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Fluorogenic ph sensitive dyes and their method of use

a technology of fluorogenic ph and dyes, which is applied in the direction of diaryl/triaryl methane dyes, organic compounds of group 3/13 elements, peptides, etc., can solve the problems of limiting the applicability of dyes described, affecting the applicability of dyes, and affecting the stability of solution,

Inactive Publication Date: 2008-11-06
LIFE TECH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

One disadvantage of these sensors is that the working range is shifted to the acidic side because of the low pKa of the indicator amino group.
However, this thereby limits the applicability of the dyes described in WO 2005 / 098 at a physiological pH (e.g pH 6-7), especially in biological systems.
These prior art compounds have been found by us to have potentially inconvenient instability in solution.

Method used

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  • Fluorogenic ph sensitive dyes and their method of use
  • Fluorogenic ph sensitive dyes and their method of use
  • Fluorogenic ph sensitive dyes and their method of use

Examples

Experimental program
Comparison scheme
Effect test

example 101

Synthesis of pH Sensor 104 Having the Methoxy pKa-Enhancing Group (Scheme 101)

[0642]

[0643]Compound 104. To a stirred suspension of ketone 101 (94 mg, 0.333 mmol) in dry chloroform (10 mL), oxalyl chloride (30 μL, 0.33 mmol) was added upon cooling to 0-5° C. The resulted red solution was stirred for 1 h, then N,N-diethyl-m-anisidine (60 mg, 0.33 mmol) was added. The reaction was allowed to warm to rt, stirred for 16 h and diluted with CHCl3 (60 mL).

[0644]Chloroform solution was shaken with sat. NaHCO3 (40 mL) until water layer turned almost colorless. The organic layer was washed with sat. NaHCO3 (20 mL) and extracted with 10% HCl (2×30 mL). The combined acid extract was washed with CHCl3 (2×15 mL; discarded), the aqueous solution was saturated with sodium acetate and extracted with CHCl3 (4×30 mL). The extract was washed with brine (30 mL), and evaporated. The crude product was purified by chromatography on silica gel column (2×40 cm bed, packed with 10% MeOH and 1% AcOH in CHCl3) e...

example 102

Synthesis of pH Sensor 109 Having Two Methoxy pKa-Enhancing Groups and Compound III with a Labeling Succinimidyl Ester Moiety (Scheme 102)

[0645]

[0646](2,5-Dimethoxyphenyl)acetamide (106). To a stirred solution of 2,5-dimethoxyaniline (105) (1.00 g, 6.52 mmol) and DIEA (1.71 mL, 9.80 mmol) in CHCl3 (20 mL) was added acetic anhydride (0.93 mL, 9.8 mmol). The reaction mixture was stirred for 1.5 h, washed with water (50 mL), 5% HCl (50 mL), brine (50 mL), dried over Na2SO4, and evaporated to give acetate 106 (1.33 g, 100%) as a dark oil.

[0647]2,5-Dimethoxy-N-ethylaniline (107). To the solution of acetamide 106 (1.27 g, 6.5 mmol) in dry THF (10 mL) a borane-THF complex (1M in THF, 58.5 mL, 58.5 mmol) was added upon ice-water cooling. The mixture was allowed to warm to rt and stirred under reflux for 16 h. The mixture was cooled to 0° C. and carefully decomposed with MeOH (40 mL), and then the mixture was heated to reflux and stirred for 1 h. Upon cooling, the solution was concentrated a...

example 103

Synthesis of pH Sensor 118 Having Methoxy pKa-Enhancing Group and Compound 120 with a Labeling Succinimidyl Ester Moiety (Scheme 103)

[0652]

[0653]Ethyl 4-(4-benzyloxy-2-nitrophenyloxy)butanoate (113). 4-(Benzyloxy)-2-nitrophenol (112) (2.00 g, 9.39 mmol), K2CO3 (1.30 g, 9.42 mmol), NaI (0.70 g, 4.7 mmol) and ethyl 4-bromobutyrate (2.70 mL, 18.7 mmol) in DMF (10 mL) were stirred for 3 hrs at 70° C. The reaction mixture was cooled to rt, diluted with EtOAc (100 mL), washed with water (4×40 mL), 5% HCl (3×40 mL), brine (30 mL), dried over Na2SO4 and evaporated to give ester 113 (2.10 g, 62%) as a brown oil.

[0654]4-(4-Hydroxy-2-nitrophenyloxy)butanoic acid (114). Ester 113 (1.50 g, 4.17 mmol) was stirred under reflux with 30 mL of water and 30 mL of conc HCl for 1.5 hrs. Upon cooling the mixture was diluted with water (150 mL) and extracted with EtOAc (3×40 mL). The extract was washed with brine (50 mL), dried over Na2SO4 and evaporated. The crude product was dissolved in 1M KOH (30 mL),...

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Abstract

A new class of pH sensitive fluorescent dyes and assays relating thereto are described. The dyes and assays are particularly suited for biological applications including phagocytosis and monitoring intracellular processes. The pH sensitive fluorescent dyes of the present invention include compounds of Formula I:wherein the variables are described throughout the application.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority to U.S. Provisional Application Nos. 60 / 863,318, filed 27 Oct. 2006 and 60 / 940,323, filed 25 May 2007, both of which are incorporated by reference.FIELD OF THE INVENTION[0002]Novel pH sensitive fluorescent dyes and assays for use in a variety of applications including monitoring of intracellular processes are disclosed.BACKGROUND OF THE INVENTION[0003]pH sensitive fluorescent dyes employed in biological research and medical diagnostics belong to two groups, each distinguished by the origin of fluorescent responses to changes in pH. The first group includes compounds having fluorescence controlled by the ionization of phenolic hydroxyl groups in a fluorophore. Examples include fluorescein, carboxyfluorescein, Oregon Green®, SNARF®, SNAFL®, and HPTS indicators.[0004]U.S. Patent Publication No. 2006 / 0051874 (M. W. Reed, et al) describes fluorescein-like structures incorporated into a fluorescent detector for ...

Claims

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Application Information

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IPC IPC(8): C07D455/04C07D311/82C07D209/12C40B30/00G01N33/68G01N21/00C12Q1/02C07F5/02C07D413/10
CPCC07K1/13C09B7/00C09B11/12C09B11/28Y10T436/143333C09B69/00G01N31/22C09B11/24G01N33/5091C09B57/00C07D311/82C07D311/90C07D405/12C07D413/04C07D491/22C07D209/14C09K11/06C07F5/02G01N33/5058G01N33/5005G01N33/582G01N33/84G01N33/80G01N2333/245
Inventor BEACHAM, DANIELDZUBAY, JEFFREYGEE, KYLEMARTIN, VLADIMIRRUKAVISHNIKOV, ALEKSEY
Owner LIFE TECH CORP
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