公开(公告)号：US20110257399A1

公开(公告)日：2011-10-20

申请号：US12906987

申请日：2010-10-18

申请人： Akwasi Minta ,  Pedro Rogelio Escamilla

发明人： Akwasi Minta ,  Pedro Rogelio Escamilla

IPC分类号： C07D405/12 ,  C07D311/78

CPC分类号： C07D311/02

摘要： A new generation of fluorescent ion indicators for measuring cytosolic concentrations of calcium. These new indicators emit in the red portion of the visible spectrum and are ratiometric. The ratiometry consists of shifting in the emission upon binding calcium when exciting with an argon laser at 488 nm. These indicators also show marked increases in red fluorescence when excited with visible light at longer wavelengths. Like all calcium indicators, they comprise the usual chelating portion based on BAPTA (Bis Amino Phenyl Tetraacetic Acid) and a fluorophore portion, either a seminaphthofluorescein/seminaphthorhodamine or dinaphthofluorescein, which enable ratiometry. Unlike other naphthofluoresceins, the pKa has been adjusted on the naphtho moiety to make it suitable for biological calcium measurements. The invention relates to the structures of the indicators and to the method of synthesizing these indicators.

摘要翻译： 新一代荧光离子指示剂，用于测量细胞溶质中的钙浓度。 这些新指标发出可见光谱的红色部分，并且是比例的。 比例测量法包括当用488nm的氩激光器激发时，结合钙时发射的转移。 当在较长波长的可见光激发时，这些指标也显示红色荧光的显着增加。 像所有的钙指示剂一样，它们包括通常的螯合部分，其基于BAPTA（双氨基苯基四乙酸）和荧光团部分，即半标准荧光素/半罗丹明胺或二萘并噻吩荧光素，其能够进行比分析。 与其他萘并荧光素不同，pKa已经在萘酚部分上进行了调整，使其适合于生物钙测量。 本发明涉及指标的结构和合成这些指标的方法。

公开(公告)号：US5576433A

公开(公告)日：1996-11-19

申请号：US322059

申请日：1994-10-12

申请人： Martin F. Poenie ,  Akwasi Minta

发明人： Martin F. Poenie ,  Akwasi Minta

IPC分类号： C07D413/04 ,  C07D413/14 ,  C07D209/20 ,  C07D403/12 ,  C07D413/12

CPC分类号： C07D413/04

摘要： The present invention discloses several new calcium indicator dyes which are retained in the cytosol or which bind to cellular membranes and report calcium levels near the membrane. These indicators are analogs of a previously described tetracarboxylate calcium indicator fura-2 and indo-1 where the 5' methyl of the BAPTA moiety is replaced with a derivative of propionic acid in the form of --CH.sub.2 CH.sub.2 CON--(CH2).sub.n --N(X.sub.1)X.sub.2, where n is 4. The propionic acid derivative provides a flexible means for introducing additional functionality to the calcium indicator without greatly impacting the fluorescence and ion chelating properties of the parental indicator. When X.sub.1 is CH.sub.2 COOH and X.sub.2 is part of a ring system like piperazine, a fluorescent calcium indicator, Fura-PE3, is obtained which resists compartmentalization and leakage out of the cytosol. The tertiary amino group survives subsequent acetoxymethyl esterification of the hexacarboxylate form of the indicator without quaternization. In addition, the tertiary amino group can ionize once the indicator is inside the cell giving a zwitterion. All the zwitterionic calcium indicators reported herein resist leakage and compartmentalization. When X.sub.1 is a sufficiently hydrophobic alkyl chain, as in C.sub.12 H.sub.25, and X.sub.2 is part of a ring system like piperazine, FFP-18, the resulting indicator binds to membranes and reports calcium levels near the membranes.

摘要翻译： 本发明公开了几种新的钙指示剂染料，其保留在细胞溶质中或结合细胞膜并报告膜附近的钙水平。 这些指标是前述四羧酸钙指示剂fura-2和indo-1的类似物，其中BAPTA部分的5'甲基被-CH 2 CH 2 CON-（CH 2）n N（X 1）x 2的形式的丙酸衍生物代替 ，其中n为4.丙酸衍生物提供了一种用于在钙指示剂上引入附加功能性的灵活方式，而不会极大地影响亲本指示剂的荧光和离子螯合性质。 当X1是CH2COOH，X2是像哌嗪这样的环状体系的一部分时，得到一种荧光钙指示剂Fura-PE3，其抵抗了细胞溶质的分隔和泄漏。 叔氨基存在于指示剂的六羧酸盐形式的后续乙酰氧基甲基酯化，而没有季铵化。 此外，一旦指示剂在电池内部产生两性离子，叔氨基可以离子化。 本文报道的所有两性离子钙指示剂均可抵抗渗漏和分隔。 当X1是足够疏水的烷基链时，如在C12H25中，X2是环状体系如哌嗪FFP-18的一部分，所得指示剂与膜结合并报告膜附近的钙浓度。

公开(公告)号：US5948906A

公开(公告)日：1999-09-07

申请号：US920255

申请日：1992-07-27

申请人： Roger Yonchien Tsien ,  Akwasi Minta

发明人： Roger Yonchien Tsien ,  Akwasi Minta

IPC分类号： C07D273/08 ,  G01N31/22 ,  G01N33/58

CPC分类号： G01N33/582 ,  G01N31/22

摘要： The invention discloses fluorescent chelator compounds that are especially useful to monitor and measure cytosolic concentrations of alkali metal cations such as Na.sup.+, K.sup.+, and Li.sup.+. The new compounds are comprised of: (1) crown ethers (that may or may not have substituent groups attached to the core carbons, but will always contain at least one core nitrogen) that are linked via the core nitrogen(s) to at least one (2) fluorophore that contains an additional heteroaromatic liganding center. In the currently preferred dye, SBFI, the core compound is crown ether 1,7-diaza-4,10,13-trioxacyclopentadecane and the heteroaromatic fluorophores are benzofurans that are linked to isophthalate groups. Selectivities for Na.sup.+ over K.sup.+ of about 20 are observed, resulting in effective dissociation constants for Na.sup.+ of about 20 mM against a background of 120 mM K.sup.+. Increasing �Na.sup.+ ! increases the ratio of excitation efficiency at 330-345 nm to that at 370-390 nm with emission collected at 450-550 nm, so that ratio fluorometry and imaging work at the same wavelengths as used with the well-known Ca.sup.2+ indicator fura-2. If the macrocyclic ring is increased in size to a 1,10-diaza-4,7,13,16-tetraoxacyclooctadecane, the chelators become selective for K.sup.+ over Na.sup.+. If the ring is decreased in size, for example to a 1,7-diaza-4,10-dioxacyclododecane, the chelators become selective for Li.sup.+ over Na.sup.+.

公开(公告)号：US20050233467A1

公开(公告)日：2005-10-20

申请号：US10824786

申请日：2004-04-15

申请人： Akwasi Minta ,  Rogelio Escamilla ,  Earl Bates

发明人： Akwasi Minta ,  Rogelio Escamilla ,  Earl Bates

IPC分类号： C07D311/90 ,  G01N31/22 ,  G01N33/20

CPC分类号： C07D311/90 ,  C09B11/24 ,  G01N31/22

摘要： A new class of visible wavelength fluorescent calcium indicators with the BAPTA-like portion rendered zwitterionic by introduction of amine and carboxylic groups. “Fluo” compounds generally function with no extra ionized groups. The modified BAPTA moiety confers new properties while retaining ion selectivity and pH insensitivity. The dyes demonstrate reduced cell leakage and improved ability to study calcium near the cell membrane and preserve the fluorescent properties of “fluo” dyes. The modifications include (a) piperazinoacetic acid XIII for the leakage resistance (FLUO-LR), (b) dodecylpiperazine XIII for a near membrane indicator (FLUO-MOMO, FLUO-NOMO amphipathic “fluo” indicators which bind to cellular membranes and respond to calcium near the membrane), and (c) a propionic acid XII for general leakage resistance -FLUO-LOJO, FLUO-KOJO “fluo” indicators with an extra charge that enables them to resist leakage).

摘要翻译： 通过引入胺和羧基，具有BAPTA样部分的新一类可见波长荧光钙指示剂呈两性离子。 “Fluo”化合物通常起不到任何额外的电离基团的作用。 修饰的BAPTA部分赋予新的性质，同时保持离子选择性和pH不敏感性。 这些染料显示减少的细胞泄漏和提高在细胞膜附近研究钙的能力并保持“荧光”染料的荧光性质。 修饰包括（a）用于防漏性的哌嗪基乙酸XIII（FLUO-LR），（b）用于近膜指示剂的十二烷基哌嗪XIII（FLUO-MOMO，FLUO-NOMO两亲性“fluo”指示剂，其结合细胞膜并响应于 钙附近），和（c）具有一般泄漏性的丙酸XII -FLUO-LOJO，FLUO-KOJO“fluo”指示剂，具有额外的电荷，使其能够抵抗泄漏）。

公开(公告)号：US5134232A

公开(公告)日：1992-07-28

申请号：US391879

申请日：1989-08-09

申请人： Roger Y. Tsien ,  Akwasi Minta

发明人： Roger Y. Tsien ,  Akwasi Minta

IPC分类号： C07D273/00 ,  C07D273/08 ,  C07D413/04 ,  C07D413/14 ,  C07D491/04 ,  G01N33/84

CPC分类号： C07D273/00 ,  C07D413/04 ,  C07D413/14 ,  C07D491/04 ,  G01N33/84

摘要： The invention discloses fluorescent chelator compounds that are especially useful to monitor and measure cytosolic concentrations of alkali metal cations such as Na.sup.+, K.sup.+, and Li.sup.+. The new compounds are comprised of: (1) crown ethers (that may or may not have substituent groups attached to the core carbons, but will always contain at least one core nitrogen) that are linked via the core nitrogen(s) to at least one (2) fluorophore that contains an additional heteroaromatic liganding center. In the currently preferred dye, SBFI, the core compound is crown ether 1,7-diaza-4,10,13-trioxacyclopentadecane and the heteroaromatic fluorophores are benzofurans that are linked to isophthalate groups. Selectivities for Na.sup.+ over K.sup.+ of about 20 are observed, resulting in effective dissociation constants for Na.sup.+ of about 20 mM against a background of 120 mM K.sup.+. Increasing [Na.sup.+ ] increases the ratio of excitation efficiency at 330-345 nm to that at 370-390 nm with emission collected at 450-550 nm, so that ratio fluorometry and imaging work at the same wavelengths as used with the well-known Ca.sup.2+ indicator fura-2. If the macrocyclic ring is increased in size to a 1,10-diaza-4,7,13,16-tetraoxacyclooctadecane, the chelators become selective for K.sup.+ over Na.sup.+. If the ring is decreased in size, for example to a 1,7-diaza-4,10-dioxacyclododecane, the chelators become selective for Li.sup.+ over Na.sup.+.

摘要翻译： 本发明公开了特别可用于监测和测量碱金属阳离子如Na +，K +和Li +的胞质浓度的荧光螯合剂化合物。 新化合物包括：（1）冠醚（其可以具有或不具有连接到核心碳上的取代基但总是含有至少一个核心氮），其通过核心氮至少连接至少 一个（2）荧光团，其中包含一个额外的杂芳族结合中心。 在目前优选的染料中，SBFI，核心化合物是冠醚1,7二氮杂-4,10,13-三氧杂环十五烷，杂芳族荧光团是与间苯二酸酯基团连接的苯并呋喃。 观察到Na +在K +上约20的选择性，导致对于120mM K +背景的Na +约20mM的有效解离常数。 增加[Na +]增加了在330-345nm处的激发效率与在370-390nm的激发效率的比率，其中在450-550nm收集的发射，使得比例荧光测定和成像工作在与公知的Ca2 + 指标fura-2。 如果大环的大小增加到1,10-二氮杂-4,7,13,16-四氧杂环十八烷，则螯合剂对于Na +而言对K +变得选择性。 如果环尺寸减小，例如减小到1,7-diaza-4,10-二氧杂环十二烷，那么螯合剂对Na +的选择性变得更高。

公开(公告)号：US4689432A

公开(公告)日：1987-08-25

申请号：US648536

申请日：1984-09-07

申请人： Roger Y. Tsien ,  Grzegorz Grynkiewicz ,  Akwasi Minta

发明人： Roger Y. Tsien ,  Grzegorz Grynkiewicz ,  Akwasi Minta

IPC分类号： C07F9/09 ,  C07C67/00 ,  C07C201/00 ,  C07C205/37 ,  C07C227/00 ,  C07C227/16 ,  C07C229/16 ,  C07C229/18 ,  C07C301/00 ,  C07C309/66 ,  C07D215/40 ,  C07D317/62 ,  C07D319/18 ,  C07F9/60 ,  C09K3/00 ,  G01N33/84 ,  C07C101/26 ,  C07D215/38

CPC分类号： G01N33/84 ,  C07D317/62 ,  Y10S424/06

摘要： The present invention discloses a group of calcium chelating compounds which have a descreased affinity for calcium following illumination. These new compounds contain a photolabile nitrobenzyl derivative coupled to a tetracarboxylate Ca.sup.2+ chelating parent compound having the octacoordinate chelating groups characteristic of EGTA or BAPTA. In a first form, the new compounds are comprised of a BAPTA-like chelator coupled to a single 2-nitrobenzyl derivative, which in turn is a photochemical precursor of a 2-nitrosobenzophenone. In a second form, the new compounds are comprised of a BAPTA-like chelator coupled to two 2-nitrobenzyl derivatives, themselves photochemical prcursors of the related 2-nitrosobenzophenones.The present invention also discloses a novel method for preparing 1-hydroxy- or 1-alkoxy-1-(2-nitroaryl)-1-aryl methanes. Methanes of this type are critical to the preparation of, or actually constitute, the photolabile Ca.sup.2+ chelating compounds disclosed and claimed herein.

摘要翻译： 本发明公开了一种钙照射后对钙的亲和力降低的钙螯合化合物。 这些新化合物含有偶联到具有EGTA或BAPTA特征的八配位螯合基团的四羧酸盐Ca 2+螯合母体化合物的光不稳定的硝基苄基衍生物。 在第一种形式中，新化合物由与单一2-硝基苄基衍生物偶联的BAPTA样螯合剂组成，其又是2-亚硝基二苯甲酮的光化学前体。 在第二种形式中，新化合物由与两个2-硝基苄基衍生物偶联的BAPTA样螯合剂组成，它们本身是相关的2-亚硝基二苯甲酮的光化学原子。 本发明还公开了一种制备1-羟基 - 或1-烷氧基-1-（2-硝基芳基）-1-芳基甲烷的新方法。 这种类型的甲烷对于制备或实际构成本文公开和要求保护的光不稳定的Ca 2+螯合化合物至关重要。

公开(公告)号：US5049673A

公开(公告)日：1991-09-17

申请号：US115921

申请日：1987-10-30

申请人： Roger Y. Tsien ,  Akwasi Minta

发明人： Roger Y. Tsien ,  Akwasi Minta

IPC分类号： C07D219/06 ,  C07D219/08 ,  C07D311/80 ,  C07D311/90 ,  C07D335/14 ,  C07D471/22 ,  C07D491/16 ,  C07D495/16 ,  C09B11/24 ,  C09B57/00 ,  G01N33/48 ,  G01N33/49 ,  G01N33/533 ,  G01N33/84

CPC分类号： C09B11/24 ,  G01N33/84

摘要： The present invention discloses a new class of calcium specific fluorescent indicator dyes having visible excitation and emission wavelengths. The new fluorescent indicator dyes combine at least one tricyclic chromophore with a tetracarboxylate parent CA.sup.2+ chelating compound having the octacoordinate pattern of liganding groups characteristic of BAPTA to give a rhodamine-like or fluorescein-like fluorophore. Binding of calcium.sup.2+ increases the fluorescence of the new compounds by up to 40-fold. The calcium.sup.2+ dissociation constants are in the range 0.37-2.3 microM, so that the new indicators give better resolution of high [CA.sup.2+ ] levels than were previously obtainable with predecessor compounds such as quin-2 or fluo-2. The visible excitation wavelengths of the new compounds are more convenient for fluorescent microscopy and flow cytometry than the UV required by previous indicators.