JPS60146848A - Novel tetraammonium salt and its use - Google Patents

Abstract

NEW MATERIAL:A compound shown by the formula I (R1-R6 are H, or lower alkyl; R7 is H, OH, lower alkoxy, or lower acyloxy; R8-R11 are 1-25C alkyl; X is anion; m is strength number of X). EXAMPLE:24-Acetoxy-19, 22, 26, 29-tetramethyl-22, 26-distearyl-19, 22, 26, 29-tetraazahexatetracontane.tetrafluoroborate. USE:A cationic modifying agent of membrane and a phase transfer catalyst. PREPARATION:A compound shown by the formula II is reacted with NaI in MEK to give a compound shown by the formula III. A compound shown by the formula IV is separately reacted with a compound shown by the formula V (R' is H, or 1-24C alkyl) to give a compound shown by the formula VI, which is reacted with LiAlH4 in dioxane to give a compound shown by the formula VII (R'' is R8-R11). This compound is reacted with the above-mentioned compound shown by the formula III to give a compound shown by the formula VIII, and this compound is reacted with a quaternary ammonium salt-forming compound, to give a compound shown by the formula I .

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel tetraammonium salts and uses of hiso.

Various enzymes exist in living organisms, and many of them include enzymes that synthesize high value-added substances, which are extremely interesting not only from a biochemical perspective but also from a synthetic chemistry perspective.

Generally, when enzymes are applied to industrial reactions, their stabilization is a prerequisite, and one method for stabilizing them is membrane immobilization. When immobilizing with a covalent membrane, enzyme activity often decreases or is inactivated.
It does not necessarily lead to problem solving.

By the way, some enzymes are anionic, such as prenyltransferase and ATPase.
This type of anionic protein can be immobilized on a cationic membrane by utilizing its ionicity. Therefore, it has been proposed to modify a carrier membrane, such as a liposome, with a cationic membrane modifier to impart cationic properties.

However, since the cationic membrane modifiers proposed so far are monoamines like stearylamine, they have only one positive charge, and even if liposomes are modified with stearylamine, they have no ability to capture negatively charged proteins. It has drawbacks such as being weak, losing positive charge in a high pH region because it is a -class amine, and having high reactivity itself.

As a result of repeated research to develop a cationic membrane modifier that does not have these drawbacks, the present inventors obtained a novel tetraammonium salt and discovered that it is an excellent cationic membrane modifier. , we have completed the present invention.

That is, the first gist of the present invention is as follows.

[In the formula, K□ to R6 are the same or different and are hydrogen or a lower alkyl group; R7 is hydrogen, a hydroxy group, a lower alkoxy group, or a lower acyloxy group, and 8 to 11 are the same or different and have 1 carbon number ~25 alkyl groups x
is an anion; m represents the potency of X. ] The tetraammonium salt (1) of the present invention, which is present in the tetraammonium salts represented by the above formula, can be synthesized by a conventional method according to the following reaction formula: [In the formula, K' is hydrogen or the number of carbon atoms. an alkyl group of 1 to 24; K represents any one of 8 to 11;

kl has the same meaning as above. ] The OH group of compound (5) can be converted into an alkokene group, an acyloxy group, etc. by an appropriate treatment, such as esterification.

A compound that forms a quaternary ammonium salt with compound (5) or an appropriate derivative thereof (e.g. Me a OB
F4 methyl iodide, methyl bromide, dimethyl sulfate, etc.) or by non-anion conversion of the quaternary ammonium salts already obtained in this way, the tetraammonium salts of the present invention (moon) can be obtained. In the formula of ammonium salt (1), Can be mentioned.

Among the tetraammonium salts (1), those in which k8 to R11 have 10 to 25 carbon atoms are particularly useful as cationic membrane modifiers, and can be effectively cationized to the carrier by reacting with a carrier such as a liposome. Provides a charge and serves as an anchoring site for anionic proteins.

The reaction between tetraammonium salt (1) and egg yolk lecithin is
The reaction may be carried out by mixing and stirring in a suitable solvent (for example, chloroform), and after the reaction is completed, the solvent is distilled off. In addition, by reacting egg yolk lecithin with tetraammonium salt (1),
Cationic liposomes can also be obtained by irradiating with ultrasound. Usually, excess egg yolk lecithin is reacted with a tetraammonium salt (11), but preferably the tetraammonium salt (111,720 mol or less) is reacted with 1 mol of lecithin.

Anionic protein immobilization carriers modified with such a tetraammonium salt (1) are also objects of the present invention.

Furthermore, the anionic protein immobilized on this carrier is also a subject of the present invention.

As the immobilization carrier, the above-mentioned liposomes are particularly preferred.

Anionic proteins include anionic enzymes such as brenyltransferanase and ATPase. Usually, these anionic enzymes are immobilized at a ratio of about 1.5 ellZ per liposome.

Prenyltransferase is an enzyme with a molecular weight of approximately 86,000 (dimer) that catalyzes the following reaction: (Farnesol birophosphate) Anionic protein immobilization is facilitated by contacting a solution of the protein with a modified carrier. can be done.

In addition to the above uses, the tetraammonium salt (1) of the present invention can be used as a phase transfer catalyst in various reactions.

Next, examples will be shown to specifically explain the present invention.

Example 1 6 g of 1,3-dichloro-2-propanol (0,0
47 mol), 42 g (0,282 mol) of NaI, and 2-butanone (150+nl) were charged, and the mixture was refluxed for 24 hours with vigorous stirring. After refluxing, add 300ml of water
was added, and the extraction was repeated three times with 100 ml of chloroform. Add the chloroform layer to 200 ml of 10% sodium hydrogen carbonate aqueous solution1.5% sodium hydrogen carbonate aqueous solution 200 ml
After repeating washing three times with 200 ml of water and drying with anhydrous magnesium sulfate.

After distilling off the chloroform and drying with a vacuum pump, 12 g of 1.3-short-2-propatool was obtained in the form of a transparent and viscous liquid.
(0,0385 mol) was obtained. Yield 82%.

IR (NaCl): 3300, 1410, 1170° 1190.990, 450 cm-'.

IH-NMR (CDCI, TMS standard): δ=3°4 (d, 4H), 3.6 (n+, IH), 4.
1(s, 1 +-1).

5001111 isobaric dropping funnel with potassium hydroxide drying tube, 2 equipped with mechanical stirrer and thermometer
In a three-bottle flask, add 10 N-methylethylenediamine.
+n1 (0.11 mol), triethylamine 39ml (
0,28 mol) and benzene 4 (+ +, 1 ml
was prepared and cooled with water. From the dropping funnel 84.7 g (0.28 mol) of stearoyl chloride and 300 m of benzene
1 dropwise over 1 hour while stirring vigorously.

During this time, the temperature of the reaction vessel was always kept below 20°C.

As soon as the stearoyl chloride was added dropwise, a white precipitate formed. After dropping, the cooling bath was removed and stirring was continued at room temperature for 6 hours. Thereafter, the resulting precipitate was shaken and dissolved in chloroform 11, f), INm acid 11, water 500+n
l, 0.1M sodium bicarbonate 11 and water 500m
1 (3 times).

After confirming that the washing solution was neutral, it was dried over anhydrous magnesium sulfate, chloroform was distilled off, and dried using a vacuum pump to obtain a white solid N-methyl-N.

63 g of N'-distearoylethylenediamine was obtained.

Recrystallization from ethanol yields a white solid N-methyl-N
, N'-distearoylethylenediamine 54g (0,
089 mol) was obtained. Melting point 93-95°C0 Yield 81%
.

IR(KBr): 3330.2900,2480゜1
650.1625.1460,720cIn-'.

'H-NMR (CDCl2, TMs standard): δ=0°9(t, 6H)+1.3(m, 70H)+2.0-2.
5 (m + 48), 3.0 (s + 38), 3.4 (m, 4
H), 6.6 (1+r, LH) Calcium chloride-phosphorus pentoxide in a 11 three-bottle flask equipped with a condenser and thermometer with a drying tube. 400 ml of lithium aluminum hydride and 8.5 g (0,224 mol) of lithium aluminum hydride were charged, and the mixture was heated to 70° C. while stirring with a magnetic cooler. 1,3-short-2-propyl 7-ol 5 obtained in the same manner as in (1) while keeping the reaction solution at 70 to 80°C.
4 g (0,089 mol) was added in small portions over 1 hour.

After the addition was completed, the mixture was refluxed for 30 minutes, and disappearance of carbonyl absorption was confirmed by IR. At this time, if carbonyl absorption remained, 1 g of lithium aluminum hydride was further added and the process of refluxing for 30 minutes was repeated. While the reaction solution was refluxing, ethyl acetate was gradually added with a dropper until no more foaming (approximately 50 ml) was added to decompose the excess lithium aluminum hydride. After the reaction solution was returned to room temperature, 100 ml of water was added. After removing sooxane under reduced pressure, 21 parts of chloroform and 11 parts of 2N hydrochloric acid were added for extraction.

Because the solubility of N-methyl-N,N'-distearylethylenediamine hydrochloride is poor near room temperature, the temperature is about 30°C.
Extraction was performed. After drying over anhydrous magnesium sulfate, chloroform was distilled off and dried using a vacuum pump to obtain N-methyl-N,N'-nostearylethylenediamine hydrochloride as a pale yellow solid. Recrystallization from dichloromethane gave 40 g (0,062 mol) of the hydrochloride as a white solid. Yield 70%.

10 g (0,0154 mol) of the hydrochloride was added to 20 g of benzene.
0 ml, 200 ml of 1N sodium hydroxide was added, and the mixture was vigorously stirred for 5 minutes. The benzene layer was taken out, washed with anhydrous magnesium sulfate, the benzene was distilled off, and dried with a vacuum pump to give a pale yellow solid N-metal N,N'-'
) Stearylethylenediamine 7g (0,012 mol)
I got it. Melting point 32-34°C.

IR(KBr): 2900.2840,1460°7
20c+n'.

'H-NMR (CDC1, TMS standard): δ=0゜9(t, 61(), 1.3(m, 701(), 2.1-
2.9 (m, 1!H) N in a 50+nl eggplant flask
-Methyl-N,N'-distearylethylenediamine 5
g (8,65 mol), l.

0.9 g (2.88 mol) of 3-short-2-propertool and diglyme 2n+I were charged and reacted at 80°C for 1 hour while stirring with a magnetic stirrer. After the reaction was completed, the diglyme was distilled off under reduced pressure. Dissolve the residue in 100 ml of benzene and 100 ml of IN sodium hydroxide.
Washed with 1. After drying over anhydrous magnesium sulfate, benzene was distilled off to obtain a brown solid. This crude product was placed on a silica gel column (4 CIIlφ x 40 cm) and purified by development with methanol/diethyl ether (weight ratio 1:1) to obtain +1.53 g (0,437 mol) of the title compound. Yield 15%.

IR (KBr): 2900, 2840.1460°7
20cm-'.

'H-NMR (CDCI, TMS standard): δ=0°9 (t, 18H), 1.3 (m, 128H), 2.
3(s,6]-1), 2.2-2.9(1n.

20H), 3.7 (br, IH), 4.7 (br, IH
) Calcium chloride-phosphorus pentoxide drying tube (;I digit 50
19.29-dimethyl-22,26-distearyl-24-hydroxy obtained in (4) was placed in an eggplant flask.
19゜22.26.29-Tetraazahexatetraconcune 0.1FI (7,97x10-5 mol), acetic anhydride 1.02g (1xlO-2 mol) and triethylamine 0.5g (5xlO-3 mol) were charged. The mixture was refluxed for 4 hours while stirring with a magnetic cooler. The reaction solution was diluted with 20 ml of 5% sodium hydrogen carbonate three times with 1 ml of water.
1 and twice with 10 ml of water, dried over anhydrous magnesium sulfate, and chloroform was distilled off. The crude product was obtained by drying with a vacuum pump. After dissolving this in dichloromethane, methanol was added to perform reprecipitation to obtain 0.04 g (3,19xlo-5 mol) of the title compound. Yield 40%.

IR (KBr): 2900, 2840, 1730°1
460.1230.720cIa-'.

IH-NMR (CDCI3, TMS standard): δ = 0°9 (t, 18H), 1.3 (m, 128H), 2.0 (
s, 3H), 2.3(s, 6H).

2.2-2.9 (m, 20H), 5.0 (1+r, IH
24-7 Setoxy 19.29-methyl
22,26-distearyl-19,22,26,29-
0.2 g of tetraazahexatetracontane (10-4 mol in 1,59), ('CH3), 41 g of OBF, and 50 ml of dry dichloromethane were charged and stirred with a magnetic stirrer at room temperature for 3 hours. Next, the mixture was taken out from the dry box, and after adding 2 parts and 1 part of ethanol, the precipitated white precipitate was filtered out. The Shito solution was washed with an aqueous sodium bicarbonate solution, dried over anhydrous magnesium sulfate, chloroform was distilled off, and dried using a vacuum pump to obtain the title compound.

IR (KBr): 2900.2840.1730°1
230.720cm-'.

'H-NMR (CDCI3, rMS standard): δ=0
°9 (t, 18H), 1.3 (+b, 128H), 2.
1(s, 3H), 2.7-4.29(1+r, 38H
), 5.3 (t+r, IH) Example 2 24-acetyl-xy-19,22 obtained in Example 1 (6)
,26゜29-tetramethyl-22,26-distearyl-19,22゜26.29-tetraazahexatetrafuntane tetrafluoroborate (10+ng) and egg yolk lecithin (90mg) were dissolved in 2ml of chloroform, and the chloroform was distilled off under reduced pressure. A film-like residue was obtained. Leave it at room temperature for 1 hour at 20+Il+n11g.
Chloroform was completely removed. 20mM) Squirrel-01
5 ml of buffer (pH 7.7) was added, and the mixture was shaken under an argon atmosphere until it became a suspension. Next, ultrasonic waves were irradiated at 0°C under an argon atmosphere (5 minutes x 3
times). Then, centrifugation (19000 rpm x 30 minutes)
Insoluble materials were removed to obtain liposomes modified with the tetraammonium salt.

For liposome purification, 5 epl + adex G-200
(1.5cmφx27cm, solvent 20mM) Lith-01
The test was carried out at 4°C using a buffer (plI7.7).

When putting Fe(CN)63- into the liposome, 5
II+1) Liposome synthesis was performed using Lis-Cl pan7ar (pH 7,7) and 0.5MK, Fe(CN)6.

Example 3 Prenyltransferase solution (3xlO""M, 0.5+
nl) was added and left at 0°C for 20 minutes. This is 5ep
l+adex G-200 (1,5c+aφx27c+
n) Purified from 1 Samurai.

Example 4 Catalysis of prenyltransferase Geraniol pyrophosphate (5+oM, 0.051111),
Isobentenol pyrophosphate (5IOM, 0.
05 Sho 1), Magnesium Chloride (0,111IM, 0
．． 1ml), Tris-Cl H7r- (pH 7,7)
(0,1tvL O,2m1), distilled water (0,5i1)
and the enzyme solution (06!owl) obtained in Example 3 were mixed well and reacted at 38° C. for 10 minutes to obtain farnesol pyrophosphate. IN hydrochloric acid (0.3 ml) was added and the mixture was reacted for an additional 15 minutes to hydrolyze 7-arnesol birophosphate to 7-arnesol. IN sodium hydroxide (0,5+nl) and 2M sodium chloride (2i1) were added and the resulting 7-arnesol was diluted with 11-
Extracted with hexane (5 ml x 2). Dry the extract with anhydrous magnesium sulfate.

After concentration, the production of farnesol was confirmed by GLC using phytol as an internal standard.

GLC conditions: Column S E 30 1'm+ 0.5 atm N21
100→200°C 75°C/min temperature increase, FID detector geraniol 11.4 minutes, 7 Arnesol 18.
6 minutes, Finetor 28 minutes.

Example 5 Prenyltransferase activation, active geraniol pyrophosphate (5mM, 0.05 Inl), isobentenol pyrophosphate (5mM, 0.05 Inl)
+al), magnesium chloride (0.1mM, 0.1m
1), Tris-Cl H77-(1)87.7>(0,
1M, 0.2n+l), distilled water (0.5ml) and the enzyme solution obtained in Example 3 (0°5ml) were mixed well,
The reaction was carried out at 38° C. for 2 hours to obtain 7-flunesol birophosphate, and the same procedure as in Example 4 was repeated to obtain 7-flunesol, and the amount produced was quantified by GLC.

From this result, the activity of the enzyme was maintained even after 2 hours had passed from the start of the reaction.

Reaction time 2 hours Enzyme water 4.2xlO-”moles produced 7 Furnesol 9.4xlO-9moles raw 7 & 7y
Renesol/enzyme 22.3 (mol 1 mol) Amount of this active enzyme (K, Ogura+ "!', N15l+ino
+S, 5eto, J, Bioebem,, 64+
197° (1968)) Example 6 24-7 Setoxy 19,22.
26゜29-tetramethyl-22,26-distearyl-19,22゜26.29-tetraazahexatetraconcune tetrafluorobole) 5+ag and egg yolk lecithin 100+og were dissolved in chloroform 2I01, and chloroform was dissolved under reduced pressure. The residue was distilled off to obtain a film-like residue.

The mixture was left at room temperature with 1 g of 20 ++ uIIl for 1 hour to completely remove chloroform. 5 ml of 11M Tris-CI, pH 8,0 was added, and the mixture was shaken in an argon atmosphere until it became a turbid solution. Then, it was irradiated with ultrasound at 0°C under an argon atmosphere. I did (5
minutes x 3 times). After that, centrifugation (19000 rpm x 3
0 minutes) to remove undissolved materials to obtain liposomes modified with the tetraammonium salt.

Add to this ATPase solution 0.5161 (approximately 5xlO-8
mol), and after ultrasonic irradiation (15 seconds) again, 5e
pl+arose 4 B (2c+*φX50CII
11 solvent 20mM) Lis-01 buffer y-(pH8
,0)).

Patent applicant: Daikin Industries, Ltd. Representative, Patent attorney: Aobai Ao and two other procedural amendments (,
E) 1. Display of the case Patent Application No. 3271 of 1982 2, Name of the invention New tetraammonium salt and its uses 3, Person making the amendment Relationship to the case Patent applicant address IJ12 & 39, 1111-chome, Kin, 1h-ku, Osaka-shi, Osaka Prefecture
)+11 Hankyu Building Representative Yama 1) Catfish 4, Agent 5, Date of amendment order: Voluntary 7, The following parts are amended in the detailed explanation of the invention column of the statement of contents of the amendment.

(1) 2nd line from the bottom of page 6, [LiA deficiency H, J]
I corrected it as “LiA deficiency■4.”

(2) On page 9, line 2, "Fela 7-ase" has been corrected to "7 Erase."

that's all

Claims (1)

[Scope of Claims] [In the formula, Rt~ and 6 are hydrogen or lower alkyl groups, which are the same or different; R7 is hydrogen, a hydroxy group, a lower alkoxy group, or a lower acyloxy group; and 8~ and □ are, The same or different alkyl groups iX having 1 to 25 carbon atoms
is an anion; the river represents the titer number of X. ] Tetraammonium salt shown by. [In the formula, k□ to R6 are the same or different and hydrogen or a lower alkyl group HR7 is hydrogen, a hydroxy group, a lower alkoxy group, or a lower acyloxy group; 8 to R□□ are the same or different and have 10 carbon atoms ~25 alkyl groups;
X is an anion; m represents the titer number of X. ] A cationic membrane modifier consisting of the compound shown below. 3. Formula: [In the formula, k0 to R6 are the same or different and are hydrogen or lower alkyl groups; R7 is hydrogen, a hydroxy group, a lower alkoxy group, or a lower acyloxy group; R8 to R0 are the same or different and are carbon Alkyl group of number 1 to 25;
is an anion; m represents the titer number of X. ] A phase transfer catalyst consisting of a compound represented by. 4, Formula: [In the formula, R□ to 6 are the same or different and are hydrogen or a lower alkyl group + R7 is hydrogen, a hydroxy group, a lower alkoxy group, or a lower acyloxy group; R8 to R□1
are the same or different alkyl groups having 10 to 25 carbon atoms; X is an anion; m represents the titer number of X; ] A cationic membrane for immobilizing anionic proteins modified with a compound represented by: 5. Claim 4, wherein the immobilization carrier is a liposome
Cationic membrane as described in Section. 6. The cationic membrane according to claim 4, wherein the anionic protein is frenyltransferase or ATPase.