Wing-Leung Wong

A Highly Selective Luminescent Switch‐On Probe for Histidine/Histidine‐Rich Proteins and Its Application in Protein Staining

The luminescence sensing of histidine and histidine-rich proteins plays a pivotal role in biochemistry and molecular biology, in particular when both temporal and spatial resolution are required. An abnormal level of histidine-rich proteins is an indicator for many diseases, such as advanced liver cirrhosis, AIDS, renal disease, asthma, pulmonary disorders, thrombotic disorders, f] and malaria. Some analyses of histidine and histidine-rich proteins have been developed in conjunction with immunoassay and colorimetric detection methods. The most commonly used method for the detection of histidine and histidine-rich proteins in biological samples is chromatography, which is usually performed through the combination of an effective separation technique, such as thin-layer chromatography, gas chromatography, or HPLC, followed by UV/Vis or fluorescence spectroscopy. The use of high-performance capillary electrophoresis with a derivation reagent has also been reported. However, the aforementioned methods are generally tedious, laborious, and, most importantly, expensive for routine detection in a biochemistry laboratory. Although numerous studies have dealt with the detection of histidine or histidine-rich proteins, studies on the use of luminescent probes for this purpose remain sparse. Notable examples include research by Fabbrizzi and co-workers, who developed competitive noncovalent fluorescence turn-on probes for histidine in the form of dizinc(II) or dicopper(II) macrocyclic complexes, which recognize histidine through the formation of an imidazolate bridge between the two dizinc(II) or dicopper(II) centers; however, the resulting noncovalent ensemble may be less stable than a covalently linking sensory system, and the complexity of the synthetic process makes it difficult to implement in a convenient manner. Photoluminescent iridium(III) complexes have emerged as a topical area of interest in inorganic photochemistry and phosphorescent materials for optoelectronic and luminescence signaling applications. Significant changes in the photophysical behavior and emission properties of iridium(III) complexes may be induced by the presence of biomolecules. Luminescent transition-metal complexes for protein staining, such as the luminescent ruthenium complex known as SYPRO Ruby dye, have been reported previously. However, despite its high sensitivity and its broad dynamic range, the use of SYPRORuby dye is limited, as it is sold only as a formulated solution; therefore, it is not possible to optimize the dye for a particular electrophoresis protocol and protein. In this context, the luminescent cyclometalated iridium(III) solvent complex [Ir(ppy)2(solv)2] + (1; ppy= 2phenylpyridine, solv=H2O or CH3CN) has received particular attention for the following reasons: 1) [Ir(ppy)2(OH2)2] , which contains weakly bound solvent ligands, may bind covalently to amino acids/proteins through a ligand substitution reaction with the OH2 ligand; 2) an intriguing solvent/ media dependence of the emission properties of [Ir(ppy)2(OH2)2] + has been observed; 3) [Ir(ppy)2(OH2)2] + can be synthesized conveniently and rapidly; 4) the use of organic solvents is not required for the optimal sensing of amino acids/proteins with [Ir(ppy)2(OH2)2] , and the iridium complex is readily soluble and stable in aqueous staining solutions. In this study, [Ir(ppy)3] (2) was also prepared for comparative studies, as its binding with proteins was expected to be largely hydrophobic in nature. Herein, we describe the luminescent switch-on probe [Ir(ppy)2(solv)2] + (1) for histidine/histidinerich proteins and demonstrate its utility in protein staining. The Ir complexes 1-CF3SO3 and 2 (Figure 1a) were prepared according to a previously reported method. The structure of 1-ClO4 was established by X-ray crystallography and is depicted in Figure 1b, and the crystal-packing diagrams are given in the Supporting Information. The metal–ligand bonding parameters for 1-ClO4 are comparable to those reported previously for cyclometalated iridium(III) complexes. The complex 1-CF3SO3 (50 mm) is weakly emissive in phosphate buffered saline (PBS). In the presence of histidine (His), 1-CF3SO3 exhibits an intense emission at lmax= 505 nm, the intensity of which reaches saturation level at [His]/[Ir] 4 (Figure 2a). A plot of I/I0 versus [His]/[1-CF3SO3] (I and I0 are emission intensities with and without His) shows an up-to180-fold intensity enhancement at ratios [His]/[1-CF3SO3] 4:1. The luminescence response of 1-CF3SO3 to various other [*] Dr. D.-L. Ma, Dr. W.-L. Wong, W.-H. Chung, F.-Y. Chan, P.-K. So, Dr. T.-S. Lai, Z.-Y. Zhou, Prof. Y.-C. Leung, Prof. K.-Y. Wong Department of Applied Biology and Chemical Technology, Central Laboratory of the Institute of Molecular Technology for Drug Discovery and Synthesis, The Hong Kong Polytechnic University Hung Hom, Kowloon, Hong Kong (China) Fax: (+1)852-2364-9932 E-mail: bcedmond@polyu.edu.hk bckywong@inet.polyu.edu.hk

A novel chiral terpyridine macrocycle as a fluorescent sensor for enantioselective recognition of amino acid derivativesElectronic supplementary information (ESI) available: plots for estimation of binding constants for tpy macrocycle 1 with (R)-PhEtOMe. See http://www.rsc.org/suppdata/cc/b3/b313960c/

Terpyridine macrocycle 1 is shown to be a strong chelating agent for organic ammonium salts and also a useful chromophore in fluorescent sensing. It exhibits very good enantioselectivity (K(obs)(S)/K(obs)(R)= 3.8) in chiral discrimination of alpha-phenylglycine methyl ester hydrochloride (PhEtOMe).

Benzothiazole-substituted benzofuroquinolinium dye: a selective switch-on fluorescent probe for G-quadruplex

A new switch-on fluorescent probe containing the natural product cryptolepine analogue benzofuroquinolinium moiety (binding scaffold) and a benzothiazole moiety (signalling unit) shows a remarkable fluorescence enhancement selective for the G-quadruplex nucleic acid structure. Binding studies revealed that the highly selective response of the fluorescent probe arises from end-stack binding to G-quadruplex.

New chiral 2,2′:6′,2″-terpyridine ligands from the chiral pool: synthesis, crystal structure of a rhodium complex and uses in copper- and rhodium-catalyzed enantioselective cyclopropanation of styrene

Abstract A number of chiral C 2 -symmetric 2,2′:6′,2″ terpyridines L 1 – L 4 were synthesized in moderate to good yields from commercially available chiral materials. Copper(II) and rhodium(III) chloride complexes of these ligands were prepared in good yields. The Rh( L 2 )Cl 3 complex was isolated as a yellow crystalline solid and characterized by X-ray crystallography. Both Cu( L )(OTf) 2 and Rh( L )(OTf) 3 were found to be active catalysts in the cyclopropanation of styrene with ethyl diazoacetate. Enantioselectivity up to 82% e.e. was observed.

A Simple and Effective Catalytic System for Epoxidation of Aliphatic Terminal Alkenes with Manganese(II) as the Catalyst

A simple catalytic system that uses commercially available manganese(II) perchlorate as the catalyst and peracetic acid as the oxidant is found to be very effective in the epoxidation of aliphatic terminal alkenes with high product selectivity at ambient temperature. Many terminal alkenes are epoxidised efficiently on a gram scale in less than an hour to give excellent yields of isolated product (>90 %) of epoxides in high purity. Kinetic studies with some C9-alkenes show that the catalytic system is more efficient in epoxidising terminal alkenes than internal alkenes, which is contrary to most commonly known epoxidation systems. The reaction rate for epoxidation decreases in the order: 1-nonene>cis-3-nonene>trans-3-nonene. ESI-MS and EPR spectroscopic studies suggest that the active form of the catalyst is a high-valent oligonuclear manganese species, which probably functions as the oxygen atom-transfer agent in the epoxidation reaction.

Detection of cancer biomarkers by piezoelectric biosensor using PZT ceramic resonator as the transducer.

A novel piezoelectric biosensor using lead titanate zirconate (PZT) ceramic resonator as transducer was developed for label-free, cost-effective, and direct detection of cancer biomarkers. We designed a dual sensing scheme where two ceramic resonators were connected in parallel, in which one resonator was used as the sensing unit and the other as the control unit, in order to minimize environment influences including temperature fluctuation and to achieve the required frequency stability for biosensing applications. Detection of selected cancer biomarkers, such as prostate specific antigen (PSA) and α-fetoprotein (AFP) was carried out to evaluate the performance of the biosensor. The device showed high sensitivity (0.25 ng/ml) and fast detection (within 30 min) with small amount of sample (1 μl), which is compatible to that required by clinical measurements. The results also showed that the ceramic resonator-based piezoelectric biosensor platform could be utilized with different chemical interfaces, and the miniaturized size of the ceramic resonators makes it suitable for fabricating sensor arrays for multiplex detection.

Molecular Engineering of Thiazole Orange Dye: Change of Fluorescent Signaling from Universal to Specific upon Binding with Nucleic Acids in Bioassay.

The universal fluorescent staining property of thiazole orange (TO) dye was adapted in order to be specific for G-quadruplex DNA structures, through the introduction of a styrene-like substituent at the ortho-position of the TO scaffold. This extraordinary outcome was determined from experimental studies and further explored through molecular docking studies. The molecular docking studies help understand how such a small substituent leads to remarkable fluorescent signal discrimination between G-quadruplex DNA and other types of nucleic acids. The results reveal that the modified dyes bind to the G-quadruplex or duplex DNA in a similar fashion as TO, but exhibit either enhanced or quenched fluorescent signal, which is determined by the spatial length and orientation of the substituent and has never been known. The new fluorescent dye modified with a p-(dimethylamino)styryl substituent offers 10-fold more selectivity toward telomeric G-quadruplexes than double-stranded DNA substrates. In addition, native PAGE experiments, FRET, CD analysis, and live cell imaging were also studied and demonstrated the potential applications of this class of thiazole-orange-based fluorescent probes in bioassays and cell imaging.

Synthesis of new chiral terpyridine mono-N-oxide and di-N-oxide ligands and their applications in copper-catalyzed asymmetric cyclopropanation

Abstract New families of chiral terpyridine mono- N -oxides L 1 – L 3 and di- N -oxides L 4 – L 6 , were synthesized in moderate to good yields by simple oxidation of their corresponding terpyridines with m -CPBA. The copper(II) triflate complexes of these ligands were found to be highly effective catalysts for asymmetric cyclopropanation of styrene with ethyl diazoacetate. The isolated yields of cyclopropane were excellent and the enantiomeric excesses of up to 83% were observed. Competition experiments with substituted styrenes showed good σ + correlations with ρ =−0.70 for mono- N -oxide L 3 and ρ =−0.69 for di- N -oxide L 6 .

New pyridinium-based fluorescent dyes: A comparison of symmetry and side-group effects on G-Quadruplex DNA binding selectivity and application in live cell imaging.

A series of C1-, C2-and C3-symmetric pyridinium conjugates with different styrene-like side groups were synthesized and were utilized as G-quadruplex selective fluorescent probes. The new compounds were well-characterized. Their selectivity, sensitivity, and stability towards G-quadruplex were studied by fluorescence titration, native PAGE experiments, FRET and circular dichroism (CD) analyses. These new compounds investigated in the fluorescence assays were preferentially bound with G-quadruplex DNA compared with other type of nucleic acids and it is fascinating to realize the effects of molecular symmetry and associated side groups showing unexpectedly great influence on the fluorescent signal enhancement for the discrimination of G-quadruplexes DNA from other nucleic acids. This may correlate with the pocket symmetry and shape of the G-quadruplex DNA inherently. Among the compounds, a C2-symmetric dye (2,6-bis-((E)-2-(1H-indol-3-yl)-vinyl)-1-methylpyridin-1-ium iodide) with indolyl-groups substituted was screened out from the series giving the best selectivity and sensitivity towards G-quadruplexes DNA, particularly telo21, due to its high equilibrium binding constant (K=2.17×10(5)M(-1)). In addition, the limit of detection (LOD) of the dye to determine telo21 DNA in bioassays was found as low as 33nM. The results of the study give insight and certain crucial factors, such as molecular symmetry and the associated side groups, on developing of effective fluorescent dyes for G-quadruplex DNA applications including G-quadruplex structure stabilization, biosensing and clinical applications. The compound was also demonstrated as a very selective G-quadruplex fluorescent agent for living cell staining and imaging.

Label-free detection of endocrine disrupting chemicals by integrating a competitive binding assay with a piezoelectric ceramic resonator

A piezoelectric biosensor for detection of endocrine disrupting chemicals (EDCs) was developed by incorporating chemical/biochemical recognition elements on the ceramic resonator surface for competitive binding assays. A facile electrodeposition was employed to modify the sensor surface with Au nanoparticles, which increased the surface area and enhanced the binding capacity of the immobilized probes. Thiol-labeled long chain hydrocarbon with bisphenol A (BPA) as head group was synthesized and self-assembled on the Au nanoparticle surface as the sensing probes, which showed a linear response upon the binding of estrogen receptor (ER-α) ranging from 1 to 30 nM. Detection of estrone, 17β-estradiol and BPA was achieved by integrating a competitive binding assay with the piezoelectric sensor. In this detection scheme, different concentrations of EDCs were incubated with 30 nM of ER-α, and the un-bounded ER-α in the solution was captured by the probes immobilized on the ceramic resonator, which resulted in the frequency changes for different EDCs. The biosensor assay exhibited a linear response to EDCs with a low detection limit of 2.4-2.9 nM (S/N=3), and required only a small volume of sample (1.5 µl) with the assay time of 2h. The performance of the biosensor assay was also evaluated for rapid and facile determination of EDCs of environmental relevant concentrations in drinking water and seawater, and the recovery rate was in the range between 94.7% and 109.8%.