Jun-Long Zhang

Rational design of true monomeric and bright photoactivatable fluorescent proteins

Monomeric (m)Eos2 is an engineered photoactivatable fluorescent protein widely used for super-resolution microscopy. We show that mEos2 forms oligomers at high concentrations and forms aggregates when labeling membrane proteins, limiting its application as a fusion partner. We solved the crystal structure of tetrameric mEos2 and rationally designed improved versions, mEos3.1 and mEos3.2, that are truly monomeric, are brighter, mature faster and exhibit higher photon budget and label density.

A unique series of reversibly switchable fluorescent proteins with beneficial properties for various applications

Reversibly switchable fluorescent proteins (RSFPs) have attracted widespread interest for emerging techniques including repeated tracking of protein behavior and superresolution microscopy. Among the limited number of RSFPs available, only Dronpa is widely employed for most cell biology applications due to its monomeric and other favorable photochemical properties. Here we developed a series of monomeric green RSFPs with beneficial optical characteristics such as high photon output per switch, high photostability, a broad range of switching rate, and pH-dependence, which make them potentially useful for various applications. One member of this series, mGeos-M, exhibits the highest photon budget and localization precision potential among all green RSFPs. We propose mGeos-M as a candidate to replace Dronpa for applications such as dynamic tracking, dual-color superresolution imaging, and optical lock-in detection.

Constructing single-chain magnets by supramolecular π-π stacking and spin canting: a case study on manganese(III) corroles.

A single-chain magnet (SCM) was constructed from manganese(III) 5,10,15-tris(pentafluorophenyl)corrole complex [Mn(III) (tpfc)] through supramolecular π-π stacking without bridging ligands. In the crystal structures, [Mn(tpfc)] molecules crystallized from different solvents, such as methanol, ethyl acetate, and ethanol, exhibit different molecular orientations and intermolecular π-π interaction or weak Mn⋅⋅⋅O interaction to form a supramolecular one-dimensional motif or dimer. These three complexes show very different magnetic behaviors at low temperature. Methanol solvate 1 shows obvious frequency dependence of out-of-phase alternating-current magnetic susceptibility below 2 K and a magnetization hysteresis loop with a coercive field of 400 Oe at 0.5 K. It is the first example of spin-canted supramolecular single-chain magnet due to weak π-π stacking interaction. By fitting the susceptibility data χ(M) T (20-300 K) of 1 with the spin Hamiltonian expression H = -2J Σ(i=1)(n-1) S(Ai) S(Ai+1) + D Σ(i) S((iZ)(2)), the intrachain magnetic coupling parameter transmitted by π-π interaction of -0.31 cm(-1) and zero field splitting parameter D of -2.59 cm(-1) are obtained. Ethyl acetate solvate 2 behaves as an antiferromagnetic chain without ordering or slow magnetic relaxation down to 0.5 K. The magnetic susceptibility data χ(M) T (20-300 K) of 2 was fitted by assuming the spin Hamiltonian H = -2JΣ(i=1)(n-1) S(Ai) S(Ai+1), and the intrachain antiferromagnetic coupling constant of -0.07 cm(-1) is much weaker than that of 1. Ethanol solvate 3 with a dimer motif shows field-induced single-molecule magnet like behavior below 2.5 K. The exchange coupling constant J within the dimer propagated by π-π interaction is -0.14 cm(-1) by fitting the susceptibility data χ(M) T (20-300 K) with the spin Hamiltonian H = -2J S(A) S(B) + β(S((A)g(A)) + S((B)g(B)))H. The present studies open a new way to construct SCMs from anisotropic magnetic single-ion units through weak intermolecular interactions in the absence of bridging ligands.

Copper‐Catalyzed Hydrodefluorination of Fluoroarenes by Copper Hydride Intermediates

C F bond activation attracts much attention because of the rapid growth of fluorocarbons in pharmaceuticals, agrochemicals, and new materials. Such transformations not only provide new routes to the fluorinated organics, but also offer a fundamental understanding for C F bond cleavage and functionalization. Previous experimental and theoretical studies demonstrated the effectiveness of late transition metals (group 8–10) such as Fe, Ru, Os, Co, Rh, Ir, Ni, Pd, and Pt for their electron-rich nature and the relatively weak M F bonds, which are important for catalytic functionality. Recently, we explored the reactivity of group 11 metal complexes and found that gold(I) complexes have good catalytic reactivity toward hydrodefluorination (HDF) of fluoroarenes. Despite the tremendous progress made, most catalytic systems suffered from either low catalyst turnovers, limited substrate scope, or harsh reaction conditions. For example, gold(I) complexes are reactive only for the substrates with strong electron-withdrawing substituents, and their efficiency was greatly dependent on the electronicdonating additives. To address these issues, we continued to explore the more-active group 11 metals such as copper, as we were inspired by the success of gold. In this context, we report herein the first example of a copper(I)-catalyzed HDF of fluoroarenes, and it features high reactivity, good regioselectivity, and a broad substrate scope. Since Subramanian and Manzer reported the CuF2mediated fluorination of aromatics, copper-catalyzed C F bond formation has been developed by the groups of Hartwig, Lectka, and others. In contrast, for catalytic C F bond activation, copper has been rarely studied. Ribas and coworkers recently showed that a Cu/Cu redox cycle activated C X bonds (X=halogens) using triazamacrocyclic ligand, thus indicating an oxidative addition mechanism. Herein, we found that for HDF reactions, copper hydrides exhibited an unprecedented reactivity toward C F bonds. Moreover, density functional theory (DFT) calculations suggest that C F bond activation by copper hydrides proceeds through a mechanism involving nucleophilic attack, and represents an alternative strategy for copper activating C F bonds. At the outset of our study, we searched for optimal HDF reaction conditions for perfluoronitrobenzene (PFNB). We first applied the our previous catalytic gold system but replaced gold(I) with [Cu(MeCN)4PF6]. Screening silanes (see Table S1 in the Supporting Information) such as HSi(OMe)3, HSiEt3, PMHS, H2SiPh2, and HSiMe2Ph, showed that HSiMe2Ph was the best hydrogen source (conv. 40%). Solvent screening resulted THF providing the highest yield (60%; see Table S2 in the Supporting Information). We then examined ligand effects and the results are listed in Table 1. In

A Water-Soluble Ruthenium Glycosylated Porphyrin Catalyst for Carbenoid Transfer Reactions in Aqueous Media with Applications in Bioconjugation Reactions

Water-soluble [Ru(II)(4-Glc-TPP)(CO)] (1, 4-Glc-TPP = meso-tetrakis(4-(beta-D-glucosyl)phenyl)porphyrinato dianion) is an active catalyst for the following carbenoid transfer reactions in aqueous media with good selectivities and up to 100% conversions: intermolecular cyclopropanation of styrenes (up to 76% yield), intramolecular cyclopropanation of an allylic diazoacetate (68% yield), intramolecular ammonium/sulfonium ylide formation/[2,3]-sigmatroptic rearrangement reactions (up to 91% yield), and intermolecular carbenoid insertion into N-H bonds of primary arylamines (up to 83% yield). This ruthenium glycosylated porphyrin complex can selectively catalyze alkylation of the N-terminus of peptides (8 examples) and mediate N-terminal modification of proteins (four examples) using a fluorescent-tethered diazo compound (15). A fluorescent group was conjugated to ubiquitin via 1-catalyzed alkene cyclopropanation with 15 in aqueous solution in two steps: (1) incorporation of an alkenic group by the reaction of N-hydroxysuccinimide ester 19 with ubiquitin and (2) cyclopropanation of the alkene-tethered Lys(6) ubiquitin (23) with the fluorescent-labeled diazoacetate 15 in the presence of a catalytic amount of 1. The corresponding cyclopropanation product (24) was obtained with approximately 55% conversion based on MALDI-TOF mass spectrometry. The products 23, 24, and the N-terminal modified peptides and proteins were characterized by LC-MS/MS and/or SDS-PAGE analyses.

Chiral ruthenium porphyrin encapsulated in ordered mesoporous molecular sieves (MCM-41 and MCM-48) as catalysts for asymmetric alkene epoxidation and cyclopropanationElectronic supplementary information (ESI) available: experimental section. See http://www.rsc.org/suppdata/cc/b2/b209276j/

Encapsulation of chiral ruthenium porphyrin [RuII(D4-Por*)CO] in modified mesoporous silica supports such as MCM-41 and MCM-48 achieves active catalysts for asymmetric epoxidation of alkenes by 2,6-dichloropyridine N-oxide and intramolecular cyclopropanation, which is the first example of chiral metalloporphyrin supported on ordered molecular sieves.

Rational design of ZnSalen as a single and two photon activatable fluorophore in living cells

Rational design of effective photoactivatable/photoswitchable fluorophores, by introducing appropriate photoreactions to tune the electron transfer processes of the ground or/and excited states and switch fluorescence off/on, is crucial to achieve high temporal and spatial resolution in live cell (organism) imaging. Besides one photon activatable fluorophores, it is highly desirable to develop two photon activatable fluorophores using light in the NIR or IR region, which reduces photodamage and allows deep penetration into cells or tissues. In this work, we describe the design of one and two photon activatable ZnSalen by incorporating thioether moieties in the 3,3′-positions which quenches the fluorescence as a result of a PET process. Through one or two photon irradiation, the thioethers can be oxidized to sulfoxides and the fluorescence of ZnSalen “switched on”, due to the electron-withdrawing sulfoxides, which perturbs the PET process. We further demonstrate the application of this ZnSalen as a photoactivatable fluorophore in living cells using one and two photon fluorescence microscopies. In two photon microscopy, a high signal to noise contrast was achieved by irradiation with an 840 nm laser. Moreover, this photoactivatable ZnSalen was successfully applied in bioimaging in a model living organism Caenorhabditis elegans (C. elegans), and ca. 5 times fluorescence intensity increase was observed after one and two photon irradiation. This paradigm by modulation of the PET process in ZnSalen provides a promising methodology for the design of photoactivatable fluorophores in further applications in super resolution molecular imaging.

PEG-Linked luminescent platinum(ii) complex as aqueous polymeric molecular light switch for protein binding reactionsElectronic supplementary information (ESI) available: general experimental procedure, synthesis and characterization of 1 and 2, titration experiments and urea unfolding of BSA. See http://www.rsc.org/suppdata/cc/b2/b207395a/

Attachment of poly(ethylene glycol) (PEG) to [Pt(4-HOPh-C⁁N⁁N)Cl] via covalent etheric bonds yields the luminescent polymer PEG-[Pt] (2), the photoluminescence of which is enhanced in hydrophobic regions of protein molecules (binding constant up to 104 M−1) in aqueous solution; complex 2 can function as a luminescent probe for protein denaturation due to urea unfolding reaction.

Protein scaffold of a designed metalloenzyme enhances the chemoselectivity in sulfoxidation of thioanisole.

We demonstrate that incorporation of MnSalen into a protein scaffold enhances the chemoselectivity in sulfoxidation of thioanisole and find that both the polarity and hydrogen bonding of the protein scaffold play an important role in tuning the chemoselectivity.

Combining myeloperoxidase (MPO) with fluorogenic ZnSalen to detect lysosomal hydrogen peroxide in live cells

Accumulating evidence suggests that lysosomal H2O2 is closely associated with autophagy and apoptosis in normal and pathological processes. Imaging H2O2 in lysosomes is a powerful tool to elucidate its diverse roles, however, is limited by the lack of fluorescent probes capable of specifically detecting H2O2 under acidic conditions (pH 4.5–6). Herein we report the design and application of the “MPO-J-S” probe, a new combination of “peroxidase/fluorogenic substrate”, which is membrane-permeable and can be used to visualize exogenous or endogenous H2O2 confined to lysosomes via one and two photon fluorescence microscopies. The features of lysosomal H2O2 selectivity, high sensitivity, and live-cell compatibility offer new opportunities for luminescent ZnSalens and the related hypochlorite responsive fluorophores, combined with MPO, to decipher the physiological roles of lysosomal H2O2 in live cells.