EP0710225A1

EP0710225A1 - Fluorescence determination of the activity of lipolytic enzymes

Info

Publication number: EP0710225A1
Application number: EP95920070A
Authority: EP
Inventors: Albin Hermetter; Martin Duque; Fritz Paltauf
Original assignee: Progen Biotechnik GmbH
Current assignee: Progen Biotechnik GmbH
Priority date: 1994-05-19
Filing date: 1995-05-19
Publication date: 1996-05-08
Also published as: JPH09501185A; ATA102994A; WO1995032181A1

Abstract

A fluorometric method was developed to determine the activity of lipases of animal, vegetable and microbial origin, in particular of lipases detectable in serum. For that purpose, fluorescent triglyceride analogues were synthesised in which an acyl or alkyl chain bears a fluorophore at the omega -end and another acyl or alkyl chain bears a fluorescence quencher. The fluorescent lipid is solubilised with amphiphiles in the form of liposomes, vesicles, micelles or emulsions in order to determine lipase activity. The fluorescent substrate may also be dissolved as such in an aqueous medium or be dissolved in organic solvents to be presented to the enzyme. The invention for the first time describes complexes of fluorescent triglycerides with appropriate proteins (for example albumin) in an aqueous medium as new substrate solubilisation forms for lipase analysis. The activity of lipolytic enzymes is determined through hydrolysis of the fluorescent substrate that results in a continuous increase in fluorescence intensity.

Description

"Fluorescence Determination of the Activity of Lipolytic Enzymes"

description

A method based on fluorimetry was developed to determine the activity of lipases of animal, plant and microbial origin (fungal and bacterial lipases) and lipases detectable in serum [lipoprotein lipase (LPL), hepatic lipase (HL), pancreatic lipase (PL)] . For this purpose, fluorogenic triglyceride analogs have been synthesized in which an acyl or

Alkyl chain at the tu end carries a fluorophore and another carries a fluorescence quencher (see formula I and II). The third substituent can be an aliphatic hydrocarbon residue. To determine the activity, the fluorogenic lipid is solubilized with amphiphiles in the form of liposomes, vesicles, micelles or emulsions. The fluorogenic substrate can also be used as such in aqueous

Medium distributed or dissolved in organic solvents are offered to the enzyme. Complexes of fluorogenic triglycerides with suitable proteins (e.g. albumin) in an aqueous medium are described for the first time as a new form of solubilization for substrates in lipase analysis. The hydrolysis of the fluorogenic substrate induced by the fat-splitting enzymes leads to a spatial separation of the fluorophore and quencher-substituted hydrocarbon chain and thus to an increase in fluorescence.

Rι ** - - X - CH ₂ |

R ₂ - Y - CH 1

R ₃ - Z - CH ₂ formula

An essential part of the invention is the complexation of the fluorogenic

Lipids with albumin in an aqueous medium and subsequent lyophilization, which gives a solid "water-soluble" substrate for fluorimetric lipase analysis. The proposed method is suitable for numerous practical applications fertilize such as for lipase analysis in medical diagnostics, biotechnological enzyme production, the quality control of lipases used in organic chemistry, the detection of lipases or lipase inhibitors in food, and the enzyme screening of biological material. The stereoisomers of each of the fluorogenic lipids were prepared and used in various substrate forms (see above) for lipase determination. Different lipases showed very different stereospecificities on the fluorogenic substrates. The described method is therefore suitable for the discrimination of lipases in natural enzyme mixtures (eg serum) as well as for the screening (genetically modified) of modified enzymes.

State of the art:

The following methods for determining the activity of lipases are known (Lipases, Borgström & Brockman, 1984):

1) radiometric methods (Clin. Chem. 30, 748 (1984))

2) titrimetric methods (Methods in Enzymology, Vol. 71, 619-627 (1981))

3) Spectroscopic methods: a) photometric b) fluorometric

ad1) The radiometric methods are discontinuous methods in which the release of radioactively labeled fatty acids from a triglyceride is determined after separation from the unreacted starting compound. These methods are very complex due to the use of radioactivity and the poor reproducibility.

ad2) The release of fatty acids from triglycerides is determined by means of alkali titration. However, large amounts of enzyme and substrate must be used for accurate measurements. This method is very prone to failure. In particular, this method is not well applicable to certain "substrate forms", for example if the substrate is solubilized with detergents or dissolved in organic solvents. ad 3a) In this case, the formation of chromogenic carboxylic acids from their (glycerol) esters is determined photometrically (and fluorimetrically) (Boehringer Mannheim GmbH, European Patent 0207252B1). The sensitivity of these methods is not very high, but the fluorimetric methods are more sensitive and the substrates are more diverse.

ad 3b) Known fluorescence methods (see also 3a) have been described based on the following principles (KSV-Chemicals, Oy Valimotie 7, European Patent 0037583B1). Tripyrenacyl-substituted triglycerides (Biochemica et Biophysica Acta, 963 (1988) 340-348) are described as substrates which show intra- or intermolecular excimer fluorescence in the presence of amphiphiles. In the hydrolytic cleavage of a pyrenacyl chain, the pyrene monomer fluorescence increases at the expense of excimer fluorescence. A second concept is structured similarly. A labeled substrate contains a fluorescent fatty acid (e.g. pyrenebutyric acid), the fluorescence of which is suppressed by a quencher fatty acid adjacent to the molecule (e.g. bromohexanoic acid). Substrate cleavage leads to de-quenching and thus to an increase in fluorescence. Both methods only work if both fluorophores (excimer technology) or fluorophore and quencher (bromine acts at a few tenths of a nm distance) are closely adjacent. As shown below, this is not the case in most cases. At the Third Conference on Methods and Applications of Fluorescence Spectroscopy in Prague / Czech Republic from October 18-21, 1993 ("Conformational effects on the fluorescence of pyrene-labeled alkyldiacyl glycerols in different model membranes", Peer reviewed articles, Plenum Press, in press) we have already reported that di-pyrenacylglycerols show "unfavorable" conformations in micelles, emulsions, in the presence of albumin and in organic solvents and only form intramolecular excimer fluorescence in vesicles. When using bromoacyl-substituted pyrentriglycerides in micelles, emulsions, albumin and in organic solvents, the required neighborhood of bromoacyl and pyrenacyl residue becomes the same

Reasons not reached and the desired quench effect is not achieved. The previously known substrates can at best be used in vesicles for determining the activity of enriched and pure enzymes. With serum

ERSÄΓZBLÄΓT (RULE 26) Analyzes, however, lead to an association of pyrentriglyceride with apolipoproteins and albumin, which leads to the problem mentioned above. A substrate is therefore required which contains fluorophore and quencher in the same molecule and in which the quench effect does not or hardly depends on the conformation. Such quenching can be done by Resonance Energy Transfer (RET)

(the absorption spectrum of the quencher overlaps with the emission spectrum of the fluorophore) (Principles of Fluorescence Spectroscopy, J.R. Lakowicz, Plenum Press, 1983). It is important that the fluorophore and the quencher are bound to the ω-end of the hydrophobic side chains, since in this case no direct interference with the ester hydrolysis by the enzyme can occur.

In addition to chromogenic triglycerides, fatty acid esters of fluorophores (e.g. methylumbelliferone; Analytic Sciences February 1991, Vol. 7, 15-18) have also been described, which fluoresce little intact and, after hydrolysis cleavage, fluoresce less (Biochemica et Biophysica Acta, 1006 (1989) 84-88 ). In general, very high amounts of substrate are required here, which can lead to artifacts when analyzing native biological material.

In all of the methods known to date, detergent is required for the solubilization of the lipophilic substrate. This often destroys the native biological milieu (e.g. solubilization of lipoproteins and denaturation of proteins in human serum) (Human Serum Lipoproteins, Fruchart & Shepherd, de Gruyter 1989).

The present invention describes the chemical synthesis of new fluorogenic lipase substrates (lipid analogs). The optical measurement signal is independent of the conformation of the lipid substrate and thus also of the shape of the substrate. Furthermore, a new substrate form for the solubilization of the substrate is described, which can be used universally for the determination of lipase activities in biological and artificial environments.

ERSÄΓZBLÄΓT (RULE 26) Detailed description:

The substances used for a fluorometric measurement are compounds with the following structure:

RT— X - CH-

R, - Y -— CH

R ₃ - Z ': H, formula

The compounds of the stated structure can be derived from sn-glycerol, where X, Y, Z can be 0. At least one of the radicals R _{1 # 2 # 3} is an acyl or alkyl group which contains a fluorescent radical at the end and at least one further radical R _{1 i2i3} is an acyl or alkyl radical which contains a chromophore bonded at the ω end and which quenches the fluorescence of the fluorophore via resonance energy transfer (RET). At least one of the hydrocarbon chains which contain quenchers or fluorophore must preferably be bound to a primary alcohol function of the glycerol or propane structure via an acyl or thioester bond. A radical R, _{2 3} can be an aliphatic hydrocarbon. However, X, Y or Z can also be S, NH or CH ₂ .

Fluorophore-substituted side chains R _{1> 2 3} can be: I. preferably pyrene bonded to the ω-end of an acyl or alkyl chain with a chain length of C ₂ - C ₂₀ with 0 to 6 double bonds. II. Instead of pyrene, one of the following fluorophores can be used: perylene, anthryl, anthraniloyl, saliciloyl, tetracene, anthracene, phenanthrene, naphthalene, coumarone, coumarin, acridine, aminonaphtolsulfonic acid, phenyloxadiazole, diphenyloxazole, alloxenzophone, stilbene , Fluorene, fluorenone, p-quinone, methylumbelliferone, trinitrophenylamine, phenazine, phenylindole, quinoline, diphenylacetylene, benzothiophene, cyanothiocarbonyls, 1, 3,5,7-decatetraene, rhodamine, diphenylhexatriene, dansyl, 7-nitrobenz-2-oxa -1, 3-diazole (NBD), benzocarbazone, dibenzofuran, parinaric acid, 4,4-difluoro-4-bora-3a, 4a-diaza-5- indacen.

Quencher-substituted side chains R _{1 <2} . ₃ can be:

I. for pyrene as fluorophore, preferably trinitrophenylamine bonded to the ω end of an acyl or alkyl chain with a chain length of C ₂ -C ₂₀ with 0 to 6 double bonds.

II. Instead of trinitrophenylamine, all chromogenic or fluorogenic compounds can be used whose absorption spectrum (acceptor) overlaps with the emission spectrum (donor) of the fluorophore measured. All suitable combinations of fluorophores (see above, II.) Can be used as suitable donor-acceptor combinations.

The 1 -O-hexadecyl-2-pyrendecanoyl-3-trinitrophenylaminododecanoyl-sn-glycerol shown in the next structure serves as an example of the type of compound described.

Formula II

The starting compound for the fluorogenic alkyldiacylglycerol is a 1-O-alkylglycerol, which by known methods (see Hermetter & Paltauf) in ether lipids; Biochemical and Biomedical Aspects (H. Mangold «St F. Paltauf, Editors). Academic Press, 1983, p. 389) is converted into a 1-O-alkyl-2-acyl-3-O-tritylglycerol (acylation protocol: F. Paltauf & A. Hermetter, Methods in Enzymology 197 (1 991) 134), the 2-acyl group the fluorophore or the Quencher can wear. According to a process which has already been described for the synthesis of phospholipids (Chem. Phys. Lipids, 50 (1989) 57-62) and which is described here for the first time for triglyceride synthesis, the trityl group is replaced by a further acyl radical which is complementary to Position 2 carries a quencher or fluorophore. This method can also be used to make up the substituted 1,2-dialkyl-3-O-trityl, 1,2-acyl-2-O-alkyl-3-O-trityl and 1,2-diacyl-3-O-tritylglycerols to produce corresponding dialkylacyl and triacylglycerols, one side chain of the starting compound containing fluorophor or quencher and an acyl residue which contains quencher or fluorophore complementary to it being introduced via the trity exchange. Likewise, the corresponding 1,3-diacyl-2-acylamino-propanediols, which are a fluorophore and a quencher, can be prepared by 1-acyl-2-acylamino-3-O-trityl-1,3-propanediols by trityl group exchange by this method -substituted acyl radical contain.

For the fluorimetric determination of lipases, the new substrates can be distributed by all conventional methods in an aqueous environment (Lipases, Borgström & Brockman (Editors), Elsevier, 1984), specifically in phospholipid vesicles, in detergent micelles and emulsions, or dissolved in organic solvents.

A new solubilization (substrate) form is described here, the substrate being distributed in a defined molar ratio in the aqueous phase by complexing with a suitable protein, preferably bovine serum albumin (BSA). For this purpose, an ethanolic solution of the lipid analog is injected into an aqueous buffer solution with a defined pH, which already contains BSA. The substrate form (lipid-protein complex) obtained in this way can be used both in biological and in artificial environments. It should be emphasized in particular that the native composition of sensitive biological systems, e.g. of human serum, not destroyed and the lipase activity of these samples can take place in the presence of their intact components (proteins, lipoproteins!). Lipid-binding proteins, in particular the following substances, can generally be used as complexing protein: BSA, HSA (human

ERSÄΓZBLÄΓT (RULE 26) serum albumin), casein, lactalbumin, ovalbumin, water-soluble apolipoproteins and modified forms of the aforementioned proteins (methylated, carboxylated, acetylated and proteins modified by chemical or genetic methods). Both the fat-free and the commercially available proteins (only partially defatted) were examined. The complexes shown are associations of lipid and protein in a molar ratio of 1/1.

The water-soluble lipid-protein complex can be freeze-dried using a lyophilizer and isolated as a solid powder. After redissolving in aqueous media, these preparations are suitable for measuring lipase activities. The lyophilization of the lipid-protein complex can be carried out in the absence or in the presence of buffers or mineral salts and of low or high molecular weight additives, preferably carbohydrates. Lyophilization is preferably carried out from a solution of 10 millimolar N- (2-hydroxyethyl) piperazine-N '- (2-ethanesulfonic acid) / NaOH (HEPES-

NaOH), pH 7.4, and glycogen or PBS (2 g KCI, 2 g KH ₂ PO ₄ , 80 g NaCI and 21, 6 g Na ₂ HPO ₄ ^• H ₂ O in 1 liter H ₂ O) and starch. For the determination of lipase, the lyophilisate can preferably be redissolved in distilled water if the lyophilization was carried out from buffer salt solution, or in aqueous buffer solution which preferably contains HEPES-NaOH or phosphate or Tris-HCl, pH 7.4.

To prepare the complex, the solution of the fluorogenic triglyceride corresponding to formula I or II, preferably in ethanol, is stirred into an aqueous solution of protein, preferably albumin, which may contain buffer or mineral salts, at temperatures, preferably between 20 and 37 ° C, injected. The ethanol-water ratio is less than 1/100, v / v. This preparation can be used immediately for a lipase determination or, advantageously, after an equilibration phase of approximately 20 hours. After adding lipase or a preparation containing lipase, the activity of the lipase can be determined continuously via the time-dependent increase in the fluorescence intensity (FIG. 1). The lipase (s) in the sample cleaves the lipid analog and leads by spatial separation of fluorophore and quencher to an increase in fluorescence linearly dependent on the extent of the hydrolysis. The low initial intensity of the fluorophore in the intact substrate is caused by the presence of fluorophore and quencher together on one molecule. This base value is therefore an important indicator for the quenching of the fluorophore in the substrate and thus for the quality and

Integrity of the test substance.

The lipase determination can also be carried out as an end point method, with a simple reading of the increase in fluorescence intensity taking place after a defined incubation time. The fluorescence measurement itself can be carried out in a continuous or discontinuous form with a fluorescence spectrometer, fluorescence microscope or plate reader. The lipase-induced cleavage of the fluorogenic substrate can be calculated quantitatively from the increase in the fluorescence intensity with the aid of a calibration curve (FIG. 2), which was created using unquenched pyrene-substituted triglycerides.

The substrate conversion, which is obtained from the time-dependent increase in fluorescence, corresponds to the increase in the fluorescent cleavage products which, after thin-layer chromatographic separation on silica gel (mobile phase: petroleum ether / chloroform / glacial acetic acid 96/4/1) and elution with chloroform / methanol 2/1 fluorimetric were determined. There is a linear relationship between the increase in fluorescence and the amount or activity of lipase in the test mixture (FIG. 3). This linearity has been commonly found for animal and microbial lipases. In particular, the following were examined: lipoprotein lipase in human serum, in cow's milk, in human breast milk; enriched and pure lipoprotein lipase; Pancreatic lipase in the presence and absence of

Colipase; hepatic lipase; as well as lipases from Pseudomonas species, Chromobacterium viscosum, Candida rugosa, Rhizopus arrhizus, Geotrichυm candidum.

The present invention can be put into practical use in the following important areas of lipase analysis.

1) Medical diagnostics: The determination of lipase activity in human serum is important when examining pathological cases such as fat metabolism disorders (hyperlipidemia, obesity), as well as destructive processes.

SPARE BLADE (RULE 26) sen, which lead to organ damage, whereby enzymes are released into the serum, which either do not occur at all or only in a very low concentration (eg in the case of carcinomas or inflammatory processes of the liver and the pancreas, or damage to these organs in the case of alcoholism ). 2) Screening of lipases in microorganisms and plants as well as chemically and genetically modified enzymes.

3) Stereoisomers of the substances of the formulas I and II can be used for the screening of different stereoselectivities of natural and chemically and genetically modified lipases. For example, the sn-1 acyl isomer of compound II of lipoprotein lipase is converted about 25 times faster than the sn-3 acyl isomer. Other lipases, which can be detectable in the serum, such as, for example, pancreatic lipase, show lower stereophonic preferences. The proportion of lipoprotein lipase in the total lipase activity of the serum can therefore be determined from the degree of the stereo preference measured. 4) Activity screening for the isolation and purification of natural and genetically engineered and biotechnologically produced lipases, which are purified using various methods such as column chromatography, HPLC, gel electrophoresis, ultrafiltration. After electrophoretic separation, the enzyme detection can be carried out directly on the gel. 5) Determination of lipases and lipase inhibitors from food.

According to the invention, a kit for determining the activity of lipases, in particular lipoprotein, hepatic and pancreatic lipases, is also provided, comprising a lipase, in particular a lipoprotein, hepatic and / or pancreatic lipase, as a positive control and one or more compounds according to one of claims 1 to 1 5 and usual auxiliary substances.

The following examples further illustrate the invention:

example 1

1 -0-hexadecyl-2-pyrendecanoyl-3- (2 ', 4', 6'-trinitrophenylaminododecanoyl) -sn.-

ERSÄΓZBLÄΓT (RULE 26) glycerol

1 -Q-Hexadecyl-sn-glycerol is tritylated with trityl chloride at the sn-3 position (see Hermetter & Paltauf in Etherlipids; Biochemical and Biomedical Aspects (H. Mangold & F. Paltauf, Editors), Academic Press, 1983, p. 389) and the resulting alkyltritylglycerol with pyrendecanoic acid on the still free sn-2 OH-

Group esterified to 1 -O-hexadecyl-2-pyrendecanoyl-3-O-trityl-.sn-glycerol (acylation protocol: F. Paltauf & A. Hermetter, Methods in Enzymology 197 (1991) 134). A solution of the resulting 1 -0-hexadecyl-2-pyrendecanoyl-3-O-trityl-sn-glycerol, 150 mg (109.5 / mol), and of 280 mg (335 μmol) trinitrophenylaminododecanoic anhydride and 500 μ \ boron trifluoride etherate

(50%) in 2 ml of methylene chloride is stirred for half an hour at 0 ° C and two more hours at room temperature. The reaction mixture is neutralized by adding NaHC0 ₃ with stirring. The solution of the crude product in the organic solvent is separated off and concentrated to 1 ml in a stream of N ₂ . Then 10 ml of petroleum ether are added. The precipitate is discarded and the supernatant is applied to a silica gel column. Elution of the column with petroleum ether / ethyl acetate 9/1 (v / v) yields 86.3 mg (79.9 mol) of the product (73% yield), which is uniform on silica gel by thin layer chromatography. TLC: Rf = 0.79 (petroleum ether / chloroform 1: 4)

Example 2

1 - (2 ', 4', 6'-trinitrophenylaminododecanoyl) -2-pyrendecanoyl-3-0-hexadecyl-sn_-glycerol Preparation analogous to Example 1: from 3-0-hexadecyl-.sn-glycerol, from which initially

1 -0-trityl-2-pyrendecanoyl-3-0-hexadecyl-sn_-glycerol was obtained. From the latter compound (300 mg, 328, 4 μmol), 1 -trinitrophenylaminododecanoyl-2-pyrendecanoyl-3-0-hexadecyl-srvglycerol (212.7 mg, 197, 1 / mol, 60% yield) was obtained after column chromatography. TLC: Rf = 0.79 (petroleum ether / chloroform 1: 4)

SPARE BLADE (RULE 26) Example 3

1 -0-Dodecyl-2-pyrendecanoyl-3- (2 ', 4', 6'-trinitrophenylaminododecanoyl) -sn.-glycerol

Preparation analogous to Example 1: from 100 mg (103.2 // mol) 1 -0-dodecyl-2-pyrenecanoyl-3-O-trityl-sn-glycerol 72.9 mg (71.2 // mol, 69% yield) 1-

Obtained 0-dodecvl-2-pvrendecanovl-3- (2 ', 4', 6 ^, -trinitrophenylaminododecanoyl) -sn-glycerol.

TLC: Rf = 0.75 (petroleum ether / chloroform 1: 4)

Example 4

1- (2 ', 4', 6'-trinitrophenylaminododecanoyl) -2-pyrendecanoyl-3-0-dodecyl-sjι- glycerol Preparation analogous to Example 1: from 100 mg (103.2 // mol) 1 -0-trityl- 2-pyrendecanoyl-3-O-dodecyl-.sn-glycerol were 63.4 mg (61, 9 // mol, 60% yield) 1 - (2 ^, .4 ^, .6 ^, -trinitrophenvlaminododecanoyl) -2- pyrendecanovl-3-O-dodecyl-sn-glycerol obtained. TLC: Rf = 0.75 (petroleum ether / chloroform 1: 4)

Example 5

1 -0-tetradecyl-2-0-pyrendecyl-3- (2 ', 4', 6'-trinitrophenylaminohexanoyl) -sn.- glycerol 1 -Q-tetradecyl-sn-glycerol is mixed with trityl chloride into 1 -Q-tetradecyl- 3-Q-trityl-sn-glycerol converted (see Hermetter & Paltauf in Etherlipids; Biochemical and Biomedical Aspects (H. Mangold & F. Paltauf, Editors), Academic Press, 1983, p. 389). Alkylation of the latter substance with pyrenhexyl mesylate, which was obtained from pyrenhexanol and mesyl chloride (see Hermetter & Paltauf in Ether lipids; Biochemical and Biomedical Aspects (H. Mangold & F. Paltauf, Editors),

Academic Press, 1983, p. 389), provides 1 -0-tetradecyl-2-0-pyrendecyl-3-0-trityl-stvglycerol. The dialkyltritylglycerol (250 mg, 286.9 μmol) is trinitrophenylaminohexanoic anhydride (573.7 mg, 860.7 / mol) in the presence of Boron trifluoride etherate (50%), analogous to the process in Example 1, converted into the end product (191.4 mg, 200.8 / mol, 70% yield). TLC: Rf = 0.85 (petroleum ether / chloroform 1: 4)

Example 6

1 - (2 ', 4', 6'-trinitrophenylaminohexanoyl) -2-0-pyrendecyl-3-0-tetradecyl-s_n-glycerol

Preparation analogous to Example 5: from 200 mg (229.5 // mol) 1 -0-trityl-2-0-pyrenecyl-3-O-tetradecyl-.sn-glycerol, 148.8 mg (156.1 / mol, 68% yield)

1 -Trinitrophenylaminohexanoyl-2-0-pyrendecyl-3-0-tetradecyl-srι-glycerol. DC: Rf = 0.85 (petroleum ether / chloroform 1: 4)

Example 7

rac.1-0leoyl-2-pyrendodecanoyl-3- (2 ', 4', 6'-trinitrophenylaminododecanoyl) glycerol

Analogously to the process in Example 1, rac.1 -oloyl-glycerol rac.1 -oloyl-3-0-trityl-glycerol is produced from rac.1- which contains pyrendodecanoic anhydride in rac.1-

Oleoyl-2-pyrendodecanoyl-3-0-tritylglycerol is converted. The conversion of the diacyltritylglycerol (200 mg, 209.8 // mol) with trinitrophenylaminododecanoic anhydride (525.5 mg, 629.4 // mol) in the presence of boron trifluoride etherate (50%) gives rac.1-oleoyl -2-pyrendodecanoyl-3- (2 ', 4', 6'-trinitrophenylamino-dodecanoyD-glycero! (122, 1 mg, 109, 1 / mol, 52% yield).

TLC: Rf = 0.75 (petroleum ether / chloroform 1: 4)

Example 8

1 - (2 ', 4', 6'-trinitrophenylaminododecanoyl) -2-pyrendodecanoyl-3-oleoyl-srι-glycerol

Analogous to the procedure in Example 7, 3-oleoyl- ^j > n-glycerol is firstly converted into 1 -0-trityl-2-pyrendodecanoyl-3-oleoyl-sn-glycerol via the oleoyltrityl-sjvglycerol

SPARE BLADE (RULE 26) converted, which gives the final product (50% yield) with trinitrophenylaminododecanoic anhydride in the presence of boron trifluoride etherate. TLC: Rf = 0.75 (petroleum ether / chloroform 1: 4)

Example 9

1-Oleovl-2-pvrendodecanoyl-3- (2 ', 4', 6'-trinitrophenvlaminododecanoyl) -sn-glycerol

Analogously to the process in Example 8, 1-oleoyl-2-pyrendodecanoyl-3-O-trityl-sn-glycerol is converted into the end product (yield 56%).

TLC: Rf = 0.75 (petroleum ether / chloroform 1: 4)

Example 10

rac.1-pyrendecanoyl-2-0-hexadecyl-3- (2 ', 4', 6'-trinitrophenylaminododecanoyl) glycerol

2-O-Hexadecylglycerol (see Hermetter & Paltauf in Etherlipids; Biochemical and Biomedical and Biomedical Aspects (H. Mangold & F. Paltauf, Editors), Academic Press, 1983, p. 389) (100 mg 31 5 // mol) acylated with pyrendecanoic acid in the presence of dimethylaminopyridine (85.5 mg, 100 // mol) and dicyclohexylcarbodimide (75 mg, 350 mol) in 5 ml of CHCI ₃ ^' (acylation protocol: F. Paltauf & A. Hermetter, Methods in Enzymology 197 (1991) 134). The rac.1 - pyrendecanoyl-2-O-hexadecyl-glycerol formed was chromatographed on silica gel (elution with a gradient of petroleum ether-ether). The pure product was obtained in 87% yield (146 mg); TLC on silica gel: Rf = 0.40 (chloroform / acetone / glacial acetic acid 96: 4: 1). The acylalkylglycerol (146 mg, 188 // mol) was then trinitrophenylaminododecanoic acid (100 mg, 230 // mol) in the presence of 52 mg dimethylaminopyridine (423 // mol) and 42 mg dicyclohexylcarbodimide (200 / mol) in 5 ml Acylated chloroform. The rac.1 -pyrendecanoyl-2-0-hexadecyl-3- (2 ', 4', 6'-trinitrophenylaminododecanoyl) glycerol formed was turned on

Silica gel (elution with a gradient of petroleum ether-ether (silica gel, chloroform / acetone / glacial acetic acid 96: 4: 1) purified (137 mg, 68% yield). TLC: Rf = 0.80

ERSÄΓZBLÄΓT (RULE 26) Example 1 1

rac.1 -Tetradecanoylamino-2-pyrendecanoyl-3- (2 ', 4', 6'-trinitrophenylaminodode-canoyl) propanediol The synthesis starts from rac.1 -Tetradecanoylamino-3-0-tritylpropanediol, which was prepared according to the literature procedure (R. Dijkman, N. Dekker, and GH Haas, Biochim.Biophys.Acta 1043 (1 990) 67-74). The acyltritylpropanediol was esterified with pyrendecanoic acid to rac.1-tetradecanoylamino-2-pyrendecanoyl-3-O-tritylpropanediol (the same method was used for the acylation of alkyltritylglycerols, see Hermetter & Paltauf in ether lipids;

Biochemical and Biomedical Aspects (H. Mangold & F. Paltauf, Editors), Academic Press, 1983, p. 389. This compound (200 mg, 286.5 // mol) was converted into 1 53.3 mg (177.6 // mol, 62% yield) by trityl group exchange with trinitrophenylaminododecanoic anhydride, analogously to the process given in Example 1. of the final product.

TLC: Rf = 0.70 (petroleum ether / chloroform 1: 4)

Example 12

In a solution of 9 nmol (0.6 mg) bovine serum albumin (BSA) in 9 ml 10 mM

Tris / HCl, pH 7.4, a solution of 9 nmol (9 // g) 1 -0-hexadecyl-2-pyrendecanoyl-3- (2 ', 4 ', 6'-trinitrophenylaminododecanoyl) - ^ rvglycerol (Example 1) injected in 30 μ \ ethanol at room temperature. 3 ml of a solution of a BSA-triglyceride complex obtained in this way are mixed with 40 μl of lipase sample (lipoprotein lipase). The reaction with a fluorimeter at 37 ° C. is followed via the time-dependent increase in fluorescence at 380 nm (excitation 342 n; slit width: excitation 10 nm emission 10 nm). The lipase activity of the sample in pmol converted substrate / test volume / time unit (here 2.1 pmol / ml / min) is determined from the slope of the increase in fluorescence (FIG. 1) and via a calibration line

(Fig. 2) determined which was obtained for known concentrations of hexadecylpyrenecanoyltritylglycerol (HPTG) in aqueous albumin solution. The lipase activities determined are linearly related to the

ERSÄΓZBLAΓT (RULE 26) set amount of enzyme (in μg) (Fig. 3).

Example 13

As described in Example 12, the activity of 1 ug Lipoprotein¬ lipase (from cow's milk) to an albumin-substrate complex of BSA and 1- ^(2, ^4, ^6, -Trinitrophenvlaminododecanovl) -2-pyrendecanovl-3- 0-hexadecyl-sn-glycerol (Example 2) determined to be 52.3 pmol / ml / min.

Example 14

As described in Example 12, the activity of 1 μg lipoprotein lipase (from cow's milk) on an albumin-triglyceride complex of BSA and 1-O-dodecyl-2-pyrendecanoyl-3- (2 ', 4', 6 ' -trinitrophenylaminododecanoyl) -sn_-glycerol (Example 3) determined to 6 pmol / ml / min.

Example 15

As described in Example 12, the activity of 1 μg lipoprotein lipase (from cow's milk) on an albumin-triglyceride complex of BSA and 1-

(2 ', 4', 6'-trinitrophenylaminododecenoyl) -2-pyrendecanoyl-3-0-dodecyl-sπ-glycerol (Example 4) determined to be 150 pmol / ml / min.

Example 16

The use of various complexing proteins for fluorogenic substrates of lipoprotein lipase gave the results summarized in Table 1. A complex of protein and 1 -0-hexadecyl-2-pyrendecanoyl-3- (2 ', 4', 6'-trinitro-phenylaminododecanoyl) -. Sn-g ^| ycerol was prepared according to Example 12, using the proteins listed in the table.

The proteins listed were purchased (BSA: Boehringer Mannheim GmbH; the rest from Sigma).

The lipoprotein lipase activity was determined according to the example 1 2 Regulation determined.

Table 1

Protein activity [pmol / ml / min]

BSA (bovine serum albumin) 18

BSA-palmitoylated 8

BSA acetylated 13

HSA (human serum albumin) 13

Ovalbumin 4

Casein 5

Lactalbumin 13

Globulin 1, 3

Example 17

For various lipases on a complex of 1 -0-dodecyl-2-pyrenecanoyl-3- (2 ', 4', 6'-trinitrophenylaminododecanoyl) - ^ rι-glycerol and BSA activities were measured which depend linearly on the enzyme concentration were. Preparation of the complex, activity measurement and evaluation were carried out as described in Example 1 2. No activities were found with pork liver esterase and pancreatic phospholipase A ₂ (both from Boehringer Mannheim).

The following lipases showed clear activities: swine pancreatic lipase (PL) (from Boehringer Mannheim GmbH), lipoprotein lipase (LPL) from human serum and cow's milk, as well as human serum and breast milk; microbial lipases from: Chromobacterium viscosum, Candida rugosa, Rhizopus arrhizus, Pseudomonas species (from Nagase Biochemicals, Ltd., Japan), Geotrichum candidυm.

SPARE BLADE (RULE 26) Example 18

The influence of salts and detergents on the lipase activity of lipoprotein lipase was determined.

Complex used: preparation and composition as described in Example 17.

Lipoprotein lipase used: LPL from cow's milk.

The results are summarized in Table 2:

Table 2

Additive activity [pmol / ml / min] without 18

0.1 M NaCI 26

10 mM CaCl ₂ 30

20 mM Triton 22

10mM CaCl ₂ + 20mM Triton 40

Example 19

As described under Example 13, the activities of 20 μ \ human plasma before and after heparinization were a normolipemic

Donors (60 U / kg body weight, blood draw after 25 min) performed. Serum lipase activity before heparinization: low to no activity Serum lipase activity before heparinization: 60 pmol / ml / min

Example 20

The complex of fluorogenic substrate and bovine serum albumin obtained in Example 12 in an aqueous medium is frozen at -75 ° C. and then lyophilized.

Example 21

As described in Example 20, a lyophilisate was prepared from albumin and fluorogenic substrate, a solution of 25 mg glycogen in 3 ml of 10 mM HEPES, pH 7.4 (pH adjustment with NaOH) being used as the aqueous medium.

Example 22

As described in Example 20, a lyophilizate was prepared from albumin and fluorogenic substrate, a solution of 25 mg of sucrose in 3 ml of 10 mM Na phosphate buffer, pH 7.4, being used as the aqueous medium.

Example 23

As described in Example 20, a lyophilizate was prepared from albumin and fluorogenic substrate, a solution of

25 mg starch in 3 ml PBS (2 g KCl, 2 g KH ₂ P0 ₄ , 80 g NaCl and 21, 6 g NA ₂ HP0 ₄ Η ₂ 0 in 1 liter H ₂ 0), pH 7.4, was used.

Example 24

The lyophilisates prepared under Example 20-23 were dissolved in 9 ml of _distilled H ₂ O. 3 ml aliquots each were used for the activity determination of LPL as described under Example 11. The results can be found in Table 3: Table 3

Lyophilisate activity (pmol / ml / min) from Tris / HCl 7 from HEPES + glycogen 1 2 from phosphate buffer + sucrose 10 from PBS + starch 13

Claims

1 . Racemates and enantiomers of the general formula

R A— X— CH-

R, _ B - Y— CH

R., C— Z— CH,

wherein R _{1 #} R ₂ and R ₃ alkyl or alkenyl chains with 2 to 26 carbon atoms, where the number of double bonds can be 0-6, X, Y and Z -S-, -0-, -NH- or also -CH ₂ -, A, B, C, 0

-C- or -CH ₂ -, characterized in that one of the side chains RR ₂ and R _{3 contains} a fluorophore as a molecular optical sensor and another radical R _{1 #} R ₂ and R ₃ contains a quencher, all of which are chromogenic and fluorogenic Substituents can be quenchers whose absorption spectrum overlaps the emission spectrum of the fluorophore, which is used as a molecular optical sensor, and which are therefore suitable as resonance energy transfer (RET) acceptors for the sensor fluorophore (RET donor).

2. Compounds according to claim 1, characterized in that as fluorescent groups pyrene, perylene, anthryl, anthraniloyi, saliciloyl, tetracene, anthracene, phenantrene, naphthalene, coumarone, coumarin, acridine, aminonaphtol sulfonic acid, phenyloxadiazole, diphenyloxazole, alloxa- oxazole, alloxa- zin, stilbene, dibenzopyrone, fluorene, fluorenone, p-quinone, methylumbelliferone, trinitrophenylamine, phenazine, phenylindole, quinoline, diphenylacetylene, benzothiophene, cyanothiocarbonyls, 1, 3,5,7-decatetraene, rhodamine,

Diphenylhexatriene, dansyl, 7-nitrobenz-2-oxa-1,3-diazole (NBD), benzocarbazone, dibenzofuran, parinaric acid, 4,4-difluoro-4-bora-3a-4a-diaza-5-

-indacen be used. 3. Compounds according to claim 1 and 2, characterized in that as quenching groups trinitrophenylamine or pyrene, perylene, anthryl, anthraniloyl, saliciloyl, tetracene, anthracene, phenanthrene, naphthalene, coumarone, coumarin, acridine, aminonaphtholsulfonic acid, phenyloxoxaziazole, diphenylazine, diphenyl Stilbene, dibenzopyrone, fluorene, fluorenone, p-quinone, methylumbelliferone, phenazine, phenylindole, quinoline, diphenylacetylene, benzothiophene, cyanothiocarbonyls, 1, 3,5,7-decatetraene, rhodamine, diphenylhexatriene, dansyl, 7-nitrobenzene 2-oxa-1,

3-diazole (NBD), benzocarbazone, dibenzofuran, parinaric acid, 4,

4-Difluoro-4-bora-3a, 4-diaza-5-indacen can be used, the absorption spectra of which must meet the conditions of claim 1.

Racemate and enantiomers of O-alkyl-diacyl-glycerols, di-O-alkyl-acyl-glycerols and triacylglycerols according to Claim 1, 2 and 3.

5. Racemate and enantiomers of the compound according to claim 1, 2 and 3, characterized in that they have the formula

Has.

1 -0-dodecyl-2-pyrendecanoyl-3- (2 ', 4',

6'-trinitrophenylaminododecanoyl) - sn-glycerol.

SPARE BLADE (RULE 26)

7. 1 - (2 ', 4', 6'-trinitrophenylaminododecanoyl) -2-pyrendecanoyl-3-0-dodecyl-sn-glycerol.

8. 1 -0-Tetradecyl-2-0-pyrendecyl-3- (2 ', 4', 6'-trinitrophenylaminohexanoyD-sn-glycerol.

9. 1 - (2 ', ^4,, 6'-Trinitrophevlaminohexanovl) -2-0-pyrendecvl-3-0-tetradecyl-sn-glycerol.

10. rac.1-oleoyl-2-pyrendodecanoyl-3- (2 ', 4', 6'-trinitrophenylaminododecanoyl) glycerol.

1 1. 1 - (2 ', 4', 6'-trinitrophenylaminododecanoyl) -2-pyrendodecanoyl-3-oleoyl-sn-glycerol.

12. 1-Oleoyl-2-pyrendodecanoyl-3- (2 ', 4', 6'-trinitrophenylaminododecanoyl) - sn-glycerol.

13. rac.1-pyrendecanoyl-2-0-hexadecyl-3- (2 ', 4', 6'-trinitrophenylaminododecanoyl) glycerol.

14. rac.1-tetradecanoylamino-2-pyrendecanoyl-3- (2 ', 4', 6'-trinitrophenylaminododecanoyU-propanediol.

15. Racemates and enantiomers of alkyl acyl, dialkyl and diacyltrityl glycerols according to claims 1, 2, 3 and 4, which have the formula

R _T - O— CH

R, • O— CH

Tr - 0— CH,

Tr = trityl (triphenylmethyl)

SPARE BLADE (RULE 26) have, characterized in that a radyl radical R or R _{2 contains} a fluorophore and another radyl radical R ₂ or R contains a quencher covalently bonded.

1 6. Process for the preparation of triacyl, alkyldiacyl and dialkylacylglycerols, characterized in that in the corresponding alkylacyltritylglycerols, dialkyltritylglycerols or diacyltritylglycerols the trityl group is replaced by an acyl group in the presence of boron trifluoride etherate as a catalyst in a one-step reaction becomes.

17. Process for the preparation of the new enantiomeric triglyceride analogues 1--0-hexadecvl-2-pvrendecanovl-3-trinitrophenvlaminododecanoyl-sn-glycerol, 1-0-hexadecvl-2-pyrendecyl-3-trinitrophenvlaminododececoyl-sn-glycole Hexadecarιoyl-2-pyrendecanoyl-3-trinitrophenylaminododecanoyl-sn-glycerol, characterized in that in the corresponding

Alkylacyltritylglycerols, dialkyltritylglycerols or diacyltritylglycerols the trityl group is replaced by an acyl group in the presence of boron trifluoride etherate as a catalyst in a one-step reaction.

18. Water-soluble complexes of fluorogenic compounds according to one of the

Claims 1 to 1 5, characterized in that the fluorescent lipid with water-soluble lipid-binding proteins, preferably with Rinder¬ serum albumin, human serum albumin, ovalbumin, lactalbumin, casein, apolipoproteins, or proteins modified by acylation, alkylation, hydroxymethylation, carboxymethylation or carboxylation aqueous solution is solubilized.

1 9. Lyophilisate of water-soluble lipid-protein complexes according to claim 1 8.

20. Water-soluble complexes of the enantiomeric triglyceride analogues 1 -0-hexadecyl-2-pyrendecanoyl-3-trinitrophenylamino-dodecanoyl-_sn, -glycerol, 1--0-hexadecyl-2-pyrendecyl-3-trinitrophenylaminododecanoycerol and 1 -0-hexadecanoyl-2-pyrendecanoyl-3-trinitrophenylaminododecanoyl-sn-glycerol, characterized in that these lipids with water-soluble lipid-binding proteins, preferably with bovine serum albumin, human serum albumin, ovalbumin, lactalbumin, casein, apolipoproteins Alkylation, hydroxymethylation, carboxymethylation or carboxylation of modified proteins are solubilized in aqueous solution.

21. Lyophilisates of water-soluble lipid-protein complexes according to claim 20.

22. Lyophilisates according to claims 19 and 21, which are prepared in the presence of additives, preferably starch, glycogen, organic and inorganic salts, sucrose.

23. Fluorimetric determination of lipase activities, characterized by offering complexes of the fluorogenic substrates with water-soluble proteins according to claims 18 and 20 or solutions of the lyophilisates according to claims 19 and 21 in aqueous buffer solution to a fat-splitting enzyme and the enzyme activity the change in a fluorescence parameter, preferably determined via the increase in the fluorescence intensity of the sensor fluorophore.

24. Fluorimetric determination of lipase activities, characterized in that compounds according to one of claims 1 to 15 solubilized in phospholipid liposomes, vesicles, detergent micelles or emulsions, solutions in organic solvents or mixtures of organic solvents with water of the lipase and the Enzyme activity determined by changing a fluorescence parameter, preferably by increasing the fluorescence intensity of the sensor fluorophore.

25. Use of the determination methods according to claim 23 or 24 in medical diagnostics for clinical lipase analysis preferably in human serum, plasma and adipose tissue after or without heparinization of the donor, in the case of pathological manifestations such as carcinoma, obesity, hyperlipidemia, alcoholism, hepatitis, pancreatitis and in healthy donors, for lipase analysis in biotechnological lipase production, for the screening of lipases from animal, vegetable and microbial material, as well as chemically and genetically modified or produced lipases, as well as in nutritional and food chemistry studies, preferably for the screening of lipases and lipase inhibitors.

26. A method for screening the stereospecificity of lipases, characterized in that the activity of the lipases on both enantiomers of the fluorogenic compounds according to one of claims 1 to 1 5 is determined using a method according to claim 22 or 23.

27. Kit for determining the activity of lipases, in particular lipoprotein, hepatic and pancreatic lipases, comprising a lipase, in particular a lipoprotein, hepatic and / or pancreatic lipase, as a positive control and one or more compounds according to one of claims 1 to 15 and usual auxiliary substances.

SPARE BLADE (RULE 26)