基于噁唑烷酮的不对称催化合成构建多官能化手性氨基酸类化合物

多官能化手性氨基酸及其衍生物是一类重要的手性药物以及合成手性药的关键中间体,如现在大量用于临床的左甲状腺素、赖诺普利、阿莫西林、缬沙坦、头孢氨苄以及青霉素等。进行多官能化手性氨基酸类化合物的不对称催化合成,可为新型化学药的设计与发现开辟新的视野。噁唑烷酮(Azlactone)被证明是合成四取代氨基酸衍生物的优秀底物。可通过不对称催化手段向其中引入需要的基团,再经多取代的噁唑烷酮直接开环得到一系列的目标化合物。本文主要综述了近年来基于恶唑烷酮的不对称催化反应构建四取代氨基酸类化合物的研究。     

Quantitative analysis of the oxidative DNA lesion, 2,2-diamino-4-(2-deoxy-beta-D-erythro-pentofuranosyl)amino]-5(2H)-oxazolone (oxazolone), in vitro and in vivo by isotope dilution-capillary HPLC-ESI-MS/MS

A major DNA oxidation product, 2,2-diamino-4-[(2-deoxy-beta-D-erythro-pentofuranosyl)amino]-5(2H)-oxazolone (oxazolone), can be generated either directly by oxidation of dG or as a secondary oxidation product with an intermediate of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG). Site-specific mutagenesis studies indicate that oxazolone is a strongly mispairing lesion, inducing approximately 10-fold more mutations than 8-oxo-dG. While 8-oxo-dG undergoes facile further oxidation, oxazolone appears to be a stable final product of guanine oxidation, and, if formed in vivo, can potentially serve as a biomarker of DNA damage induced by oxidative stress. In this study, capillary liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) methods were developed to enable quantitative analysis of both 8-oxo-dG and oxazolone in DNA from biological sources. Sensitive and specific detection of 8-oxo-dG and oxazolone in enzymatic DNA hydrolysates was achieved by isotope dilution with the corresponding 15N-labeled internal standards. Both nucleobase adducts were formed in a dose-dependent manner in calf thymus DNA subjected to photooxidation in the presence of riboflavin. While the amounts of oxazolone continued to increase with the duration of irradiation, those of 8-oxo-dG reached a maximum at 20 min, suggesting that 8-oxo-dG is converted to secondary oxidation products. Both lesions were found in rat liver DNA isolated under carefully monitored conditions to minimize artifactual oxidation. Liver DNA of diabetic and control rats maintained on a diet high in animal fat contained 2-6 molecules of oxazolone per 10(7) guanines, while 8-oxo-dG amounts in the same samples were between 3 and 8 adducts per 10(6) guanines. The formation of oxazolone lesions in rat liver DNA, their relative stability in the presence of oxidants and their potent mispairing characteristics suggest that oxazolone may play a role in oxidative stress-mediated mutagenesis.     

Synthesis,biological evaluation and structure-activity relationships of 5-arylidene tetramic acids with antibacterial activity against methicillin-resistant Staphylococcus aureus

The steady rise of the antimicrobial resistance is a major global threat to human health that requires the urgent need for novel antibiotics. In this work we report the synthesis of a small library of 3-subsituted-5-arylidene tetramic acids in order to investigate the scope of our previously established methodology via an intermediate oxazolone and their antimicrobial activity. From this series of 14 tetramic acids, 11 derivatives are novel and one of them is a Schiff base, which was structurally characterized with single-crystal X-ray analysis and NMR spectroscopy. The compounds incorporating a lipophilic acyl group at carbon-3 of the ring showed moderate to high activity with minimum inhibitory activity of 4–32 μg/mL against methicillin-resistant Staphylococcus aureus (MRSA), accompanied by no human cell toxicity and hemolytic activity within the tested concentration range. The substituent at para position of the aryl ring seemed to have no or little effect on the antimicrobial activity of these compounds.     

Partitioning of 14C-Photosynthate of Leaves in Roots,Rhizome, and in Essential Oil and Curcumin in Turmeric (Curcuma longa L.)

Incorporation of photosynthetically fixed ¹⁴C was studied at different time intervals of 12, 24, and 36 h in various plant parts—leaf 1 to 4 from apex, roots, and rhizome—into primary metabolites—sugars, amino acids, and organic acids, and secondary metabolites—essential oil and curcumin—in turmeric. The youngest leaves were most active in fixing ¹⁴C at 24 h. Fixation capacity into primary metabolites decreased with leaf position and time. The primary metabolite levels in leaves were maximal in sugars and organic acids and lowest in amino acids. Roots as well as rhizome received maximum photoassimilate from leaves at 24 h; this declined with time. The maximum metabolite concentrations in the roots and rhizome were high in sugars and organic acids and least in amino acids. ¹⁴C incorporation into oil in leaf and into curcumin in rhizome was maximal at 24 h and declined with time. These studies highlight importance of time-dependent translocation of ¹⁴C-primary metabolites from leaves to roots and rhizome and their subsequent biosynthesis into secondary metabolite, curcumin, in rhizome. This might be one of factors regulating the secondary metabolite accumulation and rhizome development.     

Influence of environmental conditions on the production of phenazine-1-carboxamide by Pseudomonas chlororaphis PCL1391

Pseudomonas chlororaphis PCL1391 produces the secondary metabolite phenazine-1-carboxamide (PCN), which is an antifungal metabolite required for biocontrol activity of the strain. Identification of conditions involved in PCN production showed that some carbon sources and all amino acids tested promote PCN levels. Decreasing the pH from 7 to 6 or decreasing the growth temperature from 21 to 16 degrees C decreased PCN production dramatically. In contrast, growth at 1% oxygen as well as low magnesium concentrations increased PCN levels. Salt stress, low concentrations of ferric iron, phosphate, sulfate, and ammonium ions reduced PCN levels. Fusaric acid, a secondary metabolite produced by the soilborne Fusarium spp. fungi, also reduced PCN levels. Different nitrogen sources greatly influenced PCN levels. Analysis of autoinducer levels at conditions of high and low PCN production demonstrated that, under all tested conditions, PCN levels correlate with autoinducer levels, indicating that the regulation of PCN production by environmental factors takes place at or before autoinducer production. Moreover, the results show that autoinducer production not only is induced by a high optical density but also can be induced by certain environmental conditions. We discuss our findings in relation to the success of biocontrol in the field.     

Identification of the degradation products of aspergillic acid by Trichoderma koningii M 102

Isolation and identification of the degradation products of aspergillic acid, a secondary metabolite of fungi, by Trichoderma koningii M 102 were undertaken. ¹⁴C-labeling experiments indicated that aspergillic acid was broken-down to a water-soluble degradation product and a chloroform-soluble one. Consequently, leucine and a new microbial metabolite, 2-hydroxyimino-3-methyl-1-pentanol, were isolated and identified as the degradation products formed by cleavage of the pyrazine ring of aspergillic acid by T. koningii M 102.     

A comparison of methanobactins from Methylosinus trichosporium OB3b and Methylocystis strain Sb2 predicts methanobactins are synthesized from diverse peptide precursors modified to create a common core for binding and reducing copper ions

Methanobactins (mb) are low-molecular mass, copper-binding molecules secreted by most methanotrophic bacteria. These molecules have been identified for a number of methanotrophs, but only the one produced by Methylosinus trichosporium OB3b (mb-OB3b) has to date been chemically characterized. Here we report the chemical characterization and copper binding properties of a second methanobactin, which is produced by Methylocystis strain SB2 (mb-SB2). mb-SB2 shows some significant similarities to mb-OB3b, including its spectral and metal binding properties, and its ability to bind and reduce Cu(II) to Cu(I). Like mb-OB3b, mb-SB2 contains two five-member heterocyclic rings with associated enethiol groups, which together form the copper ion binding site. mb-SB2 also displays some significant differences compared to mb-OB3b, including the number and types of amino acids used to complete the structure of the molecule, the presence of an imidazolone ring in place of one of the oxazolone rings found in mb-OB3b, and the presence of a sulfate group not found in mb-OB3b. The sulfate is bonded to a threonine-like side chain that is associated with one of the heterocyclic rings and may represent the first example of this type of sulfate group found in a bacterially derived peptide. Acid-catalyzed hydrolysis and decarboxylation of the oxazolone rings found in mb-OB3b and mb-SB2 produce pairs of amino acid residues and suggest that both mb-OB3b and mb-SB2 are derived from peptides. In support of this, the gene for a ribosomally produced peptide precursor for mb-OB3b has been identified in the genome of M. trichosporium OB3b. The gene sequence indicates that the oxazolone rings in mb-OB3b are derived from the combination of a cysteine residue and the carbonyl from the preceding residue in the peptide sequence. Taken together, the results suggest methanobactins make up a structurally diverse group of ribosomally produced, peptide-derived molecules, which share a common pair of five-member rings with associated enethiol groups that are able to bind, reduce, and stabilize copper ions in an aqueous environment.     

Mining novel biosynthetic machineries of secondary metabolites from actinobacteria

ABSTRACT

Secondary metabolites produced by actinobacteria have diverse structures and important biological activities, making them a useful source of drug development. Diversity of the secondary metabolites indicates that the actinobacteria exploit various chemical reactions to construct a structural diversity. Thus, studying the biosynthetic machinery of these metabolites should result in discovery of various enzymes catalyzing interesting and useful reactions. This review summarizes our recent studies on the biosynthesis of secondary metabolites from actinobacteria, including the biosynthesis of nonproteinogenic amino acids used as building blocks of nonribosomal peptides, the type II polyketide synthase catalyzing polyene scaffold, the nitrous acid biosynthetic pathway involved in secondary metabolite biosynthesis and unique cytochrome P450 catalyzing nitrene transfer. These findings expand the knowledge of secondary metabolite biosynthesis machinery and provide useful tools for future bioengineering.     

Secondary metabolite biosynthesis: the first century

An account of work on the biosynthesis of secondary metabolites up to 1965 is presented. The earliest suggestions for three of the four major pathways were speculative; for the isoprene rule, hypotheses date to 1877, for the polyketide rule to 1907, and for a role for amino acids in alkaloid biosynthesis to 1910. The fourth major pathway based on intermediates of the shikimic acid pathway has a much shorter history because shikimic acid itself was only identified as a primary metabolite in 1951. In addition to speculation, biomimetic syntheses were carried out in which chemists attempted to duplicate possible biosynthetic pathways in vitro. The classic example was Robinson's synthesis of tropinone in 1917. Direct examination of secondary metabolite biosynthesis was possible with the use of the isotopic tracer technique. This methodology, applied extensively to primary metabolism beginning in 1935 and to secondary metabolism from about 1950, was facilitated by the increasing availability of the 14C isotope. With the use of isotopes as tracers, the broad outlines of secondary metabolite biosynthesis, reviewed here, were established in the period 1950 to 1965.     

13C nuclear magnetic resonance analysis of glucose and citrate end products in an ldhL-ldhD double-knockout strain of Lactobacillus plantarum.

We have examined the metabolic consequences of knocking out the two ldh genes in Lactobacillus plantarum using 13C nuclear magnetic resonance. Unlike its wild-type isogenic progenitor, which produced lactate as the major metabolite under all conditions tested, ldh null strain TF103 mainly produced acetoin. A variety of secondary end products were also found, including organic acids (acetate, succinate, pyruvate, and lactate), ethanol, 2,3-butanediol, and mannitol.     

Microbiological degradation of bile acids. Ring a cleavage and 7α,12α-dehydroxylation of cholic acid by Arthrobacter simplex

The metabolism of cholic acid by Arthrobacter simplex was investigated. This organism effected both ring a cleavage and elimination of the hydroxyl groups at C-7 and C-12 and gave a new metabolite, (4R)-4-[4alpha-(2-carboxyethyl)-3aalpha-hexahydro-7abeta-methyl-5-oxoindan-1beta-yl]valeric acid, which was isolated and identified through its partial synthesis. A degradative pathway of cholic acid into this metabolite is tentatively proposed, and the possibility that the proposed pathway could be extended to the cholic acid degradation by other microorganisms besides A. simplex is discussed. The possibility that the observed reactions in vitro could occur during the metabolism of bile acids in vivo is considered.     

Racemization in the Coupling Reaction with the Several Methods beside the Activated Ester Methods

The degree of racemization in the coupling reaction, Pro-Val + Pro, with the several other methods than the activated ester methods was measured and the results were compared with that in the coupling reaction, Leu-Phe + Val, as well as in the previous paper.¹⁾ In the azide and the mixed anhydride methods, no or almost no racemization was detected, whereas in the other tested methods of peptide synthesis (Pachornik-, DCCD- and phosphazo-methods) the significantly large racemization was observed. It can be attributed to the strong nucleo- philic N-atom in the penultimate amino acids (Pro) and the steric hindrance of C-terminal amino acid (Val), which are favourable to the formation of the oxazolone ring.

This assumption was further systematically confirmed in the synthesis of the other several tripeptides with the DCCD method. The separation of the diastereoisomers (LLL and LDL) of the resulting tripeptides by gas chromatography with a packed column was also here reported.     

Metabolic profiles of sunflower genotypes with contrasting response to Sclerotinia sclerotiorum infection

We report a comprehensive primary metabolite profiling of sunflower (Helianthus annuus) genotypes displaying contrasting behavior to Sclerotinia sclerotiorum infection. Applying a GC-MS-based metabolite profiling approach, we were able to identify differential patterns involving a total of 63 metabolites including major and minor sugars and sugar alcohols, organic acids, amino acids, fatty acids and few soluble secondary metabolites in the sunflower capitulum, the main target organ of pathogen attack. Metabolic changes and disease incidence of the two contrasting genotypes were determined throughout the main infection period (R5.2-R6). Both point-by-point and non-parametric statistical analyses showed metabolic differences between genotypes as well as interaction effects between genotype and time after inoculation. Network correlation analyses suggested that these metabolic changes were synchronized in a time-dependent manner in response to the pathogen. Concerted differential metabolic changes were detected to a higher extent in the susceptible, rather than the resistant genotype, thereby allowing differentiation of modules composed by intermediates of the same pathway which are highly interconnected in the susceptible line but not in the resistant one. Evaluation of these data also demonstrated a genotype specific regulation of distinct metabolic pathways, suggesting the importance of detection of metabolic patterns rather than specific metabolite changes when looking for metabolic markers differentially responding to pathogen infection. In summary, the GC-MS strategy developed in this study was suitable for detection of differences in carbon primary metabolism in sunflower capitulum, a tissue which is the main entry point for this and other pathogens which cause great detrimental impact on crop yield.     

Thermodynamic and solution state NMR characterization of the binding of secondary and conjugated bile acids to STARD5

STARD5 is a member of the STARD4 sub-family of START domain containing proteins specialized in the non-vesicular transport of lipids and sterols. We recently reported that STARD5 binds primary bile acids. Herein, we report on the biophysical and structural characterization of the binding of secondary and conjugated bile acids by STARD5 at physiological concentrations. We found that the absence of the 7α-OH group and its epimerization increase the affinity of secondary bile acids for STARD5. According to NMR titration and molecular modeling, the affinity depends mainly on the number and positions of the steroid ring hydroxyl groups and to a lesser extent on the presence or type of bile acid side-chain conjugation. Primary and secondary bile acids have different binding modes and display different positioning within the STARD5 binding pocket. The relative STARD5 affinity for the different bile acids studied is: DCA > LCA > CDCA > GDCA > TDCA > CA > UDCA. TCA and GCA do not bind significantly to STARD5. The impact of the ligand chemical structure on the thermodynamics of binding is discussed. The discovery of these new ligands suggests that STARD5 is involved in the cellular response elicited by bile acids and offers many entry points to decipher its physiological role.     

Evolution of the biphenyl dioxygenase BphA from Burkholderia xenovorans LB400 by random mutagenesis of multiple sites in region III

It is now established that several amino acids of region III of the biphenyl dioxygenase (BPDO) alpha subunit are involved in substrate recognition and regiospecificity toward chlorobiphenyls. However, the sequence pattern of the amino acids of that segment of seven amino acids located in the C-terminal portion of the alpha subunit is rather limited in BPDOs of natural occurrence. In this work, we have randomly mutated simultaneously four residues (Thr(335)-Phe(336)-Ile(338)-Ile(341)) of region III of Burkholderia xenovorans LB400 BphA. The library was screened for variants able to oxygenate 2,2'-dichlorobiphenyl (2,2'-CB). Replacement of Phe(336) with Met or Ile with a concomitant change of Thr(335) to Ala created new variants that transformed 2,2'-CB into 3,4-dihydro-3,4-dihydroxy-2,2'-dichlorobiphenyl, which is a dead end metabolite that was not cleaved by BphC. Replacement of Thr(335)-Phe(336) with Ala(335)-Leu(336) did not cause this type of phenotypic change. Regiospecificity toward congeners other than 2,2'-CB that were oxygenated more efficiently by variant Ala(335)-Met(336) than by LB400 BPDO was similar for both enzymes. Thus structural changes that altered the regiospecificity toward 2,2'-CB did not affect the metabolite profile of other congeners, although it affected the rate of conversion of these congeners. It was especially noteworthy that both LB400 BPDO and the Ala(335)-Met(336) variant generated 2,3-dihydroxy-2',4,4'-trichlorobiphenyl as the sole metabolite from 2,4,2',4'-CB and 4,5-dihydro-4,5-dihydroxy-2,3,2',3'-tetrachlorobiphenyl as the major metabolite from 2,3,2',3'-CB. This shows that 2,4,2',4'-CB is oxygenated principally onto vicinal ortho-meta carbons 2 and 3 and that 2,3,2',3'-CB is oxygenated onto meta-para carbons 4 and 5 by both enzymes. The data suggest that interactions between the chlorine substitutes on the phenyl ring and specific amino acid residues of the protein influence the orientation of the phenyl ring inside the catalytic pocket.     

Altered profile of secondary metabolites in the root exudates of Arabidopsis ATP-binding cassette transporter mutants

Following recent indirect evidence suggesting a role for ATP-binding cassette (ABC) transporters in root exudation of phytochemicals, we identified 25 ABC transporter genes highly expressed in the root cells most likely to be involved in secretion processes. Of these 25 genes, we also selected six full-length ABC transporters and a half-size transporter for in-depth molecular and biochemical analyses. We compared the exuded root phytochemical profiles of these seven ABC transporter mutants to those of the wild type. There were three nonpolar phytochemicals missing in various ABC transporter mutants compared to the wild type when the samples were analyzed by high-performance liquid chromatography-mass spectrometry. These data suggest that more than one ABC transporter can be involved in the secretion of a given phytochemical and that a transporter can be involved in the secretion of more than one secondary metabolite. The primary and secondary metabolites present in the root exudates of the mutants were also analyzed by gas chromatography-mass spectrometry, which allowed for the identification of groups of compounds differentially found in some of the mutants compared to the wild type. For instance, the mutant Atpdr6 secreted a lower level of organic acids and Atmrp2 secreted a higher level of amino acids as compared to the wild type. We conclude that the release of phytochemicals by roots is partially controlled by ABC transporters.     

Difference in conformational diversity between nucleic acids with a six-membered 'sugar' unit and natural 'furanose' nucleic acids

Natural nucleic acids duplexes formed by Watson-Crick base pairing fold into right-handed helices that are classified in two families of secondary structures, i.e. the A- and B-form. For a long time, these A and B allomorphic nucleic acids have been considered as the 'non plus ultra' of double-stranded nucleic acids geometries with the only exception of Z-DNA, a left-handed helix that can be adopted by some DNA sequences. The five-membered furanose ring in the sugar-phosphate backbone of DNA and RNA is the underlying cause of this restriction in conformational diversity. A collection of new Watson-Crick duplexes have joined the 'original' nucleic acid double helixes at the moment the furanose sugar was replaced by different types of six-membered ring systems. The increase in this structural and conformational diversity originates from the rigid chair conformation of a saturated six-membered ring that determines the orientation of the ring substituents with respect to each other. The original A- and B-form oligonucleotide duplexes have expanded into a whole family of new structures with the potential for selective cross-communication in a parallel or antiparallel orientation, opening up a new world for information storage and for molecular recognition-directed self-organization.     

Involvement of the cannabinoid CB2 receptor and its endogenous ligand 2-arachidonoylglycerol in oxazolone-induced contact dermatitis in mice

The possible involvement of 2-arachidonoylglycerol (2-AG), an endogenous ligand for the cannabinoid receptors (CB1 and CB2), in contact dermatitis in mouse ear was investigated. We found that the level of 2-AG was markedly elevated in the ear following a challenge with oxazolone in sensitized mice. Of note, the swelling following the challenge was suppressed by either the administration of SR144528, a CB2 receptor antagonist, immediately after sensitization, or the administration of SR144528 upon the challenge. The effect of AM251, a CB1 receptor antagonist, was marginal in either case. It seems apparent, therefore, that the CB2 receptor and its endogenous ligand 2-AG are closely involved in both the sensitization phase and the elicitation phase of oxazolone-induced contact dermatitis. In line with this, we found that Langerhans cells (MHC class II(+)) contain a substantial amount of CB2 receptor mRNA, whereas keratinocytes (MHC class II(-)) do not. We also obtained evidence that the expression of mRNAs for proinflammatory cytokines following a challenge with oxazolone was markedly suppressed by treatment with SR144528. We next examined whether the CB2 receptor and 2-AG participate in chronic contact dermatitis accompanied by the infiltration of tissues by eosinophils. The amount of 2-AG in mouse ear dramatically increased following repeated challenge with oxazolone. Importantly, treatment with SR144528 attenuated both the recruitment of eosinophils and ear swelling in chronic contact dermatitis induced by repeated challenge with oxazolone. These results strongly suggest that the CB2 receptor and 2-AG play important stimulative roles in the sensitization, elicitation, and exacerbation of allergic inflammation.