Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
1999-02-22
2002-04-30
Woodward, Michael P. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S034000, C549S429000, C549S475000
Reexamination Certificate
active
06379911
ABSTRACT:
The invention relates to an enzyme detection method and to its use in detection of parasites.
BACKGROUND ART
Protozoan parasites have an adverse effect on the health of human and animal populations in a large number of countries. Problems presented by protozoan parasites are particularly of concern in tropical areas of the world where modern diagnostic methods are often not easily accessible.
Nucleoside hydrolases are enzymes which hydrolyse nucleosides between the ribose or deoxyribose and the purine or pyrimidine base groups. A number of different types of nucleoside hydrolases are known. N-Ribohydrolases hydrolyse ribonucleosides and N-deoxyribohydrolases hydrolyse deoxyribonucleosides. Within each of these two general groups of enzymes are enzymes of differing specificities.
Among the well-characterized N-ribohydrolases, specificity is high for the ribosyl group but varies for the leaving group purine or pyrimidine. The inosine-uridine nucleoside hydrolase (IU-nucleoside hydrolase) from the trypanosome
Crithidia fasciculata
hydrolyzes all of the naturally occurring purine and pyrimidine nucleosides with similar catalytic efficiencies. The guanosine-inosine enzyme (GI-nucleoside hydrolase) from the same organism has a strong preference for the eponymous substrates and is nearly inert with the pyrimidine nucleosides
1,2,3,4
. AMP Nucleosidase from bacterial sources is highly specific for the adenine base, and the 5′-phosphoryl is required for significant hydrolytic rates
5,6
. Nucleoside phosphorylases have a similar mechanism to nucleoside hydrolases and, for example, purine nucleoside phosphorylase is specific for inosine and guanosine substrates and activates phosphate or arsenate anions to attack C1 of the nucleosides.
It is an object of the invention to provide a method of detecting and/or assaying for the presence of certain enzymes, especially those of parasites in samples taken from parasitised humans and animals.
SUMMARY OF THE INVENTION
In one aspect the invention provides a method of detecting and/or assaying nucleoside hydrolases using a chromogenic substrate.
Preferably the chromogenic substrates have the formula:
where X is OH or H, and Y is the residue of Y—OH where Y—OH is a chromophore or a compound readily converted to a chromophore and the substrates are hydrolysed by the nucleoside hydrolase to yield ribose or 2-deoxyribose plus Y—OH.
Preferably the chromogenic substrates are of Formula I wherein X and Y are as defined and the substrates are phosphorylysed by the nucleoside phosphorylase to yield ribose-1-phosphate or 2-deoxyribose-1-phosphate plus Y—OH.
Y may be chosen so that Y—OH is a compound absorbing in the visible or TV light, readily measured at wavelengths greater than 300 nm, preferably greater than 340nm.
Preferably Y—OH is 6-hydroxynicotinamide or 2-hydroxypyridine -4-carboxamide.
Y may be chosen so that Y—OH is a chemiluminescent compound eg luminol which when released and oxidized by chemical or enzymatic means emits light.
Alternatively Y—OH may be a compound readily converted to a coloured compound eg by reaction with a diazonium salt eg &agr;-naphthol.
More preferably Y—OH is a fluorescent compound eg 4-methylumbelliferone or fluoroscein.
Most preferably Y—OH is a coloured compound eg phenolphthalein, p-nitrophenol, thymolphthalein, 2-nitrophenol, 2-hydroxy-5-nitropyridine.
It is preferred that Y—OH can be measured in the presence of the compound of Formula I by virtue of Y—OH absorbing or fluorescing to a greater extent than the compound of Formula I at certain wavelengths. Generally this will be by virtue of Y—OH ionising and being in equilibrium with quinonoid-like forms.
A particularly preferred substrate is p-nitrophenyl &bgr;-D-ribofuranoside. Also particularly preferred is 4-pyridyl &bgr;-D-ribofuranoside. Further particularly preferred substrates are 4-methylumbelliferyl &bgr;-D-ribofuranoside (4-methylcoumarin-7-yl &bgr;-D-ribofuranoside) and 2-(5-nitropyridyl) &bgr;-D-ribofuranoside.
According to another aspect of the invention, there is provided a method for detecting and/or assaying for parasites especially protozoa in samples obtained usually from humans or animals using a chromogenic substrate, preferably of Formula I.
More preferably the chromogenic substrate is one where Y—OH is fluorescent or coloured.
Another preferred type of substrate is one where Y—OH is chemiluminescent eg luminol.
According to a further aspect of the invention there is provided a kit containing materials for detection or assay of hydrolysis of the chromogenic substrate, preferably of Formula I, by enzymes in a biological sample. Preferred kits comprise the chromogenic substrate in dry form, together with a buffer. Other components eg a cell-lysing agent may also be included.
According to a further aspect of the invention there is provided a dipstick containing a chromogenic substrate, preferably of Formula I, for use in detecting nucleoside hydrolases.
According to a further aspect of the invention there are provided novel compounds of the invention of formula I. In this aspect of the invention X and Y are as previously defined except the known compounds &agr;-naphthyl &bgr;-D-ribofuranoside, 4-methylcoumarin-7-yl 2-deoxy-p-&bgr;-ribofuranoside, &bgr;-nitrophenyl &bgr;-D-ribofuranoside, p-aminophenyl &bgr;-D-ribofuranoside, 5-amino-6-chloro-3-pyridazinyl &bgr;-D-ribofuranoside, 4-chlorophenyl &bgr;-D-ribofuranoside, phenyl &bgr;-D-ribofuranoside, 4-methoxyphenyl &bgr;-D-ribofuranoside, 4-hydroxyphenyl &bgr;-D-ribofuranoside, 4-(N,N,N-trimethylammonio )phenyl &bgr;-D-ribofuranoside, 4-acetylphenyl &bgr;-D-ribofuranoside and the &bgr;-D-ribofuranosides of L-DOPA and L-&agr;-methyl-DOPA and the N-acetyl methyl esters of the abovementioned DOPA derivatives are not included in this aspect. The exceptions are not known as chromogenic substrates or for use in assay of parasites. Generally the chromogenic group is not coloured when present in the compound of Formula I but is readily detectable when that compound is hydrolysed to give Y—OH.
Preferred compounds are those defined above in which Y is an optionally substituted pyridyl group or a nitrophenyl group.
Particularly preferred novel compounds of the invention include 3-trifluoroacetamidophenyl &bgr;-D-ribofuranoside, 3-aminophenyl- &bgr;-D-ribofuranoside, 1-tetralone-5-yl &bgr;-D-ribofuranoside, 3-( 4-hydroxyphenyl)-1-(3H)-isobenzofuranone-3-(phen-4-yl) &bgr;-D-ribofuranoside, 2-nitrophenyl &bgr;-D-ribofuranoside, 4-methylcoumarin-7-yl &bgr;-D-ribofuranoside, 3-pyridyl &bgr;-D-ribofuranoside, 4-pyridyl &bgr;-D-ribofuranoside, 2-(5-nitropyridyl) &bgr;-D-ribofuranoside, 5-quinolyl &bgr;-D-ribofuranoside, the &bgr;-D-ribofuranoside of luminol, p-nitrophenyl 2-deoxy- &bgr;-D-erythro-pentofuranoside, 3-carboxamido-6-pyridyl &bgr;-D-ribofuranoside, and 4-forymylphenyl &bgr;-D-ribofuranoside.
In a further aspect of the invention there is provided a method of preparing a chromogenic substrate of the invention.
According to a further aspect the invention may be directed to a method to detect or assay for the presence of nucleoside phosphorylases using a chromogenic substrate.
DETAILED DESCRIPTION OF THE INVENTION
One group of preferred chromogenic substrates have a &bgr;-D-ribofuranosyl group attached through a &bgr;-O-ribosidic linkage to a chromogenic group.
In a preferred embodiment the chromogenic substrate is a compound of Formula I wherein X═OH. The group Y is as defined under Formula I. Preferably the chromophore Y—OH is selected from the group of phenolphthalein, p-nitrophenol, 4-methylumbelliferone, &agr;-naphthol, thymolphthalein, 2-nitrophenol, 2-hydroxy-5-nitropyridine, 6-hydroxynicotinamide, 2-hydroxypyridine-4-carboxamide and fluorescein. Other chromophores as will be known in the art may also be used.
In this specification the term “chromogenic group” is used to refer to a group in a nucleoside hydrolase substrate bound to the sugar moiety which, when enzymnatically removed from the substrate, forms a compound which is readily detectable by its visible or UV absorption or by its fl
Clinch Keith
Furneaux Richard Hubert
Schramm Vern L.
Tyler Peter Charles
Albert Einstein College of Medicine of Yeshiva University
Moran Marjorie A.
Woodward Michael P.
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