JPH0547559B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention disperses amphiphilic peptides containing special amino acid residues in water together with potassium cations or barium cations, and ripens them at room temperature. Molecular association occurs, and the length is approx.
The present invention relates to a method for producing a short fiber type helical molecular association of 15 μm or less. The industrial fields of application of this invention are wide-ranging, from bioindustry, pharmaceutical industry, fine chemical industry, to academic research fields, which utilize micromolecular aggregates with special shapes.

BACKGROUND ART Conventional techniques include a method for producing spherical molecular aggregates (liposomes) using naturally derived phospholipids. In other words, there are the thin film method, thermal dispersion method, solution injection method, cholic acid method, reverse layer evaporation method, etc. (For example, Keizo Inoue, "Biofilm Experimental Methods (Part 2)", edited by Akamatsu et al., 1974; Kyoritsu Publishing, p.185). However, the molecular aggregate obtained by the above method is
They are spherical unilamellar vesicles or spherical multilamellar vesicles. Furthermore, it is known that when a synthetic amphiphilic compound is dispersed in water and aged, a long fiber-type spiral or rod-like molecular association with an average length of 50 μm or more can be obtained [for example, journal-like molecular aggregates].・Jourmal of American Chemical Society
Vol. 107, 509-510 (1985)]. However, the shapes of aggregates obtained by these production methods are limited to spherical, long-fiber spirals, or long-fiber rods, and the shapes are unstable, which hinders the effective use of molecular aggregates. ing.

Problems to be Solved by the Invention The present inventor has developed a simple method for producing short fiber-type molecular aggregates, which cannot be produced from natural phospholipids and have a length of about 15 μm or less and whose form is stable for a long time. As a result of extensive research to develop this product, we discovered that by aging an amphipathic peptide consisting of three proline residues and a special metal cation in water, it could be suitable for this purpose. This invention was made based on this knowledge.

Means for solving the problems That is, the present invention solves the general formula (In the formula, X is a chlorine atom and R is a dodecyl group) An amphiphilic peptide represented by
Dispersed in water in the range of 10 ^-5 to 10 ^-1 mol/
The present invention provides a method for producing a short fiber type helical molecular association characterized by ripening at room temperature.

X in this general formula is a chlorine atom. R
is a dodecyl group having 12 carbon atoms. When the number of carbon atoms is 12 or less, the associative force in water becomes weak, and conversely, when the number of carbon atoms is 12 or more, it becomes insoluble without being dispersed in water.

The compound represented by this general formula is a new compound that has not been published in any literature, for example, the N-terminus is protected and the C
The ends are formed by coupling an oligopeptide consisting of three free proline residues and glutamic acid didodecylamide hydrobromide with diethylphosfluorocyanidate (DEPC) or diphenylphosphoryl azide (DPPA). You can get it. The obtained compound is solid at room temperature, and distilled water is added to this to give a concentration in the range of 10 ^-5 to ^{10 -1} mol/. If it is more than 10 ^-1 mol/, it will take time to disperse or it will be unsuitable due to the presence of insoluble parts, and if it is less than 10 ^-5 mol/, it will not form a molecular association and will be a single molecule. It is unsuitable because it disperses and dissolves. An appropriate amount of potassium cations or barium cations is added to this aqueous dispersion.
At this time, instead of adding each cation directly, distilled water in which an appropriate amount of potassium cation or barium cation is dissolved in advance may be added to the amphipathic peptide. Potassium cations include potassium chloride, potassium carbonate, potassium sulfate, etc.
As the barium cation, barium chloride, barium carbonate, barium sulfate, etc. are readily available and inexpensive, and therefore preferred. An aqueous dispersion containing an amphiphilic peptide and potassium or barium cations is subjected to ultrasonic treatment using a bath-type or probe-type ultrasonic irradiation device. Thereafter, the mixture is aged at room temperature for several days to obtain a colloidal or transparent aqueous dispersion containing a short fiber type helical molecular association having a length of 15 μm or less and stable in its form for a long time.
The ultrasonic treatment temperature is higher than the gel-liquid crystal phase transition temperature of the amphipathic peptide compound, i.e. 70 to 80°C.
is desirable. The length and morphology of the molecular aggregates can be easily confirmed using an optical microscope equipped with a dark field condenser.

Glutamic acid didodecylamide hydrobromide, which is used as a raw material compound when synthesizing this amphiphilic peptide, is produced by, for example, reacting glutamic acid with a protected amino group with hydroxysuccinimide to form a bifunctional ester, and then converting it into a bifunctional ester. It is obtained by reacting with an amine and finally eliminating the amino group.

In addition, the other raw material compound tripeptide is produced by first reacting proline with a protected amino group with proline with a protected carboxyl group to form a dipeptide, then removing the amino protecting group, and then adding the amino group to the tripeptide. is obtained by reacting protected proline to form a tripeptide, and then removing the C-terminal protecting group of this tripeptide.

Effects of the Invention The control method of the present invention makes it possible to obtain approximately
A short fiber type helical molecular association having a length of 15 μm or less can be obtained. Precision industrial parts, pharmaceuticals, dyes,
By adding metal ions, cosmetics, and other useful chemical substances, a helical molecular association in which these useful substances are introduced into the molecular association can be obtained.

Next, the present invention will be explained in more detail with reference to Examples. The Rf value of thin layer chromatography is
The value when chloroform/methanol (5/1, volume ratio) mixed solvent is used as the developing solvent is Rf1, chloroform/methanol/acetic acid (95/5/1, volume ratio)
The value when a mixed solvent was used as the developing solvent was set as Rf2, and the value when a mixed solvent of n-butanol/acetic acid/water (4/1/2, volume ratio) was used as the developing solvent was set as Rf3.

Reference Example 1 (A) Production of t-butyloxycarbonyl-L-prolyl-L-prolyl-L-proline t-butyloxycarbonyl-L-proline
0.64 g (0.00295 mol) and 0.33 ml (0.00295 mol) of N-methylmorpholine in 15 ml of chloroform
After cooling to -20℃, add 0.39 ml of isobutyl chloroformate while stirring.
(0.00295 mol) was added. Stir for 3 minutes, L
-Prolyl-L-proline benzyl ester hydrochloride 1.00 g (0.00295 mol), followed by 0.33 ml (0.00295 mol) N-methylmorpholine,
The mixture was stirred and reacted at -20°C for 6 hours and then at room temperature overnight. The chloroform solution containing the reaction mixture was washed repeatedly with a 4% aqueous sodium bicarbonate solution, a 10% aqueous citric acid solution, and a saturated saline solution until a thin layer chromatogram showed a single spot. Thereafter, it was washed with distilled water and then dried with anhydrous sodium sulfate. The solvent was then removed under reduced pressure, leaving 0.94 g of a white semisolid (64% yield).
I got it. This t-butyloxycarbonyl-L
-Prolyl-L-prolyl-L-proline benzyl ester 0.94g tert-butyl alcohol 30g
ml, and catalytic hydrogen reduction was performed for 4 hours using 5% palladium/carbon as a catalyst. After filtering the catalyst and removing the solvent under reduced pressure, the colorless transparent oil was solidified with petroleum ether to obtain 0.63 g of the desired compound (yield: 82%) as a white solid with a melting point of 189-191°C.
was gotten. The physical properties of this material are as follows.

Rf value of thin layer chromatography Rf1 = 0.17, Rf2 = 0 Elemental analysis value (as C ₂₀ H ₃₁ O ₆ N ₃ 1/3H ₂ O) C H N Calculated value (%) 57.82 7.68 10.11 Actual value (%) ) 57.93 7.43 10.04 (B) Production of L-glutamic acid didodecylamide hydrobromide Dissolve 5 g (0.0178 mol) of benzyloxycarbonyl-L-glutamic acid and 4.09 g (0.0356 mol) of N-hydroxysuccinimide in 30 ml of dimethylformamide. 8.07 g of dicyclohexylcarbodiimide while stirring at 0°C.
(0.039 mol) dissolved in dimethylformamide was added. Stir overnight at 0° C., filter the insoluble by-products, remove the filtrate under reduced pressure and solidify the colorless transparent foam with ether.
1 g of hydroxysuccinimide ester derivative of L-glutamic acid (melting point 73-74°C) obtained by recrystallization from ethyl acetate/isopropanol
(0.0021 mol) and dodecylamine 0.78 g (0.0042
mol) in 10 ml of chloroform and for 2 days.
It was left at room temperature. The reaction solution was washed with a 4% aqueous sodium hydrogen carbonate solution and distilled water, and the solvent was removed under reduced pressure to obtain a white solid. Compound 1.17, washed with ether and methanol, melting point 138-139 °C
g (yield 91%) was obtained. 0.83g of this compound
(0.00135 mol) in 25% hydrogen bromide/acetic acid solution 4.2
ml was reacted for 2 hours, the resulting precipitate was dissolved in ether, the solvent was removed, and the residue was recrystallized from a mixed solvent of water/chloroform/methanol to obtain the target compound having a melting point of 118-122°C.

Rf value of thin layer chromatography RF1=0.49, RF2=0.03 Elemental analysis value (as C ₂₉ H ₆₀ O ₂ N ₃ Br) C H N Calculated value (%) 61.90 10.75 7.47 Actual value (%) 61.57 10.69 7.61 ( C) Production of L-prolyl-L-prolyl-L-prolyl-L-glutamic acid didodecylamide hydrochloride t-butyloxycarbonyl-L-prolyl-L-prolyl-L-proline 0.30 g
(0.000733 mol) and L-glutamic acid didodecylamide hydrobromide 0.49 g (0.000879 mol)
was dissolved in 50 ml of dimethylformamide and heated to 0°C.
While stirring, 2 ml of a dimethylformamide solution containing 0.15 g (0.000879 mol) of diethyl fluorocyanidate was added, followed by 2 ml of a dimethylformamide solution containing 0.23 ml of triethylamine. After stirring at 0°C for 6 hours, the mixture was stirred at room temperature overnight. Add 100ml of chloroform to the reaction solution, add 10% citric acid aqueous solution,
After washing twice each with % sodium bicarbonate aqueous solution, saturated saline, and distilled water, the chloroform layer was dried over anhydrous sodium sulfate, the solvent was removed, and the remaining oil was purified by silica gel column chromatography. 0.47 g of the obtained colorless syrup (Rf1 = 0.61, Rf3 = 0.64) was dispersed in chloroform and mixed with 4N hydrogen chloride/ethyl acetate.
ml for 1 hour at room temperature. The colorless syrup obtained by removing the solvent was recrystallized from water/methanol/chloroform to obtain 410 mg (93% yield) of the target compound as a white solid with a melting point of 89-91°C.
The physical properties of this material are as follows.

Rf value of thin layer chromatography RF1=0.30, RF3=0.10 Elemental analysis value (as C ₄₄ H ₈₁ O ₅ N ₆ C ・H ₂ O) C H N Calculated value (%) 63.86 10.11 10.15 Actual value (%) 63.61 9.95 10.03 Example 1 L-prolyl-L-prolyl-L-prolyl-
L-glutamic acid didodecylamide hydrochloride 0.1g
(1.24×10 ^-4 mol) was placed in a beaker, and 100 ml of distilled water in which 20 mg of potassium chloride had been dissolved was added. This was treated for 5 minutes using a bath-type ultrasonic device (output 80 W) and left at room temperature for 5 days. In this way, microscopic observation revealed that a colloidal solution in which short fiber-type helical molecular associations with a length of about 15 μm or less were dispersed was obtained.

Example 2 L-prolyl-L-prolyl-L-prolyl-
L-glutamic acid didodecylamide hydrochloride 0.1g
(1.24×10 ^-4 mol) was placed in a beaker, and 100 ml of distilled water in which 30 mg of potassium chloride had been dissolved was added. This was subjected to ultrasonic treatment at 70° C. for 1 minute using a probe type ultrasonic device (output 40 W) and left at room temperature for 7 days. In this way, microscopic observation revealed that a colloidal solution in which short fiber-type helical molecular associations with a length of about 15 μm or less were dispersed was obtained.

Example 3 L-prolyl-L-prolyl-L-prolyl-
L-glutamic acid didodecylamide hydrochloride 0.1g
(1.24×10 ^-4 mol) was placed in a beaker, and 100 ml of distilled water in which 50 mg of potassium chloride had been dissolved was added. This was subjected to ultrasonic treatment at 70° C. for 1 minute using a probe type ultrasonic device (output 40 W) and left at room temperature for 2 days. In this way, microscopic observation revealed that a colloidal solution in which short fiber-type helical molecular associations with a length of about 15 μm or less were dispersed was obtained.

Example 4 L-prolyl-L-prolyl-L-prolyl-
L-glutamic acid didodecylamide hydrochloride 0.1g
(1.24×10 ^-4 mol) was placed in a beaker, and 100 ml of distilled water in which 10 mg of barium chloride had been dissolved was added. This was subjected to ultrasonic treatment at 70° C. for 1 minute using a probe type ultrasonic device (output 40 W) and left at room temperature for 2 days. In this way, microscopic observation revealed that a colloidal solution in which short fiber-type helical molecular associations with a length of about 15 μm or less were dispersed was obtained.

Example 5 L-prolyl-L-prolyl-L-prolyl-
L-glutamic acid didodecylamide hydrochloride 0.1g
(1.24×10 ⁻⁴ mol) was placed in a beaker, and 100 ml of distilled water in which 20 mg of barium chloride had been dissolved was added. This was treated at 70° C. for 5 minutes using a bath-type ultrasonic device (output 80 W) and left at room temperature for 5 days. In this way, from microscopic observation, the length was approximately 15 μm.
A colloidal solution in which the following short fiber type helical molecular association was dispersed was obtained.

Example 6 L-prolyl-L-prolyl-L-prolyl-
L-glutamic acid didodecylamide hydrochloride 0.1g
(1.24×10 ⁻⁴ mol) was placed in a beaker, and 100 ml of distilled water in which 50 mg of barium chloride had been dissolved was added. This was treated at 70° C. for 5 minutes using a bath-type ultrasonic device (output 80 W) and left at room temperature for 5 days. In this way, from microscopic observation, the length was approximately 15 μm.
A colloidal solution in which the following short fiber type helical molecular association was dispersed was obtained.

Claims (1)

[Claims] 1. General formula (In the formula, X is a chlorine atom and R is a dodecyl group.) An amphiphilic peptide represented by
1. A method for producing a short fiber type helical molecular association, which comprises dispersing it in water at a concentration of 10 ^-5 to 10 ^-1 mol/molar and aging it at room temperature.