JPWO2016006678A1 - ジペプチジルペプチダーゼiv検出用蛍光プローブ - Google Patents
ジペプチジルペプチダーゼiv検出用蛍光プローブ Download PDFInfo
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- JPWO2016006678A1 JPWO2016006678A1 JP2016532980A JP2016532980A JPWO2016006678A1 JP WO2016006678 A1 JPWO2016006678 A1 JP WO2016006678A1 JP 2016532980 A JP2016532980 A JP 2016532980A JP 2016532980 A JP2016532980 A JP 2016532980A JP WO2016006678 A1 JPWO2016006678 A1 JP WO2016006678A1
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- 125000003386 piperidinyl group Chemical group 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
- LJCNRYVRMXRIQR-OLXYHTOASA-L potassium sodium L-tartrate Chemical class [Na+].[K+].[O-]C(=O)[C@H](O)[C@@H](O)C([O-])=O LJCNRYVRMXRIQR-OLXYHTOASA-L 0.000 description 1
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- 230000000306 recurrent effect Effects 0.000 description 1
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- MYFATKRONKHHQL-UHFFFAOYSA-N rhodamine 123 Chemical compound [Cl-].COC(=O)C1=CC=CC=C1C1=C2C=CC(=[NH2+])C=C2OC2=CC(N)=CC=C21 MYFATKRONKHHQL-UHFFFAOYSA-N 0.000 description 1
- 238000007142 ring opening reaction Methods 0.000 description 1
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- 125000000020 sulfo group Chemical group O=S(=O)([*])O[H] 0.000 description 1
- 208000024891 symptom Diseases 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
- BCNZYOJHNLTNEZ-UHFFFAOYSA-N tert-butyldimethylsilyl chloride Chemical compound CC(C)(C)[Si](C)(C)Cl BCNZYOJHNLTNEZ-UHFFFAOYSA-N 0.000 description 1
- UGNWTBMOAKPKBL-UHFFFAOYSA-N tetrachloro-1,4-benzoquinone Chemical compound ClC1=C(Cl)C(=O)C(Cl)=C(Cl)C1=O UGNWTBMOAKPKBL-UHFFFAOYSA-N 0.000 description 1
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Abstract
Description
本明細書中において、「ハロゲン原子」とは、フッ素原子、塩素原子、臭素原子、又はヨウ素原子を意味する。
本発明の蛍光プローブは、キサンテン骨格に、ジペプチジルペプチダーゼIVの基質となるジペプチド部位を導入した構造を有することを特徴とするものであって、一態様において、以下の式(I)で表される構造を有する化合物を含むものである。
上記式(I)で示される化合物は、その高い水溶性、高い蛍光量子収率等から、バイオイメージングに広く利用されているキサンテン系蛍光色素の骨格であるが、キサンテン骨格上部が閉環状態では、中性領域(例えばpH5ないし9の範囲)において当該蛍光プローブ自体は実質的に無吸収・無蛍光(蛍光応答がOffの状態)である。これに対し、B−A−NHにおけるジペプチド部位がジペプチジルペプチダーゼIVにより加水分解され、キサンテン骨格から切断されると速やかに開環した互変異性体となって強蛍光性の下記化合物が生じる。
かかる発光機構に従い、本発明のジペプチジルペプチダーゼIVの検出方法は、上記の蛍光プローブを生体外において試料と接触させる工程、及び、当該試料中に含まれるジペプチジルペプチダーゼIVと蛍光プローブの反応による蛍光応答を観測する工程を含むことを特徴とする。かかる方法によってジペプチジルペプチダーゼIVを検出することにより、ジペプチジルペプチダーゼIVが発現している標的細胞を検出することも可能となる。好ましくは、上記標的細胞は癌細胞であり、より好ましくは、食道癌細胞である。本明細書において「検出」という用語は、定量、定性など種々の目的の測定を含めて最も広義に解釈されるべきである。
本発明の検出方法においては、上記蛍光プローブを含むジペプチジルペプチダーゼIV(DPP−IV)検出用キットを用いることが好ましい。特に、上記プロテアーゼがキモトリプシンである場合、上記蛍光プローブとトリプシンを含み、測定が行われるまでの期間において蛍光プローブとトリプシンが混合することなく格納されていることが好ましい。当該キットにおいて、通常、本発明の蛍光プローブは溶液として調製されているが、例えば、粉末形態の混合物、凍結乾燥物、顆粒剤、錠剤、液剤など適宜の形態の組成物として提供され、使用時に注射用蒸留水や適宜の緩衝液に溶解して適用することもできる。
以下のスキームに従って、種々のジペプチド部位を有するヒドロキシメチルローダミングリーン(HRMG)を合成した。なお、化合物A7の合成の際に用いるFmoc-アミノ酸を種々変えることによって、異なるジペプチド部位を有する化合物1〜6を得た。
Rhodamine 110 Chloride 1127 mg (3.12 mmol, 1eq) をメタノール 180 mLに溶解し、硫酸 9 mLを加えた後にアルゴン雰囲気下で80 ℃で一晩撹拌した。室温まで冷却した後、反応溶媒を減圧除去し、残渣に飽和炭酸水素ナトリウム水溶液を加えて中和後、濾過した。濾過して得られた個体をメタノールに溶解させ回収し、メタノールを減圧除去した。残渣をテトラヒドロフラン 200 mLに溶解させ、氷浴下で撹拌させながら水素化リチウムアルミニウム 1545 mg (40.7 mmol, 13 eq) を加えアルゴン雰囲気下、アルミホイルで遮光して一晩室温で撹拌した。反応溶液を氷浴下で撹拌しながらメタノール30 mLを加えた後、反応溶媒を減圧除去した。残渣に飽和ロッシェル塩水溶液 200 mLと酢酸エチル100 mLを加えてアルゴン雰囲気下、アルミホイルで遮光して一晩撹拌した。反応溶液から分液操作で酢酸エチル層を抽出した後に酢酸エチルを減圧除去し、残渣をシリカゲルクロマトグラフィーで精製して (ジクロロメタン/メタノール=90/10) 目的化合物 (557 mg, 56%)を得た。
1H NMR (300 MHz, CD3OD): δ 4.63 (s, 2H), 5.38 (s, 1H), 6.30 (dd, 2H, J = 2.1, 9.0 Hz), 6.41 (d, 2H, J = 2.1 Hz), 6.64 (d, 2H, J = 9.0 Hz), 7.04-7.08 (m, 1H), 7.14-7.16 (m, 2H), 7.39-7.42 (m, 1H)
13C NMR (100 MHz, CD3OD): δ 40.2, 63.0, 103.4, 112.2, 115.4, 127.3, 128.7, 128.9, 131.2, 131.9, 145.7, 148.5, 152.8
HRMS (ESI+): calcd for [M+H]+, 319.14018 ; found, 319.14465 (-4.48 mmu)
化合物A1 557 mg (1.75 mmol, 1eq)、t-ブチルジメチルシリルクロライド 402 mg (2.66 mmol, 1.5eq)、イミダゾール 242 mg (3.55 mmol, 2eq)をN.N’-ジメチルホルムアミド 25 mLに溶解させ、アルゴン雰囲気下、アルミホイルで遮光して4時間撹拌した。水を100 mL加えた後、酢酸エチルを加え分液操作で酢酸エチル層を抽出した。酢酸エチルを減圧除去し、残渣をシリカゲルカラムクロマトグラフィーで精製して(酢酸エチル/ヘキサン=2/3)目的化合物 (713 mg, 94%)を得た。
1H NMR (400 MHz, aceton-d6 ): δ 0.11 (s, 6H), 0.98 (s,9H), 4.72 (s, 4H), 4.79 (s, 2H), 5.44 (s, 1H), 6.35 (dd, 2H, J = 2.9, 10.7 Hz), 6.46 (d, 2H, J = 2.9 Hz), 6.71 (d, 2H, J = 10.7 Hz), 7.22-7.29 (m, 3H), 7.53-7.57 (m, 1H)
13C NMR (75 MHz, CDCl3): δ -5.47, 18.3, 25.9, 39.1, 62.9, 102.0, 110.6, 114.1, 126.4, 127.2, 127.4, 130.4, 130.7, 138.5, 143.9, 146.0, 151.3
HRMS (ESI+): calcd for [M+H]+, 433.23113 ; found, 433.23114 (0.01 mmu)
化合物A2 311 mg (0.720 mmol, 2eq)、Fmoc-プロリン 124 mg (0.368 mmol, 1eq)、HATU 137 mg (0.360 mmol, 1eq)をN,N’-ジメチルホルムアミド 5 mLに溶解させた後、N,N-ジイソプロピルエチルアミン 128 μL (0.722 mmol, 2eq)を加えてアルゴン雰囲気下にて50 ℃で90分間撹拌した。反応溶媒を減圧除去した後、残渣を酢酸エチルに溶解し、飽和食塩水を加えて分液操作を行った。酢酸エチル層を抽出し、減圧除去によって得られた残渣をシリカゲルカラムクロマトグラフィー(酢酸エチル/ヘキサン=1/1)で精製し、目的化合物(167 mg, 62%)を得た。
HRMS (ESI+): calcd for [M+H]+, 752.35197 ; found, 752.35316 (1.19 mmu)
化合物A3 167 mg (0.222 mmol, 1.5eq)をジクロロメタン1.6 mL、N,N’-ジメチルホルムアミド 0.4 mLに溶解させた後、2-クロロトリチルクロライドレジン 102 mg (0.145 mmol, 1eq)、N,N-ジエチルイソプロピルアミン 200 μL (1.13 mmol, 7.8eq)を加え、アルゴン雰囲気下、アルミホイルで遮光して24時間撹拌した。濾過により反応溶媒を取り除いた後、レジンをジクロロメタン/メタノール/N,N-ジイソプロピルエチルアミン=17/2/1混合液およびジクロロメタンで洗浄した。得られたレジンを6等分した。
得られたA4を6等分し、各々に、N,N’-ジメチルホルムアミド 1.1 mLを加え、1時間振とう後濾過により溶液を除き、テトラクロロ-p-ベンゾキノン120 mM N,N’-ジメチルホルムアミド溶液 1.6 mLを加え30分振とうした。溶液を濾過によって除き、N,N’-ジメチルホルムアミド 1.1 mLを加え1分間振とうし、溶液を濾過によって除いた。
20%ピペリジン/N,N’-ジメチルホルムアミド溶液(v/v) 800 μLを加えて3分間振とうし、溶液を濾過によって除き、再び20%ピペリジン/N,N’-ジメチルホルムアミド溶液(v/v) 800 μLを加えて12分間振とうし、溶液を濾過によって除いた。N,N’-ジメチルホルムアミド 900 μLを加え1分間振とうし、溶液を除く作業を6度行った。
440 mM HATU/N,N’-ジメチルホルムアミド溶液 440 μL、2M N,N-ジエチルイソプロピルアミン/ N-メチルピロリドン溶液 200 μL、480 mM Fmoc-アミノ酸(1 ; Fmoc-グリシン、2 ; Fmoc-t-ブチル-グルタミン酸、3 ; Fmoc-Boc-リシン、4 ; Fmoc-t-ブチル-チロシン、5 ; Fmoc-ロイシン、6 ; Fmoc-プロリン)/ N,N’-ジメチルホルムアミド溶液 400 μL 2時間振とうした。濾過により溶液を除き、N,N’-ジメチルホルムアミド 900 μLを加え1分間振とうし、溶液を除いた。再び440 mM HATU/N,N’-ジメチルホルムアミド溶液 440 μL、2M N,N-ジエチルイソプロピルアミン/ N-メチルピロリドン溶液 200 μL、480 mM Fmoc-アミノ酸(化合物1〜6に応じて、それぞれ以下を用いた。1 ; Fmoc-グリシン、2 ; Fmoc-t-ブチル-グルタミン酸、3 ; Fmoc-Boc-リシン、4 ; Fmoc-t-ブチル-チロシン、5 ; Fmoc-ロイシン、6 ; Fmoc-プロリン)/ N,N’-ジメチルホルムアミド溶液 400 μL 2時間振とうした。濾過により溶液を除き、N,N’-ジメチルホルムアミド 900 μLを加え1分間振とうし、溶液を除いた。三たび440 mM HATU/N,N’-ジメチルホルムアミド溶液 440 μL、2M N,N-ジエチルイソプロピルアミン/ N-メチルピロリドン溶液 200 μL、480 mM Fmoc-アミノ酸(1 ; Fmoc-グリシン、2 ; Fmoc-t-ブチル-グルタミン酸、3 ; Fmoc-Boc-リシン、4 ; Fmoc-t-ブチル-チロシン、5 ; Fmoc-ロイシン、6 ; Fmoc-プロリン)/ N,N’-ジメチルホルムアミド溶液 400 μL 1時間振とうした。濾過により溶液を除き、N,N’-ジメチルホルムアミド 900 μLを加え1分間振とうし、溶液を除いた。20%ピペリジン/N,N’-ジメチルホルムアミド溶液(v/v) 800 μLを加えて3分間振とうし、溶液を濾過によって除き、再び20%ピペリジン/N,N’-ジメチルホルムアミド溶液(v/v) 800 μLを加えて12分間振とうし、溶液を濾過によって除いた。N,N’-ジメチルホルムアミド 900 μLを加え1分間振とうし、溶液を除く作業を6度行った。
得られたレジンを30 mLバイアル瓶に移し、トリフルオロ酢酸 2 mL、水 200 μL、トリエチルシラン200 μLを加え2時間撹拌した。濾過によってレジンを除き、アセトニトリルで洗いこんだ後、濾液を減圧蒸留し残渣をジエチルエーテル 40 mLに加えて3,000 rpmで10分間遠心した。ジエチルエーテルを除き、再びジエチルエーテル 40 mLを加えて3,000 rpmで10分間遠心し、ジエチルエーテルを除いた。残渣を一晩風乾させた後、HPLCを用いて精製を行った。化合物1-4,6はHPLC(eluent A (H2O 0.1% TFA) and eluent B (CH3CN 80%, H2O 20%, 0.1% TFA) (A/B = 80/20 to 25/75 45 min))で精製し目的化合物を得た。 化合物5はHPLC(eluent A (H2O 0.1% TFA) and eluent B (CH3CN 80%, H2O 20%, 0.1% TFA) (A/B = 80/20 to 25/75 45 min))で精製し、さらにHPLC(eluent A (H2O 0.1% TEAA) and eluent B (CH3CN 80%, H2O 20%, 0.1% TEAA) (A/B = 80/20 to 25/75 45 min))で精製し、最後にHPLC(eluent A (H2O 0.1% TFA) and eluent B (CH3CN 80%, H2O 20%, 0.1% TFA) (A/B = 80/20 to 25/75 45 min))で精製し目的化合物を得た。
[収量及び収率]化合物1 ; 4.7 mg, 42%、化合物2 ; 3.2 mg, 25%、化合物3 ; 5.1 mg, 39%、化合物4 ; 3.2 mg, 23%、化合物5 ; 4.3 mg, 34%、化合物6 ; 6.7 mg, 55%)
実施例1で合成したグリシン−プロリンのジペプチド部位を有する化合物1(GP−HRMG)及びジペプチド部位を有しないヒドロキシメチルローダミングリーン(HRMG)の吸収スペクトル及び蛍光スペクトルをそれぞれ測定した。結果を表1に示す。
ヒト食道扁平上皮癌細胞6種類に、GP−HMRG(化合物1)を滴下した後、経時的にイメージングを行った。
術前に上部内視鏡検査を施行した症例に対して、腫瘍部と非腫瘍部から各々1個ずつ生体サンプルを採取し、各種蛍光プローブ(化合物1〜6)を滴下した後、経時的にイメージング及び蛍光強度を測定した。
食道癌の手術ならびに内視鏡的粘膜下層剥離術(ESD)を施行した症例に対して、それぞれの施術において摘出した直後の食道検体(それぞれ手術検体、ESD検体という)をイメージング室に持ち込み、検体に直接各種蛍光プローブ(化合物1〜6)を噴霧した後、経時的にイメージングを行った。手術検体には、各種蛍光プローブのDMSO溶液 (10mM) のうち10μLを2mL のRPMI1640 (phenolred-free)に溶かし (最終プローブ濃度50μM)、内視鏡用の散布チューブを用いて検体に蛍光プローブを噴霧した後、Maestro In Vivo Imaging System Exを用いてイメージングを行った。ESD検体には、各種蛍光プローブのDMSO溶液 (10mM)のうち3μLを600μL のRPMI1640 (phenolred-free)に溶かし (最終プローブ濃度50μM)、検体に蛍光プローブを滴下した後、Maestro In Vivo Imaging System Exを用いてイメージングを行った。
Claims (15)
- Aが、プロリン又はアラニンから選択されるアミノ酸残基である、請求項1に記載の蛍光プローブ。
- Bが、グリシン、グルタミン酸、リシン、チロシン、ロイシン、又はプロリンから選択されるアミノ酸残基である、請求項2に記載の蛍光プローブ。
- Aがプロリン残基であり、Bがグリシン残基である、請求項3に記載の蛍光プローブ。
- R1、R2、R3、R4、R5、R6、R7、R8及びR9が水素原子であり、Xがメチレン基である、請求項1〜4のいずれか1項に記載の蛍光プローブ。
- 請求項1〜6のいずれか1項に記載の蛍光プローブを生体外において試料と接触させる工程、及び、当該試料中に含まれるジペプチジルペプチダーゼIV(DPP−IV)と前記蛍光プローブの反応による蛍光応答を観測する工程を含むことを特徴とする、ジペプチジルペプチダーゼIVの検出方法。
- 蛍光イメージング手段を用いて前記蛍光応答を可視化することを更なる特徴とする、請求項7に記載の方法。
- 請求項1〜6のいずれか1項に記載の蛍光プローブを用いて、ジペプチジルペプチダーゼIV(DPP−IV)が発現している標的細胞を検出する方法。
- 前記標的細胞が、癌細胞である、請求項9に記載の方法。
- 前記癌細胞が、食道癌細胞である、請求項10に記載の方法。
- 請求項1〜6のいずれか1項に記載の蛍光プローブを含む、ジペプチジルペプチダーゼIV(DPP−IV)検出用キット。
- 請求項1〜6のいずれか1項に記載の蛍光プローブを含む薬液を測定対象の試料と接触させるための装置であって、前記蛍光プローブを含む薬液を収容する薬液収容部と、前記薬液を前記試料に噴霧可能に構成された薬液噴霧部を備える、該装置。
- 前記試料中に含まれるジペプチジルペプチダーゼIV(DPP−IV)と前記蛍光プローブの反応による蛍光応答を観測するための蛍光イメージング手段をさらに備える、請求項13に記載の装置。
- 前記装置が内視鏡である、請求項13又は14に記載の装置。
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