CN101679849B - 荧光细胞标记物 - Google Patents
荧光细胞标记物 Download PDFInfo
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- CN101679849B CN101679849B CN200780037489.8A CN200780037489A CN101679849B CN 101679849 B CN101679849 B CN 101679849B CN 200780037489 A CN200780037489 A CN 200780037489A CN 101679849 B CN101679849 B CN 101679849B
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- cell markers
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- fluorescein
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Abstract
描述了荧光细胞标记物的制备和用途,该荧光细胞标记物具有F-S1-S2-L结构,其中F是荧光团,S1-S2是连接F至L的间隔团,L是二酰基脂。
Description
技术领域
本发明涉及荧光细胞标记物。本发明特别涉及含有荧光素,BODIPY或者它们的衍生物之一的荧光团的荧光细胞标记物。
背景技术
化合物荧光素,BODIPY,以及它们的衍生物包含荧光团。
荧光素是水溶性的。使用荧光素作为细胞标记物要求其结合至活性基团,例如异硫氰酸酯。异硫氰酸荧光素(FITC)的异硫氰酸酯基可以和蛋白质的胺基反应。
FITC通过与表面表达蛋白质结合,用于标记细胞。被标记的细胞即可以通过荧光-激活细胞分选法(FACS)分类。
BODIPY的荧光团具有有利的超过荧光素的荧光团的光谱特性。BODIPY的衍生物还通过和表面表达蛋白质结合而用于标记细胞。
通过荧光团和表面表达蛋白质结合进行细胞标记可能影响细胞功能。此外,细胞膜两维中的荧光团的移动性必然依赖于所结合蛋白质的移动性。
因此需要有可供选择的细胞标记方法,其可以避免影响细胞功能,并且提供细胞膜两维之中的荧光团的独立的移动性。
本发明目的在于,提供一种可供选择的细胞标记方法,或者至少提供一种有用的选择。
发明内容
本发明第一方面提供一种荧光细胞标记物,结构为:
F-S1-S2-L
其包括亚结构:
其中
F是荧光团;
S1-S2是连接F至L的间隔团;
L是选自二酰基-与二烷基-甘油酯的脂,包括甘油磷脂;
m与n各自为3至6;
R1是O或者S;以及
*不为H。
选择间隔团(S1-S2),以提供水溶性的细胞标记物。
优选地,F选自下组:荧光素、俄勒冈绿、宾夕法尼亚绿、东京绿、曙红、BODIPY、BODIPY TR、Alexa Fluor350、Alexa Fluor405、Alexa Fluor488、Alexa Fluor568、Alexa Fluor594、德克萨斯红、萤光黄、四甲基若丹明和它们的衍生物的荧光团。最优选地,F选自下组:荧光素、BODIPY和它们的衍生物的荧光团。
优选地,m与n之和为6至9,且*是C或者N。
优选地,当F是荧光素或者其衍生物之一的荧光团,则S1是选自1,3-二氨基丙基、1,4-二氨基丁基或者1,5-二氨基戊基衍生物的C3-5-二氨基烷基衍生物。更优选地,当F是荧光素或者其衍生物之一的荧光团,则S1是C3-5-氨基烷基硫脲基(aminoalkylthioureidyl)。最优选地,当F是荧光素或者其衍生物之一的荧光团,S1是5-((5-氨基戊基)硫脲基。
优选地,当F是荧光素或者其衍生物之一的荧光团,则S2选自下组:-CO(CH2)3CO-,-CO(CH2)4CO-(己二酸酯),-CO(CH2)5CO-,和-CO(CH2)5NHCO(CH2)5CO-。更优选地,当F是荧光素或者其衍生物之一的荧光团,则S2是-CO(CH2)4CO-(己二酸酯)。
优选地,当F是荧光素或者其衍生物之一的荧光团,该结构包括亚结构:
或者
其中m和n各自为3至5,且*不为H。
优选地,当F是BODIPY或者其衍生物之一的荧光团,则S1是3-5-alkionyldiamine。更优选地,如果F是BODIPY或者其衍生物之一的荧光团,则S1是丙酰基乙二胺。
优选地,当F是BODIPY或者其衍生物之一的荧光团,则S2选自:
-CO(CH2)3CO-、-CO(CH2)4CO-(己二酸酯)和-CO(CH2)5CO-。更优选地,当F是BODIPY或者其衍生物之一的荧光团,则S2的-CO(CH2)4CO-(己二酸酯)。
优选地,当F是BODIPY或者其衍生物之一的荧光团,该结构包括亚结构:
其中p,q和r各自为3至5,且*不为H。更优选地,p,q和r之和为8。最优选地,p为2,q为2,r为4。
优选地,L是选自二酰基-和二烷基-甘油酯的脂,包括甘油磷脂。更优选地,L选自下组:二酰基甘油脂、磷脂酸酯、磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰丝氨酸、磷脂酰肌醇、磷脂酰甘油和双磷脂酰甘油,其衍生自一种或多种反式-3-十六烯酸、顺式-5-十六烯酸、顺式-7-十六烯酸、顺式-9-十六烯酸、顺式-6-十八碳烯酸、顺式-9-十八碳烯酸、反式-9-十八碳烯酸、反式-11-十八碳烯酸、顺式-11-十八碳烯酸、顺式-11-二十碳烯酸或者顺式-13-二十二碳烯酸。更优选地,该脂衍生自一种或多种顺式-不饱和 脂肪酸。最优选地,L选自下组:1,2-邻-二油酰基-sn-丙三氧基-3-磷酯酰乙醇胺(DOPE)、1,2-邻-二硬脂酰基-sn-丙三氧基-3-磷酯酰乙醇胺(DSPE)和rac-1,2-二油酰基丙三醇(DOG)。
在第一方面的第一实施方式中,本发明提供一种具有如下结构的细胞标记物:
其称为KODE-荧光素(I)。
在第一方面的第二实施方式中,本发明提供一种具有如下结构的细胞标记物:
其称为KODE-俄勒冈绿(II)。
在第一方面的第三实施方式中,本发明提供一种具有如下结构的细胞标记物:
其称为KODE-东京绿(III)。
在第一方面的第四实施方式中,本发明提供一种具有如下结构的细胞标记物:
其称为KODE-宾夕法尼亚绿(IV)。
在第一方面的第五实施方式中,本发明提供一种具有如下结构的细胞标记物:
其称为KODE-BODIPY(V)。
典型地,M是H,但也可以被另外的一价阳离子例如Na+、K+或者NH4 +替代。
在本发明第二方面提供细胞标记方法,包括下列步骤:
使细胞悬浮液与本发明第一方面的细胞标记物接触。
本发明第三方面提供一种合并了本发明第一方面的细胞标记物的细胞。
本发明第四方面提供一种通过本发明第二方面的方法生产的细胞。
在说明书和权利要求中:
“BODIPY”表示化学文摘社(CAS)登记号为138026-71-8的化合物,CA索引名:硼,二氟[2-[(2H-吡咯-2-叉-κ N)甲基-1H-pyrrolato-κ N]-,(T-4)-(9CI)。
“荧光素”表示化学文摘社(CAS)登记号为518-47-8的化学结构,CA索引名:螺[异苯并呋喃-1(3H),9′-[9H]呫吨-3-酮,3′,6′-二羟基-,钠盐(1∶2)。
“荧光团”表示荧光分子的具有荧光性能的亚结构或者部分。
“或者其衍生物之一”表示化学结构的化学修饰,以提供具有基本上相同物理-化学性质、以及改变的光谱特性的荧光团。
“水溶性的”表示细胞标记物在不存在有机溶剂或者洗涤剂的情况下与水或者盐水(例如PBS)接触时,可形成稳定的单相系统,术语“溶液” 具有相应含义。
发明详述
国际申请PCT/NZ2005/000052(公开号WO 2005/090368)的说明书描述了水溶性的合成分子,其为F-S1-S2-L结构。
在这些结构中,F是糖类,其自发且稳定地合并入的脂质双层,所述脂质双层包括细胞膜。
该国际申请的说明书描述的优选结构包含亚结构:
其中n=3至5,X是H或者C,且*不为H。
典型地,M是H,但也可以被另外的一价阳离子例如Na+、K+或者NH4 +替代。
F是本发明结构中的荧光团,具有不同于糖类的物理化学性质。选择间隔团(S1-S2),以提供易于分散于水性媒介物,例如盐水中的结构。
不希望受理论限制,据信本发明的细胞标记物可通过它们的二酰基脂尾部合并入细胞膜的脂质双层中。因此荧光团部分被表达在细胞表面。本发明的细胞标记物可用于标记细胞,而不会改变细胞表面表达的蛋白质。
表达在细胞表面的蛋白质介导细胞功能的可能性降低。此外,细胞标记物均匀分布在脂质双层的两维中的可能性增加。荧光团的移动性不取决于细胞表面表达的蛋白质的移动性,荧光团可能另外结合至所述蛋白质。
预期可产生其他的优点,因为细胞标记物可以容许与蛋白质功能和循环无关的细胞膜动力学的研究。使用本发明的细胞标记物标记的细胞仍然可以通过传统方法识别,并用于建立生物学方法,例如荧光激活细胞分选(FACS)系统。
对于KODE-荧光素(I)的制备,FITC首先与二胺例如1,5-戊二胺(尸胺)结合。然后使结合的FITC与按照国际申请PCT/NZ2005/000052所述方法制备的活化脂质(L-A)反应。
许多荧光化合物可商业获得,如尸胺衍生物。可以制备F为表1所示荧光团之一的细胞标记物。
表1.荧光团(表示为中性电荷的质子化物质)。
对于KODE-BODIPY(V)的制备,BODIPY可以选择性地与alkionyldiamine例如3-丙酰基乙二胺(BODIPY FL EDA)结合。然后使结合的BODIPY与根据国际申请PCT/NZ2005/000052的说明书所述方法制备的活化脂质(L-A)反应。
本发明的示范性实施方式将参照附图进行描述。
附图说明
图1:接触细胞标记物(I)之后用荧光显微镜在470纳米,250x放大倍数下看到的红血球。
图2:细胞标记物KODE-荧光素(I)的结构。
图3:细胞标记物KODE-俄勒冈绿(II)的结构。
图4:细胞标记物KODE-东京绿(III)的结构。
图5:细胞标记物KODE-宾夕法尼亚绿(IV)的结构。
图6:细胞标记物KODE-BODIPY(V)的结构。
图7:细胞标记物KODE-BODIPY(V)的1H-NMR谱。
具体实施方式
制备活化的1,2-邻-二硬脂酰基-sn-丙三氧基-3-磷酯酰乙醇胺(DSPE)和活化的1,2-邻-二油酰基-sn-丙三氧基-3-磷酯酰乙醇胺(DOPE)(L-A)。
向二(N-羟基琥珀酰基)己二酸酯(A)(70毫克,205μmol)的干N,N-二甲基甲酰胺(1.5ml)溶液加DOPE或者DSPE(L)(40μmol)的氯仿(1.5ml)溶液,随后加三乙胺(7μL)。混合物在室温下保持2小时,然后用乙酸中和,并在真空中部分浓缩。
残余物柱色谱法(Sephadex LH-20,1∶1氯仿-甲醇,0.2%乙酸)得到无色浆状的活化脂质(L-A)(37mg,95%);TLC(氯仿-甲醇-水,6∶3∶0.5)∶Rf=0.5(DOPE-A),Rf=0.55(DSPE-A)。
1H NMR(CDCl3/CD3OD,2∶1),δ:
DSPE-A-5.39(m,1H,-OCH2-CHO-CH2O-),4.53(dd,1H,J=3.42,J=11.98,-CCOOHCH-CHO-CH2O-),4.33(dd,1H,J=6.87,J=11.98,-CCOOHCH-CHO-CH2O-),4.23(m,2H,PO-CH2-CH2-NH2),4.15(m,2H,-CH2-OP),3,61(m,2H,PO-CH2-CH2-NH2),3.00(s,4H,ONSuc),2.81(m,2H,-CH2-CO(Ad),2.48(m,4H,2x(-CH2-CO),2.42(m,2H,-CH2-CO(Ad),1.93(m,4H,COCH2CH2CH2CH2CO),1.78(m,4H,2x(COCH2CH2-),1,43,1.47(2b s,40H,20CH2),1.04(m,6H,2CH3).
DOPE-A-5.5(m,4H,2x(-CH=CH-),5.39(m,1H,-OCH2-CHO-CH2O-),4.58(dd,1H,J=3.67,J=11.98,-CCOOHCH-CHO-CH2O-),4.34(dd,1H,J=6.61,J=11.98,-CCOOHCH-CHO-CH2O-),4.26(m,2H,PO-CH2-CH2-NH2),4.18(m,2H,-CH2-OP),3.62(m,2H,PO-CH2-CH2-NH2),3.00(s,4H,ONSuc),2.8(m,2H,-CH2-CO(Ad),2.50(m,4H,2x(-CH2-CO),2.42(m,2H,-CH2-CO(Ad),2.17(m,8H,2x(-CH2-CH=CH-CH2-),1.93(m,4H,COCH2CH2CH2CH2CO),1.78(m,4H,2x(COCH2CH2-),1,43,1.47(2bs,40H,20CH2),1.04(m,6H,2CH3).
DOPE-A与5-((5-氨基戊基)硫脲)荧光素(尸胺荧光素)的缩合。
向活化DOPE(L-A)(5mg,5.2(μmol)的N,N-二甲基甲酰胺(0.5ml)溶液加入3mg(4.6μmol)荧光素尸胺二氢溴化物盐和5μl三乙胺。混合物在室温下保持2小时,然后加入10μl3%的NH3水溶液,混合物在室温下保持 1小时。
混合物柱色谱法(Sephadex LH-20,1∶1氯仿-甲醇,随后硅胶,乙酸乙酯-异丙醇-水,6∶3∶1)得到4.2mg(67%)KODE-荧光素(I),Rf 0.5(乙酸乙酯-异丙醇-水,6∶3∶1)。
1H NMR(CDCl3/CD3OD,1∶1),δ:
KODE-荧光素(I)-8.38(bs,1H,荧光素的芳香质子),8.15(dd,1H,J=1.7,J=8.3,荧光素的芳香质子)7.30(d,1H,J=8.3,荧光素的芳香质子),6.87(m,4H,荧光素的芳香质子),6.72(dd,2H,J=2.4,J=8.8,荧光素的芳香质子),5.50(m,4H,2×(-CH=CH-),5,38(m,1H,-OCH2-CHO-CH2O-),4.58(dd,1H,J=6.6,Jgem=11.8,HHC-O-C(O)-),4.34(dd,1H,J=3.2,Jgem=11.8,HHC-O-C(O)-),4.14(m,2H,-OCH-CH 2 -O-P-)(4.1(m,2H,-P-O-CH 2 -CH2-NH-)3.80(m,2H,N-CH2(CH2)3-CH 2 NH-C=S)3.39and 3.58(2m,2x2H,N-CH 2 -CH2-O-P-andN-CH 2 -(CH2)3-CH2NH-C=S)2.48(m,4H,2×(-CH 2 -CO),2.39(m,4H,COCH 2 CH2CH2CH 2 CO),2.19(m,8H,2×(-CH 2 CH=CH-CH 2 -),1.84(m,2H,CH 2 -荧光素尸胺),1.8(m,10H,COCH2CH 2 CH 2 CH2CO,2×(COCH2CH 2 -,and CH 2 --荧光素尸胺),1.62(m,2H,CH 2 -荧光素尸胺)1,42,1.46(2b s,40H,20CH2),1.05(m,6H,2CH3).
KODE-荧光素(I)与细胞膜的结合
KODE-荧光素(I)易于和红血球的膜结合。当浓度大于0.1mg/ml的分子分散体接触红血球的悬浮液时,观察到分子插入。
使细胞发出强荧光的介质用来指示分子在细胞膜上的均匀分布(图1)。当细胞存储于黑暗中时,该结合和分布在至少40天期间显得稳定。
实施例2
如实施例1所述,制备活化1,2-邻-二油酰基-sn-丙三氧基-3-磷酯酰乙醇胺(DOPE)(L-A)。
DOPE-A与4,4-二氟-5,7-二甲基-4-bora-3a,4a-二氮-s-indacene-3-丙酰 基乙二胺,盐酸盐(BODIPY FL EDA)的缩合
向15mg(15.5μmol)活化DOPE的CH2Cl2(0.5ml)溶液添加5mg(13.5μmol)BODIPY FL EDA的N,N-二甲基甲酰胺(0.3ml)溶液,以及5μl三乙胺。混合物在室温下保持2小时。
混合物柱色谱法(Sephadex LH-20,1∶1氯仿-甲醇)得到14.2mg(75%)KODE-BODIPY(I),Et3N-盐;分子量1289.6,Rf 0.3(乙酸乙酯-异丙醇-水,6∶3∶1)。
1H NMR(CDCl3/CD3OD,1∶1):δ7.40(s,1H,BODIPY的芳香质子),7.12(d,1H,J=3.8BODIPY的芳香质子),6.47(d,1H,J=3.8BODIPY的芳香质子),6.32(s,1H,BODIPY的芳香质子),5.50(m,4H,2x(-CH=CH-),5.38(m,1H,-OCH2-CHO-CH2O-),4.58(dd,1H,J=3.2,Jgem=11.8,HHC-O-C(O)-),4.33(dd,1H,J=6.6,Jgem=11.8,HHC-O-C(O)-),4.16(t,2H,J=5.6,P-O-CH 2-CH2-NH-),4.1(m,2H,--OCH-CH 2-O-P-),3.60(t,2H,P-O-CH2-CH 2-NH-),3.46,3.42and 2.8(3m,4H,2H,2H,-CH 2-CH 2-C(O)NH(CH 2)2-NH of BODIPY),2.70(s,3H,CH3 of BODIPY),2.48(m,4H,2x(-CH2-CO),2.45(s,3H,CH3 of BODIPY),2.37(m,4H,COCH 2CH2CH2CH 2CO),2.19(m,8H,2x(-CH 2-CH=CH-CH 2-),1.8(m,8H,COCH2CH2CH 2CH 2CO,2x(COCH2CH 2-)),1.46,1.43(2b s,40H,20CH2),1.05(m,6H,2CH3);3.31(q,6H,J=7.4,3xCH2 of Et3N),1.50(t,9H,J=7.4,3xCH3 of Et3N).
尽管本发明已经通过示范性的实施方式进行描述,应当理解,在不脱离本发明范围的前提下可以作出变化和改进。此外如果具体的特征有已知的等价物存在,此类等价物如同特别提及一般合并于说明书中。
Claims (19)
2.根据权利要求1的荧光细胞标记物,其中m和n之和为6至9,且*为C或N。
3.根据权利要求2的荧光细胞标记物,其中F选自下组:荧光素、俄勒绿、宾夕法尼亚绿、东京绿、曙红、BODIPY、BODIPY TR、Alexa Fluor350、Alexa Fluor405、Alexa Fluor488、Alexa Fluor568、Alexa Fluor594、德克萨斯红、萤光黄、四甲基若丹明和它们的衍生物的荧光团。
4.根据权利要求3的荧光细胞标记物,其中F是荧光素或者其衍生物之一的荧光团,该结构包括亚结构:
其中m和n各自为3至5。
5.根据权利要求4的荧光细胞标记物,其中S1是选自1,3-二氨基丙基、1,4-二氨基丁基或1,5-二氨基戊基衍生物的C3-5-二氨基烷基衍生物。
6.根据权利要求4的荧光细胞标记物,其中S1是C3-5-氨基烷基硫脲基。
7.根据权利要求6的荧光细胞标记物,其中S1是5-(5-氨基戊基)硫脲基。
8.根据权利要求4的荧光细胞标记物,其中S2是-CO(CH2)3CO-、-CO(CH2)4CO-或-CO(CH2)5CO-。
9.根据权利要求8的荧光细胞标记物,其中S2是-CO(CH2)4CO-。
10.根据权利要求1的荧光细胞标记物,其中L衍生自一种或多种顺式不饱和脂肪酸。
11.根据权利要求10的荧光细胞标记物,其中L是1,2-邻-二油酰基-sn-丙三氧基-3-磷脂酰乙醇胺(DOPE)。
13.荧光细胞标记物,具有如下结构:
称为KODE-俄勒冈绿(II)。
16.荧光细胞标记物,具有如下结构:
称为KODE-BODIPY(V),其中M为一价阳离子。
17.标记细胞或者多-细胞结构的方法,包括下列步骤:
在足以使标记物结合入细胞或者多-细胞结构的膜的温度下,使细胞或者多-细胞结构的悬浮液接触权利要求1的细胞标记物一段时间。
18.结合权利要求1的细胞标记物的细胞或者多-细胞结构。
19.通过权利要求17的方法生产的细胞或者多-细胞结构。
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Non-Patent Citations (4)
Title |
---|
Molecular Probes, Inc.Phospholipids.《MOLECULAR PROBES, PRODUCT INFORMATION》.2003, * |
MolecularProbes Inc.Phospholipids.《MOLECULAR PROBES |
Piotr W. Rzepecki 等.Synthesis of Hybrid Lipid Probes: Derivatives of Phosphatidylethanolamin-Extended Phosphatidylinositol 4,5-Bisphosphate (Pea-PIP2).《J. Org. Chem.》.2002,第67卷(第16期), * |
Thomas Haselgrubler 等.Synthesis and Applications of a New Poly(ethylene glycol) derivative for the crosslinking of amines with thiols.《Bioconjugate Chem.》.1995,第6卷(第3期), * |
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