WO1979000255A1 - Enzyme assays - Google Patents
Enzyme assays Download PDFInfo
- Publication number
- WO1979000255A1 WO1979000255A1 PCT/GB1978/000033 GB7800033W WO7900255A1 WO 1979000255 A1 WO1979000255 A1 WO 1979000255A1 GB 7800033 W GB7800033 W GB 7800033W WO 7900255 A1 WO7900255 A1 WO 7900255A1
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- WO
- WIPO (PCT)
- Prior art keywords
- reagent
- enzyme
- compound
- assay
- formula
- Prior art date
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- 238000001952 enzyme assay Methods 0.000 title claims abstract description 9
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 74
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- 238000011534 incubation Methods 0.000 claims abstract description 24
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- 125000003118 aryl group Chemical group 0.000 claims abstract description 8
- 238000000034 method Methods 0.000 claims description 40
- 229930182470 glycoside Natural products 0.000 claims description 38
- -1 sulphate ester Chemical class 0.000 claims description 29
- 239000000758 substrate Substances 0.000 claims description 23
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- 150000002338 glycosides Chemical class 0.000 claims description 20
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- MCSAJNNLRCFZED-UHFFFAOYSA-N nitroethane Chemical compound CC[N+]([O-])=O MCSAJNNLRCFZED-UHFFFAOYSA-N 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- RZWZRACFZGVKFM-UHFFFAOYSA-N propanoyl chloride Chemical compound CCC(Cl)=O RZWZRACFZGVKFM-UHFFFAOYSA-N 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- NASFKTWZWDYFER-UHFFFAOYSA-N sodium;hydrate Chemical compound O.[Na] NASFKTWZWDYFER-UHFFFAOYSA-N 0.000 description 1
- 239000011343 solid material Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 125000005504 styryl group Chemical group 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 108060007951 sulfatase Proteins 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- ORHBXUUXSCNDEV-UHFFFAOYSA-N umbelliferone Chemical compound C1=CC(=O)OC2=CC(O)=CC=C21 ORHBXUUXSCNDEV-UHFFFAOYSA-N 0.000 description 1
- HFTAFOQKODTIJY-UHFFFAOYSA-N umbelliferone Natural products Cc1cc2C=CC(=O)Oc2cc1OCC=CC(C)(C)O HFTAFOQKODTIJY-UHFFFAOYSA-N 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- PIAOLBVUVDXHHL-VOTSOKGWSA-N β-nitrostyrene Chemical class [O-][N+](=O)\C=C\C1=CC=CC=C1 PIAOLBVUVDXHHL-VOTSOKGWSA-N 0.000 description 1
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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/08—Esters of oxyacids of phosphorus
- C07F9/09—Esters of phosphoric acids
- C07F9/12—Esters of phosphoric acids with hydroxyaryl compounds
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/805—Test papers
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/806—Fertility tests
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/81—Packaged device or kit
Definitions
- ENZYME ASSAYS This invention relates to enzyme assays, and in particular to reagents for the detection and determination of enzymes.
- NAG N-acetyl- ⁇ -D glucosaminidase
- a nitrophenyl glycoside substrate p-nitrophenyl-2-acetamido-2-deoxy- ⁇ -D-glucopyranoside
- a nitrophenyl glycoside substrate p-nitrophenyl-2-acetamido-2-deoxy- ⁇ -D-glucopyranoside
- enzyme to release p-nitrophenol which is monitored colorimetrically.
- Both these assays require the use of sophisticated spectrophotometric apparatus and thus are only suitable for use when there is access to well-equipped laboratory facilities.
- an enzyme assay reagent comprises a substance which is capable of interaction with the enzyme to give rise to the formation of a compound of formula X-A-Y-NO 2 , wherein A comprises an aromatic nucleus, X comprises an auxochromic group and Y comprises an unsaturated group which is capable of transmitting electron resonance between the aromatic nucleus and the nitro substituent , said compound per se being capable of producing a visual signal which is easily discernible by eye.
- the invention also includes a method for the assay of an enzyme which comprises incubating a sample containing the enzyme with a reagent according to the invention to give rise to the production of a compound of formula X-A-Y-NO 2 as hereinbefore defined, and, if necessary, subjecting the compound to further treatment to develop a visual signal which is easily discernible by eye.
- the reagent typically comprises an enzyme substrate portion and a further portion which gives rise to the formation of the compound of formula X-A-Y.
- the reagent of the invention comprises the compound X-A-Y-NO 2 , or a simple precursor therefor, in combination with the substrate portion, and on interaction with the enzyme the substance releases the compound or simple precursor.
- the compound or precursor is usally attached to the substrate portion at or adjacent to the side within the substance at which enzyme activity takes place.
- the enzyme substrate portion and further portion of the reagent are conveniently linked through the auxochromic group X and the substance has the general formula S-X-A-Y-NO 2 , wherein S comprises the enzyme substrate portion of the substance and X, A and Y are as previously defined, and such reagents release the compound on intereaction with the enzyme.
- the reagents, substances and method of the present invention are widely applicable to the assay of enzymes in general, the enzyme substrate portion of the substance being varied in accordance with the particular enzyme which it is desired to assay.
- the enzymes which may be assayed by the invention are catabolic enzymes, such as those which play an important role in the breakdown of macromolecules in cellular tissues.
- enzymes which may be assayed include carboxylate esterases and lipases, acid and alkaline phosphatases, phosphodiesterases and sulphatases, and thus corresponding reagents may comprise appropriate carboxylate, phosphate, diphosphate and sulphate esters.
- the invention is applicable to the assay of glycosidases in which case the substrate portion S of the reagent characteristically comprises a corresponding glycoside carbohydrate substituent, such as an ⁇ or ⁇ -glucopyranosyl, -galactopyranosyl, or -mannopyranosyl substitufent.
- the invention has been found to be particularly suitable for application to the assay of N-acetyl- ⁇ -D-glucosaminidase (NAG).
- NAG N-acetyl- ⁇ -D-glucosaminidase
- the invention may be employed to assay for the presence of enzymes in general and to monitor their concentration levels; for instance, for diagnosis of a corresponding defect or illness.
- the NAG content of urine may be assayed for early detection of kidney disease, or in the specific case as an early indicator of kidney rejection in "kidney transplant" patients.
- the invention may be used to determine the absence of enzymes which may, for instance, be indicative of certain genetic disorders.
- the reagents and method of the invention may provide convenient tools for use in biochemical and medical research.
- the compound X-A-Y-NO 2 is capable of producing a colour change which is easily discernible to the eye, and in this latter respect the colour produced is typically substantially different from background colouration of the sample.
- reagents of the invention for use with urine samples such as reagents for assay of NAG, typically release substances capable of producing colours substantially different from the background colour of urine.
- the reagents of the invention release compounds which are capable of producing distinctive colours, preferably reds or blues.
- suitable released compounds usually have a ⁇ max under alkaline conditions in the range from about 450 up to about 700, preferably from about 500 up to about 600, especially in combination with a high extinction co-efficient e.g. ⁇ greater than about 20,000.
- the group A comprises a monocyclic aromatic nucleus, especially a benzene nucleus.
- the group Y may comprise any suitable unsaturated group which is capable of transmitting electron resonance between A and the NO 2 substituent, and may include systems containing heteroatoms and/or annulated systems.
- the group A may comprise a benzene ring which is one ring of a naphthyl nucleus, the other ring of the naphthyl nucleus providing the group Y.
- the group Y may comprise acetylenic unsaturation. More preferably, however, the group Y comprises ethylenic unsaturation which is conjugated with respect to the aromatic nucleus A e.g. an ethenyl, butadienyl or hexatrienyl substituent.
- the reagent of the invention is capable of interacting with the enzyme to give rise to a compound of formula I
- the auxochromic group X may comprise any suitable auxochromic group such as an amino group (NH 2 ) or preferably a hydroxyl group (-OH).
- X is a hydroxyl group and A comprises a styryl group.
- particularly preferred reagents according to the invention are those which on interaction with the enzyme give rise to the formation of a 4-hydroxy- ⁇ -nitrostyrene compound, i.e. of the formula II below,
- R 1 , R 2 , R 3 and R 4 are as previously defined.
- the compounds of formula II in general exhibit surprising and highly desirable colour properties which make them particularly suitable for use as a component of the reagent of the invention. It has been found that the compounds of formula II are typically strongly red in colour under alkaline conditions as compared with the weak orange-yellow colour of para nitrophenol, and also that they have absorbance maxima ( ⁇ max) which are unexpectedly significantly higher than that of para nitrophenol ( ⁇ 400 under alkaline conditions). It is now believed that these enhanced colour properties are due to the formation of a radical ion resulting from an oxidative process, as shown below
- the mono-methoxy compound III and the di-methoxy compound IV have absorbance maxima ( ⁇ max) of 506 and 530 with extinction co-efficients ( ⁇ ) of 24,600 and 22,300 respectively in buffer at pH 8.8, and exhibit satisfactory scarlet-red colourations under these conditions.
- auxochromic group X e.g. -OH
- the auxochromic group X may provide the linkage between the compound X-A-Y-NO 2 and the substrate portion of the reagent, such that the compound is released on interaction with the enzyme.
- reagents according to the invention are given below by formulae A to E, referring, for the sake of simplicity, only to reagents comprising a simple 4-hydroxy- ⁇ -nitrostyryl substituent. It will be appreciated, however, that this latter substituent may be replaced by a substituent relating to the compounds X-A-Y-NO 2 in general.
- R Z in formula E denotes a carbohydrate substituent, such as an ⁇ or ⁇ glucopyranosyl.galactopyranosyl or manriopyranosyl
- the preferred nitrostyryl substituted reagents may be prepared from vanillin or similar hydroxy benzaldehyde based materials such as p-hydroxybenzaldehyde or salicylaldehyde by nitromethylation of the corresponding substrate-vanillin or -similar compound adduct.
- Preferably mildly alkaline conditions are employed during nitromethylation.
- similar reagents may be prepared by correspondingly similar routes.
- Samples which may be assayed are conveniently in fluid form and include samples comprising physiological fluids, such as serum or urine. Other suitable samples may also be assayed including samples derived from cell homogenates, such as those samples commonly used in the study of genetic disorders.
- the reagent and sample are incubated together; for instance, sample and solution containing the reagent are mixed and allowed to interact under appropriate conditions for a suitable period of time.
- the temperature used during incubation is greater than about 30oC e.g. from about 30 up to about 40oC, and under these conditions an incubation period of up to about thirty minutes e.g. from about 10 to 15 up to about thirty minutes, is usually satisfactory.
- the reagent may be used in association e.g. absorbed, with a suitable solid phase material, such as cellulose particles, e.g. microcrystalline cellulose, which may advantageously overcome problems arising from the insolubility or poor solubility of the reagent.
- the reagent may be used in association with a porous solid support material, such as paper, to provide a convenient test paper or dip stick for a simple dip test.
- a porous solid support material such as paper
- Such products comprising the reagent of the invention in association with a suitable inert solid material or with a porous solid support material are included within the scope of the invention.
- the reagent On incubation the reagent releases,or more generally gives rise to the formation of, a compound which may without further treatment provide the visual signal e.g. colour, which is indicative of the presence of the enzyme in the sample.
- the compound may require further treatment to develop the visual signal, and such treatment typically comprises activation to develop the inherent visual signal of the compound.
- This further treatment does not include steps of synthetic or preparative chemistry, such as the diazonium coupling procedures employed with some prior art enzyme assay reagents.
- the treatment comprises alkaline treatment, which may lead to salt formation permitting delocalisation of electronic charge, for instance, alkaline treatment of preferred conjugated unsaturated chromophore substances advantageously giving rise to the development of coloured visual signals.
- Alkaline treatment may comprise addition of alkali to solution containing the released compound, or preferably solution containing the compound is brought into contact with a solid phase reagent e.g. an absorbent, which simultaneously presents an alkaline environment to the compound.
- a solid phase reagent e.g. an absorbent
- the solution containing the compound is contacted with an anionic exchange material, which is preferably of neutral colour e.g. white or light-coloured, and which is in its free base form e.g. hydroxyl (OH ) form.
- the anionic exchange material may comprise DEAE cellulose, or a similar light coloured anion exchange material in free base form.
- the use of an absorbent anionic exchange material may be particularly advantageous, absorbing and concentrating the released substance into a narrow band for enhanced ease of monitoring.
- DEAE cellulose and similar materials in this latter respect is believed to be novel, and this use, in general, of these materials is included within the scope of the invention.
- the method of the invention comprises incubating sample comprising the enzyme with reagent associated with solid phase material e.g. an inert absorbent or a porous support material, so as to give rise to the formation of a compound X-A-Y-NO 2 , and contacting solution containing the compound with a solid phase reagent which presents an alkaline environment to the substance and thereby produces a colour change or other visual signal which is easily discernible by eye.
- solid phase material e.g. an inert absorbent or a porous support material
- apparatus for carrying out the method of the preferred embodiment usually in the form of a through-flow device normally comprising two compartments, an incubation compartment containing reagent associated with solid phase material, e.g. microcrystalline cellulose, and a visualisation compartment containing a solid phase reagent which presents an alkaline environment to the substance e.g. DEAE cellulose or a similar substance.
- the compartments are typically interconnectable e.g. by a suitable connecting passageway, to permit flow of liquid containing the released compound from the incubation to the visualisation compartment.
- the two compartments are preferably, however, capable of isolation from one another, for instance to allow for retention of the sample in the incubation compartment for the required incubation period.
- the passageway between the two compartments may be provided with an appropriate tap or other barrier which may be conveniently opened or removed after the required incubation period to permit the liquid to pass into the visualisation compartment, e.g. to pass through the preferred DEAE cellulose absorbent.
- the through-flow device may consist of two compartments attached at interfacial surfaces by a pivot, both surfaces provided with outlets at a fixed radius from the pivot such that mutual rotation of the compartments brings the outlets into coincidence with one another providing a passageway between the compartments.
- the passageway between the compartments may be blocked with a flimsy barrier which can be ruptured as desired, for instance by bringing the two compartments together e.g. by a screwthreaded action.
- concentration of reagent used e.g. present in association with inert solid phase material may be varied as desired, for instance, in accordance with the threshold concentration of enzyme which it is desired to monitor; increases in concentration of reagent generally permitting detection of lower threshold concentrations of enzyme.
- a solid phase reagent such as DEAE cellulose
- to visualise the released substance advantageously permits detection of very small quantities of released substance, owing to concentration of the colour produced in a narrow absorbed band. In this respect, it has been found possible to detect the presence of the substance 2-methoxy-4-( ⁇ -nitroethenyl) phenol at concentrations of 1-2 ⁇ 10 -6 molar by contact of the solution with DEAE cellulose.
- NAG detecting glycoside (E) on an inert cellulose solid phase in combination with DEAE cellulose visualisation is capable of detecting a threshold concentration of NAG in urine from about 40 - 50 n mol/ml after incubation at 37 for twenty minutes; whereas, use of cellulose containing 8% of the glycoside in the same system gives a thresold of detection of about 20 - 30 n mol/ml. Both systems give broad intense bands for abnormal urines which are easily distinguished from those given by normal urines.
- the method of the invention may be conveniently performed by patients themselves in their homes using a suitably prepared through-flow device and requires minimal analytical skills.
- the patient may simply add a fixed quantity of fresh urine to the incubation compartment e.g. up to a fixed level indicated on the side of the compartment, and after a standard incubation period, which will usually depend upon the ambient temperature, connect the incubation and visualisation compartments permitting incubated urine to flow through the solid phase visualising reagent.
- the visual signal, e.g. colour change, produced by the released substance advantageously provides a simple and quick means of detecting the presence of the enzyme in the sample as compared with the methods previously used which rely upon sophisticated monitoring apparatus.
- the intensity of the visual signal e.g. colour, produced may conveniently be used as a rough visual indicator of the concentration of enzyme present in the sample. If more accurate measurements are required, however, the reagents of the invention may be monitored spectrophotometrically, for instance in the conventional manner.
- the colour produced may be estimated using a colourimeter, usually after adjustment of pH to 8.5 - 9.5 with an appropriate buffer, and, in such methods, the present reagents may be more suitable than corresponding prior art reagents, such as p-nitrophenolic and o- nitrophenolic substrates.
- Figure 1 represents one form of flow-through device according to the invention.
- Figure 2 represents an alternative form of flow-through device according to the invention.
- Example I Preparation of nitrostyryl glycosides for detection and determination of corresponding .glycosidases
- nitrostyryl glycoside enzyme substrates As a first stage in the preparation of nitrostyryl glycoside enzyme substrates according to the invention the appropriate hydroxy-benzaldehyde glycosides are prepared.
- reaction mixture described above was refluxed for 15 - 45 minutes, affording similar yields of products though often of improved purity.
- glycosides prepared above are suitable for use in the detection and determination of the corresponding glycosidases.
- Reagents comprising these glycosides are included within the invention and may be used in the method of the invention.
- the glycosides release the corresponding hydroxy-nitrostyryl compound, on interaction with the appropriate enzyme, which provides the easily discernible visual signal, usually a red colouration.
- An inert solid phase material may be impregnated with the glycosides prepared above or other enzyme substrates according to the invention.
- the requisite amount of microcrystalline cellulose (Avicell) is added to the eluate from the silica gel, as prepared above, evaporated to dryness in a rotary evaporator and the resulting solid ground to a fine powder with a mortar and pestle.
- Vicell microcrystalline cellulose
- a range of ester derivatives of the 4-hydroxy- ⁇ -nitrostyrenes was prepared for use in reagents according to the invention for detection and determination of corresponding esterases.
- Vannillyl acetate (19-4g, 100 mmole) was reacted with nitromethane, ammonium acetate and acetic acid, as in method A part III of Example 1, for a period of 2-3 days.
- the red coloured reaction mixture was then diluted with water and the resultant oil layer extracted with chloroform.
- the chloroform extract was dried (MgSO 4 ) and evaporated to dryness, and the resulting solid recrystallised from chloroform-ethanol to give the acetate, m.p. 158-162.2o, (20g, 17%).
- the 3-methoxy- ⁇ -nitrostyryl acetate was prepared from the corresponding hydroxy-nitrostynene.
- ⁇ nitrostyryl acetate is suitable for use in reagents and the method of the invention for detection and determination of acetate esterases, and on interaction with such enzymes gives rise to the release of 4-hydroxy-3-methoxy- ⁇ -nitrostyrene which exhibits a strong red colouration under alkaline conditions.
- the propionate is suitable for use in the detection of propionate esterases and similarly give rise to the formation of the 4-hydroxy-3-methoxy- ⁇ -nitrostyrene on interaction with the enzyme.
- Palmitoyl chloride (3 «6g) was added to dry pyridine (20 ml); there was an immediate precipitation. To this mixture was added 4-hydroxy- ⁇ -nitrostyrene (1.65 g, 10 mmole) and the mixture heated at reflux for 1-2 min. The mixture was then poured into ice-water and the yellow amorphous precipitate filtered off. A solution of the precipitate in a mixture of chloroform and DMSO afforded seed crystals of the ester and the bulk was crystallised by dissolving in boiling methylene chloride and adding ethanol.
- a range of hydroxy- ⁇ -nitrostyrenes was also prepared to determine and compare the colour properties of these compounds being examples of compounds falling within the general formula X-A-Y-NO 2 i.e. compounds whioh may be formed as a result of interaction of the reagent of the invention with the corresponding enzyme.
- the general preparative procedure used was as follows. The corresponding hydroxybenzaldehyde (4 ⁇ mmole) was dissolved in ethanol (200 ml) and ammonium acetate (12 g), nitromethane (32 ml) and acetic acid was added. The reaction mixture was either stirred overnight at room temperature or heated under reflux for 15-60 minutes. In some cases the W-nitrostyrene crystallised from the cooled reaction mixture, or crystallised when water was added to the cooled reaction mixture. In cases where no crystalline product could be isolated by these methods, the product was extracted with ether, which was washed well with water, dried (MgSO 4 ) and taken to dryness.
- the yields, melting points and spectral properties of the various hydroxy- ⁇ -nitrostyrene products were determined by conventional methods and are given below, the spectral properties being. determined in terms of the absorbance ( ⁇ max) in ethanol and TRIS buffer of pH 8.8 and the extinction coefficient ( ⁇ ) at pH 8.8.
- 2-methoxy-4-( ⁇ -methyl- ⁇ -nitroethenyl)-phenol was also prepared by a variation of the above procedure in which vanillin was treated with nitroethane instead of nitromethane.
- the conditions employed for the reaction were essentially the same as those described above for nitromethylation.
- the ⁇ -methyl- ⁇ -nitroethenyl phenol product was obtained in 80% yield with m.p. 110-112 and had ⁇ max (ethanol) of 368 and ⁇ max (TRIS 8.8) of 475 with e of 13,100.
- the hydroxy ⁇ -nitrostyrenes, as prepared above, are typically coloured compounds which may provide the easily discernible visual signal of the reagents of the invention.
- Particularly preferred compounds are the 3-methoxy and 3.5-dimethoxy compounds, 1 and 3. which provide distinctive, deep red colourations under alkaline conditions.
- the ⁇ -nitrostyrene compounds exhibit markedly improved colour properties over the pale orange-yellow colouration of paranitrophenol ( ⁇ max ethanol 320, ⁇ . max TRIS 8.8 400 with ⁇ of 15,000), which it is believed is due to the formation radical ions with the nitrostyryl compounds. The existence of such radical ions is supported by the e.s.r. spectra of the nitrostyryl compounds.
- microcrystalline cellulose containing about 3% of the 4-(2-acetamido-2-deoxy- ⁇ -D-glucopyranosyloxy)-3-methoxy- ⁇ -nitrostyrene, the substrate for NAG, as prepared in Example 1, is treated in a small plastic sample tube with fresh urine (l ml) at body temperature, or stored urine pre-heated to
- the red band forms at the top surface interface of the DEAE cellulose and is more dispersed in nature. This latter effect can be overcome by incorporation of appropriate quantities of electrolytes into the reagent. In all cases, however, the width and intensity of the red band is approximately proportional to the concentration of NAG in the urine.
- Both devices comprise cylindrical upper incubation 1 and lower visualisation 2 compartments, and in both cases the lower visualisation compartment 2 contains a quantity of fibrous DEAE cellulose (Whatman DE 22) 3 sandwiched between upper 4 and lower 5 layers of cotton wool or a similar substance.
- the lower ends of the visualisation compartments 2 have outlets 6 for draining of fluid and are provided with a skirt or legs 7 to allow free standing of the devices in a vertical orientation.
- the walls of the lower visualisation compartments 2 are of clear material, such as clear plastic, to permit observation of the DEAE cellulose and any coloured band absorbed thereon.
- the upper incubation compartments 1 contain an appropriate quantity of the glycoside substance (not shown) either in a suitable solvent or absorbed into cellulose or other suitable absorbent e.g.
- the incubation compartments 1 are fitted with screw caps and have thick PVC or polystyrene walls 9 to insulate the urine during incubation, the walls 9 being marked with a line 10 to indicate the quantity of urine which is to be introduced for incubation.
- the compartments 1 and 2 are held together at interfacial surfaces by pivot pin 11.
- the base of the incubation compartment 1 has an eccentrically located outlet 12 and the top of the visualisation compartment has an inlet 13 eccentrically located at the same radius from the pivot 11 as outlet 12.
- Mutual rotation of the upper 1 and lower 2 compartments brings the outlet 12 and inlet 13 into coincidence with one another providing a passageway connecting the compartments through which incubated urine can flow.
- fresh urine is introduced into compartment 1 up to the level of the mark 10, outlet 12 and inlet 13 being out of coincidence with one another.
- the screw cap 8 is screwed in place and the urine is incubated for a period which depends upon the temperature though is usually about 15 to 20 minutes.
- compartments 1 and 2 are held together in screw-threaded engagement by screw thread 16 around the bottom of compartment 1 and screw thread 17 around the inside of tubular extension 18 projecting from the top of compartment 2.
- a passageway between compartments 1 and 2 is provided by tubular extension 14 which projects from the bottom of compartment 1. Prior to use passageway 14 is closed by its abutment with the weakened section 15 in the top of compartment 2.
- urine is introduced to compartment 1 and incubated as for the device of Figure 1. On completion of incubation, however, compartments 1 and 2 are screwed together and the tubular extension 14 breaks through the weakened section 15 permitting incubated urine to flow into compartment 2. Visualisation of the phenol released by the substrate is as for the device of Figure 1.
- Both devices provide simple means by which "kidney transplant" patients may test themselves in their own homes, or general screening tests may be easily conducted, without access to sophisticated monitoring apparatus.
- Other enzymes besides NAG may be assayed using similar techniques and devices.
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Abstract
An enzyme assay reagent comprises a substance which is capable of interaction with the enzyme to give rise to the formation of a compound of formula X-A-Y-NO2, wherein A comprises an aromatic nucleus, X comprises an auxochromic group and Y comprises a unsaturated group which is capable of transmitting electron resonance between the aromatic nucleus and the nitro substituent, said compound per se being capable of producing a visual signal which is easily discernible by eye. The reagent may be used in a through-flow device comprising an upper incubation compartment (1) and a lower visualisation compartment (2) held together by pivot pin (11); the lower compartment contains a quantity of fibrous cellulose (3) sandwiched between upper (4) and lower (5) layers of cotton wool.
Description
ENZYME ASSAYS This invention relates to enzyme assays, and in particular to reagents for the detection and determination of enzymes.
The presence or absence of certain enzymes in physiological samples, such as urine or serum samples from human patients, is a valuable indicator of illness or deficiencies in the organism concerned e.g. organ malfunctions in human patients. For example, increases in the level of the enzyme N-acetyl-β-D glucosaminidase (NAG) excreted in the urine of "kidney transplant" patients is an early sign of impending rejection of the transplanted kidney, besides being a general indicator of renal and other diseases. At present the customary clinical assay for NAG involves incubation of a sample of urine with the 4-methylumbelliferyl glycoside substrate of the enzyme which interacts to release the corresponding umbelliferone which is then monitored fluorimetrically. Alternatively, a nitrophenyl glycoside substrate, p-nitrophenyl-2-acetamido-2-deoxy-β-D-glucopyranoside, may be used, interacting with the enzyme to release p-nitrophenol which is monitored colorimetrically. Both these assays, however, require the use of sophisticated spectrophotometric apparatus and thus are only suitable for use when there is access to well-equipped laboratory facilities.
New enzyme assay reagents have now been devised which, in some cases, may be employed without requiring the use of sophisticated monitoring apparatus.
According to the present invention an enzyme assay reagent comprises a substance which is capable of interaction with the enzyme to give rise to the formation of a compound of formula X-A-Y-NO2, wherein A comprises an aromatic nucleus, X comprises an auxochromic group and Y comprises an unsaturated group which is capable of transmitting electron resonance between the aromatic nucleus and the nitro substituent , said compound per se being capable of producing a visual signal which is easily discernible by eye. The invention also includes a method for the assay of an enzyme which comprises incubating a sample containing the enzyme with a reagent according to the invention to give rise to the production of a compound of formula X-A-Y-NO2 as hereinbefore defined, and, if necessary, subjecting the compound to further treatment to develop a visual signal which is easily discernible by eye.
The reagent typically comprises an enzyme substrate portion and a further portion which gives rise to the formation of the compound of formula X-A-Y. Usually the reagent of the invention comprises the compound X-A-Y-NO2, or a simple precursor therefor, in combination with the substrate portion, and on interaction with the enzyme the substance releases the compound or simple precursor. Thus the compound or precursor is usally attached to the substrate portion at or adjacent to the side within the substance at which enzyme activity takes
place. In one embodiment the enzyme substrate portion and further portion of the reagent are conveniently linked through the auxochromic group X and the substance has the general formula S-X-A-Y-NO2, wherein S comprises the enzyme substrate portion of the substance and X, A and Y are as previously defined, and such reagents release the compound on intereaction with the enzyme.
The reagents, substances and method of the present invention are widely applicable to the assay of enzymes in general, the enzyme substrate portion of the substance being varied in accordance with the particular enzyme which it is desired to assay. Typically, however, the enzymes which may be assayed by the invention are catabolic enzymes, such as those which play an important role in the breakdown of macromolecules in cellular tissues. For example, enzymes which may be assayed include carboxylate esterases and lipases, acid and alkaline phosphatases, phosphodiesterases and sulphatases, and thus corresponding reagents may comprise appropriate carboxylate, phosphate, diphosphate and sulphate esters. Also, in particular, the invention is applicable to the assay of glycosidases in which case the substrate portion S of the reagent characteristically comprises a corresponding glycoside carbohydrate substituent, such as an α or β-glucopyranosyl, -galactopyranosyl, or -mannopyranosyl substitufent. For example,
the invention has been found to be particularly suitable for application to the assay of N-acetyl-β-D-glucosaminidase (NAG). The invention may be employed to assay for the presence of enzymes in general and to monitor their concentration levels; for instance, for diagnosis of a corresponding defect or illness. For example, the NAG content of urine may be assayed for early detection of kidney disease, or in the specific case as an early indicator of kidney rejection in "kidney transplant" patients. Alternatively, the invention may be used to determine the absence of enzymes which may, for instance, be indicative of certain genetic disorders. Generally, also, the reagents and method of the invention may provide convenient tools for use in biochemical and medical research.
Preferably the compound X-A-Y-NO2 is capable of producing a colour change which is easily discernible to the eye, and in this latter respect the colour produced is typically substantially different from background colouration of the sample. For example, reagents of the invention for use with urine samples, such as reagents for assay of NAG, typically release substances capable of producing colours substantially different from the background colour of urine. Preferably the reagents of the invention release compounds which are capable of producing distinctive colours, preferably reds or blues. Thus suitable released compounds usually have a λ max under alkaline conditions in the range from about 450 up to about 700,
preferably from about 500 up to about 600, especially in combination with a high extinction co-efficient e.g. ε greater than about 20,000.
It will be appreciated that within the general formula of the compounds X-A-Y-NO there exist many typical coloured and dye compounds and suitable compounds for providing the easily discernible visual signal will be apparent to skilled workers in the art. Preferably, however, the group A comprises a monocyclic aromatic nucleus, especially a benzene nucleus. Also the group Y may comprise any suitable unsaturated group which is capable of transmitting electron resonance between A and the NO2 substituent, and may include systems containing heteroatoms and/or annulated systems. Thus, for example, the group A may comprise a benzene ring which is one ring of a naphthyl nucleus, the other ring of the naphthyl nucleus providing the group Y. Also the group Y may comprise acetylenic unsaturation. More preferably, however, the group Y comprises ethylenic unsaturation which is conjugated with respect to the aromatic nucleus A e.g. an ethenyl, butadienyl or hexatrienyl substituent. Thus in a preferred embodiment the reagent of the invention is capable of interacting with the enzyme to give rise to a compound of formula I
and R 4 = H, OMe, NO2, CH3, -CH = CR'R" or CO2H. The groups R' and R" of the substituent -CH = CR'R" may represent groups similar to R 1, R 2, R 3 and R 4, preferably H, NO2 or further conjugated unsaturated substitution e.g. -CH = CRxRy. The auxochromic group X may comprise any suitable auxochromic group such as an amino group (NH2) or preferably a hydroxyl group (-OH).
In particularly preferred embodiments X is a hydroxyl group and A comprises a styryl group. Thus particularly preferred reagents according to the invention are those which on interaction with the enzyme give rise to the formation of a 4-hydroxy-α-nitrostyrene compound, i.e. of the formula II below,
wherein R 1, R 2, R 3 and R 4 are as previously defined. The compounds of formula II in general exhibit surprising and
highly desirable colour properties which make them particularly suitable for use as a component of the reagent of the invention. It has been found that the compounds of formula II are typically strongly red in colour under alkaline conditions as compared with the weak orange-yellow colour of para nitrophenol, and also that they have absorbance maxima (λ max) which are unexpectedly significantly higher than that of para nitrophenol (~ 400 under alkaline conditions). It is now believed that these enhanced colour properties are due to the formation of a radical ion resulting from an oxidative process, as shown below
The existence of electron donating substituents, such as alkoxy groups e.g. methoxy, adjacent to the hydroxyl substituent appears to stabilise the formation of this radical ion, as is borne out by the colour properties of the 3-methoxy and especially the 3.5-dimethoxy analogues shown below as
formulae III and IV respectively, upon which particularly preferred reagents are based.
Thus the mono-methoxy compound III and the di-methoxy compound IV have absorbance maxima (λ max) of 506 and 530 with extinction co-efficients (ε) of 24,600 and 22,300 respectively in buffer at pH 8.8, and exhibit satisfactory scarlet-red colourations under these conditions.
Conveniently the auxochromic group X e.g. -OH, may provide the linkage between the compound X-A-Y-NO2 and the substrate portion of the reagent, such that the compound is released on interaction with the enzyme. Thus examples of reagents according to the invention are given below by formulae A to E, referring, for the sake of simplicity, only to reagents comprising a simple 4-hydroxy- ω-nitrostyryl substituent. It will be appreciated, however, that this latter substituent may be replaced by a substituent relating to the compounds X-A-Y-NO2 in general.
being reagents suitable for assay of (A) esterases or upases, (B) acid or alkaline phosphatases, (C) phosphodiesterases, (D) arylsulphatases and (E) glycosidases respectively. RZ in formula E denotes a carbohydrate substituent, such as an α or β glucopyranosyl.galactopyranosyl or manriopyranosyl
In accordance with the invention, it has been found that the preferred nitrostyryl substituted reagents may be prepared from vanillin or similar hydroxy benzaldehyde based materials such as p-hydroxybenzaldehyde or salicylaldehyde by nitromethylation of the corresponding substrate-vanillin or -similar compound adduct. Preferably mildly alkaline conditions are employed during nitromethylation. It is believed that similar reagents may be prepared by correspondingly similar routes. Samples which may be assayed are conveniently in fluid form and include samples comprising physiological fluids, such as serum or urine. Other suitable samples may also be assayed including samples derived from cell homogenates, such as those samples commonly used in the study of genetic disorders. The reagent and sample are incubated together; for instance, sample and solution containing the reagent are mixed and allowed to interact under appropriate conditions for a suitable period of time. Preferably the temperature used during incubation is greater than about 30ºC e.g. from about 30 up to about 40ºC, and under these conditions an incubation period of up to about thirty minutes e.g. from about 10 to 15
up to about thirty minutes, is usually satisfactory. As an alternative to the use of reagent in solution, the reagent may be used in association e.g. absorbed, with a suitable solid phase material, such as cellulose particles, e.g. microcrystalline cellulose, which may advantageously overcome problems arising from the insolubility or poor solubility of the reagent. Also the reagent may be used in association with a porous solid support material, such as paper, to provide a convenient test paper or dip stick for a simple dip test. Such products comprising the reagent of the invention in association with a suitable inert solid material or with a porous solid support material are included within the scope of the invention.
On incubation the reagent releases,or more generally gives rise to the formation of, a compound which may without further treatment provide the visual signal e.g. colour, which is indicative of the presence of the enzyme in the sample. Often, however, the compound may require further treatment to develop the visual signal, and such treatment typically comprises activation to develop the inherent visual signal of the compound. This further treatment does not include steps of synthetic or preparative chemistry, such as the diazonium coupling procedures employed with some prior art enzyme assay reagents. Commonly the treatment comprises alkaline treatment, which may lead to salt formation permitting
delocalisation of electronic charge, for instance, alkaline treatment of preferred conjugated unsaturated chromophore substances advantageously giving rise to the development of coloured visual signals. Alkaline treatment may comprise addition of alkali to solution containing the released compound, or preferably solution containing the compound is brought into contact with a solid phase reagent e.g. an absorbent, which simultaneously presents an alkaline environment to the compound. For example, the solution containing the compound is contacted with an anionic exchange material, which is preferably of neutral colour e.g. white or light-coloured, and which is in its free base form e.g. hydroxyl (OH ) form. For example, the anionic exchange material may comprise DEAE cellulose, or a similar light coloured anion exchange material in free base form. The use of an absorbent anionic exchange material may be particularly advantageous, absorbing and concentrating the released substance into a narrow band for enhanced ease of monitoring. The use of DEAE cellulose and similar materials, in this latter respect is believed to be novel, and this use, in general, of these materials is included within the scope of the invention.
In a preferred embodiment, therefore, the method of the invention comprises incubating sample comprising the enzyme with reagent associated with solid phase material e.g. an
inert absorbent or a porous support material, so as to give rise to the formation of a compound X-A-Y-NO2, and contacting solution containing the compound with a solid phase reagent which presents an alkaline environment to the substance and thereby produces a colour change or other visual signal which is easily discernible by eye.
There is also provided apparatus for carrying out the method of the preferred embodiment usually in the form of a through-flow device normally comprising two compartments, an incubation compartment containing reagent associated with solid phase material, e.g. microcrystalline cellulose, and a visualisation compartment containing a solid phase reagent which presents an alkaline environment to the substance e.g. DEAE cellulose or a similar substance. The compartments are typically interconnectable e.g. by a suitable connecting passageway, to permit flow of liquid containing the released compound from the incubation to the visualisation compartment. The two compartments are preferably, however, capable of isolation from one another, for instance to allow for retention of the sample in the incubation compartment for the required incubation period. The passageway between the two compartments may be provided with an appropriate tap or other barrier which may be conveniently opened or removed after the required incubation period to permit the liquid to pass into the visualisation compartment, e.g. to pass through the preferred DEAE cellulose absorbent.
For example, the through-flow device may consist of two compartments attached at interfacial surfaces by a pivot, both surfaces provided with outlets at a fixed radius from the pivot such that mutual rotation of the compartments brings the outlets into coincidence with one another providing a passageway between the compartments. Alternatively the passageway between the compartments may be blocked with a flimsy barrier which can be ruptured as desired, for instance by bringing the two compartments together e.g. by a screwthreaded action.
The concentration of reagent used e.g. present in association with inert solid phase material, may be varied as desired, for instance, in accordance with the threshold concentration of enzyme which it is desired to monitor; increases in concentration of reagent generally permitting detection of lower threshold concentrations of enzyme. Furthermore, use of a solid phase reagent, such as DEAE cellulose, to visualise the released substance advantageously permits detection of very small quantities of released substance, owing to concentration of the colour produced in a narrow absorbed band. In this respect, it has been found possible to detect the presence of the substance 2-methoxy-4-(β-nitroethenyl) phenol at concentrations of 1-2 × 10-6 molar by contact of the solution with DEAE cellulose. Also it has been found, for example, that use of 3% of the appropriate NAG detecting glycoside (E)
on an inert cellulose solid phase in combination with DEAE cellulose visualisation is capable of detecting a threshold concentration of NAG in urine from about 40 - 50 n mol/ml after incubation at 37 for twenty minutes; whereas, use of cellulose containing 8% of the glycoside in the same system gives a thresold of detection of about 20 - 30 n mol/ml. Both systems give broad intense bands for abnormal urines which are easily distinguished from those given by normal urines. It will be appreciated, therefore, having regard to the effect of substrate concentration and the ease of visualisation, that the method of the invention may be conveniently performed by patients themselves in their homes using a suitably prepared through-flow device and requires minimal analytical skills. For example, the patient may simply add a fixed quantity of fresh urine to the incubation compartment e.g. up to a fixed level indicated on the side of the compartment, and after a standard incubation period, which will usually depend upon the ambient temperature, connect the incubation and visualisation compartments permitting incubated urine to flow through the solid phase visualising reagent.
The visual signal, e.g. colour change, produced by the released substance advantageously provides a simple and quick means of detecting the presence of the enzyme in the sample as compared with the methods previously used which
rely upon sophisticated monitoring apparatus. Also the intensity of the visual signal e.g. colour, produced may conveniently be used as a rough visual indicator of the concentration of enzyme present in the sample. If more accurate measurements are required, however, the reagents of the invention may be monitored spectrophotometrically, for instance in the conventional manner. For example, the colour produced may be estimated using a colourimeter, usually after adjustment of pH to 8.5 - 9.5 with an appropriate buffer, and, in such methods, the present reagents may be more suitable than corresponding prior art reagents, such as p-nitrophenolic and o- nitrophenolic substrates.
The invention is further described by way of illustration only in the following examples and description which relate to the preparation of enzyme assay reagents according to the invention and components thereof and to their use in the method of the invention, the description also referring to the accompanying diagrams in which:-
Figure 1 represents one form of flow-through device according to the invention; and
Figure 2 represents an alternative form of flow-through device according to the invention.
Example I Preparation of nitrostyryl glycosides for detection and determination of corresponding .glycosidases I Preparation of hydroxy-b-anzaLdehyde glycosides
As a first stage in the preparation of nitrostyryl glycoside enzyme substrates according to the invention the appropriate hydroxy-benzaldehyde glycosides are prepared.
A solution of the appropriate glycopyranosyl halide (70 mmole) in acetone (200 ml) was treated with a solution of the phenol (100 mmole) in M-sodium hydroxide, the .resulting mixture being maintained at room temperature with stirring for a period of about 16 hours. The reaction mixture was then diluted with water until the product separated. Crystalline glycoside products were filtered off directly, and non-crystalline products were isolated by chloroform extraction in the usual manner.
After crystalline products have been filtered off, further product may be recovered from the mother liquors by extraction with chloroform or dichloromethane.
The results obtained during the preparation of a range of hydroxy-benzaldehyde glycosides are given below, (a) 4-(2-acetamido-3,4,6,tri-O-acetyl-2-deoxy-α-D-gluco- pyranosyloxy)-3-methoxybenzaldehyde was obtained in 55% yield from the 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-glucopyranosyl chloride and had m.p. 220-221 (ethanol), [α]D - 13.5°(c 1, DMSO).
(b) 4-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyloxy) benzaldehyde was obtained similarly in 47% yield, [α]D -19° (c 1, CHCl3) .
(c) 4-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glycopyranosyloxy)-3,5-dimethoxybenzaldehyde was obtained similarly in 15% yield m.p. 239 - 240° (methanol), [α]D + 3.8° (c12 DMSO)
(d) 4-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyloxy)-3-methoxybenzaldehyde was obtained from 2,3 4,6-tetra-O-acetylα-D-glucopyranosyl bromide in 62% yield, m.p. 135 - 136 (ethanol), [α]D -24.2° (c 1.1, CHCl3) .
(e) 4-0(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyloxy)-3-methoxybenzaldehyde was obtained from 2,3,4,6-tetra-O-acetylα-D-galactopyranosyl bromide initially as a syrup (47%) which crystallised and was recrystallised with difficulty from ethanol-ether-light petroleum, m.p. 148- 149 , [α]D - 4. 4º (c 1.1, CHCl3).
(f) 4-(2,3-4,6-tetra-O-acetyl-α-D-mannopyranosyloxy)-3-methoxybenzaldehyde was obtained as a syrup in 14% yield from 2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl bromide (15), [α]D + 15-9° (c 1.4, CHCl3).
II O-Deacetylation of hydroxy-benzaldehyde glycosides
In the next stage of the preparation of the nitrostyryl enzyme substrates certain of the hydroxy-benzaldehyde glycosides are O-deactylated by treatment with methanolic
sodium methoxide. Solutions of the acetylated glycosides
(15 m mole) in methanol (50 - 500 ml) were treated with M-methanolic sodium methoxide (0.5 - 2 ml) and the solutions allowed to stand at room temperature for 20 - 30 minutes, or until reaction had been completed as indicated by t.l.c. determination. The solutions were then passed through pads of silica gel to remove sodium ions, and then evaporated to dryness.
The results obtained for various glycosides are given below. (a) 4-(2-acetamido-2-deoxy-β-D-glucopyranosyloxy)-3-methoxy-benzaldehyde crystallised directly from the reaction mixture in 90% yield and had m.p. 199 - 200° (dec), [α ]D - 20°
(c 1, DMSO)
(b) 4-(β-D-glucopyranosyl)-3-methoxy-benzaldehyde was obtained in 85% yield as a white powder, m.p. 188 - 190 , [α]D + 2.2 (c 0.7, DMSO)
(c) 4-(α-D-galactopyranosyloxy)-3-methoxybenzaldehyde was obtained as a white powder in 92% yield, m.p. 204 - 206º, [α]D + 4.8° (c 1.1, DMSO) (d) 4-(α-D-mannopyranosyl)-3-methoxy-benzaldehyde was obtained crystalline in 64% yield, m.p. 196 - 197 (ethanol), [α]D
+ 119.6° (c 1, DMS0)
III Nitromethylation of hydroxy-benzaldehyde glycosides
Both acetylated and deacetylated hydroxy-benzaldehyde glycosides, as prepared above, were next nitromethylated to the corresponding nitrostyryl glycosides.
Nitromethane (16 ml), ammonium acetate (8 g) and acetic acid (4 ml) were added to a stirred solution or suspension of the appropriate glycoside (20 m mole) in ethanol (150 - 200 ml), and the mixture stirred at room temperature for a period of from about 20 up to about 250 hours, usually about 20 hours. In most cases the product separated from the reaction mixture and was collected from the reaction mixture, though in some cases the product was precipitated by addition of water followed by either filtration or extraction by chloroform or other suitable solvent.
Alternatively the reaction mixture described above was refluxed for 15 - 45 minutes, affording similar yields of products though often of improved purity.
The results obtained on preparation of various nitrostyryl glycosides are given below, indicating the method used, either method (A), the room temperature method or method (B), the reflux method. (a) 4-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyloxy)-3-methoxy-ω-nitrostyrene was prepared in 88% by method (A) and 76% by method (B), as a yellow crystalline solid, m.p. 239 - 240°, [α ]D -6.9° (c 1.15, DMSO). (b) 4-(2-acetamido-2-deoxy-β-D-glucopyranosyloxy)-3-methoxy-ω-nitrostyrene was prepared by method (B) in 87% yield as a pale yellow solid, m.p. 200 - 201.5 (dec), [α]D - 1.4° (c 0.9, DMSO)
(c ) 4-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D glucopyranosyloxy)-ω-nitrostyrene was prepared by method (B) as a very pale yellow solid, m.p. 258-259º [α]D-14.3 (c 1.1, CHCl3). (d) 4-(2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyloxy)-3-methoxy-ω-nitrostyrene was prepared by method (A) as a creamy yellow solid, in 96% yield, m.p. 192-194°[α]D-12.4° (c 1.2, CHCl3).
(e) 4-(2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyloxy)-3-roethoxy-ω-nitrostyrene was prepared by method (a) in 25% yield after extraction from the reaction mixture with dichloromethane and chromatography on a silica gel column using ether-light petroleum as eluent. It had m.p. 148-149º[α]D-4.4 (c 1.1, CHCl3). (f) 4-β-D-galactopyranosyloxy-3-methoxy-ω-nitrostyrene was obtained by method (A) as pale yellow crystals in 69% yield, m.p. 212-214°, [α]D + 1-3° (c 1-5, DMSO).
(g) 4-(2,3,4,5-Tetra-O-acetyl-α-D-mannopyranosyloxy)-3-methoxy-O-nitrostyrene was obtained by method (A) as a pale yellow amorphous solid, m.p. 131-132°, [α]D -15.7° (£0.8, CHCl3). IV. O-deacectylation of nitrostyryl glycosides
Finally O-acetylated nitrostyryl glycosides, as prepared above, are deacetylated.
A stirred solution or suspension of the appropriate 0-acetylated nitrostyryl glycoside (2mmole) in methanol (150 ml) was treated with M-methanolic sodium methoxide (2.5 ml). The
mixture was stirred at room temperature 10-20 min, and then passed through a wad of silica gel to remove sodium ions. The silica gel was washed well with methanol and the combined filtrate and washings evaporated to dryness. The resulting solid was then filtered off with the aid of a little ethanol. The results obtained for the various nitrostyryl glycosides prepared are given below.
(a) 4-(2-acetamido-2-deoxy-β-D-glucopyranosyloxy)-3-methoxy-ω-nitrostyrene was obtained as a pale yellow amorphous powder in quantitative yield, which could not be recrystallised satisfactorily without causing some decomposition. It had m.p. 198-199° (dec), [α ]D -7-8 (c 1.4, DMSO).
(b) 4-β-D-glucopyranosyloxy-3-methoxy-ω-nitrostyrene was prepared in 77% yield as yellow crystals, m.p. 140-141º, [α]D -8.7° (c 1, DMSO).
(c) 4-α-D-mannopyranosyloxy-3-methoxy-ω-nitrostyrene
(23) was obtained as an amorphous hydroscopic orange solid, [α]D + 80.1° (c 0.8, DMSO).
The glycosides prepared above are suitable for use in the detection and determination of the corresponding glycosidases. Reagents comprising these glycosides are included within the invention and may be used in the method of the invention. Typically the glycosides release the corresponding hydroxy-nitrostyryl compound, on interaction with the
appropriate enzyme, which provides the easily discernible visual signal, usually a red colouration.
An inert solid phase material may be impregnated with the glycosides prepared above or other enzyme substrates according to the invention. For example, the requisite amount of microcrystalline cellulose (Avicell) is added to the eluate from the silica gel, as prepared above, evaporated to dryness in a rotary evaporator and the resulting solid ground to a fine powder with a mortar and pestle. Example 2. Preparation of Ester Derivations of 4-hydroxy-ω-nitrostyrenes for Detection and Determination of corresponding
Esterases.
A range of ester derivatives of the 4-hydroxy-ω-nitrostyrenes was prepared for use in reagents according to the invention for detection and determination of corresponding esterases.
( l ) 4-Acetoxy-3-methoxy-ω-nitrostyrene
Vannillyl acetate (19-4g, 100 mmole) was reacted with nitromethane, ammonium acetate and acetic acid, as in method A part III of Example 1, for a period of 2-3 days. The red coloured reaction mixture was then diluted with water and the resultant oil layer extracted with chloroform. The chloroform extract was dried (MgSO4) and evaporated to dryness, and the resulting solid recrystallised from chloroform-ethanol to give the acetate, m.p. 158-162.2º, (20g, 17%).
Alternatively the 3-methoxy-ω-nitrostyryl acetate was prepared from the corresponding hydroxy-nitrostynene. 4-hydroxy-3-methoxy-ω-nitrostyrene (15g, 0.77 mmole) was added to acetic anhydride (15 ml) and the mixture heated under reflux for 1½h. The mixture was then poured into ice-water and the precipitated solid filtered off and recrystallised from chloroform-ethanol to give the acetate (1.7g, 93%), m.p. 163-165º identical to that prepared above.
The above ω nitrostyryl acetate is suitable for use in reagents and the method of the invention for detection and determination of acetate esterases, and on interaction with such enzymes gives rise to the release of 4-hydroxy-3-methoxy-ω-nitrostyrene which exhibits a strong red colouration under alkaline conditions.
(2) 4-Propyloxy-3-methoxy-ω-nitrostyrene 4-hydroxy-3-methoxy-ω-nitrostyrene (3.9g. 20 mmole) was dissolved in dry pyridine (15 ml) and propionyl chloride (2.8g, c 30 mmole) added. The reaction mixture became warm and a dark red precipitate was formed. After ca 5 min. the mixture was poured into ice-water and the resulting yellow precipitate filtered off, washed well with ethanol and recrystallised from chloroform-ethanol to give the propionate (4.8g, 95%), m.p. 101-104.5º. The propionate is suitable for use in the detection of propionate esterases and similarly give rise to the formation of the 4-hydroxy-3-methoxy-ω-nitrostyrene on interaction with the enzyme.
(3) 4-(β-nitroethenyl)-3-methoxyphenyl palmitate 4-hydroxy-3-methoxy-ω-nitrostyrene (5.2g, 25 mmole) was dissolved in pyridine (20 ml) and palmitoyl chloride (8g, 29 mmole) added. A yellow solid was formed immediately and the reaction mixture was then heated at reflux for c 5 min; the reaction mixture was poured into ice-water and the resulting yellow solid filtered off. The product was stirred with dilute (10 molar) sodium hydroxide to remove free phenol, filtered off, washed well with water and ethanol, and then recrystallised from chloroform-ethanol, m.p. 96-99º (9.1 g, 84%). This product is suitable for use in the detection and determination of palmitate esterases, releasing 4-hydroxy-3-methoxy-nitrostyrene on interaction with the enzyme.
(4) 4-(β-nitroethenyl)-phenyl palmitate Palmitoyl chloride (3«6g) was added to dry pyridine (20 ml); there was an immediate precipitation. To this mixture was added 4-hydroxy-ω-nitrostyrene (1.65 g, 10 mmole) and the mixture heated at reflux for 1-2 min. The mixture was then poured into ice-water and the yellow amorphous precipitate filtered off. A solution of the precipitate in a mixture of chloroform and DMSO afforded seed crystals of the ester and the bulk was crystallised by dissolving in boiling methylene chloride and adding ethanol. When all the methylene chloride had evaporated, the ethanolic solution was seeded and gave the palmitate as pale fawn crystals, m.p. 83-84 (2 g, 50%).
Similarly this product is suitable for detection and determination of palmitate esterases, though releases the 4-hydroxy-ω-nitrostyrene on interaction with the enzyme.
(5) 4-(β-nitroetheny)-3-methoxyphenyl phosphate (as monosodium salt mono hydrate)
To vigorously stirred ice-cooled solution of phosphoryl chloride (6.2 g, 40 mmole) in pyridine (4θ ml) was added dropwise over 45 min a solution of 4-hydroxy-3-methoxy-ω-nitrostyrene (7.8 g, 40 mmole) in pyridine (20 ml). The reaction mixture was then decomposed by the dropwise addition of aqueous pyridine (50 ml, containing 10% water) during which some solid separated. Dropwise addition of 2M-sodium hydroxide (20 ml, 40 mmole) initially resulted in a clear solution, followed the separation of an amorphous mono sodium salt, as the hydrate (7.1 g, 59%), m.p. > 250º. The salt had a low solubility in water, and is suitable for detection and determination of phosphatases.
(6) 4-(β-nitroethenyl)-3-methoxyphenyl phosphate (as its disodium salt). The above reaction (5) is repeated except that the decomposed reaction mixture was poured into a concentrated solution of caustic soda (6g) in water. The product initially separated out as an oil which solidified to an amorphous solid (11g, 86%) m.p. > 250º . It was more soluble in water than the
monosodium salt and dissolved easily in boiling water, with a little decomposition, and reprecipitated on cooling to give a pale yellow solid. A 2mιmolar solution of the disodium salt could be made readily. Example 3
A range of hydroxy-ω-nitrostyrenes was also prepared to determine and compare the colour properties of these compounds being examples of compounds falling within the general formula X-A-Y-NO2 i.e. compounds whioh may be formed as a result of interaction of the reagent of the invention with the corresponding enzyme.
The general preparative procedure used was as follows. The corresponding hydroxybenzaldehyde (4θ mmole) was dissolved in ethanol (200 ml) and ammonium acetate (12 g), nitromethane (32 ml) and acetic acid was added. The reaction mixture was either stirred overnight at room temperature or heated under reflux for 15-60 minutes. In some cases the W-nitrostyrene crystallised from the cooled reaction mixture, or crystallised when water was added to the cooled reaction mixture. In cases where no crystalline product could be isolated by these methods, the product was extracted with ether, which was washed well with water, dried (MgSO4) and taken to dryness. The yields, melting points and spectral properties of the various hydroxy-ω-nitrostyrene products were determined by conventional methods and are given below, the spectral properties being.
determined in terms of the absorbance (λmax) in ethanol and TRIS buffer of pH 8.8 and the extinction coefficient (ε) at pH 8.8.
(1) 4-hydroxy-3-methoxy-ω-nitrostyrene had m.p. 164-166ºC (ethanol), was obtained in 75% yield, and hadλmax (ethanol) of 380 and λmax (TRIS 8.8) of 506 with ε 20,900.
(2) 4-hydroxy-ω-nitrostyrene had m.p. 185-186º (aqueous ethanol), was obtained in 52% yeild, and had λ max (ethanol) of 358 and λmax (TRIS 8.8) of 460 with ε of 24,627. (3) 4-hydroxy-3,5-dimethoxy-ω-nitrostyrene had m.p.
184-186 (ethanol), was obtained in 72% yield, and had λmax (ethanol) of 385 and λmax (TRIS 8.8) of 530 with ε of 22,330.
(4) 4-hydroxy-3-nitro-ω-nitrostyren*eϊhad m.p. 132-133, was obtained in 57% yield, and hadλmax (ethanol) of 320 and λmax (TRIS 8.8) of 405 with ε of 7,400.
(5) 4-hydroxy-3-methoxy-2-nitro-ω-nitrostyrene had m.p. 188-189 (aqueous acetic acid) was obtained in 42% yield, and had λmax (ethanol) of 400 and λ max (TRIS 8.8) of 436 with ε of 14,000. (6) 4-hydroxy-3-methoxy-5-nitro-ω-nitrostyrsne had m.p. 170-171 (aqueous acetic acid), was obtained in 67% yield and hadλmax (ethanol) of 334 and λ max (TRIS 8.8) of 415 with ε of 5,200.
(7) 2-hydroxy-3-methoxy-ω-nitrostyrene had m.p. 138-139 (aqueous ethanol), was isolated in 38% yield and had λ max
(ethanol) of 322 and λ max. (TRIS 8.8) of 492 with extinction coefficient ε of 7,38θ.
2-methoxy-4-( α-methyl-β-nitroethenyl)-phenol was also prepared by a variation of the above procedure in which vanillin was treated with nitroethane instead of nitromethane. The conditions employed for the reaction, however, were essentially the same as those described above for nitromethylation. The α-methyl-β-nitroethenyl phenol product was obtained in 80% yield with m.p. 110-112 and had λ max (ethanol) of 368 and λ max (TRIS 8.8) of 475 with e of 13,100.
The hydroxy ω-nitrostyrenes, as prepared above, are typically coloured compounds which may provide the easily discernible visual signal of the reagents of the invention. Particularly preferred compounds are the 3-methoxy and 3.5-dimethoxy compounds, 1 and 3. which provide distinctive, deep red colourations under alkaline conditions. Generally, the ω-nitrostyrene compounds exhibit markedly improved colour properties over the pale orange-yellow colouration of paranitrophenol ( λ max ethanol 320, λ. max TRIS 8.8 400 with ε of 15,000), which it is believed is due to the formation radical ions with the nitrostyryl compounds. The existence of such radical ions is supported by the e.s.r. spectra of the nitrostyryl compounds. For example, the 3.5-dimethoxy compound (3), in solution in DMSO to which one drop of M sodium methoxide has been added, gave a strong e.s.r. spectrum comprising 7 signals for the six methoxy protons in the ratio
1:6:15:20:15:6:1 with a calculated splitting of 0.680 gauss, and 3 signals for the two aromatic protons in the ratio 1:2:1 with a calculated splitting of 1.74 gauss, no signals being observed for the ethyl protons. Example 4 Assay for N-Acetylglucosaminidase (NAG) in urine
100 mg of microcrystalline cellulose containing about 3% of the 4-(2-acetamido-2-deoxy-β-D-glucopyranosyloxy)-3-methoxy-ω-nitrostyrene, the substrate for NAG, as prepared in Example 1, is treated in a small plastic sample tube with fresh urine (l ml) at body temperature, or stored urine pre-heated to
35-38ºC. The contents of the tube are incubated for 15-30 minutes at 30-35ºC e.g. 20 minutes at 37ºC, and then allowed to percolate through a short column (about 3 × 0.6 cm) of fibrous DEAE cellulose (Whatman DE22) contained in a Pasteur pipette blocked at the bottom with cotton wool. The presence of NAG in the urine sample is indicated by the development of a crimson red band about 0.5 cm. below the top of the DEAE cellulose column. Sometimes a broad light brown band forms above the crimson red band due to urochrome present in the urine, and in a few patients who have had kidney transplants a fast moving brown-mauve band is observed probably due to the medication the patient is receiving. Further, for diabetics or infants, who have low electrolyte concentrations in their urine, the red band forms at the top surface interface of the DEAE cellulose and is more dispersed in nature. This
latter effect can be overcome by incorporation of appropriate quantities of electrolytes into the reagent. In all cases, however, the width and intensity of the red band is approximately proportional to the concentration of NAG in the urine.
Using 3% glycoside on cellulose, normal urine (about 40-75 n mol/ml of NAG) gave a narrow faint compact red band, but abnormal urines ( > 200 n mol/ml NAG) gave intense broader bands which intensified with increasing NAG concentrations. Under the same conditions of testing, water and urine which has been boiled to destroy NAG, gave negative results.
In a modification of the basic assay, 8% glycoside on cellulose is used in place of the 3% glycoside material, and estimated to have a NAG detection threshold of from about 20-30 n mol/ml. In the modified procedure normal urine gives more intense bands than are observed above indicating the way in which it is possible to choose a threshold of detection by variation of the quantity of glycoside substrate used, and also paying attention to the temperature and time of incubation. Description of a through-flow device according to the invention. With reference to Figures 1 and 2 the ω-nitrostyrylglycosides, as prepared in Example 1, may be incorporated into through-flow devices according to the invention. Both devices comprise cylindrical upper incubation 1 and lower visualisation 2 compartments, and in both cases the lower visualisation
compartment 2 contains a quantity of fibrous DEAE cellulose (Whatman DE 22) 3 sandwiched between upper 4 and lower 5 layers of cotton wool or a similar substance. The lower ends of the visualisation compartments 2 have outlets 6 for draining of fluid and are provided with a skirt or legs 7 to allow free standing of the devices in a vertical orientation. The walls of the lower visualisation compartments 2 are of clear material, such as clear plastic, to permit observation of the DEAE cellulose and any coloured band absorbed thereon. The upper incubation compartments 1 contain an appropriate quantity of the glycoside substance (not shown) either in a suitable solvent or absorbed into cellulose or other suitable absorbent e.g. 100 mg of microcrystalline cellulose (Avicell) containing 3% glycoside. The incubation compartments 1 are fitted with screw caps and have thick PVC or polystyrene walls 9 to insulate the urine during incubation, the walls 9 being marked with a line 10 to indicate the quantity of urine which is to be introduced for incubation.
In diagram, Figures 1 and 2, the upper and lower compartments 1 and 2 are shown without a connecting passageway between them.
With reference to Figure 1, the compartments 1 and 2 are held together at interfacial surfaces by pivot pin 11. The base of the incubation compartment 1 has an eccentrically located outlet 12 and the top of the visualisation compartment
has an inlet 13 eccentrically located at the same radius from the pivot 11 as outlet 12. Mutual rotation of the upper 1 and lower 2 compartments brings the outlet 12 and inlet 13 into coincidence with one another providing a passageway connecting the compartments through which incubated urine can flow. In use, fresh urine is introduced into compartment 1 up to the level of the mark 10, outlet 12 and inlet 13 being out of coincidence with one another. The screw cap 8 is screwed in place and the urine is incubated for a period which depends upon the temperature though is usually about 15 to 20 minutes. The compartments are then mutually rotated bringing outlet 12 and inlet 13 into register with one another and incubated urine drains from the incubation compartment 1 to the visualisation compartment 2. 2-methoxy-4-(2-nitro-(E)-ethenyl) phenol released by the glycoside substrate as a result of NAG present in the urine gives rise to the formation of a bright scarlet red band about .5cm below the top of the DEAE cellulose column 3.
With reference to Figure 2, compartments 1 and 2 are held together in screw-threaded engagement by screw thread 16 around the bottom of compartment 1 and screw thread 17 around the inside of tubular extension 18 projecting from the top of compartment 2. A passageway between compartments 1 and 2 is provided by tubular extension 14 which projects from the bottom of compartment 1. Prior to use passageway 14 is closed by its
abutment with the weakened section 15 in the top of compartment 2. In use, urine is introduced to compartment 1 and incubated as for the device of Figure 1. On completion of incubation, however, compartments 1 and 2 are screwed together and the tubular extension 14 breaks through the weakened section 15 permitting incubated urine to flow into compartment 2. Visualisation of the phenol released by the substrate is as for the device of Figure 1.
Both devices provide simple means by which "kidney transplant" patients may test themselves in their own homes, or general screening tests may be easily conducted, without access to sophisticated monitoring apparatus. Other enzymes besides NAG may be assayed using similar techniques and devices.
Claims
1. An enzyme assay reagent comprising a substance which is capable of interaction with the enzyme to give rise to the formation of a compound of formula X-A-Y-NO2, wherein A comprises an aromatic nucleus, X comprises an auxochromic group and Y comprises an unsaturated group which is capable of transmitting electron resonance between the aromatic nucleus and the nitro substituent, said compound per se being capable of producing a visual signal which is easily discernible by eye.
2. A reagent according to Claim 1, comprising a substance of formula S-X-A-Y-NO2, wherein S comprises the enzyme substrate portion of the reagent.
3. A reagent according to Claim 1 or 2, comprising an appropriate carboxylate, phosphate, diphosphate, or sulphate ester for assay of corresponding carboxylate esterases and lipases, acid and alkaline phosphatases, phosphodiesterases or sulphatases.
4. A reagent according to Claim 1 or 2, comprising a glycoside for assay of the corresponding glycosidase.
5. A reagent according to Claim 4, comprising an α or β-glucopyranosyl, -glactopyranosyl, or -mannopyranosyl glycoside for assay of the corresponding glycosidase.
6. A reagent according to Claim 5, for assay of N-acetyl- β-D-glucosaminidase comprising the corresponding glucopyranosyl glycoside.
7. A reagent according to any of the preceding claims, in which the group A comprises a benzene nucleus.
8. A reagent according to Claim 7, comprising a substrate for the enzyme which, on interaction with the enzyme, gives rise to the formation of a compound of formula I
R3, R4= H, OMe, NO2,CH3,CH = CR'R" or CO2H,
9. A reagent according to any oftthe preceding claims, in which the auxochromic group X of the substance comprises an amino or a hydroxyl group.
10. A reagent according to any of the preceding claims, which, on interaction with the enzyme releases the compound 4-hydroxy-3-ttiethoxy-ω-nitrostyrene or 4-hydroxy-3,5-dimethoxy-ω-nitrostyrene.
11. A process for the production of an enzyme substrate for use in a reagent according to any of Claims 1 to 10, in which the enzyme substrate is prepared from vanillin or a similar hydroxybenzaldehyde based starting material by nitromethylation, preferably under mildly alkaline conditions, of the corresponding enzyme substrate-vanillin or -similar compound adduct.
12. A method for the assay of an enzyme comprising incubating a sample containing the enzyme with a reagent according to any of Claims 1 to 10, so as to give rise to the production of a compound of formula X-A-Y-NO2, and, if necessary, subjecting the compound to further treatment to develop a visual signal which is easily discernible by eye.
13. A method according to Claim 12, in which a sample and reagent are incubated together at a temperature of at least about 30 for a period of up to about 30 minutes.
14. A method according to Claim 12 or 13, in which the reagent is used in association with a suitable solid phase material.
15. A method according to any of Claims 12 to 14, in which further treatment to develop a visual signal comprises alkaline treatment.
16. A method according to Claim 15, in which the released compound of formula X-A-Y-NO is brought into contact with a solid phase reagent which simultaneously presents an alkaline environment to the compound.
17. A product for use in a method according to Claim 12, comprising a reagent according to any of Claims 1-10 in association with a suitable solid phase material.
18. A product according to Claim 17, in which the solid phase material comprises a porous solid support material.
19. Apparatus for use in carrying out a method according to
Claim 12, in the form of a through-flow device comprising two interconnectable compartments, an incubation compartment containing reagent associated. with solid phase material, and a visualisation compartment containing a solid phase reagent which presents an alkaline environment to the compound of formula X-A-Y-NO2.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE782857145T DE2857145T1 (en) | 1977-11-01 | 1978-10-31 | ENZYME ASSAYS |
JP54500039A JPH0238199B2 (en) | 1977-11-01 | 1978-10-31 |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB45390/77 | 1977-11-01 | ||
GB4539077 | 1977-11-01 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1979000255A1 true WO1979000255A1 (en) | 1979-05-17 |
Family
ID=10437052
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB1978/000033 WO1979000255A1 (en) | 1977-11-01 | 1978-10-31 | Enzyme assays |
Country Status (12)
Country | Link |
---|---|
US (1) | US4318986A (en) |
EP (1) | EP0007372A1 (en) |
JP (1) | JPH0238199B2 (en) |
BE (1) | BE871717A (en) |
CA (2) | CA1190223A (en) |
CH (2) | CH651068A5 (en) |
DE (2) | DE2857145T1 (en) |
DK (1) | DK484278A (en) |
GB (2) | GB2079450B (en) |
IT (1) | IT1109621B (en) |
NL (1) | NL7810846A (en) |
WO (1) | WO1979000255A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1989002473A1 (en) * | 1987-09-10 | 1989-03-23 | King's College London | Substrates for the assay of enzymes |
WO1992018874A1 (en) * | 1991-04-15 | 1992-10-29 | The London Hospital Medical College | Assessment of renal malfunction by esr spectroscopy |
Families Citing this family (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FI864875A0 (en) * | 1986-11-28 | 1986-11-28 | Orion Yhtymae Oy | NYA FARMAKOLOGISKT AKTIVA FOERENINGAR, DESSA INNEHAOLLANDE KOMPOSITIONER SAMT FOERFARANDE OCH MELLANPRODUKTER FOER ANVAENDNING VID FRAMSTAELLNING AV DESSA. |
US5283352A (en) * | 1986-11-28 | 1994-02-01 | Orion-Yhtyma Oy | Pharmacologically active compounds, methods for the preparation thereof and compositions containing the same |
YU213587A (en) * | 1986-11-28 | 1989-06-30 | Orion Yhtymae Oy | Process for obtaining new pharmacologic active cateholic derivatives |
US5489614A (en) * | 1987-11-27 | 1996-02-06 | Orion-Yhtyma Oy | Catechol derivatives, their physiologically acceptable salts, esters and use |
MTP1031B (en) * | 1987-12-24 | 1990-10-04 | Orion Yhtymae Oy | New use of cathecol-o-methyl transferase (comt) inhibitors and their physiologically acceptable salts and esters |
US4962024A (en) * | 1988-08-11 | 1990-10-09 | Becton, Dickinson And Company | Signal enhancement in assay for an enzyme |
US4948561A (en) * | 1989-02-09 | 1990-08-14 | Eastman Kodak Company | Multiple level filter device and kit containing same |
US4968486A (en) * | 1989-07-14 | 1990-11-06 | Eastman Kodak Company | Device for absorbing shock to a container |
US5416003A (en) * | 1993-04-14 | 1995-05-16 | Litmus Concepts, Inc. | Reporter enzyme release technology: methods of assaying for the presence of aspartic proteases and other hydrolytic enzyme activities |
US6512100B1 (en) * | 1997-10-27 | 2003-01-28 | Ibbex, Inc. | Chromogenic substrates of sialidase and methods of making and using the same |
US6667161B1 (en) * | 1997-10-27 | 2003-12-23 | Ibbex, Inc. | Chromogenic substrates of sialidase of bacterial, viral, protozoa, and vertebrate origin and methods of making and using the same |
JP2003522113A (en) * | 1998-10-27 | 2003-07-22 | ユーエービー リサーチ ファンデイション | Chromogenic substrate for sialidase and its production and use |
CN104297181A (en) * | 2014-10-10 | 2015-01-21 | 宁波大学 | Detection reagent of N-acetyl-beta-D-glucosaminidase |
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US3968011A (en) * | 1973-02-16 | 1976-07-06 | Alza Corporation | Test implement and test method for colorimetrically determining whether a female is fertile or pregnant |
FR2358419A1 (en) * | 1976-07-13 | 1978-02-10 | Du Pont | NITROAROMATIC GLYCOSIDES AND THEIR PREPARATION |
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US3410756A (en) * | 1966-05-27 | 1968-11-12 | Army Usa | Naphthyl esters as fluorogenic substrates for enzymes |
US3616251A (en) * | 1968-11-08 | 1971-10-26 | Miles Lab | Test device |
US3875013A (en) * | 1973-02-16 | 1975-04-01 | Alza Corp | Article for detecting the fertile period and method for using same |
US4039388A (en) * | 1976-06-04 | 1977-08-02 | The United States Of America As Represented By The Government | Diagnostic test for Niemann-Pick disease |
US4082781A (en) * | 1976-06-04 | 1978-04-04 | The United States Of America As Represented By The Department Of Health, Education And Welfare | Synthesis of 2-alkanoylamino-4-nitrophenyl phosphorylcholine-hydroxide |
US4223090A (en) * | 1978-07-13 | 1980-09-16 | American Hospital Supply Corporation | Reagents for the enzymatic determination of triglycerides |
-
1978
- 1978-10-26 CA CA000425734A patent/CA1190223A/en not_active Expired
- 1978-10-30 DK DK484278A patent/DK484278A/en not_active Application Discontinuation
- 1978-10-31 WO PCT/GB1978/000033 patent/WO1979000255A1/en unknown
- 1978-10-31 BE BE191496A patent/BE871717A/en not_active IP Right Cessation
- 1978-10-31 CH CH4978/84A patent/CH651068A5/en not_active IP Right Cessation
- 1978-10-31 NL NL7810846A patent/NL7810846A/en not_active Application Discontinuation
- 1978-10-31 GB GB8101547A patent/GB2079450B/en not_active Expired
- 1978-10-31 JP JP54500039A patent/JPH0238199B2/ja not_active Expired - Lifetime
- 1978-10-31 DE DE782857145T patent/DE2857145T1/en active Granted
- 1978-10-31 CH CH6129/79A patent/CH651319A5/en not_active IP Right Cessation
- 1978-10-31 DE DE2858733A patent/DE2858733C2/de not_active Expired - Lifetime
- 1978-10-31 GB GB7842617A patent/GB2008103B/en not_active Expired
- 1978-11-02 IT IT69517/78A patent/IT1109621B/en active
-
1979
- 1979-05-22 EP EP78900219A patent/EP0007372A1/en not_active Withdrawn
- 1979-06-29 US US06/127,242 patent/US4318986A/en not_active Expired - Lifetime
-
1983
- 1983-04-12 CA CA000425735A patent/CA1181331A/en not_active Expired
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US3968011A (en) * | 1973-02-16 | 1976-07-06 | Alza Corporation | Test implement and test method for colorimetrically determining whether a female is fertile or pregnant |
FR2358419A1 (en) * | 1976-07-13 | 1978-02-10 | Du Pont | NITROAROMATIC GLYCOSIDES AND THEIR PREPARATION |
Non-Patent Citations (1)
Title |
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CHEMICAL ABSTRACTS, Vol. 86, No 23, 6th June 1977, (Columbus, Ohio, USA), N. TATSUSHI, "Serum N-acetyl-beta-0-glucosaminidase", see page 185, 1st column, Abstract No 166848h. * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1989002473A1 (en) * | 1987-09-10 | 1989-03-23 | King's College London | Substrates for the assay of enzymes |
US5221606A (en) * | 1987-09-10 | 1993-06-22 | King's College London | Reagent and kit for enzyme assay comprising a substrate consonant with given enzyme to be assayed |
WO1992018874A1 (en) * | 1991-04-15 | 1992-10-29 | The London Hospital Medical College | Assessment of renal malfunction by esr spectroscopy |
Also Published As
Publication number | Publication date |
---|---|
CH651068A5 (en) | 1985-08-30 |
NL7810846A (en) | 1979-05-03 |
BE871717A (en) | 1979-02-15 |
DE2857145C2 (en) | 1988-10-06 |
JPH0238199B2 (en) | 1990-08-29 |
DE2858733C2 (en) | 1990-03-01 |
JPS54500072A (en) | 1979-11-29 |
CH651319A5 (en) | 1985-09-13 |
IT1109621B (en) | 1985-12-23 |
GB2079450B (en) | 1982-09-02 |
GB2079450A (en) | 1982-01-20 |
CA1181331A (en) | 1985-01-22 |
CA1190223A (en) | 1985-07-09 |
DK484278A (en) | 1979-05-02 |
GB2008103B (en) | 1982-08-25 |
IT7869517A0 (en) | 1978-11-02 |
EP0007372A1 (en) | 1980-02-06 |
DE2857145T1 (en) | 1980-12-11 |
GB2008103A (en) | 1979-05-31 |
US4318986A (en) | 1982-03-09 |
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