JP2012056899A - Method for searching anti-helicobacter pylori agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an anti-Helicobacter pylori agent having high selectivity.SOLUTION: An aminodeoxyfutalosine de-adenine enzyme on a new menaquinone biosynthetic path present in Helicobacter pylori uses aminodeoxyfutalosine that is a new compound specifically present in H. pylori as a substrate but does not use futalosine. Thereby, by selecting a substance that specifically inhibits the aminodeoxyfutalosine de-adenine enzyme, a substance specifically inhibiting growth of H pylori can be efficiently found.

Description

The present invention relates to a novel compound that is an intermediate of a novel menaquinone biosynthetic pathway of Helicobacter pylori , and selection of a compound having anti-pylori activity using the compound and an enzyme using the compound as a specific substrate. Regarding the method.

Menaquinone biosynthesis is an 8-step reaction via o-succinylbenzoate, starting with chorismate, an intermediate in the biosynthetic pathway of the aromatic amino acids phenylalanine, tyrosine, and tryptophan (shikimic acid pathway) in Escherichia coli and Bacillus subtilis. (FIG. 1, A).
On the other hand, recently, it has been reported that there is another completely new biosynthetic pathway other than the above known pathway as the biosynthetic pathway of menaquinone (Patent Document 1). In this new route, chorismic acid is used as a starting material as in the known route, but then menaquinone is biosynthesized in a completely different route. The first step is catalyzed by, for example, the enzyme (MqnA) of the gene number (locus tag) SCO4506 of the actinomycete Streptomyces coelicolor and its ortholog, and details are still unclear, but chorismate, phosphoenolpyruvate or its analogs , Phthalosin is produced from inosine or its analogs. Next, the enzyme futalosine hydrolase (MqnB, EC 3.2.2.26) of S. coelicolor gene number SCO4327 and its ortholog may be dehypoxanthinized to dehypoxanthinylphthalosin (hereinafter abbreviated as DHFL). ). DHFL is converted to cyclic dehypoxanthine phthalosin (cyclic DHFL) by S. coelicolor gene number SCO4550 enzyme (MqnC) and its ortholog, then S. coelicolor gene number SCO4326 enzyme (MqnD) and its The ortholog is converted to 1,4-dihydroxy-6-naphthoate. Although there are no reports on biosynthetic pathways after this compound, it is thought that menaquinone is finally produced by the catalytic action of prenyltransferase, 2-demethylmenaquinone methyltransferase, as in the known pathway (Fig. 1, B).

  Using the base sequences of the enzyme genes of the above-mentioned new pathway as a query, searching for organisms having these sequences in the genetic information database and examining their distribution in the organism, It was revealed that some pathogenic bacteria such as Helicobacter, Campylobacter, and Chlamydia and food poisoning bacteria were included.

There are some known drugs such as macrolide clarithromycin and cefam amoxicillin, which have antibacterial activity against the above pathogenic and food poisoning bacteria, but they have a wide antibacterial spectrum and are effective against most microorganisms. Since a useful intestinal bacterium is killed, a highly selective drug is desired. Since menaquinone is essential for the growth of microorganisms, a specific inhibitor of the novel menaquinone biosynthetic pathway is considered to be useful as a more specific antibacterial agent against the above-mentioned pathogenic bacteria and food poisoning bacteria.
However, as a result of recent analysis of genome DNA of healthy Japanese intestinal flora by metagenomic analysis that does not require bacterial culture, about 80% of bacterial species are still unanalyzed (undiscovered). Has been reported (Non-patent Document 1). Therefore, it is desired to develop an antibacterial agent more specific to the above-mentioned pathogenic bacteria and food poisoning bacteria without affecting the intestinal bacterial flora.

The base sequence of the locus tag HP0089 of Helicobacter pylori is known (Non-patent Document 2), but its function is unknown.

JP 2009-114124 A

DNA Research, 2007, 14: 169-181 Nature, 1997, 388: 539-547

  An object of the present invention is to provide a method for searching for a more specific antibacterial agent against pathogenic bacteria and food poisoning bacteria.

The present inventors have found that the second biosynthetic intermediate of the novel biosynthetic pathway of menaquinone possessed by Helicobacter pylori is not phthalosin but aminodeoxyphthalosin in which the inosine of phthalosin is replaced with adenine. Furthermore, in H. pylori , MqnB ortholog (encoded by locus tag HP0089), which acts on aminodeoxyphthalosin and generates adenine and DHFL (encoded by aminodeoxyphthalosin deadenase), It was found that it did not act at all and acted specifically only on aminodeoxyphthalosin. Based on these findings, the present inventors have found that it is possible to search for anti-pylori agents by screening compounds that inhibit the enzyme, and have completed the present invention.

That is, the present invention relates to the following (1) to (5).
(1) A compound represented by the following formula (I).

(2) A compound represented by formula (I), aminodeoxyphthalosin deadenine enzyme, and a test substance are contacted in an aqueous medium, and the amount of adenine or dehypoxanthinylfutalosine (DHFL) produced in the aqueous medium is determined. A method for selecting a compound having anti-pylori activity, comprising measuring and selecting a substance that suppresses the production of adenine or DHFL from a test substance.
(3) The method according to (2) above, wherein the aminodeoxyphthalosin deadenine enzyme is a protein derived from Helicobacter pylori .
(4) The method according to (2) above, wherein the aminodeoxyphthalosin deadenine enzyme is the protein according to any one of [1] to [3] below.
[1] a protein having the amino acid sequence represented by SEQ ID NO: 2 [2] the amino acid sequence represented by SEQ ID NO: 2 consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted or added; Protein having aminodeoxyphthalosin deadenine enzyme activity [3] Protein having amino acid sequence represented by SEQ ID NO: 2 of 80% or more and having aminodeoxyphthalosin deadenine enzyme activity (5) The test substance inhibits the growth of microorganisms belonging to the genus Bacillus having a novel menaquinone biosynthetic pathway (a pathway including the MqnA, MqnB, MqnC and MqnD enzymes), and the conventional menaquinone biosynthetic pathway (MenF, MenD, MenC) Any one of the above (2) to (4), which is a substance that does not inhibit the growth of a microorganism belonging to the genus Bacillus having the enzyme group of MenE, MenB, MenA, and MenG) The method described.

FIG. 1 is a diagram showing two types of menaquinone biosynthesis pathways in microorganisms. A represents a known menaquinone synthesis pathway, and B represents a novel menaquinone synthesis pathway. FIG. 2 shows a synthetic reaction scheme of aminodeoxyphthalosin. The reaction conditions in the steps a to j in FIG. 2 are as follows. (a) 2,2-dimethoxypropane, acetone, p-toluenesulfonic acid, room temperature, over night (b) (1) trimethylsilyl chloride, pyridine, room temperature, 30 minutes; (2) Benzoyl chloride, room temperature, 3 hours (c) N 'N'-dicyclohexylcarbodiimide, P ₂ O ₅ , DMSO, room temperature, 24 hours (d) MeOH, H ₂ SO ₄ , room temperature, over night (e) CuBr ₂ , EtOAc, reflex, 3 hours (f) triphenylphosphine, benzene, Room temperature, over night (g) pyridine, room temperature, 24 hours (h) Pd / C, room temperature, 3-7 days (i) MeOH, K ₂ CO ₃ , room temperature, over night (j) 90% aq. TFA, room temperature , 3 hours

1. Compound of the Present Invention Synthesis and structure determination of aminodeoxyphthalosin, which is an intermediate of a novel biosynthetic pathway of menaquinone, which is the compound of the present invention, can be performed as follows.
(1) Synthesis of aminodeoxyphthalosin 2 ', 3'-O, O-Isopropylidene-6-N with adenosine as a starting material and protected with a functional group other than the hydroxyl group at the 5' position by acetonidation or dibenzoate formation N-dibenzoyladenosine is synthesized (compound shown in FIGS. 2 and 2). This compound is aldehyded by Pfitzner-Moffatt oxidation and reacted with Wittig reagent (methyl-3-triphenylphosphoranylidene acetylbenzoate) starting from m-acetylbenzoic acid. Thereafter, the double bond generated by Wittig condensation is reduced, and a deprotection reaction is performed to synthesize aminodeoxyphthalosin represented by the formula (II).
The method used for synthesizing the compound of the present invention can apply general techniques of organic chemistry, and the reaction conditions and the like can be appropriately changed.

Confirmation of the production of aminodeoxyphthalosin can be performed using known methods and equipment, for example, to have the following physicochemical properties.
Property: White powder Molecular formula: C ₁₉ H ₂₀ N ₅ O ₆
Molecular weight: obsd. 414.1397, calcd. 414.1414
Solubility: Soluble in water, hardly soluble in chloroform and hexane.

2. Aminodeoxyphthalosin deadenine enzyme used in the method of the present invention The aminodeoxyphthalosin deadenine enzyme used in the method of the present invention is particularly limited as long as it has an activity of degrading aminodeoxyphthalosin to adenine and dehypoxanthinylfutalosine (DHFL) However, it is preferable that phthalosin cannot be used as a substrate, more preferably an aminodeoxyphthalosin deadenine enzyme derived from H. pylori , more preferably any one of [1] to [3] below Protein,
[1] a protein having the amino acid sequence represented by SEQ ID NO: 2 [2] the amino acid sequence represented by SEQ ID NO: 2 consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted or added; Protein having aminodeoxyphthalosin deadenine enzyme activity [3] A protein having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2 and having aminodeoxyphthalosin deadenine enzyme activity can be mentioned. it can.
Further, it is possible to use non-pathogenic Sulfurimonas denitrificans belonging to Epsilonproteobacteria genus, Sulfurospirillum deleyianum, Nitratiruptor bacteria, the Sulfurovum bacteria or Nautilia genus selected method of bacterial amino deoxy phthaloyl thin deadenylated enzymes also present invention.

In the amino acid sequence represented by SEQ ID NO: 2 in [2] above, it consists of an amino acid sequence in which 1 to 20 amino acid residues are deleted, substituted or added, and has aminodeoxyphthalosin deadenine enzyme activity Proteins include Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press (1989) (hereinafter abbreviated as Molecular Cloning 3rd Edition), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997) ( (Hereinafter abbreviated as Current Protocols in Molecular Biology), Nucleic Acids Research, 10 , 6487 (1982), Proc. Natl. Acad. Sci. USA, 79 , 6409 (1982), Gene, 34 , 315 (1985), Nucleic Acids Research, 13 , 4431 (1985), Proc. Natl. Acad. Sci. USA, 82 , 488 (1985), etc. Encodes a protein comprising the amino acid sequence represented By introducing site-specific mutations in DNA, it can be obtained.
The number of amino acid residues to be deleted, substituted or added is 1 to 20, preferably 1 to 10, and more preferably 1 to 5.
The deletion, substitution or addition of 1 to 20 amino acid residues in the amino acid sequence represented by SEQ ID NO: 1 means that one or more amino acid residues are deleted at any position in the same sequence. , May be substituted or added.

  Examples of amino acid positions at which amino acid residues can be deleted or added include 10 amino acid residues on the N-terminal side and C-terminal side of the amino acid sequence represented by SEQ ID NO: 2.

Deletions, substitutions or additions may occur simultaneously, and the amino acids substituted or added may be natural or non-natural. Natural amino acids include L-alanine, L-asparagine, L-aspartic acid, L-arginine, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L -Methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cysteine and the like.
Examples of amino acids that can be substituted with each other are shown below. Amino acids included in the same group can be substituted for each other.
Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, O-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine Group B: aspartic acid, glutamic acid, isoaspartic acid, Isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid Group C: asparagine, glutamine Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid Group E: proline, 3 -Hydroxyproline, 4-hydroxyproline Group F: serine, threonine, homoserine Group G: phenylalanine, tyrosine

  Further, the protein having aminodeoxyphthalosin deadenine activity is 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more, with the amino acid sequence represented by SEQ ID NO: 2. Particularly preferred is a protein having an amino acid sequence having 98% or more, most preferably 99% or more identity, and having aminodeoxyphthalosin deadenine activity.

For the identity of amino acid sequences and nucleotide sequences, the algorithm BLAST [Pro. Natl. Acad. Sci. USA, 90 , 5873 (1993)] by FA and [STAS Enzymol., 183 , 63 (1990)] by Karlin and Altschul is used. Can be determined. Based on this algorithm BLAST, programs called BLASTN and BLASTX have been developed [J. Mol. Biol., 215 , 403 (1990)]. When a base sequence is analyzed by BLASTN based on BLAST, the parameters are, for example, Score = 100 and wordlength = 12. Further, when an amino acid sequence is analyzed by BLASTX based on BLAST, parameters are set to score = 50 and wordlength = 3, for example. To obtain gapped alignment, Gapped BLAST can be used as described in Altschul et al. (1997, Nucleic Acids Res. 25: 3389-3402). Alternatively, PSI-Blast or PHI-Blast can be used to perform an iterated search that detects the positional relationship (Id.) Between molecules and the relationship between molecules that share a common pattern. When using BLAST, Gapped BLAST, PSI-Blast, and PHI-Blast programs, the default parameters of the respective programs can be used (see http://www.ncbi.nlm.nih.gov.).

  In the present invention, aminodeoxyphthalosin deadenine activity refers to the activity of degrading aminodeoxyphthalosin into adenine and DHFL.

  As a means for confirming that the protein of [2] or [3] used in the selection method of the present invention is a protein having aminodeoxyphthalosin deadenine enzyme activity, for example, using a DNA recombination method, A transformant that expresses a protein is prepared, and the protein is purified using the transformant. Then, the purified protein and aminodeoxyphthalosin are present in an aqueous medium, and adenine or DHFL is present in the aqueous medium. Use HPLC-mass spectrum analysis to confirm whether the product is produced or accumulated using a commercially available sample for adenine, and using a sample that can be prepared by a known method (JP 2008-11854) for DHFL. be able to.

3. Method for Producing Aminodeoxyphthalosin Deadenine Enzyme Aminodeoxyphthalosin deadenine enzyme can be produced using cells transformed with DNA encoding the enzyme. As the DNA,
[4] DNA having the base sequence represented by SEQ ID NO: 1,
[5] DNA encoding a protein having a base sequence having 80% or more identity with the base sequence represented by SEQ ID NO: 1 and having aminodeoxyphthalosin deadenine activity, and [6] SEQ ID NO: A DNA that hybridizes with a DNA having a base sequence complementary to the base sequence represented by 1 under a stringent condition and encodes a protein having aminodeoxyphthalosin deadenine enzyme activity;
Can give.

  The term “hybridize” as used herein refers to a step in which DNA hybridizes to DNA having a specific base sequence or a part of the DNA. Accordingly, the DNA having the specific base sequence or a part of the base sequence of the DNA is useful as a probe for Northern or Southern blot analysis, or DNA of a length that can be used as an oligonucleotide primer for PCR analysis. May be. As DNA used as a probe for Northern or Southern blot analysis, DNA of at least 100 bases or more, preferably 200 bases or more, more preferably 500 bases or more can be mentioned, and DNA used as oligonucleotide primers is at least 10 bases. As mentioned above, DNA having 15 bases or more can be mentioned.

  Methods for DNA hybridization experiments are well known. For example, Molecular Cloning 2nd Edition, 3rd Edition (2001), Methods for General and Molecular Bacteriology, ASM Press (1994), Immunology methods manual, Academic press ( In addition to those described in Molecular), hybridization conditions can be determined and experiments can be performed according to a number of other standard textbooks.

  The above stringent conditions include, for example, a DNA-immobilized filter and probe DNA of 50% formamide, 5 × SSC (750 mM sodium chloride, 75 mmol / L sodium citrate), 50 mmol / L After incubation overnight at 42 ° C. in a solution containing sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate, and 20 μg / L denatured salmon sperm DNA, for example about 65 ° C. The conditions for washing the filter in a 0.2 × SSC solution can be raised, but lower stringent conditions can also be used. Stringent conditions can be changed by adjusting the concentration of formamide (the lower the formamide concentration, the lower the stringency), and changing the salt concentration and temperature conditions. As low stringent conditions, for example, 6 × SSCE (20 × SSCE is 3 mol / L sodium chloride, 0.2 mol / L sodium dihydrogen phosphate, 0.02 mol / L EDTA, pH 7.4), 0.5% Conditions in which SDS, 30% formamide, 100 μg / L of denatured salmon sperm DNA are incubated overnight at 37 ° C, and then washed with 1X SSC, 0.1% SDS solution at 50 ° C Can give. Further, examples of lower stringent conditions include conditions in which hybridization is performed using a solution having a high salt concentration (for example, 5 × SSC) under the above-described low stringency conditions and then washed.

  Various conditions described above can also be set by adding or changing a blocking reagent used for suppressing the background of the hybridization experiment. The addition of the blocking reagent described above may be accompanied by a change in hybridization conditions in order to adapt the conditions.

The DNA capable of hybridizing under the above stringent conditions includes, for example, the base of the DNA described in [4] above when calculated based on the above parameters using a program such as the above BLAST and FASTA. Examples thereof include DNA having at least 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, particularly preferably 99% or more, to the sequence.
The identity of the base sequence can be determined using a program such as BLAST or FASTA described above.

  The DNA that hybridizes with the above-described DNA under stringent conditions is a DNA that encodes a protein having aminodeoxyphthalosin deadenine enzyme activity. A recombinant DNA that expresses the DNA is prepared, Purifying the protein from a culture obtained by culturing a transformant obtained by introducing recombinant DNA into a host cell, allowing the purified protein and aminodeoxyphthalosin to exist in an aqueous medium, and the aqueous medium Whether or not adenine or DHFL is produced and accumulated therein can be confirmed by a method of analyzing by HPLC or the like.

The transformant producing the above-mentioned aminodeoxyphthalosin deadenine enzyme is not particularly limited as long as it can produce aminodeoxyphthalosin deadenine enzyme, as long as it is a transformant, but the above [1] to [ 3] Using a transformant expressing the protein according to any one of [3] and a recombinant DNA containing the DNA according to any of [4] to [6] above, a host cell is transformed by a known method The transformant obtained in this way can be mentioned. Examples of host cells include prokaryotes such as bacteria, and preferably include microorganisms belonging to the genus Escherichia .

DNA encoding the amino deoxy phthaloyl thin deadenylated enzyme, using a probe or a primer which can be designed based on the nucleotide sequence represented by SEQ ID NO: 1, a microorganism belonging to the Helicobacter genus, preferably Helicobacter pylori, and more preferably H It can be obtained by Southern hybridization to the chromosome of pylori 26695 strain or PCR [PCR Protocols, Academic Press (1990)] using the chromosome as a template.

  In addition, 80% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more of the nucleotide sequence of the DNA encoding the amino acid sequence represented by SEQ ID NO: 2 with respect to various gene sequence databases Particularly preferably, a sequence having 99% or more identity is searched, and based on the base sequence obtained by the search, the method of the present invention is carried out by the above-described method from the chromosomal DNA, cDNA library, etc. of the organism having the base sequence. DNA used in the production method can also be obtained.

The obtained DNA is cut as it is or with an appropriate restriction enzyme, and incorporated into a vector by a conventional method. After the obtained recombinant DNA is introduced into a host cell, a commonly used nucleotide sequence analysis method such as the dideoxy method [ Proc. Natl. Acad. Sci., USA, 74 , 5463 (1977)] or by using a base sequence analyzer such as ABI3700 DNA analyzer (Applied Biosystems) to determine the base sequence of the DNA. can do.
If the obtained DNA has a partial length as a result of determining the base sequence, the full-length DNA can be obtained by Southern hybridization to a chromosomal DNA library using the partial length DNA as a probe. .

  Furthermore, based on the determined base sequence of DNA, the target DNA can be prepared by chemical synthesis using a 8905 type DNA synthesizer manufactured by Perceptive Biosystems.

  Examples of the DNA obtained as described above include DNA having the base sequence represented by SEQ ID NO: 1.

Examples of vectors that incorporate DNA encoding aminodeoxyphthalosin deadenine enzyme include pBluescriptII KS (+) (Stratagene), pDIRECT [Nucleic Acids Res., 18 , 6069 (1990)], pCR-Script Amp SK ( +) (Stratagene), pT7Blue (Novagen), pCR II (Invitrogen), pCR-TRAP (Gen Hunter), and the like.

Examples of host cells include microorganisms belonging to the genus Escherichia. Examples of microorganisms belonging to the genus Escherichia include, for example, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli ATCC 12435, Escherichia coli W1485, Escherichia coli.・ Cori JM109, Escherichia coli HB101, Escherichia coli No.49, Escherichia coli W3110, Escherichia coli NY49, Escherichia coli MP347, Escherichia coli NM522, Escherichia coli BL21, Escherichia coli ME8415, etc. it can.

Production of recombinant DNA expressing aminodeoxyphthalosin deadenine enzyme can be performed, for example, as follows. First, based on the DNA encoding the enzyme protein, a DNA fragment of an appropriate length containing a protein encoding portion is prepared as necessary. In addition, a transformant with an improved production rate of the protein can be obtained by substituting the base in the base sequence of the protein-encoding portion so as to be an optimal codon for host expression.
A recombinant DNA is prepared by inserting the DNA fragment downstream of the promoter of an appropriate expression vector.

By introducing the recombinant DNA into a host cell suitable for the expression vector, a transformant that produces aminodeoxyphthalosin deadenine enzyme can be obtained.
As the expression vector, one that can autonomously replicate in the host cell or can be integrated into a chromosome and contains a promoter at a position where the DNA of the present invention can be transcribed is used.

  Recombinant DNA used for obtaining transformants is a set consisting of a promoter, a ribosome binding sequence, a DNA encoding a protein having peptide synthesis activity, and a transcription termination sequence that can autonomously replicate in prokaryotes. It is preferably a recombinant DNA. A gene that controls the promoter may also be included.

The expression vectors include pBTrp2, pBTac1, pBTac2, pHelix1 (all manufactured by Roche Diagnostics), pKK233-2 (manufactured by Amersham Pharmacia Biotech), pSE280 (manufactured by Invitrogen), pGEMEX-1 (Promega) Ltd.), manufactured by pQE-8 (QIAGEN), manufactured by pET-3 (Novagen), PKYP10 (JP 58-110600), pKYP200 [Agric. Biol . Chem., 48, 669 (1984)], pLSA1 [ Agric. Biol. Chem., 53 , 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci., USA, 82 , 4306 (1985)], pBluescriptII SK (+), pBluescript II KS (-) (Strata Gene), pTrS30 [prepared from Escherichia coli JM109 / pTrS30 (FERM BP-5407)], pTrS32 [prepared from Escherichia coli JM109 / pTrS32 (FERM BP-5408)], pPAC31 (WO98 / 12343), pUC19 [ Gene, 33 , 103 (1985)], pSTV28 (manufactured by Takara Bio Inc.), pUC118 (manufactured by Takara Bio Inc.), pPA1 (Japanese Patent Laid-Open No. Sho 63-233798), and the like.

Any promoter can be used as long as it functions in a host cell such as Escherichia coli. For example, trp promoter (P _trp), cited lac promoter (P _lac), P _L promoter, P _R promoter and P _SE promoter, promoters derived from Escherichia coli or phage, such as, SPO1 promoter, SPO2 promoter, the penP promoter and the like be able to. In addition, artificially designed and modified promoters such as a promoter in which two P _trp are connected in series, tac promoter, lacT7 promoter, let I promoter, and the like can also be used.

Furthermore, xylA promoter [Appl. Microbiol. Biotechnol., 35 , 594-599 (1991)] for expression in microorganisms belonging to the genus Bacillus and P54- for expression in microorganisms belonging to the genus Corynebacterium 6 Promoters [Appl. Microbiol. Biotechnol., 53 , 674-679 (2000)] can also be used.

  Moreover, it is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 bases).

  In a recombinant DNA in which a DNA encoding an aminodeoxyphthalosin deadenine enzyme is bound to an expression vector, a transcription termination sequence is not necessarily required, but it is preferable to place a transcription termination sequence immediately below the structural gene.

As the host cell, any prokaryotic organism such as a bacterium can be used. The prokaryotic organisms, Escherichia, Bacillus, Brevibacterium (Brevibacterium), Corynebacterium, micro Agrobacterium (Microbacterium) genus Serratia (Serratia) genus Pseudomonas (Pseudomonas) genus, Agrobacterium (Agrobacterium) genus Alicyclobacillus Bacillus (Alicyclobacillus) genus Anabaena (Anabena) genus Anashisutisu (Anacystis) genus Arthrobacter (Arthrobacter) genus Azotobacter (Azotobacter) genus Kuromachiumu (Chromatium) genus Erwinia (Erwinia) genus, methylol bacterium (Methylobacterium) genus, Phormidium (Phormidium) genus Rhodobacter (Rhodobacter) genus, Rhodopseudomonas (Rhodopseudomonas) genus, Rodosupiriumu (Rhodospirillum) genus Scenedesmus (Scenedesmus) genus Streptomyces (Streptomyces) , Shinekokkasu (Synechoccus) genus Zymomonas (Zymomonas) microorganism belonging to the genus, such as, for example, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli DH5α, Escherichia coli MC1000, Escherichia Coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No.49, Escherichia coli W3110, Escherichia coli NY49, Escherichia coli MP347, Escherichia coli NM522, Escherichia coli NM522, Bacillus subtilis (Bacillus subtilis) ATCC33712, Bacillus megaterium (Bacillus megaterium), Brevibacterium ammonia monocytogenes (Brevibacterium ammoniagenes), Brevibacterium Lee Mario film (Brevibacterium immariophilum) ATCC14068, shake Bibakuteriumu Sacca Lori tee cam (Brevibacterium saccharolyticum) ATCC14066, Brevibacterium flavum (Brevibacterium flavum) ATCC14067, Brevibacterium lactofermentum (Brevibacterium lactofermentum) ATCC13869, Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032, Corynebacterium glutamicum ATCC14297, Corynebacterium acetamide A Sid film (Corynebacterium acetoacidophilum) ATCC13870, micro Corynebacterium ammoniagenes film (Microbacterium ammoniaphilum) ATCC15354, Serratia Fikaria (Serratia ficaria), Serratia Fonchikora (Serratia fonticola), Serratia Rikefashiensu (Serratia liquefaciens), Serratia marcescens (Serratia marcescens), Pseudomonas sp. (Pseudomonas sp.) D-0110 , UGG Bacterium radiobacter (Agrobacterium radiobacter), Agrobacterium Rizzo jeans (Agrobacterium rhizogenes), Agrobacterium ruby (Agrobacterium rubi), Anabaena-Shirindorika (Anabaena cylindrica), Anabaena-Doriorumu (Anabaena doliolum), Anabaena floss aqua ( Anabaena flos-aquae ), Arthrobacter aurescens , Arthrobacter citreus , Arthrobacter globformis , Arthrobacter globformis , Arthrobacter hydrocarba glutamicus ( Arthrobacter hydrocarboglutamicus), Arthrobacter Misorensu (Arthrobacter mysorens), Arthrobacter Nicotiana (Arthrobacter nicotianae), Arthrobacter paraffineus (Arthrobacter paraffineus), ground Enterobacter Proto folder Mie (Arthrobacter protophormiae), Arthrobacter Rose opal Rafi eggplant (Arthrobacter roseoparaffinus), Arthrobacter sulphureus (Arthrobacter sulfureus), Arthrobacter urea tumefaciens (Arthrobacter ureafaciens), Kuromachiumu-Buderi (Chromatium buderi), Kuromachiumu-Tepidamu (Chromatium tepidum), Kuromachiumu-Binosamu (Chromatium vinosum), Kuromachiumu-Wamingi (Chromatium warmingii), Kuromachiumu-Furubiatatire (Chromatium fluviatile), Erwinia Uredobara (Erwinia uredovora), Erwinia Karotobara (Erwinia carotovora) , Erwinia Anas (Erwinia ananas), Erwinia Herikora (Erwinia herbicola), Erwinia Pankutata (Erwinia punctata), Erwinia terreus (Erwinia t erreus), Methylobacterium-Rodeshianamu (Methylobacterium rhodesianum), Methylobacterium-exo torque Enns (Methylobacterium extorquens), Phormidium sp (Phormidium sp.) ATCC29409, Rhodobacter Kapusuratasu (Rhodobacter capsulatus), Rhodobacter sphaeroides (Rhodobacter sphaeroides), Rod Pseudomonas Burasuchika (Rhodopseudomonas blastica), Rod Pseudomonas Marina (Rhodopseudomonas marina), Rod Pseudomonas pulse tris (Rhodopseudomonas palustris), Rodosupiriumu-Riburamu (Rhodospirillum rubrum), Rodosupiriumu-Sarekishigensu (Rhodospirillum salexigens), Rodosupiriumu · Sarinaramu (Rhodospirillum salinarum), Streptomyces ambofaciens (Streptomyces ambofaciens), Streptomyces Over Leo tumefaciens (Streptomyces aureofaciens), Streptomyces aureus (Streptomyces aureus), Streptomyces Funjishidikasu (Streptomyces fungicidicus), Streptomyces Gris Theo black Moge eggplant (Streptomyces griseochromogenes), Streptomyces griseus (Streptomyces griseus ), Streptomyces lividans , Streptomyces olivogriseus , Streptomyces rameus , Streptomyces tanashiensis , Streptomyces vinashius Zymomonas mobilis and the like.

As a method for introducing recombinant DNA, any method can be used as long as it is a method for introducing DNA into the host cell. For example, a method using calcium ions [Proc. Natl. Acad. Sci., USA, 69 , 2110 (1972)], protoplast method (Japanese Patent Laid-Open No. 63-248394), electroporation method [Nucleic Acids Res., 16 , 6127 (1988)] and the like.

In addition, a transformant that expresses a protein having aminodeoxyphthalosin deadenine activity can be obtained by incorporating a DNA encoding a protein having aminodeoxyphthalosin deadenine activity into any position of the chromosomal DNA. You can also.
As a method of incorporating DNA encoding a protein having aminodeoxyphthalosin deadenine enzyme activity into an arbitrary position of the chromosomal DNA of a microorganism, a method utilizing homologous recombination can be mentioned, and E. When using Escherichia coli , the method described in Proc. Natl. Acad. Sci. US A., 97 , 6640 (2000) can be used.

  The aminodeoxyphthalosin deadenine enzyme can be obtained by culturing the transformant obtained by the above method in a medium and isolating the protein from the culture.

The transformant is cultured using a natural or synthetic medium containing a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the transformant, and capable of efficiently culturing the transformant. Can do.
Any carbon source may be used as long as it can be assimilated by the microorganism, such as glucose, fructose, sucrose, xylose, arabinose, molasses containing these, saccharified liquid of cellulosic biomass, carbohydrates such as glycerol, starch or starch hydrolysate. Further, organic acids such as acetic acid and propionic acid, alcohols such as ethanol and propanol, and the like can be used.
Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium salts of organic acids such as ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, casein A hydrolyzate, soybean meal, soybean meal hydrolyzate, various fermented cells, digests thereof, and the like can be used.
As the inorganic salt, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like can be used.

The culture is usually carried out under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is preferably 15 to 40 ° C., and the culture time is usually 5 hours to 7 days. During the culture, the pH is maintained at 3.0 to 9.0. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like.
Moreover, you may add antibiotics, such as an ampicillin and a tetracycline, to a culture medium as needed during culture | cultivation.

When cultivating a transformant having an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, isopropyl-β-D-thiogalactopyranoside is used when cultivating a transformant having an expression vector using the lac promoter, and indole acrylic is used when culturing a transformant having an expression vector using the trp promoter. An acid or the like may be added to the medium.

Recombinant aminodeoxyphthalosin deadenine protein production methods include host cell production methods, host cell secretion methods, or host cell outer membrane production methods, depending on the method selected. Can change the structure of the protein to be produced.
When aminodeoxyphthalosin deadenine protein is naturally produced in the host cell or on the host cell outer membrane, the method of Paulson et al. [J. Biol. Chem., 264 , 17619 (1989)], the method of Rowe et al. [Proc. Natl. Acad. Sci., USA, 86 , 8227 (1989), Genes Develop., 4 , 1288 (1990)], or Japanese Patent Application Laid-Open No. 05-336963, WO94 / 23021, etc. Thus, the protein can be actively secreted out of the host cell.
That is, the protein can be actively secreted outside the host cell by using a gene recombination technique and producing it in a form in which a signal peptide is added in front of the protein containing the active site of the protein.

  Further, in accordance with the method described in JP-A-2-27075, the production amount can be increased using a gene amplification system using a dihydrofolate reductase gene or the like.

As a method for isolating and purifying the above-mentioned protein having aminodeoxyphthalosin deadenine enzyme activity produced by using the transformant, usual enzyme isolation and purification methods can be used.
For example, when the protein is produced in a dissolved state in cells, the cells are collected by centrifugation after culturing, suspended in an aqueous buffer solution, an ultrasonic crusher, a French press, a Manton Gaurin homogenizer. Then, the cells are disrupted with dynomill or the like to obtain a cell-free extract.
From the supernatant obtained by centrifuging the cell-free extract, an ordinary enzyme isolation and purification method, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, diethylamino Anion exchange chromatography using a resin such as ethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), a cation using a resin such as S-Sepharose FF (manufactured by GE Healthcare Biosciences) Exchange chromatography method, hydrophobic chromatography method using resin such as butyl sepharose, phenyl sepharose, gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, electrophoresis method such as isoelectric focusing etc. These methods can be used alone or in combination to obtain a purified preparation.

In addition, when the above protein 2 is produced by forming an insoluble substance in the cells, the cells are similarly collected, disrupted and centrifuged from the precipitate fraction obtained by centrifugation according to a conventional method. After recovering the protein, the insoluble material of the protein is solubilized with a protein denaturant.
The solubilized solution is diluted or dialyzed into a dilute solution that does not contain a protein denaturing agent or the concentration of the protein denaturing agent does not denature the protein, and the protein is constituted into a normal three-dimensional structure. A purified sample can be obtained by the same isolation and purification method.

When the protein 2 is secreted extracellularly, the protein or a derivative such as a sugar adduct thereof can be recovered in the culture supernatant.
That is, a soluble fraction is obtained by treating the culture by a technique such as centrifugation as described above, and a purified preparation is obtained from the soluble fraction by using the same isolation and purification method as described above. be able to.

  Examples of the protein thus obtained include a protein consisting of the amino acid sequence represented by SEQ ID NO: 2.

In addition, the above protein 2 can be produced as a fusion protein with another protein, and purified using affinity chromatography using a substance having affinity for the fused protein. For example, the method described in Law et al. [Proc. Natl. Acad. Sci., USA, 86 , 8227 (1989), Genes Develop., 4 , 1288 (1990)], JP-A-5-336963, WO94 / 23021 According to the above, the above-mentioned 2 protein can be produced as a fusion protein with protein A and purified by affinity chromatography using immunoglobulin G.
In addition, the above two proteins are produced as a fusion protein with Flag peptide, and affinity chromatography using an anti-Flag antibody [Proc. Natl. Acad. Sci., USA, 86 , 8227 (1989), Genes Develop., 4 , 1288 (1990)], or can be purified by affinity chromatography using a metal coordination resin produced as a fusion protein with polyhistidine and having a high affinity with polyhistidine. Furthermore, it can also be purified by affinity chromatography using an antibody against the protein itself.

  Based on the amino acid sequence information of the protein obtained above, the protein of the present invention is produced by chemical synthesis methods such as Fmoc method (fluorenylmethyloxycarbonyl method) and tBoc method (t-butyloxycarbonyl method). be able to. Chemical synthesis can also be performed using peptide synthesizers such as Advanced ChemTech, Perkin Elmer, Pharmacia, Protein Technology Instrument, Synthecell-Vega, PerSeptive, Shimadzu Corporation.

4). Method for selecting anti-pylori agent of the present invention As a method for selecting an antibacterial agent having high selectivity against H. pylori using the aminodeoxyphthalosin of the present invention, aminodeoxyphthalosin, aminodeoxyphthalosin deadenine enzyme, and A test substance is contacted in an aqueous medium, the amount of adenine or DHFL produced in the aqueous medium is measured, and a substance that suppresses the production of adenine or DHFL from the test substance is aminodeoxyphthalosin deadenine The method of selecting as a substance with an enzyme inhibitor activity can be mentioned.

  The test substance may be either artificially synthesized or a natural product. As the artificially synthesized substance, a structure-similar substance of aminodeoxyphthalosin is preferable.

The design of the structure analogs of amino deoxy phthaloyl thin to be used as test substance, compound obtained by replacing the elements constituting the amino deoxy phthaloyl thin, for example, replace the carbon to oxygen (-CH ₂ - and -O-, etc.), Replace all possible elemental substitutions, such as replacing nitrogen with carbon (-NH- to -CH _2-, etc.), nitrogen to oxygen (-NH- to -O-, etc.), and vice versa. Including. Also, functional group substitution and introduction, for example, methylene group ((-CH _2- ) hydroxylation (-CHOH-), amination (-CHNH _2- ), carbonylation (-CO-), hydrogen methyl group Such structurally similar compounds may be prepared by an appropriate method such as chemical synthesis or microbial conversion and used for screening.

The amount of the enzyme in the reaction solution can be 0.01-100 mg, preferably 0.1 mg-10 mg, per 1 mg of the substrate.
The concentration of the test substance can be 0.001-10000 μg / ml, preferably 0.002-1000 μg / ml.

The aqueous medium may be any aqueous medium as long as it does not inhibit the enzyme reaction in the state containing no test substance, for example, water, phosphate buffer, borate buffer, acetate buffer, carbonate buffer. Solution, citrate buffer, Tris buffer and the like.
The aqueous medium may contain an organic solvent such as alcohols and ketones.

  The reaction time can be 1-30 minutes, preferably 2-15 minutes, more preferably 3-10 minutes. The reaction temperature is not limited as long as the enzyme activity can be maintained, The temperature is preferably 30-40 ° C, more preferably 37 ° C.

The produced adenine and DHFL can be measured by reverse phase chromatography or the like.
A compound having anti-H. Pylori activity can be obtained by selecting a test substance that produces less adenine and DHFL than a reaction solution that does not contain the test substance to be compared.

In light of the object of the present invention to provide an antibacterial agent highly specific to Helicobacter pylori, the test substance is a known menaquinone biosynthetic pathway (MenF, MenD, MenC, MenE, MenB, MenA, and MenG enzymes). It is desirable that it does not inhibit (including pathways). In addition, recently discovered novel menaquinone biosynthetic pathways (including pathways of MqnA, MqnB, MqnC and MqnD enzymes), in particular, substances that inhibit MqnB may also be able to inhibit aminodeoxyphthalosin deadenine enzyme. . Therefore, by using as a test substance a compound that inhibits the growth of a microorganism belonging to the genus Bacillus having a novel menaquinone biosynthesis pathway and does not inhibit the growth of a microorganism belonging to the genus Bacillus having a conventional menaquinone biosynthesis pathway, It is possible to efficiently select a compound exhibiting antibacterial activity against H. pylori without affecting the growth of a microorganism having a menaquinone biosynthesis pathway.
Therefore, the present invention also provides a novel menaquinone biosynthetic pathway by contacting a microorganism belonging to the genus Bacillus having a novel menaquinone biosynthetic pathway and a microorganism belonging to the genus Bacillus having a conventional menaquinone biosynthetic pathway with a test substance. After selecting a test substance that inhibits a microorganism belonging to the genus Bacillus and does not inhibit the growth of a microorganism belonging to the genus Bacillus having a conventional menaquinone biosynthesis pathway, the selected test substance is selected from aminodeoxyphthalosin and Provided is a method for selecting a compound having anti-pylori activity by contacting aminodeoxyphthalosin deadenine enzyme in an aqueous medium and measuring the amount of adenine or DHFL produced in the aqueous medium in the same manner as described above.
Examples of the microorganism belonging to the genus Bacillus having a novel menaquinone biosynthetic pathway include Basillus clausii and Bacillus halodurans . On the other hand, examples of microorganisms belonging to the genus Bacillus having a conventional menaquinone biosynthetic pathway include, but are not limited to, Bacillus subtilis. Since these microorganisms belonging to the genus Bacillus are non-pathogenic, they are safer and easier to handle than pathogenic microorganisms such as H. pylori , and are therefore suitable for use as primary screening systems for test substances.

  Examples of the present invention are shown below, but the present invention is not limited to these Examples. Unless otherwise specified, the gene recombination experiments shown in the Examples are the methods described in Molecular Cloning 2nd Edition and Practical Streptomyces Genetics, T. Kieser et al., The John Innes Foundation, Norwich (hereinafter referred to as conventional methods). Was used).

Synthesis of aminodeoxyphthalosin and determination of structure The synthesis scheme of aminodeoxyphthalosin is as shown in FIG. 2, and the following compound names and numbers are the same as those in FIG.
(A) Synthesis of 2 ', 3'-O-isopropylidene adenosine (1) Add adenosine (1 g, 3.7 mmol) and 2,2-dimethoxypropane (1.5 g, 14.8 mmol) and dissolve in acetone (70 mL). , P-toluenesulfonic acid (1.4 g, 7.4 mmol) was added and stirred at room temperature overnight. After completion of the reaction, the mixture was neutralized with saturated sodium hydrogen carbonate, the organic layer was evaporated with an evaporator, and extracted with chloroform. Thereafter, recrystallization with methanol was performed to obtain white needle crystals. Yield 79%.
Molecular formula: C ₁₃ H ₁₇ N ₅ O ₄
¹ H-NMR (400 MHz, CD ₃ Cl) δ (ppm): 8.29 (s, 1H), 7.81 (s, 1H), 6.53 (d, J = 11.5 Hz, 1H), 5.93 (br s, 2H) , 5.83 (d, J = 4.9 Hz, 1H), 5.19 (t, J = 5.3 Hz, 1H), 5.09 (dd, J = 5.3 Hz, 1.0 Hz, 1H), 4.52 (m, 1H), 3.95 (m , 2H), 3.77 (m, 1H), 1.62 (s, 3H), 1.35 (s, 3H)

(B) Synthesis of 2 ', 3'-O, O-Isopropylidene-N6, N6-dibenzoyladenosine (2)
(1) was dissolved in pyridine, 5 molar equivalents of TMS-Cl was added, and the mixture was stirred at room temperature for 30 minutes. Thereafter, 2.6 molar equivalents of benzoyl chloride was added and stirred at room temperature for 3 hours, and then about half the volume of benzoyl chloride was added, and the organic layer was evaporated by an evaporator. Chloroform was added, and the mixture was separated with cooled 2 N aqueous sulfuric acid and saturated aqueous sodium hydrogen carbonate, washed with water, and isolated with a silica gel column (CHCl ₃ : MeOH = 50: 1). Yield 75%.
Molecular formula: C ₂₇ H ₂₅ N ₅ O ₆
¹ H-NMR (400 MHz, CD ₃ Cl) δ (ppm): 8.50 (s, 1H), 8.06 (s, 1H), 7.79 (d, J = 7.2 Hz, 4H), 7.43 (t, J = 7.2 Hz, 2H), 7.30 (m, 4H), 5.87 (d, J = 4.8 Hz, 1H), 5.19 (m, 1H), 5.04 (t, J = 3.0 Hz), 4.48 (m, 1H), 3.80 ( m, 2H), 3.73 (m, 1H), 1.58 (s, 3H), 1.32 (s, 3H)

(C) Synthesis of (3)
Lyophilized in a 200 mL eggplant-shaped flask (2) 1 g was dissolved in DMSO (15 mL) dried with Molecular Sieves 4A, 1 mM anhydrous phosphoric acid (1.5 mL) was added, and lyophilized DCC (3.4 g, 16.25 mmol) dried by the method was added and stirred for 24 hours. Since the product was unstable, the reaction solution after completion of the reaction was used as it was after confirming the progress of the reaction by TLC.

(D) Synthesis of methyl 3-acetylbenzoate (4)
3-acetylbenzoic acid (5 g, 30.5 mmol) was added to a 200 mL eggplant-shaped flask, dissolved in methanol (70 mL), concentrated sulfuric acid (0.3 mL) was added, and the mixture was refluxed overnight. After completion of the reaction, the reaction mixture was neutralized with saturated aqueous sodium hydrogen carbonate solution, concentrated under reduced pressure, diluted with water, extracted three times with ethyl acetate, and the organic layer was dried under reduced pressure to give white needle crystals. Yield 93.2%.
¹ H-NMR (400 MHz, CDCl ₃ ) δ (ppm): 8.56 (m, 1H, Ar2H), 8.20 and 8.13 (2ddd, 2H, Ar4 and Ar6H), 7.53 (ddd, 1H, Ar5H), 3.93 (s, 3H, COOMe), 2.63 (s, 3H, COMe)
¹³ C-NMR (100 MHz, CDCl ₃ ) δ (ppm): 197.4 (s), 167.5 (s), 166.5 (s), 137.5 (s), 134.1 (d), 132.5 (d), 130.9 (s) , 129.8 (d), 129.1 (d), 52.6 (q), 27.0 (q)
C ₁₀ H ₁₁ O ₃ by TOF-MS 179.0703 [M + H] ⁺ (calcd. 179.0708)
IR (KBr) cm ^-1 = 1700

(E) Synthesis of methyl 3-bromoacetylbenzoate (5)
A 200 mL eggplant type flask was charged with methyl 3-acetylbenzoate ((4), 1 g, 5.62 mmol) and copper (II) bromide (2.5 g, 11.2 mmol; 2 eq.) And ethyl acetate (50 mL). ) And refluxed for 3 hours. After completion of the reaction, the deposited precipitate was filtered, and the filtrate was washed with water. The organic layer was dried under reduced pressure to obtain a white solid. Yield 90.4%.
¹ H-NMR (400 MHz, CDCl ₃ ) δ (ppm): 8.61 (m, 1H, Ar2H), 8.28 and 8.19 (2ddd, 2H, Ar4 and Ar6H), 7.60 (ddd, 1H, Ar5H), 4.49 (s , 2H, CH ₂ Br), 3.97 (s, 3H, COOMe)
¹³ C-NMR (100 MHz, CDCl ₃ ) δ (ppm): 190.5 (s), 165.9 (s), 135.0 (s), 134.6 (d), 132.9 (d), 130.8 (s), 129.9 (d) , 129.1 (d), 52.5 (q), 30.7 (q)
IR (KBr) cm ^-1 = 1700

(F) Synthesis of methyl 3- (triphenylphosphoranylidene) acetyl benzoate (6)
Triphenylphosphine (MW = 262) (1 g, 3.82 mmol; ca. 1 eq.) Was placed in a 200 mL eggplant-shaped flask and dissolved in benzene (5 mL) dried with Molecular Sieves 3A. To this was added methyl 3-bromoacetylbenzoate ((5), 1 g, 3.89 mmol) dissolved in benzene (5 mL), which was also dried, and stirred overnight at room temperature under water-free conditions. After completion of the reaction, the reaction solution was filtered to obtain a white purple precipitate. The precipitate was dissolved in a mixture of water: methanol = 1: 1, neutralized with a 0.5 N aqueous sodium hydroxide solution, and stirred for 10 minutes after the resulting turbidity did not disappear. This was concentrated under reduced pressure, extracted with ethyl acetate, and the organic layer was dried under reduced pressure to obtain a yellow powder. Yield 89.5%.
¹ H-NMR (400 MHz, CDCl ₃ ) δ (ppm): 9.01-7.50 (m, 19H, 4Ar), 6.53 (d, J = 12 Hz, 1H, CH), 3.90 (s, 3H, COOMe)
C ₂₈ H ₂₄ O ₃ P by TOF-MS 439.1462 [M + H] ⁺ (calcd. 439.1463)

(G) Synthesis of (7) Pyridine (1 mL) was added to the reaction flask of (c), and (6) (850 mg, 1.63 mmol; 0.5 eq.) Was added and stirred overnight. After completion of the reaction, the reaction solution was filtered through Celite to remove DCU, and the filtrate was diluted with ethyl acetate and washed with ice water, 1 N aqueous hydrochloric acid solution and saturated brine. The precipitate formed in the liquid separation was removed by celite filtration. The organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and isolated by silica gel column chromatography using hexane: ethyl acetate = 1: 2 as a mobile solvent. Yield 24%. (TLC is hexane: ethyl acetate = 2: 5, Rf 0.66).
Molecular formula: C ₃₇ H ₃₁ N ₅ O ₈
¹ H-NMR (400 MHz, CDCl ₃ ) δ: 8.60 (1.0H, s), 8.47 (1.0H, t, J = 1.69 Hz), 8.20 (1.0H, dt, J = 7.77, 1.39 Hz), 8.15 (1.0H, s), 7.95 (1.1H, dt, J = 7.83, 1.46 Hz), 7.81 (5.0H, dd, J = 8.33, 1.19 Hz), 7.52-7.47 (1.2H, m), 7.45 (2.3 H, ddd, J = 9.91, 5.06, 2.18 Hz), 7.32 (5.0H, t, J = 7.73 Hz), 7.10 (1.0H, dd, J = 15.42, 5.01 Hz), 7.01 (1.0H, dd, J = 15.46, 1.29 Hz), 6.21 (1.0H, d, J = 2.28 Hz), 5.50 (1.0H, dd, J = 6.34, 2.28 Hz), 5.15 (1.0H, dd, J = 6.34, 3.97 Hz), 4.94 (1.0H, t, J = 3.97 Hz), 3.91 (3.0H, s), 1.65 (3.2H, s), 1.40 (3.2H, s)
¹³ C-NMR (100 MHz, CDCl ₃ ) δ: 188.7, 172.1, 166.1, 152.4, 152.2, 144.0, 143.5, 137.4, 133.9, 133.1, 132.7, 130.8, 130.0, 129.6, 129.4, 129.0, 128.8, 128.4, 127.9 , 127.7, 125.8, 115.3, 90.6, 86.3, 84.0, 83.8, 52.4, 29.7, 27.2, 25.4

(H) Synthesis of (8) 1 g of condensate (7) was dissolved in 100 ml of ethanol, 25 mg of PtO ₂ was added, and shaken at 4 atm for 3 days with a Parr hydrogenator, Thereafter, the mixture was dried by Celite filtration and an evaporator. Yield 65%
Molecular formula: C ₃₇ H ₃₃ N ₅ O ₈
¹ H-NMR (CDCl ₃ ) δ (ppm): 8.64 (0.9H, s), 8.54 (1.0H, t, J = 1.54 Hz), 8.20 (1.0H, dt, J = 7.73, 1.44 Hz), 8.12 (1.0H, s), 8.07 (0.9H, dt, J = 7.83, 1.51 Hz), 7.83 (3.9H, dd, J = 8.33, 1.29 Hz), 7.50 (1.0H, t, J = 7.78 Hz), 7.45 (2.2H, tt, J = 7.44, 1.42 Hz), 7.33 (4.2H, t, J = 7.73 Hz), 6.06 (1.0H, d, J = 2.68 Hz), 5.45 (1.0H, dd, J = 6.54, 2.68 Hz), 4.89 (1.0H, dd, J = 6.54, 4.06 Hz), 4.32-4.28 (1.0H, m), 3.92 (3.1H, s), 3.19-3.04 (2.0H, m), 2.28 -2.10 (2.0H, m), 1.59 (3.1H, s), 1.37 (3.1H, s)
¹³ C-NMR (100 MHz, CDCl ₃ ) δ (ppm): 197.9, 172.2, 166.2, 152.4, 152.1, 144.0, 136.9, 134.0, 134.0, 133.0, 132.1, 130.7, 130.1, 129.5, 129.1, 128.9, 128.7, 128.4, 128.0, 115.1, 90.4, 85.9, 83.9, 52.4, 34.4, 27.3, 27.2, 25.4

(I) Synthesis of (9) which is a debenzoylated form of (8)
90% aqueous methanol was added to (8), 815 mg of K ₂ OCO ₃ was added to 1 g of (8), and the mixture was stirred overnight at room temperature. Thereafter, the mixture was dried with an evaporator and isolated with LH-20 (MeOH). Yield 38%.
¹ H-NMR (400MHz, CD ₃ OD) δ (ppm): 8.42 (1.0H, d, J = 1.19 Hz), 8.16 (1.0H, s), 8.09 (1.1H, s), 8.04 (1.0H, dd, J = 7.53, 1.19 Hz), 7.82 (1.0H, dd, J = 7.93, 1.19 Hz), 7.34 (1.0H, t, J = 7.73 Hz), 6.03 (1.0H, t, J = 1.19 Hz) , 5.41-5.39 (1.0H, m), 4.87 (0.9H, dd, J = 6.34, 3.57 Hz), 4.18 (1.0H, dd, J = 10.31, 6.74 Hz), 3.04 (2.0H, td, J = 6.84, 2.64 Hz), 2.04-1.98 (2.0H, m), 1.47 (3.0H, s), 1.27 (3.1H, s)
¹³ C-NMR (100 MHz, CDCl ₃ ) δ (ppm): 201.2, 173.3, 157.3, 154.0, 150.3, 141.8, 138.6, 137.8, 134.9, 130.9, 130.1, 129.3, 120.5, 115.8, 91.0, 87.2, 85.4, 85.2, 35.5, 28.8, 27.5, 25.6

(J) Synthesis of aminodeoxyphthalosin
(9) was dissolved in 10% TFA water and stirred at room temperature for 4 hours. Thereafter, it was concentrated by an evaporator, and when recrystallized, it was isolated by filtration. If it was not recrystallized, it was dried and isolated with LH-20 (MeOH). Yield 43%.

Expression of HP0089 of H. pylori HP0089 was expressed as follows using Escherichia coli as a host cell.
First, based on the genomic information of H. pylori (GenBank accession No. AE000511.1), the HP0089 5'-side primer (DNA consisting of the nucleotide sequence represented by SEQ ID NO: 3) and 3'-side primer (SEQ ID NO: 3) DNA having a base sequence represented by 4) was prepared. Using these commercially available H. pylori HB8 chromosomal DNA (Takara Shuzo, Code 3071) as a template, these primers, TaKaRa LA-PCR ^™ Kit Ver.2 (Takara Shuzo), Expand ^™ High-Fidelity PCR System (Roche) Or Taq DNA polymerase (manufactured by Promega) and PCR was performed with a DNA amplification apparatus (manufactured by MJ Research). PCR was performed under the conditions of 30 cycles at 95 ° C. for 30 seconds, 60 ° C. for 1 minute, and 72 ° C. for 2 minutes, followed by 30 cycles followed by reaction at 72 ° C. for 10 minutes.
After completion of PCR, the amplified fragment was cleaved with BamHI and PstI, and after agarose gel electrophoresis, a DNA fragment of about 700 bp was purified and obtained. The DNA fragment obtained above is mixed with pUC118 vector cleaved with BamHI and PstI, and then ethanol precipitation is performed. The resulting DNA precipitate is dissolved in 5 μl of distilled water and recombination reaction is performed. Body DNA was obtained.
Using the recombinant DNA, E. coli DH5α strain (purchased from Toyobo) was transformed according to a conventional method, and the transformant was applied to an LB agar medium containing 50 μg / ml of ampicillin and overnight at 37 ° C. Cultured. Several colonies of ampicillin-resistant transformants that had grown were cultured with shaking at 37 ° C. for 16 hours in 5 ml of LB liquid medium containing 50 μg / ml of ampicillin. The cells were obtained by centrifuging the obtained culture solution. A plasmid was isolated from the cells according to a conventional method. The plasmid isolated by this method was cleaved with various restriction enzymes, the structure was examined, and the nucleotide sequence was determined to confirm that the plasmid had the target DNA fragment inserted.
The target gene fragment inserted into the resulting plasmid vector is expressed in E. coli using the pMAL system (New England Biolabs, catalog No. N8076S), and the fusion part is further cleaved according to the pMAL system. As a result, a purified product of the enzyme protein that is the product of the HP0089 gene was obtained.

Conversion reaction of aminodeoxyphthalosin to adenine and dehypoxanthinylfutalosine (DHFL) using recombinant HP0089 The aminodeoxyphthalosin synthesized in Example 1 and the enzyme protein of H. pylori HP0089 prepared in Example 2 are as follows: The sample was subjected to an enzyme reaction at 37 ° C. for 10 minutes.

Aminodeoxyphthalosin (10 mM) 5 μL
Purified enzyme (0.1 mg / ml) 5 μL
100 mM potassium phosphate buffer (pH 6.0) 25 μL
Distilled water 15 μL
50 μL total

  The reaction solution prepared by the above reaction system was subjected to reverse phase HPLC, that is, ODS (Mightysil RP-18GP Aqua 250-4.6, manufactured by Kanto Chemical Co., Ltd.) as a column and 20 mM potassium phosphate buffer (pH 2.5) / acetonitrile (acetonitrile as a moving bed. (Concentration 0 min / 0% → 12 min / 40% → 13 min / 80% → 14 min / 0% → 25 min / 0%). Two new peaks (substances) generated by the enzyme reaction were confirmed by measuring the absorbance. By analyzing these peaks by LC-MS, a commercially available sample is used for adenine, and a sample that can be prepared by a known method (JP 2008-11854) for DHFL. Peak (M + 1) was compared and it was concluded that the purified compounds were adenine and DHFL.

Moreover, the same reaction was performed using the phthalosin prepared by the well-known method (Unexamined-Japanese-Patent No. 2008-11854).

Phthalosine (10 mM) 5 μL
Purified enzyme (0.1 mg / ml) 5 μL
100 mM potassium phosphate buffer (pH 6.0) 25 μL
Distilled water 15 μL
50 μL total

The reaction solution prepared by the above reaction system was analyzed by the reverse phase HPLC, but the substrate was present as it was, and neither hypoxanthine nor DHFL was produced.
From the above, in H. pylori , it was revealed that aminodeoxyphthalosin, which is a novel compound, is not an intermediate of the novel menaquinone biosynthetic pathway, not phthalosin.

Search for anti-pylori agents (1)
The enzyme protein of HP0089 prepared in Example 2, the aminodeoxyphthalosin prepared in Example 1, and the test substance were each subjected to 0, 5, 10, and 20 μg of the following reaction systems, and the enzyme reaction was performed at 37 ° C. for 10 minutes. Went.

Aminodeoxyphthalosin (10 mM) 20 μL
Purified enzyme (0.1 mg / ml) 40 μL
100 mM potassium phosphate buffer (pH 6.0) 100 μL
Test substance 0 ~ 20 μg
Distilled water remaining <br/> 200 μL total

  The amount of phthalosine produced in the reaction solution was analyzed by HPLC according to Example 3, and an aminodeoxyphthalosin, which is a product of HP0089, was selected from the test substances by identifying test plots with a small amount of phthalosin produced compared to the control. Compounds that show deadenine inhibitory activity can be screened.

Search for anti-pylori agents (2)
Originally, H. pylori , which should be used to evaluate its antibacterial activity, is a highly pathogenic bacterium, so it must be handled with great care. Therefore, from the viewpoint of maintaining a safe environment at the screening stage, primary screening is performed using Bacillus halodurans that have a novel pathway for menaquinone biosynthesis and Bacillus subtilis that has only a known pathway, with no known pathogenicity. be able to.
B. halodurans , which is a novel pathway-retaining bacterium, and Bacillus subtilis , which is a known pathway-retaining bacterium, are layered on a petri dish using soft agar. Place a paper disc coated with actinomycetes, mold culture solution, chemically synthesized natural product, non-natural product, etc. on the medium and incubate at 30 ° C overnight, specific to B. halodurans , a new pathway-retaining bacterium A candidate compound is obtained by screening a halo-formed sample.
Next, B. halodurans which is a novel pathway-holding bacterium is liquid-cultured in a medium to which only the candidate compound is added and the other is added with both the candidate compound and menaquinone. In this experiment, growth was inhibited by the addition of only the candidate compound, and the candidate compound whose inhibition was restored by the addition of menaquinone is subjected to the following secondary screening.
In the above screening, it can be confirmed that the candidate compound inhibits the novel menaquinone biosynthetic pathway, but it is unknown whether the target aminodeoxyphthalosin deadenine enzyme is inhibited. Therefore, the target substance can be screened by conducting an in vitro enzyme assay by the method shown in Example 4 and examining whether aminodeoxyphthalosin deadenine enzyme is actually inhibited.

The method for searching for anti-H. Pylori using the compound of the present invention makes it possible to select a drug having high specificity for H. pylori.

SEQ ID NO: 3 Description of artificial sequence-synthetic DNA
SEQ ID NO: 4 Description of artificial sequence-synthetic DNA

Claims (5)

A compound represented by the following formula (I):

  A compound represented by formula (I), aminodeoxyphthalosin deadenine enzyme, and a test substance are contacted in an aqueous medium, and the amount of adenine or dehypoxanthinylfutalosine (DHFL) produced in the aqueous medium is measured; A method for selecting a compound having anti-pylori activity, wherein a substance that suppresses the production of adenine or DHFL is selected from test substances. The method according to claim 2, wherein the aminodeoxyphthalosin deadenine enzyme is a protein derived from Helicobacter pylori . The method according to claim 2, wherein the aminodeoxyphthalosin deadenine enzyme is a protein according to any one of the following (1) to (3).
(1) a protein having the amino acid sequence represented by SEQ ID NO: 2 (2) consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2; A protein having aminodeoxyphthalosin deadenine enzyme activity (3) A protein having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2 and having aminodeoxyphthalosin deadenine enzyme activity The test substance inhibits the growth of a microorganism belonging to the genus Bacillus having a menaquinone biosynthetic pathway including the enzyme group of MqnA, MqnB, MqnC and MqnD, and the enzyme of MenF, MenD, MenC, MenE, MenB, MenA and MenG The method according to any one of claims 2 to 4, which is a substance that does not inhibit the growth of a microorganism belonging to the genus Bacillus having a menaquinone biosynthetic pathway including the group.

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