WO1991009971A1 - Substrats d'acide neuraminique chromogene modifie en position 5 et methodes de diagnostic de la grippe humaine les utilisant - Google Patents

Substrats d'acide neuraminique chromogene modifie en position 5 et methodes de diagnostic de la grippe humaine les utilisant Download PDF

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Publication number
WO1991009971A1
WO1991009971A1 PCT/US1990/007678 US9007678W WO9109971A1 WO 1991009971 A1 WO1991009971 A1 WO 1991009971A1 US 9007678 W US9007678 W US 9007678W WO 9109971 A1 WO9109971 A1 WO 9109971A1
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WIPO (PCT)
Prior art keywords
bromo
chloro
naphthyl
nitrophenyl
substrate
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PCT/US1990/007678
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English (en)
Inventor
Gregory A. Turner
James F. Maher
C. Worth Clinkscales
Michael D. Roark
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Symex Corp.
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Publication of WO1991009971A1 publication Critical patent/WO1991009971A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/02—Heterocyclic radicals containing only nitrogen as ring hetero atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
    • C07H17/06—Benzopyran radicals
    • C07H17/065—Benzo[b]pyrans
    • C07H17/075—Benzo[b]pyran-2-ones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H7/00—Compounds containing non-saccharide radicals linked to saccharide radicals by a carbon-to-carbon bond
    • C07H7/02—Acyclic radicals
    • C07H7/027—Keto-aldonic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/11—Orthomyxoviridae, e.g. influenza virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)

Definitions

  • the present invention relates to reagents and assays for diagnosing human influenza. More specifically it relates to novel chromogenic 5-position modified neuraminic acid substrates that are useful in the diagnosis of influenza through the detection of the enzymatic activity of human influenza neuraminidase (NA) .
  • NA neuraminidase
  • Influenza virus averages 30-50 million infec ⁇ tions annually in the United States alone. Epidemiologic studies of influenza epidemics estimate the incidence of infection to be 25% in the general population and higher in school age children. researchers have estimated that up to half the infected persons would see a physician because of the illness. In 1986, the Center for Disease Control (CDC) estimated that influenza epidemics have been associated with 10,000 or more excess deaths in 18 of the preceding 28 years. CDC studies indicate influenza as the fifth leading cause of death in the United States. Antigenic variations in the surface glycoproteins of influenza A and B account for their continued epidemics.
  • Influenza viruses possess surface glycoproteins that have NA activity. These glycoproteins are members of a family of neuraminidases that are found in viruses, bacteria, mycoplasmas, and animal tissues. They hydrolyze substrates that contain alpha-ketosidically linked N-acetylneuraminic acid (Neu5Ac; referred to previously as "NANA") . In viruses, NA typically constitutes 5-10% of the viral protein and exists as a mushroom-shaped spike on the envelope. Viral NA is composed of a hydrophilic area which includes the catalytic site of the enzyme and a hydrophobic area that is inserted into the viral envelope anchoring the enzyme to the virus. Various assays for NA activity are described in the literature.
  • the neuraminic acid is often referred to as 3-deoxy-D-glycero-beta-D-galacto-2-nonulopyranosonic acid (KDN) .
  • KDN is a known naturally-occurring compound, and its 4-methylumbelliferyl and 4-nitrophenyl derivatives have been reported by Zbiral, E. , et al., Liebigs Ann. Chem. 519-526 (1989); Nakamura, M. , et al. , Chem. Pharm. Bull. 36(12) :4807-4813 (1988); and Nakamura, M. , et al., Chem. Pharm. Bull.
  • One aspect of the invention is a method of detecting human influenza neuraminidase activity in a clinical sample suspected of having such activity comprising: (a) incubating the sample with a chromogenic modified neuraminic acid of the formula:
  • R represents azido, fluorine or hydroxy
  • X represents a chromogenic group that exhibits distinct color when cleaved from the derivative or a salt of said derivative
  • step (b) detecting neuraminidase activity by observing whether the sample-derivative mixture exhibits said color after step (a) .
  • Another aspect of the invention is a method of selectively detecting a specific type (e.g., A or B) of human influenza neuraminidase activity in a clinical sample suspected of having human influenza neuraminidase activity from activity exhibited by other types of human influenza neuraminidase comprising:
  • R represents azido, fluorine or hydroxy and X represents a chromogenic group that exhibits distinct color when cleaved from the derivative or a salt of said derivative;
  • step (b) observing the color exhibited by the sample-derivative mixture after step (a) ; and (c) comparing said color to colors exhibited by activity standards of human influenza neuraminidase of said specific type and other types of human influenza neuraminidase on said derivative.
  • Yet another aspect of the invention is a modified neuraminic acid chromogenic substrate useful for detecting human influenza neuraminidase activity in a clinical sample suspected of having such activity, said substrate having the formula:
  • R is hydroxy and X is selected from the group consisting of 3-cyanoumbelliferyl, 2-nitrophenyl, 3-resorufin, 4-nitrophenylazophenyl, 4-nitro- phenylazoresorcinyl, 5-bromo-4-chloro-3-indolyl, 3- methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-l-naphthyl and 6-bromo-2-naphthyl.
  • Yet another aspect of the invention is a modified neuraminic acid chromogenic substrate useful for detecting human influenza neuraminidase activity in a clinical sample suspected of having such activity, said substrate having the formula:
  • R is azido or fluorine and X is a chromogenic group that exhibits a distinct color when cleaved from the substrate and salts of said substrate.
  • Figure 1 is a schematic diagram depicting the synthesis procedure described in Example 1.
  • Figure 2 is a schematic diagram depicting the synthesis procedure described in Example 2.
  • Figure 3 is a schematic diagram depicting the synthesis procedure described in Example 3.
  • Figure 4 is a schematic diagram depicting the synthesis procedure described in Example 4.
  • Figure 5 is a schematic diagram depicting the synthesis procedure described in Example 5.
  • the chromogenic modified neuraminic acid substrates of the invention and the methods employing them are useful for detecting human influenza neuraminidase activity in clinical samples or specimens and for determining the type of human influenza neuraminidase present in the sample. Accordingly, these substrates and methods are useful for diagnosing influenza infection generally as well as the type of influenza infection present in the human patient from whom the clinical sample was collected.
  • influenza is intended to include influenza types A and B and parainfluenza types 1, 2, and 3.
  • selective detect intends the ability to detect
  • NA activity of one type of influenza virus as compared to the activity of other types of influenza virus.
  • the clinical samples that are tested in the invention will typically be pharyngeal, nasopharyngeal or respiratory secretions collected from patients suffering from influenza as wash, swab, or expectorate specimens.
  • the wash, expectorate, or swab will preferably be combined with an aqueous buffer solution containing a stabilizer prior to mixing with the substrate.
  • the buffer solution contains a buffer that maintains the pH at about 4 to 7, preferably 5.5 to 6.5, optionally about 0.1% to 10% by weight nonionic detergent, a small amount (1-20 mM) of alkaline earth metal cation (Ca, Mg, preferably Ca) , and a sufficient amount of a stabilizer selected from the group consisting of polyhydric sugar alcohols, simple sugars, and disaccharide sugars to enhance the thermal stability of the NA in the sample.
  • the volume of buffer solution combined with the specimen will normally be 0.1 to 2 ml.
  • the buffer may be organic or inorganic.
  • suitable buffers are conventional buffers of organic acids and salts thereof such as citrate buffers (e.g. monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.), acetate buffers (e.g., acetic acid-sodium acetate mixture), succinate buffers (e.g. succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate buffers (e.g.
  • citrate buffers e.g. monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.
  • acetate buffers e.g., acetic acid-sodium acetate mixture
  • succinate buffers e.g. succinic acid-monosodium succinate
  • tartaric acid-tartrate mixture tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture etc.
  • fumarate buffers e.g. fumaric acid-monosodium fumarate mixture, fumaric acid-disodium fumarate mixture, monosodium fumaric acid-disodium fumarate mixture
  • gluconate buffers e.g. gluconic acid-sodium gluconate mixture, gluconic acid-sodium hydroxide mixture, gluconic acid-potassium gluconate mixture, etc.
  • oxalate buffers e.g.
  • oxalic acid-sodium oxalate mixture oxalic acid- sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.
  • lactate buffers e.g. lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture, etc.
  • acetate buffers e.g. acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc.
  • malate buffers e.g., D,L-malic acid-disodium malate mixture
  • phosphate buffers e.g.
  • non-ionic detergents useful in the buffer solution are the Pluronics, such as Polysorbate 20 and Polysorbate 80, Triton X-100, NP-40, and alkyl glucosides such as C 8 -C 8 alkyl glucoside.
  • the detergent is an optional component and facilitates release of the NA from the viral envelope.
  • the stabilizers that are used in the buffer solution are trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, mannitol, the simple sugars glucose and fructose and the disaccharride sucrose. These polyhydric sugar alcohols, and simple and disaccharride sugars can be used alone or in combination.
  • the polyhydric sugar alcohols or simple and disaccharride sugars are added to the liquid formulation/excipient system in an amount from 0.2 M to 2.1 M and preferably, 0.6 M to 2.0 M.
  • the sample may be stored for prolonged periods, preferably at 2°C to 8°C without significant loss of NA activity.
  • the substrate that is combined with the buffered, stabilized specimen is a chromogenic neuraminic acid that is modified in the 5 position.
  • These substrates may be represented by the following chemical formula:
  • R and X are as defined previously.
  • X represents 4-methylumbelliferyl, 3-cyanoumbelliferyl, 2-nitrophe ⁇ yl, 4-nitrophenyl, 3-resorufin, 5-bromo-4- chloro-3-indolyl, nitrophenylazophenyl, nitrophenylazo- resorcinyl, 3-methoxyphenyl, 3-dimethylaminophenyl, 4- chloro-1-naphthyl, or 6-bromo-2-naphthyl.
  • Simple salts 4-methylumbelliferyl, 3-cyanoumbelliferyl, 2-nitrophe ⁇ yl, 4-nitrophenyl, 3-resorufin, 5-bromo-4- chloro-3-indolyl, nitrophenylazophenyl, nitrophenylazo- resorcinyl, 3-methoxyphenyl, 3-dimethylaminophenyl, 4- chloro-1-naphthyl,
  • the substrate such as the Na, K, or NH. salts, may also be used.
  • chromogen is intended to include, without limitation, molecules that exhibit fluorescence.
  • color is likewise intended to include, without limitation, fluorescence.
  • Examples of the 5-modified neuraminic acid derivatives falling within the above formula are 4- methylumbellifery1-3-deoxy-D-glycero-beta-D-galacto-2- nonulopyranosonic acid-alpha-ketoside, 3- cyanoumbelliferyl-3-deoxy-D-glycero-beta-D-galacto-2- nonulopyranosonic acid-alpha-ketoside, 2-nitrophenyl-3- deoxy-D-glycero-beta-D-galacto-2-nonulopyranosonic acid- alpha-ketoside, 4-nitrophenyl-3-deoxy-D-glycero-beta-D- galacto-2-nonulopyranosonic acid-alpha-ketoside, 3-resorufin-3-deoxy-D-glycero-beta-D-galacto-2- nonulopyranosonic acid-alpha-ketoside, 5- bromo-4-chloro
  • modified neuraminic acid derivatives are generally made by protecting the hydroxy groups of the neuraminic acid at the 1, 2, 4, 7, 8, and 9 positions, modifying the 5 position as indicated, deprotecting the other positions and coupling the 5- modified neuraminic acid with the chromogen. Details of these reactions are provided in the Examples, infra.
  • the substrate will normally be added to the buffered, stabilized sample in amounts ranging between 0.05 mM and 0.5 mM.
  • the mixture is incubated at ambient temperature to physiological temperature (i.e., about 22°C to 37°C) for a time sufficient to permit any NA in the sample to react with the derivative. That time will normally be in the range of 20 to 120 minutes, more usu ⁇ ally 30 to 60 minutes.
  • the chromogenic group will be cleaved from the derivative and the liberated chromogen will impart a characteristic color to the mixture.
  • the derivatives of the invention may exhibit different reactivity to the different human influenza NAs
  • the specific type of influenza infection may be determined by comparing the color of the sample mixture with the color of standard reaction mixtures for each influenza NA type. For instance, influenza A may be distinguished from influenza B on the basis of the derivatives' reactivity with the NAs of these influenza viruses.
  • the following table indicates the color generated when NA reacts with the chromogenic neuraminic acid derivatives and releases the chromogen. Released Type of Chromogen Detection Color
  • the present invention provides a simple and rapid technique for selectively diagnosing influenza that may be carried out in the clinic or physician's office and enable the physician to prescribe the appropriate therapy to treat the infection and/or the appropriate prophylactic treatment to persons in close contact with the infected patient.
  • KDN is per-acetylated with acetic anhydride/pyridine and catalytic dimethylaminopyridene at room temperature (RT) overnight.
  • the benzyl ester is treated overnight with excess acetyl chloride to form the glycosyl chloride.
  • the glycosyl chloride is then coupled with nitrophenylazophenol via reaction with the sodium salt, of 4-nitrophenylazophenol in DMF at RT for 2 hr. Standard base deprotection yields 2-[4-(4-nitro ⁇ phenylazo)phenyl-KDN-alpha-ketoside (sodium salt) .
  • Glucose ⁇ -methyl glycoside is converted to the 4,6-benzylidene by treatment with benzaldehyde/zinc shloride. Triflation occurs selectively at C-2 upon treatment with trifluoromethanesulfonyl chloride in pyridine. This group is then displaced with sodium azide to give the (mannose configuration) 2-azide. Hydrolysis with dilute acid affords 2-azido mannose. This carbohydrate may then be converted to azidoneuraminic acid via an enzymatic catalyzed step (aldolase and pyruvate) or by the Ccrnforth procedure (oxalacetic acid) . 3. Synthesis of 3-Dimethyaminophenyl-5-Deoxy-5-Azido KDN The synthesis scheme for this compound is shown in Figure 3.
  • Neu5 Azide is converted to its methyl ester by treatment in methanol with trifluoroacetic acid.
  • Benzylidene glucose ⁇ -methyl glycoside is prepared as described for Neu5 azide. Treatment with diethylamin sulfur trifluoride (DAST) selectively fluorinates the C-2 alcohol. Hydrolysis affords 2-fluoro mannose which may be converted to 5-fluoroneuraminic acid via the enzymatic (aldolase) or chemical (Cornforth) procedure.
  • DAST diethylamin sulfur trifluoride

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Abstract

Dérivés d'acide neuraminique chromogène modifié en position 5 utilisés comme substrats dans les analyses colorimétriques de l'activité de neuraminidase de la grippe humaine sur des échantillons cliniques, afin de diagnostiquer sélectivement une infection grippale. Ces dérivés peuvent présenter différents types de réactivité en fonction des différents types de neuraminidase de la grippe, ce qui permet de distinguer le type spécifique d'infection grippale et de prescrire un traitement adapté et/ou un traitement de soutien approprié.
PCT/US1990/007678 1990-01-05 1990-12-27 Substrats d'acide neuraminique chromogene modifie en position 5 et methodes de diagnostic de la grippe humaine les utilisant WO1991009971A1 (fr)

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US46067990A 1990-01-05 1990-01-05

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997032214A1 (fr) * 1996-03-01 1997-09-04 Biota Scientific Management Pty. Ltd. Procede de detection du virus de la grippe, et composes mis en oeuvre a cet effet
WO1998013372A1 (fr) * 1996-09-25 1998-04-02 Oklahoma Medical Research Foundation Derives d'acide 4,7-dialcoxy n-acetylneuraminique et procedes de detection des types a et b du virus de la grippe dans des prelevements cliniques
JP2003522113A (ja) * 1998-10-27 2003-07-22 ユーエービー リサーチ ファンデイション シアリダーゼの発色基質とその製造法および使用法
US10732135B2 (en) 2015-06-16 2020-08-04 Multicore Technologies, Llc System and method for determining one or more fluid concentrations in a fluid stream

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4668622A (en) * 1983-12-17 1987-05-26 Boehringer Mannheim Gmbh Phenolsulphonphthaleinyl-β-D-galactosides and diagnostic agents containing them

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4668622A (en) * 1983-12-17 1987-05-26 Boehringer Mannheim Gmbh Phenolsulphonphthaleinyl-β-D-galactosides and diagnostic agents containing them

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF CLINICAL MICROBIOLOGY, Vol. 26, No. 12, December 1988, PACHUCKI et al., "Early Detection of Influenza Virus by Using a Fluorometric Assay of Infected Tissue Culture", p. 2664-2666. *
JOURNAL OF INFECTIOUS DISEASE, Vol. 142, No. 4, issued October 1980, YOLKEN et al., "Fluorometric Assay for Measurement of Viral Neuraminidase Application to the Rapid Detection of Influenza Virus in Nasal Wash Specimens", pages 516-523. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997032214A1 (fr) * 1996-03-01 1997-09-04 Biota Scientific Management Pty. Ltd. Procede de detection du virus de la grippe, et composes mis en oeuvre a cet effet
US6242582B1 (en) 1996-03-01 2001-06-05 Biota Scientific Management Pty Ltd. Method of detection of influenza virus and compounds for use therein
WO1998013372A1 (fr) * 1996-09-25 1998-04-02 Oklahoma Medical Research Foundation Derives d'acide 4,7-dialcoxy n-acetylneuraminique et procedes de detection des types a et b du virus de la grippe dans des prelevements cliniques
JP2003522113A (ja) * 1998-10-27 2003-07-22 ユーエービー リサーチ ファンデイション シアリダーゼの発色基質とその製造法および使用法
US10732135B2 (en) 2015-06-16 2020-08-04 Multicore Technologies, Llc System and method for determining one or more fluid concentrations in a fluid stream

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