Reaction-based small-molecule fluorescent probes for chemoselective bioimaging

Chemically diverse small molecule fluorescent chemosensors for copper ion

Abnormal enzymatic activities are directly related to the development of cancers. Identifying the location and expression levels of these enzymes in live cancer cells have considerable importance in early-stage cancer diagnoses and monitoring the efficacy of therapies. Small-molecule fluorescent probes have become a powerful tool for the detection and imaging of enzymatic activities in biological systems by virtue of their higher sensitivity, nondestructive fast analysis, and real-time detection abilities. Moreover, due to their structural tailorability, numerous small-molecule enzymatic fluorescent probes have been developed to meet various demands involving real-time tracking and visualizing different enzymes in live cancer cells or in vivo. In this review, we provide an overview of recent advances in small-molecule enzymatic fluorescent probes mainly during the past decade, including the design strategies and applications for various enzymes in live cancer cells. We also highlight the challenges and opportunities in this rapidly developing field of small-molecule fluorescent probes for interventional surgical imaging, as well as cancer diagnosis and therapy.

Recent advances in mitochondria- and lysosomes-targeted small-molecule two-photon fluorescent probes

The concentrations of reactive oxygen/nitrogen species (ROS/RNS) are critical to various biochemical processes. Small-molecule fluorescent probes have been widely used to detect and/or quantify ROS/RNS in many redox biology studies and serve as an important complementary to protein-based sensors with unique applications. Recent Advances: New sensing reactions have emerged in probe development, allowing more selective and quantitative detection of ROS/RNS, especially in live cells. Improvements have been made in sensing reactions, fluorophores, and bioavailability of probe molecules. In this review, we will not only summarize redox-related small-molecule fluorescent probes but also lay out the challenges of designing probes to help redox biologists independently evaluate the quality of reported small-molecule fluorescent probes, especially in the chemistry literature. We specifically highlight the advantages of reversibility in sensing reactions and its applications in ratiometric probe design for quantitative measurements in living cells. In addition, we compare the advantages and disadvantages of small-molecule probes and protein-based probes. The low physiological relevant concentrations of most ROS/RNS call for new sensing reactions with better selectivity, kinetics, and reversibility; fluorophores with high quantum yield, wide wavelength coverage, and Stokes shifts; and structural design with good aqueous solubility, membrane permeability, low protein interference, and organelle specificity. Antioxid. Redox Signal. 29, 518-540.

As the cardinal support of innumerable biological processes, biomacromolecules such as proteins, nucleic acids and polysaccharides are of importance to living systems. The key to understanding biological processes is to realize the role of these biomacromolecules in thte localization, distribution, conformation and interaction with other molecules. With the current development and adaptation of fluorescent technologies in biomedical and pharmaceutical fields, the fluorescence imaging (FLI) approach of using small-molecule fluorescent probes is becoming an up-to-the-minute method for the detection and monitoring of these imperative biomolecules in life sciences. However, conventional small-molecule fluorescent probes may provide undesirable results because of their intrinsic deficiencies such as low signal-to-noise ratio (SNR) and false-positive errors. Recently, small-molecule fluorescent probes with a photoinduced electron transfer (PET) "on/off" switch for biomacromolecules have been thoroughly considered. When recognized by the biomacromolecules, these probes turn on/off the PET switch and change the fluorescence intensity to present a high SNR result. It should be emphasized that these PET-based fluorescent probes could be advantageous for understanding the pathogenesis of various diseases caused by abnormal expression of biomacromolecules. The discussion of this successful strategy involved in this review will be a valuable guide for the further development of new PET-based small-molecule fluorescent probes for biomacromolecules.

Bacterial infections remain as a prevailing situation in our today’s society. Owing to the evolving pathogenicity of bacteria, whereby more drug-resistant bacteria has been emerging and bacteria developing their own defense mechanism, traditional antimicrobial approaches like antibiotics treatment have been clinically ineffective. Early and accurate strategies are off dire need to subvert the rapid colonization of bacteria. Hence, the employment of fluorescence imaging for specific bacterial labeling and infection diagnosis could be prospective in providing physiological information in real time for the diagnosis and therapeutic intervention of bacterial infections. Small-molecule fluorescence probes have been an attractive tool in fluorescence imaging due to their inherent properties such as highly tunable emissions, high quantum yield and good biocompatibility. Among the numerous types of small-molecule fluorescence probes, the enzymatic responsive probes have been one that researchers are particularly interested in as such probes were reported to possess great specificity and high signal to noise ratio. Furthermore, bacterial enzymes are found to be closely related to the invasion of host immune system, which makes them a viable target to establish enzyme-activated fluorescent probes for specific bacterial imaging. In chapter 1, we will be giving a brief background on bacterial infections, introducing the principles and techniques used in the design of small-molecule fluorescence probes, and followed by providing some of the reported examples of the recent and typical strategies for enzyme-activated small-molecule fluorescence probes. In chapter 2, we explore the specific proteolytic activity of an outer membrane bacterial protease that is present in all wild-type Escherichia coli (E. coli) and another bacterial protease located in the periplasmic region of E. coli to synthesize a near infrared fluorescence probe for the real-time imaging of urinary tract bacterial infection in vitro and in vivo. In chapter 3, the experimental section and supplementary figures are presented.

Small luminescent molecular probe for developing as assay for alkaline phosphatase

The first small-molecule fluorescent turn-on probes for detecting PDEδ protein were rationally designed, showing reasonable fluorescent properties and the fluorescent ability has been applied for visualization of the PDEδ protein in living cells and at tissue levels. The qPCR results showed that the mRNA expression of KRAS, PDEδ, AKT1, MAPK1, MEK7, RAF1, and mTOR were downregulated by probes 1-3 through PI3K/AKT/mTOR and MAPK signal pathways. The probes also can downregulate the protein level of pErk and tErk. Therefore, these small-molecule fluorescent probes are expected to be used in the screening of antipancreatic cancer drugs targeting the PDEδ protein, as well as in obtaining a better understanding of the pathological and physiological roles of PDEδ protein.

<p indent="0mm">Fluorescence imaging technology based on small molecule probes has the advantages of high sensitivity, simple operation, fast response speed, small sample damage and high spatial and temporal resolution, and is a powerful tool for obtaining biochemical information at the cellular and living level. Among them, the orderly assembled fluorescent small molecule probes have better imaging performance than traditional small-molecule fluorescent probes (such as strong anti-diffusion ability, high imaging contrast, and good photostability due to their unique assembly methods and recognition modes). This article summarizes the applications of several common organic dye aggregates and corresponding small molecule fluorescent probes in the field of bioimaging. Subsequently, the design and optimization strategies of organic dye assemblies represented by HPQ and its derivatives with hydrogen-bond-driven ordered assembly and their applications in the biomedical field are highlighted. Finally, the problems existing in the design and application of ordered assembled fluorescent small molecule probes are discussed, which provides ideas for the design of new ordered assembled fluorescent small molecule probes.

Small-molecule fluorescent probes for H2S detection: Advances and perspectives

Reaction-based small-molecule fluorescent probes for dynamic detection of ROS and transient redox changes in living cells and small animals

Because of the limited knowledge on the relationship between molecular structure and analytical performance, developing a small molecule fluorescent probe with desirable response properties is usually a laborious work. On the other hand, the application of small molecule fluorescent probe in biological samples is always limited due to the unwanted interaction between dyes and biomacromolecules. Polymer micelles, thanks to its unique core-shell structure, may have the potential to improve these situations. However, utilization of polymer micelles to improve these situations is rarely explored. Herein, we engineered the first micellar SO2 nanoprobe Nano-Cz by self-assembly of a carbazole-based SO2 small molecule probe and an amphiphilic copolymer (DSPE-mPEG2000). The optical and cell imaging experiments revealed that Nano-Cz can work in 100% aqueous environment and act as an effective mitochondrial-targeting ratio SO2 nanoprobe. Compared with the single small molecule probe, Nano-Cz showed extraordinary large dynamic response range (0-0.7 mM vs 0-50 μM), eliminated signal interference from DNA and superior cellular imaging performance. These results clearly show the ability of polymer micelles in modulating sensors' analytical performance and reducing the signal interference from the unwanted interaction between small molecule probe and biomacromolecule, indicating that polymer micelles encapsulating single small molecule probe can provide us an alternative strategy to explore sensors with various performance and promote the biological application of fluorescent sensors. In addition, we hope that more and more polymer micelles would be used to construct biosensors in the future.

Activatable near-infrared (NIR) fluorescent probes possess advantages of high selectivity, sensitivity, and deep imaging depth, holding great potential in the early diagnosis and prognosis assessment of tumors. However, small-molecule fluorescent probes are largely limited due to the rapid diffusion and metabolic clearance of activated fluorophores in vivo. Herein, we propose an efficient and reproducible novel strategy to construct activatable fluorescent nanoprobes through bioorthogonal reactions and the strong gold-sulfur (Au-S) interactions to achieve an enhanced permeability and retention (EPR) effect, thereby achieving prolonged and high-contrast tumor imaging in vivo. To demonstrate the merits of this strategy, we prepared an activatable nanoprobe, hCy-ALP@AuNP, for imaging alkaline phosphatase (ALP) activity in vivo, whose nanoscale properties facilitate accumulation and long-term retention in tumor lesions. Tumor-overexpressed ALP significantly increased the fluorescence signal of hCy-ALP@AuNP in the NIR region. More importantly, compared with the small-molecule probe hCy-ALP-N3, the nanoprobe hCy-ALP@AuNP significantly improved the distribution and retention time in the tumor, thus improving the imaging window and accuracy. Therefore, this nanoprobe platform has great potential in the efficient construction of biomarker-responsive fluorescent nanoprobes to realize precise tumor diagnosis in vivo.

Peroxynitrite (ONOO- ) as a major reactive oxygen species plays important roles in cellular signal transduction and homeostatic regulation. Precise detection of ONOO- in biological systems is vital for exploring its physiological and pathological function. Among numerous detection methods, fluorescence imaging technology using fluorescent probes offers some advantages, including simple operation, high sensitivity and selectivity, as well as real-time and nondestructive detection. In particular, ratiometric fluorescent probes, in which the built-in calibration of the two emission bands prevents interference from the biological environment, have been extensively employed to monitor the fluctuation of bioactive species. In this review, we will discuss small-molecule ratiometric fluorescent probes for ONOO- in live cells or in vivo, which involves chemical structures, response mechanisms, and biological applications. Moreover, the challenges and future prospects of ONOO- -responsive ratiometric fluorescent probe are also proposed.

Fluorescence imaging is a powerful tool that permits visualization of specific cell states within a population; however, existing methods for fluorescence labeling cannot be easily applied in many biological systems. Unlike antibodies, small molecule proteins can be cell permeable and therefore useful in live-cell and in vivo imaging experiments; moreover, small molecule probes do not require genetic manipulation of cells. Protein kinases are in many ways ideal targets for the development of selective fluorescent small molecule probes. This is because protein kinases are involved in most cellular processes and changes in their localization, accessibility, and abundance are associated with changes in cellular state. In addition, drug discovery and chemical biology efforts have in recent decades produced many selective, cell permeable small molecule ligands of specific cellular kinases. Here we describe our attempts to leverage existing, well-characterized kinase inhibitors to develop fluorescent small molecule probes for use as imaging tools in cancer biology. BODIPYconjugated kinase inhibitors, such as Mps1-IN-1 and BI2536 were synthesized. Their inhibition ability and immunofluorescence staining were tested. We demonstrated the utility of BI-BODIPY as a cell permeable probe for monitoring PLK localization. This result serves as the foundation for more sophisticated live-cell and in vivo imaging experiments that we are currently pursuing. This study also provides proof of concept for extension of this strategy to convert other small molecule kinase inhibitors to probes that can analogously be used to monitor localization of their respective kinases. 1. N. Kwiatkowski, N. Jelluma, P. Filippakopoulos, M. Soundararajan, M. S. Manak, M. Kwon, H. G. Choi, T. Sim, Q. L. Deveraux, S. Rottmann, D. Pellman, J. V. Shah, G. J. Kops, S. Knapp and N. S. Gray, Nat. Chem. Biol, 2010, 359. 2. P. Lenart, M. Petronczki, M. Steegmaier, B. Di Fiore, J. J. Lipp, M. Hoffmann, W. J. Rettig, N. Kraut and J. M. Peters, Curr. Biol, 2007, 304. 3. Z. Zhang, N. Kwiatkowski, H. Zeng, S. M. Lim, N. S. Gray, W. Zhang, P. L. Yang, Mol. BioSystems 2012, 8, 2523. N N N N O N H O N H O N N O N N B F F