JP2011088892A - 新規な蛍光クエンチャー分子とこれを利用する方法及び使用 - Google Patents
新規な蛍光クエンチャー分子とこれを利用する方法及び使用 Download PDFInfo
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- JP2011088892A JP2011088892A JP2010234727A JP2010234727A JP2011088892A JP 2011088892 A JP2011088892 A JP 2011088892A JP 2010234727 A JP2010234727 A JP 2010234727A JP 2010234727 A JP2010234727 A JP 2010234727A JP 2011088892 A JP2011088892 A JP 2011088892A
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- MGPYJVWEJNTXLC-UHFFFAOYSA-N [6-[6-[2-cyanoethoxy-[di(propan-2-yl)amino]phosphanyl]oxyhexylcarbamoyl]-6'-(2,2-dimethylpropanoyloxy)-3-oxospiro[2-benzofuran-1,9'-xanthene]-3'-yl] 2,2-dimethylpropanoate Chemical compound C12=CC=C(OC(=O)C(C)(C)C)C=C2OC2=CC(OC(=O)C(C)(C)C)=CC=C2C11OC(=O)C2=CC=C(C(=O)NCCCCCCOP(N(C(C)C)C(C)C)OCCC#N)C=C21 MGPYJVWEJNTXLC-UHFFFAOYSA-N 0.000 description 1
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- 125000003538 pentan-3-yl group Chemical group [H]C([H])([H])C([H])([H])C([H])(*)C([H])([H])C([H])([H])[H] 0.000 description 1
- 150000002972 pentoses Chemical class 0.000 description 1
- NMHMNPHRMNGLLB-UHFFFAOYSA-N phloretic acid Chemical compound OC(=O)CCC1=CC=C(O)C=C1 NMHMNPHRMNGLLB-UHFFFAOYSA-N 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
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- ADXCEOBGDCQCKM-UHFFFAOYSA-N quinoline-2,3-dione Chemical group C1=CC=CC2=NC(=O)C(=O)C=C21 ADXCEOBGDCQCKM-UHFFFAOYSA-N 0.000 description 1
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- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 1
- 125000002088 tosyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1C([H])([H])[H])S(*)(=O)=O 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
- C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Plural Heterocyclic Compounds (AREA)
- Nitrogen Condensed Heterocyclic Rings (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
【解決手段】 本発明は、新規ピリジニル−イソキノリン−ジオン誘導体、該誘導体の製造方法、新規ピリジニル−イソキノリン−ジオン誘導体と、(i)固体支持体又は(ii)生体分子とを含むコンジュゲート、該コンジュゲートの製造方法、並びに蛍光共鳴エネルギー移動(FRET)におけるクエンチャーとしての上記コンジュゲートの使用に関する。
【選択図】なし
Description
R1及びR2の一方は、水素、C1-C6アルキル又はハロゲンであり、
他方は-Q-Yであり、
Qは、1〜10個の線状に共有結合した原子を含む連結基を表し、Yは官能基であり、
特にQは、直鎖状又は分枝状の飽和又は不飽和の置換又は非置換C1-C10炭化水素鎖であり、Yは、ヒドロキシル、カルボキシル、及びアミノからなる群より選択され;
R3及びR4は、互いに独立して、-NR5R6により表され、R5及びR6は、互いに独立して、水素又は置換若しくは非置換アリールである〕。
以下の工程:
(a)式R5-N=C(OH)-C(OH)=N-R5’の二置換シュウ酸ジアミドを五塩化リンと反応させて、式R5-N=CCl-ClC=N-R5’のシュウ酸のビス−イミドイルクロリドを取得する工程;
(b)工程(a)で取得したシュウ酸のビス−イミドイルクロリドを2-アミノメチルピリジンと反応させて、式IIIの二置換ピリド[1,2-a]ピラジン:
を含み、
ここでR1、R2、R5は、適用可能な限り、本発明の化合物について既に詳しく定義した通りであり、
R5’は、適用可能な限り、本発明の化合物についてR5として既に詳しく定義した通りである、上記方法に関する。
シュウ酸ジアミド(20 mmol)を乾燥トルエン(200ml)中に五塩化リン(40 mmol)と共に懸濁し、透明な暗黄色溶液が得られ、ガス発生が終了するまで還流した。反応終了後、溶媒を減圧留去し、残渣をn-ヘプタンから再結晶した。
2-アミノメチルピリジン(10 mmol)をトリエチルアミン(20 mmol)と共にTHF(50 ml)に溶解し、対応のビス-イミドイルクロリド(10 mmol)の溶液を滴下した。続いて、溶液を約4時間還流し、冷却後に溶媒を減圧留去した。残渣を少量の冷メタノールで洗浄した後、残渣をアセトニトリル又はTHFから再結晶した。
キノン(1当量)及びピリド[1,2-a]ピラジン(1当量)を乾燥ジクロロメタン(不活性雰囲気下ではない)(溶媒約30ml /1mmolキノン)に溶解し、数時間(典型的には3〜12時間)還流するか又は室温で(典型的には2〜3日)撹拌した。反応の進行を薄層クロマトグラフィーを用いてモニターした。反応終了後、反応混合物を濃縮乾固し、カラムクロマトグラフィー(SiO2/トルエン:酢酸エチル又はクロロホルム/メタノール)を用いて精製した。2種の位置異性体、例えばTWDQ 9 A及びBが形成された。
水25 ml中のペルオキソ二硫酸アンモニウム(27 mmol)の溶液を、水40 ml中の1,4-ベンゾキノン(20 mmol)、アジピン酸(40 mmol)及び硝酸銀(6 mmol)の溶液に、60〜65℃の温度で強く撹拌しながら45分以内に滴下した。撹拌を10分間継続した後、溶液を0℃に冷却し、ろ過し、残渣をソックスレー装置においてベンゼンを用いて抽出した。
収量:約35〜40%、文献:42%
融点:108〜109℃。
60℃で撹拌しながら、水(10ml)中のペルオキソ二硫酸ナトリウム(0.01 mol)の溶液を、水(40 ml)中の硝酸銀(0.2 g)とヘキサン(5 ml)中の1,4-ベンゾキノン(0.01 mol)及び1-ヘキサノール(0.04 mol)との不均質混合物に添加した。ジエチルエーテルで抽出した後、真空で蒸発させて、得られた残渣をシリカゲルを用いたカラムクロマトグラフィー(ヘキサン/ジエチルエーテルで溶出)により精製した。
1H-NMR (250 MHz、CDCl3中): 0.85 (t, 3H); 1.25 (t, 2H); 1.53-1.63 (m, 4H); 2.25 (s, 3H); 2.34 (s, 3H); 2.36 (s, 1H); 2.95 (m, 1H); 3.60 (t, 2H); 6.59 (s, 1H); 6.90-7.26 (m, 9H); 7.35 (t, 1H); 7.60 (d, 1H); 7.86 (t, 1H); 8.63 (d, 1H); 10.47 (s, 1H)
UV/Vis (CHCl3中): λmax (lgε) = 586 nm (3.9)
MS (DEI) = 546 (M+)。
収量:約25%
1H-NMR (250 MHz、CDCl3中): 0.70 (t, 3H); 0.83 (d, 2H); 1.51-1.61 (m, 4H); 2.36 (s, 1H); 2.88 (m, 1H); 3.51 (t, 2H); 6.64 (s, 1H); 3.90-7.30 (m, 12H); 7.67 (d, 1H); 7.88 (t, 1H); 8.56 (d, 1H); 70.27 (s, 1H)
UV/Vis (CHCl3中): λmax (lgε) = 550 nm (3.9)
MS (DEI) = 518 (M+)。
収量:約40%
1H-NMR (CDCl3中): 0.84 (t, 3H); 0.90 (d, 2H); 1.51-1.61 (m, 4H); 2.37 (s, 1H); 2.88 (m, 1H); 3.51 (t, 2H); 6.61 (s, 1H); 7.00-7.39 (m, 12H); 7.63 (d, 1H); 7.86 (t, 1H); 8.63 (d 1H); 10.35 (s, 1H)
UV/Vis (CHCl3中): λmax (lgε) = 555 nm (3.9)
MS (DEI) = 518 (M+)。
収量:約20%
1H-NMR (250 MHz、CDCl3中): 1.27 (m, 2H); 1.64 (m, 2H); 2.26 (s, 3H); 2.30 (s, 3H); 2.34 (m, 2H); 2.47 (m, 2H); 6.65 (s, 1H); 6.90-7.30 (m, 9H); 7.42 (t, 1H); 7.72 (d, 1H); 7.95 (t, 1H); 8.76 (d, 1H); 10.45 (s, 1H)
UV/Vis (CHCl3中): λmax (lgε) = 558 nm (3.9)
MS (DEI) = 546 (M+)。
収量:約35%
1H-NMR (250 MHz、CDCl3中): 1.26 (m, 2H); 1.60 (m, 2H); 2.24 (s, 3H); 2.28 (s, 3H); 2.31 (m, 2H); 2.45 (m, 2H); 6.60 (s, 1H); 6.90-7.25 (m, 9H); 7.35 (t, 3H); 7.60 (d, 1H); 7.85 (t, 1H); 8.64 (d, 1H); 10.41 (s, 1H)
UV/Vis (CHCl3中): λmax (lgε) = 568 nm (3.9)
MS (DEI) = 546 (M+)。
TDWQ 9A又はTDWQ 9Bの遊離体(educt)をDMF(3 ml中25 mg)に溶解し、6 mgのN-ヒドロキシスクシンイミド、15 mgのHBTU及び11μlのモルホリノ-エチル-イソシアニドを添加する。溶液を3時間撹拌する。エバポレーション後、粗製混合物を分取HPLCにより精製する。収量:81%。
TWDQ9のクエンチ効率を、加水分解プローブ検出技術を利用するラムダDNAリアルタイムPCRアッセイにおいて評価した。
プライマーは、ABI(登録商標)394 DNAシンセサイザー(Applied Biotechnology Institute, Inc.、Corporation Iowaの商標、Building 36, Cal Poly State University San Luis Obispo, California 93407, USA)において、標準的なホスホロアミダイト化学を利用して1μmolスケールで合成した(全ての試薬は、例えばSigma-Aldrich又はGlen Researchから入手可能である)。プライマーを水酸化アンモニウムにより55℃で8時間かけて脱保護した。アンモニア性溶液をエバポレートし、粗製オリゴヌクレオチドを、強アニオン交換HPLCカラムを用いて、高pHで塩化ナトリウムの線形勾配で精製した。生成物であるオリゴヌクレオチドを含む画分をプールし、脱塩し、10 mM Tris, pH 8.0中に調製した。純度及び光学密度を測定した。
加水分解プローブは、ABI(登録商標)394 DNAシンセサイザー(Applied Biotechnology Institute, Inc.、Corporation Iowaの商標、Building 36, Cal Poly State University San Luis Obispo, California 93407, USA)において、標準的なホスホロアミダイト化学を利用して1μmolスケールで合成した。標準的なdTホスホロアミダイトtac-dA、tac-dC及びtac-dGに加えて、保護デオキシヌクレオチドホスホロアミダイト(Sigma-Aldrich、カタログ番号T111031、A112031、C112031、G112031)を使用した。さらに、JA270ホスホロアミダイト(Roche Applied Science、マテリアル番号4906802)標識及びBlack Hole Quencher(BHQ-2)クエンチャー(Biosearch Technologies Inc.、カタログ番号BNS-5052)をホスホロアミダイト試薬を用いて組み込んだ。3'-リン酸を3'-Extension Blocker CPG(Clontech Inc.、カタログ番号PT3357-2)を用いて導入した。オリゴヌクレオチドを水酸化アンモニウムにより周囲温度で一晩かけて脱保護した。アンモニア性溶液をエバポレートし、粗製オリゴヌクレオチドを、逆相HPLCを用いて、0.1 M酢酸トリエチルアンモニウム(pH 7)バッファー中のアセトニトリルの量を増加させた勾配で精製した。生成物であるオリゴヌクレオチドを含む画分をプールし、脱塩し、10 mM Tris, pH 8.0中に調製した。純度及び光学密度を測定した。
加水分解プローブは、ABI(登録商標)394 DNAシンセサイザー(Applied Biotechnology Institute, Inc.、Corporation Iowaの商標、Building 36, Cal Poly State University San Luis Obispo, California 93407, USA)において、標準的なホスホロアミダイト化学を利用して1μmolスケールで合成した。標準的なdTホスホロアミダイトtac-dA、tac-dC及びtac-dGに加えて、保護デオキシヌクレオチドホスホロアミダイト(Sigma-Aldrich、カタログ番号T111031、A112031、C112031、G112031)、TFA保護3’-アミノ修飾用ホスホロアミダイト(3-アミノ-1,2-プロパンジオールに基づくアミノ修飾用ホスホロアミダイトの調製、米国特許第6,031,091号)を使用した。さらに、FAM(5’-フルオレセインホスホロアミダイト、Glen Research、カタログ番号10-5901)又はJA270ホスホロアミダイト(EP 0 962 497)標識を5’-末端に組み込んだ。3'-リン酸を3'-Extension Blocker CPG(Clontech Inc.、カタログ番号PT3357-2)を用いて導入した。オリゴヌクレオチドを水酸化アンモニウムにより周囲温度で一晩かけて脱保護した。アンモニア性溶液をエバポレートし、粗製オリゴヌクレオチドを、逆相HPLCを用いて、0.1 M酢酸トリエチルアンモニウム(pH 7)バッファー中のアセトニトリルの量を増加させた勾配で精製した。主ピークを含む画分を回収し、脱塩し、エバポレートした。アミノ修飾オリゴヌクレオチドを0.1 Mホウ酸ナトリウムバッファー(pH 8.5)に溶解し、DMFに溶解した2 mgのTWDQ9-NHSエステルを2回添加した(2回目の部分は5時間後に)。周囲温度で一晩(18時間)反応後、溶液を透析により脱塩し、続いて逆相HPLCを用いて、0.1 M酢酸トリエチルアンモニウム(pH 7)バッファー中のアセトニトリルの量を増加させた勾配で再度精製した。生成物であるオリゴヌクレオチドを含む画分をプールし、脱塩し、10 mM Tris, pH 8.0中に調製した。純度及び光学密度を測定した。
材料及び方法:
−LC480装置(96穴)(Roche Applied Science、カタログ番号04640268001)
−LC TaqMaster Roche(Roche Applied Science、カタログ番号04535286001)
−DNA lambda Roche(Roche Applied Science、カタログ番号10745782001, [c = 6.25ng/ml])
−ラムダリバースプライマー:
5’- GTC GCT TTT TGC CCC ACA GTA-3’(配列番号6)
BMO 07.442983 lot ah_PP_48_A12-H12 [c = 10μM](実施例1より)
−ラムダフォワードプライマー:
5’- AAC AAA AAC GGG GTT TAC CTT A-3’(配列番号1)
BMO 07.442982 lot ah_PP_A11-H11 [c = 10μM](実施例1より)
−種々のレポーター/クエンチャーの組合せを有する3つのラムダプローブ [c = 5μM](実施例2及び3より):
5'- R TCG GTA CGG ATA CCG CGA AAG AGC Q PO4 -3'(配列番号3〜5)
例A
ラムダ加水分解プローブ1(GO 2986):
5’- X TCG GTA CGG ATA CCG CGA AAG AGC Y PO4-3’(配列番号3)
X = JA270 Y = BHQ2
ラムダ加水分解プローブ2(GO 3014):
5’- X TCG GTA CGG ATA CCG CGA AAG AGC Y PO4-3’(配列番号4)
X = JA270 Y = TWDQ9
例B
ラムダ加水分解プローブ1(GO 2986):
5’- X TCG GTA CGG ATA CCG CGA AAG AGC Y PO4-3’(配列番号3)
X = JA270 Y = BHQ2
ラムダ加水分解プローブ2(GO 3014):
5’- X TCG GTA CGG ATA CCG CGA AAG AGC Y PO4-3’(配列番号4)
X = JA270 Y = TWDQ9
また、以下の配列を有し、TWDQ9によりクエンチされる、フルオレセインリポーター色素で標識した加水分解プローブを用いて、リアルタイムPCR実験を行った:
ラムダ加水分解プローブ3(HO 1214):
5’- X TCG GTA CGG ATA CCG CGA AAG AGC Y PO4-3’(配列番号5)
X = FAM Y = TWDQ9
配列番号3〜5:DNAプローブ
Claims (15)
- Qが、直鎖状又は分枝状の飽和又は不飽和の置換又は非置換C2-C8炭化水素鎖である、請求項1に記載の化合物。
- Yがヒドロキシル又はカルボキシル基である、請求項1又は2に記載の化合物。
- R3及び/又はR4が-NR5Hである、請求項1〜3のいずれか1項に記載の化合物。
- R3及びR4の各々が-NR5R6である、請求項1〜4のいずれか1項に記載の化合物。
- R5が置換又は非置換アリールである、請求項5に記載の化合物。
- 置換又は非置換アリールがフェニル又はトルイルである、請求項5又は6に記載の化合物。
- アリールが、芳香族C6H5、C10H7、又はC14H9炭化水素残基である、請求項1〜7のいずれか1項に記載の化合物。
- R1及びR2の一方が、1-ヒドロキシ-4-エチル-ブチル残基又はn-ペンタン酸残基であり、他方が水素であり、
R5が4-トルイル又はフェニル残基である、
請求項1〜8のいずれか1項に記載の化合物。 - Yが、(i)固体支持体、又は(ii)生体分子に結合することができる、請求項1〜9のいずれか1項に記載の化合物。
- 式IIの化合物:
(a)式R5-N=C(OH)-C(OH)=N-R5’の二置換シュウ酸ジアミドを五塩化リンと反応させて、式R5-N=CCl-ClC=N-R5’のシュウ酸のビス-イミドイルクロリドを取得する工程;
(b)工程(a)で取得したシュウ酸のビス-イミドイルクロリドを2-アミノメチルピリジンと反応させて、式IIIの二置換ピリド[1,2-a]ピラジン:
(c)工程(b)で取得した二置換ピリド[1,2-a]ピラジンを式IVの一置換キノン:
を含み、
R1、R2、R5は、請求項1、3〜7、9及び10のいずれか1項で定義した通りであり、
R5’は、請求項1、3〜7、9及び10のいずれか1項でR5として定義した通りである、
上記方法。 - 請求項1〜10のいずれか1項に記載の化合物と、(i)固体支持体又は(ii)生体分子とを含むコンジュゲートであって、該化合物が、連結基Qを介して該支持体又は生体分子に結合されている、上記コンジュゲート。
- 請求項12に記載のコンジュゲートを製造する方法であって、請求項1〜10のいずれか1項に記載の化合物を、(i)固体支持体又は(ii)生体分子に結合させる工程を含む方法。
- 蛍光ドナーのクエンチャーとしての、請求項1〜10のいずれか1項に記載の化合物又は請求項12に記載のコンジュゲートの使用。
- コンジュゲートが、蛍光共鳴エネルギー移動(FRET)におけるクエンチャーとして使用される、請求項14に記載の使用。
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