JP2007161690A - COMPOUND HAVING beta-N-ACETYLGLUCOSAMINIDASE INHIBITORY ACTIVITY - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a new compound which exhibits insecticidal/germicidal effect and is useful as a raw material for a safe agricultural and horticultural chemical and a food additive. <P>SOLUTION: The compound is represented by formula (X<SP>-</SP>is a counteranion; D is hydrogen H or deuterium D) in which GlcNAc-(β1,4)-GlcNAc-(β1,4)-GlcNAc is bonded to N-trimethylglucosamine by an α-1-4 bond. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a compound having β-N-acetylglucosaminidase (hereinafter also abbreviated as GlcNAcase) inhibitory activity, an insecticidal / bactericidal composition containing the same, an agrochemical, a gardening material, a food additive, and a food, and β-N -It is related with the manufacturing method of the compound which has acetylglucosaminidase inhibitory activity.

One of the physiological phenomena unique to insects in the process of insect growth is molting. For the molting, degradation metabolism of chitin constituting the epidermis is indispensable, and chitinase and β-N-acetylglucosaminidase are known as enzymes involved in the degradation metabolism. Exploratory research for GlcNAcase inhibitors has been extensive so far, and many compounds have already been found (see, for example, Non-Patent Document 1).
Several inhibitory substances against the above two enzymes have been reported, but they have also been reported to have no specificity because they also inhibit enzymes derived from species other than insects.
Pharmacol. Ther. , 76, 287-218, (1997)

  If we can find compounds that specifically inhibit insect β-N-acetylglucosaminidase, such compounds will inhibit molting of insect pests and specifically prevent insect growth. Therefore, we focused on the possibility of developing safe insect control agents for human livestock. Thus, the original object of the present invention was to find compounds that specifically inhibit such insect β-N-acetylglucosaminidase.

（式I中、ｎは０〜５の整数、Ｘ⁻は対アニオンを表す。）
で示される４種類の化合物を４種類の混合物として単離することに成功した。また、式Ｉで示される化合物を重メタノール処理後、還元することによって、式（II−１）

（式II−１中、ｎは０〜５の整数、Ｘ⁻は対アニオン、Ｒは水素Ｈまたは重水素Ｄを表す。）
および
式（II−２）

（式II−２中、ｎは０〜５の整数、Ｘ⁻は対アニオン、Ｒは水素Ｈまたは重水素Ｄを表す。）
で示される化合物の製造に成功するとともに、これらの化合物が昆虫のβ−Ｎ−アセチルグルコサミニダーゼに対して特異的に優れた阻害作用を有することを知見した。本発明者らはまたこれらの化合物が人畜に対して低毒または無毒であることを見出した。 The present inventors have extensively searched for a large number of various microorganisms, particularly microorganisms belonging to the genus Streptomyces, Actinomycete, Paecilomyces, etc., for example, filamentous fungi, actinomycetes.

(In formula I, n represents an integer of 0 to 5, and X ⁻ represents a counter anion.)
Was successfully isolated as a mixture of four compounds. Further, the compound represented by the formula I is treated with deuterated methanol and then reduced to reduce the formula (II-1)

(In formula II-1, n represents an integer of 0 to 5, X ⁻ represents a counter anion, and R represents hydrogen H or deuterium D.)
And formula (II-2)

(In formula II-2, n represents an integer of 0 to 5, X ⁻ represents a counter anion, and R represents hydrogen H or deuterium D.)
In addition to the successful production of the compounds represented by the formula (1), it has been found that these compounds have a particularly excellent inhibitory action on insect β-N-acetylglucosaminidase. The inventors have also found that these compounds are low or non-toxic to livestock.

  Furthermore, the present inventors have found that these compounds have an excellent bactericidal action. Furthermore, the present inventors have found that these compounds or compositions containing these compounds are useful as agricultural and horticultural drugs and food additives.

（式I中、ｎは０〜５の整数、Ｘ⁻は対アニオンを表す。）
または、式II

（式II中、ｎは０〜５の整数、Ｘ⁻は対アニオン、Ｒは水素Ｈまたは重水素Ｄを表す。）
で示される化合物、
（２）式Ｉ

（式I中、ｎは０〜５の整数、Ｘ⁻は対アニオンを表す。）
または、式II

（式II中、ｎは０〜５の整数、Ｘ⁻は対アニオン、Ｒは水素Ｈまたは重水素Ｄを表す。）
で示される化合物を含有する殺虫性または殺菌性組成物、
（３）農薬用または園芸用であることを特徴とする、上記（２）に記載の組成物、
（４）食品添加剤用であることを特徴とする、上記（２）に記載の組成物、
（５）上記（１）に記載の化合物を含有することを特徴とする食品、および
（６）上記（１）に記載の化合物を産生し得る微生物を培養し、その培養物から上記（１）に記載の化合物を取得することを特徴とする、上記（１）に記載の化合物の製造方法
に関する。 That is, the present invention
(1) Formula I

(In formula I, n represents an integer of 0 to 5, and X ⁻ represents a counter anion.)
Or the formula II

(In formula II, n represents an integer of 0 to 5, X ⁻ represents a counter anion, and R represents hydrogen H or deuterium D.)
A compound represented by
(2) Formula I

(In formula I, n represents an integer of 0 to 5, and X ⁻ represents a counter anion.)
Or the formula II

(In formula II, n represents an integer of 0 to 5, X ⁻ represents a counter anion, and R represents hydrogen H or deuterium D.)
An insecticidal or bactericidal composition comprising a compound represented by:
(3) The composition according to (2) above, which is used for agricultural chemicals or horticulture,
(4) The composition as described in (2) above, which is for food additives,
(5) A food containing the compound according to (1) above, and (6) a microorganism capable of producing the compound according to (1) above, and culturing the above-mentioned (1) from the culture The compound according to (1) is obtained, wherein the compound according to (1) is obtained.

  The compound of the present invention exhibits an insecticidal action and a bactericidal action, and is useful as an agricultural or horticultural drug or food additive.

The compounds of the present invention are represented by the above formulas (I-1), (I-2), (I-3), (I-4), (II-1), and (II-2).
In the above formula, the counter anion represented by X ⁻ may be any anion as long as it is an anion, and is not particularly limited. Specifically, for example, halogen, hydroxyl group, carbonate ion, bicarbonate ion, sulfate ion, Organic or inorganic anions such as bisulfate ion, oxalate ion, acetate ion and formate ion can be mentioned, and hydrogen carbonate ion is preferred.
The four types of compounds represented by Formula I are isomers, respectively. In Formulas (I-1) and (I-2) and Formulas (I-3) and (I-4), the first position of the reducing end is It is in the relationship of anomer (α, β). Further, in the formulas (I-1) and (I-3) and the formulas (I-2) and (I-4), the second position of the reducing end is an epimer, and the formulas (I-1) and (I -2) is glucosamine type, and formulas (I-3) and (I-4) are mannosamine type. Any of these four isomers and any mixtures thereof shall belong to the present invention.

The compounds of the above formula I are, for example, filamentous fungi or actinomycetes, for example microorganisms belonging to the genus Streptomyces, Actinomycete, Paecilomyces, preferably microorganisms belonging to the species of Streptomycins species, more preferably microorganisms such as Streptomyces anthulus NBRC13 , By obtaining a compound of formula I from the culture.
The microorganism is cultured according to a method known per se. The medium used for the culture can be cultured using a normal nutrient medium containing, for example, a carbon source, a nitrogen source, an inorganic salt, and the like. As the carbon source, for example, glucose, molasses and the like can be used. However, when culturing the above Streptomyces anuratus NBRC13369 strain, it is preferable to add chitin, preferably colloidal chitin as the carbon source. As the nitrogen source, for example, ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, urea and the like can be used alone or in combination. As inorganic salts, for example, potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate and the like can be used. In addition, nutrients such as various vitamins such as agar, amino acid, peptone, meat extract, yeast extract, corn steep liquor, casamino acid, biotin, thiamine and the like can be appropriately added to the medium as necessary. When culturing Streptomyces anuratus NBRC13369 strain, the compound represented by the formula I may not be produced in the rich medium, so it is necessary to select the medium appropriately.

Culturing can be usually performed at a temperature of about 20 ° C. to about 40 ° C., preferably about 25 ° C. to about 40 ° C. under aerobic conditions such as aeration agitation and shaking, or anaerobic conditions. The pH during the culture is in the range of 4 to 10, preferably 5 to 8, and the pH during the culture can be adjusted by adding an acid or an alkali. Further, the culture period is preferably half a day to 20 days.
After culturing, the cells are separated from the culture filtrate by filtration or centrifugation.
The obtained filtrate is subjected to purification means such as chromatography or solvent extraction. The purification means may be known means, for example, activated carbon column chromatography, cation exchange column chromatography, anion exchange column chromatography, Sephadex LH-20 column chromatography, Asipak ES 502C column chromatography and the like are preferable. Take as an example. All fractions obtained by chromatography are screened using biological activity as an indicator. As the biological activity, the enzyme inhibition rate by the GlcNAcase enzyme inhibition test is used as an index. Examples of such enzyme inhibition tests include (A) Spodoptera litura GlcNAcase inhibition test, (B) Calf kidney GlcNAcase inhibition test, (C) Human placenta GlcNAcase inhibition test, and (D) Aspergillus oryzae GlcNAcase inhibition test. For example, the fraction that is positive in the test (A) means that a compound that inhibits the growth of insects that molt is included, and a compound that exhibits such an inhibitory action is useful as an insecticide for molting insects. is there. Fractions that are negative for the other tests (B) and (C) indicate that they are harmless to human GlcNAcase. (D) A fraction that is positive in the test means that it exhibits an antibacterial action against turfgrass pathogens. Thus, fractions that are positive for (A) or (D) and negative for (B) and (C) test are insecticidal or bactericidal against harmful insects or fungi and are beneficial to beneficial animals. Fractions containing such compounds are collected, meaning that they can contain compounds that are safe.

Further, the fraction collected in this manner is subjected to purification means such as transfer dissolution, concentration, chromatography, crystallization, recrystallization, distillation, etc., and the intended agriculture, horticultural, etc., which peels off the target, for example, Spodoptera litura Compounds of formula (I-1), formula (I-2), formula (I-3), and formulas that inhibit the growth of pests or sanitary pests and phytopathogenic fungi and are harmless to other useful animals (I-4) can be obtained. Further, the compound represented by the formula I thus obtained exhibits an excellent antibacterial effect against, for example, turfgrass pathogens. These compounds exhibit a bactericidal action and do not exhibit activity against GlcNAcase of human livestock, that is, they are harmless to human livestock, and thus are useful, for example, as agricultural or horticultural insecticides or bactericides and food additives. Furthermore, by reducing the compound of the formula I, the compound formulas (II-1) and (II-2) represented by the formula II can be obtained. The compounds represented by the formula II are represented by the formula I It exhibits the same biological activity as the compound represented by N in these formulas is represented by 0 to 5, and n = 2 is preferable. In the examples described below, compounds with n = 2 are obtained, but compounds other than n = 2 may be obtained depending on the culture conditions and the conditions for isolating the active substance from the culture. In addition, by subjecting the obtained compound to a known hydrolysis means using an enzyme or an acid, a compound having a smaller value of n than the value of n of the compound produced in the culture solution can also be obtained. it can.
The reduction of the compound of formula I can be carried out by methods known per se. For example, conventional reduction means such as catalytic reduction using a platinum catalyst, palladium catalyst or the like as a catalyst, for example, reduction using a reducing agent such as sodium borohydride (SBH), sodium thiosulfate, etc. can be conveniently employed.

  The compound of the present invention or a salt thereof can be used as an agrochemical excellent in safety, for example, an insecticide or a fungicide. In particular, it has low toxicity to mammals and fish and shellfish, and does not pollute the environment, and can be used extremely safely as an insecticide or fungicide for paddy fields, fields, orchards or non-agricultural land.

  When the compound of the present invention or a salt thereof is used as an agrochemical, in particular, an insecticide or a fungicide, a form that can be taken by a general agrochemical, that is, one or more of the compound or a salt thereof is suitable depending on the purpose of use. Dissolved or dispersed in a liquid carrier, or mixed or adsorbed with a suitable solid carrier, for example, emulsion, oil, spray, wettable powder, powder, DL (driftless) type powder, granule, fine granule, Used as preparations such as fine granules F, flowables, dry flowables, jumbo granules, tablets. These preparations may contain, for example, an emulsifier, a spreading agent, a penetrating agent, a wetting agent, a mucilage, a stabilizer and the like, and can be prepared by a method known per se.

  In the composition of the present invention, for example, one or more active ingredients of the compounds represented by the above formula I or formula II are diluted with an appropriate inert liquid or solid carrier depending on the kind of the preparation, and if necessary, Then, a surfactant, a dispersing agent or an adjuvant is blended to produce the above preparation. Suitable carriers here include, for example, talc, bentonite, clay, kaolin, diatomaceous earth, vermiculite, acid white clay, talc powder, wax stone powder, diatomaceous earth, mica powder, alumina, sulfur powder, activated carbon, calcium carbonate, white carbon, Examples thereof include solid carriers such as vermiculite, slaked lime, silica sand, ammonium sulfate, urea, tobacco powder, and wood powder, and liquid carriers such as isopropyl alcohol, xylene, cyclohexanone, methylnaphthalene, fatty acid ester, vegetable oil, mineral oil, animal oil, and water. These carriers are used for preparing a preparation by mixing one or more kinds in an appropriate ratio.

Surfactants and dispersants include, for example, polyoxyethylene fatty acid ester, polyoxyethylene castor oil, polyoxyn ethylene alkyl phenyl ether, polyoxyethylene alkyl ether sulfate, alkyl benzene sulfonate, naphthalene sulfonate formalin condensate, polyoxyethylene alkyl phenyl. Examples include ether sulfonate.
Examples of the auxiliary agent include carboxymethyl cellulose, polyvinyl alcohol, polyethylene glycol, condensed phosphate and the like.
The agricultural and horticultural preparation of the present invention is produced by mixing the above components. These preparations are sprayed after being diluted to an appropriate concentration or applied directly.

  Specifically, the composition of the present invention can be applied to, for example, the following pest control. That is, the rice black bug (Scotinophara lurida), Nashigunbai (Stephanitis nashi), small brown planthopper (Laodelphax striatellus), brown planthopper (Nilaparvata lugens), green rice leafhopper (Nephotettix cincticeps), Unaspis yanonensis (Unaspis yanonensis), soybean aphid (Aphis glycines), radish Aphids (Brevicorine brassicae), cotton aphids (Aphis gossypii), white-tailed planthopper (Sogatella furcifera), tea-nosed leafhopper (Empoasca onukii), staghorn beetle (Ps) eudococus comstocki), common cutworm (Spodoptera litura), diamondback moth (Plutella xylostella), cabbage butterfly (Pieris rapae crucivora), rice stem borer (Chilo supppressalis), Tamanagin'uwaba (Autographa nigrisigna), tobacco budworm (Helicoverpa assulta), armyworm (Pseudaletia separata), cabbage armyworm (Mamestra brassicae), weevil (Echinocnemus squameus), rice weevil (Lissohoptrus oryzophilus), cotton weevil (Anthonomus grandis), Zukizoumushi (Callosobruchus chinensis), grass reed weevil (Sphenophorus venatus), Japanese beetle (Popillia japonica), cupreous chafer (Anomala cuprea), corn root worm fellow (Diabrotica spp.), Colorado potato beetle (Leptinotarsa decemlineata), fellow of click beetles (Agriotes spp .), Musca domestica, Culex popiens pallens, German cockroach (Blatella germanica), Black cockroach (Peripraneta friginosa), Japanese cockroach (Petella japonica) ponica), American cockroach (Periplaneta americana), rice Shin Galle nematode (Aphelenchoides besseyi), strawberry menu nematode (Nothotylenchus acris), Formosan subterranean termite (Coptotermes formosanus), Yamato termite (Reticulitermes speratus), Taiwan termites (Odontotermes formosanus), Daikoku termites (Cryptotermes It is particularly effective for controlling pests such as domesticus).

  In addition, since the compound of the present invention exhibits an antibacterial action against turfgrass pathogens, diseases such as helmintosporium, fairy ring, dollar spot, psium, brown patch, rust disease caused by the fungus are observed. It is effective for the treatment and prevention of yellow patches.

  For example, when spraying on the soil before sowing or planting, the preparation of the present invention per 10a may be sprayed about 0.8 to 30 kg about 30 days before the day of sowing or planting. Moreover, what is necessary is just to spray to the soil in which the crop is growing at intervals of 10 to 20 days with the amount of the preparation of the present invention per 10a being about 1 kg to 50 kg.

  Since the compound of the present invention is harmless to humans and exhibits an excellent bactericidal effect, it is useful as a food additive and can be added to foods to significantly extend the storage period of foods. Any one or more of the compounds represented by Formula I or Formula II may be added directly to the food, or the compound may be diluted with a suitable diluent and added to the food. Examples of the food to which the compound of the present invention is added include frozen surimi, salmon, bamboo rings, deep-fried sweet potato, fish paste such as fish sausage, meat products such as ham and sausage, green tea, oolong tea, wheat tea, mixed tea (blend tea), Coffee, coffee milk drink, cafe au lait, tea, milk tea, cocoa, milk cocoa, milkshake, low acid beverages such as juice, functional drinks including fruit juices, flavors, functional ingredients, sports drinks, nutritional drinks, etc. Acidic drinks, potato salad, macaroni salad, dumplings, shumai, thick-boiled eggs, side dishes such as seasoning, boiled food, pickles such as shallow pickles, cooked rice / porridge, tofu / deep-fried dishes, raw noodles, boiled noodles, Noodles such as steamed noodles, rice cakes such as red bean rice cake, potato rice cake, chestnut rice cakes, creams such as flower paste and custard cream, hamburger, meat dumplings, etc. Minced meat processed products, Negitoro, fish processed products, such as assault, curry donuts, core ingredient such as Chinese bun (buns), oyakodon, beef bowl, and a bowl of such katsudon.

  The compound represented by formula (I-1), formula (I-2), formula (I-3), and formula (I-4), or formula (II-1) and formula (II-2) of the present invention In the following, the compound in which n = 2 is described with reference to examples, but the present invention is not limited thereto. In addition, the enzyme inhibition test method employ | adopted in the Example of this invention is as follows. The% described below is (w / w)%.

[Test Example 1]
Enzyme Inhibition Test Method (I) Lotus moth GlcNAcase Inhibition Test Method First, the citrus moth GlcNAcas enzyme solution was added to the “K. Kawazu, S. Ohnishi, H. Kanzaki and A. Kobayashi: Z. Naturefor 7: Z. Naturfor7. 1996) ”. Specifically, it is as follows.
50 g of Spodoptera litura was ground in 50 mL of 14.3 mM citrate / phosphate / borate buffer (pH 7.0) containing 0.01% phenylthiourea (PTU) and filtered. The filtrate was centrifuged at 20,000 g and 4 ° C. for 30 minutes, and the supernatant was further ultracentrifuged at 100,000 g and 4 ° C. for 60 minutes. To the supernatant, ammonium sulfate was gradually added so as to be 60% saturated, and stirred at 4 ° C. for 1 hour. The solution was centrifuged at 20,000 g and 4 ° C. for 15 minutes, and the supernatant was dialyzed at 4 ° C. The dialyzed external solution at this time was 2 L of 14.3 mM citrate / phosphate / borate buffer (pH 7.0) containing 0.01% PTU. The dialyzed internal solution was further dialyzed under the same conditions as described above, and the resulting dialyzed internal solution was centrifuged at 20,000 g, 4 ° C. for 15 minutes, and the supernatant was used as a cedar moth GlcNAcas enzyme solution.
Moreover, as buffer solution, 643 mM citrate / phosphate / borate buffer solution (pH 6.0) 24 μL, as substrate 5 mM p-nitrophenyl N-acetyl-β-D-glucosaminide aqueous solution 16 μL, test sample aqueous solution 80 μL, Alternatively, a 14.3 mM citrate / phosphate / borate buffer solution (pH 6.0) of GlcNAcase enzyme solution prepared from Spodoptera litura as described above was added to a mixture obtained by adding 80 μL of a solution containing water not containing a test sample as a control. ) 40 μL of diluted solution was added, stirred well, and reacted at 37 ° C. for 60 minutes. After completion of the reaction, 100 μL of 1.3 M aqueous sodium hydroxide solution was added, and after stirring well, the absorbance (a) at 415 nm was measured immediately using a spectrophotometer. At the same time, the absorbance (b) of the control group not containing the test sample was measured. Here, the amount of the GlcNAcase enzyme solution added to the control group was such that the absorbance (b) at 415 nm was 0.500. The GlcNAcase inhibition rate (%) was calculated by [1- (a) / (b)] × 100.

(B) Calf kidney GlcNAcase inhibition test method The buffer solution and the enzyme solution were changed as follows according to the method of (A). Specifically, bovine kidney-derived β-N-acetylglucosaminidase (SIGMA) was used as the GlcNAcase enzyme solution, and 250 mM citrate buffer (pH 5.0) containing 0.025% BSA and 250 mM NaCl was used as the buffer. Other conditions are the same as (a).

(C) Human placenta GlcNAcase inhibition test method The buffer solution and the enzyme solution were changed as follows according to the method of (a). Specifically, human placenta-derived β-N-acetylglucosaminidase (SIGMA) was used as a GlcNAcase enzyme solution, and a 250 mM citrate buffer (pH 4.3) containing 0.025% BSA and 250 mM NaCl was used as a buffer. Other conditions are the same as (a).

(D) Penicillium oxalicum GlcNAcase inhibition test method The buffer solution and the enzyme solution were changed as follows according to the method of (a). Specifically, Penicillium oxalicum-derived β-N-acetylhexosaminidase (Seikagaku Corporation) was used as the GlcNAcase enzyme solution, and 250 mM citrate buffer (pH 4.5) was used as the buffer. Other conditions are the same as (a).

(E) Aspergillus oryzae GlcNAcase inhibition test method The buffer solution and the enzyme solution were changed as follows according to the method of (a). That is, Aspergillus oryzae-derived β-N-acetylhexosaminidase (SIGMA) as a GlcNAcase enzyme solution, and 250 mM citrate buffer (pH 5.0) containing 0.025% BSA and 250 mM NaCl as a buffer solution. Using. Other conditions are the same as (a).

(F) Tachinata Bean (Jack bean) GlcNAcase Inhibition Test Method The buffer solution and the enzyme solution were changed as follows according to the method (i). That is, Jack bean-derived β-N-acetylglucosaminidase (SIGMA) was used as the GlcNAcase enzyme solution, and 250 mM citrate buffer (pH 5.0) containing 0.025% BSA and 250 mM NaCl was used as the buffer. . Other conditions are the same as (a).

Tables 1 to 3 summarize these six enzyme inhibition test methods.

[Example 1]
Screening 916 strains of filamentous fungi and 39 strains of actinomycetes are arbitrarily collected and cultured under appropriate medium and culture conditions, and the culture (culture filtrate or organic solvent extract of the culture; acetone / methanol as the organic solvent) Mixed solvents (1: 1, v / v) or n-butanol were used for enzyme inhibition tests. As a primary screening, a sample that inhibits GlcNAcase derived from the insect moth Spodoptera litura was selected. Next, these active samples were subjected to β-N-acetylglucosaminedase (GlcNAcase) inhibition test derived from calf kidney, which is a mammal, and samples that did not inhibit this enzyme were selected. By such a screening system, an active strain that specifically inhibits insect GlcNAcase, Streptomyces anuratus NBRC13369 strain (strain purchased for research) was found.

[Example 2]
Fermentative production of active substances All media were sterilized at 120 ° C. for 20 minutes before use.
(I) Strain preservation medium As a strain preservation medium, 1 g of L-Asparagine, 10 g of Glycerol, 1 g of K ₂ HPO _4, 1 L of distilled water, 0.001 g of FeSO ₄ .7H ₂ O, 0.001 g of MnCl ₂ .4H ₂ O, ZnSO _A medium composed of 0.001 g of 4.7H ₂ O and 20 g of agar was used. The medium before sterilization was adjusted to pH 7.2.
(Ii) Fermentation production Streptomyces anuratus NBRC 13369 strain preserved one platinum ear, glucose 10g, NZ Amine typeA 2.0g (manufactured by Wako Pure Chemical Industries, Ltd.), yeast extract 1.0 g (manufactured by Nacalai), beaf
Extract 1.0 g (manufactured by DIFCO) and 10 mL (pH 7.0) of a liquid medium composed of 1 L of distilled water was inoculated and subjected to reciprocal shaking culture at 28 ° C. and 300 strokes / min for 2 days to obtain seed culture. Next, as production media, colloidal chitin 5.0 g, NZ Amine type A 2.0 g (manufactured by Wako Pure Chemical Industries, Ltd.), yeast extract 1.0 g (manufactured by Nakarai Co., Ltd.), beef extract 1.0 g (manufactured by DIFCO), A medium consisting of 1 L of distilled water was dispensed in a volume of 300 mL into a 1 L Sakaguchi flask, the whole amount of the seed medium was added, and cultured under conditions of 28 ° C. and 123 strokes / min for 14 days. After completion of the culture, 6.9 L of the culture solution was filtered and separated into cells and culture filtrate.

  Incidentally, the colloidal chitin (colloidal chitin) is, "Improved bioassay method for Spodoptera litura chitinase inhibitors using acolloidal chitin powder with a uniform particle size as substrate. T. Nitoda, H, Kurumatani, H. Kanzaki, and K. Kawazu , Pesticide Science, Vol. 55, 563-565 ", with some modifications and prepared as follows. That is, 2.5 g of chitin powder (Wako Pure Chemicals, grade 1, Lot. 0796) was added in small portions over 4 to 5 minutes while stirring with a stirrer to 100 mL of cooled (in ice, cryogen) 35% hydrochloric acid. . Further, the liquid was stirred with a stirrer for 10 minutes, taken out of ice, and warmed to room temperature while stirring. Furthermore, the liquid was stirred with a stirrer at 30 ° C. for 60 minutes, and suction filtered (SHIBATA3G3 glass filter, pore size: 40 to 100 μm). The filtrate was poured into 1 L of demineralized water (2 ° C.) while stirring with a stirrer and allowed to stand at 4 ° C. overnight. After removing the supernatant by decantation (volume of about 300 mL), the solution was neutralized with 0.5N sodium hydroxide to pH 7.0 in ice, and suction filtered (SHIBATA25GP100 glass filter, pore size: 40 to 100 μm). The residue was collected, suspended in tap water, stirred with a stirrer for 3 minutes, and dialyzed overnight against tap water (running water, room temperature) (Viskase Companies, Inc. 36/32 (φ27 mm)). Furthermore, 2 L of demineralized water was dialyzed at 4 ° C. for 3 hours as an external solution, and further dialyzed overnight at 4 ° C. using 2 L of demineralized water as an external solution. Then, chitin was collect | recovered by centrifuging a dialyzed internal solution at 13,000 rpm for 10 minutes (RPR20-2). The recovered chitin was lyophilized for 18 hours to obtain 1.448 g of colloidal chitin powder.

Example 3
Isolation of active substance (i) Activated carbon column 5.5 L of the culture filtrate was subjected to a column (6.5 × 33 cm) of activated carbon for column chromatography (manufactured by Nacalai), and 1.1 L of demineralized water The active ingredient was eluted with 2.5 L of 30% acetone containing 0.01% HCl, and the fraction showing the inhibitory activity was concentrated under reduced pressure to distill off the acetone to obtain a 1.4 L aqueous solution. . When a part of this aqueous solution was freeze-dried, the total weight of the crude active substance in this aqueous solution was calculated to be 4.7 g (brown hygroscopicity).
(Ii) Cation exchange column and activated carbon column (desalting)
The total amount (1.4 L) of the aqueous solution in which the crude active substance was dissolved was applied to an Amberlite CG-50 (H ⁺ type) column (6.5 × 31 cm), washed with 2 L of demineralized water, and then 0.1 M NaCl. The active ingredient was eluted with an aqueous solution, and fractions showing inhibitory activity were combined. This combined fraction is applied to a column (3.4 × 11 cm) of activated carbon for column chromatography (manufactured by Nakarai Co., Ltd.), washed with 400 mL of demineralized water, and then 500 mL of 30% acetone containing 0.01% HCl. The active ingredient was eluted with, the fraction showing inhibitory activity was concentrated under reduced pressure, and acetone was distilled off to obtain 75 mL of an aqueous solution. The total amount of this aqueous solution was lyophilized to obtain 215 mg of a brown hygroscopic crude active substance.
(Iii) Sephadex LH-20 column The total amount (215 mg) of the above crude active substance was dissolved in a small amount of methanol, and the column (1.0 × 50 cm) of Sephadex LH-20 (Pharmacia) equilibrated in advance with methanol. Then, the fraction showing inhibitory activity by developing with methanol was concentrated to dryness to obtain 97.8 mg of a brown hygroscopic crude active substance.
(Iv) Asahipak ES 502C HPLC
The total amount of the crude active substance (97.8 mg) was dissolved in 860 mL of water, and divided into 12 portions and subjected to the following column chromatography. That is, a column for high performance liquid chromatography (HPLC) (Shodex, Asahipak ES 502C 7C, 7.6 × 100 mm, flow rate 0.8 mL / min) previously equilibrated with 7.5 mM ammonium carbonate aqueous solution was added to the aqueous solution about 70 μL is applied, the active ingredient is eluted with 7.5 mM ammonium carbonate aqueous solution, and the fraction showing inhibitory activity is subjected to lyophilization, whereby 40 mg of pure active substance as white powder (molecular formula: C ₃₃ H ₅₉ O ₂₀ N ₄ , molecular weight: 831 FAB-MS (positive) m / z 831 (M) ⁺ ) was obtained. This compound is hereinafter referred to as active substance A.

[Test Example 2]
Structural analysis of active substance A Active substance A is represented by formula (I-1), formula (I-2), formula (I-3), and formula (I-4), and at least 4 where n = 2 An equilibrium mixture of different isomers. The information related to structural analysis is shown below.
(I) Sugar composition analysis of active substance A Active substance A (20 μg) was subjected to methanol decomposition, and the decomposition product was N-acetylated and then TMS-derivatized. When this was subjected to GC-FID analysis, about 1 molecule of GlcNAc (N-acetylglucosamine) was detected from 1 molecule of active substance A. Therefore, the presence of GlcNAc as a constituent sugar of active substance A was clarified.
(Ii) Analysis of reducing end of active substance A (pyridylamination)
Active substance A was subjected to a reductive pyridylamination (PA) reaction to prepare PA-activated active substance A in which the reducing end was converted to PA. This was subjected to acid hydrolysis, N-acetylation, and then subjected to normal phase HPLC and reverse phase HPLC analysis. As a result, PA-GlcNAc was identified. Therefore, the sugar residue at the reducing end of active substance A was identified as GlcNAc.
(Iii) NMR analysis of active substance A 36 mg of active substance A was dissolved in 750 μl of CD ₃ OD (D is deuterium) and subjected to NMR analysis at 30 ° C. for 2 days. Yes, only the following information was obtained.
(1) There are three N-acetyl groups.
(2) Considering that there are four types of anomeric protons, it is an oligosaccharide having a degree of polymerization of 4.
Interestingly, when the active substance A after the completion of this experiment was subjected to FAB-MS analysis, its molecular weight was observed as m / z 832 [M] ⁺ . That is, the molecular weight increased by 1. Considering the fact that CD ₃ OD is used as the NMR measurement solvent, the possibility that one proton of the active substance A is substituted with deuterium was strongly suggested.
Next, based on the fact that the active substance A is a reducing oligosaccharide, this was subjected to SBH reduction, and the corresponding alditol was prepared to proceed with the structural analysis. In the subsequent structural analysis experiments, deuterated active substance A (molecular weight 832) is used.

Example 4
Preparation of active substance A alditol body 5.18 mg of active substance A was subjected to SBH reduction, and the reaction product was roughly purified on an activated carbon column and then purified sequentially by cation exchange HPLC and graphite carbon HPLC. Two kinds of compounds considered to be a body, active substance A-1-ol (0.86 mg) and active substance A-2-ol (2.88 mg) were obtained. That is, the active substance A gives two types of reductants. Therefore, it is considered that the intact active substance A is an equilibrium mixture of at least four isomers, and this equilibrium is stopped by the reduction reaction to give two types of reductants.

(I) Sugar composition analysis of active substance A-1-ol and active substance A-2-ol and identification of alditol moiety 40 μg of active substance A-1-ol and 40 μg of active substance A-2-ol were decomposed with methanol, respectively. The degradation product was subjected to N-acetylation and then converted to TMS, which was subjected to GC-FID analysis. As a result, GlcNAc (N-acetylglucosamine) and ManNAc (N-acetylmannosamine) alditol were detected from the active substance A-1-ol at a ratio of 2: 1. On the other hand, from the active substance A-2-ol, GlcNAc and ManNAc alditol were detected at a ratio of 1: 1. Therefore, it was found that these two types of alditols differ in the structure of the alditol moiety derived from the reducing end. Furthermore, in view of the fact that the ratio of detected GlcNAc differs between these two types of alditols, it was strongly suggested that the stability of glycosidic bonds may also be different.

(Ii) MS analysis of active substance A-2-ol Among these two types of alditols, quantitatively active substance A-2-ol was subjected to LR-FAB MS, HR-FAB MS analysis, and the following results Got.
LR-FAB MS m / z 834 [M] ⁺ , HR-FAB MS calcd for C ₃₃ H ₆₀ D ₁ O ₂₀ N ₄ [M] ⁺ 834.3394, found 834.3396
That is, a molecular weight and a composition formula corresponding to the alditol form of the deuterium substitution product of the active substance A were obtained.
Next, NMR analysis of the active substance A-2-ol was performed.

つまり、活性物質Ａ−２−ｏｌは、Ｎ−ｔｒｉｍｅｔｈｙｌ ｇｌｕｃｏｓａｍｉｎｅにＧｌｃＮＡｃ−（β１，４）−ＧｌｃＮＡｃ−（β１，４）−ＧｌｃＮＡｃがα−１−４結合した化合物の還元体であり、さらに、そのアルジトール部分の２位のメチンプロトンは重水素で置換されている事実が判明した。 (Iii) NMR analysis of active substance A-2-ol Active substance A-2-ol, 2 mg was subjected to various NMR measurements in CD ₃ OD and D ₂ O to obtain the following structural formula.

That is, the active substance A-2-ol is a reduced form of a compound in which GlcNAc- (β1,4) -GlcNAc- (β1,4) -GlcNAc is α-1-4 linked to N-trimethyl glucosamine, It was found that the methine proton at the 2-position of the alditol moiety was substituted with deuterium.

を得た。
つまり、活性物質Ａ−１−ｏｌは、Ｎ−ｔｒｉＭｅ−ｇｌｕｃｏｓａｍｉｎｅにＧｌｃＮＡｃ−（β１，４）−ＧｌｃＮＡｃ−（β１，４）−ＭａｎＮＡｃがα−１−４結合した化合物の還元体であり、さらに、そのＭａｎＮＡｃアルジトール部分の２位のメチンプロトンは重水素で置換されている事実が判明した。 (Iv) NMR analysis of active substance A-1-ol
Next, a small amount of the active substance A-1-ol, 0.7 mg was subjected to various NMR measurements in D ₂ O, and the following structural formula

Got.
That is, the active substance A-1-ol is a reduced form of a compound in which GlcNAc- (β1,4) -GlcNAc- (β1,4) -ManNAc is α-1-4 linked to N-triMe-glucosamine. It was found that the methine proton at the 2-position of the ManNAc alditol moiety was replaced with deuterium.

Next, the flow of the above analysis is shown.

Active substance A is a very interesting new compound from the following three points.
(1) A compound isolated for the first time from nature as an oligosaccharide containing an N-triMe-glucosamine residue.
(2) N-triMe-glucosamine itself, which is an abnormal sugar moiety, has never been reported from nature.
(3) This is the first demonstration demonstrating that the N-acetylglucosamine moiety at the reducing end can be isomerized under natural conditions.

[Test Example 3]
Enzyme inhibitory activity of active substance A Using the intact active substance A in which deuterium substitution did not occur, the presence or absence of enzyme inhibitory activity against various species-derived GlcNAcase was determined. As a control compound, nagstatin produced by Streptomyces amakusaensis was used. The enzyme inhibition test method is the same as that of Test Example 1.
Table 4 shows the measurement results. As is clear from Table 4, active substance A specifically inhibits GlcNAcase from insects and filamentous fungi, whereas nagstatin, a known GlcNAcase inhibitor, is GlcNAcase from insects, filamentous fungi, mammals and plants. Showed a tendency to obstruct widely. Since there has been no report on a GlcNAcase inhibitor exhibiting such a narrow inhibitory specificity, it can be said that the active substance A is a novel compound in terms of both its structure and physiological activity.

[Test Example 4]
Enzyme inhibitory activity of active substance A-1-ol and active substance A-2-ol In the same manner as in Test Example 3, the enzyme inhibitory activity of active substance A-1-ol and active substance A-2-ol The inhibitory activity was measured, and the results are shown in Table 5.

The present invention provides a novel compound which exhibits an insecticidal action and a bactericidal action and is useful as a raw material for safe agricultural and horticultural drugs or food additives.

Claims (6)

Formula I

(In formula I, n represents an integer of 0 to 5, and X ⁻ represents a counter anion.)
Or the formula II

(In formula II, n represents an integer of 0 to 5, X ⁻ represents a counter anion, and R represents hydrogen H or deuterium D.)
A compound represented by Formula I

(In formula I, n represents an integer of 0 to 5, and X ⁻ represents a counter anion.)
Or the formula II

(In formula II, n represents an integer of 0 to 5, X ⁻ represents a counter anion, and R represents hydrogen H or deuterium D.)
An insecticidal or bactericidal composition containing the compound represented by:   The composition according to claim 2, wherein the composition is for agricultural chemicals or horticulture.   The composition according to claim 2, wherein the composition is for food additives.   A food comprising the compound according to claim 1. A method for producing the compound according to claim 1, wherein a microorganism capable of producing the compound according to claim 1 is cultured, and the compound according to claim 1 is obtained from the culture.