Electronic Supplementary Material (ESI) for RSC Advances. This journal is The Royal Society of Chemistry 2016 SUPPORTING INFORMATION Synthesis, Characterization and Biological Activity of Fluorescently Labeled Bedaquiline Analogues Jeroen A. Rombouts, Richard M.P. Veenboer, Cristina Villellas, Ping Lu, Andreas W. Ehlers, Koen Andries, Anil Koul, Holger Lill, Eelco Ruijter, Romano V. A. Orru, Koop Lammertsma, Dirk Bald,* J. Chris Slootweg* TABLE OF CONTENTS Purity Analysis of Fluorescent Bedaquiline Derivatives using HPLC Photochemical Characterization of Fluorescent Bedaquiline Derivatives Photochemical Characterization of Unbound Fluorophore B2 NMR Spectroscopy Data S2 S3 S4 S5 S1
Purity Analysis of Fluorescent Bedaquiline Derivatives using HPLC Using a HPLC system equipped with a Dr. Maisch chiral stationary phase, the presence of unreacted Bedaquiline was investigated. A 95:5 heptane:isopropanol solvent system at 0.7 ml min -1 flow rate, using a fixed column temperature of 25 C was found to give good resolution in time between peaks. A calibration curve was prepared in duplo using dilutions of Bedaquiline in the concentration range [0.000, 0.005, 0.025, 0.050, 0.250, 0.500] mol ml -1 to give a total of 12 data points, all within the linear response regime of the instrument. Fitting the instrument response I to the linear equation I = ac + b using linear regression gave I = 9.214. 10 8 C + 8.627. 10 2 with C the Bedaquiline concentration (in mol ml -1 ) and coefficient of determination R 2 = 99.92%. This linear equation has been used to calculate the concentration of unreacted Bedaquiline in the fluorescently labeled compounds. Results are as follows: Calculated concentration Bedaquiline freebase in C1: 0.085 mol%, n = 4, st. dev. = 0.039 mol% Calculated concentration Bedaquiline freebase in C2: 0.058 mol%, n = 4, st. dev. = 0.007 mol% Retention times under the given HPLC parameters are: Retention time Bedaquiline free base (from J&J, see General Information): 5 minutes 42 seconds Retention time Bedaquiline-butynoxy-BODIPY, C1: 15 minutes 15 seconds Retention time Bedaquiline-pentynoxy-eg 4 -BODIPY, C2: 63 minutes 8 seconds S2
Photochemical Characterization of Fluorescent Bedaquiline Derivatives Determination of absorption and emission spectra was performed using a Shimadzu UV-2501PC spectrophotometer (absorption measurements) and a Jobin Yvon Fluorolog spectrophotometer (fluorescence measurements). The solvent used was degassed, HPLC grade acetonitrile. Using measurements on an external standard (Aqueous fluorescein in 0.1 M NaOH), the quantum yield of fluorescence for C1 and C2 was calculated according to the following formula, where is quantum yield, Int is the fluorescence intensity (integrated area under curve of emission intensity, in arbitrary units), A is the sample absorbance at the exciting wavelength and is the refractive index of the solvents used. Superscript ref refers to values for the external standard. Absorbance measurements were performed in the linear regime of the spectrophotometer; emission measurements were checked to avoid emission detector saturation. = ref * Int Int ref * A ref A * ref Table 1: Calculation of Quantum Yield of fluorescence sample for C1 and C2, in acetonitrile sample ref Int (au) Int ref (au) A ref A ref C1 0.685 0.79 12361 6826 0.0356 0.0760 1.346 1.330 C2 0.696 0.79 11233 6826 0.0356 0.0680 1.346 1.330 To assess the usability of C1 and C2 in protic media, the quantum yield of fluorescence was determined using methanol as the solvent. For these experiments, aqueous Fluorescein 27 (CAS 76-54-0, obtained from Exciton Dye company) was used as external fluorescence standard. For completeness, normalized absorption and emission spectra in methanol are shown in Figure 1. Table 2: Calculation of Quantum Yield of fluorescence sample for C1 and C2, in methanol sample ref Int (au) Int ref (au) A ref A ref C1 0.632 0.87 44628 49874 0.00779 0.00955 1.327 1.330 C2 0.618 0.87 40679 49874 0.00779 0.00890 1.327 1.330 abs nm em nm QY C1 499 509 0.632 C2 499 508 0.618 Figure 1: Normalized absorption and emission wavelengths for C1 and C2, recorded in methanol. The inset tabulates absorption and emission wavelength maxima and the quantum yield of fluorescence. S3
Photochemical Characterization of Unbound Fluorophore B2 Quantum yield of fluorescence of compound B2 was measured to verify that the fluorophore properties are not in any way modified by the covalent attachment of Bedaquiline. For this experiment, aqueous Fluorescein 27 (CAS 76-54-0, obtained from Exciton Dye company) was used as external fluorescence standard. For ompleteness, normalized absorption and emission spectra of B2 recorded in acetonitrile are shown in Figure 2. Table 3: Calculation of Quantum Yield of fluorescence sample for B2, in acetonitrile sample ref Int (au) Int ref (au) A ref A ref B2 0.786 0.87 69530 49874 0.00779 0.01231 1.346 1.330 abs em QY nm nm B2 500 509 0.786 Figure 2: Normalized absorption and emission wavelengths for B2, recorded in acetonitrile. The inset tabulates absorption and emission wavelength maxima and the quantum yield of fluorescence. S4
NMR Spectroscopy Data, 1H NMR; 1H NMR Spectrum of 4-(but-3-ynoxy)benzaldehyde (A1) S5
1H NMR Spectrum of Butynoxy-BODIPY (B1) S6
1H NMR Spectrum of Bedaquiline-butynoxy-BODIPY (C1) S7
1H NMR Spectrum of Pentynoxy-eg4-OH S8
1H NMR Spectrum of Pentynoxy-eg4-benzaldehyde (A2) S9
1H NMR Spectrum of Pentynoxy-eg4-BODIPY (B2) S10
1H NMR Spectrum of Bedaquiline-pentynoxy-eg4-BODIPY (C2) S11
NMR Spectroscopy Data, 13C NMR; 13C NMR Spectrum of 4-(but-3-ynoxy)benzaldehyde (A1) S12
13C NMR Spectrum of Butynoxy-BODIPY (B1) S13
13C NMR Spectrum of Bedaquiline-butynoxy-BODIPY (C1) S14
13C NMR Spectrum of Pentynoxy-eg4-OH S15
13C NMR Spectrum of Pentynoxy-eg4-benzaldehyde (A2) S16
13C NMR Spectrum of Pentynoxy-eg4-BODIPY (B2) S17
13C NMR Spectrum of Bedaquiline-pentynoxy-eg4-BODIPY (C2) S18