Characterization of β2‐microglobulin conformational intermediates associated to different fibrillation conditions

β2‐Microglobulin (β2m) is the light chain of the class‐I major histocompatibility complex, being also the causing agent of dialysis‐related amyloidosis, which results from its accumulation as amyloid material in the skeletal joints. This study describes conformational properties of β2m under two distinct, in vitro amyloidogenic conditions: neutral pH in the presence of 20% 2,2,2‐trifluoroethanol (TFE) and acidic pH in the absence of TFE. Species distribution analysis by electrospray ionization–mass spectrometry (ESI‐MS) is combined with information obtained by ion mobility–mass spectrometry (IM‐MS), fluorescence and circular dichroism (CD) spectroscopy. It is shown that β2m populates quite different conformational ensembles under the two conditions, but both ensembles display a minor fraction of the population in a partially folded state. In spite of similar compactness, these two partially folded forms display different conformations: helical secondary structure is predominant in the species at pH 7.4, 20% TFE, while the low‐pH form is mainly random coil. As temperature is increased, the TFE intermediate looses helical structure becoming more similar to the low‐pH intermediate. The existence of different conformational ensembles may rationalize the different aggregation propensity displayed by β2m under the two fibrillation conditions analyzed here. Copyright © 2011 John Wiley & Sons, Ltd.     

Synthesis of 3‐aryl‐5‐styryl‐2‐pyrazolines by the reaction of (E,E)‐cinnamylideneacetophenones with hydrazines and their oxidation into pyrazoles

New 3‐aryl‐5‐styryl‐2‐pyrazolines have been synthesized by the reaction of (E,E)‐cinnamylideneace‐tophenones with hydrazines. These 2‐pyrazolines have also been converted into the corresponding pyrazoles by oxidation with chloranil. Structures of all new compounds have been elucidated by elemental analysis, mass spectrometry, ir and nmr spectroscopic measurements.     

Bioactivity‐integrated ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry for the identification of nuclear factor‐κB inhibitors and β2 adrenergic receptor agonists in Chinese medicinal preparation Chuanbeipipa dropping pills

A simple and dual‐target method based on ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry combined with dual‐bioactive [nuclear factor‐κB (NF‐κB) and β₂‐adrenergic receptor] luciferase reporter assay systems was developed to rapidly characterize the chemical structure of various bioactive compounds of TCM preparations. Chuanbeipipa dropping pills, a traditional Chinese medicine preparation used for the clinical therapy of chronic obstructive lung disease and cough caused by bronchial catarrh, was analyzed with this method. Potential anti‐inflammatory and spasmolytic constituents were screened using NF‐κB and β₂‐adrenergic receptor activity luciferase reporter assay systems and simultaneously identified according to the time‐of‐flight mass spectrometry data. One β₂‐adrenergic receptor agonist (ephedrine) and two structural types of NF‐κB inhibitors (platycosides derivatives and ursolic acid derivatives) were characterized. Platycodin D3 and E were considered new NF‐κB inhibitors. Further cytokine and chemokine detection confirmed the anti‐inflammatory effects of the potential NF‐κB inhibitors. Compared with conventional fingerprints, activity‐integrated fingerprints that contain both chemical and bioactive details offer a more comprehensive understanding of the chemical makeup of plant materials. This strategy clearly demonstrated that multiple bioactivity‐integrated fingerprinting is a powerful tool for the improved screening and identification of potential multi‐target lead compounds in complex herbal medicines. Copyright © 2013 John Wiley & Sons, Ltd.     

Cu‐Catalyzed Aerobic Oxidative Cyclizations of 3‐N‐Hydroxyamino‐1,2‐propadienes with Alcohols,Thiols, and Amines To Form α‐O‐, S‐, and N‐Substituted 4‐Methylquinoline Derivatives

A one‐pot, two‐step synthesis of α‐O‐, S‐, and N‐substituted 4‐methylquinoline derivatives through Cu‐catalyzed aerobic oxidations of N‐hydroxyaminoallenes with alcohols, thiols, and amines is described. This reaction sequence involves an initial oxidation of N‐hydroxyaminoallenes with NuH (Nu=OH, OR, NHR, and SR) to form 3‐substituted 2‐en‐1‐ones, followed by Brønsted acid catalyzed intramolecular cyclizations of the resulting products. Our mechanistic analysis suggests that the reactions proceed through a radical‐type mechanism rather than a typical nitrone‐intermediate route. The utility of this new Cu‐catalyzed reaction is shown by its applicability to the synthesis of several 2‐amino‐4‐methylquinoline derivatives, which are known to be key precursors to several bioactive molecules.     

Isomer differentiation via collision‐induced dissociation: The case of protonated α‐, β2‐ and β3‐phenylalanines and their derivatives

A combination of electrospray ionisation (ESI), multistage and high‐resolution mass spectrometry experiments is used to examine the gas‐phase fragmentation reactions of the three isomeric phenylalanine derivatives, α‐phenylalanine, β²‐phenylalanine and β³‐phenylalanine. Under collision‐induced dissociation (CID) conditions, each of the protonated phenylalanine isomers fragmented differently, allowing for differentiation. For example, protonated β³‐phenylalanine fragments almost exclusively via the loss of NH₃, only β²‐phenylalanine via the loss of H₂O, while α‐ and β²‐phenylalanine fragment mainly via the combined losses of H₂O + CO. Density functional theory (DFT) calculations were performed to examine the competition between NH₃ loss and the combined losses of H₂O and CO for each of the protonated phenylalanine isomers. Three potential NH₃ loss pathways were studied: (i) an aryl‐assisted neighbouring group; (ii) 1,2 hydride migration; and (iii) neighbouring group participation by the carboxyl group. Finally, we have shown that isomer differentiation is also possible when CID is performed on the protonated methyl ester and methyl amide derivatives of α‐, β²‐ and β³‐phenylalanines. Copyright © 2010 John Wiley & Sons, Ltd.     

Gas‐fragmentation study of the novel synthetic zwitterionic drug 3‐methyl‐9‐(2‐oxa‐2λ5‐2H‐1,3,2‐oxazaphosphorine‐2‐cyclohexyl)‐3,6,9‐triazaspiro[5,5]undecane chloride (SLXM‐2) by electrospray ionization tandem mass spectrometry

The zwitterionic drug 3‐methyl‐9‐(2‐oxa‐2λ5‐2H‐1,3,2‐oxazaphosphorine‐2‐cyclohexyl)‐3,6,9‐triazaspiro[5,5]undecane chloride (SLXM‐2) is a novel synthetic compound which has shown anticancer activity and low toxicity in vivo. In this study, the various gas‐phase fragmentation routes were analyzed by electrospray ionization mass spectrometry (positive ion mode) in conjunction with tandem mass spectrometry (ESI‐MSⁿ) for the first time. In ESI‐MS the fragment ion at m/z 289 (base peak) was formed by loss of the chlorine anion from the zwitterionic precursor SLXM‐2. The fragment ion at m/z 232 was formed from the ion at m/z 289 by loss of 1‐methylaziridine. The detailed gas‐phase collision‐induced dissociation (CID) fragmentation mechanisms obtained from the various precursor ions extracted from the zwitterionic SLXM‐2 drug was obtained by tandem mass spectrometry analyses. Copyright © 2010 John Wiley & Sons, Ltd.     

Synthesis,characterization and antibacterial activity of 3‐(2‐methoxyphenyl)‐2‐sulfanylpropenoic acid and di‐isopropylammonium [3‐(2‐methoxyphenyl)‐2‐sulfanylpropenoato] triphenylstannate(IV). The crystal structure of [HQ][SnPh3(o‐mpspa)]

Reaction of 3‐(2‐methoxyphenyl)‐2‐sulfanylpropenoic acid [H₂(o‐mpspa)] with SnPh₃OH in the presence of di‐isopropylamine resulted in the formation of the complex [HQ][SnPh₃(o‐mpspa)] (where HQ = di‐isopropylammonium cation and o‐mpspa = 3‐(2‐methoxyphenyl)‐2‐sulfanylpropenoato), which was characterized by mass spectrometry and vibrational spectroscopy, as well as by ¹H, ¹³C and ¹¹⁹Sn NMR spectroscopy. The single‐crystal X‐ray structural analysis of the new complex shows a trigonal‐bipyramidal coordination geometry around the Sn atom where o‐mpspa behaves as a bidentate chelating ligand. Dimeric units arise from the existence of N? H…O hydrogen bonds between the NH₂ group of the di‐isopropylammonium cation and the oxygen atoms of the two neighbouring carboxylato groups. The bacteriostatic activity of the complex is also reported. Copyright © 2007 John Wiley & Sons, Ltd.     

Theoretical Study on Reactivity of Electron Transfer in Model—System of Oxidation of α—Amino Carbon—centered Radical by O2

Electron transfer reaction between a simplified model model molecule of α－amino carbon-centered radical and O2 has studied with ab initio calculations at the MP2/6-31 G^**//UHF/6-31 G^** level,The reactant complex and the ion pair complex have been optimized and employed to perform calculation of the reaction heat and the reorganization energy,Solvent effects have been considered by applyning the conductor-like screening model,Theoretical results show that the highly endothermic charge separation process ,in which one electron transfers from the α－amino carbon-centered radical to O2,so as to form an ion pair complex,is difficult to occur in gas-phase,By apply-ing an external electronic field to prepare the charge-locallized molecular orbitals,the charge-separated state has been obtained using the initial-guess-induced self-consistent field technique,The theoretical investigations indicate that the solvent effect in the process of the oxidation of α-animo carbon-centered radical by O2 is remarkable.From the rate constant estima-tion ,it can be predicted that the oxidation of the model donor molecule by O2 can proceed,but not very fast.A peroxyl radi-cal compound has been found to be a competitive intermediate in the oxidation process.     

PPh3⋅HBr‐DMSO Mediated Expedient Synthesis of γ‐Substituted β,γ‐Unsaturated α‐Ketomethylthioesters and α‐Bromo Enals: Application to the Synthesis of 2‐Methylsulfanyl‐3(2 H)‐furanones

An efficient chemoselective general procedure for the synthesis of γ‐substituted β,γ‐unsaturated α‐ketomethylthioesters from α,β‐unsaturated ketones has been achieved through an unprecedented PPh₃?HBr‐DMSO mediated oxidative bromination and Kornblum oxidation sequence. The newly developed reagent system serves admirably for the synthesis of α‐bromoenals from enals. Furthermore, AuCl₃‐catalyzed efficient access to 3(2H)‐furanones from the above intermediates under extremely mild conditions are described.     

Differentiation of Boc‐protected α,δ‐/δ,α‐ and β,δ‐/δ,β‐hybrid peptide positional isomers by electrospray ionization tandem mass spectrometry

Two new series of Boc‐N‐α,δ‐/δ,α‐ and β,δ‐/δ,β‐hybrid peptides containing repeats of L ‐Ala‐δ⁵‐Caa/δ⁵‐Caa‐L ‐Ala and β³‐Caa‐δ⁵‐Caa/δ⁵‐Caa‐β³‐Caa (L ‐Ala = L ‐alanine, Caa = C‐linked carbo amino acid derived from D ‐xylose) have been differentiated by both positive and negative ion electrospray ionization (ESI) ion trap tandem mass spectrometry (MS/MS). MSⁿ spectra of protonated isomeric peptides produce characteristic fragmentation involving the peptide backbone, the Boc‐group, and the side chain. The dipeptide positional isomers are differentiated by the collision‐induced dissociation (CID) of the protonated peptides. The loss of 2‐methylprop‐1‐ene is more pronounced for Boc‐NH‐L ‐Ala‐δ‐Caa‐OCH₃ (1), whereas it is totally absent for its positional isomer Boc‐NH‐δ‐Caa‐L ‐Ala‐OCH₃ (7), instead it shows significant loss of t‐butanol. On the other hand, second isomeric pair shows significant loss of t‐butanol and loss of acetone for Boc‐NH‐δ‐Caa‐β‐Caa‐OCH₃ (18), whereas these are insignificant for its positional isomer Boc‐NH‐β‐Caa‐δ‐Caa‐OCH₃ (13). The tetra‐ and hexapeptide positional isomers also show significant differences in MS² and MS³ CID spectra. It is observed that ‘b’ ions are abundant when oxazolone structures are formed through five‐membered cyclic transition state and cyclization process for larger ‘b’ ions led to its insignificant abundance. However, b₁⁺ ion is formed in case of δ,α‐dipeptide that may have a six‐membered substituted piperidone ion structure. Furthermore, ESI negative ion MS/MS has also been found to be useful for differentiating these isomeric peptide acids. Thus, the results of MS/MS of pairs of di‐, tetra‐, and hexapeptide positional isomers provide peptide sequencing information and distinguish the positional isomers. Copyright © 2010 John Wiley & Sons, Ltd.     

Coordination chemistry of organometallic polydentate ligand Reactive chemistry of the tridentate ligand trans‐Fe(Ph2PQu‐P)2‐(CO)3 [PhzPQu=2‐diphenyl‐phosphino‐4‐methylquinoline] and molecular structure of [Fe(CO)3(μ‐Ph2PQu)2HgI]+ [HgI3]

Reaction of a new type of bidentate ligand PhPQu [PhPQu = 2‐diphenylphosphino‐4‐methylquinoline] with Fe(CO)₅ in butanol gave trans‐Fe(FpPQu‐P)(CO)₃ (1). Compound 1, which can act as a neutral tridentate organometallic ligand, was reacted with I B, II B metal compounds and a rhodium complex to give six binuclear complexes with Fe? M bonds, Fe(CO)₃ (μ‐Ph₂PQu)MX_n (2–7) [M= Zn(II), Cd(II), Hg(II), Cu(I), Ag(I), Rh(I)], and an ion‐pair complex [Fe(CO)₃ (μ‐Ph₂PQu)₂HgI][HgI₃]^? (8). The structure of 8 was determined by X‐ray crystallography. Complex 8 crystallizes in the space group P‐1 with a = 1.0758(3), b = 1.6210(4), c=1.7155(4)nm; a=75.60(2), β=71.81(2), γ=81.78(2)° and Z = 2 and its structure was refined to give agreement factors of R=0.050 and R_w = 0.057. The Fe‐Hg bond distance is 0.2536nm.     

MALDI imaging mass spectrometry of β‐ and γ‐crystallins in the ocular lens

Lens crystallin proteins make up 90% of expressed proteins in the ocular lens and are primarily responsible for maintaining lens transparency and establishing the gradient of refractive index necessary for proper focusing of images onto the retina. Age‐related modifications to lens crystallins have been linked to insolubilization and cataractogenesis in human lenses. Matrix‐assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) has been shown to provide spatial maps of such age‐related modifications. Previous work demonstrated that, under standard protein IMS conditions, α‐crystallin signals dominated the mass spectrum and age‐related modifications to α‐crystallins could be mapped. In the current study, a new sample preparation method was optimized to allow imaging of β‐ and γ‐crystallins in ocular lens tissue. Acquired images showed that γ‐crystallins were localized predominately in the lens nucleus whereas β‐crystallins were primarily localized to the lens cortex. Age‐related modifications such as truncation, acetylation, and carbamylation were identified and spatially mapped. Protein identifications were determined by top‐down proteomics analysis of lens proteins extracted from tissue sections and analyzed by LC‐MS/MS with electron transfer dissociation. This new sample preparation method combined with the standard method allows the major lens crystallins to be mapped by MALDI IMS.     

Tandem IBX‐Promoted Primary Alcohol Oxidation/Opening of Intermediate β,γ‐Diolcarbonate Aldehydes to (E)‐γ‐Hydroxy‐α,β‐enals

A tandem IBX‐promoted oxidation of primary alcohol to aldehyde and opening of intermediate β,γ‐diolcarbonate aldehyde to (E)‐γ‐hydroxy‐α,β‐enal has been developed. Remarkably, the carbonate opening delivered exclusively (E)‐olefin and no over‐oxidation of γ‐hydroxy was observed. The method developed has been extended to complete the stereoselective total synthesis of both (S)‐ and (R)‐coriolides and d ‐xylo‐ and d ‐arabino‐C‐20 guggultetrols.     

Inclusion of Ethyl Acetoacetate Bearing 7‐Hydroxycoumarin Dye by β‐Cyclodextrin and its Cooperative Assembly with Mercury(II) Ions: Spectroscopic and Molecular Modeling Studies

The inclusion of the fluorescent organic dye, ethyl 3‐(7‐hydroxy‐2‐oxo‐2H‐chromen‐3‐yl)‐3‐oxopropanoate ( 1 ) by the host β‐cyclodextrin (β‐CD), and its response toward mercuric ions (Hg²⁺), was studied by UV/Vis, fluorescence, and ¹H NMR spectroscopic analyses, mass spectrometry and molecular modeling studies. ¹H NMR measurements together with molecular modeling studies for dye 1 demonstrate that it exhibits two tautomeric forms (keto and enol); however, when the dye is included into the β‐CD cavity, the enol form predominates. Moreover, by using spectroscopic and spectrometry techniques, a 1:1 stoichiometry was determined for the complexes formed between dye 1 (enol form) and β‐CD, with a binding constant (K_b1=1.8×10⁴ m ^?1) and for the dye 1 (keto form)‐Hg²⁺ (K_b2=2.3×10³ m ^?1). Interestingly, in the presence of 1 –β‐CD complex and mercuric ions, a ternary supramolecular system (Hg– 1 –β‐CD complex) was established, with a 1:1:1 stoichiometry and a K_b3 value of 4.3×10³ m ^?1, with the keto form of the dye being the only one present in this assembly. The three‐component system provides a starting point for the development of novel and directed supramolecular assemblies.     

Identification of bioactive compounds in Shaoyao‐Gancao decoction using β2‐adrenoceptor affinity chromatography

Shaoyao‐Gancao decoction, a Chinese herbal formula, is composed of Paeoniae Radix alba and Glycyrrhiza Radix et rhizoma . It has been widely used to treat muscle spasms and asthma. However, little is known about the bioactive components of Shaoyao‐Gancao decoction. In the present study, the bioactive compounds in water‐extract of Shaoyao‐Gancao decoction were separated by the immobilized β₂‐adrenoceptor affinity column and identified using quadrupole time‐of‐flight mass spectrometry. The affinity constants of the separated compounds that bind to β₂‐adrenoceptor were determined by frontal analysis. Compound bioactivity was tested in a rat tracheal smooth muscle relaxation assay. We identified the bioactive compounds in the water extract of Shaoyao‐Gancao decoction that bound to the β₂‐adrenoceptor as paeoniflorin and liquiritin. Paeoniflorin and liquiritin had only one binding site on the immobilized β₂‐adrenoceptor, and the affinity constants were (2.16 ± 0.10) × 10⁴ M⁻¹ and (2.95 ± 0.15) × 10⁴ M⁻¹, respectively. Both compounds induced a concentration‐dependent relaxation of tracheal smooth muscle following K⁺‐stimulated contraction, and the relaxation effects were abrogated by the β₂‐adrenoceptor antagonist, ICI 118551. Therefore, paeoniflorin and liquiritin are bioactive compounds in Shaoyao‐Gancao decoction and the β₂‐adrenoceptor affinity chromatography is a useful tool for identifying potential β₂‐adrenoceptor ligands in natural products used in traditional Chinese medicine.     

Precursor‐Directed Biosynthesis: Stereospecificity for Branched‐Chain Diketides of the β‐Ketoacyl‐ACP Synthase Domain 2 of 6‐Deoxyerythronolide B Synthase

Modular polyketide synthases such as 6‐deoxyerythronolide B synthase (DEBS) catalyze the biosynthesis of structurally complex natural products. Streptomyces coelicolor CH999/pJRJ2 harbors a plasmid encoding DEBS(KS1⁰), a mutant form of 6‐deoxyerythronolide B synthase that is blocked in the formation of 6‐deoxyerythronolide B ( 1 , 6‐dEB) due to a mutation in the active site of the ketosynthase (KS1) domain that normally catalyzes the first polyketide chain‐elongation step of 6‐dEB biosynthesis. Administration of (2S,3R,4S)‐ and (2S,3R,4R)‐3‐hydroxy‐2,4‐dimethylhexanoic acid N‐acetylcysteamine (SNAC) thioesters (= S‐[2‐(acetylamino)ethyl] (2S,3R,4S)‐ and (2S,3R,4R)‐3‐hydroxy‐2,4‐dimethylhexanethioates) 3 and 4 in separate experiments to cultures of Streptomyces coelicolor CH999/pJRJ2 led to production of the corresponding (14S)‐ and (14R)‐14‐methyl analogues of 6‐dEB, 10 and 11 , respectively. Unexpectedly, when a 3 : 2 mixture of 4 and 3 was fed under the same conditions, exclusively branched‐chain macrolactone 11 was isolated. In similar experiments, feeding of 3 and 4 to S. coelicolor CH999/pCK16, an engineered strain harboring DEBS1+TE(KS1⁰), resulted in formation of the branched‐chain triketide lactones 13 and 14 , while feeding of the 3 : 2 mixture of 4 and 3 gave exclusively 14 . The biochemical basis for this stereochemical discrimination was established by using purified DEBS module 2+TE to determine the steady‐state kinetic parameters for 3 and 4 , with the k_cat/K_M for 4 shown to be sevenfold greater than that of 3 .     

Ring‐opening polymerization of ε‐caprolactone via the bismuth‐2‐mercaptoethanol complex

A complex consisting of one Bi³⁺ ion and two 2‐mercaptoethanol units (BiME₂) was used as initiator for the ring‐opening polymerization of ε‐caprolactone in bulk. A kinetic comparison showed that BiME₂ is as reactive as initiator as Sn‐octanoate and more reactive than Bi‐hexanoate. The difference to BiHex₃ decreased at higher temperatures and upon addition of an alcohol as coinitiator. When tetra(ethylene glycol) was used as coinitiator, it was completely incorporated into the poly(εCL) chain, so that telechelic polylactones having two OH‐endgroups were formed. In the absence of a coinitiator, 2‐mercaptoethanol or its disulfide were incorporated in the form of ester groups. Furthermore, it was found by MALDI‐TOF mass spectrometry that small amounts of cyclic oligolactones (detected up to a degree of polymerization of 17) were formed under all reaction conditions. Higher temperatures and longer times favored a higher content of cycles. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 3175–3183, 2006     

Screening and identification of Caulis Sinomenii bioactive ingredients with dual‐target NF‐κB inhibition and β2‐AR agonizing activities

Caulis Sinomenii (CS) is a valuable traditional medicine in China. Its extract can act as an anti‐inflammatory agent and a vascular smooth muscle relaxant. However, the underlying mechanisms remain unknown. In this study, we developed a simple dual‐target method based on ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry combined with a dual‐target bioactive screening assay for anti‐inflammatory and antispasmodic activities to characterize the chemical structure of various bioactive compounds of CS rapidly. Seven potential NF‐κB inhibitors were identified, including laudanosoline‐1‐O‐xylopyranose, 6‐O‐methyl‐laudanosoline‐1‐O‐glucopyranoside, menisperine, sinomenine, laurifoline, magnoflorine and norsinoacutin. Furthermore, IL‐6 and IL‐8 assays confirmed the anti‐inflammatory effects of these potential NF‐κB inhibitors, in which laudanosoline‐1‐O‐d ‐xylopyranose and menisperine were revealed as novel NF‐κB inhibitors. Among the seven identified alkaloids, three potential β₂‐adrenergic receptor agonists, including sinomenine, magnoflorine and laurifoline, were characterized using a luciferase reporter system to measure for the activity of β₂‐adrenergic receptor agonists. Finally, sinomenine, magnoflorine and laurifoline were identified not only as potential NF‐κB inhibitors but also as potential β₂‐adrenegic receptor agonists, which is the first time this has been reported. Molecular dynamic simulation and docking results suggest that the three dual‐bioactive constituents could not only inhibit Pseudomonas aeruginosa PAK strain‐induced inflammatory responses via a negative regulation of the Braf protein that participates in MAPK signaling pathway but also activate the β₂‐adrenegic receptor. These results suggest that CS extract has dual signaling activities with potential clinical application as a novel drug for asthma.     

Analysis of E. coli soluble proteins by non‐denaturing micro 2‐DE/3‐DE and MALDI‐MS‐PMF

Escherichia coli (strain K‐12)‐soluble proteins were analyzed by nondenaturing micro 2‐DE and MALDI‐MS‐PMF. The reported conditions of nondenaturing IEF in agarose column gels [Jin, Y., Manabe, T., Electrophoresis 2009, 30, 939–948] were modified to optimize the resolution of cellular soluble proteins. About 300 CBB‐stained spots, the apparent molecular masses of which ranged from ca. 6000 to 10 kDa, were detected. All the spots on two reference 2‐DE gels (one for wide mass range and one for low‐molecular‐mass range) were numbered and subjected to MALDI‐MS‐PMF for the assignment of constituting polypeptides. Most of the spots (310 spots out of 329) provided significant match (p<0.05) with polypeptides in Swiss‐Prot database and totally 228 polypeptide species were assigned. Activity staining of enzymes such as alkaline phosphatase and catalases was performed on the 2‐DE gels and the locations of the activity spots matched well with those of the MS‐assigned polypeptides of the enzymes. Most of the polypeptides with subunit information in Swiss‐Prot (119 polypeptides as homo‐multimers and 25 as hetero‐multimers out of the 228), such as pyruvate dehydrogenase complex which is composed of three enzymatic components, were detected at the apparent mass positions of their polymers, suggesting that the proteins were separated retaining their subunit structures. When a nondenaturing 2‐DE gel was vertically cut into 2 mm strips and one of the strips was subjected to a third‐dimension micro SDS‐PAGE (micro 3‐DE), about 190 CBB‐stained spots were detected. The assignment of the polypeptides separated on the 3‐DE gel would further provide information on protein/polypeptide interactions.     

Evidence of a Photo‐Radical Pathway Responsible of the Rearrangement of a Manganese(III)‐Schiff Base Complex in Solution

A new Mn^III‐Schiff base complex, [MnL(OH₂)](ClO₄) ( 1 ) (H₂L = N, N′‐bis‐(3‐Br‐5‐Cl‐salicylidene)‐1, 2‐diimino‐2‐methylethane), an inorganic model of the catalytic center (OEC, Oxygen Evolving Complex) in photosystem II (PSII), has been synthesized and characterized by elemental analysis, IR and EPR spectroscopy, mass spectrometry, magnetic susceptibility measurement and the study of its redox properties by cyclic and normal pulse voltammetry. This complex mimics reactivity (showing a relevant photolytic activity), and also some structural characteristics (parallel‐mode Mn^III EPR signal from partially assembled OEC cluster) of the natural OEC. The complex 1 was found to rearrange in solution into a crystallographically solved square‐pyramidal complex, [MnLL′] ( 2 ) (HL′ = 6‐bromo‐4‐chloro‐2‐cyanophenol), through a process, which probably liberates radical species (detected by EPR), and provokes a C—N bond cleavage in the ligand. A photo‐radical mechanism is discussed to explain this rearrangement.