This journal is © The Royal Society of Chemistry 2015 Chem. Commun., 2015, 51, 881--884 | 881 Cite this: Chem. Commun., 2015, 51, 881 Development of 3-alkyl-6-methoxy-7-hydroxy- chromones (AMHCs) from natural isoflavones, a new class of fluorescent scaffolds for biological imaging† Jianzhuang Miao,‡ a Huaqing Cui,‡* a Jing Jin, a Fangfang Lai, a Hui Wen, b Xiang Zhang, b Gian Filippo Ruda, c Xiaoguang Chen a and Dali Yin* a Starting from 7-hydroxyisoflavones, we developed a new class of fluorescent scaffolds, 3-alkyl-6-methoxy-7-hydroxy-chromones (AMHCs, M W B 205.19, k ab B 350 nm, k em B 450 nm) via a trial and error process. AMHCs have the advantages of being a small mole- cular moiety, having strong fluorescence in basic buffers, reasonable solubility and stability, non-toxicity, and are conveniently linked to pharmacophores. AMHCs were successfully used in fluorescence microscopy imaging of cells and tissues. Fluorescent dyes have been widely used in biological research for analytical sensing and biological imaging. 1–9 In spite of the increasing demand, the library of conventional fluorophores only contains a limited number of scaffolds, including naphthalimide, styryl, xanthone, coumarin, dapoxyl, BODIPY, rhodol and tricarbo- cyanine. 3,4,7,8 Efforts to diversify the fluorophore library include both the de novo synthesis of new scaffolds and the optimization of known scaffolds. 8,10–17 However, due to the complex of photo- physical properties of fluorophores, it is difficult to predict the emission wavelength or quantum yield of a fluorogenic scaffold, and most of these studies are performed empirically. 8,10–19 Natural products are important sources of new scaffolds for fluorophores. 1,20 Natural 7-hydroxyisoflavones and synthetic 3-hydroxyflavones have been reported to have weak fluorescence in biological buffers. This limits their application in biological imaging (Fig. S1 and S2, ESI†). 21–29 Efforts have been made to circumvent these problems. For example, 3-hydroxyflavone 1 has been further developed into a series of moderate to strong fluorophores in ethanol, such as 3 (F = 0.48 in 95% ethanol) (Fig. 1). This optimisation has increased the fluorescence, and the size of the molecule was also increased. 30,31 To our knowledge, 7-hydroxyisoflavone 2 has not yet been used as a lead for the development of new fluorophores. Nevertheless, 7-hydroxyisoflavone is able to exhibit fluores- cence, albeit weakly in a biological buffer, and has superior aqueous solubility compared to 3-hydroxyflavone (Fig. S3a and b, ESI†). Thus, in this study we opted to optimize the photophysical properties of isoflavones in order to develop a new class of fluorescent dyes. Considering the difficulty in theoretical prediction of optical properties, 1 our optimisation was carried out by a trial and error process. We investigated the effect of various substituents at different positions of the isoflavone core on its photophysical properties. Subsequently, favourable modifications were com- bined to design new fluorophores. In addition, given the future application for biological imaging, possible properties were also designed into final fluorophores. During our first round of optimisation, we developed a specific library of isoflavone analogues via chemical synth- esis. 32 After fully evaluating the fluorescence properties of the Fig. 1 Previous development of flavone 1 based fluorophore 3 and the desired optimization of isoflavone 2 in this study. a State Key Laboratory of Bioactive Substances and Function of Natural Medicine, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, 1 Xiannongtan Street, Beijing, 100050, China. E-mail: yindali@imm.ac.cn, hcui@imm.ac.cn b Beijing Key Laboratory of Active Substances Discovery and Drugability Evaluation, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, 1 Xiannongtan Street, Beijing, 100050, China c TDI-SGC Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford, OX37FZ, UK † Electronic supplementary information (ESI) available: Experimental procedures, photophysical properties, and data of compounds. See DOI: 10.1039/c4cc06762b ‡ J.M. and H.C. contributed equally. Received 27th August 2014, Accepted 21st November 2014 DOI: 10.1039/c4cc06762b www.rsc.org/chemcomm ChemComm COMMUNICATION