KR102308535B1 - Fluorescent probe for detecting cellular polarity and medical uses - Google Patents
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Abstract
본 발명은 세포내 극성 유무에 따라 형광이 변화하는 신규 화합물, 이를 이용한 세포내 극성 검출용 형광프로브 및 이를 이용한 의학적 용도에 관한 것으로, 보다 상세하게는 본 발명에서는 세포내 극성 유무를 감지할 수 있는 신규 형광프로브를 개발함으로써 세포내 미세환경 뿐 아니라 소기관의 극성 유무도 감지할 수 있고, 특히 세포내 극성 시 턴온 되는 형광프로브와 세포내 극성 시 턴오프 되는 형광프로브를 연결한 신규 형광프로브를 통해 비율계량적으로 세포내 극성 유무를 정량할 수 있다.The present invention relates to a novel compound whose fluorescence changes depending on the presence or absence of intracellular polarity, a fluorescent probe for detecting intracellular polarity using the same, and a medical use using the same. By developing a new fluorescent probe, not only the intracellular microenvironment but also the presence or absence of organelle polarity can be detected. In particular, the ratio through a new fluorescent probe that connects a fluorescent probe that turns on when intracellular polarity and a fluorescent probe that turns off when intracellular polarity occurs. The presence or absence of intracellular polarity can be quantitatively quantified.
Description
본 발명은 세포내 극성 유무에 따라 형광이 변화하는 신규 화합물, 이를 이용한 세포내 극성 검출용 형광프로브 및 이를 이용한 의학적 용도에 관한 것이다.The present invention relates to a novel compound whose fluorescence changes depending on the presence or absence of intracellular polarity, a fluorescent probe for detecting intracellular polarity using the same, and a medical use using the same.
세포내 세포들의 분포, 공간배열 및 조성이 이질적이기 때문에 세포 내 환경에서 극성, 점도, 온도, 산화 환원 상태 및 pH 파라미터는 생체분자의 물리화학적 거동의 개시 및 유지에 중요한 요인이다. Because the distribution, spatial arrangement and composition of intracellular cells are heterogeneous, polarity, viscosity, temperature, redox state, and pH parameters in the intracellular environment are important factors for the initiation and maintenance of physicochemical behavior of biomolecules.
특히, 세포내 극성은 세포 증식, 면역시스템 조절, 국소 막의 수의 증가, 세포 이동의 자극과 세포층을 가로질러 분자의 벡터 전송과 같은 다양한 세포성 과정에서 중요하며, 세포의 각 소기관은 그 역할에 따라 최적의 극성을 가지며 극성은 세포내 환경 변화에 따라 실시간 변화한다. In particular, intracellular polarity is important in a variety of cellular processes such as cell proliferation, regulation of the immune system, increase in the number of local membranes, stimulation of cell migration and vector transport of molecules across cell layers, and each organelle of the cell plays a role in its role. It has an optimal polarity according to the polarity, and the polarity changes in real time according to changes in the intracellular environment.
세포의 병리학적 활동은 극성에 따라 변화하고 비정상적인 극성은 당뇨, 신경계 질환 및 암과 관련된다고 보고되어 있다. 따라서, 세포내 극성의 검출은 병리학적, 생리학적 현상에 대한 연구에 매우 중요하다.It has been reported that the pathological activity of cells changes according to the polarity, and the abnormal polarity is associated with diabetes, neurological diseases and cancer. Therefore, the detection of intracellular polarity is very important for the study of pathological and physiological phenomena.
현재 세포내 극성을 관찰할 수 있는 유일한 방법은 광학 이미지를 이용하는 것이다. 이에 미세환경의 극성을 검출하기 위해 다양한 형광프로브가 개발되어 왔다. 지금까지 알려진 대부분의 극성 프로브는 분자내 전하 이동(intramolecular charge transfer; ICT)에 근간을 두었고 세포의 주위환경의 극성에 대한 변화에 근거한 방출 파장의 시프트를 나타낸다. 그러나, 이들 프로브의 대부분은 용매화 변색이며, 용매 극성이 증가할수록 형광 효율이 급격히 감소하여 소수성 환경에서 검출 범위가 제한되어 문제가 있다. 게다가 알려진 프로브는 단지 미토콘드리아, 리소좀, 소포체(ER), 지질 방울 및 세포막과 같은 세포 내 특정 구조만을 검출할 수 있다. 따라서, 이들 프로브는 특정 소기관의 극성을 검출하는 데 한계가 있을 뿐 아니라, 세포 전체의 극성 분포를 이미지화 하는 데에도 한계가 있었다.Currently, the only way to observe intracellular polarity is through optical imaging. Accordingly, various fluorescent probes have been developed to detect the polarity of the microenvironment. Most of the polarity probes known so far are based on intramolecular charge transfer (ICT) and exhibit a shift in emission wavelength based on changes in the polarity of the cell's surrounding environment. However, most of these probes are solvated discoloration, and as the polarity of the solvent increases, the fluorescence efficiency rapidly decreases, thereby limiting the detection range in a hydrophobic environment. Moreover, known probes can only detect specific structures within cells, such as mitochondria, lysosomes, endoplasmic reticulum (ER), lipid droplets and cell membranes. Therefore, these probes have limitations not only in detecting the polarity of specific organelles, but also in imaging the polarity distribution of the entire cell.
이에, 세포내 소기관의 극성 뿐 아니라 세포 전체의 극성 분포를 감지할 수 있고, 더 나아가 비율계량적으로 세포내 극성 분포를 정량적으로 감지할 수 있는 형광프로브의 개발이 필요한 실정이다. Accordingly, there is a need to develop a fluorescent probe capable of detecting the polarity distribution of the entire cell as well as the polarity of the intracellular organelle, and further quantitatively detecting the intracellular polarity distribution ratiometrically.
본 발명의 목적은 세포내 미세환경의 극성 유무를 감지할 수 있는 형광프로브 및 이를 이용한 세포내 극성유무 검출방법을 제공하는 데에 있다.An object of the present invention is to provide a fluorescent probe capable of detecting the presence or absence of polarity in the intracellular microenvironment and a method for detecting the presence or absence of intracellular polarity using the same.
본 발명은 화학식 1 내지 화학식 3으로 표시되는 화합물을 각각 제공한다.The present invention provides compounds represented by
[화학식 1][Formula 1]
[화학식 2][Formula 2]
[화학식 3][Formula 3]
또한, 본 발명은 화학식 1 내지 화학식 3으로 표시되는 화합물을 포함하는 세포내 극성 또는 무극성 검출용 형광프로브를 각각 제공한다.In addition, the present invention provides a fluorescent probe for intracellular polarity or non-polarity detection, each comprising a compound represented by Chemical Formulas 1 to 3.
또한, 본 발명은 상기 형광프로브를 포함하는 세포내 극성 또는 무극성 검출용 조성물을 각각 제공한다.In addition, the present invention provides a composition for detecting intracellular polarity or non-polarity comprising the fluorescent probe, respectively.
또한, 본 발명은 화학식 3으로 표시되는 화합물로 이루어진 형광프로브를 이용한 세포내 극성 유무에 대한 비율계량 검출용 조성물을 제공한다.In addition, the present invention provides a composition for ratiometric detection of the presence or absence of intracellular polarity using a fluorescent probe composed of a compound represented by Chemical Formula 3.
본 발명에 따르면, 세포내 극성 유무를 감지할 수 있는 신규 형광프로브를 개발함으로써 세포내 미세환경 뿐 아니라 소기관의 극성 유무도 감지할 수 있고, 특히 세포내 극성 시 턴온 되는 형광프로브와 세포내 극성 시 턴오프 되는 형광프로브를 연결한 신규 형광프로브를 통해 비율계량적으로 세포내 극성 유무를 정량할 수 있다.According to the present invention, by developing a novel fluorescent probe capable of detecting the presence or absence of intracellular polarity, not only the intracellular microenvironment but also the presence or absence of organelle polarity can be detected. The presence or absence of intracellular polarity can be quantitatively quantified through a novel fluorescent probe connected to a turned-off fluorescent probe.
도 1은 다양한 극성 및 무극성 용매에서 Dye1의 (a) 흡광도 스펙트럼과 (b) 형광 스펙트럼을 나타내며, 5회 주기 동안 메탄올과 THF 용매 간의 극성 변화에 따른 Dye1의 (c) 형광 스펙트럼과 (d) 형광 강도를 나타낸다(모든 농도는 1 μM, 여기 파장은 552 nm, 형광 강도는 573 nm에서 얻음).
도 2는 Dye2의 (a) 흡광도 스펙트럼과 (b) 형광 스펙트럼을 나타낸다.
도 3은 CDCl3, CDCl3 : CD3OD = 1 : 1 (v/v) 및 CD3OD : D2O = 1 : 1 (v/v) 용매 상에서 Dye1의 1H NMR 스펙트럼 (δ 0.0-4.0 영역)을 나타낸다.
도 4는 (a) Lyso-Tracker Green을 갖는 Dye1과 (b) Mito-Tracker Green을 갖는 Dye2에 관한 HeLa 세포에서의 공동 국재화 분석 결과를 나타낸다.
도 5는 Dye3의 (a) 흡광도 스펙트럼과 (b) 형광 스펙트럼을 나타낸다.
도 6은 다양한 극성 및 무극성 용매에서 RPS-1의 (a) 흡광도 스펙트럼과 (b) 형광 스펙트럼(3 μM)을 나타내고, (c) Fyellow/Fred 대 EN T 값 간의 상관관계와 (d) 5회 주기 동안 메탄올과 THF 용매 간의 극성 가역적 변화에 따른 RPS-1의 평균 Fyellow/Fred 강도를 나타낸다.
도 7은 30분 동안 RPS-1 (3 μM)로 표지한 HeLa 세포의 형광 이미지와 유사색 비례계량 이미지를 나타낸 것으로, (a) Fyellow (565-585 nm) 이미지, (b) Fred의 (630-680 nm) 이미지, (c) 비례계량 이미지, (d) 확대된 명-시야 이미지, (e) 확대된 비례계량 이미지를 나타낸다.
도 8은 RPS-1 (3 μM) 및 상업적으로 판매되는 소기관 마커 (1 μM)을 이용한 공동-국재화 분석 결과를 나타낸다(소기관 마커: 500-540 nm; RPS-1(노란색): 565-585 nm; RPS-1(붉은색): 630-680 nm).
도 9는 30분 동안 RPS-1 (3 μM)로 표지한 (a) Chang, (b) Huh7, (c) SW837 및 (d) HeLa 세포의 유사색 비례계량 이미지와 (e) 각 해당 이미지의 평균 Fyellow/Fred 강도 비율을 나타낸다(여기파장: 552 nm, 방출 윈도우: 565-585 nm (노란색), 630-680 nm (붉은색)).
도 10은 Dye1, Dye2 및 RPS-1 형광프로브를 처리한 HeLa 세포의 생존율을 MTT 분석한 결과를 나타낸다.
도 11은 다양한 물질과 함께 존재하는 RPS-1의 형광 강도 비율(Fyellow/Fred)을 나타낸 것으로, RPS-1의 선택성 분석 결과를 나타낸다[(1) control; 200 μM, (2) TBHP, (3) O2-, (4) ·OH, (5) ·OtBu, (6) H2O2, (7) NO·, (8) ONOO-; 1 mM, (9) Lys, (10) Arg, (11) His, (12) Asp, (13) Glu, (14) glucose, (15) GSH; 1 unit mL-1, (16) amidase, (17) NTR, (18) ALP, (19) CE1, (20) CE2, (21) NQO1, 이때 여기파장은 552 nm].
도 12는 RPS-1 (3 μM)로 표지한 HeLa 세포의 광안정성 분석 결과로서, (a) 조사 전과 60분 조사 후 RPS-1의 형광 이미지 및 (b) xyt 모드를 이용하여 60분 동안 2.00s 간격으로 시간 함수로 상대적인 형광 강도를 분석한 결과를 나타낸다.1 shows (a) absorbance spectrum and (b) fluorescence spectrum of Dye1 in various polar and non-polar solvents, (c) fluorescence spectrum and (d) fluorescence of Dye1 according to polarity change between methanol and THF solvent for 5 cycles Intensities are indicated (all concentrations were obtained at 1 µM, excitation wavelength at 552 nm, and fluorescence intensity at 573 nm).
2 shows (a) an absorbance spectrum and (b) a fluorescence spectrum of Dye2.
3 is a 1 H NMR spectrum of Dye1 (δ 0.0- ) on CDCl 3 , CDCl 3 :CD 3 OD = 1:1 (v/v) and CD 3 OD: D 2 O = 1:1 (v/v) solvents 4.0 area).
4 shows the results of co-localization analysis in HeLa cells for (a) Dye1 with Lyso-Tracker Green and (b) Dye2 with Mito-Tracker Green.
5 shows (a) an absorbance spectrum and (b) a fluorescence spectrum of Dye3.
6 shows (a) absorbance spectrum and (b) fluorescence spectrum (3 μM) of RPS-1 in various polar and non-polar solvents, (c) F yellow / F red vs. E N T Correlation between values and (d) average F yellow /F red intensity of RPS-1 according to reversible polarity change between methanol and THF solvent for 5 cycles.
7 shows a fluorescence image and a similar color proportional measurement image of HeLa cells labeled with RPS-1 (3 μM) for 30 minutes, (a) F yellow (565-585 nm) image, (b) F red (630-680 nm) image, (c) proportionality image, (d) magnified bright-field image, (e) magnified proportionalmetric image.
8 shows the results of co-localization assays using RPS-1 (3 μM) and commercially available organelle markers (1 μM) (organelle markers: 500-540 nm; RPS-1 (yellow): 565-585 nm; RPS-1 (red): 630-680 nm).
Fig. 9 shows pseudocolor proportionalmetric images of (a) Chang, (b) Huh7, (c) SW837 and (d) HeLa cells labeled with RPS-1 (3 μM) for 30 min and (e) images of each corresponding image. The average F yellow /F red intensity ratio is shown (excitation wavelength: 552 nm, emission window: 565-585 nm (yellow), 630-680 nm (red)).
10 shows the results of MTT analysis of the viability of HeLa cells treated with Dye1, Dye2, and RPS-1 fluorescent probes.
11 shows the fluorescence intensity ratio (F yellow /F red ) of RPS-1 present together with various substances, and shows the results of the selectivity analysis of RPS-1 [(1) control; 200 μM, (2) TBHP, (3) O 2 - , (4) ·OH, (5) · OtBu, (6) H 2 O 2 , (7) NO ·, (8) ONOO − ; 1 mM, (9) Lys, (10) Arg, (11) His, (12) Asp, (13) Glu, (14) glucose, (15) GSH; 1 unit mL -1 , (16) amidase, (17) NTR, (18) ALP, (19) CE1, (20) CE2, (21) NQO1, where the excitation wavelength is 552 nm].
12 is a photostability analysis result of HeLa cells labeled with RPS-1 (3 μM), (a) a fluorescence image of RPS-1 before and after 60 minutes irradiation, and (b) 2.00 for 60 minutes using xyt mode. The results of analyzing the relative fluorescence intensity as a function of time at s intervals are shown.
이하, 본 발명을 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명자는 세포내 소기관의 극성 뿐 아니라 세포 전체의 극성 분포를 감지할 수 있고, 더 나아가 비율계량적으로 세포내 극성 분포를 정량적으로 감지할 수 있는 신규 형광프로브의 개발을 위해 예의노력한 결과, 세포내 극성 시 턴온 되는 신규 형광프로브, 세포내 극성 시 턴오프 되는 신규 형광프로브 및 상기 2개의 형광프로브를 연결하여 비율계량적으로 세포내 극성 유무를 정량할 수 있는 신규 형광프로브를 각각 개발하여 본 발명을 완성하였다.The present inventors made diligent efforts to develop a novel fluorescent probe that can detect not only the polarity of intracellular organelles, but also the polarity distribution of the entire cell, and further quantitatively detect the intracellular polarity distribution ratiometrically. The present invention by developing a novel fluorescent probe that turns on when internal polarity, a new fluorescent probe that turns off when intracellular polarity, and a novel fluorescent probe that can quantitatively quantify intracellular polarity ratiometrically by connecting the two fluorescent probes was completed.
이에, 본 발명은 하기 화학식 1로 표시되는 화합물을 제공한다:Accordingly, the present invention provides a compound represented by the following formula (1):
[화학식 1][Formula 1]
또한, 본 발명은 상기 화학식 1로 표시되는 화합물을 포함하는 세포내 극성 검출용 형광프로브를 제공한다.In addition, the present invention provides a fluorescent probe for detecting intracellular polarity comprising the compound represented by
또한, 본 발명은 상기 형광프로브를 포함하는 세포내 극성 검출용 조성물을 제공한다.In addition, the present invention provides a composition for detecting intracellular polarity comprising the fluorescent probe.
상기 극성은 세포내 환경이 친수성인 것을 특징으로 하며, 상기 화합물은 세포내 환경이 친수성 환경일 때 형광이 증가하는 턴온(turn-on) 염료일 수 있다.The polarity is characterized in that the intracellular environment is hydrophilic, and the compound may be a turn-on dye that increases fluorescence when the intracellular environment is a hydrophilic environment.
또한, 본 발명은 하기 화학식 2로 표시되는 화합물을 제공한다:In addition, the present invention provides a compound represented by the following formula (2):
[화학식 2][Formula 2]
또한, 본 발명은 상기 화학식 2로 표시되는 화합물을 포함하는 세포내 무극성 검출용 형광프로브를 제공한다.In addition, the present invention provides a fluorescent probe for detecting non-polarity in cells comprising the compound represented by
또한, 상기 형광프로브를 포함하는 세포내 무극성 검출용 조성물을 제공한다.In addition, there is provided a composition for detecting non-polarity in cells comprising the fluorescent probe.
상기 무극성은 세포내 환경이 소수성인 것을 특징으로 하며, 상기 화합물은 세포내 환경이 친수성 환경일 때 형광이 감소하는 턴오프(turn-off) 염료일 수 있다.The non-polarity is characterized in that the intracellular environment is hydrophobic, and the compound may be a turn-off dye whose fluorescence decreases when the intracellular environment is a hydrophilic environment.
또한, 본 발명은 하기 화학식 3으로 표시되는 화합물을 제공한다:In addition, the present invention provides a compound represented by the following formula (3):
[화학식 3][Formula 3]
또한, 본 발명은 상기 화학식 3으로 표시되는 화합물을 포함하는 세포내 극성 유무에 대한 비율계량 검출용 형광프로브를 제공한다.In addition, the present invention provides a fluorescent probe for ratiometric detection of the presence or absence of intracellular polarity comprising the compound represented by
또한, 본 발명은 상기 형광프로브를 포함하는 세포내 극성 유무에 대한 비율계량 검출용 조성물을 제공한다.In addition, the present invention provides a composition for ratiometric detection of the presence or absence of intracellular polarity comprising the fluorescent probe.
또한, 본 발명은 인간으로부터 분리된 생체 시료에 화학식 1 내지 화학식 3으로 표시되는 화합물 중 하나 이상의 형광프로브를 처리하는 단계; 상기 형광프로브가 처리된 시료에 여기광을 조사하는 단계; 및 상기 여기광이 조사된 시료에서 발생하는 색의 강도 및 변화를 측정하여 비율계량 형광을 분석하는 단계를 포함하는 생체 시료의 극성 유무 분석방법을 제공한다.In addition, the present invention comprises the steps of treating a biological sample isolated from a human with one or more fluorescent probes among the compounds represented by
상기 여기광은 400 내지 600 nm 범위의 파장을 가질 수 있다.The excitation light may have a wavelength in the range of 400 to 600 nm.
또한, 본 발명은 상기 화학식 1로 표시되는 화합물을 포함하는 세포내 리소좀 전달용 약학조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for intracellular lysosomal delivery comprising the compound represented by Formula 1 above.
또한, 본 발명은 상기 화학식 2로 표시되는 화합물을 포함하는 세포내 지방방울 전달용 약학조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for intracellular fat drop delivery comprising the compound represented by Formula 2 above.
상기 약학조성물은 약물 또는 생리활성물질을 세포내 리소좀 또는 지방방울과 같은 소기관으로 특이적으로 전달할 수 있다.The pharmaceutical composition can specifically deliver a drug or a physiologically active substance to an organelle such as intracellular lysosomes or fat droplets.
본 발명의 약학조성물은 약학적으로 허용 가능한 담체를 더 포함할 수 있다. 상기 약학적으로 허용가능한 담체는 예를 들어, 부형제, 희석제 등이 있으며, 당해 기술분야에 잘 알려져있다. 담체의 선택은 제조하고자 하는 구체적인 제형 및 그 조성물의 구체적인 투여방법에 따라 결정될 수 있다. 따라서, 본 발명의 약학조성물은 매우 다양한 형태의 제제로서 존재할 수 있다. 또한, 본 발명의 약학조성물은 그 용도 또는 활성 성분의 종류에 따라 적절한 투여방법으로 투여될 수 있다. The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier includes, for example, an excipient, a diluent, and the like, and is well known in the art. The selection of the carrier may be determined according to the specific dosage form to be prepared and the specific administration method of the composition. Accordingly, the pharmaceutical composition of the present invention may exist as a formulation in a wide variety of forms. In addition, the pharmaceutical composition of the present invention may be administered by an appropriate administration method according to its use or the type of active ingredient.
본 발명의 약학조성물의 투여량은 진단 또는 치료하고자 하는 질병의 종류, 질병의 정도, 진단하고자 하는 대상 조직 또는 기관 및 진단 장치의 특성에 따라 달라질 수 있으며, 또한 상기 투여량은 투여 대상의 나이, 성별, 체중, 인종 등에 따라 증감할 수 있다.The dosage of the pharmaceutical composition of the present invention may vary depending on the type of disease to be diagnosed or treated, the severity of the disease, the target tissue or organ to be diagnosed, and the characteristics of the diagnostic device, and the dosage may vary depending on the age of the administration subject, It can increase or decrease according to gender, weight, race, etc.
본 발명의 약학조성물의 바람직한 투여방법은 비경구적, 예를 들어 볼루스 주입, 정맥내 주사, 동맥내 주입, 또는 폐가 조영되어야 하는 경우 스프레이, 예를 들어 연무제 분사가 있고, 경구 또는 직장 투여도 이용할 수 있으나, 공지된 조영제 투여방법으로도 가능하다. 비경구적 투여 제제는 무균이어야 하고, 생리학적으로 허용되지 않는 물질 및 상자성, 초상자성, 강자성, 또는 준강자성 오염물질이 없어야 하며, 방부제, 항균제, 비경구적 용액에 통상적으로 사용되는 완충액 및 항산화제, 부형제를 함유할 수 있으며, 분자영상에 방해가 되지 않는 다른 임의의 첨가제를 더 함유할 수도 있다.A preferred method of administration of the pharmaceutical composition of the present invention is parenteral, for example, bolus injection, intravenous injection, intra-arterial injection, or a spray, for example, spraying of an aerosol if the lungs are to be contrasted, and oral or rectal administration is also available. It can be used, but it is also possible with a known contrast agent administration method. Formulations for parenteral administration must be sterile and free from physiologically unacceptable substances and paramagnetic, superparamagnetic, ferromagnetic, or semiferromagnetic contaminants, including preservatives, antibacterial agents, buffers and antioxidants commonly used in parenteral solutions; It may contain excipients, and may further contain other optional additives that do not interfere with molecular imaging.
이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, to help the understanding of the present invention, examples will be described in detail. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.
<< 합성예Synthesis example > > Dye1Dye1 , , Dye2Dye2 , , Dye3 및 RPSDye3 and RPS -1 합성-1 synthesis
화합물 1, 2, 3, 5 및 8이 이미 문헌(Org. biomol. Chem., 2017, 15, 8091-8101; J. Phys. Chem. A, 2004, 108, 6452-6454; J. Am. Chem. Soc., 2017, 139, 17301-17304; Org. Lett., 2013, 15, 2730-2733; J. Mater. Chem. C, 2014, 2, 8515-8524)에 알려진 방법에 따라 준비되었고, 반응식 1의 방법으로 Dye1, Dye2, Dye3 및 RPS-1을 합성하였다.
[반응식 1][Scheme 1]
1. 2-(4-(1. 2-(4-( 디에틸아미노diethylamino )-2-)-2- 하이드록시스티릴hydroxystyryl )-1,3,3-)-1,3,3- 트리메틸trimethyl -3H-인돌-1--3H-Indole-1- 이움Ium 아이오다이드iodide [2-(4-(diethylamino)-2-hydroxystyryl)-1,3,3-trimethyl-3H-indol-1-ium iodide; Dye1] [2-(4-(diethylamino)-2-hydroxystyryl)-1,3,3-trimethyl-3H-indol-1-ium iodide; Dye1]
4-(디에틸아미노)-2-하이드록시벤즈알데히드 (100 mg, 0.517 mmol) 및 화합물 1 (156 mg, 0.517 mmol)을 에탄올 (20 mL)에 용해시켜 12시간 동안 환류시켰다. 이렇게 환류 반응시킨 혼합물을 감압 하에서 농축하고 컬럼 크로마토그래피 (CHCl3/MeOH = 9:1)로 정제하여 짙은 녹색의 반고상 물질인 Dye1 (145 mg)을 얻었다.4-(diethylamino)-2-hydroxybenzaldehyde (100 mg, 0.517 mmol) and compound 1 (156 mg, 0.517 mmol) were dissolved in ethanol (20 mL) and refluxed for 12 hours. The refluxed mixture was concentrated under reduced pressure and purified by column chromatography (CHCl 3 /MeOH = 9:1) to obtain Dye1 (145 mg) as a dark green semi-solid material.
수율: 59 %; 1H NMR (CDCl3, 600 MHz): δ (ppm) 7.16 (t, J = 8.3 Hz, 1H), 7.07 (d, J = 6.2 Hz, 1H), 6.85 (d, J = 8.3 Hz, 1H), 6.82 (t, J = 7.9 Hz, 1H), 6.74 (d, J = 10.3 Hz, 1H), 6.52 (d, J = 7.6 Hz, 1H), 6.13 (dd, J = 8.3, 2.8 Hz, 1H), 6.05 (d, J = 2.8 Hz, 1H), 5.37 (d, J = 10.3 Hz, 1H), 3.26 (q, J = 7.1 Hz, 4H), 2.74 (s, 3H), 1.32 (s, 3H), 1.14 (s, 3H), 1.10 (t, J = 7.2 Hz, 6H); 13C NMR (CDCl3, 150 MHz): δ (ppm) 156.17, 149.39, 148.63, 137.35, 129.40, 127.58, 127.47, 121.61, 118.89, 113.53, 107.79, 106.83, 104.61, 103.50, 97.53, 51.45, 44.42, 29.05, 26.07, 20.21, 12.77; HRMS (ESI+): m/z found for [C23H29ON2]+: 349.2269.Yield: 59%; 1 H NMR (CDCl 3 , 600 MHz): δ (ppm) 7.16 (t, J = 8.3 Hz, 1H), 7.07 (d, J = 6.2 Hz, 1H), 6.85 (d, J = 8.3 Hz, 1H) , 6.82 (t, J = 7.9 Hz, 1H), 6.74 (d, J = 10.3 Hz, 1H), 6.52 (d, J = 7.6 Hz, 1H), 6.13 (dd, J = 8.3, 2.8 Hz, 1H) , 6.05 (d, J = 2.8 Hz, 1H), 5.37 (d, J = 10.3 Hz, 1H), 3.26 (q, J = 7.1 Hz, 4H), 2.74 (s, 3H), 1.32 (s, 3H) , 1.14 (s, 3H), 1.10 (t, J = 7.2 Hz, 6H); 13 C NMR (CDCl 3 , 150 MHz): δ (ppm) 156.17, 149.39, 148.63, 137.35, 129.40, 127.58, 127.47, 121.61, 118.89, 113.53, 107.79, 106.83, 104.61, 105.50, 97.53, 51.45, 44.42, 51.45 , 26.07, 20.21, 12.77; HRMS (ESI + ): m/z found for [C 23 H 29 ON 2 ] + : 349.2269.
2. 2-(4-(2. 2-(4-( 디에틸아미노diethylamino )-2-)-2- 메톡시스티릴Methoxystyryl )-1,3,3-)-1,3,3- 트리메틸trimethyl -3H-인돌-1--3H-Indole-1- 이움Ium 아이오다이드iodide [2-(4-(diethylamino)-2-methoxystyryl)-1,3,3-trimethyl-3H-indol-1-ium iodide; Dye2] [2-(4-(diethylamino)-2-methoxystyryl)-1,3,3-trimethyl-3H-indol-1-ium iodide; Dye2]
Dye1의 합성방법과 동일한 방법으로 화합물 1과 화합물 2로부터 짙은 녹색의 반고상 물질인 Dye2를 얻었다.Dye2, a dark green semi-solid material, was obtained from
수율: 61 %; 1H NMR (CDCl3, 600 MHz): δ (ppm) 8.30 (brs, 1H), 8.05 (brs, 1H), 7.35 (d, J = 7.6 Hz, 1H), 7.31 (t, J = 7.6 Hz, 1H), 7.20-7.26 (m, 2H), 6.98 (d, J = 12.4 Hz, 1H), 6.40 (d, J = 9.0 Hz, 1H), 5.93 (s, 1H), 3.89 (s, 3H), 3.87 (s, 3H), 3.43 (q, J = 6.9 Hz, 4H), 1.60 (s, 6H), 1.14 (t, J = 6.9 Hz, 6H); 13C NMR (CDCl3, 150 MHz): δ (ppm) 178.10, 164.17, 155.90, 148.07, 141.85, 141.37, 133.44, 128.92, 126.82, 122.68, 113.16, 112.17, 107.73, 102.41, 92.68, 56.58, 50.25, 45.21, 34.63, 29.48, 28.04, 12.88; HRMS (ESI+): m/z found for [C24H31ON2]+: 363.2426.Yield: 61%; 1 H NMR (CDCl 3 , 600 MHz): δ (ppm) 8.30 (brs, 1H), 8.05 (brs, 1H), 7.35 (d, J = 7.6 Hz, 1H), 7.31 (t, J = 7.6 Hz, 1H), 7.20-7.26 (m, 2H), 6.98 (d, J = 12.4 Hz, 1H), 6.40 (d, J = 9.0 Hz, 1H), 5.93 (s, 1H), 3.89 (s, 3H), 3.87 (s, 3H), 3.43 (q, J = 6.9 Hz, 4H), 1.60 (s, 6H), 1.14 (t, J = 6.9 Hz, 6H); 13 C NMR (CDCl 3 , 150 MHz): δ (ppm) 178.10, 164.17, 155.90, 148.07, 141.85, 141.37, 133.44, 128.92, 126.82, 122.68, 113.16, 112.21, 107.73, 102.41, 92.68, 45.58, 50.25, 56.58 , 34.63, 29.48, 28.04, 12.88; HRMS (ESI + ): m/z found for [C 24 H 31 ON 2 ] + : 363.2426.
3. 5-3. 5- 카르복시carboxy -2-(4-(-2-(4-( 디에틸아미노diethylamino )-2-)-2- 하이드록시스티릴hydroxystyryl )-1,3,3-)-1,3,3- 트리메틸trimethyl -3H-인돌-1--3H-Indole-1- 이움Ium 아이오다이드iodide [5-carboxy-2-(4-(diethylamino)-2-hydroxystyryl)-1,3,3-trimethyl-3H-indol-1-ium iodide; 4] [5-carboxy-2-(4-(diethylamino)-2-hydroxystyryl)-1,3,3-trimethyl-3H-indol-1-ium iodide; 4]
Dye1의 합성방법과 동일한 방법으로 4-(디에틸아미노)-2-하이드록시벤즈알데히드와 화합물 3로부터 짙은 녹색의 반고상 물질인 화합물 4를 얻었다.
수율: 58 %; 1H NMR (CDCl3, 600 MHz): δ (ppm) 8.05 (d, J = 8.3 Hz, 1H), 7.82 (s, 1H), 6.89 (d, J = 9.0 Hz, 1H), 6.79 (d, J = 9.6 Hz, 1H), 6.54 (d, J = 8.3 Hz, 1H), 6.18 (d, J = 9.0 Hz, 1H), 6.06 (s, 1H), 5.37 (d, J = 10.3 Hz, 1H), 3.29 (q, J = 7.1 Hz, 4H), 2.84 (s, 3H), 1.38 (s, 3H), 1.18 (s, 3H), 1.12 (t, J = 7.2 Hz, 6H); 13C NMR (CDCl3, 150 MHz): δ (ppm) 172.13, 155.91, 152.80, 149.74, 137.24, 132.15, 129.83, 127.62, 123.96, 119.26, 112.45, 107.43, 105.78, 104.63, 103.66, 97.34, 50.93, 44.28, 28.71, 25.94, 19.95, 12.74.Yield: 58%; 1 H NMR (CDCl 3 , 600 MHz): δ (ppm) 8.05 (d, J = 8.3 Hz, 1H), 7.82 (s, 1H), 6.89 (d, J = 9.0 Hz, 1H), 6.79 (d, J = 9.6 Hz, 1H), 6.54 (d, J = 8.3 Hz, 1H), 6.18 (d, J = 9.0 Hz, 1H), 6.06 (s, 1H), 5.37 (d, J = 10.3 Hz, 1H) , 3.29 (q, J = 7.1 Hz, 4H), 2.84 (s, 3H), 1.38 (s, 3H), 1.18 (s, 3H), 1.12 (t, J = 7.2 Hz, 6H); 13 C NMR (CDCl 3 , 150 MHz): δ (ppm) 172.13, 155.91, 152.80, 149.74, 137.24, 132.15, 129.83, 127.62, 123.96, 119.26, 112.45, 107.43, 105.78, 104.28.63, 103.66, 93.66, 97.34.66, , 28.71, 25.94, 19.95, 12.74.
4. 5-(4-(4. 5-(4-( 터트turt -- 부톡시카르보닐Butoxycarbonyl )피페라진-1-카르보닐)-2-(4-()piperazine-1-carbonyl)-2-(4-( 디에틸아미노diethylamino )-2-)-2- 하이드록시스티릴hydroxystyryl )-1,3,3-트리메틸-3H-인돌-1-이움 )-1,3,3-trimethyl-3H-indol-1-ium 아이오다이드iodide [5-(4-( [5-(4-( terttert -- butoxycarbonylbutoxycarbonyl )) piperazinepiperazine -1-carbonyl)-2-(4-(diethylamino)-2-hydroxystyryl)-1,3,3-trimethyl-3H-indol-1-ium iodide; 6]-1-carbonyl)-2-(4-(diethylamino)-2-hydroxystyryl)-1,3,3-trimethyl-3H-indol-1-ium iodide; 6]
화합물 4 (95 mg, 0.182 mmol), N,N'-디사이클로헥실카르보디이미드 (DCC, 75 mg, 0.363 mmol) 및 하이드록시벤조트라이졸 (HOBt, 49 mg, 0.363 mmol)을 DMF (10 mL)에 용해시켜 실온에서 2시간 동안 교반하였다. 이렇게 얻어진 혼합물에 화합물 5 (68 mg, 0.363 mmol)를 첨가하고 12시간 동안 교반하였다. 그 후 용매를 증발시키고 반응 혼합물을 CH3CN에 용해시킨 후 여과를 통해 부산물인 우레아를 제거하였다. 이렇게 얻어진 여과물을 감압 하에서 농축하고 컬럼 크로마토그래피 (CHCl3/MeOH = 9:1)로 정제하여 짙은 녹색의 반고상 물질인 화합물 6 (81 mg)을 얻었다. Compound 4 (95 mg, 0.182 mmol), N,N'-dicyclohexylcarbodiimide (DCC, 75 mg, 0.363 mmol) and hydroxybenzotriazole (HOBt, 49 mg, 0.363 mmol) were mixed with DMF (10 mL) ) and stirred at room temperature for 2 hours. Compound 5 (68 mg, 0.363 mmol) was added to the mixture thus obtained and stirred for 12 hours. Then, the solvent was evaporated and the reaction mixture was dissolved in CH 3 CN and filtered to remove urea as a by-product. The filtrate thus obtained was concentrated under reduced pressure and purified by column chromatography (CHCl 3 /MeOH = 9:1) to obtain Compound 6 (81 mg) as a dark green semi-solid material.
수율: 65 %; 1H NMR (CDCl3, 600 MHz): δ (ppm) 7.24 (dd, J = 8.3, 1.4 Hz, 1H), 7.14 (d, J = 1.4 Hz 1H), 6.83 (d, J = 8.3 Hz, 1H), 6.72 (d, J = 9.6 Hz, 1H), 6.44 (d, J = 8.3 Hz, 1H), 6.12 (dd, J = 8.3, 2.1 Hz, 1H), 5.98 (d, J = 2.1 Hz 1H), 5.31 (d, J = 9.6 Hz, 1H), 3.61 (brs, 4H), 3.44 (brs, 4H), 3.24 (q, J = 7.3 Hz, 4H), 2.74 (s, 3H), 1.45 (s, 9H), 1.29 (s, 3H), 1.11 (s, 3H), 1.08 (t, J = 6.9 Hz, 6H); 13C NMR (CDCl3, 150 MHz): δ (ppm) 171.74, 155.86, 154.74, 150.35, 149.49, 137.49, 129.68, 128.15, 127.58, 125.08, 121.76, 112.88, 107.59, 105.71, 104.61, 103.81, 97.41, 80.24, 51.32, 44.39, 34.01, 28.88, 28.47, 26.00, 20.16, 12.75.Yield: 65%; 1 H NMR (CDCl3, 600 MHz): δ (ppm) 7.24 (dd, J = 8.3, 1.4 Hz, 1H), 7.14 (d, J = 1.4 Hz 1H), 6.83 (d, J = 8.3 Hz, 1H) , 6.72 (d, J = 9.6 Hz, 1H), 6.44 (d, J = 8.3 Hz, 1H), 6.12 (dd, J = 8.3, 2.1 Hz, 1H), 5.98 (d, J = 2.1 Hz 1H), 5.31 (d, J = 9.6 Hz, 1H), 3.61 (brs, 4H), 3.44 (brs, 4H), 3.24 (q, J = 7.3 Hz, 4H), 2.74 (s, 3H), 1.45 (s, 9H) ), 1.29 (s, 3H), 1.11 (s, 3H), 1.08 (t, J = 6.9 Hz, 6H); 13 C NMR (CDCl3, 150 MHz): δ (ppm) 171.74, 155.86, 154.74, 150.35, 149.49, 137.49, 129.68, 128.15, 127.58, 125.08, 121.76, 112.88, 107.59, 105.71, 104.61, 103.81, 97.41, 80.24, 51.32, 44.39, 34.01, 28.88, 28.47, 26.00, 20.16, 12.75.
5. 2-(4-(5. 2-(4-( 디에틸아미노diethylamino )-2-)-2- 하이드록시스티릴hydroxystyryl )-1,3,3-)-1,3,3- 트리메틸trimethyl -5-(피페라진-1-카르보닐)-3H-인돌-1-이움 -5-(piperazine-1-carbonyl)-3H-indol-1-ium 아이오다이드iodide [2-(4-( [2-(4-( diethylaminodiethylamino )-2-)-2- hydroxystyrylhydroxystyryl )-1,3,3-)-1,3,3- trimethyltrimethyl -5-(-5-( piperazinepiperazine -1-carbonyl)-3H-indol-1-ium iodide; 7]-1-carbonyl)-3H-indol-1-ium iodide; 7]
화합물 6 (90 mg, 0.131 mmol)을 TFA (2 mL) 및 DCM (2 mL)의 공용매에 용해시킨 후 실온에서 12시간 동안 교반하였다. 이렇게 얻어진 반응혼합물을 10 % Na2CO3 (10 mL) 및 소금물 (10 mL x 2)로 세정하였다. 유기상을 감압 하에서 농축하고 컬럼 크로마토그래피 (CHCl3/MeOH = 8:2)로 정제하여 짙은 녹색의 반고상 물질인 화합물 7 (69 mg)을 얻었다. Compound 6 (90 mg, 0.131 mmol) was dissolved in a cosolvent of TFA (2 mL) and DCM (2 mL) and stirred at room temperature for 12 hours. The reaction mixture thus obtained was washed with 10 % Na 2 CO 3 (10 mL) and brine (10 mL×2). The organic phase was concentrated under reduced pressure and purified by column chromatography (CHCl 3 /MeOH = 8:2) to obtain Compound 7 (69 mg) as a dark green semi-solid material.
수율: 90 %; 1H NMR (CDCl3, 600 MHz): δ (ppm) 7.26 (d, J = 9.0 Hz, 1H), 7.16 (s, 1H), 6.86 (d, J = 8.3 Hz, 1H), 6.75 (d, J = 10.3 Hz, 1H), 6.46 (d, J = 7.6 Hz, 1H), 6.14 (d, J = 9.0 Hz, 1H), 6.00 (s, 1H), 5.32 (d, J = 9.6 Hz, 1H), 3.85 (d, J = 5.5 Hz, 4H), 3.27 (q, J = 7.3 Hz, 4H), 3.08 (s, 4H), 2.77 (s, 3H), 1.30 (s, 3H), 1.13 (s, 3H) 1.10 (t, J = 7.2 Hz, 6H); 13C NMR (CDCl3, 150 MHz): δ (ppm) 171.60, 155.88, 150.27, 149.45, 137.47, 129.68, 128.10, 127.58, 125.13, 121.73, 112.91, 107.57, 105.74, 104.62, 103.75, 97.39, 51.35, 45.83, 44.41, 31.02, 28.92, 26.02, 20.18, 12.75.Yield: 90%; 1 H NMR (CDCl 3 , 600 MHz): δ (ppm) 7.26 (d, J = 9.0 Hz, 1H), 7.16 (s, 1H), 6.86 (d, J = 8.3 Hz, 1H), 6.75 (d, J = 10.3 Hz, 1H), 6.46 (d, J = 7.6 Hz, 1H), 6.14 (d, J = 9.0 Hz, 1H), 6.00 (s, 1H), 5.32 (d, J = 9.6 Hz, 1H) , 3.85 (d, J = 5.5 Hz, 4H), 3.27 (q, J = 7.3 Hz, 4H), 3.08 (s, 4H), 2.77 (s, 3H), 1.30 (s, 3H), 1.13 (s, 3H) 1.10 (t, J = 7.2 Hz, 6H); 13 C NMR (CDCl 3 , 150 MHz): δ (ppm) 171.60, 155.88, 150.27, 149.45, 137.47, 129.68, 128.10, 127.58, 125.13, 121.73, 112.91, 107.57, 105.74, 104.62, 103.75, 97.83. , 44.41, 31.02, 28.92, 26.02, 20.18, 12.75.
6. 7-6. 7- 메틸벤조[c][1,2,5]티아디아졸Methylbenzo[c][1,2,5]thiadiazole -4--4- 카르보니트릴carbonitrile [7- [7- methylbenzo[c][1,2,5]thiadiazolemethylbenzo[c][1,2,5]thiadiazole -4-carbonitrile; 9]-4-carbonitrile; 9]
DMF (15 mL)에 화합물 8 (1.00 g, 4.38 mmol)을 용해시킨 용액을 원통형 유리 압력 튜브 CuCN (1.23 g, 13.7 mmol)에 첨가하고 150 ℃에서 16 시간 동안 교반하였다. 실온으로 냉각시킨 후, 수용성 15% NH3 (50 mL)을 첨가하고 1시간 동안 교반하였다. 이렇게 얻어진 침전물을 여과하고 DCM으로 세정하였다. 여과물을 DCM으로 3회 추출하고, 유기용매를 증발시켜 얻은 물질을 용출물로 DCM을 이용한 실리카겔 컬럼 상에서 정제하여 하얀 솜털같은 고상 물질인 화합물 9 (0.57 g)를 얻었다.A solution of compound 8 (1.00 g, 4.38 mmol) in DMF (15 mL) was added to a cylindrical glass pressure tube CuCN (1.23 g, 13.7 mmol) and stirred at 150° C. for 16 hours. After cooling to room temperature, aqueous 15% NH 3 (50 mL) was added and stirred for 1 hour. The precipitate thus obtained was filtered and washed with DCM. The filtrate was extracted three times with DCM, and the material obtained by evaporating the organic solvent was purified on a silica gel column using DCM as an eluent to obtain Compound 9 (0.57 g) as a white fluffy solid.
수율: 74 %; 1H NMR (CDCl3, 600 MHz): δ (ppm) 7.94 (d, J = 6.9 Hz, 1H), 7.44 (d, J = 8.3 Hz, 1H), 2.82 (s, 3H); 13C NMR (CDCl3, 150 MHz): δ (ppm) 154.85, 152.90, 138.58, 136.20, 127.46, 115.67, 103.44, 18.56.Yield: 74%; 1 H NMR (CDCl 3 , 600 MHz): δ (ppm) 7.94 (d, J = 6.9 Hz, 1H), 7.44 (d, J = 8.3 Hz, 1H), 2.82 (s, 3H); 13 C NMR (CDCl 3 , 150 MHz): δ (ppm) 154.85, 152.90, 138.58, 136.20, 127.46, 115.67, 103.44, 18.56.
7. 7-7. 7- (브로모메틸)벤조[c][1,2,5]티아디아졸(bromomethyl)benzo[c][1,2,5]thiadiazole -4--4- 카르보니트릴carbonitrile [7-(bromomethyl)benzo[c][1,2,5]thiadiazole-4-carbonitrile; 10] [7-(bromomethyl)benzo[c][1,2,5]thiadiazole-4-carbonitrile; 10]
건조 CHCl3 (60 mL)에 화합물 9 (1.36 g, 7.78 mmol), 2,2’-아조비스(2-메틸프로피오니트닐) (AIBN, 50 mg, 0.304 mmol) 및 N-브로모숙신이미드 (NBS, 1.66 g, 9.33 mmol)를 넣어 교반시킨 용액에 0.5 mL of 33 % HBr (in acetic acid)을 첨가하고 75 ℃에서 16 시간 동안 교반하였다. TLC로 반응 완료를 확인한 후, 반응혼합물을 CHCl3 (100 mL)에 용해시키고 물로 세정한 후, 유기층을 Na2SO4으로 건조하고 여과한 후 농축하였다. 조생성물을 컬럼 크로마토그래피로 (hexane/EA = 9:1)로 정제하여 하얀 고상 물질인 화합물 10 (1.53 g)을 얻었다.Compound 9 (1.36 g, 7.78 mmol), 2,2'-azobis(2-methylpropionitnyl) (AIBN, 50 mg, 0.304 mmol) and N-bromosuccinic in dry CHCl 3 (60 mL) Mid (NBS, 1.66 g, 9.33 mmol) was added and 0.5 mL of 33 % HBr (in acetic acid) was added to the stirred solution, and the mixture was stirred at 75 °C for 16 hours. After confirming the completion of the reaction by TLC, the reaction mixture was dissolved in CHCl 3 (100 mL), washed with water, and the organic layer was dried over Na 2 SO 4 , filtered, and concentrated. The crude product was purified by column chromatography (hexane/EA = 9:1) to obtain Compound 10 (1.53 g) as a white solid.
수율: 78 %; 1H NMR (CDCl3, 600 MHz): δ (ppm) 8.02 (d, J = 7.6 Hz, 1H), 7.74 (d, J = 7.6 Hz, 1H), 4.98 (s, 2H); 13C NMR (CDCl3, 150 MHz): δ (ppm) 153.11, 152.87, 136.70, 135.94, 128.46, 115.09, 106.02, 26.84.Yield: 78%; 1 H NMR (CDCl 3 , 600 MHz): δ (ppm) 8.02 (d, J = 7.6 Hz, 1H), 7.74 (d, J = 7.6 Hz, 1H), 4.98 (s, 2H); 13 C NMR (CDCl 3 , 150 MHz): δ (ppm) 153.11, 152.87, 136.70, 135.94, 128.46, 115.09, 106.02, 26.84.
8. 7-8. 7- (6-(디에틸아미노)벤조퓨란-2-일)벤조(6-(diethylamino)benzofuran-2-yl)benzo [c][1,2,5][c][1,2,5] 티아디아졸thiadiazole -4--4- 카르보니트릴carbonitrile [7- [7- (6-(diethylamino)(6-(diethylamino) benzofuran-2-yl)benzo[c][1,2,5]thiadiazole-4-carbonitrile; 11]benzofuran-2-yl)benzo[c][1,2,5]thiadiazole-4-carbonitrile; 11]
화합물 10 (500 mg, 1.97 mmol), 4-(디에틸아미노)살리실알데히드 (390 g, 2.02 mmol), 및 K2CO3 (1.70 g, 12.3 mmol)을 DMF (10 mL)에 넣고 125 ℃에서 16시간 동안 교반하였다. 어두운 반응혼합물을 물로 희석하고 EA로 추출하였다. 유기용매를 증발시키고 컬럼 크로마토그래피 (hexane/EA = 9:1)로 정제하여 다크 바이올렛의 고상 물질인 화합물 11 (151 mg)을 얻었다.Compound 10 (500 mg, 1.97 mmol), 4-(diethylamino)salicylaldehyde (390 g, 2.02 mmol), and K 2 CO 3 (1.70 g, 12.3 mmol) were placed in DMF (10 mL) at 125 °C stirred for 16 hours. The dark reaction mixture was diluted with water and extracted with EA. The organic solvent was evaporated and purified by column chromatography (hexane/EA = 9:1) to obtain Compound 11 (151 mg) as a dark violet solid.
수율: 23 %; 1H NMR (CDCl3, 600 MHz): δ (ppm) 8.17 (s, 1H), 8.05 (d, J = 7.6 Hz, 1H), 8.02 (d, J = 7.6 Hz, 1H), 7.48 (d, J = 8.3 Hz, 1H), 6.75 (s, 1H), 6.73 (dd, J = 8.3, 2.1 Hz, 1H), 3.45 (q, J = 7.1 Hz, 4H), 1.24 (t, J = 7.2 Hz, 6H); 13C NMR (CDCl3, 150 MHz): δ (ppm) 158.28, 153.88, 150.33, 148.37, 147.80, 136.36, 128.30, 122.81, 121.10, 118.71, 116.17, 113.62, 110.44, 101.74, 92.81, 45.10, 12.71.Yield: 23%; 1 H NMR (CDCl 3 , 600 MHz): δ (ppm) 8.17 (s, 1H), 8.05 (d, J = 7.6 Hz, 1H), 8.02 (d, J = 7.6 Hz, 1H), 7.48 (d, J = 8.3 Hz, 1H), 6.75 (s, 1H), 6.73 (dd, J = 8.3, 2.1 Hz, 1H), 3.45 (q, J = 7.1 Hz, 4H), 1.24 (t, J = 7.2 Hz, 6H); 13 C NMR (CDCl 3 , 150 MHz): δ (ppm) 158.28, 153.88, 150.33, 148.37, 147.80, 136.36, 128.30, 122.81, 121.10, 118.71, 116.17, 113.62, 110.44, 101.74, 92.81, 45.10, 12.71.
9. 7-9. 7- (6-(디에틸아미노)벤조퓨란-2-일)벤조(6-(diethylamino)benzofuran-2-yl)benzo [c][1,2,5][c][1,2,5] 티아디아졸thiadiazole -4--4- 카르복실산carboxylic acid [7- [7- (6-(diethylamino)(6-(diethylamino) benzofuran-2-yl)benzo[c][1,2,5]thiadiazole-4-carboxylic acid; Dye3]benzofuran-2-yl)benzo[c][1,2,5]thiadiazole-4-carboxylic acid; Dye3]
화합물 11 (100 mg, 0.287 mmol) 및 18-크라운-6 (100 mg, 0.379 mmol)을10 % NaOH (5 mL) 및 1,4-디옥산 (5 mL)의 공용매에 용해시킨 후, 100 ℃에서 밤새도록 교반하였다. 용매를 증발시킨 반응혼합물을 빙조에서 HCl로 pH가 2가 되도록 산성화시켰다. EA (20 mL)로 희석시킨 후, 유기상을 감압 하에서 농축하고 컬럼 크로마토그래피 (DCM/methanol = 94:6)로 정제하여 어두운 보라색의 반고상 물질인 화합물 9 (31 mg)을 얻었다.Compound 11 (100 mg, 0.287 mmol) and 18-crown-6 (100 mg, 0.379 mmol) were dissolved in a cosolvent of 10% NaOH (5 mL) and 1,4-dioxane (5 mL), followed by 100 Stir overnight at °C. The reaction mixture after evaporation of the solvent was acidified to
수율: 29 %; 1H NMR (DMSO, 600 MHz): δ (ppm) 8.35 (d, J = 6.2 Hz, 1H), 8.07 (s, 1H), 8.06 (d, J = 6.6 Hz, 1H), 7.51 (d, J = 8.2 Hz, 1H), 6.81 (s, 1H), 6.73 (d, J = 9.0 Hz, 1H), 3.39 (q, J = 7.3 Hz, 4H), 1.11 (t, J = 6.9 Hz, 6H); 13C NMR (CDCl3, 150 MHz): δ (ppm) 165.86, 157.80, 152.95, 151.31, 148.17, 134.40, 126.83, 123.16, 122.01, 121.59, 118.23, 112.10, 110.65, 92.99, 44.78, 12.97.Yield: 29%; 1 H NMR (DMSO, 600 MHz): δ (ppm) 8.35 (d, J = 6.2 Hz, 1H), 8.07 (s, 1H), 8.06 (d, J = 6.6 Hz, 1H), 7.51 (d, J) = 8.2 Hz, 1H), 6.81 (s, 1H), 6.73 (d, J = 9.0 Hz, 1H), 3.39 (q, J = 7.3 Hz, 4H), 1.11 (t, J = 6.9 Hz, 6H); 13 C NMR (CDCl 3 , 150 MHz): δ (ppm) 165.86, 157.80, 152.95, 151.31, 148.17, 134.40, 126.83, 123.16, 122.01, 121.59, 118.23, 112.10, 110.65, 92.99, 44.78, 12.97.
10. 2-(4-(10. 2-(4-( 디에틸아미노diethylamino )-2-)-2- 하이드록시스티릴hydroxystyryl )-5-(4-(7-)-5-(4-(7- (6-(디에틸아미노)벤조퓨란-2-일)벤조(6-(diethylamino)benzofuran-2-yl)benzo [c][1,2,5]티아디아졸-4-카르보닐)피페라진-1-카르보닐)-1,3,3-트리메틸-3H-인돌-1-이움 [c][1,2,5]thiadiazole-4-carbonyl)piperazine-1-carbonyl)-1,3,3-trimethyl-3H-indol-1-ium 아이오다이드iodide [2-(4-(diethylamino)-2-hydroxystyryl)-5-(4-(7-(6-(diethylamino)benzofuran-2-yl)benzo[c][1,2,5]thiadiazole-4-carbonyl)piperazine-1-carbonyl)-1,3,3-trimethyl-3H-indol-1-ium iodide; RPS-1] [2-(4-(diethylamino)-2-hydroxystyryl)-5-(4-(7-(6-(diethylamino)benzofuran-2-yl)benzo[c][1,2,5]thiadiazole-4- carbonyl) piperazine-1-carbonyl)-1,3,3-trimethyl-3H-indol-1-ium iodide; RPS-1]
Dye3 (12 mg, 0.028 mmol), DCC (11 mg, 0.55 mmol) 및 HOBt (7 mg, 0.055 mmol)을 DMF (3 mL)에 용해시켜 실온에서 2시간 동안 교반하였다. 이렇게 얻어진 반응혼합물에 화합물 7 (15 mg, 0.028 mmol)을 첨가하고 12시간 동안 교반하였다. 그 후 용매를 증발시키고 반응 혼합물을 CH3CN에 용해시킨 후 여과를 통해 부산물인 우레아를 제거하였다. 이렇게 얻어진 여과물을 감압 하에서 농축하고 컬럼 크로마토그래피 (CHCl3/MeOH = 9:1)로 정제하여 짙은 보라색의 반고상 물질인 RPS-1 (9 mg)을 얻었다. Dye3 (12 mg, 0.028 mmol), DCC (11 mg, 0.55 mmol) and HOBt (7 mg, 0.055 mmol) were dissolved in DMF (3 mL) and stirred at room temperature for 2 hours. Compound 7 (15 mg, 0.028 mmol) was added to the reaction mixture thus obtained and stirred for 12 hours. Then, the solvent was evaporated and the reaction mixture was dissolved in CH 3 CN and filtered to remove urea as a by-product. The filtrate thus obtained was concentrated under reduced pressure and purified by column chromatography (CHCl 3 /MeOH = 9:1) to obtain RPS-1 (9 mg) as a dark purple semi-solid material.
수율: 34 %; 1H NMR (CDCl3, 600 MHz): δ (ppm) 8.11 (d, J = 7.6 Hz, 1H), 8.05 (s, 1H), 7.76 (d, J = 6.9 Hz, 1H), 7.48 (d, J = 9.0 Hz, 1H), 7.29 (d, J = 8.3 Hz, 1H), 7.48 (d, J = 9.0 Hz, 1H), 6.85 (d, J = 9.0 Hz, 1H), 6.80 (s, 1H), 6.74 (d, J = 10.8 Hz, 1H), 6.72 (d, J = 8.4, 1H) 6.47 (d, J = 8.3 Hz, 1H), 6.13 (s, 1H), 6.00 (d, J = 9.0 Hz, 1H), 5.32 (d, J = 10.3 Hz, 1H), 3.64-3.96 (m, 8H), 3.44 (q, J = 7.1 Hz, 4H), 3.25 (q, J = 7.3 Hz, 4H), 2.76 (s, 3H), 1.30 (s, 3H), 1.23 (t, J = 7.6 Hz, 6H), 1.13 (s, 3H), 1.09 (t, J = 7.5 Hz, 6H); 13C NMR (CDCl3, 150 MHz): δ (ppm) 171.85, 166.77, 157.66, 155.85, 152.15, 150.87, 150.52, 149.48, 148.52, 147.72, 137.56, 129.71, 129.54, 128.32, 127.58, 126.27, 125.38, 124.69, 122.31, 121.85, 118.82, 112.82, 110.90, 110.11, 107.54, 105.76, 104.61, 103.77, 97.39, 93.33, 51.33, 45.10, 44.41, 29.78, 28.91, 26.02, 22.78, 20.17, 14.22, 12.74 HRMS (ESI+): m/z found for [C47H52O4N7S]+: 810.3788.Yield: 34%; 1 H NMR (CDCl 3 , 600 MHz): δ (ppm) 8.11 (d, J = 7.6 Hz, 1H), 8.05 (s, 1H), 7.76 (d, J = 6.9 Hz, 1H), 7.48 (d, J = 9.0 Hz, 1H), 7.29 (d, J = 8.3 Hz, 1H), 7.48 (d, J = 9.0 Hz, 1H), 6.85 (d, J = 9.0 Hz, 1H), 6.80 (s, 1H) , 6.74 (d, J = 10.8 Hz, 1H), 6.72 (d, J = 8.4, 1H) 6.47 (d, J = 8.3 Hz, 1H), 6.13 (s, 1H), 6.00 (d, J = 9.0 Hz) , 1H), 5.32 (d, J = 10.3 Hz, 1H), 3.64-3.96 (m, 8H), 3.44 (q, J = 7.1 Hz, 4H), 3.25 (q, J = 7.3 Hz, 4H), 2.76 (s, 3H), 1.30 (s, 3H), 1.23 (t, J = 7.6 Hz, 6H), 1.13 (s, 3H), 1.09 (t, J = 7.5 Hz, 6H); 13 C NMR (CDCl 3 , 150 MHz): δ (ppm) 171.85, 166.77, 157.66, 155.85, 152.15, 150.87, 150.52, 149.48, 148.52, 147.72, 137.56, 129.71, 129.54, 128.32, 127.58, 126.27, 125.38, 124.27. , 122.31, 121.85, 118.82, 112.82 , 110.90, 110.11, 107.54, 105.76, 104.61, 103.77, 97.39, 93.33, 51.33, 45.10, 44.41, 29.78, 28.91, 26.02, 22.78, 20.17, 14.22, 12.74 HRMS (ESI +): m/z found for [C 47 H 52 O 4 N 7 S] + : 810.3788.
<실험예> <Experimental example>
하기의 실험예들은 본 발명에 따른 각각의 실시예에 공통적으로 적용되는 실험예를 제공하기 위한 것이다.The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.
1. 분광학 분석1. Spectroscopy Analysis
흡수 분석과 형광 분석은 UV-Vis 분광분석기 (S-3100)와 형광 분광분석기 (FS-2)로 각각 분석하였다. 형광 양자 수율은 9,10-디페닐안트라센 (시클로헥산에서 Φ = 0.93)을 이용하여 측정하였다. 1H NMR 스펙트럼은 600 MHz NMR 분광계 (JNM-ECZR)를 사용하여 기록하였다. 형광 이미지는 스펙트럼 공초점 현미경 (Leica TCS SP8)으로 얻었다.Absorption analysis and fluorescence analysis were performed with a UV-Vis spectrometer (S-3100) and a fluorescence spectrometer (FS-2), respectively. The fluorescence quantum yield was measured using 9,10-diphenylanthracene (Φ = 0.93 in cyclohexane). 1 H NMR spectra were recorded using a 600 MHz NMR spectrometer (JNM-ECZR). Fluorescence images were obtained with a spectral confocal microscope (Leica TCS SP8).
2. 세포 이미지 분석2. Cell Image Analysis
이미지 분석 전 각 세포를 이틀 동안 배양하였다. 스트렙토마이신 (100 ㎍/mL), 페니실린 (mL 당 100 unit) 및 10 % 소태아혈청(FBS)을 모든 배양배지에 첨가하였다. 배양배지를 무혈청 배지로 교체하고 각각의 합성된 형광프로브를 30분 동안 염색하였다. 살아있는 세포 이미지 분석은 생세포 기기(Chamlide IC)를 사용하여 장기간 노출을 위해 적절한 온도, 습도 및 pH를 유지하면서 수행하였다. 비율 이미지 프로세싱 및 분석 (Ratiometric image processing and analysis)은 MetaMorph 소프트웨어를 사용하여 분석을 수행하였다.Each cell was incubated for two days before image analysis. Streptomycin (100 μg/mL), penicillin (100 units per mL) and 10% fetal bovine serum (FBS) were added to all culture media. The culture medium was replaced with a serum-free medium and each synthesized fluorescent probe was stained for 30 minutes. Live cell image analysis was performed using a live cell instrument (Chamlide IC) while maintaining appropriate temperature, humidity and pH for long-term exposure. Ratiometric image processing and analysis was performed using MetaMorph software.
3. 공동 국재화 시험(Co-localization experiments)3. Co-localization experiments
공동 국재화 시험은 합성된 형광프로브와 각종 상업적으로 판매되는 소기관 추적기(리소좀: LysoTracker Green DND-2; 지질방울: BODIPY 493/503; 미토콘드리아: MitoTracker Green FM; 소포체: ER-Tracker Green)를 함께 염색하여 수행하였다. 이때, 발광 파장은 각각 488 nm (소기관 추적기) 및 552 nm (극성 형광프로브)이었고, 피어슨 공배치 상관계수 (Pearson's colocalization coefficient; A)는 AutoQuant X2 소프트웨어를 이용하여 산출하였다.The co-localization test was performed with a synthetic fluorescent probe and various commercially available organelle tracers (lysosome: LysoTracker Green DND-2; lipid droplet: BODIPY 493/503; mitochondria: MitoTracker Green FM; endoplasmic reticulum: ER-Tracker Green). was performed. At this time, the emission wavelengths were 488 nm (organelle tracer) and 552 nm (polar fluorescent probe), respectively, and Pearson's colocalization coefficient (A) was calculated using AutoQuant X2 software.
4. 세포생존율 분석4. Cell viability analysis
MTT 키트 (AbCareBio CL) 분석을 통해 세포독성을 판단하였다. HeLa 세포를 96-웰 플레이트 상에서 24시간 동안 배양한 후, 다양한 농도의 형광프로브를 첨가하였다. 2시간 동안 배양한 후, 배양배지를 10% MTT를 함유하는 무혈청 배지로 교체한 후 2시간 더 배양하였다. MTT 함유 배지를 제거하고 DMSO를 첨가하여 형성된 포마잔 침전물을 용해시킨 후, 600 nm에서 흡광도를 측정하였다.Cytotoxicity was determined through MTT kit (AbCareBio CL) analysis. After HeLa cells were cultured on a 96-well plate for 24 hours, various concentrations of fluorescent probes were added. After culturing for 2 hours, the culture medium was replaced with a serum-free medium containing 10% MTT, and then cultured for another 2 hours. After removing the MTT-containing medium and adding DMSO to dissolve the formed formazan precipitate, the absorbance was measured at 600 nm.
5. 선택성 분석5. Selectivity Analysis
각각의 종들 (200 μM ROS 및 RNS; 1mM 아미노산, 글루코오스 및 GSH; 1 unit mL-1 효소)을 3 μM RPS-1 (in PBS buffer; 10 mM, pH 7.4)에 첨가하고, 시간에 따른 형광 분석을 측정하였다. 이때, 온도는 37 ℃에서 2시간 동안 유지시켰다.Each species (200 μM ROS and RNS; 1 mM amino acids, glucose and GSH; 1 unit mL −1 enzyme) was added to 3 μM RPS-1 (in PBS buffer; 10 mM, pH 7.4) and fluorescence analysis over time was measured. At this time, the temperature was maintained at 37 °C for 2 hours.
이때, 사용된 터트부틸 하이드록시퍼옥사이드 (TBHP, 416665), KO2 (278904), 각종 아미노산들 (LAA21), 글루코오스 (G7528), 글루타치온 (GSH, G6013), 아미데이즈 (A6691), 니트로리덕테이즈 (NTR, N9284), 알칼린 포스파테이즈 (ALP, P7640), 카르복시에스테레이즈 1 (CE1, E0287), 카르복시에스테레이즈 2 (CE2, E0412), 퀴논 리덕테이즈 (NQO1, D1315)는 Sigma-Aldrich로부터 구매하였다. 하이드록시 라디칼 (·OH) 및 터트부톡시 라디칼 (·OtBu)은 FeSO4에 의해 TBHP 및 H2O2로부터 얻었다. 퍼옥시니트리트 (ONOO-)는 이미 보고된 방법(J. Immunol. 1998, 161, 1422-1427)에 따라 준비하였다.At this time, the used tertbutyl hydroxyperoxide (TBHP, 416665), KO 2 (278904), various amino acids (LAA21), glucose (G7528), glutathione (GSH, G6013), amidase (A6691), nitroreducte Is (NTR, N9284), alkaline phosphatase (ALP, P7640), carboxyesterase 1 (CE1, E0287), carboxyesterase 2 (CE2, E0412), quinone reductase (NQO1, D1315) is Sigma- Purchased from Aldrich. The hydroxy radical (·OH) and tertbutoxy radical (·OtBu) were obtained from TBHP and H 2 O 2 with FeSO 4 . Peroxynitrite (ONOO − ) was prepared according to a previously reported method (J. Immunol. 1998, 161, 1422-1427).
6. 광안정성 (Photostability) 분석6. Photostability analysis
RPS-1의 광안정성은 RPS-1 표지 (3 μM) HeLa 세포에서 3개의 지정된 위치에서 시간에 따른 형광 강도의 변화로서 측정하였다. The photostability of RPS-1 was measured as the change in fluorescence intensity with time at three designated locations in RPS-1 labeled (3 μM) HeLa cells.
<실시예 1> 합성 화합물의 흡광도 및 형광강도 분석<Example 1> Analysis of absorbance and fluorescence intensity of synthetic compounds
다양한 극성을 갖는 용매 즉, 톨루엔에서 물까지의 용매를 이용하여 Dye1의 흡광도 및 형광강도를 측정하였고, 도 1과 같이 Dye1의 전하 이동은 폐쇄-고리 구조로 인해 무극성 용매에서는 차단된 반면, 에탄올과 같은 높은 극성을 갖는 용매에서는 Dye1의 폐쇄 고리 구조의 고리가 개열되어 결합다리가 증가하여 디에틸아닐린으로부터 인돌 염으로 전하 이동을 회복시켰다(반응식 2 참조).The absorbance and fluorescence intensity of Dye1 were measured using solvents with various polarities, that is, from toluene to water. In a solvent having the same high polarity, the closed ring structure of Dye1 was cleaved and the bond bridge increased, thereby restoring charge transfer from diethylaniline to the indole salt (refer to Scheme 2).
[반응식 2][Scheme 2]
그 결과, 도 1a과 같이 흡광도는 328 nm에서 점차적으로 감소하지만, 549 nm의 긴 파장에서는 증가하였다. 유사하게, 도 1b와 같이 무극성 용매에서 552 nm의 여기파장에서 Dye1에 대한 어떠한 형광 강도가 없었지만, 극성이 높은 환경에서 573 nm에서 강한 형광이 발생하였다.As a result, as shown in FIG. 1A , the absorbance gradually decreased at 328 nm, but increased at a long wavelength of 549 nm. Similarly, there was no fluorescence intensity for Dye1 at an excitation wavelength of 552 nm in a non-polar solvent as shown in FIG. 1B, but strong fluorescence occurred at 573 nm in a highly polar environment.
한편, Dye2는 Dye1의 하이드록시기 위치에 메틸기를 도입하여 고리화 반응을 억제하기 때문에 극성과 상관없이 모든 용매에서 대략 550 nm에서 강한 흡광도를 나타내며, 328 nm의 파장에서는 어떠한 흡광도도 나타내지 않았고, 552 nm 여기 소스에서 Dye2의 형광강도는 모든 용매에서 580 nm에서 관찰되었다(도 2 참조).On the other hand, Dye2 exhibits strong absorbance at approximately 550 nm in all solvents regardless of polarity because it inhibits the cyclization reaction by introducing a methyl group at the hydroxyl group position of Dye1, and does not show any absorbance at a wavelength of 328 nm, 552 The fluorescence intensity of Dye2 in the nm excitation source was observed at 580 nm in all solvents (see Fig. 2).
Dye1의 분자간 고리화와 개열이 용매 극성에 따라 변화하여 역전되는지 확인하기 위해 Dye1을 메탄올과 THF 용매에 5회 첨가하여 형광 스펙트럼을 측정하였고, 그 결과 도 1c와 도 1d와 같이 Dye1의 고리화와 개열이 용매 극성의 변화에 따라 역전되는 것을 확인하였다.To confirm that the intermolecular cyclization and cleavage of Dye1 is reversed by changing depending on the solvent polarity, the fluorescence spectrum was measured by adding Dye1 to methanol and THF solvent 5 times. It was confirmed that the cleavage was reversed with the change of solvent polarity.
또한, 도 3은 CDCl3, CDCl3 : CD3OD = 1 : 1 (v/v) 및 CD3OD : D2O = 1 : 1 (v/v) 용매 상에서 Dye1의 1H NMR 스펙트럼 (δ 0.0-4.0 영역)을 나타낸 것으로, 극성 및 무극성 용매 상에서 Dye1의 고리화-개열 시스템을 확인할 수 있었다.In addition, FIG. 3 is a 1 H NMR spectrum (δ ) of Dye1 on CDCl 3 , CDCl 3 : CD 3 OD = 1:1 (v/v) and CD 3 OD: D 2 O = 1:1 (v/v) solvents (δ 0.0-4.0 region), confirming the cyclization-cleavage system of Dye1 in polar and non-polar solvents.
한편, 벤조티아디아졸 유도체인 Dye3는 Dye1과 유사한 흡수 파장을 가지고 있지만, 더 큰 스토크 쉬프트(stoke shift)로 인해 근적외선 영역의 긴 파장 영역에서 방출된 형광이 2개 염료 사이의 형광 간섭을 최소화하였다. Dye1과 달리, Dye3는 용매 극성이 증가할수록 형광 강도가 감소하는 턴오프 프로브이다(도 5 참조).On the other hand, Dye3, a benzothiadiazole derivative, has an absorption wavelength similar to that of Dye1, but due to a larger Stoke shift, fluorescence emitted in a long wavelength region of the near-infrared region minimized fluorescence interference between the two dyes. . Unlike Dye1, Dye3 is a turn-off probe whose fluorescence intensity decreases with increasing solvent polarity (see Fig. 5).
그리고, Dye1과 Dye3를 피페라진 링커로 연결시켜 환경적 극성에 따라 반대로 반응하는 2개 염료를 결합시킨 비율계량 극성 형광프로브인 RPS-1은 도 6a와 같이 Dye3에 근거하여 톨루엔과 에테르와 같은 무극성 용매에서 510 nm에서 약한 흡광도를 나타내는 반면, Dye1에 근거하여 메탄올과 물과 같은 극성 용매에서 550 nm에서 강한 흡광도를 나타내었다.And, RPS-1, a ratiometric polar fluorescent probe in which two dyes that react oppositely depending on environmental polarity are combined by connecting Dye1 and Dye3 with a piperazine linker, based on Dye3 as shown in FIG. 6a, non-polar such as toluene and ether The solvent showed weak absorbance at 510 nm, while strong absorbance at 550 nm was shown in polar solvents such as methanol and water based on Dye1.
또한, 도 6b와 같이 극성이 증가함에 따라 무극성 용매에서 650 nm 근처 형광 강도가 점차적으로 감소하고 580 nm 근처 형광이 증가하였다. 비율 형광 측정을 위한 2개 영역을 565-585 nm (Fyellow)과 630-680 nm (Fred)로 정의하였고 Fyellow/Fred 형광 비율과 용매 극성 파라미터인 EN T 값 간의 높은 상관관계(R2 = 0.993)가 확인되었다(도 6c). 게다가 RPS-1은 용매 극성에 따라 Fyellow/Fred 형광 비율이 가역적 변화를 나타내어 Dye1과 유사한 거동을 나타내었다(도 6d).In addition, as the polarity increased, as shown in FIG. 6b, the fluorescence intensity near 650 nm gradually decreased and the fluorescence near 580 nm increased in the non-polar solvent. Ratio was defined two regions for fluorescence measurements with 565-585 nm (yellow F) and 630-680 nm (red F) F yellow / red phosphorus F E N T fluorescence ratio as a solvent polarity parameter A high correlation between the values (R 2 =0.993) was confirmed (Fig. 6c). In addition, RPS-1 exhibited a reversible change in the F yellow / F red fluorescence ratio according to the solvent polarity, showing a similar behavior to that of Dye1 (Fig. 6d).
<실시예 2> 공동 국재화 시험<Example 2> Co-localization test
합성한 형광프로브를 HeLa 세포와 30분 동안 배양하였고, 552 nm 여기 파장 하에서 Dye1과 Dye2는 밝은 형광을 방출하지만 각 형광프로브의 세포내 위치는 달랐다. 도 4와 같이, Dye1은 세포 내 특정 소포를 염색하는 반면, Dye2는 미토콘드리아일 것으로 추정되는 세포의 넓은 영역을 염색하였다. The synthesized fluorescent probe was incubated with HeLa cells for 30 min. Under the 552 nm excitation wavelength, Dye1 and Dye2 emitted bright fluorescence, but the intracellular location of each fluorescent probe was different. As shown in FIG. 4 , Dye1 stained specific intracellular vesicles, whereas Dye2 stained a large area of cells presumed to be mitochondria.
도 3a와 같이 Dye1은 피어슨 계수값이 0.94로 의미있게 중첩되어 Dye1 프로브가 위치하는 소기관이 리소좀임을 제안하는 반면, 도 3b와 같이 Dye2는 LysoTracker Green과 중첩되지 않아 LysoTracker와 낮은 피어슨 계수 값을 나타내며 대신 MitoTracker Green과 높은 중첩을 나타내었다. 이러한 결과로부터, 리소좀 극성이 다른 세포내 소기관에 비해 특히 높은 것으로 확인되었다.As shown in Fig. 3a, Dye1 significantly overlaps with a Pearson coefficient value of 0.94, suggesting that the organelle in which the Dye1 probe is located is a lysosome, whereas, as shown in Fig. 3b, Dye2 does not overlap with LysoTracker Green, indicating a low Pearson coefficient value with LysoTracker. It showed high overlap with MitoTracker Green. From these results, it was confirmed that the lysosomal polarity was particularly high compared to other intracellular organelles.
또한, 도 8과 같이 RPS-1의 세포내 극성 이미지는 다양한 상업적 소기관 마커를 이용한 공동 국재화 시험을 통해 확인되었다. 실시예 3에서 확인된 비율계량 이미지에서 가장 높은 극성을 갖는 붉은 영역은 리소좀과 중첩된 반면, 가장 낮은 극성을 갖는 파란 영역은 지방방울 마커와 중첩되었다. 중간정도 극성을 갖는 녹색 영역은 미토콘드리아와 ER 마커와 중첩되어 이 영역을 세포질로 정의하였다. In addition, as shown in FIG. 8 , the intracellular polarity image of RPS-1 was confirmed through a co-localization test using various commercial organelle markers. In the ratiometric image identified in Example 3, the red region with the highest polarity overlapped with the lysosome, while the blue region with the lowest polarity overlapped with the fat droplet marker. The green region with moderate polarity overlapped with the mitochondrial and ER markers, defining this region as the cytoplasm.
<실시예 3> 비율계량 이미지 세포 이미지<Example 3> Ratiometric image Cell image
RPS-1이 세포 내 다양한 구획에서 극성 차이를 반영할 수 있는지 확인하기 위하여, Fyellow 및 Fred의 2가지 채널을 이용하여 비율계량 이미지를 얻었다. 그 결과, 도 7a와 도 7b와 같이 30분 동안 RPS-1 (3 μM)로 표지한 HeLa 세포에서 Fyellow 및 Fred의 형광 사이에 분명한 차이가 나타났다. Dye1의 세포 이미지와 유사하게, Fyellow 채널은 세포 내 특정 소포에서 강한 형광을 나타내었지만, Fred 채널의 형광은 동일한 Fyellow 소포 외 다른 소포에서도 관찰되어 세포 전체에서 가닥 형성으로 염색되었다. Fyellow 및 Fred의 형광 이미지를 유사색 비율계량 이미지(Fyellow/Fred)로 처리하였을 때 세포 각 영역의 극성 분포가 한 눈에 확인될 수 있을 것이다(도 7c). In order to confirm whether RPS-1 can reflect the polarity difference in various compartments in the cell, ratiometric images were obtained using two channels of F yellow and F red. As a result, there was a clear difference between the fluorescence of F yellow and F red in HeLa cells labeled with RPS-1 (3 μM) for 30 minutes as shown in FIGS. 7A and 7B . Similar to the cell image of Dye1, F yellow The channel showed strong fluorescence in specific intracellular vesicles, but the fluorescence of the F red channel was observed in other vesicles other than the same F yellow vesicles, and was stained with strand formation throughout the cell. When the fluorescence images of F yellow and F red were treated with a similar color ratiometric image (F yellow /F red ), the polarity distribution of each cell region could be confirmed at a glance (FIG. 7c).
도 7d 및 도 7e와 같이, 확대 이미지에서 리소좀, 세포질 및 지방 방울 영역을 보다 명확하게 구분되며, 비율계량 이미지에서 파란색 지방 방울은 정확하게 명-시야 이미지에서 시각적으로 관찰할 수 있는 지방 방울의 영역과 일치하였다.7d and 7e, the lysosome, cytoplasm, and fat droplet regions are more clearly distinguished in the enlarged image, and the blue fat droplet in the ratiometric image accurately corresponds to the visually observable area of the fat droplet in the bright-field image. matched.
HeLa 세포 외 Chang, Huh7 및 SW837와 같은 다양한 세포에서 세포 내 극성 분포를 관찰한 결과, 도 9와 같이 리소좀의 극성이 모든 세포에서 가장 높게 관찰되었다. Chang, Huh7, SW837 및 HeLa 세포에서의 Fyellow/Fred 형광 비율은 각각 1.9, 1.7, 1.9 및 1.7이었다. 모든 세포에서 리소좀의 극성은 메탄올과 물의 극성값 사이의 값과 같이 높았으나, 지방 방울은 다른 소기관에 비해 더 낮은 극성을 나타내었다. As a result of observing intracellular polarity distribution in various cells such as HeLa extracellular Chang, Huh7 and SW837, the lysosome polarity was highest in all cells as shown in FIG. 9 . The F yellow /F red fluorescence ratios in Chang, Huh7, SW837 and HeLa cells were 1.9, 1.7, 1.9 and 1.7, respectively. In all cells, the polarity of the lysosome was as high as the value between the polarity values of methanol and water, but the fat droplet showed a lower polarity compared to other organelles.
이러한 결과로부터 세포 내 극성은 이질적이며, 각 소기관의 극성은 리소좀에서 세포질, 지방 방울로 갈수록 점차적으로 감소하는 것을 확인할 수 있었다.From these results, it was confirmed that the intracellular polarity is heterogeneous, and the polarity of each organelle gradually decreases from the lysosome to the cytoplasm and the fat droplets.
<실시예 4> 세포생존율 분석<Example 4> Cell viability analysis
MTT 분석을 통한 세포생존율 실험 결과, 도 10과 같이 합성된 형광프로브들은 HeLa 세포의 생존율에 영향을 미치지 않아 세포독성이 없는 것으로 확인되었다.As a result of the cell viability experiment through the MTT analysis, it was confirmed that the fluorescent probes synthesized as shown in FIG. 10 did not affect the viability of HeLa cells and thus had no cytotoxicity.
<실시예 5> 선택성 분석<Example 5> Selectivity analysis
다양한 물질과 함께 존재하는 RPS-1의 형광 강도 비율(Fyellow/Fred)을 분석하여 RPS-1의 선택성을 확인하였다. 그 결과, 도 11과 같이 RPS-1의 형광 강도 비율(Fyellow/Fred)은 다른 방해 물질이 존재해도 방해받지 않고 분석이 잘 되었다 [(1) control; 200 μM, (2) TBHP, (3) O2-, (4) ·OH, (5) ·OtBu, (6) H2O2, (7) NO·, (8) ONOO-; 1 mM, (9) Lys, (10) Arg, (11) His, (12) Asp, (13) Glu, (14) glucose, (15) GSH; 1 unit mL-1, (16) amidase, (17) NTR, (18) ALP, (19) CE1, (20) CE2, (21) NQO1, 이때 여기파장은 552 nm].The selectivity of RPS-1 was confirmed by analyzing the fluorescence intensity ratio (F yellow /F red ) of RPS-1 present together with various substances. As a result, as shown in FIG. 11, the fluorescence intensity ratio (F yellow /F red ) of RPS-1 was well analyzed without being disturbed even in the presence of other interfering substances [(1) control; 200 μM, (2) TBHP, (3) O 2 - , (4) ·OH, (5) · OtBu, (6) H 2 O 2 , (7) NO ·, (8) ONOO − ; 1 mM, (9) Lys, (10) Arg, (11) His, (12) Asp, (13) Glu, (14) glucose, (15) GSH; 1 unit mL -1 , (16) amidase, (17) NTR, (18) ALP, (19) CE1, (20) CE2, (21) NQO1, where the excitation wavelength is 552 nm].
<실시예 6> 광안정성 분석<Example 6> Analysis of photostability
RPS-1의 광안정성을 조사 전과 60분 조사 후 RPS-1의 형광 이미지와(도 12a), xyt 모드를 이용하여 60분 동안 2.00s 간격으로 시간 함수로 상대적인 형광 강도(도 12b)를 분석하였고, 그 결과 도 12와 같이 60분 동안 형광 강도가 변화하지 않아 RPS-1는 높은 광안정성을 나타내었다.The light stability of RPS-1 was analyzed before and after 60 minutes of irradiation with the fluorescence image of RPS-1 (Fig. 12a), and the relative fluorescence intensity (Fig. 12b) as a function of time at intervals of 2.00 s for 60 minutes using xyt mode. , As a result, as shown in FIG. 12, the fluorescence intensity did not change for 60 minutes, so RPS-1 exhibited high photostability.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
Claims (17)
[화학식 1]
[Formula 1]
[화학식 2]
[Formula 2]
[화학식 3]
[Formula 3]
상기 형광프로브가 처리된 시료에 여기광을 조사하는 단계; 및
상기 여기광이 조사된 시료에서 발생하는 색의 강도 및 변화를 측정하여 비율계량 형광을 분석하는 단계를 포함하는 생체 시료의 극성 유무 분석방법.processing the fluorescent probe according to claim 12 on a biological sample isolated from a human;
irradiating excitation light to the sample treated with the fluorescent probe; and
and analyzing the ratiometric fluorescence by measuring the intensity and change of color generated in the sample irradiated with the excitation light.
[화학식 1]
[Formula 1]
A pharmaceutical composition for intracellular fat droplet delivery comprising the compound according to claim 6.
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