JP5550035B2 - 波長が制御されたルシフェラーゼの発光基質および製造方法 - Google Patents
波長が制御されたルシフェラーゼの発光基質および製造方法 Download PDFInfo
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- JP5550035B2 JP5550035B2 JP2009064595A JP2009064595A JP5550035B2 JP 5550035 B2 JP5550035 B2 JP 5550035B2 JP 2009064595 A JP2009064595 A JP 2009064595A JP 2009064595 A JP2009064595 A JP 2009064595A JP 5550035 B2 JP5550035 B2 JP 5550035B2
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- Prior art keywords
- luciferin
- mmol
- compound
- luminescent
- nmr
- 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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- 239000000758 substrate Substances 0.000 title claims description 49
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- 238000004519 manufacturing process Methods 0.000 title description 18
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- 150000001875 compounds Chemical class 0.000 claims description 81
- 125000000217 alkyl group Chemical group 0.000 claims description 13
- 229910052799 carbon Inorganic materials 0.000 claims description 13
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- 229910052757 nitrogen Inorganic materials 0.000 claims description 7
- 238000000504 luminescence detection Methods 0.000 claims description 2
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- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 29
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- FIQMHBFVRAXMOP-UHFFFAOYSA-N triphenylphosphane oxide Chemical compound C=1C=CC=CC=1P(C=1C=CC=CC=1)(=O)C1=CC=CC=C1 FIQMHBFVRAXMOP-UHFFFAOYSA-N 0.000 description 6
- QIJRTFXNRTXDIP-YBBRRFGFSA-N (2s)-2-amino-3-sulfanylpropanoic acid;hydrate;hydrochloride Chemical compound O.Cl.SC[C@@H](N)C(O)=O QIJRTFXNRTXDIP-YBBRRFGFSA-N 0.000 description 5
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- 235000011006 sodium potassium tartrate Nutrition 0.000 description 1
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 description 1
- 235000019345 sodium thiosulphate Nutrition 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 238000005556 structure-activity relationship Methods 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- ISIJQEHRDSCQIU-UHFFFAOYSA-N tert-butyl 2,7-diazaspiro[4.5]decane-7-carboxylate Chemical compound C1N(C(=O)OC(C)(C)C)CCCC11CNCC1 ISIJQEHRDSCQIU-UHFFFAOYSA-N 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
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 125000002769 thiazolinyl group Chemical group 0.000 description 1
- 150000003613 toluenes Chemical class 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000002834 transmittance Methods 0.000 description 1
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
- C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
- C07D277/08—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
- C07D277/12—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
- C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
- C07D277/62—Benzothiazoles
- C07D277/64—Benzothiazoles with only hydrocarbon or substituted hydrocarbon radicals attached in position 2
- C07D277/66—Benzothiazoles with only hydrocarbon or substituted hydrocarbon radicals attached in position 2 with aromatic rings or ring systems directly attached in position 2
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
- C09K2211/1037—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with sulfur
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Thiazole And Isothizaole Compounds (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
近年、生物学的事象および現象の可視化が重要視され、可視化のための材料の拡大が望まれてきている。これに伴い、標識技術にも多様化が求められている。特に分子イメージングのための標識技術は、診断および検査機器の進歩と相まって大きく発展している。たとえば、癌や心疾患などに対する個別化医療などの先端技術に応用するための標識技術が精力的に研究されている。また、計測技術の進歩に伴い、より高感度および高性能な機器や標識材料に対する需要が急速に高まっている。
多現象を観測するために、標識を利用した検出系においても、多色発光が求められている。このため、検出系に利用できる標識材料の波長域は、幅広い方が望ましい。また、生体内深部標識における用途では、短波長光よりも長波長光のほうが優れた光透過性を有するという観点から、赤色発光標識材料が望まれている。たとえば、多色発光を利用した研究には、標識として450nm以下程度〜650nm以上程度の波長にわたる発光を有する標識材料が準備されることが望ましい。
1.プロメガ社:Chroma-Luc:約613nm(非特許文献1)
この系は、ヒカリコメツキ虫(クリックビートル)の突然変異体および天然型ホタル発光基質を利用した系である。
2.東洋紡績(株):MultiReporter Assay System-Tripluc: 約630nm(非特許文献2)
この系は、鉄道虫赤色発光酵素および天然型ホタル発光基質を利用した系である。緑色発光ルシフェラーゼ(SLG、最大発光波長550nm)、橙色発光ルシフェラーゼ(SLO、580nm)および赤色発光ルシフェラーゼ(SLR、630nm)の色のルシフェラーゼ遺伝子を使用して発光色を変化させている。これは、異なる発光色を与える発光酵素を利用している。
3.東京大学:アミノルシフェリン:約610nm(特許文献1)
これは、ルシフェリン誘導体を開示している。
4.プロメガ社:Chroma-Luc:約480nm(非特許文献3)
この系は、セレンテラジンおよびウミシイタケルシフェラーゼを利用した系である。
5.ATTO社:ウミホタル生物発光 約460nm(非特許文献4)
セレンテラジン系基質およびウミホタルルシフェラーゼを利用した系である。
pH測定:東洋濾紙株式会社製pH試験紙UNIVを使用して測定した。また、pHメータとして、堀場社製pH/ION METER F-23を使用して測定した。
分析用薄層クロマトグラフィー(TLC):E. Merck社製のTLCプレート、シリカゲル60F254(Art.5715)厚さ0.25mmを使用した。TLC上の化合物の検出はUV照射(254nmまたは365nm)および発色剤に浸した後に加熱して発色させることによって行った。発色剤としてはp-アニスアルデヒド(9.3ml)と酢酸(3.8ml)をエタノール(340ml)に溶解し、濃硫酸(12.5ml)を添加したものを使用した。
反応溶液の冷却は、冷媒を満たしたジュワー瓶に反応容器を浸して行った。室温〜4℃では、氷水、4〜-90℃では、液体窒素−アセトンを冷媒として用いた。反応後の抽出溶液の乾燥は、飽和食塩水にて洗浄後、無水硫酸ナトリウムまたは無水硫酸マグネシウムを加えることで行った。反応後の中和を樹脂で行ったものについては、オルガノ株式会社製陽イオン交換樹脂アンバーライトIR120B NAまたは陰イオン交換樹脂アンバーライトIRA400 OH AGを使用した。溶液の減圧濃縮は、アスピレーターの減圧下(20〜30mmHg)、ロータリーエバポレーターを使用して行った。痕跡量の溶媒の除去は、液体窒素浴で冷却したトラップを装着させた真空ポンプ(約1mmHg)を使用して行った。溶媒の混合比は全て体積比で表した。
蒸留水は、アドバンテック東洋株式会社製GS-200型蒸留水製造装置を使用して蒸留およびイオン交換処理したものを使用した。
1H NMR (270MHz, CDCl3)
δ3.01(6H, s), 3.50(1H, dd, J = 9.2, 11Hz), 3.61(1H, dd, J = 9.2, 11Hz), 5.00(1H, t, J=9.2Hz), 6.71(2H, dd, J = 2.4, 7.0Hz), 7.71(2H, dd, J = 2.4, 7.0Hz)。
ナフタレン類似体1の合成
1H NMR(270MHz, CD3OD)
δ 3.73(2H, dd, J=3.9, 8.9Hz), 5.33(1H, t, J=8.9Hz), 7.20〜8.20(6H, complex)。
1H NMR(270MHz, CDCl3)
δ 0.01(6H, s), 0.74(9H, s), 6.80〜7.80(6H, complex)。
1H NMR(270MHz, CDCl3)
δ 0.28(6H, s), 1.03(9H, s), 7.00〜7.80(6H, complex), 10.10(1H, s)。
1H NMR(270MHz, CDCl3)
δ 0.25(6H, s), 1.00(9H, s), 1.43(3H, t, J=7.0Hz), 4.23(2H, q, J=7.1Hz), 5.95(1H, d, J=12.5Hz), 6.47(1H, d, J=16.1Hz), 7.01〜7.80(7H, complex)。
1H NMR(270MHz, CD3OD)
δ 6.47(1H, d, J=15.8Hz), 7.00〜7.90(7H, complex)。
IR(neat) 3380, 3320, 1740, 1600cm-1
1H NMR(270MHz, CDCl3)
δ2.47(1H, dd, J=8.1, 13Hz), 2.60(1H, dd, J=5.1, 13Hz), 3.21(1H, dd, J=5.1, 8.1Hz), 3.66(3H, s), 7.18-7.32(9H, complex), 7.40-7.54(6H, complex)
13C NMR(67.8MHz, CD3OD)
δ36.90(t), 52.16(q), 53.78(d), 66.83(s), 126.8(d)×3, 127.9(d)×6, 129.6(d)×6, 144.5(s)×3, 174.2(s)。
1H NMR(270MHz, CDCl3)
δ 2.75(2H, dd, J=4.6, 7.9Hz), δ3.75(3H, s), 4.77(1H, dd, 2.7, 7.9Hz), 6.35(1H, d, J=16.1Hz), 6.90〜7.80(22H, complex)。
1H NMR(270MHz, CD3OD)
δ3.63(2H, dd, J=3.1, 8.9Hz), 3.8 1(3H, s), 5.27(1H, t, J=8.9Hz), 7.07〜7.13(2H, complex), 7.33(1H, d, J=16.1Hz), 7.55〜7.83(4H, complex)。
1H NMR(270MHz, CD3OD)
δ 3.61(2H, dd, J=3.1, 8.9Hz), 5.09(1H, t, J=8.9Hz), 7.06〜7.17(2H, complex), 7.30(1H, dd, J=16.1Hz), 7.65〜7.87(4H, complex)。
mp 200-207℃
IR(film) 2680(br.), 1610, 1580, 1510cm-1
1H NMR(270MHz, CDCl3)
δ 3.70(1H, d, J=8.6Hz), 3.78(1H, d, J =8.6Hz), 5.23(1H, t, J =8.6Hz), 6.79(2H, d, J =8.9Hz), 7.66(2H, d, J =8.9Hz),
13C NMR(67.8MHz, CDCl3)
δ35.95(t), 77.97(d), 116.5(d)×2, 124.5(s), 131.8(d)×2, 163.0(s), 173.9(s), 174.6(s)
MS(EI)m/z 223(M+・, ), 178(100%), 137(13%), 121(19%)。
mp 182-183℃
IR(film) 3050, 1740, 1680, 1630cm-1
1H NMR(270MHz, CDCl3)
δ2.32(3H, s), 6.41(1H, d, J=16Hz), 7.15(2H, d, J=8.6Hz), 7.57(2H, d, J=8.6Hz), 7.76(1H, d, J=16Hz)
1H NMR(270MHz, CD3OD)
δ2.28(3H, s), 6.46(1H, d, J=16Hz), 7.15(2H, d, J=8.6Hz), 7.62-1.65(3H, complex)
1H NMR(270MHz, ACETN)
δ2.28(3H, s), 6.52(1H, d, J=16Hz), 7.21(2H, d, J=8.4Hz), 7.66-7.76(3H, complex)
13C NMR(67.8MHz, CDCl3)
δ20.95(q), 121.0(d), 123.4(d)×2, 123.2(d)×2, 133.9(s), 144.2(d), 153.6(s), 170.9(s), 171.2(s)
MS(EI)m/z 206(M+,14), 164(100), 147(20), 119(12), 92(14)。
IR(neat) 3280, 1760, 1740, 1660, 1630cm-1
1H NMR(270MHz, CDCl3)
δ2.32(3H, s), 2.70(1H, dd, J=4.6, 12Hz), 2.78(1H, dd, J=5.4, 12Hz), 3.72(3H, s), 4.76(1H, dd, J=4.6, 5.4Hz), 7.10-7.31(9H, complex), 7.36-7.41(6H, complex), 7.52(2H, d, J=8.4Hz), 7.57(1H, d, J=15Hz)
13C NMR(67.8MHz, CD3OD)
δ 21.17(q), 33.93(t), 51.19(d), 52.76(q), 66.99(s), 120.0(d), 122.1(d)×2, 126.9(d)×3, 128.0(d)×6, 129.0(d)×2, 129.5(d)×6, 132.4(s), 140.8(d), 144.3(s), 151.8(s), 165.1(s), 169.3(s), 170.9(s)。
mp 118-121℃
IR(film) 1760, 1720cm-1
1H NMR(270MHz, CDCl3)
δ2.31(3H, s), 3.56(1H, dd, J=9.2, 11Hz), 3.70(1H, dd, J=9.2, 11Hz), 3.85(3H, s), 5.22(1H, t, J=9.2Hz), 7.03(1H, d, J=16Hz)7.13(2H, d, J=8.6Hz), 7.15(1H, d, J=16Hz), 7.50(2H, d, J=8.6Hz)
13C NMR(67.8MHz, CD3OD)
δ 21.15(q), 34.64(t), 52.89(q), 77.96(d), 122.2(d)×2, 122.4(d), 128.7(d)×2, 132.8(s), 141.1(d), 151.7(s)×2, 169.2(s), 132.4(s), 170.0(s), 171.2(s)
MS(EI)m/z 305(M+,44), 263(88), 205(100), 177(69), 163(15)。
mp 138-140℃
IR(film) 3150, 1630, 1570cm-1
1H NMR(270MHz, CD3OD)
δ3.54(1H, dd, J=9.2, 11Hz), 3.59(1H, dd, J=9.2, 11Hz), 5.02(1H, t, J=9.2Hz), 6.79(1H, d, J=8.4Hz), 6.92(1H, d, J=16Hz), 7.12(1H, d, J=16Hz), 7.43(2H, d, J=8.4Hz)
13C NMR(67.8MHz, CD3OD)
δ 36.54(t), 81.19(d), 116.9(d)×2, 119.7(d), 128.0(s), 130.5(d)×2, 132.8(s), 143.7(d), 160.7(s), 172.0(s), 177.5(s)。
IR(neat) 1699cm-1
1H NMR(270MHz, CDCl3)
δ0.01(6H, s), 0.76(9H, s), 6.70(2H, dd, J=6.8, 16Hz), 7.55(2H, dd, J=6.8, 16Hz)
MS(EI)m/z 236(M+・, 43), 179(100)。
IR(KBr錠剤法) 1716cm-1
1H NMR(270MHz, CDCl3)
δ0.01(6H, s), 0.77(9H, s), 1.11(3H, t, J=7.3Hz), 4.04(2H, q, J=7.3Hz), 6.08(1H, d, J=16Hz), 6.62(2H, dd, J=1.9, 6.5Hz), 7.20(2H, dd, J=1.9, 6.5Hz), 7.42(1H, q, J=16Hz)
MS(EI)m/z 306(M+・, 53), 261(9), 249(100), 203(22)。
IR(neat) 3388cm-1
1H NMR(270MHz, CDCl3)
δ0.01(6H, s), 0.98(9H, s), 4.30(2H, t, J=5.9Hz), 6.25(2H, dt, J=5.9, 16Hz), 6.56(2H, d, J=8.6Hz), 6.80(2H, d, J=8.6Hz), 7.28(2H, d, J=8.6Hz)
1H NMR(270MHz, CD3OD)
δ0.01(6H, s), 0.80(9H, s), 4.01(2H, d, J=5.9Hz), 6.03(2H, dt, J=5.9, 16Hz), 6.35(2H, d, J=16Hz), 6.60(2H, dd, J=1.9, 8.6Hz), 7.11(2H, dd, J=1.9, 8.6Hz)
MS(EI)m/z 264(M+,54), 207(100), 189(20), 115(20), 75(22)。
IR(KBr錠剤法) 1675cm-1
1H NMR(270MHz, CDCl3)
δ0.01(6H, s), 0.77(9H, s), 6.37(1H, dd, J=7.8, 16Hz), 6.56(2H, d, J=8.6Hz), 6.66(2H, d, J=8.6Hz), 7.20(1H, d, J=16Hz), 7.25(2H, d, J=8.6Hz), 9.43(1H, d, J=7.8Hz),
MS(EI)m/z 262(M+,47), 207(25), 206(100)。
IR(neat) 1705cm-1
1H NMR(270MHz, CDCl3)
δ0.01(6H, s), 0.78(9H, s), 1.11(3H, t, J=7.3Hz), 4.04(2H, q, J=7.3Hz), 5.74(1H, d, J=7.8, 15Hz), 6.55-6.68(4H, complex), 7.15(2H, dd, J=2.2Hz), 7.25(1H, d, J=15Hz),
MS(EI)m/z 332(M+・, 41), 275(22), 218(100), 173(28), 145(92)。
IR(KBr錠剤法) 3311, 1670cm-1
1H NMR(270MHz, CD3OD)
δ5.74(1H, d, J=7.8, 15Hz), 6.55-6.68(4H, complex), 7.38(2H, d, J=8.9Hz), 7.45(1H, d, J=15Hz)
MS(EI)m/z 190(M+, 9), 183(100), 149(43), 105(58)。
IR(neat) 3290, 1739, 1652cm-1
1H NMR(270MHz, CD3OD)
δ5.91(1H, d, 15Hz), 6.76-6.92(4H, complex), 1.11(3H, t, J=7.3Hz), 4.04(2H, q, J=7.3Hz), 5.74(1H, d, J=7.8, 15Hz), 6.55-6.68(4H, complex), 7.38(2H, d, J=8.9Hz), 7.45(1H, d, J=15Hz)。
1H NMR(270MHz, CDCl3)
δ2.71(2H, d, 8.9Hz), 3.66(3H, s), 4.72(1H, t, J=8.9Hz), 5.85(2H, d, J=15Hz), 6.21-7.46(22H, d, complex)。
IR(KBr錠剤法) 3396, 1596cm-1
1H NMR(270MHz, CD3OD)
δ4.25(2H, d, 7.3Hz), 5.21(1H, t, J=7.3Hz), 6.52(2H, d, J=15Hz), 6.75-7.56(7H, complex), 6.76(2H, d, J=8.9Hz), 7.37(2H, d, J=8.9Hz)
MS(ESI) [M+H]+ ;m/z 276.0694。
1H NMR(270MHz, CDCl3)
δ3.01(6H, s), 3.(1H, dd, J=9.2, 11Hz), 3.(1H, dd, J=9.2, 11Hz), 5.00(1H, t, J=9.2Hz), 6.71(2H, dd, J=2.4, 7.0Hz), 7.71(2H, dd, J=2.4, 7.0Hz)。
1H NMR(270MHz, CDCl3)
δ2.72(2H, t, J=5.1Hz), 2.99(6H, s), 3,71(3H, s), 4.78(1H, dd, J=5.1, 7.8Hz), 6.05(1H, d, J=7.8Hz), 6.15(1H, d, J=15Hz), 6.68(1H, d, J=8.6Hz), 7.10-7.31(9H, complex), 7.35-7.41(6H, complex), 7.53(1H, d, J=15Hz)。
1H NMR(270MHz, CDCl3)
δ3.02(6H, s), 3.57(1H, d, J=8.6Hz), 3.59(1H, d, J=8.6Hz), 3,84(3H, s), 5.19(1H, t, J=8.6Hz), 6.67(1H, d, J=8.9Hz), 6.92(1H, d, J=16Hz), 7.08(1H, d, J=16Hz), 7.39(2H, d, J=8.9Hz)。
1H NMR(270MHz, CDCl3)
δ3.02(6H, s), 3.57(1H, d, J=8.6Hz), 3.59(1H, d, J=8.6Hz), 3,80(3H, s), 5.19(1H, t, J=8.6Hz), 6.73(1H, d, J=8.9Hz), 6.87(1H, d, J=16Hz), 7.23(1H, d, J=16Hz), 7.45(2H, d, J=8.9Hz)
MS(ESI) [M+H]+ ;m/z 277.1011。
1H NMR(270MHz, CD3Cl3)
δ1.57(3H, s), 3.00(6H, s), 4.21(2H, q), 5.87(1H, d, J=15Hz), 6.67(2H, d, J=8.9Hz), 6.83(1H, d, J=15Hz), 7.37(2H, d, J=8.9Hz), 7.42(1H, d, J=15Hz), 7.46(1H, d, J=15Hz)
MS(EI)m/z 245(M+・, 100%)。
1H NMR(270MHz, CD3OD)
δ2.98(6H, s), 5.86(1H, d, J=15Hz), 6.70-6.90(3H, complex), 6.72(2H, d, J=8.9Hz), 7.37-7.45(4H, complex), 7.38(2H, d, J=8.9Hz),
MS(EI)m/z 217(M+・, 100%)。
1H NMR(270MHz, CD3OD)
δ2.69(1H, dd, J=4.8, 13.1Hz), 2.85(1H, dd, J=8.8, 13.1Hz), 3.66(3H, s), 4.43(1H, dd, J=4.8, 8.8Hz), 6.84(1H, dd, J=2.0, 8.6Hz), 6.97(1H, br.s), 7.20-7.31(9H, complex), 7.38-7.41(7H, complex), 7.51(1H, d, J=8.6Hz)
13C NMR(67.8MHz, CD3OD)
δ34.32(t), 53.03(q), 53.18(d), 68.31(s), 98.70(d), 112.49(d), 114.95(d), 121.05(s), 124.13(d), 127.95(d), 129.01(d), 130.72(d), 145.87(s), 148.01(s), 158.03(s), 159.63(s), 161.04(s), 172.02(s)。
1H NMR(270MHz, CDCl3)
δ3.00(6H, s), 3.54(1H, dd, J=8.9,11Hz), 3.57(1H, dd, J=8.9,11Hz), 5.17(1H, t, J=8.9Hz), 6.54(1H, d, J=15Hz), 6.65-6.80(4H, complex), 6.94(1H, dd, J=8.9,15Hz) 7.36(2H, d, J=8.9Hz)。
1H NMR(270MHz, CD3OD)
δ2.98(6H, s), 3.54(1H, d, J=8.9Hz), 3.57(1H, d, J=8.9Hz), 5.19(1H, t, J=8.9Hz), 6.47(1H, d, J=15Hz), 6.70-7.06(5H, complex), 7.38(2H, d, J=8.9Hz)
mp 155-170℃ decomp.
IR(film)3178, 2225 cm-1
1H NMR(270 MHz, CD3OD):δ 7.17(1H, dd, J = 2.6, 8.9Hz), 7.40(1H, d, J = 2.6Hz), 7.99(1H, d, J = 8.9Hz)
13C NMR(67.8 MHz, CD3OD):δ 107.00(d), 114.29(s), 119.61(d), 126.58(d), 133.91(s), 139.01(s), 147.29(s), 160.32(s)
MS(EI)m/z 176(M+・, 100), 124(5)。
1H NMR(270 MHz, DMSO-d6):δ 3.66(1H, dd, J = 8.2, 11.2Hz), 3.67(1H, dd, J = 9.9, 11.2Hz), 5.40(1H, dd, J = 8.2, 9.9Hz), 7.06(1H, dd, J = 2.6, 8.9Hz), 7.51(1H, d, J = 2.6Hz), 7.96(1H, d, J = 8.9Hz), 10.24(1H, br.s, OH)
13C NMR(67.8 MHz, DMSO-d6):δ 34.5(t), 78.0(d), 106.7(d), 117.0(d), 124.8(d), 137.1(s), 146.1(s), 157.3(s), 159.8(s), 164.3(s), 171.1(s)。
1)測定装置
高速液体クロマトグラフィー(HPLC)
アジレント・テクノロジー株式会社製のAgilent 1100 seriesHPLC を使用した。装置の内訳は、デガッサー、クォータナリポンプ、マニュアルインジェクター、カラムコンパートメント、ダイオードアレイ検出器、蛍光検出器、ケミステーション(PC用ソフトウェア)である。用いたカラムはダイセル化学工業株式会社製CHIRALCEL OD-RH(内径0.46cm、長さ15cm)である。
堀場製作所製F-23型ガラス電極式水素イオン濃度指示計を使用して行った。
ATTO株式会社製Luminescencer-PSN AB-2200を使用して測定した。
ATTO株式会社製微弱発光蛍光スペクトル装置AB-1850を使用して測定した。測定したスペクトルは全て検出器の特性を補正したスペクトルである。
超純水は、MILLIPORE製Milli-RX12αから採水したものを使用した。メタノール、t-ブタノールは、関東化学株式会社製の特級溶媒を使用した。
基質溶液
基質を電子天秤で秤量し希釈した。溶媒として、生物発光の測定の場合はリン酸緩衝液(50mM, pH 6.0)を、化学発光測定の場合はt-ブタノールを使用した。
ルシフェラーゼを1μg/μlになるようにTris-HCl緩衝液(50mM, pH8.0)で希釈し小分けにした。これをストック溶液とし、必要量をその都度希釈して用いた。なお、ストック溶液は-80℃の冷凍庫に保存した。
ATP-Mgを超純水で希釈した。
200μLポリスチレンチューブ内で、リン酸カリウム緩衝液(0.5 M, pH8.0, 20μl)基質溶液(2.5mM, 20μl)、酵素溶液(20μl)、次いでATP-Mg溶液(10mM, 40μl)を混合して発光スペクトル測定を行った。酵素溶液の濃度は、17μMのものを使用した。ただし、ホタルルシフェリン(1)は、1.7μM、フェノール型ルシフェリンは、170μMの酵素をそれぞれ使用した。また、発光スペクトル測定の露光時間は、60秒とした。ただし、ホタルルシフェリンは、5秒で行った。
上記手順を使用して、図1に示した化合物の発光波長を測定した。図1に示した化合物は、それぞれの化合物の下に記載された発光波長を有していた。
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