A Comprehensive Studies of an Indolizine-Based Seoul-Fluor System

Abstract

Fluorescent quantum yield is one of the most important photophysical properties, governing the brightness of a given fluorescent molecule [1]. Understanding the relationship between the structure and the quantum yield of a certain fluorophore system often elicits the rational design of novel turn-on fluorescent biosensors [2]. Although there are dozens of reported fluorophore systems [3], only a few studies have systematically elucidated the change of fluorescent quantum yields from a structural point of view [4].

Experimental Section

2.1.1 General Experimental Information

The density functional theory (DFT) calculation in Sect. 2.1 was performed using the Materials Studio® 4.2 program (Accelrys Software Inc.). A generalized gradient approximation (GAA) for the exchange correlation function of Perdew, Burke, and Ernzerhof (PBE) was used together with double numerical with polarization basis set (DNP), which is implemented in DMol3.

¹H and ¹³C NMR spectra in Sect. 2.2 were recorded on Agilent 400-MR (Agilent Technologies) and Varian Inova-500 (Varian Associates), and chemical shifts were measured in ppm downfield from internal tetramethylsilane (TMS) standard. Multiplicity was indicated as follows: s (singlet); d (doublet); t (triplet); q (quartet); m (multiplet); dd (doublet of doublet); dt (doublet of triplet); br s (broad singlet), etc. Coupling constants were reported in Hz. Routine mass analyses were performed on LC/MS system equipped with a reverse phase column (C-18, 50 × 2.1 mm, 5 μm) and photodiode array detector using electron spray ionization (ESI) or atmospheric pressure chemical ionization (APCI). The identity of desired fluorescence compounds were further confirmed by high-resolution mass spectrometry (HRMS). The HRMS analyses were conducted at the Mass Spectrometry Laboratory of Seoul National University by direct injection on a JEOL JMS 600 W spectrometer using electron impact (EI) or chemical ionization (CI), and JEOL JMS AX505WA spectrometer using fast atom bombardment (FAB) method. Excitation maxima and emission maxima were measured by Cary Eclipse Fluorescence spectrophotometer (Varian Assocociates) and absolute fluorescent quantum yield of all compounds in this chapter was measured by QE-1000 (OTSUKA Electronics). Triethylamine, diisobutylaluminium hydride, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), sodium borohydride, bromoacetyl bromide, sodium hydride, 4-acetyl pyridine, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), trifluoroacetic acid, acetic acid, anhydrous dimethyl formamide (DMF) were purchased from Sigma-Aldrich and Tokyo Chemical Industry Co., LTD. The progress of reaction was monitored using thin-layer chromatography (TLC) (silica gel 60, F₂₅₄ 0.25 mm), and components were visualized by observation under UV light (254 and 365 nm) or by treating the TLC plates with anisaldehyde, KMnO₄, and ninhydrin followed by heating. Solvents were purchased from commercial venders and used without further purification. Distilled water was polished by ion exchange and filtration. Biochemical reagents were purchased from Sigma-Aldrich. Ez-cytox kit was purchased from Daeil Co. and was used for the cell viability test. Commercial dyes Nile Red were purchased from Invitrogen.

Fluorescence microscope and analysis program for Bio-Imaging experiment.

Fluorescence microscopy studies for mammalian LDs were conducted with Olympus Inverted Microscope Model IX71, equipped for epi-illumination using a halogen bulb (Philips No. 7724). Emission signal of each experiments were observed at two spectral setting: green channel, using a 450–480 band pass exciter filter, a 500 nm center wavelength chromatic beam splitter, a 515 nm–long pass barrier filter (Olympus filter set U-MWB2); and red channel using a 510–550 band pass exciter filter, a 570 nm center wavelength chromatic beam splitter, a 590 nm–long pass barrier filter (Olympus filter set U-MWG2). Emission signals of each experiment were detected with 12.5 M pixel recording digital color camera (Olympus, DP71) Algae study was carried out using a FluoView FV1000 confocal laser scanning unit with Olympus Inverted Microscope Model IX81 and Confocal PMT detector, equipped for Multi-line Ar Laser (488 nm). Fluorescence images were analyzed and quantified by Image-Pro Plus^® 6.2 program, and all graphs were figured by GraphPad Prism 5. Algal figures were processed by IMARIS software (Bitplane). The quantified data are processed via the mean measurement of 40–50 cells from at least three different independent experiments and SEM.

Cell culture

HeLa cell line was obtained from American Type Culture Collection. HeLa cells were cultured in RPMI 1640 [GIBCO, Invitrogen] supplemented with heat-inactivated 10% (v/v) fetal bovine serum [GIBCO, Invitrogen] and 1% (v/v) antibiotic-antimycotic solution [GIBCO, Invitrogen]. Cells were maintained in a humidified atmosphere of 5% CO₂ incubator at 37 °C, and cultured in 100 mm cell culture dish [CORNING]. Chlamydomonas reinhardtii cell was obtained from Chlamydomonas Resource Center. Chlamydomonas cell strain number was CC-503 cw 92 mt + . Cell was cultured in 500 mL glass flask with stir incubator at 25 °C and normal atmosphere. Algae logarithmic phase was maintained with tris-acetate-phosphate (TAP) medium, and then cell grown under nitrogen privation condition media in 6 days.

2.1.2 Experimental Procedure

Image-based screening of SF44 analogues with HeLa cells.

HeLa cells were treated for 15 min with 5 μM solutions of series of Seoul-Fluor-based bioprobes in RPMI media, supplemented with 10% (v/v) FBS and 1% (v/v) antibiotic-antimycotic solution. Without media washing, fluorescent images of stained cells were captured using fluorescence microscopy and their fluorescent intensity were determined from the region of interest (ROI) and was quantified by Image-Pro Plus^® 6.2 program

Image-based screening of SF44 analogues with chlamydomonas reinhardtii.

Nitrogen-starved cells for 6 days (density $2. 4\; \times \; 10^{ 6}$ cells/ml) were treated with 5 μM solution of series of Seoul-Fluor-based bioprobe in 2% DMSO solution. Nitrogen starvation facilitates the formation of lipid droplets in microalgae. After 25 min incubation, microalga were observed by Z-depth-controlled image captured in confocal fluorescent microscopy.

In Vitro cytotoxicity test.

Cell viability was measured by the EZ-Cytox assay kit, and the experimental procedure was based on the manufacturer’s manual. HeLa cells were cultured into 96-well plates at a density of 3 × 10³ cells/well for 24 h, followed by the treatment of compounds in various concentrations. After 12 h of incubation with increasing concentration, 10 μL of WST-1 solution, (2-(4-nitrophenyl)-5-(2-sulfophenyl)-3-[4-(4-sulfophenylazo)-2-sulfophenyl]-2H-tetrazolium disodium salt, was added to each well, and plates were incubated for an additional 1 h at 37 °C. To measure the viability in microalgae, cells were seeded in 96 well plate and treated with series of Seoul-Fluor-based bioprobes or Nile Red. Final concentration of fluorescent probe was 5 μM (Seoul-Fluor-based bioprobes) in 2% DMSO solution or 0.1 μg/mL (Nile Red) in 20% DMSO solution. After 12, 24 and 36 h incubation, cell viability was measured by identical protocol for assay with HeLa cells. The resulting signal was observed after 90 min incubation at 25 °C. Absorbance in 455 nm was measured by microplate reader. The percentage of cell viability was calculated by following formula: % cell viability = (mean absorbance in test wells)/(mean absorbance in control well) × 100. Each experiment was performed in triplicate.

2.1.3 Compound Characterization Data

7-Acetyl-9-(4-(dimethylamino)phenyl)-2-(1-hydroxypropan-2-yl)-1 H -pyrrolo[3,4-β]indolizin-3(2 H )-one (SF55)

¹H NMR (500 MHz, CDCl₃) δ 8.46 (dd, J = 1.0, 7.5 Hz, 1H), 8.33 (s, 1H), 7.42 (d, J = 8.5 Hz, 2H), 7.19 (dd, J = 1.5, 7.0 Hz, 1H), 6.86 (dd, J = 2.0, 7.0 Hz, 2H), 4.52 (q, J = 16.5 Hz, 2H), 4.41 (m, 1H), 3.92–3.75 (m, 2H), 3.03 (s, 6H), 2.57 (s, 3H), 1.35 (d, J = 7.0 Hz, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 195.7, 162.4, 134.5, 134.1, 128.4, 124.5, 122.9, 121.8, 121.5, 114.8, 113.3, 113.2, 109.2, 70.7, 65.6, 50.9, 43.8, 40.7, 26.2, 15.5; HRMS (FAB): m/z calcd for C₂₃H₂₅N₃O₃ [M]⁺: 391.1896; found: 391.1893.

7-Acetyl-2-(3-aminopropyl)-9-(4-(diethylamino)phenyl)-1 H -pyrrolo[3,4-β]indolizin-3(2 H )-one (SF56)

¹H NMR (400 MHz, CDCl₃) δ 8.51 (dd, J = 0.8, 7.2 Hz, 1H), 8.39 (s, 1H), 7.41 (d, J = 9.2 Hz, 2H), 7.23 (dd, J = 1.8, 7.4 Hz, 1H), 6.81 (d, J = 9.2 Hz, 2H), 4.48 (s, 2H), 3.71 (t, J = 6.8 Hz, 2H), 3.43 (q, J = 7.1 Hz, 4H), 2.77 (t, J = 6.6 Hz, 2H), 2.60 (s, 3H), 1.82 (pentet, J = 6.7 Hz, 2H), 1.23 (t, J = 7.0 Hz, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 195.6, 161.8, 146.8, 134.0, 133.9, 128.7, 128.3, 124.4, 122.8, 122.0, 120.2, 115.0, 112.2, 109.1, 46.5, 44.4, 40.1, 39.0, 32.3, 26.0, 12.7; HRMS (FAB): m/z calcd for C₂₅H₃₁N₄O₂ [M + H]⁺ 419.2447, found 419.2445.

7-Acetyl-9-(4-(diethylamino)phenyl)-2-(3-morpholinopropyl)-1H-pyrrolo[3,4-β]indolizin-3(2 H )-one (SF57)

¹H NMR (500 MHz, CDCl₃) δ 8.57 (d, J = 7.5 Hz, 1H), 8.38 (s, 1H), 7.73 (d, J = 8.5 Hz, 2H), 7.68 (d, J = 8.5 Hz, 2H), 7.36 (d, J = 7.0 Hz, 1H), 4.58 (s, 2H), 4.00–3.90 (m, 4H), 3.75 (t, J = 6 Hz, 2H), 3.62 (q, J = 6.5 Hz, 4H), 3.52 (d, J = 11 Hz, 2H), 3.13 (t, J = 8 Hz, 2H), 2.88 (br s, 2H), 2.65 (s, 3H), 2.24 (pentet, J = 7.1 Hz, 2H), 1.23 (t, J = 7.0 Hz, 6H); ¹³C NMR (125 MHz, CDCl₃) δ 195.4, 161.9, 136.0, 135.7, 135.3, 135.2, 130.4, 129.2, 125.0, 123.5, 122.3, 120.4, 112.1, 110.3, 63.8, 55.1, 53.4, 52.3, 46.1, 39.6, 26.2, 23.1, 10.4; HRMS (FAB): m/z calcd for C₂₉H₃₇N₄O₃ [M + H]⁺ 489.2866, found 489.2864.

tert -Butyl (2-(7-acetyl-9-(4-(diethylamino)phenyl)-3-oxo-1H-pyrrolo[3,4-β]indolizin-2(3 H )-yl)ethyl)carbamate (SF44)

¹H NMR (500 MHz, CDCl₃) δ 8.50 (dd, J = 1.0, 7.5 Hz, 1H), 8.39 (s, 1H), 7.41 (d, J = 9.0 Hz, 2H), 7.23 (dd, J = 1.7, 7.2 Hz, 1H), 6.81 (d, J = 9.0 Hz, 2H), 4.97 (br s, 1H), 4.56 (s, 2H), 3.73 (t, J = 5.7 Hz, 2H), 3.45–3.41 (m, 6H), 2.60 (s, 3H), 1.35 (s, 9H), 1.23 (t, J = 7.0 Hz, 6H); ¹³C NMR (125 MHz, CDCl₃) 195.6, 162.2, 156.3, 146.8, 134.3, 133.9, 128.6, 128.2, 124.3, 122.4, 122.0, 120.1, 115.0, 112.3, 108.9, 79.5, 47.3, 44.5, 43.1, 39.8, 28.4, 26.0, 12.7; HRMS (FAB): m/z calcd for C₂₉H₃₆N₄O₄ [M]⁺ 504.2737, found 504.2743.

Benzyl (3-(7-acetyl-9-(4-(diethylamino)phenyl)-3-oxo-1H-pyrrolo[3,4-β]indolizin-2(3 H )-yl)propyl)carbamate (SF58)

¹H NMR (500 MHz, CDCl₃) δ 8.47 (dd, J = 0.75, 7.3 Hz, 1H), 8.38 (s, 1H), 7.40 (d, J = 9 Hz, 2H), 7.37–7.26(m, 5H), 7.22 (dd, J = 1.5, 7.0 Hz, 1H), 6.81 (d, J = 9 Hz, 2H), 5.82 (br s, J = 5.75 1H), 5.10 (s, 2H), 4.47 (s, 2H), 3.68 (t, 6 Hz, 2H), 3.43 (q, J = 7.2 Hz, 4H), 3.23 (t, J = 5.8 Hz, 2H), 2.60 (s, 3H), 1.84 (pentet, J = 5.75 Hz, 2H), 1.23 (t, J = 7.0 Hz, 6H); ¹³C NMR (75 MHz, CDCl₃) δ 195.9, 162.5, 156.9, 147.1, 137.1, 134.5, 134.3, 129.0, 128.77, 128.75, 128.3, 128.0, 124.8, 122.7, 122.3, 120.3, 115.4, 112.6, 109.5, 66.9, 47.0, 44.8, 40.3, 37.9, 28.9, 26.3, 13.0; HRMS (FAB) m/z calcd for C₃₃H₃₆N₄O₄ [M]⁺ 552.2737, found 552.2744.