KR20210089986A - Antimicrobial surfactant compounds based on amino acids and core-shell nanoparticle composites - Google Patents

Abstract

The present invention relates to an amino acid-based antibacterial antibiotic surfactant compound and an antibacterial antibiotic nanocomposite coated with the same on gold or silica nanoparticles, wherein antibacterial antibiotic surfactant compound is based on natural and non-natural amino acids, uses natural extract-derived nanoparticles, can be synthesized in an environmentally friendly way, and has increased water solubility, decreased cytotoxicity, increased bioavailability, and excellent antibacterial properties to be usefully used as a novel antibacterial agent, surfactant, preservative, bio-nano material and the like.

Description

Antimicrobial surfactant compounds based on amino acids and core-shell nanoparticle composites containing amino acid-based antimicrobial surfactant compounds and core-shell structure

The present invention relates to an amino acid-based antibacterial antibiotic surfactant compound and an antibacterial antibiotic nanocomposite coated with the same on gold or silica nanoparticles.

Surfactants have actions such as surface wetting, penetration, foaming, defoaming, emulsification, solubilization, dispersion, aggregation and cleaning due to the ability to lower the surface tension through the activation of the interface between the phases. It is an essential main raw material used in all industries including cosmetics.

However, due to recent changes in environmental awareness and raised biosafety standards, industrial fields such as cosmetics are carefully selecting surfactants, and due to issues of regulation and environmental protection, the development of surfactants is an important issue. In addition, as environmental pollution or harm to human body due to chemical substances has emerged, in order to suppress them, environmental load substances or hazardous chemical substances are designated and their use is restricted by various laws and regulations ranging from manufacturing process, handling and disposal.

Recently, natural or biosynthetic bio-surfactants obtained by extracting natural substances are expected to gradually replace conventional synthetic surfactants as amphiphilic, biodegradable, eco-friendly functional materials.

In addition, the safety problem for the human body is a major problem to be solved. It is reported that small molecules have strong penetrating power, which stimulates the skin to cause atopic dermatitis and may cause disease or cancer if it enters the organs through capillaries, and there is a lot of controversy about this. It's happening.

Therefore, there is an urgent need to develop a material that can reduce environmental pollution as well as solve human safety problems, and can also achieve properties such as increased water solubility, reduced cytotoxicity, increased bioavailability, and antibacterial and antibacterial properties.

In particular, preservatives and antibacterial antibiotic surfactants are currently in a situation that requires the development of agents with new mechanisms due to the problems caused by resistance and the emergence of super bacteria, and attempts to solve the problems by using candidate substances with various antibacterial activities are difficult. is being done

Therefore, the present invention intends to provide the possibility of application of novel biomaterials through enhanced antibacterial properties and thermal stability of nano-modified materials, and for this purpose, natural and non-natural amino acid-based antibacterial antibiotic surfactants, nanoparticles of naturally-derived materials, and It is intended to provide a nanocomposite used together.

Through this, new materials with increased physiological activity and physical performance are synthesized in an eco-friendly manner to develop new antibacterial agents, surfactants, preservatives, and bio-nano materials.

Accordingly, the present invention provides an antibacterial antibiotic surfactant compound based on natural, non-natural amino acids having excellent antibacterial and antioxidant performance in an environmentally friendly way to solve the above problems.

In addition, a nanocomposite that can be used as an antibacterial antibiotic surfactant of a core-shell structure in which the compound is applied to gold (Au) synthesized using a natural extract or silica (SiO _{2 ) nanoparticles synthesized using an eco-friendly method} would like to provide

It is formed into a nanocomposite using silica (SiO ₂ ) or gold (Au) and when used at a high temperature of 100 ~ 200 ℃ or higher, it can solve the problem of preventing aggregation of the antibacterial antibiotic compound, and the silica (SiO ₂ ) or gold (Au) of the core ) The functionality of the material can be dramatically improved by the nanoparticle properties.

To this end, the present invention provides an amino acid-based antibacterial antibiotic surfactant compound represented by the following [Formula 1] to [Formula 12].

[Formula 1] [Formula 2]

[Formula 3] [Formula 4]

[Formula 5] [Formula 6]

[Formula 7] [Formula 8]

[Formula 9] [Formula 10]

[Formula 11] [Formula 12]

Specific structures of the [Formula 1] to [Formula 12], definitions of each substituent, and specific compounds implemented thereby will be described later.

In addition, the present invention provides an antibacterial antibiotic nanocomposite comprising the amino acid-based antibacterial antibiotic surfactant compound and gold (Au) or silica (SiO _{2 ) nanoparticles.}

The antibacterial antibiotic nanocomposite according to the present invention is characterized in that the gold (Au) or silica (SiO ₂ ) nanoparticles are coated with an amino acid-based antibacterial antibiotic surfactant compound to form a core-shell structure.

The surfactant compound having antibacterial and antibiotic performance according to the present invention and the nanocomposite using the same are natural, non-natural amino acid-based antibacterial antibiotic surfactant compounds, and also using nanoparticles derived from natural extracts, and can be synthesized in an environment-friendly manner. , increased water solubility, reduced cytotoxicity, increased bioavailability, and excellent antibacterial properties, so it can be usefully used as a novel antibacterial agent, surfactant, preservative, bio-nano material, etc.

1 is a view showing the synthesis results of controlling the shape and size of nanoparticles according to the present invention according to the concentration of rice bran extract, HAuCl _{4 and the presence or absence of surfactant.}
2 is a view showing the synthesis result of controlling the shape and size of the nanoparticles according to the present invention according to the concentration of spirulina extract, HAuCl _{4 .}
3 and 4 are UV confirmation results and PL confirmation results for gold nanoparticles synthesized according to an embodiment of the present invention, respectively.
_{5 is a TEM image of silica (SiO 2} ) nano airgel particles synthesized according to an embodiment of the present invention (hydrophobic nanoporous silica aerogel).
_{6 is a result of confirming the silica (SiO 2} ) nano aerogel particles (hydrophobic nanoporous silica aerogel) synthesized according to an embodiment of the present invention through SEM EDS.
7 is a TEM image and EDS confirmation result of a nanocomposite in which a surfactant (D-LAE) is applied to _{silica (SiO 2} ) nano airgel particles according to an embodiment of the present invention.
8 is a test result for antibacterial (E. coli, Staphylococcus aureus, Candida) for the nanocomposite antibacterial and antibiotic composition according to an embodiment of the present invention.
9 is a result of comparing the MICs of D-type LAE and L-type LAE in E. coli, and it can be confirmed that the MIC is increased 4 times compared to the L-type, and the properties are changed only by changing the chirality in the same structure.
10 is a result of confirming the MIC in E. coli in the case of D-type LAE and DL LAE, the activity can be confirmed at the same MIC concentration (32 ug/mL), and the difference in absorbance at different concentrations (16 ug/mL) is 2 You can confirm that the boat is flying.
11 is a result of confirming the MICs of D, DL, and L-type EUA, and it can be seen that the absorbance constantly increases in the order of L, DL, and D types at the same MIC (64 ug/mL).
12 is a result of confirming the MIC values of D-type proline and D-type proline, when salt treatment with HCl by substituting a Dodecyl group for the carboxyl acid of D-type alanine, the difference is more than 8 times, and in the case of D-type proline, D-type LAE and D-type LAE It can be confirmed by showing the same MIC.
13 is an evaluation result of chirality of LAE and toxicity in kidney and keratinocytes according to concentration.
14 shows a cytotoxicity evaluation process using Cell Counting Kit-8 (CCK-8) according to an experimental example of the present invention.
15 and 16 are TEM images confirming that a surfactant compound is applied to gold nanoparticles according to an embodiment of the present invention, respectively.
17 is a TEM EDX showing that the nanocomposite synthesized according to the present invention is coated on gold nanoparticles.
18 is a result confirming through TEM that the nanocomposite synthesized by applying a compound to gold nanoparticles according to an embodiment of the present invention has absorption and effectiveness in microorganisms.
19 and 20 are MIC results confirming that the effect of nanocomposites synthesized by applying a compound to gold nanoparticles according to the present invention is increased when absorbed into microorganisms, respectively.

Hereinafter, the present invention will be described in more detail.

One aspect of the present invention synthesizes a compound in which a salt is substituted or unsubstituted for an amine group of an amino acid or a carboxylic acid based on a new amino acid synthesis strategy (L, R, racemic type of 20 amino acids) as a basic skeleton, and an amine of an amino acid It is a novel antibacterial antibiotic surfactant compound in which a fatty acid, an alkyl group, and a protecting group are substituted for a group and a carboxylic acid.

The amino acid-based antibacterial antimicrobial surfactant compound according to the present invention is characterized in that it is represented by the following [Formula 1] to [Formula 12].

[Formula 1] [Formula 2]

[Formula 3] [Formula 4]

[Formula 5] [Formula 6]

[Formula 7] [Formula 8]

[Formula 9] [Formula 10]

[Formula 11] [Formula 12]

In the [Formula 1] to [Formula 12],

R _{1 is any one selected} from hydrogen, alkyl, carboxylic acid, amine, alcohol, guanidine, phenyl, phenol, indole, imidazole and D-glucose.

R ₂ is any one selected from hydrogen, alkyl, D-glucose, t-butyl carbamate, benzyl carbamate, 9-fluorenylmethyl carbamate and acetamide.

R ₃ is any one selected from t-butyl carbamate, benzyl carbamate, 9-fluorenylmethyl carbamate and acetamide.

X ₁ is any one selected from N, O, P and S, and X ₂ is a salt, and is any one selected from sodium, magnesium, potassium and calcium ions; Any one selected from chloride, bromine, iodine, sulfonate and acetate ions.

According to a specific embodiment of the present invention, [Formula 1] to [Formula 12] may be any one selected from the following [Formula 1a] to [Formula 20a] compounds, provided that according to this, the scope of the present invention is limited it is not

[1a]

[2a]

[3a]

[4a]

[5a]

[6a]

[7a]

[8a]

[9a]

[10a]

[11a]

[12a]

[13a]

[14a]

[15a]

[16a]

[17a]

[18a]

[19a]

[20a]

In addition, another aspect of the present invention relates to an antibacterial antibiotic nanocomposite comprising the amino acid-based antimicrobial antimicrobial surfactant compound and gold (Au) or silica (SiO _{2 ) nanoparticles.}

The antibacterial antibiotic nanocomposite according to the present invention is characterized in that the gold (Au) or silica (SiO ₂ ) nanoparticles are coated with an amino acid-based antibacterial antibiotic surfactant compound to form a core-shell structure.

Hereinafter, the present invention will be described in more detail through Examples and Experimental Examples. These Examples and Experimental Examples are only for specifically illustrating the present invention, and the scope of the present invention is not limited by these Examples and Experimental Examples.

< Synthesis example 1>

To a stirred solution of D-amino acid (5 g, 56.1 mmol) in toluene (100 mL) was added dodecanol (9.42 g, 50.5 mmol) in one lot, Toluene-4-sulfonic acid monohydrate (pTSA, toluene-4) After addition of -sulfonic acid monohydrate) (11.75 g, 61.7 mmol), the temperature of the reaction mixture was slowly raised to reflux, water was azeotropically separated, and the reaction mixture was monitored by TLC. The reaction mixture was concentrated under vacuum, and the obtained residue was taken up in ethyl acetate (200 mL) _{and washed with 5% Na 2} CO ₃ (3×50 mL) aqueous solution and then with brine solution. The organic layer was dried over NaS and concentrated in vacuo to give the dodecyl compound (14.4 g, yield: 100%) as a liquid. A stirred solution of compound (5 g, 17.54 mmol) in ethylacetate/hexanes/MeOH (10:10:1 mL) was cooled to 0 °C. The reaction mixture was stirred with 1N HCl for 60 min at 50 °C, and the reaction mixture was monitored by TLC. The reaction mixture was concentrated in vacuo, and the obtained residue was hushing with ethyl acetate (3 x 52 mL) followed by hexane (5 x 50 mL) to give the wet compound (5.5 g) as a semi-solid. The semi-solid was placed in ethyl acetate/hexane (10:10 mL), heated to reflux, and stirred at reflux for 30 minutes. The reaction mixture was slowly cooled to room temperature and then cooled to 0 °C. The obtained solid was filtered under nitrogen and dried under vacuum to give the ester salt (3 g yield 53.5%) as a white hygroscopic solid.

< Synthesis example 2>

A mixture of amino acid (12.1 mmol), pTSA (2.30 g, 12.1 mmol) and alcohol (C2-C12, 14.5 mmol) in toluene (100 mL) was heated using a Dean-Stark apparatus for 48 h. The crude product was purified by column chromatography on silica gel using EtOAC/hexanes (1:2) to give the ester as an oil. The ester was then converted to the N,N-diethyl derivative using _{EtBr/K 2} CO ₃ at 90° C. in a sealed tube. The corresponding N,N-diethyl derivative with excess MeI was then synthesized by heating at 90° C. for 18 h in a sealed tube, the solvent was removed under vacuum and diethylether was added to the desired quaternary ammonium The compound was precipitated.

< Synthesis example 3>

In 100 mL of two necks round bottom flask, 21 g of D-Arginine 2HCl and CaCl ₂ After adding 20 g, 200 mL of EtOH was added, followed by stirring for 10 minutes, H ₂ SO ₄ 10 mL was added, and reflux was performed at 100 °C for 8 hours. After cooling to room temperature, the mixture was concentrated under reduced pressure to remove EtOH, and 200 mL of distilled water was added to the reaction precipitate, followed by stirring and dispersion. 52 g of NaHCO ₃ was slowly added, and then 200 mL of ethyl acetate (EA) was added and stirred, and 22 mL of lauroyl chloride was slowly added and stirred at room temperature for 3 hours. While stirring the reaction mixture, adjust the pH between 4 and 5 with concentrated hydrochloric acid, add 200 mL of EA, separate the organic layer _{, remove moisture from the organic layer with MgSO 4} , filter, and then concentrate under reduced pressure to form a white powder of Etyhl lauroyl arginate HCl (95%, 40 g).

(1) glycine ethyl ester hydrochloride (1a, Yield 97%)

¹ H NMR (DMSO- d ₆ ), δ (ppm): 1.21-25 (t, 3H, CH ₃ ), 3.74 (s, 2H, -NH-CHCOO-), 4.16-20 (t, 2H, CH _{2 )} O), 8.61 (br s, 3H, NH ₃ ⁺ ).

¹³ C NMRδ(ppm): 14.1 (CH ₃ ), 22.7-31.9 (CH ₂ ) ₉ , 36.5 (CH ₂ Ph), 54.3 (CH ), 66.6 (OCH ₂ ), 127.5, 128.8, 129.6, 134.3 (C _{6 )} H ₄ ), 169.0 (C=O).

(2) N-α-Lauroyl-D-Arginine Ethyl ester hydrochloride (2a, Yield 95%).

White solid, melting point 57 ℃, Exact MASS 384.3100 ESI-MS; m/z 385.3172 (m+H);

¹ H NMR: δH (DMSO- d ₆ ), 0.83-87 [t, 3H, (CH ₃ alkylchain)], 1.29 [m, 18H, (9CH ₂ , alkylchain)], 1.62-1.78 [m, 4H, ( -CH ₂ -CH ₂ -CONH-)], (CH ₂ -CH ₂ -NH-C(=NH)-NH ₂ )], 2.03 [s, 1H, (-NH-C(=NH)-NH ₂ )], 2.29 [t, 2H, (-CH ₂ CONH-)], 3.08-3.10 [m, 2H, (CH ₂ -NH-C(=NH)-NH ₂ )]], 4.05 to 09 [m, 2H, (-OCH ₂ -CH ₃ )], 4.18 [1H, (-NH-CHCOO-)], 7.04 to 39 [3H, (-NH-C(=NH)-NH ₂ )], 7.87 [1H, (-NH-C(=NH)-NH ₂ )], 8.27 [1H, (-NH-CH-COO)]

(3) N-α-Undecenonyl-D-Arginine Ethyl ester hydrochloride (3a, Yield 82%).

¹ H NMR: δH (DMSO- d ₆ ), 0.83-87 [t, 3H, (CH ₃ alkylchain)], 1.29 [m, 18H, (9CH ₂ , alkylchain)], 1.62-1.78 [m, 4H, ( -CH ₂ -CH ₂ -CONH-), (CH ₂ -CH ₂ -NH-C(=NH)-NH ₂ )], 2.03 [s, 1H, (-NH-C(=NH)-NH ₂ ) ], 2.29 [t, 2H, (-CH ₂ CONH-)], 3.08-3.10 [m, 2H, (CH ₂ -NH-C(=NH)-NH _{2 )], 4.05-4.09 [m, 2H,} (-OCH ₂ -CH ₃ )], 4.18 [1H, (-NH-CHCOO-)], 7.04-7.39 [3H, (-NH-C(=NH)-NH ₂ )], 7.87 [1H, (- NH-C(=NH)-NH ₂ )], 8.27 [1H, (-NH-CH-COO)]

(4) D-Prolinr dodecyl ester hydrochloride (4a, Yield 87%)

¹ H NMR (CDCl ₃ ),δ(ppm): 0.87 (t, 3H, CH ₃ ), 1.15-1.19 (t, 3H, CH ₃ ), 1.23-1.27 (m, 18H, (CH ₂ ) ₉ ), 1.56-1.73 (m, 2H, CH ₂ CH ₂ O), 3.35 (dd, H, J 15.0 Hz, 6.5 Hz, CHCHPh), 3.43 (m, 2H, CHCHPO), 4.05 (t, 2H, J 7.5CH ₂ CH ₂ O), 4.33 (t, H, J 7.0 Hz, CH), 7.26-7.29 (m, 5H, Ph), 8.61 (br s, 3H, NH ₃ ⁺ ).

¹³ C NMRδ(ppm): 14.1 (CH ₃ ), 22.7-31.9 (CH ₂ ) ₉ , 36.5 (CH ₂ Ph), 54.3 (CH ), 66.6 (OCH ₂ ), 127.5, 128.8, 129.6, 134.3 (C _{6 )} H ₄ ), 169.0 (C=O).

(5) D-N,N-dimethylpyrrolidine-2-carboxamide hydrochloride (5a, Yield 76%)

¹ H NMR (CDCl ₃ ),δ(ppm): 0.87 (t, 3H, J 7.7 Hz, CH ₃ ), 1.28 (m, 18H, (CH ₂ ) ₉ ), 1.48 (m, 2H, CH ₂ CH _{2 )} O), 3.35 (dd, H, J 15.0 Hz, J 6.5 Hz, CHCHPh), 3.43 (dd, H, J 15.0 Hz, J 6.5 Hz, CHCHPh), 4.05 (t, 2H, J 7.5CH ₂ CH ₂ O ), 4.33 (t, H, J 7.0 Hz, CH), 7.26-7.29 (m, 5H, Ph), 8.61 (brs, 3H, NH ₃ ⁺ ).

¹³ C NMRδ(ppm): 14.1 (CH ₃ ), 22.7-31.9 (CH ₂ ) ₉ , 36.5 (CH ₂ Ph), 54.3 (CH ), 66.6 (OCH ₂ ), 127.5, 128.8, 129.6, 134.3 (C _{6 )} H ₄ ), 169.0 (C=O).

(6) D-methionine dodecyl ester hydrochloride (6a, Yield 83%)

¹ H NMR (CDCl ₃ ), δ(ppm): 0.87 (t, 3H, J 7.7 Hz, CH ₃ ), 1.28 (m, 18H, (CH ₂ ) ₉ ), 1.48 (m, 2H, CH ₂ CH _{2 )} O), 3.35 (dd, H, J 15.0 Hz, J 6.5 Hz, CHCHPh), 3.43 (dd, H, J 15.0 Hz, J 6.5 Hz, CHCHPh), 4.05 (t, 2H, J 7.5CH ₂ CH ₂ O ), 4.33 (t, H, J 7.0 Hz, CH), 7.26-7.29 (m, 5H, Ph), 8.61 (brs, 3H, NH ₃ ⁺ ).

¹³ C NMRδ (ppm): 14.1 (CH ₃ ), 22.7-31.9 (CH ₂ ) ₉ , 36.5 (CH ₂ Ph), 54.3 (CH ), 66.6 (OCH ₂ ), 127.5, 128.8, 129.6, 134.3 (C _{6 )} H ₄ ), 169.0 (C=O).

(7) D-Alanine dodecyl ester hydrochloride (7a, Yield 93%)

¹ H NMR (CDCl ₃ ), δ(ppm): 0.87 (t, 3H, J 7.7 Hz, CH ₃ ), 1.28 (m, 18H, (CH ₂ ) ₉ ), 1.48 (m, 2H, CH ₂ CH _{2 )} O), 3.35 (dd, H, J 15.0 Hz, J 6.5 Hz, CHCHPh), 3.43 (dd, H, J 15.0 Hz, J 6.5 Hz, CHCHPh), 4.05 (t, 2H, J 7.5CH ₂ CH ₂ O ), 4.33 (t, H, J 7.0 Hz, CH), 7.26-7.29 (m, 5H, Ph), 8.61 (br s, 3H, NH ₃ ⁺ ).

¹³ C NMRδ(ppm): 14.1 (CH ₃ ), 22.7-31.9 (CH ₂ ) ₉ , 36.5 (CH ₂ Ph), 54.3 (CH ), 66.6 (OCH ₂ ), 127.5, 128.8, 129.6, 134.3 (C _{6 )} H ₄ ), 169.0 (C=O).

(8) D-Leucine dodecyl ester hydrochloride (8a, Yield 90%)

¹ H NMR (CDCl ₃ ), δ(ppm): 0.87 (t, 3H, J 7.7 Hz, CH ₃ ), 1.28 (m, 18H, (CH ₂ ) ₉ ), 1.48 (m, 2H, CH ₂ CH _{2 )} O), 3.35 (dd, H, J 15.0 Hz, J 6.5 Hz, CHCHPh), 3.43 (dd, H, J 15.0 Hz, J 6.5 Hz, CHCHPh), 4.05 (t, 2H, J 7.5 CH ₂ CH ₂ O ), 4.33 (t, H, J 7.0 Hz, CH), 7.26-7.29 (m, 5H, Ph), 8.61 (br s, 3H, NH ₃ ⁺ ).

¹³ C NMR δ(ppm): 14.1 (CH ₃ ), 22.7-31.9 (CH ₂ ) ₉ , 36.5 (CH ₂ Ph), 54.3 (CH ), 66.6 (OCH ₂ ), 127.5, 128.8, 129.6, 134.3 (C ₆ H ₄ ), 169.0 (C=O).

(9) D-Tyrosine dodecyl ester hydrochloride (9a, Yield 84%)

¹ H NMR (CDCl ₃ ), δ(ppm): 0.87 (t, 3H, J 7.7 Hz, CH ₃ ), 1.28 (m, 18H, (CH ₂ ) ₉ ), 1.48 (m, 2H, CH ₂ CH _{2 )} O), 3.35 (dd, H, J 15.0 Hz, J 6.5 Hz, CHCHPh), 3.43 (dd, H, J 15.0 Hz, J 6.5 Hz, CHCHPh), 4.05 (t, 2H, J 7.5 CH ₂ CH ₂ O ), 4.33 (t, H, J 7.0 Hz, CH), 7.26-7.29 (m, 5H, Ph), 8.61 (br s, 3H, NH ₃ ⁺ ).

¹³ C NMR δ(ppm): 14.1 (CH ₃ ), 22.7-31.9 (CH ₂ ) ₉ , 36.5 (CH ₂ Ph), 54.3 (CH ), 66.6 (OCH ₂ ), 127.5, 128.8, 129.6, 134.3 (C ₆ H ₄ ), 169.0 (C=O).

(10) D-Phenylalanine dodecyl ester hydrochloride (10a, Yield 89%)

¹ H NMR (CDCl ₃ ), δ(ppm): 0.87 (t, 3H, J 7.7 Hz, CH ₃ ), 1.28 (m, 18H, (CH ₂ ) ₉ ), 1.48 (m, 2H, CH ₂ CH _{2 )} O), 3.35 (dd, H, J 15.0 Hz, J 6.5 Hz, CHCHPh), 3.43 (dd, H, J 15.0 Hz, J 6.5 Hz, CHCHPh), 4.05 (t, 2H, J 7.5 CH ₂ CH ₂ O ), 4.33 (t, H, J 7.0 Hz, CH), 7.26-7.29 (m, 5H, Ph), 8.61 (br s, 3H, NH ₃ ⁺ ).

¹³ C NMR δ(ppm): 14.1 (CH ₃ ), 22.7-31.9 (CH ₂ ) ₉ , 36.5 (CH ₂ Ph), 54.3 (CH ), 66.6 (OCH ₂ ), 127.5, 128.8, 129.6, 134.3 (C ₆ H ₄ ), 169.0 (C=O).

(11) N,N-Dimethylglycine ethyl ester hydrochloride (11a, Yield 78%)

¹ H NMR (CDCl ₃ ), δ(ppm): 0.87 (t, 3H, J 7.7 Hz, CH ₃ ), 1.28 (m, 18H, (CH ₂ ) ₉ ), 1.48 (m, 2H, CH ₂ CH _{2 )} O), 3.35 (dd, H, J 15.0 Hz, J 6.5 Hz, CHCHPh), 3.43 (dd, H, J 15.0 Hz, J 6.5 Hz, CHCHPh), 4.05 (t, 2H, J 7.5 CH ₂ CH ₂ CO ), 4.33 (t, H, J 7.0 Hz, CH), 7.26-7.29 (m, 5H, Ph), 8.61 (brs, 3H, NH ₃ ⁺ ).

¹³ C NMR δ(ppm): 14.1 (CH ₃ ), 22.7-31.9 (CH ₂ ) ₉ , 36.5 (CH ₂ Ph), 54.3 (CH ), 66.6 (OCH ₂ ), 127.5, 128.8, 129.6, 134.3 (C ₆ H ₄ ), 169.0 (C=O).

(12) D-Serine dodecyl ester hydrochloride (12a, Yield 82%)

¹ H NMR (CDCl ₃ ), δ(ppm): 0.87 (t, 3H, J 7.7 Hz, CH ₃ ), 1.28 (m, 18H, (CH ₂ ) ₉ ), 1.48 (m, 2H, CH ₂ CH _{2 )} O), 3.35 (dd, H, J 15.0 Hz, J 6.5 Hz, CHCHPh), 3.43 (dd, H, J 15.0 Hz, J 6.5 Hz, CHCHPh), 4.05 (t, 2H, J 7.5 CH ₂ CH ₂ CO ), 4.33 (t, H, J 7.0 Hz, CH), 7.26-7.29 (m, 5H, Ph), 8.61 (brs, 3H, NH ₃ ⁺ ).

¹³ C NMR δ(ppm): 14.1 (CH ₃ ), 22.7-31.9 (CH ₂ ) ₉ , 36.5 (CH ₂ Ph), 54.3 (CH ), 66.6 (OCH ₂ ), 127.5, 128.8, 129.6, 134.3 (C ₆ H ₄ ), 169.0 (C=O).

(13) D-Alanine ethyl ester hydrochloride (13a, Yield 97%)

¹ H NMR (CDCl ₃ ), δ(ppm): 0.87 (t, 3H, J 7.7 Hz, CH ₃ ), 1.28 (m, 18H, (CH ₂ ) ₉ ), 1.48 (m, 2H, CH ₂ CH _{2 )} O), 3.35 (dd, H, J 15.0 Hz, J 6.5 Hz, CHCHPh), 3.43 (dd, H, J 15.0 Hz, J 6.5 Hz, CHCHPh), 4.05 (t, 2H, J 7.5 CH ₂ CO), 4.33 (t, H, J 7.0 Hz, CH), 7.26-7.29 (m, 5H, Ph), 8.61 (brs, 3H, NH ₃ ⁺ ).

¹³ C NMR δ(ppm): 14.1 (CH ₃ ), 22.7-31.9 (CH ₂ ) ₉ , 36.5 (CH ₂ Ph), 54.3 (CH ), 66.6 (OCH ₂ ), 127.5, 128.8, 129.6, 134.3 (C ₆ H ₄ ), 169.0 (C=O).

(14) D-Alanine Isopropyl ester hydrochloride (14a, Yield 93%)

¹ H NMR (CDCl ₃ ), δ(ppm): 0.87 (t, 3H, J 7.7 Hz, CH ₃ ), 1.28 (m, 18H, (CH ₂ ) ₉ ), 1.48 (m, 2H, CH ₂ CH _{2 )} O), 3.35 (dd, H, J 15.0 Hz, J 6.5 Hz, CHCHPh), 3.43 (dd, H, J 15.0 Hz, J 6.5 Hz, CHCHPh), 4.05 (t, 2H, J 7.5 CH ₂ CO), 4.33 (t, H, J 7.0 Hz, CH), 7.26-7.29 (m, 5H, Ph), 8.61 (brs, 3H, NH ₃ ⁺ ).

¹³ C NMR δ(ppm): 14.1 (CH ₃ ), 22.7-31.9 (CH ₂ ) ₉ , 36.5 (CH ₂ Ph), 54.3 (CH ), 66.6 (OCH ₂ ), 127.5, 128.8, 129.6, 134.3 (C ₆ H ₄ ), 169.0 (C=O).

(15) D-Proline Methyl ester hydrochloride (15a, Yield 96%)

¹ H NMR (CDCl ₃ ), δ(ppm): 0.87 (t, 3H, J 7.7 Hz, CH ₃ ), 1.28 (m, 18H, (CH ₂ ) ₉ ), 1.48 (m, 2H, CH ₂ CH _{2 )} O), 3.35 (dd, H, J 15.0 Hz, J 6.5 Hz, CHCHPh), 3.43 (dd, H, J 15.0 Hz, J 6.5 Hz, CHCHPh), 4.05 (t, 2H, J 7.5 CH ₂ CO), 4.33 (t, H, J 7.0 Hz, CH), 7.26-7.29 (m, 5H, Ph), 8.61 (brs, 3H, NH ₃ ⁺ ).

¹³ C NMR δ(ppm): 14.1 (CH ₃ ), 22.7-31.9 (CH ₂ ) ₉ , 36.5 (CH ₂ Ph), 54.3 (CH ), 66.6 (OCH ₂ ), 127.5, 128.8, 129.6, 134.3 (C ₆ H ₄ ), 169.0 (C=O).

(16) N-α-Lauroyl-D-Arginine Ethyl ester (16a, Yield 92%)

¹ H NMR (CDCl ₃ ), δ(ppm): 0.87 (t, 3H, J 7.7 Hz, CH ₃ ), 1.28 (m, 18H, (CH ₂ ) ₉ ), 1.48 (m, 2H, CH ₂ CH _{2 )} O), 3.35 (dd, H, J 15.0 Hz, J 6.5 Hz, CHCHPh), 3.43 (dd, H, J 15.0 Hz, J 6.5 Hz, CHCHPh), 4.05 (t, 2H, J 7.5 CH ₂ CO), 4.33 (t, H, J 7.0 Hz, CH), 7.26-7.29 (m, 5H, Ph), 8.61 (brs, 3H, NH ₃ ⁺ ).

¹³ C NMR δ(ppm): 14.1 (CH ₃ ), 22.7-31.9 (CH ₂ ) ₉ , 36.5 (CH ₂ Ph), 54.3 (CH ), 66.6 (OCH ₂ ), 127.5, 128.8, 129.6, 134.3 (C ₆ H ₄ ), 169.0 (C=O).

(17) Nitro Ethyl ester (17a, Yield 97%)

¹ H NMR (CDCl ₃ ), δ(ppm): 0.87 (t, 3H, J 7.7 Hz, CH ₃ ), 1.28 (m, 18H, (CH ₂ ) ₉ ), 1.48 (m, 2H, CH ₂ CH _{2 )} O), 3.35 (dd, H, J 15.0 Hz, J 6.5 Hz, CHCHPh), 3.43 (dd, H, J 15.0 Hz, J 6.5 Hz, CHCHPh), 4.05 (t, 2H, J 7.5 CH ₂ CO), 4.33 (t, H, J 7.0 Hz, CH), 7.26-7.29 (m, 5H, Ph), 8.61 (brs, 3H, NH ₃ ⁺ ).

¹³ C NMR δ(ppm): 14.1 (CH ₃ ), 22.7-31.9 (CH ₂ ) ₉ , 36.5 (CH ₂ Ph), 54.3 (CH ), 66.6 (OCH ₂ ), 127.5, 128.8, 129.6, 134.3 (C ₆ H ₄ ), 169.0 (C=O).

(18) (S)-3-acetyl-N-methylimidazolidine-4-carboxamide (Aza-pro-6) (18a, Yield 71%)

¹ H NMR (CDCl ₃ ), δ(ppm): 0.87 (t, 3H, J 7.7 Hz, CH ₃ ), 1.28 (m, 18H, (CH ₂ ) ₉ ), 1.48 (m, 2H, CH ₂ CH _{2 )} O), 3.35 (dd, H, J 15.0 Hz, J 6.5 Hz, CHCHPh), 3.43 (dd, H, J 15.0 Hz, J 6.5 Hz, CHCHPh), 4.05 (t, 2H, J 7.5 CH ₂ CO), 4.33 (t, H, J 7.0 Hz, CH), 7.26-7.29 (m, 5H, Ph), 8.61 (brs, 3H, NH ₃ ⁺ ).

¹³ C NMR δ(ppm): 14.1 (CH ₃ ), 22.7-31.9 (CH ₂ ) ₉ , 36.5 (CH ₂ Ph), 54.3 (CH ), 66.6 (OCH ₂ ), 127.5, 128.8, 129.6, 134.3 (C ₆ H ₄ ), 169.0 (C=O).

(19) Glycine Dodecyl ester hydrochloride (19a, Yield 95%)

¹ H NMR (CDCl ₃ ), δ(ppm): 0.87 (t, 3H, J 7.7 Hz, CH ₃ ), 1.28 (m, 18H, (CH ₂ ) ₉ ), 1.48 (m, 2H, CH ₂ CH _{2 )} O), 3.35 (dd, H, J 15.0 Hz, J 6.5 Hz, CHCHPh), 3.43 (dd, H, J 15.0 Hz, J 6.5 Hz, CHCHPh), 4.05 (t, 2H, J 7.5 CH ₂ CO), 4.33 (t, H, J 7.0 Hz, CH), 7.26-7.29 (m, 5H, Ph), 8.61 (brs, 3H, NH ₃ ⁺ ).

¹³ C NMR δ(ppm): 14.1 (CH ₃ ), 22.7-31.9 (CH ₂ ) ₉ , 36.5 (CH ₂ Ph), 54.3 (CH ), 66.6 (OCH ₂ ), 127.5, 128.8, 129.6, 134.3 (C ₆ H ₄ ), 169.0 (C=O).

(20) N,N-dimethyldodecan-1-amine (20a, Yield 93%)

¹ H NMR (CDCl ₃ ), δ(ppm): 0.87 (t, 3H, J 7.7 Hz, CH ₃ ), 1.28 (m, 18H, (CH ₂ ) ₉ ), 1.48 (m, 2H, CH ₂ CH _{2 )} O), 3.35 (dd, H, J 15.0 Hz, J 6.5 Hz, CHCHPh), 3.43 (dd, H, J 15.0 Hz, J 6.5 Hz, CHCHPh), 4.05 (t, 2H, J 7.5 CH ₂ CO), 4.33 (t, H, J 7.0 Hz, CH), 7.26-7.29 (m, 5H, Ph), 8.61 (brs, 3H, NH ₃ ⁺ ).

¹³ C NMR δ(ppm): 14.1 (CH ₃ ), 22.7-31.9 (CH ₂ ) ₉ , 36.5 (CH ₂ Ph), 54.3 (CH ), 66.6 (OCH ₂ ), 127.5, 128.8, 129.6, 134.3 (C ₆ H ₄ ), 169.0 (C=O).

<Gold nanoparticles Synthesis example >

Crush 10 g of natural product, put it in 100 mL of distilled water, heat at 70 ~ 80 ℃ for 1 hour, cool to room temperature, filter the extraction mixture under reduced pressure using 110 mm filter paper, and centrifuge at 14,000 rpm for 30 minutes. After centrifugation, the precipitate and the filtrate are separated, and the filtrate is used as a material. Store this sample at 4℃ or less and use it within 1 week.

Extract 5 mL 1 mM-HAuCl ₄ Add to 95 mL of aqueous solution and react at 70 °C for 1 hour. Observe the color change after reaction to determine whether particles are formed. The completion of the reaction is confirmed using UV, PL, FT-IR, SEM, TEM, EDS, and SEAD patterns.

1 is a synthesis result of controlling the shape and size of nanoparticles according to the present invention according to the concentration of rice bran extract, HAuCl _{4 and the presence or absence of surfactant, A) 1.0 mM Au Conc.} in H ₂ O 0.7 mL + rice bran extract 0.3 mL, B) 1.0 mM Au Conc. in H ₂ O 0.5 mL + rice bran extract 0.5 mL, C) 1.0 mM Au Conc. in H ₂ O 0.7 mL + rice bran extract 0.3 mL + surfactant 0.1 mL, D) 1.0 mM Au Conc. in H ₂ O 0.3 mL + rice bran extract 0.7 mL, E) 1.0 mM Au Conc. in H ₂ O 0.5 mL + rice bran extract 0.5 mL + surfactant 0.1 mL, F) 1.0 mM Au Conc. in H ₂ O 0.1 mL + rice bran extract 0.9 mL, G) 1.0 mM Au Conc. in H ₂ O 0.9 mL + rice bran extract 0.1 mL, H) 1.0 mM Au Conc. in H ₂ O 0.5 mL + rice bran extract 0.5 mL + NaOH 2M 1 mL.

Figure 2 is a spirulina extract, a synthesis result of controlling the shape and size of the nanoparticles according to the present invention according to the concentration of _{HAuCl 4 A) Plant extract (10%) in H 2} O, B) 10 mM-HAuCl ₄ ( 90%) in H ₂ O, C) 0.1 mmol, D) 0.5 mmol, E) 1 mmol, F) 5 mmol, G) 10 mmol.

3 is a UV confirmation result, and FIG. 4 is a PL confirmation result.

<Silica ( SiO ₂ ) Nano airgel particles (hydrophobic nanoporous silica aerogel ( HNSA ))of Synthesis example >

Silica gel60 (230 ~ 400 mesh) (1 equivalent) in distilled water, NaHCO ₃ (2.1 equivalents) was added and stirred at 50 °C for 1 hour. After the reaction, after cooling to room temperature, phosphoric acid or citric acid is added to adjust the pH to 4 ~ 5. The completion of the reaction is confirmed using UV, PL, FT-IR, SEM, TEM, EDS. 5 is a TEM image of silica nanoparticles, and FIG. 6 is a result of confirming silica nanoparticles through SEM EDS.

<Silica ( SiO ₂ ) Nano airgel Particle + Surfactant (D- LAE ) applied Synthesis example >

Silica gel60 (230 ~ 400 mesh) (1 equivalent) in distilled water, NaHCO ₃ (2.1 equivalents) was added and stirred at 50 °C for 1 hour. After the reaction, the synthesized D-LAE was added at a concentration of 0.4% of the silica solution, and then stirred for about 1 hour. After cooling to room temperature slowly, add phosphoric acid or citric acid to adjust the pH to 4 ~ 5. The completion of the reaction is confirmed using UV, PL, FT-IR, SEM, TEM, EDS. 7 is a result of confirming the TEM image and EDS of the nanocomposite particles synthesized according to the above synthesis example.

< Experimental example >

(1) Silica (SiO ₂ ) nanocomposite according to the present invention applied with D-LAE to nano airgel particles as a liquid sample LSNp preservative as shown in the figure below, respectively, for E. coli, Staphylococcus aureus and Candida respectively, 『KCL-FIR The results of the antibacterial test according to the test method of -1002:2018 are shown in [Table 1] below.

Test Items Test Methods Test result Environment initial concentration
(CFU/mL) 24 hours later
(CFU/mL) Germ
decrease rate antibacterial test
coli BLANK KCL-FIR-1002
:2018 1.8×10 ⁴ 1.2×10 ⁵ - 37±0.2
(℃) Sample LSNp 1.8×10 ⁴ <10 99.9 Staphylococcus aureus BLANK 3.5×10 ⁴ 5.9×10 ⁴ - Sample LSNp 3.5×10 ⁴ <10 99.9 candida BLANK 1.9×10 ⁴ 1.4×10 ⁵ - Sample LSNp 1.9×10 ⁴ <10 99.9

The result photos for [Table 1] are shown in Figure 8, respectively, and in [Table 1] and Figure 8, the antibacterial performance of the nanocomposite according to the present invention in which D-LAE is applied _{to the silica (SiO 2 ) nano airgel particles is} You can see it's very good.

(2) Antimicrobial activity measurement experiment - Paper disc assay (disc diffusion method)

The antibacterial activity measurement method for the composition of the present invention was a commonly used Paper disk assay (disk diffusion method), and the antibacterial activity was measured for 14 strains of the following [Table 2]. 20 ul of the culture medium cultured in the liquid medium was spread evenly on the plate medium and absorbed. After placing a sterilized 8 mm diameter sterile paper disc (Advantec paper disc) on the surface of the medium, 20 μg of the compound was applied to the sterile paper disc under aseptic conditions. After culturing according to the incubation temperature, the diameter of the inhibition zone around it including the diameter of the paper disc was measured.

division Gram Type Species strain media One negative V. cholerae N169961 ATCC 39315 LB 2 negative S. typhimurium CDC 6516-60 ATCC 14028 LB 3 negative E. coli 0157:H7 ATCC 43889 LB 4 negative E. coli 0157:H7 ATCC 43890 LB 5 negative E. coli 0157:H7 ATCC 35150 LB 6 negative S. boydii NCCP 14745 TSB 7 negative S. flexneri NCCP 14744 TSB 8 negative P. aeruginosa PA14 ATCC 15692 TSB 9 positive E. faecalis MMH594 TSB 10 positive L. monocytogenes Scott A ATCC 45954 TSB 11 positive S. mutans MT8148 TSB 12 positive S. aureus (MSSA) RN6630 TSB 13 positive S. aureus (MRSA) MW2 TSB 14 positive B. subtilis CCARM 0003 TSB

Diameter of
inhibitory
zone (mm) D-LAE L-LAE DL-LAE D-pro-dode L-pro-dode V. cholerae X X X 14.0 14.5±0.5 S. typhimurium 10.0±0.5 11.0±0.5 12.0±0.5 11.0 11.0 E. coli (43889) 11.0±0.5 11.0±0.5 10.5±0.5 10.0 10.0 E. coli (35150) 13.0±0.5 12.5±0.5 12.25±0.25 11.0 11.0 E. coli (43890) 11.25±0.25 10.5 10.5±0.5 10.0 10.0 S. boydii 11.8±0.2 11.3±0.3 11.5±0.5 X X S. flexneri 11.5±0.5 11.6±0.5 11.0 10.0 10.0 P. aeruginosa 11.0±0.5 10.6±0.6 11.0 11.75±0.25 11.0 E. faecalis 13.0 12.7±0.3 12.7±0.3 13.75±0.25 13.0 L. monocytogenes 15.0±0.5 14.5±0.5 14.0±0.5 14.5±0.5 13.0 S. mutans 16.0±0.5 15.5±0.5 15.5±0.5 16.0 15.0 S. aureus (MSSA) 16.5±0.5 16.5±0.5 16.25±0.25 14.0 13.5±0.5 S. aureus (MRSA) 15.5±0.5 15.5±0.5 15.5±0.5 13.0 12.0 B. subtilis 14.25±0.25 13.0±0.5 13.0±0.5 12.5 12.0

Diameter of
inhibitory
zone (mm) D-meth
-dode D-ala
-dode D-leu
-dode D-tyr
-dode D-phala
-dode V. cholerae X 13.0 11.0 X X S. typhimurium X 11.0 X X X E. coli (43889) X 11.5±0.5 X 10.5±0.5 X E. coli (35150) X 11.0 X X X E. coli (43890) X 11.0 X 10.0 10.0 S. boydii X 10.0 X X X S. flexneri X 10.0 X X X P. aeruginosa X 10.0 X X X E. faecalis 10.0 13.0 11.0 X 10.0 L. monocytogenes 10.0 12.0 11.0 X X S. mutans 10.0 15.5±0.5 12.5 X X S. aureus (MSSA) 10.0 13.0 11.0 X X S. aureus (MRSA) 10.0 14.5±0.5 11.0 X X B. subtilis X 10.0 10.0 X X

In the case of the Diameter of inhibitory zone (mm) in [Table 3], in the case of a compound having antibacterial activity of each strain, it shows activity of a certain diameter, however, in the case of D, L, DL type LAE marked with X, V. cholerae did not show antibacterial activity, and in the case of D and L-type pro-dode, it did not show any activity against S. boydii. [Table 4] Again, the part marked with X is a case in which antibacterial activity is not shown for each strain.

As shown in [Table 3], the compounds D-LAE, L-LAE, and DL-LAE exhibited the highest antibacterial activity, and it can be confirmed that the antibacterial activity of Gram positive bacteria is particularly high.

In addition, as shown in [Table 4], while D-aladode showed antibacterial activity against Gram positive and Gram negative bacteria, it can be confirmed that D-methodode and D-leudode had antibacterial activity only against Gram positive bacteria.

(3) Minimum inhibitory concentration (MIC) measurement

By performing growth curves of various bacteria, the minimum inhibitory concentration (MIC) of the compound according to the present invention was measured through a 2-fold serial dilution method. Cultures of bacteria were diluted 100-fold in 96-well plates, and compounds were aliquoted at various concentrations. Only DMSO as a control and gentamicin as a positive control, bacterial growth was measured after 24 hours. All experiments were repeated three times. The lower the MIC value, the higher the antibiotic sensitivity, and the higher the antibiotic sensitivity, the higher the antibacterial activity.

MIC (ug/mL) D-LAE L-LAE DL-LAE D-pro-dode L-pro-dode V. cholerae X X X 25-50 50 S. typhimurium 25 50 25-50 50 50 E. coli (43889) 25-50 50 50 50 50 E. coli (35150) 25 50 50 25 25-50 E. coli (43890) 25 25 25 50 50 S. boydii 25 50 50 X X S. flexneri 50 50 50 100 100 P. aeruginosa 12.5 12.5 12.5 50 50-100 E. faecalis 25 25 25 25 25-50 L. monocytogenes 12.5 12.5-25 12.5-25 25-50 25-50 S. mutans 12.5 12.5 12.5 12.5 12.5 S. aureus (MSSA) 6.25 6.25 6.25 12.5-25 25 S. aureus (MRSA) 3.13 6.25 6.25 25 25 B. subtilis 12.5 12.5 12.5 50 50

MIC (ug/mL) D-meth-
dode D-ala-
dode D-leu-
dode D-tyr-
dode D-phala-
dode Gentamicin V. cholerae X 50 X X X 12.5-25 S. typhimurium X 50 X X X 25 E. coli (43889) X 100 X X X 6.25 E. coli (35150) X 25 X X X 12.5 E. coli (43890) X 25-50 X X X 6.25 S. boydii X 50 X X X 6.25 S. flexneri X 50 X X X 6.25 P. aeruginosa X 50 X X X 25 E. faecalis X 25 X X X X L. monocytogenes X 25-50 X X X 3.13 S. mutans X 12.5 X X X 50 S. aureus (MSSA) X 12.5-50 X X X 1.56 S. aureus (MRSA) X 12.5 X X X 1.56 B. subtilis X 100 X X X 3.13

The [Table 5] and [Table 6] are results showing the MIC of the compound according to the present invention, and the part marked with X is a case where the MIC value is 256 ug/mL or more, or does not show antibacterial activity.

As shown in [Table 5], the compounds D-LAE, L-LAE, and DL-LAE showed the highest antibacterial activity, and in particular, the antibacterial activity of Gram positive bacteria containing methicillin-resistant Staphylococcus aureus (MRSA) was high. It can be seen that the highest

In addition, as shown in [Table 6], only D-aladode showed antibacterial activity against Gram positive and Gram negative bacteria.

(4) MBC (Minimum Bactericidal Concentration) Minimum Bactericidal Concentration Analysis

For MIC analysis, the cultured medium was visually observed, and a medium in which the growth of the strain was completely inhibited was selected and spread on a 96-well plate. The number of live bacteria in the plated medium was measured. Like the MIC, the lower the MBC level, the higher the antibiotic sensitivity and the higher the antibacterial activity.

MBC (ug/mL) D-LAE L-LAE DL-LAE D-pro-dode L-pro-dode V. cholerae X X X 50 50 S. typhimurium 25 50 50 50 50 E. coli (43889) 50 100 50 50 50 E. coli (35150) 50 50 50 50 25 E. coli (43890) 25 25 25 50 50 S. boydii 50 50 100 X X S. flexneri 50 100 50 100 100 P. aeruginosa 50 100 50 50 100 E. faecalis 50 50 25 25 50 L. monocytogenes 25 25 25 50 50 S. mutans 50 50 50 12.5 12.5 S. aureus (MSSA) 25 50 25 25 25 S. aureus (MRSA) 12.5 50 25 25 25 B. subtilis 12.5 12.5 12.5 50 50

MBC (ug/mL) D-meth-
dode D-ala-
dode D-leu-
dode D-tyr-
dode D-phala-
dode V. cholerae X 50 X X X S. typhimurium X 50 X X X E. coli (43889) X X X X X E. coli (35150) X 25 X X X E. coli (43890) X 50 X X X S. boydii X 50 X X X S. flexneri X 50 X X X P. aeruginosa X 50 X X X E. faecalis X 50 X X X L. monocytogenes X 50 X X X S. mutans X 25 X X X S. aureus (MSSA) X 50 X X X S. aureus (MRSA) X 50 X X X B. subtilis X X X X X

The [Table 7] and [Table 8] are the results showing the MBC of the compound according to the present invention, and the part marked with X is the case where the MBC value is 256 ug/mL or more, or does not show antibacterial activity .

(5) The strain was used for the test by adjusting the standard turbidity Mcfarland (0.08 ~ 0.1 = 1 × 10 ⁸ CFU/mL), and the concentration of the test sample was 4, 8, 16, 32, 64, 128, 256, 512 ug/mL was prepared and used, and the test sample was diluted with sterile LB BROTH. As for the concentration of the strain used in the test, a strain adjusted to standard turbidity was ^{used to prepare 1 × 10 6} CFU/mL, and E-coli (ATCC 8739) was used as the strain. The medium used was BD DIFCO's LB BROTH MILLER, all sterilized and applied to the test, and the micro plate reader was PERKIN ELMER's VICTORX3 model.

For the test method, 50 uL of each test sample is added to a 96-well plate, ^{and 50 uL of a strain with a concentration of 1 × 10 6} CFU/mL is added and diluted, diluted to 1/2 the concentration of each test sample and strain, and cultured for 24 hours. did. The concentration of the test sample is 2, 4, 8, 16, 32, 64, 128, and 256 ug/mL, respectively, and the concentration of the strain is 5 × 10 ⁵ CFU/mL. A strain having a concentration of 5 × 10 ⁵ CFU/mL was used as a negative control, and sterilized LB BROTH was cultured together as a positive control and used for measurement. The turbidity was measured by measuring the cultured test sample at 570 nm with a micro plate reader.

(6) When the MICs of D-type LAE and L-type LAE were compared in E. coli, it was increased 4 times compared to L-type, and it was confirmed that the properties changed only by changing the chirality in the same structure, which can be confirmed in FIG. 9 .

(7) As a result of confirming the MIC in E. coli for D-type LAE and DL-type LAE, the activity can be confirmed at the same MIC concentration (32 ug/mL), but the difference in absorbance at different concentrations (16 ug/mL) is 2 It was confirmed that the ship is flying, and this can be confirmed in FIG. 10 .

(8) As a result of confirming the MIC of D, DL, and L-type EUA, it was confirmed that the absorbance constantly increased in the order of L, DL, and D-type at the same MIC (64 ug/mL), which can be confirmed in FIG. can

(9) As a result of salt treatment with HCl by substituting a Dodecyl group for the carboxyl acid of D-type proline and D-type alanine, the difference in MIC values was more than 8 times, and in the case of D-type proline, the same MIC as D-type LAE was observed. was confirmed, and this can be confirmed in FIG. 12 .

(10) The following FIG. 13 is a result of evaluation of the chirality of LAE and toxicity in kidney and keratinocytes by concentration. The cytotoxicity evaluation method using this Cell Counting Kit-8 (CCK-8) is shown in FIG. 14 as an iterative procedure.

A more detailed manipulation procedure for cell counting and cell proliferation and cytotoxicity analysis of the CCK-8 kit is as follows.

- Cell number determination protocol

1) Put cell suspension (100 μl/well) in 96-well plate. Also prepare wells containing a known number of viable cells (draw the calibration curve in step 5). Pre-incubate the plate in a humidified incubator (37 °C, 5% CO _{2 ).}

2) Thaw CCK-8 at 37°C if frozen on a tabletop or in a water bath.

3) Add 10 μl of CCK-8 solution to each well of the plate.

4) Incubate the plate for 1-4 hours in the incubator.

5) Measure absorbance at 450 nm using a microplate reader, and prepare a calibration curve using data obtained from wells containing a known number of viable cells.

- Cell proliferation and cytotoxicity assay protocol

1) Dispense 100 μl of cell suspension (5000 cells/well) in 96-well plate.

2) Pre-incubate the plate for 24 h in a humidified incubator (37 °C, 5% CO _{2 ).}

3) Add 10 μl of various concentrations of toxic substances to the culture medium of the plate.

4) Incubate the plate for an appropriate time (24, 48, 72 h) in the incubator.

5) Thaw CCK-8 at 37°C if frozen on the bench top or water bath.

6) Add 10 μl of CCK-8 solution to each well of the plate.

7) Incubate the plate for 1-4 h in the incubator. Measure absorbance at 450 nm using a microplate reader.

<Analysis of microbial images using nanoparticles>

(1) After applying the compound (surfactant) according to the present invention to the selected active substance at a concentration of 0.1 to 1 mM gold particles of 10 to 20 nm or less, treated with E. Absorption and action sites were confirmed using TEM. It can be seen that the surfactant compound is applied to the gold nanoparticles through FIGS. 15 and 16 below. In addition, it can be confirmed through the TEM EDX of FIG. 17 that the nanocomposite synthesized according to the present invention is applied to the gold nanoparticles.

(2) As described above, it can be confirmed through the TEM of FIG. 18 that the nanocomposite synthesized by applying the compound to gold nanoparticles according to the present invention has absorption and effectiveness in microorganisms.

(3) In addition, it can be confirmed through the MIC results of FIGS. 19 and 20 that the effect of the nanocomposite synthesized by applying the compound to gold nanoparticles according to the present invention is increased when absorbed into microorganisms.

Claims (3)

An amino acid-based antibacterial antibiotic surfactant compound represented by the following [Formula 1] to [Formula 12]:
[Formula 1] [Formula 2]

[Formula 3] [Formula 4]

[Formula 5] [Formula 6]

[Formula 7] [Formula 8]

[Formula 9] [Formula 10]

[Formula 11] [Formula 12]

In the [Formula 1] to [Formula 12],
R _{1 is any one selected} from hydrogen, alkyl, carboxylic acid, amine, alcohol, guanidine, phenyl, phenol, indole, imidazole and D-glucose,
R ₂ is any one selected from hydrogen, alkyl, D-glucose, t-butyl carbamate, benzyl carbamate, 9-fluorenylmethyl carbamate and acetamide;
R ₃ is any one selected from t-butyl carbamate, benzyl carbamate, 9-fluorenylmethyl carbamate and acetamide,
X ₁ is any one selected from N, O, P and S,
X ₂ is a salt, and is any one selected from sodium, magnesium, potassium and calcium ions; Any one selected from chloride, bromine, iodine, sulfonate and acetate ions. According to claim 1,
The following [Formula 1] to [Formula 12] is an amino acid-based antibacterial antibiotic surfactant compound, characterized in that any one selected from compounds:
[1a]

[2a]

[3a]

[4a]

[5a]

[6a]

[7a]

[8a]

[9a]

[10a]

[11a]

[12a]

[13a]

[14a]

[15a]

[16a]

[17a]

[18a]

[19a]

[20a]

The amino acid-based antibacterial antibiotic surfactant compound according to _{claim 1} and gold (Au) or silica (SiO 2 ) nanoparticles,
An antibacterial antibiotic nanocomposite, characterized in that the gold (Au) or silica (SiO ₂ ) nanoparticles are coated with an amino acid-based antibacterial antibiotic surfactant compound to form a core-shell structure.

Images