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Imaging dynamics of membrane proteins of live cells in a wash-free and real-time manner has been a challenging task. Herein, we report unprecedented applications of malachite green (MG), an organic dye widely used in pigment industry, as a switchable fluorophore to monitor membrane enzymes or non-catalytic proteins in live cells. Conformationally flexible MG is non-fluorescent in aqueous solution, yet covalent binding with endogenous proteins of cells significantly enhances its fluorescence at 670 nm by restricting flexibility of dye. Integrating a phosphate-caged quinone methide precursor with MG yielded a covalent labeling fluorogenic probe, allowing real-time imaging of membrane alkaline phosphatase (ALP, a model catalytic protein) activity in live cells with over 100-fold enhancement of fluorescence intensity. Moreover, MG is also applicable to image non-catalytic protein by conjugation with protein-specific ligand. A fluorogenic probe consisted of c-RGDfK peptide and MG proved to be compatible with wash-free and real-time visualization of non-catalytic integrin αvβ3 in live cells with high contrast.

Reported herein is a regioselective difluoromethane sulfonylation or triflylation of resorufin derivatives, which allows easy access to 2-difluoromethane sulfonylated or triflylated resorufin derivatives in good yields. The installation of a difluoromethane sulfonyl group significantly increases the solubility of the chromophore and expands its Stokes shift. A difluoromethane sulfonylated resorufin-based fluorogenic probe proved to be able to image enzyme activity in live cells with a stronger fluorescence signal compared with its resorufin counterpart.

A fluorogenic probe for the specific detection of IMP-1 β-lactamase activity has been developed. This imaging reagent features a unique trans-acetylamino cephalosporin as an enzymatic recognition moiety, exhibiting excellent selectivity to IMP-1 β-lactamase over other β-lactamases, including serine- and metallo-β-lactamases. The selective activation of the probe by IMP-1 β-lactamase leads to over 30-fold enhancement in the fluorescence intensity, which allows enzyme activity to be reported with high sensitivity.

The β-lactam drugs resistance poses a serious threat to human health throughout the world. Klebsiella pneumoniae carbapenemase 2 (KPC-2) is a carbapenemase that produced in bacteria can hydrolyze carbapenems, which typically considered as the antibiotics of last resort. Therefore, there is an urgent need to quickly and accurately detect whether bacteria express KPC-2. In this paper, a PDMS/glass microfluidic biochip integrated with aptamer-modified Ag10NPs nano-biosensors was developed for rapid, simple and specific pathogenic bacteria detection, more importantly, the biochip was combined with bright field imaging, then the captured bacteria could be observed and counted directly without using extra chemical labeling. KPC-2-expressing Escherichia coli (KPC-2 E.coli) was used as the target bacterium with a detected limit of 102 CFU and capture efficiency exceeded 90%. This method is remarkably specific towards KPC-2 E.coli over other non-resistant bacteria, and pathogen assay only takes ∼1 h to complete in a ready-to-use microfluidic biochip. Furthermore, the effective capture and fast counting of microfluidic biochip system demonstrates its potential for the rapid detection of antibiotic-resistant bacteria.

Reported herein is a self-immobilizing near-infrared fluorogenic probe that can be used to image extracellular enzyme activity in vivo. Using a fluorophore as a quinone methide precursor, this probe covalently anchors at sites of activation and greatly enhances the fluorescence intensity at 710 nm upon enzymatic stimulus, significantly boosting detection sensitivity in a highly dynamic in vivo system.

Reported herein is a fluorogenic probe for the detection of carbapenemase activity. This reagent features carbapenem as an enzyme recognition motif and a carbon-carbon double bond between carbapenem and the fluorophore, exhibiting high specificity to all carbapenemases, including metallo carbapenemases and serine carbapenemases, over other β-lactamases.

6-[18F]fluoro-L-DOPA is a radiotracer widely used in the diagnosis of a range of diseases, including neuro-oncology, endocrinology, and Parkinson’s disease. To meet the fast growing clinical need for this radioactive compound, this study reports an optimized radiosynthsis of this molecule, which proved to be highly reliable and compatible with different types of automated radiosynthesizers. Moreover, with 6-[18F]fluoro-L-DOPA, the PET/CT imaging of a total of 23 patients has been conducted, further demonstrating this radiotracer as a clinically valuable reagent to diagnose congenital hyperinsulinism (CHI) of infancy and, more importantly, localize the exact lesion on pancreas.

A fluorogenic probe for the specific detection of IMP-1 β-lactamase activity has been developed. This imaging reagent features a unique

Antibiotic resistance caused by β-lactamases, particularly metallo-β-lactamases, has been a major threat to public health globally. New Delhi metallo-β-lactamase-1 (NDM-1) represents one of the most important metallo-β-lactamases; the production of NDM-1 in bacterial pathogen significantly reduces the efficacy of β-lactam antibiotics, including life-saving carbapenems. Herein, we have demonstrated stereochemically altered cephalosporins as potent inhibitors against NDM-1, as well as mutants of NDM. The structure and activity relationship (SAR) study on over twenty cephalosporin analogues discloses the stereochemistry and the substituents on 7-position and 3'-position of cephalosporin are critical to suppress the activity of NDM-1 and the optimal compound 1u exhibited an IC

Reported herein is a novel 4-OTBS benzyl-based protective group for carboxylic acids. This protective group can be removed in the presence of TBAF or TFA with high efficiency, which makes it compatible with base-sensitive or acid-sensitive substrates. With this protective group, a near-infrared fluorogenic probe for the detection of γ-glutamyltranspeptidase activities was readily prepared.

Reported herein is a novel p-quinone methide-based self-immobilizing fluorogenic probe for the visualization of β-galactosidase activities in live cells. This easily prepared imaging reagent massively increases the fluorescence intensity and covalently links to the activation site with high efficiency upon enzymatic trigger.

An efficient copper-mediated ortho-C(sp2)-H thiolation of aromatic amides directed by a novel directing group [4-chloro-2-(1H-pyrazol-1-yl)phenyl]amine has been developed without the need of other additives or oxidants, allowing for an increased usefulness. With the high compatibility of sterically demanding substrates, this reaction is scalable and can tolerate a wide scope of functional groups to provide alkyl and aryl thioethers in good to excellent yields (up to 93%). Furthermore, the protocol has been successfully implemented for the selenylation as well.

γ-Glutamyl transpeptidase (GGT), a type of cell membrane-bound enzyme, is closely involved in a wide range of physiological and pathological processes, and a large number of fluorogenic probes have been developed to detect the activity of GGT. However, the use of these imaging reagents to visualize GGT activity in vivo is largely limited because of rapid diffusion and clearance of activated fluorophores. Herein, by merging quinone methide and a fluorogenic enzyme substrate, we report an activatable self-immobilizing near-infrared probe for the in vitro and in vivo imaging of GGT activity. This probe is initially fluorescently silent, but the selective activation by GGT is able to significantly increase its fluorescence intensity at 714 nm and covalently anchor activated fluorophores at the site of interest. We have shown that this probe induced a much stronger fluorescence on live GGT-overexpressing cells compared to regular fluorogenic probes and allowed wash-free and real-time imaging of enzyme activity. More importantly, the use of this probe in the imaging of GGT activity in U87MG tumor-bearing mice by i.v. administration indicates that this self-immobilizing reagent is capable of efficiently enhancing its retention at the detection target and thus leads to much improved detection sensitivity compared to regular fluorogenic probes. This study demonstrates the advantage of fluorogenic probes with activatable anchors in the noninvasive imaging of enzyme activity in highly dynamic in vivo systems.

Klebsiella pneumoniae carbapenemase 2 (KPC-2) is a serine β-lactamase that can hydrolyze almost all β-lactam antibiotics. The drug resistant problem of bacteria expressing carbapenemases is currently a global problem, therefore, rapid and specific detection of pathogenic bacteria is urgent. In order to obtain an aptamer that can specifically recognize bacteria expressing KPC-2, we have established a method called Precision-SELEX. Precision-SELEX combined protein SELEX and bacterium SELEX. In this method, KPC-2 was used as a target protein, and Escherichia coli expressing KPC-2 (KPC-2 E. coli) was used as a target bacterium. After precision-SELEX, the same aptamer named XK-10 that can recognize KPC-2 and KPC-2 E. coli was obtained while the screening process could be shortened to 4 rounds. Dissociation equilibrium constants were calculated as 0.81 nM by SPR. In addition, XK-10 could specifically bind to KPC-2 E. coli, which was confirmed through flow cytometry and molecular Docking Simulations. The high-content imaging method could detect KPC-2 E. coli. In all, the Precision-SELEX provides an accurate and efficient method to screening aptamers for bacteria.

Reported herein is a general approach to optically active isoflavanes based on a chiral amine-catalyzed [4 + 2] asymmetric annulation of o-quinone methides and aldehydes. A number of naturally occurring isoflavanes, including equol, sativan, isosativan, vestitol and medicarpin, as well as isoflavane analogues were readily prepared with good to excellent enantioselectivities.

Methicillin-resistant Staphylococcus aureus (MRSA) has been one of the major nosocomial pathogens to cause frequent and serious infections that are associated with various biomedical surfaces. This study demonstrated that surface modified with host defense peptide-mimicking β-peptide polymer, has surprisingly high bactericidal activities against Escherichia coli (E. coli) and MRSA. As surface-tethered β-peptide polymers cannot move freely to adopt the collaborative interactions with bacterial membrane and are too short to penetrate the cell envelop, we proposed a mode of action by diffusing away the cell membrane-stabilizing divalent ions, Ca2+ and Mg2+. This hypothesis was supported by our study that Ca2+ and Mg2+ supplementation in the assay medium causes up to 80% loss of bacterial killing efficacy and that the addition of divalent ion chelating ethylenediaminetetraacetic acid into the above assay medium leads to significant recovery of the bacterial killing efficacy. In addition to its potent bacteria...

Reported herein is a chiral Brønsted acid-catalyzed asymmetric intramolecular allylic amination reaction, allowing facile access to a range of biologically interesting chiral 2-substituted hydroquinolines in up to 90% yield and with up to 93% ee. Furthermore, a significant effect of an N-protecting group was observed in this asymmetric process.

The dissemination of antimicrobial resistance around the world is one of the biggest threats to global public health. The acquisition and expression of extended-spectrum β-lactamases (ESBLs) in pathogenic bacterial are mainly responsible for bacterial resistance to third-generation cephalosporins. Reported herein is a ratiometric fluorescent probe for the detection of the activity of ESBLs. This imaging reagent adopts the core structure of cefotaxime as an enzymatic recognition moiety, and exhibits excellent selectivity to ESBLs over other β-lactamases. The specific activation of this sensor by ESBLs can lead to over 2500-fold changes in the fluorescent ratio, which is independent of the concentration of the probe and environmental conditions. Further experiments have demonstrated that this ratiometric fluorescent probe can distinguish bacteria with extended-spectrum antibiotic resistance from a group of clinically important pathogens within a short period of time.

Reported herein is a conceptually novel organocatalytic strategy for the formylation of boronic acids. New reactivity is engineered into the α-amino-acid-forming Petasis reaction occurring between aryl boronic acids, amines, and glyoxylic acids to prepare aldehydes. The operational simplicity of the process and its ability to generate structurally diverse and valued aryl, heteroaryl, and α,β-unsaturated aldehydes containing a wide array of functional groups, demonstrates the practical utility of the new synthetic strategy.

Quinone methide (QM) as a latent trapping unit has been widely explored in activity-based self-immobilizing reagents. However, further application of this strategy has been largely hampered by the limited labeling efficiency to proteins. In this study, a thorough investigation on the labeling efficiency and the structure of QM-based trapping unit is presented, from which a QM with multiple leaving groups was identified as an optimal trapping unit. An alkaline phosphatase (ALP) immobilizing reagent featured with this multiple-labeling trapping unit exhibited lower nonspecific binding and, remarkably, a significantly higher labeling efficiency over other immobilizing reagents upon enzymatic activation. The utility of this imaging reagent was further demonstrated with the in vitro and in vivo visualization of the ALP activities. Furthermore, the multiple functional trapping unit may find greater value in the other activity-based immobilizing probes.

Reported herein is a relebactam-derived fluorogenic reagent for covalent labeling of serine β-lactamases (SBLs), which are the major causes of bacterial resistance to β-lactam antibiotics. This highly selective imaging reagent generates over 300-fold stronger near-infrared fluorescence signals upon covalently bonding to SBLs, allowing wash-free visualization of live antimicrobial-resistant bacteria.

A [4+2] annulation of aldehydes and o-quinone methides catalyzed by a secondary amine is developed. This process leads to biologically important dihydrocoumarins in moderate to good yields after oxidation. In addition, the employment of a chiral secondary amine catalyst allows access to optically active dihydrocoumarins with up to 64% ee.

The spread of antibiotic resistance in pathogenic bacteria has become one of the major concerns to public health. Improved monitoring of drug resistance is of high importance for infectious disease control. One of the major mechanisms for bacteria to overcome treatment of antibiotics is the production of β-lactamases, which are enzymes that hydrolyze the β-lactam ring of the antibiotic. In this study, we have developed a self-immobilizing and fluorogenic probe for the detection of β-lactamase activity. This fluorogenic reagent, upon activation by β-lactamases, turns on a fluorescence signal and, more importantly, generates a covalent linkage to the target enzymes or the nearby proteins. The covalent labeling of enzymes was confirmed by SDS-PAGE analysis and MALDI-TOF mass spectrometry. The utility of this structurally simple probe was further confirmed by the fluorescent labeling of a range of β-lactamase-expressing bacteria.

Tuberculosis is a pulmonary disease with an especially high mortality rate in immuno-compromised populations, specifically children and HIV positive individuals. The causative agent, Mycobacterium tuberculosis (Mtb), is a very slow growing and difficult organism to work with, making both diagnosis and development of effective treatments cumbersome. We utilize a fiber-optic fluorescence microendoscope integrated with a whole-body imaging system for in vivo Mtb detection. The system exploits an endogenous enzyme of Mtb (β-lactamase, or BlaC) using a BlaC-specific NIR fluorogenic substrate. In the presence of BlaC, this substrate is cleaved and becomes fluorescent. Using intravital illumination of the lung to excite this probe, sensitivity of the optical system increases over trans- and epi-illumination methods of whole-body fluorescence imaging. We demonstrate that integration of these imaging technologies with BlaC-specific fluorescent reporter probe improves the level of detection to ∼100 colony forming units, a 100× increase in sensitivity in comparison to epi-illumination and a 10× increase in sensitivity in comparison to previous work in intravital excitation of tdTomato-expressing Mtb. This lower detection threshold enables the study of early stage bacterial infections with clinical strains of Mtb and longitudinal studies of disease pathogenesis and therapeutic efficacy with multiple time points in a single animal.

A proline-mediated inverse electron demand Diels–Alder reaction of aldehyde and tetrazine is disclosed. This biocompatible reaction is very fast (with apparent k2 up to 13.8 M−1 s−1) and has been used in the labeling of bovine serum albumin in aqueous medium.

A highly enantioselective addition of alcohols to cyclic trifluoromethyl ketimines is developed catalyzed by quinine-thiourea, giving biologically interesting N,O-ketals in up to 99% yield and 96% ee.

A ligand-free Cu-catalyzed [3 + 2] cycloaddition of ethyl 2-(quinolin-2-yl)acetates, ethyl 2-(isoquinolin-1-yl)acetates, and ethyl 2-(pyridin-2-yl)acetates with (E)-chalcones for a "one-pot" synthesis of pyrrolo[1,2-a]quinolines, pyrrolo[2,1-a]isoquinolines and indolizines has been developed. The annulation products were isolated in moderate to good yields with air as the sole oxidant under mild conditions.

Metallo-β-lactamase is one of the major clinical threats because this β-lactam-hydrolyzing enzyme confers significant resistance to most β-lactam antibiotics, including carbapenems, among bacterial pathogens. Reported herein is a novel fluorogenic sensor for the specific detection of metallo-β-lactamase activities. This carbapenem-based reagent exhibits excellent selectivity to metallo-β-lactamase over other serine-β-lactamases, including serine carbapenemases. The usefulness of this probe was further demonstrated in the rapid identification of metallo-β-lactamase-expressing pathogenic bacteria.

A Pd(OAc)2-catalyzed divergent synthesis of multisubstituted imidazoles and quinazolines from N-allylamidines has been developed. In the presence of oxidant O2 from air and/or a ligand and Pd(OAc)2, imidazoles were obtained. Nonetheless, under microwave (MW) irradiation in a sealed system, quinazolines as major products were produced.

An unprecedented chiral secondary amine-catalyzed formal [4 + 2] annulation of aldehydes and oxidation-generated β-unsubstituted o-QMs is reported. This asymmetric protocol allows direct functionalization of the benzylic C–H bonds and furnishes [4 + 2] cycloadducts, chromanols, with excellent enantioselectivity and in up to 92% yield. The usability of this approach was further demonstrated by the enantioselective synthesis of anticancer Rhinacanthins derivative NKPLS8.

Current methods for the detection of Mycobacterium tuberculosis (Mtb) are either time consuming or require expensive instruments and are thus are not suitable for point-of-care diagnosis. The design, synthesis, and evaluation of fluorogenic probes with high specificity for BlaC, a biomarker expressed by Mtb, are described. The fluorogenic probe CDG-3 is based on cephalosporin with substitutions at the 2 and 7 positions and it demonstrates over 120 000-fold selectivity for BlaC over TEM-1 Bla, the most common β-lactamase. CDG-3 can detect 10 colony-forming units of the attenuated Mycobacterium bovis strain BCG in human sputum in the presence of high levels of contaminating β-lactamases expressed by other clinically prevalent bacterial strains. In a trial with 50 clinical samples, CDG-3 detected tuberculosis with 90 % sensitivity and 73 % specificity relative to Mtb culture within one hour, thus demonstrating its potential as a low-cost point-of-care test for use in resource-limited areas.

Antibiotic resistance has become a major challenge to public health worldwide. This crisis is further aggravated by the increasing emergence of bacterial resistance to carbapenems, typically considered as the antibiotics of last resort, which is mainly due to the production of carbapenem-hydrolyzing carbapenemases in bacteria. Herein, the carbapenem-based fluorogenic probe CB-1 with an unprecedented enamine-BODIPY switch is developed for the detection of carbapenemase activity. This reagent is remarkably specific towards carbapenemases over other prevalent β-lactamases. Furthermore, the efficient imaging of live clinically important carbapenemase-producing organisms (CPOs) with CB-1 demonstrates its potential for the rapid detection of antibiotic resistance and timely diagnosis of CPO infections.

A ligand-free Cu-catalyzed [3 + 2] cycloaddition of ethyl 2-(quinolin-2-yl)acetates, ethyl 2-(isoquinolin-1-yl)acetates, and ethyl 2-(pyridin-2-yl)acetates with (E)-chalcones for a “one-pot” synthesis of pyrrolo[1,2-a]quinolines, pyrrolo[2,1-a]isoquinolines and indolizines has been developed. The annulation products were isolated in moderate to good yields with air as the sole oxidant under mild conditions.

A highly enantioselective addition of alcohols to cyclic trifluoromethyl ketimines is developed catalyzed by quinine-thiourea, giving biologically interesting N,O-ketals in up to 99% yield and 96% ee.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains one of the most frequent causes of death worldwide. The slow growth rate of Mtb limits progress toward understanding tuberculosis including diagnosis of infections and evaluating therapeutic efficacy. Development of near-infrared (NIR) β-lactamase (BlaC)-specific fluorogenic substrate has made a significant breakthrough in the whole-animal imaging to detect Mtb infection. The reporter enzyme fluorescence (REF) system using a BlaC-specific fluorogenic substrate has improved the detection sensitivity in whole-animal optical imaging down to ~104 colony forming units (CFU) of bacteria, about 100-fold improvement over recombinant strains. However, improvement of detection sensitivity is strongly needed for clinical diagnosis of early stage infection at greater tissue depth. In order to improve detection sensitivity, we have integrated a fiber-based microendoscpe into a whole-animal imaging system to transmit the excitation light from the fiber bundle to the fluorescent target directly and measure fluorescent level using BlaC-specific REF substrate in the mouse lung. REF substrate, CNIR800, was delivered via aerosol route to the pulmonary infected mice with M. bovis BCG strain at 24 hours post-infection and groups of mice were imaged at 1–4 hours post-administration of the substrate using the integrated imaging system. In this study we evaluated the kinetics of CNIR800 substrate using REF technology using the integrated imaging system. Integration of these technologies has great promise for improved detection sensitivity allowing pre-clinical imaging for evaluation of new therapeutic agents.

The procedure allows the access to biologically important chiral dihydroquinazolinones bearing a -CF3 function at the quaternary carbon.

An entirely metal-free catalyst system consisting of an easily prepared recyclable new TEMPO derived sulfonic salt catalyst, and mineral acids (NaNO2 and HCl) has been developed for selective aerobic oxidation of structurally diverse benzylic sp3 C–H bonds of ethers and alkylarenes. The mild reaction conditions allow for the generation of synthetically and biologically valued isochromanones and xanthones from readily accessible alkyl aromatic precursors in good yields.

The reaction of 1-naphthol (I) and substituted analogues (VI) with various unsaturated aldehydes under conditions A) in the presence of piperidine affords exclusively C-4 substituted, highly functionalized derivatives as (E) isomers (E:Z < 95:5).

A mild and highly efficient amine-catalyzed, IBX-mediated oxidation of aldehydes to (E) selective α,β-unsaturated aldehydes has been achieved in good yields. The process features a new oxidation of enamines to iminium ions in a catalytic fashion.

This communication describes a novel protein labeling method that uses a single amino-acid tag — N-terminal cysteine residue — and small-molecule probes carrying the cyanobenzothiazole unit for specific labeling of proteins in vitro and at the surface of live cells. This simple ligation reaction proceeds with a high degree of specificity in physiological conditions, and should offer an important alternative to currently available protein labeling methods.

A direct preparation of synthetically useful α,β-unsaturated aldehydes from readily available aldehydes has been developed. The process is effectively cocatalyzed by an amine-palladium acetate to give rise to α,β-unsaturated aldehydes in moderate to good yields (41―62%). The reaction features the use of unmodified aldehydes rather than enol silyl ethers, which are used in a typical Saegusa oxidation reaction.