公开(公告)号：WO1993004074A1

公开(公告)日：1993-03-04

申请号：PCT/US1992007069

申请日：1992-08-21

Applicant: MOLECULAR PROBES, INC.

Inventor： MOLECULAR PROBES, INC. ,  HAUGLAND, Richard, P.

IPC: C07H01/00

CPC classification number: C12Q1/34 ,  C07H17/00 ,  C12Q2334/00 ,  G01N2333/924

Abstract: The claimed invention relates to a substrate for evaluating glycosidic enzymes comprising a resorufin derivative of general formula (I), wherein Gly is a carbohydrate bonded to resorufin by a glycosidic linkage; where at least one of substituents R1, R2, R4, R6, R8, and R9 is a lipophilic residue of the formula -L(CH2)nCH3, where n is greater than 3 and less than 22, and where L is a methylene -CH2-, an amide -NHCO-, a sulfonamide -NHSO2-, a carboxamide -CONH-, a carboxylate ester -COO-, a urethane -NHCOO-, a urea -NHCONH-, or a thiourea -NHCSNH-; and where the remainder of substituents R1, R2, R4, R6, R8, and R9, which may be the same or different, are hydrogen, halogen, or other lipophilic residues, which may be the same or different, containing from about 1 to about 22 carbon atoms of the formula -L'(CH2)mCH3, where m is less than 22, and where L' is a methylene -CH2-, an amide -NHCO-, a sulfonamide -NHSO2-, a carboxamide -CONH-, a carboxylate ester -COO-, a urethane -NHCOO-, a urea -NHCONH-, or a thiourea -NHCSNH-. A preferred embodiment of the invention is a nonfluorescent substrate specifically hydrolyzable by a glycosidase inside a cell to yield, after greater than about 2 minutes, an orange to red fluorescent detection product which is retained inside a viable cell more than about 2 hours at greater than about 15 °C and which is nontoxic to the cell. The substrates are used for evaluating a glycosidic enzyme in living plant or animal cells whether the enzyme is present endogenously; present as a result of manipulation of the cell's genome, or added to the cell exogenously, such as by covalently binding the enzyme to a protein to form an enzyme-protein complex that enters the cell.

公开(公告)号：WO1993023492A1

公开(公告)日：1993-11-25

申请号：PCT/US1993004334

申请日：1993-05-07

Applicant: MOLECULAR PROBES, INC.

Inventor： MOLECULAR PROBES, INC. ,  BRINKLEY, John, M. ,  HAUGLAND, Richard, P. ,  SINGER, Victoria, L.

IPC: C09K11/06

CPC classification number: C12Q1/6818 ,  C09K11/06 ,  G01N33/533 ,  G01N33/585 ,  Y10S436/80 ,  Y10S436/805 ,  Y10T428/2982

Abstract: The invention relates to microparticles incorporating a series of two or more fluorescent dyes having overlapping excitation and emission spectra allowing efficient energy transfer from the excitation wavelength of the first dye in the series, transfer through the dyes in the series and re-emitted as an optical signal at the emission wavelength of last dye in the series, resulting in a desired effective Stokes shift which is controlled through selection of appropriate dyes.

Abstract translation: 本发明涉及包含一系列具有重叠激发和发射光谱的两种或更多种荧光染料的微粒，其允许从串联中的第一染料的激发波长进行有效能量转移，通过串联中的染料转移并作为光学重新发射 信号在串联中的最后染料的发射波长，产生期望的有效的斯托克斯位移，其通过选择合适的染料来控制。

公开(公告)号：WO1993009185A1

公开(公告)日：1993-05-13

申请号：PCT/US1992008350

申请日：1992-09-30

Applicant: MOLECULAR PROBES, INC.

Inventor： MOLECULAR PROBES, INC. ,  KANG, Hee, Chol ,  HAUGLAND, Richard, P.

IPC: C09B57/00

CPC classification number: C07H13/04 ,  C07F5/022 ,  C07H19/10 ,  C07H21/00 ,  C09B57/00 ,  C09K11/06 ,  G01N33/533

Abstract: This invention relates to derivatives of dipyrrometheneboron difluoride fluorescent dyes that have an absorption maximum at wavelengths longer than about 525 nm, and are chemically reactive with nucleic acids, proteins, carbohydrates, and other biologically derived or synthetic chemical materials. The dyes generally have structure (I), wherein at least one of the substituents R1-R7, is a reactive functional group, and at least one of the substituents R1-R7 contains a bathochromic moiety. The bathochromic moiety is an unsaturated organic group, preferably heteroaryl or alkenyl. The remaining substituents, which may be the same or different, are hydrogen, halogen, alkyl (containing 1-5 carbon atoms), aryl, arylalkyl, or sulfo. The dyes are used to make novel conjugates with members of specific binding pairs that are ligands or receptors.

公开(公告)号：WO1997039064A1

公开(公告)日：1997-10-23

申请号：PCT/US1997006090

申请日：1997-04-11

Applicant: MOLECULAR PROBES, INC.

Inventor： MOLECULAR PROBES, INC. ,  GEE, Kyle, R. ,  POOT, Martin ,  KLAUBERT, Dieter, H. ,  SUN, Wei-Chuan ,  HAUGLAND, Richard, P. ,  MAO, Fei

IPC: C09B11/00

CPC classification number: C09B11/00 ,  C07C37/055 ,  C07C41/18 ,  C07C201/08 ,  C07C201/12 ,  C07C205/26 ,  C07C205/37 ,  C07C209/10 ,  C07C213/02 ,  C07C213/08 ,  C07C245/20 ,  C07D311/82 ,  C09B11/24 ,  G01N1/30 ,  Y10S430/116 ,  Y10S430/126 ,  C07C39/245 ,  C07C211/52 ,  C07C217/84 ,  C07C215/76 ,  C07C43/225

Abstract: The family of dyes of the invention are fluoresceins and rhodols that are directly substituted on one or more aromatic carbons by fluorine. These fluorine-substituted fluorescent dyes possess greater photostability and have lower sensitivity to pH changes in the physiological range of 6-8 than do non-fluorinated dyes, exhibit less quenching when conjugated to a substance, and possess additional advantages. The dyes of the invention are useful as detectable tracers and for preparing conjugates of organic and inorganic substances.

Abstract translation: 本发明的染料族是在一个或多个芳族碳上被氟直接取代的荧光素和二醇。 与非氟化染料相比，这些氟取代的荧光染料具有更高的光稳定性并且对生理范围内的pH变化具有较低的敏感性，当与物质缀合时表现出较少的淬灭，并且具有额外的优点。 本发明的染料可用作可检测的示踪剂并用于制备有机和无机物质的共轭物。

公开(公告)号：WO1993006482A1

公开(公告)日：1993-04-01

申请号：PCT/US1992007867

申请日：1992-09-16

Applicant: MOLECULAR PROBES, INC.

Inventor： MOLECULAR PROBES, INC. ,  YUE, Stephen, T. ,  JOHNSON, Iain, D. ,  HAUGLAND, Richard, P.

IPC: G01N33/48

CPC classification number: C09B23/02 ,  G01N33/52

Abstract: The invention relates to dimers of unsymmetrical cyanine dyes, typically dimers of benzthiazole or benzoxazole derivatives, that exhibit enhanced fluorescence on binding with DNA or RNA. The dimers generally have formula (1), where R and R , which may be the same or different, are alkyl groups having 1-6 carbons; X is O, S, or N-R , where R is H or an alkyl group having 1-6 carbons; Z is O, S, or N-R , where R is H or an alkyl group having 1-6 carbons; n and s, which may be the same or different, = 0, 1, or 2; Y is HC=CH; and p, m, q, and r = 0 or 1, such that p + m = 1 and q + r = 1; and where -BRIDGE- has general formula (2), where alpha , beta , gamma , and delta , which may be the same or different, are integers greater than 1 and less than 5; I and II, which may be the same or different, = 0 or 1; and A , A , and A , which may be the same or different, are independently O; S; (CH2) mu where mu = 0 or 1; -(NR )- where R is H or an alkyl group having 1-6 carbons; or -(N R R )- where R and R , which may be the same or different, are independently hydrogen or an alkyl group having 1-6 carbons.

Abstract translation: 本发明涉及不对称花青染料的二聚体，通常是苯并噻唑或苯并恶唑衍生物的二聚体，其在与DNA或RNA结合时表现出增强的荧光。 二聚体通常具有式（1），其中R 1和R 2可以相同或不同，是具有1-6个碳原子的烷基; X是O，S或N-R 3，其中R 3是H或具有1-6个碳的烷基; Z是O，S或N-R 4，其中R 4是H或具有1-6个碳的烷基; n和s可以相同或不同，= 0,1或2; Y为HC = CH; 和p，m，q和r = 0或1，使得p + m = 1和q + r = 1; 其中-BRIDGE-具有通式（2），其中可以相同或不同的α，β，γ和δ是大于1且小于5的整数; I和II可以相同或不同，= 0或1; 和A 1，A 2和A 3可以相同或不同，独立地为O; S; （CH 2）m，其中mu = 0或1; - （NR 5） - 其中R 5是H或具有1-6个碳的烷基; 或 - （N + R 6 R 7） - 其中R 6和R 7可以相同或不同，独立地是氢或具有1-6个碳的烷基。

公开(公告)号：WO1996036882A1

公开(公告)日：1996-11-21

申请号：PCT/US1996007297

申请日：1996-05-20

Applicant: MOLECULAR PROBES, INC.

Inventor： MOLECULAR PROBES, INC. ,  HAUGLAND, Richard, P. ,  SINGER, Victoria, L. ,  JONES, Laurie, Jeanne ,  STEINBERG, Thomas, H.

IPC: G01N33/68

CPC classification number: G01N27/44726 ,  G01N33/6839 ,  Y10T436/25125 ,  Y10T436/25375

Abstract: This invention describes the use of merocyanine dyes, including styryl dyes, for detecting and quantifying poly(amino acids) by absorbance or fluorescence. Poly(amino acids) are detected in solution, in electrophoretic gels, or on solid supports.

Abstract translation: 本发明描述了部花青染料（包括苯乙烯基染料）通过吸光度或荧光检测和定量聚（氨基酸）的用途。 聚（氨基酸）在溶液中，电泳凝胶中或固体支持物上检测。

公开(公告)号：WO1996013552A2

公开(公告)日：1996-05-09

申请号：PCT/US1995013706

申请日：1995-10-27

Applicant: MOLECULAR PROBES, INC.

Inventor： MOLECULAR PROBES, INC. ,  YUE, Stephen, T. ,  SINGER, Victoria, L. ,  ROTH, Bruce, L. ,  MOZER, Thomas, J. ,  MILLARD, Paul, J. ,  JONES, Laurie, J. ,  JIN, Xiaokui ,  HAUGLAND, Richard, P. ,  POOT, Martin

IPC: C09B23/02

CPC classification number: C07D417/14 ,  C07D215/18 ,  C07D413/06 ,  C07D417/06 ,  C09B23/02 ,  C12Q1/6816 ,  G01N15/1459 ,  G01N2015/1402 ,  Y10S436/80 ,  Y10T436/143333 ,  C12Q2563/173

Abstract: The invention describes the preparation and use of fluorescent stains for nucleic acids derived from unsymmetrical cyanine dyes comprising a substituted benzazolium ring system linked by a methine bridge to a pyridinium or quinolinium ring system. The cyanine dyes of the invention possess a high sensitivity to oligonucleotides and larger nucleic acid polymers in a wide range of cells and gels, and are useful for the analysis of cell structure, membrane integrity or function, and determination of cell cycle distribution.

Abstract translation: 本发明描述了衍生自不对称花青染料的核酸的荧光染料的制备和用途，其包括通过次甲基桥与吡啶鎓或喹啉环体系连接的取代的苯并唑鎓环系统。 本发明的花青染料在宽范围的细胞和凝胶中对寡核苷酸和较大的核酸聚合物具有高灵敏度，并且可用于细胞结构，膜完整性或功能的分析以及细胞周期分布的确定。

公开(公告)号：WO1994024213A1

公开(公告)日：1994-10-27

申请号：PCT/US1994004127

申请日：1994-04-13

Applicant: MOLECULAR PROBES, INC.

Inventor： MOLECULAR PROBES, INC. ,  ROTH, Bruce, L. ,  MILLARD, Paul, J. ,  YUE, Stephen, T. ,  WELLS, K., Sam ,  HAUGLAND, Richard, P.

IPC: C09B23/02

CPC classification number: C07D211/76 ,  C07D215/18 ,  C07D215/227 ,  C07D417/06 ,  C07D417/14 ,  C09B23/02 ,  Y10S436/80 ,  Y10T436/143333

Abstract: The invention describes the preparation and use of fluorescent stains for nucleic acids derived from unsymmetrical cyanine dyes comprising a substituted benzazolium ring system linked by a methine bridge to a pyridinium or quinolinium ring system having at least one substituent that is a saturated or unsaturated cyclic substituent. Superior fluorescent characteristics when complexed with nucleic acids give the dyes utility for the detection of oligonucleotides and nucleic acids in cells, gels, and solutions. The presence of the cyclic substituent results in improved permeability in a wide range of cells and gels, resulting in improved detection of nucleic acids. Combination with additinal dyes permits analysis of cell membrane integrity, Gram sign, or cell structure and function.

Abstract translation: 本发明描述了衍生自不对称花青染料的核酸的荧光染料的制备和用途，其包括通过次甲基桥连接至具有至少一个为饱和或不饱和环状取代基的取代基的吡啶鎓或喹啉环体系的取代的苯并唑鎓环系统。 当与核酸复合时，优异的荧光特性使得染料可用于检测细胞，凝胶和溶液中的寡核苷酸和核酸。 环状取代基的存在导致在宽范围的细胞和凝胶中改善的渗透性，从而改进了核酸的检测。 与添加染料组合允许分析细胞膜完整性，革兰氏标志或细胞结构和功能。

公开(公告)号：WO1993004192A1

公开(公告)日：1993-03-04

申请号：PCT/US1992007068

申请日：1992-08-21

Applicant: MOLECULAR PROBES, INC.

Inventor： MOLECULAR PROBES, INC. ,  HAUGLAND, Richard, P.

IPC: C12Q01/00

CPC classification number: C12Q1/04 ,  C12Q1/34 ,  C12Q2334/00 ,  C12Q2334/20 ,  C12Q2334/40 ,  C12Q2337/20 ,  G01N33/535 ,  G01N2333/924 ,  Y10S530/802

Abstract: The subject invention provides a method for analyzing the metabolic activity in cells by improving the retention of a detectable reporter molecule only in intact cells where a particular enzyme is present. In particular, improved retention results from a two part process involving conjugation of haloalkyl-substituted derivatives of a reporter molecule with intracellular cysteine-containing peptides while unblocking the reporter molecule. The method for analyzing metabolic activity of cells involves the use of a substrate having the form XR-REPORTER-BLOCK wherein -BLOCK is a group selected to be removable by action of a specific analyte, to give REPORTER spectral properties different from those of the substrate, -REPORTER- is a molecule that, when no longer bound to BLOCK by a BLOCK-REPORTER bond, has spectral properties different from those of the substrate, and XR- is a haloalkyl moiety that can covalently react with an intracellular thiol (Z-S-H) to form a thioether conjugate (Z-S-R-). After the substrate enters the cells, the analyte removes BLOCK to make REPORTER detectable by the change in spectral properties, and the haloalkyl XR reacts with the intracellular thiol to form the thioether conjugate inside the cells, which is well-retained in the cells.

Abstract translation: 本发明提供了通过仅在存在特定酶的完整细胞中改善可检测报道分子的保留来分析细胞中的代谢活性的方法。 特别地，涉及将报道分子的卤代烷基取代的衍生物与含细胞内半胱氨酸的肽结合的两部分方法的改进的保留结果，同时解除报道分子的阻断。 用于分析细胞代谢活性的方法涉及使用具有XR-REPORTER-BLOCK形式的底物，其中-BLOCK是通过特定分析物的作用选择为可除去的基团，以产生与底物相反的REPORTER光谱性质 ，-REPORTER-是当不再通过BLOCK-REPORTER键与BLOCK结合的分子时，具有与底物不同的谱特性，XR-是可与胞内硫醇（ZSH）共价反应的卤代烷基部分， 以形成硫醚缀合物（ZSR-）。 在底物进入细胞后，分析物除去BLOCK以通过光谱性质的变化使REPORTER可检测到，并且卤代烷基XR与细胞内硫醇反应以在细胞内形成硫醚缀合物，其在细胞中良好保留。

公开(公告)号：WO1993004077A1

公开(公告)日：1993-03-04

申请号：PCT/US1992006957

申请日：1992-08-21

Applicant: MOLECULAR PROBES, INC.

Inventor： MOLECULAR PROBES, INC. ,  HAUGLAND, Richard, P.

IPC: C07H15/26

CPC classification number: C07D263/57 ,  C07D235/18 ,  C07D239/91 ,  C07D277/66 ,  C07F9/65128 ,  C07F9/6541 ,  C07H15/26 ,  C12Q1/34 ,  C12Q1/42 ,  C12Q2334/00

Abstract: Novel fluorescent precipitating substrates made from a class of fluorophores, generally including quinazolinones (quinazolones), benzimidazoles, benzothiazoles, benzoxazoles, quinolines, indolines, and phenanthridines, having general formula (I), where carbon atoms of -C1=C2- are further joined so as to complete a first 5- or 6-membered aromatic ring which may contain at least one of the hetero atoms N, O or S, where carbon atoms of -C4-N=C3- are further joined so as to complete a second 5- or 6-membered aromatic ring that contains at least the nitrogen between C?3 and C4¿ and may contain at least one additional hereto atom N, O or S, where the first and second aromatic rings may be joined by a 5- or 6-membered bridging ring that contains at least the C2 from the first aromatic ring and the C3 from the second aromatic ring, which bridging ring may be saturated or unsaturated and may contain a hetero atom N, O, or S, where each of the first and second aromatic rings may be fused to at least one additional aromatic ring that may contain at least one of the hetero atoms N, O or S, and where each of said aromatic rings may be further modified by substitution of any hydrogens on an aromatic carbon by substituents that are halogen, nitro, cyano, aryl, lower alkyl (1-4 carbons), perfluoroalkyl (1-4 carbons), or alkoxy (1-4 carbons), or any combination thereof; and X¿fl? is covalently linked to the oxygen -O- at C1.