ES2281274A1 - Antimicrobial cyclic peptides - Google Patents

Abstract

The invention relates to novel cyclic peptides with antimicrobial properties. The inventive peptides contain an even number of between 4 and 10 amino acids and a have a molar lysine concentration of between 40 % and 60 %. The invention also relates to the synthesis and use of the aforementioned peptides as antimicrobial agents against bacterial and fungi that are pathogenic to plants. The invention further relates to compositions containing said peptides and an auxiliary agent and to a method for preventing and treating infections and diseases in plants caused by pathogenic bacteria and fungi.

Description

Antimicrobial cyclic peptides.

Technical field

The present invention relates to peptides cyclics that are have antimicrobial properties, and that are particularly effective against pathogenic bacteria and fungi to the plants.

Prior art

Agriculture is a process for production of food by growing certain plants. The passage of the subsistence agriculture in which only what was produced necessary to meet the needs of the farmer and his family, to industrial agriculture that implies the existence of large cultivated extensions, has led to the need for control plant diseases effectively to large scale.

A common problem in agriculture is the reduction in yield caused by attacks by pathogenic microorganisms for plants, for example bacteriosis caused by the bacteria Erwinia amylovora, Pseudomonas syringae, Xanthomonas vesicatoria , or Clavibacter michiganensis , or mycosis caused by various fungi as Fusarium oxysporum .

Currently in Europe they are only authorized some products for plant protection against bacterial diseases based mainly on derivatives Cupric with insufficient control efficacy.

Antibiotics, such as kasugamycin or streptomycin, which until a few years ago were authorized in Some countries are currently not authorized in Europe. The antibiotics streptomycin and tetracycline are authorized in countries like the US, but in many cases it has revealed the emergence of resistant strains of pathogens that make their use ineffective, or that may involve a risk of transfer of said resistance to other bacteria pathogens In the case of fungal mycoses phytopathogens, although there are active substances available, continually require new fungicides, partly because of the appearance of resistances with a certain frequency.

In addition, many of the antifungal compounds and most effective known antibacterials have persistence in the environment that is not desirable.

Another way to control diseases caused by plant pathogenic microorganisms it consists of the use of compounds isolated from the own nature, for example peptides, which are present in plants and animals, and some of which have properties antimicrobial

In Broekaert et al , Plant. Physiol., 1995, 108, 1353-1358, describes a family of peptides called plant defensins that have a high antifungal activity. Said peptides are constituted by sequences comprising between 45 and 54 amino acids, and have disulfide bridges formed between two cysteine amino acids located at different points of the structure.

In the patent application WO-A-95/11306 describes a peptide of 30 amino acids isolated from beet, which has properties bactericides and fungicides against pathogenic microorganisms of vegetables.

In patent application WO-A-99/59412 a peptide extracted from Poenibacillus polymyxa is described which inhibits fungal diseases in plants. Said peptide consists of a ring formed by six amino acids, and a side chain formed by two amino acids.

In the patent application WO-A-97/30082 describes the application for the use in agriculture of a peptide formed by 25 amino acids isolated from a scorpion.

Another way to act against microorganisms Plant pathogens consist of the use of compounds resulting from the modification of peptides or proteins antimicrobials isolated from nature. For example how to described in:

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the patent application EP-A-0472987, which refers to linear peptides of 23 amino acids, which are modifications of natural peptides belonging to the group of the magainin,

-

the patent application WO-A-02/06324, which refers to linear peptides of 44 amino acids derived from natural peptide called heliomycin, or

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the US patent US6015941, which refers to linear peptides formed by 17 amino acids derived from taquiplesin.

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As described in WO-A-96/008264, one of the problems of the use of antifungal and antibacterial compounds of natural origin is the limitation in terms of quantities commercials available, in part due to procurement problems and purification.

Another approach to dispose of peptides Antimicrobials consist of preparing them synthetically.

In the context of the development of peptides with broad spectrum antimicrobial activity, in the application for WO-A-98/03192 patent are described cyclic peptides formed by a number of amino acids comprised between 10 and 30, which can be applied for the treatment of infections and microbial diseases in plants caused by bacteria and fungi Peptides formed by sequences of 10 amino acids comprise combinations of amino acids: alanine, arginine, cysteine, phenylalanine, glycine, leucine, lysine, Ornithine, proline, serine, tyrosine, threonine, tryptophan, valine and D-arginine However, activity tests antimicrobial are only performed against pathogenic bacteria to the human being, and its activity against bacteria and / or is not described pathogenic fungi for plants.

Within the framework of designing new antimicrobial peptides, in Frecer et al , Antimicrob. Agents Chemother., 2004, 48, 3349-3357, peptides consisting of 19 amino acids are described, which comprise the glycine-serinaglycine amino acid cluster at the center of the molecule, and have a cyclic structure due to the formation of a disulfide bridge between the Cysteine amino acids that are in the terminal position. These peptides have a good efficacy against gram-positive bacteria, which makes them susceptible to being applied in medicine, but their activity against fungi or pathogenic bacteria of plants is not described.

The need to have compounds that are effective against infections and diseases of plants caused by microorganisms such as bacteria and fungi, and that are easy to synthesize.

The authors of the present invention have developed peptides that are easy to prepare and that they have a high efficacy to inhibit the growth of pathogenic microorganisms of plants.

Object of the invention

The object of the present invention is provide cyclic peptides that have properties antimicrobial

Another object of the invention is the use of said peptides as antimicrobial agents.

It is also the object of the invention a phytosanitary composition comprising the peptides of the invention.

It is also part of the object of the invention a method to prevent and treat infections and diseases of the Plants caused by bacteria and fungi.

Description of the figures

Figure one

Figure 1 shows the structure of the 56 cyclic decapeptides belonging to the prepared library using the Multipin ™ procedure, in which they are used SynPhase? Lanterns marketed by the company Mimotopes (Australia), as solid support.

Detailed description of the invention Peptides

The object of the present invention is provide cyclic peptides that have a molar content of lysine between 40% and 60%, and that respond to the general formula (I):

(I) cycle [(X) n - Y - (X) m - Gln)]

where:

X is Lys or Leu,

And it's Phe or D-Phe,

n = 1, 3, or 5,

m = 1, 3, or 5, and

n + m = 2, 4, 6 or 8.

The term "cycle" found at principle of the amino acid sequence in the general formula (I) means that the peptide is cyclic, so that the group α-carboxylic acid amino acid Gln is linked through a peptide bond with the group α-amino of the first amino acid X belonging to the group (X) n.

Abbreviations used for amino acids in this description they follow the regulations of the Commission for the Biochemical Nomenclature of the IUPAC-IUB, as described in J. Biol. Chem., 1972, 247, 977-983. From this way, Lys means L-lysine, Leu is L-leucine, Phe is L-phenylalanine, D-Phe is D-phenylalanine, and Gln is L- glutamine

Compounds that belong to this group of Peptides have antimicrobial activity.

This activity is particularly effective against pathogenic microorganisms of plants such as the fungus Fusarium oxysporum , and the bacteria Erwinia amylovora, Clavibacter michiganensis, Xanthomonas vesicatoria , and Pseudomonas syringae .

Preferably the peptides respond to the general formula (I):

(I) cycle [(X) n - Y - (X) m - Gln]

where:

X is Lys or Leu,

And it's Phe, and

m = 1, and n = 1, or

m = 3, and n = 1, 3, or 5.

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More preferably the peptides respond to the general formula (I):

(I) cycle [(X) n - Y - (X) m - Gln]

where:

X is Lys or Leu,

And it's Phe, and

m = 3, and n = 5.

Among the peptides of the invention that result be preferred are those defined by the sequences of amino acids SEQ_ID_NO: 1 to SEQ_ID_NO: 23, and whose structure is Sample in Table I:

TABLE I

one

2

The peptides object of the invention are cyclic and comprise between 4 and 10 amino acids, and have a molar content of lysine between 40% and 60%.

This means that in the case of a peptide of 4, 6 or 8 amino acids, the peptide contains two, three or four lysine residues respectively, which represents 50% molar of Lysine in all cases.

In the case of a 10 amino acid peptide, the peptide may contain 4, 5 or 6 lysine residues, which responds respectively 40%, 50% or 60% molar lysine.

To describe the structure of the peptides of the invention will be used as an example the peptide cycle (Lys-Phe-Lys-Gln), which corresponds to the general formula (I) in which n = m = 1, n + m = 2, X is Lys, and Y is Phe.

It is a cyclic peptide comprising 4 amino acids. The peptide bonds that form the structure of this peptide in the direction from left to right are the following:

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He α-carboxylic group of the first lysine is bound with the α-amino group of the phenylalanine

-

He α-carboxylic group of phenylalanine is linked to the α-amino group of the second lysine

-

He α-carboxylic group of the second lysine is bound to the α-amino group of the glutamine

-

He α-carboxylic group of glutamine forms a peptide bond with the α-amino group of the first lysine, thus closing the ring.

A characteristic of the peptides of the Invention is its cyclic structure. As described in WO-A-98/03192, cyclic peptides they are more resistant to proteolytic processes compared to its linear counterparts. This way you can extend more time its presence in the plant and also its antimicrobial activity. This fact does not translate into a negative persistence in the middle environment, since these peptides eventually degrade by the usual biological routes.

In the general formula (I) of the peptides of the In the present invention, amino acid Y can be Phe or D-Phe, that is, L-phenylalanine or D-phenylalanine

As described in WO-A-96/08264, the introduction of D-series amino acids in a peptide contribute to increase the stability of said peptide, since it is less sensitive to routes biological factors that degrade the amino acids of the L series. However, there are other alternative routes that even degrade amino acids from the D series, so this greater stability does not imply a persistence of said peptides in the environment.

The peptides described in the invention have a length between 4 and 10 amino acids that makes them appropriate to be prepared using the procedures usual solid phase peptide synthesis. Among sayings Procedures can be mentioned:

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he resin using procedure 4-methylbenzydrylamine (MBHA) functionalized with amino groups, as a solid support to carry out the reactions successive coupling between the different amino acids that they integrate the peptide and the cyclization stage, and

-

the Multipin? process, in which the SynPhase? Lanterns marketed by the company Mimotopes (Australia) are used. On the website http://www.mimotopes.com/ , information on this technique can be found.

Both procedures are well known by the expert in the field and are described for example in S.A. Kates, F. Albericio Eds., Solid-Phase Synthesis. A practical guide, Marcel Dekker, New York, 2000 (ISBN: 0-8247-0359-6), or in  K. Burguess Eds., Solid-Phase Organic Synthesis, Wiley, John & Sons, New York, 1999 (ISBN: 0471318256).

In the technique that uses MBHA resin as solid support, a resin is usually incorporated into the resin acid-sensitive bifunctional spacer, which allows the de-anchoring of the peptide synthesized under mild conditions.

For example an appropriate bifunctional spacer is the Fmoc-Rink-Linker (CAS number: 145469-56-3) marketed by the Senn Chemicals company (Switzerland).

Said spacer, once you have joined with your carboxylic group to an amino group of the MBHA resin, has a free amino group that allows the anchoring of an amino acid that has a free carboxylic group.

The commercial SynPhase? Lanterns used are of the Rink amida type and have already incorporated this bifunctional spacer.

If the amino acid has a carboxylic group free in the side chain, such as glutamic acid or aspartic acid, then it can be attached to the solid support to through said side chain, keeping the other available carboxylic group and the amino group for the formation of new peptide bonds.

The Multipin ™ technique can be used to building peptide libraries so very convenient, as described in the Examples.

A procedure for the preparation of peptides of the invention may be for example the one described in continuation.

In this procedure the chemistry of the Fmoc group (9-fluorenylmethoxycarbonyl) as α-amino group protector of amino acids that are used to prepare the peptides of the invention.

In the first stage we proceed to anchor the glutamic acid to the resin, or to SynPhase? Lantern, to through the free carboxylic group of its side chain. The others two reactive groups of glutamic acid remain protected to avoid side reactions: for example, the group α-carboxylic acid in the form of an allyl ester, and the  α-amino group protected by the group Fmoc.

The protective group is then removed Fmoc of the α-amino group of glutamic acid, and the next amino acid of the peptide having the α-amino group protected by the Fmoc group. He link is established between the carboxylic group of the new amino acid and the glutamic acid amino group.

This process of eliminating the protective group Fmoc and the incorporation of the new amino acid is repeated until it is complete a peptide with a linear structure.

In the case of the amino acid lysine, the amino group of the side chain is protected by the Boc group (t-butyloxycarbonyl), which is stable at conditions of elimination of the Fmoc group.

Once the couplings are finished, it is eliminated the allyl ester that protects the group α-carboxylic acid glutamic acid, and delete the Fmoc group that protects the group α-amino of the last amino acid Incorporated.

Then the cyclization between the α-carboxylic group of glutamic acid and the α-amino group of the last amino acid Incorporated. In this way a cyclic peptide bound to the solid support, either resin or SynPhase ™ Lantern.

The cleavage of the solid support peptide is performs in acidic conditions and implies the breaking of the link between the amino group and the benzidrile group of the linker, leading to the formation of the glutamine residue (Gln), which is present in the peptide of the invention, as can be seen in the formula general (I). At the same time that the support peptide is released Solid Boc groups are removed from lysine residues.

After a conventional isolation process, the Peptides of the invention are obtained in good yield, usually between 70% and 80%, in the form of powdery solids, and are characterized by analysis by HPLC, mass spectrometry (ESI-MS, MALDI-TOF or FAB-MS).

Surprisingly, it has been found that the peptides of the invention have an antimicrobial activity, which is particularly effective against pathogenic microorganisms of plants such as the fungus Fusarium oxysporum , and the bacteria Erwinia amylovora, Clavibacter michiganensis, Xanthomonas vesicatoria , and Pseudomonas syringae .

Also part of the invention is the use of cyclic peptides that have a molar content of lysine between 40% and 60% and that respond to general formula (I):

(I) cycle [(X) n - Y - (X) m - Gln]

where:

X is Lys or Leu,

And it's Phe or D-Phe,

n = 1, 3, or 5,

m = 1, 3, or 5, and

n + m = 2, 4, 6 or 8

as agents antimicrobials

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Preferably the peptides respond to the general formula (I):

(I) cycle [(X) n - Y - (X) m - Gln]

where:

X is Lys or Leu,

And it's Phe, and

m = 1, and n = 1, or

m = 3, and n = 1, 3, or 5

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More preferably the peptides respond to the general formula (I):

(I) cycle [(X) n - Y - (X) m - Gln]

where:

X is Lys or Leu,

And it's Phe, and

m = 3, and n = 5

Among the peptides of the invention that result be preferred the peptides defined by the sequences are counted of amino acids SEQ_ID_NO: 1 to SEQ_ D_NO: 23.

Preferably the peptides of the invention are used as antimicrobial agents against bacteria and fungi pathogens for plants.

Preferably, the pathogenic bacteria for the plants are selected from the group consisting of Erwinia amylovora, Clavibacter michiganensis, Xanthomonas vesicatoria , and Pseudomonas syringae .

These bacteria turn out to be indicators appropriate for determining the antimicrobial activity of compounds that are likely to be used to fight infections and diseases caused by bacteria in plants.

Erwinia amylovora is a gram-negative bacterium that causes the disease known as Bacterial Fire that affects plants of the Rosaceae family, among which are fruit trees of great economic importance such as apple and pear, and ornamental plants such as rowan , hawthorn, mustard, etc. In Europe it is listed as a quarantine bacterium in agriculture. Bacterial fire can affect virtually all organs of the plant and often involves the death of sick trees or shrubs. Currently there are no effective methods for the control of bacterial fire and it is necessary to combine different measures aimed at eliminating or reducing the inoculum.

Clavibacter michiganensis is a gram-positive bacterium that causes the potato's angular rot ( C. michiganensis subsp. Sepedonicum ) and the bacterial chancre of tomato ( C. michiganensis subsp. Michiganensis ). These are quarantine diseases that are very difficult to control, since the pathogen remains viable in the soil in remains of infected material and is transmitted by seeds.

Xanthomonas vesicatoria is a gram-negative bacterium that causes the disease called Bacterial Spot in plants of the Solanaceae family that have great economic importance such as tomatoes and peppers. This disease affects leaves, stems and fruits causing wilt or death.

Pseudomonas syringae is a gram-negative bacterium that causes a large number of diseases called bacterial necrosis in horticultural and woody crop plants such as fruit trees. The pathogenicity of P. syringae resides in many cases in its ice nucleating activity (INA +) that enhances frost damage and the production of various phytotoxins such as syringomycins.

Preferably, the pathogenic fungus for plants is Fusarium oxysporum .

Fusarium is a genus of soil and ubiquitous fungi, which includes many races and phytopathogenic species that cause numerous diseases of economic importance mainly in horticultural crops. The vascular diseases they cause cause loss of plant turgidity and often death due to the collapse of the conductive system. The main diseases are caused by the genus Fusarium oxysporum that is subdivided into various special forms or races attending to the affected host plant, such as lycopersici that affects tomato, melonis to melon, conglutinans to various cucurbitaceae, cubense to banana, dianthi to carnation, etc. Being a soil pathogen it is very difficult to control, mainly because the classical methods are based on disinfection by methyl bromide, whose use is currently prohibited, since in general existing fungicides are ineffective in the control of said pathogen.

Preferably the plants that can be treated with the peptides of the invention are selected from the group formed by fruit trees, horticultural plants, and plants Ornamental

Among the fruit trees can be mentioned those of pepita (apple tree, pear tree), and bone (peach tree, apricot tree, plum, cherry) and banana.

Among the horticultural plants are the solánaceas (tomato, pepper, potato) and cucurbitaceae (melon, watermelon).

Examples of ornamental plants are rowan, Carnation, hawthorn and mustard.

Phytosanitary compositions

It is also part of the object of the invention a phytosanitary composition comprising:

-

a peptide having a lysine molar content comprised between the 40% and 60%, and that is defined by the general formula (I)

(I) cycle [(X) n - Y - (X) m - Gln]

where:

X is Lys or Leu,

And it's Phe or D-Phe,

m = 1, 3, or 5,

n = 1, 3, or 5, Y

n + m = 2, 4, 6 or 8, and

an agent assistant.

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Preferably the peptides respond to the general formula (I):

(I) cycle [(X) n - Y - (X) m - Gln]

where:

X is Lys or Leu,

And it's Phe, and

m = 1, and n = 1, or

m = 3, and n = 1, 3, or 5

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More preferably the peptides respond to the general formula (I):

(I) cycle [(X) n - Y - (X) m - Gln]

where:

X is Lys or Leu,

And it's Phe, and

m = 3, and n = 5

Among the peptides of the invention that result be preferred the peptides defined by the sequences are counted of amino acids SEQ_ID_NO: 1 to SEQ_ID_NO: 23.

Usually in the compositions antimicrobials combine several active ingredients to obtain greater efficiency or a wider spectrum of action. In this case, the phytosanitary compositions of the invention also may comprise other antibacterial or antifungal agents for achieve greater antimicrobial efficacy, such as combination of several peptides of the invention, or their combination with other different active matters.

The amount of peptide that is part of the phytosanitary composition can be variable depending on factors such as the type of plant, the concentration of bacteria and pathogenic fungi in the plant, the extent of the disease, etc. The effective peptide concentration can be determined and adjusted. easily by well-known routine methods in Microbiology by the expert in the field. Usually the concentration in active matter is between 0.1 and 0.5 g / l

The auxiliary agent that accompanies the peptides of the invention in said phytosanitary compositions may have several functions, such as facilitating the dosing of peptide, provide an easily manipulated product, improve the  Wet plants.

Said auxiliary agent can be selected. among the group consisting of: solvents, diluents, fillers inert, wetting surfactants, buffering agents, dispersing agents, anti-caking agents, lubricants, filters of ultraviolet radiation, and / or mixtures thereof.

Preferably the auxiliary agent is selected between the group consisting of solvents, surfactants, agents buffers, ultraviolet radiation filters and / or their mixtures

More preferably the solvent is water.

The compositions can be presented in form liquid or in solid form. For example, in the case of formulations liquid, the composition of the invention can be presented in the form of a diluted composition ready to be used, or in form of a concentrated composition, which requires dilution before be used, usually with water.

In the case of aqueous liquid compositions, the presence of wetting surfactants facilitates wetting of plants when sprayed with said composition.

An advantage of the peptides of the invention is that can be dissolved in water easily, and can be formulated generally as aqueous solutions without using additional organic solvents.

Compositions in solid form may be in form of granules, or powders, in which the peptides of the invention are mixed with finely divided inert loads such as, for example, kaolin, diatomaceous earth, dolomite, carbonate Calcium, or talc. They can also be presented in the form of powders or dispersible granules, comprising a wetting agent for facilitate the dispersion of dust or granules in the liquid.

It is also part of the object of the invention a method to prevent and treat infections and diseases of plants caused by bacteria and fungi comprising putting in contact the plant with a phytosanitary composition that includes the peptides of the invention.

The phytosanitary composition of the invention is You can apply preventively to plants to avoid occurrence of infections and diseases caused by bacteria and mushrooms. Preventive treatment is based on the fact that peptides of the invention inhibit the growth of bacteria and fungi pathogens for plants.

It can also be used to treat these infections and diseases once its presence has been detected in plants, since the peptides of the invention inhibit the growth of bacteria and fungi causing these infections and diseases

In this description the term infection is refers to the invasion and destruction of an organ, for example, leaves, flowers, or fruits, by pathogenic microorganisms. Therefore, it is a localized process. Instead, the term disease refers to a process that affects the entire plant, leading to symptoms.

Preferably the method of the invention is used to prevent and treat infections and diseases caused by pathogenic bacteria for plants selected from the group formed by Erwinia amylovora, Clavibacter michiganensis, Xanthomonas vesicatoria , and Pseudomonas syringae .

Preferably, the pathogenic fungus for plants is Fusarium oxysporum .

Preferably the plants that can be treated with the peptides of the invention are selected from the group formed by fruit trees, horticultural plants, and plants Ornamental

In the method of the invention, the composition is can put in contact with the parts of the selected plants between the group consisting of seeds, roots, stems, leaves, or fruits, or with the soil or any means of growth that surrounds The roots of the plants.

In the method of the invention, the composition phytosanitary can be contacted with the plant by any conventional technique, among which, the spraying, the immersion or irrigation.

For example, a solution can be prepared aqueous of the peptides of the invention and proceed to the spraying of affected or susceptible parts of the plant be affected If it's the fruits of a fruit tree, then For example, they can also be treated by spraying or immersion before harvesting or in post-harvest

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Root treatment can lead to carried out for example using a solid composition in which peptides are dispersed in an inert load, or by spraying with said aqueous solution, or by means of its irrigation application.

Biological tests

The antimicrobial activity of the peptides of the invention against bacteria and pathogenic fungi for plants has been evaluated by determining the minimum peptide concentration necessary to inhibit the growth of microorganisms. Usually such concentration is called the minimum inhibitory concentration.
(CIM).

This type of test is common in microbiology and is well known to the person skilled in the art. A description of the methodology used is for example in M.J. Pelczar Jr, E.C.S. Chan, N.R. Krieg, Microbiology: Concepts and Applications New York: McGraw-Hill, 1997.

It has been found that the compositions comprising the peptides of the invention dissolved in water at concentrations between 3.12 and 6.25 µM are effective in inhibiting the growth of bacteria such as Xanthomonas vesicatoria , and Pseudomonas syringae , at concentrations between 6, 25 and 12.5 µM inhibit the growth of the bacteria Erwinia amylovora and Clavibacter michiganensis , and at concentrations below 6.25 µM they inhibit the growth of fungi such as Fusarium oxysporum .

These results demonstrate the efficacy of the antimicrobial activity of the peptides of the invention, since in Avrahami et al. , Biochemistry, 2001, 40, 12591-12603, it is described that a minimum inhibitory concentration of less than 50 µM is considered significant.

The hemolytic activity of the peptides is a characteristic that is usually determined for those compounds that may come into contact with the body human. This would be the case if fruits or vegetables treated with peptides of the invention contained residues thereof and were consumed by people, or when manipulated by operators during its application or preparation.

The said hemolytic activity has been evaluated by determining the release of hemoglobin that is produced by contacting a solution of said peptides in TRIS buffer with 5% volume / volume red blood cell suspensions from fresh human blood. The result of this determination is expressed as the percentage of hemolysis that occurs for a given concentration of peptide. A description of the methodology used for the determination of hemolytic activity is found in Oren et al , Biochemistry, 2000, 39, 6103-6114, and in Raguse et al , J. Am. Chem. Soc., 2002, 124, 12774 -12785.

It has been proven that the peptides of the invention have a hemolytic activity that can be classified as low, since most of them have an HD_ {50} greater than 375 µM. HD50 is the concentration of peptide at which 50% release of hemoglobin occurs in the bioassay referred. It is observed, then, that the concentration at which peptides would have a significant hemolytic activity is between ten hundred times higher than the concentration at which they are active to inhibit the growth of pathogenic bacteria and fungi to the plants.

The examples set out below are they must interpret as an auxiliary means for a better understanding of the invention, and not as limitations to the object thereof.

Examples

The following examples use the following abbreviations: Al: allyl; Boc: tert-butyloxycarbonyl; DIEA: N, N-diisopropylethylamine; DMF: N, N-dimethylformamide; EDTA, acid ethylenediaminetetraacetic; ESI-MS: spectrometry of electrospray ionization masses; FAB-MS: mass spectromet fa by rapid atom bombardment (Fast Atom Bombardment - Mass Spectrometry); Fmoc: 9-fluorenylmethoxycarbonyl; HBTU: hexafluorophosphate of N-oxide N - [(1 H -benzotriazol-1-yl) (dimethylamino) methylene] -N-methylmethanamine; HOBt: 1-hydroxybenzo-triazole; HPLC: high performance liquid chromatography; MALDI-TOF: ionization laser desorption assisted by a matrix - Time of flight (Matrix-Assisted Laser Desorption Ionization-Time-of-Flight); MBHA: 4-methylbenzydrylamine; MWI: radiation by microwave; NMM: N-methylmorpholine; NMP: N-methylpyrrolidone; Ph: phenyl; Py-BOP: hexafluorophosphate benzotriazol-1-yl-N-oxitris (pyrrolidino) phosphonium; TFA: trifluoroacetic acid; THF: tetrahydrofuran; TIS: trisisopropylsilane; UV: ultraviolet.

The products Fmoc-Lys (Boc) -OH, Fmoc-Phe-OH, Fmoc-Glu-OAI, Fmoc-Leu-OH, MBHA resin functionalized with amino groups, the bifunctional spacer Fmoc-Rink- Linker (CAS number: 145469-56-3), HBTU, PyBOP, and HOBt se obtained from Senn Chemicals. SynPhase? Lanterns of the type Rink polystyrene amide D series were obtained from the company Mimotopes (Clayton, Australia). The products TFA, NMP, Pd (PPh3) 4, N, N-diethyldithiocarbamate sodium, and TIS were obtained from Aldrich. Piperidine products, NMM, and DIEA were obtained from Fluka.

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The following indications about the procedure for the preparation of the peptides are of a character general:

-

They have employed amino acids that have the group α-amino protected with the Fmoc group.

-

The tert- butyloxycarbonyl (Boc) group has been used to protect the lysine side chain.

-

In the reactions carried out on resin support were used 5 or 10 ml syringes that had a microporous filter incorporated Polypropylene

-

In the reactions carried out with the SynPhase ™ Lanterns of the type Rink amida of the company Mimotopes, containers of 5 to 50 ml glass, and 10 to 50 ml plastic syringes.

-

All transformations and washes were carried out at 25 ° C, unless otherwise indicated.

-

The microwave radiation experiments were carried out in the Ethos SEL device from Milestone, equipped with a system 1,600 watt dual magnetron. The weather, the temperature of reaction, and the power was controlled by the package EasyControl software that is incorporated into the microwave equipment. The temperature was controlled through an automatic system of fiber optic temperature control, ATC-400F0, submerged in a reference vessel Milestone

-

He HPLC analysis was carried out at 1.0 mllmin using a column Kromasil reverse phase (4.6 x 40 mm; 3.5 particle size p.m). Linear gradients with 0.1% aqueous TFA and TFA were used in 0.1% acetonitrile, with a ratio between 0.98: 0.02 and 0.98: 0.1 over a period of 7 minutes with UV detection at a wavelength of 220 nm.

-

The ESI-MS spectra were acquired using a Quadrupole Navigator instrument; the instrument was operated on the positive ion mode (ES +) with a sample voltage of 3 kV

-

The MALDI-TOF spectra were acquired through a Ultra-flex company instrument Bruker

-

All the relationships between solvents are volume / volume, unless otherwise specified

Example 1 Preparation of cycle (Lys-Leu-Lys-Leu-Lys-Phe-Lys-Leu-Lys-Gln) in solid phase using MBHA resin as solid support

In a syringe of 5 ml capacity, equipped with a microporous filter at the bottom, 200 mg were placed MBHA resin with a functionalization of 0.3 mmol / g, equivalent at 0.06 mmol of amino groups, and the syringe was filled with di- solvent to swell-wash the resin according to the following sequence: CH 2 Cl 2 (5 x 0.5 min), a mixture of TFA and CH 2 Cl 2 (4: 6, 1 x 20 min), CH 2 Cl 2 (5 x 0.5 min), a mixture of DIEA and CH 2 Cl 2 (5:95, 3 x 1 min), CH 2 Cl 2 (5 x 0.5 min), and NMP (5 x 0.5 min). The expression CH 2 Cl 2 (5 x 0.5 min) refers to the fact that 5 washed with methylene chloride of 0.5 minutes each. After each washing step the solvent is removed by a filter Vacuum Manifold Vacuum Manifold by Promega Distributor).

The bifunctional spacer was coupled to the resin by treatment with 97 mg of Fmoc-Rink-Linker (0.18 mmol), 68 mg of HBTU (0.18 mmol) and 31 µl of DIEA (0.18 mmol) in 1 ml of NMP. It was maintained overnight and the ninhydrin test was found to be negative (Kaiser et al , Anal. Biochem., 1970, 34, 595-598).

After washing the resin with NMP (5 x 2 min), This was treated with a mixture of piperidine and NMP (3: 7, 2 x 8 min) to eliminate the Fmoc group present in the amino group of the bifunctional spacer, and then washed with NMP (5 x 2 min)

The resin was then treated with 74 mg of Fmoc-Glu-OA1 (0.18 mmol), 68 mg of HBTU (0.18 mmol) and 31 µl of DIEA (0.18 mmol) in 1 ml of NMP. After 4 h it was found that the ninhydrin test was negative. It was then washed with NMP (5 x 2 min).

The elimination of the Fmoc group and washes Subsequent were performed as already described previously.

The cycle of coupling-checkout was repeated for the rest of the amino acids protected with the group N? -Fmoc: Fmoc-Lys (Boc) -OH, Fmoc-Phe-OH, and Fmoc-Leu-OH. At each stage it proceeded to wash with NMP (5 x 2 min).

The C-terminal allyl ester of glutamic acid incorporated into the peptide was removed under atmosphere of nitrogen per treatment with 347 mg of Pd (PPh3) 4 (0.3 mmol) in 3 ml of a mixture of chloroform, acetic acid and NMM (37: 2: 1) for 5 h under agitation. After this time the resin was washed with THF (3 x 2 min), NMP (3 x 2 min), a mixture of DIEA and CH2Cl2 (1:19, 3 x 2 min), N, sodium N-diethyldithiocarbamate (0.03 M in NMP, 3 x 15 min), NMP (10 x 1 min), CH 2 Cl 2 (3 x 2 min), and NMP (3 x 1 min).

After removing the last Fmoc group amino acid coupled and proceed to washings as already described previously, the linear peptide was obtained Lys-Leu-Lys-Leu-Lys-Phe-Lys-Leu-Lys-Glu bonded to the resin. At this stage, the peptide still contains the glutamic acid residue, since glutamine does not form until excision of the cyclic peptide from the resin.

Peptide cyclization was carried out by treatment with 156 mg of PyBOP (0.3 mmol), 40 mg of HOBt (0.3 mmol), and 104 µl of DIEA (0.6 mmol) in 1 ml of NMP. After 24 hours at 25 ° C, the ninhydrin test was negative.

The resin was then washed with NMP (6 x 1 min) and CH 2 Cl 2 (6 x 1 min), and the peptide was cleaved cyclic of the resin by a treatment with 2 ml of a mixture of TFA, water and TIS (95: 2.5: 2.5) for 2 hours. The ones were collected filtered in a vial by a positive nitrogen pressure. The resin was washed with a mixture of TFA, water and TIS (95: 2.5: 2.5, 2 x 0.5 ml) The filtrates were combined and evaporated almost to dryness. under a stream of nitrogen until an oil is obtained. Saying oil was precipitated with diethyl ether, centrifuged, and the ether was of singing. This process was repeated 3 or 4 times. Finally the Solid product obtained was dissolved in water, and lyophilized.

A powdery solid was obtained whose structure was analyzed by HPLC and confirmed by ESI-MS, MALDI-TOF or FAB-MS.

Example 2 Preparation of cycle (Lys-Leu-Lys-Leu-Lys-Phe-Lys-Leu-Lys-Gln) in solid phase using a SynPhase? as solid support Lantern

It was split from a SynPhase? Lantern of the Rink type polystyrene amide D series consisting of nine disks joined together, equivalent to 35 µmoles. He cut a fragment of Lantern that included five discs, equivalent to 19.44 µmoles, which was placed in a container provided with a stopper and swelled with CH2Cl2 (5 min). The elimination of Fmoc protecting group present in the solid support was carried out by treatment with 1 ml of a mixture of piperidine, CH 2 Cl 2 and NMP (1: 2: 2, 2 x 45 min). The washes were taken out in 10 ml syringes, covering the fragment with NMP (3 x 5 min) and CH 2 Cl 2 (2 x 5 min). After each washing stage the solvent is removed by a vacuum filter multiple.

The Lantern fragment was placed next in a dinner vessel containing 1 ml of a solution in NMP of 49 mg of Fmoc-Glu-OAl (120 mM), 46 mg of HBTU (120 mM), and 42 µL DIEA (240 mM). After 24 hours, the Lantern fragment was washed with NMP (3 x 5 min), and CH 2 Cl 2 (2 x 5 min).

The following stages of coupling with the remaining amino acids were carried out by repeated cycles of elimination of the Fmoc group, coupling and washing, as already described above

Once the coupling stages are finished, the C-terminal alnico ester was removed under nitrogen atmosphere per treatment for 6 h with 87 mg of Pd (PPh3) 4 (75 mM) in 1 ml of a mixture of chloroform, acetic acid and NMM (37: 2: 1). Lantern's fragment it was washed with a mixture of chloroform, acetic acid and NMM (37: 2: 1, 3 x 2 min), a mixture of DIEA and CH2Cl2 (1:19, 3 x 5 min), N, sodium N-diethyldithiocarbamate (0.03 M in NMP, 3 x 15 min), NMP (5 x 5 min), and CH 2 Cl 2 (5 x 5 min)

After removing the Fmoc group and proceed to the washes as described above, a linear peptide bound to SynPhase? Lantern.

Peptide cyclization was carried out by treatment with 162 mg of PyBOP (311 µmoles), 42 mg of HOBt (311 µmoles), and 109 µl of DIEA (622 µmoles) in 1 ml of NMP. After 24 hours at 25 ° C, the Lantern fragment was washed with NMP (3 x 5 min) and CH 2 Cl 2 (3 x 5 min), and it was cleaved the cyclic peptide of said fragment by treatment with a mixture of TFA, water and TIS (95: 2.5: 2.5) for 1 hour. He Lantern fragment was removed with tweezers and the acidolytic mixture  evaporated almost to dryness under a stream of nitrogen until Get an oil. Said oil was precipitated with diethyl ether, centrifuged, and the ether was decanted. This process was repeated 3 or 4 times. Finally, the solid product obtained was dissolved in water, and It was lyophilized.

A powdery solid was obtained, whose structure was analyzed by HPLC and confirmed by ESI-MS, MALDI-TOF or FAB-MS

Example 3 Preparation of cycle (Lys-Leu-Lys-Leu-Lys-Phe-Lys-Leu-Lys-Gln) in solid phase using a SynPhase? as solid support Lantern and cycling using radiation from microwave

It was split from a SynPhase? Lantern of 35 Rink type amide polystyrene D series, of the that a fragment was cut comprising five disks joined between yes, equivalent to 19.44 µmol, which was placed in a container of glass provided with a plug and swelled with CH2Cl2 (5 min) The removal of the Fmoc protective group was carried out by treatment with 1 ml of a mixture of piperidine, CH2Cl2 and NMP (1: 2: 2, 2 x 45 min). The washes were carried out immersing the fragment in NMP (3 x 5 min) and CH 2 Cl 2 (2 x 5 min) using 10 ml syringes. After each washing stage the solvent is removed by a vacuum filter multiple.

The Lantern fragment was placed next in a dinner vessel containing 1 ml of a solution in NMP of 49 mg of Fmoc-Glu-OAI (120 mM), 46 mg of HBTU (120 mM), and 42 µL DIEA (240 mM). After 24 hours, the fragment was washed with NMP (3 x 5 min), and CH 2 Cl 2 (2 x 5 min)

The following stages of coupling with the rest of the amino acids were carried out by cycles repeated elimination of the Fmoc group, coupling and washing, as described above.

Once the coupling stages are finished, the C-terminal allyl ester was removed under nitrogen atmosphere per treatment for 6 h with 87 mg of Pd (PPh3) 4 (75 mM) in 1 ml of a mixture of chloroform, acetic acid and NMM (37: 2: 1). Lantern's fragment it was washed with a mixture of chloroform, acetic acid and NMM (37: 2: 1, 3 x 2 min), a mixture of DIEA and CH2Cl2 (1:19, 3 x 5 min), N, sodium N-diethyldithiocarbamate (0.03 M in NMP, 3 x 15 min), NMP (5 x 5 min), and CH 2 Cl 2 (5 x 5 min)

After removing the Fmoc group and proceed to the washes as described above, a linear peptide bound to SynPhase? Lantern.

Peptide cyclization was carried out placing the Lantern fragment in a glass vial and treating it with 162 mg of PyBOP (311 µmoles), 42 mg of HOBt (311 µmoles), and 109 µl of DIEA (622 µmoles) in 1 ml of NMP

The vial was placed in an appropriate container for microwave radiation. It was first applied for 5 minutes a microwave radiation ramp up to a maximum of 500 watts, to reach a temperature of 50ºC. The power of the microwave radiation was regulated by a thermostat that set at a maximum temperature of 50 ° C. Irradiation was continued to maintain the temperature at 50 ° C for 15 minutes. The mass of the reaction was cooled to room temperature, and two were applied 15 minute cycles of microwave radiation.

Once the irradiation period is over, The solvent was removed and the Lantern fragment was washed in NMP (3 x 5 min) and CH 2 Cl 2 (3 x 5 min). The cyclic peptide is excised the Lantern fragment by treatment with a mixture of TFA, water and TIS (95: 2.5: 2.5) for 1 hour. The fragment de Lantern was removed with tweezers and the acidolytic mixture was evaporated almost to dryness under a stream of nitrogen until Get an oil. Said oil was precipitated with diethyl ether, centrifuged, and the ether was decanted. This process was repeated 3 or 4 times. Finally, the solid product obtained was dissolved in water, and It was lyophilized.

A powdery solid was obtained, whose structure was analyzed by HPLC and confirmed by ESI-MS, MALDI-TOF or FAB-MS

Examples 4 to 6

Preparation of solid phase peptides of the invention using  as a solid support a SynPhase? Lantern and performing the cyclization using microwave radiation

Following a procedure similar to that described in Example 3, the cyclic peptides showing the Table II:

TABLE II

4

In all cases the cyclic peptides are analyzed by HPLC and its structure was confirmed by ESI-MS, MALDI-TOF or FAB-MS

Example 7 Preparation of a phase cyclic peptide library solid using SynPhase? Lanterns as solid support

A 56 peptide library was prepared cyclic 10 amino acids from 28 SynPhase? Lanterns Rink type polystyrene amide D series commercial, each one of which was divided into two.

Lantern fragments were marked with "spindles" and "cogs" of colors, marketed by Mimotopes, to identify the positions that contained a residue of leucine.

All Lantern fragments were placed in a single glass container provided with a cap and covered with CH 2 Cl 2 for 5 minutes to swell them. For remove the Fmoc protecting group were treated with 30 ml of a mixture of piperidine-CH 2 Cl 2 -NMP (1: 2: 2, 2 x 45 min).

The washings were carried out by submerging the fragments in NMP (3 x 5 min) and CH 2 Cl 2 (2 x 5 min) using plastic syringes. After each washing stage Remove the solvent using a multiple vacuum filter.

Next the Lantern fragments are placed in a container with a cap containing 30 ml of a solution of 1.47 g of Fmoc-Glu-OAl (120 mM), 1.36 g of HBTU (120 mM), and 1.25 ml of DIEA (240 mM) in NMP. After 24 hours, the fragments were washed with NMP (3 x 5 min) and CH 2 Cl 2 (2 x 5 min).

Subsequent links were taken to carried out by repeated cycles of elimination of the Fmoc group, coupling with a new amino acid, and washes, as already previously described.

For each link with Fmoc-Lys (Boc) -OH and Fmoc-Leu-OH, the fragments of Lantern were classified and separated into two groups, so that 21  fragments were treated with a solution containing Fmoc-Leu-OH, and 35 fragments with Fmoc-Lys (Boc) -OH.

After each coupling stage, the Lantern fragments were introduced into a single syringe of plastic for washing.

Then they separated again into two groups for subsequent coupling of Fmoc-Lys (Boc) -OH or Fmoc-Leu-OH, or were placed in a same vessel for coupling Fmoc-Phe-OH.

Once all the couplings are finished for to obtain linear peptides of 10 amino acids, we proceeded to removal of the C-terminal allyl ester, at removal of the Fmoc protective group, and washings following the procedure described in Example 2. All these steps are performed by placing all the Lantern fragments in a single container or syringe

The cyclization of each of the peptides is carried out in individual containers following the treatment described in Example 2.

Then the excision of the cyclic peptide of the solid support, and to the isolation of each of the peptides following the procedure described in Example 2. To do this, each fragment of Lantern was placed in a container individual.

The peptides obtained were analyzed by HPLC and its structure was confirmed by ESI-MS, MALDI-TOF or FAB-MS.

Figure 1 includes the structures of the 56 cyclic decapeptides synthesized in this Example 7.

Example 8 Antimicrobial activity assays

The following strains of pathogenic bacteria were used to assess the antimicrobial effect of the peptides of the invention: Erwinia amylovora PMW6076 (INRA, Angers, France), Clavibacter michiganensis, Xanthomonas vesicatoria 2323-3 (IVIA, Valencia, Spain), and Pseudomonas syringae 94 (INTEA, University of Girona, Spain).

All bacteria were conserved at one temperature of -80ºC in Luria-Bertani medium (LB) supplemented with 20% glycerin in volume / volume.

The samples of E. amylovora PMV6076 and P. syringae were obtained after 24 hour growth at 25 ° C, and the sample of X. vesicatoria 2323-3 was collected after 48 hours at 25 ° C on LB agar. In all cases they were resuspended in sterile water to obtain a suspension adjusted to an absorbance of 0.2 to 600 nm, corresponding to approximately 10 8 colony forming units / ml.

To determine the inhibitory concentration minimum (MIC), the peptides were solubilized in sterile water milli-Q to a final concentration of 1000 µM and filtered through a 0.22 µm pore filter.

Dilutions of the peptides of the invention to obtain solutions at concentrations 750, 500, 250, 125, 62.5, and 31.2 µM.

20 µl of each dilution was mixed with 20 µl of the suspension of the corresponding bacterial indicator, 160 µL of TSB (Triptone Soy Broth) liquid medium, up to total volume of 200 µl in each well of a microplate. So the effective concentrations to which the suspensions were subjected of bacteria were 75, 50, 25, 12.5, 6.25 and 3.12 µM.

Three replicates were carried out for each strain, concentration and peptide in assay.

Positive controls contained water instead of peptide, and the negative controls contained the peptides without the bacterial suspension

Microbial growth was determined automatically by measuring the optical density at 600 rim using the Microbiology Analyzer Bioscreen C (Labsystems).

The microplates were incubated at 25 ° C for 48 hours with a stirring of 20 seconds before measuring the absorbance every hour. Each experiment was performed in triplicate. As CIM, the lowest peptide concentration at which no Bacterial growth occurred at the end of the experiment.

Table III shows the range of concentrations between the MIC, expressed in µM, after 48 hours of incubation at 25 ° C, for different peptides of the invention against the bacteria Erwinia amylovora, Clavibacter michiganensis, Xanthomonas vesicatoria , and Pseudomonas syringae :

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TABLE III

5

6

7

It can be seen that the peptides of the invention have a good efficacy to inhibit the growth of the bacteria Erwinia amylovora, Clavibacter michiganensis, Xanthomonas vesicatoria , and Pseudomonas syringae , which are representative of pathogenic bacteria for plants.

In addition to the results of antibacterial activity presented by the peptides of Table 3, the rest of the decapeptides of the library prepared in Example 7, and whose structure is in Figure 1, has been tested against Erwinia amylovora, Xanthomonas vesicatoria , and Pseudomonas syringae , and all have been shown to inhibit the growth of at least two of said bacterial species.

To determine the antifungal activity, the strain of the fungus Fusarium oxysporum f.sp. lycopersici FOL 3 race 2 (ATCC 201829, isolated in Spain), first in the middle of agar-potato-dextrose (PDA). The microspores were obtained after a growth in dextrose and potato broth (PDB) for 7 days at 25 ° C on a rotary shaker at 125 rpm. The spore suspension was filtered and adjusted to 10 4 microspores / mi using the Thoma counting chamber.

The peptides of the invention were solubilized in sterile milli-Q water to a final concentration 1000 µM and filtered through a 0.22 µm filter of pore.

With this solution dilutions of the peptides to obtain solutions at concentrations 750, 500, 250, 125, 62.5, and 31.2 µM.

Then 20 µl of each was mixed dilution of peptide with 80 µl of the fungus suspension, and with 100 µl of double concentration PDB medium, until obtaining a total volume of 200 µl in a well of a plate. In this way spore suspensions exposed to the concentrations of 75, 50, 25, 12.5, 6.25 and 3.12 µM. Plates they were incubated at 22 ° C for 144 hours with 1 minute series of stirring followed by 9 minutes of rest.

Positive controls contained water instead of peptide, and the negative controls contained the peptides without the spore suspension.

The intensity of growth was determined using the area under the curve and the calculations were performed following the procedures described in K.L. Reynolds, and D.A. Enher, Statistical comparison of epidemics p. 34-37, in D.A. Francich Neher (Eds.), Exercises in plant disease epidemiology. APS Press 1997, Minnesotta.

Table IV shows the range of concentrations among which is the minimum inhibitory concentration (MIC), expressed in µM, after 144 hours of incubation for several peptides of the invention against the pathogenic fungus for Fusarium oxysporum plants:

TABLE IV

8

It can be seen that the peptides of the invention inhibit the growth of the pathogenic fungus for Fusarium oxysporum plants.

Example 8 Hemolytic activity assays

The hemolytic activity of the peptides of the invention was evaluated by determining the release of hemoglobin that is produced by contacting a solution of the peptides with a 5% volume red cell suspension from fresh human blood.

Blood was collected aseptically using a Vacuntainer® K2E system (Belliver, Great Britain) with EDTA, and stored at 4 ° C for a period of less than 2 hours.

The blood was centrifuged at 6,000 g for 5 minutes to separate the erythrocytes. These were washed three times. with TRIS buffer (10 mM TRIS, 150 mM NaCl, pH 7.2), and were suspended in TRIS buffer until a suspension containing 10% in erythrocyte volume

The peptides were solubilized in TRIS buffer to a final concentration 125, 250, 500 and 750 µM.

Three replicates were used for each peptide and concentration.

65 µl of the suspension of 10% erythrocytes by volume with 65 µl of the solution of peptide in each well of a 96-well plate Micro-Amp® (Applied Biosystems, USA) (5% in erythrocyte volume), and the mixture was incubated for 1 h at 37 ° C under agitation Thus the erythrocyte suspensions are subjected to peptide concentrations of 62.5, 125, 250 and 375 µM.

The plates were then centrifuged at 3,500 g for 10 minutes, and 80 aliquots were transferred µl of the supernatant to 100-well microplates (Bioscreen), which were diluted with 80 µl of milli-Q water.

The degree of hemolysis was determined from the absorbance at 540 nm with a Bioscreen plate reader.

The positive complete hemolysis control is determined in a TRIS buffer solution containing melitin (Sigma-Aldrich, Spain).

The percentage of hemolysis (H) was determined using the equation:

H = 100x [(Op-Ob) / (Om-Ob)]

where Op was the optical density measured for a given peptide concentration, Ob was the optical density of the buffer solution, and Om was the optical density for positive control with melitina

Table V presents the activity results hemolphic for different concentrations of peptides from the invention, expressed as the percentage of hemolysis calculated according to the previous equation:

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TABLE V

9

10

In all cases it is observed that the concentration at which hemolysis would occur significant is between 10 and 100 times higher than the concentration in which the peptide has antimicrobial activity.

Claims (20)

1. Cyclic peptides characterized by they have a lysine molar content between 40% and 60%, and that respond to the general formula (I): (I) cycle [(X) n - Y - (X) m - Gln)] where: X is Lys and / or Leu, And it's Phe or D-Phe, n = 1, 3, or 5, m = 1, 3, or 5, and n + m = 2, 4, 6 or 8. 2. Cyclic peptides according to claim 1 characterized in that in the general formula (I) X is Lys and / or Leu, And it's Phe, and m = 1, and n = 1, or m = 3, and n = 1, 3, or 5. 3. Cyclic peptides according to claim 2 characterized in that in the general formula (I) X is Lys and / or Leu, And it's Phe, and m = 3, and n = 5. 4. Cyclic peptides according to claims 1 and 2 characterized in that they are defined by the amino acid sequences SEQ_ID_NO: 1 to SEQ_ID_NO: 3. 5. Cyclic peptides according to claims 1 to 3 characterized in that they are defined by the amino acid sequences SEQ_ID_NO: 4 to SEQ_ID_NO: 23. 6. Use of the peptides according to claims 1 to 5 for the preparation of a composition antimicrobial 7. Use according to claim 6 as antimicrobial agents against bacteria and pathogenic fungi for the plants. 8. Use according to claim 7, characterized in that the pathogenic bacteria for the plants are selected from the group formed by Erwinia amylovora, Clavibacter michiganensis, Xanthomonas vesicatoria , and Pseudomonas syringae . 9. Use according to claim 7, characterized in that the pathogenic fungus for plants is Fusarium oxysporum . 10. Use according to claims 6 to 9 characterized in that the plants are selected from the group consisting of fruit trees, horticultural plants, and ornamental plants. 11. Phytosanitary composition characterized in that it comprises a peptide according to claims 1 to 5, and an auxiliary agent. 12. Composition according to claim 11 characterized in that the peptide is at a concentration between 0.1 g / l and 0.5 g / l. 13. Composition according to claims 11 and 12 characterized in that the auxiliary agent is selected from the group consisting of solvents, surfactants, buffering agents, ultraviolet radiation filters and / or mixtures thereof. 14. Composition according to claim 13 characterized in that the solvent is water. 15. Method for preventing and treating infections and diseases of plants caused by bacteria and fungi characterized in that it comprises contacting the plant with a phytosanitary composition according to claims 11 to 14. 16. Method according to claim 15 characterized in that the pathogenic bacteria for the plants are selected from the group formed by Erwinia amylovora, Clavibacter michiganensis, Xanthomonas vesicatoria , and Pseudomonas syringae . 17. Method according to claim 15 characterized in that the pathogenic fungus for plants is Fusarium oxysporum . 18. Method according to claims 15 to 17 characterized in that the plants are selected from the group consisting of fruit trees, horticultural plants, and ornamental plants. 19. Method according to claims 15 to 18, characterized in that the composition is brought into contact with the selected plant parts among the group consisting of seeds, roots, stems, leaves, or fruits, or with the soil or any growth medium that surround the roots of the plants. 20. Method according to claims 15 to 19 characterized in that the composition is brought into contact with the plant by spraying, immersion or irrigation.