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WO1991009975A1 - Chromogenic 7- or 8-position modified n-acetylneuraminic acid substrates and methods for diagnosing human influenza therewith - Google Patents

Chromogenic 7- or 8-position modified n-acetylneuraminic acid substrates and methods for diagnosing human influenza therewith Download PDF

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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
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Prior art keywords
nitrophenyl
chloro
bromo
naphthyl
group
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PCT/US1990/007677
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French (fr)
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 WO1991009975A1 publication Critical patent/WO1991009975A1/en

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    • 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.

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Abstract

Chromogenic derivatives of N-acetylneuraminic acid modified in the 7- or 8-position are used as substrates in colorimetric assays for human influenza neuraminidase activity in clinical specimens for the purpose of selectively diagnosing influenza infection. The substrates may exhibit different reactivity with the different types of influenza neuraminidases, thus enabling one to discern the specific type of influenza infection and prescribe appropriate treatment and/or supportive therapy therefor.

Description

CHROMOGENIC 7- OR 8-POSITION MODIFIED
N-ACETYLNEURAMINIC ACID SUBSTRATES AND METHODS FOR DIAGNOSING HUMAN INFLUENZA THEREWITH
Technical Field 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) .
Background of the Invention
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. , et al., Biochimica et Biophysica Acta 523:435-442 (1978), describes a colori- metric assay for NA using 2-(3-methoxyphenyl)-N- acetyl-alpha-D neuraminic acid as a substrate and 4-aminoantipyrine in the presence of an oxidizing agent to measure the enzymatically released methoxyphenol. Myers, R.W. , et al., Analytical Biochemistry 101:166-174 (1980) , describes the use of the 4-methylumbelliferyl- alpha-ketoside of Neu5Ac in a fluorometric assay for NA. This chromogenic derivative of Neu5Ac was also used in studies of the NA activity of influenza viruses by Yolken, R.H., et al., J. Infectious Diseases 142:5116-523 (1980); Clinical Chemistry 27:1490-1498 (1981); and Reviews of Infectious Diseases 4:35-68 (1982); and by Kiyotani et al., Hiroshima J. Medical Sciences 33:287-292 (1984); Zbl Bakt Hyg A260-273-285 (1985); Microbiol. Immun. 31:1131-1135 (1987). Despite the availability of these prior NA assays, however, physicians currently still diagnose influenza solely on the basis of symptom- ology. This is in part due to the fact that these prior assays were complicated and/or required equipment not typically found in a clinical setting. Another short¬ coming of these prior assays is that they were unable to discriminate between influenza type. That ability is particularly important to enable physicians to prescribe the appropriate chemotherapy and/or supportive therapy to combat the infection.
Prior workers have investigated the relation¬ ship between the chemical structure of Neu5Ac and its biological function as a substrate for non-influenza NA. Gross, H.J., et al., Biochemistry 27:4279 (1988), examined benzyl-alpha-glycosides of N-acetyl-4-epi-D- neuraminic acid as a substrate for three different bacterial NAs (C^ perfrin ens, A. ureafaciens. and Vj. cholera) and found significant differences in reactivity. After 22 hrs, the C^ perfrinqens NA cleaved 100% of the substrate while the A_j_ ureafaciens and V. cholera NAs cleaved only 50% and 11% of the substrate, respectively. Kim et al., J. Am. Chem. Soc. 110:6481- 6486 described the structural characteristics of substrates accepted by NeuSAc aldolase, its use in the synthesis of NeuSAc, and its chemical conversion to the 2-deoxy derivatives, and additionally reported that work was in progress to determine the biological activity of the 2-deoxy derivatives. Brossmer et al., Helv. Chim. Acta 69:2127 (1986); Glycoconjugates 4:145 (1987) reported that the methyl-alpha-glycoside of 4-deoxy NeuSAc was a good substrate for fowl plague viral NeuSAc, but not for the three bacterial NAs mentioned above. Additionally, Schauer, R. , et al., Eur. J. Biochem.
106:531 (1980), reported that 4-methoxy Neu5Ac was an excellent substrate for fowl plague viral NA but not for V. cholera NA. The 4-methylumbelliferyl derivative of 4-deoxy NeuSAc is also described in the literature (Helv. Chim. Acta. 69:1927 (1986)). Zbiral et al., Monatsheft fur Chemie 119:127-141 (1988) described the synthesis of 7- and 8-deoxy NeuSAc. Zbiral et al., Liebigs Ann Chem, 519-526 described the synthesis of the 4-methyl- umbelliferyl-2-α glycosides of 7-epi, 8-epi, 7,8-bis- epi, 8-deoxy, 9-deoxy, and 4,7-dideoxy NeuSAc and investigated the behavior of those compounds as inhibitors of the sialidase for V. cholera. Gross, H.J. , et al., Eur. J. Biochemistry 106:531 (1987) refers to the 9-azido and 9-fluoro analogs of Neu5Ac and the 7-epi and 7,8-bis-epi analogs of NeuSAc.
Dislosure of the Invention
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 N-acetylneuraminic acid substrate of the formula:
Figure imgf000006_0001
where Ac represents acetyl, R represents hydrogen, fluorine, hydroxy, azido or cyano, R 2 represents hydroxy, hydrogen, fluorine, oxo (=0) , or azido with the proviso that one of R 1 and R2 must be hydroxyl but not both of R1
2 and R are hydroxyl, and X represents a chromogenic group that exhibits distinct color when cleaved from the substrate or a salt of said substrate; and
(b) detecting neuraminidase activity by observing whether the sample-substrate 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:
(a) incubating the sample with a chromogenic modified N-acetylneuraminic acid substrate of the formula:
1
Figure imgf000007_0001
where Ac represents acetyl, R represents hydrogen, fluorine, hydroxy, azido or cyano, R 2 represents hydroxy, hydrogen, fluorine, oxo (=0) , or azido with the proviso that one of R 1 and R2 must be hydroxyl but not both of R1
2 and R are hydroxyl, and X represents a chromogenic group that exhibits distinct color when cleaved from the substrate or a salt of said substrate;
(b) observing the color exhibited by the sample-substrate 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 substrate.
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:
Figure imgf000008_0001
where Ac represents acetyl, R represents fluorine, hydroxy, azido or cyano, R 2 represents hydroxy, hydrogen, fluorine, oxo (=0) , or azido with the proviso that one of
R 1 and R2 must be hydroxyl but not both of Rl and R? are hydroxyl, and 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:
Figure imgf000009_0001
where Ac represents acetyl, R represents hydrogen, R2 represents hydroxyl, and 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.
Brief Description of the Drawings In the drawings:
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.
Modes for Carrying Out the Invention 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. In this regard, 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. Examples of 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. monosodium phosphate-disodium phosphate mixture, monosodium phosphate-sodium hydroxide mixture, trisodium phosphate-hydrochloric acid mixture, etc.), 2-(N-morpholino)ethanesulfonc acid, [bis-(2- hydroxyethyl)imino]tris(hydroxymethyl)methane, N-2-aceta- midoiminodiacetic acid, 1,3-bis[tris(hydroxymethyl)- methylamino]propane, piperazine-N,N*-bis(2-ethane- sulfonic acid) , N-2-acetamido-2-aminoethanesulfonic acid, 3-(N-morpholino)-2-hydroxypropanesulfonic acid, N-N-bis-(2-hydroxyethyl)2-aminoethanesulfonic acid, 3-(N-morpholino)propanesulfonic acid, 2-[tris(hydroxy¬ methyl)methylamino]ethanesulfonic acid, N-2-hydroxy- ethylpiperazine-N'-2-ethanesulfonic acid, 3-{[tris-
(hydroxymethy) ethyl]amino}-2-hydroxypropane-sulfonic acid.
Examples of 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.
Examples of 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. In order to stabilize the activity of the neuraminidase-containing viruses, 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. Once mixed with the buffer solution, 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:
Figure imgf000013_0001
where R 1, R2, X and Ac are as defi.ned previ.ously. Preferably 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.
As used herein the term "chromogen" is intended to include, without limitation, molecules that exhibit fluorescence. The term "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-ketoside, 2-[4-(4-nitrophenylazo)resorcinyl]-7-deoxy-N-acetyl¬ neuraminic acid-alpha-ketoside, 3-methoxyphenyl-7-deoxy- N-acetylneuraminic acid-alpha-ketoside, 3-dimethylamino- phenyl-7-deoxy-N-acetylneuraminic acid-alpha-ketoside, 4-chloro-l-naphthyl-7-deoxy-N-acetyl-neuraminic acid- alpha-ketoside, 6-bromo-2-naphthyl-7-deoxy-N-acetyl- neuraminic acid-alpha-ketoside, 4-methylumbelliferyl-7- fluoro-N-acetylneuraminic acid-alpha-ketoside, 3-cyano- umbelliferyl-7-fluoro-N-acetylneuraminic acid-alpha- ketoside, 2-nitrophenyl-7-fluoro-N-acetylneuraminic acid-alpha-ketoside, 4-nitrophenyl-7-fluoro-N-acetyl- neuraminic acid-alpha-ketoside, 3-resorufin-7-fluoro-N- acetylneuraminic acid-alpha-ketoside, 5-bromo4-chloro- 3-indolyl-7-fluoro-N-acetylneuraminic acid-alpha- ketoside, 2-[4-(4-nitrophenylazo)phenyl]-7-fluoro-N- acetylneuraminic acid-alpha-ketoside, 2-[4-(4-nitro¬ phenylazo)resorcinyl]-7-fluoro-N-acetylneuraminic acid- alpha-ketoside, 3-methoxyphenyl-7-fluoro-N-acetyl- neuraminic acid-alpha-ketoside, 3-dimethylaminophenyl-7- fluoro-N-acetyl-neuraminic acid-alpha-ketoside, 4-chloro- l-naphthyl-7-fluoro-N-acetyl-neuraminic acid-alpha- ketoside, 6-bromo-2-naphthyl-7-fluoro-N-acetylneuraminic acid-alpha-ketoside, 4-methylumbelliferyl-7-azido-N- acetylneuraminic acid-alpha-ketoside, 3-cyanoumbelli- feryl-7-azido-N-acetylneuraminic acid-alpha-ketoside, 2-nitrophenyl-7-azido-N-acetylneuraminic acid-alpha- ketoside, 4-nitrophenyl-7-azido-N-acetylneuraminic acid-alpha-ketoside, 3-resorufin-7-azido-N-acety1- neuraminic acid-alpha-ketoside, 5-bromo4-chloro- 3-indolyl-7-azido-N-acetylneuraminic acid-alpha- ketoside, 2-[4-(4-nitrophenylazo)phenyl]-7-azido-N- acetylneuraminic acid-alpha-ketoside, 2-[4-(4-nitro¬ phenylazo)resorcinyl]-7-azido-N-acetylneuraminic acid-alpha-ketoside, 3-methoxyphenyl-7-azido-N-acetyl- neuraminic acid-alpha-ketoside, 3-dimethylaminophenyl-7- azido-N-acetylneuraminic acid-alpha-ketoside, 4-chloro- l-naphthyl-7-azido-N-acetylneuraminic acid-alpha- ketoside, 6-bromo-2-naphthyl-7-azido-N-acetylneuraminic acid-alpha-ketoside, 4-methylumbelliferyl-7-keto-N- acetylneuraminic acid-alpha-ketoside, 3-cyanoumbelli- fery1-7-keto-N-acetylneuraminic acid-alpha-ketoside, 2-nitrophenyl-7-keto-N-acetylneuraminic acid-alpha- ketoside, 4-nitropheny1-7-keto-N-acetylneuraminic acid-alpha-ketoside, 3-resorufin-7-keto-N-acetyl¬ neuraminic acid-alphaketoside, 5-bromo-4-chloro- 3-indolyl-7-keto-N-acetylneuraminic acid-alpha-ketoside, 2-[4-(4-nitrophenylazo)phenyl]-7-keto-N-acetylneuraminic acid-alpha-ketoside, 2-[4-(4-nitrophenylazo)resorcinyl]- 7-keto-N-acetylneuraminic acid-alpha-ketoside, 3-methoxy- pheny1-7-keto-N-acetylneuraminic acid-alpha-ketoside, 3-dimethylaminopheny1-7-keto-N-acetylneuraminic acid- alpha-ketoside, 4-chloro-l-naphthyl-7-azido-N-acetyl- neuraminic acid-alpha-ketoside, 6-bromo-2-naphthyl-7- keto-N-acetylneuraminic acid-alpha-ketoside, 4-me hy1- umbelliferyl-8-deoxy-N-acetylneuraminic acid-alpha- ketoside, 3-cyanoumbelliferyl-8-deoxy-Nacetylneuraminic acid-alpha-ketoside, 2-nitropheny1-8-deoxy-N-acetyl- neuraminic acid-alpha-ketoside, 4-nitropheny1-8-deoxy-N- acetylneuraminic acid-alphaketoside, 3-resorufin-
8-deoxy-N-acetylneuraminic acid-alpha-ketoside, 5-bromo- 4-chloro-3-indolyl8-deoxy-N-acetylneuraminic acid- alpha-ketoside, 2-[4-(4-nitrophenylazo)phenyl]-8-deoxy- N-acetylneuraminic acid-alpha-ketoside, 2-[4-(4-nitro- phenylazo) esorcinyl]-8-deoxy-N-acetylneuraminic acid- alphaketoside, 3-methoxyphenyl-8-deoxy-N-acetylneuraminic acid-alpha-ketoside, 3-dimethylaminophenyl-8-deoxy-N- acetylneuraminic acid-alpha-ketoside, 4-chloro-l- naphthyl-7-azido-N-acetylneuraminic acid-alpha-ketoside, 6-bromo-2-naphthyl-8-deoxy-N-acetylneuraminic acid-alpha¬ ketoside, 4-methylumbelliferyl-8-fluoro-N-acetyl¬ neuraminic acid-alpha-ketoside, 3-cyanoumbelli- feryl-8-fluoro-N-acetylneuraminic acid-alpha-ketoside, 2-nitropheny1-8-fluoro-N-acetylneuraminic acid-alpha- ketoside, 4-nitropheny1-8-fluoro-N-acetylneuraminic acid-alphaketoside, 3-resorufin-8-fluoro-N-acetyl¬ neuraminic acid-alpha-ketoside, 5-bromo-4-chloro- 3-indolyl-8-fluoro-N-acetylneuraminic acid-alpha¬ ketoside, 2-[4-(4-nitrophenylazo)phenyl]-8-fluoro-N- acetylneuraminic acid-alpha-ketoside, 2-[4-(4-nitro¬ phenylazo)resorcinyl]-8-fluoro-N-acetylneuraminic acid-alpha-ketoside, 3-methoxyphenyl-8-fluoro-N-acetyl¬ neuraminic acid-alpha-ketoside, 3-dimethylaminophenyl-8- fluoro-N-acetylneuraminic acid-alpha-ketoside, 4-chloro- l-naphthyl-7-azido-N-acetylneuraminic acid-alpha¬ ketoside, 6-bromo-2-naphthy1-8-fluoro-N-acetylneuraminic acid-alpha-ketoside, 4-methylumbellifery1-8-azido-N- acetylneuraminic acid-alpha-ketoside, 3-cyanoumbelli- feryl8-azido-N-acetylneuraminic acid-alpha-ketoside, 2-nitropheny1-8-azido-N-acetylneuraminic acid-alpha¬ ketoside, 4-nitrophenyl-8-azido-N-acetylneuraminic acid- alphaketoside, 3-resorufin-8-azido-N-acetylneuraminic acid-alpha-ketoside, 5-bromo-4-chloro-3-indolyl-8- azido-N-acetylneuraminic acid-alpha-ketoside, 2-[4-(4-nitrophenylazo)phenyl]-8-azido-N-acetylneuraminic acid-alpha-ketoside, 2-[4-(4-nitrophenylazo)resorcinyl]- 8-azido-N-acetylneuraminic acid-alphaketoside, 3-methoxy- phenyl-8-azido-N-acetylneuraminic acid-alpha-ketoside, 3-dimethylaminophenyl-8-azido-N-acetylneuraminic acid- alpha-ketoside, 4-chloro-l-naphthyl-7-azido-N-acetyl- neuraminic acid-alpha-ketoside, 6-bromo-2-naphthyl-8- azido-N-acetylneuraminic acid-alpha-ketoside, 4-methyl- umbelliferyl-8-cyano-N-acetylneuraminic acid-alpha¬ ketoside, 3-cyanoumbelliferyl-8-cyanoN-acetylneuraminic acid-alpha-ketoside, 2-nitropheny1-8-cyano-N-acetyl- neuraminic acid-alpha-ketoside, 4-nitropheny1-8-cyano-N- acetylneuraminic acid-alphaketoside, 3-resorufin-8- cyano-N-acetylneuraminic acid-alpha-ketoside, 5-bromo- 4-chloro3-indolyl-8-cyano-N-acetylneuraminic acid-alpha- ketoside, 2-[4-(4-nitrophenylazo)phenyl]-8-cyano-N- acetylneuraminic acid-alpha-ketoside, 2-[4-(4-nitro¬ phenylazo)resorcinyl]-8-cyano-N-acetylneuraminic acid- alphaketoside, 3-methoxyphenyl-8-cyano-N-acetylneuraminic acid-alpha-ketoside, 3-dimethylaminophenyl-8-cyano-N- acetylneuraminic acid-alpha-ketoside, 4-chloro-l- naphthyl-7-azido-N-acetylneuraminic acid-alpha-ketoside and 6-bromo-2-naphthyl-8-cyano-N-acetylneuraminic acid- alpha-ketoside.
The above-described NeuSAc derivatives are generally made by protecting the functional groups of
NeuSAc at the 1, 2, 4, 7 or 8 (as the case may be) , and 9 positions, modifying the 7 or 8 position as indicated, deprotecting the other positions, and coupling the 7- or 8-modified NeuSAc with the chromogen. Details of these reactions are provided in the Examples, infra. The
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. Since the substrates 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 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.
Released Type of Chromogen Detection Color
5-bromo-4- colorimetric/ blue/purple in chloro-3- visual the presence of indolol nitroblue tetrazolium
4-methyl- fluorometric fluorescent umbelliferone emission at 450 nm after excit¬ ation at 360 nm
3-cyanoumbelli- fluorometric fluorescent ferone emission at 454 nm after excit¬ ation at 415 nm resorufin colorimetric/ pink/red visual
2-nitrophenol colorimetric/ yellow visual
4-nitrophenol colorimetric/ yellow visual
nitrophenylazo- colorimetric/ orange phenol visual
nitrophenylazo- colorimetric/ green blue resorcinol visual (presence of Mg++)
3-methoxyphenol colorimetric/ red to blue visual after reaction with diazonium salt
3-dimethyl- colorimetric/ red to blue aminophenol visual after reaction with diazonium salt
6-bromo-2- colorimetric/ red to blue naphthol visual after reaction with diazonium salt 4-chloro-l- colorimetric/ red to blue naphthol visual after reaction with diazonium salt
Accordingly, 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.
Examples
l. Synthesis of 7-Keto NeuSAc
The synthesis scheme for this compound is shown in Figure 1.
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. 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.
2. Synthesis of Chromogenic 7-Keto NeuSAc
The synthesis scheme for this compound is shown in Figure 2.
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.
3. Synthesis of 7-Azido NeuSAc The synthesis scheme for this compound is shown in Figure 3.
8,9-isopropylidene protected NeuSAc-MEMK is formed according to the procedure detailed in Example 1. Treatment with 1 eq. TBDMS-Cl, imidazole and 4-dimethylaminopyridine in DMF at 65-70°C affords
8,9-isopropylidene-4-0-TBDMS NeuSAc methyl ester methyl ketoside. Treatment of this compound with triethylamine and methanesulfonyl (Ms) chloride in methylene chloride forms the 8,9-isopropylidene-4-0-TBDMS-7-Ms NeuSAc methyl ester methyl ketoside. Reaction of this compound with sodium azide in methyl ethyl ketone at 100°C will form the corresponding 7-azido-8,9-isopropylidene-4-0-TBDMS Neu5Ac. 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.
4. Synthesis of Chromogenic 7-Azido NeuSAc
The synthesis scheme for this compound is depicted in Figure 4.
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. First 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
2-(3-resorufin)-7-azido-N-acetylneuraminic acid. 5. Synthesis of 7-Deoxy NeuSAc
The synthesis scheme for this compound is also shown in Figure 3.
4-0-TBDMS-7-mesyl-8,9-isopropylidene NeuSAc methyl ester methyl ketoside is prepared according to the procedure given above. Treatment of this compound in sodium borohydride in DMF gives the reduction product. Full deprotection by treatment with sodium hydroxide followed by Dowex-50W (H ) , then treatment with tetrabutylammonium fluoride in THF, and finally treatment with dilute HCl/Dowex-50W (H+) affords 7-deoxy-N-acetylneuraminic acid.
6. Synthesis of Chromogenic 7-Deoxy NeuSAc The synthesis scheme for this compound is shown in
Figure 5.
7-deoxy NeuSAc is converted to its methyl ester and then to the peracetylated glycosyl chloride using excess acetyl chloride overnight. Coupling for 2 hr with the sodium salt of resorufin takes place in DMF. The coupled product will then be deprotected by treatment with sodium methoxide in methanol followed by sodium hydroxide to form the sodium salt of
2-(3-resorufin)-7-deoxy-N-acetylneuraminic acid.
7. Synthesis of 8-Azido NeuSAc
The synthesis scheme for this compound is shown in Figure 6.
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.
8. Synthesis of Chromogenic 8-Azido NeuSAc
The synthesis scheme for this compound is shown in Figure 7.
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. The desired product, 2-[3-(4-chloro-5- bromo)-indolyl]-8-azido-N-acetylneuraminic acid-alpha¬ ketoside (sodium salt) , is obtained after deprotection with base, fluoride ion and dilute acid.
9. Synthesis of 8-Cyano NeuSAc
The synthesis scheme for this compound is shown in Figure 8. The 8-tosylated 4,9-di-O-TBDMS Neu5Ac-MEMK is prepared as in the synthesis of 8-azido NeuSAc. The tosyl group is displaced with sodium cyanide in acetone (100°C) to give the 8-cyano derivative. The final desired product, 8-cyano-N-acetylneuraminic acid, is obtained after deprotection with base, fluoride ion and dilute acid. 10. Synthesis of Chromogenic 8-Cyano Neu5Ac
The synthesis scheme for this compound is shown in
Figure 9.
The synthesis of 5-bromo-4-chloro-3-indolyl-8-cyano Neu5Ac is done in the same manner as the corresponding
5-bromo-4-chloro-3-indolyl-8-azido NeuSAc (see Example
8) , only the displacement reaction is done with sodium cyanide rather than sodium azide.
11. Synthesis of 8-Deoxy Neu5Ac
The synthesis scheme for this compound is shown in Figure 10.
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.
12. Synthesis of Chromogenic 8-Deoxy NeuSAc
The synthesis scheme for this compound is shown in Figure 11.
The 4,9-di-0-TBDMS-8-deoxy Neu5Ac-MEMK intermediate is deprotected with dilute hydrochloric acid and the free acid reesterified with methanol/trifluoroacetic acid. As before, the corresponding glycosyl chloride peracetate is formed by treatment in excess acetyl chloride and coupled with the sodium salt of nitrophenylazophenol in DMF. Standard base and acid deprotection affords the adduct
2-[4-(4-nitrophenylazo)-phenyl]-8-deoxy-N-acetylneuranimi c acid-alpha-ketoside (sodium salt) . 13. Synthesis of 7-Fluoro Neu5Ac
The synthesis scheme for this compound is depicted in Figure 12-
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.
14. Synthesis of Chromogenic 7-Fluoro NeuSAc The synthesis scheme for this compound is depicted in Figure 13.
The synthesis of 4-chloro-l-naphthyl-7-flouro Neu5Ac is done in the same manner as in Example 12 except that the sodium salt of 4-chloro-l-naphthol is used.
15. Enzymatic testing of 7-epi-Neu5Ac
50 μl of an influenza virus was mixed with a reaction mixture containing 50 μl of the substrate 4- methylumbelliferyl NeuSAc at various concentrations in the submillimolar to millimolar range, 150 μl of the inhibitor 7-epi-Neu5Ac at various concentrations in the submillimolar to millimolar range, and 50 μl 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 μl 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 μl 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:
Figure imgf000027_0001
(The native substrate, Neu5Ac, had a K^=0.626 mM when the Influenza A (H1N1) virus was used.)
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.
Modifications of the above-described modes for carrying out the invention that are obvious to those of skill in the fields of organic chemistry, virology, biochemistry, medical diagnostics, and related fields are intended to be within the scope of the following claims.

Claims

Claims
1. 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 N-acetylneuraminic acid substrate of the formula:
Figure imgf000028_0001
where Ac represents acetyl, R represents hydrogen, fluorine, hydroxy, azido or cyano, R 2 represents hydroxy, hydrogen, fluorine, oxo, or azido with the proviso that one of R 1 and R2 must be hydroxyl but not both of R1 and
R 2 are hydroxyl, and X represents a chromogenic group that exhibits distinct color when cleaved from the substrate or a salt of said substrate; and
(b) detecting neuraminidase activity by observing whether the sample-substrate mixture exhibits said color after step (a) .
2. The method of claim l wherein the clinical sample is a pharyngeal, nasopharyngeal or respiratory secretion.
3. The method of claim 1 or 2 wherein R
2 represents hydrogen, R represents hydroxy, and X is selected from the group consisting of 4-methylumbelli- feryl, 3-cyanoumbelliferyl, 2-nitrophenyl, 4-nitrophenyl, 3-resorufin, 5-bromo-4-chloro-3-indolyl, 4-nitrophenyl¬ azoresorcinyl, 4-nitrophenylazophenyl, 3-methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-l-naphthyl and 6-bromo- 2-naphthyl.
4. The method of claim 1 or 2 wherein R represents fluorine, R 2 represents hydroxy, and X is selected from the group consisting of 4-methylumbelli- feryl, 3-cyanoumbelliferyl, 2-nitrophenyl, 4-nitrophenyl, 3-resorufin, 5-bromo-4-chloro-3-indolyl, 4-nitrophenyl¬ azoresorcinyl, 4-nitrophenylazophenyl, 3-methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-l-naphthyl and 6-bromo- 2-naphthyl.
5. The method of claim 1 or 2 wherein R represents azido, R 2 represents hydroxy, and X is selected from the group consisting of 4-methylumbelli- feryl, 3-cyanoumbelliferyl, 2-nitrophenyl, 4-nitrophenyl, 3-resorufin, 5-bromo-4-chloro-3-indolyl, 4-nitrophenyl¬ azoresorcinyl, 4-nitrophenylazophenyl, 3-methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-l-naphthyl and 6-bromo- 2-naphthyl.
6. The method of claim 1 or 2 wherein R represents cyano, R 2 represents hydroxy, and X is selected from the group consisting of 4-methylumbelli- feryl, 3-cyanoumbelliferyl, 2-nitrophenyl, 4-nitrophenyl, 3-resorufin, 5-bromo-4-chloro-3-indolyl, 4-nitrophenyl¬ azoresorcinyl, 4-nitrophenylazophenyl, 3-methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-l-naphthyl and 6-bromo- 2-naphthyl.
7. The method of claim 1 or 2 wherein R
2 represents hydroxy, R represents hydrogen, and X is selected from the group consisting of 4-methylumbelli- feryl, 3-cyanoumbelliferyl, 2-nitrophenyl, 4-nitrophenyl, 3-resorufin, 5-bromo-4-chloro-3-indolyl, 4-nitrophenyl¬ azoresorcinyl, 4-nitrophenylazophenyl, 3-methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-l-naphthyl and 6-bromo- 2-naphthyl.
8. The method of claim 1 or 2wherein R represents hydroxy, R 2 represents fluorine, and X is selected from the group consisting of 4-methylumbelliferyl, 3-cyano¬ umbelliferyl, 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.
9. The method of claim 1 or 2 wherein R represents hydroxy, R 2 represents oxo, and X is selected from the group consisting of 4-methylumbelliferyl, 3-cyanoumbelliferyl, 2-nitrophenyl, 4-nitrophenyl, 3-resorufin, 5-bromo-4-chloro-3-indolyl, 4-nitrophenyl¬ azoresorcinyl, 4-nitrophenylazophenyl, 3-methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-l-naphthyl and 6-bromo- 2-naphthyl.
10. The method of claim 1 or 2 wherein R
2 represents hydroxy, R represents azido, and X is selected from the group consisting of 4-methylumbelli¬ feryl, 3-cyanoumbelliferyl, 2-nitrophenyl, 4-nitrophenyl, 3-resorufin, 5-bromo-4-chloro-3-indolyl, 4-nitrophenyl¬ azoresorcinyl, 4-nitrophenylazophenyl, 3-methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-l-naphthyl and 6-bromo- 2-naphthyl.
11. A method of selectively detecting a specific type of human influenza neuraminidase activity in a clinical sample suspected of having human influenza neuraminidase activity comprising:
(a) incubating the sample with a chromogenic modified N-acetylneuraminic acid substrate of the formula:
Figure imgf000031_0001
1 where Ac represents acetyl, R represents hydrogen, fluorine, hydroxy, azido or cyano, R 2 represents hydroxy, hydrogen, fluorine, oxo, or azido with the proviso that one of R 1 and R2 must be hydroxyl but not both of Rl and
2 R are hydroxyl, and X represents a chromogenic group that exhibits distinct color when cleaved from the substrate or a salt of said substrate;
(b) observing the color exhibited by the sample-substrate 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 substrate.
12. The method of claim 11 wherein the specific type of human influenza neuraminidase activity is human influenza A neuraminidase activity or influenza B neuraminidase activity, or parainfluenza neuraminidase activity.
13. The method of claim 11 or 12 wherein the clinical sample is a pharyngeal, nasopharyngeal, or respiratory secretion.
14. The method of claim 11, 12 or 13 wherein R
2 represents hydrogen, R represents hydroxy, and X is selected from the group consisting of 4-methylumbelli¬ feryl, 3-cyanoumbelliferyl, 2-nitrophenyl, 4-nitrophenyl, 3-resorufin, 5-bromo-4-chloro-3-indolyl, 4-nitrophenyl¬ azoresorcinyl, 4-nitrophenylazophenyl, 3-methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-l-naphthyl and 6-bromo- 2-naphthy1.
15. The method of claim 11, 12 or 13 wherein R represents fluorine, R 2 represents hydroxy, and X is selected from the group consisting of 4-methylumbelli- feryl, 3-cyanoumbelliferyl, 2-nitrophenyl, 4-nitrophenyl 3-resorufin, 5-bromo-4-chloro-3-indolyl, 4-nitrophenyl¬ azoresorcinyl, 4-nitrophenylazophenyl, 3-methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-l-naphthyl and 6-bromo- 2-naphthy1.
16. The method of claim 11, 12 or 13 wherein R represents azido, R 2 represents hydroxy, and X is selected from the group consisting of 4-methylumbelli¬ feryl, 3-cyanoumbelliferyl, 2-nitrophenyl, 4-nitrophenyl 3-resorufin, 5-bromo-4-chloro-3-indolyl, 4-nitrophenyl¬ azoresorcinyl, 4-nitrophenylazophenyl, 3-methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-l-naphthyl and 6-bromo- 2-naphthy1.
17. The method of claim 11, 12 or 13 wherein R 2 represents cyano, R represents hydroxy, and X is selected from the group consisting of 4-methylumbelli¬ feryl, 3-cyanoumbelliferyl, 2-nitrophenyl, 4-nitrophenyl, 3-resorufin, 5-bromo-4-chloro-3-indolyl, 4-nitrophenyl¬ azoresorcinyl, 4-nitrophenylazophenyl, 3-methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-l-naphthyl and 6-bromo- 2-naphthy1.
18. The method of claim 11, 12 or 13 wherein R
2 represents hydroxy, R represents hydrogen, and X is selected from the group consisting of 4-methylumbelli¬ feryl, 3-cyanoumbelliferyl, 2-nitrophenyl, 4-nitrophenyl, 3-resorufin, 5-bromo-4-chloro-3-indolyl, 4-nitrophenyl¬ azoresorcinyl, 4-nitrophenylazophenyl, 3-methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-l-naphthyl and 6-bromo- 2-naphthy1.
19. The method of claim 11, 12 or 13 wherein R
2 represents hydroxy, R represents fluorine, and X is selected from the group consisting of 4-methylumbelli¬ feryl, 3-cyanoumbelliferyl, 2-nitrophenyl, 4-nitrophenyl, 3-resorufin, 5-bromo-4-chloro-3-indolyl, 4-nitropheny1- azoresorcinyl, 4-nitrophenylazophenyl, 3-methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-l-naphthyl and 6-bromo- 2-naphthy1.
20. The method of claim 11, 12 or 13 wherein R1
2 represents hydroxy, R represents oxo, and X is selected from the group consisting of 4-methylumbelliferyl, 3-cyanoumbelliferyl, 2-nitrophenyl, 4-nitrophenyl, 3-resorufin, 5-bromo-4-chloro-3-indolyl, 4-nitrophenyl¬ azoresorcinyl, 4-nitrophenylazophenyl, 3-methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-l-naphthyl and 6-bromo- 2-naphthyl.
21. The method of claim 11, 12 or 13 wherein R represents hydroxy, R 2 represents azido, and X is selected from the group consisting of 4-methylumbelli¬ feryl, 3-cyanourabelliferyl, 2-nitrophenyl, 4-nitrophenyl, 3-resorufin, 5-bromo-4-chloro-3-indolyl, 4-nitrophenyl¬ azoresorcinyl, 4-nitrophenylazophenyl, 3-methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-l-naphthyl and 6-bromo- 2-naphthy1.
22. A chromogenic substrate useful for detecting human influenza neuraminidase activity in a clinical sample suspected of having such activity, said substrate having the formula:
Figure imgf000034_0001
where Ac represents acetyl, R represents hydrogen, fluorine, hydroxy, azido or cyano, R 2 represents hydroxy, hydrogen, fluorine, oxo, or azido with the proviso that one of R 1 and R2 must be hydroxyl but not both of R1 and R 2 are hydroxyl, and X is a chromogenic group that exhibits a distinct color when cleaved from the substrate and salts of said substrate.
23. The chromogenic substrate of claim 22 wherein
R 1 represents fluori.ne, R2 represents hydroxy, and X is selected from the group consisting of 4-methylumbelli¬ feryl, 3-cyanoumbelliferyl, 2-nitrophenyl, 4-nitrophenyl, 3-resorufin, 5-bromo-4-chloro-3-indolyl, 4-nitrophenyl¬ azoresorcinyl, 4-nitrophenylazophenyl, 3-methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-l-naphthyl and 6-bromo- 2-naphthy1.
24. The chromogenic substrate of claim 22 wherein
R 1 represents azi.do, R2 represents hydroxy, and X is selected from the group consisting of 4-methylumbelli¬ feryl, 3-cyanoumbelliferyl, 2-nitrophenyl, 4-nitrophenyl, 3-resorufin, 5-bromo-4-chloro-3-indolyl, 4-nitropheny1- azoresorcinyl, 4-nitrophenylazophenyl, 3-methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-l-naphthyl and 6-bromo- 2-naphthyl.
25. The chromogenic substrate of claim 22 wherein R 1 represents cyano, R2 represents hydroxy, and X is selected from the group consisting of 4-methylumbelli¬ feryl, 3-cyanoumbelliferyl, 2-nitrophenyl, 4-nitrophenyl, 3-resorufin, 5-bromo-4-chloro-3-indolyl, 4-nitrophenyl¬ azoresorcinyl, 4-nitrophenylazophenyl, 3-methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-l-naphthyl and 6-bromo- 2-naphthyl.
26. The chromogenic substrate of claim 22 wherein
R 1 represents hydroxy, R2 represents hydrogen, and X is selected from the group consisting of 4-methylumbelli¬ feryl, 3-cyanoumbelliferyl, 2-nitrophenyl, 4-nitrophenyl, 3-resorufin, 5-bromo-4-chloro-3-indolyl, 4-nitrophenyl¬ azoresorcinyl, 4-nitrophenylazophenyl, 3-methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-l-naphthyl and 6-bromo- 2-naphthy1.
27. The chromogenic substrate of claim 22 wherein
Rl represents hydroxy, R2 represents fluori.ne, and X is selected from the group consisting of 4-methylumbelli¬ feryl, 3-cyanoumbelliferyl, 2-nitrophenyl, 4-nitrophenyl, 3-resorufin, 5-bromo-4-chloro-3-indolyl, 4-nitrophenyl¬ azoresorcinyl, 4-nitrophenylazophenyl, 3-methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-l-naphthyl and 6-bromo- 2-naphthy1.
28. The chromogenic substrate of claim 22 wherein
R 1 represents hydroxy, R2 represents oxo, and X is selected from the group consisting of 4-methylumbelli¬ feryl, 3-cyanoumbelliferyl, 2-nitrophenyl, 4-nitrophenyl, 3-resorufin, 5-bromo-4-chloro-3-indolyl, 4-nitrophenyl¬ azoresorcinyl, 4-nitrophenylazophenyl, 3-methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-l-naphthyl and 6-bromo- 2-naphthy1.
29. The chromogenic substrate of claim 22 wherein
R 1 represents hydroxy, R2 represents azido, and X is selected from the group consisting of 4-methylumbelli¬ feryl, 3-cyanoumbelliferyl, 2-nitrophenyl, 4-nitrophenyl, 3-resorufin, 5-bromo-4-chloro-3-indolyl, 4-nitropheny1- azoresorcinyl, 4-nitrophenylazophenyl, 3-methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-l-naphthyl and 6-bromo- 2-naphthyl.
30. A chromogenic substrate useful for detecting human influenza neuraminidase activity in a clinical sample suspected of having such activity, said substrate having the formula:
Figure imgf000037_0001
where Ac represents acetyl, R 1 represents hydrogen, R2 represents hydroxy, and X is selected from the group consisting of 3-cyanoumbelliferyl, 2-nitrophenyl,
4-nitrophenyl, 3-resorufin, 5-bromo-4-chloro-3-indolyl,
4-nitrophenylazoresorcinyl, 4-nitrophenylazophenyl, 3- methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-l-naphthyl and 6-bromo-2-naphthy1, and salts of said substrate.
31. A modified N-acetylneuraminic acid having the formula:
1
Figure imgf000037_0002
where Ac represents acetyl, R represents fluorine, hydroxy, azido or cyano, R 2 represents hydroxy, fluorine, oxo or azido with the proviso that one of R 1 and R2 must be hydroxyl but not both 1 and 2 are hydroxyl.
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