JP4601060B2 - アジド化アミノ糖ヌクレオチド及びその応用 - Google Patents
アジド化アミノ糖ヌクレオチド及びその応用 Download PDFInfo
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- JP4601060B2 JP4601060B2 JP2005091056A JP2005091056A JP4601060B2 JP 4601060 B2 JP4601060 B2 JP 4601060B2 JP 2005091056 A JP2005091056 A JP 2005091056A JP 2005091056 A JP2005091056 A JP 2005091056A JP 4601060 B2 JP4601060 B2 JP 4601060B2
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- JP
- Japan
- Prior art keywords
- sugar
- reaction
- azidoacetyl
- amino
- diphosphate
- 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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- -1 Azido amino Chemical group 0.000 title claims description 65
- 239000002773 nucleotide Substances 0.000 title claims description 51
- 125000003729 nucleotide group Chemical group 0.000 title claims description 46
- 239000001177 diphosphate Substances 0.000 claims description 30
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 claims description 30
- 235000011180 diphosphates Nutrition 0.000 claims description 30
- 229950006780 n-acetylglucosamine Drugs 0.000 claims description 19
- 239000002777 nucleoside Substances 0.000 claims description 17
- 150000003833 nucleoside derivatives Chemical class 0.000 claims description 15
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- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 claims description 13
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 claims description 9
- 239000003112 inhibitor Substances 0.000 claims description 4
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- 238000006243 chemical reaction Methods 0.000 description 55
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- 125000004432 carbon atom Chemical group C* 0.000 description 13
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- 238000002360 preparation method Methods 0.000 description 9
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- 238000005160 1H NMR spectroscopy Methods 0.000 description 7
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- 125000006239 protecting group Chemical group 0.000 description 7
- 125000001424 substituent group Chemical group 0.000 description 7
- 239000000758 substrate Substances 0.000 description 7
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- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 7
- 238000012546 transfer Methods 0.000 description 7
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 206010028980 Neoplasm Diseases 0.000 description 6
- 125000000217 alkyl group Chemical group 0.000 description 6
- WGQKYBSKWIADBV-UHFFFAOYSA-N benzylamine Chemical compound NCC1=CC=CC=C1 WGQKYBSKWIADBV-UHFFFAOYSA-N 0.000 description 6
- 201000011510 cancer Diseases 0.000 description 6
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 6
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- 150000008064 anhydrides Chemical class 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- MSWZFWKMSRAUBD-QZABAPFNSA-N beta-D-glucosamine Chemical compound N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-QZABAPFNSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 1
- 229910052794 bromium Inorganic materials 0.000 description 1
- 125000005998 bromoethyl group Chemical group 0.000 description 1
- 125000005997 bromomethyl group Chemical group 0.000 description 1
- 125000004799 bromophenyl group Chemical group 0.000 description 1
- 159000000007 calcium salts Chemical class 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000005907 cancer growth Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 229920001429 chelating resin Polymers 0.000 description 1
- 238000012824 chemical production Methods 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 125000000068 chlorophenyl group Chemical group 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 235000021310 complex sugar Nutrition 0.000 description 1
- 125000004093 cyano group Chemical group *C#N 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 1
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 1
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 1
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 1
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 125000001028 difluoromethyl group Chemical group [H]C(F)(F)* 0.000 description 1
- UNXNGGMLCSMSLH-UHFFFAOYSA-N dihydrogen phosphate;triethylazanium Chemical compound OP(O)(O)=O.CCN(CC)CC UNXNGGMLCSMSLH-UHFFFAOYSA-N 0.000 description 1
- 125000002147 dimethylamino group Chemical group [H]C([H])([H])N(*)C([H])([H])[H] 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- JOZGNYDSEBIJDH-UHFFFAOYSA-N eniluracil Chemical compound O=C1NC=C(C#C)C(=O)N1 JOZGNYDSEBIJDH-UHFFFAOYSA-N 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 125000001301 ethoxy group Chemical group [H]C([H])([H])C([H])([H])O* 0.000 description 1
- HVJJYOAPXBPQQV-UHFFFAOYSA-N ethyl 2-azidoacetate Chemical compound CCOC(=O)CN=[N+]=[N-] HVJJYOAPXBPQQV-UHFFFAOYSA-N 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 1
- 125000000031 ethylamino group Chemical group [H]C([H])([H])C([H])([H])N([H])[*] 0.000 description 1
- 125000002534 ethynyl group Chemical group [H]C#C* 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- XRECTZIEBJDKEO-UHFFFAOYSA-N flucytosine Chemical compound NC1=NC(=O)NC=C1F XRECTZIEBJDKEO-UHFFFAOYSA-N 0.000 description 1
- 229960004413 flucytosine Drugs 0.000 description 1
- 229910052731 fluorine Inorganic materials 0.000 description 1
- 239000011737 fluorine Substances 0.000 description 1
- 125000004216 fluoromethyl group Chemical group [H]C([H])(F)* 0.000 description 1
- 125000002536 galactosaminyl group Chemical group 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 230000002140 halogenating effect Effects 0.000 description 1
- 238000003505 heat denaturation Methods 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 125000002349 hydroxyamino group Chemical group [H]ON([H])[*] 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000005040 ion trap Methods 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229910003002 lithium salt Inorganic materials 0.000 description 1
- 159000000002 lithium salts Chemical class 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 229940087646 methanolamine Drugs 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 125000000250 methylamino group Chemical group [H]N(*)C([H])([H])[H] 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- ZURGFCUYILNMNA-UHFFFAOYSA-N n-(7h-purin-6-yl)acetamide Chemical compound CC(=O)NC1=NC=NC2=C1NC=N2 ZURGFCUYILNMNA-UHFFFAOYSA-N 0.000 description 1
- HYZHKBPZFYNYNH-UHFFFAOYSA-N n-(trifluoromethyl)-7h-purin-6-amine Chemical compound FC(F)(F)NC1=NC=NC2=C1NC=N2 HYZHKBPZFYNYNH-UHFFFAOYSA-N 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- UQELSLLOPRNRNF-UHFFFAOYSA-N o-(7h-purin-6-yl)hydroxylamine Chemical compound NOC1=NC=NC2=C1NC=N2 UQELSLLOPRNRNF-UHFFFAOYSA-N 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- OJMIONKXNSYLSR-UHFFFAOYSA-N phosphorous acid Chemical class OP(O)O OJMIONKXNSYLSR-UHFFFAOYSA-N 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 230000000865 phosphorylative effect Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 229920001467 poly(styrenesulfonates) Polymers 0.000 description 1
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- UORVCLMRJXCDCP-UHFFFAOYSA-N propynoic acid Chemical compound OC(=O)C#C UORVCLMRJXCDCP-UHFFFAOYSA-N 0.000 description 1
- 125000002568 propynyl group Chemical group [*]C#CC([H])([H])[H] 0.000 description 1
- 235000011962 puddings Nutrition 0.000 description 1
- 150000003212 purines Chemical class 0.000 description 1
- VTGOHKSTWXHQJK-UHFFFAOYSA-N pyrimidin-2-ol Chemical compound OC1=NC=CC=N1 VTGOHKSTWXHQJK-UHFFFAOYSA-N 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000012134 supernatant fraction Substances 0.000 description 1
- 150000003536 tetrazoles Chemical class 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 125000003944 tolyl group Chemical group 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 150000003852 triazoles Chemical group 0.000 description 1
- 125000002023 trifluoromethyl group Chemical group FC(F)(F)* 0.000 description 1
- 150000004043 trisaccharides Chemical class 0.000 description 1
- 239000000439 tumor marker Substances 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 229940075420 xanthine Drugs 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
Description
本発明化合物は、前記式(I)で表されるアジド化アミノ糖ヌクレオチドである。
Bで示される「核酸塩基」とは、核酸に含まれるピリミジン塩基またはプリン塩基をいう。具体的には、ピリミジン塩基としては、4−アミノ−ピリミジン−2−オン(シトシン)、4−ヒドロキシ−5−メチル−ピリミジン−2−オン(チミン)、4−ヒドロキシ−ピリミジン−2−オン(ウラシル)等が挙げられる。プリン塩基としては、6−アミノプリン(アデニン)、2−アミノ−6−ヒドロキシプリン(グアニン)、2,6−ジヒドロキシプリン(キサンチン)、6−ヒドロキシプリン(ヒポキサンチン)等が挙げられる。
このような本発明化合物の中でも、Bがウラシル−1−イルである化合物、具体的には、ウリジン5’−(N−アジドアセチル−α−D−グルコサミニル)ジホスフェート、並びにウリジン5’−[2−{1H−[1,2,3]−4−カルボキシ−トリアゾリル}アセトアミド−2−デオキシ−α−D−ガラクトピラノシル]ジホスフェートが好適である。
本発明化合物は、式(III)化合物で示される単糖からベンジルアミンにより選択的に1位を脱保護することにより式(IV)化合物を得(第1工程)、得られた式(IV)化合物を2−クロロ−4H−1,3,2−ベンゾジオキサホスホリン−4−オンを用いてα選択的に亜リン酸化して式(V)化合物を得(第2工程)、得られた式(V)化合物を酸化反応に付してリン酸へと変換して式(VI)化合物を得(第3工程)、得られた式(VI)化合物とヌクレオシドモノホスホモルホリデート・4−モルホリン−N,N’−ジシクロヘキシルカルボジイミド塩と用いてカップリング反応により保護基を有する式(VII)で表されるアジド化アミノ糖ヌクレオチドを得(第4工程)、保護基を脱保護して本発明のアジド化アミノ糖ヌクレオチド(式(I)化合物)を得る(第5工程)方法により合成することができる。
出発物質として用いる式(III)化合物のアジドアセチル化グルコサミン又はアジドアセチル化ガラクトサミンは、ハロゲン化酸無水物を用いてグルコサミン又はガラクトサミンのアミノ基をハロゲン化アセチル化した後、アジド基へと変換することで調製することが可能である(Journal of the American Chemical Society (2002), 124, 14893-14902)。
第2工程は、リン酸化剤2−クロロ−4H−1,3,2−ベンゾジオキサホスホリン−4−オンを用いた亜リン酸化反応により式(IV)化合物から式(V)化合物を得る工程である。
第3工程は、式(V)化合物をテトラブチルヒドロペルオキシドを用いた酸化反応に付して式(VI)化合物を得る工程である。
第4工程は、式(VI)化合物とヌクレオシドモノホスフェート誘導体とのカップリング反応により保護基を有する式(VII)で表されるアジド化アミノ糖ヌクレオチドを得る工程である。
第5工程は、式(VII)化合物の保護基を脱保護して本発明のアジド化アミノ糖ヌクレオチド(式(I)化合物)を得る工程である。
保護基の除去は、使用した保護基で常用されている方法に従って行うことができる。
本発明化合物の酵素的な製造法は、酵素だけを使用する方法と酵母菌体と酵素を併用する方法の2つの方法に分類される。
すなわち、ヌクレオシド5’−トリリン酸とN−アジドアセチルグルコサミン又はN−アジドアセチルガラクトサミンから下記式(I)で表されるアジド化アミノ糖ヌクレオチドを酵素的に製造するにあたり、酵素としてキナーゼ、ムターゼ及び糖ヌクレオチドピロホスホリラーゼから選ばれた2種以上の酵素を使用することを特徴とする、アジド化アミノ糖ヌクレオチドの酵素的製造法に関するものである。
また、酵素の遺伝子がクローン化されている場合には、そのクローン化された酵素遺伝子を用いて常法により大腸菌などを宿主として大量生産させ、当該組換え菌より当該酵素を調製することも可能である。
すなわち、N−アジドアセチルグルコサミン又はN−アジドアセチルガラクトサミンの簡便な調製法としては、2−アジド酢酸エチルをエタノールなどのアルコール系有機溶媒に溶解し、水酸化ナトリウムなどのアルカリを添加後、この液にグルコサミン又はガラクトサミンを添加し反応させるという簡便な方法で、目的とするN−アジドアセチルグルコサミン又はN−アジドアセチルガラクトサミンを調製することができる。
本発明のアジド化アミノ糖ヌクレオチド及びその誘導体は、通常の糖供与体と競合するため、糖転移酵素による糖鎖伸長が阻害され、このメカニズムにより、たとえば癌細胞の増殖を防ぐ阻害剤として充分な効果が期待できるものである。
受容体糖及び糖供与体としてのアジド化アミノ糖ヌクレオチドの使用濃度としては、約1〜200mMが好ましい。
(1)1,3,4,6−テトラ−O−アセチル−N−アジドアセチル−D−グルコサミンの合成
2−アジド酢酸エチル(1.3g、10mmol)をエタノール(20ml)に溶解し、0.1M水酸化ナトリウム水溶液(100ml)を添加した後、室温で10分間攪拌した。反応溶液にグルコサミン塩酸塩(2.2g、10mmol)を添加し、さらに30分攪拌した。反応溶液の溶媒を減圧濃縮することにより除去し、さらにDMFを加え減圧濃縮することを三回繰り返すことで水を除去した。反応残留物をDMF(10ml)に溶解し、トリエチルアミン(1.4ml、10mmol)とジフェニルフォスフォリルアジド(2.2ml、10mmol)を加え、室温で20時間攪拌した。反応溶液にピリジン(20ml)、4ジメチルアミノピリジン(1g、8.2mmol)を加え、さらに無水酢酸(10ml)をゆっくり添加し室温で5時間攪拌した。反応溶液にメタノールを加え反応を停止し、反応溶媒を減圧濃縮により除去した。シリカゲルクロマトグラフィ(展開溶媒:ヘキサン/酢酸エチル=2/1〜1/2)で精製することにより標記化合物(2.9g,収率67%)を得た。
1,3,4,6−テトラ−O−アセチル−N−アジドアセチルグルコサミン(980mg,2.3mmol)をテトラヒドロフラン(10ml)に溶解し、ベンジルアミン(300μl,2.7mmol)を添加し室温で1日攪拌した。反応溶液の溶媒を減圧濃縮することにより除去し、残留物をシリカゲルクロマトグラフィ(展開溶媒:ヘキサン/酢酸エチル=1/1〜1/2)で精製することにより標記化合物(850mg,収率96%)を得た。
N−アジドアセチル−3,4,6−トリ−O−アセチル−グルコサミン(590mg、1.5mmol)をテトラヒドロフラン(10ml)に溶解し、トリエチルアミン(422μl、3.0mmol)と2−クロロ−4H−1,3,2−ベンゾジオキサホスホリン−4−オン(338mg、1.7mmol)を加え、室温で1時間攪拌した。反応溶液を減圧濃縮し、残留物にテトラヒドロフランを加え、沈殿物をろ過により除去した。ろ液を減圧濃縮し溶媒を除去した後、残留物をシリカゲルクロマトグラフィ(展開溶媒:クロロホルム/メタノール=5/1)で精製することにより標記化合物(689mg、収率83%)を得た。
N−アジドアセチル−3,4,6−トリ−O−アセチル−α−D−グルコサミニルホスファイト(350mg、0.63mmol)をテトラヒドロフランに溶解し、イオン交換カラムクロマトグラフィ(Amberlite IR120、H+フォーム)によりリン酸塩を遊離した。溶出液を減圧濃縮し、残留物をテトラヒドロフラン(15ml)に溶解した。5.0Mテトラブチルヒドロペルオキシド溶液(600μl,3mmol)、ヨウ素(10mg,0.039mmol)添加し、室温で11時間攪拌した。硫化ジメチルを加えることにより反応を停止し、混合物を減圧濃縮することにより溶媒を除去した。残留物をシリカゲルクロマトグラフィ(展開溶媒:クロロホルム/メタノール/トリエチルアミン=100/100/1)で精製することによりN−アジドアセチル−3,4,6−トリ−O−アセチル−α−D−グルコサミニルリン酸2トリエチルアミン塩(266mg,収率63%)を得た。
N−アジドアセチル−3,4,6−トリ−O−アセチル−α−D−グルコサミニルリン酸2トリエチルアミン塩(26mg,0.039mmol)にピリジン(2ml)に溶解した。ウリジンモノホスホモルホリデート・4−モルホリン−N,N’−ジシクロヘキシルカルボジイミド塩(32mg,0.47mmol)とテトラゾール(3mg,0.47mmol)を加え室温で2日間攪拌した。水を加えた後減圧濃縮することにより反応溶媒を除去し、イオン交換カラムクロマトグラフィ(現アマシャムバイオサイエンス社製デアエセルロース)(展開溶媒:炭酸水素アンモニウム緩衝液=0.01M〜0.2M)により分取を行い、減圧濃縮した。残留物をゲル濾過クロマトグラフィ(セファデックスG−10)により脱塩処理を行い、ジアンモニウムウリジン5’−(N−アジドアセチル−3,4,6−トリ−O−アセチル−α−D−グルコサミニル)ジホスフェートを(19mg,収率60%)得た。
ジアンモニウムウリジン5’−(N−アジドアセチル−3,4,6−トリ−O−アセチル−α−D−グルコサミニル)ジホスフェート(75mg,0.093mmol)を水(5ml)に溶解し、25%アンモニア水溶液を(5ml)を加え室温で3時間攪拌した。減圧濃縮することにより反応溶媒を除去しジアンモニウムウリジン5’−(N−アジドアセチル−α−D−グルコサミニル)ジホスフェートを(63mg,収率100%)得た。
ジアンモニウムウリジン5’−[2−{1H−[1,2,3]−4カルボキシ−トリアゾリル}アセトアミド−2−デオキシ−α−D−グルコピラノシル]ジホスフェートの合成
ジアンモニウムウリジン5’−(N−アジドアセチル−α−D−グルコサミニル)ジホスフェート(6.4mg,0.093mmol)をMeOH(3ml)に溶解し、ジイソプロピルエチルアミン(35μl、0.2mmol)、プロピオール酸(6μl、0.097mmol)、ヨウ化銅(2mg、0.011mmol)を加え室温で3時間攪拌した。反応溶液を減圧濃縮し、残留物をゲル濾過クロマトグラフィ(セファデックスG−10)により精製することでジアンモニウムウリジン5’−(2−{1H−[1,2,3]−4カルボキシ−トリアゾリル}アセトアミド−2−デオキシ−α−D−グルコピラノシル)ジホスフェートを(4.1mg,収率50%)得た。
(1)Haemophilus ducreyi β1,3−N−アセチルグルコサミン転移酵素をコードするlgtA遺伝子のクローニング
Haemophilus ducreyi 35000HPの染色体DNA(ATCC 700724D)を鋳型として、以下に示す2種類のプライマーDNAを常法に従って合成し、PCR法によりHaemophilus ducreyi lgtA遺伝子(EMBL / GENEBANK / DDBJ DATA BANKS、Accession No. AF536817)を増幅した。
プライマー(B):5'- atgtcgaccatgctgatttggaataacggg -3'
プラスミドpTrc-HDGnTを保持する大腸菌JM109菌を、100μg/mlのアンピシリンを含有する2xYT培地 50mlに植菌し、37℃で振とう培養した。1×108個/mlに達した時点で、培養液に最終濃度0.5 mMになるようにIPTGを添加し、20℃で20時間振とう培養を続けた。培養終了後、遠心分離(9,000xg, 20分)により菌体を回収し、5mlの緩衝液(50mM トリス塩酸(pH 7.5)、0.5M塩化ナトリウム、5mMメルカプトエタノール、10%(w/v)グリセロール、0.2%(w/v)CHAPS)に懸濁した。超音波処理を行って菌体を破砕し、さらに遠心分離(20,000xg、10分)により菌体残さを除去した。
10mM塩化マグネシウム、10mM塩化マンガン、20mMラクトース、UDP−N−アセチルグルコサミンを含有する100mM トリス塩酸緩衝液(pH7.5)に、β1,3−N−アセチルグルコサミン転移酵素を添加して30℃で4.5時間反応させた。また、β1,3−N−アセチルグルコサミン転移酵素の代わりにpTrc99Aを保持する大腸JM109株の菌体破砕液を用い同様の反応を行い、これをコントロールとした。
N−アジドアセチル−α−D−グルコサミニルβ1−3ラクトースの酵素合成
10mM塩化マグネシウム、10 mM塩化マンガン、20mM ラクトース、10mMジアンモニウムウリジン5’−(N−アジドアセチル−α−D−グルコサミニル)ジホスフェートを含む100mMトリス塩酸塩緩衝液(pH7.5)に、上記実施例2により調製したβ1,3−N−アセチルグルコサミン転移活性を有する酵素液(0.012units/ml反応液)を添加し、30℃で、24時間反応を行った。
反応液を90℃、3分間の熱処理を行った後、遠心分離(20,000xg、10分)により不溶性画分を除去した。Dionex DX−300による分析を行ったところ、糖受容体であるラクトースの減少から約3mMのN−アジドアセチル−α−D−グルコサミニルβ1−3ラクトースの生成が推定された。さらに当該処理液をダウエックス50W(H+)(ダウケミカル社製)、IRA410(OH−)(ローム・アンド・ハース社製)樹脂に通液し、減圧乾燥により乾固させた後、蒸留水で再度溶解させた試料をESI−イオントラップ質量分析装置(日立ハイテクノロジー社製)を用いて分析を行った結果、[M+Na]+(m/z610)及び[M+K]+(m/z626)のピークを検出したころから、N−アジドアセチル−α−D−グルコサミニルβ1−3ラクトースが生成したことを確認した。
(1)酵素液の調製
3種類の酵素(枯草菌グルコキナーゼ、酵母N−アセチルグルコサミン(GlcNAc)リン酸ムターゼ、ならびに大腸菌UDP−GlcNAcピロホスホリラーゼ)を含む酵素液を文献(Okuyama, K., et al., Biosci. Biotechnol. Biochem.,64(2),386-392(2000))に記載の方法で調製した。なお、各酵素活性も前述の文献記載の方法で測定した。
2−アジド酢酸エチル(5.3g、41mmol)をエタノール(41mL)に溶解し、1M水酸化ナトリウム水溶液(41mL)を添加した後、室温で10分間攪拌した。反応溶液にグルコサミン塩酸塩(8.0g、37mmol)を添加し、さらに30分攪拌した。反応溶液の溶媒を減圧濃縮することにより除去し、さらにジメチルホルムアミドを加え減圧濃縮することを三回繰り返すことで水を除去した。反応残留物をDMF(50mL)に溶解し、トリエチルアミン(5.7mL、41mmol)とジフェニルフォスフォリルアジド(8.7mL、41mmol)を加え、室温で1時間攪拌した。反応溶媒を減圧濃縮により除去し、残留物を逆相カラムクロマトグラフィー(WakogelR 50C18、20mL、展開溶媒:水)で精製することにより標記化合物(5.6g,収率58%)を得た。
100mMトリス塩酸緩衝液(pH7.8)、10mM塩化マグネシウム、0.1mM EDTA、5mM 5’−UTP・3Na、5mM N−アジドアセチル−D−グルコサミン、5mM 5’−ATP・3Na、20μMグルコース−1,6−二リン酸、3.7ユニット/mLグルコキナーゼ、2.5ユニット/mL GlcNAcリン酸ムターゼ、および1.1ユニット/mL UDP−GlcNAcピロホスホリラーゼを含む溶液0.5mLを調製し、37℃で反応を行った。
400mMグルコース、100mM N−アジドアセチル−D−グルコサミン、100mM 5’−UMP、200mM リン酸カリウム(pH8.0)、10mM塩化マグネシウム、5%(w/v)乾燥パン酵母(オリエンタル酵母工業)、4.9ユニット/mLグルコキナーゼ、3.3ユニット/mL GlcNAcリン酸ムターゼ、および1.5ユニット/mL UDP−GlcNAcピロホスホリラーゼを含む溶液5mLを調製し、撹拌しながら23℃で合成反応を実施した。反応開始16,24,40,48時間目に2Mのグルコース溶液を0.1mLずつ反応液に添加した。経時的に反応液の一部を採取し、HPLC分析を行った。ウリジン5’−(N−アジドアセチル−α−D−グルコサミニル)ジホスフェートの合成量は反応開始から経時的に増加し、反応64時間で54.6mMに達した(対N−アジドアセチル−D−グルコサミンモル収率54.6%)。
Claims (2)
- 前記N−アセチルグルコサミン転移酵素阻害剤が、β−1,3−N−アセチルグルコサミン転移酵素阻害剤である請求項1記載の阻害剤。
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