Cyanine dyes and their usage for in vivo staining of microorganisms and other living cells

Abstract

The invention provides new cyanine dyes for staining living cells, providing fluorescence emission in red, green and yellow, thus allowing “multichannel” staining. The dyes are binding to nucleic acids and allow the observation of the stained cells, for the staining does not negatively interfere with the viability of the stained cells. The inventive dyes thus can advantageously be used for pathogen-host investigations or any other type of investigation of cell-cell-interactions, for the natural behavior of the stained cells (microorganisms, pathogens etc.) can easily be observed.

Claims

1. A method for staining living cells characterized in that the method comprises the following steps: i) at least one cyanine dye is applied to at least one sample of cells by adding a solution of the at least one cyanine dye to the at least one sample of cells, whereat the at least one cyanine dye is not reducing the viability of the stained cells substantially, whereat the non-reduction of the viability of the living cells through the at least one cyanine dye is defined by comparable microbial growth measurement against DMSO under identical breeding conditions whereat the cell-count in the sample with the cyanine dye after 10 h breeding under identical conditions at dye concentration of 2.5 μM is at least 94% compared to DMSO-reference and/or at dye concentration of 5 μM is at least 80% compared to DMSO-reference and/or comparable viability measurements of eukaryotic cells under identical breeding conditions at dye concentration of 5 μM is at least 85% compared to reference, whereat the reference is either viability of the eukaryotic cells in presence of acetonitrile or viability of the pure cells or viability of the cells, grown in water, thus providing at least one sample of cells, being stained with at least one cyanine dye; ii) incubating the at least one sample of cells according to step i) for at least 15 minutes, so that stained living cells are existent.

2. The method for staining living cells according to claim 1, characterized in that the method comprises the following steps i) and ii) and the additional step iii): i) at least two cyanine dyes are applied to at least two separate samples of cells whereat each sample is stained with one of the at least two cyanine dyes by adding a solution of the cyanine dye to the sample of cells, whereat each cyanine dye exerts a fluorescence emission wavelength different from the fluorescence emission wavelengths of all other cyanine dyes being applied and each cyanine dye is not reducing the viability of the stained cells substantially, whereat the non-reduction of the viability of the living cells through the cyanine dye is defined for each cyanine dye by comparable microbial growth measurement against DMSO under identical breeding conditions whereat the cell-count in the sample with the cyanine dye after 10 h breeding under identical conditions at dye concentration of 2.5 μM is at least 94% compared to DMSO-reference and/or at dye concentration of 5 μM is at least 80% compared to DMSO-reference and/or comparable viability measurements of eukaryotic cells under identical breeding conditions at dye concentration of 5 μM is at least 85% compared to reference, whereat the reference is either viability of the eukaryotic cells in presence of acetonitrile or viability of the pure cells or viability of the cells, grown in water, thus providing at least two separate samples of cells, each one being stained with at least one cyanine dye; ii) each sample of cells according to modified step i) is incubated for at least 15 minutes, so that stained living cells are existent; iii) the at least two separate samples of cells provided according to step ii), each one being stained with at least one cyanine dye, are combined within one sample vessel with each other thus forming one combined sample.

3. The method according to claim 1, characterized in that the cyanine dye is a nucleic acid binding cyanine dye.

4. The method according to claim 1, characterized in that the method comprises the following additional preparation step before step i): a) obtaining and isolation of at least one sample of cells to be introduced into step i), whereat this obtaining and isolation-step may include one or more steps for concentrating and/or breeding of the cells.

5. A cyanine dye for usage in claim 1, the chemical structure of the cyanine dye being according to general formula (I), ##STR00017## wherein R′ is a moiety according to formula (II) or a moiety according to formula (III), ##STR00018## R.sub.2 is selected from the list comprising H, methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, tert-butyl, alkyl, alkinyl, alkylidene insofar as substituent R.sub.2 is present according to the choice of R′, R.sub.3 and R.sub.4 are independently from each other selected from the list comprising H, methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, tert-butyl, alkyl, alkinyl, alkylidene insofar as substituents R.sub.3, R.sub.4 are present according to the choice of R′, R.sub.5 to R.sub.20 are independently from each other selected from the list comprising the substituents H, halogen, methyl. ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy, amino, methylamino, ethylamino, monoalkylamino, dialkylamino, a nitro group, a sulfo group insofar as any one of substituents R.sub.5 to R.sub.20 is present according to the choice of R′, Z.sub.1 is S and Z.sub.2 is N, X.sup.− is selected from the list comprising halogenate, sulfate, triflate, trifluoro acetate, difluoro acetate or fluoro acetate, n is 1 to 3, m is 6, characterized by R.sub.1 is independently chosen from the list comprising the substituents triphenylphosphine, substituted triphenylphosphine, 1,2,3-triazol-1-yl, 1,2,3-triazol-2-yl, 1,2,3-triazol-4-(aminomethyl)-1-yl, mitochondria penetrating peptide—whereat the mitochondria penetrating peptide is chemically bound to the alkyl-chain of length m via peptide-bond or via sulfide-bond or via ester-bond or via thioester-bond- if R′ is chosen to be a moiety according to formula (II); or R.sub.1 is independently chosen from the list comprising the substituents OH, SH, N.sub.3, NH.sub.2, NH.sub.3+, NHR.sub.3, NH.sub.2R.sub.3.sup.+, NR.sub.21R.sub.22, NR.sub.21R.sub.22H.sup.+—whereat R.sub.21 and R.sub.22 are independently from each other chosen from the list comprising methyl, ethyl or propyl —, substituted or non-substituted heterocyclic structure, substituted or non-substituted cyclic structure, triphenylphosphine, substituted triphenylphosphine, 1,2,3-triazol-1-yl, 1,2,3-triazol-2-yl, substituted 1,2,3-triazol-1-yl, substituted 1,2,3-triazol-2-yl, mitochondria penetrating peptide—whereat the mitochondria penetrating peptide is chemically bound to the alkyl-chain of length m via peptide-bond or via sulfide-bond or via ester-bond or via thioester-bond- if R′ is chosen to be a moiety according to formula (III).

6. The cyanine dye according to claim 5, characterized in that the mitochondria penetrating peptide has the amino acid sequence H2N-Cys-Cha-dArg-Cha-dArg-COOH according to SEQ ID 1 or the amino acid sequence according to SEQ ID 2 or the amino acid sequence according to SEQ ID 3 or the amino acid sequence according to SEQ ID 4, whereat each mitochondria penetrating peptide is acetylated at the N-terminus.

7. The A cyanine dye according to claim 5, characterized in that it has the chemical structure according to formula (A), ##STR00019## or according to formula (B) ##STR00020## or according to formula (C), ##STR00021##

8. The cyanine dye according to claim 5 in its protonated or deprotonated form, whereat the counterion, resp. counterions, of the protonated or deprotonated form is, resp. are, independently selected from the list comprising halogenate, sulfate, triflate, trifluoro acetate, difluoro acetate or fluoro acetate.

9. The cyanine dye according to claim 5, characterized in that it is for use in binding-to oligomeric DNA or primer-DNA or DNA-aptamers.

10. A method of staining nucleic acid within a living cell comprising applying the cyanine dye according to claim 5 to the living cell.

11. A kit for carrying out the method according to claim 1, the kit comprising the cyanine dye and optionally supportive substances.

12. The synthesizing method of a cyanine dye according to claim 5, characterized in that the synthesizing method comprises the following steps: a) performing a substitution reaction on a precursor compound according to formula (IV), ##STR00022## whereat the substituent Y in formula (IV) is selected from the list comprising the substituents chlorine, bromine, iodine, mesylate, tosylate, fluorinated mesylate, fluorinated tosylate and all other substituents in formula (IV) have the same meaning and are chosen in the same way as is denoted in claim 5 regarding formula (I) and indices m and n have the same values as are denoted in claim 5 regarding formula (I), by reacting the precursor compound according to formula (IV) in a reaction mixture with a nucleophilic compound, leading to the substitution of substituent Y by the nucleophilic compound; b) extracting the reaction product from the reaction mixture from step a) and c) purifying the reaction product retrieved from step b).

13. The method according to claim 12 characterized in that the nucleophilic compound in step a) is triphenylphosphine or 1,2,3-triazole or a mitochondria penetrating peptide.

14. The method according to claim 12 characterized in that the nucleophilic compound in step a) is the azide ion and the reaction product from step a) or the extracted reaction product from step b) or the purified reaction product from step c) is further reacted in an additional step d), the additional step d) being inserted between steps a) and b) or between steps b) and c) or being placed after step c).

15. The method according to claim 14, characterized in that the additional step d) comprises the reaction of a precursor compound according to formula (V), ##STR00023## whereat in this case R′ is restricted to a moiety according to formula (III), with substituted or unsubstituted Tris((1-benzyl-4-triazolyl)methyl)amine, “TB TA”, thus forming a cyanine dye according claim 5 having a substructure where R′ is a moiety according to formula (III) and R.sub.1 is a substituent with a substituted or non-substituted heterocyclic structure.

16. The method according to claim 14, characterized in that the additional step d) comprises the reaction of a precursor compound according to formula (V), ##STR00024## whereat R′ is a moiety according to formula (II) or according to formula (III), whereat n is 1 or n is 2 or n is 3, in a copper-catalyzed azide-alkyne cycloaddition reaction with a substituted or unsubstituted alkyne, thus forming a cyanine dye according to claim 5, whereat R.sub.1 is a substituent with a substituted or non-substituted heterocyclic structure.

Description

DETAILED EMBODIMENTS OF THE INVENTION

[0095] All experimental data revealed herein is provided by way of example only and is not to be understood as being confining the scope of the invention.

Discussion of Toxicity of Some Exemplary Compounds

[0096] Exemplary Growth Curves of Some Sample Microorganisms, Resp. Sample Cells Exemplarily Stained

[0097] The triazole amine derivatives 6-TramTO-1 (1), 6-TramTAS (2) and 6-TramTO-3 (3) do not interfere with the growth of Escherichia coli (E. coli) NEB5α (New England Biolabs), while the previously described dyes, without the triazole amine side chain (4-6) do. Even at 5 μM concentration no toxicity is observed regarding compounds 1-3, clearly proving that TramTOs and TramTAS (1-3) are non-toxic to microorganisms (cf. FIG. 2). FIG. 3 exemplarily shows the viability of HeLa cells upon staining with cyanine dyes. HeLa cells are eukaryotic cells, therefore these experiments show the widespread applicability of the invention.

[0098] Experimental Procedure for Measuring of Cell Viability

[0099] HeLa cells are grown in a humidified cell-culture incubator (New Brunswick Scientific, USA) at 37° C. under CO.sub.2 (g) (5%, v/v). Growth medium is DMEM nutrient medium supplemented with fetal bovine serum (FBS) (10%, v/v), penicillin (100 units/mL), and streptomycin (100 μg/mL). HeLa cells (2×10.sup.4) are seeded into black 96-well microtiter plates (Greiner Bio-One, Austria) in DMEM (200 μL, 2.5% FBS). For assessing the viability stocks of the compounds of interest in milliQ water are added at different concentrations and incubated for 24 h. Resazurin fluorescence-based cell viability assay is performed to evaluate cell viability.

[0100] Measurements of cell viability is exemplarily performed with compounds 18, 19 and 20: HeLa cells are treated with different concentrations (10 μM, 7.5 μM, 5 μM, 2.5 μM, 1 μM and 0.5 μM) of the dyes (18, 19, 20) in triplicated. Viability is then examined after 21 h incubation time by resazurin-based assay. The results of the tests are depicted in FIGS. 10A and 10B.

[0101] Experimental Procedure for Measuring Bacterial Growth Curves

[0102] E. coli NEB5α are cultured in lysogeny broth (LB) liquid medium and incubated at 37° C., 200 rpm. In clear 96-well microtiter plates (Nunc, USA) in a final volume of 250 μL 5 μL of cell suspension at exponential growth phase were added and stocks of the compounds of interest in milliQ water, DMSO or MeCN (final vehicle concentration 0.8%, v/v). Bacterial growth is monitored at OD.sub.600, utilizing a microplate reader (Tecan, Switzerland).

[0103] Fluorescence Titrations

[0104] The affinity of the TramTO derivatives towards dsDNA is usually higher than the affinity of the derivatives without the modified side chain according to this invention. Usually the TramTOs according to the invention were the stronger binders compared to the corresponding controls (4-6) with the similar fluorescence, however the green control CH.sub.3TO-1 (4) is a stronger binder then the 6-TramTAS, the 6-TramTO-1 is superior to 6-TramTO-3 (about ×2) and 6-TramTAS (about ×3). K.sub.D values are determined as is well known to any person being of ordinary skill in the art. Table 2 shows the results obtained.

TABLE-US-00002 TABLE 2 Exemplary results of fluorescence titrations (affinity towards dsDNA) RFU −/+ DNA Slits (nm) K.sub.D 6-TramTO-1 (1) 1.2/460 5/5  8.63 ± 4.34 μM CH.sub.3TO-1 (4) 0.2/125 10/10 (for 5/5: 20.56 ± 6.67 μM ~10 RFU) 6-TramTAS (2) 2.9/90  10/10 (for 5/5: 30.09 ± 4.61 μM or ~8 RFU) 28.07 ± 2.33 μM CH.sub.3TAS (5) 2.3/33  10/10 (for 5/5: Not calculable ~2 RFU) 6-TramTO-3 (3) 11.8/280  5/5 22.96 ± 1.81 μM/ 19.5 ± 2.6 μM (literature value) CH.sub.3TO-3 (6) 6.8/210 5/5 44.98 ± 1.05 μM

[0105] Flow Cytometry

[0106] The flow cytometric measurements are performed by use of the LSR Fortessa cell analyser (BD Biosciences, USA) or other commercially available apparatuses (e.g. FACSAria III (BD Biosciences, USA) and Guava easyCyte (Merck Millipore, USA)). Any person of ordinary skill in the art knows how to adjust the different apparatuses commercially available in order to perform comparable experiments exerting the experimental parameters described herein. A minimum of 10000 events per sample is analysed. The measurements are each performed in at least two independent experiments. Bacteria are labelled for 15 min with 5 μM of the corresponding dyes. All dyes showed >99.9% labelling. FIG. 4 shows graphs of the results, exemplarily obtained with E. Coli (NEB5α) and B. Subtilis (NCIB 3610).

[0107] For 6-TramTO-1 (1) one can see clear labelling when 5 μM of the dye are added for only 5 min before the measurements, dead cells (treated with 70% isopropanol for 1 h, then washed with saline 3×) being labelled about 10× stronger: MFI 135 vs 1337. FIGS. 5A-5C show additional data.

[0108] As can obviously be seen from the data shown in FIG. 6, co-staining with PI is not necessary upon usage of the inventive cyanine dyes in order to distinguish live from dead cells. The green (Blue C) channel gave the same results than the orange/PI channel (YellowGreen B) in comparison with commercially available Kit BacLight™ of ThermoFisher.

[0109] Dual and Triple Labelling

[0110] Experimental data shows that dual and even triple labelling is also possible with the TramTO dyes (examples presented in FIG. 7 using Bacillus subtilis and in FIG. 8 using Klebsiella pneumonia).

[0111] Exemplary Phagocytosis Studies with Different Bacterial Strains Exemplarily Labelled with 1 or 3

[0112] The applicability of the invention is proven by exemplary Phagocytosis studies as is described below (cf. FIG. 9A, FIG. 9B and FIG. 9C):

[0113] FACS data is recorded using a Guava easyCyte flow cytometer (Millipore). Bacteria are labelled for 15 min with 5 μM of the corresponding dye, washed 3 times with DPBS. After the last wash bacteria are brought to an OD.sub.600 nm of 2.0 and 2.3 μL in such a way that an equivalent of bacteria are added to each well to achieve a multiplicity of infection (MOI) of 5 (MOI=5+5 for experiments with two bacterial strains). Culture plates are then centrifuged for 10 min at 250 g and 37° C. to establish physical contact between macrophages and bacteria. Subsequently, culture plates are transferred back into a 37° C. incubator with CO.sub.2 atmosphere for 50 min. After the stimulation period the cells are washed once with DPBS prior to flow cytometry. Macrophages are detached carefully from the culture dishes by using a rubber scraper.

[0114] Nuclear Magnetic Resonance Spectroscopy (NMR)

[0115] All NMR spectra are automatically measured at 300 K either in a Bruker AV III HD 300 MHz at a frequency of 300 MHz (.sup.1H) or 75 MHz (.sup.13C) or in a Bruker AV III HD 500 MHz at a frequency of 500 MHz (.sup.1H) or 125 MHz (.sup.13C). All chemical shifts (δ) are relative to tetramethylsilane (TMS), i.e. δ(TMS)=0 ppm. As internal standards, deuterated chloroform (CDCl.sub.3), dimethyl sulfoxide (DMSO) with TMS are used. Solvent shifts (ppm): δ(CHCl.sub.3)=7.26 ppm (.sup.1H), δ(CHCl.sub.3)=77.16 ppm (.sup.13C), δ(DMSO)=2.50 ppm (.sup.1H), δ(DMSO)=39.52 ppm (.sup.13C). The assignment of each signal is based on two-dimensional nuclear magnetic resonance spectroscopy (2D NMR), i.e. heteronuclear single quantum coherence (HSQC), heteronuclear multiple bond correlation spectroscopy (HMBC) and correlation spectroscopy (COSY).

[0116] Mass Spectrometry

[0117] Electrospray ionization mass spectrometry (ESI-MS) is performed on a Finnigan LTQ-FT (Thermo Fischer Scientific). EI-MS is performed on an AccuTOF GCv (JEOL).

[0118] Analytic HPLC

[0119] Characterization is performed via analytical RP-HPLC-MS on an Agilent 1260 Infinity II HPLC-System (Agilent Technologies). Agilent eclipse XDB-C18 column (5 μm, 4.6×150 mm) using isocratic regime during the first 5 min, followed by a linear gradient over 30 min of solvent B as stated below with a flow rate of 0.2 mL/min at 55° C. The detection was carried out by monitoring the absorbance at 220 nm. The eluents are ultrapure H.sub.2O (solvent A) with addition of 0.05% TFA and MeCN (solvent B) with addition of 0.03% TFA. [0120] Gradient 1: 5% to 75% of solvent B [0121] Gradient 2: 5% to 95% of solvent B

Synthetic Procedures

[0122] Scheme 5 (FIG. 10) shows the general retrosynthetic pathway of the production of the cyanine dyes according to the invention.

Synthesis of 6-TramTO-1

[0123] This synthesis is based on the scaffold of thiazole orange (TO), known within the state of the art, but proceeding beyond the state of the art (cf. FIG. 11, scheme 8).

[0124] (Z)-1-ethyl-4-((3-(6-iodohexyl)benzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium (6-lodoTO-1, 21): Benzothiazolium (30, 521 mg, 1.07 mmol) and quinolinium (24, 342 mg, 1.07 mmol) are suspended in EtOH (HPLC-grade, 16.0 mL) and DIPEA (410 μL, 2.35 mmol) added. The solution is stirred in the dark at r.t. for 1 h, the solvent removed and the crude purified by flash column chromatography (allox, MeOH in CH.sub.2Cl.sub.2: 0%.fwdarw.0.2%.fwdarw.0.5%.fwdarw.0.7%) to yield the desired product (21, 359 mg, 0.56 mmol, 52%) as red solid. R.sub.f=0.45 (CH.sub.2Cl.sub.2/MeOH 10:1). .sup.1H-NMR (500 MHz, DMSO-d.sub.6, δ): 8.75 (d, 1H, J=8.7 Hz, H-16), 8.67 (d, 1H, J=7.3 Hz, H-13), 8.16 (d, 1H, J=8.6 Hz, H-19), 8.05 (d, 1H, J=7.2 Hz, H-4), 8.03-7.99 (m, 1H, H-17), 7.81-7.76 (m, 2H, H-1, H-18), 7.63-7.59 (m, 1H, H-2), 7.44-7.39 (m, 2H, H-3, H-12), 6.93 (s, 1H, H-11), 4.70-4.60 (m, 4H, H-14, H-5), 3.24 (t, 2H, J=6.9 Hz, H-10), 1.83-1.76 (m, 2H, H-9), 1.76-1.70 (m, 2H, H-6), 1.50-1.45 (m, 5H, H-8, H-15), 1.45-1.39 (m, 2H, H-7). .sup.13C-NMR (125 MHz, DMSO-d.sub.6, δ): 159.4 (C.sub.q), 148.7 (C.sub.q), 144.0 (CH), 140.0 (C.sub.q), 136.9 (C.sub.q), 133.3 (CH), 128.3 (CH), 127.0 (CH), 125.7 (CH), 124.6 (CH), 124.3 (C.sub.q), 123.9 (C.sub.q), 123.0 (CH), 118.1 (CH), 113.0 (CH), 108.3 (CH), 87.7 (CH), 49.5 (CH.sub.2), 45.6 (CH.sub.2), 32.7 (CH.sub.2), 29.6 (CH.sub.2), 26.7 (CH.sub.2), 24.9 (CH.sub.2), 14.8 (CH.sub.3), 8.8 (CH.sub.2). HRMS-ESI.sup.+ (m/z): [M].sup.+ calcd for C.sub.25H.sub.28IN.sub.2S.sup.+, 515.1012; found, 515.1023.

[0125] (Z)-1-ethyl-4-((3-(6-iodohexyl)benzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium (6-AzTO-1, 12): Iodo derivative 6-IodoTO-1 (s, 13.1 mg, 23.8 μmop and NaN.sub.3 (2.00 mg, 30.9 μmop are dissolved in DMF (500 μL) and stirred for 25 h at 50° C. in the dark. The solvent is removed and the crude purified by flash column chromatography (allox basic, MeOH in CH.sub.2Cl.sub.2: 0%.fwdarw.2.0%) to yield the desired product (12, 10.8 mg, 19.9 μmol, 84%) as red solid. R.sub.f=0.43 (CH.sub.2Cl.sub.2/MeOH 10:1). .sup.1H-NMR (500 MHz, DMSO-d.sub.6, δ): 8.76 (d, 1H, .sup.3J=8.4 Hz, H-9), 8.67 (d, 1H, .sup.3J=7.2 Hz, H-10), 8.17 (d, 1H, .sup.3J=8.6 Hz, H-6), 8.06 (dd, 1H, .sup.3J=7.9 Hz, .sup.4J=1.0 Hz, H-4), 8.03-7.99 (m, 1H, H-8), 7.81-7.76 (m, 2H, H-1, H-7), 7.62 (td, 1H, .sup.3J=7.8 Hz, .sup.4J=1.1 Hz, H-2), 7.45-7.40 (m, 2H, H-3, H-11), 6.94 (s, 1H, H-5), 4.69-4.62 (m, 4H, H-12, H-14), 3.32-3.27 (m, 2H, H-19), 1.84-1.77 (m, 2H, H-15), 1.54-1.44 (m, 9H, H-13, H-16, H-17, H-18) ppm. .sup.13C-NMR (75 MHz, DMSO-d.sub.6, 5): 159.4 (C.sub.q), 148.7 (C.sub.q), 144.1 (C-10), 140.0 (C.sub.q), 136.9 (C.sub.q), 133.4 (C-8), 128.3 (C-2), 126.9 (C-7), 125.8 (C-9), 124.6 (C-3), 124.3 (Cq), 123.9 (Cq), 123.0 (C-4), 118.1 (C-6), 113.1 (C-1), 108.3 (C-11), 87.7 (C-5), 50.5 (C-19), 49.5 (C-12), 45.6 (C-14), 28.1 (C-18), 26.8 (C-15), 25.9 (C-17), 25.5 (C-16), 14.8 (C-13) ppm. HRMS-ESI.sup.+ (m/z): [M].sup.+ calcd for C.sub.25H.sub.28N.sub.5S.sup.+, 430.2060; found, 430.2063.

[0126] (Z)-4-((3-(6-(4-(ammoniomethyl)-1H-1,2,3-triazol-1-yl)hexyl)benzo[d]thiazol-2(3H)-ylidene)methyl)-1-ethylquinolin-1-ium (6-TramTO-1, 1): Under inert atmosphere, azido derivative 6-AzTO-1 (12, 60.0 mg, 108 μmop is dissolved in ethanol (1.13 mL) and propargylamine (14.2 μL, 222 μmop, copper powder (8.12 mg, 128 μmop and copper sulfate (100 mM, 53.8 μL, 5.38 μmop are added. The resulting mixture is stirred for 20 h, filtered and the solvent removed. The product is purified by preparative HPLC (Gilson 5-75%) to yield the desired product (1, 38.0 mg, 53.3 μmol, 50%) as red solid. R.sub.t=20.2 min. .sup.1H-NMR (500 MHz, DMSO-d.sub.6, δ): 8.75 (d, 1H, J=8.8 Hz, H-16), 8.67 (d, 1H, J=7.2 Hz, H-13), 8.29 (br s, 3H, NH.sub.3.sup.+), 8.17 (d, 1H, J=8.8 Hz, H-19), 8.10 (s, 1H, H-20), 8.05 (dd, 1H, J=7.9 Hz, J=0.9 Hz, H-4), 8.03-7.99 (m, 1H, H-17), 7.80-7.75 (m, 2H, H-1, H-18), 7.64-7.59 (m, 1H, H-2), 7.45-7.41 (m, 2H, H-3, H-12), 6.94 (s, 1H, H-11), 4.66 (q, 2H, J=7.2 Hz, H-14), 4.63 (t, 2H, J=7.4 Hz, H-5), 4.37 (t, 2H, J=7.0 Hz, H-10), 4.12-4.06 (m, 2H, H-21), 1.83-1.75 (m, 4H, H-6, H-9), 1.54-1.46 (m, 5H, H-8, H-15), 1.36-1.28 (m, 2H, H-7). .sup.13C-NMR (125 MHz, DMSO-d.sub.6, δ): 159.4 (C.sub.q), 148.7 (C.sub.q), 144.0 (CH), 140.0 (C.sub.q), 136.9 (C.sub.q), 133.3 (CH), 128.2 (CH), 126.9 (CH), 125.7 (CH), 124.5 (CH), 124.3 (C.sub.q), 124.1 (2C, CH, C.sub.q), 123.9 (C.sub.q), 123.0 (CH), 118.0 (CH), 113.0 (CH), 108.3 (CH), 87.6 (CH), 49.5 (CH.sub.2), 49.3 (CH.sub.2), 45.6 (CH.sub.2), 33.9 (CH.sub.2), 29.6 (CH.sub.2), 26.8 (CH.sub.2), 25.6 (CH.sub.2), 25.4 (CH.sub.2), 14.7 (CH.sub.3). HRMS-ESI.sup.+ (m/z): [M].sup.+ calcd for C.sub.28H.sub.33N.sub.6S.sup.+, 485.2482; found, 485.2487.

Synthesis of 6-xxxTO-1 and of 6-xxxTO-3

[0127] This synthesis is based on the nucleophilic substitution of the iodo substituent of 6-IodoTO-1 and 6-IodoTO-3 (cf. FIG. 15, scheme 9).

[0128] (Z)-1-ethyl-4-((3-(6-(piperazin-1-ium-1-yl)hexyl)benzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium (6-PipTO-1, 14): Iodo derivative 6-IodoTO-1 (21, 100 mg, 156 μmop and Na.sub.2CO.sub.3 (24.5 mg, 231 μmop are suspended in MeCN (320 μL) and piperazine (48.9 μL, 624 μmop added. The reaction mixture is stirred for 4 h at r.t., water added and the product extracted with CH.sub.2Cl.sub.2 and dried over MgSO.sub.4. The product is purified by preparative HPLC (Gilson 5-75%) to yield the desired product (14, 38.3 mg, 54.7 μmol, 35%) as red solid. R.sub.t=18.0 min. .sup.1H-NMR (500 MHz, DMSO-d.sub.6, δ): 9.32 (br s, 2H, NH.sub.2.sup.+), 8.76 (d, 1H, J=8.0 Hz, H-16), 8.67 (d, 1H, J=7.3 Hz, H-13), 8.17 (d, 1H, J=8.6 Hz, H-19), 8.06 (dd, 1H, J=7.8 Hz, J=0.6 Hz, H-4), 8.03-7.99 (m, 1H, H-17), 7.80-7.75 (m, 2H, H-1, H-18), 7.64-7.59 (m, 1H, H-2), 7.46-7.41 (m, 2H, H-3, H-12), 6.95 (s, 1H, H-11), 4.69-4.62 (m, 4H, H-14, H-5), 3.49-3.09 (m, 8H, H-20, H-20′, H-21, H-21′), 3.05-2.95 (m, 2H, H-10), 1.86-1.77 (m, 2H, H-9), 1.63-1.55 (m, 2H, H-6), 1.52-1.45 (m, 5H, H-8, H-15), 1.40-1.33 (m, 2H, H-7). .sup.13C-NMR (125 MHz, DMSO-d.sub.6, δ): 159.4 (C.sub.q), 148.8 (C.sub.q), 144.0 (CH), 140.0 (C.sub.q), 136.9 (C.sub.q), 133.3 (CH), 128.2 (CH), 126.9 (CH), 125.7 (CH), 124.5 (CH), 124.3 (C.sub.q), 123.9 (C.sub.q), 123.0 (CH), 118.0 (CH), 113.0 (CH), 108.3 (CH), 87.6 (CH), 55.8 (CH.sub.2), 49.5 (CH.sub.2), 48.1 (CH.sub.2), 45.6 (CH.sub.2), 40.5 (CH.sub.2), 26.8 (CH.sub.2), 23.3 (CH.sub.2), 25.8 (CH.sub.2), 25.6 (CH.sub.2), 14.7 (CH.sub.3). HRMS-ESI.sup.+ (m/z): [M].sup.+ calcd for C.sub.29H.sub.37N.sub.4S.sup.+, 473.2733; found, 473.2731.

[0129] 1-ethyl-4-((1E,3Z)-3-(3-(6-(piperazin-1-yl)hexyl)benzo[d]thiazol-2(3H)-ylidene)prop-1-en yl)quinolin-1-ium (6-PipTO-3, 10): The iodo derivate 23 (100 mg, 150 μmol, 1.00 eq) and Na.sub.2CO.sub.3 (24.5 mg, 231 μmol, 1.54 eq) are suspended in acetonitrile (320 μL) and piperazine (53.8 mg, 624 mmol, 4.16 eq) is added. The reaction mixture is stirred for 6 h at room temperature. Distilled water (1.0 mL) is added, and the product is extracted with CH.sub.2Cl.sub.2 and dried over MgSO.sub.4. The solvent is evaporated under reduced pressure, and the crude is purified using a Büchi pure system with MeCN in H.sub.2O (0%.fwdarw.45%) as a solvent to yield the desired product 12 (40.3 mg, 64.3 μmol, 43%) as a blue solid. .sup.1H-NMR (500 MHz, DMSO-d.sub.6, δ): 8.51-8.44 (m, 2H, H-11, H-12), 8.16 (t, 1H, .sup.3J=12.7 Hz, H-6), 8.12-8.08 (m, 1H, H-8), 7.97 (td, 1H, .sup.3J=7.9 Hz, .sup.4J=1.3 Hz, H-10), 7.91-7.87 (m, 2H, H-4, H-13), 7.74-7.69 (m, 1H, H-9), 7.63-7.58 (m, 1H, H-1), td (7.49, 1H, .sup.3J=7.8 Hz, .sup.4J=1.1 Hz, H-3), 7.34-7.27 (m, 1H, H-2), 7.19-7.13 (m, 1H, H-7), 6.53 (d, 1H, .sup.3J=12.2 Hz, H-5), 4.61 (q, 2H, .sup.3J=7.2 Hz, H-14), 4.26-4.21 (m, 2H, H-16), 3.14-2.99 (m, 3H, H-21), 1.78-1.70 (m, 2H, H-20), 1.70-1.57 (m, 2H, H-17), 1.49-1.42 (m, 4H, H-15, H-19), 1.41-1.35 (m, 2H, H-18) ppm. .sup.13C-NMR (75 MHz, DMSO-d.sub.6, 5): 160.8 (C.sub.q), 158.4 (C.sub.q), 158.1 (C.sub.q), 150.5 (C.sub.q), 141.5 (C.sub.q), 137.8 (C.sub.q), 144.0 (C-8), 142.3 (C-12), 133.5 (C-10), 127.7 (C-3), 126.7 (C-9), 125.3 (C-11), 124.3 (C-2), 122.6 (C-13), 118.1 (C-6), 112.5 (C-1), 110.1 (C-4), 109.6 (C-7), 98.5 (C-5), 55.7 (C-22, C-26), 49.4 (C-14), 47.9 (C-23, C-25), 45.5 (C-16), 26.9 (C-19), 25.8 (C-18), 25.7 (C-17), 14.8 (0-15) ppm. HRMS-ESI.sup.+ (m/z): [M+2H].sup.+ calcd. for C.sub.31H.sub.39N.sub.4S.sup.+499.2890; found 501.2684.

[0130] (Z)-4-((3-(6-(1H-1,2,3-triazol-1-yl)hexyl)benzo[d]thiazol-2(3H)-ylidene)methyl)-1-ethylquinolin-1-ium (6-Tri.sub.(asym.)TO-1, 13a) and (Z)-4-((3-(6-(2H-1,2,3-triazol-2-yl)hexyl)benzo[d]thiazol-2(3H)-ylidene)methyl)-1-ethylquinolin-1-ium (6-Tri.sub.(sym.)TO-1, 13b): Iodo derivative 6-IodoTO-1 (21, 150.0 mg, 0.23 mmol) and sodium carbonate (99.0 mg, 0.93 mmol) are dissolved in THF (467 μL) and 1-H-1,2,3-triazole (16, 83.1 μL, 0.47 mmol) is added. The reaction mixture is stirred in the dark for 3 h at 55° C. 1-H-1,2,3-triazole (16, 83.1 μL, 0.47 mmol) is added and the mixture is stirred overnight under the same conditions. The solution is washed with distilled water. The aqueous layer is extracted with CH.sub.2Cl.sub.2 and the organic phase is dried over magnesium sulfate. The crude products are purified by preparative HPLC (Gilson 5-75%) to yield 6-Tri.sub.(asym.)TO-1 (13a, 69.6 mg, 0.12 mmol, 51%) and 6-Tri.sub.(sym.)TO-1 (13b, 31.7 mg, 0.05 mmol, 23%) as pink solids.

[0131] 6-Tri.sub.(asym)TO-1 (13a):

[0132] R.sub.t=21.6 min (gradient 2). .sup.1H-NMR (300 MHz, DMSO-d.sub.6, δ): 8.73 (d, 1H, J=8.5 Hz, H-16), 8.66 (d, 1H, J=7.2 Hz, H-13), 8.17 (d, 1H, J=8.6 Hz, H-19), 8.10-7.97 (m, 3H, H-4, H-17, H-20), 7.82-7.74 (m, 2H, H-1, H-18), 7.67 (s, 1H, H-21), 7.65-7.58 (m, 1H, H-2), 7.47-7.39 (m, 2H, H-3, H-12), 6.93 (s, 1H, H-11), 4.70-4.59 (m, 4H, H-14, H-5), 4.35 (m, 2H, J=7.0 Hz, H-10), 1.87-1.74 (m, 4H, H-9, H-6), 1.54-1.43 (m, 5H, H-8, H-15), 1.36-1.26 (m, 2H, H-7). .sup.13C-NMR (75 MHz, DMSO-d.sub.6, δ): 159.4 (C.sub.q), 148.7 (C.sub.q), 144.0 (CH), 140.0 (C.sub.q), 136.8 (C.sub.q), 133.3 (CH), 133.1 (CH), 128.2 (CH), 126.9 (CH), 125.7 (CH), 124.5 (2C, CH), 124.3 (C.sub.q), 123.9 (C.sub.q), 122.9 (CH), 118.0 (CH), 112.9 (CH), 108.3 (CH), 87.6 (CH), 49.5 (CH.sub.2), 49.0 (CH.sub.2), 45.6 (CH.sub.2), 29.5 (CH.sub.2), 26.7 (CH.sub.2), 25.6 (CH.sub.2), 25.4 (CH.sub.2), 14.6 (CH.sub.3). HRMS-ESI.sup.+ (m/z): [M].sup.+ calcd for C.sub.27H.sub.30N.sub.5S.sup.+, 456.2216; found, 456.2212.

[0133] 6-Tri.sub.(sym)TO-1 (13b):

[0134] R.sub.t=23.6 min (gradient 2). .sup.1H-NMR (300 MHz, DMSO-d.sub.6, δ): 8.72 (d, 1H, J=8.2 Hz, H-16), 8.66 (d, 1H, J=7.2 Hz, H-13), 8.16 (d, 1H, J=8.6 Hz, H-19), 8.07-7.97 (m, 2H, H-4, H-17), 7.80-7.73 (m, 2H, H-1, H-18), 7.70 (s, 2H, H-20, H-20′), 7.64-7.57 (m, 1H, H-2), 7.45-7.38 (m, 2H, H-3, H-12), 6.91 (s, 1H, H-11), 4.70-4.56 (m, 4H, H-14, H-5), 4.38 (m, 2H, J=6.9 Hz, H-10), 1.88-1.73 (m, 4H, H-9, H-6), 1.52-1.42 (m, 5H, H-8, H-15), 1.34-1.26 (m, 2H, H-7). .sup.13C-NMR (75 MHz, DMSO-d.sub.6, δ): 159.4 (C.sub.q), 148.7 (C.sub.q), 144.0 (CH), 140.0 (C.sub.q), 136.9 (C.sub.q), 134.0 (2C, CH), 133.3 (CH), 128.2 (CH), 126.9 (CH), 125.7 (CH), 124.5 (CH), 124.3 (C.sub.q), 123.9 (C.sub.q), 122.9 (CH), 118.0 (CH), 112.9 (CH), 108.3 (CH), 87.6 (CH), 53.8 (CH.sub.2), 49.4 (CH.sub.2), 45.6 (CH.sub.2), 28.9 (CH.sub.2), 26.7 (CH.sub.2), 25.6 (CH.sub.2), 25.3 (CH.sub.2), 14.6 (CH.sub.3). HRMS-ESI.sup.+ (m/z): [M].sup.+ calcd for C.sub.27H.sub.30N.sub.5S.sup.+, 456.2216; found, 456.2213.

[0135] (Z)-1-ethyl-4-((3-(6-(triphenylphosphonio)hexyl)benzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium (6-PPh.sub.3TO-1, 15): Iodo derivative 6-IodoTO-1 (21, 118 mg, 184 μmop and PPh.sub.3 (145 mg, 551 μmop are suspended in MeCN (3.00 mL) and stirred fort h at r.t. and 13.5 h at 85° C. The solvent is removed and the crude filtered over whool. The product is purified by preparative HPLC (Gilson 5-75%) to yield the desired product (15, 3.50 mg, 3.88 μmol, 2%) as red solid. R.sub.t=25.1 min (gradient 1). HRMS-ESI.sup.+ (m/z): [M].sup.2+ calcd for C.sub.43H.sub.43N.sub.2PS.sup.2+, 325.1437; found, 325.1437.

[0136] 1-ethyl-4-((1 E,3Z)-3-(3-(6-(tri phenyl phosphonio)hexyl)benzo[d]thiazol-2(3H)-yl idene)prop-1-en-1-yl)quinolin-1-ium (6-PPh.sub.3TO-3, 17): derivative 6-IodoTO-3 (23, 49.9 mg, 74.7 μmop and PPh.sub.3 (58.9 mg, 224 μmop are suspended in MeCN (3.00 mL) and stirred 16 h at 82° C. The solvent is removed and the crude filtered over whool. The product is purified by preparative HPLC (Gilson 5-75%) to yield the desired product (17, 1.90 mg, 2.10 μmol, 44%) as blue solid. R.sub.r=26.9 min (gradient 1); .sup.1H-NMR (500 MHz, DMSO-d.sub.6) 5=8.45 (d, .sup.3J.sub.H—H=7.1 Hz, 1H, H23), 8.42 (d, .sup.3J.sub.H—H=8.2 Hz, 1H, H26), 8.14 (t, .sup.3J.sub.H—H=12.8 Hz, 1H, H18), 8.09 (d, .sup.3J.sub.H—H=8.8 Hz, 1H, H29), 7.95 (t, .sup.3J.sub.H—H=7.3 Hz, 1H, H31), 7.87 (m, 5H, H3, H24, H37, H42, H47), 7.76 (m, 12H, H35, H36, H38-41, H43-46, H48, H49), 7.67 (t, .sup.3J.sub.H—H=7.7 Hz, 1H, H30), 7.56 (d, .sup.3J.sub.H—H=8.3 Hz, 1H, H6), 7.46 (t, .sup.3J.sub.H—H=7.3 Hz, 1H, H2), 7.30 (t, .sup.3J.sub.H—H=7.8 Hz, 1H, H1), 7.11 (d, .sup.3J.sub.H—H=13.3 Hz, 1H, H19), 6.50 (d, .sup.3J.sub.H—H=12.3 Hz, 1H, H10), 4.61 (q, .sup.3J.sub.H—H=7.1 Hz, 2H, H21), 4.19 (t, .sup.3J.sub.H—H=7.4 Hz, 2H, H11), 3.59 (m, 2H, H16), 1.68 (m, 2H, H12), 1.59 (s, 4H, H13, H14), 1.34 (t, .sup.3J.sub.H—H=7.1 Hz, 5H, H15, H20) ppm; .sup.13C-NMR (126 MHz, DMSO-d.sub.6) 5=160.9 (s, 1C, C.sub.25), 150.4 (s, 1C, C.sub.8), 143.9 (s, 1C, C18), 142.3 (s, 1C, C23), 141.5 (s, 1C, C27), 137.7 (s, 1C, C4), 134.9 (s, 3C, C.sub.37, C.sub.42, C.sub.47), 133.6 (d, 6C, C.sub.35, C.sub.39, C.sub.40, C.sub.44, C.sub.45, C.sub.49), 133.5 (s, 1C, C.sub.31), 130.3 (d, 3C, C.sub.36, C.sub.38, C.sub.41, C.sub.43, C.sub.46, C.sub.48), 127.7 (s, 1C, C.sub.2), 126.7 (s, 1C, C.sub.30), 125.2 (s, 1C, C.sub.26), 124.8 (s, 1C, C1), 124.3 (s, 1C, C.sub.5), 122.7 (s, 1C, C.sub.24), 118.9 (s, 3C, C.sub.32-34), 118.2 (s, 1C, C.sub.29), 112.4 (s, 1C, C.sub.6), 110.0 (s, 1C, C.sub.3), 109.5 (s, 1C, C.sub.19), 98.5 (s, 1C, C10), 49.4 (s, 1C, C.sub.21), 45.4 (s, 1C, C.sub.11), 29.4 (s, 1C, C.sub.13), 26.6 (s, 1C, C.sub.12), 25.0 (s, 1C, C.sub.15), 21.6 (s, 1C, C.sub.14), 20.1 (s, 1C, C.sub.16), 14.7 (s, 1C, C.sub.20) ppm. HRMS-ESI.sup.+ (m/z): [M].sup.2+ calcd for C.sub.45H.sub.45N.sub.2PS.sup.2+, 338.1515; found, 338.1516.

Synthesis of CH.SUB.3.TAS

[0137] The synthesis of the CH.sub.3TAS (5) is known to the person skilled in the art.

Synthesis of CH.SUB.3.TO dyes 4 and 6

[0138] The Synthesis route of dyes CH.sub.3TO-1 (4) and CH.sub.3TO-3 (6), starting from benzothiazole 37, is depicted in FIG. 21 (scheme 10).

(Z)-1-ethyl-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium (4)

[0139] Benzothiazolium 37 (117 mg, 400 μmol, 1.00 eq) and quinolinium 24 (128 mg, 400 μmol, 1.00 eq) are suspended in ethanol (6.0 mL), diisopropylethylamine (0.15 mL, 884 μmol, 2.21 eq) is added and the solution stirred at room temperature in the dark for 2 h. The solvent is evaporated, and the crude purified by flash column chromatography (basic allox, MeOH in CH.sub.2Cl.sub.2: 0%.fwdarw.0.2%.fwdarw.0.5%.fwdarw.0.7%) to yield the desired product 4 (156 mg, 350 μmol, 87%) as red solid. R.sub.f=0.39 (CH.sub.2Cl.sub.2/MeOH 10:1+1 drop formic acid). .sup.1H-NMR (500 MHz, DMSO-d.sub.6, δ): 8.76 (d, 1H, .sup.3J=8.3 Hz, H-16), 8.64 (d, 1H, .sup.3J=7.1 Hz, H-21), 8.10 (d, 1H, .sup.3J=8.6 Hz, H-18), 8.01 (d, 1H, .sup.3J=7.8 Hz, H-3), 7.96 (t, 1H, .sup.3J=7.9 Hz, H-20), 7.75-7.70 (m, 2H, H-19, H-6), 7.56 (t, 1H, .sup.3J=7.5 Hz, H-1), 7.37 (t, 1H, .sup.3J=7.5 Hz, H-2), 7.29 (d, 1H, .sup.3J=7.1 Hz, H-17), 6.85 (s, 1H, H-10), 4.61 (q, 2H, .sup.3J=7.2 Hz, H-22), 3.97 (s, 3H, H-11), 1.46 (t, 3H, .sup.3J=7.2 Hz, H-23) ppm. .sup.13C-NMR (75 MHz, DMSO-d.sub.6, δ): 159.8 (C-12), 148.4 (C-8), 143.93 (C-21), 140.4 (C-14), 136.8 (C-4), 133.3 (C-20), 128.1 (C-1), 126.8 (C-19), 125.9 (C-16), 124.4 (C-2), 124.2 (C-13), 123.8 (C-5), 122.9 (C-3), 118.0 (C-18), 112.9 (C-6), 108.0 (C-17), 88.0 (C-10), 49.4 (C-22), 33.9 (C-11), 14.7 (C-23) ppm. HRMS-ESI.sup.+ (m/z): [M].sup.+ calcd. for C.sub.20H.sub.19N.sub.2S.sup.+: 319.1263, found: 319.1259.

(Z)-1-ethyl-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium (6)

[0140] Benzothiazolium 37 (147 mg, 505 μmol, 1.26 eq) and quinilinium 38 (161 mg, 400 μmol, 1.00 eq) are dissolved in CH.sub.2Cl.sub.2/Methanol (1:1, 4 mL), Acetic anhydride (0.4 mL) and triethylamine (0.4 mL) are added and the reaction is stirred for 3.5 h at room temperature. The crude dye is precipitated by pouring the reaction mixture in diethylether. The precipitate is washed with diethylether and purified by flash column chromatography (basic allox, MeOH in CH.sub.2Cl.sub.2: 0%.fwdarw.0.2%.fwdarw.0.5%.fwdarw.0.7%) to yield the desired product 6 (178 mg, 377 μmol, 75%) as dark blue solid. R.sub.f=0.31 (CH.sub.2Cl.sub.2/MeOH 10:1+1 drop formic acid). .sup.1H-NMR (500 MHz, DMSO-d.sub.6, δ): 8.44 (d, 1H, .sup.3J=8.6 Hz, H-23), 8.42 (d, 1H, .sup.3J=7.2 Hz, H-17), 8.11 (t, 1H, .sup.3J=12.8 Hz, H-11), 8.06 (d, 1H, .sup.3J=8.8 Hz, H-20), 7.94 (t, 1H, .sup.3J=7.5 Hz, H-22), 7.84 (t, 2H, .sup.3J=7.0 Hz, H-18, H-3), 7.69 (t, 1H, .sup.3J=7.7 Hz, H-21), 7.54 (d, 1H, .sup.3J=8.2 Hz, H-6), 7.44 (t, 1H, .sup.3J=7.3 Hz, H-2), 7.27 (t, 1H, .sup.3J=7.5 Hz, H-1), 7.08 (d, 1H, .sup.3J=13.3 Hz, H-12), 6.47 (d, 1H.sup.3J=12.3 Hz, H-10), 4.58 (q, 2H, .sup.3J=7.0 Hz, H-24), 3.71 (s, 3H, H-19), 1.44 (t, 3H, .sup.3J=7.0 Hz, H-25) ppm. .sup.13C-NMR (75 MHz, DMSO-d.sub.6, 5): 161.5 (C-13), 150.3 (C-8), 143.7 (C-11), 142.2 (C-17), 141.9 (C-15), 137.7 (C-4), 133.4 (C-22), 127.6 (C-2), 126.7 (C-21), 125.2 (C-23), 124.2 (C-14), 124.1 (C-1), 122.5 (C-18), 117.8 (C-20), 112.4 (C-6), 109.3 (C-12), 98.8 (C-10), 49.3 (C-24), 32.9 (C-19), 14.7 (C-25) ppm. HRMS-ESI.sup.+ (m/z): [M].sup.+ calcd. for C.sub.22H.sub.21N.sub.2S.sup.+: 345.1420, found: 345.1417.

Synthesis of 6-Tram TAS

[0141] This synthesis is based on the scaffold of cyanine dyes described within the state of the art (cf. FIG. 24, scheme 11).

(E)-2-(4-(dimethylamino)styryl)-3-(6-iodohexyl)benzo[d]thiazol-3-ium (6-IodoTAS, 22)

[0142] Benzothiazolium (30, 1.00 g, 2.05 mmol) and dimethyl aminobenzaldehyd (306 mg, 2.05 mmol) are dissolved in Ac.sub.2O (26.8 mL) and the mixture stirred at 120° C. for 1 h. Water (26.8 mL) is added and the mixture stirred at 100° C. for 30 min. The reaction mixture was cooled to 25° C., the solvent removed, the product is filtered off and washed with acetone to yield the desired product (22, 1.03 mg, 1.67 mmol, 81%) as purple solid. R.sub.f=0.42 (CH.sub.2Cl.sub.2/MeOH 10:1). .sup.1H NMR (300 MHz, DMSO-d.sub.6, δ): 8.30 (d, 1H, J=7.9 Hz, CH-4), 8.16-8.05 (m, 2H, CH-1, CH-11), 7.92 (d, 2H, J=8.9 Hz, CH-14, CH-14′), 7.82-7.74 (m, 1H, CH-2), 7.72-7.64 (m, 1H, CH-3), 7.60 (d, 1H, J=15.3 Hz, CH-12), 6.86 (d, 2H, J=8.9 Hz, CH-13, CH-13′), 4.79 (t, 2H, J=7.3 Hz, CH.sub.2-5), 3.25 (t, 2H, J=6.8 Hz, CH.sub.2-10), 3.12 (s, 6H, CH.sub.3-15, CH.sub.3-15′), 1.88-1.78 (m, 2H, CH.sub.2-6), 1.78-1.67 (m, 2H, CH.sub.2-9), 1.53-1.35 (m, 4H, CH.sub.2-7, CH.sub.2-8). .sup.13C NMR (75 MHz, DMSO-d.sub.6, δ): 171.0 (C.sub.q), 153.5 (C.sub.q), 150.5 (CH), 141.0 (C.sub.q), 132.9 (2C, CH), 128.9 (CH), 127.3 (CH), 127.0 (C.sub.q), 123.9 (CH), 121.4 (C.sub.q), 115.8 (CH), 111.9 (2C, CH), 105.6 (CH), 47.8 (CH.sub.2), 39.7 (2C, CH.sub.3), 32.6 (CH.sub.2), 29.4 (CH.sub.2), 28.1 (CH.sub.2), 24.6 (CH.sub.2), 8.6 (CH.sub.2). HRMS-ESI.sup.+ (m/z): [M].sup.+ calcd for C.sub.23H.sub.28IN.sub.2S.sup.+, 491.1012; found, 491.1008.

(E)-3-(6-azidohexyl)-2-(4-(dimethylamino)styryl)benzo[d]thiazol-3-ium (6-azindoTAS, 34)

[0143] Iodo-derivative (22, 500 mg, 0.81 mmol) and sodium azide (210 mg, 3.24 mmol) are dissolved in acetone (3.3 mL) and water (3.3 mL) and the mixture is stirred at r.t. for 17 h. Sodium azide (105 mg, 1.62 mmol) is added and the mixture is stirred at r.t. 25 h. Sodium azide (210 mg, 3.24 mmol) is added and the mixture is stirred at 50° C. for 3 h. The solvent is evaporated and the crude extracted in CH.sub.2Cl.sub.2 and water. The organic layers are dried over MgSO.sub.4 and purified by flash column chromatography (allox, MeOH in CH.sub.2Cl.sub.2: 0.2%.fwdarw.0.5%.fwdarw.1.0%.fwdarw.5.0%.fwdarw.10%) to yield the desired product (34, 350 mg, 0.65 mmol, 80%) as purple solid. R.sub.f=0.38 (CH.sub.2Cl.sub.2/MeOH 10:1). .sup.1H NMR (300 MHz, DMSO-d.sub.6, δ): 8.31 (d, 1H, J=7.9 Hz, CH-4), 8.17-8.05 (m, 2H, CH-1, CH-11), 7.93 (d, 2H, J=8.8 Hz, CH-14, CH-14′), 7.82-7.74 (m, 1H, CH-2), 7.72-7.57 (m, 2H, CH-3, CH-12), 6.85 (d, 2H, J=8.8 Hz, CH-13, CH-13′), 4.80 (t, 2H, J=7.1 Hz, CH.sub.2-5), 3.31-3.24 (m, 2H, CH.sub.2-10), 3.12 (s, 6H, CH.sub.3-15, CH.sub.3-15′), 1.89-1.74 (m, 2H, CH.sub.2-6), 1.53-1.36 (m, 6H, CH.sub.2-7, CH.sub.2-8, CH.sub.2-9). .sup.13C NMR (75 MHz, DMSO-d.sub.6, δ): 171.1 (C.sub.q), 153.5 (C.sub.q), 150.5 (CH), 141.1 (C.sub.q), 132.9 (2C, CH), 128.9 (CH), 127.4 (CH), 127.0 (C.sub.q), 123.9 (CH), 121.4 (C.sub.q), 115.9 (CH), 111.9 (2C, CH), 105.7 (CH), 50.5 (CH.sub.2), 47.8 (CH.sub.2), 39.7 (2C, CH.sub.3), 28.2 (CH.sub.2), 28.0 (CH.sub.2), 25.7 (CH.sub.2), 25.2 (CH.sub.2). HRMS-ESI.sup.+ (m/z): [M].sup.+ calcd for C.sub.23H.sub.28N.sub.5S.sup.+, 406.2060; found, 406.2059.

[0144] (E)-3-(6-(4-(aminomethyl)-1H-1,2,3-triazol-1-yl)hexyl)-2-(4-(dimethylamino)styryl)benzo[d]thiazol-3-ium (6-TramTAS, 2): 6-azindoTAS (34, 41.9 mg, 78.5 μmol, 1.00 equiv.), copper sulfate pentahydrate (13.4 mg, 53.7 μmol, 0.68 equiv.), sodium ascorbate (10.6 mg, 53.5 μmol, 0.68 equiv.) and TBTA (35, 7.6 mg, 14.3 μmol, 0.18 equiv.) are dissolved in water (0.8 mL) and DMF (2.4 mL). Propargylamine (48 μL, 749 μmol, 9.54 equiv.) is added and the reaction mixture is stirred at room temperature in the dark for 2 h. Afterward, the reaction mixture is treated with aqueous sodium sulfide solution and filtered over Celite. After removal of the solvent under reduced pressure the crude is purified by preparative HPLC (Varian 5-35%) to yield the desired product (2, 3.3 mg, 5.74 μmol, 7%) as dark red solid. R.sub.t=19.7 min (gradient 1); .sup.1H-NMR (500 MHz, DMSO-d.sub.6) δ=8.30 (dd, .sup.3J.sub.H—H=8.1 Hz, .sup.4J.sub.H—H=0.8 Hz, 1H, H3), 8.28 (s, 2H, H33), 8.10 (m, 3H, H1, H2, H3), 7.92 (d, .sup.3J.sub.H—H=9.0 Hz, 2H, H17, H21), 7.78 (m, 1H, H20), 7.68 (t, .sup.3J.sub.H—H=7.7 Hz, 1H, H18), 7.60 (d, .sup.3J.sub.H—H=15.2 Hz, H6), 6.85 (d, .sup.3J.sub.H—H=9.0 Hz, 2H, H22, H23), 4.79 (t, .sup.3J.sub.H—H=7.4 Hz, 2H, H10), 4.37 (t, .sup.3J.sub.H—H=7.1 Hz, 2H, H15), 4.10 (q, .sup.3J.sub.H—H=5.6 Hz, 2H, H32), 3.12 (s, 6H, H25, H26), 1.78 (dt, .sup.3J.sub.H—H=14.6 Hz, .sup.3J.sub.H—H=7.2 Hz, 4H, H11, H14), 1.46 (m, 2H, H12), 1.30 (m, 2H, H13) ppm; .sup.13C-NMR (125.76 MHz, DMSO-d.sub.6) δ=171.2 (s, 10, C8), 153.6 (s, 1C, C19), 150.6 (s, 1C, C1), 141.2 (s, 1C, C4), 140.0 (s, 1C, C31), 133.0 (s, 10, C2), 128.9 (s, 1C, C5), 127.5 (s, 1C, C18), 127.1 (s, 1C, C20), 124.1 (s, 2C, C17, C21), 124.0 (s, 1C, C16), 121.4 (s, 1C, C3), 115.9 (s, 1C, C30), 112.0 (s, 2C, C22, C23), 105.7 (s, 1C, C6), 49.3 (s, 1C, C15), 47.8 (s, 1C, C10), 40.0 (s, 2C, C25, C26), 33.9 (s, 1C, C32), 29.6 (s, 1C, C11), 28.2 (s, 1C, C14), 25.5 (s, 1C, C13), 25.2 (s, 1C, C12) ppm; HRMS (ESI.sup.+) m/z: calculated for C.sub.26H.sub.33N.sub.6S: 461.2500; found: 461.2481.

Synthesis of yellow PPh3 dye 16

[0145] Under exclusion of light 88.0 mg of 22 (0.14 mmol, 1.00 eq.) is dissolved in 1.8 mL MeCN. 112 mg of PPh.sub.3 (0.43 mmol, 3.00 eq.) is added. The reaction mixture is heated to 85° C. for 3 h. After concentrating to dryness, the crude product is purified by preparative HPLC (gradient: 25-40% MeCN in water over 40 min). 90 mg of 16 (0.10 mmol, 72%) were obtained as dark red solid. R.sub.f=0.22 (DCM/MeOH 10:1); .sup.1H-NMR (500 MHz, DMSO-d.sub.6) δ=8.31 (d, .sup.3J.sub.H—H=8.1 Hz, 1H, H3), 8.09 (t, .sup.3J.sub.H—H=11.6 Hz, 2H, H18, H22), 7.91 (d, .sup.3J.sub.H—H=9.0 Hz, 2H, H19, H21), 7.87 (m, 3H, H33, H38, H43), 7.75 (m, 12H, H31, H32, H34-37, H39-42, H44, H45), 7.72 (d, .sup.3J.sub.H—H=3.9 Hz, 1H, H2), 7.68 (m, 1H, H1), 7.58 (d, .sup.3J.sub.H—H=15.2 Hz, 1H, H6), 6.81 (d, .sup.3J.sub.H—H=9.1 Hz, 2H, H23, H24), 4.76 (t, .sup.3J.sub.H—H=7.4 Hz, 2H, H10), 3.54 (t, .sup.3J.sub.H—H=11.3 Hz, 2H, H15), 3.11 (s, 6H, H26, H27), 1.74 (m, 2H, H11), 1.47 (m, 6H, H12-14) ppm; .sup.13C-NMR (126 MHz, DMSO-d.sub.6) 5=170.8 (s, 1C, C8), 153.2 (s, 1C, C20), 150.6 (s, 1C, C22), 141.0 (s, 1C, C4), 134.9 (d, 3C, C33, C38, C43), 133.6 (d, 6C, C31, C35, C36, C40, C41, C45), 133.0 (s, 2C, C19, C21), 130.3 (d, 6C, C32, C34, C37, C39, C42, C44), 129.0 (s, 1C, C2), 127.5 (s, 1C, C1), 129.0 (s, 1C, C5), 127.1 (s, 1C, C17), 124.0 (s, 1C, C3), 119.1 (s, 30, C28-030), 115.9 (s, 1C, C18), 111.9 (s, 2C, C23, C24), 105.7 (s, 1C, C6), 47.8 (s, 1C, C10), 39.8 (s, 2C, C26, C27), 29.4 (d, 1C, C12), 28.1 (s, 1C, C11), 24.8 (s, 1C, C13), 21.6 (d, 1C, C14), 20.1 (d, 1C, C15) ppm; HRMS (ESI.sup.+) m/z: calculated for C.sub.41H.sub.42N.sub.2PS: 626.2900; found: 625.2806.

Synthesis of Yellow 6-AzTO-2 (40)

[0146] ##STR00014##

[0147] Yellow 6-AzTO-2 (40): Under exclusion of light and N.sub.2-atmosphere 1.13 g of 39 (1.82 mmol, 1.00 eq.) and 1.18 g NaN.sub.3 (18.2 mmol, 10.0 eq.) was dissolved in in 14.0 mL acetone/water (1:1) and stirred for 24 h at room temperature. The reaction mixture was heated to 50° C. for 1 h and further 2.00 eq. NaN.sub.3 (0.24 g, 3.64 mmol) was added. The dark red solution was stirred at 50° C. for 1.5 h, 16 h at room temperature and again at 50 CC for 3 h. The solvent was evaporated and the residue dissolved in DCM and an aqueous NaCO.sub.3 solution (1:1, 20 mL). The aqueous layer was extracted with DCM (2×10 mL). The combined organic layers F as washed with 5 mL brine, dried over MgSO.sub.4, and concentrated to dryness under reduced pressure. The crude product was purified by flash column chromatography (gradient of MeOH in DCM: 0%.fwdarw.0.2%.fwdarw.0.5%.fwdarw.1%.fwdarw.5%.fwdarw.10%). 0.8 g of 40 (14.9 mmol, 82%) were obtained as dark red solid. R.sub.F=0.64 (DCM/MeOH 10:1): .sup.1H-NMR (500 MHz, DMSO-d.sub.6) δ=8.31 (d, .sup.3J.sub.H—H=8.0 Hz, 1H, H3), 8.14 (d, =8.4 Hz, 1H, H22), 8.09 (d. .sup.3J.sub.H—H=15.2 Hz, 1H, H18), 7.93 (d, .sup.3J.sub.H—H=8.8 Hz, 2H, H19, H21), 7.78 (t, .sup.3J.sub.H—H=7.7 Hz, 1H, H2), 7.68 (d, .sup.3J.sub.H—H=7.6 Hz, 1H, H1), 7.61 (d, .sup.3J.sub.H—H=15.2 Hz, 1H, H6), 6.85 (d, =8.8 Hz, 2H, H2.3, H24), 4.80 (t, .sup.3J.sub.H—H=7.3 Hz, 2H, H10), 3.29 (t, .sup.3J.sub.H—H=6.9 Hz, 2H, H15), 3.12 (s, 6H, H26, H27), 1.81 (m, 4H, H11), 1.45 (m, 4H, H12-14) ppm; .sup.13C-NMR (500 MHz, DMSO-d.sub.6) δ=171.1 (s, 1C, C8), 153.6 (s, 1C, C20), (s, 1C, C18): 141.1 (s, 1C, C4), 132.9 (s, 2C, C19, C21) 128.9 (s, 1C, C2), 127.4 (s, 1C, C1), 127.1 (s, 1C, C17), 123.9 (s, 1C, (73), 121.4 (s, 1C, C5), 115.9 (s, 1C, C22), 111.9 (s, 2C, C23, C24), 105.7 (s, 1C, C6), 50.5 (s, 2C, C26, C27), 47.8 (s, 1C, C10), 40.0 (s, 1C, C15), 28.2 (s, 1C, C11), 28.0 (s, 1C, C14), 25.8 (s, 1C, C12), 25.2 (s, 1C, C13) ppm; HRMS (ESI.sup.+) m/z: calculated for C.sub.23H.sub.28N.sub.5S: 406.21007, found: 406.2061.

Synthesis of TBTA (54)

[0148] ##STR00015##

[0149] Tris(benzyltriazolmethyl)amine (TBTA, 54): 7.5 mL MeCN and 13.1 mg CuOAc.sub.2. H.sub.2O (66.0 μmol, 0.02 eq.) was stared at room temperature until a blue solution was obtained. After addition of 0.7 mL benzylazide (56, 0.74 g, 5.6 mmol, 1.70 eq.) and 0.46 mL tripropargylamine (55, 430 mg, 3.28 mmol, 1.00 eq.) in 2.5 mL MeCN the reaction mixture was stirred for further 5 min at room temperature. 13.0 mg sodium ascorbate. (66.0 μmol, 0.02 eq.) was added and the solution was stirred at room temperature for 30 min and then at 45° C. for 4 h. After addition of further 0.7 mL benzylazide (56, 0.74 g, 5.6 mmol. 1.70 eq.) the yellowish solution was stirred for 16 h at 45° C. The reaction mixture was concentrated to dryness and the residue was dissolved in 90 mL DCM/NH.sub.4OH (2:1). The aqueous layer was extracted with DCM (2×20 mL) and the combined organic layers was washed with NH.sub.4OH/brine 1:1 (2×20 mL) and dried over MgSO.sub.4. After filtration the solvent was concentrated to dryness under reduced pressure. The crude product was dissolved in a 5 mL DCM and 10 mL DEE was added. The resulting suspension was centrifuged and the precipitate was dried under reduced pressure. 880 mg TBTA (54, 1.66 mmol, 51%) was obtained as a colorless solid. R.sub.F=0.60 (DCM/MeOH 10:1); .sup.1H-NMR (300 MHz, CDCl.sub.3) δ=7.47 (s, 3H, H4), 7.08 (ddd, 15H, .sup.3J.sub.H—H=8.4 Hz. .sup.3J.sub.H—H=5.9 Hz, .sup.4J.sub.H—H=2.0 Hz. X10-14), 5.28 (s, 6H, H8), 3.51 (s, SH, H2) ppm; .sup.13C-NMR (75.48 MHz, CDCl.sub.3) δ=144.0 (s, 3C, C9), 134.9 (s, 3C, C3), 129.2 (s, 6C, C11, C13), 128.8 (s, 3C, C12), 128.1 (s, 6C, C10, C14), 124.1 (s, 3C, C4), 54.3 (s, 3C, C8), 47.2 (s, 3C, C2) ppm.

Synthesis of Yellow 6-TramTO (42)

[0150] ##STR00016##

[0151] Yellow 6-TramTO (42); Under exclusion of light and stirring 6.9 mg CuSO.sub.4.5 H.sub.2O (28.0 μmol, 0.13 eq.) and 17.7 mg 54 (33.0 mmol, 0.15 eq.) were dissolved in mL DMF/water (3:1). After 5 min 10 μL propargylamine (12.2 mg, 0.22 mmol, 1.00 eq.) was added. After further 5 min 119 mg of 40 (0.22 mmol, 11.00 eq.) was added to the green/blue solution. After further 5 min 11.0 mg sodium ascorbate (56.0 μmol, 0.25 eq.) was added. The dark red solution was stirred for 5 h at room temperature. The solvent was evaporated and the residue was dissolved in MeCN. The red solution was filtered over Celite and the solvent was evaporated. The crude product was purified by preparative HPLC (MeCN in H.sub.2O (gradient: 15-35% over 40 min)). The different fractions were lyophilized to obtain 24.8 mg of product 42 (42.1 μmol, 21%) as a red solid. R.sub.F=0.60 (DCM/MeOH 10:1); .sup.1H-NMR (500 MHz, DMSO-d.sub.6) δ=8.30 (dd, =8.1 Hz. .sup.4J.sub.H—H=0.8 Hz, 1H, H3), 8.28 (s, 2H, H33), 8.10 (m, 3H, H1, H2, H3), 7.92 (d, .sup.3J.sub.H—H=9.0 Hz, 2H, H17, H21), 7.78 (m, 1H, H20), 7.68 (t, .sup.3J.sub.H—H=7.7 Hz, 1H, H18), 7.60 (d, .sup.3J.sub.H—H=15.2 Hz, H6), 6.85 (d, .sup.3J.sub.H—H=9.0 Hz, 2H, H22, H23), 4.79 (t, .sup.3J.sub.H—H=7.4 Hz, 2H, H10), 4.37 (t, .sup.3J.sub.H—H=7.1 Hz, 2H, H15) 4.10 (q, .sup.3J.sub.H—H=5.6 Hz, 2H, H32), 3.12 (s, 6H, H25, H26), 1.78 (dt, .sup.3J.sub.H—H=14.6 Hz, .sup.3J.sub.H—H=7.2 Hz, 4H, H11, H14), 1.46 (m, 2H, H12), 1.30 (m, 2H, H13) ppm, .sup.13C-NMR (125.76 MHz, DMSO-d.sub.6) δ=171.2 (s, 1C C8), 153.6 (s, 1C, C19), 150.6 (s, 1C, C1), 141.2 (s, C4), 140.0 (s, 1C, C31), 133.0 (s, 1C, C2), 128.9 (s, 1C, C5), 127.5 (s, 1C, C18), 127.1 (s, 1C, C20), 124.1 (s, 2C, C17, C21), 124.0 (s, 1C, C16), 121.4 (s, 1C, C3), 115.9 (s, 1C, C30), 112.0 (s, 2C, C22, C23), 105.7 (s, 1C, C6), 49.3 (s, 1C, C15), 47.8 (s, 1C, C10), 40.0 (s, 2C, C25, C26) 33.9 (s, 1C, C32), 29.6 (s, 1C, C11), 28.2 (s, 1C, C14), 25.5 (s, 1C, C13), 25.2 (s, 1C, C12) ppm; HRMS (ESI.sup.+) m/z: calculated for C.sub.26H.sub.33N.sub.6S: 461.2500: found: 461.2481.

Synthesis of Mitochondria penetrating peptides (MPPs)

[0152] The following exemplary synthesis route is based on the nucleophilic attack of the cysteine in peptide 36 at the iodine substituted carbon atom of the alkyl chain of the dye-derivative (21, 22, 23) (cf. FIG. 29, scheme 12).

[0153] Ac-Cys-Cha-dArg-Cha-dArg-OH (36): For loading of the resin with the first amino acid, 2-chlorotrityl chloride resin (CTC resin, 50.0 mg, loading by supplier 0.50 mmol/g) is swollen in DMF for 30 min and a solution of the first amino acid (Fmoc-D-Arg(Pbf)-OH, 130 mg, 200 μmol 8.00 equiv.) and DI PEA (136 μL, 800 μmol, 32 equiv.) in NMP (286 μL is added to the resin which is shaken at room temperature for 90 min. After the coupling, the resin is washed with DMF (5×) and CH.sub.2Cl.sub.2 (5×). A loading of f=0.35 mmol/g is determined by UV/Vis spectroscopy (1.00 equiv.=16.5 μmop. The resin is washed with DMF (5×) and capping is performed by adding 2,6-lutidine/Ac.sub.2O/DMF (6:5:89, 500 μL) to the resin and shaking for 5 min. After washing with DMF (5×), CH.sub.2Cl.sub.2 (5×), DMF (5×), the first coupling cycle begins mainly analogous to the synthesis described for peptide 26. Deprotecting of the Fmoc-groups is performed by adding piperidine (20% in DMF, 2×500 μL) and shaking for 2×5 min. The resin is washed with DMF (5×), CH.sub.2Cl.sub.2 (5×), DMF (5×) and the coupling solution consisting of the Fmoc-amino acid (66.0 μmol, 4.00 equiv.), Oxyma (9.4 mg, 66.0 μmol, 4.00 equiv.) and DIC (10 μL, 66.0 μmol, 4.00 equiv.) in DMF (200 μL) is added to the resin which is shaken at room temperature for 45 min. After the coupling, the resin is washed with DMF (5×), CH.sub.2Cl.sub.2 (5×), DMF (5×) and capped by adding 2,6-lutidine/Ac.sub.2O/DMF (6:5:89, 500 μL) to the resin and shaking for 5 min. After washing with DMF (5×), CH.sub.2Cl.sub.2 (5×), DMF (5×), the next cycle begins. After the last coupling cycle, the final Fmoc group is deprotected as described above and the resin is washed with DMF (5×), CH.sub.2Cl.sub.2 (5×), DMF (5×) and capped as described above. After washing with DMF (5×), CH.sub.2Cl.sub.2 (5×), DMF (5×), the peptide is cleaved from the resin by adding 1.5 mL SH-cleavage mix (2.5% DODT, 2.5% H.sub.2O, 1% TIPS, 94% TFA) and shaking for 2.5 h. Afterward, the resin is washed with cleavage mix (500 μL) and TFA (2×500 μL) and the combined solutions are drained into ice-cold Et.sub.2O (˜25 mL). After centrifugation, the precipitate is washed with cold Et.sub.2O (2×) and the obtained pellet is dried in vacuo. After purification by preparative HPLC (Gilson, 15-75%), the desired peptide (36, 6.0 mg, 5.32 μmol, 32%) is obtained as a white solid. R.sub.t=19.1 min (gradient 1). HRMS (ESI): m/z calc. for C.sub.35H.sub.64N.sub.11O.sub.7S [M+H].sup.+ 782.4708, found 782.4730. This synthesis of the MPP's is provided by way of example. Any person of ordinary skill in the art knows that the amino acid sequence of MPP's may be varied in order to adjust the penetrating ability of the MPP's. Thus all MPP's are comprised by the scope of the invention.

[0154] Red MPP (20): Peptide (36, 0.9 mg, 801 nmol, 1.00 equiv.) and Iodo derivative 6-IodoTO-3 (14, 1.5 mg, 2.24 μmol, 2.80 equiv.) are dissolved in DMF (100 μL), DIPEA (1.6 μL, 8.91 μmol, 11.1 equiv.) is added and the reaction mixture is stirred at room temperature for 18 h. Afterward, the solvent is removed via centrifugation in vacuo and the crude is purified by preparative HPLC (Varian, 35-95%) to yield the desired Red MPP (20, 44 nmol, 6%) as a blue solid. R.sub.t=23.4 min (gradient 1). HRMS (ESI): m/z calc. for C.sub.62H.sub.94N.sub.13O.sub.7S.sub.2.sup.3+ [M+2H].sup.30 398.89, found 399.42.

[0155] Green MPP (18): Peptide (36, 0.9 mg, 801 nmol, 1.00 equiv.) and Iodo derivative 6-IodoTO-1 (5, 1.0 mg, 1.56 μmol, 1.95 equiv.) are dissolved in DMF (100 μL), DIPEA (1.6 μL, 8.91 μmol, 11.1 equiv.) is added and the reaction mixture is stirred at room temperature for 18 h. Afterward, the solvent is removed via centrifugation in vacuo and the crude is purified by preparative HPLC (Varian, 35-95%) to yield the desired Green MPP (18, 116 nmol, 15%) as a red solid. R.sub.t=22.3 min (gradient 1). HRMS (ESI): m/z calc. for C.sub.60H.sub.90NDO.sub.7S.sub.2.sup.+[M].sup.+1168.65, found 1168.69.

[0156] Yellow MPP (19): Peptide (36, 0.9 mg, 801 nmol, 1.00 equiv.) and Iodo derivative 22 (1.0 mg, 1.62 μmol, 2.02 equiv.) are dissolved in DMF (100 μL), DIPEA (1.6 μL, 8.91 μmol, 11.1 equiv.) is added and the reaction mixture is stirred at room temperature for 18 h. Afterward, the solvent is removed via centrifugation in vacuo and the crude is purified by preparative HPLC (Varian, 35-95%) to yield the desired Yellow MPP (19, 44 nmol, 6%) as a red solid. R.sub.t=16.9 min (gradient 1). HRMS (ESI): m/z calc. for C.sub.58H.sub.90N.sub.13O.sub.7S.sub.2.sup.+ [M].sup.+ 1144.65, found 1144.69.

List of Abbreviations

[0157] Besides abbreviations, well known to any person of ordinary skill in the art, following table 3 lists some further abbreviations used herein.

TABLE-US-00003 TABLE 3 Abbreviations used herein Cha L-cyclohexylalanine dArg D-arginine MFI mean fluorescence intensity MOI multiplicity of infection MPP mitochondria penetrating peptide NT nucleoli tracker RFU relative fluorescence untis TBTA tris((1-benzyl-4-triazolyl)methyl)amine TO thiazole orange TAS benzoThiazole AminoStyryl

DESCRIPTION OF THE DRAWINGS

[0158] FIG. 1: FIG. 1 exemplarily shows Normalized spectral properties of TramTO dyes (1-3) in 10 mM Tris buffer, 50 mM KCl, pH=7.6; DNA is dsDNA.sub.CT double-stranded calf thymus DNA.

[0159] FIG. 2: FIG. 2 exemplarily shows bacterial growth curves at 2.5 μM and 5 μM dye concentration and with DMSO control (final DMSO concentration 0.4%). Mean of three measurements in triplicate.

[0160] FIG. 3: FIG. 3 exemplarily shows the viability of HeLa cells stained with cyanine dyes.

[0161] FIG. 4: FIG. 4 exemplarily shows the results of flow-cytometric measurements with E. Coli and B. Subtilis. Each experiment is labelled via subtitle, the subtitles indicating the following parameter settings (using equipment LSR Fortressa cell analyser): “Blue B-A” indicates an excitation wavelength of 488 nm, emission filter of 575/25 nm; [0162] “Blue Green A-A” indicates an excitation wavelength of 514 nm, emission filter of 610/20 nm; [0163] “Yellowgreen A-A” indicates an excitation wavelength of 561 nm, leading to an emission filter 685/35 nm;

[0164] FIG. 5A: The graphs of FIG. 5A show that for 6-TramTO-1 (1) there is clear labelling when added 5 μM of the dye for only 5 min before the measurements, dead cells (treated with 70% isopropanol for 1 h, then washed with saline 3×) are labelled about 10× stronger: MFI 135 vs 1337. [0165] Additionally to the parameter settings regarding excitation/emission described in the legend to FIG. 4 the following parameter settings are used: [0166] “Blue A-A” indicates an excitation wavelength of 488 nm, emission filter of 670/30 nm; [0167] “Blue C-A” indicates an excitation wavelength of 488 nm, emission filter of 510/20 nm; [0168] “Blue Green B-A” indicates an excitation wavelength of 514 nm, emission filter of 542/27 nm; [0169] “Yellow Green B-A” indicates an excitation wavelength of 561 nm, emission filter of 632/22 nm.

[0170] FIG. 5B: The graphs of FIG. 5B show flow cytometric data of mixtures of dead and live E. Coli treated with left graph: PI (20 μM) and right graph: 6-TramTO-1 (1, 5 μM) for 5 min showing that the TramTO and PI give the same results. [0171] The parameter settings regarding excitation/emission (as far as they are applied) are equivalent to those described within the legend of FIG. 5A.

[0172] FIG. 5C: The graphs of FIG. 5C show dead bacteria treated with the two on the left 6-TramTO-1 (1) only and the two on the right PI only. (DMSO treated cells MFI: Blue C-A 4.26 and Yellow Green B-A 2.96). The value “262144” on the x-axis of each graph of FIGS. 5B and 5C is always referring to the rightmost scale mark.

[0173] The parameter settings regarding excitation/emission (as far as they are applied) are equivalent to those described within the legend of FIG. 5A.

[0174] FIG. 6: FIG. 6 exemplarily shows data regarding comparatively staining mixtures of dead and live cells with PI and 6-TramTO-1 (1). Upper density plot with green (Blue C-A) and orange (Yellow Green B-A) labelling in the axis and lower plots: all cells are labelled (right plot) compared to DMSO control (left plot). [0175] The parameter settings regarding excitation/emission (as far as they are applied) are equivalent to those described within the legend of FIG. 5A.

[0176] FIG. 7: FIG. 7 exemplarily shows experimental data proving that dual and even triple labelling is also possible with the TramTO dyes, using Bacillus subtilis. Suspensions of bacteria are labelled for 15 min with the different dyes at 5 μM, and are mixed in ratio 1:1 for dual labelling and in ratio 1:1:1 for the triple labelling experiments. (LSR Fortessa cell analyser). The value “262144” on the axes of each graph is always referring to the rightmost resp. uppermost scale mark. [0177] The parameter settings regarding excitation/emission (as far as they are applied) are equivalent to those described within the legend of FIG. 5A. The dyes applied are denoted on top of each graph, e.g. “1+2”.

[0178] FIG. 8: FIG. 8 shows further examples of labelling, using Klebsiella pneumoniae. There the same protocol is applied (15 min labelling at 5 μM concentration). BD FACS Aria III. 100,000 events per sample are analysed. Left signal in each graph: [0179] DMSO/background fluorescence control. Channels: Alexa Fluor 488 (excitation wavelength of 488 nm, emission filter of 530/30 nm), PE-Texas Red (excitation wavelength of 561 nm, emission filter of 610/20 nm) and APC (excitation wavelength of 633 nm, emission filter of 660/20 nm).

[0180] FIG. 9A: FIG. 9A shows the results of phagocytosis studies with different bacterial strains labelled with 1 or 3. Here the following channels were used (apparatus “Guava easyCyte”): [0181] Green: excitation wavelength of 488 nm, emission filter of 525/30 nm; [0182] Yellow: excitation wavelength of 488 nm, emission filter of 582/26 nm; [0183] Red: excitation wavelength of 488 nm, emission filter of 611/319 nm; [0184] Red2: excitation wavelength of 640 nm, emission filter of 680/30 nm.

[0185] FIG. 9B: FIG. 9B shows examples of fluorescence microscopy pictures of stained HeLa cells; [0186] left: “green” 6-TramTO-1 (1), center: “yellow” 6-TramTAS (2); right: “red” 6-TramTO-3 (3), each one showing stained nuclei of fixed HeLa cells. (3 μM, 20 min)

[0187] FIG. 9C: FIG. 9C shows further examples of fluorescence microscopy pictures of stained HeLa cells (dyes in complete live cells: PPh.sub.3-TOs (15-17) in living HeLa cells (staining being done with dye concentration=3 μM (in FluoroBrite media), duration of staining being 20 min, the cells being rinsed afterwards and the images being acquired after further 30 min)). left: “green” cyanine dye 15, center: “yellow” cyanine dye 16; right: “red” cyanine dye 17.

[0188] FIG. 9D: FIG. 9D shows exemplarily fluorescence microscopy pictures of bacterial labelling (5 μM, 15 min for TramTO dyes, 1 μM for DAPI) using exemplarily E. Coli and B. Subtilis.

[0189] FIG. 10: Scheme 5 shows the general retrosynthetic approach for the preparation of different TO-derivatives (e.g. 6-xxxTO-1 and 6-xxxTO-3 derivatives, “xxx” denoting “Tram” or “Iodo” or “Tri” or “PPh.sub.3”).

[0190] FIG. 11: Scheme 8 shows the synthesis route of 6-TramTO-1 (1) starting from benzothiazolium (30).

[0191] FIG. 12A: HSQC-NMR spectrum of 6-IodoTO-1 (21).

[0192] FIG. 12B: COSY-NMR spectrum of 6-IodoTO-1 (21).

[0193] FIG. 12C: HMBC-NMR spectrum of 6-IodoTO-1 (21).

[0194] FIG. 13A: HSQC-NMR spectrum of 12.

[0195] FIG. 13B: HMBC-NMR spectrum of 12.

[0196] FIG. 13C: COSY-NMR spectrum of 12.

[0197] FIG. 14A: HSQC-NMR spectrum of 6-TramTO-1 (1).

[0198] FIG. 14B: COSY-NMR spectrum of 6-TramTO-1 (1).

[0199] FIG. 14C: HMBC-NMR spectrum of 6-TramTO-1 (1).

[0200] FIG. 15: Scheme 9 shows the synthesis routes of 6-PipTO-1 (14), 6-PipTO-3 (10), 6-TriTO-1 (13), 6-PPh3TO-1 (15) and 6-PPh3TO-3 (17) starting from 6-IodoTO-1 (21) or 6-IodoTO-3 (23).

[0201] FIG. 16A: HSQC-NMR spectrum (500 MHz, 125 MHz) of 6-PipTO-1 (14).

[0202] FIG. 16B: COSY-NMR spectrum (300 MHz, 300 MHz) of 6-PipTO-1 (14).

[0203] FIG. 16C: HMBC-NMR spectrum (500 MHz, 125 MHz) of 6-PipTO-1 (14).

[0204] FIG. 17A: HSQC-NMR spectrum of 10 in DMSO-d6.

[0205] FIG. 17B: HMBC-NMR spectrum of 10 in DMSO-d6.

[0206] FIG. 17C: COSY-NMR spectrum of 10 in DMSO-d6.

[0207] FIG. 18A: HSQC-NMR spectrum (300 MHz, 75 MHz) of 6-Tri(asym.)TO-1 (13a).

[0208] FIG. 18B: COSY-NMR spectrum (300 MHz, 300 MHz) of 6-Tri(asym.)TO-1 (13a).

[0209] FIG. 19A: HSQC-NMR spectrum (300 MHz, 75 MHz) of 6-Tri(sym.)TO-1 (13b).

[0210] FIG. 19B: COSY-NMR spectrum (300 MHz, 300 MHz) of 6-Tri(sym.)TO-1 (13b).

[0211] FIG. 20A: COSY-NMR spectrum of 6-PPh3TO-3 (17).

[0212] FIG. 20B: HSQC-NMR spectrum of 6-PPh3TO-3 (17).

[0213] FIG. 20C: HMBC-NMR spectrum of 6-PPh3TO-3 (17).

[0214] FIG. 21: Scheme 10 shows the synthesis route of dyes CH.sub.3TO-1 (4) and CH.sub.3TO-3 (6), starting from benzothiazole 37.

[0215] FIG. 22A: HSQC-NMR spectrum of 4 in DMSO-d.sub.6.

[0216] FIG. 22B: HMBC-NMR spectrum of 4 in DMSO-d.sub.6.

[0217] FIG. 22C: COSY-NMR spectrum of 4 in DMSO-d.sub.6.

[0218] FIG. 23A: HSQC-NMR spectrum of 6 in DMSO-d.sub.6.

[0219] FIG. 23B: HMBC-NMR spectrum of 6 in DMSO-d.sub.6.

[0220] FIG. 23C: COSY-NMR spectrum of 6 in DMSO-ds.

[0221] FIG. 24: Scheme 11 shows the Synthesis route of “yellow” 6-TramTAS (2) starting from benzothiazole (30).

[0222] FIG. 25A: HSQC-NMR spectrum of 6-IodoTAS (22).

[0223] FIG. 25B: COSY-NMR spectrum of 6-IodoTAS (22).

[0224] FIG. 25C: HMBC-NMR spectrum of 6-IodoTAS (22).

[0225] FIG. 26A: HSQC-NMR spectrum of 6-azindoTAS (34).

[0226] FIG. 26B: COSY-NMR spectrum of 6-azindoTAS (34).

[0227] FIG. 26C: HMBC-NMR spectrum of 6-azindoTAS (34).

[0228] FIG. 27A: HSQC-NMR spectrum of 2.

[0229] FIG. 27B: HMBC-NMR spectrum of 2.

[0230] FIG. 27C: COSY-NMR spectrum of 2.

[0231] FIG. 28A: COSY-NMR spectrum of 16.

[0232] FIG. 28B: HSQC-NMR spectrum of 16.

[0233] FIG. 28C: HMBC-NMR spectrum of 16.

[0234] FIG. 29: Scheme 12 shows the synthesis route of Red—(20), Green—(18) and Yellow-MPP (19) starting from Ac-Cys-Cha-dArg-Cha-dArg-OH (36).

[0235] FIG. 30A: Table showing experimentally measured values of viability of HeLa cells (acquired via resazurin-based assay) under different growth conditions: pure and in H.sub.2O (both serving as possible reference values) and under different dye concentrations (examples: dyes 18, 19 and 20).

[0236] FIG. 30B: Graphical depictions of experimental results of viability-testing, exemplarily obtained by use of compounds 18, 19 and 20 (graphical depictions of data presented in FIG. 30A).

[0237] FIG. 31A: .sup.1H-NMR spectrum of 40.

[0238] FIG. 31B: .sup.13C-NMR spectrum of 40.

[0239] FIG. 31C: HSQC-NMR spectra of 40.

[0240] FIG. 31D: HMBC-NMR spectra of 40.

[0241] FIG. 31E: COSY-NMR spectra of 40.

[0242] FIG. 32A: .sup.1H-NMR spectrum of TBTA (54).

[0243] FIG. 32B: .sup.13C-NMR spectrum TBTA (54).

[0244] FIG. 33A: .sup.1H-NMR spectrum of 42.

[0245] FIG. 33B: .sup.13C-NMR spectrum of 42.

[0246] FIG. 33C: HSQC-NMR spectra of 42.

[0247] FIG. 33D: HMBC-NMR spectra of 42.

[0248] FIG. 33E: COSY-NMR spectra of 42.