WO2014058535A1 - Lysosomal targeting probes - Google Patents
Lysosomal targeting probes Download PDFInfo
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- WO2014058535A1 WO2014058535A1 PCT/US2013/055578 US2013055578W WO2014058535A1 WO 2014058535 A1 WO2014058535 A1 WO 2014058535A1 US 2013055578 W US2013055578 W US 2013055578W WO 2014058535 A1 WO2014058535 A1 WO 2014058535A1
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Classifications
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/056—Triazole or tetrazole radicals
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B11/00—Diaryl- or thriarylmethane dyes
- C09B11/04—Diaryl- or thriarylmethane dyes derived from triarylmethanes, i.e. central C-atom is substituted by amino, cyano, alkyl
- C09B11/10—Amino derivatives of triarylmethanes
- C09B11/24—Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
- C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
- C09B23/08—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
Definitions
- Lysosomes are membrane-bound organelles found in mammalian cells.
- the lysosomal lumen is more acidic (pH 4.0-6.0) than the cytosol ( ⁇ pH 7.0) and contains different types of proteases, including the capthesins.
- Lysosomes play a critical role in cellular metabolism, and act as important sites for the degradation of excess organelles, engulfed virus or bacteria, and other foreign materials.
- the dysfunction of the lysosome has been implicated in many diseases, including inflammation, cancer, neurodegenerative diseases and various lysosomal storage diseases.
- probes that are used to stain and detect lysosomes in cells are either designed to target the acidic environment of the lysosome, or to take advantage of the ability of large molecules to enter the cell via endocytosis.
- the probe DAMP targets acidic environments such as those found in lysosomes, but it is not fluorescent and must be used in conjunction with anti-DNP antibodies conjugated to detection moieties.
- Fluorophores such as neutral red and acridine orange are also used to stain acidic organelles, but lack specificity.
- LysoTracker ® probes are fluorescent acidotropic compounds that may selectively target acidic organelles, but upon long-term incubation within cells may induce an increase in lysosomal pH, which may lead to fluorescence quenching.
- fluorescent compounds that can be used to selectively stain and detect lysosomes, particularly over longer periods of time. Such compounds may facilitate understanding of intracellular metabolism, cell membrane recycling, and drug and gene delivery systems.
- this disclosure provides a compound of formula (I):
- A is a fluorescent moiety
- L is a linker
- B is a monosaccharide moiety selected from the group consisting of mannose, N- acetyl glucosamine, fucose, galactose and sialic acid;
- n 1 or 2.
- this disclosure provides a method of selectively staining a lysosome in a cell, comprising contacting the cell with an effective amount of a compound of formula (I).
- this disclosure provides a method of selectively detecting a lysosome in a cell, comprising:
- this disclosure provides a method of detecting a cancerous cell in a sample, comprising:
- this disclosure provides a kit comprising a compound of formula (I).
- FIG. 1 shows absorbance spectra for compounds of formula (I).
- FIG. 2 shows fluorescence emission spectra for compounds of formula (I).
- FIG. 3 shows confocal laser-scanning fluorescence images of HeLa cells stained with Probe A ((A) and (E)), MitoTracker (B), LysoTracker (F), Hoechst 33342 ((C) and (G)).
- (D) is an overlay of (A), (B) and (C) while (H) is an overlay of (E), (F) and (G).
- FIG. 4 shows confocal laser-scanning fluorescence images of HeLa cells stained with Probe B ((A) and (E)), MitoTracker (B), LysoTracker (F), Hoechst 33342 ((C) and (G)).
- (D) is an overlay of (A), (B) and (C) while (H) is an overlay of (E), (F) and (G).
- FIG. 5 shows confocal laser-scanning fluorescence images of HeLa cells stained with Probe C ((A) and (E)), MitoTracker (B), LysoTracker (F), Hoechst 33342 ((C) and (G)).
- (D) is an overlay of (A), (B) and (C) while (H) is an overlay of (E), (F) and (G).
- FIG. 6 shows confocal laser-scanning fluorescence images of HeLa cells stained with Probe D ((A) and (E)), MitoTracker (B), LysoTracker (F), Hoechst 33342 ((C) and (G)).
- (D) is an overlay of (A), (B) and (C) while (H) is an overlay of (E), (F) and (G).
- FIG. 7 shows confocal laser-scanning fluorescence images of HeLa cells stained with Probe E ((A) and (E)), MitoTracker (B), LysoTracker (F), Hoechst 33342 ((C) and (G)).
- (D) is an overlay of (A), (B) and (C) while (H) is an overlay of (E), (F) and (G).
- FIG. 8 shows confocal laser-scanning fluorescence images of HeLa cells stained with Probe F ((A) and (E)), MitoTracker (B), LysoTracker (F), Hoechst 33342 ((C) and (G)).
- (D) is an overlay of (A), (B) and (C) while (H) is an overlay of (E), (F) and (G).
- FIG. 9 shows confocal laser-scanning fluorescence images of HeLa cells stained with Probe H ((A) and (E)), MitoTracker (B), LysoTracker (F), Hoechst 33342 ((C) and (G)).
- (D) is an overlay of (A), (B) and (C) while (H) is an overlay of (E), (F) and (G).
- FIG. 10 shows confocal laser-scanning fluorescence images of HeLa cells stained with Probe I ((A) and (E)), MitoTracker (B), LysoTracker (F), Hoechst 33342 ((C) and (G)).
- (D) is an overlay of (A), (B) and (C) while (H) is an overlay of (E), (F) and (G).
- FIG. 1 1 shows confocal laser-scanning fluorescence images of Probes A-F in HeLa cells after 18 hours.
- FIG. 12 shows confocal laser-scanning fluorescence imaging of HeLa cells incubated with Probe B (20 ⁇ ) in media for 2 hours, following washing, incubation with 1.4 ⁇ nigericin for 30 min, and imaging in buffers having pH values of: A) pH 4.4, B) pH 5.0, C) pH 5.5, and D) pH 6.0.
- FIG. 13 shows confocal laser-scanning fluorescence images of Probes J, K and L in HeLa cells.
- FIG. 14 shows confocal laser-scanning fluorescence images of freshly frozen colon tumor tissue-slices from a patient following incubation of Probe A (20 ⁇ ) (A-C), and Probe B (20 ⁇ ) (D-F) for 2h, respectively.
- FIG. 15 shows confocal laser-scanning fluorescence images of HeLa cells stained with Probe M ((A) and (E)), MitoTracker (B), LysoTracker (F), Hoechst 33342 ((C) and (G)).
- (D) is an overlay of (A), (B) and (C) while (H) is an overlay of (E), (F) and (G).
- FIG. 16 shows confocal laser-scanning fluorescence images of HeLa cells stained with Probe N ((A) and (E)), MitoTracker (B), LysoTracker (F), Hoechst 33342 ((C) and (G)).
- (D) is an overlay of (A), (B) and (C) while (H) is an overlay of (E), (F) and (G).
- FIG. 17 shows confocal laser-scanning fluorescence images of HeLa cells stained with Probe O ((A) and (E)), MitoTracker (B), LysoTracker (F), Hoechst 33342 ((C) and (G)).
- (D) is an overlay of (A), (B) and (C) while (H) is an overlay of (E), (F) and (G).
- FIG. 18 shows confocal laser-scanning fluorescence images of HeLa cells stained with Probe P ((A) and (E)), MitoTracker (B), LysoTracker (F), Hoechst 33342 ((C) and (G)).
- (D) is an overlay of (A), (B) and (C) while (H) is an overlay of (E), (F) and (G).
- FIG. 19 shows confocal laser-scanning fluorescence images of HeLa cells stained with Probe Q ((A) and (E)), MitoTracker (B), LysoTracker (F), Hoechst 33342 ((C) and (G)).
- (D) is an overlay of (A), (B) and (C) while (H) is an overlay of (E), (F) and (G).
- Described herein are compounds, compositions and methods for selectively targeting, staining and detecting a lysosome.
- Current compounds that are used to detect lysosomes in cells may lack specificity and/or photostability, or may require multiple steps to use (e.g., incubation with a non-fluorescent compound, followed by an antibody labeled with a detection moiety).
- the compounds described herein may selectively target lysosomes to allow for selective detection in one step.
- the compounds described herein include a fluorophore, a linker, and a monosaccharide moiety selected from the group consisting of galactose, mannose, N-acetyl glucosamine, fucose and sialic acid.
- Compounds described herein are stable and sensitive markers for selectively staining lysosomes, which may enable selective detection of lysosomes over long time periods.
- each intervening number there between with the same degree of precision is explicitly contemplated.
- the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are explicitly contemplated.
- contacting a cell refers to contacting a sample or cell directly or indirectly, in vitro or ex vivo. Contacting a sample may include addition of a compound to a sample (e.g., a culture of cells).
- an "effective amount” of a compound refers to an amount of a compound or a composition effective for eliciting a desired effect.
- an "effective amount" of a compound may be an amount that allows for visualization of a fluorescent signal that is localized to a lysosome, using a method such as fluorescence microscopy.
- fluorophore fluorescent moiety
- fluorescent label fluorescent dye
- fluorescent dye refers to a molecule or a portion thereof that absorbs a quantum of electromagnetic radiation at one wavelength, and emits one or more photons at a different, typically longer, wavelength.
- Numerous fluorescent dyes of a wide variety of structures and characteristics are suitable for use in the compounds of the present disclosure. Suitable fluorophores are described in the Handbook of Fluorescent Probes and Research Chemicals (6th Ed., Molecular Probes, Inc., Eugene Oreg.).
- a "linker,” as used herein, refers to an atom or a group of atoms that links a fluorescent moiety to one or more monosaccharide moieties.
- a divalent linker may link a fluorescent moiety to one monosaccharide moiety (i.e. will provide one point of attachment to the fluorescent moiety and one point of attachment to the monosaccharide moiety).
- a trivalent linker may link a fluorescent moiety to two monosaccharide moieties (i.e. will provide one point of attachment to the fluorescent moiety, one point of attachment to a first monosaccharide moiety and one point of attachment to a second monosaccharide moiety).
- member atom refers to a polyvalent atom (e.g., a C, O, N, P or S atom) in a chain or ring system that constitutes a part of the chain or ring.
- a polyvalent atom e.g., a C, O, N, P or S atom
- two carbon atoms and three nitrogen atoms are member atoms of the ring.
- a linker -(CH ⁇ CH ⁇ O ⁇ - four carbon atoms and two oxygen atoms are member atoms of the linker.
- Member atoms will be substituted up to their normal valence.
- the five carbon atoms will each be further substituted with a hydrogen or another substituent (e.g., an alkyl group).
- the term "monosaccharide moiety,” as used herein, refers to a radical of a monosaccharide, where an atom or a group of atoms is removed.
- the radical serves as a point of attachment to another molecule.
- a hydrogen atom from a hydroxy group can be removed from a monosaccharide to provide a monosaccharide moiety that is attached to another molecule via an oxygen atom, or a hydroxy radical may be removed to provide a monosaccharide moiety that is attached to another molecule via a carbon atom.
- reactive group refers to a chemical moiety that is part of a first molecule that is capable of reacting with a "complementary group” that is part of a second molecule, to form a covalent bond between the first molecule and the second molecule.
- a complementary group may also be called a "complementary reactive group”.
- a reactive group is an electrophilic group and a complementary group is a nucleophilic group.
- a reactive group is a nucleophilic group and a complementary group is an electrophilic group.
- the reactive group is an azide and the complementary group is an alkynyl group. In some embodiments the reactive group is an alkynyl group and the complementary group is an azide.
- acyl refers to an alkylcarbonyl, cycloalkylcarbonyl, heterocyclylcarbonyl, arylcarbonyl or heteroarylcarbonyl substituent, any of which may be further substituted (e.g., with one or more substituents).
- alkyl refers to a straight or branched saturated hydrocarbon chain.
- Alkyl groups may include a specified number of carbon atoms.
- C1-C12 alkyl indicates that the alkyl group may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms.
- An alkyl group may be, e.g., a C1-C12 alkyl group, a C1-C1 0 alkyl group, a Ci-Cs alkyl group, a C1-C6 alkyl group or a C1-C4 alkyl group.
- exemplary C1-C4 alkyl groups include methyl, ethyl, w-propyl, isopropyl, w-butyl, sec-butyl, isobutyl and tert- butyl groups.
- An alkyl group may be optionally substituted with one or more substituents.
- alkenyl refers to a straight or branched hydrocarbon chain having one or more double bonds.
- Alkenyl groups may include a specified number of carbon atoms.
- C2-C12 alkenyl indicates that the alkenyl group may have 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 carbon atoms.
- An alkenyl group may be, e.g., a C2-C12 alkenyl group, a C2-C1 0 alkenyl group, a C2-C 8 alkenyl group, a C2-C6 alkenyl group or a C2-C4 alkenyl group.
- alkenyl groups include but are not limited to allyl, propenyl, 2- butenyl, 3-hexenyl and 3-octenyl groups.
- One of the double bond carbons may optionally be the point of attachment of the alkenyl substituent.
- An alkenyl group may be optionally substituted with one or more substituents.
- alkynyl refers to a straight or branched hydrocarbon chain having one or more triple bonds.
- Alkynyl groups may include a specified number of carbon atoms.
- C2-C12 alkynyl indicates that the alkynyl group may have 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 carbon atoms.
- An alkynyl group may be, e.g., a C2-C12 alkynyl group, a C2-C1 0 alkynyl group, a C2-C 8 alkynyl group, a C2-C6 alkynyl group or a C2-C4 alkynyl group.
- alkynyl groups include but are not limited to ethynyl, propargyl, and 3-hexynyl.
- One of the triple bond carbons may optionally be the point of attachment of the alkynyl substituent.
- An alkynyl group may be optionally substituted with one or more substituents.
- aryl refers to an aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring system, wherein any ring atom capable of substitution can be substituted (e.g., with one or more substituents).
- aryl moieties include but are not limited to phenyl, naphthyl, and anthracenyl.
- Aryl groups may be optionally substituted with one or more substituents.
- cycloalkyl refers to non-aromatic, saturated or partially unsaturated monocyclic, bicyclic, tricyclic or poly cyclic hydrocarbon groups having 3 to 12 carbons. Any ring atom can be substituted (e.g., with one or more substituents). Cycloalkyl groups can contain fused rings. Fused rings are rings that share one or more common carbon atoms.
- cycloalkyl groups include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, methylcyclohexyl, adamantyl, norbornyl, norbornenyl, tetrahydronaphthalenyl and dihydroindenyl. Cycloalkyl groups may be optionally substituted with one or more substituents.
- Cycloalkenyl refers to a non-aromatic monocyclic, bicyclic, tricyclic or polycyclic hydrocarbon group having one or more double bonds (e.g., cyclohexenyl or cyclohexadienyl).
- Cycloalkynyl refers to a non-aromatic monocyclic, bicyclic, tricyclic or polycyclic hydrocarbon group having one or more triple bonds (e.g., cyclooctynyl), which may be optionally substituted with one or more substituents (e.g., with one or more halo groups, e.g., to generate a difluorcyclooctynyl group).
- halo or halogen, refers to any radical of fluorine, chlorine, bromine or iodine.
- haloalkyl refers to an alkyl group as defined herein, in which one or more hydrogen atoms are replaced with halogen atoms, and includes alkyl moieties in which all hydrogens have been replaced with halogens (e.g., perfluoroalkyl such as CF 3 ).
- heteroalkyl refers to an alkyl, alkenyl or alkynyl group as defined herein, wherein at least one carbon atom of the alkyl group is replaced with a heteroatom.
- Heteroalkyl groups may contain from 1 to 18 non-hydrogen atoms (carbon and heteroatoms) in the chain, or 1 to 12 atoms, or 1 to 6 atoms, or 1 to 4 atoms.
- Heteroalkyl groups may be straight or branched, and saturated or unsaturated. Unsaturated heteroalkyl groups have one or more double bonds and/or one or more triple bonds. Heteroalkyl groups may be unsubstituted or substituted.
- heteroalkyl groups include but are not limited to alkoxyalkyl (e.g., methoxymethyl), and aminoalkyl (e.g., alkylaminoalkyl and dialkylaminoalkyl). Heteroalkyl groups may be optionally substituted with one or more substituents.
- heteroaryl refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms independently selected from O, N, S, P and Si (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms independently selected from O, N, S, P and Si if monocyclic, bicyclic, or tricyclic, respectively). Any ring atom can be substituted (e.g., with one or more
- Heteroaryl groups can contain fused rings, which are rings that share one or more common atoms.
- Examples of heteroaryl groups include, but are not limited, to radicals of pyridine, pyrimidine, pyrazine, pyridazine, pyrrole, imidazole, pyrazole, triazole, oxazole, isoxazole, furan, thiazole, isothiazole, thiophene, quinoline, isoquinoline, quinoxaline, quinazoline, cinnoline, indole, isoindole, indolizine, indazole, benzimidazole, phthalazine, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, phenazine, naphthyridines and purines. Heteroaryl groups may be optionally substituted with one or more substituents.
- heteroatom refers to a non-carbon or hydrogen atom such as a nitrogen, sulfur, oxygen, silicon or phosphorus atom. Groups containing more than one heteroatom may contain different heteroatoms.
- heterocyclyl refers to a nonaromatic, saturated or partially unsaturated 3-10 membered monocyclic, 8-12 membered bicyclic, or 1 1-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, S, Si and P (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, S, Si and P if monocyclic, bicyclic, or tricyclic, respectively). Any ring atom may be substituted (e.g., with one or more substituents).
- Heterocyclyl groups can contain fused rings, which are rings that share one or more common atoms.
- heterocyclyl groups include, but are not limited to, radicals of tetrahydro furan, tetrahydrothiophene, tetrahydropyran, oxetane, piperidine, piperazine, morpholine, pyrroline, pyrimidine, pyrrolidine, indoline, tetrahydropyridine, dihydropyran, thianthrene, pyran, benzopyran, xanthene, phenoxathiin, phenothiazine, furazan, lactones, lactams such as azetidinones and pyrrolidinones, sultams, sultones, and the like. Heterocyclyl groups may be optionally substituted with one or more substituents.
- hydroxy refers to an -OH radical.
- alkoxy refers to an -O-alkyl radical.
- aryloxy refers to an -O- aryl radical.
- mercapto or "thiol,”as used herein, each refer to an -SH radical.
- thioalkoxy or “thioether,” as used herein, each refer to an -S-alkyl radical.
- thioaryloxy refers to an -S-aryl radical.
- substituted refers to a group “substituted” on an alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl group at any atom of that group. Any atom can be substituted.
- substituents on a group are independently any one single, or any combination of the aforementioned substituents.
- a substituent may itself be substituted with any one of the above substituents.
- groups and substituents thereof may be selected in accordance with permitted valence of the atoms and the substituents, and such that the selections and substitutions result in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
- substituent groups are specified by their conventional chemical formulae, written from left to right, they optionally encompass substituents resulting from writing the structure from right to left, e.g., -CH 2 0- optionally also recites -OCH 2 -.
- A is a fluorescent moiety
- L is a linker
- B is a monosaccharide moiety selected from the group consisting of mannose, N- acetyl glucosamine, fucose, galactose and sialic acid;
- n 1 or 2.
- A is a fluorescent moiety.
- Suitable fluorescent moieties include rhodamines, fluoresceins, coumarins, cyanines, and boron-dipyrromethenes (also known as BODIPYs), as well as derivatives thereof.
- known rhodamine derivatives include amine-conjugated rhodamine compounds.
- a fluorescent moiety may be incorporated into a compound of formula (I), for example, by using a reagent that comprises a fluorophore and a reactive group such as a carboxylic acid, an isothiocyanate, a maleimide, an alkynyl group, an azide, an amine, a thiol, or an ester such as a succinimidyl, sulfodichlorophenol, pentafluorophenyl or
- tetrafluorophenyl ester Such groups may react with a complementary group, such as one present on a linker precursor compound, to attach the fluorophore to the remainder of the molecule of formula (I).
- Reagents comprising fluorophores and reactive groups may be commercially available, or may be synthesized according to methods described herein or other methods known to those skilled in the art.
- reagents comprising fluorophores which may be used to prepare compounds of formula (I), are known in the art.
- reagents comprising fluorophores that are commercially available include, but are not limited to: 5- and 6- carboxyfluoresceins and esters thereof; fluorescein-5 -isothiocyanate and fluorescein-6- isothiocyanate; BODIPY® dyes commercially available from Life TechnologiesTM, such as BODIPY® succinimidyl esters; Alexa Fluor® dyes commercially available from Life TechnologiesTM, such as Alexa Fluor® succinimidyl, tetrafluorphenyl and
- fluorophores available from PerkinElmer, such as VivoTagTM succinimidyl esters and maleimides.
- the fluorescent moiety may include the fluorophore itself, as well as additional atoms or groups of atoms, such as atoms or groups of atoms derived from reactive groups or complementary groups that serve to link the fluorescent moiety to the remainder of the compound of formula (I).
- Certain fluorescent moieties can be selected for incorporation into compounds of formula (I), based on the particular application of interest.
- fluorophores having particular excitation/emission profiles may be selected, and may, for example, be orthogonal to other fluorophores being used in a particular application.
- certain live-cell imaging experiments may be conducted using DAPI as a nuclear stain, which has an absorption maximum of about 350 nm and an emission maximum of about 460 nm.
- a fluorescent moiety for a compound of formula (I) may be selected to have excitation and emission properties that are different from those of the DAPI stain.
- rhodamine B has an absorption maximum of about 540 nm and an emission maximum of about 625 nm. (As those skilled in the art appreciate, these absorption and emission values are approximate and depend on the particular environment including solvent, pH, etc.)
- L is a linker.
- linker refers to an atom or a group of atoms that link a fluorescent moiety (A-) to one or more monosaccharide moieties (-B).
- a divalent linker allows for linkage of one -B moiety, while a trivalent linker allows for linkage of two -B moieties.
- a linker may be a straight chain, a branched chain, or may include one or more ring systems.
- Nonlimiting exemplary linkers include polyethylene glycol linkers and triazole-containing linkers, and linkers including both ethylene glycol units and triazole rings.
- a linker may include from 1 to about 50 member atoms, not including substituents.
- a linker may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 atoms, or any range therebetween.
- Illustrative linkers include, but are not limited to -(CH 2 ) c -D e -(CH 2 ) f - and -(CH 2 ) P - M r -C(0)-K s -(CH 2 ) q - where c is 0 to 8; D is O, NH, or S; e is 0 or 1; f is 0 to 8; p is 0 to 8; M is NH or O; K is NH or O; q is 0 to 8, and r and s are each independently 0 or 1.
- Illustrative linkers also include those having ring structures, such as aryl, heteroaryl, cycloalkyl or heterocyclyl rings.
- linkers may include a heteroaryl ring, such as a triazole.
- a linker may include a 1,2, 3 -triazole, which may be a product of the reaction between a reagent comprising a fluorophore and an alkyne, and a molecule comprising an azide.
- a 1,2,3-triazole may be a product of the reaction between a reagent comprising a fluorophore and an azide, and a molecule comprising an alkynyl group.
- Exemplary linkers comprising ring structures, such as 1 ,2,3- triazole groups include the followin
- Illustrative linkers also include those having both triazole rings and other groups, such as ethylene glycol units, e.g., one or more units having the formula -(CH2CH2O), and amide units (-C(O)NH-).
- ethylene glycol units e.g., one or more units having the formula -(CH2CH2O), and amide units (-C(O)NH-).
- exemplary linkers including such units include the following:
- the linker may be hydrolytically stable.
- linkers can be used to link the fluorescent moiety and the monosaccharide moiety in the compounds of formula (I).
- B is a monosaccharide moiety derived from a monosaccharide selected from the group consisting of galactose (Gal), mannose (Man), N- acetylglucosamine (GlcNAc), fucose (Fuc) and sialic acid (also known as N- acetylneuraminic acid, NeuAc).
- suitable monosaccharide moieties include, but are not limited to, the following:
- a monosaccharide moiety may be linked to the compound of formula (I) by replacing any suitable atom or group of atoms of a monosaccharide with the remainder of the compound of formula (I).
- a 1 -hydroxy group, 2-hydroxy group, or a 6-hydroxy group may be replaced to yield a deoxymonosaccharide radical, where the radical serves as the point of attachment to the remainder of the compound of formula (I),
- fluorescein may exist in neutral, monoanionic, dianionic and lactone forms, as illustrated below.
- Compounds described herein that include fluorescein moieties encompass all of these forms and mixtures thereof
- rhodamine B may exist in cationic, zwitterionic and lactone forms, as illustrated below.
- Compounds described herein that include rhodamine moieties encompass all of these forms and mixtures thereof
- compounds described herein bear one or more charges, it will be understood that they will also include one or more associated anions or cations to balance the charges.
- a salt may be formed with a suitable cation.
- suitable inorganic cations include, but are not limited to, alkali metal ions such as Na + and K + , alkaline earth cations such as Ca 2+ and Mg 2+ , and other cations.
- Suitable organic cations include, but are not limited to, ammonium ion (i.e., NH 4 + ) and substituted ammonium ions (e.g., NH3R1 , NH2R2 , NHR 3 , NR4 ).
- suitable substituted ammonium ions are those derived from: ethylamine, diethylamine,
- dicyclohexylamine triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine.
- a salt may be formed with a suitable anion.
- suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous.
- Suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric.
- RG and RG' are independently reactive groups, and CG and CG' are independently complementary groups that react with RG and RG' respectively.
- B represents a monosaccharide moiety.
- L 1 , L 2 , L 3 , L 4 and L 5 are independently linkers, which may be the same or different.
- L 2 may be a linker comprising residual atoms from the reaction of RG and CG.
- L 1 is -CH 2 -
- RG is -C ⁇ CH
- CG is -N 3
- L 4 may include atoms derived from L 1 , residual atoms from the reaction of RG and CG, and atoms derived from L 3 . Further methods of preparing compounds described herein are shown in the Examples.
- Compounds can be evaluated using a number of methods. Following structural characterization using methods known in the art, such as nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS), the absorbance and emission spectra can be evaluated. Absorbance spectra can be determined using an ultraviolet-visible (UV-vis) spectrometer, and fluorescence emission spectra can be obtained using a fluorometer. The absorbance and emission spectra can further be evaluated as a function of pH. For example, certain compounds may be fluorescent under acidic conditions, but the fluorescence may be diminished or nearly eliminated under basic conditions.
- NMR nuclear magnetic resonance
- MS mass spectrometry
- compounds possess suitable absorbance and emission characteristics they may be further evaluated in cells.
- Compounds may be incubated with cultured living cells for a period of time, followed by washing to remove excess extracellular compounds. Examination using standard fluorescence microscopy or confocal laser scanning fluorescence microscopy, can indicate the intracellular localization of the compounds. The localization can be confirmed using compounds that are known to localize to particular organelles. For example, LysoTracker® and MitoTracker® probes are commercially available from Life TechnologiesTM, and localize to the lysosome and mitochondria, respectively.
- Nuclei may be stained using compounds such as 4',6-diamidino-2-phenylindole (DAP I) or a Hoechst stain (e.g., Hoechst 33258, Hoechst 33342, or Hoechst 34580). Bright-field examination can be used to evaluate cellular viability following incubation of the cells with the compound.
- DAP I 4',6-diamidino-2-phenylindole
- Hoechst stain e.g., Hoechst 33258, Hoechst 33342, or Hoechst 34580.
- Bright-field examination can be used to evaluate cellular viability following incubation of the cells with the compound.
- Compounds of formula (I) may be used in a variety of methods, such as methods of selectively staining or detecting a lysosome in a cell, or methods of detecting a cancerous cell in a sample.
- Lysosomes include membrane proteins that are highly glycosylated, including glycans such as N-linked glycans.
- glycans include monosaccharides, including Gal, Man, GlcNAc, Fuc and NeuAc. While not wishing to be limited by theory, the presence of the monosaccharide moieties on the compounds of formula (I) may promote their uptake into lysosomes.
- this disclosure provides a method of selectively staining a lysosome in a cell, comprising contacting the cell with an effective amount of a compound of formula (I).
- the cell may be, for example, in a culture of cells.
- the compound of formula (I) may selectively localize to an acidic organelle such as a lysosome, thereby staining the lysosome.
- the disclosure provides a method of selectively detecting a lysosome in a cell, comprising contacting the cell with an effective amount of a compound of formula (I), and detecting a signal from the compound.
- the signal may be a fluorescence signal, which may be detected using a variety of instruments such as fluorescence microscope, a flow cytometer, a fluorometer, a fluorescence plate reader, or any combination thereof.
- the cell may be, for example, in a culture of cells.
- the compound of formula (I) may selectively localize to an organelle such as a lysosome, thereby allowing for selective detection of a lysosome.
- the method may further include a step of washing the cells prior to detection, to remove any free compound of formula (I).
- the cells may not be fixed prior to detection, in order to prevent cell-fixation artifacts.
- compounds of formula (I) may have increased stability compared to current probes.
- known lysosomal targeting probes may only provide stable fluorescence signals for periods of time up to about 1 hour to about 2 hours. The probes may decompose in the acidic environment of the lysosome, or may leak out of the lysosome or the cell.
- compounds of formula (I) may provide a stable fluorescence signal in living cells for at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, or about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours.
- Such properties may allow for monitoring of lysosomal morphology and trafficking in intact cells.
- experiments may be performed in any suitable cell or cell line of interest.
- the disclosure provides a method of selectively detecting a cancerous cell in a sample, comprising contacting the sample with an effective amount of a compound of formula (I), and detecting a signal from the compound.
- the sample may be an in vitro sample, such as a cell or tissue extract.
- the sample is a cell culture.
- the sample may be a culture of cells such as cancer cells.
- the sample may be a biological sample from a subject, such as a human.
- the biological sample is selected from the group consisting of a tissue sample, bodily fluid, whole blood, plasma, serum, urine,
- the methods may further involve providing or obtaining a biological sample from the subject, which can be obtained by any known means including needle stick, needle biopsy, swab, and the like.
- a signal from the fluorescent moiety may be quantitated, for example, by comparing the quantity of the signal to that of a reference sample.
- a cancerous cell may be detected if a fluorescence signal from the cell is higher than that of a signal from a reference cell.
- the cancer may be any type of cancer, such as a cancer recognized by the National Cancer Institute.
- this disclosure provides a kit, which may be used for selectively staining or detecting a lysosome in a cell, or for detecting a cancerous cell in a sample.
- kits will include a compound of formula (I) as described herein.
- a kit may also include instructions for use of the compound of formula (I). Instructions included in kits can be affixed to packaging material or can be included as a package insert. While the instructions are typically written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD, DVD), and the like. As used herein, the term "instructions" includes the address of an internet site that provides the instructions.
- the kit may comprise instructions for selectively detecting a lysosome in a cell by fluorescence detection, e.g., using a fluorescence microscope, a flow cytometer, a fluorometer, a fluorescence plate reader, or a combination thereof.
- the kit may further comprise a calibrator or control, and/or at least one container (e.g., a tube, a microtiter plate and/or a strip) for conducting the assay, and/or a buffer, such as an assay buffer or a wash buffer, either one of which can be provided as a concentrated solution.
- the kit comprises all components, i.e., reagents, standards, buffers, diluents, etc., which are necessary for conducting a particular experiment.
- the instructions also may include instructions for generating a standard curve or a reference standard for purposes of quantification.
- the kit also may optionally include other reagents required to conduct an assay or facilitate quality control evaluations, such as buffers, salts, enzymes, enzyme co-factors, substrates, detection reagents, and the like.
- Other components such as buffers and solutions for the isolation and/or treatment of a test sample (e.g., pretreatment reagents), also may be included in the kit.
- the kit additionally may include one or more other controls.
- One or more of the components of the kit may be lyophilized, in which case the kit further may comprise reagents suitable for the reconstitution of the lyophilized components.
- kits for holding or storing a sample (e.g., a container or cartridge for a sample).
- a sample e.g., a container or cartridge for a sample
- the kit optionally also may contain reaction vessels, mixing vessels, and other components that facilitate the preparation of reagents or the test sample.
- the kit also may include one or more instrument for assisting with handling a sample, such as a syringe, pipette, or the like.
- Fluorescence spectra were obtained by using a Horiba Jobin Yvon Fluoromax ® -4 spectrofluorometer. The slit width was 5 nm for both excitation and emission. All samples were excited at 480 nm. A 10 x 10 mm quartz cell was used for all fluorescence
- the final concentration of the probe was about 1 ⁇ .
- the slit width was 5 nm for both excitation and emission. All the samples were excited at 540 nm. A 10x10 mm quartz cell was used for each fluorescence measurement. Relative quantum yields of Probes A-G were compared to Rhodamine B. The following equation was used in order to determine relative quantum yields:
- F is the area under the emission curve for each probe, and A is the absorbance value at the excitation wavelength.
- A is the absorbance value at the excitation wavelength.
- HepG2 cells human hepatocellular liver carcinoma cell line
- HeLa cells human cervical cancer cells
- ACC American Type Cell Culture collection
- HepG2 and HeLa cells were grown in Eagle's Minimal Essential Medium (EMEM) with 10% FBS (Sigma- Aldrich, heat inactivated). All cells were maintained in a 5% CO2 humidified atmosphere at 37°C.
- EMEM Eagle's Minimal Essential Medium
- FBS FBS
- Cells were grown in 35 mm glass bottom dish for 24 h in media. The media was removed and the cells were washed three times with IX DPBS without Ca 2+ or Mg 2+ (Hyclone, Fisher Scientific). Probes A-I were incubated with cells in non-FBS media. After each step, cells were washed with DPBS buffer. Then, they were imaged in different pH buffers or in media. More detailed information about incubation times and media were used for imaging are provided below.
- Probes A-F To characterize the intracellular localization of Probes A-F, various localization markers were employed. Following several hours of incubation, intracellular probe distribution suggested localization in either acidic vesicles or potentially within the mitochondria. To further examine this, a double stain of HeLa cells with MitoTracker and Probes A-F was undertaken. A 45-minute incubation with Probes A-F, resulted in a distinct labeling pattern and an absence of colocalization of the probes with the mitochondria (Figs. 3-8, A-D). To further characterize the pattern of probe localization, we employed confocal laser scanning fluorescent microscopy and a double staining with LysoTracker Green (a lysosome selective stain) in HeLa cells.
- LysoTracker Green a lysosome selective stain
- Figure 3 shows confocal laser-scanning fluorescence images of Probe A in HeLa cells.
- Probe A (30 ⁇ , green, shown in Fig. 3A) was incubated with HeLa cells in non-FBS DMEM media for 2 hours and counterstained with MitoTracker (80 nM, red, shown in Fig. 3B), and Hoechst 33342 (1 ⁇ g/mL, blue, shown in Fig. 3C).
- An overlay of the images shown in Figs. 3A, 3B and 3C is shown in Fig. 3D.
- HeLa cells were also incubated with Probe A (20 ⁇ , shown in Fig. 3E) in media for 2h, followed by counterstain with LysoTracker (2 ⁇ , shown in Fig.
- Figure 4 shows confocal laser-scanning fluorescence images of Probe B in HeLa cells.
- Probe B (30 ⁇ , green, shown in Fig. 4A) was incubated with HeLa cells in non-FBS DMEM media for 2 hours and counterstained with MitoTracker (80 nM, red, shown in Fig. 4B), and Hoechst 33342 (1 ⁇ g/mL, blue, shown in Fig. 4C).
- An overlay of the images shown in Figs. 4A, 4B and 4C is shown in Fig. 4D.
- HeLa cells were also incubated with Probe B (20 ⁇ , shown in Fig. 4E) in media for 2h, followed by counterstain with LysoTracker (2 ⁇ , shown in Fig.
- Figure 5 shows confocal laser-scanning fluorescence images of Probe C in HeLa cells.
- Probe C (20 ⁇ , green, shown in Fig. 5A) was incubated with HeLa cells in non-FBS DMEM media for 45-min and counterstained with Mito-Tracker (40 nM, red, shown in Fig. 5B), and Hoechst 33342 (1 ⁇ g/mL, blue, shown in Fig. 5C).
- An overlay of the images shown in Figs. 5A, 5B and 5C is shown in Fig. 5D.
- HeLa cells were also incubated with Probe C (20 ⁇ , shown in Fig.
- Figure 6 shows confocal laser-scanning fluorescence images of Probe D in HeLa cells.
- Probe D (30 ⁇ , green, shown in Fig. 6A) was incubated with HeLa cells in non-FBS DMEM media for 45-min and counterstained with Mito-Tracker (40 nM, red, shown in Fig. 6B), and Hoechst 33342 (1 ⁇ g/mL, blue, shown in Fig. 6C).
- An overlay of the images shown in Figs. 6A, 6B and 6C is shown in Fig. 6D.
- HeLa cells were also incubated with Probe D (20 ⁇ , shown in Fig.
- Figure 7 shows confocal laser-scanning fluorescence images of Probe E in HeLa cells.
- Probe E (30 ⁇ , green, shown in Fig. 7A) was incubated with HeLa cells in non-FBS DMEM media for 45-min and counterstained with MitoTracker (40 nM, red, shown in Fig. 7B), and Hoechst 33342 (1 ⁇ g/mL, blue, shown in Fig. 7C).
- An overlay of the images shown in Figs. 7A, 7B and 7C is shown in Fig. 7D.
- HeLa cells were also incubated with Probe E (20 ⁇ , shown in Fig.
- Figure 8 shows confocal laser-scanning fluorescence images of Probe F in HeLa cells.
- Probe F (30 ⁇ , green, shown in Fig. 8A) was incubated with HeLa cells in non-FBS DMEM media for 45-min and counterstained with Mito-Tracker (40 nM, red, shown in Fig. 8B), Hoechst 33342 (1 ⁇ g/mL, blue, shown in Fig. 8C).
- An overlay of the images shown in Figs. 8A, 8B and 8C is shown in Fig. 8D.
- HeLa cells were also incubated with Probe F (20 ⁇ , shown in Fig.
- Probes A-F may exist primarily in their un-ionized, membrane-permeable forms when present in the essentially neutral cell cytosol. However, once the probes cross the membrane lipid bilayer and reach organelles with acidic luminal pH (e.g., lysosomes), then the probes may convert to an almost exclusively ionized, membrane-impermeable form, which will be trapped within the organelle. Consistent with this hypothesis, the intracellular distribution of fluorescence was punctate, as opposed to diffuse, a finding consistent with fluorescence activation occurring within intracellular compartments such as the lysosomes, and not in the cytosol. The punctuate staining patterns were preserved after incubation for 48 hours.
- Control Probes H and I include lactose moieties rather than galactose, glucose or N- acetylglucosamine moieties of Probes A-F.
- Figure 9 shows confocal laser-scanning fluorescence images of Probe H in HeLa cells.
- Probe H (20 ⁇ , green, shown in Fig. 9A) was incubated with HeLa cells in non-FBS DMEM media for 45-min and counterstained with MitoTracker (40 nM, red, shown in Fig. 9B), and Hoechst 33342 (1 ⁇ g/mL, blue, shown in Fig. 9C).
- An overlay of the images shown in Figs. 9A, 9B and 9C is shown in Fig. 9D.
- HeLa cells were also incubated with Probe H (20 ⁇ , shown in Fig.
- Figure 10 shows confocal laser-scanning fluorescence images of Probe I in HeLa cells.
- Probe I (20 ⁇ , green, shown in Fig. 10A) was incubated with cells in non-FBS DMEM media for 45-min and counterstained with MitoTracker (40 nM, red, shown in Fig. 10B), Hoechst 33342 (1 ⁇ g/mL, blue, shown in Fig. IOC).
- An overlay of the images shown in Figs. 10A, 10B and IOC is shown in Fig. 10D.
- HeLa cells were also incubated with Probe I (20 ⁇ , E) in media for 15-min, followed by counterstain with LysoTracker (2 ⁇ , shown in Fig.
- Figure 1 1 shows confocal laser-scanning fluorescence images of Probes A-F in HeLa cells.
- Panels 1A-1F show results for Probe A.
- Panels 2A-2F show results for Probe B.
- Panels BASF show results for Probe C.
- Panels 4A-4F show results for Probe D.
- Panels 5A-5F show results for Probe E.
- Panels 6A-6F show results for Probe F.
- Probes A-F (20 ⁇ , green, shown in Fig. 1 1 panels 1C, 2C, 3C, 4C, 5C and 6C) were incubated with cells in non-FBS DMEM media for 15 min, and then counterstained with Lyso-Tracker (2 ⁇ , shown in Fig. 11 panels IB, 2B, 3B, 4B, 5B and 6B), and Hoechst 33342 (1 ⁇ g/mL, blue, shown in Fig. 1 1 panels 1A, 2A, 3A, 4A, 5A and 6A).
- DIC images are shown in panels ID, 2D, 3D, 4D, 5D and 6D. Overlays of panels A, B, C and D are shown in panel E. Overlays of panels A, B and C are shown in panel F. All images were acquired with 60 X objective after 18 hours.
- FIG. 12 shows confocal laser-scanning fluorescence imaging of HeLa cells incubated with Probe B (20 ⁇ ) in media for 2 hours. After washing three times with corresponding pH buffers, cells were further incubated with 1.4 ⁇ nigericin for 30 min and then imaged at pH 4.4 (Fig. 12A), pH 5.0 (Fig. 12B), pH 5.5 (Fig. 12C) and pH 6.0 (Fig. 12D) buffers. All images were acquired with 60 X objective
- Probes J, K and L (20 ⁇ , red, shown in Fig. 13 panels 1A-3A respectively) was incubated with HeLa cells in media for 15 min, followed by counterstain with LysoTracker (2 ⁇ , panels 1B-3B respectively), Hoechst 33342 (1 ⁇ g/mL, panels 1C-3C respectively). Overlays of panels A, B and C are shown in panels 1D-3D respectively. All images were acquired with 60 X objective.
- Lysosome targeting of fluorescent probes J, K and L was observed by co- localization with LysoTracker (Life Technologies, Inc.). A high proportion of overlay was not observed in a control compound, a cyanine-lactose conjugate (without N-glycan moiety).
- FIG. 14 shows confocal laser-scanning fluorescence images of freshly frozen colon tumor tissue-slices from a patient following incubation of Probe A (20 ⁇ ) (A-C), and Probe B (20 ⁇ ) (D-F) for 2h, respectively.
- Probe A and Probe B fluorescence images are displayed in green (A, D), with nuclei counterstained by Hoechst 33342 and displayed in blue (B, E), and overlay images (C, F).
- Probe M To compound 1.4 (75 mg, 0.049 mmol) in methanol (5 mL) at room temperature was added a catalytic amount of NaOMe. The reaction was allowed to stir at room temperature for 6 h. The solution was filtered and then the volatiles were removed in vacuum to give Probe M as a yellow solid (46 mg, 74%).
- Probe N To compound 1.10 (77 mg, 0.05 mmol) in methanol (5 mL) at room temperature was added a catalytic amount of NaOMe. The reaction was allowed to stir at room temperature for 6 h. The solution was filtered and then the volatiles were removed in vacuum to give the Probe N as colorless syrup. Probe N: MS [M+Hf found: 1294.
- Compound 2.2 To a mixture of rhodamine 101 (compound 2.1, 270 mg, 0.55 mmol) and Azido-PEG 3 -amine (109 mg, 0.5 mmol) in DCM (20 mL) was sequentially added HBTU (260 mg, 1.2 mmol) and Ets (0.5 mL). The reaction mixture was stirred overnight at room temperature.
- Probe O To compound 2.4 (80 mg, 0.051 mmol) in methanol (5 mL) at room temperature was added a catalytic amount of NaOMe. The reaction was allowed to stir at room temperature for 6 h. The solution was filtered and then the volatiles were removed in vacuum to give the compound Probe O as a yellow solid (53 mg, 81%). Probe O: MS
- Probe P To compound 2.10 (80 mg, 0.051 mmol) in methanol (5 mL) at room temperature was added a catalytic amount of NaOMe. The reaction was allowed to stir at room temperature for 6 h. The solution was filtered and then the volatiles were removed in vacuum to give the compound Probe P as colorless syrup. Probe P: MS [M+H] + found: 1342. P
- Probe Q To compound 2.12 (80 mg, 0.051 mmol) in methanol (5 mL) at room temperature was added a catalytic amount of NaOMe. The reaction was allowed to stir at room temperature for 6 h. The solution was filtered and then the volatiles were removed in vacuum to give the compound Probe Q as syrup. Compound Probe Q: MS [M+H] + found: 1233.
- Figure 15 shows confocal laser-scanning fluorescent images of Probe M in HeLa cells.
- Probe M (30 ⁇ , red, shown in Fig. 15A) was incubated with cells in non-FBS DMEM media for 45min and counterstained with MitoTracker (80 nM, green, shown in Fig. 15B), and Hoechst 33342 (1 ⁇ g/mL, blue, shown in Fig. 15C).
- MitoTracker 80 nM, green, shown in Fig. 15B
- Hoechst 33342 (1 ⁇ g/mL, blue, shown in Fig. 15C).
- An overlay of the images shown in Figs. 15A, 15B and 15C is shown in Fig. 15D.
- HeLa cells incubated with compound 1.5 (20 ⁇ , shown in Fig. 15E) in media for 15min, followed by counterstain with LysoTracker (2 ⁇ , shown in Fig.
- Figure 16 shows confocal laser-scanning fluorescent images of Probe N in HeLa cells.
- Probe N (30 ⁇ , red, shown in Fig. 16A) was incubated with cells in non-FBS DMEM media for 45min and counterstained with MitoTracker (80 nM, green, shown in Fig. 16B), and Hoechst 33342 (1 ⁇ g/mL, blue, shown in Fig. 16C).
- MitoTracker 80 nM, green, shown in Fig. 16B
- Hoechst 33342 (1 ⁇ g/mL, blue, shown in Fig. 16C).
- An overlay of the images shown in Figs. 16A, 16B and 16C is shown in Fig. 16D.
- HeLa cells incubated with compound 1.1 1 (20 ⁇ , shown in Fig. 16E) in media for 15min, followed by counterstain with LysoTracker (2 ⁇ , shown in Fig.
- Figure 17 shows confocal laser-scanning fluorescent images of Probe O in HeLa cells.
- Probe O (30 ⁇ , red, shown in Fig. 17A) was incubated with cells in non-FBS DMEM media for 45min and counterstained with MitoTracker (80 nM, green, shown in Fig. 17B), and Hoechst 33342 (1 ⁇ g/mL, blue, shown in Fig. 17C).
- MitoTracker 80 nM, green, shown in Fig. 17B
- Hoechst 33342 (1 ⁇ g/mL, blue, shown in Fig. 17C).
- An overlay of the images shown in Figs. 17A, 17B and 17C is shown in Fig. 17D.
- HeLa cells incubated with compound 2.5 (20 ⁇ , shown in Fig. 17E) in media for 15min, followed by counterstain with LysoTracker (2 ⁇ , shown in Fig.
- Figure 18 shows confocal laser-scanning fluorescent images of Probe P in HeLa cells.
- Probe P (30 ⁇ , red, shown in Fig. 18A) was incubated with cells in non-FBS DMEM media for 45min and counterstained with MitoTracker (80 nM, green, shown in Fig. 18B), and Hoechst 33342 (1 ⁇ g/mL, blue, shown in Fig. 18C).
- MitoTracker 80 nM, green, shown in Fig. 18B
- Hoechst 33342 (1 ⁇ g/mL, blue, shown in Fig. 18C).
- An overlay of the images shown in Figs. 18A, 18B and 178C is shown in Fig. 18D.
- HeLa cells incubated with compound 2.11 (20 ⁇ , shown in Fig. 18E) in media for 15min, followed by counterstain with LysoTracker (2 ⁇ , shown in Fig.
- Figure 19 shows confocal laser-scanning fluorescence images of Probe Q in HeLa cells.
- Probe M (30 ⁇ , red, shown in Fig. 19A) was incubated with HeLa cells in non-FBS DMEM media for 45 min and counterstained with MitoTracker (80 nM, green, shown in Fig. 19B), and Hoechst 33342 (1 ⁇ g/mL, blue, shown in Fig. 19C).
- An overlay of the images shown in Figs. 19A, 19B and 19C is shown in Fig. 19D.
- HeLa cells incubated with Probe M (20 ⁇ , shown in Fig. 19E) in media for 15 min, followed by counterstain with LysoTracker (2 ⁇ , shown in Fig.
Abstract
Described herein are compounds that may selectively stain lysosomes in cells, which may be used in methods of selectively staining and selectively detecting lysosomes in cells. The compounds are fluorescently labelled carbohydrates in which one or two monosaccharides selected from the group mannose, N- acetyl glucosamine, fucose, galactose and sialic acid are linked via a linker to a fluorescent probe such as rhodamine or cyanine or related probes.
Description
LYSOSOMAL TARGETING PROBES
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to United States Provisional Patent Application Nos. 61/684,791 filed on August 19, 2012, 61/704,649 filed on September 24, 2012, and
61/781,312 filed on March 14, 2013, the entire contents of which are hereby incorporated by reference.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with U.S. Government support awarded by the National Aeronautics and Space Administration, Grant No. MSGC R85197, and the National Science Foundation, Grant No. CHE-9512445. The U.S. Government has certain rights in this invention.
BACKGROUND
[0003] Lysosomes are membrane-bound organelles found in mammalian cells. The lysosomal lumen is more acidic (pH 4.0-6.0) than the cytosol (~pH 7.0) and contains different types of proteases, including the capthesins. Lysosomes play a critical role in cellular metabolism, and act as important sites for the degradation of excess organelles, engulfed virus or bacteria, and other foreign materials. The dysfunction of the lysosome has been implicated in many diseases, including inflammation, cancer, neurodegenerative diseases and various lysosomal storage diseases.
[0004] Current probes that are used to stain and detect lysosomes in cells are either designed to target the acidic environment of the lysosome, or to take advantage of the ability of large molecules to enter the cell via endocytosis. For example, the probe DAMP targets acidic environments such as those found in lysosomes, but it is not fluorescent and must be used in conjunction with anti-DNP antibodies conjugated to detection moieties. Fluorophores such as neutral red and acridine orange are also used to stain acidic organelles, but lack specificity. LysoTracker® probes are fluorescent acidotropic compounds that may selectively target acidic organelles, but upon long-term incubation within cells may induce an increase in lysosomal pH, which may lead to fluorescence quenching. Large compounds, such as fluorescently-labeled dextrane and bovine serum albumin, have also been used to target lysosomes, but may have low photostability that limits their use over longer time periods.
[0005] There is a continuing need for fluorescent compounds that can be used to selectively stain and detect lysosomes, particularly over longer periods of time. Such compounds may facilitate understanding of intracellular metabolism, cell membrane recycling, and drug and gene delivery systems.
SUMMARY
[0006] In one aspect, this disclosure provides a compound of formula (I):
A-L-(B)n (I)
wherein:
A is a fluorescent moiety;
L is a linker;
B is a monosaccharide moiety selected from the group consisting of mannose, N- acetyl glucosamine, fucose, galactose and sialic acid; and
n is 1 or 2.
[0007] In another aspect, this disclosure provides a method of selectively staining a lysosome in a cell, comprising contacting the cell with an effective amount of a compound of formula (I).
[0008] In another aspect, this disclosure provides a method of selectively detecting a lysosome in a cell, comprising:
a) contact the cell with an effective amount of a compound of formula (I); and b) detecting a signal from the compound.
[0009] In another aspect, this disclosure provides a method of detecting a cancerous cell in a sample, comprising:
a) contact the sample with an effective amount of a compound of formula (I); and b) detecting a signal from the compound.
[0010] In another aspect, this disclosure provides a kit comprising a compound of formula (I).
[0011] Other aspects and embodiments will become apparent in light of the following disclosure and drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 shows absorbance spectra for compounds of formula (I).
[0013] FIG. 2 shows fluorescence emission spectra for compounds of formula (I).
[0014] FIG. 3 shows confocal laser-scanning fluorescence images of HeLa cells stained with Probe A ((A) and (E)), MitoTracker (B), LysoTracker (F), Hoechst 33342 ((C) and (G)).
(D) is an overlay of (A), (B) and (C) while (H) is an overlay of (E), (F) and (G).
[0015] FIG. 4 shows confocal laser-scanning fluorescence images of HeLa cells stained with Probe B ((A) and (E)), MitoTracker (B), LysoTracker (F), Hoechst 33342 ((C) and (G)).
(D) is an overlay of (A), (B) and (C) while (H) is an overlay of (E), (F) and (G).
[0016] FIG. 5 shows confocal laser-scanning fluorescence images of HeLa cells stained with Probe C ((A) and (E)), MitoTracker (B), LysoTracker (F), Hoechst 33342 ((C) and (G)).
(D) is an overlay of (A), (B) and (C) while (H) is an overlay of (E), (F) and (G).
[0017] FIG. 6 shows confocal laser-scanning fluorescence images of HeLa cells stained with Probe D ((A) and (E)), MitoTracker (B), LysoTracker (F), Hoechst 33342 ((C) and (G)).
(D) is an overlay of (A), (B) and (C) while (H) is an overlay of (E), (F) and (G).
[0018] FIG. 7 shows confocal laser-scanning fluorescence images of HeLa cells stained with Probe E ((A) and (E)), MitoTracker (B), LysoTracker (F), Hoechst 33342 ((C) and (G)).
(D) is an overlay of (A), (B) and (C) while (H) is an overlay of (E), (F) and (G).
[0019] FIG. 8 shows confocal laser-scanning fluorescence images of HeLa cells stained with Probe F ((A) and (E)), MitoTracker (B), LysoTracker (F), Hoechst 33342 ((C) and (G)).
(D) is an overlay of (A), (B) and (C) while (H) is an overlay of (E), (F) and (G).
[0020] FIG. 9 shows confocal laser-scanning fluorescence images of HeLa cells stained with Probe H ((A) and (E)), MitoTracker (B), LysoTracker (F), Hoechst 33342 ((C) and (G)).
(D) is an overlay of (A), (B) and (C) while (H) is an overlay of (E), (F) and (G).
[0021] FIG. 10 shows confocal laser-scanning fluorescence images of HeLa cells stained with Probe I ((A) and (E)), MitoTracker (B), LysoTracker (F), Hoechst 33342 ((C) and (G)).
(D) is an overlay of (A), (B) and (C) while (H) is an overlay of (E), (F) and (G).
[0022] FIG. 1 1 shows confocal laser-scanning fluorescence images of Probes A-F in HeLa cells after 18 hours.
[0023] FIG. 12 shows confocal laser-scanning fluorescence imaging of HeLa cells incubated with Probe B (20 μΜ) in media for 2 hours, following washing, incubation with 1.4 μΜ nigericin for 30 min, and imaging in buffers having pH values of: A) pH 4.4, B) pH 5.0, C) pH 5.5, and D) pH 6.0.
[0024] FIG. 13 shows confocal laser-scanning fluorescence images of Probes J, K and L in HeLa cells.
[0025] FIG. 14 shows confocal laser-scanning fluorescence images of freshly frozen colon tumor tissue-slices from a patient following incubation of Probe A (20 μΜ) (A-C), and Probe B (20 μΜ) (D-F) for 2h, respectively.
[0026] FIG. 15 shows confocal laser-scanning fluorescence images of HeLa cells stained with Probe M ((A) and (E)), MitoTracker (B), LysoTracker (F), Hoechst 33342 ((C) and (G)).
(D) is an overlay of (A), (B) and (C) while (H) is an overlay of (E), (F) and (G).
[0027] FIG. 16 shows confocal laser-scanning fluorescence images of HeLa cells stained with Probe N ((A) and (E)), MitoTracker (B), LysoTracker (F), Hoechst 33342 ((C) and (G)).
(D) is an overlay of (A), (B) and (C) while (H) is an overlay of (E), (F) and (G).
[0028] FIG. 17 shows confocal laser-scanning fluorescence images of HeLa cells stained with Probe O ((A) and (E)), MitoTracker (B), LysoTracker (F), Hoechst 33342 ((C) and (G)).
(D) is an overlay of (A), (B) and (C) while (H) is an overlay of (E), (F) and (G).
[0029] FIG. 18 shows confocal laser-scanning fluorescence images of HeLa cells stained with Probe P ((A) and (E)), MitoTracker (B), LysoTracker (F), Hoechst 33342 ((C) and (G)).
(D) is an overlay of (A), (B) and (C) while (H) is an overlay of (E), (F) and (G).
[0030] FIG. 19 shows confocal laser-scanning fluorescence images of HeLa cells stained with Probe Q ((A) and (E)), MitoTracker (B), LysoTracker (F), Hoechst 33342 ((C) and (G)).
(D) is an overlay of (A), (B) and (C) while (H) is an overlay of (E), (F) and (G).
DETAILED DESCRIPTION
[0031] Described herein are compounds, compositions and methods for selectively targeting, staining and detecting a lysosome. Current compounds that are used to detect lysosomes in cells may lack specificity and/or photostability, or may require multiple steps to use (e.g., incubation with a non-fluorescent compound, followed by an antibody labeled with a detection moiety). The compounds described herein may selectively target lysosomes to allow for selective detection in one step. The compounds described herein include a fluorophore, a linker, and a monosaccharide moiety selected from the group consisting of galactose, mannose, N-acetyl glucosamine, fucose and sialic acid. Compounds described herein are stable and sensitive markers for selectively staining lysosomes, which may enable selective detection of lysosomes over long time periods.
1. Definitions
[0032] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and the appended claims,
the singular forms "a," "and" and "the" include plural references unless the context clearly dictates otherwise.
[0033] Section headings as used in this section and the entire disclosure herein are not intended to be limiting.
[0034] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are explicitly contemplated.
[0035] As used herein, the term "about" is used synonymously with the term
"approximately." Illustratively, the use of the term "about" indicates that values slightly outside the cited values. Variation may be due to conditions such as experimental error, manufacturing tolerances, variations in equilibrium conditions, and the like. In some embodiments, the term "about" includes the cited value plus or minus 10%.
[0036] The term "contacting," as used herein, e.g., as in "contacting a sample" or
"contacting a cell" refers to contacting a sample or cell directly or indirectly, in vitro or ex vivo. Contacting a sample may include addition of a compound to a sample (e.g., a culture of cells).
[0037] The term "effective amount," as used herein, refers to an amount of a compound or a composition effective for eliciting a desired effect. For example, in methods of selectively detecting a lysosome in a cell, an "effective amount" of a compound may be an amount that allows for visualization of a fluorescent signal that is localized to a lysosome, using a method such as fluorescence microscopy.
[0038] The terms "fluorophore," "fluorescent moiety," "fluorescent label" and "fluorescent dye," are used interchangeably herein and refer to a molecule or a portion thereof that absorbs a quantum of electromagnetic radiation at one wavelength, and emits one or more photons at a different, typically longer, wavelength. Numerous fluorescent dyes of a wide variety of structures and characteristics are suitable for use in the compounds of the present disclosure. Suitable fluorophores are described in the Handbook of Fluorescent Probes and Research Chemicals (6th Ed., Molecular Probes, Inc., Eugene Oreg.).
[0039] A "linker," as used herein, refers to an atom or a group of atoms that links a fluorescent moiety to one or more monosaccharide moieties. A divalent linker may link a fluorescent moiety to one monosaccharide moiety (i.e. will provide one point of attachment to the fluorescent moiety and one point of attachment to the monosaccharide moiety). A trivalent linker may link a fluorescent moiety to two monosaccharide moieties (i.e. will
provide one point of attachment to the fluorescent moiety, one point of attachment to a first monosaccharide moiety and one point of attachment to a second monosaccharide moiety).
[0040] The term "member atom," as used herein, refers to a polyvalent atom (e.g., a C, O, N, P or S atom) in a chain or ring system that constitutes a part of the chain or ring. For example, in a triazole ring, two carbon atoms and three nitrogen atoms are member atoms of the ring. In an exemplary linker -(CH^CH^O^-, four carbon atoms and two oxygen atoms are member atoms of the linker. Member atoms will be substituted up to their normal valence. For example, in pyridine, the five carbon atoms will each be further substituted with a hydrogen or another substituent (e.g., an alkyl group).
[0041] The term "monosaccharide moiety," as used herein, refers to a radical of a monosaccharide, where an atom or a group of atoms is removed. The radical serves as a point of attachment to another molecule. For example, a hydrogen atom from a hydroxy group can be removed from a monosaccharide to provide a monosaccharide moiety that is attached to another molecule via an oxygen atom, or a hydroxy radical may be removed to provide a monosaccharide moiety that is attached to another molecule via a carbon atom.
[0042] The term "reactive group," as used herein, refers to a chemical moiety that is part of a first molecule that is capable of reacting with a "complementary group" that is part of a second molecule, to form a covalent bond between the first molecule and the second molecule. A complementary group may also be called a "complementary reactive group". In some embodiments, a reactive group is an electrophilic group and a complementary group is a nucleophilic group. In some embodiments, a reactive group is a nucleophilic group and a complementary group is an electrophilic group. In some embodiments, the reactive group is an azide and the complementary group is an alkynyl group. In some embodiments the reactive group is an alkynyl group and the complementary group is an azide.
[0043] For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March 's Advanced Organic Chemistry, 5tb Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.
[0044] The term "acyl," as used herein, refers to an alkylcarbonyl, cycloalkylcarbonyl, heterocyclylcarbonyl, arylcarbonyl or heteroarylcarbonyl substituent, any of which may be further substituted (e.g., with one or more substituents).
[0045] The term "alkyl," as used herein, refers to a straight or branched saturated hydrocarbon chain. Alkyl groups may include a specified number of carbon atoms. For example, C1-C12 alkyl indicates that the alkyl group may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. An alkyl group may be, e.g., a C1-C12 alkyl group, a C1-C10 alkyl group, a Ci-Cs alkyl group, a C1-C6 alkyl group or a C1-C4 alkyl group. For example, exemplary C1-C4 alkyl groups include methyl, ethyl, w-propyl, isopropyl, w-butyl, sec-butyl, isobutyl and tert- butyl groups. An alkyl group may be optionally substituted with one or more substituents.
[0046] The term "alkenyl," as used herein, refers to a straight or branched hydrocarbon chain having one or more double bonds. Alkenyl groups may include a specified number of carbon atoms. For example, C2-C12 alkenyl indicates that the alkenyl group may have 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 carbon atoms. An alkenyl group may be, e.g., a C2-C12 alkenyl group, a C2-C10 alkenyl group, a C2-C8 alkenyl group, a C2-C6 alkenyl group or a C2-C4 alkenyl group. Examples of alkenyl groups include but are not limited to allyl, propenyl, 2- butenyl, 3-hexenyl and 3-octenyl groups. One of the double bond carbons may optionally be the point of attachment of the alkenyl substituent. An alkenyl group may be optionally substituted with one or more substituents.
[0047] The term "alkynyl," as used herein, refers to a straight or branched hydrocarbon chain having one or more triple bonds. Alkynyl groups may include a specified number of carbon atoms. For example, C2-C12 alkynyl indicates that the alkynyl group may have 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 carbon atoms. An alkynyl group may be, e.g., a C2-C12 alkynyl group, a C2-C10 alkynyl group, a C2-C8 alkynyl group, a C2-C6 alkynyl group or a C2-C4 alkynyl group. Examples of alkynyl groups include but are not limited to ethynyl, propargyl, and 3-hexynyl. One of the triple bond carbons may optionally be the point of attachment of the alkynyl substituent. An alkynyl group may be optionally substituted with one or more substituents.
[0048] The term "aryl," as used herein, refers to an aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring system, wherein any ring atom capable of substitution can be substituted (e.g., with one or more substituents). Examples of aryl moieties include but are not limited to phenyl, naphthyl, and anthracenyl. Aryl groups may be optionally substituted with one or more substituents.
[0049] The term "azide," as used herein, refers to a group of the formula -N3.
[0050] The term "cycloalkyl," as used herein, refers to non-aromatic, saturated or partially unsaturated monocyclic, bicyclic, tricyclic or poly cyclic hydrocarbon groups having 3 to 12 carbons. Any ring atom can be substituted (e.g., with one or more substituents). Cycloalkyl groups can contain fused rings. Fused rings are rings that share one or more common carbon atoms. Examples of cycloalkyl groups include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, methylcyclohexyl, adamantyl, norbornyl, norbornenyl, tetrahydronaphthalenyl and dihydroindenyl. Cycloalkyl groups may be optionally substituted with one or more substituents. "Cycloalkenyl," as used herein, refers to a non-aromatic monocyclic, bicyclic, tricyclic or polycyclic hydrocarbon group having one or more double bonds (e.g., cyclohexenyl or cyclohexadienyl). "Cycloalkynyl," as used herein, refers to a non-aromatic monocyclic, bicyclic, tricyclic or polycyclic hydrocarbon group having one or more triple bonds (e.g., cyclooctynyl), which may be optionally substituted with one or more substituents (e.g., with one or more halo groups, e.g., to generate a difluorcyclooctynyl group).
[0051] The term "halo" or "halogen," as used herein, refers to any radical of fluorine, chlorine, bromine or iodine.
[0052] The term "haloalkyl," as used herein, refers to an alkyl group as defined herein, in which one or more hydrogen atoms are replaced with halogen atoms, and includes alkyl moieties in which all hydrogens have been replaced with halogens (e.g., perfluoroalkyl such as CF3).
[0053] The term "heteroalkyl," as used herein, refers to an alkyl, alkenyl or alkynyl group as defined herein, wherein at least one carbon atom of the alkyl group is replaced with a heteroatom. Heteroalkyl groups may contain from 1 to 18 non-hydrogen atoms (carbon and heteroatoms) in the chain, or 1 to 12 atoms, or 1 to 6 atoms, or 1 to 4 atoms. Heteroalkyl groups may be straight or branched, and saturated or unsaturated. Unsaturated heteroalkyl groups have one or more double bonds and/or one or more triple bonds. Heteroalkyl groups may be unsubstituted or substituted. Exemplary heteroalkyl groups include but are not limited to alkoxyalkyl (e.g., methoxymethyl), and aminoalkyl (e.g., alkylaminoalkyl and dialkylaminoalkyl). Heteroalkyl groups may be optionally substituted with one or more substituents.
[0054] The term "heteroaryl," as used herein, refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms independently selected from O, N, S, P and Si (e.g., carbon atoms and 1-3, 1-6,
or 1-9 heteroatoms independently selected from O, N, S, P and Si if monocyclic, bicyclic, or tricyclic, respectively). Any ring atom can be substituted (e.g., with one or more
substituents). Heteroaryl groups can contain fused rings, which are rings that share one or more common atoms. Examples of heteroaryl groups include, but are not limited, to radicals of pyridine, pyrimidine, pyrazine, pyridazine, pyrrole, imidazole, pyrazole, triazole, oxazole, isoxazole, furan, thiazole, isothiazole, thiophene, quinoline, isoquinoline, quinoxaline, quinazoline, cinnoline, indole, isoindole, indolizine, indazole, benzimidazole, phthalazine, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, phenazine, naphthyridines and purines. Heteroaryl groups may be optionally substituted with one or more substituents.
[0055] The term "heteroatom," as used herein, refers to a non-carbon or hydrogen atom such as a nitrogen, sulfur, oxygen, silicon or phosphorus atom. Groups containing more than one heteroatom may contain different heteroatoms.
[0056] The term "heterocyclyl," as used herein, refers to a nonaromatic, saturated or partially unsaturated 3-10 membered monocyclic, 8-12 membered bicyclic, or 1 1-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, S, Si and P (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, S, Si and P if monocyclic, bicyclic, or tricyclic, respectively). Any ring atom may be substituted (e.g., with one or more substituents). Heterocyclyl groups can contain fused rings, which are rings that share one or more common atoms. Examples of heterocyclyl groups include, but are not limited to, radicals of tetrahydro furan, tetrahydrothiophene, tetrahydropyran, oxetane, piperidine, piperazine, morpholine, pyrroline, pyrimidine, pyrrolidine, indoline, tetrahydropyridine, dihydropyran, thianthrene, pyran, benzopyran, xanthene, phenoxathiin, phenothiazine, furazan, lactones, lactams such as azetidinones and pyrrolidinones, sultams, sultones, and the like. Heterocyclyl groups may be optionally substituted with one or more substituents.
[0057] The term "hydroxy," as used herein, refers to an -OH radical. The term "alkoxy," as used herein, refers to an -O-alkyl radical. The term "aryloxy," as used herein, refers to an -O- aryl radical.
[0058] The term "oxo," as used herein, refers to an oxygen atom, which forms a carbonyl when attached to carbon, an N-oxide when attached to nitrogen, and a sulfoxide or sulfone when attached to sulfur (i.e. =0).
[0059] The terms "mercapto" or "thiol,"as used herein, each refer to an -SH radical. The terms "thioalkoxy" or "thioether," as used herein, each refer to an -S-alkyl radical. The term "thioaryloxy," as used herein, refers to an -S-aryl radical.
[0060] The term "substituents," as used herein, refers to a group "substituted" on an alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl group at any atom of that group. Any atom can be substituted. Suitable substituents include, without limitation: acyl, acylamido, acyloxy, alkoxy, alkyl, alkenyl, alkynyl, amido, amino, carboxy, cyano, ester, halo, hydroxy, imino, nitro, oxo (e.g., C=0), phosphonate, sulfinyl, sulfonyl, sulfonate, sulfonamino, sulfonamido, thioamido, thiol, thioxo (e.g., C=S), and ureido. In some embodiments, substituents on a group are independently any one single, or any combination of the aforementioned substituents. In some embodiments, a substituent may itself be substituted with any one of the above substituents.
[0061] The above substituents may be abbreviated herein. For example, the abbreviations Me, Et, Ph and Bn represent methyl, ethyl, phenyl and benzyl, respectively. A more comprehensive list of standard abbreviations used by organic chemists appears in a table entitled Standard List of Abbreviations of the Journal of Organic Chemistry. The abbreviations contained in this list are hereby incorporated by reference.
[0062] For compounds described herein, groups and substituents thereof may be selected in accordance with permitted valence of the atoms and the substituents, and such that the selections and substitutions result in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
[0063] Where substituent groups are specified by their conventional chemical formulae, written from left to right, they optionally encompass substituents resulting from writing the structure from right to left, e.g., -CH20- optionally also recites -OCH2-.
is used in structural formulae herein to depict the bond that is the point of attachment of the moiety or substituent to the core or backbone structure.
2. Compounds
[0065] Compounds that may be used in the compositions and methods described herein include those having the following formula (I):
A-L-(B)n (I)
wherein:
A is a fluorescent moiety;
L is a linker;
B is a monosaccharide moiety selected from the group consisting of mannose, N- acetyl glucosamine, fucose, galactose and sialic acid; and
n is 1 or 2.
a. Fluorescent moieties
[0066] In the compounds of formula (I), A is a fluorescent moiety. Suitable fluorescent moieties include rhodamines, fluoresceins, coumarins, cyanines, and boron-dipyrromethenes (also known as BODIPYs), as well as derivatives thereof. For example, known rhodamine derivatives include amine-conjugated rhodamine compounds.
[0067] A fluorescent moiety may be incorporated into a compound of formula (I), for example, by using a reagent that comprises a fluorophore and a reactive group such as a carboxylic acid, an isothiocyanate, a maleimide, an alkynyl group, an azide, an amine, a thiol, or an ester such as a succinimidyl, sulfodichlorophenol, pentafluorophenyl or
tetrafluorophenyl ester. Such groups may react with a complementary group, such as one present on a linker precursor compound, to attach the fluorophore to the remainder of the molecule of formula (I). Reagents comprising fluorophores and reactive groups may be commercially available, or may be synthesized according to methods described herein or other methods known to those skilled in the art.
[0068] Suitable reagents comprising fluorophores, which may be used to prepare compounds of formula (I), are known in the art. For example, reagents comprising fluorophores that are commercially available include, but are not limited to: 5- and 6- carboxyfluoresceins and esters thereof; fluorescein-5 -isothiocyanate and fluorescein-6- isothiocyanate; BODIPY® dyes commercially available from Life Technologies™, such as BODIPY® succinimidyl esters; Alexa Fluor® dyes commercially available from Life Technologies™, such as Alexa Fluor® succinimidyl, tetrafluorphenyl and
sulfodichlorophenol esters; CyDye fluors commercially available from GE Healthcare Biosciences, such as CyDye succinimidyl esters and maleimides; and VivoTag™
fluorophores available from PerkinElmer, such as VivoTag™ succinimidyl esters and maleimides.
[0069] It will be understood by the skilled artisan that the fluorescent moiety may include the fluorophore itself, as well as additional atoms or groups of atoms, such as atoms or groups
of atoms derived from reactive groups or complementary groups that serve to link the fluorescent moiety to the remainder of the compound of formula (I).
[0070] Certain fluorescent moieties can be selected for incorporation into compounds of formula (I), based on the particular application of interest. For example, fluorophores having particular excitation/emission profiles may be selected, and may, for example, be orthogonal to other fluorophores being used in a particular application. In an exemplary embodiment, certain live-cell imaging experiments may be conducted using DAPI as a nuclear stain, which has an absorption maximum of about 350 nm and an emission maximum of about 460 nm. For applications employing DAPI stain, a fluorescent moiety for a compound of formula (I) may be selected to have excitation and emission properties that are different from those of the DAPI stain. For example, rhodamine B has an absorption maximum of about 540 nm and an emission maximum of about 625 nm. (As those skilled in the art appreciate, these absorption and emission values are approximate and depend on the particular environment including solvent, pH, etc.)
:
[0072] In the compounds of formula (I), L is a linker. As used herein, the term "linker" refers to an atom or a group of atoms that link a fluorescent moiety (A-) to one or more monosaccharide moieties (-B). A divalent linker allows for linkage of one -B moiety, while a trivalent linker allows for linkage of two -B moieties.
[0073] Many linkers are known in the art. For example, a linker may comprise one or more groups selected from -CH2-, -CH=, -C≡, -NH-, -N=, 0-, -S-, -C(O)-, -C(S)-, -S(O)-, -S(0)2-, or any combination thereof. Any of the H atoms may be replaced by a suitable substituent, such as an alkyl group. It will be appreciated that a linker comprising more than one of the above groups will be selected such that the linker is stable; for example, a linker may not include two adjacent -O- groups, which would generate an unstable peroxide linkage. A
linker may be a straight chain, a branched chain, or may include one or more ring systems. Nonlimiting exemplary linkers include polyethylene glycol linkers and triazole-containing linkers, and linkers including both ethylene glycol units and triazole rings.
[0074] A linker may include from 1 to about 50 member atoms, not including substituents. For example, a linker may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 atoms, or any range therebetween.
[0075] Illustrative linkers include, but are not limited to -(CH2)c-De-(CH2)f- and -(CH2)P- Mr-C(0)-Ks-(CH2)q- where c is 0 to 8; D is O, NH, or S; e is 0 or 1; f is 0 to 8; p is 0 to 8; M is NH or O; K is NH or O; q is 0 to 8, and r and s are each independently 0 or 1. Other illustrative linker groups include, but are not limited to, -0-, -S-,-ΝΗ-, -N(alkyl)-, -C(O)-, - S(O)-, -S(0)2-, -S(0)2-NH-, -CH=CH-, -OCH=CH-, -C(0)-NH-, -NH-C(0)-NH-, -NH-C(S)- NH-, -0-C(0)-NH-, -C(S)-, -CH2-, -CH2-CH2-, -CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-, -O- CH2-, -0-CH2-CH2-, -CH2-0-CH2-, -0-CH2-CH2-CH2-, -CH2-0-CH2-CH2-, -0-CH2-CH2- CH2-CH2-, -CH2-0-CH2-CH2-CH2-, -CH2-CH2-0-CH2-CH2-, -S-CH2-, -S-CH2-CH2-, -CH2- S-CH2-, -S-CH2-CH2-CH2-, -CH2-S-CH2-CH2-, -S-CH2-CH2-CH2-CH2-, -CH2-S-CH2-CH2- CH2-, -CH2-CH2-S-CH2-CH2-, -C(0)-NH-CH2-, -C(0)-NH-CH2-CH2-, -CH2-C(0)-NH-CH2-, -C(0)-NH-CH2-CH2-CH2-, -CH2-C(0)-NH-CH2-CH2-, -C(0)-NH-CH2-CH2-CH2-CH2-, - CH2-C(0)-NH-CH2-CH2-CH2-, -CH2-CH2-C(0)-NH-CH2-CH2-, -CH2-CH2-CH2-C(0)-NH- CH2-CH2-, -CH2-CH2-CH2-CH2-C(0)-NH-, -NH-C(0)-CH2-C(0)-NH-, -NH-C(0)-CH2- CH2-C(0)-NH-, -NH-C(0)-CH2-CH2-CH2-C(0)-NH-, -NH-C(0)-CH2-CH2-CH2-CH2-C(0)- NH-, -NH-C(0)-CH=CH-C(0)-NH-C(0)-0-CH2-, -CH2-C(0)-0-CH2-, -CH2-CH2-C(0)-0- CH2-, -C(0)-0-CH2-CH2-, -NH-C(0)-CH2-, -NH-C(0)-CH2-CH2-, -CH2-NH-C(0)-CH2- CH2-, -0-C(0)-NH-CH2-, -0-C(0)-NH-CH2-CH2-, -NH-CH2-, -NH-CH2-CH2-, -CH2-NH- CH2-, -CH2-CH2-NH-CH2-, -C(0)-CH2-, -C(0)-CH2-CH2-, -CH2-C(0)-CH2-, -CH2-CH2- C(0)-CH2-, -CH2-CH2-C(0)-CH2-CH2-, -CH2-CH2-CH2-C(0)-NH-CH2-CH2-NH-, -CH2- CH2-CH2-C(0)-NH-CH2-CH2-NH-C(0)-, -CH2-CH2-CH2-C(0)-NH-CH2-CH2-NH-C(0)- CH2-, and -CH2-CH2-CH2-C(0)-NH-CH2-CH2-NH-C(0)-CH2-CH2-, or any combination thereof.
[0076] Illustrative linkers also include those having ring structures, such as aryl, heteroaryl, cycloalkyl or heterocyclyl rings. For example, linkers may include a heteroaryl ring, such as a triazole. In exemplary linkers, a linker may include a 1,2, 3 -triazole, which may be a product of the reaction between a reagent comprising a fluorophore and an alkyne, and a molecule comprising an azide. Alternatively, a 1,2,3-triazole may be a product of the
reaction between a reagent comprising a fluorophore and an azide, and a molecule comprising an alkynyl group. Exemplary linkers comprising ring structures, such as 1 ,2,3- triazole groups, include the followin
[0077] Illustrative linkers also include those having both triazole rings and other groups, such as ethylene glycol units, e.g., one or more units having the formula -(CH2CH2O), and amide units (-C(O)NH-). Exemplary linkers including such units include the following:
[0078] In some embodiments, the linker may be hydrolytically stable.
[0079] A skilled artisan will appreciate that a wide variety of linkers can be used to link the fluorescent moiety and the monosaccharide moiety in the compounds of formula (I).
c. Monosaccharide Moieties
[0080] In the compounds of formula (I), B is a monosaccharide moiety derived from a monosaccharide selected from the group consisting of galactose (Gal), mannose (Man), N- acetylglucosamine (GlcNAc), fucose (Fuc) and sialic acid (also known as N- acetylneuraminic acid, NeuAc). It will be understood by those skilled in the art that the monosaccharide moiety will be based on a monosaccharide selected from Gal, Man, GlcNAc, Fuc and NeuAc, where one or more atoms of the monosaccharide is replaced to provide a point of attachment to the remainder of the compound of formula (I). For example, suitable monosaccharide moieties include, but are not limited to, the following:
[0081] A monosaccharide moiety may be linked to the compound of formula (I) by replacing any suitable atom or group of atoms of a monosaccharide with the remainder of the compound of formula (I). For example, a 1 -hydroxy group, 2-hydroxy group, or a 6-hydroxy
group may be replaced to yield a deoxymonosaccharide radical, where the radical serves as the point of attachment to the remainder of the compound of formula (I),
d. Compound Forms
[0082] It will be understood by those skilled in the art that compounds described herein may be present in multiple forms. When a compound described herein is illustrated in one form, it is expressly understood that this reference includes all forms of the compound such as tautomeric forms, prototropic forms, salt forms, and the like.
[0083] For example, it is well appreciated that fluorescein may exist in neutral, monoanionic, dianionic and lactone forms, as illustrated below. Compounds described herein that include fluorescein moieties encompass all of these forms and mixtures thereof
monoanionic dianionic lactone
[0084] For example, it is also well appreciated that rhodamine B may exist in cationic, zwitterionic and lactone forms, as illustrated below. Compounds described herein that include rhodamine moieties encompass all of these forms and mixtures thereof
cationic zwitterionic lactone
[0085] When compounds described herein bear one or more charges, it will be understood that they will also include one or more associated anions or cations to balance the charges. For example, if the compound is anionic, or has a functional group which may be anionic (e.g., -COOH may be -COO"), then a salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na+ and K+, alkaline earth cations such as Ca2+ and Mg2+, and other cations. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH4 +) and substituted ammonium ions (e.g., NH3R1 , NH2R2 , NHR3 , NR4 ). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine,
dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine,
diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine.
[0086] If the compound is cationic, or has a functional group that may be cationic (e.g., - H2 may be -ΝΙ¾+), then a salt may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous.
[0087] Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric.
e. Preparation of Compounds
[0088] Compounds described herein may be prepared according to a variety of methods. A general representative synthesis of exemplary compounds of formula (I) in which the fluorophore is based on rhodamine B is illustrated in Scheme 1.
Scheme 1. Exemplary synthesis
[0089] In Scheme 1, RG and RG' are independently reactive groups, and CG and CG' are independently complementary groups that react with RG and RG' respectively. B represents a monosaccharide moiety. L1, L2, L3, L4 and L5 are independently linkers, which may be the
same or different. For example, L2 may be a linker comprising residual atoms from the reaction of RG and CG. For example, if L1 is -CH2-, RG is -C≡CH and CG is -N3, then L2 would have the following formula:
As those skilled in the art appreciate, reactions between alkynyl groups and azide groups form 1,2,3-triazole moieties in a reaction known as a "click" reaction.
[0090] Similarly, L4 may include atoms derived from L1, residual atoms from the reaction of RG and CG, and atoms derived from L3. Further methods of preparing compounds described herein are shown in the Examples.
[0091] As can be appreciated by the skilled artisan, further methods of synthesizing the compounds of the formulae herein will be evident to those of ordinary skill in the art.
Additionally, the various synthetic steps may be performed in an alternate sequence or order to give the desired compounds. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds described herein are known in the art and include, for example, those that are described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents or Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.
e. Evaluation of Compounds
[0092] Compounds can be evaluated using a number of methods. Following structural characterization using methods known in the art, such as nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS), the absorbance and emission spectra can be evaluated. Absorbance spectra can be determined using an ultraviolet-visible (UV-vis) spectrometer, and fluorescence emission spectra can be obtained using a fluorometer. The absorbance and emission spectra can further be evaluated as a function of pH. For example, certain compounds may be fluorescent under acidic conditions, but the fluorescence may be diminished or nearly eliminated under basic conditions.
[0093] If compounds possess suitable absorbance and emission characteristics, they may be further evaluated in cells. Compounds may be incubated with cultured living cells for a period of time, followed by washing to remove excess extracellular compounds.
Examination using standard fluorescence microscopy or confocal laser scanning fluorescence microscopy, can indicate the intracellular localization of the compounds. The localization can be confirmed using compounds that are known to localize to particular organelles. For example, LysoTracker® and MitoTracker® probes are commercially available from Life Technologies™, and localize to the lysosome and mitochondria, respectively. Nuclei may be stained using compounds such as 4',6-diamidino-2-phenylindole (DAP I) or a Hoechst stain (e.g., Hoechst 33258, Hoechst 33342, or Hoechst 34580). Bright-field examination can be used to evaluate cellular viability following incubation of the cells with the compound.
[0094] Further description of methods used to evaluate compounds can be found in the Examples.
3. Methods of use
[0095] Compounds of formula (I) may be used in a variety of methods, such as methods of selectively staining or detecting a lysosome in a cell, or methods of detecting a cancerous cell in a sample.
a. Staining and Detecting Lysosomes
[0096] Lysosomes include membrane proteins that are highly glycosylated, including glycans such as N-linked glycans. Such glycans include monosaccharides, including Gal, Man, GlcNAc, Fuc and NeuAc. While not wishing to be limited by theory, the presence of the monosaccharide moieties on the compounds of formula (I) may promote their uptake into lysosomes.
[0097] In some aspects, this disclosure provides a method of selectively staining a lysosome in a cell, comprising contacting the cell with an effective amount of a compound of formula (I). The cell may be, for example, in a culture of cells. The compound of formula (I) may selectively localize to an acidic organelle such as a lysosome, thereby staining the lysosome.
[0098] In another aspect, the disclosure provides a method of selectively detecting a lysosome in a cell, comprising contacting the cell with an effective amount of a compound of formula (I), and detecting a signal from the compound. The signal may be a fluorescence signal, which may be detected using a variety of instruments such as fluorescence microscope, a flow cytometer, a fluorometer, a fluorescence plate reader, or any combination thereof. The cell may be, for example, in a culture of cells. The compound of formula (I) may selectively localize to an organelle such as a lysosome, thereby allowing for selective detection of a lysosome. The method may further include a step of washing the cells prior to detection, to remove any free compound of formula (I). In some embodiments, the cells may not be fixed prior to detection, in order to prevent cell-fixation artifacts.
[0099] When used in living cells, compounds of formula (I) may have increased stability compared to current probes. For example, known lysosomal targeting probes may only provide stable fluorescence signals for periods of time up to about 1 hour to about 2 hours. The probes may decompose in the acidic environment of the lysosome, or may leak out of the lysosome or the cell. By contrast, compounds of formula (I) may provide a stable fluorescence signal in living cells for at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, or about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours. Such properties may allow for monitoring of lysosomal morphology and trafficking in intact cells.
[00100] In the methods described herein, experiments may be performed in any suitable cell or cell line of interest.
b. Detecting Cancerous Cells
[00101] It is well known that tumor pH plays a significant role in the response to cancer intervention. For example, it has been reported that cancer cells produce the bulk of their energy requirement through glycolysis, which generates considerable lactic acid and a lowered pH associated with cancerous tissue. Additionally, increased acidity reduces oxygen content in tissue, resulting in oxygen-poor, acid-rich environment in which malignant cells thrive with a heightened resistance to therapeutic interventions. Compounds of formula (I) that are sensitive to pH (i.e., provide a fluorescence signal under acidic conditions) may therefore be useful in detecting a cancerous cell in a sample.
[00102] Furthermore, in comparison to non-neoplastic cells, glycosis is enhanced in cancer cells, which results in an upregulation of the galactose transporter. Accordingly, compounds of formula (I) in which B is a galactose moiety could be employed as substrates in cancer cells to donate galactose residues for synthesis of glycosylated lysosomal proteins. Such compounds may therefore be capable of selective accumulation in tumors.
[00103] Accordingly, in an aspect, the disclosure provides a method of selectively detecting a cancerous cell in a sample, comprising contacting the sample with an effective amount of a compound of formula (I), and detecting a signal from the compound. In some embodiments, the sample may be an in vitro sample, such as a cell or tissue extract. In some embodiments,
the sample is a cell culture. For example, the sample may be a culture of cells such as cancer cells.
[00104] In some embodiments, the sample may be a biological sample from a subject, such as a human. In some embodiments, the biological sample is selected from the group consisting of a tissue sample, bodily fluid, whole blood, plasma, serum, urine,
bronchoalveolar lavage fluid, and a cell culture suspension or fraction thereof. In embodiments in which a cancerous cell is detected in a biological sample from a subject, the methods may further involve providing or obtaining a biological sample from the subject, which can be obtained by any known means including needle stick, needle biopsy, swab, and the like.
[00105] A signal from the fluorescent moiety may be quantitated, for example, by comparing the quantity of the signal to that of a reference sample. A cancerous cell may be detected if a fluorescence signal from the cell is higher than that of a signal from a reference cell. In some embodiments, the cancer may be any type of cancer, such as a cancer recognized by the National Cancer Institute.
4. Kits
[00106] In some aspects, this disclosure provides a kit, which may be used for selectively staining or detecting a lysosome in a cell, or for detecting a cancerous cell in a sample.
[00107] A kit will include a compound of formula (I) as described herein. A kit may also include instructions for use of the compound of formula (I). Instructions included in kits can be affixed to packaging material or can be included as a package insert. While the instructions are typically written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD, DVD), and the like. As used herein, the term "instructions" includes the address of an internet site that provides the instructions.
[00108] For example, the kit may comprise instructions for selectively detecting a lysosome in a cell by fluorescence detection, e.g., using a fluorescence microscope, a flow cytometer, a fluorometer, a fluorescence plate reader, or a combination thereof. The kit may further comprise a calibrator or control, and/or at least one container (e.g., a tube, a microtiter plate and/or a strip) for conducting the assay, and/or a buffer, such as an assay buffer or a wash buffer, either one of which can be provided as a concentrated solution. Suitably, the kit comprises all components, i.e., reagents, standards, buffers, diluents, etc., which are
necessary for conducting a particular experiment. The instructions also may include instructions for generating a standard curve or a reference standard for purposes of quantification.
[00109] The kit also may optionally include other reagents required to conduct an assay or facilitate quality control evaluations, such as buffers, salts, enzymes, enzyme co-factors, substrates, detection reagents, and the like. Other components, such as buffers and solutions for the isolation and/or treatment of a test sample (e.g., pretreatment reagents), also may be included in the kit. The kit additionally may include one or more other controls. One or more of the components of the kit may be lyophilized, in which case the kit further may comprise reagents suitable for the reconstitution of the lyophilized components.
[00110] The various components of the kit optionally are provided in suitable containers as necessary, e.g., a microtiter plate. The kit further may include containers for holding or storing a sample (e.g., a container or cartridge for a sample). Where appropriate, the kit optionally also may contain reaction vessels, mixing vessels, and other components that facilitate the preparation of reagents or the test sample. The kit also may include one or more instrument for assisting with handling a sample, such as a syringe, pipette, or the like.
[00111] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the compounds and methods of the present disclosure described herein are readily applicable and appreciable, and may be made using suitable equivalents without departing from the scope of the present disclosure or the aspects and embodiments disclosed herein. Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples, which are merely intended only to illustrate some aspects and embodiments of the disclosure, and should not be viewed as limiting to the scope of the disclosure. The disclosures of all journal references, U.S. patents and publications referred to herein are hereby incorporated by reference in their entireties.
Example 1
Synthesis of Compounds
General experimental and analytical details
[00112] Reagents and solvents available from commercial sources were used as received, unless otherwise noted. Thin layer chromatography (TLC) was performed using Sigma- Aldrich TLC plates, silica gel 60F-254 over glass support, 0.25 μιη thickness. Flash column
chromatography was performed using Alfa Aesar silica gel, particle size 230-400 mesh. XH and 13C NMR spectra were measured by using a Varian UNITY INOVA instrument at 400 MHz and 100 MHz respectively. The chemical shifts (δ) were reported in reference to solvent peaks (residue CHC13 at δ 7.24 ppm for ¾ and CDC13 at δ 77.00 ppm for 13C). High- resolution mass spectra (HR-MS) were obtained on a JEOL JMS HX 110A mass
spectrometer.
3',6'-bis(diethylamino)-2-(prop-2-ynyl)spiro[isoindoline-l,9,-xanthen]-3-one (2)
[00113] To a solution of rhodamine B (5.76g, 12mmol) in 20mL anhydrous dichloromethane was sequentially added 2-(lH-Benzotriazol-l-yl)-l, l,3,3-tetramethyluronium hexafluoro- phosphate, HBTU) (5.45g, 14.4mmol), propargylamine (0.925mL, 14.4 mmol) and tri- ethylamine (2.5mL, 14.4mmol). The reaction mixture was stirred at room temperature overnight until TLC indicated that the starting material had substantially disappeared. The reaction mixture was diluted with dichloromethane and then washed with brine. The combined organic layers were dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. The crude residue was purified by flash column chromatography on silica gel (Hexane:EtOAc, 4: 1) to yield compound 2 as light pink solid (4.25g, 8.85mmol) in 74 % yield. 'H NMR (400 MHz, CDC13) δ ppm 1.14 (t, J= 7.2, 12 H), 1.74 (t, J= 2.4, 1H), 3.32 (q, J= 7.2 Hz, 8H), 3.93 (d, J= 2.4 Hz, 2H), 6.25 (dd, J= 2.8, 8.8, 2H), 6.37 (d, J= 2.8 Hz, 2H), 6.45 (d, J= 8.8 Hz, 2H), 7.10 (m, 1H), 7.41 (m, 2H), 7.91 (m, 1H). 13C NMR (100 MHz, CDC13) δ ppm 12.5, 28.5, 44.3, 64.8, 70.0, 78.2, 97.8, 105.1, 108.0, 123.0, 123.7, 127.9, 129.1, 130.4, 132.6, 148.8, 153.4, 153.7, 167.4.
2-azidoethyl 4-methylbenzenesulfonate (3)
N3^0TS
3
[00114] To a stirring solution of bromoethanol (12.5g, lOOmmol) in 30 mL water was added sodium azide (7.8g, 120mmol). The reaction mixture was gently refluxed with stirring for 16 hours and TLC indicated that the starting material was completely consumed. The reaction mixture was extracted with dichloromethane (25 mLx3). The combined organic layers were
dried over anhydrous a2S04, filtered and concentrated. The crude residue was directly used for the next reaction step without further purification. To a solution of 2-azidoethanol in dichloromethane (50mL) was added triethylamine (20mL, 140mmol), and toluenesulfonyl chloride (19.6g, lOOmmol). The reaction mixture was stirred at room temperature for 4h. The solution was washed with IN NaOH (100mLx2) and dried over anhydrous a2S04, filtered and concentrated. The crude residue was purified by flash column chromatography on silica gel (Hexane:EtOAc, 5: 1) to yield the title compound as colorless oil (19.2g, 80mmol, 80 % yield). XH NMR (400 MHz, CDC13) δ ppm 2.43 (s, 3H), 3.45 (t, 2H, J= 4.8 Hz), 4.13 (t, 2H, J= 4.8 Hz), 7.35-7.33 (d, 2H, J= 8.0 Hz), 7.80-7.78 (d, 2H, J= 8.0 Hz). 13C NMR (100 Mhz, CDC13) δ ppm 21.6, 49.5, 68.0, 127.9, 129.9, 132.5, 145.2.
2-(4-((3',6'-bis(diethylamino)-3-oxospiro[isoindoline-l, 9'-xanthene]-2-yl)methyl)-lH-l, 2, 3-triazol-l-yl)ethyl 4-methylbenzenesulfonate (4)
[00115] To a stirring solution of rhodamine alkyne derivative 2 (2g, 4.16mmol) and 2- azidoethyl 4-methylbenzene-sulfonate (3, 1.8g, 5mmol) in 6mL of tert-butanol: water (1 : 1) was added copper (II) sulfate (0.21mmol, 40mg) and sodium ascorbate (0.42 mmol, 82mg). Stirring of the reaction mixture was continued at room temperature for 6h and TLC indicated that the starting material disappeared. The reaction was washed with saturated aq NaHC03 and then extracted with ethyl acetate (3 x 30mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated. The crude residue was purified by flash column chromatography on silica gel (Hexane:EtOAc, 1 :4) to yield the title compound as colorless foam (2.48g) in 84.5% yield. XH NMR (400 MHz, CDC13) δ ppm 1.09 (t, J= 7.0 Hz, 12H), 2.32 (s, 3H), 3.26 (q, J= 7.0 Hz, 8H), 4.21 (t, J= 5.0 Hz, 2H), 4.29 (t, J= 5.0 Hz, 2H), 4.37 (s, 1H), 6.12 (dd, J= 8.8, 2.0 Hz, 2H), 6.24 (d, J= 8.8 Hz, 2H), 6.31 (d, J= 2.0 Hz, 2H), 6.84 (s, 1H), 7.10-6.94 (m, 1H), 7.21 (d, J= 8.5 Hz, 2H), 7.47-7.29 (m, 2H), 7.65-7.46 (m, 2H), 8.09-7.73 (m, 1H). 13C NMR (100 MHz, CDCI3) δ ppm 12.81, 21.84,35.38, 44.53, 48.60, 65.19, 67.48, 76,92, 98.01, 105.50, 108.13, 123.09, 123.40, 124.14, 127.98, 128.32, 129.03, 130.26, 131.19, 132.23, 132.79, 144.76, 145.64, 148.95, 153.61, 153.64, 168.03.
2-((l-(2-azidoethyl)-lH-l, 2, 3-triazol-4-yl) methyl)-3', 6'-bis(diethylamino) spiro- [isoindoline-1, 9'-xanthen]-3-one (5)
[00116] To a stirring solution of compound 4 (3.29g, 4.65mmol) in DMF (20ml) was added a 3 (453mg, 6.97mmol). The reaction mixture was gently refluxed for 6h. The reaction was washed with saturated aq aHC03 and extracted with EtOAc (30mL x 3). The combined organic layers were dried over anhydrous Na2S04, filtered and concentrated. The crude residue was purified by flash column chromatography on silica gel (Hexane/EtOAc, 1/3) to yield compound 5 as white foam (95.7%, 2.6g). XH NMR (400 MHz, CDC13) δ ppm 1.14 (t, J = 7.2 Hz, 12H), 3.30 (q, J= 7.2 Hz, 8H), 3.60 (t, J= 6.1 Hz, 2H), 4.20 (t, J= 6.1 Hz, 2H), 4.47 (s, 2H), 6.15 (dd, J= 8.8, J=2.3 Hz, 2H), 6.30 (d, J= 8.8 Hz, 2H), 6.33 (s, 2H), 7.06 (s, 1H), 7.18-7.06 (m, 1H), 7.60-7.36 (m, 2H), 7.90-7.94 (m, 1H). 13C MR (100 MHz, CDC13) 5 ppm 12.8, 35.5, 44.5, 48.9, 50.6, 65.2, 98.1, 105.6, 108.0, 123.1, 123.2, 124.1, 128.3, 129.0, 131.2, 132.8, 144.9, 148.9, 153.6, 153.7, 168.1. HR/MS [M+H]+ calcd 592.3148 found 592.3160.
3', 6'-bis(diethylamino)-2-((l-(2-(4-((diprop-2-ynylamino) methyl)-lH-l, 2, 3-triazol-l- yl) ethyl)-lH-l,2,3-triazol-4-yl) methyl) spiro[isoindoline-l, 9'-xanthen]-3-one (6)
[00117] To a stirring solution of 5 (4g, 6.92mmol) and tripropargylamine (1.8g, 13.8mmol) in 6mL of t-BuOH: water (1: 1) was added CuS04 (5%mol, 60mg) and sodium ascorbate (10%mol, 160mg). The reaction mixture was stirred at room temperature for 6h. The reaction mixture was washed with saturated aq. aHC03 and then extracted with EtOAc (30mL x 3). The combined organic layers were dried over anhydrous Na2S04, filtered and concentrated. The crude residue was purified by flash column chromatography on silica gel
(EtOAc:MeOH, 20: 1) to yield 6 as white foam (28%, 1.4g). H NMR (400 MHz, CDC13) δ ppm 1.11 (t, J = 7.0 Hz, 12H), 2.18 (q, J = 2.2 Hz, 2H), 3.27 (q, J= 7.0 Hz, 8H), 3.33 (d, J =
2.2 Hz, 4H), 3.71 (s, 2H), 4.37 (s, 2H), 4.52 (t, J= 6.0 Hz, 2H), 4.68 (t, J= 6.0 Hz, 2H), 6.13 (dd, J= 8.9, J=2.4 Hz, 2H), 6.24 (d, J= 8.9 Hz, 2H), 6.30 (s, 2H), 6.62 (s, 1H), 7.03-7.06 ( m, 1H), 7.15 (s, 1H), 7.65-7.34 (m, 2H), 7.84-7.991 (m, 1H). 13C MR (100 MHz, CDC13) δ ppm 12.7, 35.2, 42.1, 44.5, 47.8, 49.4, 49.5, 65.1, 73.7, 78.7, 97.9, 105.6, 108.1, 123.1, 123.4, 124.1, 124.2, 128.4, 129.1, 131.7, 132.8, 144.8, 144.9, 148.9, 153.4, 153.6, 168.0. HR/MS [M+H]+ calcd 723.3883 found 723.3911.
p-Toluenesulfonyl-l,2:3,4-di-0-isopropyliden-D-galactopyranose (7)
7
[00118] l,2:3,4-Di-0-isopropyliden-D-galactopyranose (1.8g, 70 mmol) were dissolved in pyridine(anhydrous, 8ml) and DCM (anhydrous, 4 ml) under nitrogen gas (oxygen free). Then p-toluenesulfonyl chloride (2.8 g, 147 mmol), which was dissolved in DCM
(anhydrous, 6 ml), was added to a round bottom flask dropwise. Finally, DMAP (a catalytic amount) was added, and the resulting reaction mixture was refluxed overnight. After that, water (1 mL) and toluene were added to the reaction mixture and evaporated to remove excess pyridine. Then, the resulting residue was dissolved in DCM and extracted with saturated aqueous NaHC03. The organic layer was dried over Na2S04, and then filtered and concentrated with an evaporator. The resulting composition was purified by 1 :3 EtOAc: Hexane (2.27g, 78.5% yield). XH NMR (400 MHz, CDC13) δ ppm 1.25 (s, 3H), 1.29 (s, 3H), 1.32 (s, 3H), 1.47 (s, 3H), 2.42 (s, 3H), 4.06 ( m, 2H), 4.18 ( m, 2H), 4.27 (m, 1H), 4.56 (m, 1H), 5.43 (d, J = 4.87 Hz, 1H), 7.30 (d, J = 8.58 Hz, 2H), 7.78 (dd, J = 8.26 Hz, J =1.32, 2H).
6-Azido-6-deoxy-l,2:3,4-di-0-isopropylidene-a-D-galactopyranose (8)
7 8
[00119] To a stirring solution of compound 7 (2.27g, 55 mmol) in DMF (anyhdrous, 20ml), NaN3 (712 mg, 1 11 mmol) was added and the resulting reaction mixture was refluxed under nitrogen atmosphere for 18h. The reaction mixture was extracted with NaHC03 and DCM.
Later, the organic layer was separated and dried over a2S04. The reaction mixture was isolated (lg, 64% yield) with EtOAc:Hex (1 :4). The product was a light yellow syrup that solidified upon waiting. XH NMR (400 MHz, CDC13) δ ppm 1.29 (d, J = 2.27 Hz, 6H), 1.41 (s, 3H), 1.50 (s, 3H), 3.31 (dd, J = 12.71, 5.28 Hz, 1H), 3.46 (dd, J = 12.70, 7.90 Hz, 1H), 3.86 (ddd, J = 7.34, 5.28, 1.92 Hz, 1H), 4.14 (dd, J = 7.88, 1.92 Hz, 1H), 4.29 (ddd, J = 5.03, 2.49, 0.74 Hz, 1H), 4.58 (dd, J = 7.88, 2.46 Hz, 1H), 5.50 (d, J = 5.02 Hz, 1H). 13C NMR (100 MHz, CDC13) δ ppm 24.6, 25.1, 26.1, 26.2, 50.8, 67.2, 70.5, 70.9, 71.3, 96.5, 108.9, 109.8.
6-azido-6-deoxy-D-galactopyranose (9)
[00120] Compound 8 (200mg, 70mmol) was stirred in 80% TFA for 30 min. After that, the reaction mixture was evaporated until dryness, and then co-evaporated three times with ¾0, EtOAc respectively to afford compound 9 as an off-white solid (140 mg, 68mmol, 97% yield). 'H NMR (400 MHz, D20) δ ppm 3.66-3.13 (m, 2H), 3.83 (ddd, J = 25.27, 10.34, 3.52 Hz, 2H), 3.96 (d, J = 2.95 Hz, 1H), 4.20 (dd, J = 7.91, 4.68 Hz, 1H), 5.27 (d, J = 3.74 Hz, 1H).
Probe A
[00121] Compound 2(lmmol) was dissolved in a solution of DMSO: t-BuOH: H20 (8:4:7). Then, CuI(O.Olmmol) and TBTA(O.Olmmol) were added to the reaction mixture. Then, compound 9 (1.2mmol) was added to the reaction mixture and stirred overnight. The pH was neutralized and extracted with DCM. The organic layer was dried over Na2S04 and evaporated. The resulting composition was then purified on Florisil® with EtOAc:Hex: DCM (3 : 1 : 1), then 100% DCM and then l :25MeOH:DCM. A light orange solid was afforded with 71% yield. ¾ NMR (400 MHz, CD3OD) δ ppm 1.11 (t, J = 6.87 Hz, 12H), 3.31 (q, 8H), 3.42-3.48 (m, 1H), 3.73 (s, 2H), 4.12-4.64 (m, 4H), 6.18 (bs, 4H), 6.34 (bs, 2H), 6.94 (d, J =
6.68 Hz, 1H), 7.21-7.30 (m, 1H), 7.42-7.49 (m, 2H), 7.84-7.90 (m, 1H) C NMR (100 MHz, CD3OD) δ ppm 1 1.8, 34.5, 44.2, 51.1, 65.7, 68.8,68.9, 69.4,69.7, 70.2, 72.2, 73.5, 93.03, 97.4, 97.8, 104.7, 108.1, 108.2, 122.6, 123. 9, 128.4, 128.5, 128.6, 130.7, 133.1, 149.1, 153.6, 153.7, 168.4. HR/MS [M+H]+ calcd 685.3350 found 685.3358.
Probe B
[00122] Compound 6 (lmmol) was dissolved in a solution of DMSO: t-BuOH: H20 (8:4:7). Then, Cul(0.02mmol) and TBTA(0.02mmol) were added to the reaction mixture. Then, compound 9 (2.5mmol) was added to the reaction mixture and stirred overnight. The pH was neutralized and concentrated. The resulting composition was then purified on Florisil® (60- 100 mesh, fisher scientific) with 1 :25 MeOH: DCM and 50:50 MeOH: DCM. A light orange syrup was obtained with 25% yield. ¾ NMR (400 MHz, CD3OD) δ ppm 1.12 (t, J = 6.96 Hz, 12H), 3.32 (q, J= 7.34 Hz, 8H), 3.50 (m, 1H), 3.63 (s, 6H), 3.77 (dd, J = 4.63, 3.04 Hz, 2H), 3.84 (d, J = 3.06 Hz, 1H), 3.94 (m, 1H), 4.27 (s, 2H), 4.38 (dd, J = 8.64, 5.14 Hz, 2H), 4.60 (m, 6H), 4.76 (m, 2H), 5.12 (d, J = 3.25 Hz, 1H), 6.19 (dd, J = 28.17, 9.51 Hz, 4H), 6.34 (d, J = 2.47 Hz, 2H), 7.02 (m, 2H), 7.49 (m, 2H), 7.65 (m, 1H), 7.84 (m, 1H), 7.93 (m, 2H). 13C NMR (100 MHz, CD3OD) δ ppm 1 1.7, 34.3, 44.2, 65.6, 68, 9, 69.0, 69.1, 69.4, 69.7, 70.1, 72.3, 73.5, 91.4, 92.5, 93.1, 97.5, 97.8, 104.7, 108.2, 122.6, 123.7, 123.8, 124.9 125.2, 125.4, 130.6, 133.1, 149.1, 153.6, 153. 7, 168.4. MS [M+H]+ found 1133.5374.
Probe C
[00123] Compound 2 (0.643 mmol) was dissolved in a solution of DMSO: t-BuOH: H20 (8:4:7). Then, CuS04, Na ascorbate and TBTA(0.06 mmol) were added to the reaction mixture. Then, l,3,4,6-tetra-0-acetyl-2-azido-2-deoxy-a-D-mannopyranose (10, TCI America, 0.536 mmol) was added to the reaction mixture, which was stirred overnight. The pH was neutralized and extracted with DCM. The organic layer was dried over a2S04 and evaporated. The resulting composition was then purified on Florisil® with EtOAc:Hex (1 :2). An off-white solid (compound 11) was obtained with 32 % yield. XH NMR (400 MHz, CDC13) δ ppm 1.03 (m, 12H), 1.74 (d, J = 20.3 Hz, 3H), 1.90 (d, J = 3.70 Hz, 3H), 2.03 (s, 3H), 2.10 (d, J = 3.6 Hz, 3H), 3.19 (m, 8H), 4.11 ( m, 3H), 4.43 (t, J = 4.3 Hz, 2H), 5.06 (d, J = 5.1 Hz, 1H), 5.13 (t, J = 10.2 Hz, 1H), 5.27 (dd, J = 10.2, 5.1 Hz, 1H), 6.18 ( m, 7H), 6.95 (dd, J = 5.94, 2.71 Hz, 1H), 7.32 (m, 2H), 7.55 (s, 1H), 7.79 (dd, J = 7.5, 4.0 Hz, 1H). 13C NMR (100 MHz, CD3CI3) δ ppm 12.8, 20.7, 35.4, 58.9, 61.5, 62.8, 64.3, 65.0, 66.0, 68.5, 70.8,71.6, 81.7, 90.8, 92.2, 98.0, 98.3, 104.9, 105.3, 105.8, 108.0, 108.3, 122.1, 123.1, 124.1, 128.1, 128.7, 129.1, 130.7, 132.7, 145.1, 146.8, 148.8, 153.6, 154.1, 169.2, 170.3, 170.9, 171.1.
[00124] The pure product 11 was dissolved in anhydrous MeOH, and then NaOMe (10% by weight) was added. After stirring overnight, the reaction mixture was neutralized with Dowex® 50WX8 proton exchange resin and filtered. This solution further was purified by using Florisil® to afford Probe C as white solid.
Probe D
[00125] Compound 6 (0.387 mmol) was dissolved in a solution of DMSO: t-BuOH: H20 (8:4:7). Then, CuS04,Na ascorbate and TBTA (0.004 mmol) were added to the reaction mixture. Then, l,3,4,6-tetra-0-acetyl-2-azido-2-deoxy-a-D-mannopyranose (10, TCI America, 0.804 mmol) was added to the reaction mixture, which was stirred overnight. The pH was neutralized and extracted with DCM. The organic layer was dried over a2S04 and evaporated. The resulting composition was later purified on Florisil® with MeOH:EtOAc (1 : 10). An off-white solid (compound 12) was obtained with 38 % yield. XH NMR (400 MHz, CDC13) δ ppm 1.11 (t, J = 7.0 Hz, 12H), 1.92 (s, 6H), 2.05 (s, 6H), 2.01 (s, 6H), 2.19 (s, 6H), 3.28 (q, J = 7.0 Hz, 8H), 3.68 (s, 2H), 3.82 (dd, J = 23.5, 14.2 Hz, 4H), 4.18 (ddd, J = 8.21, 4.46, 2.28 Hz, 4H), 4.32 (dd, J = 12.25, 3.87 Hz, 4H), 4.39 (s, 2H), 4.60 (td, J = 13.73, 7.42 Hz, 4H), 5.34 (m, 2H), 5.48 (dd, J = 9.90, 5.10 Hz, 2H), 6.14 (m, 2H), 6.24 (s, 2H), 6.27 (s, 2H), 6.30 (d, J = 2.36 Hz, 2H), 6.36 (s, 2H), 6.82 (s, 1H), 7.06 (ddd, J = 5.62, 3.13, 0.67 Hz, 1H), 7.41 (dd, J = 5.62, 3.12 Hz, 2H), 7.56 (s, 1H), 7.87 (ddd, J = 5.62, 3.13, 0.67 Hz, 1H),
8.07 (s, 2H). ljC NMR (100 MHz, CD3C13) δ ppm 12.8, 20.8, 21.0, 35.2, 44.5, 47.2, 48.8, 49.2, 59.5, 61.9, 64.9, 68.6, 70.9, 90.8, 97.9, 105.6, 108.0, 123.2, 123.6, 124.1, 128.3, 129.1, 131.2, 132.7, 144.6, 144.7, 145.1, 148.9, 153.4, 153.6, 167.8, 168.0, 169.4, 170.3, 170.7. The pure product was dissolved in anhydrous MeOH, and NaOMe (10% by weight) was added. After stirring overnight, the reaction mixture was neutralized with Dowex® 50WX8 proton exchange resin and filtered. This solution further was purified by using Florisil® to afford Probe D.
Probe E
Probe E
[00126] Compound 2(0.967 mmol) was dissolved in a solution of DMSO: t-BuOH: H20 (8:4:7). Then, CuS04, Na ascorbate and TBTA(O.Olmmol) were added to the reaction mixture. Then, 2-acetamido-3,4,6-tri-0-acetyl-2-deoxy- -D-glucopranosyl azide (13, TCI America, 0.793 mmol) was added to the reaction mixture, which was stirred overnight. The pH was neutralized and extracted with DCM. The organic layer was dried over a2S04 and evaporated. The resulting composition was later purified on Florisil® with EtOAc:Hex (1 :2). An off-white solid (compound 14) was obtained with 46 % yield. XH NMR (400 MHz, CDCl3) 5 ppm 1.12 (m, 12H), 1.65 (s, 3H), 1.96 (s, s, s, too close peaks, 9H), 3.31 (q, J = 5.86 Hz, 8H), 4.19 ( m, 6H), 5.04 (t, J = 9.70 Hz, 1H), 5.53 (t, J = 9.70 Hz, 1H), 6.02 (d, J = 9.95 Hz, 1H), 6.18 (dd, J = 8.80, 2.03 Hz, 1H), 6.35 (m, 1H), 6.97 (s, 1H), 7.04 (dd, J = 5.06, 2.19 Hz, 1H), 7.23 (dd, J = 13.49, 5.35 Hz, 1H), 7.36 (m, 2H), 7.76 (dd, J = 6.50, 4.54 Hz, 1H). 13C NMR (100 MHz, CD3C13) δ ppm 12.8, 12.9, 20.8, 22.9, 35.9, 44.5, 53.6, 62.5 65.5,
68.7, 72.8, 74.6, 85.1,98.0, 98.3, 105.3, 108.4, 108.5, 121.7, 122.8, 124.1, 128.2, 129.1, 129.4, 131.1, 132.7, 144.8, 149.0, 153.6, 168.0, 169.7, 170.4, 170.8.
[00127] The pure product was dissolved in anhydrous MeOH, and NaOMe (10% by weight) was added. After stirring overnight, the reaction mixture was neutralized with Dowex® 50WX8 proton exchange resin and filtered. This solution further was purified by using Florisil® to afford Probe E as off-white solid. ¾ NMR (400 MHz, CD3OD) δ ppm 1.14 (dt, J = 7.00, 3.38 Hz, 12H), 1.71 (s, 3H), 1.99 (s, 1H), 3.35 (dd, J = 13.78, 7.0 Hz, 8H), 3.46 (s, 2H), 3.67 (m, 2H), 3.86 (dd, J = 12.41, 1.46 Hz, 1H), 3.94 (t, J = 9.83 Hz, 1H), 4.22 (d, J = 15.97 Hz, 1H), 4.42 (d, J = 15.59 Hz, 1H), 5.59 (d, J = 9.83 Hz, 1H), 6.28 (m, 4H), 6.41 (dd, J = 18.78, 2.09 Hz, 2H), 7.04 (dd, J = 6.85, 2.34 Hz, 1H), 7.38 (s, 1H), 7.50 (m, 2H), 7.85 (dd, J = 6.31, 1.66 Hz, 1H). HR/MS [M+H]+ calcd 725.3537 found 725.3563.
Probe F
[00128] Compound 6 (0.339 mmol) was dissolved in a solution of DMSO: t-BuOH: H20 (8:4:7). Then, CuS04, Na ascorbate and TBTA(O.Olmmol) were added to the reaction mixture. Then, 2-acetamido-3,4,6-tri-0-acetyl-2-deoxy- -D-glucopranosyl azide (13, TCI America, 0.672 mmol) was added to the reaction mixture, which was stirred overnight. The pH was neutralized and extracted with DCM. The organic layer was dried over Na2S04 and evaporated. The resulting composition was then purified on Florisil® with EtOAc:MeOH (1 : 10). A yellow solid (compound 15) was obtained with 43.5 % yield. XH NMR (400 MHz,
CDCI3) δ ppm 1.10 (dt, J = 7.05, 3.16 Hz, 12H), 1.67 (s, 6H), 1.99 (m, 18H), 3.27 (dd, J = 7.02, 3.40 Hz, 8H), 3.63 (s, 6H), 4.1 1 (dd, J = 11.62, 5.09 Hz, 4H), 4.25 (dd, J = 13.86, 6.14 Hz, 2H), 4.31 (s, 1H), 4.57 ( m, 7H), 5.21 (t, J = 9.72 Hz, 2H), 5.64 (t, J = 9.72 Hz, 2H), 6.10 (dd, J = 8.99, 2.51 Hz, 1H), 6.15 (dd, J = 8.99, 2.56 Hz, 1H), 6.25 ( m, 6H), 6.64 (s, 1H), 7.04 (dd, J = 6.17, 3.00 Hz, 1H), 7.39 ( m, 5H), 7.86 (dd, J = 6.15, 2.91 Hz, 1H), 7.93 (s, 2H). 13C NMR (100 MHz, CD3CI3) δ ppm 12.8, 14.4, 20.8, 23.1, 34.7, 44.5, 47.8, 49.3, 53.7, 60.6, 62.2, 65.2, 68.7, 72.7, 74.8, 85.6, 97.7, 105.3, 108.1, 122.9, 123.1, 123.2, 124.1, 124.5, 128.5, 129.2, 131.1, 132.9, 144.5, 144.8, 145.5, 148.9, 149.0, 153.4, 153.5, 153.6, 167.8, 169.6, 170.6, 170.9, 171.0.
[00129] The pure product was dissolved in anhydrous MeOH, and NaOMe (10% by weight) was added. After stirring overnight, the reaction mixture was neutralized with Dowex® 50WX8 proton exchange resin and filtered. This solution further was purified by using Florisil® to afford Probe F as a yellow solid. XH NMR (400 MHz, CD3OD) δ ppm 1.13 (t, J =
7.05 Hz, 12H), 1.71 (s, 6H), 1.87 (s, 2H), 3.34 (m, 8H), 3.66 (m, 12H), 3.89 (d, J = 12.01, 2H), 4.26 (t, J = 10.01 Hz, 2H), 4.31 (d, J = 2.46 Hz, 2H), 4.64 (t, J = 5.6 Hz, 2H), 4.75 (t, J=5.6 Hz, 4H), 5.77 (d, J = 9.78 Hz, 2H), 6.21-6.25 ( m, 4H), 6.36 (t, J = 2.35 Hz, 2H), 7.01-
7.06 (m, 2H), 7.51 (ddd, J = 6.08, 4.29, 1.50 Hz, 2H), 7.77 (s, 1H), 7.87 (dd, J = 6.46, 2.90 Hz, 1H), 8.15 (s, 1H).
Probe G
[00130] Compound 6 (0.339 mmol) was dissolved in a solution of DMSO: t-BuOH: H20 (8:4:7). Then, CuS04, Na ascorbate and TBTA(O.Olmmol) were added to the reaction mixture. Then, 2-acetamido-3,4,6-tri-0-acetyl-2-deoxy- -D-glucopranosyl azide (13, TCI America, 0.672 mmol) was added to the reaction mixture, which was stirred overnight. The pH was neutralized and extracted with DCM. The organic layer was dried over a2S04 and evaporated. The resulting composition was then purified on Florisil® with EtOAc:MeOH (1 : 10). A white solid (compound 16) was obtained with 27 % yield. The pure product was dissolved in anhydrous MeOH, and NaOMe (10% by weight) was added. After stirring overnight, the reaction mixture was neutralized with Dowex® 50WX8 proton exchange resin and filtered. This solution further was purified by using Florisil® to afford Probe G as a yellow solid. 'H NMR (400 MHz, CD3OD) δ ppm 1.13 (t, J = 7.04 Hz, 12H), 1.72 (s, 3H), 3.34 (q, J = 7.03 Hz, 8H), 3.55 (d, J = 7.53 Hz, 2H), 3.72 (m, 6H), 3.88 (dd, J = 12.18, 1.82 Hz, 1H), 4.21 (t, J = 10.03 Hz, 1H), 4.33 (s, 2H), 4.63 (t, J = 5.79 Hz, 2H), 4.74 (t, J = 5.79 Hz, 2H), 5.75 (d, J = 11.40 Hz, 1H), 6.21 (m, 4H), 6.36 (dd, J = 2.45, 1.12 Hz, 2H), 6.99 (s, 1H), 7.04 (m, 2H), 7.52 (ddd, J = 6.47, 4.58, 1.41 Hz, 2H), 7.62 (s, 1H), 7.88 (m, 1H), 8.06 (s, 1H).
Probe H
Probe H
[00131] Compound 2 (0.839 mmol) was dissolved in a solution of DMSO: t-BuOH: H20 (8:4:7). Then, CuS04, Na ascorbate and TBTA (0.08 mmol) were added to the reaction mixture. Then, l-azido-l-deoxy- -D-lactopyranoside (17, Sigma Aldrich, 0.700 mmol) was added to the reaction mixture, which was stirred overnight. The pH was neutralized and extracted with DCM. The organic layer was dried over a2S04 and evaporated. The resulting composition was then purified on Florisil® with MeOH:DCM (1 :20-1 : 10) to afford Probe H (74% yield). A syrup was obtained, and was then turned into a light orange solid upon waiting under vacuum. ¾ NMR (400 MHz, CD3OD) δ ppm 1.14 (dt, J = 6.96, 2.10 Hz,
12H), 3.36 ( m, 8H), 3.66 ( m, 12H), 4.36 (m, 3H), 5.38 (d, J = 8.82 Hz, 1H), 6.27 (m, 4H), 6.40 (m, 2H), 7.05 (dd, J = 6.46, 2.12 Hz, 1H), 7.28 (s, 1H), 7.50 ( m, 2H), 7.86 (dd, J = 6.11, 1.91 Hz, 1H). HR/MS [M+H]+ calcd 846.3800 found 847.3779.
Probe I
[00132] Compound 6 (0.439 mmol) was dissolved in a solution of DMSO: t-BuOH: H20 (8:4:7). Then, CuS04, Na ascorbate and TBTA (0.05 mmol) were added to the reaction mixture. Then, l-azido-l-deoxy- -D-lactopyranoside (17, Sigma Aldrich, 0.929 mmol) was added to the reaction mixture, which was stirred overnight. The pH was neutralized and extracted with DCM. The organic layer was dried over a2S04 and evaporated. The resulting composition was then purified on Florisil® with MeOH:DCM (1 :20-1 : 10-50:50) to afford Probe I (25% yield). A light orange syrup was obtained, and was then turned into a light orange solid upon waiting under vacuum. XH NMR (400 MHz, DMSO) δ ppm 1.03 (t, J = 6.90 Hz, 12H), 3.55 ( m, 24H), 3.75 (d, J = 10.63 Hz, 2H), 3.84 (t, J = 9.07 Hz, 2H), 4.18 (s, 2H), 4.23 (d, J = 7.01 Hz, 2H), 4.62 (td, J = 14.63, 6.73 Hz, 4H), 5.60 (d, J = 8.56 Hz, 2H),
6.17 (td, J = 21.88, 5.55 Hz, 4H), 6.27 (d, J = 2.19 Hz, 2H), 6.97 (m, IH), 7.32 (s, IH), 7.47 (dd, J = 5.71, 3.09 Hz, 2H), 7.78 (dd, J = 6.14, 2.88 Hz, IH), 7.91 (s, IH), 8.19 (s, 2H).
Cyanine dyes - Probes J, K and L
Scheme 2
[00133] Reagents and conditions in Scheme 2: i. C2H5I, reflux, 2d; ii. Br-(CH2)5-COOH, 120 °C, 3d; Hi. Ph-N=CH-CH2-CH=N-Ph-HCl, Ac20, AcOH, 120 °C, 30 min; iv. Et3N, EtOH, reflux, 30 min; v. Propargylamine, HBTU, CH2C12, 3 h; vi. N3-R, CuS04, Sodium ascorbate, THF:H20:t-BuOH 3: 1 : 1, 3 h.
l-ethyl-2,3,3-trimethyl-3H-indol-l-ium iodide (2)
[00134] 2,3,3-trimethyl-3H-indole (1, 15 g, 94.3 mmol) was refluxed in iodoethane (44 g, 283 mmol) for 2 d. On cooling, diethyl ether (50 mL) was added. The precipitates were filtered and dried in oven under 50-60 °C afforded the target compound (2) as light yellow powder (12.18 g, 27.4%). ¾ NMR (400 MHz, DMSO) δ ppm 8.00-7.95 (m, 1H), 7.85-7.81 (m, 1H), 7.61-7.55 (m, 2H), 4.49 (q, J= 7.3 Hz, 2H), 2.85 (s, 3H), 1.52 (s, 6H), 1.42 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO) δ ppm 196.73, 142.63, 141.38, 130.05, 129.63, 124.30, 116.06, 54.85, 43.97, 22.65, 15.04, 13.53.
l-(5-carboxypentyl)-2,3,3-trimethyl-3H-indol-l-ium bromide (3)
[00135] To a stirring suspension of 2,3,3-trimethyl-3H-indole (1, 4 g, 25.15 mmol) and 1,2- dichloro-benzene (20 mL), was added 6-bromohexanoic acid (7.36g, 37.74 mmol). The mixture was stirred at 120-130 °C for 3 d. On cooling to room temperature, (¾(¾ was added, and the precipitates formed were filtered and dried in oven under 50-60 °C afforded the target compound (3) as off white powder (3.54 g, 44.6%). ¾ NMR (400 MHz, DMSO) δ ppm 7.95 (dd, J=5.9, 3.1 Hz, 1H), 7.81 (dd, J = 6.0, 2.8 Hz, 1H), 7.61-7.57 (m, 2H), 4.46 - 4.39 (m, 2H), 2.81 (s, 3H), 2.20 (t, J=7.2 Hz, 2H), 1.81 (dt, J=15.4, 7.6 Hz, 2H), 1.58-1.46 (m, 8H), 1.39 (dt, J=15.0, 7.3 Hz, 2H). 13C NMR (100 MHz, DMSO) δ ppm 174.96, 142.55, 130.06, 129.61, 124.20, 1 16.19, 54.85, 48.15, 34.06, 27.63, 26.10, 24.71, 22.70, 14.78.
l-ethyl-3,3-dimethyl-2-((lE,3E)-4-(N-phenylacetamido)buta-l,3-dien-l-yl)-3H-indol-l- ium iodide (4)
[00136] To a stirring suspension of Malonaldehyde bis(phenylimine) monohydrochloride (5.18 g, 20 mmol) in acetyl anhydride (60 mL), was added l-ethyl-2,3,3-trimethyl-3H-indol- 1-ium iodide (2, 6 g, 16.8 mmol). The mixture was stirred at 120 °C for 30 min. On cooling, diethyl ether was added, and the precipitates formed were filtered and dried in oven under 50- 60 °C afforded the target compound (4) as dark brown powder (6.49 g, 70.1%). XH NMR (400 MHz, DMSO) δ ppm 8.88 (d, J=13.2 Hz, 1H), 8.51 (dd, J=15.0, 1 1.2 Hz, 1H), 7.76 (ddd, J=19.1, 5.1, 4.2 Hz, 2H), 7.65-7.38 (m, 7H), 6.86 (d, J=15.1 Hz, 1H), 5.52 (dd, J =13.1, 1 1.3 Hz, 1H), 4.31 (q, J=7.2 Hz, 2H), 2.01 (s, 3H), 1.66 (s, 6H), 1.23 (t, J=7.2 Hz, 3H). 13C NMR (100 MHz, DMSO) δ ppm 180.70, 170.44, 158.10, 143.80, 141.10, 138.47, 131.08, 130.28, 129.10, 128.84, 123.63, 1 14.66, 113.52, 51.90, 41.63, 26.50, 23.97, 13.82. l-(5-carboxypentyl)-2-(5-(l-ethyl-3,3- dimethylindolin- 2-ylidene)penta-l,3-dien-l-yl)- 3,3- dimethyl- 3H- indol-l-ium bromide (5)
[00137] To a stirring suspension of l-(5-carboxypentyl)-2,3,3-trimethyl-3H-indol-l-ium bromide(4, 3.98 g 1 1.24 mmol) and l-ethyl-3,3-dimethyl-2-((lE,3E)-4-(N-
phenylacetamido)buta- 1,3-dien-l-yl)- 3H- indol-1 -ium iodide (4, 5.46 g, 11.24 mmol) in anhydrous ethanol (90 mL), triethylamine (2.26 g, 22.48 mmol) was added. The mixture was refluxed for 0.5 h, and the solvent was removed on vaccum evaporator. Flash column chromatography of the residue on silica gel (CH2Ci2:MeOH:AcOH 10: 1:0.1) afforded the desired compound (5) as a blue foam (6.05 g, 93.3%). ¾ NMR (400 MHz, CDC13) δ ppm 8.04 (td, J=13.0, 4.2 Hz, 2H), 7.30 (td, J =7.7, 5.2 Hz, 4H), 7.15 (dd, J=14.7, 7.4 Hz, 2H), 7.07 (t, J=8.4 Hz, 2H), 6.79 (t, J=12.5 Hz, 1H), 6.26 (dd, J=19.3, 13.6 Hz, 2H), 4.11 (q, J =7.2 Hz, 2H), 4.01 (t, J =7.5 Hz, 2H), 2.36 (t, J =7.2 Hz, 2H), 1.76 (dt, J =15.6, 8.0 Hz, 2H), 1.73-1.61 (m, 12H), 1.49 (dt, J=15.0, 7.7 Hz, 2H), 1.36 (t, J=7.2 Hz, 3H). 13C MR (100 MHz, CDC13) δ ppm 172.90, 172.78, 153.79, 153.56, 142.15, 141.77, 141.60, 141.41, 128.84, 126.38, 125.37, 125.27, 122.54, 110.76, 110.69, 103.77, 66.02, 53.75, 49.67, 49.58, 44.45, 39.84, 34.38, 28.39, 28.27, 27.18, 26.45, 24.61, 15.43, 12.78.
2-(5-(l-ethyl-3,3-dimethylindolin-2-ylidene)penta-l,3-dien-l-yl)-3,3-dimethyl-l-(6-oxo- 6-(prop-2-yn-l-ylamino)hexyl)-3H-indol-l-ium bromide (6)
[00138] To a stirring solution of l-(5-carboxypentyl)-2-(5-(l-ethyl-3,3- dimethylindolin- 2- ylidene)penta-l,3-dien-l-yl)-3,3- dimethyl- 3H- indol-1 -ium bromide (5, 3.71 g, 6.42 mmol), propargylamine (0.53 g, 9.63 mmol) and triethylamine (1.39 g, 12.84 mmol) in anhydrous CH2CI2 (60 mL), HBTU (3.65 g, 9.63 mmol) was added. The solution was stirred for 2 h at room temperature. CH2CI2 (60 mL) was added to the reaction mixture, and washed with saturated NaHC03 solution (50 mL), 1 N HC1 (50 mL) and water (2 50 mL). The solution was dried on NaS04, filtered and concentrated. Flash column chromatography of the residue on silica gel (CH2Ci2:MeOH 20: 1) afforded the desired compound (6) as a blue foam (3.25 g, 82.2%). ¾ NMR (400 MHz, CDC13) δ ppm 7.89 (td, J=13.0, 4.4 Hz, 2H), 7.34- 7.25 (m, 4H), 7.17-7.09 (m, 2H), 7.06 (t, J=8.3 Hz, 2H), 6.60 (t, J=12.5 Hz, 1H), 6.52 (t, J=5.5 Hz, 1H), 6.09 (dd, J=21.3, 13.6 Hz, 2H), 4.00 (q, J=7.1 Hz, 2H), 3.91 (m, 4H), 2.21 (t, J=7.3 Hz, 2H), 2.09 (t, J=2.5 Hz, 2H), 1.73 (dt, J=12.6, 5.9 Hz, 2H), 1.69-1.55 (m, 8H), 1.44 (dt, J = 15.3, 7.5 Hz, 2H), 1.34 (t, J= 7.2 Hz, 3H). 13C MR (100 MHz, CDC13) δ 173.19, 173.11, 172.91, 153.59, 153.39, 142.13, 141.74, 141.59, 141.42, 128.89, 126.01, 125.45, 125.36, 122.51, 122.44, 110.83, 110.68, 103.43, 103.34, 80.42, 71.04, 53.94, 49.62, 49.56, 44.24, 39.38, 35.89, 29.08, 28.02, 27.91, 27.18, 26.52, 25.22, 12.46.
Probe L (7)
[00139] To a stirring solution of 6-azido-galactose (100 mg, 0.49 mmol) and (6) (200 mg, 0.33 mmol) in THF:H20:t-BuOH 3: 1 :1 (15 mL), was added CuS04 (4 mg, 0.025 mmol) and sodium ascorbate (10 mg, 0.05 mmol). The mixture was stirred for 2 h at room temperature
and concentrated. The residue was diluted in CH2CI2 (50 mL), washed with brine (2x 15 mL), dried on anhydrous a2S04, filtered and concentrated. Flash column chromatography of the residue on silica gel (CH2Ci2:MeOH 5: 1) afforded the desired compound (7) as a blue foam (50 mg, I S.5%).1H NMR (400 MHz, Acetonitrile) δ 8.06 (dd, J=18.2, 8.0 Hz, 2H), 7.72 (d, J = 7.4 Hz, 1H), 7.48 (t, J=7.0 Hz, 2H), 7.40 (dd, J=15.7, 8.0 Hz, 2H), 7.25 (dd, J= 16.9, 8.8 Hz, 4H), 7.03 (d, J=5.6 Hz, 1H), 6.54 (t, J=12.5 Hz, 1H), 6.21 (dd, J=13.7, 8.6 Hz, 2H), 5.11 (d, J=14.9 Hz, 1H), 4.52 (dd, J=14.9, 6.9 Hz, 2H), 4.40-4.25 (m, 2H), 4.07 (dd, J =14.1, 6.9 Hz, 2H), 4.02-3.94 (m, 2H), 3.85-3.75 (m, 2H), 3.74-3.59 (m, 4H), 3.52 (m, 1H), 3.44-3.22 (m, 2H), 2.17 (t, J=7.4 Hz, 2H), 1.95 (m, 2H), 1.75 (m, 2H), 1.71-1.55 (m, 5H), 1.43 (m, 2H), 1.34 (t, J=7.2 Hz, 3H). 13C MR (100 MHz, CD3OD) δ ppm 174.12, 173.05, 171.03, 155.76, 154.25, 142.43, 141.76, 141.08, 140.1 1, 126.58, 125.36, 123.01, 122.49, 122.17, 110.87, 108.52, 103.43, 102.68, 78.16, 75.37, 74.35, 61.56, 52.33, 52.01, 48.54, 48.12, 47.84, 47.63, 47.51, 47.24, 47.09, 43.88, 37.60, 34.46, 34.32, 26.98, 26.32, 25.45, 25.32, 25.01, 22.38, 11.78.
Probe K (8)
[00140] 3,4,6-Tri-0-acetyl-2-acetamido-glucopyranosyl azide (220 mg, 0.59 mmol) was stirred in 1 M NaOMe (1.5 mL) for 3 h at room temperature. The solution was adjusted with Dowex® 50W-X8 H+ ion exchange resin to neutral and filtered. The filtrate was concentrated to dryness and used in next reaction without further purification.
[00141] To a stirring solution of above residue and (6) (242 mg, 0.39 mmol) in THF:H20:t- BuOH 3 : 1 : 1 (15 mL), was added CuS04 (5 mg, 0.03 mmol) and sodium ascorbate (12 mg, 0.06 mmol). The mixture was stirred for 2 h at room temperature and concentrated. The residue was diluted in CH2CI2 (50 mL), washed with brine (2x 15 mL), dried on anhydrous a2S04, filtered and concentrated. Flash column chromatography of the residue on silica gel (CH2Cl2:MeOH 5: 1) afforded the desired compound (8) as a blue foam (80 mg, 23.8%). XH NMR (400 MHz, CD3OD) δ ppm 8.21 (t, J=13.0 Hz, 2H), 7.46 (dd, J=7.4, 4.6 Hz, 2H), 7.42-7.33 (m, 2H), 7.32-7.17 (m, 4H), 6.63 (t, J=12.4 Hz, 1H), 6.28 (dd, J=13.7, 7.8 Hz, 2H), 5.46 (d, J= 9.9 Hz, 1H), 4.46 (t, J= 10.0 Hz, 1H), 4.23 (q, J=14.6 Hz, 2H), 4.16-4.01 (m, 2H), 3.79 (qd, J=10.8, 5.9 Hz, 2H), 3.71-3.60 (m, 2H), 3.58-3.50 (m, 2H), 3.46 (m, 2H), 2.19 (t, J= 7.3 Hz, 2H), 1.85-1.73 (m, 5H), 1.73-1.58 (m, 8H), 1.45 (m, 2H), 1.36 (t, J= 7.2 Hz, 3H). 13C NMR (100 MHz, CD3OD) δ ppm 174.02, 173.40, 173.05, 172.00, 156.76, 154.22, 142.33, 141.90, 141.59, 141.43, 128.58, 125.48, 125.01, 122.24, 122.17, 110.90, 110.59, 103.18, 102.90, 79.36, 75.79, 70.18, 61.46, 54.53, 49.33, 48.47, 48.25, 48.04, 47.83,
47.62, 47.40, 47.19, 43.59, 38.80, 35.45, 34.18, 26.93, 26.76, 26.67, 26.12, 25.30, 21.72, 11.40.
Probe J (9)
[00142] l,3,4,6-Tetra-0-acetyl-2-azido-2-deoxy-a-D-mannopyranose (100 mg, 0.27 mmol) was stirred in 1 M NaOMe (1 mL) for 3 h at room temperature. The solution was adjusted with Dowex® 50W-X8 H+ ion exchange resin to neutral and filtered. The filtrate was concentrated to dryness and used in next reaction without further purification.
[00143] To a stirring solution of above residue and (6) (182 mg, 0.29 mmol) in THF:H20:t- BuOH 3: 1: 1 (15 mL), was added CuS04 (5 mg, 0.03 mmol) and sodium ascorbate (12 mg, 0.06 mmol). The mixture was stirred for 2 h at room temperature and concentrated. The residue was diluted in CH2C12 (50 mL), washed with brine (2x 15 mL), dried on anhydrous a2S04, filtered and concentrated. Flash column chromatography of the residue on silica gel (CH2Cl2:MeOH 5:1) afforded the desired compound (9) as a blue foam (50 mg, 21.0%). XH NMR (400 MHz, CD3OD) δ ppm 8.29-8.16 (m, 2H), 7.97 (s, 1H), 7.47 (dd, J =7.1, 3.8 Hz, 2H), 7.43-7.33 (m, 2H), 7.32-7.18 (m, 4H), 6.61 (t, J=12.6 Hz, 1H), 6.28 (t, J= 13.5 Hz, 2H), 5.26 (d, J=3.3 Hz, 1H), 5.09 (d, J=7.9 Hz, 1H), 4.61 (dd, J=l 1.0, 3.4 Hz, 1H), 4.23 (dd, J= 11.0, 8.7 Hz, 1H), 4.19-4.00 (m, 4H), 3.98-3.85 (m, 2H), 3.84-3.65 (m, 4H), 3.61- 3.38 (m, 2H), 2.23 (t, J=7.2 Hz, 2H), 1.85-1.74 (m, 2H), 1.68 (m, 8H), 1.44 (m, 2H), 1.36 (t, J=7.2 Hz, 3H). 13C MR (100 MHz, CD3OD) 5 ppm 173.15, 172.05, 172.03, 154.76, 153.22, 142.03, 141.86, 141.39, 140.43, 127.58, 125.36, 124.01, 122.09, 121.17, 110.87, 109.59, 103.13, 102.78, 78.36, 75.67, 70.35, 61.46, 52.53, 47.73, 48.57, 48.15, 47.94, 47.83, 47.52, 47.34, 47.08, 43.66, 37.80, 34.55, 34.01, 26.83, 26.67, 26.45, 25.62, 25.11, 21.34, 12.31.
Cy5-lactose (10)
[00144] To a stirring solution of l-azido-l-deoxy- -D-lactopyranoside (150 mg, 0.41 mmol) and (6) (251 mg, 0.41 mmol) in THF:H20:t-BuOH 3: 1: 1 (15 mL), was added CuS04 (4 mg, 0.02 mmol) and sodium ascorbate (9 mg, 0.04 mmol). The mixture was stirred for 2 h at room temperature and concentrated. The residue was diluted in CH2C12 (50 mL), washed with brine (2x 15 mL), dried on anhydrous Na2S04, filtered and concentrated. Flash column chromatography of the residue on silica gel (CH2Cl2:MeOH 5:1) afforded the desired compound (10) as a blue foam (70 mg, 17.4%). ¾ NMR (400 MHz, CD3OD) δ ppm 8.21 (t, J=13.0 Hz, 2H), 7.46 (dd, J=7.0, 5.5 Hz, 2H), 7.42-7.34 (m, 2H), 7.30-7.19 (m, 4H), 6.63 (t, J=12.5 Hz, 1H), 6.28 (dd, J=13.8, 7.8 Hz, 2H), 5.28 (d, J=9.1 Hz, 1H), 4.40 (dd, J=11.9, 6.8 Hz, 2H), 4.28-4.18 (m, 2H), 4.17-4.02 (m, 2H), 4.02-3.85 (m, 1H), 3.81 (dd, J=7.1, 3.7
Hz, 2H), 3.79 -3.68 (m, 4H), 3.64-3.52 (m, 4H), 3.48 (dd, J =9.8, 3.3 Hz, 2H), 2.19 (t, J=7.3 Hz, 2H), 1.77 (m, 2H), 1.69 (m, 8H), 1.44 (m, 8H), 1.36 (t, J=7.2 Hz, 3H). 13C NMR (100 MHz, CD3OD) 5 ppm 173.02, 170.84, 154.22, 142.44, 142.33, 141.94, 141.90, 141.64, 141.58, 141.45, 141.42, 128.58, 125.00, 122.16, 110.59, 103.94, 77.77, 75.88, 73.57, 71.34, 69.13, 61.32, 51.10, 49.33, 48.47, 48.25, 48.04, 47.99, 47.98, 47.96, 47.94, 47.83, 47.76, 47.75, 47.62, 47.40, 47.19, 43.59, 38.79, 35.48, 26.94, 26.76, 26.66, 26.12, 25.29, 1 1.41.
Example 2
Spectroscopic Characterization
[00145] For absorbance and fluorescence spectroscopy measurements, probes were dissolved in DMSO and diluted with water to prepare stock solutions. Later, the stock solutions were diluted with different pH buffers to the final concentration of measurement. UV-vis spectra were recorded by using a Perkin Elmer® Lambda 35 UV/Vis Spectrometer equipped with a PTP 1+1 Peltier Temperature Programmer instrument at room temperature. The slit width was 4 nm. A 10 x 10 mm quartz cell was used for all absorbance measurements. The final concentration for absorbance was about 10 μΜ.
[00146] Fluorescence spectra were obtained by using a Horiba Jobin Yvon Fluoromax®-4 spectrofluorometer. The slit width was 5 nm for both excitation and emission. All samples were excited at 480 nm. A 10 x 10 mm quartz cell was used for all fluorescence
measurements. The final concentration of the probe was about 1 μΜ.
[00147] For quantum yield experiments, the slit width was 5 nm for both excitation and emission. All the samples were excited at 540 nm. A 10x10 mm quartz cell was used for each fluorescence measurement. Relative quantum yields of Probes A-G were compared to Rhodamine B. The following equation was used in order to determine relative quantum yields:
(f)F = (A standard/A sample ) (F sample/F standard) (n sample n standard) (jjF" standard (Eq. 1)
4>F* (Rhodamine B) = 0.69 in EtOH
[00148] F is the area under the emission curve for each probe, and A is the absorbance value at the excitation wavelength. The same solution was used during the measurements for both the probes and for RhB, thereby eliminating the refractive index ratio (which was equal to 1) from the equation. The quantum yields are summarized in Table 1.
Table 1. Quantum Yields
[00149] Absorbance spectra as a function of pH for Probes A-G are shown in Figure 1. As is evident, the absorbance
[00150] Fluorescence spectra for Probes A-G are shown in Figure 2.
Example 3
Cell Imaging
General experimental details
[00151] HepG2 cells (human hepatocellular liver carcinoma cell line) and HeLa cells (human cervical cancer cells). (CCL-2) were obtained from the American Type Cell Culture collection (ATCC). HepG2 and HeLa cells were grown in Eagle's Minimal Essential Medium (EMEM) with 10% FBS (Sigma- Aldrich, heat inactivated). All cells were maintained in a 5% CO2 humidified atmosphere at 37°C.
[00152] Cells were grown in 35 mm glass bottom dish for 24 h in media. The media was removed and the cells were washed three times with IX DPBS without Ca2+ or Mg2+ (Hyclone, Fisher Scientific). Probes A-I were incubated with cells in non-FBS media. After each step, cells were washed with DPBS buffer. Then, they were imaged in different pH buffers or in media. More detailed information about incubation times and media were used for imaging are provided below.
Intracellular Localization of Probes A-F
[00153] To characterize the intracellular localization of Probes A-F, various localization markers were employed. Following several hours of incubation, intracellular probe distribution suggested localization in either acidic vesicles or potentially within the mitochondria. To further examine this, a double stain of HeLa cells with MitoTracker and
Probes A-F was undertaken. A 45-minute incubation with Probes A-F, resulted in a distinct labeling pattern and an absence of colocalization of the probes with the mitochondria (Figs. 3-8, A-D). To further characterize the pattern of probe localization, we employed confocal laser scanning fluorescent microscopy and a double staining with LysoTracker Green (a lysosome selective stain) in HeLa cells. These investigations indicated colocalization of Probes A-F in acidic vesicles, especially lysosomes, after 15-minute of intracellular incubation (Figs. 3-8, E-H). Nuclear staining with Hoechst 33342 verified that HeLa cells were viable throughout the imaging experiments.
[00154] Figure 3 shows confocal laser-scanning fluorescence images of Probe A in HeLa cells. Probe A (30 μΜ, green, shown in Fig. 3A) was incubated with HeLa cells in non-FBS DMEM media for 2 hours and counterstained with MitoTracker (80 nM, red, shown in Fig. 3B), and Hoechst 33342 (1 μg/mL, blue, shown in Fig. 3C). An overlay of the images shown in Figs. 3A, 3B and 3C is shown in Fig. 3D. HeLa cells were also incubated with Probe A (20 μΜ, shown in Fig. 3E) in media for 2h, followed by counterstain with LysoTracker (2 μΜ, shown in Fig. 3F), and Hoechst 33342 (1 μg/mL, shown in Fig. 3G). An overlay of the images shown in Figs. 3E, 3F and 3G is shown in Fig. 3H. All images were acquired with 60 X objective.
[00155] Figure 4 shows confocal laser-scanning fluorescence images of Probe B in HeLa cells. Probe B (30 μΜ, green, shown in Fig. 4A) was incubated with HeLa cells in non-FBS DMEM media for 2 hours and counterstained with MitoTracker (80 nM, red, shown in Fig. 4B), and Hoechst 33342 (1 μg/mL, blue, shown in Fig. 4C). An overlay of the images shown in Figs. 4A, 4B and 4C is shown in Fig. 4D. HeLa cells were also incubated with Probe B (20 μΜ, shown in Fig. 4E) in media for 2h, followed by counterstain with LysoTracker (2 μΜ, shown in Fig. 4F), and Hoechst 33342 (1 μg/mL, shown in Fig. 4G). An overlay of the images shown in Figs. 4E, 4F and 4G is shown in Fig. 4H. All images were acquired with 60 X objective.
[00156] Figure 5 shows confocal laser-scanning fluorescence images of Probe C in HeLa cells. Probe C (20 μΜ, green, shown in Fig. 5A) was incubated with HeLa cells in non-FBS DMEM media for 45-min and counterstained with Mito-Tracker (40 nM, red, shown in Fig. 5B), and Hoechst 33342 (1 μg/mL, blue, shown in Fig. 5C). An overlay of the images shown in Figs. 5A, 5B and 5C is shown in Fig. 5D. HeLa cells were also incubated with Probe C (20 μΜ, shown in Fig. 5E) in media for 15-min, followed by counterstain with LysoTracker (2 μΜ, shown in Fig. 5F), and Hoechst 33342 (1 μg/mL, shown in Fig. 5G). An overlay of
the images shown in Figs. 5E, 5F and 5G is shown in Fig. 5H. All images were acquired with 60 X objective.
[00157] Figure 6 shows confocal laser-scanning fluorescence images of Probe D in HeLa cells. Probe D (30 μΜ, green, shown in Fig. 6A) was incubated with HeLa cells in non-FBS DMEM media for 45-min and counterstained with Mito-Tracker (40 nM, red, shown in Fig. 6B), and Hoechst 33342 (1 μg/mL, blue, shown in Fig. 6C). An overlay of the images shown in Figs. 6A, 6B and 6C is shown in Fig. 6D. HeLa cells were also incubated with Probe D (20 μΜ, shown in Fig. 6E) in media for 15-min, followed by counterstain with LysoTracker (2 μΜ, shown in Fig. 6F), and Hoechst 33342 (1 μg/mL, shown in Fig. 6G). An overlay of the images shown in Figs. 6E, 6F and 6G is shown in Fig. 6H. All images were acquired with 60 X objective.
[00158] Figure 7 shows confocal laser-scanning fluorescence images of Probe E in HeLa cells. Probe E (30 μΜ, green, shown in Fig. 7A) was incubated with HeLa cells in non-FBS DMEM media for 45-min and counterstained with MitoTracker (40 nM, red, shown in Fig. 7B), and Hoechst 33342 (1 μg/mL, blue, shown in Fig. 7C). An overlay of the images shown in Figs. 7A, 7B and 7C is shown in Fig. 7D. HeLa cells were also incubated with Probe E (20 μΜ, shown in Fig. 7E) in media for 2h, followed by counterstain with LysoTracker (2 μΜ, shown in Fig. 7F), and Hoechst 33342 (1 μg/mL, shown in Fig. 7G). An overlay of the images shown in Figs. 7E, 7F and 7G is shown in Fig. 7H. All images were acquired with 60 X objective.
[00159] Figure 8 shows confocal laser-scanning fluorescence images of Probe F in HeLa cells. Probe F (30 μΜ, green, shown in Fig. 8A) was incubated with HeLa cells in non-FBS DMEM media for 45-min and counterstained with Mito-Tracker (40 nM, red, shown in Fig. 8B), Hoechst 33342 (1 μg/mL, blue, shown in Fig. 8C). An overlay of the images shown in Figs. 8A, 8B and 8C is shown in Fig. 8D. HeLa cells were also incubated with Probe F (20 μΜ, shown in Fig. 8E) in media for 15-min, followed by counterstain with LysoTracker (2 μΜ, shown in Fig. 8F), Hoechst 33342 (1 μg/mL, shown in Fig. 8G). An overlay of the images shown in Figs. 8E, 8F and 8G is shown in Fig. 8H. All images were acquired with 60 X objective.
[00160] Probes A-F may exist primarily in their un-ionized, membrane-permeable forms when present in the essentially neutral cell cytosol. However, once the probes cross the membrane lipid bilayer and reach organelles with acidic luminal pH (e.g., lysosomes), then the probes may convert to an almost exclusively ionized, membrane-impermeable form,
which will be trapped within the organelle. Consistent with this hypothesis, the intracellular distribution of fluorescence was punctate, as opposed to diffuse, a finding consistent with fluorescence activation occurring within intracellular compartments such as the lysosomes, and not in the cytosol. The punctuate staining patterns were preserved after incubation for 48 hours.
Intracellular Localization of Probes H and I
[00161] Control Probes H and I include lactose moieties rather than galactose, glucose or N- acetylglucosamine moieties of Probes A-F.
[00162] Figure 9 shows confocal laser-scanning fluorescence images of Probe H in HeLa cells. Probe H (20 μΜ, green, shown in Fig. 9A) was incubated with HeLa cells in non-FBS DMEM media for 45-min and counterstained with MitoTracker (40 nM, red, shown in Fig. 9B), and Hoechst 33342 (1 μg/mL, blue, shown in Fig. 9C). An overlay of the images shown in Figs. 9A, 9B and 9C is shown in Fig. 9D. HeLa cells were also incubated with Probe H (20 μΜ, shown in Fig. 9E) in media for lh, followed by counterstain with LysoTracker (2 μΜ, shown in Fig. 9F), and Hoechst 33342 (1 μg/mL, shown in Fig. 9G). An overlay of the images shown in Figs. 9E, 9F and 9G is shown in Fig. 9H. All images were acquired with 60 X objective.
[00163] Figure 10 shows confocal laser-scanning fluorescence images of Probe I in HeLa cells. Probe I (20 μΜ, green, shown in Fig. 10A) was incubated with cells in non-FBS DMEM media for 45-min and counterstained with MitoTracker (40 nM, red, shown in Fig. 10B), Hoechst 33342 (1 μg/mL, blue, shown in Fig. IOC). An overlay of the images shown in Figs. 10A, 10B and IOC is shown in Fig. 10D. HeLa cells were also incubated with Probe I (20 μΜ, E) in media for 15-min, followed by counterstain with LysoTracker (2 μΜ, shown in Fig. 10F), Hoechst 33342 (1 μg/mL, shown in Fig. 10G). An overlay of the images shown in Figs. 10E, 10F and 10G is shown in Fig. 10H. All images were acquired with 60 X objective.
[00164] Interestingly, the intracellular staining patterns from the control Probes H and I, which include lactose moieties rather than monosaccharides found on N-linked glycans, were quite different. The presence of N-linked glycan moieties on Probes A-F may facilitate selective accumulation within acidic vesicles (lysosomes), and may also enhance intracellular probe retention.
Fluorescence Properties Over Time for Probes A-F
[00165] Probes A-F were retained well in living cells after significant periods of time.
Figure 1 1 shows confocal laser-scanning fluorescence images of Probes A-F in HeLa cells. Panels 1A-1F show results for Probe A. Panels 2A-2F show results for Probe B. Panels BASF show results for Probe C. Panels 4A-4F show results for Probe D. Panels 5A-5F show results for Probe E. Panels 6A-6F show results for Probe F.
[00166] Probes A-F (20 μΜ, green, shown in Fig. 1 1 panels 1C, 2C, 3C, 4C, 5C and 6C) were incubated with cells in non-FBS DMEM media for 15 min, and then counterstained with Lyso-Tracker (2 μΜ, shown in Fig. 11 panels IB, 2B, 3B, 4B, 5B and 6B), and Hoechst 33342 (1 μg/mL, blue, shown in Fig. 1 1 panels 1A, 2A, 3A, 4A, 5A and 6A). DIC images are shown in panels ID, 2D, 3D, 4D, 5D and 6D. Overlays of panels A, B, C and D are shown in panel E. Overlays of panels A, B and C are shown in panel F. All images were acquired with 60 X objective after 18 hours.
[00167] No fluorescence signal was detectable from LysoTracker after 18 hours, as shown in Fig. 11 panels IB, 2B, 3B, 4B, 5B and 6B. By contrast, green fluorescence is from Probes A- F is still visible after 18 hours, as shown in panels 1C, 2C, 3C, 4C, 5C and 6C. These data demonstrate that Probes A-F have excellent retention properties.
[00168] Further experiments showed that no signal was detectable from LysoTracker after 1 h.
Detection of pH Values
[00169] Intracellular microscopic examination of Probe B within cells was performed to attempt to detect changes in regional pH values. Cell treatment with nigericin, an H+/K+ antiporter, was followed by fluorescent imaging of Probe B-loaded HeLa cells, with analyses performed at differing pH values. Figure 12 shows confocal laser-scanning fluorescence imaging of HeLa cells incubated with Probe B (20 μΜ) in media for 2 hours. After washing three times with corresponding pH buffers, cells were further incubated with 1.4 μΜ nigericin for 30 min and then imaged at pH 4.4 (Fig. 12A), pH 5.0 (Fig. 12B), pH 5.5 (Fig. 12C) and pH 6.0 (Fig. 12D) buffers. All images were acquired with 60 X objective
[00170] As depicted in Figure 12, intracellular pH responsiveness of Probe B is inversely correlated with pH (e.g., lower pH, higher intracellular probe accumulation). In these studies, the intracellular pH was in equilibrium with the extracellular pH. Additionally, HeLa cells maintained at an acidic extracellular pH exhibited altered lysosome morphology, where lysosomes appeared more dispersed and simultaneously located toward the cell periphery.
Conversely, lysosomes in control cells maintained at pH 7.4 were primarily localized to the peri-nuclear region (Figures 3-8, E-H).
Intracellular Localization of Probes J, K and L
[00171] Intracellular localization studies were carried out with Probes J, K and L as described above. Figure 13 shows confocal laser-scanning fluorescence images of Probes J, K and L in HeLa cells. Panels 1A-1F show results for Probe J. Panels 2A-2F show results for Probe K. Panels 3A-3F show results for Probe L.
[00172] Probes J, K and L (20 μΜ, red, shown in Fig. 13 panels 1A-3A respectively) was incubated with HeLa cells in media for 15 min, followed by counterstain with LysoTracker (2 μΜ, panels 1B-3B respectively), Hoechst 33342 (1 μg/mL, panels 1C-3C respectively). Overlays of panels A, B and C are shown in panels 1D-3D respectively. All images were acquired with 60 X objective.
[00173] Lysosome targeting of fluorescent probes J, K and L was observed by co- localization with LysoTracker (Life Technologies, Inc.). A high proportion of overlay was not observed in a control compound, a cyanine-lactose conjugate (without N-glycan moiety).
Example 4
Fluorescence Microscopy of Tumor Slices
[00174] Fluorescence microscopy of fresh tumor slices was performed following incubation with Probe A or Probe B. Figure 14 shows confocal laser-scanning fluorescence images of freshly frozen colon tumor tissue-slices from a patient following incubation of Probe A (20 μΜ) (A-C), and Probe B (20 μΜ) (D-F) for 2h, respectively. Probe A and Probe B fluorescence images are displayed in green (A, D), with nuclei counterstained by Hoechst 33342 and displayed in blue (B, E), and overlay images (C, F).
[00175] As shown in Figure 14, the fluorescence intensities of Probes A and B were more pronounced in selected tumor areas, although present in all tumor regions to varying extents. This result may be explained by confocal laser-scanning increased protein glycosylation in the more aggressive tumor areas as compared to those areas that are less aggressively expanding. These compounds may therefore be useful for diagnosis of tumors in their early stages, as well as assisting in differentiation of more aggressive lesions through evaluation of fluorescence intensity.
Example 5
Synthesis of Probes M-Q
Probe M
Probe M
[00176] Compound 1.2: To a mixture of rhodamine B (compound 1.1, 264 mg, 0.55 mmol) and azido-PEG3-amine (109 mg, 0.5 mmol) in DCM (20 mL) was sequentially added HBTU (260 mg, 1.2 mmol) and Ets (0.5 mL). The reaction mixture was stirred overnight at room temperature. The solvent was then removed by distillation under reduced pressure, and the crude residue was purified via flash chromatograph (EtOAc: Hexane, 4: 1) to obtain the pure product 1.2 as a colorless solid (248 mg, 73.2%); 'H NMR (400 MHz, CDC13) δ: 1.14 (m, 12H), 3.14 (m, 2H), 3.13-3.37 (m, 14H), 3.48 (m, 2H), 3.55-3.62 (m, 6H), 6.23 (m, 2H), 6.34 (s, 2H), 6.40 (m, 2H), 7.04 (m, 1H), 7.40 (m, 2H), 7.86 (m, 1H).
[00177] Compound 1.3: To a stirred solution of compound 1.2 (248 mg, 0.36 mmol) in THF: H20: ?-BuOH (40 mL, 3: 1: 1) was sequentially added tripropargylamine (95 mg, 0.72
mmol),Na L-ascorbate (178 mg, 0.9 mmol) and CuS04. The reaction was allowed to stir overnight at room temperature. After evaporation, the residue was dissolved in EtOAc and washed with water. The combined organic layers were dried over a2S04 and filtered. The filtrate was concentrated to dryness under reduced pressure and the residue was purified via flash chromatograph (EtOAc: Hexane: MeOH, 16:4: 1) to give the desired compound 1.3 as a purple solid (118 mg, 41 %); XH NMR (400 MHz, CDC13) : 1.10 (m, 12H), 2.29 (s, 2H), 3.08 (m, 4H), 3.26 (m, 12H), 3.41 (m, 4H), 3.49 (s, 4H), 3.78 (m, 4H), 4.46 (m, 2H), 6.15- 6.38 (m, 6H), 7.00 (m, 1H), 7.36 (m, 2H), 7.71(m, 1H), 7.80 (m, 1H).
[00178] Compound 1.4: To a stirred solution of compound 1.3 (110 mg, 0.14 mmol) in DMSO: H20: ?-BuOH (19 mL, 8:7:4) was sequentially added 2-Acetamido-3,4,6-tri-0- acetyl- 2-deoxy- β-D-glucopyranosyl azide (116 mg, 0.30 mmol), Cul and TBTA. The reaction was allowed to stir overnight at room temperature. The reaction mixture was washed with aqueous aHC03 and then extracted with EtOAc. The combined organic layers were dried over Na2S04 and filtered. The filtrate was concentrated to dryness under reduced pressure and the residue was purified via flash chromatograph (DCM: MeOH, 20: 1) to give the desired compound 1.4 as a purple solid (86 mg, 40 %); XH NMR (400 MHz, CDC13) δ: 1.10 (m, 12H), 1.68 (s, 6H), 2.02 (m, 18H), 3.09 (m, 2H), 3.26-3.31 (m, 12H), 3.40 (m, 6H), 3.49 (m, 4H), 3.78(m, 6H), 4.07-4. l l(m, 4H), 4.24 (m, 2H), 4.47 (m, 2H), 4.60 (m, 2H), 5.21 (m, 2H), 5.59 (s, 2H), 6.20 (m, 4H), 6.33-6.40 (m, 4H), 7.04 (m, 1H), 7.40 (m, 3H), 7.86- 7.7.92 (m, 2H), 8.07 (s, 2H).
[00179] Probe M: To compound 1.4 (75 mg, 0.049 mmol) in methanol (5 mL) at room temperature was added a catalytic amount of NaOMe. The reaction was allowed to stir at room temperature for 6 h. The solution was filtered and then the volatiles were removed in vacuum to give Probe M as a yellow solid (46 mg, 74%).
Probe
[00180] Compound 1.6: To A mixture of Rhodamine B (compound 1.1, 192 mg, 0.4 mmol) and Azido-PEG3 -Amine (109 mg, 0.44 mmol) in DCM (20 mL) was sequentially added HBTU (182 mg, 0.48 mmol) and Ets (0.5 mL). The reaction mixture was stirred overnight at room temperature. The solvent was then removed by distillation under reduced pressure, and the crude residue was purified via flash chromatograph (EtOAc: Hexane, 4: 1) to obtain the pure product 1.6 as a colorless solid (172 mg, 64%); XH NMR (400 MHz, CDC13) δ 1.22 (m, 12H), 1.42 (s, 9H), 3.06 (m, 2H), 3.31-3.37 (m, 14H), 3.46 (m, 4H), 4.97 (s, 1H, NH), 6.23 (m, 2H), 6.39 (m, 4H), 7.05 (m, 1H), 7.40 (m, 2H), 7.85 (m, 1H).
[00181] Compound 1.7: To a stirred solution of compound 1.6 (172 mg, 0.25 mmol) in DCM (3 mL) was added TFA (3 mL). The reaction mixture was stirred for 2h until TCL
indicated that the starting material disappeared. The mixture was diluted with DCM and then washed with saturated aHC03 and brine. The combined organic layers were dried over Na2S04 and filtered. The filtrate was concentrated to dryness under reduced pressure to give the desired compound 1.7 as a purple oil (85 mg, 60 %); XH NMR (400 MHz, CDC13) δ 1.12 (m, 12H), 2.96 (m, 2H), 3.21 (m, 4H), 3.28-3.32 (m, 10H), 3.49 (m, 2H), 3.72 (m, 2H), 6.26 (m, 2H), 6.39 (m, 4H), 7.05 (m, 1H), 7.40 (m, 2H), 7.83 (m, 1H), 8.27 (s, 2H, NH2).
[00182] Compound 1.8: To A mixture of compound 1.7, (185 mg, 0.32 mmol) and 3- azidoprpanoic acid (53 mg, 0.46 mmol) in DCM (20 mL) was sequentially added HBTU (370 mg, 1 mmol) and EtsN (0.3 mL). The reaction mixture was stirred overnight at room temperature. The solvent was then removed by distillation under reduced pressure, and the crude residue was purified via flash chromatograph (EtOAc: Hexane, 4: 1) to obtain the pure product 1.8 as a colorless solid (171 mg, 80 %); XH NMR (400 MHz, CDC13) δ: 1.09 (m, 12H), 2.46 (m, 2H), 2.98 (m, 2H), 3.26-3.31 (m, 10H), 3.35 (m, 2H), 3.44 (m, 4H), 3.50-3.57 (m, 4H), 6.19 (m, 2H), 6.37 (m, 4H), 6.96 (m, 1H), 7.03 (m, 1H), 7.42 (m, 2H), 7.81 (m, 1H).
[00183] Compound 1.9: To a stirred solution of compound 1.8 (165 mg, 0.25 mmol) in THF: H20: ?-BuOH (20 mL, 3: 1: 1) was sequentially added tripropargylamine (77 mg, 0.60 mmol),Na L-ascorbate (178 mg, 0.9 mmol) and CuS04. The reaction was allowed to stir overnight at room temperature. After evaporation, the residue was dissolved in EtOAc and washed with water. The combined organic layers were dried over Na2S04 and filtered. The filtrate was concentrated to dryness under reduced pressure and the residue was purified via flash chromatograph (EtOAc: Hexane: MeOH, 16:4: 1) to give the desired compound 1.9 as a colorless solid (112 mg, 56 %); XH NMR (400 MHz, DC )5: 1.09 (m, 12H), 2.20 (m, 2H), 2.83 (m, 2H), 3.09 (m, 2H), 3.25-3.43 (m, 22H), 3.77 (m, 2H), 4.64 (m, 2H), 6.20 (m, 2H), 6.31-6.38 (m, 4H), 6.99 (m, 2H), 7.37 (m, 2H), 7.65 (s, 1H), 7.78 (m, 1H).
[00184] Compound 1.10: To a stirred solution of compound 1.9 (103 mg, 0.13 mmol) in DMSO: H20: ?-BuOH (19 mL, 8:7:4) was sequentially added 2-Acetamido-3,4,6-tri-0- acetyl-2-deoxy- - D-glucopyranosyl azide (116 mg, 0.30 mmol), Cul and TBTA. The reaction was allowed to stir overnight at room temperature. The reaction mixture was washed with aqueous NaHC03 and then extracted with EtOAc. The combined organic layers were dried over Na2S04 and filtered. The filtrate was concentrated to dryness under reduced pressure and the residue was purified via flash chromatograph (DCM: MeOH, 20: 1) to give the desired compound 1.10 as a purple solid. Compound 1.10 MS [M+H]+ found: 1546.
[00185] Probe N: To compound 1.10 (77 mg, 0.05 mmol) in methanol (5 mL) at room temperature was added a catalytic amount of NaOMe. The reaction was allowed to stir at
room temperature for 6 h. The solution was filtered and then the volatiles were removed in vacuum to give the Probe N as colorless syrup. Probe N: MS [M+Hf found: 1294.
Probe O
Probe O
[00186] Compound 2.2: To a mixture of rhodamine 101 (compound 2.1, 270 mg, 0.55 mmol) and Azido-PEG3-amine (109 mg, 0.5 mmol) in DCM (20 mL) was sequentially added HBTU (260 mg, 1.2 mmol) and Ets (0.5 mL). The reaction mixture was stirred overnight at room temperature. The solvent was then removed by distillation under reduced pressure, and the crude residue was purified via flash chromatograph (EtOAc: Hexane, 4: 1) to obtain the pure product 2.2 as a colorless solid (251 mg, 72%); XH NMR (400 MHz, CDC13) δ 1.82 (m, 4H), 2.02 (m, 4H), 2.45 (m, 4H), 2.86 (m, 4H), 3.14-3.16 (m, 10H), 3.26-3.33 (m, 4H), 3.40 (m, 2H), 3.49 (m, 2H), 3.58-3.62 (m, 6H), 5.96 (s, 2H), 7.03 (m, 1H), 7.37 (m, 2H), 7.85 (m, 1H).
[00187] Compound 2.3: To a stirred solution of compound 2.2 (251 mg, 0.36 mmol) in THF: H20: ?-BuOH (20 mL, 3: 1: 1) was sequentially added tripropargylamine (95 mg, 0.72 mmol),Na L-ascorbate (178 mg, 0.9 mmol) and CuS04. The reaction was allowed to stir overnight at room temperature. After evaporation, the residue was dissolved in EtOAc and washed with water. The combined organic layers were dried over a2S04 and filtered. The filtrate was concentrated to dryness under reduced pressure and the residue was purified via flash chromatograph (EtOAc: Hexane: MeOH, 16:4: 1) to give the desired compound 2.3 as a purple solid (132 mg, 43 %); 'H NMR (400 MHz, CDC13) δ 1.81 (m, 4H), 2.01 (m, 4H), 2.38 (s, 2H), 2.43 (m, 4H), 2.85 (m, 4H), 3.03-3.15 (m, 10H), 3.28 (m, 2H), 3.33-3.48 (m, 8H), 3.52 (s, 4H), 3.77 (m, 4H), 4.46 (m, 2H), 5.95 (s, 2H), 7.02 (m, 1H), 7.37 (m, 2H), 7.65 (s, 2H), 7.85 (m, 1H).
[00188] Compound 2.4: To a stirred solution of compound 2.3 (130 mg, 0.16 mmol) in DMSO: H20: ?-BuOH (19 mL, 8:7:4) was sequentially added 2-Acetamido-3,4,6-tri-0- acetyl-2-deoxy- β-D-glucopyranosyl azide (148 mg, 0.40 mmol), Cul and TBTA. The reaction was allowed to stir overnight at room temperature. The reaction mixture was washed with aqueous aHC03 and then extracted with EtOAc. The combined organic layers were dried over Na2S04 and filtered. The filtrate was concentrated to dryness under reduced pressure and the residue was purified via flash chromatograph (DCM: MeOH, 20: 1) to give the desired compound 2.4 as a purple solid (89 mg, 36 %); XH NMR (400 MHz, CDC13) δ: 1.68 (s, 6H), 1.80 (m, 4H), 2.09 (m, 22H), 2.43 (m, 4H), 2.83 (m, 4H), 3.03-3.15 (m, 10H), 3.42-3.55 (m, 12H), 3.78 (m, 8H), 4.11 (m, 4H), 4.24 (m, 2H), 4.48 (m, 2H), 4.6 l(m, 2H), 5.19 (m, 2H), 5.58 (m, 2H), 5.92 (s, 2H), 6.23 (m, 1H), 7.02 (m, 1H), 7.38 (m, 3H), 7.86-7.92 (m, 2H), 8.08 (s, 2H).
[00189] Probe O: To compound 2.4 (80 mg, 0.051 mmol) in methanol (5 mL) at room temperature was added a catalytic amount of NaOMe. The reaction was allowed to stir at room temperature for 6 h. The solution was filtered and then the volatiles were removed in vacuum to give the compound Probe O as a yellow solid (53 mg, 81%). Probe O: MS
[M+H]+ found: 1315.
[00190] Compound 2.6: To a mixture of Rhodamine 101 (compound 2.1, 550 mg, 0.1 1 mmol) and Amine-PEG3-N-Boc (284 mg, 1 mmol) in DCM (20 mL) was sequentially added HBTU (520 mg, 2.4 mmol) and Ets (0.5 mL). The reaction mixture was stirred overnight at room temperature. The solvent was then removed by distillation under reduced pressure, and the crude residue was purified via flash chromatograph (EtOAc: Hexane, 4: 1) to obtain the pure product 2.6 as a colorless solid (616 mg, 84%); XH NMR (400 MHz, CDC13) δ: 1.40 (s, 9H), 1.80 (m, 4H), 1.98 (m, 4H), 2.41 (m, 4H), 2.85 (m, 4H), 2.99-3.11 (m, 10H), 3.29 (m, 4H), 3.35 (m, 2H), 3.48 (m, 4H), 5.19 (s, 1H), 5.92 (s, 2H), 7.03 (m, 1H), 7.37 (m, 2H), 7.78 (m, 1H).
[00191] Compound 2.7: To a stirred solution of compound 2.6 (600 mg, 0.83 mmol) in DCM (3 mL) was added TFA (3 mL). The reaction mixture was stirred for 2h until TCL indicated that the starting material disappeared. The mixture was diluted with DCM and then washed with saturated aHC03 and brine. The combined organic layers were dried over Na2S04 and filtered. The filtrate was concentrated to dryness under reduced pressure to give the desired compound 2.7 as a purple oil (274 mg, 53 %);δ: 1.80 (m, 4H), 2.00 (m, 4H), 2.41
(m, 4H), 2.85 (m, 4H), 3.00-3.12 (m, 12H), 3.29 (m, 4H), 3.47 (m, 2H), 3.58 (m, 2H), 5.09 (s, 2H), 5.94 (s, 2H), 7.03 (m, 1H), 7.38 (m, 2H), 7.78 (m, 1H).
[00192] Compound 2.8: To a mixture of compound 2.7, (270 mg, 0.44 mmol) and 3- azidoprpanoic acid (60 mg, 0.52 mmol) in DCM (20 mL) was sequentially added HBTU (370 mg, 1 mmol) and EtsN (0.3 mL). The reaction mixture was stirred overnight at room temperature. The solvent was then removed by distillation under reduced pressure, and the crude residue was purified via flash chromatograph (EtOAc: Hexane, 4: 1) to obtain the pure product 2.8 as a colorless solid (241 mg, 76%); ¾ NMR (400 MHz, CDC13) : 1.80 (m, 4H), 1.99 (m, 4H), 2.38 (m, 4H), 2.44 (m, 4H), 2.81 (m, 4H), 2.90-3.07 (m, 10H), 3.31 (m, 4H), 3.38 (m, 2H), 3.46 (m, 4H), 3.54 (m, 2H), 5.92 (s, 2H), 6.99 (m, 2H), 7.33 (m, 2H), 7.77 (m, 1H).
[00193] Compound 2.9: To a stirred solution of compound 2.8 (230 mg, 0.32 mmol) in THF: H20: ?-BuOH (20 mL, 3: 1: 1) was sequentially added tripropargylamine (90 mg, 0.70 mmol), Na L-ascorbate (178 mg, 0.9 mmol) and CuS04. The reaction was allowed to stir overnight at room temperature. After evaporation, the residue was dissolved in EtOAc and washed with water. The combined organic layers were dried over a2S04 and filtered. The filtrate was concentrated to dryness under reduced pressure and the residue was purified via flash chromatograph (EtOAc: Hexane: MeOH, 16:4: 1) to give the desired compound 2.9 XH NMR (400 MHz, CDC13) δ 2.65 (m, 3H), 3.28 (m, 1H), 3.87 (m, 2H), 3.61 (m, 1H), 3.37(m, 1H), 3.80 (m, 2H), 3.54 (m, 1H), 2.76 (m, 4H), 2.00 (m, 4H), 6.76 (m, 2H), 7.70 (m, 1H), 7.90 (m, 1H), 7.20 (m, 1H), 3.40 (m, 3H), 7.60 (m, 1H).
[00194] Compound 2.10: To a stirred solution of compound 2.9 (170mg, 0.20 mmol) in DMSO: H20: ?-BuOH (19 mL, 8:7:4) was sequentially added 2-Acetamido-3,4,6-tri-0- acetyl-2-deoxy- β-D-glucopyranosyl azide (148 mg, 0.40 mmol), Cul and TBTA. The reaction was allowed to stir overnight at room temperature. The reaction mixture was washed with aqueous NaHC03 and then extracted with EtOAc. The combined organic layers were dried over Na2S04 and filtered. The filtrate was concentrated to dryness under reduced pressure and the residue was purified via flash chromatograph (DCM: MeOH, 20: 1) to give the desired compound 2.10 as a purple solid. Compound 2.10: MS [M+H]+ found: 1594.
[00195] Probe P: To compound 2.10 (80 mg, 0.051 mmol) in methanol (5 mL) at room temperature was added a catalytic amount of NaOMe. The reaction was allowed to stir at room temperature for 6 h. The solution was filtered and then the volatiles were removed in vacuum to give the compound Probe P as colorless syrup. Probe P: MS [M+H]+ found: 1342.
P
[00196] Compound 2.12: To a stirred solution of compound 2.3 (130 mg, 0.16 mmol) in DMSO: H20: i-BuOH (19 mL, 8:7:4) was sequentially added l,3,4,6-tetra-0-acetyl-2-azido- 2-deoxy-a- D-mannopyranose (0.536 mmol), Cul and TBTA. The reaction was allowed to stir overnight at room temperature. The reaction mixture was washed with aqueous aHC03 and then extracted with EtOAc. The combined organic layers were dried over a2S04 and filtered. The filtrate was concentrated to dryness under reduced pressure and the residue was purified via flash chromatograph (DCM: MeOH, 20: 1) to give the desired compound 2.12 as a purple solid.
[00197] Probe Q: To compound 2.12 (80 mg, 0.051 mmol) in methanol (5 mL) at room temperature was added a catalytic amount of NaOMe. The reaction was allowed to stir at room temperature for 6 h. The solution was filtered and then the volatiles were removed in vacuum to give the compound Probe Q as syrup. Compound Probe Q: MS [M+H]+ found: 1233.
[00198] Fluorescence spectra for Probes A-G are shown in Figure 2.
Example 6
Cell Imaging - Intracellular Localization of Probes M-Q
[00199] To characterize the intracellular localization of Probes M-l, various localization markers were employed as described above in Example 3.
[00200] Figure 15 shows confocal laser-scanning fluorescent images of Probe M in HeLa cells. Probe M (30 μΜ, red, shown in Fig. 15A) was incubated with cells in non-FBS DMEM media for 45min and counterstained with MitoTracker (80 nM, green, shown in Fig. 15B), and Hoechst 33342 (1 μg/mL, blue, shown in Fig. 15C). An overlay of the images shown in Figs. 15A, 15B and 15C is shown in Fig. 15D. HeLa cells incubated with compound 1.5 (20 μΜ, shown in Fig. 15E) in media for 15min, followed by counterstain with LysoTracker (2 μΜ, shown in Fig. 15F), and Hoechst 33342 (1 μg/mL, shown in Fig. 15G). An overlay of the images shown in Figs. 15E, 15F and 15G is shown in Fig. 15H. All images were acquired with 60 X objective.
[00201] Figure 16 shows confocal laser-scanning fluorescent images of Probe N in HeLa cells. Probe N (30 μΜ, red, shown in Fig. 16A) was incubated with cells in non-FBS DMEM media for 45min and counterstained with MitoTracker (80 nM, green, shown in Fig. 16B), and Hoechst 33342 (1 μg/mL, blue, shown in Fig. 16C). An overlay of the images shown in Figs. 16A, 16B and 16C is shown in Fig. 16D. HeLa cells incubated with compound 1.1 1 (20 μΜ, shown in Fig. 16E) in media for 15min, followed by counterstain with LysoTracker (2 μΜ, shown in Fig. 16F), and Hoechst 33342 (1 μg/mL, shown in Fig. 16G). An overlay of the images shown in Figs. 16E, 16F and 16G is shown in Fig. 16H. All images were acquired with 60 X objective.
[00202] Figure 17 shows confocal laser-scanning fluorescent images of Probe O in HeLa cells. Probe O (30 μΜ, red, shown in Fig. 17A) was incubated with cells in non-FBS DMEM media for 45min and counterstained with MitoTracker (80 nM, green, shown in Fig. 17B), and Hoechst 33342 (1 μg/mL, blue, shown in Fig. 17C). An overlay of the images shown in Figs. 17A, 17B and 17C is shown in Fig. 17D. HeLa cells incubated with compound 2.5 (20
μΜ, shown in Fig. 17E) in media for 15min, followed by counterstain with LysoTracker (2 μΜ, shown in Fig. 17F), and Hoechst 33342 (1 μg/mL, shown in Fig. 17G). An overlay of the images shown in Figs. 17E, 17F and 17G is shown in Fig. 17H. All images were acquired with 60 X objective.
[00203] Figure 18 shows confocal laser-scanning fluorescent images of Probe P in HeLa cells. Probe P (30 μΜ, red, shown in Fig. 18A) was incubated with cells in non-FBS DMEM media for 45min and counterstained with MitoTracker (80 nM, green, shown in Fig. 18B), and Hoechst 33342 (1 μg/mL, blue, shown in Fig. 18C). An overlay of the images shown in Figs. 18A, 18B and 178C is shown in Fig. 18D. HeLa cells incubated with compound 2.11 (20 μΜ, shown in Fig. 18E) in media for 15min, followed by counterstain with LysoTracker (2 μΜ, shown in Fig. 18F), and Hoechst 33342 (1 μg/mL, shown in Fig. 18G). An overlay of the images shown in Figs. 18E, 18F and 18G is shown in Fig. 18H. All images were acquired with 60 X objective.
[00204] Figure 19 shows confocal laser-scanning fluorescence images of Probe Q in HeLa cells. Probe M (30 μΜ, red, shown in Fig. 19A) was incubated with HeLa cells in non-FBS DMEM media for 45 min and counterstained with MitoTracker (80 nM, green, shown in Fig. 19B), and Hoechst 33342 (1 μg/mL, blue, shown in Fig. 19C). An overlay of the images shown in Figs. 19A, 19B and 19C is shown in Fig. 19D. HeLa cells incubated with Probe M (20 μΜ, shown in Fig. 19E) in media for 15 min, followed by counterstain with LysoTracker (2 μΜ, shown in Fig. 19F), and Hoechst 33342 (1 μg/mL, shown in Fig. 19G). An overlay of the images shown in Figs. 19E, 19F and 19G is shown in Fig. 19H. All images were acquired with 60 X objective.
Example 7
Additional Compounds
[00205] Additional compounds will be prepared according to Schemes 3-8 below.
Scheme 3.
2.1 2.22 2.23
60
61
Claims
1. A compound of formula (I):
A-L-(B)n (I)
wherein:
A is a fluorescent moiety;
L is a linker;
B is a monosaccharide moiety selected from the group consisting of mannose, N- acetyl glucosamine, fucose, galactose and sialic acid; and
n is 1 or 2.
2. The compound of claim 1, wherein A is selected from a rhodamine moiety and a cyanine moiety.
3. The compound of claim 1, wherein A is selected from the group consisting of:
4. The compound of any of claims 1-3, wherein L comprises at least one triazole moiety.
5. The compound of any of claims 1-3, wherein L comprises at least one repeat unit of the formula -(CH2CH20)-.
6. The compound of any of claims 1-5, wherein L is a divalent linker and n is 1.
8. The compound of any of claims 1-5, wherein L is a trivalent linker and n is 2.
9. The compound of claim 8, wherein L is selected from the group consisting of:
10. The compound of any of claims 1-9, wherein each B independently comprises a galactose moiety.
12. The compound of any of claims 1-9, wherein each B independently comprises a mannose moiety.
14. The compound of any of claims 1-9, wherein each B independently comprises an N- acetylglucosamine moiety.
16. The compound of claim I, wherein the compound is selected from the group consisting of:
17. A method of selectively staining a lysosome in a cell, comprising contacting the cell with an effective amount of a compound of any of claims 1-16.
18. A method of selectively detecting a lysosome in a cell, comprising:
a) contact the cell with an effective amount of a compound of any of claims 1-16; and b) detecting a signal from the compound.
19. The method of claim 17, wherein the signal is detected from the compound is a fluorescence signal.
20. The method of claim 18, wherein the fluorescent signal is detected using a fluorescence microscope, a flow cytometer, a fluorometer, a fluorescence plate reader, or a combination thereof.
21. The method of any of claims 18-20, further comprising washing the cells prior to the detecting step.
22. A method of selectively detecting a cancerous cell in a sample, comprising:
a) contact the sample with an effective amount of a compound of any of claims 1-16; and
b) detecting a signal from the compound.
23. The method of claim 22, wherein the signal is detected from the compound is a fluorescence signal.
24. The method of claim 23, wherein the fluorescent signal is detected using a fluorescence microscope, a flow cytometer, a fluorometer, a fluorescence plate reader, or a combination thereof.
25. The method of any of claims 22-24, further comprising washing the cells prior to the detecting step.
26. The method of any of claims 22-25, wherein the sample is a tissue sample from a subject.
27. The method of any of claims 22-25, wherein the sample is a culture of cells.
28. A kit comprising a compound of any of claims 1-16.
29. The kit of claim 28, further comprising a buffer, instructions for detecting a lysosome in a cell, or a combination thereof.
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