Patent Application
20230096750
The present application claims priority to European application no. 21192451.9, filed on Aug. 20, 2021, which is incorporated by reference in its entirety.
The invention relates to cyanine dyes, the production of cyanine dyes and their use for in vivo-staining of microorganisms, especially pathogens, and other living cells.
Several important microorganisms are still difficult to modify for GFP expression for the purpose of investigating them. This is particularly true for the growing number of multi-drug resistant bacteria, posing an emerging threat to patients in clinics worldwide. Specifically, these strains may acquire resistances to the antibiotics used to select transformants carrying the GFP transgene. Thus, the investigation of host cell interplay with drug-resistant bacteria by means of fluorescent labelling using GFP is not applicable generally. The expression “in vivo”-staining” as is used herein has the meaning of staining living cells without major negative interference on their viability so that their live behaviour, e.g. their interactions with other cells may be observed by methods known to the person skilled in the art after being stained, e.g. via flow cytometry or fluorescence microscopy etc. The surprising effect that the in vivo-staining is not negatively interfering with the viability of the stained cells is experimentally proven by comparative growth studies of stained cells at distinct dye-concentration in comparison with reference compounds, e.g. DMSO (preferred method with respect to microbial cells) and/or by application of resazurin-based assay to the cells several hours after being stained (preferred method for non-microbial cells). These comparative viability studies resp. resazurin-based assays are performed either with the cells one wants to investigate by use of in vivo staining or with HeLa cells or other standard cell lines known to the person skilled in the art (or, for example, E. Coli as standard microbial model organism with respect to microbial cells).
In vivo staining of cells with cyanine dyes as is defined herein (especially multiple-colour staining of one or more samples of cells), exerting the surprising effect that the staining with cyanine dyes does not negatively influence the viability of the stained cells, is unknown heretofore. Interference of staining with the viability of the stained cells is mostly not addressed at all, as can be exemplarily seen within the documents referenced below.
US 2016/340717 A1 reveals a dye resp. probe for detecting bacterial or viral endonucleases which is composed of an oligonucleotide, a fluorophore connected to the oligonucleotide and a fluorescence quencher, also being connected to the oligonucleotide. Fluorescence is emitted only upon cleavage of the dye/probe. US 2016/340717 A1 nowhere reveals that it is intended to keep cells living after being stained by the dye/probe. So, US 2016/340717 A1 is not addressing the problem to be solved by the invention revealed herein.
Jieqiong Qiu et al reveal combination probes with intercalating anchors and proximal fluorophores for DNA and RNA detection (cf. Nucleic Acis Research, 2016, Vol. 44, No. 17-doi: 10.1093/narlgkw579). This article describes the usage of an anchor-dye and a reporter dye, i.e. two dyes intercalating into DNA/RNA for achieving the desired staining. Nowhere is revealed that the dyes applied do not negatively interfere with the viability of the stained cells. The technical teaching of this document is completely different from the invention revealed herein below.
US 2011/0262904 A1 describes dyes having a good solubility in water and a good live cell permeability. But nowhere within US 2011/0262904 A1 it is stated or claimed that the dyes do not negatively interfere with the viability of the stained cells. Staining cells without negatively affecting their viability is not addressed at all in US 2011/0262904 A1, indicating that this problem is not concerned about. In US 2011/0262904 A1 it is only important that the dyes can permeate the membranes of live cells for staining, but the destiny of the cells, their viability upon being stained is of no importance. The technical teaching of this document is also completely different from the invention revealed herein below.
Similarly, WO 2013/189043 A1 and US 2015/0185182 A1 only describe live cells being stained, but the effect of the staining upon viability of the stained cells is not addressed at all. The technical teaching of this document is also completely different from the invention revealed herein below.
US 2010/0151509 A1 is revealing staining and counting leucocytes, for which purpose it is not necessary that stained cells stay alive after having been stained. Consequently interference of staining with the viability of the stained cells (leucocytes) is not addressed at all in US 2010/0151509 A1. Leucocytes by nature cannot multiply (grow) like other cells or microorganisms, so that this document is dealing with a totally different subject for which the parameter “viability” is not applicable at all. Thus, the technical teaching of this document is also completely different from the invention revealed herein below.
Tinghan Zhao et al. describe a very special dye being composed of a tetramethylrhodamin moiety being chemically bound to a specific MPP (mitochondria penetrating protein), rFrFrF.
Rhodamine dyes are known mitochondria markers by themselves, e.g. tetramethylrhodamine ethyl ester (TMRE), which is commercially available for the purpose of staining mitochondria in living cells. It is structural very close to TAMRA. The known applicability of TMRE for staining of living cells is misleading to the application of dyes of similar structure—like TAMRA. Also, TMRE is not interacting with the DNA and it is not a light-up dye, so background is always higher than with cyanine dyes. Thus, application of cyanine dyes for staining according to the invention will improve the background-to-signal ratio.
The article (cf. Bioconjugate Chem. 2019, 30, 2312-2316) provides only one specific dye-component (TAMRA), belonging to a class of dyes which is known to be usable for marking/staining mitochondria, and no hint at all that conjugates for staining mitochondria may be possible to be composed of cyanine dyes and MPP's. Cyanine dyes were unknown heretofore to be applicable for marking/staining mitochondria in live cells without negatively interfering with the viability of the stained cells.
Patent application EP 3 572 468 A1 reveals cyanine dyes binding to DNA and exerting fluorescence upon excitation with light of suitable wavelength, e.g. cyanine dye 3, exerting red fluorescence. Heretofore these compounds were only applied solely.
Now, it is found and revealed herein that it is possible to combine multiple DNA binding cyanine dyes exerting different fluorescence colours within one experiment without negatively interfering with the viability of the stained cells, thus providing the possibility of multichannel observation (red, green and yellow fluorescence) of interactions of different cells with each other. Also, new cyanine dyes of different structure, also binding to DNA, have been found, synthesized and investigated, which are also exerting red, yellow resp. green fluorescence upon excitation with light of suitable wavelength, e.g. cyanine dyes 1, exerting green fluorescence, 2, exerting yellow fluorescence, 15, exerting green fluorescence, 16, exerting yellow fluorescence, 17, exerting red fluorescence, 18, exerting green fluorescence, 19, exerting yellow fluorescence and 20, exerting red fluorescence.
An objective of the present invention is to provide a method for staining living cells in such a way that different sample populations of cells are stained with different (at least two) cyanine dyes (most preferred cyanine dyes which are binding to nucleic acid) which are not reducing the viability of the stained cells, so that interaction of the stained population of cells can be observed via multichannel fluorescence microscopy.
A second objective of the present invention is to provide substances (cyanine dyes) that can be used for in vivo-staining of cells, especially microorganisms, without significantly reducing the viability of the stained cells (e.g. microbes, yeasts) according to the inventive method for staining living cells. The term “microorganism” (and all of its grammatical forms) as used with regard to the present invention comprehends not only all types of bacteria, fungi etc., but also pathogenic structures of all types. The terms “microorganism”, “pathogen”, “bacteria” etc. are therefore synonymously used throughout this whole revelation of the current invention. The term “cell” (and all of its grammatical forms) as used with regard to the present invention comprehends not only cells of microorganisms and its synonyms but also cells of vertebrates and plants.
In order to achieve the first objection a method for staining living cells is revealed herein applying at least two nucleic acid binding cyanine dyes with different fluorescence emission wavelengths which are not reducing the viability of the stained cells substantially.
A cyanine dye (most preferredly a cyanine dye which is binding to nucleic acid) is not reducing the viability of cells substantially according to the invention if: a) microbial (e.g. E. Coli) growth after 10 h under identical breeding conditions at dye concentration of 2.5 μM is at least 90% compared to DMSO-reference and at dye concentration of 5 μM is at least 70% compared to DMSO-reference and/or b) growth of HeLa cells under identical breeding conditions at dye concentration of 10 μM is at least 50% of cell viability compared to reference (for example: concentration of 10 μM TriTO-1 leads to viability of about 62%, concentration of 10 μM 6-TramTO-1 (1) leads to viability of about 97%, concentration of 10 μM 6-TramTO-3 (3) leads to viability of about 97%).
It is well known to the person of ordinary skill in the art how to perform experimental measurements of microbial growth or cell viability as are described in the proceeding paragraph, for example regarding choosing the suitable microorganism or cell type, breeding conditions (growth medium, breeding temperature, surrounding atmosphere etc.), choice and applied amount of reference compounds if necessary (e.g. DMSO, MeCN), defining further reference conditions etc.
In order to achieve the second objective, the present invention provides new types of cyanine dyes of formula (I),
allowing in vivo staining of pathogens without negatively interfering with the viability of the pathogens.
The above objectives are achieved through the independent claims 1, 4 and 9 through 12. The dependent claims reveal additional advantageous embodiments of the invention.
The cyanine dyes from previous research exhibited mainly red fluorescence, enabling them to be detected in the far-red fluorescence channels of fluorescence microscopes or other fluorescence detection apparatuses only. The work revealed herein has led to new compounds (e.g. cyanine dyes 1, 2) exerting excellent characteristics for live-staining (in vivo-staining) of cells, i.e. the new compounds exert distinctively less interference with the viability of cells upon staining in that they can also be used for staining cells of vertebrates, e.g. HeLa cells. The new compounds exert red, green and yellow fluorescence, thus providing the possibility of staining live cells with several cyanine dyes exerting different fluorescence colours at the same time and thus allowing a multichannel fluorescence observation of the behaviour of, for example, living microbial cells, to a much greater extent than previously known. Alternatively different species of microbial cells may be stained with different cyanine dyes, exerting different fluorescence, and being simultaneously observed within the same sample at the same time. This process of staining cells, basically without reducing the viability of the cells, is herein referred to as “in vivo staining”.
Scheme 2 shows cyanine dyes according to the state-of-the-art exerting green, yellow and red fluorescence upon excitation with light of suitable wavelength, which are used as control samples within the experiments. Scheme 3 shows further examples of cyanine dyes according to the state of the art, being modified regarding substituent X, whereat general formula Ia represents examples of red fluorescent cyanine dyes and general formula IIa represents examples of green fluorescent cyanine dyes.
The scope of the invention is comprising compounds where substituent R″ is (for example): I—(CH2)5—PPh3 (e.g. 15 (green fluorescence), 16 (yellow fluorescence), 17 (red fluorescence)). The substituent —PPh3 at the end of the side chain attached to the N-atom of the thiazole ring is introducing the possibility of staining organelles, especially e.g. mitochondria. This effect was heretofore unknown to be working in combination with cyanine dyes, thus this development, revealed herein, provides the possibility of staining organelles with cyanine dyes. Moreover, the scope of the invention even expands to cyanine dyes having peptide side chains as are exemplarily shown in Scheme 4 (substituent R1 according to formula I being for example MPP i.e. mitochondria penetrating peptide, e.g. the peptide H2N-Cys(Rb)-Cha-dArg-Cha-dArg-COOH (SEQ ID 1)), whereat the substituent Rb is denoting the chemical bonding site to the core structure of the cyanine dyes, i.e. here the MPP is exemplarily connected via the thioether-bridge from cysteine to the core structure of the cyanine dye (cf. compounds 18, 19 and 20)). Other examples of MPP's being comprised by the scope of the invention are: H2N-Cha-dArg-Cha-dArg-COOH (SEQ ID 2), H2N-dArg-Cha-dArg-Cha-dArg-Cha-COOH (SEQ ID 3), H2N-dArg-Phe-dArg-Phe-dArg-Phe-COOH (SEQ ID 4). In order to connect them to the core of the cyanine dye according to the invention, first cysteine is attached via SPPS (solid phase peptide synthesis) accordingly and then the cystein-modified MPP connected to the prepared cyanine dye core via substitution reaction, as is known to the person skilled in the art. As can be seen exemplarily from scheme 4, any MPP to be applied may be acetylated at the N-terminus, whereat the examples are not meant to be confining the scope of the invention to the use of acetylated or otherwise at the N-terminus substituted MPP's.
Following table 1 shows some of the molecular extinction coefficients that are exemplarily measured.
Therefore, the subject matter of the invention may be summarised as follows.
The subject matter of the invention is a method for staining living cells characterized in that the method comprises the following steps:
The cells to be stained for investigation (=“sample of cells”) and those for determining the non-interference of viability (=“eukaryotic cells, for example HeLa cells”) may be of the same type or of different type.
The subject matter of the invention also comprises a method for staining living cells as described before, characterized in that the method comprises the following steps i) and ii) and the additional step iii):
The cells to be stained for investigation (=“samples of cells”) and those for determining the non-interference of viability (=“eukaryotic cells, for example HeLa cells”) may be of the same type or of different type.
The subject matter of the invention also comprises any one method as described before, characterized in that the cyanine dye is a cyanine dye for use in binding to nucleic acid.
The subject matter of the invention also comprises any one method as described before, characterized in that the method comprises the following additional preparation step before step i):
The subject matter of the invention also comprises a cyanine dye for usage in any one method as is described before, the chemical structure of the cyanine dye being according to general formula (I),
wherein
characterized by
or
The subject matter of the invention also comprises a cyanine dye as is described before, 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.
The subject matter of the invention also comprises a cyanine dye as is described before, characterized in that it has the chemical structure according to formula (A),
or according to formula (B)
or according to formula (C),
The subject matter of the invention also comprises any cyanine dye as is described before 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.
The subject matter of the invention also comprises any cyanine dye as is described before, characterized in that it is for use in binding to oligomeric DNA or primer-DNA or DNA-aptamers.
The subject matter of the invention also comprises the usage of a cyanine dye as is described before for staining nucleic acid within a living cell.
The subject matter of the invention also comprises a kit for carrying out any one of the methods described above, the kit comprising the cyanine dye and optionally supportive substances.
The subject matter of the invention also comprises the synthesizing method of a cyanine dye as is described above, characterized in that the synthesizing method comprises the following steps:
The subject matter of the invention also comprises a method as described before, characterized in that the nucleophilic compound in step a) is triphenylphosphine or 1,2,3-triazole or a mitochondria penetrating peptide.
The subject matter of the invention also comprises a method as described above, 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).
The subject matter of the invention also comprises a method as described before, characterized in that the additional step d) comprises the reaction of a precursor compound according to formula (V),
The subject matter of the invention also comprises a method as described above, characterized in that the additional step d) comprises the reaction of a precursor compound according to formula (V),
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.
Exemplary Growth Curves of Some Sample Microorganisms, Resp. Sample Cells Exemplarily Stained
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.
Experimental Procedure for Measuring of Cell Viability
HeLa cells are grown in a humidified cell-culture incubator (New Brunswick Scientific, USA) at 37° C. under CO2 (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 (2×104) 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.
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
Experimental Procedure for Measuring Bacterial Growth Curves
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 OD600, utilizing a microplate reader (Tecan, Switzerland).
Fluorescence Titrations
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 CH3TO-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). KD values are determined as is well known to any person being of ordinary skill in the art. Table 2 shows the results obtained.
Flow Cytometry
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.
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.
As can obviously be seen from the data shown in
Dual and Triple Labelling
Experimental data shows that dual and even triple labelling is also possible with the TramTO dyes (examples presented in
Exemplary Phagocytosis Studies with Different Bacterial Strains Exemplarily Labelled with 1 or 3
The applicability of the invention is proven by exemplary Phagocytosis studies as is described below (cf.
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 OD600 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 CO2 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.
Nuclear Magnetic Resonance Spectroscopy (NMR)
All NMR spectra are automatically measured at 300 K either in a Bruker AV III HD 300 MHz at a frequency of 300 MHz (1H) or 75 MHz (13C) or in a Bruker AV III HD 500 MHz at a frequency of 500 MHz (1H) or 125 MHz (13C). All chemical shifts (δ) are relative to tetramethylsilane (TMS), i.e. δ(TMS)=0 ppm. As internal standards, deuterated chloroform (CDCl3), dimethyl sulfoxide (DMSO) with TMS are used. Solvent shifts (ppm): δ(CHCl3)=7.26 ppm (1H), δ(CHCl3)=77.16 ppm (13C), δ(DMSO)=2.50 ppm (1H), δ(DMSO)=39.52 ppm (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).
Mass Spectrometry
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).
Analytic HPLC
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 H2O (solvent A) with addition of 0.05% TFA and MeCN (solvent B) with addition of 0.03% TFA.
Scheme 5 (
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.
(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 CH2Cl2: 0%→0.2%→0.5%→0.7%) to yield the desired product (21, 359 mg, 0.56 mmol, 52%) as red solid. Rf=0.45 (CH2Cl2/MeOH 10:1). 1H-NMR (500 MHz, DMSO-d6, 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). 13C-NMR (125 MHz, DMSO-d6, δ): 159.4 (Cq), 148.7 (Cq), 144.0 (CH), 140.0 (Cq), 136.9 (Cq), 133.3 (CH), 128.3 (CH), 127.0 (CH), 125.7 (CH), 124.6 (CH), 124.3 (Cq), 123.9 (Cq), 123.0 (CH), 118.1 (CH), 113.0 (CH), 108.3 (CH), 87.7 (CH), 49.5 (CH2), 45.6 (CH2), 32.7 (CH2), 29.6 (CH2), 26.7 (CH2), 24.9 (CH2), 14.8 (CH3), 8.8 (CH2). HRMS-ESI+ (m/z): [M]+ calcd for C25H28IN2S+, 515.1012; found, 515.1023.
(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 NaN3 (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 CH2Cl2: 0%→2.0%) to yield the desired product (12, 10.8 mg, 19.9 μmol, 84%) as red solid. Rf=0.43 (CH2Cl2/MeOH 10:1). 1H-NMR (500 MHz, DMSO-d6, δ): 8.76 (d, 1H, 3J=8.4 Hz, H-9), 8.67 (d, 1H, 3J=7.2 Hz, H-10), 8.17 (d, 1H, 3J=8.6 Hz, H-6), 8.06 (dd, 1H, 3J=7.9 Hz, 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, 3J=7.8 Hz, 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. 13C-NMR (75 MHz, DMSO-d6, 5): 159.4 (Cq), 148.7 (Cq), 144.1 (C-10), 140.0 (Cq), 136.9 (Cq), 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+ (m/z): [M]+ calcd for C25H28N5S+, 430.2060; found, 430.2063.
(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. Rt=20.2 min. 1H-NMR (500 MHz, DMSO-d6, 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, NH3+), 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). 13C-NMR (125 MHz, DMSO-d6, δ): 159.4 (Cq), 148.7 (Cq), 144.0 (CH), 140.0 (Cq), 136.9 (Cq), 133.3 (CH), 128.2 (CH), 126.9 (CH), 125.7 (CH), 124.5 (CH), 124.3 (Cq), 124.1 (2C, CH, Cq), 123.9 (Cq), 123.0 (CH), 118.0 (CH), 113.0 (CH), 108.3 (CH), 87.6 (CH), 49.5 (CH2), 49.3 (CH2), 45.6 (CH2), 33.9 (CH2), 29.6 (CH2), 26.8 (CH2), 25.6 (CH2), 25.4 (CH2), 14.7 (CH3). HRMS-ESI+ (m/z): [M]+ calcd for C28H33N6S+, 485.2482; found, 485.2487.
This synthesis is based on the nucleophilic substitution of the iodo substituent of 6-IodoTO-1 and 6-IodoTO-3 (cf.
(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 Na2CO3 (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 CH2Cl2 and dried over MgSO4. 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. Rt=18.0 min. 1H-NMR (500 MHz, DMSO-d6, δ): 9.32 (br s, 2H, NH2+), 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). 13C-NMR (125 MHz, DMSO-d6, δ): 159.4 (Cq), 148.8 (Cq), 144.0 (CH), 140.0 (Cq), 136.9 (Cq), 133.3 (CH), 128.2 (CH), 126.9 (CH), 125.7 (CH), 124.5 (CH), 124.3 (Cq), 123.9 (Cq), 123.0 (CH), 118.0 (CH), 113.0 (CH), 108.3 (CH), 87.6 (CH), 55.8 (CH2), 49.5 (CH2), 48.1 (CH2), 45.6 (CH2), 40.5 (CH2), 26.8 (CH2), 23.3 (CH2), 25.8 (CH2), 25.6 (CH2), 14.7 (CH3). HRMS-ESI+ (m/z): [M]+ calcd for C29H37N4S+, 473.2733; found, 473.2731.
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 Na2CO3 (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 CH2Cl2 and dried over MgSO4. The solvent is evaporated under reduced pressure, and the crude is purified using a Büchi pure system with MeCN in H2O (0%→45%) as a solvent to yield the desired product 12 (40.3 mg, 64.3 μmol, 43%) as a blue solid. 1H-NMR (500 MHz, DMSO-d6, δ): 8.51-8.44 (m, 2H, H-11, H-12), 8.16 (t, 1H, 3J=12.7 Hz, H-6), 8.12-8.08 (m, 1H, H-8), 7.97 (td, 1H, 3J=7.9 Hz, 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, 3J=7.8 Hz, 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, 3J=12.2 Hz, H-5), 4.61 (q, 2H, 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. 13C-NMR (75 MHz, DMSO-d6, 5): 160.8 (Cq), 158.4 (Cq), 158.1 (Cq), 150.5 (Cq), 141.5 (Cq), 137.8 (Cq), 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+ (m/z): [M+2H]+ calcd. for C31H39N4S+499.2890; found 501.2684.
(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(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(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 CH2Cl2 and the organic phase is dried over magnesium sulfate. The crude products are purified by preparative HPLC (Gilson 5-75%) to yield 6-Tri(asym.)TO-1 (13a, 69.6 mg, 0.12 mmol, 51%) and 6-Tri(sym.)TO-1 (13b, 31.7 mg, 0.05 mmol, 23%) as pink solids.
6-Tri(asym)TO-1 (13a):
Rt=21.6 min (gradient 2). 1H-NMR (300 MHz, DMSO-d6, 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). 13C-NMR (75 MHz, DMSO-d6, δ): 159.4 (Cq), 148.7 (Cq), 144.0 (CH), 140.0 (Cq), 136.8 (Cq), 133.3 (CH), 133.1 (CH), 128.2 (CH), 126.9 (CH), 125.7 (CH), 124.5 (2C, CH), 124.3 (Cq), 123.9 (Cq), 122.9 (CH), 118.0 (CH), 112.9 (CH), 108.3 (CH), 87.6 (CH), 49.5 (CH2), 49.0 (CH2), 45.6 (CH2), 29.5 (CH2), 26.7 (CH2), 25.6 (CH2), 25.4 (CH2), 14.6 (CH3). HRMS-ESI+ (m/z): [M]+ calcd for C27H30N5S+, 456.2216; found, 456.2212.
6-Tri(sym)TO-1 (13b):
Rt=23.6 min (gradient 2). 1H-NMR (300 MHz, DMSO-d6, 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). 13C-NMR (75 MHz, DMSO-d6, δ): 159.4 (Cq), 148.7 (Cq), 144.0 (CH), 140.0 (Cq), 136.9 (Cq), 134.0 (2C, CH), 133.3 (CH), 128.2 (CH), 126.9 (CH), 125.7 (CH), 124.5 (CH), 124.3 (Cq), 123.9 (Cq), 122.9 (CH), 118.0 (CH), 112.9 (CH), 108.3 (CH), 87.6 (CH), 53.8 (CH2), 49.4 (CH2), 45.6 (CH2), 28.9 (CH2), 26.7 (CH2), 25.6 (CH2), 25.3 (CH2), 14.6 (CH3). HRMS-ESI+ (m/z): [M]+ calcd for C27H30N5S+, 456.2216; found, 456.2213.
(Z)-1-ethyl-4-((3-(6-(triphenylphosphonio)hexyl)benzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium (6-PPh3TO-1, 15): Iodo derivative 6-IodoTO-1 (21, 118 mg, 184 μmop and PPh3 (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. Rt=25.1 min (gradient 1). HRMS-ESI+ (m/z): [M]2+ calcd for C43H43N2PS2+, 325.1437; found, 325.1437.
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-PPh3TO-3, 17): derivative 6-IodoTO-3 (23, 49.9 mg, 74.7 μmop and PPh3 (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. Rr=26.9 min (gradient 1); 1H-NMR (500 MHz, DMSO-d6) 5=8.45 (d, 3JH—H=7.1 Hz, 1H, H23), 8.42 (d, 3JH—H=8.2 Hz, 1H, H26), 8.14 (t, 3JH—H=12.8 Hz, 1H, H18), 8.09 (d, 3JH—H=8.8 Hz, 1H, H29), 7.95 (t, 3JH—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, 3JH—H=7.7 Hz, 1H, H30), 7.56 (d, 3JH—H=8.3 Hz, 1H, H6), 7.46 (t, 3JH—H=7.3 Hz, 1H, H2), 7.30 (t, 3JH—H=7.8 Hz, 1H, H1), 7.11 (d, 3JH—H=13.3 Hz, 1H, H19), 6.50 (d, 3JH—H=12.3 Hz, 1H, H10), 4.61 (q, 3JH—H=7.1 Hz, 2H, H21), 4.19 (t, 3JH—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, 3JH—H=7.1 Hz, 5H, H15, H20) ppm; 13C-NMR (126 MHz, DMSO-d6) 5=160.9 (s, 1C, C25), 150.4 (s, 1C, C8), 143.9 (s, 1C, C18), 142.3 (s, 1C, C23), 141.5 (s, 1C, C27), 137.7 (s, 1C, C4), 134.9 (s, 3C, C37, C42, C47), 133.6 (d, 6C, C35, C39, C40, C44, C45, C49), 133.5 (s, 1C, C31), 130.3 (d, 3C, C36, C38, C41, C43, C46, C48), 127.7 (s, 1C, C2), 126.7 (s, 1C, C30), 125.2 (s, 1C, C26), 124.8 (s, 1C, C1), 124.3 (s, 1C, C5), 122.7 (s, 1C, C24), 118.9 (s, 3C, C32-34), 118.2 (s, 1C, C29), 112.4 (s, 1C, C6), 110.0 (s, 1C, C3), 109.5 (s, 1C, C19), 98.5 (s, 1C, C10), 49.4 (s, 1C, C21), 45.4 (s, 1C, C11), 29.4 (s, 1C, C13), 26.6 (s, 1C, C12), 25.0 (s, 1C, C15), 21.6 (s, 1C, C14), 20.1 (s, 1C, C16), 14.7 (s, 1C, C20) ppm. HRMS-ESI+ (m/z): [M]2+ calcd for C45H45N2PS2+, 338.1515; found, 338.1516.
The synthesis of the CH3TAS (5) is known to the person skilled in the art.
The Synthesis route of dyes CH3TO-1 (4) and CH3TO-3 (6), starting from benzothiazole 37, is depicted in
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 CH2Cl2: 0%→0.2%→0.5%→0.7%) to yield the desired product 4 (156 mg, 350 μmol, 87%) as red solid. Rf=0.39 (CH2Cl2/MeOH 10:1+1 drop formic acid). 1H-NMR (500 MHz, DMSO-d6, δ): 8.76 (d, 1H, 3J=8.3 Hz, H-16), 8.64 (d, 1H, 3J=7.1 Hz, H-21), 8.10 (d, 1H, 3J=8.6 Hz, H-18), 8.01 (d, 1H, 3J=7.8 Hz, H-3), 7.96 (t, 1H, 3J=7.9 Hz, H-20), 7.75-7.70 (m, 2H, H-19, H-6), 7.56 (t, 1H, 3J=7.5 Hz, H-1), 7.37 (t, 1H, 3J=7.5 Hz, H-2), 7.29 (d, 1H, 3J=7.1 Hz, H-17), 6.85 (s, 1H, H-10), 4.61 (q, 2H, 3J=7.2 Hz, H-22), 3.97 (s, 3H, H-11), 1.46 (t, 3H, 3J=7.2 Hz, H-23) ppm. 13C-NMR (75 MHz, DMSO-d6, 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+ (m/z): [M]+ calcd. for C20H19N2S+: 319.1263, found: 319.1259.
Benzothiazolium 37 (147 mg, 505 μmol, 1.26 eq) and quinilinium 38 (161 mg, 400 μmol, 1.00 eq) are dissolved in CH2Cl2/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 CH2Cl2: 0%→0.2%→0.5%→0.7%) to yield the desired product 6 (178 mg, 377 μmol, 75%) as dark blue solid. Rf=0.31 (CH2Cl2/MeOH 10:1+1 drop formic acid). 1H-NMR (500 MHz, DMSO-d6, δ): 8.44 (d, 1H, 3J=8.6 Hz, H-23), 8.42 (d, 1H, 3J=7.2 Hz, H-17), 8.11 (t, 1H, 3J=12.8 Hz, H-11), 8.06 (d, 1H, 3J=8.8 Hz, H-20), 7.94 (t, 1H, 3J=7.5 Hz, H-22), 7.84 (t, 2H, 3J=7.0 Hz, H-18, H-3), 7.69 (t, 1H, 3J=7.7 Hz, H-21), 7.54 (d, 1H, 3J=8.2 Hz, H-6), 7.44 (t, 1H, 3J=7.3 Hz, H-2), 7.27 (t, 1H, 3J=7.5 Hz, H-1), 7.08 (d, 1H, 3J=13.3 Hz, H-12), 6.47 (d, 1H3J=12.3 Hz, H-10), 4.58 (q, 2H, 3J=7.0 Hz, H-24), 3.71 (s, 3H, H-19), 1.44 (t, 3H, 3J=7.0 Hz, H-25) ppm. 13C-NMR (75 MHz, DMSO-d6, 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+ (m/z): [M]+ calcd. for C22H21N2S+: 345.1420, found: 345.1417.
This synthesis is based on the scaffold of cyanine dyes described within the state of the art (cf.
Benzothiazolium (30, 1.00 g, 2.05 mmol) and dimethyl aminobenzaldehyd (306 mg, 2.05 mmol) are dissolved in Ac2O (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. Rf=0.42 (CH2Cl2/MeOH 10:1). 1H NMR (300 MHz, DMSO-d6, 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, CH2-5), 3.25 (t, 2H, J=6.8 Hz, CH2-10), 3.12 (s, 6H, CH3-15, CH3-15′), 1.88-1.78 (m, 2H, CH2-6), 1.78-1.67 (m, 2H, CH2-9), 1.53-1.35 (m, 4H, CH2-7, CH2-8). 13C NMR (75 MHz, DMSO-d6, δ): 171.0 (Cq), 153.5 (Cq), 150.5 (CH), 141.0 (Cq), 132.9 (2C, CH), 128.9 (CH), 127.3 (CH), 127.0 (Cq), 123.9 (CH), 121.4 (Cq), 115.8 (CH), 111.9 (2C, CH), 105.6 (CH), 47.8 (CH2), 39.7 (2C, CH3), 32.6 (CH2), 29.4 (CH2), 28.1 (CH2), 24.6 (CH2), 8.6 (CH2). HRMS-ESI+ (m/z): [M]+ calcd for C23H28IN2S+, 491.1012; found, 491.1008.
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 CH2Cl2 and water. The organic layers are dried over MgSO4 and purified by flash column chromatography (allox, MeOH in CH2Cl2: 0.2%→0.5%→1.0%→5.0%→10%) to yield the desired product (34, 350 mg, 0.65 mmol, 80%) as purple solid. Rf=0.38 (CH2Cl2/MeOH 10:1). 1H NMR (300 MHz, DMSO-d6, 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, CH2-5), 3.31-3.24 (m, 2H, CH2-10), 3.12 (s, 6H, CH3-15, CH3-15′), 1.89-1.74 (m, 2H, CH2-6), 1.53-1.36 (m, 6H, CH2-7, CH2-8, CH2-9). 13C NMR (75 MHz, DMSO-d6, δ): 171.1 (Cq), 153.5 (Cq), 150.5 (CH), 141.1 (Cq), 132.9 (2C, CH), 128.9 (CH), 127.4 (CH), 127.0 (Cq), 123.9 (CH), 121.4 (Cq), 115.9 (CH), 111.9 (2C, CH), 105.7 (CH), 50.5 (CH2), 47.8 (CH2), 39.7 (2C, CH3), 28.2 (CH2), 28.0 (CH2), 25.7 (CH2), 25.2 (CH2). HRMS-ESI+ (m/z): [M]+ calcd for C23H28N5S+, 406.2060; found, 406.2059.
(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. Rt=19.7 min (gradient 1); 1H-NMR (500 MHz, DMSO-d6) δ=8.30 (dd, 3JH—H=8.1 Hz, 4JH—H=0.8 Hz, 1H, H3), 8.28 (s, 2H, H33), 8.10 (m, 3H, H1, H2, H3), 7.92 (d, 3JH—H=9.0 Hz, 2H, H17, H21), 7.78 (m, 1H, H20), 7.68 (t, 3JH—H=7.7 Hz, 1H, H18), 7.60 (d, 3JH—H=15.2 Hz, H6), 6.85 (d, 3JH—H=9.0 Hz, 2H, H22, H23), 4.79 (t, 3JH—H=7.4 Hz, 2H, H10), 4.37 (t, 3JH—H=7.1 Hz, 2H, H15), 4.10 (q, 3JH—H=5.6 Hz, 2H, H32), 3.12 (s, 6H, H25, H26), 1.78 (dt, 3JH—H=14.6 Hz, 3JH—H=7.2 Hz, 4H, H11, H14), 1.46 (m, 2H, H12), 1.30 (m, 2H, H13) ppm; 13C-NMR (125.76 MHz, DMSO-d6) 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+) m/z: calculated for C26H33N6S: 461.2500; found: 461.2481.
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 PPh3 (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. Rf=0.22 (DCM/MeOH 10:1); 1H-NMR (500 MHz, DMSO-d6) δ=8.31 (d, 3JH—H=8.1 Hz, 1H, H3), 8.09 (t, 3JH—H=11.6 Hz, 2H, H18, H22), 7.91 (d, 3JH—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, 3JH—H=3.9 Hz, 1H, H2), 7.68 (m, 1H, H1), 7.58 (d, 3JH—H=15.2 Hz, 1H, H6), 6.81 (d, 3JH—H=9.1 Hz, 2H, H23, H24), 4.76 (t, 3JH—H=7.4 Hz, 2H, H10), 3.54 (t, 3JH—H=11.3 Hz, 2H, H15), 3.11 (s, 6H, H26, H27), 1.74 (m, 2H, H11), 1.47 (m, 6H, H12-14) ppm; 13C-NMR (126 MHz, DMSO-d6) 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+) m/z: calculated for C41H42N2PS: 626.2900; found: 625.2806.
Yellow 6-AzTO-2 (40): Under exclusion of light and N2-atmosphere 1.13 g of 39 (1.82 mmol, 1.00 eq.) and 1.18 g NaN3 (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. NaN3 (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 NaCO3 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 MgSO4, and concentrated to dryness under reduced pressure. The crude product was purified by flash column chromatography (gradient of MeOH in DCM: 0%→0.2%→0.5%→1%→5%→10%). 0.8 g of 40 (14.9 mmol, 82%) were obtained as dark red solid. RF=0.64 (DCM/MeOH 10:1): 1H-NMR (500 MHz, DMSO-d6) δ=8.31 (d, 3JH—H=8.0 Hz, 1H, H3), 8.14 (d, =8.4 Hz, 1H, H22), 8.09 (d. 3JH—H=15.2 Hz, 1H, H18), 7.93 (d, 3JH—H=8.8 Hz, 2H, H19, H21), 7.78 (t, 3JH—H=7.7 Hz, 1H, H2), 7.68 (d, 3JH—H=7.6 Hz, 1H, H1), 7.61 (d, 3JH—H=15.2 Hz, 1H, H6), 6.85 (d, =8.8 Hz, 2H, H2.3, H24), 4.80 (t, 3JH—H=7.3 Hz, 2H, H10), 3.29 (t, 3JH—H=6.9 Hz, 2H, H15), 3.12 (s, 6H, H26, H27), 1.81 (m, 4H, H11), 1.45 (m, 4H, H12-14) ppm; 13C-NMR (500 MHz, DMSO-d6) 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+) m/z: calculated for C23H28N5S: 406.21007, found: 406.2061.
Tris(benzyltriazolmethyl)amine (TBTA, 54): 7.5 mL MeCN and 13.1 mg CuOAc2. H2O (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/NH4OH (2:1). The aqueous layer was extracted with DCM (2×20 mL) and the combined organic layers was washed with NH4OH/brine 1:1 (2×20 mL) and dried over MgSO4. 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. RF=0.60 (DCM/MeOH 10:1); 1H-NMR (300 MHz, CDCl3) δ=7.47 (s, 3H, H4), 7.08 (ddd, 15H, 3JH—H=8.4 Hz. 3JH—H=5.9 Hz, 4JH—H=2.0 Hz. X10-14), 5.28 (s, 6H, H8), 3.51 (s, SH, H2) ppm; 13C-NMR (75.48 MHz, CDCl3) 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.
Yellow 6-TramTO (42); Under exclusion of light and stirring 6.9 mg CuSO4.5 H2O (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 H2O (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. RF=0.60 (DCM/MeOH 10:1); 1H-NMR (500 MHz, DMSO-d6) δ=8.30 (dd, =8.1 Hz. 4JH—H=0.8 Hz, 1H, H3), 8.28 (s, 2H, H33), 8.10 (m, 3H, H1, H2, H3), 7.92 (d, 3JH—H=9.0 Hz, 2H, H17, H21), 7.78 (m, 1H, H20), 7.68 (t, 3JH—H=7.7 Hz, 1H, H18), 7.60 (d, 3JH—H=15.2 Hz, H6), 6.85 (d, 3JH—H=9.0 Hz, 2H, H22, H23), 4.79 (t, 3JH—H=7.4 Hz, 2H, H10), 4.37 (t, 3JH—H=7.1 Hz, 2H, H15) 4.10 (q, 3JH—H=5.6 Hz, 2H, H32), 3.12 (s, 6H, H25, H26), 1.78 (dt, 3JH—H=14.6 Hz, 3JH—H=7.2 Hz, 4H, H11, H14), 1.46 (m, 2H, H12), 1.30 (m, 2H, H13) ppm, 13C-NMR (125.76 MHz, DMSO-d6) 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+) m/z: calculated for C26H33N6S: 461.2500: found: 461.2481.
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.
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 CH2Cl2 (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/Ac2O/DMF (6:5:89, 500 μL) to the resin and shaking for 5 min. After washing with DMF (5×), CH2Cl2 (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×), CH2Cl2 (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×), CH2Cl2 (5×), DMF (5×) and capped by adding 2,6-lutidine/Ac2O/DMF (6:5:89, 500 μL) to the resin and shaking for 5 min. After washing with DMF (5×), CH2Cl2 (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×), CH2Cl2 (5×), DMF (5×) and capped as described above. After washing with DMF (5×), CH2Cl2 (5×), DMF (5×), the peptide is cleaved from the resin by adding 1.5 mL SH-cleavage mix (2.5% DODT, 2.5% H2O, 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 Et2O (˜25 mL). After centrifugation, the precipitate is washed with cold Et2O (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. Rt=19.1 min (gradient 1). HRMS (ESI): m/z calc. for C35H64N11O7S [M+H]+ 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.
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. Rt=23.4 min (gradient 1). HRMS (ESI): m/z calc. for C62H94N13O7S23+ [M+2H]30 398.89, found 399.42.
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. Rt=22.3 min (gradient 1). HRMS (ESI): m/z calc. for C60H90NDO7S2+[M]+1168.65, found 1168.69.
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. Rt=16.9 min (gradient 1). HRMS (ESI): m/z calc. for C58H90N13O7S2+ [M]+ 1144.65, found 1144.69.
Besides abbreviations, well known to any person of ordinary skill in the art, following table 3 lists some further abbreviations used herein.
The parameter settings regarding excitation/emission (as far as they are applied) are equivalent to those described within the legend of
| Number | Date | Country | Kind |
|---|---|---|---|
| 21192451.9 | Aug 2021 | EP | regional |
