JPH036222A - Thin film of optically active polypeptide - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention relates to an ultra-thin film composed of oriented molecular aggregates, and in particular to a super-thin film composed of an optically active amino acid having a peptide bond that is excellent in terms of biocompatibility. It concerns ultra-thin films of coalescence.

(Background technology) Monolayers with molecular alignment and monolayers formed by stacking multiple monolayers
Molecular assemblies such as ayers = lIuHilayer) take advantage of their ultra-thinness and denseness to be used as materials for electronic devices,
In addition to surface protection materials, it is expected to have a wide range of applications as ultrafiltration membranes that utilize selective permeability of gas molecules and ions, thin membranes for sensing, and permeation control membranes for material delivery.

The Langmuir-Prodgett (LB) method is generally known as a method for accumulating a monomolecular film of amphiphilic molecules formed at the gas-liquid interface on a support, and various LB films prepared by this method are Its use as an organic ultra-thin film has expanded in recent years [see Solid State Physics 17 (12) 45 (19B2), etc.].

Although molecular assemblies including LB films exhibit various functionalities based on molecular orientation and ultra-thinness, they are physically delicate, so the film structure is easily destroyed (and some compounds may cause structural defects in the film). It has the disadvantage that high density cannot be obtained because there are many

Physically strengthening the membrane structure of these molecular assemblies and providing membranes with excellent density is a challenge required for all purposes.

One of the effective means for physically reinforcing the membrane structure of molecular assemblies is crosslinking or polymerization of molecules.

Regarding the polymerization of molecular aggregates such as LB membranes and liposomes, conventional polymerizable compounds and polymerization modes are described in 11Bader et al., Advances in Polymer 5cie.
nce, vol. 64, p. 1 (1985) and R, B1
15chl et al., Macromol, Chem, 5u
pp1. + Volume 6, page 245 (1984).

Research on polymerizable amphiphilic compounds became active in 19
It was introduced in the 1980s, and initially unsaturated vinyl, diene, and diacetylene compounds were used as polymerizable compounds, and polymerization was performed by cleaving the unsaturated bonds with radiation such as ultraviolet rays (UV) or gamma rays. The method was widely adopted. However, although most of these polymerization methods improve the robustness of the membrane, it is difficult to maintain the orderliness of the molecular arrangement due to the strain caused by polymerization. A.Lasch
ewsky and H. Ringsdorb, Macrom
Oleculc, Volume 21, 1936 True (1988)
As pointed out in the above, the orientation of the double film is greatly influenced by the length of the alkyl chain and the type of terminal hydrophilic group, and as a result, the number of polymerizable unsaturated compounds with good order is limited to a small number.

^, Laschewsky et al. J, Am, Che
m, Soc, vol. 109, p. 788 (1987)
discloses the need for polymerizable groups to be supported via spacer groups in order to maintain order in amphiphilic compounds having various unsaturated bonds useful for radiation polymerization and the like. Furthermore, JP-A-57159506
The issue contains unsaturated compounds (surfactants) produced by radiation polymerization.
Examples of using polymeric films of monomolecular and cumulative films as ultrafiltration membranes are shown.

Conventional techniques in which compounds having these unsaturated bonds are polymerized by radiation have the following disadvantages.

That is, first, disorder of the arrangement structure or disordered aggregation/precipitation of molecules is likely to occur due to polymerization, and special molecular design such as insertion of a spacer group is required to avoid this. A second problem is that irradiation with ultraviolet rays or gamma rays causes decomposition or denaturation of various additive substances that often coexist with polymerizable amphiphilic molecules. Thirdly, membranes obtained by this type of polymerization usually have very poor biocompatibility, which limits their application to living tissues as permeation control membranes for drugs and the like.

Therefore, as a polymerization method that does not use radiation, for example, a method of forming disulfide bonds by oxidative polymerization of dithiol is shown in J. Am. Chem. A method in which a compound having an unsaturated bond is radically polymerized in the presence of an initiator is useful. However, these methods require an initiator during polymerization, which requires a step to remove it from livestock after the polymerization is complete, and the impact of initiators, including redox agents, on coexisting substances is also a problem. .

Maeromol and CheIl are methods for self-condensation polymerization of molecular membranes of long-chain alkyl amino acid derivatives that further improve the polymerization form and biocompatibility.

Rapid Col1v+un, +Volume 3, page 167 (
1982), Th1n 5olid Fi1ms+ Vol. 133, p. 39 (1985), and a method of condensation polymerization of similar derivatives using carbodiimide as a condensing agent is described in J. Am. Chem.

Soc, Vol. 108, p. 487 (1986). However, in these methods, the progress of the condensation reaction is extremely slow in self-condensation polymerization, and in the methods using a condensing agent, there is a problem in that the condensing agent and by-products remain.

J, Am, Chem, Soc,, vol. 111, 1
115 (1989) and the same magazine, No. 111S, 143
6 (1989) describes a method for producing a monolayer film using optically active amino acids and derivatives of their esters, and it has been shown that molecular alignment films made of these optically active substances can provide excellent orientation and density. Suggested. However, no method has been disclosed for rapidly self-polymerizing a film of these amino acid derivatives to strengthen the structure and produce a thin film of an optically active polypeptide.

(Problems to be Solved by the Invention) Therefore, the purpose of the present invention is to solve the above-mentioned problems of the conventional method,
The first purpose is to provide a polymerized thin film with good molecular alignment that can be polymerized without using radiation or a polymerization initiator.
Second, the purpose is to provide a polymerized thin film in which polymerization spontaneously proceeds at high speed and high yield through self-polymerization, and thirdly, it is made of an optically active substance and has excellent density and biocompatibility. The purpose of the present invention is to provide a polymerizable thin film that is excellent in other aspects as well.

(Means for Solving the Problems) An object of the present invention is to obtain a monolayer film or a cumulative film thereof consisting of an amphiphilic long-chain alkyl derivative of an optically active α-amino acid ester with the same three-dimensional structure. This was achieved by thin films of optically active long-chain alkylated polypeptides.

The polymerized monolayer or cumulative film of the present invention is an ultra-thin film supported on a support by a monolayer coating method including the LB method, and the main chain of the polymerization is an optically active polypeptide, that is, an amide bond of an optically active amino acid. It is characterized by being composed of a chain. That is, the polymer membrane of the present invention is obtained by condensation polymerization of an amphipathic amino acid derivative having a reactive, that is, electrophilic, ester group through the following reaction to form an amide bond skeleton.

0 111 (II!N-C11-C-XR')n -(-C
l-1-C-Jl+.

Here, n represents an integer of 2 or more, and R and R'X will be described later.

The method for forming the polymerized thin film of the present invention will be explained.

To form the polymerized FillQ, either of two types, polymerization at the gas-liquid interface and polymerization on the support, can be used.

In order to perform polymerization at the gas-liquid interface, the amphiphilic amino acid ester derivative monomer of the present invention must be used! The molecular film is placed in a suitable aqueous solution on the subphase of a trough for monomolecular film production. It can be prepared by developing it from a 9 medium and left on the water surface for a period of time required for polymerization. As the aqueous phase, pure water or a salt solution such as a buffer can be used, and the pH thereof is preferably controlled within the range of 5 to 9 depending on the equilibrium constant of ester decomposition of the monomer used.

The temperature of the aqueous phase is preferably in the range from room temperature to 60°C, and a high temperature is selected to accelerate the reaction rate.The surface pressure of the monomolecular film during the reaction is preferably maintained at 5 to 40 dyne/Cm. It is more preferable to maintain it at 10 to 25 dyne/1. Surface pressure is usually controlled to a constant value, but
The polymer film on the water surface may be increased or decreased as the reaction progresses. After the reaction is completed, the polymer film on the water surface is
A polymerized monomolecular film or a polymerized cumulative film is formed by sequentially transferring one or more layers onto a hydrophilic or hydrophobic support by a method such as the dgeLt method (vertical dipping method) or the horizontal deposition method.

The second method is to form a monomolecular film of the amphipathic amino acid ester derivative monomer on the water surface, transfer it onto a support by the method described above, and then leave the cumulative film on this support. This is a method of advancing polymerization. In order to accumulate a monolayer on the support prior to reaction in this method, it is necessary to maintain the aqueous phase at conditions that control the polymerization reaction, such as low pH below pH 6 and low temperature conditions. . The monomers accumulated on the support are exposed to conditions that promote polymerization, such as heating or alkaline gas (e.g. NH
, etc.) or immersed in an alkaline aqueous solution.

Of these two polymerization methods, the former method, polymerization on the gas-liquid interface, is preferred in terms of reaction tolerance. However, in terms of reaction efficiency and degree of polymerization, this is not necessarily the case (and can be used depending on the stability of the monomer used).

The amphipathic optically active amino acid ester used in the present invention is represented by the following general formula (1).

(1), II Hl N-CH-C-XR' In the formula, R is a long-chain alkyl group (preferably a carbon number of 12 to 2
0 long-chain alkyl group), and XR'
is a leaving group whose conjugate acid has a pKa of preferably 14 or less. X represents 10--8- or -NCR'')- (R'' is a hydrogen atom, an alkyl group, an aryl group. R2
may be linked with R1 to form a ring. The ring may further contain heteroatoms such as nitrogen, and may contain unsaturated bonds). X is preferably 10-. Examples of R' include an aryl group (including substituted aryl groups, such as phenyl and naphthyl).

Examples of substituents include nitro group, halogen atom), alkyl group (including substituted alkyl groups; for example, methyl group, monochloromethyl, dichloromethyl, trichloromethyl),
Acylamino group (e.g. N-methylacetylamino group,
N-methylbenzoylamino group), -N=CR'
(R'> (Here, R1R4 represents a hydrogen atom, an alkyl group, an aryl group. These alkyl groups and aryl groups include those having a substituent), an alkenyl group (e.g. allyl group), an alkynyl group ( For example, an acetyl group), among which an alkyl group, an alkynyl group, and an aryl group are preferable.

The long-chain alkyl group of R preferably has 16 to 20 carbon atoms, and when the alkyl group is bonded to an amino acid residue via a linking group, the linking group is -NHCO--
NHCOO--NHCONH--NHCO-3--0-
-3-COO-0PO03- or a combination of these and an alkyl group is preferred.

Preferred specific examples of the amino acid ester (monomer) of the present invention will be listed below, but the invention is not limited thereto.

-t C=0 Ko NHC+J! 3 -2 C=O NtlC+Jzs -3 -6 C=0 N) IC+bH, 13 C+mHsri -4 -5 -O NHC+Jsz -8 Ni+ O=C CIsHsco I4HtQ N)l O=C C10) 131 1-9 ■ -10 -11 ■-12 −1÷ too C■bH3ko C■6H33 C+Jlsco ■-13 -14 The synthesis method of the present invention is roughly divided into two.

One method is to use an optically active amino acid as a starting material and convert it into the target product while retaining the chilasoti, and the other is to proceed with the synthesis in the racemic form and use a resolving agent such as brucine to obtain the optically active form.

Typical examples will be described below, but the method is not limited to this method.

1 1 step I Boc-j! - Seritz methyl ester 1 21g (96
mmojri, DHP40g (480mmof)CH
Cjlt 450ml mixed night PTS200IIv (
110+++moji) 2mj THF solution was added dropwise and stirred for 3 hours. DHP, CHClff (7) After distillation under reduced pressure, add CH (1!ff) and wash with Na HCOs aq.
After washing with water and drying with N az SO4, CHClff
was distilled off under reduced pressure to obtain a quantitative amount, mainly as a solid. The main substance obtained in this way was dissolved in MeOH500n/93%.
NaOH4, 5g (100mm), Hi 08
0IB7 solution was added and left overnight. After the solvent was distilled off, water was added and the aqueous layer was washed with Ac0Et, and then Ac0Et was added again.

After making the aqueous layer acidic (about pH 3) with dilute HCf, Ac0E
Extracted with water, washed with water, and dried with NaIS Os.
c0Et was distilled off under reduced pressure to obtain 21 g (73 m
Mojyl was obtained as a highly viscous liquid.

Step 2 Carboxylic acid 2'2.80g (9.7mmof),
Carbonyldiimidazole 1.57g (9,711I
IIlo1) THE70-The mixture was stirred for 1 hour to obtain 0.91 g (9,711 Imol) of phenol.
F30-solution was added. Stir at r, t for 1 hour 1-5
After stirring under reflux for one hour, the mixture was allowed to stand overnight, and the solvent was distilled off under reduced pressure. Add Ac0Et to the residual scent, wash with water 3 times, and use NazSO.
4. Drying and distillation under reduced pressure were performed. The residual aroma was purified by silica gel chromatography, and 1.37 g (3.8 mmof) of phenyl ester 1 was obtained as a colorless solid, hexane/A.
c OE t = 9/1 and a yield of 39% was obtained.

The structure of Y was confirmed by IR,'H-NMRSMASS.

IR(KB r) 3300cm-', 1780(J
-'1700as-', 1230clll-Step 3 5.5g (15mlIo1), MeOH200-
PTS950w (5--Oi
) and stirred for 2 hours at r.

Next, PT3950111g (5a+5o1) was added, and the mixture was further stirred for 2 hours, and the solvent was distilled off under reduced pressure. The residual fragrance was purified by silica gel chromatography and ↓ 1.2g (4.2mnoJ
) Hexane/Ac0Et=7/3, yield 28% was obtained.

The soil structure was confirmed by IR,'H-NMRSMASS.

In addition, CHClz of ↓? Na HCOs aq
When I washed it with water, it disassembled.

IR (KBr) 3500 (J-', 3240cm-'1
780cm-', 1700. :n-'Step 4 In CHzC1t50td, TCFl, ・2g (6°Q
mmojri, zero-order Boa-j in which phosgene generated by activated carbon 300+w was blown in under water cooling! -Serine phenyl ester 4 1.18g (4.2mmol1)
, Et+ No, 42g (4.2ma+ojri CHI
C1t 25ffi7 mixed solution was added dropwise under water cooling (
30 minutes). After stirring for 3 hours, the phosgene was driven out by blowing with N, and 1.14 g of stearylamine (4
, 2 mm, EL3 No., 42 g (4,2 mm
A mixture of 50 Hz and 50 Hz was added. -After standing overnight, water was added to wash the machine layer, dried with N az SO , distilled off under reduced pressure, and subjected to silica gel chromatography to obtain 1.18 g of hexane/ac OE as a fraction containing l.
L = 8 / 2゜Of this, 400■ is Ac0Et
Recrystallize l by 0°32 g (0,56
mmol) The obtained structure is IR,,'H-NMRSM
Confirmed by ASS.

In addition, 1 is partially decomposed by silica gel chromatography.

I R (KB r) 3250c+a-', 1780
cm1750cn-', 1695cm-X step 5 5 200g (0,35mmojl) T F A
4 ml of the mixture was dissolved in a CH□C1,4-mixture and stirred for 30 minutes.

Thereafter, the solvent was distilled off under reduced pressure, and CH and ca were further added and distilled off under reduced pressure, which was repeated twice and dried under reduced pressure to quantitatively obtain 1-1. Of these, 100■ (0゜17g mojri
HCj! 3, treated with cold NaHCO3, dried NazSO4 of the a-layer, and distilled off under reduced pressure to obtain the desired fat Xl-160 (0°13 mm).

The structure of 1-1 was confirmed by IRX'H-NMR, MASS.

[α]. -+6. 0 (C=l. 0, CH
Cβ, ) IR (KB r) 3500c1m-', 1765
cm-'1695c+i- The optical resolution method using brucine for α-aminostearic acid, which is the starting material for the synthesis of Exemplary Compounds 1-9 to ■-11, is described in the following two documents, and these are referred to. I went there.

1. A. Mills, A. E. Wilde
r Sm1th, )Ielv.

Chtll, Acta, volume 43, page 1915,
1960 2, E. M., Landan, S.
Grayer 14o1f, L.

Leiseroi+itz+ M, Lahaw, J
, Sagiv, J., Am., Chew.

Sac,, vol. 111, p. 1436, 1989 Mu1 1 Bethep 1 49.4 g (0.14 mol) of the compound was dissolved in 300 methanol, and 14.1 g of triethylamine (
0.14 mol) and stirred at room temperature.

24.3 g (0.14 mol) of tert-butyl carbonate (Tokyo Kasei Products) was added thereto, and the mixture was stirred at room temperature for 10 hours.

After the reaction was completed, tetrahydrofuran was distilled off under reduced pressure, and 200 mf of ethyl acetate and 200 ml of water were added for extraction. This process was repeated twice, and the resulting organic layer was saturated with Na.
Wash once with C4 water and Nag So.

It was dried.

Yes 11a? When the solvent 8 was distilled off under reduced pressure, white crystals were obtained.

When this was recrystallized from an ethanol/hexane system, 41 g of the target compound was obtained.

m, p, 85-88'' I R3350CII-' (NH) (Nujob) 1760 (J-' (ester) 1
720 cm-' (Ure97) Step 2 10 g (0,025 mol) of compound 2 was dissolved in 200 I of a mixed solution of tetrahydrofuran: CH,0H=2:1.
2 g of sodium hydroxide (0,0
An aqueous solution 10- of 5 mol) was added dropwise.

The mixture was stirred at room temperature for 12 hours and acidified to around pH=4 with dilute hydrochloric acid while cooling in a water bath. Water 200＠
In addition, the mixture was extracted three times with ethyl acetate (100%), and the organic layer was washed with water and dried over pJ a 2 SOa.

Crystals are obtained by distilling off the organic solvent under reduced pressure. Recrystallization from ethyl acetate/hexane yields the desired compound 7.
2g was obtained.

m, I), 121~124@I R3400cm-' (NH) (Nu jol) 2800cm-'~260
0cm-' (carboxylic acid 0H) 1720C11-' (carbonyl carboxylate) 1700c+++-' (urethane) Step 3 120g of compound (0.05 mol) and 4.7 phenol
g (0.05 mol) in ethyl acetate and stirred at 0°C.

Among them, DCC (dicyclohexylcarbodiimide>1
Add 0.3 g (0.05 mol) and stir at room temperature for 10 hours.

The precipitate was removed, and the residue was separated and purified by silica gel column chromatography (eluent cohexane: ethyl acetate = 4:1) to yield the desired compound as a colorless oil.

IR(Neat) 3450cm-', 1780I
J-'1700cm-' Step 4 110g (0.02 mol) of the compound was added to CIl□C11z
100°C and cooled to -10°C. Add trifluoroic acid 6120- and stir at 0°C for 30 minutes.

After distilling off volatile components under reduced pressure, the resulting residue was dissolved in ethyl acetate and diluted with 5% Na HCO! Wash the organic layer three times with aqueous solution. After drying over N az SOa, the solvent was distilled off under reduced pressure to obtain the desired I-9,1-11.

Because it was unstable, it was used in the membrane manufacturing process without being purified.

1-9: (α) .+14° (C=0.5CH1l!
ff (TFA salt): IR=3400c+a-', 1765cm (KB
r) 1-11: (α].+12″’ (C=0.5CH
C1゜(TFA salt): IR=3350c+m-', 1760cm-1(
KBr) Other compounds can also be synthesized in a similar manner.

In the present invention, various organic resin materials and inorganic materials having hydrophilic or hydrophobic surfaces are used for the support (substrate) covering the monomolecular film or the cumulative film.

These may be flat or may have a porous or fibrous three-dimensional network structure.

Flat materials include conductive materials such as various metals, inorganic insulators such as glass and alumina, various inorganic and organic crystals, and inorganic semiconductors (Snot, Irlz Oz, Z
Various materials are used, such as nO, TiOx, WOi, Ga3%Si, etc.), organic semiconductors, a conductors, polymer materials, and composite materials of these materials. The material may be an electrode or sensing device (such as a field effect transducer) connected to an external electrical circuit.

Porous materials are mainly useful as supports when used as pass-through membranes or filters, and include, for example, organic and inorganic microporous filter cellulose resin films and various other porous polymer films.

As developing solvents for monomolecular films used in the present invention, in addition to commonly used continuous non-polar organic solvents such as chloroform, dichloromethane, heinine, toluene, and ether, mixtures of these with polar solvents such as alcohol and water can be used. is also used.

Various deposition methods can be used to coat the monolayer on the water surface onto the surface of a substrate or support, including the LB method. The LB method, which is a vertical deposition method, is described in, for example, the Journal of the American Chemical Society (J
, 8m, Chem, Soc,) Volume 57, 1007
Page (1935), Gaines (G.L., Ga1ns,
Insolub Jr.
le Monolayers at Liquid-G
asInterfaces) J, (In
terscience), New York (
(1966) or Kiyonari Fukuda, Materials Technology, Vol. 4, p. 261 (1986).

Other coating methods include horizontal deposition method and rotational deposition method (
For example, JP-A-60-189929, JP-A No. 61-4239
Various methods such as No. 4) are applied. A cumulative film is obtained by repeatedly performing the operation of coating a substrate with a monomolecular film.

For efficient accumulation, the improved horizontal adhesion method described in Japanese Patent Application No. 63-54680 and Japanese Patent Application Laid-Open No. 60-20924 are recommended.
It is also possible to use the continuous cumulative method described in No. 5 and the like.

Examples of the present invention are shown below, but the embodiments of the present invention are not limited thereto.

(Example 1) Exemplary compound 1-6 as an amphipathic amino acid phenyl ester was dissolved in dichloromethane at a concentration of 1 mM to prepare a developing solution. This solution was developed on an aqueous phase of 10-'M phosphate buffer solution at pH 7.4 using a Langmuir film balance to prepare a monomolecular film. Immediately after production, this monomolecular film was compressed using a belt drive type barrier at a speed of 10 CIIZ, and the surface pressure-molecular occupied area (π-formula) characteristics of the monomolecular film at 20°C were measured, and the results shown in A in Figure 1 were obtained. I got it.

Polymerization of this monolayer was carried out for 15 dy on this buffered aqueous solution.
This was done by leaving the monolayer under a constant surface pressure of ne/cm for about 1 h. After this polymerization, π is added again.
-As a result of measuring the A characteristic, the characteristic B in Fig. 1 was obtained,
It is clear that the membrane area shrunk by about half as a result of polymerization, the molecules became denser, and the membrane's rupture pressure increased. The polymerization was carried out by accumulating the monomolecular film after polymerization on a silicon wafer substrate by the LB method, and by transmitting the F of the accumulated film by the permeation method.
As a result of measuring the T-IR absorption spectrum, the characteristic absorption of the ester (~1750 cm-') decreased, and the absorption of the amide bond derived from the polypeptide (~l 650 C'') decreased.
This was confirmed by the appearance of Based on these absorption intensities, the reaction rate after about 1 hour of polymerization was estimated to be 8090%.

In order to evaluate the orientation of molecules after polymerization, we compared the FT-IR absorption spectra between transmission measurement and reflection absorption measurement and investigated the orientation of functional groups. It was confirmed that the polymer molecules were oriented vertically. Furthermore, the thickness of the cumulative film formed on the silicon wafer substrate was measured using ellipsometry.
The membrane thickness of 20±5 people per sample was measured, suggesting that the membranes were arranged with a thickness close to the molecular length.

Next, in order to examine the morphology of the polymerized film, one layer of the polymerized monomolecular film coated on the silicon wafer was observed using an SEM. At this time, for comparison, a polymer film prepared by polymerizing optically inactive long-chain alkyl derivatives of the following amino acid esters (a mixture of 6 and 2 amino acids) on the water surface under the same conditions as Compound 1-6 was also used. SEM observation was performed.

C+5Hit CH NH It was observed that the polymerized film using the optically active amino acid ester of the present invention had a more dense structure than the film polymerized using the optically inactive amino acid ester.

Furthermore, the relative degree of polymerization of these polypeptide films was investigated using a combination of fluorescent labeling method and ninhydrin colorimetric method according to the 4M quantitative method for the purpose of quantifying primary amino groups. It was shown that the peptide membrane had a degree of polymerization approximately 1.5 times higher than that of the optically inactive polypeptide membrane.

[Brief explanation of drawings]

FIG. 1 is a graph showing the π-A characteristics of Compound 2-6 at 20°C, where A shows the curve before polymerization and BL shows the curve after polymerization.

Claims (1)

[Claims] (1) A thin film of an optically active long-chain alkylated polypeptide obtained by polymerizing a monomolecular film or a cumulative film thereof comprising an amphiphilic long-chain alkyl derivative of an optically active α-amino acid ester with an equivalent three-dimensional structure.