WO1991010744A1 - Chromogenic 9-position modified n-acetylneuraminic acid substrates and methods for diagnosing human influenza therewith - Google Patents

Chromogenic 9-position modified n-acetylneuraminic acid substrates and methods for diagnosing human influenza therewith Download PDF

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Publication number
WO1991010744A1
WO1991010744A1 PCT/US1990/007679 US9007679W WO9110744A1 WO 1991010744 A1 WO1991010744 A1 WO 1991010744A1 US 9007679 W US9007679 W US 9007679W WO 9110744 A1 WO9110744 A1 WO 9110744A1
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bromo
nitrophenyl
chloro
naphthyl
substrate
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PCT/US1990/007679
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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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    • 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
    • C07H15/20—Carbocyclic rings
    • C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/02—Heterocyclic radicals containing only nitrogen as ring hetero atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
    • C07H17/06—Benzopyran radicals
    • C07H17/065—Benzo[b]pyrans
    • C07H17/075—Benzo[b]pyran-2-ones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/11—Orthomyxoviridae, e.g. influenza virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)

Definitions

  • the present invention relates to reagents and assays for diagnosing human influenza. More specifically it relates to novel chromogenic 9-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 influenza as the fifth leading cause of death in the United States. Antigenic variations in the surface glycoproteins of influenza A and B account for their continued epidemics.
  • Influenza viruses possess surface glycoproteins that have NA activity. These glycoproteins are members of a family of neuraminidases that are found in viruses, bacteria, mycoplasmas, and animal tissues. They hydrolyze substrates that contain alpha-ketosidically linked N-acetylneuraminic acid (Neu5Ac; previously referred to 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 colorimetric assay for NA using
  • 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:
  • 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:
  • Still 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:
  • X is a chromogenic group 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- resorufin, 5-bromo-4-chloro-3-indolyl, 4- nitrophenylazoresorcinyl, 4-nitrophenylazophenyl, 3- methoxyphenyl, 3-dimethylaminophenyl, 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 procedure described in Example 3.
  • Figure 4 is a schematic diagram depicting the synthesis procedure described in Example 4.
  • Figure 5 is a schematic diagram depicting the synthesis procedure described in Example 5.
  • Figure 6 is a schematic diagram depicting the synthesis procedure described in Example 6.
  • Figure 7 is a schematic diagram depicting the synthesis procedure described in Example 7.
  • Figure 8 is a schematic diagram depicting the synthesis procedure described in Example 8.
  • Figure 9 is a schematic diagram depicting the synthesis procedure described in Example 9.
  • Figure 10 is a schematic diagram depicting the synthesis procedure described in Example 10.
  • the chromogenic 9-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. monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.), acetate buffers (e.g., acetic acid-sodium acetate mixture), succinate buffers (e.g. succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate buffers (e.g.
  • tartaric acid-tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture etc. e.g. fumaric acid-monosodium fumarate mixture, fumaric acid-disodium fumarate mixture, monosodium fumaric acid-disodium fumarate mixture
  • fu arate 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.
  • N-2-acetamido-2-aminoethanesulfonic acid 3-(N-morpho ⁇ lino)-2-hydroxypropanesulfonic acid, N-N-bis-(2-hydroxy ⁇ ethyl)2-aminoethanesulfonic acid, 3-(N-morpholino)propanesulfonic acid, 2-[tris(hydroxy- methyl)methylamino]ethanesulfonic acid,
  • N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid 3- ⁇ [tris-(hydroxymethyl)methyl]amino ⁇ -2-hydroxypropane- sulfonic acid.
  • 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 Montgomery-C q 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 Neu5Ac derivative that is modified in the 9-position.
  • These substrates may be represented by the following chemical formula:
  • R, X and Ac are as defined previously.
  • X represents 4-methylumbelliferyl, 3-cyanoumbelliferyl, 2-nitrophenyl, 4-nitrophenyl, 3-resorufin, 5-bromo-4-chloro-3-indolyl, nitrophenylazophenyl, nitro- phenylazoresorcinyl, 3-methoxyphenyl, 3- dimethylaminophenyl, 4-chloro-l-naphthyl, 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.
  • 9-modified chromogenic Nue5Ac derivatives falling within the above formula are 4- methylumbelliferyl-9-deoxy-N-acetylneuraminic acid- alpha-ketoside, 3-cyanoumbelliferyl-9-deoxy-N- acetylneuraminic acid-alpha-ketoside, 2-nitrophenyl-9- deoxy-N-acetylneuraminic acid-alpha-ketoside, 4- nitrophenyl-9-deoxy-N-acetylneuraminic acid-alpha- ketoside, 3-resorufin-9-deoxy-N-acetylneuraminic acid- alpha-ketoside, 5-bromo-4-chloro-3-indolyl-9-deoxy-N- acetylneuraminic acid-alpha-ketoside, 2-[4-(4- nitrophenylazo)phenyl]-9-deoxy-N-acetylneuraminic acid- alpha-ketoside
  • Neu5Ac derivatives are generally made by protecting the functional groups of NeuSAc at the 1, 2, 4, 7 and 8 positions, modifying the 9 position as indicated, deprotecting the other positions, and coupling the 9-modified Neu5Ac with the chromogen.
  • 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 usu ⁇ ally 30 to 60 minutes. If there is NA activity in the sample, the chromogenic group will be cleaved from the substrate and the liberated chro ogen 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.
  • N-acetylneuraminic acid is protected as the methyl ester methyl ketoside (NeuSAc-MEMK) by treatment with methanol under Dowex 50W ion-exchange resin acid catalysis.
  • NeuSAc-MEMK is tosylated with tosyl chloride/pyridine (5°C for 2 hr) to give, selectively, the 9-tosyl compound.
  • Displacement of the tosyl group with sodium iodide (40°C) affords the 9-iodo compound.
  • Displacement of the terminal iodine is done with sodium nitrite in acetone (50°C) after which the 9-nitro compound is reduced with Zn and acetic acid (1 hr) .
  • Standard base and acid deprotection give the final product, 9-hydroxyimino-N-acetylneuraminic acid (N-acetylneuraminic acid 9-oxime) .
  • the 9-nitro NeuSAc intermediate of Example 1 is converted to its methyl ester as in Example 1 and then to its peracetate glycosyl chloride by treatment with excess acetyl chloride overnight. Coupling with o.-nitrophenol is done in dimethylformamide (DMF) and is followed by standard base/acid deprotection. Then the 9-nitro group is reduced via the Zn/acetic acid method described above to yield the final product, 2-(2-nitrophenyl)-N-acetyl ⁇ neuraminic acid-9-oxime-alpha-ketoside (sodium salt) .
  • DMF dimethylformamide
  • Neu5Ac-MEMK is tosylated with tosyl o chloride/pyridine (5 C for 2 hr) to give, selectively, the 9-tosyl compound.
  • Displacement of the tosyl group with sodium azide in acetone (40 C) is followed by base ( (NNaaOOHH)) aanndd aacciidd ((ddiilluuttee HHCCll,, DDoowweexx 5500 H ) deprotection to yield 9-azido-N-acetylneuraminic acid.
  • NeuSAc is converted to the corresponding methyl ester by stirring at room temperature (RT) with trifluoroacetic acid in methanol.
  • the methyl ester is treated overnight with excess acetylchloride to form its glycosyl chloride peracetate.
  • This intermediate is then coupled with o-nitrophenol by treatment with the sodium salt of o-nitrophenol in DMF at RT for 2 hr.
  • NeuSAc-MEMK is tosylated as in Example 5. Reduc ⁇ tion of the tosyl group with sodium borohydride in dimethylsulfoxide (4 hr) followed by base and acid deprotection as in Example 3 yields 9-deoxy-N-acety1- neuraminic acid.
  • Example 7 The 9-deoxy Neu5Ac of Example 7 is converted to its corresponding glycosyl chloride peracetate as described in Example 4. Coupling with sodium cyanoumbelliferone is done in DMF. Standard base deprotection yields 2-(3-cyanoumbelliferyl)- 9-deoxy-N-acetylneuraminic acid-alpha-ketoside (sodium salt) .
  • N-Acetylmannosamine methyl glycoside is treated with triphenylmethyl (trityl) chloride to form the 9- tritylated derivative.
  • the remaining free alcohol groups are then acetylated with acetic anhydride in pyridine to form the per-acetate.
  • the trityl group is selectively removed under catalytic hydrogenation conditions or with trimethylsilyl iodide (TMSI) .
  • TMSI trimethylsilyl iodide
  • the freed primary OH group is then converted to the fluoride with DAST and 9-fluoro N-acetylmannosamine obtained after deprotection with methoxide and mild acid treatment. Either the enzymatic catalyzed step (aldolase and pyruvate) or the chemical Cornforth procedure (oxalacetic acid) effects conversion to 9-fluoro-N-acetyneuraminic acid.
  • the 9-fluoro Neu5Ac is converted to its methyl ester by treatment in methanol with trifluoroacetic acid. Reaction with excess acetyl chloride acetylates the free alcoholic groups and effects conversion to the glycosyl chloride simultaneously.
  • Deprotection by treatment with sodium methoxide and sodium hydroxide affords the sodium salt of 6-bromo-2-naphthyl-9-fluoro Neu5Ac.

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Abstract

Chromogenic derivatives of N-acetylneuraminic acid modified in the 9-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 9-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 9-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 influenza as the fifth leading cause of death in the United States. Antigenic variations in the surface glycoproteins of influenza A and B account for their continued epidemics.
Influenza viruses possess surface glycoproteins that have NA activity. These glycoproteins are members of a family of neuraminidases that are found in viruses, bacteria, mycoplasmas, and animal tissues. They hydrolyze substrates that contain alpha-ketosidically linked N-acetylneuraminic acid (Neu5Ac; previously referred to 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 colorimetric 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 as¬ says, however, physicians currently still diagnose influenza solely on the basis of symptomology. 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 shortcoming 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 effect of certain chemical modifications of NeuSAc at the 4- ar___ 9-positions on the function of the molecule, 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. perfrinqens. A. ureafaciensf and V_s_ cholera) and found significant differences in reactivity. After 22 hours, the C_i_ perfrincrens NA cleaved 100% of the substrate while the Ai ureafaciens and Vj. 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 ethyl-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 NeuSAc 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, and 4,7-dideoxy Neu5Ac. Zbiral et al. , Liebigs Ann Chem, 519-526, described the synthesis of the 4- methylumbelliferyl-2-α glycosides of 7-epi, 8-epi, 7,8- bis-epi, 8-deoxy, 9-deoxy, and 4,7-dideoxy Neu5Ac 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 NeuSAc and the 7-epi and 7,8-bis-epi analogs of Neu5Aσ.
Disclosure 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:
1
Figure imgf000006_0001
where Ac represents acetyl, R represents hydrogen, fluorine, hydroxyimino, azido or cyano, 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:
Figure imgf000007_0001
where Ac represents acetyl, R represents hydrogen, fluorine, hydroxyimino, azido or cyano, 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, hydroxyimino, azido or cyano, 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 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_0002
where Ac represents acetyl, R represents hydrogen, and X is a chromogenic group 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- resorufin, 5-bromo-4-chloro-3-indolyl, 4- nitrophenylazoresorcinyl, 4-nitrophenylazophenyl, 3- methoxyphenyl, 3-dimethylaminophenyl, 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 procedure described in Example 3.
Figure 4 is a schematic diagram depicting the synthesis procedure described in Example 4.
Figure 5 is a schematic diagram depicting the synthesis procedure described in Example 5.
Figure 6 is a schematic diagram depicting the synthesis procedure described in Example 6. Figure 7 is a schematic diagram depicting the synthesis procedure described in Example 7.
Figure 8 is a schematic diagram depicting the synthesis procedure described in Example 8.
Figure 9 is a schematic diagram depicting the synthesis procedure described in Example 9.
Figure 10 is a schematic diagram depicting the synthesis procedure described in Example 10.
Modes for Carrying Out the Invention The chromogenic 9-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. monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.), acetate buffers (e.g., acetic acid-sodium acetate mixture), succinate buffers (e.g. succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate buffers (e.g. tartaric acid-tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture etc.) , fu arate 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)ethanesulfonic acid, [bis-(2-hydroxyethyl)imino]tris(hydroxymethyl)methane, N-2-acetamidoiminodiacetic acid, 1,3-bis[tris(hydroxymethyl)methylamino]propane, piper- azine-N,N'-bis(2-ethanesulfonic acid),
N-2-acetamido-2-aminoethanesulfonic acid, 3-(N-morpho¬ lino)-2-hydroxypropanesulfonic acid, N-N-bis-(2-hydroxy¬ ethyl)2-aminoethanesulfonic acid, 3-(N-morpholino)propanesulfonic acid, 2-[tris(hydroxy- methyl)methylamino]ethanesulfonic acid,
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 3-{[tris-(hydroxymethyl)methyl]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„-Cq 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 Neu5Ac derivative that is modified in the 9-position. These substrates may be represented by the following chemical formula:
1
Figure imgf000012_0001
where R, X and Ac are as defined previously. Preferably X represents 4-methylumbelliferyl, 3-cyanoumbelliferyl, 2-nitrophenyl, 4-nitrophenyl, 3-resorufin, 5-bromo-4-chloro-3-indolyl, nitrophenylazophenyl, nitro- phenylazoresorcinyl, 3-methoxyphenyl, 3- dimethylaminophenyl, 4-chloro-l-naphthyl, 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 9-modified chromogenic Nue5Ac derivatives falling within the above formula are 4- methylumbelliferyl-9-deoxy-N-acetylneuraminic acid- alpha-ketoside, 3-cyanoumbelliferyl-9-deoxy-N- acetylneuraminic acid-alpha-ketoside, 2-nitrophenyl-9- deoxy-N-acetylneuraminic acid-alpha-ketoside, 4- nitrophenyl-9-deoxy-N-acetylneuraminic acid-alpha- ketoside, 3-resorufin-9-deoxy-N-acetylneuraminic acid- alpha-ketoside, 5-bromo-4-chloro-3-indolyl-9-deoxy-N- acetylneuraminic acid-alpha-ketoside, 2-[4-(4- nitrophenylazo)phenyl]-9-deoxy-N-acetylneuraminic acid- alpha-ketoside, 2-[4-(4-nitrophenylazo)resorcinyl]-9- deoxy-N-acetylneuraminic acid-alpha-ketoside, 3- methoxyphenyl-9-deoxy-N-acetylneuraminic acid-alpha- ketoside, 3-dimethylaminophenyl-9-deoxy-N- acetylneuraminic acid-alpha-ketoside, 4-chloro-l- naphthyl-9-deoxy-N-acetyl-neuraminic acid-alpha- ketoside, 6-bromo-2-naphthyl-9-deoxy-N-acetylneuraminic acid-alpha-ketoside, 4-methylumbelliferyl-9-fluoro-N- acetylneuraminic acid-alpha-ketoside, 3- cyanoumbelliferyl-9-fluoro-N-acetylneuraminic acid-alpha- ketoside, 2-nitrophenyl-9-fluoro-N-acetylneuraminic acid- alpha-ketoside, 4-nitrophenyl-9-fluoro-N-acetylneuraminic acid-alpha-ketoside , 3-resorufin-9-fluoro-N- acetylneuraminic acid-alpha-ketoside, 5-bromo-4-chloro- 3-indolyl-9-fluoro-N-acetylneuraminic acid-alpha- ketoside, 2-[4-(4-nitrophenylazo)phenyl]-9-fluoro-N- acetylneuraminic acid-alpha-ketoside, 2-[4-(4- nitrophenylazo)resorcinyl]-9-fluoro-N-acetylneuraminic acid-alpha-ketoside, 3-methoxyphenyl-9-fluoro-N- acetylneuraminic acid-alpha-ketoside, 3- dimethylaminophenyl-9-fluoro-N-acetylneuraminic acid- alpha-ketoside, 4-chloro-l-naphthyl-9-fluoro-N-acetyl- neuraminic acid-alpha-ketoside, 6-bromo-2-naphthyl-9- fluoro-N-acetylneuraminic acid-alpha-ketoside, 4- methylumbelliferyl-9-azido-N-acetylneuraminic acid-alpha- ketoside, 2-nitrophenyl-9-azido-N-acetylneuraminic acid- alpha-ketoside, 4-nitrophenyl-9-azido-N-acetylneuraminic acid-alpha-ketoside, 3-cyanoumbelliferyl-9-azido-N- acetylneuraminic acid-alpha-ketoside, 3-resorufin-9- azido-N-acetylneuraminic acid-alpha-ketoside, 5-bromo-4- chloro-3-indolyl-9-azido-N-acetylneuraminic acid-alpha- ketoside, 2-[4-(4-nitrophenylazo) henyl]-9-azido-N- acetylneuraminic acid-alpha-ketoside, 2-[4-(4- nitrophenylazo)resorcinyl]-9-azido-N-acetylneuraminic acid-alpha-ketoside, 3-methoxyphenyl-9-azido-N- acetylneuraminic acid-alpha-ketoside, 3- dimethylaminophenyl-9-azido-N-acetylneuraminic acid- alpha-ketoside, 4-chloro-l-naphthyl-9-azido-N- acetylneuraminic acid-alpha-ketoside, 6-bromo-2-naphthyl- 9-azido-N-acetylneuraminic acid-alpha-ketoside, 4- methylumbelliferyl-9-hydroxyimino-N-acetylneuraminic acid-alpha-ketoside, 3-cyanoumbelliferyl-9-hydroxyimino- N-acetylneuraminic acid-alpha-ketoside, 2-nitrophenyl-9- hydroxyimino-N-acetylneuraminic acid-alpha-ketoside, 4- nitrophenyl-9-hydroxyimino-N-acetylneuraminic acid- alpha-ketoside, 3-resorufin-9-hydroxyimino-N- acetylneuraminic acid-alpha-ketoside, 5-bromo-4-chloro- 3-indolyl-9-hydroxyimino-N-acetylneuraminic acid-alpha- ketoside, 2-[4-(4-nitrophenylazo)phenyl]-9-hydroxyimino- N-acetylneuraminic acid-alpha-ketoside, 2-[4-(4- nitrophenylazo)resorcinyl]-9-hydroxyimino-N- acetylneuraminic acid-alpha-ketoside, 3-methoxyphenyl-9- hydroxyimino-N-acetylneuraminic acid-alpha-ketoside, 3- dimethylaminophenyl-9-hydroxyimino-N-acetylneuraminic acid-alpha-ketoside, 4-chloro-l-naphthyl-9-hydroxyimino- N-acetylneuraminic acid-alpha-ketoside, 6-bromo-2- naphthyl-9-hydroximino-N-acetylneuraminic acid-alpha- ketoside, 4-methylumbelliferyl-9-cyano-N- acetylneuraminic acid-alpha-ketoside, 3- cyanoumbelliferyl-9-cyano-N-acetylneuraminic acid-alpha- ketoside, 2-nitrophenyl-9-cyano-N-acetylneuraminic acid- alpha-ketoside, 4-nitrophenyl-9-cyano-N-acetylneuraminic acid-alpha-ketoside, 3-resorufin-9-cyano-N- acetylneuraminic acid-alpha-ketoside, 5-bromo-4-chloro- 3-indolyl-9-cyano-N-acetylneuraminic acid-alpha-ketoside, 2-[4-(4-nitrophenylazo)phenyl]-9-cyano-N-acetylneuraminic acid-alpha-ketoside, 2-[4-(4-nitrophenylazo)resorcinyl]- 9-cyano-N-acetylneuraminic acid-alpha-ketoside, 3- methoxyphenyl-9-cyano-N-acetylneuraminic acid-alpha- ketoside, 3-dimethylaminophenyl-9-cyano-N- acetylneuraminic acid-alpha-ketoside, 4-chloro-l- naphthyl-9-cyano-N-acetylneuraminic acid-alpha-ketoside, and 6-bromo-2-naphthyl-9-cyano-N-acetylneuraminic acid- alpha-ketoside.
The above-described Neu5Ac derivatives are generally made by protecting the functional groups of NeuSAc at the 1, 2, 4, 7 and 8 positions, modifying the 9 position as indicated, deprotecting the other positions, and coupling the 9-modified Neu5Ac with the chromogen.
Details of these reactions are provided in the Examples, infra.
The substrate will normally be added to the buffered, stabilized sample in amounts ranging between 0.05 mM and 0.5 mM. The mixture is incubated at ambient temperature to physiological temperature (i.e., about 22°C to 37°C) for a time sufficient to permit any NA in the sample to react with the substrate. That time will normally be in the range of 20 to 120 minutes, more usu¬ ally 30 to 60 minutes. If there is NA activity in the sample, the chromogenic group will be cleaved from the substrate and the liberated chro ogen 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-chloro- colorimetric/ blue/purple in the 3-indolol visual presence of nitroblue tetra- zolium
4-methylumbe11i- fluorometric fluorescent ferone emission at 450 nm after exci¬ tation at 360 nm
3-cyanoumbelli- fluorometric fluorescent ferone emission at 454 nm after exci¬ tation at 415 nm resorufin colorimetric/ pink/red visual 2-nitrophenol colorimetric/ yellow visual 4-nitrophenol colorimetric/ yellow visual nitrophenyl- colorimetric/ orange azophenol visual nitrophenyl- colorimetric/ green blue azoresorcinol visual (presence of Mg ++,) 3-methoxyphenol colorimetric/ red to blue visual after reaction with diazoriium salt
3-dimethylamino- colorimetric/ red to blue phenol visual after reaction with diazonium salt
6-bromo-2-naphthol colorimetric/ red to blue 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
1. Synthesis of 9-Hydroxyimino NeuSAc
The synthesis scheme for this compound is depicted in Figure 1.
N-acetylneuraminic acid is protected as the methyl ester methyl ketoside (NeuSAc-MEMK) by treatment with methanol under Dowex 50W ion-exchange resin acid catalysis. NeuSAc-MEMK is tosylated with tosyl chloride/pyridine (5°C for 2 hr) to give, selectively, the 9-tosyl compound. Displacement of the tosyl group with sodium iodide (40°C) affords the 9-iodo compound. Displacement of the terminal iodine is done with sodium nitrite in acetone (50°C) after which the 9-nitro compound is reduced with Zn and acetic acid (1 hr) . Standard base and acid deprotection give the final product, 9-hydroxyimino-N-acetylneuraminic acid (N-acetylneuraminic acid 9-oxime) .
2. Synthesis of Chromogenic 9-Hydroxyimino NeuSAc
The synthesis scheme for this compound is shown in Figure 2.
The 9-nitro NeuSAc intermediate of Example 1 is converted to its methyl ester as in Example 1 and then to its peracetate glycosyl chloride by treatment with excess acetyl chloride overnight. Coupling with o.-nitrophenol is done in dimethylformamide (DMF) and is followed by standard base/acid deprotection. Then the 9-nitro group is reduced via the Zn/acetic acid method described above to yield the final product, 2-(2-nitrophenyl)-N-acetyl¬ neuraminic acid-9-oxime-alpha-ketoside (sodium salt) .
3. Synthesis of 9-Azido NeuSAc
The synthesis scheme for this compound is shown in Figure 3. Neu5Ac-MEMK is tosylated with tosyl o chloride/pyridine (5 C for 2 hr) to give, selectively, the 9-tosyl compound. Displacement of the tosyl group with sodium azide in acetone (40 C) is followed by base ( (NNaaOOHH)) aanndd aacciidd ((ddiilluuttee HHCCll,, DDoowweexx 5500 H ) deprotection to yield 9-azido-N-acetylneuraminic acid.
4. Synthesis of Chromogenic 9-Azido NeuSAc
The synthesis scheme for this compound is shown in Figure 4.
NeuSAc is converted to the corresponding methyl ester by stirring at room temperature (RT) with trifluoroacetic acid in methanol. The methyl ester is treated overnight with excess acetylchloride to form its glycosyl chloride peracetate. This intermediate is then coupled with o-nitrophenol by treatment with the sodium salt of o-nitrophenol in DMF at RT for 2 hr.
Deprotection of the acetates is accomplished with methoxide ion. The 9-position is then selectively tosylated as before in Example 3. Displacement of the tosyl group with sodium azide (40°C) is followed by standard base deprotection to afford 2-(2-nitrophenyl)- 9-azido-N-acetylneuraminic acid-alpha-ketoside (sodium salt) .
5. Synthesis of 9-Cvano NeuSAc
The synthesis scheme for this compound is shown in Figure 5. Neu5Ac-MEMK is tosylated with tosyl chloride/pyridine (5°C for 2 hr) to give selectively the 9-tosyl compound. Displacement of the tosyl group with sodium cyanide in acetone (40°C) is followed by base and acid deprotection as in Example 3 to yield 9-cyano-N-acetylneuraminic acid. 6. Synthesis of Chromogenic 9-Cyano NeuSAc
The synthesis scheme for this compound is shown in Figure 6. The 9-cyano NeuSAc of Example 5 is converted to its corresponding glycosyl chloride peracetate as described in Example 4. Coupling with sodium cyanoumbelliferone is done in DMF. Standard base deprotection yields 2-(3-cyanoumbelliferyl)- 9-cyano-N-acetylneuraminic acid-alpha-ketoside (sodium salt) .
7. Synthesis of 9-Deoxy Neu5Ac
The synthesis scheme for this compound is shown in Figure 7. NeuSAc-MEMK is tosylated as in Example 5. Reduc¬ tion of the tosyl group with sodium borohydride in dimethylsulfoxide (4 hr) followed by base and acid deprotection as in Example 3 yields 9-deoxy-N-acety1- neuraminic acid.
8. Synthesis of Chromogenic 9-Deoxy NeuSAc
The synthesis scheme for this compound is shown in Figure 8.
The 9-deoxy Neu5Ac of Example 7 is converted to its corresponding glycosyl chloride peracetate as described in Example 4. Coupling with sodium cyanoumbelliferone is done in DMF. Standard base deprotection yields 2-(3-cyanoumbelliferyl)- 9-deoxy-N-acetylneuraminic acid-alpha-ketoside (sodium salt) .
9. Synthesis of 9-Fluoro NeuSAc
The synthesis scheme for this compound is shown in Figure 9. N-Acetylmannosamine methyl glycoside is treated with triphenylmethyl (trityl) chloride to form the 9- tritylated derivative. The remaining free alcohol groups are then acetylated with acetic anhydride in pyridine to form the per-acetate. The trityl group is selectively removed under catalytic hydrogenation conditions or with trimethylsilyl iodide (TMSI) . The freed primary OH group is then converted to the fluoride with DAST and 9-fluoro N-acetylmannosamine obtained after deprotection with methoxide and mild acid treatment. Either the enzymatic catalyzed step (aldolase and pyruvate) or the chemical Cornforth procedure (oxalacetic acid) effects conversion to 9-fluoro-N-acetyneuraminic acid.
10. Synthesis of Chromogenic 9-Fluoro NeuSAc
The synthesis scheme for this compound is shown in Figure 10.
The 9-fluoro Neu5Ac is converted to its methyl ester by treatment in methanol with trifluoroacetic acid. Reaction with excess acetyl chloride acetylates the free alcoholic groups and effects conversion to the glycosyl chloride simultaneously. Treatment of the glycosyl chloride with the sodium salt of 6-bromo-naphthol in DMF affords the coupled chromogenic compound. Deprotection by treatment with sodium methoxide and sodium hydroxide affords the sodium salt of 6-bromo-2-naphthyl-9-fluoro Neu5Ac.
Modifications of the above-described modes for carrying out the invention that are obvious to those of skill in the fields of chemistry, virology, biochemistry, organic chemistry, 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:
1
Figure imgf000022_0001
where Ac represents acetyl, R represents hydrogen, fluorine, hydroxyimino, azido or cyano, 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 1 wherein the clinical sample is a pharyngeal, nasopharyngeal or respiratory secretion.
3. The method of claim 1 or 2 wherein R represents hydrogen, 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-nitrophenylazoresorcinyl, 4-nitrophenylazophenyl, 3- ethoxyphenyl, 3-dimethylaminophenyl, 4-chloro-l- naphthyl, and 6-bromo-2-naphthyl.
4. The method of claim 1 or 2 wherein R represents fluorine, 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-nitrophenylazoresorcinyl, 4-nitrophenylazophenyl, 3- methoxyphenyl, 3-diraethylaminophenyl, 4-chloro-l- naphthyl, and 6-bromo-2-naphthyl.
5. The method of claim 1 or 2 wherein R represents azido, 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-nitrophenylazoresorcinyl, 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, 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-nitrophenylazoresorcinyl, 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 represents hydroxyimino, 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-nitrophenylazoresorcinyl, 4-nitrophenylazophenyl, 3-methoxyphenyl, 3- dimethylaminophenyl, 4-chloro-l-naphthyl, and 6-bromo-2- naphthyl.
8. 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 imgf000024_0001
where Ac represents acetyl, R represents hydrogen, fluorine, hydroxyimino, azido or cyano, 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.
9. The method of claim 8 wherein the specific type of human influenza neuraminidase activity is human influenza A neuraminidase activity or human influenza B neuraminidase activity.
10. The method of claim 8 where the specific type of human neuraminidase activity is human parainfluenza 1 neuraminidase activity, human parainfluenza 2 neuraminidase activity or human parainfluenza 3 neuraminidase activity.
11. The method of claim 8, 9 or 10 wherein the clinical sample is a pharyngeal, nasopharyngeal, or respiratory secretion.
12. The method of claim 8, 9, 10 or 11 wherein R represents hydrogen, 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-nitrophenylazoresorcinyl, 4-nitrophenylazophenyl, 3-methoxyphenyl, 3- dimethylaminophenyl, 4-chloro-l-naphthyl, and 6-bromo-2- naphthyl.
13. The method of claim 8, 9, 10 or 11 wherein R represents fluorine, 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-nitrophenylazoresorcinyl, 4-nitrophenylazophenyl, 3-methoxyphenyl, 3- dimethylaminophenyl, 4-chloro-l-naphthyl, and 6-bromo-2- naphthyl.
14. The method of claim 8, 9, 10 or 11 wherein R represents azido, 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-nitrophenylazoresorcinyl, 4-nitrophenylazophenyl, 3-methoxyphenyl, 3- dimethylaminophenyl, 4-chloro-l-naphthyl, and 6-bromo-2- naphthyl.
15. The method of claim 8, 9, 10 or 11 wherein R represents cyano, 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-nitrophenylazoresorcinyl, 4-nitrophenylazophenyl, 3-methoxyphenyl, 3- dimethylaminophenyl, 4-chloro-l-naphthyl, and 6-bromo-2- naphthyl.
16. The method of claim 8, 9, 10 or 11 wherein R represents hydroxyimino, 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-nitrophenylazoresorcinyl, 4-nitropheny1azopheny1, 3-methoxypheny1, 3- dimethylaminophenyl, 4-chloro-l-naphthyl, and 6-bromo-2- naphthyl.
17. A chromogenic substrate useful for detecting human influenza neuraminidase activity in a clinical sample suspected of having such activity, said substrate having the formula:
I
Figure imgf000026_0001
where Ac represents acetyl, R represents fluorine, hydroxyimino, azido or cyano, and X is a chromogenic group that exhibits a distinct color when cleaved from the substrate and salts of said substrate.
18. The chromogenic substrate of claim 17 wherein R represents fluorine, 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-nitrophenylazoresorcinyl, 4-nitrophenylazophenyl, 3-methoxyphenyl, 3- dimethylaminophenyl, 4-chloro-l-naphthyl, and 6-bromo-2- naphthyl.
19. The chromogenic substrate of claim 17 wherein R represents azido, 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-nitrophenylazoresorcinyl, 4-nitrophenylazophenyl, 3-methoxyphenyl, 3- dimethylaminophenyl, 4-chloro-l-naphthyl, and 6-bromo-2- naphthyl.
20. The chromogenic substrate of claim 17 wherein R represents cyano, 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-nitrophenylazoresorcinyl , 4-nitrophenylazophenyl, 3-methoxyphenyl, 3- dimethylaminophenyl, 4-chloro-l-naphthyl, and 6-bromo-2- naphthyl.
21. The chromogenic substrate of claim 17 wherein R represents hydroxyimino, 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-nitrophenylazoresorcinyl, 4-nitrophenylazophenyl, 3- methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-l- naphthyl, and 6-bromo-2-naphthyl.
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 imgf000028_0001
where Ac represents acetyl, R represents hydrogen, 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-naphthyl.
23. A modified N-acetylneuraminic acid having the formula:
Figure imgf000029_0001
where Ac represents acetyl and R represents hydrogen, fluorine, hydroxyimino, or cyano.
PCT/US1990/007679 1990-01-10 1990-12-27 Chromogenic 9-position modified n-acetylneuraminic acid substrates and methods for diagnosing human influenza therewith WO1991010744A1 (en)

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EP0728763A1 (en) * 1994-08-16 1996-08-28 Daikin Industries, Limited Ganglioside gm3 analog having sialic acid residue fluorinated at the 9-position and intermediate therefor
EP0728763A4 (en) * 1994-08-16 1996-12-11 Daikin Ind Ltd Ganglioside gm3 analog having sialic acid residue fluorinated at the 9-position and intermediate therefor
US5792842A (en) * 1994-08-16 1998-08-11 Daikin Industries Ltd. Ganglioside GM3 derivative having fluorine atom at 9-position of sialic acid and intermediates therefor
WO1997032214A1 (en) * 1996-03-01 1997-09-04 Biota Scientific Management Pty. Ltd. Method of detection of influenza virus and compounds for use therein
US6242582B1 (en) 1996-03-01 2001-06-05 Biota Scientific Management Pty Ltd. Method of detection of influenza virus and compounds for use therein
WO1998013372A1 (en) * 1996-09-25 1998-04-02 Oklahoma Medical Research Foundation 4,7-dialkoxy-n-acetylneuraminic acid derivatives and methods for detection of influenza type a and b viruses in clinical specimens
AU716275B2 (en) * 1996-09-25 2000-02-24 Oklahoma Medical Research Foundation 4,7-dialkoxy N-acetylneuraminic acid derivatives and methods for detection of influenza type A and B viruses in clinical specimens
JP2003522113A (en) * 1998-10-27 2003-07-22 ユーエービー リサーチ ファンデイション Chromogenic substrate for sialidase and its production and use

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