WO2017130191A1 - Chemiluminescent probes for diagnostics and in vivo imaging - Google Patents

Chemiluminescent probes for diagnostics and in vivo imaging Download PDF

Info

Publication number
WO2017130191A1
WO2017130191A1 PCT/IL2017/050088 IL2017050088W WO2017130191A1 WO 2017130191 A1 WO2017130191 A1 WO 2017130191A1 IL 2017050088 W IL2017050088 W IL 2017050088W WO 2017130191 A1 WO2017130191 A1 WO 2017130191A1
Authority
WO
WIPO (PCT)
Prior art keywords
alkyl
compound
group
independently
alkylene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/IL2017/050088
Other languages
English (en)
French (fr)
Inventor
Doron Shabat
Ronit Satchi-Fainaro
Nir HANANYA
Ori GREEN
Tal EILON
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ramot at Tel Aviv University Ltd
Original Assignee
Ramot at Tel Aviv University Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to US16/072,848 priority Critical patent/US10660974B2/en
Priority to CN201780020284.2A priority patent/CN108884386B/zh
Priority to ES17743844T priority patent/ES2866900T3/es
Priority to JP2018539034A priority patent/JP6915943B2/ja
Priority to KR1020187024288A priority patent/KR102717614B1/ko
Priority to CA3011328A priority patent/CA3011328C/en
Priority to EP17743844.7A priority patent/EP3408349B1/en
Priority to BR112018015334-6A priority patent/BR112018015334B1/pt
Application filed by Ramot at Tel Aviv University Ltd filed Critical Ramot at Tel Aviv University Ltd
Publication of WO2017130191A1 publication Critical patent/WO2017130191A1/en
Anticipated expiration legal-status Critical
Priority to US16/850,333 priority patent/US11179482B2/en
Priority to US17/451,536 priority patent/US11931429B2/en
Priority to US18/428,591 priority patent/US12357709B2/en
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D321/00—Heterocyclic compounds containing rings having two oxygen atoms as the only ring hetero atoms, not provided for by groups C07D317/00 - C07D319/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0052—Small organic molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/66—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety
    • C07C69/73—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of unsaturated acids
    • C07C69/732—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of unsaturated acids of unsaturated hydroxy carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
    • C07F5/02—Boron compounds
    • C07F5/025—Boronic and borinic acid compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
    • C07F5/02—Boron compounds
    • C07F5/04—Esters of boric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic Table
    • C07F7/02—Silicon compounds
    • C07F7/08—Compounds having one or more C—Si linkages
    • C07F7/18—Compounds having one or more C—Si linkages as well as one or more C—O—Si linkages
    • C07F7/1804—Compounds having Si-O-C linkages
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/09—Esters of phosphoric acids
    • C07F9/091—Esters of phosphoric acids with hydroxyalkyl compounds with further substituents on alkyl
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/09—Esters of phosphoric acids
    • C07F9/12—Esters of phosphoric acids with hydroxyaryl compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/655—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms
    • C07F9/6551—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a four-membered ring
    • C07F9/65512—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a four-membered ring condensed with carbocyclic rings or carbocyclic ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings
    • 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
    • C09B11/00—Diaryl- or thriarylmethane dyes
    • C09B11/04—Diaryl- or thriarylmethane dyes derived from triarylmethanes, i.e. central C-atom is substituted by amino, cyano, alkyl
    • C09B11/10—Amino derivatives of triarylmethanes
    • C09B11/24—Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
    • 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
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/0066—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain being part of a carbocyclic ring,(e.g. benzene, naphtalene, cyclohexene, cyclobutenene-quadratic acid)
    • 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
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
    • C09B23/04—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups one >CH- group, e.g. cyanines, isocyanines, pseudocyanines
    • 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
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
    • C09B23/08—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines
    • C09B23/083—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines five >CH- groups
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent materials, e.g. electroluminescent or chemiluminescent
    • C09K11/06—Luminescent materials, e.g. electroluminescent or chemiluminescent containing organic luminescent materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E10/00—Energy generation through renewable energy sources
    • Y02E10/50—Photovoltaic [PV] energy
    • Y02E10/549—Organic PV cells

Definitions

  • the present invention provides various dioxetane-based chemiluminescence probes and compositions thereof.
  • Chemiluminescence assays are widely utilized in various chemical and biological applications due to their sensitivity and high signal-to-noise ratio (Roda and Guardigli, 2012; Roda et al., 2005). Unlike fluorescence-based assays, in chemiluminescence no light excitation is required. Therefore, background signal arising from autofluorescence does not exist when chemiluminescence is used. Such circumstance makes chemiluminescence especially useful for tissue and whole-body imaging (Gross et al., 2009; Zhang et al., 2013; Van de Bittner et al., 2013; Porterfield et al., 2015).
  • chemiluminescent compounds are activated by oxidation; i.e., a stable precursor is oxidized usually by hydrogen peroxide, to form an oxidized high-energy intermediate, which then decomposes to generate an excited species. The latter decays to its ground state by either light emission or by energy transfer.
  • Common probes that act on such chemiluminescence mechanism are usually based on luminol (Merenyi et al., 1990) and oxalate esters (Silva et al., 2002).
  • ROS reactive oxygen species
  • Removal of the protecting group by the analyte of interest generates an unstable phenolate-dioxetane species II, which decomposes through a chemiexcitation process to produce the excited intermediate benzoate ester III and adamantanone.
  • the excited intermediate decays to its ground-state (benzoate ester IV) through emission of a blue light photon.
  • Schaap's dioxetanes suffer from one major limitation; their chemiluminescence efficiency decreases significantly through non- radiative energy transfer processes (quenching) by interaction with water molecules (Matsumoto, 2004).
  • a common way to amplify the chemiluminescence signal of Schaap's dioxetanes is achieved through energy transfer from the resulting excited species (benzoate ester III) to a nearby acceptor, which is a highly emissive fluorophore under aqueous conditions (Park et al, 2014; Tseng and Kung, 2015).
  • a surfactant-dye adduct is usually added in commercial chemiluminescent immunoassays.
  • the surfactant reduces water-induced quenching by providing a hydrophobic environment for the excited chemiluminescent probe, which transfers its energy to excite a nearby fluorogenic dye. Consequently, the low-efficiency luminescence process is amplified up to 400-fold in aqueous medium (Schaap et al, 1989).
  • the surfactant mode-of-action relies on micelles formation, its functional concentration is relatively high (above the critical micelle concentration) (Dominguez et al., 1997).
  • the surfactant-dye adduct approach is not practical for in vivo detection or imaging of biological activity generated by enzymes or chemical analytes (Torchilin, 2001).
  • tethering of dioxetane with fluorophore also allows color modulation and red- shifting of the emitted light; a significant requirement for bioimaging applications (Matsumoto et al., 2008; Loening et al., 2010; Branchini et al., 2010; McCutcheon et al, 2012; Jathoul et al, 2014; Steinhardt et al, 2016).
  • Study 1 shows a simple and practical synthetic route for preparation of such fluorophore-tethered dioxetane chemiluminescent probes.
  • the effectiveness of the synthesis is based on a late-stage functionalization of a dioxetane precursor by Hartwig- Miyaura C-H borylation, followed by subsequent Suzuki coupling and oxidation to dioxetane.
  • the obtained intermediate is composed of a reactive NHS-ester-dioxetane ready for conjugation with any fluorophore- amine derivative.
  • the chemiluminescent emission of the fluorophore-tethered dioxetane probes was significantly amplified in comparison to a classic dioxetane probe through an energy transfer mechanism.
  • the synthesized probes produced light of various colors that matched the emission wavelength of the excited tethered fluorophore.
  • two fluorophore-tethered dioxetane probes designed for activation by ⁇ -galactosidase and conjugated with green (fluorescein) and NIR (QCy) fluorescent dyes were synthesized.
  • Both probes were able to provide chemiluminescence in vivo images following subcutaneous injection after activation by ⁇ -galactosidase; however, a chemiluminescence image following intraperitoneal injection was observed only by the NIR probe. These are the first in vivo images produced by Schaap' s dioxetane-based chemiluminescence probes with no need of any additive.
  • the NIR probe was also able to image cells by chemiluminescence microscopy, based on endogenous activity of ⁇ -galactosidase.
  • Study 2 shows the preparation of such chemiluminescence probes with high efficiency yield under physiological conditions.
  • the chemiluminescence quantum yield of the best probe was greater than three orders of magnitude in comparison to the standard commercially available adamantylidene-dioxetane probe.
  • one of the probes prepared was able to provide high quality chemiluminescence cell images based on endogenous activity of ⁇ -galactosidase, demonstrating for the first time cell-imaging achieved by non-luciferin small molecule based probe with direct chemiluminescence mode of emission.
  • the chemiluminescence probes shown in this Study are referred to herein as compounds of the formula IVa/IVb.
  • the present invention thus provides a fluorophore-tethered dioxetane-based chemiluminescence probe of the formula Illa/IIIb as defined herein, as well as intermediates for the preparation thereof referred to herein as compounds of the formulas I and Ila/IIb as defined herein; and a ⁇ * acceptor group-containing dioxetane based chemiluminescence probe of the formula IVa/IVb as defined herein.
  • the present invention provides a composition
  • a composition comprising a carrier, e.g., a pharmaceutically acceptable carrier, and either a conjugate of the formula Illa/IIIb or a compound of the formula IVa/IVb.
  • a carrier e.g., a pharmaceutically acceptable carrier
  • the composition of the invention may be used for diagnostics as well as for in vivo imaging of reporter genes, enzymes, and chemical analytes.
  • Fig. 1 shows chemiluminescent emission spectrum of ⁇ Probe 1, nm, recorded in PBS, pH 7.4, in the presence of 1.5 units/mL ⁇ -galactosidase.
  • Fig. 2 shows decomposition of Probes 1, 2 and 3 under normal room illumination conditions. Probes (300 ⁇ ) were incubated in PBS (100 mM), pH 7.4, at ambient temperature.
  • Fig. 3 illustrates a proposed pathway for visible-light-induced decomposition of dioxetane-fluorophore conjugates.
  • Fig. 4 shows chemiluminescence emission spectra of 1 ⁇
  • Spectra were recorded in PBS (100 mM), pH 7.4, in the presence of 1.5 units/mL ⁇ -galactosidase (continuous lines). Dotted line is the fluorescence emission spectrum (DCL - direct chemiluminescence).
  • FIG. 5 shows (panels A-C) chemiluminescent kinetic profiles of 1 ⁇
  • A Probe 1,
  • B Probe 2, and
  • C Probe 3 in PBS (100 mM), pH 7.4, in the presence of 1.5 units/mL ⁇ - galactosidase and in the absence of ⁇ -galactosidase.
  • Panels D-F show total photon counts emitted from (D) Probe 1, (E) Probe 2, and (F) Probe 3 in the presence of ⁇ -galactosidase.
  • Fig. 6 shows total light emitted from 10 ⁇ Probe 2 in PBS (100 mM), pH 7.4, over a period of 1 h, with different concentrations of ⁇ -galactosidase.
  • the inset focuses on light emitted from Probe 2 upon incubation with the lowest concentrations of ⁇ - galactosidase.
  • Figs. 7A-7D show (7A) solution images obtained from 1 ⁇ Probes 1, 2 and 3 incubated in PBS, pH 7.4, in the presence and in the absence of ⁇ -galactosidase (gal); (7B) quantification of signal intensities obtained in solution in the presence of ⁇ -galactosidase; (7C) whole-body images obtained 15 min following subcutaneous injection of Probes 2 and 3 [50 ⁇ , 1 ⁇ in PBS (100 mM), pH 7.4, after 30 min pre-incubation with or without 1.5 units/mL ⁇ -galactosidase]; and (7D) quantification of signal intensities in whole-body images in the presence of ⁇ -galactosidase (quantitative data are based on repeated imaging experiments with three mice).
  • Fig. 8 shows whole-body images obtained 15 min following intraperitoneal injection of Probes 2 and 3 [50 ⁇ , 1 ⁇ in PBS (100 mM), pH 7.4, after a 30 min preincubation with or without 1.5 units/ mL ⁇ -galactosidase].
  • FIG. 9 shows transmitted light image (panel a) and chemiluminescence microscopy of HEK293-LacZ stable cells (panel b); and transmitted light image (panel c) and chemiluminescence microscopy of HEK293-WT cells (panel d). Images were obtained following 20 min incubation with cell culture medium containing Probe 3 (5 ⁇ ).
  • FIG. 10 shows transmitted light image (panel a) and chemiluminescence microscopy of HEK293-LacZ stable cells, fixed with formaldehyde (4% for 20 min) (panel b). Images were obtained following 20 min incubation with cell culture medium containing Probe 3 (5 ⁇ ).
  • Figs. 12A-12B show chemiluminescence kinetic profiles of Probes 5, 6, 7, 8 and 9
  • Figs. 13A-13B show chemiluminescence kinetic profiles of Probe 5 [1 ⁇ ] in the presence of 1.5 units/mL ⁇ -galactosidase, with and without Emerald-IITM enhancer (10%) in PBS 7.4 (10% DMSO) (left panel), and count of total emitted photon (right panel) (13A); and chemiluminescence kinetic profiles of Probe 5 [1 ⁇ ] with Emerald-IITM enhancer (10%) and Probe 9 [1 ⁇ ] in PBS 7.4 (10% DMSO) in the presence of 1.5 units/mL ⁇ -galactosidase (13B) (left panel), and count of total emitted photon (right panel).
  • Fig. 14 shows water soluble chemiluminescence probes for detection of hydrogen peroxide (Probe 10) and alkaline-phosphatase (AP) (Probe 11), which produce visible bright green luminescence under aqueous conditions.
  • Probe 10 hydrogen peroxide
  • AP alkaline-phosphatase
  • Probe 12 is a chemiluminescence for detection of GSH.
  • Figs. 15A-15B show (15A) total light emitted from Probe 10 (100 ⁇ ), Probe 11 (10 ⁇ ) and Probe 12 (10 ⁇ ) in the presence of hydrogen peroxide (1 mM), alkaline phosphatase (AP) (1.5 EU/ml) or glutathione (1 mM). Measurements were conducted in PBS (100 mM), pH 7.4, with 10% DMSO at RT; and (15B) total light emitted from Probe 10 (500 ⁇ ), Probe 11 (500 ⁇ ) and Probe 12 (10 ⁇ ) in PBS (100 mM), pH 7.4 with 10% DMSO over a period of 1 h, with various concentration of the corresponding stimulus. A detection limit (blank control+3 SD) was determined for each probe.
  • Fig. 16 shows (a) transmitted light image and (b) chemiluminescence microscopy of HEK293-LacZ stable cells; and (c) transmitted light image and (d) chemiluminescence microscopy of HEK293-WT cells. Images were obtained following 20 min incubation with cell culture medium containing Probe 7 (5 ⁇ ). Images were taken by the LV200 Olympus-microscope using 60x objective and 40s exposure time. DETAILED DESCRIPTION
  • the present invention provides a compound of the formula I:
  • R 1 is selected from a linear or branched (Ci-Ci 8 )alkyl, or (C3-C7)cycloalkyl;
  • R 2 and R 3 each independently is selected from a branched (C 3 -Ci 8 )alkyl or (C 3 - )cycloalkyl, or R 2 and R 3
  • R 5 and R 6 each independently is selected from H, (Ci-Cis)alkyl, (C2-Ci 8 )alkenyl, (C2-Ci 8 )alkynyl, (C 3 -C7)cycloalkyl, or aryl, or R 5 and R 6 together with the oxygen atoms to which they are attached form a heterocyclic ring;
  • R 10 each independently is H or -(Ci-Cis)alkyl
  • alkyl typically means a linear or branched hydrocarbon radical having, e.g., 1-18 carbon atoms and includes methyl, ethyl, n-propyl, isopropyl, n-butyl, sec -butyl, isobutyl, ie/t-butyl, n-pentyl, 2,2-dimethylpropyl, n-hexyl, n-heptyl, n-octyl, and the like.
  • alkenyl and alkynyl typically mean linear and branched hydrocarbon radicals having, e.g., 2-18 carbon atoms and one or more double or triple bond, respectively, and include ethenyl, propenyl, 3-buten-l-yl, 2-ethenylbutyl, 3-octen-l-yl, and the like, and propynyl, 2-butyn-l-yl, 3-pentyn-l-yl, and the like.
  • alkylene refers to a linear or branched divalent hydrocarbon radical having, e.g., 1-18 carbon atoms; and the terms “alkenylene” and “alkynylene” typically mean linear or branched divalent hydrocarbon radicals having, e.g., 2-18 carbon atoms, and one or more double or triple bonds, respectively.
  • alkylenes include, without being limited to, methylene, ethylene, propylene, butylene, 2-methylpropylene, pentylene, 2-methylbutylene, hexylene, 2-methylpentylene, 3-methylpentylene, 2,3- dimethylbutylene, heptylene, octylene, n-tridecanylene, n-tetradecanylene, n- pentadecanylene, n-hexadecanylene, n-heptadecanylene, n-octadecanylene, n- nonadecanylene, icosanylene, henicosanylene, docosanylene, tricosanylene, tetracosanylene, pentacosanylene, and the like.
  • Non-limiting examples of alkenylenes include 2-, 3-, 4-, 5- and 6-tridecenylene, tetradecenylenes such as myristoleylene, 2-, 3-, 4-, 5-, 6- and 7-pentadecenylene, hexadecenylenes such as palmitoleylene, 2-, 3-, 4-, 5-, 6-, 7- and 8-heptadecenylene, octadecenylenes such as oleylene, linoleylene, a-linoleylene, and the like; and non-limiting examples of alkynylenes include tridec-6-ynylene, undec-4- ynylene, and the like.
  • cycloalkyl means a mono- or bicyclic saturated hydrocarbyl group having, e.g., 3-7 carbon atoms such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like, that may be substituted, e.g., by one or more alkyl groups.
  • cycloalkylene and cycloalkenylene mean mono- or bicyclic hydrocarbyl groups having, e.g., 3-7 carbon atoms, and one or more double or triple bonds, respectively.
  • heterocyclic ring denotes a mono- or poly-cyclic non- aromatic ring of, e.g., 5-12 atoms containing at least two carbon atoms and at least three heteroatoms selected from sulfur, oxygen, nitrogen and boron, which may be saturated or unsaturated, i.e., containing at least one unsaturated bond. Preferred are 5- or 6-membered heterocyclic rings.
  • the heterocyclic ring may be substituted at any of the carbon atoms of the ring, e.g., by one or more alkyl groups.
  • Non-limiting examples of such radicals include 4,5-di-ie/t-butyl-l,3,2-dioxaborolanyl and 4,4,5,5-tetramethyl-l,3,2-dioxaborolanyl.
  • aryl denotes an aromatic carbocyclic group having, e.g., 6-14, carbon atoms consisting of a single ring or condensed multiple rings such as, but not limited to, phenyl, naphthyl, phenanthryl, and biphenyl.
  • the aryl may optionally be substituted by one or more groups each independently selected from halogen, (Ci-C 8 )alkyl, -0-(Ci-C 8 )alkyl, - COO(Ci-C 8 )alkyl, -CN, and -N0 2 .
  • arylene-diyl refers to a divalent radical derived from an aryl as defined herein by removal of a further hydrogen atom from any of the ring atoms, e.g., phenylene and naphthylene.
  • heteroaryl refers to a radical derived, e.g., from a 5-10-membered mono- or poly-cyclic heteroaromatic ring containing one to three, preferably 1-2, heteroatoms selected from N, O, or S.
  • mono-cyclic heteroaryls include, without being limited to, pyrrolyl, furyl, thienyl, thiazinyl, pyrazolyl, pyrazinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl, 1,2,3- triazinyl, 1,3,4-triazinyl, and 1,3,5-triazinyl.
  • Polycyclic heteroaryl radicals are preferably composed of two rings such as, but not limited to, benzofuryl, isobenzofuryl, benzothienyl, indolyl, quinolinyl, isoquinolinyl, imidazo[l,2-a]pyridyl, benzimidazolyl, benzthiazolyl, benzoxazolyl, pyrido[l,2-a]pyrimidinyl and 1,3-benzodioxinyl.
  • the heteroaryl may optionally be substituted by one or more groups each independently selected from halogen, (Ci-C 8 )alkyl, -0-(Ci-C 8 )alkyl, -COO(Ci-C 8 )alkyl, -CN, and -N0 2 . It should be understood that when a polycyclic heteroaryl is substituted, the substitution may be in any of the carbocyclic and/or heterocyclic rings.
  • heteroarylenediyl denotes a divalent radical derived from a "heteroaryl” as defined herein by removal of a further hydrogen atom from any of the ring atoms.
  • halogen refers to a halogen and includes fluoro, chloro, bromo, and iodo, but it is preferably fluoro or chloro.
  • amino acid refers to an organic compound comprising both amine and carboxylic acid functional groups, which may be either a natural or non- natural amino acid.
  • the twenty two amino acids naturally occurring in proteins are aspartic acid (Asp), tyrosine (Tyr), leucine (Leu), tryptophan (Trp), arginine (Arg), valine (Val), glutamic acid (Glu), methionine (Met), phenylalanine (Phe), serine (Ser), alanine (Ala), glutamine (Gin), glycine (Gly), proline (Pro), threonine (Thr), asparagine (Asn), lysine (Lys), histidine (His), isoleucine (He), cysteine (Cys), selenocysteine (Sec), and pyrrolysine (Pyl).
  • Non-limiting examples of other amino acids include citrulline (Cit), diaminopropionic acid (Dap), diaminobutyric acid (Dab), ornithine (Orn), aminoadipic acid, ⁇ -alanine, 1-naphthylalanine, 3-(l-naphthyl)alanine, 3-(2-naphthyl)alanine, ⁇ - aminobutiric acid (GABA), 3-(aminomethyl) benzoic acid, /?-ethynyl-phenylalanine, p- propargly-oxy-phenylalanine, m-ethynyl-phenylalanine, /?-bromophenylalanine, p- iodophenylalanine, /?-azidophenylalanine, /?-acetylphenylalanine, norleucine (Nle), azidonorleucine, 6-ethyny
  • peptide refers to a short chain of amino acid monomers (residues), e.g., a chain consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more amino acid residues, linked by peptide bonds, i.e., the covalent bond formed when a carboxyl group of one amino acid reacts with an amino group of another.
  • peptide moiety refers to a moiety of a peptide as defined herein after removal of the hydrogen bond from a carboxylic group, i.e., either the terminal or a side chain carboxylic group, thereof.
  • peptide moieties include, without being limited to, peptide moieties comprising the amino sequence Phe-Lys, Cit-Val, Gly-Phe-Leu-Gly, Asp-Glu-Val-Asp-, or Gly-Gly-Pro-Nle, or the modified amino acid sequence carboxybenzyl (Cbz) protected- Ala-Ala-Asn-ethylenediamine.
  • protecting group refers to an alcohol protecting group such as, without limiting, benzoyl, benzyl, methoxymethyl ether, ⁇ -methoxyethoxymethyl ether, methoxytrityl (4-methoxyphenyl) diphenylmethyl), dimethoxytrityl (bis-(4-methoxyphenyl)phenylmethyl), /?-methoxybenzyl ether, methylthiomethyl ether, pivaloyl, trityl (triphenylmethyl radical), 2-nitro-4,5- dimethoxybenzyl, and silyl ethers, e.g., trimethylsilyl (TMS), TBDMS, tri-iso- propylsilyloxymethyl (TOM), triisopropylsilyl (TIPS), and TBDPS ethers.
  • TMS trimethylsilyl
  • TBDMS tri-iso- propylsilyloxymethyl
  • TOM triisopropylsilyl
  • electron acceptor group refers to a group of atoms with a high electron affinity.
  • the invention provides a compound of the formula I, wherein R 1 is a linear or branched (Ci-C 8 )alkyl, preferably (Ci-C 4 )alkyl, more preferably methyl or ethyl.
  • the invention provides a compound of the formula I, wherein R 2 and R 3 each independently is a branched (C 3 -Ci 8 )alkyl or (C 3 -C7)cycloalkyl.
  • R 2 and R 3 each independently is a branched (C 3 -Ci 8 )alkyl or (C 3 - C7)cycloalkyl, and together with the carbon atom to which they are attached form a fused, spiro or bridged polycyclic ring.
  • R 2 and R 3 together with the carbon atom to which they are attached form adamantyl.
  • the invention provides a compound of the formula I, wherein R 5 and R 6 each independently is (Ci-C 8 )alkyl, preferably (C 3 -C 6 )alkyl, more preferably isopropyl, and together with the oxygen atoms to which they are attached form a heterocyclic ring.
  • R 5 and R 6 each is isopropyl and together with the oxygen atoms to which they are attached form 4,4,5, 5-tetramethyl- 1,3,2- dioxaborolanyl.
  • the invention provides a compound of the formula I, wherein at least one (i.e., one, two or three) of R 7 , R 8 and R 9 is H, and the other of R 7 , R 8 and R 9 each independently is an electron acceptor group as defined above. In certain particular such embodiments, R 7 , R 8 and R 9 each is H.
  • R 7 is an electron acceptor group as defined above, and R 8 and R 9 each is H; or R 8 is an electron acceptor group as defined above, and R 7 and R 9 each is H; or R 9 is an electron acceptor group as defined above, and R 7 and R 8 each is H, wherein said electron acceptor group is particularly halogen, -N0 2 or -CN.
  • the invention provides a compound of the formula I, wherein R 1 is a linear or branched (Ci-C 8 )alkyl, preferably (Ci-C 4 )alkyl, more preferably methyl or ethyl; R 2 and R 3 each independently is a branched (C 3 -Ci 8 )alkyl or (C 3 - C7)cycloalkyl, and together with the carbon atom to which they are attached form a fused, spiro or bridged polycyclic ring; R 5 and R 6 each independently is (Ci-C 8 )alkyl, preferably (C 3 -C6)alkyl, more preferably isopropyl, and together with the oxygen atoms to which they are attached form a heterocyclic ring; and at least one of R 7 , R 8 and R 9 is H, and the other of R 7 , R 8 and R 9 each independently is an electron acceptor group selected from halogen, -
  • R 2 and R 3 together with the carbon atom to which they are attached form adamantyl; or R 5 and R 6 each is isopropyl and together with the oxygen atoms to which they are attached form 4,4,5,5-tetramethyl-l,3,2- dioxaborolanyl.
  • R 1 is methyl; R 2 and R 3 together with the carbon atom to which they are attached form adamantly; R 5 and R 6 each is isopropyl and together with the oxygen atoms to which they are attached form 4,4,5,5-tetramethyl-l,3,2- dioxaborolanyl; R 7 , R 8 and R 9 are H; and Q is TBDMS (compound 1-1).
  • the invention provides a compound of the formula Ila or lib:
  • R 1 is selected from a linear or branched (Ci-Ci 8 )alkyl, or (C3-C7)cycloalkyl;
  • R 2 and R 3 each independently is selected from a branched (C 3 -Ci 8 )alkyl or (C 3 - R 2 and R 3
  • R 4 is a protecting group, such as those shown in Table 1 below;
  • Pep is a peptide moiety consisting of at least two amino acid residues and linked via a carboxylic group thereof;
  • L is absent or is a linker of the formula LI, L2 or L3, optionally substituted at the aromatic ring with one or more substituents each independently selected from (Q- Cis)alkyl or (C 3 -C7)cycloalkyl, wherein M is absent or is -O- or -NH-, and the asterisk represents the point of attachment to the group Y, provided that M is -O- or -NH- unless R 4 is 4,4,5, 5-tetramethyl-l,3,2-dioxaborolanyl or -B(OH) 2 ;
  • Y is absent or is -0-, provided that Y is -O- unless R 4 is 4,4,5,5-tetramethyl- 1,3,2-dioxaborolanyl or -B(OH) 2 , and L is absent;
  • R 10 each independently is H or -(Ci-Ci 8 )alkyl
  • X is a linker of the formula -X 1 -X 2 -, wherein X 1 is selected from (Q- Ci 8 )alkylene, (C 2 -Ci 8 )alkenylene, (C 2 -Ci 8 )alkynylene, (C 3 -C7)cycloalkylene, (C 3 - C7)cycloalkenylene, (C 6 -C 14 )arylene-diyl, (C i-C i 8 )alkylene-(C6-C 14 )arylene-diyl, heteroarylenediyl, or (Ci-Ci 8 )alkylene-heteroarylenediyl, said (Ci-Cis)alkylene, (C 2 - Ci 8 )alkenylene, (C 2 -Ci 8 )alkynylene, (C 3 -C7)cycloalkylene, (C 3 -C7)cycloalkenylene, (C 6 - Ci 4
  • R 14 is a reactive group such as -0-(Ci-Ci 8 )alkyl, -N 3 , -C ⁇ CH, N-succinimidyloxy, 3-sulfo-N-succinimidyloxy, pentafluorophenyloxy, 4-nitrophenyloxy, N-imidazolyl, and N-iH-benzo[d] [l,2,3]triazoloxy.
  • protecting group refers to an alcohol protecting group as defined with respect to the compound of the formula I, as well as to certain cleavable groups including enzyme cleavable groups such as monosaccharide moieties linked through a carbon atom thereof.
  • This protecting group is further referred to herein, with respect to the compounds of the formula Illa/IIIb and IVa/IVb, as a "caging group”. Particular protecting/caging groups are those shown in Table 1.
  • reactive group refers to any group capable of reacting with a functional group (i.e., amine, carboxylic acid, sulfhydryl, hydroxyl, or aldehyde group) of a fluorophore.
  • Examples of such groups include -0-(Ci-Ci 8 )alkyl, -N 3 , -C ⁇ CH, N-succinimidyloxy, 3-sulfo-N-succinimidyloxy, pentafluorophenyloxy, 4-nitrophenyloxy, N-imidazolyl, and N-iH-benzo[d] [ 1,2,3] triazoloxy (see Table 2).
  • the invention provides a compound of the formula Ila or lib, wherein R 1 is a linear or branched (Ci-C 8 )alkyl, preferably (Ci-C 4 )alkyl, more preferably methyl or ethyl.
  • the invention provides a compound of the formula Ila or
  • R and R each independently is a branched (C 3 -Ci 8 )alkyl or (C 3 -
  • R and R each independently is a branched (C 3 - Ci 8 )alkyl or (C 3 -C7)cycloalkyl, and together with the carbon atom to which they are 2 attached form a fused, spiro or bridged polycyclic ring. In particular such embodiments, R and R together with the carbon atom to which they are attached form adamantyl.
  • the invention provides a compound of the formula Ila or lib, wherein at least one (i.e., one, two or three) of R 7 , R 8 and R 9 is H, and the other of R 7 ,
  • R 8 and R 9 each independently is an electron acceptor group as defined above.
  • R 7 , R 8 and R 9 each is H.
  • R 7 is an electron acceptor group as defined above, and R 8 and R 9 each is H; or R 8 is an electron acceptor group as defined above, and R 7 and R 9 each is H; or R 9 is an electron acceptor group as defined above, and R 7 and R 8 each is H, wherein said electron acceptor group is particularly halogen, -N0 2 or -CN.
  • X 1 is (C 6 - Ci 4 )arylene-diyl or (Ci-C 4 )alkylene-(C 6 -Ci 4 )arylene-diyl, wherein said (C 6 -Ci 4 )arylene- diyl is, e.g., phenylene, naphthylene, phenanthrylene, or biphenylene; and X 2 is -C(O)- linked to an carbon atom of the arylene-diyl.
  • X is the linker: , i.e., X 1 is -(CH 2 )-/?ara-phenylene and X 2 is -C(O)-.
  • the invention provides a compound of the formula Ila or lib, wherein R 14 is N-succinimidyloxy, or 3-sulfo-N-succinimidyloxy.
  • the invention provides a compound of the formula Ila or lib, wherein R 1 is a linear or branched (Ci-C 8 )alkyl, preferably (Ci-C 4 )alkyl, more preferably methyl or ethyl; R 2 and R 3 each independently is selected from a branched (C 3 - Ci 8 )alkyl or (C 3 -C7)cycloalkyl, and together with the carbon atom to which they are attached form a fused, spiro or bridged polycyclic ring; at least one of R 8° and R 9 y is H, and the other of R 7 , R 8 and R 9 each independently is an electron acceptor group selected from halogen, -N0 2 or -CN; X is a linker of the formula -X 1 -X 2 -, wherein X 1 is (Q- Ci 8 )alkylene, (C 6 -C 14 )arylene-diyl, or (
  • R 2 and R 3 together with the carbon atom to which they are attached form adamantyl; or X is a linker of the formula - ⁇ - ⁇ 2 -, wherein Xi is (C 6 -Ci 4 )arylene-diyl or (Ci-C 4 )alkylene-(C 6 -Ci 4 )arylene-diyl, wherein said (C 6 - Ci 4 )arylene-diyl is phenylene, naphthylene, phenanthrylene, or biphenylene; and X 2 is - C(O)- linked to any carbon atom of the arylene-diyl.
  • Xi is (C 6 -Ci 4 )arylene-diyl or (Ci-C 4 )alkylene-(C 6 -Ci 4 )arylene-diyl, wherein said (C 6 - Ci 4 )arylene-diyl is phenylene, naphthy
  • R 2 and R 3 together with the carbon atom to which they are attached form adamantyl; and/or Xi is -(CH 2 )-/?ara-phenylene and X 2 is -C(O)-.
  • R 1 is methyl
  • R 2 and R 3 together with the carbon atom to which they are attached form adamantyl
  • X 1 is -(CH 2 )-/?ara-phenylene
  • X 2 is -C(O)-, e.g., such compounds wherein at least one of R 7 , R 8 and R 9 y is H, and the other of R 7 , R 8 and R 9 each independently is a halogen.
  • the invention provides a compound of the formula Ila or lib as defined in any one of the embodiments above, wherein (i) Y is -0-, L is absent or a linker of the formula LI, L2 or L3, wherein M is -O- or -NH-, and R 4 is a protecting group; or (ii) Y is absent, L is absent, and R 4 is 4,4,5,5-tetramethyl-l,3,2-dioxaborolanyl or - B(OH) 2 .
  • R 1 is methyl; R 2 and R 3 together with the carbon atom to which they are attached form adamantyl; R 7 , R 8 and R 9 are H; Y is -0-; L is absent; R 4 is TBDMS; Xi is -(CH 2 )-/?ara-phenylene; X 2 is -C(O)-; and R 14 is N- succinimidyloxy (compounds IIa-1 and IIb-1, Table 3).
  • Table 3 Specific compounds of the formula Ila/IIb described herein
  • the present invention provides a conjugate of the formula Ilia or Illb:
  • R 1 is selected from a linear or branched (Ci-Ci 8 )alkyl, or (C 3 -C 7 )cycloalkyl;
  • R 2 and R 3 each independently is selected from a branched (C 3 -Ci 8 )alkyl or (C 3 - cloalkyl, or R 2 and R 3
  • R 4 is a caging group, such as those shown in Table 1;
  • Pep is a peptide moiety consisting of at least two amino acid residues and linked via a carboxylic group thereof;
  • L is absent or is a linker of the formula LI, L2 or L3, optionally substituted at the aromatic ring with one or more substituents each independently selected from (Ci- Ci 8 )alkyl or (C 3 -C 7 )cycloalkyl, wherein M is absent or is -O- or -NH-, and the asterisk represents the point of attachment to the group Y, provided that M is -O- or -NH- unless R 4 is 4,4,5, 5-tetramethyl-l,3,2-dioxaborolanyl or -B(OH) 2 ;
  • Y is absent or is -0-, provided that Y is -O- unless R 4 is 4,4,5, 5-tetramethyl- 1,3,2- dioxaborolanyl or -B(OH) 2 , and L is absent;
  • R 10 each independently is H or -(Ci-Cis)alkyl
  • X is a linker of the formula - ⁇ - ⁇ 2 -, wherein Xi is selected from (Ci- Ci 8 )alkylene, (C 2 -Ci 8 )alkenylene, (C 2 -Ci 8 )alkynylene, (C3-C 7 )cycloalkylene, (C 3 - C 7 )cycloalkenylene, (C 6 -C i 4 )arylene-diyl, (C i-C i 8 )alkylene-(C 6 -C i 4 )arylene-diyl, heteroarylenediyl, or (Ci-Cis)alkylene-heteroarylenediyl, said (Ci-Cis)alkylene, (C 2 - Ci 8 )alkenylene, (C 2 -Ci 8 )alkynylene, (C 3 -C 7 )cycloalkylene, (C 3 -C 7 )cycloalkeny
  • Z is a moiety of a fluorophore or a derivative thereof.
  • fluorophore refers to a fluorescent chemical compound, typically containing several combined aromatic groups, or plane or cyclic molecules having several ⁇ bonds, which can re-emit light upon light excitation.
  • fluorophores include fluorescein-based compounds (fluorescein analogues), rhodamine-based compounds (rhodamine analogues), coumarin-based compounds (coumarin analogues), cyanines such as Cy5, Cy5.5, Cy5.18, Cy7, Cy7.18, and QCy, and boron-dipyrromethene (BODIPY)-based compounds.
  • the invention provides a conjugate of the formula Ilia or Illb, wherein R 1 is a linear or branched (Ci-C 8 )alkyl, preferably (Ci-C 4 )alkyl, more preferably methyl or ethyl.
  • the invention provides a conjugate of the formula Ilia or
  • R 2 and R 3 each independently is a branched (C 3 -Ci 8 )alkyl or (C 3 -
  • R and R each independently is a branched (C 3 - Cis)alkyl or (C3-C7)cycloalkyl, and together with the carbon atom to which they are attached form a fused, spiro or bridged polycyclic ring. In particular such embodiments, R and R together with the carbon atom to which they are attached form adamantyl.
  • the invention provides a conjugate of the formula Ilia or
  • R 8 and R 9 each independently is an electron acceptor group as defined above.
  • R 7 , R 8 and R 9 each is H.
  • R 7 is an electron acceptor group as defined above, and R 8 and R 9 each is H; or R 8 is an electron acceptor group as defined above, and R 7 and R 9 each is H; or R 9 is an electron acceptor group as defined above, and R 7 and R 8 each is H, wherein said electron acceptor group is particularly halogen, -N0 2 or -CN.
  • Xi is (C 6 - Ci 4 )arylene-diyl or (Ci-C 4 )alkylene-(C 6 -Ci 4 )arylene-diyl, wherein said (C 6 -Ci 4 )arylene- diyl is, e.g., phenylene, naphthylene, phenanthrylene, or biphenylene; and X 2 is -C(O)- linked to any carbon atom of the arylene-diyl.
  • Xi is -(CH 2 )-/?ara- phenylene and X 2 is -C(O)-.
  • the invention provides a conjugate of the formula Ilia or Illb, wherein the fluorophore Z is selected from the BODIPY derivative identified herein as Zl, the fluorescein derivative identified herein as Z2, the Cy5 derivative identified herein as Z3, or the QCy derivative identified herein as Z4 (Table 4).
  • the invention provides a conjugate of the formula Ilia or Illb, wherein R 1 is a linear or branched (Ci-C 8 )alkyl, preferably (Ci-C 4 )alkyl, more preferably methyl or ethyl; R 2 and R 3 each independently is selected from a branched (C 3 - Ci 8 )alkyl or (C 3 -C7)cycloalkyl, and together with the carbon atom to which they are 7 8 9 attached form a fused, spiro or bridged polycyclic ring; at least one of R ⁇ R° and R y is H,
  • R , R and R each independently is an electron acceptor group selected from halogen, -N0 2 or -CN;
  • R and R together with the carbon atom to which they are attached form adamantyl; or X is a linker of the formula - ⁇ - ⁇ 2 -, wherein X 1 is (C 6 -Ci 4 )arylene-diyl or (Ci-C 4 )alkylene-(C 6 -Ci 4 )arylene-diyl, wherein said (C 6 - Ci 4 )arylene-diyl is phenylene, naphthylene, phenanthrylene, or biphenylene; and X 2 is - C(O)- linked to any carbon atom of the arylene-diyl.
  • X 1 is (C 6 -Ci 4 )arylene-diyl or (Ci-C 4 )alkylene-(C 6 -Ci 4 )arylene-diyl, wherein said (C 6 - Ci 4 )arylene-diyl is phenylene, naphthylene,
  • R and R together with the carbon atom to which they are attached form adamantyl; and/or Xi is -(CH 2 )-/?izra-phenylene and X 2 is -C(O)-. Specific examples of such
  • embodiments are those wherein R is methyl; R and R together with the carbon atom to which they are attached form adamantyl; X 1 is -(CH 2 )-/?izra-phenylene; and X 2 is -C(O)-,
  • R 7 8 9 7 8 e.g., such compounds wherein at least one of R , R and R y is H, and the other of R , R and R 9 each independently is a halogen.
  • the invention provides a compound of the formula Ilia or Illb as defined in any one of the embodiments above, wherein (i) Y is -0-, L is absent or a linker of the formula LI, L2 or L3, wherein M is -O- or -NH-, and R 4 is a caging group; or (ii) Y is absent, L is absent, and R 4 is 4,4,5, 5-tetramethyl- l,3,2-dioxaborolanyl or -B(OH) 2 .
  • R is methyl; R and R together with the carbon atom to which they are attached form adamantly; Xi is -(CH 2 )-/?ara-phenylene; X 2 is - C(O)-; Z is selected from groups Zl, 72, Z3 or Z4; and (i) R 7 , R 8 and R 9 are H; Y is -0-; L is absent; and R 4 is TBDMS (compounds IIIa- 1-4 wherein Z is Z1-Z4, respectively; and IIIb-1-4 wherein Z is Z1-Z4, respectively); or (ii) R 7 is CI; R 8 and R 9 are H; Y is -0-; L is absent; and R 4 is galactosyl (compounds IIIa-5-8 wherein Z is Z1-Z4, respectively; and IIIb-5-8 wherein Z is Z1-Z4, respectively).
  • R 1 is selected from a linear or branched (Ci-Ci 8 )alkyl, or (C3-C7)cycloalkyl;
  • R 2 and R 3 each independently is selected from a branched (C 3 -Ci 8 )alkyl or (C 3 -
  • R 4 is H, or a caging group such as those shown in Table 1;
  • Pep is a peptide moiety consisting of at least two amino acid residues and linked via a carboxylic group thereof;
  • L is absent or is a linker of the formula LI, L2 or L3, optionally substituted at the aromatic ring with one or more substituents each independently selected from (Q- Cis)alkyl or (C 3 -C7)cycloalkyl, wherein M is absent or is -O- or -NH-, and the asterisk represents the point of attachment to the group Y, provided that M is -O- or -NH- unless R 4 is 4,4,5, 5-tetramethyl-l,3,2-dioxaborolanyl or -B(OH) 2 , and when R 4 is H, L is absent;
  • Y is absent or is -0-, provided that Y is -O- unless R 4 is 4,4,5, 5-tetramethyl- 1,3,2- dioxaborolanyl or -B(OH) 2 , and L is absent;
  • R 10 each independently is H or -(Ci-Ci 8 )alkyl
  • Table 6 Certain ⁇ * acceptor groups with respect to compounds of the formula IVa or IVb
  • ⁇ * acceptor group refers to any group containing a ⁇ acceptor system capable of accepting electrons.
  • the invention provides a compound of the formula IVa or IVb, wherein R 1 is a linear or branched (Ci-C 8 )alkyl, preferably (Ci-C 4 )alkyl, more preferably methyl or ethyl.
  • the invention provides a compound of the formula IVa or
  • R 2 and R 3 each independently is a branched (C3-Ci 8 )alkyl or (C 3 -
  • R and R each independently is a branched (C 3 - Ci 8 )alkyl or (C 3 -C 7 )cycloalkyl, and together with the carbon atom to which they are attached form a fused, spiro or bridged polycyclic ring.
  • R and R each independently is a branched (C 3 - Ci 8 )alkyl or (C 3 -C 7 )cycloalkyl, and together with the carbon atom to which they are attached form a fused, spiro or bridged polycyclic ring.
  • R 2 and R 3 together with the carbon atom to which they are attached form adamantyl.
  • the invention provides a compound of the formula IVa or IVb, wherein R is H, or an electron acceptor group selected from halogen or -CN attached either ortho or para to the -Y-L-R 4 group.
  • R 7 is halogen, e.g., CI, or -CN, attached ortho to the -Y-L-R 4 group.
  • E is -CN, -COOH, -COOCH 3 , -COOC 2 H 5 , -COOC 3 H 7 , -COOCH(CH 3 ) 2 , or -COOC(CH 3 ) 3 .
  • R 1 is methyl; R 2 and R 3 together with the carbon atom to which they are attached form adamantyl; R is H, or is an electron acceptor group selected from halogen or -CN, attached ortho to the -Y-L-R 4 group; and E is -CN, -COOH, or -COO(Ci-C 4 )alkyl such as - COOCH 3 , -COOC2H5, -COOC3H7, -COOCH(CH 3 ) 2 , or -COOC(CH 3 ) 3 .
  • E is -CN, -COOH, -COOCH 3 , or -COOC(CH 3 ) 3 .
  • A is acrylonitrile, acrylic acid, methylacrylate or tert-buty ⁇ acrylate substituent, respectively, attached ortho to the -Y-L-R 4 group.
  • the invention provides a compound of the formula IVa or IVb as defined in any one of the embodiments above, wherein (i) Y is -0-; L is absent; and R 4 is H; (ii) Y is -0-; L is either absent or a linker of the formula LI, L2 or L3 as defined above, wherein M is -O- or -NH-; and R 4 is a caging group such as those shown in Table 1, e.g., phosphonate, but provided that said caging group is not 4,4,5, 5-tetramethyl-l, 3,2- dioxaborolanyl or -B(OH) 2 ; (iii) Y is -0-; L is a linker of the formula LI, L2 or L3 as defined above, wherein M is absent; and R 4 is 4,4,5, 5-tetramethyl-l, 3,2-dioxaborolanyl or -B(OH) 2 ; or (iv) Y is absent;
  • the present invention provides a composition
  • a composition comprising a carrier, and a dioxetane-based chemiluminescence probe as disclosed herein, i.e., either a fluorophore-tethered dioxetane-based chemiluminescence probe of the formula Illa/IIIb or a ⁇ * acceptor group-containing chemiluminescence probe of the formula IVa/IVb, each as defined in any one of the embodiments above.
  • the composition of the present invention comprises a chemiluminescence probe of the formula Illa/IIIb selected from those listed in Table 5, or a chemiluminescence probe of the formula IVa/IVb selected from those listed in Table 7.
  • the composition of the present invention may thus be used for diagnostics and/or in vivo imaging.
  • Triggered chemiluminescence emission can provide a highly sensitive readout of biological analytes. Chemiluminescence does not require light excitation, thereby drastically reducing background from autofluorescence and photoactivation of functional groups.
  • bioluminescence i.e., chemiluminescence derived from living systems that express bioluminescent enzymes such as luciferase
  • small molecule chemiluminescence can be used with wild-type animals and opens up exciting opportunities for clinical imaging.
  • compositions of the present invention may thus be inter alia pharmaceutical compositions, wherein said carrier is a pharmaceutically acceptable carrier.
  • chemiluminescence probes of the formulas Illa/IIIb and rVa/IVb as disclosed herein have a cleavable caging group (R 4 ), e.g., an enzyme cleavable group, wherein removal of said cleavable group by the analyte of interest, e.g., in the presence of an enzyme capable of cleaving said enzyme cleavable group, generates an unstable phenolate-dioxetane species that decomposes through a chemiexcitation process to produce the excited intermediate, which then further decays to its ground-state through emission of light.
  • R 4 cleavable caging group
  • chemiluminescence probes exemplified herein having ⁇ -galactosyl as the caging group, are the fluorophore-tethered dioxetane-based chemiluminescence Probes 2 and 3, and the ⁇ * acceptor group -containing chemiluminescence Probes 6-9, and their chemiluminescent kinetic profiles in the presence vs. absent of ⁇ -galactosidase is shown in Studies 1 and 2.
  • Additional probes exemplified in Study 2 are the ⁇ * acceptor group- containing chemiluminescence Probes 11 and 12, having phosphonate or 2,4- dinitrobenzene sulfonate as the caging group, which are capable of detecting alkaline- phosphatase and GSH, respectively; and the ⁇ * acceptor group-containing chemiluminescence Probe 10, having 4,4,5,5-tetramethyl-l,3,2-dioxaborolanyl as the caging group, which is capable of detecting hydrogen peroxide.
  • chemiluminescence probes may have a caging group comprising a peptide moiety consisting of two or more amino acid residues, e.g., a peptide moiety-containing caging group as shown in Table 1.
  • Such peptide moieties may comprise an amino acid sequence cleavable by a specific enzyme, and probes containing such caging groups may thus be used for detecting the presence of said enzyme.
  • cathepsin B a lysosomal cysteine protease that plays an important role in intracellular proteolysis and is overexpressed in premalignant lesions and various pathological conditions, as well as in cancers, e.g., in tumor endothelial cells and many other tumor cells in the lysosome (Miller et al., 2009).
  • Cathepsin B- cleavable peptides include, without limiting, peptides comprising the amino acid sequence Phe-Lys, Cit-Val, or Gly-Phe-Leu-Gly.
  • cathepsin K a lysosomal cysteine protease involved in bone remodeling and resorption, which is expressed predominantly in osteoclasts and overexpressed extracellularly in bone neoplasms (Segal et ah, 2009).
  • Cathepsin K-cleavable peptides include, without being limited to, peptides comprising the amino acid sequence Gly-Gly-Pro-Nle.
  • a further particular such enzyme is legumain, a lysosomal enzyme overexpressed in tumor cells (Stern et ah, 2009).
  • Legumain-cleavable peptides include, without limiting, peptides comprising the modified amino acid sequence Cbz-Ala-Ala-Asn-ethylenediamine.
  • compositions according to the present invention may be prepared by conventional techniques, e.g., as described in Remington: The Science and Practice of Pharmacy, 19 th Ed., 1995.
  • the compositions can be prepared, e.g., by uniformly and intimately bringing the active agent, i.e., the dioxetane-based chemiluminescence probe, into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulation.
  • the compositions may be in liquid, solid or semisolid form and may further include pharmaceutically acceptable fillers, carriers, diluents or adjuvants, and other inert ingredients and excipients.
  • the pharmaceutical composition of the present invention is formulated as nanoparticles.
  • a pharmaceutical composition according to the present invention can be formulated for any suitable route of administration, e.g., for parenteral administration such as intravenous, intraarterial, intrathecal, intrapleural, intratracheal, intraperitoneal, intramuscular or subcutaneous administration, topical administration, oral or enteral administration, or for inhalation.
  • parenteral administration such as intravenous, intraarterial, intrathecal, intrapleural, intratracheal, intraperitoneal, intramuscular or subcutaneous administration, topical administration, oral or enteral administration, or for inhalation.
  • such a composition is formulated for intravenous or intraperitoneal administration, or for subcutaneous administration, e.g., by an alzet pump implanted subcutaneous.
  • the pharmaceutical composition of the invention may be in the form of a sterile injectable aqueous or oleaginous suspension, which may be formulated according to the known art using suitable dispersing, wetting or suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent.
  • Acceptable vehicles and solvents include, e.g., water, Ringer's solution and isotonic sodium chloride solution.
  • chemiluminescence emission of the probes of the present invention can be detected utilizing any technique or procedure known in the art.
  • Optical molecular imaging is a promising technique that provides a high degree of sensitivity and specificity in tumor margin detection. Furthermore, existing clinical applications have proven that optical molecular imaging is a powerful intraoperative tool for guiding surgeons performing precision procedures, thus enabling radical resection and improved survival rates.
  • An example of a clinically approved instrument for minimally invasive surgical procedures under fluorescence guidance is the da Vinci Surgical System (Haber et ah, 2010).
  • This instrument is featured with a 3D HD vision system for a clear and magnified view inside a patient's body and allows surgeons to perform complex and routine procedures through a few small openings, similar to traditional laparoscopy.
  • the following systems have already been applied in surgeries for breast cancer, liver metastases and bypassing graft surgery: The Hamamatsu's Photodynamic Eye (PDETM), ArtemisTM and Novadaq SPYTM (Novadaq Technologies Inc., Toronto, Canada) (Chi et ah, 2014).
  • PDETM The Hamamatsu's Photodynamic Eye
  • ArtemisTM ArtemisTM
  • Novadaq SPYTM Novadaq Technologies Inc., Toronto, Canada
  • Several existing intraoperative NTR fluorescence molecular imaging systems were evaluated in clinical trials; including, Fluobeam ® , FLARETM and GXMI Navigator. They have played an important role in operation convenience, improving image assessment and increasing detection depth (Chi et ah, 2014).
  • Probe 1 Enol ether lg (100 mg, 0.157 mmol) and few milligrams of methylene blue were dissolved in 10 ml of DCM. Oxygen was bubbled through the solution while irradiating with yellow light. Reaction was monitored by TLC. Upon completion, the solvent was concentrated under reduced pressure and the crude product was passed through silica gel column (Hex:EtOAc 60:40) to remove methylene blue. The solvent was evaporated and the product was dissolved in MeOH (3ml).
  • Probe 2 Amine-functionalized fluorescein 2c (41 mg, 0.073 mmol) and NHS ester lk (65.5 mg, 0.073 mmol) were dissolved in 1 ml of DMF. The flask was kept in the dark by covering it with an aluminum foil and 2 drops of Et 3 N were added. Reaction was stirred at RT and was monitored by RP-HPLC. Upon completion, the solvent was evaporated under reduced pressure and the resulting yellow solid was dissolved in 1.5 ml of MeOH. Potassium carbonate (40 mg, 0.29 mmol) was added and removal of sugar acetates was monitored by RP-HPLC.
  • Compound 3b Compound 3a (Karton-Lifshin et ah , 2012) (180 mg, 0.30 mmol), N-Boc-ethylenediamine (96 mg, 0.60 mmol) and 2-(lH-benzotriazol-l-yl)-l, 1,3,3- tetramethyluronium hexafluorophosphate (HBTU) (228 mg, 0.60 mmol) were dissolved in 3 ml of DMF. Triethylamine (120 ⁇ , 0.86 mmol) was added and reaction mixture was stirred at RT. Reaction was monitored by RP-HPLC (10-90% ACN in water, 20 min).
  • Probe 3 Amine-functionalized QCy 3c (60 mg, 0.081 mmol) and NHS ester lk (72.5 mg, 0.081 mmol) were dissolved in 1 ml of DMF. The flask was kept in the dark by covering it with an aluminum foil and 2 drops of Et 3 N were added. Reaction was stirred at RT and was monitored by RP-HPLC. Upon completion, the solvent was evaporated under reduced pressure and the resulting yellow solid was dissolved in 1.5 ml of MeOH. Potassium carbonate (45 mg, 0.33 mmol) was added and removal of sugar acetates was monitored by RP-HPLC.
  • mice Six 7-weeks old BALB/c female mice (Harlan Laboratories Israel Ltd., Jerusalem, Israel) were anesthetized using a mixture of ketamine (100 mg/kg) and xylazine (12 mg/kg) injected subcutaneously. Then, mice were injected intraperitoneally or subcutaneously with 50 of the probe solution, previously incubated in PBS 7.4 (in the presence or absence of ⁇ -galactosidase) for 30 minutes. The mice were imaged and chemiluminescence was monitored for up to 15 min by intravital non-invasive bioluminescence imaging system (Photon Imager; Biospace Lab, Paris, France). Images were obtained by Photo-Acquisition software (Biospace Lab) and analyzed by M3Vision Software (Biospace Lab).
  • Optical imaging holds several advantages over other imaging modalities (e.g., radiography, magnetic resonance imaging and ultrasound). Fluorescent molecular probes at the NIR range, possess good spatial resolution and greater depth penetration than other wavelengths. In vivo imaging is required for determining the limit of detection and signal penetration in live tissues. This data cannot be obtained by in vitro methods. These preliminary experiments use the minimal number of animals to evaluate our new probe in terms of proof of concept (Redy-Keisar et ah, 2015b). At the end of the experiment, mice were euthanized by cervical dislocation.
  • Chemiluminescence images were acquired using Olympus LV200 inverted microscope fitted with an EMCCD camera (Hamamatsu C9100-13).
  • HEK293 LacZ stable cells (amsbio SC003) and HEK293-WT cells (control) were grown on 35mm glass bottom petri dishes at 37°C for 24 h.
  • Cell culture medium was changed to Molecular Probes ® Live Cell Imaging Solution containing 5 ⁇ of Probe 3. Cells were incubated for another 20 minutes at 37°C. Thereafter, images were recorded with 20 minutes exposure time.
  • Dioxetane is usually prepared by reaction of singlet oxygen with a double bond. Since conditions for production of singlet oxygen are not always compatible with the presence of a fluorophore, we developed a late-stage functionalization chemistry that allows attachment of the fluorophore after preparation of the dioxetane.
  • the synthesis of a dioxetane- fluorophore conjugate designed for activation by ⁇ -galactosidase (as a model enzyme) is shown in Scheme 9.
  • Probes 2 and 3 are composed of dioxetane tethered with the fluorogenic dyes fluorescein and QCy (Karton-Lifshin et ah, 2011; Karton-Lifshin et ah, 2012), respectively.
  • Probe 1 is a basic Schaap-dioxetane without a tethered dye. The chlorine substituent on the phenolic ring was introduced in order to decrease the pKa.
  • Probe 1 was unaffected by light over a 12 h period.
  • Probe 3 exhibited significantly higher photostability than Probe 2.
  • the light-induced decomposition half-life of Probe 2 was 45 min, whereas the half-life of Probe 3 was about 6 h. No decomposition of any of the probes was observed when the solutions were kept in the dark.
  • a possible mechanism for lightinduced decomposition of the dioxetane-fluorophore conjugates might involve electron transfer from the excited dye to the peroxy-dioxetane bond (Wakimoto et ah, 2015).
  • Fig. 3 illustrates a possible light-induced decomposition mechanism.
  • the fluorophore of conjugate I is excited by visible light to form excited species II.
  • Electron transfer from the LUMO of the excited fluorophore to the antibonding ⁇ * orbital of 0-0 peroxide bond results in bond cleavage and subsequent decomposition of the dioxetane into benzoate III and adamantanone.
  • the observed light instability of Probes 2 and 3 underlines the advantage of our late-stage functionalization strategy over previous reported synthetic methods for dioxetane-fluorophore conjugates.
  • the oxidation of the enol ether to the dioxetane is usually performed by singlet oxygen generated from oxygen by a light source and a photosensitizer. Such conditions, if applied after the conjugation of the fluorophore, could lead to decomposition of the dioxetane.
  • Probe 1 (dioxetane-fiuorescein conjugate), upon activation by ⁇ -galactosidase, decomposed to emit greenish light with maximum emission wavelength of 535 nm exclusively (Fig. 4, panel B).
  • the observed chemiluminescence spectrum of the probe was almost identical to its fluorescence spectrum (dotted line); indicating a complete energy transfer to the fluorescein acceptor.
  • QCy only blue emission with maximum wavelength of 470 nm was obtained for the dioxetane-dye mixture (Fig. 4, panel C).
  • Probe 3 (QCy- tethered dioxetane) decomposed to emit NIR light with maximum emission wavelength of 714 nm (Fig. 4, panel D).
  • the chemiluminescence spectrum of Probe 3 was found to be almost identical to its fluorescence spectrum (dotted line). This observation clearly supports the energy transfer mechanism illustrated in Scheme 8, and properly demonstrates the significance of covalent conjugation between the dioxetane and the dye.
  • ⁇ CL chemiluminescence quantum yield
  • Probe 2 exhibited brighter chemiluminescence than the other probes since its energy transfer is resulted with an excited fluorescein species (a dye with 90% fluorescence quantum yield). We therefore selected Probe 2 and demonstrated its ability to detect ⁇ - galactosidase (Fig. 6). The probe was incubated with different concentrations of ⁇ - galactosidase and total chemiluminescence emission was collected over 1 h period. Linear correlation was observed between enzyme concentrations and integrated chemiluminescence signal, enabling quantification of enzyme concentration. We determined a detection limit (blank control+3 SD) of 4.0x10 " units/mL.
  • Probes 2 and 3 were selected for further evaluation in vivo; notably, Probe 3 emits light within the NIR region.
  • HEK293 transfected by LacZ
  • HEK293-WT control cells were incubated with Probe 3 and then imaged by the LV200 (Fig. 9) using a 20x objective (NA 0.75).
  • Probe 3 was able to produce chemiluminescence images of the HEK293-LacZ cells (Fig. 9, panel b), while no chemiluminescence signal at all was observed by the HEK293- WT cells (Fig. 9, panel d).
  • the HEK293-LacZ cells were fixed by using 4% formaldehyde and permeabilized with 0.1% Triton X-100. The cells were then incubated with Probe 3 and imaged by the microscope using a 60x objective (NA 1.42). As can be seen in Fig. 10 (panel a, transmitted light; panel b, chemiluminescence), the cells became visible, exhibiting a clear chemiluminescence emission.
  • the chemiluminescent emission of the fluorophoretethered dioxetane probes was significantly amplified in comparison to a classic dioxetane probe through an energytransfer mechanism.
  • the synthesized probes produced light of various colors that matched the emission wavelength of the excited tethered fluorophore.
  • Benzoate 5a 2-Chloro-3-hydroxybenzaldehyde (la, 312 mg, 2 mmol) was dissolved in MeOH (5 mL). Oxone (615 mg, 2 mmol) and In(OTf) 3 (112 mg, 0.22 mmol) were added at RT. The reaction mixture was heated to reflux and monitored by RP-HPLC. After the reaction was completed the mixture was filtered and the filtrate was concentrated using a rotary evaporator. Purification by column chromatography (Hex:EtOAc 30:70) afforded benzoate 5a as a white solid (339 mg, 92% yield).
  • Phenol 5b Enol ether If (100 mg 0.3 mmol) and few milligrams of methylene blue were dissolved in 20 ml of DCM. Oxygen was bubbled through the solution while irradiating with yellow light. The reaction was monitored by RP-HPLC. After completion, the reaction mixture was concentrated by evaporation under reduced pressure. The crude product was purified by preparative RP-HPLC (gradient of ACN in water). The product was obtained as a white solid.
  • Phenol 7b Compound 7c (60 mg, 0.15 mmol) was reacted according to the general procedure. The product was obtained as a white solid (20 mg, 31% yield).
  • Phenol 9b Compound 9c (120 mg, 0.35 mmol) was reacted according to the general procedure. The product was obtained as a white solid (52 mg, 38% yield).
  • Probe 5 was synthesized from compound lg in Scheme 9, by deoxygenation of the double bond and removal of the acetyl groups from the galactose moiety.
  • Scheme 21 Molecular structure of Probe 5
  • Probe 6. Compound 6d (133 mg, 0.22 mmol) was reacted according to general procedure. The product obtained as a white solid (64 mg, 63% yield).
  • Probe 7. Compound 7d (273 mg 0.33 mmol) was reacted according to general procedure. The product obtained as a white solid (90 mg, 40% yield).
  • 1H NMR 400 MHz, MeOD
  • ⁇ 7.87 (d, 7 8.4 Hz, 1H)
  • 7.83 (d, 7 16.3 Hz, 1H)
  • 7.74 (d, 7 8.4 Hz, 1H)
  • 7.33 (d, 7 8.5 Hz, 2H)
  • 7.09 (d, 7 8.4 Hz, 2H)
  • Probe 8. Compound 8d (130 mg 0.17 mmol) was reacted according to general procedure. The product obtained as a white solid (69 mg, 65% yield).
  • Probe 9. Compound 9d (160 mg, 0.2 mmol) was reacted according to general procedure. The product obtained as a white solid (61 mg, 46% yield).
  • Probe 10 Borane 10b (441 mg, 0.95 mmol), NaOH 114 mg, 2.8 mmol) were dissolved in 5 ml of 4: 1 solution THF:H 2 0. Reaction mixture was stirred at 40°C overnight and was monitored by RP-HPLC. Upon completion, the reaction mixture diluted with EtOAc (100 ml) and was washed with saturated solution of 0.5M HC1 (100 ml). The organic layer was separated, washed with brine, dried over Na 2 S0 4 and evaporated under reduced pressure. The crude residue and few milligrams of methylene blue were dissolved in 20 ml of DCM. Oxygen was bubbled through the solution while irradiating with yellow light.
  • Probe 11 Phosphate 11a (166 mg, 0.3 mmol) was dissolved in 1 ml of ACN. Pyrrolidine (0.153 ml, 1.86 mmol), triphenyl phosphine (16 mg, 0.06 mmol), tetrakis(triphenylphosphine) palladium(O) (17 mg, 0.015 mmol) was added and the solution was stirred at RT. After completion the precipitant was filtered and washed 3 times with ACN to give a yellowish solid. The crude solid and NaOH (30 mg, 0.76 mmol) were dissolved in 2 ml of 4: 1 THF:H 2 0 solution.
  • Probe 12 Compound 12a (274 mg, 0.39 mmol) and few milligrams of methylene blue were dissolved in 20 ml of DCM. Oxygen was bubbled through the solution while irradiating with yellow light. The reaction was monitored by TLC (Hex:EtOAc 80:20). Upon completion, the reaction mixture was concentrated by evaporation under reduced pressure. The crude product was purified by column chromatography on silica gel (Hex:EtOAc 80:20) to afford Probe 12 (255 mg, 89% yield).
  • Chemiluminescence images were acquired using Olympus LV200 inverted microscope fitted with an EMCCD camera (Hamamatsu C9100-13).
  • HEK293 LacZ stable cells (amsbio SC003) and HEK293-WT cells (control) were grown on 35mm glass bottom petri dishes at 37°C for 24 h.
  • Cell culture medium was changed to Molecular Probes ® Live Cell Imaging Solution containing 5 ⁇ of probe 4. Cells were incubated for another 20 minutes at 37°C. Thereafter, images were recorded with 40 seconds exposure time.
  • chemiluminescent probes based on the Schapp's adamantylidene- dioxetane probe (Scheme 1), in which the phenolate donor is substituted at the ortho position of phenolic ring with a ⁇ * acceptor group such as methyl- acrylate and acrylonitrile, i.e., an electron acceptor or electron-withdrawing group, and optionally further substituted at the other ortho position of the phenolic ring with chlorine, were designed and synthesized. Based on the teaching of Karton-Lifshin et al. (2012) it has been postulated that such donor- acceptor pair design should potentially increase the emissive nature of the benzoate species.
  • Scheme 1 Schapp's adamantylidene- dioxetane probe
  • This electron-withdrawing substituent decreases the /?Ka of the phenol released after cleavage of the protecting group and thereby enriches the relative concertation of the phenolate species in physiological pH.
  • the methyl-acrylate and acrylonitrile substituents induced the highest increase in fluorescence emission of their corresponded phenol-benzoates.
  • the absorbance and fluorescence spectra of selected phenol-benzoate derivatives are shown in Fig. 11, and their molecular structure and spectroscopic parameters are summarized Table 9.
  • the dioxetane-luminophores exhibited a chemiluminescent exponential decay kinetic profile of with varied ⁇ ⁇ - Dioxetane 5b, was used as a reference compound as its chemiluminescence quantum yield under aqueous conditions is known (3.2x10 " %) (Trofimov et al., 1996; Edwards et al., 1994).
  • the chemiluminescence emission of dioxetane 5b in water is extremely weak; however, dioxetane-luminophores 6b, 7b, 8b and 9b exhibited remarkably strong chemiluminescence emission signal upon their deprotonation in PBS 7.4.
  • Luminophore 6b (with methyl-acrylate substituent) showed emission signal, which is about 700-fold stronger than that of dioxetane 5b with chemiluminescence quantum yield of 2.3%.
  • Luminophore 7b (with the methyl-acrylate and additional chlorine substituent) showed similar signal enhancement with faster kinetic profile relative to luminophore 6b ( ⁇ 2/2 of 7 min vs. 23 min).
  • Luminophore 9b (with the acrylonitrile and additional chlorine substituent) showed the highest enhancement of chemiluminescence emission; about 3000-fold in comparison to that of dioxetane 5b with chemiluminescence quantum yield of 9.8%. Similar faster kinetic profile was observed when the chlorine substituent was present on the luminophore ( ⁇ 2 2 of 10 min for dioxetane 9b vs. 22 min for dioxetane 8b).
  • Turn-ON chemiluminescence probes can be simply prepared by masking of the phenol functional group of the dioxetane-luminophores with an enzyme responsive substrate.
  • an enzyme responsive substrate for evaluation this option, we synthesized five different adamantylidene-dioxetane probes, using dioxetane-luminophores 5b-9b, where the phenol is masked with a triggering substrate suitable for activation by ⁇ -galactosidase (Probes 5-9, see Schemes 21-22).
  • the probes exhibited a typical chemiluminescent kinetic profile in the presence of ⁇ -galactosidase with an initial signal increase to a maximum followed by a slow decrease to zero. While Probes 6-9 exhibited remarkably strong chemiluminescence emission signal under aqueous conditions in the presence of ⁇ -galactosidase, Probe 5 produced extremely weak emission (Fig. 12, inset). Probes 6 showed emission signal, which is about 500-fold stronger than that of Probe 5. Probe 7 (with the chlorine substituent) showed similar signal enhancement with faster kinetic profile relative to Probe 6. Similar faster kinetic profile was observed for Probe 9 in comparison to that of Probe 8. Probe 9 showed the highest enhancement of chemiluminescence emission; about 1800-fold, in comparison to that of Probe 5.
  • the surfactant reduces water-induced quenching by providing a hydrophobic environment for the chemiluminescent reaction that transfers the emitted light to excite the nearby fluorogenic dye. Consequently, the low-efficiency luminescence process is amplified significantly in aqueous medium (Schaap et ah, 1989).
  • Emerald-IITM enhancer (10%) was added to Probe 5 in the presence of ⁇ -galactosidase (in PBS 7.4) and its chemiluminescence emission was compared to that of Probe 6. The obtained results are presented in Fig. 13. Emerald-IITM enhancer amplifies the chemiluminescence emission of Probe 5 by 248-fold (Fig. 13A).
  • the chemiluminescence emission signal obtained by Probe 9 is more than 8-fold stronger than that of Probe 5 with the Emerald-IITM enhancer (Fig. 13B) under physiological conditions.
  • This unprecedented result suggests that a simple small molecule dioxetane compound like Probe 9 can produce chemiluminescence emission, which is about one order of magnitude stronger than the signal produced by a two component-system (Probe 5 and Emerald-IITM enhancer). Since our probes produce under aqueous conditions relatively highly emissive benzoate species, addition of the Emerald-IITM enhancer had only mild amplification effect on their chemiluminescence emission.
  • Probe 10 was equipped with boronic-ester as a substrate for hydrogen peroxide, Probe 11 with phosphate group as a substrate for alkaline-phosphatase and Probe 12 with dinitro- benzene-sulfonyl group as a substrate for GSH (Fig. 14).
  • the probes were prepared with an acrylic acid or methyl acrylate substituent at the ortho position of the phenolic oxygen.
  • the presence of an ionizable carboxylic acid group has increased significantly their aqueous solubility of Probes 10 and 11 and enabled to conduct evaluation test at a relative high concentration. At concentration of 1 mM (pH 10), Probes 10 and 11 have produced bright green luminescence upon reaction with their analyte/enzyme.
  • Probe 12 has relatively moderate aqueous solubility with an applicable concertation range between 1-10 ⁇ .
  • Probes 10, 11 and 12 To evaluate the sensitively and selectivity of Probes 10, 11 and 12 to detect their corresponded analyte/enzyme, we determined the probes' limit of detection (LOD). The probes exhibited very good selectivity towards their analyte of choice under physiological conditions (Fig. 15). Probe 10 could detect hydrogen peroxide with an LOD value of 30 nM, Probe 11 could detect alkaline-phosphatase with an LOD value of 3.9 ⁇ /ml and Probe 12 could detect GSH with an LOD value of 1.7 ⁇ .
  • LOD limit of detection
  • Probe 7 was able to produce high quality chemiluminescence images of the HEK293-LacZ cells already with 20 second exposure time (Fig. 16b). No chemiluminescence signal at all was observed by the HEK293-WT cells (Fig. 16d).
  • the pKa. value of phenolic benzoates is another factor that significantly affects the chemiluminescence emission of our probes.
  • the pKa. of the obtained phenol should be around 8.5 or lower.
  • a lower pKa. value for the phenol may be especially important for a probe aimed for in vitro use, when the probe penetrates into the cell through endocytosis mechanism (the pH in the endosome is known to be around 6.5 or lower).
  • Probe 7 was selected for cell imaging evaluation as it is composed of a phenol with a pKa. value of approximately 7.0.
  • chemiluminescence probes with high efficiency yield under physiological conditions.
  • the methodology is based on the fluorescence emission effect of a substituent on the benzoate species obtained during the chemiexcitation pathway of the Schapp's adamantylidene- dioxetane probe.
  • a striking substituent effect on the chemiluminescence efficiency of the probes was obtained when acrylate and acrylonitrile electron-withdrawing groups were installed.
  • the chemiluminescence quantum yield of the best probe was greater than three orders of magnitude in comparison to standard commercial available adamantylidene- dioxetane probe.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Materials Engineering (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • General Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Heterocyclic Compounds That Contain Two Or More Ring Oxygen Atoms (AREA)
  • Saccharide Compounds (AREA)
  • Nitrogen Condensed Heterocyclic Rings (AREA)
  • Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
  • Plural Heterocyclic Compounds (AREA)
PCT/IL2017/050088 2016-01-26 2017-01-24 Chemiluminescent probes for diagnostics and in vivo imaging Ceased WO2017130191A1 (en)

Priority Applications (11)

Application Number Priority Date Filing Date Title
EP17743844.7A EP3408349B1 (en) 2016-01-26 2017-01-24 Chemiluminescent fluorophor-linked adamantylene-dioxetane probes as diagnostics or in-vivo imaging sensors
ES17743844T ES2866900T3 (es) 2016-01-26 2017-01-24 Sondas de adamantileno-dioxetano unido a fluoróforo quimioluminiscentes como sensores de diagnóstico o de obtención de imágenes in vivo
JP2018539034A JP6915943B2 (ja) 2016-01-26 2017-01-24 診断およびインビボイメージングのための化学発光プローブ
KR1020187024288A KR102717614B1 (ko) 2016-01-26 2017-01-24 진단 및 인비보 영상용 화학발광 프로브
CA3011328A CA3011328C (en) 2016-01-26 2017-01-24 Chemolight Probes for In Vivo Diagnostics and Imaging
US16/072,848 US10660974B2 (en) 2016-01-26 2017-01-24 Chemiluminescent probes for diagnostics and in vivo imaging
CN201780020284.2A CN108884386B (zh) 2016-01-26 2017-01-24 用于诊断和体内成像的化学发光探针
BR112018015334-6A BR112018015334B1 (pt) 2016-01-26 2017-01-24 Composto de fórmula iva ou ivb e composição
US16/850,333 US11179482B2 (en) 2016-01-26 2020-04-16 Chemiluminescent probes for diagnostics and in vivo imaging
US17/451,536 US11931429B2 (en) 2016-01-26 2021-10-20 Chemiluminescent probes for diagnostics and in vivo imaging
US18/428,591 US12357709B2 (en) 2016-01-26 2024-01-31 Chemiluminescent probes for diagnostics and in vivo imaging

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201662287127P 2016-01-26 2016-01-26
US62/287,127 2016-01-26
US201662431618P 2016-12-08 2016-12-08
US62/431,618 2016-12-08

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US16/072,848 A-371-Of-International US10660974B2 (en) 2016-01-26 2017-01-24 Chemiluminescent probes for diagnostics and in vivo imaging
US16/850,333 Division US11179482B2 (en) 2016-01-26 2020-04-16 Chemiluminescent probes for diagnostics and in vivo imaging

Publications (1)

Publication Number Publication Date
WO2017130191A1 true WO2017130191A1 (en) 2017-08-03

Family

ID=59397688

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IL2017/050088 Ceased WO2017130191A1 (en) 2016-01-26 2017-01-24 Chemiluminescent probes for diagnostics and in vivo imaging

Country Status (8)

Country Link
US (4) US10660974B2 (https=)
EP (1) EP3408349B1 (https=)
JP (1) JP6915943B2 (https=)
KR (1) KR102717614B1 (https=)
CN (1) CN108884386B (https=)
CA (1) CA3011328C (https=)
ES (1) ES2866900T3 (https=)
WO (1) WO2017130191A1 (https=)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018216012A1 (en) 2017-05-24 2018-11-29 Ramot At Tel-Aviv University Ltd. Chemiluminescent probes for imaging/detection of proteases
WO2019224339A1 (en) 2018-05-25 2019-11-28 Biosynth Ag Long wavelength emitting chemiluminescent probes
WO2019224338A1 (en) 2018-05-25 2019-11-28 Biosynth Ag Dioxetane compounds and their use for the detection of microorganisms
CN111073633A (zh) * 2018-10-22 2020-04-28 北京工商大学 一种萘环类苯硫酚荧光探针
WO2020159448A1 (en) * 2019-02-01 2020-08-06 Nanyang Technological University Molecular renal probes for detecting acute kidney injury
WO2021086977A1 (en) * 2019-10-28 2021-05-06 Beckman Coulter, Inc. Rapid, high-intensity chemiluminescent dioxetanes
US11241507B2 (en) 2017-05-24 2022-02-08 Ramot At Tel-Aviv University Ltd. Near-infrared chemiluminescent probes for in-vivo imaging
WO2023018377A3 (en) * 2021-08-11 2023-05-19 Nanyang Technological University Ultrabright chemiluminescent probes for detection and imaging
WO2024147125A1 (en) * 2023-01-04 2024-07-11 Ramot At Tel-Aviv University Ltd. Cyclobutyl-substituted phenoxy-dioxetanes and uses thereof
US12281092B2 (en) 2022-01-28 2025-04-22 Sysmex Corporation Compound or salt thereof, composition, chemiluminescence method, method for measuring chemiluminescence signal, reagent, reagent kit, and method for assaying analyte
WO2026047187A1 (en) 2024-08-30 2026-03-05 Biosynth Ag Method of making chemiluminescent 1,2-dioxetanes

Families Citing this family (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110437274A (zh) * 2019-08-28 2019-11-12 南京工业大学 一种用于检测氟离子的探针、制备方法及应用
CN110804009B (zh) * 2019-10-10 2025-12-12 华东理工大学 一类化学发光强度高、波长长、稳定性好的化学发光底物及其制备方法和应用
KR102332454B1 (ko) * 2019-12-18 2021-11-29 고려대학교 산학협력단 암 세포 검출용 발광 프로브 화합물 및 이를 포함하는 암 세포 검출용 발광 센서
CN114315791B (zh) * 2021-12-22 2023-12-08 东南大学 用于实现手术导航和微小转移瘤成像的小分子化学发光探针及其制备方法和用途
CN114478473B (zh) * 2022-01-25 2024-05-03 南方医科大学 一种亮氨酸氨基肽酶化学发光检测试剂的合成及应用
CN115417863B (zh) * 2022-09-30 2024-02-02 徐州医科大学 一种二氧化硫近红外化学发光探针及其制备方法和应用
CN115894581B (zh) * 2022-10-26 2024-07-09 徐州医科大学 一种β-葡萄糖醛酸苷酶化学发光探针及其制备方法和应用
CN115819416B (zh) * 2022-11-29 2024-06-25 遵义医科大学 一种用于检测和区分的多模态化学发光分子及其应用
CN116217515B (zh) * 2022-12-20 2024-06-18 华南师范大学 一种亚甲基蓝型探针及其制备方法和在检测半胱氨酸中的应用
CN116606220A (zh) * 2023-03-21 2023-08-18 华东理工大学 一种单组分有机余辉化合物及其生物检测的应用
CN116410064A (zh) * 2023-04-13 2023-07-11 西安交通大学 一种合成2-(α-甲氧基-3-羟基苯亚甲基)金刚烷类化合物的方法
CN116655716A (zh) * 2023-04-27 2023-08-29 湖南师范大学 一种检测β-半乳糖苷酶的化学发光探针、制备方法及其应用
US12393660B2 (en) 2023-05-17 2025-08-19 The Government of the United States of America, as represented by the Secretary of Homeland Security DNA access control systems
CN117059804B (zh) * 2023-10-13 2024-03-19 瑞浦兰钧能源股份有限公司 一种化学预锂剂、锂离子电池及其制备方法
CN117442269B (zh) * 2023-12-22 2024-12-20 中日友好医院(中日友好临床医学研究所) 一种手术缝合针的搜寻系统及方法
CN119409748A (zh) * 2025-01-06 2025-02-11 四川省医学科学院·四川省人民医院 一种N-乙酰-β-D-氨基葡萄糖酶的化学发光探针、试剂盒及其制备方法和应用
CN119798211A (zh) * 2025-01-23 2025-04-11 西安交通大学 一种巯基化合物激活型化学发光探针及其合成方法和应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990007511A1 (en) 1988-12-27 1990-07-12 Board Of Governors Of Wayne State University Enhanced chemiluminescence from 1,2-dioxetanes through energy transfer to tethered fluorescers
WO2006073424A2 (en) * 2004-04-14 2006-07-13 Giri Brij P New ultra-sensitive chemiluminescent substrates for enzymes and their conjugates

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4983779A (en) * 1986-07-17 1991-01-08 The Board Of Governors Of Wayne State University Process for the preparation of vinyl ethers
EP0709466B1 (en) * 1994-10-28 2006-09-27 Gen-Probe Incorporated Compositions and methods for the simultaneous detection and quantification of multiple specific nucleic acid sequences
US5783381A (en) * 1995-10-19 1998-07-21 Tropix, Inc. Chemiluminescent 1,2-dioxetanes
JP3852140B2 (ja) * 1996-09-11 2006-11-29 東ソー株式会社 １，２−ジオキセタン誘導体
US6602679B2 (en) * 2000-01-28 2003-08-05 Brij Giri Stabilized formulations for chemiluminescent assays
US10128449B2 (en) 2014-01-08 2018-11-13 Sumitomo Chemical Company, Limited Metal complex and light emitting device using the same
KR102332743B1 (ko) 2014-03-17 2021-11-30 스미또모 가가꾸 가부시키가이샤 금속 착체 및 그것을 사용한 발광 소자

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990007511A1 (en) 1988-12-27 1990-07-12 Board Of Governors Of Wayne State University Enhanced chemiluminescence from 1,2-dioxetanes through energy transfer to tethered fluorescers
WO2006073424A2 (en) * 2004-04-14 2006-07-13 Giri Brij P New ultra-sensitive chemiluminescent substrates for enzymes and their conjugates

Non-Patent Citations (72)

* Cited by examiner, † Cited by third party
Title
"Remington: The Science and Practice of Pharmacy", 1995
AMIR, R.J.PESSAH, N.SHAMIS, M.SHABAT, D.: "Self-immolative dendrimers", ANGEW. CHEM. INT. ED., vol. 42, no. 37, 2003, pages 4494, XP008035926, DOI: 10.1002/anie.200351962
BAUER, C.R, G. I. T. IMAGING & MICROSCOPY, vol. 4, 2013, pages 32
BRANCHINI, B.R.ABLAMSKY, D.M.ROSENBERG, J.C., BIOCONJUGATE CHEM., vol. 21, 2010, pages 2023
BRONSTEIN, I.EDWARDS, B.VOYTA, J.C., J. BIOLUMIN. CHEMILUMIN., vol. 4, no. 1, 1989, pages 99
CAO, J.CAMPBELL, J.LIU, L.MASON, R.P.LIPPERT, A.R., ANAL. CHEM., vol. 88, no. 9, 2016, pages 4995
CAO, J.LOPEZ, R.THACKER, J.M.MOON, J.Y.JIANG, C.MORRIS, S.N.BAUER, J.H.TAO, P.MASON, R.P.LIPPERT, A.R., CHEM SCI, vol. 6, no. 3, 2015, pages 1979
CHI, C.DU, Y.YE, J.KOU, D.QIU, J.WANG, J.TIAN, J.CHEN, X., THERANOSTICS, vol. 4, no. 11, 2014, pages 1072 - 1084
CHO, S.HWANG, O.LEE, I.LEE, G.YOO, D.KHANG, G.KANG, P.M.LEE, D., ADV. FUNCT. MATER., vol. 22, no. 19, 2012, pages 4038
CLOUGH, J.M.BALAN, A.VAN DAAL, T.L.J.SIJBESMA, R.P., ANGEW. CHEM. INT. ED., vol. 55, no. 4, 2016, pages 1445
DOMINGUEZ, A.FERNANDEZ, A.GONZALEZ, N.IGLESIAS, E.MONTENEGRO, L., J. CHEM. EDUC., vol. 74, 1997, pages 1227
EDWARDS, B.SPARKS, A.VOYTA, J.C.BRONSTEIN, I.: "Bioluminescence and chemiluminescence: fundamentals and applied aspects", 1994, WILEY, pages: 56
GNAIM, S.SHABAT, D., ACC. CHEM. RES., vol. 47, no. 10, 2014, pages 2970
GOPINATH, R.HAQUE, S.J.PATEL, B.K., J ORG CHEM, vol. 67, no. 16, 2002, pages 5842 - 5845
GROSS, S.GAMMON, S.T.MOSS, B.L.RAUCH, D.HARDING, J.HEINECKE, J.W.RATNER, L.PIWNICA-WORMS, D., NAT. MED., vol. 15, no. 4, 2009, pages 455
HABER, G.P.WHITE, M.A.AUTORINO, R.ESCOBAR, P.F.KROH, M.D.CHALIKONDA, S.KHANNA, R.FOREST, S.YANG, B.ALTUNRENDE, F., UROLOGY, vol. 76, 2010, pages 1279 - 1282
HAGIWARA, H.WATANABE, N.IJUIN, H. K., HETEROCYCLES, vol. 87, no. 1, 2013, pages 65
HANANYA, N.ELDAR BOOCK, A.BAUER, C.R.SATCHI-FAINARO, R.SHABAT, D., J. AM. CHEM. SOC., vol. 138, no. 40, 2016, pages 13438
HANANYA, NIR ET AL.: "Remarkable Enhancement of Chemiluminescent Signal by Dioxetane- Fluorophore Conjugates: Turn-ON Chemiluminescence Probes with Color Modulation for Sensing and Imaging", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 138, no. Issue 40, 21 September 2016 (2016-09-21), pages 13438 - 13446, XP055401975, Retrieved from the Internet <URL:http://medicine.mytau.org/satchi-fainaro/wp-content/uploads/sites/5/2011/08/Hananya-et-al-JACS-2016.pdf> [retrieved on 20160921] *
JACOBSON, K.A.FURLANO, D.C.KIRK, K.L., J. FLUORINE CHEM., vol. 39, 1988, pages 339 - 347
JATHOUL, A.P.GROUNDS, H.ANDERSON, J.C.PULE, M.A., ANGEW. CHEM., INT. ED., vol. 53, 2014, pages 13059
KARTON-LIFSHIN, N.ALBERTAZZI, L.BENDIKOV, M.BARAN, P.S.SHABAT, D., J. AM. CHEM. SOC., vol. 134, no. 50, 2012, pages 20412
KARTON-LIFSHIN, N.SEGAL, E.OMER, L.PORTNOY, M.SATCHI-FAINARO, R.SHABAT, D., J. AM. CHEM. SOC., vol. 133, no. 28, 2011, pages 10960
KIELLAND, A.BLOM, T.NANDAKUMAR, K.S.HOLMDAHL, R.BLOMHOFF, R.CARLSEN, H., FREE RADICAL BIOL. MED., vol. 47, 2009, pages 760
KISIN-FINFER, E.FERBER, S.BLAU, R.SATCHI-FAINARO, R.SHABAT, D., BIOORG. MED. CHEM. LETT., vol. 24, 2014, pages 2453
LEE, D.KHAJA, S.VELASQUEZ-CASTANO, J. C.DASARI, M.SUN, C.PETROS, J.TAYLOR, W.R.MURTHY, N., NAT MATER, vol. 6, no. 10, 2007, pages 765
LEE, E.S.DEEPAGAN, V.G.YOU, D.G.JEON, J.YI, G.R.LEE, J.Y.LEE, D.S.SUH, Y.D.PARK, J.H., CHEM. COMMUN. (CAMB.), vol. 52, no. 22, 2016, pages 4132
LEE, Y.D.LIM, C.K.SINGH, A.KOH, J.KIM, J.KWON, I.C.KIM, S., ACS NANO, vol. 6, no. 8, 2012, pages 6759
LIM, C.K.LEE, Y.D.NA, J.OH, J.M.HER, S.KIM, K.CHOI, K.KIM, S.KWON, I.C., ADV. FUNCT. MATER., vol. 20, no. 16, 2010, pages 2644
LIU, L.MASON, R.P., PLOS ONE, vol. 5, 2010, pages e12024
LOENING, A.M.DRAGULESCU-ANDRASI, A.GAMBHIR, S.S., NAT. METHODS, vol. 7, 2010, pages 5
MATSUMOTO, M., J. PHOTOCHEM. PHOTOBIOL., C, vol. 5, 2004, pages 27
MATSUMOTO, M.AKIMOTO, T.MATSUMOTO, Y.WATANABE, N., TETRAHEDRON LETT., vol. 46, no. 36, 2005, pages 6075
MATSUMOTO, M.ARAI, N.WATANABE, N., TETRAHEDRON LETT., vol. 37, no. 47, 1996, pages 8535
MATSUMOTO, M.MIZOGUCHI, Y.MOTOYAMA, T.WATANABE, N., TETRAHEDRON LETT., vol. 42, no. 50, 2001, pages 8869
MATSUMOTO, M.SAKUMA, T.WATANABE, N., LUMINESCENCE, vol. 16, no. 4, 2001, pages 275
MATSUMOTO, M.SAKUMA, T.WATANABE, N., TETRAHEDRON LETT., vol. 43, no. 49, 2002, pages 8955
MATSUMOTO, M.WATANABE, N.HOSHIYA, N.IJUIN, H.K., CHEM. REC., vol. 8, 2008, pages 213
MERENYI, G.LIND, J.ERIKSEN, T.E., J. BIOLUMIN. CHEMILUMIN., vol. 5, no. 1, 1990, pages 53
MIEOG, J.S.TROYAN, S.L.HUTTEMAN, M.DONOHOE, K.J.VAN DER VORST, J.R.STOCKDALE, A.LIEFERS, G.J.CHOI, H.S.GIBBS-STRAUSS, S.L.PUTTER, , ANNALS OF SURGICAL ONCOLOGY, vol. 18, 2011, pages 2483 - 2491
MILLER, K.EREZ, R.SEGAL, E.SHABAT, D.FAINARO, R., ANGEW CHEM INT ED ENGL., vol. 48, no. 16, 2009, pages 2949 - 2954
PARK, J.Y.GUNPAT, J.LIU, L.EDWARDS, B.CHRISTIE, A.XIE, X.J.KRICKA, L.J.MASON, R.P., LUMINESCENCE, vol. 29, 2014, pages 553
PARTEARROYO, M.A.OSTOLAZA, H.GONI, F.M.BARBERA-GUILLEM, E., BIOCHEM. PHARMACOL., vol. 40, no. 6, 1990, pages 1323
PORTERFIELD, W.B.JONES, K.A.MCCUTCHEON, D.C.PRESCHER, J.A., J. AM. CHEM. SOC., vol. 137, 2015, pages 8656
REDY-KEISAR, O.FERBER, S.SATCHI-FAINARO, R.SHABAT, D., CHEMMEDCHEM, vol. 10, no. 6, 2015, pages 999
REDY-KEISAR, O.HUTH, K.VOGEL, U.LEPENIES, B.SEEBERGER, P.H.HAAG, R.SHABAT, D., ORG. BIOMOL. CHEM., vol. 13, 2015, pages 4727
REDY-KEISAR, O.KISIN-FINFER, E.FERBER, S.SATCHI-FAINARO, R.SHABAT, D., NAT. PROTOC., vol. 9, no. 1, 2014, pages 27 - 36
RICHARD, J.A.JEAN, L.ROMIEU, A.MASSONNEAU, M.NOACK-FRAISSIGNES, P.RENARD, P.Y., ORG. LETT., vol. 9, no. 23, 2007, pages 4853
RICHARD, J.A.JEAN, L.SCHENKELS, C.MASSONNEAU, M.ROMIEU, A.RENARD, P.Y., ORG. BIOMOL. CHEM., vol. 7, no. 14, 2009, pages 2941
RODA, A.GUARDIGLI, M., ANAL. BIOANAL. CHEM., vol. 402, 2012, pages 69
RODA, A.GUARDIGLI, M.PASINI, P.MIRASOLI, M.MICHELINI, E.MUSIANI, M., ANAL. CHIM. ACTA, vol. 541, 2005, pages 25
SABELLE, S.RENARD, P.Y.PECORELLA, K.DE SUZZONI-DEZARD, S.CREMINON, C.GRASSI, J.MIOSKOWSKI, C., J. AM. CHEM. SOC., vol. 124, no. 17, 2002, pages 4874
SAGI, A.WEINSTAIN, R.KARTON, N.SHABAT, D., J. AM. CHEM. SOC., vol. 130, no. 16, 2008, pages 5434
SCHAAP, A.P.AKHAVAN, H.ROMANO, L.J., CLIN. CHEM., vol. 35, no. 9, 1989, pages 1863
SCHAAP, A.P.CHEN, T.S.HANDLEY, R.S.DESILVA, R.GIRI, B.P., TETRAHEDRON LETT., vol. 28, no. 11, 1987, pages 1159
See also references of EP3408349A4
SEGAL, E.PAN, H.OFEK, P.UDAGAWA, T.KOPECKOVA, P.KOPECEK, J.SATCHI-FAINARO, R., PLOS ONE, vol. 4, no. 4, 2009, pages e5233
SHUHENDLER, A.J.PU, K.CUI, L.UETRECHT, J.PRAO, J., NAT. BIOTECHNOL., vol. 32, no. 4, 2014, pages 373
SILVA, S.M.CASALLANOVO, F.OYAMAGUCHI, K.H.CISCATO, L.F.L.M.STEVANI, C.V.BAADER, W.J., LUMINESCENCE, vol. 17, no. 5, 2002, pages 313
STEINHARDT, R.C.O'NEILL, J.M.RATHBUN, C.M.MCCUTCHEON, D.C.PALEY, M.A.PRESCHER, J.A., CHEM. EUR. J., vol. 22, 2016, pages 3671
STERN, L.PERRY, R.OFEK, P.MANY, A.SHABAT, D.SATCHI-FAINARO, R., BIOCONJUG CHEM., vol. 20, no. 3, 2009, pages 500 - 510
STEVENSON, J.D.DIETEL, A.THOMAS, N.R., CHEM. COMMUN., vol. 20, 1999, pages 2105 - 2106
TORCHILIN, V.P., J. CONTROLLED RELEASE, vol. 73, 2001, pages 137
TROFIMOV, A.V.MIELKE, K.VASIL'EV, R.F.ADAM, W., PHOTOCHEM. PHOTOBIOL., vol. 63, no. 4, 1996, pages 463
TROYAN, S.L.KIANZAD, V.GIBBS-STRAUSS, S.L.GIOUX, S.MATSUI, A.OKETOKOUN, R.NGO, L.KHAMENE, A.AZAR, F.FRANGIONI, J.V., ANNALS OF SURGICAL ONCOLOGY, vol. 16, 2009, pages 2943 - 2952
TSENG, J.C.KUNG, A.L., J. BIOMED. SCI., vol. 22, 2015, pages 45
TURAN, I.S.AKKAYA, E.U., ORG. LETT., vol. 16, no. 6, 2014, pages 1680
VAN DE BITTNER, G.C.BERTOZZI, C.R.CHANG, C.J., J. AM. CHEM. SOC., vol. 135, 2013, pages 1783
WAKIMOTO, R.KITAMURA, T.ITO, F.USAMI, H.MORIWAKI, H., APPL. CATAL., B, vol. 166-167, 2015, pages 544 - 550
WATANABE, N.KINO, H.WATANABE, S.IJUIN, H. K.YAMADA, M.MATSUMOTO, M., TETRAHEDRON, vol. 68, 2012, pages 6079
WEISSLEDER, R., NAT. BIOTECHNOL., vol. 19, 2001, pages 316
ZHANG, N.FRANCIS, K. P.PRAKASH, A.ANSALDI, D., NAT. MED., vol. 19, no. 4, 2013, pages 500

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11241507B2 (en) 2017-05-24 2022-02-08 Ramot At Tel-Aviv University Ltd. Near-infrared chemiluminescent probes for in-vivo imaging
US12173350B2 (en) 2017-05-24 2024-12-24 Ramot At Tel-Aviv University Ltd. Chemiluminescent probes for imaging/detection of proteases
WO2018216012A1 (en) 2017-05-24 2018-11-29 Ramot At Tel-Aviv University Ltd. Chemiluminescent probes for imaging/detection of proteases
CN112204020B (zh) * 2018-05-25 2023-11-14 尼米斯技术公司 二氧杂环丁烷化合物及其用于检测微生物的用途
US11649475B2 (en) * 2018-05-25 2023-05-16 Ramot At Tel-Aviv University Ltd. Dioxetane compounds and their use for the detection of microorganisms
CN112204020A (zh) * 2018-05-25 2021-01-08 尼米斯技术公司 二氧杂环丁烷化合物及其用于检测微生物的用途
WO2019224339A1 (en) 2018-05-25 2019-11-28 Biosynth Ag Long wavelength emitting chemiluminescent probes
JP2021525273A (ja) * 2018-05-25 2021-09-24 ネミス テクノロジーズ アクチェンゲゼルシャフト ジオキセタン化合物及び微生物検出のためのそれらの使用
JP7462574B2 (ja) 2018-05-25 2024-04-05 ネミス テクノロジーズ アクチェンゲゼルシャフト ジオキセタン化合物及び微生物検出のためのそれらの使用
WO2019224338A1 (en) 2018-05-25 2019-11-28 Biosynth Ag Dioxetane compounds and their use for the detection of microorganisms
CN111073633A (zh) * 2018-10-22 2020-04-28 北京工商大学 一种萘环类苯硫酚荧光探针
WO2020159448A1 (en) * 2019-02-01 2020-08-06 Nanyang Technological University Molecular renal probes for detecting acute kidney injury
US20220096662A1 (en) * 2019-02-01 2022-03-31 Nanyang Technological University Molecular renal probes for detecting acute kidney injury
US12296027B2 (en) * 2019-02-01 2025-05-13 Nanyang Technological University Molecular renal probes for detecting acute kidney injury
WO2021086977A1 (en) * 2019-10-28 2021-05-06 Beckman Coulter, Inc. Rapid, high-intensity chemiluminescent dioxetanes
WO2023018377A3 (en) * 2021-08-11 2023-05-19 Nanyang Technological University Ultrabright chemiluminescent probes for detection and imaging
US12281092B2 (en) 2022-01-28 2025-04-22 Sysmex Corporation Compound or salt thereof, composition, chemiluminescence method, method for measuring chemiluminescence signal, reagent, reagent kit, and method for assaying analyte
WO2024147125A1 (en) * 2023-01-04 2024-07-11 Ramot At Tel-Aviv University Ltd. Cyclobutyl-substituted phenoxy-dioxetanes and uses thereof
WO2026047187A1 (en) 2024-08-30 2026-03-05 Biosynth Ag Method of making chemiluminescent 1,2-dioxetanes

Also Published As

Publication number Publication date
KR20180108697A (ko) 2018-10-04
CA3011328A1 (en) 2017-08-03
BR112018015334A2 (pt) 2018-12-18
EP3408349A4 (en) 2019-10-23
US20200237932A1 (en) 2020-07-30
US20240197924A1 (en) 2024-06-20
CN108884386B (zh) 2022-03-15
US20190290787A1 (en) 2019-09-26
EP3408349A1 (en) 2018-12-05
CA3011328C (en) 2024-09-17
US10660974B2 (en) 2020-05-26
US12357709B2 (en) 2025-07-15
US11931429B2 (en) 2024-03-19
JP6915943B2 (ja) 2021-08-11
EP3408349B1 (en) 2021-03-31
JP2019509981A (ja) 2019-04-11
US20220047725A1 (en) 2022-02-17
KR102717614B1 (ko) 2024-10-16
US11179482B2 (en) 2021-11-23
CN108884386A (zh) 2018-11-23
ES2866900T3 (es) 2021-10-20

Similar Documents

Publication Publication Date Title
US12357709B2 (en) Chemiluminescent probes for diagnostics and in vivo imaging
US11241507B2 (en) Near-infrared chemiluminescent probes for in-vivo imaging
JP7553615B2 (ja) プロテアーゼのイメージング／検出のための化学発光プローブ
CN113979912B (zh) 两种前列腺特异性膜抗原靶向荧光探针及其制备方法与应用
US6403625B1 (en) Fluorescent labeling reagents
WO2019224339A1 (en) Long wavelength emitting chemiluminescent probes
CN115160391B (zh) 一种靶向性过氧化亚硝基荧光探针的制备和应用
CN109929548B (zh) 一种用于羧肽酶a检测的新型近红外荧光探针
KR101842633B1 (ko) 카텝신 b에 의해 활성화되는 암의 진단 또는 치료용 조성물, 이를 이용한 암의 근적외선 형광 이미징 및 광선 치료
US7968735B2 (en) Light-emitting biomarker
WO2024147125A1 (en) Cyclobutyl-substituted phenoxy-dioxetanes and uses thereof
BR122022024635B1 (pt) CONJUGADO DE FÓRMULA IIIa OU IIIb E COMPOSIÇÃO
BR112018015334B1 (pt) Composto de fórmula iva ou ivb e composição
BR122022024633B1 (pt) Composto de fórmula i, composto da fórmula iia ou iib, e composição
CN115724803B (zh) 一种用于检测羧酸酯酶的荧光探针及其制备方法和应用
WO2020179865A1 (ja) シアリダーゼ活性検出蛍光プローブ

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17743844

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 3011328

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 2018539034

Country of ref document: JP

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 122022024633

Country of ref document: BR

NENP Non-entry into the national phase

Ref country code: DE

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112018015334

Country of ref document: BR

ENP Entry into the national phase

Ref document number: 20187024288

Country of ref document: KR

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 1020187024288

Country of ref document: KR

WWE Wipo information: entry into national phase

Ref document number: 2017743844

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2017743844

Country of ref document: EP

Effective date: 20180827

ENP Entry into the national phase

Ref document number: 112018015334

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20180726