KR102052417B1 - Fluorescent probe compound for detecting nitroxyl in mitochondria and a nitroxyl detection fluorescence sensor comprising the same - Google Patents
Fluorescent probe compound for detecting nitroxyl in mitochondria and a nitroxyl detection fluorescence sensor comprising the same Download PDFInfo
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- KR102052417B1 KR102052417B1 KR1020180003487A KR20180003487A KR102052417B1 KR 102052417 B1 KR102052417 B1 KR 102052417B1 KR 1020180003487 A KR1020180003487 A KR 1020180003487A KR 20180003487 A KR20180003487 A KR 20180003487A KR 102052417 B1 KR102052417 B1 KR 102052417B1
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- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/655—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms
- C07F9/6552—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a six-membered ring
- C07F9/65522—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a six-membered ring condensed with carbocyclic rings or carbocyclic ring systems
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- 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
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- 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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N2021/7769—Measurement method of reaction-produced change in sensor
- G01N2021/7786—Fluorescence
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Abstract
본 발명은 하기 [화학식 1]로 표시되는 니트록실 검출용 형광 프로브 화합물에 관한 것이다:
[화학식 1]
본 발명에 따른 니트록실 검출용 형광 프로브 화합물은 타 분석물 대비 니트록실에 대해서 우수한 선택성으로 반응하며, 니트록실에 대해서 높은 검출 감도를 나타내는 바, 턴-온 방식에 의해서 니트록실을 단순하고 효과적으로 검출해낼 수 있어서, 생물학 및 환경 분야에서 매우 높은 효용성을 나타낸다. 또한, 본 발명에 따른 니트록실 형광 프로브 화합물은 세포 외부에서 도입된 니트록실 뿐만 아니라 세포 내부에서 생성된 니트록실까지 감지할 수 있어 미토콘드리아 내의 니트록실 형성과정을 실시간으로 모니터링할 수 있도록 한다.The present invention relates to a fluorescent probe compound for detecting nitroxyl represented by the following [Formula 1]:
[Formula 1]
The fluorescent probe compound for nitroxyl detection according to the present invention reacts with excellent selectivity to nitroxil compared to other analytes, and exhibits high detection sensitivity for nitroxil, thus detecting nitroxyl simply and effectively by a turn-on method. It can be pulled out, showing very high utility in biology and environment. In addition, the nitroxyl fluorescent probe compound according to the present invention can sense not only nitroxyl introduced from outside the cell but also nitroxyl generated inside the cell, thereby real-time monitoring of nitroxyl formation in the mitochondria.
Description
본 발명은 미토콘드리아에서 니트록실을 검출하기 위한 형광 프로브 화합물 및 이를 포함하는 니트록실 검출 형광 센서에 관한 것이다.The present invention relates to a fluorescent probe compound for detecting nitroxyl in mitochondria and a nitroxyl detecting fluorescent sensor comprising the same.
니트록실 (NO-/HNO)은 산화질소의 일-전자 환원된 (및 양성화된) 형태이다. 이러한 호환성이 있는 한 쌍은 신경 시스템에서 중추적인 역할을 한다(비특허문헌 1). HNO는 독특하고, 뚜렷하며 이의 산화된 유사체인 NO와 종종 반대의 약리학적 특성을 보인다(비특허문헌 2). 예를 들어, NO 공여체는 β-아드레날린작용성-유도된 수축성을 감소시켜 β-작용제의 사용을 최소화시키는 반면, HNO는 β-작용제 도부타민에 대한 첨가제로 사용된다(비특허문헌 3). HNO는 예컨대 중합효소, 구리 티올레이트 전사 인자 Ace1, 알데하이드 탈수소효소 및 징크 핑거 단백질 폴리(ADPribose) 등 티올을 포함하는 다양한 효소들의 활성을 억제한다(비특허문헌 4-6). 또한, 티올과의 결합을 통해 N-메틸-D-아스파라트산염 채널의 활성을 변화시킨다(비특허문헌 7). 또한, HNO는 생물학적 환경에서 다양한 중요 효소의 티올 잔기와 반응할 수 있다. 예를 들면, HNO는 2 x 106 M- 1 s-1의 속도로 글루타티온 (GSH)과 반응하고, 세포 산화환원계에서 조절인자로서 GSH의 기능을 대폭 감소시킨다(비특허문헌 8-9). 질소계 산화물 및 초과산화물로부터의 전환 공정이 HNO 형성 경로의 예로서 보고되어 왔지만(도 6), 니트록실의 내생적 형성(endogenous formation)에 대해서는 주변 생체분자와의 높은 반응성으로 인해 여전히 불명확하게 남아있다.Nitroxyl (NO - / HNO) is one of a nitric oxide-an electron (a and protonated) reduced form. This compatible pair plays a pivotal role in the nervous system (Non-Patent Document 1). HNO often exhibits pharmacological properties that are distinct from NO, a distinctive, distinct, and oxidized analog thereof (Non-Patent Document 2). For example, NO donors reduce β-adrenergic-induced contractility, thereby minimizing the use of β-agents, while HNO is used as an additive to β-agent dobutamine (Non-Patent Document 3). HNO inhibits the activity of various enzymes including thiols such as polymerase, copper thiolate transcription factor Ace1, aldehyde dehydrogenase and zinc finger protein poly (ADPribose) (Non-Patent Documents 4-6). In addition, the activity of the N-methyl-D-aspartate channel is changed by binding to thiol (Non-Patent Document 7). In addition, HNO can react with thiol residues of various important enzymes in a biological environment. For example, HNO is at a speed of 2 x 10 6 M - 1 s -1 Reacts with glutathione (GSH) and greatly reduces the function of GSH as a regulator in cellular redox systems (Non Patent Literature 8-9). Although conversion processes from nitrogen-based oxides and superoxides have been reported as examples of HNO formation pathways (FIG. 6), the endogenous formation of nitroxyls remains unclear due to high reactivity with surrounding biomolecules. have.
또한, HNO는 NO보다 세포독성이 있고 생체 내에서 잠재적으로 생성될 수 있으며, NO 및 퍼옥시나이트릴의 일부 형질과 생물학적 반응을 일으키기 때문에 HNO를 감지하는 것은 매우 중요하다. 종래기술로서, 형광 탐지체를 이용해 HNO를 감지하기 위해 Cu2 +로부터 Cu+ 또는 나이트록실사이드로부터 하이드록실아민의 환원을 이용하는 방법이 보고된바 있다(비특허문헌 10-11). 그러나, 이러한 탐지체들은 생체 시스템에서 몇몇의 매우 풍부한 생물학적 환원제, 예컨대 GSH 및 아스코르브산염에 의한 간섭을 받으며, 또한, 니트록실의 탐지 한계가 평가되지 않았다는 문제가 있다.In addition, it is very important to detect HNO because HNO is more cytotoxic than NO and can potentially be produced in vivo and causes biological reactions with some traits of NO and peroxynitrile. As prior art, there is a method using a fluorescence detection element from Cu + or nitroxyl side from Cu 2 + HNO used to detect the reduction of the hydroxylamine reported (Non-Patent Document 10-11). However, these detectors suffer from interference by some very abundant biological reducing agents such as GSH and ascorbate in the biological system, and also have a problem that the detection limit of nitroxyl has not been evaluated.
이에, 본 발명에서는 니트록실이 존재하는 조건하에서 턴-온(turn-on) 프로브 형광 반응을 일으킴으로써, 세포 내 미트콘드리아에서 형성되는 니트록실을 선택적으로 검출하는 것이 가능한 니트록실 검출용 형광 프로브 화합물 및 이를 포함하는 니트록실 검출용 형광 센서를 제공하고자 한다.Accordingly, in the present invention, a fluorescence probe for nitroxyl detection capable of selectively detecting nitroxyls formed in mitochondria in a cell by causing a turn-on probe fluorescence reaction under the presence of nitroxyls. It is to provide a compound and a fluorescent sensor for detecting nitroxyl containing the same.
본 발명은 상기 과제를 해결하기 위하여,The present invention to solve the above problems,
하기 [화학식 1]로 표시되는 니트록실 검출용 형광 프로브 화합물을 제공한다.It provides a fluorescent probe compound for detecting nitroxyl represented by the following [Formula 1].
[화학식 1][Formula 1]
본 발명에 따르면, 상기 화합물은 니트록실과 반응하면 형광발광을 일으킬 수 있다.According to the present invention, the compound may cause fluorescence when reacted with nitroxyl.
본 발명에 따르면, 상기 니트록실은 미토콘드리아에 형성되는 것일 수 있다.According to the invention, the nitroxyl may be formed in the mitochondria.
또한, 본 발명은 상기 형광 프로브 화합물을 포함하는 니트록실 검출용 형광 센서를 제공한다.In addition, the present invention provides a fluorescence sensor for nitroxyl detection comprising the fluorescent probe compound.
본 발명에 따른 니트록실 검출용 형광 프로브 화합물은 타 분석물 대비 니트록실에 대해서 우수한 선택성으로 반응하며, 니트록실에 대해서 높은 검출 감도를 나타내는 바, 턴-온 방식에 의해서 니트록실을 단순하고 효과적으로 검출해낼 수 있어서, 생물학 및 환경 분야에서 매우 높은 효용성을 나타낸다.The fluorescent probe compound for nitroxyl detection according to the present invention reacts with excellent selectivity to nitroxil compared to other analytes, and exhibits high detection sensitivity for nitroxil, thus detecting nitroxyl simply and effectively by a turn-on method. It can be pulled out, showing very high utility in biology and environment.
또한, 본 발명에 따른 니트록실 형광 프로브 화합물은 세포 외부에서 도입된 니트록실 뿐만 아니라 세포 내부에서 생성된 니트록실까지 감지할 수 있어 미토콘드리아 내의 니트록실 형성과정을 실시간으로 모니터링할 수 있도록 한다.In addition, the nitroxyl fluorescent probe compound according to the present invention can sense not only nitroxyl introduced from outside the cell but also nitroxyl generated inside the cell, thereby real-time monitoring of nitroxyl formation in the mitochondria.
도 1 본 발명에 따른 니트록실 형광 프로브 화합물(Mito-1)의 형광 반응을 나타낸 것으로, (A)는 Angeli’s salt(AS)의 부존재 또는 존재하 pH 7.4 PBS 완충액 (0.5% DMSO) 중 Mito-1 (5.0 μM)의 형광 스펙트럼을 나타낸다. AS와 함께 30분 동안 Mito-1을 인큐베이션 한 후에 스펙트럼을 기록하였다. (B)는 다양한 농도의 AS의 존재하에서 Mito-1의 (5.0 μM)의 반응-시간 프로파일을 나타낸 것이다. pH 7.4 PBS 완충액 (0.5% DMSO) 중에서 452 nm에서의 형광 강도를 시간 간격을 두고 연속적으로 모니터링하였다. (C)는 pH 7.4 PBS 완충액 중에서 다양한 관련 종 (30 μM)에 대한 Mito-1 (5.0 μM)의 형광 반응을 나타낸다(Emission at 452 nm). (D)는 AS (30 μM)의 부존재 및 존재하에서 다양한 pH 값에 따른 Mito-1 (5.0 μM)의 형광 강도 의존성을 나타낸다.
도 2는 외생적(exogeneous) HNO의 형광 이미지로, Mito-1 형광의 투여량 의존성을 공초점 현미경 이미지(배율, ×20)로 나타낸 것이다(λex 405 nm; λem 470-500 nm).
도 3은 외생적 HNO 감지 조건 하에서의 형광 및 공존 이미지를 나타낸 것이다. (A)는 세포기관 특이적 트래커 (미토콘드리아, 소포체 및 리소좀)를 지닌 Mito-1 (5.0 μM)의 공초점 현미경 공존 이미지이다. (B)에서는 Z-영역 이미지를 사용하여 미토콘드리아에서 Mito-1 탐지체의 위치를 확인한 것으로, (a)는 세포의 Z-영역 이미지, (b)는 미토콘드리아 트래커 및 탐지체와의 공존, (c)는 공존 산점도(배율, ×63)를 나타낸다(λex 405 nm; λem 470-500 nm).
도 4는 탐지체 및 미토콘드리아 세포기관 트래커와 내생적 HNO의 공초점 현미경 공존 이미지를 나타낸 것으로, 공초점 레이저 주사 현미경의 Z-영역 이미지를 사용함으로써 미토콘드리아에 대한 Mito-1 (5.0 mM)의 공존을 확인하였다. (A)는 세포의 3D 이미지를 나타내고, (B)는 미토콘드리아 트래커 및 탐지체와의 공존을 나타내며, (C)는 공존 산점도를 나타낸다(λex 405 nm; λem 470-500 nm).
도 5는 (A) HNO의 부재 하 및 (B) 외생적 HNO의 존재하에서 Mito-1 (10 μM)의 이광자 형광 현미경 이미지이다(λex 740 nm; λem 420-520 nm).
도 6은 세포 시스템에서의 니트록실 형성의 생화학적 경로를 도식화한 것이다(J. M. Fukuto, C. J. Cisneros and R. L. Kinkade, J. Inorg . Biochem ., 2013, 118, 201).
도 7은 pH 7.4 PBS 완충액 중 AS (30 μM)의 부존재 및 존재 하에서의 대조예 화합물 1 (10 μM) 및 Mito-1 (10 μM)의 UV-Vis 스펙트럼을 나타낸 것이다. 25 ℃에서 30분 동안 인큐베이션 한 후 데이터를 수득하였다.
도 8은 DMSO-PBS 완충액 (0.01 M, pH 7.4) (V/V= 2:98) 중 0에서 0.15 μM으로 변화하는 HNO의 농도에 대한 450 nm에서 Mito-1 (2 μM)의 형광 강도의 변화를 나타낸 것이다(슬릿폭 5/5 nm).
도 9는 종래 보고된 니트록실 탐지 한계를 나타낸 것이다.
도 10은 회귀 방정식을 적용하여 대조예 화합물 1과 니트록실 (HNO) 간 반응 속도 상수의 추정치를 나타낸 것이다.
도 11은 pH 7.4 PBS 완충액 중에서 AS (30 μM/6eq) 및 고농도의 GSH (각각 1 mM/200eq, 2 mM/400eq) 존재하에서 Mito-1 탐지체 (5.0 μM)의 형광 반응을 나타낸 것이다(Emission at 451 nm).
도 12는 Mito-1의 GSH 의존성 형광 변화를 나타낸 것으로, 탐지체를 AS 및 GSH와 함께 20분 동안 인큐베이션 한 후 AS (30 μM) 및 다양한 농도의 GSH (0 μM, 10 μM, 20 μM)를 갖는 pH 7.4 PBS 완충액 (0.5% DMSO)에서 탐지체 (5.0 μM)의 형광 스펙트럼을 기록하였다.
도 13은 HNO (AS)로 처리된 Mito-1의 LC-MS 데이터를 나타낸 것이다.
도 14는 AS로 처리된 탐지체 Mito-1의 고해상도 질량 스펙트럼을 나타낸 것이다. HRMS (ESI): C30H24NO4P에 대한 계산치=494.15, 실측치 m/z=494.050
도 15는 탐지체 및 다양한 세포기관 트래커를 지닌 내생적 HNO의 공초점 현미경 이미지, 세포기관 특이적 트래커 (미토콘드리아, 소포체 및 리소좀)를 지닌 탐지체의 공초점 현미경 공존 이미지를 나타낸다.
도 16은 세포 생존율 시험 결과를 나타낸 것이다. 96 웰 마이크로플레이트 (SPL Life Science, Gyeonggi-do, Korea) 상에 1 × 104 세포들을 뿌렸고 24시간 동안 배양하였다. 배양 후에, 다양한 조건으로 DMSO 및 탐지체로 5분 동안 세포들을 처리하였다. 세포 상의 탐지체의 세포독성을 분석하기 위해, SensoLyte® Cell Cytotoxicity Assay Kit (AnaSpec, Fremont, California)을 사용하여 세포독성 분석을 수행하였다. SPECTRA MAX GEMINI EM 마이크로플레이트 리더기 (Molecular Devices, Sunnyvale, California)를 사용하여 형광 수준을 분석하였다. 여기 및 방출 파장은 각각 544 nm 및 590 nm으로 설정하였다.Figure 1 shows the fluorescence reaction of the nitroxyl fluorescent probe compound (Mito-1) according to the present invention, (A) Mito-1 in pH 7.4 PBS buffer (0.5% DMSO) in the absence or presence of Angeli's salt (AS) A fluorescence spectrum of (5.0 μM) is shown. Spectra were recorded after incubation of Mito-1 for 30 minutes with AS. (B) shows the response-time profile of (5.0 μM) of Mito-1 in the presence of various concentrations of AS. The fluorescence intensity at 452 nm in pH 7.4 PBS buffer (0.5% DMSO) was continuously monitored over time. (C) shows the fluorescence response of Mito-1 (5.0 μM) against various related species (30 μM) in pH 7.4 PBS buffer (Emission at 452 nm). (D) shows the fluorescence intensity dependence of Mito-1 (5.0 μM) with various pH values in the absence and presence of AS (30 μM).
Figure 2 is a fluorescence image of exogeneous HNO, showing the dose dependence of Mito-1 fluorescence as confocal microscopy image (magnification, x20) (λ ex 405 nm; λ em 470-500 nm).
3 shows fluorescence and coexistence images under exogenous HNO sensing conditions. (A) is a confocal microscopic coexistence image of Mito-1 (5.0 μM) with organelle specific trackers (mitochondria, endoplasmic reticulum and lysosomes). In (B), the location of the Mito-1 detector in the mitochondria was determined using a Z-region image, (a) Z-region image of the cell, (b) coexisting with the mitochondrial tracker and the detector, (c ) Represents the co-scatter scattering plot (magnification, x 63) (λ ex 405 nm; λ em 470-500 nm).
FIG. 4 shows confocal microscopy images of endogenous HNO and detector and mitochondrial organelle trackers, illustrating the co-existence of Mito-1 (5.0 mM) against mitochondria by using Z-region images of confocal laser scanning microscope. Confirmed. (A) shows the 3D image of the cell, (B) shows the coexistence with the mitochondrial tracker and the detector, and (C) shows the coexistence scatter plot (λ ex 405 nm; λ em 470-500 nm).
5 is a two-photon fluorescence microscopy image of Mito-1 (10 μM) in the absence of (A) HNO and (B) exogenous HNO (λ ex 740 nm; λ em 420-520 nm).
Figure 6 illustrates the biochemical pathways of nitroxyl formation in cellular systems (JM Fukuto, CJ Cisneros and RL Kinkade, J. Inorg . Biochem . , 2013, 118 , 201).
FIG. 7 shows
8 shows the fluorescence intensity of Mito-1 (2 μM) at 450 nm for the concentration of HNO varying from 0 to 0.15 μM in DMSO-PBS buffer (0.01 M, pH 7.4) (V / V = 2: 98). The change is shown (slit
9 shows the previously reported nitroxyl detection limits.
FIG. 10 shows an estimate of the reaction rate constant between
FIG. 11 shows the fluorescence response of Mito-1 detector (5.0 μM) in the presence of AS (30 μM / 6eq) and high concentrations of GSH (1 mM / 200eq, 2 mM / 400eq, respectively) in pH 7.4 PBS buffer (Emission at 451 nm).
Figure 12 shows the GSH dependent fluorescence change of Mito-1, after incubating the detector with AS and GSH for 20 minutes, AS (30 μM) and various concentrations of GSH (0 μM, 10 μM, 20 μM) The fluorescence spectra of the detectors (5.0 μΜ) in pH 7.4 PBS buffer (0.5% DMSO) having were recorded.
Figure 13 shows LC-MS data of Mito-1 treated with HNO (AS).
14 shows a high resolution mass spectrum of a detector Mito-1 treated with AS. HRMS (ESI): calcd for C 30 H 24 NO 4 P = 494.15, found m / z = 494.050
15 shows confocal microscopy images of endogenous HNO with detector and various organelle trackers, confocal microscopy images of detector with organelle specific trackers (mitochondria, endoplasmic reticulum and lysosomes).
16 shows the cell viability test results. 1 × 10 4 cells were sprinkled on 96 well microplates (SPL Life Science, Gyeonggi-do, Korea) and incubated for 24 hours. After incubation, cells were treated with DMSO and the detector for 5 minutes under various conditions. To analyze the cytotoxicity of the detector on the cells, cytotoxicity assays were performed using the SensoLyte® Cell Cytotoxicity Assay Kit (AnaSpec, Fremont, California). Fluorescence levels were analyzed using a SPECTRA MAX GEMINI EM microplate reader (Molecular Devices, Sunnyvale, California). The excitation and emission wavelengths were set to 544 nm and 590 nm, respectively.
이하, 실시예를 통해서 본 발명을 더욱 상세하게 설명하기로 하되, 하기 실시예는 본 발명의 이해를 돕기 위한 것일 뿐, 본 발명의 범위를 제한하는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples, but the following Examples are only intended to help the understanding of the present invention and do not limit the scope of the present invention.
실험방법Experiment method
재료, 방법 및 기기장치Materials, methods and equipment
2,4-디하이드록시벤즈알데하이드 (TCI), 디에틸말로네이트 (Avra, India), 2-(디페닐포스피노) 벤조산 (Alfa-aesar), HATU (Alfa-aesar), DIPEA (Alfa- aesar), THF (Merck), 메탄올 (Merck), DMF (Aldrich), 디에틸 에테르 (Loba chem., India), 수산화나트륨 (Loba chem., India) 및 DCM (Loba chem. India)을 상업적으로 구매하였고 추가의 정제 없이 사용하였다. UV/Vis 및 형광 분광법을 위한 모든 재료, Angeli's salt (Cayman Chemical), L-시스테인(Acros), 초과산화 칼륨 (Aldrich), 황화 나트륨 (Aldrich), 차아염소산 나트륨 (Alfa Aesar), L글루타티온 (Aldrich), 과산화수소 (Samchun), 호모시스테인 (Aldrich), 아질산 나트륨 (Aldrich), 질산 나트륨 (Aldrich), DEA NONOate (Cayman Chemical), 2,2'-아조비스(2-메틸프로피온아미딘) 디하이드로클로라이드 (Aldrich), DMSO (J.T.Baker), 수산화나트륨 (Samchun)을 상업적 공급자로부터 구매하였고 추가의 정제 없이 사용하였다. 실리카 겔 (100-200 메쉬)을 사용하여 플래쉬 컬럼 크로마토그래피를 수행하였고 실리카 겔 60 (0.25 mm 두께로 사전 코팅된 시트)을 사용하여 분석 박층 크로마토그래피를 수행하였다. 음이온 SpecHiResESI 질량 분광계 상에 질량 스펙트럼을 기록하였다. 400MHz 분광계 (Bruker, Germany) 상에 NMR 스펙트럼을 수집하였다.2,4-dihydroxybenzaldehyde (TCI), diethylmalonate (Avra, India), 2- (diphenylphosphino) benzoic acid (Alfa-aesar), HATU (Alfa-aesar), DIPEA (Alfa- aesar) ), THF (Merck), methanol (Merck), DMF (Aldrich), diethyl ether (Loba chem., India), sodium hydroxide (Loba chem., India) and DCM (Loba chem. India) were purchased commercially Used without further purification. All materials for UV / Vis and fluorescence spectroscopy, Angeli's salt (Cayman Chemical), L-cysteine (Acros), potassium superoxide (Aldrich), sodium sulfide (Aldrich), sodium hypochlorite (Alfa Aesar), L glutathione (Aldrich) ), Hydrogen peroxide (Samchun), homocysteine (Aldrich), sodium nitrite (Aldrich), sodium nitrate (Aldrich), DEA NONOate (Cayman Chemical), 2,2'-azobis (2-methylpropionamidine) dihydrochloride ( Aldrich), DMSO (JTBaker), sodium hydroxide (Samchun) were purchased from commercial suppliers and used without further purification. Flash column chromatography was performed using silica gel (100-200 mesh) and analytical thin layer chromatography using silica gel 60 (precoated sheet to 0.25 mm thickness). Mass spectra were recorded on an anion SpecHiResESI mass spectrometer. NMR spectra were collected on a 400 MHz spectrometer (Bruker, Germany).
[화학식 Formula 1]로1] 표시되는 화합물( The compound represented ( MitoMito -- 1)의1) of 합성 synthesis
하기 [합성 경로]에 표시된 방법에 따라, 본 발명의 [화학식 1]로 표시되는 화합물(Mito-1)을 합성하였다.According to the method shown in the following [synthetic route], the compound (Mito-1) represented by [Formula 1] of the present invention was synthesized.
[합성 경로][Synthetic path]
(1) A의 합성(1) Synthesis of A
종래 보고된 방법에 따라 화합물 A를 합성하였다(J. Liu, F. Wu, L. Chen, L. Zhao, Z. Zhao, M. Wang and S. Lei, Food Chem., 2012, 135, 2872.)Compound A was synthesized according to the previously reported method (J. Liu, F. Wu, L. Chen, L. Zhao, Z. Zhao, M. Wang and S. Lei, Food Chem., 2012, 135 , 2872. )
(2) B의 합성(2) synthesis of B
종래 보고된 방법에 따라 화합물 A를 합성하였다(B. E. Maryanoff, A. B. Reitz and B. A. Duhl-Emswiler, J. Am. Chem. Soc., 1985, 107, 217.)Compound A was synthesized according to the previously reported method (BE Maryanoff, AB Reitz and BA Duhl-Emswiler, J. Am. Chem. Soc. , 1985, 107 , 217.)
(3) 화합물 1의 합성(3) Synthesis of
0℃ DIPEA (5.22 mL, 29.13 mmol)에서 DMF (10 mL) 중 화합물 A (1 g, 4.855 mmol)의 용액에, 화합물 B (2.73 g, 5.825 mmol) 및 HATU (2.77 g, 7.282 mmol)을 첨가하였다. RT에서 12시간 동안 교반하여 반응을 계속하였다. 반응을 TLC에 의해 모니터링하였다. 반응 완료 후에, 반응 혼합물을 물 (50 mL) 내로 붓고 에틸 아세테이트 (3 x 25 mL)로 추출하였다. 유기층을 물 (2 x 50 mL), 소금물 (1 x 50 mL)로 세척하였고, 무수 황산 나트륨을 통해 건조 및 감압하에서 농축시켰다. 용리제로서 DCM (1:9) 중의 메탄올을 사용하여 실리카 겔 (100-200 메쉬) 상에서 컬럼 크로마토그래피에 의해 미정제 생성물을 정제하여 옅은 노랑의 고형물 (1.8 g; 64.47%)인 화합물 1을 수득하였다. 1H-NMR (400 MHz, DMSO-d6): δ 11.10 (s, 1H); 8.96 (s, 1H); 7.89-7.74 (m, 11H); 7.73 (d, J = 2.80 Hz, 2H); 6.90 (q, 1H); 6.81 (d, J = 1.60 Hz, 1H); 3.87 (m, 2H); 3.71 (d, J = 2.4 Hz, 2H); 13C-NMR (100 MHz, DMSO-d6): 164.35, 163.40, 160.09, 157.10, 149.09, 135.01, 134.10, 133.99, 130.25, 119.51, 115.20, 113.09, 111.25, 101.99; ESI- HRMS m/z (M+H): 계산치. 494. 15157, 실측치 494.15048.To a solution of compound A (1 g, 4.855 mmol) in DMF (10 mL) in 0 ° C. DIPEA (5.22 mL, 29.13 mmol), add compound B (2.73 g, 5.825 mmol) and HATU (2.77 g, 7.282 mmol) It was. The reaction was continued by stirring at RT for 12 h. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was poured into water (50 mL) and extracted with ethyl acetate (3 x 25 mL). The organic layer was washed with water (2 x 50 mL), brine (1 x 50 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure. Purification of the crude product by column chromatography on silica gel (100-200 mesh) using methanol in DCM (1: 9) as eluent gave
(4) (4) MitoMito -1의 합성Synthesis of -1
0℃ DIPEA (0.4 mL, 2.234 mmol)에서 DMF (10 mL) 중 2-(디페닐포스피노) 벤조산 (375 mg, 1.23 mmol)의 용액에, 화합물 1 (640 mg, 1.117 mmol) 및 HATU (640 mg, 1.675 mmol)을 첨가하였다. RT에서 12시간 동안 교반하여 반응을 계속하였고 반응을 TLC에 의해 모니터링하였다. 반응 완료 후에, 반응 혼합물을 물로 희석하였고 15분 동안 교반시켰다. 침전된 고형물을 여과하였고, 물로 세척하고, 진공 속에서 건조시켰다. 용리액으로서 DCM (0.5:9.5) 중 메탄올을 사용하여 실리카 겔 (100-200 메쉬) 상에서 컬럼 크로마토그래피에 의해 미정제 생성물을 정제하여 옅은 노랑 고형물 (320 mg; 33.20%)로서 본 발명에 따른 탐지체인 Mito-1을 수득하였다. 1H-NMR (400 MHz, CDCl3): δ 9.05 (d, J= 8.0 Hz, 1H); 8.80 (s, 1H); 8.29 (d, J= 2.0 Hz, 1H); 8.02 (t, J= 2.04 Hz, 1H); 7.99-7.62 (m, 17H); 7.40-7.35 (m, 7H); 7.32-7.06 (m, 5H); 6.92 (d, J= 3.0 Hz, 1H); 3.87 (t, 2H); 3.72 (t, J= 3.2 Hz, 2H); 13C-NMR (100 MHz, DMSO-d6): 165.02, 162.15, 159.05, 154.05, 148.05, 141.02, 137.20, 135.10, 134.00, 132.50, 130.99, 129.60, 119.99, 118.50, 118.25, 117.59, 116.20, 110.79; ESI-MS m/z (M+H): 계산치. 782.22, 실측치 783.00; HRMS (M + 산소 부가생성물):798.21234; HPLC~96.04%.To a solution of 2- (diphenylphosphino) benzoic acid (375 mg, 1.23 mmol) in DMF (10 mL) at 0 ° C. DIPEA (0.4 mL, 2.234 mmol), Compound 1 (640 mg, 1.117 mmol) and HATU (640 mg, 1.675 mmol) were added. The reaction was continued by stirring at RT for 12 h and the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with water and stirred for 15 minutes. The precipitated solid was filtered off, washed with water and dried in vacuo. Purification of the crude product by column chromatography on silica gel (100-200 mesh) using methanol in DCM (0.5: 9.5) as eluent gave the detector according to the invention as a pale yellow solid (320 mg; 33.20%). Mito-1 was obtained.OneH-NMR (400 MHz, CDCl3): δ 9.05 (d,J= 8.0 Hz, 1H); 8.80 (s, 1 H); 8.29 (d,J= 2.0 Hz, 1H); 8.02 (t,J= 2.04 Hz, 1H); 7.99-7.62 (m, 17 H); 7.40-7.35 (m, 7 H); 7.32-7.06 (m, 5 H); 6.92 (d, J = 3.0 Hz, 1H); 3.87 (t, 2 H); 3.72 (t, J = 3.2 Hz, 2H);13C-NMR (100 MHz, DMSO-d6): 165.02, 162.15, 159.05, 154.05, 148.05, 141.02, 137.20, 135.10, 134.00, 132.50, 130.99, 129.60, 119.99, 118.50, 118.25, 117.59, 116.20, 110.79; ESI-MSm / z(M + H): calculated. 782.22, found 783.00; HRMS (M + oxygen adduct): 798.21234; HPLC-96.04%.
UV/UV / VisVis 및 형광분석법(Fluorescence Spectroscopy) And Fluorescence Spectroscopy
DMSO 중에 Mito-1을 용해시켜 Moito-1의 1ⅹ10-3 M 저장 용액을 제조하였다. 흡수 스펙트럼을 S-3100 (Scinco) 분광광도계 상에 기록하였고, 제논 램프가 갖춰진 RF-5301 PC 분광형광계 (Shimadzu)를 사용하여 형광 스펙트럼을 기록하였다. 형광 방출 스펙트럼을 398 nm의 여기 파장에서 기록하였고, 400-600 nm 범위의 파장 (em = 452 nm)을 통해 방출을 모니터링하였다. 증류수 중 Hcy, GSH, Cys, KO2, NaNO2, NaNO3, NaClO, H2O2 (1ⅹ10-3 M)의 저장 용액; NaOH (1ⅹ10-2 M) 중 DEA NONOate, Na2S, Angeli's salt (1ⅹ10-3 M); DMSO 중 KO2 (1ⅹ10-3 M)을 또한 제조하였다. 샘플들의 흡수 및 방출 측정 (4 mL 부피)을 위해 쿼츠 큐벳 (Quartz cuvettes)을 사용하였다. 생리적 조건의 pH (PBS 완충액, pH 7.4) 하에서 모든 분광기적 측정을 수행하였다.Dissolved Mito-1 in DMSO to prepare a 1ⅹ10 -3 M stock solution of Moito-1. Absorption spectra were recorded on an S-3100 (Scinco) spectrophotometer and fluorescence spectra were recorded using an RF-5301 PC spectrofluorometer (Shimadzu) equipped with a xenon lamp. Fluorescence emission spectra were recorded at excitation wavelengths of 398 nm and emission was monitored through wavelengths in the range 400-600 nm (em = 452 nm). Stock solution of Hcy, GSH, Cys, KO 2 , NaNO 2 , NaNO 3 , NaClO, H 2 O 2 (1 × 10 −3 M) in distilled water; DEA NONOate, Na 2 S, Angeli's salt (1 × 10 −3 M) in NaOH (1 × 10 −2 M); KO 2 (1 × 10 −3 M) in DMSO was also prepared. Quartz cuvettes were used for absorption and release measurements (4 mL volumes) of the samples. All spectroscopic measurements were performed under physiological pH (PBS buffer, pH 7.4).
세포 배양Cell culture
인간 자궁경부암 세포 (HeLa)의 세포 배양 및 데이터 분석은 Korean Cell Line Bank (Seoul, Republic of Korea)로부터 구매하였다. 37 ℃ 5% CO2 하에서, 10% 소태아 혈청 (PAN Biotech GmbH, Aidenbach, Germany) 및 1% 페니실린/스트렙토마이신 (Hyclone)이 보충된 고 글루코오스 (GE Hyclone, Logan, UT, USA)를 포함하는 둘베코 수정 이글 배지에서 세포들을 배양하였다.Cell culture and data analysis of human cervical cancer cells (HeLa) were purchased from Korean Cell Line Bank (Seoul, Republic of Korea). With high glucose (GE Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (PAN Biotech GmbH, Aidenbach, Germany) and 1% penicillin / streptomycin (Hyclone) at 37 ° C. 5% CO 2 Cells were cultured in Dulbecco's Modified Eagle's medium.
이광자 형광 현미경Two-photon fluorescence microscope
Delta T Dishes (Bioptechs) 상에 1ⅹ105 세포들을 뿌렸다. 70~85% 융합성을 갖는 세포들을 PBS로 세척하였고 37 ℃, 5% CO2 하에서 5분 동안 탐지체 Mito-1 (PBS 중 10 μM)와 함께 배양하였다. 그 다음에, PBS 중에서 30분 동안 50 μM AS로 세포들을 처리하였고 PBS로 세척하였다. a ×100 (NA = 1.30 OIL) 대물 렌즈를 지닌 스펙트럼 공초점 및 다광자 현미경 (Leica TCS SP2)로 탐지체 라벨링된 HeLa 세포들의 이광자 형광 현미경 이미지를 수득하였다. 740 nm 파장에서 설정된 모드-잠금된 티타늄-사파이어 레이저원 (Coherent Chameleon, 90 MHz, 200 fs)으로 탐지체를 여기시켜 DM IRE2 Microscope (Leica)으로 이광자 형광 현미경 이미지를 수득하였다. 420-520 nm 범위에서 이미지를 수득하기 위해, 내부 PMT를 사용하여 8 비트 무부호 512 × 512 픽셀 at 400 Hz 스캔 속도로 신호를 수집하였다.1 × 10 5 cells were sprinkled onto Delta T Dishes (Bioptechs). Cells with 70-85% confluency were washed with PBS and the detector Mito-1 for 5 minutes at 37 ° C., 5% CO 2 . (10 μM in PBS). The cells were then treated with 50 μM AS for 30 minutes in PBS and washed with PBS. Two-photon fluorescence microscopy images of detector-labeled HeLa cells were obtained by spectral confocal and multiphoton microscopy (Leica TCS SP2) with a × 100 (NA = 1.30 OIL) objective lens. Two-photon fluorescence microscopy images were obtained with a DM IRE2 Microscope (Leica) by exciting the detector with a mode-locked titanium-sapphire laser source (Coherent Chameleon, 90 MHz, 200 fs) set at a wavelength of 740 nm. To obtain images in the 420-520 nm range, signals were collected at 8-bit unsigned 512 x 512 pixels at 400 Hz scan rate using an internal PMT.
일광자Daylight 형광 현미경 Fluorescence microscope
동일한 조건으로 공초점 레이저 주사 현미경 (Carl-Zeiss LSM 700 Exciter, Oberko, Germany)을 사용하여 형광 이미지를 수득하였다. Si 레이저로 405 nm에서 형광 채널을 여기시켰고, 470-500 nm 밴드 패스 필터에 의해 방출을 수집하였다. 탐지체 위치를 추적하기 위해, Mito-Track Red (Invitrogen, Carlsbad, CA, USA), Lyso-Tracker Red (Invitrogen), 및 ER-Tracker Red (Invitrogen)를 사용하였다. 공초점 레이저 주사 현미경 (Cal Zeiss)을 사용하여 형광 이미지를 수득하였다. Fluorescence images were obtained using confocal laser scanning microscope (Carl-
내생적 Endogenous HNOHNO 감지 조건 Detection condition
DMEM 배지를 함유하는 35 mm-공초점 디쉬 (SPL Life Sciences, Seoul, Republic of Korea) 상에 1ⅹ105 세포들을 뿌렸고, 37 ℃, 5% CO2 하에서 10시간 동안 배양하였다. 그 다음에, 200 μM DEA NONOate로 20시간 동안 세포들을 처리하였다. 세포들을 PBS로 세척하였고 DMEM 배지에서 0.03 mM NEM으로 처리하였으며, 37 ℃, 5% CO2 하에서 10분 동안 배양하였다. NEM을 사전처리한 후에, 세포들을 PBS로 세척하였고, 탐지체 Mito-1(5 μM)로 처리하였으며 5분 동안 배양하였다. LSM 700 공초점 현미경 (Carl Zeiss, Oberkochen, Germany)을 사용하여 세포 모폴로지를 수득하였다. NaAsc 처리된 군을 위해, 5분동안 탐지체 Mito-1 처리 후에, 세포들을 PBS로 세척하였고 1.5 mM NaAsc로 처리하였으며, 37 ℃, 5% CO2 하에서 20분 동안 배양하였다. 그 다음에, 세포들을 세척하였고, 형광 현미경을 사용하여 분석하였다. 1 × 10 5 cells were sprinkled on a 35 mm-confocal dish containing DMEM medium (SPL Life Sciences, Seoul, Republic of Korea) and incubated for 10 hours at 37 ° C., 5% CO 2 . The cells were then treated with 200 μM DEA NONOate for 20 hours. Cells were washed with PBS and treated with 0.03 mM NEM in DMEM medium and incubated for 10 minutes at 37 ° C., 5% CO 2 . After pretreatment with NEM, cells were washed with PBS, treated with detector Mito-1 (5 μM) and incubated for 5 minutes. Cell morphology was obtained using an
외생적 Exogenous HNOHNO 감지 조건 Detection condition
35 mm-공초점 디쉬 (SPL Life Science) 상에 1ⅹ105 세포들을 뿌렸다. 70~85% 융합성을 갖는 세포들을 PBS로 세척하였고, DMEM 배지에서 0.03 mM NEM으로 10분 동안 처리하였다. 그 다음에, 5% FBS를 함유하는 PBS 중에서 탐지체 Mito-1 (5 μM)으로 5분 동안 세포들을 처리하였다. 탐지체의 처리 후에, 세포들을 PBS로 세척하였고, 현미경을 이용한 형광 이미지를 위해 PBS 중 300 μM Angeli's Salt를 30분 동안 첨가하였다. 1 × 10 5 cells were sprinkled on a 35 mm-confocal dish (SPL Life Science). Cells with 70-85% confluence were washed with PBS and treated with 0.03 mM NEM in DMEM medium for 10 minutes. Next, the detector Mito-1 in PBS containing 5% FBS Cells were treated for 5 minutes at (5 μΜ). After treatment of the detector, cells were washed with PBS and 300 μM Angeli's Salt in PBS was added for 30 minutes for fluorescence microscopy images.
세포의 세포독성 시험Cytotoxicity Test of Cells
96 웰 마이크로플레이트 (SPL Life Science, Gyeonggi-do, Korea) 상에 대략 세포 1 × 104를 뿌렸고 24시간 동안 배양하였다. 배양 후에, 다양한 조건으로 5분 동안 DMSO 및 탐지체로 세포들을 처리하였다. 세포 상의 탐지체의 세포독성을 분석하기 위해, SensoLyte® Cell Cytotoxicity Assay Kit (AnaSpec, Fremont, California)를 사용하여 세포독성 분석을 수행하였다. SPECTRA MAX GEMINI EM 마이크로플레이트 리더기 (Molecular Devices, Sunnyvale, California)를 사용하여 형광 수준을 분석하였다. 여기 및 방출 파장은 각각 544 nm 및 590 nm으로 설정하였다. Approximately 1 × 10 4 cells were sprinkled onto 96 well microplates (SPL Life Science, Gyeonggi-do, Korea) and incubated for 24 hours. After incubation, cells were treated with DMSO and the detector for 5 minutes under various conditions. To analyze the cytotoxicity of the detector on the cells, cytotoxicity assays were performed using the SensoLyte® Cell Cytotoxicity Assay Kit (AnaSpec, Fremont, California). Fluorescence levels were analyzed using a SPECTRA MAX GEMINI EM microplate reader (Molecular Devices, Sunnyvale, California). The excitation and emission wavelengths were set to 544 nm and 590 nm, respectively.
선형 범위 및 검출 한계Linear range and detection limits
검출 한계를 계산하기 위해 형광 적정(Fluorescence titration)을 사용하였다. 탐지체 Mito-1 (2.0×10-6 M)의 형광 방출 스펙트럼을 10회 측정하였고 공시료의 표준 편차를 수득하였다. 452 nm에서의 형광 강도를 HNO의 농도로서 플롯팅하였다. 하기 식을 이용하여 검출 한계를 계산하였다.Fluorescence titration was used to calculate the detection limit. The fluorescence emission spectrum of the detector Mito-1 (2.0 × 10 −6 M) was measured ten times and a standard deviation of the blanks was obtained. Fluorescence intensity at 452 nm was plotted as the concentration of HNO. The detection limit was calculated using the following formula.
검출 한계 3σ/k:Detection limit 3σ / k:
여기서, σ는 공시료(blank measurement)의 표준 편차이고, k는 형광 강도 vs. HNO 농도 간의 기울기이다.Where σ is the standard deviation of the blank measurement and k is the fluorescence intensity vs. The slope between HNO concentrations.
결과 및 고찰Results and Discussion
본 발명의 [화학식 1]로 표시되는 니트록실 검출용 형광 프로브 화합물(Mito-1)은 상기 합성 경로에 표시된 바와 같이 아미드 형성 후 에스터화를 통한 2개의 연속 단계로 합성된다. 본 발명에서는 광학 관섭을 극복하기 위해 높은 양자 수율 및 이광자 특성을 가진 쿠마린을 형광단으로 사용하였다. The fluorescence probe compound (Mito-1) for detecting nitroxyl represented by [Formula 1] of the present invention is synthesized in two successive steps through esterification after amide formation as indicated in the synthesis route. In the present invention, coumarin having high quantum yield and two-photon characteristics was used as a fluorophore to overcome optical interference.
다음으로, 본 발명에 따른 Mito-1의 HNO에 대한 민감도를 측정하기 위해, 생리적 조건하에서 HNO 생성제로 잘 알려진 Angeli’s salt (AS)의 존재하에서 Mito-1의 UV-흡수 및 형광 변화를 기록하였다. 도 7에 나타나는 바와 같이, Mito-1의 UV-흡수는 AS (3 eq.)의 존재 하 398 nm에서 ~30배 증가하였다. Mito-1의 λem 452 nm에서 형광 강도는 AS (0-100.0 mM)의 농도가 증가함에 따라 점차적으로 향상되었고, 그 다음에 포화에 도달하였다 (도 1A). 도 1A에 나타난 바와 같이 Mito-1의 형광 강도가 HNO (AS) (100.0 mM)의 존재하에서 ~45배 증가함을 확인하였다. 또한, 회귀 방정식을 적용한 결과, HNO에 대한 검출 한계가 18.0 nM (도 8)으로 계산되었으며, 이는 종래 보고된 탐지체들보다 상당히 낮은 수치이다(도 9).Next, to measure the sensitivity of Mito-1 to HNO according to the present invention, UV-absorption and fluorescence changes of Mito-1 were recorded in the presence of Angeli's salt (AS), which is well known as a HNO generator under physiological conditions. As shown in FIG. 7, the UV-absorption of Mito-1 increased ˜30 fold at 398 nm in the presence of AS (3 eq.). At λ em 452 nm of Mito-1 the fluorescence intensity gradually improved with increasing concentration of AS (0-100.0 mM), and then reached saturation (FIG. 1A). As shown in FIG. 1A, the fluorescence intensity of Mito-1 was found to increase ˜45-fold in the presence of HNO (AS) (100.0 mM). In addition, applying the regression equation, the detection limit for HNO was calculated to be 18.0 nM (FIG. 8), which is significantly lower than the previously reported detectors (FIG. 9).
다음으로, AS의 처리와 함께 HNO의 존재하에서 Mito-1의 시간-의존적 형광 강도 변화를 기록하였다. 도 1B의 결과를 통해 HNO (30.0 mM)의 존재 하에서 Mito-1의 형광 강도가 20분 내로 최대치에 도달함을 확인하였다. Mito-1과 니트록실 간 반응의 속도 상수는 1.0 x 10-3 M-1 s- 1 (도 10)로 계산되었다. 이러한 결과는 Mito-1이 단기간 내에 세포 미세환경에서 HNO의 탐지를 가능하게 함을 시사하는 것이다.Next, the time-dependent fluorescence intensity change of Mito-1 in the presence of HNO with AS treatment was recorded. The results of FIG. 1B confirmed that the fluorescence intensity of Mito-1 reached the maximum within 20 minutes in the presence of HNO (30.0 mM). The rate constant of the reaction between Mito-1 and nitroxyl was calculated as 1.0 × 10 −3 M −1 s − 1 (FIG. 10). These results suggest that Mito-1 enables the detection of HNO in the cellular microenvironment in a short time.
다음으로, 다른 생물학적으로 관련된 반응성 종, 예컨대 Cys, GSH, Hcy, Na2S, H2O2, NO, NO3 -, NO2 -, O2 -, ROO-, 및 ClO-로부터의 임의의 간섭 없이 Mito-1이 HNO와 반응할 수 있는지를 확인하였다. 이를 위해 전술한 피분석물과 함께 25 ℃에서 20분 동안의 배양에 따른 Mito-1의 형광 강도의 변화를 모니터링하였다. 도 1C에 나타난 바와 같이, Mito-1의 형광 강도는 다른 생물학적으로 관련된 피분석물의 존재 하에서 거의 변함 없이 유지되었는데 반해, Mito-1은 HNO (AS)의 존재 하에서만 형광 향상을 나타내었다.Next, the reactive species associated with other biological, e.g., Cys, GSH, Hcy, Na 2 S, H 2
HNO가 GSH와 비교하여 훨씬 덜 발현된 생물학적 시스템에서 탐지체의 가능성을 입증하기 위해, 상당히 상이한 농도하에서 기질들을 시험하였다. 도 11은 400 eq.의 GSH 및 6 eq.의 HNO가 처리된 경우의 결과를 나타낸다. GSH가 Mito-1과 특정한 범위의 반응성을 나타내지만, HNO는 그의 상당히 낮은 농도에도 불구하고 우수한 형광 강도를 보여준다. 이러한 Mito-1의 정교한 민감성은 탐지체가 다른 단수명 화학적 개체의 존재 하에서조차 세포의 미소구획에서 HNO를 감지하기 위한 우수한 도구임을 시사하는 것이다.Substrates were tested at significantly different concentrations to demonstrate the potential of the detector in biological systems in which HNO was much less expressed compared to GSH. 11 shows the results when 400 eq. Of GSH and 6 eq. Of HNO were treated. While GSH shows a certain range of reactivity with Mito-1, HNO shows good fluorescence intensity despite its fairly low concentration. This sophisticated sensitivity of Mito-1 suggests that the detector is an excellent tool for detecting HNO in microcompartments of cells, even in the presence of other short-lived chemical entities.
이러한 결과가 Angel’s salt의 다른 부산물 대신에 HNO로 인한 것이라는 것을 명확하게 하기 위해, GSH, HNO의 스캐빈저의 존재하에서 AS와 Mito-1의 반응을 수행하였다. GSH 농도가 0에서 20 mM로 증가함에 따라, 형광 강도는 예상한 대로, 감소하였고, 이를 통해 HNO가 형광 강도의 증가를 일으킨 것임을 확인하였다(도 12). To clarify that this result is due to HNO instead of other by-products of Angel's salt, the reaction of AS and Mito-1 was carried out in the presence of a scavenger of GSH and HNO. As GSH concentration increased from 0 to 20 mM, fluorescence intensity decreased as expected, thereby confirming that HNO caused an increase in fluorescence intensity (FIG. 12).
다음으로, 다양한 pH 범위에서 Mito-1의 안정성과 HNO에 대한 반응성을 분석하였다. 도 1D의 결과는 Mito-1이 생리학적 pH 범위 (pH 4 ~ 8)에서 매우 안정적이며 HNO에 대한 반응성이 형광 강도가 최대에 도달 할 때 pH 8에서 최적화됨을 나타낸다. 미토콘드리아 매트릭스가 약간 알칼리성이기 때문에 (pH ~8), 본 발명에 따른 Mito-1이 미토콘드리아에서 내생적 HNO의 탐지를 가능하게 할 것으로 예상된다.Next, the stability of Mito-1 and its reactivity to HNO were analyzed at various pH ranges. The results in FIG. 1D show that Mito-1 is very stable in the physiological pH range (pH 4-8) and the reactivity to HNO is optimized at
세포 환경에서 외생적/내생적 HNO의 감지를 위한 Mito-1의 적용을 진행하기 전에, 하기 [Mito-1과 HNO의 반응 메커니즘]에서 나타낸 바와 같이, Mito-1과 HNO의 반응 메커니즘을 평가하였다. 아세토니트릴 중에서 Mito-1로 처리된 Angeli’s salt (AS)에 대해 액체 크로마토그래피-질량 분석법 (LC-MS) 및 HR-MS 분석을 하였다. 도 13의 LC-MS 데이터는 2개의 주요한 성분들이 크로마토그램에 제공되었음을 나타낸다. 322.1 및 494.2에서의 피크는 부산물 (M + 1) 및 유리 형광단 1(화합물 1)과 각각 매칭된다. 또한, HR-MS 데이터 (도 14)에서 주요 피크가 형광단 (1)과 대응되는 494.05에서 나타났다. 이러한 결과는 분자내 스타우딩거 반응 메커니즘을 강력하게 입증하는 것이다.Before proceeding with the application of Mito-1 for the detection of exogenous / endogenous HNO in the cellular environment, the reaction mechanism of Mito-1 and HNO was evaluated, as shown in [Reaction Mechanism of Mito-1 and HNO] below. . Angeli's salt (AS) treated with Mito-1 in acetonitrile was subjected to liquid chromatography-mass spectrometry (LC-MS) and HR-MS analysis. LC-MS data in FIG. 13 indicate that two major components were provided in the chromatogram. The peaks at 322.1 and 494.2 match with by-product (M + 1) and free fluorophore 1 (compound 1), respectively. In addition, the main peak in HR-MS data (FIG. 14) was shown at 494.05, corresponding to fluorophore (1). These results strongly demonstrate the intramolecular Stoudinger reaction mechanism.
[Mito-1과 HNO의 반응 메커니즘][Reaction Mechanism of Mito-1 and HNO]
다음으로, SensoLyte® Cell Cytotoxicity Assay Kit를 사용하여 HeLa 세포에서 Mito-1의 세포독성을 평가하였으며, 측정 결과 10 mM에서 현저한 세포독성이 관찰되지 않았고, 이에 따라 Mito-1의 우수한 생체호환성을 확인하였다(도 16). Angeli’s salt-사전처리된 HeLa 세포를 가변적인 농도의 Mito-1과 함께 배양하였다. 도 7에 나타난 바와 같이, 세포라벨링의 범위는 투여량-의존적인 방식으로 증가하였다.Next, the cytotoxicity of Mito-1 was evaluated in HeLa cells using the SensoLyte® Cell Cytotoxicity Assay Kit. As a result, no significant cytotoxicity was observed at 10 mM, thereby confirming the excellent biocompatibility of Mito-1. (FIG. 16). Angeli's salt-pretreated HeLa cells were incubated with variable concentrations of Mito-1. As shown in FIG. 7, the range of cell labeling increased in a dose-dependent manner.
Mito-1로부터의 형광 신호를 모니터링함으로써 미토콘드리아에서 외생적 니트록실을 평가하였으며, 그 결과를 도 3에 나타내었다. 이를 통해 다른 세포기관, 예컨대 소포체 (ER) 및 리소좀 (Lyso)과 비교하여 미토콘드리아에서 Mito-1이 대부분 국소화됨(localized)을 확인하였다. 또한, 공초점 Z-영역 이미지 및 공존 산점도는 Mito-1이 Mito-트래커와 잘 공존함을 의미한다. 이러한 결과는 Mito-1이 살아있는 세포의 미토콘드리아에서 외생적 니트록실을 추적하는 능력이 있음을 시사하는 것이다.Exogenous nitroxyls were assessed in mitochondria by monitoring fluorescence signals from Mito-1, and the results are shown in FIG. 3. This confirmed that Mito-1 was mostly localized in the mitochondria compared to other organelles such as endoplasmic reticulum (ER) and lysosome (Lyso). In addition, the confocal Z-region image and co-scatter scatter plot mean that Mito-1 coexists well with the Mito-tracker. These results suggest that Mito-1 has the ability to track exogenous nitroxyls in the mitochondria of living cells.
다음으로, Mito-1의 형광 변화를 모니터링함으로써 미토콘드리아에서 내생적 HNO 형성을 추적하였다. NO를 HNO로 전환할 수 있는 환원제로 알려진, 아스코르브산 나트륨의 존재하에서 HeLa 세포를 DEA NONOate (NO 공여체)로 처리하였다. 3-차원 공존 이미지는 Mito-1이 다른 세포기관, 예컨대 리소좀 (Lyso) 및 소포체 (ER)을 거쳐 미토콘드리아에 대부분 국소화됨(localized)을 암시하였다 (도 9 및 도 15). 이러한 결과는 Mito-1이 미토콘드리아에서 외생적 HNO 및 내생적 HNO 형성 둘 모두를 감지할 수 있음을 시사하는 것이다.Next, endogenous HNO formation was traced in the mitochondria by monitoring the fluorescence change of Mito-1. HeLa cells were treated with DEA NONOate (NO donor) in the presence of sodium ascorbate, known as a reducing agent capable of converting NO to HNO. Three-dimensional coexistence images suggested that Mito-1 was mostly localized to the mitochondria via other organelles such as Lysosomes and endoplasmic reticulum (ER) (FIGS. 9 and 15). These results suggest that Mito-1 can detect both exogenous HNO and endogenous HNO formation in mitochondria.
마지막으로, 세포 환경에서 유비쿼터스 생물학적 개체로부터의 형광 간섭을 극복하게 해주는 Mito-1의 이광자 형광 특성을 평가하였다. 도 5에 나타낸 바와 같이 HNO (AS) 전처리 된 HeLa 세포는 420-520 nm 범위에서 형광으로 표지되었으며, Mito-1의 존재하에 lex 740 nm에서 여기되었다. 이러한 결과는 Mito-1이 고유한 생물학적 개체로부터의 임의의 형광 간섭 없이 미토콘드리아의 내생적 및 외생적 HNO 형성을 보장하는 능력을 가짐을 암시하는 것이다.Finally, we evaluated the two-photon fluorescence properties of Mito-1, which overcomes fluorescence interference from ubiquitous biological entities in the cellular environment. As shown in FIG. 5, HNO (AS) pretreated HeLa cells were fluorescently labeled in the range of 420-520 nm and excited at lex 740 nm in the presence of Mito-1. These results suggest that Mito-1 has the ability to ensure the endogenous and exogenous HNO formation of mitochondria without any fluorescence interference from native biological entities.
결론적으로, 본 발명에서는 미토콘드리아에서 니트록실 (HNO)을 감지하기 위한 무금속 화학도시미터 턴-온 형광 탐지체인 Mito-1을 제공한다. Mito-1의 λabs 398 nm에서의 UV 흡수 및 λem 452 nm에서의 형광 강도는 HNO (100 mM)의 존재하에서 ~25배 및 ~45배 향상되었다. 또한, 니트록실에 대한 Mito-1의 민감성은 18 nM 정도로 낮았으며, 생리적 pH 범위에서 매우 안정한 것으로 나타났다. 또한, Mito-1은 '턴-온' 형광 신호를 제공함으로써 미토콘드리아에 국소화된(localized) 내생적 및 외생적 니트록실의 시각화를 가능하게 한다. 이처럼, 본 발명에 따른 형광 탐지체의 살아있는 세포에서 HNO의 존재하에서 이광자 ‘턴-온’ 형광 반응은 생체 시스템에서 편재하는 개체로부터의 간섭을 극복할 수 있게 하여, 미토콘드리아에서 니트록실을 탐지하는 유망한 도구가 될 것으로 예상된다.In conclusion, the present invention provides Mito-1, a metal-free chemical dosimeter turn-on fluorescence detector for detecting nitroxyl (HNO) in the mitochondria. UV absorption at λ abs 398 nm and fluorescence intensity at λ em 452 nm of Mito-1 were enhanced ˜25 and ˜45 fold in the presence of HNO (100 mM). In addition, the sensitivity of Mito-1 to nitroxyl was as low as 18 nM and appeared to be very stable in the physiological pH range. Mito-1 also provides a 'turn-on' fluorescence signal, allowing visualization of endogenous and exogenous nitroxyls localized to mitochondria. As such, the two-photon 'turn-on' fluorescence response in the presence of HNO in the living cells of the fluorescence detector according to the present invention allows to overcome interference from ubiquitous individuals in biological systems, thus promising for detecting nitroxyl in mitochondria. It is expected to be a tool.
Claims (4)
[화학식 1]
[Formula 1]
상기 화합물은 니트록실과 반응하면 형광발광을 일으키는 것을 특징으로 하는 니트록실 검출용 형광 프로브 화합물.The method of claim 1,
The compound is a fluorescent probe compound for nitroxyl detection, characterized in that for reacting with nitroxyl to cause fluorescence.
상기 니트록실은 미토콘드리아에 형성되는 것을 특징으로 하는 니트록실 검출용 형광 프로브 화합물.The method of claim 1,
The nitroxyl is formed in the mitochondria fluorescence probe compound for nitroxil detection.
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| Bioorg. Med. Chem. 13 (2005) 1131-1139 |
| J. Am. Chem. Soc., 2013, 135, 4567. |
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