KR102112719B1 - Fluorescence Compounds and Preparation Method Therof - Google Patents

Fluorescence Compounds and Preparation Method Therof Download PDF

Info

Publication number
KR102112719B1
KR102112719B1 KR1020170135622A KR20170135622A KR102112719B1 KR 102112719 B1 KR102112719 B1 KR 102112719B1 KR 1020170135622 A KR1020170135622 A KR 1020170135622A KR 20170135622 A KR20170135622 A KR 20170135622A KR 102112719 B1 KR102112719 B1 KR 102112719B1
Authority
KR
South Korea
Prior art keywords
compound
formula
fluorescence
biomolecule
mmol
Prior art date
Application number
KR1020170135622A
Other languages
Korean (ko)
Other versions
KR20190043711A (en
Inventor
박진우
김기원
신경림
Original Assignee
(주)바이오액츠
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by (주)바이오액츠 filed Critical (주)바이오액츠
Priority to KR1020170135622A priority Critical patent/KR102112719B1/en
Publication of KR20190043711A publication Critical patent/KR20190043711A/en
Application granted granted Critical
Publication of KR102112719B1 publication Critical patent/KR102112719B1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/12—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains three hetero rings
    • C07D491/14—Ortho-condensed systems
    • C07D491/147—Ortho-condensed systems the condensed system containing one ring with oxygen as ring hetero atom and two rings with nitrogen as ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Materials Engineering (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Plural Heterocyclic Compounds (AREA)

Abstract

본 발명의 목적은 형광강도, 상대양자효율, 표지율 면에서 우수하여 조영제 조성물로도 이용될 수 있는 형광 화합물, 이를 이용한 표지방법 등을 제공하고자 한다. 본 발명에 따른 형광 화합물은 형광강도, 상대양자효율, 표지율 면에서 우수하여 타겟 물질의 표지 및 염색에 보다 효과적으로 활용될 수 있다. 또한, 광학 안정성이 우수하여 장시간의 염색에도 안정적 형광을 나타내며, 체내에 투여시 축적되지 않으면서도 형광 강도가 우수하여 종래의 염료에 비하여 소량의 사용에도 염색 및 체내 영상화가 용이하여 경제적으로 이용이 가능하다.An object of the present invention is to provide a fluorescent compound, a labeling method using the same, which is excellent in fluorescence intensity, relative quantum efficiency, and labeling rate and can be used as a contrast agent composition. The fluorescent compound according to the present invention is excellent in terms of fluorescence intensity, relative quantum efficiency, and labeling rate, and thus can be more effectively used for labeling and dyeing a target material. In addition, it has excellent optical stability and shows stable fluorescence even for long-term dyeing, and it has excellent fluorescence intensity without accumulating when administered in the body, so it is easy to dye and image in the body even with a small amount of use compared to conventional dyes. Do.

Description

형광 화합물 및 이의 제조방법{Fluorescence Compounds and Preparation Method Therof}Fluorescence Compounds and Preparation Method Therof

본 발명은 형광 화합물에 관한 것이다.The present invention relates to fluorescent compounds.

생체 물질 자체는 가시광 및 근적외 영역의 형광이 미약하거나 없으므로 바이오 분야에서는 생체 내/외에서 세포 및 세포 이하 단계에서의 생물학적인 현상을 관찰하거나 생체 내로 투영되어 조영 및 질환부위의 광학 영상을 얻기 위하여 생체 물질에 형광 염료 또는 형광 염료가 미리 표지된 특정 생체 물질을 광학장비와 함께 활용하는 다양한 기법을 통해 영상화한 자료를 얻고 있다.Since the biological material itself has little or no fluorescence in the visible and near-infrared regions, in the bio field, in order to observe biological phenomena in the cells and sub-cell stages in vivo / externally or to project them in vivo, to obtain optical images of contrast and disease sites Imaging data is obtained through various techniques that utilize fluorescent dyes or specific biomaterials pre-labeled with fluorescent dyes with optical equipment.

바이오 분야에서 사용되는 다양한 광학 분석(optical anylsis) 장비들은 내장된 광원 및 필터에 따라 형광을 관찰하기에 적합한 여기 파장(excitation wavelength) 및 형광 파장(emission wavelength)를 가진 형광 염료를 기본 소재나 시약으로 선택하게 된다.Various optical analysis equipments used in the bio field are fluorescent dyes with excitation wavelength and emission wavelength suitable for fluorescence observation according to built-in light sources and filters as basic materials or reagents. You will choose.

주로 사용되는 광학 분석 장비로는 세포 관찰을 위한 형광현미경(fluorescece microscope), 공초점현미경(confocal microscope), 유세포분석기(flowcytometer), 마이크로어레이(microarray), 정량 중합효소연쇄반응 장치(qualitative PCR system), 핵산 및 단백질 분리, 분석을 위한 전기영동(electrophoresis) 장치, 실시간 생체내 영상 장비(in vivo imaging system) 등 연구 목적의 장비 외에도, 면역 분석 기법(immnuno assay)이나 PCR 분석 및 통계 기술이 접목된 핵산 및 단백질 진단 키트(또는 바이오칩) 기반 체외 진단(in vitro diagnosis) 장비와 의료 영상 수술(image-guided surgery)을 위한 수술대 및 내시경 장비 등의 진단 및 치료를 위한 것들이 알려져 있으며, 지속적으로 새로운 응용 분야 및 더 높은 수준의 해상도 및 데이터 처리 능력을 가진 장비가 개발되고 있다.Mainly used optical analysis equipment include fluorescent microscopes for cell observation, confocal microscopes, flowcytometers, microarrays, and quantitative PCR systems. In addition to equipment for research purposes, such as nucleic acid and protein separation, electrophoresis device for analysis, real-time in vivo imaging system, immununo assay or PCR analysis and statistical technology Nucleic acid and protein diagnostic kits (or biochips) based in vitro diagnostic equipment and medical imaging surgery (image-guided surgery), such as operating table and endoscopic equipment for diagnosis and treatment are known, constantly new applications And equipment with higher levels of resolution and data processing capabilities.

일반적으로 단백질 또는 펩타이드 등 생체 분자의 표지를 위해 사용되는 형광염료(fluorescent dye)는 대부분 안트라닐레이트(anthranilate), 1-알킬틱 이소인돌(1-alkylthic isoindoles), 피롤리논(pyrrolinones), 비메인(bimanes), 벤즈옥사졸(benzoxazole), 벤즈이미다졸(benzimidazole), 벤조퓨란(benzofurazan), 나프탈렌(naphthalenes), 쿠마린(coumarins), 시아닌(cyanine), 스틸벤(stilbenes), 카바졸(carbazoles), 페난트리딘(phenanthridine), 안트라센(anthracenes), 보디피(bodipy), 플로세인(fluoresceins), 에오신(eosins), 로다민(rhodamines), 피렌(pyrenes), 크리센(chrysenes) 및 아크리딘(acridines) 등의 구조가 포함되어 있다.Fluorescent dyes commonly used for labeling biomolecules such as proteins or peptides are mostly anthranilate, 1-alkylthic isoindoles, pyrrololinones, and ratios. Mains (bimanes), benzoxazole, benzimidazole, benzofurazan, naphthalenes, coumarins, cyanines, stilbenes, carbazoles ), Phenanthridine, anthracenes, bodipy, fluoresceins, eosins, rhodamines, pyrenes, chrysenes and acres Includes structures such as acridines.

상기에서 예시한 다수의 형광 발색단 중에서 바이오 분야에서 이용 가능한 형광 염료 구조를 선별하는 경우, 일반적으로는 대부분의 생체 분자들이 존재하는 매질, 즉, 수용액 및 수용성 버퍼 내에 존재할 때 강한 형광을 내는 것과 형광 장비에 맞는 여기 및 형광 파장을 갖는 것이 중요하다.When selecting the fluorescent dye structure that can be used in the bio field among the plurality of fluorescent chromophores exemplified above, in general, a medium in which most biomolecules are present, that is, a strong fluorescence when present in an aqueous solution and an aqueous buffer, and a fluorescent device It is important to have a suitable excitation and fluorescence wavelength.

바이오 분야에서 주로 적용될 수 있는 염료는 가급적 수용액이나 친수성 조건에서 광표백(photobleaching) 및 소광(quenching) 현상이 적고, 다량의 빛을 흡수할 수 있도록 몰흡광계수(molecular extinction coefficient)가 커야 하며, 생체 분자 자체의 형광 범위와 멀리 떨어진 500 nm 이상의 가시광선 영역이나 근적외선 영역에 있어야 하고, 다양한 pH 조건에서 안정하여야 하나, 상기 제한 사항을 만족할 수 있는 생체 분자 표지용으로 사용 가능한 염료의 구조는 한정되어 있다.Dye that can be mainly applied in the bio field has as little photobleaching and quenching as possible in aqueous solution or hydrophilic condition, and must have a large molecular extinction coefficient to absorb a large amount of light. It should be in the visible or near infrared region of 500 nm or more away from its fluorescence range, but must be stable under various pH conditions, but the structure of the dye that can be used for biomolecule labeling that can satisfy the above limitation is limited.

이러한 요구 조건에 부합하는 형광 색원체로는 시아닌, 로다민, 플로세인, 보디피, 쿠마린, 아크리딘, 피렌 유도체들이 있는데, 염료 단독 또는 생체 분자 구조 내의 특정 치환기와 결합이 가능하도록 반응기를 도입시키기도 하며, 그 중 잔텐(xanthane) 계열의 플로세인 및 로다민과, 폴리메틴(polymethine) 계열의 시아닌 유도체 염료 화합물들이 주로 상품화되어 있다.Fluorescent chromogens meeting these requirements include cyanine, rhodamine, flocein, vodpi, coumarin, acridine, and pyrene derivatives. Introducing the reactor to allow dyes alone or to bind to specific substituents within the biomolecular structure Among them, xanthane-based flocein and rhodamine, and polymethine-based cyanine derivative dye compounds are mainly commercialized.

특히 시아닌 발색단을 가진 염료 화합물은 다양한 흡수/여기 파장의 화합물을 합성하기 용이하다는 장점 외에도, 일반적으로 광학 및 pH 안정성이 탁월하고, 좁은 흡수 및 발광 파장 범위를 가지며, 500 내지 800 nm의 형광 영역을 갖기 때문에 생체 분자의 자체 형광 영역과 중첩되지 않아 분석이 용이하며, 용매 및 용해도 특성에 따라 다소 차이는 있지만, 높은 몰흡광계수를 나타내는 등 많은 장점이 있어 생물학적 응용에 많이 이용된다.In particular, dye compounds having a cyanine chromophore, in addition to the advantage of being easy to synthesize compounds of various absorption / excitation wavelengths, generally have excellent optical and pH stability, have a narrow absorption and emission wavelength range, and provide a fluorescence region of 500 to 800 nm. Because it does not overlap with the self-fluorescence region of the biomolecule, it is easy to analyze, and although there are some differences depending on the solvent and solubility characteristics, it has many advantages such as exhibiting a high molar extinction coefficient, so it is widely used in biological applications.

그 이외에도, 시아닌 발색단을 가진 염료 화합물은 화상표시장치용 광학필터나 레이저 용착용 수지 조성물의 용도로 유용하게 이용될 수도 있다. 특정한 광에 강도가 큰 흡수를 가지는 화합물은 액정표시장치, 플라즈마 디스플레이 패널, 전계발광디스플레이, 음극관 표시장치, 형광 표시관 등의 화상표시장치용 광학필터나 DVDㅁR 등의 광학 기록 매체의 광학 요소로서 널리 이용되고 있다. 광학 필터에는 불필요한 파장의 광들을 선택적으로 흡수하는 기능이 요구되는데, 동시에 형광등 등의 외광의 반사나 글레어를 방지하기 위해서는 480~500 nm 및 540~560 nm의 파장광 흡수가 요구되며, 화상품질을 높이기 위해서는 근적외선의 파장을 선택적으로 흡수하는 기능이 요구되고 있다.In addition to this, the dye compound having a cyanine chromophore may be usefully used as an optical filter for an image display device or a resin composition for laser welding. Compounds with high absorption of specific light intensity include liquid crystal display devices, plasma display panels, electroluminescent displays, cathode ray tube displays, optical filters for image display devices such as fluorescent tubes, and optical elements of optical recording media such as DVD ㅁ R It is widely used as. The optical filter is required to selectively absorb unnecessary wavelengths of light. At the same time, it is required to absorb wavelengths of 480 to 500 nm and 540 to 560 nm to prevent reflection or glare of external light such as fluorescent lamps. In order to increase, a function of selectively absorbing the wavelength of near infrared rays is required.

상기와 같이, 산업적으로 유용하게 적용하기 위해서는 광학 및 pH 안정성이 우수하면서도 특정 파장 범위에서 좁은 흡수/발광 파장 범위를 가지면서도 높은 몰흡광계수를 나타내는 신규한 염료의 개발이 지속적으로 요구되는 바이다.As described above, in order to be useful in industrial applications, it is necessary to continuously develop new dyes having excellent optical and pH stability, but having a narrow absorption / emission wavelength range in a specific wavelength range, and exhibiting a high molar absorption coefficient.

한국 공개특허 제10-2010-0094034호Korean Patent Publication No. 10-2010-0094034 한국 공개특허 제10-2013-0005381호Korean Patent Publication No. 10-2013-0005381 한국 공개특허 제10-2011-0136367호Korean Patent Publication No. 10-2011-0136367 한국 공개특허 제10-2011-0122314호Korean Patent Publication No. 10-2011-0122314

본 발명의 목적은 형광강도, 상대양자효율, 표지율 면에서 우수하여 조영제 조성물로도 이용될 수 있는 형광 화합물, 이를 이용한 표지방법 등을 제공하고자 한다.An object of the present invention is to provide a fluorescent compound, a labeling method using the same, which is excellent in fluorescence intensity, relative quantum efficiency, and labeling rate and can be used as a contrast agent composition.

이러한 과제를 달성하기 위하여, 본 발명은 하기 화학식 1로 표시되는 화합물을 제공한다.In order to achieve this problem, the present invention provides a compound represented by the formula (1).

[화학식 1][Formula 1]

Figure 112017103017497-pat00001
Figure 112017103017497-pat00001

상기 A1 및 상기 A2는 서로 동일하거나 상이하고, 각각 독립적으로 SO3 -이거나 SO3H이며,A 1 and A 2 are the same as or different from each other, and each independently is SO 3 - or SO 3 H,

상기 A3은 CO2 -이거나 CO2H이고,A 3 is CO 2 - or CO 2 H,

상기 Z는 OH이거나 NH(CH2)mNH-Q이며,Z is OH or NH (CH 2 ) m NH-Q,

상기 m은 1 내지 5이고,M is 1 to 5,

상기 Q는 하기 화학식의 구조를 가지며,Q has the structure of the following formula,

[화학식 1a][Formula 1a]

Figure 112017103017497-pat00002
Figure 112017103017497-pat00002

상기 X는 할라이드이고,X is a halide,

상기 Y는 NH(CH2)nCO2-LY is NH (CH 2 ) n CO 2 -L

상기 n은 3 내지 7이고,N is 3 to 7,

상기 L은 H이거나 하기 화학식의 구조를 가진다.L is H or has the structure of the formula

[화학식 1b][Formula 1b]

Figure 112017103017497-pat00003
Figure 112017103017497-pat00003

또한, 본 발명은 본 발명의 여러 구현예에 따른 화합물을 포함하는 형광 표지용 조성물을 제공한다.In addition, the present invention provides a composition for a fluorescent label comprising a compound according to various embodiments of the present invention.

또한, 본 발명은 본 발명의 여러 구현예에 따른 화합물과 표지 대상 물질과 접촉시키는 단계를 포함하는 형광 표지 방법을 제공한다. In addition, the present invention provides a fluorescent labeling method comprising the step of contacting a compound according to various embodiments of the present invention with a target substance for labeling.

본 발명에 따른 형광 화합물은 형광강도, 상대양자효율, 표지율 면에서 우수하여 타겟 물질의 표지 및 염색에 보다 효과적으로 활용될 수 있다. 또한, 광학 안정성이 우수하여 장시간의 염색에도 안정적 형광을 나타내며, 체내에 투여시 축적되지 않으면서도 형광 강도가 우수하여 종래의 염료에 비하여 소량의 사용에도 염색 및 체내 영상화가 용이하여 경제적으로 이용이 가능하다.The fluorescent compound according to the present invention is excellent in terms of fluorescence intensity, relative quantum efficiency, and labeling rate, and thus can be more effectively used for labeling and dyeing a target material. In addition, it has excellent optical stability and shows stable fluorescence even for long-term dyeing, and it has excellent fluorescence intensity without accumulating when administered in the body, so it is easy to dye and image in the body even with a small amount of use compared to conventional dyes. Do.

도 1은 화합물 1-3과 대조형광염료의 형광강도를 비교한 그래프이다.
도 2는 화합물 1-3과 대조형광염료의 상대양자효율을 비교한 그래프이다.
도 3은 화합물 1-3과 대조형광염료를 사용하여 단백질 반응물 간 형광강도를 비교한 그래프이다.
도 4는 FOBI 분석 결과 이미지이다.
도 5a 내지 5c는 Plate reader 측정 결과를 보여주는 그래프이다.1 is a graph comparing the fluorescence intensities of Compound 1-3 and the control fluorescent dye.
2 is a graph comparing the relative quantum efficiency of compound 1-3 and the control fluorescent dye.
3 is a graph comparing fluorescence intensities between protein reactants using Compound 1-3 and a control fluorescent dye.
4 is an image of FOBI analysis results.
5A to 5C are graphs showing plate reader measurement results.

이하에서, 본 발명의 여러 측면 및 다양한 구현예에 대해 더욱 구체적으로 살펴보도록 한다.Hereinafter, various aspects and various embodiments of the present invention will be described in more detail.

본 발명의 일 측면은 하기 화학식 1로 표시되는 화합물에 관한 것이다.One aspect of the present invention relates to a compound represented by Formula 1 below.

[화학식 1][Formula 1]

Figure 112017103017497-pat00004
Figure 112017103017497-pat00004

상기 A1 및 상기 A2는 서로 동일하거나 상이하고, 각각 독립적으로 SO3 -이거나 SO3H이며,A 1 and A 2 are the same as or different from each other, and each independently is SO 3 - or SO 3 H,

상기 A3은 CO2 -이거나 CO2H이고,A 3 is CO 2 - or CO 2 H,

상기 Z는 OH이거나 NH(CH2)mNH-Q이며,Z is OH or NH (CH 2 ) m NH-Q,

상기 m은 1 내지 5이고,M is 1 to 5,

상기 Q는 하기 화학식의 구조를 가지며,Q has the structure of the following formula,

[화학식 1a][Formula 1a]

Figure 112017103017497-pat00005
Figure 112017103017497-pat00005

상기 X는 할라이드이고,X is a halide,

상기 Y는 NH(CH2)nCO2-LY is NH (CH 2 ) n CO 2 -L

상기 n은 3 내지 7이고,N is 3 to 7,

상기 L은 H이거나 하기 화학식의 구조를 가진다.L is H or has the structure of the formula

[화학식 1b][Formula 1b]

Figure 112017103017497-pat00006
Figure 112017103017497-pat00006

일 구현예에 있어서, 제1항에 있어서, 상기 m은 2 또는 3이고, 상기 X는 Cl 또는 F이며, 상기 n은 5이다.In one embodiment, the m is 2 or 3, the X is Cl or F, and the n is 5.

다른 구현예에 있어서, 상기 형광 화합물은 하기 화합물 중 하나이다.In other embodiments, the fluorescent compound is one of the following compounds.

[화학식 2a][Formula 2a]

Figure 112017103017497-pat00007
Figure 112017103017497-pat00007

[화학식 2b][Formula 2b]

Figure 112020004547499-pat00040
Figure 112020004547499-pat00040

[화학식 2c][Formula 2c]

Figure 112020004547499-pat00041
Figure 112020004547499-pat00041

[화학식 2d][Formula 2d]

Figure 112020004547499-pat00042
Figure 112020004547499-pat00042

본 발명의 다른 측면은 본 발명의 여러 구현예에 따른 화합물을 포함하는 형광 표지용 조성물에 관한 것이다.Another aspect of the present invention relates to a composition for a fluorescent label comprising a compound according to various embodiments of the present invention.

이때, 상기 표지의 대상 물질은 섬유, 생체분자, 나노입자, 유기화합물 또는 이들 2종 이상의 조합일 수 있다.At this time, the target material of the label may be fibers, biomolecules, nanoparticles, organic compounds, or a combination of two or more of these.

또한, 상기 생체분자의 예에는 단백질, 펩타이드, 탄수화물, 당, 지방, 항체, 프로테오글라이칸, 글라이코프로틴 및 siRNA 등이 포함되나, 이에 한정되지 않는다.Further, examples of the biomolecule include, but are not limited to, protein, peptide, carbohydrate, sugar, fat, antibody, proteoglycan, glycoprotein and siRNA.

본 발명의 또 다른 측면은 본 발명의 다양한 구현예에 따른 화합물과 표지 대상 물질과 접촉시키는 단계를 포함하는 형광 표지 방법에 관한 것이다.Another aspect of the invention relates to a fluorescent labeling method comprising the step of contacting a compound according to various embodiments of the invention with a target substance for labeling.

이때, 상기 표지 대상 물질은 섬유, 생체분자, 나노입자, 유기화합물 또는 이들 2종 이상의 조합일 수 있다.At this time, the target material may be a fiber, biomolecule, nanoparticle, organic compound, or a combination of two or more of these.

일 구현예에 따르면, 상기 생체분자는 인체 외 생체분자이거나 또는 인간을 제외한 포유동물의 생체분자이다.According to one embodiment, the biomolecule is a biomolecule other than a human body or a mammalian biomolecule other than a human.

또한, 상기 생체분자의 예에는 단백질, 펩타이드, 탄수화물, 당, 지방, 항체, 프로테오글라이칸, 글라이코프로틴 및 siRNA 등이 포함되나, 이에 한정되지 않는다.
Further, examples of the biomolecule include, but are not limited to, proteins, peptides, carbohydrates, sugars, fats, antibodies, proteoglycans, glycoproteins and siRNAs.

이하에서 실시예 등을 통해 본 발명을 더욱 상세히 설명하고자 하며, 다만 이하에 실시예 등에 의해 본 발명의 범위와 내용이 축소되거나 제한되어 해석될 수 없다. 또한, 이하의 실시예를 포함한 본 발명의 개시 내용에 기초한다면, 구체적으로 실험 결과가 제시되지 않은 본 발명을 통상의 기술자가 용이하게 실시할 수 있음은 명백한 것이며, 이러한 변형 및 수정이 첨부된 특허청구범위에 속하는 것도 당연하다.Hereinafter, the present invention will be described in more detail through examples and the like, but the scope and content of the present invention may be reduced or limited by the following examples and the like and cannot be interpreted. In addition, if it is based on the disclosure of the present invention including the following examples, it is obvious that a person skilled in the art can easily carry out the present invention in which experimental results are not specifically presented, and patents to which such modifications and corrections are attached Naturally, it is within the scope of the claims.

또한 이하에서 제시되는 실험 결과는 상기 실시예 및 비교예의 대표적인 실험 결과만을 기재한 것이며, 아래에서 명시적으로 제시하지 않은 본 발명의 여러 구현예의 각각의 효과는 해당 부분에서 구체적으로 기재하도록 한다.In addition, the experimental results presented below are only representative of experimental results of the examples and comparative examples, and the effects of various embodiments of the present invention, which are not explicitly presented below, will be described in detail in the corresponding parts.

실시예Example

실시예 1 : 화합물 3-1의 합성Example 1: Synthesis of Compound 3-1

(1) 화합물 2-1의 합성(1) Synthesis of Compound 2-1

Figure 112017103017497-pat00011
Figure 112017103017497-pat00011

m-아니시딘(m-Anisidine) (24.6 g, 0.2 mol, 1 eq, Sigma-Aldrich)와 p-톨루엔술폰산(p-toluenesulfonic acid) 2.6 g을 사이클로헥산(cyclohexane) 200 mL에 넣고 분산하였다. 용액을 80 내지 90 ℃로 가열한 후 아세톤 42 mL을 반응액에 8 내지 10 시간 동안 세류한 후, 온도를 유지하며 일야교반을 진행하였다. 반응 완료 후 70 ℃로 냉각한 후 소다회 0.6 g을 증류수 20 mL에 녹여 반응액에 투입한 후 2 시간 동안 상온 교반하였다. 반응액을 증류수를 사용하여 추출한 후 유기층을 회수하여 감압 건조하였다. MC를 사용하여 실리카겔 크로마토그래피로 정제한 후 건조하여 화합물 2-1을 얻었다(19.2 g, 43%).m-Anisidine (24.6 g, 0.2 mol, 1 eq, Sigma-Aldrich) and 2.6 g of p-toluenesulfonic acid were added to 200 mL of cyclohexane and dispersed. After heating the solution to 80 to 90 ° C., 42 mL of acetone was washed in the reaction solution for 8 to 10 hours, followed by overnight stirring while maintaining the temperature. After the reaction was completed, after cooling to 70 ° C, 0.6 g of soda ash was dissolved in 20 mL of distilled water, added to the reaction solution, and stirred at room temperature for 2 hours. The reaction solution was extracted with distilled water, and then the organic layer was recovered and dried under reduced pressure. Purified by silica gel chromatography using MC and dried to obtain compound 2-1 (19.2 g, 43%).

Rf = 0.65 (실리카겔, MC)Rf = 0.65 (silica gel, MC)

(2) 화합물 2-2의 합성(2) Synthesis of Compound 2-2

Figure 112017103017497-pat00012
Figure 112017103017497-pat00012

화합물 2-1 16 g (78.3 mmol, 1 eq)를 아이오도메탄 (Iodomethane) (166 g, 1.174 mol, 15 eq)에 투입한 후, 12 시간 동안 가열환류 반응을 진행하였다. 반응 완료 후 감압건조하여 아이오도메탄을 제거하였다. MC를 사용하여 실리카겔 크로마토그래피로 정제한 후 건조하여 화합물 2-2를 얻었다(14.1 g, 83%).Compound 2-1 16 g (78.3 mmol, 1 eq) was added to Iodomethane (166 g, 1.174 mol, 15 eq), followed by heating to reflux for 12 hours. After completion of the reaction, iodomethane was removed by drying under reduced pressure. After purification by silica gel chromatography using MC and dried, compound 2-2 was obtained (14.1 g, 83%).

Rf = 0.8 (실리카겔, MC)Rf = 0.8 (silica gel, MC)

(3) 화합물 2-3의 합성(3) Synthesis of Compound 2-3

Figure 112017103017497-pat00013
Figure 112017103017497-pat00013

화합물 2-2 14.11 g (64.9 mmol)을 아세트산 50 mL와 브롬산 50 mL 혼합용액에 넣고 가열환류 반응을 진행하였다. 상온으로 냉각 후 MC 300 mL, 증류수 200 mL을 투입한 후 30 분 동안 교반하였다. 교반 후 30% 가성소다용액을 투입하여 중화하였다. 반응액을 추출하여 유기층을 회수한 후 감압건조 진행하였다(11.4 g, 86%).Compound 2-2 14.11 g (64.9 mmol) was added to a mixed solution of 50 mL of acetic acid and 50 mL of bromic acid, and a heating and reflux reaction was performed. After cooling to room temperature, 300 mL of MC and 200 mL of distilled water were added, followed by stirring for 30 minutes. After stirring, 30% caustic soda solution was added to neutralize it. The reaction solution was extracted and the organic layer was recovered, followed by drying under reduced pressure (11.4 g, 86%).

Rf = 0.2 (실리카겔, MC)Rf = 0.2 (silica gel, MC)

(4) 화합물 2-4의 합성(4) Synthesis of Compound 2-4

Figure 112017103017497-pat00014
Figure 112017103017497-pat00014

화합물 2-3 (11.4 g, 56.1 mmol, 2 eq)과 트리멜리틱 무수화물 (Trimellitic anhydride) (5.38 g, 28 mmol, 1 eq)과 p-톨루엔술폰산 (p-Toluenesulfonic acid) 1 g을 프로판산 (Propionic acid) 60 mL에 투입한 후, 12 시간 동안 가열환류 반응을 진행하였다. 반응 완료 후 감압건조하여 용매를 제거한 후, 실리카겔 크로마토그래피를 사용하여 정제를 진행하여 화합물 2-4를 얻었다(6.4 g, 41%).Compound 2-3 (11.4 g, 56.1 mmol, 2 eq) and trimellitic anhydride (5.38 g, 28 mmol, 1 eq) and 1 g of p-Toluenesulfonic acid (propanoic acid) (Propionic acid) was added to 60 mL, and then heated to reflux for 12 hours. After completion of the reaction, the mixture was dried under reduced pressure to remove the solvent, followed by purification using silica gel chromatography to obtain compound 2-4 (6.4 g, 41%).

Rf = 0.2 (RP-C18, 아세토니트릴/물 1:1 v/v)Rf = 0.2 (RP-C18, acetonitrile / water 1: 1 v / v)

LC/MS, 계산치 C35H35N2O5 563.66, 측정치 567.0LC / MS, calculated C35H35N2O5 563.66, found 567.0

(5) 화합물 3-1의 합성(5) Synthesis of Compound 3-1

Figure 112017103017497-pat00015
Figure 112017103017497-pat00015

3-13-1

화합물 2-4 (3 g, 5.32 mmol)에 냉각된 진한 황산 40 mL을 투입하였다. 0 ℃로 냉각한 후 2 시간 동안 반응한 후에 상온에서 12 시간 동안 반응을 진행하였다. 반응 완료 후 다이옥산 (Dioxane) 60 mL을 투입한 후, 디에틸이더(diethylether) 2 L를 투입하여 입자를 생성하였다. 뷰흐너 깔때기에 규조토를 넣고 여과한 후, 걸러진 고체를 물에 분산하여, 소듐 바이카보네이트 (Sodium bicarbonate)를 투입하여 중화하였다. 반응액을 뷰흐너 깔때기로 여과한 후 여액을 감압건조 진행하였다. 실리카겔 크로마토그래피를 사용하여 정제를 진행한 후 감압건조 진행하여 화합물 1을 얻었다(610 mg, 16%).To compound 2-4 (3 g, 5.32 mmol) was added 40 mL of concentrated concentrated sulfuric acid. After cooling to 0 ° C. and reacting for 2 hours, the reaction was performed at room temperature for 12 hours. After completion of the reaction, 60 mL of dioxane was added, and then 2 L of diethylether was added to generate particles. After adding diatomaceous earth to a Buchner funnel and filtering it, the filtered solids were dispersed in water and neutralized by adding sodium bicarbonate. The reaction solution was filtered through a Buchner funnel, and the filtrate was dried under reduced pressure. Purification was performed using silica gel chromatography, followed by drying under reduced pressure to obtain compound 1 (610 mg, 16%).

Rf = 0.6 (RP-C18, 아세토니트릴/물 1:5 v/v)Rf = 0.6 (RP-C18, acetonitrile / water 1: 5 v / v)

LC/MS, 계산치 C35H33N2O11S2 721.77, 측정치 720.6
LC / MS, calculated C35H33N2O11S2 721.77, found 720.6

실시예 2 : 화합물 1-1의 제조Example 2: Preparation of compound 1-1

(1) 화합물 3-2의 합성(1) Synthesis of Compound 3-2

Figure 112017103017497-pat00016
Figure 112017103017497-pat00016

화합물 3-1 (250 mg, 0.346 mmol, 2 eq)을 DMF (12 mL)와 DW 5 mL 혼합용액에 넣고 완용하였다. 0 ℃로 냉각한 후, O-(N-숙신이미딜)-N,N,N',N'-테트라메틸우로늅 테드라플르오로보레이트 (O-(N-Succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate, TSTU) (312 mg, 1.038 mmol, 3 eq, Sigma-Aldrich)을 투입하여 2시간 동안 교반하였다. 반응 완료 후 동결건조하여 입자를 생성한 후, 아세토니트릴과 물을 사용하여 역상 크로마토그래피로 정제하여 화합물 3-1을 얻었다(40 mg, 14%).Compound 3-1 (250 mg, 0.346 mmol, 2 eq) was added to a mixed solution of DMF (12 mL) and DW 5 mL and completed. After cooling to 0 ° C., O- (N-succinimidyl) -N, N, N ', N'-tetramethyluropene tedrafluoroborate (O- (N-Succinimidyl) -N, N, N ', N'-tetramethyluronium tetrafluoroborate, TSTU) (312 mg, 1.038 mmol, 3 eq, Sigma-Aldrich) was added and stirred for 2 hours. After the reaction was completed, lyophilized to produce particles, and then purified by reverse phase chromatography using acetonitrile and water to obtain compound 3-1 (40 mg, 14%).

Rf = 0.4 (RP-C18, 아세토니트릴/물 1:3 v/v)Rf = 0.4 (RP-C18, acetonitrile / water 1: 3 v / v)

LC/MS, 계산치 C39H38N3O13S2 820.86, 측정치 817.9LC / MS, calculated C39H38N3O13S2 820.86, found 817.9

(2) 화합물 3-3의 합성(2) Synthesis of Compound 3-3

Figure 112017103017497-pat00017
Figure 112017103017497-pat00017

화합물 3-1 (2 g, 2.77 mmol, 1 eq)을 DMF 100 mL에 완용하였다. 디(N-숙신이미딜)카보네이트(Di(N-succinimidyl) Carbonate, DSC) (2.13 g, 8.3 mmol, 3 eq, TCI)을 용액에 투입한 후, N,N-디이소프로필에틸아민(N,N-diisopropylethylamine, DIPEA) (4.83 mL, 27.7 mmol, 10 eq, Sigma-Aldrich)을 투입하였다. 40 ℃로 승온하여 2 시간 동안 반응을 진행한 후, 디에틸이더를 투입하여 입자를 석출시켰다. 입자를 여과 및 건조 진행한 후, 다시 DMF를 투입하여 용해시켰다. 에틸렌디아민(Ethylenediamine) (500 mg, 8.3 mmol, 3 eq)을 투입하여 상온에서 2시간 동안 반응시켰다. 반응액에 디에틸이더를 투입하여 입자를 석출시킨 후, 여과 및 건조를 진행하였다. 건조 완료 후 아세토니트릴과 증류수를 사용하여 역상크로마트그래피로 정제를 진행하였다. 정제 완료 후 건조를 진행하여 화합물 3-3을 얻었다(297 mg, 14%).Compound 3-1 (2 g, 2.77 mmol, 1 eq) was completely dissolved in 100 mL of DMF. Di (N-succinimidyl) Carbonate (DSC) (2.13 g, 8.3 mmol, 3 eq, TCI) was added to the solution, and then N, N-diisopropylethylamine (N , N-diisopropylethylamine, DIPEA) (4.83 mL, 27.7 mmol, 10 eq, Sigma-Aldrich) was added. After raising the temperature to 40 ° C. for 2 hours, diethyl ether was added to precipitate particles. After the particles were filtered and dried, DMF was added again to dissolve them. Ethylenediamine (500 mg, 8.3 mmol, 3 eq) was added and reacted at room temperature for 2 hours. Diethyl ether was added to the reaction solution to precipitate particles, followed by filtration and drying. After completion of the drying, purification was performed by reverse phase chromatography using acetonitrile and distilled water. After completion of purification, drying was performed to obtain compound 3-3 (297 mg, 14%).

Rf = 0.2 (RP-C18, 아세토니트릴/물 1:3 v/v)Rf = 0.2 (RP-C18, acetonitrile / water 1: 3 v / v)

LC/MS, 계산치 C37H39N4O10S22- 763.86, 측정치 763.1LC / MS, calculated C37H39N4O10S22- 763.86, found 763.1

(3) 화합물 1-1의 합성(3) Synthesis of Compound 1-1

Figure 112020004547499-pat00043
Figure 112020004547499-pat00043

1-11-1

화합물 3-3 (297 mg, 0.388 mmol)을 증류수 30 mL에 완용한 후, 냉장보관하였다. 시아누릭 클로라이드 (Cyanuric chloride, CNC) (215 mg, 1.16 mmol, 3 eq)을 아세토니트릴 30 mL에 완용한 후 DIPEA (135 μL, 0.776 mmol, 2 eq)을 투입하여 냉동보관하였다. CNC 용액에 화합물 3-3 용액을 투입한 후 상온에서 1 시간 동안 반응시켰다.Compound 3-3 (297 mg, 0.388 mmol) was completely dissolved in 30 mL of distilled water, and then kept refrigerated. Cyanuric chloride (Cyanuric chloride, CNC) (215 mg, 1.16 mmol, 3 eq) was dissolved in 30 mL of acetonitrile, and DIPEA (135 μL, 0.776 mmol, 2 eq) was added to the product and stored frozen. After adding the compound 3-3 solution to the CNC solution, the mixture was reacted at room temperature for 1 hour.

반응 확인 후, 6-아미노헥사노익 산(6-aminohexanoic acid) (152 mg., 1.16 mmol, 3 eq)을 증류수 15 mL에 녹인 후 반응액에 투입하였다. DIPEA (405 μL, 2.328 mmol, 6 eq)을 투입한 후 40℃에서 12시간 동안 반응시켰다. 반응 완료 후 동결건조하여 얻어진 화합물을 역상크로마토그래피로 정제하여 화합물 1-1을 얻었다(168 mg, 43%).After confirming the reaction, 6-aminohexanoic acid (152 mg., 1.16 mmol, 3 eq) was dissolved in 15 mL of distilled water and added to the reaction solution. DIPEA (405 μL, 2.328 mmol, 6 eq) was added and then reacted at 40 ° C. for 12 hours. After completion of the reaction, the compound obtained by lyophilization was purified by reverse phase chromatography to obtain compound 1-1 (168 mg, 43%).

Rf = 0.3 (RP-C18, 아세토니트릴/물 1:2 v/v)Rf = 0.3 (RP-C18, acetonitrile / water 1: 2 v / v)

LC/MS, 계산치 C46H50ClN8O12S22- 1006.52 측정치 1005.2
LC / MS, calculated C46H50ClN8O12S22-1006.52, measured 1005.2

실시예 3 : 화합물 1-2의 제조Example 3: Preparation of compound 1-2

(1) 화합물 2-5의 합성(1) Synthesis of Compound 2-5

Figure 112017103017497-pat00019
Figure 112017103017497-pat00019

6-Aminohexanoic acid (1 g, 7.624 mmol)을 증류수 200 mL에 완용 한 후 냉장보관하였다. CNC (4.22 g, 22.87 mmol, 3 eq)을 아세토니트릴 200 mL에 완용 후 DIPEA (2.66 mL, 15.25 mmol, 2 eq)을 투입하여 냉동보관하였다. CNC 용액에 6-Aminohexanoic acid 용액을 투입한 후 상온에서 1시간 동안 반응시켰다.6-Aminohexanoic acid (1 g, 7.624 mmol) was completely dissolved in 200 mL of distilled water and stored in the refrigerator. After completion of CNC (4.22 g, 22.87 mmol, 3 eq) in 200 mL of acetonitrile, DIPEA (2.66 mL, 15.25 mmol, 2 eq) was added and stored frozen. 6-Aminohexanoic acid solution was added to the CNC solution, and then reacted at room temperature for 1 hour.

반응 확인 후, 1,3-Propanediamine (1.69g, 22.87 mmol, 3 eq)을 CNC 반응액에 투입 후 DIPEA (7.97 mL, 45.74 mmol, 6 eq)을 투입하여 50℃에서 2시간 반응시켰다. 반응 완료 후 동결건조하여 얻어진 화합물을 역상크로마토그래피로 정제하여 화합물 2-5을 얻었다(1.13 g, 47%).After confirming the reaction, 1,3-Propanediamine (1.69 g, 22.87 mmol, 3 eq) was added to the CNC reaction solution, and DIPEA (7.97 mL, 45.74 mmol, 6 eq) was added to react at 50 ° C. for 2 hours. After completion of the reaction, the compound obtained by lyophilization was purified by reverse phase chromatography to obtain compound 2-5 (1.13 g, 47%).

Rf = 0.5 (RP-C18, 아세토니트릴/물 1:2 v/v)Rf = 0.5 (RP-C18, acetonitrile / water 1: 2 v / v)

LC/MS, 계산치 C12H21ClN6O2 316.79, 측정치 315.0LC / MS, calculated C12H21ClN6O2 316.79, found 315.0

(2) 화합물 1-2의 합성(2) Synthesis of Compound 1-2

Figure 112020004547499-pat00044
Figure 112020004547499-pat00044

1-21-2

화합물 3-1 (2 g, 2.77 mmol, 1 eq)을 DMF 100 mL에 완용하였다. DSC (2.13 g, 8.3 mmol, 3 eq)을 용액에 투입한 후, DIPEA (4.83 mL, 27.7 mmol, 10 eq)을 투입하였다. 40 ℃로 승온하여 2 시간 동안 반응을 진행한 후, 디에틸이더를 투입하여 입자를 석출시켰다. 입자를 여과 및 건조 진행한 후, 다시 DMF를 투입하여 용해시켰다. 용액에 화합물 2-5 (2.63 g, 8.3 mmol, 3 eq)을 투입한 후 DIPEA (4.83 mL, 27.7 mmol, 10 eq)을 투입하고 50 ℃에서 2 시간 동안 반응시켰다. 반응액에 디에틸이더를 투입하여 입자를 석출시킨 후 여과 및 건조 진행하였다. 건조 완료 후 아세토니트릴과 증류수를 사용하여 역상 크로마트그래피로 정제를 진행하였다. 정제 완료 후 건조를 진행하여 화합물 1-2을 얻었다. (415 mg, 15%).Compound 3-1 (2 g, 2.77 mmol, 1 eq) was completely dissolved in 100 mL of DMF. DSC (2.13 g, 8.3 mmol, 3 eq) was added to the solution, followed by DIPEA (4.83 mL, 27.7 mmol, 10 eq). After raising the temperature to 40 ° C. for 2 hours, diethyl ether was added to precipitate particles. After the particles were filtered and dried, DMF was added again to dissolve them. Compound 2-5 (2.63 g, 8.3 mmol, 3 eq) was added to the solution, followed by DIPEA (4.83 mL, 27.7 mmol, 10 eq), and reacted at 50 ° C. for 2 hours. Diethyl ether was added to the reaction solution to precipitate particles, followed by filtration and drying. After completion of the drying, purification was performed by reverse phase chromatography using acetonitrile and distilled water. After the purification was completed, drying was performed to obtain compound 1-2. (415 mg, 15%).

Rf = 0.2 (RP-C18, 아세토니트릴/물 1:2 v/v)Rf = 0.2 (RP-C18, acetonitrile / water 1: 2 v / v)

LC/MS, 계산치 C47H53ClN8O12S22- 1021.55 측정치 1020.0
LC / MS, calculated C47H53ClN8O12S22-1021.55 found 1020.0

실시예 4 : 화합물 1-3의 제조Example 4: Preparation of compound 1-3

(1) 화합물 1-3의 합성(1) Synthesis of Compound 1-3

Figure 112020004547499-pat00045
Figure 112020004547499-pat00045

1-31-3

화합물 1-1 (165 mg, 0.164 mmol, 1 eq)을 DMF 20 mL에 완용하였다. DSC (126 mg, 0.491 mmol, 3 eq)을 용액에 투입한 후, DIPEA (285 μL, 1.638 mmol, 10 eq)을 투입하였다. 40 ℃로 승온하여 2 시간 동안 반응을 진행한 후, 디에틸이더를 투입하여 입자를 석출시켰다. 입자를 여과 및 건조 진행한 후, 아세토니트릴과 증류수를 사용하여 역상크로마트그래피로 정제를 진행하였다. 정제 완료 후 건조를 진행하여 화합물 1-3을 얻었다. (31 mg, 17%).Compound 1-1 (165 mg, 0.164 mmol, 1 eq) was slowly dissolved in 20 mL of DMF. DSC (126 mg, 0.491 mmol, 3 eq) was added to the solution, followed by DIPEA (285 μL, 1.638 mmol, 10 eq). After raising the temperature to 40 ° C. for 2 hours, diethyl ether was added to precipitate particles. After the particles were filtered and dried, purification was performed by reverse phase chromatography using acetonitrile and distilled water. After the purification was completed, drying was performed to obtain compound 1-3. (31 mg, 17%).

Rf = 0.2 (RP-C18, 아세토니트릴/물 1:2 v/v)Rf = 0.2 (RP-C18, acetonitrile / water 1: 2 v / v)

LC/MS, 계산치 C50H53ClN9O14S22- 1103.59, 측정치 1103.0
LC / MS, calculated C50H53ClN9O14S22-1103.59, found 1103.0

실시예 5 : 화합물 1-4의 제조Example 5: Preparation of compound 1-4

Figure 112017103017497-pat00022
Figure 112017103017497-pat00022

화합물 1-3 (30 mg, 0.0272 mmol, 1 eq)을 DMF 10 mL에 완용하였다. 2-(2'-클로로에틸설포닐)에틸아민 염산염 (2-(2-chloroethylsulfonyl)ethanamine hydrochloride) (6 mg, 0.0272 mmol, 1 eq)을 용액에 투입한 후, DIPEA (47 μL, 0.272 mmol, 10 eq)을 투입하였다. 40 ℃ 로 승온하여 4 시간 동안 반응을 진행한 후, 디에틸에테르를 투입하여 입자를 석출시켰다. 입자의 여과 및 건조를 진행한 후, 아세토니트릴과 증류수를 사용하여 역상 크로마트그래피로 정제를 진행하였다. 정제 완료 후 건조를 진행하여 화합물 1-4을 얻었다. (15 mg, 49 %)Compound 1-3 (30 mg, 0.0272 mmol, 1 eq) was slowly dissolved in 10 mL of DMF. 2- (2'-chloroethylsulfonyl) ethylamine hydrochloride (2- (2-chloroethylsulfonyl) ethanamine hydrochloride) (6 mg, 0.0272 mmol, 1 eq) was added to the solution, followed by DIPEA (47 μL, 0.272 mmol, 10 eq). After raising the temperature to 40 ° C. for 4 hours, diethyl ether was added to precipitate particles. After filtration and drying of the particles, purification was performed by reverse phase chromatography using acetonitrile and distilled water. After the purification was completed, drying was performed to obtain compound 1-4. (15 mg, 49%)

Rf = 0.3 (RP-C18, 아세토니트릴/물 1:4 v/v)Rf = 0.3 (RP-C18, acetonitrile / water 1: 4 v / v)

LC/MS, 계산치 C50H60ClN9O13S3 1126.71, 측정치 1125.9
LC / MS, calculated C 50 H 60 ClN 9 O 13 S 3 1126.71, found 1125.9

시험예 1: 화합물의 광학특성 평가Test Example 1: Evaluation of the optical properties of the compound

(1) 화합물 1-3과 대조형광염료의 최대 흡ㅇ형광 파장 및 몰 흡광계수 분석(1) Analysis of maximum absorption and fluorescence wavelengths and molar extinction coefficients of Compound 1-3 and the control fluorescent dye.

화합물 1-3과 대조형광염료 (Alexa Fluor?? 594 NHS ester)의 기본 광학특성을 분석하였다. Powder 상태의 2 종 염료에 DMF를 넣어 각각 10 mg/mL로 Stock solution을 제조하였고, 이후 pH 7.4 10 mM Phosphate buffered saline (이하 1x PBS)을 이용하여 각 15.1 μM 농도의 희석 샘플을 준비하였다. 해당 샘플들에 대하여 Agilent 사의 Cary 8454 UV/Vis Spectrophotometer 기기를 활용하여 흡광 측정을 진행하였고, 샘플 당 1/2 dilution하여 1/8 희석 샘플까지 총 4 회씩 분석하였다(Blank : 1x PBS).Basic optical properties of Compound 1-3 and the control fluorescent dye (Alexa Fluor® 594 NHS ester) were analyzed. DMF was added to two types of dyes in powder form to prepare a stock solution at 10 mg / mL, respectively, and then diluted samples of 15.1 μM concentration were prepared using pH 7.4 10 mM Phosphate buffered saline (hereinafter 1x PBS). Absorption measurements were performed on the samples using Agilent's Cary 8454 UV / Vis Spectrophotometer instrument, and 1/2 dilution per sample was performed to analyze the total of 4 times to 1/8 diluted samples (Blank: 1x PBS).

분석 결과를 통해 우선 화합물 1-3 및 대조형광염료의 여기 파장(최대 흡수 파장)을 순서대로 593, 591 nm로 확인하였고, 해당 파장 설정 하에 PerkinElmer의 LS 55 Fluorescence spectrometer를 이용하여 형광을 분석하였다. 형광 측정은 화합물 1-3 및 대조형광염료에 대해 0.013 μM의 동일 몰 농도 샘플을 제조하여 진행하였다.Through the analysis results, first, the excitation wavelengths (maximum absorption wavelengths) of Compound 1-3 and the control fluorescent dye were sequentially confirmed to be 593 and 591 nm, and fluorescence was analyzed using PerkinElmer's LS 55 Fluorescence spectrometer under the corresponding wavelength setting. Fluorescence measurements were made by preparing samples of the same molar concentration of 0.013 μM for Compound 1-3 and the control fluorescent dye.

표 1에 광학특성 분석 결과를 나타내었고, 몰 흡광계수의 경우 염료 당 흡광 4 회 분석 결과를 토대로 Beer's Law (A=Ebc)에 따라 산출하였다.Table 1 shows the results of the optical properties analysis, and the molar extinction coefficient was calculated according to Beer's Law (A = Ebc) based on the results of four times of absorption per dye.

Figure 112017103017497-pat00023
Figure 112017103017497-pat00023

(2) 화합물 1-3과 대조형광염료의 형광강도 비교(2) Comparison of fluorescence intensity of compound 1-3 and the control fluorescent dye

화합물 1-3과 대조형광염료(Alexa Fluor?? 594 NHS ester)의 형광강도를 비교하였다. 시험예 1-(1)에서 동일 몰 농도(0.013 μM)로 제조한 샘플과 더불어, 대조형광염료에 대하여 화합물 1-3과 동일 무게만큼 사용하였을 때를 가정하여 샘플 하나를 추가 제조(Alexa Fluor?? 594 NHS ester, 몰 농도 환산 시 0.0175 μM)하여 함께 비교하였다.The fluorescence intensities of Compound 1-3 and the control fluorescent dye (Alexa Fluor® 594 NHS ester) were compared. In addition to the samples prepared at the same molar concentration (0.013 μM) in Test Example 1- (1), one sample was additionally prepared (Alexa Fluor? 594 NHS ester, 0.0175 μM in terms of molar concentration), and compared together.

여기파장 591 nm 설정 하에 LS 55 Fluorescence spectrometer를 이용하여 측정하였다. 도 1과 같이 동일 중량 및 동일 몰 농도 분석 조건 모두에서 화합물 1-3의 형광강도가 가장 강함을 알 수 있다.It was measured using an LS 55 Fluorescence spectrometer under an excitation wavelength of 591 nm. As shown in Figure 1 it can be seen that the fluorescence intensity of the compound 1-3 is the strongest under both the same weight and the same molar concentration analysis conditions.

(2) 상대양자효율 (Relative quantum yield) 비교(2) Comparison of relative quantum yield

Rhodamine 6G (TCI)를 기준으로, 화합물 1-3과 대조형광염료 (Alexa Fluor?? 594 NHS ester)의 상대양자효율을 측정하였다. Rhodamine 6G 역시 DMF를 넣어 10 mg/mL Stock solution을 제조한 후 사용하였다. pH 7.4 1x PBS을 이용하여 10 μM의 농도로 희석한 후 흡광 및 형광을 측정하였다. 10 μM 농도로부터 1/2 dilution을 시행하여 1/1024x 농도 샘플까지 총 11 회씩 흡ㅇ형광 측정을 하였다.On the basis of Rhodamine 6G (TCI), the relative quantum efficiency of Compound 1-3 and the control fluorescent dye (Alexa Fluor® 594 NHS ester) was measured. Rhodamine 6G was also used after preparing 10 mg / mL Stock solution by adding DMF. After dilution to a concentration of 10 μM using pH 7.4 1x PBS, absorbance and fluorescence were measured. 1/2 dilution was performed from a concentration of 10 μM, and absorption fluorescence was measured 11 times to a sample of 1 / 1024x concentration.

수학식 1에 측정값을 대입하여 상대양자효율을 분석하였고, 표 2 및 도 2와 같은 결과를 얻었다. 화합물 1-3의 상대 양자효율이 대조형광염료보다 높음을 확인할 수 있다.The relative quantum efficiency was analyzed by substituting the measured value in Equation 1, and the results shown in Table 2 and FIG. 2 were obtained. It can be seen that the relative quantum efficiency of compound 1-3 is higher than that of the control fluorescent dye.

Figure 112017103017497-pat00024
Figure 112017103017497-pat00024

Figure 112017103017497-pat00025
Figure 112017103017497-pat00025

시험예Test example 2 : 단백질에 labeling한 후의 성능 비교 2: Performance comparison after labeling the protein

(1) 단백질 반응물(Conjugates) 간 형광강도 비교(1) Comparison of fluorescence intensity between protein reactants

화합물 1-3과 대조형광염료 (Alexa Fluor?? 594 NHS ester)를 항체 (Goat anti-Mouse IgG H+L Secondary Ab, 140 kDa, Thermo)에 표지한 후 반응물 (Conjugates) 간의 형광강도를 비교하였다. 항체 0.5 mg에, Alexa Fluor?? 594 NHS ester 기준 25 fold (molar) 양인 0.0732 mg으로 동일 중량의 염료를 IgG와 각각 반응하였고 (즉, 'IgG 0.5 mg+화합물 1-3 0.0732 mg', 'IgG 0.5 mg + 대조염료 0.0732 mg'. 단, 염료들이 10 mg/mL stock solution 상태임에 따라 7.32 μL 씩 사용), Fluorescence spectrometer (LS 55, PerkinElmer)와 FOBI (Fluorescence-labeled Organism Bioimaging Instrument, NeoScience), 그리고 Multi-label plate reader (Enspire 2300, PerkinElmer) 기기를 활용하여 삼중 분석하였다.Compound 1-3 and the control fluorescent dye (Alexa Fluor ?? 594 NHS ester) were labeled with an antibody (Goat anti-Mouse IgG H + L Secondary Ab, 140 kDa, Thermo), and the fluorescence intensity between reactants was compared. . At 0.5 mg of antibody, Alexa Fluor ?? The same weight of dye was reacted with IgG in 25 fold (molar) amount based on 594 NHS ester with IgG (i.e., 'IgG 0.5 mg + compound 1-3 0.0732 mg', 'IgG 0.5 mg + control dye 0.0732 mg'. , As dyes are in a 10 mg / mL stock solution, 7.32 μL is used), Fluorescence spectrometer (LS 55, PerkinElmer), FOBI (Fluorescence-labeled Organism Bioimaging Instrument, NeoScience), and Multi-label plate reader (Enspire 2300, PerkinElmer) instrument was used for triple analysis.

결과에 앞서 반응 조건으로는, Coupling buffer의 pH가 8.3~8.5가 되도록 pH 7.4 1x PBS와 1 M pH 9.5 Sodium carbonate-Bicarbonate buffer를 적정 비로 혼합하여 사용하였고, 항체의 최종 반응 농도가 2 mg/mL이 되도록 설계하였다. 상온 (25 ℃), 암실 환경에서 1 시간 동안 자석 교반하며 반응시켰고, 이후 1x PBS로 미리 Buffer equilibrium 해 둔 PD-10 Column (GE Healthcare)을 이용하여 정제 해 반응물을 획득하였다.As a reaction condition prior to the results, pH 7.4 1x PBS and 1 M pH 9.5 Sodium carbonate-Bicarbonate buffer were mixed in an appropriate ratio so that the pH of the coupling buffer was 8.3 to 8.5, and the final reaction concentration of the antibody was 2 mg / mL. It was designed to be. At room temperature (25 ° C), the mixture was reacted with magnetic stirring for 1 hour in a dark environment, and then purified using PD-10 Column (GE Healthcare) previously buffered with 1x PBS to obtain a reactant.

수득한 반응물을 활용하여 우선 FL spectrometer로 형광강도를 비교 분석하였다. 도 3이 그 결과로, 각 반응물 4 μL를 3 mL의 1x PBS에 섞어 희석한 후 Excitation 592 nm 설정 하에 측정하였다.Using the obtained reactants, fluorescence intensity was first analyzed by FL spectrometer. As a result of FIG. 3, 4 μL of each reactant was mixed in 3 mL of 1x PBS, diluted, and then measured under Excitation 592 nm.

이어서, FOBI 및 Plate reader 분석을 진행하였다. 각 반응물의 원액과 1/2, 1/4. 1/10 희석 샘플 (1x PBS 이용), 그리고 Blank로 1x PBS를 96-well Black plate에 순서대로 well 당 100 μL 씩 주입하고 분석하였다. FOBI Imaging은 기기 상 Light source Green (520 nm) channel 및 Green Emission filter를 적용, Exposure time 500 ms, Gain 3 x 설정 하에 수행하였고, Plate reader 분석의 경우 ① 대조형광염료 (Alexa Fluor?? 594 NHS ester)의 해당 사 분석 증명서 상 Specification 기준 (최대 흡ㅇ형광 파장 590/617 nm)을 기기 프로토콜 설정에 적용하여 한 차례, ② IgG와 대조형광염료 반응물의 실제 최대 흡ㅇ형광 파장을 적용하여 한 차례 (591/619 nm), ③ IgG와 화합물 1-3 반응물의 실제 최대 흡ㅇ형광 파장을 적용하여 한 차례 (593/619 nm)로 총 3 회에 걸쳐 확인하였다. 세 차례 분석에서 Wavelength 이외의 기기 설정은 모두 동일하게 하였다 (Measurement height 9.5 mm, Number of flashes 50 등).Subsequently, FOBI and Plate reader analysis was performed. The stock solution of each reactant and 1/2, 1/4. A 1/10 diluted sample (using 1x PBS) and 1x PBS as a blank were sequentially injected into a 96-well black plate and analyzed at 100 μL per well. FOBI Imaging was performed under the light source green (520 nm) channel and green emission filter on the device under exposure time of 500 ms and gain 3 x. For plate reader analysis: ① Fluorescent dye (Alexa Fluor ?? 594 NHS ester) ), The specification standards (maximum absorption fluorescence wavelength 590/617 nm) are applied once in the instrument protocol setting, ② once by applying the actual maximum absorption fluorescence wavelength of the IgG and control fluorescent dye reactant ( 591/619 nm), ③ The actual maximum absorption fluorescence wavelength of the reactant of IgG and compound 1-3 was applied to confirm them three times in total (593/619 nm). In all three analyzes, the instrument settings except Wavelength were all the same (Measurement height 9.5 mm, Number of flashes 50, etc.).

도 4에 FOBI Image를, 도 5에 Plate reader 측정 결과 그래프를 제시하였다. 도 5의 형광강도는 Blank 보정 된 결과로서, Blank well의 형광수치가 매우 낮고 (8~14 FU), 모든 결과 치에 동일하게 적용이 되기 때문에 보정 전과 경향에 있어서의 차이는 없다. 도 3 내지 도 5로부터 단백질 (IgG)에 표지 한 이후의 형광강도에 있어서도 화합물 1-3의 Conjugate가 더 강한 형광세기를 띠는 것을 확인할 수 있다. 도 4의 경우 기기 옵션의 한계로 인해 여기 파장과 완벽히 일치하는 Laser line 설정 하에 분석을 진행하지는 못하였으나, 가장 유사한 환경에서 분석을 진행한 결과 앞선 내용과 동일한 경향을 나타냄을 알 수 있다. 도 3에서는 화합물 1-3의 단백질 반응물이 대조형광염료의 단백질 Conjugate에 비해 150% 수준의 형광강도를, 도 5에서는 모든 희석 배율에서 30~45% 정도 더 높은 강도를 갖는 것으로 분석 되었다. 이는 화합물 1-3의 분자량이 대조형광염료에 비해 약 35%가 더 큼에도 대조형광염료와 같은 중량만큼의 염료를 사용 (대조형광염료 기준 25 fold로, 화합물 1-3 기준으로는 약 18.6 fold 수준)하여 표지했을 때의 결과에 해당된다. 도 5에서 세 가지 파장 조건 설정으로 측정을 진행한 결과에 있어서는 예상했던 대로 ②, ③ 번 그래프와 같이 각 Conjugates의 실제 파장 영역에서 해당 반응물의 형광강도가 약간씩 더 높게 확인되었고, 전반적인 비교 경향은 유사하였다.The FOBI image in FIG. 4 and the graph of the plate reader measurement results in FIG. 5 are presented. The fluorescence intensity of FIG. 5 is a blank-corrected result, and the fluorescence value of the blank well is very low (8-14 FU), and since it is applied equally to all results, there is no difference in pre-correction and trend. It can be seen from FIG. 3 to FIG. 5 that the conjugate of Compound 1-3 has a stronger fluorescence intensity in the fluorescence intensity after labeling the protein (IgG). In the case of Figure 4, due to the limitation of the device option, the analysis could not be performed under the laser line setting that perfectly matches the excitation wavelength, but it can be seen that the analysis shows the same tendency as the previous ones in the most similar environment. In FIG. 3, the protein reactant of Compound 1-3 has a fluorescence intensity of 150% level compared to the protein conjugate of the control fluorescent dye, and in FIG. 5, it is analyzed that it has a higher intensity of 30 to 45% at all dilution ratios. It uses the same amount of dye as the control fluorescent dye even though the molecular weight of the compound 1-3 is about 35% greater than the control fluorescent dye (25 fold based on the control fluorescent dye, about 18.6 fold based on the compound 1-3) Level). In the results of measuring with three wavelength conditions in FIG. 5, as expected, the fluorescence intensities of the corresponding reactants were slightly higher in the actual wavelength region of each conjugate, as shown in the graphs ② and ③, and the overall comparison tendency It was similar.

Claims (10)

하기 화학식 1로 표시되는 화합물:
[화학식 1]
Figure 112020004547499-pat00026

상기 A1 및 상기 A2는 서로 동일하거나 상이하고, 각각 독립적으로 SO3 -이거나 SO3H이며,
상기 A3은 CO2 -이거나 CO2H이고,
상기 Z는 NH(CH2)mNH-Q이며,
상기 m은 1 내지 5이고,
상기 Q는 하기 화학식의 구조를 가지며,
[화학식 1a]
Figure 112020004547499-pat00027

상기 X는 할라이드이고,
상기 Y는 NH(CH2)nCO2-L
상기 n은 3 내지 7이고,
상기 L은 H이거나 하기 화학식의 구조를 가진다.
[화학식 1b]
Figure 112020004547499-pat00028
Compound represented by the formula (1):
[Formula 1]
Figure 112020004547499-pat00026

A 1 and A 2 are the same as or different from each other, and each independently is SO 3 - or SO 3 H,
A 3 is CO 2 - or CO 2 H,
Z is NH (CH 2 ) m NH-Q,
M is 1 to 5,
Q has the structure of the following formula,
[Formula 1a]
Figure 112020004547499-pat00027

X is a halide,
Y is NH (CH 2 ) n CO 2 -L
N is 3 to 7,
L is H or has the structure of the formula
[Formula 1b]
Figure 112020004547499-pat00028
 제1항에 있어서, 상기 m은 2 또는 3이고,
상기 X는 Cl 또는 F이며,
상기 n은 5인 것을 특징으로 하는 화합물.The method according to claim 1, wherein m is 2 or 3,
X is Cl or F,
The n is 5, characterized in that the compound. 제1항에 있어서, 상기 형광 화합물은 하기 화합물 중 하나인 것을 특징으로 하는 화합물.
[화학식 2b]
Figure 112020004547499-pat00046

[화학식 2c]
Figure 112020004547499-pat00047

[화학식 2d]
Figure 112020004547499-pat00048
The compound according to claim 1, wherein the fluorescent compound is one of the following compounds.
[Formula 2b]
Figure 112020004547499-pat00046

[Formula 2c]
Figure 112020004547499-pat00047

[Formula 2d]
Figure 112020004547499-pat00048
 제1항 내지 제3항 중 어느 한 항에 따른 화합물을 포함하는 형광 표지용 조성물.A fluorescent labeling composition comprising a compound according to any one of claims 1 to 3. 제4항에 있어서, 상기 표지의 대상 물질은 섬유, 인체 외 생체분자, 인간을 제외한 포유동물의 생체분자, 나노입자, 유기화합물 또는 이들 2종 이상의 조합인 것을 특징으로 하는 형광 표지용 조성물.According to claim 4, The target material of the label is a fiber, a non-human biomolecule, biomolecules of mammals other than humans, nanoparticles, organic compounds, or a combination of two or more of these, characterized in that the fluorescent labeling composition. 제5항에 있어서, 상기 인체 외 생체분자, 또는 인간을 제외한 포유동물의 생체분자는 단백질, 펩타이드, 탄수화물, 당, 지방, 항체, 프로테오글라이칸, 글라이코프로틴 및 siRNA 중에서 선택되는 것을 특징으로 하는 형광 표지용 조성물.The method of claim 5, wherein the non-human biomolecule, or a mammalian biomolecule other than human, is selected from proteins, peptides, carbohydrates, sugars, fats, antibodies, proteoglycans, glycoproteins, and siRNAs. Fluorescent labeling composition. 제1항 내지 제3항 중 어느 한 항에 따른 화합물과 표지 대상 물질과 접촉시키는 단계를 포함하는 형광 표지 방법.A fluorescent labeling method comprising the step of contacting a compound according to any one of claims 1 to 3 with a substance to be labeled. 제7항에 있어서, 상기 표지 대상 물질은 섬유, 인체 외 생체분자, 인간을 제외한 포유동물의 생체분자, 나노입자, 유기화합물 또는 이들 2종 이상의 조합인 것을 특징으로 하는 형광 표지 방법.The fluorescent labeling method according to claim 7, wherein the labeling target material is a fiber, a biomolecule other than a human body, a biomolecule of a mammal other than a human, a nanoparticle, an organic compound, or a combination of two or more of these. 삭제delete 제8항에 있어서, 상기 인체 외 생체분자, 또는 인간을 제외한 포유동물의 생체분자는 단백질, 펩타이드, 탄수화물, 당, 지방, 항체, 프로테오글라이칸, 글라이코프로틴 및 siRNA 중에서 선택되는 것을 특징으로 하는 형광 표지 방법.The method of claim 8, wherein the non-human biomolecule, or a mammalian biomolecule other than human, is selected from proteins, peptides, carbohydrates, sugars, fats, antibodies, proteoglycans, glycoproteins, and siRNAs. Fluorescent labeling method.
KR1020170135622A 2017-10-19 2017-10-19 Fluorescence Compounds and Preparation Method Therof KR102112719B1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
KR1020170135622A KR102112719B1 (en) 2017-10-19 2017-10-19 Fluorescence Compounds and Preparation Method Therof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
KR1020170135622A KR102112719B1 (en) 2017-10-19 2017-10-19 Fluorescence Compounds and Preparation Method Therof

Publications (2)

Publication Number Publication Date
KR20190043711A KR20190043711A (en) 2019-04-29
KR102112719B1 true KR102112719B1 (en) 2020-05-19

Family

ID=66282617

Family Applications (1)

Application Number Title Priority Date Filing Date
KR1020170135622A KR102112719B1 (en) 2017-10-19 2017-10-19 Fluorescence Compounds and Preparation Method Therof

Country Status (1)

Country Link
KR (1) KR102112719B1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117777151B (en) * 2024-02-27 2024-05-07 深圳创元生物医药科技有限公司 Preparation method of AF594TSA

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101057176B1 (en) 2009-02-18 2011-08-16 충남대학교산학협력단 Water Soluble Conjugated Polymer Compound, Method for Producing It, and Protein Pattern Biosensor Using Electrostatic Self-Assembly
KR101125057B1 (en) * 2009-09-25 2012-03-21 주식회사 디케이씨코포레이션 Compound for labeling material, intermediate therefor and process for producing the same
KR101110170B1 (en) 2010-05-04 2012-02-09 충남대학교산학협력단 Method for selectively detecting cysteine via water-soluble fluorescent hyperbranched conjugated polymer-metal ions complexation
KR101121513B1 (en) 2010-06-15 2012-03-06 충남대학교산학협력단 Method for selectively detecting Cysteine via Conjugated Fluorescent Polyelectrolyte-Mercury-Thymine Complexation
KR101286697B1 (en) 2011-07-06 2013-07-16 충남대학교산학협력단 Water-soluble conjugated polymer and method for selective detection of cysteine using a water-soluble conjugated polymer in the film state
KR101478505B1 (en) * 2011-08-10 2015-01-06 주식회사 디케이씨코포레이션 A process of labeling biomolecules by using dye compounds with vinylsulfone group

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Bioconjugate Chem. 2008, 19, 279-289. 1부.*

Also Published As

Publication number Publication date
KR20190043711A (en) 2019-04-29

Similar Documents

Publication Publication Date Title
Gui et al. AIE-active theranostic system: selective staining and killing of cancer cells
Würth et al. Integrating sphere setup for the traceable measurement of absolute photoluminescence quantum yields in the near infrared
EP2755019B1 (en) Biological substance detection method
KR101944912B1 (en) Fluorescence Compounds and Preparation Method Therof
KR101846075B1 (en) Detection method of biomolecule using linear upconversion fluorescence
KR102112719B1 (en) Fluorescence Compounds and Preparation Method Therof
KR101980292B1 (en) Fluorescence Compounds and Preparation Method Therof
KR101921662B1 (en) Fluorescence Compounds and Preparation Method Therof
KR101695617B1 (en) Benzindocyanine compound for labeling material, intermediate therefore, and process for producing the same
KR102066344B1 (en) Novel fluorescent compound for labelling nucleic acids and the preparation method thereof
KR102121965B1 (en) Fluorescence compounds, complex nanoparticles comprising the same, and preparation method thereof
KR102414574B1 (en) Fluorescent compound for detecting biological materials and the preparation method thereof
KR102414552B1 (en) Fluorescent compound for detecting biological materials and the preparation method thereof
US20220229063A1 (en) Fluorescent compound with cyanuric-hydroxide and preparation method thereof
KR102414554B1 (en) Fluorescent compound for detecting biological materials and the preparation method thereof
KR102399778B1 (en) Fluorescent compound for detecting biological materials and the preparation method thereof
KR101915744B1 (en) Dye compound and preparation method thereof
US10294204B2 (en) Fluorescence compounds and preparation method thereof
KR20170072854A (en) Novel cyanine compound for labeling biomolecule and preparation method thereof
KR101953819B1 (en) Novel cyanine compound for labeling biomolecule and preparation method thereof
KR20160140556A (en) Novel cyanine compound for labeling biomolecule and preparation method thereof
KR20110122784A (en) Novel cyanine compound for labeling biomolecule and preparation method thereof
KR101792214B1 (en) Dye compounds and preparation method thereof
KR20240077254A (en) Fluorescent compound for immunohistochemical staining and fluorescent diagnosis composition comprising the same
WO2023244963A2 (en) Bicyclononyne reagents for cell imaging

Legal Events

Date Code Title Description
A201 Request for examination
E902 Notification of reason for refusal
E701 Decision to grant or registration of patent right
GRNT Written decision to grant