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公开(公告)号：US4603209A
公开(公告)日：1986-07-29
申请号：US648649
申请日：1984-09-07
IPC分类号： A61K38/00 , A61K38/22 , A61P5/00 , C07C229/18 , C07C229/52 , C07C237/30 , C07C255/59 , C07C309/49 , C07C311/39 , C07C317/32 , C07D209/08 , C07D209/14 , C07D215/40 , C07D307/79 , C07D307/82 , C07D413/04 , C07K7/23 , G01N21/78 , G01N33/84 , C07C101/447 , C07D307/87
CPC分类号： C07D307/79 , C07D209/08 , C07D215/40 , C07D413/04 , Y10S436/80
摘要： The present invention discloses a new class of highly fluorescent indicator dyes that are specific for calcium ions. The new fluorescent indicator dyes combine a stilbene-type fluorophore with a tetracarboxylate parent Ca.sup.2+ chelating compound having the octacoordinate pattern of liganding groups characteristic of EGTA and BAPTA. Preferred forms contain extra heterocyclic bridges to reinforce the ethylenic bond of the stilbene and to reduce hydrophobicity. Compared to their widely used predecessor, "quin2", the new dyes offer up to thirty-fold brighter fluorescence, major changes in wavelength (not just intensity) upon Ca.sup.2+ binding, slightly lower affinities for Ca.sup.2+, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca.sup.2+ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca.sup.2+, make the dyes useful indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues.The present invention also discloses an improved method for synthesizing alpha-acyloxyalkyl bromides wherein the bromides so synthesized are free of contaminating bis(1-bromoalkyl)ether. The improved method is exemplified herein in the synthesis of acetoxymethyl bromide, a compound useful in preparing the acetoxymethyl esters disclosed herein as novel Ca.sup.2+ specific fluorescent indicators.
摘要翻译： 本发明公开了一类对钙离子具有特异性的高荧光指示剂染料。 新的荧光指示剂染料将二苯乙烯型荧光团与具有EGTA和BAPTA特征的连接组的八配位图案的四羧酸母体Ca 2+螯合化合物结合。 优选的形式包含额外的杂环桥，以加强1,2-二苯乙烯的烯键并降低疏水性。 与其广泛使用的前身“quin2”相比，新型染料提供高达三十倍的荧光，Ca2 +结合时的波长（不仅仅是强度）的主要变化，Ca2 +的亲和力略低，激发波长稍长， 提高Ca2 +对其他二价阳离子的选择性。 这些性质，特别是对Ca2 +的波长敏感性，使得染料对于许多细胞内应用尤其是在单细胞，贴壁细胞层或体组织中是有用的指标。 本发明还公开了一种合成α-酰氧基烷基溴化物的改进方法，其中如此合成的溴化物没有污染的双（1-溴烷基）醚。 改进的方法在本文中示例性地合成乙酰氧甲基溴，其是可用于制备本文公开的乙酰氧基甲酯作为新型Ca 2+特异性荧光指示剂的化合物。
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公开(公告)号：US4689432A
公开(公告)日：1987-08-25
申请号：US648536
申请日：1984-09-07
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。
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公开(公告)号：US09651494B2
公开(公告)日：2017-05-16
申请号：US14118836
申请日：2012-05-21
申请人： Evan Walker Miller , Roger Y. Tsien
发明人： Evan Walker Miller , Roger Y. Tsien
IPC分类号： G01N21/64 , G01N33/50 , G01N33/542 , C09K11/06
CPC分类号： G01N21/6486 , C09K11/06 , C09K2211/1007 , C09K2211/1011 , C09K2211/1088 , G01N33/5008 , G01N33/542 , G01N2500/10
摘要： Compounds and methods for determining transmembrane potential, monitoring changes in transmembrane potential, and/or drug screening are provided. In one aspect, compounds of the invention have a structure according to the formula: E-M-A, wherein A is a fluorophore, selected from xanthenes, coumarins, cyanines, bimanes, and difluoroboradizaindacenes, charged at physiological pH; M is a molecular wire; and E is a hydrophobic moiety, wherein A and E are capable of being involved in a photo-induced, intramolecular electron transfer that quenches the fluorescence of A in response to a voltage condition. When in use, compounds of the invention are membrane-impermeant and oriented within the cell membrane such that the charged moiety localizes at the outer leaflet of the lipid bilayer and the hydrophobic moiety and molecular wire associate with the hydrophobic portion of the lipid bilayer. The rate of electron transfer, fluorescence intensity, and quenching are altered in response to changes in transmembrane potential.
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4.发明授权公开(公告)号：US08993256B2
公开(公告)日：2015-03-31
申请号：US12443411
申请日：2007-09-28
申请人： Michael Z. Lin , Roger Y. Tsien
发明人： Michael Z. Lin , Roger Y. Tsien
CPC分类号： C12Q1/37 , G01N33/582
摘要： The disclosure provides method and compositions for visualizing protein turnover. In particular, the disclosure provides methods and compositions useful for measuring the age of particular proteins or the dynamics of localized protein translation.
摘要翻译： 本公开提供了用于可视化蛋白质周转的方法和组合物。 特别地，本公开提供了可用于测量特定蛋白质的年龄或局部蛋白质翻译的动力学的方法和组合物。
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公开(公告)号：US08759492B2
公开(公告)日：2014-06-24
申请号：US13588957
申请日：2012-08-17
申请人： John Yu-luen Lin , Roger Y. Tsien
发明人： John Yu-luen Lin , Roger Y. Tsien
CPC分类号： C07K19/00 , A61K48/005 , A61N5/062 , C07K14/705 , C07K2319/00 , C12N15/62 , C12N15/85 , C12N15/864 , C12N2740/15043 , C12N2740/15071 , C12N2750/14143 , C12N2750/14171
摘要： The invention provides engineered red-shifted channelrhodopsin variants. In some embodiments, the channelrhodopsin variants are characterized by improved membrane trafficking, expression, and/or unique spectral and kinetic properties.
摘要翻译： 本发明提供工程化的红移通道视紫红质变体。 在一些实施方案中，通道视紫红质变体的特征在于改进的膜运输，表达和/或独特的光谱和动力学性质。
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6.发明申请公开(公告)号：US20140093907A1
公开(公告)日：2014-04-03
申请号：US14118836
申请日：2012-05-21
申请人： Evan Walker Miller , Roger Y. Tsien
发明人： Evan Walker Miller , Roger Y. Tsien
IPC分类号： G01N21/64
CPC分类号： G01N21/6486 , C09K11/06 , C09K2211/1007 , C09K2211/1011 , C09K2211/1088 , G01N33/5008 , G01N33/542 , G01N2500/10
摘要： Compounds and methods for determining transmembrane potential, monitoring changes in transmembrane potential, and/or drug screening are provided. In one aspect, compounds of the invention have a structure according to the formula: E-M-A, wherein A is a fluorophore, selected from xanthenes, eoumarins, cyanines, bimanes, and difluoroboradizaindacenes, charged at physiological pH; M is a molecular wire; and E is a hydrophobic moiety, wherein A and E are capable of being involved in a photo-induced, intramolecular electron transfer that quenches the fluorescence of A in response to a voltage condition. When in use, compounds of the invention are membrane-impermeant and oriented within the cell membrane such that the charged moiety localizes at the outer leaflet of the lipid bilayer and the hydrophobic moiety and molecular wire associate with the hydrophobic portion of the lipid bilayer. The rate of electron transfer, fluorescence intensity, and quenching are altered in response to changes in transmembrane potential.
摘要翻译： 提供了确定跨膜电位的化合物和方法，监测跨膜电位的变化和/或药物筛选。 在一个方面，本发明的化合物具有下式的结构：其中A是荧光团，选自在生理pH下加入的呫吨，香豆素，花青，双马来酰亚胺和二氟硼烷二恶烷; M是分子线; 并且E是疏水部分，其中A和E能够参与光诱导的分子内电子转移，其响应于电压条件淬灭A的荧光。 当使用时，本发明的化合物是膜不渗透的并且在细胞膜内定向，使得带电部分定位在脂质双层的外部小叶上，并且疏水部分和分子线与脂质双层的疏水部分缔合。 响应于跨膜电位的变化，电子转移速率，荧光强度和淬灭速率发生变化。
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公开(公告)号：US20110076711A1
公开(公告)日：2011-03-31
申请号：US12861791
申请日：2010-08-23
IPC分类号： C12Q1/02
CPC分类号： G01N33/582 , C12Q1/682 , G01N33/5005 , G01N33/542 , Y10S436/80 , Y10S436/809 , Y10T436/13 , C12Q2563/107 , C12Q2527/125
摘要： The present invention provides a method for reducing undesirable light emission from a sample using at least one photon producing agent and at least one photon reducing agent (e.g. dye-based photon reducing agents). The present invention further provides a method for reducing undesirable light emission from a sample (e.g., a biochemical or cellular sample) with at least one photon producing agent and at least one collisional quencher. The present invention also provides a method for reducing undesirable light emission from a sample (e.g., a biochemical or cellular sample) with at least one photon producing agent and at least one quencher, such as an electronic quencher. The present invention also provides a system and method of screening test chemicals in fluorescent assays using photon reducing agents. The present invention also provides compositions, pharmaceutical compositions, and kits for practicing these methods.
摘要翻译： 本发明提供一种使用至少一种光子产生剂和至少一种光子还原剂（例如基于染料的光子还原剂）来减少来自样品的不希望的光发射的方法。 本发明进一步提供了用至少一种光子产生剂和至少一种碰撞猝灭剂减少来自样品（例如生物化学或细胞样品）的不希望的光发射的方法。 本发明还提供了用至少一种光子产生剂和至少一种猝灭剂（例如电子猝灭剂）减少来自样品（例如，生物化学或细胞样品）的不希望的光发射的方法。 本发明还提供了使用光子还原剂在荧光测定中筛选测试化学品的系统和方法。 本发明还提供了用于实施这些方法的组合物，药物组合物和试剂盒。
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公开(公告)号：US07854898B2
公开(公告)日：2010-12-21
申请号：US12271762
申请日：2008-11-14
申请人： Andrew A. Pham , Peter J. Coassin , Alec Tate Harootunian , Peter N. Pham , Harry Stylli , Roger Y. Tsien
发明人： Andrew A. Pham , Peter J. Coassin , Alec Tate Harootunian , Peter N. Pham , Harry Stylli , Roger Y. Tsien
IPC分类号： B01L3/00
CPC分类号： B01L3/5085 , B01J2219/00315 , B01J2219/00317 , B01J2219/00659 , B01J2219/00707 , B01J2219/0072 , B01L3/50855 , B01L9/523 , B01L2300/0654 , B01L2300/0829 , C12M23/12 , C40B60/14
摘要： The present invention relates to multi-well platforms, lids, caddies and any combination thereof. The multi-well platforms comprise a plurality of wells that can be of any shape and can be arranged in any ornamental pattern. The lid comprises an area corresponding to the well field. The caddy has a footprint approximately that of a standard 96-well microtiter plate. When the multi-well platform is engaged with the caddy, the wells of the well-field of the multi-well platform are preferably not obscured by the caddy when viewed from the bottom of the combination.
摘要翻译： 本发明涉及多孔平台，盖子，球童及其任何组合。 多井平台包括可以是任何形状的多个井，并且可以以任何装饰图案排列。 盖子包括对应于井场的区域。 球罐的尺寸大约是标准96孔微孔板的面积。 当多孔平台与球童接合时，当从组合的底部观察时，多孔平台的井场的井优选地不被球童遮蔽。
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9.发明申请公开(公告)号：US20080293068A1
公开(公告)日：2008-11-27
申请号：US12124888
申请日：2008-05-21
申请人： Roger Y. Tsien , Lei Wang
发明人： Roger Y. Tsien , Lei Wang
CPC分类号： C12P21/02
摘要： Methods using somatic hypermutation (SHM) for producing polypeptide and nucleic acid variants, and nucleic acids encoding such polypeptide variants are disclosed. Such variants may have desired properties. Also disclosed are novel polypeptides, such as improved fluorescent proteins, produced by the novel methods, and nucleic acids, vectors, and host cells comprising such vectors.
摘要翻译： 公开了使用体细胞超突变（SHM）用于产生多肽和核酸变体的方法，以及编码这种多肽变体的核酸。 这样的变体可以具有期望的性质。 还公开了通过新方法产生的新型多肽，例如改良的荧光蛋白，以及包含这种载体的核酸，载体和宿主细胞。
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公开(公告)号：US07173130B2
公开(公告)日：2007-02-06
申请号：US09967772
申请日：2001-09-28
IPC分类号： C07D403/06
CPC分类号： G01N33/502 , C07D209/08 , C07D215/227 , C07D239/60 , C07D239/62 , C07D239/66 , C07D239/80 , C07D241/44 , C07D265/36 , C07D279/16 , C07D311/16 , C07D487/04 , C07F5/003 , C07F5/02 , C07F5/027 , C07F9/10 , C07F9/5728 , C07K2319/033 , C09B23/02 , G01N33/5005 , G01N33/5008 , G01N33/5044 , G01N33/5058 , G01N33/5061 , G01N33/5076 , G01N33/52 , G01N33/542 , G01N33/582 , G01N33/6872 , G01N33/92 , G01N2405/04 , G01N2500/10 , Y10S436/80 , Y10S436/805
摘要： Methods and compositions are provided for detecting changes in membrane potential in membranes biological systems. In one aspect, the method comprises; a) providing a living cell with a first reagent comprising a charged hydrophobic molecule which is typically a fluorescence resonance energy transfer (FRET) acceptor or donor, or is a quencher and is capable of redistributing within the membrane of a biological membrane in response to changes in the potential across the membrane; b) providing the cell with a second reagent that can label the first face or the second face of a biological membrane within the cell; c) detecting light emission from the first reagent or the second reagent. One aspect of this method involves monitoring membrane potential changes in subcellular organelle membranes in a living cells.Another aspect of the invention is the use of certain embodiments of the method for the screening of test chemicals for activity to modulate the activity of a target ion channel.Another aspect of the present invention is a transgenic organism comprising a first reagent that comprises a charged hydrophobic fluorescent molecule, and a second reagent comprising a bioluminescent or naturally fluorescent protein.
摘要翻译： 提供了用于检测膜生物系统中膜电位变化的方法和组合物。 一方面，该方法包括： a）向活细胞提供包含通常为荧光共振能量转移（FRET）受体或供体的带电荷的疏水性分子的第一试剂，或者是猝灭剂，并且能够响应于变化而重新分布在生物膜的膜内 在膜的潜力; b）向细胞提供可以标记细胞内生物膜的第一面或第二面的第二试剂; c）检测来自第一试剂或第二试剂的发光。 该方法的一个方面涉及监测活细胞中亚细胞细胞器膜中的膜电位变化。 本发明的另一方面是该方法的某些实施方案用于筛选测试化学品以调节靶离子通道的活性的活性。 本发明的另一方面是转基因生物，其包含含有带电荷的疏水荧光分子的第一试剂和包含生物发光或天然荧光蛋白的第二试剂。
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