Classifications

• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
  • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
  • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D219/00—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems
  • C07D219/04—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the ring system
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

• the present invention relates to chemiluminescent derivatives of acridinium as well as a conjugate comprising one of these derivatives, linked to a specific biological component.
• Another aspect of the invention relates to the diagnostic and / or assay kit comprising this conjugate or one of these derivatives, and the use of the conjugate or of one of these chemiluminescent derivatives for the assay and / or detection of a specific biological compound.
• Chemiluminescence is a formation of light obtained by a chemical reaction.
• the general mechanism of this reaction has been described in particular by Schuster et al (Advances in Physical organic Chemistry,
• Compound A by a chemical, physical or biochemical reaction (most often by oxidation with a peroxide) gives a product in an excited state ("B *") which returns to the ground state by the emission of light
• Patent applications EP-A-273115, EP-A-257541 and EP-A-263657 describe heterocyclic chemiluminescent compounds derived from acridinium, quinolenium phenantridiniumn and isoquinolenium and their isomers, possibly conjugated to antigens, antibodies or nucleic acids for stretch used in chemiluminescence tests.
• chemiluminescent power of these derivatives is calculated by the emission of light generated in contact with hydrogen peroxide in an alkaline medium.
• Studies by F. McCapra (Accounts of Chemical Research (1976), 9, 6, p. 201-209) have established a mechanism for chemiluminescence of acridinium compounds. He postulates the formation of an excited reaction intermediate (dioxetane) which breaks down into C0 2 and methylacridone. It also involves the displacement by H 2 0 2 of a leaving group Y. This displacement would, according to generally accepted theory, be possible for derivatives having a group Y having a pKa lower than that of hydrogen peroxide (pKa ⁇ 12 ).
• Patent application WO95 / 19976 describes heterocyclic derivatives of acridinium, phenanthridinium, quinolinium and isoquinolenium as well as their isomers and any derivatives obtained by substitution, improved, in particular stable derivatives and / or having a high chemiluminescence, in particular when the pKa value of the leaving group is high, preferably when the pKa is greater than 12.
• the object of the present invention is to obtain acridinium derivatives chemically related to those described in the abovementioned patent application 095/19976, which are easy to synthesize, which also offer numerous possibilities for adaptation, notably including a series of chemical modifications.
• grafting an arm (spacer) the aim being inter alia to orient the coupling towards functions that are usually difficult to access when labeling proteins such as phenol and thiol groups.
• the present invention also aims to obtain chemiluminescent derivatives characterized by a dose-response curve allowing their use in the assay and / or diagnosis over a wide concentration range.
• a final object of the present invention is to obtain a diagnostic kit comprising said active and / or stable chemiluminescent derivative. Characteristic elements of the invention.
• the present invention relates to chemiluminescent acridinium derivatives of formula:
• R x to R 13 are substituents which do not prevent the expression of chemiluminescence, it being understood that at least one of the substituents R 12 and R 13 comprises, as a single element or as a binding element to the derivative of acridinium, an atom other than carbon.
• A is a complementary ion chosen from the halogen, nitrate, sulfate, iodomercurate, trifluoromethanesulfonate, tartrate and phthalate ions, R x to R 13 .
• R 12 and R 13 are hydrogen or radicals of type R 14 or BR 14 where B represents a group or a connecting atom and R X4 represents a hydrocarbon radical which may contain one or more heteroatoms
• R 12 and R 13 are identical or different substituents whose function relates to chemiluminescence and to the coupling, one of the substituents R 12 and R 13 comprising as a single element a halogen atom or comprising as connecting element to the acridinium derivative an atom chosen from azotides and chalcogens, while the other of the substituents R 12 and R 13 is similar to the substituents R x to R X1 , with the exception of hydrogen.
• B represents in the formula BR 14 , a group - (CH 2 ) n - where n represents an integer from 1 to 5, B also represents a group containing an azotide or represents a chalcogen, preferably a nitrogen group or twice-linked oxygen, R X4 being as defined above.
• R 14 is a radical chosen from the group consisting of al yl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl and heteroarylkyl optionally substituted.
• R x and R lx are hydrogen, one of the substituents R 12 and R 13 is a substituent chosen from halogens and BR 14 groups, the other of the substituents R 12 and R 13 being similar to BR 14 or R 14 , B and R 14 being as defined above.
• the chemiluminescent derivatives are the compounds
• Another aspect of the invention relates to the conjugate comprising a chemiluminescent derivative according to the invention linked to a specific biological component.
• specific biological compound means any biological molecule (lipid, saccharide, protein, peptide, nucleic acid, ...) or set of biological molecules, specific to a species (viral, bacterial, plant, animal or other) , an individual, a pathology (such as cancer or caused by a viral, bacterial or other agent), an activity or a biochemical system (such as an enzyme reaction, ...) .
• Said biological component is capable of being detected and / or measured, or capable of being used for assaying and / or detecting a specific biological component.
• this specific biological compound is chosen from the group consisting of antibodies, haptens, antigens, nucleic acids, agonists, cell transporters, fatty acids, lipids and / or a mixture of them.
• the present invention also relates to the diagnostic and / or assay kit comprising the conjugate or one of the chemiluminescent derivatives according to the invention and the use of the conjugate and / or of a chemiluminescent derivative according to the invention for the assay and / or the detection of a specific biological compound.
• FIG. 1 represents a general diagram for the preparation of the OX derivatives.
• FIG. 2 represents the diagram of synthesis of the derivative 0X7.
• FIG. 3 illustrates the kinetics of the Ab (antibodies) labeled with 0X3, 0X5, 0X6 and 0X7.
• FIG. 4 to 6 illustrate the stability of the Ac marked with 0X6, 0X7 and 0X5 respectively.
• FIG. 7 represents the evolution of the concentration of product 0x7 as a function of time in a buffer at pH 8.
• FIG. 8 represents an example of TSH assay with Ac labeled with 0x7.
• Figure 9 shows the infrared spectrum of pentafluorobenzaldehyde.
• Figure 10 shows the infrared spectrum of pentafluorobenzaldoxime.
• Figure 11 shows the infrared spectrum of chlorinated pentafluorobenzaldoxime.
• Figure 12 shows the infrared spectrum of the coupling product of chlorinated pentafluorobenzaldoxime with 9-acridine carboxylic acid chloride, being 1-chloro-1-perfluorophenyl-1-iminomethane 9-acridine carboxylate.
• FIGS. 13 to 15 represent the infrared spectra of the markers 0X7, 0X5 and 0X6.
• Figures 16 to 18 show the mass spectra of the markers 0X5, 0X6 and 0X7.
• the OX compounds were all prepared by following the general preparation scheme described in FIG. 1. Their syntheses required the synthesis of the starting aldehyde (II) when the latter does not was not directly available.
• the first step is to form the oxime (III) by reaction between the aldehyde (II) and hydroxylamine. The reaction is carried out in a buffer at pH 4.5-5.
• Chloroxime (IV) is prepared by bubbling chlorine through an oxime solution.
• the chloro-derivatives were obtained directly by coupling of chloroxime to the acridine nucleus.
• the other 3 derivatives were previously treated with an equivalent of sodium alcoholate before the step of coupling to the acridine nucleus.
• the alcoholate is prepared from sodium and the corresponding alcohol.
• Compounds IV or IVb are coupled to the carboxylic acid chloride in an organic solvent supplemented with NET 3 .
• the products V or Vb are methylated by the pair of reagents CH 3 I / HgCl 2 or methyl triflate.
• EXAMPLE 1 OPERATING MODES COMMON TO CERTAIN DERIVATIVES (FIG. 1). 1. Synthesis of aldehyde II (for OX6 and OX7 derivatives).
• the aldehydes used in the synthesis of the 0X6 and 0X7 derivatives were produced by reduction of the corresponding acid chlorides (2,4,5-trifluorobenzoic acid chloride or 2,3,4,5,6-pentafluorobenzoic acid).
• the protocol used is as follows: one equivalent of acid chloride is placed in the presence of 1.1 equivalents of Bu 3 SnH in THF. After reaction, the THF is evaporated in vacuo and the residue treated with water. The latter is then extracted with petroleum ether (Eb 40 ° C) and then passed over charcoal. The aldehyde is obtained by evaporation under vacuum.
• the chloroxime is prepared by bubbling chlorine into an oxime solution in a CHCl 3 / dioxane mixture previously cooled to 0 ° C. The reaction is complete and the chloroxime (III) is directly obtained by evaporation of the solvents followed, when the chloroxime is solid at room temperature, recrystallization by adding 40 ° C petrolein (the chloroxime of derivatives 0X6 and 0X7 is liquid).
• the alcoholate is prepared by reaction between an equivalent of sodium and an equivalent of alcohol (ethanol or phenol). The reaction is carried out in alcohol when possible (ethanol) or in acetone
• the product formed is either crystallized by adding petroleum ether (Eb 40 ° C) to the solution (0X1, 0X2, 0X3, 0X4 and 0X5) or chromatographed on a silica column using a toluene / ethyl acetate phase / acetic acid in propotions 2/1 / 0.01 (0X6 and 0X7).
• the fractions corresponding to the first product leaving the column are recovered and concentrated in vacuo.
• the addition of petroleum ether (Eb 40 ° C) causes the product to crystallize.
• the products obtained in step 5 are methylated with the pair of reactants CH 3 I / HgCl 2 .
• the reaction is carried out on an intimate mixture of 50 mg of product with 50 mg of HgCl 2 added with 1 ml of CH 3 I. It takes place in a bomb brought to 110 ° C for 2 h 30 min. After reaction, the bomb is refrigerated at -20 ° C. After cooling and decanting of the contents of the bomb, the supernatant is removed and the crystals of methylated product are washed successively with CH 3 I and then with diethyl ether. The methylated product can optionally be recrystallized from an acetone / diethyl ether mixture.
• EXAMPLE 2 SYNTHESIS OF THE 0X7 PROBE (FIG. 2) OR
• the chemiluminescence yield of the OX derivatives cannot be determined when the molecules are in the free state. It is measured after coupling of these products to a protein. For example, for the derivatives 0X5, 0X6 and 0X7, the rate of incorporation on the protein is measured by a fluorescent method.
• chloroximes appear by far the most stable in the presence of proteins. Measurements carried out by mass spectrometry on 0x7 show no significant variation in the spectrum of the molecule after an incubation of 45 minutes in an acetonitrile / water mixture (proportions 50/50). This chemical stability makes it possible to envisage coupling the probe to the proteins via the halogenated function of the molecule. All the stability measurements carried out show that the oximes 0X5, 0X6 and 0X7 greatly increase their stability in an aqueous medium after coupling to the proteins. The first results of a stability study on antibodies labeled with these probes are given in Figures 4, 5 and 6.
• the tracer 0X7 appears as being the most stable of the three, and this more particularly at pH 5. This important stability at pH 5 makes it possible to envisage a long conservation of the tracer at 4 ° C. On the other hand, the stability of the latter at pH 7 allows to consider all possible immunoassays at this pH.
• the stability of the 0X7 probe is very sensitive to pH. In an acid medium (pH 5) the stability of the compound is much higher than in a basic medium (pH 8). When the probe is attached to a protein, its stability greatly increases regardless of the pH tested. 3.1 STABILITY OF THE UNCoupled OX7 PROBE AT PH 8.
• the stability of the free probe was tested at 25 ° C in a medium identical to that used in the labeling reactions (pH of the medium: 8).
• the degradation of the product was followed by HPLC analysis using as a chromatographic support a column (4x125 mm) of RP-18 Lichrospheres (5 ⁇ m) and as mobile phase an acetonitrile / water mixture added with decane sulfonic acid, hydroxide tetramethyl ammonium and trifluoroacetic acid (final pH: 2.5).
• FIG. 7 shows the evolution of the concentration of OX7 product as a function of time in a buffer at pH 8.
• the stability of the OX7 probe coupled to an antibody was tested at pH 5, 6 and 7. It was evaluated by measuring the chemiluminescent activity of samples of labeled protein aged in phosphate / NaCl buffers supplemented with proteins. Parametric adjustment of the data collected at pH 6 and 7 leads to half-life times of the order of 600 and 375 hours. On the other hand, at pH 5, the half-life time is much longer (the small signal variations recorded over a period of approximately 2 months do not allow a valid parametric adjustment).
• the medium is chromatographed on a column (50 ⁇ 1 cm) of Sephadex G25.
• the chromatographic column is balanced and eluted with a 0.1 M phosphate buffer, 0.15 M NaCl. 0.1% NaN 3 containing BSA (lg / 1) and adjusted to p 5.0.
• the chromatographic fractions are read by measurement of chemiluminescence.
• the fractions corresponding to the antibody peak are pooled and stored in the purification buffer at a temperature of 4 ° C.
• a non-optimized TSH assay was carried out following a protocol identical to a commercial IRMA assay with anti-TSH antibodies labeled with the 0X7 probe.
• the protocol used includes the following steps:
• the calibration curve obtained shows that antibodies labeled with the 0X7 probe can be perfectly suitable as a chemiluminescent tracer in the context of an assay requiring high sensitivity.

Landscapes

• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Organic Chemistry (AREA)
• Health & Medical Sciences (AREA)
• Urology & Nephrology (AREA)
• Biomedical Technology (AREA)
• Hematology (AREA)
• Immunology (AREA)
• Molecular Biology (AREA)
• Cell Biology (AREA)
• Microbiology (AREA)
• Food Science & Technology (AREA)
• Medicinal Chemistry (AREA)
• Physics & Mathematics (AREA)
• Analytical Chemistry (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Pathology (AREA)
• Biotechnology (AREA)
• Materials Engineering (AREA)
• Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Applications Claiming Priority (3)

Publications (1)

Family

ID=3890565

Family Applications (1)

Country Status (3)

Families Citing this family (5)

Family Cites Families (3)

• 1997
  • 1997-06-11 BE BE9700503A patent/BE1011206A4/fr not_active IP Right Cessation
• 1998
  • 1998-06-10 WO PCT/BE1998/000087 patent/WO1998056765A1/fr not_active Application Discontinuation
  • 1998-06-10 EP EP98925343A patent/EP0988288A1/de not_active Withdrawn

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events