Patent Application
20220330548
This application includes a Sequence Listing submitted electronically in ASCII format. The ASCII copy of the Sequence Listing, created on Mar. 2, 2022, is named 19720-121_sequence-listing.txt and is 7,125 bytes in size. The ASCII copy of the Sequence Listing is expressly incorporated herein by this reference.
The present invention relates to novel analogue peptides of the natural peptaboil trichogin GA IV and their use as plant protection products for the treatment and/or prevention of diseases caused by pathogenic micro-organisms to the plants.
The present invention also relates to a plant protection composition comprising at least one of such peptides and its use as a plant protection product against pathogenic micro-organisms of the plants.
The present invention relates to the field of fighting biological pathogen agents of plant crops, in particular biological agents such as fungi, oomycetes and/or phytopathogenic bacteria.
In order to protect and prevent the damaging effects of biological agents on plants, numerous types of agrochemicals and biocides are in use.
The term agrochemical refers to a chemical compound, natural or synthetic, used in agriculture to fight plant pests and diseases, to protect crops from harmful agents, and to improve their productivity.
The term biocide, on the other hand, refers to:
Although the use of biocides and agrochemicals, especially synthetic ones, is aimed at ensuring human well-being and the conservation/development of many plant species, the chemicals contained therein can have harmful effects on human health, plant and animal organisms and, last but not least, on the environment. Their release into the environment, for example, can lead to accumulation phenomena in surface and groundwaters, soil and air.
The active ingredients contained in the agrochemicals are therefore subject to a strict evaluation regime and included in a list of substances permitted throughout the European Union.
The agrochemicals, moreover, can lose efficacy over time due to the emergence of resistant strains.
Therefore, those eco-sustainable agricultural models that offer productive solutions with a reduced environmental impact and increased healthiness of the products themselves are increasingly encouraged.
The improvement interventions that can be carried out in the development of new formulations of agrochemicals and biocides, in order to make them efficient and safe, are referable to the three macro-areas described below:
The use of analogue peptides of the natural peptaboil trichogin GA IV and their use as plant protection products are part of this improvement perspective.
As known from document WO2020003220 on behalf of the applicant, peptaboils are secondary metabolites produced by fungi belonging to the genus Trichoderma. These peptaboils are known for their ability to protect plants from pest attacks as they possess antimicrobial activity; some peptaboils act as plant defence stimulants and induce volatile compounds in plants that attract natural enemies of herbivorous insects (Vos C M F et al, Mol Plant Pathol, 2015).
Peptaibols are peptides produced by fungi in a non-ribosomal manner. They are peptides rich in Aib (alpha-aminoisobutyric acid) amino acid residues and have an aminoalcohol as the C-terminus group. There is also an acyl group at the N-terminus end (a 1-octanoyl group in the case of the peptaibol trichogin GA IV).
Eight peptide analogues of peptaboils having plant protection activity have been described in the mentioned document.
These peptides are advantageous in that they can be used in purified form, are water-soluble and are stable to solar irradiation.
However, these known peptides have some drawbacks.
In particular, such peptides show limited efficacy against certain pathogenic micro-organisms of cereal crops, such as Pyricularia oryzae.
Still disadvantageously, such peptides are not particularly active in fighting infections caused on plants by Gram-negative phytopathogenic bacteria.
Moreover, inconveniently, such peptides are particularly expensive to produce due to the complexity of their sequence.
The object of the present invention is therefore to provide new peptides derived from peptaibols to be used as plant protection products.
In particular, an object of the present invention is that such peptides are suitable for use in fighting pathogenic micro-organisms of the plants, in particular against phytopathogenic bacteria.
Furthermore, an object of the present invention is that such peptides are alternative to those known, in particular are alternative to those described in document WO2020003220, and exhibit a higher plant protection activity than the latter and overcome the known drawbacks thereof.
Again, an object of the present invention is that such peptides can be easily manipulated and used by operators, thus limiting or completely eliminating health risks for agricultural operators and/or end users of plants treated with such peptides.
Furthermore, an object of the present invention is that such peptides exhibit a wide range of efficacy.
Further, an object of the present invention is that such peptides, after carrying out their plant protection activity, are adapted to be degraded into non-toxic amino acids, thus limiting the environmental impact of their use on plants, in particular on agricultural crops. Again, an object of the present invention is that such peptides are stable under extreme temperature, irradiation and pH conditions, so as to be suitable for use in the field, regardless of the atmospheric and environmental conditions.
Last but not least, an object of the present invention is that such peptides are easily producible, so as to reduce their production cost.
The aforesaid objects are achieved by a peptide as set forth in claim 1 and by the use of such a peptide or of a salt thereof as a plant protection product against pathogenic micro-organisms of the plants, as described in claim 10.
In particular, the present invention relates to the use of a peptide or of a salt thereof as a plant protection product against pathogenic micro-organisms of the plants, wherein such a peptide has general formula (I)
1-octanoyl-X-Aib-Y-Z (I),
Furthermore, the objects are also achieved by a plant protection composition and the use of said plant protection composition as a plant protection product against pathogenic micro-organisms of the plants, as set forth in claims 8 and 10, respectively.
Further characteristics of the peptide, the use of the peptide, the plant protection composition and the use of the plant protection composition are set forth in the dependent claims.
In addition, other advantages and features of the peptide, the use of the peptide, the plant protection composition and the use of the plant protection composition will be apparent to a person skilled in the art from the following description of some preferred embodiments of the invention which are given by way of indication but not of limitation.
It should be noted that in the present document, where the use of the peptide, of a salt thereof or of a plant protection composition as a plant protection product against pathogenic micro-organisms of the plants is set forth, such use is intended to correspond to a method for treating a pathogenic micro-organism of the plants wherein such method provides for contacting the pathogenic micro-organism with an effective amount of the peptide of the invention or of a salt thereof.
In this document, the term “effective amount” refers to that amount of a peptide or of a salt thereof or of a plant protection composition which, when applied to a plant, is sufficient to eliminate, inhibit or otherwise control the pathogenic micro-organism of the plant. Thus, for example, an effective amount of peptide as described herein is an amount of peptide sufficient to cause inhibition of the pathogenic micro-organism such that the effects of the latter in the plant are prevented, reduced or alleviated, as will be shown in the figures and examples given below.
As mentioned above, the present invention relates to the use of a peptide or of a salt thereof as a plant protection product against pathogenic micro-organisms of the plants, wherein such a peptide has general formula (I)
1-octanoyl-X-Aib-Y-Z (I),
In particular, the use of a peptide having a sequence selected from 1-octanoyl-Aib-Gly-Leu-Aib-Lys(HCl)-Lys(HCl)-Leu-Aib-Gly-Ile-Lol, 1-octanoyl-Aib-Gly-Leu-Aib-Lys(HCl)-Lys(HCl)-Aib-Gly-Ile-Leu-NH2, 1-octanoyl-Aib-Lys(HCl)-Lys(HCl)-Leu-Aib-Gly-Ile-Lol and 1-octanoyl-Aib-Lys(HCl)-Lys(HCl)-Leu-Aib-Gly-Ile-Leu-NH2 is to be considered excluded from the present invention.
It should be noted that the peptides having general formula (I) are different from those known, in particular from those described in document WO2020003220, because none of the known peptides have a sequence that is included in the general formula of the present invention and which is to be considered with the exclusions indicated above.
The peptides of the present invention are thus new compared to those described in this document, and, in addition, they show a greater plant protection activity than such known peptides, as will be apparent to a person skilled in the art from the examples given below.
It is specified that, in this document:
With regard to the other amino acids described, they are indicated by their three-letter code: Leu is leucine, Gly is glycine, Ile is isoleucine.
Preferably, the chiral amino acids are in L configuration, unless otherwise specified.
It is further specified that the peptide of the invention may be used alone or in combination with other peptides of the present invention and, in addition, may be prepared in the form of a composition, as will be set forth in more detail later.
According to one aspect of the invention, the aforesaid peptide is used to fight and/or prevent infections caused by oomycetes, fungi and phytopathogenic bacteria in plants.
In particular, the aforesaid peptide is used to fight and/or prevent infections in plants caused by Botrytis cinerea, Plasmopara viticola, Fusarium graminearum, Pyricularia oryzae and Xanthomonas campestris.
Preferably, such a peptide is used to fight and/or prevent infections caused by phytopathogenic bacteria, preferably Gram negative bacteria, more preferably Xanthomonas campestris.
More preferably, the peptide is used to fight and/or prevent infections caused by pathogenic micro-organisms on vine plants, cereals and horticultural crops.
According to one aspect of the invention, the use of such a peptide provides for applying such a peptide to the plant(s) to be treated.
Preferably, the use of the peptide provides for dissolving the aforesaid peptide in an aqueous solvent prior to application on the plant(s) to be treated so as to favour the dispersion thereof on the same plant(s).
Preferably, the aforesaid aqueous solvent is water.
Advantageously, in fact, the peptide of the invention is soluble in water at a concentration >10 mM.
It should be noted that this peptide is also soluble in polar or protic organic solvents.
Still preferably, the peptide of the invention is applied at a concentration comprised between 10-100 μM on each of the aforesaid plants, more preferably at a concentration of about 50 μM.
However, it is not excluded that the peptide is used and applied on plants at a concentration other than that indicated.
In such a case, an expert in the field is to be considered capable of assessing, depending on the type of plant disease and the severity thereof, what will be the optimal concentration of the peptide for its use according to the invention and the frequency of application thereof on the plants to be treated.
Advantageously, the peptide of the invention applied on the plant shows excellent efficiency in preventing infection by blocking the growth of the pathogenic micro-organism.
These characteristics make its use on plants, especially on crops, very advantageous, especially for the preventive use on plants, especially on vine plants, cereals and horticultural crops.
Without wishing to be bound by any theory, from the results of the experiments carried out, the inventors believe that at least some of the efficacy of the peptide of the invention is due to its ability to cause damages to the cell membranes of the pathogenic micro-organism, inducing cell death.
Advantageously, moreover, the aforesaid peptide is a product that is fully degradable into non-toxic amino acids.
The peptide of the invention was in fact found to be completely degraded into amino acids by the action of the enzyme trypsin.
Still advantageously, the peptide of the invention is water-soluble, making its use particularly practical in the field.
Another advantage of using the peptide as a plant protection product is that it is completely harmless to plants at even very high concentrations (1 mM).
Preferably, the peptide of the invention is selected from:
According to another aspect of the invention, the peptide has general formula (I) wherein X is selected from Aib-Lys(HCl)-Leu and Aib-Gly-Leu; Y is selected from Lys(HCl)-Gly-Leu-Aib-Lys(HCl), Lys(HCl)-Lys(HCl)-Leu-Aib-Lys(HCl), Gly-Gly-Leu-Aib-Lys(HCl) and Lys(HCl)-Lys(HCl)-Leu-Aib-Gly; Z is selected from Ile-Lol and Ile-Leu-NH2.
Preferably, according to this aspect of the invention, such a peptide is selected from the group consisting of the peptides having SEQ. ID. Nos. from 1 to 8.
Even more preferably, the peptide is selected from the peptides having SEQ. ID. Nos. 2, 4, 6 and 8.
According to a further aspect of the invention, the peptide has general formula (I) wherein X is Leu or X is absent; Y is Lys(HCl) and wherein Z is selected from Lol, Ilol, Ile-NH2, Leu-NH2, Ile-Lol and Ile-Leu-NH2.
In particular, according to this further aspect of the invention, the peptide preferably has a sequence selected from the group consisting of SEQ. ID. Nos. from 9 to Advantageously, the peptides having SEQ. ID. Nos. from 9 to 16 are particularly suitable for being synthesised by synthetic strategies in solution which, as is known, represent techniques that are easier to carry out and cheaper than solid-phase peptide synthesis.
It is not excluded that, according to embodiment variants, one or more of the peptides of the present invention are made by solid-phase techniques or by techniques other than that indicated.
Therefore, an object of the present invention is also a peptide having general formula (I)
1-octanoyl-X-Aib-Y-Z (I),
In particular, the peptide having a sequence selected from 1-octanoyl-Aib-Gly-Leu-Aib-Lys(HCl)-Lys(HCl)-Leu-Aib-Gly-Ile-Lol, 1-octanoyl-Aib-Gly-Leu-Aib-Lys(HCl)-Lys(HCl)-Aib-Gly-Ile-Leu-NH2, 1-octanoyl-Aib-Lys(HCl)-Lys(HCl)-Leu-Aib-Gly-Ile-Lol and 1-octanoyl-Aib-Lys(HCl)-Lys(HCl)-Leu-Aib-Gly-Ile-Leu-NH2 is to be considered excluded from the present invention.
Preferably, the peptide according to the invention has general formula (I) having a sequence selected from the group consisting of SEQ. ID. Nos. from 1 to 16.
According to one aspect of the present invention, the aforesaid peptide has general formula (I) wherein X is selected from Aib-Lys(HCl)-Leu and Aib-Gly-Leu; Y is selected from Lys(HCl)-Gly-Leu-Aib-Lys(HCl), Lys(HCl)-Lys(HCl)-Leu-Aib-Lys(HCl), Gly-Gly-Leu-Aib-Lys(HCl) and Lys(HCl)-Lys(HCl)-Leu-Aib-Gly; Z is selected from Ile-Lol and Ile-Leu-NH2.
According to this aspect of the invention, the peptide is selected from the peptides having SEQ. ID. Nos. from 1 to 8.
Preferably, the aforesaid peptide is selected from the group consisting of the peptides having SEQ. ID. Nos. 2, 4, 6 and 8.
According to another aspect of the invention, the peptide of the invention has general formula (I) wherein X is Leu or X is absent; Y is Lys(HCl) and Z is selected from Lol, Ilol, Ile-NH2, Leu-NH2, Ile-Lol and Ile-Leu-NH2.
Preferably, this peptide is selected from the peptides having SEQ. ID. Nos. from 9 to 16.
More preferably, such a peptide is the peptide having SEQ. ID. No. 10.
According to one aspect of the invention, the peptide of the present invention is 1-octanoyl-Aib-Lys(HCl)-Leu-Aib-Lys(HCl)-Gly-Leu-Aib-Lys(HCl)-Ile-Lol (SEQ. ID. No. 1 or “K259G6”).
According to one aspect of the invention, the peptide of the present invention is 1-octanoyl-Aib-Lys(HCl)-Leu-Aib-Lys(HCl)-Gly-Leu-Aib-Lys(HCl)-Ile-Leu-NH2 (SEQ. ID. No. 2 or “K259G6-NH2”).
According to one aspect of the invention, the peptide of the present invention is 1-octanoyl-Aib-Lys(HCl)-Leu-Aib-Lys(HCl)-Lys(HCl)-Leu-Aib-Lys(HCl)-Ile-Lol (SEQ. ID. No. 3 or “K2569”).
According to one aspect of the invention, the peptide of the present invention is 1-octanoyl-Aib-Lys(HCl)-Leu-Aib-Lys(HCl)-Lys(HCl)-Leu-Aib-Lys(HCl)-Ile-Leu-NH2 (SEQ. ID. No. 4 or “K2569-NH2”).
According to one aspect of the invention, the peptide of the present invention is 1-octanoyl-Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Lys(HCl)-Ile-Lol (SEQ. ID. No. 5 or “K9”).
According to one aspect of the invention, the peptide of the present invention is 1-octanoyl-Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Lys(HCl)-Ile-Leu-NH2 (SEQ. ID. No. 6 or “K9-NH2”).
According to one aspect of the invention, the peptide of the present invention is 1-octanoyl-Aib-Lys(HCl)-Leu-Aib-Lys(HCl)-Lys(HCl)-Leu-Aib-Gly-Ile-Lol (SEQ. ID. No. 7 or “K256”).
According to one aspect of the invention, the peptide of the present invention is 1-octanoyl-Aib-Lys(HCl)-Leu-Aib-Lys(HCl)-Lys(HCl)-Leu-Aib-Gly-Ile-Leu-NH2 (SEQ. ID. No. 8 or “K256-NH2”).
According to one aspect of the invention, the peptide of the present invention is 1-octanoyl-Leu-Aib-Lys(HCl)-Ile-Lol (SEQ. ID. No. 9).
According to one aspect of the invention, the peptide of the present invention is 1-octanoyl-Leu-Aib-Lys(HCl)-Ile-Leu-NH2 (SEQ. ID. No. 10).
According to one aspect of the invention, the peptide of the present invention is 1-octanoyl-Leu-Aib-Lys(HCl)-Lol (SEQ. ID. No. 11).
According to one aspect of the invention, the peptide of the present invention is 1-octanoyl-Leu-Aib-Lys(HCl)-Leu-NH2 (SEQ. ID. No. 12).
According to one aspect of the invention, the peptide of the present invention is 1-octanoyl-Leu-Aib-Lys(HCl)-Ilol (SEQ. ID. No. 13).
According to one aspect of the invention, the peptide of the present invention is 1-octanoyl-Leu-Aib-Lys(HCl)-Ile-NH2 (SEQ. ID. No. 14).
According to one aspect of the invention, the peptide of the present invention is 1-octanoyl-Aib-Lys(HCl)-Ile-Lol (SEQ. ID. No. 15).
According to one aspect of the invention, the peptide of the present invention is 1-octanoyl-Aib-Lys(HCl)-Ile-Leu-NH2 (SEQ. ID. No. 16).
As indicated above, the present invention also relates to a plant protection composition comprising at least one peptide of the invention, or a salt thereof, as defined above, including variants, and a phytopharmaceutically acceptable carrier and/or excipient.
Preferably, the aforesaid carrier is water.
According to the invention, the plant protection composition comprises at least one peptide of the invention, or a salt thereof, selected from the peptides having SEQ. ID. Nos. from 1 to 16.
According to one aspect of the plant protection composition of the invention, the plant protection composition comprises at least one peptide of the invention, or a salt thereof, having general formula (I) wherein X is selected from Aib-Lys(HCl)-Leu and Aib-Gly-Leu; Y is selected from Lys(HCl)-Gly-Leu-Aib-Lys(HCl), Lys(HCl)-Lys(HCl)-Leu-Aib-Lys(HCl), Gly-Gly-Leu-Aib-Lys(HCl) and Lys(HCl)-Lys(HCl)-Leu-Aib-Gly, and wherein Z is selected from Ile-Lol and Ile-Leu-NH2.
Preferably, such at least one peptide is selected from the peptides having SEQ. ID. Nos. from 1 to 8.
More preferably, according to this aspect of the composition of the invention, the aforesaid composition comprises at least one peptide selected from the peptides having SEQ. ID. Nos. 2, 4, 6 and 8.
According to another aspect of the invention, the plant protection composition comprises at least one peptide of the invention, or a salt thereof, having general formula (I) wherein X is Leu or X is absent; Y is Lys(HCl) and wherein Z is selected from Lol, Ilol, Ile-NH2, Leu-NH2, Ile-Lol and Ile-Leu-NH2.
Preferably, such at least one peptide is selected from the peptides of the invention having SEQ. ID. Nos. from 9 to 16.
It should be noted that the plant protection composition of the invention may comprise one or more peptides of the present invention described above.
Additionally, the plant protection composition of the invention may further comprise one or more excipients of the known type which will be selected according to usual practice.
Such excipients may include, among others, co-formulant compounds that serve to reduce the concentration of the peptide, such as inert substances and diluents.
In addition, these excipients may include adjuvant compounds, which are intended to increase the efficacy of the peptide and promote its distribution.
Examples of such adjuvants comprise synergists, emulsifiers, wetting agents, adhesives, humectants, propellants for aerosol, inert diluents, anti-drift, anti-foaming, preservative formulations, and the like.
The plant protection composition of the present invention may be in liquid or solid form.
Examples of liquid formulations comprise aqueous solutions in which the peptide is finely dissolved in water to form a stable, diluted or concentrated solution.
Optionally, this solution is also mixed with wetting, dispersing, inert and other per se known excipients.
The formulation of the plant protection composition of the invention in liquid form is particularly preferred for ease of application on the plant by spraying.
Examples of solid formulations of the plant protection composition of the invention comprise granules and dry powders.
Although the peptide of the invention is already itself absorbable by the plant on which it is applied, it is not excluded that the plant protection composition of the invention comprising this peptide is used in the form of a formulation for endotherapic treatments, i.e., by injection of the same into the trunk of the plant.
In such endotherapic formulation, the plant protection composition will comprise adjuvants especially formulated and known in the state of the art for being injected along the xylematic vessels and spreading along them.
The plant protection composition of the invention is advantageously suitable for use as a plant protection product in plants.
In particular, the aforesaid composition is particularly suitable for use for the treatment of vine plants, cereals and horticultural crops, preferably for the treatment and/or prevention of plants against infections caused by Botrytis cinerea, Plasmopara viticola, Fusarium graminearum, Pyricularia oryzae or Xanthomonas campestris.
The use of the previously defined plant protection composition, including variants, as a plant protection product against pathogenic micro-organisms of plants, preferably as a plant protection product for the treatment and/or prevention of infections in plants caused by oomycetes, fungi and phytopathogenic bacteria of plants, such plants being preferably vine plants, cereals, horticultural crops is therefore also part of the present invention.
Further characteristics and advantages of the peptides, the plant protection composition and their use will be apparent to a person skilled in the art from the examples below, which are provided for the purpose of a better understanding of what is described and are not intended as limitations of the claims.
The peptide of the present invention can be synthesised following a manual, semi-automatic or automatic solid phase, solution synthesis methodology. The chiral amino acids used have an L configuration, unless otherwise specified.
Synthesis in solution takes place from the primary amide of the last amino acid of the desired sequence.
An exemplary synthetic protocol in solution starts from the synthesis of H-Leu-NH2, from H-Leu-OCH3 (leucine methyl ester) and ammonia in methanol. The synthesis proceeds with the condensation, using appropriate activating reagents, of the subsequent amino acid, protected to the amino function, e.g., as benzyloxycarbonyl (Z). The removal of the protective group (e.g., by catalytic hydrogenation) closes the cycle. The subsequent residues are incorporated by a similar process. Each intermediate is isolated and characterised. The amino acid lysine is inserted protected in side chain (e.g., as tert-butoxycarbonyl). This protective group is removed as the last stage of the synthesis, by acid treatment (e.g., with a mixture of trifluoroacetic acid in dichloromethane).
The raw peptides are obtained with a degree of purity greater than 70% and purified at >95% by chromatography, flash chromatography on silica, semi-automatic medium pressure chromatography or preparative high pressure liquid chromatography (H PLC).
The characterisation of the peptides and the determination of the degree of purity are obtained by high-resolution mass spectrometry analysis (electron spray ionization time-of-fly, ESI-TOF), analytical HPLC, nuclear magnetic resonance (NMR).
For the chemoenzymatic peptide synthesis, protected residues and/or segments of the sequence are synthesised in solution which are then condensed by means of enzymes such as the enzyme papain (Kayo T. et al, ACS Biomaterials Science & Engineering, 2020), trypsin or others. Chemoenzymatic synthesis, for example, provides for the preparation of protected residues such as Z and benzyl esters or as amides. Each residue may have one or more enzymes that catalyse the formation of the peptide bond.
An example of a synthetic solid-phase protocol uses “amide Rink” resins or 2-chlorotritic resin preloaded with aminoalcohol L-leucinol or Isoleucinol. Both are commercial resins and used at 100-200 or 200-400 mesh and different loading degree.
The synthesis proceeds starting from the C-terminus amino acid towards the N-terminus amino acid with a step-by-step process.
The protocol provides for the use of the fluorenylmethyloxycarbonyl- (Fmoc-) protective group for the protection of the amino group in alpha. This protective group is removed in basic conditions, for example by treatment with piperidine (PIP) 20% in dimethylformamide (DMF) or other secondary amines in different percentages and in other organic solvents. The protective group used for the protection of the amino group in the side chain is tert-butyloxycarbonyl (Boc), removable by acid treatment, for example with a solution of HCl 3M in methanol or other organic solvents, or trifluoroacetic acid in different percentages in dichloromethane or other organic solvents.
The formation reactions of the amide bond take place through activation of the carboxyl group of the incoming amino acid (protected in its amine function and possible side chains) through the use of different activating agents depending on the amino acid.
By way of non-limiting example, it is possible to use: (i) a 1:1 mixture of 1-hydroxy-1,2,3-benzotriazole (HOBt) and O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro phosphate (HBTU); (ii) a 1:1 mixture of 1-hydroxy-1,2,3-benzotriazole (HOBt) and O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU); (iii) O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU); (iv) ethyl cyano(hydroxyimino)acetate (Oxyma pure); (v) K-Oxyma; (vi) a 1:1 mixture of N-ethyl-N′-(3-dimethylamino)propylcarbodiimide (EDC) or diisopropylcarbodiimide (DIC) or dicyclohexylcarbodiimide (DCC) and HOBt; (vii) a 1:1 mixture of EDC or DIC or DCC and HOAt; again in DMF solution.
The incoming amino acid excess varies between 1.5 and 3 equivalents and the activating reactants are added in an equimolar quantity with respect to the incoming amino acid. The tertiary base is added in twice the amount of the incoming amino acid when HATU or HBTU are agents used as activators. Base addition is not necessary with OXYMA PURE or K-Oxyma.
As a tertiary base it can used (by way of non-limiting example): diisopropylethylamine, triethylamine, or N-methylmorpholine.
The n-octanoyl group is inserted at the N-terminus end using the same methods of activation of the carboxylic group previously described. The formation reactions of the amide bond also take place through the use of the enzyme papaine. The peptide is released from the resin, previously dried with dichloromethane washes and permanence under vacuum, by acid treatment.
By way of example: from the “amide Rink” resin, the peptide can be released by treatment with a mixture of trifluoroacetic acid (TFA) 95%, water 2.5% and triisopropylsilane 2.5%. The collected solution is brought to dryness. The solid or oily precipitate obtained is then washed several times with diethyl ether; from the 2-chlorotritilic resin, the peptide is released with repeated treatments of variable duration from one hour to overnight with a solution of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) 30% in dichloromethane (DCM). Alternatively, a solution with various percentages of TFA in DCM can be also used. When using the 2-chlorotryl resin, the protective group Boc is removed in solution by the treatments described above, e.g., by treatment with 3M hydrochloric acid in methanol. In some cases, the protected peptides Boc are purified by aqueous acid and/or basic washes, followed by anhydrification over anhydrous Na2SO4 or MgSO4.
The raw peptides are obtained with a degree of purity greater than 70% and purified at >95% by chromatography, flash chromatography on silica, semi-automatic medium pressure chromatography or preparative high pressure liquid chromatography (H PLC).
The characterisation of the peptides and the determination of the degree of purity are obtained by high-resolution mass spectrometry analysis (electron spray ionization time-of-fly, ESI-TOF), analytical HPLC, nuclear magnetic resonance (NMR).
Botrytis cinerea (strain PM10) was cultivated at 25° C. on potato dextrose agar (PDA). Pyricularia oryzae (strain IT10) was initially cultivated on PDA culture medium at 25° C. and then on oat meal agar (OMA). F. graminearum (strain 8/1) was cultivated on PDA at 25° C. and subsequently on synthetic nutrient agar (SNA). After a few days the spores (conidia) of each plate were collected in 6 mL of sterile water containing glycerol (10% v/v).
The antifungal activity of the peptides was determined against B. cinerea, P. oryzae and F. graminearum in microtitre plates containing in each well: 200 μL of potato dextrose broth (PDB; pH 6.9), the peptide of interest and, alternatively to each other, spores of B. cinerea (5×105 mL−1), F. graminearum (5×105 mL−1) or P. oryzae (1×105 mL−1). Peptide-free controls were also prepared. Each peptide was tested in three independent replicates.
After 96 hours of incubation in the dark at 25° C., growth was measured spectrophotometrically at 450 nm and fungal growth was expressed as a percentage of the absorbance values of each well compared to the maximum absorbance measured in the control wells not inoculated with peptides.
The peptides were assayed in microtitre plates against a strain of X. campestris pv. campestris isolated from cauliflower. Each well contained 200 μL of LB substrate, 1×106 CFU/mL of bacterium and the peptide of interest (15 μM). The reduction in growth was determined after 48 h by spectrophotometrically measuring the reduction in turbidity (A600 nm) of the wells containing the peptides compared to the wells without peptides. Each peptide was assayed in 3 different wells.
Microscopic observations of the peptide-treated spores were performed by optical microscopy (Laborlux 12) after 24 hours suspension of the spores in PDB. The spores of P. oryzae were also observed up to 48 h by fluorescence microscopy (Leica DM 4000B).
Infection tests were performed on vine leaf disks (cv. Glera) of 1 cm in diameter. Twenty leaf discs obtained from randomly collected leaves from different plants were placed with the lower side facing upwards on moistened sterile paper placed in Petri dishes (15 cm diameter). Approximately 0.1 mL of each aqueous solution containing the peptide (50 μM) was distributed on each leaf disc. Aqueous suspensions of P. viticola sporangia, collected from infected vine plants, were diluted to obtain 3-5×105 sporangia/mL and distributed on the leaf discs. The plates containing the leaf discs were incubated in the dark at room temperature (22-23° C.). The incidence was calculated 12 days after inoculation as the ratio of the number of sporulating discs to the total number of discs inoculated. Control plates contained water-treated leaf discs. Two or three plates were used for each treatment. The data obtained were statistically analysed by applying the Anova and Bonferroni-Holmes one-way test.
The first leaves of 6-day-old barley seedlings were inoculated at two points by pipetting 10 μL of a suspension containing the peptide at 50 μM and 1×103 spores of P. oryzae strain IT10. For each peptide, at least five leaves were inoculated in each experiment and the experiment was performed at least three times. Water-treated leaves inoculated with fungal conidia were used as a positive control. The lesion area was calculated after 7 days by Assess© Software (APS).
Flowering ears of wheat were inoculated by spraying with a solution containing 50 μM of each peptide and 5×105 spores mL−1 of F. graminearum. At least eight ears were inoculated for each treatment in each experiment. Ears sprayed with water and fungal spores were used as a positive control. The percentage of infection was determined by counting the number of visually symptomatic spikelets compared to the total number of spikelets of the respective ear. The experiments with each peptide were repeated at least three times.
| Number | Date | Country | Kind |
|---|---|---|---|
| 102021000005057 | Mar 2021 | IT | national |
