THEREOF

RELATED PATENT APPLICATION(S)

This application is a divisional application of Indian Patent application number 202041023813 dated June 06, 2020.

FIELD OF INVENTION

The invention relates to a method of synthesis of trimeric amino acid conjugate of trimeric amino acid conjugate of Tris(hydroxymethyl)Phosphine (THP). The present invention further relates to a one pot synthesis of the Novel amino acid conjugate of THP. It further relates to the metal nanoparticles stabilized by biopolymer, preferably silver nanoparticles prepared by reduction of silver salt with A conjugate of THP as reducing agent. Additionally the invention relates to silver nanoparticles stabilized by the biopolymer as antimicrobial agent.

BACKGROUND OF INVENTION

Despite the ever-growing percentage of antibiotic-resistant bacterial pathogens isolated from hospitals from all across the world, fundamental and applied research campaigns for the discovery of novel drug leads to remedy these bacterial resistant infections are utterly failing to produce sufficient leads to combat this deadly public health problem. This shortfall is not so much due to lack of effort by screening programs, which continue to screen hundreds of thousands of small molecules for antibacterial activity. Complicated organic synthesis has often resulted poor yields of useful antibiotic compounds making commercial viability less attractive to pharmaceutical companies involved in antibiotic drug development. The fast mutation of bacteria to existing and third/fourth generation new antibiotics is further exacerbating the scientific difficulties, money and time investment required to bring novel antibiotic products to market.

Background art discloses as well as explored silver nanoparticles for their antimicrobial properties. Silver has been known to possess strong antimicrobial properties. Silver ions such as silver nitrate and sulfadiazine had been used for the treatment of burns, wounds and several bacterial infections. Their use was largely discontinued in the 1940s, due to the development of modern antibiotics and side effects due to the presence of ionic silver. Silver nanoparticles have unique optical and electrical properties. Colloidal silver is one of the mostly used substrates for Surface Enhanced Raman Spectroscopy (SERS) for single molecule detection. The highly reflective silver nanoparticles have also been used in metal film plasmonic solar cells to improve the conversion efficiency from photos to electrons. The capability of making highly conductive traces and films at low temperatures is of enormous commercial interest to the electronics industry. Silver nanoparticles can be produced by various processes such as chemical reduction of silver salts in an aqueous and organic solution, radiation assisted- chemical and photoreduction in reverse micelles, thermal decomposition of silver compounds, evaporation and condensation of silver metal, etc. The chemical reduction methods are based on reduction of silver salt with a number of reducing agents including sodium citrate, sodium borohydride, hydroxylamine hydrochloride, hydrazine, and ethylenediaminetetraacetic acid (EDTA), ascorbic acid, polyol, etc. A stabilizing agent needs to be added to the reaction mixture to prevent the aggregation of the silver nanoparticles formed unless the reducing agent itself is a stabilizing agent (such as citrate).

US20190000759 discloses an antimicrobial gel comprising nano-sized particles of metallic silver (Ag), a polymer comprising carboxylate groups, carboxylate molecules comprising at least one group capable of binding to Ag, and metal ions, where the gel is useful as a topically applied antimicrobial agent. The carboxylate molecules are selected from 6-mercaptohexanoic acid, 8- mercaptooctanoic acid, mercaptosuccinic acid, 4-mercaptobenzoic acid, 4-mercaptophenylacetic acid, lipoic acid, dihydrolipoic acid, glutathione, penicillamine, 5-(4-amino-6-hydroxy-2-mercapto- 5-pyrimidinyl)pentanoic acid, and 2-mercapto-4-methyl-5-thiazoleacetic acid. It is preferably a topical anti-bacterial agent.

WO2014052973A1 disclosed a method of making silver nanoparticles, using an ascorbic acid derivative or an alpha-hydroxyl carboxylic acid derivative as a reducing agent. The silver nanoparticles may be coated onto micro particles, embedded in hydrogel particles or coated with polysaccharide. The silver nanoparticles may be used in a wound dressing, a bandage, a fungal treatment product, a deodorant, a floss product, a toothpick, a dietary supplement, dental X-ray, a mouthwash, a toothpaste, acne or wound treatment product, skin scrub, and skin defoliate agent.

US8563020B2 discloses a composition having antimicrobial activity comprising particles comprising at least one inorganic copper salt; and at least one functionalizing agent in contact with the particles, the functionalizing agent stabilizing the particle in a carrier such that an anti- microbially effective amount of ions are released into the environment of a microbe. The average size of the particles ranges from about 1000 nm to about 4 nm. Preferred copper salts include copper iodide, copper bromide and copper chloride. Preferred functionalizing agents include amino acids, thiols, hydrophilic polymers emulsions of hydrophobic polymers and surfactants.

US9005663B2 discloses method of making silver nanoparticles, by reacting silver salt with a phosphene aminoacid. The silver nanoparticles are prepared with trimeric amino acid conjugate of alanine and have an average size of 10-20 nm.

Genevieve et al., (2014) disclosed antibacterial activity and inhibition of biofilm formation of silver nanoparticles (AgNPs) against Escherichia coli (MG 1655), Bacillus subtilis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and Janthinobacterium lividum. The AgNPs utilized in this study were prepared through one-pot microwave-assisted syntheses guided by principles of green chemistry. The AgNPs were synthesized in three different schemes by reducing Ag ⁺ ions (from AgNCh) with reducing agents dextrose, arabinose, and soluble starch. Formation of AgNPs occurred in less than 15 min, and nanoparticles had diameters of 30 nm or less. [ACS Sustainable Chem. Eng., 2014, 2 (4), pp 590-598, Investigation of Antibacterial Activity by Silver Nanoparticles Prepared by Microwave-Assisted Green Syntheses with Soluble Starch, Dextrose, and Arabinose].

There is an attempt in the present invention to provide metal nanoparticles stabilized by polymers as anti-microbial agents. The present invention discloses the method of formation of metal nanoparticles with anti-microbial activity and high stability, by formation using natural biopolymers as stabilizing agents. Further, the present invention also discloses a method of synthesis of novel phosphine amino acid conjugate of THP or tris(hydroxymethyl)phosphine (THP).

Present invention explores the possibility of metal nanoparticles and exemplary silver nanoparticles on their efficacy against a broad spectrum of bacterial and fungal pathogens viz Candida albicans, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Enterobacter areogenes, Acinetobacter baumannii, Bacillus subtilis and the like. OBJECTS OF INVENTION

It is a primary object of the invention to provide a process for preparation of novel phosphine amino acid conjugate (Katti Peptide).

It is another object of the invention to provide a process for preparation of amino acid conjugates of Tris(hydroxymethyl)Phosphine (THP).

It is another object of the invention to provide a synthetic route to ‘Katti Peptide’ without the use of triethylamine.

It is another object of the invention to provide a synthetic route to ‘Katti Peptide’ which is environmentally friendly, industrially more convenient and safer alternative for THP synthesis.

It is another object of the present invention to provide metal nanoparticles with anti-microbial activity and high stability

It is another object of the present invention discloses a method of synthesis of novel phosphine amino acid conjugate of THP or tris(hydroxymethyl)phosphine (THP).

It is another object of the present invention to provide a one pot method of synthesis of the Novel amino acid conjugate of THP without usage of triethylamine.

It is another object of the present invention to provide metal nanoparticles stabilized by biopolymer.

It is another object of the present invention to provide silver nanoparticles prepared by reduction of silver salt with a conjugate of THP as reducing agent.

It is another object of the present invention to provide silver nanoparticles stabilized by the biopolymer as antimicrobial agent.

It is another object of the present invention to provide antimicrobial nanoparticles with activity against a broad spectrum of bacterial and fungal pathogens. SUMMARY OF THE INVENTION

Thus, according to the present invention, there is provided a process for preparation of amino acid conjugates of Tris(hydroxymethyl)Phosphine (THP) of Formula 1, 2 or 3 or pharmaceutically acceptable salts thereof, comprising of Steps:

Formula (1 ) Formula (2) Formula (3) reacting an aqueous solution of tetrakis(hydroxymethyl)phosphonium hallide (THPX) with a base to yield a reaction mixture;

ΊΉRC base THP addition of an amino acid solution to the reaction mixture to yield the monomeric (Formula 1), dimeric (Formula 2) or trimeric amino acid conjugate of Formula (3)

Formula 3 wherein the amino acid is added in a stoichiometric ratio of 1-3 moles to yield monomeric aminoacid conjugate (Formula 1) at a stoichiometric concentration of 1 mole, dimeric amino acid (Formula 2) at a stoichiometric concentration of 12 moles and trimeric amino acid conjugate (Formula 3) at a stoichiometric concentration of 3 moles. It is another aspect of the present invention to provide a process for preparation of amino acid conjugates of Tris(hydroxymethyl)Phosphine (THP) given in Formula 1, 2 or 3 or pharmaceutically acceptable salts, wherein addition of base to tetrakis(hydroxymethyl)phosphonium hallide (THPX) is at a temperature range of 23-25°C under Nitrogen or any other noble gas with stirring.

It is another aspect of the present invention to provide a process for preparation of amino acid conjugates of Tris(hydroxymethyl)Phosphine (THP) given in Formula 1, 2 or 3 or pharmaceutically acceptable salts thereof, wherein addition of amino acid solution to the reaction mixture is performed at a temperature of 23-25°C, for a time period of 1.5-2.5 hours under Nitrogen.

It is another aspect of the present invention to provide a process for preparation of amino acid conjugates of Tris(hydroxymethyl)Phosphine (THP) of Formula 1, 2 or 3 or pharmaceutically acceptable salts thereof, wherein A is the residue of an amino acid.

It is another aspect of the present invention to provide a process of preparation of a monomeric, dimeric and trimeric amino acid conjugate compound given in Formula 1, 2 or 3 or pharmaceutically acceptable salt thereof as, wherein the residue of amino acid is an alkylidenyl or substituted alkylidenyl residue of glycine, alanine, serine, threonine, valine, leucine, isoleucine, phenylalanine or tryptophan.

It is another aspect of the present invention to provide a process of preparation of amino acid conjugates of Tris(hydroxymethyl)Phosphine (THP) given in Formula 1, 2 or 3 or pharmaceutically acceptable salts thereof as claimed in claim 1, wherein the residue (A) is an alkylidenyl or substituted alkylidenyl residue of Glycine, Arginine or Alanine.

It is another aspect of the present invention to provide a process for preparation of amino acid conjugates of Tris(hydroxymethyl)Phosphine (THP) given in Formula 1, 2 or 3 or pharmaceutically acceptable salts thereof, wherein the halide in tetrakis(hydroxymethyl)phosphonium halide is selected from Cl, Br, F, or I.

It is another aspect of the present invention to provide a process for preparation of amino acid conjugates of Tris(hydroxymethyl)Phosphine (THP) given in Formula 1, 2 or 3 or pharmaceutically acceptable salts thereof, wherein the base is selected from Sodium Hydroxide (NaOH) and Potassium hydroxide (KOH).

It is another aspect of the present invention to provide a process for preparation of amino acid conjugates of Tris(hydroxymethyl)Phosphine (THP) given in Formula 1, 2 or 3 or pharmaceutically acceptable salts thereof, wherein the pH of the reaction mixture ranges from 4.0 to 7.0.

It is another aspect of the present invention to provide a process for preparation of amino acid conjugates of Tris(hydroxymethyl)Phosphine (THP) given in Formula 1, 2 or 3 or pharmaceutically acceptable salts thereof, wherein the base is dissolved in a solvent comprising aprotic or polar solvent.

It is another aspect of the present invention to provide a metal nanoparticle composition comprising: metal nanoparticles and biopolymer, wherein the metal nanoparticles comprises nanoparticles as colloidal solution obtained from the reduction of a metal salt with a reducing agent and wherein the stabilizing agent is biopolymer, and the reducing agent is amino acid conjugates of Tris(hydroxymethyl)Phosphine (THP) given in Formula 1, 2 or 3.

It is yet another aspect of the present invention wherein the metal nanoparticle composition comprises of any natural biopolymer, preferably selected from Gum arabic.

It is yet another aspect of the present invention wherein the metal nanoparticle composition comprises of metal selected from one or more of Silver, Gold, Palladium, Platinum, Zinc and Iron, most preferably silver.

It is another aspect of the present invention wherein the antimicrobial metal nanoparticle composition of an amino acid conjugate, wherein A the residue of an amino acid in the Trimeric amino acid conjugate of Tris(hydroxymethyl)Phosphine (Formula 3) is an alkylidenyl or substituted alkylidenyl residue of glycine, alanine, serine, threonine, valine, leucine, isoleucine, phenylalanine or tryptophan.

It is another aspect of the present invention to provide a process of preparing a metal nanoparticle composition, comprising of steps: preparation of an aqueous solution of trimeric amino acid conjugate compound of formula 3 of 0.1 M concentration, by dissolving in water; preparation of a 0.1M concentration of metal salt solution; dissolving polymer in water by continuous stirring at a temperature of 90-100°C to yield polymer solution; addition of metal salt solution (0.1M) to the biopolymer solution at 100°C; addition of aqueous solution of amino acid conjugate compound of formula 1 (0.1M) to the polymer solution with metal salt solution to yield a mixture; heating of the mixture for 5-15 minutes at 80-100°C and removing of the heat, and stirring of the mixture for a time period of 60-90 min to yield metal nanoparticles stabilized by biopolymer, wherein the aqueous solution of trimeric amino acid conjugate compound (Formula 3) is reduces the metal salt to metal nanoparticles, and the ratio of metal salt solution to aqueous amino acid solution is 5:1 by volume.

It is another aspect of the present invention to provide a process of preparing a metal nanoparticle composition, wherein A the residue of an amino acid in the trimeric amino acid conjugate compound of formula 3 is an alkylidenyl or substituted alkylidenyl residue of glycine, alanine, serine, arginine, threonine, valine, leucine, isoleucine, phenylalanine or tryptophan and the metal salt solution is 0.1 M silver nitrate.

It is another aspect of the present invention to provide a process of preparing a metal nanoparticle composition, wherein A is residue of an amino acid and is an alkylidenyl or substituted alkylidenyl residue of glycine, arginine or alanine.

It is another aspect of the present invention to provide a metal nanoparticle composition, wherein the silver nanoparticle has antimicrobial activity is against Gram positive and Gram negative bacteria.

It is another aspect of the present invention to provide a silver nanoparticle composition, wherein the minimum inhibitory concentration when A the residue of amino acid is alkylidenyl or substituted alkylidenyl residue of glycine is 5 to 20 pg/ml It is another aspect of the present invention to provide a silver nanoparticle composition, wherein the minimum inhibitory concentration when A the residue of amino acid is alkylidenyl or substituted alkylidenyl residue of Arginine is 0.63 to 20 pg/ml.

It is another aspect of the present invention to provide a silver nanoparticle composition, wherein the minimum inhibitory concentration when A the residue of amino acid is alkylidenyl or substituted alkylidenyl residue of Alanine is 5-10 pg/ml.

BRIEF DESCRIPTION OF DRAWINGS:

The annexed drawings show an embodiment of the present invention, wherein:

FIGURE 1: Formation of Silver Nanoparticles (X) and their stabilization by biopolymers, Gum Arabic stabilized Al-NP’s (X-GA).

FIGURE 2: 3 IP NMR of THPA1 in D ₂ 0.

FIGURE 3: 1H NMR of THPA1 in D ₂ 0 (before drying so methanol seen).

FIGURE 4: 13C NMR (not calibrated) of THPA1 in D20 (before drying so methanol seen). FIGURE 5: TEM image of Gum Arabic stabilized Arginine katti peptide nanoparticle.

FIGURE 6: illustrates inhibition of the respiratory chain of bacteria by the biopolymer stabilized metal nanoparticles.

FIGURE 7: Antibacterial efficacy of Alanine katti peptide silver nanoparticle against Bacteria gram-negative Escherichia coli.

FIGURE 8: Antibacterial efficacy of Alanine katti peptide silver nanoparticle against Bacteria gram-negative Pseudomonas aeruginosa.

FIGURE 9: Antibacterial efficacy of Alanine katti peptide silver nanoparticle against Bacteria gram-negative Enterobacter aerogenes.

FIGURE 10: Antibacterial efficacy of Alanine katti peptide silver nanoparticle against Bacteria gram-negative Acinetobacter baumannii

FIGURE 11: Antibacterial efficacy of Alanine katti peptide silver nanoparticle against Bacteria gram-positive Staphylococcus aureus.

FIGURE 12: Antibacterial efficacy of Alanine katti peptide silver nanoparticle against grampositive Staphylococcus epidermidis

FIGURE 13: Antibacterial efficacy of Alanine katti peptide silver nanoparticle against Bacteria gram-positive Bacillus subtilis. FIGURE 14: Antibacterial efficacy of Alanine katti peptide silver nanoparticle against Bacteria gram-positive Enterococcus faecalis .

FIGURE 15: Antibacterial efficacy of Alanine katti peptide silver nanoparticle against opportunistic pathogenic yeast Candida albicans.

DETAILED DESCRIPTION OF THE INVENTION ACCOMPANYING FIGURES

An exemplary embodiment of the present invention discloses an antimicrobial metal nanoparticle composition comprising of metal nanoparticles stabilized by a biopolymer and the formation of the metal nanoparticles by reduction of a metal salt with a reducing agent, resulting in nanoparticles. The resultant colloidal solution of metal nanoparticles is stable and exhibit antimicrobial property against a plethora of gram positive and gram negative bacteria.

The Alanine Katti Peptide (Citation by the National Academy of Inventors, 2014) was first reported by the Katti group in 2003. Addition of 1 equivalence of tris(hydroxymethyl)phosphine (THP) to 3 equivalence of the amino acid alanine (D or L isomer) yields the Alanine Katti Peptide, a Mannich- type condensation product. The starting material THP can be obtained commercially (Acros Organics or Sigma- Aldrich) and costs between $25 and $60 per gram. THP can also be synthesized in the laboratory using tetrakis(hydroxymethyl)phosphonium chloride (THPC1). It is a much cheaper starting material in comparison to THP. THPC1 can also be obtained commercially (Sigma-Aldrich, Fisher Scientific or Cytec Canada Inc.) and costs between 27 cents and 44 cents per gram. THPC1 decomposition by a base like sodium hydroxide (NaOH) or triethylamine (NEt3) can be utilized for cost-effective THP synthesis as shown in the reaction scheme. NEt3 has a strong fishy odor similar to ammonia and is unpleasant to work with. Triethylamine is a federal hazardous air pollutant and was identified as a toxic air contaminant. There was some evidence of carcinogenic activity in female mice exposed to triethylamine based on increased incidences of hepatocellular adenoma. Exposure to triethylamine by dermal application resulted in increased incidences of eosinophilic focus of the liver in males and females. Dosed mice developed treatment-related non-neoplastic lesions at the site of application. ECOTOXICITY STUDIES: Triethylamine may produce potential acute, sub-chronic and chronic toxicity effects in aquatic species. It is also a skin and eye irritant and causes respiratory distress.

Therefore, it was important to develop a synthetic route to ‘Katti Peptide’ without the use of triethylamine. Our invention uses sodium hydroxide (NaOH) in place of the toxic triethylamine as an environmentally friendly and industrially more convenient and safer alternative for THP synthesis. The Alanine Katti Peptide synthesis starting from THPC1 entails an additional step, cost and time in the overall process as well. Therefore, the feasibility of a one pot synthesis was explored utilizing 1 equivalence of THPC1, 1 equivalence of NaOH and 3 equivalence of alanine. This proved to be a cost effective one step synthesis with excellent yield (~90 %) and purity. With no remnants of starting materials, solvent or oxidized impurities after work-up this one pot synthesis is a fairly straight forward reaction carried out in environmentally benign water as a solvent. The process also showed excellent scalability (from few grams to quarter kilogram) and is commercially viable.

The invention further discloses a process of synthesis of the metal nanoparticles wherein the reducing agent is a phosphene amino acid and preferably a conjugate of amino acid of phosphine. The invention specifically discloses a novel method of synthesis of amino acid conjugate of THP tetrakis(hydroxymethyl)phosphonium chloride (THPC1) exemplified by compound of Formula 3.

Metal Nanoparticles and method of preparation:

Process of synthesis of the metal nanoparticles wherein the reducing agent is a phosphene amino acid and preferably a conjugate of amino acid of phosphine (Formula 1, 2 and 3). Background art discloses traditional methods of product of metal nanoparticles using harmful chemicals, like hydrazine, sodium borohydrates, and DMF or dimethyl formamide. They lack in both being environmentally toxic and harsh on handling. The object of the present invention is to prepare metal nanoparticles, the phosphene amino acid conjugate preferably is a trimeric amino acid conjugate consisting of trimeric amino acid and one phosphine group. Metal-containing nanoparticles refer to metallic silver nanoparticles, silver-containing alloy nanoparticles, metallic gold nanoparticles, palladium nanoparticles, Zinc nanoparticles, platinum and Iron. Preferably the metal nanoparticles are silver nanoparticles exhibiting anti-microbial activity. The nanoparticles are further stabilized by stabilizing agents for improved shelf life. The compositions can be employed in the form of particles, films, coatings, fibers, or bulk materials. The compositions can be used for inhibiting microorganism growth or killing microorganisms.

Present invention relates to a process of synthesis of the metal nanoparticles comprising: reacting a metal compound or a metal salt with a reducing agent comprising an amino acid conjugate of phosphine, in the presence of a stabilizer in a reaction mixture comprising the metal salt/ compound, the reducing agent, the stabilizer, an aqueous solvent preferably water, to form a plurality of metal- containing nanoparticles with molecules of the stabilizer on the surface of the metal-containing nanoparticles. The metal is preferably Silver as silver nanoparticles exhibit excellent anti-microbial activity. The stabilizer is aqueous soluble and is preferably Gum arabic. Amino acids are selected from one or more of the L-amino acid is selected from a group comprising of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalaine, proline, serine, threonine, tryptophan, tyrosine, and valine.

SYNTHESIS OF BIOPOLYMER STABILIZED METAL NANO PARTICLES I. One Pot Synthesis of Amino acid Conjugate of THP from Tetrakis (hydroxymethyl)phosphonium chloride (THPC1) and Sodium Hydroxide (NaOH)

An embodiment of the present invention discloses a synthetic route to Amino acid conjugate of THP (Formula 3) also referred here as ‘Katti Peptide’ here without the use of triethylamine. The present invention uses sodium hydroxide (NaOH) in place of the toxic triethylamine as an environmentally friendly and industrially more convenient and safer alternative for THP synthesis. Process for preparation of amino acid conjugates of Tris(hydroxymethyl)Phosphine (THP) of Formula 1, 2 or 3 or pharmaceutically acceptable salts thereof, comprising of Steps:

1. Reacting an aqueous solution of tetrakis(hydroxymethyl)phosphonium hallide (THPX) with a base to yield a reaction mixture; x= Cl, F, I

2. Addition of an amino acid solution to the reaction mixture to yield the monomeric (Formula 1), dimeric (Formula 2) or trimeric amino acid conjugate of Formula (3)

amino acid is added in a stoichiometric ratio of 1-3 moles to yield monomeric amino acid conjugate (Formula 1) at a stoichiometric concentration of 1 mole, dimeric amino acid (Formula 2) at a stoichiometric concentration of 2 moles and trimeric amino acid conjugate (Formula 3) at a stoichiometric concentration of 3 moles. Addition of base i.e. NaOH to tetrakis(hydroxymethyl)phosphonium hallide (THPX) is at a temperature range of 23-25°C under Nitrogen or any other noble gas with stirring. The base is chosen from sodium hydroxide or potassium hydroxide. Here A is the residue of an amino acid and is an alkylidenyl or substituted alkylidenyl residue of glycine, alanine, serine, threonine, valine, leucine, isoleucine, phenylalanine or tryptophan. According to an exemplified embodiment of the present invention A is an alkylidenyl or substituted alkylidenyl residue of Glycine, Arginine or Alanine. Example 1 discloses A as alanine and is referred as Alanine Katti Peptide. Where A is Glycine it is referred as Glycine Katti Peptide, and where A is Arginine it is referred as Arginine Katti Peptide. THPX, X i.e the halide in tetrakis(hydroxymethyl)phosphonium halide is selected from Cl, Br, F, or I. Exemplified embodiments disclose it as THPC1. Base dissolved in aprotic or polar solvent, preferably water is added drop wise to a solution of THPX or THPC1 at 23-25 degree centigrade under an atmosphere of Nitrogen gas or nobel gas. This yields a reaction mixture comprising of THP, sodium chloride, methanol and water. pH of the reaction mixture ranges from 4.0 to 7.0. The base is dissolved in a solvent comprising aprotic or polar solvent. The amino acid is dissolved in water or aprotic solvent and was added drop- wise to the reaction mixture. Excess solvent is removed under vacuum to obtain straw color solution. It was kept in ice bath at 0°C, then layered with alcohol preferably methanol about 20-50 ml and left overnight to yield a white solid and clear solution, which is filtered and dried to yield THP-AA or (Amino acid conjugate of THP).

EXAMPLE 1: Katti peptide Alanine

Synthesis of Amino acid Conjugate of THP: Amino acid is Alanine.

Materials and Methods

All reactions were carried out under nitrogen atmosphere using standard Schlenk line techniques. 80% aqueous solution of tetrakis(hydroxymethyl)phosphonium chloride (THPC1, density = 1.341 g/mol) was obtained from Cytec Canada Inc. L-alanine (Al) was purchased from Acros Organics. Sodium hydroxide (NaOH) was purchased from Fisher Scientific. All reagents were used without further purification. Degassed (by bubbling nitrogen) ultrapure water (18.2 MW·ah at 25 °C, Millipore Corporation) and methanol (HPLC grade, Fisher Scientific) were used as solvents. 1H, 13C, and 3 IP Nuclear magnetic resonance (NMR) spectra were recorded at 300, 75 and 121 MHz, respectively on a Bruker ARX300 spectrometer. The 1H chemical shifts are reported relative to internal D2O and 3 IP NMR chemical shifts are reported relative to an external standard of 85% H3P04.lt is here referred as Alanine Katti peptide.

Reaction Scheme for Alanine Katti Peptide Synthesis from THP The synthesis of amino acid conjugate of THP (Formula 3) where the A is an alkylidenyl or substituted alkylidenyl residue of glycine, alanine, serine, threonine, valine, leucine, isoleucine, phenylalanine or tryptophan residue of an amino acid. Preferably, residue (A) is an alkylidenyl or substituted alkylidenyl residue of Glycine, Arginine or Alanine. When Alanine, herein referred as Alanine Katti Peptide, and when arginine referred as Arginine Katti Peptide.

In the synthesis of Alanine Katti Peptide, starting from THPC1 entails an additional step, cost and time in the overall process as well. Therefore, the feasibility of a one pot synthesis was explored utilizing 1 equivalence of THPC1, 1 equivalence of NaOH and 3 equivalence of Alanine. This proved to be a cost effective one step synthesis with excellent yield (~90 %) and purity. With no remnants of starting materials, solvent or oxidized impurities after work-up this one pot synthesis is a fairly straight forward reaction carried out in environmentally benign water as a solvent. The process also showed excellent scalability (from few grams to quarter kilogram) and is commercially viable.

One Pot synthesis of Alanine Katti Peptide:

NaOH (1.295g, 32.4 mmol) dissolved in 20 mL water was added drop wise to 5.2 ml of 80% THPC1 (5.579 g, 29.3 mmol) at 25 °C under stirring and nitrogen atmosphere. Then (L)-alanine (7.811 g, 87.7 mmol) dissolved in 55 mL of water was added to the reaction mixture dropwise. It was then stirred under nitrogen for 2 hours. The reaction mixture pH was ~4.5. Excess solvent was then removed in vacuo to obtain ~20 ml of clear straw colored solution. This was kept in an ice bath (0 °C), then layered with ~30 ml methanol and left overnight to yield a white solid and a clear solution. The white solid was then filtered off and washed with methanol (5 X -lOrnl) and dried in vacuo to obtain 8.576 g of pure THPA1 with 87% yield.

Reaction Scheme for One Pot Synthesis of Alanine Katti Peptide

Alanine Katti Peptide (THPA1) Mass Spectrometry (ESI-MS) and Nuclear Magnetic Resonance (NMR) Characterization

Molecular Formula: C12H24N306P Theoretical m/z = 337.30 Observed m/z = 337.30

Table 2: Mass spectrometry results of Katti Peptide-Alanine

Synthesis of metal nanoparticles of Alanine Katti Peptide: A process of preparing a metal nanoparticle composition, comprising of steps:

Preparation of an aqueous solution of trimeric amino acid conjugate compound of formula 3 of 0.1M concentration, by dissolving in water. Preparation of a 0.1M concentration of metal salt solution, dissolving polymer in water by continuous stirring at a temperature of 90-100°C to yield polymer solution; addition of metal salt solution (0.1M) to the biopolymer solution at 100°C, addition of aqueous solution of amino acid conjugate compound of formula 1 (0.1M) to the polymer solution with metal salt solution to yield a mixture, heating of the mixture for 5-15 minutes at 80- 100°C and removing of the heat, and stirring of the mixture for a time period of 60-90 min to yield metal nanoparticles stabilized by biopolymer, wherein the aqueous solution of trimeric amino acid conjugate compound of formula 3 reduces the metal salt to metal nanoparticles, and the ratio of metal salt solution to aqueous amino acid solution is 5:1 by volume. Preferably the biopolymer is Gum Arabic.

Example 2: (Figure 1) Katti peptide alanine silver nano synthesis Katti peptide silver nano synthesis: To a 20-ml vial, added 12mg of Gum Arabic and 6ml of DI water and stir it on a pre -heated (100 °C) hot plate. Once the GA solution reaches 100°C, added 100 micro liters of 0.1M solution of AgNCh (16.9 mg/ml) and then added 20 micro liters of pre-made 0.1M (33.7mg/ml, molecular weight of THP Alanine (Katti peptide - Alanine) is 337gm) solution (clear solution) of THPAlanine (Katti peptide - Alanine). Heated the solution for 10 more minutes, then turn off the heating but continue stirring for 60-90 minutes on the hot plate.

Characterization of Alanine Katti Peptide Silver nanoparticles stabilized by Gum Arabic is illustrated in Table 1A.

Table 1A: Alanine Katti peptide silver nanoparticles stabilized by Gum arabic

TEM images show that silver nanoparticles of Katti peptide Alanine are spherical in shape with the size range of 20-30 nm (Table 1A).

EXAMPLE 3: Synthesis of Katti peptide Arginine One Pot synthesis of Arginine Katti Peptide:

NaOH (1.295g, 32.4 mmol) dissolved in 20 mL water was added drop wise to 5.2 ml of 80% THPC1 (5.579 g, 29.3 mmol) at 25 °C under stirring and nitrogen atmosphere. Then (L)-Arginine (7.811 g, 44.9 mmol) dissolved in 55 mL of water was added to the reaction mixture drop wise. It was then stirred under nitrogen for 2 hours. The reaction mixture pH was ~4.5. Excess solvent was then removed in vacuo to obtain ~20 ml of clear straw colored solution. This was kept in an ice bath (0 °C), then layered with ~30 ml methanol and left overnight to yield a white solid and a clear solution. The white solid was then filtered off and washed with methanol (5 X ~10ml) and dried in vacuum to obtain 7.72 g of pure THPAr with 87% yield.

Reaction Scheme Arginine Katti Peptide (THPAr) Mass Spectrometry (ESI-MS) and Nuclear Magnetic Resonance (NMR) Characterization

Molecular Formula: C21H45N1206P Theoretical m/z = 593.34 Observed m/z = 593.34 Table 2: Mass spectrometry results of Katti Peptide- Arginine

Example 4

Katti peptide Arginine silver nano synthesis

Synthesis of Gum Arabic stabilized THP-Ar silver nanoparticles (GA T.Arg AgNPs) To a 20 mL vial, 12 mg of Gum Arabic (VI) was dissolved in 6 mL of doubly ionized water. The reaction mixture was stirred continuously at 90-100°C. To the hot stirring mixture, 100 pL of 0.1M AgN0 ₃ was added and 40 pL of 0.1M THP Arginine thereafter. The color of the solution turned to dark brown from colorless in about 5 minutes indicating the formation of silver nanoparticle formation. The reaction was stirred at 60-65°C for 10 minutes and at room temperature for 60-90 minutes. Then the nanoparticles were characterized by UV-Vis spectroscopy, zeta-sizer and transmission electron microscopy (TEM) analysis (Figure 5).

Characterization of Arginine-Katti peptide Silver nanoparticles stabilized by Gum Arabic.

Table 1: Arginine katti peptide silver nanoparticles stabilized by Gum arabic

TEM images show that silver nanoparticles of Katti peptide Arginine are spherical in shape with the size range of 3-8 nm (Table 1).

Antimicrobial efficacy of silver nanoparticles:

The detailed antibacterial results as outlined in this invention show that the natural biopolymers, Gum Arabic encapsulated metal nanoparticles, herein exemplified as silver nanoparticles (Formula X-GA as illustrated in Figure 1) show antibacterial efficacy far superior than the well-known antibiotics currently used in treating bacterial infections in humans as well as those currently used cosmetics products in treating dandruff and various skin/dermal microbes.

Silver nanoparticles as outlined in this invention have demonstrated minimum inhibitory concentrations (MIC) and Minimal Bactericidal Concentration (MBC: lowest concentration of silver nanoparticles to prevent growth) in the range 0.3 to 10 micrograms/ml thus provide first scientific evidence on the realistic applications of nanoayurvedic medicine -based silver nanoparticles as a new generation of antibiotics for treating various common and deadly infections in humans and animals. The highly potent nano-ayurvedic-medicine based silver nanoparticles have also provided the first scientific evidence for their use in the development of a new generation of antibacterial consumer products in various sectors including cosmetics/beauty products, maintaining deadly bacteria free environment in house hold, public spaces and hospitals.

Mechanism of Antibacterial / Antifungal activity of the Silver Nanoparticles positive and gram-negative bacteria,

Exemplary silver nanoparticles of the present invention are multipronged and multimodal which eliminate possibilities for the bacteria to mutate and become drug resistant. The silver nanoparticles possess active surface allowing for efficient reactions with nucleophilic amino acid residues with surface proteins, which are inherent with all the pathogens (gram positive and gram-negative bacteria). In addition, silver has the highest affinity to bond and react with soft sulfur functionalities of sulphydryl groups of proteins present on the bacterial surface thus leading to instant death of deadly bacteria. Additionally, the highly reactive silver atoms of the silver nanoparticles interact with the plethora of amino, imidazole, phosphate and carboxyl groups present on and within the bacterial domains causing multi-pronged choking effects-thus leaving no chance for the bacterial survival. These multipronged interactions of silver nano-particulate surface with bacterial cell surface functionalities, singularly or collectively, cause bacterial cell wall damage and disruption of cytoplasmic membrane leading to leaching of metabolites, direct interference with DNA synthesis, concomitantly causing denaturing of bacterial proteins and enzymes (dehydrogenases). The series of silver nanoparticle interactions with bacterial domains cause binding to ribosomes thus inhibiting protein synthesis, and also thereby interfering with electron transport in cytochrome system which is involved in the production of ROS (reactive oxygen species). Figure 22 shows mechanism of bacterial cell death due to the accumulation of the silver nanoparticles antibiotics agents (KPR-AgNP) within the cell membrane. Encapsulation of the silver nanoparticles with gum arabic has proven to be effective process to rapidly increasing cell permeability and ultimately, leading to bacterial cell death. Figure 6 shows the cumulative effects of various interactions of the silver nanoparticles, as described above, to effectively attack the respiratory chain in bacterial mitochondria thus causing the complete bacterial cell death in various gram positive and gram-negative bacteria. Respiratory machinery of pathogenic cells is vitally important for bacterial cell metabolic activity as it drives obtaining energy to perform all the energy -demanding life processes. Energy generation relies on the respiratory enzyme complexes associated with the respiratory chain. The ability of silver nanoparticulate surface to react with soft sulfur functionalities of sulphydryl groups of proteins, present on the bacterial surface, as well as the highly reactive silver atoms of our silver nanoparticles which interact with the plethora of amino, imidazole, phosphate and carboxyl groups present on and within the bacterial domains synergistically cause multi-pronged choking effects on the respiratory mechanism thus leaving no chance for the bacterial survival. It is also known that binding silver atoms with amino acid functional groups of pathogenic enzymes inhibit the efficient electron transport via the respiratory chain. These effects hinder electron transport and thus result in blockage of phosphorylation of ADP to ATP. Sustained release of silver ions by the silver nanoparticles inside the bacterial cells (in an environment with lower pH) will create cascade of free radicals and thus induce oxidative stress causing enhancement in bactericidal activity. Overall, there are no antibiotic agents in background art, which are able to act in a multipronged and multimodal fashion similar to that is illustrated in Figure 6. Therefore, the silver nanoparticles of the present invention are capable of destroying plethora drug resistant bacteria. The multimodal bacterial killing mechanisms provide compelling rationale that our antibiotic technology will not allow bacteria to mutate and will therefore establish new industry standards in antibiotic products for treating deadly bacterial infections in human patients. Our new antibiotic technology is also highly relevant toward the development of a new generation of antibacterial consumer products in various sectors including cosmetics/beauty products, maintaining deadly bacteria free environment in house hold, public spaces and hospitals.

METHOD:

Determination of Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC)

A broth micro-dilution methodology was used to assess the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of silver nanoparticles as exemplary compounds in solution. Solution growth for MIC determinations was quantified by optical density (600 nm).

RESULTS: (Example 5 to Example 13)

1. Obtained isolated colonies of bacterial strains to test.

2. Combined 4-5 colonies and culture overnight in rich media broth.

3. After overnight incubation rich broth with appropriate dilution series of test antibiotic to test tubes is added. The bacteria are inoculated to a final density of 5x105 cfu/ml.

4. Plane aliquot of growth control (i.e., no antibiotic added) to verify cfu/ml counts of viable bacteria. It is incubated overnight and colonies were counted

5. After overnight incubation the cultures are checked for growth. The MIC is the lowest concentration of antibiotic that prevents visible growth.

No visible growth for each silver NPs compounds were subcultured out onto petrifilms and incubated overnight to determine the MBC (i.e., lowest concentration of silver nanoparticles to prevent growth).

Panel of Microorganism used:

1. Candida albicans ATCC 10231

2. Escherichia coli ATCC 12435

3. Pseudomonas aeruginosa ATCC 15442

4. Staphylococcus aureus ATCC 25923

5. Staphylococcus epidermidis ATCC 35984

6. Enterococcus faecalis ATCC 29212

7. Enterobacter areogenes ATCC 29212

8. Acinetobacter baumannii ATCC 19606 9. Bacillus subtilis ATCC 6051

RESULTS

Example 5: Antibacterial efficacy of Gum Arabica stabilized Katti Peptide Alanine silver nanoparticles on gram-negative Bacteria Escherichia coli as Illustrated in Figure 7 and Table 3.

Figure 7 illustrates the antibacterial efficacy against gram-negative Bacteria Escherichia coli.

Table 3: Antibacterial efficacy against gram-negative Bacteria Escherichia coli

Example 6:

Antibacterial efficacy of Gum Arabica stabilized Katti Peptide Alanine silver nanoparticles on gram-negative Bacteria Pseudomonas aeruginosa as Illustrated in Figure 8 and Table 4.

Table 4. Antibacterial efficacy against gram-negative Bacteria Pseudomonas aeruginosa.

Example 7 :

Antibacterial efficacy of Gum Arabica stabilized Katti Peptide Alanine silver nanoparticles on gram-negative Bacteria Enterobacter aerogenes as illustrated in Figure 9 and Table 5 Figure 9. Antibacterial efficacy against gram-negative Bacteria Enterobacter aerogenes Table 5. Antibacterial efficacy against gram-negative Bacteria Enterobacter aerogenes

Example 8:

Antibacterial efficacy of Gum Arabica stabilized Katti Peptide Alanine silver nanoparticles against gram-negative Bacteria Acinetobacter baumannu as illustrated in Figure 10 and table 6. Figure 10. Antibacterial efficacy against gram-negative Acinetobacter baumannu

Table 6. Antibacterial efficacy against gram-negative Bacteria Acinetobacter baumannu

Example 9:

Antibacterial efficacy Antibacterial efficacy of Gum Arabica stabilized Katti Peptide Alanine silver nanoparticles against gram-positive Bacteria Staphylococcus aureus as illustrated in Figure 11 and Table 7.

Figure 11. Antibacterial efficacy against gram-positive Bacteria Staphylococcus aureus Table 7: Antibacterial efficacy against gram-positive Bacteria Staphylococcus aureus Example 10:

Antibacterial efficacy of Gum Arabica stabilized Katti Peptide Alanine silver nanoparticles against gram-positive Bacteria Staphylococcus epidermidis as illustrated in Figure 12 and Table 8 Figure 12: Antibacterial efficacy against gram-positive Bacteria Staphylococcus epidermidis Table 8: Antibacterial efficacy against gram-positive Bacteria Staphylococcus epidermidis

Example 11:

Antibacterial efficacy of Gum Arabica stabilized Katti Peptide Alanine silver nanoparticles against gram-positive Bacteria Bacillus subtilis as illustrated in Figure 13 and Table 9 Figure 13: Antibacterial efficacy against gram-positive Bacteria Bacillus subtilis

Table 9. Antibacterial efficacy against gram-positive Bacteria Bacillus subtilis.

Example 12: Antibacterial efficacy of Gum Arabica stabilized Katti Peptide Alanine silver nanoparticles against gram-positive Bacteria Enterococcus faecalis as illustrated in Figure 4 and Table 10. Figure 14: Antibacterial efficacy against gram-positive Bacteria Enterococcus faecalis Table 10. Antibacterial efficacy against gram-positive Bacteria Enterococcus faecalis

Example 13:

Antifungal efficacy of Gum Arabica stabilized Katti Peptide Alanine silver nanoparticles against opportunistic pathogenic yeast Candida albicans as illustrated in Figure 15 and Table 11. Figure 15: Antifungal efficacy against Yeast Candida albicans Table 11. Antifungal efficacy against Yeast Candida albicans