Dual-fluorescence dye compounds based on 2 chromophores covalently bonded to one another

Abstract

The invention relates to a dual-fluorescence dye compound, comprising a fluorescent dye A and a fluorescent dye B, wherein: - fluorescent dye A and fluorescent dye B are covalently bonded to one another; - the emission maxima of fluorescent dye A and fluorescent dye B are separated by at least 100 nm, measured at 23°C, pH 5.8; - fluorescent dye A is pH sensitive and at pH 7.4 has at most 20% of the fluorescence intensity it has at pH 5; - the dual-fluorescence dye compound is lipophilic.

Description

[0001] Double fluorescent dye compounds based on two chromophore covalently bonded to each other

[0002] The present invention relates to double fluorescent dye compounds, their preparation and use.

[0003] It is a common procedure in cell biology to examine cells and tissue using fluorescence. Systems exist that stain cells as a whole, such as in FACS (Fluorescence Activating Cell Sorting). There are also known methods in which individual cell components are stained with fluorescent dyes. For example, coupling fluorescent dyes with antibodies is common, allowing specific structures in cells to be observed using fluorescence microscopy.

[0004] Although a variety of fluorescent dyes and methods are known, there is a need for further options for analyzing cells and tissues.

[0005] The task is solved by a double fluorescent dye comprising a fluorescent dye A and a fluorescent dye B where

[0006] Fluorescent dye A and fluorescent dye B are covalently bonded to each other, the emission maxima of fluorescent dye A and fluorescent dye B are separated by at least 100 nm, measured at 23°C, pH 5.8

[0007] Fluorescent dye A is pH-sensitive and at pH 7.4 has a maximum of 20% of the fluorescence intensity of pH 5. The double fluorescent dye compound is lipophilic.

[0008] According to the invention, fluorescent dye A and fluorescent dye B are covalently linked. This can be done directly via functional groups of the fluorescent dyes or by means of a linker.

[0009] The two fluorescent dyes A and B have emission maxima separated by at least 100 nm. To determine this separation, both dyes are measured in an aqueous solution with pH 5.8 at 23 °C. These measurement conditions are used only to characterize the dyes. Typically, emission maxima of fluorescent dyes are also known.

[0010] In the double fluorescent dye according to the invention, fluorescent dye A is pH-sensitive, i.e., the fluorescence intensity changes depending on the pH. At pH 7.5, fluorescent dye A exhibits a maximum of 20% of the fluorescence intensity it exhibits at pH 5. Preferably, the fluorescence intensity at pH 7.4 is a maximum of 10%, preferably a maximum of 5%, and even more preferably a maximum of 1% of the fluorescence intensity at pH 5.

[0011] The double fluorescent dye compound is lipophilic.

[0012] A typical measure of lipophilicity is the calculation of the so-called logP of a compound. The logP determines the partition coefficient of a compound between octanol and water, see, for example, Gerd Klebe, Wirkstoffdesign, 2nd edition, Spektrum Akademischer Verlag, Heidelberg 2009, p. 267. It can now be predicted using computer methods. Corresponding methods are known to those skilled in the art as AlogP, XlogP, and MlogP. In case of doubt, logP values ​​are calculated using the QikProp module from Schrödinger's Maestro program [Schrödinger Release 2022-3: QikProp, Schrödinger, LLC, New York, NY, 2021]. Preferably, the logP of the compound is > 3, even more preferably > 4.

[0013] The distance between the emission maxima of fluorescent dyes A and B is preferably chosen so that there is no fluorescence transfer, ie that the emission wavelength of one dye does not interact with the excitation wavelength of the other dye, ie that the two dyes, in particular the chromophores, behave separately.

[0014] In principle, the skilled person is familiar with numerous fluorescent dyes from which to choose. A preferred dye for fluorescent dye A is a xanthene or rhodamine analogue. A preferred dye for fluorescent dye B is a boron dipyrromethene (BODIPY) dye.

[0015] If the basic structures of such compounds are not lipophilic enough, for example, it may be useful to introduce one or more lipophilic side groups into at least one of the fluorescent dyes A or B. Suitable lipophilic side chains typically have 5 to 22 carbon atoms, which may optionally also contain silicon groups (silanyl residues). The side chain should typically contain no more than 10 silicon atoms. Typical side groups are pentyl, hexyl, octyl, or decyl groups, which are coupled, for example, as ethers or esters. These can, for example, have trimethylsilyl side chains.

[0016] In one embodiment of the invention, the double fluorescent dye compound has the following structure and where the substituents have the following meaning:

[0017] R a , R b , R c , R d , R e , R f= independently H, alkyl, aryl, alkenyl, alkynyl, halogen, alkoxy, trialkylsilyl, trialkylsilyloxy

[0018] R 1 = alkyl, aryl, silanylalkyl, silyloxyalkyl

[0019] R 2 = H, alkyl, aryl, silanylalkyl, silyloxyalkyl

[0020] XY = a linker with 0-20 C atoms

[0021] Z a ' Z b , Z c , Z d = independently H, alkyl, acyl, silyl, alkoxy, silyloxy, halogen, sulfonyl; where R a , R b , R c , R d , R e , R f , Z a ' Z b , Z c , Z d each with a maximum of 20 C atoms and R 1 , R 2 each contains a maximum of 16 C atoms.

[0022] The substituents mentioned can also be branched and / or substituted.

[0023] The linker XY serves to connect the dye molecules. It can be omitted if suitable (directly linkable) functional groups are present on the fluorescent dyes. In addition to C atoms, it can contain other atoms, particularly N, S, O, Si, and H. The linker itself can comprise carbocyclic and heterocyclic systems. Ether or ester groups, for example, can be used to couple the linker to the fluorescent dyes.

[0024] Preferred is a connection in which

[0025] R a , R f Alkyl, alkenyl or aryl are

[0026] R b , R c , R d , R e H are,

[0027] R 1 Alkyl is

[0028] R 2 H is,

[0029] Z a ' Z b , Z c , Z d H are.

[0030] Particularly preferred compounds are called Lipo-Fluddy 1, 2 and 3 and have the following structure

[0031] The process for preparing the double fluorescent dye compound typically involves synthesizing the two fluorescent dyes and then linking them together, optionally with the aid of a linker. The dyes according to the invention can be brought into contact with cells. After excitation, the emission of fluorescent dyes A and B can be measured. The measurement is preferably carried out with spatial resolution, for example, by fluorescence microscopy.

[0032] In a preferred method, this is done for imaging and / or quantifying lipophagy in cells.

[0033] The autophagic process is divided into several steps. First, the material to be degraded is enveloped by membranes that form an autophagosome. The autophagosome fuses with a lysosome to form an autolysosome with an acidic pH. This is where further degradation takes place.

[0034] Autophagocytosis is also essential during food deprivation. It therefore increases during stressful conditions, such as food deprivation. Autophagocytosis alterations have been found in tumor cell metastasis, as well as in ALS, Alzheimer's disease, Huntington's disease, SENDA, multiple sclerosis, Crohn's disease, and systemic lupus erythematosus. It would be desirable to be able to study these processes in more detail.

[0035] According to the invention, the pH value changes due to the formation of the autolysosome, since lysosomes provide an acidic environment with a pH of approximately 4.5 to 5. This changes the fluorescence intensity of dye A according to the invention.

[0036] The double fluorescent compounds according to the invention can therefore be used to determine where the dye molecule is located and whether it is in a neutral or acidic environment, because fluorescent dye A is only visible in an acidic environment.

[0037] Typically, cells are put into a "starvation" mode to increase autophagy. By measuring cells at two different excitation wavelengths and overlaying the images, autophagy can be observed particularly well. Figure 1: Mechanism of the imaging study according to the invention or for quantifying lipophagy in cells.

[0038] Figure 2: The bottom row shows the signal collected at 445(±30)+615(±35) nm after excitation with a 561 nm laser. The middle row shows the signal collected at 525(±25) nm after excitation with a 488 nm laser. The top row shows the merge of the two images.

[0039] The invention is further illustrated by the following examples.

[0040] Example 1 Syntheses

[0041] General synthesis methods

[0042] Syntheses involving moisture- or air-sensitive substances were carried out in flame-dry, vacuum-evacuated glassware under an inert atmosphere. Argon BIP (Air Products) with a purity of 99.9997% was used as the protective gas without further purification. Near-vacuum pressures were generated using oil pumps to evacuate the glassware and dry the reaction products. Solids were added to reactions under argon countercurrent; liquids and solutions were added via plastic syringes with steel cannulas that had been pre-dried at 80 °C. Reactions at low temperatures were carried out using ice-water mixtures for 0 °C and dry ice-isopropanol mixtures for temperatures down to -78 °C. Preparative electrolysis was performed using Didac-Tec 48x28 mm graphite plate electrodes and a Voltcraft VLP-1602 Pro power supply.The solvents and reagents used for the syntheses were purchased from Acros Organics, AK Scientific, Alfa Aesar, Cambridge Isotope Labs, Eastman Organic Chemicals, Fisher Chemical, TCI, Sigma-Aldrich, and Strem Chemicals with purities of >97%. Anhydrous THF was prepared from HPLC-grade THF, which was dried over sodium in the presence of benzophenone and freshly distilled under an argon atmosphere. Anhydrous CH2Cl2 was prepared from HPLC-grade CH2Cl2, which was dried over CaH2 and freshly distilled under an argon atmosphere. Unless otherwise stated, all commercially available chemicals were used without further purification. The moisture-sensitive catalysts were stored in an mBraun LABmaster 130 glove box under an argon atmosphere at <1 ppm O2 and <1 ppm H2O. The concentrations of vinylmagnesium bromide solutions (Sigma-Aldrich, solution in THF) were determined by titration against iodine.

[0043] Thin-layer chromatography (TLC) was performed using Merck TLC Silica Gel 60 F254 plates with a thickness of 0.25 mm. The separated substances were visualized either by their fluorescence under UV light (254 nm) or with a potassium permanganate staining solution (1.5 g KMnÖ4, 10 g K2CO3, and 1.25 mL 10% NaOH in 200 mL water). The ratios of the solvents used are given in volume per volume.

[0044] Acros silica gel for chromatography (60 Å, 0.035 - 0.070 mm) was used for column chromatography. The eluent flow rate was increased by pressure. The ratios of the solvents used are given in volume per volume.

[0045] Gas chromatograms (GC) were recorded using an Agilent HP6890 gas chromatograph coupled to an MSD 5937 N mass detector. A Macherey-Nagel Optima-l-MS (30 m x 0.25 mm Ø) capillary column was used. Hydrogen served as the carrier gas. The temperature program used was 50–300 °C (17 min). The signals indicated refer to the m / z ratio, and the intensities are given relative to the highest signal (100%).

[0046] For nuclear magnetic resonance spectroscopy (NMR), the Bruker spectrometers Avance-I 300 CH: 300 MHz, 13 C: 75 MHz), Avance-II 500 CH: 500 MHz, 13 C: 126 MHz, n B: 160 MHz, 19 F: 471 MHz), Avance-III 499 CH: 500 MHz, 13 C: 126 MHz) and Avance-II+ 600 CH: 600 MHz, 13C: 151 MHz). All spectra were recorded at 298 K. The chemical shift δ is given in parts per million (ppm) with respect to the signal of tetramethylsilane TMS [δ CH-NMR) = 0.00 ppm]. The solvent used was deuterated chloroform CDC [δ CH) = 7.26 ppm; δ ( 13 C) = 77.16 ppm]. The multiplicities of the signals in the ^-spectra are given and abbreviated as s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublets, bs = broad singlet, and m = multiplet. 13C spectra were recorded as β-decoupled and DEPT (Distortionless Enhancement by Polarization Transfer) experiments. All spectra were analyzed using the MestReNova software. The chosen numbering of the atomic signals for the assignment of the NMR signals does not comply with IUPAC rules. Each signal was assigned by comparison with the literature or via two-dimensional NMR spectra obtained by H,H-COSY, HMBC, and HMQC experiments.

[0047] High-resolution (HR) mass spectra (MS) were recorded by ESI using a THERMO Scientific LTQ Orbitrap XL spectrometer and the signals were compared with the calculated values ​​(error < 1 ppm).

[0048] All spectra were recorded using a Perkin Elmer Spectrum Two Fourier Transform Infrared Spectrometer (FT-IR) with ATR technology. The positions of the absorption bands are given in wavenumbers (cm' 1), their intensities are abbreviated as s = strong, m = medium, w = weak and b = broad.

[0049] The melting points (MP) were determined using a melting point meter from Wepa Apotec.

[0050] UV / VIS absorption spectra were recorded using a Perkin Elmer Lambda 35 UV / VIS spectrometer with a 1 nm slit width and a scan speed of 500 nm / s. Fluorescence spectra were recorded using a Perkin Elmer LS 55 fluorescence spectrometer at excitation wavelengths corresponding to the absorption maxima of the respective compounds, with an excitation slit width of 15 nm, an emission slit width of 5 nm, and a scan speed of 500 nm / s. All measurements were performed at room temperature in 10 mm wide glass cuvettes using 10 pM solutions of the respective compounds in ethanol or a 1:1 mixture of ethanol and acetic acid.

[0051] Synthesis instructions

[0052] Di-(TBSO-ethyl)-fluorescein (2)

[0053] 1 2

[0054] To a solution of 2.00 g (6.02 mmol, 1.00 eq.) of fluorescein 1 in 60 mL of anhydrous DMF under argon atmosphere, 2.50 g (18.1 mmol, 3.00 eq.) of K2CO3 was added, and the mixture was stirred at room temperature for 1 hour. Then, 387 mL (18.1 mmol, 3.00 eq.) of (2-bromoethoxy)-tert-butyldimethylsilane was added dropwise over 10 minutes. The reaction mixture was heated to 70 °C and stirred for 23 hours. Once the reaction was complete, 120 mL of ice-cold water was added, and stirring was continued at 0 °C for 30 minutes. The reaction mixture was extracted three times with 100 mL of EtOAc each, the combined organic phases were washed with water, dried with MgSO, and the solvent was removed under reduced pressure to obtain 3.75 g (5.78 mmol, 96%) of alkylated fluorescein 2 as an orange solid.

[0055] M (C36H48O7S12): 648.29 g / mol.

[0056] Ausbeute: 3.75 g (5.78 mmol, 96%).

[0057] DC: (SiO2, EtOAc / cHex 1:1) R f = 0.13.

[0058] MP: 165 °C.

[0059] 1 H-NMR: (600 MHz, CDCI3) 5 [ppm] = 8.26 (dd, 3 J H H = 7.9 Hz, 4 J H H = 1.3 Hz, 1H, H-3),

[0060] 7.73 (td, 3 J H H = 7.5 Hz, 4 J H H = 1.4 Hz, 1H, H-5), 7.66 (td, 3 J H H = 7.7 Hz, 4JHH = 1.3 Hz, 1H, H-4), 7.30 (dd, 3 JHH = 7.6 Hz, 4 J HH = 1.2 Hz, 1H, H-6), 6.97 (d, 4 J H H = 2.4 Hz, 1H, H-18), 6.87 (d, 3 J H H = 9.0 Hz, 1H, H-15), 6.84 (d, 3 J H H = 9.7 Hz, 1H, H-24), 6.73 (dd, 3 J H H = 8.9 Hz, 4 JHH = 2.4 Hz, 1H, H-16), 6.52 (dd, 3 J H H = 9.7 Hz, 3 J HH = 1.9 Hz, 1H, H-23), 6.44 (d, 3 J H H = 2.0 Hz, 1H, H- 21), 4.14 - 4.12 (m, 2H, H-8), 4.09 - 4.06 (m, 2H, H-26), 4.01 - 4.00 (m, 2H, H-9), 3.61 - 3.54 (m, 2H, H-27), 0.90 (s, 9H, H-12), 0.83 (s, 9H, H-30), 0.10 (s, 6H, H-10), -0.01 (s, 6H, H-28). 13 C-NMR: (151 MHz, CDCI3) ö [ppm] = 185.8 (C-17), 165.2 (C-7), 163.6 (C-22), 159.1

[0061] (C-20), 154.3 (C-19), 150.2 (C-13), 134.8 (C-2), 132.8 (C-5), 131.3 (C-3), 130.7 (C-6), 130.6 (C-24), 130.4 (C-l), 130.1 (C-23), 129.8 (C-4), 129.0 (C- 15), 117.8 (C-25), 115.0 (C-14), 113.8 (C-16), 105.9 (C-21), 101.2 (C-18),

[0062] 70.3 (C-8), 66.5 (C-26), 61.8 (C-9), 60.9 (C-27), 26.0 (C-12), 25.9 (C-30), 18.5 (C-ll), 18.4 (C-29), -5.1 (C-10), -5.2 (C-28).

[0063] HRMS (ESI): m / z berechnet für [M+H] + : 649.3011; gefunden: 649.3010. m / z berechnet für [M+Na] + : 671.2831; gefunden: 671.2829.

[0064] FT-IR: (ATR) v [cm- 1] = 3059 (w), 2953 (w), 2929 (w), 2856 (w), 1727 (m), 1644

[0065] (m), 1594 (s), 1572 (w), 1544 (w), 1517 (s), 1482 (m), 1463 (m), 1414 (w), 1377 (w), 1361 (w), 1346 (w), 1256 (s), 1215 (m), 1191 (w), 1104 (s), 1079 (m), 1006 (w), 993 (w), 960 (m), 853 (s), 834 (s), 777 (s), 759 (m), 721 (w), 661 (w), 615 (w), 591 (w).

[0066] TBSO-ethyl-hydrofluorescein (3)

[0067] To a solution of 3.72 g (5.73 mmol, 1.00 eq.) of alkylated fluorescein 2 in 70 mL of anhydrous THF under an argon atmosphere, 653 mg (17.2 mmol, 3.00 eq.) of UAIH4 was added at 0 °C. The reaction mixture was stirred for 2 hours at 0 °C and then for 1 hour at room temperature. Once the reaction was complete by TLC, 10.3 g (32.1 mmol, 5.60 eq.) of sodium sulfate decahydrate was added at 0 °C and stirring was continued for 30 minutes at room temperature. The reaction mixture was filtered through a frit containing Celite, which was washed with CH2Cl2. The filtrate was concentrated under reduced pressure, the residue was dissolved in 70 mL MeOH, and 4.23 g (17.2 mmol, 3.00 eq.) of choranil was added. This mixture was stirred for 1 hour at room temperature and then filtered through a Celite filter, which was washed with CH2Cl2.After removal of the solvent under reduced pressure, the residue was purified by column chromatography (SiO2, EtOAc / cHex 1:4) to obtain 2.23 g (4.69 mmol, 82%) of reduced fluorescein 3 as a yellow oil.

[0068] M (C28H32O5S12): 476.64 g / moi.

[0069] Yield: 2.23 g (4.69 mmol, 82%).

[0070] TLC: (SiO2, EtOAc / cHex 1:1) R f = 0.48.

[0071] *H-NMR: (500 MHz, CDCI3) 5 [ppm] = 7.40 - 7.35 (m, 2H, H-3, H-4), 7.30 - 7.26 (m,

[0072] 1H, H-5), 6.94 - 6.89 (d, 3 J H H = 7.3 Hz, 1H, H-6), 6.85 - 6.80 (m, 2H, H-10, H-19), 6.75 - 6.72 (m, 1H, H-16), 6.65 - 6.59 (m, 2H, H-13, H-18), 6.53 - 6.48 (m, 1H, H-ll), 5.24 (s, 1H, OH), 4.05 (t, 3 J H H = 5.2 Hz, 2H, H-21), 4.00 - 3.95 (m, 2H, H-22), 0.91 (s, 9H, H-25), 0.10 (s, 6H, H-23).

[0073] 13 C-NMR: (126 MHz, CDCI3) 5 [ppm] = 159.5 (0-12), 157.4 (C-17), 151.7 (0-14), 151.6

[0074] (0-15), 143.3 (0-1), 139.1 (0-2), 130.2 (0-10), 129.8 (0-19), 128.3 (0-5), 128.2 (C-4), 123.9 (0-6), 120.4 (C-3), 117.1 (0-20), 116.9 (0-9) 112.0 (C- 18), 111.6 (0-11), 102.8 (0-16), 101.2 (C-13), 83.1 (C-8), 69.6 (0-21), 61.9

[0075] (C-22), 25.9 (0-25), 18.4 (C-24) -5.2 (C-23).

[0076] HRMS (ESI): m / z berechnet für [M+H] + : 477.2092; gefunden: 477.2090. m / z berechnet für [M+Na] + : 499.1911; gefunden: 499.1912.

[0077] FT-IR: (ATR) v [cm- 1 ] = 3676 (w), 3280 (w), 3074 (w), 2955 (m), 2928 (m),

[0078] 2902 (m), 2857 (m), 2104 (w), 1689 (w), 1632 (m), 1607 (m), 1501 (m), 1459 (s), 1426 (m), 1394 (m), 1347 (m), 1255 (s), 1214 (m), 1182 (s), 1152 (m), 1107 (s), 1066 (m), 1057 (m), 1027 (m), 1001 (m), 954 (w), 885 (w), 835 (s), 805 (m), 778 (m), 757 (m), 725 (w), 679 (w), 665 (w), 633 (w), 616 (w), 600 (w).

[0079] TBSO-ethyl-hydrofluorescein triflate (4)

[0080] To a solution of 2.20 g (4.62 mmol, 1.00 eq.) of phenol 3 in 23 mL of anhydrous CH2Cl2 under an argon atmosphere, 1.49 mL (18.5 mmol, 4.00 eq.) of pyridine was added at 0 °C. The mixture was stirred for 15 minutes at 0 °C, then 1.55 mL (9.23 mmol, 2.00 eq.) of trifluoroanhydride was added dropwise at 0 °C. The mixture was warmed to room temperature and stirred for 3 hours. Once completion of the reaction was confirmed by TLC, the reaction was quenched with 60 mL of water and extracted three times with 50 mL of CH2Cl2 each time. The organic phase was washed with 100 mL each of saturated NH4Cl(aq), water and saturated NaCl(aq), dried with MgSO4, the solvent was removed under reduced pressure and the residue was purified by column chromatography (SiO2, EtOAc / cHex 1:9) to obtain 1.95 g (3.20 mmol, 69%) of triflate 4 as a yellow oil.

[0081] M (C2gH 31F3O7SSi): 608.70 g / mol.

[0082] Ausbeute: 1.95 g (3.20 mmol, 69%).

[0083] DC: (SiO2, EtOAc / cHex 1:4) R f = 0.53.

[0084] 1 H-NMR: (600 MHz, CDCI3) ö [ppm] = 7.41 - 7.35 (m, 2H, H-3, H-4), 7.30 - 7.27 (m,

[0085] 1H, H-5), 7.16 (d, 4 J H H = 2.5 Hz, 1H, H-13), 7.06 (d, 3 J H H = 8.7 Hz, 1H, H- 10), 6.93 (dd, 3 J H H = 8.7 Hz, 3 J H H = 2.5 Hz, 1H, H-ll), 6.90 - 6.84 (m, 2H, H-6, H-19), 6.75 (d, 4 J H H = 2.5 Hz, 1H, H-16), 6.67 (dd, 3 J H H = 8.8 Hz, 4 J H H = 2.5 Hz, 1H, H-18), 5.37 - 5.26 (m, 2H, H-7), 4.08 - 4.02 (m, 2H, H-22), 4.00 - 3.95 (m, 2H, H-23), 0.91 (s, 9H, H-26), 0.10 (s, 6H, H-24).

[0086] 13 C-NMR: (151 MHz, CDCI3) ö [ppm] = 160.1 (C-17), 151.3 (C-15), 151.0 (C-14),

[0087] 149.3 (C-12), 144.5 (C-l), 138.9 (C-2), 131.0 (C-10), 129.8 (C-19), 128.8 (C-5), 128.7 (C-4), 125.5 (C-20), 123.9 (C-6), 121.0 (C-3), 116.6 (C-9),

[0088] 116.4 (C-ll), 112.4 (C-18), 110.0 (C-13), 101.4 (C-16), 83.2 (C-8), 72.7 (C-7), 69.8 (C-22), 62.0 (C-23), 26.1 (C-26), 18.6 (C-25), -5.0 (C-24).

[0089] HRMS ( ESI): m / z berechnet für [M+H ] + : 609.1585; gefunden : 609.1591. m / z berechnet für [M+Na] + : 631.1404; gefunden: 631.1412.

[0090] FT-IR: (ATR) v [cm- 1 ] = 2930 (w), 2857 (w), 1771 (w), 1611 (m), 1579 (w),

[0091] 1505 (w), 1488 (m), 1421 (s), 1255 (m), 1237 (m), 1213 (s), 1164 (m), 1140 (s), 1105 (s), 1014 (w), 991 (m), 941 (w), 864 (w), 837 (s), 810 (w), 779 (w), 758 (w), 728 (w), 662 (w), 612 (w), 546 (w).

[0092] N-hexyl-O-(TBSO-ethyl)-hydrorhodol (5) To a solution of 1.92 g (3.15 mmol, 1.00 eq.) of Triflat 4 and 8.3 mL (63 mmol, 20 eq.) of n-hexylamine in 47 mL of anhydrous toluene under argon atmosphere were added 289 mg (315 pmol, 10 mol%) of Pd2(dba)3, 292 mg (505 pmol, 16 mol%) of Xantphos, and 3.08 g (9.46 mmol, 3.00 eq.) of CsCC, and the mixture was stirred for 4 hours at 105 °C. Once the reaction was complete by TLC, the mixture was filtered through a frit containing Celite, which was washed with CH2Cl2. The filtrate was concentrated under reduced pressure and the residue was purified by column chromatography (SiO2, EtOAc / cHex 1:20) to obtain 1.07 g (1.91 mmol, 61%) of nhexyl-rhodol 5 as a red oil.

[0093] M (C34H45NO4S1): 559.31 g / mol.

[0094] Yield: 1.07 g (1.91 mmol, 61%).

[0095] TLC: (SiO2, EtOAc / cHex 1:4) R f = 0.57. -NMR: (500 MHz, CDCI3) 5 [ppm] = 7.35 - 7.33 (m, 2H, H-3, H-4), 7.26 - 7.23 (m,

[0096] 1H, H-5), 6.91 (d, 3 JH H = 7.6 Hz, 1H, H-6), 6.81 (d, 3 J H H = 8.7 Hz, 1H, H- 19), 6.71 - 6.69 (m, 2H, H-10, H-16), 6.58 (dd, 3 J H H = 8.7 Hz, 3 J H H = 2.5 Hz, 1H, H-18), 6.37 - 6.35 (m, 1H, H-13), 6.31 - 6.26 (m, 1H, H-ll), 5.25 (s,

[0097] 2H, H-7), 4.06 - 4.01 (m, 2H, H-27), 3.99 - 3.95 (m, 2H, H-28), 3.11 (t, 3JHH = 7.1 Hz, 2H, H-21), 1.64 - 1.59 (m, 2H, H-22), 1.42 - 1.36 (m, 2H, H-23), 1.35 - 1.30 (m, 4H, H-24, H-25), 0.91 - 0.90 (m, 12H, H-26, H-31),

[0098] 0.10 (s, 6H, H-29).

[0099] 13 C-NMR: (126 MHz, CDCI3) ö [ppm] = 159.6 (C-17), 152.0 (C-14), 151.8 (C-15),

[0100] 149.6 (C-12), 145.2 (C-2), 139.6 (C-l), 130.0 (C-19), 129.7 (C-10), 128.3 (C-5), 128.0 (C-3), 124.2 (C-6), 120.7 (C-4), 117.6 (C-20), 113.3 (C-9),

[0101] 111.3 (C-18), 110.1 (C-ll), 101.3 (C-16), 98.2 (C-13), 84.0 (C-8), 71.8 (C- 7), 69.6 (C-27), 62.1 (C-28), 44.0 (C-21), 31.8 (C-24), 29.5 (C-22), 27.0 (C-23), 26.1 (C-31), 22.8 (C-25), 18.5 (C-30), 14.2 (C-26), -5.0 (C-29).

[0102] HRMS (ESI): m / z berechnet für [M+H] + : 560.3191; gefunden: 560.3182.

[0103] FT-IR: (ATR) v [CUT 1 ] = 3676 (w), 3369 (w), 2955 (s), 2928 (s), 2857 (m),

[0104] 1731 (w), 1634 (m), 1615 (s), 1572 (w), 1520 (m), 1501 (m), 1460 (m), 1417 (m), 1394 (w), 1349 (w), 1317 (w), 1256 (s), 1196 (s), 1161 (w), 1109 (s), 1078 (m), 1066 (m), 1057 (m), 1027 (m), 1013 (m), 957 (w), 898 (w), 834 (s), 778 (m), 756 (m), 725 (w), 663 (w), 633 (w). N-hexyl-O-(2-hydroxyethyl)-hydrorhodol (6)

[0105] To a solution of 990 mg (1.77 mmol, 1.00 eq.) of TBS-protected rhodol 5 in 47 mL of anhydrous THF was added 2.12 mL (2.12 mmol, 1.20 eq.) of a 1 M solution of TBAF in THF, and the mixture was stirred for 2 hours at room temperature. Once the reaction was complete by TLC, 200 mL of water was added, and the mixture was extracted three times with 200 mL of EtOAc each. The combined organic phases were dried with MgSO4, the solvent was removed under reduced pressure, and the residue was purified by column chromatography (SiCl, EtOAc / cHex 1:4) to afford 629 mg (1.41 mmol, 80%) of the alcohol 6 as a colorless oil.

[0106] M (C28H31NO4): 445.23 g / mol.

[0107] Yield: 629 mg (1.41 mmol, 80%).

[0108] TLC: (SiO2, EtOAc / cHex 1:1) R f = 0.29.

[0109] 1 H-NMR: (500 MHz, CDCI3) ö [ppm] = 7.37 - 7.33 (m, 2H, H-3, H-4), 7.24 (m, 1H, H-

[0110] 5), 6.91 (d, 3 J H H = 7.6 Hz, 1H, H-6), 6.84 (d, 3 J H H = 8.7 Hz, 1H, H-19), 6.72 - 6.70 (m, 2H, H-10, H-16), 6.60 (dd, 3 J H H = 8.7 Hz, 4 J H H = 2.5 Hz, 1H, H-18), 6.37 (d, 4 J H H = 2.3 Hz, 1H, H-13), 6.29 (dd, 3 J H H = 8.5 Hz, 4 J H H = 2.4 Hz, 1H, H-ll), 5.25 (s, 2H, H-7), 4.11 - 4.06 (m, 2H, H-27), 3.99 - 3.91 (m, 2H, H- 28), 3.78 (s, 1H, OH), 3.11 (t, 3 J H H = 7.1 Hz, 2H, H-21), 1.64 - 1.58 (m, 2H,

[0111] H-22), 1.42 - 1.36 (m, 2H, H-23), 1.35 - 1.30 (m, 4H, H-24, H-25), 0.93 - 0.88 (m, 3H, H-26).

[0112] 13 C-NMR: (126 MHz, CDCI3) ö [ppm] = 159.2 (C-17), 152.0 (C-14), 151.8 (C-15), 149.7

[0113] (C-12), 145.2 (C-2), 139.6 (C-l), 130.1 (C-19), 129.7 (C-10), 128.4 (C-5), 128.0 (C-3), 124.1 (0-6), 120.7 (C-4), 118.0 (C-20), 113.2 (C-9), 111.2 (C- 18), 110.1 (C-ll), 101.4 (C-16), 98.1 (C-13), 83.9 (C-8), 71.8 (C-7), 69.5 (C- 27), 61.5 (C-28), 44.0 (C-21), 31.7 (C-24), 29.5 (C-22), 26.9 (C-23), 22.8 (C- 25), 14.2 (C-26).

[0114] HRMS (ESI): m / z berechnet für [M+H] + : 446.2326; gefunden : 446.2325.

[0115] UV / VIS: Absorption (EtOH / AcOH 1:1): A max = 501 nm. Fluoreszenz (EtOH / AcOH 1:1): A max = 531 nm.

[0116] FT-IR: (ATR) v [cm 3 ] = 3365 (w), 2928 (w), 2856 (w), 1633 (m), 1615 (s), 1570 (w),

[0117] 1521 (m), 1501 (m), 1457 (w), 1418 (m), 1350 (w), 1245 (m), 1193 (s), 1161 (w), 1112 (m), 1078 (w), 1026 (w), 1010 (m), 904 (w), 831 (w), 797 (w), 757 (m), 725 (m), 634 (w).

[0118] Methyl-4-formylbenzoate (8) 8

[0119] To a solution of 5.00 g (33.3 mmol, 1.00 eq.) of 4-formylbenzoic acid (7) in 135 mL of CH2Cl2 were added 4.00 mL (99.9 mmol, 3.00 eq.) of MeOH, 814 mg (6.66 mmol, 0.20 eq.) of DMAP, and 4.50 g (66.6 mmol, 2.00 eq.) of imidazole. This solution was cooled to 0 °C and stirred for 15 minutes, then a suspension of 9.60 g (50.0 mmol, 1.50 eq.) of EDC in 50 mL of CH2Cl2 was added dropwise. The mixture was warmed to room temperature and stirred for a further 17 hours. Once the reaction was confirmed by TLC, the reaction mixture was washed with 70 mL of 0.5 M HCl(aq), 70 mL of saturated NaHCO3(aq), 70 mL of saturated NaCl(aq), and dried with MgSO4. The solvent was evaporated under reduced pressure to afford 5.83 g (35.5 mmol, quant.) of crude methyl 4-formylbenzoate 8 as a colorless solid, which was used directly in the next step without further purification.

[0120] M (CgHsO3): 164.16 g / mol.

[0121] Ausbeute: 5.83 g (35.5 mmol, quant.).

[0122] DC: (SiO2, EtOAc / cHex 1:2) R f = 0.43.

[0123] MP: 33 °C. -NMR: (500 MHz, CDCI3) Ö [ppm] = 10.09 (s, 1H, H-6), 8.19 (d, 3 JHH = 8.3 Hz, 2H, H- 3), 7.95 (d, 3 J H H = 8.3 Hz, 2H, H-4), 3.95 (s, 3H, H-7).

[0124] 13 C-NMR: (126 MHz, CDCI3) ö [ppm] = 191.8 (C-6), 166.2 (C-l), 139.3 (C-2), 135.2 (C- 5), 130.3 (C-3), 129.6 (C-4), 52.7 (C-7).

[0125] Methyl-4-( dipyrromethenjyl-benzoat (10)

[0126] To 300 mL of 0.05 M HCl(aq) were added 5.83 g (35.5 mmol, 1.00 eq.) of methyl 4-formylbenzoate (8) and 7.00 mL (101 mmol, 2.85 eq.) of pyrrole (9), and the mixture was stirred at room temperature for 4.5 hours. The filtrate was diluted with 150 mL of CH2Cl2, the phases were separated, and the organic phase was washed twice with 300 mL of water each time, dried with MgSO4, and the solvent was removed under reduced pressure. The residue was dissolved in 120 mL of THF. 6.10 g (24.8 mmol, 1.11 eq.) of chloranil was added to this solution, and the mixture was stirred at room temperature for 6 hours. After the completion of the reaction was confirmed by TLC, the solvent was removed under reduced pressure and the residue was purified by column chromatography (SiO2, EtOAc / cHex 1:4) to obtain 6.12 g (22.0 mmol, 62%) of dipyrromethene 10 as a green solid.

[0127] M (C 17 HI4N2O2): 278.31 g / mol.

[0128] Ausbeute: 6.12 g (22.0 mmol, 62° / o).

[0129] DC: (SiO2, EtOAc / cHex 1 :4) R f = 0.27.

[0130] MP: 147 °C. -NMR: (500 MHz, CD2CI2) ö [ppm] = 8.11 (d, 3 J H H = 8.4 Hz, 2H, H-3), 7.66 (s, 2H,

[0131] H-10), 7.57 (d, 3 J H H = 8.4 Hz, 2H, H-4), 6.53 (dd, 3 JHH = 4.2 Hz, 3 J H H = 1.0 HZ, 2H, H-9), 6.41 (dd, 3 J HH = 4.3 Hz, 4 JHH = 1.5 Hz, 2H, H-8), 3.95 (s, 3H, H-ll). 13 C-NMR: (126 MHz, CD2CI2) ö [ppm] = 167.1 (Cl), 144.6 (C-10), 142.3 (C-2),

[0132] 141.0 / 140.9 (C-6, C-7), 131.3 (C-4), 131.3 (C-5), 129.3 (C-3), 119.6 (C-9),

[0133] 118.4 (C-8), 52.7 (C-ll).

[0134] HRMS (ESI): m / z berechnet for [M+H] + : 279.1128; gefunden: 279.1129.

[0135] FT-IR: (ATR) v [crrr 1 ] = 3113 (w), 2951 (w), 2846 (w), 1723 (s), 1608 (w),

[0136] 1572 (s), 1559 (m), 1505 (w), 1434 (m), 1416 (m), 1405 (m), 1384 (s), 1353 (m), 1338 (m), 1328 (m), 1311 (m), 1276 (s), 1220 (w), 1179 (m), 1111 (m), 1096 (m), 1044 (m), 1020 (w), 1006 (s), 987 (w), 938 (m), 876 (m), 814 (m), 777 (m), 759 (s), 721 (s), 651 (w), 637 (w), 597 (w),

[0137] 523 (w).

[0138] Meth yl-4-(BODIPY) -benzoat (11) 11

[0139] To a solution of 4.00 g (14.4 mmol, 1.00 eq.) of dipyrromethene (10) in 50 mL of CH2Cl2, 14.2 mL (83.4 mmol, 5.80 eq.) of DIPEA and 8.00 mL (63.2 mmol, 4.40 eq.) of BF3-0Et2 were added, and the mixture was stirred at room temperature for 1.5 hours. The reaction mixture was diluted with 200 mL of CH2Cl2, the phases were separated, and the organic phase was washed with 400 mL each of 0.1 M NaOH(aq), 0.1 M HCl(aq), and water, dried with MgSO4, and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography (SiO2, EtOAc / cHex 1:6) to obtain 850 mg (2.61 mmol, 18%) of BODIPY 11 as a blue solid.

[0140] M (CI7H 13 BF2N2O2): 326.11 g / mol.

[0141] Yield: 850 mg (2.61 mmol, 18%).

[0142] TLC: (SiO2, EtOAc / cHex 1:4) R f = 0.33.

[0143] MP: 204 °C.

[0144] 1 H-NMR: (600 MHz, CDCI3) ö [ppm] = 8.20 (d, 3JHH = 8.3 Hz, 2H, H-3), 7.97 (s, 2H,

[0145] H-10), 7.64 (d, 3 J H H = 8.3 Hz, 2H, H-4), 6.88 (d, 3 J H H = 4.1 Hz, 2H, H-8), 6.56 (d, 3 J H H = 3.8 Hz, 2H, H-9), 3.99 (s, 3H, H-ll). 13 C-NMR: (151 MHz, CDCI3) ö [ppm] = 166.4 (C-l ), 145.9 (C-2), 145.0 (C-10), 138.1 (C-5), 134.9 (C-7), 132.3 (C-6), 131.6 (C-8), 130.6 (C-4), 129.7 (C-3), 119.1 (C-9), 52.7 (C-l 1).

[0146] 21 B-NMR: (160 MHz, CDCI3) ö [ppm] = 0.26 (t, 2 J BF = 28.6 Hz).

[0147] 19 F-NMR: (471 MHz, CDCI3) ö [ppm] = -145.1 (q, 2 J FB = 28.6 Hz).

[0148] HRMS (ESI): m / z berechnet für [M+H] + : 327.1111; gefunden: 327.1111. m / z berechnet für [M+H] + : 349.0930; gefunden: 349.0929.

[0149] FT-IR: (ATR) v [cm- 1 ] = 3113 (w), 2953 (w), 1721 (m), 1572 (m), 1542 (s),

[0150] 1479 (w), 1436 (w), 1412 (m), 1386 (s), 1355 (w), 1311 (w), 1278 (m), 1258 (s), 1225 (m), 1156 (m), 1107 (s), 1075 (s), 1048 (m), 1019 (m), 982 (m), 954 (w), 913 (m), 877 (w), 854 (w), 824 (w), 785 (m), 751 (m), 730 (s), 698 (w), 644 (w), 622 (w), 606 (w), 593 (w), 581 (w).

[0151] Methyl-4-( dichloro-BODIPYj-benzoat (12)

[0152] 11 12 13

[0153] 950 mg (2.91 mmol, 1.00 eq.) of BODIPY 11, 4.17 g (14.6 mmol, 5.00 eq.) of tBu4NCI, and 10.8 g (29.1 mmol, 10.0 eq.) of Cu(0Tf)2 were suspended in 120 mL of acetonitrile, and then 0.35 mL (5.8 mmol, 2.0 eq.) of ethanolamine was added. The reaction mixture was heated to 105 °C for 10 minutes, stirred at 105 °C for 110 minutes, and cooled to room temperature for 15 minutes. The reaction mixture was diluted with 300 mL of CH2Cl2 and washed three times with 200 mL of water each time. The aqueous phase was extracted with 100 mL of CH2Cl2, and the combined organic phases were dried with MgSO. The solvent was removed under reduced pressure, and the residue was purified by column chromatography (SiCl, EtOAc / cHex 1:8) to afford 603 mg (1.50 mmol, 52%) of a 5:1 mixture of dichloride 12 and trichloride 13 as a pink solid, which was used in the subsequent reaction without further separation. Yield of 12: 495 mg (1.25 mmol, 43%).

[0154] MP: 207 °C. tH-NMR: (500 MHz, CDCI3) ö [ppm] = 8.19 (d, 3 J H H = 8.2 Hz, 2H, H-3), 7.57 (d, 3 J H H = 8.2 Hz, 2H, H-4), 6.79 (d, 3 JHH = 4.2 Hz, 2H, H-8), 6.45 (d, 3JHH = 4.4 Hz, 2H, H-9), 3.99 (s, 3H, H-ll).

[0155] 13 C-NMR: (126 MHz, CDCI3) ö [ppm] = 166.2 (C-l), 145.9 (C-10), 142.4 (C-2), 136.7 (C-5), 133.7 (C-7), 132.5 (C-6), 131.6 (C-8), 130.6 (C-4), 129.9 (C-3), 119.4 (C-9), 52.7 (C-ll). nB-NMR: (160 MHz, CDCI3) ö [ppm] = 0.55 (t, 2 J BF = 27.9 Hz).

[0156] 19 F-NMR: (471 MHz, CDCI3) ö [ppm] = -148.2 (g, 2 J FB = 27.7 Hz).

[0157] HRMS (ESI): m / z berechnet für [M+H] + : 417.0151; gefunden: 417.0151.

[0158] FT-IR: (ATR) v[cm-‘] = 3676 (w), 3134 (w), 2988 (m), 2972 (m), 2902 (w), 1724 (m), 1572 (m), 1549 (m), 1453 (w), 1436 (w), 1407 (m), 1393 (s), 1315 (m), 1263 (m), 1195 (m), 1106 (s), 1077 (m), 1066 (m), 1057 (m), 1020 (m), 998 (w), 985 (m), 884 (w), 825 (w), 789 (w), 747 (w), 727 (m), 662 (w), 526 (w).

[0159] Ausbeute von 13: 0.108 g (0.25 mmol, 9%).

[0160] Methyl-4-( diiodo-BODIPY)-benzoat (14)

[0161] To 9.80 mL of a saturated solution of sodium iodide in propionitrile, 603 mg (1.50 mmol, 1.00 eq.) of a 5:1 mixture of dichloride 12 and trichloride 13 in 50 mL of CH2Cl2 was added. The resulting suspension was heated to 107 °C and stirred for 24 hours. After the reaction mixture had cooled to room temperature, 120 mL of CH2Cl2 was added, and the organic phase was washed twice with 120 mL of water each time. The combined aqueous phases were extracted with 150 mL of CH2Cl2, and the combined organic phases were dried with MgSO4. The solvent was removed under reduced pressure and the residue was purified by column chromatography (SiC; cHexiCl-FC 1:2) to obtain 460 mg (796 pmol, 64%) of diiodide 14 as a violet solid and 91 mg (129 pmol, 51%) of triiodide 15 also as a violet solid.

[0162] Yield of 14: 460 mg (796 pmol, 64%).

[0163] M (C 17 HnBF2l2N2O2): 577.90 g / mol.

[0164] DC: (SiO2, cHex:CH2CI21:2) R f = 0.50.

[0165] MP: 268 °C (Decomposition).

[0166] 1 H-NMR: (500 MHz, CDCI3) ö [ppm] = 8.17 (d, 3 J H H = 8.4 Hz, 2H, H-3), 7.57 (d,

[0167] 3 JHH = 8.4 Hz, 2H, H-4), 6.72 (d, 3 J H H = 4.3 Hz, 2H, H-8), 6.61 (d, 3 J H H = 4.2 Hz, 2H, H-9), 3.98 (s, 3H, H-ll).

[0168] 13 C-NMR: (126 MHz, CDCI3) ö [ppm] = 166.3 (C-3), 139.8 (C-6), 137.8 (C-7),

[0169] 137.0 (C-2), 132.4 (C-5), 131.7 (C-9), 130.4 (C-8), 130.4 (C-4), 129.8 (C-3), 104.1 (C-10), 52.7 (C- 11).

[0170] 11 B-NMR: (160 MHz, CDCI3) ä [ppm] = 0.72 (t, 2 J BF = 29.4 Hz).

[0171] 19 F-NMR: (471 MHz, CDCI3) ö [ppm] = -144.7 (q, 2 J FB = 29.6 Hz).

[0172] HRMS (ESI): m / z calculated for [M+H] + : 578.9043; found: 578.9045. m / z calculated for [M+Na] + : 600.8863; found: 600.8866.

[0173] FT-IR: (ATR) v [cm' 1 ] = 2951 (w), 1720 (w), 1569 (m), 1544 (s), 1438 (w),

[0174] 1406 (f), 1368 (m), 1302 (m), 1277 (m), 1243 (m), 1174 (m), 1141 (f), 1093 (s), 1019 (f), 990 (f), 970 (f), 912 (f), 881 (f), 854 (f), 824 (f), 789 (f), 727 (m).

[0175] Yield of 15: 91 mg (129 pmol, 51%).

[0176] General Working Procedure 1 (AAV1): Cross-coupling of BODIPY-

[0177] Diiodide 15

[0178] Diiodide 15 (1.00 eq.), boronic acids of type 16 (4.00 eq.), Pd(dba)2 (8 mol%), XPhos (8 mol%), and K3PO4·7H2O (4.00 eq.) were added to a Schlenk flask under an argon atmosphere. THF (80 mL / mmol 15) and water (12 mL / mmol 15) were both degassed individually and then added to the Schlenk flask. The mixture was stirred at 50 °C for 20 hours, then diluted with CH2Cl2 (200 mL / mmol 15) and washed three times with water (200 mL / mmol 15). The combined aqueous phases were extracted with CH2Cl2 (500 mL / mmol 15), the combined organic phases were dried with MgSO4, and the solvent was removed under reduced pressure. The residue was purified by column chromatography on silica gel to obtain Suzuki coupling products of type 17.

[0179] Methyl 4-(distyryl-BODIPY) benzoate (18)

[0180] According to AAV1, 60.0 mg (104 pmol, 1.00 eq.) of diiodide 15 was coupled to 61.5 mg (415 pmol, 4.00 eq.) of styrylboronic acid 16a using 4.8 mg (8.3 pmol, 8.0 mol%) of Pd(dba)2, 4.0 mg (8.3 pmol, 8.0 mol%) of XPhos, and 140 mg (415 pmol, 4.00 eq.) of K3PO4.7H2O in a mixture of 8.0 mL of degassed THF and 1.2 mL of degassed water. Purification by column chromatography (SiO2, EtOAc / cHex 1:20) afforded 41 mg (77 pmol, 75%) of BODIPY 18 as a blue solid.

[0181] M (C33H25BN2O2F2): 530.38 g / mol.

[0182] Yield: 41 mg (77 mol, 75%).

[0183] TLC: (SiO2, EtOAc / cHex 1:4) R f = 0.20.

[0184] MP: 153 °C.

[0185] 1 H-NMR: (600 MHz, CDCI3) 5 [ppm] = 8.18 (d, 3 J H H = 8.4 Hz, 2H, H-3), 7.79 (d,

[0186] 3 JHH = 16.3 Hz, 2H, H-12), 7.67 (d, 3 Jun = 7.4 Hz, 4H, H-14), 7.61 (d,

[0187] According to AAV1, 60.0 mg (104 pmol, 1.00 eq.) of diiodide 15 was coupled to 50.7 mg (415 pmol, 4.00 eq.) of phenylboronic acid 16b, containing 4.8 mg (8.3 pmol, 8.0 mol%) of Pd(dba)2, 4.0 mg (8.3 pmol, 8.0 mol%) of X-phos, and 140 mg (415 pmol, 4.00 eq.) of K3PO4.7H2O in a mixture of 8.0 mL of degassed THF and 1.2 mL of degassed water. Purification by column chromatography (SiCh, CH2Cl2) afforded 29.5 mg (0.062 mmol, 59%) of BODIPY 19 as a blue solid. M (C29H21BF2N2O2): 478.31 g / mol.

[0188] Yield: 29.5 mg (0.062 mmol, 59%).

[0189] TLC: (SiO2, cHex:CH2Cl21:2) R f = 0.75.

[0190] MP: 198 °C.

[0191] 1 H-NMR: (500 MHz, CDCI3) <5 [ppm] = 8.21 (d, 3 J H H = 8.3 Hz, 2H, H-3), 7.88 - 7.86

[0192] (m, 4H, H-12), 7.68 (d, 3 J H H = 8.3 Hz, 2H, H-4), 7.46 - 7.42 (m, 6H, H-13, H-14), 6.84 (d, 3 J H H = 4.3 Hz, 2H, H-8), 6.64 (d, 3 JH H = 4.2 Hz, 2H, H-

[0193] 9), 4.00 (s, 3H, H-15).

[0194] 13 C-NMR: (126 MHz, CDCI3) ö [ppm] = 166.5 (C-l ), 159.4 (C-10), 142.6 (C-6), 138.9 (C-2), 136.2 (C-7), 132.6 (C-ll), 131.8 (C-5), 130.7 (C-8) ,130.7 (C-4), 129.8 (C-14), 129.6 (C-12), 129.6 (C-3), 128.4 (C-13), 121.4 (C-9), 52.6 (C-15).

[0195] “B-NMR: (160 MHz, CDCI3) 5 [ppm] = 1.41 (t, 2 JBF = 31.6 Hz).

[0196] 19 F-NMR: (471 MHz, CDCI3) ö [ppm] = -132.5 (q, 2 J FB = 31.6 Hz).

[0197] HRMS (ESI): m / z berechnet für [M+Na] + : 501.1556; gefunden: 501.1560.

[0198] UV / VIS: Absorption (EtOH): A max = 557 nm.

[0199] Fluoreszenz (EtOH): A max = 594 nm.

[0200] FT-IR: (ATR) v [cm- 1 ] = 3063 (w), 2952 (w), 1722 (m), 1601 (w), 1570 (s),

[0201] 1546 (s), 1512 (f), 1469 (m), 1451 (s), 1398 (f), 1356 (f), 1281 (s), 1272 (s), 1219 (m), 1190 (f), 1138 (s), 1107 (m), 1070 (s), 1019 (m), 1000 (m), 983 (m), 945 (f), 909 (f), 884 (f), 855 (f), 823 (f), 801 (f), 764 (m), 749 (f), 728 (s), 695 (m), 649 (f), 618 (f).

[0202] Methyl 4-(dinaphthyl-BODIPY) benzoate (20)

[0203] According to AAV1, 60.0 mg (104 pmol, 1.00 eq.) of diiodide 15 was coupled to 714 mg (415 pmol, 4.00 eq.) of naphthylboronic acid 16c, yielding 4.8 mg (8.3 pmol, 8.0 mol / mol) of Pd(dba)z, 4.0 mg (8.3 pmol, 8.0 mol / mol) of X-Phos, and 140 mg (415 pmol, 4.00 eq.) of K3PO4.7H2O in a mixture of 8.0 mL of degassed THF and 1.2 mL of degassed water. Purification by column chromatography (SiO2, EtOAc / cHex 1:7) afforded 52 mg (90 pmol, 87%) of BODIPY 20 as a blue solid.

[0204] M (C37H25BF2N2O2): 578.43 g / mol.

[0205] Yield: 52 mg (90 pmol, 87%).

[0206] TLC: (SiO2, EtOAc / cHex 1:4) R f= 0.37.

[0207] MP: 206 °C.

[0208] 1 H-NMR: (500 MHz, CDCI3) ö [ppm] = 8.39 (d, 4 J HH = 0.8 Hz, 2H, H-12), 8.24 (d,

[0209] 3 JHH = 8.6 Hz, 2H, H-3), 8.01 (dd, 3 J H H = 8.6 Hz, 4 J H H = 1.8 Hz, 2H, H-20), 7.93 - 7.89 (m, 2H, H-14), 7.87 (d, 3 J H H = 8.6 Hz, 2H, H-19), 7.85 - 7.80 (m, 2H, H-17), 7.72 (d, 3 J H H = 8.6 Hz, 2H, H-4), 7.53 - 7.44 (m, 4H, H- 15, H-16), 6.89 (d, 3 JHH = 5.6 Hz, 2H, H-9), 6.78 (d, 3 J H H = 5.6 Hz, 2H, H-8), 4.01 (s, 3H, H-21).

[0210] 13C-NMR: (126 MHz, CDCI3) ö [ppm] = 166.6 (C-l ), 159.6 (C-10), 142.2 (C-2), 139.0

[0211] (C-5), 136.5 (C-7), 133.9 (C-18), 133.1 (C-13), 131.8 (C-6), 130.8 (C-4), 130.7 (C-9), 130.0 (C-ll), 129.8 (C-12), 129.7 (C-3), 129.1 (C-14), 128.0 (C-19), 127.8 (C-17), 127.2 (C-16), 126.8 (C-20), 126.5 (C-15), 121.8 (C-8), 52.7 (C-21).

[0212] ^B-NMR: (160 MHz, CDCI3) 5 [ppm] = 1.60 (t, 2 J BF = 31.8 Hz).

[0213] 19 F-NMR: (471 MHz, CDCI3) ö [ppm] = -132.2 (q, 2 J FB = 31.7 Hz).

[0214] HRMS (ESI): m / z calculated for [M+H] + : 579.2050; found: 579.2045. m / z calculated for [M+Na] + : 601.1869; found: 601.1873.

[0215] UV / VIS: Absorption (EtOH): A max = 579 nm.

[0216] Fluorescence (EtOH): A max = 624 nm.

[0217] FT-IR: (ATR) v [erm 1 ] = 3056 (w), 2951 (w), 2926 (w), 2853 (w), 2073 (w),

[0218] 1922 (f), 1722 (m), 1628 (f), 1598 (f), 1571 (s), 1545 (s), 1489 (m),

[0219] 1459 (m), 1438 (m), 1418 (w), 1404 (w), 1353 (w), 1332 (w), 1272 (s),

[0220] 1246 (m), 1222 (w), 1195 (w), 1180 (w), 1139 (s), 1126 (s), 1109 (s),

[0221] 1072 (s), 1056 (s), 1019 (w), 986 (m), 939 (w), 909 (w), 886 (w),

[0222] 861 (w), 823 (w), 796 (m), 750 (m), 727 (s), 694 (w), 648 (w), 612 (w), 600 (w), 477 (w), 445 (w). 4-( Distyryl-BODIPY)-4-benzoesäure (21)

[0223] To a solution of 375 mg (707 pmol, 1.00 eq.) of methyl ester 18 in 71 mL of MeOH was added 7.1 mL of 0.5 M NaOH(aq). The mixture was heated to 80 °C and stirred for 18 hours. The solvent was removed under reduced pressure, and the residue was dissolved in 50 mL of 10% KHSO4(aq). This solution was extracted three times with 50 mL of CH2Cl2 each time. The combined organic layers were dried with MgSO4, and the solvent was removed under reduced pressure. The residue was purified by column chromatography (SiCl2, EtOAc) to afford 210 mg (407 pmol, 61%) of acid 21 as a blue solid.

[0224] M (C32H23BN2O2F2): 516.35 g / mol.

[0225] Yield: 210 mg (407 pmol, 61%).

[0226] TLC: (SiO2, EtOAc) R f = 0.15.

[0227] MP: 218 °C.

[0228] 1 H-NMR: (500 MHz, CDCI3) ä [ppm] = 8.25 (d, 3 JHH = 8.2 Hz, 2H, H-3), 7.97 - 7.80

[0229] (m, 2H, H-12), 7.68 - 7.64 (m, 6H, H-4, H-14), 7.44 - 7.31 (m, 8H, H-ll, H-15, H-16), 6.98 - 6.96 (m, 1H, H-9), 6.80 (d, 3 JHH = 4.4 Hz, 1H, H-8).

[0230] 13 C-NMR: (126 MHz, CDCI3) ö [ppm] = 169.8 (C-l ), 155.6 (C-10), 139.6 (C-2), 137.8

[0231] (C-7), 137.4 (C-ll), 136.6 (C-13), 136.5 (C-5), 130.8 (C-6), 130.7 (C-4), 130.2 (C-3), 129.5 (C-8), 129.3 (C-16) 129.0 (C-15), 127.9 / 127.9 (C-16),

[0232] 119.4 (C-2), 116.8 (C-9).

[0233] 11 B-NMR: (160 MHz, CDCI3) ö [ppm] = 1.32 (t, 2 J BF = 33.1 Hz).

[0234] 19 F-NMR: (471 MHz, CDCI3) ö [ppm] = -139.6 (q, 2 J FB = 33.4 Hz).

[0235] HRMS (ESI): m / z berechnet für [M+Na] + : 539.1713; gefunden: 539.1709. UV / VIS: Absorption (EtOH): Ä max = 636 nm.

[0236] Fluoreszenz (EtOH): Ä max = 655 nm.

[0237] FT-IR: (ATR) v [cm- 1 ] = 3676 (w), 2970 (m), 2922 (m), 2114 (w), 1691 (m),

[0238] 1610 (w), 1570 (m), 1537 (m), 1498 (m), 1471 (m), 1439 (m), 1407 (m), 1395 (m), 1378 (m), 1344 (m), 1311 (m), 1285 (m), 1231 (s), 1178 (m), 1088 (s), 1066 (s), 1058 (s), 1028 (s), 1014 (s), 958 (m), 903 (m), 887 (m), 845 (m), 789 (m), 767 (m), 752 (m), 725 (s), 691 (s).

[0239] Lipo Fluddy 1

[0240] Lipo Fluddy 1

[0241] To a solution of 33 mg (64 pmol, 1.0 eq.) of BODIPY 21 and 28 mg (64 pmol, 1.0 eq.) of Rhodol 6 in 2 mL of anhydrous CH2Cl2, 9.4 mg (77 pmol, 1.2 eq.) of DMAP and 20 mg (105 pmol, 1.7 eq.) of EDC were added, and the mixture was stirred at room temperature for 16 hours. The solvent was removed under reduced pressure, and the residue was purified by column chromatography (SiO2, EtOAc / cHex 1:9) to afford 24 mg (25 pmol, 39%) of Lipo-Fluddy 1 as a blue solid.

[0242] M (C60H52BN3O5F2): 943.90 g / mol.

[0243] Ausbeute: 24 mg (25 pmol, 39%).

[0244] DC: (SiO2, EtOAc / cHex 1:2) R f = 0.42.

[0245] MP: 87 °C.

[0246] ^-NMR: (600 MHz, CDCI3) ä [ppm] = 8.18 (d, 3 JHH = 8.4 Hz, 2H, H-31 ), 7.96 - 7.79

[0247] (m, 2H, H-40), 7.66 (d, 3 J H H = 7.3 Hz, 4H, H-42), 7.60 (d, 3 JHH = 8.4 Hz,

[0248] 2H, H-32), 7.44 - 7.32 (m, 10H, H-3, H-4, H-39, H-43, H-44), 7.25 - 7.23 (m, 1H, H-5), 6.96 - 6.93 (m, 2H, H-37), 6.90 (d, 3 J H H = 7.7 Hz, 1H, H- 6), 6.87 - 6.84 (m, 1H, H-19), 6.78 - 6.73 (m, 3H, H-16, H-36), 6.70 (d, 3JHH = 8.5 Hz, 1H, H-10), 6.67 - 6.64 (m, 1H, H-18), 6.37 - 6.36 (m, 1H, H-13), 6.30 - 6.29 (m, 1H, H-ll), 5.25 (s, 2H, H-7), 4.74 - 4.70 (m, 2H, H-28), 4.37 - 4.34 (m, 2H, H-27), 3.10 (t, 3 JHH = 7.1 Hz, 2H, H-21), 1.63

[0249] - 1.59 (m, 2H, H-22), 1.41 - 1.36 (m, 2H, H-23), 1.33 - 1.29 (m, 4H, H- 24, H-25), 0.91 - 0.88 (m, 3H, H-26).

[0250] 13 C-NMR: (151 MHz, CDCI3) ö [ppm] = 166.0 (C-29), 159.2 (C-17), 155.5 (C-38), 152.0 (C-14), 151.9 (C-15), 149.7 (C- 12), 145.2 (C-2), 139.6 (C-l), 139.1 (C-30), 137.9 (C-35), 137.3 (C-39), 136.5 (C-41), 136.1 (C-33), 131.2 (C-34), 130.6 (C-32), 130.2 (C-19), 129.9 (C-31), 129.8 (C-10), 129.5 (C-36), 129.4 (C-44), 129.0 / 128.9 (C-43), 128.4 (C-4), 128.0 (C-3), 127.9 (C-42), 124.1 (C-6), 120.8 (C-4), 119.4 (C-40), 118.3 (C-20), 116.9 (C-37), 113.2 (C-9), 111.3 (C-18), 110.2 (C-ll), 101.6 (C-16), 98.1 (C- 13), 83.9 (C-8), 71.8 (C-7), 66.3 (C-27), 63.8 (C-28), 44.0 (C-21), 31.8 (C-24), 29.5 (C-22), 27.0 (C-23), 22.8 (C-25), 14.2 (C-26). nB-NMR: (160 MHz, CDCI3) ä [ppm] = 1.31 (t, 2 J B F = 32.9 Hz).

[0251] 19 F-NMR: (471 MHz, CDCI3) ä [ppm] = -139.5 (q, 2 J FB = 32.8 Hz).

[0252] HRMS (ESI): m / z berechnet für [M+H] + : 944.4041; gefunden: 944.4054.

[0253] FT-IR: (ATR) v [cm- 1 ] = 2926 (w), 2855 (w), 1722 (w), 1615 (m), 1571 (m),

[0254] 1540 (s), 1499 (m), 1473 (m), 1440 (m), 1423 (m), 1346 (w), 1311 (m), 1272 (m), 1240 (m), 1196 (m), 1120 (s), 1068 (w), 1014 (m), 959 (w), 905 (w), 846 (w), 793 (w), 754 (w), 726 (m), 693 (w), 600 (w). 4-( Diphenyl-BODIPY)-4-benzoesäure (22) 22

[0255] To a solution of 22 mg (45 gmoi, 1.0 eq.) of methyl ester 19 in 4.5 mL MeOH was added 0.45 mL of 0.2 M NaOH(aq). The mixture was heated to 90 °C and stirred for 19 hours. The solvent was removed under reduced pressure, and the residue was dissolved in 10 mL of 10% KHSO4(aq). This solution was extracted three times with 20 mL of CH2Cl2 each. The combined organic phases were washed with 10 mL of 10% KHSO4(aq) and water, dried with MgSO4, and the solvent was removed under reduced pressure to afford 15 mg (32 pmol, 72%) of the crude acid 22, which was used directly in the next step without further purification.

[0256] M (C2SH 19 BN2O2F2): 464.28 g / mol.

[0257] Yield: 15 mg (32 gmol, 72%).

[0258] TLC: (SiO2, EtOAc / cHex / AcOH 1:4:0.1) R f = 0.09.

[0259] Lipo Fluddy 2

[0260] Lipo Fluddy 2

[0261] To a solution of 15 mg (32 pmol, 1.0 eq.) of acid 22 and 18 mg (40 pmol, 1.0 eq.) of rhodol 6 in 1.2 mL of anhydrous CH2Cl2, 6.1 mg (50 pmol, 1.6 eq.) of DMAP and 13 mg (67 pmol, 2.1 eq.) of EDC were added, and the mixture was stirred at room temperature for 18 hours. The solvent was removed under reduced pressure, and the residue was purified by column chromatography (SiO2, EtOAc / cHex 1:4) to afford 21 mg (24 pmol, 75%) of Lipo-Fluddy 2 as a purple oil.

[0262] M (C56H48BF2N3O5): 891.82 g / mol.

[0263] Yield: 21 mg (0.024 mmol, 75%).

[0264] TLC: (SiO2, EtOAc / cHex 1:4) R f = 0.15.

[0265] ^-NMR: (600 MHz, CDCI3) ö [ppm] = 8.21 (d, 3 JHH = 7.9 Hz, 2H, H-3), 7.93 - 7.86

[0266] (m, 4H, H-12), 7.68 - 7.66 (m, 2H, H-4), 7.45 - 7.38 (m, 6H, H-13, H-14), 7.36 - 7.34 (m, 2H, H-27, H-28), 7.25 - 7.24 (m, 1H, H-26), 6.91 (d, 3 JHH = 7.5 Hz, 1H, H-25), 6.87 (d,3 JHH = 8.7 Hz, 1H, H-21 ), 6.83 - 6.80 (m, 2H, H-8), 6.78 (d, 4 JHH = 1.5 Hz, 1H, H-18), 6.71 (d, 3 JHH = 8.5 Hz, 1H, H-32), 6.66 (dd, 3 J H H = 8.6 Hz, 4 JHH = 2.0 Hz, 1H, H-22), 6.63 - 6.62 (m, 2H, H- 9), 6.37 (s, 1H, H-35), 6.29 (dd, 3 JHH = 8.5 Hz, 4 J H H = 1.8 Hz, 1H, H-33), 5.26 (s, 2H, H-30), 4.75 - 4.74 (m, 2H, H-15), 4.38 - 4.37 (m, 2H, H-16), 3.10 (t, 3 JHH = 7.0 Hz, 2H, H-37), 1.63 - 1.58 (m, 2H, H-38), 1.41 - 1.38 (m, 2H, H-39), 1.32 - 1.31 (m, 4H, H-40, H-41), 0.90 (t, 3 J H H = 6.7 Hz, 3H, H-42).

[0267] 13 C-NMR: (151 MHz, CDCI3) ö [ppm] = 165.9 (C-l), 159.6 (C-10), 159.2 (C-17), 152.0 (C-36), 151.9 (C-19), 149.7 (C-34), 145.2 (C-29), 142.5 (C-6),

[0268] 139.6 (C-24), 139.1 (C-2), 136.2 (C-7), 132.6 (C-ll), 131.5 (C-5), 130.8 (C-4), 130.7 (C-8), 130.2 (C-21), 129.8 - 129.6 (C-3, C-12, C-14, C-32, C-33), 128.4 (C-13), 128.2 (C-26), 128.0 (C-28), 124.1 (C-25), 121.5 (C- 9), 120.8 (C-27), 118.3 (C-20), 113.2 (C-31 ), 111.4 (C-22), 110.2 (C-33),

[0269] 101.6 (C-18), 98.1 (C-35), 83.9 (C-23), 71.8 (30), 66.3 (C-16), 63.9 (C-15), 44.0 (C-37), 31.7 (C-40), 29.5 (C-38), 26.9 (C-39), 22.8 (C-41), 14.2 (C-42). nB-NMR: (193 MHz, CDCI3) ö [ppm] = 1.41 (t, 2 J B F = 63.3 Hz).

[0270] 19 F-NMR: (376 MHz, CDCI3) ö [ppm] = -132.5 (q, 2 J FB = 31.7 Hz).

[0271] HRMS (ESI): m / z berechnet für [M+Na] + : 914.3553; gefunden: 914.3552.

[0272] FT-IR: (ATR) v [cm- 1 ] = 3416 (w), 3065 (w), 2954 (w), 2928 (w), 2856 (w),

[0273] 1722 (m), 1633 (f), 1615 (m), 1571 (s), 1548 (s), 1502 (m), 1470 (m), 1452 (s), 1418 (m), 1398 (f), 1355 (f), 1271 (s), 1217 (m), 1195 (m), 1138 (s), 1109 (s), 1070 (s), 1017 (m), 1000 (m), 964 (w), 945 (f), 909 (f), 885 (f), 849 (f), 800 (f), 763 (m), 727 (m), 696 (m), 649 (f), 636 (w).

[0274] 4-(Dinaphtyl-BODIPY)-4-benzoic acid (23)

[0275] To a solution of 35 mg (61 gmol, 1.0 eq.) methyl ester 20 in 6.1 mL MeOH was heated to 60 °C and stirred for 18 hours. The solvent was evaporated under reduced pressure, and the residue was dissolved in 20 mL of 10% KHSO 4 (aq). This solution was extracted three times with 30 mL of CH 2 Cl 2 each time. The combined organic phases were dried with MgSO 3 , and the solvent was removed under reduced pressure to yield 30 mg (53 pmol, 88%) of the crude acid 23, which was used directly in the next step without further purification.

[0276] M (C36H23BN2O2F2): 564.18 g / mol.

[0277] Yield: 30 mg (53 gmol, 88%).

[0278] TLC: (SiO2, EtOAc / cHex / AcOH 1:4:0.1) R f = 0.17.

[0279] Lipo Fluddy 3

[0280] Lipo Fluddy 3

[0281] To a solution of 30 mg (53 pmol, 1.0 eq.) of acid 23 and 25 mg (56 pmol, 1.1 eq.) of rhodol 6 in 1.6 mL of anhydrous CH2Cl2, 7.8 mg (64 pmol, 1.2 eq.) of DMAP and 17 mg (88 pmol, 1.7 eq.) of EDC were added, and the mixture was stirred at room temperature for 16 hours. The solvent was removed under reduced pressure, and the residue was purified by column chromatography (SiO2, EtOAc / cHex 1:9) to afford 40 mg (40 pmol, 76%) of Lipo-Fluddy 3 as a blue solid.

[0282] M (C54H52BF2N3O5): 991.40 g / mol.

[0283] Yield: 40 mg (40 pmol, 76%).

[0284] TLC: (SiO2, cHex:EtOAc 1:4) R f = 0.44.

[0285] MP: 114 °C.

[0286] 1 H-NMR: (500 MHz, CDCI3) ö [ppm] = 8.38 (d, 4 J H H = 1.8 Hz, 2H, H-40), 8.23 (d,

[0287] 3 JHH = 8.3 Hz, 2H, H-31), 8.00 (dd, 3 J H H = 8.6 Hz, 4 J H H = 1.8 Hz, 2H, H-48), 7.92 - 7.79 (m, 6H, H-42, H-45, H-47), 7.70 (d, 3 JHH = 8.3 Hz, 2H, H-32), 7.53 - 7.44 (m, 4H, H-43, H-44), 7.38 - 7.32 (m, 2H, H-3, H-4), 7.25 - 7.23 (m, 1H, H-5), 6.91 (d, 3 J HH = 7.6 Hz, 1H, H-6), 6.89 - 6.85 (m, 3H, H-19, H-37), 6.80 - 6.74 (m, 3H, H-16, H-36), 6.71 (d, 3 J HH = 8.5 Hz, 1H, H-10), 6.67 (dd, 3 JHH = 8.7 Hz, 4 JHH = 2.5 Hz, 1H, H-18), 6.37 (d, 4JHH = 2.3 Hz, 1H, H-13), 6.29 (dd, 3 J H H = 8.6 Hz, 4 J H H = 2.4 Hz, 1H, H-ll), 5.26 (s, 2H, H-7), 4.75 (t, 3 J H H = 4.7 Hz, 2H, H-28), 4.38 (t, 3JHH = 4.7 Hz, 2H, H-27), 3.10 (t, 3JHH = 7.1 Hz, 2H, H-21), 1.63 - 1.58 (m, 2H, H-22), 1.41 - 1.36 (m, 2H, H-23), 1.34 - 1.27 (m, 6H, H-24, H-25), 0.90 - 0.88 (m, 3H, H-26).

[0288] 13 C-NMR: (126 MHz, CDCI3) ö [ppm] = 166.0 (C-29), 159.5 (C-38) , 159.2 (C-17),

[0289] 152.0 (C-14), 151.9 (C-15), 149.7 (C-12), 145.2 (C-2), 142.2 (C-30), 139.6 (C-l), 139.2 (C-33), 136.5 (C-35), 133.9 (C-46), 133.1 (C-41),

[0290] 131.5 (C-34), 130.8 (C-32), 130.7 (C-37), 130.2 (C-19), 130.1 (C-39),

[0291] 129.8 (C-40), 129.7 (C-10, C-31), 129.1 (C-42), 128.4 (C-5), 128.0 (C-

[0292] 47), 128.0 (C-3), 127.8 (C-45), 127.2 (C-44), 126.8 (C-48), 126.4 (C-43), 124.1 (C-6), 121.8 (C-36), 120.7 (C-4), 118.3 (C-20), 113.2 (C-9), 111.4 (C-18), 110.2 (C-ll), 101.6 (C-16), 98.1 (C-13), 83.9 (C-8), 71.8 (C-7), 66.3 (C-27), 63.9 (C-28), 44.0 (C-21), 31.7 (C-24), 29.5 (C-22), 27.0 (C- 23), 22.8 (C-25), 14.2 (C-26). nB-NMR: (160 MHz, CDCI3) ö [ppm] = 1.59 (t,2 J BF = 31.8 Hz).

[0293] 19 F-NMR: (471 MHz, CDCI3) ö [ppm] = -132.1 (q, 2 J FB = 31.9 Hz).

[0294] HRMS (ESI): m / z calculated for [M+H] + : 992.4041; found: 992.4053. m / z calculated for [M+Na] + : 1014.3860; found: 1014.3869.

[0295] FT-IR: (ATR) v [cm- 1 ] = 2925 (m), 1723 (w), 1615 (m), 1572 (s), 1548 (s),

[0296] 1490 (m), 1459 (m), 1439 (m), 1417 (f), 1376 (f), 1270 (s), 1196 (m), 1139 (s), 1127 (s), 1109 (s), 1072 (s), 1057 (s), 1018 (m), 939 (f), 887 (f), 797 (m), 752 (f), 727 (f), 478 (f). Example 4

[0297] Measurement of autophagy

[0298] Lipophagy measurement

[0299] HeLa cells were plated in serum-containing medium to prevent activation of endogenous lipophagy and maintained at 37°C and 5% CO2. After 24 hours, oleic acid was added to the lipid droplets of the cells at a final concentration of 400 μM for 1 hour to facilitate staining of the lipid droplets. The cells were stained with Lipo-Fluddyl resuspended in PBSlx at a concentration of 10 μM for 10 minutes at 37°C and 5% CO2. After staining, the cells were maintained in serum-containing medium as a reference (tO) or in HBSS-deficient medium for 15, 30, or 60 minutes to induce different stages of lipophagy. Live imaging was performed at the indicated time points. An increase in the green signal over time after lipophagy induction is evident, demonstrating the functionality of the probe.

Claims

Patent claims 1. A double fluorescent dye compound comprising a fluorescent dye A and a fluorescent dye B, wherein Fluorescent dye A and fluorescent dye B are covalently bonded to each other, the emission maxima of fluorescent dye A and fluorescent dye B are separated by at least 100 nm, measured at 23°C, pH 5.8 Fluorescent dye A is pH-sensitive and at pH 7.4 has a maximum of 20% of the fluorescence intensity of pH 5. The double fluorescent dye compound is lipophilic.

2. Double fluorescent dye compound according to claim 1, wherein fluorescent dye A and fluorescent dye B are covalently linked to each other by means of a linker.

3. A double fluorescent dye compound according to claim 1 or 2, wherein fluorescent dye A is a xanthene or rhodamine analogue.

4. Double fluorescent dye compound according to any one of the preceding claims, wherein fluorescent dye B is a boron dipyrromethene (BODIPY) dye.

5. Double fluorescent dye compound according to one of the preceding claims, wherein at least one of the fluorescent dyes A and fluorescent dyes B has a lipophilic side chain with 5 to 22 C atoms, which optionally comprises silyl groups.

6. Double fluorescent dye compound according to one of the preceding claims, wherein the double fluorescent dye compound has the following structure, and where the substituents have the following meaning: R a , R b , R c , R d , R e , R f = independently H, alkyl, aryl, alkenyl, alkynyl, halogen, alkoxy, trialkylsilyl, trialkylsilyloxy R 1 = alkyl, aryl, silanylalkyl, silyloxyalkyl R 2= H, alkyl, aryl, silanylalkyl, silyloxyalkyl XY = a linker with a chain length of 0-20 C atoms Z a ' Z b , Z c , Z d = independently H, alkyl, acyl, silyl, alkoxy, silyloxy, halogen, sulfonyl; where R a , R b , R c , R d , R e , R f , Z a ' Z b , Z c , Z d maximum of 20 C atoms each and R 1 , R 2 contain a maximum of 16 C atoms each.

7. Double fluorescent dye compound according to one of the preceding claims, wherein R a , R f Alkyl, alkenyl or aryl are R b , R c , R d , R e H are, R 1 Alkyl is R 2 H is, Z a ' Z b , Z c , Z d H are.

8. Double fluorescent dye compound according to one of the preceding claims, wherein the double fluorescent dye compound has one of the following structures:

9. A process for preparing the double fluorescent dye compound according to any one of claims 1 to 8, wherein dye A and dye B are coupled together.

10. A method for examining cells, comprising the steps of: contacting cells with a double fluorescent dye compound according to any one of the preceding claims Excitation of the fluorescent dyes A and B. Measurement of the emission of the fluorescent dyes A and B.

11. The method according to claim 10, wherein the measurement of the emission is spatially resolved.

Images