WO2017190345A1 - Molecular fluorophores and preparation method thereof and use for short wavelength infrared imaging - Google Patents

Molecular fluorophores and preparation method thereof and use for short wavelength infrared imaging Download PDF

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WO2017190345A1
WO2017190345A1 PCT/CN2016/081265 CN2016081265W WO2017190345A1 WO 2017190345 A1 WO2017190345 A1 WO 2017190345A1 CN 2016081265 W CN2016081265 W CN 2016081265W WO 2017190345 A1 WO2017190345 A1 WO 2017190345A1
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compound
independently
unit
mmol
shielding
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PCT/CN2016/081265
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English (en)
French (fr)
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Yongye Liang
Huasen WANG
Qinglai YANG
Meijie TANG
Su Zhao
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South University Of Science And Technology Of China
Nirmidas Biotech, Inc.
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Priority to PCT/CN2016/081265 priority Critical patent/WO2017190345A1/en
Priority to CN201680087445.5A priority patent/CN109641921B/zh
Publication of WO2017190345A1 publication Critical patent/WO2017190345A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D517/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having selenium, tellurium, or halogen atoms as ring hetero atoms
    • C07D517/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having selenium, tellurium, or halogen atoms as ring hetero atoms in which the condensed system contains two hetero rings
    • C07D517/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0058—Antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D498/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D513/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00
    • C07D513/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00 in which the condensed system contains two hetero rings
    • C07D513/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

  • Organic molecules dyes could be a better alternative as they can be made to be more biocompatible and easily excreted from the body than inorganic nanomaterials.
  • Molecular fluorophore properties could be easily tuned by structure engineering.
  • the general molecular fluorescent agents such as indocyanine green (ICG) , methylene blue (MB) and fluorescein isothiocyanate (FITC)
  • ICG indocyanine green
  • MB methylene blue
  • FITC fluorescein isothiocyanate
  • Some polymethine dyes, such as IR-26, IR-1051 and IR-1100 could have emission in the SWIR region. But these dyes have never been used for biological imaging as none of them is water-soluble.
  • SWIR fluorophores Although small molecule dye and polymers have been used as SWIR fluorophores, they have to be encapsulated in a hydrophilic polymer matrix because of their low solubility in aqueous solution. Such encapsulation significantly increases the particle size, prevents rapid excretion and lowers the quantum yield. Recently, a molecular dye CH1055 has been reported with SWIR emission and demonstrated renal excretion. However, the emission quantum yield of such dye is too low (less than 0.2%) for real-time imaging, the size (molecular weight 9.7 k Da) is relatively large and the fluorescence is limited below ⁇ 1200 nm.
  • SWIR fluorophores with good aqueous solubility and biocompatibility, high quantum yield, fluorescence emission at longer wavelength than previously reported and highly efficient conjugation to biological molecules, which is of vital importance to fully develop SWIR fluorescence-based imaging methods for research and potential clinical translation.
  • This invention comprises design, synthesis and application of molecular fluorophores for bioimaging in the SWIR or SWIR window. Some molecular fluorophores can be extended to NIR window. Embodiments of the present disclosure seek to solve at least one of the problems existing in the related art to at least some extent, which include: 1) low emission quantum yield. 2) limited solubility of molecular fluorophores in aqueous and biological solutions. 3) low efficiency of conjugation to biological molecules including targeting ligands or antibodies. 4) the fluorescence is limited below ⁇ 1200 nm. In the present invention, a donor-acceptor-donor structure and a strong acceptor unit are employed to afford low band gap of the molecular fluorophores.
  • SWIR fluorophores with fluorescence > 1200nm, which is superior to the CH1055 dye with fluorescence ⁇ 1200nm and can afford much reduced scattering effects in vivo and thus deeper tissue imaging depth.
  • SWIR dyes also contain azide groups to facilitate bio-conjugation with ultrahigh efficiency superior to N-hydroxysuccinimide (NHS) esters based conjugation.
  • Embodiments of a first broad aspect of the present disclosure provide a compound, comprising:
  • a shielding unit shielding the electron accepting aromatic unit and/or the electron donating aromatic unit from intermolecular interactions
  • n1 is an integer ranging from 1 to 12.
  • each R 1 is independently H, C n2 H 2n2+1 , or tert-butyloxycarbonyl,
  • each one of Y 1 and Y 2 is independently H, OC n2 H 2n2+1 , C n2 H 2n2+1 , OC n2 H 2n2 B, or C n2 H 2n2 Z,
  • each B is independently Br, I, OTs, OMs, ONs, N3, or OMe,
  • each Z is independently Br, or N3,
  • each n2 is independently an integer ranging from 1 to 20,
  • each p is independently an integer ranging from 1 to 20,
  • the shielding unit has a formula of any one selected from the group consisting of:
  • each R 2 is independently OC n3 H 2n3+1 , C n3 H 2n3+1 , OC n3 H 2n3 W, or C n3 H 2n3 W,
  • each n3 is independently an integer ranging from 0 to 20,
  • each W is independently H, Br, I, OH, OTs, N 3 ,
  • each X 1 is independently Si, Ge, or C,
  • n5 is an integer ranging from 1 to 20,
  • n is an integer ranging from 4 to 120
  • the compound comprises two shielding units and two electron donating aromatic units, and the compound has a formula of : S’1-D1-A-D2-S’2, wherein
  • S’1 represents a first shielding unit
  • D1 represents a first electron donating aromatic unit
  • D2 represents a second electron donating aromatic unit
  • A represents the electron accepting aromatic unit.
  • the compound has a formula of: S’-D-A, wherein
  • the compound comprises two electron accepting aromatic units, three electron donating aromatic units, and two shielding units, and the compound has a formula of : S’3-D3-A1-D5-A2-D4-S’4, wherein
  • S’3 represents a third shielding unit
  • S’4 represents a forth shielding unit
  • D3 represents a third electron donating aromatic unit
  • D4 represents a forth electron donating aromatic unit
  • D5 represents a forth electron donating aromatic unit
  • A1 represents a first electron accepting aromatic unit
  • A2 represents a second electron accepting aromatic unit.
  • the compound comprises one electron accepting aromatic unit, two shielding units, and the compound has a formula of : S’5-A-S’6, wherein
  • S’6 represents a sixth shielding unit
  • A represents the electron accepting aromatic unit.
  • the compound comprises one electron accepting aromatic unit, four shielding units, and the compound has a formula of : S’7-S’8-A-S’9-S’10, wherein
  • S’7 represents a seventh shielding unit
  • S’8 represents an eighth shielding unit
  • S’9 represents a ninth shielding unit
  • S’10 represents a tenth shielding unit
  • the compound having a formula of any one selected from the group consisting of:
  • each one of Y 1 and Y 2 is independently H, C n2 H 2n2+1 , OC n2 H 2n2+1 , OC n2 H 2n2 B,
  • the compound having a formula of any one selected from the group consisting of:
  • Embodiments of a third broad aspect of the present disclosure provide use of the compound or the kit mentioned above in labeling or conjugating to a biomolecule.
  • the biomolecule and the compound mentioned above are combined together through click chemistry with azide groups on the compound mentioned above or intermolecular forces to form strong, non-covalent complexes through simple mixing or mixing followed by heating to 40-70 degree Celsius.
  • the biomolecule includes a small bioactive molecule (folic acid, tretinoin, cholic acid, galactose, biotin, etc. ) , a peptide (decapeptide: synB3, ovarian cancer specific binding peptide: OSBP-1 and OSBP-S, etc.
  • a small bioactive molecule folic acid, tretinoin, cholic acid, galactose, biotin, etc.
  • a peptide decapeptide: synB3, ovarian cancer specific binding peptide: OSBP-1 and OSBP-S, etc.
  • an antibody (erbitux, anti-SA2, Herceptin, secondary antibody against human or animal antibodies, abciximab, adalimumab, alemtuzumab, basiliximab, belimumab, bevacizumab, brentuximab vedotin, canakinumab, cetuximab, certolizumab pegol, daclizumab, daratumumab, denosumab, eculizumab, efalizumab, golimumab, ibritumomab tiuxetan, infliximab, ipilimumab, muromonab-CD3, natalizumab, nivolumab, ofatumumab, omalizumab, palivizumab, panitumumab, Pembrolizumab, ranibizumab, rituximab, tocilizumab,
  • Embodiments of a forth broad aspect of the present disclosure provide a conjugate of the compound mentioned above to a biomolecule, wherein the biomolecule comprising a small bioactive molecule, a peptide, an antibody, a protein, an affibody, a nucleic acid, and an aptamer modified with terminal alkynyl.
  • the biomolecule and the compound mentioned above are conjugated together through click chemistry with azide groups on the compound mentioned above or intermolecular forces to form strong, non-covalent complexes through simple mixing or mixing followed by heating to 40-70 degree Celsius.
  • the biomolecule described here is the same with the biomolecule mentioned above, and will not be described in detail again.
  • the biomolecule described here is the same with the biomolecule mentioned above, and will not be described in detail again.
  • Embodiments of a sixth broad aspect of the present disclosure provide a method of biomedical imaging, comprising:
  • the subject comprising an animal, a human, a tissue, a cell, a 3D organoid or a spheroid.
  • the compound, the kit or the conjugate mentioned above are administrated into a blood vessel, a tissue, an organ, or a lymph node of the subject.
  • the NIR light source is a laser light source or a light emitting diode, and the wavelength of the NIR light source is 780nm, 808nm, or 980 nm.
  • imaging blood vessels with fluorophore circulation in brain, eye or other organs of a body of human or animals sentinel lymph node (SLN) mapping to image lymph nodes proximal to tumor for diagnosis of cancer metastasis, or molecular imaging of cancer though fluorophore-biomolecule targeted homing to cancer cells in the body.
  • SSN sentinel lymph node
  • Embodiments of a seventh broad aspect of the present disclosure provide a method of imaging guided tumor surgery, comprising:
  • Embodiments of an eighth broad aspect of the present disclosure provide a method for labeling a biomolecule, comprising:
  • the biomolecule includes a small bioactive molecule (folic acid, tretinoin, cholic acid, galactose, biotin, etc. ) , a peptide (decapeptide: synB3, ovarian cancer specific binding peptide: OSBP-1 and OSBP-S, etc.
  • a small bioactive molecule folic acid, tretinoin, cholic acid, galactose, biotin, etc.
  • a peptide decapeptide: synB3, ovarian cancer specific binding peptide: OSBP-1 and OSBP-S, etc.
  • biomolecule is used in combination of an SWIR dye as described here that is linked to a molecule or molecular fragment that specifically binds to a marker of interest in a target.
  • SWIR dye as described here that is linked to a molecule or molecular fragment that specifically binds to a marker of interest in a target.
  • Some specific peptide receptors were also displayed the highest binding affinity and specificity with their “peptides” ligands by their ligand receptor, which can directionally deliver peptides-dye conjugation ligands to targeted cell and tissue.
  • Antibody molecule is any immunoglobulin, including antibodies and fragments, its binds to a specific antigen, which can contemplate recombinant generated intact immunoglobulin molecules and immunologically active portions of an immunoglobulin molecule.
  • the targeted bio-imaging or molecular imaging can be achieved in vivo, in vitro or ex vivo.
  • the compounds mentioned above in the present invention can be used as molecular SWIR fluorophores with enhanced quantum yield and good biocompatibility. These compounds exhibit fluorescence in the range from 900-1700 nm under the excitation of light in the range of 400-1000 nm, the intermolecular and intramolecular interactions of the conjugated backbone are reduced and the quantum yield is enhanced. Meanwhile, the molecular fluorophores (the compounds mentioned above) exhibit good water solubility and the dynamic range of the dyes in aqueous solution is small enough to ensure rapid urine or fecal excretion through the renal or biliary system and little toxicity. The high quantum yield and biocompatibility of the molecular dyes along with their SWIR emission opens up the opportunities of SWIR imaging using molecular fluorophores for in-vivo applications.
  • any embodiment disclosed herein can be combined with other embodiments as long as they are not contradictory to one another, even though the embodiments are described under different aspects of the invention.
  • any technical feature in one embodiment can be applied to the corresponding technical feature in other embodiments as long as they are not contradictory to one another, even though the embodiments are described under different aspects of the invention.
  • Fig 2 shows SWIR imaging of a mouse’s brain blood vessels with the IRETBN-PEG1700 fluorophore circulating in the blood flow of the mouse through tail vein injection.
  • Fig 3 shows SWIR fluorescence images of a 4T1 tumor bearing mouse after injection of IREFNS solution.
  • the fluorophores accumulate in the tumor through EPR effect, allowing for tumor imaging with high tumor/normal tissue signal ratio.
  • Fig 4 shows a schematic of conjugation between alkyne functional biomolecules and the azide functionalized SWIR fluorophore.
  • Fig 5 shows fluorescence of samples after density gradient ultra-centrifugation (DGU) separation of fluorophore-protein conjugate and free fluorophore excited by an 808 nm laser.
  • DGU density gradient ultra-centrifugation
  • grammatical articles “a” , “an” and “the” are intended to include “at least one” or “one or more” unless otherwise indicated herein or clearly contradicted by the context.
  • the articles are used herein to refer to one or more than one (i.e. at least one) of the grammatical objects of the article.
  • a component means one or more components, and thus, possibly, more than one component is contemplated and may be employed or used in an implementation of the described embodiments.
  • Embodiments of a first broad aspect of the present disclosure provide a compound, comprising:
  • a shielding unit shielding the electron accepting aromatic unit and/or the electron donating aromatic unit from intermolecular interactions
  • each one of Z 1 , and Z 2 is independently O, S, Se, or NR,
  • each R is independently H, C 2n+1 H 2n+1 , or tert-butyloxycarbonyl,
  • n1 is an integer ranging from 1 to 12.
  • the electron donating aromatic unit has a formula of any one selected from the group consisting of:
  • each X is independently S, Se, NR 1 , or O,
  • each B is independently Br, I, OTs, OMs, ONs, N3, or OMe,
  • each m is independently an integer ranging from 0 to 6
  • each n2 is independently an integer ranging from 1 to 20,
  • each p is independently an integer ranging from 1 to 20.
  • substitution of the “D” unit (the electron donating aromatic unit) close to the “A” unit (the electron accepting aromatic unit) side is preferred, which can improve the quantum yield of molecular dyes in aqueous solution, possibly due to the reduced intermolecular and intramolecular interactions.
  • substitution of the “D” unit (the electron donating aromatic unit) close to the “A” unit (the electron accepting aromatic unit) side is preferred, which can improve the quantum yield of molecular dyes in aqueous solution, possibly due to the reduced intermolecular and intramolecular interactions.
  • the shielding unit has a formula of any one selected from the group consisting of:
  • each R 2 is independently OC n3 H 2n3+1 , C n3 H 2n3+1 , OC n3 H 2n3 W, or C n3 H 2n3 W,
  • each n3 is independently an integer ranging from 0 to 20,
  • each p1 is independently an integer ranging from 1 to 20,
  • each one of Z 5 , Z 6 , and Z 7 is independently S, Se, O, or NR 2 ’,
  • R 2 ’ is H, C n5 H 2n5+1 , or tert-butyloxycarbonyl
  • each X 1 is independently Si, Ge, or C,
  • each b is independently an integer ranging from 1 to 6.
  • the shielding unit contains side chains extended out of the plane of conjugated backbone (the electron accepting aromatic unit and/or the electron donating aromatic unit) , which can reduce the stacking of the molecular conjugated backbone.
  • the terminals of the side chains are functionalized with polyethylene glycol (PEG) , ionic group (such as quaternary ammonium salt) , which can enhance aqueous solubility.
  • PEG polyethylene glycol
  • ionic group such as quaternary ammonium salt
  • the terminals of the side chains are functionalized with azide group (N3) for further conjugation with targeting ligands.
  • the compound comprises two shielding units and two electron donating aromatic units
  • the compound has a formula of : S’1-D1-A-D2-S’2, wherein S’1 represents a first shielding unit, S’2 represents a second shielding unit, D1 represents a first electron donating aromatic unit, D2 represents a second electron donating aromatic unit, A represents the electron accepting aromatic unit.
  • S’1 represents one shielding unit of the two shielding units
  • S’2 represents the other shielding unit of the two shielding units
  • D1 represents one electron donating aromatic unit of the two electron donating aromatic units
  • D2 represents the other electron donating aromatic unit of the two electron donating aromatic units.
  • the compound has a formula of: S’-D-A, wherein S’represents the shielding unit, D represents the electron donating aromatic unit, A represents the electron accepting aromatic unit.
  • the compound comprises two electron accepting aromatic units, three electron donating aromatic units, and two shielding units, and the compound has a formula of : S’3-D3-A1-D5-A2-D4-S’4, wherein S’3 represents a third shielding unit, S’4 represents a forth shielding unit, D3 represents a third electron donating aromatic unit, D4 represents a forth electron donating aromatic unit, D5 represents a fifth electron donating aromatic unit, A1 represents a first electron accepting aromatic unit, A2 represents a second electron accepting aromatic unit.
  • S’3 represents one shielding unit of the two shielding units
  • S’4 represents the other shielding unit of the two shielding units.
  • the compound comprises one electron accepting aromatic unit, two shielding units, and the compound has a formula of : S’5-A-S’6, wherein
  • S’6 represents a sixth shielding unit
  • A represents the electron accepting aromatic unit.
  • the compound having a formula of any one selected from the group consisting of:
  • each one of Y 1 and Y 2 is independently H, C n2 H 2n2+1 , OC n2 H 2n2+1 , OC n2 H 2n2 B,
  • the compound having a formula of any one selected from the group consisting of:
  • -PEG600 represents the formula of 600, 700, and 1000 represent the weight average molecular weight, and the value of n depends on the weight average molecular weight.
  • the biomolecule and the compound mentioned above are combined together through click chemistry with azide groups on the compound mentioned above or intermolecular forces to form strong, non-covalent complexes through simple mixing or mixing followed by heating to 40-70 degree Celsius.
  • the biomolecule includes a small bioactive molecule (folic acid, tretinoin, cholic acid, galactose, biotin, etc. ) , a peptide (decapeptide: synB3, ovarian cancer specific binding peptide: OSBP-1 and OSBP-S, etc.
  • a small bioactive molecule folic acid, tretinoin, cholic acid, galactose, biotin, etc.
  • a peptide decapeptide: synB3, ovarian cancer specific binding peptide: OSBP-1 and OSBP-S, etc.
  • a protein streptavidin, etc.
  • an affibody a nucleic acid, and an aptamer, which are modified with terminal alkynyl and then click reacted with fluorophores’azide.
  • Embodiments of a forth broad aspect of the present disclosure provide a conjugate of the compound mentioned above to a biomolecule, wherein the biomolecule comprising a small bioactive molecule, a peptide, an antibody, a protein, an affibody, a nucleic acid, and an aptamer modified with terminal alkynyl.
  • the conjugate can be used as molecular fluorescence for biomedical imaging, such as blood vessel imaging (brain vessel for TBI, tumor vessel) , tumor imaging and so on.
  • the biomolecule and the compound mentioned above are conjugated together through click chemistry with azide groups on the compound mentioned above or intermolecular forces to form strong, non-covalent complexes through simple mixing or mixing followed by heating to 40-70 degree Celsius.
  • the biomolecule described here is the same with the biomolecule mentioned above, and will not be described in detail again.
  • Embodiments of a fifth broad aspect of the present disclosure provide use of the compound, the kit, or the conjugate mentioned above in biomedical imaging.
  • the compound, or the kit, or the conjugate mentioned above can be used as molecular fluorescence for biomedical imaging with enhanced quantum yield and good biocompatibility.
  • the biomolecule described here is the same with the biomolecule mentioned above, and will not be described in detail again.
  • Embodiments of a sixth broad aspect of the present disclosure provide a method of biomedical imaging, comprising: administrating the compound, the kit or the conjugate to a subject, irradiating the subject at a site of interest by NIR light source, and recording an image by a camera.
  • the quantum yield and biocompatibility are improved significantly by using the compound or the conjugate mentioned above as molecular fluorescence.
  • the site of interest comprising vessels (brain vessels, tumor vessels, etc. ) , tumor, lymphatic system, and so on.
  • the subject comprising an animal, a human, a tissue, a cell, a 3D organoid or a spheroid.
  • the method of the present invention can be effectively used for non-invasive biomedical imaging of animal or human body, such as targeted tumor imaging, vascular imaging, lymph system imaging, and other targets in various parts of the animal or human body, which can be used for clinical diagnosis.
  • the compound or the conjugate mentioned above are administrated into a blood vessel, a tissue, an organ or a lymph node of the subject.
  • the compound or the conjugate are able to accumulate in the target area, then the image of the target area can be obtained easily.
  • the camera comprising an InGaAs camera for imaging in 900nm-1700nm, a Si charge coupled device (CCD) or camera with or without NIR enhanced detector for imaging in 800-1100nm range.
  • CCD Si charge coupled device
  • imaging blood vessels with fluorophore circulation in brain, eye or other organs of a body of human or animals sentinel lymph node (SLN) mapping to image lymph nodes proximal to tumor for diagnosis of cancer metastasis, or molecular imaging of cancer though fluorophore-biomolecule targeted homing to cancer cells in the body.
  • SSN sentinel lymph node
  • Embodiments of a seventh broad aspect of the present disclosure provide a method of imaging guided tumor surgery, comprising: recording an image of the targeted area by method of biomedical imaging mentioned above, using the image to guide tumor removal.
  • the method of the present invention can reduce tumor surgery difficulty, and improve the accuracy of the operation.
  • Embodiments of an eighth broad aspect of the present disclosure provide a method for labeling a biomolecule, comprising: making the compound or the kit mentioned above reacting with the biomolecule, or mixing the compound or the kit mentioned above with the biomolecule with or without heating to 40-70 degree Celsius.
  • the biocompatibility and fluorescence intensity are improved significantly by using the compound mentioned above as molecular fluorescence.
  • the biomolecule includes a small bioactive molecule (folic acid, tretinoin, cholic acid, galactose, biotin, etc. ) , a peptide (decapeptide: synB3, ovarian cancer specific binding peptide: OSBP-1 and OSBP-S, etc.
  • a small bioactive molecule folic acid, tretinoin, cholic acid, galactose, biotin, etc.
  • a peptide decapeptide: synB3, ovarian cancer specific binding peptide: OSBP-1 and OSBP-S, etc.
  • an affibody an antibody (erbitux, anti-SA2, Herceptin, secondary antibody against human or animal antibodies, abciximab, adalimumab, alemtuzumab, basiliximab, belimumab, bevacizumab, brentuximab vedotin, canakinumab, cetuximab, certolizumab pegol, daclizumab, daratumumab, denosumab, eculizumab, efalizumab, golimumab, ibritumomab tiuxetan, infliximab, ipilimumab, muromonab-CD3, natalizumab, nivolumab, ofatumumab, omalizumab, palivizumab, panitumumab, Pembrolizumab, ranibizumab, rituximab,
  • the compounds mentioned above in the present invention can be used as molecular SWIR fluorophores with enhanced quantum yield and good biocompatibility. These compounds exhibit fluorescence in the range from 900-1700 nm under the excitation of light in the range of 400-1000 nm, the intermolecular and intramolecular interactions of the conjugated backbone are reduced and the quantum yield is enhanced. Meanwhile, the molecular fluorophores (the compounds mentioned above) exhibit good water solubility and the dynamic range of the dyes in aqueous solution is small enough to ensure rapid urine or fecal excretion through the renal or biliary system and little toxicity. The high quantum yield and biocompatibility of the molecular dyes along with their SWIR emission opens up the opportunities of SWIR imaging using molecular fluorophores for in-vivo applications.
  • Example 2 Synthesis of IREF-PEG600 and IREFN-PEG600.
  • IREF-PEG600 (250 mg) was afforded as a green oil.
  • MALDI-TOF-MS expected M.W. about 3,700, measured M.W. 3,700.
  • IREFN-PEG600 was prepared as green oil with the same method by changing the amount of HO-PEG-Alkyne-0.6k to 85 mg.
  • MALDI-TOF-MS expected M.W. about 2,800, measured weight average M.W. about 2,800.
  • MALDI-TOF-MS expected M.W. about 5,208, measured M.W. 5,210. And IRDTN-PEG1000 was afforded as green oil with the same method but changing the amount of w-alkynyl-PEG-hydroxyl to 85 mg.
  • MALDI-TOF-MS expected M.W. about 3,198, measured weight average M.W. about 3,200.
  • UV-Vis-NIR spectrophotometer (UV 3600) with background correction was employed to measure the optical absorption spectra in water in the range of 300-1,200 nm.
  • a home build setup was used to measure the fluorescence spectrum of IR-E1 in the region of 900-1,600 nm using an array detector (Princeton OMA-V) and a spectrometer (Acton SP2300i) under an 808-nm diode laser (RMPC lasers) excitation (160 mW) .
  • RMPC lasers 808-nm diode laser
  • emission filters an 850-nm (Thorlabs) , 1,000-nm (Thorlabs) , 1,100-nm (Omega) and 1,300-nm short-pass filter (Omega) were used as excitation filters and 900-nm long-pass filter (Thorlabs) was used as emission filter.
  • the obtained emission spectra were further corrected by the detector sensitivity profile and the absorbance features of the filter.
  • Fig 1a Absorption and emission spectra of IRETBN-PEG1700 in aqueous solution were shown in Fig 1a, fluorescence intensity of IRETBN-PEG1700 in water and PBS measured over 1 week were shown in Fig 1b.
  • IRETBN-PEG1700 In aqueous solution, IRETBN-PEG1700 exhibited an absorption peak at 830 nm, while the fluorescence emission spectrum showed emission range from 1000 to 1400 nm with a main emission peak at around 1080 nm (Fig. 1a) .
  • the weight average molecular weight (MW) of IRETBN-PEG1700 was ⁇ 4.5 kDa with a hydrodynamic size of ⁇ 3.6 nm in aqueous solution.
  • the commercial SWIR fluorescent IR-26 dye was used as the reference sample with the quantum yield of 0.5%.
  • the IR-26 was dissolved in 1, 2-dichloroethane (DCE) , and diluted to different concentration with absorbance value at 808 nm of ⁇ 0.067, ⁇ 0.029, ⁇ 0.014 and ⁇ 0.002 using a ultraviolet-visible-near-infrared absorbance spectrometer.
  • the fluorescence spectra in the range of 900-to 1,600-nm was collected (900-nm long-pass filter) under the 808-nm diode laser (RMPC lasers) excitation.
  • the absorption and emission of compounds of examples 1 to 6 in water were measured using same method with IR-26.
  • QY stands for quantum yields
  • n refractive index of the solvent
  • A absorbance of the solution
  • I fluorescence intensity
  • Example 16 Non-invasive SWIR fluorescence imaging for brain blood vessels
  • the PBS solution of IRETBN-PEG1700 (1 mg/ml, 200 ⁇ L) was injected into hair removed mouse for SWIR imaging.
  • An 808 nm laser was used as excitation (140 mW ⁇ cm -2 ) filtered through 850 nm short-pass filter.
  • Dynamic imaging was done with a 2D InGaAs camera (Princeton Instrument 2D-OMA V: 320) with exposure time of 300 ms in the > 1300 nm range (by collecting emission through 1, 300-nm long-pass filter) .
  • SWIR fluorescence signals in the inferior cerebral vein, transverse sinus and middle cerebral vessels in the contralateral hemisphere showed up immediately within 1 s post injection, and these signals rapidly increased and plateaued at ⁇ 10 s (Figure 2) .
  • SWIR imaging of a mouse’s brain blood vessels with the IRETBN-PEG1700 fluorophore were shown in Figure 2.
  • the PBS solution of IREFNS (0.3 mg/ml, 350 ⁇ L) was injected intravenously into a mouse with a subcutaneous xenograft 4T1 murine tumor located on the left and right hind limbs.
  • An 808 nm laser was used as excitation (140 mW ⁇ cm -2 ) filtered through 850 nm short-pass filter.
  • Dynamic imaging was done with a 2D InGaAs camera (Princeton Instrument 2D-OMA V: 320) with exposure time of 300 ms in the > 1100 nm range (by collecting emission through 1, 100-nm long-pass filter) .

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