DE102022120408A1 - Cycloacetamide, new natural product from Mortierella alpina, process for its production, its use and Mortierella alpina DSM 34346 - Google Patents

Abstract

The invention relates to the natural substance cycloacetamide A to F as a new matebolite from Mortierella alpina, a process for its production, its use and the strain Mortierella alpina DSM 34346. The object of the present invention is a new natural substance (bioactive substance) from a group that has hardly been researched to date of basal evolutionary fungi, such as Mortierella spec., through isolation and identification, which represents a new pharmaceutical lead structure, to provide a biological process for its economical and time-saving production with high product yield and without any special equipment effort and alternatively to provide a chemical synthesis process for its production and its Use (bioactivity) is solved by providing cycloacetamides A to F, which correspond to the general formula cyclo-(-N-Ac-L-Thr-A-B-C-O-), where L-Thr represents L-configured threonine and , in which A, B and C independently represent identical or different L- or D-configured amino acids with non-polar side chains, in particular alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met) , phenylalanine (Phe), tyrosine (Tyr) or tryptophan (Trp), whereby the fungus Mortierella alpina is cultivated in nutrient medium and cycloacetamides A to F are formed, which are extracted from the nutrient medium and as a concentrated extract by preparative and semi-preparative HPLC into pure substances of cycloacetamide A to F is separated, whereby cycloacetamides A to F act as an insecticide and the producer strain is Mortierella alpina DSM 34346, or a chemical synthesis route is shown as an alternative to the biological production of cycloacetamides A to F, whereby cycloacetamides A to F can be used as an insecticide are.

Description

The invention relates to the natural substance cycloacetamide, in particular the cycloacetamides A to F, which is synthesized as a natural substance by the fungus Mortierella alpina during fermentation and released into the culture supernatant or the mycelium, a manufacturing process and the biological effectiveness of the cycloacetamides A to F and the Producer strain / fungus strain Mortierella alpina DSM 34346.

In natural product research as well as in pharmacy, new active ingredients are traditionally isolated from bacteria or higher fungi (Asco- or Basidiomycetes). However, the same substances or substance classes often re-identify. It is therefore expedient to look for new active ingredient producers that have not previously been the focus of natural product research.

Natural substances have been isolated from microorganisms for hundreds of years and form the basis for more than a third of all antibiotics, immunosuppressants and cytostatics available on the market.

Natural products are low-molecular chemical compounds that come from secondary metabolism and are formed by plants but also by microorganisms. The most important pharmaceutical producers are bacteria and higher fungi from the Ascomycetes and Basidiomycetes divisions.

Basal fungi, i.e. fungi that developed early in evolution, such as Mortierella alpina, have not yet been the focus of pharmaceutical drug research because they were subject to the dogma that they cannot produce natural substances.

Basal fungi - grouped in the paraphylum “Zygomycetes” until 2016 - have only come into the focus of natural products chemists in the last few years because, contrary to previous dogma, some representatives are able to form secondary metabolites. The representative Mortierella alpina belongs to the subphylum of Mortierellomycotina and is already used as a GRAS organism (Generally Recognized As Safe) in the food and feed industry for the production of oils with polyunsaturated fatty acids such as arachidonic acids and γ -Linolenic acid fermented on an industrial scale.

It has only been known for a few years that Mortierella alpina also produces peptidic natural products, namely the biosurfactants malpinin AD (hexapeptide), the antimycobacterial substance calpinactam (hexapeptide) and the antibiotic malpicycline AD (cyclopentapeptide) ( Baldeweg F, Warncke P, Fischer D, Gressler M. Fungal biosurfactants from Mortierella alpina. Org Lett. 2019 Mar 1;21(5):1444-1448 . doi: 10.1021/acs.orglett.9b00193. Epub 2019 Feb 21. PMID: 30789272; Wurlitzer JM, Stanišić A, Wasmuth I, Jungmann S, Fischer D, Kries H, Gressler M. Bacterial-Like Nonribosomal Peptide Synthetases Produce Cyclopeptides in the Zygomycetous Fungus Mortierella alpina. Appl Environ Microbiol. 2021 Jan 15;87(3):e02051-20 . doi: 10.1128/AEM.02051-20. PMID: 33158886; PMCID: PMC7848919; Koyama N, Kojima S, Nonaka K, Masuma R, Matsumoto M, Omura S, Tomoda H. Calpinactam, a new anti-mycobacterial agent, produced by Mortierella alpina FKI-4905. J Antibiot (Tokyo). 2010 Apr;63(4):183-6 . doi: 10.1038/ja.2010.14. Epub 2010 Feb 26. PMID: 20186169.).

In addition, the fungus Mortierella alpina also produces malpibaldine AC, hydrophobic cyclopentapeptides with unknown function ( Baldeweg F, Warncke P, Fischer D, Gressler M. Fungal biosurfactants from Mortierella alpina. Org Lett. 2019 Mar 1;21 (5): 1444-1448 . doi: 10.1021/acs.orglett.9b00193. Epub 2019 Feb 21. PMID: 30789272).

Mortierella alpina occurs ubiquitously and can be isolated from the soil primarily in the temperate, subpolar and polar zones.

A few years ago, Mortierella species were isolated from Antarctica that have an insecticidal effect on the pupae of the common housefly and on the larvae of the wax moth, although the reason for this phenomenon has not been described ( Edgington S, Thompson E, Moore D, Hughes KA, Bridge P. Investigating the insecticidal potential of Geomyces (Myxotrichaceae: Helotiales) and Mortierella (Mortierellacea: Mortierellales) isolated from Antarctica. Springer plus. 2014 Jun 9,3:289. doi: 10.1186/2193-1801-3-289 . PMID: 25013747; PMCID: PMC4071458.).

A large number of Mortierella alpina isolates have been deposited in the strain collections of the Jena Microbial Resource Collection (JMRC) and were genetically re-identified as such in 2013 ( Wagner L, Stielow B, Hoffmann K, Petkovits T, Papp T, Vägvölgyi C, de Hoog GS, Verkley G, Voigt K. A comprehensive molecular phylogeny of the Mortierellales (Mortierellomycotina) based on nuclear ribosomal DNA. Persoonia. 2013; 30:77-93 . doi: 10.3767/003158513X666268. PMID: 24027348; PMCID: PMC3734968).

Insecticides are pesticides that inhibit growth and kill adult insects or their developmental stages (egg, larva, pupa). There are currently a lot of insecticides on the market, which are often produced in an energy-intensive manner through multi-stage synthesis and whose toxic (halogenated or sulfurized) by-products then have to be disposed of at high cost. Since insects are also eukaryotes, common insecticides are often toxic to higher organisms (animals and humans). In accordance with the specifications of the Insecticide Resistance Action Committee (IRAC), an association of well-known insecticide producing companies (https://irac-online.org/), insecticides are divided into 26 classes according to their active target and chemical structure.

Most of the 26 synthetic compounds have a non-specific effect (i.e. across species) and contain halogens (fluorine, chlorine) or sulfur, which reduces environmental compatibility and increases toxicity for humans.

Proteins or peptides, which are completely underrepresented in the above-mentioned substance classes, therefore represent an alternative.

Proteins such as the Bacillus thuringensis toxins mentioned above are already used in the agricultural industry as anti-feeding agents, but are chemically less stable because they can be hydrolytically broken down by proteases ( Palma L, Muñoz D, Berry C, Murillo J, Caballero P. Bacillus thuringiensis toxins: an overview of their biocidal activity. Toxins (Basel). 2014 Dec 11;6(12):3296-325 . doi: 10.3390/toxins6123296. PMID: 25514092; PMCID: PMC4280536.).

To date, only a few insecticides, albeit of pharmaceutical/agricultural significance, have been obtained from microorganisms, plants or worms. These include the avermectins (macrolides from Streptomyces avermitilis), milbemycins (macrolides from Streptomyces hygroscopicus), spinosyne (cyclic polyketide from the bacterium Saccharopolyspora spinosa), nereis toxin (lower organodisulfide from the annelid Lumbriconereis heteropoda) and the above-mentioned Bacillus thuringensis toxins (small ribosomal proteins formed).

Cyclic oligopeptides with insecticidal activity are very rare. The non-ribosomally formed cyclodepsioctapeptide bassianolide from Beauveria bassiana shows, in addition to insecticidal activity, inhibition of acetylcholine-induced smooth muscles as well as antiplasmodial and antimycobacterial activities.

Bassianolide therefore has a rather unspecific effect ( Xu Y, Orozco R, Kithsiri Wijeratne EM, Espinosa-Artiles P, Leslie Gunatilaka AA, Patricia Stock S, Molnar I. Biosynthesis of the cyclooligomer depsipeptide bassianolide, an insecticidal virulence factor of Beauveria bassiana. Fungal Genet Biol. 2009 May;46(5):353-64 . doi: 10.1016/j.fgb.2009.03.001. Epub 2009 Mar 11. PMID: 19285149).

The closely related compound PF1022A, isolated from Mycelia sterila, has anthelmintic but not insecticidal effects ( Harder A, Schmitt-Wrede HP, Krücken J, Marinovski P, Wunderlich F, Willson J, Amliwala K, Holden-Dye L, Walker R. Cyclooctadepsipeptides--an anthelmintically active class of compounds exhibiting a novel mode of action. Int J Antimicrob Agents. 2003 Sep;22(3):318-31 . doi: 10.1016/s0924-8579(03)00219-x. PMID: 13678839.).

The cyclic hexadepsipeptides beauvericin from Beauveria bassiana and enniatin from Fusarium spec. are insecticidal and also show a very broad biological activity ( Xu Y, Zhan J, Wijeratne EM, Bums AM, Gunatilaka AA, Molnár I. Cytotoxic and antihaptotactic beauvericin analogues from precursor-directed biosynthesis with the insect pathogen Beauveria bassiana ATCC 7159. J Nat Prod. 2007 Sep;70(9):1467 -71 . doi: 10.1021/np070262f. Epub 2007 Sep 6. PMID: 17803266 and Firäkovä S, Proksa B, Sturdikova M. Biosynthesis and biological activity of enniatins. Pharmacy. 2007 Aug;62(8):563-8 . PMID: 17867547).

The best-studied cyclopeptides are the destruxins AF from Metarhizium anisopliae/ Oospora destructor ( Dornetshuber-Fleiss R, Heffeter P, Mohr T, Hazemi P, Kryeziu K, Seger C, Berger W, Lemmens-Gruber R. Destruxins: fungal-derived cyclohexadepsipeptides with multifaceted anticancer and antiangiogenic activities. Biochem Pharmacol. 2013 Aug 1;86(3):361-77 . doi: 10.1016/j.bcp.2013.05.022. Epub 2013 Jun 6. PMID: 23747344; PMCID: PMC5850990).

They have an insecticidal effect on the silk moth (Bombyx mori), the common rose beetle (Cetonia aurata), the Mexican bean beetle (Epilachna sparsa), the large wax moth (Galleria mellonella), the cotton boll borer (Heliothis virescens), the common housefly (Musca domestica), the Asian rhinoceros beetle (Oryctes rhinoceros), the mustard beetle (Phaedon cochleariae) and the diamondback moth (Plutella xylostella), but also show broad phytotoxic effects on Brassica spec., Camelina sativa, Capsella bursa-pastoris, Capsicum annuum, Chenopodium quinoa, Citrullus lanatus, Diplotaxis erucoides, Eruca sativa, Hirschfeldia incana, Hordeum vulgare, Lycopersicon esculentum, Nicotiana spec., Phaseolus vulgaris, Raphanus sativus, Sinapis spec., Sisymbrium officinalis, Solanum tuberosum, Thlaspi arvense, Triticum aestivum etc. ( Pedras MS, Irina Zaharia L, Ward DE. The destruxins: synthesis, biosynthesis, biotransformation, and biological activity. Phytochemistry. 2002 Mar;59(6):579-96 . doi: 10.1016/s0031-9422(02)00016-x. PMID: 11867090.).

As a conclusion to the prior art listed above, it can be stated that no selective insecticidal cyclopeptides have been discovered to date.

The invention is therefore based on the task of developing a new natural substance (bioactive substance) from a previously poorly researched group of basal evolutionary fungi such as Mortierella spec. through isolation and identification, which represents a new pharmaceutical lead structure, to provide a biological process for its economical and time-saving production with high product yield and without any special equipment expenditure and alternatively to provide a chemical synthesis process for its production and to explain its use (bioactivity).

According to the invention, this object is achieved by the characterizing features of the first patent claim. Further favorable design options for the invention are specified in the subordinate patent claims.

This task is solved by growing several Mortierella spec. isolates in culture medium and analyzing their extracts chromatographically.

The two Mortierella alpina strains JMRC:SF:10519 and JMRC:SF:10520 from the JMRC were preferably cultivated in order to enable the production, isolation and active ingredient analysis of the new natural product.

It was thus possible to identify six novel substances, cycloacetamide A-F, from the strains JMRC:SF:10519 and JMRC:SF:10520, of which the two representatives cycloacetamide A and B were isolated, structure elucidated and tested for bioactivity in a screening process . Cycloacetamide A-F has an insecticidal effect against fruit fly larvae.

The Mortierella alpina strain DSM 34346 is particularly preferably cultivated in order to enable the production, isolation and active ingredient analysis (structure elucidation) of the new natural product in the form of the structure-elucidated cycloacetamide A and B.

Cycloacetamides A and B have insecticidal activity against fly larvae but show little bioactivity against other organisms.

Cycloacetamides belong to the group of cyclodepsitetrapeptides. Cyclodepsitetrapeptides are characterized by a canonical peptide-linked chain of amino acids that are closed into a ring by an ester bond. This ring closure requires nucleophilic amino acids that carry a free hydroxyl or thiol group (such as threonine, serine and cysteine).

To date, many cyclodepsitetrapeptides are known from bacteria that have diverse biological functions. These include the antibiotic xenamid A from Xenorhabdus nematophila, the antibiotic teixobactin from Eleftheria terrae as well as the cyclodepsit fatflabet A and xeneprotide A with unknown biological function (isolated from Xenorhabdus and Photorhabdus spec.). The branching amino acid in the cycloacteamides is L-threonine, which has an acetyl group amidated at the N-terminus. In addition to the cyclopeptides mentioned above, there are numerous bioactive, threonine-branching cyclopenta to decapeptides from bacteria - among them the antiprozoic Taxlllaid A from Xenorhabdus indica, the antibiotic Tumescenamide C from Streptomyces spec. and the commercially used antibiotic daptomycin from Streptomyces roseo.

Cyclic peptides from fungi are rather rare: These include the immunomodulating aspergillicin F from Aspergillus flavus and the cytotoxic zygosporamide from Zygosporium masonii.

With the present method, a novel family of six peptide active ingredients, the cycloacetamides, is produced from the basal evolutionary fungus Mortierella alpina strains JMRC:SF:10519 and JMRC:SF: 10520, with the producer strain JMRC:SF:10519 being deposited as DSM 34346. isolated, characterized and tested for biological activity/efficacy.

The active ingredients/natural substances cycloacetamides A to F are formed continuously, regardless of the culture medium used, which enables sustainable production even using organic industrial waste as a carbon source. The substances can be isolated both in the mycelium of the fungus and from the culture filtrate, the latter containing the substances in greater purity. Cycloacetamides A to F, especially A and B, are characterized by a selective insecticidal effect.

In the process for the production of cycloacetamides A and B by the mucoromycete Mortierella alpina, the mushroom mycelium is grown on an agar surface in a manner known for mushroom cultures and homogenized (mechanically crushed), the homogenized mushroom mycelium is transferred into a nutrient solution for a preculture, which becomes preculture incubated or shaken and, if necessary, crushed again. To produce the natural product cycloacetamide, especially cycloacetamide A and B, the preculture is transferred to a production vessel and exposed to another nutrient solution. The mixture of the natural substances cycloacetamides A to F is harvested in the production vessel or separated from the nutrient solution after they have been removed.

Surprisingly, it has been shown that the new natural product cycloacetamide A and the new natural product cycloacetamide B (in the mixture A-F) can be obtained with the mucoromycete Mortierella alpina without the need for fixation to a carrier in known production vessels, such as stirred tank reactors, fermenters, etc , without reducing the shear force load during the formation of natural substances in the nutrient solution. This means that the fungus Mortierella alpina does not have any special requirements for the production of the pre-culture or for the form and function of the production vessel. While known technical solutions for the fermentation of mushrooms sometimes require very special and complex structures for storage, treatment and circulation in order to avoid or at least reduce the harmful effects of shear forces on the mycelium that produces natural substances, the production solution according to the invention uses production vessels without that their size, shape and function negatively influence the consistency of the nutrient solution and the formation of the natural product cycloacetamides A to F. This enables a large throughput and more effective formation of the natural product cycloacetamides A to F. The use of conventional fungal media and the implementation of cyclic or continuous natural product extraction processes have proven to be particularly advantageous. This enables the extraction of natural substances in conventional production vessels, with very good product yield and time effectiveness.

The separation/isolation of cycloacetamide A and cycloacetamide B from the mixture A to F takes place after liquid-liquid extraction, separation of the organic phase of the extraction, evaporation and lyophilization of the concentrated extract using known methods of chromatography, in the form of preparative HPLC and semi-preparative HPLC. The scheme for the separation/isolation of the individual cycloacetamides A and B from the mixture A to F is shown schematically in shown.

In one application of the method, the fungal strain Mortierella alpina (DSM 34346) was cultivated in a simple stirred tank reactor in a nutrient solution volume of 30 liters. The cultivation period was 5-8 days. Surprisingly, culture volumes that were up to 30 times higher could be used to produce the mixture of the natural substances cycloacetamides A to F by fermentation, although this is due to the use of disc stirrers with relatively high shear forces. In another process, the space-time yield was significantly increased through the targeted exchange of the production medium.

By changing media up to ten times, the fungal culture of Mortierella alpina (DSM 34346) can surprisingly be kept in a productive state for several weeks and so can An active culture filtrate containing natural substances can be obtained from a culture up to ten times the usual fermentor volume, with the natural substance also being detectable in the mycelium.

The invention is explained below using the schematic and Tables 1 to 4 and the exemplary embodiment are explained in more detail without being limited to this.

• : die Strukturformeln der erfindungsgemäßen Cycloacetamid-Derivate A bis F,
• : das ESI-MS/MS-Spektrum des erfindungsgemäßen Cycloacetamid-Derivates A gemäß (Fragmentierung mit einer Kollisionsdissoziationsenergie von 20%),
• : das ESI-MS/MS-Spektrum des erfindungsgemäßen Cycloacetamid-Derivates B gemäß (Fragmentierung mit einer Kollisionsdissoziationsenergie von 20%),
• : des ESI-MS/MS- Spektrum des erfindungsgemäßen Cycloacetamid-Derivates C gemäß (Fragmentierung mit einer Kollisionsdissoziationsenergie von 20%),
• : ESI-MS/MS-Spektrum des erfindungsgemäßen Cycloacetamid-Derivates D gemäß (Fragmentierung mit einer Kollisionsdissoziationsenergie von 20%),
• : das ESI-MS/MS-Spektrum des erfindungsgemäßen Cycloacetamid-Derivates E gemäß (Fragmentierung mit einer Kollisionsdissoziationsenergie von 20%),
• : das ESI-MS/MS-Spektrum des erfindungsgemäßen Cycloacetamid-Derivates F gemäß (Fragmentierung mit einer Kollisionsdissoziationsenergie von 20%),
• : ein Schema zur Isolation von Cycloacetamid-Derivaten A bis E aus Kulturen von Mortierella alpina,
• : ein Schema zur chemischen Darstellung von Cycloacetamiden,
• : ein ITS-Sequenz-Vergleich zwischen dem Mortierella alpina Referenzstamm ATCC32222 und den beiden Cycloacetamid-produzierenden Mortierella alpina-Stämmen JMRC:SF:10519 und JMRC:SF10520
• : die konzentrationsabhängige Induktion der Mortalität von Larven der Schwarzbäuchigen Fruchtfliege (Drosophila melanogaster) durch Cycloacetamid A (linkes Säulendiagramm) sowie Cycloacetamid B (rechtes Säulendiagramm) sowie

Show it

• : the structural formulas of the cycloacetamide derivatives A to F according to the invention,
• : the ESI-MS/MS spectrum of the cycloacetamide derivative A according to the invention (fragmentation with a collision dissociation energy of 20%),
• : the ESI-MS/MS spectrum of the cycloacetamide derivative B according to the invention (fragmentation with a collision dissociation energy of 20%),
• : the ESI-MS/MS spectrum of the cycloacetamide derivative C according to the invention (fragmentation with a collision dissociation energy of 20%),
• : ESI-MS/MS spectrum of the cycloacetamide derivative D according to the invention according to (fragmentation with a collision dissociation energy of 20%),
• : the ESI-MS/MS spectrum of the cycloacetamide derivative E according to the invention according to (fragmentation with a collision dissociation energy of 20%),
• : the ESI-MS/MS spectrum of the cycloacetamide derivative F according to the invention (fragmentation with a collision dissociation energy of 20%),
• : a scheme for the isolation of cycloacetamide derivatives A to E from cultures of Mortierella alpina,
• : a scheme for the chemical representation of cycloacetamides,
• : an ITS sequence comparison between the Mortierella alpina reference strain ATCC32222 and the two cycloacetamide-producing Mortierella alpina strains JMRC:SF:10519 and JMRC:SF10520
• : the concentration-dependent induction of mortality of larvae of the black-bellied fruit fly (Drosophila melanogaster) by cycloacetamide A (left bar diagram) and cycloacetamide B (right bar diagram) as well

• Table 1: the physicochemical parameters of the cycloacetamide derivatives A to F according to ,
• Table 2: the NMR data of 1 (in DMSO-d ₆ ), where * overlapping signals, ** signals only detectable in DEPT NMR spectra, chemical shift (δ) in ppm, multiplicity (s: singlet, d: doublet, t: triplet, q: quartet, m multiplet), coupling constants (J) in Hz,
• Table 3: the NMR data of 2 (in DMSO-d ₆ ), where * overlapping signals, ** signals only detectable in DEPT NMR spectra, chemical shift (δ) in ppm, multiplicity (s: singlet, d: doublet, t: triplet, q: quartet, m multiplet), coupling constants (J) in Hz,
• Table 4: the NMR data of 2 (in CD ₃ OD), where * overlapping signals. ** Signals only detectable in DEPT-NMR spectra, chemical shift (δ) in ppm, multiplicity (s: singlet, d: doublet, t: triplet, q: quartet, m multiplet), coupling constants (J) in Hz,
• Table 5: Overview of the semi-preparative HPLC methods for the isolation of the cycloacetamide derivatives A to E from cultures of Mortierella alpina according to and
• Table 6: Data from the determination of the biological activities of cycloacetamides A and B with a) agar diffusion test against Bacillus subtilis, Staphylococcus aureus (MRSA, VRSA), Escherichia coli, Pseudomonas aeruginosa; b) MIC determination against Mycobacterium smegmatis; c) Agar diffusion test against Streptomyces prunicolor, Streptomyces canus, Streptomyces fradiae; d) agar diffusion test against Sporidiobolus salmonicolor, Candida albicans, Penicillium notatum, e) cytotoxicity against HeLa, U-937, HUVEC, K-562; f) pro/anti-inflammatory release of prostaglandins by monocyte-derived M2 macrophages; g) ATP-based luciferase viability assay with Trypanosoma brucei, h) Caenorhabditis elegans. n.b., not determined.

The structural formulas of the six newly provided substances Cylcoacetamide A to F are in the shown.

With the structures described (see ) are structural isomers of cyclodepsitetrapeptides with N-terminally acetylated threonine (position 1). While all other amino acids have the canonical L configuration, only the amino acid at position 2 is D-configured (D-Leu in 1-3 and D-Phe in 4 and 5). At position 3 there is either L-Leu (in 1), L-Phe (in 2, 3, 4 and 5) or L-Tyr (in 6). At position 4 is L-Leu (in 1, 2, 4 and 6) or L-Phe (in 3 and 5). The configurations of the stereocenters in Figure 6 have not been determined.

The molecular formulas, the double bond equivalents (DBE) and the exact mass of the cylcoacetamides A to F (=compounds 1-6) are shown in Table 1.

The structure was determined using HPLC-MS/MS (see also ) and the configuration of the amino acids was determined using Marfey's analysis.

For the cylcoacetamides A and B (=compounds 1 and 2), the structure was confirmed by 1D and 2D NMR analyzes in deuterated dimethyl sulfoxide and methanol (see Tables 2-4).

The cylcoacetamides A and B (=compounds 1 and 2) are protease-stable and cannot be broken down by chymotrypsin, for example.

All cylcoacetamides A to F (=compounds 1-6) are unknown until they are made available, as they have not yet been stored in any chemical database (SciFinder, Reaxys, Dictionary of Natural Products by Chapman and Hall).

Example 1

- Precultures (2,000 ml Erlenmeyer flasks with 500 ml medium each) -

Mortierella alpina (DSM 34346) is used as a strain. The producing organism Mortierella alpina (DSM 34346) is cultured on MEP agar plates (30 g/L malt extract, 3 g/L soy peptone, 18 g/L agar, pH 5.6) at 25°C for 7 days and then for 1 Stored in the refrigerator (4-7°C) for 2 weeks to stimulate lipid synthesis and sporulation. The plates can be stored like this for up to 2 months.

2,000 ml Erlenmeyer flasks containing 500 ml of medium each are inoculated with 3-6 overgrown MEP agar blocks from the Mortierella alpina culture agar plate (DSM 34346) and then incubated at 25°C and 130 rpm for at least 10 days.

The yields of cycloacetamide B (compound 2) in MEP are about 0.5-0.7 mg per g of dry biomass after 7 days and about 1.2-1.3 mg per g of dry biomass after 10 days. A significant increase in product is not expected after 10 days.

Various complex liquid media such as MEP (30 g/L malt extract, 3 g/L soy peptone pH 5.6) or YPD (20 g/L soy peptone, 20 g/L glucose, 10 g/L yeast extract, pH) are used for metabolite production 6-7) in question. The yield is usually a factor of 2 higher in MEP medium. In principle, various other solid media (including organic kitchen or industrial waste) can also be used, as production is independent of the medium. Only growth on inorganic nitrate or ammonium salts as the sole source of nitrogen should be avoided, as these reduce the growth rate enormously.

In order to stimulate the production of specific cycloacetamides, it is recommended to add the amino acids to be incorporated after autoclaving the media (121°C, 20 min) (10 mM L-leucine for compound 1, 10 mM L-phenylalanine for compounds 2 - 5 and 5 mM L-tyrosine for compound 6).

Cultivation is necessarily carried out aerobically (emers or submersed with shaking in flasks or alternatively in a fermenter with oxygen or air supply).

Since the producer is a psychrotolerant organism, cultivation can take place between 4°C and 30°C. Higher temperatures should be avoided.

Tolerated pH ranges are between pH 5 and pH 9, ideal at pH 6 - pH 7.

Cultivation times vary between 3 and 21 days depending on the culture medium. In MEP, 10 days is ideal.

Cylcoacetamides A to F (=compounds 1-6) are contained both in the culture filtrate and in the mycelium and the isolation of the compound according to the invention is carried out using known methods according to known physical-chemical separation processes and the biological activity of the active ingredients (see scheme in ).

The cultivation success can be monitored based on the increase in biomass or, better, based on extracted culture filtrate aliquots and their subsequent chromatographic analysis.

Example 2

- Scale up a 5 liter fermenter

The precultures (2,000 ml Erlenmeyer flasks with 500 ml medium each) of Mortierella alpina (DSM 34346) are carried out as described in Example 1.

To scale up, three of these pre-cultures of Mortierella alpina (DSM 34346) are used as production strains to inoculate a 5 liter fermenter with a disc stirrer (culture duration 10 days, 25 ° C and 130 rpm).

The cultivation conditions correspond to those according to exemplary embodiment 1.

After the cultivation period of 10 days in the 5 liter fermenter has been reached, stirring and ventilation are switched off (approx. 30 minutes).

After this time, the fungal biomass has settled at the bottom of the fermenter. The supernatant of the culture medium containing natural substances is separated off in a suitable manner and the mycelium remains in the 5 liter fermenter. New, sterile medium is filled. After 10 days, the culture is harvested again. In this way the procedure continues (as long as necessary). A maximum of 4.5 liters of culture solution containing natural substances can be obtained per cycle.

The yields of cycloacetamide B (compound 2) correspond to the yields according to exemplary embodiment 1. Here too, a significant increase in product is no longer to be expected after 10 days.

The cycloacetamides A to E (= compounds 1-6) are contained both in the culture filtrate and in the mycelium and the isolation of the compound according to the invention is carried out using known methods in accordance with known physical-chemical separation processes and the biological activity of the active ingredients (see scheme in ).

Example 3

-Isolation of cycloacetamides A to E from the MEP culture medium-

The Mortierella alpina (DSM 34346) mycelium is removed by centrifugation or filtration, for example via Miracloth.

The culture filtrate (13-15 liters) is adjusted to pH 2 with 3 M hydrochloric acid and extracted three times with the same volume of a water-insoluble solvent (preferably ethyl acetate, chloroform or dichloromethane).

The solvent is removed using a rotary evaporator and lyophilization.

The residue containing cycloacetamide is taken up in 150 ml of methanol.

The crude extract is centrifuged for 15 min at 5,000 x g to remove cell debris and then further purified using semi-preparative HPLC.

The cycloacetamide A and the cycloacetamide B (=compounds 1 and 2) are isolated using the HPLC method 1 and subsequently method 2 (see Table 5).

The cycloacetamides C to E (= compounds 3 - 5) are pre-purified using HPLC method 3 (see Table 5).

The pure compound 5 is separated from 3 and 4 using HPLC method 4. The enantioselective separation of 3 and 4 is achieved using HPLC method 5 (see Table 5).

Since the substances are hardly UV-absorbing, analysis should be carried out using mass spectrometry.

Example 4

Process for the chemical synthesis of cycloacetamides A-F (compounds 1-6)

mit Aminosäure A mit Rest R² Aminosäure B mit Rest R³ Aminosäure C mit Rest R⁴ With the chemical synthesis process according to the invention, the cycloacetamides A to F (compounds 1-6) have the general formula cyclo-(-N-Ac- _L -Thr-ABCO-) can be produced, where _L -Thr represents _L -configured threonine and in which A, B and C independently represent identical or different _L - or _D -configured amino acids with non-polar side chains, in particular alanine (Ala), valine (Val), leucine (Leu ), isoleucine (Ile), methionine (Met), phenylalanine (Phe), tyrosine (Tyr) or tryptophan (Trp). cyclo-(- N -Ac-L-Thr-ABCO-)

with Amino acid A with residue R ² Amino acid B with residue R ³ Amino acid C with residue R ⁴

The compounds of the formula cyclo-(-N-Ac- _L -Thr-ABCO-) are each a sequence of amino acids which are linked to one another by peptide bonds and have an N and a C terminus. The amino acid sequence described in brackets is cyclized with the C-terminal carboxy group of the amino acid C by an intramolecular ester via the hydroxy group of the N-acetylated threonine. At the formula cyclo-(-N-Ac-L-Thr-ABCO-) These are cyclotetradepsipeptides that can be synthesized in solution using methods known in peptide chemistry, such as those found in the literature in standard works (Houben Weyl, Methods of Organic Chemistry, Georg-Thieme-Verlag, Stuttgart) or review articles (such as Y. Okada , Synthesis of Peptides by Solution Methods, Current Organic Chemistry).

1. a) lineare Kopplung der jeweils geeignet geschützten AminosäureDerivate
2. b) lineare Veresterung der mit einer Aminoschutzgruppe X protektierten Aminosäure C mit N-Ac-L-Thr -O, wobei Y eine Alkylhydroxyschutzgruppe darstellt
3. c) Abspaltung der Amino- bzw. Carboxylschutzgruppen der jeweils erhaltenen Peptide
4. d) Makrolaktamisierung des finalen linearen Depsipeptids.

The important basic steps of the synthesis are:

1. a) linear coupling of the suitably protected amino acid derivatives
2. b) linear esterification of the amino acid C protected with an amino protecting group X with N-Ac-L-Thr -O, where Y represents an alkylhydroxy protecting group
3. c) cleavage of the amino or carboxyl protective groups of the peptides obtained
4. d) Macrolactamization of the final linear depsipeptide.

Suitable amino protective groups These primarily include urethane derivatives such as the tert-butyloxycarbonyl (Boc) group. The blocking of the carboxyl function of the amino acids occurs as alkyl esters Y, for example as methyl esters, which in turn are deprotected by alkaline or nucleophilic dealkylation. This corresponds to the general peptide protecting group chemistry as can be found, for example, in Kocienski, Protecting Groups, Georg-Thieme-Verlag, Stuttgart.

What is special about this technical solution is that the non-canonical liquid phase synthesis of a cyclotetradepsipeptide is carried out using a combination of standard and non-standard deprotection strategies to ensure the integrity of the intramolecular ester of the peptide. In particular, the combination of i) the rarely used deprotection of the methyl ester with trimethyltin hydroxide ( K. Nicolaou et al., A mild and selective method for the hydrolysis of esters with trimethyltin hydroxide. Applied Chemistry 44, 1378-1382 and HA Grab, Total Synthesis of the Cyclic Depsipeptide Vioprolide D via its (Z)-Diastereoisomer. Applied Chemistry - International Edition 59, 12357-12361 ), ii) the deprotection of the Boc group by trimethylsilyl iodide (M. Jung and M. Lyster, Conversion of Alkyl Carbamates into Amines via Treatment with Trimethylsilyl Iodide, Journal of the Chemical Society, Chemical Communications) and iii) the final macrolactamization - lactonization represents a novelty.

The synthesis strategy is therefore to produce the complete linear depsipeptide with subsequent final macrolactamization, as in presented as an overview diagram.

The esterification required for this takes place according to the example of Steglich ( B. Neises and W. Steglich, Simple Method for the Esterification of Carboxylic Acids, Angewandte Chemie 17, 522-524 ), but using the coupling reagent EDC-HCl (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) instead of DCC (N,N'-dicyclohexylcarbodiimide) ( M. Tsakos et al., Ester coupling reactions-an enduring challenge in the chemical synthesis of bioactive natural products, Natural Product Reports 32, 605-632 ).

In the The examples listed as an overview scheme refer as an example to the synthesis steps of cyclo-(N-Ac-L-Thr-L-Leu-L-Phe-D-Leu-O-) (compound 2) and are intended to explain the invention below.

Example 1 in Figure 9:

To form a new peptide bond, the deprotected acid (e.g. N-Boc-D-Leu-COOH) is dissolved in dichloromethane (DCM) to form a 0.2 M solution. This is incubated with 4 equivalents of DIPEA (N,N-diisopropylethylamine) and 1.1 equivalents of TBTU (2-(1H-benzotriazol-1-yl)-1,1,1,3,3-tetramethylaminium tetrafluoroborate) for five minutes. To generate the tetrapeptide, DIPEA can be omitted to obtain the intramolecular ester. A solution of the amino acid to be coupled (eg NH ₂ -L-Phe-OMe-HCl) or the peptide to be coupled in DCM is then added in order to obtain a final concentration of 0.1 M of the respective substrates. The reaction is stopped after 3.5 - 22 h by washing the mixture three times with 10 - 15 mL of saturated ammonium chloride solution and once with 10 - 15 mL of water. The combined aqueous phases are counter-extracted with 50 mL dichloromethane and all organic phases obtained are combined and concentrated under reduced pressure. The dipeptides are purified by flash chromatography on an FP Si 12 g column at a flow rate of 30 L/min and DCM as eluent A and methanol as eluent B. The synthesized dipeptide NH ₂ -D-Leu-L-Phe-OMe is resuspended in 5 mL DCM and injected onto the mentioned column and a gradient of 0 - 6.4 min: 0% B, 6.4 - 9.5 min: 20% B, 9.5 - 12.1 min: 40% B, 12.1 - 19.6 min: 100% B used.

The linear tetrapeptides obtained are purified using flash chromatography on an FP Si column with hexane as eluent A and ethyl acetate as eluent B. For example, N-Boc-D-Leu-O-(N-Ac-L-Thr)-L-Leu-L-Phe-OMe is purified using an FP Si 12 g column with a flow rate of 30 mL/min . To do this, the peptide is dispersed in 5 mL of DCM and analyzed by flash chromatography with a gradient of 0 - 2 min: 0% B, 2 - 4 min: 20% B, 4 - 6 min: 40% B, 6 - 14 min: 60 - 80% B, 14 - 23 min: 100% B purified.

Example 2 in Figure 9:

The resulting dipeptide (e.g. N-Boc-D-Leu-L-Phe-OMe) is mixed with 2 equivalents of anisole in dichloromethane (DCM) and trifluoroacetic acid is added dropwise until a 20% solution is achieved. The reaction is stopped after about 2 - 2.5 h at room temperature by neutralization with 3 M potassium hydroxide solution and washed with saturated ammonium chloride solution. The aqueous ammonium chloride phase is again counter-extracted three times with dichloromethane and all organic phases are combined and concentrated under vacuum.

Example 3 in Figure 9:

One equivalent of the free acid, e.g. N-Boc-L-Leu-COOH (0.2M), is placed in 8 mL of DCM and mixed for 30 min at room temperature with 1.5 equivalents of EDC-HCl and 0.3 equivalents of 4-(dimethylamino)-pyridine (DMAP). allow to pre-react. 1 equivalent (0.2 M) of N-Ac-L-Thr-OMe in 8 mL of DCM is then added to the reaction at initially 0 °C and allowed to incubate for 18 h. The product is washed with 30 mL of 1 M hydrochloric acid, twice 30 mL of ammonium chloride solution and 30 mL of water. The aqueous phases were counter-extracted with 50 mL DCM. The combined organic phases were dried over sodium sulfate and in vacuo.

Example 4 in Figure 9:

The resulting ester, for example N-Boc-L-Leu-O-(N-Ac-L-Thr-OMe), is dissolved in dichloroethane (DCE) and 6 equivalents of trimethyltin hydroxide are added. The reaction is stopped after 26 - 64 h at 80 ° C under an argon atmosphere by cooling and the reaction mixture is dried in vacuo. The dried reaction product is taken up again in 50 mL of ethyl acetate and washed three times with 50 mL of 1 M HCl and once with 50 mL of saturated NaCl solution. The combined aqueous phases were counter-extracted again with ethyl acetate and all ethyl acetate phases were combined, dried with sodium sulfate and concentrated on a rotary evaporator under reduced pressure.

Example 5 in Figure 9:

To deprotect the linear tetrapeptide, trimethylsilyl iodide (TMS-I) is used as a non-hydrolytic deprotection agent due to the acid and base lability of the intramolecular ester. The peptide, for example N-Boc-L-Leu-O-(N-Ac-L-Thr-)-D-Leu-L-Leu-COOH, is dissolved in 3 mL of deuterated chloroform (CDCl ₃ ) with 1.5 equivalents of TMS I moved. The reaction is ended under an argon atmosphere after 3.5 h at 50 ° C by adding an excess of methanol and drying in vacuo. Purification is then carried out using flash chromatography using an FP ID C18 12 g column. The samples are resuspended in methanol/water (83/17) and 4.8 mL are injected with water as eluent A and acetonitrile as eluent B and a flow rate of 30 mL/min using the following gradient: 0 - 2.5 min: 0% B , 2.5 - 5 min: 20% B, 5 - 7.5 min: 40% B, 7.5 - 9.5 min: 60% B, 9.5 - 11.5 min: 80% B, 11.5 - 15.5 min: 100% B.

Example 6 in Figure 9:

The final cyclization of the depsipeptides occurs with 2.5 mM of the deprotected linear peptide in a mixture of DCM/dimethyl sulfoxide (DMSO) (95/5) and is treated with 3 equivalents of O-(7-azabenzotriazol-1-yl)-N,N,N ',N'-tetramethyluronium hexafluorophosphate (HATU) started.

If substrate is still visible after monitoring the course of the reaction using LC-MS, HATU is added again. The reaction is stopped after a maximum of 48 h by concentrating the DCM in vacuo and the residue is resuspended in a total of 5 mL DMSO and purified using flash chromatography on an FP Si C18 12 g column at a flow rate of 30 L/min. The following gradient is used with water as eluent A and acetonitrile as eluent B: 0 - 2.5 min: 0% B, 2.5 - 5 min: 20% B, 5 - 7.5 min: 40% B, 7.5 - 10 min: 60% B, 10 - 12.5 min: 80% B, 12.5 - 17.2 min: 100% B.

The combined collected fractions are concentrated in vacuo and resuspended in 1.5 mL MeOH and further purified by semipreparative HPLC. For this purpose, an Agilent 1200 system is used with an Agilent Eclipse a column temperature of 12 °C was used. 50 µL are injected per run and detected at 220 nm. A gradient of 0 - 1.5 min: 30 is created % B, 1.5 - 16.5 min: 30 - 100% B, 16.5 - 19 min: 100% B, 19 - 19.5 min: 100 - 30% B, 19.5 - 22 min: 30% B used.

5. Embodiment

-Mortierella alpina- isolates JMRC: SF:10519 and JMRC:SF:10520-

Of a total of 23 strains examined, the cycloacetamides could only be isolated from the two strains JMRC:SF:10519 and JMRC:SF:10520, where JMRC:SF:10519 = DSM 34346 = is the producer strain. Both strains come from an alpine region and were collected between 2004 and 2011.

The strains were deposited in the Jena Microbial Resource Collection (JMRC) and classified in 2013 into group 6 of the Mortierellacea family by maximum likelihood analyzes based on their LSU (large ribosomal subunits) and ITS (internal transcribed spacer) sequences (Wagner L, Stielow B, Hoffmann K, Petkovits T, Papp T, Vägvölgyi C, de Hoog GS, Verkley G, Voigt K. A comprehensive molecular phylogeny of the Mortierellales (Mortierellomycotina) based on nuclear ribosomal DNA. Persoonia. 2013; 30:77-93 . doi: 10.3767/003158513X666268. PMID: 24027348; PMCID: PMC3734968.).

Before natural product analysis, the strains were re-identified as Mortierella alpina JMRC:SF:10519 (= DSM 34346) and JMRC:SF:10520 based on their ITS region amplified using PCR technology.

Both Mortierella alpina isolates have an identical ITS sequence and are in turn 96.4% identical to the reference genome strain M. alpina ATCC32222 ( Wang L, Chen W, Feng Y, Ren Y, Gu Z, Chen H, Wang H, Thomas MJ, Zhang B, Berquin IM, Li Y, Wu J, Zhang H, Song Y, Liu X, Norris JS, Wang S , Du P, Shen J, Wang N, Yang Y, Wang W, Feng L, Ratledge C, Zhang H, Chen YQ. Genome characterization of the oleaginous fungus Mortierella alpina. PLoS One. 2011;6(12):e28319 . doi: 10.1371/journal.pone.0028319. Epub 2011 Dec 8. PMID: 22174787; PMCID: PMC3234268.). The ITS sequence alignment of the two production strains is shown below shown.

In terms of natural product chemistry, the 2 Mortierella alpina strains can be characterized by the fact that, in addition to the above-mentioned cycloacetamide AF (= compounds 1-6), they also contain malpinin A (m/z 859.4827 [M+H] ⁺ ), malpibaldin A (m/z 626.4062 [ M+H] ⁺ ) and malpicycline C (m/z 629.4343 [M+H] ⁺ ) produce.

While Mortierella alpina isolate JMRCSF:10519 additionally produces calpinactam (m/z 768.4330 [M+H] ⁺ ), the calpinactam production levels of Mortierella alpina isolate JMRC:SF:10520 are below the detection level.

Therefore, Mortierella alpina isolate JMRC:SF:1010519 was selected as producer strain DSM 34346.

6. Embodiment

Bioactivity of cycloacetamides A and B (=compounds 1 and 2)-

The cycloacetamides A and B (=compounds 1 and 2) were subjected to intensive bioactivity screening.

Cycloacetamides A and B (=compounds 1 and 2) show an insecticidal effect on larvae of the black-bellied fruit fly (Drosophila melanogaster) from 60 µg/ml (see ).

A repellent effect on insects cannot be determined, so a toxic effect can be assumed.

For cycloacetamides A and B (=compounds 1 and 2) no antimicrobial (fungi/bacteria), cytotoxic or hormone-releasing effects could be determined (see Table 5). The substances are also not effective against trypanosomes or nematodes (each >100 µg/ml).

It is particularly advantageous that the cycloacetamides provided here are halogen-free cyclooligopeptides, which are particularly advantageously produced by a fungus Mortierella spec., in particular Mortierella alpina are naturally formed in the producer strain Mortierella alpina DSM 34346 and are protease stable. They therefore represent a sustainable, ecologically compatible alternative to conventional insecticides.

1 Cycloacetamid A D-Leu L-Leu L-Leu 482.3104 6 C₂₄H₄₂N₄O₆ 2 Cycloacetamid B D-Leu L-Phe L-Leu 516.2948 10 C₂₇H₄₀N₄O₆ 3 Cycloacetamid C D-Leu L-Phe L-Phe 550.2791 14 C₃₀H₃₈N₄O₆ 4 Cycloacetamid D D-Phe L-Phe L-Leu 550.2791 14 C₃₀H₃₈N₄O₆ 5 Cycloacetamid E D-Phe L-Phe L-Phe 584.2635 18 C₃₃H₃₆N₄O₆ 6 Cycloacetamid F Leu Tyr Leu 532.2897 10 C₂₇H₄₀N₄O₇ Tabelle 2 δ ¹³C [ppm] type δ ¹H [ppm], M (J [Hz]) COSY HMBC Acetat 1 170.11 C=O 2 22.22* CH₃ 1.96 1 L-Threonin 3 169.51 C=O 4 55.16 CH 4.62, dd (9.4, 4.4) 4-NH, 5 1, 3 NH 8.05, d (9.3) 4 1, 4 5 71.02 CH 5.19, dq (6.4; 4.6) 4, 6 3*, 6, 19* 6 15.00 CH₃ 1.18, d (6.5) 5 4, 5 D-Leucin 7 171.44 C=O 8 51.44 CH 4.36, m 8-NH, 9 3, 7, 9, 10 NH 7.84, d (8.2) 8 3, 8 9 38.03 CH₂ a: 1.65, m 8, 10* 3, 8, 10* b: 1.45, m 8 8, 10* 10 24.33* CH 1.46, m* 11*, 12* 11 21.99 CH₃ 0.85, d (6.5) 10* 9, 10, 12 12 22.56 CH₃ 0.89, d (6.6) 10* 9, 10 L-Leucin 13 171.24 C=O 14 49.94 CH 4.20, m 14-NH, 15 7*, 13*, 15, 16* NH 7.48, d (7.8) 14 7, 14 15 39.63** CH₂ a: 1.63, m 14 13*, 14, 16* b: 1.42, m 14 14, 16* 16 24.43* CH 1.51*, m 17*, 18* 17 22.06 CH₃ 0.86, d (6.6) 16* 15, 16 18 22.49 CH₃ 0.87, d (6.9) 16* 15, 16 L-Leucin 19 170.13 C=O 20 52.61 CH 4.18, m 20-NH, 21 19*, 21, 22* NH 8.30, d (8.7) 20 13, 20 21 39.24** CH₂ a: 1.56 20 20, 22, 24 b: 1.52 20 20, 22 22 24.13 CH 1.60 23, 24 23 22.56 CH₃ 0.88, d (7.2) 22 21, 22 24 21.12 CH₃ 0.81, d (6.5) 22 21, 22 Tabelle 3 δ ¹³C [ppm] type δ ¹H [ppm], M (J [Hz]) COSY HMBC Acetat 1 170.30 C=O 2 22.50 CH₃ 1.95, s L-Threonin 3 169.80 C=O 4 55.13 CH 4.61, dd (9.4, 4.3) 4-NH, 5 3, 5, 6 NH 8.0, d (9.4) 4 2*, 4, 5 5 71.23 CH 5.18, dq (6.4, 4.6) 4, 6 6 6 15.15 CH₃ 1.17, d (6.5) 5 4, 5 D-Leucin 7 171.11 C=O 8 51.40 CH 4.35, m 8-NH, 9 9 NH 7.89, d (8.3) 8 3, 8 9 37.71 CH₂ a: 1.56, m 8, 9b 8 b: 1.31, m 8, 9a 7, 8 10 24.20 CH 1.20, m 11, 12 8 11 21.97 CH₃ 0.73*, d (6.6) 10 9, 10, 12 12 21.87 CH₃ 0.81 *, d (6.6) 10 9 L-Phenylalanin 13 170.42 C=O 14 55.66 CH 4.35, m 14-NH 13*, 15, 16 NH 8.34, d (9.1) 14, 15 7, 14, 15 15 36.20 CH₂ a: 3.00, dd (13.6, 5.6) 14,15b 14, 16, 17, 21 b: 2.81, dd (13.5, 10.2) 14,15a 14, 16, 17, 21 16 137.87 C (ar) 17,21 128.90 CH (ar) 7.23*, m 17, 19, 21 18,20 128.02 CH (ar) 7.25*, m 16, 18, 20 19 126.25 CH (ar) 7.18, m 17, 21 L-Leucin 22 170.39 C=O 23 50.19 CH 4.12, dt (7.9, 6.2) 23-NH, 24 24, 25 NH 7.70, d (7.7) 23 13*, 23 24 39.29** CH₂ a: 1.60, m 23, 24b 23 b: 1.50, m 23, 24a 23, 25 25 23.99 CH 1.43, m 26, 27 23, 24, 26, 27 26 22.89 CH₃ 0.86, d (6.5)* 25 24, 25 27 22.67 CH₃ 0.84, d (6.5)* 25 24, 26 Tabelle 4 δ ¹³C [ppm] type δ ¹H [ppm], M (J [Hz]) COSY HMBC Acetat 1 174.51 C=O 2 22,63 CH₃ 2.10, s 1 L-Threonin 3 172.16 C=O 4 57.43 CH 4.74, d (2.8) 5 1, 3, 5, 6 5 73.67 CH 5.44, dq (6.3, 2.9) 4, 6 4, 6, 22 6 16.03 CH₃ 1.24, d (6.4) 5 4, 5 D-Leucin 7 174.32 C=O 8 53.15 CH 4.50, (t, 7.8 Hz) 9 7, 9 9 38.77 CH₂ a: 1.64, m 8, 9b 7, 8 b: 1.46, m 8, 9a 7, 8 10 25.70* CH 1.38, m 11, 12 11, 12 11 22.59* CH₃ 0.88, m* 10 12 22.53* CH₃ 0.84, m* 10 L-Phenylalanin 13 172.86 C=O 14 57.40 CH 4.57, (dd, 8.6, 7.4) 7, 13, 15, 16, 22 15 37.73 CH₂ a: 3.10, dd (13.8, 7.2) 14, 15b 13, 14, 16, 17, 21 b: 2.87, dd (13.6, 9.0) 14, 15a 13, 14, 16, 17, 16 138.17 C (ar) 21 17,21 130.05 CH (ar) 7.25*, m 17, 19, 21 18,20 129.51 CH (ar) 7.27*, m 16, 18, 20 19 127.86 CH (ar) 7.21, m 17, 21 L-Leucin 22 171.66 C=O 23 52.63 CH 4.11, dt (t, 7.2 Hz) 24 22, 24, 25 24 40.32** CH₂ a: 1.66, m 23, 24b 22, 23, 25, 26, 27 b: 1.58, m 23, 24a 22, 23, 25 25 25.93 * CH 1.36*, m 26, 27 26 23.12* CH₃ 0.86*, m 25 27 22.67* CH₃ 0.82*, m 25 Tabelle 5 Methode 1 Methode 2 Methode 3 Methode 4 Methode 5 HPLC-System^® Präparative HPLC Präparative HPLC Präparative HPLC Präparative HPLC Präparative HPLC Agilent 1260 Agilent 1260 Agilent 1260 Agilent 1260 Agilent 1260 Lösungsmittel A H₂O + 0.1 % TFA H₂O + 0.1 % TFA H₂O + 0.1% TFA H₂O + 0.1% TFA H₂O + 0.1% TFA Lösungsmittel B Acetonitril Acetonitril Acetonitril Acetonitril Acetonitril Gradient für B 0-0.5 min: 35%, 0-0.5min: 45%, 0-0.5 min: 35%, 0-0.5 min: 70%, 0-0.5 min: 80%, 0.5-13.5 min: 35-80 %, 0.5-20 min: 45-100 %, 0.5-12.5 min: 35-80 %, 0.5-6 min: 70-85 %, 0.5-10.5 min: 80-100 %, 13.5-17 min: 80-100 %, 10-11 min: 100 %, 12.5-16 min: 80-100 %, 6-13.5 min: 85-100 %, 10.5-16.0 min: 100 %, 17-19 mm: 100 %, 11-11.5 min: 100-45 %, 16-17 min: 100 %, 13.5-14.5 min: 100 %, 16-16.5 min: 100-80 %, 19-19.5 min: 100-35 %, 11.5-13 min: 45 % 17-17.5 min: 100-35 %, 14.5-15 min: 100-70 %, 16.5-21 min: 80% 19.5-21 min: 35 % 17.5-19 min: 35 min 15-16.5 min: 70 % Temperatur Fließgeschwindigkeit Säule 12 °C 12 °C 12 °C 12 °C 12 °C 25 mL/min 2 mL/min 2 mL/min 2 mL/min 3.5 mL/min Agilent Eclipse XDB-C18 Thermo Fisher Hypercarb Agilent Eclipse XDB-C18 Phenomenex SynergiHydro-RP Thermo Fisher Hypercarb Säulenmaße 250 × 9.4 mm, 5 µm 150 × 4.6 mm, 5µm 250 × 9.4 mm, 5 µm 250 × 10 mm, 4 µm 150 × 4.6 mm, 5 µm Tabelle 6 Vbdg. pathogene Bakterien ^a Mycobakterien ^b filamentöse Bakterien ^c Pilze ^d Säugerzelllinien ^e menschliche Makrophagen ^f Trypanosomen ^g Nematoden ^h 1 >1000 µg mL^-1 >125 µg mL^-1 n.b. >1000 µg mL ^-1 >100 µg mL^-1 >14.4 µg mL^-1 >150 µg mL^-1 >100 µg mL^-1 2 >1000 µg mL^-1 >125 µg mL^-1 > 516 µg mL^-1 >1000 µg mL^-1 >100 µg mL^-1 >15.5 µg mL^-1 >150 µg mL^-1 >100 µg mL^-1 All features presented in the description, the exemplary embodiments and the following claims can be essential to the invention both individually and in any combination with one another. Table 1 # Surname R2 ^_ R3 ^_ R4 ^_ [ M +H] ⁺ DBE Molecular formula

1 Cycloacetamide A D-Leu L-Leu L-Leu 482.3104 6 _{C24 H42 N4 O6} 2 Cycloacetamide B D-Leu L-Phe L-Leu 516.2948 10 _{C27 H40 N4 O6} 3 Cycloacetamide C D-Leu L-Phe L-Phe 550.2791 14 _{C30 H38 N4 O6} 4 Cycloacetamide D D-Phe L-Phe L-Leu 550.2791 14 _{C30 H38 N4 O6} 5 Cycloacetamide E D-Phe L-Phe L-Phe 584.2635 18 _{C33 H36 N4 O6} 6 Cycloacetamide F Leu Tyr Leu 532.2897 10 _{C27 H40 N4 O7} Table 2 δ ¹³ C [ppm] type.type δ ¹ H [ppm], M (J [Hz]) COZY HMBC acetate 1 170.11 C=O 2 22.22* _CH3 1.96 1 L-threonine 3 169.51 C=O 4 55.16 Ch 4.62, dd (9.4, 4.4) 4-NH, 5 1, 3 NH 8.05, d (9.3) 4 1, 4 5 71.02 Ch 5.19, dq (6.4; 4.6) 4, 6 3*, 6, 19* 6 3 p.m _CH3 1.18, d (6.5) 5 4, 5 D-leucine 7 171.44 C=O 8th 51.44 Ch 4.36, m 8-NH, 9 3, 7, 9, 10 NH 7.84, d (8.2) 8th 3, 8 9 38.03 _CH2 a: 1.65, m 8, 10* 3, 8, 10* b: 1.45, m 8th 8, 10* 10 24.33* Ch 1.46, m* 11*, 12* 11 21.99 _CH3 0.85, d (6.5) 10* 9, 10, 12 12 22.56 _CH3 0.89, d (6.6) 10* 9, 10 L-Leucine 13 171.24 C=O 14 49.94 Ch 4.20, m 14-NH, 15 7*, 13*, 15, 16* NH 7.48, d (7.8) 14 7, 14 15 39.63** _CH2 a: 1.63, m 14 13*, 14, 16* b: 1.42, m 14 14, 16* 16 24.43* Ch 1.51*, m 17*, 18* 17 June 22nd _CH3 0.86, d (6.6) 16* 15, 16 18 22.49 _CH3 0.87, d (6.9) 16* 15, 16 L-Leucine 19 170.13 C=O 20 52.61 Ch 4.18, m 20-NH, 21 19*, 21, 22* NH 8.30, d (8.7) 20 13, 20 21 39.24** _CH2 a: 1.56 20 20, 22, 24 b: 1.52 20 20, 22 22 24.13 Ch 1.60 23, 24 23 22.56 _CH3 0.88, d (7.2) 22 21, 22 24 12/21 _CH3 0.81, d (6.5) 22 21, 22 Table 3 δ ¹³ C [ppm] type.type δ ¹ H [ppm], M (J [Hz]) COZY HMBC acetate 1 170.30 C=O 2 22.50 _CH3 1.95, p L-threonine 3 169.80 C=O 4 55.13 Ch 4.61, dd (9.4, 4.3) 4-NH, 5 3, 5, 6 NH 8.0, d (9.4) 4 2*, 4, 5 5 71.23 Ch 5.18, dq (6.4, 4.6) 4, 6 6 6 15.15 _CH3 1.17, d (6.5) 5 4, 5 D-leucine 7 171.11 C=O 8th 51.40 Ch 4.35, m 8-NH, 9 9 NH 7.89, d (8.3) 8th 3, 8 9 37.71 _CH2 a: 1.56, m 8, 9b 8th b: 1.31, m 8, 9a 7, 8 10 24.20 Ch 1.20, m 11, 12 8th 11 21.97 _CH3 0.73*, d (6.6) 10 9, 10, 12 12 21.87 _CH3 0.81 *, d (6.6) 10 9 L-Phenylalanine 13 170.42 C=O 14 55.66 Ch 4.35, m 14-NH 13*, 15, 16 NH 8.34, d (9.1) 14, 15 7, 14, 15 15 36.20 _CH2 a: 3.00, dd (13.6, 5.6) 14.15b 14, 16, 17, 21 b: 2.81, dd (13.5, 10.2) 14.15a 14, 16, 17, 21 16 137.87 C (ar) 17.21 128.90 CH (ar) 7.23*, m 17, 19, 21 18.20 128.02 CH (ar) 7.25*, m 16, 18, 20 19 126.25 CH (ar) 7.18, m 17, 21 L-Leucine 22 170.39 C=O 23 50.19 Ch 4.12, dt (7.9, 6.2) 23-NH, 24 24, 25 NH 7.70, d (7.7) 23 13*, 23 24 39.29** _CH2 a: 1.60, m 23, 24b 23 b: 1.50, m 23, 24a 23, 25 25 23.99 Ch 1.43, m 26, 27 23, 24, 26, 27 26 22.89 _CH3 0.86, d (6.5)* 25 24, 25 27 22.67 _CH3 0.84, d (6.5)* 25 24, 26 Table 4 δ ¹³ C [ppm] type.type δ ¹ H [ppm], M (J [Hz]) COZY HMBC acetate 1 174.51 C=O 2 22.63 _CH3 2.10, p 1 L-threonine 3 172.16 C=O 4 57.43 Ch 4.74, d (2.8) 5 1, 3, 5, 6 5 73.67 Ch 5.44, dq (6.3, 2.9) 4, 6 4, 6, 22 6 16.03 _CH3 1.24, d (6.4) 5 4, 5 D-leucine 7 174.32 C=O 8th 53.15 Ch 4.50, (t, 7.8 Hz) 9 7, 9 9 38.77 _CH2 a: 1.64, m 8, 9b 7, 8 b: 1.46, m 8, 9a 7, 8 10 25.70* Ch 1.38, m 11, 12 11, 12 11 22.59* _CH3 0.88, m* 10 12 22.53* _CH3 0.84, m* 10 L-Phenylalanine 13 172.86 C=O 14 57.40 Ch 4.57, (dd, 8.6, 7.4) 7, 13, 15, 16, 22 15 37.73 _CH2 a: 3.10, dd (13.8, 7.2) 14, 15b 13, 14, 16, 17, 21 b: 2.87, dd (13.6, 9.0) 14, 15a 13, 14, 16, 17, 16 138.17 C (ar) 21 17.21 130.05 CH (ar) 7.25*, m 17, 19, 21 18.20 129.51 CH (ar) 7.27*, m 16, 18, 20 19 127.86 CH (ar) 7.21, m 17, 21 L-Leucine 22 171.66 C=O 23 52.63 Ch 4.11, dt (t, 7.2 Hz) 24 22, 24, 25 24 40.32** _CH2 a: 1.66, m 23, 24b 22, 23, 25, 26, 27 b: 1.58, m 23, 24a 22, 23, 25 25 25.93 * Ch 1.36*, m 26, 27 26 23.12* _CH3 0.86*, m 25 27 22.67* _CH3 0.82*, m 25 Table 5 Method 1 Method 2 Method 3 Method 4 Method 5 HPLC system ^® Preparative HPLC Preparative HPLC Preparative HPLC Preparative HPLC Preparative HPLC Agilent 1260 Agilent 1260 Agilent 1260 Agilent 1260 Agilent 1260 Solvent A _H2O + 0.1% TFA _H2O + 0.1% TFA _H2O + 0.1% TFA _H2O + 0.1% TFA _H2O + 0.1% TFA Solvent B Acetonitrile Acetonitrile Acetonitrile Acetonitrile Acetonitrile Gradient for B 0-0.5min: 35%, 0-0.5min: 45%, 0-0.5min: 35%, 0-0.5min: 70%, 0-0.5min: 80%, 0.5-13.5 min: 35-80%, 0.5-20 min: 45-100%, 0.5-12.5 min: 35-80%, 0.5-6 min: 70-85%, 0.5-10.5 min: 80-100%, 13.5-17 min: 80-100%, 10-11 min: 100%, 12.5-16 min: 80-100%, 6-13.5 min: 85-100%, 10.5-16.0 min: 100%, 17-19mm: 100%, 11-11.5 min: 100-45%, 16-17 min: 100%, 13.5-14.5 min: 100%, 16-16.5 min: 100-80%, 19-19.5 min: 100-35%, 11.5-13 min: 45% 17-17.5 min: 100-35%, 14.5-15 min: 100-70%, 16.5-21 min: 80% 19.5-21 min: 35% 17.5-19 min: 35 min 15-16.5 min: 70% Temperature flow rate column 12°C 12°C 12°C 12°C 12°C 25 mL/min 2mL/min 2mL/min 2mL/min 3.5 mL/min Agilent Eclipse XDB-C18 Thermo Fisher Hypercarb Agilent Eclipse XDB-C18 Phenomenex SynergiHydro-RP Thermo Fisher Hypercarb Column dimensions 250 × 9.4 mm, 5 µm 150 × 4.6mm, 5µm 250 × 9.4 mm, 5 µm 250 × 10mm, 4µm 150 × 4.6mm, 5µm Table 6 Vbdg. pathogenic bacteria ^a Mycobacteria ^b filamentous bacteria ^c Mushrooms ^d Mammalian cell lines ^e human macrophages ^f Trypanosomes ^g Nematodes ^h 1 >1000 µg mL ^-1 >125 µg mL ^-1 nb >1000 µg mL ^-1 >100 µg mL ^-1 >14.4 µg mL ^-1 >150 µg mL ^-1 >100 µg mL ^-1 2 >1000 µg mL ^-1 >125 µg mL ^-1 > 516 µg mL ^-1 >1000 µg mL ^-1 >100 µg mL ^-1 >15.5 µg mL ^-1 >150 µg mL ^-1 >100 µg mL ^-1

QUOTES INCLUDED IN THE DESCRIPTION

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Claims (11)

Cycloacetamides A to F with the general formula cyclo-(-N-Ac-L-Thr-ABCO-) where L-Thr represents L-configured threonine and, in which A, B and C independently represent the same or different L- or D-configured amino acids with non-polar side chains, in particular alanine (Ala), valine (Val), leucine (Leu) , isoleucine (Ile), methionine (Met), phenylalanine (Phe), tyrosine (Tyr) or tryptophan (Trp). Cycloacetamides A to F according to Claim 1 , where

is. Process for the production of the natural substances cycloacetamides A to F using Mortierella alpina, in which Mortierella alpina mycelium is transferred to a nutrient solution and then cultivated, the natural substances cycloacetamides A to F being separated from the Mortierella alpina mycelium by filtration with the nutrient solution, whereby the natural substances cycloacetamides A to F from the fungus Mortierella alpina are produced without fixation on a carrier in a known production vessel in the nutrient solution without reducing the shear force load, then • the separated filtrate is acidified, • a liquid-liquid extraction, • evaporation and • is subjected to lyophilization, whereby • the dissolved lyophilisate is subjected as a concentrated extract to a preparative and then semi-preparative HPLC, so that • after the 1st and 2nd semi-preparative HPLC, the cycloacetamide A and the cycloacetamide B are present in pure form, • after the 3rd and 4th semi-preparative HPLC the cycloacetamide E and • After the 5th semi-preparative HPLC, the cycloacetamide C and the cycloacetamide D are present in pure form. Procedure according to Claim 3 , characterized in that the fungus Mortierella alpina is cultivated in a shaken Erlenmeyer flask, a stirred tank reactor or a stirred fermenter to obtain the natural substances cycloacetamides A to F. Procedure according to Claim 3 or 4 , characterized in that the incubated preculture of the fungus Mortierella alpina for the production of the natural substance cycloacetamide A to F is fed to an MEP nutrient solution with 30 g/L malt extract and 3 g/L soy peptone, the temperature being between 4 ° C and 30 °C, the pH value is between pH 5 and pH 9 and the cultivation time is between 3 and 21 days. Procedure according to Claim 3 , 4 or 5 , characterized in that the fungus Mortierella alpina is reused for renewed cycloacetamide A to F production in the production vessel, the fungus being separated from the nutrient solution in the production vessel and remaining therein for reuse and being supplied with new nutrient medium. Procedure according to Claim 3 , 4 , 5 or 6 , characterized in that for a cyclic cycloacetamide A to F extraction process, the nutrient solution containing cycloacetamides A to F is removed from the production vessel at intervals of 10 days and replaced with new nutrient solution. Process for the chemical, non-canonical liquid phase synthesis of cycloacetamides A to F with the production of a complete linear depsipeptide with subsequent final macrolactamization comprising the substeps: a) linear coupling of the suitably protected amino acid derivatives, b) linear esterification of the amino acid C protected with an amino protecting group X according to the general formula Claim 1 with TV-Ac-Z-Thr -O, where Y represents an alkylhydroxy protecting group, c) cleavage of the amino or carboxyl protecting groups of the peptides obtained in each case and d) macrolactamization of the final linear depsipeptide, where the amino protecting group is X-protecting groups that are the existing Protect amino group from chemical reactions and can be split off again at will by acid or alkaline hydrolysis, reduction or nucleophilic attack, the blocking of the carboxyl function of the amino acids takes place as alkyl ester Y, which in turn is deprotected by alkaline or nucleophilic dealkylation. Procedure according to Claim 8 , characterized in that • the deprotection of the methyl ester is carried out with trimethyltin hydroxide, • the deprotection of the Use of cycloacetamides A to F produced or chemically synthesized by Mortierella alpina as an insecticide. Mortierella alpina DSM 34346 as a producer strain for cycloacetamide A and cycloacetamide B.

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