An Activatable Photosensitizer Targeted to γ‐Glutamyltranspeptidase

We adopted a spirocyclization‐based strategy to design γ‐glutamyl hydroxymethyl selenorhodamine green (gGlu‐HMSeR) as a photo‐inactive compound that would be specifically cleaved by the tumor‐associated enzyme γ‐glutamyltranspeptidase (GGT) to generate the potent photosensitizer HMSeR. gGlu‐HMSeR has a spirocyclic structure and is colorless and does not show marked phototoxicity toward low‐GGT‐expressing cells or normal tissues upon irradiation with visible light. In contrast, HMSeR predominantly takes an open structure, is colored, and generates reactive oxygen species upon irradiation. The γ‐glutamyl group thus serves as a tumor‐targeting moiety for photodynamic therapy (PDT), switching on tumor‐cell‐specific phototoxicity. To validate this system, we employed chick chorioallantoic membrane (CAM), a widely used model for preliminary evaluation of drug toxicity. Photoirradiation after gGlu‐HMSeR treatment resulted in selective ablation of implanted tumor spheroids without damage to healthy tissue.     

Direct Monitoring of γ‐Glutamyl Transpeptidase Activity In Vivo Using a Hyperpolarized 13C‐Labeled Molecular Probe

The γ‐glutamyl transpeptidase (GGT) enzyme plays a central role in glutathione homeostasis. Direct detection of GGT activity could provide critical information for the diagnosis of several pathologies. We propose a new molecular probe, γ‐Glu‐[1‐¹³C]Gly, for monitoring GGT activity in vivo by hyperpolarized (HP) ¹³C magnetic resonance (MR). The properties of γ‐Glu‐[1‐¹³C]Gly are suitable for in vivo HP ¹³C metabolic analysis since the chemical shift between γ‐Glu‐[1‐¹³C]Gly and its metabolic product, [1‐¹³C]Gly, is large (4.3 ppm) and the T₁ of both compounds is relatively long (30 s and 45 s, respectively, in H₂O at 9.4 T). We also demonstrate that γ‐Glu‐[1‐¹³C]Gly is highly sensitive to in vivo modulation of GGT activity induced by the inhibitor acivicin.     

Development of a LC–MS/MS method for the determination of isomeric glutamyl peptides in food ingredients

A liquid chromatography with tandem mass spectrometry method was developed for the determination of 27 glutamyl di‐ and tripeptides in food ingredients. Such compounds are of importance for the food industry, as they can modulate the perception of basic tastes (sweet, salty, and umami). Due to their high polarity, the hydrophilic interaction chromatography mode was selected to have sufficient retention on the column and the best separation was obtained on an amide hybrid silica stationary phase packed with 1.7 μm particles. Thorough optimization of the mobile phase was performed as the start‐composition had to be free of ammonium to avoid on‐column cis–trans isomerization of the first eluting proline dipeptide. A baseline separation was achieved for all α and γ isomers whereas only a partial resolution was obtained for γ‐Glu‐Leu and γ‐Glu‐Ile, for which only the position of a methyl group differs. A fast sample preparation, based on successive dilutions, was performed before injection into the liquid chromatography with tandem mass spectrometry system. The developed method was then applied for the semi‐quantification of glutamyl di‐ and tri‐peptides in four different food ingredients. The methodology will further support the optimization of production processes to select the conditions for which the peptide concentrations would be the highest.     

Structure of the Sulfoxide Synthase EgtB from the Ergothioneine Biosynthetic Pathway

The non‐heme iron enzyme EgtB catalyzes O₂‐dependent C? S bond formation between γ‐glutamyl cysteine and N‐α‐trimethyl histidine as the central step in ergothioneine biosynthesis. Both, the catalytic activity and the architecture of EgtB are distinct from known sulfur transferases or thiol dioxygenases. The crystal structure of EgtB from Mycobacterium thermoresistibile in complex with γ‐glutamyl cysteine and N‐α‐trimethyl histidine reveals that the two substrates and three histidine residues serve as ligands in an octahedral iron binding site. This active site geometry is consistent with a catalytic mechanism in which C? S bond formation is initiated by an iron(III)‐complexed thiyl radical attacking the imidazole ring of N‐α‐trimethyl histidine.     

Leveraging γ‐Glutamyl Transferase To Direct Cytotoxicity of Copper Dithiocarbamates against Prostate Cancer Cells

A prodrug approach is presented to direct copper‐dependent cytotoxicity to prostate cancer cells. The prochelator GGTDTC requires activation by γ‐glutamyl transferase (GGT) to release the metal chelator diethyldithiocarbamate from a linker that masks its thiol reactivity and metal binding properties. In vitro studies demonstrated successful masking of copper binding as well as clean liberation of the chelator by GGT. GGTDTC was stable to non‐specific degradation when incubated with a series of prostate cancer and normal cell lines, with selective release of diethyldithiocarbamate only occurring in cells with measurable GGT activity. The antiproliferative efficacy of the prochelator correlated with cellular GGT activity, with 24 h inhibitory concentrations ranging from 800 nm in prostate cancer lines 22Rv1 and LNCaP to over 15 μm in normal prostate PWR‐1E cells. These findings underscore a new strategy to leverage the amplified copper metabolism of prostate cancer by conditional activation of a metal‐binding pharmacophore.     

Enzyme‐Triggered Fluorescence Turn‐on: A Probe for Specifically Imaging Ovarian‐Cancer‐Related γ‐Glutamyltranspeptidase

A fluorescent turn‐on probe for specifically targeting γ ‐glutamyltranspeptidase (GGT ) was designed and synthesized by integrating boron‐dipyrromethene (BODIPY ) as a chromophore and glutathione (GSH ) as the GGT substrate. GGT ‐catalyzed the cleavage of the γ ‐glutamyl bond and generated the aromatic hydrocarbon transfer between the sulfur and the nitrogen atom in BODIPY , leading to distinct optical changes. Such specific responsiveness provides an easily distinguishable fluorescence signal to visualize the GGT activity in living cells and differentiate GGT ‐positive cancer cells from GGT ‐negative cells.     

Novel 3D Neuron Regeneration Scaffolds Based on Synthetic Polypeptide Containing Neuron Cue

Neural tissue engineering has become a potential technology to restore the functionality of damaged neural tissue with the hope to cure the patients with neural disorder and to improve their quality of life. This paper reports the design and synthesis of polypeptides containing neuron stimulate, glutamic acid, for the fabrication of biomimetic 3D scaffold in neural tissue engineering application. The polypeptides are synthesized by efficient chemical reactions. Monomer γ‐benzyl glutamate‐N‐carboxyanhydride undergoes ring‐opening polymerization to form poly(γ‐benzyl‐l ‐glutamate), then hydrolyzes into poly(γ‐benzyl‐l ‐glutamate)‐r‐poly(glutamic acid) random copolymer. The glutamic acid amount is controlled by hydrolysis time. The obtained polymer molecular weight is in the range of 200 kDa for good quality of fibers. The fibrous 3D scaffolds of polypeptides are fabricated using electrospinning techniques. The scaffolds are biodegradable and biocompatible. The biocompatibility and length of neurite growth are improved with increasing amount of glutamic acid in scaffold. The 3D scaffold fabricated from aligned fibers can guide anisotropic growth of neurite along the fiber and into 3D domain. Furthermore, the length of neurite outgrowth is longer for scaffold made from aligned fibers as compared with that of isotropic fibers. This new polypeptide has potential for the application in the tissue engineering for neural regeneration.     

Detection of miRNA in Live Cells by Using Templated RuII‐Catalyzed Unmasking of a Fluorophore

Reactions templated by cellular nucleic acids are attractive for nucleic acid sensing or responsive systems. Herein we report the use of a photocatalyzed reductive cleavage of an immolative linker to unmask a rhodamine fluorophore, and its application to miRNA imaging. The reaction was found to proceed with a very high turnover (>4000) and provided reliable detection down to 5 pM of template by using γ‐serine‐modified peptide nucleic acid (PNA) probes. The reaction was used for the selective detection of miR‐21 in BT474 cells and miR‐31 in HeLa cells following irradiation for 30 min. The probes were introduced by using reversible permeation with streptolysin‐O (SLO) or a transfection technique.     

Bioconjugatable Azo‐Based Dark‐Quencher Dyes: Synthesis and Application to Protease‐Activatable Far‐Red Fluorescent Probes

We describe the efficient synthesis and one‐step derivatization of novel, nonfluorescent azo dyes based on the Black Hole Quencher‐3 (BHQ‐3) scaffold. These dyes were equipped with various reactive and/or bioconjugatable groups (azido, α‐iodoacetyl, ketone, terminal alkyne, vicinal diol). The azido derivative was found to be highly reactive in the context of copper‐catalyzed azide–alkyne cycloaddition (CuAAC) reactions and allowed easy synthetic access to the first water‐soluble (sulfonated derivative) and aldehyde‐modified BHQ‐3 dyes, the direct preparation of which failed by means of conventional azo‐coupling reactions. The aldehyde‐ and α‐iodoacetyl‐containing fluorescence quenchers were readily conjugated to aminooxy‐ and cysteine‐containing peptides by the formation of a stable oxime or thioether linkage, respectively. Further fluorescent labeling of the resultant peptide conjugates with red‐ or far‐red‐emitting rhodamine or cyanine dyes through sequential and/or one‐pot bioconjugations, led to novel Förster resonance energy transfer (FRET) based probes suitable for the in vivo detection and imaging of urokinase plasminogen activator, a key protease in cancer invasion and metastasis.     

Preparation,mechanical properties,and in vitro biocompatibility of novel nanocomposites based on polyhexamethylene carbonate fumarate and nanohydroxyapatite

In the current study a new biodegradable nanocomposite based on poly hexamethylene carbonate fumarate (PHMCF) and nano‐sized hydroxyapatite (nano‐HA) has been developed. A silane coupling agent γ‐methacryloxypropyltrimethoxy silane, was used to achieve a good interfacial adhesion between nano‐HA and PHMCF matrix. PHMCF with different nano‐HA contents were characterized using dynamical mechanical thermal analysis (DMTA) and hardness test. The effect of frequency on storage modulus, glass transition temperature (T_g) and the damping were investigated. In vitro cytotoxicity and proliferation were performed using G292 cell lines by MTT assay. The addition of nano‐HA resulted in an increment on the storage modulus and decrement on the damping. Along with improvement in mechanical properties of composites, the addition of nano‐HA resulted in enhanced cell proliferation. Following these results, the newly developed nano‐PHMCF composite scaffold may be considered for bone tissue engineering applications. Copyright © 2009 John Wiley & Sons, Ltd.     

Single‐Flask Multicomponent Palladium‐Catalyzed α,γ‐Coupling of Ketone Enolates: Facile Preparation of Complex Carbon Scaffolds

A three‐component palladium‐catalyzed reaction sequence has been developed in which γ‐substituted α,β‐unsaturated products are obtained in a single flask by an α‐alkenylation with either a subsequent γ‐alkenylation or γ‐arylation of a ketone enolate. Coupling of a variety of electronically and structurally different components was achieved in the presence of a Pd/Q‐Phos catalyst (2 mol %), usually at 22 °C with yields of up to 85 %. Most importantly, access to these products is obtained in one simple operation in place of employing multiple reactions.     

Synthesis of a Far‐Red Photoactivatable Silicon‐Containing Rhodamine for Super‐Resolution Microscopy

The rhodamine system is a flexible framework for building small‐molecule fluorescent probes. Changing N‐substitution patterns and replacing the xanthene oxygen with a dimethylsilicon moiety can shift the absorption and fluorescence emission maxima of rhodamine dyes to longer wavelengths. Acylation of the rhodamine nitrogen atoms forces the molecule to adopt a nonfluorescent lactone form, providing a convenient method to make fluorogenic compounds. Herein, we take advantage of all of these structural manipulations and describe a novel photoactivatable fluorophore based on a Si‐containing analogue of Q‐rhodamine. This probe is the first example of a “caged” Si‐rhodamine, exhibits higher photon counts compared to established localization microscopy dyes, and is sufficiently red‐shifted to allow multicolor imaging. The dye is a useful label for super‐resolution imaging and constitutes a new scaffold for far‐red fluorogenic molecules.     

A Lysosome‐Compatible Near‐Infrared Fluorescent Probe for Targeted Monitoring of Nitric Oxide

The requirement for nitric oxide (NO) of lysosomes has motivated the development of a sophisticated fluorescent probe to monitor the distribution of this important biomolecule at the subcellular level in living cells. A near‐infrared (NIR) fluorescent Si‐rhodamine (SiRB)‐NO probe was designed based on the NO‐induced ring‐opening process of Si‐rhodamine. The probe exhibits fast chromogenic and fluorogenic responses, and high sensitivity and selectivity toward trace amounts of NO. Significantly, the spirolactam in Si‐rhodamine exhibits very good tolerance to H⁺, which in turn brings extremely low background fluorescence not only in the physiological environment but also under acidic conditions. The stability of the highly fluorescent product in acidic solution provides persistent fluorescence emission for long‐term imaging experiments. To achieve targeted imaging with improved spatial resolution and sensitivity, an efficient lysosome‐targeting moiety was conjugated to a SiRB‐NO probe, affording a tailored lysosome‐targeting NIR fluorescent Lyso‐SiRB‐NO probe. Inheriting the key advantages of its parent SiRB‐NO probe, Lyso‐SiRB‐NO is a functional probe that is suited for monitoring lysosomal NO with excellent lysosome compatibility. Imaging experiments demonstrated the monitoring of both exogenous and endogenous NO in real time by using the Lyso‐SiRB‐NO probe.     

A study on the system of nonaqueous microchip electrophoresis with on‐line peroxyoxalate chemiluminescence detection

This article describes a further development of our previously reported miniaturized analysis system of microchip electrophoresis with on‐line chemiluminescence detection. The system, developed first time for nonaqueous microchip electrophoresis with peroxyoxalate chemiluminescence detection, consists of a suction pressure device for sample or reagent introduction, a constant voltage supplied for electrophoretic separation, an either hydrophilic or hydrophobic porous polymer plug for preventing chemiluminescence reagent flowing upstream and a spiral detection channel for enhancement of both detection sensitivity and reproducibility. Especially, by using organic solvent as BGE medium, the developed system avoided the interface problem between aqueous running buffer and low‐water‐content chemiluminescence solvent in previous reports. The influencing factors on chemiluminescence signal were optimized using rhodamine 6G as model molecule. The system performance was further investigated in the experiment of separation of hydrophilic rhodamine dyes and analysis of hydrophobic polycyclic aromatic hydrocarbon, providing the detection limit (S/N = 3) of 3.5 nmol/L for rhodamine 123, 6.8 nmol/L for rhodamine 6G, and 60 nmol/L for 1‐aminopyrene, respectively. The experimental results showed the system offered a number of benefits, including compact structure, high sensitivity, good reproducibility, and a wide range of application prospect.     

Synthesis,Photophysics and Nonlinear Optical Properties of Stilbenoid Pyrimidine‐Based Dyes Bearing Methylenepyran Donor Groups

The nonlinear properties and the photophysical behavior of two π‐conjugated chromophores that incorporate an electron‐deficient pyrimidine core (A) and γ‐methylenepyrans as terminal donor (D) groups have been thoroughly investigated. Both dipolar and quadrupolar branching strategies are explored and rationalized on the basis of the Frenkel exciton model. Even though a cooperative effect is clearly observed if the dimensionality is increased, the nonlinear optical (NLO) response of this series is moderate if one considers the nature of the D/A couple and the size of the chromophores (as measured by the number of π electrons). This effect was attributed to a disruption in the electronic conjugation within the dyes’ scaffold for which the geometry deviates from planarity owing to a noticeable twisting of the pyranylidene end‐groups. This latter structural parameter also has a strong influence on the excited‐state dynamics, which leads to a very efficient fluorescence quenching.     

Enhanced Targeted Delivery of Cyclodextrin‐Based Supermolecules by Core–Shell Nanocapsules for Magnetothermal Chemotherapy

In this study, double‐emulsion capsules (DECs) capable of concealing drug‐incorporated targeted‐supermolecules are developed to achieve “on‐demand” supermolecule release and enhanced sequential targeting for magneto‐chemotherapy. These water‐in‐oil‐in‐water DECs less than 200 nm in diameter are synthesized using a single component of PVA (polyvinyl alcohol) polymer and the magnetic nanoparticles, which are capable of encapsulating large quantities of targeted supermolecules composed of palitaxel‐incorporated beta‐cyclodextrin decorated by hyaluronic acid (HA, a CD44‐targeting ligand) in the watery core. The release profiles (slow, sustained and burst release) of the targeted supermolecules can be directly controlled by regulating the high‐frequency magnetic field (HFMF) and polymer conformation without sacrificing the targeting ability. Through an intravenous injection, the positive targeting of the supermolecules exhibited a 20‐fold increase in tumor accumulation via the passive targeting and delivery of DECs followed by positive targeting of the supermolecules. Moreover, this dual‐targeting drug‐incorporated supermolecular delivery vehicle at the tumor site combined with magneto‐thermal therapy suppressed the cancer growth more efficiently than treatment with either drug or supermolecule alone.

     

Total Synthesis of (+)‐Dendrowardol C

The first total synthesis of the tetracyclic sesquiterpenoid (+)‐dendrowardol C is described. It relies on an intramolecular aldol reaction to forge the central bicyclic scaffold and subsequent cyclobutane formation by cyclization of a γ‐triflyloxy ketone. Key is the treatment of the latter with lithium naphthalenide. Finally, the diastereoselective hydroboration of a 1,1‐disubstituted double bond is enabled by a chiral Co^I catalyst.     

Fast,simple, and sensitive high‐performance liquid chromatography method for measuring vitamins A and E in human blood plasma

Vitamins A and E are fat‐soluble vitamins that play important roles in several physiological processes. Monitoring their concentrations is needed to detect deficiency and guide therapy. In this study, we developed a high‐performance liquid chromatography method to measure the major forms of vitamin A (retinol) and vitamin E (α‐tocopherol and γ‐tocopherol) in human blood plasma. Vitamins A and E were extracted with hexane and separated on a reversed‐phase column using methanol as the mobile phase. Retinol was detected by ultraviolet absorption, whereas tocopherols were detected by fluorescence emission. The chromatographic cycle time was 4.0 min per sample. The analytical measurement range was 0.03–5.14, 0.32–36.02, and 0.10–9.99 mg/L for retinol, α‐tocopherol, and γ‐tocopherol, respectively. Intr‐aassay and total coefficient of variation were <6.0% for all compounds. This method was traceable to standard reference materials offered by the National Institute of Standards and Technology. Reference intervals were established using plasma samples collected from 51 healthy adult donors and were found to be 0.30–1.20, 6.0–23.0, and 0.3–3.2 mg/L for retinol, α‐tocopherol, and γ‐tocopherol, respectively. In conclusion, we developed and validated a fast, simple, and sensitive high‐performance liquid chromatography method for measuring the major forms of vitamins A and E in human plasma.     

Determination of hexabromocyclododecane isomers in sewage sludge by LC‐MS/MS

A method has been developed to determine α, β and γ diastereoisomers of hexabromocyclododecane (HBCD), a brominated flame retardant, in sewage sludge, based on the ultrasonic‐assisted extraction (UAE) of samples with dichloromethane–ACN (1:1) and the subsequent clean‐up of extracts by dispersive solid phase extraction with primary–secondary amine. Levels of HBCD diastereoisomers were determined by LC coupled with ESI MS/MS. Evaluation of the matrix effect showed a high ion suppression for all the diastereoisomers studied, which was counteracted by using a mixture of labelled HBCD diastereoisomers as internal standards. This method yielded recoveries in the range of 79.6–112.5% with SDs equal or lower than 9.1 The limits of detection were 0.3 ng/g for α‐ and β‐HBCD and 0.2 ng/g for γ‐HBCD. The developed method was successfully applied to 19 sludge samples collected from the province of Madrid (Spain). In most of the samples, β‐HBCD was below the method detection limit, whereas α‐ and γ‐HBCD were quantified in all of the sludge samples, and γ‐HBCD was the predominant diastereoisomer in 63% of the analyzed samples and α‐HBCD predominated in the rest.