JP7092308B2 - Lipid A - Google Patents
Lipid A Download PDFInfo
- Publication number
- JP7092308B2 JP7092308B2 JP2019501132A JP2019501132A JP7092308B2 JP 7092308 B2 JP7092308 B2 JP 7092308B2 JP 2019501132 A JP2019501132 A JP 2019501132A JP 2019501132 A JP2019501132 A JP 2019501132A JP 7092308 B2 JP7092308 B2 JP 7092308B2
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- JP
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- Prior art keywords
- lipid
- lps
- derived
- chain
- fatty acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- GZQKNULLWNGMCW-PWQABINMSA-N lipid A (E. coli) Chemical compound O1[C@H](CO)[C@@H](OP(O)(O)=O)[C@H](OC(=O)C[C@@H](CCCCCCCCCCC)OC(=O)CCCCCCCCCCCCC)[C@@H](NC(=O)C[C@@H](CCCCCCCCCCC)OC(=O)CCCCCCCCCCC)[C@@H]1OC[C@@H]1[C@@H](O)[C@H](OC(=O)C[C@H](O)CCCCCCCCCCC)[C@@H](NC(=O)C[C@H](O)CCCCCCCCCCC)[C@@H](OP(O)(O)=O)O1 GZQKNULLWNGMCW-PWQABINMSA-N 0.000 title claims description 143
- 239000002158 endotoxin Substances 0.000 claims description 125
- 229920006008 lipopolysaccharide Polymers 0.000 claims description 123
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- 239000000203 mixture Substances 0.000 claims description 56
- 239000002671 adjuvant Substances 0.000 claims description 53
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 claims description 31
- 229960002442 glucosamine Drugs 0.000 claims description 31
- QFLWZFQWSBQYPS-AWRAUJHKSA-N (3S)-3-[[(2S)-2-[[(2S)-2-[5-[(3aS,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]-3-methylbutanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-4-[1-bis(4-chlorophenoxy)phosphorylbutylamino]-4-oxobutanoic acid Chemical compound CCCC(NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@@H](NC(=O)CCCCC1SC[C@@H]2NC(=O)N[C@H]12)C(C)C)P(=O)(Oc1ccc(Cl)cc1)Oc1ccc(Cl)cc1 QFLWZFQWSBQYPS-AWRAUJHKSA-N 0.000 claims description 20
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- 125000002252 acyl group Chemical group 0.000 description 31
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 30
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 24
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 11
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
- C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
- C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
- C07H13/06—Fatty acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
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- Saccharide Compounds (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Description
本発明は、新規構造からなるリピドＡ（lipid A）に関し、さらには当該リピドＡを含むリポ多糖（Lipopolysaccharide: LPS）に関する。そして、当該リピドＡを含むアジュバント組成物に関する。 The present invention relates to Lipid A having a novel structure, and further to Lipopolysaccharide (LPS) containing the Lipid A. Then, the present invention relates to an adjuvant composition containing the Lipid A.
本出願は、参照によりここに援用されるところの日本出願特願２０１７－３０１７９号優先権を請求する。 This application claims the priority of Japanese application Japanese Patent Application No. 2017-30179, which is incorporated herein by reference.
アジュバント（Adjuvant）はワクチンの効果を増強する因子の総称であり、抗原と共に投与されることで、その抗原の抗原性を増強する。アジュバントとしては、抗原が吸着する無機物の懸濁剤である沈降性アジュバントと、抗原水溶液を鉱油で包みミセルを形成し乳化する油乳剤である油性アジュバントなどに分類される。沈降性アジュバントの例としては水酸化ナトリウム、水酸化アルミニウム（Alum）、リン酸カルシウム、リン酸アルミニウム、ミョウバン、ペペス、カルボキシビニルポリマーなどが挙げられる。水酸化アルミニウム等のアルミニウム塩はB型肝炎ワクチン等に用いられ、誘導される獲得免疫は抗体やTh2型である。水酸化アルミニウムでは、液性免疫は誘導されるが、細胞性免疫の誘導が低いこと、発熱やアレルギー反応誘導（IgE誘導）などの副反応の問題も挙げられている。油性アジュバントの例としては、流動パラフィン、ラノリン、フロイント（Freund）などが挙げられる。フロイントアジュバントでは、パラフィンとアラセルの混合物である不完全フロイントアジュバント（Incomplete Freund's adjuvant: IFA）や、IFAに死滅したミコバクテリア又は結核菌の死菌を加え、抗原性をさらに増強させた完全フロイントアジュバント（Complete Freund's adjuvant: CFA）などが挙げられる。CFA中には熱殺菌マイコバクテリウム、LPS、ムラミルジペプチド、並びに百日咳毒素（Pertussis toxin: PT）、コレラ毒素（Cholera Toxin）及び大腸菌（E. Coli）腸毒素等の微生物産物が含まれ、免疫調節特性を有することが知られている。大腸菌由来LPSはアジュバント試薬として使用されているが、毒性が強いためヒトへの投与はできない。 An adjuvant is a general term for factors that enhance the effect of a vaccine, and when administered together with an antigen, it enhances the antigenicity of the antigen. The adjuvant is classified into a precipitating adjuvant which is a suspending agent for an inorganic substance to which an antigen is adsorbed, and an oil-based adjuvant which is an oil emulsion in which an aqueous antigen is wrapped in mineral oil to form micelles and emulsified. Examples of precipitating adjuvants include sodium hydroxide, aluminum hydroxide (Alum), calcium phosphate, aluminum phosphate, alum, pepes, carboxyvinyl polymers and the like. Aluminum salts such as aluminum hydroxide are used for hepatitis B vaccine and the like, and the induced acquired immunity is antibody or Th2 type. Although aluminum hydroxide induces humoral immunity, it also raises the problem of low induction of cell-mediated immunity and side reactions such as fever and allergic reaction induction (IgE induction). Examples of oil-based adjuvants include liquid paraffin, lanolin, Freund and the like. Freund's adjuvant includes Incomplete Freund's adjuvant (IFA), which is a mixture of paraffin and alasel, and complete Freund's adjuvant (IFA), which is further enhanced in antigenicity by adding killed mycobacteria or tubercle bacilli. Complete Freund's adjuvant: CFA) and so on. CFA contains heat-sterilized mycobacteria, LPS, muramildipeptides, and microbial products such as pertussis toxin (PT), cholera toxin, and E. coli enterotoxin, and is immune. It is known to have regulatory properties. Although E. coli-derived LPS is used as an adjuvant reagent, it cannot be administered to humans due to its high toxicity.
LPSは、グラム陰性菌の細胞壁を構成するリポ多糖（Lipopolysaccharide）であり、エンドトキシンともいわれる。LPSは主にO特異多糖、コア多糖及びリピドＡから構成されている。LPSの生理作用発現は、宿主細胞の細胞膜表面に存在するトール様受容体（Toll-like Receptor: TLR）4を介して行われる。TLRは自然免疫に関係し、病原微生物を認識する受容体であり、TLR4は感染防御やワクチン等における抗原の免疫原性の増強に重要な役割を担う自然免疫系を活性化する代表的な受容体の１つである。TLR4の活性化は免疫反応を引き起こすことから、TLR4を活性化させる作用を有する化合物等は、免疫賦活剤、免疫不全症の治療薬、癌細胞の増殖を抑制する医薬等となり得る。TLR4アゴニストとしてLPSやLPS断片等が知られている。 LPS is a lipopolysaccharide that constitutes the cell wall of Gram-negative bacteria, and is also called endotoxin. LPS is mainly composed of O-specific polysaccharides, core polysaccharides and Lipid A. The expression of the physiological action of LPS is mediated by Toll-like Receptor (TLR) 4 present on the cell membrane surface of the host cell. TLR is a receptor that is involved in innate immunity and recognizes pathogenic microorganisms, and TLR4 is a typical receptor that activates the innate immune system, which plays an important role in defense of infection and enhancement of the immunogenicity of antigens in vaccines and the like. It is one of the bodies. Since activation of TLR4 induces an immune reaction, compounds having an action of activating TLR4 can be immunostimulators, therapeutic agents for immunodeficiency, drugs for suppressing the growth of cancer cells, and the like. LPS and LPS fragments are known as TLR4 agonists.
リピドＡは糖鎖に脂質が結合した構造を有し、二糖鎖の1位及び4'位の親水基や2位、3位、2'位や3'位に結合する脂肪酸鎖の種類と位置に多様性がある。リピドＡはLPSの活性中心であり、きわめて多彩な生物活性を有する。 Lipid A has a structure in which lipids are bound to sugar chains, and includes hydrophilic groups at the 1st and 4'positions of disaccharide chains and types of fatty acid chains that bind to the 2nd, 3rd, 2'and 3'positions. There is variety in position. Lipid A is the active center of LPS and has an extremely diverse biological activity.
大腸菌やサルモネラ属菌（Salmonella spp.）のLPSに含まれるリピドＡの構造は既に解明されている。非特許文献１には、リピドＡ類似体を合成したことが報告されている。非特許文献２には、アルカリゲネス属（Alcaligenes）のリピドＡが、緑膿菌のリピドＡに似た構造的特徴を有することが報告されているが、その構造の詳細については明示されていない。腸管内の主要な常在菌であるバクテロイデス属（Bacteroides）、歯周病の原因であるポルフィロモナス属（Porphyromonas）等の菌が有するLPSの生物活性は、大腸菌やサルモネラ属菌が有するLPSの100分の1以下と報告されており、ピロリ菌LPSは発熱活性、致死活性が大腸菌由来LPSのそれぞれ1000分の1以下、500分の1以下であるといわれている。菌が有するLPSに含まれるリピドＡの構造は解明されていないものも多く、またその作用についても不明である。
The structure of Lipid A contained in LPS of Escherichia coli and Salmonella spp. Has already been elucidated. Non-Patent
先天性免疫を増強しワクチンの効力を増強するための、効力が高く安全な新しい免疫活性化能を有する組成物として、リポタンパク質を含むアジュバントについて報告がある。前記アジュバントに含まれるリポタンパク質が少なくとも１つの五量体単位を含み、前記リポタンパク質が重量／体積比で前記アジュバントの少なくとも10％を含むリポタンパク質であることを特徴とする。ここでは、免疫活性化組成物について、タンパク質、LPS、及びペプチド等を含むことが開示されている。LPSとして、例えばフレクスナー赤痢菌（Shigella flexneri）若しくはプレジオモナス・シゲロイデス（Plesiomonas shigelloides）等の第2のグラム陰性細菌、又は他のグラム陰性細菌の内因性のLPSであってもよいことが示され、グラム陰性細菌の一例として、アルカリゲネス属が例示されている（特許文献１、２）。しかしながら、アルカリゲネスは数多くの菌の一例として一行記載されているのみであり、アルカリゲネス由来LPSの構造が具体的に示されていない。
There is a report on an adjuvant containing lipoprotein as a highly effective and safe composition having a new immunostimulatory ability for enhancing innate immunity and enhancing the efficacy of a vaccine. It is characterized in that the lipoprotein contained in the adjuvant contains at least one pentamer unit, and the lipoprotein is a lipoprotein containing at least 10% of the adjuvant by weight / volume ratio. Here, it is disclosed that the immunostimulatory composition contains proteins, LPS, peptides and the like. It has been shown that the LPS may be a second Gram-negative bacterium, such as Shigella flexneri or Plesiomonas shigelloides, or an endogenous LPS of another Gram-negative bacterium. The genus Shigella is exemplified as an example of Gram-negative bacteria (
本発明は、新規構造からなるリピドＡを提供することを課題とする。より詳しくは、優れた免疫活性化能を維持したまま、アレルギー反応や炎症等の副作用が軽減化された、アジュバントに使用可能なリピドＡを提供することを課題とする。さらには、当該リピドＡを含むアジュバント組成物を提供することを課題とする。 An object of the present invention is to provide Lipid A having a novel structure. More specifically, it is an object of the present invention to provide Lipid A which can be used as an adjuvant and has reduced side effects such as allergic reaction and inflammation while maintaining excellent immunostimulatory ability. Further, it is an object to provide an adjuvant composition containing the Lipid A.
本発明者らは、アルカリゲネス由来LPSに含まれるリピドＡの構造を解析した結果、当該リピドＡを構成するグルコサミン二糖鎖の2位及び2'位に3-ヒドロキシ脂肪酸鎖が結合されており、さらに前記2位及び2'位に結合する3-ヒドロキシ脂肪酸鎖の少なくとも一方に3-ヒドロキシ脂肪酸鎖からなる二次脂肪酸鎖を含むことを初めて見出した。そして、上記課題を解決するために鋭意研究を重ねた結果、前記リピドＡを含むLPSをアジュバントとして使用した場合に、アレルギー反応や炎症等の副作用が低く、より効果的な免疫活性化作用を示すことが確認され、本発明を完成した。 As a result of analyzing the structure of lipid A contained in LPS derived from alkaligenes, the present inventors have found that a 3-hydroxy fatty acid chain is bonded to the 2- and 2'-positions of the glucosamine disaccharide chain constituting the lipid A. Furthermore, it was discovered for the first time that at least one of the 3-hydroxy fatty acid chains bound to the 2-position and the 2'-position contains a secondary fatty acid chain composed of a 3-hydroxy fatty acid chain. As a result of diligent research to solve the above problems, when LPS containing Lipid A is used as an adjuvant, side effects such as allergic reaction and inflammation are low, and more effective immunostimulatory action is exhibited. It was confirmed that the present invention was completed.
本発明は、すなわち以下よりなる。
１．グルコサミン二糖鎖と脂肪酸鎖の複合体からなるリピドＡであって、グルコサミン二糖鎖の2位及び2'位に3-ヒドロキシ脂肪酸鎖が結合しており、当該3-ヒドロキシ脂肪酸鎖の少なくとも一方に、3-ヒドロキシ脂肪酸鎖からなる二次脂肪酸鎖がさらに結合していることを特徴とするリピドＡ。
２．さらに、グルコサミン二糖鎖の3位及び3'位の少なくとも１つに3-ヒドロキシ脂肪酸鎖が結合していることを特徴とする、前項１に記載のリピドＡ。
３．脂肪酸鎖が、3-ヒドロキシミリスチン酸鎖、3-ヒドロキシラウリン酸鎖及びカプリン酸鎖から選択されるいずれかである、前項１又は２に記載のリピドＡ。
４．グルコサミン二糖鎖の2位及び2'位と、3位及び3'位の少なくとも１つに結合する脂肪酸鎖が3-ヒドロキシミリスチン酸である、前項１～３のいずれかに記載のリピドＡ。
５．グルコサミン二糖鎖の2位及び2'位に結合する少なくとも一方の二次脂肪酸鎖が3-ヒドロキシラウリン酸鎖である、前項１～４のいずれかに記載のリピドＡ。
６．グルコサミン二糖鎖と脂肪酸鎖の複合体からなるリピドＡであって、グルコサミン二糖鎖の2位、2'位、3位及び3'位に3-ヒドロキシミリスチン酸鎖が結合しており、当該グルコサミン二糖鎖の2位に結合する3-ヒドロキシミリスチン酸鎖にカプリン酸鎖からなる二次脂肪酸鎖が結合しており、当該グルコサミン二糖鎖の2'位に結合する3-ヒドロキシミリスチン酸鎖に3-ヒドロキシラウリン酸鎖からなる二次脂肪酸鎖が結合していることを特徴とするリピドＡ。
７．グルコサミン二糖鎖の4'位にリン酸基が結合してなる前項１～６のいずれかに記載のリピドＡ。
８．リピドＡが、以下の化１１に示す化合物１１である、前項１～７のいずれかに記載のリピドＡ。
１０．アルカリゲネス属由来のリポ多糖に含まれるリピドＡであって、グルコサミン二糖鎖の2位及び2'位に結合する２種の脂肪酸鎖が、二次脂肪酸鎖を含む脂肪酸鎖であることを特徴とするリピドＡ。
１１．リピドＡが、化学合成方法により作製されたリピドＡである前項１～８のいずれかに記載のリピドＡ。
１２．前項１～１１のいずれかに記載のリピドＡを含むリポ多糖。
１３．前項１～１１のいずれかに記載のリピドＡを含むアジュバント組成物及び／又は免疫賦活化剤。
１４．前項１３に記載のアジュバント組成物及び／又は免疫賦活化剤を含むワクチン組成物。
１５．前項１～１１のいずれかに記載のリピドＡの作製方法。That is, the present invention comprises the following.
1. 1. Lipid A, which is a complex of glucosamine disaccharide chain and fatty acid chain, has a 3-hydroxy fatty acid chain bonded to the 2nd and 2'positions of the glucosamine disaccharide chain, and at least one of the 3-hydroxy fatty acid chains. Lipid A is characterized by further binding to a secondary fatty acid chain consisting of a 3-hydroxy fatty acid chain.
2. 2. Further, the lipid A according to the
3. 3. The lipid A according to
4. The lipid A according to any one of the
5. The lipid A according to any one of the
6. Lipid A, which is a complex of a glucosamine disaccharide chain and a fatty acid chain, has a 3-hydroxymyristic acid chain bonded to the 2-, 2', 3-, and 3'-positions of the glucosamine disaccharide chain. A secondary fatty acid chain consisting of a capric acid chain is bound to the 3-hydroxymyristic acid chain that binds to the 2-position of the glucosamine disaccharide chain, and the 3-hydroxymyristic acid chain that binds to the 2'position of the glucosamine disaccharide chain. Lipid A, characterized in that a secondary fatty acid chain consisting of a 3-hydroxylauric acid chain is bonded to.
7. The lipid A according to any one of the
8. The lipid A according to any one of the
10. Lipid A contained in lipopolysaccharide derived from the genus Alkalinegenes is characterized in that the two fatty acid chains bound to the 2nd and 2'positions of the glucosamine disaccharide chain are fatty acid chains containing the secondary fatty acid chain. Lipid A to do.
11. The lipid A according to any one of the
12. The lipopolysaccharide containing Lipid A according to any one of the
13. The adjuvant composition and / or immunostimulatory agent containing Lipid A according to any one of the
14. A vaccine composition containing the adjuvant composition and / or immunostimulatory agent according to the
15. The method for producing Lipid A according to any one of the
本発明のリピドＡの構造は、新規な構造を有する。本発明のリピドＡを含むLPSをアジュバントとして使用した場合、アジュバントとして汎用される水酸化アルミニウムと比較して優れており、大腸菌由来LPSと同程度の免疫誘導能を示すことが確認された。一方、毒性が強い大腸菌由来LPSに比べ、本発明のリピドＡを含むLPSは、アレルギー反応や炎症等の副作用が低いことが確認された。これにより、本発明のリピドＡを含むLPSは、優れた免疫活性化能を維持したまま、アレルギー反応や炎症等の副作用が低い、安全性が高く有効なアジュバントとして使用することができる。 The structure of Lipid A of the present invention has a novel structure. It was confirmed that when LPS containing Lipid A of the present invention was used as an adjuvant, it was superior to aluminum hydroxide, which is widely used as an adjuvant, and exhibited immunostimulatory ability comparable to that of Escherichia coli-derived LPS. On the other hand, it was confirmed that the LPS containing Lipid A of the present invention has lower side effects such as allergic reaction and inflammation than the highly toxic E. coli-derived LPS. As a result, the LPS containing Lipid A of the present invention can be used as a highly safe and effective adjuvant with low side effects such as allergic reaction and inflammation while maintaining excellent immunostimulatory ability.
背景技術の欄にも示したように、LPSはグラム陰性菌の細胞壁外膜を構成する成分である。LPSは主にO特異多糖、コア多糖及びリピドＡから構成されている（図１参照）。本明細書において、「リピドＡ」とは糖鎖に脂質が結合した構造を有し、リン酸基が結合したグルコサミン二糖鎖と脂肪酸鎖の複合体からなる物質をいう。リピドＡの基本骨格における脂肪酸鎖の結合様式は、リピドＡの生物活性と密接な関係がある。リピドＡとして、現在は大腸菌に代表される６個のアシル基を含むグループ、クロモバクテリウムや緑膿菌に代表される６個のアシル基、５個のアシル基、及び４個のアシル基等を含むグループが知られている。これらは、いずれもグルコサミン二糖鎖を基本骨格とし、2位、3位、2'位及び3'位に3-ヒドロキシ脂肪酸鎖を結合している点で共通している。 As shown in the background technique section, LPS is a component that constitutes the outer membrane of the cell wall of Gram-negative bacteria. LPS is mainly composed of O-specific polysaccharides, core polysaccharides and Lipid A (see FIG. 1). As used herein, "lipid A" refers to a substance having a structure in which a lipid is bound to a sugar chain and consisting of a complex of a glucosamine disaccharide chain to which a phosphate group is bound and a fatty acid chain. The binding mode of fatty acid chains in the basic skeleton of Lipid A is closely related to the biological activity of Lipid A. Lipid A is currently a group containing 6 acyl groups represented by Escherichia coli, 6 acyl groups represented by Chromobacterium and Pseudomonas aeruginosa, 5 acyl groups, 4 acyl groups, etc. Groups including are known. All of these have a glucosamine disaccharide chain as a basic skeleton, and are common in that a 3-hydroxy fatty acid chain is bonded to the 2-position, 3-position, 2'-position and 3'-position.
本発明のリピドＡは、４～６個のアシル基を含み、好ましくは６個である。具体的にはグルコサミン二糖鎖の2位及び2'位には各々結合する２個の脂肪酸鎖を含み、3位及び3'位には各々結合する０～２個、好ましくは２個の脂肪酸鎖を含み、グルコサミン二糖鎖の2位及び2'位に結合する２個のヒドロキシ脂肪酸鎖の水酸基にさらに飽和脂肪酸がそれぞれ０～１個、好ましくは１個結合した二次脂肪酸鎖を含む脂肪酸鎖であることを特徴とする（図１参照）。本明細書において、一次脂肪酸鎖とは、グルコサミンと直接結合している脂肪酸鎖をいい、二次脂肪酸鎖とは当該一次脂肪酸鎖に結合している脂肪酸鎖をいう。二次脂肪酸鎖が結合している脂肪酸鎖は分枝状の脂肪酸鎖を示す。 Lipid A of the present invention contains 4 to 6 acyl groups, preferably 6 groups. Specifically, the 2nd and 2'positions of the glucosamine disaccharide chain contain two fatty acid chains that bind to each other, and the 3rd and 3'positions each contain 0 to 2 fatty acids, preferably 2 fatty acids that bind to each other. A fatty acid containing a secondary fatty acid chain containing a chain and further having 0 to 1 saturated fatty acid, preferably 1 saturated fatty acid, attached to the hydroxyl groups of the two hydroxy fatty acid chains bonded to the 2nd and 2'positions of the glucosamine disaccharide chain. It is characterized by being a chain (see FIG. 1). In the present specification, the primary fatty acid chain means a fatty acid chain directly bonded to glucosamine, and the secondary fatty acid chain means a fatty acid chain bonded to the primary fatty acid chain. The fatty acid chain to which the secondary fatty acid chain is bound indicates a branched fatty acid chain.
すなわち本発明のリピドＡは、グルコサミン二糖鎖と脂肪酸鎖の複合体からなるリピドＡであって、グルコサミン二糖鎖の2位及び2'位に3-ヒドロキシ脂肪酸鎖が結合しており、当該3-ヒドロキシ脂肪酸鎖の少なくとも一方に、3-ヒドロキシ脂肪酸鎖からなる二次脂肪酸鎖がさらに結合していることを特徴とする。さらに、グルコサミン二糖鎖の3位及び3'位の少なくとも一方に3-ヒドロキシ脂肪酸鎖が結合していることを特徴とする。 That is, Lipid A of the present invention is Lipid A composed of a complex of a glucosamine disaccharide chain and a fatty acid chain, and a 3-hydroxy fatty acid chain is bonded to the 2nd and 2'positions of the glucosamine disaccharide chain. It is characterized in that a secondary fatty acid chain composed of a 3-hydroxy fatty acid chain is further bonded to at least one of the 3-hydroxy fatty acid chains. Furthermore, it is characterized in that a 3-hydroxy fatty acid chain is bound to at least one of the 3-position and the 3'-position of the glucosamine disaccharide chain.
本発明のリピドＡを構成する一次脂肪酸鎖の炭素数は、各脂肪酸鎖ごとに１２～１６であり、好ましくは１４（ミリスチン酸）である。本発明のリピドＡを構成するグルコサミンの2位及び2'位に各々結合する２個の脂肪酸鎖のいずれか少なくとも一方に二次脂肪酸鎖が結合している。各二次脂肪酸鎖の炭素数は１０～１２であり、好ましくは１０（カプリン酸）であり、2'位に結合する２個の脂肪酸鎖における二次脂肪酸鎖の炭素数は１２（ラウリン酸）である。例えば、本発明のリピドＡを構成するグルコサミン二糖鎖の2位及び2'位と、3位及び3'位の少なくとも１つに結合する脂肪酸鎖は、3-ヒドロキシミリスチン酸であるのが好適である。グルコサミン二糖鎖の2位及び2'位に結合する少なくとも一方の二次脂肪酸鎖は、3-ヒドロキシラウリン酸鎖であるのが好適である。具体的には、本発明のリピドＡはグルコサミン二糖鎖と脂肪酸鎖の複合体からなり、グルコサミン二糖鎖の2位、2'位、3位及び3'位に3-ヒドロキシミリスチン酸鎖が結合しており、当該グルコサミン二糖鎖の2位に結合する3-ヒドロキシミリスチン酸鎖にカプリン酸鎖からなる二次脂肪酸鎖が結合しており、当該グルコサミン二糖鎖の2'位に結合する3-ヒドロキシミリスチン酸鎖に3-ヒドロキシラウリン酸鎖からなる二次脂肪酸鎖が結合しているのが最も好適である（図１参照）。 The number of carbon atoms of the primary fatty acid chains constituting Lipid A of the present invention is 12 to 16 for each fatty acid chain, preferably 14 (myristic acid). The secondary fatty acid chain is bound to at least one of the two fatty acid chains bound to the 2-position and the 2'-position of glucosamine constituting Lipid A of the present invention, respectively. The carbon number of each secondary fatty acid chain is 10 to 12, preferably 10 (capric acid), and the carbon number of the secondary fatty acid chain in the two fatty acid chains bonded to the 2'position is 12 (lauric acid). Is. For example, the fatty acid chain that binds to at least one of the 2-position and 2'-position and the 3-position and 3'-position of the glucosamine disaccharide chain constituting Lipid A of the present invention is preferably 3-hydroxymyristic acid. Is. It is preferable that at least one secondary fatty acid chain attached to the 2-position and the 2'-position of the glucosamine disaccharide chain is a 3-hydroxylauric acid chain. Specifically, Lipid A of the present invention is composed of a complex of a glucosamine disaccharide chain and a fatty acid chain, and 3-hydroxymyristic acid chains are present at the 2-position, 2'-position, 3-position and 3'-position of the glucosamine disaccharide chain. A secondary fatty acid chain consisting of a capric acid chain is bound to the 3-hydroxymyristinic acid chain that is bound and binds to the 2-position of the glucosamine disaccharide chain, and is bound to the 2'position of the glucosamine disaccharide chain. It is most preferable that a secondary fatty acid chain consisting of a 3-hydroxylauric acid chain is bonded to the 3-hydroxymyristic acid chain (see FIG. 1).
本発明のリピドＡ分子において、グルコサミン二糖鎖の還元端（4'）又は非還元端（1）にはリン酸基を含むことができる。特にグルコサミン二糖鎖の4'位にリン酸基が結合していることが好ましい。本発明のリピドＡに、O特異多糖やコア多糖を結合し、LPSを作製することもできる。ここで使用可能なO特異多糖やコア多糖は、LPSを構成する多糖であればよく特に限定されない。アルカリゲネス由来、例えば、アルカリゲネス属フェカーリス（Alcaligenes faecalis）由来のO特異多糖やコア多糖が好適である。具体的には、図１左側に示す糖鎖を例示することができる。O特異多糖やコア多糖は、グルコサミン二糖鎖の6'部位に結合可能である。本発明は、本発明のリピドＡとO特異多糖やコア多糖を含むLPSにも及ぶ。 In the lipid A molecule of the present invention, the reducing end (4') or the non-reducing end (1) of the glucosamine disaccharide chain can contain a phosphate group. In particular, it is preferable that the phosphate group is bonded to the 4'position of the glucosamine disaccharide chain. LPS can also be prepared by binding O-specific polysaccharide or core polysaccharide to Lipid A of the present invention. The O-specific polysaccharides and core polysaccharides that can be used here are not particularly limited as long as they are polysaccharides constituting LPS. O-specific polysaccharides and core polysaccharides derived from Alcaligenes, for example, Alcaligenes faecalis, are suitable. Specifically, the sugar chain shown on the left side of FIG. 1 can be exemplified. O-specific polysaccharides and core polysaccharides can bind to the 6'site of the glucosamine disaccharide chain. The present invention also extends to LPS containing the lipid A and O specific polysaccharides and core polysaccharides of the present invention.
本発明のリピドＡは、化学合成により作製することもできるし、アルカリゲネス由来のLPSを抽出し、さらにリピドＡを分離して作製することもできる。本発明のリピドＡは、アルカリゲネス由来のリピドＡに限定されず、化学合成により作製されたリピドＡであってもよい。 Lipid A of the present invention can be produced by chemical synthesis, or can be produced by extracting LPS derived from Alcaligenes and further separating Lipid A. Lipid A of the present invention is not limited to Lipid A derived from Alcaligenes, and may be Lipid A produced by chemical synthesis.
本発明のリピドＡを、アルカリゲネス由来のLPSを抽出し、さらにリピドＡを分離して作製する場合、菌体からのLPSの抽出は、自体公知の方法又は今後開発されるあらゆる方法により行うことができる。具体的には、以下の２種類の手法により乾燥菌体よりLPSを抽出可能である。
１）市販のLPS抽出キット（iNtRON Biotechnology Inc.）をアルカリゲネスの乾燥菌体に対して使用する。
２）アルカリゲネスの乾燥菌体を石油エーテルとクロロホルムとフェノールの混合溶媒や加熱されたフェノールと水の混合溶媒を用いてLPSを抽出した後、デオキシリボヌクレアーゼ、リボヌクレアーゼ、プロテイナーゼによって処理し、透析及び体積排除カラム（General Electric Company: Sephacryl S-400 HR）によって精製する。
抽出したアルカリゲネス由来LPSを、例えば酢酸水溶液で処理し、生じた沈殿物を水で洗浄することでアルカリゲネスのリピドＡを得ることが可能である。When Lipid A of the present invention is prepared by extracting LPS derived from Alcaligenes and further separating Lipid A, the extraction of LPS from the cells may be carried out by a method known per se or any method developed in the future. can. Specifically, LPS can be extracted from dried cells by the following two methods.
1) Use a commercially available LPS extraction kit (iNtRON Biotechnology Inc.) for dried cells of Alcaligenes.
2) LPS is extracted from dried cells of alkaline genesis using a mixed solvent of petroleum ether, chloroform and phenol, or a mixed solvent of heated phenol and water, and then treated with deoxyribonuclease, ribonuclease, and proteinase for dialysis and volume exclusion. Purify by column (General Electric Company: Sephacryl S-400 HR).
It is possible to obtain Lipid A of Alcaligenes by treating the extracted LPS derived from Alcaligenes with, for example, an aqueous acetic acid solution and washing the resulting precipitate with water.
本発明のリピドＡを、化学合成により作製する場合は、以下に示す方法によることができる。本発明のリピドＡは、グルコサミン塩酸塩を出発原料として化合物１を経ることで合成可能である（図１９参照）。この化合物１はそれぞれ異なる条件で切断可能な保護基で保護されているため、様々な種類の脂肪酸鎖及びリン酸基を持つリピドＡの合成に適用できる。
When Lipid A of the present invention is produced by chemical synthesis, it can be produced by the method shown below. Lipid A of the present invention can be synthesized by using glucosamine hydrochloride as a starting material and passing through compound 1 (see FIG. 19 ). Since this
本発明のリピドＡは、具体的には以下の方法で合成することができる（図１９参照）。
化合物１の3位にミリスチン酸誘導体２を2-メチル-6-ニトロ安息香酸無水物（MNBA）とN,N-ジメチル-4-アミノピリジン（DMAP）、N,N-ジイソプロピルエチルアミン（DIPEA）を用いて導入し、化合物３を得る。化合物３の2'位のトリクロロエトキシカルボニル（Troc）基を亜鉛-銅合金を用いて切断した後、遊離となったアミノ基に、二次脂肪酸鎖としてラウリン酸を有するミリスチン酸誘導体４を、1-[ビス（ジメチルアミノ）メチレン]-1H-1,2,3-トリアゾロ[4,5-b]ピリジニウム 3-オキシドヘキサフルオロホスフェート（HATU）とDMAPを用いることで導入し、化合物５を得る。化合物５の2位のアリルオキシカルボニル基（Alloc）をテトラキス（トリフェニルホスフィン）パラジウム（0）とN-（トリメチルシリル）ジメチルアミン（TMSDMA）を用いて切断する。続いて、遊離となったアミノ基に、二次脂肪酸鎖としてカプリン酸を有するミリスチン酸誘導体である化合物６を、HATUとDMAPを用いて導入し、化合物７を得る。化合物７の3'位のパラメトキシベンジル基（MPM）を、2,3-ジクロロ-5,6-ジシアノ-p-ベンゾキノン（DDQ）と2,6-ジ-tert-ブチルピリジンによって切断し、遊離となった水酸基にミリスチン酸誘導体２をMNBAとDMAP及びDIPEAを用いて導入し、化合物８を得る。化合物８の4'位及び6'位のベンジリデン基にトリフルオロ酢酸（TFA）を作用させて切断し、トリチルクロライドとピリジンを用いて6'位にトリチル基を導入し、化合物９を得る。化合物９の1位のアリル基を、（1,5）-（シクロオクタジエン）-ビス（メチルジフェニルホスフィン）イリジウム（1）ヘキサフルオロホスフェートを用いて異性化させた後、ヨウ素と水を作用させることで切断し、化合物１０を得る。化合物１０に対し、ジベンジル-N,N-ジイソプロピルホスホロアミダイト、テトラゾール、続いて、ジメチルジオキシラン（DMDO）を作用させることで1位と4'位にリン酸基を導入した。最終脱保護として、水酸化パラジウムを用いた接触水素化を行うことで、全てのベンジル基及びトリチル基を切断し、アルカリゲネス由来リピドＡと同じ構造からなる化合物１１を得ることができる。
Specifically, Lipid A of the present invention can be synthesized by the following method (see FIG. 19 ).
本発明のリピドＡは、例えばモノホスホリルリピドＡ（MPL）、リピドＡ誘導体（例えば低毒性リピドＡ誘導体）、3-O-脱アシル化MPL等の処理を行うことなく、アジュバント組成物又は免疫賦活化剤に適用することができる。本発明は、本発明のリピドＡを含むアジュバント組成物又は免疫賦活化剤にも及ぶ。 Lipid A of the present invention is an adjuvant composition or immunostimulatory composition without treatment with, for example, monophosphoryl lipid A (MPL), lipid A derivative (for example, low-toxicity lipid A derivative), 3-O-deacylated MPL, or the like. It can be applied to agents. The present invention also extends to an adjuvant composition or immunostimulatory agent containing Lipid A of the present invention.
前記アジュバント組成物は、主にTh1型及びTh2型の免疫応答を誘導する。一方、水酸化アルミニウムや抗原のみの場合は、Th2型の免疫応答のみを誘導する。ここで、Th2型の免疫応答は、アレルギーに関与するともいわれており、本発明のリピドＡを含むアジュバント組成物は、優れた免疫誘導能を保持しつつ、体重減少やアレルギー等の副作用が軽減化された優れた効果を有する。高レベルのTh1型サイトカイン（例えばIFN-γ、TNF-α、IL-2及びIL-12）は、投与される抗原に対する細胞介在免疫応答の誘導を促進する傾向にあると考えられるため、本発明のアジュバント組成物は、免疫賦活化剤としても使用することができる。 The adjuvant composition mainly induces Th1 type and Th2 type immune responses. On the other hand, when only aluminum hydroxide or antigen is used, only Th2 type immune response is induced. Here, the Th2-type immune response is also said to be involved in allergies, and the adjuvant composition containing Lipid A of the present invention reduces side effects such as weight loss and allergies while maintaining excellent immune-inducing ability. It has an excellent effect. High levels of Th1-type cytokines (eg IFN-γ, TNF-α, IL-2 and IL-12) are thought to tend to promote the induction of cell-mediated immune responses to the antigens administered, and thus the present invention. The adjuvant composition of can also be used as an immunostimulatory agent.
本発明のアジュバント組成物又は免疫賦活化剤は、免疫応答を誘導又は増強しうる医薬組成物とすることができる。医薬組成物の例として、例えば抗原と本発明のアジュバント組成物と薬学的に許容される担体とを含む、ワクチン組成物とすることができる。免疫賦活化剤には、特定の抗原を含まず、薬学的に許容される担体を含むことができる。上記薬学的に許容される担体としては、リン酸カルシウム、水中油型エマルジョン、油中水型エマルジョン、リポソーム、及び微粒子等が例示されるが、これらに限定されるものではない。これらの組成物は、宿主において免疫応答を誘導することができる。別の特定の実施形態では、この免疫応答は、前記抗原に特異的である。前記特定の実施形態のいずれかによれば、前記抗原は、宿主において、体液性応答及び細胞性応答から選択される免疫応答を誘導することができる。前記特定の実施形態のいずれかによれば、前記組成物は、宿主において、Th1型Ｔリンパ球応答、Th2型Ｔリンパ球応答、細胞傷害性Ｔリンパ球（CTL）応答、抗体応答、サイトカイン応答、リンホカイン応答、ケモカイン応答、ならびに炎症性応答から選択される少なくとも１種の免疫応答を誘導することができる。前記組成物は、宿主においてインターフェロン－γ（IFN-γ）、腫瘍壊死因子－α（TNF-α）、IL-1、IL-2、IL-3、IL-4、IL-6、IL-8、IL-10、IL-12、IL-13、IL-16、IL-18、IL-23、MIP-1α、MIP-1β、RANTES、CCL4及びCCL5から選択されるサイトカイン等を産生することができる。さらに記憶Ｔ細胞応答、記憶Ｂ細胞応答、エフェクターＴ細胞応答、細胞傷害性Ｔ細胞応答、エフェクターＢ細胞応答から選択されるリンパ球応答、から選択される少なくとも１種の免疫応答を誘導することができる。 The adjuvant composition or immunostimulatory agent of the present invention can be a pharmaceutical composition capable of inducing or enhancing an immune response. As an example of the pharmaceutical composition, it can be a vaccine composition containing, for example, an antigen, an adjuvant composition of the present invention, and a pharmaceutically acceptable carrier. The immunostimulatory agent does not contain a specific antigen and may contain a pharmaceutically acceptable carrier. Examples of the pharmaceutically acceptable carrier include, but are not limited to, calcium phosphate, oil-in-water emulsion, water-in-oil emulsion, liposomes, and fine particles. These compositions can induce an immune response in the host. In another particular embodiment, this immune response is specific for said antigen. According to any of the particular embodiments, the antigen is capable of inducing an immune response selected from humoral and cellular responses in a host. According to any of the particular embodiments, the composition comprises a Th1 type T lymphocyte response, a Th2 type T lymphocyte response, a cytotoxic T lymphocyte (CTL) response, an antibody response, a cytokine response in a host. , Lymphocyte response, chemokine response, and at least one immune response selected from inflammatory responses can be induced. The composition comprises interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), IL-1, IL-2, IL-3, IL-4, IL-6, IL-8 in the host. , IL-10, IL-12, IL-13, IL-16, IL-18, IL-23, MIP-1α, MIP-1β, RANTES, CCL4, CCL5 and the like. .. Furthermore, it is possible to induce at least one immune response selected from a memory T cell response, a memory B cell response, an effector T cell response, a cytotoxic T cell response, and a lymphocyte response selected from the effector B cell response. can.
本発明のワクチン組成物に含みうる抗原、又は免疫賦活化剤が応答しうる抗原としては特に限定されないが、例えば細菌、ウイルス及び真菌等の感染病原体やその断片が挙げられる。他の態様によれば、少なくとも１種のがん細胞に由来する抗原であってもよい。さらに他の実施形態によれば、自己免疫疾患に関連する少なくとも１種のエピトープ、生体分子、細胞又は組織に由来するか、あるいはこれらと免疫学的に交差反応性である物質であってもよい。自己免疫疾患に関連する抗原として、具体的には、前記自己免疫疾患が全身性エリテマトーデスの場合はsnRNPから選択され、前記自己免疫疾患がグレーブス病の場合はチログロブリン、チロトロピン受容体及び甲状腺上皮細胞のうちの少なくとも１種であり、前記自己免疫疾患が血小板減少性紫斑病の場合は血小板であり、前記自己免疫疾患が天疱瘡の場合は天疱瘡抗原、デスモグレイン-3、デスモプラキン、エンボプラキン及び水疱性類天疱瘡抗原のうちの少なくとも１種であり、前記自己免疫疾患が多発性硬化症の場合はミエリン塩基性タンパク質であり、前記自己免疫疾患が1型糖尿病の場合は膵島β細胞であり、そして前記自己免疫疾患が重症無力症の場合はアセチルコリン受容体等が挙げられる。
The antigen that can be contained in the vaccine composition of the present invention or the antigen that the immunostimulatory agent can respond to is not particularly limited, and examples thereof include infectious pathogens such as bacteria, viruses and fungi, and fragments thereof. According to another aspect, it may be an antigen derived from at least one type of cancer cell. According to still other embodiments, it may be a substance derived from or immunologically cross-reactive with at least one epitope, biomolecule, cell or tissue associated with an autoimmune disease. .. Specifically, the antigens associated with the autoimmune disease are selected from snRNP when the autoimmune disease is systemic erythematosus, and tyroglobulin, tyrotropin receptor and thyroid epithelial cells when the autoimmune disease is Graves disease. At least one of these, platelets if the autoimmune disease is thrombocytopenic purpura, and vesicular antigen, desmograin-3, desmoprakin, emboplakin and blisters if the autoimmune disease is vesicles. It is at least one of the sex blemishes antigens, a myelin basic protein when the autoimmune disease is multiple sclerosis, and pancreatic islet β cells when the autoimmune disease is
本発明の理解を助けるために、以下に実施例及び実験例を示して具体的に本発明を説明するが、本発明は本実施例等に限定されるものでないことはいうまでもない。 In order to help understanding of the present invention, the present invention will be specifically described with reference to Examples and Experimental Examples, but it goes without saying that the present invention is not limited to the present examples and the like.
（実施例１）アルカリゲネス由来LPSを構成するリピドＡの構造解析
本実施例では、アルカリゲネス属フェカーリス（Alcaligenes faecalis）のLPSを構成するリピドＡの構造解析を行った。前記アルカリゲネスの乾燥菌体を石油エーテル、クロロホルム及びフェノールの混合溶媒や加熱されたフェノールと水の混合溶媒を用いて抽出し、デオキシリボヌクレアーゼ、リボヌクレアーゼ、プロテイナーゼによって処理し、透析及び体積排除カラム（General Electric Company: Sephacryl S-400 HR）によってLPSを分離精製した。分離したLPSを、酢酸水溶液にて処理し、生じた沈殿物を水で洗浄することでリピドＡを得た。(Example 1) Structural analysis of Lipid A constituting LPS derived from Alcaligenes In this example, the structural analysis of Lipid A constituting LPS of Alcaligenes faecalis was performed. The dried cells of alkaligenes are extracted using a mixed solvent of petroleum ether, chloroform and phenol or a mixed solvent of heated phenol and water, treated with deoxyribonuclease, ribonuclease, and proteinase, and dialed and volume-removed column (General Electric). LPS was separated and purified by Company: Sephacryl S-400 HR). The separated LPS was treated with an aqueous acetic acid solution, and the resulting precipitate was washed with water to obtain Lipid A.
上記方法より得たリピドＡを、質量分析により構造解析した。質量分析による解析結果ａ～ｊを、図２～１２に示した。ａ（図２）に示すとおりアルカリゲネス由来リピドＡの分子イオンピークがm/z=1756, 1602, 1376に観察され、脂肪酸鎖部分に構造多様性を有することが示された。m/z=1756は６個の脂肪酸鎖、m/z=1602は５個の脂肪酸鎖、m/z=1376は４個の脂肪酸鎖から成る。さらに詳細な解析を行ったところ、ｂ１（図３）に示す解析では、m/z=552.3, 574.3, 588.3に２価の分子イオンが観測され、それぞれ脂肪酸鎖組成の異なる３個の脂肪酸鎖から成る。ａ～ｂ（図２～４）に示すようにアルカリゲネス由来リピドＡの脂肪酸鎖の数は３～６個であり、脂肪酸鎖の種類にも多様性を有することが考えられた。脂肪酸鎖としては、C14は3-ヒドロキシミリスチン酸、C12は3-ヒドロキシラウリン酸、C10はカプリン酸をそれぞれ示す。３～５個の脂肪酸鎖を有するリピドＡの骨格は、６個の脂肪酸鎖を持つリピドＡの部分構造であることが推測されたため、６個の脂肪酸鎖を持つアルカリゲネス由来リピドＡ（m/z=1756）を中心に更なる構造解析を進めた。 Lipid A obtained by the above method was structurally analyzed by mass spectrometry. The analysis results a to j by mass spectrometry are shown in FIGS. 2 to 12. As shown in a (Fig. 2), the molecular ion peak of lipid A derived from alkaligenes was observed at m / z = 1756, 1602, 1376, indicating that the fatty acid chain portion had structural diversity. m / z = 1756 consists of 6 fatty acid chains, m / z = 1602 consists of 5 fatty acid chains, and m / z = 1376 consists of 4 fatty acid chains. As a result of further detailed analysis, in the analysis shown in b1 (Fig. 3), divalent molecular ions were observed at m / z = 552.3, 574.3, 588.3, and from three fatty acid chains with different fatty acid chain compositions, respectively. Become. As shown in a to b (FIGS. 2 to 4), the number of fatty acid chains of Alcaligenes-derived Lipid A was 3 to 6, and it was considered that the types of fatty acid chains also had diversity. As the fatty acid chain, C14 indicates 3-hydroxymyristic acid, C12 indicates 3-hydroxylauric acid, and C10 indicates capric acid. Since the skeleton of Lipid A having 3 to 5 fatty acid chains was presumed to be a partial structure of Lipid A having 6 fatty acid chains, Alkaline Genes-derived Lipid A (m / z) having 6 fatty acid chains Further structural analysis was carried out centering on = 1756).
ｃ（図５）に示す様に、６個の脂肪酸鎖を持つリピドＡの1位のリン酸基が脱離したピークが観測された（m/z=1677）。ｄ（図６）は、前述のm/z=1677のピークをプリカーサーイオンとしてMS/MSを行った結果である。3-ヒドロキシラウリン酸が脱離し、m/z=1460のフラグメントイオンピークを与えた。この3-ヒドロキシラウリン酸は、容易に脱離したことから、二次脂肪酸鎖として結合していると考えられた。ｅ（図７）は、ｄ（図６）の結果からさらにm/z=1460をプリカーサーイオンとして、タンデム質量分析（MS/MS）を行った結果である。3-ヒドロキシミリスチン酸が脱離し、m/z=1216のフラグメントイオンピークを与えた。ｆ（図８）は、ｅ（図７）の結果からさらにm/z=1216をプリカーサーイオンとしてMS/MSを行った結果である。3-ヒドロキシミリスチン酸が脱離し、m/z=972のフラグメントイオンピークを与えた。ｇ（図９）は、ｆ（図８）の結果からさらにm/z=972をプリカーサーイオンとしてMS/MSを行った結果である。カプリン酸が脱離し、m/z=799のフラグメントイオンピークを与えた。 As shown in c (FIG. 5), a peak in which the phosphate group at the 1-position of Lipid A having 6 fatty acid chains was eliminated was observed (m / z = 1677). d (FIG. 6) is the result of performing MS / MS using the above-mentioned peak of m / z = 1677 as a precursor ion. 3-Hydroxylauric acid was desorbed, giving a fragment ion peak of m / z = 1460. Since this 3-hydroxylauric acid was easily desorbed, it was considered that it was bound as a secondary fatty acid chain. e (FIG. 7) is the result of tandem mass spectrometry (MS / MS) using m / z = 1460 as a precursor ion from the result of d (FIG. 6). 3-Hydroxymyristic acid was desorbed, giving a fragment ion peak of m / z = 1216. f (FIG. 8) is the result of performing MS / MS using m / z = 1216 as a precursor ion from the result of e (FIG. 7). 3-Hydroxymyristic acid was desorbed, giving a fragment ion peak of m / z = 972. g (FIG. 9) is the result of performing MS / MS using m / z = 972 as a precursor ion from the result of f (FIG. 8). Capric acid was desorbed, giving a fragment ion peak of m / z = 799.
以上の結果から、アルカリゲネス由来リピドＡを構成する脂肪酸鎖は3-ヒドロキシミリスチン酸が４個、3-ヒドロキシラウリン酸が１個、カプリン酸が１個であり、かつ2位及び2'位には二次脂肪酸鎖が結合した3-ヒドロキシミリスチン酸が結合していると考えられた。 From the above results, the fatty acid chains constituting the alkaligenes-derived lipid A are 4-hydroxymyristic acid, 1 3-hydroxylauric acid, 1 capric acid, and in the 2nd and 2'positions. It was considered that 3-hydroxymyristic acid to which the secondary fatty acid chain was bound was bound.
ｈ（図１０）とｉ（図１１）は、ａ（図２）のそれぞれm/z=1376と1602をプリカーサーイオンとしてMS/MSを測定した結果であり、どちらもm/z=710.4にグリコシド結合の切断によって生成した還元末端側フラグメントイオンピークを与えた。この結果から、2位に3-ヒドロキシミリスチン酸、3位に3-ヒドロキシミリスチン酸が結合し、かつ2'位に3-ヒドロキシラウリン酸を二次脂肪酸鎖として持つ3-ヒドロキシミリスチン酸、3'位に3-ヒドロキシミリスチン酸が結合していると考えられた。 h (FIG. 10) and i (FIG. 11) are the results of MS / MS measurement using m / z = 1376 and 1602 of a (FIG. 2) as precursor ions, respectively, and both are glycosides at m / z = 710.4. The reduced end-side fragment ion peak generated by the cleavage of the bond was given. From this result, 3-hydroxymyristic acid, 3', which has 3-hydroxymyristic acid at the 2-position, 3-hydroxymyristic acid at the 3-position, and 3-hydroxylauric acid at the 2'position as a secondary fatty acid chain. It was considered that 3-hydroxymyristic acid was bound to the position.
ｊ（図１２）はm/z=1676をプリカーサーイオンとしてMS/MSを行った結果であり、2位はカプリン酸を二次脂肪酸鎖として持つ3-ヒドロキシミリスチン酸が結合していると考えられた。以上の結果から、2位はカプリン酸を二次脂肪酸鎖として持つ3-ヒドロキシミリスチン酸、3位は3-ヒドロキシミリスチン酸、2'位は3-ヒドロキシラウリン酸を二次脂肪酸鎖として持つ3-ヒドロキシミリスチン酸、3'位は3-ヒドロキシミリスチン酸がそれぞれ結合した構造を有していると考えられた。 j (Fig. 12) is the result of MS / MS with m / z = 1676 as a precursor ion, and it is considered that 3-hydroxymyristic acid having capric acid as a secondary fatty acid chain is bound at the 2-position. rice field. From the above results, the 2-position has 3-hydroxymyristic acid having capric acid as a secondary fatty acid chain, the 3-position has 3-hydroxymyristic acid, and the 2'position has 3-hydroxylauric acid as a secondary fatty acid chain. It was considered that hydroxymyristic acid and 3-hydroxymyristic acid were bound to each other at the 3'position.
さらにNMRによる詳細な構造決定を行い、アルカリゲネス由来LPSの構造を決定した。 Further, detailed structure determination was performed by NMR, and the structure of Alcaligenes-derived LPS was determined.
（実施例２）アルカリゲネス由来LPSの作用
本実施例では、アルカリゲネス属フェカーリス（Alcaligenes faecalis）からLPSを抽出し、各種作用を確認した。本実施例では、市販のLPS抽出キット（iNtRON Biotechnology Inc.）を用いてLPSを抽出し、LPS試料とした。比較例として使用する大腸菌由来のLPSは大腸菌O111由来LPSを用いた。(Example 2) Action of LPS derived from Alcaligenes In this example, LPS was extracted from Alcaligenes faecalis, and various actions were confirmed. In this example, LPS was extracted using a commercially available LPS extraction kit (iNtRON Biotechnology Inc.) and used as an LPS sample. As the LPS derived from Escherichia coli used as a comparative example, LPS derived from Escherichia coli O111 was used.
１．アルカリゲネス由来LPSの免疫活性１
アルカリゲネス由来LPSのTLR4アゴニスト活性を、TLR4ノックアウト（KO）マウスの骨髄由来樹状細胞を用いて確認した。また、TLR2 KOマウスについても確認した。
野生型マウスとして、１０週齢の雌のBalb/c（ニホンクレア）を用いた。TLR2 KOマウス又はTLR4 KOマウスとして、１０週齢の雌のC57/BL6（オリエンタルバイオ）を用いた。1. 1. Immune activity of LPS derived from
The TLR4 agonist activity of Alcaligenes-derived LPS was confirmed using bone marrow-derived dendritic cells of TLR4 knockout (KO) mice. We also confirmed TLR2 KO mice.
As a wild-type mouse, 10-week-old female Balb / c (Japanese Claire) was used. As TLR2 KO mice or TLR4 KO mice, 10-week-old female C57 / BL6 (Oriental Bio) was used.
各マウスの骨髄から細胞を回収し、赤血球を溶血した後、GM-CSF入りのRPMI1640培地で６日間培養し、骨髄由来樹状細胞に分化、誘導した。樹状細胞はCD11c抗体が結合したMACSビーズで単離精製し、実験に供した。1×105個の樹状細胞を含む培養液に、各濃度のLPSを加えて３７℃で４８時間培養し、その後培養上清を回収した。培養液として、10%のウシ胎児血清（FCS）入りのRPMI1640培地を用いた。Mockとして、リン酸緩衝生理食塩水（PBS）を用いた。培養液中のIL-6量を、R&D Systems社の ELISAキットで測定した。Cells were collected from the bone marrow of each mouse, erythrocytes were hemolyzed, and then cultured in RPMI1640 medium containing GM-CSF for 6 days to differentiate and induce bone marrow-derived dendritic cells. Dendritic cells were isolated and purified from MACS beads bound to the CD11c antibody and used in the experiment. LPS of each concentration was added to the culture medium containing 1 × 10 5 dendritic cells, and the cells were cultured at 37 ° C. for 48 hours, and then the culture supernatant was collected. RPMI1640 medium containing 10% fetal bovine serum (FCS) was used as the culture medium. Phosphate buffered saline (PBS) was used as the Mock. The amount of IL-6 in the culture medium was measured with an ELISA kit from R & D Systems.
上記の結果、野生型マウス樹状細胞に対し、アルカリゲネス由来LPSは大腸菌由来LPSと同様にLPSの濃度依存的にTLR4に作用し、IL-6の産生を認めた。TLR4 KOマウス由来樹状細胞に各LPSで処理しても殆どIL-6の産生を認めなかった（図１３）。一方、TLR2 KOマウス由来樹状細胞に各LPSを加えた場合は、添加するLPSの濃度依存的にIL-6の産生を認めた（図１３）。このことから、アルカリゲネス由来LPSは大腸菌由来LPSと同様にTLR4に作用し、TLR4アゴニストとして機能することが確認された。 As a result of the above, on wild-type mouse dendritic cells, alkaline Genes-derived LPS acted on TLR4 in a concentration-dependent manner, similar to Escherichia coli-derived LPS, and IL-6 production was observed. Even when TLR4 KO mouse-derived dendritic cells were treated with each LPS, almost no IL-6 production was observed (Fig. 13). On the other hand, when each LPS was added to TLR2 KO mouse-derived dendritic cells, IL-6 production was observed depending on the concentration of the added LPS (Fig. 13). From this, it was confirmed that Alcaligenes-derived LPS acts on TLR4 in the same manner as Escherichia coli-derived LPS and functions as a TLR4 agonist.
２．アルカリゲネス由来LPSの免疫活性２
大腸菌由来LPSによる樹状細胞でのIL-6の産生に対して、アルカリゲネス由来LPSが及ぼす影響を確認した。上記１の野生型マウスの樹状細胞に各濃度のアルカリゲネス由来LPSを加え、さらに100pg/mLの大腸菌由来LPSを加えて３７℃で４８時間培養し、IL-6の産生を上記１と同手法により測定した。その結果、アルカリゲネス由来LPSは大腸菌由来LPSによるIL-6の産生を阻害せず、大腸菌由来LPSに対するアンタゴニスト作用がないことが確認された（図１４）。2. 2. Immune activity of LPS derived from
We confirmed the effect of Alcaligenes-derived LPS on the production of IL-6 in dendritic cells by E. coli-derived LPS. LPS derived from Alkaline Genesis at each concentration was added to the dendritic cells of the wild-type mouse described in 1 above, and LPS derived from Escherichia coli at 100 pg / mL was further added and cultured at 37 ° C. for 48 hours to produce IL-6 by the same method as in 1 above. Measured by. As a result, it was confirmed that Alcaligenes-derived LPS did not inhibit the production of IL-6 by E. coli-derived LPS and had no antagonistic effect on E. coli-derived LPS (Fig. 14).
３．アルカリゲネス由来LPSのアジュバント活性
1μgの卵白アルブミン（Ovalbumin：Sigma, A5503、以下単に「OVA」ともいう）を抗原とし、アジュバントとしてアルカリゲネス又は大腸菌由来のLPS（100μg）、あるいは200μLのアラムアジュバント（Imject Alum Adjuvant：Thermo Fisher Scientific Inc, 77161、以下単に「Alum」ともいう）を８週齢（雌）のBalb/cマウス（ニホンクレア）に各々投与したときのOVA特異抗体産生能を調べた。回収した血清中のOVA特異的抗体を、ELISA法にて測定した。3. 3. Alcaligenes-derived LPS adjuvant activity
1 μg of egg white albumin (Ovalbumin: Sigma, A5503, hereinafter simply referred to as “OVA”) is used as an antigen, and as an adjuvant, Alkalinegenes or LPS derived from Escherichia coli (100 μg) or 200 μL of Alum Adjuvant (Thermo Fisher Scientific Inc,) The OVA-specific antibody-producing ability when 77161 (hereinafter, also simply referred to as “Alum”) was administered to an 8-week-old (female) Balb / c mouse (Japanese Claire) was investigated. The OVA-specific antibody in the collected serum was measured by the ELISA method.
上記の結果、抗体産生能は大腸菌由来LPSをアジュバントとしたときに最も高く、アルカリゲネス由来LPSのアジュバント活性はAlumと同程度であった。産生するIgGサブタイプについても確認した。IgG1産生能は大腸菌由来LPSが最も高く、アルカリゲネス由来LPSはAlumよりも高い値であった。一方、IgG2aについてはAlumでも抗原のみでも殆ど産生されなかったが、大腸菌由来LPSの場合が最も高く、アルカリゲネス由来LPSの場合でも産生が認められた。これにより、アルカリゲネス由来LPSは、アジュバントとして抗原と共に使用したときに、IgG1及びIgG2aについて産生を認め、Th1型及びTh2型応答を誘導することが確認された（図１５）。 As a result of the above, the antibody-producing ability was highest when E. coli-derived LPS was used as an adjuvant, and the adjuvant activity of Alcaligenes-derived LPS was similar to that of Alum. The IgG subtype produced was also confirmed. E. coli-derived LPS had the highest IgG1 production capacity, and Alcaligenes-derived LPS had a higher value than Alum. On the other hand, IgG2a was hardly produced by either Alum or the antigen alone, but it was the highest in the case of E. coli-derived LPS, and the production was also observed in the case of Alcaligenes-derived LPS. From this, it was confirmed that Alcaligenes-derived LPS, when used together with an antigen as an adjuvant, showed production of IgG1 and IgG2a and induced Th1 type and Th2 type responses (FIG. 15).
４．副作用の確認１
10μgのOVAと、100μgのアルカリゲネス若しくは大腸菌由来のLPS、又は200μLのAlum（Thermo Fisher Scientific Inc）を８週齢（雌）のBalb/cマウス（ニホンクレア）の背部皮下に投与し、各マウスの体重の変化及びIgE産生量を確認した。体重は経時的に測定し、７日後に血清を回収し、ELISA法にてOVA特異的IgE抗体を、DSマウスIgE ELISA（OVA）キット（DSファーマメディカル株式会社）を用いて測定した。4. Confirmation of
10 μg of OVA and 100 μg of Alcaligenes or E. coli-derived LPS, or 200 μL of Alum (Thermo Fisher Scientific Inc) were administered subcutaneously to the back of 8-week-old (female) Balb / c mice (Japanese Claire). Changes in body weight and IgE production were confirmed. The body weight was measured over time, and after 7 days, the serum was collected, and the OVA-specific IgE antibody was measured by the ELISA method using the DS mouse IgE ELISA (OVA) kit (DS Pharma Medical Co., Ltd.).
上記の結果、大腸菌由来LPSを投与した場合に、投与２日目で体重が大幅に減少した。一方、アルカリゲネス由来LPSを投与した場合の体重変化はPBSや抗原単独投与の場合と殆ど差を認めなかった（図１６Ａ）。IgEの誘導量はAlumが最も高く、アジュバントとして使用した場合に抗原特異的なアレルギー症状を示すおそれが高かった。一方、アルカリゲネス由来LPSのアジュバントは、IgE誘導量が最も低かった（図１６Ｂ）。これらの結果より、アルカリゲネス由来LPSのアジュバントは、大腸菌由来LPSやAlumに比べて副作用が少なく安全性が高いことが確認された。 As a result of the above, when E. coli-derived LPS was administered, the body weight was significantly reduced on the second day of administration. On the other hand, the change in body weight when LPS derived from Alcaligenes was administered was almost the same as that when PBS or antigen alone was administered (FIG. 16A). The amount of IgE induced was highest in Alum, and there was a high risk of exhibiting antigen-specific allergic symptoms when used as an adjuvant. On the other hand, the adjuvant of LPS derived from Alcaligenes had the lowest amount of IgE induced (Fig. 16B). From these results, it was confirmed that the adjuvant of Alcaligenes-derived LPS has fewer side effects and higher safety than E. coli-derived LPS and Alum.
５．副作用の確認２
１０週齢（雌）のBalb/cマウス（ニホンクレア）に、1.5 mgの大腸菌由来又はアルカリゲネス由来のLPSを腹腔内投与したときの直腸体温を直腸温度計で毎時間測定した。さらに、各LPS投与した７時間後に肺組織を摘出し、HE染色にて肺組織に及ぼす影響を確認した。PBSで処理したものをMockとした。5. Confirmation of
The rectal body temperature of 10-week-old (female) Balb / c mice (Japanese Claire) after intraperitoneal administration of 1.5 mg of LPS derived from Escherichia coli or alkaligenes was measured every hour with a rectal thermometer. Furthermore, the lung tissue was removed 7 hours after each LPS administration, and the effect on the lung tissue was confirmed by HE staining. Mock was treated with PBS.
上記の結果、大腸菌由来LPSを投与した場合に体温の低下が最も激しかったのに対し、アルカリゲネス由来LPSを投与した場合には、やや低下するにとどまった（図１７）。肺組織では、大腸菌由来LPSを投与した場合に炎症所見が確認されたのに対し、アルカリゲネス由来LPSを投与した場合にはMockの場合と殆ど差を認めなかった（図１８）。これらの結果より、アルカリゲネス由来LPSは、大腸菌由来LPSに比べて副作用が少なく安全性が高いことが確認された。 As a result of the above, the decrease in body temperature was the most severe when E. coli-derived LPS was administered, whereas it was only slightly decreased when Alcaligenes-derived LPS was administered (Fig. 17). In the lung tissue, inflammatory findings were confirmed when E. coli-derived LPS was administered, whereas there was almost no difference from Mock when Alcaligenes-derived LPS was administered (Fig. 18). From these results, it was confirmed that Alcaligenes-derived LPS has fewer side effects and higher safety than Escherichia coli-derived LPS.
（実施例３）本発明のリピドＡの合成
本実施例では、本発明のリピドＡの合成方法について説明する。グルコサミン塩酸塩を出発原料として化合物１を経ることで本発明のリピドＡを合成した（図１９参照）。(Example 3) Synthesis of Lipid A of the present invention In this example, a method for synthesizing Lipid A of the present invention will be described. Lipid A of the present invention was synthesized by passing through
本発明のリピドＡの合成法は以下の通りである。化合物１の3位にミリスチン酸誘導体２をMNBAとDMAP、DIPEAを用いて導入し、化合物３を得た。化合物３の2'位のTroc基を亜鉛-銅合金を用いて切断した後、遊離となったアミノ基に、二次脂肪酸鎖としてラウリン酸を有するミリスチン酸誘導体４をHATUとDMAPを用いることで導入し、化合物５を得た。化合物５の2位のAllocをテトラキス（トリフェニルホスフィン）パラジウム（0）とTMSDMAを用いて切断した。続いて遊離となったアミノ基に、二次脂肪酸鎖としてカプリン酸を有するミリスチン酸誘導体６をHATUとDMAPを用いて導入し、化合物７を得た。化合物７の3'位のMPMを、DDQと2,6-ジ-tert-ブチルピリジンによって切断し、遊離となった水酸基にミリスチン酸誘導体２をMNBAとDMAP及びDIPEAを用いて導入し、化合物８を得た。化合物８の4'位及び6'位のベンジリデン基をTFAを作用させることで切断し、トリチルクロライドとピリジンを用いて6'位にトリチル基を導入し、化合物９を得た。化合物９の1位のアリル基を、（1,5）-（シクロオクタジエン）-ビス（メチルジフェニルホスフィン）イリジウム（1）ヘキサフルオロホスフェートを用いて異性化させた後、ヨウ素と水を作用させることで切断し、化合物１０を得た。化合物１０に対し、ジベンジル-N,N-ジイソプロピルホスホロアミダイト、テトラゾール、続いて、DMDOを作用させることで1位と4'位にリン酸基を導入した。最終脱保護として、水酸化パラジウムを用いた接触水素化を行うことで、全てのベンジル基及びトリチル基を切断し、アルカリゲネス属リピドＡと同じ構造からなる化合物１１を得た。
The method for synthesizing Lipid A of the present invention is as follows.
NMRによる構造解析結果
1H NMRスペクトル測定はJEOL ECA 500 MHz NMR spectrometerを用いて、指定した溶媒中、３０℃で行った。化学シフトはテトラメチルシラン（0 ppm）を基準物質としてδ値で表した。質量分析はmicrOTOF-QIII/compact（BRUKER）又はOrbitrap XL（登録商標）（Thermo Fisher Scientific）をそれぞれ用いて測定した。薄層クロマトフラフィーはKieselgel 60F254 Plates（Merck, 0.25 mm thickness）を用いた。中圧シリカゲルカラムクロマトグラフィーは、指定した溶媒系でKieselgel 60（Merck, 0.040-0.063 mm）を用い、0.1-0.3 MPaにて行った。ゲル浸透クロマトグラフィーは指定した溶媒系でSephadex LH-20 を用い、大気圧下にて行った。特に言及しない限り、非水中の反応はアルゴン雰囲気下で行った。Structural analysis results by NMR
1 H NMR spectrum measurement was performed using a
既知の化合物１（500 mg, 532μmol）と(R)-3-（ベンジルオキシ）ミリスチン酸２（267 mg, 799 μmol）をジクロロメタン（10 mL）とN,N-ジメチルホルムアミド（10 mL） の混合溶媒に溶解し、DIPEA（139 μL, 799 μmol）、MNBA（367.0 mg, 1.07 mmol）とDMAP（6.59 mg, 53.9μmol）を加え室温で１５時間撹拌した。反応溶液をクロロホルムで希釈した後、10%クエン酸水溶液を加えて撹拌し、クロロホルムで抽出した。有機層を10%クエン酸水溶液、飽和炭酸水素ナトリウム水溶液と飽和塩化ナトリウム水溶液で順次洗浄し、無水硫酸ナトリウムで乾燥した。乾燥剤を瀘去して減圧濃縮し、得られた残渣を中圧シリカゲルカラムクロマトグラフィー（トルエン／酢酸エチル = 97 : 3）で精製し、上記式で表される化合物３を白色固体（662 mg, 収率98%）として得た。
Mixing known compound 1 (500 mg, 532 μmol) and (R) -3- (benzyloxy) myristic acid 2 (267 mg, 799 μmol) with dichloromethane (10 mL) and N, N-dimethylformamide (10 mL) It was dissolved in a solvent, DIPEA (139 μL, 799 μmol), MNBA (367.0 mg, 1.07 mmol) and DMAP (6.59 mg, 53.9 μmol) were added, and the mixture was stirred at room temperature for 15 hours. After diluting the reaction solution with chloroform, a 10% aqueous citric acid solution was added, the mixture was stirred, and the mixture was extracted with chloroform. The organic layer was washed successively with 10% aqueous citric acid solution, saturated aqueous sodium hydrogen carbonate solution and saturated aqueous sodium chloride solution, and dried over anhydrous sodium sulfate. The desiccant was removed and concentrated under reduced pressure, and the obtained residue was purified by medium pressure silica gel column chromatography (toluene / ethyl acetate = 97: 3) to obtain
1H-NMR (CDCl3) δ: 7.51 (dd, J = 8.0, 2.0 Hz, 2H, C6H5-CH), 7.40-7.19 (m, 15H, C6H5-CH, C6H5-CH2, CH3O-C6H4-CH2), 6.85 (d, J = 8.5 Hz, 2H, CH3O-C6H4-), 5.85-5.79 (m, 2H, OCH2-CH=CH2 of Alloc and allyl groups), 5.57 (s, 1H, C6H5-CH), 5.35 (dd, J = 9.5, 11.0 Hz, 1H, H-3), 5.23 (ddd, J = 1.0, 10.0, 34.0 Hz, 2H, OCH2-CH=CH2 of Alloc group), 5.22 (d, J = 17.0, 33.5 Hz, 2H, OCH2-CH=CH2 of Allyl group), 5.02 (d, J = 14.0 Hz, 1H, 2-NH), 4.94 (br s, 1H, 2'-NH), 4.87 (d, J = 3.5 Hz, 1H, H-1), 4.81 (d, J = 11.0 Hz, 1H, CH3O-C6H4-CH2-), 4.65-4.43 (m, 10H, H-1' CH3O-C6H4-CH2-, OCH2-CH=CH2 of Alloc group, -COO-CH2-CCl3, CH2-C6H5), 4.33 (dd, J = 11.5, 5.0 Hz, 1H, H-3'), 4.15 (dd, J = 5.0, 12.0, Hz, 1H, OCH2-CH=CH2 of allyl group), 4.05 (d, J = 9.0 Hz, 1H, H-6a), 3.96-3.92 (m, 3H, H-4', H-2, OCH2-CH=CH2 of allyl group), 3.88-3.77 (m, 3H, β-CH of acyl chain, H-5, H-6'a), 3.79 (s, 3H, CH3O-C6H4-) 3.77-3.63 (m, 3H, H-6'b, H-6b, H-4), 3.45-3.33 (m, 2H, H-2', H-5'), 2.50 (ddd, J = 81.0, 15.5, 6.5 Hz, 2H, α-CH2 of acyl chain), 1.64-1.49 (m, 2H, γ-CH2 of acyl chain), 1.40-1.20 (m, 18H, acyl chain), 0.88 (t, J = 7.0 Hz, 3H, CH2-CH3 of acyl chain).
MS (ESI-LIT-orbitrap MS, positive) Calcd. for C65H83Cl3N2O16 [M+Na]+ : 1275.4700, Found 1275.4723 1 H-NMR (CDCl 3 ) δ: 7.51 (dd, J = 8.0, 2.0 Hz, 2H, C 6 H 5 -CH), 7.40-7.19 (m, 15H, C 6 H 5 -CH, C 6 H 5 -CH 2 , CH 3 OC 6 H 4 -CH 2 ), 6.85 (d, J = 8.5 Hz, 2H, CH 3 OC 6 H 4- ), 5.85-5.79 (m, 2H, OCH 2 -CH = CH 2 of Alloc and allyl groups), 5.57 (s, 1H, C 6 H 5 -CH), 5.35 (dd, J = 9.5, 11.0 Hz, 1H, H-3), 5.23 (ddd, J = 1.0, 10.0, 34.0 Hz, 2H, OCH 2 -CH = CH 2 of Alloc group), 5.22 (d, J = 17.0, 33.5 Hz, 2H, OCH 2 -CH = CH 2 of Allyl group), 5.02 (d, J = 14.0 Hz, 1H, 2-NH), 4.94 (br s, 1H, 2'-NH), 4.87 (d, J = 3.5 Hz, 1H, H-1), 4.81 (d, J = 11.0 Hz, 1H, CH 3 OC 6 H 4 -CH 2- ), 4.65-4.43 (m, 10H, H-1'CH 3 OC 6 H 4 -CH 2- , OCH 2 -CH = CH 2 of Alloc group, -COO-CH 2 -CCl 3 , CH 2 -C 6 H 5 ), 4.33 (dd, J = 11.5, 5.0 Hz, 1H, H-3'), 4.15 (dd, J = 5.0, 12.0, Hz, 1H, OCH 2 -CH = CH 2 of allyl group), 4.05 (d, J = 9.0 Hz, 1H, H-6a), 3.96-3.92 (m, 3H, H-4', H-2, OCH 2 -CH = CH 2 of allyl group) , 3.88-3.77 (m, 3H, β-CH of acyl chain, H-5, H-6'a), 3.79 (s, 3H, CH 3 OC 6 H 4- ) 3.77-3.63 (m, 3H, H -6'b, H-6b , H-4), 3.45-3.33 (m, 2H, H-2', H-5'), 2.50 (ddd, J = 81.0, 15.5, 6.5 Hz, 2H, α-CH 2 of acyl chain), 1.64 -1.49 (m, 2H, γ-CH 2 of acyl chain), 1.40-1.20 (m, 18H, acyl chain), 0.88 (t, J = 7.0 Hz, 3H, CH 2 -CH3 of acyl chain).
MS (ESI-LIT-orbitrap MS, positive) Calcd. For C 65 H 83 Cl 3 N 2 O 16 [M + Na] + : 1275.4700, Found 1275.4723
亜鉛粉末（1.5 g）を水に懸濁させ、１時間超音波処理を行った後、硫酸銅水溶液を加え、濾取することで亜鉛/銅を得た。化合物３（200 mg, 159μmol）を1,4-ジオキサン（10 mL）と酢酸（10 mL）の混合溶媒に溶解し、亜鉛／銅を加え室温で７時間撹拌した。亜鉛／銅をメンブレンフィルターにより瀘去し、トルエンを加えて減圧濃縮した。得られた残渣をクロロホルムに溶解し、飽和炭酸水素ナトリウム水溶液と飽和塩化ナトリウム水溶液で順次洗浄し、無水硫酸ナトリウムで乾燥した。乾燥剤を瀘去して減圧濃縮した。得られた残渣と(R)-3-[[(R)-3-(ベンジルオキシ)ラウロイル]オキシ]ミリスチン酸４（93.3 mg, 175μmol）をクロロホルム（10 mL）とN,N-ジメチルホルムアミド（10 mL）の混合溶媒に溶解し、HATU（90.9 mg, 239μmol）とDMAP（1.95 mg, 15.9μmol）を加え、室温で１９時間撹拌した。反応溶液をクロロホルムで希釈した後、10%クエン酸水溶液を加えて撹拌し、有機層を10%クエン酸水溶液、飽和炭酸水素ナトリウム水溶液と飽和塩化ナトリウム水溶液で順次洗浄し、無水硫酸ナトリウムで乾燥した。乾燥剤を瀘去して減圧濃縮し、得られた残渣を中圧シリカゲルカラムクロマトグラフィー（クロロホルム）で精製し、上記式で表される化合物５を白色固体（223 mg, 収率88%）として得た。
Zinc powder (1.5 g) was suspended in water, ultrasonically treated for 1 hour, an aqueous solution of copper sulfate was added, and the mixture was collected by filtration to obtain zinc / copper. Compound 3 (200 mg, 159 μmol) was dissolved in a mixed solvent of 1,4-dioxane (10 mL) and acetic acid (10 mL), zinc / copper was added, and the mixture was stirred at room temperature for 7 hours. Zinc / copper was removed by a membrane filter, toluene was added, and the mixture was concentrated under reduced pressure. The obtained residue was dissolved in chloroform, washed successively with saturated aqueous sodium hydrogen carbonate solution and saturated aqueous sodium chloride solution, and dried over anhydrous sodium sulfate. The desiccant was removed and concentrated under reduced pressure. The obtained residue and (R) -3-[[(R) -3- (benzyloxy) lauroyl] oxy] myristic acid 4 (93.3 mg, 175 μmol) were added to chloroform (10 mL) and N, N-dimethylformamide ( It was dissolved in a mixed solvent of 10 mL), HATU (90.9 mg, 239 μmol) and DMAP (1.95 mg, 15.9 μmol) were added, and the mixture was stirred at room temperature for 19 hours. After diluting the reaction solution with chloroform, 10% aqueous citric acid solution was added and stirred, and the organic layer was washed successively with 10% aqueous solution of citric acid, saturated aqueous sodium hydrogen carbonate solution and saturated aqueous sodium chloride solution, and dried over anhydrous sodium sulfate. .. The desiccant was removed and concentrated under reduced pressure, and the obtained residue was purified by medium pressure silica gel column chromatography (chloroform) to prepare
1H-NMR (CDCl3) δ: 7.47 (m, 2H, C6H5-CH), 7.40-7.19 (m, 20H, C6H5-CH, C6H5-CH2, CH3O-C6H4-CH2), 6.80 (d, J = 8.5 Hz, 2H, CH3O-C6H4-), 5.87-5.79 (m, 3H, 2'-NH, OCH2-CH=CH2 of Alloc and allyl groups), 5.52 (s, 1H, C6H5-CH), 5.35 (dd, J = 9.5, 11.0 Hz, 1H, H-3), 5.30-5.16 (m, 4H, OCH2-CH=CH2 of Alloc group, OCH2-CH=CH2 of Allyl group), 5.08 (t, J = 5.0, β-CH of acyl chain) 5.03 (d, J = 9.5 Hz, 1H, H-1'), 4.97 (d, J = 8.0, 1H, 2-NH), 4.85 (d, J = 3.5 Hz, 1H, H-1), 4.76 (d, J = 11.5 Hz, 1H, CH3O-C6H4-CH2-), 4.61-4.42 (m, 9H, CH3O-C6H4-CH2-, OCH2-CH=CH2 of Alloc group, CH2-C6H5), 4.31 (dd, J = 10.0, 5.5 Hz, 1H, H-3'), 4.22 (t, J = 5.0, β-CH of acyl chain), 4.16 (dd, J = 5.0, 12.0, Hz, 1H, OCH2-CH=CH2 of allyl group), 3.99 (d, J = 10.0 Hz, 1H, H-6a), 3.96-3.92 (m, 2H, H-2, OCH2-CH=CH2 of allyl group), 3.88-3.77 (m, 4H, β-CH of acyl chain, H-4', H-5, H-6'a), 3.80 (s, 3H, CH3O-C6H4-) 3.77-3.63 (m, 3H, H-6'b, H-6b, H-4), 3.48-3.33 (m, 2H, H-2', H-5'), 2.59-2.19 (m, 6H, α-CH2 of acyl chain), 1.64-1.49 (m, 6H, γ-CH2 of acyl chain), 1.40-1.20 (m, 50H, acyl chain), 0.88 (t, J = 7.0 Hz, 9H, CH2-CH3 of acyl chain).
MS (ESI-LIT-orbitrap MS, positive) Calcd. for C95H136N2O18 [M+Na]+ : 1615.9680, Found 1615.9742 1 H-NMR (CDCl 3 ) δ: 7.47 (m, 2H, C 6 H 5 -CH), 7.40-7.19 (m, 20H, C 6 H 5 -CH, C 6 H 5 -CH 2 , CH 3 OC 6 H 4 -CH 2 ), 6.80 (d, J = 8.5 Hz, 2H, CH 3 OC 6 H 4- ), 5.87-5.79 (m, 3H, 2'-NH, OCH 2 -CH = CH 2 of Alloc and allyl groups), 5.52 (s, 1H, C 6 H 5 -CH), 5.35 (dd, J = 9.5, 11.0 Hz, 1H, H-3), 5.30-5.16 (m, 4H, OCH 2 -CH = CH 2 of Alloc group, OCH 2 -CH = CH 2 of Allyl group), 5.08 (t, J = 5.0, β-CH of acyl chain) 5.03 (d, J = 9.5 Hz, 1H, H-1'), 4.97 (d, J = 8.0, 1H, 2-NH), 4.85 (d, J = 3.5 Hz, 1H, H-1), 4.76 (d, J = 11.5 Hz, 1H, CH 3 OC 6 H 4 -CH 2- ), 4.61-4.42 (m, 9H, CH 3 OC 6 H 4 -CH 2- , OCH 2 -CH = CH 2 of Alloc group, CH 2 -C 6 H 5 ), 4.31 (dd, J = 10.0 , 5.5 Hz, 1H, H-3'), 4.22 (t, J = 5.0, β-CH of acyl chain), 4.16 (dd, J = 5.0, 12.0, Hz, 1H, OCH 2 -CH = CH 2 of allyl group), 3.99 (d, J = 10.0 Hz, 1H, H-6a), 3.96-3.92 (m, 2H, H-2, OCH 2 -CH = CH 2 of allyl group), 3.88-3.77 (m, 4H, β-CH of acyl chain, H-4', H-5, H-6'a), 3.80 (s, 3H, CH 3 OC 6 H 4- ) 3.77-3.63 (m, 3H, H-6) 'b, H-6b, H-4), 3.48-3.33 (m, 2H, H-2', H-5'), 2.59-2.19 (m, 6H, α-CH 2 of acyl chain), 1.64-1.49 (m, 6H, γ-CH 2 of acyl) chain), 1.40-1.20 (m, 50H, acyl chain), 0.88 (t, J = 7.0 Hz, 9H, CH 2 -CH 3 of acyl chain).
MS (ESI-LIT-orbitrap MS, positive) Calcd. For C 95 H1 36 N 2 O 18 [M + Na] + : 1615.9680, Found 1615.9742
化合物５（210 mg, 132μmol）をクロロホルム（3 mL）に溶解し、TMSDMAとテトラキス（トリフェニルホスフィン）パラジウム（0）を加えて室温で８０分間撹拌した。反応溶液に水を加えて撹拌した後、飽和炭酸水素ナトリウム水溶液と飽和塩化ナトリウム水溶液で順次洗浄し、無水硫酸ナトリウムで乾燥した。乾燥剤を瀘去して減圧濃縮した。得られた残渣と(R)-3-（カプロイルオキシ）ミリスチン酸６（57.8 mg, 145μmol）をクロロホルム（3 mL）とN,N-ジメチルスルホキシド（3 mL）の混合溶媒に溶解し、トリメチルシリルジメチル HATU（75.1 mg, 198μmol）とDMAP（1.61 mg, 13.2μmol）を加え、室温で１８時間撹拌した。反応溶液をクロロホルムで希釈した後、10%クエン酸水溶液を加えて撹拌し、有機層を10%クエン酸水溶液、飽和炭酸水素ナトリウム水溶液と飽和塩化ナトリウム水溶液で順次洗浄し、無水硫酸ナトリウムで乾燥した。乾燥剤を瀘去して減圧濃縮し、得られた残渣を中圧シリカゲルカラムクロマトグラフィー（クロロホルム）で精製し、上記式で表される化合物７を白色固体（193 mg, 収率78%）として得た。
Compound 5 (210 mg, 132 μmol) was dissolved in chloroform (3 mL), TMSDMA and tetrakis (triphenylphosphine) palladium (0) were added, and the mixture was stirred at room temperature for 80 minutes. Water was added to the reaction solution and the mixture was stirred, washed successively with saturated aqueous sodium hydrogen carbonate solution and saturated aqueous sodium chloride solution, and dried over anhydrous sodium sulfate. The desiccant was removed and concentrated under reduced pressure. The obtained residue and (R) -3- (caproyloxy) myristic acid 6 (57.8 mg, 145 μmol) were dissolved in a mixed solvent of chloroform (3 mL) and N, N-dimethyl sulfoxide (3 mL), and trimethylsilyldimethyl HATU. (75.1 mg, 198 μmol) and DMAP (1.61 mg, 13.2 μmol) were added, and the mixture was stirred at room temperature for 18 hours. After diluting the reaction solution with chloroform, 10% aqueous citric acid solution was added and stirred, and the organic layer was washed successively with 10% aqueous solution of citric acid, saturated aqueous sodium hydrogen carbonate solution and saturated aqueous sodium chloride solution, and dried over anhydrous sodium sulfate. .. The desiccant was removed and concentrated under reduced pressure, and the obtained residue was purified by medium pressure silica gel column chromatography (chloroform) to prepare
1H-NMR (CDCl3) δ: 7.47 (m, 2H, C6H5-CH), 7.46-7.17 (m, 20H, C6H5-CH, C6H5-CH2, CH3O-C6H4-CH2), 6.79 (d, J = 8.0 Hz, 2H, CH3O-C6H4-), 5.88-5.79 (m, 3H, 2-NH, 2'-NH, OCH2-CH=CH2 of allyl groups), 5.52 (s, 1H, C6H5-CH), 5.33-5.20 (m, 3H, H-3, OCH2-CH=CH2 of Allyl group), 5.07 (m, 2H, β-CH of acyl chain) 4.97 (d, J = 8.0 Hz, 1H, 1'-H), 4.81 (d, J = 3.5, 1H, H-1), 4.76 (d, J = 11.0 Hz, 1H, CH3O-C6H4-CH2-), 4.61-4.42 (m, 7H, CH3O-C6H4-CH2-, CH2-C6H5), 4.31 (dd, J = 10.5, 5.0 Hz, 1H, H-3'), 4.24-4.20 (m, 2H, β-CH of acyl chain), 4.16 (dd, J = 5.0, 12.0, Hz, 1H, OCH2-CH=CH2 of allyl group), 3.98 (d, J = 10.0 Hz, 1H, H-6a), 3.93 (dd, J = 9.0, 5.0 Hz, 1H, OCH2-CH=CH2 of allyl group), 3.88-3.77 (m, 3H, H-4', H-5, H-6'a), 3.80 (s, 3H, CH3O-C6H4-) 3.77-3.63 (m, 3H,H-2, H-6'b, H-6b, H-4), 3.48-3.33 (m, 2H, H-2', H-5'), 2.59-2.17 (m, 10H, α-CH2 of acyl chain), 1.61-1.49 (m, 8H, γ-CH2 of acyl chain), 1.40-1.20 (m, 82H, acyl chain), 0.88 (t, J = 7.0 Hz, 15H, CH2-CH3 of acyl chain).
MS (ESI-LIT-orbitrap MS, positive) Calcd. for C115H176N2O19 [M+Na]+ : 1912.2760, Found 1912.2801 1 H-NMR (CDCl 3 ) δ: 7.47 (m, 2H, C 6 H 5 -CH), 7.46-7.17 (m, 20H, C 6 H 5 -CH, C 6 H 5 -CH 2 , CH 3 OC 6 H 4 -CH 2 ), 6.79 (d, J = 8.0 Hz, 2H, CH 3 OC 6 H 4- ), 5.88-5.79 (m, 3H, 2-NH, 2'-NH, OCH 2 -CH = CH 2 of allyl groups), 5.52 (s, 1H, C 6 H 5 -CH), 5.33-5.20 (m, 3H, H-3, OCH 2 -CH = CH 2 of Allyl group), 5.07 (m, 2H , β-CH of acyl chain) 4.97 (d, J = 8.0 Hz, 1H, 1'-H), 4.81 (d, J = 3.5, 1H, H-1), 4.76 (d, J = 11.0 Hz, 1H) , CH 3 OC 6 H 4 -CH 2- ), 4.61-4.42 (m, 7H, CH 3 OC 6 H 4 -CH 2- , CH 2 -C 6 H 5 ), 4.31 (dd, J = 10.5, 5.0 Hz, 1H, H-3'), 4.24-4.20 (m, 2H, β-CH of acyl chain), 4.16 (dd, J = 5.0, 12.0, Hz, 1H, OCH 2 -CH = CH 2 of allyl group ), 3.98 (d, J = 10.0 Hz, 1H, H-6a), 3.93 (dd, J = 9.0, 5.0 Hz, 1H, OCH 2 -CH = CH 2 of allyl group), 3.88-3.77 (m, 3H) , H-4', H-5, H-6'a), 3.80 (s, 3H, CH 3 OC 6 H 4- ) 3.77-3.63 (m, 3H, H-2, H-6'b, H -6b, H-4), 3.48-3.33 (m, 2H, H-2', H-5'), 2.59-2.17 (m, 10H, α-CH 2 of acyl chain), 1.61-1.49 (m, 8H, γ-CH 2 of acyl chain), 1.40-1.20 (m, 82H, acyl chain) , 0.88 (t, J = 7.0 Hz, 15H, CH 2 -CH 3 of acyl chain).
MS (ESI-LIT-orbitrap MS, positive) Calcd. For C 115 H 176 N 2 O 19 [M + Na] + : 1912.2760, Found 1912.2801
化合物７（150 mg, 79.4μmol）をジクロロメタン（3 mL）に溶解し、2,6-ジ-tert-ブチルピリジン（53.4 μL, 238μmol）を加えて０℃で３０分間撹拌した。この溶液にDDQ（27.0 mg, 119μmol）と水（100μL）を加えて０℃で２０時間撹拌した。反応溶液をクロロホルムで希釈した後、飽和炭酸水素ナトリウム水溶液を加えて撹拌し、クロロホルムで抽出した。有機層を飽和炭酸水素ナトリウム水溶液と飽和塩化ナトリウム水溶液で順次洗浄し、無水硫酸ナトリウムで乾燥した。乾燥剤を瀘去して減圧濃縮し、得られた残渣を中圧シリカゲルカラムクロマトグラフィー（クロロホルム）で精製し、白色固体を得た。この白色固体と(R)-3-（ベンジルオキシ）ミリスチン酸２（24.9 mg, 74.6μmol）をジクロロメタン（2 mL）とN,N-ジメチルホルムアミド（2 mL）の混合溶媒に溶解し、DIPEA（21.7μL, 124μmol）、MNBA（32.1 mg, 93.2 μmol）とDMAP（0.76 mg, 6.2μmol）を加え４０℃で４４時間撹拌した。反応溶液をクロロホルムで希釈した後、10%クエン酸水溶液を加えて撹拌し、クロロホルムで抽出した。有機層を10%クエン酸水溶液、飽和炭酸水素ナトリウム水溶液と飽和塩化ナトリウム水溶液で順次洗浄し、無水硫酸ナトリウムで乾燥した。乾燥剤を瀘去して減圧濃縮し、得られた残渣を中圧シリカゲルカラムクロマトグラフィー（クロロホルム）で精製し、上記式で表される化合物８を白色固体（108 mg, 収率67%）として得た。
Compound 7 (150 mg, 79.4 μmol) was dissolved in dichloromethane (3 mL), 2,6-di-tert-butylpyridine (53.4 μL, 238 μmol) was added, and the mixture was stirred at 0 ° C. for 30 minutes. DDQ (27.0 mg, 119 μmol) and water (100 μL) were added to this solution, and the mixture was stirred at 0 ° C. for 20 hours. After diluting the reaction solution with chloroform, saturated aqueous sodium hydrogen carbonate solution was added, the mixture was stirred, and the mixture was extracted with chloroform. The organic layer was washed successively with saturated aqueous sodium hydrogen carbonate solution and saturated aqueous sodium chloride solution, and dried over anhydrous sodium sulfate. The desiccant was removed and concentrated under reduced pressure, and the obtained residue was purified by medium pressure silica gel column chromatography (chloroform) to obtain a white solid. This white solid and (R) -3- (benzyloxy) myristic acid 2 (24.9 mg, 74.6 μmol) were dissolved in a mixed solvent of dichloromethane (2 mL) and N, N-dimethylformamide (2 mL), and DIPEA ( 21.7 μL, 124 μmol), MNBA (32.1 mg, 93.2 μmol) and DMAP (0.76 mg, 6.2 μmol) were added, and the mixture was stirred at 40 ° C. for 44 hours. After diluting the reaction solution with chloroform, a 10% aqueous citric acid solution was added, the mixture was stirred, and the mixture was extracted with chloroform. The organic layer was washed successively with 10% aqueous citric acid solution, saturated aqueous sodium hydrogen carbonate solution and saturated aqueous sodium chloride solution, and dried over anhydrous sodium sulfate. The desiccant was removed and concentrated under reduced pressure, and the obtained residue was purified by medium pressure silica gel column chromatography (chloroform) to prepare
1H-NMR (CDCl3) δ: 7.38 (m, 2H, C6H5-CH), 7.31-7.18 (m, 23H, C6H5-CH, C6H5-CH2), 5.90 (d, J = 9.5 Hz, 1H, 2'-NH), 5.88-5.83 (m, 1H, OCH2-CH=CH2 of allyl groups), 5.77 (d, J = 8.5 Hz, 1H, 2-NH) 5.39 (s, 1H, C6H5-CH), 5.39 (t, J = 10.0 Hz, 1H, H-3), 5.34-5.18 (m, 3H, OCH2-CH=CH2 of Allyl group, β-CH of acyl chain), 5.07 (quint, J = 6.0 Hz, 1H, β-CH of acyl chain), 5.00 (quint, J = 6.0 Hz, 1H, β-CH of acyl chain), 4.80 (d, J = 4.0 Hz, 1H, H-1), 4.74 (d, J = 8.0, 1H, H-1'), 4.59-4.38 (m, 8H, CH2-C6H5), 4.30 (dd, J = 10.5, 5.5 Hz, 1H, H-3'), 4.20 (dt, J = 5.0, 12.0 Hz, 1H, H-5'), 4.15 (dd, J = 5.0, 12.0, Hz, 1H, OCH2-CH=CH2 of allyl group), 3.97 (d, J = 9.5 Hz, 1H, H-6a), 3.93 (dd, J = 9.0, 5.0 Hz, 1H, OCH2-CH=CH2 of allyl group), 3.94-3.77 (m, 5H, H-2, H-4', H-6'a, β-CH of acyl chain), 3.77-3.57 (m, 4H, H-2, H-4', H-6'b, H-6b,), 3.47 (dt, J = 5.0, 12.0 Hz, 1H, H-2'), 2.72-2.03 (m, 12H, α-CH2 of acyl chain), 1.61-1.41 (m, 10H, γ-CH2 of acyl chain), 1.40-1.20 (m, 100H, acyl chain), 0.88 (m, 18H, CH2-CH3 of acyl chain).
MS (ESI-LIT-orbitrap MS, positive) Calcd. for C128H200N2O20 [M+Na]+ : 2108.4587, Found 2108.4644 1 H-NMR (CDCl 3 ) δ: 7.38 (m, 2H, C 6 H 5 -CH), 7.31-7.18 (m, 23H, C 6 H 5 -CH, C 6 H 5 -CH 2 ), 5.90 ( d, J = 9.5 Hz, 1H, 2'-NH), 5.88-5.83 (m, 1H, OCH 2 -CH = CH 2 of allyl groups), 5.77 (d, J = 8.5 Hz, 1H, 2-NH) 5.39 (s, 1H, C 6 H 5 -CH), 5.39 (t, J = 10.0 Hz, 1H, H-3), 5.34-5.18 (m, 3H, OCH 2 -CH = CH 2 of Allyl group, β -CH of acyl chain), 5.07 (quint, J = 6.0 Hz, 1H, β-CH of acyl chain), 5.00 (quint, J = 6.0 Hz, 1H, β-CH of acyl chain), 4.80 (d, J) = 4.0 Hz, 1H, H-1), 4.74 (d, J = 8.0, 1H, H-1'), 4.59-4.38 (m, 8H, CH 2 -C 6 H 5 ), 4.30 (dd, J = 10.5, 5.5 Hz, 1H, H-3'), 4.20 (dt, J = 5.0, 12.0 Hz, 1H, H-5'), 4.15 (dd, J = 5.0, 12.0, Hz, 1H, OCH 2 -CH = CH 2 of allyl group), 3.97 (d, J = 9.5 Hz, 1H, H-6a), 3.93 (dd, J = 9.0, 5.0 Hz, 1H, OCH 2 -CH = CH 2 of allyl group), 3.94 -3.77 (m, 5H, H-2, H-4', H-6'a, β-CH of acyl chain), 3.77-3.57 (m, 4H, H-2, H-4', H-6 'b, H-6b,), 3.47 (dt, J = 5.0, 12.0 Hz, 1H, H-2'), 2.72-2.03 (m, 12H, α-CH 2 of acyl chain), 1.61-1.41 (m) , 10H, γ-CH 2 of acyl chain), 1. 40-1.20 (m, 100H, acyl chain), 0.88 (m, 18H, CH 2 -CH 3 of acyl chain).
MS (ESI-LIT-orbitrap MS, positive) Calcd. For C 128 H 200 N 2 O 20 [M + Na] + : 2108.4587, Found 2108.4644
化合物８（20 mg, 9.58μmol）をジクロロメタン（1 mL）と水（0.1 mL）とTFA（0.1 mL）に溶解し、０℃で２０分間撹拌した。反応溶液をトルエンで希釈した後、減圧濃縮した。得られた残渣をジクロロメタン（1 mL）に溶解し、ピリジン（7.7μL, 95.8μmol）と塩化トリチル（2.94 mg, 10.54μmol）を加えて室温で６時間撹拌した。反応溶液をクロロホルムで希釈した後、10%クエン酸水溶液を加えて撹拌し、クロロホルムで抽出した。有機層を10%クエン酸水溶液、飽和炭酸水素ナトリウム水溶液と飽和塩化ナトリウム水溶液で順次洗浄し、無水硫酸ナトリウムで乾燥した。乾燥剤を瀘去して減圧濃縮し、得られた残渣を中圧シリカゲルカラムクロマトグラフィー（クロロホルム）で精製し、上記式で表される化合物９を白色固体（14.6 mg, 収率68%）として得た。
Compound 8 (20 mg, 9.58 μmol) was dissolved in dichloromethane (1 mL), water (0.1 mL) and TFA (0.1 mL), and the mixture was stirred at 0 ° C. for 20 minutes. The reaction solution was diluted with toluene and then concentrated under reduced pressure. The obtained residue was dissolved in dichloromethane (1 mL), pyridine (7.7 μL, 95.8 μmol) and trityl chloride (2.94 mg, 10.54 μmol) were added, and the mixture was stirred at room temperature for 6 hours. After diluting the reaction solution with chloroform, a 10% aqueous citric acid solution was added, the mixture was stirred, and the mixture was extracted with chloroform. The organic layer was washed successively with 10% aqueous citric acid solution, saturated aqueous sodium hydrogen carbonate solution and saturated aqueous sodium chloride solution, and dried over anhydrous sodium sulfate. The desiccant was removed and concentrated under reduced pressure, and the obtained residue was purified by medium pressure silica gel column chromatography (chloroform) to prepare
MS (ESI-LIT-orbitrap MS, positive) Calcd. for C140H210N2O20 [M+Na]+ : 2252.5369, Found 2252.5422MS (ESI-LIT-orbitrap MS, positive) Calcd. For C 140 H 210 N 2 O 20 [M + Na] + : 2252.5369, Found 2252.5422
（1,5）-（シクロオクタジエン）-ビス（メチルジフェニルホスフィン）イリジウム（1）ヘキサフルオロホスフェートをアルゴン雰囲気下でテトラヒドロフラン（0.5 mL）に溶解させた後、アルゴンを水素に置換した。溶液が赤色から黄色に変化したことを確認した後、水素をアルゴンに置換することで活性化イリジウム錯体のテトラヒドロフラン溶液を得た。化合物９（10 mg, 4.49μmol）をテトラヒドロフラン（0.5 mL）に溶解し、活性化イリジウム錯体のテトラヒドロフラン溶液を加え室温で１６時間撹拌した。反応溶液に水（50μL）とヨウ素（3.42 mg, 13.5μmol）を加え、室温で３０分間撹拌した。反応溶液をクロロホルムで希釈した後、20%チオ硫酸ナトリウム水溶液を加え撹拌し、クロロホルムで抽出した。有機層を20%チオ硫酸ナトリウム水溶液、飽和炭酸水素ナトリウム水溶液と飽和塩化ナトリウム水溶液で順次洗浄し、無水硫酸ナトリウムで乾燥した。乾燥剤を瀘去して減圧濃縮し、得られた残渣を中圧シリカゲルカラムクロマトグラフィー（クロロホルム）で精製し、上記式で表される化合物１０を白色固体（11.0 mg, quant.）として得た。
(1,5-)-(Cyclooctadiene) -bis (methyldiphenylphosphine) iridium (1) Hexafluorophosphate was dissolved in tetrahydrofuran (0.5 mL) under an argon atmosphere, and then argon was replaced with hydrogen. After confirming that the solution changed from red to yellow, a tetrahydrofuran solution of the activated iridium complex was obtained by substituting hydrogen with argon. Compound 9 (10 mg, 4.49 μmol) was dissolved in tetrahydrofuran (0.5 mL), a solution of the activated iridium complex in tetrahydrofuran was added, and the mixture was stirred at room temperature for 16 hours. Water (50 μL) and iodine (3.42 mg, 13.5 μmol) were added to the reaction solution, and the mixture was stirred at room temperature for 30 minutes. After diluting the reaction solution with chloroform, a 20% aqueous sodium thiosulfate solution was added, the mixture was stirred, and the mixture was extracted with chloroform. The organic layer was washed successively with 20% aqueous sodium thiosulfate solution, saturated aqueous sodium hydrogen carbonate solution and saturated aqueous sodium chloride solution, and dried over anhydrous sodium sulfate. The desiccant was removed and concentrated under reduced pressure, and the obtained residue was purified by medium pressure silica gel column chromatography (chloroform) to obtain
MS (ESI-LIT-orbitrap MS, positive) Calcd. for C137H206N2O20 [M+Na]+ : 2222.5056, Found 2222.5033MS (ESI-LIT-orbitrap MS, positive) Calcd. For C 137 H 206 N 2 O 20 [M + Na] + : 2222.5056, Found 2222.5033
化合物１０（6.0 mg, 2.73μmol）をジクロロメタン（1.0 mL）に溶解し、モレキュラーシーブス4A、テトラゾール（1.3 mg, 19.1μmol）とジベンジル-N,N-ジイソプロピルホスホロアミダイト（4.52μL, 13.6μmol）を加え室温で１６時間撹拌した。反応溶液にDMDOを加え、室温で２時間撹拌した。反応溶液をクロロホルムで希釈した後、飽和炭酸水素ナトリウム水溶液を加え撹拌し、クロロホルムで抽出した。有機層を飽和炭酸水素ナトリウム水溶液と飽和塩化ナトリウム水溶液で順次洗浄し、無水硫酸ナトリウムで乾燥した。乾燥剤を瀘去して減圧濃縮し、得られた残渣を中圧シリカゲルカラムクロマトグラフィー（クロロホルム／アセトン=99:1）で精製した。得られた白色固体をテトラヒドロフラン（2 mL）と水（0.2 mL）と酢酸（0.1 mL）の混合溶媒に溶解し、水酸化パラジウム／炭素（3.0 mg）を加え水素雰囲気下（20 kgf/cm2）室温で１９時間撹拌した。反応溶液にトリエチルアミンを加えて撹拌した後、水酸化パラジウム/炭素をメンブレンフィルターにより瀘去し、減圧濃縮した。得られた残渣を1,4-ジオキサンに溶解させ、凍結乾燥を行うことにより上記式で表される本発明のリピドＡである化合物１１を白色固体として得た。Compound 10 (6.0 mg, 2.73 μmol) was dissolved in dichloromethane (1.0 mL) and molecular sieves 4A, tetrazole (1.3 mg, 19.1 μmol) and dibenzyl-N, N-diisopropylphosphoroamidite (4.52 μL, 13.6 μmol) were added. In addition, the mixture was stirred at room temperature for 16 hours. DMDO was added to the reaction solution, and the mixture was stirred at room temperature for 2 hours. After diluting the reaction solution with chloroform, saturated aqueous sodium hydrogen carbonate solution was added, the mixture was stirred, and the mixture was extracted with chloroform. The organic layer was washed successively with saturated aqueous sodium hydrogen carbonate solution and saturated aqueous sodium chloride solution, and dried over anhydrous sodium sulfate. The desiccant was removed and concentrated under reduced pressure, and the obtained residue was purified by medium pressure silica gel column chromatography (chloroform / acetone = 99: 1). The obtained white solid is dissolved in a mixed solvent of tetrahydrofuran (2 mL), water (0.2 mL) and acetic acid (0.1 mL), palladium hydroxide / carbon (3.0 mg) is added, and a hydrogen atmosphere (20 kgf / cm 2 ) is added. ) Stirred at room temperature for 19 hours. After adding triethylamine to the reaction solution and stirring, palladium / carbon hydroxide was removed by a membrane filter and concentrated under reduced pressure. The obtained residue was dissolved in 1,4-dioxane and freeze-dried to obtain
MS (ESI-LIT-orbitrap MS, negative) Calcd. for C90H170N2O26P2 [M-2H]2- : 877.5688, Found 877.5755MS (ESI-LIT-orbitrap MS, negative) Calcd. For C 90 H 170 N 2 O 26 P 2 [M-2H] 2- : 877.5688, Found 877.5755
（実施例４）アルカリゲネス由来LPSによる卵白アルブミン（OVA）特異的免疫応答
本実施例では、実施例２に記載の方法と同手法により抽出したアルカリゲネス属フェカーリス由来LPSについて、マウスでのOVA特異的免疫応答の増強を確認した。比較例として使用する大腸菌由来LPSは大腸菌O111由来LPSを用いた。(Example 4) Ovalbumin (OVA) -specific immune response by Alcaligenes-derived LPS In this example, OVA-specific immunity of Alcaligenes-derived Fecarlis-derived LPS extracted by the same method as that described in Example 2 in mice. Enhanced response was confirmed. As the Escherichia coli-derived LPS used as a comparative example, Escherichia coli O111-derived LPS was used.
10μg/doseのOVA（Sigma, A5503）を抗原とし、アジュバントとしてアルカリゲネス又は大腸菌由来のLPS（100μg）、あるいは200μLのAlum（Thermo Fisher Scientific Inc）を８週齢の雌BALB/c（ニホンクレア）に皮下注射で２回投与した（０日目と７日目）。最終免疫から７日目に脾臓を回収し、脾臓細胞からCD4マイクロビーズ（Miltenyi biotec, 130-049-201）を用いてCD4陽性細胞を分離した。また脾臓細胞に放射線（30 Gy）を照射し、抗原提示細胞として用いた。CD4陽性細胞（2×105 cells/well）及び抗原提示細胞（2×105 cells/well）を96穴プレート（Thermo, 163320）に播種し、OVA（1 mg/mL）を添加して３７℃、5% CO2環境下で培養した。培養４日目に培養上清を回収し、Mouse Th1/TH2/Th17 cytokine kit（BD biosciences, 560485）を用いて培養上清中のIL-17A濃度を測定した。10 μg / dose OVA (Sigma, A5503) is used as an antigen, and alkaline Genes or E. coli-derived LPS (100 μg) or 200 μL Alum (Thermo Fisher Scientific Inc) is used as an adjuvant for 8-week-old female BALB / c (Japanese Claire). It was administered twice by subcutaneous injection (
その結果、脾臓細胞の培養上清中のIL-17Aについて大腸菌由来のLPSで処理した細胞が最も高い値を示した（図２０）。このことから、アルカリゲネス由来LPSはTh1, Th2に加え、Th17細胞も誘導できることが示された。 As a result, IL-17A in the culture supernatant of spleen cells showed the highest value in the cells treated with LPS derived from Escherichia coli (Fig. 20). From this, it was shown that Alcaligenes-derived LPS can induce Th17 cells in addition to Th1 and Th2.
（実施例５）本発明のリピドＡによる卵白アルブミン（OVA）特異的免疫応答
本実施例では、リピドＡについてマウスでのOVA特異的抗体産生能の増強を確認した。本実施例のリピドＡとして実施例３で合成されたリピドＡ（化合物１１）を用いた。なお、リピドＡ（化合物１１）はアルカリゲネス属フェカーリス由来リピドＡと同等の構造を有する。(Example 5) Egg white albumin (OVA) -specific immune response by Lipid A of the present invention In this example, it was confirmed that Lipid A enhanced the OVA-specific antibody-producing ability in mice. Lipid A (Compound 11) synthesized in Example 3 was used as Lipid A of this example. Lipid A (Compound 11) has a structure equivalent to that of Lipid A derived from Alcaligenes Fecaris.
10μg/doseのOVA（Sigma, A5503）を抗原とし、アジュバントとしてリピドＡ（1 μg）を８週齢の雌BALB/c（ニホンクレア）に皮下注射で２回投与した（０日目と１０日目）。最終免疫から７日目に採血し、採取した血液は氷上で３０分間静置した後に遠心分離（3,000×g、１０分間、４℃）を行い、血清を回収し、－３０℃で保管した。
Lipid A (1 μg) was administered subcutaneously twice to 8-week-old female BALB / c (Nihon Claire) using 10 μg / dose OVA (Sigma, A5503) as an antigen (
ELISA法によって血清中のOVA特異的なIgG抗体を検出した。OVAをPBS（ナカライテスク, 27575-31）に1 mg/mLで溶解し、96穴プレート（Thermo, 442404）に0.1 mL/well加え、４℃で一晩保管し、固相化した。溶液を捨て、1% BSA（ナカライテスク, 01859-47）添加PBSを0.1 mL/well加えた。室温で２時間反応させた後に溶液を捨て、0.05% Tween20（ナカライテスク, 28353-85）添加PBSで３回洗浄した。1% BSA-0.05% Tween20添加PBSで血清を希釈し、0.1 mL/well添加した。室温で２時間反応させた後に溶液を捨て、0.05% Tween20添加PBSで３回洗浄した。1% BSA-0.05% Tween20添加PBSで4,000倍に希釈した抗体（抗マウスIgG抗体、SouthernBiotech）を0.1 mL/well添加した。室温で１時間反応させた後に溶液を捨て、0.05% Tween20添加PBSで３回洗浄した。TMB（3,3',5,5'-tetramethylbenzidine：KPL, 50-76-03）を0.1 mL/well加え、室温で２分間反応させた後に0.5N HCl（ナカライテスク, 37345-15）を0.05 mL/well添加した。マイクロプレートリーダー（バイオラッド, iMark）を用いてOD450 nmの波長で測定した。OVA-specific IgG antibody in serum was detected by ELISA method. OVA was dissolved in PBS (Nacalai Tesque, 27575-31) at 1 mg / mL, 0.1 mL / well was added to a 96-well plate (Thermo, 442404), and the mixture was stored overnight at 4 ° C. to solidify. The solution was discarded and 0.1 mL / well of PBS with 1% BSA (Nacalai Tesque, 01859-47) was added. After reacting at room temperature for 2 hours, the solution was discarded and washed 3 times with PBS containing 0.05% Tween20 (Nacalai Tesque, 28353-85). Serum was diluted with PBS containing 1% BSA-0.05
その結果、リピドＡを投与したマウスの方が、血清中のOVA特異的なIgG抗体が高い値を示した（図２１）。このことから、リピドＡは、アルカリゲネス属フェカーリス由来LPSと同様にアジュバントとして使用可能であることが示された。 As a result, the mice administered with Lipid A showed higher values of OVA-specific IgG antibody in serum (FIG. 21). From this, it was shown that Lipid A can be used as an adjuvant as well as LPS derived from Alcaligenes Fecaris.
（実施例６）本発明のリピドＡによるマウス樹状細胞の活性化
本実施例では実施例５に示したリピドＡ（化合物１１）について、マウス樹状細胞の活性化を確認した。活性化はサイトカイン（IL-6）の産生能により確認した。(Example 6) Activation of mouse dendritic cells by Lipid A of the present invention In this example, activation of mouse dendritic cells was confirmed for Lipid A (Compound 11) shown in Example 5. Activation was confirmed by the ability to produce cytokine (IL-6).
細胞の培養は10% 牛胎児血清（Gibco, 10437-028）、1% ピルビン酸ナトリウム（ナカライテスク, 06977-34）、0.1% 2-メルカプトエタノール（Gibco, 21985-023）、1% ペニシリン及びストレプトマイシン（ナカライテスク, 26253-84）添加RPMI 1640（Sigma, R8758）で行った（以下、「cRPMI培地」）。マウス樹状細胞（BMDC：Bone marrow-derived dendritic cell）は、４～６週齢の雌BALB/cの大腿骨及び下腿骨から骨髄細胞を採取し、当該骨髄細胞をGM-CSF（10μg/mL）添加cRPMI培地で６日間培養後、CD11c MACSビーズ（Miltenyi biotec, 130-052-001）を用いてCD11c陽性細胞を分離し、調製した。６日間の培養中、２日に１回培地を半量交換した。当該CD11c陽性マウス樹状細胞を、以下「BMDC」と称する。BMDC（1×105 cells/well）を96穴プレート（Thermo, 163320）に播種し、リピドＡを0.1～10 pg/mLの濃度で添加して３７℃、5% CO2環境下で培養した。培養４８時間後に培養上清を回収し、Mouse Th1/TH2/Th17 cytokine kit（BD Biosciences）を用いて培養上清中のIL-6の濃度を測定した。Cell cultures are 10% fetal bovine serum (Gibco, 10437-028), 1% sodium pyruvate (Nakalitesk, 06977-34), 0.1% 2-mercaptoethanol (Gibco, 21985-023), 1% penicillin and streptomycin. (Nakalaitesk, 26253-84) added RPMI 1640 (Sigma, R8758) (hereinafter, "cRPMI medium"). For mouse dendritic cells (BMDC: Bone marrow-derived dendritic cells), bone marrow cells are collected from the femoral and lower leg bones of female BALB / c at 4 to 6 weeks of age, and the bone marrow cells are collected from GM-CSF (10 μg / mL). ) After culturing in the added cRPMI medium for 6 days, CD11c-positive cells were isolated and prepared using CD11c MACS beads (Miltenyi biotec, 130-052-001). During the 6-day culture, half the medium was replaced once every 2 days. The CD11c-positive mouse dendritic cells are hereinafter referred to as "BMDC". BMDC (1 × 10 5 cells / well) was seeded on a 96-well plate (Thermo, 163320), Lipid A was added at a concentration of 0.1-10 pg / mL, and the cells were cultured at 37 ° C. in a 5% CO 2 environment. .. After 48 hours of culturing, the culture supernatant was collected, and the concentration of IL-6 in the culture supernatant was measured using a Mouse Th1 / TH2 / Th17 cytokine kit (BD Biosciences).
その結果、リピドＡの添加濃度依存的にIL-6の産生量が増加した（図２２）。このことから、前記リピドＡによりマウス樹状細胞が活性化されることが確認された。 As a result, the amount of IL-6 produced increased depending on the concentration of Lipid A added (Fig. 22). From this, it was confirmed that the mouse dendritic cells were activated by Lipid A.
（実施例７）本発明のリピドＡによるヒト抹消血単核細胞の活性化
本実施例では実施例５に示したリピドＡ（化合物１１）について、ヒトの末梢血単核細胞の活性化を確認した。末梢血単核細胞の活性化はサイトカイン（IL-6、IL-1β）の産生能により確認した。(Example 7) Activation of human peripheral blood mononuclear cells by Lipid A of the present invention In this example, activation of human peripheral blood mononuclear cells was confirmed for Lipid A (Compound 11) shown in Example 5. did. The activation of peripheral blood mononuclear cells was confirmed by the ability to produce cytokines (IL-6, IL-1β).
2.8×105 cells/wellのヒト末梢血単核細胞（Wako, 555-24481）を96穴プレート（Thermo, 163320）に播種し、リピドＡ（1 ng/mL）を添加して３７℃、5% CO2環境下で培養した。培養２４時間後に培養上清を回収し、Human inflammatory cytokine kit（BD biosciences、551811）を用いて上清中のIL-6とIL-1βの濃度を測定した。Human peripheral blood mononuclear cells (Wako, 555-24481) of 2.8 × 10 5 cells / well were seeded on a 96-well plate (Thermo, 163320), and Lipid A (1 ng / mL) was added at 37 ° C., 5 Cultivated in a% CO 2 environment. After 24 hours of culturing, the culture supernatant was collected, and the concentrations of IL-6 and IL-1β in the supernatant were measured using a Human inflammatory cytokine kit (BD biosciences, 551811).
その結果、リピドＡの添加によりIL-6及びIL-1βの産生量の増加が確認された（図２３）。このことから、前記リピドＡによりヒトの末梢血単核細胞が活性化されることが確認された。 As a result, it was confirmed that the addition of Lipid A increased the production of IL-6 and IL-1β (Fig. 23). From this, it was confirmed that the above Lipid A activates human peripheral blood mononuclear cells.
以上詳述したように、本発明のリピドＡは新規な構造からなる。本発明のリピドＡを含むアジュバント組成物又は免疫賦活化剤は、優れた免疫活性化能を維持したまま、アレルギー反応や炎症等の副作用が軽減化された、安全性が高く有効な医薬組成物として使用することができる。 As described in detail above, Lipid A of the present invention has a novel structure. The adjuvant composition or immunostimulatory agent containing Lipid A of the present invention is a highly safe and effective pharmaceutical composition in which side effects such as allergic reaction and inflammation are reduced while maintaining excellent immunostimulatory ability. Can be used as.
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| Microbiol. Immunol., 1988, Vol.32, No.7, pp.653-666 |
| Mikrobiologicheskii Zhurnal, 1979, Vol.41, No.5, pp.549-551 |
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