JP4590625B2 - New isocoumarin fluorescent substance - Google Patents

Description

  The present invention relates to a novel fluorescent material and a method for producing the same.

The fluorescent substance can be used for a fluorescent brightener, a fluorescent probe, and the like.
Since some of the bacteria belonging to the genus Legionella emit blue-white fluorescence, there is a possibility that a novel fluorescent substance can be isolated from the Legionella bacterium. The present inventors have so far attempted to isolate a new fluorescent substance from bacteria belonging to the genus Legionella, but have not yet identified the fluorescent substance.

Junko Maekawa, Fumiaki Kura, Haruo Watanabe: Analysis of blue-white fluorescence of Legionella dumoffii. Abstracts of the 75th Annual Meeting of the Japanese Society for Bacteriology, 2002.

  An object of the present invention is to provide a novel fluorescent material.

で表される化合物。 (1) The following formula (I):

A compound represented by

で表される化合物を生産する能力を有する細菌を培養し、培養物から上記式（I）で表される化合物を採取することを特徴とする上記式（I）で表される化合物の製造方法。 (2) It belongs to the Legionella genus, and the following formula (I):

A method for producing the compound represented by the formula (I), comprising culturing a bacterium having the ability to produce the compound represented by the formula and collecting the compound represented by the formula (I) from the culture. .

  INDUSTRIAL APPLICABILITY According to the present invention, a novel fluorescent material that emits blue fluorescence of 450 nm when excited by long wave ultraviolet light having a wavelength of 340 nm and a method for producing the same are provided. This novel fluorescent substance can be used for fluorescent whitening agents, fluorescent probes, and the like.

で表される新規イソクマリン系化合物およびその製造方法に関する。 The present invention provides the following formula (I):

And a method for producing the same.

The compound is excited by long wave ultraviolet light having a wavelength of 340 nm and emits blue fluorescence of 450 nm.
As a naturally derived compound having an isocoumarin skeleton and having fluorescence, Leprocybin (Liebigs Ann. Chem. 1982, 1280-1296) is known, but is a compound different from the compound of the present invention.

  The compound represented by the formula (I) is a bacterium belonging to the genus Legionella and has the ability to produce the compound represented by the formula (I), and the compound represented by the formula (I) is collected from the culture. Can be manufactured.

  Bacteria belonging to the genus Legionella and having the ability to produce a compound represented by formula (I) include, for example, Legionella dumoffii, Legionella bozemanii, Legionella gormanii, Legionella・ Legionella anisa, Legionella tucsonensis, Legionella cherrii, Legionella parisiensis, Legionella lytica, Legionella steiger Examples include Legionella rowbothamii having the ability to produce the compound represented by formula (I), and Legionella Dumofi is particularly preferable. These bacteria may be appropriately modified according to the purpose. Examples of the modification method include a method of introducing a plasmid vector into which a transcription factor (for example, Legionella dumofi gacA gene) that promotes overproduction of the compound represented by formula (I) is cloned into a bacterium belonging to the genus Legionella. It is done.

  More specifically, as a bacterium belonging to the genus Legionella and having the ability to produce the compound represented by the formula (I), the gacA gene amplified by PCR is added to the plasmid vector pMMB207c that can grow in Legionella. A plasmid prepared by cloning: pMMB207c-gacA introduced into Legionella Dumofi NY23 strain (ATCC 33279) (hereinafter referred to as “fluorescent substance mass production strain”). This fluorescent substance mass production strain was rejected by the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (Certificate of Acceptance Refusal No. 15 Production No. 1922). Is guaranteed by the applicant. Here, the gacA gene is a gene encoding a factor that regulates transcription of various operons. The DNA sequence containing the gacA gene is shown in SEQ ID NO: 1. In this DNA sequence, the coding region of the protein (including the termination codon) is numbered 395 to 1054. The amino acid sequence of the protein encoded by the gacA gene is shown in SEQ ID NO: 2. Overproduction of the gacA gene increases the production amount of the novel fluorescent substance of the present invention several times. This is considered to be a result of producing a large amount of the biosynthetic enzyme (s) of the novel fluorescent substance of the present invention. This fluorescent substance mass-producing strain can be concentrated in 10% skim milk and stored at -80 ° C.

  In the present invention, a mutant strain obtained by mutagenesis treatment of the bacterial strain described above (including those modified like a fluorescent substance mass-producing strain) may be used. Mutagenesis treatment can be performed using any suitable mutagen. Here, the term “mutagen” should be understood in a broad sense to include not only a drug having a mutagenic effect but also a treatment having a mutagenic effect such as UV irradiation. Examples of suitable mutagens include ethyl methanesulfonate, UV irradiation, N-methyl-N'-nitro-N-nitrosoguanidine, nucleotide base analogs such as bromouracil, and acridines, but any other effect A typical mutagen may be used.

  As a method for culturing the bacterium used in the present invention, culture can be performed in a liquid medium or a solid medium, and culture conditions suitable for the growth of the bacterium used and suitable for production of the compound of the present invention are selected. It is generally advantageous to perform the culture under aerobic conditions, the culture temperature is 30 ° C. to 37 ° C., preferably 35 ° C. to 36 ° C., and the culture time varies depending on the medium used and the culture conditions. Usually 2 to 7 days.

  The medium used for culturing the bacteria used in the present invention may be any medium containing nutrients that can be used by the bacteria. For example, amino acids can be used as carbon and nitrogen sources, and for example, yeast extract can be used as a source of amino acids. Among amino acids, since L-cysteine is particularly demanding, it is more preferable to add L-cysteine to the medium. In addition, inorganic salts can be added to the medium as necessary. Since the requirement for iron is particularly high as an inorganic substance, it is preferable to add inorganic salts containing iron to the medium. Furthermore, an organic compound or an inorganic compound that promotes the growth of bacteria or the production of the compound of the present invention can be appropriately added. Further, when culturing in a solid medium using agar, since impurities (fatty acids and the like) contained in the agar may inhibit growth, it is preferable to add starch or activated carbon to adsorb the impurities. The culture pH of Legionella bacteria used in the present invention is 6.5 to 7.5, preferably 6.85 to 6.95. Thus, since the culture pH range is very narrow, it is preferable to adjust the pH by adding a buffer to the medium. As the buffering agent, ACES (N- (2-acetamido) -2-aminoethanesulfonic acid) is preferably used. For the culture of the bacterium of the present invention, BCYEα medium (Buffered Charcoal-Yeast Extract 0.1% α-ketogulutalate) can be suitably used as the solid medium, and BYE medium (Buffered Yeast Extract) can be suitably used as the liquid medium.

  In collecting the compound of the present invention from the culture, ordinary purification / collection means such as solvent extraction, adsorption column chromatography using ion exchange resin or the like, distribution column chromatography, gel filtration, dialysis, centrifugation, etc. are used alone. Or can be used in appropriate combination. A particularly preferred method is as follows.

  The cultured cells of bacteria belonging to the genus Legionella are collected by centrifugation, lipids are extracted from the cells using an organic solvent, the extracted lipids are fractionated on a silica gel column, and a fraction containing a fluorescent substance is further collected. The compound of the present invention is collected by subjecting it to a preparative HPLC to fractionate a fraction emitting 450 nm fluorescence with an excitation wavelength of 340 nm.

The compound represented by the above formula (I) can also be synthesized by a usual chemical synthesis method.
Next, the present invention will be described in more detail with reference to examples.

Preparation of the strain The PCR-amplified gacA gene is cloned into the plasmid vector pMMB207c (see Hales, LM, and HA Shuman. 1999. Infect. Immun. 67: 3662-3666) to produce a plasmid, pMMB207c-gacA did. Primers used for PCR were set outside so that the entire gacA gene could be amplified. The primer sequences are 5′-TGCTGCAACCGGTTTATTTCG-3 ′ (SEQ ID NO: 3) and 5′-GGAACGATAGCTTTCTATGCG-3 ′ (SEQ ID NO: 4). PCR was performed using Ampli Taq (Applied Biosystems) as the Taq polymerase by the usual method recommended by the manufacturer. The PCR product was purified, blunt-ended, and inserted into the SmaI site of pMMB207c.

  The pMMB207c-gacA thus obtained was introduced into Legionella dumofi NY23 strain (ATCC 33279) by electroporation using Gene Pulser II (Bio-Rad). A 0.1 cm cuvette was used under the conditions of 2.0 kV, 100Ω, and 2.5 μF.

  The obtained fluorescent substance mass-produced strain was stored at −80 ° C. with the cells suspended in 10% skim milk until it was used in the following experiment.

Spread 1 platinum ear of the above-mentioned fluorescent substance mass-produced strain that has been cultured and collected and collected on a BCYEα agar medium (Buffered Charcoal-Yeast Extract 0.1% α-ketogulutalate, Difco) in a 9 cm petri dish and grow at 35 ° C for 3 days I let you. Inoculate all densely grown colonies into 1.8 L of BYE medium (Buffered Yeast Extract) (see Ristroph, JD, KW Hedlund, and RG Allen. J Clin Microbiol. 11: 19-21) The culture was shaken overnight at 0 ° C. After culturing, the cells were collected by a high-speed centrifuge to obtain cells having a wet weight of 6 g. It was washed twice with distilled water.

Extraction and purification of fluorescent substances
Lipids were extracted from the cells by the modified Bligh-Dyer method (see Lipid Chemistry, Biochemistry Laboratory, Tokyo Kagaku Dojin, 1974). 6 g of the cells were suspended in 120 mL of methanol: chloroform (1: 1), stirred for 1 hour, and then centrifuged (5500 g, 10 minutes). The obtained supernatant was dried under reduced pressure to obtain 200 mg of an extract. Distilled water (30 mL), methanol (75 mL), and chloroform (37.5 mL) were sequentially added to the extract, and the mixture was stirred for 3 minutes and then allowed to stand for 15 minutes. Further, 37.5 mL of chloroform was added and stirred for 1 minute, and then 37.5 mL of distilled water was added and stirred for 30 minutes. Centrifugation (6000 g, 30 minutes) was performed until the organic solvent layer and the aqueous layer were completely separated. The obtained organic solvent layer (lower layer) was dried under reduced pressure. Thus, 100 mg of lipid was obtained from 6 g of cells.
The above operation was performed twice and the obtained lipids were pooled.

  Next, the silica gel column was used to fractionate lipids (see Lipid Research Method, Biochemical Experiment Method, Tokyo Chemical Dojin, 1975). 200 mg of the lipid obtained above was dissolved in 5 mL of hexane. This solution was loaded onto a column (2 cm inner diameter) packed with 12 g of silica gel swollen with hexane, 45 mL of hexane, 95 mL of hexane: diethyl ether (99: 1), 60 mL of hexane: diethyl ether (95: 5), hexane : 115 mL of diethyl ether (92: 8) and 115 mL of hexane: diethyl ether (85:15) were sequentially eluted as an elution solvent. The eluate was irradiated with long-wave ultraviolet light, and fluorescence emission was monitored. When the elution solvent is hexane: diethyl ether (99: 1) or hexane: diethyl ether (95: 5), fluorescence is observed in the eluted fraction, and almost no fluorescence is observed in the other fractions. There wasn't. Therefore, fractions eluted when hexane: diethyl ether (99: 1) or hexane: diethyl ether (95: 5) were collected, and the solvent was distilled off under reduced pressure.

  The eluate containing the fluorescent substance thus obtained was dissolved in hexane and purified by HPLC. Using a silica gel column (Inertsil 5 μm, inner diameter 4.6 mm × 25 cm, GL Sciences Inc.), solvent is 10% ether / hexane, flow rate is 1 ml / min, and 450 nm fluorescence with 340 nm light is monitored for fractionation. A single fluorescent material (about 100 μg) was obtained.

The physicochemical properties of the obtained single substance were as follows.
(1) Shape: Yellow powder (2) Solubility: Soluble in chloroform, hexane, methanol, DMSO, dimethyl ether, acetone, poorly soluble in water (3) Molecular formula: C ₁₉ H1 ₄ O ₃
(4) Mass spectrum (EI, m / z, relative intensity): 290 (M ⁺ , 100), 215 (14), 149 (32), 145 (13), 138 (17), 115 (25), 105 (13), 57 (13)
(5) As a result of the high-resolution mass spectrum, the molecular weight is 290.0942. (6) ¹ H-NMR (500 MHz, CDCl ₃ ): δ 10.97 (s, 1H), 7.54 (t, 1H, J = 7.5 Hz), 7.44 (d, 2H, J = 7.5Hz), 7.33 (t, 2H, J = 7.5Hz), 7.26 (t, 1H, J = 7.5Hz), 7.22 (dd, 1H, J = 11.0, 15.0Hz), 6.91 (d, 1H, J = 7.5Hz), 6.89 (dd, 1H, J = 11.0, 15.5Hz), 6.85 (d, 1H, J = 7.5Hz), 6.80 (d, 1H, J = 15.5Hz), 6.37 (s, 1H), 6.23 (d, 1H, J = 15.0Hz)
(7) ¹³ C-NMR (125 MHz, CDCl ₃ ): δ 166.0, 161.8, 152.1, 138.0, 137.3, 137.0, 136.6, 133.8, 128.8, 128.4, 127.5, 126.8, 122.4, 116.3, 115.3, 106.4, 106.1
(8) This material further exhibited the 1H-1H-COSY spectrum shown in FIG. 1, the HMQC spectrum shown in FIG. 2, and the HMBC spectrum shown in FIG.

で表される、イソクマリン骨格を持つ化合物であると特定された。この化合物は新規な化合物である。 From the above physicochemical properties, this fluorescent substance has the following formula (I):

It was identified as a compound having an isocoumarin skeleton represented by: This compound is a novel compound.

  When the excitation spectrum and fluorescence spectrum of this compound were measured, the maximum excitation wavelength was 340 nm and the maximum fluorescence wavelength was 450 nm (FIG. 4). In FIG. 4, the horizontal axis indicates the wavelength, the vertical axis indicates the relative intensity, Ex indicates the excitation spectrum, and Em indicates the fluorescence spectrum.

1 is a 1H-1H-COSY spectrum of a compound represented by formula (I). 3 is an HMQC spectrum of a compound represented by formula (I). 2 is an HMBC spectrum of a compound represented by formula (I). It is a figure which shows the spectral characteristic of the compound represented by Formula (I).

Sequence number 3: Synthetic DNA
Sequence number 4: Synthetic DNA

Claims (2)

Formula (I):

A compound represented by Legionella Dumofi NY strain (ATCC 33279) , into which the gacA gene containing the nucleotide sequence shown in SEQ ID NO: 1 from No. 395 to No. 1054 has been introduced, is cultured, lipids are extracted from the cultured cells , and formulas are derived from the lipids. (I) :

A method for producing a compound represented by the above formula (I), wherein the compound represented by formula (I) is collected.

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