Fluorescent Probe as Dual-Organelle Localizer Through Differential Proton Gradients Between Lipid Droplets and Mitochondria

Dual-organelle molecular localizers represent powerful new tools allowing the exploration of interorganelle physical contacts and subcellular chemical communication. Here, we describe new dynamic molecular probes to localize mitochondria and lipid droplets taking advantage of the differential proton gradients present in these organelles as well as the activity of mitochondrial esterase. We unveil their potential utility when organelle retention mechanisms and proton gradients are synchronized, an insight that has not been documented previously. Our discoveries indicate that dual-organelle probes serve as a valuable multiplexing assay during starvation-induced autophagy. The pioneering molecular mechanism they employ opens doors to avoid using labile esters such as acetoxymethyl derivatives which are not optimal in imaging microscopy assays.


5) Compound synthesis and chemical characterization S6
Experimental Procedures

Materials, physical measurements, and cell culture methods
Commercially available starting materials, components of buffer solutions (CHES, MOPS, MES from Sigma, Mexico) and solvents were used as supplied. 1H and 13 C NMR spectra were recorded at room temperature on a 500 MHz Bruker unity spectrometer.Chemical shifts (ppm) are relative to (CH3)4Si.High resolution mass spectrometry (ESI-TOF) was obtained by using an Agilent Technologies 6530 Accurate-Mass Q-TOF LC/MS equipment.
Fluorescence experiments were measured either on a FS5 spectrofluorometer from Edinburgh Instruments or in a Cary Eclipse fluorimeter from Agilent, UV-Vis absorption spectra were taken on a Thermo Scientific Evolution diode array UV-Vis spectrophotometer.
For light irradiation sources for the photochemical reactions we used both, xenon lamp 300 W irradiation or LED-based portable light for PDT VL400-EMITTER and LED PAR38 lamp (19W, 2700K, 1380 lumen).

Determination of the fluorescence quantum yield
Fluorescence quantum yield for AztecM of 0.32 and AztecM-LD of 0.41 were determined by using Rhodamine B ( != 0.490 in ethanol) 1 as a fluorescence standard.The quantum yield was calculated using the following equation (S1): where  ! is the fluorescence quantum yield, A is the absorbance at the excitation wavelength, F is the area under the corrected emission curve, and n is the refractive index of the solvents used.Subscripts S an X refer to the standard and to the unknown, respectively.Finally, Log P values were measured via octanol partitioning by a modification of the shakeflask method and as previously described (reference #5 main text).An aliquot of 100 ml of 300 mM of the probe in Tris buffer (10 mM, pH 7.4) and 100 ml 1-octanol (Aldrich) were added to a 0.5 ml microtube.Buffer was employed in order to measure log P of the probes at physiological pH where AztecM, AztecH-1and AztecM-LD exist in its neutral form.The tubes were vortexed for 1 min and centrifuged; 25 ml of each layer was removed and diluted in 100 ml 3:1 methanol:Tris or methanol:octanol for a final composition of 3:1:1 methanol:octanol:Tris.The aqueous layer was diluted an additional 4-fold.Three dilutions were prepared per layer, 100 ml of each dilution was pipetted into a 96 well plate, and the absorbance read at 488 nm and 625 nm wavelengths.The mean A 500 of three dilutions was calculated for each layer.The log (A 500 of the organic layer/A 500 of the aqueous layer) yielded log P. All absorbance measurements used were within the linear range of the instrument.

Yeast cells maintenance and imaging
Two strains of yeast were used, Saccharomyces cerevisiae W303 (MATa/MATa {Leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his 3-11,15} [phi + ]) and Saccharomyces cerevisiae from a commercial strain (La Azteca, México).After obtaining the culture in YPD media at 30 °C, it was left fasting for 24 hours, then a 50% aqueous suspension was prepared by weight, and a temporal monitoring of fluorescence emission was carried out by adding different solutions to a stock mixture (1.83 mL of 0.1M MES-TEA buffer, 2 µL of 10 mM BaCl2, and 40 µL of 1M glucose) at pH 6, further culture details can be found in the ESI file.

Compound synthesis and chemical characterization
Scheme S1 describes the synthetic methodology followed to prepare fluorophores.
Scheme S1.General synthetic methodology to obtain molecules I-VI and fluorescent probes.

Synthesis of I (8-Hydroxyjulolidine)
3-aminophenol (5 g, 45.82 mmol), 1-bromo-3-chloropropane (15 mL, 151.49mmol) and NaHCO3 (14 g, 166.65 mmol) were dissolved in 30 mL of anhydrous DMF.The mixture was stirred and heated at 90 °C for 24 hours.It was allowed to cool and AcOEt: H2O extraction was performed.The organic phase was recovered and purified by column chromatography (hexane:AcOEt).A white solid was obtained.Yield: 35%.Compound II (2.9 g, 13.34 mmol) and diethyl malonate (4.23 mL, 30.39 mmol) were dissolved into 20 mL ethanol, then 1.27 mL piperidine (13.66 mmol) was added.The mixture was stirred and refluxed for 24h.After that, the solvent was removed by vacuum to obtain a solid.Subsequently, 25 mL of concentrated hydrochloric acid (295.9mmol) and 20 mL glacial acetic acid (349.7 mmol) were added sequentially to a flask with the solid.Then mixture was stirred and heated to 80 ℃ for 12 h.After the reaction was accomplished, the solution was cooled to room temperature.The pH of the solution was adjusted to 7.0 after pouring the solution into 400 mL ice water and adding 20 % NaOH solution.Yellow precipitate slowly appeared in the solution along with stirring for 30 min.Afterwards, the mixture was filtered, washed with pure water and a yellow precipitated was obtained.Yield: 62%.Compound IV was done following the same procedure of compound II but modifying the reflux time (overnight) and the product was purified by chromatography column with hexane:AcOEt 1:1.An orange solid was obtained.Yield: 45%.

Synthesis of probes AztecM and AztecM-LD
Fluorescent probes were carried out using the same methodology and only changing the quaternary salt.Compound IV (100 mg, 0.37 mmol) were dissolved in 10 mL of ethanol, 1.5 equivalents of the corresponding salt (V or VI) and piperidine (10 μL, 0.1 mmol) were added.The mixture was refluxed for 12 hours.The products were purified by column chromatography using DCM:MeOH as eluent

Figure S1. 1 HSynthesis
Figure S1. 1 H NMR spectrum (400 MHz, CDCl3) of compound I.Synthesis of II (8-Hydroxyjulolidine-9-carboxaldehyde)In an ice bath, 2.4 mL of POCl3 (25.67 mmol) were added dropwise to 3 mL of DMF (38.75 mmol), this mixture was left stirring for 30 min.The compound I (3.03 g, 16 mmol) was dissolved in 4 mL of DMF and added to the previous mixture keeping the ice bath, the new mixture was stirred for another 30 min at room temperature.The mixture was refluxed for one hour and subsequently allowed to cool.200 g of ice were added and was left stirring until the formation of a precipitate was observed.It was filtered and allowed to dry to obtain a blue-green solid which was thus used for the next reaction.Yield: 90% Synthesis of III (2,3,6,7-Tetrahydro-1H,5H,11H-[1]benzopyrano[6,7,8-ij]quinolizin-11-one)

Figure S15 .
Figure S15.Co-localization imaging in live SK-Lu-1 cells for (A) synthesized spyrocyclic AztecM (red channel: lexc = 647 nm, lem = 700 nm) contrasted with MitoLite Blue lipid droplets co-localizer (blue channel, lexc = 410 nm, lem = 440 nm).The estimated Pearson's coefficient (PC) was calculated from scatter plot.To avoid blue signal contamination to the red channel, laser powers were maintained at 0.05 mW (0.2% from a 25 mW laser) and untreated cells were first recorded in order to subtract any native emission signal.Scale bars represent 20 µm.

Table S1 .
Relevant physicochemical characterization values for the Aztec Fluors probes.