WO2007074185A1

WO2007074185A1 - Antimicrobial cyclic peptides

Info

Publication number: WO2007074185A1
Application number: PCT/ES2006/000686
Authority: WO
Inventors: Eduard BARDAJÍ RODRÍGUEZ; Emili MONTESINOS SEGUÍ; Marta Planas Grabuleda; Esther Badosa Romacho; Lidia Feliu Soley; Sylvie Martine Colette Monroc
Original assignee: Universitat De Girona
Priority date: 2005-12-21
Filing date: 2006-12-14
Publication date: 2007-07-05
Also published as: ES2281274B2; ES2281274A1

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 cyclic peptides that are have antimicrobial properties, and that are particularly effective against pathogenic bacteria and fungi for plants.

Prior art

Agriculture is a process for food production by growing certain plants. The transition from subsistence agriculture in which only what was necessary to meet the needs of the farmer and his family was produced, to industrial agriculture that implies the existence of large cultivated areas, has led to the need to control the diseases of Plants effectively on a 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 only some products are authorized for the protection of plants against bacterial diseases based mainly on cupric derivatives 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 such as the US, but in many cases the emergence of resistant strains of the pathogen that make their use ineffective, or that may pose a risk of transfer of such resistance to other pathogenic bacteria. In the case of mycoses caused by phytopathogenic fungi, although there are active substances available, new fungicides are continually required, partly due to the occurrence of resistance with a certain frequency. In addition, many of the most effective known antifungal and antibacterial compounds have a persistence in the environment that is undesirable.

Another way of controlling the diseases caused by the pathogenic microorganisms of plants is the use of isolated compounds of their own nature, for example peptides, which are present in plants and animals, and some of which have antimicrobial properties.

In Broekaert et al, Plant. Physiol., 1995, 108, 1353-1358, is described

'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.

Patent application WO-A-95/11306 describes a 30 amino acid peptide isolated from beet, which has bactericidal and fungicidal properties against pathogenic microorganisms of plants.

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

Another way of acting against plant pathogenic microorganisms is the use of compounds resulting from the modification of peptides or antimicrobial proteins isolated from nature. For example as described in:

- patent application EP-A-0472987, which refers to linear peptides of 23 amino acids, which are modifications of natural peptides belonging to the magainin group, patent application WO-A-02/06324, which refers to linear peptides of 44 amino acids derived from the natural peptide called heliomycin, or

- US patent US6015941, which refers to linear peptides formed by 17 amino acids derived from taquiplesin. 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 as to the commercial quantities available, partly due to problems of obtaining and purification. Another approach to dispose of antimicrobial peptides is to prepare them synthetically.

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

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-serine-glycine amino acid cluster at the center of the molecule, and have cyclic structure due to the formation of a disulfide bridge between the cistern 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. There remains a need for 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 have a high efficacy to inhibit the growth of plant pathogenic microorganisms. Object of the invention

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

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

A phytosanitary composition comprising the peptides of the invention is also the subject of the invention.

Also part of the object of the invention is a method for preventing and treating infections and diseases of plants caused by bacteria and fungi.

Description of the figures

Figure 1

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

Detailed description of the invention

Peptides The object of the present invention is to provide cyclic peptides that have a Usine molar content between 40% and 60%, and that respond to the general formula (I): cycle [(X) _n -Y- (X ) _m - GIn)] (I) where: X is Lys or Leu, Y is 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 the beginning of the amino acid sequence in the general formula (I) means that the peptide is cyclic, so that the α-carboxylic group of the amino acid GIn is linked by a peptide bond with the α group -amino of the first amino acid X belonging to group (X) _n . The abbreviations used for the amino acids in this description follow the regulations of the Commission for the Biochemical Nomenclature of the IUPAC-

IUB, as described in J. Biol. Chem., 1972, 247, 977-983. In this way, Lys means L-lysine, Leu is L-leucine, Phe is L-phenylalanine, D-Phe is D-phenylalanine, and GIn is L-glutamine.

Compounds belonging to this group of peptides have antimicrobial activity.

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

Preferably the peptides respond to the general formula (I): cycle [(X) _n -Y- (X) _m -GIn] (I) where: X is Lys or Leu, Y is Phe, ym = l, and n = l , om = 3, and n = 1, 3, or 5. More preferably the peptides respond to the general formula (I): cycle [(X) _n -Y- (X) _m -GIn] (I) where: X is Lys or Leu,

Y is Phe, and m = 3, and n = 5.

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

TABLE I

The peptides object of the invention are cyclic and comprise between 4 and amino acids, and have a Usine molar content of between 40% and%. This means that in the case of a 4, 6 or 8 amino acid peptide, the peptide contains two, three or four lysine residues respectively, which represents 50% molar lysine in all cases.

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

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

- The α-carboxylic group of the first lysine is linked to the α-amino group of phenylalanine. - The α-carboxylic group of phenylalanine is linked to the α-amino group of the second lysine.

- The α-carboxylic group of the second lysine is linked to the α-amino group of glutamine.

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

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

In the general formula (I) of the peptides of the present invention, the amino acid Y may 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 into a peptide contributes to increasing the stability of said peptide, since it is less sensitive to the biological pathways that degrade the L-series amino acids. 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 which makes them suitable for being prepared using the usual methods of solid phase peptide synthesis. Among these procedures can be mentioned:

- the process using the 4-methylbenzydrylamine resin (MBHA) functionalized with amino groups, as a solid support to carry out the successive coupling reactions between the different amino acids that make up the peptide and the cyclization step, and

- the Multipin ™ procedure, in which 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 to those skilled in the art 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 the MBHA resin as a solid support, a bifunctional spacer sensitive to the acid medium is usually incorporated into the resin, which allows the peptide synthesized to be synthesized under mild conditions.

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

Said spacer, once linked with its carboxylic group to an amino group of the MBHA resin, has a free amino group that allows the anchoring of an amino acid having a free carboxylic group.

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

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

The Multipin ™ technique can be used to construct peptide libraries in a very convenient manner, as described in the Examples. A process for the preparation of the peptides of the invention may be, for example, the one described below.

In this process, the chemistry of the Fmoc group (9-fluorenylmethoxycarbonyl) is used as a protector of the α-amino group of the amino acids used to prepare the peptides of the invention. In the first stage, the glutamic acid is anchored to the resin, or to the SynPhase ™ Lantern, through the free carboxylic group of its side chain. The other two reactive groups of glutamic acid are kept protected to avoid side reactions: for example, the α-carboxylic group in the form of an allyl ester, and the α-amino group protected by the Fmoc group. The Fmoc protecting group is then removed from 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 is incorporated. The bond is established between the carboxylic group of the new amino acid and the amino group of glutamic acid.

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

In the case of the amino acid lysine, the amino group of the side chain is protected by the Boc (t-butyloxycarbonyl) group, which is stable to the removal conditions of the Fmoc group. Once the couplings are finished, the allyl ester that protects the α-carboxylic group from glutamic acid is removed, and the Fmoc group that protects the α-amino group of the last incorporated amino acid is removed.

Next, the cyclization between the α-carboxylic group of glutamic acid and the α-amino group of the last amino acid incorporated is carried out. In this way a cyclic peptide bound to the solid support is obtained, either the resin or the SynPhase ™ Lantern.

The cleavage of the solid support peptide is carried out under acidic conditions and involves breaking the bond between the amino group and the benzidrile group of the linker, leading to the formation of the glutamine residue (CiIn), which is present in the peptide of the invention, as can be seen in the general formula (I). At the same time that the peptide is released from the solid support, the Boc groups are removed from the 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 powder solids, and are characterized by HPLC analysis, mass spectrometry (ESI- MS, MALDI-TOF or FAB-MS).

Surprisingly, it has been proven that the peptides of the invention have an antimicrobial activity, which is particularly effective against plant pathogenic microorganisms such as the Fusarium oxysporum fungus, 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 lysine molar content of between 40% and 60% and that respond to the general formula (I): cycle [(X) _n -Y- (X ) _m - GIn] (I) where: X is Lys or Leu,

Y is Phe or D-Phe, n = 1, 3, or 5, m = 1, 3, or 5, and n + m = 2, 4, 6 or 8 as antimicrobial agents.

Preferably the peptides respond to the general formula (I): cycle [(X) _n -Y- (X) _m -GIn] (I) where: X is Lys or Leu,

Y is Phe, ym = l, yn = 1, om = 3, and n = I, 3, or 5 More preferably the peptides respond to the general formula (I): cycle [(X) _n - Y - (X) _m - GIn] (I) where: X is Lys or Leu,

And it's Phe, and m = 3, and n = 5

Among the peptides of the invention that are preferred are the peptides defined by the amino acid sequences SEQ_ID_NO: 1 to SEQ_ID_NO: 23. Preferably the peptides of the invention are used as antimicrobial agents against bacteria and fungi 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 appropriate indicators to determine 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 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 cucurbits, cover 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 consisting of fruit trees, horticultural plants, and ornamental plants.

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

Among the horticultural plants are the Solanaceae (tomato, pepper, potato) and cucurbitaceae (melon, watermelon). Examples of ornamental plants are rowan, carnation, sea-buckthorn and mustard.

Phytosanitary compositions

Also part of the object of the invention is a phytosanitary composition comprising:

- a peptide having a molar content of Usina between 40% and 60%, and which is defined by the general formula (I) cycle [(X) _n - Y - (X) _m - GIn] (I) where: X is Lys or Leu, Y is Phe or D-Phe, m = l, 3, or 5, n = 1, 3, or 5, and n + m = 2, 4, 6 or 8, and an auxiliary agent .

Preferably the peptides respond to the general formula (I): cycle [(X) _n -Y- (X) _m -GIn] (I) where: X is Lys or Leu, Y is Phe, ym = l, and n = 1 , om = 3, and n = 1, 3, or 5 More preferably the peptides respond to the general formula (I): cycle [(X) _n -Y- (X) _m -Gln] (I) where: X is Lys or Leu,

Y is Phe, and m = 3, and n = 5

Among the peptides of the invention that are preferred are peptides defined by amino acid sequences SEQ_ID_NO: 1 to SEQ_ID_NO: 23.

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

The amount of peptide that is part of the phytosanitary composition can vary depending on factors such as the type of plant, the concentration of pathogenic bacteria and fungi in the plant, the extent of the disease, etc. The effective concentration of the peptide can be easily determined and adjusted by routine methods well known in microbiology by the person skilled in the art. Usually the concentration in active matter is between 0.1 and 0.5 g / 1. The auxiliary agent that accompanies the α peptides of the invention in said phytosanitary compositions can have several functions, such as facilitating the dosage of the peptide, providing an easily manipulated product, improving wetting of plants. Said auxiliary agent can be selected from the group consisting of: solvents, diluents, inert fillers, wetting surfactants, buffering agents, dispersing agents, anti-caking agents, lubricants, ultraviolet radiation filters, and / or mixtures thereof.

Preferably the auxiliary agent is selected from the group consisting of solvents, surfactants, buffering agents, ultraviolet radiation filters and / or mixtures thereof.

More preferably the solvent is water.

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

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

An advantage of the peptides of the invention is that they can be dissolved in water with ease, and can generally be formulated as aqueous solutions without the need for additional organic solvents.

Compositions in solid form may be in the form of granules, or powders, in which the peptides of the invention are mixed with finely divided inert fillers such as, for example, kaolin, diatomaceous earth, dolomite, calcium carbonate, or talc. They can also be presented in the form of dispersible powders or granules, which comprise a wetting agent to facilitate dispersion of the powder or granules in the liquid. Also part of the object of the invention is a method for preventing and treating infections and diseases of plants caused by bacteria and fungi which comprises contacting the plant with a phytosanitary composition that includes the peptides of the invention. The phytosanitary composition of the invention can be applied preventively to plants to prevent the occurrence of infections and diseases caused by bacteria and fungi. Preventive treatment is based on the fact that the peptides of the invention inhibit the growth of pathogenic bacteria and fungi for plants.

It can also be used to treat such infections and diseases once its presence in plants has been detected, since the peptides of the invention inhibit the growth of the bacteria and fungi causing said infections and diseases. In this description the term infection 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, giving rise 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.

In the method of the invention, the composition can be contacted with the selected plant parts among the group consisting of seeds, roots, stems, leaves, or fruits, or with the soil or any growth medium surrounding the roots. of the plants.

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

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

Root treatment can be carried out, for example, by using a solid composition in which the peptides are dispersed in an inert filler, or by spraying with said aqueous solution, or by applying it by irrigation.

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. This concentration is usually called the minimum inhibitory concentration (MIC).

This type of assay is common in microbiology and is well known to the person skilled in the art. A description of the methodology used is found for example in MJ.Pelczar Jr, E.C.S.Chan, N.R. Krieg, Microbiology: Concepts and

Applications New York: McGraw-Hill, 1997.

It has been found that 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 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 generally determined for those compounds that may come into contact with the human body. This would be the case if fruits or vegetables treated with the peptides of the invention contained traces thereof and were consumed by people, or when manipulated by operators during their 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 found 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. HD ₅₀ is the concentration of peptide at which 50% release of hemoglobin occurs in the referred bioassay. It is observed, then, that the concentration at which the peptides would have a significant hemolytic activity is between ten and one hundred times higher than the concentration at which they are active to inhibit the growth of pathogenic bacteria and fungi for plants.

The examples set forth below should be interpreted as an auxiliary means for a better understanding of the invention, and not as limitations on the object thereof.

Examples

The following abbreviations are used in the following examples: Al: allyl; Boc: tert-butyloxycarbonyl; DIEA: N, N-diisopropylethylamine; DMF: N ₅ N-dimethylformamide; EDTA, ethylenediaminetetraacetic acid; ESI-MS: mass spectrometry with electrospray ionization; FAB-MS: fast atom bombardment mass spectrometry (Fast Atom Bombardment - Mass Spectrometry); Fmoc: 9-fluorenihnetoxycarbonyl; HBTU: N - [(1 H- benzotriazol-l-yl) (dimethylamino) methylene] -N-methylmethanamine N-oxide hexafluorophosphate; HOBt: 1-hydroxybenzotriazole; HPLC: high performance liquid chromatography; MALDI-TOF: matrix-assisted laser desorption ionization - Flight time (Matrix-Assisted Laser Desorption Ionization - Time-of-Flight); MBHA: 4-methylbenzydrylamine; MWI: microwave radiation; NMM: N-methylmorpholine; NMP: N-methylpyrrolidone; Ph: phenyl; PyBOP: benzotriazol-l-yl-N-oxitris (pyrrolidine) phosphonium hexafluorophosphate; TFA: trifluoroacetic acid; THF: tetraMdrofuran; TlS: trisisopropylsilane; UV: ultraviolet.

Fmoc-Lys (Boc) -OH, Fmoc-Phe-OH, Fmoc-Glu-OAl, Fmoc-Leu-OH products, MBHA resin functionalized with amino groups, Fmoc-Rink bifunctional spacer

Linker (CAS number: 145469-56-3), HBTU ₅ PyBOP, and HOBt were obtained from Senn

Chemicals SynPhase ™ Lanterns of the Rink polystyrene amide type D series were obtained from Mimotopes (Clayton, Australia). TFA products,

NMP, Pd (PPh ₃ ) ₄ , N, sodium N-diethyldithiocarbamate, and TIS were obtained from Aldrich. Piperidine, NMM, and DIEA products were obtained from Fluka.

The following indications on the procedure for the preparation of the peptides are of a general nature:

- Amino acids having the α-amino group protected with the Fmoc group have been used.

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

- In the reactions carried out on resin support, syringes of 5 or 10 ml were used which had a microporous polypropylene filter incorporated. - In the reactions carried out with the SynPhase ™ Lanterns of the Rmk amide type of the Mimotopes company, glass containers of 5 to 50 ml, and plastic syringes of 10 to 50 ml were used.

- All transformations and washes were carried out at 25 ° C, unless otherwise indicated. - The experiments with microwave radiation were carried out in the apparatus

Ethos SEL from Milestone, equipped with a 1,600-watt dual magnetron system. Time, reaction temperature, and power were controlled by the EasyControl computer package that is incorporated into the microwave equipment. The temperature was controlled by an automatic fiber optic temperature control system, ATC-400FO, immersed in a Milestone reference vessel.

- HPLC analysis was carried out at 1.0 ml / min using a Kromasil reverse phase column (4.6 x 40 mm; particle size of 3.5 μm). Be they used linear gradients with 0.1% aqueous TFA and 0.1% acetonitrile TFA, 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. - ESI-MS spectra were acquired using a quadrupole instrument

Navigator; The instrument was operated in the positive ion mode (ES +) with a voltage in the sample of 3 kV. - MALDI-TOF spectra were acquired through an instrument

Ultraflex of the Bruker company. - All relationships between solvents are volume / volume, unless otherwise specified.

Example 1.- Preparation of cycleCLvs-Leu-Lvs-Leu-Lvs-Phe-Lys-Leu-Lvs-

GIn) 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 of MBHA resin with a functionalization of 0.3 mmol / g, equivalent to 0.06 mmol of amino groups, were placed and filled the syringe with 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 5 washes with methylene chloride of 0.5 minutes each. After each washing step the solvent is removed by a multiple vacuum filter (Vac Man Laboratory Vacuum Manifold from Promega Distribuidora).

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), it was treated with a mixture of piperidine and NMP (3: 7, 2 x 8 min) to remove 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-OAl (0.18 immoles), 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 removal of the Fmoc group and subsequent washes were performed as previously described.

The coupling-deprotection cycle was repeated for the rest of the amino acids protected with the group N ^α -Fmoc: Fmoc-Lys (Boc) -OH, Fmoc-Phe-OH, and

Fmoc-Leu-OH. In each stage the NMP was washed (5 x 2 min). The C-terminal allyl ester of glutamic acid incorporated into the peptide was removed under a nitrogen atmosphere by treatment with 347 mg of Pd (PPh ₃ ) ₄ (0.3 mmol) in 3 ml of a mixture of chloroform, acetic acid and NMM ( 37: 2: 1) for 5 h under stirring. After this time the resin was washed with THF (3 x 2 min), NMP (3 x 2 min), a mixture of DIEA and CH ₂ Cl ₂ (1:19, 3 x 2 min), N, N-diethyldithiocarbamate sodium (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 Fmoc group from the last coupled amino acid and proceeding to the washes as described above, 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 is not formed until the 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 resin cyclic peptide was cleaved by a 2 ml treatment of a mixture of TFA, water and TIS (95: 2.5: 2.5) for 2 hours. The filtrates were collected 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 was obtained. 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 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 cycloofLvs-Leu-Lvs-Leu-Lys-Phe-Lvs-Leu-Lys-

GIn) in solid phase using a SvnPhase ™ Lantern as solid support

It was based on a SynPhase ™ Lantern of the Rink amide type polystyrene D series consisting of nine disks joined together, equivalent to 35 μmoles. A fragment of Lantern comprising five discs, equivalent to 19.44 μmoles, was cut into a container provided with a cap and swollen with CH ₂ Cl ₂ (5 rnin). The removal of the 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 washings were carried out in 10 ml syringes, covering the fragment with NMP (3 x 5 min) and CH ₂ CI ₂ (2 x 5 min). After each washing step the solvent is removed by a multiple vacuum filter.

The Lantern fragment was then placed in a closed container containing 1 ml of a 49 mg NMP solution 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 coupling steps 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 steps were completed, the C-terminal allyl ester was removed under nitrogen atmosphere by treatment for 6 h with 87 mg of Pd (PPh ₃ ) ₄ (75 mM) in 1 ml of a mixture of chloroform, acetic acid and NMM (37: 2: 1). The Lantern fragment was washed with a mixture of chloroform, acetic acid and NMM (37: 2: 1, 3 x 2 min), a mixture of DIEA and CH ₂ Cl ₂ (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 proceeding to the washes as described above, a linear peptide bound to the SynPhase ™ Lantern was obtained.

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 the cyclic peptide of said fragment was cleaved by treatment with a mixture of TFA, water and TIS (95: 2.5: 2.5) for 1 hour. The Lantern fragment was removed with tweezers and the acidolytic mixture was evaporated almost to dryness under a stream of nitrogen until an oil was obtained. 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 lyophilized.

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

Example 3.- Preparation of the cycleCLvs-Leu-Lvs-Leu-Lvs-Phe-Lys-Leu-Lvs-

GIn) in solid phase using a SvnPhase ™ Lantern as a solid support and cycling using microwave radiation

It was split from a SynPhase ™ Lantern of 35 μmoles of the Rink amide type polystyrene D series, from which a fragment comprising five disks joined together, equivalent to 19.44 μmoles, was cut into a container of glass provided with a stopper and swollen with CH ₂ Cl ₂ (5 min). The removal of the Fmoc protecting group 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). Washing was carried out by immersing the fragment in NMP (3 x 5 min) and CH ₂ CI ₂ (2 x 5 min) using 10 ml syringes. After each washing step the solvent is removed by a multiple vacuum filter. The Lantern fragment was then placed in a closed container containing 1 ml of a 49 mg NMP solution of Fmoc-Glu-OAl (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 coupling steps with the rest of the amino acids were carried out by repeated cycles of elimination of the Fmoc group, coupling and washing, as already described above.

After completion of the coupling steps, the C-terminal allyl ester was removed under a nitrogen atmosphere by treatment for 6 h with 87 mg of

Pd (PPh ₃ ) ₄ (75 mM) in 1 ml of a mixture of chloroform, acetic acid and NMM

(37: 2: 1). The Lantern fragment was washed with a mixture of chloroform, acetic acid and NMM (37: 2: 1, 3 x 2 min), a mixture of DIEA and CH ₂ Cl ₂ (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 proceeding to the washes as described above, a linear peptide bound to the SynPhase ™ Lantern was obtained.

Peptide cyclization was carried out by 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 My from NMP.

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

Once the irradiation period was 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 was cleaved from the Lantern fragment by treatment with a mixture of

TFA, water and TIS (95: 2.5: 2.5) for 1 hour. The Lantern fragment was removed with tweezers and the acidolytic mixture was evaporated almost to dryness under a stream of nitrogen until an oil was obtained. 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 lyophilized.

A powdery solid was obtained, the structure of which was analyzed by HPLC and confirmed by ESI-MS, MALDI-TOF or FAB-MS. Examples 4 to 6.- Preparation of peptides of the invention in solid phase using a SynPhase ™ Lantern as solid support and cyclizing using microwave radiation

Following a procedure analogous to that described in Example 3, the cyclic peptides shown in Table II were prepared:

TABLE II

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

Example 7.- Preparation of a solid phase cyclic peptide library using SvnPhase ™ Lanterns as solid support

A library of 56 cyclic peptides of 10 amino acids was prepared from 28 SynPhase ™ Lanterns of the commercial R-type polystyrene Rink amide type, each of which was divided into two.

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

All Lantern fragments were placed in a single glass container provided with a stopper and covered with CH ₂ Cl ₂ for 5 minutes to swell them. To remove the Fmoc protecting group, they were treated with 30 ml of a mixture of piperidine-CH ₂ Cl ₂ -NMP (1: 2: 2, 2 x 45 min). Washing was carried out by immersing the fragments in NMP (3 x 5 min) and CH ₂ Cl ₂ (2 x 5 min) using plastic syringes. After each washing step the solvent is removed by a multiple vacuum filter.

The Lantern fragments were then placed in a container provided 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 couplings were carried out by repeated cycles of elimination of the Fmoc group, coupling with a new amino acid, and washes, as already described above.

For each coupling with Fmoc-Lys (Boc) -OH and Fmoc-Leu-OH, the Lantern fragments 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 stage of coupling, the Lantern fragments were introduced into a single plastic syringe to perform the washes.

They were then separated again into two groups for the subsequent coupling of Fmoc-Lys (Boc) -OH or Fmoc-Leu-OH, or placed in the same container for the coupling of Fmoc-Phe-OH. Once all the couplings were finished to obtain linear peptides of 10 amino acids, the C-terminal allyl ester was removed, the Fmoc protective group was removed, and the washes were followed following the procedure described in Example 2. All these steps were They performed by placing all the Lantern fragments in a single container or syringe. The cyclization of each of the peptides was carried out in individual containers following the treatment described in Example 2.

Subsequently, the cyclic peptide was excised from the solid support, and the isolation of each of the peptides following the procedure described in Example 2. For this, each Lantern fragment was placed in an individual container.

The peptides obtained were analyzed by HPLC and their structure was confirmed by ESI-MS, MALDI-TOF or FAB-MS. The structures of the 56 cyclic decapeptides synthesized in this Example 7 are included in Figure 1.

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 stored at a temperature of -80 ° C in Luria-Bertani (LB) medium supplemented with 20% glycerin in volume / volume.

Samples of E. amylovora PMV6076 and P. syringae were obtained after 24 hour growth at 25 ° C, and the X.vesicatoria sample 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 minimum inhibitory concentration (MIC), the peptides were solubilized in sterile milli-Q water to a final concentration of 1000 μM and filtered through a 0.22 μm pore filter.

Dilutions of the peptides of the invention were made 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 corresponding bacterial indicator suspension, 160 μl of TSB liquid medium (Triptone Soy Broth), to a total volume of 200 μl in each well of a microplate. Thus the effective concentrations to which the bacterial suspensions were subjected were 75,

50, 25, 12.5, 6.25 and 3.12 μM.

Three replicates were carried out for each strain, concentration and peptide tested. Positive controls contained water instead of peptide, and negative controls contained peptides without bacterial suspension. Microbial growth was determined automatically by measuring the optical density at 600 nm using the Microbiology Analyzer Bioscreen C (Labsystems).

The microplates were incubated at 25 ° C for 48 hours with a shaking of 20 seconds before measuring the absorbency 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 was taken.

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 michiganensi $, Xanthomonas vesicatoria, and Pseudomonas syringae:

TABLE III

(N.D.: Not determined)

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 / ml using the Thoma counting chamber.

The peptides of the invention were solubilized in sterile milli-Q water to a final concentration of 1000 μM and filtered through a 0.22 μm pore filter. With said solution dilutions of the peptides were made to obtain solutions at concentrations 750, 500, 250, 125, 62.5, and 31.2 μM.

Then 20 µl of each dilution of peptide was mixed with 80 µl of the mushroom suspension, and with 100 µl of PDB medium at double concentration, until a total volume of 200 µl was obtained 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 were obtained. The plates 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 negative controls contained peptides without spore suspension.

The intensity of growth was determined by the area under the curve and the calculations were performed following the procedures described in KX.Reynolds, and D.A. Enher, Statistical comparison of epidemics p. 34-37, in D.A. Franclch Neher (Eds.), Exercises inplant 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

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 tests

The hemolytic activity of the peptides of the invention was evaluated by determining the release of hemoglobin that occurs upon contacting a solution of the peptides with a 5% volume red cell suspension from fresh human blood. Blood was collected aseptically using a Vacuntainer ^® system

K2E (Belliver, Great Britain) with EDTA, and was stored at 4 ^o 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 suspended in TRIS buffer until a suspension containing 10% by volume of erythrocytes was obtained.

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 10% volume red cell suspension was mixed with 65 µl of the peptide solution in each well of a 96-well Micro-Amp ^® plate (Applied Biosystems, USA) (5% by volume of erythrocytes), and the mixture was incubated for 1 h at 37 ° C with stirring. Thus the erythrocyte suspensions were 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 μl aliquots of the supernatant were transferred 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 control of complete hemolysis was determined in a TRIS buffer solution containing melitin (Sigma-Aldrich, Spain).

The percentage of hemolysis (H) was determined using the equation: H = 1 OQxKQp-ObV (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 the positive control with melitin.

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

TABLE V

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

Claims

REΓVINDICACIOΓNES

L- Cyclic peptides characterized in that they have a lysine molar content between 40% and 60%, and that they respond to the general formula (I): cycle [(X) _n - Y - (X) _1n - GIn)] (I) where: X is Lys and / or Leu,

Y is Phe or D-Phe, n = 1, 3, o5, m = 1.3, or 5, and n + m = 2.4.6o8.

2. Cyclic peptides according to claim 1 characterized in that in the general formula (I)

X is Lys and / or Leu, Y is Phe, and m = l, 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,

Y is 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 an antimicrobial composition.

7. Use according to claim 6 as antimicrobial agents against bacteria and fungi pathogenic to 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 / 1 and 0.5 g / 1.

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, Clavihacter 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 contacted with the parts of the plants selected from the group consisting of seeds, roots, stems, leaves, or fruits, or with the soil or any growth medium surrounding the roots of 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.