JP6483250B2 - エルゴチオネインおよび関連化合物の合成方法 - Google Patents
エルゴチオネインおよび関連化合物の合成方法 Download PDFInfo
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- JP6483250B2 JP6483250B2 JP2017515766A JP2017515766A JP6483250B2 JP 6483250 B2 JP6483250 B2 JP 6483250B2 JP 2017515766 A JP2017515766 A JP 2017515766A JP 2017515766 A JP2017515766 A JP 2017515766A JP 6483250 B2 JP6483250 B2 JP 6483250B2
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- ergothioneine
- compound
- sulfoxide
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- 229940093497 ergothioneine Drugs 0.000 title claims description 71
- 238000000034 method Methods 0.000 title claims description 50
- SSISHJJTAXXQAX-ZETCQYMHSA-N L-ergothioneine Chemical compound C[N+](C)(C)[C@H](C([O-])=O)CC1=CNC(=S)N1 SSISHJJTAXXQAX-ZETCQYMHSA-N 0.000 title claims description 44
- 150000001875 compounds Chemical class 0.000 title claims description 32
- 230000002194 synthesizing effect Effects 0.000 title claims description 6
- -1 β-amino-β-carboxyethyl Chemical group 0.000 claims description 79
- UCKMPCXJQFINFW-UHFFFAOYSA-N Sulphide Chemical compound [S-2] UCKMPCXJQFINFW-UHFFFAOYSA-N 0.000 claims description 44
- 150000003462 sulfoxides Chemical class 0.000 claims description 31
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 27
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 claims description 27
- 108090000790 Enzymes Proteins 0.000 claims description 25
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- 235000007682 pyridoxal 5'-phosphate Nutrition 0.000 claims description 22
- 239000011589 pyridoxal 5'-phosphate Substances 0.000 claims description 22
- 229960001327 pyridoxal phosphate Drugs 0.000 claims description 22
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 claims description 19
- 229910052757 nitrogen Inorganic materials 0.000 claims description 18
- 229960002885 histidine Drugs 0.000 claims description 16
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- 150000003457 sulfones Chemical class 0.000 claims description 14
- PCLIMKBDDGJMGD-UHFFFAOYSA-N N-bromosuccinimide Chemical compound BrN1C(=O)CCC1=O PCLIMKBDDGJMGD-UHFFFAOYSA-N 0.000 claims description 13
- 150000002596 lactones Chemical class 0.000 claims description 13
- 239000000203 mixture Substances 0.000 claims description 10
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- 125000002883 imidazolyl group Chemical group 0.000 claims description 7
- RAKGGEXKAWATLR-AWEZNQCLSA-N C(C1=CC=CC=C1)N1C=NC(=C1)C[C@@H](C(=O)O)N(C)C Chemical compound C(C1=CC=CC=C1)N1C=NC(=C1)C[C@@H](C(=O)O)N(C)C RAKGGEXKAWATLR-AWEZNQCLSA-N 0.000 claims description 6
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 5
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- 230000002378 acidificating effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000009056 active transport Effects 0.000 description 1
- 238000011949 advanced processing technology Methods 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- 125000004429 atom Chemical group 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- AGEZXYOZHKGVCM-UHFFFAOYSA-N benzyl bromide Chemical compound BrCC1=CC=CC=C1 AGEZXYOZHKGVCM-UHFFFAOYSA-N 0.000 description 1
- 150000001576 beta-amino acids Chemical class 0.000 description 1
- 229960003237 betaine Drugs 0.000 description 1
- 230000003592 biomimetic effect Effects 0.000 description 1
- 230000002051 biphasic effect Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- TWFZGCMQGLPBSX-UHFFFAOYSA-N carbendazim Chemical compound C1=CC=C2NC(NC(=O)OC)=NC2=C1 TWFZGCMQGLPBSX-UHFFFAOYSA-N 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 239000012468 concentrated sample Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000005100 correlation spectroscopy Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 238000007257 deesterification reaction Methods 0.000 description 1
- MHDVGSVTJDSBDK-UHFFFAOYSA-N dibenzyl ether Chemical group C=1C=CC=CC=1COCC1=CC=CC=C1 MHDVGSVTJDSBDK-UHFFFAOYSA-N 0.000 description 1
- 235000015872 dietary supplement Nutrition 0.000 description 1
- IJKVHSBPTUYDLN-UHFFFAOYSA-N dihydroxy(oxo)silane Chemical compound O[Si](O)=O IJKVHSBPTUYDLN-UHFFFAOYSA-N 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 101150003448 egtB gene Proteins 0.000 description 1
- 101150065774 egtC gene Proteins 0.000 description 1
- 101150005220 egtD gene Proteins 0.000 description 1
- 238000010931 ester hydrolysis Methods 0.000 description 1
- 125000006125 ethylsulfonyl group Chemical group 0.000 description 1
- 230000001815 facial effect Effects 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000008098 formaldehyde solution Substances 0.000 description 1
- 125000002642 gamma-glutamyl group Chemical group 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-L glutamate group Chemical group N[C@@H](CCC(=O)[O-])C(=O)[O-] WHUUTDBJXJRKMK-VKHMYHEASA-L 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000005570 heteronuclear single quantum coherence Methods 0.000 description 1
- 150000002411 histidines Chemical class 0.000 description 1
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 238000005984 hydrogenation reaction Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 1
- 230000000155 isotopic effect Effects 0.000 description 1
- 238000001948 isotopic labelling Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000000622 liquid--liquid extraction Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 230000000269 nucleophilic effect Effects 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000000197 pyrolysis Methods 0.000 description 1
- 125000001453 quaternary ammonium group Chemical group 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000010282 redox signaling Effects 0.000 description 1
- 238000006268 reductive amination reaction Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 239000011877 solvent mixture Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 125000000446 sulfanediyl group Chemical group *S* 0.000 description 1
- 125000004434 sulfur atom Chemical group 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 150000003568 thioethers Chemical class 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical compound CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 description 1
- 201000008827 tuberculosis Diseases 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
- C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
- C07D233/66—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D233/84—Sulfur atoms
-
- 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
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/4164—1,3-Diazoles
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Low-Molecular Organic Synthesis Reactions Using Catalysts (AREA)
- Enzymes And Modification Thereof (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Description
nは0、1または2であり、
RはHまたは
またはその生理学的に許容される塩、互変異性体、立体異性体、もしくは立体異性体の混合物の合成方法であって、
a)式11のN−ベンジル保護ヒスチジンを脱保護して式12のN−ベンジルヒスチジンを形成する工程、
f)式15の化合物を式IIのスルホキシドに変換する工程
g)式15の化合物を式IIIのスルホンに変換する工程
h)式15の化合物を式IVのエルゴチオネイン(ESH)に変換する工程、
nが0の場合、
Rは
nが1の場合、
Rは
nが2の場合、
Rは
nが0の場合、
RはHであってよい。
nは0、1または2であり、
RはHまたは
またはその生理学的に許容される塩、互変異性体、立体異性体、もしくは立体異性体の混合物の合成方法を提供する。
a)式11のN−ベンジル保護ヒスチジンを脱保護して式12のN−ベンジルヒスチジンを形成する工程、
f)式15の化合物を式IIのスルホキシドに変換する工程
g)式15の化合物を式IIIのスルホンに変換する工程
h)式15の化合物を式IVのエルゴチオネイン(ESH)に変換する工程、
本出願人は、N−ベンジル保護ヘルシニン中間体を1当量のN−ブロモスクシンイミド(NBS)で臭素化することにより、溶媒としてDMFを使用して、より安定なN−ベンジル脱保護5−ブロモへルシニンラクトン誘導体が収率90%(w/w)で得られることを見出した。この方法の成功は、他の既知の方法と比較して、位置選択的C−5臭素化にある。しかしながら、さらに驚くべきことに、反応物に対して2モル当量のNBS試薬を使用すると、N−ベンジル基の前例のない脱保護が起こり、その結果、N−ベンジル基の新たなin situ脱保護が起こる。その後の方法工程は、この後者の中間体を介して進行する場合、ほぼ定量的であり、比較的簡単であり、すべて室温で、短縮され、80%(w/w)の全合成収率が可能になる。したがって、本発明の方法は、以前に公開されたいずれの方法よりも少なくとも2倍優れた全収率を提供することができる。最終工程は、化学的、生合成的または微生物的手段のいずれかで成し遂げることができる。化学変換は熱分解C−S切断を伴う。20 あるいは、スルフィド基質またはスルホキシド基質の生体模倣ピリドキサールリン酸(PLP)媒介切断や、M.smegmatisの粗酵素抽出物によってもESHが得られた。(スキーム3および4)。
すべての溶媒は適切な技法によって乾燥させ、使用前に新たに蒸留した。市販の試薬はすべてSigma−AldrichおよびMerckから購入し、さらに精製することなく使用した。
S−(β−アミノ−β−カルボキシエチル)エルゴチオネインスルホキシドまたは(2S)−N,N,N−2−トリメチルアンモニウム−3−[2−((2R)−2−アミノ−2−ヒドロキシカルボニル)エチルスルフィニル)−1H−イミダゾール−4−イル]プロパン酸(II)26
Mc2155(M.smegmatis)の増殖条件
M.smeg培養液(800ml)を指数期まで増殖させた後、乾燥させて乾燥菌体10gを得た。得られたM.smeg細胞のペレットを、その後、必要になるまで−80℃で保存した。
M.smeg細胞を4℃で35分間超音波処理し(25台のパルサー)、続いてリン酸カリウム緩衝液(60ml;pH7)を加えた。この溶液を4℃で10分間撹拌させ、その後3000rpmで20分間遠心分離した。上清を回収し、測定し、次いで適切な量の硫酸アンモニウムを4℃で一晩撹拌しながら徐々に加えて60〜70%飽和させた。28
ピリドキサールリン酸(10ml;20μM)、リン酸カリウム緩衝液(8ml;50mM;pH7)および(2ml;1mM EDTA)を含有する緩衝液混合物(20ml;pH7)に、複合総タンパク質アンモニウム塩沈殿物を再懸濁させた。
総タンパク質濃度を決定するために、タンパク質DcアッセイおよびBradfordアッセイを使用した。Bradfordの検量線は、この場合にはタンパク質Dcよりも正確であることが判明した(図13)。
材料および方法
分析は、UHPLC Agilent 1290 Infinity Series(ドイツ)、すなわちAgilentジェット流イオン化源(正イオン化モード)(ESI+)およびカラム(Polaris 3 C18 Ether 100×2mm、粒子サイズ3μm、Agilent、ドイツ)を備えた正確な質量分析計Agilent 6530 Qradrupole Time Of Flight(QTOF)で行った。
すべての代謝産物中に存在する第四級アンモニウム基の極性および電荷のために、UHPLC(Eclipse+C18 RRHD 1.8μm、2.1×50)カラム上でESHが良好に保持されないことが観察された(保持時間=0.8分)。これ以降の分析はすべて、セクションS3.1に記載されているように、改善されたカラムを使用して行った(保持時間=1.5分)(図14〜図16)。
実験を三連で行い、数回(4回以上)繰り返したところ、これらの結果は再現可能であることが判明した。
ESHの定量のための検量線を作成するために、8つの異なる濃度(0.78、1.56、3.125、6.25、12.5、25、50、100ng/ml)のESHを三連調製し、ESHについて0.78ng/mlの定量限界(LOQ)を得た。これは、L−Z Wangらが見出したものと同様であった。15 ESHの検出限界(LOD)は9pg/mLであった。ESHの保持時間は1.5分であった。良好な対称ピークが、ESH標準試料および分析された反応試料の両方で得られた(図17〜図19)。
20mMのTris HCl(pH=7.4)、20mMのNaCl、0.2MmのFeSO4・7H2O、0.5mMのメルカプトエタノール、83μlの粗酵素、および50mMの(1)S−(β−アミノ−β−カルボキシエチル)エルゴチオネインスルフィド(15)、(2)S−(β−アミノ−β−カルボキシエチル)エルゴチオネインスルホキシド(II)、(3)S−(β−アミノ−β−カルボキシエチル)エルゴチオネインスルホン(III)または(4)対照(粗酵素のみ基質なし)のいずれかを含む100μlの反応物(1〜4)3セット。粗酵素反応物を37℃で1日間インキュベートした。混合物を90℃で2分間加熱することによって反応を停止させ、続いて凍結乾燥し、その後LC緩衝液中で再構築してからLC/MSによる分析を行った(図20)。
20mMのTris HCl(pH=7.4)、ならびに50mMの(1)S−(β−アミノ−β−カルボキシエチル)エルゴチオネインスルフィド(15)およびPLP、(2)S−(β−アミノ−β−カルボキシエチル)エルゴチオネインスルホキシド(II)およびPLP、または(3)S−(β−アミノ−β−カルボキシエチル)エルゴチオネインスルホン(III)およびPLPのいずれかを含む100μlの反応物(1〜3)3セット。非酵素反応物を37℃で1日間インキュベートし、続いて凍結乾燥し、その後LC緩衝液中で再構築してからLC/MSによる分析を行った(図2および図3)。
ヘルシニン−d3(7)は、市販のL−ヒスチジンから出発する2段階反応で合成した(スキーム2)。第1段階は、ホルムアルデヒド水溶液およびトリアセトキシ水素化ホウ素ナトリウムを使用してN,N−ジメチルヒスチジン(6)を得る還元的アミノ化を含んだ。第2段階は、塩基性条件下でd3ヨウ化メチルを使用して粗N,N−ジメチルヒスチジン(6)を四級化してヘルシニン−d3(7)を得ることを含んだ。
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Claims (19)
- 式V
nは0、1または2であり、
RはHまたは
またはその生理学的に許容される塩、互変異性体、立体異性体、もしくは立体異性体の混合物の合成方法であって、
a)式11のN−ベンジル保護ヒスチジンを脱保護して式12のN−ベンジルヒスチジンを形成する工程、
要すれば、工程(f)〜(h):
f)式15の化合物を式IIのスルホキシドに変換する工程
g)式15の化合物を式IIIのスルホンに変換する工程
h)式15の化合物を式IVのエルゴチオネイン(ESH)に変換する工程、
- nは0であり、
RはHである、請求項1に記載の方法。 - 式11の化合物が、Nα−Boc−N(im)−ベンジル保護L−ヒスチジンである、請求項1に記載の方法。
- 工程(d)において、ジメチルホルムアミド(DMF)およびN−ブロモスクシンイミド(NBS)を使用して5−ブロモへルシニンラクトンを形成する、請求項1に記載の方法。
- 工程(d)において、化合物14に対して少なくとも2モル当量のNBSを使用する、請求項8に記載の方法。
- 5−ブロモへルシニンラクトンが、工程(e)を実施する前に、工程(d)の間に形成される反応性中間体である、請求項1に記載の方法。
- 工程(e)において、システインを使用して式15の化合物を形成する、請求項1に記載の方法。
- 工程(d)および(e)がワンポット合成で行われる、請求項1に記載の方法。
- 工程(h)において、ピリドキサール−5リン酸(PLP)を使用して式IVのエルゴチオネインを形成する、請求項1に記載の方法。
- 工程(f)で形成された式IIのスルホキシドをさらに式IVのエルゴチオネインに変換する、請求項1に記載の方法。
- 式IIのスルホキシドをegtE遺伝子によってコードされる酵素と接触させて式IVのエルゴチオネインを形成する、請求項14に記載の方法。
- 式IIのスルホキシドをEgtE酵素と接触させて式IVのエルゴチオネインを形成する、請求項14に記載の方法。
- 工程(e)で形成された式15のスルフィド、または工程(a)〜(d)で形成された化合物のいずれか1つが、安定同位体で標識されている、請求項1に記載の方法。
- 前記同位体が重水素である、請求項17に記載の方法。
- 標識されている化合物が5−ブロモヘルシニンラクトンである、請求項17に記載の方法。
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