WO1991009975A1 - Substrats d'acide n-acetylneuraminique chromogene modifie dans les positions 7 ou 8 utilises dans des procedes de diagnostic de la grippe chez l'homme - Google Patents

Substrats d'acide n-acetylneuraminique chromogene modifie dans les positions 7 ou 8 utilises dans des procedes de diagnostic de la grippe chez l'homme Download PDF

Info

Publication number
WO1991009975A1
WO1991009975A1 PCT/US1990/007677 US9007677W WO9109975A1 WO 1991009975 A1 WO1991009975 A1 WO 1991009975A1 US 9007677 W US9007677 W US 9007677W WO 9109975 A1 WO9109975 A1 WO 9109975A1
Authority
WO
WIPO (PCT)
Prior art keywords
nitrophenyl
chloro
bromo
naphthyl
group
Prior art date
Application number
PCT/US1990/007677
Other languages
English (en)
Inventor
Gregory A. Turner
James F. Maher
C. Worth Clinkscales
Michael D. Roark
Original Assignee
Symex Corp.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Symex Corp. filed Critical Symex Corp.
Publication of WO1991009975A1 publication Critical patent/WO1991009975A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
    • 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
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • 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
    • 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
    • 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
    • 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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • 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 7- or 8-position modified N-acetylneuraminic 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 influ ⁇ enza as the fifth leading cause of death in the United States. Antigenic variations in the surface glyco- proteins 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. Santer, U.V.
  • One aspect of the invention is a method of detecting human influenza neuraminidase activity in a clinical sample suspected of having such activity comprising:
  • R represents hydrogen, fluorine, hydroxy, azido or cyano
  • X represents a chromogenic group that exhibits distinct color when cleaved from the substrate or a salt of said substrate
  • 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 hydrogen, fluorine, hydroxy, azido or cyano
  • X represents a chromogenic group that exhibits distinct color when cleaved from the substrate or a salt of said substrate;
  • step (b) observing the color exhibited by the sample-substrate mixture after step (a) ;
  • Yet another aspect of the invention is a modified NeuSAc chromogenic substrate useful for detecting human influenza neuraminidase activity in a clinical sample suspected of having such activity, said substrate having the formula:
  • R represents fluorine, hydroxy, azido or cyano
  • R 1 and R2 must be hydroxyl but not both of Rl and R? are hydroxyl
  • X is a chromogenic group that exhibits a distinct color when cleaved from the substrate and salts of said substrate.
  • Still another aspect of the invention is a chromogenic substrate useful for detecting human influenza neuraminidase activity in a clinical sample suspected of having such activity, said substrate having the formula:
  • X is a chromogenic group selected form the group consisting of 3-cyanoumbelli- feryl, 2-nitrophenyl, 4-nitrophenyl, 3-resorufin, 5-bromo-4-chloro-3-indolyl, 4-nitrophenylazoresorcinyl, 4-nitrophenylazophenyl, 3-methoxyphenyl, 3-dimethyl- aminophenyl, 4-chloro-l-naphthyl and 6-bromo-2-naphthyl.
  • 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 procedures described in Examples 3 and 5.
  • 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 6.
  • Figure 6 is a schematic diagram depicting the synthesis procedure described in Example 7.
  • Figure 7 is a schematic diagram depicting the synthesis procedure described in Example 8.
  • Figure 8 is a schematic diagram depicting the synthesis procedure described in Example 9.
  • Figure 9 is a schematic diagram depicting the synthesis procedure described in Example 10.
  • Figure 10 is a schematic diagram depicting the synthesis procedure described in Example 11.
  • Figure 11 is a schematic diagram depicting the synthesis procedure described in Example 12.
  • Figure 12 is a schematic diagram depicting the synthesis procedure described in Example 13.
  • Figure 13 is a schematic diagram depicting the synthesis procedure described in Example 14.
  • the chromogenic modified N-acetylneuraminic 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.
  • the term "influenza” is intended to include influenza types A and B and parainfluenza types 1, 2, and 3.
  • the term "selectively 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. monosodiu 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.
  • 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_-C 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 NeuSAc derivative that is modified in the 7- or 8-positions (but not both positions) .
  • These substrates may be represented by the following chemical formula:
  • R 1, R2, X and Ac are as defi.ned previ.ously.
  • X represents 4-methylumbelliferyl
  • 3-cyanoumbelliferyl 2-nitrophenyl, 4-nitrophenyl, 3-resorufin, 5-bromo-4-chloro-3-indolyl, nitrophenyl- azophenyl, nitrophenylazoresorcinyl, 3-methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-l-napthyl or 6-bromo-2- naphthyl.
  • Simple salts of 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 7- or 8-modified chromogenic NeuSAc derivatives falling within the above formula are 4-methylumbelliferyl-7-deoxy-N-acetylneuraminic acid-alpha-ketoside, 3-cyanoumbelliferyl-7-deoxy-N- acetylneuraminic acid-alpha-ketoside, 2-nitrophenyl-7- deoxy-N-acetylneuraminic acid-alphaketoside, 4-nitro- phenyl-7-deoxy-N-acetylneuraminic acid-alpha-ketoside, 3-resorufin-7-deoxy-N-acetylneuraminic acid-alpha- ketoside, 5-bromo-4-chloro3-indolyl-7-deoxy-N-acetyl- neuraminic acid-alpha-ketoside, 2-[4-(4-nitrophenylazo)- phenyl]-7-deoxy-N-acetylneuraminic acid-alpha-
  • NeuSAc derivatives are generally made by protecting the functional groups of
  • Neu5Ac derivatives modified in the 7- or 8- positions with azido or cyano groups may be produced as epimeric mixtures due to the mechanism of the reactor involved coupled with the configuration of neighboring group(s).
  • the epimeric mixture may be used or the epimers may be separated and used separately.
  • 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 substrate. That time will normally be in the range of 20 to 120 minutes, more usually 30 to 60 minutes. If there is NA activity in the sample, the chromogenic group will be cleaved from the substrate and the liberated chromogen will impart a characteristic color to the mixture.
  • 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 substrate reactivity with the NAs of these influenza viruses.
  • the following table indicates the color generated when NA reacts with a modified NeuSAc and releases the chromogen.
  • 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.
  • the invention is further illustrated by the fol- lowing examples. These examples are not intended to limit the invention in any manner.
  • N-acetylneuraminic acid is protected as the methyl ester methyl ketoside (NeuSAc-MEMK) by treatment with methanol under Dowex SOW ion-exchange resin acid catalysis. Subsequent reaction with acetone and catalytic p-toluenesulfonic acid for 4 hr affords the 8,9-isopropylidene adduct.
  • This intermediate is treated with one equivalent of t-butyldimethylsilyl (TBDMS) chloride, imidazole and a catalytic amount of dimethylaminopyridine to selectively yield the 4-silylated derivative.
  • TDMS t-butyldimethylsilyl
  • Oxidation of the lone 7-alcoholic group with pyridiniu -dichromate gives the 7-keto adduct.
  • Saponification of the ester and deprotection of the TBDMS group and acid-labile ketal and ketoside groups is then accomplished with hydroxide solution, followed by tetrabutylammonium fluoride and dilute acid to afford 7-keto-N-acetylneuraminic acid.
  • the 8,9-isopropylidene-4-TBDMS-7-keto NeuSAc-MEMK intermediate from the previous synthesis is treated with dilute hydrochloric acid solution to deprotect the 4,8 and 9 positions and the resulting free acid esterified with methanol/trifluoroacetic acid.
  • Formation of the glycosyl chloride with concomitant acetylation of all free OH groups is accomplished by treatment in excess acetyl chloride overnight.
  • Coupling of this intermediate with the sodium salt of nitrophenylazoresorcinol (NAR) is done in dimethylformamide (DMF) solution (2 hr) .
  • the final product 2-[4-(4-nitrophenylazo)resorcinyl]- 7-keto-N-acetylneuraminic acid-alpha-ketoside (sodium salt) , is obtained by deprotecting the alcohol groups with methoxide ion and saponification of the methyl ester under base catalysis.
  • Deprotection of this compound will consist of treatment with sodium hydroxide followed by Dowex-50W (H ) then treatment with tetrabutylammonium fluoride in THF to remove the silyl group, and finally treatment with dilute HCl/Dowex-50W (H + ) to afford 7-azido-N-acetylneuraminic acid.
  • Neu5Ac methyl ester is formed through the usual route, as is the glycosyl chloride which is coupled to the sodium salt of resorufin in DMF (2 hr) .
  • Deprotection (deacetylation) is accomplished by treatment with sodium ethoxide in methanol.
  • the 7-azido group is then introduced.
  • the 8 and 9-hydroxy groups will be protected as the isopropylidene by treatment with excess acetone and a catalytic amount of p-toluenesulfonic acid at room temperature.
  • the 4-hydroxy group will then be protected as the O-TBDMS by treatment with 5 equivalents of imidazole, 1 equivalent of t-buyldimethylsilyl chloride, and a catalytic amount of dimethylaminopyridine in DMF, at 65°C.
  • the 7-hydroxy will then be mesylated by treating the compound with methanesulfonyl chloride and triethylamine in methylene chloride at 0°C.
  • the azido group will be substituted on the 7-position by treating the mesylate with sodium azide at 100°C.
  • the molecule will then be fully deprotected by treating with p-toluenesulfonic acid, tetrabutylammonium fluoride, and finally sodium hydroxide to give
  • N-acetylneuraminic acid is protected as NeuSAc-MEMK, after which the 4,9-disilylated intermediate is obtained by treatment with 2 equivalents of TBDMS-C1 with imidazole/dimethylaminopyridine in DMF.
  • the more reactive 8-alcohol group is tosylated with tosyl chloride/pyridine (5°C for 7 hr) and subsequently displaced by sodium azide in acetone at 100°C.
  • the desired compound, 8-azido-N-acetylneuraminic acid is obtained after deprotection with base, fluoride ion and dilute acid.
  • Neu5Ac methyl ester is prepared by treating with methanol under trifluroacetic acid catalysis and converted to the glycosyl chloride in excess acetyl chloride. Coupling with 5-bromo-4-chloro-3-indolol is done in DMF with 1 equivalent of sodium hydroxide. Protection of the 4 and 9-alcohol groups is done with 2.5 eq. of TBDMS-C1, imidazole and catalytic dimethylaminopyridine. Tosylation of the 8-alcohol group and displacement with azide ion is performed as described previously.
  • This compound is prepared in an identical manner to that of 8-azido NeuSAc or 8-cyano NeuSAc, only the 8-tosyl intermediate is reduced with sodium borohydride (4 hr) in dimethylsulfoxide to give the corresponding deoxy derivative.
  • the final product, 8-deoxy-N-acetylneuraminic acid is obtained after deprotection with base, fluoride ion and dilute acid.
  • Neu5Ac-MEMK 8,9-isopropylidene is treated with one equivalent of tert-butyldimethylsilyl chloride to obtain the corresponding 4-TBDMS derivative.
  • Oxidation with PDC affords the 7-keto compound.
  • Reduction with borane- ammonia gives primarily the 7-epimeric alcohol which may then be converted to the corresponding 7-fluoride with DAST (original stereochemistry) .
  • Deprotection with dilute base and acid will afford 7-fluoro NeuSAc after chromotography on Dowex 1 (formate form) or cellulose.
  • 50 ⁇ l of an influenza virus was mixed with a reaction mixture containing 50 ⁇ l of the substrate 4- methylumbelliferyl NeuSAc at various concentrations in the submillimolar to millimolar range, 150 ⁇ l of the inhibitor 7-epi-Neu5Ac at various concentrations in the submillimolar to millimolar range, and 50 ⁇ l of 100 mM CaCl-- All solutions were made up in a 50 mM sodium acetate buffer, pH 5.9. After incubation at 37°C for 15 to 30 minutes (depending on virus strain) , the reaction was terminated by adding 500 ⁇ l of 1 M Tris, pH 9.0, with 1.33% ethanol.
  • the fluorescence intensity was measured at an excitation wavelength of 360 nm and an emission wavelength of 450 nm with a fluorescence spectro- photometer (Hitachi Model 3010) .
  • 4-methylumbelliferone in 1 M Tris, pH 9.0, with 1.33% ethanol served as a standard.
  • Enzyme activity was expressed as mM of Neu5Ac liberated per minute per 50 ⁇ l of virus.
  • a plot of 1/v vs. 1/[S] for varying concentrations of substrate and inhibitor showed typical competitive inhibition. Plotting the slopes of the 1/v vs. 1/[S] plot versus the inhibitor concentration allowed for the calculation of K- for 7-epi-Neu5Ac as follows:
  • the K- for 7-epi-Neu5Ac indicates how the compound interacts with the enzyme as well as the rate at which it interacts. In general, the lower the K ⁇ , the greater the degree of inhibition at any given substrate and inhibitor concentration. It is also desirable to have a modified compound which can interact with an enzyme in a similar manner as the native compound without compromising its ability as a substrate.
  • the K ⁇ gives a first indication of the compound's interaction with the enzyme.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Virology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

On utilise des dérivés chromogènes d'acide N-acétylneuraminique modifié dans les positions 7 ou 8, en tant que substrats dans des analyses colorimétriques déterminant une activité de neuraminidase de la grippe chez l'homme, dans des échantillons, afin de diagnostiquer sélectivement une infection grippale. Les substrats peuvent présenter une réactivité différente selon les divers types de neuramidases de la grippe, ce qui permet de discerner le type spécifique d'infection grippale et de prescrire un traitement approprié et/ou un traitement de soutien à cet effet.
PCT/US1990/007677 1989-12-29 1990-12-27 Substrats d'acide n-acetylneuraminique chromogene modifie dans les positions 7 ou 8 utilises dans des procedes de diagnostic de la grippe chez l'homme WO1991009975A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US45908189A 1989-12-29 1989-12-29
US459,081 1989-12-29

Publications (1)

Publication Number Publication Date
WO1991009975A1 true WO1991009975A1 (fr) 1991-07-11

Family

ID=23823331

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1990/007677 WO1991009975A1 (fr) 1989-12-29 1990-12-27 Substrats d'acide n-acetylneuraminique chromogene modifie dans les positions 7 ou 8 utilises dans des procedes de diagnostic de la grippe chez l'homme

Country Status (2)

Country Link
AU (1) AU7176091A (fr)
WO (1) WO1991009975A1 (fr)

Cited By (8)

* 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
WO2001025246A2 (fr) * 1999-10-05 2001-04-12 Ibbex, Inc. Substrats chromogeniques de sialidases d'origine bacterienne, virale, protozoaire, et vertebree, et procede de fabrication et d'utilisation de ceux-ci
US6512100B1 (en) 1997-10-27 2003-01-28 Ibbex, Inc. Chromogenic substrates of sialidase and methods of making and using the same
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
EP1520858A1 (fr) * 2002-07-05 2005-04-06 Otsuka Chemical Company, Ltd. Procede de production d'un peptide de sucre comprenant une chaine de sucre d'asparagine et peptide de sucre resultant
WO2010029302A2 (fr) * 2008-09-11 2010-03-18 The University Of Bath Composés pour traiter des infections virales
WO2015123756A1 (fr) * 2014-02-18 2015-08-27 The University Of British Columbia Dérivés d'acide sialique résistants à l'hydrolyse et leurs méthodes d'utilisation
US20190233404A1 (en) * 2018-01-26 2019-08-01 The University Of British Columbia Compositions and methods for neuraminidase detection and quantitification

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991009972A1 (fr) * 1989-12-29 1991-07-11 Symex Corp. Procedes de diagnostic de la grippe chez l'homme et substrats d'acide n-acetylneuraminique chromogene modifie en position 4 utilises dans lesdits procedes

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3950322A (en) * 1973-08-27 1976-04-13 Research Corporation Fluorogenic substrate glycosides
US4316954A (en) * 1980-04-18 1982-02-23 Eastman Kodak Company Assay for neuraminic acids
US4675391A (en) * 1985-03-06 1987-06-23 Kanto Ishi Pharmaceutical Co., Ltd. Glycoside derivatives
US4731324A (en) * 1984-05-21 1988-03-15 Cooper-Lipotech, Inc. Viral lysis assay
US4810636A (en) * 1986-12-09 1989-03-07 Miles Inc. Chromogenic acridinone enzyme substrates

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3950322A (en) * 1973-08-27 1976-04-13 Research Corporation Fluorogenic substrate glycosides
US4316954A (en) * 1980-04-18 1982-02-23 Eastman Kodak Company Assay for neuraminic acids
US4731324A (en) * 1984-05-21 1988-03-15 Cooper-Lipotech, Inc. Viral lysis assay
US4675391A (en) * 1985-03-06 1987-06-23 Kanto Ishi Pharmaceutical Co., Ltd. Glycoside derivatives
US4810636A (en) * 1986-12-09 1989-03-07 Miles Inc. Chromogenic acridinone enzyme substrates

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
J. KEESEY, "BIOCHEMICAL INFORMATION", Published 1987, by BOEHRINGER MANNHEIM BIOCHEMICALS, (Indianapolis, Indiana), see page 176. *

Cited By (22)

* 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
US6812332B2 (en) 1997-10-27 2004-11-02 Ibbex, Inc. Chromogenic substrates of sialidase and methods of making and using the same
US6844346B2 (en) 1997-10-27 2005-01-18 Ibbex, Inc. Chromogenic substrates of sialidase and methods of making and using the same
US6512100B1 (en) 1997-10-27 2003-01-28 Ibbex, Inc. Chromogenic substrates of sialidase and methods of making and using the same
US6667161B1 (en) 1997-10-27 2003-12-23 Ibbex, Inc. Chromogenic substrates of sialidase of bacterial, viral, protozoa, and vertebrate origin and methods of making and using the same
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
AU781391B2 (en) * 1999-10-05 2005-05-19 Ibbex, Inc. Chromogenic substrates of sialidase of bacterial, viral, protozoa, and vertebrate origin and methods of making and using the same
WO2001025246A2 (fr) * 1999-10-05 2001-04-12 Ibbex, Inc. Substrats chromogeniques de sialidases d'origine bacterienne, virale, protozoaire, et vertebree, et procede de fabrication et d'utilisation de ceux-ci
WO2001025246A3 (fr) * 1999-10-05 2001-11-29 Ibbex Inc Substrats chromogeniques de sialidases d'origine bacterienne, virale, protozoaire, et vertebree, et procede de fabrication et d'utilisation de ceux-ci
US7943763B2 (en) 2002-07-05 2011-05-17 Otsuka Chemical Holdings Co., Ltd. Process for preparing glycopeptides having asparagine-linked oligosaccharides, and the glycopeptides
EP1520858A4 (fr) * 2002-07-05 2007-12-26 Otsuka Chemical Co Ltd Procede de production d'un peptide de sucre comprenant une chaine de sucre d'asparagine et peptide de sucre resultant
EP1520858A1 (fr) * 2002-07-05 2005-04-06 Otsuka Chemical Company, Ltd. Procede de production d'un peptide de sucre comprenant une chaine de sucre d'asparagine et peptide de sucre resultant
US8063202B2 (en) 2002-07-05 2011-11-22 Otsuka Chemical Co., Ltd. Process for preparing glycopeptides having asparagine-linked oligosaccharides, and the glycopeptides
EP2412720A2 (fr) * 2002-07-05 2012-02-01 Otsuka Chemical Co., Ltd. Procédé de préparation de glycopeptides dotés d'oligosaccharides à asparagine liée et glycopeptides
EP2412720A3 (fr) * 2002-07-05 2012-06-13 Otsuka Chemical Co., Ltd. Procédé de préparation de glycopeptides dotés d'oligosaccharides à asparagine liée et glycopeptides
WO2010029302A2 (fr) * 2008-09-11 2010-03-18 The University Of Bath Composés pour traiter des infections virales
WO2010029302A3 (fr) * 2008-09-11 2010-05-06 The University Of Bath Composés pour traiter des infections virales
WO2015123756A1 (fr) * 2014-02-18 2015-08-27 The University Of British Columbia Dérivés d'acide sialique résistants à l'hydrolyse et leurs méthodes d'utilisation
US10023601B2 (en) 2014-02-18 2018-07-17 The University Of British Columbia Hydrolysis resistant sialic acid derivatives and methods for their use
US20190233404A1 (en) * 2018-01-26 2019-08-01 The University Of British Columbia Compositions and methods for neuraminidase detection and quantitification
US11639348B2 (en) * 2018-01-26 2023-05-02 The University Of British Columbia Compositions and methods for neuraminidase detection and quantification

Also Published As

Publication number Publication date
AU7176091A (en) 1991-07-24

Similar Documents

Publication Publication Date Title
US5663055A (en) Methods for diagnosing human influenza and 4-position modified chromogenic N-acetylneuraminic acid substrated for use therein
EP0888371B1 (fr) 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
US7081352B2 (en) Chemiluminescent influenza diagnostic kit
EP0507854B1 (fr) Methode de diagnostic de la grippe chez l'homme et substrats d'acide n-acetylneuraminique chromogene modifie en position 4 utilises dans lesdites methodes
CN107602643B (zh) 一种基于萘酰亚胺的β-葡萄糖醛酸苷酶的荧光探针及其应用
WO1991009975A1 (fr) Substrats d'acide n-acetylneuraminique chromogene modifie dans les positions 7 ou 8 utilises dans des procedes de diagnostic de la grippe chez l'homme
JP3947218B2 (ja) ４−アルコキシ−ｎ−アセチルノイラミン酸の合成
US8795979B2 (en) Hydrolase enzyme substrates and uses thereof
WO1991010744A1 (fr) Substrats chromogeniques d'acide n-acetylneuraminique modifie en position 9 et procedes de diagnostic de la grippe chez l'homme utilisant de tels substrats
AU781391B2 (en) Chromogenic substrates of sialidase of bacterial, viral, protozoa, and vertebrate origin and methods of making and using the same
WO1991009971A1 (fr) Substrats d'acide neuraminique chromogene modifie en position 5 et methodes de diagnostic de la grippe humaine les utilisant
US11639348B2 (en) Compositions and methods for neuraminidase detection and quantification
US6420552B1 (en) Syntheses of 4-alkyl chromogenic glycosides and 7-alkyl chromogenic glycosides of N-acetylneuraminic acids
JPH01131192A (ja) 色原体アクリジノン酵素基質及びその製造方法
MXPA98004081A (en) 4,7-dialkoxy-n-acetylneuraminic acid derivatives and methods for detection of influenza type a and b viruses in clinical specimens
Cui Synthetic Glycans Analogues for the Precise Point-of-care Detection of Pathogens
JP5167451B2 (ja) 蛍光性アミラーゼ基質及び該基質を用いたアミラーゼ活性測定方法
JPH0653076B2 (ja) グリコシル化合物加水分解酵素活性測定試薬

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AT AU BB BG BR CA CH DE DK ES FI GB HU JP KP KR LK LU MC MG MW NL NO RO SD SE SU US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE BF BJ CF CG CH CM DE DK ES FR GA GB GR IT LU ML MR NL SE SN TD TG

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: CA