Substrates for phospholipases.

Phospholipases (PL) catalyze the hydrolysis of ester groups of the sn-3-phosphoglycerides.

The substrates form micelles in aqueous solution and the enzymes act at the lipid-water interface.

A number of phospholipases are known to be of particular interest, especially PL C and PL A₂ (the best studied PL of the human body). With certain diseases, such as pancreatitis, infectious diseases, autoimmune diseases, allergies, the PL A₂ concentration in the blood and other body fluids increases. The determination of PL A₂ activity is therefore of considerable diagnostic importance.

PL C, in particular also phosphatidyl-inositol-specific PL C = PInase has recently become increasingly important. The regulator system phosphatidyl-inositol is intensively worked on by many research groups. In addition, PL C also plays a role in the detachment of membrane-bound proteins.

PL A₂ is also contained in some snake venoms (cobra, rattlesnake), an increased concentration is life-threatening. PL A₂ is one of the digestive enzymes and the determination plays an important role not only in clinical chemistry, but also in biochemistry, pharmaceutical chemistry and food chemistry (GEHoffmann, Dt.Ges.f.Klin.Chem. EV - Mitteilungen<u style="single">4</u>, 196 (86)).

Several phospholipase measurement methods are already known, such as, for example, the titrimetric determination of the fatty acid released during the ester cleavage (Figarella, Scand.J. Gastroent. <u style="single">6</u>, 133 (71)) or the measurement of the radiolabelled released fatty acid (Shakir, Anal.Biochem. <u style="single">114</u>, 64 (81)). However, these methods are very complex and prone to failure for routine purposes.

Photometric determination methods are also known:<ul id="ul0001" list-style="none"><li>a) Hendrickson J. Lipid Res. <u style="single">24</u>, 1532 (83),</li><li>b) Hoffmann, Dt.Ges.f.Klin.Chem., Mitteilungen <u style="single">4</u>, 201 (86)</li></ul>

to a):

It is based on the release of an -SH group and its determination with DTNB, but was found to be too insensitive.

to b):

The fully enzymatic method for determining the fatty acid according to Wako, Japan is complex and highly susceptible to disruption (numerous pipetting steps). NEFA-C test

Fluorescent phospholipids are also known as substrates (Thuren, Clin.Chem. <u style="single">31</u>, 714 (85)). However, the method is very susceptible to interference and only a few laboratories are equipped with fluorescence measuring devices.

Immunological methods for the determination of PL A are also described: Radioimmunoassay: Nishijima, J.Biochem. <u style="single">94</u>, 137 (83) and fluorescence immunoassay: Eskola, Clin.Chem. <u style="single">29</u>, 1777 (83), which are sufficient in terms of sensitivity, but cannot distinguish between the active phospholipase A and its inactive precursor.

Only a few methods are known for determining phospholipase C. It is known, after cleavage of the substrate by PL C with lipase as the auxiliary enzyme, to release glycerol and then to carry out a fully enzymatic determination of the released glycerol (Wahlefeld in Bergmeyer: Methods of Enzymatic Analysis, 3rd Edition, Vol. II, Verlag Chemie Weinheim 1974, 1974 , P.1878). However, this method is very prone to failure and time-consuming.

Furthermore, the determination of the PL C by using radioactively labeled substrates is published (Waku, J.Biochem. <u style="single">72</u>, 149 (72)). However, this method is cumbersome and insensitive.

There is therefore a need for a color test that can be carried out with simple equipment and can be checked directly visually.

The invention is therefore based on the object of providing a substrate and a color test for determining the phospholipase using this substrate, which does not have the disadvantages of the known tests, provides accurate results, is easy to use, has high sensitivity and is only low lag phase, so that the adaptation to the various analyzer systems is not difficult.

This object is achieved according to the invention by a phospholipase substrate of the general formula<chemistry id="chem0001" num="0001"><img file="EP0331167A2_D0001.tif" /></chemistry> wherein A is an alkylene or alkenylene group with 1 to 16 carbon atoms, RH or an alkyl, alkenyl or acyl group with 1 to 20 C atoms or an optionally alkyl-substituted aryl or aralkyl group with 1 to 8 C atoms in the alkyl radical, X is the residue of an aromatic hydroxy or thiol compound and each Y is independently -S- or -O-, Z -SO₃^⊖ or<chemistry id="chem0002" num="0002"><img file="EP0331167A2_D0002.tif" /></chemistry> mean, where R¹ can be a hydrogen atom or the group (CH₂)_nNO<maths id="math0001" num=""><math display="inline"><mrow><mfrac linethickness="0"><mrow><mtext>2</mtext></mrow><mrow><mtext>3</mtext></mrow></mfrac></mrow></math><img file="EP0331167A2_D0003.tif" /></maths>, in which n is a number from 2 to 4 and R² is H or CH₃, or inositol, or serine<chemistry id="chem0003" num="0003"><img file="EP0331167A2_D0004.tif" /></chemistry> or glycerin.

Under the action of the phospholipase, the phospholipase substrate according to the invention is cleaved to liberate the aromatic hydroxy or thiol compound corresponding to the radical X, which is either determined directly optically or coupled with a suitable chromophore or fluorophore and the coupling product is measured or possibly measured after the addition of an auxiliary enzyme.

R preferably has 6 to 20 C atoms, particularly preferably 12 to 18 C atoms. Surprisingly, the compounds with R = alkyl, alkenyl or aralkyl have proven to be good phospholipase substrates, although the natural substrates carry acyl groups.

Examples of R are methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tetradecyl, hexadecyl and octadecyl radicals as alkyl groups and the corresponding acyl groups such as the acetyl, propionyl, butyryl, valeryl, capronyl, capryl, caprinyl, lauryl, myristyl, palmityl and stearyl group, the oleyl, crotonyl, linolyl group, phenyl, benzyl group or octylphenyl group.

The phospholipase substrate according to the invention further contains the remainder of a dicarboxylic acid COOH-A-COOH, in which A preferably has 3 to 7 carbon atoms. Examples of acids from which A is derived are malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, nonandicarboxylic acid, decanedicarboxylic acid and undecanedicarboxylic acid. The acids from glutaric acid to azelaic acid are preferred. For A with more than 4 C atoms, the addition of an auxiliary enzyme is recommended.

X can be an aromatic hydroxy or thiol compound which is a chromophore or can only be converted into a dye by a subsequent reaction. Typical examples are phenol, thiophenol, naphthol, thionaphthol and their derivatives and the chromogenic compounds such as the resorufin, chlorophenol red, indoxyl or thiofluorescein residue. An exhaustive list of the suitable hydroxy or thiol compounds is not possible because of their large number, but the directly chromophoric or convertible aromatic hydroxy or thiol compounds are known to the person skilled in the art.

As compounds of the general formula (I) or (II) 1-O-octadecyl-2-glutaric acid-methylresorufine ester-sn-glycero-3-phosphocholine, 1-O-octadecyl-2-glutaric acid-p-nitrophenyl ester-sn-glycero-3-phosphocholine, 1-O-dodecyl-2-glutaric acid-nitrophenyl ester-rac-glycero-3-sulfate, 1-O-dodecyl-2-adipic acid-nitrophenyl ester-rac-glycero-3-sulfate or / and 1-O-Hexadecyl-2-glutaric acid-nitrophenyl ester-rac-glycero-3-sulfate is preferred.

The above-mentioned reaction to the dye can be carried out either by direct coupling (for example with a diazonium salt such as 4-chloro-2-methylbenzene diazonium salt (Fast Red), 4-benzamido-2-methoxy-5-methylbenzene diazonium salt (Fast Violet), diazotized sulfanilic acid, 2,4- and 2,5-substituted phenyldiazonium salts, for example 2,4-dichlorophenyldiazonium-1,5-naphthalenedisulfonic acid), or by an oxidative coupling, for example with 4-aminoantipyrine or other aminopyrazolones (such as trimethylaminopyrazolone, diaminoantipyrine) or MBTHS (3-methyl-2-benzothiazolinone hydrazone-6-sulfonic acid).

Chromophores which have a low polarity and are lipophilic are preferred. However, water solubility should still be guaranteed.

The lipophilic character of the above chromophores can be positively influenced by suitable substitution, such as with alkyl groups. The methyl, dimethyl and ethyl groups and the substitution with bromine, among others, have proven to be suitable substituents for the resorufin radical.

The compounds of the invention are new. They have an asymmetry center and are therefore optically active. Both the racemates obtained in the customary production methods and the optical isomers can be used as the phospholipase substrate. However, the optical isomers are preferred.

The phospholipase substrates according to the invention can be produced by methods known per se. Suitable processes for the synthesis of the ether-glycero- and acyl-glycerophosphocholines can be found, for example, in Methods in Enzymology<u style="single">98</u>, 623 (1983) and Biochim.Biophys.Acta <u style="single">666</u>, 230 (81). The synthesis of sulfates is described, for example, in Beilstein 2, EII, 356.

Starting from the described 1-alkyl or 1-acyl-3-O-trityl-glycerol compounds, the corresponding glycerodicarboxylic acid monoesters are then obtained by reaction with the corresponding dicarboxylic acid anhydrides in an anhydrous medium such as chloroform / pyridine. Suitable methods for the preparation of the dicarboxylic acid anhydrides are described in Houben-Weyl-Müller "Methods of Organic Chemistry", Volume IV / 4, page 786.

Instead of the 3-O-trityl group, another protective group can also be used, for example benzyl.

The esterification of the monoester with the aromatic hydroxy or thiol compound from which the radical X is derived can be carried out, for example, by directly reacting the dicarboxylic acid monoester with the aromatic alcohol or thiol in the presence of a dehydrating agent such as dicyclohexylcarbodiimide. Alternatively, the dicarboxylic acid monoester is first converted to an activated ester, for example the hydroxysuccinimide ester or the imidazolide, and the activated ester is then reacted with the aromatic alcohol or thiol.

Likewise, it is also possible first to prepare a monoester of dicarboxylic acid with the aromatic alcohol or thiol, for example the mononitrophenyl adipate or monophenyl glutarate, and then to esterify it with the alkyl or acylglycerol, for example via the intermediate formation of an acid chloride or anhydride or activated ester. The dicarboxylic acid monoesters with the aromatic alcohol or thiol can be prepared, for example, from acid anhydride and aromatic compound in a molar ratio of 1: 1 or from dicarboxylic acid and aromatic compound in a molar ratio of 2: 1 or from a dicarboxylic acid monoester with an easily removable protective group and the aromatic compound. A suitable method is, for example, in Arch. Pharm.<u style="single">287</u>, 514 (1954).

Alternatively, the 1-O-alkyl or 1-O-acyl-3-O-trityl-glycerodicarboxylic acid monoester can also be prepared by first of all creating a dicarboxylic acid monoester from the dicarboxylic acid and an easily removable alcohol, such as. B. benzyl alcohol or 2,2,2-trichloroethyl alcohol and the acid thus obtained is then esterified with the alkyl or acyl-3-O-trityl glycerol mentioned. The protective group is then removed and the reaction with the aromatic alcohol or thiol is carried out as described above.

Protective groups must then be split off in the products described above and the C-3-OH position sulfated or phosphorylated.

Another method of preparation consists in first reacting a protected glycerol such as 1,2-isopropylidene-glycerol with a dicarboxylic acid monoester to form the corresponding protected glycero-3-dicarboxylic acid diester, then removing the protective group of the glycerol and removing the 1-OH- Group and the liberated 2-OH group alkylated or acylated. Finally, the first introduced monoester group (carboxyl protecting group) is split off and reacted with the aromatic alcohol or thiol. After the protective group has been cleaved at C-1-OH, phosphating or sulfation is carried out.

In addition, the desired substrate can be prepared from appropriate lyso compounds (for example 1-alkyl-glycero-3-sulfate), for example by reaction with activated dicarboxylic acid monoester.

The preparation of the substrates according to the invention described above is not exhaustive and a number of further methods known per se are available to the person skilled in the art which enable him to easily produce any of the compounds according to the invention. If desired, the pure optical isomers can be obtained from the racemic products obtained by the processes described above by known separation processes. Likewise, the isomers can also be obtained by stereospecific synthesis using methods which are also known per se.

The method according to the invention for the optical determination of the phospholipase is characterized in that a phospholipase substrate according to the invention is subjected to the action of the sample containing phospholipase and the amount of the aromatic hydroxy or thiol compound released is determined optically or directly after coupling with a suitable chromogen, the color formed therefrom. It may be necessary to add one or two auxiliary enzymes.

Another object of the invention is a simple and long-lasting reagent for the optical determination of phospholipase, which in addition to a phospholipase substrate and buffer substance according to the invention also contains a surface-active agent, such as in particular bile acid salt, a chromogenic coupler and / or a salt such as calcium chloride. In addition, the reagent can expediently also contain preservatives and / or activator.

In a preferred composition, this contains reagent 0.05-10 mg / ml substrate, particularly preferably 0.5-10 mg / ml substrate, 2 - 50 mg / ml bile acid, 0.5 - 10 mM / l calcium chloride (activator), 0 - 10 g / l detergent, 20 - 250 mM / l buffer substance each based on the ready-to-use solution in the test.

Suitable surface-active agents of the bile acid group are cholic acid, taurocholic acid, deoxycholic acid, taurodeoxycholic acid, glycodesoxycholic acid or their alkali metal salts, in particular the sodium salt. The preferred amount is 0.5 to 1.5 mM / l. Alternatively or additionally, the reagent can also contain one or more nonionic detergents.

Both ionic and nonionic detergents can be used as detergents. Nonionic detergents such as Triton® (alkylaryl polyether) are preferably used. A particularly suitable concentration range is between 0-10 g / l. The concentration range between 1-5 g / l is particularly preferably used.

Suitable buffer substances are buffers which are able to set a pH between 6.0 and 10.5 in the context of the reagent according to the invention. The preferred pH range is between 7.0 and 9.5. Examples of suitable buffers are diethanolamine buffers, triethanolamine buffers, tris buffers and good buffers such as Hepes buffers (well suited for addition before lyophilization), taps buffers, CHES buffers (2- (cyclohexylamino) ethanesulfonic acid) and bicine. Tris buffer is particularly preferred. The preferred amount of buffer substance is between 20 and 250 mM / l.

The preservatives used in the context of the invention are those which do not impair the enzymatic activity of the phospholipase to be determined. Alkali azides, in particular sodium azide, are particularly suitable. Other preservatives, such as However, thiozide and other sulfur-containing preservatives are also suitable. The preferred amount of preservative is 0.001 to 2 mg / ml.

Alkaline earth ions, preferably calcium ions, are suitable as activators. Since these form compounds insoluble with deoxycholic acid, taurodeoxycholic acid is preferred in the presence of calcium as bile acid, since this permits higher calcium concentrations in the range from 1 to 5 mmol.

If the reagent according to the invention is used in a dry or concentrated form for dilution to the final composition, it contains the substances mentioned in appropriate proportions, and preferably protective colloid.

The protective colloid includes the substances known to the chemist, such as polyhydroxy compounds, serum albumin, polyvinylpyrrolidone, solid polyethylene oxides and the like. Polyhydroxy compounds, in particular monomeric or polymeric pentose or hexose with 1 to 10 pentose or hexose units in the molecule and / or polyethylene glycol which is solid at room temperature, are preferred. Preferred examples of suitable polyhydroxy compounds are mannitol and similar sugar alcohols, oligosaccharide of glucose, mannose, maltoheptaose, polyethylene glycol with an average molecular weight between 3500 and 7000 u. Other protective colloids that can be used are e.g. B. amino acids, such as alanine, plant gums, such as gum arabic, etc. The preferred amount of protective colloid or a mixture of protective colloids is 20 to 90 wt .-%. A mixture of sugar alcohol and polyalkylene glycol proved to be particularly suitable.

The reagent according to the invention can also be impregnated on a suitable carrier material. Both an absorbent carrier material and a swellable, soluble film-forming carrier material can be considered. In this form, the reagent according to the invention enables the production of test strips which can be evaluated directly visually or by means of suitable measuring devices.

The color test according to the invention for determining the phospholipases provides very precise results with high sensitivity. It is very easy to use and is even suitable for test strips. Since it has only a very small or no lag phase, it can be easily adapted to the various analyzer systems.

The determination itself can be carried out both as an end point determination and kinetically. Compared to many known processes, the kinetic feasibility has the advantage that the reaction product formed does not have to be stopped or shaken out.

The following examples further illustrate the invention.

example 1

1-O-octadecyl-2-glutaric acid-p-nitrophenyl ester-sn-glycero-3-phosphocholine

a) 1-O-octadecyl-2-glutaric acid-sn-glycero-3-phosphocholine

1 g 1-O-octadecyl-sn-glycero-3-phosphocholine, 0.73 g glutaric anhydride, 0.2 g dimethylaminopyridine are heated in 30 ml pyridine at 50 ° C for 70 h. The solvent is then stripped off and the residue is chromatographed on LH 20 (eluent: chloroform / methanol 1: 1). Then it is chromatographed on RP 18 (mobile phase: isopropanol / water 9: 1). Yield: 0.87 g DC: R_f = 0.26 (silica gel; methanol) Spray reagent: Hanes-Isherwood reagent 1 H-NMR (CDCl₃): δ [ppm]: 0.87 (t, 3H); 1.26 (m, 32H); 1.90 (m, 2H); 2.40 (m, 4H); 3.1-3.6 (m, 13H); 3.78 (m. 2H); 4.02 (m, 2H); 4.25 (m. 2H); 5.18 (m, 1H).

b) 1-O-octadecyl-2-glutaric acid-p-nitrophenyl ester-sn-glycero-3-phosphocholine

310 mg 1a) are dissolved in a mixture of water / tetrahydrofuran 1: 1 and mixed with 70 mg p-nitrophenol, 480 mg N-ethyl-N'-dimethylaminopropylcarbodiimide. The mixture is then stirred at 60 ° C for 40 h. After evaporation of the solvent, it is chromatographed on RP 18 (eluent: isopropanol / water 8: 2). DC: R_f = 0.21 (RP18; isopropanol / water 8: 2) 1 H-NMR (CDCl₃): δ [ppm]: 0.88 (t, 3H); 1.25 (m, 32H); 2.04 (m, 2H); 2.2-2.8 (m, 4H); 3.1-3.6 (m, 13H); 3.80 (m, 2H); 3.96 (m, 2H); 4.28 (m. 2H); 5.15 (m. 1H); 7.30 (d. 2H); 8.26 (d. 2H).

Example 2

1-O-octadecyl-2-glutaric acid-methylresorufine ester-sn-glycero-3-phosphocholine

Preparation analogous to 1b) from 31 mg 1a), 10 ml water / tetrahydrofuran 1: 1, 110 mg 4 methylresorufin, 48 mg N-ethyl-N'-dimethylaminopropylcarbodiimide. DC: R_f = 0.20 (RP 18; isopropanol / water 8: 2) 1 H NMR (d₄-methanol): δ [ppm]: 0.90 (t, 3H); 1.28 (m, 32H); 1.92 (m, 2H); 2.16 (s. 3H); 2.44 (m, 4H); 3.51 (m. 2H); 3.69 (m, 11H); 4.00 (m, 2H); 4.29 (m. 2H); 4.47 (m. 2H); 5.2 (m, 1H); 6.7-7.9 (m, 5H).

Example 3

1-O-dodecyl-2-glutaric acid p-nitrophenyl ester glycero-3-sulfate

a) 1-O-Dodecyl-2-glutaric acid-3-O-trityl-glycerol

20.1 g of 1-O-dodecyl-3-O-trityl-glycerol, 9.2 g of glutaric anhydride and 0.4 g of dimethylaminopyridine are heated to 50 ° C. in 100 ml of pyridine for 8 hours. The solvent is removed, the residue is taken up in ethyl acetate and shaken with 0.05 N hydrochloric acid. After the ethyl acetate phase has been dried over sodium sulfate, the solvent is evaporated off and the residue is chromatographed on silica gel (mobile phase: ethyl acetate / petroleum ether 1: 4). Yield: 14 g DC: R_f = 0.20 (silica gel; ethyl acetate / petroleum ether 1: 1) 1 H-NMR (CDCl₃): δ [ppm]: 0.88 (t, 3H); 1.25 (m, 20H); 1.99 (q, 2H); 2.43 (t, 4H); 3.15-3.45 (m, 4H); 3.59 (d. 2H); 5.22 (m, 1H); 7.3 (m, 15H).

b) 1-O-Dodecyl-2-glutaric acid-p-nitrophenyl ester-3-O-trityl-glycerol

8.6 g 3a) are dissolved in 150 ml chloroform and 1.94 g p-nitrophenol and 14.4 g dicyclohexylcarbodiimide are added in succession. After stirring for 12 hours at room temperature, the precipitate is filtered off and the filtrate is concentrated. The residue is chromatographed on silica gel (eluent: ethyl acetate / petroleum ether 1: 4). Yield: 8.8 g DC: R_f = 0.42 (silica gel; ethyl acetate / petroleum ether 1: 4) 1 H-NMR (CDCl₃): δ [ppm]: 0.88 (t, 3H); 1.25 (m, 20H); 2.12 (q, 2H); 2.53 (t, 2H); 2.71 (t, 2H); 3.2-3.5 (m, 4H); 3.59 (d. 2H); 5.28 (m, 1H); 7.18 (d. 2H); 7.30 (m, 15H); 8.20 (d. 2H).

c) 1-O-Dodecyl-2-glutaric acid p-nitrophenyl ester glycerol

6 g 3b) are dissolved in petroleum ether and applied to silica gel treated with boric acid. The product is washed down with petroleum ether / ethyl acetate 8: 2. Yield: 2.7 g DC: R_f = 0.21 (silica gel; chloroform / acetone 49: 1) 1 H-NMR (CDCl₃): δ [ppm]: 0.87 (t, 3H); 1.25 (m, 20H); 2.10 (m, 2H); 2.53 (t, 2H); 2.71 (t, 2H); 3.45 (t, 2H); 3.63 (d. 2H); 3.82 (d. 2H); 5.04 (m, 1H); 7.28 (d. 2H); 8.26 (d. 2H).

d) 1-O-Dodecyl-2-glutaric acid-p-nitrophenyl ester-glycero-3-sulfate.

500 mg 3c) are taken up in 4 ml of chloroform and mixed with 0.22 ml of pyridine. 0.18 ml of chlorosulfonic acid in 2 ml of chloroform is added dropwise with ice cooling. Then the reaction solution is stirred for 2 h at 0 ° C and 1 h at room temperature. After adding 2 drops of water, the mixture is concentrated and the residue is taken up in chloroform. After drying over sodium sulfate, the solvent is evaporated off and the residue is chromatographed on silica gel (mobile solvent: methylene chloride / methanol 8: 1). Yield: 180 mg DC: R_f = 0.26 (silica gel; methylene chloride / methanol 6: 1) 1 H-NMR (CDCl₃): δ [ppm]: 0.89 (t, 3H); 1.21 (m; 20H); 2.02 (m, 2H); 2.54 (m, 4H); 3.45 (m, 4H); 4.19 (m. 2H); 5.33 (m, 1H); 7.28 (d. 2H); 8.22 (d. 2H).

Example 4

1-O-Hexadecyl-2-glutaric acid p-nitrophenyl ester glycero-3-sulfate.

a) 1-O-Hexadecyl-2-glutaric acid-3-O-trityl-glycerol

Preparation analogous to 3a) from 15 g of 1-O-hexadecyl-3-O-trityl-glycerol, 50 ml of pyridine, 6.2 g of glutaric anhydride, 0.4 g of dimethylaminopyridine. Yield: 7.14 g DC: R_f = 0.35 (silica gel; ethyl acetate / petroleum ether 2: 3) 1 H-NMR (CDCl₃): δ [ppm]: 0.87 (t, 3H); 1.1-1.65 (m, 28H); 2.0 (m, 2H); 2.43 (t, 4H); 3.32 (m, 4H); 3.59 (d. 2H); 5.21 (m, 1H); 7.33 (m, 15H).

b) 1-O-Hexadecyl-2-glutaric acid-p-nitrophenyl ester-3-O-trityl-glycerol.

Preparation analogous to 3b) from 3.5 g 4a), 50 ml chloroform, 0.84 g p-nitrophenol, 6.18 g dicyclohexylcarbodiimide. Yield: 2.85 g DC: R_f = 0.47 (silica gel; ethyl acetate / petroleum ether 1: 4) 1 H-NMR (CDCl₃): δ [ppm]: 0.88 (t, 3H); 1.25 (m, 28H); 2.13 (q, 2H); 2.53 (t, 2H); 2.7 (t, 2H); 3.33 (m, 4H); 3.59 (d. 2H); 5.28 (m, 1H); 7.17 (d. 2H); 7.33 (m, 15H), 8.20 (d, 2H).

c) 1-O-Hexadecyl-2-glutaric acid p-nitrophenyl ester glycerol

Production analogous to 3c) from 2.8 g 4b). Yield: 1.8 g DC: R_f = 0.22 (silica gel; chloroform / acetone 49: 1) 1 H-NMR (CDCl₃): δ [ppm]: 0.87 (t, 3H); 1.25 (m, 28H); 2.12 (q, 2H); 2.55 (m, 4H); 3.35-4.0 (m, 6H); 5.1 / m, 1H); 7.28 (d. 2H); 8.27 (d. 2H).

d) 1-O-Hexadecyl-2-glutaric acid p-nitrophenyl ester glycero-3-sulfate.

Production analogous to 3d) from 500 mg 4c). Yield: 310 mg DC: R_f = 0.30 (silica gel; methylene chloride / methanol 8: 1) 1 H-NMR (CDCl₃): δ [ppm]: 0.87 (t, 3H); 1.24 (m, 28H); 2.03 (m, 2H); 2.3-2.8 (m, 4H); 3.20-3.70 (m, 4H); 4.19 (m. 2H); 5.31 (m, 1H); 7.29 (d. 2H); 8.23 (d. 2H).

Example 5

1-O-Dodecyl-2-adipic acid p-nitrophenyl ester glycero-3-sulfate

a) 1-O-Dodecyl-2-O-benzyl-glycero-3-sulfate

28 g of 1-O-dodecyl-2-O-benzylglycerol are dissolved in 200 ml of chloroform, 20 ml of pyridine are added and a solution of 11.2 ml of chlorosulfonic acid in 80 ml of chloroform is added dropwise at 0.degree. The mixture is then stirred at room temperature for 3 h and then the mixture is poured onto ice. The water phase is shaken 3 times with chloroform and the organic phase is concentrated after drying over sodium sulfate. The residue is chromatographed on silica gel (mobile phase: chloroform / methanol 4: 1). Yield: 23 g DC: R_f = 0.25 (silica gel; methylene chloride / methanol 9: 1) Spray reagent: dichlorofluorescein 1 H-NMR (CDCl₃): δ [ppm]: 0.88 (t, 3H); 1.24 (m, 20H); 3.15-3.50 (m, 4H); 3.74 (m, 1H); 4.0 (s, 1H); 4.22 (m. 2H); 4.59 (s, 1H); 4.64 (s, 1H); 7.26 (m, 5H).

b) 1-O-dodecyl-glycero-3-sulfate

22.8 g 5a) are dissolved in 500 ml methanol and hydrogenated on 2.3 g Pd / C. The course of the reaction is controlled by DC. After the reaction has ended, the mixture is filtered and concentrated. The residue is slurried in acetone and filtered off. Yield: 15.6 g DC: R_f = 0.34 (silica gel; methylene chloride / methanol 4: 1) 1 H-NMR (d₄-methanol): δ [ppm]: 0.89 (t, 3H); 1.28 (m, 20H); 3.4-3.6 (m, 4H); 3.9-4.1 (m. 3H).

c) p-nitrophenyl adipic acid anhydride

1 g of p-nitrophenyl adipate are stirred in 40 ml of acetic anhydride at 80 ° C. for 2 h. The solvent is then stripped off and the residue is dried under high vacuum. The raw product will continue to be implemented. DC: R_f = 0.6 (silica gel; ethyl acetate)

d) 1-O-dodecyl-2-adipic acid p-nitrophenyl ester glycero-3-sulfate.

0.44 g 5b), 5c) and 100 mg dimethylaminopyridine are stirred in 50 ml pyridine at 80 ° C for 5 h. After the solvent has been stripped off, it is chromatographed on silica gel (mobile phase: chloroform / methanol 6: 1). Yield: 100 mg DC: R_f = 0.29 (silica gel; chloroform / methanol 6: 1) 1 H-NMR (CDCl₃): δ [ppm]: 0.86 (t, 3H); 1.23 (m, 20H); 1.73 (m, 4H); 2.20-2.75 (m, 4H); 3.2-3.7 (m, 4H); 4.16 (m. 2H); 5.31 (m, 1H); 7.29 (d. 2H); 8.24 (d. 2H).

Example 6

Solution 1:

125 mM Tris buffer pH 7.1, 4.0 mM calcium chloride, 250 µl / 100 ml Triton X-100, 41 mg / 100 ml sodium deoxycholate.

Solution 2:

2 mg substrate 1-O-hexadecyl-2-glutaric acid-nitrophenyl ester-rac-glycero-3-sulfate are stirred into 1 ml of solution 1 with gentle heating until an emulsion is formed. For this, 20 µl of the sample (enzyme solution) are added. The course of the reaction is monitored photometrically at λ = 405 nm.

When evaluated using a standard known phospholipase activity, the phospholipase activity of the sample is calculated as follows:<maths id="math0002" num=""><img file="EP0331167A2_D0005.tif" /></maths>

The phospholipase activity of the sample can also be calculated using the following formula:<maths id="math0003" num=""><img file="EP0331167A2_D0006.tif" /></maths> V_total      : Total volume of the test batch [cm³] V_sample      : Volume of sample [cm³] ε: extinction coefficient of the chromogen at 405 nm d: layer thickness of the cuvette [cm] ΔE / min: change in extinction per minute at 405 nm

Under the reaction conditions mentioned, the extinction coefficient is ε = 9.0 · l · cm² · µmol⁻¹.