Synthesis of novel N-acetylneuraminic acid derivatives as substrates for rapid detection of influenza virus neuraminidase

Abstract

Two novel N-acetylneuraminic acid derivatives, luciferyl N-acetylneuraminic acid (1) and luciferyl 4,7-di-O-methyl-N-acetylneuraminic acid (2), were designed and synthesized as substrates for the rapid detection of influenza virus neuraminidase. The sensitivity and specificity of the assays with compound 1 or 2 as the substrate for detection of neuraminidases from influenza virus (H1N1 and H5N1) and bacteria (A. ureafaciens and C. perfringens) were evaluated. Compound 1 was sensitive to neuraminidases from both influenza virus and bacteria. Biochemiluminescent assays with this compound with H1N1 and H5N1 neuraminidases were approximately 20- and 16-fold more sensitive, respectively, than the fluorescent method with the commercial substrate 4-MUNANA. In contrast, compound 2 was only sensitive to the neuraminidases from influenza virus, showing approximately 10- and 8-fold greater sensitivity than 4-MUNANA for the detection of H1N1 and H5N1 neuraminidases, respectively. The data showed that compound 2 could be used in assays that detect of influenza viral neuraminidase.

Bioluminescence-Based Neuraminidase Inhibition Assay for Monitoring Influenza Virus Drug Susceptibility in Clinical Specimens

“…The elevated zanamivir IC 50 suggests the presence of an exogenous source of NA (e.g., bacteria) in clinical sample 11, similarly to previous reports (30). To avoid such false positives that may arise from bacterial contamination, a second generation of the QFlu substrate (mentioned above) with a modified chemical structure (21) specific to influenza NA has recently been developed. On the other hand, despite similar H275Y content (ϳ90%), clinical sample 12 had a 3-fold-lower oseltamivir IC 50 than that of its matching isolate.…”

“…1, protocol A). The prototype QFlu kit used in this study contained a master mix including the NA substrate, luciferyl N-acetylneuraminic acid (21), and luciferase. NeuAc cleavage from the substrate releases luciferin, the substrate for luciferase.…”

“…Besides longer turnaround time, virus culturing can introduce changes that alter susceptibility to NA inhibitors (19,20). Recent advances in the development of new and more-sensitive methods for measuring NA activity, intended for detection of drug resistance at point of care (POC) (21), prompted the present study. Here, we assessed a prototype of the bioluminescence-based NI assay kit (QFlu) as a potential tool for monitoring drug susceptibility in influenza viruses.…”

Electrochemical Behavior of Zanamivir at Gold-Modified Boron-Doped Diamond Electrodes for an Application in Neuraminidase Sensing

“…8 The qualitative NA detection is very important to monitor the spread of pathogen microbes or viruses, while the quantification is also crusial for vaccine composition investigation. Previously, NA analysis was already developed based on enzymatic reaction, 9,10 ELISA, 11 RT-PCR, 12,13 and LC/MS/MS. 14 These methods generally require a several types of chemicals, special instruments, and highly skilled analysts.…”

Proximity Ligation‐Based Fluorogenic Imaging Agents for Neuraminidases

“…Based on the report that introduction of methyl groups to the C4 and C7 positions of the sialoside moiety can greatly enhance the selectivity of substrate-based inhibitors and probes of influenza NA, with comparatively small effect on binding [16,17] we sought to develop a virus-specific reagent for imaging influenza NA activities. Such a reagent could prove useful in specifically localizing viral NA activity, without interference from endogenous sialidase activities.…”