Method of analysis of lipase

专利摘要:
This invention relates to analytical chemistry. The purpose of the invention is to increase the sensitivity. The method of determining lipase by affecting the substrate of the lipase to form the chromogen involves the use of a substrate of the general formula X-CO-A-CO-Y-CH 2 -CH (YR) -CH 2 -ZR 1 , where A is an alkyl or alkylene group with 2 - 12 C-atoms, R and R 1 , which may be the same or different, each represent an alkyl group with 6 to 18 C-atoms, one of the residues R and R 1 also represents a hydrogen atom. X - resorufin, 6-methylresorufin, 4,6-dimethylresorufin, 1,6-dimethylresorufin, 8-ethylresorufin and 8-bromresorufin and each Y and Z, independently of one another, denote CH 2 -. The amount of released aromatic hydroxyl or thiol compound, either directly or after combination with a suitable chromogen, is optically determined by the resulting color. 3 tab.
公开号:SU1604167A3
申请号:SU864027411
申请日:1986-04-30
公开日:1990-10-30
发明作者:Нойманн Ульрих；Юниус Мартина；Батц Ханс-Георг
申请人:Берингер Маннхайм Гмбх (Фирма)；
IPC主号:

专利说明:

The invention relates to analytical chemistry and is of paramount importance in clinical chemistry, biochemistry, pharmaceutical chemistry, and food chemistry.
The purpose of the invention is to increase the detection sensitivity.
. The invention provides for the use of a compound of the general formula as a substrate for lipase.
00
and ti HjC-Y-C-A-C-X
HC-Y-R
I
H C-Z-KI
where A is an alkyl or alkylene group with 2-12 carbon atoms;
about g
Ot) SI

s
R and
R is the same or different, each means an alkyl group with 6-18 carbon atoms, one of the residues R and R is also a hydrogen atom;
X - resorufin, 6-methylresorufin, I, 6-dimethylresorufin, 8 ethyl resorufin, 8-bromresorufin; Y and Z - each independently of the other means -S- or
-O-, Z is also -CH-, and the optical determination by coloring of the amount of the released aromatic hydroxyl or thiol compound directly, or after combining with a suitable chromogen.
For R and R, alkyl groups are preferred as opposed to acyl groups, since they are not sensitive to hydrolysis.
Examples for R and / or R are methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, dedyl, undecyl, decyl, tetradecyl, hexadecyl and octadecyl residues as alkyl groups, as well as the corresponding acyl groups. - Pyi, as grtsshpy acetyl, propionyl, butyryl, valeryl, capronyl, capryl, capril, lauryl, myristyl, palmityl and stearyl, oleyl, crotonyl, linole, phenyl, benzyl or octylphenyl.
Further, the proposed lipase substrate contains a dicarboxylic acid residue COOH-A-COOH, in which A has preferably 3-7 C-atoms. The acids forming A are malonic, amber, glutaric, adipic, pimelinic, corkova azelaicic, sebacinic, nonanedicarboxylic, decandicarboxylic to undecane dicarboxylic acid. Prefer acid from glutaric acid to azelaic acid, which correspond to A with 3-7 C-atoms.
X can be an aromatic hydroxy or thiol compound, which is a chromophore or can only be converted into a coloring matter by a subsequent reaction. Typical pri- measures are phenol, thiophenol, naphthol, thionaphthol and their derivatives, as well as chromogenic compounds such as resorufin residue, 6-methylresorufin, | 4, b-diketylresorufin, 1,6-dimethylpremide

Q
15
jQ
25 Q 35

five
zorufin and 8-ztilresorufin, and 8-bromresorufin, chlorophenol red, indoxyl or thiofluorescein.
Chromophores are preferred which have a small polarity and are liposoluble. However, the water-solubility must also be guaranteed.
The proper dissolution with alkyl groups contributes to the fat dissolving of the mentioned chromophores. Thus, methyl, dimethyl and ethyl groups were found for the resorufin residue, and the bromine substitution also appeared for the residue.
The compounds of the invention are new. They have an asymmetric center and are therefore optically active. Lipases can be used as substrates for both racemates and optical isomers formed by the usual methods of preparation.
The substrates of lipaea according to the invention can be obtained by known methods.
The method involves the use of a reagent in the analyte, preferably containing mg / ml: substrate 0.05-10, deoxycholate 2-50, colipase 0.001-0.01, urea 1-100, NaCl 0.1-10, buffer 1-50, setting the pH from 6 to 10.5, respectively, in terms of usable solution.
These examples illustrate both the preparation of substrates and methods for determining lipase using them.
Example 1. a) 1,2-0-dihexyl-rac-glycero-3-glutaric acid mono-ester.
In a solution of 3.3 g (11.5 mmol) of 1,2-O-dihexyl-glycerol in 30 ml of chloroform, 2.5 MP of pyridine are added successively, at the tip of a spatula 4-dimers of aminopyridine and 2.6 g (23 mmol) of anhydride glutaric acidic acid The mixture is heated IOh under reflux and after cooling is diluted with 200 ml of chloroform. The chloroform phase is shaken with J n. hydrochloric acid and dried. After filtering off the drying agent, the solvent is removed and the residue is purified on a column of silica gel (eluent: ethyl acetate - petroleum ether 1: 1).
DS: Rf 0.45 (acetic ester - petroleum ether 1: 2 +% glacial acetic acid).
1,2-0-dihexyl-rac-glycero-3-glutaric acid complex resorufinic ester.
B) 1.4 g (3.7 mmol) of the solution according to method 1a is dissolved in 20 ml of chloroform and 2 ml (23.3 mmol) of oxalyl chloride are instilled with ice-cooling. The ice bath is removed and the solution is stirred for 12 hours at room temperature. The solvent is then poured, the residue is taken up in toluene and again evaporated. The oil thus obtained is used without further purification.
c) About 8 g (3.7 mmol) of resorufin is suspended in 40 ml of dimethylformamide with the addition of 1.1 ml of pyridine and at the tip of a spatula 4-dimethylaminopyridine. The solution of method 1b is instilled into 20 ml of dimethylformamide. After one- and two-day stirring at room temperature, filter and drain the solvent. The residue is taken up in ethyl acetate, the insoluble components are filtered off and the filtrate is stirred in 1N. hydrochloric acid, then with water. After drying the organic phase and distilling off the solvent, an oily residue is obtained, which is purified by chromatography on a column of silica gel (eluent: ethyl acetate – 1: 1 petroleum ether)
DS: Rf 0.70 (ethyl acetate - hexane 1: 1).
Example 2. a). Monoester 1,2-0-dioctyl-rac-glycero-3-glutaric acid.
Preparation is analogous to example 1a of 13 g (41 mmol) of 1,2 0-dioctyl-glycerin, 150 ml of chloroform, 10 ml of pyridine and 6.8 g (59.5 mmol) of glutaric anhydride. Yield 6.5 g (37%).
DS: Rf 0.31 (acetic ester - 1: 1 ether).
Complex resorufinic ester 1,2-0-dioctyl-rac-glycero-3-glutaric acid.
b). Preparation as in Example Ib of 2 g (4.5 mmol) of Example 2a. with). 0.96 g (4.5 mmol) of resorufin is dissolved in 50 ml of chloroform with the addition of 0.75 ml (5 mmol) of 1,8-diaza bicyclo- (5,4,0) -undec-7-ene and 0 , 1 g of 4-dimethylaminopyridine. The solution according to example 2b is instilled into 20 ml of chloroform. After one and two days 04167
- ten
stirring at room temperature is filtered and the solvent is removed. Recycling is similar to example 1c.
DS: Rf 0.66 (ethyl acetate 1: 1 hexane).
Froze, a). Mono ester of 1,2-0-dioctyl-rac-glycero-3-pymelic acid.
Preparation is analogous to example 1a of 3.2 g (10 mmol) of 1,2-0-dioctyl-glycerol and 2.1 g (15 mmol) of pimelic acid anhydride.
DS: Rf 0.61 (ethyl acetate 1: 1 petroleum ether).
IR (cm -): (film): 1740; 1710.
Complex resorufinic ester 1,2-0-dioctyl-rac-glycero-3-pimelic
acid.
B). Preparation as in Example 1b out of 2.2 g (4.7 mmol) for.
with). Preparation is analogous to Example 2c from 1 g (4.7 mmol) of resorufin, 25 0.7 ml of 1,8-diazabicyclo- (5.4.0) -undec-7-ene and 3b.
DS: Rf 0.78 (ethyl acetate - hexane 1: 2).
Example4. but). 1,2-0-dioctyl-rac-glycero-3-azelaic acid mono-ester.
15
20
thirty
0
five
Preparation is analogous to Example 1a from 6.3 g (20 mmol) of 1.2-0-dioctyl-glycerol, 80 ml of chloroform, 5 ml of pyridine and 5.2 g (30 mmol) of azelaic anhydride.
Complex resorufinic ester 1,2-0-dioctyl-rac-glycero-3-azelaic
acid.
B) Preparation is analogous to example b from 3.2 g (6.5 mmol) 4a and 3 ml of oxalyl chloride.
with). Preparation is analogous to example 2c from 1.4 g (6.5 mmol) of resorufin, 65 ml of chloroform, 1.1 ml of 1.8-diazabicyclo- (5.4.4) -undec-7-ene and 4b.
DS: Rf 0.86 (ethyl acetate - hexane 1: 2).
IR (cm-): (film) 1762; 1736.
Example5. but). Mono ester of 1,2-0-didecyl-rac-glycero-3-glutaric acid.
Preparation is analogous to example 1a from 3.7 g (10 mmol) of 1.2-0-didecyl-glycerol, 40 ml of chloroform, 2.5 ml of pyridine and 1.8 g (15.8 mmol) of glutaric anhydride.
0
five

DS: Rf 0.77 (ethyl acetate - hexane 1: 2)
Complex resorufinic ester 1,2-0 didecyl-rac-glycero-3-glutaric acid,
b). Preparation as in Example 1 2.5 g (3 mmol) 5a, 50 ml of chloroform and 2.2 ml of oxalyl chloride.
with). The preparation is similar to the example of 2c from 1.1 g (5 mmol) of resorufin, 1 ml of 1,8-diazabicyclo- (5,4,0) - undec-7-ene and 5b.
DS: Rf 0.70 (ethyl acetate - hexane 1: 1).
PRI me R b. but). Mono-ester 1,2-0 diundecyl-rac-glycero Z-glutaric acid.
Preparation as in Example 1a. from 2 g to 5 mmol) 1,2-0-diundec1-glycerol, 25 ml of chloroform, 1.4 ml of pyridine and 0.9 g (7.5 mmol) of glutaric anhydride.
DS: Rf 0.55 (ethyl acetate - petroleum ether 1: 2).
IR (cm): (film) 1740; 1710.
Complex resorufinic ester 1,2-0 diundecyl-rac-glycero-3-glutaric acid.
B) Preparation is similar to the example of pyjb from 2 g (3.9 mmol) 6a 5 ml of chloroform and 1.8 oxalyl chloride
with). Preparation is analogous to example 2c from 0.83 g (3.9 mmol) of resorbine, 0.61 ml of 1,8-diazabicyclo (5.4 0) -undec-7-ene and 6b.
DS: Rf 0.47 (RP 18, ethanol - acetone 2: 1).
PRI me R 7. a). Mono ester, 2-0 -dilaura1-rac-glycero-3-glutaric acid.
Preparation is analogous to Example 1a from 10.7 g (25 mmol) of 1.2-0-dilauryl-glycerol, 70 ml of chloroform, 5.5 pyridine, and 3.3 g (29 mmol) of glutaric anhydride.
DS: Rf 0.33 (ethyl acetate – peroleic ether 1: 1).
IR () (film): 1741; 1708.
Complex resorufinic ester 1,2-0-dilauryl-rac-glydero-3-glutaric acid
B) „Preparation as in example 1b from 5.5 g (10 1 hmol) 7a, 50 ml of chloroform and 4.3 ml of oxalyl chloride
CT Ilech, as in Example 2c, of 2.2 g (10 mmol) of resorufin, 100 ml of chloroform and 1.5 ml of 1.8-diaz bicyclo (5 94.0) -undec-7-ene and 7b.
eight
DS: Rf 0.78 (1: 1 ethyl acetate / petroleum ether).
IR (cm-O (film): 1765; 1720.
PRI me R 8. a). 1,2-0-dilaura-1-rac-glycero-3-pimelic acid mono-ester.
Preparation is analogous to Example 1a and 8.6 g (20 mmol) of 1.2-0-dilauryl-glycerol, 50 ml of chloroform, 15 ml of pyridine and 4.3 g (30 mmol) of pimelic anhydride.
DS: Rf 0.5 (ethyl acetate - petroleum ether 1: 2).
IR () (film): 1740; 1710. Complex resorufinic ester 1,2-0 diluryl-rac-glycero-3-pimelic acid.
B) Preparation as in Example Ib from 1.66 g (3 mmol) 8a and 1.3 ml of oxalyl chloride.
with). Preparation is analogous to Example 2c from 0.65 g (3 mmol) of resorufin, 30 ml of chloroform, 0.5 ml of 1.8-diaza-bicyclo- (5.4.4) -undec-7-ene and & b.
DS: Rf 0.75 (ethyl acetate - 1 hexane 1: 1).
IR (cm-) (film): 1768; 1739.
Pr them 8 p 9. a). Mono-ester of 1,2-0-ditetradech1-rac-glycero-3-glutaric acid.
Preparation is analogous to example 1a of 14.6 g (30 mmol), 1,2-0-ditertradecyl-glycerol, 150 ml of chloroform, 8.2 ml of pyridine, and 5.1 g (45 mmol) of glutaric anhydride.
DS: Rf 0.42 (ethyl acetate - petroleum ether 1: 2).
IR () (KBr): 1740; 1710.
1,2-0-ditetradecyl-rac-glycero-3-glutaric acid complex resorufinic ester.
B) Preparation is analogous to Example 1b from 3 g (5 mmol) 9a and 2.2 ml of oxalyl chloride.
with). Preparation is analogous to example 2c from 1.06 g (5 mmol) of resorufin, 50 ml of chloroform, 0.75 ml of 1,8-diaza-Ibicyclo- (5,4,0) -undec-7-ene and 9b. DS: Rf 0.34 (RP18, acetonitrile dichloromethane 1: 1).
IR (cm IkVg): 1763, 1735.
PRI me R 10. a). Monoester 1, 2-0-ditetradelen-rac-glycero-3-pimelic acid.
Preparation analogously to example 9a of 6.4 g (45 mmol) of pimelic acid anhydride.
DS: Rf 0.45 (acetic ether - petroleum ether 1: 2).
IR () (film): 1740; 1708.
1,2-0-ditetradecyl-rac-glycero-3-pimelic acid acid resorufinic ester.
B) Preparation is analogous to example 1b from 3.1 g (5 mmol) 10a and 2.2 ml of oxalyl chloride.
with). Preparation as in Example 2c from 1.06 g (5 mmol) of resorufin, 50 ml of chloroform and 0.78 ml of 1.8-diazabicyclo- (5.4.4) -undec-7-ene
and lOb,
DS: Rf 0.71 (ethyl acetate - petroleum ether 1: 2)
IR (cm -) (film): 1755; 1734.
Example 11. a). Mono ester of 1,2-0-dihexadecyl-8n-glyceroglutaric acid.
Preparation is analogous to Example 1a from 2.7 g (5 mmol) of 1.2-0-dihexadecyl-3p glycerol, 50 ml of chlorofom, 3 ml of pyridine and 1.5 g (13 mmol of glutaric anhydride.
DS: Rf 0.65 (ethyl acetate - petroleum ether 151)
IR (cm-) (KBG): .1740; 1710.
Complex resorufinic ester 1,2-0 dihexadecyl-8p-glycero-3-glutarovo
acid.
B) Preparation is analogous to example 1b from 2.2 g (3.3 mmol) Pa and 1 ml of oxalyl chloride.
with). Preparation is analogous to Example 1c from 0.71 g (3.3 mmol) of resorufin, 20 ml of dimethylformamide, 0.5 ml of pyridine and Pb.
DS: Rf 0.72 (acetic ester - petroleum ether 1: 2).
Example 12. a). Monoester 1,2-0-dibenzyl-rac-glycero-3-glutaric acid
Preparation is analogous to example 1a of 3 g (11 mmol) of 1,2-0-dibenzsh1-glycerin, 30 ml of chloroform, 2.5 ml of pyridine and 1.8 g (16 mmol) of glutaric anhydride.
DS: Rf 0.39 (acetic ester - petroleum ether 1: G + 1% glacial acetic acid) .t
Complex resorufinic ester 1,2-0-dibenzyl-rac-glycero-3-glutaric
acid.
B) Preparation is analogous to example 1b from 2.9 g (7.5 mmol 12a), 30 mp of chloroform and 3.3 ml of oxalyl chloride.
five
0
five
with) . Preparation is analogous to the example, 2c of 1.6 g (7.5 mmol) of resorufia, 75 MP of chloroform, 1.2 ml of 1.8 diazabicyclo- (5.4.4) -undec-7-ene and 12b.
DS: Rf 0.48 (acetic ester - hexane 1: 1).
Example 13. a). Monoester 1-0-Octadecid-2-0-benzyl-5n-glycero-3-glutaric acid.
Preparation is analogous to example 1a of 2.2 g (15 mmol of 1-0-octadecyl-2-O-benzyl-3p glycerol, 50 ml of chloroform, 3 MP of pyridine and 1.5 g (13 mmol) of glutaric anhydride.
IR () (film): 1730; 1700
Resorufinic ester of 1-0-octadecyl-2-0-benzyl-8n-glycero-3glutaric acid.
B) Preparation is analogous to example 1b from 1.7 g (3.1 mmol) 1 Over and 1.3 ml of oxalyl chloride.
with). Preparation is analogous to Example 1c from 0.8 g (37 mmol) of resorufin, 20 ml of dimethylformamide, 0.7 ml of pyridine and 1Zb.
DS: Rf 0.68 (acetic ester - petroleum eLir 1: 1).
IR () (KBr): 1768; 1739.
PRI me R 14.a). Complicated mono. 1,2-dioctanoyl-5n-glycero-3-glutaric acid ester.
Preparation is analogous to example 1a of 6.8 g (20 mmol) of 1,2-dioctanoyl, Sn-glycerol, 100 ml of chloroform, 12.5 ml of pyridine and 5.8 g (50 mmol) of an1: glutaric acid idride.
DS: Rf 0.49 (ethyl acetate 1: 1 petroleum ether).
1,2-dioctanos 1-8p-glycero-3-glutaric acid complex resorufinic ester.
B) Preparation is analogous to Example 1b from 2.2 g (5 mmol) 14a and 2.2 ml of d5 oxalyl chloride.
with). Preparation is analogous to example 1c from 1.05 g (5 mmol) of resorufin, 30 ml of dimethylformamide, 0.75 ml of pyridine and 14b.
DS: Rf 0.86 (RP18, acetonitrile - dichloromethane 1: 2).
Example 15. a). 1,2-Dioleyl-rac-glycero-3-glutaric acid monoester.
Preparation is analogous to example 1a of 3.1 g (5. mmol) of diolein, 40 ml of chloroform, 3 ml of pyridine and 1.5 g (13 mmol) of glutaric anhydride.
0
40
50
55
eleven
DS: Rf 0.32 (ethyl acetate - 1: 1 role ether).
IR () (film): 1740; 1706. Diolein can be obtained in pure form from technical diolein by chromatography on a column of silica gel using acetic ether – petroleum ether 1: 3 as a solvent.
1,2-dioleyl-rac-glycero-3-glutaric acid complex resorufinic ester.
B) Preparation is analogous to Example 1b from 3.5 g (4.8 mmol) of 15a and 1.3 m of oxalyl chloride.
with). Preparation is analogous to Example 1c with 0.9 g (4.2 mmol) of resorufin, 20 ml of dimethylformamide, 1 ml of pyridine 15b.
DS: Rf 0.78 (ethyl acetate - petroleum ether 1: 1).
Example 16. 1,2-0-ditetradecyl-rac-glyceropimelic acid acid naphthyl ester.
Preparation was obtained analogously to Example 1 Ob and with from 0.72 g (5 mmol) of 1-naphthol. Purification by flash chromatography on silica gel with ethyl acetate: hexane 1I3.
DS: Rf 0.89 (ethyl acetate - petroleum ether 1: 5).
Example 17. 1,2-diolesh-1-rac-glidero-3-glutaric acid ester-ester ester.
10.3 g (14.2 mmol) of 1,2-dioleyl-3-glycero-glutaric acid monospher monosfere as in Example 15a, 3.2 g (15 mmol) resorufin, 6.2 g (30 mmol) dicyclohexylcarbodiimide and at the tip of the spatula 4-dimethyl-aminopyridine is stirred in 75 ml of dimethylformamide for 2-3 days at room temperature. It is then diluted with ethyl acetate and the precipitate is filtered off. The phase of ethyl acetate is shaken with 1 n. hydrochloric acid and dried over sodium sulfate. After distilling off the solvent, an oily residue remains, which is purified by chromatography on a column of silica gel (solvent: ethyl acetate – petroleum ether 1: 1).
In this way, the corresponding chlorophenol red derivative is obtained.
DS: Rf 0.69 (RP18; isopropanol: methanol 1: 2).
)
041
0
15
20
25
thirty
,,
Q j l
five
6712
Example 18. Preparation of 1,2-0-dioctyl-3-pimelic acid monoester.
Stage I: synthesis of benzyl monoether pimelic acid.
Step 2: as in Example 1b out of 2.5 g (10 mmol) of pimelic acid benzyl ester; the resulting oil is instilled into a solution of 3.2 g (10 mmol) of 1,2-0-dioct-1-glycerin, 7 ml of pyridine; processing is similar to the example. 1s.
Step 3: This product is dissolved in 20 ml of tetrahydrofuran and, after addition of 0.4 g of palladium-activated carbon, is hydrogenated. The crude product is purified on a column of silica gel; acetic acid solvent - petroleum ether 1: 1.
DS: Rf 0.61 (ethyl acetate - petroleum ether 1: 1)
IR () (film): 1740; 1710.
Example 19. Synthesis of 1,2-dioleyl-glycero-3-glutaric acid monoester.
but). Trichlorostilled ester of 1,2-0-isopropylidene-glidero-3-glutaric acid.
Stage 1: 5.55 g (42 mmol) of isopropylidene glyderol and 12 g (45.3 mmol) of 2,2,2-trichloroethyl-hydro-glutarate are dissolved in ethylene glycol dimethyl ether and taken up from 10.5 g (51 mmol) didiclohexylcarbodiimide, after two days of stirring, filtered and subjected to distillation; boil; (0.1 torr), colorless oil.
DS: Rf 0.82 (adetone - chloroform 1: 1).
Step 2: The resulting oil is dissolved in P ml of ether, mixed with 3 ml of methanol and 3 ml of 3N. hydrochloric acid and stirred for 12 hours at room temperature; the organic phase is shaken with an aqueous solution of sodium bicarbonate, then with a saturated solution of sodium chloride and szppat; after removal of the solvent, an oily residue remains.
DS: Rf 0.23 (ethyl acetate 1: 1 petroleum ether).
B) Trichloroethyl ester 1,2-diolesh1-glidero-3-glutaric acid.
Step 3: 6.1 g (18 mmol) of the obtained product and 7.84 g (38 mmol) of oleic acid are dissolved in
100 ml of ethylene glycol dimethyl ether and a solution of 10.5 g (37 mmol) of dicyclohexylcarbodiimide are instilled in 50 ml of ethylene glycol dimethyl ether; after stirring for 12 hours at room temperature, filter and shake in succession with 3N. hydrochloric acid with sodium bicarbonate solution, then with water. After drying and concentrating the organic phase, the residue is chromatographed on silica gel.
Stage 4: the monoester of 1,2-diolsh1-glycero-3-glutaric acid cleavage of the protective group of trichloroethyl is carried out by a known method. .
 PRI me R 20. a). Mono ester of I, 2-0-didecyl-rac-glycero-3-pimelic acid.
Preparation is analogous to Example 1a from 5.6 g (15 mmol) of 1.2-0-didecyl-glycerol, 100 ml of chloroform, 3.2 ml of pyridine and 4.0 g (28 mmol) of pimelic anhydride.
DS: Rf 0.36 (ethyl acetate - hexane 1: 1).
IR (cm -) (film): 1735; 1710.
1,2-0-didecyl-rac-glider-3-pimelic acid acid resorufinic ester.
B) Preparation as in Example 1b out of 3 tons (5.8 mmol) of 20a, 50 ml of chloroform and 2.2 ml of oxalyl chloride
with). Preparation as in Example 2c from 1.1 g (5 mmol) of resorufin, 1 ml (6.4 mmol) of 1,8-diazabicyclo- (5,4,0) -undec-7-ene and 20b.
DS; Rf 0.71 (ethyl acetate - hexane 1: 1).
IR (cm -) (film): 1755; 1720.
PRI me R 21. a). Mono ester of 1,2-0-dilauryl-rac-glycero-3-azelaic acid.
;
.
ten
15
-
604167
Preparation is analogous to example 1a of 6.7 g (15.6 mmol) of 1.2-0-dilauryl-glycerol, 100 ml of chloroform, 3.2 ml of pyridine and 4.0 g (23 mmol) of angdridi a azelaic acid .
DS: Rf 0.22 (ethyl acetate - hexane 1: 1)
IR () (film): 1738; 1710.
Complex resorufinic ester 1,2-0-dilauryl-rac-glycero-3-azelaic acid
B) Preparation as in Example 1b out of 1.5 g (2.5 mmol) 21a 30 ml. chloroform and 1.5 ml of oxalyl chloride.
with). Preparation is analogous to example 2c from 0.55 g (2.5 mmol) of resorufin, 25 ml of chloroform, 0.5 ml of 20 (3.2 mmol) of 1,8-diazabicyclo- (5.4.4) —undec- 7th and 21b.
DS: Rf 0.78 (ethyl acetate - hexane 1: 1).
IR () (film) :, 1762; 1740.
EXAMPLE 22 60 ml of distilled water are dissolved with stirring 1., 2 g of sodium deoxycholate and 0.15 ml of colipase (from pig). With vigorous stirring, 70 mg of the 1,2-0 -dioctyl-rac-glycero-3-azelaic acid resorphine ester substrate lipase is injected as thinly as possible in a 1.7 ml of 1.7 ml of p-propanol under pressure. The solution, which in 200 ml of distilled water contains in solution 1.5 g of urea, 1 g of sodium deoxycholate, 200 mg of sodium chloride, 800 mg of TRIS and 107 mg of TRIS - HC1 buffer, is well mixed with the Bbmie emulsion.
2.5 ml of the solution prepared in this way is mixed with 100 ml of the sample (serum). The reaction is carried out at 578 mm Hg. with indications on the photometer.
With standard lipase activity, the sample lipase activity is calculated as follows:
25
thirty
35
40
45

activity
activity
D U / min
(samples
active writing / V-U .- ,,
, i Z§SB§2l2- iE ° b 2.
 l. "g"
D U / min
The calculation of sample lipase activity is also possible by the following formula:
activity
(samples)
(unit / 1)
 lOOO - V-n1kr «« V
D U / min
U
l. "g"
D U / min
(standard)
where v
ges
five
- total rise of the tested initial mixture, cm;
I
gz
V samples - sample volume, cm
 - extinction coefficient
chromogen at 578 nm; d is the thickness of the cuvette layer, cm;
AE / min, the extinction change for 1 min at 578 them.
Under the reaction conditions mentioned, the extinction coefficient is 60.65 cm.cmol.
Example 23. For different lipase substrates, changes in the blank test, the sensitivity of esterase and lipase, and the correlation to the nephelometric test with turbidity were determined.
The change in the value of the blank experiment was determined with the reagent of Example 1 and various lipase substrates. Instead of the sample, 100 ml of water was added and a change in extinction at 578 (mU / min) was observed using a photometer.
Dp of determining the sensitivity of esterase was added instead of a sample of 100 ml of carboxylesterase (EC 3.1.1.1 about 20000 units / l of Beringer Mannheim GmbH, definition number 10 46 98) and the extrusions were described as described above.
To determine the sensitivity of lipase, 100 ml of lipase (EC 3.1.1.3, about 100 U / L, Beringer Mannheim GmbH, definition number .41 45 90) was added instead of the sample and the change in extinction was observed, as described above,
To determine the correlation to the felometric test with clouding, we performed with increasing amounts of lipase (0-1000 units / l) test with turbidity (Beringer Mannheim GmbH, definition number 26 23 58), compared with the color test of Example 22, and the correlation coefficient was determined ,
The results are shown in table 1.
Example 24 8.5 g of deoxychol sodium, 0.05 g colipase, 20 g mannit, 0.05 g calcium chloride, 80 g sodium chloride, 2.7 g TRIS, and 0.4 g TRIS-HCl are dissolved in 200 ml of distilled water. With stirring, a solution is injected, which contains in dissolved form 0.35 g of naphthol ester, 1.2-0 DIettetradextra-rac-glycero-3-pimelic acid, in 7 ml of propanol. The emulsion thus obtained is frozen at -40 ° C and lyophilized.
70 g of the obtained lyophilisate is dissolved in 2 ml of distilled water.
0
five
0
five
0
five
0
five
and mixed with 100 ml of a TR solution of red paranitroaniline (red paranitroaniline-4-chloro-3-methylbenzen-diazonium-naphthalene-1,5-disulfonate) (231 mg in 10 ml of distilled water).
After the addition of 100 ml of serum, the reaction at 405 nm is monitored using a photometer with
The concentration of lipase was determined by a calibration curve as in Example 22.
Example 25. To a solution of 3.04 g of taurodeozoxycholate, 2.7 g of a polivosk 4000, 7 mg of calcium chloride, 0.2 mg of colipase (from a pig) in 120 distilled water, 150 mg of the resorufinic ester 1,2-1 lipase substrate is injected with stirring 0-didecyl-rac-glycero-3-glutaric acid in 3.5 ml of p-propanol.
To the emulsion thus obtained, add the following solution and mix well
10 g of taurodesoxycholate, 6.4 g of a polosk, 50 g of mannitol, 14 g of urea, 800 mg of sodium chloride, and 15 g of TR.IS are dissolved in 300 ml of distilled water. The solution is adjusted to pH 7.5 with the help of solution acid. After this, the solution is supplemented to 400 ml with distilled water.
2.5 ml of the thus obtained reaction mixture is mixed with 100 ml of serum. The reaction is observed at 578 nm by photometer.
The evaluation is carried out as in Example 22.
EXAMPLE 26 A solution of 3.04 g of taurodesoxycholate, 2.7 g of a polosk 4000, 7 mg of calcium chloride, 0.2 mg of colipase (from a pig) injected 150 ml of complex lipase substrate into 120 ml of distilled water. 1,2-0-didecyl-rac-glycero-3-glutaric acid resorufinic ester in 3.5 ml of p-propanol.
To the emulsion thus obtained, the next solution is added and mixed well.
10 g of taurodeoxycholate, 6.4 polivosk, 50 g of mannitol, 14 g of urea, 800 mg of sodium chloride, 28 g of a buffer solution of CHES in 300 ml of distilled water. The solution is adjusted to pH 8.5 with hydrochloric acid solution. Then add the solution to 400 ml with distilled water.
1716041
2.5 ml of the thus obtained reaction mixture is mixed with 100 ml of serum. The reaction is observed at 578 them according to the photometer.
Evaluation was performed as indicated in Example 22.
PRI me R 27, In a solution of 4.0 g of taurodesoxycholate Na, 0.06 g of calcium chloride, 0.2 mg of colipase (from pig Q), 5.0 g of mannitol and 2.0 g of a polosk 4000 per 100 ml of distilled water is injected under stirring with 150 ml of naphthol ester 1,2-0-ditetradec-1-rac-glycero-3-pi-5-melinic acid, which is dissolved in 4 ml of p-propanol. With good cooling, the emulsion obtained in this way is treated with ultrasound for several minutes. 20
The second solution contains, in 100 ml, 2.4 g of sodium taurodesoxycholate, 2.0 g of TRIS, 12.0 g of mannitol, 3.5 g of urea, and 0.5 g of sodium chloride. The pH is adjusted to 8.3 with hydrochloric acid.
The third solution contains 1 g of red paranitroanaline TR (red para-nitroanaline TR 4-chloro-methyl-benzene-, diazonium-naphthalene-1,5-disulfonate) in DL 40 ml of distilled zoda in dissolved form,
5 parts of solution 1 and 6 parts of solution 2 are mixed with part 1 of solution 3. The resulting solution is impregnated with suction paper suitable for the preparation of test strips, for example, grade, VS532 from Schlieher und Schulle and dried safely in a chamber dryer. with air circulation at a temperature of from 30 to 654.
Until further use, storage in the presence of a dehydration agent is recommended.
35
For testing serum lipase
a small amount (a few drops) is applied to the strip.
On the basis of the yellow staining curve, a conclusion can be drawn about the lipase activity of the test sample,
PRI me R 28. a). Mono-ester of 1,2-0-dilauryl-rac-glycero-3-tetradecanedioic acid.
Preparation analogously to example 1a of 8.6 g (20 mmol) of 1,2-0-dilauryl-. glycerol, 100 ml of chloroform, 4 ml of pyridine, 0.4 g of dimethylaminopyridine
Q 5 0
five
ABOUT

five
five
on, 9.6 g (40 mmol) of dibasic tetradecanoic anhydride.
DS: R 0.45 (ethyl acetate - petroleum ether 1: 5).
The 1,2-0-dilaurip-rac-glycero-3-tetradecanoic acid (6-methylresorufinic) ester.
B) Preparation is analogous to example 1b from 3.35 g (5 mmol) 28a and 2.2 ml of oxalyl chloride.
with). Preparation is analogous to example 2c of 1.2 g (5 mmol) of 4-methyresoronane, 20 ml of chloroform, 0.75 ml of 1.8-diazabicyclo- (5.4.0) -undec-7-ene, 0.1 g dimethylaminopyridine and 28b. DS: R 0.63 (ethyl acetate - hexal 1: 4).
d). An emulsion is obtained as indicated in Example 22. However, instead of the above-mentioned lipase substrate, a solution of 70 mg of the 1,2-0-dilaural-rac-glycero-3-tetradecanoic acid complex (6-methylresorufinic) ester substrate lipase is used. , 7 ml of p-propanol.
The following test-specific parameters were obtained (cf. example 23 and table 1): the idle test value was 0.2 mU / min; esterase activity 0.7 mU / min; total lipase content of 23.6 mU / min per 100 units / l; the correlation coefficient is 0.09509.
PRI me R 29. Complex (6-methyl-resorufinic) ester 1,2-0-d1-aryl-rac-glycero-3-glutaric acid,
but). Preparation as in examples 7d, -c 2.3 g (10 mmol). 4-meter-ruzo.
DS: R, 0.68 (ethyl acetate - hexane 1: 2).
B) B, 60 ml of distilled water was dissolved with stirring, 0.9 g of sodium taurodesoxycholate and 0.3 g of a colloid (from a pig). With good stirring, a solution of 1,2-0-dilauryl-rac-gl1-stero-3-glutaric acid complex (6-methylresorufinic) ester, 70 mg in 1.7 ml of p-propanol is injected there. Then a solution (200 ml) is added, which contains 200 ml of 0.5 g of urea, 1 g of sodium taurodeoxycholate, 200 ml of sodium chloride and 2.9 g of TRIS, and its pH is set to 7.5. After stirring well, 2.5 ml of the solution thus obtained is mixed with 100 ml of sample (serum). The reaction is observed at 578 nm Hg. by photo19 rating,
16 as indicated, produced
but in example 22,
Testospecific parameters (cf. example 23 and table 1): the value of the blank experiment P, 4 mU / min; lipase sensitivity of 28 mU / min per 100 mU / min; the correlation coefficient is 0.988.
Example 30. 4-Methyl Resorufin. It is obtained from 7.5 g of 2-metsh-1-4-nitro-zorezorezin, 4.2 g of resorcinol, 3.6 g of pyrolusite, 4.3 MP of sulfuric acid, 75 ml of methanol, 4 g of zinc powder and 18 ml of 25% ammonia.
DS: R, 0.70 (ethanol - acetone 2: 1).
UY / YIS (0.1 M potassium phosphate buffer solution, pH 8.5), (o, x 579 nm.
2-Methyl-nitrozorezoretsin receive according to the German patent 23 11 574, AI.
Example 31. Compound (4-methyl-umbilliferyl) ester of 1,2-0-d1a-p IL-pic-glycero-3-hydrochloric acid,
but). 2.75 g (5 mmol) of 7a and 1 g (5 mmol) of 4-methylumbelliferone TET in 30 ml of tetrahydrofuran and then 2 g (10 mmol) of dicyclohexylcarbodiimide and 0.15 g of dimethylamino-pyridine are added. After stirring for 24 hours at room temperature, filter and thicken. The crude product is flash chromatographed.
DS: R 0.40 (Acetic ether - petroleum ether 1: 3).
B) The emulsion is prepared as indicated in Example 22, but instead of the lipase substrate used there, 35 mg of 1,2-d1-tri1-aryl-rac-glycero-3-glutaric acid complex (4-methylumbelliferic ester) dissolved in 1.7 ml of n- propanol about
 The reaction is observed on a fluorometer at 25 ° C, namely, at an excitation wavelength of 364 nm, a slit width of 5 nm and a radiation wavelength of 448 nm and a slit width of 10 nmc
Test-specific parameters (cf. example 23 and table 1): the value of the idle test is 0.4 mU / min; lipase sensitivity of 28 mU / min per 100 units / l; correlation coefficient 0,9942
PRI me R 32. 1,2-0-dilapyl-pats-glycero-3-hydrofluoric acid (p-nitrothiophenyl) ester.
Getting 32-b is similar to examples 7a-b.
5 o
five
0
20
with). Preparation is analogous to 1 s of 1.4 g (10 mmol) of p-nitrothiophenol, 100 ml of dimethylformamide, 2.4 ml of pyridine, 0.2 g of dimethylaminopyridine and 32b.
DS: R 0.76 (ethyl acetate - hexane 1: 4).
d). The following compounds are dissolved in 27 MP of distilled water with good stirring: 4.83 mg. deoxycholate sodium; 28.00 mg CHES-2- (cyclohexylamino) ethane-sulphonic acid, 17.50 g of urea, 2.13 mg of sodium chloride, 0.16 mg of pig colipase, 0.33 mg of calcium chloride, 152.00 mg of sodium taurodeoxycholate ,
The pH of the solution is 8.3. With good stirring, a solution of 1,2-0-dilauryl-rac-glycero-3-glutaric acid complex (p-nitrothiophenyl) ester is injected into this solution. Then the solution was treated by cooling with ultrasound (medium intensity) for 2 minutes.
In 1 MP of the thus obtained emulsion solution, 50 ml of sample (containing human serum lipase) were added and mixed. The reaction was observed at 405 nm Hg. by photometer and carried out the calculation, as indicated in example 22.
Testospecific parameters: the value of the blank test is 0.3 mU / min; lipase sensitivity of 12.2 mU / min at 100 units / l; the correlation coefficient is 0.998.
PRI me R 33. a). 1-O- (2-methoxy-octadecyl) -2-0-methyl rac-glycero-3-glutaric acid monoester.
Preparation analogously to example 1a of 3 g (7.7 mmol) of 1-0- (2-methoxy-octadecyl) -2-0-methyl-glycerol, 25 ml of chloroform, 1.8 ml of pyridine, 0.1 g of dimethylaminopyridine and 1.75 (15 mmol) glutaric anhydride. Yield 1.5 g (39%).
DS: R, 0.68 (RP18, acetone - ethanol 1: 2).
Compound (6-methylresorinofine) 1-0- (2-methoxy-octadec1) 2-methylmethyl-rac-glycero-3-glutaric acid ester.
B) Preparation is analogous to Example 1b from 1.5 g (3 mmol) of ZZa and 1.5 ml of oxalyl chloride.
with). Preparation analogously to example 2c from 0.68 g (3 mmol) 4-methyl g
one
zorufina, 0.45 ml of 1, 8-diazabicyclo (5.4.0) -undec-7-ene and ZZb.
DS: R 0.86 (acetic ester), d). A test emulsion is prepared as indicated in Example 22, however, a complex (6-methyl resorufinic J ester 1 (2-methoxy-octadecyl) - 2-0-methyl-rac glycero 3-glutaric acid.
The distribution of lipase concentration is made through a calibration straight line from two standards of different lipase concentration, at which an extinction change is made in 1 minute relative to an extinction change.
The value of the blank experiment is 8.4 mU / m, the sensitivity of esterase is 25.3 mU / m, the sensitivity of lipase is 19.0 mU / min per 100 units / l; the correlation coefficient is 0.9483.
PRI me R 34. a). Mono ester of 1,2-0-dilauryl-rac-glycero-3-succinic acid.
Preparation is analogous to example 1a of 8.56 g (20 mmol) of I, 2-0-dilaursh1-glycerol, 4 g (40 mmol) of succinic anhydride, 60 mp of chloroform, 4.6 ml of pyridine and 0.24 g of dimethylaminopyridine . The product is crystallized from hexane. M.p. 41-43 ° C.
DS: R, 0.26 (ethyl acetate - hexane 1: 4).
Complex resorufinic ester 1,2-0-
Dilauryl-rac-glycero-3-succinic acid.
B) Preparation as in Example 17 of 2.65 g (5 mmol) of 34a, 1.06 g (5 mmol) of resorufin, 6.18 g (30 mmol of dicyclohexylcarbodiimide, 0.1 g of dimethylaminopyridine and 50 ml of dimethylformamide
DS: R 0.47 (acetic ether - petroleum ether 1: 3).
with). An emulsion is obtained as indicated in Example 22, however, in this case, 70 mg of 1,2-0-dy-1-aryl-rac-glycero-3-succinic acid, dissolved in 1.7 ml of p-propanol, are used as the lipase substrate substrate.
At the same time, the following test-specific parameters (cf, example 23 and Table 1): the value of the blank experiment is 3.8-4.0 mU / min; esterase sensitivity not determined; lipase sensitivity of 8.7 mU / min per 100 units / l; the correlation coefficient is 0.8793.
6722
PRI me R 35. a). Monoester 2-0-lauryl-octadecanediol (1,2) glutaric acid.,
Preparation is analogous to example 1a of 4.6 g (10 mmol) of 2-0-lauryl-octa-decanediol (1.2), 29 ml of chloroform, 2.3 ml of pyridine, 0.12 g of dimethylamino pyridine, 2.3 g ( 20 mmol) glutaric anhydride.
DS: R 0,54 (petroleum ether - ethyl acetate 4: 1)
Complex (f-methylresorufinic) ester 2-0-lauryl-octadecanediol (1,2) glutaric acid.
B) Preparation is analogous to Example 1b. Of 1.15 g (2 mol) of 35a and 0.88 ml of rxalil chloride.
with). Preparation analogously to example 2c from 0.45 ml (2 mmol) of 4-methylresorufin, 20 ml of chloroform, 0.3 ml of 1,8-diazabicyclo- (5,4,0) -undec-7-ene, 40 mg of dimethylaminopyridine and 35b „
DS: R, 0.37 (petroleum ether - ethyl acetate 5: 1).
d). An emulsion is obtained as indicated in Example 22, however, 70 mg of 2-0-laur-1-octadecanediol-1,2-1-glutaric acid dissolved in 1.7 ml of p is used as a substrate of lipase. -propanolao
In accordance with Example 23, Table 1, the following test-specific parameters are obtained: the idle test value is 0.3-1.0 mU / min; esterase sensitivity 1.0 mU / min; lipase sensitivity 25.5 mU / mink henna 100 u / lo, correlation coefficient 0.996 s
PRI me R 36 "a). Mono ester of 1,2-0-dilauryl-rac-3-thioglycero-3-glutaric acid.
Preparation is analogous to example 1a of 2.5 g (4.8 mmol) of 1,2-0-dilauuryl-3-3-thioglycerol 14 ml of chloroform 1 MP. pyridine and 1.1 g (9.6 mmol) of glutaric anhydride.
DS: RI 0.5 (hexane - tetrahydrofuran 1: 4).
The 1,2-0-dilaurash1-rac-3-thioglycero-3-5-glutaric acid (6-methylresorufinic) ester.
B) Preparation is analogous to Example 1b from 0s 74 g (1.3 mmol) of Zba and 0.6 ml of oxalyl chloride.
with). Preparation analogously to example 2c of 0.3 g (1.3 mmol) of 4-methylreso
Rufina, 13 ml of chloroform, 0.2 ml
1,8-diazobicyclo- (5,4,0) -undec-7-ene
27 mg of dimethylaminopyridine and Zb.
DS: R 0.38 (petroleum ether
Acetate 17: 3).
As a starting material, 1,2-0-dilauryl-rac-3-thioglycerol is prepared in a known manner as follows.
Conversion of 1,2-0-dilaurylglycerol with toluenesulfonic acid chloride to dilauryl-glycero-3-toluene sulfonate, then reaction with thiourea to the corresponding isothiourea salt, subsequent hydrolysis with hydrochloric acid with
DS: R 0.52 (petroleum ether - ethyl acetate 49: 1).
d). An emulsion is obtained, as indicated in Example 22. However, instead of the substrate used for the lipase used there, a solution of 70 mg of 1,2-0-dv1auril rat-3-thioglycero-3-5-glutaric acid 1.7 is used. ml of p-propanol
Testospecific parameters: the value of the idle test of 0.2 - 0.9 mU / min; estrarase sensitivity is 1.3 mU / min; The sensitivity of lipase is 4.1 mU / min per 100 units / l., the correlation coefficient is 0.857.
PRI me R 37. a). 1,2-8-dilaural-rac-1,2-dithio-glycero-3-glutaric acid monoester.
Preparation is analogous to example 1a from 3 g (6.5 mmol) of 1,2-8-dilauryl-1,2-dithioglycerol, 30 ml of pyridine and 1.5 g (13 mmol) of glutaric anhydride.
DS: Rf 0.43 (petroleum ether - ethyl acetate 1: 1).
DS: Rf 0.37 (ethyl acetate - petroleum ether 1: 4).
1,2-8-dilauuryl-rac-1,2-dithioglycero-3-glutaric acid (6-methylresorufinov) ester.
B) The decay is analogous to Example 1b out of 1.2 g (2 mmol) 37a and 1 ml of oxalyl chloride.
with). Preparation is analogous to example 2c of 0.46 g (2 mmol) of 4-methylresorufin, 20 ml of chloroform, 0.3 ml of 1, diazabicyclo- (5,4,0) -undec-7-ene, and 37b.
The 1,2-8-dilauryl-rac-1,2-dioglycerol used as the starting material was prepared as follows.
24
In a solution of 9 g (160 mmol) of potassium hydroxide in 250 ml of ethanol instilled
0
five
ten).
at room temperature, 10 g (80 mmol) of 2,3-dimercaptopropanol in 100 ml of ethanol. After one hour of stirring, a solution of 40 g (160 mmol) of dodecyl bromide in 100 ml of ethanol is instilled. To supplement the reaction, it is moved for two more days, then filtered and the filtrate is mixed with ice. After acidification 2 n. hydrochloric acid is extracted three times with ether, the organic phase is dried and concentrated. The residue is purified on a column of silica gel (solvent: ethyl acetate - petroleum ether 1: DS: Rf 0.54 (ethyl acetate - petroleum ether 1:10).
d). An emulsion is obtained as indicated in Example 22. Instead of the lipase substrate used there, the complex (6-methylresorufinic) ester 1,2-5-d1-aryl-rac-1,2-dithio-5 glycero-3-5-glutaric is used. acids, 70 mg, dissolved in 1.7 ml of p-propanol.
Idle experience 3 mU / min; esterase sensitivity 3.4 mU / min; lipase sensitivity of 24 mU / min; the correlation coefficient is 0.9943.
PRI me R 38. 4,6-Dimethylresorufinic ester 1j 2-0-dilauryl-rac-glycero-3-glutaric acid.
This compound was prepared analogously to Examples 7a-c, using 2.4 g (U mmol) of 4,6-dimethylresofurin as a starting material. Thin layer chromatography: Rf 0.63 (a mixture of ethyl acetate and hexane in a 1: 2 ratio).
thirty
35
40
five
0
five
PRI me R 39. 8-Bromo-resorufinic ester of 1,2-0-dilauryl-rac-glycero-3-glutaric acid.
This compound was prepared analogously to Examples 7a-c, using 2.7 g (5 mmol) of 2-bromoresofurin as a starting material.
Thin layer chromatography: Rf 0.6 (mixture of ethyl acetate and hexane in a ratio of 1: 2)
EXAMPLE 40 8-Etsh1-resorbic ester of 1,2-0-dilauryl-rac-glycero-3-glutaric acid.
This compound was prepared analogously to Examples 7a-c, using 1.2 g (5 mmol) of 2-ethylresorufin as the starting material.
ten
20
25
Thin layer chromatography: Rf 0.51 (mixture of ethyl acetate and petroleum ether in a ratio of 1: 3)
EXAMPLE 41. 1-, 6-Dimethyl-esterophinic ester 1,2-0-dilauryl-rac. -Glycero-3-glutaric acid.
This compound was prepared analogously to Examples 7a-c, using 1.2 (5 mmol) 1.6 dimethylresorufin as a starting material.
Thin layer chromatography: Rf 0.73 (mixture of ethyl acetate and petroleum ether in the ratio 1: 2)
PRI me R 42. 4,6-Dimethylresor-jj fin.
This compound was prepared in the same manner as described in Example 30, using as starting materials 60 g of 2-methyl-4-nitrosorzorezin, 43.5 g of 2-methyl-resorcin, 34 g of manganese dioxide, 40 ml of sulfuric acid, 400 ml of 25% ammonia solution and 40 g of zinc dust.
UV / VIS. (O, 1 M potassium phosphate buffer with pH 8.5) (cc 585 nm.
PRI amper 43. 2-Bromresorufin.
This compound was prepared in the same manner as described in Example 30, using as starting materials 19 g of 4-bromoresorcine, and g of 4-nitrosorezorecin, 9.7 g of manganese dioxide, 11.5 ml of sulfuric acid, 115 ml 25 % ammonia solution and 1.5 g of zinc dust.
UV / VIS (0.1 M potassium phosphate buffer with pH 8.5), ax 576 nm.
PRI me R 44. 2-Etilresorufin.
This compound was cited in the same manner as described in Example 30, using as starting materials 5 g of 4-ZTIL-6-NIT-rozoresorcin, 3.3 g of resorcin, 3 g of manganese dioxide, 3.4 ml of sulfuric acid, 50 ml of 25% aqueous solution of ammonia and 4 g of zinc drink.
UV / VIS (O, M potassium phosphate buffer, pH 8.5); L „o (Cc 573 nm.
PRI me R 45. 1,6-Dimethylresorufin.
Acquired compound is obtained in the same manner as described in c. of example 30, using as starting materials 7.2 g of 3.5-dioxytoluene, 7.7 g of nitrosorezorecin, 4.9 g of manganese dioxide, 6.8 ml of ser30
35
40
45
50
55
ten
20
25
jj
thirty
hydrochloric acid, 60 ml of a 25% aqueous solution of sulfuric acid and 6 g of zinc dust.
UV / VIS (O, 1 M potassium phosphate buffer with pH 8.5) cp 574 nm. Color test for lipase (P 2711.
The results of the determination of lipase activity with the resorufin-substituted synthetic derivatives of Examples 38-41 are presented in Table 2.
A comparison of the correlation of the method according to the invention and that of Kursok is shown in Table 3.
I

权利要求:
Claims (1)
[1]
Invention Formula
The method of determining lipase, which involved the interaction, was known with the lipase substrate, leading to the formation of a chromogen, followed by an optical method, characterized in that, in order to increase the sensitivity, a common vegetation was used as a substrate. formulas
00
n 1}
HjC-y-C-A-C-X
Where
35
40
45
50
HC-Y-R
I
Hjc-z-ki
R and R X 5
- Alkyl or alkylene groups with 2-12 carbon atoms; the same or different, each represents an alkyl group with 6-18 carbon atoms, with one of R and R representing hydrogen;
Resorufin, b-methylresor fin residue, 4,6-dimethyl resorufin, 1,6-dimethyl resorufin, 8-ethidre: zorufin and 8-bromresor fin;
Ouya Z - each, independently of one another, means —S or -0-;
Z-CHji-,
and the amount of released hydroxyl or thiol compound is directly determined by optical method after combination with the corresponding chromogen.
Correlation with reference method (coefficient of
correlation)
Table3

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引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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DE19853516001|DE3516001A1|1985-05-03|1985-05-03|LIPASE COLOR TEST|

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