Electronic Supplementary Information. For. A turn-on-and-off ph sensitive BODIPY fluorescent probe for imaging E. coli cells

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1 Electronic Supplementary Material (ESI) for New Journal of Chemistry. This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 218 Electronic Supplementary Information or A turn-on-and-off ph sensitive BODIPY fluorescent probe for imaging E. coli cells Dijo Prasannan and Chellaiah Arunkumar* Bioinorganic Materials Research Laboratory, Department of Chemistry, National Institute of Technology Calicut, Kozhikode, Kerala, India ; arunkumarc@nitc.ac.in Contents Experimental Materials and methods All the chemicals used for the synthesis were reagent grade unless otherwise specified. Pyrrole and Borontrifluoride etherate (B 3 OEt 2 ) purchased from Spectrochem (India) was distilled over CaH 2 before use. 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) was obtained from Sigma Aldrich were used as such. Trifluoroacetic acid (TA) was purchased from Merck (India) and was distilled over P 2 O 5. Triethylamine (Et 3 N) purchased from Qualigens was used as such. Sodium chloride, Potassium chloride, Sodium hydrogen phosphate (Na 2 HPO 4 ), Monopotassium phosphate (KH 2 PO 4 ) and Dimethylsulphoxide (DMSO) and dimethylformamide (DM) were also purchased from Merck (India) and was used as such. Tetrabutylammonium hexafluorophosphate (NBu 4 P 6 ) was purchased from Alfa Aeser. Chloroform, dichloromethane, hexane, ethanol and methanol were purchased from Avra Synthesis Pvt. Ltd. and were purified by distilling over K 2 CO 3. Toluene and acetonitrile were purchased from ischer Scientific. TH was obtained from SRL Chemicals Ltd. India. 1 H NMR spectra were recorded with a Bruker 4 MHz T-NMR spectrometer in CDCl 3 using tetramethylsilane as the internal reference. IR data was collected on a JASCO T/IR-47 with KBr pellets. UV-Visible spectra were recorded on a Shimadzu double beam spectrometer 245 instrument using 1 cm matched quartz cuvettes at room temperature. luorescence spectra were recorded on a Perkin-Elmer LS-55 Luminescence spectrophotometer with a slit width of 9 at 48 nm excitation wavelength and emission from 5 to 75 nm. The fluorescence quantum yield (Φ f ) was computed using the below equation,

2 Φ f = Φ R f S A R ɳ 2 S/ R A S ɳ 2 R, where S and R are the integrated fluorescence intensities of the sample and reference, A S and A R are the absorbance of the sample and reference at excitation wavelength, ɳ S and ɳ R are the refractive indexes of the solvents used for the sample and reference. luorescein in.1 M NaOH solution was used as the reference (Φ f =.9, λ ex = 47 nm). All Φ values are corrected for changes in refractive index. Both the BODIPYs and reference solutions were prepared with the same absorbance at the excitation wavelength (between.1 and.5 in a 1 cm quartz cell). Microorganism and growth conditions Escherichia coli (MTCC 1847) were purchased from Institute of Microbial Technology (IMTECH), Chandigarh, India. The bacterial strain was maintained in viable state via inoculation on Mueller-Hinton broth (MHB) and overnight incubation at 37 C. Intracellular localization of BODIPYs by fluorescence microscopy Initially, E. coli were grown in the liquid broth for 3 minutes in the presence of compounds 1 and 4 at desired ph in a shaking incubator at 37 C. The cells were harvested and washed twice with 1X PBS in order to remove the uninternalized compounds. The cells were then resuspended in PBS buffer, fixed with 3 % glutaraldehyde (grade I) for 3 minutes, mounted on clean glass slides with a drop of glycerol and sealed for microscopic analysis. The compounds were excited using a green filter (49 51 nm) to observe the intracellular localization of the BODIPY. The fluorescence imaging was performed using an Olympus- IX73 microscope attached with a digital camera at 1X magnification. The images were processed using cellsens software. Synthesis of 2 NMe 2 HCl N Et 3 N, 2 min, B 3 OEt 2, 1 h N DM, 8 O C, 41h DCM, RT NH N NH N N B 2 N

3 Pentafluoro dipyrromethane was converted to meso-(2,3,4,5,6-pentafluorophenyl) dipyrrin via DDQ oxidation. The dipyrrin was purified by following the standard protocols. The dipyrrin was refluxed with dimethylamine hydrochloride (25 equiv.) at 8 C for 41 h in DM. urther solvent was evaporated, extracted with DCM and the combined organic layers were washed with water; dried over Na 2 SO 4, filtered and concentrated in vacuum. The crude product was purified by silica gel column chromatography in hexane/ chloroform to yield meso-(6-n,ndimethyl)-(2,3,4,5-tetrafluorophenyl)dipyrrin in 12 % yield. This dipyrrin were then reacted with Et 3 N for 2 min followed by complexation with B 3 OEt 2 (1 h) at room temperature. Column chromatography on silica gel yielded the meso-substituted BODIPY dye 2 in 45 % yield. 1 H NMR (4 MHz, CDCl 3, δ in ppm): 3.9 (6H, t), 6.54 (2H, d), 6.8 (2H, d), 7.93 (2H, s). IR (KBr, cm 1 ): 3459, 2926, 271, 2372, 1738, 1644, 1546, 1483, 1429, 1414, 1387, 1351, 1251, 1221, 118, 112, 166, 121, 977, 859, 8, 772, 754. Mass (m/z = 383.1, observed = ). Preparation of stock solution Stock solutions of the BODIPYs dyes (1 mm) were prepared in ethanol. urther, 1 to 4 µl of this was pipette into respective ph solution (3 ml) to prepare the desired working solutions to record the absorption or emission spectra. All optical measurements were performed at room temperature in phosphate-buffered saline (PBS), ph 7.4. igure S1. 1 H NMR spectrum of meso-(6-n,n-dimethyl)-(2,3,4,5-tetrafluorophenyl)dipyrrin. igure S2. 1 H NMR spectrum of 2. igure S3. 13 C NMR spectrum of 2. igure S4. Mass spectrum of 2. igure S5. IR spectrum of 2. igure S6. Overlaid of IR spectra of compound 4 (red) and 4-OH + 2 (black). igure S7. Overlaid of 1 H NMR spectra of compound 4 and 4-OH + 2. igure S8. Normalized optical and fluorescence profiles of 2 in toluene at 298 K, λ ex = 48 nm. igure S9. (a) luorescence profiles of 1-4, (4 x 1-6 M) at 298 K in solvents of variable polarity, λ ex = 48 nm.

4 igure S1. (a) ph responsive absorbance spectra of 1-4 (4 x 1-6 M) at 298 K in ph ranging from 1 to14. igure S11. ph responsive fluorescence spectra of 3 (4 x 1-6 M) at 298 K. igure S12. luorescent intensity of 1 in EtOH / water at ph.5 and 5. in the presence of diverse metal ions. igure S13. luorescent intensity of 4 in EtOH / water at ph.5 and 5. in the presence of diverse metal ions. igure S14. (a) Hirshfeld surfaces with normalized contact distance ranging from -.84 Å (red) to Å (blue) and (b) 2D fingerprint plots of all the intermolecular contacts with d i and d e ranging from 1. to Å for 1 and 2. igure S15. Percentage interactions of various intermolecular contacts for 1 and 2. igure S16. Molecular orbital diagrams of compounds, 1-4. Table S1. Photophysical data of 1-4 (λ ex = 48 nm). Table S2. Calculated electronic excitation energies, oscillator strengths and wavelength.

5 igure S1. 1 H NMR spectrum of meso-(6-n,n-dimethyl)-(2,3,4,5-tetrafluorophenyl)dipyrrin.

6 igure S2. 1 H NMR spectrum of 2.

7 igure S3. 13 C NMR spectrum of 2.

8 igure S4. Mass spectrum of 2.

9 % Transmittance % Transmittance 1 NMe 2 T Wavenumber (cm -1 ) igure S5. IR spectrum of 2. Wavenumber (cm -1 ) igure S6. Overlaid of IR spectra of compound 4 (red) and 4-OH 2 + (black). 4: IR (KBr, cm -1 ): 349, 3133, 381, 2484, 164, 1558, 1538, 1478, 1412, 1397, 126, 18, 861, 742, 727, OH 2 + : IR (KBr, cm -1 ): 3735, 3397, 2372, 1631, 1558, 1538, 1479, 1413, 1398, 126, 181, 861, 742, 728, 666.

10 Absorption a.u luorescence Intensity a.u 4-OH OH igure S7. Overlaid of 1 H NMR spectra of compound 4 and 4-OH Wavelength (nm) igure S8. Normalized optical and fluorescence profiles of 2 in toluene at 298 K, λ ex = 48 nm.

11 luorescence Intensity luorescence Intensity a.u luorescence intensity a.u luorescence intensity a.u EtOH MeOH MeCN DM DMSO EtOH MeOH MeCN DM DMSO Wavelength (nm) Wavelength (nm) EtOH MeOH MeCN DM DMSO EtOH MeOH MeCN DM DMSO Wavelength (nm) Wavelength (nm) igure S9. (a) luorescence profiles of 1-4, (4 x 1-6 M) at 298 K in solvents of variable polarity, λ ex = 48 nm.

12 luorescence Intensity Absorbance a.u Absorbance a.u Absorbance a.u Absorbance a. u Wavelength (nm) Wavelength (nm) Wavelength (nm) Wavelength (nm) igure S1. ph responsive absorbance spectra of 1-4 (4 x 1-6 M) at 298 K in ph ranging from 1 to ph igure S11. ph responsive fluorescence spectra of 3 (4 x 1-6 M) at 298 K.

13 luorescence intensity a.u luorescence intensity a.u NM 3 Mn 2+ Na + Mg 2+ Ba 2+ Ca 2+ K + Cd 2+ Cu 2+ Ni 2+ Hg 2+ Co 2+ e 3+ Al Al 3+ Zn 2+ Cr Cr 3+ A igure S12. luorescent intensity of 1 in EtOH / water at ph.5 and 5. in the presence of diverse metal ions Br2PHMn 4 2+ Na + Mg 2+ Ba 2+ Ca 2+ K + Cd 2+ Cu 2+ Ni 2+ Hg 2+ Co 2+ e 3+ Al 3+ Zn 2+ Cr 3+ igure S13. luorescent intensity of 4 in EtOH / water at ph.5 and 5. in the presence of diverse metal ions.

14 igure S14. (a) Hirshfeld surfaces with normalized contact distance ranging from -.84 Å (red) to Å (blue) and (b) 2D fingerprint plots of all the intermolecular contacts with d i and d e ranging from 1. to Å for 1 and 2. 2 CC HH H CH Others % Interaction igure S15. Percentage interactions of various intermolecular contacts for 1 and 2.

15 Energy Energy (ev) (ev) HOMO HOMO igure S16. Molecular orbital diagrams of compounds,

16 Table S1. Photophysical data of 1-4 (λ ex = 48 nm). Cpd Solvent λ abs (nm) λ em (nm) Stokes shift (nm) Cpd Solvent λ abs (nm) λ em (nm) Stokes shift (nm) MeOH Toluene a MeCN EtOH TH DM DCM b DMSO Toluene MeOH EtOH MeCN DM TH DMSO DCM c 4 MeOH Toluene MeCN EtOH TH DM DCM DMSO Toluene MeOH EtOH MeCN DM Phenyl- TH DMSO BODIP DCM c MeOH a Y Toluene d MeCN a EtOH TH DM DCM c DMSO a J. Org. Chem., 25, 7, ; b Eur. J. Org. Chem., 211, , c RSC Adv., 216, 6, ; d J. Phys. Chem. B, 213, 117,

17 Table S2. Calculated electronic excitation energies, oscillator strengths and wavelength. Compound Excited State Oscillator strength (f) Excitation energy (ev) Wavelength (nm) Transition Coefficient of transition Percentage contribution HOMO HOMO HOMO HOMO HOMO HOMO HOMO HOMO HOMO HOMO HOMO HOMO HOMO HOMO HOMO HOMO HOMO HOMO HOMO HOMO HOMO HOMO HOMO

18 HOMO HOMO HOMO-2 HOMO HOMO HOMO-4 HOMO HOMO-3 HOMO-1 HOMO HOMO HOMO HOMO-3 HOMO-2 HOMO HOMO-4 HOMO-3 HOMO HOMO-4 HOMO