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WO2004041840A2 - Chromogenes a liaison peptidique servant a determiner des activites enzymatiques - Google Patents

Chromogenes a liaison peptidique servant a determiner des activites enzymatiques Download PDF

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Publication number
WO2004041840A2
WO2004041840A2 PCT/EP2003/012463 EP0312463W WO2004041840A2 WO 2004041840 A2 WO2004041840 A2 WO 2004041840A2 EP 0312463 W EP0312463 W EP 0312463W WO 2004041840 A2 WO2004041840 A2 WO 2004041840A2
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Prior art keywords
crs
atoms
compound
amino
peptide
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PCT/EP2003/012463
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German (de)
English (en)
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WO2004041840A3 (fr
Inventor
Stefan Spichiger
Ursula Spichiger-Keller
Michael Linnhoff
Gleb Zhylyak
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C-Cit Ag
Fluka Gmbh
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Application filed by C-Cit Ag, Fluka Gmbh filed Critical C-Cit Ag
Priority to AU2003286158A priority Critical patent/AU2003286158A1/en
Publication of WO2004041840A2 publication Critical patent/WO2004041840A2/fr
Publication of WO2004041840A3 publication Critical patent/WO2004041840A3/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/081—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/13—Labelling of peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2337/00—N-linked chromogens for determinations of peptidases and proteinases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/50—Lipopolysaccharides; LPS
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • 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.
  • protease activities is also important for the diagnosis of carcinoma cells or the development of inhibitors.
  • active substances are tested for their inhibitory effects from protease activities.
  • 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.
  • LAL Limulus amebocyte lysate
  • endotoxin of gram-negative bacteria can be detected enzymatically by determining the proteolytic activity of the enzymes used.
  • 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 ⁇ -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.
  • a disadvantage of p-nitroaniline and 7-amino-4-methylcoumarin (AMC) labeled chromogens 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.
  • 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 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 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 -, ⁇ -, or ⁇ -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.
  • 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.
  • linker L 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.
  • 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.
  • 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.
  • the linker has at least the length of an ethyl, in particular at least one, propyl radical.
  • linker significantly longer or to introduce a further aromatic compound or a further amino acid or an oligopeptide.
  • linkers which can be used according to the invention are hydroquinones, quinones, aminoquinones, quinolines, aminocarboxylic acids, dicarboxylic acids (or their anhydrides), ⁇ -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.
  • 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.
  • pKa degree of protonation
  • 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.
  • this bathochromic “shift” comprises at least 30 nm, preferably at least 50 (in particular i and more preferably at least 100 nm.
  • 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
  • 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
  • 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.
  • the structural element CRS can be neutral, anionic, cationic or zwitterionic.
  • the protonated form is preferably long-wave absorbent.
  • the deprotonated, negatively charged form is preferably long-wave absorbent.
  • 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).
  • 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).
  • the structural element CRS is neutral or basic. It advantageously has a pKa value of 6-13.
  • 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).
  • 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);
  • calmagite as calmagite (absorption max: 665 nm)
  • naphthol blue black asbsorption 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.
  • 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):
  • 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,
  • radical R 9 influences the solubility of the compound, which can be adjusted in accordance with the respective radical R 9 .
  • 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.
  • “low water solubility” means values in the range from about 50 mg / 1 to about 1 mg / 1.
  • 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:
  • 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 ⁇ -, ⁇ - or ⁇ -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,
  • 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.
  • CRS-LC where C is arginine, lysine, tyrosine, phenylalanine or tryptophan or other amino acids and their stereoisomers or their respective homologues
  • 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:
  • Aerosol mixture or pure substance
  • 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
  • peptides are not bioactive in that they can convert other molecules or promote specific bonds to other molecules.
  • biosensors on the one hand the so-called bioaffinity sensors and on the other hand the so-called bioaffinity sensors
  • bioaffinity sensors generate a signal by complexing bioactive molecules to form an analyte.
  • enzyme activities are to be determined, the enzymes causing a change in the absorption spectrum of the substrate by proteolysis of the corresponding substrate.
  • 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.
  • 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.
  • the chip surface is chemically modified and functionalized using two different methods:
  • 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.
  • 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.
  • 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.
  • Nile blue as a chromogen which is coupled to 4-aminobenzoic acid (as a linker) in the form of a carboxylamide.
  • 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.
  • 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.
  • 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.
  • the amino-1,4-naphthoquinones are optionally linked to the peptide sequence via a linker.
  • Step A 4-nitro-N- (9-diethylamino-benzo [a] phenoxazin-5-ylidene) benzoic acid amide
  • Step B 4-amino-N- (9-diethylamino-benzo [a] phenoxazin-5-ylidene) benzoic acid amide
  • Step B 4- (L-arginylamido) - N- (9-diethylamino-benzo [a] phenoxazin-5-ylidene) benzoic acid amide
  • Step D 4 - ((Benzyl-L-serinyl-L-glycyl) -L-arginylamido) -N- (9-diethylamino-benzo [a] phenoxazin-5-ylidene) benzoic acid reamide
  • Step B 4-amino-N- (9-dimethylamino-benzo [a] phenoxazin-5-ylidene) aniline
  • Step B 4- (L-arginylamido) -N- (9-dimethylamino-benzo [a] phenoxazin-5-ylidene) aniline
  • Step D 4- (fBenzyl-L-serinyl-L-glycyl) -L-arginylamido) -N- (9-diethylamino-benzo [a] phenoxazin-5-ylidene) aniline
  • 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.
  • 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.
  • D3 glass suction filter
  • 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).
  • Trypsin 1 mg trypsin (bovine pancreas) dissolved in 100 ml 0.001 M HC1,
  • 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.
  • the measuring solutions were prepared directly in the measuring cuvettes in such a way that 2 ml total volume contained 25 ⁇ g / 1 trypsin, 100 ⁇ l 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.

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Abstract

L'invention concerne un composé chimique de formule générale (I) : X-A-B-C-L-CRS. Dans ladite formule : X représente hydrogène ou un groupe de protection de groupes amino terminaux courant en chimie des peptides ; A et B représentent un α-, β-, ou η-amino-acide comportant 2 à 15 atomes de C, jusqu'à 4 atomes de N, jusqu'à 2 atomes de S et jusqu'à 6 atomes d'O, A et B pouvant être identiques ou différents et B représentant éventuellement un dipeptide formé par lesdits amino-acides ; C représente arginine, lysine, tyrosine, phénylalanine ou tryptophane ou leurs homologues respectifs ; L représente un lieur pouvant former une liaison peptidique avec C ; CRS représente un système cyclique carboné conjugué relié au radical -L-C-B-A-X par l'intermédiaire d'un atome d'azote. Selon l'invention, ledit composé de formule générale (I) et CRS présentent un maximum d'absorption à une longueur d'onde supérieure à 550 nm.
PCT/EP2003/012463 2002-11-07 2003-11-07 Chromogenes a liaison peptidique servant a determiner des activites enzymatiques WO2004041840A2 (fr)

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AU2003286158A AU2003286158A1 (en) 2002-11-07 2003-11-07 Peptide-bound chromogens used for determining enzymatic activities

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DE10251894.7 2002-11-07
DE10251894A DE10251894A1 (de) 2002-11-07 2002-11-07 Peptidgebundene Chromogene zur Bestimmung von Enzymaktivitäten

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WO2004041840A2 true WO2004041840A2 (fr) 2004-05-21
WO2004041840A3 WO2004041840A3 (fr) 2004-07-01

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7671139B1 (en) 2004-06-18 2010-03-02 Bridgestone Corporation Functionalized polymers and tires made therefrom
EP2465941A1 (fr) * 2010-12-20 2012-06-20 Siemens Healthcare Diagnostics Products GmbH Procédé de détermination simultanée de plusieurs protéases de coagulation

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FR2875242B1 (fr) * 2004-09-10 2006-11-17 Biomerieux Sa Nouveaux substrats enzymatiques derives de phenoxazinone et leur utilisation comme revelateur dans la detection de microorganismes a activite peptidase
EP1674580A1 (fr) * 2004-12-23 2006-06-28 F. Hoffmann-La Roche Ag Procédé d'identification d'activateurs et/ou d'inhibiteurs d'activité enzymatique

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FI860043A7 (fi) * 1986-01-06 1987-07-07 Orion Yhtymae Oy Peptidisubstraatit sekä menetelmä endotoksiinin kvantitatiiviseksi määrittämiseksi.
DE3629175A1 (de) * 1986-08-28 1988-03-17 Behringwerke Ag Chromogene verbindungen, verfahren zu deren herstellung und ihre verwendung
US5175089A (en) * 1988-07-15 1992-12-29 The Trustees Of Columbia University In The City Of New York Method for monitoring periodontal disease by monitoring endotoxins and inflammatory agents
SE8904188D0 (sv) * 1989-12-12 1989-12-12 Kabivitrum Ab Chromogenic substrate

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7671139B1 (en) 2004-06-18 2010-03-02 Bridgestone Corporation Functionalized polymers and tires made therefrom
EP2465941A1 (fr) * 2010-12-20 2012-06-20 Siemens Healthcare Diagnostics Products GmbH Procédé de détermination simultanée de plusieurs protéases de coagulation
EP2465942A1 (fr) * 2010-12-20 2012-06-20 Siemens Healthcare Diagnostics Products GmbH Procédé de détermination simultanée de plusieurs protéases de coagulation
US8932826B2 (en) 2010-12-20 2015-01-13 Siemens Healthcare Diagnostics Products Gmbh Method for simultaneously determining multiple coagulation proteases
US9482673B2 (en) 2010-12-20 2016-11-01 Siemens Healthcare Diagnostics Products Gmbh Method for simultaneously determining multiple coagulation proteases

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AU2003286158A1 (en) 2004-06-07
WO2004041840A3 (fr) 2004-07-01

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