CA1242707A - PHENOLSULPHONPHTHALEINYL-.beta.-D-GALACTOSIDES, A PROCESS FOR THE PREPARATION THEREOF AND DIAGNOSTIC AGENTS CONTAINING THEM - Google Patents
PHENOLSULPHONPHTHALEINYL-.beta.-D-GALACTOSIDES, A PROCESS FOR THE PREPARATION THEREOF AND DIAGNOSTIC AGENTS CONTAINING THEMInfo
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- CA1242707A CA1242707A CA000470093A CA470093A CA1242707A CA 1242707 A CA1242707 A CA 1242707A CA 000470093 A CA000470093 A CA 000470093A CA 470093 A CA470093 A CA 470093A CA 1242707 A CA1242707 A CA 1242707A
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- Prior art keywords
- beta
- galactoside
- formula
- galactosidase
- hydrogen
- Prior art date
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/20—Carbocyclic rings
- C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
-
- 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
-
- 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
- C12Q2334/00—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
- C12Q2334/40—Triphenylmethane dye chromogens, e.g. fluorescein derivatives
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
Abstract
ABSTRACT OF THE DISCLOSURE
Phenolsulphonphthaleinyl-.beta.-D-galactosides of the general formula (I):
Phenolsulphonphthaleinyl-.beta.-D-galactosides of the general formula (I):
Description
q~e present invention i~ concerned with new phenol~ulphonphthaleinyl-~-D-galacto~ides, a proces~
for the preparation thereof and the use thereof for the determination of ~-D-galactosidaSe.
Oligo~ or polysaccharides which contain D-galactose with ~-glycosidic bonding occur in almo3t all organisms. Consequently, the corresponding ~-D-galactosidases (EC 3.2.1.23) are also widely occurring and can be detected in numerous micro-organisms, animals and plants~
~ -D-galactosidase fulfils a multiple physio-logical function in mammals. Thus, it plays an important part in carbohydrate metabolism since it brings about the hydrolysis of lactose. Furthermore, ~-D-galactosidase is a key enzyme in the breakdown of - glycolipid~, mucopolysaccharides and glycoproteins In recent years, ~-D-galactosidase has achieved importance in the field of diagnosis due to its physiological importance. Thus, for cxa~ple, this enzyme is employed to an increasing extent a~ an indicator enzyme for Pnzyme immunoassays (see, for exarnple, ~nnals of Clinical BiochemistryJ 16, 221 240/1979 ~ ~
Con~equently, the determination of the activity of ~-D-galactosidase is of increasing importance not only in clinical chemi~try but also in diagnosis~
For thi~ purpose, quit~ generally, a galactosidase-~, P
,~
containing qample i~ mixed with an appropriate ~-D-galactosidase substrate, the ~ub~trate being split by the enzyme, one of the fis~ion product~ then being detected in an appropriate manner. rhere can be mea~ured either the glycone liberated by action of the enz~ne or the aglyconeS A~ a rule, the latter is detenmined. As substrate, there can be used the natural sub~trate lactose, as well as especially a chromogenic galactoside.
Thu~, in Biochem. ZO~ 333, 209/1960, there are described phenyl-~-D-galactoside, ~s well as some further derivatives substituted on the aromatic ring, for example o-nitrophenyl- and ~-nitrophenyl-~-D-galactoside, as substrates of ~D-galactosidase. The phenols liberated by hydrolysis are determined photo~
metrically in the W range or, in the case of the nitrophenols, in the short-waved, visib:le wavelength range. ~n oxidatlve coupling with aminoan-tipyrine can al~o fQllow as indicator reaction (see Analytical Bioch~m., 40, 281/1971).
For histochemical investigations, there are used, on the on~ hand, naphthyl-~-D-galacto~ide~, thus, for example, the l-naphthyl compound in Hi~tochemie, 35, lgg/1973, the 6-bromo-2~naphthyl derivative in J. Biol. Chem., 195, 239/1952 and the naphthol-AS-BI-~-D-galacto3ide in Hintochemie, 37, 89/1973. For vi~ualisation, the naphthols thereby re~ulting are reacte~ with various diammonium salt~
to give azo dyestuff~.
Furthermore, 5-bromo-~-chloroindoxyl-~-D-galacto3ide i3 Xnown a~ a substrate of ~-yalacto3id-ase. The indicator reaction .is here the oxidativedimerisation of the resulting indoxyl to give indiyo (Histochemie, 23, 266/1970) or coupling with diazonium salts to give indoxyl azo dyestuffs (Mistochemie, 57, 323/197~).
The described method~ of determination display considerable disadvantage~q on the one hand, they are too insensitive. On the other hand, the substrates used in the histochemical detection are very poorly soluble.
Substantially more sensiti~le tests re~ult when, as ~ubstrate~, galactosides are used, the aglycone of which can be detected fluorometrically. Thus, in Proc. Nat. Acad~ Sci. US, 47, 1981/1961, fluorescein di-~-D-galactoside is described as a suhstrate~
Furthenmore, u~e can be made of 2-naphthyl-~-D-galactoside (Analytical Biochem., 42, 275flg71) or of 4-methyl-umbelLiferyl-~-D-galactoside (Biochem. J., 02, 525/1967).
A di~adva~tage of the fluorometrical methods i~ the con~iderable expense of thP apparatu~ which ha~ to be u~ed.
~2~
~, There~ore, there is ~till a need ~or ~ub~trates with which ~-D-~alactosida~e can be determined 3imply, quickly and dependablyO
We have now found that ~-D-galactosida3e c~n be detected very sensitively and vi~ually in the visible spectral range or with a simple ~pectral photometer apparatus when using ~ulphonphthaleinyl-~D-galactosides as substrates~ Furthermore, these com-pounds have the advantage that they are very easily water-soluble.
Consequently, according to the present invention, there are provided sulphonphthaleinyl~
galactosides of the general ~ormula:-HO~ o~ R R ~ O
R7 ~ ~ ~ R
~ ~ S3-wherein Rl to R4, which can be the same or different, are hydrogen or halogen atoms or nitro or amino groups, R5 to Rl~, which can be the ~me or dif~erent, are hydrogen or halogen atoms or lower alkyl, hydroxyl, lower alko.~y, carboxyl or nitro groups and ~ i3 a 7~
proton or an alkali metal, alkaline earth metal or ammonium ion.
All the ~ulphonphthaleinyl-~-D-galactoside~
of the general fonmula I are new compounds. They can be prepared by methods known from carbohydrate chemistry~ which methods form another aspect of the invention.
Preferably, phenolsulphonphthaleins of the general formula:-HO ~ R5 R9 ~ OH
R7 ~ C ~ ~ Rll R~ ¦ \ O R (II) R ~ ~ S02 R3 ~ `R
w~erein R to R12 have the above-given meanings, are reacted in known manner with per-O-substituted l-halogend ~-D-galactoses of the general formula:-R O ~ O
(III) ~X
oR13 in which X is a leaving group and R13 is a protecti.ve ~roup conventional in carbohydrate chemistry, with ~i~d~d 1~3 6 ~
Walden inversion of the C-l atorn of the ~ugar re~idue to give per-O-substituted sulphonphthaleinyl-~ D-galactosides of the general formula:-CH 2 R6 Rl o 13 ~ Rll OR R8 ¦ R12 (IV) 4 1 _ +
R ~ S03 M
R3 ~ Rl and the protective groups R 3 are split off from thela~ter in known manner.
In particular X i9 a leaving group displaceable by a phenolic hydroxyl group in a phenolsulphon~
phthale:in of formula (II) :in an SN nuc:Leophilic substitut:ion reaction. Most suitably X is a ha:Logen atom.
The reaction of the compounds of general forrnulae II and III to give galactosides of general formula IV is preferably carried out in the presence of an acid acceptor, for example, alkali metal hydroxide or carbonate, in aqueous acetone or, under phase transfer conditions, in a water/benzene or water/chloroform rnixture.
Furthermore, the galactosides of general formula IV can be prepared by first converting the lû phenolsulphonphthaleins of formula II by means of an alkali metal hydroxide or alcoholate into a di-alkali metal salt or by means of an optionally substituted amine into an ~mmonium salt, whexeafter these are then reacted in a dipolar aprotic ~olvent, for example,acetone, dimethyl sulphoxide, dichloromethane, tetrahydrofuran or dimethylformamide, with the per-0-substituted l~halogenogalactoses of general formula III.
Furthermore, in the case of the synthesis ofgalacto~ides of general formula IV from the phenol-sulphonphthaleins of general fonmula II and the 1-halogalactoses of general formula III, additions of individual silver salts or mixtures of silver salta (silver oxide, silver carbonate, silver carbonate on Celite~ silver triflate or silver salicylate) and/or of individual mercury salts or mixtures of mercury salts (mercury bromide, cyanide, acetate or oxide), optionally with the use of drying agents, rOr example, - calcium chloride or Drieri~e~ in solvents, for example, methylene chlorlde, chloroform, benzene, toluene or dioxan, have proved to be useful.
The ~o-obtained per-0 sub~tituted sulphon-phthaleinyl-~-D-galactosides of gener~l formula IV
are also new compounds.
The splitting off of the protective groups R 3 from the per-0-substituted ~ulphonphthaleinyl-~-D-galacto3ide~ of general fonmula IV to give the sulphonphthaleinyl-~-D-galactoaide~ of general fonmula I i~ carried out according to the conventional method~
in carbGhydrate chemi3try (~ee, for example, Advanced ~ trade mark _9~
Carbohydrate Chem., 12, 1S7/1957), for example in the case of acyl protective groups by means of sodium methylate or barium methylate or ammonia in methanol.
The phenolsulphonphthaleins of the general formula II are either known, co~ercially available substances or can be prepared by known proce~ses from the corresponding phenol and the corresponding o-sulphonbenzoic acid (see, for example, D.S 7 Breslow and H. Skolnik,in A.Weissberger~ The Chemistry of Hetero-cyclic Compounds, Interscience Publishers, New York, 1966, Volume 21t p. 118) or, starting from known sulphon-phthaleins, by subsequent derivatisation, for example by halogenation or nitration (cf., for example, D.S. Bre~low and H. Skolnik, ibid., pp. 141 and 144).
The per-0-substituted l-halogeno-a-D-galactoses of general formula III employed as staxting materials are al~o known compounds. They are described, for example, in Chern. Ber., 35~ 836/1902, Nature, 165, 369/1950: Acta chem. Scand., Ser. B, 33, 116/1979, J. Chem. Soc~, 1419/1965; and Carbohydr. Res., 11, 85/1969~
By halogen in the definitions of Rl to ~12 and X is to be understood fluorins, chlorine, bromine and iodine and, in the case of Rl to R12, preferably fluorine, chlorine and bromine and, in the case of X~
preferably chlorins and bromine.
3~
The lower alkyl radical in the definitions of R5 to R suitably contains l to 5 and preferably l to 3 carbon atoms, the methyl and isopropyl radicals being especially preferred.
The lower alkoxy radical in the definitions of R5 to R suitably contains l to 5 and preferably l to 3 carbon atoms, the methoxy radical being especially preferred.
The alkali metal ion in the definition of M is to be understood to be the lithium, sodium or potassium ion9 lithium and sodium ions being preferred.
The alkaline earth metal ion in the definition of M signifies the magnesium9 calcium or barium iOIl, the calcium ion being preferred.
The ammonium ion in the definition of M is to be understood to be a radical of` general formula 14 15Rl6Rl7]~ wherein R14 to R , which can be same or dirfcr-ent, slgnify hydrogen atoms or lower a]kyl radicals suitably containing l to 4 and preferably l or
for the preparation thereof and the use thereof for the determination of ~-D-galactosidaSe.
Oligo~ or polysaccharides which contain D-galactose with ~-glycosidic bonding occur in almo3t all organisms. Consequently, the corresponding ~-D-galactosidases (EC 3.2.1.23) are also widely occurring and can be detected in numerous micro-organisms, animals and plants~
~ -D-galactosidase fulfils a multiple physio-logical function in mammals. Thus, it plays an important part in carbohydrate metabolism since it brings about the hydrolysis of lactose. Furthermore, ~-D-galactosidase is a key enzyme in the breakdown of - glycolipid~, mucopolysaccharides and glycoproteins In recent years, ~-D-galactosidase has achieved importance in the field of diagnosis due to its physiological importance. Thus, for cxa~ple, this enzyme is employed to an increasing extent a~ an indicator enzyme for Pnzyme immunoassays (see, for exarnple, ~nnals of Clinical BiochemistryJ 16, 221 240/1979 ~ ~
Con~equently, the determination of the activity of ~-D-galactosidase is of increasing importance not only in clinical chemi~try but also in diagnosis~
For thi~ purpose, quit~ generally, a galactosidase-~, P
,~
containing qample i~ mixed with an appropriate ~-D-galactosidase substrate, the ~ub~trate being split by the enzyme, one of the fis~ion product~ then being detected in an appropriate manner. rhere can be mea~ured either the glycone liberated by action of the enz~ne or the aglyconeS A~ a rule, the latter is detenmined. As substrate, there can be used the natural sub~trate lactose, as well as especially a chromogenic galactoside.
Thu~, in Biochem. ZO~ 333, 209/1960, there are described phenyl-~-D-galactoside, ~s well as some further derivatives substituted on the aromatic ring, for example o-nitrophenyl- and ~-nitrophenyl-~-D-galactoside, as substrates of ~D-galactosidase. The phenols liberated by hydrolysis are determined photo~
metrically in the W range or, in the case of the nitrophenols, in the short-waved, visib:le wavelength range. ~n oxidatlve coupling with aminoan-tipyrine can al~o fQllow as indicator reaction (see Analytical Bioch~m., 40, 281/1971).
For histochemical investigations, there are used, on the on~ hand, naphthyl-~-D-galacto~ide~, thus, for example, the l-naphthyl compound in Hi~tochemie, 35, lgg/1973, the 6-bromo-2~naphthyl derivative in J. Biol. Chem., 195, 239/1952 and the naphthol-AS-BI-~-D-galacto3ide in Hintochemie, 37, 89/1973. For vi~ualisation, the naphthols thereby re~ulting are reacte~ with various diammonium salt~
to give azo dyestuff~.
Furthermore, 5-bromo-~-chloroindoxyl-~-D-galacto3ide i3 Xnown a~ a substrate of ~-yalacto3id-ase. The indicator reaction .is here the oxidativedimerisation of the resulting indoxyl to give indiyo (Histochemie, 23, 266/1970) or coupling with diazonium salts to give indoxyl azo dyestuffs (Mistochemie, 57, 323/197~).
The described method~ of determination display considerable disadvantage~q on the one hand, they are too insensitive. On the other hand, the substrates used in the histochemical detection are very poorly soluble.
Substantially more sensiti~le tests re~ult when, as ~ubstrate~, galactosides are used, the aglycone of which can be detected fluorometrically. Thus, in Proc. Nat. Acad~ Sci. US, 47, 1981/1961, fluorescein di-~-D-galactoside is described as a suhstrate~
Furthenmore, u~e can be made of 2-naphthyl-~-D-galactoside (Analytical Biochem., 42, 275flg71) or of 4-methyl-umbelLiferyl-~-D-galactoside (Biochem. J., 02, 525/1967).
A di~adva~tage of the fluorometrical methods i~ the con~iderable expense of thP apparatu~ which ha~ to be u~ed.
~2~
~, There~ore, there is ~till a need ~or ~ub~trates with which ~-D-~alactosida~e can be determined 3imply, quickly and dependablyO
We have now found that ~-D-galactosida3e c~n be detected very sensitively and vi~ually in the visible spectral range or with a simple ~pectral photometer apparatus when using ~ulphonphthaleinyl-~D-galactosides as substrates~ Furthermore, these com-pounds have the advantage that they are very easily water-soluble.
Consequently, according to the present invention, there are provided sulphonphthaleinyl~
galactosides of the general ~ormula:-HO~ o~ R R ~ O
R7 ~ ~ ~ R
~ ~ S3-wherein Rl to R4, which can be the same or different, are hydrogen or halogen atoms or nitro or amino groups, R5 to Rl~, which can be the ~me or dif~erent, are hydrogen or halogen atoms or lower alkyl, hydroxyl, lower alko.~y, carboxyl or nitro groups and ~ i3 a 7~
proton or an alkali metal, alkaline earth metal or ammonium ion.
All the ~ulphonphthaleinyl-~-D-galactoside~
of the general fonmula I are new compounds. They can be prepared by methods known from carbohydrate chemistry~ which methods form another aspect of the invention.
Preferably, phenolsulphonphthaleins of the general formula:-HO ~ R5 R9 ~ OH
R7 ~ C ~ ~ Rll R~ ¦ \ O R (II) R ~ ~ S02 R3 ~ `R
w~erein R to R12 have the above-given meanings, are reacted in known manner with per-O-substituted l-halogend ~-D-galactoses of the general formula:-R O ~ O
(III) ~X
oR13 in which X is a leaving group and R13 is a protecti.ve ~roup conventional in carbohydrate chemistry, with ~i~d~d 1~3 6 ~
Walden inversion of the C-l atorn of the ~ugar re~idue to give per-O-substituted sulphonphthaleinyl-~ D-galactosides of the general formula:-CH 2 R6 Rl o 13 ~ Rll OR R8 ¦ R12 (IV) 4 1 _ +
R ~ S03 M
R3 ~ Rl and the protective groups R 3 are split off from thela~ter in known manner.
In particular X i9 a leaving group displaceable by a phenolic hydroxyl group in a phenolsulphon~
phthale:in of formula (II) :in an SN nuc:Leophilic substitut:ion reaction. Most suitably X is a ha:Logen atom.
The reaction of the compounds of general forrnulae II and III to give galactosides of general formula IV is preferably carried out in the presence of an acid acceptor, for example, alkali metal hydroxide or carbonate, in aqueous acetone or, under phase transfer conditions, in a water/benzene or water/chloroform rnixture.
Furthermore, the galactosides of general formula IV can be prepared by first converting the lû phenolsulphonphthaleins of formula II by means of an alkali metal hydroxide or alcoholate into a di-alkali metal salt or by means of an optionally substituted amine into an ~mmonium salt, whexeafter these are then reacted in a dipolar aprotic ~olvent, for example,acetone, dimethyl sulphoxide, dichloromethane, tetrahydrofuran or dimethylformamide, with the per-0-substituted l~halogenogalactoses of general formula III.
Furthermore, in the case of the synthesis ofgalacto~ides of general formula IV from the phenol-sulphonphthaleins of general fonmula II and the 1-halogalactoses of general formula III, additions of individual silver salts or mixtures of silver salta (silver oxide, silver carbonate, silver carbonate on Celite~ silver triflate or silver salicylate) and/or of individual mercury salts or mixtures of mercury salts (mercury bromide, cyanide, acetate or oxide), optionally with the use of drying agents, rOr example, - calcium chloride or Drieri~e~ in solvents, for example, methylene chlorlde, chloroform, benzene, toluene or dioxan, have proved to be useful.
The ~o-obtained per-0 sub~tituted sulphon-phthaleinyl-~-D-galactosides of gener~l formula IV
are also new compounds.
The splitting off of the protective groups R 3 from the per-0-substituted ~ulphonphthaleinyl-~-D-galacto3ide~ of general fonmula IV to give the sulphonphthaleinyl-~-D-galactoaide~ of general fonmula I i~ carried out according to the conventional method~
in carbGhydrate chemi3try (~ee, for example, Advanced ~ trade mark _9~
Carbohydrate Chem., 12, 1S7/1957), for example in the case of acyl protective groups by means of sodium methylate or barium methylate or ammonia in methanol.
The phenolsulphonphthaleins of the general formula II are either known, co~ercially available substances or can be prepared by known proce~ses from the corresponding phenol and the corresponding o-sulphonbenzoic acid (see, for example, D.S 7 Breslow and H. Skolnik,in A.Weissberger~ The Chemistry of Hetero-cyclic Compounds, Interscience Publishers, New York, 1966, Volume 21t p. 118) or, starting from known sulphon-phthaleins, by subsequent derivatisation, for example by halogenation or nitration (cf., for example, D.S. Bre~low and H. Skolnik, ibid., pp. 141 and 144).
The per-0-substituted l-halogeno-a-D-galactoses of general formula III employed as staxting materials are al~o known compounds. They are described, for example, in Chern. Ber., 35~ 836/1902, Nature, 165, 369/1950: Acta chem. Scand., Ser. B, 33, 116/1979, J. Chem. Soc~, 1419/1965; and Carbohydr. Res., 11, 85/1969~
By halogen in the definitions of Rl to ~12 and X is to be understood fluorins, chlorine, bromine and iodine and, in the case of Rl to R12, preferably fluorine, chlorine and bromine and, in the case of X~
preferably chlorins and bromine.
3~
The lower alkyl radical in the definitions of R5 to R suitably contains l to 5 and preferably l to 3 carbon atoms, the methyl and isopropyl radicals being especially preferred.
The lower alkoxy radical in the definitions of R5 to R suitably contains l to 5 and preferably l to 3 carbon atoms, the methoxy radical being especially preferred.
The alkali metal ion in the definition of M is to be understood to be the lithium, sodium or potassium ion9 lithium and sodium ions being preferred.
The alkaline earth metal ion in the definition of M signifies the magnesium9 calcium or barium iOIl, the calcium ion being preferred.
The ammonium ion in the definition of M is to be understood to be a radical of` general formula 14 15Rl6Rl7]~ wherein R14 to R , which can be same or dirfcr-ent, slgnify hydrogen atoms or lower a]kyl radicals suitably containing l to 4 and preferably l or
2 carbon atoms, or benzyl radicals.
The protective group R 3 conventional iD carbo-hydrate chemistry is especially preferably an acetyl, benzoyl, benzyl or trimethylsilyl radical.
The present invention is also concerned with the use of the new sulphonphthaleinyl-~D-galactosides of general formula I for the determination of the activity of ~D-galacto idase. Furthermore, the present invention provides diagnostic agents for the detenmination of the activity of ~-D-galactosid~
ases, which agents contain the new sulphonphthaleinyl-~-D-galactosides of general formula I.
The use of the new sulphonphthaleinyl-~-D-galactosides as substrates for ~-D-galactosidase gives ~-D-galactosidase test systems which are much more sensitive than those previously known. The new substrates can be advantageously used for th~ deter-mination of the activity of ~-D-galactosidases, not only in the biochemical field but also in the clinical-chemical field since they are more sensitive.
From this result several advantages:
a) Smaller ~-D-galactosidase activities can be measured.
b) Small~r amounts o~ sample can be employed.
c) The determi.nation of the ~-D-galactosidas~ activity can take place in a considerably shorter time.
d) Furthermore, the small sample use and the favour-able measurement wavelength reduce the suscept-ibility to disturbance of the method due to other components present in the sample~
We have found that the new substrates can be used for the determination of the activity of ~-D-galactosida~es of any origin and which can differ in their optimum pH v~lue. In such ca~es, too, ~, .......................... .
-12~
diagnostic ~gents con-taining substrates of general formula I react much more sensitively than the previously known test agents.
The sulphonphthaleinyl-~-D-galactosides of general formula I can also be used or immunological methods of determination in which ~-D-galactosidase is used as indicator enzyme, the activity of which must be determined after carrying out of the immunological reaction. Such immunological methods of determination with an enzymatic indicator reaction are known as enzyme immunoassays. These methods serve for the determination of the concentration of proteins, polysaccharides, honmones, pharmaceuticals and other low molecular substances in the range of 10 5 tu 10 12 mole/litre. Depending upon the require-ment of phase separation steps, a differentiation ~s made between a homogeneous and a heterogeneou3 carrying out of the tests. A further subdivision can take place into competitive and non-competitive test principles.
However, all test principles work with enzyme-antigen or enzyme-antibody conjugates. The enzymatic indicator reaction is common to all enzyme immuno-as~ays.
An indicator enzyme which can be u~ed for such purposes is ~-D-galactosidase. The determination of ~-D-galactosidase in such enzyme immunoassays usually takes place by adding an appropriate ~-D-galactosida~e substrate thereto, which is enzymatically split and measured photometrically in the usual way.
Consequently, an improvement of the ~-D-galactosidase test system also leads to considerableadvantage6 in the case o such enzyme immunoassays:
1. Here, too, a higher sensitivity makes possible a further lowering of ~he detection limits, shorter reaction times and smaller sample use and thus also smaller disturbances by other components of the sample.
2. The more favourable measurement wavelength reduces, in ~he case of certain carryings out of reaction, the susceptibility to disturbance of the methods by insoluble components, for example by turbidities.
Besides one OI' more of the substrates of gen~ral fonmula I according to the present invention, the d.iagno~tic agent contains an appropriate buffer ~ys-tem, as well as poss.ibly further appropriate additional materials conventionally used for such diagnostic agents, for example wetting agents, stabilisers and the like. ~he diagnostic agent can be present in the form of a solution, a lyophilisa~e, a powder mixture or a reaysnt tablet or can ke applied to an absorbent carrier.
~ ~ *~r~ ff The diagno~tic agent according to the pre~ent invention in the form of a ~olution preferably con-tain~ all reagents required for the test7 As solvent, there can be used water or a mixture of water with a water-~oluble organic solvent, for example methanol, ethanol, acetone or dimethylformamide. For reason~
of storage stability, it can be advantageou~ to divide up the reagents required for the test into two or more solutions which are first mixed immediately before carrying out the actual inve~tigation.
For the preparation of the diagnostic agent in the form of a lyophilisate with a total weight of, in each case, about 5 to 20 mgO and preferably of about 10 mg., a solution i5 dried which, besides all the reagents needed for the te~t, contains conventional structural formers, Eor example polyvinylpyrrolidone, and optionally ~urther filler materials, for example mannitol, ~orbitQI or xylitol.
A diagnostic agPnt in the form of a powder mixture or of a reagent tablet can be produced by mixing the components of the test with conventional galenical additive materials and granulated~ Additive materials of thi~ Xind include, for example, carbo-hydrates, ~uch a mono-, oligo- and poly3accharide~, sugar alcohol~, such a3 mannitol, sorbitol and xylitol, and other ~oluble inert compound~, ~uch a~ polyethylene glycols and polyvinylpyrrolidone. ln general, the s~ j/ il ~A 1i~3 ~5-powder mixtures or reagent tablets have an end weight of about 30 to 200 mg. and preferably of 50 to 80 mg.
For the production of the diagnostic agents in the fonm of a test strip, an absorbent carrier, preferably filter paper, cellulose or synthetic fibre fleece, is impregnated with solutions of the necess-ary reagents usually-employed for the production of test strips in readily volatile solvents, for example water, methanol, ethanol or acetone. This can take place in one impregnation step. However, it is often preferable to carry out the impregnation in several steps, solutions being used which each contain some of the components of the diagnostic agent. Thus, for example, in a firs~ step, impregnation can be carried out with an aqueous solution which contains the buffer and other water-soluble additive materials and then, in a second step, with a solution which contains khe ~--D-galactQsida~e substrate. The finished test papers can be used a3 such o~, in known manner, can be stuck on to handLes or preferably sealed between synthetic materials and fine meshworks according to Federal Republic of Germany Patent Specification No.2/118,455O
The following Examples illustrate some of the numerous process variants which can be used for the ~ynthesis of the new compounds according to the present invention, a~ well as, by way of example, the us~ of the new sulphonphthaleinyl-~-D-galactosides for the . ~
763~
determination of the activity of ~-D-galactosidase.
The following abbreviations are used in the Examples:
HEPES 2-~4-~2-hydroxyethyl)-1-piperazinyl~-ethane-sulphonic acid BSA bovine serum albumin Tween-20* polyoxyethylene(20~sorbitan monolaurate Tricin [N-tris-(hydroxymethyl)J-methyl-glycine~
Example 1.
The protective group R 3 conventional iD carbo-hydrate chemistry is especially preferably an acetyl, benzoyl, benzyl or trimethylsilyl radical.
The present invention is also concerned with the use of the new sulphonphthaleinyl-~D-galactosides of general formula I for the determination of the activity of ~D-galacto idase. Furthermore, the present invention provides diagnostic agents for the detenmination of the activity of ~-D-galactosid~
ases, which agents contain the new sulphonphthaleinyl-~-D-galactosides of general formula I.
The use of the new sulphonphthaleinyl-~-D-galactosides as substrates for ~-D-galactosidase gives ~-D-galactosidase test systems which are much more sensitive than those previously known. The new substrates can be advantageously used for th~ deter-mination of the activity of ~-D-galactosidases, not only in the biochemical field but also in the clinical-chemical field since they are more sensitive.
From this result several advantages:
a) Smaller ~-D-galactosidase activities can be measured.
b) Small~r amounts o~ sample can be employed.
c) The determi.nation of the ~-D-galactosidas~ activity can take place in a considerably shorter time.
d) Furthermore, the small sample use and the favour-able measurement wavelength reduce the suscept-ibility to disturbance of the method due to other components present in the sample~
We have found that the new substrates can be used for the determination of the activity of ~-D-galactosida~es of any origin and which can differ in their optimum pH v~lue. In such ca~es, too, ~, .......................... .
-12~
diagnostic ~gents con-taining substrates of general formula I react much more sensitively than the previously known test agents.
The sulphonphthaleinyl-~-D-galactosides of general formula I can also be used or immunological methods of determination in which ~-D-galactosidase is used as indicator enzyme, the activity of which must be determined after carrying out of the immunological reaction. Such immunological methods of determination with an enzymatic indicator reaction are known as enzyme immunoassays. These methods serve for the determination of the concentration of proteins, polysaccharides, honmones, pharmaceuticals and other low molecular substances in the range of 10 5 tu 10 12 mole/litre. Depending upon the require-ment of phase separation steps, a differentiation ~s made between a homogeneous and a heterogeneou3 carrying out of the tests. A further subdivision can take place into competitive and non-competitive test principles.
However, all test principles work with enzyme-antigen or enzyme-antibody conjugates. The enzymatic indicator reaction is common to all enzyme immuno-as~ays.
An indicator enzyme which can be u~ed for such purposes is ~-D-galactosidase. The determination of ~-D-galactosidase in such enzyme immunoassays usually takes place by adding an appropriate ~-D-galactosida~e substrate thereto, which is enzymatically split and measured photometrically in the usual way.
Consequently, an improvement of the ~-D-galactosidase test system also leads to considerableadvantage6 in the case o such enzyme immunoassays:
1. Here, too, a higher sensitivity makes possible a further lowering of ~he detection limits, shorter reaction times and smaller sample use and thus also smaller disturbances by other components of the sample.
2. The more favourable measurement wavelength reduces, in ~he case of certain carryings out of reaction, the susceptibility to disturbance of the methods by insoluble components, for example by turbidities.
Besides one OI' more of the substrates of gen~ral fonmula I according to the present invention, the d.iagno~tic agent contains an appropriate buffer ~ys-tem, as well as poss.ibly further appropriate additional materials conventionally used for such diagnostic agents, for example wetting agents, stabilisers and the like. ~he diagnostic agent can be present in the form of a solution, a lyophilisa~e, a powder mixture or a reaysnt tablet or can ke applied to an absorbent carrier.
~ ~ *~r~ ff The diagno~tic agent according to the pre~ent invention in the form of a ~olution preferably con-tain~ all reagents required for the test7 As solvent, there can be used water or a mixture of water with a water-~oluble organic solvent, for example methanol, ethanol, acetone or dimethylformamide. For reason~
of storage stability, it can be advantageou~ to divide up the reagents required for the test into two or more solutions which are first mixed immediately before carrying out the actual inve~tigation.
For the preparation of the diagnostic agent in the form of a lyophilisate with a total weight of, in each case, about 5 to 20 mgO and preferably of about 10 mg., a solution i5 dried which, besides all the reagents needed for the te~t, contains conventional structural formers, Eor example polyvinylpyrrolidone, and optionally ~urther filler materials, for example mannitol, ~orbitQI or xylitol.
A diagnostic agPnt in the form of a powder mixture or of a reagent tablet can be produced by mixing the components of the test with conventional galenical additive materials and granulated~ Additive materials of thi~ Xind include, for example, carbo-hydrates, ~uch a mono-, oligo- and poly3accharide~, sugar alcohol~, such a3 mannitol, sorbitol and xylitol, and other ~oluble inert compound~, ~uch a~ polyethylene glycols and polyvinylpyrrolidone. ln general, the s~ j/ il ~A 1i~3 ~5-powder mixtures or reagent tablets have an end weight of about 30 to 200 mg. and preferably of 50 to 80 mg.
For the production of the diagnostic agents in the fonm of a test strip, an absorbent carrier, preferably filter paper, cellulose or synthetic fibre fleece, is impregnated with solutions of the necess-ary reagents usually-employed for the production of test strips in readily volatile solvents, for example water, methanol, ethanol or acetone. This can take place in one impregnation step. However, it is often preferable to carry out the impregnation in several steps, solutions being used which each contain some of the components of the diagnostic agent. Thus, for example, in a firs~ step, impregnation can be carried out with an aqueous solution which contains the buffer and other water-soluble additive materials and then, in a second step, with a solution which contains khe ~--D-galactQsida~e substrate. The finished test papers can be used a3 such o~, in known manner, can be stuck on to handLes or preferably sealed between synthetic materials and fine meshworks according to Federal Republic of Germany Patent Specification No.2/118,455O
The following Examples illustrate some of the numerous process variants which can be used for the ~ynthesis of the new compounds according to the present invention, a~ well as, by way of example, the us~ of the new sulphonphthaleinyl-~-D-galactosides for the . ~
763~
determination of the activity of ~-D-galactosidase.
The following abbreviations are used in the Examples:
HEPES 2-~4-~2-hydroxyethyl)-1-piperazinyl~-ethane-sulphonic acid BSA bovine serum albumin Tween-20* polyoxyethylene(20~sorbitan monolaurate Tricin [N-tris-(hydroxymethyl)J-methyl-glycine~
Example 1.
3,3'-DichlorophenolsulphonPhthaleinyl-~-D-~actoside sodium salt.
a) A solution of 45 g. (0.11 mole) 2,3,4,6-tetra-O-acetyl-a-D-galactopyranosyl bromide in 450 ml.
chloroform is warmed to 60C. While stirring at this temperature, there are added a solution of 29.9 g.
(0.11 mole~ benzyl triethylammonium bromide in 114 ml.
1.25N aqueous sodium hydroxide solution (0.142 mole), followed by 46.5 g. (0.11 mole) 3,3'-dichlorophenol-sulphonphthalein (chlorophenol red). Residues of the dyestuffs are rinsed down from the walls of the reaction vessel used with some water and a furthex 114 ml. 1.25N aqueous sodium hydroxide solution.
The reaction mixture is boiled under reflux for 12 hours and thereafter left to stand for 8 hour~ at ambie~t temperature. ~he organic phase ~ separated off and the aqueous phase is shaken several times with chloroform. For the removal of starting material * trade mark still present, the combi~ed organic phases are shaken several times with O.lN aqueous sodium hydroxide solution.
After washing the chloroform phase with water and drying with anhydrous sodium sulphate, the organic solvent is evaporated. The residue is triturated with diethyl ether to give 46 g. 3,3'-dichlorophenol-sulphonphthaleinyl-2,3,4,6-tetra-0-acetyl-~-D
galactoside sodium salt as a yellow amorphous material (yield: 54% of theory), m.p. 190Co ~decomp.).
NMR: (DMSO~d6): 1095 (s~ 3H), 1.99 (s, 3H), 2.02 ts, 3H), 2.12 (s, 3H), 4.0-4.6 ~m, 4H), 5~1-5~7 (m, 3H), 6~1-6~8 ~m, lH), 6~9-7~7 (m, 8H), 7~8-8~0 (m, lH).
b) ~ solutlon of 28 g~ (0~036 mole) of ~le tetra-acetylgalactoside prepared according to a~ in 270 r~.
anhydrous methanol is cooled to 0 - 5C~ For des-acetylation, 72 ml. of a 1 rnolar (0.072 mole) sodium methylate solution in methanol are added thereto, while stirring at this temperature.
After 15 minutes at 0 - 5 CO~ the solution is mixed with about 300 ml. Amberlite~IRC 50 for the removal of excess sodium ions and the mixture is stirred for 2 hours at 5C. After filtering off the ion exchanc,~er with suction, this is wa3hed several times with methanol.
* trade marlc 7J~
Ater evaporation of the combined filtrates, the residue ;3 purified by column chromatography on silica gel with methylene chloride/methanol (~/1 v/v) to give 12 g. 3,3'-dichlorophenolsulphonphthaleinyl-~-D-galactoside sodium salt as a yellow, amorphous powder (yield: 55% of theory), m.p. 210C. tdecomp~).
NMR: ~DMS0-d6): 3O3-3~7 (m, 6H), 3.9-5.0 (m, 4H), 5.1 (d, J = 7 Hz, lH), 6.1-6.8 (m, lH~, 6.9-7.6 (m, 3H), 7.8-8.0 (m, lH).
Example 2 o In a manner analogous to that described in Example 1, by the reaction of 2,3,4,6-tetra-0-acetyl-~-D-galactopyranosyl bromide with the phenolsulphon-phthaleins given below undex "starting material", there are preparedJ via the corresponding peracetylated galactosides, the ~-D-galactosides given under "end product":
starting mater:ial end product m.p. in C~
~____ 1) phenol red phenolsulphonphthal- 218-220 einyl-~-D-galactoside sodium salt 2) fluorophenol 3,3'-difluorophenol- glass-red sulphonphthaleinyl-~- like D-galactoside sodium salt 3) chlorophenol 3,3',5,5'-tPtrachloro- 145-150 blue phenolsulphonphthal-einyl-~-D-galactoside sodium salt "
3~
~_,1
a) A solution of 45 g. (0.11 mole) 2,3,4,6-tetra-O-acetyl-a-D-galactopyranosyl bromide in 450 ml.
chloroform is warmed to 60C. While stirring at this temperature, there are added a solution of 29.9 g.
(0.11 mole~ benzyl triethylammonium bromide in 114 ml.
1.25N aqueous sodium hydroxide solution (0.142 mole), followed by 46.5 g. (0.11 mole) 3,3'-dichlorophenol-sulphonphthalein (chlorophenol red). Residues of the dyestuffs are rinsed down from the walls of the reaction vessel used with some water and a furthex 114 ml. 1.25N aqueous sodium hydroxide solution.
The reaction mixture is boiled under reflux for 12 hours and thereafter left to stand for 8 hour~ at ambie~t temperature. ~he organic phase ~ separated off and the aqueous phase is shaken several times with chloroform. For the removal of starting material * trade mark still present, the combi~ed organic phases are shaken several times with O.lN aqueous sodium hydroxide solution.
After washing the chloroform phase with water and drying with anhydrous sodium sulphate, the organic solvent is evaporated. The residue is triturated with diethyl ether to give 46 g. 3,3'-dichlorophenol-sulphonphthaleinyl-2,3,4,6-tetra-0-acetyl-~-D
galactoside sodium salt as a yellow amorphous material (yield: 54% of theory), m.p. 190Co ~decomp.).
NMR: (DMSO~d6): 1095 (s~ 3H), 1.99 (s, 3H), 2.02 ts, 3H), 2.12 (s, 3H), 4.0-4.6 ~m, 4H), 5~1-5~7 (m, 3H), 6~1-6~8 ~m, lH), 6~9-7~7 (m, 8H), 7~8-8~0 (m, lH).
b) ~ solutlon of 28 g~ (0~036 mole) of ~le tetra-acetylgalactoside prepared according to a~ in 270 r~.
anhydrous methanol is cooled to 0 - 5C~ For des-acetylation, 72 ml. of a 1 rnolar (0.072 mole) sodium methylate solution in methanol are added thereto, while stirring at this temperature.
After 15 minutes at 0 - 5 CO~ the solution is mixed with about 300 ml. Amberlite~IRC 50 for the removal of excess sodium ions and the mixture is stirred for 2 hours at 5C. After filtering off the ion exchanc,~er with suction, this is wa3hed several times with methanol.
* trade marlc 7J~
Ater evaporation of the combined filtrates, the residue ;3 purified by column chromatography on silica gel with methylene chloride/methanol (~/1 v/v) to give 12 g. 3,3'-dichlorophenolsulphonphthaleinyl-~-D-galactoside sodium salt as a yellow, amorphous powder (yield: 55% of theory), m.p. 210C. tdecomp~).
NMR: ~DMS0-d6): 3O3-3~7 (m, 6H), 3.9-5.0 (m, 4H), 5.1 (d, J = 7 Hz, lH), 6.1-6.8 (m, lH~, 6.9-7.6 (m, 3H), 7.8-8.0 (m, lH).
Example 2 o In a manner analogous to that described in Example 1, by the reaction of 2,3,4,6-tetra-0-acetyl-~-D-galactopyranosyl bromide with the phenolsulphon-phthaleins given below undex "starting material", there are preparedJ via the corresponding peracetylated galactosides, the ~-D-galactosides given under "end product":
starting mater:ial end product m.p. in C~
~____ 1) phenol red phenolsulphonphthal- 218-220 einyl-~-D-galactoside sodium salt 2) fluorophenol 3,3'-difluorophenol- glass-red sulphonphthaleinyl-~- like D-galactoside sodium salt 3) chlorophenol 3,3',5,5'-tPtrachloro- 145-150 blue phenolsulphonphthal-einyl-~-D-galactoside sodium salt "
3~
~_,1
4) pyrocatechol 3,3'-dihydroxyphenol- 115-120 violet ',sulphonphthaleinyl-~-D-galactoside sodium :salt 55~ iodophenol l3,3',5,5'-tetraiodo- 210-215 blue lphenolsulphonphthal-~einyl-~-D-galacto~ide jsodium salt 6~ m-cresol l2,2'-dimethylphenol- 205-209 10purple ¦sulphonphthaleinyl-~-jD-galactoside sodium !salt 7) bromocresol 3,3'-dibromo-5,5'- 200-203 purple dimethylphenolsulphon-phthaleinyl-~-D-galactoside sodium salt 8) o-cresol red l3,3l-dimethylphenol- 200-204 ¦sulphonphthaleinyl-~-D-Igalactoside sodium salt 209) thymol blue ! 3,3'-diisopropyl-6,6'- 205-209 dimethylphenolsulphon-phkhaleinyl-~-D~
galactoside sodium salt 10) hromothymol 3,3'~dihromo-5,5'-diiso- lgo-195 25 blu~ propyl-2,2'~dimethyl-phenolsulphonphthal-einyl-~-D-galactoside sodium salt 11) salicyl red . 3,3'-dicarboxyphenol- 178-180 sulphonphthaleinyl-~-D-galactoside sodium salt 12) 3,3',5,5'- 3,3',5,5'-tetrabromo- 100-103 tetrabromo- 2,2'-dimethylphenol-2,2'-dimethyl- ~ulphonphthaleinyl- .
phenolsulphon- ~-D-galactoside sodium phthalein salt 13) 3,3'-dinitro 3,3l-dinitrophenol- 167-~70 phenol- sulphonphthaleinyl-sulphon- ~ D-gala~to~ide sodium 40phthalein ~alt ~ ~.3~
--~o--1~) 3,3'-dichloro- 3,3'-dichloro-5,5'- 115-118
galactoside sodium salt 10) hromothymol 3,3'~dihromo-5,5'-diiso- lgo-195 25 blu~ propyl-2,2'~dimethyl-phenolsulphonphthal-einyl-~-D-galactoside sodium salt 11) salicyl red . 3,3'-dicarboxyphenol- 178-180 sulphonphthaleinyl-~-D-galactoside sodium salt 12) 3,3',5,5'- 3,3',5,5'-tetrabromo- 100-103 tetrabromo- 2,2'-dimethylphenol-2,2'-dimethyl- ~ulphonphthaleinyl- .
phenolsulphon- ~-D-galactoside sodium phthalein salt 13) 3,3'-dinitro 3,3l-dinitrophenol- 167-~70 phenol- sulphonphthaleinyl-sulphon- ~ D-gala~to~ide sodium 40phthalein ~alt ~ ~.3~
--~o--1~) 3,3'-dichloro- 3,3'-dichloro-5,5'- 115-118
5,5'-dinitro dinitrophenolsulphon-phenolsulphon phthaleinyl-~-D-phthalein .galacto~ide sodium salt 15) 3,3'-dimethyl- l3,3'-dimethyl-5,5'- 155~158 ' 5,5'-dinitro dinitrophenolsulphon-phenolsulphon !phthaleinyl-~-D-phthalein Igalactoside ~odium salt 16) 3,3' dimethoxy-¦3,3'-dimethoxyphenol-phenolsulphon- ,sulphonphthaleinyl-phthalein l~-D-galactoside 17~ 3,3'-difluoro- 13,3'-difluorophenyl-phenyl-3",4", l3",4",5",6"-tetrabromo-5",6"-tetra- ¦sulphonphthaleinyl~
bromosulphon- ID-galactoside phthalein 18) 2,2'-dimethyl- ¦2,2'-dimethyl-3,3"-3,3'-dinitro- ¦dinitrophenolsulphon-phenolsulphon- Iphthaleinyl-~-D-phthalein galactoside 19) 2,2'--dimethyl- 2,2'-dimethyl-5,5'-5,5'-dinitro dinitrophenolsulphon phenol~ulphon- phthaleinyl-~-D-phthalein galactoside ~0) phenol 4"- phenol-4"-nitrosulphon- 300 nitrosulphon phthalelnyl-~-D-phthalein galactoside sodium salt 21) phenol-5"- phenol-5"-nitro~ulphon-nitrosulphon- phthaleinyl-~-D-phthalein galactoside 22) 3,3'-dichloro- 3,3'-dichlorophenol-4"-160/300 phenol-4"- nitrosulphonphthaleinyl nitrosulphon- ~-D-galactoside sodium phthalein salt 23~ 3,3'-difluoro- 3,3'-difluorophenol-4 n_ phenol-~"- nitrosulphonphthaleinyl-nitrosulphon- ~ galactoside phthalein 24) 3,3',4"-tri- 3,3',4n-trinitrophenol- 300 nitrophenol~ sulphon~hthalei~yl-~
sulphon- D-galactoside sodium salt phthalein 3~
-2~-____~ _ ~
25) phenol-4"-amino~ phenol-4"-aminosulphon- amorphou~
sulphonphthalein phthaleinyl-~-D-ga~actoside sodium , . . _ _ _ ~ ~_ ~ æ~
qalactoside sodium salt.
a) 6.2 g. ~0.016 mole~ 3,3'-difluorophenolsulphon-phthalein (fluorophenol red) are dissolved in 170 ml.
anhydrous methanol. For the formation of the disodium salt, 32 ml. (0.032 mole) of a 1 molar sodium methylate solution in methanol are added thereto. The solution is then evaporated to dryness. For dissolving the residue in 140 ml. anhydrous dimethylformamide, 7.3 g.
(0.0176 mole) 2,3,4,6-tetra-0-acetyl-a-D-galacto-pyranosyl bromide are added thereto, followed by stirring for 6 hours at ambient temperature. Ater ~uctlon filtration, the f:iltrate is evaporated at ambient temperature under oil pump vacuum. q~e ~0 residue is tritura~ed with diethyl ether, filtered off with suction and dried to give 6.9 g. 3,3'-difluorophenolsulphonphthaleinyl-2,3,4,6-tetra-0-acetyl-~-D-galactoside sodium salt as an orange-coloured amorphous material ~yield 53~ of theory), m.p. 215C. (decomp.).
NMR: ~DMSO-d6): 1.94 (s, 3~ 96 ~s, 3Hl, 1.99 (s, 3H), 2.15 (~, 3~), 3.9-4.7 ~m, 4H), 7~3'~
~22-S.0-5.6 (m, 3H), 6.2-6.6 (m, lH), 7.0-7.6 (m, 8H), 7.9-8.2 (m, lH~.
b) A solution of 3.5 g. (0O005 mole) of the tetra-acetylgalactoside prepared according to a) in 750 ml~
anhydrous methanol is mi~ed at ambient temperature with 1.5 ml. (0.0015 mole) of a 1 molar sodium methylate solution in methanol. After standing over~
night, the solution is evaporated. The residue is purified by column chromatography on silica gel with methylene chloride/methanol- ~5/1 v/v) to give 1.2 g.
3,3'-difluorophenolsulphonphthaleinyl-~-D-galactoside sodium salt as an orange-red, hygroscopic, a~orphous powder (yield 41% of theory).
NMR: (DMSO-d6): 3.1~3.9 (m, 6H), 4.1-5.3 (m, 4H), 4.95 (d/ J = 7 Hz, lH), 6.2-6.6 (m, lH),
bromosulphon- ID-galactoside phthalein 18) 2,2'-dimethyl- ¦2,2'-dimethyl-3,3"-3,3'-dinitro- ¦dinitrophenolsulphon-phenolsulphon- Iphthaleinyl-~-D-phthalein galactoside 19) 2,2'--dimethyl- 2,2'-dimethyl-5,5'-5,5'-dinitro dinitrophenolsulphon phenol~ulphon- phthaleinyl-~-D-phthalein galactoside ~0) phenol 4"- phenol-4"-nitrosulphon- 300 nitrosulphon phthalelnyl-~-D-phthalein galactoside sodium salt 21) phenol-5"- phenol-5"-nitro~ulphon-nitrosulphon- phthaleinyl-~-D-phthalein galactoside 22) 3,3'-dichloro- 3,3'-dichlorophenol-4"-160/300 phenol-4"- nitrosulphonphthaleinyl nitrosulphon- ~-D-galactoside sodium phthalein salt 23~ 3,3'-difluoro- 3,3'-difluorophenol-4 n_ phenol-~"- nitrosulphonphthaleinyl-nitrosulphon- ~ galactoside phthalein 24) 3,3',4"-tri- 3,3',4n-trinitrophenol- 300 nitrophenol~ sulphon~hthalei~yl-~
sulphon- D-galactoside sodium salt phthalein 3~
-2~-____~ _ ~
25) phenol-4"-amino~ phenol-4"-aminosulphon- amorphou~
sulphonphthalein phthaleinyl-~-D-ga~actoside sodium , . . _ _ _ ~ ~_ ~ æ~
qalactoside sodium salt.
a) 6.2 g. ~0.016 mole~ 3,3'-difluorophenolsulphon-phthalein (fluorophenol red) are dissolved in 170 ml.
anhydrous methanol. For the formation of the disodium salt, 32 ml. (0.032 mole) of a 1 molar sodium methylate solution in methanol are added thereto. The solution is then evaporated to dryness. For dissolving the residue in 140 ml. anhydrous dimethylformamide, 7.3 g.
(0.0176 mole) 2,3,4,6-tetra-0-acetyl-a-D-galacto-pyranosyl bromide are added thereto, followed by stirring for 6 hours at ambient temperature. Ater ~uctlon filtration, the f:iltrate is evaporated at ambient temperature under oil pump vacuum. q~e ~0 residue is tritura~ed with diethyl ether, filtered off with suction and dried to give 6.9 g. 3,3'-difluorophenolsulphonphthaleinyl-2,3,4,6-tetra-0-acetyl-~-D-galactoside sodium salt as an orange-coloured amorphous material ~yield 53~ of theory), m.p. 215C. (decomp.).
NMR: ~DMSO-d6): 1.94 (s, 3~ 96 ~s, 3Hl, 1.99 (s, 3H), 2.15 (~, 3~), 3.9-4.7 ~m, 4H), 7~3'~
~22-S.0-5.6 (m, 3H), 6.2-6.6 (m, lH), 7.0-7.6 (m, 8H), 7.9-8.2 (m, lH~.
b) A solution of 3.5 g. (0O005 mole) of the tetra-acetylgalactoside prepared according to a) in 750 ml~
anhydrous methanol is mi~ed at ambient temperature with 1.5 ml. (0.0015 mole) of a 1 molar sodium methylate solution in methanol. After standing over~
night, the solution is evaporated. The residue is purified by column chromatography on silica gel with methylene chloride/methanol- ~5/1 v/v) to give 1.2 g.
3,3'-difluorophenolsulphonphthaleinyl-~-D-galactoside sodium salt as an orange-red, hygroscopic, a~orphous powder (yield 41% of theory).
NMR: (DMSO-d6): 3.1~3.9 (m, 6H), 4.1-5.3 (m, 4H), 4.95 (d/ J = 7 Hz, lH), 6.2-6.6 (m, lH),
6.8-7~6 (m, 8~1), 7.8-8.0 (m, lH)~
X~ .
In a manner analogous to that described in Example 3, from 2,3,4,6~tetra-0-acetyl-a-D-yalacto~
pyranosyl bromide and the phenolsulphonphthaleins stated below under "starting material", there are prepared the ~D~galactosides mentioned under "end product":
~tarting materiall end product I m~pO ~C.
_ _ 1) phenol red phenolsulphonphthal- 208-212 einyl-~-D-galactoside sodium salt 2) o-cresol red 3,3'-dimethylp~enol- 202-205 sulphonphthaleinyl-~-D-galactoside sodiur~l 33 bromocresol 3,3'-dîbromo-5,5'- 198-202 purple dimethylp~enolsulphon-phthaleinyl-~-D-. galactoside sod~um ~_ _ ~.
3,3'-Dibrom~ 5' ~ thalei~y~
~.
a) A solution of 11.03 g. (0.027 mole3 2,3,4,6-tetra-0-acetyl-a-D-galactopyranosyl bromide and 5.6 gO
(0.007 mole) bromocresol purple tribenzylammonium salt in 60 ~1. dichloromethane is mixed with 3.1 g~
(0.013 mole~ ~ilver oxide and 3.7 g. (0.013 mole) silver carbonate and stirred ~or 1~ hours at ambient temperature. After filtering off the precipitate, the filtrate is evaporated and the residue is purified by column chromatography on silica gel with toluene/
ethyl acetate/methanol (1/1/0.2 v/v/v). Evaporation of th~ appropriate fraction give~ 4~4 g. 3,3'-dibromo-5,5'-dimethylphenolsulphonphthaleinyl-2,3,4,6-tetra-0-acetyl-~-D-galacto~ide tribenæylammonium ~alt a~ ~ yellow, amorphou~ material ~yield 54% of theory).
-2~-NMR: ~DMS0-d6): 1.8-2.3 (m, 18H), 3.8-4.4 (m, 4H), 5.2-5.6 (m, 9H), 6.6-8.1 (m, 23H~.
b) A solution of 4 g. (0.0035 mole) of the tetra-acetyl-galactoside prepared according to a3 in 40 ml.
anhydrous methanol is cooled to -40C. and, for desacetylation, mixed with 15.5 r~. of a lM (0.015 mole) sodium methylate solution.
After one hour, the solution is neutralised by treatment with about 30 ml. Amberlite~ LRC 50 (H form) and evaporated~ The residue is purified by column chromatography on silica gel with methylene chloride/
methanol/acetone ( 6/2/1 v/v~v ~ to give 2 g. 3,3'-dibromo-5,5'-dimethylphenolsulphonphthaleinyl-~-D-galactoside sodium salt as a yellow, amorphous powder (yield 62% of theory~, m.p. 200 - 203C. (decomp~).
NMR: (DMSO-d6): 1~9-2.4 (m, 6H), 3.2-4.Q (m, 6H), 4.4 ~m, 2H), 4.8 (m, 2EI) [after deuterium exchallge: 4.9 (d, ~ _ 7 Hz, 15:5)], 6.7-~.1 (m, 8H).
~ y~
In a manner analogous to that described in Exampl~ 5, there are prepared from 2,3,4,6-t~tra-0-acetyl-a-D-galactopyranosyl bromide and 1) fluorophenol red hygroscopic glass * trade mark 2) phenol-3",4",5",6"~tetrabromosulphonphthalein phenol-3",4",5ll6 -tetr m.p. 215C. (decomp.) 3) 3,3',5,5'-tetrachlorophenol~3",4",5",6"-tetrabromo-sulphonphthalein 3,3',515'-tetrachlorophenol-3l,4",5",6"-tetrabromo-m.p. 150C. (decomp.) 4) phenol-4"-nitrosulphonphthalein m.p. ? 300 C.
5) 3,3'-dichlorophenol-4"-nitrosulphonphthalein double m.p. 160C./ ~ 300C.
6) 3,3'-dimethylphenol-4"-nitro~ulphonphthalein ~0 m.p. 210 - 220C.
X~ .
In a manner analogous to that described in Example 3, from 2,3,4,6~tetra-0-acetyl-a-D-yalacto~
pyranosyl bromide and the phenolsulphonphthaleins stated below under "starting material", there are prepared the ~D~galactosides mentioned under "end product":
~tarting materiall end product I m~pO ~C.
_ _ 1) phenol red phenolsulphonphthal- 208-212 einyl-~-D-galactoside sodium salt 2) o-cresol red 3,3'-dimethylp~enol- 202-205 sulphonphthaleinyl-~-D-galactoside sodiur~l 33 bromocresol 3,3'-dîbromo-5,5'- 198-202 purple dimethylp~enolsulphon-phthaleinyl-~-D-. galactoside sod~um ~_ _ ~.
3,3'-Dibrom~ 5' ~ thalei~y~
~.
a) A solution of 11.03 g. (0.027 mole3 2,3,4,6-tetra-0-acetyl-a-D-galactopyranosyl bromide and 5.6 gO
(0.007 mole) bromocresol purple tribenzylammonium salt in 60 ~1. dichloromethane is mixed with 3.1 g~
(0.013 mole~ ~ilver oxide and 3.7 g. (0.013 mole) silver carbonate and stirred ~or 1~ hours at ambient temperature. After filtering off the precipitate, the filtrate is evaporated and the residue is purified by column chromatography on silica gel with toluene/
ethyl acetate/methanol (1/1/0.2 v/v/v). Evaporation of th~ appropriate fraction give~ 4~4 g. 3,3'-dibromo-5,5'-dimethylphenolsulphonphthaleinyl-2,3,4,6-tetra-0-acetyl-~-D-galacto~ide tribenæylammonium ~alt a~ ~ yellow, amorphou~ material ~yield 54% of theory).
-2~-NMR: ~DMS0-d6): 1.8-2.3 (m, 18H), 3.8-4.4 (m, 4H), 5.2-5.6 (m, 9H), 6.6-8.1 (m, 23H~.
b) A solution of 4 g. (0.0035 mole) of the tetra-acetyl-galactoside prepared according to a3 in 40 ml.
anhydrous methanol is cooled to -40C. and, for desacetylation, mixed with 15.5 r~. of a lM (0.015 mole) sodium methylate solution.
After one hour, the solution is neutralised by treatment with about 30 ml. Amberlite~ LRC 50 (H form) and evaporated~ The residue is purified by column chromatography on silica gel with methylene chloride/
methanol/acetone ( 6/2/1 v/v~v ~ to give 2 g. 3,3'-dibromo-5,5'-dimethylphenolsulphonphthaleinyl-~-D-galactoside sodium salt as a yellow, amorphous powder (yield 62% of theory~, m.p. 200 - 203C. (decomp~).
NMR: (DMSO-d6): 1~9-2.4 (m, 6H), 3.2-4.Q (m, 6H), 4.4 ~m, 2H), 4.8 (m, 2EI) [after deuterium exchallge: 4.9 (d, ~ _ 7 Hz, 15:5)], 6.7-~.1 (m, 8H).
~ y~
In a manner analogous to that described in Exampl~ 5, there are prepared from 2,3,4,6-t~tra-0-acetyl-a-D-galactopyranosyl bromide and 1) fluorophenol red hygroscopic glass * trade mark 2) phenol-3",4",5",6"~tetrabromosulphonphthalein phenol-3",4",5ll6 -tetr m.p. 215C. (decomp.) 3) 3,3',5,5'-tetrachlorophenol~3",4",5",6"-tetrabromo-sulphonphthalein 3,3',515'-tetrachlorophenol-3l,4",5",6"-tetrabromo-m.p. 150C. (decomp.) 4) phenol-4"-nitrosulphonphthalein m.p. ? 300 C.
5) 3,3'-dichlorophenol-4"-nitrosulphonphthalein double m.p. 160C./ ~ 300C.
6) 3,3'-dimethylphenol-4"-nitro~ulphonphthalein ~0 m.p. 210 - 220C.
7) 3,3',4"-trinitrophenolsulphonphthal~in 9~
m.p. ~ 300C.
-2~~
m.p. ~ 300C.
-2~~
8~ phenol~ amino~ulphonphthalein henol 4"-aminosul hon hthalein l-B-D- alactoside sodi~m alt lyophilisate (amorphous) ~ .
A solution of 6.77 g. (0~01 mole) fluorophenol red tribenzylammonium salt and 5.3 g. (0~01 mole) per-0-trimethylsilyl-a-D-galactopyranosyl bromide in 70 ml. dichloromethane are mixed with 1.15 g. (0.005 mole) silver oxide and 1.4 g. (0.005 mole~ silver carbonate and stirred for 18 hours with the exclusion of moisture. After filtering off the precipitate, the filtrate is evaporated and the residue is, for spli~ting off the protective groups, taken up in 60 ml. methanol and kept for 12 hours at arnbient tempexature. For purification, it is chromatographed over silica gel with dichloromethane/methanol (5~1 v/v) 20 to give 1.2 g. 3,3'-difluorophenolsulphonphthaleinyl-~-D~galactoside tribenzylammonium salt as an orange coloured, amorphous powder ~yield 14% of theory), m.p. 157 - 165C~
~MR: (DMSO-d6): 3.1-3.9 (m, 6H), 4.2~5.2 ~m, llH), 6.3 (m, lH), 6.8-7.6 (m, 23H), 7.9 (m, 1~).
E ~ o ~.
1.5 g. (0.0025 mole) 3,3'-dichlorophenol~ulphon- , phthaleinyl-~-D-galactoside ~odium salt, prepared accordi.ng to Ex~nple 1, is dissolved in a little water.
The soluti~n is applied to a column filled with ~nberlite IR 120 (Li form) . Lyophilisation of the eluate gives 1.4 g. 3,3'-dichlorophenolsulphon-phthaleinyl-~-D-galactoside lithium salt as an orange coloured, amorphous powder (yield: 96% of theory), m.p. 190Co (decomp.).
~MR: (DMSO-d6): 3.3-3.8 (m, 6Hl, 4.3-4.9 (m, 4H), 5~07 (s~ J = 7 Hz, lH), 6~1-7~7 (m~ 9H)~
7~8-8~1 (m, lH).
In a manner analogous to that described in the Example 8, there are prepared from 3,3'-~ichloro-phenolsulphonphthale.inyl-~-D-galactoside sodium salt (~ee Excample 1) a) by exchange chromatography on Amberlite*IR 120 in the Ca fonm orange-red, amorphous product (yield: 78~o of theory), m.p. 250C. (decomp.) * trade ~ark ~l~ql2~
~28-NMR: (DMSO-d6): 3.2 3~8 (m, 6H), 4.4-5.1 (m, 4H), 5.1 (s, J = 7 Hz, lH), 6~2~7a6 ~m, 9H~, 7.8-8.0 (m, lH)o b) By exchange chromatography on Amberlite* IR 120 in 5 the (H3c)4N f~rm yellow, amorphous product (yield 85% of theory) m.p. 190 - 195Co NMR: (DMSO-d6): 3.2 (s, 12H), 3.3-4.0 ~m, 6H), 4~1-5~3 (m, 4H), 5.1 (d, J = 7 Hz, lH), 6~4 (m, lH~, 7.0-7.7 (m, 8H), 7.9 (m, lH).
~.
tribenz lammonium salt.
1.5 g~ (0~0025 mole3 3,3'-dichlorophenolsulphon-phthal~inyl-~D~galacko~ide sodium ~alt (see Example 1) is dissolved in a little wa-ter and pa~sed through a column filled with Amberlite~IR 120 ~H formj. The eluate is mixed with a stoichiometric (Oa72 g~) amount of triben~ylamine dissolved in 15 ml. ethanol and evaporated~ There is obtained 1~7 g. 3,3'-dichloro-ph~nolsulphonphthaleinyl-~-D-galactoside triben~yl-ammonium salt as a yellow, amorphou~ materi~l ~yield 78% of theory)a, m.p. 140 - 150C.
~.
In a manner analogou~ to that described in * trade mark D A ~1 ~W
~1;~ a ~J 6' Exam~le 10, there is prepared ~rom 3,3'-dichloro-phenolsulphonphthaleinyl-~-D-galactoside sodium salt (see Example 1), with the use of benzyldiethylamine, the corresponding yellow, amorphous powder tyield 69% of theory), m.p.
245 - 248C.
~.
Detenmination of the activlty of @-D= lalactosidase a) _~r ,'Y51~
HEPES 100 mmol~litre sodium chloride154 mmol/litre m~gnesium L-aspartate2 mmol~litre BSA 10 g./litre T~een-20* 0.5 y./litre pH value ~adjusted w.ith aqueous sodium hydroxide7~3 ~37C.) solution ~0~ :
In the a~ove-described buffer solution are dissolved 5 mmol/litre phenolsulphonphthaleinyl-~~D-galactoside sodium salt~ ~he pH value is adjusted with aqueous sodium hydroxide solution to pH 8.5 (37C.~.
* trade mark ~, nt 5~ t~ 2~
-In the above-described buffer solution are dissolved 5 mmol/litre 3,3'-difluorophenolsulphon-phthaleinyl-~-D-galactoside sodium salt. The pH value is ad~usted with aqueous sod:ium hydroxide solution to pH 7.5 (37C.).
_ ~ r~ g~
In the above-described buffer solution are dissolved 5 mmol/litre 3,3'-dichlorophenolsulphon-phthaleinyl-~-D-galactoside sodium salt. The pH value of the buffer solution of 7.3 (37C.) is maintained.
Rea ent Solution 4~
q _ _.
In the above-described buffer solution are dissolved 5 mmol/litre 3,3',5,5'-tetrachlorophenol-3",4",5",6"-tetrabromosulphonphthaleinyl-~-D-galactoside sodium salt. The pH value of the buffer solution of 7.3 ~37C.) is maintained.
The substrate concentration and the pH values are to be optimised for each substrate used. There-fore, quite knowingly, different values for substrateconcentrations or pH values can occur in the individual reagent solutions.
Enzy~ solution Commercially available ~-D~galactosidase from Escherichia coli is dissolved in the above-mentioned buffer solution. The activity of this ~olution is about 0.08 U/ml. (referr d to the statements of t~e manufacturer).
~ ~a C' ~ ~
b~ ~
The measurement takes place photometrically, in each case at the wavelength given below.
950 ~1. of reagent are mixed in a 1 cm. cuvette at 37C. with 50 ~1~ of enzyme solution. As a m~asure for the reaction, there is determined the extinction increase per unit time in [mExt/min]. It is calculated from -the measured extinction hy division with the - reaction time.
The following Table gives the measurement values found:
Reagent No. Measurement reaction i wavelength ~mExt/min~
[nm~
1 5~0 ~5 3 S7~3 123 57~ 121 ~ ~___ ~.
a) HEPES 50 mmol/litre citric acid 50 mmol/litre tricin 50 ~nol/litr2 sodium chloride 154 mmol~litre magnesium L-aspartate1 mmol/litre BSA 10 g./litre pH value (adjusted with aqueous 6.9 (37C.3 sodium hydroxide solution) ~gL____ olution 1:
In the above-described buffer solution are dissolved 5 mmol/litre 3,3'-dimethylphenolsulphon-phthaleinyl-~-D-galactoside.
Reaqent Solution 2-In the above-described buffer solution are dissolved S mmol/litre 3,3'-dihydroxyphenolsulphon-phthaleinyl-~-D-galactoside.
~=:
Commercially available ~-D-galactosidase from Escherichia coli i5 dissolved in the above-mentioned buffer solution. The activity of khis solut.ion is about 0 . 08 U/ml . ~ referred to the staternents oE the manuf ac tu re r ) ~
b~ r ~ _ :r~ out o Ie ~-c~c~:~r~:s 950 ~1~ of reagent are mixed in a 1 cm. cuvette at 37 C. with 50 ~1. of enzyme solution. After 10 minutes reaction time, it is adjusted to pH 10 with aqueous sodium hydroxide solution and the extinction measured. The same procedure is used for a blank containing buffer instead of en~yme solution~ During th~ reaction and the mea~urement, the temperature is kept at 37C. From the extinctions determined on th~
~33 ~
batches with and without enzyme, there i~ calculat.ed the extinction difference. By division of this extinction difference by the reaction time, there i5 given, as mea~ure for the reaction, the extinction increase per unit time in [mExt/min].
The following Table gives the measurement values found:
Reagent No. Measur~mentreaction wavelength [nm~[r~xt/min~
~ _ _ .
2 5g3 6 ~ _-- .
.
~ "'C~15 ~
There are used cor~nercially available ~-D~
galactosida~e preparations of differing oriyin. As a characteristic feature, the~e manife~t their maximurn act.iv.ity at differing pH values:
20 Statements of the manufacturer:
from beans (jack beans) p~ 3.5 from ~ ~ P
from Escherichi6 coli pH 6Og from bovine liver pH 7.3 a3 ~r~FLarat__ __ _ h~_ ~l~tic~ used:
25 ~uLI- ' HEPES 50 mmol/litre citric acid 50 mmol/litre ~S',~ .k~ ~ 3 ~r _3d~_ tricin 50 r~nol/litre sodium chloride154 ~nol/litre magnesiurn L-aspartate1 mmol/litre BSA 10 g./litre Solutions are prepared of the above composition with differing pH values according to the pH optima of the ~-D-galactosidases (pH 3~ 5/4.0/6.9/7~3), the pH values being adjusted at 37C. with hydrochloric acid or aqueous sodium hydroxide solution.
1 0 _~5 ~ ~ j D
In the above-mentioned buffer solutions with the differing pH values of 3~5/4~0~6D9/7~3 are dissolved, in each case, 5 ~nol/litre 3,3l-dichloro-phenolsulphonphthaleinyl-~-D-galactoside sodi~n salt.
_ = ' The ~-D-galactosidases are dissolved in the buffers with, in each case, the optirnurn pH values:
from bean~ (jack beans) in buffer of pH 3.5 from ~ ~g~ in buffer of pH 4.0 from E:cherichia coli in buffer of pH 6 9 from bovine liver in buffer of pH 7D 3 The activity of these solutions is about 0.08 U/ml~ (referred to the statements of the manufactu~r).
b~ ~D
The enzyme reaction takes place during a defirlite reaction time at the optimum pH value for ~ t~ 7 the particular enzyme. 1000 ~1. of reagent are mixed in a 1 cm~ cuvette at 37C. ~th 30 ~1. of an enzyme solution~ After 15 minutes reaction time, it is adjusted with aqueous sodium hydroxide solution to pH 8.5 and the extinction deterrnined at 578 nm.
~uring the reaction and the measurement, the temper-ature is kept constant at 37C. In the same way, for each measurement a blank is carried out. For this purpose, instead of the enzyme solution, there are used 30 ~1. of buffer solution.
c) Evaluation __ .
First, there is obtained the difference between the measurement value with the enzyme and the measure-ment value of the hlank. By division of this differ-ence by the reaction time, there is determined, asmeasure for the reaction, the extinction increase per unit time :in [mExt/min~.
measurement value (with enzyme) - measurement value (blank) = ~ measurement value ~ measurement value _ - reaction [mExt/min]
reaction time ~ he measurement values found for the reaction of the individual enzymes are given in the following Table:
.3 enzyme from pH value reaction s~eed [rnExt/minJ
beans (jack beans) 3.5 71 ~ger 4.0 112 Escherichia coli 6.9 74 bovine liver 7.3 32 _ . _ . _ _ The above-described experimental results show that the sulphonphthaleinyl-~-D~galactosides can be used as substrates for ~-D-galactosidases of any origin.
Example 15.
I~ L=3_ ~:t~
a) ~tl~ s~
HEPE5 100 ~nol/litre sodium chloride154 r~ol/litre magnesium ~-aspartate2 mmol/litre BSA 10 g./litre Tween 20* 0.5 g./litre pH value (adjusted with a~ueous 7.3 ~37C.) sodium hydroxide solution) ~D~ t o~:ti~:
In the above-described buffer ~olu~ion are dissolved 5 r~nol/litre 3,3'-dichloropherlol~ulphon-* trade mark 12~2~d~:3~
-37~
phthaleinyl-~-D-galactoside ~odium ~altO The pH
value o the buffer ~olution of 7.3 (37 C.~ i~
maintained.
~ ti~:
Commercially available ~-D~galactosidase from Escherichia coli is dissol~ed in buffer~ The activity of this solution is about 0.08 U~ml. (referred to the statements of the manufacturer~.
A ~-D-galactosidase-antibody preparation is used. ~he preparation of such an enzyme-antihody conjugate is known. It is described, for example, in Biochem. Biophys. Acta 612, 40-49/1980. The prepar-ation is so diluted with buffer that there is obtained an activity approximatel~ comparable to that of the above-described enzyme ~olution.
b) Carr in out of the mea~urement:
'~he measurement takes place photometrically at 578 nm. 950 ~1. of reagent solution are, in each case, mixed in a 1 cm. cuvet~e at 37C. with 50 ~1. of enæyme solution or with 50 ~1. of enzyme conjugate solution. As a measure for the r~action, there is detenmined the extinction increase per unit time in [mExt/min].
For the reaction with the ree ~-D-galacto~idase there are mea~uxed 124 mExt/min ancl for the reaction with ~-~galactosida~e-antibody conjugate 120 rr~Ext/min.
-3~-Both measurement values show that a very readily measurable extinctlon ~ifference is found not only with free but also with conjugated ~-D-galactosidase.
From this it follows that the new sulphonphthaleinyl-~-D-galactosides can be used in the same way as sub-strates not only for free ~-D-galactosidase but also for ~-D-galactosidase conjugates. Thus, the new substrates can be used not only as diagnostic agents for the determination of free ~-D-galactosidases but can also be used in an advantageous manner in the case of enæyme immunoassays in which ~-D-galactosidase is used as indicator enzyme.
German Patent 2,118,455 referred to herein is further identified below:
German Patent 2,118,455, Hans Lange et al issued to Boehringer Mannheim GmbH, Apr:il 12, 1973.
A solution of 6.77 g. (0~01 mole) fluorophenol red tribenzylammonium salt and 5.3 g. (0~01 mole) per-0-trimethylsilyl-a-D-galactopyranosyl bromide in 70 ml. dichloromethane are mixed with 1.15 g. (0.005 mole) silver oxide and 1.4 g. (0.005 mole~ silver carbonate and stirred for 18 hours with the exclusion of moisture. After filtering off the precipitate, the filtrate is evaporated and the residue is, for spli~ting off the protective groups, taken up in 60 ml. methanol and kept for 12 hours at arnbient tempexature. For purification, it is chromatographed over silica gel with dichloromethane/methanol (5~1 v/v) 20 to give 1.2 g. 3,3'-difluorophenolsulphonphthaleinyl-~-D~galactoside tribenzylammonium salt as an orange coloured, amorphous powder ~yield 14% of theory), m.p. 157 - 165C~
~MR: (DMSO-d6): 3.1-3.9 (m, 6H), 4.2~5.2 ~m, llH), 6.3 (m, lH), 6.8-7.6 (m, 23H), 7.9 (m, 1~).
E ~ o ~.
1.5 g. (0.0025 mole) 3,3'-dichlorophenol~ulphon- , phthaleinyl-~-D-galactoside ~odium salt, prepared accordi.ng to Ex~nple 1, is dissolved in a little water.
The soluti~n is applied to a column filled with ~nberlite IR 120 (Li form) . Lyophilisation of the eluate gives 1.4 g. 3,3'-dichlorophenolsulphon-phthaleinyl-~-D-galactoside lithium salt as an orange coloured, amorphous powder (yield: 96% of theory), m.p. 190Co (decomp.).
~MR: (DMSO-d6): 3.3-3.8 (m, 6Hl, 4.3-4.9 (m, 4H), 5~07 (s~ J = 7 Hz, lH), 6~1-7~7 (m~ 9H)~
7~8-8~1 (m, lH).
In a manner analogous to that described in the Example 8, there are prepared from 3,3'-~ichloro-phenolsulphonphthale.inyl-~-D-galactoside sodium salt (~ee Excample 1) a) by exchange chromatography on Amberlite*IR 120 in the Ca fonm orange-red, amorphous product (yield: 78~o of theory), m.p. 250C. (decomp.) * trade ~ark ~l~ql2~
~28-NMR: (DMSO-d6): 3.2 3~8 (m, 6H), 4.4-5.1 (m, 4H), 5.1 (s, J = 7 Hz, lH), 6~2~7a6 ~m, 9H~, 7.8-8.0 (m, lH)o b) By exchange chromatography on Amberlite* IR 120 in 5 the (H3c)4N f~rm yellow, amorphous product (yield 85% of theory) m.p. 190 - 195Co NMR: (DMSO-d6): 3.2 (s, 12H), 3.3-4.0 ~m, 6H), 4~1-5~3 (m, 4H), 5.1 (d, J = 7 Hz, lH), 6~4 (m, lH~, 7.0-7.7 (m, 8H), 7.9 (m, lH).
~.
tribenz lammonium salt.
1.5 g~ (0~0025 mole3 3,3'-dichlorophenolsulphon-phthal~inyl-~D~galacko~ide sodium ~alt (see Example 1) is dissolved in a little wa-ter and pa~sed through a column filled with Amberlite~IR 120 ~H formj. The eluate is mixed with a stoichiometric (Oa72 g~) amount of triben~ylamine dissolved in 15 ml. ethanol and evaporated~ There is obtained 1~7 g. 3,3'-dichloro-ph~nolsulphonphthaleinyl-~-D-galactoside triben~yl-ammonium salt as a yellow, amorphou~ materi~l ~yield 78% of theory)a, m.p. 140 - 150C.
~.
In a manner analogou~ to that described in * trade mark D A ~1 ~W
~1;~ a ~J 6' Exam~le 10, there is prepared ~rom 3,3'-dichloro-phenolsulphonphthaleinyl-~-D-galactoside sodium salt (see Example 1), with the use of benzyldiethylamine, the corresponding yellow, amorphous powder tyield 69% of theory), m.p.
245 - 248C.
~.
Detenmination of the activlty of @-D= lalactosidase a) _~r ,'Y51~
HEPES 100 mmol~litre sodium chloride154 mmol/litre m~gnesium L-aspartate2 mmol~litre BSA 10 g./litre T~een-20* 0.5 y./litre pH value ~adjusted w.ith aqueous sodium hydroxide7~3 ~37C.) solution ~0~ :
In the a~ove-described buffer solution are dissolved 5 mmol/litre phenolsulphonphthaleinyl-~~D-galactoside sodium salt~ ~he pH value is adjusted with aqueous sodium hydroxide solution to pH 8.5 (37C.~.
* trade mark ~, nt 5~ t~ 2~
-In the above-described buffer solution are dissolved 5 mmol/litre 3,3'-difluorophenolsulphon-phthaleinyl-~-D-galactoside sodium salt. The pH value is ad~usted with aqueous sod:ium hydroxide solution to pH 7.5 (37C.).
_ ~ r~ g~
In the above-described buffer solution are dissolved 5 mmol/litre 3,3'-dichlorophenolsulphon-phthaleinyl-~-D-galactoside sodium salt. The pH value of the buffer solution of 7.3 (37C.) is maintained.
Rea ent Solution 4~
q _ _.
In the above-described buffer solution are dissolved 5 mmol/litre 3,3',5,5'-tetrachlorophenol-3",4",5",6"-tetrabromosulphonphthaleinyl-~-D-galactoside sodium salt. The pH value of the buffer solution of 7.3 ~37C.) is maintained.
The substrate concentration and the pH values are to be optimised for each substrate used. There-fore, quite knowingly, different values for substrateconcentrations or pH values can occur in the individual reagent solutions.
Enzy~ solution Commercially available ~-D~galactosidase from Escherichia coli is dissolved in the above-mentioned buffer solution. The activity of this ~olution is about 0.08 U/ml. (referr d to the statements of t~e manufacturer).
~ ~a C' ~ ~
b~ ~
The measurement takes place photometrically, in each case at the wavelength given below.
950 ~1. of reagent are mixed in a 1 cm. cuvette at 37C. with 50 ~1~ of enzyme solution. As a m~asure for the reaction, there is determined the extinction increase per unit time in [mExt/min]. It is calculated from -the measured extinction hy division with the - reaction time.
The following Table gives the measurement values found:
Reagent No. Measurement reaction i wavelength ~mExt/min~
[nm~
1 5~0 ~5 3 S7~3 123 57~ 121 ~ ~___ ~.
a) HEPES 50 mmol/litre citric acid 50 mmol/litre tricin 50 ~nol/litr2 sodium chloride 154 mmol~litre magnesium L-aspartate1 mmol/litre BSA 10 g./litre pH value (adjusted with aqueous 6.9 (37C.3 sodium hydroxide solution) ~gL____ olution 1:
In the above-described buffer solution are dissolved 5 mmol/litre 3,3'-dimethylphenolsulphon-phthaleinyl-~-D-galactoside.
Reaqent Solution 2-In the above-described buffer solution are dissolved S mmol/litre 3,3'-dihydroxyphenolsulphon-phthaleinyl-~-D-galactoside.
~=:
Commercially available ~-D-galactosidase from Escherichia coli i5 dissolved in the above-mentioned buffer solution. The activity of khis solut.ion is about 0 . 08 U/ml . ~ referred to the staternents oE the manuf ac tu re r ) ~
b~ r ~ _ :r~ out o Ie ~-c~c~:~r~:s 950 ~1~ of reagent are mixed in a 1 cm. cuvette at 37 C. with 50 ~1. of enzyme solution. After 10 minutes reaction time, it is adjusted to pH 10 with aqueous sodium hydroxide solution and the extinction measured. The same procedure is used for a blank containing buffer instead of en~yme solution~ During th~ reaction and the mea~urement, the temperature is kept at 37C. From the extinctions determined on th~
~33 ~
batches with and without enzyme, there i~ calculat.ed the extinction difference. By division of this extinction difference by the reaction time, there i5 given, as mea~ure for the reaction, the extinction increase per unit time in [mExt/min].
The following Table gives the measurement values found:
Reagent No. Measur~mentreaction wavelength [nm~[r~xt/min~
~ _ _ .
2 5g3 6 ~ _-- .
.
~ "'C~15 ~
There are used cor~nercially available ~-D~
galactosida~e preparations of differing oriyin. As a characteristic feature, the~e manife~t their maximurn act.iv.ity at differing pH values:
20 Statements of the manufacturer:
from beans (jack beans) p~ 3.5 from ~ ~ P
from Escherichi6 coli pH 6Og from bovine liver pH 7.3 a3 ~r~FLarat__ __ _ h~_ ~l~tic~ used:
25 ~uLI- ' HEPES 50 mmol/litre citric acid 50 mmol/litre ~S',~ .k~ ~ 3 ~r _3d~_ tricin 50 r~nol/litre sodium chloride154 ~nol/litre magnesiurn L-aspartate1 mmol/litre BSA 10 g./litre Solutions are prepared of the above composition with differing pH values according to the pH optima of the ~-D-galactosidases (pH 3~ 5/4.0/6.9/7~3), the pH values being adjusted at 37C. with hydrochloric acid or aqueous sodium hydroxide solution.
1 0 _~5 ~ ~ j D
In the above-mentioned buffer solutions with the differing pH values of 3~5/4~0~6D9/7~3 are dissolved, in each case, 5 ~nol/litre 3,3l-dichloro-phenolsulphonphthaleinyl-~-D-galactoside sodi~n salt.
_ = ' The ~-D-galactosidases are dissolved in the buffers with, in each case, the optirnurn pH values:
from bean~ (jack beans) in buffer of pH 3.5 from ~ ~g~ in buffer of pH 4.0 from E:cherichia coli in buffer of pH 6 9 from bovine liver in buffer of pH 7D 3 The activity of these solutions is about 0.08 U/ml~ (referred to the statements of the manufactu~r).
b~ ~D
The enzyme reaction takes place during a defirlite reaction time at the optimum pH value for ~ t~ 7 the particular enzyme. 1000 ~1. of reagent are mixed in a 1 cm~ cuvette at 37C. ~th 30 ~1. of an enzyme solution~ After 15 minutes reaction time, it is adjusted with aqueous sodium hydroxide solution to pH 8.5 and the extinction deterrnined at 578 nm.
~uring the reaction and the measurement, the temper-ature is kept constant at 37C. In the same way, for each measurement a blank is carried out. For this purpose, instead of the enzyme solution, there are used 30 ~1. of buffer solution.
c) Evaluation __ .
First, there is obtained the difference between the measurement value with the enzyme and the measure-ment value of the hlank. By division of this differ-ence by the reaction time, there is determined, asmeasure for the reaction, the extinction increase per unit time :in [mExt/min~.
measurement value (with enzyme) - measurement value (blank) = ~ measurement value ~ measurement value _ - reaction [mExt/min]
reaction time ~ he measurement values found for the reaction of the individual enzymes are given in the following Table:
.3 enzyme from pH value reaction s~eed [rnExt/minJ
beans (jack beans) 3.5 71 ~ger 4.0 112 Escherichia coli 6.9 74 bovine liver 7.3 32 _ . _ . _ _ The above-described experimental results show that the sulphonphthaleinyl-~-D~galactosides can be used as substrates for ~-D-galactosidases of any origin.
Example 15.
I~ L=3_ ~:t~
a) ~tl~ s~
HEPE5 100 ~nol/litre sodium chloride154 r~ol/litre magnesium ~-aspartate2 mmol/litre BSA 10 g./litre Tween 20* 0.5 g./litre pH value (adjusted with a~ueous 7.3 ~37C.) sodium hydroxide solution) ~D~ t o~:ti~:
In the above-described buffer ~olu~ion are dissolved 5 r~nol/litre 3,3'-dichloropherlol~ulphon-* trade mark 12~2~d~:3~
-37~
phthaleinyl-~-D-galactoside ~odium ~altO The pH
value o the buffer ~olution of 7.3 (37 C.~ i~
maintained.
~ ti~:
Commercially available ~-D~galactosidase from Escherichia coli is dissol~ed in buffer~ The activity of this solution is about 0.08 U~ml. (referred to the statements of the manufacturer~.
A ~-D-galactosidase-antibody preparation is used. ~he preparation of such an enzyme-antihody conjugate is known. It is described, for example, in Biochem. Biophys. Acta 612, 40-49/1980. The prepar-ation is so diluted with buffer that there is obtained an activity approximatel~ comparable to that of the above-described enzyme ~olution.
b) Carr in out of the mea~urement:
'~he measurement takes place photometrically at 578 nm. 950 ~1. of reagent solution are, in each case, mixed in a 1 cm. cuvet~e at 37C. with 50 ~1. of enæyme solution or with 50 ~1. of enzyme conjugate solution. As a measure for the r~action, there is detenmined the extinction increase per unit time in [mExt/min].
For the reaction with the ree ~-D-galacto~idase there are mea~uxed 124 mExt/min ancl for the reaction with ~-~galactosida~e-antibody conjugate 120 rr~Ext/min.
-3~-Both measurement values show that a very readily measurable extinctlon ~ifference is found not only with free but also with conjugated ~-D-galactosidase.
From this it follows that the new sulphonphthaleinyl-~-D-galactosides can be used in the same way as sub-strates not only for free ~-D-galactosidase but also for ~-D-galactosidase conjugates. Thus, the new substrates can be used not only as diagnostic agents for the determination of free ~-D-galactosidases but can also be used in an advantageous manner in the case of enæyme immunoassays in which ~-D-galactosidase is used as indicator enzyme.
German Patent 2,118,455 referred to herein is further identified below:
German Patent 2,118,455, Hans Lange et al issued to Boehringer Mannheim GmbH, Apr:il 12, 1973.
Claims (23)
1. A phenolsulphonphthaleinyl-.beta.-D-galactoside of formula (I):
(I) wherein R1 and R4, which can be the same or different, are hydrogen or halogen atoms or nitro or amino groups, R5 to R12, which can be the same or different, are hydrogen or halogen atoms or lower alkyl, hydroxyl, lower alkoxy, carbonyl or nitro groups and M+ is a proton, an alkali metal, alkaline earth metal or ammonium ion.
(I) wherein R1 and R4, which can be the same or different, are hydrogen or halogen atoms or nitro or amino groups, R5 to R12, which can be the same or different, are hydrogen or halogen atoms or lower alkyl, hydroxyl, lower alkoxy, carbonyl or nitro groups and M+ is a proton, an alkali metal, alkaline earth metal or ammonium ion.
2. A phenolsulphonphthaleinyl-.beta.-D-galactoside of formula (I), as defined in claim 1, wherein R1 to R4 which can be the same or different are selected from hydrogen, fluorine, chlorine and bromine atoms, nitro or amino; R5 to R12, which can be the same or different are selected from hydrogen, fluorine, chlorine or bromine atoms, lower alkyl of 1 to 3 carbon atoms, hydroxyl, lower alkoxy of 1 to 3 carbon atoms, carboxyl or nitro, and M+ is selected from the group consisting of a proton, Li+, Na+ and Ca2+.
3. Phenolsulphonylphthaleinyl-.beta.-D-galactoside sodium salt.
4. 3,3'-Difluorophenolsulphonphthaleinyl-.beta.-D-galactoside sodium salt.
5. 3,3'-Dichlorophenolsulphonphthaleinyl-.beta.-D-galactoside sodium salt.
6. 3,3',5,5'-Tetrachlorophenol-3",4",5",6"-tetra-bromosulphonphthaleinyl-.beta.-D-galactoside sodium salt.
7. 3,3'-Dimethylphenolsulphonphthaleinyl-.beta.-D-galactoside.
8. 3,3'-Dihydrophenolsulphonphthaleinyl-.beta.-D-galactoside.
9. A process for the preparation of a phenol-sulphonphthaleinyl-.beta.-D-galactoside of the formula (I):
(I) in which R1 to R4, which can be the same or different are hydrogen or halogen atoms or nitro or amino groups; R5 to R12, which can be the same or different, are hydrogen or halogen atoms or lower alkyl, hydroxyl, lower alkoxy, carboxyl or nitro groups and M+ is a proton or an alkali metal, alkaline earth metal or ammonium ion, comprising:
reacting a compound of formula (II):
(II) in which R1 to R12 have the above-given meanings, with a per-O-substituted l-halogeno-.alpha.-D-galactose of the formula (III):
(III) in which X is a leaving group and each R13 is a protective group, with Walden inversion on the C-1 atom of the sugar residue to produce a per-O-sub-stituted sulphonphthaleinyl-.beta.-D-galactoside of the formula (IV):
(IV) and splitting off the protective groups R13 from the galactoside of formula (IV).
(I) in which R1 to R4, which can be the same or different are hydrogen or halogen atoms or nitro or amino groups; R5 to R12, which can be the same or different, are hydrogen or halogen atoms or lower alkyl, hydroxyl, lower alkoxy, carboxyl or nitro groups and M+ is a proton or an alkali metal, alkaline earth metal or ammonium ion, comprising:
reacting a compound of formula (II):
(II) in which R1 to R12 have the above-given meanings, with a per-O-substituted l-halogeno-.alpha.-D-galactose of the formula (III):
(III) in which X is a leaving group and each R13 is a protective group, with Walden inversion on the C-1 atom of the sugar residue to produce a per-O-sub-stituted sulphonphthaleinyl-.beta.-D-galactoside of the formula (IV):
(IV) and splitting off the protective groups R13 from the galactoside of formula (IV).
10. A process according to claim 9, wherein X is a halogen atom.
11. A process according to claim 9, wherein X is chlorine or bromine.
12. A process according to claim 9, 10 or 11, wherein each protective group R13 is selected from acetyl, benzoyl, benzyl and trimethylsilyl.
13. A diagnostic agent for the detection of .beta.-D-galactosidase, containing at least one chromo-genic substrate and an appropriate buffer substance wherein the at least one chromogenic substrate is selected from phenolsulphonphthaleinyl-.beta.-D-galactosides according to claim 1.
14. A diagnostic agent according to claim 13, further containing conventional adjuvants.
15. A diagnostic agent according to claim 14, wherein the adjuvants are selected from wetting agents, oxidation agents, galenical additive mate-rials and structural formers.
16. A diagnostic agent for the detection of .beta.-D-galactosidase in the form of a test strip com-prising an absorbent carrier impregnated with ingredients comprising a buffer solution and at least one galactoside of formula (I), as defined in claim 1.
17. A diagnostic agent for the detection of .beta.-D-galactosidase in the form of a lyophilisate of ingredients comprising a buffer solution, at least one galactoside of formula (I), as defined in claim 1, and a structural former.
18. A diagnostic agent for the detection of .beta.-D-galactosidase comprising a granulated mass of ingredients comprising a buffer solution, galenical additives and at least one galactoside of formula (I), as defined in claim 1.
19. A diagnostic agent according to claim 18, wherein said mass is formed into a tablet.
20. In a method for the determination of activity of .beta.-D-galactosidase in which a .beta.-D-galactosidase substrate is enzymatically split with .beta.-D-galactosidase and the reaction is determined photometrically, the improvement wherein the sub-strate is at least one galactoside of formula (I), as defined in claim l.
21. A method according to claim 20, wherein the photometric determination comprises determining the extinction increase per unit time.
22. In an enzyme immunoassay for the deter-mination of the concentration of a low molecular weight substance in the range of 10-5 to 10-12 mole/litre employing .beta.-D-galactosidase as enzyme indicator with photometric determination of .beta.-D-galactosidase with a .beta.-D-galactosidase substrate after the immunological reaction, the improvement wherein said substrate is at least one galactoside of formula (I), as defined in claim 1.
23. A process for the preparation of a phenol-sulphonphthaleinyl-.beta.-D-galactoside of formula (I), as defined in claim 1, comprising reacting a compound of formula (II), as defined in claim 9, with:
i) an alkali metal hydroxide or alcoholate to produce a corresponding dialkali metal salt, or ii) with a substituted amine to produce a corresponding ammonium salt, and reacting the salt from i) or ii) in a dipolar aprotic solvent with a per-O-substituted l-halogenogalactose of formula (III), as defined in claim 9.
i) an alkali metal hydroxide or alcoholate to produce a corresponding dialkali metal salt, or ii) with a substituted amine to produce a corresponding ammonium salt, and reacting the salt from i) or ii) in a dipolar aprotic solvent with a per-O-substituted l-halogenogalactose of formula (III), as defined in claim 9.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP3345748.4 | 1983-12-17 | ||
| DE19833345748 DE3345748A1 (en) | 1983-12-17 | 1983-12-17 | PHENOLSULPHONPHTHALEINYL-SS-D-GALACTOSIDE, METHOD FOR THE PRODUCTION THEREOF AND THE USE THEREOF FOR DETERMINING THE SS-D-GALACTOSIDASE |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1242707A true CA1242707A (en) | 1988-10-04 |
Family
ID=6217269
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000470093A Expired CA1242707A (en) | 1983-12-17 | 1984-12-13 | PHENOLSULPHONPHTHALEINYL-.beta.-D-GALACTOSIDES, A PROCESS FOR THE PREPARATION THEREOF AND DIAGNOSTIC AGENTS CONTAINING THEM |
Country Status (15)
| Country | Link |
|---|---|
| US (1) | US4668622A (en) |
| EP (1) | EP0146866B1 (en) |
| JP (1) | JPS60192767A (en) |
| AR (1) | AR240606A1 (en) |
| AT (1) | ATE64600T1 (en) |
| AU (1) | AU549177B2 (en) |
| CA (1) | CA1242707A (en) |
| CS (1) | CS268664B2 (en) |
| DD (1) | DD234432A5 (en) |
| DE (2) | DE3345748A1 (en) |
| DK (1) | DK162231C (en) |
| ES (1) | ES538621A0 (en) |
| SU (1) | SU1393322A3 (en) |
| YU (1) | YU43390B (en) |
| ZA (1) | ZA849747B (en) |
Families Citing this family (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0309525A1 (en) | 1987-04-10 | 1989-04-05 | The Flinders University Of South Australia | Determination of ions in fluids |
| DE3714147A1 (en) * | 1987-04-28 | 1988-11-17 | Boehringer Mannheim Gmbh | IMMUNCHEMICAL METHOD AND REAGENT FOR DETERMINING A POLYVALENT ANTIGENT IN A LIQUID SAMPLE |
| IL85581A (en) * | 1987-05-21 | 1992-05-25 | Technicon Instr | Substrate for beta-galactosidase comprising derivatives of 4-nitrophenyl-beta-d-galactopyranoside and beta-galactosidase immunoassay containing said substrate |
| US5068180A (en) * | 1987-05-21 | 1991-11-26 | Technicon Instruments Corporation | Substrates for β-galactosidase |
| JPH0650991B2 (en) * | 1987-06-11 | 1994-07-06 | 塩野義製薬株式会社 | Reagent for measuring NAGase activity and measuring method |
| US5151348A (en) * | 1988-12-23 | 1992-09-29 | E. I. Du Pont De Nemours And Company | Enzyme-linked immunoassay for measurement of cyclosporin a levels in whole blood samples |
| US5122602A (en) * | 1989-02-13 | 1992-06-16 | Miles Inc. | Chromogenic merocyanine enzyme substrates |
| US5191073A (en) * | 1989-02-13 | 1993-03-02 | Miles Inc. | Chromogenic merocyanine enzyme substrates |
| US5444161A (en) * | 1989-08-16 | 1995-08-22 | Microgenics Corporation | Substrates for β-galactosidase |
| AU7176191A (en) * | 1990-01-05 | 1991-07-24 | Symex Corp. | Chromogenic 5-position modified neuraminic acid substrates and methods for diagnosing human influenza therewith |
| WO1991010744A1 (en) * | 1990-01-10 | 1991-07-25 | Symex Corp. | Chromogenic 9-position modified n-acetylneuraminic acid substrates and methods for diagnosing human influenza therewith |
| US5210022A (en) * | 1990-04-20 | 1993-05-11 | Rcr Scientific, Inc. | Method test media and chromogenic compounds for identifying and differentiating general coliforms and Escherichia coli bacteria |
| DE4217474A1 (en) * | 1992-05-27 | 1993-12-02 | Boehringer Mannheim Gmbh | Method and means for determining an analyte |
| NL9300829A (en) * | 1993-05-13 | 1994-12-01 | Netagco Holding Bv | Method for controlling an agricultural machine as well as an agricultural machine. |
| US6262674B1 (en) * | 2000-08-10 | 2001-07-17 | Honeywell International Inc. | Aircraft display with potential thrust indicator |
| US6534637B2 (en) | 2001-02-12 | 2003-03-18 | Beckman Coulter, Inc. | Synthesis of chlorophenol red glucuronic acid |
| KR101259452B1 (en) * | 2010-10-18 | 2013-04-29 | (주) 바이오앤텍 | Nanoemulsion emulsifier and cosmetic composition comprising the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3953422A (en) * | 1973-08-17 | 1976-04-27 | Smithkline Corporation | Deoxyglucose derivatives |
| JPS5163826A (en) * | 1974-07-10 | 1976-06-02 | Iatron Lab | Fuenoorufutarein beeta dd monogurukuronidonatoriumuenno goseihoho |
| DE2453069A1 (en) * | 1974-11-06 | 1976-05-13 | Schering Ag | 4-(Cyclohexylidene-(4-hydroxyphenyl)methyl)phenyl glucopyranosides - which are anti-oestrogens useful e.g. as post-coital contraceptives |
| DE2510633C3 (en) * | 1975-03-12 | 1978-07-13 | Boehringer Mannheim Gmbh, 6800 Mannheim | Diagnostic agent for the detection of protein in body fluids and indicator dyes suitable therefor |
| JPS51114990A (en) * | 1975-03-31 | 1976-10-09 | Alza Corp | Instruments of colorimetric determination of enzyme and testing method of the same |
| JPS58994A (en) * | 1981-03-17 | 1983-01-06 | Shionogi & Co Ltd | Novel n-acetyl-beta-d-glucosamine derivative and method of determining the activity of n-acetyl-beta-d-glucosaminidase using the same |
| CA1219201A (en) * | 1983-03-07 | 1987-03-17 | Albert E. Chu | Microorganism detection test |
| JPH0650991B2 (en) * | 1987-06-11 | 1994-07-06 | 塩野義製薬株式会社 | Reagent for measuring NAGase activity and measuring method |
-
1983
- 1983-12-17 DE DE19833345748 patent/DE3345748A1/en active Granted
-
1984
- 1984-12-11 AT AT84115131T patent/ATE64600T1/en not_active IP Right Cessation
- 1984-12-11 DE DE8484115131T patent/DE3484735D1/en not_active Expired - Lifetime
- 1984-12-11 DD DD84270601A patent/DD234432A5/en not_active IP Right Cessation
- 1984-12-11 EP EP84115131A patent/EP0146866B1/en not_active Expired - Lifetime
- 1984-12-13 US US06/681,009 patent/US4668622A/en not_active Expired - Lifetime
- 1984-12-13 JP JP59261949A patent/JPS60192767A/en active Granted
- 1984-12-13 CA CA000470093A patent/CA1242707A/en not_active Expired
- 1984-12-14 ES ES538621A patent/ES538621A0/en active Granted
- 1984-12-14 ZA ZA849747A patent/ZA849747B/en unknown
- 1984-12-14 YU YU2130/84A patent/YU43390B/en unknown
- 1984-12-17 SU SU843826173A patent/SU1393322A3/en active
- 1984-12-17 DK DK604184A patent/DK162231C/en not_active IP Right Cessation
- 1984-12-17 AU AU36842/84A patent/AU549177B2/en not_active Ceased
- 1984-12-17 CS CS849877A patent/CS268664B2/en unknown
- 1984-12-17 AR AR29897884A patent/AR240606A1/en active
Also Published As
| Publication number | Publication date |
|---|---|
| ATE64600T1 (en) | 1991-07-15 |
| AU549177B2 (en) | 1986-01-16 |
| ES8600777A1 (en) | 1985-11-01 |
| JPS621978B2 (en) | 1987-01-17 |
| DK162231C (en) | 1992-03-16 |
| CS268664B2 (en) | 1990-04-11 |
| DK604184D0 (en) | 1984-12-17 |
| JPS60192767A (en) | 1985-10-01 |
| EP0146866A2 (en) | 1985-07-03 |
| DD234432A5 (en) | 1986-04-02 |
| US4668622A (en) | 1987-05-26 |
| DE3345748A1 (en) | 1985-08-29 |
| YU213084A (en) | 1986-12-31 |
| EP0146866A3 (en) | 1987-05-13 |
| DE3484735D1 (en) | 1991-07-25 |
| ZA849747B (en) | 1985-08-28 |
| CS987784A2 (en) | 1989-07-12 |
| EP0146866B1 (en) | 1991-06-19 |
| DK604184A (en) | 1985-06-18 |
| ES538621A0 (en) | 1985-11-01 |
| AR240606A1 (en) | 1990-06-30 |
| DK162231B (en) | 1991-09-30 |
| DE3345748C2 (en) | 1992-02-13 |
| SU1393322A3 (en) | 1988-04-30 |
| YU43390B (en) | 1989-06-30 |
| AU3684284A (en) | 1985-07-04 |
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