JP2003064095A - Peptide lipid compound - Google Patents

Abstract

(57) [Summary] PROBLEM TO BE SOLVED: To provide a novel peptide lipid compound
More specifically, biodegradable films and fluoroplastics
The present invention relates to a peptide lipid compound useful as an adhesive. The present invention relates to at least three leucines.
Having a hydrophilic group and a hydrophobic group at both ends of the peptide
Is a tide lipid compound, that is, the following chemical formula R¹-C_nH
_2n-CONH-R²-COO-C_mH_{2m + 1} (formula
Medium, R¹Is -N⁺X⁻H_i(C_jH_2jOH)_k(C
H₃)_3-ik(Wherein X represents a halogen atom, i
Is 0 to 3, k is 0 or 1, provided that i + k is 3 or less and j is 2
~ 4. ), Hydroxyl group, carboxylic acid group, or halogen
Represents an atom, R²Contains 3 to 6 consecutive leucines
And may contain one or two other amino acids at each end.
Represents a good polypeptide, n is 2 to 12, m is 12 to 1
6 is represented. ) Is a peptide lipid compound. This
Here -C_mH_{2m + 1}Is a portion showing hydrophobicity, and R¹
Is a portion showing hydrophilicity.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel peptide lipid compound, and more particularly to a peptide lipid compound containing at least three leucines useful as an adhesive for biodegradable films and fluororesins.

[0002]

2. Description of the Related Art Various resin products, which are indispensable to our daily lives, are made of polymer compounds. A polymer compound has excellent mechanical strength and stability by controlling the structural regularity of the molecule, but these characteristics are conversely a problem when recycling or disposal is considered. On the other hand, the inventors have reported that an amphipathic molecule having a peptide group as a partial structure undergoes self-association in water or an organic solvent (J.
Am. Chem. Soc., 120, 12192-12199 (1998), Journal of Japan Oil Chemists' Society Vol. 49, No. 5 (2000)). Without exception, these aggregates form parallel β-sheets in the peptide part, so that the molecular arrangement is highly ordered with respect to the hydrogen bond chain direction. Therefore, it is considered that if the β-sheets can be immobilized by some interaction, the molecules can be regularly arranged in the direction orthogonal to the chain direction of hydrogen bonds. A general parallel β-sheet has a pleated structure. In this structure, the amino acid side chains alternately extend orthogonally to the front and back of the plane of the sheet.
Therefore, if leucine is used as the amino acid, the β-sheet is fixed by the leucine zipper.

[0003]

When a tripeptide-type amphipathic molecule and a peptide-type amphipathic molecule having a larger number of amino acid residues are used, a fibrous aggregate can be formed in a nonpolar organic solvent. Stable, parallel to the peptide part β-
Form a sheet. Since at least three leucine residues must be linked in order to form a leucine zipper, a molecule containing a Leu-Leu-Leu group was synthesized and investigated. The inventors have found a polypeptide-containing short molecule useful as an adhesive and completed the present invention.

[0004]

[Means for Solving the Problems] It is considered that what a certain short molecule needs to form a film is how to fix the molecule three-dimensionally. In a conventional polymer, monomer units are covalently aligned in the polymer main chain direction (one-dimensionally fixed), and the string-like molecules are entangled to form a resin. Two-dimensional or three-dimensional fixation is possible if the monomer has three or more reactive atomic groups and is covalently bonded to three or more monomers. Well-known means in the polymer industry such as vulcanization are operations of fixing the main chains to each other with sulfur, which is used for the purpose of fixing the structure in two or more dimensions. Although the strength of the resin is improved by immobilizing the molecules in such a multidimensional manner, in the case of a polymer, it is not known where the molecules are crosslinked.

In the present invention, the polypeptide-containing short molecule x
It is intended to fix the axial direction by "hydrogen bond" and the y-axis direction by "leucine zipper". The leucine zipper is a special effect possessed only by leucine (Leu), which is one of the amino acids, as is known as one of the elements that determine the tertiary structure of proteins in biochemistry. Since leucine has a side chain in the shape of the letter "Y" of the alphabet, when a peptide having a plurality of amino acids faces each other, the "Y" s are caught by each other and bound like a fastener (zipper). This state is shown in FIG. Just as the zipper is longer, the leucine zipper is stronger as the number of “Y”, ie, leucine is larger. Further, as the number of leucines increases, the number of peptide bonds (amide bonds) inevitably increases, so that the strength of hydrogen bonds also increases.

The strength of hydrogen bonds is about 10 to 30 kJ / mol. Since the covalent bond has a strength of several hundred kJ / mol, it is stronger than the hydrogen bond by one order or more, but if there are four hydrogen bonds instead of one, it is possible to obtain the same strength as a weak covalent bond. Become. This will fix the molecules in the x and y directions. As for the z-axis, if the molecules are placed on a flat surface, they are forced to line up flat. Therefore, the flat surface may be used as a template. However, at this time, since the molecules need to be aligned in the same direction, it is necessary to greatly change the structures at both ends of the molecule. For example, if hydrophilic and hydrophobic groups are placed at both ends of the peptide group,
When the molecule is placed on a hydrophilic surface, hydrophilic groups adhere to the surface.

From the above, the following three structural elements are indispensable for such molecules to form a film. 1. Have multiple peptide bonds. It is enough that three or more pieces have been obtained from the studies so far. 2. Having leucine. A minimum of 3 leucines is required to form the zipper. This point is important. The use of amino acids such as valine and isoleucine, which are very similar in structure to leucine, is not effective and the film produced from such molecules will break (brittle fracture). 3. The peptide group has functional groups with completely different properties at both ends. Based on such findings, the present inventors have reached a peptide lipid compound containing at least three leucines and having a hydrophilic group and a hydrophobic group at both ends of the peptide, and completed the present invention.

That is, the present invention has the following chemical formula R¹-C_nH_2n-CONH-R^Two-COO-C_mH
_{2m + 1} It is a peptide lipid compound represented by. This hydrocarbon chain
-C_nH_2n-And-C_mH_{2m + 1}Is a straight-line
Although it may be branched, it is a molecule that is linear.
Is preferable from the viewpoint of forming an aggregate. Where −
C_mH_{2m + 1}Is the hydrophobic portion of this compound
It On the other hand, in the formula, R¹Is the hydrophilic portion of the compound
Yes, -N⁺X⁻H_i(C _jH_2jOH)_k(CH_Three)
_3-i-k(In the formula, X is a halogen atom, preferably chlorine.
Or represents bromine, i is 0 to 3, preferably 0 or
3, k is 0 or 1, preferably 0, provided that i + k is 3 or more.
Below, j represents 2 to 4, preferably 2. ), Hydroxyl group,
Rubonic acid group or halogen atom (preferably chlorine or
Represents bromine).

R ² is a polypeptide residue having a bond at both ends, and it is necessary that R ² contains 3 to 6 consecutive leucines. When three or more leucines are not contained, the molecule cannot form a leucine zipper, so that this molecule cannot form an aggregate as in the present invention, and thus it does not function as a film or an adhesive. Absent. On the other hand, when 7 or more leucines are contained, the molecules cannot be crystallized and cannot function as a film or an adhesive. R
² is preferably a peptide chain consisting only of this continuous leucine, but it may contain 1 to 2 other amino acids at both ends. Particularly, a peptide in which glutamic acid or aspartic acid is bound to the carboxyl group-side end of this peptide is preferable from the viewpoint of forming an aggregate because it can have two hydrocarbon chains via an ester bond as a hydrophobic group. .

N represents 2 to 12, and m represents 12 to 16. Further, ^{R 1} is _{^{-N + (CH 3) 3 Br}} - represents, ^{R 2} is -
It is preferable to represent Leu-Leu-Leu-Glu-, in which the carboxylic acid of Glu has an ester chain in the form of COOC ₁₂ H ₂₅ , n represents 10 and m represents 12.
The present invention also provides a film made of this peptide lipid compound, or a fluororesin adhesive containing this peptide lipid compound as an active ingredient.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The peptide lipid compound of the present invention is
It may be prepared by any known method, for example, by synthesizing an ester of a higher alcohol and an amino acid by acid-catalyzed esterification and condensing an amino group-protected (for example, Boc) peptide to the obtained amino acid ester, After that, the amino-protecting group is removed, and a carboxylic acid having an appropriate carbon chain is condensed to obtain the compound.

The peptide lipid compound of the present invention is dissolved in any solvent in any ratio. This peptide lipid compound forms a fibrous aggregate in a nonpolar organic solvent having a dielectric constant of 2.0 to 2.3 such as carbon tetrachloride, cyclohexane, benzene, and toluene. The formation of aggregates can be easily detected by measuring the Fourier transform infrared spectrum and examining the formation of hydrogen bonds (J. Am. Chem. Soc., 120, 12192-12.
199 (1998)). On the other hand, water and chloroform do not form an aggregate because they are polar solvents. When a carbon tetrachloride solution that forms an aggregate and a chloroform solution that does not form an aggregate are prepared, and a cast film is made from both of them, a chloroform solution that does not contain an aggregate is also used as a cast film to form a hydrogen bond. . Atomic force microscopy (AFM) observations of the surface structures of the two solutions show that the cast films from the carbon tetrachloride solution are aggregates of fibers and chloroform solution despite the formation of hydrogen bonds in the same manner. The cast film from is a flat sheet structure. That is, the cast film has a surface structure that reflects the structure of the aggregates contained in the solution. Therefore, in order to obtain a flat surface structure, it is preferable that the solution before casting is an isotropic solution, that is, a solution of a polar solvent such as chloroform containing no aggregate. The concentration is arbitrary as it is naturally dried and concentrated after casting. A thin solution results in a thin film and a thick solution results in a thick film. The temperature at that time is also arbitrary. Higher temperatures lead to faster film formation (due to faster evaporation), and lower temperatures lead to slower film formation.

[0013]

EXAMPLES The present invention will be illustrated below with reference to Examples.
It is not intended to limit the light.EXAMPLE ^{_{1 Br - (CH 3) 3}} N + C 10 H 20 (C = O) -Leu-Leu-Leu-Glu
Synthesis of (OC ₁₂ H ₂₅ ) ₂ Boc-Leu-Leu-Leu-OH (Mw
 = 457.60) 1.04 g (2.03 × 10^-3 mol), L-glutamine
Acid didodecyl ester hydrochloride (Mw = 520.23) 1.06
g (2.03 x 10^-3 mol) and weigh it in 70 ml of dehydrated THF.
I understand. Dehydrated triethylamine (Mw = 10
1.19) 0.25g (2.44 x 10^-3 mol) and then DEPC (M
w = 163.11) 0.40g (2.44 x 10^-3 mol) dehydrated THF 30
 The solution dissolved in ml was slowly added dropwise. Ice for a while
Stir cold, then cover with aluminum foil and stir at room temperature for 4 days
did. After stirring, a white solid precipitated (triethylamine salt
The acid salt) was filtered off. The solvent was distilled off under reduced pressure.
), Dissolve the residue in 100 ml of ethyl acetate, and separate it with 200 ml.
Transfer to a funnel and saturate with saturated aqueous sodium bicarbonate twice.
It was washed twice with an aqueous solution of citric acid and twice with a saturated saline solution.
(50 ml each). The organic layer is anhydrous magnesium sulfate
It was dehydrated for about 30 minutes and then naturally filtered. Evaporate the solvent under reduced pressure
As a result, an oily residue was obtained. Leave it in the refrigerator
Solidify and recrystallize twice from acetone to N- (N-tert-but
Xycarbonyl-tri-L-leucyl) -L-glutamic acid zido
Decyl ester (Boc-Leu-Leu-Leu-Glu- (OC₁₂H_{twenty five})₂)of
A colorless solid was obtained. ・ Yield 1.67 g (theoretical yield 1.98 g) ・ Yield 85% ・ M.p. 55.2-63.1 ℃ ・ TLC (using Merck Kieselgel 60 F254, chloro
Formed in a mixed solvent of form: methanol = 10: 1; Rf value 0.89
(Single spot)) ・ IR (CCl_FourMethod) 3300 cm^-1(Ν N-H), 2920 cm^-1(Νas
 CH₂), 2850 cm^-1(Νs CH₂), 1740 cm^-1(Ν C = O (d
Stell)), 1695cm^-1(Ν C = O (urethane)) 1640 cm^-1
(Ν C = O (amide I)), 1540 cm^-1(Ν δN-H (amide I
I))

Next, in a 100 ml Erlenmeyer flask with rubbing,
c-Leu-Leu-Leu-Glu- (OC ₁₂ H ₂₅ ) ₂ (Mw = 457.60) 1.87 g (2.
(03 × 10 ⁻³ mol) was weighed and dissolved in 10 ml of chloroform. 25% HBr / acetic acid 13.14 g (Boc-Leu-Leu-
Leu-Glu- (OC ₁₂ H ₂₅ ) ₂ (20 times mol) was slowly added dropwise, and the mixture was stirred at room temperature for 5 hours. After confirming the reaction process by TLC (confirming that TLC is at the origin), saturated aqueous sodium hydrogen carbonate solution was slowly added to neutralize under ice cooling. Transfer to a 500 ml separatory funnel and add 3 to 50 ml of chloroform.
The extract was extracted twice, and washed three times with saturated aqueous sodium hydrogen carbonate solution and once with saturated brine (70 ml each). The organic layer was dehydrated with anhydrous magnesium sulfate for about 30 minutes and naturally filtered. The solvent was distilled off under reduced pressure to obtain an oily residue. It was left to stand in the refrigerator to solidify and recrystallized twice from ethyl acetate containing a small amount of methanol to give N- (tri-L-leucyl) -L-glutamic acid didodecyl ester (H-Leu-Leu-Leu-Glu- (OC
A colorless solid of ₁₂ H ₂₅ ) ₂ ) was obtained.・ Yield 1.05 g (theoretical yield 1.52 g) ・ Yield 70.1% ・ TLC (using Merck Kieselgel 60 F254, developed with a mixed solvent of chloroform: methanol = 10: 1; Rf value 0.49
(Single spot) ・ IR (CCl ₄ method) 3350cm ^-1 (ν NH), 2920cm ^-1 (νas
^{_{CH 2), 2850cm -1 (νs}} CH 2), 1740cm -1 (ν C = O ( ^{ester)), 1620cm -1 (ν C} = O ( amide I)), 1540 cm
^-1 (ν δN-H (amide II))

Next, H-Le was placed in a 100 ml rubbed eggplant flask.
u-Leu-Leu-Glu- (OC ₁₂ H ₂₅ ) ₂ (Mw = 823.24) 0.60 g (7.29 ×
10 ^-4 mol), 11-bromoundecanoic acid (Mw = 265.19) 0.1
9 g (7.29 × 10 ⁻⁴ mol) was weighed and dissolved in 20 ml of dehydrated THF. Dehydrated triethylamine (Mw = 1
01.19) 0.08 g (8.25 × 10 ^-4 mol) was added, and DEPC was added.
(Mw = 163.11) 0.14 g (8.25 × 10 ^-4 mol) dehydrated TH
The solution dissolved in 20 ml of F was slowly added dropwise. The mixture was stirred with ice cooling for a while, then covered with an aluminum foil to protect it from light, and stirred at room temperature for 4 days. After stirring, the precipitated white solid was filtered off. The solvent was evaporated under reduced pressure, the residue was dissolved in 100 ml of chloroform, transferred to a 200 ml separatory funnel, and washed twice with a saturated aqueous sodium hydrogen carbonate solution, twice with a saturated citric acid aqueous solution, and once with a saturated saline solution. (50 ml each). The organic layer was dehydrated with anhydrous magnesium sulfate for about 30 minutes and naturally filtered.
When the solvent was distilled off under reduced pressure, an oily residue was obtained. When left in the freezer overnight, it solidified. This was recrystallized once with acetone single solvent and once with methanol single solvent,
N- [N- (11-Bromoundecanoyl) -tri-L-leucyl] -L-
Glutamic acid didodecyl ester (Br-C ₁₀ H ₂₀ (C = O) -Le
u-Leu-Leu-Glu- was obtained (OC ₁₂ H _{25) 2)} of a colorless solid. -Yield 1.00 g (theoretical yield 0.78 g) -Yield 128.2% (It seems that the solvent and trace impurities are included.)-Mp 53.9-66.5 ° C-TLC (Merck Kieselgel 60 F254 used, chloroform: methanol = 10: 1 developed with mixed solvent; Rf value 0.93
(Single spot)) · IR (CCl ₄ ^{method) 3350cm -1 (ν NH),} 2930cm -1 (νas
CH ₂ ), 2850cm ^-1 (νs CH ₂ ), 1735cm ^-1 (ν C = O (ester)), 1625cm ^-1 (ν C = O (amide I)), 1545cm
^-1 (ν δN-H (amide II))

Next, Br-C was placed in a 100 ml rubbed eggplant flask.
₁₀ H ₂₀ (C = O) -Leu-Leu-Leu-Glu- (OC ₁₂ H ₂₅ ) ₂ (Mw = 1070.4
1) Weigh 1.00 g (9.34 × 10 ^-4 mol) and dry THF 60
dissolved in ml. Approximately 1 trimethylamine gas under ice cooling
After bubbling 5 ml, it was sealed and stored in the dark for 2 weeks. The solvent was distilled off under reduced pressure in a fume hood to give a colorless solid (N- [N
-(11-Trimethylammonioundecanoyl) -tri-L-leucyl] -L-glutamic acid didodecyl ester bromide) was obtained.

[0017]Example 2 Colorless solid prepared in Example 1 (N- [N- (11-trimethyl
Ammonium undecanoyl) -tri-L-leucyl] -L-gluta
Chloromic acid didodecyl ester bromide) at room temperature
Dissolve in formol at a concentration of 10 ml / liter and add
Cast on release paper made of paper, air-dried,
A film was obtained by peeling it off. Film obtained
Has high transparency and returns to its original shape even when bent. That is,
It does not cause brittle fracture or plastic deformation. The feel is
Similar to "parafilm". Also, this film in water
When thrown in, the film is quite flexible.
Despite being a bull, adding water would make the baraba
(No need for heating. This change occurs at room temperature.
). This is because it does not melt,
Divided into pieces. Mold grows and biodegrades within a few days
It turned out to have sex. In addition, the
Colored solid (N- [N- (11-trimethylammonioundecano
Ill) -tri-L-leucyl] -L-didodecyl es-glutamate
Terbromide) in chloroform at room temperature 10 ml / liter
Calcium Fluoride Substrate
To form a film.
Spectra with a conversion infrared spectroscope (Nicolet, Nexus 670)
The result of the tor analysis is shown in FIG. Fourier transform red in Figure 2
From the outer spectrum, as described previously (J. Am. C
hem. Soc., 120, 12192-12199 (1998)), 1632cm^-1near
Amide I absorption band and 1543 cm^-1Amide II absorption band of
Which indicates that a parallel β-sheet is formed.
However, these absorptions are sharp absorptions with a full width at half maximum.
It means that the structure of β-sheet is very well arranged.
Which indicates that. That is, the film obtained here is a molecule
It can be seen that the array is two-dimensionally arranged.

[0018]Example 3 Colorless solid prepared in Example 1 (N- [N- (11-trimethyl
Ammonium undecanoyl) -tri-L-leucyl] -L-gluta
Chloromic acid didodecyl ester bromide) at room temperature
Fluorine was dissolved in formol at a concentration of 10 ml / liter.
Resin sheet (Polytetrafluoroe manufactured by Nichias Corporation)
Tylene, film thickness 0.05 mm (JIS K6887, approud No. 785
7)) was applied on top of this fluororesin sheet
Since it does not peel off, this peptide lipid compound
It was found that the coating property of the surface of the base resin is excellent
It was Next, 2 pieces of this fluororesin sheet 2.5 cm x 5.0 cm
Prepare the solution and apply the above solution to each 2.5 cm × 3.0 cm area.
I was just. Align the cast parts in a semi-dry state, and
I crimped strongly. Clip for 30 minutes at room temperature
Support one side with and attach a clip with a weight to the other side.
I installed it. Weights are 100 g, 200 g and 500 g
However, the adhesive part did not come off. If you peel it off with your fingers,
Can be divided into fluororesin sheets, but at least
The shear strength was quite high.

[0019]

INDUSTRIAL APPLICABILITY As described above, the peptide lipid compound of the present invention forms a relatively firm film, while it is biodegradable and easily decomposes in water. Further, it has a remarkable property of adhering a fluororesin. Due to such properties, the peptide-lipid compound of the present invention can be used as a film or coating agent required for biodegradation, or an adhesive for fluororesins. As described above, it was revealed that the peptide-lipid compound of the present invention has some such interesting and useful properties, but since the full range of these properties has not yet been elucidated, it depends on future development. There seems to be ample room for finding industrially useful applications.

[Brief description of drawings]

FIG. 1 is a diagram showing how the peptide-lipid compound of the present invention associates.

FIG. 2 is a diagram showing a Fourier transform infrared spectrum of a film prepared from the peptide lipid compound of the present invention.

Front page continuation (51) Int.Cl. ⁷ Identification code FI theme code (reference) // C08L 89:00 C08L 89:00 F term (reference) 4F071 AA70 AF52 BB02 BC01 4H045 AA10 AA20 AA30 BA12 BA13 BA14 BA55 EA61 EA65 FA10 4J040 BA151 GA03 GA05 GA07 MA10

Claims (4)

[Claims] 1. The following chemical formula R¹-C_nH_2n-CONH-R^Two-COO-C_mH
_{2m + 1} (In the formula, R¹Is -N⁺X⁻H_i(C_jH_2jOH)_k
(CH_Three)_3-i-k(In the formula, X represents a halogen atom.
Where i is 0 to 3, k is 0 or 1, and i + k is 3 or less,
j represents 2 to 4. ), A hydroxyl group, a carboxylic acid group, or
Rogen atom, R^TwoContinuously leucine 3-6
Including 1 and 2 other amino acids at each end
May be a polypeptide, where n is 2 to 12 and m is 12
Represents ~ 16. ) A peptide lipid compound represented by: 2. The R¹Is -N⁺(CH_Three)_ThreeBr⁻To
Represent, R^TwoRepresents -Leu-Leu-Leu-Glu-
The carboxylic acid of the Glu is COOC₁₂H ₂₅In the form of
A contract having an ester chain, in which n represents 10 and m represents 12.
The peptide lipid compound according to claim 1. 3. A film comprising the peptide lipid compound according to claim 1 or 2. 4. A fluororesin adhesive containing the peptide lipid compound according to claim 1 or 2 as an active ingredient.