GB2096989A - Choline derivatives and a process for their preparation - Google Patents

Abstract

A compound of the formula:- <IMAGE> [wherein A represents a hydrogen atom or a methyl or methoxy group in the p-position of the benzene ring and B represents a group of the formula -R-CO- (wherein -R- represents an alkenyl group with 2 to 6 carbon atoms); or A represents a methyl group in either or both o-positions of the benzene ring and B represents a group of the formula -CO-; X represents a stabilising anion and n represents the charge on the ion] may be used to determine cholinesterase activity. Cholinesterase determinations using the compounds of formula I ensure a high resistance of saturation, good stability of the compounds used as substrates, a conveniently low reaction rate and a high degree of accuracy. The preparation of the compounds of formula I is described and exemplified and may be effected, for example, by reaction of a halogenated choline and an acid derivative.

Description

SPECIFICATION Choline derivatives and a process for their preparation The present invention relates to choline derivatives and to a process for their preparation. The choline derivatives of the present invention are of particular interest for use in determining cholinesterase activity.

Various methods for determining the activity of cholinesterase are known, and all of these known methods use choline derivatives as a substrate for reaction with cholinesterase. It is also known that choline and an acid derived from the choline derivative are formed by the action of cholinesterase on the substrate, which appears to effect hydrolysis.

According to known methods, for example, acetyl choline, butyryl choline, acetyl thiocholine, propyl thiocholine, o-nitrophenyl butyrate or indophenyl acetate may be used as substrates, and buffer solutions containing such substrates at a given concentration may be used for reaction with cholinesterase. Reaction with cholinesterase results in the formation of an acid which causes the pH of the buffer solution to be lowered. Cholinesterase activity is determined with reference to the variation of pH (ApH) over a given time [Glass electrode method, Baum G.: Clin. Chimica Acta, 36, 405 (1972)].

However, such choline derivatives possess poor stability and it is thus difficult to conduct the necessary measurements accurately and accordingly it is also difficult to determine the activity of cholinesterase with good results.

It is also known to use benzoyl choline as the substrate (Kalow method) [Kalow W., Genest. K.: Canad. J. Biochem. Physiol. 35, 341(1957)]. This method includes (i) a process for determining cholinesterase activity by reference to the benzoic acid which is formed when the benzoyl choline used as substrate is decomposed by the action of cholinesterase and (ii) a process for determining cholinesterase activity by reference to the choline which is formed, the amount of choline formed being estimated by the use of choline oxidase which is added to the reaction system (Japanese Patent Application as laid open to public inspection No. 130,984/77 of Kokai Koho). It is, however, difficult to carry out the determination accurately by such processes as a result of the poor stability of the benzoyl choline used as substrate.

Moreover, the determination of cholinesterase activity in blood presents some special difficulties.

For example, when the concentration of benzoyl choline used as substrate is too low, the hydrolyzing action of cholinesterase on the substrate proceeds very quickly so that it is very difficult to trace this reaction exactly by manual or mechanical operation. It is thus difficult to determine the amount of cholinesterase present with any degree of accuracy. Moreover, when choline oxidase is used to estimate the choline produced by the action of cholinesterase, the presence of a certain amount of oxygen dissolved in the reaction system is necessary in addition to the formed choline and choline oxidase, but the amount of such oxygen present is usually very low. Furthermore, in such cases, it is not only difficult but also impracticable to enhance the amount of oxygen, for example, by introducing, e.g.

blowing, air or oxygen into the reaction system. On the other hand, choline is liberated very quickly by the action of cholinesterase on benzoyl choline used as the substrate and is converted into betaine aldehyde in the presence of oxygen by the action of choline oxidase. As a result, an equimolar amount of hydrogen peroxide is formed which is introduced into a colour-developing solution to determine the amount of choline formed as a result of the cholinesterase activity. These reactions are, however, discontinued as a result of the consumption of oxygen in the reaction system. It is thus difficult to accurately determine the amount of choline formed.

It is thus necessary to perform the measurement over the shortest possible time, before the consumption of the oxygen dissolved in the reaction solution, when benzoyl choline is used as the substrate.

On the other hand, when the concentration of benzoyl choline is too high, it is difficult to determine exactly the amount of cholinesterase as benzoyl choline per se serves to inhibit the cholinesterase in blood and the reaction is discontinued.

It is also known to determine cholinesterase activity by using butyl thiocholine as the substrate [Szasz, G.: Clin. Chim. Acta, 79. 191(1968)]. The cholinesterase activity is determined by forming thiocholine as a reaction product fro-m butyl thiocholine and cholinesterase, subjecting the thiocholine formed to reaction with a compound relating to 5,5'-dithiobisbenzoic acid (e.g. 2,2'- or 4,4'dithiopyridine) to obtain a coloured substance, and measuring the extinction coefficient of this substance. However, both butyl thiocholine and 5,5'-dithiobisbenzoic acid and related substances used for this process are very unstable and undergo natural oxidation.Also, the reaction performed by this process is the same as the reaction of various thiol derivatives in blood (e.g. glutathione) so that again it is not possible to make any measurement with accuracy.

We have found that the various disadvantages detailed above of known methods of determining cholinesterase activity may, at least in part, be overcome by the use of certain choline derivatives which exhibit good stability and also decompose by the action of cholinesterase at a conveniently slow reaction rate. In addition to the above, the use of the compounds to determine cholinesterase activity ensures a high resistance to saturation. Saturation is a difficulty which is described hereinafter and which gives rise to inaccurate results.

The present invention is based on the discovery of certain novel choline derivatives of formula I hereinafter defined which possess the above-mentioned advantages and which may be used to determine cholinesterase activity with improved accuracy. In our copending Patent Application No.

7912994 (Serial No. 2,018,988) we describe and claim a method for determining cholinesterase activity as well as a reagent for use in determining cholinesterase activity which method and reagent involve inter alia the use of the novel compounds of formula I hereinafter defined.

Thus according to one feature of the present invention there is provided a compound of the formula:

wherein A represents a hydrogen atom or a methyl or methoxy group in the p-position of the benzene ring and B represents a group of the formula -R-CO- (wherein -R- represents an alkenyl group with 2 to 6 carbon atoms): or A represents a methyl group in either or both o-positions of the benzene ring and B represents a group of the formula -CO-; X represents a stabilising anion and n represents the charge on the ion.

The compounds of formula I in which X represents a halide ion, e.g. a chloride ion, and n is 1 are preferred, by virtue of their use as substrates in determining cholinesterase activity.

Thus according to the present invention, a compound of formula (I) as hereinbefore defined may be used as substrate for reaction with cholinesterase to form choline and/or an acid. Cholinesterase activity is determined by means of the choline and/or acid formed for example, by direct measurement of the choline and/or acid formed e.g. by referring to the variation of pH of a solution containing the formed acid.

It is also possible to determine the amount of choline formed, for example, by subjecting the choline to the action of choline dehydrogenase in the presence of NAD and feeding the same into a colour-developing solution. It is also possible to subject the formed choline to the action of choline oxidase to produce hydrogen peroxide which is then added to a colour-developing solution. Various known methods (e.g. the method proposed by Kalow et al and the method proposed by Takahashi et a/) may be used to determine the variation of pH caused by the acid formed.

Especially preferred compounds of formula (I) are listed below together with their physical properties in Table 1, TABLE 1

Structlral formula Name 11.P.

O P-methylcinnamoyl 170 Ir + CH ' , choline cbloride 170 tH C- O -CH=CH-C-0-CH2-CHz-N CH33| f CH9 C1 1740C ~ CH9 (Compound No. I) O P-metho: :ycinn2moyl a + ~CH,] choline chloride 183 [r?co-Clr=CH-C-o-CH,-CH2-Nf CI-IJ I C1- 154 OC CH33 CH33 Cl (Compound No.11) lst0c CH 0 CM3 0 o-metbylbenzoyl h + ,C143 + CH 1 choline chloride 142 [ O - C-0-CH2-CH2-N tCh~ Cl (Compound No.111) 143 C o j (Compaund Mo.III) I-CH = CH - e - O- CH2-CH2-NLCH3 C113J Cinnamoylcholine 196 chloride 1970C (Compound No. IV) Compound N.M.R. (in CD3OD) IR(KBr) cam~' 2.00 s 3H; 3.58 s 9H; 3.78 m 2H; 1711; 1625; 4.68 m 2H; 6.48 d=18Hzl H; 7.18 d=8Hz2H; 7.50 d=8Hz2H; 7.72 d=18Hz1 H II 3.30 s 9H; 3.83 s 3H; 3.87 m 2H; 1710; 1640; 4.68 m 2H; 6.42 d=l 8Hzl H; 6.93 1605; 1175; d=1 OHz2H;.758 d=1 OHz2H; 7.72 d=1 8Hz1 H 825; 805 Ill 2.60 s 3H; 3.33 s 9H; 3.93 m 2H; 1730; 742 4.81 s 2H; 7.32 m3H;.795 m 1H IV 3.35 s 9H; 3.95 m 2H; 4.75 m 2H; 1720; 1635; 7.18 d=8Hz 2H; 7.22 d=8H 2H; 7.60 m 5H 775 Table 2 indicates the reaction rates of compounds of formula I as hereinbefore defined.

The reaction rate is indicated by reference to the reaction rate of benzoyl choline which is a known substrate. The reaction rate was determined in the following manner.

Each (0.2 mg) of the substrates shown in Table 2 was added to a tris HCI buffer solution (3 mi; pH 7.5) containing the following ingredients: 4-Aminoantipyrine 3 mg Phenol 2.8 mg Choline oxidase (prepared in Reference 2) 7.05 Unit Peroxidase (Worthington Corpn., U.S.A.) 9.1 Unit Each solution was preliminarily heated at 370for 10 minutes and a standard serum (each 20 microl) was added thereto. The variation of OD was then measured continuously to determine the rate of increase in OD in a unit of time (min). In Table 2, the rate of increase of OD of benzoyl choline in a unit of time is referred to as 1.

TABLE 2

Compound In Formula (I) Reaction rate A B Benzoyl choline 0 1 chloride O -C 0 0 II 0.043 cS oline chloride CH3 t CH C 0 050 D-methoxycirmamayl H C0- O - -CH=CH-X- 0.017 Sholine chloride 3 t--/ 0.019 o-methylbenzoyl CM3 C 0.32 choline chloride O - - 0.37 0 Cinnamoyl choline II 0.06 chloride -CM=CM-C- 0.07 The solution stabilities of the compounds (substrates) of the present invention as well as the solution stabilities of the known substrates were determined and shown in Table 3, in which the decomposition rate of benzoyl choline is referred to as 1. The decomposition rate was determined in the following manner.

A tris HCI buffer solution (0.05M; pH 7.5) containing a specified compound of formula 1 (0.615 millomol/3 ml) was prepared and allowed to stand at 370C for 3 days. After this, the decomposition rate was determined on the basis of the amount of choline produced from this compound. In a similar manner to that described above, hydrogen peroxide which had been produced by the action of choline oxidase was treated with 4-aminoantipyrine to form a quinoneimine-type pigment, and its OD at 500 nm was measured to determine the amount of choline formed. The amount of this pigment depends upon the amount of choline produced so that a smaller amount of the pigment formed indicates a lower decomposition rate.

As is apparent from Table 3, the known substrates such as benzoyl choline and acetyl choline are liable to decompose relatively rapidly with time, and their blank values are also liable to increase over a period of time after the preparation of reagents containing such substrates. On the other hand, the substrates of the present invention decompose slowly and have better preservability and such properties are clearly advantageous for the determination of cholinesterase activity.

Structural formula(I ) Decomposition rate Compound A B ~~~~~~~~~~~~~~ Benzoyl choline O -C- 1 chloride - II Acetyl choline chloride CH3- -C- 2 Propionyl choline CH3-CH3- -ICI- 2.3 chloride 0 p-methylcinnamoyl 0 choline chloride CH3 e -CM=CM-C- 0.1 0 P-methoxycinnamoyl u -CM=CM-- 0.05 choline chloride MC0 o-metthylbenzoyl 0 0.002 choline chlorideCH3 -e Cinnamoyl choline 0 0.26 chloride V rL The compounds of formula 1, may be produced in a similar manner to that applicable to the production of known choline derivatives, for example by using an acid derivative and a halogenated choline.

Thus according to a further feature of the present invention there is provided a process for the preparation of a compound of formula I as hereinbefore defined which comprises reacting an acid derivative of a compound of the formula

(wherein A and B are as hereinbefore defined and Y represents a leaving atom or group) with a compound of the formula {(CH3)3NCH2CH2oH1 xne (VI) (wherein X and n are as hereinbefore defined) whereby a compound of formula I as hereinbefore defined is obtained.

The reaction between the acid derivative e.g. a compound of formula V in which Y represents a chlorine atom and the halogenated choline e.g. choline hydrochloride may, for example, be effected in the absence of a solvent, in which case the reaction is preferably effected at a temperature of from 100--2000C, conveniently for 2-8 hours. When the reaction is effected in the presence of an appropriate solvent such as for example chloroform, toluene, benzene and the like, the reaction is preferably effected at a temperature of from 20-600C, conveniently for 8-20 hours. Isolation and purification of the desired compound may for example be effected in the usual manner applicable to the synthesis of organic compounds.

Experiment 1 Determination of cholinesterase activity by using a substrate of the present invention and choline oxidase: (A) Preparation of reagent In conventional manner, a reagent is prepared by using a 0.05M tris-HCI buffer solution (pH 7.5) containing the following ingredients: 4-Aminoantipyrine 3 mg Phenol 2.8 mg Choline oxidase prepared in Reference 7.05 U Peroxidase (commercial product of Worthington 9.1 U Biochemical Corpn., U.S.A.) Substrate (o-methylbenzoyl choline chloride) 0.2 mg (B) Analysis procedure Each of the test serums and Q-PAKI standard serum having a cholinesterase activity of 1 458mU/ml (commercial product of Highland Div. Travenol Laboratories Inc., U.S.A.) is added to the thus-prepared reagent (3 ml) and incubated at 370C for a pre-determined time (cf. Table 4).The reaction is discontinued by addition of neostigmine (2 mg). Table 4 indicates various optical densities measured at 500 nm by using a spectrometer. For comparison, the substrate according to the present invention is replaced by benzoyl choline, which is a known substrate, to obtain the corresponding values which are also indicated in this table.The reagent containing benzoyl choline is prepared in conventional manner by using 3 ml of a 0.05M tris-HCI buffer solution (pH 7.5) containing the following ingredients: 4-Aminoantipyrine 0.5 mg Phenol 1.4 mg Choline oxidase 7.15 Unit Peroxidase 10.3 Unit Substrate (benzoyl choline) 0.5 mg Table 4 Substrate Optical density after (min)* Serum used used 4 10 20 40 Q-PAKI Standard ill** 0.140 0.350 0.700 1.400 Serum (choline esterase activity: All*** 0.440 1.100 1.600 1.650 1458 mU/ml) Serum of patient(1) Ill 0.081 0.202 0.404 0.808 in a case of liver disease VII 0.255 0.638 1.300 1.650 Serumofpatient(2) lil 0.217 0.543 1.085 2.100 in a case of kidney disease VII 0.679 1.600 1.650 1.670 Notes:: * Reaction time ** Ortho-methylbenzoyl choline chloride (Compound lli) Benzoyl choline chloride (Compound VII) Where o-methylbenzoyl choline chloride is used, the cholinesterase activity (about 841 mU/ml) of the serum of patient (1) was determined by the following proportional distribution method (hereinafter, the optical density is referred to as OD).

Cholinesterase activity=[1 458/(OD of the standard serum)] x [OD of the serum of patient (1)] Where benzoyl choline chloride is used as the substrate, the cholinesterase activity (about 844 mU/ml) of the serum of patient (1) was similarly determined by the proportional distribution method, with reference to the values measured after 4 and 10 minutes (the values measured after 20 and 40 minutes are likely to be incorrect).

Where o-methylbenzoyl choline chloride is used as the substrate, a similar procedure to the above-mentioned gave an activity of 2260 mU/ml in respect of the serum of patient (2). Where benzoyl choline chloride is used as the substrate, an activity of 2250 mU/ml in respect of the serum of patient 2 is similarly obtained, with reference only to the O.D. (optical density) measured after 4 minutes. This result is believed to be reliable. However, other OD values are likely to be unreliable owing to their saturation. In our view, such saturation may be caused by the lack of sufficient dissolved oxygen which is required by the choline oxidase. In such cases the reaction of benzoyl choline proceeds at an excessively rapid rate. It is desirable in such cases to dilute the serum in order to obtain a reliable result (however, such dilution procedure is virtually impracticable).

Also, from practical viewpoint, the discontinuation of the measurement after 4 minutes can give rise to various disadvantages such as a large error variance.

When the substrates of the present invention are used, however, it is possible to obtain a reliable OD result, without saturation, even when the cholinesterase activity is very high and the reaction is carried out over a long period of time. Thus the substrates of the present invention are very useful for determining cholinesterase activity.

The following table shows the preferred concentrations of the ingredients of the reagent according to the present invention and of the known reagent containing benzoyl choline.

Table 5 A Reagent of the present invention B Reagent of the known type In gre dients A B 4-Aminoantipyrine 1-6 mg 0.1-1 mg Phenol 0.7-14 mg 0.7-3 mg Choline oxidase 1.4-30 Unit 0.5-10 Unit Peroxidase 0.1-20 Unit 0.550 Unit Substrate of the present 0.1-6 mg invention Substrate (benzoyl choline) - 0.1-1 mg Reaction temperature: 20---400C Reaction time: one minuteone hour As apparent from Table 5, the range of the concentration of benzoyl choline is narrower than the corresponding range of the substrate of the present invention, and thus the versatility of the former is narrower than that of the latter.

Experiment 2 OD values of the standard serum and of the serum of patient (1) (in the case of liver disease) shown in Table 6 were obtained in a similar manner to that described in Example 1 with the exception that o-methylbenzoyl choline was replaced by the other substrates of the present invention detailed in the Table.

It is apparent from these results that the OD values determined are reliable and free from the saturation difficulties referred to above when measured using the substrates of the present invention.

Table 6 Substrate OD measured after-minutes* Compound (*reaction time) Serum used No. 4 10 20 40 Q-PAKI Standard 1 0.022 0.055 0.110 0.220 serum (choline 11 0.008 0.021 0.042 0.084 esterase activity: IV 0.031 0.079 0.158 0.316 1458 mU/ml) Serum of patient (1) 1 0.012 0.032 0.063 0.126 in a case of liver 11 0.005 0.012 0.025 0.050 disease IV 0.017 0.046 0.090 0.181 Notes: Compound I p-methylcinnamoyl choline chloride il p-methoxycinnamoyl choline chloride IV Cinnamoyl choline chloride Experiment 3 Determination of cholinesterase activity using the substrate of the present invention (in a similar manner to the Shibata-Takahashi Method): 1. Preparation of reagents: (1) Veronal/p-glycerophosphoric acid buffer solution (pH 8.3): 5,5-Diethylbarbituric acid (sodium salt; 3.00 g) is dissolved in water (about 500 ml).The solution is treated with 5,5-diethylbarbituric acid (1.00 g) which is dissolved at an elevated temperature. The solution is cooled and is treated with P- glycerophosphoric acid (sodium salt, 5.00 g), followed by addition of water to make up to 1 ,000 ml.

(2) Substrate solution (A) Acetyl choline chloride (1 g) is added to water (10 ml) and dissolved.

(B) o-methylbenzoyl choline chloride (0.5 g) is added to water (10 ml) and dissolved.

(3) Phenol red solution Phenol red (100 mg) is added to a 0.1N sodium hydroxide solution (3.0 ml) and water (7.5 ml).

The solution is heated to about 600C to dissolve the additives completely. The solution is cooled and is made up with water to 250 ml.

(4) Eserine solution Physostigmine salicylate (0.1 g) is dissolved in water which is then made up to 100 ml.

(5) Phosphate buffer solution (1/1 5M) (A) Potassium dihydrogen phosphate (KH2PO4.. .9.08 g) is dissolved in water which is then made up to 1,000 ml.

(B) Sodium hydrogen phosphate (NaHPO4.2H20. . .11.88 g) is dissolved in water which is then made up to 1,000 ml.

2. Preparation of reaction solution 1) Veronal/p-glycerophosphate buffer solution 1.5 ml 2) Substrate solution 0.25 ml 3) Phenol red solution 0.1 ml 4) Water 3.05 ml The reaction solution contains the above-mentioned ingredients. The ratio is indicated by volume.

3. Preparation of calibration curve Various buffer solutions (potassium hydrogen phosphate and sodium hydrogen phosphate) are prepared and adjusted in series to different pHs within the range 5.9-8.3. Each (5.0 ml) of the buffer solutions thus-prepared is added to the phenol red solution (0.1 ml) and thoroughly mixed. Each mixture is then kept at 20+1 C and OD at 570 nm is measured with reference to water which is used for control purposes. The thus-measured OD and the indicated pH are plotted to obtain a pH- OD calibration curve.

4. Operation for determination Vessels for blank test and test sample (serum) receive the reaction solution (each 4.9 ml) and are then preliminanly kept at 370C for 5 minutes. After this, water (0.1 ml) and serum (0.1 ml) are added to the vessels for blank test and serum with agitation, followed by carrying out the reaction at 37"C for 60 minutes exactly, respectively. Each reaction mixture is treated with eserine solution (0.1 ml) and is left at room temperature, after which the mixture is allowed to stand in a water bath (2000) until the solution in the test tube is cooled to 20+20C (in about 10 minutes). After this, OD at 570 nm is measured with reference to water. The pH Blank and pH Serum are determined in accordance with the calibration curve previously prepared.The ApH (i.e. pH Blank minus pH Serum) values thus-obtained are indicated in the following table.

Table 7 A-Cholinesterase activity (ApH) of acetyl choline chloride B-Cholinesterase activity (ApH) of o-methylbenzoyl choline chloride Substrate Substrate Serum No. A B Serum No. A B 1 0.83 1.30 6 0.35 0.87 2 0.75 1.28 7 0.58 1.10 3 1.46 1.90 8 0.43 0.90 4 0.65 1.20 9 0.60 1.15 5 0.70 1.25 10 0.63 1.17 This table indicates that the correlationbetween ApH of acetyl choline chloride and ApH of omethylbenzoyl choline chloride is excellent.

Correlation coefficient 0.993 Regression line y=0.928X+0.564 (wherein y represents the value measured by using o-methylbenzoyl choline chloride, the substrate of the present invention and X represents the value measured by using acetyl choline chloride).

Thus it is possible to simply determine the cholinesterase activity with reference to the previously prepared list of correlations between the ApH of acetyl choline chloride and the cholinesterase activity (it).

Experiment 4 The results shown in Table 8 were obtained in a similar manner to that described in Experiment 3 with the exception that serum No. 5 used in Experiment 3 was repeatedly determined. As is apparent from this table, the use of the substrate of the present invention results in a very small coefficient of variation and exact analytical value.

Table 8 A-Cholinesterase activity (ApH) of acetyl choline chloride B-Cholinesterase activity (ApH) of o-methylbenzoyl choline chloride Substrate Substrate Serum No. A B Serum No. A B 1 0.68 1.24 6 0.70 1.25 2 0.73 1.26 7 0.74 1.25 3 0.69 1.25 8 0.69 1.26 4 0.70 1.25 9 0.76 1.25 5 0.65 1.23 10 0.68 1.24 Substrate used A B Mean value 0.696 1.247 Standard deviation 0.025 0.009 Coefficient of variation (%) 3.59 0.72 Example Preparation of the compounds (substrates) of the present invention: (A) Synthesis of o-methylbenzoyl choline chloride: o-methylbenzoyl chloride (25 g) and choline hydrochloride (22.4 g) are heated under reflux to 120-1 400C for 4 hours, and the reaction solution is concentrated to dryness. The solid is washed well by the addition of n-hexane (100 ml).After the removal of n-hexane by filtration, the material is added to tert-butanol (40 ml) and heated in order to dissolve it. The solution is allowed to stand in a refrigerator at 50C overnight to form crystals. The crystals are collected by filtration and recrystallized from a mixture of tert-butanol/ethanol (30 ml; 3:1 by volume), followed by drying under reduced pressure. 33 g of the desired product is obtained in a yield of about 33% (M.P. 142--1430C). The desired product is identified by reference to various properties shown in Table 1. In a similar manner to that described above, the other products indicated in Table 1 are also produced by using corresponding acid chlorides and choline hydrochloride. The properties of these products are also indicated in Table 1.

Reference Preparation of choline oxidase: Brevibacterium album KY 431 9 (FERM P-3777; NRRL B-1 1046) is used as the choline oxidase producing micro-organism. A seed medium (10 ml) containing choline chloride (2 g/dl), corn steep liquor (0.5 g/dl), yeast extract (0.5 g/dl), sodium glutamate (0.5 g/dl) and magnesium sulfate .7H,O (0.05 g/dl) is put in a test tube (capacity..70 ml) and sterilized at 1 200C for 15 minutes. After this, one platinum loop of the strain is used to inoculate the medium for culturing at 300C for 48 hours with shaking. All broths obtained are transferred to another seed medium (300 ml) containing the same ingredients as those of the first seed medium and put in an Erlenmeyerflask (capacity..2 litre) for culturing at 300C for 48 hours with shaking. After completion of the fermentation, all broths are used to inoculate a main medium (3.0 litre) having the same ingredients and put in a 5 litre jar fermentor for culturing at 300C for 24 hours with aeration (one litre/litre of the medium/min) and shaking (500 r.p.m.). The fermented liquor contains 0.62 U/ml of choline oxidase.

In order to recover the choline oxidase from the fermented liquor, microbial bodies are separated from the liquor by centrifugation and dispersed in one litre of tris buffer solution (0.05 mol/litre; pH 8.0) which is then transferred to a crusher (Dyno Laboratory mill, KDL type, commercial product of Willy A.

Bachofen Inc., Switzerland) to obtain a solution containing crushed microbial bodies. This solution is centrifuged to obtain a supernatant (the above-mentioned amount of choline oxidase produced is determined by using this supernatant as an enzyme solution for measuring the activity of choline oxidase). Ammonium sulfate is added to the supernatant to make it saturated with 30% ammonium sulfate. The precipitates are removed from the saturated solution by centrifugation, and the supernatant thus-obtained is treated with ammonium sulfate to make it saturated with 60% ammonium sulfate. The saturated solution is centrifuged to collect the precipitates. The precipitates are dissolved in a tris buffer solution (pH 8.0; 0.05 mol/litre) and subjected to dialysis overnight, by using the same tris buffer solution and a dialysing membrane in the form of tube made of cellophane.

The solution in the tube is transferred to a column packed with one litre of DEAE cellulose which has been equilibrated with a tris buffer solution (pH 8.0) containing sodium chloride (0.05 mol/litre). The column is washed with one litre of a tris buffer solution (pH 8.0) containing sodium chloride (0.05 mol/litre), and gradient elution is effected by using an eluting solution containing sodium chloride (0.05-0.45 mol/litre). Fractions containing choline oxidase are collected and combined, and the combined fractions are treated with ammonium sulfate to obtain a solution saturated with 60% ammonium sulfate. The precipitates are collected by centrifugation and dissolved in a tris buffer solution (0.05 mol/litre; pH 8.0).The solution is transferred to 500 ml of Sephadex G-150 (a molecular sieve composed of dexstran derivatives, commercial product of Pharmacia Fine Chemicals Inc., U.S.A., the word "Sephadex" being a registered Trade Mark) which has been equilibrated with a tris buffer solution (0.05 mol/litre; pH 8.0), and elution is effected by using an identical buffer solution.

Fractions containing choline oxidase are collected and combined. The combined fractions are made saturated with 60% ammonium sulfate. The precipitates are collected by centrifugation and dissolved in a tris buffer solution (0.05 mol/litre; pH 8.0). The solution is subjected to dialysis overnight, by using the same buffer solution and a dialysing membrane in the form of tube made of cellophane.

The dialysed solution is transferred to 500 ml of Sephadex A-50 (weakly basic anion exchange resin, commercial product of Pharmacia Fine Chemicals Inc., U.S.A., the word "Sephadex" being a registered Trade Mark) which has been equilibrated with a tris buffer solution (0.05 mol/litre; pH 8.0) containing sodium chloride (0.1 mol/litre), and is washed with 500 ml of a tris buffer solution (0.05 mol/litre; pH 8.0) containing sodium chloride (0.1 mol/litre). Then gradient elution is effected by using an eluting solution containing sodium chloride (0.1--0.5 mol/litre). Fractions containing choline oxidase are collected and combined. The combined fractions are subjected to dialysis using a tris buffer solution (0.05 mol/litre; pH 8.0) and a tube made of cellophane. The dialysed solution is freeze-dried to obtain choline oxidase with a yield of about 10% (activity...2.2 U/mg of protein).

Claims (11)

Claims

1. A compound of the formula:

wherein A represents a hydrogen atom or a methyl or methoxy group in the p-position of the benzene ring and B represents a group of the formula -R-CO- (wherein -R- represents an alkenyl group with 2 to 6 carbon atoms); or A represents a methyl group in either or both o-positions of the benzene ring and B represents a group of the formula -CO-; X represents a stabilising anion and n represents the charge of the ion.

2. A compound as claimed in claim 1 wherein X represents a halide ion and n is 1.

3. Compounds as claimed in claim 2 wherein X represents a chloride ion.

4. p-Methylcinnamoyl choline chloride.

5. p-Methoxycinnamoyl choline chloride.

6. o-Methylbenzoyl choline chloride.

7. Cinnamoyl choline chloride.

8. A process for the preparation of a compound of formula I as defined in claim 1 which comprises reacting an acid derivative of a compound of the formula:

(wherein A and B are as defined in claim 1 and Y represents a leaving atom or group) with a compound of the formula.

[CH3)3N-CH2-CH2-oH]# xne (VI) (wherein X and n are as defined in claim 1) whereby a compound of formula I as defined in claim 1 is obtained.

9. A process as claimed in claim 8 substantially as herein described.

10. A process for the preparation of a compound of formula I as defined in claim 1 substantially as herein described in the Example.

11. A compound of formula I as defined in claim 1 when prepared by a process as claimed in any one of claims 8 to 10.