Monomethyl Ester Research Articles

Spontaneous reaction of malonyl peroxides with methanol

The spontaneous reaction of disubstituted malonyl peroxides (MPOs) with methanol affording monopermalonic acid monomethyl esters is fast (minutes) for lower homologues but is sharply decelerated (days) for the higher ones. Spirocyclopropyl-MPO is an exception in which the nucleophilic opening of the spiroactivated cyclopropane ring leads to 2,4-dimethoxy-2-carboxybutanoic acid.

Overexpression of an EaZIP gene devoid of transit peptide sequence induced leaf variegation in tobacco.

Leaf variegation is an ornamental trait that is not only biologically but also economically important. In our previous study, a Mg-protoporphyrin IX monomethyl ester cyclase homologue, EaZIP (Epipremnum aureum leucine zipper) was found to be associated with leaf variegation in Epipremnum aureum (Linden & Andre) G.S. Bunting. The protein product of this nuclear-encoded gene is targeted back to chloroplast involving in chlorophyll biosynthesis. Based on a web-based homology analysis, the EaZIP was found to lack a chloroplast transit peptide (cTP) sequence. In the present study, we tested if overexpression of the EaZIP cDNA with or without the cTP sequence could affect leaf variegation. Transgenic tobacco plants overexpressing EaZIP genes with (EaZIPwcTP) and without (EaZIPwocTP) cTP sequence were generated. Many plant lines harboring EaZIPwocTP showed variegated leaves, while none of the plant lines with EaZIPwcTP produced such a phenotype. Molecular analysis of T0 plants and selfed T1 progeny, as well as observations of tagged marker GFP (green fluorescent protein) did not show any other difference in patterns of gene integrity and expression. Results from this study indicate that transgenic approach for expressing EaZIPwocTP could be a novel method of generating variegated plants even through the underlying mechanisms remain to be elucidated.

Mediating gold nanoparticle growth in nanoreactors: Role of template-metal interactions and external energy

The unique properties of gold at the nanoscale have favored application in a variety of fields from catalysis to targeted drug delivery; moreover, the reactive nature of gold nanoparticles allows its use as an active center for nanoreactors. This study presents the controlled synthesis of unsupported nanoreactors with self-assembled polymers as template and gold incorporated as active centers. Gold (I) chloride was found to react with the hydrophilic domain of the template and produce metal gold sheets of 30–50 nm in size, as well as with the hydrophobic cavity to produce metal gold clusters, 2–3 nm in size. The size of gold nanostructures was characterized by High Resolution TEM, while the interaction between gold and polymer was confirmed by molecular modelling. The careful spectroscopic characterization including UV–Vis and IR allowed to differentiate clusters and sheets and to follow the growth of the gold particles. The control of the environment surrounding the active center (hydrophilic versus hydrophobic domains) is essential for the development of tailored nanoreactors. The control of the interaction between the polymer and gold was obtained by modifying the nature of the polymer and external energy. It was found that the presence of monomethyl ester in the polymer intensifies the interaction of gold on the hydrophilic domain while producing larger gold sheets (both hpc and fcc) and slightly smaller gold clusters. Sonication was found to disrupt the whole self-assembly process and completely eliminate the interaction of gold with the hydrophobic cavity producing exclusively atomically thin layers of hpc gold. The ability to control the interaction of gold with the hydrophobic or hydrophilic cavity offers tremendous potential for many applications from drug delivery to nanocatalysis in hydrophilic and hydrophobic confinement.

Effects of Light-Emitting Diode Irradiation on Growth Characteristics and Regulation of Porphyrin Biosynthesis in Rice Seedlings.

We examined the effects of light quality on growth characteristics and porphyrin biosynthesis of rice seedlings grown under different wavelengths from light emitting diodes (LEDs). After 10 days of exposure to various wavelengths of LEDs, leaf area and shoot biomass were greater in seedlings grown under white and blue LEDs than those of green and red LEDs. Both green and red LED treatments drastically decreased levels of protoporphyrin IX (Proto IX) and Mg-porphyrins compared to those of white LED, while levels of Mg-Proto IX monomethyl ester and protochlorophyllide under blue LED were decreased by 21% and 49%, respectively. Transcript levels of PPO1 were greatly upregulated in seedlings grown under red LED compared to white LED, whereas transcript levels of HO2 and CHLD were upregulated under blue LED. Overall, most porphyrin biosynthetic genes in the Fe-porphyrin branch remained almost constant or upregulated, while most genes in the Mg-porphyrin branch were downregulated. Expression levels of nuclear-encoded photosynthetic genes Lhcb and RbcS noticeably decreased after exposure to blue and red LEDs, compared to white LED. Our study suggests that specific wavelengths of LED greatly influence characteristics of growth in plants partly through altering the metabolic regulation of the porphyrin biosynthetic pathway, and possibly contribute to affect retrograde signaling.

Methylation of mono- and dicarboxylic acids with dimethyl carbonate catalyzed with binder-free zeolite NaY

Synthesis of methyl mono- and dicarboxylates was developed consisting in treating the corresponding acids with dimethyl carbonate in the presence of a heterogenic catalyst, crystalline aluminosilicate whose mechanically strong granules to 90–95% were built of crystal aggregates of zeolite Y with modulus of about 5.0 in the Na-form. Optimum catalyst and reagents ratio and the reaction conditions were found for the preparation in high yields of methyl esters of mono- and dicarboxylic acids.

Mutation in Mg-Protoporphyrin IX Monomethyl Ester Cyclase Decreases Photosynthesis Capacity in Rice.

In photosynthesis, the pigments chlorophyll a/b absorb light energy to convert to chemical energy in chloroplasts. Though most enzymes of chlorophyll biosynthesis from glutamyl-tRNA to chlorophyll a/b have been identified, the exact composition and regulation of the multimeric enzyme Mg-protoporphyrin IX monomethyl ester cyclase (MPEC) is largely unknown. In this study, we isolated a rice pale-green leaf mutant m167 with yellow-green leaf phenotype across the whole lifespan. Chlorophyll content decreases 43–51% and the granal stacks of chloroplasts becomes thinner in m167. Chlorophyll fluorescence parameters, including Fv/Fm (the maximum quantum efficiency of PSII) and quantum yield of PSII (Y(II)), were lower in m167 than those in wild type plants (WT), and photosynthesis rate decreases 40% in leaves of m167 mutant compared with WT plants, which lead to yield reduction in m167. Genetic analysis revealed that yellow-green leaf phenotype of m167 is controlled by a single recessive genetic locus. By positional cloning, a single mutated locus, G286A (Alanine 96 to Threonine in protein), was found in the coding sequence of LOC_Os01g17170 (Rice Copper Response Defect 1, OsCRD1), encoding a putative subunit of MPEC. Expression profile analysis demonstrated that OsCRD1 is mainly expressed in green tissues of rice. Sequence alignment analysis of CRD1 indicated that Alanine 96 is very conserved in all green plants and photosynthetic bacteria. OsCRD1 protein mainly locates in chloroplast and the point mutation A96T in OsCRD1 does not change its location. Therefore, Alanine96 of OsCRD1 might be fundamental for MPEC activity, mutation of which leads to deficiency in chlorophyll biosynthesis and chloroplast development and decreases photosynthetic capacity in rice.

Process Development of the Soft Histone Deacetylate Enzyme Inhibitor SHP-141: Acylation of Methyl Paraben and Suberyl Hydroxamic Acid Formation

SHP-141 (1) is a hydroxamic acid-based inhibitor of histone deacetylase enzymes which is under development for the treatment of cutaneous T-cell lymphoma. The original synthesis of 1 involved five synthetic steps beginning with suberic acid monomethyl ester. Final deprotection of the O-benzyl hydroxamate moiety using hydrogen and palladium catalyst mandated the use of metal scavengers to reduce palladium levels to within International Council for Harmonisation (ICH) guidance. Owing to the sensitivity of 1 toward self-condensation and the potential for N–O bond cleavage under hydrogenolytic conditions, we developed an alternative route to 1 which avoids Pd-mediated hydrogenation and prolonged metal scavenger treatment. This two-step process employs readily available suberic acid and methyl paraben and has successfully delivered multiple kilograms of 1 for clinical use. Importantly, crude 1 was stabilized for recrystallization in acetonitrile (ACN) solution by the addition of 0.1% citric acid and 4% water. ...

The catalytic subunit of magnesium-protoporphyrin IX monomethyl ester cyclase forms a chloroplast complex to regulate chlorophyll biosynthesis in rice.

YGL8 has the dual functions in Chl biosynthesis: one as a catalytic subunit of MgPME cyclase, the other as a core component of FLU-YGL8-LCAA-POR complex in Chl biosynthesis. Magnesium-protoporphyrin IX monomethyl ester (MgPME) cyclase is an essential enzyme involved in chlorophyll (Chl) biosynthesis. However, its roles in regulating Chl biosynthesis are not fully explored. In this study, we isolated a rice mutant yellow-green leaf 8 (ygl8) that exhibited chlorosis phenotype with abnormal chloroplast development in young leaves. As the development of leaves, the chlorotic plants turned green accompanied by restorations in Chl content and chloroplast ultrastructure. Map-based cloning revealed that the ygl8 gene encodes a catalytic subunit of MgPME cyclase. The ygl8 mutation caused a conserved amino acid substitution (Asn182Ser), which was related to the alterations of Chl precursor content. YGL8 was constitutively expressed in various tissues, with more abundance in young leaves and panicles. Furthermore, we showed that expression levels of some nuclear genes associated with Chl biosynthesis were affected in both the ygl8 mutant and YGL8 RNA interference lines. By transient expression in rice protoplasts, we found that N-terminal 40 amino acid residues were enough to localize the YGL8 protein to chloroplast. In vivo experiments demonstrated a physical interaction between YGL8 and a rice chloroplast protein, low chlorophyll accumulation A (OsLCAA). Moreover, bimolecular fluorescence complementation assays revealed that YGL8 also interacted with the other two rice chloroplast proteins, viz. fluorescent (OsFLU1) and NADPH:protochlorophyllide oxidoreductase (OsPORB). These results provide new insights into the roles of YGL8, not only as a subunit with catalytic activity, but as a core component of FLU-YGL8-LCAA-POR complex required for Chl biosynthesis.

Chemoselective Protection of Glutathione in the Preparation of Bioconjugates: The Case of Trypanothione Disulfide.

A novel synthetic route to the chemoselectively protected N,S-ditritylglutathione monomethyl ester is described involving the chemical modification of the commercially available glutathione (GSH). The synthetic value of this building block in the facile preparation of GSH bioconjugates in a satisfying overall yield was exemplified by the case of trypanothione disulfide (TS2), a GSH-spermidine bioconjugate, involved in the antioxidative stress protection system of parasitic protozoa, such as trypanosoma and leishmania parasites.

Metagenomic evidence for the presence of phototrophic Gemmatimonadetes bacteria in diverse environments

Gemmatimonadetes represents a poorly understood bacterial phylum with only a handful of cultured species. Recently, one of its few representatives, Gemmatimonas phototrophica, was found to contain purple bacterial photosynthetic reaction centres. However, almost nothing is known about the environmental distribution of phototrophic Gemmatimonadetes bacteria. To fill this gap, we took advantage of fast-growing public metagenomic databases and performed an extensive survey of metagenomes deposited into the NCBI's WGS database, the JGI's IMG webserver and the MG-RAST webserver. By employing Mg protoporphyrin IX monomethyl ester oxidative cyclase (AcsF) as a marker gene, we identified 291 AcsF fragments (24-361 amino acids long) that are closely related to G. phototrophica from 161 metagenomes originating from various habitats, including air, river waters/sediment, estuarine waters, lake waters, biofilms, plant surfaces, intertidal sediment, soils, springs and wastewater treatment plants, but none from marine waters or sediment. Based on AcsF hit counts, phototrophic Gemmatimonadetes bacteria make up 0.4-11.9% of whole phototrophic microbial communities in these habitats. Unexpectedly, an almost complete 37.9 kb long photosynthesis gene cluster with identical gene composition and arrangement to those in G. phototrophica was reconstructed from the Odense wastewater metagenome, only differing in a 7.2 kb long non-photosynthesis-gene insert. These data suggest that phototrophic Gemmatimonadetes bacteria are much more widely distributed in the environment and exhibit a higher genetic diversity than previously thought.

Synthesis of [3-13C]-2,3-dihydroxy-4-methoxybenzaldehyde

An efficient synthesis of [3-13C]-2,3-dihydroxy-4-methoxybenzaldehyde, an isotopically labelled probe of a common intermediate used in the synthesis of a number of biologically relevant molecules, has been achieved in 9 steps from an acyclic, non-aromatic precursor. A 13C label for molecular imaging was introduced in a linear synthesis from the reaction of [13C]-labelled methyl iodide with glutaric monomethyl ester chloride. Cyclisation then aromatisation gave 1,3-dimethoxybenzene and an additional methoxy group was introduced by a formylation/Baeyer–Villiger/hydrolysis/methylation sequence. Subsequent ortho-formylation and selective demethylation yielded the desired [3-13C]-2,3-dihydroxy-4-methoxybenzaldehyde.

Reversible reorganization of alkyl ester groups grafted on glassy carbon electrode: Induction by a redox probe

The oxidation of the tetrabutylammonium salt of adipic acid monomethyl ester was performed to extend the concept that the oxidation of carboxylates with terminal functional groups with electrons in π-orbitals induces the covalent grafting of carbon surfaces. When 1,4-benzoquinone was used as a redox probe to identify the compaction level of the grafted film, the voltammetric behaviour of this redox probe changed by cycling. Once a reproducible and quasi-reversible wave was obtained, the repose of the electrode in the electrolyte solution during a few minutes allowed the recovery of the original voltammogram of the redox probe on the modified electrode. This behaviour is cyclic and can be understood as the result of a reversible reorganization effect of the organic film. This reorganization phenomenon is affected by the water content in the electrochemical cell, which can be explained by hydrogen bonds forming between the ester groups present in the structure film and water.

Stereoselective synthesis of two highly potent 5-oxo-ETE receptor antagonists

Enantioselective synthesis of highly potent 5-oxo-ETE receptor antagonists 5a and 6a with a high level of enantiomeric purity is described. The main feature of this work is the simple and efficient synthesis of the bi-functionalized 3-(S)-methyl-pentanedioic acid monomethyl ester (24) with the enantiomeric ratio of S/R:98.4/1.6. We investigated the asymmetric conjugate addition of MeMgBr to 5-Benzyloxy-pent-2-enoic acid methyl ester (23) catalyzed by the CuBr/(S)-Tol-BINAP system, and optimized the reaction conditions toward regioselectivity and high enantioselectivity.

Linking chlorophyll biosynthesis to a dynamic plastoquinone pool

Chlorophylls are essential cofactors in photosynthesis. All steps in the chlorophyll pathway are well characterized except for the cyclase reaction in which the fifth ring of the chlorophyll molecule is formed during conversion of Mg-protoporphyrin IX monomethyl ester into Protochlorophyllide. The only subunit of the cyclase identified so far, is AcsF (Xantha-l in barley and Chl27 in Arabidopsis). This subunit contains a typical consensus di-iron-binding sequence and belongs to a subgroup of di-iron proteins, such as the plastid terminal oxidase (PTOX) in the chloroplast and the alternative oxidase (AOX) found in mitochondria. In order to complete the catalytic cycle, the irons of these proteins need to be reduced from Fe3+ to Fe2+ and either a reductase or another form of reductant is required. It has been reported that the alternative oxidase (AOX) and the plastid terminal oxidase (PTOX) utilize the di-iron center to oxidise ubiquinol and plastoquinol, respectively. In this paper, we have used a specific inhibitor of di-iron proteins as well as Arabidopsis and barley mutants affected in regulation of photosynthetic electron flow, to show that the cyclase step indeed is directly coupled to the plastoquinone pool. Thus, plastoquinol might act as an electron donor for the cyclase reaction and thereby fulfil the role of a cyclase reductase. That would provide a functional connection between the redox status of the thylakoids and the biosynthesis of chlorophyll.

Stability engineering of the Geobacillus stearothermophilus alcohol dehydrogenase and application for the synthesis of a polyamide 12 precursor.

The thermostable NAD(+)-dependent alcohol dehydrogenase from Geobacillus stearothermophilus (BsADH) was exploited with regard to the biocatalytic synthesis of ω-oxo lauric acid methyl ester (OLAMe), a key intermediate for biobased polyamide 12 production, from the corresponding long-chain alcohol. Recombinant BsADH was produced in Escherichia coli as a homogeneous tetrameric enzyme and showed high activity towards the industrially relevant substrate ω-hydroxy lauric acid methyl ester (HLAMe) with K M=86μM and 44Umg(-1). The equilibrium constant for HLAMe oxidation to the aldehyde (OLAMe) with NAD(+) was determined as 2.16×10(-3) from the kinetic parameters of the BsADH-catalyzed forward and reverse reactions. Since BsADH displayed limited stability under oxidizing conditions, the predominant oxidation-prone residue Cys257 was mutated to Leu based on sequence homology with related enzymes and computational simulation. This substitution resulted in an improved BsADH variant exhibiting prolonged stability and an elevated inactivation temperature. Semi-preparative biocatalysis at 60°C using the stabilized enzyme, employing butyraldehyde for in situ cofactor regeneration with only catalytic amounts of NAD(+), yielded up to 23% conversion of HLAMe to OLAMe after 30min. In contrast to other oxidoreductases, no overoxidation to the dodecanoic diacid monomethyl ester was detected. Thus, the mutated BsADH offers a promising biocatalyst for the selective oxidation of fatty alcohols to yield intermediates for industrial polymer production.

Rapid and highly sensitive analysis of chlorophylls and carotenoids from marine phytoplankton using ultra-high performance liquid chromatography (UHPLC) with the first derivative spectrum chromatogram (FDSC) technique

We developed a rapid and highly sensitive analytical method for chlorophylls and carotenoids derived from marine phytoplankton using ultra-high performance liquid chromatography (UHPLC). High-performance liquid chromatography (HPLC) has been widely used in phytoplankton pigment analysis since the 1980s for estimating the abundance, composition, and photosynthetic physiology of natural algal assemblages or laboratory cultures. However, the run-time of the HPLC analyses is generally ca. 30min or more, which is time-consuming for analysts. Our UHPLC technique enabled us to complete the separations of chlorophylls and carotenoids from marine phytoplankton within 7min with similar resolution as conventional HPLC methods. The analytical method was tested on authentic pigment standards, marine phytoplankton cultures, and field samples that were collected from the central tropical and subarctic Pacific plus the neritic Bering Sea. Critical pigment pairs that generally co-eluted as a single peak were successively resolved by obtaining the first derivative spectrum chromatograms (FDSCs) with a photodiode array (PDA) detector based on differences in pigment absorption spectra, e.g., chlorophyll (Chl) c2 and Mg 2,4 divinyl (DV) pheoporphyrin a5 monomethyl ester (MgDVP), as well as DVChl b and Chl b. Because the maximum injection volume of UHPLC is generally lower than that of HPLC to minimize the unwanted broadening of chromatographic peaks, the detection sensitivity needed to be increased, especially for oligotrophic seawater samples with low pigment concentration. To overcome this sensitivity issue, a PDA detector equipped with an 85mm path length capillary cell was used with a fluorescence detector. As a result, the limit of quantitation (LOQ) as determined by absorbance was of the order of 0.1ng for chlorophylls and carotenoids. Furthermore, a bead-beating technique using N,N-dimethylformamide (DMF) and zirconia beads was utilized to minimize the volume of the organic solvent used for pigment extraction. Our UHPLC method can replace the conventional HPLC techniques, and allows us to yield high-throughput data of the chlorophylls and carotenoids derived from marine phytoplankton.

Differential Proteomic Analysis Using iTRAQ Reveals Alterations in Hull Development in Rice (Oryza sativa L.).

Rice hull, the outer cover of the rice grain, determines grain shape and size. Changes in the rice hull proteome in different growth stages may reflect the underlying mechanisms involved in grain development. To better understand these changes, isobaric tags for relative and absolute quantitative (iTRAQ) MS/MS was used to detect statistically significant changes in the rice hull proteome in the booting, flowering, and milk-ripe growth stages. Differentially expressed proteins were analyzed to predict their potential functions during development. Gene ontology (GO) terms and pathways were used to evaluate the biological mechanisms involved in rice hull at the three growth stages. In total, 5,268 proteins were detected and characterized, of which 563 were differentially expressed across the development stages. The results showed that the flowering and milk-ripe stage proteomes were more similar to each other (r=0.61) than either was to the booting stage proteome. A GO enrichment analysis of the differentially expressed proteins was used to predict their roles during rice hull development. The potential functions of 25 significantly differentially expressed proteins were used to evaluate their possible roles at various growth stages. Among these proteins, an unannotated protein (Q7X8A1) was found to be overexpressed especially in the flowering stage, while a putative uncharacterized protein (B8BF94) and an aldehyde dehydrogenase (Q9FPK6) were overexpressed only in the milk-ripe stage. Pathways regulated by differentially expressed proteins were also analyzed. Magnesium-protoporphyrin IX monomethyl ester [oxidative] cyclase (Q9SDJ2), and two magnesium-chelatase subunits, ChlD (Q6ATS0), and ChlI (Q53RM0), were associated with chlorophyll biosynthesis at different developmental stages. The expression of Q9SDJ2 in the flowering and milk-ripe stages was validated by qRT-PCR. The 25 candidate proteins may be pivotal markers for controlling rice hull development at various growth stages and chlorophyll biosynthesis pathway related proteins, especially magnesium-protoporphyrin IX monomethyl ester [oxidative] cyclase (Q9SDJ2), may provide new insights into the molecular mechanisms of rice hull development and chlorophyll associated regulation.

Molecular characterization and primary functional analysis of PeMPEC, a magnesium-protoporphyrin IX monomethyl ester cyclase gene of bamboo (Phyllostachys edulis).

A homologous gene of MPEC from Phyllostachys edulis was isolated and characterized. Its overexpression in Arabidopsis thaliana increased chlorophyll concentration and photosynthesis efficiency, indicating it is involved in chlorophyll biosynthesis. Magnesium-protoporphyrin IX monomethyl ester cyclase (MPEC) is an essential enzyme in the biosynthesis of chlorophyll, which plays an important role in photosynthesis. However, limited information is available on the roles of MPEC gene in bamboo. A homologous gene, PeMPEC was identified from Phyllostachys edulis, which comprised 1474bp and contained an open reading frame encoding 415 amino acids. PeMPEC was transcribed abundantly in leaf blade where photosynthesis predominantly occurs, which agreed with the protein accumulation pattern confirmed by Western blotting. The PeMPEC transcription was promoted by continuous darkness for 24h, and was suppressed by increasing light intensity (100-1500 µmo1m(-2)s(-1)) and high temperature (42°C). However, transcription was induced within 0.5h and thereafter declined with prolonged treatment (up to 12h) under low temperature (4°C). Although PeMPEC expressed weakly in etiolated leaves, transcript levels increased gradually with subsequent light treatment (200µmolm(-2)s(-1)). Overexpression of PeMPEC in Arabidopsis thaliana resulted in increased chlorophyll concentration and photosynthesis efficiency in sense transgenic plants compared with a reduction in antisense transgenic plants. These changes were consistent with the transcript levels of PeMPEC. These results indicated that PeMPEC might be involved in chlorophyll biosynthesis and play important roles in maintaining the stability of photosystems, and provide a basis for the study of chlorophyll biosynthesis and dissection of photosynthesis in bamboo.

Identification of the chlE gene encoding oxygen-independent Mg-protoporphyrin IX monomethyl ester cyclase in cyanobacteria

The fifth ring (E-ring) of chlorophyll (Chl) a is produced by Mg-protoporphyrin IX monomethyl ester (MPE) cyclase. There are two evolutionarily unrelated MPE cyclases: oxygen-independent (BchE) and oxygen-dependent (ChlA/AcsF) MPE cyclases. Although ChlA is the sole MPE cyclase in Synechocystis PCC 6803, it is yet unclear whether BchE exists in cyanobacteria. A BLAST search suggests that only few cyanobacteria possess bchE. Here, we report that two bchE candidate genes from Cyanothece strains PCC 7425 and PCC 7822 restore the photosynthetic growth and bacteriochlorophyll production in a bchE-lacking mutant of Rhodobacter capsulatus. We termed these cyanobacterial bchE orthologs “chlE.”

Synthesis of first-generation calix-dendrimer from 1,3,5-tris(aminomethyl)-2,4,6-trimethylbenzene and p-tert-butylcalix[4]arene monomethyl ester

A first-generation calix-dendrimer 1 suitable as a multidentate ligand for metal-ion binding was prepared from 1,3,5-tris(aminomethyl)-2,4,6-trimethylbenzene as the dendrimer core and p-tert-butylcalix [4] arene monomethyl ester as a repetitive branch, and characterized by spectroscopic techniques.