Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-08-29
2002-07-16
Peselev, Elli (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S018500, C536S018600, C536S053000
Reexamination Certificate
active
06420552
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to an improved synthesis of derivatives of N-acetylneuraminic acid monoalkylated at either the 4- or the 7-position. The synthetic procedures represent improvements and enhancements that permit obtaining large quantities of the products suitable for commercial production. These derivatives of N-acetylneuraminic acid can be used as chromogenic substrates for the detection of viral neuraminidases.
BACKGROUND OF THE INVENTION
Viral infections are a principal cause of illness due to communicable diseases that affect the public at large. Of these, influenza viruses, including types A and B, are a significant factor responsible for causing respiratory symptoms as well as systemic malaise; other respiratory viruses include parainfluenza 1, 2, 3, and 4, respiratory syncytial virus, and adenovirus. The influenza viruses undergo rapid mutation of strains, producing pathogens with varying degrees of virulence and severity of symptoms. Recently, influenza infection has been as high as the fifth leading cause of death from acute respiratory disease in the United States (Morbidity and Mortality Weekly Report, 36 (1987) 2).
Influenza virus types A, B, and C belong to the family of Orthomyxoviridae. Influenza A and B are significant pathogens in children and adults causing sever lower respiratory tract disease, whereas influenza C can cause sporadic upper respiratory tract. illness. Influenza virus is highly contagious and can affect large proportions of the population each winter. Influenza A epidemics occur every 2-3 years, whereas influenza B epidemics appear every 4-6 years. Symptoms include moderate to high fever together with chills, headache, myalgia, rhinorrhea, among others. Importantly, virus progeny are detectable 24 hours prior to the appearance of symptoms, and virus titers peak 24-48 hours after symptoms arise.
For this reason it is important to have available ways of diagnosing the presence of an influenza infection, and of distinguishing it from related viral and bacterial infections. Particularly among infants, the elderly and those having compromised or deficient immune responses, early diagnosis of influenza can lead to appropriate symptomatic treatment to minimize the risk of morbidity.
Diagnosis of viral infection, such as infection by influenza virus, may be carried out by detecting the presence of unique moieties characteristic of the virus. Virus particles typically carry distinctive antigenic components on the exterior of the virion which may be detected by specific ligand-antiligand interactions, in particular by the use of an antibody specific for a viral epitope. Such interactions rely on the law of mass action, and for this reason may have limited sensitivity. Many virus particles additionally carry specific enzymatic activities on the virion particle. Influenza viruses, parainfluenza viruses, and mumps are examples of such viruses; they are endowed with a virus-specific surface glycoprotein with neuraminidase activity as an integral part of the virion. Utilization of the enzymatic activity for diagnostic assays in such cases offers the potential for increasing the sensitivity of a detection method. For example, influenza A and B, having neuraminidase activity, are detectable in this way, whereas influenza C is not.
N-acetylneuraminic acid (sialic acid, Neu5Ac), whose structure is shown below with atoms numbered, is the terminal saccharide residue
of many complex carbohydrate side chains of cell surface glycoproteins. In this structure the C2 position is the anomeric carbon, which is characterized by being part of a hemiketal group, or the 2-ketoside moiety. The glycosidic linkage bonding Neu5Ac with the penultimate saccharide is the substrate of the neuraminidase activity of the influenza virion. The neuraminidase hydrolyzes glycosidic linkages having the a anomeric configuration, thereby cleaving Neu5Ac from the penultimate saccharide. Consequently, suitable synthetic substrates may be derivatives of Neu5Ac in 2-ketosidic a linkage with a detectable moiety. The moiety then provides a product, when the substrate is acted upon by the viral neuraminidase activity, that signals the presence and amount of influenza virus particles in a sample. Since the viral enzyme cleaves the substrate catalytically, the sensitivity of detecting the presence of the enzyme is greatly enhanced. For this reason the overall sensitivity of detection may be comparable to, and may even be improved over, that provided by antibody binding assays.
One method for detecting the presence of a virus through the reaction of an enzyme with a chromogenic substrate for the enzyme is described in U.S. Pat. No. 5,252,458, which is incorporated herein by reference. An assay for the direct measurement of influenza neuraminidase was developed by Yolken et al. (J. Infectious Diseases 142 (1980) 516-523). Yolken et al. used the 4-methylumbelliferyl-2-ketoside of Neu5Ac as a fluorescent substrate to measure neuraminidase activity in preparations containing small quantities of cultivated virus as well as in some nasal wash specimens from human volunteers infected with the influenza virus. Yolken et al. suggested that “successful development of influenza neuraminidase might thus provide for a practical means of influenza diagnosis that is sufficiently rapid to allow for the institution of appropriate preventive and therapeutic interventions.” According to Yolken et al., colorimetric assays were insufficiently sensitive for clinical applications, suggesting instead that fluorimetric assays for influenza neuraminidase might be suitable for detecting the virus in clinical samples.
Pachucki et al. (J. Clinical Microbiology 26 (1988) 2664-2666) tested the 4-methylumbelliferyl-2-ketoside of Neu5Ac on clinical specimens collected from influenza patients. Due to its low sensitivity, the assay was not useful in detecting neuraminidase in clinical specimens. The assay did, however, identify 91% of virus-positive isolates 25 hours after inoculation of tissue cultures.
The use of modified Neu5Ac substrates can increase the specificity of the neuraminidase assay. In sialic acids, the C4 position has been reported to play an important role in enzyme-substrate interactions. Further, since it is known that salivary bacterial enzymes exhibit neuraminidase activity (Varki et al., J. Biol. Chem. 258 (1983) 12465-12471), it is essential to avoid these undesired enzymatic activities. It has, for example, been shown that ketosides of 4-methoxy-Neu5Ac are resistant towards certain bacterial sialidases (Beau et al., Eur. J. Biochem. 106 (1980) 531-540).
U.S. Pat. No. 5,252,458 to Liav et al. provides a direct chromogenic assay for detecting a virus, including influenza viruses and parainfluenza viruses, that include in the virion a characteristic enzymatic activity, such as neuraminidase activity. The method, which is implemented in a clinic or physician's office, includes incubating a clinical sample suspected of containing the virus with a solution of a chromogenic substrate. The samples typically are obtained by swabbing the pharyngeal, or nasopharyngeal surfaces. If the virus is present, a chromogen is cleaved from the substrate, the chromogen is then reacted with a precipitating agent that intensifies the color, and the colored precipitate is concentrated for detection as a colored spot. The patent also discloses a kit for use in the clinic or physician's office that includes a filtration device for concentrating the colored precipitate into a spot.
U.S. Pat. No. 5,252,458 to Liav et al. provides synthetic routes for the synthesis of a precursor for chromogenic substrates that are useful in the diagnostic assay of viruses. Specifically the patent discloses syntheses for 4-alkoxy-N-acetylneuraminic acid.
U.S. Pat. No. 5,663,055 to Turner et al. discloses 4-modified Neu5Ac chromogenic substrates of viral neuraminidases for use in assays carried out in a clinic or a physician's office. The modification at position 4 includes hydrogen, fluorine, methoxy or ethoxy, and t
Bundle David R.
Du Minghui
Hingsgaul Ole
Srivastava Geeta
Srivastava Om
Fitch Even Tabin & Flannery
Peselev Elli
Zymetx, Inc.
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