Dye with pH dependent absorption and fluorescence

Abstract

The invention relates to a dye for pH detection and having pH-dependent absorption and fluorescence, and application thereof in biology, medicine and diagnosis and treatment. In particular, the present invention relates to: compounds as shown in general formulae (I) to (IV), said compounds exhibiting pH-dependent absorption and fluorescence spectra as well as pH-dependent fluorescence quantum yields; a method for preparing the compound; and the use of said compounds as pH sensors and marker groups in the detection of analytes.

Description

TECHNICAL FIELD

[0001] The present application relates to dyes for pH detection with pH dependent absorption and fluorescence and their use in biological, medical and diagnostic applications.

[0002] In particular, the present application relates to compounds having general formula (I) to (IV) which show pH dependent absorption and fluorescence spectra and pH dependent fluorescence quantum yield; to a process for the preparation of said compounds; and to the use of said compounds as pH sensors and labelling groups in the detection of analytes. BACKGROUND

[0003] pH plays an important role in many areas of daily life, for example in food shelf life or quality, skin protection barriers and cleaning agents. Traditionally, pH is measured electrochemically using pH electrodes or optically, i.e. usually by a color change of classical indicators (e.g. litmus, phenolphthalein, etc.).

[0004] However, pH can also be determined using fluorescence. For example, fluorescent dyes can be used whose fluorescence intensity or fluorescence wavelength changes with pH. Optical measurements by fluorescence are often even more accurate because the measurement of emitted light is more sensitive than the measurement of absorption. This is particularly important when measuring biological samples, where lower concentrations or smaller amounts of pH sensors are required, or early responses are needed.

[0005] Therefore, pH can also be used to monitor the wound healing process, as detailed in the following literature: a) S.L. Percival, S. McCarty, J.A. Hunt, E.J. Woods: The effects of pH on wound healing, biofilms, and antimicrobial efficacy. Wound Rep. Reg. 22, 174 (2014); b) C. Waters, T. Yuan, K. Rumbaugh: Beneficial and deleterious bacterial-host interactions in chronic wounds pathophysiology. Chronic Wound Care Management and Research, 2, 52 (2015); or c) Z. Han, M. Yuan, L. Liu, K. Zhang, B. Zhao, B. He, Y. Liang, F. Li: pH-Responsive wound dressings; advances and prospects, Nanoscale Horizons, 8, 422 (2023).

[0006] When an acute wound occurs, the internal tissues and tissue fluid with a pH of approximately 7.4 are exposed, altering the acidic environment of normal skin. The pH will return to acidic during wound healing. Typically, the wound's pH decreases as a result during the healing process.

[0007] If the pH rises again later and remains at a high level, this strongly indicates that the wound has become infected or chronic. Chronic wounds have a pH between 7 and 9 due to the presence of substances such as blood, tissue fluid, and ammonia. Most pathogens raise the pH due to metabolites produced by bacterial growth. However, some pathogens cause the wound's pH to decrease due to their acidic metabolites, such as lactic acid and carbonic acid.

[0008] Therefore, pH is considered a reliable indicator of wound condition, and changes in pH can predict the likelihood of wound healing, infection, and deterioration.

[0009] Hospital-acquired infections (also known as "nosocomial infections") and chronic infections in immunocompromised, immunosuppressed, or diabetic patients constitute a major global challenge. These complications lead to higher mortality rates and impose significant costs on healthcare systems. Early diagnosis of pathogenic infections and differentiation from harmless subclinical bacterial colonization offers numerous advantages, such as reduced prophylactic antibiotic use, thereby lowering the risk of antibiotic resistance development, avoiding unnecessary dressing changes associated with wound reinfection, and generally reducing healthcare system costs.

[0010] Therefore, new strategies are urgently needed to detect and treat such wound infections. Standard bacterial cultures and tests are slow (up to 48 hours) and require laboratory testing far from the patient. In contrast, pH-dependent point-of-care testing methods can enable rapid diagnosis by displaying real-time pH changes within the wound dressing—and may even trigger the release of drugs or antibiotics. A simple method for detecting infection in wound dressings via pH monitoring is described in G. Panzarasa, A. Osypova, C. Toncelli, M. T. Buhmann, M. Rottmar, Q. Ren et al.: Thepyranine-benzalkonium ion pair. A promising fluorescent system for theratiometric detection of wound pH. Sensors and Actuators B: Chemical, 249, 156–160 (2017).

[0011] In summary, continuous and long-term pH monitoring is crucial for chronic wounds because the pH level is constantly changing during the healing process.

[0012] The so-called SNARF (seminaphthorhodafluorescein) dye was developed as a pH indicator for measuring intracellular pH; see US4945171A or O. Seksek, N. Henry-Toulme, F. Sureau, J. Bolard: SNARF-1 as an intracellular pH indicator in laser microspectrofluorometry. A critical assessment. Analytical Biochemistry, 1991, 193(1), 49–54 (1991). Compared to previously used fluorescein derivatives, these dyes are characterized by greater photostability and pH-dependent band shapes or wavelengths in their fluorescence spectra. This allows for more sensitive ratioometric measurements using fluorescence spectroscopy, thereby determining intracellular pH. For example, both acidic and basic forms of the dye can be excited simultaneously at a specific wavelength, and the ratio of the two fluorescence intensities is correlated with the pH of the medium. The pK of SNARF dyes... a The value is in the range of 7.5 to 7.9, which is too high to be used cost-effectively for detecting wound infections. Summary of the Invention

[0013] Therefore, one object of the present invention is to provide suitable fluorescent dyes that can be used as pH sensors and / or as labeling groups for pH detection in analyte detection steps. In particular, the fluorescent dyes have an absorption maxima, which allows absorption outside the absorption range of naturally occurring substances contained in biological samples using inexpensive light sources, exhibiting good solubility, high photostability, and / or characterized by high fluorescence quantum yield and / or high turn-on ratio, thereby at least partially overcoming the deficiencies of the prior art. In particular, one object of the present invention is to provide small organic dye molecules suitable for colorimetric or ratiometric measurements based on differences in absorption and fluorescence wavelengths and / or fluorescence quantum yields in acidic and basic forms, and having a pK value in the range of 4 to 7. a .

[0014] This objective is achieved through compounds of general formulas (I) to (IV) and their use as sensor dyes or labeling groups in the step of detecting analytes.

[0015] This invention relates to a dye having pH-dependent absorption and / or pH-dependent fluorescence, having general formulas (I), (II), (III), or (IV). in X represents CR 11 R 12 SiR 13 R 14 Sulfur, SO2 or P(O)OR 15 ; Y represents a hydrogen, nitrile, carboxyl, carboxylic acid derivative, amino, or a straight-chain, branched, or cyclic saturated or unsaturated hydrocarbon moiety having up to 40 carbon atoms. The hydrocarbon moiety may contain or be substituted with one or more heteroatoms selected from N, O, and S, and / or preferably one or more substituents selected from the following: halogen, OH, (O)alkyl, O(aryl), SH, S(alkyl), S(aryl), NH2, NH(aryl), NH(alkyl), N(aryl)2, N(alkyl)2, NO2, CHO, COOH, COO(alkyl), COO(aryl), PO3H2, SO3H, or CONR. 31 R 32 ; R1, R3, R4, R5, R6, R7, R8, R9, R 10 Independently representing a hydrogen, halogen, hydroxyl, mercapto, amino, sulfonyl, phosphoro, nitro, carbonyl, carboxyl, carboxylic acid derivative, nitrile, isonitrile, cyanate, thiocyanate, isothiocyanate, or a straight-chain, branched, or cyclic saturated or unsaturated hydrocarbon moiety having up to 20 carbon atoms, said hydrocarbon moiety may contain one or more heteroatoms selected from N, O, and S or be substituted by one or more heteroatoms selected from N, O, and S, and / or preferably one or more substituents selected from the following: halogen, OH, (O)alkyl, O(aryl), SH, S(alkyl), S(aryl), NH2, NH(aryl), NH(alkyl), N(aryl)2, N(alkyl)2, NO2, CHO, COOH, COO(alkyl), COO(aryl), PO3H2, SO3H, or CONR 31 R 32 ; R 11 R 12 R 13 R 14 R 15 R 31 R 32Independently representing hydrogen or a hydrocarbon group having 1 to 20 carbon atoms, wherein the hydrocarbon group optionally includes one or more heteroatoms selected from N, O, and S, and / or preferably one or more substituents selected from: halogen, nitrile, hydroxyl, mercapto, amino, nitro, sulfonyl, phosphoro, carbonyl, carboxyl, carboxylic acid derivatives (e.g., carboxylates, esters, halides, amides, acid anhydrides); or R 11 and R 12 Or R 13 and R 14 The atoms bonded to them together form 3- to 7-membered rings, wherein the rings may include one or more double bonds, and / or one or more heteroatoms selected from N, O, and S, and / or one or more substituents selected from the following: halogen, OH, (O)alkyl, O(aryl), SH, S(alkyl), S(aryl), NH2, NH(aryl), NH(alkyl), N(aryl)2, N(alkyl)2, NO2, CHO, COOH, COO(alkyl), COO(aryl), PO3H2, SO3H, CONR 31 R 32 and a hydrocarbon group having 1 to 20 carbon atoms, and / or may be fused with one or more 3- to 7-membered rings, wherein the hydrocarbon group optionally includes one or more heteroatoms selected from N, O, and S and / or one or more substituents selected from the following: halogen, OH, (O)alkyl, O(aryl), SH, S(alkyl), S(aryl), NH2, NH(aryl), NH(alkyl), N(aryl)2, N(alkyl)2, NO2, CHO, COOH, COO(alkyl), COO(aryl), PO3H2, SO3H, or CONR 31 R 32 ; R2 represents NR 16 R 17 Or R1 and R2 together Where R 18 R 19 and R 22 Limited to R1, and R 16 R 17 R 20 R 21 Limited to R 11 ; Or R1 to R 22 At least one of them forms a ring system with one or more adjacent parts.

[0016] Preferred embodiments of the dyes of the present invention having pH-dependent absorption and / or pH-dependent fluorescence are those embodiments given herein with respect to the compounds described herein.

[0017] The dyes described in this invention have pH-dependent absorption and / or emission properties.

[0018] In particular, the dyes described in this invention have pH-related absorption properties.

[0019] Preferably, the dye of the present invention exhibits a color change in absorption as pH changes.

[0020] The wavelength shift associated with the color change is at least higher than 50 nm, preferably higher than 75 nm, which allows for good differentiation of the color change. This color change occurs between acidic and basic forms, and is therefore related to the pK of the dye. a Related.

[0021] Preferably, the pK of the dye of the present invention a More preferably, pK is preferred within the pH range of 3 to 8. a Within the pH range of 4 to 7, and most preferably pK a Within the pH range of 5.5 to 6.5. pK within this range a The dyes described in this invention are particularly useful for physiological measurements, and most preferably for detecting wound infections.

[0022] In particular, the dyes described in this invention exhibit a maximum absorption peak in the wavelength range of 500 nm to 800 nm, especially 600 nm to 700 nm. Therefore, inexpensive light sources, such as diode lasers, can be used to excite the compounds and / or dyes. Furthermore, this maximum absorption peak allows for excitation outside the absorption range of naturally occurring substances contained in biological samples. Due to the low background emission from those biological substances, this enables more sensitive detection of emission or fluorescence.

[0023] The dyes of the present invention also preferably exhibit pH-dependent emission. In a preferred embodiment, the dyes of the present invention exhibit emission of different characteristic wavelengths according to changes in the pH of the surrounding medium, which promotes the acidic or basic form of the dye. Preferably, the emission wavelength shift between the acidic and basic forms is at least greater than 5 nm, more preferably greater than 10 nm.

[0024] The compounds and dyes described in this invention, in their basic form, preferably exhibit fluorescence quantum yields of at least 2%, more preferably at least 10%, and even more preferably at least 30%, respectively.

[0025] Furthermore, the dye of the present invention preferably exhibits a change in emission or fluorescence intensity according to changes in the pH of the surrounding medium. Therefore, the dye of the present invention can be particularly used as a fluorescence-generating or ratiometric sensor. Specifically, the dye of the present invention exhibits a high on-state ratio of at least 2, meaning that the fluorescence in the alkaline form is at least twice as high as the fluorescence in the acidic form. More preferably, the on-state ratio is at least 10, and even more preferably at least 25 and at most 50.

[0026] Preferably, the Stokes shift of the dye of the present invention, that is, the wavelength difference between the absorption peak and the fluorescence peak, is at least 30 nm, more preferably at least 50 nm, most preferably at least 60 nm, and at most 130 nm, preferably at most 120 nm, and most preferably at most 110 nm.

[0027] The dyes described in this invention also exhibit high photostability, comparable to that of the most photostable organic dyes and chromophores derived from the xaton system. Commercially available examples of such dyes include, for instance, Rhodamine 6G, ATTO 647N, and ATTO 643. They are all well-known for their exceptionally high photostability.

[0028] The compounds and dyes described in this invention also exhibit different lyotropic properties, meaning their absorption and fluorescence spectra are affected by the solvent or the surrounding medium. This photophysical property can be used to study and interpret solvent parameters such as polarity, H-bond donor or acceptor characteristics, ionic strength, and dielectric constant.

[0029] Due to the color change in absorption associated with pH, ​​the highly photostable dyes described in this invention can be used as conventional indicator dyes. Furthermore, the dyes exhibit emission at different characteristic wavelengths depending on the pH change of the surrounding medium, thus allowing them to be used as fluorescence-generating or ratiometric sensors. Additionally, due to their varying lyochromic properties, the dyes can also be used as sensors for solvent polarity.

[0030] Therefore, the dye is suitable as a pH sensor for a variety of applications, especially for biological or medical purposes. pK a Dyes close to pH 6 can be used, for example, as sensors for bacterial growth within smart wound dressings to determine chronic wound infections or bacterial biofilms.

[0031] The present invention also relates to a compound having the general formula (I), (II), (III) or (IV). in X represents CR 11 R 12 SiR13 R 14 Sulfur, SO2 or P(O)OR 15 , Y represents a hydrogen, nitrile, carboxyl, carboxylic acid derivative (e.g., carboxylates, esters, halides, amides, anhydrides), amino, or a straight-chain, branched, or cyclic saturated or unsaturated hydrocarbon moiety (e.g., alkyl, cycloalkyl, aryl, heteroaryl) having up to 40 carbon atoms. The hydrocarbon moiety may contain one or more heteroatoms selected from N, O, and S, or be substituted by one or more heteroatoms selected from N, O, and S, and / or preferably one or more substituents selected from: halogen, OH, (O)alkyl, O(aryl), SH, S(alkyl), S(aryl), NH2, NH(aryl), NH(alkyl), N(aryl)2, N(alkyl)2, NO2, CHO, COOH, COO(alkyl), COO(aryl), PO3H2, SO3H, or CONR. 31 R 32 ; R1, R3, R4, R5, R6, R7, R8, R9, R 10 Each of these groups independently represents hydrogen, halogen, hydroxyl, mercapto, amino, sulfonyl, phospho group, nitro, carbonyl, carboxyl, carboxylic acid derivatives (e.g., carboxylates, esters, halides, amides, acid anhydrides), nitrile group, isonitrile group, cyanate group, thiocyanate group, and isothiocyanate group. A group), or a straight-chain, branched, or cyclic saturated or unsaturated hydrocarbon moiety (e.g., alkyl, cycloalkyl, aryl, heteroaryl) having up to 20 carbon atoms, wherein the hydrocarbon moiety may contain one or more heteroatoms selected from N, O, and S or be substituted by one or more heteroatoms selected from N, O, and S, and / or preferably one or more substituents selected from the following: halogen, OH, (O)alkyl, O(aryl), SH, S(alkyl), S(aryl), NH2, NH(aryl), NH(alkyl), N(aryl)2, N(alkyl)2, NO2, CHO, COOH, COO(alkyl), COO(aryl), PO3H2, SO3H, or CONR 31 R 32 ; R 11 R 12 R 13 R 14 R 15 R 31 R 32Independently representing hydrogen or a hydrocarbon group having 1 to 20 carbon atoms, wherein the hydrocarbon group optionally includes one or more heteroatoms selected from N, O, and S, and / or preferably one or more substituents selected from: halogen, nitrile, hydroxyl, mercapto, amino, nitro, sulfonyl, phosphoro, carbonyl, carboxyl, carboxylic acid derivatives (e.g., carboxylates, esters, halides, amides, acid anhydrides); or R 11 and R 12 Or R 13 and R 14 The atoms bonded to them together form 3- to 7-membered rings, wherein the rings may include one or more double bonds and / or one or more heteroatoms selected from N, O, and S and / or one or more substituents selected from the following: halogen, OH, (O)alkyl, O(aryl), SH, S(alkyl), S(aryl), NH2, NH(aryl), NH(alkyl), N(aryl)2, N(alkyl)2, NO2, CHO, COOH, COO(alkyl), COO(aryl), PO3H2, SO3H, CONR 31 R 32 and a hydrocarbon group having 1 to 20 carbon atoms, and / or fused with one or more 3- to 7-membered rings, wherein the hydrocarbon group optionally includes one or more substituent heteroatoms selected from N, O, and S and / or one or more substituents selected from the following: halogen, OH, (O)alkyl, O(aryl), SH, S(alkyl), S(aryl), NH2, NH(aryl), NH(alkyl), N(aryl)2, N(alkyl)2, NO2, CHO, COOH, COO(alkyl), COO(aryl), PO3H2, SO3H, or CONR 31 R 32 ; R2 represents NR 16 R 17 Or R1 and R2 together Where R 18 R 19 and R 22 Limited to R1, and R 16 R 17 R 20 R 21 Limited to R 11 , Or R1 to R 22 At least one of them forms a ring system with one or more adjacent parts, but must meet the following conditions: In the compounds of formula (I), the following terms do not appear simultaneously: X represents C(CH3)2; R1, R3, R4, R5, R6, and R7 represent H. R2 represents N(CH3)2; and Where R 33 Indicates H or CH3; and In the compounds of formula (I), the following terms do not appear simultaneously: X represents C(CH3)2; R1, R3, R4, R5, R6, and R7 represent H. R2 represents N(CH3)2; and as well as In the compounds of formula (I), the following terms do not appear simultaneously: X represents C(CH3)2; R1, R3, R4, R5, R6, and R7 represent H. R2 represents N(CH3)2; and Where R 33 Indicates H or CH3; and In the compound of formula (I), the following terms do not appear simultaneously: X represents Si(CH3)2; R1, R3, R4, R5, R6, and R7 represent H. R2 represents NH2, NH(CH3), N(CH3)2, or NH-C(O)CH3; and Y represents 1-methyl-phenyl. And where X represents CR 11 R 12 Compounds of formula (III) include at least one sulfonyl group.

[0032] As used within the scope of this application, the term "hydrocarbon group" includes a saturated or unsaturated hydrocarbon moiety having 1 to 40 carbon atoms, preferably 1 to 20 carbon atoms, more preferably 1 to 12 carbon atoms, which may be linear, branched, or cyclic, and having a bond valence on any of the carbon atoms. Examples of hydrocarbon groups according to the invention include alkyl, cycloalkyl, alkenyl, or alkynyl groups and aryl groups.

[0033] The hydrocarbon group may include one or more heteroatoms selected from N, O, and S. Therefore, the hydrocarbon group may also be a heteroalkyl, heterocycloalkyl, or heteroaryl group. The hydrocarbon group may include one or more substituents, preferably selected from halogens, OH, (O)alkyl, O(aryl), SH, S(alkyl), S(aryl), NH2, NH(aryl), NH(alkyl), N(aryl)2, N(alkyl)2, NO2, CHO, COOH, COO(alkyl), COO(aryl), PO3H2, SO3H, and CON(alkyl)2.

[0034] As used within the scope of this application, the term "aliphatic hydrocarbon group" has the same meaning as hydrocarbon group, except for aryl or heteroaryl.

[0035] As used within the scope of this application, the term "alkyl" refers to a saturated, linear, or branched hydrocarbon moiety having 1 to 40 carbon atoms, preferably 1 to 20 carbon atoms, more preferably 1 to 12 carbon atoms, having a bond valence on any of said carbon atoms. Preferably, the alkyl group constitutes a hydrocarbon moiety having 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms. Particularly preferred alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

[0036] If the hydrocarbon portion includes one or more heteroatoms selected from N, O, and S, the portion may be a heteroalkyl group, and therefore particularly includes alkoxy groups such as methoxy, ethoxy, etc.

[0037] As used within the scope of this application, the term "cycloalkyl" refers to a saturated or unsaturated cyclic hydrocarbon moiety having 3 to 20 carbon atoms, wherein the cyclic hydrocarbon moiety has a bond valence on any of the 3 to 20 carbon atoms. Preferably, the cycloalkyl moiety constitutes a cyclic hydrocarbon moiety having 3 to 12 carbon atoms, more preferably having 3 to 8 carbon atoms. Particularly preferred cycloalkyl moieties include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In addition, the cyclic hydrocarbon moieties may be interconnected to form bicyclic or polycyclic compounds, such as norbornane, norbornene, bicyclic [2.2.2]octyl, bicyclic [3.2.2]nonyl, and adamantyl.

[0038] If the cyclic hydrocarbon portion includes one or more heteroatoms selected from N, O, and S, the portion may also be a heterocyclic alkyl group, and thus include aliphatic heterocycles such as tetrahydropyrrole, piperidine, dioxane, or tetrahydrofuran.

[0039] As used within the scope of this application, the term "alkenyl" refers to an unsaturated, linear, or branched hydrocarbon moiety having 2 to 20 carbon atoms, having a bond valence and at least one double bond on any of the 2 to 20 carbon atoms. Preferably, the alkenyl constitutes a hydrocarbon moiety having 2 to 12 carbon atoms, more preferably 2 to 6 carbon atoms. Particularly preferred alkenyl groups include vinyl, propenyl, and butenyl groups.

[0040] As used within the scope of this application, the term "alkynyl" refers to an unsaturated, linear, or branched hydrocarbon moiety having 2 to 20 carbon atoms, having a bond valence and at least one triple bond on any of the 2 to 20 carbon atoms. Preferably, the alkynyl group constitutes a hydrocarbon moiety having 2 to 12 carbon atoms, more preferably 2 to 6 carbon atoms. Particularly preferred alkynyl groups include ethynyl, propynyl, and butynyl.

[0041] As used within the scope of this application, the term "aryl" refers to an aromatic ring system having 3 to 20 ring atoms, more preferably 6 to 14 ring atoms, containing only carbon atoms, and having a bond valence on any of the carbon atoms in the 3 to 20 ring-forming atoms. Preferred aryl groups include phenyl, naphthyl, anthraceneyl, phenanthryl, and pyreneyl.

[0042] As used within the scope of this application, the term "heteroaryl" refers to an aromatic ring system having 3 to 20 ring atoms, more preferably 5 to 14 ring atoms, wherein the ring atoms, in addition to carbon atoms, include at least one heteroatom selected from N, O, and S, and have a bond valence on either a carbon atom or a nitrogen atom among the 3 to 20 ring-forming atoms. Preferred heteroaryl groups include furanyl, imidazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrazolyl, pyrroleyl, tetrazolyl, thiadiazolyl, thiazolyl, thiophenyl, triazolyl, pyrazinyl, pyridinyl, pyrimidinyl, and triazinyl.

[0043] As used within the scope of this application, the term "halogen" includes fluorine, chlorine, bromine, and iodine, particularly fluorine and chlorine.

[0044] The term "sulfo group" preferably refers to a sulfonic acid group or a sulfuric acid group. Preferably, the term sulfo group refers to an SO3H group.

[0045] The term "phosphorus group" preferably refers to a phosphonic acid group or a phosphate group.

[0046] According to the present invention, preferably, In the compound of formula (I), the following terms do not appear simultaneously: X represents C(CH3)2; R1, R3, R4, R5, R6, and R7 represent H. R2 represents N(CH3)2; and Where R 33 Indicates H or CH3; and In the compound of formula (I), the following terms do not appear simultaneously: X represents C(CH3)2; R1, R3, R4, R5, R6, and R7 represent H. R2 represents N(CH3)2; and as well as In the compound of formula (I), the following terms do not appear simultaneously: X represents C(CH3)2; R1, R3, R4, R5, R6, and R7 represent H. R2 represents N(CH3)2; and Where R 33 It represents H or CH3.

[0047] More preferably, in the compound of formula (I), the following terms do not appear simultaneously: X represents Si(CH3)2; R1, R3, R4, R5, R6, and R7 represent H. R2 represents NH2, NH(CH3), N(CH3)2, or NH-C(O)CH3; and Y represents 1-methyl-phenyl.

[0048] In a preferred embodiment of the invention, R1, R3, R4, R5, R6 and R7 represent H.

[0049] In a further preferred embodiment, at least one of R1, R3, R4, R5, R6, and R7 represents a substituent containing a COOH group, an SO3H group, and / or a halogen (especially Cl or F). More preferably, R3 is a substituent containing a COOH group, an SO3H group (especially CH2SO3H), and / or a halogen (especially Cl or F).

[0050] R2 preferably represents NR 16 R 17 , where R 16 and R 17 It is a C1-C4 alkyl group, most preferably R 16 =R 17 All of these represent methyl groups. Compounds where R2 represents N(CH3)2 are preferred because they have a relatively simple structure and are easy to prepare.

[0051] More preferably, R2 represents NR 16 R 17 , where R 17 Represents C1-C4 alkyl, most preferably, R 17 It represents CH2CH3, and R 16 Together with R3, they form a ring system with the following formula:

[0052] More preferably, R2 represents NR 16 R 17 , where R 17 Represents C1-C4 alkyl, most preferably, R 17 It represents CH2CH3, as well as R3 and R 16 Together they form a ring system with the following formula:

[0053] In a further preferred embodiment, R2 represents NR 16 R 17 , where R 16 and R 17 At least one of them is a hydrocarbon group having 1 to 20 carbon atoms, which includes one or more heteroatoms selected from oxygen, particularly wherein R 16 and R 17 At least one of the components comprises a polyether, preferably polyethylene glycol, and / or a polypropylene glycol group having 2 to 20 carbon atoms. Such a group imparts good water solubility to the compound without the addition of a further negative charge.

[0054] In a further preferred embodiment, R2 represents NR 16 R 17 , where R 16 and R 17 At least one of them is a long-chain hydrocarbon group having at least 13 carbon atoms, more preferably at least 15 carbon atoms. Particularly preferred groups are C13 to C20 alkyl groups. Such groups endow the structure with high hydrophobicity, and these compounds can be intercalated into polymers, for example.

[0055] Each of the portions present in the compounds of formulas (I) to (IV) of this invention may optionally form a ring system having one or more adjacent portions. The ring system preferably comprises 5-membered and / or 6-membered rings. Preferably, the rings are composed of R1 and R2. 16 And / or R3 and R 17 The resulting ring system yields the following structures: (A), (B), (C), (D), (E), (F), and (G): In each case, R is independently defined as R1, and the dashed line is optionally a double bond, in the presence of which the portion bound by the dashed line does not exist.

[0056] In the structures (A) to (G), adjacent substituents R may optionally form additional ring systems, which may include 5-membered and / or 6-membered rings, and the 5-membered and / or 6-membered rings may optionally contain other heteroatoms or substituents.

[0057] In the same manner, as shown in structures (A) to (G), 5-membered and / or 6-membered rings can be formed by the portions R3 and R 17 and / or R1 and R 16 The resulting structure is also a preferred embodiment of the present invention.

[0058] Examples of this structure with other rings include, in particular:

[0059] Having R1 and R 16 And / or R3 and R 17 The resulting ring system structure, compared to a structure without such a ring system, exhibits a bathochromic shift in absorption and / or fluorescence of approximately 25 nm to 50 nm. This shift to longer wavelengths is advantageous for various applications because it allows the use of inexpensive light sources and improves detection sensitivity due to lower background fluorescence caused by the absence of naturally occurring fluorophores.

[0060] A further preferred embodiment is a structure having an annulated ring system. These are preferably formed by the ring formation of portions R3 and R4 and / or R5 and R6 and / or R6 and R8 and / or R5 and R6 and R8 as described in formulas (I) to (IV). Aromatic six-membered ring systems as shown in the following examples are preferred:

[0061] The position R in each case is independently defined as R1.

[0062] According to the present invention, compounds of formula (I), (II), (III) or (IV) have been found to have absorption and / or fluorescence at relatively long wavelengths, wherein in said compounds, R1 to R 21At least one group in the compound forms a ring system with one or more adjacent moieties, and thus the ring system preferably comprises a five-membered and / or six-membered ring. That is, these compounds exhibit a shift towards the visible spectrum, and particularly towards the red spectrum. Therefore, these compounds possess advantageous properties for a wide range of applications.

[0063] According to the present invention, it is further preferred that Y in formulas (I) and (II) is an optionally substituted cycloalkyl, optionally substituted phenyl, optionally substituted naphthyl, or optionally substituted heteroaryl, having the following structures: (H), (J), (K), (L), (M), (N), and (O). in E represents oxygen, sulfur, nitrogen, or... + NR 23 , R u Indicates halogen, OR 24 NHR 25 NR 26 R 27 , Z represents hydrogen or halogen. R v Represents Z and S(CH2). x COOH, S(CH2) x SO3H, NHR 28 NR 29 R 30 ,as well as R w Indicates hydrogen, alkyl, (CH2) x COOH, (CH2) x SO3H, (CH2) x OH, (CH2) x SH, (CH2) x NH2, (CH2) x Z, where x = 1, 2, 3, 4, 5, 6, R 23 R 24 R 25 R 26 R 27 R 28 R 29 R 30Independently represents another saturated or unsaturated hydrocarbon moiety having up to 20 carbon atoms, such as alkyl, cycloalkyl, aryl, and heteroaryl, said other hydrocarbon moiety may contain one or more heteroatoms selected from N, O, and S or be substituted by one or more heteroatoms selected from N, O, and S, and / or preferably one or more substituents selected from the following: halogen, OH, (O)alkyl, O(aryl), SH, S(alkyl), S(aryl), NH2, NH(aryl), NH(alkyl), N(aryl)2, N(alkyl)2, NO2, CHO, COOH, COO(alkyl), COO(aryl), PO3H2 and SO3H or CONR 31 R 32 And the position R in each case is independently defined as R1.

[0064] Particularly preferred is Y being aryl or heteroaryl, having one or more substituents comprising a COOH group, an SO3H group, and / or a halogen (especially Cl and / or F). More preferably, Y is aryl or heteroaryl, having one or more substituents selected from COOH, C(=O)-N(CH3)-CH2-CH2-CH2-COOH, SO3H, F, and / or Cl.

[0065] The COOH group is particularly suitable for coupling the dyes described in this invention, and is particularly suitable for coupling the dyes described in this invention to surfaces, supports or analytes.

[0066] The SO3H group is particularly useful for improving the water solubility of the compound and / or adding a negative charge to the compound, which, for example, can be advantageous in electrophoretic applications for distinguishing molecules with different charges without altering the optical properties of the molecules with different charges.

[0067] Preferred groups Y include

[0068] Particularly preferred are groups Y comprising ortho-substituted aryl or heteroaryl groups. It has been found that such substituents Y promote the chemical stability and color vibrancy of the compounds in a basic pH range.

[0069] Further preferred are compounds of formulas (I) and (II), wherein Where R 34 It represents OH or N(CH3)-(CH2). x -C(O)-OR 35 Where x = 1 - 6, and R 35 It represents H or CH3. A particularly preferred value is x = 3.

[0070] A particularly preferred residue Y is one that includes an imidazole group.

[0071] Other compounds having formula (I), (II), (III) or (IV) are also preferred, having at least one halogen substituent, particularly at least one F and / or Cl substituent.

[0072] According to the invention, compounds comprising at least one SO3H group are further preferred. Such compounds are particularly advantageous due to their good water solubility.

[0073] Further preferred are compounds of formulas (I), (II), (III), or (IV) that include at least one carboxyl group (-COOH). Such carboxyl groups can be particularly used as linking groups for covalently attaching the compounds of the present invention to target molecules.

[0074] In a preferred embodiment of the present invention, X in formulas (I) to (IV) represents CR. 11 R 12 or SiR 13 R 14 Even more preferred is the corresponding structure, where R 11 =R 12 =R 13 =R 14 All are methyl, ethyl, propyl, or R 11 With R 12 and / or R 13 With R 14 Together they form quaternary, pentagonal, or hexacyclic hydrocarbon ring systems.

[0075] In a further preferred embodiment, R 11 =R 12 =R 13 =R 14 Both represent phenyl groups, thus yielding a hydrophobic structure.

[0076] In a preferred embodiment of the invention, the compound has general formula (I) or formula (II), more preferably formula (II).

[0077] In such compounds of formula (I) or (II), X is preferably CR. 11 R 12 or SiR 13 R 14 .

[0078] In the preferred embodiment, X represents CR 11 R 12 Such compounds are also known as carborhodol.

[0079] According to this preferred embodiment, R 11 and R 12 Each is preferably a C1-C4 alkyl group, and most preferably, R 11 =R 12 Both represent CH3.

[0080] In this preferred embodiment, Y is preferably a phenyl or imidazole group, said imidazole group being substituted with one or more substituents selected from the following: halogen (especially Cl and / or F), C(O)OH, NH-CH2-CH2-SO3H, S-CH2-CH2-SO3H, C(O)-N(CH3)-CH2-CH2-CH2-C(O)OH, or C(O)-(1-piperidine)-4-carboxyl, or C(O)-(1-pyrrolidinyl)-2-carboxyl. Furthermore, a Y substituent comprising at least one SO3H group is particularly preferred.

[0081] In a preferred embodiment of this embodiment, R1, R3, R4, R5, R6, and R7 represent H. In a further preferred embodiment, at least one of R1, R3, R4, R5, R6, and R7 represents a substituent containing a COOH group, an SO3H group, and / or a halogen (especially Cl or F). More preferably, R3 is a substituent containing a COOH group, an SO3H group (especially CH2SO3H), and / or a halogen (especially Cl or F).

[0082] In this preferred embodiment, R2 preferably represents NR. 16 R 17 , where R 16 and R 17 It is a C1-C4 alkyl group, most preferably R 16 =R 17 All of these represent methyl groups. Compounds where R2 represents N(CH3)2 are preferred because they have a relatively simple structure and are easy to prepare. More preferably, R2 represents NR. 16 R 17 , where R 17 Represents C1-C4 alkyl, most preferably R 17 It represents CH2CH3 and R 16 Together with R3, they form a ring system with the following formula:

[0083] More preferably, R2 represents NR 16 R 17 , where R 17 Represents C1-C4 alkyl, most preferably R 17 It represents CH2CH3 and R3 and R16 Together they form a ring system with the following formula:

[0084] Furthermore, compounds of formula (I) are preferred, wherein the following items do not appear simultaneously in compounds of formula (I): X represents C(CH3)2; R1, R3, R4, R5, R6, and R7 represent H. R2 represents N(CH3)2; and Where R 33 Indicates H or CH3; and / or In the compounds of formula (I), the following terms do not appear simultaneously: X represents C(CH3)2; R1, R3, R4, R5, R6, and R7 represent H. R2 represents N(CH3)2; and and / or In the compounds of formula (I), the following terms do not appear simultaneously: X represents C(CH3)2; R1, R3, R4, R5, R6, and R7 represent H. R2 represents N(CH3)2; and Where R 33 It represents H or CH3.

[0085] Further preferred are compounds of formula (I), wherein Y is not Where R 34 It represents OH, C(O)-N(CH3)-CH2-CH2-CH2-C(O)-OR, where R represents H or CH3; or C(O)-N(CH3)-CH2-CH2-O-CH2-CH2-O-CH2-COOR 33 , where R 33 It represents H or CH3.

[0086] In another preferred embodiment, the compound of the present invention is a compound of formula (I) or (II), wherein X represents SiR 13 R 14 These compounds are also known as silico-rhodol. In these silico-rhodols, R... 13 and R 14Each is preferably a C1-C4 alkyl group, and most preferably R. 11 =R 12 Both represent CH3.

[0087] In the silico-rhodol of this invention, Y is preferably a phenyl or imidazole group, said imidazole group being substituted with one or more substituents selected from the following: halogen (especially Cl and / or F), C(O)OH, NH-CH2-CH2-SO3H, S-CH2-CH2-SO3H, C(O)-N(CH3)-CH2-CH2-CH2-C(O)OH, or C(O)-(1-piperidine)-4-carboxyl, or C(O)-(1-pyrrolidinyl)-2-carboxyl. Furthermore, Y substituents comprising at least one SO3H group are particularly preferred.

[0088] Preferred is the silico-rhodol compound of formula (I), wherein Y is not 1-methyl-phenyl.

[0089] Further preferred are silico-rhodol compounds of formula (I), wherein the following terms do not appear simultaneously in compounds of formula (I): X represents Si(CH3)2; R2 represents NH2, NH(CH3), N(CH3)2, or NHC(O)CH3; and Y represents 1-methyl-phenyl.

[0090] In yet another embodiment, compounds having formula (III) or (IV), particularly formula (IV), are preferred.

[0091] The preferred structures of formulas (III) and (IV) include those composed of R1 and R 15 And / or R3 and R 17 The formed ring system. Examples of such preferred structures include

[0092] In compounds of formula (III) or (IV), X is preferably CR. 11 R 12 or SiR 13 R 14 .

[0093] If X represents CR 11 R 12 Compounds of this type with formula (III) or (IV) are called carbazine.

[0094] Carbazides of this type are particularly preferred, having at least one SO3H group and / or at least one COOH group.

[0095] A further preferred option is carbazide, where R2 represents NR. 16 R 17 R 16 Represents CH3 and R 17 It represents CH2-CH2-CH2-C(O)OH.

[0096] Further preferred are carbazides in which R6, R7, R8 and R9 represent H.

[0097] According to this preferred embodiment, R 11 and R 12 Each is preferably a C1-C4 alkyl group, and most preferably R. 11 =R 12 Both represent CH3. In a further preferred embodiment, R 11 =R 12 All of these represent phenyl groups, thus yielding more hydrophobic structures.

[0098] In a further preferred embodiment, R1, R3, R4, R5, R6, R7, R8, R9, R 10 This indicates a substituent containing a COOH group or an SO3H group. More preferably, R3 is a substituent containing a COOH group or an SO3H group, especially CH2SO3H.

[0099] According to the present invention, the compound of formula (III) comprises at least one sulfonyl group, wherein X represents CR 11 R 12 Preferably, the compound of formula (III) comprises at least one sulfonic acid group, wherein X represents CR. 11 R 12 More preferably, in the compounds of formula (III), where X represents CR 11 R 12 At least one of R1, R3, R4, R5, R6 or R7 represents SO3H.

[0100] Ionic salts / charged salts can be obtained from the compounds described in this invention. The solubility of these salts may be altered in polar media, particularly aqueous media. Furthermore, such salts correspond to compounds with the indicated chemical formulas.

[0101] Furthermore, the compound exists in solution in a dissolved form, particularly a hydrated form, or may contain one or more water molecules of crystallization. Both crystalline and amorphous forms correspond to compounds with the indicated chemical formulas.

[0102] Some compounds may have an asymmetric carbon atom as an optically active center, or contain double bonds. This invention covers all racemic, diastereomer, and geometric isomers resulting therefrom.

[0103] The compounds may also have non-natural isotope ratios, or may even be radiolabeled. The chemical formulas shown also cover these isotope variants, whether or not they are radioactive.

[0104] The compounds described in this invention are particularly suitable as dyes. Therefore, this invention also relates to a dye comprising the compounds as defined herein.

[0105] Surprisingly, the compounds described in this invention have pH-dependent absorption and / or pH-dependent fluorescence. Therefore, in a preferred embodiment, this invention relates to dyes having pH-dependent absorption and / or pH-dependent fluorescence.

[0106] The compounds of general formulas (I) to (IV) can be used as pH sensors because their acidic and basic forms have different absorption and fluorescence spectra and exhibit different fluorescence quantum yields. This will be demonstrated in more detail when discussing the properties of typical compounds among those described in this invention.

[0107] Therefore, the present invention also relates to pH sensors comprising the compounds and / or dyes described herein.

[0108] pH can be detected or monitored by utilizing the absorption and fluorescence of the acidic and basic forms of the compound and / or dye, or by different fluorescence quantum yields, either as a colorimetric measurement by absorption or as a ratio measurement or a fluorescence generator.

[0109] The compounds of general formulas (I) to (IV) can also be used as labeling groups in the qualitative and / or quantitative determination of analytes.

[0110] For use as a labeling group, the compound of the present invention preferably includes a group capable of linking the compound to the analyte, particularly a group capable of covalently linking the compound to the analyte. Therefore, as a labeling group, the compound of the present invention is preferably having at least one functional group capable of covalent coupling (e.g., OH, SH, NH2, COOH, olefins, alkynes, azides, tetrazines) to covalently link the compound to the analyte.

[0111] pH measurements in biological systems and fluids provide crucial information about health status—the function of cells, tissues, organs, or the entire organism. Intracellular pH changes are associated with endocytosis, calcium regulation, cell growth, chemotaxis, and cell adhesion. In individual cells or tissues, pH depends on or is related to external influences such as oxygen, light, ions, drugs, bacteria, or their metabolites.

[0112] Therefore, pH can also provide information about the shelf life, quality, and edibility of foods with different consistencies. Microbial processes initiated by bacteria, viruses, or fungi and their metabolic products can cause pH changes, which are associated with the degradation of the food.

[0113] pH sensing or measurement can preferably be performed in an aqueous fluid, such as bodily fluid samples (e.g., blood, serum, plasma, lymph, bile, cerebrospinal fluid, extracellular tissue fluid, urine, saliva, and sweat), or wastewater or food. The method can be performed as a wet assay (e.g., in a cuvette) or as a dry assay on a suitable reagent carrier. The carrier can be made of any material deemed suitable by those skilled in the art, and the material must be wettable to the sample to be tested. Examples of such carrier materials include, but are not limited to, porous glass, plastics, ion exchange resins, dextran, cellulose, cellulose derivatives, and / or hydrophilic polymers. The determination of the analyte can be performed by a single reaction or a series of reactions. The analyte is typically a biomolecule, preferably selected from antibodies, nanobodies, enzymes, peptides, polypeptides, proteins, nucleotides, nucleosides, nucleic acids, nucleic acid analogs, and / or haptens.

[0114] Therefore, the compounds of general formulas (I) to (IV) can be used in all chemical, medical, and biological detection methods known to those skilled in the art, wherein fluorescent dyes are suitable as labeling groups. For this purpose, the compounds are typically covalently coupled to a specific receptor for the analyte to be identified or to the analyte itself. For this purpose, the compounds of general formulas (I) to (IV) of the present invention preferably have at least one functional group capable of covalent coupling, such as OH, SH, NH2, and / or COOH. A well-known process involves coupling the N-hydroxysuccinimide ester of the dye to the amino group of the substrate. The specific receptor or analyte can be any suitable compound or molecule, and preferably a peptide, polypeptide, or nucleic acid. Many common coupling methods are described in Greg T. Hermanson, Bioconjugate Techniques, Academic Press, San Diego, Calif., 1996.

[0115] In addition to these conventional methods, so-called “click chemistry” has been developed in recent years. Two molecules are linked together in a bioorthogonal manner, meaning that in the presence of many other functional groups, the two reaction partners react in a directed manner without affecting the biological process. For this purpose, the compounds of general formulas (I) to (IV) described in this invention preferably have at least one functional group preferred in such “click reactions,” such as an azide, alkene, alkyne, or tetraazine. An overview of the principle can be found in K. Nwe, M.M. Brechbiel: Growing Applications of “Click Chemistry” for Bioconjugation in Contemporary Biomedical Research. Cancer Biotherapy and Radiopharmaceuticals, 24(3), 289 (2009).

[0116] The pH sensor described in this invention can be designed and used in many applications, particularly for biological or medical purposes. Specifically, the pH sensor described in this invention can be used to detect and / or measure bacterial growth.

[0117] If the pK of the substance a Using an indicator close to the pH of the medium under study allows for the highest sensitivity to even the smallest pH changes. pK values ​​within the pH range of 6 to 8 are also relevant. a Therefore, it is used for physiological measurements. In the detection of wound infection, 5 to 7 pK a Scope is crucial.

[0118] In a preferred embodiment, the present invention relates to a wound dressing comprising the compounds and / or dyes described herein, and to the use of the wound dressing to monitor the condition or healing process of a wound.

[0119] This can be achieved by incorporating a pH-sensitive dye into wound dressings, which can indicate pH in real time by changing the fluorescence color. This avoids time-consuming laboratory tests or unnecessary dressing changes.

[0120] The use of the compounds of the present invention as pH sensors in wound dressings includes: physically incorporating them into self-assembled liposomes, polymer vesicles, vesicles or capsules, or chemically coupling the pH-sensitive dye with a carrier or support.

[0121] In the first scenario, different pH-sensing mechanisms exist. For example, the compound described in this invention is trapped within the ion-permeable vesicles and responds to the pH of the surrounding wound medium in a known manner through changes in absorption and / or fluorescence. Another mechanism involves the incorporation of a pH-responsive compound at high concentrations, leading to fluorescence quenching. When the vesicle structure is opened or ruptured, for example through the opening or rupture of pathogenic metabolites or other irritants, the quenched molecules are released, resulting in a decrease in concentration and an increase in fluorescence. Such constructs can be tailored to different types of pathogens and can also be combined with the simultaneous release of drugs, therapeutics, and / or antimicrobial agents. For applications in wound dressings, these nanoscale reporter systems can be incorporated into or combined with hydrogels, materials commonly used in commercially available wound dressings.

[0122] On the other hand, the pH-responsive compound can have covalent bonds with the support material of the wound dressing. This allows for strong chemical fixation of the compound and prevents its uncontrolled release into the wound area. Such supports in dressings for chronic or complex wounds are typically chitosan- or chitin-based hydrogels; see H. Liu, C. Wang, C. Li, Y. Qin, Z. Wang, F. Yang, Z. Li, J. Wang: A functional chitosan-based hydrogel as a wound dressing and drug delivery system in the treatment of wound healing. RSC Advances 8, 7533 (2018). Other types or designs of carriers can include nanofibers, nanosheet bandages, composite films, nanoparticle clusters, microneedles, sponges, and foams.

[0123] Chitin and chitosan are generally considered to be biodegradable, biocompatible, non-antigenic, non-toxic, bioadhesive, antibacterial, bioactive, and hemostatic, making them ideal materials for wound dressings. They can be combined with other natural or synthetic polymers, such as alginate, cellulose, glycosaminoglycans, polyacrylic acid, polyethylene glycol, polyethylene oxide, polyvinyl alcohol, and polylactic acid, to enhance the stability or permeability of the tissue.

[0124] Commercially available chitosan wound dressings come in various forms, including nonwoven fabrics, hydrogels, films, and sponges. These wound dressings can also deliver antibacterial agents, growth factors, stem cells, peptides, and other active substances in a sustained-release manner, thus making them suitable for use as a diagnostic and therapeutic device. Attached Figure Description

[0125] Figure 1Compound 1, acidic form: absorption and emission spectra in a buffer solution at pH 3 at 25°C.

[0126] Figure 2 Compound 1, basic form: absorption and emission spectra in a buffer solution at pH 10 at 25°C.

[0127] Figure 3 Compound 1: By measuring the absorption spectra at different pH values, the pK values ​​in an aqueous solution at 25°C were determined. a The spectra were measured.

[0128] Figure 4 Compound 1, acidic and basic forms: fluorescence spectra, fluorescence quantum yield and on-off ratio, i.e. the ratio of fluorescence quantum yield of the basic and acidic forms (buffered in pH 10 and pH 3 at 25°C).

[0129] Figure 5 Compound 3: Relative fluorescence intensity at 25°C in aqueous buffer solutions with different pH values.

[0130] Figure 6 Compounds 1, 6 and 8, acidic form: photostability in buffer solution at 25°C and pH 3 compared to commercial dyes (ATTO 643, Fluorescein, Cy 5).

[0131] Figure 7 Compounds 1, 6 and 8, basic form: photostability in buffer solution at 25°C and pH 10 compared to commercial dyes (ATTO 643, Fluorescein, Cy 5).

[0132] Figure 8 Compound 1, basic form: lyochromic. At 25°C, according to E... T (30) Solvent polarity scaling, normalized absorption spectra in different solvents.

[0133] Figure 9 Compound 1, basic form: lyochromic. At 25°C, according to E... T (30) Solvent polarity scaling, normalized fluorescence spectra in different solvents.

[0134] Figure 10 Compared with commercial dyes (ATTO 643, ATTO 647N, Fluorescein), compounds 1, 2 and 3 are hydrophobic.

[0135] Figure 11 Compounds 10 and 8, used as chitosan membranes in wound dressing models, were moistened with aqueous solutions of different pH values. See the main text for detailed instructions. a) Under ambient light, the pH-dependent color changes of the two compounds are clearly visible. b) Under ultraviolet light (366 nm) irradiation, the fluorescence generation behavior of compound 8 is clearly visible, with an on-off ratio of approximately 29.

[0136] Figure 12 Compound 11, acidic form: absorption and emission spectra in a buffer solution at pH 3 at 25°C.

[0137] Figure 13 Compound 11, basic form: absorption and emission spectra in a buffer solution at pH 10 at 25°C.

[0138] Figure 14 Compound 11: The pK values ​​were determined by measuring the absorption spectra at different pH values ​​in an aqueous solution at 25°C. a Spectroscopic determination.

[0139] Figure 15 Compound 11, acidic and basic forms: fluorescence spectra, fluorescence quantum yield and on-off ratio, i.e., the ratio of fluorescence quantum yield of the basic and acidic forms (buffered in pH 10 and pH 3 at 25°C). Detailed Implementation

[0140] The examples in Tables 1 and 2 below are intended to help to better understand the invention, rather than to limit its scope.

[0141] Table 1: Compounds of general formula (I), where X represents CR 11 R 12 (a): Acidic form; measured in a buffer solution (citric acid, NaOH, and NaCl) at pH 3. (b): Basic form; measured in a buffer solution (boric acid, NaOH, and KCl) at pH 10.

[0142] Table 2: Compounds of general formula (III), where X represents CR 11 R 12 (a): Acidic form; measured in a buffer solution (citric acid, NaOH, and NaCl) at pH 3. (b): Basic form; measured in a buffer solution (boric acid, NaOH, and KCl) at pH 10. (c): Basic form; measured in ethanol.

[0143] The subgroups of the compounds described in this invention belong to the carborhodol class of dyes, and X in general formulas (I) and (II) represents CR. 11 R 12 One example (compound 7) was first described in M. Sednev, C. Wurm, V. Belov, S. Hell, Carborhodol: A Hybrid Fluorophore Obtained by Combination of Fluorescein and Carbopyronin Dye Cores. Bioconjugate Chemistry 24, 690 (2013). Although this literature mentions the pH equilibrium of these dyes (very similar to well-known rhodamine dyes), it makes no mention of their use as pH sensors or for ratio measurements. Nor does it mention the acidic form and pK of the dyes. a Optical data.

[0144] Compared to existing technologies, these compounds (see Table 1) do indeed possess many very interesting and advantageous properties, making them particularly suitable for use as ratiometric dyes and pH sensors. This is illustrated below.

[0145] The absorption spectra measured at different pH values ​​clearly show isoabsorption points, indicating the conversion between the two defined species, i.e., the protonated (acidic) form and the deprotonated (basic) form of compound 1:

[0146] Due to the different absorbance of the two forms, the color change according to the solution pH is visible to the naked eye. Therefore, the compound can be readily used as a pH sensor or indicator dye for colorimetric measurements (see...). Figure 3 ).

[0147] Surprisingly and unexpectedly, the compound exhibited a pK value in the range of 5.5 to 6.5. a (See Table 1), which contrasts sharply with the SNARF dyes described herein. This makes the compounds of this invention highly suitable as pH sensors for use in wound dressings, and also capable of reasonably and reliably detecting bacterial infections in chronic wounds or pathological biofilms. Interestingly, the changes in molecular structure and the introduction of different substituents only affect pK. aThere is a very slight effect. This allows the structure to operate without significantly altering the critical pK. a It can freely adapt to the required optical and chemical properties.

[0148] The basic form exhibits significantly higher fluorescence than the acidic form, resulting in an "on-off ratio" as high as 50 times (see Table 1 and...). Figure 4 ). Figure 5 The results show that as the equilibrium shifts towards the alkaline form, the fluorescence intensity increases with increasing pH. Therefore, the compound can be well used as a ratiometric or fluorescence-generating sensor to, for example, measure the pH of the surrounding medium or environment, or its changes during chemical reactions, biochemical or metabolic processes, etc.

[0149] The compound described in this invention is lyochromic, meaning that its absorption and fluorescence peaks are affected by the solvent. The color of the solution changes with the polarity of the solvent, which is very similar to the well-known Reischert dyes, which define commonly used E0. T (30) Solvent polarity scaling (see C. Reichardt, Solvatochromic Dyes as Solvent Polarity Indicators, Chem. Rev. 94(8), 2319(1994)). Since the absorption peak red-shifts with increasing polarity, this is called positive solvatochromism. Figure 8 This is demonstrated for compound 1 in its basic dye form in a variety of different solvents. Furthermore, the fluorescence of the compound also depends on the polarity of the solvent (see [link to documentation]). Figure 9 By adding Hünig's base, the dye was generated in its basic form in the solvent, in addition to an aqueous buffer solution at pH 10.

[0150] Another advantage of these compounds is their absorption and fluorescence in the red region of the visible spectrum at wavelengths above 550 nm or even above 600 nm. In this spectral region, the inherent natural fluorescence of biological samples is low. As the wavelength increases, light absorption decreases and scattering reduces, allowing the excitation light to penetrate deeper into samples such as blood and tissue. This, in turn, contributes to more rational and sensitive pH detection in biofilms or infected wounds. The absorption and fluorescence wavelengths of these compounds can be easily tuned by introducing different substituents, such as additional ring systems and / or double bonds. This can be verified by comparing compound 1 with compound 2, or compound 7 with compound 9 in Table 1.

[0151] Introducing hydrophilic or hydrophobic groups can also alter the solubility of the compounds. This is demonstrated by comparing compounds 1 (without a sulfonic acid group), 2 (containing one sulfonic acid group), and 3 (containing two sulfonic acid groups) with commercially available dyes known for their hydrophilicity. The results show that with the introduction of more sulfonic acid groups, the hydrophilicity of the compounds of this invention increases, and their solubility in water also improves. This is verified by two different measurements (see...). Figure 10 ): 1) Increased residence time in high performance reversed-phase liquid chromatography corresponds to higher hydrophobicity of the compound. 2) The partition coefficient between octanol and water, log(K) ow Octanol-water partition coefficients (Kow) vs. pH for fluorescent dye tracers (fluorescein, eosin Y), and implications for hydrologictracer tests. Geochem. J. 46, 46, 517 (2012) are widely used in the literature as a measure of hydrophilicity (see Y. Oba, SRPoulson: Octanol-water partition coefficients (Kow) vs. pH for fluorescent dye tracers (fluorescein, eosin Y), and implications for hydrologictracer tests. Geochem. J. 46, 46, 517 (2012)). Lower values ​​(negative values) correspond to hydrophilic compounds, and higher values ​​(positive values) indicate hydrophobic compounds.

[0152] For various applications, such as using polymer-bound dyes in smart wound dressings or as chemical sensors in vesicles, the difference in solubility in water or polar solvents versus nonpolar organic solvents is an important requirement. Dye molecules are released from their initial environment into the external solution as pathogenic metabolic toxins rupture the vesicle membrane. After release, the dye must be soluble in a more hydrophilic medium to achieve its sensing function (see J. Zhou, D. Yao, Z. Qian, S. Hou, L. Li, ATA Jenkins, Y. Fan: Bacteria-responsive intelligent wound dressing: simultaneous in situ detection and inhibition of bacterial infection for accelerated wound healing. Biomaterials 161, 11-23 (2018)).

[0153] The compound may possess additional coupling functionality to covalently bind to an analyte or substrate. The analyte or substrate may be, for example, biomolecules such as proteins, nanobodies, antibodies, enzymes, oligonucleotides, or polymers used in wound dressings. Those skilled in the art can readily introduce a variety of different coupling groups using active ester chemistry methods (see examples).

[0154] Even without covalent coupling groups, the dye can be used in a variety of ways, such as as a sensor for use inside vesicles, membranes, etc., or for measuring the surrounding pH.

[0155] The use of pH sensors in wound dressings can include coupling the pH-sensitive compound to a carrier material. Such carriers in dressings for chronic or complex wounds are typically chitin- or chitosan-based hydrogels.

[0156] To demonstrate the suitability and functionality of the novel compounds as pH sensors under real-world conditions, compounds 8 and 10 were coupled to chitosan via NHS esterification. Figure 11 As shown, the color differences between the acidic and basic forms of the chemically bound compounds are clearly visible to the naked eye in response to the pH of the surrounding medium. Therefore, the compounds of the present invention can be used as colorimetric or chromatographic pH monitors through their pH-dependent absorption.

[0157] In the case of compound 8 with an on / off ratio of approximately 29, the basic form exhibits significantly higher fluorescence than the acidic form. This phenomenon can be easily observed with the naked eye when illuminated with a standard ultraviolet lamp (“black light torch”), as the chitosan membrane at pH 7.4 is “much brighter” than the membrane at pH 4. Therefore, compound 8 can be used as both a colorimetric pH sensor and a fluorescence-generating pH sensor.

[0158] For clinical or medical applications, the ability to observe color changes or easily monitor fluorescence intensity changes without requiring complex technical equipment is highly beneficial. Real-time monitoring of pH changes in chronic wounds (such as those caused by pathogenic infections) is a significant advantage compared to standard bacterial culture methods that require time-consuming procedures performed in laboratories far from patients.

[0159] The compound exhibits extremely high photostability. Its photostability was measured under stress conditions by irradiating both the acidic and basic forms in an aqueous buffer solution with a 575-watt mercury halide lamp (see [link to relevant documentation]). Figure 6 and Figure 7For clarity, only compounds 2, 6, and 8 of this invention are shown, covering the observed range of photostability. Both forms exhibit photostability equivalent to the excellent photostability of the hydrophilic dye ATTO 643, and significantly superior to the photostability of the equally commercially available dyes Fluorescein and Cy 5. In particular, Fluorescein can be used as a direct comparison because it is commonly used as a pH-sensitive dye. The photostability of the compounds of this invention is significantly higher than that of Fluorescein.

[0160] This makes the compounds described in this invention ideal candidates, not only for use in wound dressings, enabling long-term use in long-term monitoring of wound pH, but also for application in high-resolution microscopy or nanomicroscopy, where high laser power is used to excite fluorescent labeling.

[0161] The compound is a xaton derivative, and therefore it is presumed to have similar low toxicity (see R. Alford, H. M. Impson, J. Duberman, G. Chill, M. Ogawa, C. Regino, H. Kobayashi, P. P. Choyke: Toxicity of Organic Fluorophores Used in Molecular Imaging. Molecular Imaging 8(6), 341(2009)).

[0162] A significant commercial advantage of this invention is that the compound is simply prepared from commercially available or economically producible precursor compounds. This one-step synthesis facilitates simple and flexible treatment of the precursor using diluted hydroxide solutions, for example, for compound 22:

[0163] For this purpose, the direct precursor compound is dissolved in an aqueous hydroxide solution. Organic solvents such as ethanol, methanol, acetone, or acetonitrile can be used as co-solvents, for example, to improve the solubility of the reactants.

[0164] For the conversion, any alkali metal or alkaline earth metal hydroxide can be used, such as lithium hydroxide, sodium hydroxide, potassium hydroxide, cesium hydroxide, calcium hydroxide, or barium hydroxide. Lithium hydroxide, sodium hydroxide, and potassium hydroxide are preferred due to their higher reactivity. Even mixtures of different hydroxides can be used.

[0165] The reaction time depends on the concentrations of the reactants (precursor and base) and the temperature. The reaction must proceed within a reasonable time, and the temperature must reach at least 0°C. It has been found that the reaction is faster and the conversion more uniform within a temperature range of 25°C to 100°C. Optimal results are obtained at temperatures between 50°C and 85°C.

[0166] The only prerequisite for this simple one-step reaction is that at least one terminal of the parent compound's chromophoric system contains a dimethylamino group, as can be seen in the following examples of precursors used to prepare the compounds described in this invention:

[0167] Table 3: Precursor compounds used to synthesize compounds of general formula (I).

[0168] The precursor compound can be prepared according to literature and steps known to those skilled in the art, for example, J. L. Bachmann, C. I. Pavlich, A. J. Boley, E. M. Marcotte, EV. Anslyn: Synthesis of Carboxy ATTO 647N Using Redox Cycling for Xanthone Access. Org. Lett. 22, 281 (2020).

[0169] Therefore, a very important advantage of this invention is that it can produce two high-value dyes almost simultaneously: the precursor "carborhodamine" and the product "carborhodol"; whereas the method described in M. Sednev, C. Wurm, V. Belov, S. Hell, Carborhodol: A Hybrid Fluorophore Obtained by Combination of Fluorescein and Carbopyronin Dye Cores, Bioconjugate Chemistry 24, 690 (2013) requires multiple steps to obtain a single target compound - carborhodol.

[0170] Another subgroup of the compound described in this invention belongs to the asymmetric carbazide dye class, where X in general formulas (III) and (IV) represents CR. 11 R 12These dyes can be prepared, for example, according to the synthetic route described in US 4892858. No other optical properties besides absorption are mentioned in that patent, nor are any fluorescent or pH-dependent optical properties of these compounds mentioned, and these compounds are used only as part of a thermosensitive transfer material.

[0171] The compounds exhibit absorption and fluorescence at wavelengths above 560 nm in the red region of the visible spectrum (see Table 2). The numerous advantages of this red spectral region have been detailed above. The absorption and fluorescence wavelengths of the compounds can be easily tuned by introducing different substituents, such as additional ring systems. This can be seen by comparing compounds 12, 19, 20, and 21 in Table 2.

[0172] Furthermore, we successfully introduced coupling groups. Compounds 15, 16, and 17 in Table 2 include carboxylic acid groups and can therefore be covalently bonded to analytes or substrates as described above.

[0173] Introducing hydrophilic or hydrophobic groups can also alter the solubility of the compound according to the specific application requirements.

[0174] Surprisingly, these compounds exhibit a large Stokes shift, i.e., a wavelength difference between the absorption and fluorescence peaks, typically ranging from approximately 50 nm to 120 nm (see Table 2). A large Stokes shift is highly advantageous in multiplex detection applications, where multiple dyes are simultaneously excited at a single wavelength, and their spectrally separated fluorescence is detected in different emission channels. For dyes with large Stokes shifts, spectral separation of the excitation light using optical filters is easier. This results in a better signal-to-noise ratio, thereby achieving higher detection sensitivity.

[0175] The absorption and fluorescence spectra of compound 11 are as follows: Figure 12 (acidic form) and Figure 13 (Alkaline form) is shown.

[0176] The absorption spectra measured at different pH values ​​clearly show isoabsorption points, indicating the conversion between the two defined species, namely, for example, the protonated (acidic) and deprotonated (basic) forms of compound 11:

[0177] Due to the different absorbance of the two forms, color changes according to the solution pH are visible to the naked eye. Therefore, the compound can be readily used as a pH sensor or indicator dye for colorimetric measurements (see...). Figure 14 ).

[0178] The fluorescence generation behavior and on-state ratio of compound 11 are shown in Figure 15The figure shows that the fluorescence quantum yield of the basic form is more than twice that of the acidic form.

[0179] The compound of the present invention is produced according to the steps described in more detail in the examples below.

[0180] Examples of preparation of the compounds described in this invention

[0181] Compounds 1 and 2 (Contains a chlorine-substituted head group)

[0182] The corresponding carboridine (compound 22 or 23) was dissolved in a 1:1 mixture of ethanol and 0.1 M LiOH aqueous solution. The reaction mixture was stirred overnight at 90 °C, during which time the color changed from green to blue within the first hour. After the reaction was complete, the organic layer was separated using dichloromethane acidified with trifluoroacetic acid. The organic layer was dried with sodium sulfate, filtered, and the solvent was evaporated. The crude product was purified by chromatography to give compound 1 (47%) or compound 2 (43%), respectively.

[0183] Compounds 3 and 4 (The part containing the fluorine-substituted head group)

[0184] Compound 24 was dissolved in DMSO, and 5 equivalents of sodium taurate or sodium 2-mercaptoethanesulfonate were added. The solution was stirred at 70°C for two hours. The intermediate compound was purified by column chromatography.

[0185] The corresponding tetrafluorosubstituted carramine was dissolved in 0.05 M LiOH solution and stirred at 50 °C for 96 hours. After approximately 4 hours, the color changed from green to blue. After the reaction was complete, the pH was adjusted to 5 by adding trifluoroacetic acid. The resulting solutions were subjected to column chromatography to give compound 3 (59%) or compound 4 (46%), respectively.

[0186] Compounds 5, 7 and 9 (Contains a carboxylic acid head group)

[0187] The corresponding carboridine (compounds 25, 26, or 27) was dissolved in a 1:1 mixture of ethanol and 2.5M NaOH aqueous solution. The resulting mixture was stirred at 85°C for 3 hours, during which time the color of the solution changed from blue to red within 15 minutes.

[0188] Upon completion of the reaction, the mixture was cooled to 0°C, and trifluoroacetic acid was added to achieve a pH of 4 to 5. After addition, the bright red color turned dark red. The product was extracted with dichloromethane, and the organic layer was dried with sodium sulfate, filtered, and evaporated to dryness. The crude products were purified by chromatography to give compounds 5 (44%), 7 (68%), or 9 (54%), respectively.

[0189] Compound 6

[0190] At 0°C, anhydrous acetonitrile (concentration 5 g / L, 10 mL) containing p-toluenesulfonic acid was added to a stirred solution (20 mg, 36 μmol) of compound 5. The reaction mixture consisted of N-hydroxysuccinimide (5.5 mg, 1.3 equivalents) and N,N'-dicyclohexylcarbodiimide (DCC) (8.05 mg, 1.05 equivalents). A precipitate formed immediately upon the addition of DCC. The reaction mixture was stirred overnight at 0°C to produce a dark orange color. The reaction mixture was filtered through a 0.2 μm membrane filter to remove solids. After evaporation of the solvent, a magenta residue was obtained. This residue was redissolved in 2 mL of acetonitrile, and the active ester of compound 5 was precipitated using diethyl ether and separated into a red powder (19 mg, 98%).

[0191] The active ester (20 mg, 38 μmol) was dissolved in 3 mL of anhydrous DMSO, and potassium 4-(methylamino)butyrate (22 mg, 95 μmol) was added. The red reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was then added to 13 mL of distilled water while stirring at 0 °C. The resulting solution was subjected to reverse column chromatography to give compound 6 (98%).

[0192] Compound 8

[0193] Compound 7 (100 mg, 0.2 mmol) was dissolved in 10 mL of dichloroethane, and POCl3 (951 μL, 10.2 mmol) was added. After stirring at 80 °C for 4 hours, all volatile substances were evaporated under vacuum, and the solid residue was dissolved in 5 mL of acetonitrile. A solution of piperidine-4-carboxylic acid (129 mg, 1 mmol) in acetonitrile (5 mL) was added, followed by triethylamine (842 μL, 6 mmol). After stirring at room temperature for 30 minutes, all volatile substances were removed under vacuum. The residue was redissolved in 20 mL of dichloromethane and washed with a saturated aqueous solution of ammonium chloride. The organic layer was separated, dried with sodium sulfate, and the organic solvent was evaporated. Compound 8 (60%) was obtained by column chromatography.

[0194] Compound 10

[0195] Following the steps for compound 8 above, this compound was synthesized starting with compound 9. Methyl 4-(methylamino)butyrate was used instead of piperidine-4-carboxylic acid.

[0196] For the hydrolysis of the ester group, the crude product was stirred at room temperature for 5 hours in a mixture of H2O:THF:1M NaOH aqueous solution (22 mL:34 mL:1.4 mL). Acetic acid (23 mL) was added, and the mixture was evaporated to dryness. The crude product was purified by column chromatography to give mixture 10 (67%).

[0197] Compound 17

[0198] 3-(hydroxydiphenylmethyl)phenol is prepared in diethyl ether by a standard Grignard reaction from ethyl 3-hydroxybenzoate and magnesium phenyl bromide.

[0199] N-methyl-4-nitroaniline and ethyl 4-bromobutyrate were refluxed together with N-ethyl-N-isopropylprop-2-amine in acetonitrile to give ethyl 4-(methyl(4-nitro-phenyl)amino)butyrate. The yellow product was separated into a solid by silica gel column chromatography using a pentane / chloroform / methanol gradient. The conversion of the nitro group to the amino group was carried out in boiling water in the presence of iron powder and a small amount of hydrochloric acid. The resulting phenylenediamine compound was purified by column chromatography.

[0200] At room temperature, equimolar amounts of 3-(hydroxydiphenylmethyl)phenol and ethyl 4-((4-aminophenyl)(methyl)amino)butyrate were mixed with sodium carbonate in a 1:5 mixture of ethanol and water. An aqueous solution of ammonium persulfate was added to promote the intermediate oxidation of the phenylenediamine. After half an hour, the deep blue solution was extracted with ethyl acetate. Treatment of the blue extract with an aqueous solution of sodium dithionite yielded the colorless leuco form of the indoleaniline intermediate. The organic phase was dried over sodium sulfate, filtered, and evaporated to dryness.

[0201] The oily residue was redissolved in chloroform, and the solution was cooled to 0°C. Sulfuric acid was added dropwise, followed by vacuum removal of the chloroform. The remaining mixture was stirred at room temperature for 1 hour to complete the ring-closing reaction. Cooled aqueous sodium hydroxide solution was added to adjust the pH to 5-6. Then, chloroform and sodium periodate aqueous solution were added to the mixture. The phases were separated, and the aqueous layer was extracted twice with chloroform. The chloroform extract was pooled, dried over sodium sulfate, filtered, and evaporated to dryness. The intermediate was purified by column chromatography.

[0202] For the hydrolysis of the ester group, the intermediate was refluxed with four molar amounts of hydrochloric acid in a 1:1 mixture of water and acetone for 5 hours. The crude product was purified by column chromatography to give mixture 17 (20%).

[0203] Compound 18

[0204] 2-(3-Dimethylamino)phenyl)prop-2-ol is prepared in diethyl ether by a standard Grignard reaction from ethyl 3-(dimethylamino)benzoate and magnesium methyl iodide.

[0205] At room temperature, equimolar amounts of 2-(3-dimethylamino)phenyl)prop-2-ol and 2,6-dichloro-4-(chloroimino)cyclohexane-2,5-dienone (Gibberellic reagent), together with sodium carbonate, were mixed in a 1:10 mixture of ethanol and water. After half an hour, the dark green solution was extracted with ethyl acetate. Treatment of the blue extract with an aqueous solution of sodium dithionite yielded the colorless leuco form of the indoleaniline intermediate. The organic phase was dried over sodium sulfate, filtered, and evaporated to dryness.

[0206] The oily residue was redissolved in chloroform, and the solution was cooled to 0°C. Sulfuric acid was added dropwise, followed by vacuum removal of the chloroform. The remaining blue-black mixture was stirred at room temperature for 2 hours to complete the ring-closure reaction. Ice and an aqueous sodium hydroxide solution were added to adjust the pH to 5-6. Then, an aqueous chloroform and sodium periodate solution were added to the mixture. The phases were separated, and the aqueous layer was extracted again with chloroform. The chloroform extract containing the target compound was pooled, dried over sodium sulfate, filtered, and evaporated to dryness. The black residue was purified by column chromatography to give compound 18 (25%).

[0207] This application also provides the following items:

[0208] Project 1. A dye having pH-dependent absorption and / or pH-dependent fluorescence, having general formula (I), (II), (III) or (IV). in X represents CR 11 R 12 SiR 13 R 14 Sulfur, SO2 or P(O)OR 15 , Y represents a hydrogen, nitrile, carboxyl, carboxylic acid derivative, amino, or a straight-chain, branched, or cyclic saturated or unsaturated hydrocarbon moiety having up to 40 carbon atoms. The hydrocarbon moiety may contain one or more heteroatoms selected from N, O, and S, or be substituted by one or more heteroatoms selected from N, O, and S, and / or be substituents selected from one or more of the following: halogen, OH, (O)alkyl, O(aryl), SH, S(alkyl), S(aryl), NH2, NH(aryl), NH(alkyl), N(aryl)2, N(alkyl)2, NO2, CHO, COOH, COO(alkyl), COO(aryl), PO3H2, SO3H, or CONR. 31 R 32 ; R1, R3, R4, R5, R6, R7, R8, R9, R 10Independently representing a hydrogen, halogen, hydroxyl, mercapto, amino, sulfonyl, phosphoro, nitro, carbonyl, carboxyl, carboxylic acid derivative, nitrile, isonitrile, cyanate, thiocyanate, isothiocyanate, or a straight-chain, branched, or cyclic saturated or unsaturated hydrocarbon moiety having up to 20 carbon atoms, said hydrocarbon moiety may contain one or more heteroatoms selected from N, O, and S or be substituted by one or more heteroatoms selected from N, O, and S, and / or preferably one or more substituents selected from the following: halogen, OH, (O)alkyl, O(aryl), SH, S(alkyl), S(aryl), NH2, NH(aryl), NH(alkyl), N(aryl)2, N(alkyl)2, NO2, CHO, COOH, COO(alkyl), COO(aryl), PO3H2, SO3H, or CONR 31 R 32 ; R 11 R 12 R 13 R 14 R 15 R 31 R 32 Independently representing hydrogen or a hydrocarbon group having 1 to 20 carbon atoms, wherein the hydrocarbon group optionally includes one or more heteroatoms selected from N, O, and S, and / or preferably one or more substituents selected from: halogen, nitrile, hydroxyl, mercapto, amino, nitro, sulfonyl, phosphoro, carbonyl, carboxyl, carboxylic acid derivatives; or R 11 and R 12 Or R 13 and R 14 The atoms bonded to them together form 3- to 7-membered rings, wherein the rings may include one or more double bonds and / or one or more heteroatoms selected from N, O, and S and / or one or more substituents selected from the following: halogen, OH, (O)alkyl, O(aryl), SH, S(alkyl), S(aryl), NH2, NH(aryl), NH(alkyl), N(aryl)2, N(alkyl)2, NO2, CHO, COOH, COO(alkyl), COO(aryl), PO3H2, SO3H, CONR 31 R 32 and a hydrocarbon group having 1 to 20 carbon atoms, and / or may be fused with one or more 3- to 7-membered rings, wherein the hydrocarbon group optionally includes one or more heteroatoms selected from N, O, and S and / or one or more substituents selected from the following: halogen, OH, (O)alkyl, O(aryl), SH, S(alkyl), S(aryl), NH2, NH(aryl), NH(alkyl), N(aryl)2, N(alkyl)2, NO2, CHO, COOH, COO(alkyl), COO(aryl), PO3H2, SO3H, or CONR31 R 32 ; R2 represents NR 16 R 17 Or R1 and R2 together Where R 18 R 19 and R 22 Limited to R1, and R 16 R 17 R 20 R 21 Limited to R 11 , Or R1 to R 22 At least one of them forms a ring system with one or more adjacent parts.

[0209] Project 2. A compound having the general formula (I), (II), (III) or (IV). in X represents CR 11 R 12 SiR 13 R 14 Sulfur, SO2 or P(O)OR 15 , Y represents a hydrogen, nitrile, carboxyl, carboxylic acid derivative, amino, or a straight-chain, branched, or cyclic saturated or unsaturated hydrocarbon moiety having up to 40 carbon atoms. The hydrocarbon moiety may contain one or more heteroatoms selected from N, O, and S, or be substituted by one or more heteroatoms selected from N, O, and S, and / or one or more substituents selected from: halogen, OH, (O)alkyl, O(aryl), SH, S(alkyl), S(aryl), NH2, NH(aryl), NH(alkyl), N(aryl)2, N(alkyl)2, NO2, CHO, COOH, COO(alkyl), COO(aryl), PO3H2, SO3H, or CONR. 31 R 32 ; R1, R3, R4, R5, R6, R7, R8, R9, R 10Independently representing a hydrogen, halogen, hydroxyl, mercapto, amino, sulfonyl, phosphoro, nitro, carbonyl, carboxyl, carboxylic acid derivative, nitrile, isonitrile, cyanate, thiocyanate, isothiocyanate, or a straight-chain, branched, or cyclic saturated or unsaturated hydrocarbon moiety having up to 20 carbon atoms, said hydrocarbon moiety may contain one or more heteroatoms selected from N, O, and S or be substituted by one or more heteroatoms selected from N, O, and S, and / or be substituted by one or more substituents selected from: halogen, OH, (O)alkyl, O(aryl), SH, S(alkyl), S(aryl), NH2, NH(aryl), NH(alkyl), N(aryl)2, N(alkyl)2, NO2, CHO, COOH, COO(alkyl), COO(aryl), PO3H2 and SO3H or CONR 31 R 32 ; R 11 R 12 R 13 R 14 R 15 R 31 R 32 Independently representing hydrogen or a hydrocarbon group having 1 to 20 carbon atoms, wherein the hydrocarbon group optionally includes one or more heteroatoms selected from N, O, and S, and / or preferably one or more substituents selected from: halogen, nitrile, hydroxyl, mercapto, amino, nitro, sulfonyl, phosphoro, carbonyl, carboxyl, carboxylic acid derivatives; or R 11 and R 12 Or R 13 and R 14 The atoms bonded to them together form 3- to 7-membered rings, wherein the rings may include one or more double bonds, and / or one or more heteroatoms selected from N, O, and S, and / or one or more substituents selected from the following: halogen, OH, (O)alkyl, O(aryl), SH, S(alkyl), S(aryl), NH2, NH(aryl), NH(alkyl), N(aryl)2, N(alkyl)2, NO2, CHO, COOH, COO(alkyl), COO(aryl), PO3H2, SO3H, CONR 31 R 32 and a hydrocarbon group having 1 to 20 carbon atoms, and / or may be fused with one or more 3- to 7-membered rings, wherein the hydrocarbon group optionally includes one or more heteroatoms selected from N, O, and S and / or one or more substituents selected from the following: halogen, OH, (O)alkyl, O(aryl), SH, S(alkyl), S(aryl), NH2, NH(aryl), NH(alkyl), N(aryl)2, N(alkyl)2, NO2, CHO, COOH, COO(alkyl), COO(aryl), PO3H2, SO3H, or CONR31 R 32 ; R2 represents NR 16 R 17 Or R1 and R2 together Where R 18 R 19 and R 22 Limited to R1, and R 16 R 17 R 20 R 21 Limited to R 11 , Or R1 to R 22 At least one of them forms a ring system with one or more adjacent parts, but must meet the following conditions: In the compound of formula (I), the following terms do not appear simultaneously: X represents C(CH3)2; R2 represents N(CH3)2; and Where R 33 Indicates H or CH3; and In the compound of formula (I), the following terms do not appear simultaneously: X represents C(CH3)2; R2 represents N(CH3)2; and as well as In the compound of formula (I), the following terms do not appear simultaneously: X represents C(CH3)2; R2 represents N(CH3)2; and Where R 33 Indicates H or CH3; and In the compound of formula (I), the following terms do not appear simultaneously: X represents Si(CH3)2; R2 represents NH2, NH(CH3), N(CH3)2, or NH-C(O)CH3; and Y represents 1-methyl-phenyl. And where X represents CR 11 R 12 Compounds of formula (III) include at least one sulfonyl group, and particularly, wherein X represents CR. 11 R 12In compounds of formula (III), at least one of R1, R3, R4, R5, R6 or R7 represents SO3H.

[0210] Item 3. The dye or compound according to Item 1 or 2, wherein R1 and R 16 And / or R3 and R 17 By forming a ring system, one of the following structures (A), (B), (C), (D), (E), (F), and (G) is obtained: In each case, R is independently defined as R1, and the dashed line is optionally a double bond, in the presence of which the portion bound by the dashed line does not exist; and thereby Adjacent substituents R may optionally form additional ring systems, including 5-membered and / or 6-membered rings, which may optionally contain other heteroatoms or substituents.

[0211] Item 4. The dye or compound according to any one of Items 1 to 3, wherein R3 and R 17 and / or R1 and R 16 Forming a ring system. In each case, R is independently defined as R1.

[0212] Item 5. A dye or compound according to any one of Items 1 to 4, wherein R3 and R4 and / or R5 and R6 and / or R6 and R8 form a cyclic system selected from: In each case, R is independently defined as R1.

[0213] Item 6. A dye or compound according to any one of Items 1 to 5, wherein Y in formulas (I) and (II) is an optionally substituted cycloalkyl, optionally substituted phenyl, optionally substituted naphthyl, or optionally substituted heteroaryl, having one of the following structures (H), (J), (K), (L), (M), (N), and (O): in E represents oxygen, sulfur, nitrogen, or... + NR 23 , R u Indicates halogen, OR24 NHR 25 NR 26 R 27 , Z represents hydrogen or halogen. R v Represents Z and S(CH2). x COOH, S(CH2) x SO3H, NHR 28 NR 29 R 30 ,as well as R w Indicates hydrogen, alkyl, (CH2) x COOH, (CH2) x SO3H, (CH2) x OH, (CH2) x SH, (CH2) x NH2, (CH2) x Z, where x = 1, 2, 3, 4, 5, 6, R 23 R 24 R 25 R 26 R 27 R 28 R 29 R 30 Independently represents another saturated or unsaturated hydrocarbon moiety having up to 20 carbon atoms, such as alkyl, cycloalkyl, aryl, and heteroaryl, which may contain one or more heteroatoms selected from N, O, and S or be substituted by one or more heteroatoms selected from N, O, and S, and / or preferably one or more substituents selected from: halogen, OH, (O)alkyl, O(aryl), SH, S(alkyl), S(aryl), NH2, NH(aryl), NH(alkyl), N(aryl)2, N(alkyl)2, NO2, CHO, COOH, COO(alkyl), COO(aryl), PO3H2, SO3H, or CONR 31 R 32 ; And R in each case is independently defined as R1.

[0214] Item 7. A dye or compound according to any one of Items 1 to 6, wherein Y is selected from...

[0215] Item 8. The dye or compound according to any one of Items 1 to 7, wherein R1, R3, R4, R5, R6 and R7 represent H.

[0216] Item 9. A dye or compound according to any one of Items 1 to 7, wherein at least one of R1, R3, R4, R5, R6 and R7 represents a substituent containing a COOH group, an SO3H group and / or a halogen (especially Cl or F).

[0217] Item 10. A dye or compound according to any one of Items 1 to 7, wherein R3 is a substituent containing a COOH group, an SO3H group (especially CH2-SO3H) and / or a halogen (especially Cl or F).

[0218] Item 11. The dye or compound according to any one of Items 1 to 10, wherein R2 is N(CH3)2.

[0219] Item 12. A dye or compound according to any one of Items 1 to 10, wherein R2 represents NR. 16 R 17 , where R 17 Represents C1-C4 alkyl, most preferably, R 17 It represents CH2CH3, as well as R3 and R 16 Together they form a ring system with the following formula:

[0220] Item 13. A dye or compound according to any one of Items 1 to 10, wherein R2 represents NR. 16 R 17 , where R 17 Represents C1-C4 alkyl, preferably, R 17 It represents CH2CH3, as well as R3 and R 16 Together they form a ring system with the following formula:

[0221] Item 14. The dye or compound according to any one of Items 1 to 13, comprising at least one SO3H group.

[0222] Item 15. The dye or compound according to any one of Items 1 to 14, comprising at least one COOH group.

[0223] Item 16. The dye or compound according to any one of Items 1 to 15, wherein Y comprises phenyl, naphthyl, or imidazole.

[0224] Item 17. A dye or compound according to any one of Items 1 to 15, wherein X represents SIR. 13 R 14 And Y is not 1-methyl-phenyl.

[0225] Item 18. Use of any dye or compound described in any one of Items 1 to 17 as a pH sensor.

[0226] Item 19. pH sensor, comprising any one of the dyes or compounds described in Items 1 to 17.

[0227] Item 20. The pH sensor according to Item 19, wherein the dye or compound is used either in solution form; or incorporated into vesicles, polymer vesicles or similar self-assembled compartments; or is covalently bound to or attached to an analyte, substrate or support.

[0228] Item 21. Wound dressing, comprising any one of items 1 to 17, the dye or compound.

[0229] Item 22. Use of any dye or compound as a labeling group in analytes in qualitative and / or quantitative samples.

[0230] Item 23. The use as described in Item 22, wherein the analyte is a biomolecule, particularly an antibody, nanobody, peptide, polypeptide, protein, nucleotide, polynucleotide, nucleoside, nucleic acid, nucleic acid analog or hapten.

[0231] Item 24. The use as described in Item 22, wherein the dye is covalently attached to the analyte.

[0232] Item 25. A method for preparing a dye or compound having formula (I), (II), (III) or (IV) as defined in any one of Items 1 to 17, comprising: using an aqueous solution of an alkali metal or alkaline earth metal hydroxide as a reaction medium, and converting the precursor compound by a one-step one-pot synthesis.

[0233] Item 26. The method according to Item 25, wherein the alkali metal or alkaline earth metal hydroxide is selected from lithium hydroxide, sodium hydroxide, potassium hydroxide, cesium hydroxide, calcium hydroxide or barium hydroxide, or a mixture of different hydroxides.

[0234] Item 27. The method according to Item 25 or 26, wherein the conversion is carried out at a temperature of at least 0°C, more preferably at a temperature in the range of 25°C to 100°C, or at a temperature in the range of 50°C to 85°C.

[0235] Item 28. The method according to any one of Items 25 to 27, wherein the conversion is carried out in the presence of a co-solvent, preferably selected from acetone, acetonitrile, ethanol or methanol.

[0236] Item 29. The method according to any one of Items 25 to 28, wherein the precursor is carboridine.

[0237] Item 30. The method according to any one of Items 25 to 29, comprising the following steps:

[0238] Item 31. The wound dressing according to Item 21, wherein the dye or compound of any one of Items 1 to 17 is incorporated into a self-assembled liposome, polymer vesicle, vesicle or capsule.

[0239] Item 32. The wound dressing according to Item 21 or 31, wherein the dye or compound of any one of Items 1 to 17 is coupled, in particular covalently coupled, to a carrier or support.

[0240] Item 33. A wound dressing according to any one of Items 21 or 31 to 32, wherein the wound dressing comprises a support material, particularly when the support material comprises chitin and / or chitosan.

[0241] Item 34. The wound dressing according to any one of Items 21 and 31 to 33, wherein the support material comprises nanofibers, nanosheet bandages, composite membranes, nanoparticle clusters, microneedles, sponges and / or foams.

[0242] Item 35. The wound dressing according to any one of items 21 and 31 to 34, wherein the wound dressing comprises a carrier comprising a chitin- or chitosan-based hydrogel.

Claims

1. A dye having pH-dependent absorption and / or pH-dependent fluorescence having the general formula (I), (II), (III) or (IV) wherein R in each case is independently defined as R1, and the dotted line is optionally a double bond, in the presence of which the moieties bound by the dotted line are not present; and whereby X represents CR 11 R 12 , SiR 13 R 14 , sulfur, SO2or P(O)OR 15 ; Y represents hydrogen, a nitrile group, a carboxyl group, a carboxylic acid derivative, an amino group, or a linear, branched or cyclic, saturated or unsaturated hydrocarbon moiety having up to 40 carbon atoms, which hydrocarbon moiety can comprise or be substituted by one or more heteroatoms selected from N, O and S, and / or which hydrocarbon moiety is preferably substituted by one or more substituents selected from halogen, OH, (O)alkyl, O(aryl), SH, S(alkyl), S(aryl), NH2, NH(aryl), NH(alkyl), N(aryl)2, N(alkyl)2, NO2, CHO, COOH, COO(alkyl), COO(aryl), PO3H2, SO3H or CONR 31 R 32 ; R1, R3, R4, R5, R6, R7, R8, R9, R 10 independently represent hydrogen, halogen, hydroxyl, thiol, amino, sulfo, phospho, nitro, carbonyl, carboxyl, carboxylic acid derivative, nitrile, isonitrile, cyanate, thiocyanate, isothiocyanate, or a linear, branched or cyclic, saturated or unsaturated hydrocarbon moiety having up to 20 carbon atoms, which hydrocarbon moiety can comprise or be substituted by one or more heteroatoms selected from N, O and S, and / or which is preferably substituted by one or more substituents selected from halogen, OH, (O)alkyl, O(aryl), SH, S(alkyl), S(aryl), NH2, NH(aryl), NH(alkyl), N(aryl)2, N(alkyl)2, NO2, CHO, COOH, COO(alkyl), COO(aryl), PO3H2, SO3H or CONR 31 R 32 ; R 11 , R 12 , R 13 , R 14 , R 15 , R 31 , R 32 independently represent hydrogen or a hydrocarbon group having 1 to 20 carbon atoms, wherein the hydrocarbon group optionally includes one or more heteroatoms selected from N, O and S, and / or is preferably substituted with one or more substituents selected from halogen, nitrile, hydroxyl, thiol, amino, nitro, sulfo, phospho, carbonyl, carboxyl, carboxylic acid derivative; or R 11 and R 12 or R 13 and R 14 together with the atoms to which they are attached form a 3- to 7-membered ring, wherein the ring can include one or more double bonds, and / or one or more heteroatoms selected from N, O and S, and / or one or more substituents selected from halogen, OH, (O)alkyl, O(aryl), SH, S(alkyl), S(aryl), NH2, NH(aryl), NH(alkyl), N(aryl)2, N(alkyl)2, NO2, CHO, COOH, COO(alkyl), COO(aryl), PO3H2, SO3H, or CONR 31 R 32 and a hydrocarbon group having 1 to 20 carbon atoms, and / or can be fused with one or more 3- to 7-membered rings, wherein the hydrocarbon group optionally includes one or more heteroatoms selected from N, O and S, and / or one or more substituents selected from halogen, OH, (O)alkyl, O(aryl), SH, S(alkyl), S(aryl), NH2, NH(aryl), NH(alkyl), N(aryl)2, N(alkyl)2, NO2, CHO, COOH, COO(alkyl), COO(aryl), PO3H2, SO3H, or CONR 31 R 32 ; R2represents NR 16 R 17 or R1and R2together are wherein R 18 , R 19 and R 22 are defined as R1, and R1is selected from the group consisting of H, F, Cl, Br, I, CN, CF3, CH3, OCH R 16 , R 17 , R 20 , R 21 are defined as R 11 ; or wherein at least one of R1to R 22 forms a ring system having one or more adjacent moieties.

2. The dye according to claim 1, wherein R1and R 16 and / or R3and R 17 form a ring system, resulting in one of the following structures (A), (B), (C), (D), (E), (F) and (G): wherein, adjacent substituents R can optionally form an additional ring system, which comprises 5- and / or 6-membered rings, which can optionally contain further heteroatoms or substituents. R in each case is independently defined as R1.

3. The dye according to claim 1 or 2, wherein R3 and R 17 and / or R1 and R 16 form a ring system: wherein, 4. The dye according to any one of claims 1 to 3, wherein R3 and R4 and / or R5 and R6 and / or R6 and R8 form a fused ring system selected from the group consisting of: R in each case is independently defined as R1. wherein, 5. The dye according to any one of claims 1 to 4, wherein Y in formula (I) and (II) is an optionally substituted cycloalkyl, an optionally substituted phenyl, an optionally substituted naphthyl, or an optionally substituted heteroaryl, having one of the following structures (H), (J), (K), (L), (M), (N) and (O): wherein Z denotes hydrogen or halogen, E represents oxygen, sulfur, nitrogen or + NR 23 , R u represents halogen, OR 24 , NHR 25 , NR 26 R 27 , and R in each case is independently defined as R1. R v represents Z, S(CH2) x COOH, S(CH2) x SO3H, NHR 28 , NR 29 R 30 , and R w represents hydrogen, alkyl, (CH2) x COOH, (CH2) x SO3H, (CH2) x OH, (CH2) x SH, (CH2) x NH2, (CH2) x Z, wherein x = 1, 2, 3, 4, 5, 6, R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 independently represent a linear, branched or cyclic, saturated or unsaturated, further hydrocarbon moiety having up to 20 carbon atoms, such as alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl, which further hydrocarbon moiety can comprise or be substituted by one or more heteroatoms selected from N, O and S, and / or is preferably substituted by one or more substituents selected from halogen, OH, (O)alkyl, O(aryl), SH, S(alkyl), S(aryl), NH2, NH(aryl), NH(alkyl), N(aryl)2, N(alkyl)2, NO2, CHO, COOH, COO(alkyl), COO(aryl), PO3H2, SO3H or CONR 31 R 32 ; 6. The dye according to any one of claims 1 to 5, wherein Y is selected from the group consisting of:

7. The dye according to any one of claims 1 to 6, wherein R2 is N(CH3)2.

8. The dye according to any one of claims 1 to 7, comprising at least one SO3H group.

9. The dye according to any one of claims 1 to 8, comprising at least one carboxyl group.

10. A compound having the general formula (I), (II), (III) or (IV) wherein in the compound of formula (I) the following do not occur simultaneously: X represents CR 11 R 12 , SiR 13 R 14 , sulfur, SO2or P(O)OR 15 ; Y represents hydrogen, a nitrile group, a carboxyl group, a carboxylic acid derivative, an amino group or a linear, branched or cyclic, saturated or unsaturated hydrocarbon moiety having up to 40 carbon atoms, which hydrocarbon moiety can comprise or be substituted by one or more heteroatoms selected from N, O and S, and / or is preferably substituted by one or more substituents selected from halogen, OH, (O)alkyl, O(aryl), SH, S(alkyl), S(aryl), NH2, NH(aryl), NH(alkyl), N(aryl)2, N(alkyl)2, NO2, CHO, COOH, COO(alkyl), COO(aryl), PO3H2, SO3H or CONR 31 R 32 ; R1, R3, R4, R5, R6, R7, R8, R9, R 10 independently represent hydrogen, halogen, hydroxyl, thiol, amino, sulfo, phospho, nitro, carbonyl, carboxyl, carboxylic acid derivative, nitrile, isonitrile, cyanate, thiocyanate, isothiocyanate, or a linear, branched, or cyclic, saturated or unsaturated hydrocarbon moiety having up to 20 carbon atoms, which hydrocarbon moiety can comprise or be substituted by one or more heteroatoms selected from N, O, and S, and / or, preferably, one or more substituents selected from halogen, OH, (O)alkyl, O(aryl), SH, S(alkyl), S(aryl), NH2, NH(aryl), NH(alkyl), N(aryl)2, N(alkyl)2, NO2, CHO, COOH, COO(alkyl), COO(aryl), PO3H2, and SO3H or CONR 31 R 32 ; R 11 , R 12 , R 13 , R 14 , R 15 , R 31 , R 32 independently represent hydrogen or a hydrocarbon group having 1 to 20 carbon atoms, wherein the hydrocarbon group optionally includes one or more heteroatoms selected from N, O and S, and / or is preferably substituted with one or more substituents selected from halogen, nitrile, hydroxyl, thiol, amino, nitro, sulfo, phospho, carbonyl, carboxyl, carboxylic acid derivative; or R 11 and R 12 or R 13 and R 14 together with the atoms to which they are attached form a 3- to 7-membered ring, wherein the ring can include one or more double bonds, and / or one or more heteroatoms selected from N, O, and S, and / or one or more substituents selected from halogen, OH, (O)alkyl, O(aryl), SH, S(alkyl), S(aryl), NH2, NH(aryl), NH(alkyl), N(aryl)2, N(alkyl)2, NO2, CHO, COOH, COO(alkyl), COO(aryl), PO3H2, SO3H, CONR 31 R 32 and a hydrocarbon group having 1 to 20 carbon atoms, and / or it can be fused with one or more 3- to 7-membered rings, wherein the hydrocarbon group optionally includes: one or more heteroatoms selected from N, O and S, and / or one or more substituents selected from halogen, OH, (O)alkyl, O(aryl), SH, S(alkyl), S(aryl), NH2, NH(aryl), NH(alkyl), N(aryl)2, N(alkyl)2, NO2, CHO, COOH, COO(alkyl), COO(aryl), PO3H2, SO3H or CONR 31 R 32 ; R2represents NR 16 R 17 or R1and R2together are wherein R 18 , R 19 and R 22 are defined as R1, and R1is selected from the group consisting of H, F, Cl, Br, I, CN, N02, CF3, CH3 R 16 , R 17 , R 20 , R 21 is defined as R 11 , or wherein at least one of R1to R 22 forms a ring system having one or more adjacent moieties, but subject to the following conditions: X denotes C(CH3)2; R2 denotes N(CH3)2; and in the compound of formula (I) the following do not occur simultaneously: wherein R 33 represents H or CH3; and X denotes C(CH3)2; R2 denotes N(CH3)2; and in the compound of formula (I) the following do not occur simultaneously: and X denotes C(CH3)2; R2 denotes N(CH3)2; and in the compound of formula (I) the following do not occur simultaneously: wherein R 33 represents H or CH3; and X denotes Si(CH3)2; R2 denotes NH2, NH(CH3), N(CH3)2 or NH-C(O)CH3; and Y denotes 1-methyl-phenyl, 11. Use of the dye according to any one of claims 1 to 9 or the compound according to claim 10 as a pH sensor. and wherein X represents CR 11 R 12 The compounds of formula (III) comprise at least one sulfo group.

12. A wound dressing comprising the dye according to any one of claims 1 to 9 and / or the compound according to claim 10.

13. Use of the dye according to any one of claims 1 to 9 or the compound according to claim 10 as a labeling group in the qualitative and / or quantitative determination of an analyte in a sample. ​ 14. Use of a dye according to any one of claims 1 to 9 or of a compound according to claim 10 as a pH sensor according to claim 11 or as a labeling group according to claim 13, which comprises binding the dye or the compound to the analyte or substrate.

15. A process for the preparation of a dye or compound of formula (I), (II), (III) or (IV) as defined in any one of claims 1 to 10, said process comprising the following steps: a) conversion of a precursor compound by a one-pot synthesis in one step using an aqueous alkali or alkaline earth hydroxide solution as reaction medium; b) using lithium hydroxide, sodium hydroxide, potassium hydroxide, cesium hydroxide, calcium hydroxide or barium hydroxide, or a mixture of different hydroxides; c) using a temperature of at least 0 °C, more preferably a temperature in the range of 25 °C to 100 °C, or even more preferably a temperature in the range of 50 °C to 85 °C; and d) employing a co-solvent, which is preferably selected from acetone, acetonitrile, ethanol or methanol. ​ ​ ​ ​

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