Halide indicators

Details Halide indicators Halide indicators

1999-03-26

2001-03-13

Lee, Howard C. (Department: 1623)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C544S251000, C544S252000, C436S172000

Reexamination Certificate

active

06201116

ABSTRACT:

INTRODUCTION TO THE INVENTION
1. Field of the Invention
The field of the invention is halide indicators and their use in detecting anion transport.
2. Background
Several halide-sensitive fluorescent indicators have been introduced to study the functional properties of Cl
−
transporters in biomembrane vesicles and living cells and epithelial tissues (for review, see Verkman, 1990; Verkman and Biwersi, 1995). The archetype indicator SPQ (6-methoxy-N-[3-sulfopropyl]quinolinium, Illsley and Verkman, 1987) is a polar quinolinium compound whose fluorescence is quenched by Cl
−
and I
−
by a collisional mechanism. SPQ and related quinolinium indicators have been useful in studying the CFTR (Cystic Fibrosis Transmembrane conductance Regulator) Cl
−
channel expressed in native and transfected cell models (e.g. Brown et al., 1996), and recently, in assaying functional CFTR delivery in human gene therapy trials (McLachlan et al., 1996; Porteous et al., 1997; Gill et al., 1997). Various SPQ derivatives have been synthesized for specific applications including cell-permeable/trappable compounds (Biwersi and Verkman, 1991), dual-wavelength Cl
−
indicators for ratio imaging (Jayaraman et al., 1999), dextran-linked conjugates (Biwersi et al., 1992), and fiberoptic halide sensors (Kao et al., 1992).
Although used in numerous studies of CFTR function in cell culture models, the existing quinolinium-based halide indicators have imperfect optical and physical properties that limit their utility in more challenging applications including the analysis of CFTR function in gene therapy trials and high-throughput drug screening. SPQ and related indicators have relatively dim fluorescence in cells (molar extinction <6000 M
−1
cm
−1
, quantum yield <0.1) and require ultraviolet excitation (excitation 320-365 nm, emission 420-460 nm). Significant technical limitations include the need for sensitive detection instrumentation with high numerical aperture optics, and the background autofluorescence resulting from ultraviolet excitation. In addition, the quinolinium halide indicators are quenched by intracellular proteins and organic anions, resulting in decreased cytoplasmic Cl
−
sensitivity and dependence of indicator fluorescence on changes in cell volume (Chao et al., 1989; Srinivas and Bonanno, 1997). We previously synthesized long-wavelength Cl
−
indicators containing the acridinium chromophore (Biwersi et al., 1994); although these indicators have been useful to study Cl
−
transport in liposomes and biomembrane vesicles, they are chemically unstable in cytoplasm—a problem that could not be overcome by derivatization.
The present invention provides bright, long-wavelength halide indicators and their uses in assays of CFTR-mediated anion conductance in living cells, particularly a sensitive, robust anion transport assay suitable for high-throughput drug screening. The specifications of the indicator(s) include: high Cl
−
and/or I
−
sensitivity, bright fluorescence with excitation wavelength >400 nm and emission wavelength >500 nm, minimal photobleaching, lack of cellular toxicity, rapid loading into cytoplasm, uniform distribution and chemical stability in cytoplasm, and low leakage out of cells.
SUMMARY OF THE INVENTION
The invention provides methods and compositions for measuring ion concentration inside a cell by measuring fluorescence of a compound of the general formula I:
wherein R
1
is selected from C and N, and
R
2
-R
9
are independently selected from the group consisting of substituted or unsubstituted alkylalkenyl, alkynyl, aryl, heteroalkyl, heteroaryl, and acyl substituents, wherein one or more of the pairs R
2
and R
3
, R
3
and R
4
, R
4
and R
5
, R
6
and R
7
, and R
8
and R
9
, may be directly or indirectly further covalently connected, to form a substituted or unsubstituted, aromatic or nonaromatic ring. The methods encompass the use of wide variety of particular embodiments, derivatives, analogs, etc. of the generic compound. In particular embodiments, the measured ion is halide, particularly iodide, the cell contains a functional anion transport protein or channel, the method measures a change in fluorescence as a function of a predetermined condition such as the presence of a predetermined amount of a candidate modulator of ion transport in the cell (e.g. for drug screening) or the expression by the cell of a transgene (e.g. to assess the efficacy of gene therapy).
The subject compositions comprise a compound comprising the general formula I, wherein (a) one or more of the pairs R
2
and R
3
, R
3
and R
4
, R
4
and R
5
, R
6
and R
7
, and R
8
and R
9
, is further covalently connected, to form a substituted or unsubstituted, aromatic or nonaromatic ring, (b) one or more of R
2
-R
7
comprises a functional group, and/or (c) R
1
is N. In particular embodiments, the functional group is a conjugation group, a polarity enhancing group or an iodide sensitivity enhancing group, and in a more particular embodiment, the conjugation group is coupled to a moiety selected from a membrane impermeable molecule, an optical sensor and a chromophore. A wide variety of additional particular embodiments of the generic compound as well as numerous exemplary compounds are disclosed. For example, preferred compounds provide one or more of: threshold molar extinctions, quantum yields, excitation wavelengths and emission wavelengths, fluorescence, relatively specific iodide quenching, relatively low cytotoxicity, relatively low photobleaching in cells, relatively uniform distribution in cells, relatively high chemical stability in cells, relatively low leakage out of cells and relatively rapid loading into cells.
DESCRIPTION OF PARTICULAR EMBODIMENTS OF INVENTION
The following descriptions of particular embodiments and examples are offered by way of illustration and not by way of limitation. Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms “a” and “an” mean one or more, the term “or” means and/or and polynucleotide sequences are understood to encompass opposite strands as well as alternative backbones described herein.
The general invention methods involve measuring ion concentration inside a cell by detecting fluorescence inside the vesicle of a compound of the general formula I. Pursuant to the general formula, R
2
-R
9
are independently selected from the group consisting of substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroaryl, and acyl substituents.
Exemplary such substituents may be independently selected from:
hydrogen,
substituted or unsubstituted (C1-C10)alkyl,
substituted or unsubstituted (C1-C10)alkoxy,
substituted or unsubstituted (C3-C6)alkenyl,
substituted or unsubstituted (C2-C6)heteroalkyl,
substituted or unsubstituted (C3-C6)heteroalkenyl,
substituted or unsubstituted (C3-C6)alkynyl,
substituted or unsubstituted (C3-C8)cycloalkyl,
substituted or unsubstituted (C5-C7)cycloalkenyl,
substituted or unsubstituted (C5-C7)cycloalkadienyl,
substituted or unsubstituted aryl,
substituted or unsubstitated aryloxy,
substituted or unsubstituted aryl-(C3-C8)cycloalkyl,
substituted or unsubstituted aryl-(C5-C7)cycloalkenyl,
substituted or unsubstituted aryloxy-(C3-C8)cycloalkyl,
substituted or unsubstituted aryl-(C1-C4)alkyl,
substituted or unsubstituted aryl-(C1-C4)alkoxy,
substituted or unsubstituted aryl-(C1-C4)heteroalkyl,
substituted or unsubstituted aryl-(C3-C6)alkenyl,
substituted or unsubstituted aryloxy-(C1-C4)alkyl,
substituted or unsubstituted aryloxy-(C2-C4)heteroalkyl,
substituted or unsubstituted heteroaryl,
substituted or unsubstituted heteroaryloxy,
substituted or unsubstituted heteroaryl-(C1-C4)alkyl,
substituted or unsubstituted heteroaryl-(C1-C4)alkoxy,
substituted or unsubstituted heteroaryl-(C1-C4)heteroalkyl,
substituted or unsubstituted heteroaryl-(C3-C6)alkenyl,
substituted or unsubstituted heteroaryloxy-(C1-C4)alkyl, and
substituted or unsubstituted heteroaryloxy-(C2-C4)heteroal

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