METHODS FOR DIAGNOSTING HUMAN INFLUENZA AND 4-POSITION MODIFIED CHROMOGENIC N-ACETYLNEURAMINIC ACID SUBSTRATES FOR USE THEREIN

Abstract: Chromogenic derivatives of N-acetylneuraminic acid modified in the 4-position by eliminating the hydroxyl group, by replacing the hydroxyl group with fluorine, or by replacing the hydrogen of the hydroxyl group with methyl 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: METHODS FOR DIAGNOSING HUMAN INFLUENZA AND 4-POSITION MODIFIED CHROMOGENIC N-ACETYLNEURAMINIC ACID SUBSTRATES FOR USE THEREIN Technical Field The present invention relates to diagnostic tests for human influenza. More specifically it relates to chromogenic 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 infections annually in the United States alone. Epidemiologic studies of influenza epidemics estimate the incidence of infection to be 25% in the general population and higher in school age children. Researchers have estimated that up to half the infected persons would see a physician because of the illness. In 1986, the Center for Disease Control (CDC) estimated that influenza epidemics have been associated with 10,000 or more excess deaths in 18 of the preceding 28 years. CDC studies indicate influenza as the fifth leading cause of death in the United States. Antigenic variations in the surface glycoproteins of influenza A and B account for their continued epidemics. Influenza viruses possess surface glycoproteins that have NA activity. These glycoproteins are members of a family of neuraminidases that are found in viruses, bacteria, mycoplasmas, and animal tissues. They hydrolyze substrates that contain alpha-ketosidically linked N-acetylneuraminic acid (Neu5Ac; referred to previously as "NANA") . In viruses, NA typically constitutes 5-10% of the viral protein and exists as a mushroom-shaped spike on the envelope. Viral NA is composed of a hydrophilic area which includes the catalytic site of the enzyme and a hydrophobic area that is inserted into the viral envelope anchoring the enzyme to the virus. Various assays for NA activity are described in the literature. 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 NeuδAc 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 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 relationship between the chemical structure of NeuSAc 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__ perfrinσens. A. ureafaciens. and ___, cholera) and found significant differences in reactivity. After 22 hrs, the C__ perfrinαens NA cleaved 100% of the substrate while the A__ ureafaciens and V-. cholera NAs cleaved only 50% and 11% of the substrate, respectively. Kim et al., J. Am. Chem. Soc. 110:6481-6486 (1988) described the structural characteristics of substrates accepted by NeuδAc aldolase, its use in the synthesis of Neu5Ac, 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 Neu5Ac was a good substrate for fowl plague viral Neu5Ac, but not for the three bacterial NAs mentioned above. Additionally, Schauer, R. , et al., Eur. J. Bioche . 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 NeuδAc and 4,7-dideoxy Neu5Ac. Zbiral et al. , Liebigs Ann Chem 119:127-141 (1989) 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 NeuSAc and investigated the behavior of those compounds as inhibitors of the sialidase from V. cholera. Applicant is unaware of any prior reports on the reactivity of 4-modified NeuSAcs with human influenza NA. 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: where Ac represents acetyl, R represents hydrogen, fluorine or methoxy, 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: where Ac represents acetyl, R represents hydrogen, fluorine or methoxy, 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 Neu5Ac chromogenic substrate useful for detecting human influenza neuraminidase activity in a clinical sample suspected of having such activity, said substrate having the formula: where Ac represents acetyl, R is methoxy or fluorine, 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: where Ac represents acetyl, R is hydrogen and X is a chromogenic group selected from the group consisting of 3-cyanoumbelliferyl, 2- or 4-nitrophenyl, 3-resorufin, 5-bromo-4-chloro-3-indolyl, 4-nitrophenylazophenyl 4-nitrophenylazoresorcinyl, 3-methoxyphenyl, 3- dimethylaminophenyl, 4-chloro-l-naphthyl, and 6-bromo-2- naphthyl and salts of said substrate. 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. 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 alditols, 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.), fumarate buffers (e.g. fumaric acid-monosodium fumarate mixture, fumaric acid-disodium fumarate mixture, monosodium fumarate 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-πιalic 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)methylaminojpropane, 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-hydroxy- ethylpiperazine-N'-2-ethanesulfonic acid, 3-{[tris- (hydroxymethyl) ethyl]a ino}-2-hydroxypropanesulfonic acid. Examples of non-ionic detergents useful in the buffer solution are the Pluronics, for example, Polysorbate 20 or Polysorbate 80, Triton X-100, NP-40, and alkyl glucosides such as C 8 -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 alditols, 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 alditols or simple sugars and disaccharrides 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 4 position by removal of the hydroxyl group at that position, by replacement of the hydroxyl group with fluorine, or by replacement of the hydrogen of the hydroxyl group with a lower alkyl group. These substrates may be represented by the following chemical formula: 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, nitrophenyl- azoresorcinyl, 3-methoxyphenyl, 3-dimethylaminophenyl, 4- chloro-1-naphthyl, or 6-bromo-2-naphthyl. Simple salts of these substrates, such as the Na, K and NH. salts, may also be used. As used herein the term "chro ogen" is intended to include, without limitation, molecules that exhibit fluorescence. The term "color" is likewise intended to include, without limitation, fluorescence. Examples of 4-modified chromogenic Neu5Ac derivatives falling within the above formula are 4- methylumbelliferyl-4-deoxy-N-acetylneuraminic acid- alpha-ketoside, 3-cyanoumbelliferyl-4-deoxy-N-acetyl- neuraminic acid-alpha-ketoside, 2-nitrophenyl-4-deoxy-N- acetylneuraminic acid-alpha-ketoside, 4-nitrophenyl-4- deoxy-N-acetylneuraminic acid-alpha-ketoside, 3- resorufin-4-deoxy-N-acetylneuraminic acid-alpha-ketoside, 5-bromo-4-chloro-3-indolyl-4-deoxy-N-acetylneuraminic acid-alpha-ketoside, 2-[4-(4-nitrophenylazo)phenyl]-4- deoxy-N-acetylneuraminic acid-alpha-ketoside, 2-[4-(4- nitrophenylazo)resorcinyl]-4-deoxy-N-acetyl-neuraminic acid-alpha-ketoside, 3-methoxyphenyl-4-deoxy-N-acetyl- neuraminic acid-alpha-ketoside, 3-dimethylaminophenyl-4- deoxy-N-acetylneuraminic acid-alpha-ketoside, 4-chloro- l-naphthyl-4-deoxy-N-acetylneuraminic acid-alpha- ketoside, 6-bromo-2-naphthyl-4-deoxy-N-acetylneuraminic acid-alpha-ketoside, 4-methylumbelliferyl-4-methoxy- N-acetylneuraminic acid-alpha-ketoside, 2-nitrophenyl- 4-methoxy-N-acetylneuraminic acid-alpha-ketoside, 4-nitrophenyl-4-methoxyN-acetylneuraminic acid-alpha-ketoside, 3-cyanoumbelliferyl- 4-methoxy-N-acetylneuraminic acid-alpha-ketoside, 3-resorufin-4-methoxy-N-acetylneuraminic acid-alpha-ketoside, 5-bromo-4-chloro-3-indolyl-4- methoxy-N-acetylneuraminic acid-alpha-ketoside, 2-[4-(4-nitrophenylazo)phenyl]-4-methoxy-N-acetyl¬ neuraminic acid-alpha-ketoside, 2-[4-(4-nitrophenyl- azo)resorcinyl]-4-methoxy-N-acetylneuraminic acid- alpha-ketoside, 3-methoxypheny1-4-methoxy-N-acetyl¬ neuraminic acid-alpha-ketoside, 3-dimethylaminophenyl-4- methoxy-N-acetylneuraminic acid-alpha-ketoside, 6-bromo- 2-naphthy1-4-methoxy-N-acetylneuraminic acid-alpha- ketoside, 4-chloro-l-naphthyl-4-methoxy-N- acetylneuraminic acid-alpha-ketoside, 4-methylumbelliferyl-4-fluoro-N-acetylneuraminic acid-alpha-ketoside, 2-nitrophenyl-4-fluoro-N- acetylneuraminic acid-alpha-ketoside, 4-nitrophenyl-4- fluoro-N-acetylneuraminic acid-alpha-ketoside, 3-cyanoum- belliferyl-4-fluoro-N-acetylneuraminic acid-alpha- ketoside, 3-resorufin-4-methoxy-N-acetylneuraminic acid-alpha-ketoside, 5-bromo-4-chloro-3-indolyl-4-fluoro- N-acetylneuraminic acid-alpha-ketoside, 2-[4-(4-nitro- phenylazo) henyl]-4-fluoro-N-acetylneuraminic acid-alpha-ketoside, 2-[4-(4-nitrophenylazo)resorcinyl]- 4-fluoro-N-acetylneuraminic acid-alpha-ketoside, 3- methoxyphenyl-4-fluoro-N-acetylneuraminic acid-alpha- ketoside, 3-dimethylaminophenyl-4-fluoro-N- acetylneuraminic acid-alpha-ketoside, 4-chloro-l- naphthyl-4-fluoro-N-acetylneuraminic acid-alpha-ketoside, and 6-bromo-2-naphthy1-4-fluoro-N-acetylneuraminic acid-alpha-ketoside. The above-described NeuSAc derivatives are generally made by protecting the functional groups of NeuSAc at the 1, 2, 7, 8, and 9 positions, modifying the 4.position as indicated, deprotecting the 1, 2, 7, 8 and 9 positions, and coupling the 4-modified NeuSAc 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 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 Neu5Ac and releases the chromogen. Released Chromogen Type of Detection Color 5-bromo-4-chloro- colorimetric/ blue/purple 3-indolol visual in the presence of nitroblue tetrazolium 4-methylumbe11i- fluorometric fluorescent ferone emission at 450 nm after excitation at 360 nm 3-cyanoumbelli- fluorometric fluorescent ferone emission at 454 nm after excitation at 415 nm resorufin colorimetric/ pink/red visual 2-nitropheno1 colorimetric/ yellow visual 4-nitrophenol colorimetric/ yellow visual nitrophenyl- colorimetric/ orange azophenol visual nitrophenyl- colorimetric/ green blue azoresorcino1 visual (presence of Mg ) 3-methoxyphenol co1orimetric/ red to blue visual after reaction with diazonium salt 3-dimethylamino- colorimetric/ red to blue phenol visual after reaction with diazonium salt 4-chloro-l- colorimetric/ red to blue naphthol visual after reaction with diazonium salt 6-bromo-2-naphthol colorimetric/ red to blue 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 following examples. These examples are not intended to limit the invention in any manner. Examples l. Synthesis of 4-Deoxy NeuSAc The reaction scheme for this synthesis is shown in Figure 1. NeuSAc (500 mg) in 100 ml of methanol and 1.25 g of Dowex-50 (H ) are heated under reflux for 20 hr., and treated with decolorizing carbon to afford the methyl ester methyl ketoside of NeuSAc (NeuSAc-MEMK) . To the ketoside in dry acetone (dried over molecular sieves) is added catalytic p-toluenesulfonic acid monohydrate and the reaction allowed to stir for 4 hr. at room temperature (RT) . Neutralization with Dowex-1 (acetate form) and solvent evaporation gives 8,9-isopropylidene Neu5Ac methyl ester methyl ketoside. The 4-mesylate- 8,9-isopropylidene NeuSAc methyl ester methyl ketoside is obtained by treating with methanesulfonyl-chloride in methylene chloride and 1.5 equivalents of triethylamine at 0°C for 30 min. The mesylate is then treated with sodium iodide in acetone or methyl ethyl ketone and heated to ca. 100°C in a sealed reaction vial to form the 4-iodo compound. Subsequent hydrogenation over palladium on carbon affords the 4-deoxy molecule. Deprotection is accomplished by treatment first with sodium hydroxide ffoolllloowweedd bbyy ttrreeaattmmeenntt wwiitthh ddiilluuttee HHCC]l/Dowex-50 (H ) to give 4-deoxy-N-acetylneuraminic acid. 2. Synthesis of 2-(p-Nitrophenyl)-4-Deoxy Neu5Ac The reaction scheme for this synthesis is shown in Figure 2. The 4-deoxy NeuSAc of Example 1 is converted to the corresponding methyl ester by stirring at room temperature (RT) with trifluoroacetic acid in methanol. The methyl ester is then treated with an excess of acetyl chloride overnight to form the fully acetylated 2-chloro-4-deoxy NeuSAc methyl ester. This intermediate is then treated with the sodium salt of the para-nitropheno1 in dimethyl formamide (DMF) at RT for 2 hr. The coupled chromogenic 4-deoxy NeuSAc is then deprotected by treatment with sodium methoxide in methanol (1 hr) followed by treatment with sodium hydroxide (2 hr) to form the sodium salt of 2-(p-nitrophenyl)-4-deoxy-N-acetylneuraminic acid. 3. Synthesis of 4-Methoxy Neu5Ac The reaction scheme for this synthesis is shown in Figure 3. NeuδAc-MEMK is treated with 1 eq. of tert-butyldimethylsilyl (TBDMS) chloride, imidazole and a o catalytic amount of 4-dimethylaminopyridine at 65 C to afford 9-O-TBDMS Neu5Ac-MEMK. Treatment of this compound with acetone and a catalytic amount of p-toluenesulfonic acid monohydrate at RT yields 9-0-TBDMS-7,8-isopropyl- idene Neu5Ac-MEMK. Treatment with diazomethane/tri- fluoroborate in ether at 0°C gives the corresponding 4-methoxy derivative. This compound is fully deprotected by treatment with sodium hydroxide followed by tetrabutylammonium fluoride in THF and finally by treatment with dilute HCl/Dowex-50W (H ) to give 4-methoxy-N-acetylneuraminic acid. 4. Synthesis of 2-(p-Nitrophenyl.-4-Methoxy NeuSAc The reaction scheme for this synthesis is shown in Figure 4. The 9-TBDMS-7,8-isopropylidene-protected 4-methoxy NeuSAc-MEMK is deprotected with tosic acid followed by tetrabutylammonium fluoride in tetrahydro- furan. This intermediate is then treated with excess acetyl chloride overnight to form fully acetylated 2-chloro-4-methoxy NeuSAc methyl ester. Coupling with the sodium salt of nitrophenol chromogen is done in dry DMF at RT for 2 hr. Deprotection and deprotonation is effected by treatment with sodium methoxide in methanol followed by treatment with sodium hydroxide to give 2-(p-nitrophenyl)-4-methoxy-N-acetylneuraminic acid, sodium salt. 5. Synthesis of 4-Fluoro NeuSAc The reaction scheme for this synthesis is shown in Figure 5. NeuSAc (500 mg) in 100 ml of methanol and 1.25 g of Dowex-50W (H ) are heated under reflux for 20 hr. , and treated with decolorizing carbon to afford the methyl ester methyl ketoside of NeuSAc (Neu5Ac-MEMK) . To the ketoside in dry acetone (dried over molecular sieves) is added catalytic p-toluenesulfonic acid monohydrate and the reaction allowed to stir for 4 hr. at room temperature (RT) . Neutralization with Dowex-1 (acetate form) and solvent evaporation gives 8,9-isopropylidene Neu5Ac methyl ester methyl ketoside. This compound is then selectively oxidized to the 4-keto derivative with either pyridinium dichromate (PDC) or ruthenuim tetra- oxide, with 4 angstrom molecular sieves in acetonitrile. Mild reduction with borane-ammonia in tetrahydrofuran affords the inverted 4-alcohol which may then be converted to the 4-fluoride (with inversion of configuration) with diethyla ino sulfur trifluoride (DAST) . Deprotection is accomplished by treatment first with sodium hydroxide followed by treatment with dilute HHCCll//DDiowex-50W (H ) to give 4-fluoro-N-acetylneuraminic acid. 6. Synthesis of 2-(4-Chloro-l-Naphthyl)-4-Fluoro NeuSAc The reaction scheme for this synthesis is shown in Figure 6. The 4-fluoro NeuSAc of Example 5 is converted to its methyl ester by treatment in methanol with trifluoroacetic acid. Reaction in excess acetyl chloride acetylates the free alcohol groups and effects conversion to the glycosyl chloride simultaneously. The sodium salt of 4-chloro-l-naphthol (4-CN) was prepared with 1 equivalent of NaOH in water. Reaction of the glycosyl chloride in chloroform with this aqueous 4-CN solution (2.5 equivalents) in the presence of benzyl triethylammonium chloride (phase transfer reagent) affords the coupled chromogenic compound. Deprotection of the acetates and methyl ester group is accomplished by treatment with sodium methoxide and sodium hydroxide which affords the sodium salt of 2-(4-chloro-l-naphthyl)- 4-fluoro NeuSAc. 7. Enzymatic testing of 4-Deoxy NeuSAc 50 μl of an influenza virus solution was mixed with a reaction mixture containing 50 μl of the substrate 4-methylumbelliferyl-Neu5Ac at various concentrations in the submillimolar to millimolar range, 150 μl of the inhibitor 4-deoxy NeuSAc at various concentrations in the submillimolar to millimolar range, and 50 μl of 100 mM CaCl 2 . All solutions were made up in 50 mM sodium acetate buffer, pH 5.9, to a final volume of 500 μl. 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 spectrophotometer (Hitachi Model 3010). 4-methyl-umbelliferone in 1 M Tris, pH 9.0 with 1.33% Ethanol served as a standard. Enzyme activity was expressed as mM of NeuSAc 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 4-deoxy NeuSAc as follows: (The native substrate, NeuSAc, had a K^=0.626 mM when the Influenza A (H1N1) virus was used.) The K^ for 4-deoxy NeuSAc 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: where Ac represents acetyl, R represents hydrogen, fluorine or methoxy, 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-nitrophenylazophenyl, 4-nitrophenylazoresorcinyl, 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 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-nitrophenylazophenyl, 4-nitrophenylazoresorcinyl, 3-methoxyphenyl, 3- dimethylaminophenyl, 4-chloro-l-naphthyl, and 6-bromo-2- naphthyl. 5. The method of claim 1 or 2 wherein R represents methoxy 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-nitrophenylazophenyl, 4-nitrophenylazoresorcinyl, 3-methoxyphenyl, 3- dimethylaminophenyl, 4-chloro-l-naphthyl and 6-bromo-2- naphthyl. 6. 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: where Ac represents acetyl, R represents hydrogen, fluorine or methoxy, 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. 7. The method of claim 6 wherein the specific type of human influenza neuraminidase activity is human influenza A neuraminidase activity, human influenza B neuraminidase activity, or parainfluenza neuraminidose activity. 8. The method of claim 6 or 7 wherein the clinical sample is a pharyngeal, nasopharyngeal, or respiratory secretion. 9. The method of claim 6, 7 or 8 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-nitrophenylazophenyl, 4-nitrophenylazoresorcinyl, 3-methoxyphenyl, 3- dimethylaminopheny1, 4-chloro-l-naphthyl, and 6-bromo-2- naphthyl. 10. The method of claim 6, 7 or 8 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-nitrophenylazophenyl, 4-nitrophenylazoresorcinyl, 3-methoxyphenyl, 3- dimethylaminophenyl, 4-chloro-l-naphthyl, and 6-bromo-2- naphthy1. 11. The method of claim 6, 7 or 8 wherein R represents methoxy 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-nitrophenylazophenyl, 4-nitrophenylazoresorcinyl, 3-methoxyphenyl, 3- dimethylaminophenyl, 4-chloro-l-naphthyl, and 6-bromo-2- naphthyl. 12. A chromogenic substrate useful for detect¬ ing human influenza neuraminidase activity in a clinical sample suspected of having such activity, said substrate having the formula: where Ac represents acetyl, R is methoxy or fluorine, and X is a chromogenic group that exhibits a distinct color when cleaved from the substrate and salts of said substrate. 13. The chromogenic substrate of claim 12 wherein R represents methoxy the chromogenic group is selected from the group consisting of 4-methylumbelliferyl, 3-cyanoumbelliferyl, 2- or 4-nitrophenyl, 3-resorufin, 5-bromo-4-chloro-3-indolyl, 4-nitrophenylazophenyl, 4-nitrophenylazoresorcinyl, 3- methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-l- naphthyl, and 6-bromo-2-naphthyl. 14. The chromogenic substrate of claim 12 wherein R represents fluorine and the chromogenic group is selected from the group consisting of 4-methylumbelliferyl, 3-cyanoumbelliferyl, 2- or 4-nitrophenyl, 3-resorufin, 5-bromo-4-chloro-3-indolyl, 4-nitrophenylazophenyl, 4-nitrophenylazoresorcinyl, 3- methoxyphenyl, 3-dimethylaminophenyl, 4-chloro-l- naphthyl, and 6-bromo-2-naphth 1. 15. A chromogenic substrate useful for detect- ing human influenza neuraminidase activity in a clinical sample suspected of having such activity, said substrate having the formula: where Ac represents acetyl, R is hydrogen, and X is a chromogenic group selected from the group consisting of 3-σyanoumbelliferyl, 2- or 4-nitrophenyl, 3-resorufin, 5-bromo-4-chloro-3-indolyl, 4-nitrophenylazophenyl, 4-nitrophenylazoresorcinyl, 3-methoxyphenyl, 3-dimethyl- aminophenyl, 4-chloro-l-naphthyl, and 6-bromo-2-naphthyl and salts of said substrate.

Inventors: CLINKSCALES C WORTH, MAHER JAMES F, ROARK MICHAEL C, TURNER GREGORY A, CLINKSCALES, C., WORTH, MAHER, JAMES, F., ROARK, MICHAEL, C., TURNER, GREGORY, A.
Applicants: SYMEX CORP, SYMEX CORP.
Application year: 1990
Main classification: C07G3
IPC codes: C07H13, C07H15, C07H17, C12Q1, G01N33