JP6145742B2 - 蛍光性質量標識プローブ - Google Patents
蛍光性質量標識プローブ Download PDFInfo
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- JP6145742B2 JP6145742B2 JP2013006647A JP2013006647A JP6145742B2 JP 6145742 B2 JP6145742 B2 JP 6145742B2 JP 2013006647 A JP2013006647 A JP 2013006647A JP 2013006647 A JP2013006647 A JP 2013006647A JP 6145742 B2 JP6145742 B2 JP 6145742B2
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- 239000000523 sample Substances 0.000 title claims description 22
- -1 diazirine compound Chemical class 0.000 claims description 16
- WBYWAXJHAXSJNI-UHFFFAOYSA-N methyl p-hydroxycinnamate Natural products OC(=O)C=CC1=CC=CC=C1 WBYWAXJHAXSJNI-UHFFFAOYSA-N 0.000 claims description 10
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- WBYWAXJHAXSJNI-SREVYHEPSA-N Cinnamic acid Chemical compound OC(=O)\C=C/C1=CC=CC=C1 WBYWAXJHAXSJNI-SREVYHEPSA-N 0.000 claims description 9
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- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 6
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical group [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 claims description 5
- 239000000470 constituent Substances 0.000 claims description 2
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- DNSISZSEWVHGLH-UHFFFAOYSA-N butanamide Chemical compound CCCC(N)=O DNSISZSEWVHGLH-UHFFFAOYSA-N 0.000 description 4
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- 238000005886 esterification reaction Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- KZENFXVDPUMQOE-UHFFFAOYSA-N ethyl 2-(triphenyl-$l^{5}-phosphanylidene)propanoate Chemical compound C=1C=CC=CC=1P(C=1C=CC=CC=1)(=C(C)C(=O)OCC)C1=CC=CC=C1 KZENFXVDPUMQOE-UHFFFAOYSA-N 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 238000000105 evaporative light scattering detection Methods 0.000 description 1
- 238000001506 fluorescence spectroscopy Methods 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000010230 functional analysis Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 125000004051 hexyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 125000005462 imide group Chemical group 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000009878 intermolecular interaction Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000003402 intramolecular cyclocondensation reaction Methods 0.000 description 1
- PGLTVOMIXTUURA-UHFFFAOYSA-N iodoacetamide Chemical compound NC(=O)CI PGLTVOMIXTUURA-UHFFFAOYSA-N 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 125000000959 isobutyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])* 0.000 description 1
- 238000006317 isomerization reaction Methods 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 150000002611 lead compounds Chemical class 0.000 description 1
- 150000007517 lewis acids Chemical class 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 239000000463 material Substances 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
- 239000012299 nitrogen atmosphere Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 125000001147 pentyl group Chemical group C(CCCC)* 0.000 description 1
- 238000000955 peptide mass fingerprinting Methods 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000007856 photoaffinity label Substances 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 229920002401 polyacrylamide Polymers 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
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000005932 reductive alkylation reaction Methods 0.000 description 1
- 239000012925 reference material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 238000001179 sorption measurement 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
- AOCSUUGBCMTKJH-UHFFFAOYSA-N tert-butyl n-(2-aminoethyl)carbamate Chemical compound CC(C)(C)OC(=O)NCCN AOCSUUGBCMTKJH-UHFFFAOYSA-N 0.000 description 1
- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 description 1
- ZGYICYBLPGRURT-UHFFFAOYSA-N tri(propan-2-yl)silicon Chemical compound CC(C)[Si](C(C)C)C(C)C ZGYICYBLPGRURT-UHFFFAOYSA-N 0.000 description 1
- PIEPQKCYPFFYMG-UHFFFAOYSA-N tris acetate Chemical compound CC(O)=O.OCC(N)(CO)CO PIEPQKCYPFFYMG-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Description
この技術は、分子間相互作用を利用し、基質(薬物)プローブ自体は無蛍光性であり、光反応で基質は切除され小さなクマリン誘導体のみ結合ドメインに標識される。この技術は、標的タンパク質の探索・同定、阻害剤スクリーニングや基質センサー、機能部位解析、細胞内物質の可視化などに応用が可能である。
以下に本発明を詳細に説明する。
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)の環化反応において、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。
reaction)に付すことにより製造することができる。
製造例1
<桂皮酸型ジアジリン活性エステルおよびアデノシン三リン酸(ATP)プローブの合成>
2-(2-methoxy-4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzylidene)butanoate
(2a)
NaH (211mg, 6.15mmol)をジクロロメタン6
mLで懸濁したものにethyl
2-(diethoxyphosphoryl)butanoate (855μL)をジクロロメタン10 mLに溶かしたものを加え、アルゴンガス雰囲気下、0℃で撹拌した。その後、化合物1 [2-methoxy-4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzaldehyde;740 mg]をジクロロメタン14mLで溶かしたものを加え、1日撹拌した。酢酸エチルを加え、水で3回、飽和食塩水で1回洗浄後、無水硫酸マグネシウムで乾燥し、減圧に溶媒を留去した。残渣をシリカゲルカラム (酢酸エチル:ヘキサン=1:10)で精製し、黄色油状物質 (化合物2a;974mg,
94%, E/Z =5/1) を得た。
CDCl3, TMS) d 7.67 (s, 1H), 7.27 (d, 1H, J = 7.8 Hz), 6.81
(d, 1H, J = 7.8 Hz), 6.61 (s, 1H), 4.28 (q, 2H, J = 7.0 Hz), 3.84
(s, 3H), 2.43 (q, 2H, J = 7.5 Hz), 1.35 (t, 3H, J = 7.1 Hz), 1.13
ppm (t, 3H, J = 7.3 Hz)
13C-NMR (100 MHz,
CDCl3, TMS) d 167.87, 157.57, 136.30, 132.91, 130.47, 129.91,
126.67, 122.04 (q, J = 275.15 Hz), 118.42, 108.21, 60.76, 55.58, 28.52
(q, J = 41.22 Hz), 21.11, 14.28, 13.93 ppm
19F-NMR (375 MHz,
CDCl3, CFCl3) d -65.46 ppm
ESI-MS (positive) [M + Na]+
= 365.1080 (理論値365.1084 C16H17F3N2NaO3).
2-(2-methoxy-4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzylidene) [3,3-2H2,4,4,4-2H3]
butanoate (2b)
上記(a)のEthyl
2-(diethoxyphosphoryl)butanoateに代えて、 [2H5]Ethyl
2-(diethoxyphosphoryl)butanoateを使用し、化合物2bを、収率
62% (E/Z = 5/1)で得た。
CDCl3, TMS) d 7.68 (s, 1H), 7.28 (d, 1H, J = 7.8 Hz), 6.81
(d, 1H, J = 7.8 Hz), 6.62 (s, 1H), 4.27 (q, 2H, J = 7.0 Hz), 3.84
(s, 3H), 1.35 ppm (t, 3H, J = 7.1 Hz)
ESI-MS (positive) [M + Na]+
= 370.1393 (理論値370.1397 C16H12 2H5F3N2NaO3).
2-(2-hydroxy-4-(3-trifluoromethyl)-3H-diazirin-3-yl)benzylidene)butanoate
(3a)
化合物2a (1.2g, 3.5mmol)をジクロロメタン20mLに溶かし、アルゴンガス雰囲気下、-40℃で撹拌しながら、三臭化ホウ素 (1mL, 11mmol)をジクロロメタン15mLで希釈したものをゆっくりと加えた。室温で1日撹拌後、氷を加え反応を停止した。続いて、水で3回、飽和食塩水で1回洗浄後、無水硫酸マグネシウムで乾燥し、減圧下に溶媒を留去した。残渣をシリカゲルカラム (酢酸エチル:ヘキサン=1:5)で精製し、黄色固体(化合物3a;560mg,
49%) を得た。
CDCl3, TMS) d 7.59 (s, 1H), 7.20 (d, 1H, J = 8.2 Hz), 6.72
(s, 1H), 6.72 (d, 1H, J = 4.6 Hz), 5.71 (s, 1H), 4.30 (q, 2H, J =
7.0 Hz), 2.40 (q, 2H, J = 7.3 Hz), 1.36 (t, 3H, J = 7.1 Hz), 1.09
ppm (t, 3H, J = 7.6 Hz)
13C-NMR (100 MHz,
CDCl3, TMS) d 168.43, 154.04, 137.87, 132.41, 130.81, 129.87,
124.28, 121.97 (q, J = 275.05 Hz), 118.14, 113.78, 61.32, 28.22 (q, J
= 41.22 Hz), 21.17, 14.16, 13.77 ppm
19F-NMR (375 MHz,
CDCl3, CFCl3) d -65.46 ppm
ESI-MS (positive) [M + H]+
= 329.1106 (理論値329.1108 C15H16F3N2O3).
2-(2-hydroxy-4-(3-trifluoromethyl)-3H-diazirin-3-yl)benzylidene) [3,3-2H2,4,4,4-2H3]
butanoate (3b)
化合物2b を用い、(c)と同様にして、化合物3bを収率 69%で得た。
CDCl3, TMS) d 7.57 (s, 1H), 7.20 (d, 1H, J = 8.3 Hz), 6.72
(d, 1H, J = 7.3 Hz), 6.72 (s, 1H), 5.39 (s, 1H), 4.30 (q, 2H, J =
7.0 Hz), 1.36 ppm (t, 3H, J = 7.1 Hz)
ESI-MS (positive) [M + H]+
= 334.1419 (理論値334.1421 C15H11 2H5F3N2O3).
acid (4a)
化合物3a (452mg, 1.38mmol)をメタノール7mLに溶かし、アルゴンガス雰囲気下、-20℃で撹拌しながら、2mol/Lの水酸化ナトリウム水溶液20mLをゆっくりと加えた。1日撹拌後、減圧に溶媒を留去した。残渣に水を加え、酢酸エチルで3回洗浄した。希塩酸を加えpH1にした後、酢酸エチルで3回抽出、飽和食塩水で1回洗浄した。有機層を合わせ、無水硫酸マグネシウムで乾燥し、減圧下に溶媒を留去した。残渣をシリカゲルカラム (酢酸エチル:ヘキサン=1:5)で精製し、黄色固体 (化合物4a; 404
mg, 98%) を得た。
CD3OD, TMS) d 7.71 (s, 1H), 7.31 (d, 1H, J = 8.2 Hz), 6.71
(s, 1H), 6.66 (d, 1H, J = 7.3 Hz), 2.43 (q, 2H, J = 7.3 Hz), 1.11
ppm (t, 3H, J = 7.3 Hz)
13C-NMR (100 MHz,
CD3OD, TMS) d 171.42, 157.34, 136.80, 134.71, 131.19, 126.24, 123.48
(q, J = 274.15 Hz), 117.84, 114.06, 29.31 (q, J = 40.21 Hz),
21.87, 14.18 ppm
19F-NMR (375 MHz,
CD3OD, CFCl3) d -65.12 ppm
ESI-MS (positive) [M + Na]+
= 323.0611 (理論値323.0614 C13H11F3N2NaO3).
[3,3-2H2,4,4,4-2H3]butanoic acid (4b)
化合物3b を用い、(e)と同様にして、化合物4bを収率 93%で得た。
CD3OD, TMS); d 7.72 (s, 1H), 7.32 (d, 1H, J = 7.8 Hz), 6.72
(s, 1H), 6.66 ppm (d, 1H, J = 8.2 Hz)
ESI-MS (positive) [M + Na]+
= 328.0927 (理論値328.0928 C13H6 2H5F3N2NaO3).
2-(2-hydroxy-4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzylidene) butanoate
(5a)
化合物4a(323mg, 1.08mmol)とN-ヒドロキシスクシンイミド (312mg, 2.71mmol)をアセトニトリル5mLに溶かし、アルゴンガス雰囲気下、0℃で撹拌した。水溶性カルボジイミド(550mg)をアセトニトリル8mLに溶かしたものを加え、室温で2 時間撹拌した。溶媒を減圧留去後、酢酸エチルを加え、水で3回、飽和食塩水で1回洗浄した。有機層を分取し、無水硫酸マグネシウムで乾燥し、減圧に溶媒を留去した。残渣をシリカゲルカラム (酢酸エチル: ヘキサン=1:2)で精製し、黄色固体
(化合物5a ;265mg, 51%)を得た。
CD3OD, TMS) d 7.88 (s, 1H), 7.26 (d, 1H, J = 7.8 Hz), 6.74
(d, 1H, J = 7.8 Hz), 6.70 (s, 1H), 5.70 (s, 1H), 2.90 (s, 4H), 2.50 (q,
2H, J = 7.3 Hz), 1.19 ppm (t, 3H, J = 7.3 Hz)
13C-NMR (100 MHz,
CDCl3, TMS) d 171.94, 164.63, 157.83, 138.77, 132.53, 132.11,
131.40, 124.94, 120.76 (q, J = 274.12 Hz), 118.00, 114.31, 29.36 (q, J
= 40.21 Hz), 26.56, 22.29, 13.82 ppm
19F-NMR (375 MHz,
CD3OD, CFCl3) d -65.06 ppm
ESI-MS (positive) [M + Na]+
= 420.0779 (理論値420.0778 C17H14F3N3NaO5).
2-(2-hydroxy-4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzylidene) [3,3-2H2,4,4,4-2H3]butanoate
(5b)
化合物4b を用い、(g)と同様にして、化合物5bを収率 53%で得た。
CD3OD, TMS) d 7.89 (s, 1H), 7.25 (d, 1H, J = 8.2 Hz), 6.73
(d, 1H, J = 8.7 Hz), 6.69 (s, 1H), 6.05 (s, 1H), 2.91 ppm (s, 4H)
ESI-MS (positive) [M + Na]+
= 425.1091 (理論値425.1092 C17H9 2H5F3N3NaO5).
ethyl) carbamate (6a)
tert-Butyl
(2-aminoethyl)carbamate(47mg, 0.29mmol)とN,N-ジイソプロピルエチルアミン(40μL, 0.23mmol)をアセトニトリル1mLに溶かし、化合物5a(88mg, 0.22mmol)のアセトニトリル溶液3mLを加えた。アルゴンガス雰囲気下、室温で1日撹拌し、減圧に溶媒を留去後、残渣に酢酸エチルを加え、飽和炭酸水素ナトリウムで2回、飽和食塩水で2回洗浄した。有機層を分取し、無水硫酸マグネシウムで乾燥後、減圧に溶媒を留去した。残渣をシリカゲルカラム(酢酸エチル:ヘキサン=1:2)で精製し、黄色固体 (化合物6a;72mg,
73%)を得た。
acetone-d6) d 7.30 (d, 1H, J = 7.8 Hz), 7.16 (s, 1H), 6.78
(s, 1H), 6.72 (d, 1H, J = 8.2 Hz), 6.22 (s, 1H), 3.39 (q, 2H, J =
5.8 Hz), 3.25 (q, 2H, J = 5.8 Hz), 2.43 (q, 2H, J = 7.3 Hz), 1.38
(s, 9H), 1.03 ppm (t, 3H, J = 7.6 Hz)
13C-NMR (100 MHz,
acetone-d6, TMS) d 170.09, 157.27, 156.52, 142.03, 131.27, 130.00,
127.00, 126.47, 123.03 (q, J = 274.15 Hz), 117.83, 114.03, 78.86, 41.10,
39.57, 29.69 (q, J = 34.48 Hz), 28.56, 21.86, 13.67 ppm
19F-NMR (375 MHz,
acetone-d6, CFCl3) d -64.90 ppm
ESI-MS (positive) [M + Na]+
= 465.1719 (理論値465.1720 C20H25F3N4NaO4).
(E)-tert-Butyl(2-(2-(2-hydroxy-4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzylidene)
[3,3-2H2,4,4,4-2H3]butanamido)ethyl)carbamate
(6b)
化合物5b を用い、(i)と同様にして、化合物6bを収率 74%で得た。
acetone-d6, TMS) d 7.33 (d, 1H, J = 8.2 Hz), 7.19 (s, 1H), 6.82
(s, 1H), 6.73 (d, 1H, J = 7.8 Hz), 6.19 (s, 1H), 3.41 (q, 2H, J =
5.8 Hz), 3.27 (q, 2H, J = 5.8 Hz), 1.40 ppm (s, 9H)
ESI-MS (positive) [M + Na]+
= 470.2033 (理論値470.2034 C20H20 2H5F3N4NaO4).
butanamide (7a)
化合物6a(34mg, 76μmol)に窒素雰囲気下0 ℃で、トリフルオロ酢酸4mLを加えた。その後室温で1時間撹拌した。減圧下に留去後、残渣をODSカラム (水:アセトニトリル=9:1)で精製し、白色固体
(化合物7a;24mg,
92%) を得た。
CD3OD, TMS) d 7.28 (d, 1H, J = 8.2 Hz), 7.24 (s, 1H), 6.72
(s, 1H), 6.67 (d, 1H, J = 7.8 Hz), 3.56 (t, 2H, J = 6.0 Hz), 3.12
(t, 2H, J = 6.0 Hz), 2.47 (q, 2H, J = 7.5 Hz), 1.08 ppm (t, 3H, J
= 7.6 Hz)
13C-NMR (125 MHz,
CD3OD) d 173.27, 157.16, 140.82, 131.35, 131.15, 126.34, 123.53 (q, J
= 274.42 Hz), 117.97, 114.05, 40.94, 38.83, 29.49 (q, J = 40.48 Hz) ppm
19F-NMR (375 MHz,
CD3OD, CFCl3) d -65.15 ppm
ESI-MS (positive) [M + H]+
= 343.1378 (理論値343.1376 C15H18F3N4O2).
(E)-N-(2-Aminoethyl)-2-(2-hydroxy-4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzylidene)butanamide
(7b)
化合物6b を用い、(k)と同様にして、化合物7bを収率 93%で得た。
CD3OD, TMS) d 7.28 (d, 1H, J = 8.2 Hz), 7.24 (s, 1H), 6.72
(s, 1H), 6.67 (d, 1H, J = 7.8 Hz), 3.56 (t, 2H, J = 6.0 Hz), 3.11
ppm (t, 2H, J = 6.0 Hz)
13C-NMR (125 MHz,
CD3OD) d 173.16, 156.98, 140.57, 131.19, 131.08, 126.19, 123.40 (q, J
= 274.04 Hz), 117.92, 114.02, 40.77, 38.57, 29.23 (q, J = 40.60 Hz) ppm
19F-NMR (375 MHz,
CD3OD, 標準物質
CFCl3) d -65.21 ppm
ESI-MS (positive) [M + H]+
= 348.1690 (理論値348.1690 C15H13 2H5F3N4O2).
50mM ATPの250mM
MES buffer pH 6.8溶液(16μL)、50mM 化合物7aの水溶液(8μL, 0.4μmol)、1M EDCI水溶液(2.0μL)、250mM
MES buffer pH6.8(2.0μL)、600 mMトリエチルアミンの250mM MES buffer pH6.8溶液(4.0μL)を混合し、室温遮光して6時間反応させた。反応混合物を逆相HPLCで精製後、凍結乾燥してEDCI付加物を得た。100mM水酸化ナトリウム水溶液を450μL加え、1時間37℃で撹拌し、再度ODS C18カラムを用いてHPLCにより精製を行い、24時間、凍結乾燥して化合物8aを得た。
CAPCELLPAK C18 φ10 mm×250 mm
溶液A:50 mM
トリエチルアミン酢酸水溶液(pH 7.0)
溶液B:50 mM
トリエチルアミン酢酸溶液(pH 7.0)/80%アセトニトリル20%水
流速 4 mL/min
グラジェント0 min (A 100%, B 0%)→0.1 min (A 100%, B 0%)→40 min (A 50%, B 50%)→
45 min (A 0%, B 100%)→50
min (A 0%, B 100%)→55 min (A 100%, B 0%)
EDCI付加物: 34.7
min, ESI-MS (positive)
[M + H]+
= 987.2648 (理論値987.2651 C33H49F3N12O14P3)
化合物7b を用い、(n)と同様にして、化合物8bを得た。
(negative) [M - H]- = 830.1088 (理論値830.1083 C25H30F3N9O14P3).
EDCI付加物: ESI-MS
(positive) [M + H]+ = 992.2967 (理論値992.2965
C33H44 2H5F3N12O14P3).
化合物8b: ESI-MS (negative) [M - H]- = 835.1400 (理論値835.1397 C25H25 2H5F3N9O14P3).
<ペプチド(SSILRAFY)プローブの合成>
3-[2-hydroxy-4-(3-trifluoromethyl-3H-diazirin-3-yl)phenyl-2-methylacrylate (9).
2-hydroxy-4-(3-trifluoromethyl-3H-diazirin-3-yl)benzaldehyde(2.0g, 8.8mmol)をベンゼン40mLに溶かし、[1-(ethoxycarbonyl)ethylidene]
triphenylphosphorane (3.5g, 9.7mmol)を加えて、アルゴンガス雰囲気下、室温で終夜撹拌した。溶媒留去後、残渣をシリカゲルカラム (酢酸エチル:ヘキサン=5:1)で精製し、薄い黄色固体 (化合物9; 2.2g,
79%) を得た。
1H-NMR (500 MHz, CDCl3, TMS) δ 7.72 (1
H, s), 7.24 (1 H, d, J 8.1), 6.75 (s, 1 H), 6.70 (1 H, d, J 8.1),
4.29 (2 H, q, J 7.3), 2.00 (3H, s), 1.35 (3 H, t, J 7.3)
13C-NMR (126 MHz, CDCl3, TMS)TM 168.8 (s), 154.2 (s), 133.1 (d),
131.3 (s), 130.8 (d), 130.4 (s), 124.4 (s), 122.6 (q, 1JC-F
273), 118.0 (d), 113.8 (d), 61.4 (t), 28.3 (q, 2JC-F
41), 14.1 (q), 14.2 (q)
19F-NMR (376 MHz, CDCl3, CFCl3)
TM-65.6 (3F, s); λmax/nm(ε) (MeOH) 352 (sh, 1,270)
HRMS
(EI) m/z [M]+ = 314.0872(理論値 314.0878 C14H13F3N2O3).
acid (10).
化合物9 (2.0g, 6.4mmol)をメタノール1mLに溶かし、3 M水酸化ナトリウム水溶液(50mL) を0℃でゆっくり加え、室温で2時間撹拌した。冷3 M塩酸をゆっくり加えて酸性にした後、酢酸エチルで抽出した。無水硫酸マグネシウムで乾燥後、溶媒を留去した。残渣をクロロホルムで再結晶して薄い黄色鱗片状結晶を得た(化合物10; 1.52 g, 83%) を得た。
1H-NMR
(500 MHz, CD3OD, TMS) δ 7.78 (1 H, s), 7.36 (1 H, d, J 8.1),
6.72 (1 H, s), 6.65 (1 H, d, J 8.1), 1.99 (3 H, s)
13C-NMR (126 MHz, CD3OD, TMS) δ171.8 (s), 157.5 (s), 135.0 (d),
131.9 (d), 131.4 (s), 130.5 (s), 126.3 (s), 123.6 (q, 1JC-F
273), 117.8 (d), 114.1 (d), 29.4 (q, 2JC-F
41), 14.4 (q)
19F-NMR (376 MHz, CD3OD, CFCl3)TM-64.9 (3F, s);λmax/nm(ε) (MeOH) 347 (sh, 1,100); HRMS (EI) m/z
[M]+ 286.0565理論値 286.0565 C12H9F3N2O3).
H-K(biotin)SSILRAFY-OHはAlko PEG
resin (0.78mmol/g, 50mg)を用いて、Fmoc固相合成法により合成機で製造した。
化合物10 (40mg, 0.14 mmol)のジメチルホルムアミド溶液1 mLに、N-hydroxysuccinimide (17mg, 0.15mmol) と1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (28mg,
0.15mL)を加えた。化合物10が消失した後、この反応液760 mLおよびトリエチルアミン(40mL)を樹脂に加え、終夜、室温で振盪した。ジメチルホルムアミドおよびメタノールで洗浄後、トリフルオロ酢酸 (340mL)溶液(m-クレゾール(20mL)、チオアニソール(20mL)およびトリイソプロピルシラン(20mL) を含む)で1時間インキュベートした。上静をエーテルで沈殿させ、沈殿物をODSを用いた逆相HPLCで精製した(リニアグラジエント2〜60% アセトニトリル・水(0.1% TFAを含む)、50min、流速1mL/min)。37.6minのピークを採取し、凍結乾燥した。
HRMS (ESI+) m/z
[MH]+1578.7294 理論値1578.7391 C73H103F3N17O17S).
<グルタミン酸脱水素酵素 (GDH)の光アフィニティーラベル、LC-MS解析>
GDH (0.2mg/mL), ATP probe
(8aおよび8b 各々0.2mM) のトリス−酢酸緩衝液(50mM, pH8.0, 25μL)を調整し、遮光下室温で1時間インキュベートした。波長365nm光を氷冷中15秒間照射した後、さらに37 ℃で15分間照射した。2×SDS-sample bufferを5μL加え、室温で1時間変性した後、サンプル全量を10%ポリアクリルアミドゲルにより電気泳動し、CBB染色を行い、対象バンドを1.5mLエッペンチューブに回収した。脱染色後、ヨウ化アセトアミドによる還元アルキル化を行い、リシルエンドペプチターゼ処理(37℃、18時間インキュベート)を行った。ゲルから消化物を抽出して、ZipTipを用いて脱塩、濃縮した(10μL)。この溶液を用い、nanoLC-MSn解析を行った。
さらに、本発明方法は、創薬以外にも、基礎生物学から医療分野に至る広範囲の学術領域に有用である。
Claims (1)
- 蛍光性質量標識プローブ作成の中間体として用いられる下記一般式で表された桂皮酸型ジアジリン化合物。
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