公开(公告)号：US08071761B2

公开(公告)日：2011-12-06

申请号：US11606642

申请日：2006-11-29

申请人： Roger Y. Tsien ,  Gregor Zlokarnik

发明人： Roger Y. Tsien ,  Gregor Zlokarnik

IPC分类号： C07D501/14

CPC分类号： C07D501/36 ,  C07D501/00 ,  C07D505/00 ,  C07D519/00 ,  C12N9/86 ,  C12N15/85 ,  C12N2800/90 ,  C12N2830/002 ,  C12P17/184 ,  C12P17/188 ,  C12P35/00 ,  C12Q1/18 ,  C12Q1/34 ,  C12Q1/6897 ,  C12Q2334/00 ,  G01N2333/986 ,  G01N2415/00 ,  Y10S435/805

摘要： Substrates for β-lactamase of the general formula I in which one of X and Y is a fluorescent donor moiety and the other is a quencher (which may or may not re-emit); R′ is selected from the group consisting of H, lower (i.e., alkyl of 1 to about 5 carbon atoms) and (CH2)nOH, in which n is 0 or an integer from 1 to 5; R″ is selected from the group consisting of H, physiologically acceptable metal and ammonium cations, —CHR2OCO(CH2)nCH3, —CHR2OCOC (CH3)3, acylthiomethyl, acyloxy-alpha-benzyl, delta-butyrolactonyl, methoxycarbonyloxymethyl, phenyl, methylsulphinylmethyl, beta-morpholinoethyl, dialkylaminoethyl, acyloxyalkyl, dialkylaminocarbonyloxymethyl and aliphatic, in which R2 is selected from the group consisting of H and lower alkyl; A is selected from the group consisting of S, O, SO, SO2 and CH2; and Z′ and Z″ are linkers for the fluorescent donor and quencher moieties. Methods of assaying β-lactamase activity and monitoring expression in systems using β-lactamase as a reporter gene also are disclosed.

摘要翻译： X和Y中的一个是荧光供体部分，另一个是猝灭剂（其可以或不再发射）的通式I的β-内酰胺酶底物; R'选自H，低级（即1至约5个碳原子的烷基）和（CH 2）nOH，其中n为0或1至5的整数; -CH 2 COO（CH 2）n CH 3，-CHR 2 OCOC（CH 3）3，酰基硫代甲基，酰氧基-α-苄基，δ-丁基内酰胺，甲氧基羰氧基甲基，苯基，甲基亚磺酰基甲基， β-吗啉基乙基，二烷基氨基乙基，酰氧基烷基，二烷基氨基羰氧基甲基和脂族基，其中R2选自H和低级烷基; A选自S，O，SO，SO 2和CH 2; 和Z'和Z“是荧光供体和猝灭剂部分的接头。 公开了使用β-内酰胺酶作为报道基因的系统中测定β-内酰胺酶活性和监​​测表达的方法。

公开(公告)号：US20100105095A1

公开(公告)日：2010-04-29

申请号：US12499047

申请日：2009-07-07

申请人： Lewis R. Makings ,  Gregor Zlokarnik

发明人： Lewis R. Makings ,  Gregor Zlokarnik

IPC分类号： C12Q1/26 ,  C07D311/02 ,  C07D265/38

CPC分类号： C07D493/10 ,  C07D219/06 ,  C07D265/38 ,  C07D311/16 ,  C07D311/82 ,  C12Q1/26 ,  G01N2333/90245 ,  G01N2500/02 ,  Y10S435/968

摘要： The invention provides a compound, useful as an optical probe or sensor of the activity of at least one cytochrome P450 enzyme, and methods of using the compound to screen candidate drugs, and candidate drugs identified by these methods. The optical probe of the invention is a compound having the generic structure Y-L-Q, wherein Y is selected from the group consisting of Q as herein defined, saturated C1-C20 alkyl, unsaturated C1-C20 alkenyl, unsaturated C1-C20 alkynyl, substituted saturated C1-C20 alkyl, substituted unsaturated C1-C20 alkenyl, substituted unsaturated C1-C20 alkynyl, C1-C20 cycloalkyl, C1-C20 cycloalkenyl, substituted saturated C1-C20 cycloalkyl, substituted unsaturated C1-C20 cycloalkenyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl; L is selected from the group of (—OCR2H)p—, wherein for each p, all R2 are separately selected from the group consisting of a hydrogen atom, saturated C1-C20 alkyl, unsaturated C1-C20 alkenyl, unsaturated C1-C20 alkynyl, substituted saturated C1-C20 alkyl, substituted unsaturated C1-C20 alkenyl, substituted unsaturated C1-C20 alkynyl, C1-C20 cycloalkyl, C1-C20 cycloalkenyl, substituted saturated C1-C20 cycloalkyl, substituted unsaturated C1-C20 cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, and p is a positive integer no greater than twelve; and Q is a chemical moiety that gives rise to optical properties in its hydroxy or hydroxylate, phenol or phenoxide form that are different from the optical properties that arise from its ether form. Most preferably, p is one, R2 is hydrogen, and Q is the ether form of a phenoxide fluorophore.

摘要翻译： 本发明提供了可用作至少一种细胞色素P450酶的活性的光学探针或传感器的化合物，以及使用该化合物筛选候选药物的方法以及通过这些方法鉴定的候选药物。 本发明的光学探针是具有一般结构YLQ的化合物，其中Y选自本文定义的Q，饱和C 1 -C 20烷基，不饱和C 1 -C 20烯基，不饱和C 1 -C 20炔基，取代的饱和C 1 -C 20烷基，取代的不饱和C 1 -C 20烯基，取代的不饱和C 1 -C 20炔基，C 1 -C 20环烷基，C 1 -C 20环烯基，取代的饱和C 1 -C 20环烷基，取代的不饱和C 1 -C 20环烯基，芳基，取代的芳基， 杂芳基; L选自（-OCR 2 H）p - ，其中对于每个p，所有的R 2分别选自氢原子，饱和C 1 -C 20烷基，不饱和C 1 -C 20烯基，不饱和C 1 -C 20炔基 取代的饱和C 1 -C 20烷基，取代的不饱和C 1 -C 20烯基，取代的不饱和C 1 -C 20炔基，C 1 -C 20环烷基，C 1 -C 20环烯基，取代的饱和C 1 -C 20环烷基，取代的不饱和C 1 -C 20环烯基，芳基， 杂芳基，取代的杂芳基，p是不大于12的正整数; 并且Q是一种化学部分，其在其羟基或羟基化物，苯酚或酚盐形式中产生与由其醚形式产生的光学性质不同的光学性质。 最优选地，p是1，R2是氢，Q是醚形式的苯氧基荧光团。

公开(公告)号：US07067324B2

公开(公告)日：2006-06-27

申请号：US09759629

申请日：2001-01-12

申请人： Tom Knapp ,  Gregor Zlokarnik ,  Paul Negulescu ,  Roger Y. Tsien ,  Tim Rink

发明人： Tom Knapp ,  Gregor Zlokarnik ,  Paul Negulescu ,  Roger Y. Tsien ,  Tim Rink

IPC分类号： C12Q1/70 ,  G01N21/76

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.

公开(公告)号：US20050136469A1

公开(公告)日：2005-06-23

申请号：US11047074

申请日：2005-01-31

申请人： Tom Knapp ,  Gregor Zlokarnik ,  Paul Negulescu ,  Roger Tsien ,  Timothy Rink

发明人： Tom Knapp ,  Gregor Zlokarnik ,  Paul Negulescu ,  Roger Tsien ,  Timothy Rink

IPC分类号： C12Q1/68 ,  G01N33/50 ,  G01N33/542 ,  G01N33/58 ,  G01N33/53 ,  G01N33/567

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。

公开(公告)号：US06214563B1

公开(公告)日：2001-04-10

申请号：US09120516

申请日：1998-07-21

申请人： Paul Negulescu ,  Gregor Zlokarnik ,  Tom Knapp ,  Roger Y. Tsien ,  Tim Rink

发明人： Paul Negulescu ,  Gregor Zlokarnik ,  Tom Knapp ,  Roger Y. Tsien ,  Tim Rink

IPC分类号： C12Q170

CPC分类号： G01N33/542 ,  C12Q1/34 ,  C12Q1/682 ,  G01N33/5005 ,  G01N2415/00 ,  Y10S436/80 ,  Y10S436/809 ,  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 further provides a method of determining bound and free analyte in a sample using at least one photon reducing agent. The present invention also provides a method of screening test chemicals in fluorescent assays using photon reducing agents. The present invention also provides 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。 本发明还提供使用至少一种光子还原剂测定样品中结合和游离分析物的方法。 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方法。 本发明还提供了用于实施这些方法的组合物和试剂盒。

公开(公告)号：US06200762B1

公开(公告)日：2001-03-13

申请号：US09118497

申请日：1998-07-17

申请人： Gregor Zlokarnik ,  Paul Negulescu ,  Tom Knapp ,  Roger Y. Tsien ,  Tim Rink

发明人： Gregor Zlokarnik ,  Paul Negulescu ,  Tom Knapp ,  Roger Y. Tsien ,  Tim Rink

IPC分类号： C12Q170

CPC分类号： G01N33/542 ,  C12Q1/682 ,  G01N33/5005 ,  Y10S436/80 ,  Y10S436/809 ,  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 further provides a method of determining bound and free analyte in a sample using at least one photon reducing agent. The present invention also provides a method of screening test chemicals in fluorescent assays using photon reducing agents. The present invention also provides 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。 本发明还提供使用至少一种光子还原剂测定样品中结合和游离分析物的方法。 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方法。 本发明还提供了用于实施这些方法的组合物和试剂盒。

公开(公告)号：US6031094A

公开(公告)日：2000-02-29

申请号：US128679

申请日：1998-07-23

申请人： Roger Y. Tsien ,  Gregor Zlokarnik

发明人： Roger Y. Tsien ,  Gregor Zlokarnik

IPC分类号： C07D205/08 ,  C07D401/04 ,  C07D403/04 ,  C07D405/04 ,  C07D405/10 ,  C07D205/085

CPC分类号： C07D401/04 ,  C07D205/08 ,  C07D403/04 ,  C07D405/04 ,  C07D405/10

摘要： ##STR1## Substrates for beta-lactamase of general formula 1, in which R
1 is a moiety, in which a primary amine attached to an aryl carbon of a fluorophore precursor was converted into the nitrogen atom of the beta-lactam ring and the fluorophore precursor is not anthranilic acid when R
2 is H; R
2 is selected from the group consisting of H and --NR
3 R
4 , in which R
3 and R
4 are selected from the group consisting of H, aliphatic, alkyl, and acyl; and the beta-lactam ring may be cleaved by a beta-lactamase enzyme. Methods of assaying beta-lactamase activity and monitoring expression in systems using beta-lactamase as a reporter gene are also disclosed.

摘要翻译： 用于通式1的β-内酰胺酶的底物，其中R1是部分，其中连接到荧光团前体的芳基碳上的伯胺转化成β-内酰胺环的氮原子和荧光团前体不是邻氨基 当R2为H时为酸; R 2选自H和-NR 3 R 4，其中R 3和R 4选自H，脂族，烷基和酰基; 并且β-内酰胺环可以被β-内酰胺酶切割。 还公开了使用β-内酰胺酶作为报告基因的系统中测定β-内酰胺酶活性和监​​测表达的方法。

公开(公告)号：US5741657A

公开(公告)日：1998-04-21

申请号：US407544

申请日：1995-03-20

申请人： Roger Y. Tsien ,  Gregor Zlokarnik

发明人： Roger Y. Tsien ,  Gregor Zlokarnik

IPC分类号： C12N15/09 ,  C07D463/00 ,  C07D471/04 ,  C07D501/00 ,  C07D501/20 ,  C07D501/59 ,  C07D505/00 ,  C12N5/02 ,  C12N5/10 ,  C12N9/86 ,  C12N15/55 ,  C12N15/85 ,  C12P17/18 ,  C12P35/00 ,  C12Q1/34 ,  C12Q1/68 ,  C07D501/14

CPC分类号： C07D501/36 ,  C07D501/00 ,  C07D505/00 ,  C07D519/00 ,  C12N15/85 ,  C12N9/86 ,  C12P17/184 ,  C12P17/188 ,  C12P35/00 ,  C12Q1/18 ,  C12Q1/34 ,  C12Q1/6897 ,  C12N2800/90 ,  C12N2830/002 ,  C12Q2334/00 ,  G01N2333/986 ,  G01N2415/00 ,  Y10S435/805

摘要： Fluorogenic substrates of the general formula I ##STR1## in which one of X and Y is a fluorescent donor moiety and the other is a quencher (which may or may not re-emit); R' is selected from the group consisting of H, lower (i.e., alkyl of 1 to about 5 carbon atoms) and (CH.sub.2 OH).sub.n OH, in which n is 0 or an integer from 1 to 5; R" is selected from the group consisting of H, physiologically acceptable metal and ammonium cations, --CHR.sup.2 OCO(CH.sub.2).sub.n CH.sub.3, --CHR.sup.2 OCOC(CH.sub.3).sub.3, acylthiomethyl, acyloxy-alpha-benzyl, delta-butyrolactonyl, methoxycarbonyloxymethyl, phenyl, methylsulphinylmethyl, beta-morpholinoethyl, dialkylaminoethyl, acyloxyalkyl, dialkylaminocarbonyloxymethyl and aliphatic, in which R.sup.2 is selected from the group consisting of H and lower alkyl; A is selected from the group consisting of S, O, SO, SO.sub.2 and CH.sub.2 ; and Z' and Z" are linkers for the fluorescent donor and quencher moieties. The substrates are useful in conjunction with .beta.-lactamase as reporter gene in a wide range of assays, for example to determine protein localization or bacterial resistance.

摘要翻译： 其中X和Y中的一个是荧光供体部分，另一个是淬灭剂（其可以或不再发射）的通式I 的荧光底物; R'选自H，低级（即1至约5个碳原子的烷基）和（CH 2 OH）nOH，其中n为0或1至5的整数; R“选自H，生理学上可接受的金属和铵阳离子，-CHR2OCO（CH2）nCH3，-CHR2OCOC（CH3）3，酰基硫代甲基，酰氧基-α-苄基，δ-丁基内酰基，甲氧基羰氧基甲基，苯基，甲基亚磺酰基甲基 ，β-吗啉代乙基，二烷基氨基乙基，酰氧基烷基，二烷基氨基羰氧基甲基和脂族基，其中R2选自H和低级烷基; A选自S，O，SO，SO 2和CH 2; Z'和Z“是荧光供体和猝灭剂部分的接头。 在广泛的测定中，底物可与β内酰胺酶作为报告基因结合使用，例如确定蛋白质定位或细菌耐药性。

公开(公告)号：US08476442B2

公开(公告)日：2013-07-02

申请号：US12728173

申请日：2010-03-19

申请人： John DeMattei ,  Adam R. Looker ,  Bobbianna Neubert-Langille ,  Martin Trudeau ,  Stefanie Roeper ,  Michael P. Ryan ,  Dahrika Milfred Lao Yap ,  Brian R. Krueger ,  Peter D. J. Grootenhuis ,  Fredrick F. van Goor ,  Martyn C. Botfield ,  Gregor Zlokarnik

发明人： John DeMattei ,  Adam R. Looker ,  Bobbianna Neubert-Langille ,  Martin Trudeau ,  Stefanie Roeper ,  Michael P. Ryan ,  Dahrika Milfred Lao Yap ,  Brian R. Krueger ,  Peter D. J. Grootenhuis ,  Fredrick F. van Goor ,  Martyn C. Botfield ,  Gregor Zlokarnik

IPC分类号： C07D215/233 ,  C07D215/00 ,  C07D307/77 ,  C07D307/00 ,  A61K31/47

CPC分类号： C07C205/43 ,  C07C68/02 ,  C07C69/96 ,  C07C201/08 ,  C07C213/02 ,  C07C229/66 ,  C07D215/233 ,  C07D215/56 ,  G01N33/5041 ,  G01N33/6872 ,  Y02P20/55

摘要： The invention provides a process for the preparation of a compound of Formula 1, comprising coupling a carboxylic acid of Formula 2 with an aniline of Formula 3 in the presence of a coupling agent.

摘要翻译： 本发明提供了制备式1化合物的方法，包括在偶联剂存在下将式2的羧酸与式3的苯胺偶联。

公开(公告)号：US20130004960A1

公开(公告)日：2013-01-03

申请号：US13437846

申请日：2012-04-02

申请人： Tom KNAPP ,  Gregor Zlokarnik ,  Paul Negulesou ,  Roger Y. Tsien ,  Timothy James Rink

发明人： Tom KNAPP ,  Gregor Zlokarnik ,  Paul Negulesou ,  Roger Y. Tsien ,  Timothy James Rink

IPC分类号： G01N21/64 ,  G01N21/75

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。