Near-infrared fluorescent sensors for biological amines

Abstract

A fluorescence sensing compound for separately detecting and visualizing one or more monoamine neurotransmitters in cells, the fluorescence sensing compound having the following formula: ##STR00001## wherein R.sub.1 and R.sub.2 are each independently selected from the group consisting of hydrogen, alkyl, alkylene, aryl, cycloalkyl; wherein R.sub.3 is selected from the group consisting of hydrogen, alkyl, alkylene, aryl, cycloalkyl, cyano, azido; and wherein R.sub.4 is selected from the group consisting of (CH.sub.3).sub.2Si, O, N, S, and CH.sub.2.

Claims

1. A method of detecting monoamine neurotransmitters, the method comprising: (a) contacting a biological sample with a fluorescence sensing compound so that the fluorescence sensing compound binds to one or more monoamine neurotransmitters that are present in in the biological sample; and (b) exposing the contacted biological sample to electromagnetic energy of one or more wavelengths capable of inducing fluorescence of bound fluorescence sending compounds and/or unbound fluorescence sensing compound, and detect the presence or absence and/or degree of fluorescence, wherein the presence or absence of fluorescence indicates the presence or absence of one or more monoamine neurotransmitters within the exposed biological sample, respectively, and the degree of fluorescence indicates the degree that one or more monoamine neurotransmitters are present within the exposed biological sample; wherein the fluorescence sensing compound has the following formula: ##STR00033## wherein R.sub.1 and R.sub.2 are each independently selected from the group consisting of hydrogen, alkyl, alkylene, aryl, cycloalkyl; wherein R.sub.3 is selected from the group consisting of hydrogen, alkyl, alkylene, aryl, cycloalkyl, cyano, azido; and wherein R.sub.4 is selected from the group consisting of (CH.sub.3).sub.2Si, O, N, S, and CH.sub.2.

2. The method of claim 1, wherein: R.sub.1 and R.sub.2 are each the alkyl moiety and are each independently selected from the group moieties consisting of haloalkane, hydroxyalkyl, tosylalkyl, triflatealkyl, alkyl azide, acetyl, trifluoroacetyl, ester, alkyl ester, alkyl sulfonate, and polyethylene glycol; and R.sub.3 is one of the following: (a) the alkyl moiety and is selected from the group of moieties consisting of (i) C.sub.nH.sub.2n+1 and (ii) alkynyl; (b) the aryl moiety and is selected from the group of moieties consisting of (i) monocyclic or polycyclic (homo- or hetero-) benzene-based moiety, (ii) monocyclic or polycyclic (homo- or hetero-) heterocyclopentadiene-based moiety having one or more heteroatoms selected from the group consisting of oxygen and sulfur and nitrogen, (iii) tetrazine, (iv) pyridine, and (v) quaternary aminobenzene; (c) the cyano; or (d) the azido.

3. The method of claim 2, wherein the heterocyclopendtadiene-based moiety is selected from the group consisting of thiophene-based moiety, furan-based moiety, pyrrole-based moiety, and azole-based moiety.

4. The method of claim 3, wherein the aryl moiety is the thiophene-based moiety, wherein the thiophene-based moiety has one of the following formulas: ##STR00034## wherein R.sub.5, R.sub.6, and R.sub.7 are each independently selected from the group consisting of hydrogen, alkyl, alkylene, aryl, cycloalkyl, halo, hydroxyl, alkoxyl, aryloxy, alkylthio, arylthio, carboxyl, alkoxycarbonyl, and a constituent of a fused aryl along with the adjacent R.sub.5, R.sub.6, or R.sub.7, as the case may be.

5. The method of claim 3, wherein the aryl moiety is the furan-based moiety, wherein the furan-based moiety has one of the following formulas: ##STR00035## wherein R.sub.5, R.sub.6, and R.sub.7 are each independently selected from the group consisting of hydrogen, alkyl, alkylene, aryl, cycloalkyl, halo, hydroxyl, alkoxyl, aryloxy, alkylthio, arylthio, carboxyl, alkoxycarbonyl, and a constituent of a fused aryl along with the adjacent R.sub.5, R.sub.6, or R.sub.7, as the case may be.

6. The method of claim 3, wherein the aryl moiety is the pyrrole-based moiety, wherein the pyrrole-based moiety has one of the following formulas: ##STR00036## wherein R.sub.5, R.sub.6, and R.sub.7 are each independently selected from the group consisting of hydrogen, alkyl, alkylene, aryl, cycloalkyl, halo, hydroxyl, alkoxyl, aryloxy, alkylthio, arylthio, carboxyl, alkoxycarbonyl, and a constituent of a fused aryl along with the adjacent R.sub.5, R.sub.6, or R.sub.7, as the case may be.

7. The method of claim 3, wherein the aryl moiety is the azole-based moiety, wherein the azole-based moiety has a has a formula selected from the group consisting of the following: ##STR00037## wherein R.sub.5 and R.sub.6 are each independently selected from the group consisting of hydrogen, alkyl, alkylene, aryl, cycloalkyl, halo, hydroxyl, alkoxyl, aryloxy, alkylthio, or arylthio, carboxyl, alkoxycarbonyl, and a constituent of a fused aryl if R.sub.5 and R.sub.6 are adjacent.

8. The method of claim 2, wherein the aryl moiety is the benzene-based moiety, wherein the benzene-based moiety has the following formula: ##STR00038## wherein R.sub.5, R.sub.6, R.sub.7, R.sub.8, and R.sub.9 are each independently selected from the group consisting of hydrogen, alkyl, alkylene, aryl, cycloalkyl, halo, hydroxyl, alkoxyl, aryloxy, alkylthio, arylthio, carboxyl, alkoxycarbonyl, and a constituent of a fused aryl along with the adjacent R.sub.5, R.sub.6, R.sub.7, R.sub.8, and R.sub.9, as the case may be.

9. The method of claim 8, wherein the benzene-based moiety is a naphthalene-based moiety having the following formula: ##STR00039## wherein R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, R.sub.10, and R.sub.11 are each independently selected from the group consisting of hydrogen, alkyl, alkylene, aryl, cycloalkyl, halo, hydroxyl, alkoxyl, aryloxy, alkylthio, arylthio, carboxyl, alkoxycarbonyl, and a constituent of a fused aryl along with the adjacent R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, R.sub.10, and R.sub.11, as the case may be.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 (A) depicts the structure of NeuroSensor 521 and (B) depicts the binding of NeuroSensor 715 to serotonin.

(2) FIG. 2 is a frontier orbital energy diagram and schematic representation of acceptor-excited PET mechanism, wherein the left column identified as a-PET Quenching is a schematic representation of the E.sub.HOMO value of the corresponding coumarin aldehyde scaffold of derivatives of the NS521 platform and the right column identified as NO a-PET Quenching is a schematic representation of the E.sub.HOMO of the corresponding modified coumarin aldehyde scaffold of NS715, both in relation to the middle column that is a schematic representation of the E.sub.HOMO value of the corresponding 5-hydroxyindole moiety of serotonin.

(3) FIG. 3 depicts the process for making an .sup.18F radiolabeled derivative of NS715.

(4) FIG. 4 depicts a synthetic scheme of NS715.

(5) FIGS. 5 (A), (B) and (C) show epinephrine-enriched cells incubated with NS715 (10 M): (A) .sub.ex=458 nm; (B) .sub.ex=633 nm; and (C) brightfield image. FIGS. 5 (D), (E), and (F) show norepinephrine-enriched cells incubated with (10 M): (D) .sub.ex=458 nm; (E) .sub.ex=633 nm; and (F) brightfield image. Fluorescence was visualized using a 650-710 nm bandpass filter. FIG. 5 (G) shows the average fluorescence intensity for norepinephrine-enriched (NE) and epinephrine-enriched (EP) cells, with NE being 12.421.75 and EP being 0.850.29 (n=20).

(6) FIG. 6 (A) is an absorbance and (B) is fluorescence spectra of NS715 (20 M) in buffer (50 mM Na.sub.2S.sub.2O.sub.3, 120 mM NaCl, pH 5.0) adding aliquots of 200 mM serotonin. Inset is the fit to a binding isotherm. .sub.em=780 nm.

(7) FIG. 7 (A) is an absorbance and (B) is fluorescence spectra of NS715 (20 M) in buffer (50 mM Na.sub.2S.sub.2O.sub.3, 120 mM NaCl, pH 5.0) adding aliquots of 200 mM norepinephrine. Inset is the fit to a binding isotherm. .sub.em=780 nm.

(8) FIG. 8 (A) is an absorbance and (B) is fluorescence spectra of NS715 (20 M) in buffer (50 mM Na.sub.2S.sub.2O.sub.3, 120 mM NaCl, pH 5.0) adding aliquots of 200 mM dopamine. Inset is the fit to a binding isotherm. .sub.em=780 nm.

DETAILED DESCRIPTION OF THE INVENTION

(9) In one embodiment, the present invention is directed to fluorescence sensing compound(s) for separately detecting and visualizing one or more monoamine neurotransmitters in cells that exhibit a turn-on NIR fluorescence response toward monoamine-neurotransmitters, such as serotonin. Said fluorescence sensing compound(s) are based upon a coumarin-3-aldehyde scaffold that is derived from a 1,2,3,4-tetrahydroquinoxaline (THQ) framework. In one embodiment, the fluorescence sensing compound has the following structure or formula:

(10) ##STR00003##

(11) wherein R.sub.1 and R.sub.2 are each independently selected from the group consisting of hydrogen, alkyl, alkylene, aryl, cycloalkyl;

(12) wherein R.sub.3 is selected from the group consisting of hydrogen, alkyl, alkylene, aryl, cycloalkyl, cyano, azido; and

(13) wherein R.sub.4 is selected from the group consisting of (CH.sub.3).sub.2Si, O, N, S, and CH.sub.2.

(14) Design

(15) The foregoing structure was developed for use as a NIR fluorescence-based, turn-on molecular sensor for the selective labeling and visualization of serotonin in synaptic vesicles of neuronal cells. Conceptually, the above-fluorescent compound may be considered as having two distinct moieties from the viewpoint of fluorescence: (i) a modified bicyclic aromatic aldehyde scaffold (also referred to as the fluorophore), wherein the aldehyde recognition element at the 3-position of the scaffold associates with the analyte amine via iminium ion formation, and wherein the rigid alkylated di-nitrogen species situated at the 6- and 7-positions of the scaffold (C6- and C7-positions) modulate the fluorescence properties of the fluorophore such that the fluorescence response is enhanced upon interaction with serotonin; and (ii) the pendant R.sub.3 group at the 4-position of the scaffold (C4-position) serves to modulate the spectroscopic and photophysical properties (i.e., absorption, emission, and quantum yield profiles) of the fluorophore.
In one embodiment, R.sub.4 of the scaffold is an oxygen (such that the scaffold may be referred to as a modified coumarin aldehyde scaffold) and R.sub.3 is a thiophene moiety as shown if FIG. 1 (B), which is referred as NeuroSensor 715 or NS715. The modified coumarin aldehyde scaffold of NS715 derives from the electron-rich 1,2,3,4-tetrahydroquinoxaline (THQ) framework.

(16) It should be noted that the terms NeuroSensor 715 and NS715 may, depending upon the context, be used to refer to the specific structure depicted in FIG. 1 (B) or to structures of the disclosed herein generally. Similarly, the terms coumarin and coumarin scaffold may, depending upon the context, be used to refer to particular embodiments in which R.sub.4 is N or to the bicyclic aromatic aldehyde scaffold or fluorophore moiety generally.

(17) The aforementioned rigid, electron-donating di-nitrogen species at the C6- and C7-positions of the core structure reinforces orbital alignment by restricting free rotation of the nitrogen atoms and impart pronounced spectroscopic and photophysical properties by simultaneously raising the calculated E.sub.HOMO value and lowering the E.sub.LUMO value beyond that of 5-hydroxyindoleamine moiety of serotonin to allow for both a substantial enhancement in fluorescence, a redshifted absorption profile, and a remarkable Stokes' shift of approximately 186 nm. For example, NS715 (which has ethyl groups for R.sub.1 and R.sub.2, oxygen for R.sub.4 at the 1-position, a thiophene moiety for R.sub.3 at the C4-position) resulted in a 8.0-fold fluorescence enhancement measured at 780 nm in the NIR spectral region with a binding affinity of 409 M.sup.1 in in vitro analyses with serotonin compared to (a) no modulation in the absorption or fluorescence spectrum of NS715 upon interaction with epinephrine due to no binding, and (b) fluorescence quenching (i.e., turn-off fluorescence) upon interaction with NS521 in in vitro analyses. Referring to FIG. 2, left column, serotonin quenched the response of all the NS521 derivatives because the particular underlying fluorophore maintained insufficient energetics (i.e., low E.sub.HOMO and E.sub.LUMO values), whereas N5715, right column, has sufficient energetics for preventing serotonin from quenching fluorescence emission. Thus, the foregoing structure represents a convenient molecular sensor that allows for integration with a tailored approach to selective labeling and visualization of serotonin in live and fixed neuronal cells.

TERMINOLOGY

(18) Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Throughout the specification and claims, a given chemical formula or name shall encompass all optical and stereoisomers as well as racemic mixtures where such isomers and mixtures exist.

(19) As used herein, the term independently with respect to references being selected from a group means that each reference may be selected from the entire list set forth as possible selections within the group without regard to the selections of other references having the same or different appellations.

(20) As used herein the term alkyl refers to C1-10 inclusive, linear, branched, or cyclic, saturated or unsaturated (i.e., alkenyl and alkynyl) hydrocarbon chains. The alkyl group can be optionally substituted with one or more alkyl group substituents which can be the same or different, where alkyl group substituent includes alkyl, amino, halo, arylamino, acyl, hydroxyl, aryloxy, alkoxyl, alkylthio, arylthio, aralkyloxy, aralkylthio, carboxy, alkoxycarbonyl, oxo, cycloalkyl, mesyl, tosyl, and triflic. There can be optionally inserted along the alkyl chain one or more oxygen, sulfur or substituted nitrogen atoms, wherein the nitrogen substituent is hydrogen, lower alkyl (also referred to herein as alkylaminoalkyl), or aryl. Branched refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to linear alkyl chain.

(21) Aryl refers to an aromatic substituent that may be a single ring or multiple rings that are fused together, linked covalently, or linked to a common group such as an ethylene, methylene or oxy moiety. The aromatic rings of the aryl group may each and optionally contain heteroatoms. The aryl group can be optionally substituted with one or more aryl group substituents which can be the same or different, where aryl group substituent includes alkyl, aryl, arylalkyl, hydroxy, alkoxyl, aryloxy, arylalkoxyl, carboxy, acyl, halo, nitro, alkoxycarbonyl, aryloxycarbonyl, arylalkoxycarbonyl, acyloxyl, acylamino, aroylamino, carbamoyl, cyano, alkylcarbamoyl, dialkylcarbamoyl, arylthio, alkylthio, alkylene, nitroso, sulfo, and NRR, where R and R can be each independently hydrogen, alkyl, aryl, and aralkyl.

(22) As used herein, the terms substituted alkyl and substituted aryl include alkyl and aryl groups, as defined herein, in which one or more atoms or functional groups of the aryl or alkyl group are replaced with another atom or functional group, including for example, halogen, aryl, alkyl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, nitroso, carboxy, and mercapto.

(23) Cyclic and cycloalkyl refer to a non-aromatic mono- or multicyclic ring system of about 3 to about 10 carbon atoms. The cycloalkyl group can be optionally partially unsaturated. The cycloalkyl group can be also optionally substituted with an alkyl group substituent as defined herein, ox and/or alkylene. There can be optionally inserted along the cyclic alkyl chain one or more oxygen, sulfur or substituted nitrogen atoms, wherein the nitrogen substituent is hydrogen, lower alkyl, or aryl, thus providing a heterocyclic group.

(24) Alkylene refers to a straight or branched bivalent aliphatic hydrocarbon group having from 1 to about 10 carbon atoms. The alkylene group can be straight, branched or cyclic. The alkylene group can be also optionally unsaturated and/or substituted with one or more alkyl group substituents. There can be optionally inserted along the alkylene group one or more oxygen, sulfur or substituted nitrogen atoms, wherein the nitrogen substituent is alkyl as previously described.

(25) Fluorophore

(26) R.sub.4 at the C1-Position

(27) In at least some of embodiments of the above-described fluorescent compound, R.sub.4 at the 1-position is oxygen (i.e., a coumarin-based structure; more particularly, a coumarin-3-aldehyde scaffold) or nitrogen (i.e., a quinolinone-based structure; more particularly, a quinolinone-3-aldehyde scaffold). Results to date have shown that selecting either an oxygen or nitrogen for R.sub.4 provides compounds with either a redshifted absorption and fluorescence profile or a molecular handle for appending additional functionalities compared to compounds having a different R.sub.4 (e.g., S, or CH.sub.2). As such, when R4 at the 1-position is N, the resulting compounds generally tend to be brighter fluorophores.

(28) That said, there may be reasons or end use applications for which one would select something other than oxygen or nitrogen for R.sub.4. For example, choosing R.sub.4 to be (CH.sub.3).sub.2Si is expected to dramatically redshift the absorption and fluorescence profiles toward the NIR spectral region, whereas selecting R.sub.4 to be sulfur is expected to blue-shift the absorption and fluorescence profiles toward the UV spectral region. Alternatively, selecting a carbon ring member with covalently bonded hydrogens (CH.sub.2) is expected to decrease the brightness (i.e., quantum yield and absorbance).

(29) Rigid Alkylated Di-Nitrogen Species at C6- and C7-Positions

(30) The aforementioned rigid alkylated di-nitrogen species at C6- and C7-positions of the scaffold comprise R.sub.1 and R.sub.2. As set forth above, R.sub.1 and R.sub.2 are each independently selected from the group consisting of hydrogen, alkyl, alkylene, aryl, cycloalkyl. The selection of R.sub.1 and R.sub.2 allows for substantial customization or variation of several properties of the fluorescent compound. For example, varying the selection of R.sub.1 and R.sub.2 allows for modification of selectivity, specificity, solubility, fluorescence, and the attachment of other small molecules, fluorophores, functionalities, biological molecules, or radionuclides/metals. Here, specificity refers to the ability of a molecule to interact with only a specified target, whereas selectivity refers to the ability of a molecule to interact with a specified target only when it is amongst other relevant molecules/systems that would not interfere with the interaction. Thus, specificity and selectivity are not the same.

(31) In an embodiment, R.sub.1 and R.sub.2 are each the alkyl moiety and are each independently selected from the group moieties consisting of haloalkane, hydroxyalkyl, tosylalkyl, triflatealkyl, alkyl azide, acetyl, trifluoroacetyl, ester, alkyl ester, alkyl sulfonate, and polyethylene glycol. Table A below contains corresponding structures for these moieties and provides a brief description of one or more property(ies) that may be affected, adjusted, modified, or imparted by selecting such a moiety for R.sub.1 and/or R.sub.2.

(32) TABLE-US-00001 TABLE A Moiety Structure Property(ies) Affected haloalkane pH-sensitivity or the ability to append to other molecules (e.g., proteins in vitro upon using a HaloTag assay) hydroxyalkyl, tosylalkyl, triflatealkyl pH-sensitivity or the ability to synthetically attach other functional elements alkyl azide Ability to covalently label DNA, RNA, proteins of interest, or pre-label cells via click or inverse electron demand Diels-Alder chemistry acetyl pKa, activation state of fluorophore, and modulation of spectral properties of fluorophore trifluoroacetyl pKa, activation state of fluorophore, and modulation of spectral properties of fluorophore ester pKa, activation state of fluorophore, and modulation of spectral properties of fluorophore alkyl ester 0 Ability to covalently attach other small-molecules or biomolecules that would allow for additional targeting or modified properties alkyl sulfonate enhances solubility in water as well as charged state of molecule polyethylene glycol methyl ether enhances solubility polar solvents

(33) Aldehyde Recognition Element

(34) The aldehyde recognition element at the C3-position of the scaffold, as mentioned above, associates with the analyte amine via iminium ion formation. Additionally, aldehyde-derived iminium ion provides hydrogen-bonding capabilities with the carbonyl group of the fluorophore, which assists in modulating the spectroscopic and photophysical profiles of the sensor. The aldehyde-derived iminium ion at the C3-position of the scaffold undergoes further restricted rotation due to the proximity to the C4-position group, which promotes the absorption and emission profiles of the sensor.

(35) Bathochromic-Shift Component: R.sub.3 at the C4-Position

(36) As disclosed above, R.sub.3 may be selected from the group consisting of hydrogen, alkyl, alkylene, aryl, cycloalkyl, cyano, azido. As with other moieties in the compound, R.sub.3 may be selected to affect, adjust, modify, or impart certain properties of, or to, the fluorescent compound.

(37) Alkyl Moiety

(38) In an embodiment, R.sub.3 is the alkyl moiety is an unsubstituted, linear alkyl, or an alkynyl moiety. In another embodiment, R.sub.3 is a cyano moiety. In another embodiment R.sub.3 is an azido moiety. Table B below contains corresponding structures or formulas for these moieties and provides a brief description of the property(ies) that may be affected, adjusted, modified, or imparted by selecting such a moiety for R.sub.3.

(39) TABLE-US-00002 TABLE B Moiety Structure Property(ies) Affected unsubstituted, (C.sub.nH.sub.2n+1) Ability to incorporate linear additional recognition alkyl elements or atoms/molecules for other labeling purposes alkynyl (or alkyne) Absorption and fluorescence profile (redshifts), ability perform click-chemistries, pKa if pH-sensitive cyano Absorption and fluorescence profile (marked redshifts), pKa if pH-sensitive azido Precursor for forming triazole in cells via click chemistry; imparts relatively long term monitoring of cells (e.g., life of vesicle) by enhancing the reactions of the compound within cells

(40) Aryl Moiety

(41) In yet another embodiment, R.sub.3 is the aryl moiety and is selected from the group of moieties consisting of (i) a benzene-based moiety (monocyclic or polycyclic; and if polycyclic, the rings may be homo- or hetero-cyclic), (ii) a heterocyclopentadiene-based moiety having one or more heteroatoms selected from the group consisting of oxygen and sulfur and nitrogen (monocyclic or polycyclic; and if polycyclic, the rings may be homo- or hetero-cyclic), (iii) tetrazine, (iv) pyridine, and (v) quaternary aminobenzene. Both the benzene-based moiety and heterocyclopentadiene-based moiety may be monocyclic or polycyclic and, if polycyclic, the rings may be homo- or hetero-cyclic. These moieties are discussed in greater detail below. Table C below contains corresponding structures or formulas for these moieties and provides a brief description of the property(ies) that may be affected, adjusted, modified, or imparted by selecting such a moiety for R.sub.3.

(42) Benzene-Based Moiety

(43) The benzene-based moiety may be represented by the following structure or formula:

(44) ##STR00016##
wherein R.sub.5, R.sub.6, R.sub.7, R.sub.8, and R.sub.9 are each independently selected from the group consisting of hydrogen, alkyl, alkylene, aryl, cycloalkyl, halo, hydroxyl, alkoxyl, aryloxy, alkylthio, arylthio, carboxyl, alkoxycarbonyl, and a constituent of a fused aryl along with the adjacent R.sub.5, R.sub.6, R.sub.7, R.sub.8, and R.sub.9, as the case may be (e.g., R.sub.5 with R.sub.6, or R.sub.6 with R.sub.7, or R.sub.7 with R.sub.8, or R.sub.8 with R.sub.9, or R.sub.5 with R.sub.6 with R.sub.7, or R.sub.5 with R.sub.6 and R.sub.7 with R.sub.8, or R.sub.5 with R.sub.6 and R.sub.8 with R.sub.9, or R.sub.5 with R.sub.6 with R.sub.7 with R.sub.8, or R.sub.5 with R.sub.6 with R.sub.7 with R.sub.8 with R.sub.9, or R.sub.6 with R.sub.7 with R.sub.8, or R.sub.6 with R.sub.7 and R.sub.8 with R.sub.9, etc.).

(45) One may select a benzene-based moiety for the bathochromic-shift component of the fluorescent compound because these moieties make it relatively easy to append other constituents to the compound to provide different, modified, adjusted, or new or additional functionality(ies) to the fluorescent compound. For example, .sup.18F may be added to facilitate use of the compound as a combined PET-Near Infrared Fluorescent Sensor such as that depicted in FIG. 3. Additionally, the selection of a benzene-based moiety may affect the wavelength and brightness of flourescence (e.g., it has been observed that selecting phenyl as R.sub.3 instead of thiophene resulted in a wavelength shift of about 5 nm shorter and an increased brightness by about 3 times).

(46) In various embodiments of the present invention, the benzene-based moiety has one of the following structures:

(47) ##STR00017##

(48) As shown in the foregoing formulas, one such moiety is naphthyl, a naphthalene-based moiety. As described in greater detail below, naphthalene-based moieties may be of particular interest. As such, in one embodiment the benzene-based moiety is a naphthalene-based moiety having the following formula:

(49) ##STR00018##
wherein R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, R.sub.10, and R.sub.11 are each independently selected from the group consisting of hydrogen, alkyl, alkylene, aryl, cycloalkyl, halo, hydroxyl, alkoxyl, aryloxy, alkylthio, arylthio, carboxyl, alkoxycarbonyl, and a constituent of a fused aryl along with the adjacent R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, R.sub.10, and R.sub.11, as the case may be (e.g., R.sub.5 with R.sub.6, or R.sub.6 with R.sub.7, or R.sub.7 with R.sub.8, or R.sub.8 with R.sub.9, or R.sub.9 with R.sub.10, or R.sub.10 with R.sub.11, or R.sub.5 with R.sub.6 with R.sub.7, or R.sub.5 with R.sub.6 and R.sub.7 with R.sub.8, or R.sub.5 with R.sub.6 and R.sub.8 with R.sub.9, or R.sub.5 with R.sub.6 with R.sub.7 with R.sub.8, or R.sub.5 with R.sub.6 with R.sub.7 with R.sub.8 with R.sub.9 with R.sub.10 with R.sub.11, or R.sub.6 with R.sub.7 with R.sub.8, or R.sub.6 with R.sub.7 and R.sub.8 with R.sub.9, etc.).

(50) Heterocyclopentadiene-Based Moiety

(51) In an embodiment, the bathochromic-shift component, R.sub.3, at the C4-position of the scaffold is a heterocyclopentadiene-based moiety. Of particular interest are heterocyclopentadiene-based moieties that are thiophene-based, furan-based, pyrrole-based, and azole-based In one such embodiment the heterocyclopentadiene-based moiety is thiophene-based. In another such embodiment the heterocyclopentadiene-based moiety is furan-based. In another such embodiment, the hetercyclopentadiene-based moiety is pyrrole-based or azole-based.

(52) Thiophene-Based Moiety

(53) In one such embodiment, the heterocyclopentadiene-based moiety is the thiophene-based moiety having the following structure or formula:

(54) ##STR00019##
wherein R.sub.5, R.sub.6, and R.sub.7 are each independently hydrogen, alkyl, alkylene, aryl, cycloalkyl, halo, hydroxyl, alkoxyl, aryloxy, alkylthio, or arylthio, carboxyl, alkoxycarbonyl, and a constituent of a fused aryl along with the adjacent R.sub.5, R.sub.6, or R.sub.7, as the case may be (i.e., R.sub.5 with R.sub.6, or R.sub.6 with R.sub.7, or R.sub.5 with R.sub.6 with R.sub.7).

(55) In another embodiment, the thiophene-based moiety has the following structure or formula:

(56) ##STR00020##
wherein R.sub.5, R.sub.6, and R.sub.7 are each independently selected from the group consisting of hydrogen, alkyl, alkylene, aryl, cycloalkyl, halo, hydroxyl, alkoxyl, aryloxy, alkylthio, or arylthio, carboxyl, alkoxycarbonyl, and a constituent of a fused aryl along with the adjacent R.sub.5, R.sub.6, or R.sub.7, as the case may be (i.e., R.sub.5 with R.sub.6).

(57) In various embodiments, the thiophene-based moiety has one of the following structures or formulas:

(58) ##STR00021##

(59) Furan-Based Moiety

(60) In one embodiment, the aryl moiety is a furan-based moiety, wherein the furan-based moiety has one of the following structures or formulas:

(61) ##STR00022##
wherein R.sub.5, R.sub.6, and R.sub.7 are each independently selected from the group consisting of hydrogen, alkyl, alkylene, aryl, cycloalkyl, halo, hydroxyl, alkoxyl, aryloxy, alkylthio, or arylthio, carboxyl, alkoxycarbonyl, and a constituent of a fused aryl along with the adjacent R.sub.5, R.sub.6, or R.sub.7, as the case may be (i.e., for the left structure, R.sub.5 with R.sub.6, or R.sub.6 with R.sub.7, or R.sub.5 with R.sub.6 with R.sub.7; and for the right structure, R.sub.5 with R.sub.6).

(62) In one embodiment the furan-based moiety has the following structure or formula:

(63) ##STR00023##

(64) Pyrrole-Based Moiety

(65) In one embodiment, the aryl moiety is a pyrrole-based moiety, wherein the pyrrole-based moiety has one of the following structures or formulas:

(66) ##STR00024##

(67) wherein R.sub.5, R.sub.6, and R.sub.7 are each independently selected from the group consisting of hydrogen, alkyl, alkylene, aryl, cycloalkyl, halo, hydroxyl, alkoxyl, aryloxy, alkylthio, or arylthio, carboxyl, alkoxycarbonyl, and a constituent of a fused aryl along with the adjacent R.sub.5, R.sub.6, or R.sub.7, as the case may be (i.e., for the left structure, R.sub.5 with R.sub.6, or R.sub.6 with R.sub.7, or R.sub.5 with R.sub.6 with R.sub.7; and for the right structure, R.sub.5 with R.sub.6).

(68) In one embodiment, the pyrrole-based moiety has the following structure or formula:

(69) ##STR00025##

(70) Azole-Based Moiety

(71) In one embodiment, the aryl moiety is the azole-based moiety, wherein the azole-based moiety has a has a structure or formula selected from the group consisting of the following:

(72) ##STR00026##

(73) wherein R.sub.3, R.sub.4, and R.sub.5, are each independently selected from the group consisting of hydrogen, alkyl, alkylene, aryl, cycloalkyl, halo, hydroxyl, alkoxyl, aryloxy, alkylthio, or arylthio, carboxyl, alkoxycarbonyl, and a constituent of a fused aryl if R.sub.5 and R.sub.6 are adjacent.

(74) In one embodiment, the azole-based moiety is a triazole moiety.

(75) Tetrazine, Pyridine, and Quaternary Aminobenzene

(76) As disclosed above, R.sub.3, may be, in different embodiments, be a tetrazine moiety, pyridine moiety, or quaternary aminobenzene. Table C below contains corresponding structures or formulas for these moieties and provides a brief description of the property(ies) that may be affected, adjusted, modified, or imparted by selecting such a moiety for R.sub.3.

(77) TABLE-US-00003 TABLE C Moiety Structure Property(ies) Affected tetrazine Enables attachment of molecules through Cu-free inverse electron demand Diels-Alder reaction pyridine pH-sensitivity, modulation of fluorescence via PeT quaternary aminobenzene Enables direct attachment of radionuclides (.sup.18F) or other atoms/ molecules via nucleophilic aromatic substitution
Use of NeuroSensor 715

(78) As indicated above, NeuroSensor 715 is a NIR fluorescence-based, turn-on molecular sensor for the selective recognition and imaging of serotonin in neurosecretory vesicles. NeuroSensor 715 is envisaged to integrate with a tailored approach to comprise a method for the selective labeling and visualization of serotonin in live and fixed cells. The method entails utilizing NeuroSensor 715 to exploit the high concentration of serotonin and acidic environment within secretory vesicles through the formation of a charged complex that prevents translocation across the vesicle membrane and accumulates within secretory vesicles.

(79) NeuroSensor 715 features an aldehyde group that associates with the analyte amine via iminium ion formation and features a pendant thiophene moiety at the 4-position of a modified coumarin aldehyde scaffold derived from the electron-rich THQ framework. The modified coumarin aldehyde scaffold and pendant thiophene moiety are key design parameters that (i) confer a fluorescence turn-on response upon binding to serotonin, and (ii) impart unique spectroscopic properties to NeuroSensor 715 that allow for direct monitoring of the unbound and bound states using a conventional confocal microscope equipped with the standard 458 nm and 559 nm lasers, respectively.

(80) NeuroSensor 715 exhibits an unprecedented 8.0-fold fluorescence enhancement at 715 nm in the NIR spectral region with a binding affinity of 409 M.sup.1 in in vitro analyses. NeuroSensor 715 binds to serotonin with a 19-fold higher binding constant compared and to typical primary amines such as glutamate.

EXAMPLES

Example 1: Synthesis of NS715

(81) FIG. 4 depicts a synthetic scheme of NS715. As shown in FIG. 4, Compound 1 was acylated and demethylated under Friedel-Crafts conditions to give intermediate 2 in high yield. A high-temperature Wittig reaction with the appropriate phosphorane gave the coumarin core (intermediate 3) in good yield. Regioselective formylation under Vilsmeier conditions completed the synthesis of NS715.

(82) Synthetic Procedures

(83) Compound 1 to Compound 2

(84) Compound 1 (250.0 mg, 1.136 mmol) was dissolved in 11.3 mL CH.sub.2Cl.sub.2 and cooled to 0 C. Aluminum chloride (605.2 mg, 4.542 mmol) was added and the solution stirred for 10 min. 2-thiophene carbonyl chloride (166.5 mg, 1.136 mmol) was then added. The mixture stirred for 5 min at 0 C. then warmed to room temperature and stirred for 4 hours while sonicating intermittently. Then 6 M HCl was slowly added to the mixture which stirred another 10 min. The crude product was extracted with CH.sub.2Cl.sub.2 (50 mL3), the organic layers combined and dried over Na.sub.2SO.sub.4, and the solvent removed in vacuo. After purifying twice by column chromatography (9:1 CH.sub.2Cl.sub.2/EtOAc then 8:2 hexanes/EtOAc), Compound 2 was isolated as a red oil (316.2 mg, 1.000 mmol, 88%): .sup.1H NMR (500 MHz, CDCl.sub.3) 12.83 (s, 1H), 7.69 (dd, 1H, J=4.0, 1.0 Hz), 7.61 (dd, 1H, J=5.0, 1.0 Hz), 7.14-7.18 (m, 1H), 7.07 (s, 1H), 6.13 (s, 1H), 3.53 (t, 2H, J=5.0 Hz), 3.41 (q, 2H, J=7.0 Hz), 3.24 (q, 2H, J=7.5 Hz), 3.17 (t, 2H, J=5.0 Hz), 1.17-1.25 (m, 6H); .sup.13C NMR (125 MHz, CDCl.sub.3) 186.8, 161.1, 144.5, 143.2, 131.6, 131.1, 127.6, 127.3, 112.1, 108.1, 96.6, 47.8, 45.7, 45.5, 44.9, 10.7, 10.3; IR (neat, cm.sup.1) 3101, 2970, 1618, 1524, 1409, 1324, 1217, 1119; HRMS calculated for C.sub.17H.sub.20N.sub.2O.sub.2SNa (M+Na.sup.+): 339.1138. Found: 339.1135.

(85) Compound 2 to Compound 3

(86) Compound 2 (72.5 mg, 0.229 mmol), (carbethoxymethylene)triphenylphosphorane (87.9 mg, 0.252 mmol), DMAP (2.8 mg, 0.023 mmol), and 2 mL o-xylene were combined in a round bottom flask and heated at 140 C. for 4 h. The solvent was boiled off and the remaining crude product was purified via column chromatography (8:2 hexanes/EtOAc) to yield Compound 3 as an orange oil (51.7 mg, 0.176 mmol, 66%): .sup.1H NMR (500 MHz, CDCl.sub.3) 7.52 (dd, 1H, J=5.0, 1.0 Hz), 7.40 (dd, 1H, J=3.5, 1.0 Hz), 7.18-7.21 (m, 1H), 6.93 (s, 1H), 6.51 (s, 1H), 6.17 (s, 1H), 3.51 (t, 2H, J=5.0 Hz), 3.41 (q, 2H, J=7.5 Hz), 3.24-3.30 (m, 4H), 1.22 (t, 3H, J=7.5 Hz), 1.17 (t, 3H, J=7.0 Hz); .sup.13C NMR (125 MHz, CDCl.sub.3) 162.2, 150.2, 147.9, 140.1, 137.8, 131.9, 128.3, 127.8, 127.7, 108.3, 107.5, 105.6, 97.0, 47.2, 45.7, 45.5, 45.2, 10.3, 10.0; IR (neat, cm.sup.1) 2966, 1716, 1699, 1608, 1538, 1418, 1373, 1334; HRMS calculated for C.sub.19H.sub.20N.sub.2O.sub.2SNa (M+Na.sup.+): 363.1138. Found: 363.1133.

(87) Compound 3 to NS715

(88) The Vilsmeier reagent was made by combining 10.8 mL DMF and 5.23 mL POCl.sub.3 at 0 C. in a flame-dried round bottom flask. The solution was stirred for 1 h. In a separate dry flask, Compound 3 (66.0 mg, 0.194 mmol) was dissolved in 2 mL DMF and purged with N.sub.2. To the starting material was added 0.5 mL of the Vilsmeier reagent and the mixture stirred at ambient temperature for 2.5 h. The mixture was poured over ice chips (100 g), basified to pH 7 with NaHCO.sub.3, and extracted with CH.sub.2Cl.sub.2 (25 mL5). The solvent was removed in vacuo without heating and the crude product purified via column chromatography (1:1 hexanes/EtOAc.fwdarw.100% EtOAc.fwdarw.100% acetone) to yield NeuroSensor 715 as a red oil (35.4 mg, 0.096 mmol, 50%): .sup.1H NMR (500 MHz, CDCl.sub.3) 9.90 (s, 1H), 7.60 (dd, 1H, J=5.0, 1.0 Hz), 7.19-7.23 (m, 1H), 7.15 (dd, 1H, J=3.5, 1.0 Hz), 6.45 (s, 1H), 6.33 (s, 1H), 3.57 (t, 2H, J=5.0 Hz), 3.46 (q, 2H, J=7.5 Hz), 3.22 (t, 2H, J=5.0 Hz), 3.13 (q, 2H, J=7.5 Hz), 1.26 (t, 3H, J=7.5 Hz), 1.05 (t, 3H, J=7.5 Hz); .sup.13C NMR (125 MHz, CDCl.sub.3) 188.2, 159.6, 153.8, 152.4, 143.8, 133.0, 132.4, 129.6, 128.2, 127.3, 113.1, 109.9, 106.5, 95.8, 47.8, 46.4, 45.4, 44.6, 10.6, 9.6; IR (neat, cm.sup.1) 1728, 1695, 1675, 1605, 1503, 1426, 1336; HRMS calculated for C.sub.20H.sub.20N.sub.2O.sub.3SNa (M+Na.sup.+): 391.0868. Found: 391.1078.

Example 2: Validation of the Utility of NS715 Using Chromaffin Cells

(89) FIG. 5 shows that NS715 may be used to differentiate between cell populations that express distinct neurotransmitter phenotypes. As shown in FIG. 5, populations of chromaffin cells are separated into distinct norepinephrine-enriched and epinephrine-enriched fractions using standard methods. NS715 binds to norepinephrine and strongly fluoresces, but not to epinephrine because it is a secondary-amine neurotransmitter. Both populations of cells were incubated with NS715 (10 M), washed, and subsequently imaged using confocal microscopy. The unbound UV .sub.max=500 nm and the bound UV .sub.max=546 nm. The unbound sensor emits at 686 nm and the bound sensor emits at 715 nm. Excitation wavelengths of 458 nm and 633 nm were used to preferentially excite the unbound and bound forms of NS715. Emission for all excitation wavelengths was measured using 650 nm-710 nm bandpass filter.

(90) When excited at 633 nm, only the norepinephrine-enriched cells revealed the punctate pattern with strong fluorescence that is expected for staining high concentrations of norepinephrine in secretory vesicles. The epinephrine-enriched cells showed marginal fluorescence, which can be attributed to NS715 binding to the very low levels of norepinephrine that is present within this type of cell population. Neither cell population showed any appreciable fluorescence upon exciting at 458 nm, thereby indicating that any potentially unbound sensor was removed during the washing step. Further analysis indicated that the average fluorescence intensity of the norepinephrine-enriched cells was 15-fold higher than that of the epinephrine-enriched cells.

Example 2: Spectroscopic Studies

(91) NeuroSensor 715 was screened with various relevant amines via absorption and fluorescence spectroscopy. Steady-state fluorescence spectroscopic studies were performed using a Horiba Scientific Fluorolog-3 Model FL3C-111 spectrofluorometer and data was collected and analyzed using HJY FluorEssence 3.5.1.20 software package. UV-visible spectra were recorded on a Varian Cary 1E UV-visible spectrophotometer at 37 C.

(92) Solution Preparations

(93) A 1 mg/mL stock solution of NeuroSensor 715 in DMSO was prepared. A stock solution of NeuroSensor 715 and derivatives in buffer (210.sup.5 M, 25 mM HEPES, 50 mM Na.sub.2S.sub.2O.sub.3, pH 5.0) was prepared. Serotonin, norepinephrine, dopamine, and glutamate stock solutions were prepared by separately dissolving the analytes at the concentration to be used in the titrations with the buffered stock solution of NeuroSensor 715 (thus avoiding dilution of NeuroSensor 715 during the experiment). NeuroSensor 715 was titrated with aliquots of the analyte solution. NeuroSensor 715 derivatives were excited at 559 nm. The slit width was 10 nm for both excitation and emission.

(94) NeuroSensor 715 binds to all primary amines via iminium ion formation, which produces a red shift in absorption from 500 to 546 nm (FIG. 6 (A)). In fluorescence mode, exciting the unbound NeuroSensor 715 at 559 nm provided fluorescence emission at 686 nm. In fluorescence mode, exciting the bound complex at 559 nm upon adding serotonin produced a marked 8.0-fold fluorescence enhancement at 780 nm (FIG. 6 (B)).

(95) Table D summarizes binding and spectroscopic data for the interaction of NeuroSensor 715 with a number of relevant amines.

(96) TABLE-US-00004 TABLE D Association constants and spectroscopic parameters for the binding of NS715 to various analytes amine guest K.sub.a (M.sup.1).sup.a I.sub.sat/I.sub.0.sup.b .sub.max, abs.sup.c (nm) serotonin 409 8.0 46 dopamine 145 4.0 37 norepinephrine 129 3.4 37 glutamate 22.3 3.0 30 .sup.aK.sub.a measured by fluorescence spectroscopy, .sub.ex = 559 nm, .sub.em = 780 nm. Error in K.sub.a values are 10% based on triplicate titrations. .sup.bI.sub.sat = fluorescence intensity at saturation taken from the theoretical fit to the binding isotherm. .sup.cBathochromic shift in absorbance upon saturating with analyte.
Glutamate binds with a low binding affinity of 22.3 M.sup.1 that provides a 3.0-fold fluorescence enhancement. The catecholamines norepinephrine and dopamine have on average 6.0-fold higher binding constants compared to glutamate with a binding affinity of 129 M.sup.1 and 145 M.sup.1 and a 3.0- and 3.4-fold fluorescence enhancement, respectively. Interestingly, the indoleamine serotonin has an 18-fold higher binding constant compared to glutamate with a binding affinity of 409 M.sup.1 and 8.0-fold fluorescence enhancement. The bathochromic shift upon analyte binding was 46 nm, 37 nm, and 30 nm for serotonin, the catecholamines, and glutamate, respectively.

(97) NeuroSensor 715 incorporates a pendant thiophene moiety and fused alkylated di-nitrogen species to that derives from the THQ framework to constitute a modified coumarin aldehyde moiety and provides pronounced spectroscopic and photophysical properties. Table E provides a comparison of the spectral characteristics between a benzene-based NeuroSensor 521 derivative, thiophene-based NeuroSensor 521 derivative, and NeuroSensor 715, and highlights the effect of both the incorporation of the thiophene moiety at the C4-position and the electron-rich THQ framework of the coumarin core that situates electron-donating nitrogen substituents at the C6- and C7-positions.

(98) TABLE-US-00005 TABLE E Sensor benzene-based NeuroSensor 521 derivative 0 thiophene-based NeuroSensor 521 derivative NS715 .sub.abs/.sub.em (nm).sup.a 452/505 462/522 500/686 .sub.abs/.sub.em (nm).sup.b 488/520 502/539 546/715 Stokes shift 53 60 186 (nm).sup.a Stokes shift 32 37 169 (nm).sup.b .sup.aSpectroscopic properties of the unbound sensor; .sup.bSpectroscopic properties of the sensor saturated with primary amine analyte.

(99) Replacing the pendant benzene moiety with a thiophene moiety on the coumarin aldehyde scaffold of the NeuroSensor 521 platform affords a reasonable bathochromic shift of 10 nm and 17 nm in absorbance and fluorescence emission, respectively, with a maximum absorbance and fluorescence emission at 462 nm and 522 nm, respectively. In doing so, both the unbound and bound Stokes shift increases 53 nm and 32 nm, respectively, for the benzene-based NeuroSensor 521 derivative to the unbound and bound Stokes shift of 60 nm and 37 nm, respectively for the thiophene-based NeuroSensor 521 derivative.

(100) Modify the coumarin aldehyde scaffold by fixing electron-donating nitrogen species at the C6- and C7-positions results in a drastic bathochromic shift in the absorption and emission profiles. The pendant thiophene and THQ-derived modified coumarin aldehyde scaffold afford a noteworthy bathochromic in both the absorption and fluorescence properties with a 500 nm and 546 nm maximum absorbance and a 686 nm and 715 nm maximum fluorescence emission for the unbound and bound complex, respectively. The Stokes shift of both the unbound and bound complex of NeuroSensor 715 increases over 125 nm compared to the thiophene-based NeuroSensor 521 derivative to 186 nm and 169 nm, respectively.

(101) While the invention has been described in connection with specific embodiments thereof, it will be understood that the inventive method is capable of further modifications. This patent application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features herein before set forth and as follows in scope of the appended claims.