WO2011059457A1 - Composés luminescents haute performance - Google Patents

Composés luminescents haute performance Download PDF

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WO2011059457A1
WO2011059457A1 PCT/US2009/064621 US2009064621W WO2011059457A1 WO 2011059457 A1 WO2011059457 A1 WO 2011059457A1 US 2009064621 W US2009064621 W US 2009064621W WO 2011059457 A1 WO2011059457 A1 WO 2011059457A1
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compound
alkyl
methylene
formula
alkyi
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PCT/US2009/064621
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English (en)
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Bradley Smith
Arunkumar Easwaran
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University Of Notre Dame Du Lac
Molecular Targeting Technologies, Inc.
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Priority to PCT/US2009/064621 priority Critical patent/WO2011059457A1/fr
Publication of WO2011059457A1 publication Critical patent/WO2011059457A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B57/00—Other synthetic dyes of known constitution
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B57/00—Other synthetic dyes of known constitution
    • C09B57/007—Squaraine dyes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2458/00—Labels used in chemical analysis of biological material
    • G01N2458/30—Electrochemically active labels

Definitions

  • Embodiments herein relate to the field of chemistry, and, more specifically, to novel luminescent compounds, synthesis thereof, and methods of using same.
  • Squaraine dyes are organic dyes with very intense
  • fluorescence spectra typically in the red and near infrared region, which makes them attractive for biological applications. They are characterized by an aromatic four-membered ring system derived from squaric acid.
  • Some squaraine dyes may be conjugated to biomolecules through a free carboxylic acid group.
  • these lipophilic squaraine dyes suffer from three problems in physiological solutions: poor solubility, massive aggregation-induced loss of fluorescence signal, and poor stability. Even sulfonated squaraine derivatives that are water-soluble are still unstable in PBS.
  • squaraines are encumbered by nucleophilic attack of the central four-membered ring, which is highly electron deficient. This encumbrance may be attenuated by trapping the squaraine inside a macrocycle to form a permanently interlocked molecule called a squaraine rotaxane. Formation of a rotaxane around the dye helps to protect it from nucleophiles, which improves the stability of squaraine dyes in physiological solutions like phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • squaraine rotaxanes still lose fluorescence signal under very strong aggregation conditions such as phosphate buffered saline, which limits their usefulness in biological applications.
  • Figure 1 illustrates the synthesis of symmetric
  • unsymmetric squaraine dyes according to various embodiments.
  • Figure 2 illustrates absorption spectra of an aggregated squaraine rotaxane 3 in DMSO: PBS (1 :4), and illustrates a strong aggregate band at 573 nm, in accordance with various embodiments.
  • Figure 3 illustrates a typical example of absorption (left peak) and emission (right peak) spectra of a non-aggregated squaraine rotaxane 4 in PBS attached with a single sulfonate group, in accordance with various embodiments.
  • Figure 4 illustrates a typical example of absorption (left peak) and emission (right peak) spectra of a non-aggregated squaraine rotaxane 5 in PBS attached with two sulfonate groups, in accordance with various embodiments.
  • Figure 5 illustrates a typical example of absorption (left peak) and emission (right peak) spectra of a non-aggregated squaraine rotaxane 6 in PBS attached with a zwitterionic group, in accordance with various embodiments.
  • Figure 6 illustrates a photobleaching profile of biotinylated fluorophores, in accordance with various embodiments.
  • Figure 7 illustrates exemplary squaraine rotaxane dyes, in accordance with various embodiments. Detailed Description of Disclosed Embodiments
  • a phrase in the form "A B” or in the form “A and/or B” means (A), (B), or (A and B).
  • a phrase in the form "at least one of A, B, and C” means (A), (B), (C), (A and B), (A and C), (B and C), or (A, B and C).
  • a phrase in the form "(A)B” means (B) or (AB) that is, A is an optional element.
  • halogen refers to fluoro, bromo, chloro and iodo substituents.
  • alkyl refers to a cyclic, branched, or straight chain alkyl group containing only carbon and hydrogen, and unless otherwise mentioned contains one to twelve carbon atoms. This term may be further exemplified by groups such as methyl, ethyl, n-propyl, isopropyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, adamantyl, and cyclopentyl.
  • Alkyl groups may either be unsubstituted or substituted with one or more substituents, for instance, halogen, alkyl, alkoxy, alkylthio, trifluoromethyl, acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryl, arylalkyl, heteroaryl, amino, alkylamino, dialkylamino, morpholino, piperidino, pyrrolidin-1-yl, piperazin-1 -yl, or other functionality.
  • substituents for instance, halogen, alkyl, alkoxy, alkylthio, trifluoromethyl, acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryl, arylalkyl, heteroaryl, amino, alkylamino, dialkylamino, morpholino, piperidino, pyrrolidin-1-yl, piperazin-1 -yl, or other functionality.
  • cycloalkyi refers to a cyclic alkyl moiety. Unless otherwise stated, cycloalkyi moieties include between 3 and 8 carbon atoms.
  • Each Q 0 is independently selected from H, alkyl, cycloalkyi, het, cycloalkenyl, and aryl.
  • the het, cycloalkyi, cycloalkenyl, and aryl being optionally substituted with 1-3 substitutuents selected from halo and Q13.
  • Each Qn is independently selected from H, halogen, alkyl, aryl, cycloalkyi, and het.
  • Each Qi 4 is independently selected from H, alkyl, cycloalkyl, phenyl, or naphthyl, each optionally substituted with 1 -4 substituents independently selected from F, CI, Br, I, -OQi 6> -SQi 6> -S(O) 2 Qi 6 , -S(0)Qi 6> - OS(O) 2 Qi6, -NQ16Q16, -C(0)Qi6, -C(S)Qi6, -C(0)OQi 6> -N0 2> -C(0)NQi 6 Qi 6 , -C(S)NQi 6 Qi6, -CN, -NQi 6 C(0)Qi6, -NQi 6 C(S)Qi 6> -NQi 6 C(O)NQi 6 Qi6, -NQi 6 C(O)NQi 6 Qi6, -S(O) 2 NQi 6 Qi6, and -NQi 6 S
  • Each Qi 6 is independently selected from H, alkyl, and cycloalkyl.
  • the alkyl and cycloalkyl optionally including 1 -3 halogens.
  • sulfone refers to a chemical compound containing a sulfonyl functional group attached to two carbon atoms.
  • the central sulfur atom is twice double bonded to oxygen and has two further hydrocarbon substituents.
  • aryl refers to phenyl and naphthyl.
  • heteroaryl refers to a mono- or bicyclic het in which one or more cyclic ring is aromatic.
  • substituted heteroaryl refers to a heteroaryl moiety substituted with one or more functional groups selected from halogen, alkyl, hydroxyl, amino, alkoxy, cyano, and nitro.
  • triazole refers to either one of a pair of isomeric chemical compounds with molecular formula C2H3N3, having a five-member ring of two carbon atoms and three nitrogen atoms.
  • sulfonate refers to an anion with the general formula RSO 2 O-. Sulfonates are the conjugate bases of sulfonic acids with formula RSO 2 OH.
  • uccinimide refers to a cyclic imide with the formula C H 5 NO 2 .
  • maleimide refers to, the chemical compound with the formula H 2 C 2 (CO) 2 NH.
  • polyethylene glycol refers to a chemical compound composed of one or more ethoxy units (-OCH 2 CH 2 -) in a repeating linear series. The series may begin or end with a hydroxyl group (-OH groups) or other functionality.
  • biotin refers to a water-soluble B- complex vitamin which is composed of an ureido (tetrahydroimidizalone) ring fused with a tetrahydrothiophene ring.
  • a valeric acid substituent is attached to one of the carbon atoms of the tetrahydrothiophene ring.
  • dimethylammonium refers to organic compounds that have four carbons, two of them methyl carbons, attached to a nitrogen atom that has a formal positive charge. Depending on the rest of the molecule there may or may not be a counter anion. It is also understood that in some embodiments the methyl groups may be substituted with longer chain alkyl groups.
  • zwitterionic group refers to a group that has both a positive charge and a negative charge when the molecule is in an environment that is close to neutral pH.
  • a molecule may have several attached “zwitterionic groups”
  • Embodiments of the present disclosure provide a new family of squaraine rotaxane dyes that solve all major problems that known squaraine rotaxanes exhibit in physiological solutions, including: poor solubility, aggregation-induced loss of fluorescence signal, poor stability, only moderate brightness, poor biodistribution in animals, or bioconjugates with greatly altered charge patterns.
  • the novel squaraine rotaxane dyes have photophysical properties that are very similar to the commonly used Cy-5 fluorophore, however, the new dyes are substantially more photostable and they resist quenching. This makes them superior substitutes for Cy-5 in many biotechnological and imaging applications.
  • the dyes may be conjugated to small and large biological molecules to make fluorescent molecular probes for various imaging applications.
  • the squaraine rotaxanes may include one or more sulfonate or phosphonate groups and also may include one or more reactive sites for bioconjugation.
  • the squaraine rotaxanes may be uncharged or nearly uncharged overall, but may include one or more zwitterionic groups for water solubility and also may include one or more reactive sites for bioconjugation.
  • Embodiments of the present disclosure encompass any racemic, optically-active, polymorphic,
  • Other embodiments include bioconjugates of the squaraine rotaxane dyes disclosed herein. Still other embodiments provide methods of synthesizing the squaraine rotaxane compounds described herein.
  • squaraine rotaxanes are made in three major steps; (1 ) fabrication of the central squaraine thread component, (2) encapsulation of this thread component inside a macrocycle to make a rotaxane, and (3) covalent modification of the rotaxane.
  • sulfonate, phophonate, or zwitterionic groups are added after the second step since their presence diminishes the synthetic yields in the second step. Reactions that directly attach sulfonate groups via electrophilic addition mechanisms do not produce high yields of products.
  • Embodiments disclose herein a much better approach for attaching a sulfonate or a phosphate group or a zwitterionic group to the squaraine rotaxane.
  • attaching a sulfonate, phophonate, or zwitterionic group to the squaraine rotaxane via amide bond formation is undesirable for bioimaging probes because amide bonds may be broken by enzymatic action.
  • Preferred embodiments involve the conjugation method of forming triazole linkages using a copper catalyzed azide/alkyne cycloaddition reaction.
  • triazoles are excellent linkages because they are resistant to protease enzyme cleavage and their formation does not produce by-products.
  • a squaraine rotaxane may have at least one sulfonate/phosphonate group and/or at least one zwitterionic group, and may have the formul
  • R 1 , R 2 , and R 3 are each independently alkyi, phenyl, polyethylene glycol, alkyl-phosphonate, alkyl-sulfonate, methylene-triazole-alkyl-sulfonate, methylene-triazole-alkyl- phosphonate, methylene-triazole-methylene-dimethylammonium-alkyl- sulfonate, or methylene-dimethylammonium-alkyl-phosphonate;
  • X alkyi, phenyl, alkyl-carboxylic acid, alkyi ester, alkyi hydroxysuccinimde ester, alkyi maleimide, alkyi isothiocyanate, alkyi azide, alky alkyne, alkyi
  • Y 1 and Y 2 are each independently H, alkoxy-t azole-methylene-sulfonate, alkoxy-triazole- methylene-dimethylammonium-alkyl-sulfonate, alkoxy-triazole-methylene- phosphonate, alkoxy-triazole-methylene-dimethylammonium-alkyl- phosphonate, t azole-methylene-sulfonate, triazole-methylene- phosphonate, triazole-methylene-dimethylammonium-alkyl-sulfonate, triazole-methylene-dimethylammonium-alkyl-phosphonate, or one of the reactive
  • the squaraine rotaxane may have the formula:
  • R 1 , R 2 , and R 3 are each independently alkyi, phenyl, polyethylene glycol, alkyl-phosphonate, alkyl-sulfonate, methylene-triazole-alkyl-sulfonate, methylene-triazole-alkyl- phosphonate, methylene-triazole-methylene-dimethylammonium-alkyl- sulfonate, or methylene-dimethylammonium-alkyl-phosphonate;
  • X alkyi, phenyl, alkyl-carboxylic acid, alkyi ester, alkyi hydroxysuccinimde ester, alkyi maleimide, alkyi isothiocyanate, alkyi azide, alky alkyne, alkyi
  • Y 1 and Y 2 are each independently H, alkoxy-triazole- methylene-sulfonate, alkoxy-triazole- methylene-dimethylammonium-alkyl-sulfonate, alkoxy-triazole-methylene- phosphonate, alkoxy-triazole-methylene-dimethylammonium-alkyl- phosphonate, triazole-methylene-sulfonate, triazole-methylene- phosphonate, triazole-methylene-dimethylammonium-alkyl-sulfonate, triazole-methylene-phosph or one of the reactive groups listed
  • the squaraine rotaxane may have the formula:
  • R 1 , R 2 , and R 3 are each independently alkyi, phenyl, polyethylene glycol, alkyl-phosphonate, alkyl-sulfonate, methylene-triazole-alkyl-sulfonate, methylene-triazole-alkyl- phosphonate, methylene-triazole-methylene-dimethylammonium-alkyl- sulfonate, or methylene-dimethylammonium-alkyl-phosphonate;
  • X alkyi, phenyl, alkyl-carboxylic acid, alkyi ester, alkyi hydroxysuccinimde ester, alkyi maleimide, alkyi isothiocyanate, alkyi azide, alky alkyne, alkyi
  • Y 1 and Y 2 are each independently H, alkoxy-triazole-methylene-sulfonate, alkoxy-triazole- methylene-dimethylammonium-alkyl-sulfonate, alkoxy-triazole-methylene- phosphonate, alkoxy-triazole-methylene-dimethylammonium-alkyl- phosphonate, triazole-methylene-sulfonate, triazole-methylene- phosphonate; triazole-methylene-dimethylammonium-alkyl-sulfonate, triazole-methylene-dim or one of the reactive groups listed as X; and W 1 , W 2 , W
  • the exemplary compounds described above may be synthesized according to the following general procedures.
  • symmetrical squaraine dyes are prepared in one step by heating two equivalents of the appropriate aniline derivative with squaric acid.
  • unsymmetrical squaraines are prepared in two steps via a stable semi squaraine intermediate (see, e.g., Figure 1 ).
  • macrocyclization step yields a squaraine rotaxane dye, as shown in the example below using an aniline-based squaraine.
  • Aniline-based squaraine rotaxanes with ortho-hydroxyl groups are a novel set of squaraine rotaxanes that exhibit highly favorable chemical and photophysical performance, due in part to the hydrogen bonding between the ortho-hydroxyl groups and the squaraine oxygens which provides structural rigidity and blocks chemical attack.
  • Another favorable attribute gained by the presence of ortho-hydroxyl groups is reduced fluorescence quenching by water. This may be demonstrated by comparing compound 1 , which has no ortho-hydroxyls attached to the anilines, with compound 2, which has two ortho-hydroxyls attached to one of the anilines.
  • a new class of squaraine rotaxane dyes may be formed by a slippage process that involves mixing the squaraine and the macrocycle components and using heat, or microwave radiation to induce the squaraine dye to slip inside the
  • the squaraine rotaxane may be modified and groups may be introduced that provide water solubility and reactivity using the conjugation method of forming triazole linkages using a copper catalyzed azide alkyne cycloaddition reaction, as shown in the following exemplary reaction.
  • a 1 , A 2 , A 3 , A 4 , and A 5 are each independently H or azido (N 3 ); T 1 , T 2 , T 3 , T 4 , and T 5 are each independently H or 1 ,3-triazole rings; and W 1 , W 2 , W 3 , and W 4 are each independently H or OH.
  • FIG. 2 shows an exemplary absorption spectrum of unmodified, lipophilic squaraine rotaxane 3 in a DMSO/PBS solvent mixture that promotes aggregation. Aggregates that exhibit the absorption band at 573 nm are not fluorescent.
  • Figure 3 illustrates an example of absorption (left peak) and emission (right peak) spectra in PBS of a squaraine rotaxane 4 attached with a single sulfonate group. There is no evidence of non-fluorescent aggregates.
  • Figure 4 shows an example of absorption (left peak) and emission (right peak) spectra in PBS of a squaraine rotaxane 5 attached with two sulfonate groups.
  • squaraine rotaxanes with one or more zwitterionic groups may not form aggregates in PBS.
  • Figure 5 shows an example of absorption (left peak) and emission (right peak) spectra of a squaraine rotaxane 6 in PBS attached with a zwitterionic group.
  • the squaraine rotaxanes are water soluble but overall uncharged because they have one or more attached polyethylene glycol or zwitterionic groups for water solubility and one or more reactive sites for bioconjugation.
  • the attachment of a dye with many anionic or cationic groups to a biological molecule sometimes changes the charge pattern of the biological molecule to such an extent that its chemical, biochemical or supramolecular properties are altered adversely.
  • the attachment of a dye having many anionic groups or many cationic groups to a biological molecule produces a bioconjugate that when introduced into an animal is not likely to be cleared primarily from the bloodstream by the kidney, which complicates imaging performance.
  • these problems are solved, in embodiments, by creating squaraine rotaxanes with one or more polyethyleneglycol or zwitterionic groups for water solubility and one or more reactive sites for bioconjugation.
  • the overall net charge of these compounds is zero or close to zero.
  • these compounds have excellent water solubility and excellent stability, do not form non-fluorescent aggregates in PBS, and have a greater tendency to clear from the bloodstream of animals through the kidney.
  • squaraine rotaxanes with one or more attached sulfonates or attached zwitterionic groups are very
  • the probe SQR-biotin is a squaraine rotaxane with an attached sulfonate that gives excellent water solubility, and an attached biotin group that has strong affinity for the protein avidin or related variants such as streptavidin.
  • the complex of this probe associated with streptavidin is very photostable compared to complexes made with other dyes.
  • the photobleaching of commercially available biotin-4-carboxyfluorescein (CF-biotin), and biotin- squaraine rotaxane (SQR-biotin) was examined by measuring the half-life of each of the probes.
  • the dyes were incubated in excess with 1 .3 ⁇ magnetic particles coated in avidin. Following the incubation, the particles were washed of any unbound fluorophore, and the samples were then imaged on an epifluorescent microscope. The samples remained under constant excitation and images were captured every 30 seconds.
  • ImageJ software a region of interest (ROI) was drawn around the imaged magnetic particles and the mean pixel intensity was measured for each image.
  • FIG. 6 shows the photobleaching profile of the two biotinylated fluorophores.
  • the biotin-4-carboxyfluorescein probe showed dramatic photobleaching, but the biotin-squaraine rotaxane probe retained
  • Figure 7 illustrates a number of specific, non-limiting examples of squaraine rotaxanes. 1. Synthesis of squaraine rotaxanes with the following general structure (or a pharmaceutically acceptable salt thereof):
  • X OH, alkoxy, aryloxy, NH-alkyl, NH-aryl, N-succinimide, NH-alkyl-maleinnide, NH-polyethylene glycol-biotin, polyethylene glycol, methylene-sulfonate, methylene-dimethylammonium- alkyl-sulfonate, methylene-phosphonate, or methylene-dimethylammonium- alkyl-phosphonate.
  • N-(ethyl)-anilino ethanol (1 mmol) is mixed with tertiary butyl bromoacetate (1 .1 mmol) in benzene.
  • a 50% NaOH solution is added to the reaction along with tetrabutyl ammonium hydrogen sulfate (phase transfer agent). Stirring continues at room temperature for 3 hours.
  • the organic layer is evaporated to dryness and the product is purified using silica gel chromatography using hexane:ethyl acetate (19:1 ).
  • Trifluoroacetic acid (TFA; 1 mL) is added to a solution of squaraine rotaxane (0.1 mmol) in CH 2 Cl2 (5 mL). The mixture is stirred at room temperature for 6 hours, and the solvent and excess TFA evaporated under reduced pressure. The residue is washed with distilled water, and extracted with excess CH 2 CI 2 . The organic layer is dried with MgSO , and the solvent is removed to afford azide-carboxylic acid rotaxane as a blue solid.
  • TFA Trifluoroacetic acid
  • Azide squaraine rotaxane (0.1 mmol) is dissolved in a mixture of THF:t-butanol:water (10 mL each). Excess of alkyne reagent is added and a mixture of CuSO 4 -5H 2 O (75 mg in 2 mL H 2 O) and sodium ascorbate (300 mg in 3 mL H 2 O) are also added. The solution is stirred overnight and the product purified by reverse-phase chromatography. (g) Synthesis of zwitterionic squaraine rotaxane that is activated for bioconjugation
  • X OH, alkoxy, aryloxy, NH-alkyl, NH-aryl, N-succinimide, NH-alkyl-maleimide, NH-polyethylene glycol-biotin, polyethylene glycol, methylene-sulfonate, methylene-dimethylammonium- alkyl-sulfonate, methylene-phosphonate, or methylene-dimethylammonium- alkyl-phosphonate.
  • N-(diethanol) aniline (1 mmol) is mixed with tertiary butyl bromoacetate (1 .1 mmol) in benzene.
  • a 50% NaOH solution is made and added to the reaction along with tetrabutyl ammonium hydrogen sulfate (phase transfer agent). Stirring continued at room temperature for 3 hours The organic layer is evaporated to dryness and the product purified using silica gel chromatography using hexane:ethyl acetate (9:1 ).
  • Tosylate is dissolved in DMF and treated with an excess of NaN 3 . This is refluxed for 16 hours. The reaction mixture was filtered and the solution was evaporated to dryness. This is purified using column chromatography using hexane: ethyl acetate (49:1 ).
  • Trifluoroacetic acid (TFA; 1 mL) is added to a solution of rotaxane (0.1 mmol) in CH 2 Cl 2 (5 mL). The mixture is stirred at room temperature for 6 hours, and the solvent and excess TFA evaporated under reduced pressure. The residue is washed with distilled water, and extracted with excess CH 2 CI 2 . The organic layer is dried with MgSO 4 and the solvent removed to afford carboxylic acid rotaxane as a blue solid.
  • TFA Trifluoroacetic acid
  • X OH, alkoxy, aryloxy, NH-alkyl, NH-aryl, N-succinimide, NH-alkyl-maleinnide, NH-polyethylene glycol-biotin, polyethylene glycol, methylene-sulfonate, methylene-dimethylammonium- alkyl-sulfonate, methylene-phosphonate, or methylene-dimethylammonium- alkyl-phosphonate.
  • Trifluoroacetic acid (1 ml_) is added to a solution of azide squaraine rotaxane (0.1 mmol) in CH 2 Cl2 (5 ml_). The mixture is stirred at room temperature for 6 hours, and the solvent and excess TFA are evaporated under reduced pressure. The residue is washed with distilled water, and extracted with excess CH2CI2. The organic layer is dried with MgSO 4 and the solvent removed to afford carboxylic acid rotaxane as a blue solid.
  • Azide squaraine rotaxane (0.1 mmol) is dissolved in a mixture of THF:t-butanol:water (10 mL each). Excess of alkyne reagent is added and a mixture of CuSO 4 -5H 2 O (75 mg in 1 mL H 2 O) and sodium ascorbate (300 mg in 3 mL H 2 O) is also added. The solution is stirred overnight. Purification in reverse phase column gives the pure product.
  • T 1 , T 2 , T 3 , T 4 , and T 5 are each independently H or 1 ,3-triazole rings; and W 1 , W 2 , W 3 , and W 4 are each independently H or OH.
  • Azide squaraine rotaxane (0.1 mmol) is dissolved in a mixture of THF:t-butanol:water (10 ml_ each). Excess of alkyne reagent is added and a mixture of CuSO -5H 2 O (75 mg in 1 ml_ H 2 O) and sodium ascorbate (300 mg in 3 ml_ H 2 O) is also added. The solution is stirred overnight.
  • T 1 , T 2 , T 3 , T 4 , T 5 are each independently H or a 1 ,3-triazole rings, and W 1 , W 2 , W 3 , and W 4 are each independently H or OH
  • Azide squaraine rotaxane (0.1 mmol) is dissolved in a mixture of THF:t-butanol:water (10 ml_ each). Excess of alkyne reagent is added and a mixture of CuSO -5H 2 O (75 mg in 1 ml_ H 2 O) and sodium ascorbate (300 mg in 3 ml_ H 2 O) is also added. The solution is stirred overnight. Purification in a reverse phase column gives the pure product.
  • Fluorescent dyes are often used to covalently label cells and allow studies of cell motility, proliferation, and migration.
  • Cells are washed and resuspended in 0.1 % (bovine serum albumin) BSA/PBS to a concentration of 10 7 cells/mL. Labeling with squaraine rotaxane N-hydroxysuccinimde ester is performed by adding an aliquot of the squaraine rotaxane N-hydroxysuccinimde ester (50 mM stock in DSMO) to a give a final concentration of 0.5 ⁇ , and incubating the cells for 30 minutes at 37°C. The cells are washed extensively and resuspended in the appropriate buffer for microscopic studies. The cells are imaged by confocal microscopy using a Cy-5 filter set. Protein Labeling
  • Fluorescent dyes are often used to covalently label proteins, especially antibodies, and allow detection assays for various applications in biotechnology, medicine, public health, and national security.
  • Anti-rabbit IgG antibodies (2 mg/nnL) in sodium bicarbonate buffer (0.1 M, pH 8.3) are labeled by adding an aliquot of squaraine rotaxane N-hydroxysuccinimde ester (50 mM stock in DSMO) to a give a final concentration of 5 mg/nnL, and incubating for one hour at 37°C in the dark. Unconjugated dye is separated from the labeled protein by size-exclusion chromatography using 30,000 MW resin and sodium phosphate buffer (50 mM, pH 7.3) as eluent.
  • the dye/protein ratio is determined spectrometrically from the molar absorptivity of 180,000 cm “1 M “1 for the dye at 640 nm, and 203,000 cm “1 M “1 for the antibody at 280 nm (this includes the contribution from attached dye).
  • Fluorescent dyes are often used to covalently label
  • oligonucleotides especially DNA
  • Oligonucleotide (2 img/mL) is desalted with a NAP 5 column and placed in sodium bicarbonate buffer (0.1 M, pH 8.3). Labeling is achieved by adding an aliquot of squaraine rotaxane N-hydroxysuccinimde ester (50 mM stock in DSMO) to a give a final concentration of 5 img/mL, and incubating for one hour at 37°C in the dark. Unconjugated dye was separated from the oligonucleotide by size-exclusion chromatography using a Sephadex 25 column followed by ethanol precipitation and the labeled and unlabeled oligonucleotides are separated by gel electrophoresis.

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Abstract

Des modes de réalisation de l'invention concernent le domaine de la chimie et de la biochimie, et plus spécifiquement, de nouveaux colorants absorbant les infrarouges proches qui sont photostables et résistants à l'extinction. Les colorants appartiennent à une nouvelle famille de rotaxanes dérivés de squaraines, et ils conviennent particulièrement bien pour être utilisés dans des applications biologiques. L'invention porte également sur des procédés de synthèses des colorants et sur des procédés d'utilisation des colorants.
PCT/US2009/064621 2009-11-16 2009-11-16 Composés luminescents haute performance WO2011059457A1 (fr)

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WO2013160701A1 (fr) * 2012-04-27 2013-10-31 University Of Bristol Macrocycles anthracényle-tétralactame et leur utilisation dans la détection d'un saccharide cible
WO2016053880A1 (fr) * 2014-09-29 2016-04-07 Northwestern University Colorants fluorescents supramoléculaires
WO2020020849A1 (fr) * 2018-07-24 2020-01-30 F. Hoffmann-La Roche Ag Réactif pour spectrométrie de masse
US11041073B2 (en) 2016-09-26 2021-06-22 Samsung Sdi Co., Ltd. Compound, core-shell dye, photosensitive resin composition including the same, and color filter
US11091645B2 (en) 2016-08-17 2021-08-17 Samsung Sdi Co., Ltd. Core-shell dye, photo-sensitive resin composition comprising same, and color filter
US11267979B2 (en) 2014-09-29 2022-03-08 Northwestern University Supramolecular encrypted fluorescent security ink compositions
US11427529B2 (en) 2016-09-28 2022-08-30 Samsung Sdi Co., Ltd. Compound, core-shell dye, photosensitive resin composition including the same, and color filter
US11845733B2 (en) 2018-07-24 2023-12-19 Roche Diagnostics Operations, Inc. Reagent for mass spectrometry
US11885818B2 (en) 2018-07-24 2024-01-30 Roche Diagnostics Operations, Inc. Reagent for mass spectrometry

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002024816A1 (fr) * 2000-09-25 2002-03-28 Basf Aktiengesellschaft Colorants de rotaxane
WO2008094637A2 (fr) * 2007-01-30 2008-08-07 Seta Biomedicals. Llc Composés luminescents

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002024816A1 (fr) * 2000-09-25 2002-03-28 Basf Aktiengesellschaft Colorants de rotaxane
WO2008094637A2 (fr) * 2007-01-30 2008-08-07 Seta Biomedicals. Llc Composés luminescents

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
ARUNKUMAR ET AL.: "Singlet oxygen generation using iodinated squaraine and squaraine-rotaxane dyes", NEW JOURNAL OF CHEMISTRY, vol. 31, 20 December 2006 (2006-12-20), pages 677 - 683 *
ARUNKUMAR ET AL.: "Squaraine-Derived Rotaxanes: Sterically Protected Fluorescent Near-IR Dyes", JOURNAL OF AMERICAN CHEMICAL SOCIETY, vol. 127, no. 10, 18 February 2005 (2005-02-18), pages 3288 - 3289, XP055033351, DOI: doi:10.1021/ja042404n *
FU ET AL.: "Squaraine Rotaxanes with Boat Conformation Macrocycles", JOURNAL OF ORGANIC CHEMISTRY, vol. 74, no. 17, 29 July 2009 (2009-07-29), pages 6462 - 6468, XP055035255, DOI: doi:10.1021/jo901298n *
GASSENSMITH ET AL.: "Self-Assembly of Fluorescent Inclusion Complexes in Competitive Media Including the Interior of Living Cells", JOURNAL OF AMERICAN CHEMICAL SOCIETY, vol. 129, no. 48, 10 November 2007 (2007-11-10), pages 15054 - 15059, XP055067880, DOI: doi:10.1021/ja075567v *
GASSENSMITH ET AL.: "Synthesis and Photophysical Investigation of Squaraine Rotaxanes by "licked Capping"", ORGANIC LETTERS, vol. 10, no. 15, 27 June 2008 (2008-06-27), pages 3343 - 3346 *
HSUEH ET AL.: "Protecting a Squaraine near-IR Dye through Its Incorporation in a Slippage-Derived [2]Rotaxane", ORGANIC LETTERS, vol. 9, no. 22, 3 October 2007 (2007-10-03), pages 4523 - 4526 *
SMITH ET AL.: "Squaraine-Derived Rotaxanes: Highly Stable, Fluorescent Near-IR Dyes", CHEMISTRY A EUROPEAN JOURNAL, vol. 12, no. 17, 31 March 2006 (2006-03-31), pages 4684 - 4690, XP008115111 *

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9610365B2 (en) 2012-04-27 2017-04-04 University Of Bristol Anthracenyl-tetralactam macrocycles and their use in detecting a target saccharide
WO2013160701A1 (fr) * 2012-04-27 2013-10-31 University Of Bristol Macrocycles anthracényle-tétralactame et leur utilisation dans la détection d'un saccharide cible
GB2517345A (en) * 2012-04-27 2015-02-18 Univ Bristol Anthracenyl-tetralactam macrocycles and their use in detecting a target saccharide
CN104428290A (zh) * 2012-04-27 2015-03-18 布里斯托尔大学 蒽基-四内酰胺大环类及它们在检测靶糖中的用途
US9937272B2 (en) 2012-04-27 2018-04-10 University Of Bristol Anthracenyl-tetralactam macrocycles and their use in detecting a target saccharide
CN104428290B (zh) * 2012-04-27 2017-11-14 布里斯托尔大学 蒽基‑四内酰胺大环类及它们在检测靶糖中的用途
CN103214410B (zh) * 2013-04-03 2016-01-06 四川大学 一系列不对称方酸菁小分子及其制备方法和应用
CN105541694A (zh) * 2013-04-03 2016-05-04 四川大学 一系列不对称方酸菁小分子及其制备方法和应用
CN103214410A (zh) * 2013-04-03 2013-07-24 四川大学 一系列不对称方酸菁小分子及其制备方法和应用
WO2016053880A1 (fr) * 2014-09-29 2016-04-07 Northwestern University Colorants fluorescents supramoléculaires
US11267979B2 (en) 2014-09-29 2022-03-08 Northwestern University Supramolecular encrypted fluorescent security ink compositions
US11091645B2 (en) 2016-08-17 2021-08-17 Samsung Sdi Co., Ltd. Core-shell dye, photo-sensitive resin composition comprising same, and color filter
US11041073B2 (en) 2016-09-26 2021-06-22 Samsung Sdi Co., Ltd. Compound, core-shell dye, photosensitive resin composition including the same, and color filter
US11427529B2 (en) 2016-09-28 2022-08-30 Samsung Sdi Co., Ltd. Compound, core-shell dye, photosensitive resin composition including the same, and color filter
US11945763B2 (en) 2016-09-28 2024-04-02 Samsung Sdi Co., Ltd. Compound, core-shell dye, photosensitive resin composition including the same, and color filter
US12110262B2 (en) 2016-09-28 2024-10-08 Samsung Sdi Co., Ltd. Compound, core-shell dye, photosensitive resin composition including the same, and color filter
WO2020020849A1 (fr) * 2018-07-24 2020-01-30 F. Hoffmann-La Roche Ag Réactif pour spectrométrie de masse
US11845733B2 (en) 2018-07-24 2023-12-19 Roche Diagnostics Operations, Inc. Reagent for mass spectrometry
US11885818B2 (en) 2018-07-24 2024-01-30 Roche Diagnostics Operations, Inc. Reagent for mass spectrometry

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