Long wavelength lipophilic fluorogenic glycosidase substrates

The claimed invention relates to a substrate for evaluating glycosidic enzymes comprising a resorufin derivative of the general formula: ##STR1## wherein Gly is a carbohydrate bonded to resorufin by a glycosidic linkage; where at least one of substituents R.sub.1, R.sub.2, R.sub.4, R.sub.6, R.sub.8, and R.sub.9 is a lipophilic residue of the formula --L(CH.sub.2).sub.n CH.sub.3, where n is greater than 3 and less than 22, and where L is a methylene --CH.sub.2 --, an amide --NHCO--, a sulfonamide --NHSO.sub.2 --, a carboxamide --CONH--, a carboxylate ester --COO--, a urethane --NHCOO--, a urea --NHCONH--, or a thiourea --NHCSNH--; and PA0 where the remainder of substituents R.sub.1, R.sub.2, R.sub.4, R.sub.6, R.sub.8, and R.sub.9, 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'(CH.sub.2).sub.m CH.sub.3, where m is less than 22, and where L' is a methylene --CH.sub.2 --, an amide --NHCO--, a sulfonamide --NHSO.sub.2 --, 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 non-fluorescent 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.degree. C. and which is non-toxic 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.

FIELD OF THE INVENTION 
This invention relates to reddish fluorogenic substrates used to analyze 
glycosidic enzyme activity. In particular, the invention relates to 
improved resorufin glycosides that incorporate a lipophilic group, useful 
in detecting cells producing enzymes that hydrolyze the glycoside. 
BACKGROUND INFORMATION 
By studying the chemical reactions that occur inside particular cells, 
scientists can learn more about those cells. It is difficult to conduct 
experiments inside cells, however. One technique shown to be useful is to 
develop a probe to enter the cell, react with a particular substance 
inside the cell, and signal that the reaction has occurred. 
Unfortunately, many tests for the presence of products inside of cells are 
destructive to the cells being tested, either killing them outright or 
preventing them from growing or reproducing as normal cells. They may have 
other disadvantages as well. For example, radioactive substances, while 
sufficiently sensitive to distinguish cells, ultimately destroy the 
viability of the cells during measurement and destroy the reproductive 
capability of the cells. In addition, radioactive reagents are dangerous 
to handle and require slow and cumbersome experimental techniques and 
disposal methods, and radioactive measurements can not be done on viable 
cells. Dyes that are chromogenic rather than fluorescent, e.g., 
5-bromo-4-chloroindolyl galactoside (X-gal) and 5-bromo-4-chloro-3-indolyl 
.beta.-D-glucuronic acid (X-GlcU) are less sensitive and require a large 
turnover of substrate or multiple reactions to obtain a signal. 
Furthermore, the hydrolysis product of X-gal (and X-GlcU) is frequently 
toxic to cells. 
In order to study living cells, tests are typically limited to only a 
portion of a cell population. If the subject cell population is a very 
small one, however, and some cells are removed for testing, sufficient 
cells may not be available for further study or use. In order to study or 
otherwise utilize a small population of cells, as living cells, it is 
essential to be able to locate and, if possible, separate those cells 
without destroying them. 
Ideally, a probe used to identify and separate a living cell which contains 
a particular substance or to localize a substance in an organelle of a 
living cell, has the following characteristics: 1) the probe enters the 
cell without damaging the cell or preventing its subsequent cloning or 
reproduction, 2) the probe reacts exclusively with the particular 
substance inside the cell to form a specific detection product, 3) the 
detection product produces a signal sufficiently intense to distinguish 
the cell from other cells that do not contain the substance or that 
contain less of the substance, and 4) the detection product is 
sufficiently well retained by the cell to permit analysis and, if desired, 
sorting of the cell. 
Fluorescent enzyme substrates generally make ideal probes. Often, a 
fluorescent substrate can enter the cell using the cell's own mechanisms. 
Once inside the cell, a fluorescent substrate usually only reacts with a 
specific enzyme. Typically, the reaction produces a change in fluorescence 
which is sufficiently distinctive to distinguish cells or organelles that 
have the enzyme from cells or organelles that do not or that have lower 
levels of the enzyme. 
Use of fluorescent substrates also permits utilization of flow cytometers. 
Flow cytometers are designed for the rapid and specific sorting of highly 
fluorescent cells from cells that have low fluorescence. Flow cytometers 
commonly use an argon laser to excite the fluorescent product inside the 
cells. Thus, excitation of fluorescence at the principal wavelengths of 
the argon laser (488 or 514 nm) or by longer wavelength excitation sources 
is a preferred characteristic of a fluorescent substrate for some 
applications. As a result, fluorescent substrates which respond poorly at 
this wavelength, such as umbelliferone (7-hydroxycoumarin) conjugates, are 
not as suitable for such applications. 
Two advantages exist for even longer wavelength probes that absorb at 
greater than about 500 nm: 1) the autofluorescence from cells generally 
decreases with increasing wavelengths and 2) longer wavelength emission 
can be detected in the presence of a second dye such as fluorescein that 
emits at a shorter wavelength, permitting measurement of two parameters 
that are detected simultaneously or sequentially at separate wavelengths. 
Quantitative imaging of fluorescence using microscopes and image 
intensifiers has been used to measure substances such as intracellular 
calcium ions and superoxide production in living cells. Methods exist for 
quantitatively measuring changes in fluorescence intensity with time, such 
as occurs in turnover of fluorescent substrates. Quantitative differences 
in the fluorescence change that result from hydrolysis of a fluorescent 
substrate may be the result of either a higher enzyme content or a faster 
enzyme turnover rate. 
Fluorescent substrates, and flow cytometry, can also be used to detect and 
separate cells which have acquired the ability to produce certain enzymes 
as a result of a gene fusion. Gene fusions are used to study or work with 
a particular gene or genetic material by inserting it into a host cell. 
Typically, the foreign genetic material is inserted into the host cell 
using a vector (transfection). Alternatively, the foreign genetic material 
enters the cell through pores created in the membrane, e.g. 
electroporation, or is microinjected into the host cell. Cells which have 
successfully incorporated the foreign genetic material are termed 
"transformed". 
One way to determine whether transformation has occurred is to test for the 
presence of a protein product resulting from the inserted genetic 
material. Depending on the nature of the foreign genetic material inserted 
into the host cell and the desired genetic characteristics of the 
transformed cell, however, testing for successful transformation can be 
expensive and time-consuming. 
Glycosidic enzymes are commonly used to differentiate cells, including 
transformed cells. For example, .beta.-galactosidase is a bacterial enzyme 
commonly found in Escherichia coli (E. coli). The enzyme is coded by the 
E. coli lacZ gene. The presence of .beta.-galactosidase activity in a 
transformed cell can be used to indicate the presence of the foreign lacZ 
gene. The lacZ gene, in turn, is used as a genetic marker to indicate that 
additional foreign genetic material, including the lacZ gene, has been 
incorporated into a host cell otherwise lacking in .beta.-galactosidase. 
The enzyme .beta.-glucuronidase, coded by the GUS gene in E. coli, is 
primarily used to detect transformation in plant cells and tissues, where 
this activity is normally lacking (Jefferson, The GUS Reporter Gene 
System, NATURE 342, 837 (1989)), but it is also useful in detecting 
transformations in mammalian cells. 
Not all glycosidic enzymes are useful as marker enzymes. Some glycosidic 
enzymes, such as .beta.-glucosidase, are intrinsically present in many 
cells. Their activity, however, may be characteristic of the cell type, of 
an organelle of the cell, or of the metabolic state of the cell. Some 
common glycosidic enzymes and representative carbohydrates cleaved by such 
enzymes are listed in Table 1. This listing is not meant to limit or 
define the extent of all glycosidic enzymes. 
TABLE 1 
______________________________________ 
SELECTED GLYCOSIDIC ENZYMES 
(from ENZYME NOMENCLATURE, 1984 (International 
Union Biochemistry, Academic Press, 1984 pages 306-26) 
CARBOHYDRATE- 
GROUP 
E.C. NO. 
ENZYME SELECTIVITY 
______________________________________ 
3.2.1.18 
Sialidase N- or O-Acetyl 
Neuraminic Acid 
(Sialic Acid) 
3.2.1.20 
.alpha.-Glucosidase 
.alpha.-D-Glucose 
3.2.1.21 
.beta.-Glucosidase 
.beta.-D-Glucose 
3.2.1.22 
.alpha.-Galactosidase 
.alpha.-D-Galactose 
3.2.1.23 
.beta.-Galactosidase 
.beta.-D-Galactose 
3.2.1.24 
.alpha.-Mannosidase 
.alpha.-D-Mannose 
3.2.1.25 
.beta.-Mannosidase 
.beta.-D-Mannose 
3.2.1.26 
.beta.-Fructofuranosidase 
.beta.-D-Fructose 
3.2.1.30 
N-Acetyl-.beta.-glucosaminidase 
.beta.-D-N-Acetyl- 
Glucosamine 
3.2.1.31 
.beta.-Glucuronidase 
.beta.-D-Glucuronic Acid 
3.2.1.38 
.beta.-D-Fucosidase 
.beta.-D-Fucose 
3.2.1.40 
.alpha.-L-Rhamnosidase 
.alpha.-L-Rhamnose 
3.2.1.43 
.beta.-L-Rhamnosidase 
.beta.-L-Rhamnose 
3.2.1.48 
Sucrose .alpha.-glucosidase 
.alpha.-D-Glucose 
3.2.1.49 
.alpha.-N-Acetylgalactosaminidase 
.alpha.-D-N-Acetyl- 
Galactosamine 
3.2.1.50 
.alpha.-N-Acetylglucosaminidase 
.alpha.-D-N-Acetyl- 
Glucosamine 
3.2.1.51 
.alpha.-L-Fucosidase 
.alpha.-L-Fucose 
3.2.1.52 
.beta.-N-Acetylhexosaminidase 
.beta.-D-N-Acetyl- 
Glucosamine 
3.2.1.53 
.beta.-N-Acetylgalactosaminidase 
.beta.-D-N-Acetyl- 
Galactosamine 
3.2.1.55 
.alpha.-L-Arabinofuranosidase 
.alpha.-L-Arabinose 
3.2.1.76 
L-Iduronidase .alpha.-L-Iduronic Acid 
3.2.1.85 
6-Phospho-.beta.-galactosidase 
6-Phospho-.beta.-D- 
Galactose 
3.2.1.86 
6-Phospho-.beta.-glucosidase 
6-Phospho-.beta.-D-Glucose 
3.2.1.88 
.beta.-L-Arabinosidase 
.beta.-L-Arabinose 
3.2.1.4 
Cellulase .beta.-Cellobiose 
______________________________________ 
When the presence of an enzyme is used to indicate gene fusion, the marker 
included with the foreign genetic material provides a relatively fast and 
inexpensive means of detecting successful transformation. Cells which have 
successfully incorporated the marker gene are called "marker" positive 
(e.g. lacZ.sup.+ or GUS.sup.+). Using the marker gene to show successful 
transformation, however, requires detecting the activity of a very small 
number of enzyme molecules, usually in the cytosol of the lacZ.sup.+ or 
GUS.sup.+ cell. The activity must be detected in a way which does not 
inhibit further use, replication or study of the living transformed cell. 
Activity of the marker enzyme is most often used to monitor: 1) promotor 
and/or repressor effectiveness; 2) the crucial sequence of the promotor 
gene after sequential or selective deletions on it; 3) the level of 
induction of the operon so as to evaluate the effectiveness of potential 
inducer(s); and 4) any possible gene expression regulation at the pro- 
and/or post-transcription or translation level. Such monitoring is done by 
the methods generally known in the art, such as described by Jarvis, 
Hagen, & Sprague, Identification of a DNA segment that is necessary and 
sufficient for .alpha.-specific gene control in Saccharomyces cerevisiae: 
implications for regulation of .alpha.-specific and a-specific genes, 
MOLEC. & CELL. BIOL. 8, 309 (1988). 
Several substrates derived from fluorescent dyes have previously been 
described for measurement of glycosidic activity both in cell extracts and 
of the purified enzyme. Among the most common fluorescent substrates for 
detection of galactosidase activity are .beta.-methylumbelliferyl 
galactoside, resorufin galactoside, fluorescein digalactoside, and 
Naphthol AS-BI galactoside. Fluorescent substrates for detection of 
glucuronidase activity include 4-methylumbelliferyl .beta.-D-glucuronic 
acid, resorufin .beta.-D-glucuronic acid, 4-trifluoromethylumbelliferyl 
.beta.-D-glucuronic acid, Naphthol AS-BI .beta.-D-glucuronide, and 
fluorescein mono-.beta.-D-glucuronide. 
Most glycosidase substrates have been designed to be water soluble to 
facilitate their use in aqueous solution. This hydrophilic character 
appears to retard passage of the substrate through the membrane of living 
cells. Legler & Liedtke, Glucosylceramidase from Calf Spleen, BIOL. CHEM., 
366, 1113 (1985) describe the use of fluorescent glucosidase substrates 
4-heptyl-, nonyl-, and -undecylumbelliferone in assaying 
glucosylceramidase purified from calf spleen. Legler & Liedtke note a 
preference of the enzyme for long aliphatic side chains in the aglycon. 
The longer alkyl chains, however, appear to interfere with fluorescence 
and solubility in the absence of detergents. Legler & Liedtke do not use 
resorufin derivatives or other derivatives that have long wavelength 
absorption or fluorescence emission and do not discuss the assay of 
glucosidase inside intact living cells, or in tissues. 
Although the use of fluorescent substrates is preferable to other methods, 
such as radioactivity, they are not entirely problem-free. In addition to 
the problems of cell leakage and cell entry discussed in greater detail 
below, some of the disadvantages of these substrates include fluorescence 
at a wavelength not well suited for flow cytometry (e.g. 
.beta.-methylumbelliferyl galactoside, 4-trifluoromethylumbelliferyl 
galactoside, naphthol AS-BI galactoside), pH sensitivity or pH change 
necessary to exhibit maximal fluorescence (e.g. .beta.-methylumbelliferyl 
galactoside), and low sensitivity or limited change in fluorescence in the 
presence of the enzyme (e.g. naphthol AS-BI galactoside). 
U.S. Pat. No. 4,812,409 to Babb et al. (1989) discloses substrates attached 
to a blocked phenalenone or benzphenalenone fluorescent moiety, which when 
cleaved from the substrate by hydrolysis at a pH of 9 or less, releases a 
fluorescent moiety excitable at a wavelength above about 530 nm with 
maximum fluorescent emission at a wavelength of at least about 580 nm. 
There is no indication in the patent that the substrate is non-toxic to 
living cells or that the fluorescent product(s) do not leak from cells 
after enzymatic turnover, and are thus amenable to in vivo detection of 
enzyme activity. 
Fluorescent compounds that are not enzyme substrates have been used to 
detect transformed cells. David W. Galbraith, active in research involving 
fluorescent dyes used with plant cells, identified four dyes used to label 
plant cell populations prior to gene fusion in Selection of Somatic Hybrid 
Cells by Fluorescence-Activated Cell Sorting, in CELL CULTURE AND SOMATIC 
CELL GENETICS OF PLANTS 1, 433, ch. 50 (1984). The four dyes, octadecanoyl 
aminofluorescein (F18), octadecyl rhodamine B (R18), fluorescein 
isothiocyanate (FITC) and rhodamine isothiocyanate (RITC) were nontoxic to 
the cells (although FITC and RITC are toxic at high levels) and did not 
leak from the cells during culturing (p. 434). The fluorescein dye was 
added to one cell population, the rhodamine dye to the other. After gene 
fusion, the presence of both dyes was used to detect the heterokaryons. 
Galbraith noted (p. 442) that the lipophilic F18 and R18 dyes were 
observed localized in the membranes of cells, whereas FITC and RITC were 
distributed through the cytoplasm. Neither set of dyes was used to 
identify specific enzymes associated with the cells. 
An article by Nolan, et al., Fluorescence-activated cell analysis and 
sorting of viable mammalian cells based on .beta.-D-galactosidase activity 
after transduction of Escherichia coli lacZ, CELL BIOLOGY 85, 2603 (1988) 
describes the measurement of galactosidase activity in lacZ.sup.+ 
transformed cells using fluorescein di-.beta.-D-galactopyranoside (FDG). 
The use of FDG to measure promoter activity is described in another 
article, Ikenaka, et al., LABORATORY METHODS: Reliable Transient Promoter 
Assay Using Fluorescein-di-.beta.-D-galactopyranoside Substrate, DNA & 
CELL BIOL. 9, 279 (1990). FDG has excellent properties for these purposes. 
Despite the advantages; however, there are at least two major drawbacks 
that are recognized in the use of FDG and all other fluorescent substrates 
for the analysis and selection of transformed cells. 
First, it is difficult to get the substrate through the outer cell membrane 
without disrupting the cell. The permeability properties of available 
substrates, such as 4-methyl umbelliferyl glucuronide, require detection 
of GUS activity in plant tissue homogenate or cell extracts. Such 
destructive assay conditions will certainly cause inaccuracy and set 
limitations when an investigator is looking for relatively rare events 
such as the regulation of transcription and/or translation or 
transformation with a chimeric gene. Nolan, et al., using FDG, reduced 
this problem for cells in suspension by using brief hypo-osmotic or 
hypotonic shock. Ikenaka, et al. used the same technique. To use 
hypo-osmotic shock, the cells are placed in a hypotonic solution causing 
the membrane to swell. Swelling of the membrane results in 
permeabilization of the substrate so that it enters the cell. If the cell 
stays too long in the dilute solution, however, it ruptures. Removal of 
the cells from the dilute solution must be carefully timed to maximize 
entry of the substrate yet minimize cell loss. 
A second and more important drawback of known fluorescent substrates is the 
problem of cell leakage. For example, following enzymatic hydrolysis of 
FDG, the resulting product (fluorescein) rapidly leaks out of the cell. 
See, e.g., FIG. 3(c) and FIG. 4; see also, Ikenaka, et al.; Nolan, et al. 
Commonly half of the fluorescein leaks from the cell in about 10 minutes 
at about 37.degree. C. Fluorescent products derived from other substrates, 
including all .beta.-methylumbelliferyl and resorufin glycosides have been 
found to leak from cells under in vivo conditions even faster than 
fluorescein. Nolan et al., and Ikenaka, et al., working with FDG, were 
able to suppress the leakage of fluorescein by quickly cooling their cells 
to 4.degree. C. Cooling the cells, however, also reduces the enzyme 
turnover rate significantly, and is not desirable when working with whole 
living organisms. Leakage of the fluorescent product, even at 4.degree. C. 
makes enzyme activity quantitation particularly difficult. It also 
increases the difficulty in differentiating weakly expressing gene 
positive cells from the background fluorescence of negative cells. 
Neither hypo-osmotic shock loading nor sudden cooling well below 
physiological temperatures is suitable for measuring enzyme activity in 
transformed living cells, tissues or organisms under physiological 
conditions (typically 37.degree. C.) such as during development and cell 
division. A copending application, LIPOPHILIC FLUORESCENT GLYCOSIDASE 
SUBSTRATES (Ser. No. 07/623,600, filed Dec. 7, 1990), describes lipophilic 
derivatives of fluorescein glycosides and related compounds that yield 
green fluorescent products suitable for detection of glycosidase activity 
in living cells. Both classes of substrates are permeant to cells under 
physiological conditions, are not toxic to living cells, and are 
nonfluorescent until specifically hydrolyzed by the glycosidase enzyme. 
Both of their fluorescent products are readily detected in single cells 
and even within specific organelles of single cells. Substrates of either 
class can be selected that yield fluorescent products that are very well 
retained in the original cell with no or minimal leakage or transfer 
between marker-positive and marker-negative cells, even through cell 
division. 
The two inventions differ, however, in the use of a different fluorophore. 
As a result, the compounds disclosed in the co-pending application have 
emission that is maximal at wavelengths less than 550 nm. The resorufin 
derivatives of this invention, in contrast, have orange to red emission 
that is maximal above 550 nm. This contrast permits, for example, the 
simultaneous detection of two different enzymes or a combination of 
activity of one enzyme and a second fluorescent label in a mixture of 
cells or even in a single cell. It also makes possible the detection of 
different genetic elements under regulation of different promoters in 
cells or tissues. 
Hofman & Sernetz, Immobilized Enzyme Kinetics Analyzed by Flow-Through 
Microfluorimetry, ANALYTICA CHIMICA ACTA 163, 67 (1984) describes the 
synthesis and enzymatic properties of resorufin .beta.-D-galactopyranoside 
but does not describe lipophilic alkylated derivatives of this compound or 
the measurement of enzyme activity inside live cells. Attempts have been 
made to use resorufin .beta.-D-galactoside to detect incorporation of the 
lacZ gene in cells, but were unsuccessful as the result of leakage of the 
hydrolysis product from the cell. 
Leakage of 7-hydroxyresorufin resulting from the use of 7-ethoxy- and 
7-pentoxyresorufin in an assay for monooxygenase activity is described by 
Reiners, et al., Fluorescence Assay for Per-Cell Estimation of Cytochrome 
P-450-Dependent Monooxygenase Activities in Keratinocyte Suspensions and 
Cultures, ANALYT. BIOCHEM. 188, 317 at p. 322 (1990). Leakage of metabolic 
products of other substrates is also described. Although Reiners, et al. 
note improved retention of 7-hydroxyresorufin in hepatocytes, only 29% of 
the intracellular product remained after 35 minutes. Furthermore, there is 
no indication that the activity of the enzyme they were studying (which is 
not a glycosidase) can be determined on a single cell basis. 
U.S. Pat. No. 3,731,222 to Drexhage (1973) describes a resorufin derivative 
with a lower alkyl (1-6 carbons) for use as a laser dye. There is no 
indication in the reference that an alkylated resorufin could be attached 
to a carbohydrate moiety for use as a glycosidase substrate, nor that that 
alkyl group(s) would provide any advantage to such a substrate. 
Resorufin glycosides with lower alkyl (1-5 carbons) substituents for use as 
glycosidase substrates are described in German Patent No. DE 3411574A1 to 
Klein, et al., 1985. The patent does not describe the use or the 
advantages of alkyl residues of more than 5 carbons to increase the time 
that the fluorescent hydrolysis product is retained in intact cells. In 
fact, the patent recites that alkyl substituents with 1-3 carbons are 
preferred.

SUMMARY OF THE INVENTION AND DESCRIPTION OF PREFERRED EMBODIMENTS 
This invention describes a class of novel fluorogenic substrates for 
measuring the presence and activity of a glycosidic enzyme and whose 
hydrolysis products have orange to red fluorescence emission that 
contrasts with the green fluorescent emission of fluorescein. 
The substrates are derivatives of resorufin of the general formula: 
##STR2## 
wherein Gly is a carbohydrate bonded to resorufin by a glycosidic linkage; 
where at least one of substituents R.sub.1, R.sub.2, R.sub.4, R.sub.6, 
R.sub.8, and R.sub.9 is a first lipophilic residue containing from about 6 
to about 22 carbon atoms of the formula --L(CH.sub.2).sub.n CH.sub.3, 
where n is greater than 3 and less than 22, and where L is a methylene 
--CH.sub.2 --, an amide --NHCO--, sulfonamide --NHSO.sub.2 --, 
carboxyamide --CONH--, carboxylate ester --COO--, urethane --NHCOO--, urea 
--NHCONH--, or thiourea --NHCSNH--; and where the remainder of the 
substituents R.sub.1, R.sub.2, R.sub.4, R.sub.6, R.sub.8, and R.sub.9, 
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'(CH.sub.2).sub.m 
CH.sub.3, where m is less than 22, and where L' is methylene --CH.sub.2 
--, an amide --NHCO--, sulfonamide --NHSO.sub.2 --, carboxyamide --CONH--, 
carboxylate ester --COO--, urethane --NHCOO--, urea --NHCONH--, or 
thiourea --NHCSNH--. Specifically the subject substrates contain the 
7-hydroxy-3H-phenoxazin-3-one structure of resorufin in which one of the 
hydroxyl residues is converted to a glycoside derived from the sugar; and 
substituted by at least one lipophilic or hydrophobic fatty alkyl residue 
containing from about 6 to about 22 carbon atoms. 
The two nonheterocyclic aromatic rings of the phenoxazine ring portion of 
resorufin may be further substituted. In one embodiment of the invention, 
the ring portion is substituted by one to four halogen atoms, which may be 
the same or different. The preferred halogens are bromine, chlorine, or 
iodine. The one or more halogens are substituted at the 2, 4, 6, or 8 
positions, or combinations thereof. 
The carbohydrate substituent (Gly) and its linkage to resorufin provide the 
resorufin-derived substrate with its specificity for a particular 
glycosidic enzyme. Gly is a hydrolyzable monovalent moiety, derived by 
removal of an anomeric hydroxyl group from a mono- or oligosaccharide, 
which is linked to the 7-oxo-position on the phenoxazine fluorophore. The 
linkage can be an .alpha.- or .beta.-glycosidic linkage. The attachment of 
Gly to the resorufin fluorophore changes the spectral 
(excitation/emission) properties of the fluorophore. Typically hydrolysis 
of Gly by action of a specific enzyme results in a shift in the absorbance 
of the hydrolysis product to longer wavelengths that are not absorbed or 
are minimally absorbed before the removal of Gly, and an increase in 
fluorescence. Quantitative or qualitative detection of the hydrolysis 
product permits detection of the corresponding enzyme. Any of the enzymes 
listed in Table 1, and similar carbohydrate substrates derived from other 
sugars or modified sugars for which hydrolytic enzymes that hydrolyze the 
glycosidic linkage exist, could be detected using resorufin glycoside 
substrates derived from the appropriate sugar specific for the enzyme. 
The first lipophilic residue, preferably an unbranched fatty alkyl residue, 
contains from about 6 to about 22 carbon atoms. Those substrates with the 
longer alkyl chain are better retained by the cell. When it is desired 
that the fluorescent product be strongly retained within a cell, the 
preferred alkyl residue contains about 12-18 carbons. Without wishing to 
be bound by theory, applicants believe that the hydrophobic or lipophilic 
residue facilitates passive diffusion of the substrate through the cell 
membrane and enhances retention of the fluorescent hydrolysis product, in 
a form where it is preferentially bound to the cell membranes rather than 
being free in the cellular cystosol. Binding of the product to a cell 
membrane considerably enhances retention of the fluorescent product by the 
cell and considerably reduces transfer of the resulting dye between marker 
positive and marker negative cells. 
Preferably, each lipophilic group is directly linked to the resorufin ring, 
in which case L or L' is a single methylene --CH.sub.2 --. Alternatively, 
other linking groups in which the alkyl group is bonded to the resorufin 
through an amide --NHCO--, sulfonamide --NHSO.sub.2 --, carboxyamide 
--CONH--, carboxylate ester --COO--, urethane --NHCOO--, urea --NHCONH--, 
or thiourea --NHCSNH-- also result in products with similar utility where 
they do not materially effect the fluorescence spectra. From ease of 
synthesis and availability of synthetic precursors it is preferred that 
the first lipophilic residue is linked to the resorufin derivative at the 
2 position. Attachment at other positions, however, or use of more than 
one lipophilic residue results in products of similar utility. Where two 
lipophilic residues are used, preferably the combined total length of the 
two alkyl chains is between 12 and 22 carbons. From ease of synthesis the 
two lipophilic residues are preferentially at equivalent positions on the 
resorufin structure, such as in the R.sub.2 and R.sub.8 positions. 
The starting material for preparation of the lipophilic resorufin glycoside 
is a lipophilic resorufin ("LR"), or its respective halogenated 
derivative. The LR starting material commonly includes at least one 
lipophilic group containing from 6 to 22 carbon atoms. Most commonly, the 
unsymmetrical alkyl resorufin is synthesized by condensation of one mole 
of an appropriately substituted resorcinol with a nitrosoresorcinol (see 
Example 2). Alternatively, for instance, lipophilic resorufin carboxamides 
can be synthesized by reaction of activated resorufin carboxylic acids 
such as those sold by Boehringer Mannheim Corporation (Indianapolis, Ind. 
USA) with an aliphatic amine containing from 4 to 22 carbon atoms. 
Halogenation can also be accomplished subsequent to formation of the 
lipophilic resorufin dye such as by bromination with liquid bromine. 
From the halogenated or non-halogenated LR starting material, a protected 
glycoside intermediate is prepared in a multi-step process. Glycosylation 
using a modified Koenigs-Knorr methodology involves treatment of a LR with 
a soft acid catalyst, an activated protected carbohydrate (APC) 
derivative, and a non-nucleophilic base, under anhydrous conditions. 
Symcollidine or quinoline are a preferred non-nucleophilic base and silver 
carbonate is a representative soft acid catalyst. 
The APC will contain one or more sugars with an activating group at the 
anomeric position of the sugar to be attached to the LR. Typically the APC 
is a halogenated sugar, where a halogen is the activating group at the 
anomeric position. Depending on the reaction conditions, the sugar 
involved, or the anomeric isomer required, other activating groups at the 
anomeric position of the APC can be used, most commonly 
trichloroacetimidate, thiophenyl, or acetate. 
Using one or more equivalents of an APC, a glycoside intermediate is 
formed. Because of the asymmetry typical of LR but recognizing the two 
resonance structures of the hydroxyphenoxazinone dye, there are two 
possible glycoside products. The predominant product when the lipophilic 
substituent (--L(CH.sub.2).sub.n CH.sub.3) is in the 2 position appears to 
be the 2--L(CH.sub.2).sub.n CH.sub.3 --3-oxo-7-O-glycoside. 
After isolation of the protected glycoside intermediate, the protecting 
groups are removed from the protected glycoside using processes 
appropriate to the protecting group(s) present. For example, catalytic 
sodium methoxide is used for removal of acetylated alcohols, aqueous 
lithium hydroxide for methyl esters, etc. Final purification and 
crystallization yields the glycoside substrate. 
Modifications of the above procedures will be obvious to a chemist skilled 
in the art of organic chemistry. 
The substrate is useful for evaluating a glycosidic enzyme, including the 
evaluation of a variety of detection, localization, monitoring, and 
quantitative parameters. The substrate provides the desired permeability, 
retention, detectability, specificity and lack of toxicity to detect 
activity of a particular gene in single cells containing the gene, by 
detecting the enzyme activity that results from the presence of the gene. 
In combination with a compound or compounds having an emission maximum 
that is less than 550 nm, the resorufin derivatives of this invention 
having an emission maximum that is greater than 550 nm can be used to 
simultaneously detect two different enzymes in a mixture of cells or even 
in a single cell. These characteristics permit analysis, sorting and 
cloning of the cells and monitoring of cell development in vitro and in 
vivo. 
To detect the presence of a glycosidic enzyme or enzymes in a substance, a 
substrate is selected that is specific for the enzyme to be detected. The 
sugar of the carbohydrate substituent and its linkage to resorufin 
provides the substrate with specificity for the particular glycosidic 
enzyme. The substrate comprising the sugar specific for the enzyme 
appropriately linked to the lipophilic resorufin derivative is combined 
with the substance being evaluated. 
The substrates are particularly effective in detecting glycosidic enzyme 
activity inside a living cell. The fluorescent enzymatic hydrolysis 
products are specifically formed and adequately retained inside living 
cells, and are nontoxic to the cells. Furthermore, the substrates can 
penetrate the cell membrane under physiological conditions. Increasing the 
lipophilicity of the fluorescent product by addition of extra methylene 
groups improves the product retention while decreasing the number of 
methylene groups accelerates the rate of product clearance from the cell. 
To detect the presence of the glycosidic enzyme in living cells, the 
substrate is added to the standard culture medium of the cells being 
evaluated. If the cells being evaluated have been subjected to 
transformation or other disruptive procedures, the cells should be allowed 
to stabilize before adding the fluorescent substrate. Typically 12-24 
hours rest is sufficient for the cells to stabilize. For adherent cells, 
growing them on several cover glasses inside a Petri dish will enable cell 
samples to be taken out at any designated time during the incubation for 
examination. 
For ease of addition to the culture medium, the substrate is dissolved in 
solution to make a labeling reagent. Preferably the substrate is dissolved 
in a polar, aprotic organic solvent sufficiently dilute not to disrupt 
cell membrane structure when the labeling reagent is added to the culture 
medium. A 10 mM solution of substrate in 20% DMSO yields an effective 
stock solution of the labeling reagent that remains stable for at least a 
month if stored 4.degree. C. in the absence of light. 
The labeling reagent is added to culture medium of the cells being 
evaluated so that the cells incubate further in a labeling culture medium. 
Some plant cells may only be penetrable as protoplasts, i.e. after 
alteration or removal of the outer membrane. The labeling reagent is added 
to the culture medium in an amount sufficient to yield a concentration of 
about 50 to about 250 .mu.M of substrate, preferably about 100 to about 
200 .mu.M of substrate, in the labeling culture medium. A sterilized 
labeling reagent is preferred so that the substrate will be taken up by 
cells in a normal physiological condition during their growth. Usually the 
labeling reagent is first added to fresh culture medium to form a fresh 
labeling culture medium. The fresh labeling culture medium is then 
filter-sterilized by passing through a low protein-binding, sterilizing 
filter, such as a 0.20 .mu.m pore size ACRODISC.TM. filter. Spent culture 
medium is removed and sterilized fresh labeling medium is added to 
culturing cells. 
The cells are incubated in the labeling culture medium for sufficient time 
for the substrate to enter the cells and to react with the enzyme to yield 
a fluorescent detection product. Commonly this time is about 1-10 hours. A 
sample of the cells is observed under a microscope equipped with a filter 
for visualizing fluorescence. Preferably, the sample of cells is exposed 
to a radiation source at a wavelength of between about 488 and about 550 
nm and the emission is detected beyond 550 nm. Washing the cell sample 
with normal culture medium before examination will reduce background 
fluorescence from broken cells or decomposed substrate. 
The fluorescent detection product is only observed inside cells producing 
the specific glycosidic enzyme. The substrates can be used to detect 
activity of the enzyme in any medium, cell-free or not, that contains the 
enzyme. The glycosidase enzyme being evaluated inside cells may be present 
endogenously, or present as a result of manipulation of the cell's genome, 
such as by transformation. In one embodiment of the invention, the 
presence of the fluorescent detection product is used to indicate the 
successful insertion of foreign genetic material responsible for the 
production of the enzyme. In another embodiment of the invention, the 
substrates are used to monitor whether and to what extent a glycosidic 
enzyme has been successfully affected by promoters and/or repressors for 
the enzyme. In such embodiments involving evaluation of genetic 
information, appropriate cells are selected for incubation with a suitable 
substrate specific for the enzyme useful to evaluate such genetic 
information. Appropriate cells are selected or prepared by means generally 
known in the art. In the case of evaluating the response of an enzyme with 
respect to an inducible promoter, the appropriate inducer is subsequently 
added to the incubated cells. 
Regulation of gene expression by inducible upstream elements can be 
evaluated using a suitable reporter gene and a substrate specific for the 
product of the reporter gene. Cells containing appropriate genetic 
material useful for the evaluation of such regulation are selected or 
prepared according to methods known in the art (Emilie, Peuchmaur, Barad, 
Jouin, Maillot, Couez, Nicolas, Malissen, EUR. J. IMMUNOL. 19, 1619 (1989) 
incorporated herein by reference). In yeast, genes that are expressed in a 
cell-type specific manner and genes whose transcription increases in 
response to peptide mating pheromones are known. Expression levels of such 
controlled genes can be related to substrate turnover levels by using lacZ 
as a reporter gene. The general procedure involves fusing the gene of 
interest adjacent to the reporter gene and using this construct, usually 
on a replicating plasmid, in transformation of selected cells. After 
appropriate growth of the selected cells, the cells are assayed for 
glycosidase activity using the substrates. The level of activity can be 
analyzed by fluorescence microscopy or video-fluorescence image analysis 
at the single cell level by methods known in the art, such as described in 
Jarvis, et al., MOLEC. & CELL. BIOL. 8, 309 (1988), incorporated herein by 
reference. Detection of such induction is facilitated by the improved cell 
retention properties of the lipophilic galactosidase substrates. 
For example, yeast promoters are made up of two components: (1) an upstream 
activation sequence (UAS) that confers the characteristic regulatory 
pattern of the gene and (2) a TATA sequence. The full length of UAS will 
drive a high level of transcription of genes encoded downstream. It is 
possible to examine whether the complete sequence of the UAS gene is 
important for the regulation of the transcription of downstream elements. 
Due to the manipulation of the promotor sequence, different levels of 
expression of the lacZ gene in each recombinant yeast strain will result 
in varied .beta.-galactosidase activities in the transformed yeast cells. 
Alternatively, the substrate can be used to evaluate an enzyme added to 
cells exogenously, for example, by a glycosidase enzyme covalently bound 
to a protein such as an antibody to form an enzyme-protein complex, 
usually as an enzyme conjugate, that binds to or enters the cell. The 
cells are incubated in a complex-containing medium. The incubated cells 
are subsequently washed and transferred to a labeling medium containing 
substrate specific for the enzyme. After incubating the cells in the 
labeling medium sufficiently long for the substrate to enter the cells, a 
portion of the cells is evaluated for fluorescence as above. The presence 
or absence of a fluorescent detection product is used to determine whether 
or not the conjugate or complex has bound to or entered the cells. 
Enzymatic generation of the fluorescence through use of a glycosidase 
conjugate of, for instance, an antibody to a cell surface receptor, 
provides a means of obtaining amplification of the fluorescence signal 
beyond that normally obtained using direct conjugates of fluorescence dyes 
with an antibody or protein that binds to or enters the cell. 
Because of the production of a unique reddish fluorescence, the substrate 
can be used in conjunction with another substrate of a dye with a 
different color fluorescence yielding a multicolor system for evaluating 
two different analytes. For such an evaluation of two different enzymes 
present in a sample, the sample is combined with 1) the novel resorufin 
substrate specific for one of the enzymes of interest and having an 
emission maximum greater than about 550 nm; and 2) a second substrate 
specific for a different enzyme having an emission maximum less than about 
550 nm. Where the sample is living cells, the second substrate should be 
equally well-retained in and nontoxic to such cells, preferably the 
substrate described in the co-pending application LIPOPHILIC FLUORESCENT 
GLYCOSIDASE SUBSTRATES (U.S. Ser. No. 07/623,600, filed Dec. 7, 1990) 
(incorporated herein by reference). After a sufficient period for reaction 
of both substrates, the cells are washed to remove fluorescent 
decomposition products and analyzed using a suitable instrument such as a 
microscope or flow cytometer commonly employing optical filters to 
separate the two colors of fluorescence. 
The invention can be used to sort cells according to their glycosidic 
enzyme activity. As the substrates generate fluorescence products that are 
well cell-retained upon the action of glycosidic enzyme inside cells, an 
easy and convenient selection/collection of gene-transferred cells of 
interest is available through a fluorescence-activated cell sorting (FACS) 
technique. In principle, the substrates are loaded into a cell and then 
converted to fluorescent products to an extent determined by the 
glycosidic enzyme activity (or amount) inside the cell. It has been 
demonstrated (e.g. Nolan, et al.) that modern flow cytometers can sort the 
cells by their fluorescence signal amplitude therein and, in turn, by cell 
enzyme activity. 
For the sorting of a cell being investigated, the cell is loaded with the 
glycosidase substrate and incubated to obtain a sufficient fluorescence 
signal which corresponds to the cell glycosidic enzyme activity being 
evaluated. The procedure for cell staining under physiological conditions 
has been described above. To discriminate cells expressing low enzyme 
activity from cells that express high levels of enzyme activity, it is 
preferred to limit the period of incubation to the minimum necessary to 
achieve the desired resolution. The appropriate balance between sufficient 
and excessive fluorescence development varies with the substrate 
concentration, the cell type in terms of membrane permeability for both 
the substrate and the hydrolysis product, and the distribution of enzyme 
activity in cell organelles. In a mammalian cell that contains a 
glycosidic enzyme, 30 to 60 minute incubation rather than hours or 
overnight, is recommended when a substrate concentration of 10 to 50 .mu.M 
is used to stain the cell. Other known automated methods of cell sorting 
may also be used. There is extensive literature published on cell analysis 
and sorting instrumentation for flow cytometry. Post-sorting cell 
examination by other techniques such as assay of enzyme activity or 
cytochemistry may be needed to confirm the sorting results or to confirm 
the success of the sorting. 
The labeling reagent shows no detectible cytotoxicity. Cells incubated with 
C.sub.12 -Resorufin-Gal (100 .mu.M) in the culture medium exhibited 
equivalent population doubling time as in controls. Cells preincubated in 
the labeled culture medium for 24 hours could be subcultured and the cells 
of the second generation were normal. The cells grown in C.sub.12 
-Resorufin-Gal (100 .mu.M) for 4 days had normal morphology and remained 
viable. 
Transfer of dye between cells was tested in a semi-confluent mixture of 
enzyme expressing (lacZ+) and nonexpressing (lacZ-) cells. Visualization 
of adjacent (visible morphological contacts formed) fluorescent and 
nonfluorescent cells indicated that the fluorescent dye did not transfer 
between cells. 
The following examples are included by way of illustration and not by way 
of limitation. 
EXAMPLE 1 
Preparation of Resorcinol Containing an 18-carbon Alkyl Chain at the 
4-position 
The following compound was prepared: 
##STR3## 
Preparation of 4-octadecanoylresorcinol 
Stearic acid (200 g, 0.70 mole) is dissolved in boron trifluoride etherate 
(275 mL). Resorcinol (100 g, 0.91 mole) is added as a solution in BF.sub.3 
etherate (75 mL) and this mixture is heated to reflux with stirring for 4 
hours. The solution is cooled to room temperature, diluted with CHCl.sub.3 
(300 mL) and poured into water (700 mL at 0.degree. C.). The organic layer 
is separated and washed with saturated sodium chloride solution 
(1.times.200 mL), dried over anhydrous Na.sub.2 SO.sub.4, filtered and 
evaporated under reduced pressure. This residue is crystallized from 
hexanes (300 mL) and then recrystallized from ethanol (300 mL) to yield 
15.8 g tan crystals. 1H NMR (DMSO-d.sub.6) .delta.:0.87(t,3H); 
1.22(m,28H); 1.62(m,2H); 2.86(t,2H); 3.82(s,2H); 6.22(d,1H); 6.33(dd,1H); 
7.67(d,1H). m.p.=78.degree. C.; SiO.sub.2 -t.l.c. (2:8 EtOAc:CHCl.sub.3) 
R.sub.f =0.88 
Preparation of 4-octadecylresorcinol 
Stearoylresorcinol (0.32 g, 0.85 mmole) and anhydrous hydrazine (0.25 mL) 
are combined in diethylene glycol (20 mL). After the stearoylresorcinol 
dissolves, potassium hydroxide pellets (0.1 g, 1.78 mmole) are added, and 
the solution is refluxed at 100.degree. C. for 1 hour (the presence of the 
hydrazone is confirmed by SiO.sub.2 t.l.c. (R.sub.f =0.45, 9:1 CHCl.sub.3 
:MeOH), with H.sub.2 O removed through use of Dean-Stark type distillation 
until the temperature reaches 115.degree. C. The temperature is then 
elevated to 180.degree.-190.degree. C. for an additional 3 hours. The 
resulting diethylene glycol solution is cooled to room temperature, 
acidified using 0.5M H.sub.2 SO.sub.4 solution (approx. 8 mL, to pH 2-3), 
diluted with water (250 mL), and the aqueous solution is extracted with 
ethyl acetate (3.times.100 mL). The combined ethyl acetate layers are 
washed with water (2.times.100 mL) and saturated NaCl solution 
(1.times.100 mL) and dried over anhydrous Na.sub.2 SO.sub.4. 
The organic layer is evaporated under reduced pressure and dried in vacuo 
to give an off-white powder (230 mg, 74%). .sup.1 H NMR (CDCl.sub.3) 
.delta.:6.97(d,1H); 6.33(dd,1H); 6.31(d,1H); 4.65(s,1H); 4.57(s,1H); 
2.50(t,2H); 1.56(m,2H); 1.24(br s,30H); 0.88(t,3H). 
m.p.=98.degree.-101.degree. C. SiO.sub.2 -t.l.c. 1:9 MeOH:CHCl.sub.3) 
R.sub.f =0.30. 
EXAMPLE 2 
Preparation of a Resorufin Containing a Twelve Carbon Fatty Alkyl Chain at 
the 2-position 
The following compound was prepared: 
##STR4## 
Preparation of 2-dodecyl-7-hydroxy-3H-phenoxazine-3-one 
("dodecylresorufin") 
A solution of 4-dodecylresorcinol (8.5 g, 30.53 mmole) (Aldrich Chemical 
Company) and 4-nitrosoresorcinol (5.0 g, 35.9 mmole) (Aldrich) in 
concentrated sulfuric acid (100 mL) is heated to 67.degree. C. with 
stirring for 5 hours. The reaction mixture is allowed to cool to room 
temperature, poured into ice-water (1800 mL) and filtered. The resulting 
precipitate is washed with water until the filtrate is neutral to pH 
paper. This precipitate is dissolved in chloroform and the combined 
aqueous filtrates are back-extracted with chloroform (6.times.250 mL) to 
recover additional product. The combined chloroform layers are dried over 
anhydrous sodium sulfate and evaporated to a dark purple solid (2.55 g), 
homogenous by SiO.sub.2 -t.l.c. (45:5:1 chloroform:methanol:triethylamine) 
R.sub.f =0.69. An additional 5.58 g of product is obtained by 
neutralization of the aqueous filtrates above with 8M KOH and extraction 
with chloroform (total yield=8.13 g, 68%). .sup.1 H NMR (d.sub.6 -DMSO) 
.delta.:7.57(d,1H); 7.27(s,1H); 6.79(D,1H); 6.66(s,1H); 6.23(s,1H); 
4.14(c,1H); 2.46(t,2H); 1.52(c,2H); 1.23(c,18H); 0.85(c,3H). 
EXAMPLE 3 
Preparation of a Substrate Having a Galactopyanoside Blocking Group at the 
8-position and a Eighteen Carbon Alkyl Chain at the 2-position of 
Resorufin 
The following compound was prepared: 
##STR5## 
Preparation of 2-octadecyl-7-(2,3,4,6-tetra-O-acetyl 
.beta.-D-galactopyranosyloxy)-3H-phenoxazin-3-one 
A mixture of 7-hydroxy-2-octadecyl-3H-phenoxazine-3-one 
("octadecylresorufin) (220 mg, 0.47 mmole), powdered, activated 4.ANG. 
molecular sieves (0.5 g), sym-collidine (125 .mu.L, 0.95 mmole), and 
silver carbonate (155 mg, 0.56 mmole) in dry dichloromethane (20 mL) is 
allowed to stir at room temperature, under an atmosphere of dry nitrogen 
gas, in the dark for 1 hour. To this mixture is added 
1-bromo-2,3,4,6-tetra-O-acetyl-.alpha.-D-galactopyranoside (389 mg, 0.95 
mmole), slowly with stirring, and the resulting heterogenous solution 
allowed to stir as above for 72 hours. After this time the reaction 
mixture is filtered through a pad of diatomaceous earth. The precipitate 
is washed with chloroform (5.times.10 mL) and the combined organic 
filtrates are extracted with 1M aqueous HCl (1.times.75 mL), saturated 
aqueous sodium bicarbonate solution (1.times.75 mL), 1M sodium thiosulfate 
(1.times.75 mL), and water (1.times.75 mL). The organic layer is dried 
over anhydrous sodium sulfate, filtered, evaporated under reduced 
pressure, and dried in vacuo to an orange-brown powder (440 mg). An 
analytical sample of the title compound can be isolated by preparative 
t.l.c. (20.times.20 cm SiO.sub.2 plate; eluent=9:1 
chloroform:ethylacetate) with the bright-orange product eluting at the 
highest R.sub.f (0.44), (50 mg from 200 mg applied). 
Preparation of 
2-octadecyl-7-(.beta.-D-galactopyranosyloxy)-3H-phenoxazin-3-one (C.sub.18 
-Resorufin-Gal) 
A suspension of dry 2-octadecyl-7-(2,3,4,6-tetra-O-acetyl 
.beta.-D-galactopyranosyloxy)-3H-phenoxazin-3-one (50 mg, 0.06 mmole) in 
anhydrous methanol (40 mL) is cooled to 0.degree. C. in an ice-bath while 
under an atmosphere of dry nitrogen gas. A solution of freshly prepared 
sodium methoxide in methanol is added (200 .mu.L, 1.13M solution) and this 
mixture is allowed to stir at 0.degree. C. for 2 hours then at ambient 
temperature for 3 hours. The red product is filtered and washed with 
methanol. The resulting red-orange solid is dried in vacuo (28 mg, 71%). 
The filtrate is neutralized with Amberlite IRC 50(H+) resin (pH 4) and 
filtered, with the resin being washed with methanol (5.times.10 mL). The 
combined filtrates are evaporated to dryness and dried in vacuo to yield a 
second crop of product (8 mg, 91% total) T.l.c. (SiO.sub.2) (8:2 ethyl 
acetate:methanol) R.sub.f =0.33. .sup.1 H-n.m.r.(d.sub.6 -DMSO) .delta.: 
7.77(d,1H); 7.30(s,1H); 7.13(s,1H); 7.10(d,1H); 6.27(s,1H); 
5.02(d,1H,H-1); 3.72(d,1H); 3.70-3.25(m,5H); 2.48(t,2H); 1.52(m,2H); 
1.26(m,30H); 0.86(t,3H). 
EXAMPLE 4 
Preparation of a Substrate Containing a Twelve Carbon Fatty Alkyl Chain at 
the 2-position and a .beta.-D-glucuronic Acid at the 7-position of 
Resorufin 
The following compound was prepared: 
##STR6## 
Preparation of the protected glucuronide of dodecyl resorufin 
2-dodecyl-7-hydroxy-3H-phenoxazine-3-one (2.0 g, 5.2 mmole) is dissolved in 
quinoline (50 mL), containing anhydrous CaSO.sub.4 powder (5 g), silver 
carbonate (2.86 g, 10 mmole) and 1-bromo-2,3,4-tri-O-acetyl 
.alpha.-D-glucopyranuronic acid, methyl ester (4.20 g, 10 mmole). This 
mixture is stirred under anhydrous conditions in the dark at ambient 
temperature for 72 hours. The reaction solution is filtered through a pad 
of diatomaceous earth and the filter bed is washed with ethyl acetate (200 
mL). The ethyl acetate solution is back-extracted with cold 3M 
hydrochloric acid (1.times.200 mL, 0.degree. C.) followed by saturated 
sodium bicarbonate solution (4.times.200 mL) and water (4.times.100 mL) 
until very little red color appears in the aqueous layer. The solution is 
dried over anhydrous sodium sulphate, evaporated, and dried in vacuo (3.36 
g). The crude product is purified by silica gel column chromatography 
using 1% methanol in toluene as eluent to give a dark red wax (0.63 g, 
17%). 
Preparation of 2-dodecylresorufin glucuronide methyl ester 
The protected dodecyl resorufin glucuronide above (0.63 g, 0.89 mmole) is 
suspended in anhydrous MeOH (60 mL) and this mixture is stirred under dry 
nitrogen at 0.degree. C. while sodium methoxide/methanol solution is added 
(0.50 mL of a 0.99M solution). The pH is maintained between 8 and 9 by 
adding more NaOMe/MeOH solution as needed. This solution is stirred for 4 
hours, neutralized to pH 5 using washed, dry Amberlite IRC 50 (H+) ion 
exchange resin, filtered, evaporated and dried in vacuo. 
Preparation of 2-dodecylresorufin .beta.-d-glucuronide (C.sub.12 -Resorufin 
GlcU) 
The above crude sample of the above 2-dodecylresorufin glucuronide methyl 
ester is dissolved in a 5:1 acetonitrile:dichloromethane solution (4 mL). 
A freshly prepared lithium hydroxide solution (10.2 mL of a 0.08M 
solution) is added slowly to the flask and stirring is continued at 
0.degree. C. for 30 minutes and at room temperature for 2 hours. The 
solution is neutralized using Amberlite IRC 50 (H+) ion exchange resin, 
filtered and evaporated to a dark red oil which is purified on by 
reverse-phase column chromatography on Sephadex.TM. LH-20 prepared in and 
eluted with water to give 206 mg of the pure free acid (0.359 mmoles, 40% 
from the fully protected material). m.p.&gt;250.degree. C. (d): SiO.sub.2 
-t.l.c. (7:1:1:1 ethyl acetate:methanol:water:acetic acid); R.sub.f =0.46. 
EXAMPLE 5 
Labeling of LacZ+Cells with the Fluorogenic Substrate 
2-Dodecyl-7-(.beta.-D-Galactopyranosyloxy)-3H-Phenoxazin-3-One (C.sub.12 
-Resorufin-Gal) 
CRE BAG 2 (lacZ+) cells were used to test the effectiveness of the 
substrate in labeling lacZ+ cells. NIH 3T3 (lacZ-) cells were used as a 
control. Both cell lines were obtained from American Type Culture 
Collection Co., Rockville, Md. The cells were grown in a humidified 
atmosphere of 5% CO.sub.2 in Dulbecco's modified Eagle's medium (DMEM) 
supplemented with 10% calf serum, 50 .mu.g/mL gentamycin, 300 .mu.g/mL 
L-glutamine and 10 mM HEPES pH 7.4 (culture medium). 
Stock solution of labeling reagent 
C.sub.12 Resorufin-Gal is dissolved in 20% DMSO to get 20 mM stock 
solution. Stock solution should be kept sealed a in brown reagent bottle 
and stored at -20.degree. C. 
Labeling culture medium 
Labeling reagent is added to fresh culture medium in an amount sufficient 
to make 100 .mu.M C.sub.12 -Resorufin-Gal labeling culture medium. 
Labeling culture medium is filter-sterilized by passing through an 
ACRODISC.TM. filter (0.20.mu. pore size). 
Examination of cells 
Cells were washed with fresh (nonlabeling) culture medium before 
examination. Cells are observed using a microscope equipped with a filter 
optimized for tetramethylrhodamine which has absorption and emission 
similar to those of resorufin. At 100 .mu.M concentration, after about 30 
minutes incubation, fluorescence is observed inside CRE BAG 2 cells but 
not in 3T3 cells. After about 2-4 hours, the fluorescence intensity in CRE 
BAG 2 cells reaches its highest level. 
EXAMPLE 6 
Analysis and Sorting of LacZ.sup.- and LacZ.sup.+ Cells by Facs Using 
Glycosidic Substrates 
The mixture of cells used for analysis and sorting (if desired) is a 
mixture of about 50-99% NIH3T3 (lacZ.sup.-) cells and about 1-50% CRE BAG 
2 (lacZ.sup.+) cells. The cell source, culturing and substrate labeling 
procedure are essentially same as EXAMPLE #5: LABELING OF LacZ.sup.+ CELLS 
WITH THE FLUOROGENIC SUBSTRATE. For positive/negative sorting, the 
substrate is incubated with cells for 5 to 6 hours to achieve a maximum 
cell fluorescence. For subclone analysis and sorting of CRE BAG 2 cells, 
the substrate incubation is limited to between 30-60 minutes to facilitate 
discrimination of low activity and high activity cells. 
Flow Cytometer Instrumentation 
Suitable instrumentation for simultaneous analysis and sorting of lacZ+ 
cells includes a FACS II.TM. cell sorter (Becton and Dickinson, Sunnyvale, 
Calif.). This instrument is typically equipped with an argon ion laser 
(488 nm, Mountain View, Calif.) and a 70 .mu.M nozzle. Cell 
autofluorescence is compensated by means of a two-color system. The 
typical cell velocity for sorting is 1000-2000 cells/second. Preferably 
the cell culture medium without substrate serves as the sheath fluid. The 
fluorescence of lacZ- cells and lacZ+ cells is typically separated by at 
least a 10 fold difference in intensities. 
Cell Collection and Examination 
The sorted cells are collected in a 96 well microtiter plate (Corning, 
N.Y.). The cell galactosidase activity is examined by means of the 
permeant fluorogenic substrate resorufin galactoside (Molecular Probes, 
Inc., Eugene, Oreg.). To a 20 .mu.L cell sample in a microliter well, is 
added 200 .mu.L of a 0.5 mM resorufin galactoside solution in 0.1M 
phosphate buffer containing 1 mM MgCl.sub.2 and 0.1% Triton X-100. After 1 
hour incubation, the plate is read in a CYTOFLUOR.TM. fluorescence plate 
reader (Millipore, Bedford, Mass.) with excitation at 560 nm, emission at 
645 nm, sensitivity=1. A fluorescence reading below 20 can be scored 
lacZ.sup.- cell and a reading higher than 60 can be scored lacZ.sup.+. The 
resorufin galactoside can detect the enzyme activity in a single 
lacZ.sup.+ cell within 1 hour. 
EXAMPLE 7 
Detection of Gus Gene Expression in Transformed Plant Cells with C.sub.12 
-Resorufin Glucuronide 
In our cellular assay, tobacco plants with and without the GUS gene 
(CaMV35S-GUS, cauliflower mosaic virus 35S promoter with the coding region 
of GUS) are used. (Ref: Jefferson, R. A., EMBO J., 1987, 6, 3901-3907). 
Sections of the stem of both plants are cultured on Murashige and Skoog 
basal medium with sucrose and agar inside of plant culture dish at 2000 
lux, 18 h day, 26.degree. C. The calli are collected from the dishes, cut 
to small pieces and transferred to liquid medium (in screw cap 50 mL 
Erlenmeyer flasks) to get a cell suspension that is shaken at 120 rpm in a 
culture room. Filtration sterilized 1 mM C.sub.12 -Resorufin-GlcU solution 
(in water) is added to the cell suspension to a final concentration of 100 
.mu.M. After incubating cells in normal conditions for 4 hours, 1 mL of 
the suspension is spun down to obtain the cells for analysis. The cells 
are resuspended in 100 .mu.L of fresh culture medium, placed onto a glass 
slide, covered with a cover glass and sealed with wax. 
Both the GUS+ and control tobacco cells are examined under a Ziess 
microscope equipped with a rhodamine filter set. Photographs can be taken 
of both fluorescent and Nomarski images (Fujichrome P1600D, color reversal 
film, daylight). Only the GUS gene positive tobacco plant cells are 
stained with C.sub.12 -Resorufin-GlcU and show bright orange red 
fluorescence that is most evident in the cell membrane. 
EXAMPLE 8 
Detecting the Induction of Two Different Promoters, A and B, During the 
Cell Cycle by Using Two Fluorogenic Substrates. 
1. Transformation 
A fibroblast cell line, such as NIH/3T3 cell, is co-transformed with two 
plasmids: #1 and #2. Plasmid #1 is constructed with an upstream promoter A 
that drives a high level expression of lacZ gene (.beta.-galactosidase). 
Plasmid #2 is constructed with an upstream promoter B that drives a high 
level expression of GUS gene (.beta.-glucuronidase). Promoters are 
selected so that promoter A will only be induced by a specific 
.alpha.-molecule existing in the G.sub.1 phase and promoter B will be 
induced by a specific .beta.-molecule existing in G.sub.2 phase. 
2. Simultaneous staining with two substrates 
The transformed cells are incubated with two lipophilic, fluorogenic 
substrates C.sub.12 FDG (ImaGene Green.TM., Molecular Probes, Eugene, 
Oreg.) and C.sub.12 -Resorufin-GlcU (50 mM each) under normal culture 
conditions for 30 minutes. The cells are then washed and processed for 
microscopic examination under a Zeiss microscope equipped with both FITC 
(EX 485 nm/EM 530 nm band pass filter) and rhodamine (EX 540 nm/EM 590 nm) 
filter sets. 
3. Results 
The cells in G1 phase show green fluorescence (with the FITC filter set) 
and those in G.sub.2 phase show red fluorescence (with the rhodamine 
filter set). After a longer incubation time, such as 8 hours, a lot of 
cells will show yellow fluorescence (i.e. a mixture of green and red 
fluorescence). 
It is to be understood that, while the foregoing invention has been 
described in detail by way of illustration and example, numerous 
modifications, substitutions, and alterations are possible without 
departing from the spirit and scope of the invention as described in the 
following claims.