Patent Application
20170276611
This disclosure relates generally to the field of chalcogenopyrylium compounds and surface-enhanced Raman spectroscopy and chalcogenopyrylium compositions used with surface-enhanced Raman spectroscopy.
Surface-enhanced Raman scattering (SERS) has been utilized as a sensitive analytical tool in the study of biological systems. The combination of a metallic nanoparticle and an organic dye as a reporter molecule provide SERS nanotags that can be used to detect target molecules using laser Raman spectroscopy or SERS microscopy. This spectroscopic technique not only has high sensitivity (10−9 M-10−12 M limits of detectability), but also the potential for multiplexing capabilities due to the unique vibrational structure of adsorbed molecules on the metallic nanoparticle. For most medical applications, the 785-nm laser has been used to excite SERS nanotags and, while systematic investigation of SERS reporter molecules has been limited, SERS reporters for this wavelength have been designed and utilized. Orders-of-magnitude higher sensitivities (10−12-10−14 M) can be achieved utilizing Raman reporters that are in resonance with the incident laser, thereby producing surface-enhanced resonance Raman scattering (SERRS) nanoprobes. [Note: SERS and SERRS are used interchangeably from this point forward.] The optical absorptance of human tissue is minimal in the 600-800-nm window and increases at longer wavelengths due to absorption by water. While the 785-nm laser operates within this window, the depth of penetration of infrared light increases at longer wavelengths due to decreased scattering, reaching a minimum near 1300 nm. The superior penetration depth of 1300-nm light vs. 800-nm light has been documented, but Raman scattering at 1300 nm is so weak that it may be impossible to use. Therefore, to exploit the advantages of the unique vibrational signatures produced by Raman scattering, surface enhancement of the signal must be used to operate at this longer wavelength of excitation. Lasers emitting in the 1500-nm to 1600-nm range are invisible to the human eye and exposure of the eye to these wavelengths is not damaging.
The region from 1000 nm to 1300 nm is of particular interest and is compatible with commercial laser excitation sources operating at 1064 and 1280 nm. SERS nanotags operating at 1064-nm have been described using crystal violet, rhodamine 6G, methylene blue, and 9-aminoacridine as reporter molecules. A direct comparison of the 1064-nm (Ti:sapphire) and 1280-nm (Cr:forsterite) lasers showed that the 1280-nm laser excitation gave reduced sample burning, limited photobleaching, reduced background fluorescence/autofluorescence, and greater penetration depth into biological tissues. The 1280-nm laser has been utilized in both optical coherence tomography and fluorescence microscopy, to take advantage of the superior penetration of 1280-nm light in turbid media such as tissue and blood. To date, there appear to be no SERS nanotags compatible with a 1280 nm excitation laser. Thus, there is great need to provide 1280-nm SERS nanotags in order to harness the significant benefits of operating at this wavelength of excitation. One possible application of SERS nanotags operating at this wavelength is human biomedical imaging of SERS nanotags targeted to specific sites such as tumors.
The present disclosure provides chalcogenopyrylium compounds having the following structure:
where each E is, at each occurrence in the compound, independently charged or neutral and is independently selected from S, Se, O, or Te, wherein at least one E is S or Se; each R1 is, at each occurrence in the compound, independently selected from the group consisting of —H, C1-8 alkyl group, halo group, —CN, aryl group, and heteroaryl group and adjacent R1 groups can combine to form C5-8 aryl groups, each R2 is, at each occurrence in the compound, independently selected from the group consisting of —H, C1-8 alkyl group, halo group, —CN, and aryl group, R2 groups beta to each other can combine to form C5-8 cycloalkyl groups, C5-8 aryl groups or C5-8 heteroaryl groups, and n is an odd number from 1 to 7; and Z is optionally present and is a counter ion.
In an embodiment, the compound does not have the following structures:
In an embodiment, the compound does not have the following structure:
where E is S or Se. For example, the compound does not have the following structure:
In various examples, the compounds have one of the following structures:
where R1, R2, and E are as defined herein.
In various examples, the compounds have one of the following structures:
The present disclosure also provides composite nanostructures. The composite nanostructures can comprises: a core comprising a nanomaterial; one or more reporter molecules having the structure as described herein, wherein each of the reporter molecules is independently, at each occurrence in the composite nanostructure, directly covalently bound to the core or covalently bound to the core via a linking group to the core; and optionally, an encapsulating material that at least partially encapsulates the core and the one or more reporter molecules. For example, the core comprises a metal nanomaterial. For example, the core is a hollow gold nanoshell. The nanomaterial can be a nanoparticle and the nanoparticle size is 15 to 300 nm. The nanostructure morphology can be selected from the group consisting of sphere, rod, star, raspberry, and hollow shell. The encapsulating material can be an inorganic material, polyethylene glycol (PEG), or organic polymer.
The composite nanostructure can further comprise one or more targeting moieties bound (e.g., covalently or non-covalently bound) to the core or bound (e.g., covalently or non-covalently bound) to the core via a linking group. The encapsulating material, if present, at least partially encapsulates the core, the one or more reporter molecules. The one or more targeting moieties, if present, are directly bound (e.g., covalently or non-covalently bound) or bound (e.g., covalently or non-covalently bound) via a linking group to an outer surface of the encapsulating material. A targeting moiety is any moiety that specifically interacts with (e.g., binds) a target molecule. Examples of targeting moieties include, but are not limited to, antibodies, aptamers, synthetic receptors, DNA sequences, proteins, peptides, and the like. Examples of suitable conjugation methods and linkers are known in the art.
The present disclosure also provides methods of making composite nanostructures. For example, a method of making a composite nanostructure comprises binding one or more reporter molecules of the present invention to a core, and optionally, encapsulating the core and reporter molecule within an encapsulating material.
The present disclosure also provides methods of using the chalcogenopyrylium compounds or composite nanoparticles comprising the chalcogenopyrylium compounds. For example, a method for detecting one or more target molecules in a sample comprises: contacting an individual with one or more composite nanostructures; obtaining surface-enhanced Raman spectroscopy data (e.g., a surface-enhanced Raman spectrum) of a portion of the individual after contact of the portion of the individual with the one or more said composite nanostructures, wherein observation of surface-enhanced Raman spectroscopy data (e.g., a surface-enhanced Raman spectrum) attributable (e.g., specifically attributable) to a particular composite nanostructure of the one or more composite nanostructures indicates the presence of the target molecule in the portion of the individual corresponding to the targeting moiety of the particular nanostructure. The method may further comprise obtaining surface-enhanced Raman spectroscopy data (e.g., a surface-enhanced Raman spectrum) of one or more additional portions of the individual after contact of the one or more additional portions of the individual with the one or more of the composite nanostructures. The method may further comprise generating an image of at least a portion of the individual using the surface-enhanced Raman spectroscopy data (e.g., a surface-enhanced Raman spectrum) from the portion and, optionally, additional portions of the individual.
(red) based SERRS-nanoprobes were performed in triplicate and determined to be 1.0 fM and 100 attomolar, respectively.
As used herein, the term “alkyl group”, unless otherwise stated, refers to branched or unbranched hydrocarbons. Examples of such alkyl groups include methyl groups, ethyl groups, propyl groups, butyl groups, isopropyl groups, tert-butyl groups, and the like. For example, the alkyl group can be a C1-C8 alkyl group including all integer numbers of carbons and ranges of numbers of carbons there between. The alkyl group can be unsubstituted or substituted with various substituents.
As used herein, the term “aryl group”, unless otherwise stated, refers to a C5-C8 aromatic carbocyclic group including all integer numbers of carbons and ranges of numbers of carbons there between. The aryl group can be unsubstituted or substituted with various substituents (e.g., as described herein) which may be the same or different. A non-limiting example of a suitable aryl group include phenyl.
As used herein, the term “halo group”, unless otherwise state, means fluoro, chloro, bromo, or iodo group. As used herein, the term “halide”, unless otherwise state, means fluoride, chloride, bromide, or iodide.
As used herein, the term “heteroaryl group”, unless otherwise stated, refers to a C5-C8 monocyclic or fused bicyclic ring system, including all integer numbers of carbons and ranges of numbers of carbons there between, wherein 1-8 of the ring atoms are selected from the group consisting of S, Se, O, P, B, and N. The heteroaryl group can be unsubstituted or substituted with various substituents (e.g., as described herein) which may be the same or different. Examples of heteroaryl groups include, benzofuranyl, thienyl, furyl, pyridyl, oxazolyl, quinolyl, thiophenyl, selenophenyl, isoquinolyl, indolyl, triazinyl, triazolyl, isothiazolyl, isoxazolyl, imidazolyl, benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, and thiadiazolyl groups.
As used herein, the term “cycloalkyl group”, unless otherwise stated, refers to a C5-C8 cyclic aliphatic group, including all integer numbers of carbons and ranges of numbers of carbons there between. Examples of cycloalkyl groups include cyclohexyl, cyclohexenyl, and cyclopentyl groups. The cycloalkyl group can be unsubstituted or substituted with various substituents.
It is an object of the present disclosure to provide surface-enhanced Raman spectroscopic (SERS) or surface-enhanced resonance Raman scattering (SERRS) active composite nanostructures, methods of fabricating these nanostructures, and methods of using these nanostructures. It is to be understood that references to SERS in this application include SERRS.
The surface-enhanced Raman spectroscopic (SERS) active composite nanostructures are comprised of a core attached (e.g., covalently or non-covalently) to at least one reporter molecule, and, optionally, an encapsulating material (i.e. a shell). The reporter molecule(s) is (are) bonded to the core directly or via a coupling agent. The reporter molecule(s) is (are) selected from the chalcogenopyrylium dyes described herein. In some embodiments, at least two distinct reporter molecules may be bonded to the core, thus allowing for detection of more than one SERS signal. The encapsulating material is disposed over the core and the reporter molecule. The reporter molecule, whether or not encapsulated, has a measurable surface-enhanced Raman spectroscopic signature. Although not intending to be bound by theory, the core optically enhances the SERS spectrum, while the reporter molecule provides a distinct spectroscopic SERS signature. Although optional, disposing the encapsulant material over the core and reporter molecule does not substantially impact the spectroscopic SERS signature of the reporter molecule, while protecting the core and reporter molecules. A preferred size range for nanoparticles is 50-100 nm, but particles in the range of 40-300 nm are also useful.
The core can be made of plasmonic materials that have a resonance in the range of 400 nm to 2000 nm, including all nm values and ranges therebetween. In an example, the plasmonic materials have a resonance in the range of 780 nm to 1600 nm. In an example, the plasmonic materials have a resonance in the range of 1000 nm to 1600 nm (e.g., 1064 nm or 1280 nm). The core can be made of nanomaterials such as, but not limited to, metals. In some embodiments, the core can be a metallic core. In particular, the core can be made of noble metals such as, but not limited to, gold, silver, copper, and combinations thereof. In other embodiments, the core can be metal-coated silica particles such as gold-coated silica particles. Suitable morphologies for such materials include, but are not limited to, spheres, rods, stars, raspberries, and hollow shells. In an embodiment, the core can be a gold core. In some examples, the core is a hollow gold nanoshell. The core can be a nanomaterial, such as, for example, a nanoparticle, and the core can have a size (e.g., longest dimension), which can be measured by electron microscopy, of 15 nm to 300 nm, including all nm values and ranges therebetween. For example, the core has a size of 40 nm to 100 nm.
Suitable encapsulating materials, if used, include but are not limited to, silica-based materials such as xerogels from tetraalkoxy silanes or organically modified xerogels from organotrialkoxy silanes and tetraalkoxy silanes; polyethylene glycol (PEG) such as PEG 500; and organic polymers such as, but not limited to, polyvinylethylene (PVE) and polyvinylpropylene (PVP). The encapsulating materials can be inorganic materials including, but not limited to, SiO2 or MnO2.
The surface-enhanced Raman spectroscopic (SERS) active composite nanostructures of the present disclosure may further comprise a coupling agent, wherein the coupling agent is bonded to the core and reporter molecule. An example of a suitable coupling agent is thiol PEG with carboxylate terminal groups.
The surface-enhanced Raman spectroscopic (SERS) active composite nanostructures of the present disclosure can be incorporated into (e.g., used in) systems such as, for example, anti-counterfeit systems, covert tagging systems, cytometry systems (e.g., a flow cytometry system), chemical array systems, biomolecule array systems, biosensing systems, bioimaging systems, biolabeling systems, high-speed screening systems, gene expression systems, protein expression systems, medical diagnostic systems, diagnostic libraries, and microfluidic systems.
It is also an object of the present disclosure to provide chalcogenopyrylium compounds. The chalcogenopyrylium compounds can be dyes that can be used as reporter molecules for surface-enhanced Raman scattering (SERS) attached to nanoparticles such as noble metal nanoparticles, for example, those comprised of gold, silver, copper or combinations thereof. It is an advantage that SERS active composite nanostructures comprising the SERS reporters of this disclosure work with excitation from light sources emitting in the near infrared region of 1000 to 1600 nm. For example, SERS reporters of the present disclosure bound to noble metal nanoparticles such as hollow gold nanoparticles work with excitation from light sources emitting in the near infrared region of 1000 to 1600 nm, for example, both/either 1064-nm and/or 1280-nm lasers.
The present disclosure also provides novel chalcogenopyrylium compositions of matter as SERS reporters attached to nanoparticles such as noble metal nanoparticles (e.g., those comprised of gold, silver, copper or combinations thereof). It is an advantage that SERS active composite nanostructures comprising novel chalcogenopyrylium compositions of matter of this disclosure work with excitation from light sources emitting in the near infrared region of 1000 to 1600 nm. For example, novel chalcogenopyrylium compositions of matter of this disclosure bound to noble metal nanoparticles such as hollow gold nanoparticles work with excitation from light sources emitting in the near infrared region of 1000 to 1600 nm, for example, both/either 1064-nm and/or 1280-nm lasers.
The chalcogenopyryliums of the present disclosure can be defined by the following generic structures:
In one embodiment, the present disclosure provides thiopyrylium dyes and selenopyrylium dyes of general structures I-VII (shown above) as SERS reporters attached to nanoparticles, for example, those comprised of gold (e.g. hollow gold nanoshells), silver, copper or combinations thereof, and methods of using these compositions for SERS and/or SERSS spectroscopy with incident light from 780 nm to 1300 nm wherein E and E′ are independently selected from the chalcogen atoms 0, S, Se, and Te wherein at least one of E or E′ is S or Se; Ar, Ar′, Ar″, and Ar′″ are independently selected from the group consisting of phenyl (substituted or unsubstituted), 2-thienyl (substituted or unsubstituted), 3-thienyl (substituted or unsubstituted), 2-selenophenyl (substituted or unsubstituted), and 3-selenophenyl (substituted or unsubstituted); and the counter ion Z is an anion. In other embodiments of the composition and method, the counter ion Z is selected from the group consisting of PF6, BF4, Cl, Br, CF3CO2, and CF3SO3.
In another embodiment, the present disclosure provides thiopyrylium dyes and selenopyrylium dyes of general structures I-VII as SERS reporters attached to noble metal nanoparticles such as those comprised of gold (e.g. hollow gold nanoparticles), silver, copper, or combinations thereof and methods of using these compositions for SERS and/or SERSS spectroscopy with incident light from a 1280-nm laser wherein E and E′ are independently selected from the chalcogen atoms 0, S, Se, and Te wherein at least one of E or E′ is S or Se; Ar, Ar′, Ar″, and Ar′″ are independently selected the group consisting of phenyl (substituted or unsubstituted), 2-thienyl (substituted or unsubstituted), 3-thienyl (substituted or unsubstituted), 2-selenophenyl (substituted or unsubstituted), and 3-selenophenyl (substituted or unsubstituted); and the counter ion Z is an anion selected from the group consisting of PF6, BF4, Cl, Br, CF3CO2, and CF3SO3.
In another embodiment, the present disclosure provides thiopyrylium dyes and selenopyrylium dyes of general structures I-III and V-VII as SERS reporters attached to nanoparticles, for example, those comprised of gold (e.g. hollow gold nanoshells), silver, copper or combinations thereof, and methods of using these compositions for SERS and/or SERSS spectroscopy with incident light from 780 nm to 1600 nm wherein E and E′ are independently selected from the chalcogen atoms 0, S, Se, and Te wherein at least one of E or E′ is S or Se; Ar, Ar′, Ar″, and Ar′″ are independently selected from the group consisting of phenyl (substituted or unsubstituted), 2-thienyl (substituted or unsubstituted), 3-thienyl (substituted or unsubstituted), 2-selenophenyl (substituted or unsubstituted), 3-selenophenyl (substituted or unsubstituted); R, R′, and R″ are independently selected from the group consisting of H, C1-8 alkyl (straight chain or branched), halides and pseudohalides; and the counter ion Z is an anion selected from Z═PF6, BF4, Cl, Br, CF3CO2, and CF3SO3. In one embodiment, R, R′, and R″ are independently selected from the group consisting of H, C1-8 alkyl (straight chain or branched), Cl, Br and CN.
In another embodiment, the present disclosure provides thiopyrylium dyes and selenopyrylium dyes of general structures I-III and V-VII as SERS reporters attached to noble metal nanoparticles such as those comprised of gold (e.g. hollow gold nanoparticles), silver, copper, or combinations thereof and methods of using these compositions for SERS and/or SERSS spectroscopy with incident light from a 1280-nm laser wherein E and E′ are independently selected from the chalcogen atoms 0, S, Se, and Te wherein at least one of E or E′ is S or Se; Ar, Ar′, Ar″, and Ar′″ are independently selected from the group consisting of phenyl (substituted or unsubstituted), 2-thienyl (substituted or unsubstituted), 3-thienyl (substituted or unsubstituted), 2-selenophenyl (substituted or unsubstituted), and 3-selenophenyl (substituted or unsubstituted); R, R′, and R″ are independently selected from the group consisting of H, C1-8 alkyl (straight chain or branched), halides and pseudohalides; and the counter ion Z is an anion selected from the group consisting of PF6, BF4, Cl, Br, CF3CO2, and CF3SO3. In one embodiment, R, R′, and R″ are independently selected from the group consisting of H, C1-8 alkyl (straight chain or branched), Cl, Br and CN.
In another embodiment, the present disclosure provides thiopyrylium dyes and selenopyrylium dyes of general structures I-III and V-VII as SERS reporters attached to nanoparticles, for example, those comprised of gold (e.g. hollow gold nanoshells), silver, copper or combinations thereof, and methods of using these compositions for SERS and/or SERSS spectroscopy with incident light from 780 nm to 1600 nm wherein E and E′ are independently selected from the chalcogen atoms S and Se; Ar, Ar′, Ar″, and Ar′″ are independently selected from the group consisting of phenyl (substituted or unsubstituted), 2-thienyl (substituted or unsubstituted), 3-thienyl (substituted or unsubstituted), 2-selenophenyl (substituted or unsubstituted), and 3-selenophenyl (substituted or unsubstituted); R, R′, and R″ are independently selected from the group consisting of H, C1-8 alkyl (straight chain or branched), halides or pseudohalides; and the counter ion Z is an anion selected from the group consisting of PF6, BF4, Cl, Br, CF3CO2, and CF3SO3.
In another embodiment, the present disclosure provides thiopyrylium dyes and selenopyrylium dyes of general structures I-III and V-VII as SERS reporters attached to noble metal nanoparticles such as those comprised of gold (e.g. hollow gold nanoparticles), silver, copper, or combinations thereof and methods of using these compositions for SERS and/or SERSS spectroscopy with incident light from a 1280-nm laser wherein E and E′ are independently selected from the chalcogen atoms S and Se; Ar, Ar′, Ar″, and Ar′″ are independently selected from the group consisting of phenyl (substituted or unsubstituted), 2-thienyl (substituted or unsubstituted), 3-thienyl (substituted or unsubstituted), 2-selenophenyl (substituted or unsubstituted), 3-selenophenyl (substituted or unsubstituted); R, R′, and R″ are independently selected from the group consisting of H, C1-8 alkyl (straight chain or branched), halides and pseudohalides; and the counter ion Z is an anion selected from the group consisting of PF6, BF4, Cl, Br, CF3CO2, and CF3SO3. In one embodiment, R, R′, and R″ are independently selected from the group consisting of H, C1-8 alkyl (straight chain or branched), Cl, Br and CN.
In another embodiment, the present disclosure provides thiopyrylium dyes and selenopyrylium dyes of general structures I-III and V-VII as SERS reporters attached to nanoparticles, for example, those comprised of gold (e.g. hollow gold nanoshells), silver, copper or combinations thereof, and methods of using these compositions for SERS and/or SERSS spectroscopy with incident light from 780 nm to 1600 nm wherein E and E′ are independently selected from the chalcogen atoms S and Se; Ar, Ar′, Ar″, and Ar′″ are independently selected from the group consisting of phenyl (substituted or unsubstituted), 2-thienyl (substituted or unsubstituted), and 2-selenophenyl (substituted or unsubstituted); R, R′, and R″ are independently selected from the group consisting of H, C1-8 alkyl (straight chain or branched), halides and pseudohalides; and the counter ion Z is an anion selected from the group consisting of PF6, BF4, Cl, Br, CF3CO2, and CF3SO3. In one embodiment, R, R′, and R″ are independently selected from the group consisting of H, C1-8 alkyl (straight chain or branched), Cl, Br and CN.
In another embodiment, the present disclosure provides thiopyrylium dyes and selenopyrylium dyes of general structures I-III and V-VII as SERS reporters attached to noble metal nanoparticles such as those comprised of gold (e.g. hollow gold nanoparticles), silver, copper, or combinations thereof and methods of using these compositions for SERS and/or SERSS spectroscopy with incident light from a 1280-nm laser wherein E and E′ are independently selected from the chalcogen atoms S and Se; Ar, Ar′, Ar″, and Ar′″ are independently selected from the group consisting of phenyl (substituted or unsubstituted), 2-thienyl (substituted or unsubstituted), and 2-selenophenyl (substituted or unsubstituted); R, R′, and R″ are independently selected from the group consisting of H, C1-8 alkyl (straight chain or branched), halides and pseudohalides; and the counter ion Z is an anion selected from the group consisting of PF6, BF4, Cl, Br, CF3CO2, and CF3SO3. In one embodiment, R, R′, and R″ are independently selected from the group consisting of H, C1-8 alkyl (straight chain or branched), Cl, Br and CN.
In another embodiment, the present disclosure provides thiopyrylium dyes and selenopyrylium dyes of general structures I-III and V-VII as SERS reporters attached to nanoparticles, for example, those comprised of gold (e.g. hollow gold nanoshells), silver, copper or combinations thereof, and methods of using these compositions for SERS and/or SERSS spectroscopy with incident light from 780 nm to 1600 nm wherein E and E′ are independently selected from the chalcogen atoms S and Se; Ar, Ar′, Ar″, and Ar′″ are independently selected from the group consisting of phenyl, 2-thienyl, and 2-selenophenyl; R R′, and R″ are independently selected from the group consisting of H, C1-8 alkyl (straight chain or branched), halides or pseudohalides; and the counter ion Z is an anion selected from the group consisting of PF6, BF4, Cl, Br, CF3CO2, and CF3SO3. In one embodiment, R, R′, and R″ are independently selected from the group consisting of H, C1-8 alkyl (straight chain or branched), Cl, Br and CN.
In another embodiment, the present disclosure provides thiopyrylium dyes and selenopyrylium dyes of general structures I-III and V-VII as SERS reporters attached to noble metal nanoparticles such as those comprised of gold (e.g. hollow gold nanoparticles), silver, copper, or combinations thereof and methods of using these compositions for SERS and/or SERSS spectroscopy with incident light from a 1280-nm laser wherein E and E′ are independently selected from the chalcogen atoms S and Se; Ar, Ar′, Ar″, and Ar′″ are independently selected from the group consisting of phenyl, 2-thienyl, and 2-selenophenyl; R, R′, and R″ are independently selected from the group consisting of H, C1-8 alkyl (straight chain or branched), halides or pseudohalides; and the counter ion Z is an anion selected from the group consisting of PF6, BF4, Cl, Br, CF3CO2, and CF3SO3. In one embodiment, R, R′, and R″ are independently selected from the group consisting of H, C1-8 alkyl (straight chain or branched), Cl, Br and CN.
In another embodiment, the present disclosure provides thiopyrylium dyes and selenopyrylium dyes of general structures I-III and V-VII as SERS reporters attached to nanoparticles, for example, those comprised of gold (e.g. hollow gold nanoshells), silver, copper or combinations thereof, and methods of using these compositions for SERS and/or SERSS spectroscopy with incident light from 780 nm to 1600 nm wherein E and E′ are independently selected from the chalcogen atoms S and Se; Ar, Ar′, Ar″, and Ar′″ are independently selected from the group consisting of phenyl, 2-thienyl, and 2-selenophenyl; R, R′ and R″ are H; and the counter ion Z is an anion selected from the group consisting of PF6, BF4, Cl, Br, CF3CO2, and CF3SO3.
In another embodiment, the present disclosure provides thiopyrylium dyes and selenopyrylium dyes of general structures I-III and V-VII as SERS reporters attached to noble metal nanoparticles such as those comprised of gold (e.g. hollow gold nanoparticles), silver, copper, or combinations thereof and methods of using these compositions for SERS and/or SERSS spectroscopy with incident light from a 1280-nm laser where E and E′ are independently selected from the chalcogen atoms S and Se; Ar, Ar′, Ar″, and Ar′″ are independently selected from the group consisting of phenyl, 2-thienyl, and 2-selenophenyl; R, R′ and R″ are H; and the counter ion Z is an anion selected from the group consisting of PF6, BF4, Cl, Br, CF3CO2, and CF3SO3.
In another embodiment, the present disclosure provides thiopyrylium dyes and selenopyrylium dyes of general structures I-III and V-VII as SERS reporters attached to nanoparticles, for example, those comprised of gold (e.g. hollow gold nanoshells), silver, copper or combinations thereof and methods of using these compositions for SERS and/or SERSS spectroscopy with incident light from 780 nm to 1600 nm wherein E and E′ are independently selected from the chalcogen atoms S and Se; Ar, Ar′, Ar″, and Ar′″ are independently selected from the group consisting of phenyl, 2-thienyl, and 2-selenophenyl; R, R′ and R″ are H; and Z is PF6.
In another embodiment, the present disclosure provides thiopyrylium dyes and selenopyrylium dyes of general structures I-III and V-VII as SERS reporters attached to noble metal nanoparticles such as those comprised of gold (e.g. hollow gold nanoparticles), silver, copper, or combinations thereof and methods of using these compositions for SERS and/or SERSS spectroscopy with incident light from, a 1280-nm laser wherein E and E′ are independently selected from the chalcogen atoms S and Se; Ar, Ar′, Ar″, and Ar′″ are independently selected from the group consisting of phenyl, 2-thienyl, and 2-selenophenyl; R, R′ and R″ are H; and Z is PF6.
In another embodiment, the present disclosure provides thiopyrylium dyes and selenopyrylium dyes of general structures I-III wherein E and E′ are independently selected from the chalcogen atoms S and Se; Ar, Ar′, Ar″, and Ar′″ are independently selected from the group consisting of phenyl, 2-thienyl, and 2-selenophenyl wherein at least two of the groups Ar, Ar′, Ar″, or Ar′″ are 2-thienyl or 2-selenophenyl; R, R′ and R″ are H; and Z is selected from the group consisting of PF6, BF4, Cl, Br, CF3CO2, and CF3SO3, which are novel compositions of matter. The subject disclosure also provides thiopyrylium dyes and selenopyrylium dyes of general structures I-III as SERS reporters attached to, for example, hollow gold, silver or copper nanoparticles and methods of using these compositions for SERS and/or SERSS spectroscopy with incident light from 780 nm to 1600 nm, wherein E and E′ are independently selected from the chalcogen atoms S and Se; Ar, Ar′, Ar″, and Ar′″ are independently selected from the group consisting of phenyl, 2-thienyl, and 2-selenophenyl wherein at least two of the groups Ar, Ar′, Ar″, or Ar′″ are 2-thienyl or 2-selenophenyl; R, R′ and R″ are H; and Z is selected from the group consisting of PF6, BF4, Cl, Br, CF3CO2, and CF3SO3. In other embodiments of the composition and the method, the incident light is from a 1280-nm laser.
In another embodiment, the present disclosure provides thiopyrylium dyes and selenopyrylium dyes of general structures I-III wherein E and E′ are independently selected from the chalcogen atoms S and Se; Ar, Ar′, Ar″, and Ar′″ are independently selected from the group consisting of phenyl, 2-thienyl, and 2-selenophenyl wherein at least two of the groups Ar, Ar′, Ar″, or Ar′″ are 2-thienyl or 2-selenophenyl; R, R′ and R″ are H; and Z is PF6−, which are novel compositions of matter. The subject disclosure also provides thiopyrylium dyes and selenopyrylium dyes of general structures I-III as SERS reporters attached to, for example, hollow gold, silver or copper nanoparticles and methods of using these compositions for SERS and/or SERSS spectroscopy with incident light from 780 nm to 1600 nm, wherein E and E′ are independently selected from the chalcogen atoms S and Se; Ar, Ar′, Ar″, and Ar′″ are independently selected from the group consisting of phenyl, 2-thienyl, and 2-selenophenyl wherein at least two of the groups Ar, Ar′, Ar″, or Ar′″ are 2-thienyl or 2-selenophenyl; R, R′ and R″ are H; and Z is PF6. In other embodiments of the composition and the method, the incident light is from a 1280-nm laser.
In another embodiment, the present disclosure provides thiopyrylium dyes and selenopyrylium dyes of general structure IV as SERS reporters attached to nanoparticles, for example, those comprised of gold (e.g. hollow gold nanoshells), silver, copper or combinations thereof, and methods of using these compositions for SERS and/or SERSS spectroscopy with incident light from 1000 nm to 1300 nm wherein E and E′ are independently selected from the chalcogen atoms 0, S, Se, and Te wherein at least one of E or E′ is S or Se; Ar, Ar′, Ar″, and Ar′″ are independently selected from the group consisting of phenyl (substituted or unsubstituted), 2-thienyl (substituted or unsubstituted), 3-thienyl (substituted or unsubstituted), 2-selenophenyl (substituted or unsubstituted), and 3-selenophenyl (substituted or unsubstituted); R is H; all R′s are H or together can form a five- or six-membered ring; R″ is selected from the group consisting of H, halides, pseudohalides alkylthio and arylthio groups; and the counter ion Z is an anion selected from the group consisting of PF6, BF4, Cl, Br, CF3CO2, and CF3SO3. In one embodiment, R″ is selected from the group consisting of H, Cl, Br, CN, alkylthio and arylthio groups.
In another embodiment, the present disclosure provides thiopyrylium dyes and selenopyrylium dyes of general structure IV as SERS reporters attached to nanoparticles, for example, those comprised of gold (e.g. hollow gold nanoshells), silver, copper or combinations thereof, and methods of using these compositions for SERS and/or SERSS spectroscopy with incident light from a 1280-nm laser wherein E and E′ are independently selected from the chalcogen atoms 0, S, Se, and Te wherein at least one of E or E′ is S or Se; Ar, Ar′, Ar″, and Ar′″ are independently selected from the group consisting of phenyl (substituted or unsubstituted), 2-thienyl (substituted or unsubstituted), 3-thienyl (substituted or unsubstituted), 2-selenophenyl (substituted or unsubstituted), and 3-selenophenyl (substituted or unsubstituted); R is H; all R′s are H or together can form a five- or six-membered ring; R″ is selected from the group consisting of H, halides, pseudohalides alkylthio and arylthio groups; and the counter ion Z is an anion selected from the group consisting of PF6, BF4, Cl, Br, CF3CO2, and CF3SO3. In one embodiment, R″ is selected from the group consisting of H, Cl, Br, CN, alkylthio and arylthio groups.
In another embodiment, the present disclosure provides thiopyrylium dyes and selenopyrylium dyes of general structure IV as SERS reporters attached to nanoparticles, for example, those comprised of gold (e.g. hollow gold nanoshells), silver, copper or combinations thereof, and methods of using these compositions for SERS and/or SERSS spectroscopy with incident light from 1000 nm to 1300 nm where E and E′ are independently selected from the chalcogen atoms S and Se; Ar, Ar′, Ar″, and Ar′″ are independently selected from the group consisting of phenyl (substituted or unsubstituted), 2-thienyl (substituted or unsubstituted), 3-thienyl (substituted or unsubstituted), 2-selenophenyl (substituted or unsubstituted), and 3-selenophenyl (substituted or unsubstituted); R is H; all R′s are H or together can form a five- or six-membered ring; R″ is selected from H, halides, pseudohalides, alkylthio and arylthio groups; and the counter ion Z is an anion selected from the group consisting of PF6, BF4, Cl, Br, CF3CO2, and CF3SO3. In one embodiment, R″ is selected from the group consisting of H, Cl, Br, CN, alkylthio and arylthio groups.
In another embodiment, the present disclosure provides thiopyrylium dyes and selenopyrylium dyes of general structure IV as SERS reporters attached to nanoparticles, for example, those comprised of gold (e.g. hollow gold nanoshells), silver, copper or combinations thereof, and methods of using these compositions for SERS and/or SERSS spectroscopy with incident light from a 1280-nm laser wherein E and E′ are independently selected from the chalcogen atoms S and Se; Ar, Ar′, Ar″, and Ar′″ are independently selected from the group consisting of phenyl (substituted or unsubstituted), 2-thienyl (substituted or unsubstituted), 3-thienyl (substituted or unsubstituted), 2-selenophenyl (substituted or unsubstituted), and 3-selenophenyl (substituted or unsubstituted); R is H; all R′s are H or together can form a five- or six-membered ring; R″ is selected from H, halides or pseudohalides, alkylthio and arylthio groups; and the counter ion Z is an anion selected from the group consisting of PF6, BF4, Cl, Br, CF3CO2, and CF3SO3. In one embodiment, R″ is selected from the group consisting of H, Cl, Br, CN, alkylthio and arylthio groups.
In another embodiment, the present disclosure provides thiopyrylium dyes and selenopyrylium dyes of general structure IV as SERS reporters attached to nanoparticles, for example, those comprised of gold (e.g. hollow gold nanoshells), silver, copper or combinations thereof, and methods of using these compositions for SERS and/or SERSS spectroscopy with incident light from 1000 nm to 1300 nm wherein E and E′ are independently selected from the chalcogen atoms S and Se; Ar, Ar′, Ar″, and Ar′″ are independently selected from the group consisting of phenyl (substituted or unsubstituted), 2-thienyl (substituted or unsubstituted), and 2-selenophenyl (substituted or unsubstituted); R is H; all R′s are H or together can form a five- or six-membered ring; R″ is selected from the group consisting of H, halides, pseudohalides alkylthio and arylthio groups; and the counter ion Z is an anion selected from the group consisting of PF6, BF4, Cl, Br, CF3CO2, and CF3SO3. In one embodiment, R″ is selected from the group consisting of H, Cl, Br, CN, alkylthio and arylthio groups. In another embodiment, the present disclosure provides thiopyrylium dyes and selenopyrylium dyes of general structure IV as SERS reporters attached to nanoparticles, for example, those comprised of gold (e.g. hollow gold nanoshells), silver, copper or combinations thereof, and methods of using these compositions for SERS and/or SERSS spectroscopy with incident light from a 1280-nm laser wherein E and E′ are independently selected from the chalcogen atoms S and Se; Ar, Ar′, Ar″, and Ar′″ are independently selected from the group consisting of phenyl (substituted or unsubstituted), 2-thienyl (substituted or unsubstituted), and 2-selenophenyl (substituted or unsubstituted); R is H; all R′s are H or together can form a five- or six-membered ring; R″ is selected from H, halides, pseudohalides alkylthio and arylthio groups; and the counter ion Z is an anion selected from the group consisting of PF6, BF4, Cl, Br, CF3CO2, and CF3SO3. In one embodiment, R″ is selected from the group consisting of H, Cl, Br, CN, alkylthio and arylthio groups.
In another embodiment, the present disclosure provides thiopyrylium dyes and selenopyrylium dyes of general structure IV as SERS reporters attached to nanoparticles, for example, those comprised of gold (e.g. hollow gold nanoshells), silver, copper or combinations thereof, and methods of using these compositions for SERS and/or SERSS spectroscopy with incident light from 1000 nm to 1300 nm wherein E and E′ are independently selected from the chalcogen atoms S and Se; Ar, Ar′, Ar″, and Ar′″ are independently selected from the group consisting of phenyl (substituted or unsubstituted), 2-thienyl (substituted or unsubstituted), and 2-selenophenyl (substituted or unsubstituted); R is H; all R′s together form a six-membered ring, R″ is Cl; and the counter ion Z is an anion selected from the group consisting of PF6, BF4, Cl, Br, CF3CO2, and CF3SO3.
In another embodiment, the present disclosure provides thiopyrylium dyes and selenopyrylium dyes of general structure IV as SERS reporters attached to nanoparticles, for example, those comprised of gold (e.g. hollow gold nanoshells), silver, copper or combinations thereof, and methods of using these compositions for SERS and/or SERSS spectroscopy with incident light from a 1280-nm laser wherein E and E′ are independently selected from the chalcogen atoms S and Se; Ar, Ar′, Ar″, and Ar′″ are independently selected from the group consisting of phenyl (substituted or unsubstituted), 2-thienyl (substituted or unsubstituted), and 2-selenophenyl (substituted or unsubstituted); R is H; all R′s together form a six-membered ring; R″ is Cl; and the counter ion Z is an anion selected from the group consisting of PF6, BF4, Cl, Br, CF3CO2, and CF3SO3.
In another embodiment, the present disclosure provides thiopyrylium dyes and selenopyrylium dyes of general structure IV wherein E and E′ are independently selected from the chalcogen atoms S and Se; Ar, Ar′, Ar″, and Ar′″ are independently selected from the group consisting of phenyl, 2-thienyl, and 2-selenophenyl wherein at least two of the groups Ar, Ar′, Ar″, or Ar′″ are 2-thienyl or 2-selenophenyl; R is H; all R′s together form a six-membered ring; R″ is Cl; and the counter ion Z is PF6, which are novel compositions of matter. The subject disclosure also provides thiopyrylium dyes and selenopyrylium dyes of general structure IV as SERS reporters attached to, for example, hollow gold, silver or copper nanoparticles, and methods of using these compositions for SERS and/or SERSS spectroscopy with incident light from 1000 nm to 1300 nm wherein E and E′ are independently selected from the chalcogen atoms S and Se; Ar, Ar′, Ar″, and Ar′″ are independently selected from the group consisting of phenyl, 2-thienyl, and 2-selenophenyl wherein at least two of the groups Ar, Ar′, Ar″, or Ar′″ are 2-thienyl or 2-selenophenyl; R is H; all R′s together form a six-membered ring; R″ is Cl; and the counter ion Z is PF6. In other embodiments of the composition and method, the incident light is from a 1280-nm laser.
In another embodiment, the present disclosure provides a thiopyrylium dye of general structure I wherein E=E′=S, Ar═Ar′═Ar″═Ar′″=2-thienyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a thiopyrylium dye of general structure I wherein E=E′=S, Ar═Ar′=Ph, Ar″=Ar′″=2-thienyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure I wherein E=Se, E′=S, Ar═Ar′═Ar″═Ar′″=2-thienyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure I wherein E=Se, E′=S, Ar═Ar′=Ph, Ar″=Ar′″=2-thienyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure I wherein E=Se, E′=S, Ar═Ar′=2-thienyl, Ar″=Ar′″=Ph, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure I wherein E=E′=Se, Ar═Ar′═Ar″═Ar′″=2-thienyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure I wherein E=E′=Se, Ar═Ar′=Ph, Ar″=Ar′″=2-thienyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a thiopyrylium dye of general structure I wherein E=E′=S, Ar═Ar′═Ar″═Ar′″=2-selenophenyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a thiopyrylium dye of general structure I wherein E=E′=S, Ar═Ar′=Ph, Ar″=Ar′″=2-selenophenyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure I wherein E=Se, E′=S, Ar═Ar′═Ar″═Ar′″=2-selenophenyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure I wherein E=Se, E′=S, Ar═Ar′=2-thienyl, Ar″=Ar′″=2-selenophenyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure I wherein E=Se, E′=S, Ar═Ar′=2-selenophenyl, Ar″=Ar′″=2-thienyl, R═H; and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure I wherein E=E′=Se, Ar═Ar′=2-thienyl, Ar″=Ar′″=2-selenophenyl, R═H; and Z═PF6.
In another embodiment, the present disclosure provides thiopyrylium dye of general structure I wherein E=E′=S, Ar═Ar′═Ar″═Ar′″=2-selenophenyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a thiopyrylium dye of general structure II wherein E=E′=S, Ar═Ar′═Ar″═Ar′″=2-thienyl, R═R′═R″=H, and Z═PF6.
In another embodiment, the present disclosure provides a thiopyrylium dye of general structure II wherein E=E′=S, Ar═Ar′=Ph, Ar″=Ar′″=2-thienyl, R═R′═R″=H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure II wherein E=Se, E′=S, Ar═Ar′═Ar″═Ar′″=2-thienyl, R═R′═R″=H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure II wherein E=Se, E′=S, Ar═Ar′=Ph, Ar″=Ar′″=2-thienyl, R═R′ ═R″=H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure II wherein E=Se, E′=S, Ar═Ar′=2-thienyl, Ar″=Ar′″=Ph, R═R′ ═R″=H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure II wherein E=E′=Se, Ar═Ar′═Ar″═Ar′″=2-thienyl, R═R′═R″=H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure II wherein E=E′=Se, Ar═Ar′=Ph, Ar″=Ar′″=2-thienyl, R═R′═R″=H, and Z═PF6.
In another embodiment, the present disclosure provides a thiopyrylium dye of general structure II wherein E=E′=S, Ar═Ar′═Ar″═Ar′″=2-selenophenyl, R═R′═R″ ═H, and Z═PF6.
In another embodiment, the present disclosure provides a thiopyrylium dye of general structure II wherein E=E′=S, Ar═Ar′=Ph, Ar″=Ar′″=2-selenophenyl, R═R′ ═R″=H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure II wherein E=Se, E′=S, Ar═Ar′═Ar″═Ar′″=2-selenophenyl, R═R′═R″=H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure II wherein E=Se, E′=S, Ar═Ar′=2-thienyl, Ar″=Ar′″=2-selenophenyl, R═R′═R″=H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure II wherein E=Se, E′=S, Ar═Ar′=2-selenophenyl, Ar″=Ar′″=2-thienyl, R═R′═R″=H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure II wherein E=E′=Se, Ar═Ar′=2-thienyl, Ar″=Ar′″=2-selenophenyl, R═R′═R″=H, and Z═PF6.
In another embodiment, the present disclosure provides a thiopyrylium dye of general structure II wherein E=E′=S, Ar═Ar′═Ar″═Ar′″=2-selenophenyl, R═R′═R″ ═H, and Z═PF6.
In another embodiment, the present disclosure provides a thiopyrylium dye of general structure III wherein E=E′=S, Ar═Ar′═Ar″═Ar′″=2-thienyl, R═H, and Z=PF6.
In another embodiment, the present disclosure provides a thiopyrylium dye of general structure III wherein E=E′=S, Ar═Ar′=Ph, Ar″=Ar′″=2-thienyl, R═H, and Z ═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure III wherein E=Se, E′=S, Ar═Ar′═Ar″═Ar′″=2-thienyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure III wherein E=Se, E′=S, Ar═Ar′=Ph, Ar″=Ar′″=2-thienyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure III wherein E=Se, E′=S, Ar═Ar′=2-thienyl, Ar″=Ar′″=Ph, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure III wherein E=E′=Se, Ar═Ar′═Ar″═Ar′″=2-thienyl, R═H, and Z PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure III wherein E=E′=Se, Ar═Ar′=Ph, Ar″=Ar′″=2-thienyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a thiopyrylium dye of general structure III wherein E=E′=S, Ar═Ar′═Ar″═Ar′″=2-selenophenyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a thiopyrylium dye of general structure III wherein E=E′=S, Ar═Ar′=Ph, Ar″=Ar′″=2-selenophenyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure III wherein E=Se, E′=S, Ar═Ar′═Ar″═Ar′″=2-selenophenyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure III wherein E=Se, E′=S, Ar═Ar′=2-thienyl, Ar″=Ar′″=2-selenophenyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure III wherein E=Se, E′=S, Ar═Ar′=2-selenophenyl, Ar″=Ar′″=2-thienyl, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure III wherein E=E′=Se, Ar═Ar′=2-thienyl, Ar″=Ar′″=2-selenophenyl, and Z═PF6.
In another embodiment, the present disclosure provides a thiopyrylium dye of general structure III wherein E=E′=S, Ar═Ar′═Ar″═Ar′″=2-selenophenyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a thiopyrylium dye of general structure IV wherein E=E′=S, Ar═Ar′═Ar″═Ar′″=2-thienyl, R═H; R′, all R′s together form a six-membered ring and R″=Cl, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure IV wherein E=E′=Se, Ar═Ar′═Ar″═Ar′″=2-thienyl, R═H; all R′s together form a six-membered ring and R″=Cl, and Z═PF6.
In another embodiment, the present disclosure provides a thiopyrylium dye of general structure IV wherein E=E′=S, Ar═Ar′═Ar″═Ar′″=2-selenophenyl, R═H; R′, all R′s together form a six-membered ring and R″=Cl, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure IV wherein E=E′=Se, Ar═Ar′═Ar″═Ar′″=2-selenophenyl, R═H; R′, all R′s together form a six-membered ring and R″=Cl, and Z═PF6.
In another embodiment, the present disclosure provides a thiopyrylium dye of general structure V wherein E=E′=S, Ar′═Ar″═Ar′″=2-thienyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a thiopyrylium dye of general structure V wherein E=E′=S, Ar′=Ph, Ar″=Ar′″=2-thienyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure V wherein E=Se, E′=S, Ar′═Ar″═Ar′″=2-thienyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure V wherein E=Se, E′=S, Ar′=Ph, Ar″=Ar′″=2-thienyl, R═H, and Z ═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure V wherein E=Se, E′=S, Ar′=2-thienyl, Ar″=Ar′″=Ph, R═H, and Z PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure V wherein E=E′=Se, Ar′═Ar″═Ar′″=2-thienyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure V wherein E=E′=Se, Ar′=Ph, Ar″=Ar′″=2-thienyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a thiopyrylium dye of general structure V wherein E=E′=S, Ar′═Ar″═Ar′″=2-selenophenyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a thiopyrylium dye of general structure V wherein E=E′=S, Ar′=Ph, Ar″=Ar′″=2-selenophenyl, R═H, and Z ═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure V wherein E=Se, E′=S, Ar′═Ar″═Ar′″=2-selenophenyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure V wherein E=Se, E′=S, Ar′=2-thienyl, Ar″=Ar′″=2-selenophenyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure V wherein E=Se, E′=S, Ar′=2-selenophenyl, Ar″=Ar′″=2-thienyl, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure V wherein E=E′=Se, Ar′=2-thienyl, Ar″=Ar′″=2-selenophenyl, and Z═PF6.
In another embodiment, the present disclosure provides a thiopyrylium dye of general structure V wherein E=E′=S, Ar′═Ar″═Ar′″=2-selenophenyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a thiopyrylium dye of general structure VI or VII wherein E=E′=S, Ar′═Ar″=2-thienyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a thiopyrylium dye of general structure VI or VII wherein E=E′=S, Ar′=Ph, Ar′″=2-thienyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure VI or VII wherein E=Se, E′=S, Ar′═Ar′″=2-thienyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure VI or VII wherein E=Se, E′=S, Ar′=Ph, Ar′″=2-thienyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure VI or VII wherein E=Se, E′=S, Ar′=2-thienyl, Ar′″=Ph, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure VI or VII wherein E=E′=Se, Ar′═Ar′″=2-thienyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure VI or VII wherein E=E′=Se, Ar′=Ph,′″=2-thienyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a thiopyrylium dye of general structure VI or VII wherein E=E′=S, Ar′═Ar′″=2-selenophenyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a thiopyrylium dye of general structure VI or VII wherein E=E′=S, Ar′=Ph, Ar′″=2-selenophenyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure VI or VII wherein E=Se, E′=S, Ar′═Ar′″=2-selenophenyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure VI or VII wherein E=Se, E′=S, Ar′=2-thienyl, Ar′″=2-selenophenyl, R═H, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure VI or VII wherein E=Se, E′=S, Ar′=2-selenophenyl, Ar′″=2-thienyl, and Z═PF6.
In another embodiment, the present disclosure provides a selenopyrylium dye of general structure VI or VII wherein E=E′=Se, Ar′=2-thienyl, Ar′″=2-selenophenyl, and Z═PF6.
In another embodiment, the present disclosure provides a thiopyrylium dye of general structure VI or VII wherein E=E′=S, Ar′═Ar′″=2-selenophenyl, R═H, and Z═PF6.
In an object, the present disclosure provides methods of preparing a nanostructure. In an embodiment, a method of preparing a nanostructure comprises: introducing a core to a reporter molecule, where the reporter molecule bonds to the core and the reporter molecule is selected from the chalcogenopyrylium dyes described herein; and optionally, disposing an encapsulating material onto the core and reporter molecule (e.g., reacting a material to form an encapsulating material), where the reporter molecule has a measurable surface-enhanced Raman spectroscopic signature. If applicable, the encapsulating material can be, for example, silica. Other suitable encapsulating materials include silica-based materials such as xerogels from tetraalkoxy silanes or organically modified xerogels from organotrialkoxy silanes and tetraalkoxy silanes; also polyethylene glycol (PEG) such as PEG 5000; and organic polymers such as, but not limited to, polyvinylethylene (PVE).
The method may further comprise conjugating (e.g., covalently or non-covalently bonding) one or more targeting moieties (which can be part of a probe molecule or probe molecules) directly to a surface of the core or to a surface of the core via a linking group. A targeting moiety is any moiety that specifically interacts with (e.g., binds) a target molecule. A probe molecule can comprise a targeting moiety. Examples of targeting moieties (e.g., probe molecules) include, but are not limited to, antibodies, aptamers, synthetic receptors, DNA sequences, proteins, peptides, and the like. Examples of suitable conjugation methods and linkers are known in the art.
In an object, the present disclosure provides uses of the composite nanostructures. The composite nanostructures can be used in methods such as, for example, anti-counterfeit methods, covert tagging methods, cytometry methods (e.g., a flow cytometry system), chemical array methods, biomolecule array methods, biosensing methods, bioimaging methods, biolabeling methods, high-speed screening methods, gene expression methods, protein expression methods, medical diagnostic methods, diagnostic methods, and microfluidic methods.
One embodiment of an exemplary method of detecting a target molecule, among others, includes: attaching a target molecule to a nanostructure as described above; exciting the reporter molecule with a source of radiation; and measuring the surface-enhanced Raman spectroscopy spectrum of the nanostructure corresponding to the reporter molecule in order to determine the presence of the target molecule.
The present disclosure provides a method of detecting one or more target molecules in a sample. The method includes attaching a target molecule (e.g., via interaction with) a probe molecule (i.e., a molecule having a targeting moiety) to the nanostructure and measuring the SERS spectrum of the nanostructure, where the detection of SERS spectrum specific for the reporter molecule indicates the presence of the target molecule specific for the probe molecule (i.e., a molecule having a targeting moiety). The SERS active composite nanostructure can be used to detect the presence of one or more target molecules in chemical array systems, bioimaging and biomolecular array systems. In addition, SERS active composite nanostructures can be used to enhance encoding and multiplexing capabilities in various types of systems.
For example, a method for detecting one or more target molecules in a sample comprises: contacting (e.g., administering to) an individual or other biological material, such as, for example, plants, bacteria, viruses, and other organisms, or a portion thereof, with one or more of the composite nanostructures of the present disclosure, and obtaining surface-enhanced Raman spectroscopy data (e.g., a surface-enhanced Raman spectrum) of a portion of the individual after contact of the portion of the individual with the one or more said composite nanostructures, where observation of surface-enhanced Raman spectroscopy data attributable (e.g., specifically attributable) to a particular composite nanostructure of the one or more said composite nanostructures indicates the presence of the target molecule in the portion of the individual corresponding to the targeting moiety of the particular nanostructure. The method may further comprises obtaining surface-enhanced Raman spectroscopy data (e.g., a surface-enhanced Raman spectrum) of one or more additional portions of the individual after contact of the one or more additional portions of the individual with the one or more said composite nanostructures. The method may further comprise generating an image of at least a portion of the individual using the surface-enhanced Raman spectroscopy data from the portion and, optionally, additional portions of the individual.
An individual may be a human or non-human animal. An individual can be contacted with (e.g., administered) composite nanostructures by methods known in the art. The composite nanostructures can be administered systemically (e.g., by intravenous delivery) or locally to a desired area of an individual. The composite nanostructures are contacted (e.g., administered) prior to obtaining surface-enhanced Raman spectroscopy data from a portion of the individual or other biological material. Composite nanostructures can accumulate in a specific portion (e.g., a specific tissue) of the individual or other biological material as a result of the targeting moiety binding to a target molecule.
Surface-enhanced Raman spectroscopy data (e.g., a surface-enhanced Raman spectrum) can be obtained by methods known in the art. For example, surface-enhanced Raman spectroscopy data (e.g., a surface-enhanced Raman spectrum) is obtained using a laser having a wavelength of 780 nm to 1600 nm, including all nm values and ranges therebetween. In another example, surface-enhanced Raman spectroscopy data (e.g., a surface-enhanced Raman spectrum) is obtained using a laser having a wavelength of 1000 nm to 1600 nm (e.g., 1064 nm or 1280 nm).
In one embodiment, a flow cytometer can be used in multiplexed assay procedures for detecting one or more target molecules using one or more SERS active composite nanostructure. Flow cytometry is an optical technique that analyzes particular particles (e.g., SERS active composite nanostructures) in a fluid mixture based on the particles' optical characteristics. Flow cytometers hydrodynamically focus a fluid suspension of SERS active composite nanostructures into a thin stream so that the SERS active composite nanostructures flow down the stream in substantially single file and pass through an examination zone. A focused light beam, such as a laser beam, illuminates the SERS active composite nanostructures as they flow through the examination zone. Optical detectors within the flow cytometer measure certain characteristics of the light as it interacts with the SERS active composite nanostructures. Commonly used flow cytometers can measure SERS active composite nanostructure emission at one or more wavelengths.
For example, a flow cytometry method comprises, subjecting a plurality of cells to flow cytometry, where the cells comprise composite nanostructures of the present disclosure; obtaining surface-enhanced Raman spectroscopy data (e.g., a surface-enhanced Raman spectrum) for individual cells; and separating the cells based the surface-enhanced Raman spectroscopy data (e.g., a surface-enhanced Raman spectrum) obtained for the individual cells.
One or more target molecules can be detected using a SERS active composite nanostructures and one or more probes having an affinity for one or more of the target molecules. Each SERS active composite nanostructure has a reporter molecule that corresponds to the probe. Prior to being introduced to the flow cytometer, the SERS active composite nanostructures specific for certain target molecules are mixed with a sample that may include one or more target molecules. The SERS active composite nanostructures interact with (e.g., bond or hybridize) the corresponding target molecules for which the probe has an affinity.
Next, the SERS active composite nanostructures are introduced to the flow cytometer. As discussed above, the flow cytometer is capable of detecting the SERS active composite nanostructure after exposure to a first energy. Detection of a certain Raman spectrum corresponding to a certain reporter molecule indicates that a target molecule is present in the sample.
Step(s) of the methods disclosed herein are sufficient to produce the compounds, composite nanostructures, or methods of using the compounds and/or composite nanostructures of the present disclosure. Thus, in various examples, any such method consists essentially of a combination of one or more of the steps of the methods disclosed herein. In various other examples, any such method consists of such step(s).
The following examples are presented to illustrate the present disclosure. They are not intended to be limiting in any manner.
Here, we describe the design of a small library of thiophene- and selenophene-substituted chalcogenopyrylium dyes 1-14 (Scheme 1) that are sensitive SERS reporters on hollow gold nanoshells operating with 1064-nm and 1280-nm excitation. The chalcogenopyrylium dyes allow fine tuning of wavelengths of absorption through the choice of chalcogen atoms in the pyrylium/pyranyl rings and the substituents at the 2- and 6-positions of these rings. Since the SERS effect decreases exponentially as a function of distance from the nanoparticle, it is important that the Raman reporter be near the Au surface. Due to this distance dependence, planar molecules capable of lying flat on the surface should experience the largest enhancement in Raman intensity. X-ray structural studies have shown that the chalcogenopyrylium/chalcogenopyranyl rings and methine carbon of chalcogenopyrylium dyes related to 1-8 are coplanar and computational studies predict similar coplanarity in chalcogenopyrylium trimethine dyes 9-14. Other structural and computational studies have shown that five-membered rings such as thiophene or selenophene can be coplanar with attached chalcogenopyrylium/chalcogenopyranyl rings. The affinity of the reporter for the surface of Au is another important consideration. Thiophenes and selenophenes are both capable of forming self-assembled monolayers on gold. Selenolates have also been shown to have greater affinity for gold than thiolates. Chalcogenopyrylium dyes 1-14 incorporate all these features. The dyes 1-14 incorporate S and Se atoms in the chalcogenopyrylium core to provide attachment to gold and the 2-thienyl and 2-selenophenyl groups provide novel attachment points to gold for Raman reporters.
Results. Synthesis and Properties of the Chalcogenopyrylium Dyes. The synthesis of dyes 1-14 is summarized in Scheme 1. 4-Methylthiopyrylium and 4-methylselenopyrylium salts 15 were prepared by the addition of MeMgBr to the corresponding chalcogenopyranone 16 followed by treatment with aqueous HPF6. Condensation of compound 15 either with the chalcogenopyranone 16 or the (chalcogenopyranyl)acetaldehyde 17 in acetic anhydride gave monomethine dyes 1-8 or trimethine dyes 9-14, respectively. Values of absorption maxima, λmax, in CH2Cl2 for 1-8 varied from 653 nm for 1 to 724 nm for 6 and values of the molar extinction coefficient, ε, were in the range of 1.1×105 to 1.5×105 M−1 cm−1 (Table 1). For trimethine dyes 9-14, values of λmax in CH2Cl2 varied from 784 nm for 10 to 826 nm for 14 while values of E were in the range of 2.0×105 to 2.8×105 M−1 cm−1 (Table 1). The interchange of S and Se atoms in the chalcogenopyrylium backbone, the use of monomethine and trimethine bridges, and the interchange of phenyl, 2-thienyl, and 2-selenophenyl substituents at the 2-,2′-, 6-, and 6′-positions allow each dye to have a unique Raman fingerprint.
In order to demonstrate the unique structure of these dyes, crystals of dye 14 were grown from acetonitrile and the chemical structure was determined by X-ray crystallographic analysis. The results are shown in
The importance of the thiophene and selenophene rings to provide an enhanced SERS spectrum is shown in
Examples of Dyes 1-14 as SERS Reporters on Hollow Gold Nanoshells (HGNs). Synthesis of HGNs for Use with 1064-nm Excitation. The HGN synthesis was carried out under inert conditions using a standard Schlenk line to prevent the cobalt nanoparticles from prematurely oxidizing. The method described was modified slightly from previous reports. In a typical synthesis, cobalt chloride hexahydrate (100 μL, 0.4M; Fisher Scientific, 99.99%) and trisodium citrate dihydrate (550 μL, 0.1 M; Sigma-Aldrich, >99%) were added into deionised water (100 mL) and degassed several times (10 mins vacuum and 15 mins argon). Sodium borohydride (1 mL, 0.1 M; Fisher Scientific, 99%) was injected into the solution and allowed to react for a further 20 minutes (under constant argon flow) until hydrogen evolution ceased, indicating complete hydrolysis of the reductant. The solution was degassed again (8 min vacuum and 10 min argon) before chloroauric acid trihydrate (33 mL, 248 μM; Fisher Scientific, ACS reagent grade) was injected. This mixture was allowed to react for an additional 10 minutes under argon with vigorous stirring. Before being exposed to air, were an obvious colour change from brown to green was observed. Finally, trisodium citrate (500 μL, 0.1 M) was added to stabilise the hollow gold nanoshell solution. Post synthesis, the HGN solution was concentrated through centrifugation (5000×g) and the precipitate was re-dispersed in trisodium citrate solution (2 mM) to give a final concentration of 2.14 nM. The HGNs had a localized surface plasmon resonance (SPR) at 690 nm.
Characterization and Use of HGNs with 1064-nm Excitation. Investigation into the SERS properties of the HGNs were carried out by mixing concentrated HGN solution (135 μL) with Raman reporter solution; namely dyes 1-14, BPE and AZPY (15 μL, 10 μM; synthesised in-house or purchased from Sigma-Aldrich) and potassium chloride (150 μL, 30 mM; Sigma-Aldrich). The Raman measurements were performed using a Real Time Analyzer FT-Raman spectrometer and a laser excitation wavelength of 1064 nm. All the measurements had a 5 second acquisition time and a laser power operating at 420 mW. Each sample was prepared in triplicate and 5 scans of each replicate were recorded. Furthermore, all the Raman spectra have been background corrected. For the SERS particle dilution study, the optimum conditions were used (as stated above) and deionised water was added to obtain subsequent concentrations, over the concentration range 1.3 nM to 1 pM. All other experimental conditions were kept the same as those stated above.
Examples of 1064-nm Excitation of Chalcogenopyrylium Dyes as SERS Reporters. A comparison of aggregated and unaggregated SERS spectra for the dye 9-HGN assemblies with 1064-nm excitation is shown in
The benzo analogues of the chalcogenopyrylium dyes were also useful as SERS reporters on HGNs with 1064-nm excitation. As shown in
Not only do the dye-HGN assemblies give readable SERS spectra, the dye-HGN assemblies give low picomolar limits of detection. Limits of detection (LOD) for dye-HGN assemblies with dyes 9 and 11-13 are shown in
Synthesis of HGNs for Use with 1280-nm Excitation. The HGN synthesis was carried out under inert conditions using a standard Schlenk line to prevent the cobalt nanoparticles from prematurely oxidizing. The method described was modified slightly from previous reports (The Journal of Physical Chemistry B, 2006, 110, 19935-19944; Nanoscale, 2013, 5, 765-771). In a typical synthesis, cobalt chloride hexahydrate (100 μL, 0.4M; Fisher Scientific, 99.99%) and trisodium citrate dihydrate (550 μL, 0.1 M; Sigma-Aldrich, >99%) were added into deionised water (100 mL) and degassed several times (10 mins vacuum and 15 mins argon). Sodium borohydride (1 mL, 0.1 M; Fisher Scientific, 99%) was injected into the solution and allowed to react for a further 20 minutes (under constant argon flow) until hydrogen evolution ceased, indicating complete hydrolysis of the reductant. The solution was degassed again (8 min vacuum and 10 min argon) before chloroauric acid trihydrate (33 mL, 248 μM; Fisher Scientific, ACS reagent grade) was injected. This mixture was allowed to react for an additional 10 minutes under argon with vigorous stirring. Before being exposed to air, were an obvious colour change from brown to green was observed. Finally, trisodium citrate (500 μL, 0.1 M) was added to stabilise the hollow gold nanoshell solution. Post synthesis, the HGN solution was concentrated through centrifugation (5000×g) and the precipitate was re-dispersed in trisodium citrate solution (2 mM) to give a final concentration of 2.97 nM. The HGNs had a localized surface plasmon resonance (SPR) at 720 nm.
Characterization and Use of HGNs with 1280-nm Excitation. Investigation into the SERS properties of the HGNs were carried out by mixing concentrated HGN solution (270 μL) with Raman reporter solution; namely dyes 1-14, BPE and AZPY (40 μL, 10 μM; synthesized in-house or purchased from Sigma-Aldrich) and potassium chloride (300 μL, 30 mM; Sigma-Aldrich). The Raman measurements were performed using a SnRI portable Raman spectrometer and a laser excitation wavelength of 1280 nm. All the measurements had a 7 second acquisition time and a laser power operating at 100 mW. Each sample was prepared in triplicate and 5 scans of each replicate were recorded. Furthermore, all the Raman spectra have been background corrected. For the SERS particle dilution study, the optimum conditions were used (as stated above) and deionised water was added to obtain subsequent concentrations, over the concentration range 1.3 nM to 80 pM. All other experimental conditions were kept the same as those stated above.
Examples of 1280-nm Excitation of Chalcogenopyrylium Dyes as SERS Reporters. The aggregated SERS spectra for the dye-HGN assemblies for dyes 1-14 with 1280-nm excitation is shown in
Dyes 13 and 14 with four 2-thienyl and four 2-selenophenyl substituents, respectively, did not require aggregation to give intense SERS signals. The unaggregated SERS spectra for the dye 13-HGN and dye 14-HGN assemblies with 1280-nm excitation is shown in
The dyes of this disclosure gave very weak SERS spectra with 1280-nm excitation on solid gold nanoparticles prepared as described above for
In imaging applications, the nanoparticle assemblies might be assembled as shown in
Phenyl, 2-thienyl and 2-selenophenyl substituents can be incorporated into chalcogenopyrylium dyes absorbing at even longer wavelengths. If dyes absorb light at the wavelength of emission of the incident laser, the Raman reporters are in resonance with the incident laser and produce surface-enhanced resonance Raman scattering (SERRS), which can be orders of magnitude greater than the SERS response. To this end, we prepared dyes 18-21 (Chart 1, intermediates shown in Chart 2) as novel compositions of matter to show the feasibility of this approach. Dyes 18-21 show absorption maxima of 943 nm, 1001 nm, 1042, and 1119 nm, respectively (Table 1). Dye 20 is the hexafluorophosphate analogue of the commercially available tetrafluoroborate salt, which is sold as IR-1061.
Chart 1 Longer-wavelength absorbing thiopyrylium and selenopyrylium dyes with four phenyl, 2-thienyl, or 2-selenophenyl substituents for use as SERS and SERRS reporters.
Aggregated and unaggregated dye 20-HGN assemblies were prepared as described for aggregated and unaggregated dye-HGN assemblies described with dyes 1-14. The aggregated and unaggregated SERS spectra for the dye 20-HGN assemblies with 1064-nm excitation are shown in
Synthetic Methods. All reactions were performed open to air unless otherwise noted. Concentration in vacuo was performed on a rotary evaporator. NMR spectra were recorded at 300 or 500 MHz for 1H and at 75.5 MHz for 13C with residual solvent signal as internal standard. UV/VIS-near-IR spectra were recorded in quartz cuvettes with a 1-cm path length. Melting points were determined with a capillary melting point apparatus and are uncorrected. Non-hygroscopic compounds have a purity of ≧95% as determined by elemental analyses for C, H, and N. Experimental values of C, H, and N are within 0.3% of theoretical values. 13C NMR was not recorded for pyrylium dyes due to limited solubility in common NMR solvents. Pyranones 16b-16d are known compounds (J. Heterocycl. Chem. 1999, 36, 707-717). The synthesis of thiopyranone 16a and 4-methylpyrylium salt 15c is shown in Scheme 2. 4-Methylpyrylium salts 15a, 15d, and 15e have been reported previously in the literature (J. Org. Chem. 1982, 47, 5235-5239; Organometallics 1988, 7, 1131-1147; Dyes Pigm. 2000, 45, 1-7).
Synthesis of selenophen-2-carbaldehyde (22). Anhydrous DMF (15.0 mL) was added to a flame dried flask under argon and cooled to 0° C. POCl3 (1.71 mL, 18.3 mmol) was added and the mixture allowed to stir for 0.5 h. The ice bath was removed, and selenophene (1.41 mL, 15.3 mmol) added. The reaction was heated to 85° C. and maintained at this temperature for 2.5 h. After cooling to ambient temperature, the mixture was poured into ice water (200 mL), neutralized with satd. NaHCO3 (100 mL) and the product extracted with EtOAc (2×75 mL). The organic layer was dried with MgSO4 and after concentration purified on SiO2 with a 25% EtOAc/hexanes eluent to yield 1.64 g (67%) of a colorless oil: 1H NMR [500 MHz, CDCl3] δ 9.83 (s, 1H), 8.50 (d, 1H, J=5.5 Hz), 8.03 (d, 1H, J=4.0 Hz), 7.49-7.47 (m, 1H).
Synthesis of (1E,4E)-1,5-di(selenophen-2-yl)penta-1,4-dien-3-one (23). Selenophen-2-carbaldehyde (1.64 g, 10.3 mmol) and acetone (0.376 mL, 5.13 mmol) were dissolved in EtOH (10 mL). KOH (0.287 g, 5.13 mmol) dissolved in H2O was added slowly to the stirring mixture and then allowed to stir for 3 hours at ambient temperature. The solution was diluted with H2O (50 mL), and the product extracted with CH2Cl2 (2×50 mL). The organic layer was dried with MgSO4, concentrated, and recrystallized from CH2Cl2/hexanes to yield 1.26 g (72%) of a yellow crystalline solid, mp 135-137° C.: 1H NMR [500 MHz, CDCl3] δ 8.09 (d, 2H, J=5.5 Hz), 7.85 (d, 2H, J=15.5 Hz), 7.52 (d, 2H, J=3.5 Hz), 7.30 (m, 2H), 6.70 (d, 2H, J=15.0 Hz); 13C NMR [75.5 MHz, CDCl3] δ 187.53, 146.19, 137.84, 135.06, 133.90, 130.69, 125.67; HRMS (ESI) m/z 342.9131 (calcd for C13H10O80Se2+H+: 342.9135).
Synthesis of 2,6-di(selenophen-2-yl)tetrahydro-4H-thiopyran-4-one (24a). (1E,4E)-1,5-Di(selenophen-2-yl)penta-1,4-dien-3-one (1.20 g, 3.51 mmol) was dissolved in THF (5.0 mL). To this mixture isopropyl alcohol (10 mL), and K2HPO4 (0.961 g, 4.21 mmol) dissolved in H2O were added followed by the addition of NaHS (0.356 g, 3.86 mmol). This was allowed to stir overnight at ambient temperature and under an argon atmosphere. The reaction was then diluted with H2O (50 mL) and the product extracted with CH2Cl2 (2×50 mL). The organic layer was dried with MgSO4, concentrated and then recrystallized from CH2Cl2/hexanes to yield 1.21 g (92%) of an off-white solid, mp 115-116° C.: 1H NMR [300 MHz, CDCl3] δ 7.97 (dd, 2H, J=5.3, 2.0 Hz), 7.21-7.17 (m, 4H), 4.71 (dd, 2H, J=12.3, 5.0 Hz), 3.14 (dd, 2H, J=13.8, 2.7 Hz), 2.95 (m, 2H); 13C NMR [75.5 MHz, CDCl3] δ 205.63, 149.27, 130.91, 129.09, 127.09, 51.99, 45.45; HRMS (EI) m/z 375.8933 (calcd for C13H12OS80Se2: 375.8934).
Synthesis of 2,6-di(selenophen-2-yl)tetrahydro-4H-selenopyran-4-one (24b). Selenium powder (0.502 g, 6.36 mmol), NaBH4 (0.481 g, 12.7 mmol), K2HPO4 (1.45 g, 6.36 mmol), H2O (7.5 mL) and iPrOH (15 mL) were combined in a flask that had been flushed with argon and stirred for 15 min. (1E,4E)-1,5-di(selenophen-2-yl)penta-1,4-dien-3-one (1.45 g, 4.24 mmol) was dissolved in THF (7.5 mL) and added slowly to the stirring mixture. This was allowed to stir at ambient temperature for 1.5 h. The reaction was then diluted with H2O (100 mL) and the product extracted with CH2Cl2 (3×50 mL). The organic layer was dried with MgSO4, concentrated and then purified on SiO2 with a CH2Cl2 eluent (Rf=0.60) to yield 1.33 g (74%) of a light yellow oil: 1H NMR [500 MHz, CDCl3] δ 7.98-7.96-7.95 (m, 2H), 7.17-7.11 (m, 4H), 4.96 (dd, 1H, 12.5, 3.0 Hz), 4.91 (t, 1H, J=6.5 Hz), 3.27-3.23 (m, 2H), 3.16 (t, 1H, J=13.0 Hz); 13C NMR [75.5 MHz, CDCl3] δ 206.98, 206.69, 152.55, 150.55, 130.85, 130.68, 129.35, 129.24, 127.50, 126.83, 52.34, 50.78, 38.08, 36.22.
Synthesis of 2,6-di(selenophen-2-yl)-4H-thiopyran-4-one (16a). 2,6-Di(selenophen-2-yl)tetrahydro-4H-thiopyran-4-one (0.450 g, 1.20 mmol) was dissolved in anhydrous toluene (6.0 mL) and placed in a flame dried flask under argon. 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (0.679 g, 2.99 mmol) was added in one portion and the reaction refluxed for 1.5 h. This was cooled to ambient temperature, diluted with CH2Cl2 (50 mL) and the mixture washed with satd. aqueous NaHCO3 (50 mL). The organic layer was separated, dried with MgSO4, and after concentration purified on SiO2 with a 20% EtOAc/CH2Cl2 eluent (Rf=0.47) to yield 0.273 g (61%) of a light brown solid, mp 138-140° C.: 1H NMR [500 MHz, CDCl3] δ 8.19 (dd, 2H, J=5.5, 1.5 Hz), 7.69 (dd, 2H, J=4.0, 1.5 Hz), 7.38 (m, 2H), 7.09 (s, 2H); 13C NMR [75.5 MHz, CDCl3] δ 182.13, 146.78, 143.61, 134.77, 130.82, 129.51, 125.67; HRMS (ESI) m/z 375.8699 (calcd for C13H8OS80Se2+H+: 372.8698).
Synthesis of 2,6-di(selenophen-2-yl)-4H-thiopyran-4-one (27). 2,6-di(selenophen-2-yl)tetrahydro-4H-selenopyran-4-one (0.386 g, 0.911 mmol) was dissolved in anhydrous toluene (7.5 mL) and placed in a flame dried flask under argon. 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (0.517 g, 2.28 mmol) was added in one portion and the reaction refluxed for 2 h. The reaction was cooled to ambient temperature, diluted with CH2Cl2 (50 mL) and the mixture washed with satd. aqueous NaHCO3 (50 mL). The organic layer was separated and the product extracted with additional CH2Cl2 (2×50 mL). The organic layer was dried with MgSO4, and after concentration purified on SiO2 with a 20% EtOAc/CH2Cl2 eluent (Rf=0.56) to yield 0.184 g (48%) of a light brown solid: 1H NMR [500 MHz, CDCl3] δ 8.19 (d, 2H, J=5.5 Hz), 7.62 (d, 2H, J=3.5 Hz), 7.38 (t, 2H, J=5.0 Hz), 7.16 (s, 2H); 13C NMR [75.5 MHz, CDCl3] δ 184.16, 147.44, 145.35, 134.65, 130.75, 129.55, 126.94.
Synthesis of 4-methyl-2,6-di(thiophen-2-yl)thiopyrylium hexafluorophosphate (15a). 2,6-Bis(thiophen-2-yl)-4H-thiopyran-4-one (0.288 g, 1.04 mmol) was dissolved in anhydrous THF (7.0 mL) in a flame dried flask under argon. 3.0 M MeMgBr (1.04 mL, 3.12 mmol) was added dropwise to this solution and allowed to stir at ambient temperature for 2 h. The solution was poured into 10% aqueous HPF6 (50 mL) and allowed to stir for 15 min before the solid was isolated by filtration. The resulting solid was dissolved in CH2Cl2, dried with Na2SO4, and the solvent removed under reduced pressure. The product was then recrystallized from CH3CN/ether to yield 0.381 g (87%) of a bright red solid, mp 190-192° C.: 1H NMR [500 MHz, CD3CN] δ 8.35 (s, 2H), 8.08-8.07 (m, 4H), 7.39 (t, 2H, J=5.0 Hz), 2.78 (s, 3H); 13C NMR [75.5 MHz, CD3CN] δ 167.63, 159.95, 137.91, 137.18, 133.96, 131.84, 131.21, 25.92; HRMS (ESI) m/z 275.0021 (calcd for C14H11S3+: 275.0017).
Synthesis of 4-methyl-2,6-bis(thiophen-2-yl)selenopyrylium hexafluorophosphate (15b). In a flame-dried flask under argon, 2,6-bis(thiophen-2-yl)-4H-selenopyran-4-one (0.200 g, 0.621 mmol), was dissolved in anhydrous THF (5.0 mL). 3.0 M MeMgBr (0.620 mL, 1.86 mmol) was added dropwise and the solution allowed to stir at ambient temperature for 0.5 h. The mixture was quenched with MeOH (1 mL), poured into 10% aqueous HPF6 (50 mL), and extracted with CH2Cl2 (3×25 mL). The organic layer was dried with Na2SO4, concentrated under reduced pressure, and then recrystallized from CH3CN/ether to yield 0.239 g (82%) of a red solid, mp 185-187° C.: 1H NMR [500 MHz, CD3CN] δ 8.23 (s, 4H), 8.11 (d, 2H, J=5.5 Hz), 8.02 (d, 2H, J=4.5 Hz), 7.39 (t, 2H, J=4.0 Hz), 2.70 (s, 3H); 13C NMR [75.5 MHz, CD3CN] δ 168.23, 167.68, 139.67, 134.04, 132.10, 131.35, 27.31; HRMS (ESI) m/z 322.9469 (calcd for C14H11S280Se+: 322.9462).
Synthesis of 4-methyl-2,6-di(selenophen-2-yl)thiopyrylium hexafluorophosphate (15c). 2,6-Di(selenophen-2-yl)-4H-thiopyran-4-one (0.300 g, 0.807 mmol) was dissolved in anhydrous THF (8.0 mL) in a flame dried flask under argon. 3.0 M MeMgBr (0.807 mL, 2.42 mmol) was added dropwise and the reaction stirred at ambient temperature for 1 h. The reaction was poured into 10% aqueous HPF6 (40 mL), and stirred for 10 min. The resulting solid was extracted with a mixture of CH2Cl2 (50 mL) and CH3CN (5.0 mL), dried with Na2SO4, and after concentration recrystallized from CH3CN/ether to yield 0.312 g (75%) of a bright red solid, mp >260° C.: 1H NMR [500 MHz, CD3CN] δ 8.78 (d, 2H, J=5.5 Hz), 8.26-8.25 (m, 4H), 7.61 (t, 2H, J=4.5 Hz), 2.76 (s, 3H); 13C NMR [75.5 MHz, CDCl3] δ 167.42, 162.17, 144.98, 142.11, 136.62, 133.88, 132.19, 25.77; HRMS (EI) m/z 370.8900 (calcd for C14H11S80Se2+: 370.8906).
Synthesis of 4-methyl-2,6-di(selenophen-2-yl)selenopyrylium hexafluorophosphate (28). 2,6-di(selenophen-2-yl)-4H-selenopyran-4-one (0.750 g, 1.79 mmol) was dissolved in anhydrous THF (9.0 mL) in a flame dried flask under argon. 3.0 M MeMgBr (1.79 mL, 3.02 mmol) was added dropwise and the reaction stirred at ambient temperature for 1 h. The reaction was poured into 10% aqueous HPF6 (50 mL), and stirred for 30 min. The resulting solid was extracted with CH2Cl2 (50 mL) with the aid of CH3CN (10 mL), dried with Na2SO4, and after concentration recrystallized from CH3CN/ether to yield 0.789 g (78%) of a dark red solid: 1H NMR [500 MHz, CD3CN] δ 8.82 (d, 2H, J=6.0 Hz), 8.19 (d, 2H, J=4.0 Hz), 8.12 (s, 2H), 7.62-7.60 (m, 2H), 2.68 (s, 3H).
Synthesis of 4-(2,6-diphenyl-4H-thiopyran-4ylidene)acetaldehyde (17a). 4-Methyl-2,6-di(phenyl)selenopyrylium hexafluorophosphate (0.200 g, 0.439 mmol), N,N-dimethylthioformamide (0.112 mL, 1.32 mmol) and Ac2O (4.0 mL) were added to a round-bottom flask and heated at 95° C. for 90 min. After cooling to ambient temperature CH3CN (4.0 mL) was added and the product precipitated by addition of ether and chilling overnight in the freezer. The iminium salt was isolated by filtration, and hydrolyzed by dissolving in CH3CN (4.0 mL), adding satd. aqueous NaHCO3 (4.0 mL) and heating the mixture to 80° C. over a 15 min period. The reaction was maintained at this temperature for 0.5 h, after which the reaction was diluted with H2O (50 mL), the product extracted with CH2Cl2 (3×30 mL), dried with MgSO4, and after concentration purified on SiO2 with first a CH2Cl2 and then a 10% EtOAc/CH2Cl2 (Rf=0.70) eluent to yield 0.122 g (82%) of a orange oil: 1H NMR [500 MHz, CDCl3] δ 10.11 (d, 1H, J=10.5 Hz), 8.32 (s, 1H), 7.62-7.46 (m, 9H), 7.00 (s, 1H), 5.88 (d, 1H, J=11.0 Hz); 13C NMR [75.5 MHz, CDCl3] δ 188.69, 148.44, 147.01, 146.03, 138.65, 138.40, 129.96, 129.90, 129.14, 126.62, 126.44, 126.37, 126.31, 125.67, 120.57, 120.48; HRMS (EI) m/z 339.0292 (calcd for C19H15O80Se: 339.0283).
Synthesis of 4-(2,6-di(thiophene-2-yl)-4H-thiopyran-4ylidene)acetaldehyde (17b). 4-Methyl-2,6-di(thiophen-2-yl)thiopyrylium hexafluorophosphate (0.350 g, 0.833 mmol), N,N-dimethylthioformamide (0.213 mL, 2.50 mmol) and Ac2O (3.0 mL) were combined in a small round bottom flask and heated at 95° C. for 1 h. After cooling to ambient temperature an additional portion of Ac2O (2.0 mL) was added and the solution diluted with ether. The formed iminium salt was allowed to precipitate in the freezer overnight, and then isolated by filtration to yield a bright orange solid. This solid was dissolved in CH3CN (3.0 mL) and satd. aqueous NaHCO3 (3.0 mL) was added. This mixture was heated to 80° C. over 15 min, and kept at that temperature for 0.5 h. After diluting with H2O (30 mL) the product was extracted with CH2Cl2 (3×50 mL), dried with Na2SO4 and purified on SiO2 with a 10% EtOAc/CH2Cl2 eluent (Rf=0.71) to yield a yellow oil that was recrystallized in CH2Cl2/hexanes to yield 0.219 g (87%) of a yellow crystalline solid, mp 143-144° C.: 1H NMR [500 MHz, CDCl3] δ 9.84 (d, 1H, J=6.0 Hz), 8.26 (s, 1H), 7.45-7.39 (m, 4H), 7.13-7.11 (m, 2H), 6.88 (s, 1H), 5.72 (d, 1H, J=6.5 Hz); 13C NMR [75.5 MHz, CDCl3] δ 188.05, 146.43, 139.36, 139.07, 137.33, 136.65, 128.16, 127.78, 127.58, 126.30, 126.01, 122.48, 117.63, 117.48; HRMS (EI) m/z 302.9971 (calcd for C15H11O1S3: 302.9967).
Synthesis of 2-(2,6-di(selenophen-2-yl)-4H-thiopyran-4-ylidene)acetaldehyde (17c). 4-Methyl-2,6-di(selenophen-2-yl)thiopyrylium hexafluorophosphate (0.150 g, 0.291 mmol), N,N-dimethylthioformamide (74.3 μL, 0.872 mmol) and Ac2O (2.0 mL) were added to a round-bottom flask and heated at 95° C. for 90 min. After cooling to ambient temperature, Ac2O (2.0 mL) was added and the product precipitated by addition of ether and chilling overnight in the freezer. The iminium salt was isolated by filtration, and hydrolyzed by dissolving in CH3CN (3.0 mL), adding satd. aqueous NaHCO3 (3.0 mL) and heating the mixture to 80° C. over a 15 min period. The reaction was maintained at this temperature for ½ h, after which the reaction was diluted with H2O (30 mL), the product extracted with CH2Cl2 (3×20 mL), dried with MgSO4, and after concentration purified on SiO2 with a 10% EtOAc/CH2Cl2 (Rf=0.62) eluent to yield 80.3 mg (69%) of a brown solid, mp 145-146° C.: 1H NMR [500 MHz, CDCl3] δ 9.95 (d, 1H, J=6.0 Hz), 8.21 (s, 1H), 8.13-8.10 (m, 2H), 7.62 (d, 1H, J=4.0 Hz), 7.57 (d, 1H, J=4.0 Hz), 7.36-7.33 (m, 2H), 6.81 (s, 1H), 5.72 (d, 1H, J=6.0 Hz); 13C NMR [75.5 MHz, CDCl3] δ 188.10, 146.72, 144.92, 144.58, 139.48, 138.77, 133.47, 133.21, 130.57, 128.62, 128.34, 123.32, 118.53, 117.38; HRMS (ESI) m/z 398.8861 (calcd for C15H10OS80Se2+H+: 398.8856).
Synthesis of 4-(2,6-diphenyl-4H-thiopyran-4-ylidene)methyl)-2,6-di(thiophen-2-yl)thiopyrylium hexafluorophosphate (Dye 1). 4-Methyl-2,6-di(thiophen-2-yl)thiopyrylium hexafluorophosphate (50.0 mg, 0.119 mmol), 2,6-bis(phenyl)-4H-thiopyran-4-one (34.6 mg, 0.131 mmol) and Ac2O (2.0 mL) were heated for 5 min at 105° C. prior to cooling to ambient temperature, diluting with CH3CN (3.0 mL) and adding ether to precipitate the product. Yielded 77.6 mg (91%) of a copper bronze solid, mp 222-223° C.: 1H NMR [500 MHz, CD2Cl2] δ 8.04 (br s, 2H), 7.90 (br s, 2H), 7.83 (d, 4H, J=8.0 Hz), 7.75-7.73 (m, 4H), 7.70-7.63 (m, 6H), 7.30 (t, 2H, J=4.5 Hz), 6.67 (s, 1H); Anal. Calcd for C31H21S4.PF6: C, 55.85; H, 3.17. Found: C, 55.90; H, 3.29; HRMS (ESI) m/z 521.0518 (calcd for C31H21S4+: 521.0521); λmax (CH2Cl2)=653 nm, ε=1.3×105 M−1 cm−1; 473 nm ε=1.6×104 M−1 cm1; 410 nm ε=1.4×104 M−1 cm1.
Synthesis of 4-((2,6-diphenyl-4H-selenopyran-4-ylidene)methyl)-2,6-di(thiophen-2-yl)thiopyrylium hexafluorophosphate (Dye 2). 4-Methyl-2,6-di(thiophen-2-yl)thiopyrylium hexafluorophosphate (50.0 mg, 0.119 mmol), 2,6-bis(phenyl)-4H-selenopyran-4-one (40.6 mg, 0.131 mmol) and Ac2O (2.0 mL) were heated for 5 min at 105° C. prior to cooling to ambient temperature, diluting with CH3CN (3.0 mL) and adding ether to precipitate the product. Yielded 77.6 mg (91%) of a copper bronze solid, mp 249-250° C.: 1H NMR [500 MHz, CD2Cl2] δ 8.02 (br s, 2H), 7.94 (s, 2H), 7.78-7.75 (m, 8H), 7.67-7.60 (m, 6H), 7.30 (t, 2H, J=5.0 Hz), 6.76 (s, 1H); Anal. Calcd for C31H21S3Se.PF6½H2O:C, 51.53; H, 3.07. Found: C, 51.55; H, 3.01; HRMS (ESI) m/z 568.9958 (calcd for C31H21S380Se+: 568.9965); λmax (CH2Cl2)=676 nm, ε=1.3×105 M−1 cm−1; 483 nm, ε=1.6×104 M−1 cm−1; 422 nm, ε=1.5×104 M−1 cm−1.
Synthesis of 4-((2,6-diphenyl-4H-selenopyran-4-ylidene)methyl)-2,6-di(thiophen-2-yl)selenopyrylium hexafluorophosphate (Dye 3). 4-Methyl-2,6-di(thiophen-2-yl)selenopyrylium hexafluorophosphate (50.0 mg, 0.107 mmol), 2,6-bis(phenyl)-4H-selenopyran-4-one (36.6 mg, 0.118 mmol) and Ac2O (4.0 mL) were heated for 90 sec at 105° C. prior to cooling to ambient temperature, diluting with CH3CN (3.0 mL) and adding ether to precipitate the product. Product was purified on SiO2 with a 10% EtOAc/CH2Cl2 eluent (Rf=0.34) to yield 35.7 mg (44%) of a copper bronze solid, mp 243-244° C.: 1H NMR [500 MHz, CD2Cl2] δ 8.06 (br s, 2H), 7.93 (s, 2H), 7.79 (d, 4H, J=7.5 Hz), 7.75 (d, 2H, J=5.5 Hz), 7.68-7.65 (m, 4H), 7.62 (t, 4H, J=7.0 Hz), 7.29 (t, 2H, J=4.5 Hz), 6.86 (s, 1H); Anal. Calcd for C31H21S2Se2.PF6: C, 48.96; H, 2.78. Found: C, 48.68; H, 2.76; HRMS (ESI) m/z 616.9402 (calcd for C31H21S280Se2+: 616.9410); λmax (CH2Cl2)=699 nm, ε=1.5×105 M−1 cm−1; 499 nm, ε=1.9×104 M−1 cm−1; 428 nm ε=1.8×104 M−1 cm−1.
Synthesis of 4-((2,6-di(thiophen-2-yl)-4H-thiopyran-4-ylidene)methyl)-2,6-di(thiophen-2-yl)thiopyrylium hexafluorophosphate (Dye 4). 4-Methyl-2,6-di(thiophen-2-yl)thiopyrylium hexafluorophosphate (50.0 mg, 0.119 mmol), 2,6-bis(thiophen-2-yl)-4H-thiopyran-4-one (36.2 mg, 0.131 mmol) and Ac2O (2.0 mL) were heated for 20 min at 105° C. prior to cooling to rt, diluting with CH3CN (3.0 mL) and adding ether to precipitate the product. Yielded 78.6 mg (97%) of a copper bronze solid, mp >260° C.: 1H NMR [500 MHz, CD2Cl2] δ 7.86 (br s, 4H), 7.75 (d, 8H, J=4.5 Hz), 7.30 (t, 4H, J=4.5 Hz), 6.61 (s, 1H); Anal. Calcd for C27H17S6.PF6: C, 47.78; H, 2.52. Found: C, 47.94; H, 2.44; HRMS (ESI) m/z 532.9628 (calcd for C27H17S6+: 532.9649); λmax (CH2Cl2)=676 nm, ε=1.2×105 M−1 cm−1; 480 nm, ε=2.7×104 M−1 cm−1.
Synthesis of 4-((2,6-di(thiophen-2-yl)-4H-thiopyran-4-ylidene)methyl)-2,6-di(thiophen-2-yl)selenopyrylium hexafluorophosphate (Dye 5). 4-Methyl-2,6-di(thiophen-2-yl)thiopyrylium hexafluorophosphate (50.0 mg, 0.119 mmol), 2,6-bis(thiophen-2-yl)-4H-selenopyran-4-one (42.3 mg, 0.131 mmol) and Ac2O (2.0 mL) were heated for 5 min at 105° C. prior to cooling to ambient temperature, diluting with CH3CN (3.0 mL) and adding ether to precipitate the product. Yielded 83.2 mg (96%) of a copper bronze solid, mp 254-256° C.: 1H NMR [500 MHz, CD2Cl2] δ 7.94 (s, 2H), 7.89 (br s, 2H), 7.83-7.87 (m, 6H), 7.71 (dd, 2H, J=4.0, 1.0 Hz), 6.73 (s, 1H); Anal. Calcd for C27H17S5Se.PF6: C, 44.69; H, 2.36. Found: C, 44.76; H, 2.49; HRMS (ESI) m/z 580.9087 (calcd for C27H17S580Se+: 580.9094); λmax (CH2Cl2)=698 nm, ε=1.1×105 M−1 cm−1; 493 nm, ε=2.5×104 M−1 cm−1.
Synthesis of 4-((2,6-di(thiophen-2-yl)-4H-selenopyran-4-ylidene)methyl)-2,6-di(thiophen-2-yl)selenopyrylium hexafluorophosphate (Dye 6). 4-Methyl-2,6-di(thiophen-2-yl)selenopyrylium hexafluorophosphate (50.0 mg, 0.107 mmol), 2,6-bis(thiophen-2-yl)-4H-selenopyran-4-one (37.9 mg, 0.118 mmol) and Ac2O (2.0 mL) were heated for 5 min at 105° C. prior to cooling to ambient temperature, diluting with CH3CN (3.0 mL) and adding ether to precipitate the product. Yielded 77.6 mg (94%) of a copper bronze solid, mp 233-234° C.: 1H NMR [500 MHz, CD2Cl2] δ 7.92 (br s, 4H), 7.79 (d, 4H, J=5.0 Hz), 7.74 (d, 4H, J=3.0 Hz), 7.33 (t, 4H, J=4.5 Hz), 6.82 (s, 1H); Anal. Calcd for C27H17S4Se2.PF6: C, 41.98; H, 2.22. Found: C, 41.69; H, 2.15; HRMS (ESI) m/z 628.8533 (calcd for C22H17S480Se2+: 628.8543); λmax (CH2Cl2)=723 nm, ε=1.3×105 M−1 cm−1; 506 nm, ε=2.9×104 M−1 cm−1.
Synthesis of 4-((2,6-diphenyl-4H-thiopyran-4-ylidene)methyl)-2,6-di(selenophen-2-yl)thiopyrylium hexafluorophosphate (Dye 7). 4-Methyl-2,6-diphenylthiopyrylium hexafluorophosphate (50.0 mg, 0.123 mmol), 2,6-di(selenophen-2-yl)-4H-thiopyran-4-one (50.1 mg, 0.135 mmol) and Ac2O (2.0 mL) were heated for 5 min at 105° C. prior to cooling to ambient temperature, diluting with CH3CN (3.0 mL) and adding ether to precipitate the product to yield 79.0 mg (85%) of a copper bronze solid, mp >260° C.: 1H NMR [500 MHz, CD2Cl2] δ 8.44 (d, 2H, J=5.0 Hz), 8.03 (br s, 2H), 7.91 (d, 2H, J=4.0 Hz), 7.83-7.82 (m, 6H), 7.69-7.62 (m, 6H), 7.51 (t, 2H, J=5.0 Hz), 6.72 (s, 1H); Anal. Calcd for C31H21S2Se2.PF6: C, 48.96; H, 2.78. Found: C, 49.09; H, 2.98; HRMS (ESI) m/z 616.9397 (calcd for C31H21S280Se2+: 616.9410); λmax (CH2Cl2)=659 nm, ε=1.4×105 M−1 cm−1; 484 nm, ε=1.8×104 M−1 cm1; 428 nm ε=1.7×104 M−1 cm1.
Synthesis of 4-((2,6-di(selenophen-2-yl)-4H-thiopyran-4-ylidene)methyl)-2,6-di(selenophen-2-yl)thiopyrylium hexafluorophosphate (Dye 8). 4-Methyl-2,6-di(selenophen-2-yl)thiopyrylium hexafluorophosphate (50.0 mg, 96.9 μmol), 2,6-di(selenophen-2-yl)-4H-thiopyran-4-one (39.6 mg, 0.107 mmol) and Ac2O (2.0 mL) were heated for 5 min at 105° C. prior to cooling to ambient temperature, diluting with CH3CN (3.0 mL) and adding ether to precipitate the product to yield 64.2 mg (76%) of a copper bronze solid, mp >260° C.: 1H NMR [500 MHz, CD3CN] δ 8.48 (d, 4H, J=6.0 Hz), 7.93 (d, 4H, J=3.5 Hz), 7.74 (s, 4H), 7.47 (t, 4H, J=5.0 Hz), 6.57 (s, 1H); Anal. Calcd for C27H17S2Se4.PF6: C, 37.43; H, 1.98. Found: C, 37.70; H, 2.06; HRMS (ESI) m/z 724.7393 (calcd for C22H17S280Se4+: 724.7427); λmax (CH2Cl2)=687 nm, ε=1.1×105 M−1 cm−1; 491 nm, ε=2.8×104 M−1 cm−1.
Synthesis of 4-(3-(2,6-diphenyl-4H-selenopyran-4-ylidene)prop-1-enyl)-2,6-diphenylselenopyrylium hexafluorophosphate (CAS Registry Number: 51848-65-8) (Dye 9). 4-Methyl-2,6-di(phenyl)selenopyrylium hexafluorophosphate (0.190 g, 0.417 mmol), 4-(2,6-diphenyl-4H-selenopyran-4ylidene)acetaldehyde (0.155 g, 0.459 mmol) and Ac2O (3.0 mL) were combined in a round bottom flask and heated at 105° C. for 10 min. The reaction was cooled to ambient temperature, precipitated with ether, and the collected solid recrystallized from CH3CN/ether to yield 0.278 g (86%) of a golden-green solid: 1H NMR [500 MHz, CD2Cl2] δ 8.59 (t, 1H, J=13.5 Hz), 8.40-7.80 (br s, 4H), 7.71 (d, 8H, J=7.0 Hz), 7.63-7.59 (m, 12H), 6.85 (d, 2H, J=13.0 Hz); Anal. Calcd for C37H27Se2.PF6: C, 57.38; H, 3.51; F, 14.72. Found: C, 57.34; H, 3.48; F, 14.76; LRMS (ESI) m/z 631.2 (calcd for C32H2280Se2: 631.0); λmax (CH2Cl2)=806 nm, ε=2.5×105 M−1 cm−1.
Synthesis of 4-(3-(2,6-diphenyl-4H-thiopyran-4-ylidene)prop-1-enyl)-2,6-diphenylselenopyrylium hexafluorophosphate (CAS Registry Number: 79054-92-5) (Dye 10). 4-Methyl-2,6-di(phenyl)thiopyrylium hexafluorophosphate (0.128 g, 0.312 mmol), 4-(2,6-diphenyl-4H-selenopyran-4ylidene)acetaldehyde (0.157 g, 0.344 mmol) and Ac2O (2.0 mL) were combined in a round bottom flask and heated at 105° C. for 10 min. The reaction was cooled to ambient temperature, CH3CN (2.0 mL) was added and ether was used to precipitate product from solution to yield 0.196 g (86%) of a copper-bronze solid: 1H NMR [500 MHz, CD2Cl2] δ 8.54 (t, 1H, J=13.0 Hz), 8.20-7.80 (br s, 4H), 7.78 (d, 4H, J=8.0 Hz), 7.70 (d, 4H, J=7.5 Hz), 7.66-7.58 (m, 12H), 6.78 (d, 2H, J=13.5 Hz); Anal. Calcd for C39H34O3Se2.PF6: C, 61.08; H, 3.74. Found: C, 61.10; H, 3.68; LRMS (ESI) m/z 583.3 (calcd for C37H27S80Se: 583.1); λmax (CH2Cl2)=784 nm, ε=2.0×105 M−1 cm−1.
Synthesis of 4-(3-(2,6-dithiophen-2-yl-4H-thiopyran-4-ylidene)prop-1-enyl)-2,6-diphenylselenopyrylium hexafluorophosphate (Dye 11). 4-Methyl-2,6-di(phenyl)selenopyrylium hexafluorophosphate (0.102 g, 0.225 mmol), 4-(2,6-(thiophene-2-yl)-4H-thiopyran-4ylidene)acetaldehyde (75.0 mg, 0.248 mmol) and Ac2O (3.0 mL) were combined in a round bottom flask and heated at 105° C. for 5 min. The reaction was cooled to ambient temperature, precipitated with ether, and the collected solid recrystallized from CH3CN/ether to yield 0.145 g (87%) of a bronze solid, mp 229-231° C.: 1H NMR [500 MHz, CD2Cl2] δ 8.46 (t, 1H, J=13.0 Hz), 7.71-7.58 (m, 18H), 7.26 (t, 2H, J=4.0 Hz), 6.77 (d, 1H, J=13.0 Hz), 6.70 (d, 1H, J=14.0 Hz); Anal. Calcd for C33H23S3Se.PF6: C, 53.59; H, 3.13; F, 15.41. Found: C, 53.79; H, 3.13; F, 15.19; HRMS (ESI) m/z 595.0125 (calcd for C33H23S380Se+: 595.0122); λmax (CH2Cl2)=810 nm, ε=2.5×105 M−1 cm−1.
Synthesis of 4-(3-(2,6-dithiophen-2-yl-4H-thiopyran-4-ylidene)prop-1-enyl)-2,6-diphenylthiopyrylium hexafluorophosphate (Dye 12). 4-Methyl-2,6-diphenylthiopyrylium hexafluorophosphate (30.0 mg, 73.0 μmol), 4-(2,6-(thiophene-2-yl)-4H-thioopyran-4ylidene)acetaldehyde (24.4 mg, 81.0 μmol) and Ac2O (1.0 mL) were combined in a round bottom flask and heated at 105° C. for 5 min. The reaction was cooled to ambient temperature, CH3CN (4.0 mL) was added and ether was used to precipitate product from solution to yield 45.0 mg (88%) of a bronze solid, mp >260° C.: 1H NMR [500 MHz, CD2Cl2] δ 8.44 (t, 1H, J=13.0 Hz), 8.40-7.80 (br s, 4H), 7.78 (d, 4H, J=7.0 Hz), 7.67-7.59 (m, 10H), 7.24 (t, 2H, J=4.5 Hz), 6.71 (d, 1H, J=13.0 Hz), 6.63 (d, 1H, J=13.5 Hz); Anal. Calcd for C33H23S4.PF6: C, 57.21; H, 3.35. Found: C, 56.97; H, 3.36; HRMS (ESI) m/z 547.0674 (calcd for C33H23S4+: 547.0677); λmax (CH2Cl2)=789 nm, ε=2.2×105 M−1 cm−1.
Synthesis of 4-(3-(2,6-dithiophen-2-yl-4H-thiopyran-4-ylidene)prop-1-enyl)-(2,6-dithiophen-2-yl)thiopyrylium hexafluorophosphate (CAS Registry Number: 95410-36-9) (Dye 13). 4-Methyl-2,6-di(thiophen-2-yl)thiopyrylium hexafluorophosphate (11.0 mg, 26.2 μmol), 4-(2,6-(thiophene-2-yl)-4H-thioopyran-4ylidene)acetaldehyde (9.5 mg, 31.4 μmol) and Ac2O (1.0 mL) were combined in a round bottom flask and heated at 105° C. for 5 min. The reaction was cooled to ambient temperature, CH2Cl2 (2.0 mL) was added and ether was used to precipitate product from solution to yield 17.8 mg (94%) of a bronze solid, mp >260° C.: 1H NMR [500 MHz, CD3CN] δ 8.32 (t, 1H, J=13.5 Hz), 7.68 (d, 2H, J=4 Hz), 7.56 (br s, 4H) 7.14 (t, 4H, J=4.5 Hz), 6.48 (d, 2H, J=13.0 Hz); Anal. Calcd for C29H19S6.PF6: C, 49.42; H, 2.72. Found: C, 49.19; H, 2.79; HRMS (ESI) m/z 558.9805 (calcd for C29H19S6+: 558.9806); λmax (CH2Cl2)=813 nm, ε=2.8×105 M−1 cm−1.
Synthesis of 4-(3-(2,6-di(selenophen-2-yl)-4H-thiopyran-4-ylidene)prop-1-en-1-yl)-2,6-di(selenophen-2-yl)thiopyrylium hexafluorophosphate (Dye 14). 4-Methyl-2,6-di(selenophen-2-yl)thiopyrylium hexafluorophosphate (47.1 mg, 91.4 μmol), 2-((2,6-di(selenophen-2-yl)-4H-thiopyran-4-ylidene)acetaldehyde (47.1 mg, 0.101 mmol) and Ac2O (2.0 mL) were heated for 5 min at 105° C. prior to cooling to ambient temperature, diluting with CH3CN (3.0 mL) and adding ether to precipitate the product to yield 71.0 mg (87%) of a copper bronze solid, mp 249-251° C.: 1H NMR [500 MHz, CD3CN] δ 8.51-8.46 (m, 5H), 7.88 (d, 4H, J=3.0 Hz), 7.71 (br s, 4H), 7.46 (t, 4H, J=4.5 Hz), 6.62 (d, 2H, J=13.0 Hz); Anal. Calcd for C29H19S2Se4.PF6: C, 39.03; H, 2.15. Found: C, 39.28; H, 2.19; HRMS (ESI) m/z 750.7560 (calcd for C29H19S280Se4+: 750.7584); λmax (CH2Cl2)=826 nm, ε=2.3×105 M−1 cm−1; 750 nm, ε=5.2×104 M−1 cm−1; 490 nm, ε=2.8×104 M−1 cm−1.
Synthesis of 4-((1E,3E)-5-(2,6-di(thiophen-2-yl)-4H-thiopyran-4-ylidene)penta-1,3-dien-1-yl)-2,6-di(thiophen-2-yl)thiopyrylium (Dye 18). 4-Methyl-2,6-di(thiophen-2-yl)thiopyrylium hexafluorophosphate (0.200 g, 0.476 mmol), N-(2-(phenylamino)ethen-1yl)methylenebenzaminium hexafluorophosphate (25, 87.6 mg, 0.238 mmol), NaOAc (39.1 mg, 0.476 mmol), AcOH (1.0 mL) and Ac2O (1.0 mL) were combined and heated at 90° C. for 15 min. The mixture was cooled to ambient temperature and diluted with H2O (50 mL). The product was extracted with a mixture of CH2Cl2 (3×20 mL) and CH3CN (3×5 mL). The organic layer was dried with Na2SO4, concentrated and the product recrystallized from hot CH3CN and an equivalent amount of ether to yield 82.9 mg (48%) of a red, metallic solid. λmax (CH2Cl2)=944 nm.
Synthesis of 4-((1E,3E)-5-(2,6-di(selenophen-2-yl)-4H-selenopyran-4-ylidene)penta-1,3-dien-1-yl)-2,6-di(selenophen-2-yl)selenopyrylium (Dye 19). 4-Methyl-2,6-di(selenophen-2-yl)selenopyrylium hexafluorophosphate (28, 0.150 g, 0.266 mmol), N-(2-(phenylamino)ethen-1yl)methylenebenzaminium hexafluorophosphate (25, 49.0 mg, 0.133 mmol), NaOAc (21.8 mg, 0.266 mmol), AcOH (1.0 mL) and Ac2O (1.0 mL) were combined and heated at 90° C. for 15 min. The mixture was cooled to ambient temperature and diluted with H2O (50 mL). The product was extracted with a mixture of CH2Cl2 (3×20 mL) and CH3CN (3×5 mL). The organic layer was dried with Na2SO4, concentrated and the crude product gave λmax (CH2Cl2)=1001 nm.
Synthesis of 4-((E)-2-((E)-2-chloro-3-(2-(2,6-diphenyl-4H-thiopyran-4-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-2,6-diphenylthiopyrylium hexafluorophosphate (Dye 20). 4-Methyl-2,6-diphenylselenopyrylium (0.100 g, 0.245 mmol), N-((E)-((E)-2-chloro-3-((phenylamino)methylene)cyclohex-1-en-1-yl)methylene)benzenaminium hexafluorophosphate (26, 43.9 mg, 0.123 mmol) and NaOAc (20.1 mg, 0.245 mmol) were stirred in a mixture of Ac2O (1.0 mL) and AcOH (1.0 mL). This mixture was heated at 95° C. for 1 h prior to cooling to ambient temperature and stirring with 10% aqueous HPF6 (30 mL) for 1 h. The product was extracted in CH2Cl2 (50 mL), the organic layer dried with Na2SO4, and after concentration purified on SiO2 with a 20% EtOAc/CH2Cl2 eluent. Fractions containing product were combined and recrystallized from CH3CN/ether to yield 39.8 mg (40%) of a copper bronze solid, mp 200-202° C.: 1H NMR [500 MHz, CD3CN] δ 8.34 (d, 2H, J=14.5 Hz), 7.80 (br s, 4H), 7.77-7.75 (m, 8H), 7.62-7.57 (m, 12H), 6.71 (d, 2H, J=14.5 Hz), 2.77 (t, 4H, J=6.5 Hz), 1.98-1.96 (m, 2H); λmax (CH2Cl2)=1042 nm, ε=1.0×105 M−1 cm−1.
Synthesis of 4-((E)-2-((E)-2-chloro-3-(2-(2,6-di(thiophen-2-yl)-4H-thiopyran-4-ylidene)ethylidene)-cyclohex-1-en-1-yl)vinyl)-2,6-di(thiophen-2-yl)thiopyrylium hexafluorophosphate (Dye 21). 4-Methyl-2,6-di(thiophen-2-yl)thiopyrylium hexafluorophosphate (0.200 g, 0.476 mmol), N-((E)-((E)-2-chloro-3-((phenylamino)methylene)cyclohex-1-en-1-yl)methylene)benzenaminium chloride (26, 85.3 mg, 0.238 mmol), NaOAc (39.1 mg, 0.476 mmol), AcOH (1.0 mL) and Ac2O (1.0 mL) were combined and heated at 95° C. for 15 min. The mixture was cooled to ambient temperature and diluted with H2O (50 mL). The product was extracted with a mixture of CH2Cl2 (3×20 mL) and CH3CN (3×5 mL). The organic layer was dried with Na2SO4, and concentrated and the crude product gave λmax (CH2Cl2)=1119 nm.
Synthesis of Benzopyrylium Derivatives. S3 and S9 were prepared in a similar manner to literature procedures (J Org. Chem. 1980, 45, 4611-15), but S3 is a novel derivative of this class of compounds. S4 and S6 were prepared in accordance with literature procedures (Organometallics 1988, 7, 1131-1147; J. Org. Chem. 1982, 47, 5235-5239; J Org Chem 2003, 68 (5), 1804-1809; Chem. Heterocycl. Compd. (N.Y.) 1998, 34, 438-443; J. Org. Chem. 1980, 45, 4611-4615). The reaction in Ac2O to form the final dye compounds S5, S8, and S10 follows literature precedent as well (J. Org. Chem. 1982, 47, 5235-5239).
In a round bottom flask under argon, diphenyl diselenide (0.734 g, 2.35 mmol) was dissolved in THF (5 mL) and NaBH4 (0.356 g, 9.41 mmol) was added in one portion. EtOH (10 mL) was added over a 10 min period until bubble formation had ceased and the solution was colorless. Ethyl 3-(thiophene-2-yl)propiolate (0.782 g, 4.71 mmol) was dissolved in THF (5 mL) and added to the reduced selenide, after which the solution was heated to reflux over a 10 min period and then 3 M aqueous KOH (20 mL) was added and the reaction refluxed overnight. After cooling to rt 3 M aqueous HCl was added to bring the solution to a pH≈1, the product extracted with EtOAc (50 mL), the organic layer dried with MgSO4 and after concentration recrystallized from EtOAc/hexanes to yield 0.939 g (65%) of an off-white solid. NMR revealed only trace (<5%) of the E isomer, mp 139.0-140.5° C.: 1H MNR [500 MHz, CDCl3] δ 7.35 (d, 2H, J=7.5 Hz), 7.21-7.17 (m, 1H), 7.15-7.12 (m, 3H), 6.76-6.71 (m, 2H), 6.54 (s, 1H); 13C NMR [75.5 MHz, CDCl3] δ 171.05, 154.03, 140.49, 136.31, 135.26, 130.02, 129.90, 129.55, 128.76, 128.07, 127.23, 126.88, 117.42, 115.13; HRMS (EI) m/z 309.9569 (calcd for C13H10O2S80Se: 309.9561).
Methanesulfonic acid (6.76 mL) and P2O5 (1.00 g, 3.52 mmol) were placed in a flame dried flask under argon and heated to 65° C. until all of the P2O5 was dissolved. (Z)-3-(thiophene-2-yl)-3-(phenylselanyl)acrylic acid (0.400 g, 1.29 mmol) was added in portions over approximately 5 min, and then allowed to stir at 65° C. for 5 min. The reaction was then quenched by pouring into satd. aqueous NaHCO3 (250 mL), and the product extracted with (3×75 mL) of CH2Cl2. The organic layer was dried with MgSO4, concentrated under reduced pressure and then purified on SiO2 with a 10% EtOAc/CH2Cl2 eluent to yield 0.154 g (41%) of a light brown solid, mp 130-132° C.: 1H MNR [500 MHz, CDCl3] δ 8.58 (dxd, 1H, J=8.0, 2.0 Hz), 7.66 (d, 1H, J=8.0 Hz), 7.56-7.50 (m, 4H), 7.39 (s, 1H), 7.16 (t, 1H, J=4 Hz); 13C NMR [75.5 MHz, CDCl3] δ 182.53, 144.96, 140.72, 135.81, 131.86, 131.69, 129.98, 129.05, 128.42, 128.04, 127.79, 127.29, 123.55; HRMS (EI) m/z 291.9465 (calcd for C13H8OS80Se: 291.9456).
2-(Thiophene-2-yl)benzoselenopyran-4-one (0.150 g, 0.515 mmol) was dissolved in anhydrous THF (5.0 mL) in a flame dried flask under argon. 3.0 M MeMgBr (0.520 mL, 1.56 mmol) was added slowly and the reaction allowed to stir at rt for ½ h. This was then poured into 10% aqueous HPF6, stirred for ½h, and the product isolated by filtration. The solid was dissolved in CH2Cl2, dried with Na2SO4, and after concentration recrystallized from CH3CN/ether to yield 0.171 g (76%) of a bright orange solid, mp 137-140° C.: 1H MNR [500 MHz, CD3CN] 8.82-8.79 (m, 1H), 8.57 (s, 1H), 8.51-8.49 (m, 1H), 8.35-8.33 (m, 2H), 8.05-8.03 (m, 2H), 7.49 (t, 1H, J=4.5 Hz), 3.07 (s, 3H); 13C NMR [75.5 MHz, CD3CN] δ 178.08, 167.28, 145.16, 142.39, 141.82, 136.60, 134.95, 133.17, 132.80, 132.11, 130.81, 130.40, 25.77; HRMS (EI) m/z 289.9663 (calcd for C14H10S80Se: 289.9663). (Phys. Med. Biol. 1994, 39, 1705-1720)
4-Methyl-2-phenylselenobenzopyrylium hexafluorophosphate (0.100 g, 0.233 mmol), N,N-Dimethylthioformamide (59.4 μL, 0.698 mmol) and Ac2O (2.0 mL) were combined in a small round bottom flask and heated at 95° C. for 1 h. After cooling to rt the solution was diluted with ether. The formed iminium salt was allowed to precipitate in the freezer overnight, and then isolated by filtration to yield a bright orange solid. This solid was dissolved in CH3CN (3.0 mL) and satd. aqueous NaHCO3 (3.0 mL) was added. This mixture was heated to 80° C. over 15 min, and kept at that temperature for ½ h. After diluting with H2O (30 mL) the product was extracted with CH2Cl2 (3×20 mL), dried with Na2SO4 and purified on SiO2 with a CH2Cl2 eluent (Rf=0.56) to give a yellow oil that was recrystallized in CH2Cl2/hexanes to yield 66.0 mg (91%) of a yellow crystalline solid, mp 89-92° C.: 1H MNR [300 MHz, CDCl3] δ 10.35 (d, 1H, J=7.0 Hz), 8.34 (s, 1H), 8.00-7.97 (m, 1H), 7.65-7.57 (m, 3H), 7.49-7.40 (m, 5H) 6.53 (d, 1H, J=6.5 Hz); 13C NMR [75.5 MHz, CDCl3] δ 189.79, 148.35, 143.18, 138.86, 132.24, 129.88, 129.69, 129.12, 128.08, 127.12, 126.86, 119.35, 119.06; HRMS (EI) m/z (calcd for C17H12O80Se:).
4-methyl-2-phenylselenobenzopyrylium hexafluorophosphate (0.200 g, 0.466 mmol), 2-phenylbenzoselenopyran-4-one (0.146 g, 0.513 mmol), and Ac2O (4.0 mL) were combined and heated at 105° C. for 10 min. The solution was diluted with CH3CN (4.0 mL) and precipitated with ether. The resulting solid was recrystallized from CHCl3/ether to yield 0.237 g (73%) of a dark green solid, mp 154-156° C.: 1H MNR [500 MHz, CD2Cl2] δ 8.73-8.72 (m, 2H), 8.57 (br. s, 2H), 8.14-8.12 (m, 3H), 7.85-7.83 (m, 4H), 7.64-7.60 (m, 6H), 7.50 (t, 4H, J=8.0 Hz); Anal. Calcd for C31H21Se2.PF6: C, 53.47; H, 3.04. Found: C, 53.40; H, 3.06; HRMS (ESI) m/z 552.9966 (calcd for C31H2180Se2: 552.9968); λmax (CH2Cl2)=748 nm, ε=7.6×104 M−1 cm−1.
4-Methyl-2-(thiophene-2yl)selenobenzopyrylium hexafluorophosphate (30.0 mg, 68.9 μmol), 2-(thiophene-2-yl)benzoselenopyran-4-one (22.1 mg, 75.8 μmol), and Ac2O (1.0 mL) were combined and heated at 105° C. for 10 min. The solution was diluted with CH3CN (3.0 mL) and precipitated with ether to yield 40.9 mg (84%) of a bronze solid, mp 199-201° C.: 1H MNR [500 MHz, CD2Cl2] δ; Anal. Calcd for C27H17S2Se2.PF6: C, 45.78; H, 2.42. Found: C, 45.65; H, 2.63; HRMS (ESI) m/z 564.9105 (calcd for C22H12S280Se2: 564.9097); λmax (CH2Cl2)=786 nm, ε=7.8×104 M−1 cm−1.
4-Nethyl-2-phenylthiochromenylium hexafluorophosphate (65.1 mg, 0.170 mmol), (E)-2-(2-phenyl-4H-thiochromen-4-ylidene)acetaldehyde (54.0 mg, 0.204 mmol), and Ac2O (2.0 mL) were combined and heated at 105° C. for 10 min. The solution was cooled to rt, diluted with CH3CN, and the product precipitated with ether to yield 94.4 mg (88%) of a copper bronze solid, mp 253-254° C.: 1H NMR [500 MHz, CD3CN] δ Due to poor solubility in common NMR solvents resolution was extremely poor; Anal. Calcd for C33H23S2.PF6: C, 63.05; H, 3.69. Found: C, 62.79; H, 3.88; LRMS (ESI) m/z 483.4 (calcd for C33H23S2+: 483.1); λmax (CH3CN)=789 nm, ε=1.5×105 M−1 cm−1.
4-Methyl-2,6-di(phenyl)thiopyrylium hexafluorophosphate (51.5 mg, 0.126 mmol), 2-phenylbenzoselenopyran-4-one (43.2 mg, 0.139 mmol), and Ac2O (2.0 mL) were combined and heated at 105° C. for 10 min. The solution was diluted with CH3CN (3.0 mL) and precipitated with ether to yield 65.3 mg (74%) of a copper bronze solid, mp >260° C.: 1H NMR [500 MHz, CD3CN] Spectrum was unresolved due to poor solubility and solution dynamics of this compound. Anal. Calcd for C35H25SSe.PF6: C, 59.92; H, 3.59. Found: C, 59.74; H, 3.48; HRMS (ESI) m/z 557.0853 (calcd for C35H25S80Se: 557.0837); λmax (CH3CN)=748 nm, ε=6.1×104 M−1 cm−1.
In this example, we describe the design and synthesis of a novel group of near infrared absorbing 2-thienyl-substituted chalcogenopyrylium dyes tailored to have high affinity for gold. When adsorbed onto gold nanoparticles, these dyes produce biocompatible SERRS-nanoprobes with attomolar limits of detection amenable to ultrasensitive in vivo multiplexed tumor and disease marker detection.
One notable feature of the pyrylium dyes is the ease in which a broad range of absorptivities can be accessed, and consequently be matched with the NIR light source by careful tuning of the dye's optical properties. Specifically, the large differences in absorption maxima introduced by switching the chalcogen atom is a useful property of this dye class. nother important consideration is the affinity of the reporter for the surface of gold. Since the SERS effect decreases exponentially as a function of distance from the nanoparticle, it is important that the Raman reporter is near the gold surface. The 2-thienyl substituent provides a novel attachment point to gold for Raman reporters. The 2-thienyl group is not only part of the dye chromophore, but also can be rigorously coplanar with the rest of the chromophore. This allows the dye molecules to be in close proximity to the nanoparticle surface, creating a more intense SERRS-signal.
Results. Chalcogenopyrylium dye synthesis and characterization. Cationic chalcogenopyrylium dyes 1-3, with absorption maxima near the 785-nm emission of the detection laser were synthesized as outlined in
SERRS-nanoprobe synthesis and characterization. Chalcogenopyrylium dyes 1-3 were dissolved in dry N,N-dimethylformamide (DMF), at a concentration between 1.0 and 10 mM, and were subsequently used to generate the SERRS-nanoprobes. The SERRS-nanoprobes consist of a gold core onto which the SERRS-reporter is adsorbed, which is then protected by an encapsulating silica layer (
Effect of counterion on colloidal stability and SERRS-signal. In previous reports, the dye counterion was shown to affect the structural and electronic properties of polymethine dyes and the solubility of chalcogenopyrylium dyes. Since SERRS is highly dependent on these factors, we evaluated the effect of the counterion (Z−) on the SERRS spectrum, intensity, and colloidal stability of the pyrylium-based SERRS-nanoprobes. We compared chloride (Cl−), bromide (Br−), perchlorate (ClO4−), and hexafluorophosphate (PF6−) as counterions for chalcogenopyrylium dye 1a. The SERRS-nanoprobes were synthesized in the presence of equimolar amounts (10 μM) of CP dye 1a.Z− (where Z−═Cl−, Br−, ClO4−, or PF6−). The counterion introduces almost no difference in optical properties (e.g. absorption maxima, extinction coefficient). Furthermore, with the exception of the chloride counter-ion, the Raman shifts and intensity of 1a were minimally affected by the different counterions (
Effect of increased affinity on colloidal stability and SERRS-signal. We also examined the SERRS-signal intensity as a function of the number of sulfur atoms in the dye. Sulfur-containing functionality has been used frequently to adhere molecules to gold, with several reports using thiol or lipoic acid functional groups to add sulfur-containing functionality. In our structures, 2-thienyl groups attached to the 2- and 6-positions of the dye were used to bind the dyes to the gold surface. We also explored the impact of the chalcogen atoms in the chalcogenopyrylium core, switching a Se (1a and 2a) to S (1b and 2b). The chalcogen switch was used to increase semi-covalent interactions with the gold surface, and also to create a chromophore that had a more resonant absorption with the 785-nm detection laser (Table 2). Chalcogenopyrylium dyes 1-3 were used at a final concentration of 1.0 μM, which prevented nanoparticle aggregation for dye 3.
Comparison of CP-dye 3 with a cyanine-based SERRS-reporter. In order to assess the quality of our optimized nanoprobe, thiopyrylium dye 3 and commercially available IR792 (
A concentration series of the as-synthesized SERRS-nanoprobes was generated in triplicate fashion to determine the limit of detection (LOD) of both nanoprobes.
In vivo comparison of EGFR-targeted CP-3- or IR792-SERRS-nanoprobes. The ability of our SERRS-nanoprobe to delineate tumor tissue in vivo was assessed by utilizing CP dye 3 and IR792-based SERRS-nanoprobes functionalized with an epidermal growth factor receptor (EGFR)-targeting antibody. Equimolar amounts (15 fmol/g) of these two EGFR-targeted nanoprobes were injected intravenously into athymic nude mice which had been inoculated two weeks prior with the EGFR-overexpressing cell line A431 (1×106 cells). After 18 hours, the skin around the tumor was carefully peeled back and multiplexed Raman imaging the tumor site and surrounding tissue was performed (
Discussion Effective biomedical imaging requires low limits of detection and high specificity for biological targets. Raman imaging has surfaced as an optical imaging modality that has the promise to enable both. While the Raman effect is relatively weak (1 in 107 photons), the Raman scattering cross section of a molecule can be massively amplified by noble metal surfaces. Here, we demonstrated that rational SERRS-reporter design afforded SERRS-nanoprobes with unprecedented limits of detection: 100 attomolar. This is to the best of our knowledge the lowest reported limit of detection at near-real-time detection (≦2.0 s acquisition times) for SERRS-nanoprobes that are compatible with a NIR light source. As a comparison non-resonant SERS-nanoprobes are in the 0.1-1.0 pM range (1,000-10,000-fold less sensitive), while reported detection limits of SERRS-nanoprobes are >17 fM at near real-time detection. Others have reported a 0.4 fM detection limit, however, this was acquired through cumulative data acquisition with an acquisition time ≧60 s, which is not practical for biomedical imaging applications.
We believe the unprecedented limit of detection of our novel SERRS-nanoprobe is due to several factors. First, we demonstrate that rational design and optimization of the SERRS-reporter is important to achieve efficient “loading” on the nanoparticle. Our results demonstrate that the counterion and gold surface affinity are important considerations. For instance, while the chaotropic PF6− anions stabilized the dye-nanoparticle system during silica shell formation in ethanol, the system becomes more destabilized with Cl− (more kosmotropic) ions present. Chloride-induced aggregation of colloidal dispersions in relation to SERS has been studied. Natan et al. demonstrated that the strongest enhancements were obtained from aggregates with effective diameters of less than 200 nm and aggregates with sizes >200 nm did not generate appreciable SERS intensities. The aggregates that were induced by the chloride counterion in our system were >200 nm, which might explain the reduced SERRS-signal when chloride is used as a counterion. Others have shown that the kosmotropic chloride-ion could induce reorientation of the dye on the surface, which could also contribute to the reduced SERRS intensities. However, while we did observe a decrease in the SERRS-signal intensity when chloride is present, we did not find any appreciable differences between the Raman spectra of the dyes when different counterions were use, which would have been expected if the molecule had reoriented on the surface. Since the most chaotropic counterion, PF6−, induced the least aggregation and generated robust SERRS-signal intensities, we used PF6− as a counterion.
Next, we showed that an increase in affinity of the SERRS-reporter for the gold nanoparticle surface via incorporation of 2-thienyl functional groups considerably increased the SERRS-signal without inducing aggregation. Others have reported the functionalization of NIR dyes with thiol or lipoic acid functional groups. In contrast to a 2-thienyl substituent, thiol and lipoic acid functional groups offer no benefit to the optical properties of the dye, and as a tether, do not allow the dye to be as close to the gold surface. Moreover, based on the absorption spectra of reported lipoic-acid modified cyanine dye-gold nanoparticle conjugates, it is clear that lipoic-acid modified dyes promote aggregation.
Finally, the strategy chosen to stabilize the SERRS-nanoprobe is a key factor. Others have reported using either surfactants or thiolated-polymers to stabilize their SERRS-nanoparticles. However, such stabilizing agents compete with the SERRS-reporter for the surface of the nanoparticle, which leads to relatively low SE(R)RS-signal. We achieved very low limits of detection by using a primerless silication procedure in which the silica not only served as a stabilizing agent, but also as a matrix to contain our optimized CP-based SERRS-reporter. Since silica has much lower affinity for the gold than the applied SERRS-reporters, attomolar limits of detection were achieved.
The chalcogenopyrylium dyes represent a new class of SERRS-reporters. Selection of the right combination of chaotropic counterions and increased affinity of the SERRS-reporter for the gold nanoparticle's surface produces stable SERRS-nanoprobes with exceptionally low limits of detection (attomolar range). The low limit of detection (i.e. close to single nanoparticle detection) in combination with the high resolution of Raman imaging, enables highly sensitive and specific, near-real-time tumor delineation and, as a result of the fingerprint like spectra of the different SERRS-nanoprobes, can offer multiplexed disease marker detection in vivo.
Methods. Dye synthesis and characterization. SERRS-nanoprobe synthesis. Gold nanoparticles were synthesized through addition of 7.5 ml 1% (w/v) sodium citrate to 1000 ml boiling 0.25 mM HAuCl4. The as-synthesized gold nanoparticles were concentrated by centrifugation (10 min, 7500×g, 4° C.) and dialyzed overnight (3.5 kDa MWCO; 5 L 18.2 MΩ·cm). The dialyzed gold nanoparticles (100 μL; 2.0 nM) were added to 1000 μL absolute ethanol in the presence of 30 μL 99.999% tetraethylorthosilicate (Sigma Aldrich), 15 μL 28% (v/v) ammonium hydroxide (Sigma Aldrich) and 1 μL chalcogenopyrylium dye (1-10 mM) in N,N-dimethylformamide. After shaking (375 rpm) for 25 min at ambient conditions in a plastic container, the SERRS-nanoprobes were collected by centrifugation, washed with ethanol, and redispersed in water to yield 2.0 nM SERRS-nanoprobes.
SERRS-nanoprobe characterization. The as-synthesized SERRS-nanoprobes were characterized by transmission electron microscopy (TEM; JEOL 1200ex-II, 80 kV, 150,000× magnification) to study the SERRS-nanoprobe structural morphology. The size and concentration of the SERRS-nanoprobes were determined on a Nanoparticle Tracking Analyzer (NTA; Malvern Instruments, Malvern, UK). Absorption spectra to determine possible nanoparticle aggregation (typically detectable at wavelengths >600 nm) were measured on an M1000Pro spectrophotometer (Tecan Systems Inc. San Jose, Calif.). Finally Raman spectra were acquired on a Renishaw InVIA system equipped with a 785-nm laser (Renishaw Inc, Hoffman Estates, Ill.). All measurements were performed at a laser power of 50 μW/cm2 (1.0 s acquisition time, 5× objective).
SERRS-nanoprobe limit of detection. SERRS-nanoprobes were synthesized as described above in the presence of an equimolar (1.0 μM) amount of 3 or IR792. SERRS imaging to determine the limit of detection was performed at 100 mW/cm2 (2.0 s acquisition time (StreamLime™), 5× objective) on a phantom that consisted of a serial diluted IR792- or chalcogenopyrylium dye (3)-based SERRS-nanoprobe redispersed in 10 μL water (concentration range 3000-0.003 fM; n=3). The Raman maps were generated by WiRE 3.4 software (Renishaw) by applying a direct classical least square (DCLS) algorithm. The Raman image was analyzed with ImageJ software and plotted in GraphPad Prism (GraphPad Software Inc., La Jolla, Calif.).
Serum stability. The SERRS-nanoprobes (2.0 nM) were incubated in triplicate in 50% mouse serum (Abd Serotec, Raleigh, N.C.) at 37° C. At the indicated time points, a Raman spectrum was taken (50 μW/cm2; 1.0 s acquisition time; 5× objective). The intensities of the 1600 cm−1 were plotted in GraphPad Prism (GraphPad Software Inc., La Jolla, Calif.).
Animal studies. All animal experiments were approved by the Institutional Animal Care and Use Committees of Memorial Sloan Kettering Cancer Center.
In vivo comparison of EGFR-targeted CP-3- or IR792-SERRS-nanoprobes. Female athymic nude mice (n=5) were inoculated with the EGFR-overexpressing cell line A431 (1×106 cells). After 2 weeks, the mice were injected with an equimolar amount (15 fmol/g) of EGFR-targeted IR792- and 3-based SERRS-nanoprobes. The EGFR-targeted SERRS-nanoprobes were synthesized as described above in the presence of an equimolar (1.0 μM) amount of 3 or IR792. The as-synthesized SERRS-nanoprobes were subsequently functionalized with sulfhydryl-groups by heating the SERRS-nanoprobes in 5 mL 2% (v/v) mercaptotrimethoxysilane (MPTMS) in ethanol at 70° C. for 2 hours. The sulfhydryl-functionalized SERRS-nanoprobes were washed and conjugated to an EGFR-targeting antibody (cetuximab; Genentech, South San Francisco, Calif.) through a 4000 Da heterobifunctional maleimide/N-hydroxysuccinimide polyethylene glycol linker. Eighteen hours later, the mice were sacrificed by CO2-asphyxiation. The tumor was exposed and scanned by Raman imaging (10 mW/cm2, 1.5 s acquisition time (StreamLime™), 5× objective). The Raman maps were generated by WiRE 3.4 software (Renishaw) by applying a direct classical least square (DCLS) algorithm.
Immunohistochemical staining. The tissues from the imaging studies were collected and fixed in 4% paraformaldehyde, 4° C. overnight and subsequently processed to be embedded in paraffin. The Discovery XT biomarker platform (Ventana, Tucson, Ariz.) was used to stain the tissue sections (5 μm). Heat-induced epitope retrieval was performed using the citrate buffer (pH 6.0). The primary anti-EGFR antibody (D38B1, Cell Signaling Technology, Danvers, Mass.) was diluted 1:150. The biotin-labeled secondary anti-rabbit antibody (BA-1000, Vector Laboratories) was diluted 1:300.
In this example, we describe the design of SERS nanotags that operate with 1280-nm excitation. The nanotags are based on hollow gold nanoshells (HGNs) and reporter molecules selected from a small library of (chalcogenopyranyl)chalcogenopyrylium monomethine (1-8) and trimethine dyes (9-14) substituted with phenyl, 2-thienyl, and 2-selenophenyl substituents at the 2- and 6-positions of the pyrylium/pyranyl rings (Scheme 1 in Example 1). Dye 14 with two sulfur atoms in the thiopyrylium/thiopyranyl core and four 2-selenophenyl substituents at the 2,2′,6,6′-positions was exceptionally bright in this library of reporters. All fourteen members of the reporter library can be uniquely identified by principal component analysis of their SERS spectra.
Results. The syntheses of 1-14 are shown in Scheme 1 and the library was constructed by condensation of 4-methylthiopyrylium and 4-methylselenopyrylium salts 15 either with chalcogenopyranones 16 or with (4-chalcogenopyranylidene)acetaldehyde derivatives 17 in acetic anhydride to give monomethine dyes 1-8 or trimethine dyes 9-14, respectively. 4-Methylthiopyrylium and 4-methylselenopyrylium salts 15 were prepared by the addition of MeMgBr to the corresponding chalcogenopyranone 16 followed by treatment with aqueous HPF6. Synthetic details are provided in the Supporting Information. Values of absorption maxima, λmax, in CH2Cl2 for 1-8 varied from 653 nm for 1 to 724 nm for 6 and values of the molar extinction coefficient, ε, were in the range of 1.1×105 to 1.5×105 M−1 cm−1. For trimethine dyes 9-14, values of λmax in CH2Cl2 varied from 784 nm for dye 10 to 826 nm for dye 14 while values of E were in the range of 2.0×105 to 2.8×105 M−1 cm−1. The interchange of S and Se atoms in the chalcogenopyrylium backbone, the use of monomethine and trimethine bridges, and the interchange of phenyl, 2-thienyl, and 2-selenophenyl substituents at the 2-,2′-, 6-, and 6′-positions allow the fine tuning of wavelengths of absorption and allow each dye to have a unique Raman fingerprint.
Raman scattering tends to be weak in the near infrared (NIR) region due to its dependence on the 4th power of the excitation frequency. However, the scattering effect can be significantly enhanced by trapping molecules close to the roughened surface of metallic nano-substrates and in this case, the gold surface of HGNs. The enhancement obtained from SERS is related to the frequency of the surface plasmon excited on the metal rather than the 4th power law. Therefore, to make SERS a viable method in the NIR region, and specifically at 1280 nm where no SERS nanotags have previously been reported to be compatible, the surface plasmon resonance (SPR) must be resonant with the NIR excitation source. We engineered the combination of HGNs and dyes 1-14 as SERS nanotags to produce SERS signals with 1280-nm excitation. The SERS spectrum of dye 14 is shown in
There are three important components which make up these '1280 SERS nanotags, the first being the SERS substrate. For this study we have chosen HGNs as these nanostructures have strong SERS properties. Also, HGNs have desirable characteristics such as small size (usually from 50-80 nm), spherical shape and a strong tunable plasmon band from the visible to the NIR region. Commonly, Ag and Au spherical nanoparticles that have plasmon bands in the visible region are used as SERS substrates. However, these nanoparticles in conjunction with dyes 1-14 produced much weaker SERS signals than the HGNs due to their lack of red-shifted SPR.
The second necessary component of SERS nanotags is the Raman reporter. The thiophene and selenophene-substituted chalcogenopyrylium dyes were specifically designed as Raman reporters for use in the NIR region. Since the SERS effect decreases exponentially as a function of distance from the nanoparticle, it is important that the Raman reporter be near the Au surface. The dyes 1-14 incorporate S and Se atoms in the chalcogenopyrylium core to provide attachment to Au and the 2-thienyl and 2-selenophenyl groups on select members of this library provide novel attachment points to Au for Raman reporters. Earlier studies have shown that thiophenes and selenophenes are both capable of forming self-assembled monolayers on Au. Selenolates have also been shown to have greater affinity for Au than thiolates.
It can be seen in
Both dye 13 and dye 14 are significantly red-shifted with light absorption maxima >800 nm, making them NIR active. Another benefit of these dyes is the multiple S and Se atoms incorporated into their structures allowing them to adsorb onto the HGN surface very strongly and experience a larger enhancement.
X-ray structural studies have shown that the chalcogenopyrylium/chalcogenopyranyl rings and the methine carbon of chalcogenopyrylium monomethine dyes related to 1-8 are coplanar and computational studies predict similar coplanarity in chalcogenopyrylium trimethine dyes 9-14. Other studies have shown that a 2-thienyl group can be coplanar with an attached thiopyranyl ring. X-ray crystallographic analysis of single crystals of dye 14 indicate that the thiopyrylium/thiopyranyl trimethine core and the four 2-selenophenyl substituents are coplanar as shown in
The third component in the SERS nanotag is the aggregating agent, usually a simple inorganic salt such as potassium chloride (KCl) that screens the Coloumbic repulsion energy between the nanoparticles, allowing the reporter molecules to adhere more closely to the nanoparticle surface. Although the aggregating agent was necessary for most of the dyes, it is important to note that with chalcogenopyrylium dyes 13 and 14, KCl was not required for intense signals to be observed. This is possibly due to a strong interaction occurring between the reporter and HGN surface inducing self-aggregation. This partial aggregation observed from these nanotags perhaps widens the scope for future SERS applications where aggregating agents are not required and the aggregation of the nanoparticles comes solely from a biological recognition event such as DNA-DNA interactions, DNA-protein interactions, peptide-protein interactions or sugar-protein interactions. Furthermore, these nanotags could be used as alternative reporters in biological applications such as photothermal ablation therapy or optical coherence tomography where there is a great need for NIR active materials.
Due to the exceptional response obtained with dye 14 and HGNs, particle dilution studies were conducted in order to calculate a limit of detection (LOD) for this dye at this extremely red-shifted laser wavelength. The LOD study was carried out by initially using the optimum conditions (those used in
In addition, the non-resonant commercial dyes BPE (1,2-bis(4-pyridyl)ethylene) and AZPY (4,4-azopyridine), which are commonly used with Au nanosubstrates for SERS analysis were also tested with the HGNs at this laser wavelength but failed to produce a SERS signal. Until now, no SERS nanotags compatible with the critical laser excitation wavelength of 1280 nm have been reported. We have demonstrated a range of nanotags that show excellent SERS properties and a LOD in the picomolar range. This work provides the basis for advancement in the SERS field with these nanotags showing promise for future use in a wide range of optical applications.
Furthermore, these dyes can be separated out and individually identified in a reproducible manner based on their unique structures and SERS spectra by performing multivariate analysis in the form of principal component analysis (PCA). PCA is employed to reduce the dimensionality of the spectroscopic data while making it easier to identify variations in the SERS spectra. PCA was carried out on 14 data sets consisting of the spectra obtained from each individual dye experiment. The resulting principal component (PC) scores plot (
A new extreme red shifted SERS nanotag was designed and synthesized to demonstrate unprecedented performance using 1280 nm excitation. This was achieved by combining a set of chalcogen dyes with hollow gold nanoshells to provide a unique performance at this longer wavelength of excitation. These dyes with the more widely used Au nanoparticles or HGNs with conventional Raman reporters such as BPE were unable to match the combined performance of the chalcogenopyrylium dyes and HGNs indicating the unexpected and superior performance of SERS nanotags based on the combination of these dyes and the tunable HGNs. This significant result now makes SERS nanotags available for use at wavelengths suitable for deep tissue analysis.
The preceding description provides specific examples of the present disclosure. Those skilled in the art will recognize that routine modifications to these embodiments can be made which are intended to be within the spirit and scope of the disclosure.
This application claims priority to U.S. Patent Application No. 62/082,554, filed Nov. 20, 2014, the disclosure of which is hereby incorporated by reference.
This invention was made with government support under contract no. CHE-1151379 awarded by the National Science Foundation. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US15/61791 | 11/20/2015 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62082554 | Nov 2014 | US |
