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 - 673 61; E-mail: 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,
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
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 = 384.11). 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.
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 1.293 Å (blue) and (b) 2D fingerprint plots of all the intermolecular contacts with d i and d e ranging from 1. to 2.627 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.
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.
% Transmittance % Transmittance 1 NMe 2 T 9 8 7 4 35 3 25 2 15 1 5 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, 666. 4-OH 2 + : IR (KBr, cm -1 ): 3735, 3397, 2372, 1631, 1558, 1538, 1479, 1413, 1398, 126, 181, 861, 742, 728, 666.
Absorption a.u luorescence Intensity a.u 4-OH 2 + 4-OH igure S7. Overlaid of 1 H NMR spectra of compound 4 and 4-OH 2 +. 1..8.6.4.2. 3 35 4 45 5 55 6 65 Wavelength (nm) igure S8. Normalized optical and fluorescence profiles of 2 in toluene at 298 K, λ ex = 48 nm.
luorescence Intensity luorescence Intensity a.u luorescence intensity a.u luorescence intensity a.u 15 1 5 EtOH MeOH MeCN DM DMSO 25 1 2 2 15 1 5 EtOH MeOH MeCN DM DMSO 5 55 6 65 7 Wavelength (nm) 1 5 55 6 65 7 Wavelength (nm) 3 7 4 8 6 6 4 EtOH MeOH MeCN DM DMSO 5 4 3 EtOH MeOH MeCN DM DMSO 2 2 1 5 55 6 65 Wavelength (nm) 5 55 6 65 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.
luorescence Intensity Absorbance a.u Absorbance a.u Absorbance a.u Absorbance a. u 1..75.5.25 1 2 3 4 5 6 7 8 9 1 11 12 13 14.25.125 1 2 3 4 5 6 7 8 9 1 11 12 13. 3 4 5 6 Wavelength (nm) 1.4 1.2 1..8.6.4.2 1 2 3 4 5 6 7 8 9 1 11 12 13 14. 3 4 5 6 Wavelength (nm). 3 4 5 6.4.3.2.1 1 2 3 4 5 6 7 8 9 1 11 12 13 14 Wavelength (nm). 3 35 4 45 5 55 6 65 Wavelength (nm) igure S1. ph responsive absorbance spectra of 1-4 (4 x 1-6 M) at 298 K in ph ranging from 1 to14. 7 6 5 4 3 2 1 2 4 6 8 1 12 14 ph igure S11. ph responsive fluorescence spectra of 3 (4 x 1-6 M) at 298 K.
luorescence intensity a.u luorescence intensity a.u 35.5 2.9 3 25 2 15 1 5 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. 12 5.5 1 8 6 4 2 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.
igure S14. (a) Hirshfeld surfaces with normalized contact distance ranging from -.84 Å (red) to 1.293 Å (blue) and (b) 2D fingerprint plots of all the intermolecular contacts with d i and d e ranging from 1. to 2.627 Å for 1 and 2. 2 CC HH H CH Others 1 2 4 6 8 1 % Interaction igure S15. Percentage interactions of various intermolecular contacts for 1 and 2.
Energy Energy (ev) (ev) 2 +1 -.71-1. -.99-2 -2-1.48-2.76-3.13-2.96-3.19-4 -4 19.19 19.7-6 HOMO -6.17 HOMO-1-7.31-6.39-6.64-6.34-7.1-6 -6.52-7.23-8 1. 1.2 1.4-8 1. 1.1 1.2 1.3 1.4-1 igure S16. Molecular orbital diagrams of compounds, 1-4. -2
Table S1. Photophysical data of 1-4 (λ ex = 48 nm). Cpd 1 2 3 Solvent λ abs (nm) λ em (nm) Stokes shift (nm) Cpd Solvent λ abs (nm) λ em (nm) Stokes shift (nm) MeOH 488 557 2538.2 Toluene 51 518 655.81 a MeCN 483 55 2522.28 EtOH 496 59 515.15 3 TH 492 543 1868.3 DM 496 511 592.15 DCM 489 52 1219.1 b DMSO 498 514 625.11 Toluene 498 626 468.21 MeOH 49 521 1214.26 EtOH 489 53 1582.1 MeCN 54 516 461.32 DM 486 515 1159.3 TH 57 526 712.72 DMSO 487 518 1229.5 DCM 51 529 74.5 c 4 MeOH 513 534 767.3 Toluene 512 532 734.127 MeCN 511 534 843.3 EtOH 487 522 1377.8 TH 514 537 833.1 DM 475 51 1445.16 DCM 516 537 758.12 DMSO 478 525 1873.12 Toluene 519 538 68.55 MeOH 497 514 665.26 EtOH 513 533 731.2 MeCN 496 513 668.3 DM 514 536 799.2 Phenyl- TH 499 516 66.34 DMSO 516 538 792.1 BODIP DCM 5 527 124.3 c MeOH 493 58 599.15 a Y Toluene 51 522 81.44 d MeCN 493 58 599.23 a EtOH 498 514 625.35 TH 495 51 594.58 DM 5 52 769.3 DCM 497 512 589.6 c DMSO 52 523 8.34 a J. Org. Chem., 25, 7, 4152 4157; b Eur. J. Org. Chem., 211, 546 5468, c RSC Adv., 216, 6, 888 8824; d J. Phys. Chem. B, 213, 117, 5533 5539.
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 1 1.2996 2.7284 454.43 HOMO-1.671689 9.2 HOMO -.2897 8.7 2.3674 2.962 418.58 HOMO-2.149181 4.5 HOMO-1.2588 8.5 HOMO.66121 87.4 3.632 3.5511 349.14 HOMO-.688252 94.7 2 HOMO -.1457 4.2 4.325 3.763 329.72 HOMO-3.7271 98.8 5.27 3.9652 312.68 HOMO-4.68832 92.7 HOMO-1.17326 6. +1 HOMO -.1973 7.8 +1 2 1.1746 2.794 444.33 HOMO-1.719 98.5 2.3753 2.8814 43.29 HOMO-3 -.1315 3.4 HOMO.689861 95.1 HOMO -.114 2.4 3.78 3.4428 36.13 HOMO-3 -.47129 44.4 HOMO-2 -.49817 49.6 HOMO -.16752 5.6 4.72 3.5336 35.87 HOMO-3.63631 72.9 HOMO-2.489619 47.9 5.312 3.6366 34.94 HOMO-4.735 98.9 3 1.3773 2.955 419.57 HOMO-2 -.15831 5. HOMO.69798 95.4
HOMO 2.27 3.2351 383.25 HOMO-1 3.729 3.546 35.18 HOMO-2 HOMO 4.384 3.7454 331.3 HOMO-3 5.25 4.465 36.4 HOMO-4 HOMO-1 +1 4 1.366 2.9279 423.45 HOMO-3 HOMO-1 HOMO HOMO 2.1737 3.2589 38.45 HOMO-1 3.664 3.4928 354.97 HOMO-3 HOMO-2 HOMO 4.136 3.6655 338.25 HOMO-4 HOMO-3 HOMO-2 5.428 3.715 334.96 HOMO-4 HOMO-3 -.1144 2.1.7158 98.4.69656 97..16119 5.2.71959 98.5.6959 96.6 -.11933 2.8 -.11579 2.7.116449 2.7.689949 95.2 -.143 2.2.7151 98.3 -.32544 21.2.6366 72.8 -.15972 5.1 -.13752 3.8.654 72.1.328881 21.6.688391 94.8.115379 2.7