Patent Publication Number: US-2003224421-A1

Title: New oxazine dyes and their use as fluorescent markers

Description:
[0001] The invention concerns new oxazine dyes which are capable of coupling and their use as fluorescent markers in conjugates.  
       [0002] Markers or labels are necessary to carry out immunological assays and in DNA analytics which allow a quantification of the analyte after an analyte-specific reaction is completed.  
       [0003] Due to their high sensitivity fluorometric markers in particular have recently gained in importance. Thus the labelling of an antibody or a nucleotide with fluorescent dyes enables a direct quantification.  
       [0004] Widespread fluorescent dyes are for example FITC (fluorescein isothiocyanate), FLUOS (fluorescein N-hydroxysuccinimide ester), resorufin and rhodamine labels which, however, require relatively sophisticated light sources such as argon lasers for their excitation.  
       [0005] The rapid development of cheap laser diodes with an emission range of 630-780 nm which in addition are excellently suitable for constructing miniaturised systems, makes it desirable to have dyes which absorb in these wavelength regions.  
       [0006] Pentacyclic rhodamine dyes are described in EP-A-0 543 333 which can be used as a label. Their absorbance lies mainly in the range up to 660 nm.  
       [0007] In WO 88/047 77 phthalocyanine dyes are described which, however, have more than one functional group so that when they are conjugated for example with antibodies this leads to cross-linking and to product mixtures which require a very time-consuming purification.  
       [0008] Pentacyclic oxazine derivatives are described as laser dyes in U.S. Pat. No. 5,149,807. However, they do not have a functional group and are thus not suitable for specific coupling to biological molecules such as proteins, haptens and nucleic acids and such an application is not referred to.  
       [0009] A tricyclic activated oxazine compound from the Dojindo Company is known whose amine substituents are only substituted with non-ring forming alkyl groups. The corresponding conjugate has a very low quantum yield.  
       [0010] The object of the present invention is to provide dyes which are suitable for coupling to biological molecules which have a high quantum yield, absorb in an absorption range of 645-700 nm and have the lowest possible unspecific binding to biological compounds or to solid phases.  
       [0011] This object is achieved by the invention as characterized in the claims.  
       [0012] The invention concerns functional oxazine derivatives capable of coupling of the general formula I  
                 
 
       [0013] in which R 1 , R 4 , R 5 , R 6 , R 7 , R 10  represent hydrogen, alkyl, alkoxy, hydroxy, halogen, carboxyl, sulfonyl or amino  
       [0014] and  
       [0015] R 2 , R 3    
       [0016] denote hydrogen, alkyl, alkoxy, polyoxyhydrocarbyl units, phenyl, phenylalkyl which can be substituted by hydroxy, halogen, sulfonyl, carboxy, amino, alkoxycarbonyl, in which R 2  with R 1  or R 3  with R 4  can form a saturated or unsaturated C2 or C3 bridge or R 2  with R 3  can form a saturated or unsaturated C4 or C5 bridge and  
       [0017] R 8 , R 9  denote hydrogen, alkyl, alkoxy, polyoxyhydrocarbyl units, phenyl, phenylalkyl which can be substituted by hydroxy, halogen, sulfonyl, carboxy, amino, alkoxycarbonyl in which R 8  with R 7  or R 9  with R 10  can form a saturated or unsaturated C2 or C3 bridge or R 8  with R 9  can form a saturated or unsaturated C4 or C5 bridge  
       [0018] and in which at least one of the residues R 2 , R 3 , R 8  or R 9  represents a non-bridge forming residue which is substituted by an activated group capable of coupling or by a group that can be activated to couple.  
       [0019] and in which at least one of the residues R 2 , R 3 , R 8  or R 9  represents a bridge-forming residue which optionally can be substituted by alkyl.  
       [0020] It is preferred when R 3  with R 4  and/or R 7  with R 8  forms a saturated or unsaturated C3 bridge.  
       [0021] It is particularly preferred when R 3  with R 4  and/or R 7  with R 8  form a C3 bridge while R 2  and/or R 9  represent non-bridge forming substituents, preferably alkyl and at least one non-bridge forming substituent is substituted with an activated group capable of coupling or with a group that can be activated.  
       [0022] The term “polyoxyhydrocarbyl units” is understood within the sense of the present invention as polymeric or oligomeric organic residues which are linked together by O bridges. In particular this term is understood as polyethers, polyoles, soluble carbohydrates, derivatives thereof or water-soluble polymers. Polyethyleneoxy groups are particularly preferred whose size is such that the molecular weight of the total compound is 800-1200, preferably about 1000. The aforementioned polyethyleneoxy groups improve the solubility, reduce the unspecific binding of the compounds to proteins and prevent a dimerization.  
       [0023] An alkyl group has 1-10 preferably 1-7 carbon atoms and can be branched or straight-chained; it especially preferably has 1 to 4 carbon atoms and is in particular for example methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl or tert. butyl.  
       [0024] A phenylalkyl group with preferably 1-3 carbon atoms in the alkyl group is in particular a phenethyl or benzyl group.  
       [0025] Halogen is understood as fluorine, chlorine, bromine or iodine and preferably chlorine.  
       [0026] An alkoxy group in an alkoxycarbonyl group has 1-10, preferably 1-4 and especially preferably 1 or 2 C atoms.  
       [0027] In the compounds according to the invention of formula I at least one of the residues R 2 , R 3 , R 8 , R 9  is preferably present as a non-bridge forming residue which is substituted with an activated group capable of coupling or with a group which can be activated for coupling. Such an activated group is in particular derived from an activatable carboxylic acid or sulfonic acid group and is for example an acid ester, an acid anhydride, an acid halogenide, preferably bromide in particular chloride or an N-hydroxysuccinimide ester. A linker compound such as DADOO may be inserted between the activated group and the non-bridge forming residue.  
       [0028] The table gives some examples for activated groups capable of coupling. A person skilled in the art knows further such groups from the synthetic chemistry for conjugates.  
                               TABLE 1                                   Activated group   Linkage with   Product                          NHS ester   amines   amide           isothiocyanate   amines   thiourea           mixed anhydride   amines   amide           maleimide   thiol   thioether           thiol   maleimide   thioethers           haloacetyl   thiol   thioethers           hydrazine   aldehyde   hydrazones           amines   aldehyde   amine (after                   reduction)           amines   reactive   amides               carboxylic acid                      
 
       [0029] Any anion suitable for charge neutralization and compatible with the cationic backbone can be used as the counterion; perchlorate is preferably used or the counterion is derived from a carboxyl or sulfonic group of one of the residues. In addition to the selection and combination of the residues, the selection of a suitable counterion enables the desired degree of lipophilicity to be optimized according to the intended application purpose.  
       [0030] Examples of particularly preferred substituents in the meaning of R 2 , R 3 , R 8  or R 9  are: hydrogen, methyl, carboxymethyl, ethyl, carboxyethyl, 3-sulfopropyl, 4-sulfobutyl, 3-carboxypropyl, 4-carboxy-butyl, 3-methoxycarbonylpropyl, 3-ethoxycarbonylpropyl, methoxyethoxyethyl, hydroxyethoxyethyl, benzyl.  
       [0031] When used as hydrophilic narkers it can be expedient to use asymmetric substituted products in which the residues R 2 , R 3  are different from R 8 , R 9  and for example denote a 3-carboxypropyl or 4-carboxybutyl group (R 2  or/and R 3 ) and a 3-sulfopropyl or 4-sulfobutyl group (R 8  and/or R 9 ).  
       [0032] Particularly preferred residues in compounds of formula I are:  
                 
 
       [0033] R 1 , R 4 , R 5 , R 6  and/or R 10 =hydrogen  
       [0034] R 3  with R 4  and/or R 8  with R 7 =(CH 2 ) 3 ,  
       [0035] R 1  with R 2  or R 9  with R 10 =(CH 2 ) 3    
       [0036] R 2 , R 3 , R 8 , R 9 =—CH 2 —CH 3 , —CH 2 —CH 2 —CH 2 —COOH  
       [0037] R 2  with R 3  or R 8  with R 9 =(CH 2 ) 4    
       [0038] The invention in addition concerns biologically active substances coupled to the fluorescent dyes according to the invention (conjugates) of formula II.  
                 
 
       [0039] in which R 1 , R 4 , R 5 , R 6 , R 7 , R 10  have the meaning stated above.  
       [0040] R 2 ′, R 3 ′ 
       [0041] denote hydrogen, alkyl, alkoxy, polyoxyhydrocarbyl units, phenyl, phenylalkyl which can be substituted by hydroxy, sulfonyl, carboxy, amino, alkoxycarbonyl in which R 2 ′ with R 1  or R 3 ′ with R 4  can form a saturated or unsaturated C4 or C5 bridge and  
       [0042] R 8 ′, R 9 ′ 
       [0043] denote hydrogen, alkyl, alkoxy, polyoxyhydrocarbyl units, phenyl, phenylalkyl which can be substituted by hydroxy, sulfonyl, carboxy, amino, alkoxycarbonyl in which R 2 ′ with R 1  or R 3 ′ with R 4  can form a saturated or unsaturated C4 or C5 bridge and in which at least one of the residues R 2 ′, R 3 ′, R 8 ′ or R 9 ′ represents a non-bridge forming residue which is coupled to a biologically active substance and in which at least one of the residues R 2 ′, R 3 ′, R 8 ′ and R 9 ′ represents a non-bridge forming residue which is optionally substituted by alkyl.  
       [0044] A biologically active substance is in particular understood as a hapten, antigen, antibody or fragment thereof, protein or mononucleotide or polynucleotide (PNA, RNA or DNA molecule).  
       [0045] The compounds for formula I can be obtained by condensation of 1,3-aminophenols of formula III with nitrosoaminophenols of formula IV.  
                 
 
       [0046] Activatable groups are activated to form groups capable of coupling according to known methods and coupled to reactive groups of biologically active molecules to form conjugates of formula II. In this process it is also possible to incorporate linkers between the activated groups and the biologically active molecules.  
       [0047] The compounds according to the invention provide new compounds which due to their spectroscopic properties (absorption maximum in the range between 645 to 700 nm) are very well suited for absorption dyes capable of coupling, in particular fluorescent dyes for application in hapten/and antibody-protein conjugates, for polynucleotide labelling and for dyeing latices (fluorescent latices). The quantum yield is high and is between 40 and 70% in ethanolic solution.  
       [0048] For application in hapten/antibody/protein or polynucleotide conjugates it is advantageous when the dyes are readily water-soluble. For this application compounds of the general formula I are preferably used in which R 2 , R 3 , R 8 , R 9  are as hydrophilic as possible. These compounds are preferably asymmetrically substituted products which for example contain carboxyl as well as sulfonic acid groups. Coupling to the conjugate is achieved via one of these activated substituents of the residues R 2 , R 3 , R 8  or R 9  and in particular via a hydroxysuccinimide group.  
       [0049] Conjugates of the fluorescent dyes with haptens such as for example theophylline, digoxin, T 3 , T 4  or protein such as for example antibodies are for example suitable for use in diagnostic systems in particular for fluorescence immunoassays.  
       [0050] A further subject matter of the present invention is a method for the determination of a first immunologically bindable substance which is characterized in that a conjugate of a compound according to the invention is used with a second immunologically bindable substance which can be the same or different from the first substance and that the change in absorbance or fluorescence or fluorescence polarization of the compound according to the invention caused by an immunological binding reaction which is specific for the first substance is determined as a measure for the amount of substance to be determined contained in the sample.  
       [0051] A further subject matter of the invention is the use of the conjugates according to the invention for immunoassays.  
       [0052] The compounds capable of coupling of formula I according to the invention are also suitable for the production of conjugates with mononucleotides or polynucleotides or PNA.  
       [0053] The invention therefore also concerns the use of these conjugates for DNA analysis.  
     
    
    
     EXAMPLE 1  
     [0054] Synthesis of I  
     [0055] 0.6 g (2.3 mmol) γ-(7-hydroxy-1,2,3,4-tetrahydroquinol-1-yl)butyric acid ethyl ester and 0.5 g (2,4 mmol) N-ethyl-7-hydroxy-6-nitroso-1,2,3,4-tetrahydroguinoline are boiled for 2 hours under reflux in 12 ml ethanol after addition of 2 ml 2.5 M hydrochloric acid. The solution is evaporated to dryness on a rotary evaporator. The residue is taken up in ethanol and chromatographically pre-purified over aluminium oxide. The ethyl ester of the target dye obtained in this manner has an absorption maximum in ethanol at 653 nm.  
     [0056] The ethyl ester is boiled for 30 min under reflux in a mixture of 30 ml acetone, 20 ml water and 1 ml 2.5 M hydrochloric acid. For purification the dye is chromatographed over silica gel (mobile solvent: firstly acetone-chloroform 3:1, then acetone and finally ethanol). 0.5 g I is obtained.  
                 
 
     EXAMPLE 2  
     [0057] Oxazine-N-hydroxysuccinimide ester II  
     [0058] 40 mg oxazine I is dissolved together with 10 mg N-hydroxysuccinimide ester and 19 mg dicyclohexyl carbodiimide in 20 ml acetonitrile. It is allowed to stir for 4 hours at room temperature and the product mixture is rotary evaporated. It is purified by means of reverse phase silica gel.  
                 
 
     [0059] Digoxin-3-carboxymethyl ether-diaminodioxooctane conjugate III (Dig-CME-DADOO)  
     [0060] The hapten-fluorescent conjugate is obtained by reacting 11 mg I and 17.5 mg Dig-DADOO for 18 hours at room temperature in acetonitrile. The mixture is rotary evaporated and subsequently purified over silica gel, eluant chloroform-methanol-acetic acid 3:1:0:1.  
     [0061] Yield: 4 mg  
     [0062] Analysis: MS corresponds  
     [0063] Labelling of proteins with II  
     [0064] 10 mg protein e.g. MAB&lt;TSH&gt;is dissolved in 1 ml sodium hydrogen phosphate buffer pH 8. A solution of a 10-fold molar excess of II dissolved in 500 μl DMSO is added to this. The reaction solution is shaken for 1 hour at room temperature. The conjugate is purified over a Sephadex G 50 column, mobile solvent buffer, dialysed three times against water and lyophilized.  
                 
 
     [0065] Synthesis of oxazines of formulae IV-X with activatable COOH groups  
     [0066] 3 mmol substituted m-aminophenol or m-aminoanisol and 33 mmol 6-nitroso-3-amino-phenol are dissolved in a mixture of 20 ml ethanol and 1 ml 2.5 N hydrochloric acid and heated to reflux. The dye formation that takes place in this process is monitored spectrometrically (λmax in a range of 650-700 nm). The reaction is terminated when the dye concentration no longer increases.  
     [0067] The solution obtained in this way is evaporated to a volume of ca. 10 ml and then added dropwise to 200 ml 10% aqueous NaBF 4  solution. The dye tetrafluoroborate precipitates completely in this process. After decanting the supernatant liquid and filtering, the residue is taken up in 100 ml dichloromethane and this solution is washed 3 tines with 100 ml water each time. The solution is dried over Na 2 SO 4  and rotary evaporated. In this process the dye accumulates as a viscous, almost black oil. Yield: 40-60%.  
     [0068] Hydrolysis:  
     [0069] The crude dye (ethyl ester) is dissolved in a mixture of 30 ml acetone, 15 ml water and 1 ml 2.5 N hydrochloric acid and heated to reflux. The hydrolysis is monitored by thin layer chromatography (silica gel, MeOH/H 2 O 3:1). After almost complete reaction, the reaction solution is rotary evaporated at ca. 30° C. and the residue is purified by chromatography.  
                                                           Reaction   Duration of                   time   hydrolysis   Purification   λmax. EtOH                                                        IV   1.5 h    7 h   —   660       V    70 min    9 h   silica gel MS =   650                   chloroform/EtOH       VI    75 min    9 h   silica gel MS =   649                   chloroform/EtOH       VII    40 min   10 h   silica gel MS =   672                   chloroform/EtOH       VIII    90 min   48 h   silica gel MS =   682                   chloroform/EtOH       IX     3 h   24 h   silica gel MS =   673                   chloroform/EtOH       X    60 min   24 h   silica gel MS =   672                   chloroform/EtOH                  
 
     [0070]