WO2004041840A2

WO2004041840A2 - Peptide-bound chromogens used for determining enzymatic activities

Info

Publication number: WO2004041840A2
Application number: PCT/EP2003/012463
Authority: WO
Inventors: Stefan Spichiger; Ursula Spichiger-Keller; Michael Linnhoff; Gleb Zhylyak
Original assignee: C-Cit Ag; Fluka Gmbh
Priority date: 2002-11-07
Filing date: 2003-11-07
Publication date: 2004-05-21
Also published as: DE10251894A1; AU2003286158A8; AU2003286158A1; WO2004041840A3

Abstract

The invention relates to a chemical compound of a general formula (I): X-A-B-C-L-CRS, wherein X is hydrogen or a protection group of amino-terminal groups which are routine for peptide chemistry; A and B are a α-, β-, or η-aminoacids containing from 2 to 15 C atoms, up to 4 N atoms, up to 2 S atoms and up to 6 O atoms, A and B can be identical or different, and B eventually being a peptide formed by said amino-acids; C is arginine, lysine, tyrosine, phenylalanine or tryptophan of the homologues respectively ; L is a linker forming a peptide liaison with C ; CRS is a combined cyclic carbon system linked to a L-C-B-A-X radical by means of a nitrogen atom. The inventive compound of said general formula (I) and CRS produce a maximum absorption having a wavelength equal to or higher than 550 nm.

Description

PEPTΓDBUNDENE CH OMOGENE FOR DETERMINING ENZYM ACTIVITIES

The determination of enzyme activities is important, for example, for the diagnosis of disease states such as thromboembolism, fibrinolysis, arthritis, wound healing or when used to monitor anticoagulant therapy in laboratories or hospitals.

The determination of protease activities is also important for the diagnosis of carcinoma cells or the development of inhibitors. In so-called drug screening, active substances are tested for their inhibitory effects from protease activities.

Furthermore, the determination of the enzymatic activity is used as a means for the detection of pyrogens, for example endotoxins, which can occur during sterilization in solutions to be injected by the so-called LAL (Limulus amebocyte lysate) test. With the LAL test, endotoxin of gram-negative bacteria can be detected enzymatically by determining the proteolytic activity of the enzymes used.

In addition to turbidimetric determination, the absorption properties of chromogens are used in the LAL test.

Chromogens are molecules that can absorb electromagnetic radiation, such as visible light, UV or IR. Usually Chromogenic characterized by extensive conjugated compound systems in which the π-lT ^"transitions are shifted into the visible.

The proteolytic activity of the enzyme is measured in the LAL test as an increase in the optical density when a peptide bond is cleaved between a specific recognition sequence of a peptide and a dissociable chromogen (Witt, I. 1991). The split the peptide bond leads, for example in the case of a chromogen labeled with p-nitroaniline, to a change in the absorption spectrum of the released chromogen compared to the chromogen bound to the peptide.

This change in absorption can be determined and thus also indirectly the proteolytic activity of the corresponding enzyme.

A disadvantage of p-nitroaniline and 7-amino-4-methylcoumarin (AMC) labeled chromogens, however, is that they have absorption maxima in the wave range of <400 nm and are evaluated at 405 nm, in which cell components, for example hemoglobin, also absorb electromagnetic radiation. It is therefore difficult to determine specific enzyme activities in biological fluids, such as blood or other body fluids, with chromogens labeled in this way.

Chromogens are also known from the prior art which have their absorption maxima in other wavelength ranges, for example in a wavelength range of> 600 nm. However, their use is problematic for the LAL detection of enzyme activities, since the enzymatic attack by the target protease in the labeled chromogen is mostly incomplete or very slow due to the size and spatial structure of the chromogen. This is because the enzyme's access to the corresponding recognition sequence is hindered by the molecular structure of such chromogens.

The measurement of changes in the absorption maxima of chromogens which are directly bound to the substrate is also problematic. These may only have a limited molecular size in order not to hinder the enzymatic attack by the target protease. Known examples are 7-amino-4-methylcurnarin (AMC), p-nitroaniline and 2-aminoacridone (2-AA). There was therefore a need for an optical detection possibility for target substances, for example endotoxins, which is not adversely affected by the presence of natural components in the blood or other body fluids.

There was also a need for compounds with which target substances, such as endotoxins, can be clearly and directly or indirectly determined even in low concentrations.

There was also a need for a method for producing such compounds and for a simple and inexpensive method with which even low concentrations of certain target substances can also be specifically detected in biological liquids.

The object of the invention was therefore to meet one or more of the above-mentioned needs.

The invention therefore relates to novel chromogens which are coupled to peptide sequences and which absorb at wavelengths in the range from approximately 530 to 700 nm, preferably from 630 nm to 700 nm.

The present invention relates to a chemical compound of the general formula (I):

X-A-B-C-L-CRS

where X is hydrogen or a protective group for terminal amino groups which is customary in peptide chemistry,

A and B stand for a natural or synthetic -, β-, or γ-amino acid and its stereoisomers / enantiomers, which have 2 to 15 C atoms and up to 4 N atoms, up to 2 S atoms and up to 6 O. -Atoms include and where A and B are identical or can be different and B optionally represents a dipeptide or oligopeptide, in particular a dipeptide to dodecapeptide, which is formed from these amino acids,

C stands for natural amino acids, in particular arginine, lysine, tyrosine, phenylalanine, glycine, methionine, lencin, sarin or tryptophan or their respective homologues and enantiomers / stereoisomers and isologues,

L stands for a linker which is capable of forming a peptide bond with C, or which is itself an amino acid or an oligopeptide.

CRS stands for a conjugated carbon ring system which is connected to the radical -L-C-B-A-X via a nitrogen atom and the compound of the general formula (I) has an absorption maximum at a wavelength of more than 530 nm.

In the context of the present invention, the conjugated carbon ring system CRS is to be understood in principle as meaning any carbon compound which has its absorption maximum in the range from about 530 nm to about 850 nm, preferably in the range from 600 to 700 nm and more preferably in the range 630 to 680 nm, which has at least four conjugated double bonds and at least one carbon ring, preferably 2, 3 or 4 carbon rings.

The carbon ring can be a continuous acyclic or aromatic carbon chain with 3 to 9 carbon atoms, but it is also possible for the ring to have one or more identical or different substituents, or heteroatoms, for example oxygen, sulfur or nitrogen. The conjugated carbon ring system according to the invention preferably contains at least one carbon ring with a continuous carbon chain.

In the context of the present invention, the term "linker" L (synonymously the term "spacer" can also be used) denotes any compound which is capable of forming a peptide bond with C and which is responsible for the spatial arrangement of CRS and the The rest of the -CBAX is affected, in particular the distance between the CRS and the rest of the -CBAX is increased. Furthermore, the linker is characterized in that it is connected to CRS via an N atom. This can also take the form of an imine or imide bond. The nature of the linker essentially only has to meet the condition that the proteolytic cleavage of the peptide bond between L and C is possible by means of a suitable enzyme.

L can therefore be, for example, a linear or branched, saturated or unsaturated alkyl (s) radical having 1 to about 40 C atoms, preferably 1 to 15, more preferably 1 to 10 C atoms, but it is also possible for L Contains cycloalkyls.

L can furthermore represent, for example, one or more, for example 2, 3, 4, 5, 6 or more amino acids, furthermore e.g. represent a p-aminobenzoic acid, a phenydiamine, diamine, amine, aniline, substituted aniline, substituted aromatics, alkaloid, thiol, thiolates, thiocyanate, aniline, aminocarboxylic acid, substituted or unsubstituted phenol, nitroaromatic or derivatives of the above-mentioned compounds.

However, the linker should have stability that does not allow all of the CRS-LCBAX compound to be exposed to air, water, alcohol and / or fats or other organic or non-organic solvents at temperatures up to about 40 ° C without the presence of enzymes reacts in such a way that the basic structure of the CRS-LCBAX connection is changed.

In the context of an embodiment of the present invention, it has proven to be advantageous if the linker has at least the length of an ethyl, in particular at least one, propyl radical.

Depending on the nature of the structural element CRS, it can also be advantageous to make the linker significantly longer or to introduce a further aromatic compound or a further amino acid or an oligopeptide.

In a particular embodiment, for example, 4-aminobenzoic acid and 1,4-phenylenediamine and their homologs and derivatives are suitable as linkers between the structural element CRS and -CBAX. Further non-limiting examples of linkers which can be used according to the invention are hydroquinones, quinones, aminoquinones, quinolines, aminocarboxylic acids, dicarboxylic acids (or their anhydrides), γ-aminobutyric acid, carboxamides, aminophenols, 2- or 3- or 4-hydroxyanilines and their homologues.

The absorption maximum of the chemical compound according to the general formula (I) is preferably at lower wavelengths than that of the free structural element CRS alone. Suitably, in a preferred embodiment, in addition to the usual selection of corresponding structural elements CRS with a suitable absorption maximum, this can also be done by choosing the linker which, in addition to the absorption maximum, also contains other physical parameters of the compound (I), such as the degree of protonation (pKa) and its photostability. The following applies in particular to the following three specific classes of compounds:

The linker has a slight influence on the UV / VIS spectrum with Nilblau and Meldolablau derivatives, but a significant influence on the pKa and the photo stability. In the case of naphthoquinone derivatives, a hypsochromic shift usually occurs due to the influence of the linker.

The compounds according to the invention are characterized by the property that the structural element CRS (-L) can be cleaved enzymatically from the rest X-A-B-C- and that after this cleavage the absorption maximum of the structural element CRS (- L) shifts bathochromically, that is to say towards longer wavelengths.

In the context of the present invention, it has proven to be advantageous if this bathochromic “shift” comprises at least 30 nm, preferably at least 50 (in particular i and more preferably at least 100 nm.

In a preferred embodiment, the bathochromic shift is about 30 to about 100 nm for naphthoquinones, for p-nitroaniline it is about 100 nm measured in absorption and for AMC derivatives 400 to about 470 nm. With fluorescence emission, this shift is about 70 nm when excited with 330 or 380 nm. The above-mentioned enzymatic cleavage of the structural element CRS (-L) from the rest of the chemical compound of the general formula (I) is carried out by enzymes as defined below.

Enzymes in the sense of the invention are highly specialized proteins that catalyze biochemical reactions without being consumed in the process. Ribozymes, which under the

The term enzymes can be subsumed include ribonucleic acid molecules. Enzymes outperform synthetic catalysts in their catalytic properties. Enzymes have a high affinity and selectivity for certain substrates. Important among the enzymes are in particular proteases, the so-called serine proteases I, such as trypsin, bacterial serine proteases II, such as subtilism,

Cysteine proteases such as papain, aspartate proteases such as penicillopepsin, metalloproteases I such as boron coride oxide peptidase A, bacterial metalloproteases II such as thermolysin. Proteases always digest at a certain point on a peptide or

Protein. Endopeptidases hydrolyze certain amide bonds within a protein, while exopeptidases C or N terminate one to three amino acids from the peptide.

The chemical compound according to the general formula (I) can comprise various structural elements CRS with regard to the electrical charge. For example, the structural element CRS can be neutral, anionic, cationic or zwitterionic. For example, in the case of the Nile and Meldola blue derivatives, the protonated form is preferably long-wave absorbent. In contrast, in the case of phenolic compounds, the deprotonated, negatively charged form is preferably long-wave absorbent.

However, the type and strength of the charge of the structural element CRS should not lead to reactions being triggered which would impair an enzymatic cleavage reaction of the structural element CRS (-L) from the chemical compound of the general formula (I). This means that the type and strength of the charge of the structural element CRS is selected according to the medium in which an enzymatic cleavage takes place (for example blood, serum, urine).

In the context of an embodiment of the present invention, the structural element CRS is neutral or basic. It advantageously has a pKa value of 6-13. Examples of suitable structural elements CRS in addition to the basic bodies Meldolablanlau and Nilblau are, for example, amino-substituted 1,4 naphthoquinones such as 6-amino-2,3-dichloro-l, 4-naphthoquinone and 6-amino-2,3-dicyano-l, 4- naphthoquinone). Also phenolic azo compounds such as calmagite (absorption max: 665 nm), naphthol blue black (absorption max: 680 nm); Brilliant yellow (absorption max: 554 nm), nitrazin yellow (absorption max: 620 nm); The 4 or 5 amino naphthoquinones can of course also be used. Other suitable chromogens are, for example, 2-anilino-3-chloro-l, 4-naphthoquinone, 2- (4-N, N-diethylamino-phenyl) -l, 4-naphthoquinone, 2,3-dicyano-l, 4-naphtholdiol , 2,3-dicyano-l, 4-naphthoquinone, 2,3-dicyano-l, 4-naphthoquinone-l-phenyleneimine, 4-nitro [2.2] paracyclophanes or other nitrated paracyclophanes and simple cyclophanes, indane derivatives such as 2 -p-amino benzylidene-l, 3-bisdicyanovinylindan and their derivatives or homologs.

The present invention furthermore relates to chemical compounds of the general formula (I) in which the structural element CRS is a compound selected from the following group of compounds (a), (b), (c) or (d):

(A)

(B)

(C)

(D)

^

where R is H, C \ to C ₈ alkyl, in particular methyl, ethyl, propyl, i-propyl, n-butyl, i-butyl, aryl, aralkyl, CN, halogen,

R 1 and R _{2 can be the} same or different and represent H, CN, halogen, aryl or aralkyl or alkoxy,

R ₃ , Ri, R ₅ , R _<5 , R ₇ and R _{8 can be the} same or different and for H, CN, NO ₂ , NH ₂ , - HSO ₃ ^" , COO ^" , -OH (-O ^" ) - SO ₃ ^" , alkyl, aralkyl, halogen, in particular CI, Br, or alkoxy,

R9 H, halogen, SH, SR, OH, OR, carboxylic acid, aminocarboxylic acid, HSO ₃ ^" , COO ^" , -OH (- O ^" ) -SO ₃ ^" , an azo or diazo group, substituted or unsubstituted aromatic such as nitroaromatic, halogenated aromatics, thiocyanate, unsubstituted or unsubstituted phenol means and with the condition for compound (c) that at least one radical from R ₃ to _{Ö is} an amino group, one nitrogen substituent of which is linked to the radical of the compound of the general formula (I).

In particular, the radical R _{9 influences} the solubility of the compound, which can be adjusted in accordance with the respective radical R ₉ .

In the context of a particular embodiment of the present invention, those chemical compounds are particularly preferred which have a structural element CRS which, after dissociation from the rest of the compound of the general formula (I), has a low water solubility. In the context of the present invention, “low water solubility” means values in the range from about 50 mg / 1 to about 1 mg / 1.

According to a particularly preferred embodiment of the present invention, the water solubility of the structural element CRS is in the range from about 10 to about 0.1 mg / l.

The present invention further provides a process for the preparation of a compound of the general formula (I) comprising the following steps:

1. Preparation of an oligopeptide XAB- under customary conditions, where X is hydrogen or a protective group which is customary in peptide chemistry for terminal amino groups, A and B are an α-, β- or γ-amino acid which are 2 to 15 C- Comprise atoms and up to 4 N atoms, up to 2 S atoms and up to 6 O atoms and where A and B can be identical or different and B optionally represents a dipeptide which is formed from these amino acids,

2. Production of CRS-L, where L stands for a linker as defined above which is capable of forming a peptide bond with C and where the linker is preferably covalently bound to an N atom of the structural element CRS and where CRS is for a is as defined above conjugated carbon ring system which has an absorption maximum at a higher wavelength than the compound XAB-CL-CRS (I), whose absorption maximum is preferably in the range of <530 nm, 3. Reacting the CRS-L thus formed with C under customary conditions to CRS-LC, where C is arginine, lysine, tyrosine, phenylalanine or tryptophan or other amino acids and their stereoisomers or their respective homologues

4. Convert X-A-B and CRS-L-C to X-A-B-C-L-CRS

In the context of the method according to the invention, steps (1) and (2) can be carried out in any order.

The present invention furthermore relates to a method for the detection of target substances, comprising the following steps:

the provision of compounds of the general formula (I),

- the determination of the wavelength at which the compounds have their absorption maximum if the wavelength is not known,

- bringing the provided connections into contact (and if necessary allowing them to act) with the solution, emulsion, gas intended for the detection,

Aerosol, mixture or pure substance,

- The determination of the wavelength at which the compounds, which were provided in the first step, after the provided compounds have been brought into contact (and allowed to act) with the solution, emulsion, gas, aerosol, mixture or pure substance intended for the detection, their absorption maximum after changing the pH with acid, base or buffer

According to the invention, biosensors are preferably used to detect the target substances. Biosensors are based on a coupling of biomolecules that recognize specific analytes as receptors in the broadest sense with physicochemical transducers that convert a biologically generated signal into electrical measurement signals. Bioactive recognition components include, but are not limited to, antibodies, enzymes, Cell organelles or whole microorganisms. Peptides and proteins are also considered

Called biomolecules. However, peptides are not bioactive in that they can convert other molecules or promote specific bonds to other molecules. at

A distinction is made between two basic types of biosensors: on the one hand the so-called bioaffinity sensors and on the other hand the so-called

Metabolism sensors. The bioaffinity sensors generate a signal by complexing bioactive molecules to form an analyte. Here lies after the

Measurement again the initial state. While the latter on a specific

Detection and subsequent implementation of substrates are based. After the measurement process, this sensor must either be discarded or the initial state restored.

In the present invention, enzyme activities are to be determined, the enzymes causing a change in the absorption spectrum of the substrate by proteolysis of the corresponding substrate.

In the context of the present invention, it has been found to be advantageous to determine the absorption changes with the so-called attenuated total reflection spectroscopy (ATRS). Furthermore, the use of a planar thin-film waveguide chip, which is chemically e.g. modified with a selective chemical polymer was suitable for an enzyme test, in particular for an endotoxin test. The coupled light travels within a planar waveguide and creates an evanescent field along the surface. The evanescent field migrates within the neighboring medium and has a penetration depth of a few nanometers (approximately 10 nm) in the Z direction of the traveling light. The absorption of light using the ATRS technique within the adjacent layer correlates with the change in the concentration of the specific analyte. The ATR technology has the advantage that CRS, CRS-L and CRS-L-C-B-A-X do not need to have different absorption wavelengths and generally do not have either. On the other hand, simple local separation is possible with pH adjustment.

In order to enable a particularly efficient detection method, chromogen-labeled substrates can be used, for example, on a chip such as that in the unpublished German patent application DE 10251893.9 is described as a so-called waveguide chip, applied and immobilized. For example, the chip surface is chemically modified and functionalized using two different methods:

a) By covalent binding (immobilization) of the above-defined chromophores and chromogenic substrates on the activated, pretreated surface of the chip, for example one using a spacer (avidin-biotin, suberic acid, hydrocarbon chains, hexylenediamine, sulfhydryl groups).

b) By nano-structured immobilization or physicochemical adsorption of chromogenic and chromophoric substrates (specific oligopeptide that is labeled with one of the aforementioned dyes) on the waveguide chip.

For example, chromogens can be coupled to a peptide, such as BOC-Ser (Bz) -Gly-Arg, via suitable linkers and immobilized on a waveguide of an optical chip at the N-terminal. The proteolysis is detected by measuring the change in absorption in the region of the evanescent field of the waveguide, which is induced by the chromogen's diffusion from the path. The chromogen only needs to have a low water solubility, since it is released in very small quantities based on the volume of the measuring cell.

Within the polymeric membrane, the chromogenic part of the chemical compound according to the general formula (I) can show a spectral shift due to various mechanisms. If the chromogenic part of the chemical compound according to the general formula (I) is cleaved from the immobilized peptide after adding the enzyme (eg serine protease), the chromogenic part (CRS or CRS-L) can change its absorption spectrum or at least its distribution and diffuse from the detection layer closer to the surface of the waveguide (diffusion-restricted process). The layers of the detection area can be applied, for example (using a "Genesis NPS" pipettor) with an active Tip M (volume) from Tecan. The substrates can be applied as optically active materials in defined areas on the surface of the optical chip.

Another object of the present invention is the use of compounds according to the invention for the detection of target substances, in particular the use for the detection of endotoxins.

The present invention is explained in more detail below by means of exemplary embodiments without restricting the invention to these.

pictures

The invention is explained below using examples and examples. It is clear that the features explained above and to be described below are claimed not only in combination but also on their own.

Fig. 1 shows a synthetic scheme for the representation of a peptide labeled with an amino naphthoquinone.

Fig. 2 shows the cleavage of the LAL substrate serine (BZ) -Gly-Arg-L-LM46 (linker: p-aminobenzoic acid, chromophore: Nile blue) in TRIS buffer pH 7 at 37 ° C with bovine trypsin 10 ^"6 M Measurement at 641 nm.

Fig. 3 shows the cleavage of the LAL substrate serine (BZ) -Gly-Arg-L-LM46 (linker: p-aminobenzoic acid, chromophore: Nile blue) in TRIS buffer pH 7 at 37 ° C with bovine trypsin 10 ^"6 M Measurement at 674 nm. Exemplary embodiments and description of the figures

For example, the following substrates were produced within the scope of the invention:

1 . BOC-Ser (Bn) -Gly-Arg (HCl) -p-NA (p-NitroaniIin): This substrate was used as a reference for enzymatic cleavage test in solutions. Measurements on a chip were not possible because the wavelength of approximately 405 nm is not accessible with the laser spectrometer.

2. Nile blue as a chromogen which is coupled to 4-aminobenzoic acid (as a linker) in the form of a carboxylamide.

3. Meldolablanlau as chromogen, which is coupled to 1,4-phenylenediamine (linker). This further leads to a 4 - ((benzyl-L-serinyl-L-glycyl) -L-arginylamindo) -N- (9-diethylamino-benzo [a] phenoxazin-5-ylidene) aniline.

Substrates 1 and 2 (hereinafter also referred to as LAL substrates) were cleaved by trypsin, although there was no spectral shift. However, a change in the absorbance at 641 and 674 nm could be detected in solution, since the chromogen was cleaved from the substrate and is almost insoluble in solution. The absorption spectra and the absorption coefficients were characterized in different solvents at a pH of 7 and a temperature of 37 ° C.

Surprisingly, it has been found that aminonaphthoquinones meet all the requirements for a chromogen that is coupled to a peptide. Examples of naphthoquinones according to the invention are: Scheme 1: 5- and 6-amino-l, 4-naphthoquinones (ANC)

Aminonaphthoquinones are a class of chromogens, the spectra of which change after enzymatic hydrolysis, in particular due to a change in the absorption maximum towards longer wavelengths. In these chromogens, the chromogen is linked via the amino-terminated group directly to the peptide (arginine) at the 6-amino position and changes its spectrum after hydrolysis of the amide group. Here too, the amino-1,4-naphthoquinones are optionally linked to the peptide sequence via a linker.

While compound 4 in ethanol has the longest wavelength absorption band at 479 nm in the uncharged state, this is bathochromically shifted by 200 nm in the protonated state. With 6-amino-2,3-dicyano-l, 4-naphthoquinone (6) there is a further bathochromic shift around 86 nm for the absorption band, due to the more electron-withdrawing cyano substituents in 5 compared to the chlorine substituents. As a result, the absorption band of 6 in ethanol is around 598 nm.

By coupling the aminonaphthoquinone (ANC) chromogen to the peptide, for example, the substrate BOC-Ser- (Bz) -Gly-Arg-6-ANC was generated; the peptide substrate showed an absorbance of A <0.06 at a wavelength λ = 479 nm and a baseline absorbance at 550 nm. The addition of trypsin at a pH of 7.4 in ethanol quickly showed an absorption maximum of free ANC at one Wavelength λ = 479 nm and a second corresponding wavelength λ = 550 nm.

By comparing the effects of electron-accepting substituents on the absorption spectra of existing chromophores (7-amino-4-methylcoumarin and 2-aminoacridone), it was found that the bathochromic effect of electron donor substituents is on the order of up to approximately 100 nm. υ i -> ΔΛIIUM 19

Abbreviations:

DMF N, N-dimethylformamide

NEt ₃ triethylamine

EE ethyl acetate

TLC thin layer chromatography

IBCF isobutyl chloroformate

AcOH glacial acetic acid iPrOH 2-propanol

BOG B enzy loxy carbonyl

MeOH methanol

RF retention factor

«BuOH ra-butanol

ANC aminonaphthoquinone

TsCC tosyl chloride

EtOH ethanol

DCM dichloromethane

FC flash chromatography

example 1

Nile blue as a chromogenic building block

Synthesis of 4 - ((benzyl-L-senήnyl-L-glycyl) ~ L ~ arginylamido) -N- (9-diethylamino-benzo [a] phenoxazin-5-ylidene) benzoic acid amide

1.1. Synthesis of 4-amino-N- (9-diethylamino-heήzo [a] phenoxazin-5-ylidene) benzoic acid amide

Step A: 4-nitro-N- (9-diethylamino-benzo [a] phenoxazin-5-ylidene) benzoic acid amide

11.3 g of Nilblau A were stirred into 140 ml of DMF and 11 g of NEt ₃ were added to the mixture, followed by 7.2 g of N-oxy-succinimidyl 4-nitrobenzoate. After stirring overnight, the solvent was removed in vacuo and under high vacuum, the residue was adsorbed on silica gel 60 and the title compound was applied to a chromatography column filled with CHC1 ₃ / EE 8: 1 (v / v) and filled with silica gel 60 with the same solvent mixture eluted. Yield: 5.6 g Purity control: DC R ^ = 0.60 (CHOb / EE 11: 1 v / v)

Step B: 4-amino-N- (9-diethylamino-benzo [a] phenoxazin-5-ylidene) benzoic acid amide

4 g of 4-nitro-N- (9-diethylamino-benzo [a] phenoxazin-5-ylidene) benzoic acid amide were dissolved in 70 ml of glacial acetic acid and 10 g of zinc dust were added with stirring. After stirring for half an hour, the solvent was largely removed in vacuo and the residue was transferred into a shaking funnel with CHCI ₃ . The organic phase was washed with 2N NaOH and dried over MgSO ₄ . After removing the solvent in vacuo, the remaining residue was purified by chromatography on silica gel 60 (CHCl ₃ / MeOH 40: 1 v / v). Yield: 2.2 g purity control: DC R _F = 0.24 1.2 Synthesis of (4 - ((Benzyl-L-serinyl-L-glycyl) -L-arginylamido) -N- (9-diethylamino-benzo [a] phenoxazin-5-ylidene) benzoic acid amide)

Step A:

4- (N _a N _& N _ω -tri-ZL-arginylamido) -N- (9-diethylamino-benzo [a] phenoxazin-5-ylidene) benzoic acid amide

2.88 g of N _α , N _δ , N _ω -tri-ZL-arginine were dissolved in 8 ml of abs. DMF dissolved in the heat. After cooling to room temperature, 0.7 ml of NEt ₃ and 0.65 ml of IBCF were added and the resulting mixture was stirred for 20 minutes. Then 1.32 g of 4-amino-N- (9-diethylamino-benzo [a] phenoxazin-5-ylidene) benzoic acid amide were added and the reaction mixture was stirred at room temperature for 12 h. The solvent was removed in a high vacuum and the residue was purified by chromatography on silica gel 60 (CHCl ₃ / EtOH 30: 1 v / v). Yield: 2.89 g purity control: DC R _F = 0.34

Step B: 4- (L-arginylamido) - N- (9-diethylamino-benzo [a] phenoxazin-5-ylidene) benzoic acid amide

750 mg of 4- (N _α , N _δ , N _ω -tri-ZL-arginylamido) - N- (9-diethylamino-benzo [a] phenoxazin-5-ylidene) benzoic acid amide were dissolved in 20 ml of dioxane and rinsed with 30 ml of MeOH was added to a pre-hydrogenated suspension of 500 mg of palladium on activated carbon (10%, about 50% water content) in 50 ml of MeOH. After 3 h of hydrogenation with vigorous stirring under 1.1 bar of H ₂ , 12.5 ml of 1% methanolic HCl were added to the reaction mixture and the same was filtered through Celite. After the filtrate had been freed from the solvent in vacuo, the residue was purified by chromatography on silica gel 60 (zPrOH / AcOH / H ₂ O 3: 2: 2 v / v / v). Yield: 250 mg purity control: TLC R ^ = 0.20 Step C: 4 - (((N _a -BOC- (benzyl-L-serinyl)) - L-glycyl) -L-arginylamido) -N- (9-diethylamino-benzo [a] phenoxazin-5-ylidene) benzoic acid amide

453 mg 4- (L-arginylamido) - N- (9-diethylamino-benzo [a] phenoxazin-5-ylidene) benzoic acid amide and 433 mg N _α -BOC- (benzyl-L-serinyl)) - L-glycyl-N - A mixture of 7.5 ml of DMF and 91 mg of NEt _{3 was} added to the oxysuccinimidyl ester and the resulting solution was stirred for 12 h. After removal of the solvent in a high vacuum, the resulting residue was purified by chromatography on silica gel 60 (z'PrOH / AcOH / H ₂ O 4: 1: 1 v / v / v). Yield: 350 mg purity control: DC R _F = 0.18

Step D: 4 - ((Benzyl-L-serinyl-L-glycyl) -L-arginylamido) -N- (9-diethylamino-benzo [a] phenoxazin-5-ylidene) benzoic acid reamide

222 mg of 4 - (((N _α -BOC- (benzyl-L-serinyl)) - L-glycyl) -L-arginylamido) -N- (9-diethylamino-benzo [a] phenoxazin-5-ylidene) benzoic acid amide in a mixture of 10 ml DCM and 10 ml TFA for 2 h at room temperature. The solvent was removed in vacuo and then in a high vacuum and the residue was purified by chromatography on silica gel 60 (CHCl ₃ / MeOH / AcOH 1: 2: 2 v / v / v). Yield: 180 mg purity control: DC R ^ = 0.17 (zPrOH / AcOH / H ₂ O 2: 1: 1 v / v / v)

Example 2

Meldola blue as a chromogenic building block

Synthesis of 4 - ((benzyl-serinyl-L-glycyl) -L-arginylamido) -N- (9-diethylamino-benzo [a] phenoxazin-5-ylidene) aniline

2.1 Synthesis of 4-amino-N- (9-dimethylamino-benzo [a] phenoxazin-5-ylidene) aniline

Step A:

4-nitro-N- (9-dimethylamino-benzo [a] phenoxazin-5-ylidene) aniline (ETH ^T 6005)

7.03 g of Meldola blue and 3.28 g of 4-nitroaniline were placed under argon. Then 172 ml of DCE dried over molecular sieves and 8 ml of NEt ₃ also dried over molecular sieves were added and the resulting suspension was refluxed for 12 h. After cooling, 110 g of neutral aluminum oxide (activity level III) were added to the RG and concentrated in vacuo and then in a high vacuum. The crude product was then chromatographed on 200 g of neutral aluminum oxide (activity level III) (gradient from hexane / DCM 1/1 to pure DCM). The resulting product was recrystallized from DCM / EE. Yield: 2.7 g purity control: DC R _F.

Step B: 4-amino-N- (9-dimethylamino-benzo [a] phenoxazin-5-ylidene) aniline

A suspension of 413 mg of 4-nitro-N- (9-dimethylamino-benzo [a] phenoxazin-5-ylidene) aniline in 8 g of glacial acetic acid was mixed with 1.1 g of zinc dust at RT with stirring. The previously deep blue suspension turned into a light green solution. After 15 min the RG was largely freed of AcOH in vacuo and into one with DCM Shake funnel transferred. It was washed with 2 N NaOH and the aqueous phase was extracted again with DCM. After drying over MgSO ₄ and concentration in vacuo, the title compound was obtained in sufficient purity for the subsequent coupling. Yield: 346 mg purity control: TLC R ^ = 0.45 (MeOH / AcOH 2: 1 v / v)

2.2 Synthesis of 4 - ((benzyl-L-serinyl-L-glycyl) -L-arginylamido) -N- (9-diethylamino-benzo [a] phenoxazin-5-ylidene) aniline

Step A:

4- (N _a , N _δ , ^~ N _ta -tri-ZL-arginylamido) - N- (9-dimethylamino-benzo [a] phenoxazin-5-ylidene) aniline

378 mg of N _α , N ₅ , N ₍₀ -tri-ZL-arginine were dissolved under argon in 1.2 ml of absolute DMF while hot. After cooling to room temperature, 66 mg of absolute NEt ₃ and 90 mg of IBCF were added and the resulting The mixture was stirred for 20 min, after which a solution of 250 mg of 4-amino-N- (9-dimethylamino-benzo [a] phenoxazin-5-ylidene) aniline in 3.5 ml of DMF was added and the reaction mixture was stirred at room temperature for 12 h The solvent was removed under high vacuum and the residue was purified by chromatography on silica gel 60 (CHCl ₃ / MeOH / AcOH 13: 1: 1 v / v / v) The resulting acetate was taken up in DCM, washed with 2 N NaOH and the organic Phase dried over K ₂ CO ₃ and concentrated in vacuo Yield: 338 mg purity control: TLC R = 0.21

Step B: 4- (L-arginylamido) -N- (9-dimethylamino-benzo [a] phenoxazin-5-ylidene) aniline

200 mg of 4- (N _α , N _δ , N _ω -tri-ZL-arginylamido) - N- (9-dimethylamino-benzo [a] phenoxazin-5-ylidene) aniline were suspended in 18 ml MeOH or partially dissolved and added a pre-hydrogenated suspension of 155 mg palladium on activated carbon (10%, about 50% Water content) in 6 ml of MeOH. After 2 hours of hydrogenation with vigorous stirring under 1.1 bar of H ₂ , a few drops of glacial acetic acid were added to the reaction mixture and the same was filtered through cellite. After the filtrate had been freed from the solvent in vacuo, the title compound was obtained in sufficient purity for the subsequent reaction. Yield: 105 mg purity control: DC R _F.

Step C:

4 - (((N _a -BOC- (benzyl-L-serinyl)) - L-glycyl) -L-arginylamido) -N- (9-dimethylamino-benzo [a] phenoxazin-5-ylidene) aniline

73 mg N _α -BOC- (benzyl-L-serinyl)) - L-glycyl-N-oxysuccinimidyl ester and 81 mg 4- (L-arginylamido) -N- (9-dimethylamino-benzo [a] phenoxazin-5-ylidene ) Aniline was mixed with a mixture of 1 ml DMF and 22 mg NEt and the resulting solution was stirred for 12 h. After removal of the solvent in a high vacuum, the resulting residue was purified by chromatography on silica gel 60 (“BuOH / AcOH / H ₂ O 5: 1: 1 v / v / v). Yield: 65 mg purity control: TLC R ^ = 0.23

Step D: 4- (fBenzyl-L-serinyl-L-glycyl) -L-arginylamido) -N- (9-diethylamino-benzo [a] phenoxazin-5-ylidene) aniline

Example 3

Naphthoquinone as a chromogenic building block

Compound 4 was first synthesized according to the method described in the literature (cf. experimental part) and then coupled to Z-Arg (Z ₂ ) -OH (13). After deprotection to 16, the synthesis of the substrate 18 BOC-Ser (Bz) -Gly-Arg-6ANC was carried out without difficulty (see Scheme 2). Scheme 2: Scheme of the syntheses of 6-amino-l, 4-naphthoquinone (4) and BOC-Ser (Bz) - Gly-Arg-6ANC (18)

C ₁₀ HgNQ 159.18

12

C ₁ 0H7NO2 173.17

Synthesis strategy to display chromogen labels 4, 5 and 6

The following path was chosen for the representation of 6:

C ₁₀ H ₅ CljNO ₂

242.06

KCN EtOH.RT.1h KCN EtOH. RT.1 h 83%? 83%?

Alternatively, the conversion from 5 to 6 took place over 25:

3.1 synthesis of 6-amino-1,4-naphthoquinone (4)

3.1.1 p-toluenesulfonic acid-β-naphthylamide (8)

55.7 g (389 mmol) of β-naphthylamine (7) were dissolved in 111 g (1.4 mol = 113 ml) of pyridine, and 84.8 g (393 mmol = 1.01 eq.) Of TsCl were added in portions with stirring at RT. The mixture was allowed to stir for about 2 h, then the RG was concentrated to the mixture of 175 ml. HC1 and

Poured 500 g of ice. The RP was filtered off and the filtrate extracted once with ether.

The RP was then dissolved in DCM and the solution was combined with the ether phase. You washed with sat. CuSO ₄ (until the color was no longer intensified) and sat.

NaCl, dried over MgSO ₄ and concentrated at RV and HV. DC (H / EE 2: 1) showed only slight reddish impurities remaining at the starting spot. Yield: 101.98 g

(342.9 mmol = 88%).

3.1.2 1, 6-Dinitro-p-toluenesulfonyl-2-naphthylamide (9)

29.7 g (100 mmol) of tosylate 8 were dissolved in 89 ml of glacial acetic acid heated to 55 ° C and slowly stirred with 20.8 ml of 72% HNO ₃ (from a mixture of 18 ml of 65% HNO ₃ and 4.5 ml of smoking HNO ₃ ) offset. The RG was allowed to cool to RT with stirring, whereupon a portion of the product crystallized out in the form of a light yellow solid, the RG was mixed with 10 ml of water and, after 10 minutes, suction filtered through a glass suction filter. The title compound was obtained in pure form by washing with a little semi-concentrated HOAc and drying on the HV. Yield: 27.9 g (72 mmol = 72%).

3.1.3 6-Nitro-2-diazo-l-naphthol (10)

13 g (33.6 mmol) 9 were concentrated in 26 ml. H ₂ SO _{4 was} stirred in and the mixture was then heated to 40 ° C. until a clear solution was obtained. The RG was then cooled to <20 ° C and concentrated with stirring with the solution of 3.12 g (45 mmol = 1.3 eq.) NaNO ₂ in 14.3 ml. H ₂ SO ₄ added. 52 ml of glacial acetic acid were then added in portions with stirring and the RG was stirred until no more phase separation was visible. The RG was then slowly added to 1.5 l of ice water (50% ice) and the whole was left to stand overnight. The product was suctioned off and dried at the HV. Yield: 6.67 g (31 mmol = 92%). 3.1.4 6-nitro-l-naphthol (11)

7.82 g (36.3 mmol) 10 were cooled (~ 10 ° C) in a mixture of 58.6 ml glacial acetic acid and conc. H ₂ SO ₄ dissolved. This solution was added over a period of about 15 minutes to the well-stirred suspension of 19.55 g of Cu ₂ O in 156 ml of EtOH, heated to 55 ° C. The RG was then heated to 80 ° C., taking care that this temperature was not exceeded, 5.45 g of Cu ₂ O were added to the RG and the mixture was stirred for a further 30 min. The RG was then filtered through a glass suction filter (D3), the residue was washed with a little boiling EtOH and the filtrate was concentrated on a RV at a bath temperature of 60 ° C. until most of the ethyl acetate formed had been removed. The same was stirred into 780 ml of ice / water mixture (2/3 ice) and the resulting mixture was left to stand at RT overnight. The next day the mixture was extracted with DCM, the extract was dried over MgSO ₄ , concentrated on a RV and the resulting brownish orange-red solid was dried on a HV. The title compound was obtained by filtration over silica gel (0 = 5 cm, h = 5 cm, hexane / ethyl acetate 3: 1) and subsequent concentration and drying in the form of an orange-red solid. Rf: 0.25 (hexane / ethyl acetate 3: 1) Yield: 2.61 g (13.8 mmol = 38%).

3.1.5 6-amino-1-naphthol (12)

2.1 g (11.1 mmol) 11 were dissolved in 21 ml iPrOH and added to the well pre-hydrogenated mixture of 5.5 ml Ra-Ni suspension and 5.5 ml iPrOH. The RG was hydrogenated with intensive stirring for 3 h at RT under 3 bar H2, then the catalyst was sucked off through a glass filter and washed with acetone. The RP was dissolved in a little acetone and filtered through silica gel (0 = 5 cm, h = 10 cm, hexane / ethyl acetate 3: 2), then dissolved in ethyl acetate and 12 were obtained from it in the form of colorless crystals after adding hexane. Rf: 0.28 (hexane / ethyl acetate 3: 2) Yield: 1.64 g (10.3 mmol = 93%). 3.1.6 6-amino-l, 4-naphthoquinone (4)

To a stirred, cooled to 10 ° C solution of 400 mg (2.52 mmol) of 6-amino-1-naphthol (12) in 24.8 ml of MeOH, which had been placed in a 500 ml two-necked flask, was added dropwise under a gentle Ar stream a 1/2 h a chilled solution (5-10 ° C) of 1.55 g (5.78 mmol = 2.3 eq.) Potassium nitrodisulfonate (Fremy salt) [14, 15] in 25 ml 1/6 M KH ₂ PO and 88 ml water , After no more educt was present in the RG, approximately after 45 min, the RG was extracted with ethyl acetate, the extract was dried over MgSO ₄ and concentrated. After purification via FC (0 = 3 cm, h = 20 cm, hexane / ethyl acetate 3: 2), 271 mg (1.56 mmol = 62%) of pure title compound were obtained in the form of dark violet crystals. Rf: 0.32 (DCM / ethyl acetate 10: 1)

3.2 Synthesis of BOC-Ser (Bz) -Gly-Arg-6ANC (18)

3.2.1 Z-Arg (Z ₂ ) -6ANC (15)

1,038 g (1.8 mmol) of Z-Arg (Z ₂ ) OH (13) were placed with a stirring magnet under Ar in a 25 ml flask which had been heated and sealed with a septum, and 2.7 ml of absolute THF were added by means of a syringe. After a clear solution had been obtained by stirring at RT, the RG was cooled to -5 ° C. and with stirring through the septum with 249 μl (181.8 mg = 1.8 mmol, Hamilton syringe) absolute NEt ₃ , followed by 234 μl ( 246.3 mg = 1.8 mmol, Hamilton syringe) freshly distilled isobutyl chloroformate. The mixture was stirred at -5 ° C. for about 1/2 h and then the solution of 156 mg (0.9 mmol = 0.5 eq.) 6-amino-1,4-naphthoquinone (4) in 1.5 ml of dry DMF was added in one shot , After stirring for 1/2 h at -5 ° C., the cooling was removed and the RG was stirred for a further 6 h at RT. The RG was concentrated on the RV and thoroughly dried on the HV. After purification via FC (0 = 3 cm, h = 20 cm, DCM / ethyl acetate 11: 1 until after the elution of excess 6ANC (4), then DCM / ethyl acetate 10: 1), 270 mg (369 μmol = 41%) 7 pure title compound in the form of a light yellow solid. Rf: Z-Arg (Z ₂ ) -6ANC (15): 0.25 (DCM / ethyl acetate 10: 1) 6ANC (4): 0.32 (DCM / ethyl acetate 10: 1) Pl (by-product): 0.53 (DCM / ethyl acetate 10: 1 3.2.2 Arg-6ANC (16)

100 mg (137 μmol) of Z-Arg (Z ₂ ) -6ANC (15) were dissolved in approx. 4 ml of 1,4-dioxane and rinsed with a little MeOH to give the prehydrated suspension of 37 mg Pd / activated carbon (10%, 50% water content) in 10 ml of MeOH. The mixture was hydrogenated with vigorous stirring for 2 1/2 hours under 1.2 bar H ₂ in a stirred autoclave. 2.28 ml of 1% methanolic HC1 (273 μmol = 2 eq.) Were added to the RG and the catalyst was sucked off through a glass filter funnel filled with Celite while rinsing with MeOH. The filtrate was evaporated to dryness at the RV and HV and purified via FC (0 = 1 cm, h = 20 cm, 1-butanol / glacial acetic acid / water 3: 1: 1). After concentration on RV and HV, 56.6 mg (126 μmol = 92%) of the title compound were obtained as diacetate in the form of a greenish-yellow solid. Rf: 0.47 (1-butanol / HOAc / water 3: 1: 1)

3.2.3 BOC-Ser (Bz) -Gly-Arg-6ANC (18)

26 mg (57.8 μmol) Arg-6ANC (16) and 36 mg (80 μmol = 1.38 eq.) BOC-Ser (Bz) -Gly-OSu (17) were dissolved in 0.4 ml dry DMF (brownish yellow solution) and 13.8 μl (10.5 mg = 1.8 eq.) NEt _{3 added} using a GC syringe. The RG was shaken overnight and the solvent was then removed at the HV. The one obtained after FC (0 = 1 cm, h = 20 cm, CHCl ₃ / MeOH / HOAc 8: 1: 1 until shortly before (clearly visible) elution of 18, then CHCl ₃ / MeOH / HOAc 7: 1: 1) The product fraction was evaporated to dryness on RV and HV and then suspended in CHC to remove residues of silica gel and filtered. After concentration, 27 mg (37.2 μmol = 64%) of the slightly apolar-contaminated title compound resulted as monoacetate in the form of a brownish-yellow powder. Rf: 0.11 - 0.25 (CHCl ₃ / MeOH / HOAc 8: 1: 1)

3.3 Synthesis of 6-amino-2,3-dichloro-l, 4-naphthoquinone (5)

3.3.1 6-nitro-2,3-dichloro-1,4-naphthoquinone (22)

5 g (22 mmol) 2,3-dichloro-l, 4-naphthoquinone were concentrated to 15 ml. H ₂ SO _{4 was} added and the mixture was heated to 80 ° C. with stirring until a clear solution was obtained. 30 ml smoking HNO ₃ were added and the RG was stirred at 80-90 ° C. for 1 h. The same was then placed in 800 ml of ice water and the precipitate was filtered off with suction. The filtrate was extracted with ether and combined with the extract with the solution of the precipitation in ether. You washed twice with sat. NaHCO ₃ and once with sat. NaCl, dried over MgSO ₄ and concentrated. The RP was drawn onto 3 large polyspoons of silica gel and purified via FC (0 = 5 cm, h = 20 cm, hexane / ethyl acetate 5: 1). This resulted in 1.12 g (4.13 mmol = 16.5%) of pure title compound in the form of lemon-yellow leaves. Rf: 5-nitro-2,3-dichloronaphthoquinone (22): 0.38 (hexane / ethyl acetate 5: 1) 5-nitro-2,3-dichloronaphthoquinone: 0.16 (hexane / ethyl acetate 5: 1)

3.3.2 6-amino-2,3-dichloro-l, 4-naphthoquinone (5)

a.) 135 mg (0.5 mmol) 6-nitro-2,3-dichloro-l, 4-naphthoquinone (22) were dissolved in 0.7 ml hot glacial acetic acid and the solution of 450 mg SnCl ₂ dihydrate (2 mmol ) in 3-4 drops of conc. HC1 added. The RG was heated to 80 ° C. while stirring under Ar until the color changed from violet to dirty yellow (a few minutes). By adding 0.7 ml of conc. HC1, the hydrochloride of 6-amino-2,3-dichloro-l, 4-naphthohydro-quinone was precipitated and suction filtered. After drying at the HV, the RP was dissolved in a little water (approx. 5 ml) and acidified with a lot of conc. HC1 on. The hydrochloride which crystallized out after about half an hour of cooling in the refrigerator was suction filtered and thoroughly dried at the HV. This resulted in 81 mg (287 μmol = 57%) of 6-amino-2,3-dichloro-l, 4-naphthohydroquinone hydrochloride in the form of a slightly yellowish powder.

b.) 22.8 mg (81 μmol) of hydrochloride were dissolved in 2 ml of water and a solution of about 500 mg of FeC _{! 3} in a few ml of water was added with stirring, whereupon the RG immediately turned deep purple. After stirring for about • h, the RG was extracted with ethyl acetate, the extract was dried over MgSO ₄ and concentrated. 19 mg (78 μmol = 97%) of pure title compound resulted in the form of deep violet crystals. Rf: 0.45 (hexane / ethyl acetate 1: 1) 3.4 Synthesis of 6-amino-2,3-dicyano-l, 4-naphthoquinone

10 g (44 mmol) 2,3-dichloro-l, 4-naphthoquinone (obtainable according to scheme 1) were carefully dissolved in 30 mml concentrated H ₂ SO at 80 ° C until a clear solution was obtained. 60 ml of smoking HnO ₃ was slowly added to this solution and the temperature was kept at approximately 80 to 90 ° C. The reaction mixture was stirred at 80 ° C for an additional hour. After one hour, the cold solution was poured into 1.6 l of ice-water mixture and the product was filtered off. The solid obtained was dissolved in ethyl acetate and saturated 4 times with 50 mml NaHCO ₃ and washed 2 times with saturated NaCl solution. 5-nitro-2,3-dichloro-l, 4-naphthoquinone (Rf: 0.16) was obtained from 6-nitro-2,3-dichloro-l, 4-naphthoquinone (R _f : 0.38) via column chromatography or separated on silica and hexane / ethyl acetate (5: 1).

1.41 g (5.2 mmol) of 6-nitro-2,3-dichloro-l, 4-naphthoquinone were placed in 20 mml of ethanol. The mixture was heated to 30 to 40 ° C and a solution of 1.7 g (5 mol equivalents) of NaCn in 5 mml of water was added dropwise and the mixture was stirred for an additional hour. The reaction mixture was cooled to room temperature and acidified with concentrated HC1. The light solid was filtered and washed with water. The solid was dissolved in 20 ml of acetic acid and a solution of 6.3 g of SnCl ₂ x2 H ₂ O in 30 ml of concentrated HCl was added. The mixture was heated and at 80 ° C for 30 min. touched. The mixture was cooled to room temperature and the light solid was filtered off. The solid (6-amino-2,3-dicyano-l, 4-naphthodiol) was suspended in a solution of 17 g FeClx6 HO in 300 ml water and at room temperature for 45 min. touched. The blue solid was filtered and dried in a desiccator. 0.114 g of 6-amino-2,3-dicyano-1,4-naphthoquinone was obtained (approximately 10% yield).

Example 4

Enzymatic cleavage of BOC-Ser (Bz) -Gly-Arg-6ANC (18) with trypsin (bovine pancreas) Stock solutions:

TRIS buffer: 4.41 g (30 mmol) CaCl ₂ . 2 H ₂ O and

(2 conc.) 1,211 g (10 mmol) TRIS

in 100 ml of water, adjusted to pH 7.4 with 0.1 M HC1

Trypsin: 1 mg trypsin (bovine pancreas) dissolved in 100 ml 0.001 M HC1,

5 ml of which diluted to 100 ml with 0.001 M HC1 => 0.5 mg / 1.

Substrate: 4.34 mg (6 μmol) dissolved in 240 ml DMSO, plus

2.4 ml TRIS buffer (2x) and 2.16 ml water.

Concentration: 1.25 mM

The stock solution was filtered twice over a little cotton in a Pasteur pipette. After leaving the residue not used for the experiments for two days, a fine but distinct sediment had formed.

Measurement solutions:

The measuring solutions were prepared directly in the measuring cuvettes in such a way that 2 ml total volume contained 25 μg / 1 trypsin, 100 μl DMSO (5%), simple TRIS buffer concentration (0.015 M CaCl ₂ , 0.005 M TRIS) and the indicated ideal substrate concentration , The trypsin solution was added after annealing the solution at 37.2 ° C. for 15 minutes immediately before the start of the measurement.

Example 5

Enzymatic cleavage of a LAL substrate which was labeled with L-MEL-46 (linker, paraaminobenzoic acid, chromophore CAS, Nile blue protonates at pH 7.5 The absorption bands of the substrate in aqueous solution (TRIS buffer pH 7.5, 10 mmol, 10% DMSO, substrate concentration: 40x10 ^"6 M correspond to 10xlO ^{" 6} M, enzymatic cleavage with trypsin): The absorption bands are in the visible range of 350 nm up to 750 nm and have the following characteristics:

Table 1

Measured absorbance values of the labeled substrate in (TRIS, pH 7.5, 10 mmol) concentration of the substrate: 10 μmol, 37 ° C in solution before the start of the cleavage reaction and the corresponding molar concentrations at corresponding wavelengths that are accessible on the chip, reproducibility test :

Table 2:

Claims

1. Chemical compound of the general formula (I):

X-A-B-C-L-CRS

A and B represent an α-, β- or γ-amino acid which comprise 2 to 15 C atoms and up to 4 N atoms, 2 S atoms and 6 O atoms and A and B are identical or different and B optionally represents a dipeptide formed from these amino acids,

C represents arginine, lysine, tyrosine, phenylalanine or tryptophan or their respective homologues,

L stands for a linker which is able to form a peptide bond with C,

CRS stands for a (neutral or positively charged) conjugated carbon ring system which is connected to the radical -L-C-B-A-X via a nitrogen atom and the compound of the general formula (I) has an absorption maximum at a wavelength of more than 550 nm.

2. Chemical compound according to claim 1, characterized in that it has an absorption maximum at lower wavelengths than CRS (and L-CRS).

3. Chemical compound according to claim 1 or 2, characterized in that the connecting part CRS is neutral or positively charged. Chemical compound according to one of the preceding claims, characterized in that the structural element CRS is a compound selected from the following group of compounds (a), (b), (c) or (d):

(A)

(B)

(C)

(D)

where R is H, C _t to C ₈ alkyl, in particular methyl, ethyl, propyl, i-propyl, n-butyl, i-butyl, aryl, aralkyl, CN, halogen,

R] and R _{2 may be the} same or different and are H, CN, halogen, alkyl. Aryl or aralkyl or alkoxy or has the meaning of R, R ₃ , R ₄ , R ₅ , R ₅ , R ₇ and R _{8 may be the} same or different and for H, CN, NO ₂ , NH ₂ , NR, -HSO3 ^" , COO ^" , -OH (-O ^" ) -SO ₃ ^" , alkyl, in particular methyl, ethyl, propyl, i-propyl, n-butyl, i-butyl, aralkyl, halogen, in particular CI, Br or alkoxy,

R ₉ H, halogen, SH, SR, OH, OR, carboxylic acid, aminocarboxylic acid, HSO ₃ ^" , COO ^" , -OH (- O ^" ) -SO ₃ ^" , an azo or diazo group, substituted or unsubstituted aromatic such as nitroaromatic , halogenated aromatics, thiocyanate, substituted or unsubstituted phenol,

and with the condition for compound (c) that at least one radical from R ₃ to Rβ is an amino group, one nitrogen substituent of which is linked to the radical of the compound of the general formula (I).

5. A process for the preparation of a compound of general formula (I) comprising the following steps:

a) the preparation of an oligopeptide X-A-B under usual conditions

A and B represent an α-, β- or γ-amino acid which comprise 2 to 15 C atoms and up to 4 N atoms, up to 2 S atoms and up to 6 O atoms and where A and B may be identical or different and B may be for one

Dipeptide, which is formed from these amino acids,

b) the production of CRS-L, where L stands for a linker compound which is capable of forming a peptide bond with C and where the linker compound is bonded to an N atom of the structural element CRS and wherein CRS stands for a conjugated carbon ring system which has an absorption maximum at a higher wavelength than the compound XABCL-CRS (I), whose absorption maximum is in the range of more than 550 nm,

c) the reaction of CRS-L with C under customary conditions to CRS-L-C, where C is arginine, lysine, tyrosine, phenylalanine or tryptophan or their respective homologues

d) the conversion of X-A-B and CRS-L-C to X-A-B-C-L-CRS

6. A method for the detection of target substances comprising the following steps:

the provision of connections according to one of claims 1 to 4,

optionally determining the wavelength at which the compounds according to one of claims 1 to 4 have their absorption maximum if the wavelength is not known,

- bringing the provided compounds into contact (and if necessary allowing them to act) with the solution, emulsion, gas, aerosol, mixture or pure substance intended for the detection,

- ^' the determination of the wavelength at which the compounds which were provided in the first step, after contacting (and allowing to act) of the compounds provided with the solution provided for the detection,

Emulsion, gas, aerosol, mixture or pure substance have their absorption maximum.

7. Use of chemical compounds according to one of claims 1 to 4 for the detection of target substances.

8. Use according to claim 7 for the detection of endotoxins.