Patent Application
20140248218
Reactive oxygen species (ROS) encompass a wide array of endogenously produced intermediates that directly result from oxygen metabolism in biological systems.[1] The chemical biology of ROS, especially hydrogen peroxide (H2O2), is rather complex, as recent studies show that controlled generation of H2O2 is necessary to maintain cellular functions such as growth, proliferation, and immune system function.[2] However, misregulation in the production of ROS can lead to significant oxidative damage due to the inability of cells to effectively manage oxidation-reduction equilibrium.[1,3] It is the specific cellular localization and concentration that alter the role of ROS from one of cell signaling to that of oxidative stress and disease.[2a,2e] H2O2 is a major ROS byproduct and has been studied as a common indicator for oxidative stress in a number of pathologies including cancer,[4] cardiovascular[5] and neurodegenerative[6] diseases, and diabetes.[7] There is a need in the art to understand the roles and implications of H2O2 generation in biological systems and for probes to detect physiological levels of H2O2. Provided herein are solutions to these and other problems in the art.
Disclosed herein, inter alia, are compositions of boronic acid-based and boronic ester-based probes that are sensitive to ROS compounds, including H2O2. The compositions include a compound having the formula:
R1 and R2 of compounds (I) and (II) are independently —B(OH)2 or
R3, R4, R5 and R6 of the compound of formula (V) are independently hydrogen, halogen, —N3, —NO2, —CF3, —CCl3, —CBr3, —CI3, —CN, —OH, —NH2, —COOH, —CONH2, —SH, —SO3H, —SO4H, —SO2NH2, —NHNH2, —ONH2, —OCH3, —NHC(O)NHNH2, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. X1 is a monovalent pro-fluorophore moiety. X2 is a divalent pro-fluorophore moiety.
Also disclosed herein, inter alia, are methods of using the probes. A method is provided for detecting a ROS compound. The method includes contacting a compound of formula (I) or formula (II) with a ROS compound. The ROS compound is allowed to react with the compound of formula (I) and remove R1. The ROS compound is allowed to react with the compound of formula (II) and remove R1 and R2. Removing R1 of the compound of formula (I) or R1 and R2 of the compound of formula (II) forms a fluorescent compound. The fluorescent compound is detected thus detecting the ROS compound.
Also provided is a method for detecting a ROS compound in vivo in a subject. The method includes administering to a subject, a compound of formula (I) or formula (II). The compound of formula (I) is allowed to react with a ROS compound remove R1. The compound of formula (II) is allowed to react with a ROS compound and remove R1 and R2. Removing R1 of the compound of formula (I) or R1 and R2 of the compound of formula (II) forms a fluorescent compound. The fluorescent compound is detected thus detecting the ROS compound in vivo in the subject.
The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.
Where substituent groups are specified by their conventional chemical formulae, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left, e.g., —CH2O— is equivalent to —OCH2—.
The term “alkyl,” by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched chain, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated (i.e., C1-C10 means one to ten carbons). An “alkyl” is not cyclized. Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. An alkoxy is an alkyl attached to the remainder of the molecule via an oxygen linker (—O—).
The term “alkylene,” by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkyl, as exemplified, but not limited by, —CH2CH2CH2CH2—. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention. A “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.
The term “heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or combinations thereof, consisting of at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N, P, S, and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. A heteroalkyl is not cyclized. Examples include, but are not limited to: —CH2—CH2—O—CH3, —CH2—CH2—NH—CH3, —CH2—CH2—N(CH3)—CH3, —CH2—S—CH2—CH3, —CH2—CH2, —S(O)—CH3, —CH2—CH2—S(O)2—CH3, —CH═CH—O—CH3, —Si(CH3)3, —CH2—CH═N—OCH3, —CH═CH—N(CH3)—CH3, —O—CH3, —O—CH2—CH3, and —CN. Up to two heteroatoms may be consecutive, such as, for example, —CH2—NH—OCH3.
Similarly, the term “heteroalkylene,” by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from heteroalkyl, as exemplified, but not limited by, —CH2—CH2—S—CH2—CH2— and —CH2—S—CH2—CH2—NH—CH2—. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula —C(O)2R′— represents both —C(O)2R′— and —R′C(O)2—. As described above, heteroalkyl groups, as used herein, include those groups that are attached to the remainder of the molecule through a heteroatom, such as —C(O)R′, —C(O)NR′, —NR′R″, —OR′, —SR′, and/or —SO2R′. Where “heteroalkyl” is recited, followed by recitations of specific heteroalkyl groups, such as —NR′R″ or the like, it will be understood that the terms heteroalkyl and —NR′R″ are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term “heteroalkyl” should not be interpreted herein as excluding specific heteroalkyl groups, such as —NR′R″ or the like.
The terms “cycloalkyl” and “heterocycloalkyl,” by themselves or in combination with other terms, mean, unless otherwise stated, cyclic versions of “alkyl” and “heteroalkyl,” respectively. Cycloalkyl and heterocycloalkyl are not aromatic. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and a “heterocycloalkylene,” alone or as part of another substituent, means a divalent radical derived from a cycloalkyl and heterocycloalkyl, respectively.
The terms “halo” or “halogen,” by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as “haloalkyl” are meant to include monohaloalkyl and polyhaloalkyl. For example, the term “halo(C1-C4)alkyl” includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.
The term “acyl” means, unless otherwise stated, refers to a moiety with formula, —C(O)R, where R is a substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
The term “aryl” means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent, which can be a single ring or multiple rings (preferably from 1 to 3 rings) that are fused together (i.e., a fused ring aryl) or linked covalently. A fused ring aryl refers to multiple rings fused together wherein at least one of the fused rings is an aryl ring. The term “heteroaryl” refers to aryl groups (or rings) that contain one or more heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. Thus, the term “heteroaryl” includes fused ring heteroaryl groups (i.e., multiple rings fused together wherein at least one of the fused rings is a heteroaromatic ring). A 5,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 5 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. Likewise, a 6,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. A 6,5-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 5 members, and wherein at least one ring is a heteroaryl ring. A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. An “arylene” and a “heteroarylene,” alone or as part of another substituent, mean a divalent radical derived from an aryl and heteroaryl, respectively.
For brevity, the term “aryl” when used in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above. Thus, the term “arylalkyl” is meant to include those radicals in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl, and the like) including those alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced by, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).
The term “oxo,” as used herein, means an oxygen that is double bonded to a carbon atom.
Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl,” and “heteroaryl”) includes both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.
Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one or more of a variety of groups selected from, but not limited to, —OR′, ═O, ═NR′, ═N—OR′, —NR′R—SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO2R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)2R′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)2R′, —S(O)2NR′R″, —NRSO2R′, —CN, and —NO2 in a number ranging from zero to (2m′+1), where m′ is the total number of carbon atoms in such radical. R′, R″, R′″, and R″″ each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected as are each R′, R″, R′″, and R″″ group when more than one of these groups is present. When R′ and R″ are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example, —NR′R″ includes, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term “alkyl” is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., —CF3 and —CH2CF3) and acyl (e.g., —C(O)CH3, —C(O)CF3, —C(O)CH2OCH3, and the like).
Similar to the substituents described for the alkyl radical, substituents for the aryl and heteroaryl groups are varied and are selected from, for example: —OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO2R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)2R′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)2R′, —S(O)2NR′R″, —NRSO2R′, —CN, —NO2, —R′, —N3, —CH(Ph)2, fluoro(C1-C4)alkoxy, and fluoro(C1-C4)alkyl, in a number ranging from zero to the total number of open valences on the aromatic ring system; and where R′, R″, R′″, and R″″ are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected as are each R′, R″, R′″, and R″″ groups when more than one of these groups is present.
Two or more substituents may optionally be joined to form aryl, heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such so-called ring-forming substituents are typically, though not necessarily, found attached to a cyclic base structure. In one embodiment, the ring-forming substituents are attached to adjacent members of the base structure. For example, two ring-forming substituents attached to adjacent members of a cyclic base structure create a fused ring structure. In another embodiment, the ring-forming substituents are attached to a single member of the base structure. For example, two ring-forming substituents attached to a single member of a cyclic base structure create a spirocyclic structure. In yet another embodiment, the ring-forming substituents are attached to non-adjacent members of the base structure.
Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally form a ring of the formula -T-C(O)—(CRR′)q—U—, wherein T and U are independently —NR—, —O—, —CRR′—, or a single bond, and q is an integer of from 0 to 3. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH2)r—B—, wherein A and B are independently —CRR′—, —O—, —NR—, —S—, —S(O)—, —S(O)2—, —S(O)2NR′—, or a single bond, and r is an integer of from 1 to 4. One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula —(CRR′)s—X′—(C″R′″)d—, where s and d are independently integers of from 0 to 3, and X′ is —O—, —NR′—, —S—, —S(O)—, —S(O)2—, or —S(O)2NR′—. The substituents R, R′, R″, and R′″ are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.
As used herein, the terms “heteroatom” or “ring heteroatom” are meant to include boron (B), oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).
A “substituent group,” as used herein, means a group selected from the following moieties:
A “size-limited substituent” or “size-limited substituent group,” as used herein, means a group selected from all of the substituents described above for a “substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C20 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C4-C8 cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 4 to 8 membered heterocycloalkyl.
A “lower substituent” or “lower substituent group,” as used herein, means a group selected from all of the substituents described above for a “substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C5-C7 cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 5 to 7 membered heterocycloalkyl.
The term “acceptable salts” is meant to include salts of the compounds disclosed herein that are prepared with acids or bases, depending on the particular substituents found on the compounds. When compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, oxalic, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge et al., “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.
Thus, the compounds of the present invention may exist as salts, such as with acceptable acids or bases. The present invention includes such salts. Examples of such salts include hydrochlorides, hydrobromides, sulfates, methanesulfonates, nitrates, maleates, acetates, citrates, fumarates, tartrates (e.g., (+)-tartrates, (−)-tartrates, or mixtures thereof including racemic mixtures), succinates, benzoates, and salts with amino acids such as glutamic acid. These salts may be prepared by methods known to those skilled in the art.
The neutral forms of the compounds are preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents.
Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.
Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, tautomers, geometric isomers, and individual isomers are encompassed within the scope of the present invention. The compounds of the present invention do not include those that are known in the art to be too unstable to synthesize and/or isolate.
The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (3H), iodine-125 (125I), or carbon-14 (4C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.
The symbol “” denotes the point of attachment of a chemical moiety to the remainder of a molecule or chemical formula.
A “reactive oxygen species” or “ROS” as used herein generally refers to free radicals, reactive anions containing oxygen atoms, or molecules containing oxygen atoms that can either produce free radicals or are chemically activated by them. Accordingly, reactive oxygen species may include, without limitation, superoxide radicals, hydrogen peroxide, peroxynitrite (e.g. ONOO−), lipid peroxides, hydroxyl radicals, thiyl radicals, superoxide anion, organic hydroperoxide, RO. alkoxy and ROO. peroxy radicals, and hypochlorite (OCl−). One of the main sources of reactive oxygen species (ROS) in vivo is aerobic respiration, although reactive oxygen species may also be produced by peroxisomal b-oxidation of fatty acids, microsomal cytochrome P450 metabolism of xenobiotic compounds, stimulation of phagocytosis by pathogens or lipopolysacchrides, arginine metabolism, tissue specific enzymes. Accumulating oxidative damage may also affect the efficiency of mitochondria and further increase the rate of ROS production.
A “pro-fluorophore moiety,” “monovalent pro-fluorophore,” “divalent pro-fluorophore,” “trivalent pro-fluorophore,” or “tetravalent pro-fluorophore,” refers to a substituent that has suppressed fluorescence when attached to the remainder of the compounds described herein relative to the fluorescence of the compound formed from the substituent when detached from the remainder of the compounds described herein. Thus, a “pro-fluorophore moiety” may be a fluorophore (fluorescent compound) in its monovalent, divalent, trivalent or tetravalent form attached to the remainder of the compounds described herein. In embodiments, the compound described herein attached to a pro-fluorophore moiety is reacted with a ROS as described herein to form a fluorescent compound (e.g. a fluorophore). Fluorophores may be detected using techniques known in the art. The terms “monovalent,” “divalent,” “trivalent,” and “tetravalent” refer to radicals derived from a fluorophore that can form one, two, three, or four bonds respectively. Fluorophores contemplated herein include but are not limited to fluorescein and its conjugates, derivatives, and analogues, including those described herein; coumarin and its conjugates, derivatives, and analogues including those described herein; dansyl, bimane, eosin, rhodamine, cyanines, nile red, xanthones, xanthenes, flazo-orange, Snarf1, resorufin, and conjugates, derivatives, and analogues thereof.
The term “fluorescein” as used herein refers to fluorescent derivatization agents. As used herein, fluorescein includes its derivatives such as, for example, fluorescein isothiocyanate (e.g. FITC), carboxyfluorescsein, succinimidyl esters of carboxyfluorescein (e.g. FAM, 6-JOE)), fluorescein-X-succinimidyl esters (e.g. SFX), and fluorescein dichlorotriazine (e.g. DTAF). In embodiments, fluorescein has the formula:
RW and RX are independently hydrogen, halogen, —N3, CF3, —CCl3, —CBr3, —CI3, CN, —CHO, —OH, —OCH3, NH2, COOH, —CONH2, NO2, SH, —SO2, —SO2Cl, —SO3H, —SO4H, —SO2NH2, —NHNH2, —ONH2, —NHC(O)NHNH2, unsubstituted C1-C10 alkyl, unsubstituted 2 to 10 membered heteroalkyl, unsubstituted 3 to 6 membered cycloalkyl, unsubstituted 3 to 6 membered heterocycloalkyl, unsubstituted 5 or 6 membered aryl, or unsubstituted 5 or 6 membered heteroalkyl. w1 and x1 are independently 1, 2, or 3. In embodiments, RW and RX are independently hydrogen, halogen or —OCH3.
RY is independently, hydrogen, halogen, —N3, CF3, —CCl3, —CBr3, —CI3, —CN, —CHO, —OH, —OCH3, NH2, COOH, —CONH2, NO2, SH, —SO2, —SO2Cl, —SO3H, —SO4H, —SO2NH2, —NHNH2, —ONH2, —NHC(O)NHNH2, unsubstituted C1-C10 alkyl, unsubstituted 2 to 10 membered heteroalkyl, unsubstituted 3 to 6 membered cycloalkyl, unsubstituted 3 to 6 membered heterocycloalkyl, unsubstituted 5 or 6 membered aryl, or unsubstituted 5 or 6 membered heteroalkyl. y1 is 1, 2, 3, or 4. In embodiments, RY is independently hydrogen, halogen, —COOH, —CH2Br, Iodoacetamido, malemide, N-succinimidyl ester, N-hydroxysuccinimidyl ester, hexanoic acid, hexanoic acid N-hydroxysuccinimide ester, or isothiocyanate.
RZ is hydrogen, halogen, —N3, —CF3, —CCl3, —CBr3, —CI3, —CN, —CHO, —OH, —OCH3, —NH2, COOH, —CONH2, NO2, SH, —SO2, —SO2Cl, —SO3H, —SO4H, —SO2NH2, —NHNH2, —ONH2, —NHC(O)NHNH2, unsubstituted C1-C10 alkyl, unsubstituted 2 to 10 membered heteroalkyl, unsubstituted 3 to 6 membered cycloalkyl, unsubstituted 3 to 6 membered heterocycloalkyl, unsubstituted 5 or 6 membered aryl, or unsubstituted 5 or 6 membered heteroalkyl. In embodiments, RZ is hydrogen, Iodoacetamido, N-succinimidyl ester, hexanoic acid, hexanoic acid N-hydroxysuccinimide ester, or isothiocyanate.
The term “coumarin” as used herein refers to fluorescent derivatization agents. As used herein, coumarin includes its derivatives such as, for example, halo-substituted coumarins (e.g. Chlorocoumarin, fluorocoumarin, bromocoumarin and its derivatives), hydroxycoumarin and its derivatives including umbelliferone and its derivatives, cyanocoumarin and its derivatives, methylcoumarin and its derivatives, ethoxycoumarin and its derivatives, benzocoumarin and its derivatives, phenylcoumarin and its derivatives, acetylcoumarin and its derivatives, and carboxylated derivatives and succinimidyl esters thereof. In embodiments, the coumarin has the formula:
RT and RU are independently hydrogen, halogen, —N3, CF3, —CCl3, —CBr3, —CI3, CN, —CHO, —OH, —OCH3, NH2, COOH, —CONH2, NO2, SH, —SO2, —SO2Cl, —SO3H, —SO4H, —SO2NH2, —NHNH2, —ONH2, —NHC(O)NHNH2, unsubstituted C1-C10 alkyl, unsubstituted 2 to 10 membered heteroalkyl, unsubstituted 3 to 6 membered cycloalkyl, unsubstituted 3 to 6 membered heterocycloalkyl, unsubstituted 5 or 6 membered aryl, or unsubstituted 5 or 6 membered heteroalkyl. t1 is 1 or 2. u1 is 1, 2, or 3. In embodiments, RT is hydrogen, halogen, —NH2, —NO2, —OH, —CN, —COOH, —CF3, —OCH3, —OCH2CH3, —CH2Br, —CH2COOH, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, or substituted or unsubstituted aryl. In embodiments, RT is formyl, methyl, ethyl, propyl, bromoacetal, acetyl, acetamide, methoxymethyl, allyloxy, propanoic acid, or succinimidyl ester. In embodiments, RU is hydrogen, halogen, —OH, —NH2, —CH3, —NO2, -EtNH3, methoxy, acetyl, allyloxy, ethoxy, or acetic acid.
“Analog,” “analogue,” or “derivative” is used in accordance with its plain ordinary meaning within Chemistry and Biology and refers to a chemical compound that is structurally similar to another compound (i.e., a so-called “reference” compound) but differs in composition, e.g., in the replacement of one atom by an atom of a different element, or in the presence of a particular functional group, or the replacement of one functional group by another functional group, or the absolute stereochemistry of one or more chiral centers of the reference compound. Accordingly, an analog is a compound that is similar or comparable in function and appearance but not in structure or origin to a reference compound.
Conjugates described herein may be synthesized using bioconjugate or conjugate chemistry. Conjugate chemistry includes coupling two molecules together to form an adduct. Conjugation may be a covalent modification. Currently favored classes of conjugate chemistry reactions available with reactive known reactive groups are those which proceed under relatively mild conditions. These include, but are not limited to nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions) and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alder addition). These and other useful reactions are discussed in, for example, March, A
Useful reactive functional groups used for conjugate chemistries herein include, for example:
(a) carboxyl groups and various derivatives thereof including, but not limited to, N-hydroxysuccinimide esters, N-hydroxybenztriazole esters, acid halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromatic esters;
(b) hydroxyl groups which can be converted to esters, ethers, aldehydes, etc.
(c) haloalkyl groups wherein the halide can be later displaced with a nucleophilic group such as, for example, an amine, a carboxylate anion, thiol anion, carbanion, or an alkoxide ion, thereby resulting in the covalent attachment of a new group at the site of the halogen atom;
(d) dienophile groups which are capable of participating in Diels-Alder reactions such as, for example, maleimido groups;
(e) aldehyde or ketone groups such that subsequent derivatization is possible via formation of carbonyl derivatives such as, for example, imines, hydrazones, semicarbazones or oximes, or via such mechanisms as Grignard addition or alkyllithium addition;
(f) sulfonyl halide groups for subsequent reaction with amines, for example, to form sulfonamides;
(g) thiol groups, which can be converted to disulfides, reacted with acyl halides, or bonded to metals such as gold;
(h) amine or sulfhydryl groups, which can be, for example, acylated, alkylated or oxidized;
(i) alkenes, which can undergo, for example, cycloadditions, acylation, Michael addition, etc;
(j) epoxides, which can react with, for example, amines and hydroxyl compounds;
(k) phosphoramidites and other standard functional groups useful in nucleic acid synthesis;
(l) metal silicon oxide bonding; and
(m) metal bonding to reactive phosphorus groups (e.g. phosphines) to form, for example, phosphate diester bonds.
(n) azides coupled to alkynes using copper catalyzed cycloaddition click chemistry.
The reactive functional groups can be chosen such that they do not participate in, or interfere with, the chemical stability of the conjugate described herein. Alternatively, a reactive functional group can be protected from participating in the crosslinking reaction by the presence of a protecting group.
Molecular imaging of H2O2 with reaction-based fluorescent probes represents a non-invasive approach to monitor the chemistry of a particular ROS (e.g. H2O2) in living systems. The specific spatial and temporal distribution of H2O2 may be analyzed within a cell or a tissue (e.g. organ). Several selective probes have been reported for the detection of a number of ROS including nitric oxide,[8] peroxynitrite,[9] superoxide,[10] singlet oxygen[11] and H2O2[11]. However, these probes fail to address visualization of nominal levels of H2O2 typically found in biological systems and may have solubility issues in aqueous solvents. Furthermore, no straightforward synthetic route exists for these probes.
Herein are disclosed novel boronic esters which in the presence of H2O2, provide for the detection of endogenous H2O2 and a one-step, highly accessible synthetic procedure that allows for gram scale preparation using routine synthetic techniques.
When designing a fluorescent probe for biological imaging, several key factors should be considered including: synthetic ease, aqueous solubility and stability, kinetic rates of deprotection, and fluorescent turn-on response (i.e. conversion from pro-fluorophore to fluorophore). Ideally, a probe should also have the ability to image biologically relevant levels of H2O2 with or without external stimulation. Current H2O2 probes fail to meet these factors as these probes detect H2O2 only after exogenous addition, or by adding H2O2 stimulants such as phorbol myristate acetate (PMA) or epidermal growth factor receptor (EGFr). Thus, while these probes can detect exogenous or upregulated levels of H2O2 (such as in oxidative-mediated cell signaling), they are unable to detect nominal levels of H2O2, including H2O2 concentrations in biological systems. The compounds herein provide for detecting of H2O2 at nominal levels, including H2O2 concentrations typically found in biological systems (e.g. about 10 μM to about 100 μM H2O2).
Provided herein are compounds having the formula:
In the compound of formula (I) or formula (II), R1 and R2 are independently —B(OH)2 or
R3, R4, R5 and R6 of the compound of formula (V) are independently hydrogen, halogen, —N3, —NO2, —CF3, —CCl3, —CBr3, —CI3, —CN, —OH, —NH2, —COOH, —CONH2, —SH, —SO3H, —SO4H, —SO2NH2, —NHNH2, —ONH2, —OCH3, —NHC(O)NHNH2, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. X1 is a monovalent pro-fluorophore moiety. X2 is a divalent pro-fluorophore moiety.
R1 and R2 may independently be —B(OH)2. In embodiments, R1 is —B(OH)2. In embodiments, R2 is —B(OH)2. In embodiments, R1 and R2 are —B(OH)2. In embodiments R1 and R2 are independently
In embodiments, R1 and R2 are the same (e.g. formula (V) for R1 and R2 is identical). In embodiments, R1 and R2 are different (e.g. formula (V) for R1 and R2 is different). In embodiments, at least one of R1 and R2 in the compound of formula (II) is a moiety having formula (V).
R3, R4, R5 and R6 of the compound of formula (V) may independently be hydrogen, halogen, —N3, —NO2, —CF3, —CCl3, —CBr3, —CI3, —CN, —OH, —NH2, —COOH, —CONH2, —SH, —SO3H, —SO4H, —SO2NH2, —NHNH2, —ONH2, —OCH3, or —NHC(O)NHNH2. R3, R4, R5 and R6 may independently be hydrogen, halogen, —N3, —NO2, —CF3, —CCl3, —CBr3, —CI3, —CN, —OH, —NH2, —COOH, —CONH2, —SH, —OCH3.
R3, R4, R5 and R6 of the compound of formula (V) may independently be hydrogen, halogen, —N3, —NO2, —CH3, —CF3, —CCl3, —CBr3, —CI3, —CN, —OH, —NH2, —COOH, —CONH2, —SH, —SO3H, —SO4H, —SO2NH2, —NHNH2, —ONH2, —OCH3, —NHC(O)NHNH2, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
R3, R4, R5 and R6 of the compound of formula (V) may independently be hydrogen, halogen, —N3, —NO2, —CH3, —CF3, —CCl3, —CBr3, —CI3, —CN, —OH, —NH2, —COOH, —CONH2, —SH, —SO3H, —SO4H, —SO2NH2, —NHNH2, —ONH2, —OCH3, or —NHC(O)NHNH2. R3, R4, R5 and R6 may independently be hydrogen, halogen, —N3, —NO2, —CH3, —CF3, —CCl3, —CBr3, —CI3, —CN, —OH, —NH2, —COOH, —CONH2, —SH, —OCH3.
In embodiments, at least one of R3, R4, R5 and R6 is an electron withdrawing group (e.g. a group that removes electron density from the moiety to which it is attached). In embodiments, none of R3, R4, R5 and R6 is an electron withdrawing group. In embodiments, at least one of R3, R4, R5 and R6 is hydrogen. In embodiments, R3, R4, R5 and R6 are hydrogen. In embodiments, R3 is not hydrogen. In embodiments, R4 is not hydrogen. In embodiments, R5 is not hydrogen. In embodiments, R6 is not hydrogen. In embodiments, none of R3, R4, R5 and R6 is hydrogen.
R3, R4, R5 and R6 may independently be substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
R3, R4, R5 and R6 may independently be substituted or unsubstituted alkyl. R3, R4, R5 and R6 may independently be R7-substituted or unsubstituted alkyl. R3, R4, R5 and R6 may independently be substituted or unsubstituted C1-C20 alkyl. R3, R4, R5 and R6 may independently be R7-substituted or unsubstituted C1-C20 alkyl. R3, R4, R5 and R6 may independently be substituted or unsubstituted C1-C10 alkyl. R3, R4, R5 and R6 may independently be R7-substituted or unsubstituted C1-C10 alkyl. R3, R4, R5 and R6 may independently be substituted or unsubstituted C1-C5 alkyl. R3, R4, R5 and R6 may independently be R7-substituted or unsubstituted C1-C5 alkyl. In embodiments, at least one of R3, R4, R5 and R6 is R7-substituted or unsubstituted C1-C5 alkyl.
In embodiments, R3, R4, R5 and R6 are independently hydrogen, methyl, substituted or unsubstituted ethyl, or substituted or unsubstituted propyl. In embodiments, R3, R4, R5 and R6 are independently methyl, substituted or unsubstituted ethyl, or substituted or unsubstituted propyl. In embodiments, R3, R4, R5 and R6 are independently hydrogen, methyl, R7-substituted or unsubstituted ethyl, or R7-substituted or unsubstituted propyl. In embodiments, R3, R4, R5 and R6 are independently methyl, R7-substituted or unsubstituted ethyl, or R7-substituted or unsubstituted propyl.
R3, R4, R5 and R6 may independently be hydrogen or methyl. R3, R4, R5 and R6 may independently be methyl. R3, R4, R5 and R6 may independently be hydrogen or unsubstituted ethyl. R3, R4, R5 and R6 may independently be unsubstituted ethyl. R3, R4, R5 and R6 may independently be hydrogen or unsubstituted propyl. R3, R4, R5 and R6 may independently be unsubstituted propyl. In embodiments, at least one of R3, R4, R5 and R6 is methyl. In embodiments, R3, R4, R5 and R6 are methyl.
In embodiments, R3, R4, R5 and R6 are independently hydrogen or substituted or unsubstituted ethyl. R3, R4, R5 and R6 may independently be substituted or unsubstituted ethyl. R3, R4, R5 and R6 may independently be hydrogen or R7-substituted or unsubstituted ethyl. R3, R4, R5 and R6 may independently be R7-substituted or unsubstituted ethyl.
R3, R4, R5 and R6 may independently be hydrogen or substituted or unsubstituted propyl. R3, R4, R5 and R6 may independently be substituted or unsubstituted propyl. R3, R4, R5 and R6 may independently be hydrogen or R7-substituted or unsubstituted propyl. R3, R4, R5 and R6 may independently be R7-substituted or unsubstituted propyl.
R3, R4, R5 and R6 may independently be substituted or unsubstituted heteroalkyl. R3, R4, R5 and R6 may independently be R7-substituted or unsubstituted heteroalkyl. R3, R4, R5 and R6 may independently be substituted or unsubstituted 2 to 20 membered heteroalkyl. R3, R4, R5 and R6 may independently be R7-substituted or unsubstituted 2 to 20 membered heteroalkyl. R3, R4, R5 and R6 may independently be substituted or unsubstituted 2 to 10 membered heteroalkyl. R3, R4, R5 and R6 may independently be R7-substituted or unsubstituted 2 to 10 membered heteroalkyl. R3, R4, R5 and R6 may independently be substituted or unsubstituted 2 to 6 membered heteroalkyl. R3, R4, R5 and R6 may independently be R7-substituted or unsubstituted 2 to 6 membered heteroalkyl.
R3, R4, R5 and R6 may independently be substituted or unsubstituted cycloalkyl. R3, R4, R5 and R6 may independently be R7-substituted or unsubstituted cycloalkyl. R3, R4, R5 and R6 may independently be substituted or unsubstituted 3 to 20 membered cycloalkyl. R3, R4, R5 and R6 may independently be R7-substituted or unsubstituted 3 to 20 membered cycloalkyl. R3, R4, R5 and R6 may independently be substituted or unsubstituted 3 to 10 membered cycloalkyl. R3, R4, R5 and R6 may independently be R7-substituted or unsubstituted 3 to 10 membered cycloalkyl. R3, R4, R5 and R6 may independently be substituted or unsubstituted 3 to 6 membered cycloalkyl. R3, R4, R5 and R6 may independently be R7-substituted or unsubstituted 3 to 6 membered cycloalkyl.
R3, R4, R5 and R6 may independently be substituted or unsubstituted heterocycloalkyl. R3, R4, R5 and R6 may independently be R7-substituted or unsubstituted heterocycloalkyl. R3, R4, R5 and R6 may independently be substituted or unsubstituted 3 to 20 membered heterocycloalkyl. R3, R4, R5 and R6 may independently be R7-substituted or unsubstituted 3 to 20 membered heterocycloalkyl. R3, R4, R5 and R6 may independently be substituted or unsubstituted 3 to 10 membered heterocycloalkyl. R3, R4, R5 and R6 may independently be R7-substituted or unsubstituted 3 to 10 membered heterocycloalkyl. R3, R4, R5 and R6 may independently be substituted or unsubstituted 3 to 6 membered heterocycloalkyl. R3, R4, R5 and R6 may independently be R7-substituted or unsubstituted 3 to 6 membered heterocycloalkyl.
R3, R4, R5 and R6 may independently be substituted or unsubstituted aryl. R3, R4, R5 and R6 may independently be R7-substituted or unsubstituted aryl. R3, R4, R5 and R6 may independently be substituted or unsubstituted 5 to 10 membered aryl. R3, R4, R5 and R6 may independently be R7-substituted or unsubstituted 5 to 10 membered aryl. R3, R4, R5 and R6 may independently be substituted or unsubstituted 5 to 8 membered aryl. R3, R4, R5 and R6 may independently be R7-substituted or unsubstituted 5 to 8 membered aryl. R3, R4, R5 and R6 may independently be substituted or unsubstituted 5 or 6 membered aryl. R3, R4, R5 and R6 may independently be R7-substituted or unsubstituted 5 or 6 membered aryl.
R3, R4, R5 and R6 may independently be substituted or unsubstituted heteroaryl. R3, R4, R5 and R6 may independently be R7-substituted or unsubstituted heteroaryl. R3, R4, R5 and R6 may independently be substituted or unsubstituted 5 to 10 membered heteroaryl. R3, R4, R5 and R6 may independently be R7-substituted or unsubstituted 5 to 10 membered heteroaryl. R3, R4, R5 and R6 may independently be substituted or unsubstituted 5 to 8 membered heteroaryl. R3, R4, R5 and R6 may independently be R7-substituted or unsubstituted 5 to 8 membered heteroaryl. R3, R4, R5 and R6 may independently be substituted or unsubstituted 5 or 6 membered heteroaryl. R3, R4, R5 and R6 may independently be R7-substituted or unsubstituted 5 or 6 membered heteroaryl.
R7 is halogen, —N3, —NO2, —CF3, —CCl3, —CBr3, —CI3, —CN, —OH, —NH2, —COOH, —CONH2, —SH, —SO3H, —SO4H, —SO2NH2, —NHNH2, —ONH2, —OCH3, —NHC(O)NHNH2, R8-substituted or unsubstituted alkyl, R8-substituted or unsubstituted heteroalkyl, R8-substituted or unsubstituted cycloalkyl, R8-substituted or unsubstituted heterocycloalkyl, R8-substituted or unsubstituted aryl, or R8-substituted or unsubstituted heteroaryl. In embodiments, R7 is halogen, —NO2, —CF3, —CN, —OH, —NH2, —COOH, —CONH2, —SH, —OCH3, or R8-substituted or unsubstituted alkyl. In embodiments, R7 is halogen, —CF3, —OH, —NH2, —COOH, —CONH2, or R8-substituted or unsubstituted alkyl. In embodiments, R7 is R8-substituted or unsubstituted alkyl, R8-substituted or unsubstituted heteroalkyl, R8-substituted or unsubstituted cycloalkyl, R8-substituted or unsubstituted heterocycloalkyl, R8-substituted or unsubstituted aryl, or R8-substituted or unsubstituted heteroaryl. In embodiments, R7 is R8-substituted or unsubstituted alkyl.
R8 is halogen, —N3, —NO2, —CF3, —CCl3, —CBr3, —CI3, —CN, —OH, —NH2, —COOH, —CONH2, —SH, —SO3H, —SO4H, —SO2NH2, —NHNH2, —ONH2, —OCH3, —NHC(O)NHNH2, R9-substituted or unsubstituted alkyl, R9-substituted or unsubstituted heteroalkyl, R9-substituted or unsubstituted cycloalkyl, R9-substituted or unsubstituted heterocycloalkyl, R9-substituted or unsubstituted aryl, or R9-substituted or unsubstituted heteroaryl. In embodiments, R8 is halogen, —N3, —NO2, —CF3, —CCl3, —CBr3, —CI3, —CN, —OH, —NH2, —COOH, —CONH2, —SH, —SO3H, —SO4H, —SO2NH2, —NHNH2, —ONH2, —OCH3, —NHC(O)NHNH2. In embodiments, R8 is halogen, —NO2, —CF3, —CN, —OH, —NH2, —COOH, —CONH2, or —OCH3. In embodiments, R7 is R8-substituted or unsubstituted alkyl,
where R8 is halogen, —NO2, —CF3, —CN, —OH, —NH2, —COOH, —CONH2, —OCH3, or R9-substituted or unsubstituted alkyl, R9-substituted or unsubstituted heteroalkyl, R9-substituted or unsubstituted cycloalkyl, R9-substituted or unsubstituted heterocycloalkyl, R9-substituted or unsubstituted aryl, or R9-substituted or unsubstituted heteroaryl. In embodiments, R7 is R8-substituted or unsubstituted alkyl, where R8 is halogen, —CF3, —OH, —NH2, —COOH, —CONH2, —OCH3.
R9 is halogen, —N3, —NO2, —CF3, —CCl3, —CBr3, —CI3, —CN, —OH, —NH2, —COOH, —CONH2, —SH, —SO3H, —SO4H, —SO2NH2, —NHNH2, —ONH2, —OCH3, —NHC(O)NHNH2, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl.
X1 may be a monovalent pro-fluorescein, monovalent pro-coumarin, monovalent pro-dansyl, monovalent pro-bimane, monovalent pro-BODIPY, monovalent pro-eosin, monovalent pro-rhodamine, monovalent pro-texas red, monovalent pro-PyMPO, monovalent pro-cyanine; monovalent pro-nile red, monovalent pro-xanthone (e.g. substituted or unsubstituted xanthone), monovalent-pro-xanthene (e.g. substituted or unsubstituted xanthene), monovalent pro-flazo orange, monovalent pro-Snarf1, or a monovalent pro-resorufin, or conjugates, derivatives, or analogues thereof.
In embodiments, monovalent pro-fluorescein (including conjugates, derivatives, and analogues thereof), monovalent pro-coumarin (including conjugates, derivatives, and analogues thereof), monovalent pro-dansyl, monovalent pro-bimane, monovalent pro-eosin, monovalent pro-rhodamine, monovalent pro-cyanine, monovalent nile red, monovalent xanthone, monovalent xanthene, monovalent flazo orange, monovalent Snarf1, or monovalent resorufin, including conjugates, derivatives and analogues thereof.
In embodiments, X1 is monovalent pro-fluorescein, monovalent pro-coumarin, monovalent pro-dansyl, monovalent pro-bimane, monovalent pro-BODIPY, monovalent pro-eosin, monovalent pro-rhodamine, monovalent pro-texas red, monovalent pro-PyMPO, or monovalent pro-cyanine, including conjugates, derivatives, and analogues thereof. X1 may be a monovalent pro-fluorescein (including conjugates, derivatives, and analogues thereof) or a monovalent pro-coumarin (including conjugates, derivatives, and analogues thereof). X1 may be a monovalent pro-fluorescein (including conjugates, derivatives, and analogues thereof). X1 may be a monovalent pro-coumarin (including conjugates, derivatives, and analogues thereof).
In embodiments, X2 is divalent pro-fluorescein, divalent pro-coumarin, divalent pro-dansyl, divalent pro-bimane, divalent pro-texas red, divalent pro-PyMPO, divalent pro-BODIPY, divalent pro-eosin, divalent pro-rhodamine, or divalent pro-cyanine, divalent nile red, divalent xanthones, divalent substituted xanthenes, divalent flazo orange, divalent Snarf1, or divalent resorufin, including conjugates, derivatives, and analogues thereof. X2 may be a divalent pro-fluorescein (including conjugates, derivatives, and analogues thereof) or a divalent pro-coumarin (including conjugates, derivatives, and analogues thereof). X2 may be a divalent pro-fluorescein (including conjugates, derivatives, and analogues thereof). X2 may be a divalent pro-coumarin (including conjugates, derivatives, and analogues thereof).
In embodiments, the compound of formula (I) has the formula:
R1 and X1 are as described herein. In embodiments, R1 is a moiety having formula (V), where R3, R4, R5 and R6 are independently C1-C5 alkyl. X1 may be a monovalent pro-fluorescein (including conjugates, derivatives, and analogues thereof) or a monovalent pro-coumarin (including conjugates, derivatives, and analogues thereof).
In embodiments, the compound of formula (I) has the formula:
R1, RW, RX, RY, RZ, w1, x1, and y1 are as described herein. In embodiments, RW and RX are independently hydrogen, halogen or —OCH3. In embodiments, RY and RZ are independently hydrogen, halogen, —OH, —OCH3, —COOH, —CH2Br, Iodoacetamido, N-succinimidyl ester, hexanoic acid, hexanoic acid N-hydroxysuccinimide ester, or isothiocyanate.
In embodiments, the compound of formula (I3) has the formula:
In embodiments, the compound of formula (I3) has the formula:
In embodiments, R3, R4, R5 and R6 are independently C1-C5 substituted or unsubstituted alkyl.
In embodiments, the compound of formula (I3) has the formula:
In embodiments, the compound of formula (I10) has the formula:
In embodiments, the compound of formula (I10) has the formula:
In embodiments, R3, R4, R5 and R6 are independently C1-C5 substituted or unsubstituted alkyl.
In embodiments, the compound of formula (I) has the formula:
R1, RT, RU, t1, and u1 are as described herein. In embodiments, RT is hydrogen, halogen, —NH2, —NO2, —OH, —CN, —COOH, —CF3, —OCH3, —OCH2CH3, —CH2Br, —CH2COOH, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, or substituted or unsubstituted aryl. In embodiments, RT is formyl, methyl, ethyl, propyl, bromoacetal, acetyl, acetamide, methoxymethyl, allyloxy, propanoic acid, or succinimidyl ester. In embodiments, RU is hydrogen, halogen, —OH, —NH2, —CH3, —NO2, -EtNH3, methoxy, acetyl, allyloxy, ethoxy, or acetic acid.
In embodiments, the compound of formula (I15) has the formula:
In embodiments, the compound of formula (I15) has the formula:
In embodiments, R3, R4, R5 and R6 are independently C1-C5 substituted or unsubstituted alkyl.
In embodiments, the compound of formula (I) has the formula:
In embodiments, the compound of formula (I22) has the formula:
In embodiments, the compound of formula (I22) has the formula:
In embodiments, R3, R4, R5 and R6 are independently C1-C5 substituted or unsubstituted alkyl.
In embodiments, the compound of formula (I) has the formula:
(i.e. FBBE).
In embodiments, the compound of formula (I) has the formula:
(i.e. CBBE).
In embodiments, the compound of formula (I) has formula:
In embodiments, the compound of formula (II) has the formula:
R1, R2, X1, and X2 are as described herein. In embodiments, R1 is a compound of formula (V), where R3, R4, R5 and R6 are independently methyl. In embodiments, R2 is a compound of formula (V), where R3, R4, R5 and R6 are independently methyl. In embodiments, R1 and R2 are identical. In embodiments, R1 and R2 are independently a compound of formula (V) or —B(OH)2. In embodiments, R1 and R2 are —B(OH)2. X1 may be a divalent pro-fluorescein (including conjugates, derivatives, and analogues thereof) or a divalent pro-coumarin (including conjugates, derivatives, and analogues thereof).
In embodiments, the compound of formula (II) has the formula:
R1, R2, RW, RX, RY, RZ, w1, x1, and y1 are as described herein. In embodiments, RW and RX are independently hydrogen, halogen or —OCH3. In embodiments, RY and RZ are independently hydrogen, halogen, —OH, —OCH3, —COOH, —CH2Br, Iodoacetamido, N-succinimidyl ester, hexanoic acid, hexanoic acid N-hydroxysuccinimide ester, or isothiocyanate.
In embodiments, the compound of formula (II3) has the formula:
In embodiments, the compound of formula (II3) has the formula:
In embodiments, the compound of formula (II) has the formula:
In embodiments, the compound of formula (II10) has the formula:
In embodiments, the compound of formula (II10) has the formula:
In embodiments, R3, R4, R5 and R6 are independently C1-C5 substituted or unsubstituted alkyl.
In embodiments, the compound of formula (II) has the formula:
(i.e. FBBBE).
In embodiments, the compound of formula (II) has the formula:
Also provided herein are compounds having formula (III):
R1 and R2 are as described herein (e.g. formula (I) and (II), including embodiments thereof). R3 is —B(OH)2 or a moiety of formula (V) as described herein, including embodiments thereof. In embodiments, R1, R2, and R3 are identical. X3 is a trivalent pro-fluorophore. In embodiments, X3 is trivalent pro-fluorescein, trivalent pro-coumarin, trivalent pro-dansyl, trivalent pro-bimane, trivalent pro-texas red, trivalent pro-PyMPO, trivalent pro-BODIPY, trivalent pro-eosin, trivalent pro-rhodamine, or trivalent pro-cyanine, trivalent nile red, trivalent xanthone, trivalent substituted xanthene, trivalent flazo orange, trivalent Snarf1, or trivalent resorufin, including conjugates, derivatives, and analogues thereof.
Also provided herein are compounds of having formula (IV):
R1, R2, and R3, are as described herein (e.g. formula (I), (II) and (III)), including embodiments thereof. R4 is —B(OH)2 or a moiety of formula (V) as described herein, including embodiments thereof. In embodiments, R1, R2, R3 and R4 are identical. X4 is a tetravalent pro-fluorophore. In embodiments, X4 is tetravalent pro-fluorescein, tetravalent pro-coumarin, tetravalent pro-dansyl, tetravalent pro-bimane, tetravalent pro-texas red, tetravalent pro-PyMPO, tetravalent pro-BODIPY, tetravalent pro-eosin, tetravalent pro-rhodamine, or tetravalent pro-cyanine, tetravalent nile red, tetravalent xanthone, tetravalent substituted xanthene, tetravalent flazo orange, tetravalent Snarf1, or tetravalent resorufin, including conjugates, derivatives, and analogues thereof.
The compounds described herein (e.g. compounds of formula (I), (II), (III), and (IV), including embodiments thereof) may be synthesized according to the scheme shown in
The compounds described herein are useful in methods of detecting a ROS compound. Such methods include contacting a compound of formula (I) or formula (II) with a ROS compound. The ROS compound is allowed to react with the compound of formula (I) and remove R1 or allowed to react with the compound of formula (II) and remove R1 and R2. The ROS compound may remove at least one of R1 and R2. Removing R1 of the compound of formula (I) or R1 and R2 of the compound of formula (II) forms a fluorescent compound. In embodiments, removing at least one of R1 and R2 of the compound of formula (II) forms a fluorescent compound. The fluorescent compound is detected thus detecting the ROS compound. The detection may be performed using techniques known in the art. In embodiments, the subject is a cell.
In embodiments, the detecting includes quantifying an amount or level of the ROS compound. The quantifying may include determining an amount of fluorescence from the fluorescent compound. The amount of the fluorescence may be linearly proportional to the concentration of the ROS species (i.e. a greater concentration of ROS species results in a greater amount of fluorescence). In embodiments, the amount is compared to a control to determine the fold increase upon conversion of the pro-fluorophore to the fluorophore (e.g. fluorescent turn-on response).
The ROS compound may be H2O2, OCl−, or ONOO−. In embodiments, the ROS compound is H2O2. In embodiments, the ROS compound is OCL. In embodiments, the ROS compound is ONOO−. R1 and X1 of the compound of formula (I) are as described herein, including embodiments thereof. In embodiments, R1 is a moiety of formula (V), where R3, R4, R5, and R6 are as described herein. In embodiments, R3, R4, R5, and R6 independently substituted or unsubstituted alkyl. R3, R4, R5, and R6 may independently be R7-substituted or unsubstituted alkyl, where R7 is as described herein, including embodiments thereof. R3, R4, R5, and R6 may independently be R7-substituted or unsubstituted C1-C5 alkyl. R3, R4, R5, and R6 may independently be methyl. In embodiments, R1 is —B(OH)2. In embodiments, X1 is monovalent pro-fluorescein (including conjugates, derivatives and analogues thereof) or monovalent pro-coumarin (including conjugates, derivatives and analogues thereof). X1 may be monovalent pro-fluorescein (including conjugates, derivatives and analogues thereof). X1 may be monovalent pro-coumarin (including conjugates, derivatives and analogues thereof).
R1, R2, and X2 of the compound of formula (II) are as described herein, including embodiments thereof. In embodiments, R1 and R2 are a moiety of formula (V), where R3, R4, R5, and R6 are as described herein. In embodiments, R1 and R2 are identical (e.g. R3, R4, R5, and R6 are identical). In embodiments, R1 and R2 are —B(OH)2. In embodiments, R3, R4, R5, and R6 independently substituted or unsubstituted alkyl. R3, R4, R5, and R6 may independently be R7-substituted or unsubstituted alkyl, where R7 is as described herein, including embodiments thereof. R3, R4, R5, and R6 may independently be R7-substituted or unsubstituted C1-C5 alkyl. R3, R4, R5, and R6 may independently be methyl. In embodiments, X2 is divalent pro-fluorescein (including conjugates, derivatives and analogues thereof). In embodiments, X2 is divalent pro-fluorescein, divalent pro-rhodamine, divalent pro-nile red, divalent xanthone (e.g. substituted or unsubstituted), divalent xanthene (e.g. substituted or unsubstituted), divalent flazo orange, divalent Snarf1, or divalent resorufin, including conjugates, derivatives and analogues thereof.
The compounds herein are also useful in detecting a ROS compound in vivo. The method includes administering to a subject, a compound of formula (I) or formula (II). The compound of formula (I) is allowed to react with a ROS compound and remove R1. The compound of formula (II) is allowed to react with a ROS compound and remove R1 and R2. The ROS compound may remove at least one of R1 and R2. Removing R1 of the compound of formula (I) or R1 and R2 of the compound of formula (II) forms a fluorescent compound. In embodiments, removing at least one of R1 and R2 of the compound of formula (II) forms a fluorescent compound. The fluorescent compound is detected thus detecting the ROS compound in vivo. The detection may be performed using techniques known in the art.
R1 and R2 are as described herein, including embodiments thereof. In embodiments, R1 and R2 are a moiety of compound (V). R1 and R2 may be the identical. In embodiments, R1 and R2 are independently —B(OH)2. X1 and X2 are as described herein, including embodiments thereof. In embodiments, X1 is monovalent pro-fluorescein (including conjugates, derivatives and analogues thereof) or monovalent pro-coumarin (including conjugates, derivatives and analogues thereof). X1 may be monovalent pro-fluorescein (including conjugates, derivatives and analogues thereof). X1 may be monovalent pro-coumarin (including conjugates, derivatives and analogues thereof). In embodiments, X2 is divalent pro-fluorescein (including conjugates, derivatives and analogues thereof). In embodiments, X2 is divalent pro-fluorescein, divalent pro-rhodamine, divalent pro-nile red, divalent xanthone (e.g. substituted or unsubstituted), divalent xanthene (e.g. substituted or unsubstituted), divalent flazo orange, divalent Snarf1, or divalent resorufin, including conjugates, derivatives and analogues thereof.
In embodiments, R1 and R2 are removed at physiological levels of H2O2. In embodiments, R1 and R2 are removed at a H2O2 concentration of about 5 μM to about 200 μM. R1 and R2 may be removed at a H2O2 concentration of about 10 μM to about 200 μM. R1 and R2 may be removed at a H2O2 concentration of about 10 μM to about 100 μM. R1 and R2 may be removed at a H2O2 concentration of about 10 μM to about 90 μM. R1 and R2 may be removed at a H2O2 concentration of about 10 μM to about 80 μM. R1 and R2 may be removed at a H2O2 concentration of about 10 μM to about 70 μM. R1 and R2 may be removed at a H2O2 concentration of about 10 μM to about 60 μM. R1 and R2 may be removed at a H2O2 concentration of about 10 μM to about 50 μM. R1 and R2 may be removed at a H2O2 concentration of about 10 μM to about 40 μM. R1 and R2 may be removed at a H2O2 concentration of about 10 μM to about 30 μM. R1 and R2 may be removed at a H2O2 concentration of about 10 μM to about 20 μM.
R1 and R2 may be removed at a H2O2 concentration of about 5 μM to about 10 μM. R1 and R2 may be removed at a H2O2 concentration of about 5 μM to about 20 μM. R1 and R2 may be removed at a H2O2 concentration of about 5 μM to about 30 μM. R1 and R2 may be removed at a H2O2 concentration of about 5 μM to about 40 μM. R1 and R2 may be removed at a H2O2 concentration of about 5 μM to about 50 μM. R1 and R2 may be removed at a H2O2 concentration of about 5 μM to about 60 μM. R1 and R2 may be removed at a H2O2 concentration of about 5 μM to about 70 μM. R1 and R2 may be removed at a H2O2 concentration of about 5 μM to about 80 μM. R1 and R2 may be removed at a H2O2 concentration of about 5 μM to about 90 μM. R1 and R2 may be removed at a H2O2 concentration of about 5 μM to about 100 μM.
R1 and R2 may be removed at a H2O2 concentration of about 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 85, 90, 95, 100, 125, 150, 175, or about 200 μM. R1 and R2 may be removed at a H2O2 concentration of at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 85, 90, 95, 100, 125, 150, 175, or 200 μM. R1 and R2 may be removed at a H2O2 concentration of at least 5 μM. R1 and R2 may be removed at a H2O2 concentration of at least 10 μM. R1 and R2 may be removed at a H2O2 concentration of at least 20 μM. R1 and R2 may be removed at a H2O2 concentration of at least 30 μM. R1 and R2 may be removed at a H2O2 concentration of at least 40 μM. R1 and R2 may be removed at a H2O2 concentration of at least 50 μM. R1 and R2 may be removed at a H2O2 concentration of at least 60 μM. R1 and R2 may be removed at a H2O2 concentration of at least 70 μM. R1 and R2 may be removed at a H2O2 concentration of at least 80 μM. R1 and R2 may be removed at a H2O2 concentration of at least 90 μM. R1 and R2 may be removed at a H2O2 concentration of at least 100 μM. R1 and R2 may be removed at a H2O2 concentration of at least 150 μM. R1 and R2 may be removed at a H2O2 concentration of at least 200 μM.
R1 and R2 may be removed at a H2O2 concentration of at least 5 μM to at least 200 μM. R1 and R2 may be removed at a H2O2 concentration of at least 5 μM to at least 100 μM. R1 and R2 may be removed at a H2O2 concentration of at least 10 μM to at least 200 μM. R1 and R2 may be removed at a H2O2 concentration of at least 10 μM to at least 100 μM.
In embodiments, amount of fluorescence of the fluorescent compound (e.g. fluorescent turn-on response) is at least 5-, 10-, 20-, 30-, 40-, 50-, 55-, 60-, 65-, 70-, 80-, 90-, 95-, or 100-fold increased after contacting with the ROS compound. The amount of fluorescence of the fluorescent compound may be at least 10-fold increased after contacting with the ROS compound. The amount of fluorescence of the fluorescent compound may be at least 20-fold increased after contacting with the ROS compound. The amount of fluorescence of the fluorescent compound may be at least 30-fold increased after contacting with the ROS compound. The amount of fluorescence of the fluorescent compound may be at least 40-fold increased after contacting with the ROS compound. The amount of fluorescence of the fluorescent compound may be at least 50-fold increased after contacting with the ROS compound. The amount of fluorescence of the fluorescent compound may be at least 55-fold increased after contacting with the ROS compound. The amount of fluorescence of the fluorescent compound may be at least 60-fold increased after contacting with the ROS compound. The amount of fluorescence of the fluorescent compound may be at least 65-fold increased after contacting with the ROS compound. The amount of fluorescence of the fluorescent compound may be at least 70-fold increased after contacting with the ROS compound. The amount of fluorescence of the fluorescent compound may be at least 80-fold increased after contacting with the ROS compound. The amount of fluorescence of the fluorescent compound may be at least 90-fold increased after contacting with the ROS compound. The amount of fluorescence of the fluorescent compound may be at least 100-fold increased after contacting with the ROS compound.
The detecting of the ROS compound may include detecting the location of the ROS compound in the subject. Thus, in embodiments, detection of fluorescence at a location in the subject using the compounds herein indicates the location has ROS activity. In embodiments, the detection of the ROS compound may indicate a particular disease state or injury. In embodiments, the detecting includes quantifying a level or amount of the ROS compound in the subject. The amount of the fluorescence may be linearly proportional to the concentration of the ROS compound (i.e. a greater concentration of ROS compound results in a greater amount of fluorescence). The detection may be performed using techniques known in the art.
In embodiments, the monovalent pro-fluorophore moiety has substantially no fluorescence (e.g. less than 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%) in the absence of a ROS compound when compared to the pro-fluorophore after contact with a ROS compound (e.g. fluorescent compound). In embodiments, the divalent pro-fluorophore moiety has substantially no fluorescence (e.g. less than 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%) in the absence of a ROS compound when compared to the pro-fluorophore after contact with a ROS compound (e.g. fluorescent compound).
All chemicals were purchased from commercial suppliers (Aldrich, Alfa Aesar, or Fisher) and used as provided. Chromatography was performed using a CombiFlash Rf-200 automated system from TeledyneISCO. 1H and 13C NMR spectra were recorded on a Varian FT-NMR instrument running at 400 MHz at the Department of Chemistry and Biochemistry, University of California, San Diego. Mass spectrometry was performed at the Molecular Mass Spectrometry Facility in the Department of Chemistry and Biochemistry at the University of California, San Diego.
3′,6′-bis((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one (FBBBE): Fluorescein (2.45 g, 7.3 mmol) was dissolved in 50 mL MeCN. To this was added Cs2CO3 (7.21 g, 22.1 mmol) followed by 4-bromomethylphenyl boronic acid pinacol ester (6.57 g, 22.1 mmol). The reaction was held at reflux for 18 h under nitrogen. The mixture was then cooled to room temperature, filtered, and rinsed with CH2Cl2. The solvent was removed and the product was purified by silica gel chromatography, eluting with 20-30% EtOAc in hexanes to afford FBBBE in 31% yield (1.72 g, 2.3 mmol). 1H NMR (400 MHz, CDCl3): δ=8.01 (d, J=7.6 Hz, 1H), 7.85 (d, J=7.6 Hz, 4H), 7.62 (m, 2H), 7.43 (d, J=7.6 Hz, 4H), 7.15 (d, J=7.6 Hz, 1H), 6.82-6.81 (m, 2H), 6.70-6.67 (m, 4H), 5.11 (s, 4H), 1.35 (s, 24H). 13C NMR (100 MHz, CDCl3): δ=169.7, 160.5, 153.4, 152.6, 139.6, 135.3, 135.2, 129.9, 129.4, 127.0, 126.7, 125.2, 124.2, 112.5, 111.7, 102.1, 84.1, 83.4, 70.4, 25.1. ESI-MS (+): m/z 765.44 [M+H]+, 787.37 [M+Na]+.
Fluorescein (0.75 g, 2.3 mmol) was dissolved in 15 mL MeCN. To this was added Cs2CO3 (2.21 g, 6.8 mmol) followed by benzyl bromide (1.1 mL, 9.0 mmol). The reaction was heated to reflux for 18 h under nitrogen. The mixture was then cooled to room temperature, filtered, and rinsed with CH2Cl2. The solvent was removed and the product was purified by silica gel chromatography, eluting with 5-25% EtOAc in hexanes to afford FBn in 50% yield (0.58 g, 1.1 mmol). 1H NMR (400 MHz, CDCl3): δ=8.03, (d, J=7.2 Hz, 1H), 7.69-7.60 (m, 2H), 7.45-7.33 (m, 10H), 7.17 (d, J=7.2 Hz, 1H), 6.85 (s, 2H), 6.71-6.79 (m, 4H), 5.10 (s, 2H). 13C NMR (100 MHz, CDCl3): δ=169.7, 160.7, 153.3, 152.7, 136.5, 135.2, 129.9, 129.4, 128.9, 128.4, 127.7, 127.1, 125.2, 124.2, 112.5, 111.7, 102.1, 83.4, 70.5. ESI-MS (+): m/z 513.15 [M+H]+.
7-hydroxycoumarin (1.25 g, 7.7 mmol) was dissolved in 15 mL of anhydrous DMF. To this was added of K2CO3 (3.20 g, 23.1 mmol) followed by 4-bromomethylphenyl boronic acid pinacol ester (6.18 g, 20.8 mmol). The reaction was heated to 80° C. under nitrogen for 18 h. The mixture was then cooled to room temperature, filtered, and rinsed with CH2Cl2. The filtrate was then washed with water and brine, dried over MgSO4, filtered, and concentrated. The resulting residue was purified via silica gel chromatography eluting with 20% EtOAc in hexanes to afford the desired product in 69% yield (2.01 g, 5.3 mmol). 1H NMR (400 MHz, CDCl3): δ=7.83 (d, J=8.1 Hz, 2H), 7.63 (d, J=9.5 Hz, 1H), 7.42 (d, J=8.1 Hz, 2H), 7.37 (d, J=8.6 Hz, 1H), 6.90 (dd, J1=8.6 Hz, J2=2.4 Hz, 1H), 6.87 (d, J=2.4 Hz, 1H), 6.25 (d, J=9.5 Hz, 1H), 5.15 (s, 2H), 1.35 (s, 12H). 13C NMR (100 MHz, CDCl3): δ=162.0, 161.4, 156.0, 143.61, 139.01, 135.4, 129.0, 126.8, 113.5, 113.4, 113.0, 102.2, 84.1, 70.6, 25.1. ESI-MS: (+) m/z: 378.95 [M+H]+, 395.79 [M+NH4]+.
7-Hydroxycoumarin (0.65 g, 4.0 mmol) was dissolved in 20 mL of MeCN. To this was added Cs2CO3 (2.61 g, 8.0 mmol) followed by benzyl bromide (1.0 mL, 8.0 mmol). The reaction was heated to 80° C. under nitrogen for 18 h.
The mixture was then cooled to room temperature, filtered, and rinsed with CH2Cl2. The solvent was removed and the resulting residue was purified by silica gel chromatography eluting with 20-50% EtOAc in hexanes to afford the purified product in 78% yield (0.78 g, 3.1 mmol). 1H NMR (400 MHz, CDCl3): δ=7.62 (d, J=9.6 Hz, 2H), 7.44-7.35 (m, 6H), 6.91 (dd, J1=8.4 Hz, J2=2.4 Hz, 1H), 6.87 (d, J=2.4 Hz, 1H), 5.12 (s, 2H). 13C NMR (100 MHz, CDCl3): δ=162.1, 161.4, 156.0, 143.6, 136.0, 129.1, 129.0, 128.6, 127.8, 113.5, 113.4, 112.9, 102.1, 70.7. ESI-MS (+): m/z 253.03 [M+H]+, 269.72 [M+NH4]+.
Fluorescein methyl ester (S. Y. Chen, et al., Bioconjugate Chem. 2010, 21, 979-987.) (2.09 g, 6.0 mmol) was dissolved in 20 mL DMF. To this was added potassium carbonate (2.50 g, 18.1 mmol) followed by 4-bromomethylphenyl boronic acid pinacol ester (3.23 g, 10.9 mmol). The reaction was held at 80° C. for 18 h under nitrogen. The mixture was then cooled to RT and filtered, rinsing with CH2Cl2. The solvent was removed and the resulting residue was purified by silica gel chromatography eluting 50-90% EtOAc in hexanes to afford 1 in 49% yield (1.66 g, 3.0 mmol). 1H NMR (400 MHz, DMSO): δ=8.20 (d, J=8.0 Hz, 1H), 7.86 (td, J1=8.0 Hz, J2=1.2 Hz, 1H), 7.77 (td, J1=8.0 Hz, J2=1.2 Hz, 1H), 7.70 (d, J=8.0 Hz, 2H), 7.48 (t, J=8.0 Hz, 3H), 7.28 (d, J=2.8 Hz, 1H), 6.95 (dd, J1=9.2 Hz, J2=2.4 Hz, 1H), 6.83 (d, J=8.8 Hz, 1H), 6.78 (d, J=9.6 Hz, 1H), 6.37 (dd, J1=9.6 Hz, J2=2.0 Hz, 1H), 6.22 (d, J=2.0 Hz, 1H), 5.32 (s, 2H), 3.57 (s, 3H), 1.28 (s, 12H). ESI-MS (+): m/z 563.2 [M+H]+.
1 (1.00 g, 1.8 mmol) was dissolved in 30 mL THF and 30 mL 4% KOH (aq). The reaction was held at RT for 12 h under nitrogen. The solvent was then removed and the resulting residue was brought up in 20 mL H2O and 20 mL 1 M HCl. An extraction with EtOAc (4×50 mL) was performed, collecting the organic layer. This layer was dried over MgSO4, filtered and concentrated. The solvent was removed and the resulting residue was purified by silica gel chromatography eluting 30-70% EtOAc in hexanes to afford FBBE in 56% yield (0.59 g, 1.1 mmol). 1H NMR (400 MHz, CDCl3): δ=8.01 (d, J=7.6 Hz, 1H), 7.83 (d, J=7.6 Hz, 1H), 7.68-7.59 (m, 2H), 7.42 (d, J=6.4 Hz, 2H), 7.16 (d, J=7.2 Hz, 1H), 6.80 (d, J=2.8 Hz, 1H), 6.73 (J=2.8 Hz, 1H), 6.67-6.65 (m, 2H), 6.58 (dd, J1=8.8 Hz, J2=0.8 Hz), 6.54-6.51 (m, 1H), 5.11 (s, 2H), 1.35 (s, 12H). ESI-MS (+): m/z 549.2 [M+H]+.
Luminescence Spectroscopy.
Fluorescence emission spectra were recorded with a Perkin-Elmer LS-55 fluorescence spectrometer. To a 2.0 mL solution of probe of varying concentrations in HEPES buffer (50 mM, pH 7.5) was added H2O2 in HEPES to monitor fluorescence turn-on. Spectra were monitored over time at room temperature with quartz cuvettes.
UV-Vis Spectroscopy.
Absorption spectra were taken on a Perkin-Elmer Lambda 25 UV-visible spectrophotometer. To a 1.0 mL solution of 50 μM probe in HEPES buffer (50 mM, pH 7.5) was added varying concentrations of H2O2 in HEPES to monitor deprotection. Spectra were monitored over time at room temperature with quartz cuvettes.
Calculation of Rate Constants.
The pseudo-first order rate constant was calculated by monitoring the absorption spectra over time in the presence of excess H2O2. To a 1.0 mL solution of 50 μM FBBBE or CBBE in HEPES buffer (50 mM, pH 7.5) was added H2O2 to a final concentration of 10 mM. Spectra were monitored over 5-15 min at room temperature with at least 100 spectra recorded. The change in absorption at 494 nm for FBBBE and 370 nm for CBBE were monitored. The rate constant (kobs) was determined by monitoring the appearance of the absorption peak by plotting the linear slope of ln [(Amax−A)/(Amax)] vs. time where Amax is the absorbance of a 50 μM sample of fluorescein (at 494 nm) for FBBBE and 7-hydroxycoumarin (at 370 nm) for CBBE. All rate constants were measured in triplicate using three independent experiments.
Calculation of Fluorescence Turn-On.
To quantitate the fluorescence turn-on, a sample of each probe (1 μM in 50 mM HEPES pH 7.5) was prepared. An excess of H2O2 (300 eq, 300 μM) was added to the latent fluorophore and spectra were recorded until no increase in fluorescence intensity was observed, signifying a complete reaction. The collected initial and final emission spectrum were integrated, and the ratio reports the fluorescence turn on.
Cell Imaging Experiments.
RAW 264.7 macrophages were grown in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 μg/mL penicillin, and 100 μg/mL streptomycin (Life Technologies, Carlsbad, Calif.). For confocal microscopy, the RAW 264.7 cells were seeded at 250,000 in glass bottom 35 mm dishes (MatTek Corporation, Ashland, Mass.) and post-adhering (5 h), some dishes were treated with 1 μg/mL of LPS (Sigma-Aldrich, St. Louis, Mo.) for 24 h. Other dishes were treated the next day after plating with either 100 μM H2O2 for 1 h or PBS for 1 h. Cells were washed twice with PBS and fixed in ice-cold 95% ethanol for 15 min. Cells were twice rinsed with PBS and incubated with either 50 μM probe (FBBBE) or 50 μM control (FBn) for 1 h at room temperature. Cells were washed twice with PBS and coverslips were mounted over cells using Aqua Mount (Thermo Scientific, Waltham, Mass.). Confocal images were obtained at 80× magnification on an Olympus FV1000 confocal laser scanning microscope. Images were processed using ImageJ software (NIH).
Immunohistological Studies.
3-4 week old female C57BL/6J mice were purchased from Jackson Laboratory (Bar Harbor, Me.). Mice were sacrificed and perfused with sterile PBS. The brain and spinal cord were harvested and imbedded in Tissue-Tek® OCT Compound (Sakura Finetek USA, Torrance, Calif.) and frozen in dry ice before storage at −80° C. All animal studies were reviewed and approved by the University of California, San Diego Institutional Animal Care and Use Committee (La Jolla, Calif.).
For confocal microscopy, the frozen tissue was cut into 10-μm sections on a Leica cryomicrotome and mounted on slides. Tissue sections were fixed in ice-cold 95% ethanol for 15 min and rehydrated in PBS for 5 min at room temperature in a humidity chamber. Tissue sections were either incubated with PBS or 100 μM H2O2 for 1 h at room temperature in a humidity chamber, followed by a 1 h incubation with either 50 μM probe (FBBBE) or 50 μM control (FBn) and a 10 min nuclear stain (DAPI, 4′,6-diamidino-2-phenylindole). The sections were rinsed in PBS and hydrated in water. Coverslips were mounted using Aqua Mount (Thermo Scientific, Waltham, Mass.). Confocal images were obtained at 40× or 80× magnification on an Olympus FV1000 confocal laser scanning microscope. Images were processed using ImageJ software.
Fluorescein was selected as an inexpensive and widely used dye for cellular imaging due to its high quantum yield, low toxicity, and excellent water solubility.[24]
A protected fluorescein compound, fluorescein bis(benzyl boronic ester (FBBBE), was synthesized in one step from commercially available starting materials as shown in
To investigate the synthetic generality of this approach, a pro-fluorophore coumarin moiety (e.g. umbelliferone or 7-hydroxycoumarin) was protected with a boronic ester motif attached via a benzyl ether linkage (
FBBBE and CBBE were evaluated for H2O2 sensitivity under simulated physiological conditions (50 mM HEPES, pH 7.5). For FBBBE, the installation of the benzyl protecting group forces the compound to remain in the closed lactone form, rendering FBBBE (and FBn) nonfluorescent. UV-Vis spectroscopy revealed a baseline absorption profile in the visible region for FBBBE (
To determine whether the cleavage of one or both benzyl ether protecting groups from FBBBE is necessary to generate fluorescence, a second fluorescein derivative (in the free carboxylic acid form, FBBE), was synthesized that showed absorption in the visible region (
The coumarin-based compound, CBBE, was nonfluorescent in the absence of H2O2. The quenched fluorescence of CBBE quickly turned on in the presence of H2O2, with a maximum λem=453 nm (
Additional studies were performed to quantify the fluorescence turn-on for each probe to directly compare the sensitivity to H2O2. FBBBE showed a 52-fold increase in fluorescence in response to excess H2O2, while CBBE showed a 57-fold increase (
FBBBE was examined for H2O2 detection in biological samples via confocal microscopy. Murine macrophage RAW 264.7 cells were treated with PBS buffer (unstimulated cells), lipopolysaccharide (LPS, stimulated cells), or with exogenous H2O2, followed by incubation with either FBBBE (
Cellular studies were extended to a more physiologically relevant model for H2O2 detection. H2O2 is particularly active in the brain and neuronal tissue, triggered by the metabolism of neurotransmitters.[28] The brain and spinal cord from 3-4 week old female C57BL/6J mice were excised, frozen in OCT medium, and 10 μm sagittal cryosections were prepared for staining with FBBBE and FBn. At this age, the mice undergo extensive neuronal development and remodeling. Such activity has been associated with higher levels of neurotransmitter activity and ROS in both neurons and microglial cells in the brain. Consistent with the previous experiments, the sections were either incubated with PBS (unstimulated) or H2O2 (
To further investigate whether FBBBE is activated by truly endogenous levels of H2O2, the probe was evaluated in the spinal cord. It is well established that the spinal cord consists primarily of white matter (myelinated axons), gray matter (axons), glial cells and some fibrous tissue with few axon terminals and activated macrophages. The presence of ROS is ubiquitous at axon terminals associated with neurotransmitter activity and metabolism.[28] Thus, in a healthy, uninjured spinal cord, the presence of ROS such as H2O2 is not typical.[27c] Spinal cord sections were either incubated with PBS (unstimulated) or H2O2, in a manner identical to the brain tissue studies. Confocal fluorescence images of the unstimulated spinal cord tissue treated with FBBBE showed that the probe remained essentially inactive with a weak fluorescence signal observed. The weak signal is attributable to tissue autofluorescence. Thus, the probe remained latent in areas of low ROS levels. The addition of exogenous H2O2 to the spinal cord tissue showed a minimal amount of fluorescence when compared to the brain tissue (
Herein are reported the synthesis, reactivity, and imaging properties of a new class of fluorescence-based molecular probes for detecting endogenous H2O2 in biological systems. By appending a H2O2-sensitive boronic ester to a fluorophore moiety via a benzyl ether linkage, these fluorescent probes can be accessed in a single synthetic step using two commercially available starting materials. It was discovered, inter alia, under simulated physiological conditions, that both fluorescein and coumarin-based probes are sensitive for the detection of H2O2 in the biologically relevant micromolar concentration range. The fluorescein-based probe was particularly effective in imaging endogenous H2O2 levels with and without stimulation in murine macrophage cells using confocal miscroscopy and ex vivo imaging of endogenous H2O2 was effectively demonstrated in mouse brain. Overall, the combined synthetic ease, stability, solubility, fast reaction kinetics, and relatively high fluorescent turn-on response of these probes in comparison to other current fluorophores illustrate that the compounds herein represent accessible tools for molecular imaging for a wide range of researchers interested in the chemistry and biology of H2O2.
This application claims priority to U.S. Provisional Application No. 61/762,706, Filed Feb. 7, 2013.
This invention was made with Government support under grant numbers DK007233 and GM098435 awarded by the National Institutes of Health. The Government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61762706 | Feb 2013 | US |
