CN111393872B - Ultraviolet Light Excited Fluorescent Dyes and Their Applications - Google Patents

Abstract

Translated fromChinese

本发明公开了一种紫外光激发荧光染料及其应用，具体涉及通式(Ⅰ)化合物，式中各基团如文中所述，本发明还涉及该染料‑偶联物，以及应用在抗体检测上。本发明的荧光染料同时存在活性基团和水溶性聚合物基团，该荧光染料可以被405nm激光激发，且具有很好的水溶性、显著降低对生物活性的影响和在较短波长内具有较高的荧光信号的效果。

The present invention discloses an ultraviolet light-excited fluorescent dye and its application, in particular to a compound of general formula (I), wherein the groups in the formula are as described in the text. The present invention also relates to the dye-conjugate, and its application in antibody detection superior. The fluorescent dye of the present invention has both an active group and a water-soluble polymer group, the fluorescent dye can be excited by a 405nm laser, has good water solubility, significantly reduces the influence on biological activity, and has a relatively low wavelength in a shorter wavelength. effect of high fluorescence signal.

Description

Translated fromChinese

紫外光激发荧光染料及其应用Ultraviolet Light Excited Fluorescent Dyes and Their Applications

技术领域technical field

本发明涉及荧光染料，特别涉及紫外光激发荧光染料及其应用，特别是其在神经病理学、免疫学、病理学和蛋白质组学上的应用。The present invention relates to fluorescent dyes, in particular to ultraviolet light excited fluorescent dyes and their applications, especially their applications in neuropathology, immunology, pathology and proteomics.

背景技术Background technique

随着新的显微技术的发展和各种合成及天然荧光分子的出现，荧光显微技术已经成为细胞生物学的基础工具。荧光标记的目标分子或细胞器可以用来研究细胞的结构和功能。荧光染料可以作为示踪剂使用荧光显微镜对生物结构进行定位，使用荧光免疫方法对分析物进行定量，使用流式细胞仪对细胞进行分析，细胞物理状态的测定及其他应用。与其它类型吸收染料相比，荧光染料通常灵敏度高，并且其吸收波长与发射波长是不同，一般通过光学激发来检测荧光发射，在大部分生物学样品中具有较低的荧光背景，其固有的光学性质例如荧光极性，荧光寿命和激发态能量转移均是可以进行测定的。目前荧光染料广泛应用于对病人的生理学功能进行日常检查或疾病诊断过程，也可以同步监控暴露在环境中的有害化学物质例如毒素或致癌农药，或者空气、泥土或饮用水中的有害无机物质对工人的影响，确定药物疗效，扫描新化合物对特殊酶的促进或抑制作用，监控基因和转基因表达，以及一些其他生物学应用例如免疫酶联反应和蛋白质印迹法(Western Blots)。With the development of new microscopy techniques and the emergence of various synthetic and natural fluorescent molecules, fluorescence microscopy has become a fundamental tool in cell biology. Fluorescently labeled target molecules or organelles can be used to study cell structure and function. Fluorescent dyes can be used as tracers to localize biological structures using fluorescence microscopy, quantify analytes using fluorescent immunoassays, analyze cells using flow cytometry, determine the physical state of cells, and other applications. Compared with other types of absorbing dyes, fluorescent dyes are usually highly sensitive, and their absorption wavelengths are different from their emission wavelengths. Fluorescence emission is generally detected by optical excitation, and has a low fluorescence background in most biological samples. Optical properties such as fluorescence polarity, fluorescence lifetime, and excited state energy transfer can all be measured. At present, fluorescent dyes are widely used in daily examinations of patients' physiological functions or in the process of disease diagnosis. They can also simultaneously monitor exposure to harmful chemicals in the environment such as toxins or carcinogenic pesticides, or harmful inorganic substances in the air, soil or drinking water. Worker impact, determine drug efficacy, scan new compounds for promotion or inhibition of specific enzymes, monitor gene and transgene expression, and several other biological applications such as immunoenzyme-linked reactions and Western Blots.

在研究和诊断过程中，需要对有机化合物、生物化合物和生物分析物进行快速高效的检测或定量。特别需要提供一种用来检测少量核酸、多肽、药物、新陈代谢产物和微组织的方法。例如用于治疗目的需要对麻醉剂、中毒、药物情况进行了解，还需要了解激素、病理学微组织、病毒、抗体、酶和核酸等所反应出的疾病状态，光学方法检测(荧光发射，紫外吸收，比色法)用于监测和测量荧光，是一种传统并且高效的检测，这些方法可以通过直接观察或者仪器检测来定性或定量检测所需要的信息。In research and diagnostics, fast and efficient detection or quantification of organic compounds, biological compounds and biological analytes is required. There is a particular need to provide a method for detecting small amounts of nucleic acids, polypeptides, drugs, metabolites and microtissues. For example, for therapeutic purposes, it is necessary to understand the situation of anesthetics, poisoning, and drugs, as well as the disease states reflected by hormones, pathological micro-tissues, viruses, antibodies, enzymes, and nucleic acids, etc., optical methods detection (fluorescence emission, ultraviolet absorption) , colorimetry) for monitoring and measuring fluorescence, is a traditional and efficient assay, these methods can qualitatively or quantitatively detect the required information by direct observation or instrumental detection.

使用荧光染料作为示踪剂，一般需要将染料和生物活性配体如细胞、组织、蛋白、抗体、药物、酶、激素、核酸、多糖、脂类或者其他生物分子之间形成化学共价键或者将染料与自然或人工合成聚合物(载体分子)相连制成合成探针(荧光探针/染料-偶联剂)，使用这些合成探针(荧光探针/染料-偶联剂)，生物分子一般会发生生物化学变化，合成探针(荧光探针/染料-偶联剂)可以对这种变化提供定性或定量检测。Using fluorescent dyes as tracers generally requires the formation of chemical covalent bonds between dyes and biologically active ligands such as cells, tissues, proteins, antibodies, drugs, enzymes, hormones, nucleic acids, polysaccharides, lipids or other biological molecules. The dyes are linked to natural or synthetic polymers (carrier molecules) to make synthetic probes (fluorescent probes/dye-coupling agents), using these synthetic probes (fluorescent probes/dye-coupling agents), biomolecules Biochemical changes typically occur and synthetic probes (fluorescent probes/dye-coupling agents) can provide qualitative or quantitative detection of such changes.

紫外激发具有其固有的弱点，如会造成光漂白和会对标记物造成损害等，但是在多色荧光应用中，如免疫荧光，核酸，蛋白质组，原位杂交和神经追踪中，蓝色荧光探针可以与绿色、黄色、橙色和红色荧光探针(染料-偶联剂)明显区分。UV excitation has inherent weaknesses such as photobleaching and label damage, but in multicolor fluorescence applications such as immunofluorescence, nucleic acids, proteomics, in situ hybridization and neural tracking, blue fluorescence The probes can be clearly distinguished from green, yellow, orange and red fluorescent probes (dye-coupling agents).

半导体激光器又称二极管，是以半导体材料为工作物质的一类激光器件，是成熟较早、进展较快的一类激光器，由于它的波长范围宽，制作简单、成本低、易于大量生产，并且由于其具有体积小、重量轻、寿命长等特点，因此，品种发展快、应用范围广和易于与各种光电子器件实现电子集成，如405nm的激光器。目前，能被405nm激光激发的合适染料则屈指可数，如Cascade Blue，Cascade Yellow和Pacific Blue和Pacific Orange等。为了在生物学应用中减少聚集，大部分染料中均含有磺酸基团，虽然磺化可减少二聚体的形成和增加染料的水溶性，但也在生物分子中引入了负电荷，因此可能增加破坏标记生物分子的生物学活性的风险。Semiconductor lasers, also known as diodes, are a class of laser devices that use semiconductor materials as the working substance. They are a class of lasers that mature earlier and progress faster. Because of their wide wavelength range, they are simple to manufacture, low in cost, and easy to mass-produce. Because of its small size, light weight, long life and other characteristics, it has fast variety development, wide application range and easy electronic integration with various optoelectronic devices, such as 405nm lasers. Currently, there are only a handful of suitable dyes that can be excited by a 405nm laser, such as Cascade Blue, Cascade Yellow, and Pacific Blue and Pacific Orange. In order to reduce aggregation in biological applications, most dyes contain sulfonic acid groups. Although sulfonation can reduce dimer formation and increase the water solubility of dyes, it also introduces negative charges into biomolecules, so it may be Increased risk of disrupting the biological activity of labeled biomolecules.

因此，目前仍然需要开发出可以被405nm激光激发的合适染料。而目前存在的紫外激光激发染料，在波长大约405nm时，一般吸收较弱(在最大吸光处的摩尔消光系数小于20000cm^-1·M^-1)、量子产率较低和在水溶液环境中溶解度差。这些缺点限制了其在生物学领域的应用。Cascade Yellow是一个普通的紫外光激发的染料，然而，这种染料与载体分子或固相支撑物结合后，容易形成二聚体并且在水溶液体系中倾向于形成聚集，而已知具有宽光谱的染料在合适的紫外光激发下(典型的为515-525nm)具有显著的荧光背景。另外，许多已知的染料在水溶液中会发生淬灭现象，从而导致荧光量子产率降低，将导致探测的灵敏度降低或者需要加大染料的用量等缺点。Therefore, there is still a need to develop suitable dyes that can be excited by 405 nm laser light. However, the existing UV laser-excited dyes generally have weak absorption at a wavelength of about 405 nm (the molar extinction coefficient at the maximum absorption is less than 20000 cm^-1 ·M^-1 ), low quantum yield and poor solubility in aqueous environment. . These shortcomings limit its application in the biological field. Cascade Yellow is a common UV-excited dye. However, when combined with carrier molecules or solid supports, this dye easily forms dimers and tends to aggregate in aqueous systems, while dyes with a broad spectrum are known Under suitable UV excitation (typically 515-525 nm) there is a significant fluorescence background. In addition, many known dyes will be quenched in aqueous solution, resulting in a decrease in fluorescence quantum yield, a decrease in detection sensitivity, or the need to increase the amount of dye.

综上所述，迫切需要开发出具有适用于紫外光激光器(例如405nm激光器)且具有活性基团的荧光染料。这种染料需要具有很好的水溶性、显著降低对生物活性的影响和在较短波长内具有较高的荧光信号的特点。In conclusion, there is an urgent need to develop fluorescent dyes with reactive groups suitable for ultraviolet lasers (eg, 405 nm lasers). Such dyes need to have good water solubility, significantly reduced effects on biological activity, and high fluorescence signals at shorter wavelengths.

发明内容SUMMARY OF THE INVENTION

鉴于上述所述，本发明公开了一类紫外光激发荧光染料及其应用。本申请的紫外光激发荧光染料具有水溶性好、能显著降低对生物活性的影响和在较短波长内具有较高的荧光信号等优点。In view of the above, the present invention discloses a class of ultraviolet light-excited fluorescent dyes and their applications. The ultraviolet light-excited fluorescent dye of the present application has the advantages of good water solubility, significantly reducing the influence on biological activity, and high fluorescence signal in a shorter wavelength.

本发明要解决的技术问题之一是提供一类新型化合物(即通式Ⅰ化合物)，此化合物可作为紫外光激发荧光染料，此荧光染料同时包含水溶性聚合物和活性基团；One of the technical problems to be solved by the present invention is to provide a new type of compound (namely the compound of general formula I), which can be used as a fluorescent dye excited by ultraviolet light, and the fluorescent dye contains a water-soluble polymer and an active group at the same time;

本发明要解决的技术问题之二是提供制备“荧光染料-偶联物”的方法；The second technical problem to be solved by the present invention is to provide a method for preparing "fluorescent dye-conjugate";

本发明要解决的技术问题之三提供此种荧光染料及“荧光染料-偶联物”的应用。The third technical problem to be solved by the present invention is to provide the application of such fluorescent dyes and "fluorescent dye-conjugates".

为达到上述目的，本发明采用如下技术方案：To achieve the above object, the present invention adopts the following technical solutions:

紫外光激发荧光染料，具有通式Ⅰ的结构：Ultraviolet light excited fluorescent dyes with the structure of general formula I:

式Ⅰ中：In formula I:

X、Y和Z各自独立选自：C、CR¹、N、NR³、S、O、Se、P、Si，As、C-C、CR¹-CR²、NR³-NR⁴、S-S,此处，X、Y和Z中至少一个不是C或C-C；X, Y and Z are each independently selected from: C, CR¹ , N, NR³ , S, O, Se, P, Si, As, CC, CR¹ -CR² , NR³ -NR⁴ , SS, where , at least one of X, Y and Z is not C or CC;

W为N或C；W is N or C;

R₁、R₂、R₃、R₄、R₅、R₆、R₇、R₉和R₁₀各自独立选自：H、C_1-8烷基、C_1-8取代烷基、C_1-8烷氧基、羟基、磺酸基、S、卤素、氨基、取代氨基、醛基、羧基、酯基、叠氮基、亚硝基、氰基、硫醚基、C_5-7环、C_5-7含杂环、取代的C_5-7环、取代的C_5-7含杂环、活性基团或水溶性聚合物基团，或者R₁、R₂、R₃、R₄、R₅、R₆、R₇、R₉和R₁₀中的R₁与R₂、R₃与R₄、R₅与R₆和R₉与R₁₀分别连接形成饱和或者不饱和的五元环、六元环或七元环；R₁ , R₂ , R₃ , R₄ , R₅ , R₆ , R₇ , R₉ and R₁₀ are each independently selected from: H, C_1-8 alkyl, C_1-8 substituted alkyl, C_{1 -8} alkoxy, hydroxyl, sulfonic acid, S, halogen, amino, substituted amino, aldehyde, carboxyl, ester, azide, nitroso, cyano, thioether, C_5-7 ring, C_5-7 containing heterocycle, substituted C_5-7 ring, substituted C_5-7 containing heterocycle, reactive group or water-soluble polymer group, or R₁ , R₂ , R₃ , R₄ , Among R₅ , R₆ , R₇ , R₉ and R₁₀ , R₁ and R₂ , R₃ and R₄ , R₅ and R₆ , and R₉ and R₁₀ are respectively connected to form a saturated or unsaturated five-membered ring , a six-membered ring or a seven-membered ring;

R₈为H、C_1-8烷基、C_1-8取代烷基、C_5-7环、C_5-7含杂环、取代的C_5-7环、取代的C_5-7含杂环、活性基团或水溶性聚合物基团，或者与R₇连接形成饱和或者不饱和的五元环、六元环或七元环；R₈ is H, C_1-8 alkyl, C_1-8 substituted alkyl, C_5-7 ring, C_5-7 heterocycle, substituted C_5-7 ring, substituted C_5-7 heterocycle Ring, reactive group or water-soluble polymer group, or connected with R₇ to form a saturated or unsaturated five-membered ring, six-membered ring or seven-membered ring;

R¹-R⁴各自独立选自：H、C_1-8烷基、C_1-8取代烷基、C_1-8烷氧基、羟基、磺酸基、卤素、氨基、取代氨基、醛基、羧基、酯基、叠氮基、硝基、亚硝基、氰基或硫醚基；R¹ -R⁴ are each independently selected from: H, C_1-8 alkyl, C_1-8 substituted alkyl, C_1-8 alkoxy, hydroxyl, sulfonic acid, halogen, amino, substituted amino, aldehyde , carboxyl, ester, azide, nitro, nitroso, cyano or thioether group;

在R₁、R₂、R₃、R₄、R₅、R₆、R₇、R₈、R₉和R₁₀中，存在至少一个活性基团和至少一个水溶性聚合物基团。Among R₁ , R₂ , R₃ , R₄ , R₅ , R₆ , R₇ , R₈ , R₉ and R₁₀ , at least one reactive group and at least one water-soluble polymer group are present.

依照本发明的一个方面，X为C或CR¹，其中，R¹为磺酸基；Y为N；Z为O或S；W为N；R₁、R₂、R₅、R₆、R₇、R₉和R₁₀全部为H；R₃、R₄、R₈互相独立选自：磺酸基、活性基团或水溶性聚合物。According to one aspect of the present invention, X is C or CR¹ , wherein R¹ is a sulfonic acid group; Y is N; Z is O or S; W is N; R₁ , R₂ , R₅ , R₆ , R₇ , R₉ and R₁₀ are all H; R₃ , R₄ , and R₈ are independently selected from each other: sulfonic acid group, active group or water-soluble polymer.

依照本发明的一个方面，所述活性基团包括：丙烯酰胺、羧酸的活化酯、羧酸酯、酰基叠氮、酰基氰、醛、卤素取代芳环、酸酐、氨基、叠氮、氮杂环丙烷、硼化物、重氮烷、卤代乙酰胺、卤代烷、卤代三嗪、肼、亚胺酯、异氰酸、异硫氰酸、马来酰亚胺、亚磷酰胺、活性钯络合物、卤代硅烷、酰卤或硫醇、四氟苯酚酯、炔烃。According to one aspect of the present invention, the reactive groups include: acrylamides, activated esters of carboxylic acids, carboxylic acid esters, acyl azides, acyl cyanides, aldehydes, halogen-substituted aromatic rings, acid anhydrides, amino groups, azides, aza Cyclopropane, borides, diazane, haloacetamides, haloalkanes, halotriazines, hydrazine, imidoesters, isocyanic acid, isothiocyanic acid, maleimides, phosphoramidites, active palladium complexes compounds, halosilanes, acid halides or thiols, tetrafluorophenol esters, alkynes.

依照本发明的一个方面，所述水溶性聚合物包括：聚环氧乙烷、糖类、多肽、聚乙二醇。According to one aspect of the present invention, the water-soluble polymer comprises: polyethylene oxide, saccharide, polypeptide, polyethylene glycol.

依照本发明的一个方面，所述活性基团为包含羧酸的丁二酰亚胺酯、肼、氨基或马来酰亚胺，所述水溶性聚合物为离散型聚乙二醇，所述离散型聚乙二醇的分子量在300-3000之间。According to one aspect of the present invention, the reactive group is a succinimide ester containing a carboxylic acid, a hydrazine, an amino group or a maleimide, the water-soluble polymer is a discrete polyethylene glycol, and the The molecular weight of discrete polyethylene glycol is between 300-3000.

依照本发明的一个方面，一种紫外光激发荧光染料与载体分子制备染料-偶联物的方法，包括以下步骤：According to one aspect of the present invention, a method for preparing a dye-conjugate from a fluorescent dye and a carrier molecule excited by ultraviolet light, comprising the following steps:

将载体分子与所述的通式Ⅰ化合物接触形成结合样品；contacting the carrier molecule with the compound of general formula I to form a binding sample;

将结合样品孵化一定时间使得所述的通式Ⅰ化合物与载体分子之间形成共价键，获得染料-偶联物的混合物；Incubating the binding sample for a certain period of time to form a covalent bond between the compound of the general formula I and the carrier molecule to obtain a dye-conjugate mixture;

将染料-偶联物的混合物中未连接载体分子的化合物和已连接在载体分子的化合物进行分离，获得染料-偶联物。The dye-conjugate is obtained by separating the compound not linked to the carrier molecule and the compound linked to the carrier molecule in the dye-conjugate mixture.

依照本发明的一个方面，所述载体分子包括氨基酸、多肽、蛋白质、多糖、核苷酸、核苷、寡核苷酸、核酸、半抗原、补骨脂素、药物、激素、合成聚合物、聚合微粒、生物细胞或病毒。According to one aspect of the invention, the carrier molecule comprises amino acids, polypeptides, proteins, polysaccharides, nucleotides, nucleosides, oligonucleotides, nucleic acids, haptens, psoralens, drugs, hormones, synthetic polymers, Aggregate particles, biological cells or viruses.

依照本发明的一个方面，所述的通式Ⅰ化合物的至少一个活性基团将通式Ⅰ化合物与载体分子进行连接。According to one aspect of the present invention, at least one reactive group of the compound of general formula I links the compound of general formula I to the carrier molecule.

依照本发明的一个方面，所述载体分子包含核苷酸或多肽。According to one aspect of the invention, the carrier molecule comprises a nucleotide or a polypeptide.

依照本发明的一个方面，所述紫外光激发荧光染料应用于细胞的免疫检测分析。According to one aspect of the present invention, the ultraviolet light-excited fluorescent dye is used for immunodetection analysis of cells.

本发明的优点：Advantages of the present invention:

(1)本发明公开的紫外光激发荧光染料，其吸收在300nm-500nm之间并且具有较大的斯托克位移(Stoke's shift),可以在特定环境下在紫外激光下激发而用于多色试验。本发明化合物母体基于Cascade Yellow荧光团，然而却比Cascade Yellow更亮、更稳定和在水中的溶解性更好。(1) The ultraviolet light-excited fluorescent dye disclosed in the present invention absorbs between 300nm and 500nm and has a large Stoke's shift, which can be excited under ultraviolet laser in a specific environment and used for multicolor test. The parent compound of the present invention is based on the Cascade Yellow fluorophore, yet is brighter, more stable and more soluble in water than Cascade Yellow.

(2)本发明公开的紫外光激发荧光染料同时具有水溶性聚合物基团和活性基团，因此，具有生物标记所需特性，如分子内迁移率受限、荧光量子产率升高、聚集降低以及在非极性有机溶剂中的溶解度升高、淬灭减少和体内及体外稳定性增加等特性，因此，可将本发明的染料标记分子和生物分子，如多肽、核苷酸和/或金属螯合剂，适用于诊断、成像系统等各个领域。(2) The ultraviolet light-excited fluorescent dye disclosed in the present invention has both a water-soluble polymer group and an active group. Therefore, it has the properties required for biomarkers, such as limited intramolecular mobility, increased fluorescence quantum yield, and aggregation. Reduced and increased solubility in non-polar organic solvents, reduced quenching, and increased in vivo and in vitro stability, therefore, the dyes of the present invention can be labeled with molecules and biomolecules, such as polypeptides, nucleotides and/or Metal chelators, suitable for various fields such as diagnostics, imaging systems, etc.

(3)本发明公开的紫外光激发荧光染料可以被405nm激光激发，由于染料聚集荧光淬灭的主要原因，本发明染料减少聚集可不用过量带负电荷的磺酸基团来实现，利用水溶性聚合物有助于减少染料上的负电荷，可显著降低聚集，是且具有很好的水溶性、显著降低对生物活性的影响和在较短波长内具有较高的荧光信号的效果。(3) The ultraviolet light-excited fluorescent dye disclosed in the present invention can be excited by a 405 nm laser. Due to the main reason for the quenching of the dye aggregation fluorescence, the dye aggregation of the present invention can be reduced without excessive negatively charged sulfonic acid groups. The polymers help to reduce negative charges on the dyes, can significantly reduce aggregation, are highly water-soluble, significantly reduce the impact on biological activity, and have the effect of higher fluorescence signals at shorter wavelengths.

(4)本发明经标记的蛋白质(比如:经标记的抗体)应用于动物体(如哺乳动物体)时,在循环中可具有更长的半衰期，因此,可适用于体内成像；经标记的生物分子在体外系统中(例如在采用血清或其它生物提取物的测定中)也可显示出更长的半衰期。例如标记抗体在细胞染色中可具有更高的信噪比。(4) When the labeled protein (such as: labeled antibody) of the present invention is applied to an animal body (such as a mammalian body), it can have a longer half-life in circulation, and therefore, can be suitable for in vivo imaging; the labeled protein Biomolecules may also exhibit longer half-lives in in vitro systems (eg, in assays using serum or other biological extracts). For example, labeled antibodies may have a higher signal-to-noise ratio in cell staining.

本发明相较于专利US8158801：Compared with the patent US8158801, the present invention:

本发明公开的紫外光激发荧光染料同时具有活性基团和水溶性聚合物基团，专利US8158801中公开的荧光染料只具有活性基团，因此，本发明公开的紫外光激发荧光染料利用水溶性聚合物有助于减少染料上的负电荷，可显著降低聚集，具有更强的荧光强度、更加稳定和在水中溶解性更好的优点。The ultraviolet light-excited fluorescent dye disclosed in the present invention has both active groups and water-soluble polymer groups, and the fluorescent dye disclosed in the patent US8158801 only has active groups. Therefore, the ultraviolet light-excited fluorescent dye disclosed in the present invention utilizes water-soluble polymerization. The compound helps reduce the negative charge on the dye, can significantly reduce aggregation, has the advantages of stronger fluorescence intensity, more stability and better solubility in water.

附图说明Description of drawings

为了更清楚地说明本发明实施例中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。In order to illustrate the technical solutions in the embodiments of the present invention more clearly, the following briefly introduces the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained from these drawings without any creative effort.

图1为化合物4的质谱图；Fig. 1 is the mass spectrum ofcompound 4;

图2为化合物5的质谱图；Fig. 2 is the mass spectrum ofcompound 5;

图3为化合物5的吸收和发射光谱图；Fig. 3 is the absorption and emission spectrogram ofcompound 5;

图4为不同DOL下，Pacific Orange-GAM的荧光强度与化合物6-GAM的荧光强度对比；Figure 4 shows the comparison of the fluorescence intensity of Pacific Orange-GAM with that of compound 6-GAM under different DOLs;

图5为化合物6A显示的免疫分型图。FIG. 5 is an immunophenotyping chart of compound 6A.

具体实施方式Detailed ways

下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

在详细描述本发明之前，描述各种术语用来帮助理解：Before describing the present invention in detail, various terms are described to aid understanding:

本申请中的术语“活性基团”指活性基团能够在载体分子上与底物或反应偶联体反应从而形成共价键的化学部分。本发明化合物可用于标记多种宽泛的载体分子。所述载体分子含有合适的反应偶联体或其衍生物为含有合适的反应偶联体。在此，通常采用而非限制，化合物上的成键基团通常指活性基团，底物或载体分子上的成键基团通常指反应偶联体。“反应底物”、“底物”和“反应偶联体”在本申请文件中通篇可互换使用The term "reactive group" in this application refers to a chemical moiety that is capable of reacting with a substrate or reactive conjugate on a carrier molecule to form a covalent bond. The compounds of the present invention can be used to label a wide variety of carrier molecules. The carrier molecule contains a suitable reaction conjugate or its derivative is a suitable reaction conjugate. Here, it is generally adopted but not limited, the bonding group on the compound usually refers to the active group, and the bonding group on the substrate or carrier molecule usually refers to the reaction conjugate. "Reaction substrate", "substrate" and "reaction conjugate" are used interchangeably throughout this application document

本申请中的术语“反应底物”，“底物”和“反应伴侣”通常可互换使用。术语“合成探针”、“荧光探针”和“染料-偶联剂”可互换使用，都表示荧光染料与载体分子进行共价键连接，其实质是同一种物质。The terms "reaction substrate", "substrate" and "reaction partner" are generally used interchangeably in this application. The terms "synthetic probe", "fluorescent probe" and "dye-coupling agent" are used interchangeably, and all refer to the covalent bond between the fluorescent dye and the carrier molecule, which are essentially the same substance.

本申请中的术语“多肽”、“肽”和“蛋白质”可互换使用，指任何长度的氨基酸聚合物，聚合物可以是线性、环状或支链的，其可包含修饰的氨基酸，也可被非氨基酸中断，还包括修饰的氨基酸聚合物，如通过磺化、糖基化、脂化、酰化、磷酸化、碘化、甲基化、氧化、蛋白水解加工、异戊烯化、外消旋化、硒化和转运RNA介导氨基酸加入蛋白质，如精氨酰化、泛素化或任何其它操作，如与标记组分偶联。本文所用的术语“氨基酸”指天然或非天然或合成氨基酸，包括甘氨酸、甘氨酸D或L光学异构体、氨基酸类似物及肽模拟物。The terms "polypeptide", "peptide" and "protein" are used interchangeably in this application to refer to a polymer of amino acids of any length, which may be linear, cyclic or branched, which may contain modified amino acids, or Can be interrupted by non-amino acids, but also includes modified amino acid polymers, such as by sulfonation, glycosylation, lipidation, acylation, phosphorylation, iodination, methylation, oxidation, proteolytic processing, prenylation, Racemization, selenylation, and transfer RNA mediate amino acid addition to proteins, such as arginylation, ubiquitination, or any other manipulation, such as conjugation to labeling components. The term "amino acid" as used herein refers to natural or unnatural or synthetic amino acids, including glycine, glycine D or L optical isomers, amino acid analogs and peptidomimetics.

本申请中的术语“抗体”指免疫球蛋白分子和免疫球蛋白分子的免疫学活性部分，即含有特异性结合抗原(“与之免疫反应”)的抗原结合位点的分子。在结构上，最简单的天然产生抗体(例如IgG)包含二硫键相互连接的4条多肽链，两条重链(H)和两条轻链(L)。免疫球蛋白包括几类分子，例如IgD、IgG、IgM和IgE的分子大家族，其实质是多肽或蛋白质，因此，本申请中的术语“免疫球蛋白分子”包括，例如杂交抗体或改变的抗体及其片段。现已证明天然产生抗体的片段可执行抗体的抗原结合功能。这些片段统称为“抗原结合单位”。根据分子结构可将抗原结合单位大致分为“单链(“Sc”)和“非单链”(“Nsc”)型。The term "antibody" in this application refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, ie, molecules that contain an antigen-binding site that specifically binds ("immunoreacts" with) an antigen. Structurally, the simplest naturally occurring antibodies (eg, IgG) comprise four polypeptide chains, two heavy (H) and two light (L) chains, interconnected by disulfide bonds. Immunoglobulins include several classes of molecules, such as the large family of molecules of IgD, IgG, IgM and IgE, which are polypeptides or proteins in nature, thus the term "immunoglobulin molecule" in this application includes, for example, hybrid antibodies or altered antibodies and its fragments. Fragments of naturally occurring antibodies have been shown to perform the antigen-binding function of the antibody. These fragments are collectively referred to as "antigen binding units". Antigen-binding units can be roughly classified into "single-chain ("Sc") and "non-single-chain" ("Nsc") types according to their molecular structure.

本文中所用的术语“核氨酸”、“多肽”、“抗体”可以互换。The terms "nucleic acid", "polypeptide", "antibody" are used interchangeably herein.

术语“抗体”还包括各种物种来源，包括无脊椎动物和脊椎动物的免疫球蛋白分子。应用于抗体或抗原结合单位的术语“人”指人基因表达的免疫球蛋白分子或其片段。应用于非人(例如，啮齿类或灵长类)抗体的术语“人源化”是含有衍生自非人免疫球蛋白的最少序列的杂交免疫球蛋白、免疫球蛋白链或其片段。人源化抗体的大部分是人免疫球蛋白(受者抗体)，其中受者的互补决定区(CDR)的残基被具有所需特异性、亲和力和性能的非人物种(供者抗体)，例如小鼠、大鼠、家兔或灵长类的CDR的残基替代。在一些情况中，人免疫球蛋白的Fv框架区(FR)残基被相应的非人残基替代。此外，人源化抗体可包含即未在受者抗体，又未在输入CDR或框架区序列中的发现的残基。作这些修饰以进一步精制和优化抗体性能并最大程度降低引入人体时的免疫原型。人源化抗体通常包含基本上所有、至少一个、通常两个可变区，其中所有或基本上所有CDR区对用于非人免疫球蛋白的那些，所有或基本上所有FR区是人免疫球蛋白序列的那些。人源性抗体还可包含免疫球蛋白恒定区(Fc)，通常是人免疫球蛋白的恒定区的至少一部分。The term "antibody" also includes immunoglobulin molecules of various species origin, including invertebrates and vertebrates. The term "human" as applied to antibodies or antigen binding units refers to human gene-expressed immunoglobulin molecules or fragments thereof. The term "humanized" as applied to non-human (eg, rodent or primate) antibodies is a hybrid immunoglobulin, immunoglobulin chain or fragment thereof containing minimal sequence derived from a non-human immunoglobulin. The majority of humanized antibodies are human immunoglobulins (recipient antibodies) in which residues in the complementarity determining regions (CDRs) of the recipient are replaced by non-human species with the desired specificity, affinity and properties (donor antibodies) , eg, substitution of residues in CDRs of mouse, rat, rabbit or primate. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, a humanized antibody may contain residues found neither in the recipient antibody nor in the imported CDR or framework region sequences. These modifications are made to further refine and optimize antibody performance and minimize immunogenicity when introduced into humans. Humanized antibodies typically comprise substantially all, at least one, usually two variable regions, wherein all or substantially all CDR regions to those used for non-human immunoglobulins, and all or substantially all FR regions are human immunoglobulins those of the protein sequence. Human antibodies may also comprise an immunoglobulin constant region (Fc), typically at least a portion of the constant region of a human immunoglobulin.

就多肽-抗体偶联物而言，本文的术语荧光“信噪比”指(偶联体的荧光信号，其中所述偶联体包括的多肽与一抗体结合，进而与本发明化合物标记的结合剂结合)/(多肽，同种型对照抗体和标记结合剂的混合物的荧光信号)之比。With respect to polypeptide-antibody conjugates, the term fluorescence "signal-to-noise ratio" as used herein refers to the fluorescence signal of a conjugate comprising a polypeptide that binds to an antibody, which in turn binds to the label of the compound of the present invention. agent binding)/(fluorescence signal of a mixture of polypeptide, isotype control antibody and labeled binding agent).

术语“标记程度”或“DOL”指每个靶分子(包括但不限于多肽和寡核苷酸)连接的染料分子的数量。例如，每个多肽，例如抗体一个染料分子标示标记程度(DOL)为1。如果平均多于一个染料分子与多肽，例如多肽，如抗体反应并与之偶联，标记程度大约1，还可以是除以整数以外的数值。DOL的数值越高，标记程度越高。The term "degree of labeling" or "DOL" refers to the number of dye molecules attached per target molecule (including but not limited to polypeptides and oligonucleotides). For example, one dye molecule per polypeptide, such as an antibody, is marked with a degree of labeling (DOL) of 1. If, on average, more than one dye molecule is reacted with and conjugated to a polypeptide, eg, a polypeptide, such as an antibody, the degree of labeling is about 1, and may also be a value other than an integer. The higher the value of DOL, the higher the degree of marking.

本发明中所述的紫外光激发荧光染料，具有通式Ⅰ的结构：The ultraviolet light-excited fluorescent dye described in the present invention has the structure of general formula I:

通式Ⅰ中，X、Y和Z各自独立选自：C、CR¹、N、NR³、S、O、Se、P、Si，As、C-C、CR¹-CR²、NR³-NR⁴、S-S,此处，X、Y和Z中至少一个不是C或C-C；作为优选地，选自C、CR¹、N、S、O；进一步优选，X优选为C或CR¹，Y优选为N，Z优选为O或S，R¹优选为磺酸基。其中，磺酸基可以是磺酸、磺酸胺或具有反应活性的磺酰胺。In general formula I, X, Y and Z are each independently selected from: C, CR¹ , N, NR³ , S, O, Se, P, Si, As, CC, CR¹ -CR² , NR³ -NR⁴ , SS, here, at least one of X, Y and Z is not C or CC; as preferably, selected from C, CR¹ , N, S, O; further preferably, X is preferably C or CR¹ , and Y is preferably N, Z are preferably O or S, and R¹ is preferably a sulfonic acid group. Among them, the sulfonic acid group can be sulfonic acid, sulfonic acid amine or reactive sulfonic acid amide.

通式Ⅰ中，W是N或C；优选为N。In formula I, W is N or C; preferably N.

通式Ⅰ中，R₁、R₂、R₃、R₄、R₅、R₆、R₇、R₉和R₁₀各自独立选自：H、C_1-8烷基、C_1-8取代烷基、C_1-8烷氧基、羟基、磺酸基、S、卤素、氨基、取代氨基、醛基、羧基、酯基、叠氮基、亚硝基、氰基、硫醚基、C_5-7环、C_5-7含杂环、取代的C_5-7环、取代的C_5-7含杂环、活性基团或水溶性聚合物基团，或者R₁、R₂、R₃、R₄、R₅、R₆、R₇、R₉和R₁₀中的R₁与R₂、R₃与R₄、R₅与R₆和R₉与R₁₀分别连接形成饱和或者不饱和的五元环、六元环或七元环；作为优选地，选自H、磺酸基、活性基团、水溶性聚合物基团；进一步优选，R₁、R₂、R₅、R₆、R₇、R₉和R₁₀优选为H，R₃和R₄各自独立优选为磺酸基、活性基团或水溶性聚合物基团。其中，磺酸基可以是磺酸、磺酸胺或具有反应活性的磺酰胺。In general formula I, R₁ , R₂ , R₃ , R₄ , R₅ , R₆ , R₇ , R₉ and R₁₀ are each independently selected from: H, C_1-8 alkyl, C_1-8 substituted Alkyl, C_1-8 alkoxy, hydroxyl, sulfonic acid, S, halogen, amino, substituted amino, aldehyde, carboxyl, ester, azide, nitroso, cyano, thioether, C_5-7 ring, C_5-7 containing heterocycle, substituted C_5-7 ring, substituted C_5-7 containing heterocycle, reactive group or water-soluble polymer group, or R₁ , R₂ , R₃ , R₄ , R₅ , R₆ , R₇ , R₉ and R₁₀ in R₁ and R₂ , R₃ and R₄ , R₅ and R₆ , and R₉ and R₁₀ are respectively connected to form a saturated or non- Saturated five-membered ring, six-membered ring or seven-membered ring; preferably, selected from H, sulfonic acid group, active group, water-soluble polymer group; more preferably, R₁ , R₂ , R₅ , R₆ , R₇ , R₉ and R₁₀ are preferably H, and R₃ and R₄ are each independently preferably a sulfonic acid group, a reactive group or a water-soluble polymer group. Among them, the sulfonic acid group can be sulfonic acid, sulfonic acid amine or reactive sulfonic acid amide.

通式Ⅰ中，R₈为H、C_1-8烷基、C_1-8取代烷基、C_5-7环、C_5-7含杂环、取代的C_5-7环、取代的C_5-7含杂环、活性基团或水溶性聚合物基团，或者与R₇连接形成饱和或者不饱和的五元环、六元环或七元环；作为优选地，选自磺酸基、活性基团、水溶性聚合物基团。其中，磺酸基可以是磺酸、磺酸胺或具有反应活性的磺酰胺。In general formula I, R₈ is H, C_1-8 alkyl, C_1-8 substituted alkyl, C_5-7 ring, C_5-7 containing heterocycle, substituted C_5-7 ring, substituted C_5-7 contains a heterocyclic ring, an active group or a water-soluble polymer group, or connects with R₇ to form a saturated or unsaturated five-membered ring, six-membered ring or seven-membered ring; preferably, selected from sulfonic acid group , active groups, water-soluble polymer groups. Among them, the sulfonic acid group can be sulfonic acid, sulfonic acid amine or reactive sulfonic acid amide.

通式Ⅰ中，R¹-R⁴各自独立选自：H、C_1-8烷基、C_1-8取代烷基、C_1-8烷氧基、羟基、-SO₃^-、卤素、氨基、取代氨基、醛基、羧基、酯基、叠氮基、硝基、亚硝基、氰基或硫醚基；作为优选地，选为-SO₃^-。In the general formula I, R¹ -R⁴ are each independently selected from: H, C_1-8 alkyl, C_1-8 substituted alkyl, C_1-8 alkoxy, hydroxyl, -SO₃^- , halogen, amino , a substituted amino group, an aldehyde group, a carboxyl group, an ester group, an azide group, a nitro group, a nitroso group, a cyano group or a thioether group; preferably, it is -SO₃^- .

在R₁、R₂、R₃、R₄、R₅、R₆、R₇、R₈、R₉和R₁₀中，存在至少一个活性基团和至少一个水溶性聚合物基团。Among R₁ , R₂ , R₃ , R₄ , R₅ , R₆ , R₇ , R₈ , R₉ and R₁₀ , at least one reactive group and at least one water-soluble polymer group are present.

活性基团可以是丙烯酰胺、羧酸的活化酯、羧酸酯、酰基叠氮、酰基氰、醛、卤素取代芳环、酸酐、氨基、叠氮、氮杂环丙烷、硼化物、重氮烷、卤代乙酰胺、卤代烷、卤代三嗪、肼、亚胺酯、异氰酸、异硫氰酸、马来酰亚胺、亚磷酰胺、活性钯络合物、卤代硅烷、酰卤或硫醇、四氟苯酚酯或炔烃；作为优选地，活性基团为包含羧酸的丁二酰亚胺酯、肼、氨基或马来酰亚胺。The reactive group can be acrylamide, activated ester of carboxylic acid, carboxylic acid ester, acyl azide, acyl cyanide, aldehyde, halogen substituted aromatic ring, acid anhydride, amino, azide, aziridine, boride, diazane , haloacetamide, haloalkane, halotriazine, hydrazine, imidoester, isocyanic acid, isothiocyanic acid, maleimide, phosphoramidite, active palladium complex, halosilane, acid halide Or thiol, tetrafluorophenol ester or alkyne; as preferably, the active group is succinimide ester containing carboxylic acid, hydrazine, amino or maleimide.

水溶性聚合物基团可以是聚环氧乙烷、糖类、多肽、聚乙二醇；作为优选地，水溶性聚合物基团为分子量为300-3000的离散型聚乙二醇。The water-soluble polymer group can be polyethylene oxide, saccharide, polypeptide, polyethylene glycol; preferably, the water-soluble polymer group is discrete polyethylene glycol with a molecular weight of 300-3000.

当上述优选条件的组合，所得到的通式Ⅰ的化合物，是本发明中关于紫外光激发荧光染料的优选化合物。应理解，本发明包括上述各优选基团的任意组合，其组合可举例为，当X优选为C，Y优选为N，Z优选为O，W优选为N，R₁、R₂、R₅、R₆、R₇、R₉和R₁₀优选为H，R₃优选为水溶性聚合物基团，R₄优选为磺酸基，R₈优选为活性基团时，所获得的化合物视为本发明代表性的优选化合物，如下结构的化合物5、化合物6A、化合物6B、化合物6C和化合物7为该组合的优选化合物五种。化合物6A、化合物6B和化合物6C都属于化合物5的衍生物，化合物5、化合物6A、化合物6B和化合物6C的区别是R₈的活性基团不同。化合物5和化合物7的区别是R₃的水溶性聚合物基团(聚乙二醇)的分子量不同，化合物5、化合物6A、化合物6B、化合物6C和化合物7的主体结构相同，所以，它们的吸收光谱和发射光谱基本一致。应当理解为该组合的优选化合物不限于此。如下为化合物5、化合物6A、化合物6B、化合物6C和化合物7的结构式：When the above-mentioned preferred conditions are combined, the obtained compound of general formula I is the preferred compound for the ultraviolet light excited fluorescent dye in the present invention. It should be understood that the present invention includes any combination of the above-mentioned preferred groups, and the combination can be exemplified, when X is preferably C, Y is preferably N, Z is preferably O, W is preferably N, R₁ , R₂ , R₅ , R₆ , R₇ , R₉ and R₁₀ are preferably H, R₃ is preferably a water-soluble polymer group, R₄ is preferably a sulfonic acid group, and R₈ is preferably an active group, the obtained compound is regarded as Representative preferred compounds of the present invention,compound 5, compound 6A, compound 6B, compound 6C and compound 7 of the following structures are the five preferred compounds of the combination. Compound 6A, compound 6B and compound 6C are all derivatives ofcompound 5. The difference betweencompound 5, compound 6A, compound 6B and compound 6C is that the active group of R₈ is different. The difference betweencompound 5 and compound 7 is that the molecular weight of the water-soluble polymer group (polyethylene glycol) of R₃ is different. The main structures ofcompound 5, compound 6A, compound 6B, compound 6C and compound 7 are the same, so their The absorption and emission spectra are basically the same. It should be understood that the preferred compounds of the combination are not limited thereto. The following are the structural formulas ofcompound 5, compound 6A, compound 6B, compound 6C and compound 7:

当X优选为C，Y优选为N，Z优选为S，W优选为N，R₁、R₂、R₅、R₆、R₇、R₉和R₁₀优选为H，R₃优选为水溶性聚合物基团，R₄优选为磺酸基，R₈优选为活性基团时，所获得的化合物视为本发明代表性的优选化合物，如下结构的化合物8为该组合的优选化合物的一种，化合物8的结构为：When X is preferably C, Y is preferably N, Z is preferably S, W is preferably N, R₁ , R₂ , R₅ , R₆ , R₇ , R₉ and R₁₀ are preferably H, and R₃ is preferably water-soluble When R₄ is preferably a sulfonic acid group, and R₈ is preferably a reactive group, the obtained compound is regarded as a representative preferred compound of the present invention, and thecompound 8 of the following structure is one of the preferred compounds of the combination species, the structure ofcompound 8 is:

活性基团active group

本发明化合物包括至少一个为活性基团。反应活性基团及其反应偶联体可以分别是亲电体和亲核体，其能够与或不与偶联剂或催化剂形成共价键。例如，所述当进行紫外光活化或光解反应活性基团时其与烃分子反应的光活化活性基团，所述反应性基团为能够与共轭二烯烃经由第尔斯-阿德反应(Diels-Alder Reaction)进行反应的亲双稀体、能够与亲双稀体反应的1，3-二烯、能够与叠氮官能团反应形成1，2，3-三唑连接键的炔烃和能够与叠氮官能团经由所谓的Staudinger反应形成酰胺连接键的2-(二苯基膦基)苯甲酸甲酯。The compounds of the present invention include at least one group that is reactive. The reactive groups and their reactive conjugates can be electrophiles and nucleophiles, respectively, which are capable of forming covalent bonds with or without a coupling agent or catalyst. For example, the photoactive active group that reacts with hydrocarbon molecules when the active group is activated by ultraviolet light or photolytically decomposed, the reactive group is capable of reacting with the conjugated diene via Diels-Alder reaction ( Diels-Alder Reaction), 1,3-dienes capable of reacting with dialnophiles, alkynes capable of reacting with azide functional groups to form 1,2,3-triazole linkages, and alkynes capable of reacting with azide functions Methyl 2-(diphenylphosphino)benzoate forming an amide linkage via a so-called Staudinger reaction with the azide function.

反应活性基团和载体分子上的反应偶联体/底物的共价连接键实例如下表1所示：Examples of covalent linkages between reactive groups and reactive conjugates/substrates on the carrier molecule are shown in Table 1 below:

表1活性基团与载体分子连接的共价键Table 1 Covalent bond between active group and carrier molecule

本领域所理解的活化酯通常具有式-COΩ,其中Ω为良好的离去基团，如琥珀酰亚胺基氧基(-OC₄H₄O₂)、磺基琥珀酰亚胺基氧基(-OC₄H₃O₂-SO₃H)或苯并三唑基-1-氧基(-OC₆H₄N₃)、芳基氧基或经吸电子取代基(如硝基、氟、氯、氰基、三氟甲基或其组合)取代一次或多次的芳基氧基和由碳二亚胺活化的羧酸，从而形成酸酐或混合酸酐-OCORa或-OCNRaNHRb，其中Ra和Rb，可以是相同的或不同的独立为C1-C6烷基、C1-C6全氟烷基或C1-C6烷氧基、环己基、3-二甲基氨基丙基或N-吗啉代乙基。Activated esters as understood in the art generally have the_formula-COΩ , where Ω is a good leaving group such as succinimidyloxy (_-OC4H4O2 ), sulfosuccinimidyloxy (-OC₄ H₃ O₂ -SO₃ H) or benzotriazolyl-1-oxyl (-OC₆ H₄ N₃ ), aryloxy or electron withdrawing substituents such as nitro, fluorine , chlorine, cyano, trifluoromethyl, or a combination thereof) substituted one or more aryloxy and carbodiimide-activated carboxylic acids to form anhydrides or mixed anhydrides -OCORa or -OCNRaNHRb, where Ra and Rb, which may be the same or different independently, are C1-C6 alkyl, C1-C6 perfluoroalkyl or C1-C6 alkoxy, cyclohexyl, 3-dimethylaminopropyl or N-morpholinoethyl base.

酰基叠氮也可重排为异氰酸酯。Acyl azides can also rearrange to isocyanates.

反应活性基团可以是与胺、硫醇、羟基或醛反应的基团。反应活性基团可以是胺-反应活性基团，如琥珀酰亚胺基酯(SE)，或硫醇-反应活性基团，如马来酰亚胺、卤代乙酰胺或甲基硫代磺酸酯(methylthiosulfonate，MTS)，醛-反应活性基团，如胺、氨基氧基(aminooxy)或酰肼。Reactive groups can be groups that react with amines, thiols, hydroxyls, or aldehydes. The reactive group can be an amine-reactive group, such as succinimidyl ester (SE), or a thiol-reactive group, such as maleimide, haloacetamide, or methylthiosulfone Methylthiosulfonate (MTS), aldehyde-reactive group such as amine, aminooxy or hydrazide.

水溶性聚合物water soluble polymer

本发明化合物还包括至少一个水溶性聚合物基团。根据本发明，水溶性聚合物基团可显著降低荧光基团核心结构的分子内移动性，以及由此可改善荧光基团的荧光量子产率。所述基团可赋予它们所连接的化合物其它特性，如荧光基团的耐光性改善，针对生物分子标记时荧光基团聚集减少，荧光标记的生物分子(如抗体)的染色特异性增加；和体内标记的生物分子(如抗体)的免疫原性和抗原性降低。The compounds of the present invention also include at least one water-soluble polymer group. According to the present invention, the water-soluble polymer group can significantly reduce the intramolecular mobility of the core structure of the fluorophore, and thus can improve the fluorescence quantum yield of the fluorophore. The groups may impart other properties to the compounds to which they are attached, such as improved light resistance of the fluorophore, reduced aggregation of the fluorophore for labeling of biomolecules, and increased staining specificity of the fluorescently-labeled biomolecules (eg, antibodies); and The immunogenicity and antigenicity of labeled biomolecules (eg, antibodies) in vivo are reduced.

各水溶性聚合物基团通常为足够大以改善化合物的荧光特性且基本上无反应活性的、水溶性部分。在该上下文中使用的术语“聚合物”不要求存在严格重复单元。就本发明的目的而言，具有足够分子大小和溶解性但不具有重复单元的分子就视为“水溶性聚合物基团”。Each water-soluble polymer group is typically a substantially non-reactive, water-soluble moiety large enough to improve the fluorescent properties of the compound. The term "polymer" used in this context does not require the presence of strictly repeating units. Molecules that have sufficient molecular size and solubility but do not have repeating units are considered "water-soluble polymer groups" for the purposes of the present invention.

水溶性聚合物基团包括但不限于有机聚合物和生物分子如多肽和碳水化合物。各水溶性聚合物可以是线性的、支链的、环状的或它们的组合。可以使用具有一条、二条、三条、四条或更多支链的水溶性聚合物。本发明化合物可包括任何数目的水溶性聚合物基团。一般而言，本发明化合物包括至少一个水溶性聚合物基团至约8个水溶性聚合物基团。在一个化合物中个水溶性聚合物基团的合适分子量，或可供选择的，所有水溶性聚合物基团的合适分子量可为约100、200、300、400、500、600、700、800、900、1000、1500、2000、2500、3000、3500、4000、4500、5000、10000、15000、20000Da或更大。在一个实施方案中，本发明的水溶性聚合物基团分子量在300和3000之间。Water-soluble polymer groups include, but are not limited to, organic polymers and biomolecules such as polypeptides and carbohydrates. Each water-soluble polymer can be linear, branched, cyclic, or a combination thereof. Water-soluble polymers with one, two, three, four or more branches can be used. The compounds of the present invention may include any number of water-soluble polymer groups. In general, the compounds of the present invention include from at least one water-soluble polymer group to about 8 water-soluble polymer groups. Suitable molecular weights for one water-soluble polymer group in a compound, or alternatively, suitable molecular weights for all water-soluble polymer groups may be about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 10000, 15000, 20000Da or larger. In one embodiment, the molecular weight of the water-soluble polymer groups of the present invention is between 300 and 3000.

在一实施方案中，本发明的水溶性聚合物为聚环氧烷。合适的聚环氧烷包括聚乙二醇(PEG)、聚丙二醇(PPG)、聚乙二醇-聚丙二醇(PEG-PPG)共聚物和含N上经取代的甲基丙烯酰胺的聚合物和共聚物。适用于本发明的各种聚环氧烷以及制备和使用所述聚环氧烷的方法描述在下列文献中：US637749；5650388；5298643；5605976；5567422；5681567；5321095；5349001；5405877；5234903；5478805；5324844；5612460；5756593；5686110；5880131；6824782；5808096；6013283；6251382。商业来源的聚环氧烷试剂包括Sigma-Aldrich、Nanocs、Creative Biochem、Pierce、Enzon、Nektar和Nippon Oils and Fats。聚环氧烷具有如下基本结构：In one embodiment, the water-soluble polymer of the present invention is a polyalkylene oxide. Suitable polyalkylene oxides include polyethylene glycol (PEG), polypropylene glycol (PPG), polyethylene glycol-polypropylene glycol (PEG-PPG) copolymers and N-substituted methacrylamide containing polymers and copolymer. Various polyalkylene oxides suitable for use in the present invention and methods of making and using them are described in the following documents: US637749; 5650388; 5298643; 5605976; 5567422; 5681567; ;5324844;5612460;5756593;5686110;5880131;6824782;5808096;6013283;6251382. Commercially available polyalkylene oxide reagents include Sigma-Aldrich, Nanocs, Creative Biochem, Pierce, Enzon, Nektar and Nippon Oils and Fats. Polyalkylene oxide has the following basic structure:

必要时聚环氧烷可经额外取代以赋予聚合物其它期望特性。所述修饰可包括例如：增加或降低聚合物的化学稳定性的化学连接键，这可允许调整聚合物的半衰期的化学或生物学稳定性。在一些情形下，聚环氧烷分子被各种基团端接或封端。所述基团的实例为羟基、烷基醚(如甲醚、乙醚、丙基醚)、羧基甲基醚、羟基乙基醚、苄基醚、二苄基亚甲基醚或二甲基胺。聚环氧烷可具有多个可能的端基中的一个，所述端基包括但不限于羟基、甲基醚、乙基醚、羧基甲基醚和羟基乙基醚。在一实施方案中，聚环氧烷为用据甲基醚端接的聚乙二醇聚合物。这样的基团被成为m PEG。m PEG通常具有式-(CH₂CH₂O)nCH₃,其中n为乙二醇的单元数目，并且由所述m PEG的大小决定。The polyalkylene oxides can be additionally substituted as necessary to impart other desirable properties to the polymer. The modifications may include, for example, chemical linkages that increase or decrease the chemical stability of the polymer, which may allow for chemical or biological stability to adjust the half-life of the polymer. In some cases, the polyalkylene oxide molecules are terminated or capped with various groups. Examples of such groups are hydroxy, alkyl ethers (eg methyl ether, diethyl ether, propyl ether), carboxymethyl ether, hydroxyethyl ether, benzyl ether, dibenzylmethylene ether or dimethylamine . The polyalkylene oxide may have one of a number of possible end groups including, but not limited to, hydroxy, methyl ether, ethyl ether, carboxymethyl ether, and hydroxyethyl ether. In one embodiment, the polyalkylene oxide is a polyethylene glycol polymer terminated with methyl ether. Such a group is called mPEG. mPEGs typically have the_formula -(_CH2CH2O )_nCH3 , where n is the number of units of ethylene glycol and is determined by the size of the mPEG.

其他合适的聚合物包括一下物质的衍生物和络合物：聚(2-羟基乙基甲基丙烯酸酯)、聚羟基丙基甲基丙烯酰胺、聚(苯乙烯磺酸)、聚(乙烯醇)或聚(碘化2-乙烯基-N-甲基吡啶鎓盐)。Other suitable polymers include derivatives and complexes of poly(2-hydroxyethyl methacrylate), polyhydroxypropyl methacrylamide, poly(styrene sulfonic acid), poly(vinyl alcohol) ) or poly(2-vinyl-N-methylpyridinium iodide).

本发明的水溶性聚合物也可以为碳水化合物。所述碳水化合物包括单糖或多糖，且可以是例如，可溶淀粉、糖原、葡萄糖、果胶、甘露聚糖、半乳聚糖、羟甲基纤维素、羟乙基纤维素和其它纤维素衍生物。当水溶性聚合物是碳水化合物时，碳水化合物中存在至少30％的羟基可被掩蔽为甲基醚、磺酸根合烷基醚和/或乙酸酯。The water-soluble polymers of the present invention may also be carbohydrates. The carbohydrates include monosaccharides or polysaccharides, and can be, for example, soluble starch, glycogen, glucose, pectin, mannan, galactan, hydroxymethyl cellulose, hydroxyethyl cellulose, and other fibers Derivatives. When the water-soluble polymer is a carbohydrate, at least 30% of the hydroxyl groups present in the carbohydrate can be masked as methyl ethers, sulfonate alkyl ethers and/or acetates.

本发明的水溶性聚合物也可为为多肽。合适的多肽可包括例如，丝氨酸、精氨酸、含有修饰的ε-氨基的多聚赖氨酸或半胱氨酸。所述多肽的其它实例在WO 2006/08249中有披露。预期的是所述多肽可用于作本发明的水溶性聚合物。The water-soluble polymer of the present invention may also be a polypeptide. Suitable polypeptides may include, for example, serine, arginine, polylysine containing a modified epsilon-amino group, or cysteine. Other examples of such polypeptides are disclosed in WO 2006/08249. It is contemplated that the polypeptides can be used as water-soluble polymers of the present invention.

本发明的水溶性聚合物还包括上述不同类型物质的组合。例如，这样的水溶性聚合物可以是与聚环氧烷连接的多肽。The water-soluble polymers of the present invention also include combinations of the different types of materials described above. For example, such a water-soluble polymer can be a polypeptide linked to a polyalkylene oxide.

水溶性聚合物通常不包括能与本发明化合物中包括的一个或多个反应性基团的化学不相容的任何一种或多种基团。例如，若反应活性基团为亲电体，则水溶性聚合物不应包括强亲核体。在更具体的实例中，若反应活性基团为N-羟基琥珀酰亚胺基酯，则水溶性聚合物不应包括伯胺或仲胺基团；当反应活性基团为马来酰亚胺时，水溶性聚合物不应包括巯基。同样的，若反应活性基团为亲核体，则水溶性聚合物基团中不应该包括强亲电体。然而，水溶性聚合物可包括最少数量的弱亲核体或做小数目的弱亲电体，从而在储存或处理期间反应活性基团的化学不受到显著影响，或本发明化合物的稳定性不受影响。弱亲核体的实例为羟基，其通常存在于碳水化合物中。由此，水溶性聚合物是碳水化合物时，碳水化合物中存在至少30％的羟基可被掩蔽为甲基醚、磺酸根合烷基醚和/或乙酸酯。Water-soluble polymers generally do not include any one or more groups that would be chemically incompatible with one or more of the reactive groups included in the compounds of the present invention. For example, if the reactive group is an electrophile, the water-soluble polymer should not include a strong nucleophile. In a more specific example, if the reactive group is an N-hydroxysuccinimidyl ester, the water-soluble polymer should not include primary or secondary amine groups; when the reactive group is a maleimide , the water-soluble polymer should not include mercapto groups. Likewise, if the reactive group is a nucleophile, the water-soluble polymer group should not include a strong electrophile. However, the water-soluble polymer may include a minimum number of weak nucleophiles or a small number of weak electrophiles so that the chemistry of the reactive groups is not significantly affected during storage or handling, or the stability of the compounds of the present invention is not Affected. An example of a weak nucleophile is a hydroxyl group, which is usually present in carbohydrates. Thus, when the water-soluble polymer is a carbohydrate, at least 30% of the hydroxyl groups present in the carbohydrate can be masked as methyl ethers, sulfonate alkyl ethers and/or acetates.

载体分子carrier molecule

在一个实施案例中，本发明染料与载体分子偶联。此处包括但不限于任何本发明所揭示化合物和任何在此处揭示的载体分子。典型的，本发明化合物含有活性基团与载体分子共价偶联。活性基团可以包含活性功能部分和一个连接部分或者仅仅是活性功能部分。本发明化合物可以应用于各种载体分子包括但不仅限于：氨基酸、多肽、蛋白质、多糖、核苷、核苷酸、寡核苷酸、核酸、抗原、类固醇、维生素、药物、代谢物、毒素、环境污染物、半抗原、脂素、药物、激素、脂质、脂质体、葡聚糖、合成聚合物、聚合微粒、生物细胞、病毒或者其组合形式。In one embodiment, the dyes of the present invention are coupled to carrier molecules. Included herein, but not limited to, any of the compounds disclosed herein and any carrier molecule disclosed herein. Typically, the compounds of the present invention contain reactive groups covalently coupled to carrier molecules. The reactive group may comprise the reactive functional moiety and a linking moiety or only the reactive functional moiety. The compounds of the present invention can be applied to various carrier molecules including but not limited to: amino acids, polypeptides, proteins, polysaccharides, nucleosides, nucleotides, oligonucleotides, nucleic acids, antigens, steroids, vitamins, drugs, metabolites, toxins, Environmental pollutants, haptens, liposomes, drugs, hormones, lipids, liposomes, dextran, synthetic polymers, polymeric microparticles, biological cells, viruses, or combinations thereof.

优先的载体分子为抗体或者部分抗体，抗原，亲和素，链酶亲和素，生物素，葡聚糖，IgG结合蛋白，荧光蛋白，琼脂糖和非生物亲和颗粒。Preferred carrier molecules are antibodies or partial antibodies, antigens, avidin, streptavidin, biotin, dextran, IgG binding proteins, fluorescent proteins, agarose and non-biological affinity particles.

在某些情况下，载体分子可能包含载体分子活性基团，包括但不限于羟基，羰基，氨基，巯基，醛基，卤素，硝基，氰基，酰胺，脲，碳酸，氨基甲酸酯，异氰酸酯，砜，磺胺，亚砜等，用来与本发明化合物共价相连。常用的活性基团在上面已经有所表述用来与这些载体分子上的活性部分相连。In some cases, the carrier molecule may contain carrier molecule reactive groups, including but not limited to hydroxyl, carbonyl, amino, sulfhydryl, aldehyde, halogen, nitro, cyano, amide, urea, carbonic acid, carbamate, Isocyanates, sulfones, sulfonamides, sulfoxides, etc., are used to covalently link the compounds of the present invention. Common reactive groups have been described above for attachment to reactive moieties on these carrier molecules.

“染料-偶联物”的制备Preparation of "dye-conjugates"

一种本发明的紫外光激发荧光染料与载体分子或者固相支撑物偶联的方法，包括如下步骤：A method for coupling an ultraviolet light-excited fluorescent dye to a carrier molecule or a solid-phase support of the present invention comprises the following steps:

(1)将载体分子或者固相支撑物与染料接触形成结合样品；(1) contacting the carrier molecule or the solid phase support with the dye to form a binding sample;

(2)将结合样品孵化一定时间使得所述的通式Ⅰ化合物与载体分子或者固相支撑物之间形成共价键，获得染料-偶联物的混合物；(2) incubating the binding sample for a certain period of time so that a covalent bond is formed between the compound of the general formula I and the carrier molecule or the solid phase support to obtain a dye-conjugate mixture;

(3)将染料-偶联物的混合物中未连接载体分子或者固相支撑物的化合物和已连接在载体分子或者固相支撑物的化合物进行分离，获得染料-偶联物。(3) The dye-conjugate mixture is separated from the compound that is not connected to the carrier molecule or the solid phase support and the compound that has been connected to the carrier molecule or the solid phase support to obtain the dye-conjugate.

染料与载体分子或固相支撑物的偶联物，例如：药物，多肽，毒素，核酸，磷脂和其它通过有机合成方法制备的有机物与本发明活性染料形成偶联物。更加优化的条件是，含有本发明的活性化合物在合适的溶剂中，本发明活性化合物与底物均是可溶的，他们之间可以形成化学键。此化学反应的进行可以在室温或者低温下同步进行。对于具有光活性的本发明化合物，偶联反应的进行需要通过合适的光照来照射反应混合物，通过光照来活化本发明活性化合物从而使其与底物形成偶联。通过化学改性得到的水不溶性底物可以在非质子溶剂例如DMF，DMSO，丙酮，乙酸乙酯，甲苯或氯仿中进行反应。通过使用具有高效活性的化合物使用相似的方法对水溶性材料的改性可以增加其在有机溶剂中的溶解性能。The conjugates of dyes and carrier molecules or solid supports, such as: drugs, polypeptides, toxins, nucleic acids, phospholipids and other organic substances prepared by organic synthesis methods, form conjugates with the reactive dyes of the present invention. A more optimized condition is that the active compound of the present invention is contained in a suitable solvent, and the active compound of the present invention and the substrate are both soluble, and a chemical bond can be formed between them. The chemical reaction can be carried out simultaneously at room temperature or low temperature. For photoactive compounds of the present invention, the coupling reaction requires that the reaction mixture is irradiated with suitable light, which activates the active compound of the present invention to form a coupling with the substrate. The water-insoluble substrates obtained by chemical modification can be reacted in aprotic solvents such as DMF, DMSO, acetone, ethyl acetate, toluene or chloroform. Modification of water-soluble materials using similar methods by using compounds with high activity can increase their solubility in organic solvents.

制备多肽或蛋白的偶联物，典型的步骤是把蛋白在室温或低温下以1-10mg/ml溶解在偶联水缓冲液中.碳酸氢盐缓冲体系(pH为8.3)特别适合与琥珀酰亚胺酯反应，磷酸盐缓冲溶液(pH为7.2-8)特别适合与硫醇-活性官能团之间的偶联，碳酸盐或硼酸盐缓冲溶液(pH为9)特别适合异硫氰酸酯和二氯三嗪的偶联。合适的活性化合物以合适的偶联度所需要的量溶解在无水溶解(常用DMSO或DMF)中，然后将其加入蛋白溶液中进行偶联。活性化合物的加入量预先是经过实验计算出所需的量，偶联产物经过柱色谱去除未偶联的产物而偶联产物可以在需要的应用中进行应用研究。To prepare conjugates of polypeptides or proteins, a typical procedure is to dissolve the protein at 1-10 mg/ml in conjugated water buffer at room temperature or low temperature. Bicarbonate buffer system (pH 8.3) is particularly suitable for succinyl Imidoester reaction, phosphate buffer solution (pH 7.2-8) is particularly suitable for coupling with thiol-reactive functional groups, carbonate or borate buffer solution (pH 9) is particularly suitable for isothiocyanate Coupling of Esters and Dichlorotriazine. The appropriate active compound is dissolved in anhydrous solubilization (usually DMSO or DMF) in the amount required for the appropriate degree of coupling, which is then added to the protein solution for coupling. The amount of active compound added is pre-calculated by experiments, and the coupled product is subjected to column chromatography to remove uncoupled products, and the coupled product can be used for application research in required applications.

把活性化合物加入组分溶液中，混合物需要经过合适时间的孵化(一般是在室温下孵化1h或者冰浴下孵化数小时)，多余的组分需要通过过滤，透析，HPLC，使用离子交换或疏水性聚合物或其它合适材料吸附。偶联物可以在溶液或冻干后应用。通过这种方式可以制备抗体，部分抗体，抗生物素蛋白，血凝素，酶，蛋白A和G,细胞蛋白，白蛋白，组蛋白，生长因子，激素和其它蛋白的偶联物。The active compound is added to the solution of the components, the mixture needs to be incubated for a suitable time (usually 1h at room temperature or several hours in an ice bath), and the excess components need to be filtered, dialysis, HPLC, using ion exchange or hydrophobic. Adsorbed by polymers or other suitable materials. Conjugates can be applied in solution or after lyophilization. In this way, conjugates of antibodies, partial antibodies, avidin, hemagglutinin, enzymes, proteins A and G, cellular proteins, albumin, histones, growth factors, hormones and other proteins can be prepared.

在一个实施例中，将与本发明的化合物反应的多肽包括3到约80个氨基酸。此类多肽的实例包括但不限于神经肽、细胞因子、毒素和肽酶或蛋白酶底物。荧光标记的神经肽、细胞因子和毒素可用于绘图或显现各肽的特异性受体的分布。作为一个实例，当用本发明的化合物标记时，鬼笔环肽(一种具有环肽结构的毒素)可以用于染色细胞中的F-肌动蛋白丝。作为另一个实例，当用本发明的荧光基团标记时，α-银环蛇毒素(一种基于肽的蛇毒素)可以用于检测乙酰胆碱受体。用本发明的荧光基团标记的肽酶或蛋白酶底物可用于测定肽酶或蛋白酶的活性，并用于筛选设计为肽酶或蛋白酶抑制剂的药物。In one embodiment, the polypeptides to be reacted with the compounds of the present invention comprise from 3 to about 80 amino acids. Examples of such polypeptides include, but are not limited to, neuropeptides, cytokines, toxins, and peptidase or protease substrates. Fluorescently labeled neuropeptides, cytokines, and toxins can be used to map or visualize the distribution of specific receptors for each peptide. As an example, phalloidin, a toxin with a cyclic peptide structure, can be used to stain F-actin filaments in cells when labeled with the compounds of the present invention. As another example, alpha-bungarotoxin, a peptide-based snake toxin, can be used to detect acetylcholine receptors when labeled with a fluorophore of the present invention. The peptidase or protease substrate labeled with the fluorophore of the present invention can be used to measure the activity of peptidase or protease, and to screen drugs designed as peptidase or protease inhibitors.

可以按照本发明制备的其它多肽偶联物包括抗体、凝集素、酶、脂蛋白、白蛋白、抗生物素蛋白、链霉亲和素、膜联蛋白、蛋白A、蛋白G、转铁蛋白、脱铁运铁蛋白、藻胆蛋白和其它荧光蛋白、毒素、生长因子、微管蛋白、激素、各种受体和离子通道的那些偶联物。Other polypeptide conjugates that can be prepared according to the present invention include antibodies, lectins, enzymes, lipoproteins, albumin, avidin, streptavidin, annexin, protein A, protein G, transferrin, Apotransferrin, phycobiliproteins and those conjugates of other fluorescent proteins, toxins, growth factors, tubulins, hormones, various receptors and ion channels.

另外，非抗体蛋白或者多肽例如G蛋白或其它合适蛋白可以单独使用或者与白蛋白结合使用，合适的白蛋白包括人或牛血清白蛋白或卵清蛋白。其中包括蛋白A,G和L或者至少一个可以与IgG蛋白结合的衍生物。例如与IgG具有亲和作用的蛋白。这些蛋白可以经过改性但是不需要特殊处理，可以与其它载体分子采用相同的方式进行偶联。In addition, non-antibody proteins or polypeptides such as G protein or other suitable proteins can be used alone or in combination with albumin, suitable albumin including human or bovine serum albumin or ovalbumin. These include proteins A, G and L or at least one derivative that can bind to IgG proteins. For example, a protein with an affinity for IgG. These proteins can be modified but do not require special handling and can be coupled in the same way as other carrier molecules.

在一个实施例中，本发明化合物可以与抗体反应。此类抗体可以是一抗或二抗，这取决于所需的应用。如果待检测的抗原以非常少的量存在，可以使用二抗，以提供信号放大。可以标记各种二抗同种型。二抗同种型的非限制性实例是抗小鼠IgG、抗小鼠IgM、抗兔IgG、抗大鼠IgG、抗大鼠IgM、抗豚鼠IgG、抗鸡IgG、抗仓鼠IgG、抗人IgG、抗人IgM、抗山羊IgG和抗绵羊IgG。In one embodiment, the compounds of the present invention may react with antibodies. Such antibodies can be primary or secondary, depending on the desired application. If the antigen to be detected is present in very small amounts, a secondary antibody can be used to provide signal amplification. Various secondary antibody isotypes can be labeled. Non-limiting examples of secondary antibody isotypes are anti-mouse IgG, anti-mouse IgM, anti-rabbit IgG, anti-rat IgG, anti-rat IgM, anti-guinea pig IgG, anti-chicken IgG, anti-hamster IgG, anti-human IgG , anti-human IgM, anti-goat IgG and anti-sheep IgG.

紫外光激发荧光染料的用途Uses of UV-Excited Fluorescent Dyes

本发明化合物存在多种用途。相关的一个应用是本发明化合物作为标记试剂的用途，所述标记试剂能够赋予特定物质组分的荧光特性。本发明化合物可用于与任何宽范围的分子反应，包括但不限于生物分子如多肽、基于多肽的毒素、氨基酸、核苷酸、多核苷酸(包括DNA和RNA)、脂质和碳水化合物或他们的任何组合。此外，本发明化合物可用于与一下物质反应：半抗原、药物、微粒、合成或天然聚合物、细胞、病毒或其它荧光分子(包括本发明的荧光分子)，或表面。底物分子通常包括一个或多个官能团，所述光能团与本发明化合物的反应活性基团反应以形成共价连接键或非共价连接键。另一方面，本发明化合物的反应活性基团为活化的酯(如琥珀酰亚胺基酯或SE)、马来酰亚胺、酰肼或氨氧基团。因此，在另一方面，来自底物分子(或反应底物)的官能团为胺、硫醇、醛或酮。得到的经荧光标记的底物分子可以被成为“染料-偶联物”。本领域实践的用于制备“染料-偶联物”的任何方法(如Brinkley，Bioconjugate Chem.3,2(1992))适用于实践本发明。The compounds of the present invention have a variety of uses. A related application is the use of the compounds of the invention as labelling reagents capable of imparting fluorescent properties to specific components of matter. The compounds of the present invention can be used to react with any wide range of molecules including, but not limited to, biomolecules such as polypeptides, polypeptide-based toxins, amino acids, nucleotides, polynucleotides (including DNA and RNA), lipids and carbohydrates or their any combination of . In addition, the compounds of the present invention can be used to react with haptens, drugs, microparticles, synthetic or natural polymers, cells, viruses or other fluorescent molecules (including fluorescent molecules of the present invention), or surfaces. Substrate molecules typically include one or more functional groups that react with reactive groups of the compounds of the present invention to form covalent or non-covalent linkages. In another aspect, the reactive groups of the compounds of the present invention are activated esters (eg, succinimidyl esters or SE), maleimides, hydrazides, or aminooxy groups. Thus, in another aspect, the functional group from the substrate molecule (or reaction substrate) is an amine, thiol, aldehyde or ketone. The resulting fluorescently labeled substrate molecules can be referred to as "dye-conjugates". Any method practiced in the art for preparing "dye-conjugates" (eg, Brinkley, Bioconjugate Chem. 3, 2 (1992)) is suitable for use in the practice of the present invention.

生物分子和本发明化合物的偶联物通常具有较高的荧光产率，同时通常保持了未标记的分子的关键参数，如溶解性、与受体或核酸选择性的结合、使具有的酶活化或抑制或掺入到生物膜中的能力。然而，具有最高标记程度的偶联物仍然可沉淀或非特异性结合。必要时，为了保持功能或结合特异性，比最大值小的标记程度是可接受的。制备本发明的偶联体可涉及优化特性的实验。进行偶联后，为偶联的标记试剂可以通过本领域已知的技术除去，所述技术如凝胶过滤、透析、偶联体沉淀和再溶解、HPLC或这些技术的组合。游离染料的存在，特别是当染料保持化学反应活性时，可使得随后与生物偶联体的实验复杂化。Conjugates of biomolecules and compounds of the present invention generally have high fluorescence yields, while generally maintaining key parameters of unlabeled molecules, such as solubility, selective binding to receptors or nucleic acids, activation of enzymes with or the ability to inhibit or incorporate into biofilms. However, conjugates with the highest degree of labeling can still precipitate or bind nonspecifically. Where necessary, in order to preserve function or binding specificity, less than the maximum degree of labeling is acceptable. Preparation of conjugates of the present invention may involve experiments to optimize properties. After conjugation, the labeled reagent for conjugation can be removed by techniques known in the art, such as gel filtration, dialysis, conjugate precipitation and re-dissolution, HPLC, or a combination of these techniques. The presence of free dyes, especially when the dyes remain chemically reactive, can complicate subsequent experiments with bioconjugates.

本发明标记的生物分子的用途Use of the labeled biomolecules of the present invention

本发明的荧光标记的生物分子为多种宽泛的生物应用提供了一种有效的工具。标记使得技术人员能够辨别涉及生物分子的相互作用，所述生物分子如蛋白质、糖蛋白、核酸和脂质，以及无机化学物质，或他们的任何组合。可以进行此类实验以辨别特异性的蛋白质-蛋白质相互作用、蛋白质-核酸相互作用、目标蛋白质与候选抑制剂或者激活剂之间的相互作用。候选抑制剂或激活剂包括但不限于反义寡核苷酸、双链RNA、核酶、核酶衍生物、抗体、脂质体、小分子、无机或者有机化合物。也可以使用本发明实验来研究酶促动力学，用于例如药物设计、筛选和/或优化，并且可以使用在溶液中或固定在固体基质上的荧光标记的多肽来进行。The fluorescently labeled biomolecules of the present invention provide an effective tool for a wide variety of biological applications. Labels allow the skilled artisan to discern interactions involving biomolecules such as proteins, glycoproteins, nucleic acids and lipids, as well as inorganic chemicals, or any combination thereof. Such experiments can be performed to discern specific protein-protein interactions, protein-nucleic acid interactions, interactions between target proteins and candidate inhibitors or activators. Candidate inhibitors or activators include, but are not limited to, antisense oligonucleotides, double-stranded RNA, ribozymes, ribozyme derivatives, antibodies, liposomes, small molecules, inorganic or organic compounds. Enzymatic kinetics can also be studied using the present assays, eg, for drug design, screening and/or optimization, and can be performed using fluorescently labeled polypeptides in solution or immobilized on solid substrates.

尤其感兴趣的是细胞表面受体与其对应的配体之间的特异性相互作用。细胞表面受体是锚定在细胞质膜上或插入其中的分子。它们是构成蛋白质、糖蛋白、多糖和脂质的大家族，其不仅充当质膜的结构组分，还作为调节多种生物功能的调控元件。另一方面，特异性的蛋白质-蛋白质相互作用涉及细胞表面受体和免疫脂质体或免疫毒素。另一方面，特异性的蛋白质-蛋白质相互作用可以涉及胞质溶胶蛋白、核蛋白、伴侣蛋白或锚定在其它细胞内膜结构上的蛋白质。另外，特异性的蛋白质-蛋白质相互作用存在于靶蛋白质)例如抗原)和该抗原的特异性抗体之间。Of particular interest are the specific interactions between cell surface receptors and their corresponding ligands. Cell surface receptors are molecules anchored to or inserted into the cytoplasmic membrane. They are a large family of proteins, glycoproteins, polysaccharides and lipids that serve not only as structural components of the plasma membrane, but also as regulatory elements regulating various biological functions. On the other hand, specific protein-protein interactions involve cell surface receptors and immunoliposomes or immunotoxins. On the other hand, specific protein-protein interactions may involve cytosolic proteins, nuclear proteins, chaperones, or proteins anchored to other intracellular membrane structures. Additionally, specific protein-protein interactions exist between a target protein (eg, an antigen) and an antibody specific for that antigen.

通过在怀疑发生相互作用的条件下混合标记多肽与相互作用实体，来测定这两种实体之间的特异性相互作用。通常，借助于光学装置来显现相互作用。需要时，这些实体可以放置在光学限制内(参见例如美国专利号7，267，673和7，170，050)。检测单分子时，各光学限制区域仅含有一种所研究的靶标。这可以通过在大体积溶液中稀释靶标而实现，或大多数限制区域将有一个靶分子分配在其中。通过任何本领域可用的方法，可将标记多肽和相互作用实体固体在光学限制区域的内表面上，此类方法包括使用由多种结合部分实现的共价和非共价连接。结合部分的选择将取决于标记多肽和/或相互作用实体的性质。固定标记多肽或蛋白质探针的一种方式涉及链霉亲和素或抗生物素蛋白/生物素结合对的使用。The specific interaction between the two entities is determined by mixing the labeled polypeptide with the interacting entity under conditions where the interaction is suspected. Usually, the interaction is visualized by means of optical means. If desired, these entities can be placed within optical confinement (see, eg, US Pat. Nos. 7,267,673 and 7,170,050). When detecting single molecules, each optical confinement region contains only one target of interest. This can be achieved by diluting the target in a large volume of solution, or most confinement regions will have one target molecule distributed in it. Labeled polypeptides and interacting entities can be solidified on the inner surface of the optical confinement region by any method available in the art, including the use of covalent and non-covalent linkages by a variety of binding moieties. The choice of binding moiety will depend on the nature of the marker polypeptide and/or interacting entity. One way of immobilizing labeled polypeptide or protein probes involves the use of streptavidin or avidin/biotin binding pairs.

光源light source

本发明化合物可在任何时候，无论是测试过程中或者是测试后，均可以使用合适光源进行照射后产生可检测的光学信号，信号强度的变化可由本领域已知的任何方法检测，且通常取决于对所使用的荧光基团的选择。这可在光学系统的辅助下进行。所述系统通常包括至少两种元件，即激发源和光子检测器。这些元件的多种实例是本领域可获得的。实例性激发源为激光器，如偏振激光器。激光的选择依赖于与探针相连的荧光基团。本领域技术人员可通过常规实验容易的确定激发给定荧光团的适当激发波长(参见例如thehandbook-a guide to fluorescent probes and label ing techmologies，tenthedition(2005)。本领域技术人员可采用其它光学系统，这些光学系统可包括元件如光学读数器、高效光子检测系统、光电倍增管、门控感应的FET、纳米管FET、光电二极管、照相机、电荷耦合器件(CCD)、电子倍增电荷耦合器件(EMCCD)、增强型电荷耦合器件(ICCD)和共聚焦显微镜。可以选择合适的设备来对本发明荧光化合物进行照射，包括的设备包括但不限于手持式紫外灯、汞弧灯、氙灯、氩激光器和YAG激光器。这些照射源可选择性的集成在激光扫描器，流式细胞仪，荧光微孔读数仪，标准或mini荧光仪或色谱检测器中。荧光发射可以使用肉眼检测或者使用下述设备进行检测：CCD相机、摄像机、胶片、激光扫描设备、荧光仪、光学二极管、量子计数器、落射荧光显微镜、扫描显微镜、流式细胞仪、酶标仪。The compounds of the present invention may, at any time, either during or after the test, generate a detectable optical signal upon irradiation with a suitable light source. Changes in signal intensity may be detected by any method known in the art and generally depend on for the choice of fluorophore used. This can be done with the aid of an optical system. The system typically includes at least two elements, an excitation source and a photon detector. Numerous examples of these elements are available in the art. Exemplary excitation sources are lasers, such as polarized lasers. The choice of laser depends on the fluorophore attached to the probe. The appropriate excitation wavelength to excite a given fluorophore can be readily determined by routine experimentation by those skilled in the art (see, e.g., the handbook-a guide to fluorescent probes and labeling technologies, tenthedition (2005). Other optical systems may be employed by those skilled in the art, These optical systems may include elements such as optical readers, high efficiency photon detection systems, photomultiplier tubes, gated sensing FETs, nanotube FETs, photodiodes, cameras, charge coupled devices (CCDs), electron multiplying charge coupled devices (EMCCDs) , Enhanced Charge-Coupled Device (ICCD) and confocal microscope. Suitable equipment can be selected to irradiate the fluorescent compounds of the present invention, including equipment including but not limited to hand-held UV lamps, mercury arc lamps, xenon lamps, argon lasers and YAG lasers These illumination sources can optionally be integrated into laser scanners, flow cytometers, fluorescence microwell readers, standard or mini fluorometers or chromatographic detectors. Fluorescence emission can be detected with the naked eye or with the following equipment: CCD cameras, video cameras, film, laser scanning equipment, fluorometers, optical diodes, quantum counters, epifluorescence microscopes, scanning microscopes, flow cytometers, microplate readers.

试剂盒Reagent test kit

试剂盒为装本发明的紫外光激发荧光染料的外包装。试剂盒一般都会给出本发明化合物的一种或者几种使用方法。一般而言，试剂盒中包括本发明活性染料化合物，染料与包含合适功能位点的任何底物的偶联方法以及回收或纯化标记后物质的方法。染料及说明书就构成了标记合适底物的试剂盒。选择性的合适底物包括但不限于大分子聚合物(例如蛋白质，寡核苷酸或碳水化合物)，聚合物树脂和塑料(例如聚苯乙烯)，金属，玻璃和其它有机或无机底物。The kit is an outer package containing the ultraviolet light-excited fluorescent dye of the present invention. The kits generally provide one or more methods of using the compounds of the present invention. In general, the kit includes a reactive dye compound of the invention, a method for coupling the dye to any substrate containing suitable functional sites, and a method for recovering or purifying the labeled material. The dye and instructions constitute a kit for labeling a suitable substrate. Selective suitable substrates include, but are not limited to, macromolecular polymers (eg, proteins, oligonucleotides, or carbohydrates), polymeric resins and plastics (eg, polystyrene), metals, glasses, and other organic or inorganic substrates.

另一方面，本发明试剂盒也包括本发明化合物-底物偶联物，本发明的试剂盒可装有一种或多种本发明化合物和指导利用所述化合物的使用说明书。典型的，试剂盒中包含染料二抗偶联物。因此，最终用户需要提供一抗，这样就可以对分析物或络合物进行检测。另外，试剂盒中包括染料二抗偶联物和染料一抗偶联物，这样可以用来检测细胞受体，酶，细胞器或其它与抗体相连的其它配体。In another aspect, kits of the present invention also include compound-substrate conjugates of the present invention, and the kits of the present invention may contain one or more compounds of the present invention and instructions for use of the compounds. Typically, a dye secondary antibody conjugate is included in the kit. Therefore, the end user needs to provide a primary antibody so that the analyte or complex can be detected. In addition, the kit includes dye secondary antibody conjugates and dye primary antibody conjugates, which can be used to detect cellular receptors, enzymes, organelles or other ligands linked to the antibody.

下面结合实施例进一步描述本发明紫外光激发荧光染料的合成方法。The synthetic method of the ultraviolet light excited fluorescent dye of the present invention is further described below in conjunction with the examples.

实施例1：化合物5的制备：Example 1: Preparation of Compound 5:

(1)化合物1的合成：(1) Synthesis of compound 1:

根据US2012004397中化合物6的合成方法，制得化合物1。According to the synthesis method ofcompound 6 in US2012004397,compound 1 was prepared.

将化合物1进行核磁共振(NMR)表征，表征结果如下：Compound 1 was characterized by nuclear magnetic resonance (NMR), and the characterization results were as follows:

化合物1:¹H NMR:1.1(t,3H),1.3-3(m,8H),4-5(m,4H),7.03(d,2H),7.1(S,1H),7.66(d,2H),7.9-8.8(m,4H)Compound 1:¹ H NMR: 1.1(t, 3H), 1.3-3(m, 8H), 4-5(m, 4H), 7.03(d, 2H), 7.1(S, 1H), 7.66(d, 2H),7.9-8.8(m,4H)

(2)化合物2的合成：(2) Synthesis of compound 2:

1g化合物1(2.63mmol)悬浮于10ml浓盐酸中，在65℃反应过夜，TLC跟踪反应，反应结束后，旋蒸除去盐酸和水，无需干燥即可用于下一步反应。取少量进行柱层析并抽干得到的固体产物，即化合物2。1 g of compound 1 (2.63 mmol) was suspended in 10 ml of concentrated hydrochloric acid, reacted at 65°C overnight, followed by TLC, after the reaction was completed, the hydrochloric acid and water were removed by rotary evaporation, and it was used in the next reaction without drying. A small amount of the obtained solid product, namelycompound 2, was subjected to column chromatography and suction-dried.

将化合物2进行NMR表征，表征结果如下：Compound 2 was characterized by NMR, and the characterization results were as follows:

化合物2:¹H NMR:1.3-3(m,8H),4-5(m,2H),7.03(d,2H),7.1(S,1H),7.66(d,2H),7.9-8.8(m,4H)Compound 2:¹ H NMR: 1.3-3(m, 8H), 4-5(m, 2H), 7.03(d, 2H), 7.1(S, 1H), 7.66(d, 2H), 7.9-8.8( m,4H)

(3)化合物3的合成：(3) Synthesis of compound 3:

400mg化合物2(1.13mmol)溶解在3ml浓硫酸中，冷却降温到0℃,加入3ml 30％发烟硫酸，温度不超过5℃,加入完毕后，自然升温过夜，将反应混合物滴入到预先冷却到0℃的200ml乙醚中，搅拌，离心后用制备液相进行分离、冻干得到的固体产物，即化合物3。400mg of compound 2 (1.13mmol) was dissolved in 3ml of vitriol oil, cooled to 0°C, added 3ml of 30% oleum, the temperature was no more than 5°C, after the addition was completed, the temperature was naturally heated overnight, and the reaction mixture was dropped into the precooled It was placed in 200 ml of ether at 0°C, stirred, centrifuged, and separated with a preparative liquid phase, and the solid product obtained by lyophilization, namely compound 3.

将化合物3进行NMR表征，表征结果如下：Compound 3 was characterized by NMR, and the characterization results were as follows:

化合物3:¹H NMR:1.3-3(m,8H),4-5(m,2H),7.5(d,1H),7.1(S,1H),8.1(d,1H)，8.3(s,1H),7.9-8.8(m,4H)Compound 3:¹ H NMR: 1.3-3(m, 8H), 4-5(m, 2H), 7.5(d, 1H), 7.1(S, 1H), 8.1(d, 1H), 8.3(s, 1H),7.9-8.8(m,4H)

(4)化合物4的合成：(4) Synthesis of compound 4:

0.5g化合物3(1.16mmol)溶解在5ml的DMF中，加入1.21g m-dPEG23-NH2(1.16mmol)(QUATA BIODESIGN LIMITED,MW:1044.28)，然后再加入0.48g HBTU(1.27mmol)及210ul DIPEA(1.27mmol)，室温反应过夜，旋蒸除去DMF，用水溶解剩余物，用液相进行制备，得到的纯品冻干，得到化合物4。0.5g compound 3 (1.16mmol) was dissolved in 5ml DMF, 1.21g m-dPEG23-NH2 (1.16mmol) (QUATA BIODESIGN LIMITED, MW: 1044.28) was added, and then 0.48g HBTU (1.27mmol) and 210ul DIPEA were added (1.27 mmol), reacted at room temperature overnight, removed DMF by rotary evaporation, dissolved the residue with water, and prepared with liquid phase, the obtained pure product was lyophilized to obtaincompound 4.

将化合物4进行质谱(MS)表征和NMR表征。MS质谱图如图1所示，由图1可知，M＝1457.27及M+1峰＝1458.43；NMR表征结果如下：Compound 4 was characterized by mass spectrometry (MS) and NMR. The MS mass spectrum is shown in Figure 1. It can be seen from Figure 1 that M=1457.27 and M+1 peak=1458.43; the NMR characterization results are as follows:

化合物4:¹H NMR:1.3-2.5(m,8H),3-5(m,97H),7.5(d,1H),7.1(S,1H),8.1(d,1H)，8.3(s,1H),7.9-8.8(m,4H)Compound 4:¹ H NMR: 1.3-2.5(m, 8H), 3-5(m, 97H), 7.5(d, 1H), 7.1(S, 1H), 8.1(d, 1H), 8.3(s, 1H),7.9-8.8(m,4H)

(5)化合物5的合成：(5) Synthesis of compound 5:

0.5g化合物4(0.343mmol)与0.67g 6-溴己酸(3.43mmol)悬浮在10ml的乙腈中，耐压瓶中120℃反应过夜后，直接旋蒸除去乙腈，剩余物用水溶解进行高效液相制备，纯品收集，冻干得到化合物5。0.5 g of compound 4 (0.343 mmol) and 0.67 g of 6-bromohexanoic acid (3.43 mmol) were suspended in 10 ml of acetonitrile, reacted at 120 °C overnight in a pressure-resistant flask, and then directly rotary evaporated to remove acetonitrile, and the residue was dissolved in water to carry out a high-efficiency solution. Phase preparation, pure product collection, and lyophilization to obtaincompound 5.

将进行MS表征、紫外分光光度计和荧光分光光度表征和NMR表征。化合物5的MS质谱图如图2所示，由图2可知，M＝1572.38及M+1峰＝1573.64；化合物5的吸收和发射光谱如图3所示；NMR表征结果如下：MS characterization, UV spectrophotometer and fluorescence spectrophotometric characterization and NMR characterization will be performed. The MS mass spectrum ofcompound 5 is shown in Figure 2. It can be seen from Figure 2 that M=1572.38 and M+1 peak=1573.64; the absorption and emission spectra ofcompound 5 are shown in Figure 3; the NMR characterization results are as follows:

化合物5:¹H NMR:1.3-2.5(m,16H),3-5(m,99H),7.5(d,1H),7.1(S,1H),8.1(d,1H)，8.3(s,1H),7.9-8.8(m,4H)Compound 5:¹ H NMR: 1.3-2.5(m, 16H), 3-5(m, 99H), 7.5(d, 1H), 7.1(S, 1H), 8.1(d, 1H), 8.3(s, 1H),7.9-8.8(m,4H)

实施例2：Example 2:

化合物6A的制备：Preparation of compound 6A:

1g化合物5(0.64mmol)溶解在20ml无水DMA中，0.16g DSC与6.2mg DMAP加入到反应溶液中，在室温下搅拌悬浮液2天。反应结束后，减压将溶剂浓缩至3ml，加入300ml乙酸乙酯并且搅拌0.5h后，收集固体，过滤用乙酸乙酯洗涤可得化合物6.1 g of compound 5 (0.64 mmol) was dissolved in 20 ml of anhydrous DMA, 0.16 g of DSC and 6.2 mg of DMAP were added to the reaction solution, and the suspension was stirred at room temperature for 2 days. After the reaction, the solvent was concentrated to 3 ml under reduced pressure, 300 ml of ethyl acetate was added and stirred for 0.5 h, the solid was collected, filtered and washed with ethyl acetate to obtaincompound 6.

将化合物6A进行NMR表征，表征结果如下：Compound 6A was characterized by NMR, and the characterization results were as follows:

化合物6A:¹H NMR:1.3-2.5(m,20H),3-5(m,99H),7.5(d,1H),7.1(S,1H),8.1(d,1H)，8.3(s,1H),7.9-8.8(m,4H)Compound 6A:¹ H NMR: 1.3-2.5(m, 20H), 3-5(m, 99H), 7.5(d, 1H), 7.1(S, 1H), 8.1(d, 1H), 8.3(s, 1H),7.9-8.8(m,4H)

实施例3Example 3

化合物6B的制备：Preparation of compound 6B:

将250mg化合物5溶解在10ml 0℃的DME中，100mg肼加入其中，在0℃反应1h。真空下抽干溶剂，剩余物经过HPLC制备可得化合物6B。250 mg ofcompound 5 was dissolved in 10 ml of DME at 0°C, 100 mg of hydrazine was added to it, and the reaction was carried out at 0°C for 1 h. The solvent was drained under vacuum, and the residue was prepared by HPLC to give compound 6B.

将化合物6B进行NMR表征，表征结果如下：Compound 6B was characterized by NMR, and the characterization results were as follows:

化合物6B:¹H NHR:1.3-2.5(m,16H),3-5(m,101H),7.5(d,1H),7.1(S,1H),8.1(d,1H)，8.3(s,1H),7.9-8.8(m,5H)Compound 6B:¹ H NHR: 1.3-2.5(m, 16H), 3-5(m, 101H), 7.5(d, 1H), 7.1(S, 1H), 8.1(d, 1H), 8.3(s, 1H),7.9-8.8(m,5H)

实施例4Example 4

化合物6C的制备：Preparation of compound 6C:

将250mg化合物5溶解在10ml 0℃DME中，加入90mg N-(2-氨基乙基)马来酰亚胺三氟乙酸盐和0.1ml三乙胺，在0℃反应1h。真空下抽干溶剂，剩余物经过HPLC制备可得化合物6C.250 mg ofcompound 5 was dissolved in 10 ml of DME at 0°C, 90 mg of N-(2-aminoethyl)maleimide trifluoroacetate and 0.1 ml of triethylamine were added, and the reaction was carried out at 0°C for 1 h. The solvent was drained under vacuum, and the residue was prepared by HPLC to obtain compound 6C.

将化合物6C进行NMR表征，表征结果如下：Compound 6C was characterized by NMR, and the characterization results were as follows:

化合物6C:¹H NHR:1.3-2.5(m,16H),3-5(m,103H),7.5(d,1H),7.1(S,1H),8.1(d,1H)，8.3(s,1H),7.9-8.8(m,6H)Compound 6C:¹ H NHR: 1.3-2.5(m, 16H), 3-5(m, 103H), 7.5(d, 1H), 7.1(S, 1H), 8.1(d, 1H), 8.3(s, 1H),7.9-8.8(m,6H)

实施例5Example 5

化合物7的制备：Preparation of compound 7:

制备方法同实施例1中化合物5的制备方法，不同点是将实施例1中第四步的m-dPEG23-NH2换成m-dPEG7-NH2，就可得到化合物7。The preparation method is the same as the preparation method ofcompound 5 in Example 1, except that m-dPEG23-NH2 in the fourth step in Example 1 is replaced with m-dPEG7-NH2, and compound 7 can be obtained.

将化合物7进行NMR表征，表征结果如下：Compound 7 was characterized by NMR, and the characterization results were as follows:

化合物7:¹H NHR:1.3-2.5(m,16H),3-5(m,35H),7.5(d,1H),7.1(S,1H),8.1(d,1H)，8.3(s,1H),7.9-8.8(m,4H)Compound 7:¹ H NHR: 1.3-2.5(m, 16H), 3-5(m, 35H), 7.5(d, 1H), 7.1(S, 1H), 8.1(d, 1H), 8.3(s, 1H),7.9-8.8(m,4H)

实施例6Example 6

化合物8的制备：Preparation of compound 8:

根据专利US8158801中关于主体结构的合成过程结合实施例1化合物5的制备方法，即制得化合物8。According to the synthesis process of the main structure in patent US8158801 combined with the preparation method ofcompound 5 in Example 1,compound 8 is prepared.

将化合物8进行NMR表征，表征结果如下：Compound 8 was characterized by NMR, and the characterization results were as follows:

化合物8:¹H NHR:1.3-2.5(m,16H),3-5(m,135H),7.5(d,1H),7.2(S,1H),8.1(d,1H)，8.3(s,1H),7.9-8.8(m,4H)Compound 8:¹ H NHR: 1.3-2.5(m, 16H), 3-5(m, 135H), 7.5(d, 1H), 7.2(S, 1H), 8.1(d, 1H), 8.3(s, 1H),7.9-8.8(m,4H)

实施例7Example 7

紫外光激发荧光染料-偶联物(荧光染料+抗体)的制备Preparation of Fluorescent Dye-Conjugate (Fluorescent Dye + Antibody) Excited by Ultraviolet Light

在0.1mM pH为8.5碳酸氢钠缓冲液中，将荧光染料化合物6A与浓度为1mg/ml的抗体(山羊抗小鼠IgG,即GAM)按照不同的比例混合，在室温孵育约1h后，使用经PBS(pH＝7.4)平衡的Sephadex G-25经凝胶过滤法分离反应混合物，得到一组染料-偶联物(荧光染料+抗体)。将该组染料-偶联物进行标记反应，得出如图4所示的不同标记程度(DOL)的化合物6A-GAM的荧光强度。其中，图4还将不同标记程度(DOL)下，化合物6A-GAM的荧光强度与pacificorange-GAM(现有技术)的荧光强度进行了对比，发现化合物6A-GAM比pacific orange-GAM的荧光强度更强。In 0.1 mM sodium bicarbonate buffer with pH 8.5, the fluorescent dye compound 6A was mixed with the antibody (goat anti-mouse IgG, GAM) at a concentration of 1 mg/ml according to different ratios, incubated at room temperature for about 1 h, and then used The reaction mixture was separated by gel filtration with Sephadex G-25 equilibrated in PBS (pH=7.4) to obtain a set of dye-conjugates (fluorescent dye + antibody). The group of dye-conjugates were subjected to labeling reaction to obtain the fluorescence intensities of compound 6A-GAM with different degrees of labeling (DOL) as shown in FIG. 4 . Among them, Figure 4 also compares the fluorescence intensity of compound 6A-GAM with the fluorescence intensity of pacificorange-GAM (prior art) under different labeling degrees (DOL), and it is found that the fluorescence intensity of compound 6A-GAM is higher than that of pacific orange-GAM stronger.

制备方法参考文件如下所示：US6974873；Haugland et al.；Meth.Mol.Biol.45,205,(1995)；Haugland et al.Current Protocols in Cell Biology，16.51.-16.5.22(2000)The reference documents for the preparation method are as follows: US6974873; Haugland et al.; Meth. Mol. Biol. 45, 205, (1995); Haugland et al.

Pacific Orange-GAM参考专利US8158801。Pacific Orange-GAM refers to patent US8158801.

实施例8Example 8

紫外光激发荧光染料6A应用于细胞的免疫分型检测分析Immunophenotyping of cells using UV-excited fluorescent dye 6A

具体的实验方法如下：The specific experimental methods are as follows:

将人的单核细胞用1％BSA/PBS进行洗涤，并以1000万/ml的浓度重悬，然后用载体分子为1微克小鼠抗人CD4和荧光染料为化合物6A制备成的染料-偶联剂进行染色30分钟。将染色后的单核细胞用1％BSA/PBS洗涤，离心并重悬于400毫升的1％BSA/PBS中，在LSR II流式细胞仪上分析样品。其中，采用的二极管激光器为405nm二极管激光器，发射滤波器使用前向散射VS侧向散点图，对淋巴细胞进行门控，并使用淋巴细胞门进行绘制，使用单色控件进行补偿。The human monocytes were washed with 1% BSA/PBS and resuspended at a concentration of 10 million/ml, and then the carrier molecule was 1 μg mouse anti-human CD4 and the fluorescent dye was a dye-coupled compound prepared from compound 6A. Binders were stained for 30 minutes. Stained monocytes were washed with 1% BSA/PBS, centrifuged and resuspended in 400 ml of 1% BSA/PBS, and samples were analyzed on an LSR II flow cytometer. Among them, the diode laser used was a 405 nm diode laser, and the forward scatter VS side scatter plot was used as the emission filter to gate the lymphocytes, and the lymphocyte gate was used for drawing, and the monochrome control was used for compensation.

经过上述操作即得到如图5所示的化合物6A显示的免疫分型。After the above operations, the immunophenotyping shown by compound 6A as shown in Figure 5 was obtained.

以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本领域技术的技术人员在本发明公开的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited to this. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, All should be included within the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims (6)

1. An ultraviolet light excited fluorescent dye having the structure of formula I:

in formula I:

x is C;

y is N;

z is O;

w is N;

R₁、R₂、R₅、R₆、R₇、R₉and R₁₀All are H;

R₃is a water-soluble polymer group of

R₄Is a sulfonic acid group;

R₈is a reactive group which is

2. The method for preparing dye-conjugate by exciting fluorescent dye with ultraviolet light and carrier molecule according to claim 1, wherein the method comprises the following steps: the method comprises the following steps:

contacting a carrier molecule with said compound of formula I to form a bound sample;

incubating the bound sample for a time sufficient to allow covalent bonds to form between said compound of formula I and the carrier molecule, to obtain a dye-conjugate mixture;

and separating the compound which is not connected with the carrier molecule and the compound which is connected with the carrier molecule in the dye-conjugate mixture to obtain the dye-conjugate.

3. The method for preparing an ultraviolet excited fluorescent dye-conjugate according to claim 2, wherein the method comprises the following steps: the carrier molecule comprises an amino acid, a polypeptide, a protein, a polysaccharide, a nucleotide, a nucleoside, an oligonucleotide, a nucleic acid, a hapten, a psoralen, a drug, a hormone, a synthetic polymer, a polymeric microparticle, a biological cell, or a virus.

4. The method for preparing an ultraviolet excited fluorescent dye-conjugate according to claim 2, wherein the method comprises the following steps: at least one active group of the compound of the general formula I connects the compound of the general formula I with a carrier molecule.

5. The method for preparing an ultraviolet excited fluorescent dye-conjugate according to claim 4, wherein the method comprises the following steps: the carrier molecule comprises a nucleotide or a polypeptide.

6. Use of a uv-excited fluorescent dye according to any one of claims 1 to 5, characterized in that: the ultraviolet light excited fluorescent dye is applied to immunodetection and analysis of cells.

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