Ansaetherone,a New Radical Scavenger from Streptomyces sp.

We identified a new radical scavenger, ansaetherone (C₂₆H₃₃NO₇), from a culture of the Streptomyces sp. USF-4727 strain. In our previous study, it was shown that this strain produced four lipoxygenase inhibitors, tetrapetalones A, B, C and D. The chemical structure of ansaetherone was elucidated by the spectroscopic method, indicating that this compound was constructed with an aglycon and a sugar moiety. This chemical structure suggested that ansaetherone was related to the tetrapetalones. This finding provided information regarding tetrapetalone biosynthesis. Ansaetherone showed radical scavenging activity with an ED₅₀ value of 300 μM in our assay.     

Towards an interpretation of 13C chemical shifts in bathorhodopsin,a functional intermediate of a G-protein coupled receptor

Photoisomerization of the membrane-bound light receptor protein rhodopsin leads to an energy-rich photostate called bathorhodopsin, which may be trapped at temperatures of 120 K or lower. We recently studied bathorhodopsin by low-temperature solid-state NMR, using in situ illumination of the sample in a purpose-built NMR probe. In this way we acquired ¹³C chemical shifts along the retinylidene chain of the chromophore. Here we compare these results with the chemical shifts of the dark state chromophore in rhodopsin, as well as with the chemical shifts of retinylidene model compounds in solution. An earlier solid-state NMR study of bathorhodopsin found only small changes in the ¹³C chemical shifts upon isomerization, suggesting only minor perturbations of the electronic structure in the isomerized retinylidene chain. This is at variance with our recent measurements which show much larger perturbations of the ¹³C chemical shifts. Here we present a tentative interpretation of our NMR results involving an increased charge delocalization inside the polyene chain of the bathorhodopsin chromophore. Our results suggest that the bathochromic shift of bathorhodopsin is due to modified electrostatic interactions between the chromophore and the binding pocket, whereas both electrostatic interactions and torsional strain are involved in the energy storage mechanism of bathorhodopsin.     

Structural analysis of mucoidan, an acidic extracellular polysaccharide produced by a pristane-assimilating marine bacterium, Rhodococcus erythropolis PR4

Rhodococcus erythropolis PR4 is a marine bacterium that can degrade various alkanes including pristane, a C(19) branched alkane. This strain produces a large quantity of extracellular polysaccharides (EPS), which are assumed to play an important role in the hydrocarbon tolerance of R. erythropolis PR4. The strain produced an acidic EPS, mucoidan, together with a fatty acid-containing EPS, PR4 FACEPS. The chemical structure of the mucoidan was determined using (1)H and (13)C NMR spectroscopy and by conducting 2D DQF-COSY, TOCSY, HMQC, HMBC, and NOESY experiments. The mucoidan was shown to consist of a pentasaccharide repeating unit with the following structure: [structure: see text].     

Structure of the O-polysaccharide from Proteus myxofaciens. Classification of the bacterium into a new Proteus-O-serogroup.

The O-polysaccharide (O-antigen) was obtained from the lipopolysaccharide of Proteus myxofaciens, a Proteus strain producing copious amounts of slime, which was isolated from the gypsy moth larvae. The structure of the polysaccharide was studied by chemical analysis and 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, ROESY and H-detected 1H,13C HMQC experiments. It was found that the polysaccharide contains an amide of glucuronic acid (GlcA) with an unusual alpha-linked amino acid, Nepsilon-[(R)-1-carboxyethyl]-l-lysine (2S,8R-alaninolysine, 2S,8R-AlaLys), and has a linear tetrasaccharide repeating unit of the following structure: This structure is unique among known bacterial polysaccharide structures. On the basis of these and serological data, it is proposed that P. myxofaciens be classified into a new Proteus serogroup, O60, of which this strain is the single representative. Structural and serological relatedness of P. myxofaciens to other AlaLys-containing O-antigens of Proteus and Providencia is discussed.     

Chemical characterization of capsular polysaccharide from Cryptococcus neoformans serotype A-D

During a study of serotyping of Cryptococcus neoformans, we found that the type strain of C. neoformans (CBS 132) was serotype A-D. This strain agglutinated with both factor 7 serum (specific for serotype A) and factor 8 serum (specific for serotype D) in our serotyping system. Therefore, we investigated the chemical structure of the antigenic capsular polysaccharide of this strain. The soluble capsular polysaccharide was obtained from the culture supernatant fluid by precipitation with ethanol. Column chromatography of the polysaccharide on DEAE-cellulose yielded three fractions (F-1 to F-3). The major antigenic activity was found in the F-3 fraction. The results obtained by methylation analysis, controlled Smith degradation-methylation analysis, partial acid hydrolysis, and other structural studies of F-3 polysaccharide indicated that the polysaccharide contains mannose, xylose, and glucuronic acid at a ratio of 7:2:2, and has a backbone of alpha (1-3)-linked D-mannopyranoside residues with a single branch of beta (1-2)-xylose and glucuronic acid. The ratio of mannose residues with or without a branch in the F-3 polysaccharide was 4:3 and its molecular weight calculated from the average of the degree of polymerization was 46,500 daltons. These results indicate that the chemical structure of the capsular polysaccharide of serotype A-D is very similar to those from serotypes A and D, suggesting that small differences in the molar ratio and pattern of linkage of monosaccharides in the branch of the polysaccharides of the three serotypes may be responsible for their different specificities.     

The Origin of Strain Effects on Sulfur Redox Electrocatalyst for Lithium Sulfur Batteries

Introducing strain is considered an effective strategy to enhance the catalytic activity of host material in lithium-sulfur batteries (LSB). However, the introduction of strain through chemical methods often inevitably leads to changes in chemical composition and phase structure, making it difficult to truly reveal the essence and root cause of catalytic activity enhancement. In this paper, strain into MoS₂ is introduced through a simple heat treatment and quenching. Experimental research and theoretical analysis show that the strain raises parts of antibonding orbitals in Mo─S bonds above the Fermi level and weakens Li─S and S─S bonds, resulting in tight anchoring and accelerating the conversion for lithium polysulfides (LiPSs). The cells based on the MoS₂ with high strain delivers an initial discharge specific capacity as high as 1265 mAh g⁻¹ under 0.2 C and a low average capacity fading of 0.041% per cycle during 1500 cycles under 1 C. This research work deeply reveals the origin of strain effects in the reaction process of LSB, providing important design principles and references for the rational design of high-performance catalytic materials in the future.     

一种被毛孢(Hirsutella sp.)培养液中自由基清除剂的分析、分离和制备

[目的]在一次对虫生真菌代谢物进行大规模的清除自由基活性物质筛选中,发现一种被毛孢(Hirsutella sp.)菌株RCEF0881发酵液中存在有较强的清除自由基活性物质.本研究目的是初步搞清这些活性成分的具体组成,并制备出一定量的纯品用于进一步的结构鉴定.[方法]用有机溶剂法提取活性成分;用二苯基苦基苯肼自由基(DPPH)酶标仪法和薄层色谱法进行活性测定;用高分辨液质联用方法进行活性成分初步分析和鉴定;用反相制备色谱法制备活性组分.[结果]提取实验结果表明具清除自由基活性的物质能较好地被乙酸乙酯提取出来;液相色谱-质谱-活性测定分析表明提取物中活性组分的可能分子式分别为C7H6O4、C8H8O3和C12H14N2O.结合色谱特性、紫外光谱特征、质谱碎片和数据库查询可初步推断它们分别为二羟基苯甲酸、羟基甲基苯甲酸和生物碱类物质,但具体结构还有待于进一步确认.从高效液相色谱和质谱离子流的峰面积可知上述3种活性物质中C12H14N2O的含量最高.本研究成功地用反相制备色谱制备出该天然活性组分的纯品.该3种清除自由基活性物质都是首次发现存在于虫生真菌的代谢物中.     

New sensitive agents for detecting singlet oxygen by electron spin resonance spectroscopy.

Free radicals are well-established transient intermediates in chemical and biological processes. Singlet oxygen, though not a free radical, is also a fairly common reactive chemical species. It is rare that singlet oxygen is studied with the electron spin resonance (ESR) technique in biological systems, because there are few suitable detecting agents. We have recently researched some semiquinone radicals. Specifically, our focus has been on bipyrazole derivatives, which slowly convert to semiquinone radicals in DMSO solution in the presence of potassium tert-butoxide and oxygen. These bipyrazole derivatives are dimers of 3-methyl-1-phenyl-2-pyrazolin-5-one and have anti-ischemic activities and free radical scavenging properties. In this work, we synthesized a new bipyrazole derivative, 4,4'-bis(1p-carboxyphenyl-3-methyl-5-hydroxyl)-pyrazole, DRD156. The resulting semiquinone radical, formed by reaction with singlet oxygen, was characterized by ESR spectroscopy. DRD156 gave no ESR signals from hydroxyl radical, superoxide, and hydrogen peroxide. DRD156, though, gives an ESR response with hypochlorite. This agent, nevertheless, has a much higher ability to detect singlet oxygen than traditional agents with the ESR technique.     

Structural study of cell wall phosphomannan of Candida albicans NIH B-792 (serotype B) strain, with special reference to 1H and 13C NMR analyses of acid-labile oligomannosyl residues

Chemical structures of manno-oligosaccharides, from biose to heptaose, released from the phosphomannan of Candida albicans NIH B-792 strain (serotype B) by mild acid hydrolysis were investigated. The results of 1H NMR, 13C NMR, and fast atom bombardment mass spectrometry analyses confirmed that these manno-oligosaccharides belong to a homologous beta-1,2-linked series. Although chemical shifts of 1H NMR patterns of these oligosaccharides were considerably too complicated to be assigned, their 13C NMR patterns were sufficiently simple to be interpreted, exhibiting a regular increase of downfield shift of ppm values of the C-1 atom from each mannopyranose residue in proportion to their molecular weights. In order to determine the whole chemical structure of the parent phosphomannan, the acid-stable domain was subjected to acetolysis and then enzymolysis with the Arthrobacter GJM-1 alpha-mannosidase and the resultant manno-oligosaccharides were investigated for their chemical structures by 1H NMR spectroscopy. The results of a precipitin-inhibition test using the beta-1,2-linked manno-oligosaccharides, from biose to hexaose, in comparison with the corresponding isomers containing alpha-1,2 linkage with small amounts of alpha-1,3 linkage, indicated that the haptens possessing the former linkage exhibited much higher inhibitory effects than the corresponding isomers containing the latter linkages did. Based on the present findings, a chemical structure of the phosphomannan of this C. albicans strain was proposed.     

Structural analysis of the lipooligosaccharide from the commensal Haemophilus somnus strain 1P

The structure of the lipooligosaccharide (LOS) from the commensal Haemophilus somnus strain 1P was elucidated. The structure of the O-deacylated LOS was established by monosaccharide analysis, NMR spectroscopy and mass spectrometry. The following structure for the O-deacylated LOS was determined on the basis of the combined data from these experiments. [chemical structure: see text] In the structure Kdo is 3-deoxy-D-manno-octulosonic acid, Hep is L-glycero-D-manno-heptose and lipid A-OH refers to O-deacylated Lipid A. The elucidation of this structure has increased our understanding of the relationship between the variability in LOS structure and the pathogenic potential of this organism. Specifically, the inability of this commensal strain to sialylate its LOS suggests that LOS sialylation could be a crucial virulence factor for H. somnus.     

Structural analysis of an acidic, fatty acid ester-bonded extracellular polysaccharide produced by a pristane-assimilating marine bacterium, Rhodococcus erythropolis PR4

Rhodococcus erythropolis PR4 is a marine bacterium that can degrade various alkanes including pristane, a C(19) branched alkane. This strain produces a large quantity of extracellular polysaccharides, which are assumed to play an important role in the hydrocarbon tolerance of this bacterium. The strain produced two acidic extracellular polysaccharides, FR1 and FR2, and the latter showed emulsifying activity toward clove oil, whereas the former did not. FR2 was composed of D-galactose, D-glucose, D-mannose, D-glucuronic acid, and pyruvic acid at a molar ratio of 1:1:1:1:1, and contained 2.9% (w/w) stearic acid and 4.3% (w/w) palmitic acid attached via ester bonds. Therefore, we designated FR2 as a PR4 fatty acid-containing extracellular polysaccharide or FACEPS. The chemical structure of the PR4 FACEPS polysaccharide chain was determined by 1D (1)H and (13)C NMR spectroscopies as well as by 2D DQF-COSY, TOCSY, HMQC, HMBC, and NOESY experiments. The sugar chain of PR4 FACEPS was shown to consist of tetrasaccharide repeating units having the following structure: [structure: see text].     

Glycosyl-sn-1,2-dimyristylphosphatidylinositol is covalently linked to Trypanosoma brucei variant surface glycoprotein

The COOH terminus of the externally disposed variant surface glycoprotein (VSG) of the eukaryotic pathogenic protozoan Trypanosoma brucei strain 427 variant MITat 1.4 (117) is covalently linked to a novel phosphatidylinositol-containing glycolipid. This conclusion is supported by analysis of the products of nitrous acid deamination or Staphylococcus aureus phosphatidylinositol-specific phospholipase C treatment of purified membrane-form VSG. Lysis of trypanosomes is accompanied by release of soluble VSG, catalyzed by activation of an endogenous phospholipase C. The only apparent difference between membrane-form VSG and soluble VSG is the removal of sn-1,2-dimyristylglycerol. The COOH-terminal glycopeptide derived by Pronase digestion of soluble VSG was characterized by chemical modification and digestion with alkaline phosphatase. The results are consistent with the single non-N-acetylated glucosamine residue being the reducing terminus of the oligosaccharide and in a glycosidic linkage to a myo-inositol monophosphate that is probably myo-inositol 1,2-cyclic monophosphate. A partial structure for the VSG COOH-terminal moiety is presented. This structure represents a new type of eukaryotic post-translational protein modification and membrane anchor. We discuss the relevance of this structure to observations that have been made with other eukaryotic membrane proteins.     

Cyclooxygenase reaction mechanism of PGHS--evidence for a reversible transition between a pentadienyl radical and a new tyrosyl radical by nitric oxide trapping

Incubation of prostaglandin H synthase-1 (PGHS-1) under anaerobic conditions with peroxide and arachidonic acid leads to two major radical species: a pentadienyl radical and a radical with a narrow EPR spectrum. The proportions of the two radicals are sensitive to temperature, favoring the narrow radical species at 22 °C. The EPR characteristics of this latter radical are somewhat similar to the previously reported narrow-singlet tyrosine radical NS1a and are insensitive to deuterium labeling of AA. To probe the origin and structure of this radical, we combined EPR analysis with nitric oxide (NO) trapping of tyrosine and substrate derived radicals for both PGHS-1 and -2. Formation of 3-nitrotyrosine in the proteins was analyzed by immunoblotting, whereas NO adducts to AA and AA metabolites were analyzed by mass spectrometry and by chromatography of ¹⁴C-labeled products. The results indicate that both nitrated tyrosine residues and NO-AA adducts formed upon NO trapping. The predominant NO-AA adduct was an oxime at C11 of AA with three conjugated double bonds, as indicated by absorption at 275 nm and by mass spectral analysis. This adduct amounted to 10% and 20% of the heme concentration of PGHS-1 and -2, respectively. For PGHS-1, the yield of NO-AA adduct matched the yield of the narrow radical signal obtained in parallel EPR experiments. High frequency EPR characterization of this narrow radical, reported in an accompanying paper, supports assignment to a new tyrosyl radical, NS1c, rather than an AA-based radical. To reconcile the results from EPR and NO-trapping studies, we propose that NS1c is in equilibrium with an AA pentadienyl radical, and that the latter reacts preferentially with NO.     

Introduction to the Thematic Minireview Series on Radical S-Adenosylmethionine (SAM) Enzymes

In the early days, radical enzyme reactions that use S-adenosylmethionine (SAM) coordinated to an Fe-S cluster, which Perry Frey described as a “poor man''s coenzyme B₁₂,” were believed to be relatively rare chemical curiosities. Today, bioinformatics analyses have revealed the wide prevalence and sheer numbers of radical SAM enzymes, conferring superfamily status. In this thematic minireview series, the JBC presents six articles on radical SAM enzymes that accomplish wide-ranging chemical transformations. We learn that despite the diversity of the reactions catalyzed, family members share some common structural and mechanistic themes. Still in its infancy, continued explorations promise to be fertile grounds for discoveries that will undoubtedly further broaden our understanding of the catalytic repertoire and deepen our understanding of the chemical strategies used by radical SAM enzymes.     

Elucidation of the O-chain structure from the lipopolysaccharide of Agrobacterium tumefaciens strain C58

A linear homopolysaccharide built of 3-alpha-L-6dTalp residues, randomly acetylated at position C-4, is described for the O-specific polysaccharide of Agrobacterium tumefaciens strain C58. This structure, determined by spectroscopical and chemical methods, is strictly correlated to that of Rhizobium loti strain NZP2213, which differs for the degree and the position of O-acetylation.     

Structure of the O-polysaccharide of a serologically separate strain of Proteus mirabilis, TG 332, from a new proposed Proteus serogroup O50

The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Proteus mirabilis TG 332 strain. The following structure of the O-polysaccharide was determined by chemical methods along with NMR spectroscopy, including 2D COSY, TOCSY, ROESY and 1H, 13C HMQC experiments: [see equation in text]. The O-polysaccharide studied has a unique structure among Proteus O-antigens. Accordingly, P. mirabilis TG 332 is serologically separate, and we propose to classify this strain into a new Proteus serogroup, O50. The nature of minor epitopes that provide a cross-reactivity of P. mirabilis TG 332 O-antiserum with the LPS of P. mirabilis O30 and Proteus penneri 34 (O60) is discussed.     

Studies on the hexose region of the lipopolysaccharide from a low virulence strain of Salmonella typhimurium

The structure of the hexose region of the lipopolysaccharide from M206 strain, a mutant of Salmonella typhimurium having reduced virulence, was partially determined. Immunological tests indicated cross-reactions of anti-(M206) antiserum with wild-type C5 and Ra mutant strains. Data obtained on chemical composition, periodate oxidation, acetolysis, methylation and analysis by gas chromatography/mass spectrometry show that M206 type lipopolysaccharide contains the common core polysaccharide of Salmonella which was substituted in position 4 of the subterminal glucose unit by a disaccharide: D-glucosyl 1----3 D-galactose. This substitution is probably related to the slight virulence of M206 strain.     

丹参醌化合物结构特征与抗氧化活性

对双氢丹参醌I和丹参醌Ⅱ在油脂中的抗氧化作用及构效关系进行了量子化学研究。结果表明 ,抗氧化剂与油脂中碳中心自由基 (R· )复合成稳定自由基 (AH -R· ) ,可能是丹参醌抑制或阻止油脂氧化酸败的主要途径 ,双氢丹参醌I也可借助释放活泼H(本身转化成稳定自由基A· )有效清除R·。丹参醌分子本身的化学活性和对应的AH -R·与A·的稳定性对此类抗氧化作用有重要影响。AH -R·和A·中自旋集居分布 ,对比较抗氧化剂的相对活性可能是一个有价值的电子结构参数。     

15N and 13C NMR studies of ligands bound to the 280,000-dalton protein porphobilinogen synthase elucidate the structures of enzyme-bound product and a Schiff base intermediate

Porphobilinogen synthase (PBGS) catalyzes the asymmetric condensation of two molecules of 5-aminolevulinic acid (ALA). Despite the 280,000-dalton size of PBGS, much can be learned about the reaction mechanism through 13C and 15N NMR. To our knowledge, these studies represent the largest protein complex for which individual nuclei have been characterized by 13C or 15N NMR. Here we extend our 13C NMR studies to PBGS complexes with [3,3-2H2,3-13C]ALA and report 15N NMR studies of [15N]ALA bound to PBGS. As in our previous 13C NMR studies, observation of enzyme-bound 15N-labeled species was facilitated by deuteration at nitrogens that are attached to slowly exchanging hydrogens. For holo-PBGS at neutral pH, the NMR spectra reflect the structure of the enzyme-bound product porphobilinogen (PBG), whose chemical shifts are uniformly consistent with deprotonation of the amino group whose solution pKa is 11. Despite this local environment, the protons of the amino group are in rapid exchange with solvent (kexchange greater than 10(2) s-1). For methyl methanethiosulfonate (MMTS) modified PBGS, the NMR spectra reflect the chemistry of an enzyme-bound Schiff base intermediate that is formed between C4 of ALA and an active-site lysine. The 13C chemical shift of [3,3-2H2,3-13C]ALA confirms that the Schiff base is an imine of E stereochemistry. By comparison to model imines formed between [15N]ALA and hydrazine or hydroxylamine, the 15N chemical shift of the enzyme-bound Schiff base suggests that the free amino group is an environment resembling partial deprotonation; again the protons are in rapid exchange with solvent. Deprotonation of the amino group would facilitate formation of a Schiff base between the amino group of the enzyme-bound Schiff base and C4 of the second ALA substrate. This is the first evidence supporting carbon-nitrogen bond formation as the initial site of interaction between the two substrate molecules.