of two Mtb mutants grown in the
Figure S6: In vitro labelling with DMN-Tre (75 µM) of three Mtb mutants grown in the presence of the identified anti-Mtb hit compounds, YA-5 or YA-1. Scale bars, 50 μm.
Figure S7: Effect of DMN-Tre on the viability of human monocyte-derived
macrophages (THP-1). Viability was determined by cell count as detected by High-Content
Screening of cells that had been grown without (vehicle) or with DMN-Tre. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) for visualization. Cells numbers were sampled from identical sections of the experimental culturing wells. Data are means + SD from six independent trials.
Figure S8: Intracellular labelling of different mycobacterial species with DMN-Tre. Images were collected in the DIC, DAPI (Hoechst fluorescence), or GFP (DMN-Tre fluorescence) channels of the Leica SP5 laser scanning confocal microscope (Nikon A1R). Magnifications 63X by oil immersion. “Overlay” represents merged images from all channels. Scale bars, 25 μm.
References
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(2) Narouz, M. R.; Soliman, S. E.; Bassily, R. W.; El-Sokkary, R. I.; Nasr, A. Z.; Nashed, M. Improved Synthesis of 6-Azido-6-Deoxy- and 6,6′-Diazido-Dideoxy-
α,α-Trehaloses. Synlett2013, (24), 2271–2273. https://doi.org/10.1055/s-0033-1339843.
(3) Kollár, J.; Hrdlovič, P.; Chmela, Š.; Sarakha, M.; Guyot, G. Synthesis and Transient Absorption Spectra of Derivatives of 1,8-Naphthalic Anhydrides and Naphthalimides Containing 2,2,6,6-Tetramethylpiperidine; Triplet Route of Deactivation. J.
Photochem. Photobiol. A Chem.2005, (170), 151–159. https://doi.org/10.1016/j.jphotochem.2004.07.021.
(4) Kamariza, M.; Shieh, P.; Ealand, C. S.; Peters, J. S.; Chu, B.; Rodriguez-Rivera, F. P.; Babu Sait, M. R.; Treuren, W. V.; Martinson, N.; Kalscheuer, R.; et al. Rapid
Detection of Mycobacterium Tuberculosis in Sputum with a Solvatochromic Trehalose Probe. Sci. Transl. Med.2018). https://doi.org/10.1126/scitranslmed.aam6310.
