Zanamivir phosphonate congeners with anti-influenza activity and determining oseltamivir susceptibility of influenza viruses

1. A method for detecting the presence of oseltamivir-resistant influenza virus using a synthetic binding molecule, the method comprising the steps of:

(a) providing a sample suspected of comprising an oseltamivir-resistant influenza-virus or virus particle;

(b) contacting the sample with a synthetic binding molecule comprising a fluorescent reporting element in the presence and absence of oseltamivir carboxylate (OC);

(c) measuring the fluorescence of the reporting element of the synthetic binding molecule in the presence and absence of said oseltamivir carboxylate (OC); and

(d) measuring a difference in fluorescence the reporting element of the synthetic binding molecule bound to the influenza virus, or viral particle, in the presence and absence of oseltamivir,

wherein a lack of fluorescence by the reporting element of the synthetic binding molecule in the presence of oseltamivir as compared to the fluorescence when contacted with a oseltamivir-sensitive influenza virus control, detects the presence of an oseltamivir-resistant influenza virus in the sample,

wherein the synthetic binding molecule comprising a fluorescent reporting element is selected from the group consisting of:

BM 2: Zanamivir-(Triazole Linker)-Fluorescein

BM 4: Zanamivir-(Ethylene Glycol Linker)-Fluorescein

BM 6: (Tamiphosphor Guanidine)-(Ethylene Glycol Linker)-Fluorescein

BM 8: PhosphaZanamivir-(Ethylene Glycol Linker)-Fluorescein

and

BM 10: (PhosphaZanamivir Monoester)-(Ethylene Glycol Linker)-Fluorescein

wherein x=1 to 4.

2. A method for detecting the presence of oseltamivir-resistant influenza virus using a synthetic binding molecule, the method comprising the steps of:

(a) providing a sample suspected of comprising an oseltamivir-resistant influenza-virus or virus particle;

(b) contacting the sample with a synthetic binding molecule comprising biotin in the presence and absence of oseltamivir carboxylate (OC);

(c) contacting the synthetic binding molecule comprising biotin with an avidin-linked or streptavidin-linked moiety comprising a fluorescent reporting element;

(d) measuring the fluorescence of the reporting element of the avidin-linked or streptavidin-linked moiety comprising a fluorescent reporting element in the presence and absence of said oseltamivir carboxylate (OC); and

(e) measuring a difference in fluorescence of the fluorescent reporting element of the avidin-linked or streptavidin-linked moiety bound to the synthetic binding molecule comprising biotin bound to the influenza virus, or viral particle, in the presence and absence of oseltamivir, wherein a lack of fluorescence by the reporting element of the avidin-linked or streptavidin-linked moiety binding in the presence of oseltamivir as compared to the fluorescence when contacted with a oseltamivir-sensitive influenza virus control, detects the presence of an oseltamivir-resistant influenza virus in the sample,

wherein the synthetic binding molecule comprising biotin is selected from the group consisting of:

BM 1: Zanamivir-(Triazole Linker)-Biotin

BM 3: Zanamivir-(Ethylene Glycol Linker)-Biotin

wherein x=1 to 4,

BM 5: (Tamiphosphor Guanidine)-(aliphatic chain Linker)-Biotin

wherein x=1 to 4,

BM 7: PhosphaZanamivir-(Triazole Linker)-Biotin

BM 9: (PhosphaZanamivir Monoester)-(Ethylene Glycol Linker)-Biotin

wherein x=1 to 4, and

wherein R is

3. A method for detecting the presence of oseltamivir-resistant influenza virus using a synthetic binding molecule, the method comprising the steps of:

(a) providing a sample suspected of comprising an oseltamivir-resistant influenza-virus or virus particle;

(b) contacting the sample with a synthetic binding molecule comprising a compound of formula (I) further comprising biotin or a fluorophore in the presence and absence of oseltamivir carboxylate (OC);

(c) when the synthetic binding molecule comprising a compound of formula (I) comprises biotin, contacting the synthetic binding molecule of formula (I) with an avidin-linked or streptavidin-linked moiety comprising a fluorescent reporting element;

(d) measuring the fluorescence of the synthetic binding molecule of formula (I) or fluorescent reporting element in the presence and absence of said oseltamivir carboxylate (OC); and

(e) measuring a difference in fluorescence of the fluorophore or reporting element of the synthetic compound of formula (I) bound to the influenza virus, or viral particle, in the presence and absence of oseltamivir,

wherein a lack of fluorescence by the fluorophore or reporting element of the synthetic compound of formula (I) in the presence of oseltamivir as compared to the fluorescence when contacted with a oseltamivir-sensitive influenza virus control, detects the presence of an oseltamivir-resistant influenza virus in the sample,

wherein the compound of formula (I) has the structure:

wherein A is PO(OR)(OR 5 ), wherein R and R 5 are independently selected from H, C1-C10 alkyl, aryl, aralkyl and X, wherein X is a cationic counterion selected from the group consisting of ammonium, methyl ammonium, dimethylammonium, trimethylammonium, tetramethylammonium, ethanol-ammonium, dicyclohexylammonium, guanidinium, ethylenediammonium cation, lithium cation, sodium cation, potassium cation, cesium cation, beryllium cation, magnesium cation, calcium cation, and zinc cation, wherein B is NHR 6 , NH 3 + Y − , R 6 N(C═NH)NH 2 or R 6 N(C═NH 2 + )NH 2 Y − , wherein R 6 represents hydrogen, C1-C10 alkyl, aryl, or aralkyl, wherein Y − is a anionic counterion selected from the group consisting of chloride, bromide, iodide, acetate, trifluoroacetate, phosphate, diphosphate, nitrate, sulfate, benzenesulfonate, benzoate, salicylate, hydroxynaphthoate, fumarate, maleate, lactate, malate, succinate, tartrate, citrate, glutamate, gluconate, and stearate, wherein R 1 is CH 3 or CF 3 ; wherein R 2 is O═C—NHR 7 , wherein R 7 represents a linker annexed with functional moiety selected from biotin, or fluorophore, and wherein R 3 and R 4 are independently hydrogen, C1-C10 alkyl or O═C—R 8 , where R 8 represents C1-C10 alkyl, aryl, or aralkyl.

4. The method of any of claim 1 , 2 , or 3 wherein the oseltamivir-resistant influenza comprises a mutation at amino acid position 274 of the neuraminidase (NA) protein of influenza.

5. The method of any of claim 1 , 2 , or 3 , wherein the influenza mutant is resistant to a neuraminidase inhibitor drug but is still sensitive to the binding molecule.

6. The method of claim 4 , wherein the influenza virus containing sample is selected from the group consisting of mucus, saliva, respiratory secretion, throat wash, nasal wash, spinal fluid, sputum, urine, semen, sweat, feces, plasma, blood, broncheoalveolar fluid, vaginal fluid, tear fluid and tissue biopsy.

7. The method of claim any of claim 1 , 2 , or 3 , wherein the binding of the synthetic binding molecule to the influenza virus is competitively inhibited by the concurrent binding by oseltamivir carboxylate.

8. The method of claim 3 , wherein the fluorophore is selected from: fluorescein, BODIPY, Alexa Fluor, Cy3, Cy5, DyLight 488, Oregon Green, tetramethylrhodamine, Rhodamine Red, Texas Red, pyridyloxazole, benzoxadiazole, NBD halides, iodoacetamides, SB, Lucifer Yellow, iodoacetamide, stilbene, coumarin, naphthalene, aziridine, dapoxyl, pyrene, bimanes, xanthene, cyanine, pyrene, phthalocyanine, phycobiliprotein, and squarene.