JP7474737B2 - Method for preparing asparagusic acid - Google Patents
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- JP7474737B2 JP7474737B2 JP2021213440A JP2021213440A JP7474737B2 JP 7474737 B2 JP7474737 B2 JP 7474737B2 JP 2021213440 A JP2021213440 A JP 2021213440A JP 2021213440 A JP2021213440 A JP 2021213440A JP 7474737 B2 JP7474737 B2 JP 7474737B2
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- AYGMEFRECNWRJC-UHFFFAOYSA-N asparagusic acid Chemical compound OC(=O)C1CSSC1 AYGMEFRECNWRJC-UHFFFAOYSA-N 0.000 title claims description 77
- 238000000034 method Methods 0.000 title claims description 10
- KRHAHEQEKNJCSD-UHFFFAOYSA-N 3-mercapto-2-mercaptomethylpropanoic acid Chemical compound OC(=O)C(CS)CS KRHAHEQEKNJCSD-UHFFFAOYSA-N 0.000 claims description 44
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 42
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 28
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 27
- 239000003960 organic solvent Substances 0.000 claims description 21
- 239000002904 solvent Substances 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- 238000006243 chemical reaction Methods 0.000 claims description 17
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 15
- 239000012044 organic layer Substances 0.000 claims description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 11
- 238000000926 separation method Methods 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 8
- 150000002825 nitriles Chemical class 0.000 claims description 6
- 230000001590 oxidative effect Effects 0.000 claims description 5
- 239000005456 alcohol based solvent Substances 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 239000004210 ether based solvent Substances 0.000 claims description 3
- 238000000622 liquid--liquid extraction Methods 0.000 claims 1
- 239000000243 solution Substances 0.000 description 26
- 239000007864 aqueous solution Substances 0.000 description 16
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 11
- 238000003860 storage Methods 0.000 description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 9
- WMRJXSKATUXDDH-UHFFFAOYSA-N 4-bromo-2-(bromomethyl)butanoic acid Chemical compound OC(=O)C(CBr)CCBr WMRJXSKATUXDDH-UHFFFAOYSA-N 0.000 description 8
- -1 amine compounds Chemical class 0.000 description 8
- 239000000203 mixture Substances 0.000 description 7
- 229920000742 Cotton Polymers 0.000 description 6
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 239000000706 filtrate Substances 0.000 description 6
- 239000010410 layer Substances 0.000 description 6
- 229920000642 polymer Polymers 0.000 description 6
- 229910021642 ultra pure water Inorganic materials 0.000 description 6
- 239000012498 ultrapure water Substances 0.000 description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 5
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 235000002597 Solanum melongena Nutrition 0.000 description 4
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 4
- 238000007254 oxidation reaction Methods 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 238000005160 1H NMR spectroscopy Methods 0.000 description 3
- ZWEHNKRNPOVVGH-UHFFFAOYSA-N 2-Butanone Chemical compound CCC(C)=O ZWEHNKRNPOVVGH-UHFFFAOYSA-N 0.000 description 3
- 241000234427 Asparagus Species 0.000 description 3
- 235000005340 Asparagus officinalis Nutrition 0.000 description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000012043 crude product Substances 0.000 description 3
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000011347 resin Substances 0.000 description 3
- 229920005989 resin Polymers 0.000 description 3
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- JYWKEVKEKOTYEX-UHFFFAOYSA-N 2,6-dibromo-4-chloroiminocyclohexa-2,5-dien-1-one Chemical compound ClN=C1C=C(Br)C(=O)C(Br)=C1 JYWKEVKEKOTYEX-UHFFFAOYSA-N 0.000 description 2
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 2
- RGSFGYAAUTVSQA-UHFFFAOYSA-N Cyclopentane Chemical compound C1CCCC1 RGSFGYAAUTVSQA-UHFFFAOYSA-N 0.000 description 2
- XTHFKEDIFFGKHM-UHFFFAOYSA-N Dimethoxyethane Chemical compound COCCOC XTHFKEDIFFGKHM-UHFFFAOYSA-N 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- OFBQJSOFQDEBGM-UHFFFAOYSA-N Pentane Chemical compound CCCCC OFBQJSOFQDEBGM-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- ZXEKIIBDNHEJCQ-UHFFFAOYSA-N isobutanol Chemical compound CC(C)CO ZXEKIIBDNHEJCQ-UHFFFAOYSA-N 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000001953 recrystallisation Methods 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 2
- SCYULBFZEHDVBN-UHFFFAOYSA-N 1,1-Dichloroethane Chemical compound CC(Cl)Cl SCYULBFZEHDVBN-UHFFFAOYSA-N 0.000 description 1
- 208000002874 Acne Vulgaris Diseases 0.000 description 1
- DKPFZGUDAPQIHT-UHFFFAOYSA-N Butyl acetate Natural products CCCCOC(C)=O DKPFZGUDAPQIHT-UHFFFAOYSA-N 0.000 description 1
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 1
- 208000001840 Dandruff Diseases 0.000 description 1
- 239000012901 Milli-Q water Substances 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 206010000496 acne Diseases 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 150000001338 aliphatic hydrocarbons Chemical class 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000012062 aqueous buffer Substances 0.000 description 1
- 229910052786 argon Inorganic materials 0.000 description 1
- 150000004945 aromatic hydrocarbons Chemical class 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000010908 decantation Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 150000004662 dithiols Chemical class 0.000 description 1
- 239000003759 ester based solvent Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 150000008282 halocarbons Chemical class 0.000 description 1
- DMEGYFMYUHOHGS-UHFFFAOYSA-N heptamethylene Natural products C1CCCCCC1 DMEGYFMYUHOHGS-UHFFFAOYSA-N 0.000 description 1
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 description 1
- 150000005605 isobutyric acids Chemical class 0.000 description 1
- 239000005453 ketone based solvent Substances 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- DUWWHGPELOTTOE-UHFFFAOYSA-N n-(5-chloro-2,4-dimethoxyphenyl)-3-oxobutanamide Chemical compound COC1=CC(OC)=C(NC(=O)CC(C)=O)C=C1Cl DUWWHGPELOTTOE-UHFFFAOYSA-N 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 150000002898 organic sulfur compounds Chemical class 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- FVSKHRXBFJPNKK-UHFFFAOYSA-N propionitrile Chemical compound CCC#N FVSKHRXBFJPNKK-UHFFFAOYSA-N 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- NVRBTKMAZQNKPX-UHFFFAOYSA-N trimethyl-(4-trimethylsilylphenyl)silane Chemical compound C[Si](C)(C)C1=CC=C([Si](C)(C)C)C=C1 NVRBTKMAZQNKPX-UHFFFAOYSA-N 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Cosmetics (AREA)
Description
本発明は、保存安定性に優れたアスパラガス酸の調製方法に関する。 The present invention relates to a method for preparing asparagusic acid with excellent storage stability.
アスパラガス酸（２－ジチオラン－４－カルボン酸）は、アスパラガスに含まれる有機硫黄化合物であり、アスパラガスの生長を促進する作用があることが知られている。また、アスパラガス酸には、ニキビ治療効果（特許文献１）やフケ防止作用（特許文献２）等の薬理作用があることも報告されている。
また、アスパラガス酸は、アミノ酸やペプチド等のアミン化合物にアミド結合することにより、当該アミン化合物の細胞膜内への取り込みを向上することが報告されている（非特許文献１）。
Asparagusic acid (2-dithiolane-4-carboxylic acid) is an organosulfur compound contained in asparagus, and is known to promote the growth of asparagus. It has also been reported that asparagus acid has pharmacological effects such as acne treatment effects (Patent Document 1) and dandruff prevention effects (Patent Document 2).
It has also been reported that asparagusic acid improves the uptake of amine compounds, such as amino acids and peptides, into cell membranes by forming an amide bond with the amine compounds (Non-Patent Document 1).
アスパラガス酸は、ハロゲン化イソ酪酸を出発物質として、化学合成できることが報告されている（非特許文献２）。すなわち、下記反応式で示されるように、３－ブロモ－２－（ブロモエチル）プロパン酸（１）にチオ酢酸カリウムを反応させて３－チオアセチル－２－（チオアセチルエチル）プロパン酸（２）を得、次いで脱アセチル化してアスパラガス酸の還元型（ジチオール）であるジヒドロアスパラガス酸（３）を得、これをジメチルスルホキシド（ＤＭＳＯ）を用いた酸化反応に付すことによりアスパラガス酸（４）を製造することができる。 It has been reported that asparagusic acid can be chemically synthesized using halogenated isobutyric acid as a starting material (Non-Patent Document 2). That is, as shown in the reaction formula below, 3-bromo-2-(bromoethyl)propanoic acid (1) is reacted with potassium thioacetate to obtain 3-thioacetyl-2-(thioacetylethyl)propanoic acid (2), which is then deacetylated to obtain dihydroasparagusic acid (3), which is the reduced form (dithiol) of asparagusic acid, and asparagusic acid (4) can be produced by subjecting this to an oxidation reaction using dimethyl sulfoxide (DMSO).
しかしながら、ジヒドロアスパラガス酸をジメチルスルホキシドを用いて酸化して製造されたアスパラガス酸は、混在する不純物を介して重合され易いという問題があった。 However, asparagusic acid produced by oxidizing dihydroasparagusic acid with dimethyl sulfoxide has the problem that it is easily polymerized through the presence of impurities.
本発明は、重合物の生成が抑制され、長期間保存した場合でも安定なアスパラガス酸を調製する方法を提供することに関する。 The present invention relates to a method for preparing asparagusic acid that suppresses the formation of polymerization products and is stable even when stored for a long period of time.
本発明者らは、上記課題に鑑み、検討した結果、酸化反応終了後のアスパラガス酸を含む反応溶液を分液処理して有機層を回収し、これを水と混和する有機溶剤及び水の順で溶媒置換した後、凍結乾燥することにより、不純物を効果的に除去でき、アスパラガス酸の保存安定性を高められることを見出した。 In view of the above problems, the present inventors conducted a study and found that impurities can be effectively removed and the storage stability of asparagusic acid can be improved by separating the reaction solution containing asparagusic acid after the oxidation reaction is completed to recover the organic layer, which is then solvent-substituted with a water-miscible organic solvent and then with water, and then freeze-drying the organic layer.
すなわち、本発明は、ジヒドロアスパラガス酸を、ジメチルスルホキシドを用いて酸化することによりアスパラガス酸を製造する方法において、アスパラガス酸を含む反応溶液を分液処理して有機層を回収し、次いで水と混和する有機溶剤及び水の順で溶媒置換した後、凍結乾燥する、アスパラガス酸の調製方法、を提供する。 That is, the present invention provides a method for producing asparagusic acid by oxidizing dihydroasparagusic acid with dimethyl sulfoxide, in which a reaction solution containing asparagusic acid is subjected to a liquid separation treatment to recover an organic layer, which is then subjected to solvent replacement with a water-miscible organic solvent and then water, and then freeze-dried.
本発明によれば、酸化反応により生成する副生成物等を効率良く除去でき、保存安定性に優れた高純度なアスパラガス酸を調製することができる。 According to the present invention, by-products generated by the oxidation reaction can be efficiently removed, and highly pure asparagusic acid with excellent storage stability can be prepared.
本発明のアスパラガス酸の調製方法は、ジヒドロアスパラガス酸をジメチルスルホキシドを用いて酸化することによりアスパラガス酸を製造する方法において、アスパラガス酸を含む反応溶液を分液処理して有機層を回収し、次いで有機溶剤及び水の順で溶媒置換した後、凍結乾燥するものである。 The method for preparing asparagusic acid of the present invention is a method for producing asparagusic acid by oxidizing dihydroasparagusic acid with dimethyl sulfoxide, in which the reaction solution containing asparagusic acid is subjected to a separation treatment to recover the organic layer, which is then subjected to solvent replacement with an organic solvent and then water, and then freeze-dried.
ジヒドロアスパラガス酸をジメチルスルホキシドを用いてアスパラガス酸に酸化する反応は、一般的に、ジヒドロアスパラガス酸からアスパラガス酸を得るための効率的な酸化法として知られている。
反応は、ジヒドロアスパラガス酸をジメチルスルホキシド中で、６０～１００℃で３～２４時間、好ましくは６５～９０℃で、１０～２０時間、撹拌することにより行われる。
なお、ジヒドロアスパラガス酸は、粗精製物であっても良いが、吸着性樹脂やシリカゲル、ＯＤＳ修飾シリカゲル等を充填したカラム精製、または再結晶精製、好ましくは再結晶精製で高純度化されているものが、重合物生成抑制の点から好ましい。
The reaction of oxidizing dihydroasparagusic acid to asparagusic acid using dimethyl sulfoxide is generally known as an efficient oxidation method for obtaining asparagusic acid from dihydroasparagusic acid.
The reaction is carried out by stirring dihydroasparagusic acid in dimethyl sulfoxide at 60 to 100° C. for 3 to 24 hours, preferably at 65 to 90° C. for 10 to 20 hours.
Dihydroasparagusic acid may be a crude product, but is preferably highly purified by column purification packed with an adsorbent resin, silica gel, ODS-modified silica gel, or the like, or by recrystallization purification, preferably by recrystallization purification, from the viewpoint of suppressing the formation of polymerization products.
本発明において、「アスパラガス酸を含む反応溶液」とは、上記酸化反応終了後の反応溶液を指す。当該反応溶液には、アスパラガス酸及び、反応副生物等の不純物（纏めて「反応物」と称する）が含まれ得る。 In the present invention, the "reaction solution containing asparagusic acid" refers to the reaction solution after the above-mentioned oxidation reaction is completed. The reaction solution may contain asparagusic acid and impurities such as reaction by-products (collectively referred to as "reactants").
分液処理は、アスパラガス酸を含む反応溶液に、水又は水溶液、及び水と混和しない有機溶剤を添加する工程、水層及び有機層に分離する工程、及び有機層を回収する工程を含む。
分液処理に用いられる有機溶剤としては、水と混和しない、脂肪族炭化水素系溶剤（例えば、ヘキサン、シクロヘキサン、ペンタン、シクロペンタン等）、芳香族炭化水素系溶剤（例えば、トルエン、キシレン、ベンゼン等）、ハロゲン化炭化水素系溶剤（例えば、ジクロロメタン、ジクロロエタン、クロロホルム、四塩化炭素等）、エーテル系溶剤（例えば、ジエチルエーテル、テトラヒドロフラン、１，２－ジメトキシエタン、ジオキサン等）、ケトン系溶剤（例えば、メチルエチルケトン、アセトン等）、エステル系溶剤（例えば、酢酸エチル、酢酸ブチル）等が挙げられる。
有機溶剤は、１種を単独で又は２種以上を組み合わせて用いてもよい。有機溶媒としては、酢酸エチルが好ましい。
The liquid separation treatment includes a step of adding water or an aqueous solution and a water-immiscible organic solvent to the reaction solution containing asparagusic acid, a step of separating the solution into an aqueous layer and an organic layer, and a step of recovering the organic layer.
Examples of organic solvents used in the liquid separation treatment include water-immiscible aliphatic hydrocarbon solvents (e.g., hexane, cyclohexane, pentane, cyclopentane, etc.), aromatic hydrocarbon solvents (e.g., toluene, xylene, benzene, etc.), halogenated hydrocarbon solvents (e.g., dichloromethane, dichloroethane, chloroform, carbon tetrachloride, etc.), ether solvents (e.g., diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, dioxane, etc.), ketone solvents (e.g., methyl ethyl ketone, acetone, etc.), ester solvents (e.g., ethyl acetate, butyl acetate), and the like.
The organic solvent may be used alone or in combination of two or more. As the organic solvent, ethyl acetate is preferred.
水溶液としては、塩酸、硫酸、硝酸等の無機酸水溶液、中性リン酸緩衝液等を含む酸性～中性水系緩衝液が挙げられる。例えば、１～０．００１Ｍ、好ましくは０．１～０．０１Ｍの塩酸水溶液が好適に挙げられる。 Examples of aqueous solutions include aqueous solutions of inorganic acids such as hydrochloric acid, sulfuric acid, and nitric acid, and acidic to neutral aqueous buffer solutions including neutral phosphate buffer solutions. For example, an aqueous hydrochloric acid solution of 1 to 0.001 M, preferably 0.1 to 0.01 M, is preferred.
水又は水溶液と有機溶剤の使用比率は、用いる有機溶剤の種類によって異なるが、例えば、水又は水溶液は、反応工程で使用したジメチルスルホキシドに対して５～２０質量倍であり、有機溶剤の比率が水又は水溶液に対して１０～７０質量％、より好ましくは１５～６５質量％、さらに好ましくは２５～５５質量％である。 The ratio of water or aqueous solution to organic solvent used varies depending on the type of organic solvent used, but for example, the amount of water or aqueous solution is 5 to 20 times the amount of dimethyl sulfoxide used in the reaction step, and the ratio of organic solvent to water or aqueous solution is 10 to 70% by mass, more preferably 15 to 65% by mass, and even more preferably 25 to 55% by mass.
水層及び有機層の分離は、特に限定されず、例えば、デカンテーションや分液漏斗を用いた分液等、公知の分液手段を用いて行うことができる。また、分離後の有機層を飽和食塩水や水で洗浄することができる。 The separation of the aqueous layer and the organic layer is not particularly limited, and can be performed using a known separation method, such as decantation or separation using a separatory funnel. In addition, the separated organic layer can be washed with saturated saline or water.
次いで、回収された有機層は、水と混和する有機溶剤及び水の順で溶媒置換される。
溶媒置換は、ロータリーエバポレーターや真空ポンプ等を用いて減圧で溶媒を除去した後、水と混和する有機溶剤、次いで水を添加することにより行われる。添加する水と混和する有機溶剤の量は、減圧濃縮後の有機溶媒に対して１～５質量倍が好ましい。次いで添加する水の量は、減圧濃縮後の水と混和する有機溶剤溶液に対して、１～５質量倍が好ましい。
減圧で溶媒を除去（留去）する際の減圧度、温度は、特に制限されないが、９０～９５ＫＰａ（絶対圧力）程度、２０～６０℃程度が好ましい。
有機溶剤の除去は、反応生成物の重合化を抑制する点から、その体積が元の体積の５％以上となるように行うのが好ましく、７～４０％となるように行うのがより好ましく、１０～２０％となるように行うのがさらに好ましい。
The recovered organic layer is then subjected to solvent replacement with a water-miscible organic solvent and then water.
The solvent replacement is carried out by removing the solvent under reduced pressure using a rotary evaporator, a vacuum pump, or the like, and then adding a water-miscible organic solvent and then water. The amount of the water-miscible organic solvent to be added is preferably 1 to 5 times by mass relative to the organic solvent after the reduced pressure concentration. The amount of water to be added next is preferably 1 to 5 times by mass relative to the water-miscible organic solvent solution after the reduced pressure concentration.
The degree of vacuum and temperature when removing (distilling off) the solvent under reduced pressure are not particularly limited, but are preferably about 90 to 95 KPa (absolute pressure) and about 20 to 60°C.
From the viewpoint of suppressing polymerization of the reaction product, the organic solvent is removed preferably so that its volume is 5% or more of the original volume, more preferably 7 to 40%, and even more preferably 10 to 20%.
水と混和する有機溶剤としては、例えば、ニトリル系溶剤、アルコール系溶剤、エーテル系溶剤、アセトン等が挙げられ、１種を単独で又は２種以上を組み合わせて用いてもよい。
ここで、ニトリル系溶剤としては、例えば、アセトニトリル、プロピオニトリル等が挙げられる。アルコール系溶剤としては、メタノール、エタノール、プロピルアルコール、イソプロピルアルコール、ノルマルブタノール、イソブタノール等が挙げられる。エーテル系溶剤としては、１，４－ジオキサン、テトラヒドロフラン等が挙げられる。
水と混和する有機溶剤としては、アルコール系溶媒やニトリル系溶剤が好ましく、具体的には、エタノール、アセトニトリルが挙げられる。
Examples of organic solvents that are miscible with water include nitrile-based solvents, alcohol-based solvents, ether-based solvents, acetone, and the like. One type may be used alone, or two or more types may be used in combination.
Here, examples of the nitrile solvent include acetonitrile, propionitrile, etc. Examples of the alcohol solvent include methanol, ethanol, propyl alcohol, isopropyl alcohol, normal butanol, isobutanol, etc. Examples of the ether solvent include 1,4-dioxane, tetrahydrofuran, etc.
As the organic solvent miscible with water, alcohol-based solvents and nitrile-based solvents are preferable, and specific examples thereof include ethanol and acetonitrile.
最後に、溶媒を水に置換し、反応物の水溶液が凍結乾燥される。
ここで、用いられる水としては、特に限定されず、水道水、蒸留水、脱イオン水、超純水（例えば、超純水装置ミリＱ（Ｍｉｌｌｉ－Ｑ）により精製されたミリＱ水）等を挙げることができる。
凍結乾燥条件は、特に限定されず、例えば、－５℃～－８０℃で６～４８時間の凍結処理を行った後、凍結乾燥機の内部の真空度を１０～５００Ｐａにまで低下させた状態で、内部の温度を１０～４０℃にし、１５～５０時間保持することが挙げられる。
Finally, the solvent is replaced with water and the aqueous solution of the reaction product is freeze-dried.
The water used here is not particularly limited, and examples thereof include tap water, distilled water, deionized water, and ultrapure water (for example, Milli-Q water purified by an ultrapure water apparatus Milli-Q).
The freeze-drying conditions are not particularly limited, and examples thereof include performing a freezing treatment at −5° C. to −80° C. for 6 to 48 hours, and then lowering the degree of vacuum inside the freeze-dryer to 10 to 500 Pa, raising the internal temperature to 10 to 40° C., and maintaining the temperature for 15 to 50 hours.
斯くして得られた凍結乾燥物は、後述する試験例に示すとおり、アスパラガス酸の重合物の生成が抑制され、優れた保存安定性を有する。 The freeze-dried product thus obtained has excellent storage stability, as shown in the test examples described below, and the formation of asparagusic acid polymers is suppressed.
参考例１ ジヒドロアスパラガス酸の合成 Reference Example 1: Synthesis of dihydroasparagusic acid
（１）５００ ｍＬナスフラスコに３－ブロモ－２－（ブロモエチル）プロパン酸（東京化成化成工業、Ｂ３０８１）３０．０３ｇ（１２２．１ｍｍｏｌ）を秤りとり、１Ｍ ＮａＯＨ水溶液１４３ｍＬに溶解し、超純水２７０ｍＬを加えた。この溶液にチオ酢酸カリウム３４．７６ｇを加え、室温で１８時間撹拌した。０℃に冷却した後、６Ｍ ＨＣｌ水溶液３４ｍＬを滴下してｐＨ１に調整し酢酸エチル２００ｍＬ、１５０ｍＬ、１５０ｍＬで順に抽出した。有機層を合わせて０．１Ｍ塩酸の飽和食塩水３００ｍＬで洗浄し、無水硫酸ナトリウムで乾燥した。綿栓ろ過した濾液を減圧濃縮し、３－チオアセチル－２－（チオアセチルエチル）プロパン酸の粗精製物２９．８３ｇを得た。 (1) 30.03 g (122.1 mmol) of 3-bromo-2-(bromoethyl)propanoic acid (Tokyo Kasei Kasei Kogyo Co., Ltd., B3081) was weighed into a 500 mL recovery flask and dissolved in 143 mL of 1 M NaOH aqueous solution, and 270 mL of ultrapure water was added. 34.76 g of potassium thioacetate was added to this solution and stirred at room temperature for 18 hours. After cooling to 0°C, 34 mL of 6 M HCl aqueous solution was added dropwise to adjust the pH to 1, and extraction was performed with 200 mL, 150 mL, and 150 mL of ethyl acetate in that order. The organic layers were combined, washed with 300 mL of saturated saline solution of 0.1 M hydrochloric acid, and dried over anhydrous sodium sulfate. The filtrate filtered through a cotton plug was concentrated under reduced pressure to obtain 29.83 g of crude 3-thioacetyl-2-(thioacetylethyl)propanoic acid.
（２）２５０ｍＬナスフラスコに３－チオアセチル－２－（チオアセチルエチル）プロパン酸の粗精製物２９．８３ｇを秤りとり、２．５Ｍ ＮａＯＨ水溶液２５０ｍＬを加え、１８時間撹拌した。反応溶液を０℃に冷却し、６Ｍ ＨＣｌ水溶液１１５ｍＬを加えｐＨ１に調整した後、酢酸エチル３００、１５０、１５０ｍＬで抽出した。有機層を合わせて０．１Ｍ塩酸の飽和食塩水２００ｍＬで洗浄し、無水硫酸ナトリウムで乾燥した。綿栓ろ過した濾液を減圧濃縮し、３－チオ－２－（チオエチル）プロパン酸（ジヒドロアスパラガス酸）の粗精製物２０．５６ｇを得た。
粗精製物のうち１／２量の１０．５６ｇをダイアイオンＨＰ－２０（三菱ケミカル社）を用いて樹脂精製し、ジヒドロアスパラガス酸５．９７ｇ（３９．７ｍｍｏｌ，２工程収率換算６５％）を得た。樹脂精製では、ダイアイオンＨＰ－２０、５００ｇを０．１体積％ぎ酸水でコンディショニングした後、ジヒドロアスパラガス酸を０．１体積％ぎ酸水：メタノール＝１：１混合液３重量倍に溶解してチャージし、０．１体積％ぎ酸水：メタノール＝１：１で溶出した。溶出画分を減圧濃縮しメタノールを除去した後、凍結乾燥することでジヒドロアスパラガス酸の白色固体を得た。
(2) 29.83 g of crude 3-thioacetyl-2-(thioacetylethyl)propanoic acid was weighed into a 250 mL recovery flask, 250 mL of 2.5 M NaOH aqueous solution was added, and the mixture was stirred for 18 hours. The reaction solution was cooled to 0°C, and 115 mL of 6 M HCl aqueous solution was added to adjust the pH to 1, followed by extraction with 300, 150, and 150 mL of ethyl acetate. The combined organic layer was washed with 200 mL of saturated saline solution of 0.1 M hydrochloric acid, and dried over anhydrous sodium sulfate. The filtrate filtered through a cotton plug was concentrated under reduced pressure to obtain 20.56 g of crude 3-thio-2-(thioethyl)propanoic acid (dihydroasparagusic acid).
Half of the crude product (10.56 g) was purified with DIAION HP-20 (Mitsubishi Chemical Corporation) to obtain 5.97 g (39.7 mmol, calculated as 65% yield for two steps) of dihydroasparagusic acid. In the resin purification, 500 g of DIAION HP-20 was conditioned with 0.1% by volume formic acid water, and then dihydroasparagusic acid was dissolved in a 3-fold mixture of 0.1% by volume formic acid water:methanol = 1:1, and charged, and eluted with 0.1% by volume formic acid water:methanol = 1:1. The eluted fraction was concentrated under reduced pressure to remove the methanol, and then freeze-dried to obtain a white solid of dihydroasparagusic acid.
＜ジヒドロアスパラガス酸＞
１Ｈ ＮＭＲ（６００ＭＨｚ，ＤＭＳＯ－ｄ６）δ＝１２．５６（ｂｒ ｓ，１Ｈ，－ＣＯＯＨ），３．２９（ｂｒ ｓ，１Ｈ，－ＳＨ），２．７９－２．７１（ｍ，４Ｈ），２．６５（ｍ，１Ｈ），２．３８（ｂｒ ｓ，１Ｈ，－ＳＨ）．
１３Ｃ ＮＭＲ（１５０ＭＨｚ，ＤＭＳＯ－ｄ６）δ＝１７３．４， ５０．５（２Ｃ），２４．０（２Ｃ）．
ＨＲＭＳ（Ｎｅｇａｔｉｖｅ）［Ｍ－Ｈ］－ｍ／Ｚ＝１５０．９９０５（ｃａｌｃｄ １５０．９８９３ ｆｏｒ Ｃ４Ｈ７Ｏ２Ｓ２）
<Dihydroasparagusic acid>
1H NMR (600MHz, DMSO- d6 ) δ = 12.56 (br s, 1H, -COOH), 3.29 (br s, 1H, -SH), 2.79-2.71 (m, 4H), 2.65 (m, 1H), 2.38 (br s, 1H, -SH).
13C NMR (150MHz, DMSO- d6 ) δ=173.4, 50.5 (2C), 24.0 (2C).
HRMS (Negative) [M-H] - m/Z = 150.9905 ( calculated 150.9893 for C4H7O2S2 )
粗精製物のうち１／４量の４．６５５３ｇを秤りとり、ｎ－ヘキサン６０ｍＬを加えて６５℃で５分撹拌し、無色透明のジヒドロアスパラガス酸の加熱ヘキサン溶液と黄色スラリー状沈殿に分離した。加熱ヘキサン溶液のみを抜き出し、室温まで放冷し無色透明平板状の結晶を析出させた後、結晶をろ紙でろ別して減圧乾燥することで、ジヒドロアスパラガス酸の再結晶精製物３．０５２８ｇ（２０．３２ｍｍｏｌ，２工程収率換算６７％）を得た。 4.6553 g, a quarter of the crude product, was weighed out, 60 mL of n-hexane was added, and the mixture was stirred at 65°C for 5 minutes, separating the mixture into a colorless, transparent heated hexane solution of dihydroasparagusic acid and a yellow slurry-like precipitate. Only the heated hexane solution was extracted and allowed to cool to room temperature to precipitate colorless, transparent, flat crystals. The crystals were then filtered off with filter paper and dried under reduced pressure to obtain 3.0528 g (20.32 mmol, calculated as a two-step yield of 67%) of recrystallized purified dihydroasparagusic acid.
実施例１～３、比較例１ アスパラガス酸の製造
以下の実施例１～３及び比較例１に示す方法で、参考例１で製造したジヒドロアスパラガス酸からアスパラガス酸を合成し、精製した。
Examples 1 to 3 and Comparative Example 1 Production of Asparagusic Acid According to the methods shown in the following Examples 1 to 3 and Comparative Example 1, asparagusic acid was synthesized from the dihydroasparagusic acid produced in Reference Example 1 and purified.
（１）実施例１
１０ｍＬナスフラスコにジヒドロアスパラガス酸再結晶精製物５２０ｍｇを秤りとり、ＤＭＳＯ ５．０ｍＬに溶解し、７５℃で１９時間撹拌した。反応溶液を酢酸エチル／０．０２Ｍ塩酸水溶液＝５０ｍＬ／５０ｍＬで液／液分配し、水層をさらに酢酸エチル２０ｍＬで２回抽出した。有機層を合わせて０．０２Ｍ塩酸の飽和食塩水２０ｍＬで洗浄し、無水硫酸ナトリウムで乾燥した。綿栓ろ過した濾液を約１５ｍＬまで減圧濃縮した。さらにアセトニトリル３０ｍＬを加えた後、約１５ｍＬまで減圧濃縮し、さらに超純水５０ｍＬを加え、約４０ｍＬまで減圧濃縮して得た水溶液を凍結乾燥することで、アスパラガス酸の淡黄色粉末５４２ｍｇ（収率 ｑｕａｎｔ．、純度約９７％）を得た。
(1) Example 1
520 mg of the recrystallized dihydroasparagusic acid was weighed into a 10 mL eggplant flask, dissolved in 5.0 mL of DMSO, and stirred at 75°C for 19 hours. The reaction solution was partitioned liquid-liquid with ethyl acetate/0.02 M aqueous hydrochloric acid solution = 50 mL/50 mL, and the aqueous layer was further extracted twice with 20 mL of ethyl acetate. The organic layers were combined, washed with 20 mL of saturated aqueous sodium chloride solution of 0.02 M hydrochloric acid, and dried over anhydrous sodium sulfate. The filtrate filtered through a cotton plug was concentrated under reduced pressure to about 15 mL. After adding 30 mL of acetonitrile, the solution was concentrated under reduced pressure to about 15 mL, and then 50 mL of ultrapure water was added. The aqueous solution obtained by concentrating under reduced pressure to about 40 mL was freeze-dried to obtain 542 mg of pale yellow powder of asparagusic acid (yield quant., purity about 97%).
＜アスパラガス酸＞
１Ｈ ＮＭＲ（６００ＭＨｚ，ＤＭＳＯ－ｄ６）δ＝１２．７１（ｂｒ ｓ，１Ｈ，－ＣＯＯＨ），３．５３－３．４８（ｍ，１Ｈ），３．４０（ｄｄ，Ｊ＝１１．３，５．１Ｈｚ，２Ｈ），３．３０（ｄｄ，Ｊ＝１１．３，７．５Ｈｚ，２Ｈ）．
１３Ｃ ＮＭＲ（１５０ＭＨｚ，ＤＭＳＯ－ｄ６）δ＝１７２．８， ５０．２（２Ｃ），４１．０（２Ｃ）．
ＨＲＭＳ（Ｎｅｇａｔｉｖｅ）［Ｍ－Ｈ］－ｍ／Ｚ＝１４８．９７４７（ｃａｌｃｄ １４８．９７３１ ｆｏｒ Ｃ４Ｈ５Ｏ２Ｓ２）
<Asparagusic acid>
1 H NMR (600 MHz, DMSO-d 6 ) δ=12.71 (br s, 1H, -COOH), 3.53-3.48 (m, 1H), 3.40 (dd, J=11.3, 5.1 Hz, 2H), 3.30 (dd, J=11.3, 7.5 Hz, 2H).
13C NMR (150MHz, DMSO- d6 ) δ=172.8, 50.2 (2C), 41.0 (2C).
HRMS (Negative) [M-H] - m/Z = 148.9747 (calculated 148.9731 for C4H5O2S2 )
（２）実施例２
１０ｍＬナスフラスコにジヒドロアスパラガス酸再結晶精製物５００．２ｍｇを秤りとり、ＤＭＳＯ ５．０ｍＬに溶解し、７５℃で１７時間撹拌した。反応溶液を酢酸エチル／０．０２Ｍ塩酸水溶液＝５０ｍＬ／５０ｍＬで液／液分配し、水層をさらに酢酸エチル２０ｍＬで２回抽出した。有機層を合わせて０．０２Ｍ塩酸の飽和食塩水２０ｍＬで洗浄し、無水硫酸ナトリウムで乾燥した。綿栓ろ過した濾液を約１０ｍＬまで減圧濃縮した。さらにエタノール３０ｍＬを加えた後、約１０ｍＬまで減圧濃縮し、さらに超純水５０ｍＬを加え、約４５ｍＬまで減圧濃縮して得た水溶液を凍結乾燥することで、アスパラガス酸の淡黄色粉末４６５．８ｍｇ（収率９３％、純度約９８％）を得た。
(2) Example 2
500.2 mg of the recrystallized dihydroasparagusic acid was weighed into a 10 mL eggplant flask, dissolved in 5.0 mL of DMSO, and stirred at 75 ° C. for 17 hours. The reaction solution was partitioned into ethyl acetate/0.02 M aqueous hydrochloric acid solution = 50 mL/50 mL, and the aqueous layer was further extracted twice with 20 mL of ethyl acetate. The organic layers were combined, washed with 20 mL of saturated aqueous sodium chloride solution of 0.02 M hydrochloric acid, and dried over anhydrous sodium sulfate. The filtrate filtered through a cotton plug was concentrated under reduced pressure to about 10 mL. After adding 30 mL of ethanol, the solution was concentrated under reduced pressure to about 10 mL, and then 50 mL of ultrapure water was added. The aqueous solution obtained by concentrating under reduced pressure to about 45 mL was freeze-dried to obtain 465.8 mg of pale yellow powder of asparagusic acid (yield 93%, purity about 98%).
（３）実施例３
１００ｍＬナスフラスコにジヒドロアスパラガス酸粗精製物（純度７５％）３．３８３ｇを秤りとり、ＤＭＳＯ ３３．６ｍＬに溶解し、７５℃で１９時間撹拌した。反応溶液を酢酸エチル／０．０２Ｍ塩酸水溶液＝３００ｍＬ／３００ｍＬで液／液分配し、水層をさらに酢酸エチル１５０ｍＬ、１００ｍＬで２回抽出した。有機層を合わせて０．０２Ｍ塩酸の飽和食塩水１５０ｍＬで洗浄し、無水硫酸ナトリウムで乾燥した。綿栓ろ過した濾液を約５０ｍＬまで減圧濃縮した。さらにアセトニトリル２００ｍＬを加えた後、約６０ｍＬまで減圧濃縮し、さらに超純水４００ｍＬを加え、約３５０ｍＬまで減圧濃縮して得た水溶液を凍結乾燥することで、アスパラガス酸の淡黄色粉末３．２３０ｇ（純度７１％、収率９３％）を得た。
(3) Example 3
3.383 g of crude dihydroasparagusic acid (purity 75%) was weighed into a 100 mL eggplant flask, dissolved in 33.6 mL of DMSO, and stirred at 75 ° C. for 19 hours. The reaction solution was liquid/liquid distributed with ethyl acetate/0.02 M hydrochloric acid aqueous solution = 300 mL/300 mL, and the aqueous layer was further extracted twice with 150 mL and 100 mL of ethyl acetate. The organic layers were combined, washed with 150 mL of saturated saline solution of 0.02 M hydrochloric acid, and dried with anhydrous sodium sulfate. The filtrate filtered through a cotton plug was concentrated under reduced pressure to about 50 mL. Further, 200 mL of acetonitrile was added, and the solution was concentrated under reduced pressure to about 60 mL, and further 400 mL of ultrapure water was added, and the aqueous solution obtained by concentrating under reduced pressure to about 350 mL was freeze-dried to obtain 3.230 g of pale yellow powder of asparagusic acid (purity 71%, yield 93%).
（４）比較例１
１００ｍＬナスフラスコにジヒドロアスパラガス酸樹脂カラム精製物１０２ｍｇを秤りとり、ＤＭＳＯ １０ｍＬに溶解し、７５℃で１５時間撹拌した。反応溶液を酢酸エチル／０．０２Ｍ ＨＣｌ水溶液＝３０ｍＬ／１００ｍＬで液／液分配し、水層をさらに酢酸エチル３０ｍＬ、３０ｍＬで２回抽出した。有機層を合わせて０．０２Ｍ塩酸の飽和食塩水４０ｍＬで洗浄し、無水硫酸ナトリウムで乾燥した。綿栓ろ過した濾液を約５０ｍＬまで減圧濃縮し、溶媒を乾固させた後、真空ポンプで減圧乾燥すると、黄色ゴム状の固体１０１ｍｇ（アスパラガス酸とその重合物の混合物）が得られた。
得られたアスパラガス酸とその重合物の混合物から成るゴム状の固体は、水、クロロホルム、酢酸エチルに溶解せず、ＤＭＳＯに溶解した。
(4) Comparative Example 1
102 mg of the dihydroasparagusic acid resin column purified product was weighed into a 100 mL eggplant flask, dissolved in 10 mL of DMSO, and stirred at 75° C. for 15 hours. The reaction solution was partitioned liquid-liquid with ethyl acetate/0.02 M HCl aqueous solution = 30 mL/100 mL, and the aqueous layer was further extracted twice with 30 mL and 30 mL of ethyl acetate. The organic layers were combined, washed with 40 mL of saturated saline solution of 0.02 M hydrochloric acid, and dried over anhydrous sodium sulfate. The filtrate filtered through a cotton plug was concentrated under reduced pressure to about 50 mL, the solvent was dried, and then dried under reduced pressure with a vacuum pump to obtain 101 mg of a yellow rubber-like solid (a mixture of asparagusic acid and its polymer).
The resulting rubbery solid consisting of a mixture of asparagusic acid and its polymer was insoluble in water, chloroform, and ethyl acetate, but was soluble in DMSO.
試験例 アスパラガス酸の保存安定性
実施例１で調製したアスパラガス酸を、－２０℃で１か月保存後、褐色サンプル瓶中でアルゴン封入下分注し、－２０℃、５℃及び室温の各温度条件で９０日又は２３０日間保存した。保存後のサンプル５．０ｍｇをＤＭＳＯ－ｄ６（内部標準１，４－ビス(トリメチルシリル)ベンゼン５０３．１ｍｇ/Ｌ添加）０．６０ｍＬに溶解し、１Ｈ ＮＭＲ（６００ＭＨｚ，ＤＭＳＯ－ｄ６）を測定して、サンプル中のアスパラガス酸純度保存安定性を評価した。また比較例１で調製したゴム状固体（アスパラガス酸とその重合物の混合物）１．０ｍｇをＤＭＳＯ－ｄ６- ０．６０ｍＬに溶解し、アスパラガス酸重合物（δ＝３．０２ｐｐｍの幅広シグナル）の確認した。図１に１Ｈ ＮＭＲチャートを示す。
図１より、保存後の実施例１記載のアスパラガス酸の純度は９５％（－２０℃、９０日間保存後）、９１％（－２０℃、２３０日間保存後）、８３％（５℃、２３０日間保存後）、８１％（室温、２３０日間保存後）であり、比較例１（調製直後）はアスパラガス酸重合物由来のδ＝３．０２ｐｐｍ幅広シグナルを確認した。
Test Example Storage Stability of Asparagusic Acid Asparagusic acid prepared in Example 1 was stored at -20°C for one month, then dispensed into brown sample bottles under argon and stored at -20°C, 5°C, and room temperature for 90 or 230 days. 5.0 mg of the sample after storage was dissolved in 0.60 mL of DMSO-d 6 (internal standard 1,4-bis(trimethylsilyl)benzene 503.1 mg/L added), and 1H NMR (600 MHz, DMSO-d 6 ) was measured to evaluate the purity and storage stability of asparagusic acid in the sample. In addition, 1.0 mg of the rubber-like solid (a mixture of asparagusic acid and its polymer) prepared in Comparative Example 1 was dissolved in 0.60 mL of DMSO-d 6 -, and the presence of asparagusic acid polymer (broad signal at δ=3.02 ppm) was confirmed. The 1H NMR chart is shown in FIG. 1.
As shown in FIG. 1 , the purity of the asparagusic acid described in Example 1 after storage was 95% (after storage at -20°C for 90 days), 91% (after storage at -20°C for 230 days), 83% (after storage at 5°C for 230 days), and 81% (after storage at room temperature for 230 days). In Comparative Example 1 (immediately after preparation), a broad signal at δ = 3.02 ppm derived from an asparagusic acid polymer was confirmed.
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