DE2259512C3 - gamma-glutamyl-4-nitranilid-3carboxylic acid, its salts, process for the preparation of these compounds and their use - Google Patents
gamma-glutamyl-4-nitranilid-3carboxylic acid, its salts, process for the preparation of these compounds and their useInfo
- Publication number
- DE2259512C3 DE2259512C3 DE19722259512 DE2259512A DE2259512C3 DE 2259512 C3 DE2259512 C3 DE 2259512C3 DE 19722259512 DE19722259512 DE 19722259512 DE 2259512 A DE2259512 A DE 2259512A DE 2259512 C3 DE2259512 C3 DE 2259512C3
- Authority
- DE
- Germany
- Prior art keywords
- glutamyl
- acid
- nitranilide
- salts
- determination
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
- 150000001875 compounds Chemical class 0.000 title claims description 8
- 239000002253 acid Substances 0.000 title claims description 5
- 238000002360 preparation method Methods 0.000 title claims description 3
- 150000003839 salts Chemical class 0.000 title claims 5
- 239000011780 sodium chloride Substances 0.000 title claims 4
- 238000000034 method Methods 0.000 title claims 2
- 239000000758 substrate Substances 0.000 claims description 8
- 101700066372 ECM38 Proteins 0.000 claims description 4
- 101700082072 GGT1 Proteins 0.000 claims description 4
- 238000006243 chemical reaction Methods 0.000 claims description 4
- 230000000694 effects Effects 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 102000006640 EC 2.3.2.2 Human genes 0.000 claims description 3
- 229940043257 Glycylglycine Drugs 0.000 claims description 3
- 108010008488 Glycylglycine Proteins 0.000 claims description 3
- 101710038776 acyI Proteins 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- YMAWOPBAYDPSLA-UHFFFAOYSA-N glycylglycine zwitterion Chemical compound [NH3+]CC(=O)NCC([O-])=O YMAWOPBAYDPSLA-UHFFFAOYSA-N 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 238000005259 measurement Methods 0.000 claims description 3
- 239000000047 product Substances 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 claims description 2
- TYMLOMAKGOJONV-UHFFFAOYSA-N 4-Nitroaniline Chemical compound NC1=CC=C([N+]([O-])=O)C=C1 TYMLOMAKGOJONV-UHFFFAOYSA-N 0.000 claims description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 2
- OAKJQQAXSVQMHS-UHFFFAOYSA-N hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 claims 6
- WPYMKLBDIGXBTP-UHFFFAOYSA-N Benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 claims 2
- 150000001412 amines Chemical class 0.000 claims 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 claims 2
- 239000003495 polar organic solvent Substances 0.000 claims 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims 2
- ICDLEMPZXFCQEB-UHFFFAOYSA-N 2-(2,6-dioxooxan-3-yl)isoindole-1,3-dione Chemical compound O=C1C2=CC=CC=C2C(=O)N1C1CCC(=O)OC1=O ICDLEMPZXFCQEB-UHFFFAOYSA-N 0.000 claims 1
- 241000272517 Anseriformes Species 0.000 claims 1
- 239000005711 Benzoic acid Substances 0.000 claims 1
- 229940105847 Calamine Drugs 0.000 claims 1
- 108090000790 Enzymes Proteins 0.000 claims 1
- 102000004190 Enzymes Human genes 0.000 claims 1
- 101700083761 GGT3 Proteins 0.000 claims 1
- 101710028470 GGT4 Proteins 0.000 claims 1
- 150000008065 acid anhydrides Chemical class 0.000 claims 1
- 125000001931 aliphatic group Chemical group 0.000 claims 1
- 125000000217 alkyl group Chemical group 0.000 claims 1
- 125000004429 atoms Chemical group 0.000 claims 1
- 235000010233 benzoic acid Nutrition 0.000 claims 1
- 239000007795 chemical reaction product Substances 0.000 claims 1
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 238000003745 diagnosis Methods 0.000 claims 1
- 239000000446 fuel Substances 0.000 claims 1
- 125000002642 gamma-glutamyl group Chemical group 0.000 claims 1
- 229910052864 hemimorphite Inorganic materials 0.000 claims 1
- 201000009673 liver disease Diseases 0.000 claims 1
- 125000000612 phthaloyl group Chemical group C(C=1C(C(=O)*)=CC=CC1)(=O)* 0.000 claims 1
- 230000002269 spontaneous Effects 0.000 claims 1
- 125000001424 substituent group Chemical group 0.000 claims 1
- 239000004094 surface-active agent Substances 0.000 claims 1
- 239000011787 zinc oxide Substances 0.000 claims 1
- 235000014692 zinc oxide Nutrition 0.000 claims 1
- CPYIZQLXMGRKSW-UHFFFAOYSA-N zinc;iron(3+);oxygen(2-) Chemical compound [O-2].[O-2].[O-2].[O-2].[Fe+3].[Fe+3].[Zn+2] CPYIZQLXMGRKSW-UHFFFAOYSA-N 0.000 claims 1
- 239000000243 solution Substances 0.000 description 8
- KZZWQCKYLNIOBT-UHFFFAOYSA-N 5-Amino-2-nitrobenzoic acid Chemical compound NC1=CC=C([N+]([O-])=O)C(C(O)=O)=C1 KZZWQCKYLNIOBT-UHFFFAOYSA-N 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 4
- LRHPLDYGYMQRHN-UHFFFAOYSA-N n-butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 4
- 210000002966 Serum Anatomy 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 238000002211 ultraviolet spectrum Methods 0.000 description 3
- 229960000583 Acetic Acid Drugs 0.000 description 2
- 229960002989 Glutamic Acid Drugs 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- IMFACGCPASFAPR-UHFFFAOYSA-N Tributylamine Chemical compound CCCCN(CCCC)CCCC IMFACGCPASFAPR-UHFFFAOYSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M buffer Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 230000024881 catalytic activity Effects 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 239000012362 glacial acetic acid Substances 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- IKDUDTNKRLTJSI-UHFFFAOYSA-N hydrazine hydrate Chemical compound O.NN IKDUDTNKRLTJSI-UHFFFAOYSA-N 0.000 description 2
- 230000003287 optical Effects 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- PPBRXRYQALVLMV-UHFFFAOYSA-N styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- QIWKCQDJZPRXNS-VIFPVBQESA-N (2S)-2-[(2-carboxybenzoyl)amino]pentanedioic acid Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)C1=CC=CC=C1C(O)=O QIWKCQDJZPRXNS-VIFPVBQESA-N 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K Aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- PRKQVKDSMLBJBJ-UHFFFAOYSA-N Ammonium carbonate Chemical compound N.N.OC(O)=O PRKQVKDSMLBJBJ-UHFFFAOYSA-N 0.000 description 1
- 102000037197 Anion exchangers Human genes 0.000 description 1
- 108091006437 Anion exchangers Proteins 0.000 description 1
- 210000001124 Body Fluids Anatomy 0.000 description 1
- 208000008313 Contusions Diseases 0.000 description 1
- FEMOMIGRRWSMCU-UHFFFAOYSA-N Ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 238000005377 adsorption chromatography Methods 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 239000001099 ammonium carbonate Substances 0.000 description 1
- 235000012501 ammonium carbonate Nutrition 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L cacl2 Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- STNNHWPJRRODGI-UHFFFAOYSA-N carbonic acid;N,N-diethylethanamine Chemical compound [O-]C([O-])=O.CC[NH+](CC)CC.CC[NH+](CC)CC STNNHWPJRRODGI-UHFFFAOYSA-N 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000002255 enzymatic Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- BDAGIHXWWSANSR-UHFFFAOYSA-M methanoate Chemical group [O-]C=O BDAGIHXWWSANSR-UHFFFAOYSA-M 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000001376 precipitating Effects 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 238000005496 tempering Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- -1 γ-glutamyl- Chemical group 0.000 description 1
Description
Die Geschwindigkeit der Freisetzung des gelbge- Keton als Lösungsmittel.The rate of release of the yellow ketone as a solvent.
färbten p-Nitroanilins kann optisch verfolgt werden Zur Isolierung des Rohproduktes bedient man sichcolored p-nitroaniline can be followed optically. The crude product is isolated
und ist ein Maß für die vorhandene Aktivität an 40 zweckmäßig chromatographischer Methoden, bei-and is a measure of the existing activity in 40 suitable chromatographic methods, both
y-GT. spielsweise der Anionenaustauscherchromatographiey-GT. for example anion exchange chromatography
Für eine routinemäßige Anwendung dieses Ver- oder/und der Adsorptionschromatographie unter Verfahrens
ist die Instabilität und schlechte Löslichkeit wendung von Adsorptionsmitteln ,vie Kieselgel oder
des Substrates y-GlutamyI-4-nitranilid ein schwerer Aluminiumhydroxyd, wie sie sich insbesondere für die
Nachteil. Die Verbindung läßt sich nur schwer in 45 Dünnschichtchromatographie eignen.
Lösung bringen und die Lösungen sind nur kurzzei- Die Löslichkeit (in Wasser bzw. 0,2 M Tris-Puffer
tig (etwa 2 Stunden) haltbar. Es war daher erforderlich, pH 8,25 bei 20 bis 25° C) der erfindungsgemäßen VerAbfüllungen
jeweils nur für relativ wenige innerhalb bindung ohne Lösungsvermittler liegt über 120 mM,
kurzer Zt it durchzuführende Bestimmungen herzustel- d. h., daß sie mindestens 20mal größer als die Löslichlen.
Eine Aufbewahrung der Reagenslösungen war 5° keit des y-Glutamyl-4-nitranilids ist.
nicht möglich. Ein weiterer Nachteil bestand darin, Die folgenden Beispiele erläutern die Herstellung der
daß die SuI stanz bei 50 bis 6O0C in Lösung gebracht erfindungsgemäßen Verbindung, ihre Eigenschaften
werden mußte, bei einem Überschreiten dieser Tempe- und ihre Verwendung als Substrat für die Bestimmung
ratur aber spontan hydiolysierte. Eine derartige der y-Glutamyltranspeptidase (y-GT).
Hydrolyse teeinflußt die optische Extinktion der 55 Bei der enzymatischen Spaltung der erfind ungsge-·
Lösungen, die für die Messung entscheidend ist, in mäßen Verbindung wird 2-Nitro-5-amino-benzoestark
em Maße. Dies ist insbesondere von großem Nach- säure freigesetzt. Deren UV-Spektrum weist folgende
teil bei der Durchführung der y-GT-Bestimmung mit Charakteristika auf:
Analysenautomaten, da infolge der dabei gebildeten χ _ ^gQ nm
hohen Absorption höhere Aktivitäten nicht exakt 60 f""" = 94 (cm^Mol) bei 405 nm
genug gemessen werden können. Infolge dessen wurde = {2 75 (crn*/(xMol) bei 380 nm;
bisher bei suboptimalen Substratkonzentrationen von = 9 83 · 10~s M
nur 3 bis 4 mM gemessen im Gegensatz zur optimalen 'For routine use of this and / or adsorption chromatography under the method, the instability and poor solubility of adsorbents, such as silica gel or the substrate y-glutamyl-4-nitranilide, is a heavy aluminum hydroxide, which is particularly disadvantageous. The compound is difficult to use in thin layer chromatography.
Bring the solution and the solutions are only stable for a short time. The solubility (in water or 0.2 M Tris buffer (approx. 2 hours). It was therefore necessary to have a pH of 8.25 at 20 to 25 ° C) of the bottlings according to the invention only for relatively few in each case within a bond without a solubilizer is more than 120 mM, short time determinations to be carried out, ie that they are at least 20 times greater than the solubles. The reagent solutions had to be kept safe for the γ-glutamyl-4-nitranilide.
not possible. A further disadvantage was The following examples illustrate the preparation of the Sui punched at 50 to 6O 0 C in solution brought compound of the invention had to be their properties, at a temperature-exceeding this temperature, and their use as a substrate for the determination but hydiolysed spontaneously. One of the γ-glutamyl transpeptidase (γ-GT).
Hydrolysis affects the optical extinction of the 2-nitro-5-amino-benzoic acid in moderate connection. In particular, this is released by large amounts of post-acidic acid. Their UV spectrum shows the following parts when carrying out the y-GT determination with characteristics:
Analyzers, because as a result of the χ _ ^ gQ nm
high absorption higher activities not exactly 60 f """ = 94 ( cm ^ mol) at 405 nm
enough can be measured. As a result, = { 2 75 (cm * / (xM ol) at 380 nm;
so far at suboptimal substrate concentrations of = 9 83 · 10 ~ s M
only 3 to 4 mM measured in contrast to the optimal '
Konzentraticn bei etwa 6 mM (Clin. Chim. Acta, 31 Die erfindungsgemäße Verbindung weist nicht nurConcentrate at about 6 mM (Clin. Chim. Acta, 31) The compound according to the invention not only exhibits
[1971], 175 bis 179). 65 eine bessere Löslichkeit als das y-Glutamyl-4-nitrani-[1971], 175 to 179). 65 a better solubility than the y-glutamyl-4-nitrani-
Die aufgeführten Nachteile konnten teilweise be- lid auf, es erfordert auch beim Lösen kein Erwärmen,The listed disadvantages could partly be resolved, it does not require heating even when dissolving,
seitigt werden durch das in der DT-AS 20 42 829 be- so daß höhere Blindextinktionen vermieden, werden,are eliminated by the in the DT-AS 20 42 829 so that higher blind absorbances are avoided,
schriebene Reagens, bei welchem in Gegenwart eines Die Stabilität in Lösung ist gut, bei der Optima listeningwritten reagent, in which in the presence of a The stability in solution is good, with Optima listening
der Testbedingungen zur Bestimmung der y-GT treten keine Probleme auf, was für die Erfordernisse der klinischen Chemie von großer Bedc'itung ist. Die Hersteilung der Verbindung ist einfach und billig, die Empfindlichkeit der Meßanordnung entspricht der bei Verwendung von y-Gluta.;iyl-4-nitran:lid.the test conditions for determining the y-GT occur presents no problems, which is of great concern to the requirements of clinical chemistry. The manufacture the connection is simple and cheap, the sensitivity of the measuring arrangement corresponds to that of Use of y-gluta.; Iyl-4-nitran: lid.
Herstellung von y-Glutamyl-^nitranilidO-carbonsäure (GLUPA-3-carbonsäure)Production of γ-glutamyl- ^ nitranilidO-carboxylic acid (GLUPA-3-carboxylic acid)
9,1 g 2-Nitro-5-amino-benzoesäure (50 mMol) und 14,2 g Phthaloylglutaminsäureanhydrid (55 mMol) werden in 50 ml absolutem Dioxan nach Zusatz von 13 ml Tributylamin gelöst. Das Reaktionsgemisch wird 2 Stunden unter Rückfluß gekocht. Nach dem Abkühlen werden dem Reaktionsgemisch 100 ml 2 N-Ammoniak zugesetzt und das sich abscheidende Tributylamin mit Äther ausgeschüttelt. Die wäßrige Lösung wird zur Trockne eingeengt, in Methanol gelöst und mit 80%igem Hydrazinhydrat auf pH 10 gestellt. Die Reaktionslösung bleibt etwa 20 Stunden bei Raumtemperatur stehen. Teilweise scheidet sich dabei ein Produkt ab, das nach dem Abfiltrieren und nach dem Waschen mit Methanol verworfen wird. Das methanolische Filtrat wird bis zur Trockene konzentriert, und das zurückbleibende Öl in etwa 300 ml destilliertes Wasser gelöst.9.1 g of 2-nitro-5-aminobenzoic acid (50 mmol) and 14.2 g phthaloylglutamic acid anhydride (55 mmol) are dissolved in 50 ml of absolute dioxane after adding 13 ml of tributylamine. The reaction mixture will Boiled under reflux for 2 hours. After cooling, 100 ml of 2N ammonia are added to the reaction mixture added and the precipitating tributylamine shaken out with ether. The aqueous solution is concentrated to dryness, dissolved in methanol and adjusted to pH 10 with 80% hydrazine hydrate. The reaction solution remains at room temperature for about 20 hours. Partly there is a divorce a product which, after filtering off and washing with methanol, is discarded. The The methanolic filtrate is concentrated to dryness and the remaining oil is distilled into about 300 ml Dissolved in water.
Diese wäßrige Lösung wird über eine Säule mit 100 ml eines stark basischen Anionenaustauschers auf Styrolbasis mit einer Korngröße zwischen 0,15 und 0,30 mm in Formiat-Form Chromatographien, mit 300 ml destilliertes Wasser gewaschen und graduierend mit 2 1 Wasser gegen 2 1 0,5 N-Ammoniumcarbonatlösung eluiert. Das Eluat wird in 100-ml-Fraktionen aufgefangen und dünnschichtchromatographisch (Kieselgelplatten) im System Butanol/Eisessig/Wasser im Verhältnis 50:15:15 untersucht (Entwicklung der Chromatogramme: im UV-Licht blaue Flecke oder nach Besprühen mit Ninhydrin und anschließendem Erhitzen, braune Flecke der aminosäurehaltigen Zonen). Die Fraktionen, die das Produkt y-Glutamyl-4-nitranilid-3· carbonsäure enthalten tRf-Wert 0,25) werden gesammelt und im Vakuum bis zur Trockene konzentriert. Das zurückbleibende Pulver wird mit absolutem Alkohol verrührt, abnitriert und im Vakuum über Calciumchlorid getrocknet.This aqueous solution is chromatographed over a column with 100 ml of a strongly basic anion exchanger based on styrene with a grain size between 0.15 and 0.30 mm in formate form, washed with 300 ml of distilled water and graduated with 2 l of water against 2 1 0 , 5 N ammonium carbonate solution eluted. The eluate is collected in 100 ml fractions and examined by thin layer chromatography (silica gel plates) in the system butanol / glacial acetic acid / water in a ratio of 50:15:15 (development of the chromatograms: bruises in UV light or after spraying with ninhydrin and subsequent heating, brown spots of the amino acid-containing zones). The fractions which contain the product γ-glutamyl-4-nitranilide-3 · carboxylic acid (tR f value 0.25) are collected and concentrated to dryness in vacuo. The remaining powder is stirred with absolute alcohol, nitrided off and dried in vacuo over calcium chloride.
Ausbeute: 7 g y-GlutamyM-nitranilid-S-carbonsäure,
Di-Ammoniumsalz (58% der Theorie).
Elektrophoretische Mobilität:
V = konstant; 1120 VoSt, etwa 35 mA,
0,05 M Triäthylammonium-carbonat-Puffer pH 7,5 Laufzeit: 45 min bei Raumtemperatur,
Laufstrecke:Yield: 7 g of γ-glutamym-nitranilide-S-carboxylic acid, di-ammonium salt (58% of theory).
Electrophoretic mobility:
V = constant; 1120 VoSt, about 35 mA,
0.05 M triethylammonium carbonate buffer pH 7.5 running time: 45 min at room temperature,
Running route:
bei Bezug auf die Laufstrecke der Glutaminsäure (= 1,0) ergibt sich ein Verhältnis der Laufstrecken
für y-Glutamyl-4-nitranilid-3-carbonsäure: 0,82,
für 4-Nitranilin-3-carbonsäure: 0,88, jeweils anodisch. With reference to the running distance of glutamic acid (= 1.0), the ratio of the running distances for y-glutamyl-4-nitranilide-3-carboxylic acid is 0.82,
for 4-nitroaniline-3-carboxylic acid: 0.88, each anodic.
i?/-Werte im System Butanol/Eisessig/Wa&ser (Verhältnis 50: 15: 15):i? / - values in the system butanol / glacial acetic acid / water (ratio 50: 15: 15):
y-GiutamyM-nitranilid-S-carbonsäure: 0,25;
i-Nitranilin-S-carbonsäure: 0,6;
Glutaminsäure: 0,18.γ-GiutamyM-nitroanilide-S-carboxylic acid: 0.25;
i-nitroaniline-S-carboxylic acid: 0.6;
Glutamic acid: 0.18.
Der optische Drehwert *D in 10°/oiger Lösung (bidestilliertes
Wasser bei 25c C) in einem 50 mm langen Rohr beträgt 37,6°.
ίο UV-Spektrum:The optical rotation D * 10 ° / cent solution (twice-distilled water at 25 c C) in a 50 mm long pipe is 37, 6 °.
ίο UV spectrum:
Arnax = 317 nm;Arnax = 317 nm;
ε = 11,7 (Cm2VMoI) bei 317 nm. ε = 11.7 (Cm 2 VMoI) at 317 nm.
Das UV-Spektrum ist in der Zeichnung wiedergegeben, zusammen mit dem Spektrum der bei der enzymatischen
Spaltung freigesetzten 2-Nitro-5-aminobenzoesäure in 0,2 M 2-Amino-2-(hydroxymethyi)-l,3-propandiol
(Tris-Puffer) und 45 mM Glycylglycin pH 8,25.
10 The UV spectrum is shown in the drawing, together with the spectrum of the 2-nitro-5-aminobenzoic acid released in the enzymatic cleavage in 0.2 M 2-amino-2- (hydroxymethyi) -l, 3-propanediol (Tris- Buffer) and 45 mM glycylglycine pH 8.25.
10
Verwendung der GLUPA-3-carbonsäure zur
Bestimmung der y-GTUse of GLUPA-3-carboxylic acid for
Determination of the y-GT
A. Durchführung der MessungA. Performing the measurement
In eine Küvette pipettieren:
0,1 M TRIS-Puffer, 4OmM Glycylglycin,
pH = 8,25 2,8 mlPipette into a cuvette:
0.1 M TRIS buffer, 40 mM glycylglycine, pH = 8.25 2.8 ml
130 mM GLUPA-3-carbonsäure in130 mM GLUPA-3-carboxylic acid in
o. g. Puffer 0,2 mlo. g. Buffer 0.2 ml
Temperieren auf 25°C, Bestimmung
starten durch Zugabe von
Humanserum 0,2 mlTempering to 25 ° C, determination
start by adding
Human serum 0.2 ml
mischen, Extinktion bei 405 nm ablesen, gleichzeitig Stoppuhr starten. Nach genau 1,2 und 3 min Ablesung wiederholen.Mix, read the absorbance at 405 nm, start the stopwatch at the same time. After exactly 1, 2 and 3 minutes of reading repeat.
Aus den Extinktionsdifferenzen pro min (A £/min) Mittelwert bilden und diesen in die Berechnung einsetzen. Calculate the mean value from the absorbance differences per min (A £ / min) and use this in the calculation.
B. BerechnungB. Calculation
Eine internationale Einheit (U) ist die Enzymaktivi-An international unit (U) is the enzyme activity
!ät, die bei 25°C 1 μΜοΙ Substrat in 1 min umsetzt. Be-! ät that converts 1 μΜοΙ substrate in 1 min at 25 ° C. Loading
zogen wird auf 1 ml Körperflüssigkeit, z. B. Serum. Für die Berechnung der Enzymaktivitäten pro ml (A) gilt allgemein die Formelis drawn to 1 ml of body fluid, e.g. B. Serum. The formula generally applies to the calculation of the enzyme activities per ml (A)
_/· 1000
min ■ e ■ el ■ ν _ / · 1000
min ■ e ■ el ■ ν
Δ £(mU/ml). Δ £ (mU / ml).
Der Extinktionskoeffizient (e) von 4-Nitranilin-3-carbonsäure beträgt unter den Testbedingungen bei 405 nm 9,4 cm2/VMol. Die Schichtdicke (</) der Küvette ist 1 cm, ν ist das Volumen des eingesetzten Serums(0,2 ml), Kist dieSummederVoUimina(3,2mi). Die Messung der Extinktion E erfolgt in Abständen von 1 min. Daraus ergibt sichThe extinction coefficient (e) of 4-nitroaniline-3-carboxylic acid is 9.4 cm 2 / VMol under the test conditions at 405 nm. The layer thickness (</) of the cuvette is 1 cm, ν is the volume of the serum used (0.2 ml), K is the sum of the VoUimina (3.2 mi). The measurement of the extinction E takes place at intervals of 1 min
A=JE (405 nm)/min ■ 1702 (mU/ml). A = JE (405 nm) / min ■ 1702 (mU / ml).
Hierzu 1 Blatt Zeichnungen1 sheet of drawings
Claims (3)
aminsäureanhydrid und anschließend das Reak- io Die erfindungsgemaße Verbindung beseitigt diese tionsprodukt mit Hydrazin umsetzt und y-Glut- Nachteile und weist einerseits ausgezeichnete Löslich- «myl-4-nitranilid-3^rbonsäure, gegebenenfalls in keitseigenschaften auf und bildet gleichzeitig ein gute«= Salzform, in üblicher Weise isoliert. Substrat für das Enzym y-GT. Besonders letzteres Ist 2. Process for the preparation of the compounds case, however, is still a heating to 50 to according to claim 1, characterized ^ indicates that 6O 0 C required with the risk of spontaneous Hyman in each case in a known manner initially drolysis and the inaccuracy caused thereby. g-2-nitro-5-aminobenzoic acid with phthaloyl gluteness of the measurement. ...
amic acid anhydride and then the reac- io The compound according to the invention eliminates this ionization product, reacts with hydrazine and y-glow disadvantages and, on the one hand, has excellent solubility «myl-4-nitranilid-3 ^ rboxylic acid, possibly in keitse features and at the same time forms a good« = Salt form, isolated in the usual way. Substrate for the enzyme y-GT. Especially the latter is
Priority Applications (28)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19722259512 DE2259512C3 (en) | 1972-12-05 | gamma-glutamyl-4-nitranilid-3carboxylic acid, its salts, process for the preparation of these compounds and their use | |
| DE19732333798 DE2333798C3 (en) | 1973-07-03 | gamma-glutamyl-4-nitranilide-3sulfonic acid, its salts, process for the preparation of these compounds and their use | |
| AT910973A AT331994B (en) | 1972-12-05 | 1973-10-29 | METHOD FOR DETERMINATION OF DELTA-GLUTAMYL TRANSPEPTIDASE |
| IT31264/73A IT1012529B (en) | 1972-12-05 | 1973-11-13 | DERIVATIVES OF THE GLUTAMIL P NITRO ANILIDE RANGE |
| NL7315657.A NL166463C (en) | 1972-12-05 | 1973-11-15 | PROCESS FOR PREPARING A GAMMA-GLUTAMYL-P-NITROANILIDE DERIVATIVE. |
| CA186,729A CA1000721A (en) | 1972-12-05 | 1973-11-26 | DERIVATIVES OF .alpha.-GLUTAMYL-4-NITROANILIDE |
| US05/419,455 US3979447A (en) | 1972-12-05 | 1973-11-27 | γ-Glutamyl-4-nitroanilide compounds |
| DK642273A DK151232C (en) | 1972-12-05 | 1973-11-28 | GAMMA-GLUTAMYL-P-NITROANILINE DERIVATIVES USED AS SUBSTRATE FOR DETERMINATION OF GAMMA-GLUTAMYL TRANSPEPTIDASE |
| FI3670/73A FI59790C (en) | 1972-12-05 | 1973-11-28 | NY-GLUTAMYL-P-NITROANILIDE DERIVATIVES SOM AER ANVAENDBARA SOM SUBSTRAT VID BESTAEMNING AV NY-GLUTAMYLTRANSPEPTIDAS |
| FR7342515A FR2208886B1 (en) | 1972-12-05 | 1973-11-29 | |
| CH1687073A CH605670A5 (en) | 1972-12-05 | 1973-11-30 | |
| AU63115/73A AU485187B2 (en) | 1972-12-05 | 1973-11-30 | Glutamyl-p-nitroanilide-derivative |
| BE138358A BE808035A (en) | 1972-12-05 | 1973-11-30 | GAMMA-GLUTAMYL-P-NITRANILIDE DERIVATIVES |
| GB5599173A GB1397008A (en) | 1972-12-05 | 1973-12-03 | Derivatives ofypsilon-glutamyl-4-nitroanilide |
| IL43735A IL43735A (en) | 1972-12-05 | 1973-12-03 | Derivatives of gamma-glutamyl-4-nitroanilide and process for the preparation thereof |
| SU1974541A SU1253439A3 (en) | 1972-12-05 | 1973-12-04 | Method of determining gamma-glutamyltranspeptidase |
| HUBO1472A HU168735B (en) | 1972-12-05 | 1973-12-04 | |
| YU3132/73A YU39232B (en) | 1972-12-05 | 1973-12-04 | Process for obtaining new gama-glutamyl-p-nitroanilide comounds |
| SE7316353A SE404791B (en) | 1972-12-05 | 1973-12-04 | GAMMA -GLUTAMYL-P-NITROANILIDE DERIVATIVES USED FOR DETERMINATION OF ACTIVITIES OF GAMMA-GLUTAMYL TRANSPPTIDASES |
| AR251367A AR199011A1 (en) | 1972-12-05 | 1973-12-05 | DERIVATIVES OF BETA-GLUTAMIL-P-NITRO-ANILIDE, FOR THE DETERMINATION OF BETA-GLUTAMILTRANSPEPTIDASE |
| DD175142A DD109377A5 (en) | 1972-12-05 | 1973-12-05 | |
| ZA739238A ZA739238B (en) | 1972-12-05 | 1973-12-05 | Gamma-glutamyl-p-nitroanilid-derivate |
| BR956173A BR7309561D0 (en) | 1972-12-05 | 1973-12-05 | PROCESS FOR OBTAINING NEW DERIVATIVES FROM THE GAMMA-GLUTAMIL-4-NITROANILIDE AND PROCESS FOR ANALYSIS OF GAMMA-GLU-TAMIL- TRANSEPTIDASE ACTIVITY WITH THE USE OF THE NEW COMPOUND |
| JP13601673A JPS547781B2 (en) | 1972-12-05 | 1973-12-05 | |
| US05/589,653 US4049702A (en) | 1972-12-05 | 1975-06-23 | γ-Glutamyl-4-nitroanilide compounds |
| US05/601,775 US3986931A (en) | 1972-12-05 | 1975-08-04 | γ-GLUTAMYL-4-NITROANILIDE COMPOUNDS AND THEIR USE IN DETERMINING γ-GLUTAMYL TRANSPEPTIDASE |
| AT827975A AT347431B (en) | 1972-12-05 | 1975-10-30 | PROCESS FOR THE PRODUCTION OF NEW DELTA-GLUTAMYL-P-NITROANILIDES |
| SE7610106A SE420322B (en) | 1972-12-05 | 1976-09-13 | USE OF NEW GAMMA-GLUTAMYL-4-NITROANILD DERIVATIVES FOR DETERMINATION OF GAMMA-GLUTMYL TRANSPEPTIDAS |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19722259512 DE2259512C3 (en) | 1972-12-05 | gamma-glutamyl-4-nitranilid-3carboxylic acid, its salts, process for the preparation of these compounds and their use | |
| DE19732333798 DE2333798C3 (en) | 1973-07-03 | gamma-glutamyl-4-nitranilide-3sulfonic acid, its salts, process for the preparation of these compounds and their use |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| DE2259512A1 DE2259512A1 (en) | 1974-06-27 |
| DE2259512B2 DE2259512B2 (en) | 1975-07-31 |
| DE2259512C3 true DE2259512C3 (en) | 1976-03-18 |
Family
ID=
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