Nam-Young Kang

Diversity-driven chemical probe development for biomolecules: beyond hypothesis-driven approach

Bioprobes are indispensable tools for biological study and clinical diagnosis. A conventional strategy for probe development is hypothesis-driven approach based on known molecular mechanisms of recognition for individual analytes. However, even the most sophisticated rational design does not always guarantee the applicability of probes in complex biological systems, therefore the efficiency and scope of probe development has been intrinsically limited. Diversity-driven approach is a rapidly emerging alternative and has been employed for the development of new probes even in the absence of the knowledge about target recognition mechanism. This tutorial review summarizes the recent advances in probe development along with conceptual advantages and perspectives of the diversity-driven approach.

A Fluorescent Rosamine Compound Selectively Stains Pluripotent Stem Cells

Stem cells, which are capable of self-renewing and differ-entiating into various types of cells, have captured greatinterest as a valuable resource for regenerative medicine anddevelopmental biology research. Technical progress duringthe last decade has enabled the isolation of stem cells from awide range of tissues, their differentiation into specific typesof cells, and the generation of induced pluripotent stem cells(iPSC) from somatic cells. The recent success of patient-specific iPSC generation

Neural stem cell specific fluorescent chemical probe binding to FABP7.

Fluorescent small molecules have become indispensable tools for biomedical research along with the rapidly developing optical imaging technology. We report here a neural stem cell specific boron-dipyrromethane (BODIPY) derivative compound of designation red 3 (CDr3), developed through a high throughput/content screening of in-house generated diversity oriented fluorescence library in stem cells at different developmental stages. This novel compound specifically detects living neural stem cells of both human and mouse origin. Furthermore, we identified its binding target by proteomic analysis as fatty acid binding protein 7 (FABP7), also known as brain lipid binding protein) which is highly expressed in neural stem cells and localized in the cytoplasm. CDr3 will be a valuable chemical tool in the study and applications of neural stem cells.

Embryonic and induced pluripotent stem cell staining and sorting with the live-cell fluorescence imaging probe CDy1

Detecting and isolating specific types of cells is crucial to understanding a variety of biological processes, including development, aging, regeneration and pathogenesis; this understanding, in turn, allows the use of cells for therapeutic purposes, for which stem cells have emerged recently as invaluable materials. The current methods of isolation and characterization of stem cells depend on cell morphology in culture or on immunostaining of specific markers. These methods are, however, time consuming and involve the use of antibodies that may often make the cells unsuitable for further study. We recently developed a fluorescent small molecule named CDy1 (compound of designation yellow 1) that selectively stains live embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). This protocol describes detailed procedures for staining ESC and iPSC in live conditions and for fluorescence-activated cell sorting (FACS) of ESC using CDy1. Cell staining, image acquisition and FACS can be done within 6 h.

meso-Ester and carboxylic acid substituted BODIPYs with far-red and near-infrared emission for bioimaging applications.

A series of meso-ester-substituted BODIPY derivatives 1-6 are synthesized and characterized. In particular, dyes functionalized with oligo(ethylene glycol) ether styryl or naphthalene vinylene groups at the α positions of the BODIPY core (3-6) become partially soluble in water, and their absorptions and emissions are located in the far-red or near-infrared region. Three synthetic approaches are attempted to access the meso-carboxylic acid (COOH)-substituted BODIPYs 7 and 8 from the meso-ester-substituted BODIPYs. Two feasible synthetic routes are developed successfully, including one short route with only three steps. The meso-COOH-substituted BODIPY 7 is completely soluble in pure water, and its fluorescence maximum reaches around 650 nm with a fluorescence quantum yield of up to 15 %. Time-dependent density functional theory calculations are conducted to understand the structure-optical properties relationship, and it is revealed that the Stokes shift is dependent mainly on the geometric change from the ground state to the first excited singlet state. Furthermore, cell staining tests demonstrate that the meso-ester-substituted BODIPYs (1 and 3-6) and one of the meso-COOH-substituted BODIPYs (8) are very membrane-permeable. These features make these meso-ester- and meso-COOH-substituted BODIPY dyes attractive for bioimaging and biolabeling applications in living cells.

Mitochondria-targeted fluorescent thermometer monitors intracellular temperature gradient

Intracellular thermometry at the microscopic level is currently a hot topic. Herein we describe a small molecule fluorescent thermometer targeting mitochondria (Mito thermo yellow). Mito thermo yellow successfully demonstrates the ability to monitor the intracellular temperature gradient, generated by exogenous heating, in various cells.

Solid phase combinatorial synthesis of a xanthone library using click chemistry and its application to an embryonic stem cell probe

We report the first solid phase synthesis of a xanthone library CX and its application to embryonic stem cell probe development. The CX library was further derivatised with an activated ester resin to provide an acetylated CX (CXAC) library. Screening of these libraries led to the discovery of a novel fluorescent mESC probe, CDb8.

Visualization and Isolation of Langerhans Islets by a Fluorescent Probe PiY

Pancreatic Langerhans islets are mainly composed of insulinsecreting beta cells and glucagon-secreting alpha cells, along with other minor cell types, and play a central role in the regulation of blood glucose levels. Because of this, imaging of viable pancreatic islets is an important component in research on diabetes both in clinical and experimental medicine. The conventional imaging technique for pancreatic islets is antibody-based immunostaining directly on pancreatic sections, or using transgenic mice with luminescent reporter genes linked to islet-specific promoters. Among small molecule probes, Newport Green and dithiazone (DTZ) have been used for ex vivo fluorescent staining of pancreatic islets, based on their Zn ion binding affinity, which are abundant in beta cells in complex with insulin. For in situ application, fluorescently labeled exendin-4 (a GLP1R binding peptide: M.W. is about 5 kDa) has been recently introduced for the measurement of the mass of pancreatic islet beta cells. However, small molecule probes for selective staining of beta cells in pancreatic islets of live animals have not yet been reported. We predicted that a diversity-oriented fluorescence library approach (DOFLA), an expedited bioimaging probe discovery method using high throughput synthesis and high contents screening, would be a powerful method to achieve this goal. Using a similar approach, we have previously elucidated probes for pluripotent stem cells (CDy1), muscle cells (CDy2), neuronal stem cells (CDr3), and pancreatic alpha cells (GY= glucagon yellow). Although the glucagon-targeting probe GY selectively stains alpha cells in isolated cell culture, it did not clearly mark mouse pancreatic islets in tissue, partially owing to the small population of alpha cells (around 15–20% in mouse islets). We expected that a fluorescent probe for pancreatic beta cells (with a larger population of 75–80% in mouse islets) would be more effective for visualizing pancreatic islets. As a first step, we synthesized fluorescent smallmolecule libraries composed of 1200 compounds, and screened them against beta TC-6 cells in comparison to alpha TC-1 cells and acinar cells (exocrine cells in the pancreas) as controls. The three cell types were compared in 384-well plates and incubated with the library compounds (1 mm) at incubation times ranging from one to 48 hours. The fluorescence live-cell images were acquired by an automated imaging microscope system, ImageXpress Micro. One compound from the BDNCA series (Scheme 1), BDNCA-325 (labs/lem= 558/585 nm, extinction cooefficient e= 58,000m 1 cm , quantum yield F= 0.06) was chosen as the most selective for the beta TC-6 cells (Figure 1a) in comparison to the two control cell types in terms of relative fluorescence intensity. The BDNCA library was prepared from a BODIPY-aniline (BDN) series by chloroacetylation. While BDN has very low fluorescence emission (less than 1% quantum yield) owing to photoinduced electron transfer (PET), by converting the amine to an amide, the fluorescence of BDNCA was moderately increased (F= 5–10%). Therefore, this amide motif is a modulator of the fluorescence intensity of the BDNCA series through interaction with the surrounding environment or binding partner. However, when we injected BDNCA-325 intravenously into a mouse, the pancreatic islets were not selectively stained at various incubation times and concentrations (data not shown). Because BDNCA-325 contains a chemically reactive chloroacetyl group, we hypothesized that the compound might have reacted with other tissues in the animal before reaching the pancreatic islets. Thus, BDNCA-325 was modified by removing the reactive alpha-chloride and also by [*] Dr. N.-Y. Kang, Dr. S.-C. Lee, Dr. S.-J. Park, Dr. S.-W. Yun, Dr. E. Kostromina, Dr. N. Gustavsson, Dr. Y. Ali, Y. Chandran, Dr. W. Han, Dr. G. K. Radda, Prof. Y.-T. Chang Singapore Bioimaging Consortium, Agency for Science, Technology and Research (A*STAR) 138667, Singapore (Singapore) E-mail: chmcyt@nus.edu.sg Homepage: http://ytchang.science.nus.edu.sg

Real-Time In Vivo Hepatotoxicity Monitoring through Chromophore-Conjugated Photon-Upconverting Nanoprobes

Drug toxicity is a long-standing concern of modern medicine. A typical anti-pain/fever drug paracetamol often causes hepatotoxicity due to peroxynitrite ONOO- . Conventional blood tests fail to offer real-time unambiguous visualization of such hepatotoxicity in vivo. Here we report a luminescent approach to evaluate acute hepatotoxicity in vivo by chromophore-conjugated upconversion nanoparticles. Upon injection, these nanoprobes mainly accumulate in the liver and the luminescence of nanoparticles remains suppressed owing to energy transfer to the chromophore. ONOO- can readily bleach the chromophore and thus recover the luminescence, the presence of ONOO- in the liver leads to fast restoring of the near-infrared emission. Taking advantages of the high tissue-penetration capability of near-infrared excitation/emission, these nanoprobes achieve real-time monitoring of hepatotoxicity in living animals, thereby providing a convenient screening strategy for assessing hepatotoxicity of synthetic drugs.

Detection of Pathogenic Biofilms with Bacterial Amyloid Targeting Fluorescent Probe, CDy11

Bacterial biofilms are responsible for a wide range of persistent infections. In the clinic, diagnosis of biofilm-associated infections relies heavily on culturing methods, which fail to detect nonculturable bacteria. Identification of novel fluorescent probes for biofilm imaging will greatly facilitate diagnosis of pathogenic bacterial infection. Herein, we report a novel fluorescent probe, CDy11 (compound of designation yellow 11), which targets amyloid in the Pseudomonas aeruginosa biofilm matrix through a diversity oriented fluorescent library approach (DOFLA). CDy11 was further demonstrated for in vivo imaging of P. aeruginosa in implant and corneal infection mice models.