Title of Invention

A PROCESS FOR DETERMINATION OF THE INTEGRITY OF COMPLEX PHOSPHOLIPID/LIPID STRUCTURES

Abstract

The invention relates to a simple continuous test for identifying structures that favour the arrangement of aromatics for charge-transfer complexes, e.g. complex phospholipid/lipid structures (bilayer, monolayer, aggregates, micelles), using synthetic fluorescence-marked acylglycerides, and to their use for determining the activity of lipases/lipase inhibitors, to a monoacylglyceride for use in this test and to a method for producing the same, to the substrate obtained therefrom and to a method for producing said substrate.

Full Text

Description DETERMINATION OF COMPLEX PHOSPHOLIPID/LIPID STRUCTURES WITH THE AID OF SYNTHETIC FLUORESCENCE-LABELED ACYLGLYCERIDES The invention relates to a simple continuous test for the identification of structures which favor the arrangement of aromatics to give charge-transfer complexes, such as, for example, complex phospholipid/lipid structures (bilayer, monolayer, aggregates, micelles), with the aid of synthetic fluorescence-labeled acylglycerides, and its use for determination of the activity of lipases/lipase inhibitors. Lipases, phospholipases and other lipolytic enzymes have great importance in the biotechnological and medical field. In certain metabolic disorders, increased lipase activity in the fatty tissue can be detected, which is held partly responsible for the pathogenesis of this disease. The greatest part of the energy reserves of the body is stored in cells of the fatty tissue as fatty acids of the triglycerides. The essential anabolic processes caused by insulin include the stimulation of the uptake of substrates for triglyceride synthesis and the increase in lipogenesis. A further important process caused by insulin is the inhibition of lipolysis, the process by means of which catabolic hormones, primarily catecholamines, stimulates the hydrolysis of triglycerides and thereby induce the release of fatty acids. An important problem which is linked with noninsulin-dependent diabetes (NIDDM) has its cause in the uninhibited lipolysis of the fat cells, which leads to increased levels of unesterified fatty acids in the plasma. According to a present idea, the fatty acids stimulate gluconeogenesis in the liver and decrease the glucose utilization in the skeletal muscle by means of still poorly characterized molecular mechanisms. In fact, it was possible to show that the suppression of lipolysis in fat cells by inhibitors of lipolysis, such as agonists of the nicotinic acid receptor of the fat cell, lowers both the fatty acid concentrations in the plasma and raised blood sugar in diabetic animals and patients. Unfortunately, these beneficial effects are not particularly strongly pronounced and only of relatively short duration. This may be based on a physiological counterregulation caused by intervention in the regulatory mechanism of the rate-determining enzyme of lipolysis, the hormone-sensitive lipase (HSL). There are good reasons to assume that the inhibition of the lipolytic reaction will lead to an improved therapy of NIDMM, at least with respect to the suppression of the fatty acid release from the fat cells. The direct inhibition of HSL by suitable inhibitors should in this case get around the obvious difficulties of an intervention into the complex regulation of HSL The activity of lipolytic enzymes is traditionally investigated using radiometric, titrimetric, enzymatic or fluorimetric/photometric methods. Radiometric assays are the most sensitive, but they require expensive radiolabeled substrates, are discontinuous and require the separation of the radiolabeled substrate from the radiolabeled product. Such separations are often troublesome and the avoidance/reduction of radioactive waste is of increasing importance (especially relevant if there are a large number of tests). Titrimetric tests are continuous and can be carried out both with natural and synthetic substrates, but they frequently suffer from a fairly low sensitivity and are susceptible to conditions which influence the amount of protons released. Enzymatic or chromatographic methods for the detection of one of the products of the lipolytic reaction (e.g. glycerol) are very sensitive and relatively robust, but are also involved in terms of handling, as they demand the working-up of the incubation batch of the lipase reaction before the actual enzymatic/chromatographic detection. The coupling of an enzyme test allows only endpoint measurements ("time-stop" measurement). Furthermore, in the investigation of unknown substances (e.g. searching for potential inhibitors), an effect on the enzymes of the detection reaction cannot be excluded in principle and therefore necessitates appropriate controls. These considerations gave the impetus to the development of fluorimetric/photometric processes. In principle, these achieve the sensitivity of radiometric methods but necessitate the use of synthetic substrates or samples modified with fluorophores or chromophores. Traditional fluorimetric/photometric methods, like the radiometric procedures, are discontinuous in course and necessitate the separation of the substrate from the product. Recently, continuous fluorimetric/ photometric assays have been developed (S. Hendrickson, Analyt. Biochem 219 (1994) 1-8), which are based on a shift in the fluorescence or extinction maximum of the product in comparison with the substrate. However, all these processes are restricted to the detection of phospholipases, lipoprotein lipase, cholesterol esterase, sphingomyelinase and glucosylceramide glucosidase. Substrates having fluorophoric/chromophoric groups, suitable for the continuous activity measurement of tryglyceride-cleaving enzymes (e.g. hormone-sensitive lipase, monoglyceride lipase, diglyceride lipase, triglyceride lipase, lipoprotein lipase, pancreatic lipase, hepatic lipase, bacterial lipase, PLA2, PLC, cholesterol esterase), are as yet unknown. It is therefore the aim of the invention to develop a simple continuous test for the identification of structures which favor the arrangement of aromatics to give charge-transfer complexes, such as, for example, complex phospholipid/lipid structures (bilayer, monolayer, aggregates, micelles), with the aid of synthetic fluorescence-labeled acylglycerides, and a process for determination of the activity of lipid-binding proteins, such as lipases. Lipid transporters are proteins which recognize lipids and do not cleave like lipases, but instead transport through biological membranes. Lipases are understood here as meaning biologically relevant endogenous lipases, such as are defined, for example, in R.D. Schmid, R. Verger, Angew. Chem. 110(1998) 1694-1720. A hormone-sensitive enzyme is understood as meaning an enzyme which is influenced in its activity by secondary messengers (e.g. cAMP) of dependent phosphorylation or by means of other allosteric mechanisms (e.g. protein-protein interaction) which are under hormone control. Hormones which regulate the cAMP level are, for example, adrenalin, noradrenalin, glucagon and insulin. The invention relates to a process for the preparation of a substrate, which comprises a) reacting a fatty acid provided with a fluorescent label with 2,3-epoxypropanol to give a monoacylglyceride in alcoholic solution, such as, for example, C-i-C^alkanol, preferably methanol, at room temperature with addition of a base, such as, for example, a non nucleophilic inorganic base, preferably alkali metal carbonates and alkali metal Ct-C^alkanolates, particularly preferably methanolates, such as sodium methanolate or potassium methanolate, b) subjecting this monoacylglyceride to ultrasonic treatment with phospholipids in the ratio (mg/ml) 1:10 to 10:1, preferably 1:2 to 3:1 and particularly preferably 1:1 to 1.5:1, from which the substrate results, which is recognizable by a color change from yellow to red. A fluorescent label is defined as a chemical group within a molecule which, after excitation by light, is itself capable of emitting light. Such groups are employed here in order to prepare substances which themselves are still detectable in lowest concentrations of about 1nM. Mention may be made, for example, of N,N-dimethylaminosulfonic acid (dansyl) or NBD, preferably NBD. A fatty acid is understood, for example, as meaning a long-chain carboxylic acid, which is saturated or unsaturated and has a chain length of C-8 to C-20, preferably C-12, C-14, C-16 and C-18 which is saturated or unsaturated, particularly preferably C-12 and saturated. A fatty acid provided with a fluorescent label was coupled to a monoacylglyceride using 2,3-epoxypropanol. From this synthetic substrate and phospholipids, such as, for example, phosphatidylinositol and phosphatidylcholine on their own or together, optionally in a ratio by weight of 10:1 to 1:10, preferably 3:1 to 1:3, particularly preferably 2:1, micelles or vesicles which serve as a substrate of the lipase to be investigated were formed by ultrasonic treatment which lasts, for example, for about 1 to 10 minutes, preferably 1 to 6 minutes, particularly preferably 4 minutes. This incorporation into micelles or vesicles is associated with a color change from yellow to red, based on a charge-transfer complex of the aromatics which are spatially closely adjacent in this structure. Incubation with lipase leads to the removal of the fatty acids with release of labeled fatty acid and glycerol. As phospholipids, for example, phosphatidylcholine (6 mg) and phosphatidylinositol (6 mg) are dissolved in chloroform (1 ml each). For the preparation of the substrate, two parts of phosphatidylinositol solution (e.g. 83.5 fj\) and one part of phosphatidylcholine solution (e.g. 41.5//I) and 100 /vl of NAG solution (10 mg in 1 ml of chloroform) are pipetted together (final concentration in the test: 0.0375 mg of phospholipid/ml; 0.05 mg/ NAG/ml). After removal of the chloroform, 20 ml of 25 mM tris/HCI, pH 7.4; 150 mM NaCI are added and two ultrasonic treatments are carried out using an ultrasonic probe (Branson Sonifier type II, standard microtip, 25 W): 1st treatment: setting 2, 2 x 1 min, in between 1 min each on ice; 2nd treatment: setting 4, 2 x 1 min, in between 1 min each on ice. During this procedure, the color of the substrate solution changes from yellow (extinction maximum 481 nm) to red (extinction maximum 550 nm) due to intercalation of NAG between the phospholipid molecules of the vesicles/micelles. The free fatty acid forms no micelles or vesicles. Therefore a color change from red to yellow is observed during the removal of the fatty acid from the micelles/vesicles. Thus the destruction of the micelles/vesicles and thus the enzyme activity of the lipase is measurable, either visually (at 481 nm, or at 550 nm), by means of the color change from red to yellow, with the aid of a cuvette photometer (e.g. DU-640 from Beckman (Munich)) or of a microtiter plate reader (e.g. MicroBeta from Wallac (Turku, Finland) or alternatively fluorimetrically with the aid of a phosphoimager (e.g. Storm 840 from Molecular Dynamics (Krefeld)), of a fluorescence scanner (e.g. DA-2 from Shimadzu (Osaka, Japan)) or of an image analysis process (e.g. ArrayScan from Molecular Devices (USA)) which is based on a CCD camera (charge-coupled device), which has an integrated circuit for the processing of electric and optical signals in which the information is stored and transmitted in the form of electrical charges. All these methods are preferably employed in combination with high-throughput screening (HTS). Further processes which are based on this concept are the measurement of the cytotoxicity of compounds and the action of detergents. The invention also relates to a substrate prepared by the process described above and a substrate for use in a process for the identification of structures which favor the arrangement of aromatics to give charge-transfer complexes, preferably for the identification of phospholipid/lipid structures, particularly preferably of lipases/lipase inhibitors, as described above. The process can also be employed for the destruction of mono- or bilayer structures, which are curved (e.g. micelles or vesicles) or planar (e.g. artificially produced straight bilayers), which is accompanied by a color change. The color change can be monitored visually/optically or in a fluorimetrically measurable manner, as described above. The invention further relates to a process for the preparation of the monoacyiglyceride 2,3-dihydroxypropyl 12-(7-nitrobenzo[1,2,3]oxadiazol- 4-ylamino)dodecanoate, where 12-aminolauric acid is first reacted with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and the intermediate obtained is then reacted with 2,3-epoxypropano! in alcoholic solution at room temperature with addition of a base, such as alkali metal carbonate and alkali metal C-i-C^alkanolate, preferably methanolate, such as sodium methanolate or potassium methanolate, and the monoacylglyceride 2,3-dihydroxypropyl 12-(7-nitrobenzo[1,2,3]oxadiazol-4-ylamino) dodecanoate itself. The object of the invention is achieved by a process for the identification of lipases/lipase inhibitors whose presence produces a color change, which comprises a) preparing a substrate as described above, b) incubating this substrate with a lipase (such as, for example, a hormone-sensitive lipase, monoglyceride lipase, diglyceride lipase, triglyceride lipase, lipoprotein lipase, pancreatic lipase, hepatic lipase, bacterial lipase, PLA2, PLC, cholesterol esterase, preferably a hormone-sensitive lipase and a pancreatic lipase, particularly preferably a hormone-sensitive lipase), and c) determining the color change, e.g. visually/optically or fluorimetrically. The invention further relates to lipases and lipase inhibitors which have been identified by the process described above. The invention likewise relates to a process for determination of the activity of lipases/lipase inhibitors, where a substrate such as described above is prepared, this substrate is incubated with a lipase, and the rate of color change from red to yellow or conversely from yellow to red is determined and the activity is ascertained, for example, by means of an absorption measurement with a photometer or a fluorescence measurement with a fluorimeter. A typical reaction is carried out at 30°C for 60 min, for example in 1.5 ml Eppendorf vessels or 96-hole plates. 10^1 of a test substance (e.g. inhibitors of HSL) are introduced in assay buffer (25 mM tris/HCI, pH 7.4; 150 mM NaCI) in the presence of 16.6% DMSO. 180 /J\ of the substrate solution (20/;g/ml of phosphatidylcholine, 10//g/ml of phosphatidylinositol, 50^g/ml of NAG in assay buffer) are added. After a preincubation for 15min at 30°C, 20/J\ of HSL in assay buffer are pipetted in and the extinction is immediately measured (see above) at 481 nm in a cuvette photometer (0.5 ml cuvette) or microtiter plate reader. After a certain incubation time, which is variable and depends on the chosen enzyme concentration and can be between 2 and 240 minutes, in this case incubation at 30°C for 60 min, the extinction is measured again. The increase in the extinction in the yellow region, in this case at 481 nm, is a measure of the enzyme activity. Assay systems for identification of a lipase inhibitor or for determination of the activity of a lipase/of a lipase inhibitor are likewise a subject of the invention. They comprise a substrate such as described above, an ultrasonic device and optionally a device for the visual/optical and/or fluorimetric determination of the color change from red to yellow or, in addition to a substrate as described above and an ultrasonic device, a device for determination of the rate of color change and a device for absorption or fluorescence measurement. The assay system can also be present in the form of a kit, the assay being a lipase assay. The kit contains a substrate as described above, optionally in an assay buffer, and a container for carrying out the test, such as, for example, an Eppendorf vessel or a microtiter plate, preferably a microtiter plate. Further subjects of the invention relate to processes for determination of molecular transport/transfer systems, for measurement of the detergent action of compounds or for investigation of the cytotoxicity of compounds (medicaments and the like) comprising a substrate as described above and a phospholipase, for example phospholipase A2 (from snake venom) or C (Bacillus cereus). Transporters/transfer proteins are understood as meaning proteins which themselves recognize principal nutrients such as carbohydrates, lipids and proteins and transport them through biological membranes or transfer them from a certain biological membrane to another. Transporters/transfer proteins, for example isolated from rat ileum, are functionally reconstituted (proteoliposomes) in phospholipid vesicles (liposomes) or incubated together with liposomes as soluble polypeptides and then added to a mixture of phospholipids and NBD-glyceride as described above and treated with ultrasound. The transport process or transfer process of the NBD-glyceride from the NBD-glyceride-containing micelles/vesicles into the lumen of the proteoliposomes with the aid of the transporters or into the membrane of the liposomes with the aid of the transfer proteins leads to the dissolution/destruction of the micelle structure and can in turn be monitored photometrically or fluorimetrically as described above. The detergent action of chemical compounds is based on the direct destruction of the micelles/vesicles. As biological membranes are also constructed in this way, such compounds are usually cytotoxic. Destruction of micelles can easily be detected using the present process by means of the described color change. Syntheses: A few NBD-labeled fatty acids such as 12-(7-nitrobenzo[1,2,3]oxadiazol-4-ylamino)dodecanoic acid (1) are indeed commercially available, but expensive. Although the first experiments were also carried out with commercially obtainable material, it was possible to obtain compound (1) in good yields by reaction of 12-aminolauric acid with 4-chloro-7-nitrobenzo[1,2,5]oxadiazole in MeOH. List of the compounds 1 - 9: 1 12-(7-nitrobenzo[1,2,3]oxadiazol-4-ylamino)dodecanoic acid 2 2,3-epoxypropanol 3 Ri = OH R2 = OH 4 Ri =OAc R2 = OAc 5 Ri = OOC(CH2)3CH3 R2 = OOC(CH2)3CH3 6 Ri = OOC(CH2)14CH3 R2 = OH By nucleophilic addition of (1) to 2,3-epoxypropanol (2), it was possible to obtain the monoacylglyceride (3) in good yields. Compound (3) was then acylated in order to reach the triglycerides (4) and (5). The diacylglyceride (6) was obtained by esterification of (1) with palmitin. This compound was also reacted to give the corresponding triacylglyceride (7). The same method was used in order to react the glycerol diether (8) to give the pseudotriacylglyceride (9), in which two acyl radicals are replaced by long-chain ethers. All compounds synthesized proved to be substrates of lipases, preferably of HSL, but showed considerable activity differences. The "best" substrate of the lipases proved to be the monoacylglyceride (3). The introduction of further acyl groups, as in the compounds (4), (5), (6) and (7), led to a decrease in activity. This can easily be explained by competition in the removal of the NBD acyl group by the newly introduced acyl radicals. In order to confirm this hypothesis, a pseudotriacylglyceride (9) was synthesized in which two acyl groups are replaced by hexadecyl ether units. These cannot be removed from the lipases and should therefore not compete with the NBD fatty acid ester. In biological tests, this compound indeed proved to be a substrate, but with low activity. Obviously, in addition to the catalytic region, the lipases have an extended hydrophobic binding region accessible to the long-chain ether groups, such that the addition of the fatty acid unit is impeded. As the monoacylglyceride (3) proved to be a good substrate of the lipases, it was investigated whether there is a regioselective preference for position 1 or 3. For these investigations, the enantiomeric regioisomers (3a) and (3b) were synthesized. The synthesis of the enantiomers (3a,b) starts from D- and L-1,2-0-isopropylideneglycerol, which is esterified with the NBD-labeled fatty acid (1) by dicyclohexylcarbodiimide activation. The protective group was removed using 1 N methanolic HCI. Both compounds showed identical biological activity, such that the use of enantiomerically pure compound promises no advantage. 1. 12-(7-Nitrobenzo[1,2,3]oxadiazol-4-ylamino)dodecanoic acid (1): 30% strength sodium methanolate solution (14.5 ml, 76 mmol) is added with stirring to a solution of 12-aminolauric acid (18 g, 83.7 mmol) in MeOH (300 ml). After 5 minutes, the reaction mixture becomes clear and a solution of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (15 g, 75 mmol) in MeOH (300 ml) is added. The reaction mixture, which immediately becomes dark, is stirred for 18 h at 25°C. 1 M methanolic HCI (100 ml, 100 mmol) is then added and the solvent is distilled off in vacuo. The residue is taken up in MeOH, the mixture is filtered through silica gel, the filtrate is concentrated to dryness and the residue is purified by flash chromatography (1 : 1 toluene/EtOAc). (1) is obtained as a red solid (26.4 g, 93%); RF: 0.16 (1 : 1 toluene/EtOAc). VlMMR (250 MHz, CDCI3): 8 8.5 (d, 1 H, ArH), 6.35 (m, 1 H, NH), 6.18 (d, 1 H, ArH), 3.48 (dt, 2 H, CH2NH2), 2.36 (t, 2 H, CH2COOH), 1.9-1.2 (m, 18 H, 9 CH2). MS (ESI-MS) 379.2 (M+1). 2. 2,3-Dihydroxypropyl 12-(7-nitrobenzo[1,2,3]oxadiazol-4-ylamino) dodecanoate (3): A solution of compound (1) (12 g, 31.7mmol) and 2,3-epoxypropanol (50 ml) in isopropanol (50 ml) is stirred at 50°C for 16 h. The solvent is distilled off in vacuo, and the residue is dried at 0.01 torr and purified by flash chromatography (diisopropyl ether, ether, EtOAc). Compound (3) is obtained as a red oil (10.3 g, 71.8%); RF: 0.18 (1:1 toluene/EtOAc); RF: 0.5 (30:5:1 CH2Cl2-MeOH-NH3), which crystallizes from EtOAc/diethyl ether. 1H-NMR (250 MHz, CDCI3): 5 8.5 (d, 1H, ArH), 6.35 (m, 1 H, NH), 6.18 (d, 1 H, ArH), 4.19 (dd, 2 H, H-1, H-1'), 3.94 (m, 1 H, H-2), 3.65 (dd, 2 H, H-3, H-3'), 3.48 (dt, 2 H, CH2NH2), 2.35 (t, 2 H, CH2COOH), 1.8 (m, 2 H, CH2), 1.6 (m, 2 H, CH2), 1.27-1.15 (m, 14 H, 7 CH2). MS (ESI-MS) 453.4 (M+1). 3. (S)-2,2-Dimethyl[1,3]dioxolan-4-ylmethyl 12-(7-nitrobenzo[1,2,5] oxadiazol-4-ylamino)dodecanoate (9a): A solution of compound (1) (60 mg, 159/vmol) in CH2CI2 (2 ml) is treated with dicyclohexylcarbodiimide (160 mg, 770/;mol) and stirred at 25°C for 30 min. A solution of (R)-(2,2-dimethyl[1,3]dioxolan-4-yl)methanol (100 mg, 760 fjmo\) and dimethylaminopyridine (94 mg, 770 /jmo\) in CH2CI2 (2 ml) is then added and the reaction solution is stirred for a further 4 h at 25°C. The solvent is distilled off in vacuo and the residue is purified by flash chromatography (15:1 toluene/EtOAc). Compound (9a) is obtained as a yellow fluorescent oil (46 mg, 58%). RF 0.29 (4:1 toluene/EtOAc). VlMMR (CDCI3): 5 8.5 (d, 1 H, aromat.), 6.2 (m, 1 H, NH), 6.16 (d, 1 H, aromat.), 4.31 (m, 1 H), 4.1 (m, 3 H), 3.73 (dd, 1 H), 3.48 (dt, 2 H, CH2NH2), 2.35 (t, 2 H, CO-Ch^), 2.0-1.2 (m, 18 H, 9 CH2), 1. 42 (s, 3 H, CMe2), 1.37(s, 3H, CMe2). 4. (R)-2,2-Dimethyl[1,3]dioxolan-4-ylmethyl 12-(7-nitrobenzo[1,2,5] oxadiazol-4-ylamino)dodecanoate (9b): Compound (9b) is prepared as described for compound (9a), Compound (9b) is obtained as a yellow fluorescent oil (51.4 mg, 65%). RF 0.29 (4:1 toluene/EtOAc). VlMMR (CDCI3): 5 8.5 (d, 1 H, ArH), 6.2 (m, 1 H, NH), 6.16 (d, 1 H, ArH), 4.31 (m, 1 H), 4.1 (m, 3 H), 3.73 (dd, 1 H), 3.48 (dt, 2 H, CH2NH2), 2.32 (t, 2 H, C0-CH2), 2.0-1.2 (m, 18 H, 9 CH2), 1.42 (s, 3 H, CMe2), 1.37 (s, 3 H, CMe2). 5. (S)-2,3-Dihydroxypropyl 12-(7-nitrobenzo[1,2,5]oxadiazol-4-ylamino) dodecanoate and (R)-2,3-dihydroxypropyl 12-(7-nitrobenzo[1,2,5]oxadiazol- 4-ylamino)dodecanoate (3b): Methanolic HCI (1 M, 200 )J) is added to a solution of 13.9 mg (28.2 /jmo\) of compound (9a) or 17.8 mg (36.1 /;mol) of compound (9b) in 25 ml of methanol and the mixture is stirred for 1.5 h at 25°C. The solvent is distilled off in vacuo and the residue is purified by flash chromatography (2:1, 1:1 toluene/EtOAc). The fatty acid esters (3a) and (3b) are obtained in a yield of 10.5 mg (82%), and 9.3 mg (57%) respectively. H-NMR (CDCI3) data and mass spectra are identical to compound (3). 6. 2-Acetoxy-3-[12-(7-nitrobenzo[1,2,5]oxadiazol-4-ylamino)- dodecanoyloxy]propyl acetate (4): Compound (3) (12 mg, 26.5 /jmo\) is acetylated in 2:1 pyridine/acetic anhydride (3 ml). After 8 h, the mixture is concentrated to dryness and the residue is purified by flash chromatography (1:2 toluene/EtOAc). Compound (4) is obtained as a yellow fluorescent oil (13 mg, 91%). RF 0.66 (1:1 toluene/EtOAc). 1 H-NMR (250 MHz, CDCI3): 5 8.5 (d, 1 H, ArH), 6.3 (m, 1 H, NH), 6.18 (d, I H, ArH), 5.24 (m, 1 H), 4.92 (m, 1H), 4.34 (m) 4.28, 4.16, 3.48 (dt, 2 H, CH2NH2), 2.32 (CH2COO), 2.1 (2 s, 6 H, 2 OAc) 2.0-1.0 (m, 18 H, 9 CH2). MS (ESI-MS): 537.4 (M+1). 7. 2-Hexanoyloxy-3-[12-(7-nitrobenzo[1,2,5]oxadiazol-4-ylamino)- dodecanoyloxy]propyl hexanoate (5): Hexanoic anhydride (100 //I) is added to a solution of compound (2) (5 mg, II fjmo\) in pyridine (300 JJ\) and the reaction mixture is allowed to stand at 25°C for 16 h. The solvent is distilled off in vacuo and the residue is purified by flash chromatography (9:1 toluene/EtOAc). Compound (5) is obtained as a yellow fluorescent oil (1.8 mg, 25%). RF: 0.73 (1:1 toluene/EtOAc). 1 H-NMR (250 MHz, CDCI3): 5. 8.5 (d, 1H ArH), 6.25 (m, 1 H, NH), 6.18 (d, 1 H, ArH), 5.26 (m, 1 H, H-2), 4.29 (dd, 2 H, H-3, H-3'), 4.15 (dd, 2 H, H-1, H-1'), 3.46 (dt, 2H, Chb-NH), 2.34 (t, 6 H, 3 CH2COO), 1.63-1.2 (m, 12 H, 6 CH2), 0.88 (m, 6 H, 2 CH3). MS (ESI-MS): 649.5 (M+1). 8. 2-Hydroxy-3-[12-(7-nitrobenzo[1,2,5]oxadiazol-4-ylamino)- dodecanoyloxy]propyl hexadecanoate (6): A solution of compound (1) (25 mg, 66 /vmol), 4-dimethylaminopyridine (9 mg, 73/ymol) and 1,1-carbonyldiimidazole (15 mg, 73μ mol) in CH2CI2 (5 ml) is stirred at 25°C for 30 min. The solvent is distilled off in vacuo and the residue is purified by flash chromatography (9:1 toluene/EtOAc). Compound (6) is obtained as a yellow fluorescent oil (9.5 mg, 21%). RF 0.25 (5:1 toluene/EtOAc). 1H-NMR (250 MHz, CDCI3):5. 8.54 (d, 1 H ArH), 6.3 (m, 1 H, NH), 6.19. (d, 1 H, ArH), 4.20 (dd, 2 H, H-1, H-1'), 4.15 (dd, 2 H, H-3, H-3'), 4.12 (m, 1 H, H-2), 3.5 (dt, 2H, CH2-NH), 2,36 (t, 4 H, 2 CH2COO), 1.82 (m, 2 H, CH2-CH2-NH), 1.63 (m, 4 H, 2 CH2CH2COO), 1.47 (m, 2 H, CH2-(CH2)2-NH), 1.31-1.26 (m, 22 H, 11 CH2), 1.287 (m, 2 H, CH2), 1.26 (m, 2 H, CH2), (m, 4 H, 2 CH2CH2CH2COO), 1.31-1.26 (m, 18 H, 9 CH2), 1.3 (m, 2 H, CH2), 1.26 (m, 2 H, CH2), 0.89 (t, 3 H, CH3). MS (ESI-MS): 649.5 (M+1). 9. 2-Acetyloxy-3-[12-(7-nitrobenzo[1,2,5]oxadiazol-4-ylamino)- dodecanoyloxy]propyl hexadecanoate (7): Compound (6) (6 mg, 8.7 /vmol) is acetylated and worked up as described for compound (4). The crude product is purified by flash chromatography (9:1 toluene/EtOAc). Compound (7) is obtained as a yellow fluorescent oil (5.1 mg, 95%). RF0.71 (5:1 toluene/EtOAc). 1H-NMR (250 MHz, CDCI3):5 8.5 (d, 1 H, ArH), 6.25 (m, 1H, NH), 6.17 (d, 1 H, ArH), 5.25 (m, 1 H, H-2), 4.27 (dd, 2 H, H-1, H-3), 4.15 (dd, 2 H, H-1', H-3'), 3.73 (dt, 4 H, CH2-NH), 2.3 (t, 2 H, CH2COO), 2.08 (s, 3 H, OAc), 1.8 (m, 2 H, CH2-CH2-NH), 1.6 (m, 8 H, 4 CH2), 1.4-1 (m, 32 H, 16 CH2), 0.85 (t, 3 H, CH3). MS (FAB-MS): 739 (M+Li). 10. 2,3-bis-Octadecyloxy-propyl 12-(7-nitrobenzo[1,2,5]oxadiazol-4- ylamino)dodecanoate (8): 2,3-bis-octadecyloxypropan-1-ol (5 mg, 8.37 ^mol) is added to a solution of compound (1) (3 mg, 7.9 /vmol), dimethylaminopyridine (5 mg, 41 /vmol) and dicyclohexylcarbodiimide (5 mg, 24 /vmol) in CH2CI2. The reaction mixture is stirred at 25°C for 2 h, the solvent is distilled off in vacuo and the residue is purified by flash chromatography (2:1 petroleumether/diethyl ether). Compound (8) is obtained as a yellow fluorescent oil (3.5 mg, 46%). RF 0.55 (3:7 diethyl ether/petroleum ether). MS (FAB-MS): 957.8 (M+1). Enzyme preparation: Preparation of the partially purified HSL: Isolated rat fat cells are obtained from epididymal fatty tissue from untreated male rats (Wistar, 220-250 g) by collagenase treatment according to published processes. The fat cells from 10 rats are washed three times by flotation with 50 ml each of homogenization buffer (25 ml tris/HCI, pH 7.4, 0.25 M sucrose, 1 mM EDTA, 1 mM DTT, 10 /yg/ml of leupeptin, 10 /yg/ml of antipain, 20/;g/ml of pepstatin) and finally taken up in 10 ml of homogenization buffer. The fat cells are homogenized in a Teflon-in-glass homogenizer (Braun-Melsungen) by 10 strokes at 1500 rpm and 15°C. The homogenizate is centrifuged (Sorvall SM24 tubes, 5000 rpm, 10 min, 4°C). The bottom layer between the overlying fatty layer and the pellet is removed and the centrifugation is repeated. The bottom layer resulting from this is centrifuged again (Sorvall SM24 tubes, 20,000 rpm, 45 min, 4°C). The bottom layer is removed and treated with 1 g of heparin-Sepharose (Pharmacia Biotech, CL-6B, washed 5 x with 25 mM tris/HCI, pH 7.4, 150 mM NaCI). After incubation for 60 min at 4°C (shake at intervals of 15 min), the batch is centrifuged (Sorvall SM24 tubes, 3000 rpm, 10 min, 4°C). The supernatant is brought to pH 5.2 by addition of glacial acetic acid and incubated at 4°C for 30 min. The precipitates are collected by centrifugation (Sorvall SS34, 12,000 rpm, 10 min, 4°C) and suspended in 2.5 ml of 20 mM tris/HCI, pH 7.0, 1 mM EDTA, 65 mM NaCI, 13% sucrose, 1 mM DTT, 10/;g/ml of leupeptin/pepstatin/antipain. The suspension is dialyzed overnight at 4°C against 25 mM tris/HCI, pH 7.4, 50% glycerol, 1 mM DTT, 10//g/ml of leupeptin, pepstatin, antipain and then applied to a hydroxylapatite column (0.1 g per 1 ml of suspension, equilibrated with 10 mM potassium phosphate, pH 7.0, 30% glycerol, 1 mM DTT). The column is washed with four volumes of equilibration buffer at a flow rate of 20 to 30 ml/h. The HSL is eluted with a volume of equilibration buffer which contains 0.5 M potassium phosphate, then dialyzed (see above) and concentrated 5 to 10 times by ultrafiltration (Amicon Diaflo PM 10 filter) at 4°C. The partially purified HSL can be stored at -70°C for 4 to 6 weeks. Preparation of the NAG (NBD-monoacylglyceride) substrate: 6 mg of phosphatidylcholine and 6 mg of phosphatidylinositol are dissolved in 1 ml of chloroform each. 10 mg of NAG are dissolved in 1 ml of chloroform. Two parts of phosphatidylinositol solution (e.g. 83.5 JJ\) and one part of phosphatidylcholine solution (e.g. 41.5//I) and 100//I of NAG solution are pipetted together into plastic scintillation containers (final concentration in the test: 0.0375 mg of phospholipid/ml; 0.05 mg/ NAG/ml). The chloroform (225 /vl total volume) Is completely removed by overblowing with a stream of N2. The dried substrate can be stored at 4°C for up to three days. For the preparation of the phospholipid vesicles/micelles having intercalated NAG (on the test day), the dried substrate is taken up in 20 ml of assay buffer (25 mM tris/HCI, pH 7.4; 150 mM NaCI) and two ultrasonic treatments with an ultrasonic probe (Branson Sonifier type II, standard microtip): 1st treatment setting 2, 2 x 1 min, in between in each case 1 min on ice; 2nd treatment setting 4, 2 x 1 min, in between in each case 1 min on ice. During this procedure, the color of the substrate solution changes from yellow (extinction maximum 481 nm) to red (extinction maximum 550 nm) due to intercalation of NAG between the phospholipid molecules of the vesicles/micelles. Before use as a substrate (within the next 2 h), the solution is additionally incubated on ice for 15 min. Indirect NAG assay: The assay is carried out at 30°C for 60 min in 1.5 ml Eppendorf vessels or 96-hole plates. To find inhibitors of HSL, 10 /yl of the test substance in assay buffer (25 mM tris/HCI, pH 7.4; 150 mM NaCI) are introduced in the presence of 16.6% DMSO. 180//I of the substrate solution (20/vg/mI of phosphatidylcholine, 10/vg/ml of phosphatidylinositol, 50/yg/ml of NAG in assay buffer) are added. After a preincubtion for 15 min at 30°C, 20 /J\ of the enzyme solution in assay buffer (diluted 1 to 4 times) are pipetted in and the extinction is immediately measured at 480 nm in a cuvette photometer (0.5 ml cuvette) or microtiter plate reader (microBeta, Wallac). After incubation at 30°C for 60 min, the extinction is measured again. The increase in the extinction at 480 nm is a measure of the enzyme activity. Under standard conditions, 20 \xg of partially purified HSL lead to an extinction change of 4000 arb. units. Direct NAG assay: Alternatively to the measurement of the extinction change of the substrate solution, the products of the HSL reaction are investigated by phase separation/thin-layer chromatography. For this, the incubation batch (200 JJ\ total volume, see indirect NAG assay) is mixed in 2 ml Eppendorf vessels with 1.3 ml of methanol/chloroform/heptane (10:9:7) and then with 0.4 ml of 0.1 M NaOH. After intensive mixing (10 sec), the phase separation is initiated by centrifugation (800xgr, 20 min, room temperature). Equivalent volumes (e.g. 0.4 ml) are taken from the aqueous upper phase and the extinction is determined photometrically at 481 nm. For thin-layer chromatography, the aqueous phase is dried (SpeedVac) and then taken up in 50 fj\ of tetrahydrofuran. 5/J\ samples are applied to silica gel Si-60 plates (Merck, Darmstadt). The chromatography is carried out using 78 ml of diethyl ether/22 ml of petroleum ether/1 ml of glacial acetic acid as an eluent. The amount of released fluorescent NBD fatty acid is determined by phosphorimaging (Molecular Dynamics, Storm 840 and ImageQuant software) at an excitation wavelength of 460 nm and emission wavelength of 540-560 nm. TAG assay: 3 For the preparation of the substrate, 25-50 ^Ci of [ H]trioleoylglycerol (in toluene), 6.8 /JMO\ of unlabeled trioleoylglycerol and 0.6 mg of phospholipids (phosphatidylcholine/phosphatidylinositol 3:1 w/v) are mixed, dried over N2 and then taken up in 2 ml of 0.1 M KPj (pH 7.0) by ultrasonic treatment (Branson 250, microtip, setting 1-2, 2 x 1 min at a 1 min interval). After addition of 1 ml KPj and fresh ultrasonic treatment (4 x 30 sec on ice at 30 sec intervals), 1 ml of 20% BSA (in KPj) is added (final concentration of trioleoylglycerol 1.7 mM). For the reaction, 100//I of substrate solution are pipetted into 100 fj\ of HSL solution (HSL prepared as above, diluted in 20 mM KPj, pH7.0, 1 mM EDTA, 1 mM DTT, 0.02% BSA, 20/;g/ml of pepstatin, 10/;g/ml of leupeptin) and incubated at 37°C for 30 min. After addition of 3.25 ml of methanol/chloroform/heptane (10:9:7) and of 1.05 ml of 0.1 M K2C03, 0.1 M boric acid (pH 10.5), the batch is well mixed and finally centrifuged (800xg, 20 min). After phase separation, one equivalent of the upper phase (1 ml) is taken and the radioactivity is determined by liquid scintillation measurement. PNPB assay: 10 /vl of p-nitrophenyl butyrate (PNPB)(2 mM in acetonitrile), 890/y] of 0.1 M KPj (pH 7.25), 0.9% NaCI, 1 mM DTT and 100//I of HSL (prepared as above, diluted in this buffer) are incubated at 37°C for 10min. After addition of 3.25 ml of methanol/chloroform/heptane (10:9:7, w/v) and vigorous shaking, the batch is centrifuged (800xgr, 20 min) and incubated at 42°C for 3 min. An equivalent volume of the upper phase is then taken and the absorption is determined at 400 nm. Tributyrin assay: For the preparation of the substrate, 5/vCi of [1- C]tributyrin (in toluene) are added to 1 ml of 20 mM unlabeled tributyrin (in acetonitrile). 10//I of this substrate solution are incubated at 37°C for 30 min with 390 /vl of 0.1 M KPj (pH 7.25), 0.9% NaCI, 1 mM DTT, 2% BSA and 100 fj\ of HSL (prepared as above, diluted in this buffer). After addition of 3.25 ml of methanol/chloroform/heptane (10:9:7, w/v) and of 1 ml of 0.1 M NaOH, the batch is vigorously mixed and finally centrifuged (800xg, 20 min). An equivalent volume (1 ml) of the upper phase is taken and the radioactivity is determined by liquid scintillation measurement. Analysis: Substances are customarily tested in four independent batches. The inhibition of the enzymatic activity of the HSL by a test substance is determined by comparison with an uninhibited control reaction. The IC50 value is calculated by means of an inhibition curve using at least 10 concentrations of the test substance. For the analysis of the data, the software package GRAPHIT, Elsevier-BIOSOFT (version 3.0) is used. Examples: Example 1: Kinetics of the cleavage of NAG by HSL NAG (0.05 mg/ml) is incubated with the indicated amounts of partially purified HSL protein (temperature-controlled photometer) and the extinction at 481 nm is determined at specific times. For inactivation, the HSL is incubated at 100°C for 15 min. (n = 8, mean + SD). Result: Up to an amount of protein of 20 ^g, the reaction proceeds linearly up to 60 min. The extinction difference is in this case 0.8-0.9 OD. In the case of smaller amounts of protein, linearity is afforded up to 180 min. Example 2: Dependence of the cleavage of NAG on the amount of HSL NAG (0.05 mg/ml) is incubated with the indicated amounts of partially purified HSL protein for 60 min. The increase in the extinction at 481 nm (formation of the free NBD fatty acid as a product of the HSL reaction = indirect NAG assay) or the decrease in the extinction at 550 nm (consumption of NAG as a substrate of the HSL reaction) is determined in aliquots of the reaction batches. Alternatively, further aliquots are extracted with methanol/chloroform and the released NBD fatty acid contained in the organic phase is determined (= direct NAG assay) (n = 6, mean + SD) by TLC analysis and fluorimetry (phosphorimager Storm 840, Molecular Dynamics). Result: Up to an amount of protein of 20 /vg the reaction proceeds linearly with respect to product formation (extinction increase at 481 nm or occurrence of free NBD fatty acid according to TLC analysis) and with respect to the consumption of substrate (extinction decrease at 550 nm). The agreement between indirect and direct NAG assay supports the analysis of the cleavage of NAG in the indirect assay. Example 3: Dependence of the cleavage of NAG by HSL on the substrate concentration Different amounts of NAG (with a constant ratio to the phospholipids) are incubated for 60 min with the indicated amounts of partially purified HSL and the extinction is then determined at 481 nm (n = 5, mean + SD). Result: With all three amounts of enzyme, the cleavage rate exhibits the course of a typical saturation curve. On account of the enzymatic peculiarity of the HSL reaction ("two-dimensional" substrate presentation, "interfacial activation"), however, an approximately linear dependence can be determined only with 5/yg of protein and the lowest substrate concentrations. The combination of 20 /yg of protein and 0.05 mg/ml of NAG represents a rational compromise between substrate dependence and signal strength (OD 0.6 to 0.7). Example 4: Dependence of the cleavage of NAG by HSL on the ratio of NAG and phospholipids NAG (0.05 mg/ml). is prepared as a substrate by ultrasonic treatment using different amounts of phospholipids (total amounts) with the indicated ratios of phosphatidylinositol to phosphatidylcholine and then incubated with partially purified HSL (20 //g) for 60 min. The increase in the extinction at 481 nm is determined (n = 4, mean + SD). Result: The enzyme rate is greatest with a PI/PC ratio (according to weight) of 3:1 and 0.0375 to 0.075 total phospholipid. The pronounced optimum course for the total concentration of the phospholipids and for their composition supports the importance of the presentation of the substrates of HSL (in this case NAG) in phospholipid vesicles and/or micelles or the formation of a monolayer of phospholipids on the (neutral) core of the substrate. Example 5: Comparison of the indirect/direct NAG assay with a conventional HSL assay by means of the determination of the IC50 values for various inhibitors NAG (0.05 mg/ml) is incubated with 20 /vg of partially purified HSL for 60 min in the presence of different concentrations (0.1 to 100 JJM) of various substances. The extinction is determined at 481 nm in aliquots of the batches (indirect assay) or the NBD-FA released is extracted by chloroform/methanol and determined by TLC analysis and fluorimetry 3 (direct assay). Alternatively, [ H]trioleoylglycerol is incubated with partially purified HSL according to published conditions and the radiolabeled oleate released is determined by liquid scintillation measurement after extraction with chloroform/methanol. The IC50 values are determined from the inhibition curves (n = 6, mean + SD). Result: For all substances tested, typically sigmoidal inhibition curves were determined using the three processes. In the indirect/direct NAG assay (extinction increase at 481 nm/release of the NBD fatty acid), the IC50 values were generally lower by a factor of 4 to 10 compared with the cleavage of trioleoylglycerol by HSL; the order of the inhibitors (according to their C50 values), however, is identical for all three processes. This confirms published findings that the action of inhibitors of HSL depends on the nature of the substrate and the substrate presentation. Moreover, the effective substrate concentrations (NAG and trioleoylglyceride) can also differ between the two assays (barely determinable on account of the substrate preparation as vesicles or micelles) and thus explain these differences in the case of competitive inhibitors. The nearly identical IC50 values, which were determined according to the change in extinction and the NBD fatty acid release, propose cleavage of NAG by HSL and thus release of NBD fatty acid as a cause of the increase in extinction at 481 nm, i.e. the NAG assay detects the lipolytic cleavage of lipids. Example 6: Comparison of the indirect NAG assay with the assays for TAG, PNPB and tributyrin NAG (0.05 mg/ml) is incubated with 20 fjg of partially purified HSL for 60 min in the presence of different concentrations (0.1 to 100 /vM) of various substances. The extinction at 481 nm is determined in aliquots of 3 the batches (indirect assay). Alternatively, [ H]trioleoylglycerol (TAG), 14 p-nitrophenyl butyrate (PNPB) or [ C]tributyrin is incubated with partially purified HSL and the radiolabeled oleate, p-nitrophenol or butyrate released is determined by liquid scintillation measurement or photometry after extraction with chloroform/methanol. The IC50 values are determined from the inhibition curves (n = 5, mean + SD). Results: The relative order of the inhibitors, demonstrated in the IC50 25 values, is identical for both assays with lipid substrates (NAG and TAG). This fundamentally also applies to the water-soluble substrates, tributyrin and PNPB, but some active compounds, which significantly reduce the HSL activity compared with NAG and TAG, are inactive in the inhibition of HSL compared with tributyrin and PNPB. This can be explained by an interference of these inhibitors with the lipid binding of the HSL (through the lipid binding domains), while the catalytic mechanism is not adversely affected. Water-soluble substrates are therefore cleaved from the HSL even in the presence of these inhibitors. The IC50 values for inhibitors which act even in the case of water-soluble substrates generally lie between those for NAG and TAG as a substrate. This shows that NAG behaves as a "lipid-Iike" substrate for the HSL similarly to the authentic triglycerides and the NAG assay can be employed for finding inhibitors which block lipid binding (and the catalytic mechanism) of the HSL. Example 7: Influence of various detergents and solvents on the substrate stability NAG (0.05 mg/ml) is incubated at 37°C for 180 min in the presence of increasing concentrations of various detergents and solvents and the extinction decrease at 550 nm (dissolution of the spec, substrate structure) 25 is then determined (n = 4, mean + SD). Result: The substrate (phospholipid vesicles or micelles) exhibited different sensitivity compared with the agents employed. The extinction, (i.e. the amount of substrate, decreased in the presence of 1% DMSO by 10%, in the case of ethanol or methanol by at most 20%, in the case of 1% TX-100 or SDS by at most 30%. DMF was most efficient in dissolving the vesicles/micelles, and at 1% over 80% of NAG was released from the phospholipid/vesicle structures. The order in the efficiency of the solvents and detergents employed in the dissolution of the substrate structure is compatible with a shift in the extinction maximum (from 481 nm to 550 nm) by NAG, or of the chromophoric group (NBD) by incorporation into the apolar environment of phospholipid vesicles/micelles and the removal from aqueous environment caused thereby. Release of NAG from these structures in aqueous medium by dissolution of the vesicles/micelles (e.g. by detergents) or release of the chromophoric group as NBD fatty acid by lipolytic cleavage (by HSL) of NAG leads to a lowering of the extinction at 550 nm and an increase in the extinction at 481 nm. With the stability at 1% DMSO, the NAG substrate fulfils one of the basic requirements for a robust HTS assay. Example 8: Influence of various detergents and solvents on the activity of the HSL NAG (0.05 mg/ml) is incubated for 60 min with HSL (20 /yg) in the presence of increasing concentrations of various agents. The extinction at 481 nm is determined (n = 4, mean + SD). Result: DMSO up to 1% reduced the amount of released NBD-FA by approximately 10%. As at this DMSO concentration up to 10% of NAG is released from the phospholipid vesicles/micelles by dissolution of the structures, a decrease in the enzyme activity by 20% results arithmetically. In the case of 0.5% DMSO, the reduction is still 10%. TX-100, acetone, ethanol and methanol between 0.1 and 1% cause an increase in the HSL activity, possibly produced by a more efficient substrate presentation. At high concentrations, they interfere with the activity. DMF leads to a significant loss in activity of the HSL, even at concentrations from 0.1%. Example 9: Cleavage of NAG by lipases of differing specificity NAG (0.05 mg/ml) is incubated for 60 min with 20/yg of partially purified HSL, 75 fjg of partially purified LPL from rat adipocytes, 20 mU of bacterial lipase, 50 mU of pancreatic lipase, 100 mU of PLA2 from snake venom and 0.5 U of PC-specific phospholipase from Bacillus cereus. The increase in the extinction at 481 nm is determined (n = 5, mean + SD). Result: Under the optimized conditions given for HSL, as expected the HSL (100%) and LPL (approximately 70%) exhibited the greatest activity. The bacterial and pancreatic lipase is markedly less active (25 or 1%), while the bacterial PC-specific phospholipase was virtually inactive. These strongly different activities show the specificity of the chosen conditions of the indirect NAG assay for the HSL, thus, for example, phospholipases possibly contained in coarse cell extracts are not detected. These data, however, also show the applicability in principle of the assay principle to other lipases. Example 10: Cleavage of various acylglycerides modified with NBD fatty acid by HSL NAG (0.05 mg/ml) or various NBD fatty acid-modified lipids in different concentrations are incubated with 20 /yg of partially purified HSL The extinction at 481 nm is determined at specific times (n = 5, mean + SD). Results: Example 11: Inhibition of the HSL by diisopropyl phosphofluoridate NAG (0.05 mg/ml) is incubated for 60 min with 20 /yg of partially purified HSL in the presence of increasing concentrations of diisopropyl phosphofluoridate. The increase in the extinction at 481 nm is determined in aliquots of the reaction batches (indirect NAG assay), or after extraction with chloroform/methanol the amount of released NBD-FA in the organic phase is determined by fluorimetry (direct NAG assay). Alternatively, 3 incubations of the HSL with [ Hjtrioleoylglycerol as substrate (see above) are carried out and the amount of radiolabeled oleic acid released is determined after extraction with chloroform/methanol. The cleavage activity in the absence of inhibitor is set at 100% for each assay (n = 7, mean + SD). Result: In all three assays, typically sigmoidal inhibition curves resulted for diisopropyl phosphofluoridate. The IC50 values calculated therefrom did not differ significantly from one another for the indirect (0.8 mM) or direct NAG assay (1.1 mM). A somewhat higher IC50 value of 2.1 mM is calculated for the cleavage of trioleoylglyceride. This is in accord with the differences found above in the inhibitory actions of various inhibitors which are observed with these assays (see Example 6 for possible explanations). Independently of this, the IC50 values determined for the inhibition of HSL by diisopropyl phosphofluoridate by the indirect NAG assay are very much in accord with published data (P. Stralfors, H. Olsson, P. Belfrage, The Enzymes XVIII, 1987, 147-177; P. Stralfors, P. Belfrage, J. Biol. Chem. 258, 1983, 15146-15151; P. Belfrage, B. Jergil, P. Stralfors, H. Tornquist, FEBS Lett. 75, 1977, 259-263). Example 12: Feasibility of the indirect NAG assay in the microliter plate format NAG is incubated for different times with partially purified HSL in an assay volume of 200 /J\ in wells of 96-hole microtiter plates. The extinction at 481 nm is determined in a microtiter plate reader. Result: Under the conditions chosen, the reaction is linear down to approximately 60 min. The variance (SD) is in this case between 4 and 7%. The indirect NAG assay is thus suitable for use in HT screening. Abbreviations used: arb. units arbitrary units BSA Bovine serum albumin cAMP Cyclic adenosine monophosphate DCC Dicyclohexylcarbodiimide DMF N,N-Dimethylformamide DMSO Dimethyl sulfoxide DTT Dithiothreitol EDTA Ethylenediamine-N,N,N',N'-tetraacetic acid FAB-MS Fast atom bombardment mass spectrometry HSL Hormone-sensitive lipase Kpi Potassium dihydrogenphosphate/potassium phosphate buffer LPL Lipoprotein lipase NAG NBD-monoacylglyceride: 2,3-dihydroxypropyl 12-(7-nitro benzo[1,2,3]oxadiazol-4-ylamino)dodecanoate NBD 4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole NBD-FA NBD fatty acid: 12-(7-nitrobenzo[1,2,3]oxadiazol-4-ylamino)- dodecanoic acid NIDDM non insulin-dependent diabetes meliitus PLA2 Phospholipase A2 PLC Phospholipase C PNPB p-Nitrophenyl butyrate SD Standard deviation SDS Sodium dodecyl sulfate TAG [3H]-Trioleoylglycerol Tris Tris(hydroxymethyl)aminomethane TLC Thin-layer chromatography TX-100 Triton® X-100 Patent claims: 1. A process for determination of the integrity of complex phospholipid/lipid structures, which comprises a) reacting a fatty acid provided with a fluorescent label with 2,3-epoxypropanol to give a monoacylglyceride in alcoholic solution at room temperature with addition of a base, b) subjecting this monoacylglyceride to an ultrasonic treatment with phospholipids in the ratio from 1:10 to 10:1 mg/ml, from which the substrate results, which is recognizable by a color change from yellow to red based on a charge-transfer complex, 2. The process as claimed in claim 1, where the fluorescent label is dansyl or NBD. 3. The process as claimed in claim 2, where the fluorescent label is NBD. 4. The process as claimed in any of claims 1 to 3, where the fatty acid is a saturated or unsaturated carboxylic acid having a chain length of C-8 to C-20. 5. The process as claimed in claim 4, where the fatty acid is a saturated C-12 carboxylic acid. 6. The process as claimed in any of claims 1 to 5, where the phospholipids employed are phosphatidylcholine and phosphatidylinositol on their own or together. 7. The process as claimed in claim 6, where phosphatidylcholine and phosphatidylinositol are employed together in the ratio 10:1 to 1:10. 5 8. The process as claimed in any of claims 1 to 7, where the monoacylglyceride is 2,3-dihydroxypropyl 12-(7-nitrobenzo-[1,2,3]oxadiazol-4-ylamino)-dodecanoate. ) 9. A substrate prepared by a process of claims 1 to 8. 11. The substrate as claimed in claim 9 for use in a process for the identification of phospholipid/-lipid structures. 12. The substrate as claimed in claim 9 for use in a process for the identification of lipases and lipase inhibitors. 13. A process for the preparation of the monacylglyceride 2, 3-dihydroxypropyl 12- (7-nitrobenzo[1,2,3]oxadiazol-4-ylamino) dodecanoates, which comprises reacting 12-aminolauric acid first with 7-chloro-4-nitrobenzo-2-oxa-l,3-diazole and then reacting the intermediate obtained with 2,3-epoxypropanol in alcoholic solution at room temperature with addition of a base. 14. 2,3-Dihydroxypropyl 12- (7-nitrobenzo[1,2,3] oxa-diazol-4-ylamino)dodecanoate. 15. A process for the identification of lipases and lipase inhibitors, which comprises a) preparing a substrate as claimed in claim 9, b) incubating this substrate with a lipase, and c) determining the color change. 16. A lipase/lipase inhibitor, identified by a process as claimed in claim 15. 17. High-throughput screening (HTS) for use in a process for the identification of lipases/lipase inhibitors as claimed in claim 15. 18. A process for determination of the activity of lipases/lipase inhibitors, which comprises a) preparing a substrate as claimed in claim 9, b) incubating this substrate with a lipase, c) determining the rate of color change and d) ascertaining the activity. 19. The process as claimed in claim 18, wherein the substrate is incubated with a hormone-sensitive lipase. 20. An assay system for identification of a lipase/lipase inhibitor, comprising a) a substrate as claimed in claim 9, b) an ultrasonic device and, optionally, c) a device for the visual/optical and/or fluorimetric determination of the color change. 21. An assay system for determination of the activity of a lipase/a lipase inhibitor, comprising a) a substrate as claimed in claim 9, b) an ultrasonic device, c) a device for'determination of the rate of the color change and d) a device for absorption or fluorescence measurement. 22. The assay system as claimed in claim 20 or 21 in the form of a kit. 23. The kit as claimed in claim 22, where the assay is a lipase assay. 24. The kit as claimed in claim 22, comprising a) a substrate as claimed in claim 9 and b) a container. 25. A process for determination of the detergent action or the cytotoxicity of compounds, which comprises a) preparing a substrate as claimed in claim 9, b) incubating this substrate with a test compound, and c) visually/optically or fluorimetrically determining the color change from red to yellow which identifies cytotoxic compounds and compounds with detergent action. 26. A process for determination of the activity of lipid transporters, which comprises a) preparing a substrate as claimed in claim 9, b) functionally reconstructing transporters/-transporter proteins in liposomes and c) adding the liposomes prepared according to b) to the substrate according to a) and determining the color change visually or optically. 27. A process for determination of the integrity of complex phospholipid/lipid structures substantially as herein described and exemplified 28. An assay system substantially as herein described and exemplified.

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