FULL PAPER Synthesis and Systematic Evaluation of Dark Resonance Energy Transfer (DRET)-based Library and Its Application in Cell Imaging Dongdong Su, [a],[b] Chai Lean Teoh, [b] Nam-Young Kang, [b] Xiaotong Yu, [a] Srikanta Sahu [b] and Young- Tae Chang* ,[a],[b] Abstract: In this paper, we report a new strategy for constructing large Stokes shift dye library. By coupling a dark donor with tunable high quantum yield BODIPY acceptors, a novel Dark Resonance Energy Transfer (DRET) based library, named BNM, has been synthesized. Upon excitation of the dark donor (BDN) at 490 nm, it was demonstrated that the absorbed energy was transferred to the acceptor (BDM) with high efficiency, which was tunable in a broad range from 557 nm to 716 nm, with high quantum yield of up to 0.8. It is noteworthy to mention that the majority of the non-radiative energy loss of donor was converted to the acceptor’s fluorescence output with minimum leaks of donor emission. Fluorescence imaging tested in live cells showed that the BNM compounds are cell-permeable and can also be employed for live cell imaging. This is a new library which can be excited by dark donor to emit a tunable wide range of high fluorescence emission. Thus, the BNM library is well suited for high- throughput screening or multiplexing experiments in biological methods by using a single laser excitation source. Introduction Fluorescent labelling has been developed to be a powerful tool in many biochemical and medical diagnostics, which can provide visualization of organelles as well as real-time monitoring of biochemical processes. [1] In recent years, many approaches have been explored to develop fluorescent labeling technology, such as labeling of individual cells, [2] DNA sequencing labeling [3] and proteins labeling. [4] Among all these approaches, multicolor fluorescent labeling exhibits the advantages of time resolution, however, this approach has been hampered due to the limited availability of multicolor fluorescent molecules. Up to now, antibody, [5] quantum dots [6] and organic dyes are the widely used signal reporters for specific labeling of targets, however, each of them has its drawbacks in multicolor fluorescent labeling technology. Different antibodies can provide specific labeling of cellular organelles in a multicolor format, but their relatively large size and poor cell permeability hindered their progress in practical biological applications. [7] Compared to other fluorophores, quantum dots show some advantages like having multiple colors and high photostability. [8] However, their cell toxicity and high consumption become their significant drawbacks. [9] Aside from antibody and quantum dots, small fluorescence molecules were also developed in the field of fluorescence labeling because of their sensitivity and easy visibility. [1b, 10] Till now, most of the multiplexing experiments have been achieved by using different structures of organic fluorophores for each color labeling. [11] However, using different organic dyes may suffer from photoinstability, pH and ionic sensitivity and may induce unpredictable interactions with various biopolymers during the experiments. [3c] New fluorescence libraries based on DOFLA (diversity- oriented fluorescence library approach) reported by our group have led to the discovery of a series of novel sensors. [12] The DOFLA libraries which were constructed with the same fluorescence scaffold but with different building blocks can be used for multicolor labeling. The features of DOFLA libraries can potentially overcome some of the drawbacks caused by fluorophores with different structures. [13] Single fluorescent core structure can show tunable emission and even predictable photophysical properties. [14] However, for these published libraries, it was difficult to find one single wavelength excitation suitable for all these fluorophores with tunable emission. The dye at longer absorption wavelength cannot absorb enough energy to emit strong fluorescent intensities. [15] With the involvement of Förster resonance energy transfer (FRET) approach, we can solve the above mentioned problems. [16] The FRET-based dyes are constructed with the same donor but different acceptors, which will allow us to obtain strong tunable emissions by exciting the same donor. Recently, our group reported a set of novel Dark Resonance Energy Transfer (DRET) dyes, BNM, which use low quantum yield donor (less than 1%) to emit a wide range of high fluorescence emission. [17] The results show that the absorbed energy can be transferred to the acceptor with a high energy transfer rate, before being quenched by non-radiative intramolecular rotations. Also, this new designed DRET-based dyes show unique photophysical characteristics, such as high ability of light harvesting without fluorescence leaking from the donor, tunable emission wavelength excited at a single wavelength excitation, large pseudo-Stokes shifts and emission shifts, as well as highly efficient energy transfer. Furthermore, this type of dyes shows good cell penetration, which makes them as good candidate for living cells imaging. [17] All of these great properties encourage us to develop one library of BNM compounds and further study the relationship between structures and photophysical properties and then we can design and develop new BNM dyes for practical applications. [a] D. Su, X. Yu and Prof. Y. T. Chang Department of Chemistry & MedChem Program of Life Sciences Institute National University of Singapore, 117543, Singapore Tel: (65) 6516-6774, Fax: (+65) 6779-1691 E-mail: chmcyt@nus.edu.sg [b] Dr. C. L. Teoh, Dr. N. Y. Kang, Dr. S. Sahu and Prof. Y. T. Chang Singapore Bioimaging Consortium, Agency for Science, Technology and Research (A*STAR), 138667, Singapore Supporting information for this article is given via a link at the end of the document.((Please delete this text if not appropriate))