Tracking Surface Glycans on Live Cancer Cells with Single‐Molecule Sensitivity

Using a combination of metabolically labeled glycans, a bioorthogonal copper(I)‐catalyzed azide–alkyne cycloaddition, and the controlled bleaching of fluorescent probes conjugated to azide‐ or alkyne‐tagged glycans, a sufficiently low spatial density of dye‐labeled glycans was achieved, enabling dynamic single‐molecule tracking and super‐resolution imaging of N‐linked sialic acids and O‐linked N‐acetyl galactosamine (GalNAc) on the membrane of live cells. Analysis of the trajectories of these dye‐labeled glycans in mammary cancer cells revealed constrained diffusion of both N‐ and O‐linked glycans, which was interpreted as reflecting the mobility of the glycan rather than to be caused by transient immobilization owing to spatial inhomogeneities on the plasma membrane. Stochastic optical reconstruction microscopy (STORM) imaging revealed the structure of dynamic membrane nanotubes.     

Metabolic Labeling and Imaging of N‐Linked Glycans in Arabidopsis Thaliana

Molecular imaging of glycans has been actively pursued in animal systems for the past decades. However, visualization of plant glycans remains underdeveloped, despite that glycosylation is essential for the life cycle of plants. Metabolic glycan labeling in Arabidopsis thaliana by using N‐azidoacetylglucosamine (GlcNAz) as the chemical reporter is reported. GlcNAz is metabolized through the salvage pathway of N‐acetylglucosamine (GlcNAc) and incorporated into N‐linked glycans, and possibly intracellular O‐GlcNAc. Click‐labeling with fluorescent probes enables visualization of newly synthesized N‐linked glycans. N‐glycosylation in the root tissue was discovered to possess distinct distribution patterns in different developmental zones, suggesting that N‐glycosylation is regulated in a developmental stage‐dependent manner. This work shows the utility of metabolic glycan labeling in elucidating the function of N‐linked glycosylation in plants.     

Cathepsin B‐Specific Metabolic Precursor for In Vivo Tumor‐Specific Fluorescence Imaging

Recently, metabolic glycoengineering with bioorthogonal click reactions has focused on improving the tumor targeting efficiency of nanoparticles as delivery vehicles for anticancer drugs or imaging agents. It is the key technique for developing tumor‐specific metabolic precursors that can generate unnatural glycans on the tumor‐cell surface. A cathepsin B‐specific cleavable substrate (KGRR) conjugated with triacetylated N‐azidoacetyl‐d ‐mannosamine (RR‐S‐Ac₃ManNAz) was developed to enable tumor cells to generate unnatural glycans that contain azide groups. The generation of azide groups on the tumor cell surface was exogenously and specifically controlled by the amount of RR‐S‐Ac₃ManNAz that was fed to target tumor cells. Moreover, unnatural glycans on the tumor cell surface were conjugated with near infrared fluorescence (NIRF) dye‐labeled molecules by a bioorthogonal click reaction in cell cultures and in tumor‐bearing mice. Therefore, our RR‐S‐Ac₃ManNAz is promising for research in tumor‐specific imaging or drug delivery.     

Qualitative and quantitative comparison of N‐glycans between pig endothelial and islet cells by high‐performance liquid chromatography and mass spectrometry‐based strategy

N‐glycan structures released from miniature pig endothelial and islet cells were determined by matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF), negative ion electrospray ionization (ESI) MS/MS and normal‐phase high performance liquid chromatography (NP‐HPLC) combined with exoglycosidase digestion. Totally, the identified structures were 181 N‐glycans including 129 sialylated and 18 α‐galactosylated glycans from pig endothelial cells and 80 N‐glycans including 41 sialylated and one α‐galactosylated glycans from pig islet cells. The quantity of the α‐galactosylated glycans from pig islet cells was certainly neglectable compared to pig endothelial cells. A number of NeuGc‐terminated N‐glycans (80 from pig endothelial cells and 13 from pig islet cells) are newly detected by our mass spectrometric strategies. The detailed structural information will be a matter of great interest in organ or cell xenotransplantation using α 1,3‐galactosyltransferase gene‐knockout (GalT‐KO) pig. Copyright © 2009 John Wiley & Sons, Ltd.     

Divergent Chemoenzymatic Synthesis of Asymmetrical‐Core‐Fucosylated and Core‐Unmodified N‐Glycans

A divergent chemoenzymaytic approach for the preparation of core‐fucosylated and core‐unmodified asymmetrical N‐glycans from a common advances precursor is described. An undecasaccharide was synthesized by sequential chemical glycosylations of an orthogonally protected core fucosylated hexasaccharide that is common to all mammalian core fucosylated N‐glycans. Antennae‐selective enzymatic extension of the undecasaccharide using a panel of glycosyl transferases afforded core fucosylated asymmetrical triantennary N‐glycan isomers, which are potential biomarkers for breast cancer. A unique aspect of our approach is that a fucosidase (FucA1) has been identified that selectively can cleave a core‐fucoside without affecting the fucoside of a sialyl Lewis^X epitope to give easy access to core‐unmodified compounds.     

Screening the anti‐allergic components in Saposhnikoviae Radix using high‐expression Mas‐related G protein‐coupled receptor X2 cell membrane chromatography online coupled with liquid chromatography and mass spectrometry

Saposhnikoviae Radix, the dried root of Saposhnikoviae divaricata, is commonly used in the traditional Chinese anti‐allergic preparations, like Bofutsusho‐san and Yupingfeng granules. A high‐expression Mas‐related G protein‐coupled receptor X2 cell membrane chromatography coupled online with high‐performance liquid chromatography combined with an ion trap time‐of‐flight multistage mass spectrometry system was established and used for screening and identifying the anti‐allergic components in Saposhnikoviae Radix. The system was validated for excellent specificity and suitability using the appropriate standards. Two retained fractions were obtained on the cell membrane chromatography column, and three main components were identified as prim‐O‐glucosylcimifugin, cimifugin, and 4′‐O‐β‐d ‐glucosyl‐5‐O‐methylvisamminol. Next, the molecular docking study was conducted, which confirmed that these three components could effectively bind to MRGPRX2 through hydrogen bonds with its amino acid residues. Finally, histamine release assay was performed to investigate the bioactivities of prim‐O‐glucosylcimifugin, cimifugin, and 4′‐O‐β‐d ‐glucosyl‐5‐O‐methylvisamminol. Results showed that these three components could exert anti‐allergic effects by inhibiting the histamine release in a dose‐dependent manner (from 10 to 100 µM). In conclusion, the high‐expression Mas‐related G protein‐coupled receptor X2 cell membrane chromatography is an effective tool for discovering the anti‐allergic components in Saposhnikoviae Radix.     

Imaging Glycosylation In Vivo by Metabolic Labeling and Magnetic Resonance Imaging

Glycosylation is a ubiquitous post‐translational modification, present in over 50 % of the proteins in the human genome, 1 with important roles in cell–cell communication and migration. Interest in glycome profiling has increased with the realization that glycans can be used as biomarkers of many diseases, 2 including cancer. 3 We report here the first tomographic imaging of glycosylated tissues in live mice by using metabolic labeling and a gadolinium‐based bioorthogonal MRI probe. Significant N‐azidoacetylgalactosamine dependent T₁ contrast was observed in vivo two hours after probe administration. Tumor, kidney, and liver showed significant contrast, and several other tissues, including the pancreas, spleen, heart, and intestines, showed a very high contrast (>10‐fold). This approach has the potential to enable the rapid and non‐invasive magnetic resonance imaging of glycosylated tissues in vivo in preclinical models of disease.     

The Tn antigen-structural simplicity and biological complexity

Glycoproteins in animal cells contain a variety of glycan structures that are added co‐ and/or posttranslationally to proteins. Of over 20 different types of sugar–amino acid linkages known, the two major types are N‐glycans (Asn‐linked) and O‐glycans (Ser/Thr‐linked). An abnormal mucin‐type O‐glycan whose expression is associated with cancer and several human disorders is the Tn antigen. It has a relatively simple structure composed of N‐acetyl‐D ‐galactosamine with a glycosidic α linkage to serine/threonine residues in glycoproteins (GalNAcα1‐O‐Ser/Thr), and was one of the first glycoconjugates to be chemically synthesized. The Tn antigen is normally modified by a specific galactosyltransferase (T‐synthase) in the Golgi apparatus of cells. Expression of active T‐synthase is uniquely dependent on the molecular chaperone Cosmc, which is encoded by a gene on the X chromosome. Expression of the Tn antigen can arise as a consequence of mutations in the genes for T‐synthase or Cosmc, or genes affecting other steps of O‐glycosylation pathways. Because of the association of the Tn antigen with disease, there is much interest in the development of Tn‐based vaccines and other therapeutic approaches based on Tn expression.     

A QM/MM Investigation of the Catalytic Mechanism of Metal‐Ion‐Independent Core 2 β1,6‐N‐Acetylglucosaminyltransferase

β1,6‐GlcNAc‐transferase (C2GnT) is an important controlling factor of biological functions for many glycoproteins and its activity has been found to be altered in breast, colon, and lung cancer cells, in leukemia cells, in the lymhomonocytes of multiple sclerosis patients, leukocytes from diabetes patients, and in conditions causing an immune deficiency. The result of the action of C2GnT is the core 2 structure that is essential for the further elongation of the carbohydrate chains of O‐glycans. The catalytic mechanism of this metal‐ion‐independent glycosyltransferase is of paramount importance and is investigated here by using quantum mechanical (QM) (density functional theory (DFT))/molecular modeling (MM) methods with different levels of theory. The structural model of the reaction site used in this report is based on the crystal structures of C2GnT. The entire enzyme–substrate system was subdivided into two different subsystems: the QM subsystem containing 206 atoms and the MM region containing 5914 atoms. Three predefined reaction coordinates were employed to investigate the catalytic mechanism. The calculated potential energy surfaces discovered the existence of a concerted S_N2‐like mechanism. In this mechanism, a nucleophilic attack by O6 facilitated by proton transfer to the catalytic base and the separation of the leaving group all occur almost simultaneously. The transition state for the proposed reaction mechanism at the M06‐2X/6‐31G** (with diffuse functions on the O1′, O5′, O_Glu, and O6 atoms) level was located at C1? O6=1.74 Å and C1? O1=2.86 Å. The activation energy for this mechanism was estimated to be between 20 and 29 kcal mol^?1, depending on the method used. These calculations also identified a low‐barrier hydrogen bond between the nucleophile O6H and the catalytic base Glu320, and a hydrogen bond between the N‐acetamino group and the glycosidic oxygen of the donor in the TS. It is proposed that these interactions contribute to a stabilization of TS and participate in the catalytic mechanism.     

Terminal Alkenes as Versatile Chemical Reporter Groups for Metabolic Oligosaccharide Engineering

The Diels–Alder reaction with inverse electron demand (DAinv reaction) of 1,2,4,5‐tetrazines with electron rich or strained alkenes was proven to be a bioorthogonal ligation reaction that proceeds fast and with high yields. An important application of the DAinv reaction is metabolic oligosaccharide engineering (MOE) which allows the visualization of glycoconjugates in living cells. In this approach, a sugar derivative bearing a chemical reporter group is metabolically incorporated into cellular glycoconjugates and subsequently derivatized with a probe by means of a bioorthogonal ligation reaction. Here, we investigated a series of new mannosamine and glucosamine derivatives with carbamate‐linked side chains of varying length terminated by alkene groups and their suitability for labeling cell‐surface glycans. Kinetic investigations showed that the reactivity of the alkenes in DAinv reactions increases with growing chain length. When applied to MOE, one of the compounds, peracetylated N‐butenyloxycarbonylmannosamine, was especially well suited for labeling cell‐surface glycans. Obviously, the length of its side chain represents the optimal balance between incorporation efficiency and speed of the labeling reaction. Sialidase treatment of the cells before the bioorthogonal labeling reaction showed that this sugar derivative is attached to the glycans in form of the corresponding sialic acid derivative and not epimerized to another hexosamine derivative to a considerable extent.     

Fragmentation of negative ions from N‐linked carbohydrates,Part 4. Fragmentation of complex glycans lacking substitution on the 6‐antenna

Negative ion CID spectra of N‐linked glycans released from glycoproteins contain many ions that are diagnostic for specific structural features such as the detailed arrangement of antennae and the location of fucose residues. Identification of such ions requires reference glycans that are often difficult to acquire in a pure state. The recent acquisition of a sample of N‐glycans from a patient lacking the enzyme N‐acetylglucosaminyltransferase‐2 provided an opportunity to investigate fragmentation of glycans lacking a 6‐antenna. These glycans contained one or two galactose‐N‐acetylglucosamine‐chains attached to the 3‐linked mannose residue of the trimannosyl‐chitobiose core with and without fucose substitution. The spectra from the patient sample clearly defined the antenna distribution and showed striking differences from the spectra of isomeric compounds obtained from normal subjects. Furthermore, they provided additional information on previously identified antenna‐specific fragment ions and indicated the presence of additional ions that were diagnostic of fucose substitution. Glycans obtained from such enzyme‐deficient patients can, thus, be a valuable way of obtaining spectra of specific isomers in a relatively pure state for interpretation of mass spectra. Copyright © 2010 John Wiley & Sons, Ltd.     

Chemical Remodeling of Cell‐Surface Sialic Acids through a Palladium‐Triggered Bioorthogonal Elimination Reaction

We herein report a chemical decaging strategy for the in situ generation of neuramic acid (Neu), a unique type of sialic acid, on live cells by the use of a palladium‐mediated bioorthogonal elimination reaction. Palladium nanoparticles (Pd NPs) were found to be a highly efficient and biocompatible depropargylation catalyst for the direct conversion of metabolically incorporated N‐(propargyloxycarbonyl)neuramic acid (Neu5Proc) into Neu on cell‐surface glycans. This conversion chemically mimics the enzymatic de‐N‐acetylation of N‐acetylneuramic acid (Neu5Ac), a proposed mechanism for the natural occurrence of Neu on cell‐surface glycans. The bioorthogonal elimination was also exploited for the manipulation of cell‐surface charge by unmasking the free amine at C5 to neutralize the negatively charged carboxyl group at C1 of sialic acids.     

Unraveling the Mechanism of Water Oxidation Catalyzed by Nonheme Iron Complexes

Density functional theory (DFT) is employed to: 1) propose a viable catalytic cycle consistent with our experimental results for the mechanism of chemically driven (Ce^IV) O₂ generation from water, mediated by nonheme iron complexes; and 2) to unravel the role of the ligand on the nonheme iron catalyst in the water oxidation reaction activity. To this end, the key features of the water oxidation catalytic cycle for the highly active complexes [Fe(OTf)₂(Pytacn)] (Pytacn: 1‐(2′‐pyridylmethyl)‐4,7‐dimethyl‐1,4,7‐triazacyclononane; OTf: CF₃SO₃^?) ( 1 ) and [Fe(OTf)₂(mep)] (mep: N,N′‐bis(2‐pyridylmethyl)‐N,N′‐dimethyl ethane‐1,2‐diamine) ( 2 ) as well as for the catalytically inactive [Fe(OTf)₂(tmc)] (tmc: N,N′,N′′,N′′′‐tetramethylcyclam) ( 3 ) and [Fe(NCCH₃)(^MePy₂CH‐tacn)](OTf)₂ (^MePy₂CH‐tacn: N‐(dipyridin‐2‐yl)methyl)‐N′,N′′‐dimethyl‐1,4,7‐triazacyclononane) ( 4 ) were analyzed. The DFT computed catalytic cycle establishes that the resting state under catalytic conditions is a [Fe^IV(O)(OH₂)(LN₄)]²⁺ species (in which LN₄=Pytacn or mep) and the rate‐determining step is the O?O bond‐formation event. This is nicely supported by the remarkable agreement between the experimental (ΔG^≠=17.6±1.6 kcal mol^?1) and theoretical (ΔG^≠=18.9 kcal mol^?1) activation parameters obtained for complex 1 . The O?O bond formation is performed by an iron(V) intermediate [Fe^V(O)(OH)(LN₄)]²⁺ containing a cis‐Fe^V(O)(OH) unit. Under catalytic conditions (Ce^IV, pH 0.8) the high oxidation state Fe^V is only thermodynamically accessible through a proton‐coupled electron‐transfer (PCET) process from the cis‐[Fe^IV(O)(OH₂)(LN₄)]²⁺ resting state. Formation of the [Fe^V(O)(LN₄)]³⁺ species is thermodynamically inaccessible for complexes 3 and 4 . Our results also show that the cis‐labile coordinative sites in iron complexes have a beneficial key role in the O?O bond‐formation process. This is due to the cis‐OH ligand in the cis‐Fe^V(O)(OH) intermediate that can act as internal base, accepting a proton concomitant to the O?O bond‐formation reaction. Interplay between redox potentials to achieve the high oxidation state (Fe^V?O) and the activation energy barrier for the following O?O bond formation appears to be feasible through manipulation of the coordination environment of the iron site. This control may have a crucial role in the future development of water oxidation catalysts based on iron.     

Conversion of Nitric Oxide into Nitrous Oxide as Triggered by the Polarization of Coordinated NO by Hydrogen Bonding

Reduction of the {Co(NO)}⁸ cobalt–nitrosyl N‐confused porphyrin (NCP) [Co(CTPPMe)(NO)] ( 1 ) produced electron‐rich {Co(NO)}⁹ [Co(CTPPMe)(NO)][Co(Cp*)₂] ( 2 ), which was necessary for NO‐to‐N₂O conversion. Complex 2 was NO‐reduction‐silent in neat THF, but was partially activated to a hydrogen‐bonded species 2 ??? MeOH in THF/MeOH (1:1, v/v). This species coupling with 2 transformed NO into N₂O, which was fragmented from an [N₂O₂]‐bridging intermediate. An intense IR peak at 1622 cm^?1 was ascribed to ν(NO) in an [N₂O₂]‐containing intermediate. Time–course ESI(?) mass spectra supported the presence of the dimeric [Co(NCP)]₂(N₂O₂) intermediate. Five complete NO‐to‐N₂O conversion cycles were possible without significant decay in the amount of N₂O produced.     

Twist‐chair conformation of the tetraoxepane ring remains unchanged in tetraoxaspirododecane diamines

A detailed structural analysis has been performed for N,N′‐bis(4‐chlorophenyl)‐7,8,11,12‐tetraoxaspiro[5.6]dodecane‐9,10‐diamine, C₂₀H₂₂Cl₂N₂O₄, (I), N,N′‐bis(2‐fluorophenyl)‐7,8,11,12‐tetraoxaspiro[5.6]dodecane‐9,10‐diamine, C₂₀H₂₂F₂N₂O₄, (II), and N,N′‐bis(4‐fluorophenyl)‐7,8,11,12‐tetraoxaspiro[5.6]dodecane‐9,10‐diamine, C₂₀H₂₂F₂N₂O₄, (III). The seven‐membered ring with two peroxide groups adopts a twist‐chair conformation in all three compounds. The lengths of the C—N and O—O bonds are slightly shorter than the average statistical values found in the literature for azepanes and 1,2,4,5‐tetraoxepanes. The geometry analysis of compounds (I)–(III), the topological analysis of the electron density at the (3, ?1) bond critical points within Bader's quantum theory of `Atoms in molecules' (QTAIM) and NBO (natural bond orbital) analysis at the B3LYP/6‐31G(d,2p) level of theory showed that there are n_O→σ*(C—O), n_N→σ*(C—O) and n_O→σ*(C—N) stereoelectronic effects. The molecules of compounds (I) and (III) are packed in the crystals as zigzag chains due to strong N—H…O and C—H…O hydrogen‐bond interactions, whereas the molecules of compound (II) form chains in the crystals bound by N—H…O, C—H…π and C—H…O contacts. All these data show that halogen atoms and their positions have a minimal effect on the geometric parameters, stereoelectronic effects and crystal packing of compounds (I)–(III), so that the twist‐chair conformation of the tetraoxepane ring remains unchanged.     

Modifying the 5′‐Cap for Click Reactions of Eukaryotic mRNA and To Tune Translation Efficiency in Living Cells

The 5′‐cap is a hallmark of eukaryotic mRNAs and plays fundamental roles in RNA metabolism, ranging from quality control to export and translation. Modifying the 5′‐cap may thus enable modulation of the underlying processes and investigation or tuning of several biological functions. A straightforward approach is presented for the efficient production of a range of N7‐modified caps based on the highly promiscuous methyltransferase Ecm1. We show that these, as well as N²‐modified 5′‐caps, can be used to tune translation of the respective mRNAs both in vitro and in cells. Appropriate modifications allow subsequent bioorthogonal chemistry, as demonstrated by intracellular live‐cell labeling of a target mRNA. The efficient and versatile N7 manipulation of the mRNA cap makes mRNAs amenable to both modulation of their biological function and intracellular labeling, and represents a valuable addition to the chemical biology toolbox.     

Photochemical grafting of polysulfobetaine onto polyethylene and polystyrene surfaces and investigation of long‐term stability of the polysulfobetaine layer in seawater

Low‐density polyethylene (LDPE) and polystyrene (PS) films with hydrophilic surface were prepared by photochemical grafting of sulfobetaine‐based copolymer containing photolabile moiety, and long‐term stability of the hydrophilic nature of the surfaces in seawater was proved. The sulfobetaine‐based copolymer was prepared by copolymerization of N,N‐dimethyl‐N‐(3‐(methacryloylamino)propyl)‐N‐(3‐sulfopropyl) ammonium betaine with 2 or 5 mol% of N‐methacryloyl‐4‐azidoaniline, and the resulted polymers were grafted onto the plasma pretreated LDPE and PS films. The contact angle measurements were used to prove the modification as well as to follow the changes in the hydrophilicity during storage at room temperature under air atmosphere as well as in seawater at 32°C. The stability of the polymer layer was confirmed also by FTIR and AFM. Polysulfobetaine‐modified LDPE and PS surfaces exhibited significantly higher long‐term hydrophilicity compared with only plasma treated LDPE and PS surfaces.     

Derivatization of sialic acids for stabilization in matrix‐assisted laser desorption/ionization mass spectrometry and concomitant differentiation of α(2 → 3)‐ and α(2 → 6)‐isomers

Sialylated carbohydrates usually decompose by loss of sialic acid when ionized by matrix‐assisted laser desorption/ionization (MALDI) as the result of the labile carboxylic proton. Stabilization has previously been achieved by forming methyl esters with methyl iodide, a procedure that eliminates the labile proton. In this paper, we describe an alternative procedure for methyl ester formation that provides information on the sialic acid linkage directly from the MALDI spectrum. The sugars were desalted, dissolved in methanol, and treated with 4‐(4,6‐dimethoxy‐1,3,5‐triazin‐2‐yl)‐4‐methylmorpholinium chloride (DMT‐MM). After removal of the solvent, the products were transferred directly to the MALDI target and examined from 2,5‐dihydroxybenzoic acid. Small amounts of N‐glycans derived from biological sources benefited from an additional clean‐up stage involving Nafion 117. α(2 → 6)‐Linked sialic acid produced only methyl esters whereas α(2 → 3)‐linked sialic acids were converted into their lactones providing a 32 Da difference in mass. Negative ion collision‐induced decomposition (CID) mass spectra of these neutralized glycans provided information, in many cases, on the antenna of N‐linked glycans to which the variously linked sialic acids were attached. The method was applied to N‐linked glycans released from bovine fetuin and porcine thyroglobulin. Copyright © 2008 John Wiley & Sons, Ltd.     

A Deadly Organometallic Luminescent Probe: Anticancer Activity of a ReI Bisquinoline Complex

The photophysical properties of [Re(CO)₃(L ‐N₃)]Br (L ‐N₃=2‐azido‐N,N‐bis[(quinolin‐2‐yl)methyl]ethanamine), which could not be localized in cancer cells by fluorescence microscopy, have been revisited in order to evaluate its use as a luminescent probe in a biological environment. The Re^I complex displays concentration‐dependent residual fluorescence besides the expected phosphorescence, and the nature of the emitting excited states have been evaluated by DFT and time‐dependent (TD) DFT methods. The results show that fluorescence occurs from a ¹LC/MLCT state, whereas phosphorescence mainly stems from a ³LC state, in contrast to previous assignments. We found that our luminescent probe, [Re(CO)₃(L ‐N₃)]Br, exhibits an interesting cytotoxic activity in the low micromolar range in various cancer cell lines. Several biochemical assays were performed to unveil the cytotoxic mechanism of the organometallic Re^I bisquinoline complex. [Re(CO)₃(L ‐N₃)]Br was found to be stable in human plasma indicating that [Re(CO)₃(L ‐N₃)]Br itself and not a decomposition product is responsible for the observed cytotoxicity. Addition of [Re(CO)₃(L ‐N₃)]Br to MCF‐7 breast cancer cells grown on a biosensor chip micro‐bioreactor immediately led to reduced cellular respiration and increased glycolysis, indicating a large shift in cellular metabolism and inhibition of mitochondrial activity. Further analysis of respiration of isolated mitochondria clearly showed that mitochondrial respiratory activity was a direct target of [Re(CO)₃(L ‐N₃)]Br and involved two modes of action, namely increased respiration at lower concentrations, potentially through increased proton transport through the inner mitochondrial membrane, and efficient blocking of respiration at higher concentrations. Thus, we believe that the direct targeting of mitochondria in cells by [Re(CO)₃(L ‐N₃)]Br is responsible for the anticancer activity.     

Mass spectrometric analysis of the glycosphingolipid‐derived glycans from miniature pig endothelial cells and islets: identification of NeuGc epitope in pig islets

Glycosphingolipid (GSL) is a major component of the plasma membrane in eukaryotic cells that is involved directly in a variety of immunological events via cell‐to‐cell or cell‐to‐protein interactions. In this study, qualitative and quantitative analyses of GSL‐derived glycans on endothelial cells and islets from a miniature pig were performed and their glycosylation patterns were compared. A total of 60 and 47 sialylated and neutral GSL‐derived glycans from the endothelial cells and islets, respectively, were characterized by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) and collision‐induced fragmentation using positive‐ion electrospray ionization (ESI) ion‐trap tandem mass spectrometry (MS/MS). In accordance with previous immunohistochemistry studies, the α‐Gal‐terminated GSL was not detected but NeuGc‐terminated GSLs were newly detected from miniature pig islets. In addition, the neutral GSL‐derived glycans were relatively quantified by derivatization with carboxymethyl trimethylammonium hydrazide (so called Girard's T reagent) and MALDI‐TOF MS. The structural information of the GSL‐derived glycans from pig endothelial cells and islets suggests that special attention should be paid to all types of glycoconjugates expressed on pig tissues or cells for successful clinical xenotransplantation. Copyright © 2009 John Wiley & Sons, Ltd.