Compounds capable of detecting microorganisms and their applications

Abstract

To provide compounds and methods for the rapid and sensitive detection of microorganisms. [Solution] The disclosed compound contains an artificial siderophore represented by the following formula (1) that can detect microorganisms having a mechanism to incorporate iron internally via a siderophore. TIFF2026098528000022.tif56170 Here, R1, R2, and R3 in formula (1) are each independently a catechol group or a hydroxamic acid group. M in formula (1) is any element belonging to the main group elements. X in formula (1) is a terminal portion that can be bound to a detectable label. In one preferred embodiment of the compound of this disclosure, a detectable label is further bound to X.

Description

[Technical Field]

[0001] This disclosure relates to compounds capable of detecting microorganisms and their uses, and more particularly to methods for detecting microorganisms using such compounds. [Background technology]

[0002] In fields such as food and beverage, pharmaceuticals, and medical devices, proper hygiene management is considered crucial. If microbial contamination occurs, consuming contaminated food or other products can lead to various illnesses and diseases, including food poisoning and infectious diseases. Furthermore, microbial contamination can have a significant impact on businesses due to health damage, post-incident response, and resulting economic losses. Therefore, preventing microbial contamination in the first place, or responding quickly if contamination does occur, is extremely important.

[0003] As a means of hygiene management, microbiological testing is generally cited. Conventional microbiological testing methods include, for example, a method for detecting microorganisms using a culture medium for microbial detection, disclosed in Japanese Patent Publication No. 2024-73718. In addition, Japanese Patent Publication No. 2023-113746 discloses a method for identifying microorganisms targeting 16S ribosomal RNA and 18S ribosomal RNA.

[0004] Japanese Patent Publication No. 2006-124346 discloses an immobilization material equipped with artificial siderophores for fixing predetermined target cells onto a substrate. Furthermore, Japanese Patent Publication No. 2014-181219 and Non-Patent Literature 1 disclose a microbial detection system using a complex of artificial siderophore complex and iron ions.

[0005] Incidentally, there are international standards for hygiene management in various fields such as food and beverage, pharmaceuticals, and medical devices. For example, in the food manufacturing sector, Hazard Analysis and Critical Control Point (HACCP) is adopted. From June 1, 2021, in principle, all food businesses are required to implement hygiene management based on HACCP. As a result, the burden on food manufacturers and restaurants regarding hygiene management is tending to increase. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2024-73718 [Patent Document 2] Japanese Patent Publication No. 2023-113746 [Patent Document 3] Japanese Patent Publication No. 2006-124346 [Patent Document 4] Japanese Patent Publication No. 2014-181219

[0007] [Non-Patent Document 1] Inomata et al. “Iron(III) Complexes with Hybrid-Type Artificial Siderophores Containing Catecholate and Hydroxamate Sites”Inorganic Chemistry 62,40,2023,16362-16377 [Overview of the project] [Problems that the invention aims to solve]

[0008] However, the method described in Japanese Patent Publication No. 2024-73718 requires culturing until microbial colonies form on the culture medium, which may result in a time-consuming detection process. The method described in Japanese Patent Publication No. 2023-113746 requires knowledge of microbial genes and the skills to handle them. Furthermore, implementing such microbial detection methods requires advanced testing equipment. For these reasons, there is a need for a faster and simpler detection method.

[0009] Therefore, this disclosure provides compounds and methods for the rapid and sensitive detection of microorganisms. [Means for solving the problem]

[0010] The compounds disclosed herein include artificial siderophores represented by the following formula (1) that are detectable by microorganisms having a mechanism for incorporating iron into their interior via siderophores. [ka] Here, R1, R2, and R3 in formula (1) are each independently a catechol group or a hydroxamic acid group. M in formula (1) is any element belonging to the main group elements. X in formula (1) is a terminal portion that can be bound to a detectable label. In one preferred embodiment of the compounds of this disclosure, the compound is a compound in which an artificial siderophore is bound to a detectable label. The preferred compound further includes a detectable labeling substance (hereinafter also simply referred to as "label") bound to X.

[0011] Through diligent research by the inventors, it was discovered that a complex of artificial siderophores and typical elements remarkably improves detection sensitivity. Furthermore, this artificial siderophore makes it possible to selectively detect microorganisms. This effect, through improved detection sensitivity, enables rapid and convenient detection of microorganisms.

[0012] In one aspect of the compounds disclosed herein, the above typical elements are selected from the elements belonging to Group 13 and the elements belonging to Group 14. Thereby, the detection sensitivity can be further improved.

[0013] In one aspect of the compounds disclosed herein, the above typical elements are selected from any of silicon, aluminum, and gallium. Thereby, the detection sensitivity can be more reliably improved.

[0014] In one aspect of the compounds disclosed herein, the terminal X is selected from any of an amino group, an alkylamino group, a nitro group, a phosphate group, a sulfo group, a vinyl group, an allyl group, an aldehyde group, an azide group, an ethynyl group, and a thiocyanate group. Thereby, the binding to a detectable label becomes easy. Such an effect can be expected to stabilize the compound.

[0015] In one aspect of the compounds disclosed herein, the detectable labeling substance is selected from either a fluorescent substance or an antibody. Thereby, microorganisms having a mechanism of taking in iron through a siderophore can be easily detected.

[0016] According to the present disclosure, a composition for detecting microorganisms having a mechanism of taking in iron through a siderophore is provided. In other words, as one aspect of the composition disclosed herein, it includes the compound of the present disclosure and a dispersion medium in which the above compound is dispersed.

[0017] In one aspect of the method for detecting the microorganisms disclosed herein, it includes the steps of preparing the compound of the present disclosure, introducing the compound of the present disclosure into a sample in which contamination by microorganisms is assumed and allowing the microorganisms to take it in, and detecting the label of the compound taken in by the above microorganisms. According to such a method, the detection sensitivity of microorganisms having a mechanism of taking in iron through a siderophore is improved. Thereby, microorganisms can be detected quickly and easily.

[0018] In one embodiment of the method for detecting microorganisms disclosed herein, the sample suspected to be contaminated with the above-mentioned microorganisms does not contain iron ions. This further improves the sensitivity of microorganism detection. [Brief explanation of the drawing]

[0019] [Figure 1] Figure 1 is a graph showing the fluorescence spectrum of the artificial siderophore-iron complex. [Figure 2] Figure 2 is a graph showing the fluorescence spectrum of one of the compounds disclosed herein (an artificial siderophore-silicon complex). [Figure 3A] Figure 3A is a microscopic image showing the detection results of E. coli using a catechol-type artificial siderophore-iron complex. [Figure 3B] Figure 3B is a microscopic image showing the detection results of Mycobacterium flavescens using a catechol-type artificial siderophore-iron complex. [Figure 4] Figure 4 is a microscopic image showing the detection results of Escherichia coli using one of the compounds disclosed herein (catechol-type artificial siderophore-silicon complex). [Figure 5A] Figure 5A is a microscopic image showing the detection results of Bacillus subtilis using one of the compounds disclosed herein (catechol-type artificial siderophore-silicon complex). [Figure 5B] Figure 5B is a microscopic image showing the detection results of Shewanella oneidensis using one of the compounds disclosed herein (catechol-type artificial siderophore-silicon complex). [Figure 5C] Figure 5C is a microscopic image showing the detection results of Mycobacterium flavescens using one of the compounds disclosed herein (catechol-type artificial siderophore-silicon complex). [Modes for carrying out the invention]

[0020] The following describes in detail typical embodiments of this disclosure. Matters necessary for implementation other than those specifically mentioned herein can be understood as design matters for those skilled in the art based on the prior art. This disclosure can be implemented based on the contents disclosed herein and common technical knowledge in the art. In addition, redundant explanations of members and parts that perform the same function in the following drawings may be omitted or simplified. Furthermore, the dimensional relationships (length, width, thickness, etc.) in each drawing are schematic for the purpose of clearly illustrating this disclosure and do not necessarily accurately reflect actual dimensional relationships.

[0021] In this specification, when a numerical range is described as "A to B (where A and B are arbitrary numbers)," it means "greater than or equal to A and less than or equal to B," and also encompasses the meanings of "greater than A and less than B," "greater than A and less than or equal to B," and "greater than or equal to A and less than B."

[0022] In this specification, “microorganism” can refer to microscopic organisms observed under a microscope, particularly bacteria, archaea, eukaryotes (e.g., protists, fungi, algae, etc.), and viruses. In this disclosure, “microorganism” is defined as any organism that has a mechanism for incorporating iron into its interior via siderophores. Typically, “microorganism” in this disclosure refers to bacteria, fungi, and algae. Furthermore, in this specification, “microorganism” should be understood as a plural expression.

[0023] <Siderophore> Siderophores are small molecules secreted by certain microorganisms to the extracellular space (outside the cell membrane) in order to take in iron ions into the cell (inside the cell membrane). In nature, siderophores secreted outside the cell take in trivalent iron ions (Fe) from the environment. 3+It captures the iron ion and forms a complex (siderophore-iron ion complex). This complex is recognized by membrane proteins (siderophore receptors) present on the cell membrane of microorganisms and is taken up into the cell by passing through the siderophore receptors. Furthermore, even microorganisms that do not produce or secrete siderophores themselves can take up iron ions by utilizing exogenous siderophores secreted by other microorganisms. 3+ It is reduced by iron reductase, etc., to divalent iron ions (Fe 2+ ) This weakens the bond with the siderophore, and Fe 2+ The siderophores are then released. Subsequently, the siderophores are either secreted again outside the cell or broken down by enzymes. In this way, natural siderophores are utilized by microorganisms to take up iron. In this specification, the term "natural" when referring to siderophores means siderophores that exist in nature. Examples of natural siderophores include coprogens, ferriclomes, ferrioxamine, deferoxamine, N,N,N-triacetylfusarinine C, ornibactin, rhodotrulic acid, enterobactin, batilibactin, vibriobactin, agrobactin, anguibactin, azotobactin, pioverdin, yersinia bactin, and staphyloferrin A.

[0024] <Compound> The compounds disclosed herein can detect specific microbial groups by utilizing the mechanism by which microorganisms take up iron into cells via siderophores. The compounds disclosed herein are compounds in which an artificial siderophore is bound to a detectable label. This compound contains a structural portion derived from the artificial siderophore of formula (1) below. Here, R1, R2, and R3 in formula (1) are each independently a catechol group or a hydroxamic acid group. Also, M in formula (1) is a main group element. In formula (1), X is the terminal portion that binds to the detectable label. That is, the detectable label is bound to X. The compounds disclosed herein can significantly improve the sensitivity of microbial detection. Such an effect allows for rapid and simple detection of microorganisms. [ka]

[0025] <Artificial siderophore> The siderophores constituting the compounds disclosed herein, unlike the natural siderophores described above, are artificially created siderophores that do not exist in nature. In this specification, the term "artificial" is used to distinguish siderophores from molecules with structures that already exist in nature. The artificial siderophores of this disclosure are artificially designed or fabricated and possess structures not found in natural siderophores. Specifically, the artificial siderophores disclosed herein are represented by formula (1) above. In formula (1), R1, R2, and R3 are each independently a catechol group or a hydroxamic acid group. Also, M in formula (1) is one of the main group elements. In formula (1), X is a terminal portion that can be bound to a detectable label.

[0026] Natural siderophores are expensive because they can only be isolated in small quantities and are difficult to synthesize. In contrast, the artificial siderophores of this disclosure have a simpler structure than natural siderophores and can be synthesized, making them possible to produce in large quantities at a low cost. Furthermore, their structure can be synthetically tuned. For example, the type of ligand in the artificial siderophores of this disclosure can be changed, thereby allowing for microbial selectivity. In addition, in order to bind a detectable label to a natural siderophore, it is necessary to introduce a new binding site. Since there are few types of natural siderophores that possess a binding site, their use is limited. In contrast, the artificial siderophores of this disclosure have terminal parts (in this case, functional groups) at their ends, so labels such as fluorescent substances can be easily bound to them. Therefore, one preferred embodiment of the compound disclosed herein further includes a detectable label bound to X.

[0027] The artificial siderophores of the present disclosure have a simplified structure compared to natural siderophores. Therefore, the artificial siderophores of the present disclosure can be easily synthesized, and their structures can be synthetically tuned. For example, R1 to R3 and X in the above formula (1) can be appropriately selected respectively.

[0028] The artificial siderophores disclosed herein have a chemical structure capable of coordinating with transition metal elements or typical elements such as iron ions. Specifically, the artificial siderophores of the present disclosure have three substituents (R1, R2, and R3) having catechol-type or hydroxamic acid-type ligands. These substituents can be chelating sites for iron ions and typical elements. The catechol site (catechol group) is represented by the following chemical formula (2). The hydroxy groups on catechol can coordinate bidentately to typical elements with oxygen atoms as coordinating atoms (donor atoms). The hydroxamic acid site (hydroxamic acid group) is represented by the following chemical formula (3). Here, R4 in formula (3) can typically be a lower alkyl group (for example, having 1 to 3 carbon atoms). The hydroxy group on hydroxamic acid can also coordinate bidentately to typical elements with oxygen atoms as donor atoms. That is, the artificial siderophores of the present disclosure are hexadentate ligands that form a tripod structure by the coordination bonding of three bidentate coordinating substituents to one typical element. Such a tripod-structured siderophore is preferable because it can appropriately chelate typical elements by utilizing the above catechol site and / or the above hydroxamic acid site.

Chemical formula

Chemical formula

[0029] <R1~R3: Ligand> The artificial siderophores disclosed herein have a catechol-type ligand and / or a hydroxamic acid-type ligand, as described above. Substituents R1, R2, and R3 are each independently catechol or hydroxamic acid. The artificial siderophores of this disclosure are selective for the detection of microorganisms depending on the ratio of catechol moieties to hydroxamic acid moieties. The ratio of catechol to hydroxamic acid can be appropriately selected depending on the purpose. That is, the ratio of catechol to hydroxamic acid [(catechol moieties):(hydroxamic acid moieties)] in the artificial siderophores of this disclosure may be 0:3 (in other words, all catechol moieties), 1:2, 2:1, or 3:0 (in other words, all hydroxamic acid moieties).

[0030] A preferred example of the artificial siderophore disclosed herein is the one represented by the following chemical formula (4). Formula (4) is an artificial siderophore in which R1 to R3 of formula (1) are all catechol. The ratio of catechol to hydroxamic acid in R1 to R3 [(catechol moiety):(hydroxamic acid moiety)] is 3:0. That is, formula (4) represents a catechol-type artificial siderophore. Furthermore, because the catechol moiety has a high chelating effect, it can stably retain the coordinated main group element. [ka]

[0031] Furthermore, other preferred examples of the artificial siderophore disclosed herein include, for example, the one represented by the following chemical formula (5). Formula (5) is an artificial siderophore in which R1 to R2 are catechol and R3 is hydroxamic acid in formula (1). That is, formula (5) represents a mixed ligand type artificial siderophore. The ratio of catechol to hydroxamic acid in R1 to R3 [(catechol part):(hydroxamic acid part)] should be 2:1. More specifically, the structure may be such that R1 and R2 are catechol and R3 is hydroxamic acid, or R1 and R3 are catechol and R2 is hydroxamic acid, or R2 and R3 are catechol and R1 is hydroxamic acid. Equivalent effects can be obtained with any of these combinations. [ka]

[0032] Alternatively, other preferred examples of the artificial siderophore disclosed herein include, for example, those represented by the following chemical formula (6). Formula (6) is an artificial siderophore in which R2 to R3 of formula (1) are catechol and R1 is hydroxamic acid. That is, formula (6) represents a mixed ligand type artificial siderophore. The ratio of catechol to hydroxamic acid in R1 to R3 [(catechol part):(hydroxamic acid part)] should be 1:2. More specifically, the structure may be such that R1 is catechol and R2 and R3 are hydroxamic acid, or R2 is catechol and R1 and R3 are hydroxamic acid, or R3 is catechol and R1 and R2 are hydroxamic acid. Equivalent effects can be obtained with any of these combinations. [ka]

[0033] Alternatively, as another preferred example of the artificial siderophore disclosed herein, for example, those represented by the following chemical formula (7) can be mentioned. Formula (7) is an artificial siderophore when all of R1 to R3 in the above formula (1) are hydroxamic acids. The ratio of catechol and hydroxamic acid in R1 to R3 [(catechol moiety):(hydroxamic acid moiety)] is 0:3. That is, formula (7) represents a hydroxamic acid type artificial siderophore.

Chem.

[0034] <M: Typical element> The compounds disclosed herein can be in an embodiment containing a typical element chelated to the above artificial siderophore. Also, the typical element takes the form of an ion. That is, the compounds of the present disclosure can be complexes (artificial siderophore - typical element ion complexes). According to such compounds, an artificial siderophore (complex) in which a typical element is chelated in advance is rapidly recognized by target cells and efficiently taken up in vivo. Furthermore, by using a typical element, the fluorescence intensity of the fluorescence label can be significantly improved.

[0035] M in the above-mentioned formula (1) is a typical element. The bond between R1 to R3 and the typical element means the coordination of R1 to R3 to the typical element. The typical element takes the form of an ion, but the valence is not particularly limited.

[0036] The typical elements chelated to the artificial siderophore of the present disclosure are not particularly limited as long as the effects of the technology of the present disclosure are not significantly impaired. Typically, as long as the typical element and the artificial siderophore form a complex (coordination compound) and the chelate complex (artificial siderophore-typical element ion complex) can be recognized by the siderophore receptor of the target cell. The typical elements to be chelated are preferably selected from, for example, the elements belonging to Group 13 and the elements belonging to Group 14. Specific examples of the elements belonging to Group 13 include aluminum, gallium, etc. Specific examples of the elements belonging to Group 14 include silicon, germanium, etc. From the ionic radius and the affinity with the coordinating oxygen atoms, the elements coordinated to the artificial siderophore of the present disclosure are preferably aluminum, gallium, silicon, etc. Among these, tetravalent ions are preferred. Furthermore, non-metal ions or semi-metal ions are more preferred. An example of such a suitable typical element is silicon.

[0037] <X: Terminal portion> In the above formula (1), X is a terminal portion capable of binding to a detectable label. The terminal portion can bind to a functional group contained in the label (for example, a chemical bond such as a covalent bond). That is, the artificial siderophore and the label of the present disclosure can be in a bound state. In this case, the terminal portion X can be a bonding portion that binds the artificial siderophore and the label.

[0038] Typical examples of such a terminal portion X include an amino group (-NH2), an alkylamino group (-NHR 0 ), a nitro group (-NO2), a phosphate group (-H2PO4), a sulfo group (SO3H), a vinyl group (-C=CH), an allyl group (-CH2CH=CH2), an aldehyde group (-CHO), an azido group (-N3), an ethynyl group (-C≡CH), a thiocyanate group (-NCS), etc. In the case of an alkylamino group, those having 1 to 3 carbon atoms are preferred. R 0Here, X is an alkyl group, preferably a methyl group, an ethyl group, or a butyl group. Of these, the terminal portion X is preferably an amino group or a carboxyl group from the viewpoint of ease of bonding with the label. Furthermore, the amino group is preferred because it readily bonds with the thiocyanate group on the label side.

[0039] <Detectable labels> The compounds disclosed herein include detectable labeling substances bound to the artificial siderophores described above. In this specification, “detectable label(s)” means a substance that produces a signal detectable by instrumental, measurement, or visual means, and which can constitute a part of the compounds disclosed herein. Labels may also be detectable by chemical reactions or antigen-antibody reactions, even if they are not detectable themselves. Examples of labels include chromogens, fluorescent substances, chemiluminescent substances, radioactive compounds, and antibodies.

[0040] A preferred label disclosed herein is a substance that can be bound to the terminal portion X. More specifically, the label is preferably one that has one or more chemical bonds with a functional group. The label can also be linked to the artificial siderophore via the terminal portion X. Therefore, the label and the artificial siderophore do not separate, and when the artificial siderophore is taken up by a microbial cell, the label can be taken up into the body along with the artificial siderophore. This makes it possible to confirm the amount taken up into the cell.

[0041] The compounds disclosed herein (i.e., compounds that are combinations of an artificial siderophore and a detectable label) may include structural parts derived from the label. Typical examples of functional groups that can be bonded to terminal X in such labels include thiocyanate groups, amino groups, and carboxyl groups.

[0042] <Fluorescent substance> As a label, a fluorescent substance is preferable from the viewpoint of improving detection accuracy. A fluorescent substance is a substance that is excited by irradiation with light energy of a specific wavelength and emits light at a detectable wavelength after excitation. The artificial siderophore of this disclosure can become detectable by being bound to a fluorescent substance. Fluorescent substances are preferred because they can be easily bound to artificial siderophores. Examples of fluorescent substances include fluorescein isothiocyanate (FITC), carboxyfluorescein (FAM), 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), tetrachlorofluorescein (TET), carboxy-X-rhodamine (ROX), 6-carboxytetramethylrhodamine (TAMRA), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE), phycoerythrin (PE), allophycocyanin (APC), green fluorescent protein (GFP), and their derivatives, or quantum dots. Of these, FITC is preferred.

[0043] <Antibody> Alternatively, a preferred example of a labeled substance disclosed herein is, for example, an antibody. The artificial siderophore of this disclosure can be made detectable by being linked to an antibody. Such an antibody can be detected by a known detection method (e.g., enzyme-linked immunosorbent assay: ELISA). For example, an enzymatically detectable antibody (enzyme-labeled antibody) can be linked to the artificial siderophore, and the antibody can be detected directly.

[0044] Compounds containing artificial siderophores and detectable labels as described above exhibit improved detection sensitivity. The compounds disclosed herein can detect specific microbial groups by utilizing the mechanism of iron ion uptake via siderophores in microorganisms. Specifically, the artificial siderophores are bound (coordinated) to main group elements. This alters the structure of the artificial siderophores, allowing them to be taken up by specific microorganisms. The main group elements are not reduced by intracellular iron reductases. Therefore, the original mechanism of releasing and utilizing iron ions from the siderophore complex taken up into the cell is not completed. In other words, the state in which main group elements are trapped by the artificial siderophores is easily maintained even within the cell, making it difficult for them to be secreted outside the cell.

[0045] Furthermore, the compounds of this disclosure are detectable by labeling. When siderophores are bound to transition metal elements such as iron, the detection sensitivity decreases. This is presumed to be because, for example in fluorescence detection, excited electrons are transferred to the transition metal ion coordinated to the siderophore, causing thermal vibrations and resulting in energy loss (and consequently quenching). In contrast, the artificial siderophores of the compounds of this disclosure are bound to main group elements. It has been found that the detection sensitivity of the compounds of this disclosure is significantly improved compared to those bound to transition metal elements such as iron.

[0046] <Microorganisms> The compounds disclosed herein can detect microorganisms that have a mechanism for taking iron into the cell via siderophores. More specifically, the compounds disclosed herein include artificial siderophores. These artificial siderophores can be taken into the cell of microorganisms by permeating siderophore receptors by chelating transition metal ions or main group element ions. The compounds disclosed herein include detectable labels. Microorganisms can be detected by detecting the taken-up of the compounds disclosed herein.

[0047] Microorganisms that can be detected only need to have a mechanism for taking iron into the cell via siderophores. In other words, the compounds of this disclosure can detect all microorganisms that take in siderophores. Microorganisms that can be detected using the compounds of this disclosure particularly preferably include bacteria and fungi. Examples of such bacteria include those of the genera Esherichia, Bacillus, Rhodococcus, Pseudomonas, Azotobacter, Mycobacterium, Salmonella, Vibrio, Yersinia, Klebsiella, Shigella, and Shewanella. Examples of fungi include filamentous fungi and yeasts. Examples of yeasts include those of the genera Saccharomyces and Candida. Examples of filamentous fungi include those of the genera Aspergillus and Acremonium.

[0048] Microorganisms that take up artificial siderophores can recognize and selectively take up the local structure of the artificial siderophore (catechol moiety, hydroxamic acid moiety). In other words, the compounds of this disclosure enable selective detection of microorganisms at the species level. For example, the catechol-type artificial siderophore shown in formula (4) above (i.e., a ratio [(catechol moiety):(hydroxamic acid moiety)] of 3:0) is particularly easily taken up by bacteria such as Escherichia coli and Bacillus subtilis. In other words, while other artificial siderophores (formulas (5), (6), and (7)) can also be used for E. coli and Bacillus subtilis, selecting the catechol-type artificial siderophore allows for the combined detection of coliforms or Bacillus subtilis. Furthermore, the mixed ligand type artificial siderophore shown in formula (5) above (i.e., a ratio [(catechol moiety):(hydroxamic acid moiety)] of 2:1) is readily incorporated into Rhodococcus rhodochrous, Rhodococcus erythropolis, Pseudomonas putida, and Azotobacter vinelandii. The mixed ligand type artificial siderophore shown in formula (6) above (i.e., a ratio [(catechol moiety):(hydroxamic acid moiety)] of 1:2) is readily incorporated into Rhodococcus jostii. The hydroxamic acid type artificial siderophore shown in formula (7) above (i.e., a ratio [(catechol moiety):(hydroxamic acid moiety)] of 0:3) is readily incorporated into Mycobacterium flavescens.

[0049] The ligands R1, R2, and R3 of the artificial siderophore disclosed herein can be appropriately selected depending on the purpose. For example, in the food industry, where there is a high need to confirm the presence or absence of contamination by E. coli and Bacillus subtilis, catechol-type artificial siderophore may be preferably used. As described above, the compounds disclosed herein allow for the selection of microorganisms to be detected by the ratio of the ligands of the artificial siderophore [(catechol moiety):(hydroxamic acid moiety)]. The ratio of such ligands can be appropriately determined by a person skilled in the art through preliminary tests, etc.

[0050] <Composition> A composition is provided comprising a compound of the disclosure and a dispersion medium for dispersing the compound of the disclosure. This composition makes it possible to detect microorganisms having a mechanism for incorporating iron into their interior via siderophores. The dispersion medium is not particularly limited. Examples of dispersion media include water, physiological saline, buffer solutions, and liquid culture media. Preferably, the dispersion medium is substantially free of iron ions. The absence of iron ions in the dispersion medium suppresses the binding of artificial siderophores to iron ions. This effect improves detection sensitivity by enabling a more reliable binding of artificial siderophores to typical elements. In this specification, "substantially free" means not intentionally added. Therefore, if chelateable elements are present in trace amounts due to raw materials, samples, manufacturing processes, or detection processes, these are included in the concept of "substantially free" as defined herein. For example, if the iron ion content in the above sample is 0.1 mol% or less (preferably 0.005 mol% or less, more preferably 0.01 mol% or less, even more preferably 0.0005 mol% or less, and particularly preferably 0.0001 mol% or less), it can be said that it "substantially does not contain" iron ions.

[0051] The form of the composition is not particularly limited. Examples of compositional forms include liquids, granules, powders, tablets, capsules, and the like.

[0052] <Use of Compounds: Methods for Detecting Microorganisms> This disclosure provides a method for detecting microorganisms having a mechanism for incorporating iron into their interior via siderophores. One embodiment of the method includes the step of preparing the compound described above. The method includes the step of introducing the compound into a sample suspected to be contaminated by microorganisms and allowing the microorganisms to take it up. The method also includes the step of detecting the label taken up by the microorganisms.

[0053] <Steps for preparing the compounds disclosed herein> The microbial detection method disclosed herein is a selective microbial detection method utilizing the compound described above. The microbial detection method disclosed herein first involves preparing the compound disclosed herein. The compound disclosed herein is a compound comprising an artificial siderophore, a main group element chelated to the artificial siderophore, and a detectable label. First, the artificial siderophore is synthesized. A label is then attached to this artificial siderophore. The labeled artificial siderophore is dissolved in a suitable solvent (e.g., water), and an organic solvent containing a main group element (preferably gallium, aluminum, or silicon, more preferably silicon) (e.g., methanol) is added. This yields an artificial siderophore-main group element complex (artificial siderophore-main group element complex).

[0054] <Step of adding the compound disclosed herein to a sample that is suspected to be contaminated by the target microorganism> Next, a sample is prepared in which the growth of the target microorganism (i.e., microbial contamination) is expected. The type of target microorganism can typically be determined by the attributes of the sample (e.g., food, pharmaceuticals, etc.). For example, microorganisms that are likely to be present in the sample are considered, taking into account factors such as the microorganisms that are likely to cause contamination and the severity of the effects after contamination. The compound of this disclosure, which is capable of selectively detecting this microorganism, is added to the sample.

[0055] The sample is preferably in liquid form. However, even in the case of a solid sample, it can be converted into a liquid sample by adding it to water or a liquid culture medium and vigorously stirring it with a vortex mixer. It is preferable that the sample into which the compound of this disclosure is added does not substantially contain iron ions.

[0056] Furthermore, in a preferred form of the microbial detection method disclosed herein, it is preferable to pre-treat the sample to remove iron ions when preparing the sample. This effectively eliminates the presence of iron ions in the sample, thereby suppressing the binding of artificial siderophores to iron ions. This effect improves detection sensitivity by enabling a more reliable binding of artificial siderophores to typical elements. In this pre-treatment step, the bacterial cells are typically precipitated by centrifuging the liquid sample. After centrifugation, the supernatant is removed and resuspended in physiological saline or an iron-free minimal medium. By performing this once or several times (for example, twice), iron ions in the sample can be removed.

[0057] The method for detecting microorganisms disclosed herein may include the step of introducing the compound disclosed into a sample and then allowing the microorganisms in the sample to take up the compound disclosed. The means for allowing the microorganisms to take up the compound disclosed are not particularly limited. Typically, after introducing the artificial siderophore into the sample, approximately 15 seconds or more may be allowed to pass. However, the sample may be left to stand for 1 minute or more, or for 5 minutes or more, although this is not limited to the above.

[0058] Furthermore, the method for detecting microorganisms disclosed herein may include a step of removing excess artificial siderophore main group element complexes after the microorganisms have taken up the compound disclosed herein. That is, in this step, artificial siderophore main group element complexes that were not taken up by the microorganisms are removed from the sample. Typically, the bacterial cells are precipitated by centrifuging the liquid sample. After centrifugation, the supernatant is removed and the sample is resuspended in physiological saline or phosphate buffer. By performing this once or several times (for example, twice), artificial siderophore main group element complexes that were not taken up by the microorganisms in the sample can be removed.

[0059] <Process for detecting labels incorporated into microorganisms> The method for detecting microorganisms disclosed herein includes the step of detecting a label incorporated into the microorganism. The compounds disclosed herein contain detectable labels. Therefore, by detecting the labels, microorganisms that have incorporated the compounds can be detected. The method for detecting the labels can be appropriately set depending on the type of label. The method for detecting the labels is not particularly limited, and known means for detecting the labels can be appropriately employed.

[0060] The methods for detecting microorganisms disclosed herein may include, for example, methods for detecting fluorescent substances. The methods for detecting fluorescent substances are not particularly limited, and conventionally known methods can be used. Conventionally known methods include, for example, microscopic observation (e.g., fluorescence microscopy), flow cytometry, and immunochemical methods (e.g., Western blotting, immunocytochemistry, etc.). Typically, the microorganisms can be observed by irradiating a compound (here, the label is a fluorescent substance) incorporated into the microorganisms with a specific excitation wavelength and detecting a specific fluorescence wavelength. In the methods for detecting microorganisms disclosed herein, the fluorescence intensity is significantly improved, making it easy to observe the microorganisms.

[0061] Furthermore, artificial siderophore main group element complexes are selectively taken up by specific microorganisms. Therefore, the microorganism detection method of this disclosure can be used to determine whether or not specific microorganisms are present in a sample.

[0062] The compounds of this disclosure will be specifically described below with reference to examples and comparative examples. Here, catechol-type artificial siderophores were used as the compounds of this disclosure in order to selectively detect Escherichia coli, which is a major cause of contamination in the food industry and is in high demand as a target for detection. However, the compounds of this disclosure are not limited to the following examples.

[0063] <Artificial siderophore synthesis: Example of tripod portion synthesis> The artificial siderophores disclosed herein can be synthesized by the synthesis example shown below. First, an intermediate (tripod portion) for the synthesis of the artificial siderophores was synthesized using the synthesis scheme shown below. In the scheme below, "Boc" represents a Boc group. [ka]

[0064] First, 10.0 g (164 mmol, 1.0 eq) of nitromethane and 2.74 ml (6.55 mmol, 0.04 eq) of Triton B (benzyltrimethylammonium hydroxide) were dissolved in 25 ml of 1,4-dioxane. While stirring the dioxane solution, 26.1 g (492 mmol, 3.0 eq) of acrylonitrile was added dropwise over 40 minutes under an ice bath, and the mixture was stirred at room temperature for 24 hours. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in methanol and recrystallized. In this way, 11.0 g of colorless solid crystals of compound [1] shown in the above synthesis scheme were obtained.

[0065] Under a nitrogen atmosphere, 11.0 g (50 mmol, 1.0 eq) of the above-obtained compound [1] was dissolved in 33 mL of tetrahydrofuran (THF). While stirring this THF solution, 250 mL (250 mmol, 5.0 eq) of 1 mol / L BH3·THF complex was added dropwise. The reaction mixture was refluxed for 21 hours. After the reaction mixture was cooled to room temperature, 6 M hydrochloric acid was added to bring the pH to 1. Subsequently, 6 M sodium hydroxide was added to neutralize the reaction mixture. Using a rotary evaporator, the solution was concentrated under reduced pressure, and 165 mL of methanol was added to the remainder to dissolve it. Then, 27.9 mL (200 mmol, 4.0 eq) of triethylamine and 36.0 mL (165 mmol) of di-tert-butyl dicarbonate were added, and the mixture was heated under reflux for 6 hours. After that, the reaction mixture was concentrated, acetic anhydride was added to the residue, and the insoluble matter was filtered out. The filtrate was then concentrated under reduced pressure. The concentrated residue was dissolved in 30 mL of acetic anhydride, 50.0 mL of 4N hydrochloric acid / acetic anhydride was added, and the mixture was stirred for 5 hours. The precipitated solid was then filtered to obtain 12.3 g of a colorless solid of compound [2] shown in the above synthesis scheme.

[0066] Under a nitrogen atmosphere, 3.06 g (0.009 mol) of the above-obtained compound [2] was dissolved in a small amount of methanol. Under ice cooling, a solution prepared by dissolving 3.93 g (0.018 mol) of di-tert-butyl dioxide in 200 mL of anhydrous dichloromethane was added very slowly dropwise to the solution of compound [2] while stirring. After stirring for 48 hours, the solution was dried under reduced pressure to obtain a white solid. The obtained white solid was dissolved in ethyl acetate, and after filtering off unwanted substances, it was concentrated to dryness to obtain a yellow oily substance. This was dissolved in dichloromethane, washed with 0.5 M aqueous sodium hydroxide solution, and the organic layer was separated. Further, 100 mL of saturated saline solution was added to the aqueous phase and extracted with dichloromethane. Subsequently, all the organic layers were combined and dried over anhydrous magnesium sulfate. The solvent was dried under reduced pressure to obtain a colorless oily substance, which was purified by silica gel column chromatography (eluent: chloroform / methanol = 9 / 1) to obtain the white oily substance of compound [3] shown in the above scheme. The yield at this time was 1.02g, and the yield rate was 25.8%.

[0067] <Synthesis of artificial siderophores: Example of synthesis of the hydroxamic acid moiety> An intermediate for the synthesis of artificial siderophores (the part that provides the hydroxamic acid structure) was synthesized using the synthesis scheme shown below. In the scheme below, "Bzl" represents a benzyl group and "Boc" represents a boc group. [ka]

[0068] 44.0 g (0.30 mol) of phthalic anhydride and 26.11 g (0.32 mol) of hydroxyamine hydrochloride were suspended in 100 mL of water. 20.6 g (0.19 mol) of anhydrous sodium carbonate was gradually added to this suspension. The solution was then heated at 60°C to 70°C for 1 hour and cooled to room temperature. In this manner, white needle-shaped crystals of N-hydroxyphthalimide were obtained. The yield was 28.92 g, and the yield was 84.4%.

[0069] 29.28 g (0.179 mol) of the obtained N-hydroxyphthalimide was dissolved in 200 mL of dimethyl sulfoxide (DMSO). While stirring this solution, 18.57 g (0.134 mol) of anhydrous potassium carbonate was gradually added, followed by the dropwise addition of 44.01 g (0.348 mol) of benzyl chloride. After stirring at room temperature for 24 hours, the mixture was poured into 400 mL of cold water and left at 0°C to 5°C for several minutes. The precipitated crystals were filtered and washed several times with pure water. The obtained crude crystals were recrystallized from hot ethanol to obtain white needle-shaped crystals of N-benzyl oxyphthalimide. The yield at this time was 40.09 g, and the yield was 89.9%.

[0070] 10.12 g (0.040 mol) of the obtained N-benzyloxyphthalimide and 2.77 g (0.055 mmol) of hydrazine monohydrate were dissolved in 300 mL of ethanol. This solution was heated and refluxed for 2 hours. Next, the solution was allowed to cool to room temperature, then 4.83 mL of concentrated hydrochloric acid was added, and the mixture was left in ice water for several minutes. The precipitated material was filtered and dried under reduced pressure using a rotary evaporator. Cold water was added to the residue, and granular sodium hydroxide was added until the oil phase separated. This solution was extracted with diethyl ether, and the ether phase was dried over anhydrous magnesium sulfate. This solution was filtered and concentrated under reduced pressure using a rotary evaporator to obtain oily N-benzyloxyamine. The yield at this time was 2.60 g, and the yield was 60.2%.

[0071] The obtained N-benzyloxyamine (7.56 g, 0.06 mol) and 3.35 g (0.011 mol) of 3-bromopropionic acid were dissolved in 100 mL of methanol. This solution was heated and refluxed for 5 hours. The reaction mixture was then concentrated, and a 10% aqueous sodium carbonate solution was added to the residue and dissolved. Unreacted N-benzyloxyamine was removed by extraction of this solution with diethyl ether. After adjusting the pH of the aqueous phase to 1-2 by adding hydrochloric acid, unreacted 3-bromopropionic acid was removed by extraction with diethyl ether. Next, sodium bicarbonate was added to this aqueous phase to adjust the pH to 3-4, and the target product was extracted with diethyl ether. The ether phase was dried over anhydrous magnesium sulfate and concentrated to obtain colorless oily 3-benzyloxyaminopropionic acid. The yield at this time was 5.20 g, and the yield was 43.5%.

[0072] 1.70 g (0.011 mmol) of the obtained 3-benzyloxoaminopropionic acid and 1.82 g (0.018 mmol) of triethylamine were dissolved in 150 mL of ethyl acetate. While cooling this solution on ice and stirring, 30 mL of ethyl acetate solution containing 1.70 g (0.022 mmol) of acetyl chloride was added by dropping. After removing the precipitated triethylamine hydrochloride by filtration, the reaction mixture was stirred overnight in a 10% aqueous citric acid solution. The mixture was then washed twice with 50 mL of water and twice with 50 mL of saturated saline solution. The mixture was dried over anhydrous magnesium sulfate and concentrated to obtain colorless oily 3-(N-acetyl-N-benzyloxyamino)propionic acid (the compound shown in the above synthesis scheme [4]). The yield at this time was 1.89 g, and the yield was 91.0%.

[0073] <Synthesis of artificial siderophores: Examples of catechol moiety synthesis> An intermediate for the synthesis of artificial siderophores (the part that provides the catechol structure) was synthesized using the synthesis scheme shown below. In the scheme below, "Bzl" represents a benzyl group. [ka]

[0074] 2.0 g (0.13 mol) of 2,3-dihydroxybenzoic acid was dissolved in 17 mL of methanol. 0.7 mL of concentrated sulfuric acid was added to the resulting solution. This solution was refluxed for 6 hours, and then dried under reduced pressure to dryness. The resulting solid was dissolved in 70 mL of ethyl acetate and washed with saturated sodium bicarbonate solution and saturated brine. This was dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a pale pink solid of methyl 2,3-dihydroxybenzoate. The yield at this time was 2.10 g, and the yield was 96.0%.

[0075] 1.34 g (7.96 mmol) of methyl 2,3-dihydroxybenzoate was dissolved in 10 mL of DMF. 3.43 g (0.025 mmol) of potassium carbonate and 2.43 g (0.019 mmol) of benzyl chloride were added to the resulting solution. This solution was refluxed at 120°C for 1 hour and then allowed to cool to room temperature. 100 mL of water was added to the reaction solution, and it was extracted with dichloromethane (3 × 50 mL). The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a yellow oily substance, methyl 2,3-bisbenzyloxybenzoate. The yield at this stage was 2.03 g, and the yield was 72.8%.

[0076] 2.03 g (5.83 mmol) of methyl 2,3-bisbenzyloxybenzoate was dissolved in 24 mL of methanol, and 6.0 mL of 2 M aqueous sodium hydroxide solution was added to the resulting solution. The solution was refluxed at 100°C for 1 hour and then allowed to cool to room temperature. 100 mL of water was added to the reaction solution, and 15 mL of 2 M hydrochloric acid was gradually added. The mixture was extracted with dichloromethane (3 × 50 mL). The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a white powder. The obtained powder was recrystallized from ethanol / water to obtain 2,3-bisbenzyloxybenzoic acid (the compound shown in the above synthesis scheme [5]). The yield at this time was 1.67 g, and the yield was 86.0%.

[0077] <Synthesis of artificial siderophores: Example of synthesis of hydroxam-type artificial siderophore S1> Using the compounds obtained from the above synthesis examples, hydroxam-type artificial siderophores were synthesized according to the synthesis scheme shown below. In the scheme below, "HOBt" represents 1-hydroxybenzotriazole, "EDC" represents 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and "NMM" represents N-methylmorpholine. [ka]

[0078] Under ice cooling, 0.90 g (3.79 mmol) of the above-obtained compound [2], 0.564 g (4.17 mmol) of 1-hydroxybenzotriazole, 0.922 mL (8.34 mmol) of N-methylmorpholine, and 0.80 g (4.17 mmol) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride were dissolved in 20 mL of DMF and stirred for 10 minutes. Then, 0.341 g (1.00 mmol) of the above-obtained compound [4] and 0.362 mL (3.3 mmol) of N-methylmorpholine were added and stirred for 12 hours. After that, the reaction solution was dried under reduced pressure, dissolved in 100 mL of chloroform, and washed five times each with 5% citric acid aqueous solution, saturated sodium bicarbonate aqueous solution, and saturated saline solution. This was dried over anhydrous magnesium sulfate and concentrated to obtain a yellow oily substance. This was purified using a silica gel column (eluent: chloroform / methanol = 10 / 1) to obtain compound [6]. The yield was 0.85 g and the yield was 75.6%.

[0079] 2.31 g (2.60 mmol) of the obtained compound [6] was dissolved in 40 mL of methanol / ethyl acetate mixed solution (methanol:ethyl acetate volume ratio 1:1). This was then prepared by dissolving approximately 3.04 × 10⁻⁶. 5 The compound [7] was obtained by catalytic reduction with a Pd / C catalyst for 4 hours under an H2 atmosphere at Pa (3 atm). The catalyst was removed by Celite filtration and the compound was dried under reduced pressure. The yield was 0.94 g and 52.2%.

[0080] The obtained compound [7] 1.31 g (2.12 mmol) was dissolved in 40 mL of methanol. This was then divided into approximately 3.04 × 10⁻⁶ units. 5 The sample was catalytically reduced at 50°C for 5 days using a Raney nickel catalyst under a Pa (3 atm) H2 atmosphere. The catalyst was removed by Celite filtration, and the sample was dried under reduced pressure to obtain hydroxamic acid-type artificial siderophore [S1]. The yield at this stage was 1.19 g, and the yield was 95.3%.

[0081] <Synthesis of artificial siderophores: Example of synthesis of catechol-type artificial siderophore S2> An intermediate for the synthesis of artificial siderophores (the part that provides hydromorpholine) was synthesized using the synthesis scheme shown below. In the scheme below, "HOBt" represents 1-hydroxybenzotriazole, "EDC" represents 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and "NMM" represents N-methylmorpholine. [ka]

[0082] Under ice cooling, 2.00 g (6.00 mmol) of the above-obtained compound [5], 1.01 g (6.60 mmol) of 1-hydroxybenzotriazole, 1.46 mL (13.2 mmol) of N-methylmorpholine, and 1.266 g (6.6 mmol) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride were dissolved in 50 mL of DMF and stirred for 10 minutes. Then, 0.682 g (2.00 mmol) of the above-obtained compound [2] and 0.724 mL (6.6 mmol) of N-methylmorpholine were added and stirred for 48 hours. After that, the reaction solution was dried under reduced pressure, dissolved in 100 mL of chloroform, and washed five times each with 5% citric acid aqueous solution, saturated sodium bicarbonate aqueous solution, and saturated saline solution. This was dried over anhydrous magnesium sulfate and concentrated to obtain a yellow oily substance. This was purified using a silica gel column (eluent: chloroform / methanol = 10 / 1) to obtain compound [8]. The yield was 1.98 g and the yield was 780.3%.

[0083] 1.15 g (1.00 mmol) of the obtained compound [8] was dissolved in 40 mL of a mixed solution of methanol / ethyl acetate (volume ratio of methanol:ethyl acetate 1:1). 5 This was subjected to catalytic reduction with a Pd / C catalyst for 4 hours under a H2 atmosphere of about 3.04×10

[0084] 0.23 g (0.36 mmol) of the obtained compound [9] was dissolved in 40 mL of methanol. 5 This was subjected to catalytic reduction with 0.5 g of a Raney nickel catalyst at 50 °C for 5 days under a H2 atmosphere of about 3.04×10

[0085] <Synthesis of artificial siderophore: Synthesis of mixed-type artificial siderophore S3> Using the compound obtained by the above synthesis example, a mixed-type artificial siderophore ((catechol site):(hydroxamic acid site) = 1:2) was synthesized according to the following synthesis scheme. In the following scheme, "HOBt" represents 1-hydroxybenzotriazole, "EDC" represents 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and "NMM" represents N-methylmorpholine.

Chemical formula

[0086] 1.38 g (4.12 mmol) of the compound obtained above [5] was dissolved in 40 mL of anhydrous dichloromethane. Then, under ice cooling, 0.69 g (4.53 mmol) of 1-hydroxybenztriazole, 0.87 g (4.53 mmol) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 0.995 mL (9.05 mmol) of N-methylmorpholine were added and the mixture was stirred for 10 minutes. Subsequently, 1.78 g (4.12 mmol) of the compound obtained above [3] and 0.50 mL (4.53 mmol) of N-methylmorpholine were added to this solution, and the suspension was returned to room temperature and stirred for 24 hours. The solution was then dried under reduced pressure to obtain a yellow oily substance. The obtained oily substance was dissolved in 100 mL of chloroform and washed five times each with 5% citric acid aqueous solution, saturated sodium bicarbonate aqueous solution, and saturated saline solution. The organic phase was dried over anhydrous magnesium sulfate and dried under reduced pressure to dryness. The resulting oily substance was purified using a silica gel column (eluent: chloroform / methanol = 10 / 1) to obtain compound

[10] as a white foamy substance according to the above scheme. The yield at this time was 2.28 g, and the yield was 80.0%.

[0087] 2.21 g (2.95 mmol) of the obtained compound

[10] was dissolved in 20 mL of ethyl acetate, and 15 mL of a 4 M hydrochloric acid / ethyl acetate mixture was added to this solution and stirred for 5 minutes. The solution was dried under reduced pressure to obtain the target compound

[11] as a yellow oily substance. The yield at this time was 1.81 g, and the yield was 99.0%.

[0088] 0.692 g (2.96 mmol) of the obtained compound

[11] was dissolved in 20 mL of anhydrous dichloromethane. To this solution, 0.44 g (3.26 mmol) of 1-hydroxybenztriazole, 0.62 g (3.26 mmol) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 0.72 mL (6.53 mmol) of N-methylmorpholine were added under ice cooling, and the mixture was stirred for 10 minutes. Subsequently, 0.907 g (1.46 mmol) of the above-mentioned compound [4] and 0.735 mL (3.27 mmol) of N-methylmorpholine were added, and the mixture was stirred for 24 hours after returning to room temperature. The solution was then dried under reduced pressure to obtain a pale yellow oily substance. This oily substance was dissolved in 100 mL of chloroform and washed five times each with 5% citric acid aqueous solution, saturated sodium bicarbonate aqueous solution, and saturated saline solution. The organic phase was dried over anhydrous magnesium sulfate and dried under reduced pressure to dryness. The resulting colorless oily substance was purified using a silica gel column (eluent: chloroform / methanol = 10 / 1) to obtain compound

[12] as a white foamy substance according to the above scheme. The yield at this time was 0.968 g, and the yield was 67.2%.

[0089] 0.563 g (0.57 mmol) of the obtained compound

[12] was dissolved in 40 mL of methanol / ethyl acetate mixture (methanol:ethyl acetate volume ratio 1:1). This was then divided into approximately 3.04 × 10⁻⁶ units. 5 The compound

[13] was obtained by catalytic reduction with a Pd / C catalyst for 4 hours under an H2 atmosphere at Pa (3 atm). The catalyst was removed by Celite filtration and the compound was dried under reduced pressure. The yield was 0.314 g and 88.0%.

[0090] 0.033 g (0.15 mmol) of the obtained compound

[13] was dissolved in 40 mL of methanol. This was then divided into approximately 3.04 × 10⁻⁶ units. 5 Under a Pa (3 atm) H2 atmosphere, the mixture was catalytically reduced with 0.5 g of Raney nickel catalyst at 50°C for 5 days. The catalyst was removed by Celite filtration, and the mixture was dried under reduced pressure to obtain mixed artificial siderophores [S3]. The yield at this stage was 0.065 g, and the yield was 73.0%.

[0091] <Synthesis of artificial siderophores: Synthesis of mixed-type artificial siderophore S4> Using the compounds obtained from the above synthesis examples, a mixed artificial siderophore ((catechol moiety):(hydroxamic acid moiety)=2:1) ​​was synthesized by the synthesis scheme shown below. In the scheme below, "HOBt" represents 1-hydroxybenzotriazole, "EDC" represents 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and "NMM" represents N-methylmorpholine. [ka]

[0092] 0.98 g (4.13 mmol) of compound [4] was dissolved in 40 mL of anhydrous dichloromethane. Under ice cooling, 0.69 g (4.53 mmol) of 1-hydroxybenztriazole, 0.87 g (4.53 mmol) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 0.995 mL (9.05 mmol) of N-methylmorpholine were added, and the mixture was stirred for 10 minutes. Then, 1.78 g (4.12 mmol) of compound [3] and 0.50 mL (4.53 mmol) of N-methylmorpholine were added to this solution, and the suspension was allowed to return to room temperature and stirred for 24 hours. The solution was then dried under reduced pressure to obtain a yellow oily substance. The obtained oily substance was dissolved in 100 mL of chloroform and washed five times each with 5% citric acid aqueous solution, saturated sodium bicarbonate aqueous solution, and saturated saline solution. The organic phase was dried over anhydrous magnesium sulfate and dried under reduced pressure to dryness. The resulting oily substance was purified using a silica gel column (eluent: chloroform / methanol = 10 / 1) to obtain compound

[14] as a white foam. The yield at this time was 2.50 g, and the yield was 80.0%.

[0093] 1.92 g (2.95 mmol) of the compound obtained above

[14] was dissolved in 20 mL of ethyl acetate. 15 mL of a 4 M hydrochloric acid / ethyl acetate mixture was added to this solution and the mixture was stirred for 5 minutes. The solution was then dried under reduced pressure to obtain the target compound

[15] as a yellow oily substance. The yield at this time was 1.50 g, and the yield was 97.0%.

[0094] 0.976 g (2.92 mmol) of the compound obtained above

[15] was dissolved in 20 mL of anhydrous dichloromethane. Under ice cooling, 0.44 g (3.26 mmol) of 1-hydroxybenztriazole, 0.63 g (3.26 mmol) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 0.72 mL (6.53 mmol) of N-methylmorpholine were added to this solution and stirred for 10 minutes. Then, 0.77 g (1.46 mmol) of the compound obtained above [5] and 0.735 mL (3.27 mmol) of N-methylmorpholine were added, and the mixture was stirred for 24 hours after returning to room temperature. After that, the solution was dried under reduced pressure to obtain a pale yellow oily substance. This oily substance was dissolved in 100 mL of chloroform and washed five times each with 5% citric acid aqueous solution, saturated sodium bicarbonate aqueous solution, and saturated saline solution. The organic phase was dried over anhydrous magnesium sulfate and dried under reduced pressure to dryness. The resulting colorless oily substance was purified using a silica gel column (eluent: chloroform / methanol = 10 / 1) to obtain the compound

[16] shown in the above scheme as a white foamy substance. The yield at this time was 1.11 g, and the yield was 70.2%.

[0095] 1.08 g (1.00 mmol) of the compound obtained above

[16] was dissolved in 40 mL of methanol / ethyl acetate mixed solution (methanol:ethyl acetate volume ratio 1:1). This was then dissolved in approximately 3.04 × 10⁻⁶ units. 5 The compound

[17] was obtained by catalytic reduction with a Pd / C catalyst for 4 hours under an H2 atmosphere at Pa (3 atm). The catalyst was removed by Celite filtration and the compound was dried under reduced pressure. The yield was 0.48 g and 76.0%.

[0096] 0.10 g (0.15 mmol) of the obtained compound

[17] was dissolved in 40 mL of methanol. This was then divided into approximately 3.04 × 10⁻⁶ units. 5 Under a Pa (3 atm) H2 atmosphere, 0.5 g of Raney nickel catalyst was catalytically reduced at 50°C for 5 days. The catalyst was removed by Celite filtration, and the mixture was dried under reduced pressure to obtain mixed artificial siderophores [S4]. The yield at this stage was 0.08 g, and the yield was 81.5%.

[0097] <Binding of fluorescent substances> Compound (13) was synthesized by combining the above-mentioned artificial siderophore with a fluorescent substance using the synthesis scheme shown below. Here, FITC was used as the fluorescent substance. [ka]

[0098] 0.0935 g of the above-mentioned catechol-type artificial siderophore [S2], 0.0567 g of FITC, and triethylamine (1 equivalent) were dissolved in 40 mL of N,N-dimethylformamide and stirred at room temperature for 12 hours. The reaction mixture was allowed to dry under reduced pressure and purified by HPLC in DMF using a GPC column (JAIGEL-2HR, 2.5HR, manufactured by Nippon Analytical Industries, Ltd.) to obtain compound H6L as shown in the scheme above. C3 (Catechol-type artificial siderophore-FITC) was obtained. The yield at this time was 0.137 g, and the yield was 76.7%. Similarly, mixed ligand-type artificial siderophore-FITC and hydroxam-type artificial siderophore-FITC were obtained.

[0099] <Comparative example: Iron chelate> 0.164 g (0.164 mmol) of the obtained compound (artificial siderophore-FITC) was dissolved in 20 mL of methanol. To this solution, 0.0579 g (0.164 mmol) of tris(2,4-pentanedionato)iron(trivalent) complex (Fe(acac)3) and 5.18 mL (0.498 mmol) of 0.095 M potassium hydroxide / methanol solution were added, and the mixture was stirred overnight at room temperature under a nitrogen atmosphere. The solution was dried under reduced pressure, and the resulting residue was reprecipitation with DMF / diethyl ether to obtain the compound Fe shown in the scheme below. III L C3 A black solid of -FITC (catechol-type artificial siderophore-iron complex) was obtained. The yield at this time was 0.1546 g, and the yield was 80.8%. [ka]

[0100] <Example: Silicon Chelate> 0.10 g of the obtained compound (catechol-type artificial siderophore-FITC) was dissolved in 5 mL of methanol. 5.4 μL of tetramethyl orthosilicate and 0.613 mL of 0.12 M potassium hydroxide / methanol solution were added to this solution, and the mixture was stirred at room temperature for 24 hours. The solution was dried under reduced pressure, and the resulting residue was reprecipitation with DMF / diethyl ether to obtain the compound Si shown in the scheme below. IV L C3 An orange powder of -FITC (catechol-type artificial siderophore-silicon complex) was obtained. The yield at this time was 0.088 g, and the yield was 90.0%. [ka]

[0101] <Checking Fluorescence Intensity> The fluorescence intensity of the catechol-type artificial siderophores obtained above was analyzed using a luminescence efficiency analyzer. A Hamamatsu Photonics C13534-23 high-sensitivity near-infrared PL quantum yield analyzer was used as the luminescence efficiency analyzer. For this analysis, solutions for analysis were prepared by diluting catechol-type artificial siderophore-FITC, catechol-type artificial siderophore-iron complex, catechol-type artificial siderophore-silicon complex, and FITC with 0.01 mol / L sodium phosphate buffer, to which sodium perchlorate was added to achieve an absorbance of 0.6 at the excitation wavelength of FITC (490 nm).

[0102] By irradiating with an excitation wavelength of 490 nm, fluorescence was detected in the wavelength range of 495 nm to 695 nm. Figure 1 is a graph showing the fluorescence spectrum of the catechol-type artificial siderophore-iron complex. As shown in Figure 1, FITC shows quenching upon binding to the artificial siderophore. Furthermore, the artificial siderophore-iron complex showed greater quenching than the artificial siderophore without coordinated elements. In contrast, the artificial siderophore-silicon complex showed improved fluorescence intensity. Figure 2 is a graph showing the fluorescence spectrum of the catechol-type artificial siderophore-silicon complex. The artificial siderophore-silicon complex showed improved fluorescence intensity compared to the artificial siderophore without coordinated silicon. Table 1 shows the quantum yield of each compound in flow cytometry.

[0103] [Table 1]

[0104] <Fluorescent labeling of microorganisms using artificial siderophore complexes> Escherichia coli (ATCC33475) was cultured in LB (Luria-Bertani) liquid medium at 37°C for 24 hours. The culture medium was centrifuged at 6000 rpm for 10 minutes to collect the E. coli. The supernatant was discarded, and the bacterial mass was resuspended in iron-depleted minimal liquid medium. At this point, the bacterial count was 10 6The culture medium was diluted with iron-depleted minimum liquid medium to a concentration of 1 / mL. Artificial siderophore-iron complex was added to this culture medium to a concentration of 20 μM, and the mixture was incubated at 37°C for 4 hours to allow the artificial siderophore-iron complex to be incorporated into the E. coli cells. After incubation, the E. coli cells were collected by centrifugation at 6000 rpm for 10 minutes. The supernatant was discarded, PBS buffer (pH 7.4) was added, and the cells were washed by centrifugation at 6000 rpm for 15 minutes. A further washing was performed, and the mixture was centrifuged again at 6000 rpm for 15 minutes. The supernatant was discarded, and an appropriate amount of 1% agarose gel aqueous solution was added to the remaining bacterial mass to resuspend it and prepare the sample solution. This sample solution was spotted onto a glass slide and observed using a fluorescence microscope (Nikon ECLIPSE100 inverted culture microscope, 450 nm~490 nm excitation, 505 nm fluorescence, mercury lamp). The same tests were conducted for M. flavescens, except that the culture temperature was set to 30°C.

[0105] Escherichia coli (ATCC33475) was cultured in LB (Luria-Bertani) liquid medium at 37°C for 24 hours. The culture medium was centrifuged at 6000 rpm for 10 minutes to collect the E. coli. The supernatant was discarded, and the bacterial mass was resuspended in iron-depleted minimal liquid medium. At this point, the bacterial count was 10 6The culture medium was diluted with iron-depleted minimum liquid medium to a concentration of 1 / mL. Artificial siderophore silicon complex was added to this culture medium to a concentration of 20 μM, and the medium was incubated at 37°C for 5 minutes or 15 seconds to allow the artificial siderophore silicon complex to be incorporated into the E. coli cells. After incubation, the E. coli cells were collected by centrifugation at 1000 × g for 5 minutes. The supernatant was discarded, PBS buffer (pH 7.4) was added, and the cells were washed by centrifugation at 6000 rpm for 15 minutes. A second washing was performed, and the cells were centrifuged again at 6000 rpm for 15 minutes. The supernatant was discarded, and an appropriate amount of 1% agarose gel aqueous solution was added to the remaining bacterial mass to resuspend it and prepare the sample solution. This sample solution was spotted onto a glass slide and observed using a fluorescence microscope (Nikon ECLIPSE100 inverted culture microscope, 450 nm~490 nm excitation, 505 nm fluorescence, mercury lamp). The same tests were conducted for Bacillus subtilis, Shewanella oneidensis, and M. flavescens, except that the culture time was set to 30 minutes and the culture temperature to 30°C.

[0106] The minimum iron-depletion liquid culture medium was prepared by the following method: 0.3 mg of potassium dihydrogen phosphate, 1.5 g of sodium dihydrogen phosphate, 0.1 g of ammonium chloride, and 0.5 g of sodium chloride were dissolved in 100 mL of ultrapure water. 5 mL of the resulting solution was added to a photorecorder cell. Next, artificial siderophores were added to the cell to a concentration of 1 μM. Then, 5 μL of an aqueous solution of 10 mg of thiamine hydrochloride dissolved in 10 mL of ultrapure water, 50 μL of aqueous solutions of 10 mg each of the amino acids (L-proline, L-leucine, L-tryptophan) dissolved in 10 mL of ultrapure water, 100 μL of an aqueous solution of 2.5 g of D-glucose dissolved in 10 mL of ultrapure water, and 100 μL of an aqueous solution of 1.23 g of magnesium sulfate dissolved in 10 mL of ultrapure water were added to the cell to prepare the minimum iron-depletion liquid culture medium.

[0107] Figure 3A is a microscopic image showing the detection results of Escherichia coli using a catechol-type artificial siderophore-iron complex. Figure 3B is a microscopic image showing the detection results of M. flavescens using a catechol-type artificial siderophore-iron complex. Figure 4 is a microscopic image showing the detection results of Escherichia coli using a catechol-type artificial siderophore-silicon complex. Figure 5A is a microscopic image showing the detection results of Bacillus subtilis using a catechol-type artificial siderophore-silicon complex. Figure 5B is a microscopic image showing the detection results of S. oneidensis using a catechol-type artificial siderophore-silicon complex. Figure 5C is a microscopic image showing the detection results of M. flavescens using a catechol-type artificial siderophore-silicon complex. The left side of each figure shows the image obtained by bright-field observation. The right side shows the image obtained by fluorescence observation. In Figures 5A, B, and C, "○" indicates that the artificial siderophore is readily taken up by the target microorganism, while "×" indicates that it is relatively less readily taken up by the target microorganism. As shown in Figures 3A and B, the artificial siderophore-iron complex taken up by the microorganism has weak fluorescence intensity, and there is no difference in luminosity between the artificial siderophore-iron complex and the other parts. Therefore, labeling and detection were difficult. In contrast, as shown in Figure 4, the fluorescence intensity of the artificial siderophore-silicon complex is significantly improved, making it easy to confirm (detect) the artificial siderophore-silicon complex taken up by E. coli. Furthermore, labeling was possible even in a very short time of 15 seconds after adding the artificial siderophore-silicon complex to the E. coli suspension. In addition, as shown in Figures 5A, B, and C, the catechol-type artificial siderophore-silicon complex is readily taken up by Bacillus subtilis. However, it was found that the catechol-type artificial siderophore-silicon complex was relatively poorly incorporated by S. Oneidensis and hardly incorporated at all by M. flavescens. Therefore, it was shown that the artificial siderophore has selectivity for microorganisms.

[0108] Although detailed data is not provided, it was found that mixed ligand-type artificial siderophore-silicon complexes and hydroxamic acid-type artificial siderophore-silicon complexes are relatively poorly incorporated by E. coli and Bacillus subtilis. However, hydroxamic acid-type artificial siderophore-silicon complexes were found to be relatively well incorporated by M. flavescens.

[0109] Although this disclosure has been described above with reference to excellent examples, this description is not limiting, and various modifications are possible, of course, such as the type of ligand and the type of main group element to be coordinated.

[0110] The technologies disclosed herein may be omitted or combined as appropriate, unless there is a particular problem. Furthermore, this specification includes the disclosures described in the following sections.

[0111] Item 1: A compound capable of detecting microorganisms having a mechanism for incorporating iron into its interior via a siderophore, wherein the compound is a compound in which an artificial siderophore and a detectable label are bound, the compound comprising a structural portion derived from an artificial siderophore represented by the following formula (1); where R1, R2, and R3 in formula (1) are each independently a catechol group or a hydroxamic acid group, M is an element belonging to the main group elements, and X is a terminal portion bound to the detectable label.

[0112] Item 2: The compound described in Item 1, wherein the main group element is selected from elements belonging to Group 13 and elements belonging to Group 14.

[0113] Item 3: The compound described in Item 1 or Item 2, wherein the main group element is selected from silicon, aluminum, and gallium.

[0114] Item 4: X is a compound according to any one of items 1 to 3, selected from an amino group, alkylamino group, nitro group, phosphate group, sulfo group, vinyl group, allyl group, aldehyde group, azi group, ethynyl group, and thiocyanate group.

[0115] Item 5: The detectable labeling substance is a compound according to any one of items 1 to 4, selected from either a fluorescent substance or an antibody.

[0116] Item 6: A composition for detecting microorganisms having a mechanism for incorporating iron via a siderophore, comprising a compound described in any one of items 1 to 5 and a dispersion medium for dispersing the compound.

[0117] Item 7: A method for detecting microorganisms having a mechanism for incorporating iron into their interior via siderophores, comprising the following steps: preparing a compound described in any one of items 1 to 6; introducing the compound into a sample suspected to be contaminated by microorganisms and allowing the microorganisms to take it up; and detecting the label of the compound taken up by the microorganisms.

[0118] Item 8: The method described in Item 7, wherein the sample suspected to be contaminated with the above microorganisms does not contain iron ions.

Claims

1. A compound capable of detecting microorganisms that have a mechanism for incorporating iron into their interior via siderophores, It is a compound in which an artificial siderophore is bound to a detectable label. The compound includes a structural portion derived from an artificial siderophore represented by the following formula (1); 【Chemistry 1】 Here, in equation (1) R １ , R ２ , and R ３ These are, independently, a catechol group or a hydroxamic acid group. M is one of the elements belonging to the main group of elements. X is a compound, which is a terminal portion that binds to the detectable label.

2. The compound according to claim 1, wherein the main group element is selected from elements belonging to Group 13 and elements belonging to Group 14.

3. The compound according to claim 2, wherein the main group element is selected from silicon, aluminum, and gallium.

4. The compound according to claim 1, wherein X is selected from any of the following: an amino group, an alkylamino group, a nitro group, a phosphate group, a sulfo group, a vinyl group, an allyl group, an aldehyde group, an azi group, an ethynyl group, and a thiocyanate group.

5. The compound according to claim 1, wherein the detectable labeling substance is selected from either a fluorescent substance or an antibody.

6. A compound according to any one of claims 1 to 5, A dispersion medium for dispersing the aforementioned compound, A composition for detecting microorganisms that have a mechanism for incorporating iron into their interior via siderophores, including [specific component].

7. A method for detecting microorganisms that have a mechanism for incorporating iron into their interior via siderophores, comprising the following steps: A step of preparing the compound according to claim 6; A step of introducing the compound into a sample suspected to be contaminated by microorganisms and allowing the microorganisms to take it up; A step of detecting the label of a compound taken up by the microorganism; A method of including.

8. The method according to claim 7, wherein the sample suspected to be contaminated by the aforementioned microorganisms does not contain iron ions.

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