[go: up one dir, main page]

WO2012144654A1 - Sonde fluorescente pour mesurer le sulfure d'hydrogène - Google Patents

Sonde fluorescente pour mesurer le sulfure d'hydrogène Download PDF

Info

Publication number
WO2012144654A1
WO2012144654A1 PCT/JP2012/061300 JP2012061300W WO2012144654A1 WO 2012144654 A1 WO2012144654 A1 WO 2012144654A1 JP 2012061300 W JP2012061300 W JP 2012061300W WO 2012144654 A1 WO2012144654 A1 WO 2012144654A1
Authority
WO
WIPO (PCT)
Prior art keywords
hydrogen atom
group
salt
general formula
compound represented
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/JP2012/061300
Other languages
English (en)
Japanese (ja)
Inventor
哲雄 長野
健二郎 花岡
潔 篠倉
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.)
University of Tokyo NUC
Original Assignee
University of Tokyo NUC
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
Application filed by University of Tokyo NUC filed Critical University of Tokyo NUC
Publication of WO2012144654A1 publication Critical patent/WO2012144654A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/10—Spiro-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/78—Ring systems having three or more relevant rings
    • C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
    • C07D311/82—Xanthenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • the present invention relates to a fluorescent probe for measuring hydrogen sulfide.
  • H 2 S hydrogen sulfide
  • NO nitrogen monoxide
  • CO carbon monoxide
  • H 2 S play is being studied using NaHS and Na 2 S is H 2 S donor, the roles that are best studied is an operation related to relaxation of smooth muscle.
  • H 2 S causes relaxation of vascular smooth muscle and intestinal smooth muscle, and causes symptoms such as headache and blood pressure reduction at the individual level.
  • H 2 S is involved in memory formation in nerve cells, and there is also a report that H 2 S is involved in O 2 sensing and control of insulin secretion in the carotid body.
  • H 2 S contributes to intracellular signal transduction
  • identification of molecules that are targets of H 2 S has not progressed, and there are many unresolved points in specific intracellular signal transduction mechanisms. From such a background, development of a means for visualizing H 2 S in a living body is eagerly desired.
  • This fluorescent probe can detect sulfide ions in water, and is highly specific and sensitive even in the presence of various anions (such as halogen ions, sulfate ions, acetate ions, nitrate ions, hypochlorite ions). 2 It has the characteristic that S can be measured. However, according to a follow-up test by the present inventors, this compound has low selectivity with reduced glutathione, and H 2 S present in cells or the like in vivo due to glutathione present in large quantities in the living body. There is a problem that it cannot be measured specifically and with high sensitivity. A compound using a cyclic polyamine as a zinc ion capturing group is known (dansylaminoethylcyclene: J.
  • An object of the present invention is to provide a fluorescent probe capable of specifically visualizing hydrogen sulfide in a living body and performing highly sensitive measurement.
  • R 1 represents the following formula (A): (In the formula, p, q, r, and s each independently represent an integer of 2 or 3, t represents 0 or 1, and R 11 , R 12 , and R 13 each independently represent a hydrogen atom or a carbon atom.
  • R 2 represents a hydrogen atom or 1 to 3 monovalent substituents substituted on a benzene ring
  • R 3 and R 4 represent Each independently represents a hydrogen atom or a halogen atom
  • R 5 and R 6 each independently represent a hydrogen atom, an alkylcarbonyl group, or an alkylcarbonyloxyalkyl group
  • R 7 represents a hydrogen atom, an alkyl group, or an alkoxyalkyl group.
  • R 8 and R 9 are each independently a hydrogen atom, a halogen atom, or — (CH 2 ) x —N (R 14 ) (R 15 ) (wherein x represents an integer of 1 to 4; R 14 and R 15 are each The standing - (CH 2) y -COOR 16 ( wherein, y represents an integer of 1 4, R 16 is a hydrogen atom, an alkylcarbonyl group, or an alkyl carbonyl an oxy alkyl group) a group represented by Or a salt thereof is provided.
  • p, q, r, and s are 2, t is 1, and R 11 , R 12 , and R 13 are hydrogen atoms, A compound represented by (IB) or a salt thereof; p, q, r, and s are 2, t is 1, R 11 , R 12 , and R 13 are hydrogen atoms, and R 2 is A compound represented by the above general formula (IA) or (IB) which is a hydrogen atom or a salt thereof; and R 1 present on the benzene ring is bonded to the para-position relative to the binding site of the xanthene ring, p, q, r, and s are 2, t is 1, R 11 , R 12 , and R 13 are hydrogen atoms, and R 2 is a hydrogen atom, or the above general formula (IA) or ( A compound represented by IB) or a salt thereof is provided.
  • the compound represented by the above general formula (IA) or (IB) or a salt thereof wherein R 3 and R 4 are hydrogen atoms; R 5 and R 6 are each independently a hydrogen atom, an acetyl group; Or a compound represented by the above general formula (IA) or (IB), which is an acetyloxymethyl group, or a salt thereof; R 7 is a hydrogen atom, an alkyl group, or a methoxymethyl group; And a compound represented by the above general formula (IA) or (IB) or a salt thereof, wherein both R 8 and R 9 are hydrogen atoms.
  • R 21 represents the following formula (B): (In the formula, d, e, f, and g each independently represent an integer of 2 or 3, h represents 0 or 1, and R 31 , R 32 , and R 33 each independently represent a hydrogen atom or a carbon atom.
  • R 22 represents a hydrogen atom or 1 to 3 monovalent substituents substituted on a benzene ring
  • R 23 and R 24 represent Each independently represents a hydrogen atom or a halogen atom
  • R 25 and R 26 each independently represent a hydrogen atom, an alkylcarbonyl group, or an alkylcarbonyloxyalkyl group
  • R 27 represents a hydrogen atom, an alkyl group, or an alkoxyalkyl group
  • R 28 and R 29 are each independently a hydrogen atom, a halogen atom, or — (CH 2 ) m —N (R 34 ) (R 35 ) (wherein m represents an integer of 1 to 4; R 34 as well as 35 are each independently - (CH 2) n -COOR 36 ( wherein, n represents an integer of 1 to 4, R 36 is a hydrogen atom, an alkyl group, or an alkylcarbonyloxy group) is represented by Or a salt thereof is provided.
  • d, e, f, and g are 2, h is 1, and R 31 , R 32 , and R 33 are hydrogen atoms, A compound represented by (IIB) or a salt thereof; d, e, f, and g are 2, h is 1, R 31 , R 32 , and R 33 are hydrogen atoms, and R 22 is A compound represented by the above general formula (IIA) or (IIB) which is a hydrogen atom or a salt thereof; and R 21 present on the benzene ring is bonded to the para-position relative to the binding site of the xanthene ring, wherein d, e, f, and g are 2, h is 1, R 31 , R 32 , and R 33 are hydrogen atoms, and R 22 is a hydrogen atom, the above general formula (IIA) or ( A compound represented by IIB) or a salt thereof is provided.
  • the compound represented by the above general formula (IIA) or (IIB) or a salt thereof wherein R 23 and R 24 are hydrogen atoms; R 25 and R 26 are each independently a hydrogen atom, an acetyl group; Or a compound represented by the above general formula (IIA) or (IIB), which is an acetyloxymethyl group, or a salt thereof; R 27 is a hydrogen atom, an alkyl group, or a methoxymethyl group; IIB) or a salt thereof; and a compound represented by the above general formula (IIA) or (IIB) or a salt thereof, in which R 28 and R 29 are both hydrogen atoms.
  • a reagent for measuring hydrogen sulfide containing the compound or a salt thereof is provided.
  • the present invention also provides use of the compound represented by the above general formula (IA) or (IB) or a salt thereof for the production of a fluorescent probe for measuring hydrogen sulfide or a reagent for measuring hydrogen sulfide.
  • a fluorescent probe for measuring hydrogen sulfide containing a compound represented by the above general formula (IIA) or (IIB) or a salt thereof, and a compound represented by the above general formula (IIA) or (IIB) or A reagent for measuring hydrogen sulfide containing the salt is provided.
  • the present invention also provides use of the compound represented by the above general formula (IIA) or (IIB) or a salt thereof for the production of a fluorescent probe for measuring hydrogen sulfide or a reagent for measuring hydrogen sulfide.
  • This fluorescent probe and reagent react with divalent copper ions in the measurement system to generate a compound represented by the above general formula (IA) or (IB) or a salt thereof in situ in the measurement system. It is useful as a fluorescent probe or reagent for measurement.
  • a method for measuring hydrogen sulfide comprising the following steps: (a) contacting a compound represented by the above general formula (IA) or (IB) or a salt thereof with hydrogen sulfide And (b) a method of measuring the fluorescence of the compound represented by the general formula (IIA) or (IIB) produced in the step (a) or a salt thereof.
  • a method for measuring hydrogen sulfide comprising the steps of: (a) reacting a compound represented by the general formula (IIA) or (IIB) or a salt thereof with a divalent copper ion. A step of producing a compound represented by the above general formula (IA) or (IB) or a salt thereof; (b) a compound represented by the above general formula (IA) or (IB) produced in the above step (a); A step of contacting the salt with hydrogen sulfide; and (c) a step of measuring the fluorescence of the compound represented by the general formula (IIA) or (IIB) generated in the step (b) or the salt thereof. Is provided.
  • the compound represented by the general formula (IA) or (IB) provided by the present invention or a salt thereof has high reactivity to hydrogen sulfide, and is a general formula having strong fluorescence in the presence of hydrogen sulfide. It has the property of producing a compound represented by (IIA) or (IIB) or a salt thereof.
  • the compound represented by the general formula (IA) or (IB) or a salt thereof since the compound represented by the general formula (IA) or (IB) or a salt thereof has substantially no reactivity with reduced glutathione, it can be sulfided with high sensitivity even in the presence of reduced glutathione. Hydrogen can be measured.
  • the compound represented by the general formula (IA) or (IB) or a salt thereof is specific even in the presence of various anions (halogen ion, sulfate ion, acetate ion, nitrate ion, hypochlorite ion, etc.). It can react with hydrogen sulfide with high sensitivity. Therefore, the fluorescent probe for measuring hydrogen sulfide containing the compound represented by the general formula (IA) or (IB) or a salt thereof is a fluorescence for measuring a small amount of hydrogen sulfide existing in a living body with high sensitivity and high accuracy. It is useful as a probe and extremely useful as a reagent for imaging hydrogen sulfide in cells and tissues in an in vivo environment.
  • the upper part shows before Na 2 S addition (0 sec), the middle part shows after Na 2 S addition (240 sec), and the lower part shows the change in fluorescence intensity over time.
  • 100 ⁇ M Na 2 S (red), 10 ⁇ M Na 2 S (green), or 10 mM GSH (yellow) was added, and the results of examining the reactivity with the addition-free experiment as a negative control (blue) (lower) are shown. It is a figure.
  • FIG. 5 is a diagram showing the results of examining the reactivity of an additive-free experiment as a negative control (blue).
  • R 1 represents a group represented by the formula (A).
  • p, q, r, and s each independently represent an integer of 2 or 3, preferably p, q, r, and s are all 2.
  • . t represents 0 or 1, but is preferably 1.
  • R 11 , R 12 , and R 13 each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and any or all of R 11 , R 12 , and R 13 are hydrogen atoms. It is preferable that R 11 , R 12 , and R 13 are all hydrogen atoms.
  • alkyl group includes an alkyl group composed of linear, branched, cyclic, and combinations thereof. The same applies to the alkyl part of other substituents having an alkyl part (for example, an alkylcarbonyl group or an alkoxyalkyl group).
  • R 2 represents a hydrogen atom or 1 to 3 monovalent substituents substituted on the benzene ring, preferably 1 or 2 monovalent substituents substituted on the hydrogen atom or benzene ring. More preferably, it represents one monovalent substituent substituted on a hydrogen atom or a benzene ring.
  • R 2 is a monovalent substituent
  • substituents include a halogen atom (in this specification, the term halogen atom includes a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom), a hydroxyl group, Examples thereof include, but are not limited to, an oxo group, a carboxyl group, an alkoxycarbonyl group, an acyl group, an amino group, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, and an aralkyl group. These substituents may be further substituted with another substituent. Examples of such include, but are not limited to, a fluoroalkyl group, a fluoroacetyl group, a methoxybenzyl group, and the like.
  • R 2 is particularly preferably a hydrogen atom.
  • R 3 and R 4 each independently represent a hydrogen atom or a halogen atom, but it is preferable that both R 3 and R 4 are hydrogen atoms.
  • R 5 and R 6 each independently represent a hydrogen atom, an alkylcarbonyl group, or an alkylcarbonyloxyalkyl group.
  • the alkylcarbonyl group is preferably an alkylcarbonyl group having about 2 to 7 carbon atoms, and for example, an acetyl group can be preferably used.
  • alkylcarbonyloxyalkyl group for example, an alkylcarbonyloxyalkyl group in which an alkylcarbonyloxy group having about 2 to 7 carbon atoms is substituted with an alkyl group having about 1 to 6 carbon atoms is preferable.
  • an acetoxymethyl group Etc. can be preferably used.
  • R 7 represents a hydrogen atom, an alkyl group, or an alkoxyalkyl group.
  • the alkoxyalkyl group is preferably an alkoxyalkyl group in which an alkoxy group having about 1 to 6 carbon atoms is substituted with an alkyl group having about 1 to 6 carbon atoms.
  • a methoxymethyl group or an ethoxymethyl group is preferably used. Can do.
  • R 8 and R 9 each independently represent a hydrogen atom, a halogen atom, or a group represented by — (CH 2 ) x —N (R 14 ) (R 15 ).
  • x represents an integer of 1 to 4, but x is preferably 1 or 2, and x is particularly preferably 1.
  • R 14 and R 15 each independently represent a group represented by — (CH 2 ) y —COOR 16 .
  • y represents an integer of 1 to 4, and y is preferably 1 or 2, and y is particularly preferably 1.
  • R 16 represents a hydrogen atom, an alkylcarbonyl group, or an alkylcarbonyloxyalkyl group, and the alkylcarbonyl group and the alkylcarbonyloxyalkyl group are the same as those described for R 5 above.
  • a hydrogen atom or an acetoxymethyl group can be used as R 16 .
  • R 11 , R 12 , R 13 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , x, R 14 , R 15 , n, and R 16 are the same.
  • the compound represented by general formula (IA) or (IB), or general formula (IIA) or (IIB) may form an acid addition salt or a base addition salt.
  • acid addition salts include mineral acid salts such as hydrochloride, sulfate, and nitrate, and organic acid salts such as p-toluenesulfonate, oxalate, and malate.
  • base addition salt include metal salts such as sodium salt, potassium salt, magnesium salt, or calcium salt, ammonium salts, and organic amine salts such as triethylamine salt or ethanolamine salt.
  • physiologically acceptable salts are preferable when the compound of the present invention is used as a reagent for measuring hydrogen sulfide in a living body, cell or tissue.
  • the compound represented by general formula (IA) or (IB), or general formula (IIA) or (IIB) may have one or two or more asymmetric carbons depending on the type of the substituent.
  • Arbitrary optical isomers based on these asymmetric carbons, arbitrary mixtures of optical isomers, racemates, diastereoisomers based on two or more asymmetric carbons, arbitrary mixtures of diastereoisomers, etc. Are included in the scope of the present invention. Any hydrate or solvate of the free compound or salt form of the compound is also encompassed within the scope of the present invention.
  • R 7 is a hydrogen atom
  • the carboxy group may form a lactone, but such structural isomers are also included in the scope of the present invention.
  • the compound in which R 5 is a hydrogen atom in the general formula (IA) and the compound in which R 7 is a hydrogen atom in the general formula (IB) correspond to tautomers, but such tautomers exist. Is easily understood by those skilled in the art, and any tautomer is included in the scope of the present invention.
  • a fluorescein derivative such as 4-aminofluorescein that can be used as a raw material compound can be produced according to the method described in, for example, Tetsuji Kameya, Synthetic Organic Chemistry IX, Nanedo, page 215 (1977).
  • cyclic polyamine partial structure J.A. Am. Chem. Soc. , 118, 12696, 1996, can be referred to the synthesis of the partial structure of the zinc probe.
  • the method for measuring hydrogen sulfide generally comprises (a) a step of reacting a compound represented by the general formula (IA) or (IB) or a salt thereof with hydrogen sulfide, and (b) the above step.
  • the compound represented by the general formula (IA) or (IB) is quenched by the coordination of divalent copper ions, and is itself non-fluorescent or weakly fluorescent. Copper ions react with hydrogen sulfide to form insoluble CuS, which is removed from the cyclic polyamine moiety to produce a strongly fluorescent compound represented by the general formula (IIA) or (IIB) or a salt thereof.
  • a compound represented by the general formula (IA) or (IB) in which a divalent copper ion is coordinated or a salt thereof is dissolved in an appropriate aqueous medium such as water.
  • an appropriate aqueous medium such as water.
  • a compound represented by the general formula (IIA) or (IIB) or a salt thereof and a divalent copper ion are reacted to represent in situ the general formula (IA) or (IB).
  • a salt thereof may be produced in a measurement system and the compound represented by the general formula (IA) or (IB) or a salt thereof may be reacted with hydrogen sulfide. It goes without saying that such a measuring method is also included in the scope of the present invention.
  • the fluorescence measuring means using the hydrogen sulfide measuring reagent of the present invention is not particularly limited, but a method of measuring a fluorescence spectrum in vitro, a method of measuring a fluorescence spectrum in vivo using a bioimaging technique, etc. Can be adopted. For example, when quantification is performed, it is desirable to prepare a calibration curve in advance according to a conventional method. If the reagent of the present invention is incorporated into cells by a microinjection method or the like, hydrogen sulfide localized in individual cells can be measured with high sensitivity in real time by a bioimaging technique, and a cell culture solution or tissue slice The hydrogen sulfide released from cells and living tissues can be measured by using it in a culture solution or perfusate.
  • the hydrogen sulfide measurement reagent of the present invention it is possible to measure the behavior of hydrogen sulfide in cells or living tissues in real time. In addition to elucidating the mechanism of signal transmission by hydrogen sulfide, It can be suitably used for investigating the cause of the disease and developing therapeutic agents.
  • ester group that can be hydrolyzed by esterase or the like when introduced into any one or more of R 5 , R 6 , R 7 , R 8 , or R 9 , the entire molecule becomes fat-soluble. Thus, it easily penetrates the cell membrane and reaches the cytoplasm.
  • a hydrophilic carboxyl group is generated by esterase hydrolysis in the cytoplasm, the cell membrane cannot be easily penetrated. Accordingly, such ester-introduced compounds (for example, compounds into which acetoxymethyl ester or methoxymethyl ester is introduced as an ester) are extremely useful as reagents for measuring the concentration of hydrogen sulfide in the cytoplasm.
  • the reagent of the present invention may be used as a composition by blending additives usually used in the preparation of the reagent as necessary.
  • additives such as a solubilizer, pH adjuster, buffer, and isotonic agent can be used as an additive for using the reagent in a physiological environment. Is possible.
  • These compositions are provided as a composition in an appropriate form such as a mixture in a powder form, a lyophilized product, a granule, a tablet, or a liquid.
  • Example 1 Cyclen-4-AF and Cyclen-4-AF-Cu were synthesized according to the following synthesis scheme.
  • Cyclen-4-AF Chloroacetylamido-4-aminofluorescein (106.6 mg, 251.5 ⁇ mol) was dissolved in 30 mL of dehydrated acetonitrile, cyclen (368.0 mg, 2.14 mmol) and diisopropylethylamine (DIEA, 300 ⁇ L, 1.72 mmol) were added, Stir at 70 ° C. for 8 hours under argon. After removing the solvent, the residue was purified by HPLC to obtain Cyclen-4-AF (TFA salt) (103.3 mg, 109.0 ⁇ mol, y.43%).
  • DIEA diisopropylethylamine
  • Cyclen-4-AF-Cu CuSO 4 pentahydrate was dissolved in 30 mM HEPES buffer (pH 7.4) to prepare a 1M CuSO 4 aqueous solution.
  • Cyclen-4-AF (73.8 mg, 77.9 ⁇ mol) was dissolved in an aqueous CuSO 4 solution (7792 ⁇ L, 7.79 mmol), stirred overnight at room temperature, purified by HPLC, and purified with Cyclen-4-AF-Cu ( 65.2 mg, quant yield).
  • Cyclen-4-AF-Cu diacetate Cyclen-4-AF-Cu (5.60 ⁇ mol) was dissolved in 4 mL of dehydrated acetonitrile, pyridine (4.14 mmol) and acetic anhydride (41.9 mmol) were added, and the mixture was stirred at 60 ° C. for 6 hours under argon. After removing the solvent, the residue was purified by HPLC to obtain Cyclen-4-AF-Cu diacetate (3.6 mg, 5.1 ⁇ mol, y. 91%).
  • Example 2 TACN-4-AF was synthesized by the following scheme.
  • Chloroacetylamide-4-aminofluorescein (53.8 mg, 126.9 ⁇ mol) was dissolved in 8 mL of dehydrated acetonitrile, and 1,4,7-triazacyclononane (TACN, 101.4 mg, 784.8 ⁇ mol), potassium iodide. (6.8 mg, 41.0 ⁇ mol) and K 2 CO 3 (111.0 mg, 803.1 ⁇ mol) were added and stirred overnight at room temperature under argon. After removing the solvent, the product was purified by HPLC to obtain TACN-4-AF (TFA salt) (47.4 mg, 55.2 ⁇ mol, y.44%).
  • TACN 1,4,7-triazacyclononane
  • K 2 CO 3 111.0 mg, 803.1 ⁇ mol
  • TMCyclen-4-AF and TMCyclen-4-AF-Cu were synthesized by the following scheme.
  • step (B) The compound obtained in the above step (a) (669.7 mg, 1.82 mmol) was dissolved in 5 mL of concentrated sulfuric acid and stirred at 110 ° C. for 11 hours under argon. After dilution with water, the solution was made basic with 2N NaOH solution and extracted with dichloromethane. After dehydration, the solvent was removed to obtain the desired product (348.7 mg, 1.63 mmol, y.89%).
  • TMCyclen-4-AF-Cu CuSO 4 pentahydrate was dissolved in 30 mM HEPES buffer (pH 7.4) to prepare a 1M CuSO 4 aqueous solution.
  • TMCyclen-AF (12.1 mg, 12.2 ⁇ mol) was dissolved in an aqueous CuSO 4 solution (1220 ⁇ L, 1.22 mmol), stirred at room temperature for 4 hours, purified by HPLC, and purified by TMCyclen-4-AF-Cu. (9.52 mg, y.quant) was obtained.
  • Example 6 The reactivity of Cyclen-4-AF-Cu was evaluated. 10 ⁇ M Na 2 S (red), 10 ⁇ M Na 2 S, 10 mM GSH (green), and 10 mM GSH (yellow) were added to 1 ⁇ M Cyclen-4-AF-Cu 300 seconds later, and the experiment without addition was negative control The reactivity was investigated as (blue). Excitation was performed at 491 nm in 30 mM HEPES buffer (pH 7.4), and the fluorescence intensity at 516 nm was measured. The results are shown in FIG. Cyclen-4-AF-Cu showed an increase in fluorescence after reaction with Na 2 S, indicating that it has selectivity compared to GSH. Cyclen-4-AF-Cu was also found to increase in fluorescence intensity when 10 ⁇ M Na 2 S was added even in the presence of 10 mM GSH.
  • Example 7 It was evaluated whether or not hydrogen sulfide in vivo can be measured using Cyclen-4-AF-Cu.
  • HeLa cells were cultured in a medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin, and 1% streptomycin in Dulbecco's modified Eagle medium (DMEM) at 37 ° C. in 5% CO 2 mixed air. Washed twice with Hanks balanced salt solution (HBSS). HBSS (0.1% DMSO) containing 100 ⁇ M Cyclen-4-AF-Cu was microinjected (region 1-4) into HeLa cells in HBSS. Excitation was performed at 470-490 nm with a fluorescence microscope, and the fluorescence intensity at 515-550 nm was measured.
  • FBS fetal bovine serum
  • DMEM Dulbecco's modified Eagle medium
  • FIG. 6 shows the results of measurement of hydrogen sulfide by incorporation into HeLa cells using Cyclen-4-AF-Cu diacetate.
  • Hela cells cultured as described above were incubated at 37 ° C. for 2 hours in HBSS containing 100 ⁇ M Cyclen-4-AF-Cu diacetate. Excitation was performed at 470-490 nm with a fluorescence microscope, and the fluorescence intensity at 515-550 nm was measured. 210 mM after the start of measurement, 10 mM Na 2 S was added to the extracellular fluid. Increased fluorescence intensity was observed inside and outside HeLa cells.
  • the upper part of FIG. 6 shows before Na 2 S addition (0 sec), the middle part after Na 2 S addition (240 sec), and the lower part shows the change in fluorescence intensity with time.
  • Example 8 The absorption spectrum and fluorescence spectrum of TACN-4-AF alone and TACN-4-AF added with 2 equivalents of Cu 2+ were measured. The measurement was performed in 30 mM HEPES buffer (pH 7.4), and excitation was performed at 491 nm in the measurement of the fluorescence spectrum. Addition of Cu 2+ significantly reduced the fluorescence intensity (FIG. 7, upper left: absorption spectrum, upper right: fluorescence spectrum).
  • Example 9 The absorption spectrum and fluorescence spectrum of Cyclam-4-AF alone and Cyclam-4-AF added with 2 equivalents of Cu 2+ were measured. The measurement was performed in 30 mM HEPES buffer (pH 7.4), and excitation was performed at 491 nm in the measurement of the fluorescence spectrum. Addition of Cu 2+ significantly decreased the fluorescence intensity (FIG. 8).
  • Example 10 2 ⁇ M CuSO 4 was added to 1 ⁇ M Cyclam-4-AF, and after 300 seconds, 100 ⁇ M Na 2 S (red), 10 ⁇ M Na 2 S (green), or 10 mM GSH (yellow) was added. The reactivity was investigated as blue). Excitation was performed at 491 nm in 30 mM HEPES buffer (pH 7.4), and the fluorescence intensity at 516 nm was measured. As a result, Cyclam-4-AF + Cu 2+ after the reaction with Na 2 S, shows the fluorescence increase was shown to have a selectivity compared with GSH (Fig. 9 top).
  • TMCyclen-4-AF-Cu 100 ⁇ M Na 2 S (red), 10 ⁇ M Na 2 S (green), or 10 mM GSH (yellow) was added to 1 ⁇ M TMCyclen-4-AF-Cu after 300 seconds.
  • TMCyclen-4-AF-Cu also showed an increase in fluorescence after reaction with Na 2 S, indicating that it has selectivity compared to GSH (lower part of FIG. 9). ).
  • Example 11 Detection of hydrogen sulfide using cell lysate of 3MST-expressing cells Express 3-mercaptopyruvate sulfate transferase (3MST), an enzyme that produces H 2 S using 3-mercaptopyruvate (3MP) as a substrate
  • 3MST 3-mercaptopyruvate sulfate transferase
  • the plasmid was introduced into HEK293 cells by a method described in the literature (Antioxid. Redox Signal, 11, 703, 2009). The cells were suspended in 30 mM HEPES buffer (pH 7.4) containing 100 ⁇ M dithiothreitol and 1% protease inhibitor cocktail (Sigma), lysed using an ultrasonic crusher, and used for experiments.
  • HEK293 cells into which no plasmid was introduced were also lysed and used for the experiment. Then, 1 ⁇ M Cycle-4-AF-Cu was added, and 100 ⁇ M 3MP was added after 180 seconds to evaluate the responsiveness. As a result of excitation at 491 nm and measurement of fluorescence intensity at 516 nm, it was shown that when 3MP was added to a lysate containing 3MST, the fluorescence intensity significantly increased (left figure in FIG. 10). Further, after 10 minutes in the presence of 3-mercaptopyruvic acid, a difference was observed in fluorescence intensity with a significant difference (p ⁇ 0.05) depending on the presence or absence of 3MST (the right diagram in FIG. 10).
  • Example 12 Detection of hydrogen sulfide using purified 3MST Glutathione-S-transferase (GST) -tagged 3-mercaptopyruvate sulfatransferase (3MST) purified protein and 3-mercaptopyruvate (3MP) as substrate It was evaluated whether the produced H 2 S was detectable by Cyclen-4-AF-Cu. 1 ⁇ M Cyclen-4-AF-Cu was added, and GST tag fusion 3MST (red, green) or GST tag (yellow, blue) was added 60 seconds later.
  • GST Glutathione-S-transferase
  • 3MST 3-mercaptopyruvate sulfatransferase
  • Cyclen-4-AF-Cu showed a significant increase in fluorescence intensity in response to H 2 S produced by the addition of GST-tag-fused 3MST and 3MP, which are purified proteins (left panel in FIG. 11).
  • H 2 S produced by an enzyme reaction could be detected in a 96-well plate. Then, 3MP or the same amount of HEPES buffer (pH 7.4) was added and incubated at 37 ° C.
  • the compound of the present invention can be used as a fluorescent probe capable of measuring hydrogen sulfide with high sensitivity and specificity without being affected by reduced glutathione present in large quantities in the living body.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
  • Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
  • Plural Heterocyclic Compounds (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

La présente invention concerne un composé de formule (I) (où R1 représente un groupe de formule (A) ; R2 représente un atome d'hydrogène ou un substituant univalent ; R3 et R4 représentent indépendamment un atome d'hydrogène ou un atome d'halogène ; R5 et R6 représentent indépendamment un atome d'hydrogène, un groupe alkylcarbonyle ou un groupe équivalent ; R7 représente un atome d'hydrogène, un groupe alkyle ou un groupe alcoxyalkyle ; et R8 et R9 représentent indépendamment un atome d'hydrogène, un atome d'halogène ou un groupe équivalent). Le composé de formule (I) peut être utilisé comme sonde fluorescente pour mesurer spécifiquement et avec une grande sensibilité le sulfure d'hydrogène, sans être soumis à l'influence de la glutathione réduite.
PCT/JP2012/061300 2011-04-22 2012-04-20 Sonde fluorescente pour mesurer le sulfure d'hydrogène Ceased WO2012144654A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2011-095598 2011-04-22
JP2011095598A JP2014133704A (ja) 2011-04-22 2011-04-22 硫化水素測定用蛍光プローブ

Publications (1)

Publication Number Publication Date
WO2012144654A1 true WO2012144654A1 (fr) 2012-10-26

Family

ID=47041744

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2012/061300 Ceased WO2012144654A1 (fr) 2011-04-22 2012-04-20 Sonde fluorescente pour mesurer le sulfure d'hydrogène

Country Status (2)

Country Link
JP (1) JP2014133704A (fr)
WO (1) WO2012144654A1 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103131205A (zh) * 2013-02-04 2013-06-05 大连理工大学 罗丹明荧光染料及其制备方法和应用
WO2014136781A1 (fr) * 2013-03-04 2014-09-12 国立大学法人 東京大学 Sonde fluorescente
WO2014153624A1 (fr) * 2013-03-28 2014-10-02 The University Of Sydney Composés antibactériens
CN104419401A (zh) * 2013-08-28 2015-03-18 苏州罗兰生物科技有限公司 一种荧光增强检测硫化氢荧光探针及其合成与应用
CN105038763A (zh) * 2015-06-04 2015-11-11 济南大学 一种识别溶酶体内硫化氢的荧光探针及其应用
KR20170138158A (ko) 2016-06-07 2017-12-15 경북대학교 산학협력단 황화수소 검출용 조성물 및 이를 이용한 황화수소 검출방법
CN107686479A (zh) * 2017-09-30 2018-02-13 湖南师范大学 一种近红外荧光探针化合物及其制备方法和应用
CN109265440A (zh) * 2018-12-13 2019-01-25 中国科学院合肥物质科学研究院 氮杂环类荧光探针的制备方法及在硫化氢检测中的应用
CN114539280A (zh) * 2020-11-26 2022-05-27 佳能株式会社 用于检测硫化氢的新化合物和结构体

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102280948B1 (ko) * 2019-11-28 2021-07-23 한국과학기술연구원 단백질 아세틸화 검출을 위한 형광 프로브

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007238650A (ja) * 2006-03-03 2007-09-20 Kyoto Univ 希土類金属錯体及びそれを用いた蛍光発光材料

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007238650A (ja) * 2006-03-03 2007-09-20 Kyoto Univ 希土類金属錯体及びそれを用いた蛍光発光材料

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
AITA KAZUKI ET AL.: "Development of a novel neodymium compound for in vivo fluorescence imaging", LUMINESCENCE, vol. 22, no. 5, 2007, pages 455 - 461, XP009147455, DOI: doi:10.1002/bio.984 *
ALEXANDER R. LIPPERT ET AL.: "Reaction-Based Fluorescent Probes for Selective Imaging of Hydrogen Sulfide in Living Cells", J. AM. CHEM. SOC., vol. 113, 15 June 2011 (2011-06-15), pages 10078 - 10080 *
DIEGO JIMENEZ ET AL.: "A New Chromo- chemodosimeter Selective for Sulfide Anion", J. AM. CHEM. SOC., 2003, pages 9000 - 9001 *
KIYOSHI SASAKURA ET AL.: "Development of a Highly Selective Fluorescence Probe for Hydrogen Sulfide", J. AM. CHEM. SOC., vol. 133, 14 October 2011 (2011-10-14), pages 18003 - 18005 *
MYUNG GIL CHOI ET AL.: "Sulfide-selective chemosignaling by a Cu2+ complex of dipicolylamine appended fluorescein", CHEM. COMMUN., 2009, pages 7390 - 7392 *
SHIN MIZUKAMI ET AL.: "A Fluorescent Anion Sensor That Works in Neutral Aqueous Solution for Bioanalytical Application", J. AM. CHEM. SOC., vol. 124, 2002, pages 3920 - 3925 *
TOHRU KOIKE ET AL.: "A Novel Biomimetic Zinc(II) -Fluorophore, Dansylamidoethyl-Pendant Macrocyclic Tetraamine 1,4,7,10- Tetraazacyclododecane (Cyclen)", J. AM. CHEM. SOC., vol. 118, 1996, pages 12696 - 12703, XP001042265, DOI: doi:10.1021/ja962527a *
XIAO-FENG YANG ET AL.: "A fluorescein-based fluorogenic and chromogenic chemodosimeter for the sensitive detection of sulfide anion in aqueous solution", ANALYTICA CHIMICA ACTA, vol. 631, 2009, pages 91 - 95, XP025713794, DOI: doi:10.1016/j.aca.2008.10.037 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103131205A (zh) * 2013-02-04 2013-06-05 大连理工大学 罗丹明荧光染料及其制备方法和应用
WO2014136781A1 (fr) * 2013-03-04 2014-09-12 国立大学法人 東京大学 Sonde fluorescente
WO2014153624A1 (fr) * 2013-03-28 2014-10-02 The University Of Sydney Composés antibactériens
CN104419401A (zh) * 2013-08-28 2015-03-18 苏州罗兰生物科技有限公司 一种荧光增强检测硫化氢荧光探针及其合成与应用
CN105038763A (zh) * 2015-06-04 2015-11-11 济南大学 一种识别溶酶体内硫化氢的荧光探针及其应用
KR20170138158A (ko) 2016-06-07 2017-12-15 경북대학교 산학협력단 황화수소 검출용 조성물 및 이를 이용한 황화수소 검출방법
CN107686479A (zh) * 2017-09-30 2018-02-13 湖南师范大学 一种近红外荧光探针化合物及其制备方法和应用
CN109265440A (zh) * 2018-12-13 2019-01-25 中国科学院合肥物质科学研究院 氮杂环类荧光探针的制备方法及在硫化氢检测中的应用
CN109265440B (zh) * 2018-12-13 2020-04-07 中国科学院合肥物质科学研究院 氮杂环类荧光探针的制备方法及在硫化氢检测中的应用
CN114539280A (zh) * 2020-11-26 2022-05-27 佳能株式会社 用于检测硫化氢的新化合物和结构体
US12049469B2 (en) 2020-11-26 2024-07-30 Canon Kabushiki Kaisha Compound and structural body for detecting hydrogen sulfide

Also Published As

Publication number Publication date
JP2014133704A (ja) 2014-07-24

Similar Documents

Publication Publication Date Title
WO2012144654A1 (fr) Sonde fluorescente pour mesurer le sulfure d'hydrogène
Zeng et al. A distinctive mitochondrion-targeting, in situ-activatable near-infrared fluorescent probe for visualizing sulfur dioxide derivatives and their fluctuations in vivo
Sunnapu et al. Rhodamine based effective chemosensor for Chromium (III) and their application in live cell imaging
EP3096143B1 (fr) Agent de détection d'ions de fer (ii) et procédé de détection l'utilisant
JP6351511B2 (ja) 非対称Ｓｉローダミン及びロドールの合成
Zhou et al. An activated near-infrared fluorescent probe with large Stokes shift for discrimination of bio-thiols
WO2012111818A1 (fr) Sonde fluorescente
Huang et al. A novel near-infrared fluorescent hydrogen sulfide probe for live cell and tissue imaging
Ismail et al. A julolidine-fused coumarin-NBD dyad for highly selective and sensitive detection of H2S in biological samples
Liu et al. Synthesis, crystal structure and living cell imaging of a Cu 2+-specific molecular probe
JP5228190B2 (ja) パーオキシナイトライト蛍光プローブ
Liu et al. A new coumarin-based fluorescent probe for selective recognition of Cu2+ and S2− in aqueous solution and living cells
Liu et al. A squaraine-based red emission off–on chemosensor for biothiols and its application in living cells imaging
Guo et al. A fluorescent probe derived from benzofuranone for turn-on detection of sulfite in living cells and mice
Rong et al. Double-channel based fluorescent probe for differentiating GSH and H2Sn (n> 1) via a single-wavelength excitation with long-wavelength emission
Shao et al. Identification of fatty liver disease at diverse stages using two-photon absorption of triphenylamine-based BODIPY analogues
Wu et al. A novel near-infrared xanthene-based fluorescent probe for detection of thiophenol in vitro and in vivo
Wang et al. A visible-light-excited europium (III) complex-based luminescent probe for visualizing copper ions and hydrogen sulfide in living cells
JP4402191B2 (ja) 亜鉛蛍光プローブ
JPWO2018174253A1 (ja) ニトロベンゼン誘導体またはその塩およびそれらの用途
CN113637048B (zh) 一种γ-谷氨酰转肽酶的双光子荧光探针及其制备方法和应用
Guo et al. Novel dual-site fluorescent probe for monitoring cysteine and sulfite in living cells
US7696245B2 (en) Fluorescent probe for zinc
CN110105391B (zh) 碱性磷酸酶响应型分子探针及其应用
CN114539183A (zh) 一种癌细胞组织诊断的脂滴靶向和生物硫醇敏感的荧光探针及制备和应用

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: 12774910

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12774910

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP