Invented by Chi-Huey Wong, Jim-Min Fang, Jiun-Jie Shie, Academia Sinica

Reactive labelling compounds are a class of chemicals that are widely used in various industries for labelling and tracking purposes. These compounds have unique properties that allow them to react with specific molecules or functional groups, making them ideal for use in a variety of applications. The market for reactive labelling compounds is growing rapidly, driven by the increasing demand for these compounds in the healthcare, pharmaceutical, and biotechnology industries. These industries use reactive labelling compounds for a wide range of applications, including drug discovery, diagnostics, and disease monitoring. One of the key drivers of the reactive labelling compounds market is the increasing demand for personalised medicine. As healthcare becomes more personalised, there is a growing need for labelling compounds that can be used to track and monitor individual patients. Reactive labelling compounds are ideal for this purpose, as they can be used to label specific molecules or cells in the body, allowing doctors to track the progress of a disease or monitor the effectiveness of a treatment. Another key driver of the reactive labelling compounds market is the increasing use of these compounds in drug discovery. Reactive labelling compounds are used to identify and characterise drug targets, which is essential for the development of new drugs. These compounds are also used to screen potential drug candidates, helping to identify compounds that are most likely to be effective. The use of reactive labelling compounds is not limited to the healthcare and pharmaceutical industries. These compounds are also used in the food and beverage industry, where they are used to label and track food products. They are also used in the environmental monitoring industry, where they are used to track pollutants and other environmental contaminants. The reactive labelling compounds market is highly competitive, with a large number of companies operating in this space. Some of the key players in this market include Thermo Fisher Scientific, Merck KGaA, GE Healthcare, and PerkinElmer. These companies are investing heavily in research and development to develop new and innovative labelling compounds that can be used in a wide range of applications. In conclusion, the market for reactive labelling compounds is growing rapidly, driven by the increasing demand for these compounds in the healthcare, pharmaceutical, and biotechnology industries. These compounds have unique properties that make them ideal for use in a wide range of applications, including drug discovery, diagnostics, and disease monitoring. As the demand for personalised medicine continues to grow, the market for reactive labelling compounds is expected to continue to expand in the coming years.

The Academia Sinica invention works as follows

Provided is azido-BODIPY compound of formula I, cyclooctyne based fluorogenic probes (IV) and activity-based Probes (VI). These compounds are subjected to azide-alkyne additions (AAC), which result in triazolyl products. These compounds can be used for the detection and imaging alkyne or azide containing molecules. “Disclosed are methods for detecting and imaging biomolecules with compounds from the present disclosure.

Background for Reactive Labelling Compounds and their Uses

The CuAAC (Copper-catalyzed Azide-Alkyne 1,3 Dipolar Cycloaddition) is widely used in chemical biology to label biomolecules within complex mixtures, and to image fixed cells and tissue. (Kolb, et al., Angew. Chem. Int. Ed. 2001, 40, 2004; Rostovtsev, et al., Angew. Chem. Int. Ed. Ed. The incorporation of fluorescent probes in proteins, DNA and RNA, as well as lipids and glycocans, within their native cell environments, provides opportunities to image and understand their roles in vivo. “(Best, Biochemistry, 2009, 48, 6571.).

For instance, glycans are shown on the surface of cells with implications for numerous physiological and disease processes. Pathological conditions such as cancer metastasis and inflammation are often accompanied by abnormal glycosylation of the cell surface. The altered terminal sialylation or fucosylation that is believed to be caused by changes in the expression levels and locations of sialyltransferases are linked with malignancy. Glycomics research has focused on the ability to examine glycans attached to proteins or lipids for their biological information as biomarkers. (Hsu, et al., Proc. Nat. Acad. Sci. U.S.A., 2007, 104, 2614; Sawa, et al., Proc. Nat. Acad. Sci. U.S.A., 2006, 103, 12371.)

Now it is possible to analyze changes in glycosylation patterns within living systems. (Prescher & Bertozzi, Nat. Chem. Bio. 2005, 1, 13.) The process begins with the metabolic incorporation of a non-natural carbohydrate that contains a unique functional group which acts as a bioorthogonal chemcial reporter in the cell biosynthetic apparatus. The modified glycan then undergoes processing and is constructed on the surface of the cell. The unnatural glycan is detected by a subsequent reaction with a detectable probe equipped with complementary bioorthogonal function groups. (Sletten & Bertozzi, Angew. Chem. Int. Ed. 2009, 48, 2.)

The concept bioorthogonal chemicals reporter has been applied for proteomic analysis glycosylation of proteins and chemical remodelling of cell surfaces within living systems. Other applications of bioorthogonal reactions include protein labeling and folding, protein target identification, posttranslational modification, cell proliferation monitoring, and activity-based protein fold. Cell biology has become more powerful by using bioorthogonal chemical reporters to label specific functional groups in living cells. Bioorthogonal Chemistry has made tremendous advances in the last few years. This is especially true for chemistries that show biocompatibility in living systems. The cycloadditions are ideal bioorthogonal reaction because of their inherent selectivity and tunable electronic properties. There are many challenges in the field. This is especially true from the perspective of organismal and cellular applications. Most bioorthogonal reporters strategies involve multistep procedures using fluorophroe labeled reactant partners. This can cause high background fluorescent noise, which is difficult to remove in intracellular environments and tissues. These methods also require high concentrations to produce detectable signals.

Recent efforts have focused on the development of non-or weak fluorescent probes upon CuAAC reaction with non-fluorescent azides or alkynes, which can ligate in order to provide a highly fluorescent complex (FIG. 2). (Zhou & Fahrni J. Am. Chem. Soc. 2004, 126, 8862; Sivakumar, et al., Org. Lett. 2004, 24, 4603; Sawa, et al., Proc. Nat. Acad. Sci. U.S.A., 2006, 103, 12371; Xie, et al., Tetrahedron 2008, 64, 2906; Li, et al., Org. Lett. 2009, 11, 3008; Le Droumaguet, et al., Chem. Soc. Rev. 2010, 39, 1223; Qi, et al, Bioconjugate Chem. 2011, 22, 1758; Chao, et al., Sci. China Chemistry 2012 55, 125. Herner, et al., Org. Biomol. Chem. 2013, 11, 3297.) This CuAAC reaction, which occurs with high efficiency, would have wide applications in the emerging fields of cell biology and functional protomics because of its distinct fluorescence characteristics in the formation of triazole without the background fluorescent noise from the starting materials. However, these azido- and alkynyl-functionalized probes usually require excitation in the UV region and emit blue light with poor quantum yield in aqueous solution; such optical properties are not ideal for biological applications.

The distinct enhancement of fluorescence induced by highly effective CuAAC reactions will have broad applications in emerging fields such as cell biology and functional proomics (Le Droumaguet C. Wang C. Wang Q. Soc. Rev. 2010, 39, 1233-1239; Sawa, M.; Hsu, T.-L.; Itoh, T.; Sugiyama, M.; Hanson, S. R.; Vogt, P. K.; Wong, C.-H. Proc. Natl. Acad. Sci. U.S.A 2006, 103, 12371-12376, Shie, J.-J. ; Liu, Y.-C.; Lee, Y.-M.; Lim, C.; Fang, J.-M.; Wong, C.-H. J. Am. Chem. Soc. 2014, 136, 9953-9961, Hsu, T.-L.; Hanson, S. R.; Kishikawa, K.; Wang, S.-K.; Sawa, M.; Wong, C.-H. Proc. Natl. Acad. Sci. U.S.A 2007, 104, 2614-2619, Tsai, C.-S.; Liu, P.-Y. ; Yen, H.-Y. Hsu T.-L., Wong C.H. Chem. Commun. 2010, 46, 5575-5577). Cu(I), however, is toxic and has prevented the use of CuAAC for living systems.

4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (also known as BODIPY) dyes are a type of popular fluorophores for many biological applications. BODIPY dyes are characterized by a number of advantages, including high chemical and photophysical stabilities, high fluorescence quantum yields and molar absorption coefficients. Loudet and Burgess (Chem. Rev. 2007, 107, 4891; Ulrich et al., Angew. Chem. Int. Ed. 2008, 47, 1184; Boens, et al., Chem. Soc. Rev. 2012, 41, 1130; Kamkaew, et al, Chem. Soc. Rev. 2013, 42, 77.)

Some azidoBODIPY derivates have been developed to fluorescently label CuAAC reactions. (Li, et al., J. Org. Chem. 2008, 73, 1963.) The CuAAC reaction has been shown to produce the triazole derivative with increased fluorescence from the 3-azidoBODIPY derivatives that have low fluorescence. The triazole product, although it provided a 300-fold increase in emission over azidoBODIPY, exhibited a very low fluorescence yield (?fl0.03). Furthermore, the unreacted azidoBODIPY is unstable, and does not react with alkynyl molecules under physiological conditions. (Wang, et al., Sci. China Chemistry 2012, 55, 125; Chauhan, et al. Tetrahedron Lett. 2014, 55, 244.)

Accordingly, there is an urgent need for new designs of molecular probing agents that are relevant to cell environments, for labeling cells, detecting biomolecules and/or visualizing their localization in the cell.

The present disclosure is a series of azidoBODIPY compound containing a BODIPY scaffold that emits green light and is relevant to cell environments. The BODIPY is used to start a project because of its attractive synthetic and fluorescent properties. The 8-position is a simple way to modify exemplary BODIPYs. The arylation at this position does not have a significant effect on emission and absorption wavelengths, because the BODIPY core and aryl moiety are uncoupled and twisted.

These compounds can be used to label proteins with alkyne functionalization without the need for washing, and they are also suitable for visualizing alkyne-tagged conjugates within cells using confocal microscopes. AzBOCEt can also be used to detect the probe-labeled proteins directly on SDS-PAGE.

The present disclosure also relates to cyclooctyne-based fluorogenic probes which are capable of reacting to alkyne-functionalized moieties. The cyclooctyne based fluorogenic agents can in some cases be present within a cell. In some aspects, the cyclooctyne-based fluorogenic probes can be used to detect azide-glycoconjugates in a cell.

The present disclosure also relates to the dual-imaging of both azido-functionalized glycoconjugates and alkynyl-functinonalized glyconjugates by contacting a sample with azido-BODIPY compounds of formula (I) and/or cyclooctyne-based fluorogenic probes of formula (IV) in a dual imaging mode.

The present disclosure also relates measuring the enzyme activity using probes that form a covalent link with the active site. These probes contain an alkyne moiety, which is detected by a fluorogenic azide probe. The enzyme could be a sialidase. The 3-fluorosialylfluoride can be used as an inhibitor to the fluorogenic sialidase.

The present disclosure is a description of exemplary azido-BODIPY compound formula (I) which undergoes azide-alkyne reactions (AAC). The azide alkyne reactions (AAC) may be promoted by strains or catalysts (organic or metal). In certain embodiments, the metal catalyst is used. Copper(I) is a metal catalyst in certain embodiments.

The exemplary azido-BODIPY compound described here can react with alkyne to give triazole products that are stable and have enhanced fluorescence for detection. The provided exemplary compound represents a significant advancement in cell-imaging processes without washing and is applicable to direct detection in gel of alkyne-tagged proteins from cell lysates following SDS-PAGE.

One aspect” of the disclosure is a compound with Formula (I), which is an azidoBODIPY.

or a pharmaceutically accepted salt, solvate or hydrate thereof and in which G1,G2,G3, G4a. G4b,G5, G6,G7, and G8 are as described herein.

The present disclosure also provides methods of synthesis for the preparation of azidoBODIPY compound. The present disclosure also shows that azidoBODIPY can react with alkynes organic to produce triazole products enhanced by fluorescence.

In another aspect of the present disclosure, a triazolylBODIPY-compound of Formula (III:) is provided.

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