Fluorogenic compounds

Details Fluorogenic compounds Fluorogenic compounds

2000-07-07

2002-04-16

Fonda, Kathleen K. (Department: 1623)

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

Organic compounds

Carbohydrates or derivatives

C536S017200, C536S017400, C536S018100, C536S018400, C530S300000, C530S331000, C548S100000, C548S146000, C548S200000, C548S202000, C548S215000, C548S235000, C548S300100, C548S311700, C548S333500, C548S335100, C549S029000, C549S030000, C549S070000, C549S072000, C549S218000, C549S368000, C549S402000

Reexamination Certificate

active

06372895

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to fluorogenic compounds, including novel fluorogenic compounds suitable for use in biological assays, and to methods of using the flourogenic compounds in biological assays.
BACKGROUND OF THE INVENTION
Fluorogenic and chromogenic enzyme substrates find broad utility in biological detection assays. Many of these substrates are formed by covalently linking a fluorescent or chromophoric dye to a biological molecule that is specific to an enzyme being investigated. Subsequent cleavage of the covalent linkage by the enzyme releases the dye, allowing the fluorescent or calorimetric properties of the dye to be detected visually or measured spectrophotometrically.
The challenge in using this method is finding a fluorophore or chromophore that can satisfy a wide range of conditions for the biological assay of interest. For instance, the fluorescence or the color of the cleaved product should preferably vary from the uncleaved substrate, the background fluorescence and color of the biological sample should not interfere with the detection of the cleaved product, the substrate should be stable to heat and light under the conditions required for the assay, and the substrate should not interfere with the biological activity of the enzyme.
Commonly used substrates include fluorogenic synthetic enzyme substrates derived from coumarin derivatives 4-methylumbelliferone (4-MU) or 7-amino-4-methylcoumarin (7-AMC). MU derivatives have the following structure:
The popularity of these substrates can be ascribed to availability of a wide range of enzyme cleavable R groups. Typical R groups frequently used are esters, monosaccharides, disaccharides, and phosphates. In addition, the fluorogenic synthetic enzyme substrates are popular because of their noncarcinogenicity, ease of visual detection of the products of enzyme activity with UV light sources, autoclave stability, and availability of suitable fluorometers for measurement of fluorescence in both tube and multiwell panels. Enzyme cleavage releases 4-MU, giving rise to fluorescence associated with the 4-MU anion (excitation wavelength of 365 nm, emission wavelength 440 nm). Aryl peptides of 7-AMC are also frequently used as fluorogenic enzyme substrates. The released 7-AMC shows a blue fluorescence (excitation wavelength 370 nm, emission wavelength 440 nm).
Release of 4-MU and 7-AMC can be detected visually as a blue fluorescence when irradiated with a long wavelength UV lamp (for example, &lgr;=360±20 nm). However, at these excitation and emission wavelengths, it is quite common for the biological sample to emit a significant background fluorescence of its own. In other cases, materials employed in making the plate, tube or polymeric support of an assay format can emit a significant background fluorescence. In these cases, low amounts of enzyme activity cannot be detected above background. This common problem adversely affects the sensitivity and speed of many enzyme-linked biological assays.
In contrast, at longer excitation and longer emission wavelengths, background fluorescence drops off dramatically. Therefore, it would be advantageous to develop a class of fluorogenic enzyme substrates that absorb and emit at longer wavelengths (i.e., are red-shifted) than those of the 4-MU- and 7-AMC-based derivatives, without losing the desirable properties of these dyes.
Alternative detection methods do exist. For example, esters, monosaccharides, disaccharides, and phosphates of o-nitrophenol (ONP) or p-nitrophenol (PNP) are frequently used as colorimetric enzyme substrates. Release of nitrophenol gives rise to an increase in absorbence at 405 mn and appearance of a yellow color. The absorbence of these products can be detected using, e.g., a GaN LED source. However, these products are not fluorescent, therefore the sensitivities are far less than 4-MU and 7-AMC derivatives. Aryl peptide derivatives of p-nitroanaline (p-NA) give similar appearance of yellow color. In this case, a substantial increase in sensitivity can be achieved by reacting the p-NA with a diazo dye, yielding blue to dark purple colors. Esters of indoxyl or 5-bromo-4-chloro-3-indolyl give rise to enzyme induced release of indoxyl, to form a blue color, but still do not result in sufficient fluorescence output that allows for more sensitive measurements.
Esters, monosaccharides, disaccharides, and phosphates of fluorescein (FL) and rhodamine (RH) dyes have been developed as fluorogenic enzyme substrates. Release of FL gives rise to an increase in fluorescence associated with the FL anion (excitation wavelength 490 nm, emission wavelength 514 nm). Release of RH gives rise to an increase in fluorescence associated with the RH anion (excitation wavelength 499 nm, emission wavelength 521 nm). The FL and RH anion fluorescences can easily be detected using bulky gas lasers as excitation sources, but there is currently no commercially available solid state light source for these materials. In addition, these classes of enzyme substrates are not typically autoclave stable and therefore are not appropriate for many applications. Finally, the Stokes shift (the difference between the absorbence and emission wavelength) for these materials is far less than that for the 4-MU and 7-AMC materials, requiring the use of more specialized optics to separate the emission signal from the excitation signal.
One particularly relevant application for enzyme substrate indicators is in the detection and differentiation of bacteria. Growing microcolonies will often secrete extracellular enzymes that can convert upwards of a million fluorescent indicator molecules per enzyme molecule. Because the fluorescence detection method is extremely sensitive, this provides a method to amplify the signal from a growing microcolony so that it can be detected in a shorter period of time. For example, a growing microcolony might be detected in 4 to 6 hours using fluorogenic enzyme substrates, whereas the microcolony may be detected in 24 to 48 hours using conventional chromogenic enzyme substrates. This would offer great benefit in the food processing industry, as contaminants could be discovered in eight hours or less.
An example where such methods would be useful is detection of
E. coli
or coliform.
E. coli
is an important indicator of fecal contamination in environmental and food samples, while coliform count is an important indicator of bacteriological contamination. In the quality control of water and food, it is highly important to examine both coliforms and
E. coli
. Testing procedures for coliforms commonly employ a 4-MU derivative specific for detecting &bgr;-D-galactosidase (&bgr;-Gal) activity. This substrate is 4-methylumbelliferyl-&bgr;-D-galactoside (MUGal), which is hydrolyzed by &bgr;-Gal, liberating blue fluorescent 4-MU. Testing procedures for
E. coli
commonly employ a 4-MU derivative specific for detecting &bgr;-D-glucuronidase (&bgr;-Gud) activity. This substrate is 4-methylumbelliferyl-&bgr;-D-glucuronide (MUGud), which is hydrolyzed by &bgr;-Gud, again liberating 4-MU. For selective detection of
E. coli
in primary isolation media, it is common to perform an aerobic incubation in a selective growth medium that inhibits growth of gram-positive strains. In this way, &bgr;-Gud activities from strains other than
E. coli
are suppressed. Additionally, incubation at 44° C. and detection of gas formation help in exclusive detection of
E. coli.
Several MUGud and MUGal testing procedures have been employed for identifying and enumerating total coliforms and
E. coli
. These include most probable number, membrane filtration, presence absence test, agar plate, microtitration plate, paper strip, and related techniques. Because of the thermal stability of these dyes to autoclave, they can be incorporated directly into the growth media before autoclave sterilization. This is an important advantage for commercial test kits, which are sterilized and packaged in the factory.
In a related application, it has been desirable to use th

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