JP2009254355A - Peptide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new antibacterial/bactericidal compound exhibiting antibacterial and/or bactericidal properties only for Pseudomonas aeruginosa. <P>SOLUTION: The antibacterial/bactericidal compound is a quaternary ammonium salt-peptide compound obtained by binding a thiol group of an L-cysteine residue introduced into the N-terminal or the C-terminal of a peptide having a specific amino acid sequence to a quaternary ammonium salt through a covalent bond, and is determined to be effective by a phage display method using the Pseudomonas aeruginosa. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a novel peptide, a quaternary ammonium salt-peptide compound and a method for producing the same, an antibacterial or bactericidal agent, and a disinfectant.

  Conventionally, quaternary ammonium salt compounds have an antibacterial effect effective against microorganisms such as bacteria and fungi, and thus have been widely used as microorganism control agents in various industrial fields such as the medical industry, textile industry, and household. Among the quaternary ammonium salt compounds, benzalkonium chloride is an important compound as a disinfectant having a broad antibacterial spectrum against bacteria and fungi.

  However, these quaternary ammonium salt compounds have a fatal defect that exerts an antibacterial effect against various bacterial species and kills useful environmental microorganisms regardless of whether they are gram positive bacteria or gram negative bacteria. . In addition, these quaternary ammonium salt compounds have a low antibacterial effect on some bacterial species, especially Pseudomonas aeruginosa, and therefore need to abnormally increase the concentration used, and as a result, induce drug-resistant bacteria in the environment. There is a risk.

  Under such circumstances, development of a novel antibacterial and bactericidal compound that exhibits antibacterial and / or bactericidal properties is eagerly desired only against Pseudomonas aeruginosa.

  An object of the present invention is to develop a novel antibacterial / bactericidal compound that exhibits antibacterial and / or bactericidal properties only against Pseudomonas aeruginosa.

  The inventors of the present invention have made extensive studies in order to solve the above problems. In the course of the research, we succeeded in producing a novel peptide that specifically adsorbs to Pseudomonas aeruginosa by the phage display method. The present inventors further synthesized a peptide having L-cysteine introduced at the N-terminus or C-terminus of the peptide, and covalently bound the thiol group of the cysteine residue and the quaternary ammonium salt in the peptide. It has been found that quaternary ammonium salt-peptide compounds can be the desired antibacterial and bactericidal compounds that develop antibacterial and / or bactericidal properties only against Pseudomonas aeruginosa. The present invention has been completed based on such findings.

The present invention provides peptides, DNAs, quaternary ammonium salt-peptide compounds and methods for producing the same, antibacterial or bactericidal agents, and disinfectants described in items 1 to 12 below.
Item 1. A peptide having the amino acid sequence shown in SEQ ID NO: 1.
Sequence number 1: SerIleLeuProTyrProTyr
Item 2. A peptide in which 1 to 5 amino acids other than cysteine are added to the N-terminus and / or C-terminus of the peptide according to Item 1.
Item 3. A DNA encoding the peptide according to Item 1.
Item 4. The thiol group of the L-cysteine residue introduced into the N-terminus or C-terminus of the peptide according to Item 1 or Item 2 and a quaternary ammonium salt represented by the following general formula (1) are bound by a covalent bond. Quaternary ammonium salt-peptide compound.

[Wherein, R ₁ represents a linear or branched alkyl group having 1 to 18 carbon atoms. R ₂ represents a linear or branched alkylene group having 1 to 10 carbon atoms. R ₃ represents a hydrogen atom, a halogen atom, a lower alkyl group or a lower alkoxy group. Y represents a halogen atom or an R ₄ SO ₃ group (R ₄ represents a lower alkyl group or a substituted or unsubstituted phenyl group). Z represents a halogen atom, an OH group, a CF ₃ COO group or an R ₅ SO ₃ group (R ₅ represents a lower alkyl group or a substituted or unsubstituted phenyl group). ]
Item 5. Item 4 is a compound of the general formula (2) or (3)

[Wherein, D represents an amino group or an N-acylamino group. E represents a hydroxyl group or an amino group. X represents the peptide according to Item 1 or Item 2. R ₁ , R ₂ , R ₃ and Z are as defined above. ]
Item 5. A quaternary ammonium salt-peptide compound according to Item 4, which is a compound represented by:
Item 6. Item 4 is a compound of the general formula (4)

[Wherein, R _1a represents an n-hexyl group, an n-octyl group, an n-decyl group, an n-dodecyl group, an n-tetradecyl group, an n-hexadecyl group or an n-octadecyl group. Z is as defined above. ]
Item 5. A quaternary ammonium salt-peptide compound according to Item 4, which is a compound represented by:
Item 7. Item 4 is a compound of the general formula (5)

[Wherein, R _1a represents an n-hexyl group, an n-octyl group, an n-decyl group, an n-dodecyl group, an n-tetradecyl group, an n-hexadecyl group or an n-octadecyl group. Z is as defined above. ]
Item 5. A quaternary ammonium salt-peptide compound according to Item 4, which is a compound represented by:
Item 8. A method for producing a quaternary ammonium salt-peptide compound represented by the general formula (2) or (3) according to Item 5,
General formula (1)

[Wherein, R ₁ represents a linear or branched alkyl group having 1 to 18 carbon atoms. R ₂ represents a linear or branched alkylene group having 1 to 10 carbon atoms. R ₃ represents a hydrogen atom, a halogen atom, a lower alkyl group or a lower alkoxy group. Y represents a halogen atom or an R ₄ SO ₃ group (R ₄ represents a lower alkyl group or a substituted or unsubstituted phenyl group). Z represents a halogen atom, an OH group, a CF ₃ COO group or an R ₅ SO ₃ group (R ₅ represents a lower alkyl group or a substituted or unsubstituted phenyl group). ]
And a quaternary ammonium salt represented by the general formula (6) or (7)

[Wherein, D represents an amino group or an N-acylamino group. E represents a hydroxyl group or an amino group. X represents the peptide according to Item 1 or Item 2. ]
A peptide-cysteine adduct represented by
A method for producing a quaternary ammonium salt-peptide compound.
Item 9. A method for producing a quaternary ammonium salt-peptide compound represented by the general formula (4) according to Item 6,
General formula (1a)

[Wherein, R _1a has the same _meaning as R _1a described in item 6.] Y and Z have the same meanings as Y and Z described in Item 8. ]
And a quaternary ammonium salt represented by the formula (8)

A peptide-cysteine adduct represented by
A method for producing a quaternary ammonium salt-peptide compound.
Item 10. A method for producing a quaternary ammonium salt-peptide compound represented by the general formula (5) according to Item 7,
General formula (1a)

[Wherein, R _1a has the same _meaning as R _1a described in Item 7.] Y and Z have the same meanings as Y and Z described in Item 8. ]
A quaternary ammonium salt represented by the formula (9)

A cysteine-peptide adduct represented by
A method for producing a quaternary ammonium salt-peptide compound.
Item 11. Item 8. An antibacterial or bactericidal agent containing the quaternary ammonium salt-peptide compound according to any one of Items 4 to 7 as an active ingredient.
Item 12. Item 8. A disinfectant containing the quaternary ammonium salt-peptide compound according to any one of Items 4 to 7 as an active ingredient.

Peptide The peptide of the present invention has the amino acid sequence shown in SEQ ID NO: 1. The peptide has a property of specifically adsorbing to Pseudomonas aeruginosa, and is produced by the phage display method as shown in Example 1 described later.
There are 6912 DNAs encoding the peptide having the amino acid sequence shown in SEQ ID NO: 1. That is, 6 types in Ser (TCT, TCC, TCA, TCG, AGT and AGC), 3 types in Ile (ATT, ATC and ATA), and 6 types in Leu (TTA, TTG, CTT, CTC, CTA and CTG), Since there are 4 types of Pro (CCT, CCC, CCA and CCG) and 2 types of Tyr (TAT and TAC), 6 × 3 × 6 × 4 × 2 × 4 × 2 = 6912 DNA sequences should be taken. Can do.

  The peptide of the present invention may be a peptide having 1 to 5 amino acids other than cysteine added to the N-terminal and / or C-terminal of the peptide having the amino acid sequence shown in SEQ ID NO: 1.

  Examples of amino acids added to the N-terminal and / or C-terminal of the peptide include glycine, alanine, valine, leucine, isoleucine, serine, threonine, methionine, aspartic acid, glutamic acid, lysine, arginine, phenylalanine, tyrosine, histidine, and tryptophan. And proline. These amino acids are added to 1 to 5, preferably 1 to 3 amino acids, and the types of amino acids to be added may be the same or different.

  In adding an amino acid to a peptide, known reaction conditions for peptide bond formation may be appropriately employed.

Quaternary ammonium salt-peptide compound The quaternary ammonium salt-peptide compound of the present invention has the above peptide (a peptide having the amino acid sequence shown in SEQ ID NO: 1 or an amino acid other than cysteine at the N-terminal and / or C-terminal of the peptide. A peptide in which a thiol group of an L-cysteine residue introduced at the N-terminal or C-terminal of a peptide added with ˜5 and a quaternary ammonium salt represented by the general formula (1) are bonded by a covalent bond.

In the general formula (1), examples of the linear or branched alkyl group having 1 to 18 carbon atoms represented by R ₁ include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, Isobutyl group, tert-butyl group, sec-butyl group, n-pentyl group, neopentyl group, n-hexyl group, isohexyl group, 3-methylpentyl group, n-octyl group, n-decyl group, n-dodecyl group, Examples thereof include an n-tetradecyl group, an n-hexadecyl group, and an n-octadecyl group. Among these, a linear or branched alkyl group having 6 to 18 carbon atoms is preferable, and an even-numbered carbon group is more preferable. Specifically, n-hexyl group, n-octyl group, n-decyl group, n-dodecyl group, n-tetradecyl group, n-hexadecyl group and n-octadecyl group are preferable, n-hexyl group, n-octyl group. And the group n-decyl are particularly preferred.

Examples of the linear or branched alkylene group having 1 to 10 carbon atoms represented by R ₂ include a methylene group, an ethylene group, a propylene group, an isopropylene group, a butylene group, and an isobutylene group. Among these, a methylene group is preferable.

Examples of the halogen atom represented by R ₃ , Y and Z include a chlorine atom, a bromine atom and an iodine atom. Preferred halogen atoms are a chlorine atom and a bromine atom.

Specific examples of the lower alkyl group represented by R ₃ , R ₄ and R ₅ include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, n -C1-C6 linear or branched alkyl groups, such as a pentyl group, an isopentyl group, a neopentyl group, are mentioned.

Specific examples of the lower alkoxy group represented by R ₃ include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, Examples thereof include linear or branched alkoxy groups having 1 to 6 carbon atoms such as a pentyloxy group, neopentyloxy group, and tert-pentyloxy group.

Specific examples of the substituted or unsubstituted phenyl group represented by R ₄ and R ₅ include a phenyl group, o, m or p-methylphenyl group, o, m or p-ethylphenyl group, o, m or Examples thereof include a phenyl group which may have a substituent such as a p-isopropylphenyl group, a lower alkyl group (an alkyl group having 1 to 6 carbon atoms) such as o, m or p-tert-butylphenyl group.

In the general formula (1), the Y—R ₂ — group may be substituted at any position on the pyridine ring, but the preferred substitution position is the 3 or 4 position of the pyridine ring, and the more preferred substitution position is pyridine. The 4th position of the ring.

In the general formula (1), R ₃ is preferably a hydrogen atom.

  Among the quaternary ammonium salts represented by the general formula (1), the quaternary ammonium salt represented by the general formula (1a) is preferable.

[Wherein, R _1a , Y and Z are the same as above. ]
Method for Producing Quaternary Ammonium Salt-Peptide Compound The quaternary ammonium salt-peptide compound of the present invention is obtained by reacting the above-described peptide of the present invention with L-cysteine to leave an L-cysteine residue at the N-terminus or C-terminus of the peptide. It is produced by introducing a group and then reacting the thiol group of the L-cysteine residue with a quaternary ammonium salt represented by the general formula (1).

  For the reaction between the peptide of the present invention and L-cysteine, known reaction conditions for peptide bond formation can be appropriately employed.

  The peptide-cysteine adduct of the present invention thus produced can be represented by, for example, the following general formula (5) or (6).

[Wherein, D, E and X are the same as above. ]
Preferred peptide-cysteine adducts of the present invention can be represented by, for example, the following general formulas (8) and (9).

  The reaction of the peptide-cysteine adduct and the quaternary ammonium salt represented by the general formula (1) is carried out in an appropriate solvent in the presence of imidazole, for example, as shown below.

  As the solvent, known solvents can be widely used as long as the peptide-cysteine adduct can be dissolved. The solvent is preferably, for example, water; lower alcohols such as methanol and ethanol; a mixed solvent of water and lower alcohol. Two or more kinds of solvents may be used in combination as required. The amount of the solvent used is such that the concentration of the peptide-cysteine adduct is usually 0.001 to 1.0M, preferably 0.01 to 0.1M.

  The imidazole is generally used in an amount of 1 to 6 mol, preferably 2 to 5 mol, per 1 mol of the peptide-cysteine adduct.

  The peptide-cysteine adduct is usually used in an amount of 0.1 to 1.5 mol, preferably 0.8 to 1.2 mol, per 1 mol of the quaternary ammonium salt represented by the general formula (1). .

  This reaction is usually performed at a temperature of 4 to 20 ° C., and is generally completed in 1 to 3 hours. The reaction atmosphere for the reaction is not particularly limited.

  After completion of the above reaction, the quaternary ammonium salt-peptide compound of the present invention can be produced by isolating and purifying the resulting reaction product according to known isolation and purification means.

  For example, the quaternary ammonium salt-peptide compound of the present invention can be obtained in the form of crude crystals by freeze-drying the resulting reaction product after completion of the above reaction.

  Moreover, high performance liquid chromatography can be used for purification of the obtained crude crystals. For example, an eluate containing a quaternary ammonium salt-peptide compound of the present invention separated by high performance liquid chromatography using a C18 reverse phase column is concentrated by an evaporator and lyophilized to obtain a quaternary ammonium salt-peptide. Crystals of the compound can be produced with high purity.

  The quaternary ammonium salt-peptide compound of the present invention has excellent antibacterial and bactericidal properties only against Pseudomonas aeruginosa. Utilizing this property, unlike antibacterial agents, bactericides, disinfectants, etc. that are effective against various bacterial species, it is used as an antibacterial or bactericidal agent or disinfectant that exhibits antibacterial and bactericidal properties only against Pseudomonas aeruginosa it can.

Antibacterial or bactericidal agent or disinfectant The antibacterial or bactericidal agent and disinfectant of the present invention are the other components contained in these drugs, and their combinations, except that the active ingredient is the quaternary ammonium salt-peptide compound of the present invention. The amount is the same as known antibacterial or disinfectants and disinfectants.

  The content of the quaternary ammonium salt-peptide compound in the antibacterial or disinfectant and disinfectant of the present invention is usually about 0.01 to 50% by weight, preferably about 0.1 to 1% by weight.

  The quaternary ammonium salt-peptide compound of the present invention specifically adsorbs to Pseudomonas aeruginosa and exhibits high antibacterial and bactericidal properties only for the bacterial species.

  The peptide of the present invention is extremely useful as an intermediate for producing such a quaternary ammonium salt-peptide compound.

  Since the quaternary ammonium salt-peptide compound of the present invention has the property of specifically adsorbing only against Pseudomonas aeruginosa, it can exhibit an effective antibacterial effect at a low drug use concentration, As a result, it is possible to prevent the appearance of drug-resistant bacteria due to large-scale use of the drug.

  Since the quaternary ammonium salt in the quaternary ammonium salt-peptide compound of the present invention alone has extremely low antibacterial properties, even if quaternary ammonium residues are liberated, they hardly exhibit antibacterial properties against general bacteria. . Therefore, it can be said that the antibacterial or bactericidal agent containing the quaternary ammonium salt-peptide compound of the present invention is an antibacterial or bactericidal agent that is very gentle to environmental microorganisms.

1 is a ¹ H-NMR spectrum of the quaternary ammonium salt-peptide compound obtained in Example 2, (2.3). FIG. 2 is a ¹ H-NMR spectrum of the quaternary ammonium salt-peptide compound obtained in Example 2, (2.4).

  The present invention will be further clarified by the following examples.

Example 1
As a production target microorganism of the peptide of the present invention , Pseudomonas aeruginosa was used, and a peptide having a binding property to Pseudomonas aeruginosa was screened by the phage display method as shown in the following (1) to (5).

  In the phage display method, a library in which peptides are randomly displayed on the minor protein pIII on the surface of M13 phage (a peptide library having 7 random amino acids to be displayed) was purchased from New England Bio Lab.

(1) Panning Pseudomonas aeruginosa cultured overnight in LB medium was collected by centrifugation, added to 100 μl of 10 mM HEPES (pH 7.5), gently stirred and suspended.

About 10 μl (2 × 10 ¹⁰ pfu or more / 10 μl) of phage solution was added to the resulting suspension to suspend, and incubated at room temperature for 10 minutes. After collecting the cells by centrifugation, the supernatant is discarded, washed 5 times with HEPES-Tween buffer (10 mM HEPES + 0.1% Tween 20, pH 7.5), collected again by centrifugation, and 100 μl of sterilized. Suspended with water to obtain a bound phage solution.

(2) Phage amplification / purification 90 μl of the combined phage solution obtained above was added to a 50 ml conical tube, and 100 μl of E. coli (F +) cultured overnight and tetracycline were added to a final concentration of 25 μg / ml. Infected and stirred vigorously at 37 ° C. for 4 and a half hours to amplify the phage.

After amplification, the medium is transferred to a 15 ml centrifuge tube, centrifuged (8,000 rpm × 10 minutes, 4 ° C.), the supernatant is transferred to another 15 ml centrifuge tube, and 1.6 ml of PEG / NaCl solution (20% PEG, 2.5M NaCl) was added and suspended, and incubated overnight at 4 ° C. Then, the supernatant was discarded by centrifugation (15,000 rpm × 5 minutes, 4 ° C.), and 1 ml of TBS was added to dissolve the precipitate (phage). Transfer this lysate to a 1.5 ml Eppendorf tube, add 160 μl of PEG / NaCl, mix, incubate in ice for 1 hour, centrifuge (10,000 rpm × 10 min, 4 ° C.), discard the supernatant, The precipitate (phage) was lysed with 20 μl TB + 0.02% NaN ₃ . Furthermore, it was centrifuged (10,000 rpm × 2 minutes, 4 ° C.), and the supernatant was transferred to a 0.5 ml Eppendorf tube to obtain a phage purified solution. Using this phage purification solution, the operations of panning, amplification and purification were repeated three times in the same manner, and the fourth time until the phage was eluted in the panning step, a phage eluate was obtained.

(3) Titer measurement (plaque formation)
The fourth phage eluate was diluted with TBS to prepare a 102 to 105-fold diluted solution.

  On the other hand, the LB plate medium (prepared in a plastic petri dish) added with IPTG and X-Gal was warmed at 37 ° C., and the agaro stop was dissolved in an autoclave and warmed so as not to harden at 45 ° C.

Add 10 μl of the diluted phage solution to 200 μl of E. coli (F +) cultured in LB medium until OD ₆₀₀ = 0.5, stir and incubate for 1-5 minutes, and then add to the agaro stop stored at 45 ° C. After stirring, the mixture was poured into LB plate medium supplemented with IPTG and X-Gal and incubated for about 5 minutes. When the agarostop had solidified and excess water had blown off, it was capped and incubated overnight at 37 ° C (blue plaques appeared if phage were present).

(4) Amplification of phage from plaque The appeared plaque was picked with a toothpick and picked. Picked toothpicks are placed in 2 ml of LB medium supplemented with tetracycline to a final concentration of 20 μg / ml, and 20 μl of E. coli (F +) cultured overnight is added (test tube) and shaken vigorously for 4 and a half hours. Cultured at last. After the culture, the same operation as in (2) above was performed to purify the single phage, and 86 phages were obtained.

(5) Confirmation of peptide sequence In the phage display method performed this time, since the peptide is displayed at the N-terminus of the pIII protein of the phage, reading the 5 'upstream of the phage pIIIgene (excluding the signal sequence part), the amino acid of the peptide The sequence can be determined.

  As a result, it was revealed that the obtained peptide had the amino acid sequence shown in SEQ ID NO: 1.

The peptide having the amino acid sequence shown in SEQ ID NO: 1 has the following physicochemical properties.
Isoelectric point IP = 5.65
m / z: 850.0.

Example 2
(2.1) Production of peptide- cysteine adduct represented by formula (8) Peptide synthesis was performed by a 9-fluorenylmethyloxycarbonyl (Fmoc) solid phase synthesis method using a commercially available peptide synthesizer. The condensing agent was hexafluorophosphate. According to the peptide synthesis program of the peptide synthesizer, the deprotection reaction and the condensation reaction are repeated to extend the peptide chain from the Fmoc-amino acid bound to the resin. Specifically, Fmoc, which is an amino acid protecting group, was cleaved and removed with 20% piperidine / dimethylformamide, washed with dimethylformamide, then reacted with Fmoc-amino acid and washed with dimethylformamide. After completion of the elongation reaction, the Fmoc group was cleaved with 20% piperidine / dimethylformamide and washed with dimethylformamide. Peptides were cleaved from the resin with 82.5% trifluoroacetic acid solution (trifluoroacetic acid: 1,2-ethanedithiol: m-cresol: thioanisole: water = 82.5: 2.5: 5: 5: 5) The resin was removed by filtration, precipitated in diethyl ether, and then the diethyl ether layer was removed by centrifugation. This diethyl ether washing step was performed three times. The obtained peptide-cysteine adduct was dissolved in pure water, crystallized by lyophilization, and purified by HPLC to obtain the desired peptide-cysteine adduct with a purity of> 95%.
Isoelectric point IP = 4.97
m / z: 955.5.

(2.2) Preparation of quaternary ammonium salt represented by formula (1a ) wherein R _1a is an n-octyl group and Y and Z are bromine atoms 4-hydroxylmethyl-1-octylpyridinium bromide 500% of hydrogen peroxide bromide was added and refluxed for 4 hours. After the reaction, the reaction mixture was concentrated under reduced pressure by an evaporator and purified by a silica gel open column using a chloroform-methanol mixed solvent as an eluent. R _1a was represented by the general formula (1a) in which n-octyl group, Y and Z represent bromine atoms. 95 mg (yield 77%) of quaternary ammonium salt (4-bromomethyl-1-octylpyridinium bromide) was obtained.

When the obtained compound was analyzed, it was confirmed by the ¹ H-NMR spectrum (CD ₃ OD, reference material TMS) of the obtained compound that the target compound was obtained.
¹ H-NMR (CD ₃ OD, TMS) δ ppm: 0.89 (3H, t, J = 6.9 Hz), 1.27-1.32 (8H, m), 1.36-1.40 ( 2H, m), 2.01 (2H, m), 4.61 (2H, t, J = 7.7), 4.81 (2H, s), 8.15 (2H, d, J = 6. 6), 8.96 (2H, d, J = 6.8).

(2.3) Production of quaternary ammonium salt-peptide compound
By reacting the peptide-cysteine adduct represented by the formula (8) produced in (2.1) and 4-bromomethyl-1-octylpyridinium bromide produced in (2.2) as shown below, R _1a becomes A quaternary ammonium salt-peptide compound represented by the general formula (4) in which an n-octyl group and Z represents a bromine atom was produced.

  0.0249 mmol of peptide-cysteine adduct (8) was cooled in an ice bath and dissolved in 2.4 ml of water with stirring. Next, 0.0253 mmol of 4-bromomethyl-1-octylpyridinium bromide (1a) was added, and finally 0.111 mmol of imidazole was added, and the mixture was reacted for 1 hour with stirring in an ice bath. After the reaction, the reaction mixture in the form of an aqueous solution was frozen with liquid nitrogen and lyophilized to obtain 40.0 mg of crude crystals.

  The obtained crude crystal was purified by a high performance liquid chromatograph. Specifically, precolumn (GL Science product, product name Inertsil WP300 C18, 10 mm ID × 30 mm) and C18 reverse phase column (GL Science product, product name Peptides C18, 10 mm ID × 150 mm). And a mixed solution of 0.1% trifluoroacetic acid aqueous solution and 0.1% trifluoroacetic acid-containing acetonitrile solution was used as an eluent. That is, while increasing the amount of the trifluoroacetic acid-containing acetonitrile solution contained in the eluent over time (providing a concentration gradient from 0% to 100% by volume), the column was used at a flow rate of 2 ml / min. For 27 minutes. The peptide eluted from the reverse phase column is detected at 254 nm using an ultraviolet detector and is shown as a peak on the recording chart. When the obtained compound (retention time 18.95 minutes) was analyzed, the yield was 65% and the purity was> 93%.

In addition, the mass of the eluted compound was measured using a Voyager-DE STR mass spectrometer (Applied Biosystems, USA) as MALDI-TOF / MS (Matrix-Assisted Laser Desorption Time of Flight Mass Spectrometry). Mass spectrometry). Furthermore, from the ¹ H-NMR spectrum (CD ₃ OD, reference material TMS) of the obtained compound, the proton peak (8.07 ppm, 8.86 ppm) of the pyridinium skeleton characteristic of 1-octylpyridinium salt, and n- The methylene proton peak (1.37 ppm-1.54 ppm) derived from the octyl group was detected, and it was confirmed that the 1-octylpyridinium salt was bound to the peptide chain. As a result, it was confirmed that the target quaternary ammonium salt-peptide compound was produced.
m / z: 1158.6
FIG. 1 shows a ¹ H-NMR spectrum of the obtained quaternary ammonium salt-peptide compound which is the compound 1 of the present invention.

(2.4) Production of quaternary ammonium salt-peptide compound
By reacting the peptide-cysteine adduct represented by the formula (9) produced by the same method as (2.1) and 4-bromomethyl-1-octylpyridinium bromide produced in (2.2) as shown below. A quaternary ammonium salt-peptide compound represented by the general formula (5) in which R _1a represents an n-octyl group and Z represents a bromine atom was produced.

  0.0313 mmol of cysteine-peptide adduct (9) was cooled in an ice bath and dissolved in 5.0 ml of water with stirring. Next, 0.0344 mmol of 4-bromomethyl-1-octylpyridinium bromide (1a) was added, finally 0.128 mmol of imidazole was added, and the mixture was reacted for 1.5 hours with stirring in an ice bath. After the reaction, the reaction mixture in the form of an aqueous solution was frozen with liquid nitrogen and lyophilized to obtain 55.3 mg of crude crystals.

  The obtained crude crystal was purified by a high performance liquid chromatograph. Specifically, a precolumn (GL Science product, product name Peptides C18, 20 mm ID × 50 mm) and a C18 reverse phase column (GL Science product, product name Peptides C18, 20 mm ID × 200 mm) are used. Then, a mixed solution of 0.1% trifluoroacetic acid aqueous solution and 0.1% trifluoroacetic acid-containing acetonitrile solution was used as an eluent. That is, while increasing the amount of the trifluoroacetic acid-containing acetonitrile solution contained in the eluent over time (providing a concentration gradient from 0% to 100% by volume), the column was used at a flow rate of 8 ml / min. For 45 minutes. The peptide eluted from the reverse phase column is detected at 254 nm using an ultraviolet detector and is shown as a peak on the recording chart. When the obtained compound (retention time 24-26 minutes) was analyzed, the yield was 63% and the purity was> 98%.

In addition, the mass of the eluted compound was measured using a Voyager-DE STR mass spectrometer (Applied Biosystems, USA) as MALDI-TOF / MS (Matrix-Assisted Laser Desorption Time of Flight Mass Spectrometry). Mass spectrometry). Further, from the ¹ H-NMR spectrum (CD ₃ OD, reference substance TMS) of the obtained compound, the proton peak (8.13 ppm-8.15 ppm, 8.90 ppm-) characteristic of the 1-octylpyridinium salt is obtained. 8.94 ppm) and a methylene proton peak (1.29 ppm-1.38 ppm) derived from an n-octyl group were detected, confirming that the 1-octylpyridinium salt was bound to the peptide chain. As a result, it was confirmed that the target quaternary ammonium salt-peptide compound was produced.
m / z: 1158.6
FIG. 2 shows a ¹ H-NMR spectrum of the obtained quaternary ammonium salt-peptide compound which is the compound 2 of the present invention.

Example 3
The quaternary ammonium salt-peptide compound (Compound 1 of the present invention) obtained in (2.3) of Example 2 has a 96-well bactericidal power (minimum bactericidal concentration: MBC) against gram-negative and gram-positive bacteria described below. (Well) The specific bactericidal performance against Pseudomonas aeruginosa ATCC 10145 was examined by a sterile water dilution method using a microplate.

  The same applies to the quaternary ammonium salt (4-hydroxymethyl-1-octylpyridinium bromide, comparative compound A) and the peptide (comparative compound B) of (2.1), which are the raw materials of (2.2) of Example 2. Thus, bactericidal power (minimum bactericidal concentration: MBC) against gram-negative and gram-positive bacteria was determined.

Test bacteria / gram-negative bacteria
Pseudomonas aeruginosa ATCC 10145
Klebsiella pneumoniae ATCC 4352
Escherichia coli NBRC 12713
Proteus mirabilis NBRC 3849
Serratia marcescens ATCC 13880
・ Gram negative bacteria
Kocuria rhizophila NBRC 12708
Staphylococcus aureus NBRC 12732
Staphylococcus aureus COL 1 (MRSA)
Measurement of minimum bactericidal concentration (MBC) This measurement was performed in a sterile hood (Airtech, clean bench), and the microorganisms were kept under ice cooling unless otherwise specified. L-broth (tryptone 1.0% (W / V), yeast extract 0.5% (W / V), sodium chloride 0.5% (W / V), pH 7.0 to 7.2) Pre-cultured in 5 ml at 37 ° C. for 18 hours. The precultured cells are collected by centrifuging (0 ° C., 6500 rpm × 10 minutes) with a centrifuge (manufactured by Sakuma Seisakusho, MODEL 50A-7), washed with physiological saline, and then washed with sterile water. Diluted and prepared using a spectrophotometer (manufactured by Shimadzu Corporation, model name: UV-1700) so that the bacterial suspension concentration is 1 × 10 ⁴ cells / ml (OD ₆₆₀ = 0.00001). did. For the sterilization test, a drug solution prepared using 80% ethyl alcohol was diluted 50-fold with sterile water and then diluted 10-fold twice. 25 μl of the above bacterial suspension was inoculated into 25 μl of each drug solution and contacted with the drug at 37 ° C. for 1 hour, and then 20 μl of the sample solution was collected and inoculated into 200 μl of NB medium. This NB determination medium was cultured at 37 ° C. for 24 hours, the presence or absence of growth was determined with the naked eye, and the minimum drug concentration at which no growth was observed was defined as the minimum bactericidal concentration (MBC). For the compound 1 of the present invention and the comparative compound A, the above test was performed three times or more.

  The results are shown in Table 1.

  From Table 1, the quaternary ammonium salt-peptide compound of the present invention (the present compound 1) has excellent antibacterial and bactericidal properties only against Pseudomonas aeruginosa ATCC 10145, and other bacterial species It is clear that the antibacterial effect is low. In addition, while the unbound quaternary ammonium salt (Comparative Compound A) alone had a low antibacterial activity, the antibacterial activity was remarkably increased by binding to the peptide (Comparative Compound B). It was revealed that the Pseudomonas aeruginosa adsorption specificity was sufficiently exhibited and the bacterial species specificity of the quaternary ammonium salt was imparted.

Example 4
For the quaternary ammonium salt-peptide compound (Compound 2 of the present invention) obtained in (2.4) of Example 2, the bactericidal power (minimum bactericidal concentration: MBC) against gram-negative and gram-positive bacteria described below was 96 wells. (Well) The specific bactericidal performance against Pseudomonas aeruginosa ATCC 10145 was examined by a sterile water dilution method using a microplate.

  In addition, quaternary ammonium salt (4-hydroxylmethyl-1-octylpyridinium bromide, comparative compound A), which is the raw material of Example 2.2 (2.2), was also sterilized against gram-negative and gram-positive bacteria in the same manner as described above. The force (minimum bactericidal concentration: MBC) was determined.

Test bacteria / gram-negative bacteria
Pseudomonas aeruginosa ATCC 10145
Klebsiella pneumoniae ATCC 4352
Escherichia coli NBRC 12713
Serratia marcescens ATCC 13880
・ Gram negative bacteria
Kocuria rhizophila NBRC 12708
Staphylococcus aureus NBRC 12732
Staphylococcus aureus COL 1 (MRSA)
Measurement of Minimum Bactericidal Concentration (MBC) This measurement was performed in the same manner as in Example 3, and the present test 2 and comparative compound A were subjected to the above test three times or more.

  The results are shown in Table 2.

  From Table 2, the quaternary ammonium salt-peptide compound of the present invention (present compound 2) has excellent antibacterial and bactericidal properties only against Pseudomonas aeruginosa ATCC 10145, and other bacterial species It is clear that the antibacterial effect is low. In addition, the unbound quaternary ammonium salt (Comparative Compound A) alone had a low antibacterial activity, whereas the antibacterial activity was remarkably increased by binding to the peptide. Was fully demonstrated, and it became clear that the bacterial species specificity of the quaternary ammonium salt was imparted.

Claims (12)

A peptide having the amino acid sequence shown in SEQ ID NO: 1.
Sequence number 1: SerIleLeuProTyrProTyr A peptide comprising 1 to 5 amino acids other than cysteine added to the N-terminal and / or C-terminal of the peptide according to claim 1. DNA encoding the peptide according to claim 1. A thiol group of an L-cysteine residue introduced at the N-terminus or C-terminus of the peptide according to claim 1 or claim 2 and a quaternary ammonium salt represented by the following general formula (1) are bound by a covalent bond. And quaternary ammonium salt-peptide compounds.

[Wherein, R ₁ represents a linear or branched alkyl group having 1 to 18 carbon atoms. R ₂ represents a linear or branched alkylene group having 1 to 10 carbon atoms. R ₃ represents a hydrogen atom, a halogen atom, a lower alkyl group or a lower alkoxy group. Y represents a halogen atom or an R ₄ SO ₃ group (R ₄ represents a lower alkyl group or a substituted or unsubstituted phenyl group). Z represents a halogen atom, an OH group, a CF ₃ COO group or an R ₅ SO ₃ group (R ₅ represents a lower alkyl group or a substituted or unsubstituted phenyl group). ] The compound according to claim 4 is represented by the general formula (2) or (3)

[Wherein, D represents an amino group or an N-acylamino group. E represents a hydroxyl group or an amino group. X represents the peptide according to claim 1 or claim 2. R ₁ , R ₂ , R ₃ and Z are as defined above. ]
The quaternary ammonium salt-peptide compound of Claim 4 which is a compound represented by these. The compound according to claim 4 has the general formula (4).

[Wherein, R _1a represents an n-hexyl group, an n-octyl group, an n-decyl group, an n-dodecyl group, an n-tetradecyl group, an n-hexadecyl group or an n-octadecyl group. Z is as defined above. ]
The quaternary ammonium salt-peptide compound of Claim 4 which is a compound represented by these. The compound according to claim 4 has the general formula (5)

[Wherein, R _1a represents an n-hexyl group, an n-octyl group, an n-decyl group, an n-dodecyl group, an n-tetradecyl group, an n-hexadecyl group or an n-octadecyl group. Z is as defined above. ]
The quaternary ammonium salt-peptide compound of Claim 4 which is a compound represented by these. A method for producing a quaternary ammonium salt-peptide compound represented by the general formula (2) or (3) according to claim 5,
General formula (1)

[Wherein, R ₁ represents a linear or branched alkyl group having 1 to 18 carbon atoms. R ₂ represents a linear or branched alkylene group having 1 to 10 carbon atoms. R ₃ represents a hydrogen atom, a halogen atom, a lower alkyl group or a lower alkoxy group. Y represents a halogen atom or an R ₄ SO ₃ group (R ₄ represents a lower alkyl group or a substituted or unsubstituted phenyl group). Z represents a halogen atom, an OH group, a CF ₃ COO group or an R ₅ SO ₃ group (R ₅ represents a lower alkyl group or a substituted or unsubstituted phenyl group). ]
And a quaternary ammonium salt represented by the general formula (6) or (7)

[Wherein, D represents an amino group or an N-acylamino group. E represents a hydroxyl group or an amino group. X represents the peptide according to claim 1 or claim 2. ]
A peptide-cysteine adduct represented by
A method for producing a quaternary ammonium salt-peptide compound. A method for producing a quaternary ammonium salt-peptide compound represented by the general formula (4) according to claim 6,
General formula (1a)

_{Wherein, R 1a} has the same meaning as _{R 1a} according to claim 6. Y and Z have the same meanings as Y and Z described in claim 8. ]
And a quaternary ammonium salt represented by the formula (8)

A peptide-cysteine adduct represented by
A method for producing a quaternary ammonium salt-peptide compound. A method for producing a quaternary ammonium salt-peptide compound represented by the general formula (5) according to claim 7,
General formula (1a)

_{Wherein, R 1a} has the same meaning as _{R 1a} according to claim 7. Y and Z have the same meanings as Y and Z described in claim 8. ]
A quaternary ammonium salt represented by the formula (9)

A cysteine-peptide adduct represented by
A method for producing a quaternary ammonium salt-peptide compound. An antibacterial or bactericidal agent containing the quaternary ammonium salt-peptide compound according to any one of claims 4 to 7 as an active ingredient. A disinfectant containing the quaternary ammonium salt-peptide compound according to any one of claims 4 to 7 as an active ingredient.

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