JP4923298B2 - 細胞内カルシウムのリアルタイムモニタリング用二光子プローブ - Google Patents
細胞内カルシウムのリアルタイムモニタリング用二光子プローブ Download PDFInfo
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Classifications
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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
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B57/00—Other synthetic dyes of known constitution
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
- C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
- C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C225/00—Compounds containing amino groups and doubly—bound oxygen atoms bound to the same carbon skeleton, at least one of the doubly—bound oxygen atoms not being part of a —CHO group, e.g. amino ketones
- C07C225/22—Compounds containing amino groups and doubly—bound oxygen atoms bound to the same carbon skeleton, at least one of the doubly—bound oxygen atoms not being part of a —CHO group, e.g. amino ketones having amino groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Plural Heterocyclic Compounds (AREA)
Description
R.Pethig,M.Kuhn,R.Payne,E.Adler,T.-H.Chen,L.F.Jaffe,Cell Calcium 1989,10491- 498 H.M.Kim,C.Jung,B.R.Kim,S.-Y.Jung,J.H.Hong,Y.-G.Ko,K.J.Lee,B.R.Cho,Angew.Chem.Int.Ed.2007,46,3460-3463 J.N.Demas,G.A.Crosby,J.Phys.Chem.1971,75991-1024 C.Reichardt,Chem.Rev.1994,94,2319-2358 R.Rudolf,M.Mongillo,R.Rizzuto,T.Pozzan,Nat.Rev.Mol.Cell Biol.2003,4579-586 K.A.Connors,Binding Constants;JohnWiley & Sons,Inc.:New York,1987 J.R.Long,R.S.Drago,J.Chem.Ed.1982,59,1037 K.Hirose,J.Incl.Phenom.Macrocycl.Chem.2001,39193 J.R.Long,R.S.Drago,J.Chem.Educ.1982,59,1037-1039 A Guide to Fluorescent Probes and Labeling Technologies,10thed.,(Ed.:R.P.Haugland),Molecular Probes, Eugene, OR, 2005 S.K.Lee,W.J.Yang,J.J.Choi,C.H.Kim,S.-J.Jeon,B.R.Cho,Org.Lett.2005,7323-326 H.R.Parri,T.M.Gould,V.Crunelli,Nat.Neurosci.2001,4803-812 C.M.Matias,N.C.Matos,M.Arif,J.C.Dionisio,M.E.Quinta-Ferreira,Biol.Res.2006,39521-530 M.E.Quinta-Ferreira,C.M.Matias,Brain Res.2004,1004,52-60 J.Y.Koh,S.W.Suh,B.J.Gwag,Y.Y.He,C.Y.Hsu,D.W.Choi,Science 1996,272,1013-1016 H.R.Parri,T.M.Gould,V.Crunelli,Nat.Neurosci.2001,4803-812
[製造例1] 本発明による二光子プローブの製造
本実施例では、下記の手順によって、下記式(4)の化合物を合成した。
DMF(5mL)中の前記製造例1.1で製造した式(6)の化合物(0.38g、1.48mmol)及び1−エチル−3−(3−ジメチルアミノプロピル)−カルボジイミド・HCl(0.34g、1.80mmol)の混合物を、20分間撹拌した。前記混合物に、製造例1.2で製造した式(2)の化合物(0.90g、1.64mmol)及び4−ジメチルアミノピリジン(26mg、0.22mmol)を添加し、N2雰囲気下で12時間撹拌した。得られた産物をクロロホルムで抽出し、MgSO4上で乾燥させた後、減圧して溶媒を除去した。引き続き、産物をヘキサン/エチルアセテート(2:3)を溶離液として使うカラムクロマトグラフィーによって精製し、エタノールによる再結晶でさらに精製した。
本実施例では、下記製造例によって、本発明による二光子プローブである下記式(7)の化合物を合成した。
前記式(7)の化合物に対する吸収スペクトルは、Hewlett−Packard 8453 ダイオードアレイスペクトロメータで記録し、該化合物の蛍光スペクトルは、1cm標準石英セルを備えたAmico−Bowmanシリーズ2発光スペクトロメーターを使って得た。公知文献に記載の方法で、Coumarin307を使って蛍光量子収率を測定した(非特許文献3参照)。図1には、それぞれ1,4−ジオキサン、DMF、エタノール及びH2O中の前記化合物の吸収スペクトル(a)及び発光スペクトル(b)を示す。下記表1には、多様な溶媒中での式(7)の化合物に対する最大吸収値及び最大発光値を示す。
多様な濃度の[Ca2+]を含有する一連の較正溶液を、2種類の溶液(10mM K2EGTAを含む溶液A及び10mM CaEGTAを含む溶液B)を多様な比率で混合することで製造した。溶液A及び溶液Bは、すべて、1μMの式(4)の化合物、100mM KCl、30mM MOPSを含み、pH7.2に調整されている。
総プローブ及び金属イオン濃度は、それぞれ[L]0=[L]+[LM]及び[M]0=[M]+[LM]と定義され、[L]0及び[M]0の値が与えられる場合に、Kdは下記数式4または数式5で表わされる。
また、二光子過程に対するKd TPを決定するため、780nmの波長及び1230mWの出力に設定し(焦点平面から略10mWの平均出力に相当)、設定が固定されたチタン−サファイアレーザー光源(Coherent Chameleon、90MHz、200fs)によって励起されるDM IRE2 Microscope(Leica)を使ってTPEFスペクトルを得た。これによりTPEF滴定曲線(図6)を得て、これを数式3に適合(fitting)させた。
緩衝溶液のうちCa2+イオンの式(4)との複合体及びOG1との複合体に対する二光子励起スペクトルは、式(4)の化合物−Ca2+のΦδに対して780nmで110GMを示し、これはOG1−Ca2+の場合よりさらに5倍大きい値である。従って、式(4)の化合物で染色された試料に対するTPMイメージは、通常的なプローブで染色されたものなどより遥かに明るい。これに加えて、二光子適正曲線から計算された二光子蛍光強化因子(TPEF)は44であり、これはTPMでCa2+を探知することができるということを意味する(表2)。
[b]一光子吸収及び発光スペクトルのλmaxは、nm単位である。
[c]蛍光量子収率、±10%
[d]一光子(Kd OP)及び二光子(Kd TP)過程によってμM単位で測定されたCa2+に対する解離定数、±12%
[e]一光子(FEFOP)過程及び二光子(FEFTP)過程によって測定された蛍光強化因子(F−Fmin)/Fmin
[f]nm単位の二光子励起スペクトルに対するλmax
[g]10−50cm4s/光子(GM)単位のピークTP断面、±15%
[h]二光子励起断面
[i]DMF中からΦ=0.27±0.02(式(4))及び0.22±0.02(式(7))
[j]二光子励起蛍光強度は、断面を正確に測定するには余りにも弱かった。
[k]Mg2+に対する式(4)の化合物のKd OP数値は、6.8±0.7mMであった。
[l]公知文献を参照して行った(非特許文献10参照)。
TP断面(δ)の測定は、フェムト秒(fs)蛍光測定技法(非特許文献11参照)を使って行った。
生後1日齢のラット(Sprague−Dawley、SD)の大脳皮質から得られた星状細胞を3U/mlのパパイン(Worthington Biochemical Corporation社製、NJ、米国)を含有するHank’s balanced塩溶液(HBSS;Gibco BRL、Gaithersburg、MD、米国)を入れた75mmフラスコにプレーティングした。精製した星状細胞を培養するために、フラスコを撹拌機上で37℃に6時間撹拌した後、培地内の浮遊細胞を除去した。星状細胞を5分間0.25%トリプシンで処理し、50〜100細胞/mm2となるようにポリ−D−リシンコーティングされたガラスカバースリップ上に再びプレーティングした後、ペニシリン/ストレプトマイシン及び10%牛胎児血清が添加されたDulbecco’s modified Eagle’s培地(DMEM;Gibco社製)を使って、37℃のCO2培養器内で保持した。7〜15日間にインビトロで保持した後、星状細胞は無血清培地で3回洗浄し、無血清培地中の2μM濃度の式(7)の化合物とともに37℃で20分間培養した。細胞をリン酸緩衝溶液(PBS;Gibco社製)で3回洗浄した後、15分間無色の無血清培地中でさらに培養した後に観察した。
切片は、2日齢のラット(Sprague−Dawley、SD)の海馬及び視床下部から得た。冠状切片(coronal slices)を、人工脳脊髓流体(ACSF;138.6mM NaCl、3.5mM KCl、21mM NaHCO3、0.6mM NaH2PO4、9.9mM D−glucose、1mM CaCl2、及び3mM MgCl2)中の振動刃ミクロトームを使って400umの厚さに切り出した。切片をACSF中の10μMの式(7)の化合物で、37℃で95% O2及び5% CO2で30分間気泡を吹き込みながら培養した。引き続き、切片をACSFで3回洗浄し、ガラスボトムディッシュ(MatTek社製)に移した後、スペクトル共焦点多重光子顕微鏡で観察した。
Claims (5)
- 下記式(1)で表わされることを特徴とする、細胞内カルシウムのリアルタイムモニタリング用二光子プローブ。
- 前記R3は、CH2OCOCH3であることを特徴とする請求項1記載の細胞内カルシウムのリアルタイムモニタリング用二光子プローブ。
- 下記式(1)で表わされる細胞内カルシウムのリアルタイムモニタリング用二光子プローブの製造方法であって、下記式(2)の化合物と下記式(3)の化合物とが反応する段階を含むことを特徴とする二光子プローブの製造方法。
- 請求項1または請求項2記載の二光子プローブを観察対象とする細胞内に注入した後、放出される二光子励起蛍光イメージを得る段階を含むことを特徴とする細胞内カルシウムのリアルタイムモニタリング方法。
- 前記二光子励起蛍光イメージは、500〜620nmの範囲の波長を使って得ることを特徴とする請求項4記載の細胞内カルシウムのリアルタイムモニタリング方法。
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