WO2017190345A1

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

Info

Publication number: WO2017190345A1
Application number: PCT/CN2016/081265
Authority: WO
Inventors: Yongye Liang; Huasen WANG; Qinglai YANG; Meijie TANG; Su Zhao
Original assignee: Southwest University of Science and Technology; Nirmidas Biotech Inc
Current assignee: Southwest University of Science and Technology; Nirmidas Biotech Inc
Priority date: 2016-05-06
Filing date: 2016-05-06
Publication date: 2017-11-09
Anticipated expiration: 2018-11-06
Also published as: CN109641921B; CN109641921A

Abstract

Provided is the design, synthesis and applications of molecular fluorophores for bioimaging in the short wavelength infrared window (1000-1700 nm). The molecular fluorophores compound comprise structures with electron accepting aromatic units, electron donating aromatic units, and an shielding units which shield the conjugated backbones from intermolecular interactions.

Description

Molecular Fluorophores and Preparation Method Thereof and Use for Short Wavelength Infrared Imaging

FIELD

Embodiments of the present disclosure generally relate to biomedical imaging with molecular fluorophores.

BACKGROUND

The development of biomedical imaging requires higher imaging resolution and deeper penetration depths, which are limited by photon scattering, biological tissue auto-fluorescence and water optical absorption. It’s known that photon scattering is reduced and biological tissue auto-fluorescence is lowered at longer wavelengths. At the same time, water optical absorption is low in the near-infrared region. As a result, the short wavelength infrared (SWIR, SWIR1.0-1.7 μm) has shown promise recently over the traditional near-infrared window (750-1000 nm) for in vivo optical imaging.

Inorganic nanomaterials, such as carbon nanotubes, PbS and Ag₂S quantum dots, and rare earth nanoparticles have been employed as SWIR fluorophores owing to their low band gap, high quantum yield and good stability. However, these nanomaterial-based SWIR fluorophores are generally very difficult to be cleared out from the body due to their large size and have toxicity concerns.

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. However, the general molecular fluorescent agents such as indocyanine green (ICG) , methylene blue (MB) and fluorescein isothiocyanate (FITC) , exhibit fluoresce below 900 nm. 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. 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. Thus, it remains highly challenging to develop organic molecules as 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.

SUMMARY

The following brief summary is not intended to include all features and aspects of the present invention, nor does it imply that the invention must include all features and aspects discussed in this summary.

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. Novel donor units are introduced to afford conformational distortion of the conjugated backbone, which can reduce both intermolecular and intramolecular interactions, thus enhance QY. To further lower the intermolecular interactions, inventors of the present invention introduce shielding (enveloping) units, which can sheath the conjugated backbone, at the terminals of the conjugated backbone. The shielding units, and the donor units can introduce end functionalized side chains, which can conjugate to hydrophilic unit (such as PEG) or other targeting ligands by a highly efficient click reaction (such as azide –alkyne cycloaddition) that is known to be superior to typical conjugation chemistry used for previous dyes. Importantly, by tuning the donor and shielding group, the inventors have developed 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. These 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：

an electron accepting aromatic unit,

an optional electron donating aromatic unit, and

a shielding unit shielding the electron accepting aromatic unit and/or the electron donating aromatic unit from intermolecular interactions,

wherein the electron accepting aromatic unit, the electron donating aromatic unit and the shielding unit are connected via a covalent bond.

Acording to some embodiments of the present invention, the electron accepting aromatic unit has a formula of any one selected from the group consisting of:

each one of Z₁ and Z₂ is independently O, S, Se, or NR,

each R is independently H, C_n1H_2n1+1, or tert-butyloxycarbonyl,

n1 is an integer ranging from 1 to 12.

Acording to some embodiments of the present invention, the electron donating aromatic unit has a formula of any one selected from the group consisting of:

each R₁ is independently H, C_n2H_2n2+1, or tert-butyloxycarbonyl,

each X is independently S, Se, NR₁, or O,

each one of Y₁ and Y₂ is independently H, OC_n2H_2n2+1, C_n2H_2n2+1,

OC_n2H_2n2B, or C_n2H_2n2Z,

each B is independently Br, I, OTs, OMs, ONs, N3, or OMe,

each Z is independently Br, or N3,

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,

Acording to some embodiments of the present invention, the shielding unit has a formula of any one selected from the group consisting of:

each R₂ is independently OC_n3H_2n3+1, C_n3H_2n3+1,

OC_n3H_2n3W, or C_n3H_2n3W,

each n3 is independently an integer ranging from 0 to 20,

each p is independently an integer ranging from 1 to 20,

each W is independently H, Br, I, OH, OTs, N₃,

each one of Z₅, Z₆, and Z₇ is independently S, Se, O, or NR₂’,

R₂’is H, C_n5H_2n5+1, or tert-butyloxycarbonyl,

each X₁ is independently Si, Ge, or C,

n5 is an integer ranging from 1 to 20,

n is an integer ranging from 4 to 120,

each b is independently an integer ranging from 1 to 6.

Acording to some embodiments of the present invention, 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,

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.

Acording to some embodiments of the present invention, 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.

Acording to some embodiments of the present invention, 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.

Acording to some embodiments of the present invention, 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’5 represents a fifth shielding unit,

S’6 represents a sixth shielding unit,

A represents the electron accepting aromatic unit.

Acording to some embodiments of the present invention, 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,

A represents the electron accepting aromatic unit.

Acording to some embodiments of the present invention, the compound having a formula of any one selected from the group consisting of:

each one of Y₁ and Y₂ is independently H, C_n2H_2n2+1, OC_n2H_2n2+1, OC_n2H_2n2B,

Embodiments of a second broad aspect of the present disclosure provide a kit, comprising the compound mentioned above.

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.

According to some embodiments of the present invention, 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.

According to some embodiments of the present invention, 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. ) , 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, tositumomab, trastuzumab, ustekinumab, vedolizumab, 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.

According to some embodiments of the present invention, 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. According to some embodiments of the present invention, 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.

According to some embodiments of the present invention, 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 mentioned above to a subject,

irradiating the subject at a site of interest by NIR light source,

recording a fluorescence image or video by a camera.

According to some embodiments of the present invention, the subject comprising an animal, a human, a tissue, a cell, a 3D organoid or a spheroid.

According to some embodiments of the present invention, 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.

According to some embodiments of the present invention, 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.

According to some embodiments of the present invention, 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.

According to some embodiments of the present invention, 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.

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 the method of biomedical imaging mentioned above,

using the image to guide tumor removal.

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.

According to some embodiments of the present invention, 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. ) , 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, tocilizumab, tositumomab, trastuzumab, ustekinumab, vedolizumab, etc. ) , and proteins (streptavidin, etc. ) , a nucleic acid, and an aptamer, which are modified with terminal alkynyl and then click reacted with fluorophores’azide.

The term “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. “Small bioactive molecules” as folic acid, tretinoin, cholic acid, galactose, they can bind to their specific receptors are excessively expressed on the surface of tumor cells selectively. 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. By utilizing the specificity of biomolecules, 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. In addition, 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.

The above summary of the present disclosure is not intended to describe each disclosed embodiment or every implementation of the present disclosure. The Figures and the detailed description which follow more particularly exemplify illustrative embodiments.

Additional aspects and advantages of embodiments of present disclosure will be given in part in the following descriptions, become apparent in part from the following descriptions, or be learned from the practice of the embodiments of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other aspects and advantages of embodiments of the present disclosure will become apparent and more readily appreciated from the following descriptions made with reference the accompanying drawings, in which:

Fig. 1a shows absorption and emission spectra of IRETBN-PEG1700 in aqueous solution, Fig 1b shows fluorescence intensity of IRETBN-PEG1700 in water and PBS measured over 1 week.

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.

Fig 6a shows a SWIR fluorescence image demonstrating that IRETBN-PEG1700 was accumulated in the bladder of the mouse at 6 h after injection, Fig 6b shows a SWIR image of urine collected at 3 h after injection.

DETAILED DESCRIPTION

Reference will be made in detail to embodiments of the present disclosure. The embodiments described herein with reference to drawings are explanatory, illustrative, and used to generally understand the present disclosure. The embodiments shall not be construed to limit the present disclosure. The same or similar elements and the elements having same or similar functions are denoted by like reference numerals throughout the descriptions.

The grammatical articles “a” , “an” and “the” , as used herein, are intended to include “at least one” or “one or more” unless otherwise indicated herein or clearly contradicted by the context. Thus, 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. By way of example, “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.

The term “comprising” is meant to be open ended, including the indicated component but not excluding other elements.

an electron accepting aromatic unit,

an optional electron donating aromatic unit, and

each one of Z₁, and Z₂ is independently O, S, Se, or NR,

each R is independently H, C_2n+1H_2n+1, or tert-butyloxycarbonyl,

n1 is an integer ranging from 1 to 12.

each R₁ is independently H, C_n2H_2n2+1, or tert-butyloxycarbonyl,

each X is independently S, Se, NR₁, or O,

each one of Y₁ and Y₂ is independently H, OC_n2H_2n2+1, C_n2H_2n2+1,

OC_n2H_2n2B, or C_n2H_2n2Z,

each B is independently Br, I, OTs, OMs, ONs, N3, or OMe,

each Z is independently Br, or N3,

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.

Acording to some embodiments of the present invention, 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. Such as:

each R₂ is independently OC_n3H_2n3+1, C_n3H_2n3+1,

OC_n3H_2n3W, or C_n3H_2n3W,

each n3 is independently an integer ranging from 0 to 20,

each p1 is independently an integer ranging from 1 to 20,

each W is H, Br, I, OH, OTs, N₃,

each one of Z₅, Z₆, and Z₇ is independently S, Se, O, or NR₂’,

R₂’is H, C_n5H_2n5+1, or tert-butyloxycarbonyl,

each X₁ is independently Si, Ge, or C,

each n5 is an integer ranging from 1 to 20,

each n is an integer ranging from 4 to 120,

each b is independently an integer ranging from 1 to 6.

Acording to some embodiments of the present invention, 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. Sometimes, the terminals of the side chains are functionalized with azide group (N3) for further conjugation with targeting ligands.

Acording to some embodiments of the present invention, 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, 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. In other words, 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.

Acording to some embodiments of the present invention, 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.

Acording to some embodiments of the present invention, 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. In other words, S’3 represents one shielding unit of the two shielding units, S’4 represents the other shielding unit of the two shielding units. D3 represents one electron donating aromatic unit of the three electron donating aromatic units, D4 represents another electron donating aromatic unit of the three electron donating aromatic units, D5 represents the third electron donating aromatic unit of the three electron donating aromatic units. A1 represents one electron accepting aromatic unit of the two electron accepting aromatic units, A2 represents the other electron accepting aromatic unit of the two electron accepting aromatic units.

S’5 represents a fifth shielding unit,

S’6 represents a sixth shielding unit,

A represents the electron accepting aromatic unit.

Acording to some embodiments of the present invention, 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 seventh shielding unit, S’8 represents an eighth shielding unit, S’9 represents a ninth shielding unit, S’10 represents a tenth shielding unit, A represents the electron accepting aromatic unit. In other words, A1 represents the electron accepting aromatic unit, S’7, S’8, S’9, and S’10 respectively represent one shielding unit of the four shielding units.

The term “-PEG600” , “-PEG1000” , and “-PEG1700” represent 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.

In the present invention, the meaning of

is that two of R₁, R₂, R₃, R₄ represent N3, the other two of R₁, R₂, R₃, R₄ represent

unless otherwise indicated herein or clearly contradicted by the context. Other similar descriptions are the same.

Embodiments of a second broad aspect of the present disclosure provide a kit, comprising the compound mentioned above. The kit can be used as molecular fluorescence for biomedical imaging or labeling a biomolecule.

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. Thus, the biomolecule can be tracked by the molecular fluorescence (the compound mentioned above) . According to some embodiments of the present invention, the molecular can be further combined to tumor, blood vessels, etc., thus the tumor, blood vessels, etc., can also be tracked.

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.

According to some embodiments of the present invention, 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. According to some embodiments of the present invention, 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.

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.

According to some embodiments of the present invention, the site of interest comprising vessels (brain vessels, tumor vessels, etc. ) , tumor, lymphatic system, and so on.

According to some embodiments of the present invention, 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.

According to some embodiments of the present invention, 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.

According to some embodiments of the present invention, 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. Thus, photon scattering, biological tissue auto-fluorescence and water optical absorption are reduced significantly, and QY is high.

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.

In the examples described below, all air and moisture sensitive reactions were carried out in flame-dried glassware under a nitrogen atmosphere. Reactive liquid compounds were measured and transferred by gas-tight syringes and were added in the reaction flask through rubber septa. Tetrahydrofuran (THF) was freshly distilled from sodium benzophenoneketyl. Toluene was distilled from CaH₂. Unless otherwise noted, all reagents were obtained commercially and used without further purification.

Example 1: Synthesis of IRETBN-PEG1700 and IRETBN-PEG600

(1) Synthesis of 5- (5- (2, 6-bis ( (6-bromohexyl) oxy) phenyl) thiophen-2-yl) -2, 3-dihydro-thieno [3, 4 -b] [1, 4] dioxine (compound 2) ：

To a solution of tributyl (2, 3-dihydrothieno [3, 4-b] [1, 4] dioxin-5-yl) stannane (860 mg, 2.0 mmol) and compound 1 (720 mg, 1.2 mmol) in 10 mL toluene under Ar atmosphere, Pd (PPh₃) ₄ (71 mg, 0.061 mol) was added. The mixture was stirred at 110 ℃ for 24 h. After cooling to room temperature, the mixture was poured into water and extracted twice with ethyl acetate, and the organic phase was dried with MgSO₄ and evaporated in vacuo. The crude product was subjected to column chromatography on silica gel with PE/DCM 1: 1 to afford compound 2 as a light yellow oil (590 mg, 45 ％) .

¹H NMR (400 MHz, CDCl₃) : δ 7.45 (d, J ＝ 3.9 Hz, 1H) , 7.24 (d, J ＝ 3.9 Hz, 1H) , 7.17 (t, J ＝ 8.3 Hz, 1H) , 6.61 (d, J ＝ 8.4 Hz, 2H) , 6.21 (s, 1H) , 4.37 –4.32 (m, 2H) , 4.28 –4.23 (m, 2H) , 4.01 (t, J ＝ 6.2 Hz, 4H) , 3.38 (t, J ＝ 6.9 Hz, 4H) , 1.84 (m, 7.2 Hz, 8H) , 1.52 –1.43 (m, 8H) ； ¹³C NMR (100 MHz, CDCl₃) : δ 157.1, 141.9, 137.2, 134.3, 132.6, 129.2, 128.2, 122.1, 113.0, 112.9, 105.5, 96.4, 68.9, 64.9, 64.7, 33.9, 32.7, 29.0, 27.9, 25.3；

HRMS (ESI) calcd. for C₂₈H₃₅Br₂O₄S₂ ⁺, ( [M+H⁺] ) 657.0344, Found 657.0347.

(2) Synthesis of (7- (5- (2, 6-bis ( (6-bromohexyl) oxy) phenyl) thiophen-2-yl) -2, 3-dihydro -thieno [3, 4-b] [1, 4] dioxin-5-yl) tributylstannane (compound 3) ：

To a solution of compound 2 (350 mg, 0.53 mmol) in 5 mL THF at -78 ℃ under Ar atmosphere, n-BuLi (1.6 M in hexane, 1 mL, 1.6 mmol) was added dropwise. After the mixture was stirred at this temperature for another 1.5 h, trinbutyltinchloride (330 mg, 1 mmol) was added to the solution. Then the reaction mixture was slowly warmed to room temperature and stirred for 8 h. After that the mixture was poured into water and extracted twice with ethyl acetate, the combined organic phase was dried with MgSO₄ and evaporated in vacuo without further purification.

(3) Synthesis of compound 4:

To a solution of compound a (72 mg, 0.2 mmol) and compound 3 (400 mg, 0.44 mmol) in 5 mL toluene under Ar atmosphere, Pd (PPh₃) ₂Cl₂ (35 mg, 0.03 mol) was added. The mixture was stirred at 110 ℃ for 48 h. After cooling to room temperature, the mixture was poured in to water and extracted twice with ethyl acetate. The combined organic phase was dried with MgSO₄ and evaporated in vacuo. The crude product was subjected to column chromatography on silica gel to afford compound 5 as a dark green solid (93 mg, 31 ％) .

¹H NMR (400 MHz, CDCl₃) δ 7.55 (d, J ＝ 4.0 Hz, 2H) , 7.42 (d, J ＝ 4.0 Hz, 2H) , 7.19 (t, J ＝8.3 Hz, 2H) , 6.63 (d, J ＝ 8.4 Hz, 4H) , 4.51 (m, 4H) , 4.37 (m, 4H) , 4.04 (t, J ＝ 6.3 Hz, 8H) , 3.40 (t, J ＝ 6.8 Hz, 8H) , 1.87 (m, 16H) , 1.55 –1.43 (m, 16H) ； ¹³C NMR (100 MHz, CDCl₃) : δ 157.1, 152.5, 141.6, 137.6, 134.0, 133.9, 129.4, 128.4, 123.2, 117.6, 113.0, 112.8, 108.4, 105.5, 69.0, 64.9, 64.8, 34.0, 32.8, 29.1, 28.0, 25.5；

HRMS (ESI) calcd for C₆₂H₆₇Br₄N₄O₈S₆ ⁺, ( [M+H⁺] ) 1503.0017, Found 1503.1578.

(4) Synthesis of IRETBN-1700 and IRETBN-PEG600. Compound 4 (50 mg, 0.036 mmol) was dissolved in 5 mL DMF. Then sodium azide (47 mg, 0.72 mmol) was added and the reaction solution was stirred at 60 C for 20 h. After that, a large portion of water was added, and the product was extracted twice with ethyl acetate. The combined organic phase was dried with MgSO₄ and evaporated in vacuo. The crude product was subjected to column chromatography on silica gel to afford azide product as dark green solid 43 mg (quant) .

Azide product 20 mg was dissolved in 5 mL THF and cuprous iodide 100 mg, α-alkynyl-ω-hydroxyl-poly (ethylene glycol) -1.7k (HO-PEG-Alkyne-1.7k, weight average molecular weight ＝ 1.68k, PDI ＝ 1.05) 120 mg, and triethylamine 200uL were added. The system was stirred at RT for 48 h. Then the reaction mixture was filtered with Al₂O₃, and the solution was evaporated in vacuo. When all the organic solvent was removed, the solid was dissolved in 15 mL water and it was transferred to the dialyzer (MWCO ＝ 3500) . The system was stirred at RT for 72 h with 10 times water exchange. The resulting water solution in the dialyzing membrane was lyophilized. IRETBN-1700 (100 mg) was afforded as yellow powder. MALDI-TOF-MS expected M.W. about 4,500, measured weight average M.W. about 4,500. (It suggested that two PEG chains were connected to the dye molecule by click reactions. Another two azide groups in the dye molecule were blocked by the introduced PEG chains for further click reaction. ) . And IRETBN-PEG600 was prepared as light green powder with the same method by using HO-PEG-Alkyne-0.6k (weight average molecular weight ＝ 0.60k) . MALDI-TOF-MS expected M.W. about 2,800, measured weight average M.W. about 2,800.

Optical parameters of IRETBN-1700 in H₂O: absorption peak λ ＝ 835 nm, emission peak λ_em ＝ 1098 nm, absorption coefficient K＝ 1.1 L/g. cm in 808 nm, quantum yield is 0.7％ (with 808 nm excitation)

Optical parameters of IRETBN-600 in H₂O: absorption peak λ ＝ 830 nm, emission peak λ_em ＝ 1098 nm, absorption coefficient K＝ 1.22 L/g. cm in 808 nm, quantum yield is 0.9％ (with 808 nm excitation)

Example 2: Synthesis of IREF-PEG600 and IREFN-PEG600.

(1) Synthesis of 5- (9H-fluoren-2-yl) -2, 3-dihydrothieno [3, 4-b] [1, 4] dioxine (compound 6) :

2-bromo-9H-fluorene (compound 5) (5.0 g, 20.4 mmol) and tributyl (2, 3-dihydrothieno [3, 4-b] [1, 4] dioxin-5-yl) stannane (9.2 g, 21.4 mmol) were dissolved in 40 mL toluene under protective gas atmosphere, then Pd (PPh₃) ₄ (200 mg) was added. After reflux for 6 h, the crude product was subjected to column chromatography on silica gel to afford compound 6 as a light yellow solid (5.8 g, 94 ％) .

¹H NMR (500 MHz, Chloroform-d) δ 7.94 (dd, J ＝ 3.8, 1.7 Hz, 1H) , 7.82 –7.74 (m, 3H) , 7.56 (dd, J ＝ 7.3, 2.0 Hz, 1H) , 7.40 (td, J ＝ 7.5, 2.7 Hz, 1H) , 7.36 –7.29 (m, 1H) , 6.41 –6.26 (m, 1H) , 4.37 –4.31 (m, 2H) , 4.29 –4.23 (m, 2H) , 3.95 (s, 2H) . ¹³C NMR (126 MHz, Chloroform-d) δ 37.60, 65.10, 65.41, 98.00, 118.68, 120.45, 120.62, 123.15, 125.43, 125.64, 127.22, 127.41, 132.37, 138.60, 140.84, 142.11, 142.91, 144.05, 144.30.

HRMS (ESI) calcd for C₁₉H₁₅O₂S ⁺, ( [M+H⁺] ) 307.0793, Found 307.0787.

(2) Synthesis of 5- (9, 9-bis (6-bromohexyl) -9H-fluoren-2-yl) -2, 3-dihydrothieno [3, 4-b] [1, 4] dioxine (compound 7) :

5-(9H-fluoren-2-yl) -2, 3-dihydrothieno [3, 4-b] [1, 4] dioxine (compound 6) (3.0 g, 9.8 mmol) and 1, 6-dibromohexane (9.7 g, 40 mmol) were dissolved in 50 mL THF at 0 ℃. Then potassium tert-butanolate (2.5 g, 2.1 mmol) in THF was added dropwise. After reacting for 6 h at RT, the crude product was subjected to column chromatography on silica gel to afford compound 7 as a light yellow oil (4.9 g, 81 ％) .

¹H NMR (500 MHz, Chloroform-d) δ 7.77 (dd, J ＝ 8.0, 1.6 Hz, 1H) , 7.71 (ddd, J ＝ 7.9, 4.6, 0.8 Hz, 2H) , 7.69 –7.64 (m, 1H) , 7.41 –7.29 (m, 3H) , 6.35 (s, 1H) , 4.42 –4.34 (m, 2H) , 4.34 –4.26 (m, 2H) , 3.30 (t, J ＝ 6.8 Hz, 4H) , 2.02 (dt, J ＝ 11.1, 5.7 Hz, 4H) , 1.74 –1.59 (m, 4H) , 1.29 –1.17 (m, 4H) , 1.16 –1.04 (m, 4H) , 0.78 –0.56 (m, 4H) . ¹³C NMR (126 MHz, Chloroform-d) δ23.54, 27.76, 29.06, 32.65, 34.07, 40.20, 55.01, 64.52, 64.88, 77.29, 97.35, 118.20, 119.70, 119.88, 120.23, 122.77, 124.98, 126.92, 127.02, 132.09, 138.06, 139.69, 140.86, 142.38, 150.55, 150.77.

HRMS (ESI) calcd for C₃₁H₃₇Br₂O₂S⁺, ( [M+H⁺] ) 630.0803, Found 631.0861.

(3) Synthesis of compound 8:

To a solution of compound 7 (2.0 g, 3.18 mmol) in 25 mL THF at -78 ℃ under Ar atmosphere, n-BuLi (1.6 M in hexane, 2.4 mL, 3.8 mmol) was added dropwise. After the mixture was stirred at this temperature for another 1.5 h, trinbutyltinchloride (1.3 g, 3.8 mmol) was added to the solution. Then the reaction was slowly warmed to room temperature and stirred for 8 h. After that, the mixture was poured into water and extracted twice with ethyl acetate, the combined organic phase was dried with MgSO₄ and evaporated in vacuo without further purification.

To a solution of the crude product (1.4 g, 1.5 mmol) above and compounds of a (195 mg, 0.5 mmol) in 15 mL toluene under Ar atmosphere, Pd (PPh₃) ₂Cl₂ (100 mg) was added. The mixture was stirred at 110 ℃ for 12 h. After cooling to room temperature, the mixture was poured into water and extracted twice with ethyl acetate, dried with MgSO₄ and evaporated in vacuo. The crude product was subjected to column chromatography on silica gel to afford compound 8 as a dark green solid (435 mg, 60 ％) .

¹H NMR (500 MHz, Chloroform-d) δ 7.95 (dd, J ＝ 7.8, 1.7 Hz, 2H) , 7.85 –7.73 (m, 6H) , 7.39 –7.32 (m, 6H) , 4.54 (dd, J ＝ 6.0, 2.6 Hz, 4H) , 4.48 –4.32 (m, 4H) , 3.31 (t, J ＝ 6.8 Hz, 8H) , 2.11 –2.01 (m, 8H) , 1.70 –1.66 (m, 8H) , 1.29 –1.21 (m, 8H) , 1.17 –1.10 (m, 8H) , 0.76 –0.64 (m, 8H) . ¹³C NMR (126 MHz, Chloroform-d) δ 13.71, 17.59, 23.59, 26.90, 27.81, 27.92, 29.07, 32.66, 34.12, 40.26, 55.13, 64.61, 64.79, 108.85, 113.14, 119.84, 119.95, 119.97, 120.68, 122.66, 122.80, 125.63, 127.01, 127.24, 131.79, 138.40, 140.36, 140.82, 142.00, 150.66, 150.83, 152.60.

HRMS (ESI) calcd for C₆₈H₇₁Br₄N₄O₄S₄ ⁺, ( [M+H⁺] ) 1451.1092, Found 1451.1080.

(4) Synthesis of IREF-PEG600 and IREFN-PEG600:

Compound 8 (100 mg, 0.069 mmol) was dissolved in 10 mL DMF and sodium azide (47 mg, 0.72 mmol) was added. The mixture was heated for 3 h at 70 ℃. After cooling to RT, water was added to dissolve all the solids. Then it was extracted twice with ethyl acetate, the combined organic phase was dried with MgSO₄ and evaporated in vacuo. The crude product was subjected to column chromatography on silica gel to afford dark green solid 95 mg (quant) .

The dark green solid was dissolved in 5 mL THF and CuTc (10 mg) , HO-PEG-Alkyne-0.6k (weight average molecular weight ＝ 0.60k, 170 mg) , and TBTA (10 mg) was added. The system was stirred at RT for 0.5 h. Then filtered with diatomite, and the solution was evaporated in vacuo. When all the organic solvent was removed, the solid was dissolved in 15 mL water and it was transferred to the dialyzer (MWCO ＝ 3500) . The system was stirred at RT for 72 h with 10 times water exchange. The resulting water solution in the dialyzing membrane was lyophilized. IREF-PEG600 (250 mg) was afforded as a green oil. MALDI-TOF-MS expected M.W. about 3,700, measured M.W. 3,700. And 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.

Optical parameters of IREFN-PEG600 in H₂O: absorption peak λ ＝ 793 nm, emission peak λ_em ＝ 1047 nm, absorption coefficient K＝ 2.2 L/g. cm in 808 nm, quantum yield is 2.1％ (with 808 nm excitation)

Example 3: Synthesis of IREFNS

Compound 8 (100 mg, 0.069 mmol) was dissolved in 10 mL THF and dimethylamine (2.0 M in THF, 2 mL) , then stirred for 6 h at 50 ℃. After that the solvents were evaporated in vacuo. The dark green solid and 1, 2-oxathiolane 2, 2-dioxide 122 mg (1 mmol) was dissolved in 5 mL THF, then the solution was stirred overnight. After that the solution was filtered, washed with acetone and ethyl ether for several times. IREFNS (110 mg) was afforded as a green solid.

HRMS (ESI) calcd for C₈₈H₁₂₀N₈O₁₆S₈, ( [M+H⁺] ) 1800.6588, Found 1800.6545.

Optical parameters of IREFNS in H₂O: absorption peak λ ＝ 795 nm, emission peak λ_em ＝1047 nm, absorption coefficient K＝ 6.2 L/g. cm in 808 nm, quantum yield is 0.46％ (with 808 nm excitation)

Example 4: Synthesis of IRTTBN-PEG1000

(1) Synthesis of 3- (2- (2- (2-methoxyethoxy) ethoxy) ethoxy) thiophene (compound 9) :

A dry 250 mL two-neck round bottom flask with condenser and magnetic stir bar was charged with potassium tertbutylate (10.5 g, 94 mmol) and cuprous iodide (2.34 g, 12 mmol) . Next, 10 mL pyridine and 100 mL toluene mixture solution was added and stirred until dissolved, and 15 mL triethylene glycol monomethyl ether was added. The reaction mixture was then allowed to stir at room temperature for 30 min. 3-bromothiophene (5.8 mL, 62 mmol) was then added in one portion and the reaction mixture was heated to 110 ℃ for 24 h. After cooling to room temperature, the reaction mixture was centrifuged and concentrated under vacuum. 50 mL dichloromethane was added and washed with 5 M HCl (aq) . The aqueous layers were extracted with DCM, and the combined organic portions were dried with MgSO₄ and concentrated under reduced pressure. The crude material was purified via column chromatography, eluting with 1: 1 ethyl ether in hexanes (v/v) . The desired product was isolated as a yellow tinted oil (10.35 g, 68％yield) .

¹H-NMR (400 MHz, CDCl₃) δ 7.18 (dd, J ＝ 5.3, 3.1 Hz, 1H) , 6.79 (dd, J ＝ 5.3, 1.5 Hz, 1H) , 6.28 (dd, J ＝ 3.1, 1.5 Hz, 1H) , 4.17 –4.10 (m, 2H) , 3.89 –3.83 (m, 2H) , 3.77 –3.72 (m, 2H) , 3.72 –3.64 (m, 4H) , 3.57 (dd, J ＝ 5.7, 3.6 Hz, 2H) , 3.40 (s, 3H) .

(2) Synthesis of 2-bromo-3- (2- (2- (2-methoxyethoxy) ethoxy) ethoxy) thiophene (compound 10) :

Compound 9 (2.46 g, 10 mmol) was dissolved in 15 mL DMF, and NBS (1.78 g, 10 mmol) was added in portions at 0 ℃. After 18 h at RT, it was added just as much water as needed to dissolve all solids. Then it was extracted twice with ethyl acetate, the combined organic phase was washed with Na₂SO₃ (aq) three times and dried with MgSO₄ and evaporated in vacuo without further purification and afford compound 10 as a yellow tinted oil (3.0 g, 92％yield) .

¹H NMR (400 MHz, CDCl₃) δ 7.18 (d, J ＝ 6.0 Hz, 1H) , 6.77 (d, J ＝ 5.9 Hz, 1H) , 4.28 –4.13 (m, 2H) , 3.86 –3.79 (m, 2H) , 3.76 –3.70 (m, 2H) , 3.70 –3.63 (m, 4H) , 3.55 (dd, J ＝ 5.6, 3.6 Hz, 2H) , 3.38 (s, 3H) .

(3) Synthesis of triisopropyl (3- (2- (2- (2-methoxyethoxy) ethoxy) ethoxy) thiophen-2-yl) silane (compound 11) :

To a solution of Compound 10 (3.25 g, 10 mmol) in 20 mL THF at -78 ℃ under protection gas atmosphere, n-BuLi (1.6 M in hexane, 6.25 mL, 20 mmol) was added dropwise. After the mixture was stirred at this temperature for another 2.0 h, triisopropylsilyl chloride (1.9 g, 12 mmol) was added to the solution. Then the reaction was slowly warmed to room temperature and stirred for 12 h. After that, the mixture was poured into water and extracted twice with ethyl acetate, the combined organic phase was dried with MgSO₄ and evaporated in vacuo. The crude material was purified via column chromatography, eluting with 2: 1 ethyl ether in hexanes (v/v) . The desired product was isolated as a yellow tinted oil (3.53 g, 88％yield) .

¹H NMR (500 MHz, CDCl₃) δ 7.46 (d, J ＝ 5.0 Hz, 1H) , 6.95 (d, J ＝ 5.0 Hz, 1H) , 4.16 –4.10 (m, 2H) , 3.79 (t, J ＝ 5.2 Hz, 2H) , 3.71 –3.66 (m, 6H) , 3.59 –3.55 (m, 2H) , 3.40 (s, 3H) , 1.49 –1.35 (m, 2H) , 1.10 (d, J ＝ 7.5 Hz, 19H) . ¹³C NMR (126 MHz, CDCl₃) δ 163.62, 129.85, 116.51, 108.99, 71.95, 70.72, 70.67, 70.06, 69.96, 59.07, 18.78, 12.03.

HRMS (ESI) calcd for C₂₀H₃₉O₄SSi⁺, ( [M+H⁺] ) 402.2294, Found 403.2333.

(4) Synthesis of triisopropyl (3- (2- (2- (2-methoxyethoxy) ethoxy) ethoxy) -5- (tributylstannyl) thiophen-2-yl) silane (compound 12) :

To a solution of compound 11 (402 mg, 1.0 mmol) in 5 mL THF at -78 ℃ under protection gas atmosphere, n-BuLi (1.6 M in hexane, 0.625 mL, 1.2 mmol) was added dropwise. After the mixture was stirred at this temperature for another 2.0 h, trinbutyltinchloride (390 mg, 1.2 mmol) was added to the solution. Then the reaction was slowly warmed to room temperature and stirred for 8 h. After that, the mixture was poured into water and extracted twice with ethyl acetate, the combined organic phase was dried with MgSO₄ and evaporated in vacuo without further purification.

(5) Synthesis of (5'- (2, 6-bis ( (6-bromohexyl) oxy) phenyl) -4- (2- (2- (2-methoxyethoxy) ethoxy) ethoxy) - [2, 2'-bithiophen] -5-yl) triisopropylsilane (compound 13) :

To a solution of compound 2- (2, 6-bis ( (6-bromohexyl) oxy) phenyl) -5-bromothiophene (1.19 g, 2.0 mmol) , compound 12 (1.66 g, 2.4 mmol) in 10 mL toluene under protection gas atmosphere. Pd (PPh₃) ₄ (142 mg, 0.122 mol) was added. The mixture was stirred at 110 ℃ for 18 h. After cooling to room temperature, the mixture was poured into water and extracted twice with ethyl acetate, dried with MgSO₄ and evaporated in vacuo. The crude product was subjected to column chromatography on silica gel with PE/EA 2: 1 to afford compound 13 as a light yellow oil (1.18 g, 54 ％) .

¹H NMR (400 MHz, CDCl₃) δ 7.44 (d, J ＝ 3.9 Hz, 1H) , 7.19 (dd, J ＝ 6.1, 4.5 Hz, 2H) , 7.05 (s, 1H) , 6.64 (d, J ＝ 8.4 Hz, 2H) , 4.04 (t, J ＝ 6.3 Hz, 4H) , 3.82 (t, J ＝ 5.1 Hz, 2H) , 3.76 –3.63 (m, 8H) , 3.58 (dd, J ＝ 5.7, 3.6 Hz, 2H) , 3.46 –3.34 (m, 7H) , 1.92 –1.80 (m, 8H) , 1.68 (s, 3H) , 1.55 –1.47 (m, 8H) , 1.14 (d, J ＝ 7.4 Hz, 18H) . ¹³C NMR (101 MHz, CDCl₃) δ 163.19, 157.12, 141.65, 136.97, 133.40, 129.67, 128.50, 122.15, 112.88, 112.48, 108.23, 105.46, 71.97, 70.75, 70.73, 70.62, 70.04, 68.94, 59.04, 33.80, 32.54, 29.03, 27.84, 25.42, 18.83, 12.11.

HRMS (ESI) calcd for C₄₂H₆₇O₆Br₈₁BrS₂Si, ( [M+H⁺] ) 919.24891, Found 919.24902.

(6) Synthesis of 5'- (2, 6-bis ( (6-bromohexyl) oxy) phenyl) -4- (2- (2- (2-methoxyethoxy) ethoxy) ethoxy) -2, 2'-bithiophene (compound 14) :

To a solution of compound 13 (918 mg, 1 mmol) in 4 mL THF at -78 ℃ under protection gas atmosphere, tetrabutylammonium fluoride (1.0 M in THF, 4 mL, 4 mmol) was added. After the mixture was stirred at this temperature for another 1.0 h, the reaction was slowly warmed to room temperature and stirred for 4 h. After that, the mixture was poured into water and extracted twice with ethyl acetate, the combined organic phase was dried with MgSO₄ and evaporated in vacuo. The crude material was purified via flash column chromatography, eluting with hexanes to ethyl acetate. The desired product was isolated as a yellow tinted oil (722 mg, 95 ％) .

¹H NMR (400 MHz, CDCl₃) δ 7.43 (d, J ＝ 3.9 Hz, 1H) , 7.30 (d, J ＝ 3.9 Hz, 1H) , 7.19 (t, J ＝8.3 Hz, 1H) , 7.05 (d, J ＝ 5.5 Hz, 1H) , 6.91 (d, J ＝ 5.5 Hz, 1H) , 6.63 (d, J ＝ 8.4 Hz, 2H) , 4.02 (t, J ＝ 6.3 Hz, 4H) , 3.93 –3.84 (m, 2H) , 3.75 (dd, J ＝ 5.8, 3.5 Hz, 2H) , 3.71 –3.60 (m, 4H) , 3.54 (dd, J ＝ 5.7, 3.6 Hz, 2H) , 3.44 –3.34 (m, 7H) , 1.90 –1.78 (m, 8H) , 1.56 –1.44 (m, 8H) . ¹³C NMR (101 MHz, CDCl₃) δ 157.15, 151.93, 134.56, 132.35, 129.03, 128.23, 122.23, 121.03, 117.11, 113.30, 105.56, 71.93, 71.17, 70.91, 70.68, 70.57, 70.06, 68.94, 59.01, 33.88, 32.70, 29.04, 27.86, 25.41.

HRMS (ESI) calcd for C₃₃H₄₇O₆Br₈₁BrS₂, ( [M+H⁺] ) 763.1155, Found 763.1154.

(7) Synthesis of 4, 8- (di-2, 6-bis ( (6-bromohexyl) oxy) phenyl) -4- (2- (2- (2-methoxyethoxy) ethoxy) ethoxy) -2, 2'-bithiophene) -1H, 5H-benzo [1, 2-c: 4, 5-c'] bis ( [1, 2, 5] thiadiazole) (compound 15) :

To a solution of compound 14 (1.52 g, 2 mmol) in 15 mL THF at -78 ℃ under protection gas atmosphere, n-BuLi (1.6 M in hexane, 1.5 mL, 2.4 mmol) was added dropwise. After the mixture was stirred at this temperature for another 2.0 h, trinbutyltinchloride (0.812 mg, 2.5 mmol) was added to the solution. Then the reaction was slowly warmed to room temperature and stirred for 8 h. After that, the mixture was poured into water and extracted twice with ethyl acetate, the combined organic phase was dried with MgSO₄ and evaporated in vacuo without further purification.

To a solution of the crude product above (2 mmol) and 4, 8-dibromo-1H, 5H-benzo [1, 2-c: 4, 5-c'] bis ( [1, 2, 5] thiadiazole) (compound a, 234 mg, 0.67 mmol) in 15 mL toluene under protection gas atmosphere, Pd (PPh₃) ₂Cl₂ (150 mg) was added. The mixture was stirred at 110 ℃ for 12 h. After cooling to room temperature, the mixture was poured into water and extracted twice with ethyl acetate, dried with MgSO₄ and evaporated in vacuo. The crude product was subjected to column chromatography on silica gel with PE/EA 3: 1 to afford compound 15 as a dark green solid (505 mg, 43 ％) .

¹H NMR (400 MHz, CDCl₃) δ 7.56 (dd, J ＝ 8.6, 4.4 Hz, 2H) , 7.33 (dd, J ＝ 3.7, 1.4 Hz, 2H) , 7.23 (dd, J ＝ 15.5, 4.8 Hz, 4H) , 6.70 –6.59 (m, 4H) , 4.41 (d, J ＝ 4.2 Hz, 4H) , 4.07 (t, J ＝ 6.2 Hz, 8H) , 3.72 (t, J ＝ 5.0 Hz, 4H) , 3.63 –3.47 (m, 16H) , 3.42 (t, J ＝ 6.7 Hz, 8H) , 3.36 (s, 6H) , 1.97 –1.83 (m, 16H) , 1.60 –1.45 (m, 16H) .

¹³C NMR (125 MHz, CDCl₃ ) δ 159.25, 152.93, 150.90, 149.60, 140.11, 138.00, 135.70, 129.38, 124.74, 123.08, 122.44, 120.18, 118.16, 109.81, 102.14, 73.68, 70.37 (d, J ＝ 1.6 Hz) , 69.96, 69.54, 69.25, 57.85, 33.30, 31.58, 30.68, 28.80, 26.58.

HRMS (ESI) calcd for C₇₂H₉₁Br₄N₄O₁₂S₆ ⁺, ( [M+H⁺] ) 1716.1639, Found 1716.1618.

(8) Synthesis of IRTTBN-PEG1000:

Compound 15 (100 mg, 0.058 mmol) and sodium azide (47 mg, 0.72 mmol) were dissolved in 10 mL DMF and heated for 3 h at 70 ℃. A large amount of water was added to dissolve all the solids. Then it was extracted twice with ethyl acetate, the combined organic phase was dried with MgSO₄ and evaporated in vacuo. The crude product was subjected to flash column chromatography on silica gel to afford dark green solid 97 mg.

The dark green solid was dissolved in 5 mL THF and CuTc (10 mg) , w-alkynyl-PEG-hydroxyl (M_w ＝ 1000, 116 mg) , and TBTA (5 mg) were added. The system was stirred at RT for 1.0 h. Then the reaction mixture was filtered with diatomite, and the solvent was evaporated in vacuo. When all the organic solvent was removed, the crude product was purified by thin layer chromatography eluting with DCM/MeOH 10: 1. IRTTBN-PEG1000 (142 mg) was afforded as a green oil.

Optical parameters of IRTTBN-PEG1000 in H₂O: absorption peak λ ＝ 830 nm, emission peak λ_em ＝ 1098 nm, absorption coefficient K＝ 1.22 L/g. cm in 808 nm, quantum yield is 0.93％(with 808 nm excitation)

Example 5: Synthesis of IRTBBN-PEG1000

(1) Synthesis of (5- (2', 6'-bis ( (6-bromohexyl) oxy) - [1, 1'-biphenyl] -4-yl) -3- (2- (2- (2-methoxyethoxy) ethoxy) ethoxy) thiophen-2-yl) triisopropylsilane (compound 16) :

To a solution of compound 4'-bromo-2, 6-bis ( (6-bromohexyl) oxy) -1, 1'-biphenyl (1.18 g, 2.0 mmol) , compound 12 (1.66 g, 2.4 mmol) in 10 mL toluene under protection gas atmosphere, Pd (PPh₃) ₄ (142 mg, 0.122 mol) was added. The mixture was stirred at 110 ℃ for 18 h. After cooling to room temperature, the mixture was poured into water and extracted twice with ethyl acetate, dried with MgSO₄ and evaporated in vacuo. The crude product was subjected to column chromatography on silica gel with PE/EA 2: 1 to afford compound 16 as a light yellow oil (1.04 g, 57 ％) .

¹H NMR (500 MHz, CDCl₃) δ 7.62 (d, J ＝ 8.2 Hz, 2H) , 7.37 (d, J ＝ 8.2 Hz, 2H) , 7.27 –7.20 (m, 2H) , 6.64 (d, J ＝ 8.4 Hz, 2H) , 4.22 (t, J ＝ 5.1 Hz, 2H) , 3.91 (t, J ＝ 6.2 Hz, 4H) , 3.76 –3.65 (m, 8H) , 3.59 –3.55 (m, 4H) , 3.40 (s, 3H) , 3.33 (t, J ＝ 6.8 Hz, 4H) , 1.86 –1.73 (m, 4H) , 1.68 –1.60 (m, 4H) , 1.49 –1.41 (m, 3H) , 1.41 –1.30 (m, 8H) , 1.14 (d, J ＝ 7.1 Hz, 18H) . ¹³C NMR (126 MHz, CDCl₃) δ 163.63, 157.17, 148.10, 133.74, 132.47, 131.61, 128.65, 123.99, 119.99, 112.47, 108.72, 105.72, 71.97, 70.64, 70.06, 68.52, 59.07, 33.83, 32.69, 28.94, 27.68, 25.19, 18.86, 17.99, 12.12.

HRMS (ESI) calcd for C₄₄H₆₉O₆Br₈₁BrSSi, ( [M+H⁺] ) 913.29249, Found 913.29279.

(2) Synthesis of 2- (2', 6'-bis ( (6-bromohexyl) oxy) - [1, 1'-biphenyl] -4-yl) -4- (2- (2- (2-methoxyethoxy) ethoxy) ethoxy) thiophene (compound 17) :

To a solution of compound 16 (912 mg, 1 mmol) in 4 mL THF at -78 ℃ under protection gas atmosphere, tetrabutylammonium fluoride (1.0 M in THF, 4 mL, 4 mmol) was added. After the mixture was stirred at this temperature for another 1.0 h, the reaction was slowly warmed to room temperature and stirred for 3 h. After that, the mixture was poured into water and extracted twice with ethyl acetate, the combined organic phase was dried with MgSO₄ and evaporated in vacuo. The crude material was purified via flash column chromatography, eluting with hexanes to ethyl acetate. The desired product was isolated as a yellow tinted oil (695 mg, 92 ％) .

¹H NMR (500 MHz, CDCl₃) δ 7.70 –7.51 (m, 2H) , 7.42 –7.32 (m, 2H) , 7.27 –7.22 (m, 1H) , 7.09 (d, J ＝ 1.6 Hz, 1H) , 6.65 (d, J ＝ 8.4 Hz, 2H) , 6.24 (d, J ＝ 1.6 Hz, 1H) , 4.21 –4.16 (m, 2H) , 3.95 –3.90 (m, 4H) , 3.80 –3.76 (m, 2H) , 3.75 –3.68 (m, 4H) , 3.61 –3.56 (m, 2H) , 3.41 (s, 3H) , 3.35 (t, J ＝ 6.8 Hz, 4H) , 1.83 –1.76 (m, 4H) , 1.69 –1.61 (m, 4H) , 1.41 –1.30 (m, 8H) . ¹³C NMR (126 MHz, CDCl₃) δ 157.32, 157.15, 143.05, 133.94, 132.29, 131.02, 128.70, 124.09, 119.85, 115.32, 105.66, 96.84, 71.96, 70.84, 70.84, 70.66, 69.72, 69.16, 68.51, 59.09, 33.89, 32.66, 28.90, 27.67, 25.17, 18.86, 12.12.

HRMS (ESI) calcd for C₃₅H₄₉O₆Br₈₁BrS, ( [M+H⁺] ) 757.15906, Found 757.15814

(3) Synthesis of 4, 8- (di-2- (2', 6'-bis ( (6-bromohexyl) oxy) - [1, 1'-biphenyl] -4-yl) -4- (2- (2- (2-methoxy ethoxy) ethoxy) ethoxy) thiophene) -1H, 5H-benzo [1, 2-c: 4, 5-c'] bis ( [1, 2, 5] thiadiazole) (compound 18) :

To a solution of compound 17 (1.51 g, 2 mmol) in 15 mL THF at -78 ℃ under protection gas atmosphere, n-BuLi (1.6 M in hexane, 1.5 mL, 2.4 mmol) was added dropwise. After the mixture was stirred at this temperature for another 2.0 h, trinbutyltinchloride (0.812 mg, 2.5 mmol) was added to the solution. Then the reaction was slowly warmed to room temperature and stirred for 8 h. After that, the mixture was poured into water and extracted twice with ethyl acetate, the combined organic phase was dried with MgSO₄ and evaporated in vacuo without further purification.

To a solution of the crude product above (2 mmol) and 4, 8-dibromo-1H, 5H-benzo [1, 2-c: 4, 5-c'] bis ( [1, 2, 5] thiadiazole) (compound a, 234 mg, 0.67 mmol) in 15 mL toluene under protection gas atmosphere, Pd (PPh₃) ₂Cl₂ (150 mg) was added. The mixture was stirred at 110 ℃ for 12 h. After cooling to room temperature, the mixture was poured into water and extracted twice with ethyl acetate, dried with MgSO₄ and evaporated in vacuo. The crude product was subjected to column chromatography on silica gel with PE/EA 3: 1 to afford compound 18 as a dark green solid (433 mg, 38 ％) .

¹H NMR (500 MHz, CDCl₃) δ 7.74 (d, J ＝ 7.7 Hz, 4H) , 7.53 –7.39 (m, 6H) , 7.32 –7.17 (m, 2H) , 6.67 (d, J ＝ 8.3 Hz, 4H) , 4.51 –4.42 (m, 4H) , 3.95 (t, J ＝ 5.7 Hz, 8H) , 3.74 (t, J ＝ 4.8 Hz, 4H) , 3.67 –3.47 (m, 16H) , 3.45 –3.27 (m, 16H) , 1.78 –1.63 (m, 16H) , 1.48 –1.33 (m, 16H) .

¹³C NMR (125 MHz, CDCl₃) δ 159.98, 152.93, 152.27, 150.90, 144.66, 134.00, 133.34, 132.53, 130.26, 127.74, 125.41, 124.74, 119.65, 105.93, 103.87, 73.68, 70.37 (d, J ＝ 1.6 Hz) , 69.96, 69.54, 69.25, 57.85, 33.30, 31.58, 30.68, 28.80, 26.58. HRMS (ESI) calcd for C₇₆H₉₅Br₄N₄O₁₂S₄ ( [M+H⁺] ) 1704.4600, Found 1704.4611.

(4) Synthesis of IRTBBN-PEG1000:

Compound 18 (100 mg, 0.058 mmol) was dissolved in 10 mL DMF and heated for 3 h at 70 ℃. A large amount of water was added to dissolve all solids. Then it was extracted twice with ethyl acetate, the combined organic phase was dried with MgSO₄ and evaporated in vacuo. The crude product was subjected to flash column chromatography on silica gel to afford dark green solid 97 mg.

The dark green solid was dissolved in 5 mL THF and CuTc 10 mg, w-alkynyl-PEG-hydroxyl (M_w ＝1000, 117 mg) , and TBTA (5 mg) was added. The system was stirred at RT for 1.0 h. The reaction mixture was filtered with diatomite, and the solution was evaporated in vacuo. When all the organic solvent was removed, the crude product was purified by thin layer chromatography eluting with DCM/MeOH 10: 1. IRTBBN-PEG1000 (160 mg) was afforded as a green oil.

Optical parameters of IRTBBN-PEG1000 in H₂O: absorption peak λ＝ 730 nm, emission peak λ_em ＝ 1047 nm, absorption coefficient K＝ 1.19 L/g. cm in 808 nm.

Example 6: Synthesis of IRTFN-PEG600

(1) Synthesis of (5- (9, 9-bis (6-bromohexyl) -9H-fluoren-2-yl) -3- (2- (2- (2-methoxyethoxy) ethoxy) ethoxy) thiophen-2-yl) triisopropylsilane (compound 19) :

To a solution of compound 2-bromo-9, 9-bis (6-bromohexyl) -9H-fluorene (1.14 g, 2.0 mmol) , compound 12 (1.66 g, 2.4 mmol) in 10 mL toluene under protection gas atmosphere, Pd (PPh₃) ₄ (142 mg, 0.122 mol) was added. The mixture was stirred at 110 ℃ for 24 h. After cooling to room temperature, the mixture was poured into water and extracted twice with ethyl acetate, dried with MgSO₄ and evaporated in vacuo. The crude product was subjected to column chromatography on silica gel with PE/EA 2: 1 to afford compound 19 as a light yellow oil (856 mg, 48 ％) .

¹H NMR (500 MHz, CDCl₃) δ 7.70 (dd, J ＝ 10.3, 7.4 Hz, 2H) , 7.62 (dd, J ＝ 7.9, 1.6 Hz, 1H) , 7.54 (d, J ＝ 1.3 Hz, 1H) , 7.35 (tt, J ＝ 6.6, 3.6 Hz, 3H) , 7.27 (s, 1H) , 4.25 (t, J ＝ 5.3 Hz, 2H) , 3.85 (t, J ＝ 5.2 Hz, 2H) , 3.77 –3.65 (m, 6H) , 3.59 (dd, J ＝ 5.6, 3.7 Hz, 2H) , 3.41 (s, 3H) , 3.29 (t, J ＝6.8 Hz, 4H) , 2.06 –2.00 (m, 4H) , 1.71 –1.63 (m, 4H) , 1.47 (dt, J ＝ 14.8, 7.5 Hz, 3H) , 1.25 –1.05 (m, 26H) , 0.61 –0.68 (m, 4H) . ¹³C NMR (126 MHz, CDCl₃) δ 163.66, 151.09, 150.48, 148.34, 140.79, 140.70, 133.38, 127.23, 126.99, 124.29, 122.77, 119.11, 112.59, 109.05, 71.97, 70.77, 70.64, 70.07, 70.01, 59.09, 55.10, 40.30, 34.02, 32.65, 29.04, 27.78, 23.50, 18.87, 12.14.

HRMS (ESI) calcd for C₄₅H₆₉Br₂O₄SSi, ( [M+H⁺] ) 893.2987, Found 893.3026.

(2) Synthesis of 2- (9, 9-bis (6-bromohexyl) -9H-fluoren-2-yl) -4- (2- (2- (2-methoxyethoxy) ethoxy) ethoxy) thiophene (compound 20) :

To a solution of compound 19 (893 mg, 1 mmol) in 4 mL THF at -78 ℃ under protection gas atmosphere, tetrabutylammonium fluoride (1.0 M in THF, 4 mL, 4 mmol) was added. After the mixture was stirred at this temperature for another 1.0 h, the reaction was slowly warmed to room temperature and stirred for 3 h. After that, the mixture was poured into water and extracted twice with ethyl acetate, the combined organic phase was dried with MgSO₄ and evaporated in vacuo. The crude material was purified via flash column chromatography, eluting with hexanes to ethyl acetate. The desired product was isolated as a yellow tinted oil (691 mg, 94 ％) .

¹H NMR (500 MHz, CDCl₃) δ 7.70 (t, J ＝ 7.4 Hz, 2H) , 7.56 (d, J ＝ 8.0 Hz, 1H) , 7.51 (s, 1H) , 7.35 (d, J ＝ 7.1 Hz, 3H) , 7.11 (s, 1H) , 6.26 (d, J ＝ 1.3 Hz, 1H) , 4.24 –4.17 (m, 2H) , 3.93 –3.86 (m, 2H) , 3.78 (dd, J ＝ 5.8, 3.5 Hz, 2H) , 3.71 (ddd, J ＝ 9.3, 5.0, 2.9 Hz, 4H) , 3.59 (dd, J ＝ 5.6, 3.7 Hz, 2H) , 3.41 (s, 3H) , 3.29 (t, J ＝ 6.8 Hz, 4H) , 2.00 (t, J ＝ 8.2 Hz, 4H) , 1.73 –1.61 (m, 4H) , 1.23 –1.05 (m, 8H) , 0.76 –0.57 (m, 4H) . ¹³C NMR (126 MHz, CDCl₃) δ 157.54, 151.08, 150.54, 143.31, 140.96, 140.58, 133.17, 127.29, 127.00, 124.41, 122.79, 120.10, 119.83 , 119.51, 115.53, 96.88, 71.96, 70.84, 70.70, 70.61, 69.71, 69.42, 59.09, 55.03, 40.23, 33.99, 32.64, 29.05, 27.76, 23.53.

HRMS (ESI) calcd for C₃₆H₄₈Br₂O₄S, ( [M+H⁺] ) 737.1653, Found 737.1692.

(3) Synthesis of 4, 8- (di-5- (9, 9-bis (6-bromohexyl) -9H-fluoren-2-yl) -3- (2- (2- (2-methoxyethoxy) ethoxy) ethoxy) thiophene) -1H, 5H-benzo [1, 2-c: 4, 5-c'] bis ( [1, 2, 5] thiadiazole) (compound 21) :

To a solution of compound 20 (1.47 g, 2 mmol) in 15 mL THF at -78 ℃ under protection gas atmosphere, n-BuLi (1.6 M in hexane, 1.5 mL, 2.4 mmol) was added dropwise. After the mixture was stirred at this temperature for another 2.0 h, trinbutyltinchloride (0.812 mg, 2.5 mmol) was added to the solution. Then the reaction was slowly warmed to room temperature and stirred for 8 h. After that, the mixture was poured into water and extracted twice with ethyl acetate, the combined organic phase was dried with MgSO₄ and evaporated in vacuo without further purification.

To a solution of the crude product above (2 mmol) and 4, 8-dibromo-1H, 5H-benzo [1, 2-c: 4, 5-c'] bis ( [1, 2, 5] thiadiazole) (compound a, 234 mg, 0.67 mmol) in 15 mL toluene under protection gas atmosphere then Pd (PPh₃) ₂Cl₂ 150 mg was added. The mixture was stirred at 110 ℃ for 12 h. After cooling to room temperature, the mixture was poured in to water and extracted twice with ethyl acetate, dried with MgSO₄ and evaporated in vacuo. The crude product was subjected to column chromatography on silica gel with DCM/EA 5: 1 to afford compound 21 as a dark green solid (565 mg, 52 ％) .

¹H NMR (500 MHz, CDCl₃) δ 7.75 (m, 6H) , 7.67 (s, 2H) , 7.49 (s, 2H) , 7.38 (m, 6H) , 4.49 (t, J ＝ 5.1 Hz, 4H) , 3.76 (t, J ＝ 5.1 Hz, 4H) , 3.66 –3.56 (m, 12H) , 3.54 (dd, J ＝ 5.8, 3.5 Hz, 4H) , 3.38 (s, 6H) , 3.31 (t, J ＝ 6.8 Hz, 8H) , 2.07 (t, J ＝ 8.1 Hz, 8H) , 1.69 (dd, J ＝ 14.6, 7.0 Hz, 8H) , 1.28 –1.20 (m, 8H) , 1.16 –1.09 (m, 8H) , 0.75 –0.63 (m, 8H) . ¹³C NMR (126 MHz, CDCl₃) δ156.62, 152.79, 151.23, 150.60, 146.70, 141.55, 140.56, 133.06, 127.49, 127.08, 124.60, 122.82, 120.24, 119.94, 119.66, 113.98, 113.77, 71.91, 71.15, 70.72, 70.60, 70.55, 70.06, 59.05, 55.18, 40.33, 34.03, 32.65, 29.06, 27.81, 23.56.

HRMS (ESI) calcd for C₇₈H₉₄O₈N₄Br₄NaS₄ ⁺, ( [M+Na⁺] ) 1681.2537, Found 1681.2580.

(4) Synthesis of IRTFN-PEG600:

Compound 21 (100 mg, 0.06 mmol) and sodium azide (47 mg, 0.72 mmol) was dissolved in 10 mL DMF and heated for 3 h at 70 ℃. Then a large amount of water was added to dissolve all solids. Then it was extracted twice with ethyl acetate, the combined organic phase was dried with MgSO₄ and evaporated in vacuo. The crude product was subjected to flash column chromatography on silica gel to afford a dark green solid (96 mg) . The dark green solid was dissolved in 5 mL THF and CuTc 10 mg, w-alkynyl-PEG-hydroxyl (M_w ＝ 600, 72 mg) , and TBTA (5 mg) was added. The system was stirred at RT for 0.5 h. Then the reaction mixture was filtered with diatomite, and the solution was evaporated in vacuo. When all the organic solvent was removed, the crude product was purified by thin layer chromatography, eluting with DCM/MeOH 10: 1. IRTFN-PEG600 (130 mg) was afforded as a green oil.

Optical parameters of IRTFN-PEG600 in H₂O: absorption peak λ ＝ 750 nm, emission peak λ_em ＝ 1047 nm, absorption coefficient K＝ 2.85 L/g. cm at 808 nm, quantum yield is 2.49％ (with 808 nm excitation)

Example 7: Synthesis of IRDTNS

(1) Synthesis of 4, 4-bis (6-bromohexyl) -4H-cyclopenta [2, 1-b: 3, 4-b'] dithiophene (compound 23)

4H-cyclopenta [2, 1-b: 3, 4-b'] dithiophene (2.0 g, 11.23 mmol) and 1, 6-Dibromhexane (13.27 g, 55 mmol) were dissolved in 30 mL DMSO at 10 ℃. Then KOH (1.8 g, 33 mmol) was added in several portions. After stirring for 24 h, the mixture was poured into water and extracted twice with ethyl acetate, the combined organic phase was dried with MgSO₄ and the crude product was subjected to column chromatography on silica gel to afford compound 23 as a light yellow oil (4.8 g, 86 ％) .

¹H NMR (400 MHz, CDCl₃) δ 7.53 (d, 2H) , δ 6.90 (d, 2H) , 3.52 –3.41 (t, 4H) , 1.80 –1.68 (m, 4H) , 1.43 (d, J ＝ 4.0 Hz, 4H) , 1.33 –1.23 (m, 4H) , 1.15 (m, 4H) , 0.89 –0.85 (m, 4H) .

(2) Synthesis of compound (24) .

To a solution of compound 23 (2.0 g, 4.0 mmol) in 25 mL THF at -78 ℃ under Ar atmosphere, n-BuLi (1.6 M in hexane, 2.5 mL, 4.0 mmol) was added dropwise. After the mixture was stirred at this temperature for another 1.5 h, trinbutyltinchloride (1.5 g, 4.4 mmol) was added to the solution. Then the reaction was slowly warmed to room temperature and stirred for 8 h. After that the mixture was poured into water and extracted twice with ethyl acetate, the combined organic phase was dried with MgSO₄ and evaporated in vacuo without further purification.

To a solution of the above crude product (320 mg, 0.4 mmol) and compounds of a (50 mg, 0.13 mmol) in 5 mL toluene under Ar atmosphere, Pd (PPh₃) ₄ (10 mg) was added. The mixture was stirred at 110 ℃ for 12 h. After cooling to room temperature, the mixture was poured into water and extracted twice with ethyl acetate, dried with MgSO₄ and evaporated in vacuo. The crude product was subjected to column chromatography on silica gel and yielded compound 24 as a dark brown solid (88 mg, 58 ％) .

¹H NMR (400 MHz, CDCl₃) δ 7.35 (d, J ＝ 4.8 Hz, 2H) , 7.04 (d, J ＝ 4.8 Hz, 2H) , 3.33 (t, J ＝6.8 Hz, 9H) , 2.05 (td, J ＝ 10.4, 5.0 Hz, 9H) , 1.83 –1.69 (m, 10H) , 1.40 (d, J ＝ 7.4 Hz, 7H) , 1.17 –1.04 (m, 8H) , 0.95 (t, J ＝ 7.3 Hz, 10H) .

(3) Synthesis of compound IRDTNS

Compound 24 (50 mg, 0.042 mmol) was dissolved in 10 mL THF and dimethylamine (2.0 M in THF, 5 mL) , then stirred for 6 h at 50 ℃. After that, the solution was evaporated in vacuo. The dark brown solid and 1, 2-oxathiolane 2, 2-dioxide 122 mg (1 mmol) were dissolved in 5 mL THF, then the solution was stirred overnight. The solution was filtered, washed with acetone and ethyl ether for several times. IRDTNS (40 mg) was afforded as a dark brown solid.

HRMS (ESI) calcd for C₆₈H₁₀₃N₈O₁₂S₁₀, ( [M+H⁺] ) 1543.4903, Found 1543.4908.

Optical parameters of IRDTNS in H₂O: absorption peak λ ＝ 930 nm, emission peak λ_em ＝1127 nm, absorption coefficient K＝ 10.2 L/g. cm at 980 nm.

Example 8: synthesis of IRDT-PEG1000 and IRDTN-PEG1000

Compound 24 (50 mg, 0.042 mmol) was dissolved in 10 mL DMF and sodium azide (47 mg, 0.72 mmol) and heated for 3 h at 70 ℃. Then a large amount of water was added to dissolve all the solids. The mixture was extracted twice with ethyl acetate, the combined organic phase was dried with MgSO₄ and evaporated in vacuo. The crude product was subjected to column chromatography on silica gel to afford a dark brown solid 95 mg (quant) .

The dark brown solid was dissolved in 5 mL THF and CuTc (10 mg) , w-alkynyl-PEG-hydroxyl (Mw ＝ 1000, 170 mg) , and TBTA (10 mg) was added. The system was stirred at RT for 0.5 h. Then the reaction was filtered with diatomite, and the solution was evaporated in vacuo. When all the organic solvent was removed, the solid was dissolved in 15 mL water and it was transferred to the dialyzer (MWCO ＝ 3500) . The system was stirred at RT for 72 h with 10 times water exchange. The resulting water solution in the dialyzing membrane was lyophilized. IRDT-PEG1000 (250 mg) was afforded as a green oil. 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.

Optical parameters of IRDTN-PEG1000 in H₂O: absorption peak λ ＝ 930 nm, emission peak λ_em ＝ 1127 nm, absorption coefficient K＝ 1.4 L/g. cm at 808 nm.

Example 9: synthesis of IRTSBN-PEG1000

(1) Synthesis of (5- (5- (2, 6-bis ( (6-bromohexyl) oxy) phenyl) selenophen-2-yl) -3- (2- (2- (2-methoxyethoxy) ethoxy) et hoxy) thiophen-2-yl) triisopropylsilane (compound 26) :

To a solution of compound 2- (2, 6-bis ( (6-bromohexyl) oxy) phenyl) -5-bromoselenophene (1.28 g, 2.0 mmol) and compound 12 (1.66 g, 2.4 mmol) in 10 mL toluene under protection gas atmosphere, Pd (PPh₃) ₄ (142 mg, 0.122 mol) was added. The mixture was stirred at 110 ℃ for 18 h. After cooling to room temperature, the mixture was poured into water and extracted twice with ethyl acetate, and the organic phase was dried with MgSO₄ and evaporated in vacuo. The crude product was subjected to column chromatography on silica gel to afford compound 26 as a light yellow oil (1.18 g, 54 ％) .

¹H NMR (400 MHz, CDCl₃) δ 7.44 (d, J ＝ 3.9 Hz, 1H) , 7.19 (dd, J ＝ 6.1, 4.5 Hz, 2H) , 7.05 (s, 1H) , 6.64 (d, J ＝ 8.4 Hz, 2H) , 4.04 (t, J ＝ 6.3 Hz, 4H) , 3.82 (t, J ＝ 5.1 Hz, 2H) , 3.76 –3.63 (m, 8H) , 3.58 (dd, J ＝ 5.7, 3.6 Hz, 2H) , 3.46 –3.34 (m, 7H) , 1.92 –1.80 (m, 8H) , 1.68 (s, 3H) , 1.55 –1.47 (m, 8H) , 1.14 (d, J ＝ 7.4 Hz, 18H) .

(2) Synthesis of 2- (5- (2, 6-bis ( (6-bromohexyl) oxy) phenyl) selenophen-2-yl) -4- (2- (2- (2-methoxyethoxy) ethoxy) eth oxy) thiophene (compound 27) :

To a solution of compound 26 (1.0 g, 1 mmol) in 4 mL THF at -78 ℃ under protection gas atmosphere, tetrabutylammonium fluoride (1.0 M in THF, 8 mL, 8 mmol) was added. After the mixture was stirred at this temperature for another 1.0 h, the reaction was slowly warmed to room temperature and stirred for 4 h. After that the mixture was poured into water and extracted twice with ethyl acetate, the combined organic phase was dried with MgSO₄ and evaporated in vacuo. The crude material was purified via flash column chromatography, eluting with hexanes to ethyl acetate. The desired product was isolated as a yellow tinted oil (722 mg, 95 ％) .

¹H NMR (400 MHz, CDCl₃) δ 7.43 (d, J ＝ 3.9 Hz, 1H) , 7.30 (d, J ＝ 3.9 Hz, 1H) , 7.19 (t, J ＝8.3 Hz, 1H) , 7.05 (d, J ＝ 5.5 Hz, 1H) , 6.91 (d, J ＝ 5.5 Hz, 1H) , 6.63 (d, J ＝ 8.4 Hz, 2H) , 4.02 (t, J ＝ 6.3 Hz, 4H) , 3.93 –3.84 (m, 2H) , 3.75 (dd, J ＝ 5.8, 3.5 Hz, 2H) , 3.71 –3.60 (m, 4H) , 3.54 (dd, J ＝ 5.7, 3.6 Hz, 2H) , 3.44 –3.34 (m, 7H) , 1.90 –1.78 (m, 8H) , 1.56 –1.44 (m, 8H) .

(3) Synthesis of compound 28:

To a solution of compound 27 (400 mg, 0.5 mmol) in 15 mL THF at -78 ℃ under protection gas atmosphere, n-BuLi (1.6 M in hexane, 1 mL, 1.6 mmol) was added dropwise. After the mixture was stirred at this temperature for another 1.0 h, trinbutyltinchloride (0.812 mg, 2.5 mmol) was added to the solution. Then the reaction was slowly warmed to room temperature and stirred for 8 h. After that, the mixture was poured into water and extracted twice with ethyl acetate, the combined organic phase was dried with MgSO₄ and evaporated in vacuo without further purification.

To a solution of the above crude product (0.25 mmol) and 4, 8-dibromo-1H, 5H-benzo [1, 2-c: 4, 5-c'] bis ( [1, 2, 5] thiadiazole) (compound a, 32 mg, 0.08 mmol) in 5 mL toluene under protection gas atmosphere, Pd (PPh₃) ₂Cl₂ (15 mg) was added. The mixture was stirred at 110 ℃ for 12 h. After cooling to room temperature, the mixture was poured into water and extracted twice with ethyl acetate. The organic phase was dried with MgSO₄ and evaporated in vacuo. The crude product was subjected to column chromatography on silica gel to afford compound 28 as a dark green solid (86 mg, 61 ％) .

(4) Synthesis of IRTTBN-PEG1000:

Compound 28 (100 mg, 0.058 mmol) was dissolved in 10 mL DMF and sodium azide (47 mg, 0.72 mmol) and heated for 3 h at 70 ℃. A large amount of water was added to dissolve all the solids. Then it was extracted twice with ethyl acetate, and the combined organic phase was dried with MgSO₄ and evaporated in vacuo. The crude product was subjected to flash column chromatography on silica gel to afford a dark green solid 97 mg. The dark green solid was dissolved in 5 mL THF and CuTc 10 mg, w-alkynyl-PEG-hydroxyl (M_w ＝ 1000, 52 mg) , and TBTA (5 mg) were added. The system was stirred at RT for 1.0 h. Then the reaction was filtered with diatomite, and the solution was evaporated in vacuo. When all the organic solvent was removed, the solid was dissolved in 15 mL water and it was transferred to the dialyzer (MWCO ＝3500) . The system was stirred at RT for 72 h with 10 times water exchange. The resulting water solution in the dialyzing membrane was lyophilized. IRTSBN-PEG1000 (140 mg) was afforded as a green oil.

Optical parameters of IRTSBN-PEG1000 in H₂O: absorption peak λ ＝ 800 nm, absorption coefficient K＝ 0.85 L/g. cm at 808 nm.

Example 10: synthesis of IRESBN-PEG1000

(1) Synthesis of 5- (5- (2, 6-bis ( (6-bromohexyl) oxy) phenyl) selenophen-2-yl) -2, 3-dihydrothieno [3, 4-b] [1, 4] dioxine (compound 29) ：

To a solution of tributyl (2, 3-dihydrothieno [3, 4-b] [1, 4] dioxin-5-yl) stannane (860 mg, 2.0 mmol) and compound 25 (720 mg, 1.1 mmol) in 10 mL toluene under Ar atmosphere, Pd (PPh₃) ₄ (71 mg, 0.061 mol) was added. The mixture was stirred at 110 ℃ for 24 h. After cooling to room temperature, the mixture was poured into water and extracted twice with ethyl acetate, and the organic phase was dried with MgSO₄ and evaporated in vacuo. The crude product was subjected to column chromatography on silica gel to afford compound 29 as a light yellow oil (468 mg, 60 ％) .

¹H NMR (400 MHz, CDCl₃) : δ 7.45 (d, J ＝ 3.9 Hz, 1H) , 7.24 (d, J ＝ 3.9 Hz, 1H) , 7.17 (t, J ＝ 8.3 Hz, 1H) , 6.61 (d, J ＝ 8.4 Hz, 2H) , 6.21 (s, 1H) , 4.37 –4.32 (m, 2H) , 4.28 –4.23 (m, 2H) , 4.01 (t, J ＝ 6.2 Hz, 4H) , 3.38 (t, J ＝ 6.9 Hz, 4H) , 1.84 (m, 7.2 Hz, 8H) , 1.52 –1.43 (m, 8H) ；

(2) Synthesis of (7- (5- (2, 6-bis ( (6-bromohexyl) oxy) phenyl) selenophen-2-yl) -2, 3-dihydrothieno [3, 4-b] [1, 4] dioxin-5-yl) tributylstannane (compound 30) ：

To a solution of compound 29 (350 mg, 0.53 mmol) in 5 mL THF at -78 ℃ under Ar atmosphere, n-BuLi (1.6 M in hexane, 1 mL, 1.6 mmol) was added dropwise. After the mixture was stirred at this temperature for another 1.5 h, trinbutyltinchloride (330 mg, 1 mmol) was added to the solution. Then the reaction mixture was slowly warmed to room temperature and stirred for 8 h. After that, the mixture was poured into water and extracted twice with ethyl acetate, the combined organic phase was dried with MgSO₄ and evaporated in vacuo without further purification.

(3) Synthesis of compound 31:

To a solution of compound a (72 mg, 0.2 mmol) and compound 30 (500 mg, 0.47 mmol) in 5 mL toluene under Ar atmosphere, Pd (PPh₃) ₂Cl₂ (35 mg, 0.03 mol) was added. The mixture was stirred at 110 ℃ for 48 h. After cooling to room temperature, the mixture was poured into water and extracted twice with ethyl acetate. The combined organic phase was dried with MgSO₄ and evaporated in vacuo. The crude product was subjected to column chromatography on silica gel to afford compound 31 as a dark green solid (93 mg, 31 ％) .

¹H NMR (400 MHz, CDCl₃) δ 7.55 (d, J ＝ 4.0 Hz, 2H) , 7.42 (d, J ＝ 4.0 Hz, 2H) , 7.19 (t, J ＝8.3 Hz, 2H) , 6.63 (d, J ＝ 8.4 Hz, 4H) , 4.51 (m, 4H) , 4.37 (m, 4H) , 4.04 (t, J ＝ 6.3 Hz, 8H) , 3.40 (t, J ＝ 6.8 Hz, 8H) , 1.87 (m, 16H) , 1.55 –1.43 (m, 16H) ；

(4) Synthesis of IRESBN-1700. Compound 31 (50 mg, 0.036 mmol) was dissolved in 5 mL DMF. Then sodium azide (47 mg, 0.72 mmol) was added and the reaction solution was stirred at 60 ℃ for 20 h. After that, a large portion of water was added, and the product was extracted twice with ethyl acetate. The combined organic phase was dried with MgSO₄ and evaporated in vacuo. The crude product was subjected to column chromatography on silica gel to afford azide product as dark green solid 43 mg (quant) .

The azide product (20 mg) was dissolved in 5 mL THF and cuprous iodide (100 mg) , α-alkynyl-ω-hydroxyl-poly (ethylene glycol) (Mw ＝ 1.68k, 120 mg) , and triethylamine 200μL were added. The system was stirred at RT for 48 h. Then the reaction mixture was filtered with Al₂O₃, and the solution was evaporated in vacuo. When all the organic solvent was removed, the solid was dissolved in 15 mL water and it was transferred to the dialyzer (MWCO ＝ 3500) . The system was stirred at RT for 72 h with 10 times water exchange. The resulting water solution in the dialyzing membrane was lyophilized, IRETBN-1700 (100 mg) was afforded as yellow powder.

Example 11: Synthesis of IREDTN-PEG1700

(1) Synthesis of (4, 4-bis (6-bromohexyl) -4H-cyclopenta [2, 1-b: 3, 4-b'] dithiophen-2-yl) triisopropylsilane (compound 32) .

4, 4-bis (6-bromohexyl) -4H-cyclopenta [2, 1-b: 3, 4-b'] dithiophene (compound 23) (3.0 g, 6.0 mmol) was dissolved in 40 mL dry THF at -78 ℃ under Ar atmosphere. n-BuLi (1.6 M in hexane, 4 mL, 6.4 mmol) was added dropwise. After the mixture was stirred at this temperature for another 1.5 h, TIPSCl (1.5 g, 7.0 mmol) was added to the solution. Then the reaction mixture was slowly warmed to room temperature and stirred for 8 h. After that, the mixture was poured into water and extracted twice with ethyl acetate, the combined organic phase was dried with MgSO₄ and evaporated in vacuo without further purification.

¹H NMR (400 MHz, CDCl₃) δ 7.53 (d, 1H) , δ 6.90 (d, 2H) , 3.52 –3.41 (t, 4H) , 1.92 –1.80 (m, 8H) , 1.68 (s, 3H) , 1.55 –1.47 (m, 8H) , 1.14 (d, J ＝ 7.4 Hz, 18H) .

(2) Synthesis of (6-bromo-4, 4-bis (6-bromohexyl) -4H-cyclopenta [2, 1-b: 3, 4-b'] dithiophen-2-yl) triisopropylsilane (compound 33) .

(4, 4-bis (6-bromohexyl) -4H-cyclopenta [2, 1-b: 3, 4-b'] dithiophen-2-yl) triisopropylsilane (compound 32) (2.1 g, 3.18 mmol) was dissolved in 40 mL dry THF at -78 ℃ under Ar atmosphere. Then n-BuLi (1.6 M in hexane, 3 mL, 4.8 mmol) was added dropwise. After the mixture was stirred at this temperature for another 1.5 h, CBr₄ (1.68 g, 4.77 mmol) was added to the solution. Then the reaction mixture was slowly warmed to room temperature and stirred overnight. The mixture was poured into water and extracted twice with ethyl acetate, and the organic phase was dried with MgSO₄ and evaporated in vacuo. The crude product was subjected to column chromatography on silica gel to afford compound 33 as a light red oil (1.88 g, 80 ％) .

¹H NMR (400 MHz, CDCl₃) δ 6.90 (d, 2H) , 3.52 –3.41 (t, 4H) , 1.92 –1.80 (m, 8H) , 1.68 (s, 3H) , 1.55 –1.47 (m, 8H) , 1.14 (d, J ＝ 7.4 Hz, 18H) .

(3) Synthesis of (4, 4-bis (6-bromohexyl) -6- (2, 3-dihydrothieno [3, 4-b] [1, 4] dioxin-5-yl) -4H-cyclopenta [2, 1-b: 3, 4-b'] dithiophen-2-yl) triisopropylsilane (compound 34)

To a solution of tributyl (2, 3-dihydrothieno [3, 4-b] [1, 4] dioxin-5-yl) stannane (860 mg, 2.0 mmol) and (6-bromo-4, 4-bis (6-bromohexyl) -4H-cyclopenta [2, 1-b: 3, 4-b'] dithiophen-2-yl) triisopropylsilane (compound 33) (1.15 g, 1.6 mmol) in 10 mL toluene under Ar atmosphere, Pd (PPh₃) ₄ (71 mg, 0.061 mol) was added. The mixture was stirred at 110 ℃ for 24 h. After cooling to room temperature, the mixture was poured into water and extracted twice with ethyl acetate, and the organic phase was dried with MgSO₄ and evaporated in vacuo. The crude product was subjected to column chromatography on silica gel to afford compound 34 as a red oil (868 mg, 68 ％) .

¹H NMR (400 MHz, CDCl₃) δ 6.90 (d, 2H) , 4.28 (s, 2H) , 4.25 (s, 2H) , 3.52 –3.41 (t, 4H) , 1.92 –1.80 (m, 8H) , 1.68 (s, 3H) , 1.55 –1.47 (m, 8H) , 1.14 (d, J ＝ 7.4 Hz, 18H) .

(4) Synthesis of compound 35.

To a solution of compound 34 (400 mg, 0.5 mmol) in 15 mL THF at -78 ℃ under protection gas atmosphere, n-BuLi (1.6 M in hexane, 1 mL, 1.6 mmol) was added dropwise. After the mixture was stirred at this temperature for another 1.0 h, trinbutyltinchloride (0.812 mg, 2.5 mmol) was added to the solution. Then the reaction was slowly warmed to room temperature and stirred for 8 h. After that, the mixture was poured into water and extracted twice with ethyl acetate, the combined organic phase was dried with MgSO₄ and evaporated in vacuo without further purification.

To a solution of the above crude product (0.25 mmol) and 4, 8-dibromo-1H, 5H-benzo [1, 2-c: 4, 5-c'] bis ( [1, 2, 5] thiadiazole) (compound a) (32 mg, 0.08 mmol) in 5 mL toluene under protection gas atmosphere, Pd (PPh₃) ₂Cl₂ (15 mg) was added. The mixture was stirred at 110 ℃ for 12 h. After cooling to room temperature, the mixture was poured into water and extracted twice with ethyl acetate, and the organic phase was dried with MgSO₄ and evaporated in vacuo. The crude product was subjected to column chromatography on silica gel to afford compound 35 as a brown solid (75 mg, 61 ％) .

(5) Synthesis of IREDTN-PEG1700.

Compound 35 (50 mg, 0.028 mmol) was dissolved in 5 mL DMF. Then sodium azide (47 mg, 0.72 mmol) was added and the reaction solution was stirred at 60 ℃ for 20 h. After that, a large portion of water was added, and the product was extracted twice with ethyl acetate. The combined organic phase was dried with MgSO₄ and evaporated in vacuo. The crude product was subjected to column chromatography on silica gel to afford the azide product as dark green solid 43 mg (quant) .

The azide product 20 mg was dissolved in 5 mL THF and cuprous iodide 100 mg, α-alkynyl-ω-hydroxyl-poly (ethylene glycol) (Mw ＝ 1.7k, 76 mg) , and triethylamine 200 μL were added. The system was stirred at RT for 48 h. Then the reaction mixture was filtered with Al₂O₃, and the solution was evaporated in vacuo. When all the organic solvent was removed, the solid was dissolved in 15 mL water and it was transferred to the dialyzer (MWCO ＝ 3500) . The system was stirred at RT for 72 h with 10 times water exchange. The resulting water solution in the dialyzing membrane was lyophilized. IRETBN-1700 (100 mg) was afforded as brown powder.

Optical parameters of IRDTNS in H₂O: absorption peak λ＝ 982 nm, absorption coefficient K＝1.3 L/g. cm at 980 nm.

Example 12: Synthesis of IRETBNS

Synthesis of IRETBNS: Compound 4 (50 mg, 0.036 mmol) was dissolved in 5 mL DMF. Then sodium azide (47 mg, 0.72 mmol) was added and the reaction solution was stirred at 60 C for 20 h. After that, a large portion of water was added, and the product was extracted twice with ethyl acetate. The combined organic phase was dried with MgSO₄ and evaporated in vacuo. The crude product was subjected to column chromatography on silica gel to afford the azide product as a dark green solid 43 mg (quant) .

The azide product 20 mg was dissolved in 5 mL DMSO and cuprous iodide 100 mg, sodium prop-2-yne-1-sulfonate 15 mg, and triethylamine 200μL were added. The system was stirred at RT for 48 h. Then the reaction mixture was filtered with diatomite, and the solution was dissolved in 15 mL water and it was transferred to the dialyzer (MWCO ＝ 3500) . The system was stirred at RT for 72 h with 10 times water exchange. The resulting water solution in the dialyzing membrane was lyophilized, IRETBNS (23 mg) was afforded as a green powder.

¹H NMR (500 MHz, D₂O) δ 8.53 (s, 4H) , 7.54 –7.46 (m, 4H) , 7.34 (d, J ＝ 7.5 Hz, 2H) , 6.80 (d, J ＝ 7.5 Hz, 4H) , 4.60 –4.42 (m, 8H) , 4.28 (s, 8H) , 4.22 –4.04 (m, 17H) , 2.12 –1.86 (m, 8H) , 1.90 –1.76 (m, 8H) , 1.54 –1.37 (m, 8H) , 1.39 –1.27 (m, 8H) .

¹³C NMR (125 MHz, D₂O) δ 159.25, 153.19, 151.34, 150.73, 147.66, 142.77, 140.11, 137.54, 129.38, 126.07, 125.39, 123.90, 123.08, 120.49, 118.16, 109.81, 101.64, 70.37, 66.98, 51.79, 42.08, 28.80, 27.64, 27.05, 26.58.

HRMS (ESI) calcd for C₇₄H₇₈O₂₀N₁₆Na₂S₁₀ ²⁺, ( [M+2H⁺] ) 938.12960, Found 938.1335.

Optical parameters of IRETBNS in H₂O: absorption peak λ ＝ 913 nm, emission peak λ_em ＝1149 nm, absorption coefficient K＝ 3.23 L/g. cm at 808 nm, quantum yield is 0.14％ (with 808 nm excitation) .

Example 13: Synthesis of IRTTEBN-PEG600

(1) Synthesis of 3, 4-bis (2- (2- (2-methoxyethoxy) ethoxy) ethoxy) thiophene (compound 36) :

A dry 250 mL two-neck round bottom flask with condenser and magnetic stir bar was charged with potassium tertbutylate (10.5 g, 94 mmol) and cuprous iodide (2.34 g, 12 mmol) . Next, 10 mL pyridine and 100 mL toluene mixture solution was added and the mixture was stirred until dissolved, and 15 mL triethylene glycol monomethyl ether was then added. The reaction mixture was allowed to stir at room temperature for 30 min. 3, 4-dibromothiophene (7.4 g, 31 mmol) was then added in one portion and the reaction mixture was heated to 115 ℃ for 24 h. After cooling to room temperature, the reaction mixture was centrifuged and concentrated under vacuum. 100 mL dichloromethane was added and washed with 5 M HCl (aq) . The aqueous layers were extracted with DCM, and the combined organic portions were dried with MgSO₄ and concentrated under reduced pressure. The crude material was purified via column chromatography, eluting with 1: 1 ethyl ether in hexanes (v/v) . The desired product was isolated as a yellow tinted oil (8.04 g, 61％yield) .

¹H NMR (500 MHz, CDCl₃) δ 6.25 (s, 2H) , 4.19 –4.10 (m, 4H) , 3.97 –3.80 (m, 4H) , 3.78 –3.60 (m, 12H) , 3.61 –3.49 (m, 4H) , 3.39 (s, 6H) .

¹³C NMR (126 MHz, CDCl₃) δ 147.10, 97.87, 71.94, 70.80, 70.66, 70.56, 58.36.

HRMS (ESI) calcd for C₁₈H₃₂O₈S⁺, ( [M+H⁺] ) 409.1854, Found 409.1817.

(2) Synthesis of (3, 4-bis (2- (2- (2-methoxyethoxy) ethoxy) ethoxy) thiophen-2-yl) tributylstannane (compound 37) :

To a solution of Compound 36 (408 mg, 1.0 mmol) in 5 mL THF at -78 ℃ under protection gas atmosphere, n-BuLi (1.6 M in hexane, 0.625 mL, 1.2 mmol) was added dropwise. After the mixture was stirred at this temperature for another 2.0 h, trinbutyltinchloride (390 mg, 1.2 mmol) was added to the solution. Then the reaction was slowly warmed to room temperature and stirred for 8 h. After that, the mixture was poured into water and extracted twice with ethyl acetate, the combined organic phase was dried with MgSO₄ and evaporated in vacuo without further purification.

(3) Synthesis of 5'- (2, 6-bis ( (6-bromohexyl) oxy) phenyl) -3, 4-bis (2- (2- (2-methoxyethoxy) ethoxy) ethoxy) -2, 2'-bithi ophene (compound 38) :

To a solution of compound 2- (2, 6-bis ( (6-bromohexyl) oxy) phenyl) -5-bromothiophene (1.19 g, 2.0 mmol) , compound 37 (1.53 g, 2.2 mmol) in 10 mL toluene under protection gas atmosphere. Pd (PPh₃) ₄ (142 mg, 0.122 mol) was added. The mixture was stirred at 110 ℃ for 18 h. After cooling to room temperature, the mixture was poured into water and extracted twice with ethyl acetate, and the organic phase was dried with MgSO₄ and evaporated in vacuo. The crude product was subjected to column chromatography on silica gel with PE/EA 2: 1 to afford compound 38 as a light yellow oil (0.89 g, 48 ％) .

¹H NMR (500 MHz, CDCl₃ ) δ 7.61 –7.38 (m, 2H) , 7.34 (d, J ＝ 15.0 Hz, 1H) , 6.80 (d, J ＝15.0 Hz, 2H) , 6.61 (s, 1H) , 4.45 –4.22 (m, 4H) , 4.22 –4.05 (m, 4H) , 3.87 –3.68 (m, 4H) , 3.63 –3.45 (m, 20H) , 3.40 (s, 6H) , 2.01 –1.70 (m, 8H) , 1.62 –1.19 (m, 8H) .

¹³C NMR (125 MHz, CDCl₃ ) δ 159.25, 147.48 (d, J ＝ 18.6 Hz) , 140.11, 137.21, 133.39, 129.38, 124.96, 123.08, 118.16, 109.81, 95.52, 73.68, 70.37 (d, J ＝ 1.6 Hz) , 69.96, 69.54, 68.97, 67.92, 57.85, 33.30, 31.58, 30.68, 28.80, 26.58.

HRMS (ESI) calcd for C₄₀H₆₁O₁₀Br₂S₂, ( [M+H⁺] ) 923.20598, Found 923.20674.

(4) Synthesis of 4, 8- (5'- (2, 6-bis ( (6-bromohexyl) oxy) phenyl) -3, 4-bis (2- (2- (2-methoxyethoxy) ethoxy) ethoxy) -2, 2'-bithiophene) -1H, 5H-benzo [1, 2-c: 4, 5-c'] bis ( [1, 2, 5] thiadiazole) (compound 39) :

To a solution of compound 38 (1.844 g, 2 mmol) in 15 mL THF at -78 ℃ under protection gas atmosphere, n-BuLi (1.6 M in hexane, 1.5 mL, 2.4 mmol) was added dropwise. After the mixture was stirred at this temperature for another 2.0 h, trinbutyltinchloride (0.812 mg, 2.5 mmol) was added to the solution. Then the reaction was slowly warmed to room temperature and stirred for 8 h. After that the mixture was poured into water and extracted twice with ethyl acetate, the combined organic phase was dried with MgSO₄ and evaporated in vacuo without further purification.

To a solution of the above crude product (2 mmol) and 4, 8-dibromo-1H, 5H-benzo [1, 2-c: 4, 5-c'] bis ( [1, 2, 5] thiadiazole) (compound a) (234 mg, 0.67 mmol) in 15 mL toluene under protection gas atmosphere, Pd (PPh₃) ₂Cl₂ (150 mg) was added. The mixture was stirred at 110 ℃ for 12 h. After cooling to room temperature, the mixture was poured into water and extracted twice with ethyl acetate, dried with MgSO₄ and evaporated in vacuo. The crude product was subjected to column chromatography on silica gel with PE/EA 2: 1 to afford compound 39 as a dark green solid (772 mg, 38 ％) .

¹H NMR (500 MHz, CDCl₃ ) δ 7.55 –7.40 (m, 4H) , 7.34 (d, J ＝ 7.5 Hz, 2H) , 6.80 (d, J ＝7.5 Hz, 4H) , 4.31 (t, J ＝ 7.0 Hz, 8H) , 4.11 (t, J ＝ 7.4 Hz, 8H) , 3.77 (t, J ＝ 7.1 Hz, 8H) , 3.64 –3.48 (m, 40H) , 3.40 (s, 12H) , 1.97 –1.62 (m, 16H) , 1.58 –1.37 (m, 8H) , 1.38 –1.26 (m, 8H) .

¹³C NMR (125 MHz, CDCl₃ ) δ 159.25, 157.63, 153.23 (d, J ＝ 10.1 Hz) , 151.34, 140.11, 137.54, 129.38, 127.88, 126.07, 123.90, 123.08, 118.16, 109.81, 104.50, 73.68, 70.37 (d, J ＝ 1.6 Hz) , 69.96, 69.54, 68.97, 57.85, 33.30, 31.58, 30.68, 28.80, 26.58.

HRMS (ESI) calcd for C₈₆H₁₁₉Br₄N₄O₂₀S₆, ( [M+H⁺] ) 2039.3356, Found 2039.3389.

(5) Synthesis of IRTTEBN-PEG600:

Compound 39 (118 mg, 0.058 mmol) and sodium azide (47 mg, 0.72 mmol) was dissolved in 10 mL DMF and heated for 3 h at 70 ℃. Then a large amount of water was added to dissolve all the solids. Then it was extracted twice with ethyl acetate, and the combined organic phase was dried with MgSO₄ and evaporated in vacuo. The crude product was subjected to flash column chromatography on silica gel to afford a dark green solid 114 mg. The dark green solid was dissolved in 5 mL THF and CuTc 10 mg, w-alkynyl-PEG-hydroxyl (M_w ＝ 600, 70 mg) , and TBTA (5 mg) was added. The system was stirred at RT for 1.0 h. The reaction mixture was filtered with diatomite, and the solution was evaporated in vacuo. When all the organic solvent was removed, the crude product was purified by thin layer chromatography eluting with DCM/MeOH 10: 1. IRTTEBN-PEG600 (118 mg) was afforded as a green oil.

Optical parameters of IRTTEBN-PEG600 in H₂O: absorption peak λ ＝ 710 nm, absorption coefficient K＝ 1.22 L/g. cm at 808 nm.

Example 14: Synthesis of IRTEFN-PEG600

(1) Synthesis of 2- (9, 9-bis (6-bromohexyl) -9H-fluoren-2-yl) -3, 4-bis (2- (2- (2-methoxyethoxy) ethoxy) ethoxy) thioph ene (compound 40) :

To a solution of compound 2-bromo-9, 9-bis (6-bromohexyl) -9H-fluorene (1.14 g, 2.0 mmol) , compound 37 (1.53 g, 2.2 mmol) in 10 mL toluene under protection gas atmosphere, Pd (PPh₃) ₄ (142 mg, 0.122 mol) was added. The mixture was stirred at 110 ℃ for 24 h. After cooling to room temperature, the mixture was poured into water and extracted twice with ethyl acetate, the organic phase was dried with MgSO₄ and evaporated in vacuo. The crude product was subjected to column chromatography on silica gel with PE/EA 2: 1 to afford compound 40 as a light yellow oil (790 mg, 44 ％) .

¹H NMR (500 MHz, CDCl₃) δ 8.20 –8.00 (m, 1H) , 7.90 (dd, J ＝ 14.8, 3.1 Hz, 1H) , 7.85 –7.75 (m, 2H) , 7.48 –7.03 (m, 3H) , 6.29 (s, 1H) , 4.31 (t, J ＝ 13.9 Hz, 4H) , 3.77 (t, J ＝ 13.9 Hz, 4H) , 3.62 –3.45 (m, 20H) , 3.40 (s, 6H) , 2.06 –1.65 (m, 8H) , 1.47 –1.11 (m, 12H) .

¹³C NMR (125 MHz, CDCl₃ ) δ 150.16, 149.92, 149.70, 148.88, 141.93, 137.29, 131.31, 129.29, 125.80, 123.74 (d, J ＝ 7.8 Hz) , 120.15 (d, J ＝ 13.1 Hz) , 118.44, 114.51, 96.83, 73.68, 70.38, 69.96, 69.54, 68.97, 67.92, 57.85, 52.54, 40.16, 33.30, 31.58, 30.68, 29.54, 25.54.

HRMS (ESI) calcd for C₄₃H₆₃O₈Br₂S, ( [M+H⁺] ) 897.26049, Found 897.25927.

(2) Synthesis of 4, 8- (di-2- (9, 9-bis (6-bromohexyl) -9H-fluoren-2-yl) -3, 4-bis (2- (2- (2-methoxyethoxy) ethoxy) ethox y) thiophene) -1H, 5H-benzo [1, 2-c: 4, 5-c'] bis ( [1, 2, 5] thiadiazole) (compound 41) :

To a solution of compound 40 (1.79 g, 2 mmol) in 15 mL THF at -78 ℃ under protection gas atmosphere, n-BuLi (1.6 M in hexane, 1.5 mL, 2.4 mmol) was added dropwise. After the mixture was stirred at this temperature for another 2.0 h, trinbutyltinchloride (0.812 mg, 2.5 mmol) was added to the solution. Then the reaction was slowed warmed to room temperature and stirred for 8 h. After that the mixture was poured into water and extracted twice with ethyl acetate, the combined organic phase was dried with MgSO₄ and evaporated in vacuo without further purification.

To a solution of the above crude product (2 mmol) and 4, 8-dibromo-1H, 5H-benzo [1, 2-c: 4, 5-c'] bis ( [1, 2, 5] thiadiazole) (compound a) (234 mg, 0.67 mmol) in 15 mL toluene under protection gas atmosphere, Pd (PPh₃) ₂Cl₂ (150 mg) was added. The mixture was stirred at 110 ℃ for 12 h. After cooling to room temperature, the mixture was poured into water and extracted twice with ethyl acetate, the organic phase was dried with MgSO₄ and evaporated in vacuo. The crude product was subjected to column chromatography on silica gel with DCM/EA 3: 1 to afford compound 41 as a dark green solid (674 mg, 34 ％) .

¹H NMR (500 MHz, CDCl₃ ) δ 8.09 (d, J ＝ 7.5 Hz, 2H) , 8.00 (d, J ＝ 1.4 Hz, 2H) , 7.90 (dd, J ＝ 7.4, 1.5 Hz, 2H) , 7.78 (dd, J ＝ 7.5, 1.4 Hz, 2H) , 7.48 (dd, J ＝ 7.4, 1.5 Hz, 2H) , 7.34 (td, J ＝ 7.5, 1.5 Hz, 2H) , 7.24 (td, J ＝ 7.5, 1.5 Hz, 2H) , 4.31 (t, J ＝ 6.9 Hz, 8H) , 3.77 (t, J ＝ 7.0 Hz, 8H) , 3.60 –3.44 (m, 40H) , 3.40 (s, 12H) , 2.02 –1.72 (m, 16H) , 1.44 –1.01 (m, 24H) .

¹³C NMR (125 MHz, CDCl₃ ) δ 159.92, 153.19, 151.88, 151.34, 150.46, 150.16, 142.59, 137.29, 129.54, 129.29, 126.07, 125.80, 124.19, 123.77, 121.16, 120.20, 119.88, 119.32, 105.62, 73.68, 70.38, 69.96, 69.54, 68.97, 57.85, 52.98, 40.16, 33.30, 31.58, 30.68, 29.54, 25.54.

HRMS (ESI) calcd for C₉₂H₁₂₂Br₄N₄O₁₆S₄ ⁺, ( [M+Na⁺] ) 2007.4477, Found 2007.4452.

(3) Synthesis of IRTEFN-PEG600:

Compound 41 (120 mg, 0.06 mmol) was dissolved in 10 mL DMF and sodium azide (47 mg, 0.72 mmol) and heated for 3 h at 70 ℃. Then a large amount of water was added to dissolve all solids. Then it was extracted twice with ethyl acetate, the combined organic phase was dried with MgSO₄ and evaporated in vacuo. The crude product was subjected to flash column chromatography on silica gel to afford a dark green solid 118 mg. The dark green solid was dissolved in 5 mL THF and CuTc 10 mg, w-alkynyl-PEG-hydroxyl (M_w ＝ 600, 72 mg) , and TBTA (5 mg) were added. The system was stirred at RT for 0.5 h. The reaction mixture was filtered with diatomite, and the solution was evaporated in vacuo. When all the organic solvent was removed, the crude product was purified by thin layer chromatography eluting with DCM/MeOH 10: 1. IRTEFN-PEG600 (153 mg) was afforded as a green oil.

Optical parameters of IRTEFN-PEG600 in H₂O: absorption peak λ ＝ 700 nm, absorption coefficient K＝ 1.35 L/g. cm at 808 nm.

Example 15: Characterization experiments

Spectral Characterization:

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) . During emission measurements, 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.

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. 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. Both the molecular weight and hydrodynamic size were below the renal excretion cutoff of 40 kD and 5.5 nm, respectively. The florescence intensity of IRETPBN-PEG1700 in water and phosphate buffer saline (PBS) didn’t show any decay after 1 week, indicating excellent stability (Fig 1b) . IRETBN-PEG1700 showed high photostability in PBS and water with negligible decay under continuous 808 nm laser at a power density of 0.33 W/cm² for 2 hour.

Determination of fluorescence quantum yield

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. Then all emission spectra of both IR-26 and the measured fluorophores were corrected by the detector sensitivity profile and the extinction profiles of the filters, and then integrated in the 900 to 1,600 nm. The integrated SWIR fluorescence intensity was plotted against absorbance at the excitation wavelength of 808 nm and fitted into a linear function. The slope of IR-E1 was contrast with dye IR-26, and the quantum yield of measured fluorophore in SWIR was determined by the equation below.

QY stands for quantum yields, n is refractive index of the solvent, A is the absorbance of the solution, and I is the fluorescence intensity.

Example 16: Non-invasive SWIR fluorescence imaging for brain blood vessels

The PBS solution of IRETBN-PEG1700 (1 mg/ml, 200 μL) 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.

Example 17: SWIR fluorescence imaging for tumors

The PBS solution of IREFNS (0.3 mg/ml, 350 μL) 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) . The dye fluorescence in the 4T1 tumors started to appear at around 10 s post injection and the intensity increased over time, suggesting the accumulation of the dye molecules in the tumors. The dye fluorescence in the tumors was stable for over 12 h and the tumor signal to noise ratio could reach over 4 (Figure 3) . SWIR fluorescence images of a 4T1 tumor bearing mouse after injection of IREFNS solution were shown in Figure 3.

Example 18. Conjugating molecular fluorophores to biomolecules

Conjugation was completed by the click reaction between alkyne functional biomolecules (such as protein and antibody) and the azide functionalized fluorophore, schematic was shown in Figure 4.

For a typical reaction for Straptavidin (SA) conjugation: 5 μL SA (90.9 μM) and 2.95μL DBCO-PEG4-NHS (3.08 mM) were added in 50μL PBS sequentially. The mix was vortexed a little bit, and reacted at shaker or stirring for 2 hours. After the reaction, the product was washed by 30 k filter for 4 times, the final volume is 100μL. Then, 29.7μL IRETBN-PEG600 (152.9 μM) was added to 100 μL SA@DBCO-PEG4 (the concentration of the dye can be tuned) . The mix was vortexed a little bit, and reacted at stirring for 6 hours. To purify the conjugate, the product was washed by 30 k filter for 3 times. And the product could be further subjected to density gradient ultracentrifugation (DGU) purification (sucrose column gradient: 10, 15, 20, 25, 30, 35, 40 ％, ultracentrifuge for 18 hours at 50000 rpm and 4 ℃) . The samples after DGU were excited by a 808 nm laser and their fluorescence was recorded, the results were shown in Figure 5. As shown in figure 5, IRETBN-PEG600 was successfully linked to SA. The fluorescence efficiency can be maintained or enhanced after their binding to biomolecules.

For a typical reaction for antibody conjugation: 72.2 μL Erbitux (13.7 μM) and 1.93 μL DBCO-PEG4-NHS (3.08 mM) were added together. The mix was vortexed a little bit, and reacted at shaker for 2 hours. After the reaction, the product was washed by 30 k filter for 4 times, the final volume is 100 μL. Then, 29.7 μL IRETBN-PEG600 (152.9 μM) was added to 100 μL Erbitux@DBCO-PEG4. The mix was vortexed a little bit, and reacted at stirring for 6 hours. The product was washed by 100 k filter for 6 times. The dye-Erbitux conjugates were observed to bind specifically on EGFR over-expressing SCC cell lysate spotted on a substrate without binding to EGFR negative SKOV cell lysate spots.

Example 19: Excretion of the molecular fluorophores

By proper design, the molecular fluorophores can be excreted in vivo, which is important to reduce the potential toxicity of fluorophores. Inventors used IRETBN-PEG1700 as an example. First, PBS solution of IRETBN-PEG1700 at a dose of 2.5 mg/kg was injected into healthy C57BL/6 mice, and whole-body imaging was performed to follow the fluorophores in vivo. It was observed that IRETBN-PEG1700 accumulated in the bladder within several hours after injection (Figure 6 a) . Urine samples at different time points were collected to check the excretion of IRETBN-PEG1700. Fluorescence method was used to evaluate the amount of the fluorophore in urine. Strong SWIR fluorescence signals in the urine samples were observed with the maximum SWIR signal at 3 h after injection (Figure 6 b) . The total amount of IRETBN-PEG1700 excreted by urine was estimated to be over 80 ％at 15 hours post injection.

Reference throughout this specification to "an embodiment, " "some embodiments, " "one embodiment" , "another example, " "an example, " "a specific example, " or "some examples, " means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present disclosure. Thus, the appearances of the phrases such as "in some embodiments, " "in one embodiment" , "in an embodiment" , "in another example, "in an example, " "in a specific examples, " or "in some examples, " in various places throughout this specification are not necessarily referring to the same embodiment or example of the present disclosure. Furthermore, the particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples.

Although explanatory embodiments have been shown and described, it would be appreciated by those skilled in the art that the above embodiments cannot be construed to limit the present disclosure, and changes, alternatives, and modifications can be made in the embodiments without departing from spirit, principles and scope of the present disclosure.

Claims

A compound, comprising：

an electron accepting aromatic unit,

an optional electron donating aromatic unit, and

an shielding unit shielding the electron accepting aromatic unit and/or the electron donating aromatic unit from intermolecular interactions,

wherein the electron accepting aromatic unit, the electron donating aromatic unit and the shielding unit are connected via a covalent bond,

the electron accepting aromatic unit has a formula of any one selected from the group consisting of:

each one of Z₁ and Z₂ is independently O, S, Se, or NR,

each R is independently H, C_n1H_2n1+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 R₁ is independently H, C_n2H_2n2+1, or tert-butyloxycarbonyl,

each X is independently S, Se, NR₁, or O,

each one of Y1 and Y2 is independently H, OC_n2H_2n2+1, C_n2H_2n2+1,
OC_n2H_2n2B, or C_n2H_2n2Z,

each B is independently Br, I, OTs, OMs, ONs, N3, or OMe,

each Z is independently H, Br, or N3,

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,

the shielding unit has a formula of any one selected from the group consisting of:

each R₂ is independently OC_n3H_2n3+1, C_n3H_2n3+1,

OC_n3H_2n3W, or C_n3H_2n3W,

each n3 is independently an integer ranging from 0 to 20,

each p1 is independently an integer ranging from 1 to 20,

each W is H, Br, I, OH, OTs, N₃,

each one of Z₅, Z₆, and Z₇ is independently S, Se, O, or NR₂’ ,

R₂’ is H, C_n5H_2n5+1, or tert-butyloxycarbonyl,

each X₁ is independently Si, Ge, or C,

each n5 is an integer ranging from 1 to 20,

each n is an integer ranging from 4 to 120,

each b is independently an integer ranging from 1 to 6.
The compound of claim 1, wherein 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,

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.
The compound of claim 1, wherein 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 of claim 1, 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 of claim 1, wherein 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’ 5 represents a fifth shielding unit,

S’ 6 represents a sixth shielding unit,

A represents the electron accepting aromatic unit.
The compound of claim 1, wherein 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,

A represents the electron accepting aromatic unit.
The compound of claim 1, wherein the compound having a formula of any one selected from the group consisting of:

each one of Y₁ and Y₂ is independently H, C_n2H_2n2+1, OC_n2H_2n2+1, OC_n2H_2n2B,
The compound of claim 1, wherein the compound having a formula of any one selected from the group consisting of:
A kit comprising the compound of any one of claims 1 to 8.
Use of the compound of any one of claims 1 to 8 or the kit of claim 9 in labeling or conjugating to a biomolecule.
The use of claim 10, wherein the biomolecule and the compound of any one of claims 1 to 8 are combined together through click chemistry with azide groups on the compound of any one of claims 1 to 8 or intermolecular forces to form strong, non-covalent complexes through simple mixing or mixing followed by heating to 40-70 degree Celsius.
A conjugate of the compound of any one of claims 1 to 8 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 of claim 12, wherein the biomolecule and the compound of any one of claims 1 to 8 are conjugated together through click chemistry with azide groups on the compound of any one of claims 1 to 8 or intermolecular forces to form strong, non-covalent complexes through simple mixing or mixing followed by heating to 40-70 degree Celsius.
Use of the compound of any one of claims 1 to 8, the kit of claim 9, or the conjugate of claim 12or 13 in biomedical imaging.
The use of claim 14, 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.
A method of biomedical imaging, comprising:

administrating the compound of any one of claims 1 to 8, the kit of claim 9, or the conjugate of claim 12 or 13 to a subject,

irradiating the subject at a site of interest by NIR light source,

recording a fluorescence image or video by a camera.
The method of claim 16, wherein the subject comprising an animal, a human, a tissue, a cell, a 3D organoid or a spheroid.
The method of claim 17, wherein the compound of any one of claims 1 to 8, the kit of claim 9, or the conjugate of claim 12 or 13 are administrated into a blood vessel, a tissue, an organ or a lymph node of the subject.
The method of claim 16, wherein 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.
The method of claim 16, wherein 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.
The method of claim 16, wherein the biomedical imaging comprising:

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.
A method of imaging guided tumor surgery, comprising:

recording an image of the targeted tumor by the method of any one of claims 16 to 21,

using the image to guide tumor removal.
A method for labeling a biomolecule, comprising:

making the compound of any one of claims 1 to 8 or the kit of claim 9 reacting with the biomolecule, or

mixing the compound of any one of claims 1 to 8 or the kit of claim 9 with the biomolecule with or without heating to 40-70 degree Celsius.
The method of claim 23, wherein the biomolecule comprising a small bioactive molecule, a peptide, an antibody, a protein, an affibody, a nucleic acid, and an aptamerul modified with terminal alkynyl.
The method of claim 24, wherein the small bioactive molecule comprising folic acid, tretinoin, cholic acid, galactose, and biotin,

the peptide comprising decapeptide: synB3, OSBP-1 and OSBP-S,

the antibody comprising 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, tositumomab, trastuzumab, ustekinumab, and vedolizumab.