JP6537068B2 - 光切断性蛍光標識プローブ - Google Patents
光切断性蛍光標識プローブ Download PDFInfo
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- JP6537068B2 JP6537068B2 JP2015167367A JP2015167367A JP6537068B2 JP 6537068 B2 JP6537068 B2 JP 6537068B2 JP 2015167367 A JP2015167367 A JP 2015167367A JP 2015167367 A JP2015167367 A JP 2015167367A JP 6537068 B2 JP6537068 B2 JP 6537068B2
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- diazirine
- coumarin
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- 239000000523 sample Substances 0.000 title description 14
- -1 thioxycarbonyl Chemical group 0.000 claims description 21
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N coumarin Chemical compound C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 claims description 20
- 125000003545 alkoxy group Chemical group 0.000 claims description 11
- 235000001671 coumarin Nutrition 0.000 claims description 10
- 229960000956 coumarin Drugs 0.000 claims description 10
- 239000003446 ligand Substances 0.000 claims description 8
- 229910052805 deuterium Inorganic materials 0.000 claims description 6
- 125000005740 oxycarbonyl group Chemical group [*:1]OC([*:2])=O 0.000 claims description 6
- 125000003917 carbamoyl group Chemical group [H]N([H])C(*)=O 0.000 claims description 4
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 4
- 125000004430 oxygen atom Chemical group O* 0.000 claims description 4
- WUUXOEFCIHOGRV-UHFFFAOYSA-N 3-(trifluoromethyl)-1h-diazirine Chemical group FC(F)(F)C1=NN1 WUUXOEFCIHOGRV-UHFFFAOYSA-N 0.000 claims 1
- 125000000332 coumarinyl group Chemical group O1C(=O)C(=CC2=CC=CC=C12)* 0.000 claims 1
- 125000004431 deuterium atom Chemical group 0.000 claims 1
- 229910052739 hydrogen Inorganic materials 0.000 description 32
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 24
- 150000001875 compounds Chemical class 0.000 description 21
- 238000006243 chemical reaction Methods 0.000 description 14
- 150000004845 diazirines Chemical group 0.000 description 13
- 102000004169 proteins and genes Human genes 0.000 description 11
- 108090000623 proteins and genes Proteins 0.000 description 11
- 238000000746 purification Methods 0.000 description 11
- 239000000243 solution Substances 0.000 description 11
- 238000004458 analytical method Methods 0.000 description 10
- 238000003776 cleavage reaction Methods 0.000 description 10
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 9
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 8
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- 239000002904 solvent Substances 0.000 description 7
- 239000000758 substrate Substances 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 108090000765 processed proteins & peptides Proteins 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- FANCTJAFZSYTIS-IQUVVAJASA-N (1r,3s,5z)-5-[(2e)-2-[(1r,3as,7ar)-7a-methyl-1-[(2r)-4-(phenylsulfonimidoyl)butan-2-yl]-2,3,3a,5,6,7-hexahydro-1h-inden-4-ylidene]ethylidene]-4-methylidenecyclohexane-1,3-diol Chemical compound C([C@@H](C)[C@@H]1[C@]2(CCCC(/[C@@H]2CC1)=C\C=C\1C([C@@H](O)C[C@H](O)C/1)=C)C)CS(=N)(=O)C1=CC=CC=C1 FANCTJAFZSYTIS-IQUVVAJASA-N 0.000 description 4
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical group [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 4
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 4
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
- 238000007876 drug discovery Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 4
- 235000019341 magnesium sulphate Nutrition 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000012044 organic layer Substances 0.000 description 4
- NROKBHXJSPEDAR-UHFFFAOYSA-M potassium fluoride Chemical compound [F-].[K+] NROKBHXJSPEDAR-UHFFFAOYSA-M 0.000 description 4
- 102000005962 receptors Human genes 0.000 description 4
- 108020003175 receptors Proteins 0.000 description 4
- 239000012925 reference material Substances 0.000 description 4
- 238000010898 silica gel chromatography Methods 0.000 description 4
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 102000013138 Drug Receptors Human genes 0.000 description 3
- 108010065556 Drug Receptors Proteins 0.000 description 3
- 150000001975 deuterium Chemical group 0.000 description 3
- 238000004949 mass spectrometry Methods 0.000 description 3
- GVOISEJVFFIGQE-YCZSINBZSA-N n-[(1r,2s,5r)-5-[methyl(propan-2-yl)amino]-2-[(3s)-2-oxo-3-[[6-(trifluoromethyl)quinazolin-4-yl]amino]pyrrolidin-1-yl]cyclohexyl]acetamide Chemical compound CC(=O)N[C@@H]1C[C@H](N(C)C(C)C)CC[C@@H]1N1C(=O)[C@@H](NC=2C3=CC(=CC=C3N=CN=2)C(F)(F)F)CC1 GVOISEJVFFIGQE-YCZSINBZSA-N 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- 238000012916 structural analysis Methods 0.000 description 3
- 125000001424 substituent group Chemical group 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- YZCKVEUIGOORGS-OUBTZVSYSA-N Deuterium Chemical compound [2H] YZCKVEUIGOORGS-OUBTZVSYSA-N 0.000 description 2
- 108010052285 Membrane Proteins Proteins 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 239000003377 acid catalyst Substances 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 239000012300 argon atmosphere Substances 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 2
- 229910052794 bromium Inorganic materials 0.000 description 2
- OSVHLUXLWQLPIY-KBAYOESNSA-N butyl 2-[(6aR,9R,10aR)-1-hydroxy-9-(hydroxymethyl)-6,6-dimethyl-6a,7,8,9,10,10a-hexahydrobenzo[c]chromen-3-yl]-2-methylpropanoate Chemical compound C(CCC)OC(C(C)(C)C1=CC(=C2[C@H]3[C@H](C(OC2=C1)(C)C)CC[C@H](C3)CO)O)=O OSVHLUXLWQLPIY-KBAYOESNSA-N 0.000 description 2
- 229940125898 compound 5 Drugs 0.000 description 2
- 239000000356 contaminant Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 2
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 2
- 229910052753 mercury Inorganic materials 0.000 description 2
- 229940098779 methanesulfonic acid Drugs 0.000 description 2
- 238000007699 photoisomerization reaction Methods 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- 239000011698 potassium fluoride Substances 0.000 description 2
- 235000003270 potassium fluoride Nutrition 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 125000006239 protecting group Chemical group 0.000 description 2
- 230000005855 radiation Effects 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000007363 ring formation reaction Methods 0.000 description 2
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 description 2
- AQTUACKQXJNHFQ-LURJTMIESA-N (2s)-2-[(2-methylpropan-2-yl)oxycarbonylamino]pentanedioic acid Chemical compound CC(C)(C)OC(=O)N[C@H](C(O)=O)CCC(O)=O AQTUACKQXJNHFQ-LURJTMIESA-N 0.000 description 1
- 125000004191 (C1-C6) alkoxy group Chemical group 0.000 description 1
- WBYWAXJHAXSJNI-VOTSOKGWSA-M .beta-Phenylacrylic acid Natural products [O-]C(=O)\C=C\C1=CC=CC=C1 WBYWAXJHAXSJNI-VOTSOKGWSA-M 0.000 description 1
- XEZNGIUYQVAUSS-UHFFFAOYSA-N 18-crown-6 Chemical compound C1COCCOCCOCCOCCOCCO1 XEZNGIUYQVAUSS-UHFFFAOYSA-N 0.000 description 1
- BZDFOBOXZAQPEH-UHFFFAOYSA-N 5-[3-(trifluoromethyl)diazirin-3-yl]benzene-1,3-diol Chemical compound OC1=CC(O)=CC(C2(N=N2)C(F)(F)F)=C1 BZDFOBOXZAQPEH-UHFFFAOYSA-N 0.000 description 1
- CFNMUZCFSDMZPQ-GHXNOFRVSA-N 7-[(z)-3-methyl-4-(4-methyl-5-oxo-2h-furan-2-yl)but-2-enoxy]chromen-2-one Chemical compound C=1C=C2C=CC(=O)OC2=CC=1OC/C=C(/C)CC1OC(=O)C(C)=C1 CFNMUZCFSDMZPQ-GHXNOFRVSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical group [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonium chloride Substances [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- VRXMDPXAPHMAAF-UHFFFAOYSA-N C(C)OC=1C=C(C=C(C=1)C1(N=N1)C(F)(F)F)O Chemical compound C(C)OC=1C=C(C=C(C=1)C1(N=N1)C(F)(F)F)O VRXMDPXAPHMAAF-UHFFFAOYSA-N 0.000 description 1
- WBYWAXJHAXSJNI-SREVYHEPSA-N Cinnamic acid Chemical compound OC(=O)\C=C/C1=CC=CC=C1 WBYWAXJHAXSJNI-SREVYHEPSA-N 0.000 description 1
- WBVGMEJQTUJDRX-UHFFFAOYSA-N ClCC1=CC(OC2=CC(=CC(=C12)OCC)C1(N=N1)C(F)(F)F)=O Chemical compound ClCC1=CC(OC2=CC(=CC(=C12)OCC)C1(N=N1)C(F)(F)F)=O WBVGMEJQTUJDRX-UHFFFAOYSA-N 0.000 description 1
- 239000004971 Cross linker Substances 0.000 description 1
- YXHKONLOYHBTNS-UHFFFAOYSA-N Diazomethane Chemical compound C=[N+]=[N-] YXHKONLOYHBTNS-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- CTAJKDSRGUWGAH-UHFFFAOYSA-N NCC1=CC(OC2=CC(=CC(=C12)OCC)C1(N=N1)C(F)(F)F)=O Chemical compound NCC1=CC(OC2=CC(=CC(=C12)OCC)C1(N=N1)C(F)(F)F)=O CTAJKDSRGUWGAH-UHFFFAOYSA-N 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 238000001261 affinity purification Methods 0.000 description 1
- 238000003450 affinity purification method Methods 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 125000001309 chloro group Chemical group Cl* 0.000 description 1
- 229930016911 cinnamic acid Natural products 0.000 description 1
- 235000013985 cinnamic acid Nutrition 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- OHLRLMWUFVDREV-UHFFFAOYSA-N ethyl 4-chloro-3-oxobutanoate Chemical compound CCOC(=O)CC(=O)CCl OHLRLMWUFVDREV-UHFFFAOYSA-N 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 238000010230 functional analysis Methods 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- JFCQEDHGNNZCLN-UHFFFAOYSA-L glutarate(2-) Chemical compound [O-]C(=O)CCCC([O-])=O JFCQEDHGNNZCLN-UHFFFAOYSA-L 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 125000005843 halogen group Chemical group 0.000 description 1
- 125000003707 hexyloxy group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])O* 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- HVTICUPFWKNHNG-UHFFFAOYSA-N iodoethane Chemical compound CCI HVTICUPFWKNHNG-UHFFFAOYSA-N 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000006317 isomerization reaction Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 150000002611 lead compounds Chemical class 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 102000006240 membrane receptors Human genes 0.000 description 1
- 108020004084 membrane receptors Proteins 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- WBYWAXJHAXSJNI-UHFFFAOYSA-N methyl p-hydroxycinnamate Natural products OC(=O)C=CC1=CC=CC=C1 WBYWAXJHAXSJNI-UHFFFAOYSA-N 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 125000005522 oxopentanoic acid group Chemical group 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 238000005222 photoaffinity labeling Methods 0.000 description 1
- 238000006349 photocyclization reaction Methods 0.000 description 1
- 238000006303 photolysis reaction Methods 0.000 description 1
- 230000015843 photosynthesis, light reaction Effects 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 230000020978 protein processing Effects 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
Landscapes
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Plural Heterocyclic Compounds (AREA)
Description
一方で、光クロスリンクと光切断反応は簡便であり、標的タンパク質同定に極めて有効な機能であることは証明されているから、上記した2つの光反応を制御し、損失を抑えることが課題である。
以下に本発明を詳細に説明する。
この反応で使用される酸触媒は、主にメタンスルホン酸や硫酸であり、それらを溶媒として利用することもできる。
この反応は、室温で、10〜20時間行えば良い。
一般式1の化合物は、例えば、非特許文献1に記載の方法またはそれに準じた方法で製造される。
この反応で使用される溶媒は、例えば、N,N−ジメチルホルムアミドが挙げられる。
この反応は、45〜55℃、12〜48時間行えばよい。
(a)0℃以下の低温下、ジアジリン基光反応により結合したタンパク質を共有結合により捕捉(クロスリンク)する。続いて、(b)30℃〜40℃で光を再照射すると、切断反応が速やかに起こり、リガンドが切断される。
ここで、光照射は、(a)ジアジリン基光反応において、0℃、350nm〜360nmで、 高圧水銀灯(250W)で10秒〜5分、あるいはブラックライトランプ(60W)で2分〜30分、(b)の切断反応において、室温から60℃、300nm〜320nmで、高圧水銀灯(250W)で10秒〜5分、あるいはブラックライトランプ(60W)では10分〜60分である。
製造例1
5-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzene-1,3-diol[化合物5]764mg(3.5mmol)をアセトン20mL中に溶解し、18-crown-6-ether 92.5mg(0.35mmol)、炭酸カリウム2.90g (21mmol)を加えた。この時溶液はオレンジ色に変化した。ヨードエタン140μL(1.75mmol)をゆっくり滴下し、室温で17時間撹拌した。反応溶液を酢酸エチルと1 M塩酸で1回、飽和食塩水で2回処理し、有機層を硫酸マグネシウムで乾燥後、減圧留去して溶媒を取り除いた。残渣をシリカゲルカラムクロマトグラフィー(ヘキサン:酢酸エチル=4:1〜1:1)で精製し、黄土色油状の化合物1a 168.8mg(0.6857mmol)を得た。また化合物5を収率71%で回収した。
化合物1a 169mg(0.688mmol)をメタンスルホン酸6.8mL中に溶解した。この時、溶液は黄色に変化した。その後、ethyl 4-chloroacetoacetate 187μL(1.37mmol)をゆっくりと滴下し、室温で15時間撹拌した。反応溶液を氷水中に注ぎ、白色沈殿の生成を確認した。沈殿物を濾取し、デシケーター内でシリカゲル顆粒と共に真空乾燥させ、白色固体の化合物3a 215mg(0.619mmol)を得た。
HRMS(ESI+) m/z 347.0410 (M-H+) C14H11ClF3N2O3 required 347.0417
化合物3a 52.0mg(0.150mmol)とBoc-L-グルタミン酸1-tert-ブチルエステル68.3mg(0.225mmol)とフッ化カリウム26.1mg(0.45mmol)をそれぞれ量りとり、乾燥N,N-ジメチルホルムアミド5mLに溶解させ、アルゴン雰囲気下、50℃で18時間撹拌した。反応溶液を酢酸エチルと水で2回、酢酸エチルと飽和食塩水で1回洗浄し、有機層を硫酸マグネシウムで乾燥後、減圧留去して溶媒を取り除いた。残渣をシリカゲルカラムクロマトグラフィー(ヘキサン:酢酸エチル=4:1〜2:1)で精製し、茶色油状の化合物1aa 87.9 mg(0.143mmol)を得た。
HRMS(ESI+) m/z 636.2139 (M-Na+) C28H34F3N3NaO9 required 636.2145
化合物1aa 33.6mg(0.055mmol)にトリフルオロ酢酸5 mLを加え、室温で1時間撹拌した。反応溶液を四塩化炭素で2回、クロロホルムで1回共沸し、薄茶色の油状物質を得た。これを高速液体クロマトグラフィーで精製し、白色個体の化合物1ab 15.2mg(0.033mmol)を得た。
HRMS(ESI+) m/z 458.1170 (M-H+) C19H19F3N3O7 required 458.1174
使用カラム:SHISEIDO C18 UG120 5μm 10mmI.D.×250 mm
A液:10% acetonitrile 89.9% H2O 0.1% TFA、B液:10%H2O 89.9% acetonitrile 0.1% TFA
流速:4 mL/min、
グラジエント:0 min(A:B=85%:15%)→30 min(A:B=20%:80%)
検出波長:215 nm, 295 nm, 360 nm, 295 nm(ex)→460 nm(em)
化合物1aa 17.4mg(0.028mmol)をメタノール3mL中に溶かした。この時、反応溶液は黄色になった。その後、1M塩酸3 mL(3.00mmol)を滴下し、室温で30分間撹拌した。反応溶液を、酢酸エチル、1M塩酸、水、飽和食塩水で各1回順次洗浄し、有機層を硫酸マグネシウムで乾燥後、減圧留去して溶媒を除去した。残渣をシリカゲルカラムクロマトグラフィー(ヘキサン:酢酸エチル=1:2)で分離精製し、白色個体の化合物3b 3.4mg(収率36%)を得た。
HRMS(ESI+) m/z 329.0744 (M-H+) C14H12F3N2O4 required 329.0749
化合物3a 52.0mg(0.15mmol)に28%アンモニア水溶液を15mL加え、アルゴン雰囲気下、50℃で4時間撹拌した。反応溶液を、酢酸エチルと1 M塩酸で2回、飽和食塩水で1回順次洗浄、有機層を硫酸マグネシウムで乾燥後、減圧下に溶媒を留去した。残渣をシリカゲルカラムクロマトグラフィー(クロロホルム 100%→クロロホルム:メタノール=10:1)で分離精製し、黄色個体の化合物3c 36.9mg(収率73%)を得た。
Claims (4)
- 一般式
で表されるクマリン型ジアジリン化合物。 - トリフルオロメチルジアジリンがクマリン環の5位〜7位に結合している請求項1のクマリン型ジアジリン化合物。
- Rが、低級アルコキシ基または低級アルコキシ基の同位体である請求項1または 2のク マリン型ジアジリン化合物。
- Aが、オキシカルボニルである請求項1〜3のクマリン型ジアジリン化合物。
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