Tat-Shing Lai

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

Discovery of a drug-like G-quadruplex binding ligand by high-throughput docking.

There has been considerable interest in the study of G-quadruplex DNA owing to its involvement in the regulation of telomerase activities. 2] Human telomeric DNA is composed of a repeating double-stranded [TTAGGG/CCCTAA]n sequence except in the 3’-terminal region, which consists of a singlestranded tandem [TTAGGG] repeat sequence over several hundred bases. In normal somatic cells, approximately 100 bases are lost in each cell division, and after the telomeres have been shortened to a critical threshold, the cell undergoes apoptosis. In cancer cells, telomeric length is maintained by telomerase, and telomerase activity is expressed in >90% of tumor cell lines, but in relatively few normal cell types. Therefore, the inhibition of telomerase activity by ligand-induced stabilization of G-quadruplexes has become an attractive strategy for developing new anticancer drugs. Planar aromatic molecules with scaffolds that have extended delocalized pelectron systems such as cationic porphyrins, 2,7] BRACO19, 9-anilinoproflavin, 7] triazines, pentacyclic acridines, and telomestatin 9b,10a] are known to bind to and stabilize Gquadruplex DNA, resulting in anti-telomerase activity. This gives rise to telomere shortening and suppression of cell growth, ultimately leading to cell death. Recently, we also demonstrated by molecular modeling studies that quindoline derivatives have the ability to stabilize the G-quadruplex structure in c-myc. However, most reported small-molecule Gquadruplex stabilizers have extended planar structures that result in poor bioavailability. Virtual screening of chemical databases by molecular docking is one of the most powerful approaches to discover smallmolecule inhibitors. The major advantage of virtual screening of drug-like compounds is that chemical diversity is generated without the need for chemical synthesis ; confirmed hits identified in a screen could be used to guide further synthesis and quantitative structure–activity relationship analysis. Abagyan and co-workers recently demonstrated the applicability of high-throughput virtual screening of a marketed drug database in the identification of anti-androgen scaffolds. Inspired by this promising result, we extended the scope of identifying G-quadruplex DNA binding ligands through the virtual screening of a drug-like compound database. To develop a highthroughput screening platform for G-quadruplex DNA stabilizing ligands, a computer model was constructed by using the X-ray crystal structure of the intramolecular human telomeric G-quadruplex DNA (PDB code: 1KF1). It is common to use X-ray crystal structures for virtual screening of novel compounds from large databases because X-ray crystallography generally provides a larger amount of high-quality experimental data than NMR spectroscopy, and thus crystal structures are thought to provide a more accurate depiction. NMR structures are solved in a more biologically relevant environment; however, they provide a dynamic representation of the biomolecule when used as a collection. In the current study, the NMR structure of the intramolecular human telomeric G-quadruplex DNA in K solution (PDB code: 2GKU) is different from the X-ray crystal structure; the DNA strands are oriented in a (3+1) direction in the NMR structure, whereas the X-ray structure shows an all parallel direction, and as such, studies on the structure of the intramolecular human telomere quadruplex in physiological K solution have raised extreme controversy. Tan and co-workers recently reported the intramolecular human telomere quadruplex to adopt a parallel-stranded conformation in the noncrystalline state in K solution under molecular crowding conditions, as the K crystal structure quadruplex does. We report herein a new drug-like compound identified through in silico screening that is an effective stabilizer of Gquadruplex DNA. This compound also possesses high selectivity for G-quadruplex versus duplex DNA. Over 100000 compounds in a drug-like database that passed the Lipinski filters were screened in silico. The continuously flexible ligands were docked to a grid representation of the receptor and assigned a score reflecting the quality of the complex according to the ICM method (Molsoft). The bestscoring molecule in this new class of drug-like hits, 1H-pyrazole-3-carboxy-4-methyl-5-phenyl-(1H-indol-3-ylmethylene)hydrazide, was evaluated for its ability to stabilize G-quadruplex DNA (Figure 1). To the best of our knowledge, compound 1 has not yet been reported to stabilize G-quadruplex DNA.

Asymmetric inter- and intramolecular cyclopropanations of alkenes catalyzed by rhodium D4-porphyrin: a comparison of rhodium- and ruthenium-centred catalysts

Abstract Iodo-(5,10,15,20-tetrakis(1,2,3,4,5,6,7,8-octahydro-1,4:5,8-dimethanoanthracen-9-yl)porphyrinatorhodium(III), designated as [Rh(P*)(I)], was prepared and its catalytic activity in the asymmetric cyclopropanation of alkenes with ethyl diazoacetate (EDA) was examined. High catalyst turnovers (TON >103) and moderate enantioselectivities (up to 68% ee) were observed. However, the obtained trans/cis ratios are low. Competition experiments revealed that electron-donating substituents on styrene accelerate the cyclopropanations. The log(kX/kH) versus σ+ plot for substituted styrenes exhibits a good linearity with a small negative ρ+ value (−0.14). [Rh(P*)(I)] is also active in the intramolecular cyclopropanation of allyl diazoacetates. A comparison between rhodium and ruthenium porphyrin complexes was made.

Ruthenium catalyzed asymmetric transfer hydrogenation based on chiral P,N,O Schiff base ligands and crystal structure of a ruthenium(II) complex bearing chiral P,N,O Schiff base ligands

Abstract A ruthenium catalyst, generated in situ by heating the chiral P,N,O Schiff base ligand L (L = (S)-Ph2PC6H4CNCHPhCH2OH) with Ru(DMSO)4Cl2 in 2-propanol, is active for asymmetric transfer hydrogenation with best enantioselectivity up to 81%. A ruthenium complex of formula RuL2Cl2 is prepared and its crystal structure revealed that the two chiral P,N,O Schiff ligands are in meridional configuration. This complex is also an active catalyst for asymmetric transfer hydrogenation. However, the ‘[Ru(DMSO)4]Cl2 + chiral P,N,O ligand’ protocol displays better enantioselectivity.

Aerobic enantioselective alkene epoxidation by a chiral trans-dioxo(D4-porphyrinato)ruthenium(VI) complex

A dioxoruthenium(VI) complex with a D4-chiral porphyrin ligand has been prepared and characterized by spectroscopic methods and X-ray crystal analysis; the complex exhibits catalytic activity towards aerobic enantioselective epoxidation of prochiral alkenes with enantioselectivity up to 73% ee at an oxygen pressure of 8 atm.

Enantioselective hydroxylation of benzylic C–H bonds by D4-symmetric chiral oxoruthenium porphyrins†

A D4-symmetric chiral dioxoruthenium(VI) porphyrin can effect stoichiometric and catalytic enantioselective hydroxylation of benzylic C–H bonds to give enantioenriched aryl alcohols, the highest ee of 76% being attained in the catalytic oxidation of 4-ethyltoluene with 2,6-dichloropyridine N-oxide as terminal oxidant; the oxidations proceed via a rate-limiting H-atom abstraction to germinate a benzylic radical intermediate.

Enantioselective epoxidation of trans-disubstituted alkenes by D2-symmetric chiral dioxoruthenium(VI) porphyrins

A series of D2-symmetric chiral trans-dioxoruthenium(VI) porphyrins can effect enantioselective epoxidation of trans-β-methylstyrene in up to 70% ee, and 76% ee is attained for the oxidation of cinnamyl chloride; the facial selection for the trans-alkenes epoxidation is explained by a ‘head-on approach’ model.

Catalytic and asymmetric aziridination of alkenes catalysed by a chiral manganese porphyrin complex

A D4-manganese(III) porphyrin is utilized to catalyze aziridination of styrene-type substrates with enantiomeric excess ranging from 43 to 68%; evidence for a MnIV reactive intermediate in the catalysis was obtained from spectroscopic studies and organic product analysis.

Remarkable axial ligand effect on regioselectivity towards terminal alkenes in epoxidation of dienes by a robust manganese porphyrin

With a highly encumbered manganese porphyrin as catalyst, significant improvements in regioselectivity towards less substituted C-C double bond in diene epoxidation were attained by simply adding organic bases as axial ligand.