WO2001090716A2 - Bras de liaison pour nanocristaux et composes de ces derniers - Google Patents

Bras de liaison pour nanocristaux et composes de ces derniers Download PDF

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WO2001090716A2
WO2001090716A2 PCT/US2001/016739 US0116739W WO0190716A2 WO 2001090716 A2 WO2001090716 A2 WO 2001090716A2 US 0116739 W US0116739 W US 0116739W WO 0190716 A2 WO0190716 A2 WO 0190716A2
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nanocrystal
compound
linker arm
derivatives
group
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PCT/US2001/016739
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WO2001090716A3 (fr
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Sandra J. Rosenthall
Ian D. Tomlinson
Tadd Kippeny
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Vanderbilt University
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Publication of WO2001090716A3 publication Critical patent/WO2001090716A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/12—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms
    • C07D295/135—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms separated by carbocyclic rings or by carbon chains interrupted by carbocyclic rings
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/10—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton
    • C07C323/11—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C323/12—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/10—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton
    • C07C323/11—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C323/16—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton containing six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
    • C07D209/14—Radicals substituted by nitrogen atoms, not forming part of a nitro radical
    • C07D209/16—Tryptamines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/22—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C07D277/24—Radicals substituted by oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/22—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C07D277/26—Radicals substituted by sulfur atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D451/00—Heterocyclic compounds containing 8-azabicyclo [3.2.1] octane, 9-azabicyclo [3.3.1] nonane, or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane or granatane alkaloids, scopolamine; Cyclic acetals thereof
    • C07D451/02—Heterocyclic compounds containing 8-azabicyclo [3.2.1] octane, 9-azabicyclo [3.3.1] nonane, or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane or granatane alkaloids, scopolamine; Cyclic acetals thereof containing not further condensed 8-azabicyclo [3.2.1] octane or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane; Cyclic acetals thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D451/00—Heterocyclic compounds containing 8-azabicyclo [3.2.1] octane, 9-azabicyclo [3.3.1] nonane, or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane or granatane alkaloids, scopolamine; Cyclic acetals thereof
    • C07D451/14—Heterocyclic compounds containing 8-azabicyclo [3.2.1] octane, 9-azabicyclo [3.3.1] nonane, or 3-oxa-9-azatricyclo [3.3.1.0<2,4>] nonane ring systems, e.g. tropane or granatane alkaloids, scopolamine; Cyclic acetals thereof containing 9-azabicyclo [3.3.1] nonane ring systems, e.g. granatane, 2-aza-adamantane; Cyclic acetals thereof
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots

Definitions

  • This invention generally relates to nanocrystals, linker arms for nanocrystals, and compounds resulting therefrom.
  • this invention relates to labeling techniques using the compounds of the present invention.
  • Fluorescent nanocrystal labeling has broad application in the biomedical sciences.
  • the labeling technique of the present invention provides improved and widely applicable methods for detecting biomolecules and for scrutinizing biomolecular processes.
  • Quantum dots are being used as fluorescent tags capable of tracing specific substances within cells. Quantum dots can be activated to glow with different colors, so it is easier to use quantum dots in tandem than combinations of conventional fluorescent dyes. See “Semiconductor Beacons Light up Cell Structures” Service, Science. Vol. 281.
  • the conventional fluorescent dye typically made from small organic dye molecules can be toxic, can quench quickly, and can be difficult to use in tandem, since typically each dye must be excited with photons at a different wavelength.
  • the quantum dots compared with conventional coloring agents such as rhodamine 6G or other organic dyes, the quantum dots produce narrower and much brighter fluorescence spectra. See “Quantum Dots Meet Biomolecules", Jacoby.
  • the absorbency onset and emission maxima shift to a higher energy with decreasing size.
  • the excitation typically tracks the absorbency, resulting in a tunable fluorophore that can be excited efficiently at any wavelength shorter than the emission peak, yet will emit with the same characteristic a narrow, symmetric spectrum regardless of the excitation wavelength. See “Semiconductor Nanocrystals as Fluorescent Biological Labels", Bruchez, et al., Science, Vol. 281, 1998.
  • the color can be changed. Additionally, a range of quantum dots of different colors may be excited with a single wavelength and detected simultaneously. See “Bright Lights for Biomolecules", Analytical Chemistry News and Features.
  • quantum dots or semiconducting nanocrystals, are much more flexible and advantageous when used in assays.
  • the attachment of biologically active ligands to nanocrystals including, for example, cadmium selenide nanocrystals is a new method of producing novel fluorescent sensors.
  • the sensors can have a variety of applications. They may be used in fundamental studies ranging from assay systems to locate the distribution and localization of membrane bound receptors, transporter proteins and channels in whole assay systems. They may also be used in novel methodologies for the development of pharmaceutically active compounds using high throughput screening.
  • the small size of the of the nanocrystal ligand conjugate offers advantages over conventional techniques that use antibodies bound to fluorescent dyes. These advantages include the small size of the drug nanocrystal conjugate which enables it to fit into the synaptic gap. Antibody-fluorescent dye systems are much larger than the nanocrystal drug conjugates of the present invention, so the antibody-fluorescent dye stems are less likely to fit into the synaptic gap. Additionally most antibodies are cell permeable. The increased photostability of the nanocrystals means that they are not as easily photo-bleached as conventional dyes. Therefore, the nanocrystal compounds of the present invention may be used in experiments that require longer periods of illumination without photo-bleaching becoming a major problem.
  • the increased brightness of the nanocrystals enhances the sensitivity of the assay systems when compared to traditional dyes. Therefore, assay systems can be developed that detect lower concentrations of the analyte.
  • U.S. Patent No. 5,990,479 to Weiss et al. discloses a luminescent nanocrystal compound that is capable of linking to an affinity molecule. Weiss et al. further describe a process for making luminescent semiconductor nanocrystal compounds and for making an organo luminescent semiconductor probe comprising the nanocrystal compound linked to an affinity molecule capable of bonding to a detectable substance and a process for using the probe to determine the presence of a detectable substance in a material.
  • U.S. Patent No. 5,751,018 to Alivisatos et al. discloses methods for attaching semiconductor nanocrystals to solid inorganic surfaces, using self-assembled bifunctional organic monolayers as bridge compounds.
  • U.S. Patent No. 5,537,000 to Alivisatos et al. which describes electroluminescent devices formed using semiconductor nanocrystals as an electron transport media and a method for making such electroluminescent devices.
  • U.S. Patent No. 5,505,928 to Alivisatos et al. discloses nanocrystals of III-V semiconductors
  • U.S. Patent No. 5,262,352 Alivisatos et al. discloses a process for forming a solid, continuos thin film of a semiconductor material on a solid support surface.
  • An embodiment of the present invention is to provide linker arms to attach organic compounds to nanocrystals, or quantum dots.
  • a linker arm of the present invention may have the following formula:
  • Y represents the attachment point to the nanocrystal and X represents the attachment point of an organic compound.
  • R is a bond or is selected from the group consisting of: SH,
  • n 1-10, with S being attached to the nanocrystal.
  • R 2 is a bond or selected from the group consisting of carbonyl, NH, S, CONH, COO, S, Ci-io alkyl, carbamate, and thiocarbamate.
  • n and p are 1 or more, the resulting carbon or carbon chain may be substituted.
  • z is O.
  • n and p are 1-5.
  • the linker arm may have the following formula:
  • Y is the attachment point for a nanocrystal
  • X is an attachment point of an organic compound.
  • R 2 is a bond or selected from the group consisting of carbonyl
  • R3 is selected from the group consisting of: SH,
  • n 1-10. S is attached to the nanocrystal.
  • the organic compound is a biologically active compound.
  • biologically active compounds of the present invention include seratonin or seratonin derivatives, cocaine analogues, phenyl tropane analogues, phenylisopropylamine derivatives, dopamine derivatives, melatonin derivatives, chlormethiazole derivatives, derivatives of RTI-4229-75, and derivatives of GBR 12935. RTI-4229-75 and GBR 12935 are further described below.
  • the preferred organic compounds attached to the nanocrystal of the present invention specifically include the following:
  • R represents the attachment point to the linker arm.
  • the R group may be "floating" when attached to the phenyl ring. That is, the R group may be attached to any available carbon atom on the ring.
  • the present invention further is directed to nanocrystal compounds, which include linker arm derivatives of the present invention. More specifically, the nanocrystal compounds of the present invention comprise a semiconducting nanocrystal and a linking arm having a first portion linked to the nanocrystal and a second portion linked to an organic compound.
  • nanocrystal compounds of the present invention include the following formulae (II), (III), (IN), (N), (VI), (VII), (X) and (XI):
  • n 0-10
  • n 2,3,4 or 5.
  • the linker arm may be attached to positions 1,2,3, or 4. Most preferably, the linker arm is attached to position 2.
  • n is 1, 2, 3, or 4 and the linker arm is attached to positions 1, 2, 3, or 4. Most preferably, positions 1, 2, or 3. Most preferably, position 2.
  • n 1, 2, 3, 4 or 5 and the linker arm is attached to positions 1, 2, 3, or 4.
  • the linker arm is attached to one of positions 1, 2, or 3.
  • X H or halogen.
  • X is H or F.
  • n 0-10
  • n is 2, 3, 4 or 5.
  • the linker arm may be attached to positions 1, 2, 3, or 4. Preferably, position 2.
  • n 0-10
  • n 2, 3, 4, or 5.
  • the linker arm may be attached to positions 1 or 2. Preferably, position 2.
  • n 0-10
  • n is 2, 3, 4 or 5.
  • the linker arm may be attached to positions 1,2, 3, or 4. Preferably, position 2.
  • n is 2, 3, 4 or 5.
  • the linker arm may be attached to positions 1, 2, 3, or 4. Preferably, position 2.
  • n 0-10
  • n is 2,3,4 or 5.
  • the linker arm may be attached to positions l,2,3,or 4. Preferably, position 2.
  • n 0-10
  • n is 2, 3, 4 or 5.
  • the linker arm may be attached to positions 1, 2, 3, or 4. Preferably, position 2.
  • n is 2,3,4 or 5.
  • the linker arm may be attached to positions 1, 2, 3, or 4. Preferably, position 2.
  • the linker arm attaching the compounds to the nanocrystal can be altered by replacing the oxygen with sulfur or NH.
  • the length of the alkyl substituent between the oxygen atoms may be increased or decreased and it may comprise of chains with lengths of 1 to 10 carbon atoms. Also the hetero atom in the chain may vary thus the chain may contain alternating NH and O functionalities or O and S functionalities.
  • the present invention relates to linker arms to which biologically active molecules can be attached to nanocrystals.
  • the nanocrystals used in conjunction with the present invention are the nanocrystals typically used in fluorescent imaging techniques.
  • the nanocrystals used in conjunction with the present invention are semiconductor nanocrystals capable of luminescence and/ or scattering or diffraction when excited by an electromagnetic radiation source (of broad or narrow bandwidth) or a particle beam, and capable of exhibiting a detectable change of absorption and/ or emitting radiation in a narrow wavelength band and/ or scattering or diffracting when excited.
  • the nanocrystals of US 5,990,479 may be used with the present invention.
  • an organic or inorganic single crystal particle having an average cross-section of about 20 nanometers (nm) or 20 x IO **9 meters (200 Angstroms), preferably no larger than about lOnm (100 Angstroms) and a minimum average cross-section of about lnm, although in some instances a smaller average cross- section nanocrystal, i.e., down to about 0.5nm (5 Angstroms), may be acceptable.
  • the nanocrystal will have an average cross-section ranging in size from about 1 nm (10 Angstroms) to about 10 nm (100 Angstroms).
  • these nanocrystals include, but not are limited to CdSe, CdS, PbSe, PbS, and CdTe.
  • CdSe CdS
  • PbSe PbS
  • CdTe CdTe
  • simultaneous localization of several different proteins in situ is currently limited by the wide emission spectra and photostabilities of fluorescent dyes traditionally used to study cell surface receptors, ion channels, and transporters.
  • the nanocrystal compounds of the present invention can overcome the above deficiencies.
  • the nanocrystal compounds comprise core (CdSe)/shell(ZnS) semiconducting nanocrystals.
  • the fluorescent wavelength of these nanocrystals are continuously tunable by size.
  • a 25 Angstrom nanocrystal of this embodiment emits at 455nm while a 60 Angstrom nanocrystal of this embodiment emits at 625 nm.
  • these nanocrystals have narrow gaussian emission spectra enabling multiplex imaging.
  • the absorption of these nanocrystals is continuous above the band-gap; hence all sizes of nanocrystals can be excited with a single excitation wavelength.
  • the nanocrystals of this embodiment are much brighter than traditional dyes, even hours after continuous illumination.
  • the present invention further relates to multiple organic compounds in combination with the linker arms of the present invention.
  • the present invention further relates to a method of attaching a linker arm to multiple organic compounds and a method of attaching a linker arm to a nanocrystal.
  • the present invention further relates to the linker arms herein described and nanocrystals attached to the linker arms herein described.
  • the present invention also relates to nanocrystals and semiconductor nanocrystals in combination with the linker arms of the present invention.
  • the present invention further relates to the attachment of a nanocrystal and a linker arm to an organic compound.
  • the present invention relates to assay systems and assay kits for CNS research, receptor purification, pathogens, environmental contaminants, toxins, and screening for drugs, insecticides, herbicides, and other biologically active substances.
  • the linker arms and linker arm compound derivatives of the present invention enhance stability and are relatively stable, including stability to biomedical degradation.
  • the linker arms and the linker arm compound derivatives of the present invention are also advantageous in that they can be synthesized at a relatively low cost.
  • the present invention relates to linker arms such as, for example, ether-containing, polyether or carbon-carbon chain linker arms by which biologically active molecules such as CNS drugs and neurotransmitters can be attached to nanocrystals.
  • the attachment of a linker arm of the present invention allows nanocrystals to be used as imaging agents in diverse applications such as biochemical research, CNS research, receptor purification, and high throughput screening for new drugs and other biologically active substances.
  • the present invention relates to linker arms such as, for example, ether containing, polyether or carbon linker arm by which biologically active molecules such as drugs, hormones, etc. can be attached to nanocrystals.
  • the linker arms of the present invention enhance water solubility of nanocrystals and allow nanocrystals to be attached to a diverse range of molecules ranging from drugs to polypeptides and neurotransmitters.
  • the linker arm compounds of the present invention allow nanocrystals to be used as imaging agents in diverse applications such as CNS research, receptor purification, assay systems for pathogens, environmental contaminants, toxins, and a high throughput assay system for new drugs and biologically active molecules.
  • the organic part of the nanocrystal compounds of the present invention are biologically active compounds.
  • the biologically active compound is one that will bind to detectable substances, if the substance is present, in the material being analyzed.
  • affinity molecules useful in the prior art in combination with a dye molecule to provide specific recognition of a detectable substance will find utility in the formation of the organo-luminescent semi conductor nanocrystal probes of the invention.
  • affinity molecules include, by way of example only, such classes of substances as monoclonal and polyclonal antibodies, nucleic acids (both monomeric and oligomeric), proteins, polysaccharides, and small molecules such as sugars, peptides, drugs, and ligands. Lists of such affinity molecules are available in the published literature such as, by way of example, the "Handbook of Fluorescent Probes and Research Chemicals", (sixth edition) by R.P Haugland, available from Molecular Probes, Inc.
  • the compounds of the present invention enable nanocrystals to be used as probes for neurotransmitters, receptors and transporter proteins.
  • seratonin (5-hydroxytriptamine) is attached to a nanocrystal.
  • Seratonin is a neurotransmitter which has been linked to the regulation of critical behaviors including sleep, appetite, and mood.
  • the seratonin transporter is a 12-transmembrane domain protein responsible for the uptake of seratonin by the cell.
  • the seratonin labeled nanocrystal compounds of the present invention have a measurable ability to block the uptake of tritiated sepatonin by the human and Drosophila seratonin transporter (hSERT and dSERT).
  • Seratonin labeled nanocrystals may be prepared by reacting trioctylphosphineoxide coated nanocrystals with seratonin and tetramethylammonium hydroxide in methanol.
  • the SNACs are isolated by precipitation and purified to remove seratonin. Linkage of the seratonin presumptively occurs through the lone pair of the hydroxyl to the Cd surface atoms of the nanocrystal.
  • hSERT and dSERT are transfected into HeLa cells via a vaccinia virus/T7 expression system.
  • Ki values the concentration at which half the SNACs are bound to the transporter, are determined by nonlinear regression.
  • Ki the concentration at which half the SNACs are bound to the transporter.
  • the present invention enables nanocrystals to be used as imaging agents, which results in an assay system that is superior to traditional immunoassay systems because, among other things, several wavelengths can be used to induce fluorescence.
  • the linker arm can be attached to a number of different ligands, thus enabling them to be used in high throughput screening and receptor purification.
  • the linker arm is stable and not as subject to enzymatic degradation as other linker arms may experience.
  • the linker arm of the present invention also enhances the solubility of the nanocrystal, and can be readily derivitised. This enables a wide range of molecules to be attached to the nanocrystals.
  • the linker arm of the present invention is not as temperature sensitive as many immunoassay systems, and thus is likely to have a longer shelf life. Further, the linker arm of the present invention is also robust and therefore not susceptible to extremes of pH that may denature and degrade peptide linkers.
  • the linker arm of the present invention may have the following formula:
  • Y represents the attachment point to the nanocrystal and X represents the attachment point of an organic compound.
  • R is a bond or is selected from the group consisting of: SH,
  • n 1-10, with S being attached to the nanocrystal.
  • R2 is a bond or selected from the group consisting of carbonyl, NH, S, CONH, COO, S, C ⁇ -10 alkyl, carbamate, and thiocarbamate.
  • n and p are 1 or more, the resulting carbon or carbon chain may be substituted.
  • z is O.
  • n and p are 1-5.
  • the linker arm may have the following formula:
  • Y is the attachment point for a nanocrystal
  • X is an attachment point of an organic compound.
  • R2 is a bond or selected from the group consisting of carbonyl
  • R 3 is selected from the group consisting of: SH,
  • n 1-10. S is attached to the nanocrystal.
  • n 1-5.
  • the length of the linker arms of the present invention may be increased or shortened in order to increase the solubility of the nanocrystal drug conjugate and increase the affinity of the ligand for its target protein.
  • the linker arms of the present invention include the following compounds: 31
  • R represents the point of attachment of an organic compound.
  • nanocrystal compounds of the present invention include the following examples, with S being the attachment point of the nanocrystal:
  • Nanocrystal compounds of the present invention include compounds that comprise of nanocrystals with the following specific and preferred features: a CdSe core, ZnS shell, generally their cores are less than 25nm, in diameter.
  • the surrounding ZnS shell is typically 10 to 20nm in thickness, and the ligand coated core shells are water solubilized by the addition of a mercapto acetic acid co-solubility ligand.
  • an embodiment of the present invention is an assay kit developed for the detection of a diverse range of substances ranging from environmental contaminant such as DDT, dioxanes, chemical warfare agents, herbicides, pesticides, and pathogenic organisms such as Ecoli 0157 and Salmonela.
  • the present invention comprises a process for treating a material, such as a biological material, to determine the presence of a detectable substance in the material.
  • the process comprises contacting the material with a nanocrystal conjugated compound of the present invention, washing unbound nanocrystal conjugated compound away, and exposing the material to energy such as an electromagnetic source or particle beam capable of exciting the nanocrystal conjugated compound of the present invention, and causing a detectable fluorescence to occur in the nanocrystal conjugated compound of the present invention.
  • energy such as an electromagnetic source or particle beam capable of exciting the nanocrystal conjugated compound of the present invention
  • nanocrystal compounds of the present invention may be used in the assays described in US Patent 5,990,479.
  • One assay system of the present invention is a high throughput fluorescence assay to identify novel ligands that might be effective antidepressants or ligands that might help combat cocaine addiction.
  • a known agonist or antagonist for the dopamine receptor or transporter is bound to nanocrystals, and incubated with cells that either naturally express or have been engineered to express dopamine receptors or transporters. After incubating for 12 hours excess ligands are removed by washing and unknown compounds are incubated with the cells for a further 12 hours. The cells are washed again with buffer and a fluorescence assay is performed. Any cells that no longer fluorescence have a high affinity ligand bound to them and this ligand may be used as a lead compound for drug discovery.
  • Such an assay system may be carried out in a conventional multiple well format system, such as the 96 well format.
  • Chart A below demonstrates another method of the present invention that may be used to detect biologically active analytes.
  • Chart A describes a sandwich assay system.
  • step 1 monoclonal or polyclonal antibodies raised against a specific analyte or groups of analytes are bound to the surface of the plate.
  • step 2 the analyte is added and binds to the antibody.
  • step 3 the unbound analyte is washed away and a nanocrystal antibody conjugated using our linker arm of the present invention is added (once again poly or monoclonal antibodies may be used).
  • step 3 the unbound nanocrystal antibody conjugates are removed by washing, and a fluorescence assay is performed to determine if the analyte is present in the sample being analyzed and its concentration as a sample with a higher concentration will produce a greater fluorescence.
  • Multiple analytes can be screened for using a conventional 96 well plate format.
  • Monoclonal or polyclonal antibodies raised against a specific analyte or group of analytes are bound to a surface.
  • O o Analyte binds to the antibody.
  • NC nanocrystal. A sandwich assay system.
  • the nanocrystal of the present invention may be used in affinity chromatography, where a compound or biological molecule of interest may be bound to a column. This may then be specifically labeled with the antibody nanocrystal conjugate, substrate nanocrystal conjugate, or drug nanocrystal conjugate of the present invention.
  • the compound could be a drug, a hormone, an enzyme, a protein, a nucleic acid or a receptor.
  • the nanocrystal conjugate Once the nanocrystal conjugate has bound to the substrate of interest, it may either remain bound to the column or be eluted with the mobile phase. This would enable the isolation and identification of the compound orbiological molecule of interest. Unlike fluorescent dyes, nanocrystals are not easily photo- bleached.
  • Such a system may be applied to several different analytes enabling the identification of several unknowns at once by using different sized nanocrystals conjugated to different ligands.
  • receptor classes or subtypes e.g. 5-HT receptor subtypes
  • the linker arm acts as a spacer and separates the ligand from the nanocrystal thus possible steric and other interactions between nanocrystals and ligand are minimized.
  • the linker arm may be an ethylene glycol moiety this helps to enhance the solubility in aqueous media. Many affinity chromatographic systems are typically run in such media.
  • the polyether linker arm is also resistant to proteolytic cleavage which may be a problem with other assay systems.
  • Nanocrystals can be attached to enzymes via linker arms of the present invention.
  • the amino derived carboxylic acid derived poly- ethers may be linked to the backbone of the peptide via a peptide bond.
  • the linker arm removes the enzyme from the immediate environment of the nanocrystal. This may be important in reducing any effects that the nanocrystal may have upon the enzymes activity. Many such instances could be envisaged particularly if the enzyme or protein undergoes a conformational change during its catalytic cycle (e.g.
  • the linker arm may increase the catalytic efficiency of the en2yme if the active site or sites are close to the enzymes surface.
  • Such a system may also be used to identify analytes in a similar manner to the nanocrystal antibody conjugates previously described. It may also be used in high throughput screening where the compounds of interest are bound to wells in plates and the enzyme nanocrystal conjugate is added. An example of this is shown in chart B below:
  • Chart B identifying active compounds in a high throughput assay system
  • the enzymes substrate or inhibitor may also be bound to the polyethylene glycol nanocrystal conjugate.
  • the linker arm of the present invention reduces steric hindrance between nanocrystal and enzyme and it enables the substrate to enter the enzymes catalytic or alosteric site, which may not be possible if the substrate were bound to the surface of the nanocrystal (particularly if the site of interest is deep within the enzyme).
  • An assay system that could use this technique as a tool for identifying new drugs is outlined in chart C, below, where compounds that will compete for the site of interest can be identified. If the nanocrystal is bound to an inhibitor via the linker arm of the present invention it is likely that this assay system could also be used to identify other inhibitors of the enzyme. Chart C:
  • Enzyme or receptor is bound to the i late. Incubate with drug nanocrystal conj igate Wash with buffer and identify novel Antagonists, etc.
  • F,J,X and Z are new chemical entities bound to nanocrystals via our linker arm.
  • Inactive compounds will not bind and will be washed away thus we have an assay system for detecting novel active compounds.
  • One specific substance may also be bound to the nanocrystal (e.g. a substrate for the enzyme) and a simple competitive assay could be performed with unknown substances in a manner similar to that shown above in chart C.
  • Any substance that has a higher affinity for the site of interest on the enzyme, protein or receptor than the ligand conjugated nanocrystal would displace the ligand conjugated nanocrystal resulting in a loss off fluorescence, thus enabling this system also to be used as a high throughput assay system as well as an analytical tool for environmental contaminates, toxins, and other unknowns.
  • This system can be applied to receptors rather than enzymes.
  • the nanocrystal is bound to an agonist, antagonist, or natural ligand for the receptor (e.g. Seratonin).
  • This system could be used as an assay system for receptor agonist or antagonist. It would be of interest in neuropharmacology where receptor location and distribution could be mapped.
  • By attaching different sized nanocrystals to different agonists, antagonists, or ligands it may be feasible to develop multiplexing assay systems, thus enabling the effects of drugs and other neurologically active agents to be monitored in whole cell assay systems.
  • Nanocrystals may be attached to DNA or RNA via the linker arm of the present invention.
  • the major role of the linker arm acts as a spacer and reduces steric hindrance.
  • the DNA or RNA conjugates may be used as a tool in molecular biology for identifying the location and frequency and rate of expression of specific gene sequences. Such a system is outlined in chart D, below:
  • a nanocrystal via the linker arm Then wash.
  • the sections of the DNA or RNA of interest will bind to our probe.
  • the nanocrystal conjugates of the present invention can also be used in assay systems in the same manner that antibody fluorescent dye conjugates, radio immuno assays, and ELISA are used.
  • Examples of the assay system include routine assays used in medical laboratories such as tests for various disease states for example HIV, Diabetes, etc.
  • Route B is a variant of Route A in which the protecting group is changed to a phalimido substituent.
  • the combined organic extracts are washed with water (1x20ml), hydrochloric acid (5%, 15ml) and brine (15ml).
  • the organic solution is dried over magnesium sulfate and the crude product is obtained as a black tar upon evaporation.
  • the product is purified using column chromatography on silica gel eluted with dichloromethane. This yields approximately 1.34g (100%) of the product as a pale yellow oil.
  • linker arm may be attached to alkyl alcohols via an ether linkage.
  • Many drugs, DNA, RNA, glycoproteins, intracellular messengers and hormones such as the steroids contain these functionalities.
  • chlormethiazole (9) has been synthesized.
  • Chart 4 (i) 4-methyl-5-thiazoleethanol, 2, KOH, tertiary butyl ammonium chloride; (ii) Mercury(II) acetate, trifluoroacetic acid, Hydrogen sulfide.
  • linker arm to attach aryl and alkyl amines via an amide linkage
  • the polyethylene glycol linker arm can be altered so that it can be attached to aryl and alkyl amines via an amide linkage.
  • the derivative of the linker arm can be readily prepared and a synthetic scheme for the derivative is outlined in chart 5, below.
  • the resulting carboxylic acid (13) can be attached to amines using a variety of reagents such as DCC or by making the acid chloride (14).
  • Two such derivatives that we have synthesized are the derivative of the cocaine analogue RTI-4229- 75 (15) and the derivative of GBR 12935 (16):
  • linker arm derivative that contains this carboxylic acid functionality may also be attached to proteins and antibodies via an amide bond, alternatively it may be attached to RNA and DNA via a ester linkage to the ribose or deoxy ribose moiety.
  • the aqueous solution is extracted with ethyl acetate (3x200ml) and this is dried over magnesium sulfate.
  • the product is purified using column chromatography on silica gel eluted with ethylacetate 92%: methanol 5%: triethylamine. This gives approximately 0.66g (78.6%) of the product as a pale yellow oil.
  • 8-(4-Methoxybenzylthio)-3,6-dioxaoctanoic acid (0.008g, 0.027 mmols) is dissolved in dry toluene (10ml), oxalyl chloride (0.0008ml is added and then dry dimethyl formamide (1 drop) . The mixture is stirred at room temperature for 1 hour and then evaporated to yield crude 8-(4-Methoxybenzylthio)-3,6- dioxaoctonyl chloride (14).
  • the 8-(4-Methoxybenzylthio)-3,6- dioxaoctonyl chloride (14) is dissolved in dry dichloromethane (20ml), 3 ⁇ -(p-Chlorophenyl)tropane-2 ⁇ -carboxylic acid p-aminophenylethyl ester (O.OlOOg, 0.025 mmols) and triethylamine (2 drops) are added. The mixture is heated at reflux for 18 hours cooled and evaporated under reduced pressure. (17) is purified using column chromatography on silica gel eluted with ethyl acetate 98%: triethyl amine. This yields approximately 0.006g (34%) of (17) as a tar.
  • the reaction mixture is heated at reflux until a cloudy grey color forms. The heat is removed and the remaining bromo benzene is added drop wise at such a rate so as to maintain reflux.
  • the solution is heated at reflux for a further hour after the addition of bromobenzene is complete. After which it is cooled to 10°C in an ice acetone bath and benzaldehyde (60ml, 62.4g, 588 mmols) in anhydrous ether 200ml is added drop wise so that the temperature of the reaction mixture does not exceed 20° C.
  • the reaction mixture is allowed to warm to room temperature after the addition of benzaldehyde and it is stirred at room temperature for a further 18 hours.
  • Piperazine hexahydrate (47g, 240 mmols) is added to toluene (100ml) and anhydrous potassium carbonate (66g, 600 mmoles) is added.
  • the mixture is heated at reflux and l, l'-[(2-Chloroethoxy)methylene]bis-benzene (20g, 80 mmols) is added drop wise over five hours.
  • the solution is allowed to cool to 70°C washed with water (5 x 250ml), dried over magnesium sulfate, filtered and evaporated.
  • the resulting yellow oil is converted to a dimaliate salt by crystallising from diethyl ether. This gives approximately 25g (50%) of the product as a colourless solid.
  • para-Nitrohyrocinnamic acid (2.8g, 9.5 mmols) is added to dry toluene (100ml), in a 250ml round bottomed flask equipped with a stirrer and a reflux condenser.
  • Oxalyl chloride (1ml) is added, after which a catalytic quantity of dry DMF (2drops) is also added and the mixture is stirred at room temperature for 2 hours. The solvent is removed by evaporation and the crude acid chloride is dissolved in dry dichloromethane (100ml).
  • the product is purified using silica gel chromatography eluted with a gradient system eluted with ethyl acetate 90%: methanol to ethyl acetate 87%: methanol 10%: triethylamine. This gives approximately 3.35g (68%) of the product as a pale yellow oil.
  • 8-(4-Methoxybenzylthio)-3,6-dioxaoctanoic acid (0.6g, 2.2 mmols) is dissolved in dry toluene (50ml) under nitrogen in a 100ml round bottomed flask equipped with a stirrer and a reflux condenser. Oxalyl chloride (0.5ml) and a catalytic quantity of dimethyl formamide (1 drop) are added. The solution is stirred at room temperature for 2 hours, then evaporated under reduced pressure.
  • the length of the linker arms of the present invention may be changed. Accordingly, at least a di and tetra polyethylene glycol linker arm may be synthesised.
  • the synthetic routes for these compounds are outlined in charts 9 and 10, below.
  • the linker arm is shortened in chart 9 and lengthened in chart 10.
  • 2-(2-chloroethoxy)ethanol (5.48g, 44 mmols) is added 30 minutes later and the mixture is heated at reflux for 18 hours.
  • the solution is cooled to room temperature and added to saturated ammonium chloride solution (100ml). It is extracted into dichloromethane (3x100ml). After drying the combined organic extracts over magnesium sulfate and filtering, the dichloromethane is removed under reduced pressure.
  • the product is purified by column chromatography on silica gel eluted with a gradient system from dichloromethane to dichloromethane 90%: methanol. This yields approximately 5.9g (60%) of the product as a colorless oil.
  • the product is purified using column chromatography on a silica column eluted with a gradient system from petroleum spirit 70%: diethyl ether to petroleum spirit 30%: diethyl ether. This yields approximately 0.13g (3.6%o) of the product as a colorless oil.
  • Tetraethylene glycol (192g, 990 mmols) is added to dry chloroform (200ml) in a IL flask equipped with a stirrer, reflux condenser and a thermometer. Dry pyridine (80ml) is added to this solution and it is cooled to 0°C. Freshly distilled thionyl chloride (73ml) is added over a 4 hour period, whilst maintaining the temperature below 10°C. After all the thionyl chloride has been added the solution is heated at reflux for 18 hours. Then the chloroform is removed under reduced pressure and the resulting residue is extracted with water (2x100ml).
  • the aqueous solution is washed with hexane's (2x100ml) and the crude product is extracted into toluene (5x100ml). Then the solvent is dried with magnesium sulfate filtered and evaporated.
  • 2-(2-(2-(2-(2-Chloroethoxy)ethoxy)ethoxy)ethanol is dissolved in dry pyridine (10ml) and cooled to 0°C, in a 50ml round bottomed flask equipped with a stirrer and a calcium chloride drying tube.
  • Para- toluene sulfonyl chloride (4.66g, 24 mmols) is added to the mixture and it is stirred for 18 hours, during this period the temperature of the reaction mixture is allowed to increase from 0°C to room temperature.
  • Water (50ml) is added to the reaction mixture and it is extracted with dichloromethane (2x50ml).
  • the linker arms of the present invention may be synthesized with other functionalities, including a chloride and an amine functionality.
  • the synthesis of these compounds is outlined in chart 11, below.
  • the amino functionality may also be attached to drugs or biologically active molecules such as cholesterol, proteins and antibodies, via an amide linker.
  • the product is purified using silica gel chromatography eluted with a gradient system from petroleum ether 60%: diethyl ether to petroleum ether 40%: diethyl ether. This yields approximately 0.39g (57%) of the product as a colorless oil.
  • a biologically active organic compound may be attached to the linker arm as follows:
  • the biologically active molecule is attached to the linker arm via a functional group or a methylene group.
  • R may be O, NH, S, CH 2 , etc.
  • PG is a protecting group and may be para-methoxy benzyl, benzyl, a thioamide, a thio ether, etc.
  • This example discloses a method of attaching linker arms of the present invention to nanocrystal core shells.
  • An example of the methodology used is outlined below:
  • trioctylphosphine oxide coated core shells 9 mg are weighed out and suspended in pyridine (2ml). The concentration and thus the number of moles of nanocrystals may be determined before hand using UV-vis spectroscopy.
  • This suspension is stirred at 60°C for 24 hours, N- (4-(3- [4- (2-Benhydryloxyethyl)piperazine- 1 -yl]propyl)phenyl- 2 - [2- (2 - mercaptoetoxy)ethoxy] acetamide (25), (lOOmg) is dissolved in dichloromethane (100ml) and 2.7ml of this solution is added to the solution of nanocrystals.
  • the water solubility of the ligand functionalised core shells may be increased if necessary by using a modification of the method of Fred Mikulec (private communication).
  • Mercaptoacetic acid (1ml) and dimethyl formamide (1ml) are added to the ligand coated core shells and stirred at room temperature under argon for 2 hours. After cooling to room temperature the solution is diluted with dimethyl formamide (100ml) and potassium teriary butoxide (l. ⁇ lg) is added. The resulting solid is collected by centrifugation and is washed with tetrahydrofuran (4 x 100ml) and methanol (7 x 100ml).
  • the product is collected by centrifugation to yield 45 mg of l-[2-bisphenylmethoxy]ethyl]-4-(3-(4-(3,6-dioxa-8- thiol)octanamidophenyl) propyl piperazine (25) coated nanocrystals. After drying the precipitate under reduced pressure for 4 days at room temperature the ligand coated cores can be dissolved in a minimum quantity of buffer in a pH range of 6 to 8.

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Abstract

L'invention concerne des composés nanocristallins, et un bras de liaison pour ces derniers, qui fixe les nanocristaux aux composés organiques. Le composé nanocristallin comprend un nanocristal, un bras de liaison et un composé organique. Ce dernier est, de préférence, un composé biologiquement actif pouvant fournir un capteur fluorescent. Le bras de liaison est, de préférence, une chaîne polyéther dotée d'un point de fixation pour le nanocristal ou un point de fixation pour le composé organique. Plus spécialement, le nanocristal est fixé au bras de liaison par l'intermédiaire d'un groupe R et le composé organique est fixé au bras de liaison par l'intermédiaire d'un groupe R2. R est une liaison ou est sélectionné dans le groupe constitué par SH, O(CH2(n)O)nSH, NH(CH2(n)O)nSH, NH(CH2(n)NH)SH, S(CH2(n)O)nSH, et S(CH2(n)S)SH, n étant compris entre 1 et 10, et S étant fixé au nanocristal. R2 est une liaison ou est sélectionné dans le groupe constitué par carbonyle, NH, S, CONH, COO, alkyle C1-10, carbamate et thiocarbamate.
PCT/US2001/016739 2000-05-24 2001-05-24 Bras de liaison pour nanocristaux et composes de ces derniers WO2001090716A2 (fr)

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WO2006118543A1 (fr) * 2005-05-04 2006-11-09 Agency For Science, Technology And Research Nanocristaux hydrosolubles innovants comprenant un reactif de revetement a faible poids moleculaire et leurs procedes de preparation

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CN110394167B (zh) * 2019-07-22 2021-10-08 中国科学院兰州化学物理研究所 四乙烯五胺碳量子点/单体共键合硅胶亲水色谱固定相的制备及应用
US11078144B2 (en) 2019-10-24 2021-08-03 Institute of Nuclear Energy Research, Atomic Energy Council, Executive Yuan, R.O.C Process for synthesizing of hydroquinone derivatives with heptadecatrienyl side chain

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US4659370A (en) * 1982-04-20 1987-04-21 Nihon Tokushu Noyaku Seizo K.K. Substituted phenoxypropionates and herbicidal compositions containing same and their herbicidal use
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