Cell-permeable probes for identification and imaging of sialidases
10214765 ยท 2019-02-26
Assignee
Inventors
- Chi-Huey Wong (Rancho Santa Fe, CA)
- Jim-Min Fang (Taipei, TW)
- Yih-Shyun E. Cheng (Taipei, TW)
- Chamg-Sheng Tsai (New Taipei, TW)
Cpc classification
C07H13/04
CHEMISTRY; METALLURGY
C12Y302/01018
CHEMISTRY; METALLURGY
C07H13/12
CHEMISTRY; METALLURGY
C12N9/2402
CHEMISTRY; METALLURGY
C12Q1/04
CHEMISTRY; METALLURGY
International classification
C07H5/00
CHEMISTRY; METALLURGY
C07H13/12
CHEMISTRY; METALLURGY
C07H13/04
CHEMISTRY; METALLURGY
Abstract
Provided herein are compounds for use as sialidase inhibitors, including alkynyl-3-fluorosialyl fluoride. The compounds, which include the compound DFSA, function by trapping a 3-fluorosialylenzyme intermediate (reporter-inhibitor-enzyme conjugate). These compounds can be conjugated with a detectable tagging moiety for isolation and identification of sialidases.
Claims
1. A compound of formula (I): ##STR00051## or a salt thereof, wherein F atom at the C3-position is axial or equatorial; R.sup.1 is H or C.sub.1-6 alkyl; R.sup.2 is OR.sup.2O, N.sub.3, N(R.sup.2N).sub.2, or NH(CNH)NH.sub.2; each instance of R.sup.2O is independently hydrogen, C.sub.1-6 alkyl, acyl, or a hydroxyl protecting group; each instance of R.sup.2N is independently hydrogen, C.sub.1-6 alkyl, acyl, or an amine protecting group; each instance of R.sup.3a and R.sup.3b is independently hydrogen, C(O)R.sup.3r, or a hydroxyl protecting group; each instance of R.sup.3r is C.sub.1-6 alkyl, aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheteroaryl, or alkylheterocyclyl; X is selected from the group consisting of O, O(CO), NH, NH(CO), (CO)NH, O(CO)NH, O(CS)NH, NH(CO)NH, and NH(CS)NH; R.sup.4 is H, C.sub.1-6 alkyl, or -L-Z; Y is CF.sub.3, C.sub.1-6 alkyl or -L-Z; each instance of L is independently selected from the group consisting of (CH.sub.2).sub.n, (CH.sub.2).sub.nCO, (CH.sub.2).sub.nNH, (CO)(CH.sub.2).sub.n, (CH.sub.2).sub.nNH(CO), (CO)(CH.sub.2).sub.nNH(CO), (CH.sub.2).sub.nSCH.sub.2(CO), and (CH.sub.2CH.sub.2O).sub.n; each instance of n is an integer from 1 to 8, inclusive; each instance of Z is alkynyl, alkenyl, halogen, N(R.sup.N).sub.2, OR.sup.O, SR.sup.S, or CO.sub.2R.sup.O; each instance of R.sup.N is independently hydrogen, C.sub.1-6 alkyl, acyl, or an amine protecting group; each instance of R.sup.O is independently hydrogen, C.sub.1-6 alkyl, acyl, or a hydroxyl protecting group; each instance of R.sup.S is independently hydrogen, C.sub.1-6 alkyl, or a thiol protecting group; provided that the compound is not of the formula ##STR00052## provided that when R.sup.4 is -L-Z, Y is C.sub.1-6 alkyl; and provided that when Y is -L-Z, R.sup.4 is H or C.sub.1-6 alkyl.
2. The compound of claim 1 of formula (II-a): ##STR00053## or a salt thereof, wherein R.sup.3c is independently hydrogen, C.sub.1-6 alkyl, acyl, or a hydroxyl protecting group.
3. The compound of claim 2 of formula (II-b): ##STR00054## or a salt thereof.
4. The compound of claim 3, having a formula (II-b1): ##STR00055## or a salt thereof.
5. The compound of claim 4, having the formula (II-b2): ##STR00056## wherein R.sup.y1 is hydrogen, halogen, or C.sub.1-6 alkyl, or a salt thereof.
6. The compound of claim 1 of formula (II-c): ##STR00057## or a salt thereof.
7. The compound of claim 6, having a formula selected from (II-c1) and (II-C2): ##STR00058## wherein R.sup.y2 is hydrogen or C.sub.1-6 alkyl, or a salt thereof.
8. The compound of claim 6, wherein Y is C.sub.1-6 alkyl.
9. The compound of claim 8, wherein Y is methyl or CF.sub.3.
10. The compound of claim 1, wherein R.sup.1 is H or methyl.
11. The compound of claim 1, wherein the F atom at the C3-position is axial.
12. The compound of claim 1, wherein the F atom at the C3-position is equatorial.
13. The compound of claim 1, wherein R.sup.3a is C(O)R.sup.3r, wherein R.sup.3r is C.sub.1-6 alkyl or aryl.
14. The compound of claim 1, wherein R.sup.3a is benzoyl.
15. The compound of claim 1, wherein R.sup.3a is CH.sub.3CO, C.sub.2H.sub.5CO, C.sub.3H.sub.7CO, t-BuCO, CF.sub.3CO, PhCH.sub.2CO, or C.sub.6H.sub.5CO.
16. The compound of claim 1, wherein the compound is one of the following formulae: ##STR00059##
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
DETAILED DESCRIPTION OF THE INVENTION
(9) This invention relates to irreversible inhibitors for sialidases. The provided irreversible inhibitors form a covalent bond with sialidases and trap the 3-fluorosialyl-enzyme intermediate. The fluorosialyl-enzyme adduct can be ligated with a detectable tagging moiety such as azide-annexed biotin (azido-biotin) via CuAAC for isolation and identification of the sialidase.
(10) The invention further provides ester-protected sialidases inhibitors as the membrane permeable precursor to improve cell uptake. (A. K. Sarkar, et al. Proc. Natl. Acad. Sci. USA 1995, 92, 3323. C. L. Jacobs, et al. Methods Enzymol. 2000, 327, 260.) The cell-permeable sialidase inhibitors have allowed, for the first time, identification and in situ imaging of the changes of sialidase activity under physiological conditions.
(11) In one aspect, the present disclosure provides novel irreversible sialidase inhibitors of formula (I):
(12) ##STR00016##
(13) or a salt thereof,
(14) wherein F atom at the C3-position is axial or equatorial; R.sup.1 is H or optionally substituted C.sub.1-6 alkyl; R.sup.2 is OR.sup.2O, N.sub.3, N(R.sup.2N).sub.2, or guanidine;
(15) each instance of R.sup.2O is independently hydrogen, optionally substituted C.sub.1-6 alkyl, optionally substituted acyl, or an oxygen protecting group;
(16) each instance of R.sup.2N is independently hydrogen, optionally substituted C.sub.1-6 alkyl, optionally substituted acyl, or a nitrogen protecting group;
(17) each instance of R.sup.3a and R.sup.3b is independently hydrogen, C(O)R.sup.3r, or an oxygen protecting group;
(18) each instance of R.sup.3r is optionally substituted C.sub.1-6 alkyl, optionally substituted aryl, optionally substituted heteroaryl, or optionally substituted heterocycle, optionally substituted alkylaryl, optionally substituted alkylheteroaryl, or optionally substituted alkylheterocycle;
(19) X is selected from the group consisting of O, O(CO), NH, NH(CO), (CO)NH, O(CO)NH, O(CS)NH, NH(CO)NH, and NH(CS)NH;
(20) R.sup.4 is H, optionally substituted C.sub.1-6 alkyl, or -L-Z;
(21) Y is optionally substituted C.sub.1-6 alkyl or -L-Z;
(22) each instance of L is independently selected from the group consisting of (CH.sub.2).sub.n, (CH.sub.2).sub.nCO, (CH.sub.2).sub.nNH, (CO)(CH.sub.2).sub.n, (CH.sub.2).sub.nNH(CO), (CO)(CH.sub.2).sub.nNH(CO), (CH.sub.2).sub.nSCH.sub.2(CO), and (CH.sub.2CH.sub.2O).sub.n;
(23) each instance of n is an integer from 1 to 8, inclusive;
(24) each instance of Z is a functional group for further ligation; and
(25) provided that the compound is not of the formula
(26) ##STR00017##
(27) As generally defined herein, R.sup.1 is H or optionally substituted C.sub.1-6 alkyl. In certain embodiments, R.sup.1 is H. In certain embodiments, R.sup.1 is optionally substituted C.sub.1-6 alkyl. In certain embodiments, R.sup.1 is methyl, ethyl, or n-propyl.
(28) As generally defined herein, R.sup.2 is OR.sup.2O, N.sub.3, N(R.sup.2N).sub.2, or guanidine. In certain embodiments, R.sup.2 is OR.sup.2O, wherein R.sup.2O is independently hydrogen, optionally substituted C.sub.1-6 alkyl, optionally substituted acyl, or an oxygen protecting group. In certain embodiments, R.sup.2 is OH. In certain embodiments, R.sup.2 is OR.sup.2O, wherein R.sup.2O is optionally substituted C.sub.1-6 alkyl. In certain embodiments, R.sup.2 is OCH.sub.3 or OC.sub.2H.sub.5. In certain embodiments, R.sup.2 is OH. In certain embodiments, R.sup.2 is OR.sup.2O, wherein R.sup.2O is optionally substituted acyl. In certain embodiments, R.sup.2 is OR.sup.2O, wherein R.sup.2O is acetyl. In certain embodiments, R.sup.2 is OH. In certain embodiments, R.sup.2 is OR.sup.2O, wherein R.sup.2O is an oxygen protecting group. In certain embodiments, R.sup.2 is N.sub.3. In certain embodiments, R.sup.2 is N(R.sup.2N).sub.2, wherein each instance of R.sup.2N is independently hydrogen, optionally substituted C.sub.1-6 alkyl, optionally substituted acyl, or a nitrogen protecting group. In certain embodiments, R.sup.2 is NH(R.sup.2N), wherein R.sup.2N is independently hydrogen, optionally substituted C.sub.1-6 alkyl, optionally substituted acyl, or a nitrogen protecting group. In certain embodiments, R.sup.2 is NH.sub.2.
(29) As generally defined herein, R.sup.3a is independently hydrogen, C(O)R.sup.3r, or an oxygen protecting group. In certain embodiments, R.sup.3a is hydrogen. In certain embodiments, R.sup.3a is C(O)R.sup.3r, wherein R.sup.3r is optionally substituted C.sub.1-6 alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycle, optionally substituted alkylaryl, optionally substituted alkylheteroaryl, or optionally substituted alkylheterocycle. In certain embodiments, R.sup.3a is C(O)R.sup.3r, wherein R.sup.3r is optionally substituted C.sub.1-6 alkyl. In certain embodiments, R.sup.3a is C(O)R.sup.3r, wherein R.sup.3r is methyl or ethyl. In certain embodiments, R.sup.3a is C(O)R.sup.3r, wherein R.sup.3r is optionally substituted aryl. In certain embodiments, R.sup.3a is C(O)R.sup.3r, wherein R.sup.3r is phenyl. In certain embodiments, R.sup.3a is C(O)R.sup.3r, wherein R.sup.3r is optionally substituted alkylaryl. In certain embodiments, R.sup.3a is C(O)R.sup.3r, wherein R.sup.3r is optionally substituted benzoyl. In certain embodiments, R.sup.3a is CH.sub.3CO, C.sub.2H.sub.5CO, C.sub.3H.sub.7CO, t-BuCO, CF.sub.3CO, PhCH.sub.2CO, or C.sub.6H.sub.5CO.
(30) As generally defined herein, R.sup.3b is independently hydrogen, C(O)R.sup.3r, or an oxygen protecting group. In certain embodiments, R.sup.3b is hydrogen. In certain embodiments, R.sup.3b is C(O)R.sup.3r, wherein R.sup.3r is optionally substituted C.sub.1-6 alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocycle, optionally substituted alkylaryl, optionally substituted alkylheteroaryl, or optionally substituted alkylheterocycle. In certain embodiments, R.sup.3b is C(O)R.sup.3r, wherein R.sup.3r is optionally substituted C.sub.1-6 alkyl. In certain embodiments, R.sup.3b is C(O)R.sup.3r, wherein R.sup.3r is methyl or ethyl. In certain embodiments, R.sup.3b is C(O)R.sup.3r, wherein R.sup.3r is optionally substituted aryl. In certain embodiments, R.sup.3b is C(O)R.sup.3r, wherein R.sup.3r is phenyl. In certain embodiments, R.sup.3b is C(O)R.sup.3r, wherein R.sup.3r is optionally substituted alkylaryl. In certain embodiments, R.sup.3b is C(O)R.sup.3r, wherein R.sup.3r is optionally substituted benzoyl. In certain embodiments, R.sup.3b is CH.sub.3CO, C.sub.2H.sub.5CO, C.sub.3H.sub.7CO, t-BuCO, CF.sub.3CO, PhCH.sub.2CO, or C.sub.6H.sub.5CO.
(31) As generally defined herein, linker X is selected from the group consisting of O, O(CO), NH, (CO)NH, NH(CO), O(CO)NH, O(CS)NH, NH(CO)NH, and NH(CS)NH. In certain embodiments, X is O. In certain embodiments, X is O(CO). In certain embodiments, X is NH(CO). In certain embodiments, X is (CO)NH. In certain embodiments, X is O(CO)NH.
(32) As generally defined herein, R.sup.4 is H, optionally substituted C.sub.1-6 alkyl, or -L-Z. In certain embodiments, R.sup.4 is H. In certain embodiments, R.sup.4 is -L-Z, wherein L is independently selected from the group consisting of (CH.sub.2).sub.n, (CH.sub.2).sub.nCO, (CH.sub.2).sub.nNH, (CO)(CH.sub.2).sub.n, (CH.sub.2).sub.nNH(CO), (CO)(CH.sub.2).sub.nNH(CO), (CH.sub.2).sub.nSCH.sub.2(CO), and (CH.sub.2CH.sub.2O).sub.n; each instance of n is an integer from 1 to 8, inclusive; and Z is a functional group for further ligation. In certain embodiments, R.sup.4 is (CH.sub.2).sub.nZ, wherein n is an integer from 1 to 8, inclusive; and Z is a functional group for further ligation. In certain embodiments, R.sup.4 is (CH.sub.2).sub.nZ, wherein n is an integer from 1 to 8, inclusive; and Z is is optionally substituted alkyne, optionally substituted alkene, halogen, N.sub.3, N(R.sup.N).sub.2, OR.sup.O, SR.sup.S, or CO.sub.2R.sup.O; wherein each instance of R.sup.N is independently hydrogen, optionally substituted C.sub.1-6 alkyl, or a nitrogen protecting group; each instance of R.sup.O is independently hydrogen, optionally substituted C.sub.1-6 alkyl, optionally substituted acyl, or an oxygen protecting group; and each instance of R.sup.S is independently hydrogen, optionally substituted C.sub.1-6 alkyl, or a sulfur protecting group.
(33) As generally defined herein, Y is optionally substituted C.sub.1-6 alkyl or -L-Z. In certain embodiments, Y is optionally substituted C.sub.1-6 alkyl. In certain embodiments, Y is substituted C.sub.1-6 alkyl. In certain embodiments, Y is CF.sub.3. In certain embodiments, Y is unsubstituted C.sub.1-6 alkyl. In certain embodiments, Y is methyl, ethyl, or n-propyl. In certain embodiments, Y is -L-Z, wherein L is independently selected from the group consisting of (CH.sub.2).sub.n, (CH.sub.2).sub.nCO, (CH.sub.2).sub.nNH, (CO)(CH.sub.2).sub.n, (CH.sub.2).sub.nNH(CO), (CO)(CH.sub.2).sub.nNH(CO), (CH.sub.2).sub.nSCH.sub.2(CO), and (CH.sub.2CH.sub.2O).sub.n; each instance of n is an integer from 1 to 8, inclusive; and Z is a functional group for further ligation. In certain embodiments, Y is (CH.sub.2).sub.nZ, wherein n is an integer from 1 to 8, inclusive; and Z is a functional group for further ligation. In certain embodiments, Y is (CH.sub.2).sub.nZ, wherein n is an integer from 1 to 8, inclusive; and Z is is optionally substituted alkyne, optionally substituted alkene, halogen, N.sub.3, N(R.sup.N).sub.2, OR.sup.O, SR.sup.S, or CO.sub.2R.sup.O; wherein each instance of R.sup.N is independently hydrogen, optionally substituted C.sub.1-6 alkyl, or a nitrogen protecting group; each instance of R.sup.O is independently hydrogen, optionally substituted C.sub.1-6 alkyl, optionally substituted acyl, or an oxygen protecting group; and each instance of R.sup.S is independently hydrogen, optionally substituted C.sub.1-6 alkyl, or a sulfur protecting group.
(34) In one embodiments for the compound of formula (I), Z is optionally substituted alkyne, optionally substituted alkene, halogen, N.sub.3, N(R.sup.N).sub.2, OR.sup.O, SR.sup.S, or CO.sub.2R.sup.O; wherein each instance of R.sup.N is independently hydrogen, optionally substituted C.sub.1-6 alkyl, or a nitrogen protecting group; each instance of R.sup.O is independently hydrogen, optionally substituted C.sub.1-6 alkyl, optionally substituted acyl, or an oxygen protecting group; and each instance of R.sup.S is independently hydrogen, optionally substituted C.sub.1-6 alkyl, or a sulfur protecting group.
(35) In some embodiments, the compound of formula (I) of formula (II-a):
(36) ##STR00018##
(37) or a salt thereof,
(38) wherein R.sup.3c is independently hydrogen, optionally substituted C.sub.1-6 alkyl, optionally substituted acyl, or an oxygen protecting group.
(39) In certain embodiments, R.sup.3c is hydrogen. In certain embodiments, R.sup.3c is optionally substituted C.sub.1-6 alkyl. In certain embodiments, R.sup.3c is methyl or ethyl. In certain embodiments, R.sup.3c is optionally substituted acyl. In certain embodiments, R.sup.3c is acetyl. In certain embodiments, R.sup.3c is an oxygen protecting group.
(40) In some embodiments, the compound of formula (I) of formula (II-b):
(41) ##STR00019##
(42) or a salt thereof.
(43) In some embodiments, the compound of formula (I) of formula (II-b1):
(44) ##STR00020##
(45) or a salt thereof.
(46) In some embodiments, the compound of formula (I) of formula (II-b2):
(47) ##STR00021##
(48) or a salt thereof,
(49) wherein R.sup.y1 is hydrogen, halogen, or optionally substituted C.sub.1-6 alkyl.
(50) As defined herein, R.sup.y1 is hydrogen, halogen, or optionally substituted C.sub.1-6 alkyl. In certain embodiments, R.sup.y1 is hydrogen. In certain embodiments, R.sup.y1 is halogen. In certain embodiments, R.sup.y1 is optionally substituted C.sub.1-6 alkyl. In certain embodiments, R.sup.y1 is methyl or ethyl.
(51) In some embodiments, the compound of formula (I) of formula (II-b3):
(52) ##STR00022##
(53) or a salt thereof.
(54) In some embodiments, the compound of formula (I) of formula (II-c):
(55) ##STR00023##
(56) or a salt thereof.
(57) In some embodiments, the compound of formula (I) of formula (II-c1):
(58) ##STR00024##
(59) or a salt thereof.
(60) In some embodiments, the compound of formula (I) of formula (II-c2):
(61) ##STR00025##
(62) or a salt thereof,
(63) wherein R.sup.y2 is hydrogen, halogen, or optionally substituted C.sub.1-6 alkyl.
(64) As defined herein, R.sup.y2 is hydrogen, halogen, or optionally substituted C.sub.1-6 alkyl. In certain embodiments, R.sup.y2 is hydrogen. In certain embodiments, R.sup.y2 is optionally substituted C.sub.1-6 alkyl. In certain embodiments, R.sup.y2 is methyl or ethyl.
(65) In some embodiments, the compound of formula (I) of formula (II-c3):
(66) ##STR00026##
(67) or a salt thereof.
(68) In some embodiments, the provided compounds have the F atom at the C3 axial position. In some embodiments, the provided compounds have the F atom at the C3 equatorial position.
(69) In another aspect, the invention provides a sialidase protein adduct comprising a compound provided herein. In certain embodiments, the sialidase protein is covalently conjugated to the compound. In certain embodiments, the protein adduct is of the formula
(70) ##STR00027##
wherein Y, X, R.sup.2, R.sup.3a, R.sup.3b, R.sup.4 are as defined herein. In certain embodiments, the compound is DFSA-5-yne or DFSA-7-yne. In certain embodiments, the compound is covalently linked to one or more tyrosine (Y) residues within any peptide of SEQ ID NOS: 1-6, wherein the peptide is a fragment of nanA, nanB, nanC, nanJ, nanI or nanH.
(71) In another aspect, the invention provides a detectable conjugate comprising a compound as described herein, wherein the compound is covalently conjugated to a detectable tagging moiety.
(72) In another aspect, the invention provides a detectable sialidase conjugate comprising a sialidase protein adduct as described herein, wherein the sialidase protein adduct is covalently conjugated to a detectable tagging moiety.
(73) The detectable tagging moiety is a functional group that enables detection of the entity to which it is conjugated to. The provided detectable conjugates have a detectable tagging moiety covalently conjugated to the compound or sialidase conjugate as described herein. The provided detectable conjugates can be ascertained for their existence and presence by detection of the signals generated from the detectable tagging moiety. The detection of the signals can be conducted by any of the chemical or physical means such as imaging or recordation of signals. In certain embodiments, the signals are detected by the streptavidin-specific reporting signals.
(74) Exemplary detectable tagging moieties include, but are not necessarily limited to, fluorescent molecules (e.g., autofluorescent molecules, molecules that fluoresce upon contact with a reagent, etc.), radioactive labels (e.g., .sup.111In, .sup.125I, .sup.131I, .sup.212B, .sup.90Y, .sup.186Rh, and the like); biotin (e.g., to be detected through reaction of biotin and avidin); fluorescent tags; imaging reagents (e.g., those described in U.S. Pat. No. 4,741,900 and U.S. Pat. No. 5,326,856), and the like. Detectable labels also include peptides or polypeptides that can be detected by antibody binding, e.g., by binding of a detectably labeled antibody or by detection of bound antibody through a sandwich-type assay. Also suitable for use are quantum dots (e.g., detectably labeled semiconductor nanocrystals, such as fluorescently labeled quantum dots, antibody-conjugated quantum dots, and the like). See, e.g., Dubertret et al. 2002 Science 298:759-1762; Chan et al. (1998) Science 281:2016-2018; U.S. Pat. No. 6,855,551; Bruchez et al. (1998) Science 281:2013-2016.
(75) Suitable fluorescent molecules (fluorophores) include, but are not limited to, fluorescein, fluorescein isothiocyanate, succinimidyl esters of carboxyfluorescein, succinimidyl esters of fluorescein, 5-isomer of fluorescein dichlorotriazine, caged carboxyfluorescein-alanine-carboxamide, Oregon Green 488, Oregon Green 514; Lucifer Yellow, acridine Orange, rhodamine, tetramethylrhodamine, Texas Red, propidium iodide, JC-1 (5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazoylcarbocyanine iodide), tetrabromorhodamine 123, rhodamine 6G, TMRM (tetramethylrhodamine-, methyl ester), TMRE (tetramethylrhodamine, ethyl ester), tetramethylrosamine, rhodamine B and 4-dimethylaminotetramethylrosamine, green fluorescent protein, blue-shifted green fluorescent protein, cyan-shifted green fluorescent protein, red-shifted green fluorescent protein, yellow-shifted green fluorescent protein, 4-acetamido-4-isothiocyanatostilbene-2,2disulfonic acid; acridine and derivatives: acridine, acridine isothiocyanate; 5-(2-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS); 4-amino-N-[3-vinylsulfonyl)phenyl]naphth-alimide-3,5 disulfonate; N-(4-anilino-1-naphthyl)maleimide; anthranilamide; 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a diaza-5-indacene-3-propioni-c acid BODIPY; cascade blue; Brilliant Yellow; coumarin and derivatives: coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcoumarin (Coumarin 151); cyanine dyes; cyanosine; 4,6-diaminidino-2-phenylindole (DAPI); 5,5-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red); 7-diethylamino-3-(4-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriaamine pentaacetate; 4,4-diisothiocyanatodihydro-stilbene-2-,2-disulfonic acid; 4,4-diisothiocyanatostilbene-2,2-disulfonic acid; 5-(dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride); 4-dimethylaminophenylazophenyl-4-isothiocyanate (DABITC); eosin and derivatives: eosin, eosin isothiocyanate, erythrosin and derivatives: erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein and derivatives: 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)amino-fluorescein (DTAF), 2,7dimethoxy-45-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4-methylumbelli-feroneortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene, pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum dots; Reactive Red 4 (Cibacron Brilliant Red 3B-A) rhodamine and derivatives: 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); N,N,N,N-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl hodamine isothiocyanate (TRITC); riboflavin; 5-(2-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS), 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL), rosolic acid; CAL Fluor Orange 560; terbium chelate derivatives; Cy 3; Cy 5; Cy 5.5; Cy 7; IRD 700; IRD 800; La Jolla Blue; phthalo cyanine; and naphthalo cyanine, coumarins and related dyes, xanthene dyes such as rhodols, resorufins, bimanes, acridines, isoindoles, dansyl dyes, aminophthalic hydrazides such as luminol, and isoluminol derivatives, aminophthalimides, aminonaphthalimides, aminobenzofurans, aminoquinolines, dicyanohydroquinones, and fluorescent europium and terbium complexes; and the like. Fluorophores of interest are farther described in WO 01/42505 and WO 01/86001.
(76) Suitable fluorescent proteins and chromogenic proteins include, but are not limited to, a green fluorescent protein (GFP), including, but not limited to, a GFP derived from Aequoria victoria or a derivative thereof, e.g., a humanized derivative such as Enhanced GFP, which is available commercially, e.g., from Clontech, Inc.; a GFP from another species such as Renilla reniformis, Renilla mulleri, or Ptilosarcus guernyi, as described in, e.g., WO 99/49019 and Peelle et al. (2001) J. Protein Chem. 20:507-519; humanized recombinant GFP (hrGFP) (Stratagene); any of a variety of fluorescent and colored proteins from Anthozoan species, as described in, e.g., Matz et al. (1999) Nature Biotechnol. 17:969-973; and the like.
(77) Suitable epitope tags include, but are not limited to, hemagglutinin; FLAG; FLAG-C; a metal ion affinity tag such as a polyhistidine tag (e.g., His.sub.6), and the like.
(78) Suitable imaging agents include positive contrast agents and negative contrast agents. Suitable positive contrast agents include, but are not limited to, gadolinium tetraazacyclododecanetetraacetic acid (Gd-DOTA); Gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA); Gadolinium-1,4,7-tris(carbonylmethyl)-10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane (GdHP-DO3A); Manganese(II)-dipyridoxal diphosphate (Mn-DPDP); Gd-diethylenetriaminepentaacetate-bis(methylamide) (Gd-DTPA-BMA); and the like. Suitable negative contrast agents include, but are not limited to, a superparamagnetic iron oxide (SPIO) imaging agent; and a perfluorocarbon, where suitable perfluorocarbons include, but are not limited to, fluoroheptanes, fluorocycloheptanes, fluoromethylcycloheptanes, fluorohexanes, fluorocyclohexanes, fluoropentanes, fluorocyclopentanes, fluoromethylcyclopentanes, fluorodimethylcyclopentanes, fluoromethylcyclobutanes, fluorodimethylcyclobutanes, fluorotrimethylcyclobutanes, fluorobutanes, fluorocyclobutanse, fluoropropanes, fluoroethers, fluoropolyethers, fluorotriethylamines, perfluorohexanes, perfluoropentanes, perfluorobutanes, perfluoropropanes, sulfur hexafluoride, and the like.
(79) In certain embodiments, the detectable tagging moiety comprises a label. In certain embodiments, the label is a fluorophore. In certain embodiments, the label is of the formula
(80) ##STR00028##
wherein L.sup.a is optionally substituted alkylene; optionally substituted alkenylene; optionally substituted alkynylene; optionally substituted heteroalkylene; optionally substituted heteroalkenylene; optionally substituted heteroalkynylene; optionally substituted arylene; optionally substituted heteroarylene; or optionally substituted acylene.
(81) In certain embodiments, the label is of the formula
(82) ##STR00029##
wherein
(83) L.sup.a is defined herein; and
(84) each instance of R.sup.t is hydrogen, halogen, optionally substituted C.sup.1-6 alkyl, optionally substituted C.sub.1-6 alkyl, optionally substituted aryl, optionally substituted heteroaryl, or optionally substituted heterocycle.
(85) As generally defined herein, L.sup.a is optionally substituted alkylene; optionally substituted alkenylene; optionally substituted alkynylene; optionally substituted heteroalkylene; optionally substituted heteroalkenylene; optionally substituted heteroalkynylene; optionally substituted arylene; optionally substituted heteroarylene; or optionally substituted acylene. In certain embodiments, L.sup.a is an optionally substituted alkylene. In certain embodiments, L.sup.a is an unsubstituted alkylene. In certain embodiments, L.sup.a is CH.sub.2. In certain embodiments, L.sup.a is (CH.sub.2).sub.2. In certain embodiments, L.sup.a is (CH.sub.2).sub.3. In certain embodiments, L.sup.a is (CH.sub.2).sub.4. In certain embodiments, L.sup.a is (CH.sub.2).sub.5.
(86) As generally defined herein, R.sup.t is hydrogen, halogen, optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.1-6 alkyl, optionally substituted aryl, optionally substituted heteroaryl, or optionally substituted heterocycle. In certain embodiments, R.sup.t is hydrogen. In certain embodiments, R.sup.s is halogen. In certain embodiments, R.sup.t is optionally substituted C.sub.1-6 alkyl. In certain embodiments, R.sup.t is methyl or ethyl.
(87) In certain embodiments, the provided detectable conjugates are of formula (X-a), (X-b), (XI-a) or (XI-b):
(88) ##STR00030##
(89) ##STR00031##
(90) or a salt thereof,
(91) wherein F atom at the C3-position is axial or equatorial; R.sup.K is H, optionally substituted C.sub.1-6 alkyl, or a sialidase protein; R.sup.2, R.sup.3a, R.sup.3b, and X are as defined herein; R.sup.4a is H, optionally substituted C.sub.1-6 alkyl, or an optionally substituted acyl; Y.sup.a is H or optionally substituted C.sub.1-6 alkyl; each instance of R.sup.p and R.sup.q is independently hydrogen, optionally substituted aliphatic; optionally substituted heteroaliphatic; substituted or unsubstituted aryl; optionally substituted heteroaryl; optionally substituted acyl; a resin; a protein; a reporter; a label optionally joined by a linker L.sup.a, wherein the linker L.sup.a is optionally substituted alkylene; optionally substituted alkenylene; optionally substituted alkynylene; optionally substituted heteroalkylene; optionally substituted heteroalkenylene; optionally substituted heteroalkynylene; optionally substituted arylene; optionally substituted heteroarylene; or optionally substituted acylene.
(92) As generally defined herein, R.sup.K is H, optionally substituted C.sub.1-6 alkyl, or a sialidase protein. In certain embodiments, R.sup.K is H. In certain embodiments, R.sup.K is optionally substituted C.sub.1-6 alkyl. In certain embodiments, R.sup.K is methyl or ethyl. In certain embodiments, R.sup.K is a sialidase protein.
(93) As generally defined herein, R.sup.4a is H, optionally substituted C.sub.1-6 alkyl, or an optionally substituted acyl. In certain embodiments, R.sup.4a is H. In certain embodiments, R.sup.4a is optionally substituted C.sub.1-6 alkyl. In certain embodiments, R.sup.4a is methyl, ethyl, or n-propyl. In certain embodiments, R.sup.4a is an optionally substituted acyl. In certain embodiments, R.sup.4a is an optionally substituted acetyl.
(94) In certain embodiments of formula (X-a), (X-b), (XI-a) or (XI-b), X is O and R.sup.4a is H, optionally substituted C.sub.1-6 alkyl, or an optionally substituted acyl acyl. In certain embodiments of formula (X-a), (X-b), (XI-a) or (XI-b), X is O and R.sup.4a is H. In certain embodiments of formula (X-a), (X-b), (XI-a) or (XI-b), X is O and R.sup.4a is optionally substituted C.sub.1-6 alkyl. In certain embodiments of formula (X-a), (X-b), (XI-a) or (XI-b), X is O and R.sup.4a is methyl or ethyl. In certain embodiments of formula (X-a), (X-b), (XI-a) or (XI-b), X is O and R.sup.4a is optionally substituted acyl. In certain embodiments of formula (X-a), (X-b), (XI-a) or (XI-b), X is O and R.sup.4a is acetyl.
(95) As generally defined herein, Y.sup.a is H or optionally substituted C.sub.1-6 alkyl. In certain embodiments, Y.sup.a is H. In certain embodiments, Y.sup.a is optionally substituted C.sub.1-6 alkyl. In certain embodiments, Y.sup.a is methyl, ethyl, or n-propyl.
(96) As generally defined herein, R.sup.p is independently hydrogen, optionally substituted aliphatic; optionally substituted heteroaliphatic; substituted or unsubstituted aryl; optionally substituted heteroaryl; optionally substituted acyl; a resin; a protein; a reporter; a label optionally joined by a linker L.sup.a, wherein the linker L.sup.a is optionally substituted alkylene; optionally substituted alkenylene; optionally substituted alkynylene; optionally substituted heteroalkylene; optionally substituted heteroalkenylene; optionally substituted heteroalkynylene; optionally substituted arylene; optionally substituted heteroarylene; or optionally substituted acylene. In certain embodiments, R.sup.p is hydrogen. In certain embodiments, R.sup.p is a reporter. In certain embodiments, R.sup.p is a reporter joined by linker L.sup.a, wherein L.sup.a is defined herein. In certain embodiments, R.sup.p is a label. In certain embodiments, R.sup.p is a label joined by linker L.sup.a, wherein L.sup.a is defined herein.
(97) As generally defined herein, R.sup.q is independently hydrogen, optionally substituted aliphatic; optionally substituted heteroaliphatic; substituted or unsubstituted aryl; optionally substituted heteroaryl; optionally substituted acyl; a resin; a protein; a reporter; a label optionally joined by a linker L.sup.a, wherein the linker L.sup.a is optionally substituted alkylene; optionally substituted alkenylene; optionally substituted alkynylene; optionally substituted heteroalkylene; optionally substituted heteroalkenylene; optionally substituted heteroalkynylene; optionally substituted arylene; optionally substituted heteroarylene; or optionally substituted acylene. In certain embodiments, R.sup.q is hydrogen. In certain embodiments, R.sup.q is a reporter. In certain embodiments, R.sup.q is a reporter joined by linker L.sup.a, wherein L.sup.a is defined herein. In certain embodiments, R.sup.q is a label. In certain embodiments, R.sup.q is a label joined by linker L.sup.a, wherein L.sup.a is defined herein.
(98) In certain embodiments, R.sup.p is hydrogen and R.sup.q is a reporter or a label optionally joined by a linker L.sup.a. In certain embodiments, R.sup.q is hydrogen and R.sup.p is a reporter or a label optionally joined by a linker L.sup.a.
(99) In certain embodiments, the provided detectable conjugates are of one of the formulae:
(100) ##STR00032## ##STR00033##
(101) In another aspect, the invention provides a method for detecting presence of a sialidase, the method comprising:
(102) (a) contacting a sample suspected of comprising a sialidase with a compound as described herein;
(103) (c) adding a reporter; and
(104) (c) detecting a signal,
(105) wherein presence of a signal indicates the presence of the sialidase in the sample.
(106) In another aspect, the invention provides a method for detecting presence of a sialidase, the method comprising:
(107) (a) contacting a sample suspected of comprising a sialidase with a detectable conjugate;
(108) (c) adding a reporter; and
(109) (c) detecting a signal,
(110) wherein presence of a signal indicates the presence of the sialidase in the sample.
(111) In certain embodiments, the sialidase is intracellular. In certain embodiments, the detectable conjugate comprises PDFSA-5-yne (IV) or PDFSA-7-yne (VII):
(112) ##STR00034##
a derivative, conjugate or ester thereof. In certain embodiments, the sample is from a mammal, fowl, or fish. In certain embodiments, the sample is from a human. In certain embodiments, the sample is suspected of containing a pathogen. In certain embodiments, the sample contains a bacterium, virus, protozo, or a fungus.
(113) As generally used herein, the sialidase is a human, viral or bacterial sialidase. In certain embodiments, the sialidase is a human sialidase. In certain embodiments, the sialidase is an influenza virus neuraminidase (NA). In certain embodiments, the sialidase is a human sialidase selected from the group consisting of Neu1, Neu2, Neu3 and Neu4. In certain embodiments, the sialidase is a bacterial sialidase selected from the group consisting of nanA, nanB, nanC, nanJ, nanI, and nanH.
(114) In certain embodiments, the method further comprises imaging the intracellular locations of sialidases in a cell.
(115) As generally used herein, the reporter refers to a chemical entity capable of forming a detectable tagging moiety with a target molecule, adduct, or conjugate. In some embodiments, the target molecule can be any compound as described herein or any a sialidase-compound conjugate. In certain embodiments, the reporter reacts with the compound or sialidase-compound conjugate via click reaction to introduce a detectable tagging moiety to the compound or sialidase-compound conjugate.
(116) Click chemistry is a chemical approach introduced by Sharpless in 2001 and describes chemistry tailored to generate substances quickly and reliably by joining small units together. See, e.g., Kolb, Finn and Sharpless Angewandte Chemie International Edition (2001) 40: 2004-2021; Evans, Australian Journal of Chemistry (2007) 60: 384-395). Exemplary coupling reactions (some of which may be classified as Click chemistry) include, but are not limited to, formation of esters, thioesters, amides (e.g., such as peptide coupling) from activated acids or acyl halides; nucleophilic displacement reactions (e.g., such as nucleophilic displacement of a halide or ring opening of strained ring systems); azide-alkyne Huisgon cycloaddition; thiol-yne addition; imine formation; and Michael additions (e.g., maleimide addition). In certain embodiments, the click reaction is carried out in the presence of copper (Kolb, et al., Angew Chem. Int. Ed., 2001, 40, 2004; Rostovtsev et al., Angew Chem. Int. Ed., 2002, 41, 2596; Wu et al, Aldrichimica Acta, 2007, 40, 7).
(117) In certain embodiments, the reporter comprises an azido group. In certain embodiments, the reporter comprises a terminal alkyne. In certain embodiments, the reporter is an azido-biotin.
(118) In certain embodiments, the method further comprises:
(119) (d) contacting a cell with a compound of PDFSA-5-yne (IV), PDFSA-7-yne (VII), or a derivative, or ester thereof;
(120) (e) allowing intracellular esterase to convert the compound to DFSA-5-yne (III) or DFSA-7-yne (VI) respectively;
(121) (f) allowing DFSA-5-yne (III) or DFSA-7-yne (VI) to covalently conjugate to one or more sialidases at an intracellular location in the cell;
(122) (g) adding a reporter;
(123) (h) obtaining an image of intracellular sialidase distribution.
(124) In another aspect, the invention provides a method for diagnosis of sialidosis, the method comprising:
(125) (a) contacting a test sample from a subject suspected of sialidosis with a compound as described herein;
(126) (b) adding a reporter;
(127) (c) detecting a signal, and
(128) (d) comparing the signal with that from a healthy subject,
(129) wherein a relative reduction of signal in the test sample indicates sialidosis.
(130) In another aspect, the invention provides a method for diagnosis of sialidosis, the method comprising:
(131) (a) contacting a test sample from a subject suspected of sialidosis with a detectable conjugate as described herein;
(132) (b) detecting a signal, and
(133) (d) comparing the signal with that from a healthy subject,
(134) wherein a relative reduction of signal in the test sample indicates sialidosis.
(135) In some embodiments of the diagnosis method, the test sample contains fibroblast. In some embodiments of the diagnosis method, the detectable conjugate comprises PDFSA-5-yne (IV), PDFSA-7-yne (VII), or a derivative, conjugate or ester thereof.
(136) In another aspect, the invention provides a method for diagnosing infection by influenza virus, the method comprising:
(137) (a) contacting a test sample from a subject suspected of influenza virus infection with the compound of any one of claims 1-23;
(138) (b) adding a reporter;
(139) (c) detecting a signal;
(140) wherein the presence of a signal indicates a possibility of infection by influenza virus.
(141) In another aspect, the invention provides a method for diagnosing infection by influenza virus, the method comprising:
(142) (a) contacting a test sample from a subject suspected of influenza virus infection with the detectable conjugate of claim 32;
(143) (b) detecting a signal;
(144) wherein the presence of a signal indicates a possibility of infection by influenza virus.
(145) In certain embodiments of the diagnostic methods, the influenza virus is extracellular. In certain embodiments of the diagnostic methods, the influenza virus is intracellular. In certain embodiments of the diagnostic methods, the detectable conjugate comprises DFSA-5-yne (II) or DFSA-7-yne (VI) or a derivative, conjugate or ester thereof. In certain embodiments of the diagnostic method, the the influenza virus is not resistant to oseltamivir (OS). In certain embodiments of the diagnostic method, the the influenza virus is sensitive to oseltamivir (OS). In certain embodiments of the diagnostic methods, the the influenza virus is resistant to oseltamivir (OS). In certain embodiments, the oseltamivir-resistant influenza virus is H1N1, H1N9, H3N1, H3N2, H5N1, H7N9. In certain embodiments, the oseltamivir-resistant influenza virus is H1N1. In certain embodiments of the diagnostic method, the influenza virus is present at a titer of 10.sup.4 or higher.
(146) In another aspect, the invention provides a method for imaging sialidase in a live cell, comprising:
(147) incubating a live cell containing sialidase with any compound as described herein under conditions allowing conjugation of the compound to the sialidase,
(148) contacting the sialidase-compound conjugate with a reporter under conditions allowing conjugation of the reporter to the compound, and
(149) detecting a signal released from the reporter that is conjugated to the compound.
(150) In certain embodiments of any of the provided methods, the detectable signal is generated by the label of the detectable conjugate. In certain embodiments of the provided methods, the detectable signal is released from the reporter conjugated with the compound. In certain embodiments, the detectable signal is released from the reporter conjugated with the compound-sialidase adduct. In certain embodiments, the reporter comprises a label. In certain embodiments, the reporter comprises an azido moity and a label. In certain embodiments, the reporter comprises an alkyne moiety and a label. In certain embodiments, the reporter comprises a biotin moiety and an alkyne moiety. In certain embodiments, the reporter comprises a biotin moiety and an azido moiety. In certain embodiments, the reporter is of the formula
(151) ##STR00035##
(152) In another aspect, the exemplary syntheses of the provided compounds such as DFSA-5-yne and PDFSA-5-yne comprise the steps of:
(153) (a) reaction of N-(pent-4-ynoyl)-mannosamine (1) (T. L. Hsu, et al. Proc. Natl. Acad. Sci. USA 2007, 104, 2614.) with 3-fluoropyruvic acid (as the sodium salt) by catalysis of N-acetylneuraminic acid aldolase (Neu5Ac aldolase, EC 4.1.3.3) to yield an adduct 2;
(154) ##STR00036##
(155) (b) esterification of adduct 2 in methanol in the presence of IR-120 resin (acid form) to give compound 3;
(156) ##STR00037##
(157) (c) acetylation of compound 3, followed by chromatographic isolation, to give peracetylated ester 4;
(158) ##STR00038##
(159) (d) selective deacetylation at the anomeric position of compound 4 by using hydrazine acetate to give compound 5;
(160) ##STR00039##
(161) (e) treatment of compound 5 with diethylaminosulfurtrifluoride (DAST) to give PDFSA-5-yne (-anomer, 60%) and its -anomer (30%);
(162) and
(163) (f) deprotection of PDFSA-5-yne under alkaline conditions, followed by purification on a reversed-phase column, to give DFSA-5-yne.
(164) In another aspect, the exemplary synthesis of the provided compounds such as DFSA-7-yne and PDFSA-7-yne further comprise steps:
(165) (g) reaction of D-mannosamine with 3-fluoropyruvic acid (as the sodium salt) by catalysis of N-acetylneuraminic acid aldolase (Neu5Ac aldolase, EC 4.1.3.3) to yield an adduct 6;
(166) ##STR00040##
(167) (h) esterification of adduct 6 in methanol in the presence of IR-120 resin (acid form) to give compound 7;
(168) ##STR00041##
(169) (i) acetylation of compound 7, followed by chromatographic isolation, to give peracetylated ester 8;
(170) ##STR00042##
(171) (j) selective deacetylation at the anomeric position of compound 8 by using hydrazine acetate to give compound 9;
(172) ##STR00043##
(173) (k) treatment of compound 9 with diethylaminosulfurtrifluoride (DAST) to give compound 10;
(174) ##STR00044##
(175) (l) treatment of compound 10 with methanesulfonic acid in methanol to give compound 11;
(176) ##STR00045##
(177) (m) treatment of compound 11 with 4-nitrophenylchloroformate in the presence of sodium bicarbonate to give compound 12;
(178) ##STR00046##
(179) (n) treatment of compound 12 with 2,2-dimethoxypropane by acid catalysis, followed by treatment with 4-nitrophenylchloroformate in pyridine to give compound 13;
(180) ##STR00047##
(181) (o) treatment of compound 13 with propargyl amine in pyridine to give compound 14;
(182) ##STR00048##
(183) (p) treatment of compound 14 with trifluoroacetic acid to give compound 15;
(184) ##STR00049##
(185) (q) treatment of compound 15 with acetic anhydride in pyridine to give compound 16;
(186) ##STR00050##
(187) (r) treatment of compound 16 with acetyl chloride in diisopropylethylamine to give PDFSA-7-yne; and
(188) (s) saponification of PDFSA-7-yne to give DFSA-7-yne.
(189) To examine the feasibility of DFSA-5-yne as an activity-based probe, the inhibition of various sialidases by DFSA-5-yne were evaluated using 2-(4-methylumbelliferyl)--D-N-acetylneuraminic acid (MUNANA) as the substrate. The sialidases used in this study are derived from a variety of species, including influenza virus (NA), bacteria (nanA, nanB, nanC, nanJ, nanI and nanH) and human (Neu1, Neu2, Neu3 and Neu4) sialidases.
(190) All tested sialidases were sensitive to DFSA-5-yne with micro- to submicromolar half maximal inhibitory concentrations (Table 1), indicating that DFSA could be a potent activity-based probe for these enzymes. In contrast to the sensitive inhibition by DFSA, the ester-protected analog PDFSA-5-yne did not inhibit these sialidases (data not shown), suggesting that esterification of the hydroxy and carboxy groups in PDFSA prevents bindings to the sialidase active sites.
(191) TABLE-US-00001 TABLE 1 IC.sub.50 values (M) of sialidase inhibition by DFSA-5-yne, DANA, Zanamivir, and Oseltamivir..sup.a Sialidase.sup.b DFSA-5-yne DANA.sup.c Zanamivir Oseltamivir NA 51 24 5.4 1.6 0.005 0.003 Neu1 10.4 2.3 >100 >100 >100 Neu2 26.1 12.8 24 2.8 17 >100 Neu3 5.3 3.2 2.4 0.6 3 >100 Neu4 59.8 1.3 4.3 1.4 30.8 >100 nanA 0.3 0.1 9.3 >100 3.8 2.6 nanB 82 22 25.4 >100 14.2 6.2 nanC 42.8 17.5 >100 >100 >100 nanJ 34.9 4.2 4.5 >100 >100 nanI 25.5 0.6 1.9 77 >100 nanH 354 146 15.7 83 >100 .sup.aA fluorescent substrate, 2-(4-methylumbelliferyl)--D-N-acetylneuraminic acid (MUNANA), was used to determine the IC.sub.50 values that are concentrations causing 50% inhibition of different sialidases. .sup.bThe sialidases used in this study are NA (influenza neuraminidase from A/WSN/1933/H1N1), Neu1-Neu4 (recombinant sialidases from human), nanA-nanC (recombinant sialidases of S. pneumoniae), and nanJ-nanH (recombinant sialidases from C. perfringens). .sup.cDANA represents 2,3-didehydro-2-deoxy-N-acetylneuraminic acid.
(192) To validate the sialidase labeling by DFSA-5-yne, formation of the fluorosialosyl-enzyme adducts by SDS-PAGE analyses were examined. All the bacterial sialidases formed DFSA adducts that were captured by azido-biotin and detected by the streptavidin specific reporting signals (
(193) The influenza neuraminidase located at the surface of influenza virus (A/WSN/1933/H1N1) also formed a DFSA adduct that could be out competed by the neuraminidase inhibitor oseltamivir acid (OS), suggesting that DFSA interacted with neuraminidase at the active site (
(194) Similar to the labeling of influenza neuraminidase by DFSA-5-yne, specific DFSA labeling was identified by four human sialidases in the crude extracts of transfected 293T cells (
(195) The disclosure reveals that the DFSA labeling of human sialidases were sensitive to pH conditions. Neu1 and Neu3 were poorly labeled at pH 9. For Neu3, weak labeling was observed even at pH 7 (
(196) Using the bacterial nanH as an example, the DFSA labeled products were roughly proportional to the amounts of sialidase (
(197) The disclosure provides LC-MS/MS analyses on the tryptic peptide fragments of the DFSA-labeled sialidases to identify the interacting amino acids. All six bacterial sialidase peptides were found to be labeled by the 3-fluorosialyl moiety at the tyrosine residues (Table 2).
(198) TABLE-US-00002 TABLE2 DFSA-labeledtrypticpeptidesfromdifferent sialidases. Modi- Siali- fied dase Trypticpeptide sites nanA .sup.723FAYNSIQEIGNGEYGIIYEHTEKGQNAYTI Y725 SFR.sup.755 (SEQIDNO:1) nanB .sup.628YHYDIDIPSYGYAYSAITEIPNHHIGVIFE Y639and K.sup.658 Y641.sup.a (SEQIDNO:2) nanC .sup.708YHHDVDYSNYGYSYSTITEIPNHEIGIMFE Y721 K.sup.738 (SEQIDNO:3) nanI .sup.649IVKPGYYAYSCITE.sup.762 Y657 (SEQIDNO:4) nanJ .sup.779TVKPGSFAYSCITEIPDGNIGIFYEGEGAG Y787 R.sup.809 (SEQIDNO:5) nanH .sup.369IGGGYSCISFK.sup.379 Y373 (SEQIDNO:6) .sup.aThe LC-MS/MS analysis showed that DFSA was covalently linked to Tyr639 and Tyr641 in a ratio of 85:15.
(199) For the profiling of intracellular sialidases, the probe needs to be cell permeable. However, being a hydrophilic compound, DFSA-5-yne is poorly permeable to cells. To enhance the cellular uptake, the ester-protected probe, PDFSA-5-yne, was used to test the labeling of intracellular sialidases expressed in 293T cells. In comparison with the sialidase labeling of cell extracts with DFSA-5-yne (
(200) The success in sialidase labeling using PDFSA-5-yne prompted us to determine the cellular localizations of the expressed sialidase activities by incubating live cells with PDFSA and examining the cellular location of the sialidase adducts in fixed and permeated cells (
(201)
(202) Analyses of sialidase activity and Neu1 in expressing cells showed high co-localization at 85%. Similar high co-localization was found in Neu2 and Neu3 expressing cells, suggesting that the observed sialidase activity profiling is correlated with the enzyme distribution.
(203) The activity and co-localization for the Neu4 expressing cells is lower at only 68%. The possible explanation is that Neu4 is expressed in long and short isoforms, differing in the presence and absence of a 12-amino-acid sequence at the N-terminus. (V. Seyrantepe, et al. J. Biol. Chem. 2004, 279, 3702. K. Yamaguchi, et al. Biochem. J. 2005, 390, 85. A. Bigi, et al. Glycobiology 2010, 20, 148.) The Neu4 long is mainly located in mitochondria and has the N-terminal flag-tag, while the Neu4 short targets membrane and does not have the flag-tag. The fact that these two expressed Neu4 sialidase forms are different in cellular localizations and flag-antigen expressions could explain the apparent low co-localization observations.
(204) The results of profiling sialidase activity in the four sialidases thus suggest that PDFSA is a useful probe for living cells.
(205) Sialidosis is an inherited lysosomal storage disease usually due to Neu1 deficiency. Neu1 activity differences in fibroblasts of normal and sialydosis patients were examined by live cell labeling using PDFSA-5-yne. The sialidase labeling was significantly reduced in the more sever sialidosis (GM02921) fibroblast cells than the milder sialydosis (GM02922) cells (
(206) The results of the sialidase activity difference observed by PDFSA labeling are consistent with the conventional activity measurement of the cell extracts using MUNANA as the substrate (
(207) DFSA was also successfully used to image the influenza virus infected cells that express neuraminidase on the cell surface accessible to DFSA labeling (
(208) To determine the sensitivity of influenza detection using DFSA, DFSA-5-yne with varied quantities of influenza viruses were incubated and then immobilized the viral samples on membrane followed by similar detection procedures used in SDS-PAGE. This procedure allowed the detection of influenza virus present at 10.sup.4 or higher titers (
(209) DFSA-5-yne is shown to bind at the active site of influenza neuraminidase, and the binding can be competitively inhibited by OS (
(210) The disclosure provides an activity-based sialidase probe DFSA-5-yne by using the 3-fluorosialyl fluoride as the mechanism-based inhibitor and by incorporating an alkyne group for reporter ligation. Biochemical analyses of the DFSA inactivated sialidases by LC-MS/MS analysis showed that the tyrosine residues in the enzyme active site were specifically labeled by DFSA. The ability of DFSA-5-yne to label all sialidases from viral, bacterial, and human enzymes suggests that DFSA-5-yne may be used as a general sialidase probe for various applications.
(211) DFSA-5-yne is advantageous as a general ABPP because of its small size. Introduction of the ester-protected PDFSA-5-yne enhances cell permeable properties and allows the profiling of intracellular sialidases. The ability of PDFSA-5-yne to probe intracellular sialidases using living cells has an added advantage over the labeling using cell lysates, particularly for unstable sialidases. Since sialidases are known to be involved in various diseases, these probes can be useful in developing sialidase-based diagnoses.
EXAMPLES
(212) Without intent to limit the scope of the invention, exemplary instruments, apparatus, methods and their related results according to the embodiments of the present invention are given below. Note that titles or subtitles may be used in the examples for convenience of a reader, which in no way should limit the scope of the invention. Moreover, certain theories are proposed and disclosed herein; however, in no way they, whether they are right or wrong, should limit the scope of the invention so long as the invention is practiced according to the invention without regard for any particular theory or scheme of action.
Example 1: Methods and Materials
(213) Unless otherwise noted, all compounds and reagents were purchased from Acros or Sigma-Aldrich. All chemicals were purchased as reagent grade and used without further purification. N-Acetylneuraminic acid aldolase was purchased from ToYoBo STC. Reactions were monitored with analytical thin-layer chromatography (TLC) in EM silica gel 60 F254 plates and visualized under UV (254 nm) and/or staining with acidic ceric ammonium molybdate or ninhydrin. Flash column chromatography was performed on silica gel 60 Geduran (35-75 m, EM Science). .sup.1H NMR spectra were recorded on a Bruker DRX-400 (400 MHz) spectrometer at 20 C. Chemical shifts were assigned according to the CHCl.sub.3 (=7.24 ppm). .sup.13C NMR spectra were obtained using Attached Proton Test (APT) on a Bruker DRX-400 (100 MHz) spectrometer and were reported using the signal of CDCl.sub.3 (=77.0 ppm of central line) for calibration. Mass spectra were obtained by the analytical services of The Scripps Research Institute (Agilent ESI-TOF) and The Genomics Research Center (Acadmia Sinica) (LTQ Orbitrap XL ETD). Fluorescence spectra were obtained on a Molecular Devices Spectramax M5 spectrometer. Protease inhibitors were purchased from Roche Applied Sciences, PVDF membranes were from Millipore. NuPAGE Bis-Tris Mini gels (4-12%), PBS and cell culture media and reagents were from Invitrogen. Protein concentration was measured by either BCA protein assay (Thermo Scientific) or Bradford assay (Bio-rad). GM02921 and GM02922 were obtained from the NIGMS Human Genetic Mutant Cell Repository. Chemiluminescence on protein blots was visualized and quantified using FUJI LAS3000 imaging system (Fujifilm). Confocal microscopy of sialidase-expressing 293T cells were obtained using Leica TCS-SP5-MP-SMD.
Example 2: Methyl 5-(pent-4-ynamido)-2,4,7,8,9-penta-O-acetyl-3,5-dideoxy-3-fluoro-D-erythro--L-manno-non-2-ulopyranosonate (4)
(214) A mixture of N-4-pentynoylmannosamine (460.0 mg, 1.78 mmol) (Z. Zhou and C. J. Fahrni, J. Am. Chem. Soc. 2004, 126, 8862.), 3-fluoropyruvic acid (as the sodium salt, 458.2 mg, 3.56 mmol), NaN.sub.3 (1%, 500 L), and N-acetylneuraminic acid aldolase (200 U), in potassium phosphate buffer (pH 7.4, 0.05 mmol/L, 25.0 mL), was incubated at room temperature for 3 days. The mixture was concentrated. The residue was applied to a Dowex column (12, 200 mesh), and eluted with water and formic acid (0.1-1.0 mol/L) sequentially. Fractions containing the desired product 2 were pooled, and concentrated under reduced pressure. The diastereomeric ratio (axial/equatorial=7:1) 2 was determined by NMR analyses.
(215) To the crude product 2 were added MeOH (30 mL) and ion exchange resin Amberlite IR 120-H (500 mg). The mixture was stirred at room temperature for 24 h, and filtered through a pad of Celite, giving ester 3. MeOH was removed, and the residue was treated with pyridine (25 mL), DMAP (10.0 mg) and Ac.sub.2O (10 mL). The mixture was stirred at room temperature for 12 h. After that, pyridine was removed under vacuum first and the residue was taken up in EtOAc (100 mL) and washed with 5% citric acid (3), 10% NaHCO.sub.3 (3) and brine. The combined organic layers were dried over anhydrous MgSO.sub.4, filtered and concentrated. The single diastereomer 4 (366.6 mg, 35% overall yield) was obtained as white foam after silica gel column chromatography eluted with EtOAc/hexane (4:1). TLC (EtOAc/hexane=3:1) R.sub.f=0.31. .sup.1H-NMR (CDCl.sub.3, 400 MHz) 5.66 (d, J=9.0 Hz, 1H), 5.55 (d, J=2.4, 10.9, 27.7 Hz, 1H), 5.34 (dd, J=1.9, 5.3 Hz, 1H), 5.11 (m, 1H), 4.92 (dd, J=2.4, 49.1 Hz, 1H), 4.51 (dd, J=2.4, 12.5 Hz, 1H), 4.25 (dd, J=1.3, 10.6 Hz, 1H), 4.17 (dd, J=6.4, 12.5 Hz, 1H), 4.13 (m, 1H), 2.53-2.39 (m, 2H), 2.37-2.26 (m, 2H), 2.16 (s, 3H), 2.13 (s, 3H), 2.07 (s, 3H), 2.01 (s, 3H), 1.99 (s, 3H), 1.97 (t, J=2.5 Hz, 1H). .sup.13C-NMR (CDCl.sub.3, 100 MHz) 171.2, 170.6, 170.5, 170.3, 170.2, 167.1, 165.1, 95.1 (d, J=29.0 Hz), 86.9 (d, J=184.0 Hz), 82.7, 71.6, 71.1, 69.6, 68.2, 68.1, 68.0, 62.1, 53.5, 45.7, 35.4, 0.8 (2), 20.7, 20.5, 14.6. .sup.19F-NMR: (CDCl.sub.3, 282.4 MHz) 209.1 (dd, J=28.0, 52.0 Hz) HR-ESI MS calcd for C.sub.25H.sub.33NO.sub.14 [M+H].sup.+: 548.1774; found: 548.1770.
Example 3: Methyl 5-(pent-4-ynamido)-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-3-fluoro-D-erythro--L-manno-2-non-2-ulopyranosonate (5)
(216) To a solution of compound 4 (165.0 mg, 0.28 mmol) in 10 mL of CH.sub.2Cl.sub.2 was added hydrazine acetate (116.0 mg, 1.26 mmol) in 2.0 mL of MeOH. The mixture was stirred at 0 C. for 8 h, and then concentrated under reduced pressure. The product 5 (110.0 mg, 72%) was obtained as an oil after silica gel column chromatography eluted with EtOAc/hexane (4:1). TLC (EtOAc/hexane=3:1) R.sub.f=0.31. .sup.1H-NMR (400 MHz, CDCl.sub.3+CD.sub.3OD) 5.46 (dd, J=2.4, 4.4 Hz, 1H), 5.39-5.26 (m, 2H), 4.97 (dd, J=2.4, 50 Hz, 1H), 4.76 (m, 1H), 4.46-4.34 (m, 2H), 4.20 (dd, J=7.4, 12.4 Hz, 1H), 3.85 (s, 3H), 2.54-2.40 (m, 2H), 2.36-2.30 (m, 2H), 2.17 (s, 3H), 2.11 (s, 3H), 2.09 (s, 3H), 2.07-2.05 (m, 4H). .sup.13C-NMR (100 Hz, CDCl.sub.3+CD.sub.3OD) 173.7, 172.4, 171.9, 171.7, 171.6, 168.7, 95.5 (d, J=20.0 Hz), 88.5 (d, J=146.0 Hz), 83.5, 72.6, 71.2, 71.1, 71.0, 70.0, 69.3, 63.6, 53.4, 45.4, 36.1, 21.1 (2), 21.0, 15.4. .sup.19F-NMR (CDCl.sub.3, 282.4 MHz) 205.3 (dd, J=28.0, 52.0 Hz). HR-ESI MS calcd for C.sub.23H.sub.31FNO.sub.13 [M+H].sup.+: 522.1618; found: 522.1211.
Example 4: Methyl 5-(pent-4-ynamido)-4,7,8,9-tetra-O-acetyl-2,3,5-trideoxy-3-fluoro-D-erythro--L-manno-non-2-ulopyranosylonate fluoride (PDFSA-5-yne)
(217) To a solution of compound 5 (75.0 mg, 0.14 mmol) in 5 mL of CH.sub.2Cl.sub.2 was added 19 L (0.19 mmol) of DAST at 30 C., and stirred for 5 h. The reaction was quenched by adding small amount of silica gel and 1.5 mL of MeOH. The mixture was concentrated under reduced pressure. PDFSA-5-yne (-anomer, 46.0 mg, 60%) and the -anomer (23.0 mg, 30%) were isolated by silica gel column chromatography eluted with EtOAc/hexane (5:1).
(218) PDFSA-5-yne (-anomer): TLC (EtOAc/hexane=3:1) R.sub.f=0.33. .sup.1H-NMR (400 MHz, CDCl.sub.3) 5.79 (d, J=8.9 Hz, 1H), 5.46 (dd, J=10.7, 25.6 Hz, 1H), 5.36-5.28 (m, 1H), 5.10 (ddd, J=2.6, 2.7, 50.7, 1H), 4.34 (d, J=10.9 Hz, 1H), 4.29 (dd, J=1.7, 12.4 Hz, 1H), 4.16 (dd, J=4.2, 12.4 Hz, 1H), 4.08 (m, 1H), 3.87 (s, 3H), 2.52-2.37 (m, 2H), 2.36-2.23 (m, 2H), 2.12 (s, 3H), 2.08 (s, 3H), 2.06 (s, 3H), 1.99 (s, 3H), 1.97 (t, J=2.5 Hz, 1H). .sup.13C-NMR (100 Hz, CDCl.sub.3) 171.3, 170.5 (2), 170.4, 170.2, 164.3 (d, J=20.0 Hz), 104.5 (dd, J=13.0, 179.0 Hz), 85.4 (dd, J=16.0, 154.0 Hz), 82.7, 72.5, 69.6, 69.0, 68.3 (d, J=5.0 Hz), 67.0, 61.8, 53.7, 45.5 (d, J=3.0 Hz), 35.4, 20.7, 20.6 (2), 20.5, 14.6. .sup.19F-NMR: (CDCl.sub.3, 282.4 MHz) 123.3 (d, J=12.0 Hz), 217.1 (ddd, J=12.0, 24.0, 52.0 Hz). ESI-HRMS calcd for C.sub.23H.sub.30F.sub.2NO.sub.12 [M+H].sup.+: 550.1730; found: 550.1736.
(219) PDFSA-5-yne (-Anomer): TLC (EtOAc/hexane=3:1) R.sub.f=0.37. .sup.1H-NMR (400 MHz, CDCl.sub.3) 5.68 (d, J=9.7 Hz, 1H), 5.42 (dd, J=2.1, 6.3 Hz, 1H), 5.38 (m, 1H), 5.25 (m, 1H), 5.10 (dd, J=2.3, 48.6 Hz, 1H), 4.48 (dd, J=2.6, 12.6 Hz, 1H), 4.43 (dd, J=10.5, 20.8 Hz, 1H), 4.32 (d, J=10.7 Hz, 1H), 4.11 (dd, J=10.5, 20.8 Hz, 1H), 3.86 (s, 3H), 2.38-2.53 (m, 2H), 2.23-2.37 (m, 2H), 2.13 (s, 3H), 2.09 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H), 1.99 (t, J=2.6 Hz, 1H). .sup.13C-NMR (100 Hz, CDCl.sub.3) 171.1, 170.6, 170.5, 170.2, 169.9, 162.6 (d, J=21.0 Hz), 105.0 (dd, J=23.0, 183.0 Hz), 84.6 (dd, J=35.0, 147.0 Hz), 82.84, 72.7 (d, J=2.0 HZ), 72.3 (d, J=2.0 Hz), 69.6, 68.4 (d, J=14.0 Hz), 67.1, 62.1, 53.7, 44.5, 35.4, 20.7 (3), 20.6, 14.6. .sup.19F-NMR: (CDCl.sub.3, 282.4 MHz) 122.4 (d, J=20.0 Hz), 207.2 (d, J=16.0 Hz).
Example 5: 5-(Pent-4-ynamido)-2,3,5-trideoxy-3-fluoro-D-erythro--L-manno-non-2-ulopyranosylonic fluoride (DFSA-5-yne)
(220) To a solution of PDFSA-5-yne (42.0 mg, 0.076 mmol) in 5 mL of CH.sub.3OH was added Na.sub.2CO.sub.3 (32.4 mg, 0.31 mmol) at room temperature for 1 h. H.sub.2O (1 mL) was added, and the mixture was left at room for 2 h. The mixture was neutralized by ion exchange resin Amberlite IR 120-H, and filtered through a pad of Celite. The filtrate was concentrated under reduced pressure, and the crude product was chromatographed on a silica gel 100 reversed-phase C18 column (H.sub.2O to 10% aqueous MeOH) to yield product DFSA (23.7 mg, 85%) as a white foam. .sup.1H-NMR (400 MHz, D.sub.2O) 5.24 (ddd, J=2.5, 2.5, 51.3 Hz, 1H), 4.12-4.37 (m, 2H), 3.82-3.93 (m, 3H), 3.61-3.72 (m, 2H), 2.51-2.60 (m, 4H), 2.42 (s, 1H). .sup.13C-NMR (150 Hz, D.sub.2O) 175.6, 168.2 (d, J=40.6 Hz), 106.1 (dd, J=15.5, 218.1 Hz), 88.4 (dd, J=18.0, 183.5 Hz), 83.3, 72.7 (d, J=3.3 Hz), 70.3 (d, J=5.6 Hz), 68.6 (dd, J=5.6, 17.8 Hz), 67.8, 63.0, 48.8, 46.8 (d, J=3.3 Hz), 34.6 (d, J=7.1 Hz), 14.5. .sup.19F-NMR (CDCl.sub.3, 282.4 MHz) 121.3 (d, J=12.0 Hz), 218.0 (ddd, J=12, 28, 52 Hz). HR-ESI MS calcd for C.sub.14H.sub.20F.sub.2NO.sub.8 [M+H].sup.+: 368.1151; found: 368.1152.
Example 6: Cloning of Bacterial Sialidases
(221) The cDNA of sialidases nanA (SP1693), nanB (SP1687) and nanC (SP1326) were amplified by PCR from Streptococcus pneumonia TIGR4 genomic DNA (ATCC ATCC BAA-334). Similarly, the cDNA of sialidases nanH (CPF 0985), nanI (CPF 0721) and nanJ (CPF 0532) were from Clostridium perfringens NCTC 8237 genomic DNA (ATCC 13124D-5) by specific primers (Table S3). The obtained cDNAs were then cloned into modified form of pET47b+ (Novagen, Madison, Wis.) for expressions in E. coli. The hydrophobic regions at the N-terminus of those sialidases predicted to be a signal peptide by SignalIP were not included in the primers during cloning. All these bacterial sialidases were expressed with N-terminal His tag for protein purification and antibody identification.
Example 7: Expression of Sialidase in E. coli and Purification of the Recombinant Sialidases
(222) All sialidase genes were obtained via PCR from genomic DNA or cDNA library by respective primer (Table S3). The PCR products were ligated into the modified form of pET47b vector and confirmed by DNA sequencing. The plasmids with correct sequences were transformed into ArcticExpress/RIL competent cells by chemical transformation method. Single colonies were picked and cultured in TB medium with kanamycin overnight. The cell cultures into fresh TB medium, were induced by 0.1 mM IPTG and to grow at 16 C. for 24 h. E. coli cells were harvested and disrupted in a buffer containing 50 mM sodium phosphate buffer, pH 8.0, 300 mM sodium chloride, and 10 mM imidazole by microfluidizer and clarified by Centrifugation. The expressed sialidases were purified by Ni-NTA agarose. The protein concentration was determined by Qubit Protein Quantitation (Invitrogen, CA), and purity was confirmed by SDS-PAGE.
Example 8: Cloning of Human Sialidase
(223) The cDNA of human sialidases, Neu1, Neu2 and Neu4 were amplified from MGC clone (Clone ID: 40004620 and 40125765, respectively) by PCR and sub-cloned into modified form of expression vector, pCMV-Tag 2 (Sigma, St. Louis, Mo.) with N-terminal FLAG tag, whereas Neu1 with both FLAG tags in N- and C-terminals (Clone ID: 3506824) by primer addition. Neu3 cDNA was synthesized according to its sequence (Genbank: BC144059.1) and cloned as other three sialidases. All clones are confirmed by DNA sequencing, and sialidase expressions confirmed by FLAG-specific antibody.
Example 9: Determination of IC50 of DFSA and PDFSA
(224) Sialidase inhibition was determined by mixing inhibitor and neuraminidase for 10 min at room temperature, followed by the addition of 200 M of substrate MUNANA. Inhibitor IC.sub.50 value was determined from the dose-response curves by plotting the percent inhibition of NA activity versus inhibitor concentrations using Graph Pad Prism 4.
Example 10: Membrane Click Reaction
(225) The PVDF membranes were blocked with blocking buffer 5% BSA/PBST (0.1% Tween 20/PBS) and streptavidin blocking buffer 0.02% streptavidin/3% BSA/PBST (0.1% Tween 20/PBS) for 1 h, respectively. The membranes were washed twice with PBS for 5 min. The protein side of the PVDF membrane was faced down to immerse in the click reaction mixture (0.1 mM azido-biotin, 0.1 mM tris-triazole ligand, 1 mM CuSO.sub.4, 2 mM sodium ascorbate; with 1 mL for a blot of a minigel size) and incubated at room temperature for 1 h. After washing with PBST twice, the membrane was probed with peroxidase-conjugated streptavidin for biotin labels on blots. The signals were detected by ECL system.
Example 11: Labeling of Bacteria Sialidase, Influenza Neuraminidase, and Recombinant Human Sialidases
(226) Purified bacteria sialidases (1 g) were incubated with DFSA (0.1 mM) at room temperature for 1 h, and separated on 4-12% NuPAGE (Invitrogen). 5105 influenza viruses were incubated with DFSA (0.1 mM) at room temperature for 1 h, and separated on 4-12% NuPAGE (Invitrogen). Sialidase transfectant 293T cells were lysed by different lysis buffers: pH4.5 (1% NP-40, 100 mM NaOAc, 150 mM NaCl, 3 mM KCl, pH 4.5, 1EDTA-free protease inhibitor cocktail from Roche), pH 7.4 buffer (1% NP-40, 25 mM Tris, 150 mM NaCl, 3 mM KCl, pH 7.4, 1EDTA-free protease inhibitor cocktail from Roche), and pH 9.0 buffer (1% NP-40, 25 mM Tris, 150 mM NaCl, 3 mM KCl, pH 9.0, 1EDTA-free protease inhibitor cocktail from Roche). The lysates were collected and incubated with DFSA (0.1 mM) at 37 C. for 1 h. Following incubation, the samples were clarified, and protein concentrations were determined by bicinchoninic acid protein assay kit (Pierce). For each sample, 20 g total lysate was separated on 4-12% NuPAGE (Invitrogen). After electrophoresis, the gels were blotted onto PVDF membranes (Millipore). Click reactions were performed on the PVDF membranes, and labeling signals were processed and analyzed by chemiluminescence detector.
Example 12: In Situ Labeling of Sialidase Expressing Cells with PDFSA
(227) Sialidase transfectant 293T cells, normal (D551) and sialidosis fibroblasts/(GM02921 and GM02922) were incubated with PDFSA (0.2 mM) at 37 C. for 15 h. Cells were lysed by lysis buffer (1% NP-40, 25 mM Tris, 150 mM NaCl, 3 mM KCl, pH 7.4, 1EDTA-free protease inhibitor cocktail from Roche) and then incubated on ice for 15 min. Following incubation, the samples were spun at 18,000g for 15 min. The supernatants were collected, and protein concentrations were determined by bicinchoninic acid protein assay kit (Pierce). For each sample, 50 g total lysate was loaded and separated on 4-12% NuPAGE (Invitrogen). After transferring proteins onto the PVDF membrane (Millipore), membrane click reaction was performed and labeling signal was analyzed by chemiluminescence detector.
(228) For confocal microscopy analysis, sialidase transfectant 293T cells were seeded onto four-well chamber slices (310.sup.5/mL per well), and were cultivated in penicillin/streptomycin-containing 10% FBS/DMEM. Growth medium was supplemented with PDSFA (0.2 mM) and cultured for 15 h. Cells were fixed with 4% paraformaldehyde for 15 min, permeabilized in 0.5% TritonX-100 for 10 min at room temperature, and subjected to the probe labeling reaction consisting 0.1 mM azide-biotin probe/0.1 mM tris-triazole ligand/1 mM CuSO.sub.4/2 mM sodium ascorbate, in PBS, at room temperature for 1 h. Subsequently, the fixed and labeled cells were rinsed with PBS and stained with Dylight 488-conjugated streptavidin (2.5 g/mL in 0.5% BSA/PBS) at room temperature for 30 min. Recombinant sialidases were detected by Alexa Fluor 594-conjugated anti-FLAG antibody (5 ug/ml in 0.5% BSA/PBS). Fluorescent images were captured by Leica TCS-SP5-MP-SMD.
Example 13: Sialidase Activity Assays
(229) Fibroblasts (from D551, GM02921, and GM02922) were fed with PDFSA (0.2 mM) at 37 C. for 15 h. Fibroblasts were lysed by lysis buffer (1% NP-40, 100 mM NaOAc, 150 mM NaCl, 3 mM KCl, pH 4.5, 1EDTA-free protease inhibitor cocktail from Roche) and then incubated on ice for 15 min. Following incubation, the samples were spun at 18,000g for 15 min. The supernatants were collected. One hundred g total lysates in a total volume of 0.1 mL were incubated with MUNANA (0.1 mM) at 37 C. for 1 h. The reaction was terminated with 0.1 mL of 0.85 M glycine-carbonate buffer (pH 9.3), and kept at 4 C. before reading fluorescence. Fluorescence was determined on a fluorometer with excitation at 365 nm and emission at 450 nm.
Example 14: Visualization of Flu Infected Cells Using DFSA
(230) The human kidney cell line, MDCK, were seeded onto six-well plates (310.sup.5/2 ml per well) containing glass coverslips, and were cultivated in 2% FCS/DMEM, and 1% P/S antibiotic-antimycotic. Cells were infected with 0.03 multiplicity of infection (MOI) of flu virus for 20 h at 35 C. and treated with 30 M of DFSA for 1 h at 35 C. Cells on coverslips were fixed with methanol for 3 min, then permeabilized with 0.05% triton-X100 in PBS for 1 min. Cells were subjected to the probe labeling reaction (0.1 mM azide-biotin probe, 0.1 mM tris-triazole ligand, 1 mM CuSO.sub.4, 2 mM sodium ascorbate in PBS) at room temperature for 30 min. Subsequently, the fixed and labeled cells were rinsed with PBS and stained with anti-NP monoclonal antibody (500 fold dilution in PBS), streptavidin-DyLight 488 (2 g/mL in 5% BSA/PBS), and 0.6 g/mL of Alexa Fluor 594 labeled Goat Anti-Mouse IgG (Invitrogen cat#A11020) at room temperature for 30 min. DAPI (10 g/mL in PBS) was used to stain nuclei. Fluorescent images were captured by Leica upright microscope DM 6000B.
Example 15: Quick Detection of OS Susceptibility of Influenza Viruses on Membrane
(231) Polyvinylidene fluoride (PVDF) membrane mounted on Bio-Dot SF of Bio-Rad Inc. (Bio-Rad, CA, USA) was wetted with methanol. Influenza viral samples that were previously treated for 1 h with either 30 M DFSA or 30 M DFSA plus OS were introduced to neighboring slots by suction. The membranes were blotted using PBS with 3% BSA and then PBS with streptavidin 5 g/mL to lower the endogenous biotin noise. Following the click reaction and then incubated with horseradish peroxidase conjugated streptavidin from KPL (Gaithersburg, Md., USA) according to the manufacturer's instruction. After additional washing using PBS with 0.05% tween-20, horseradish peroxidase substrate ECL (Calbiochem) was added for chemiluminescent development.
Example 16: Mass Spectrometric Analyses of Tryptic Peptides of DFSA-Labeled Sialidases
(232) DFSA-labeled sialidases (5 g) were dissolved in 100 mM ammonium bicarbonate and 8 mM dithiothreitol, and incubated at 65 C. for 1 h. To the protein solutions were added 4 L of 40 mM iodoacetamide, and incubated in dark at room temperature for 1 h. The protein solutions were added 1 L of 40 mM dithiothreitol at room temperature for 1 h. The sialidases samples were treated with trypsin at neutral pH for 17 h, heated to inactivate trypsin, and dried for MS analysis.
EQUIVALENTS AND SCOPE
(233) In the claims articles such as a, an, and the may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include or between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
(234) Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms comprising and containing are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.
(235) This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.
(236) Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.
REFERENCES
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(238) All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
(239) Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.