WO2023167282A1 - Sonde raman activable de type à fonction o - Google Patents

Sonde raman activable de type à fonction o Download PDF

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WO2023167282A1
WO2023167282A1 PCT/JP2023/007813 JP2023007813W WO2023167282A1 WO 2023167282 A1 WO2023167282 A1 WO 2023167282A1 JP 2023007813 W JP2023007813 W JP 2023007813W WO 2023167282 A1 WO2023167282 A1 WO 2023167282A1
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group
compound
jcr
salt
βgal
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PCT/JP2023/007813
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English (en)
Japanese (ja)
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泰照 浦野
真子 神谷
礼任 藤岡
歩弥 駒沢
泰之 小関
景文 寿
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国立大学法人 東京大学
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Publication of WO2023167282A1 publication Critical patent/WO2023167282A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/06—Peri-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06139—Dipeptides with the first amino acid being heterocyclic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • 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
    • 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
    • G01N33/483—Physical analysis of biological material

Definitions

  • the present invention relates to a novel O-function type activatable Raman probe. More specifically, the present invention relates to an O-function Raman probe having an activatable property that the Raman signal is off before reaction with the target enzyme, but is turned on by the reaction with the target enzyme. .
  • the wavelength shift amount (Raman shift value) in Raman scattering corresponds to the natural frequency of the molecule that scattered the light, it is possible to obtain information such as the type and state of the molecules contained in the sample from the Raman spectrum. Therefore, an imaging method using Raman scattering has attracted attention as a method for observing biomolecules without labels (see FIG. 1).
  • Raman scattered light is extremely weak, there are problems such as (1) low detection sensitivity and (2) low time resolution due to the need for a long observation time (several tens of minutes to several hours). Its application was limited.
  • Non-Patent Document 1 stimulated Raman scattering (SRS)
  • Non-Patent Document 2 resonant Raman scattering
  • biocompatibility has been dramatically improved by using a micro tag such as alkyne having stretching vibration in a silent region (1800-2800 cm ⁇ 1 ) where Raman signals of biomolecules do not occur (Non-Patent Document 3).
  • the Raman signal is off before encountering the target enzyme, but the absorption wavelength is lengthened due to hydrolysis by the target enzyme (non-resonance condition ⁇ early resonance condition) Raman signal
  • the absorption wavelength is lengthened due to hydrolysis by the target enzyme (non-resonance condition ⁇ early resonance condition)
  • Raman signal We have succeeded in developing an activatable Raman probe that turns on.
  • isotopic substitution of the CN group of 9CN-JCP which was found as the probe mother nucleus
  • the developed Raman probes are N-function type probes that mainly target aminopeptidases, and the kinds of enzymes that can be targeted are limited.
  • the probe does not have a single-cell level resolution because it does not have a structure that stays in cells, and when the probe is added to Drosophila tissue, target enzyme expression It was found that areas other than the region were also stained non-specifically (see FIG. 2).
  • the purpose of the present invention is to provide an activatable Raman probe that can expand targetable enzymes and has improved intracellular retention.
  • the present inventors have found that by using a rhodol-based O-function type fluorescent scaffold instead of a pyronine-based dye, the target enzyme can be expanded, and cells
  • the target enzyme can be expanded, and cells
  • an activatable Raman probe with improved internal retention can be provided, and have completed the present invention.
  • R 1 and R 2 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a halogen atom
  • R 3 and R 4 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a halogen atom
  • R 1 or R 3 may be -CH 2 -F or -CH-F 2
  • R 5 and R 6 each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, however, (1) R 5 or R 6 together with R 2 or R 4 form a 5- to 7-membered heterocyclyl or heteroaryl containing the nitrogen atom to which R 2 or R 4 is attached; or (2) R 5 and R 6 together with R 2 and R 4 , respectively, form a 5- to 7-membered heterocyclyl or heteroaryl containing the nitrogen atom to which R 2 and R 4 are
  • R1a is a natural amino acid (glycine, alanine, leucine, isoleucine, valine, lysine, cysteine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine, phenylalanine, methionine, tryptophan, tyrosine, proline) selected from the group consisting of a group constituting a side chain, a group constituting a side chain of an unnatural amino acid (citrulline, norvaline, etc.), a substituted or unsubstituted alkyl group; * represents the location where T is bonded.
  • R1a is a natural amino acid (glycine, alanine, leucine, isoleucine, valine, lysine, cysteine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine,
  • R2a is a natural amino acid (glycine, alanine, leucine, isoleucine, valine, lysine, cysteine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine, phenylalanine, methionine, tryptophan, tyrosine, proline) selected from the group consisting of a group constituting a side chain, a group constituting a side chain of an unnatural amino acid (citrulline, norvaline, etc.), a substituted or unsubstituted alkyl group; represents an amino group, an N-acetylated amino group, or a structure in which one or more amino acids are linked by peptide bonds; * represents a site where T is bonded.
  • R2a is a natural amino acid (glycine, alanine, leucine, isoleucine, valine, lysine, cysteine,
  • a method for detecting a target enzyme or reactive oxygen species in a cell or tissue comprising the step of (a) introducing a compound represented by general formula (I) or a salt thereof into the cell or tissue, and ( b) A method comprising the step of measuring Raman scattered light enhanced by reaction of the compound or its salt with target enzymes or reactive oxygen species in cells or tissues.
  • FIG. 1 shows a schematic diagram of a biological imaging method using Raman scattering. Fluorescence and SRS imaging results of Drosophila tissue spiked with ⁇ Gal-9 13 C 15 N-JCP, which is our previous study, are shown. (Wing Discs were incubated with 100 ⁇ M ⁇ Gal-9 13 C 15 N-JCP in medium containing 0.1% DMSO for 30 minutes. Scale bar: 10 ⁇ m. Acquisition time 67 seconds.) The optical properties of a series of 9CN-rhodol derivatives synthesized in Reference Examples are shown. The results of evaluating the optical properties of 9CN-JR-Bn- ⁇ Gal and 9C 15 N-JCR-Bn- ⁇ Gal are shown.
  • FIG. 11 shows the results of comparing the aggregate-forming ability. Shown are the results of target enzyme-selective ex vivo imaging of Drosophila tissue using aggregate formation with 9C 15 N-JCR-Bn- ⁇ Gal.
  • Results of simultaneous imaging of ⁇ Gal-9 13 C 15 N-JCP and 9C 15 N-JCR-Bn- ⁇ Gal using Drosophila wing discs are shown.
  • Results of simultaneous imaging of ⁇ Gal-9 13 C 15 N-JCP and 9C 15 N-JCR-Bn- ⁇ Gal using the Drosophila fat body are shown.
  • the conceptual diagram of the intracellular retention type probe is shown.
  • a labeling scheme of 9CN-2CH 2 F-JCR-Bn- ⁇ Gal upon reaction with ⁇ -galactosidase is shown.
  • FIG. 2 shows the results of evaluating the in vitro enzymatic reaction of 9CN-2CH 2 F-JCR-Bn- ⁇ Gal.
  • the results of Live-cell confocal fluorescence imaging using 9CN-2CH 2 F-JCR-Bn- ⁇ Gal are shown.
  • 9 shows the results of comparing wash resistance and fixation resistance of 9CN-2CH 2 F-JCR-Bn- ⁇ Gal and 9C 15 N-JCR-Bn- ⁇ Gal) in live-cell confocal fluorescence imaging.
  • 9 shows the results of comparing wash resistance and fixation resistance of 9CN-2CH 2 F-JCR-Bn- ⁇ Gal and 9C 15 N-JCR-Bn- ⁇ Gal) in live-cell confocal fluorescence imaging.
  • the results of two-color imaging of 9CN-2CH 2 F-JCR-Bn- ⁇ Gal and 9C 15 N-JCR-Bn- ⁇ Gal on Drosophila wing discs are shown.
  • alkyl may be straight chain, branched chain, cyclic, or an aliphatic hydrocarbon group consisting of a combination thereof.
  • the number of carbon atoms in the alkyl group is not particularly limited, but for example, 1 to 6 carbon atoms (C 1-6 ), 1 to 10 carbon atoms (C 1-10 ), ) and 1 to 20 carbon atoms (C 1-20 ).
  • the number of carbon atoms is specified, it means “alkyl” having the number of carbon atoms within the specified range.
  • C 1-8 alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neo-pentyl, n-hexyl, isohexyl, n-heptyl, n-octyl and the like are included.
  • an alkyl group may have one or more optional substituents.
  • substituents include, but are not limited to, alkoxy groups, halogen atoms, amino groups, mono- or di-substituted amino groups, substituted silyl groups, acyl, and the like.
  • alkyl group When an alkyl group has more than one substituent, they may be the same or different.
  • alkyl moieties of other substituents containing alkyl moieties eg, alkoxy groups, arylalkyl groups, etc.
  • halogen atom may be fluorine, chlorine, bromine or iodine, preferably fluorine, chlorine or bromine.
  • substituents include, but are not limited to, alkyl groups, alkoxy groups, hydroxyl groups, carboxyl groups, halogen atoms, sulfo groups, amino groups, alkoxycarbonyl groups, and oxo groups. These substituents may further have a substituent. Examples of such groups include, but are not limited to, halogenated alkyl groups, dialkylamino groups, and the like.
  • aryl may be either a monocyclic or condensed polycyclic aromatic hydrocarbon group, and a heteroatom (e.g., an oxygen atom, a nitrogen atom, or a sulfur atom) as a ring-constituting atom. etc.) may be aromatic heterocycles containing one or more. In this case, it is sometimes referred to as “heteroaryl” or “heteroaromatic.” Whether the aryl is a single ring or a condensed ring, it may be attached at all possible positions.
  • a heteroatom e.g., an oxygen atom, a nitrogen atom, or a sulfur atom
  • Non-limiting examples of monocyclic aryl include phenyl (Ph), thienyl (2- or 3-thienyl), pyridyl, furyl, thiazolyl, oxazolyl, pyrazolyl, 2-pyrazinyl. group, pyrimidinyl group, pyrrolyl group, imidazolyl group, pyridazinyl group, 3-isothiazolyl group, 3-isoxazolyl group, 1,2,4-oxadiazol-5-yl group or 1,2,4-oxadiazole-3 -yl group and the like.
  • Non-limiting examples of fused polycyclic aryl include 1-naphthyl, 2-naphthyl, 1-indenyl, 2-indenyl, 2,3-dihydroinden-1-yl, 2,3 -dihydroinden-2-yl group, 2-anthryl group, indazolyl group, quinolyl group, isoquinolyl group, 1,2-dihydroisoquinolyl group, 1,2,3,4-tetrahydroisoquinolyl group, indolyl group, isoindolyl group, phthalazinyl group, quinoxalinyl group, benzofuranyl group, 2,3-dihydrobenzofuran-1-yl group, 2,3-dihydrobenzofuran-2-yl group, 2,3-dihydrobenzothiophen-1-yl group, 2 ,3-dihydrobenzothiophen-2-yl group, benzothiazolyl group, benzimidazo
  • an aryl group may have one or more optional substituents on its ring.
  • substituents include, but are not limited to, alkoxy groups, halogen atoms, amino groups, mono- or di-substituted amino groups, substituted silyl groups, acyl groups, and the like.
  • substituents include, but are not limited to, alkoxy groups, halogen atoms, amino groups, mono- or di-substituted amino groups, substituted silyl groups, acyl groups, and the like.
  • substituents include, but are not limited to, alkoxy groups, halogen atoms, amino groups, mono- or di-substituted amino groups, substituted silyl groups, acyl groups, and the like.
  • arylalkyl represents alkyl substituted with the above aryl.
  • Arylalkyl may have one or more optional substituents. Examples of such substituents include, but are not limited to, alkoxy groups, halogen atoms, amino groups, mono- or di-substituted amino groups, substituted silyl groups, acyl groups, and the like. When an acyl group has two or more substituents, they may be the same or different.
  • Non-limiting examples of arylalkyl include benzyl, 2-thienylmethyl, 3-thienylmethyl, 2-pyridylmethyl, 3-pyridylmethyl, 4-pyridylmethyl, 2-furylmethyl, 3-furylmethyl group, 2-thiazolylmethyl group, 4-thiazolylmethyl group, 5-thiazolylmethyl group, 2-oxazolylmethyl group, 4-oxazolylmethyl group, 5-oxazolylmethyl group, 1-pyrazolylmethyl group , 3-pyrazolylmethyl group, 4-pyrazolylmethyl group, 2-pyrazinylmethyl group, 2-pyrimidinylmethyl group, 4-pyrimidinylmethyl group, 5-pyrimidinylmethyl group, 1-pyrrolylmethyl group, 2-pyrrolylmethyl group, 3-pyrrolylmethyl group , 1-imidazolylmethyl group, 2-imidazolylmethyl group, 4-imidazolylmethyl group, 3-pyridazinylmethyl group, 4-pyridazinylmethyl group,
  • alkoxy group refers to a structure in which the aforementioned alkyl group is bonded to an oxygen atom, and examples thereof include saturated alkoxy groups that are linear, branched, cyclic, or a combination thereof.
  • methoxy, ethoxy, n-propoxy, isopropoxy, cyclopropoxy, n-butoxy, isobutoxy, s-butoxy, t-butoxy, cyclobutoxy, cyclopropylmethoxy, n- Pentyloxy group, cyclopentyloxy group, cyclopropylethyloxy group, cyclobutylmethyloxy group, n-hexyloxy group, cyclohexyloxy group, cyclopropylpropyloxy group, cyclobutylethyloxy group, cyclopentylmethyloxy group and the like are preferred. Examples include:
  • alkylene refers to a linear or branched saturated hydrocarbon divalent group, such as methylene, 1-methylmethylene, 1,1-dimethylmethylene, ethylene, 1-methylethylene, 1-ethylethylene, 1,1-dimethylethylene, 1,2-dimethylethylene, 1,1-diethylethylene, 1,2-diethylethylene, 1-ethyl-2-methylethylene, trimethylene, 1 -methyltrimethylene, 2-methyltrimethylene, 1,1-dimethyltrimethylene, 1,2-dimethyltrimethylene, 2,2-dimethyltrimethylene, 1-ethyltrimethylene, 2-ethyltrimethylene, 1,1 -diethyltrimethylene, 1,2-diethyltrimethylene, 2,2-diethyltrimethylene, 2-ethyl-2-methyltrimethylene, tetramethylene, 1-methyltetramethylene, 2-methyltetramethylene, 1,1- dimethyltetramethylene, 1,2-dimethyltetram
  • a compound represented by the general formula (I) or a salt thereof is a compound represented by the following general formula (I) or a salt thereof (hereinafter also referred to as "the compound of the present invention") .
  • the compound of the present invention has a rhodol O-function type as a fluorescent core.
  • probes with an N-function type pyronine dye as a mother core were limited in the types of enzymes that can be targeted (for example, when introducing a sugar substrate into 9CN-pyronine, carbamate Although it is via a linker, it is difficult to introduce with a carbamate linker depending on the structure of the sugar substrate (for example, ⁇ -glycosidic bond)). is possible.
  • probes having a pyronine dye as a core nucleus become cationic after reaction with a target enzyme, and thus dissolve in water.
  • R 1 and R 2 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom.
  • the alkyl group has one or more halogen atoms, carboxy groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, etc. good too.
  • the alkyl groups represented by R 1 and/or R 2 may be halogenated alkyl groups, hydroxyalkyl groups, carboxyalkyl groups, and the like.
  • R 1 and R 2 are each independently a hydrogen atom or a halogen atom . more preferred.
  • R 3 and R 4 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom.
  • the alkyl group contains one or more of a halogen atom, a carboxy group, a sulfonyl group, a hydroxyl group, an amino group, an alkoxy group, etc. good too.
  • the alkyl groups represented by R3 and/or R4 may be halogenated alkyl groups, hydroxyalkyl groups, carboxyalkyl groups, and the like.
  • R 3 and R 4 are preferably each independently a hydrogen atom or a halogen atom, and when both R 3 and R 4 are hydrogen atoms, or both R 3 and R 4 are fluorine atoms or chlorine atoms more preferred.
  • R 1 or R 3 may be -CH 2 -F or -CH-F 2 .
  • introduction of —CH 2 —F or —CH—F 2 at the position of R 1 or R 3 results in reaction with the target enzyme
  • a quinone methide intermediate can be generated by elimination of the fluoride ion that accompanies (see first reaction in FIG. 15). It is expected that this quinone methide active intermediate forms a covalent bond with an intracellular protein or the like to be immobilized in the cell and further improve the intracellular retention.
  • R 5 and R 6 each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.
  • the alkyl group may contain one or more of a halogen atom, a carboxy group, a sulfonyl group, a hydroxyl group, an amino group, an alkoxy group, etc.
  • the alkyl group represented by R5 or R6 may be a halogenated alkyl group, a hydroxyalkyl group, a carboxyalkyl group, or the like.
  • R5 and R6 must satisfy the following conditions. (1) R 5 or R 6 together with R 2 or R 4 form a 5- to 7-membered heterocyclyl or heteroaryl containing the nitrogen atom to which R 2 or R 4 is attached; or (2) R5 and R6 together with R2 and R4 , respectively, form a 5- to 7-membered heterocyclyl or heteroaryl containing the nitrogen atom to which R2 and R4 are attached; ing, That is, either or both of R 5 and R 6 together with R 2 or R 4 or together with R 2 and R 4 are 5- to 7-membered heterocyclyl containing a nitrogen atom or form a heteroaryl.
  • the substituents on the nitrogen atom form a 5- to 7-membered heterocyclyl or heteroaryl ring structure that is bright (absorption) as a probe using the epr-SRS method. wavelength) and dye stability.
  • heterocyclyl or heteroaryl may contain from 1 to 3 additional heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur atoms as ring members.
  • heterocyclyl or heteroaryl is alkyl of 1 to 6 carbons, alkenyl of 2 to 6 carbons or alkynyl of 2 to 6 carbons, aralkyl of 6 to 10 carbons, 6 to 10 carbons may be substituted with an alkyl-substituted alkenyl group of Examples of heterocyclyl or heteroaryl so formed include, but are not limited to, pyrrolidine, piperidine, hexamethyleneimine, pyrrole, imidazole, pyrazole, oxazole, thiazole, and the like.
  • R5 and R6 together with R2 and R4 form a julolidine structure with the benzene ring to which the nitrogen atom is attached.
  • R 7 and R 8 each independently represent an alkyl group having 1 to 6 carbon atoms, and R 7 and R 8 each independently represent 1 carbon atom. It is preferred that there are to 3 alkyl groups, and more preferred that both R 7 and R 8 are methyl groups.
  • the alkyl group represented by R 7 and R 8 may contain one or more of halogen atom, carboxy group, sulfonyl group, hydroxyl group, amino group, alkoxy group, etc.
  • R 7 or R 8 represents Alkyl groups may be halogenated alkyl groups, hydroxyalkyl groups, carboxyalkyl groups, and the like.
  • R 7 and R 8 do not exist.
  • X represents an oxygen atom or a carbon atom.
  • X either an oxygen atom or a carbon atom can provide a suitable Raman probe.
  • T is a linker.
  • the compounds of the present invention essentially have a linker between the oxygen (O) of the fluorophore and Y, which is the site that reacts with the target molecule. It is theoretically possible to directly introduce a site (for example, sugar) that reacts with the target molecule into the oxygen (O) of the fluorescent scaffold. It was found that direct binding to the oxygen of the fluorophore lowers the stability of the compound molecule, resulting in elimination of the sugar, making it impossible to obtain the desired compound. On the other hand, by introducing a linker between the oxygen of the fluorophore and the site that reacts with the target molecule such as a sugar substrate, it has become possible to synthesize the target compound.
  • the linker for T is, for example, an alkylene group (provided that one or more —CH 2 — in the alkylene group may be substituted with —O—, —S—, —NH—, or —CO—). , arylene (including heteroarylene), cycloalkylene, alkoxyl group, polyethylene glycol chain, and a group consisting of two or more groups selected from these groups arbitrarily bonded to each other. .
  • the linker is a benzyl group optionally having a substituent (-Bz-CH 2 - * : Bz represents a benzene ring optionally having a substituent, * represents an oxygen (O) ), or a methylene group.
  • Y is a site that reacts with the target molecule.
  • Y can be selected according to the type of target molecule.
  • the target enzyme is a glycosidase
  • Y is selected from groups derived from saccharides
  • the target enzyme is a peptidase
  • Y is selected from groups derived from and containing amino acids.
  • Y is -OL, -NH-L' or a scavenging group for reactive oxygen species.
  • L is a saccharide or a partial structure of a saccharide
  • L' is a partial structure of an amino acid.
  • a target enzyme glycosidase or Before the reaction with aminopeptidase, the signal is off because the absorption wavelength is short. When a peptide is introduced) is cleaved and the absorption wavelength is lengthened, so the signal is turned on.
  • Raman probes makes it possible to detect the activity of these target enzymes even in living cells.
  • L is a saccharide or a partial structure of a saccharide.
  • the partial structure of the saccharide of L refers to a structure corresponding to the remaining partial structure after removing one hydroxyl group from the saccharide.
  • the partial structure of the saccharide, together with the O to which L is bound, constitutes the saccharide and a part of the saccharide.
  • Sugars include ⁇ -D-glucose, ⁇ -D-galactose, ⁇ -L-galactose, ⁇ -D-xylose, ⁇ -D-mannose, ⁇ -D-fucose, ⁇ -L-fucose, ⁇ -L- fucose, ⁇ -D-arabinose, ⁇ -L-arabinose, ⁇ -DN-acetylglucosamine, ⁇ -DN-acetylgalactosamine and the like, preferably ⁇ -D-galactose.
  • L' is a partial structure of an amino acid.
  • the amino acid partial structure of L' constitutes an amino acid, an amino acid residue, a peptide, a portion of an amino acid, or a portion of a peptide.
  • the "part of the amino acid” includes the structure of the portion excluding -NH when the side chain of the amino acid is bonded to -NH (for example, between the carboxyl group on the side chain side of glutamic acid and -NH A glutamic acid residue portion when forming an amide bond, etc.) are also included.
  • Part of the peptide also includes the structure of the portion excluding -NH when the side chain of one amino acid residue constituting the peptide is bonded to -NH.
  • amino acid can be any compound as long as it has both an amino group and a carboxyl group, including natural and non-natural compounds. It may be a neutral amino acid, a basic amino acid, or an acidic amino acid. In addition to amino acids that themselves function as transmitters such as neurotransmitters, physiologically active peptides (dipeptides, tripeptides, tetrapeptides, (including oligopeptides) and proteins that are constituents of polypeptide compounds, such as ⁇ -amino acids, ⁇ -amino acids, and ⁇ -amino acids. As the amino acid, it is preferable to use an optically active amino acid.
  • ⁇ -amino acids either D- or L-amino acids may be used, but it may be preferable to select optically active amino acids that function in vivo.
  • the N-terminus of the amino acid may be N-acetylated.
  • the C-terminal of the amino acid may be amidated (for example, alkylamide such as ethylamide) or esterified.
  • amino acid residue refers to a structure corresponding to a partial structure remaining after removing the hydroxyl group from the carboxyl group of an amino acid.
  • Amino acid residues include ⁇ -amino acid residues, ⁇ -amino acid residues, and ⁇ -amino acid residues.
  • peptide also includes a structure corresponding to a partial structure remaining after removing the hydroxyl group from the carboxyl group of the amino acid at the C-terminus of the peptide.
  • the target peptidase can be ⁇ -glutamyl transpeptidase (GGT), dipeptidyl peptidase 4 (DPP-4), or calpain. Therefore, when the target peptidase is ⁇ -glutamyltranspeptidase, the partial structure of the amino acid is preferably a ⁇ -glutamyl group (the structure of the portion of the formula (1) described below with —NH removed). . When the target peptidase is dipeptidyl peptidase 4, the amino acid partial structure is preferably an acyl group containing a proline residue or a peptide containing a proline residue.
  • the amino acid substructure can be, for example, an acyl group containing a cysteine residue, or Suc-Leu-Leu-Val-Tyr, known in the art as a calpain substrate. (Suc-LLVY) and AcLM can also be used.
  • Preferred amino acid partial structures include the " ⁇ -glutamyl group" of the GGT substrate, the dipeptide of the DPP-4 substrate (a dipeptide consisting of amino acids and proline; where the amino acids are, for example, glycine, glutamic acid, and proline), and the LAP substrate.
  • a leucine residue and the like can be mentioned.
  • Y is selected from any of the following.
  • * represents the location where T is bonded.
  • R1a is a natural amino acid (glycine, alanine, leucine, isoleucine, valine, lysine, cysteine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine, phenylalanine, methionine, tryptophan, tyrosine, proline), groups constituting side chains of non-natural amino acids (citrulline, norvaline, etc.), and substituted or unsubstituted alkyl groups.
  • R2a is a natural amino acid (glycine, alanine, leucine, isoleucine, valine, lysine, cysteine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine, phenylalanine, methionine, tryptophan, tyrosine, proline), groups constituting side chains of non-natural amino acids (citrulline, norvaline, etc.), and substituted or unsubstituted alkyl groups. represents a structure in which an amino group, an N-acetylated amino group, or one or more amino acids are linked by a peptide bond.
  • Y is represented below.
  • the right nitrogen atom of the proline residue is attached to the T linker.
  • the scavenging group is preferably hydroxyphenoxy, aminophenoxy, azide, or pinacolborane.
  • Z is selected from -C ⁇ N, -C ⁇ 15N , -13C ⁇ N , or -13C ⁇ 15N .
  • One preferred embodiment of the compound of the present invention is a compound represented by the following general formula (Ia) or a salt thereof.
  • R 1 , R 3 , R 7 , R 8 , T and Y are as defined in general formula (I).
  • R 9 if present, is an alkyl group of 1-3 carbon atoms.
  • R 10 if present, is an alkyl group of 1 to 3 carbon atoms.
  • the compounds represented by general formulas (I) and (Ia) can exist as acid addition salts or base addition salts.
  • Acid addition salts include, for example, mineral acid salts such as hydrochlorides, sulfates and nitrates, or organic acid salts such as methanesulfonates, p-toluenesulfonates, oxalates, citrates and tartrates.
  • base addition salts include metal salts such as sodium, potassium, calcium and magnesium salts, ammonium salts, organic amine salts such as triethylamine salts, and the like. In addition to these, it may form a salt with an amino acid such as glycine.
  • the compounds represented by formulas (I) and (Ia) or salts thereof may exist as hydrates or solvates, and these substances can also be used in the present invention.
  • the compounds represented by general formulas (I) and (Ia) may have one or more asymmetric carbon atoms depending on the type of substituent.
  • stereoisomers such as optically active isomers based on the asymmetric carbon of , and diastereoisomers based on two or more asymmetric carbons, arbitrary mixtures and racemates of stereoisomers can also be used.
  • Raman Probe of the Present Invention Another aspect of the present invention is a Raman probe containing the compound of formula (I) or a salt thereof (hereinafter also referred to as "the Raman probe of the present invention").
  • the Raman probe of the present invention is a Raman probe that can be used in the epr-SRS method.
  • the epr-SRS method is a Raman imaging method that combines the electronic pre-resonance (epr) effect and stimulated Raman scattering (SRS).
  • excitation is performed by light with a wavelength slightly longer than the electronic absorption band of the molecule (pre-resonance condition), so background rise due to photobleaching and fluorescence can be suppressed, and stimulated emission by Stokes light can further enhance sensitivity. Imaging can be realized. With conventional SRS microscopes, it takes time on the order of seconds to change the wavelength of Stokes light, so one of the problems was the long measurement time when performing spectral analysis and spectral imaging.
  • the high-speed SRS spectroscopic microscope developed by Koseki Laboratory in the Department of Electrical Engineering, graduate School of Engineering is capable of high-speed imaging that changes by 30 wavenumbers per second (Ozeki, Y., Biological Imaging Based on Stimulated Raman Scattering. Seibutsu Butsuri, 2014. 54(6): p.311-314).
  • a schematic diagram of the high-speed SRS spectroscopic microscope is shown in FIG.
  • Raman imaging using high-speed SRS spectroscopic microscopy can be performed with reference to the Ozeki paper cited above.
  • data are obtained about 5 to 10 times for in vitro measurements and about 100 to 1000 times for in cellulo measurements. averaging is preferably performed.
  • the excitation light of this high-speed SRS spectroscopic microscope of Oseki Lab is 843 nm
  • the method for detecting a target enzyme using the Raman probe of the present invention can preferably use the high-speed SRS spectroscopic microscope described above, but is not limited to the method using the high-speed SRS spectroscopic microscope.
  • one preferred aspect of the present invention is an activatable Raman probe that can be used in the epr-SRS method and contains the compound of general formula (I) or a salt thereof.
  • the method of using the Raman probe of the present invention is not particularly limited, and it can be used in the same manner as conventionally known Raman probes.
  • the compound represented by the above formula (I) is added to an aqueous medium such as physiological saline or a buffer solution, or a mixture of a water-miscible organic solvent such as ethanol, acetone, ethylene glycol, dimethylsulfoxide and dimethylformamide and an aqueous medium.
  • the compounds or their salts are dissolved, this solution is added to an appropriate buffer containing cells or tissues, and the Raman spectrum is measured.
  • the Raman probe of the present invention may be used in the form of a composition in combination with suitable additives. For example, it can be combined with additives such as buffers, solubilizers, and pH adjusters.
  • Another aspect of the present invention is a method for detecting a target enzyme in a cell or tissue, comprising: (a) a Raman probe containing a compound represented by general formula (I) or a salt thereof; and (b) measuring Raman scattered light that is enhanced by the reaction of the compound or its salt with a target enzyme in cells or tissues (hereinafter “the method of the present invention”).
  • the cells include normal cells, cancer cells, nerve cells and the like.
  • Raman scattered light is preferably measured using the epr-SRS method.
  • the compound represented by the general formula (I) or a salt thereof can be introduced into cells or tissues by spraying the compound represented by the general formula (I) or a salt thereof on a cell or tissue sample. be able to.
  • Types of cancer cells or cancer tissues to be detected by the detection method of the present invention include lung cancer, prostate cancer, ovarian cancer, breast cancer, bladder cancer, brain tumor, esophageal cancer, stomach cancer, bile duct cancer, and liver cancer.
  • cancer pancreatic cancer, head and neck cancer, renal cancer, leukemia, skin cancer, and thyroid cancer cells or tissues.
  • cancer tissue means any tissue containing cancer cells.
  • tissue should be interpreted in the broadest sense, including a part or the whole of an organ, and should not be interpreted restrictively in any way.
  • One aspect of the detection method of the present invention includes the step of (a) introducing two or more compounds represented by formula (I) or a salt thereof into a cell or tissue, and (b) the two or more compounds or A method for detecting two or more target enzymes in a cell or tissue, comprising the step of measuring Raman scattered light enhanced by the salt reacting with each of the target enzymes in the cell or tissue.
  • the detection method of the present invention and the probe of the present invention can stain tissue as well as cells
  • the detection method of the present invention and the probe of the present invention can be used in cancer diagnostic methods based on enzyme activity patterns. can also be used. That is, another embodiment of the present invention includes the steps of: (a) administering a Raman probe containing the compound of general formula (I) or a salt thereof to a subject; , a method for diagnosing cancer comprising the step of measuring Raman scattered light enhanced by reaction with a target enzyme in a tissue or organ (hereinafter also referred to as "diagnostic method of the present invention").
  • Administration methods include topical administration, oral administration, intravenous administration, and the like.
  • Types of cancer targeted by the diagnostic method of the present invention include lung cancer, prostate cancer, ovarian cancer, breast cancer, bladder cancer, brain tumor, esophageal cancer, stomach cancer, bile duct cancer, liver cancer, and pancreatic cancer. , head and neck cancer, kidney cancer, leukemia, skin cancer, and thyroid cancer.
  • Another embodiment of the present invention is a target molecule detection kit containing the Raman probe of the present invention.
  • the Raman probe of the present invention is usually prepared as a solution. It can also be applied by dissolving in distilled water for injection or an appropriate buffer solution.
  • kit may contain other reagents and the like as necessary.
  • additives such as dissolution aids, pH adjusters, buffers, tonicity agents, and the like can be used, and the amount of these additives can be appropriately selected by those skilled in the art.
  • NMR spectra were obtained on a Bruker NMR AVANCE III 400 spectrometer [ 1 H 400 MHz, 13 C 100 MHz] using deuterated solvents.
  • High resolution ESI mass spectra were obtained on a Bruker microTOF II-TM (ESI).
  • HPLC purification was performed on JASCO PU-2080 Plus pumps (GL Science Co., Ltd.) and MD-2015 with Inertstil-ODS-3 columns ( ⁇ 10 ⁇ 250 mm (semi-preparative) and ⁇ 20 ⁇ 250 mm (preparative)).
  • a detector JASCO was used.
  • Solvents used for HPLC were obtained from Wako Co., Ltd.
  • Silica gel column chromatography was performed using a medium-pressure preparative liquid chromatograph YFLC-Al560 (Yamazen Co., Ltd.). TLC was performed on silica gel plates F254 (0.25 mm (analytical); Merck, AKG).
  • UV-vis spectra were obtained on a Shimadzu UV-2450 spectrophotometer. Fluorescence spectra were acquired with F-7000 (Hitachi). The SRS spectrum and SRS image were obtained with a high-speed SRS spectroscopic microscope developed by Kozeki Laboratory, Department of Electrical Engineering, graduate School of Engineering, University of Tokyo.
  • the wavelengths of the pump light pulse and the Stokes light pulse are 843 nm and 1014-1046 nm, the pulse duration is about 5 picoseconds, and the spectral resolution is 5/cm.
  • a water immersion objective lens is used and its numerical aperture is 1.2. Images of 500 ⁇ 500 pixels were acquired at 30 frames per second by varying the wavelength of the Stokes light pulse for each frame. Data were acquired 5 times in vitro and 1000 times in cellulo and averaged to increase the signal-to-noise ratio.
  • the julolidine structure is considered to be the optimal substituent on N from the viewpoint of both brightness and stability. was selected as
  • FIG. 4 shows the results of evaluating the optical properties of 9CN-JR-Bn- ⁇ Gal and 9C 15 N-JCR-Bn- ⁇ Gal synthesized above.
  • FIG. 4(a) shows absorption spectra before and after reaction of 1 ⁇ M 9CN-JR-Bn- ⁇ Gal (upper) and 9C15N-JCR-Bn- ⁇ Gal (lower) with 1 unit ⁇ -galactosidase.
  • FIG. 5 shows the results of cell imaging using the two types of probes synthesized above, 9CN-JR-Bn- ⁇ Gal and 9C 15 N-JCR-Bn- ⁇ Gal.
  • FIG. 5(a) shows structural changes of 9CN-JR-Bn- ⁇ Gal (upper) and 9C15N-JCR-Bn- ⁇ Gal (lower) before and after reaction with ⁇ -galactosidase.
  • HEK-LacZ cells were seeded in a 35 mm glass-bottom dish (Matsunami, Glass-bottom dish Hydro) and cultured overnight. The medium was then replaced with a D-MEM solution (phenol red free) containing 20 ⁇ M 9C 15 N-JCR-Bn- ⁇ Gal and 20 ⁇ M 9CN-JR-Bn- ⁇ Gal with 0.2% DMSO as a co-solvent. and exchanged at 37° C. for 2.5 hours. After incubation, the probe solution was removed and replaced with fresh HBSS(+), and SRS measurements were performed with an SRS microscope as described above.
  • D-MEM solution phenol red free
  • the detected wavenumbers were 2190 cm ⁇ 1 and 2193 cm ⁇ 1 for 9C 15 N-JCR-Bn- ⁇ Gal and 2223 cm ⁇ 1 and 2227 cm ⁇ 1 for 9CN- JR -Bn- ⁇ Gal. Background wavenumbers were 2170 cm ⁇ 1 and 2247 cm ⁇ 1 . Scale bar: 10 ⁇ m. Acquisition time for each cell was 60 seconds.
  • (d) shows the SRS spectra of the whole field (left) and the aggregate part (right) of (c).
  • b of FIG. 5 is an experiment with the same content as that of FIG. 4c.
  • 9C 15 N-JCR-Bn- ⁇ Gal could be imaged brighter than 9CN-JR-Bn- ⁇ Gal. suggested to be better.
  • the resulting eluent was evaporated and the residue was subjected to preparative HPLC using eluent A ( H2O with 1% MeCN and 0.1% TFA) and eluent B (MeCN with 1% H2O ).
  • A/B 95/5 to 5/95 (0-3.5min), 5/95 (3.5-4.0min), 5/95 to 95/5 (4.0-4.1min), 95/5 (4.1-5.0min) ).
  • FIG. 6(a) shows a schematic diagram of the reaction of 9 13 CN-JCR-Bn-gGlu with an enzyme (GGT).
  • FIG. 6( b ) shows 1 ⁇ M of 1 ⁇ M dimethylformamide before (blue) and after (red) reaction with 1 unit of GGT, measured in PBS (pH 7.4) containing 0.1% DMSO as co-solvent.
  • 9 shows the absorption spectrum of 9 13 CN-JCR-Bn-gGlu. The reaction solution was incubated at room temperature for 20 minutes.
  • (c) of FIG. 6 shows the concentration of 200 ⁇ M before (blue) and after (red) reaction with 1 unit GGT, measured in PBS (pH 7.4, final DMSO concentration 30% (v/v)).
  • FIG. 9 shows the SRS spectrum of 9 13 CN-JCR-Bn-gGlu.
  • the reaction solution was incubated at room temperature for 1 hour.
  • the image was constructed by subtracting the image at 2100 cm ⁇ 1 as background. Scale bar: 10 ⁇ m.
  • FIG. 6(d) shows the time-dependent change in absorbance of 9 13 CN-JCR-Bn-gGlu upon addition of GGT1 unit (red, arrow indicates timing of enzyme addition) and 50 ⁇ M GGT-specific inhibitor. Time-dependent change in absorbance in the presence of GGsTop (green). The observed wavelength was 690 nm.
  • FIG. 6(e) shows the results of LC-MS analysis of the reaction solution of 1 unit of GGT and 9 13 CN-JCR-Bn-gGlu under neutral conditions. The reaction solution was incubated at room temperature for 20 minutes.
  • FIG. 7(a) shows a schematic diagram of the reaction of 9 13 CN-JCR-Bn-EP with an enzyme (DPP-4).
  • FIG. 7(b) shows before (blue) and after (red ) of 9 13 CN-JCR-Bn-EP at 1 ⁇ M. The reaction solution was incubated at room temperature for 1 hour.
  • FIG. 7(c) shows before (blue) and after (red) reaction with 0.033 units of DPP-4, measured in PBS (pH 7.4, final DMSO concentration 30% (v/v)). shows the SRS spectrum of 200 ⁇ M 9 13 C 15 N-JCR-Bn-EP of . The reaction solution was incubated at room temperature for 1 hour.
  • FIG. 7(d) shows the time dependence of the absorbance of 9 13 C 15 N-JCR-Bn-EP upon addition of 0.033 units of DPP-4 (red, arrow indicates timing of enzyme addition). changes and time-dependent changes in absorbance in the presence of 1.8 ⁇ M DPP-4 specific inhibitor; sitagliptin (green) is shown. The observed wavelength was 690 nm.
  • FIG. 7(e) shows the results of LC-MS analysis of the reaction solution of 0.033 units of DPP-4 and 9 13 C 15 N-JCR-Bn-EP under neutral conditions. The reaction solution was incubated at room temperature for 20 minutes.
  • A549 cells were purchased from the Riken Cell Bank (RCB 0098) and H226 cells were purchased from the American Type Culture Collection. A549 cells were cultured in Dulbecco's modified Eagle medium (D-MEM) (Wako) and H226 cells in RPMI 1640 (Wako) containing 10% fetal bovine serum (GIBCO), 100 U/mL, penicillin, and 100 ⁇ g/mL streptomycin. at 37°C in humidified air containing 5% CO2 .
  • D-MEM Dulbecco's modified Eagle medium
  • RPMI 1640 Wako
  • GEBCO fetal bovine serum
  • the probe solution was removed and replaced with fresh HBSS(+), and SRS measurements were performed with an SRS microscope as described above.
  • the detected wavenumbers were 2187 cm -1 and 2190 cm -1 for 9C 15 N-JCR-Bn- ⁇ Gal, 2167 cm -1 and 2170 cm -1 for 9 13 CN-JCR-Bn-gGlu, and 9 13 C 15 N-JCR-Bn-.
  • the EP was 2137 cm ⁇ 1 and 2140 cm ⁇ 1 .
  • Background wavenumbers were 2117 cm ⁇ 1 and 2210 cm ⁇ 1 .
  • Results of simultaneous imaging of three enzymatic activities in live cultured cells are shown in FIG.
  • Left panel of FIG. 8 is SRS imaging of ⁇ -Gal, GGT and DPP-4 activity in live A549 cells (upper panel) and H226 cells (lower panel). Scale bar: 10 ⁇ m. Acquisition time for each cell was 80 seconds.
  • the right panel of FIG. 8 shows SRS spectra of enzymatic activity in A549 cells (upper panel) and H226 cells (lower panel). Highlighted regions indicate peaks for each probe.
  • FIG. 10 shows the results of comparing the ability of 9CN-DEP and 9CN-DER to form aggregates.
  • FIG. 10(a) shows the chemical structures of 9CN-DEP and 9CN-DER under neutral conditions.
  • FIG. 10(b) Permeation of a 300 ⁇ M solution of 9CN-DEP (top) or 9CN-DER (bottom) in PBS (pH 7.4) containing 3%, 10%, 30% and 90% DMSO. Show the image. Scale bar is 100 ⁇ m.
  • FIG. 10(c,d) shows 9CN-DEP (c) and 9CN-DER (d) from 5 to 100 ⁇ M measured in PBS (pH 7.4) containing 0.05% to 1% DMSO as co-solvent. ) shows the normalized absorption spectrum.
  • FIG. 10(e) shows the relationship between dye concentration and normalized absorbance at the absorption maximum of a 5 ⁇ M solution.
  • the black dashed line represents the linear relationship between dye concentration and normalized absorbance.
  • FIG. 10(f,g) 300 ⁇ M 9CN-DEP (f) and 9CN-DER (g) measured in PBS (pH 7.4) containing 3%, 10%, 30% and 90% DMSO. shows the SRS spectrum of The image was constructed by subtracting the image at 2150 cm ⁇ 1 as background. Scale bar is 10 ⁇ m.
  • FIG. 11 shows the results of comparing the ability of nuclei, 9CN-JCR, to form aggregates.
  • FIG. 11 (a-d) 9C 15 N-JCR-Bn- ⁇ Gal (a) from 5 to 100 ⁇ M measured in PBS (pH 7.4) with 0.05% to 1% DMSO as co-solvent. , 9 13 CN-JCR-Bn-gGlu (b), 9 13 C 15 N-JCR-Bn-EP (c) and 9CN-JCR (d). Absorbance was normalized based on the absorbance at the absorption maximum of a 5 ⁇ M solution.
  • FIG. 11(e) shows the relationship between dye concentration and normalized absorbance at the absorption maximum of a 5 ⁇ M solution. The black dashed line represents the linear relationship between dye concentration and normalized absorbance.
  • Example 5 Based on the above results, it was examined whether it is possible to perform target enzyme-specific ex vivo imaging with the 9CN-JCR probe, which could not be performed with the conventional 9CN-JCP probe.
  • the experimental method is as follows.
  • SRS images and spectra of living cells and ex vivo tissues were created by subtracting the average image of background wavenumbers from the average image of detected wavenumbers. For detection wavenumbers, two wavenumbers were selected around the maximum of the SRS intensity of each probe. For the background wavenumber, a wavenumber 20 cm ⁇ 1 away from the detected wavenumber was chosen. If the background and detected wavenumbers were too close due to crosstalk, we chose the highest and lowest background wavenumbers as the background wavenumbers common to all peaks. Data were acquired 1000 times and averaged to enhance the signal-to-noise ratio. To measure the spectrum, the wavenumber was adjusted continuously at 3.3 cm ⁇ 1 intervals and the number of frames used for averaging at each wavenumber was 5.
  • Drosophila culture conditions and strains The Drosophila strains used in this study were en-Gal4, UAS-mCD8-GFP (from Dr. E. Kuranaga), hs- Flp122 , UAS-mCD8-GFP; , UAS-GFP, UAS-lacZ (Bloomington Drosophila Stock Center 1776), and UAS-Ggt-1.
  • Drosophila tissue was cultured in Schneider's Drosophila medium (containing 0.5%, 1% DMSO as co-solvent) with 100 ⁇ M 9C 15 N-JCR-Bn- ⁇ Gal solution, 100 ⁇ M 9C 15 N-JCR-Bn- ⁇ Gal.
  • FIG. 12 shows the results of target enzyme-selective ex vivo imaging of Drosophila tissue using aggregate formation with 9C 15 N-JCR-Bn- ⁇ Gal.
  • FIG. 12(a) is an anatomical drawing of a Drosophila larva. Ex vivo imaging of the wing disc or fat pad was performed.
  • FIG. 12(b) shows the results of fluorescence imaging of membrane-localized GFP and SRS imaging of ⁇ -galactosidase activity in the Drosophila wing disc.
  • Drosophila wing discs (genotype: en-Gal 4, UAS-mCD 8-GFP/UAS-lacZ) were incubated with 100 ⁇ M 9C 15 N-JCR-Bn in Schneider's Drosophila medium with 0.5% DMSO as co-solvent. - incubated with ⁇ Gal for 2.5 hours at room temperature.
  • (c) of FIG. 12 shows the SRS spectrum of the enzyme activity obtained from the field of view of (b).
  • FIG. 12(d) shows the results of fluorescence imaging of membrane and cytoplasmic GFP and SRS imaging of b-galactosidase activity in the Drosophila fat pad.
  • Drosophila fat bodies (genotypes: hs-Flp 122 , UAS-mCD8-GFP; Ay-Gal4, UAS-GFP/UAS-lacZ) were added as co-solvents to 100 ⁇ M in Schneider's Drosophila medium containing 0.5% DMSO. Cultured with 9C 15 N-JCR-Bn- ⁇ Gal at room temperature for 3.5 hours.
  • FIG. 12 shows the SRS spectrum of ⁇ -Gal activity obtained from the field of view (d). Scale bar: 10 ⁇ m. The image acquisition time was 133 seconds.
  • Drosophila tissue larvae in which ⁇ -galactosidase was forcibly expressed region-specifically were stained with the 9Cn-JCR probe. were confirmed in the wing disc and fat body tissues, suggesting that enzyme active region-specific detection was possible.
  • FIG. 13 shows the results of simultaneous imaging of ⁇ Gal-9 13 C 15 N-JCP and 9C 15 N-JCR-Bn- ⁇ Gal using Drosophila wing discs.
  • FIG. 13(a) shows the results of fluorescence imaging of GFP and SRS imaging of ⁇ -Gal activity in the wing disc of Drosophila (genotype: en-Gal4, UAS-mCD8-GFP/UAS-lacZ).
  • FIG. 13(b) shows the SRS spectrum of the ⁇ -Gal probe obtained from the field of view of (a). Highlighted regions indicate peaks for each probe. Green (right area): 9C 15 N-JCR-Bn- ⁇ Gal, yellow (left area): ⁇ Gal-9 13 C 15 N-JCP.
  • the red ROI (lower right box) indicates the LacZ(+) region of (b), and the blue ROI (upper left box) indicates the LacZ( ⁇ ) region of (b).
  • Probes 100 ⁇ M each. Scale bar: 10 ⁇ m.
  • the image acquisition time was 267 seconds.
  • FIG. 14 shows the results of simultaneous imaging of ⁇ Gal-9 13 C 15 N-JCP and 9C 15 N-JCR-Bn- ⁇ Gal using the Drosophila fat pad.
  • FIG. 14(a) shows fluorescence imaging of GFP and ⁇ -Gal activity in Drosophila fat pads (genotypes: hs-Flp 122 , UAS-mCD8-GFP; Ay-Gal4, UAS-GFP/UAS-lacZ). Results of SRS imaging are shown.
  • (b) of FIG. 14 shows the SRS spectrum of the ⁇ -Gal probe obtained from the field of view of (a). Highlighted regions indicate peaks for each probe.
  • the 9CN-JCP probe non-specifically stains the non-enzyme-expressing region, but the 9CN-JCR probe can specifically stain only the enzyme-expressing region in the same tissue. confirmed by imaging at
  • FIG. 15 shows a conceptual diagram of the intracellular retention type probe.
  • 9CN-2CH 2 F-JCR-Bn- ⁇ Gal (see FIG. 16) targeting ⁇ -galactosidase was synthesized according to Synthetic Example 5 described below.
  • Example 6 The in vitro enzymatic reaction of 9CN-2CH 2 F-JCR-Bn- ⁇ Gal synthesized above was evaluated. The results are shown in FIG. As shown in FIG. 16, it was observed to react rapidly with ⁇ -galactosidase to produce a long wavelength product.
  • Example 7 Cell imaging with 9CN-2CH 2 F-JCR-Bn- ⁇ Gal was then performed. At this time, imaging of 9C 15 N-JCR-Bn- ⁇ Gal as a control compound was performed in parallel for comparison. The experimental method is shown below.
  • HEK293 cells and HEK-LacZ cells were purchased from JCRM (JCRB1414/Murakami, T.).
  • HEK293 cells and HEK-LacZ cells were grown in 5% Dulbecco's modified Eagle medium (D-MEM) (Wako) containing 10% fetal bovine serum (GIBCO), 100 U/mL, penicillin, and 100 ⁇ g/mL streptomycin. was cultured at 37 °C in humidified air with 300 rpm of CO2 .
  • D-MEM Dulbecco's modified Eagle medium
  • GEBCO fetal bovine serum
  • FIG. 18 are confocal fluorescence images of ⁇ -galactosidase activity in live HEK-LacZ or HEK293 cells.
  • Cells were incubated with 10 ⁇ M 9CN-2CH 2 F-JCR-Bn- ⁇ Gal (upper panel) or 9C 15 N-JCR-Bn- ⁇ Gal (lower panel) for 30 min in HBSS(+).
  • all probes were confirmed to emit fluorescence specifically to LacZ-expressing cells. Also, the localization of both probes appeared to be almost the same.
  • Example 8 the wash resistance and fixation resistance of both probes (9CN-2CH 2 F-JCR-Bn- ⁇ Gal and 9C 15 N-JCR-Bn- ⁇ Gal) were compared.
  • the wash operation was carried out two times in total, with one operation of replacing with fresh HBSS(+) and incubating for 10 minutes, and imaging after each wash. Furthermore, after the second wash, PFA fixation (15 minutes) was performed and the final imaging was performed. The results are shown in Figures 19 and 20.
  • Figures 19 and 20 are confocal fluorescence images of ⁇ -galactosidase activity in live or fixed HEK-LacZ cells.
  • Cells were incubated with 10 ⁇ M 9CN-2CH 2 F-JCR-Bn- ⁇ Gal (FIG. 19) or 9C 15 N-JCR-Bn- ⁇ Gal (FIG. 20) for 30 min in HBSS(+), then HBSS(+ ) for 10 minutes twice. After washing, cells were fixed with PFA for 15 minutes.
  • Drosophila Culture Conditions and Strains The Drosophila strain used in this study was en-Gal4, UAS-mCD8-GFP (from Dr. E. Kuranaga), and Drosophila tissue was grown in Schneider's Drosophila medium (2% DMSO as co-solvent). containing) with a mixture of 100 ⁇ M 9C 15 N-JCR-Bn- ⁇ Gal and 100 ⁇ M 9CN-2CH 2 F-JCR-Bn- ⁇ Gal for 3 hours at room temperature. After culturing, the tissue was removed from the medium and immersed in fresh PBS(-) on the imaging chamber. SRS measurements were performed with an SRS microscope as previously described.
  • the detected wavenumbers were 2190 cm ⁇ 1 and 2193 cm ⁇ 1 for 9C 15 N-JCR-Bn- ⁇ Gal and 2220 cm ⁇ 1 and 2223 cm ⁇ 1 for 9CN-2CH 2 F-JCR-Bn- ⁇ Gal. Background wavenumbers were 2170 cm ⁇ 1 and 2243 cm ⁇ 1 . Confocal fluorescence imaging was performed after SRS image acquisition. For fluorescence imaging of GFP, the excitation wavelength was 488 nm and emission was detected in the range of 500-520 nm.
  • FIG. 21 shows confocal fluorescence and SRS images of ⁇ -galactosidase activity in the Drosophila wing disc. Scale bar: 10 ⁇ m. The average time was 1000 hours.
  • aggregates such as 9C 15 N-JCR-Bn- ⁇ Gal were also confirmed specifically in the LacZ(+) region of the SPiDER type. Although the LacZ(-) region was also stained widely and leaked out from the enzyme expression region, the signal intensity was significantly stronger with the SPiDER type, which is expected to improve retention in the entire tissue. It was thought that there was.

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Abstract

Le problème décrit par la présente invention est de fournir une sonde Raman de type activable qui permet l'expansion d'une enzyme pouvant être ciblée et qui présente des propriétés de rétention intracellulaire améliorées. À cet effet, l'invention propose un composé représenté par la formule générale (I) ou un sel de celui-ci.
PCT/JP2023/007813 2022-03-02 2023-03-02 Sonde raman activable de type à fonction o WO2023167282A1 (fr)

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