Synthesis Of Side Chain Research Articles

Enzymes Repertoires and Genomic Insights into Lycium Barbarum Pectin Polysaccharides Biosynthesis.

Lycium barbarum, a member of the Solanaceae family, represents an important eudicot lineage with homology of food and medicine. Lycium barbarum pectin polysaccharides (LBPPs) are key bioactive ingredients of Lycium barbarum, and are among the few polysaccharides with both biocompatibility and biomedical activity. While previous studies have primarily focused on the functional properties of LBPPs, the mechanisms of biosynthesis and transport by key enzymes remain poorly understood. Here, we reported the completion of a 2.18-gigabase reference genome of Lycium barbarum, reconstructed the first entire biosynthesis of pectin polysaccharides and sugar transport, and characterized the important genes responsible for backbone extending, sidechain synthesis, and modification of pectin polysaccharides. Additionally, we characterized long non-coding RNAs (lncRNAs) associated with polysaccharide metabolism and identified a specific rhamnogalacturonan I (RG-I) rhamnosyltransferase, RRT3020, which enhances RG-I biosynthesis in LBPPs. These newly identified enzymes and pivotal genes endow L. barbarum with specific pectin biosynthesis capabilities, distinguishing it from other Solanaceae species. Our findings provide a foundation for evolutionary studies and molecular breeding to enhance the diverse applications of L. barbarum.

Synthesis and Characterization of Novel Perfluoro Aromatic Side Chain Sulfonated PSU Ionomers

Synthesis and Characterization of Novel Perfluoro Aromatic Side Chain Sulfonated PSU Ionomers

The DUF579 proteins GhIRX15s regulate cotton fiber development by interacting with proteins involved in xylan synthesis

Cotton provides the most abundant natural fiber for the textile industry. The mature cotton fiber largely consists of secondary cell walls with the highest proportion of cellulose and a small amount of hemicellulose and lignin. To dissect the roles of hemicellulosic polysaccharides during fiber development, four IRREGULAR XYLEM 15 (IRX15) genes, GhIRX15-1/-2/-3/-4, were functionally characterized in cotton. These genes encode DUF579 domain-containing proteins, which are homologs of AtIRX15 involved in xylan biosynthesis. The four GhIRX15 genes were predominantly expressed during fiber secondary wall thickening, and the encoded proteins were localized to the Golgi apparatus. Each GhIRX15 gene could restore the xylan deficient phenotype in the Arabidopsis irx15irx15l double mutant. Silencing of GhIRX15s in cotton resulted in shorter mature fibers with a thinner cell wall and reduced cellulose content as compared to the wild type. Intriguingly, GhIRX15-2 and GhIRX15-4 formed homodimers and heterodimers. In addition, the GhIRX15s showed physical interaction with glycosyltransferases GhGT43C, GhGT47A and GhGT47B, which are responsible for synthesis of the xylan backbone and reducing end sequence. Moreover, the GhIRX15s can form heterocomplexes with enzymes involved in xylan modification and side chain synthesis, such as GhGUX1/2, GhGXM1/2 and GhTBL1. These findings suggest that GhIRX15s participate in fiber xylan biosynthesis and modulate fiber development via forming large multiprotein complexes.

Convergent synthesis of tetrahydropyranyl side chain of verucopeptin, an antitumor antibiotic active against multidrug-resistant cancers.

A concise synthesis of the tetrahydropyranyl side chain of verucopeptin, an antitumor antibiotic cyclodepsipeptide efficacious against MDR cancers in vivo, is achieved using 12 steps in the longest linear sequence and 21 total steps, in which Julia-Kocienski olefination for the segments coupling, asymmetric hydroxylation as well as stereoselective synthesis of stable tetrahydropyran ring from a D-isoascorbic acid derivative are key steps. This convergent synthetic strategy enables the structural modification and mechanism study of verucopeptin for its clinical applications.

Gene excavation and expression analysis of CYP and UGT related to the post modifying stage of gypenoside biosynthesis in Gynostemma pentaphyllum (Thunb.) Makino by comprehensive analysis of RNA and proteome sequencing.

Previous studies have revealed that gypenosides produced from Gynostemma pentaphyllum (Thunb.) Makino are mainly dammarane-type triterpenoid saponins with diverse structures and important biological activities, but the mechanism of diversity for gypenoside biosynthesis is still unclear. In this study, a combination of isobaric tags for relative and absolute quantification (iTRAQ) proteome analysis and RNA sequencing transcriptome analysis was performed to identify the proteins and genes related to gypenoside biosynthesis. A total of 3925 proteins were identified by proteomic sequencing, of which 2537 were quantified. Seventeen cytochrome P450 (CYP) and 11 uridine 5’-diphospho-glucuronosyltransferase (UDP-glucuronosyltransferase, UGT) candidate genes involved in the side chain synthesis and modification of gypenosides were found. Seven putative CYPs (CYP71B19, CYP77A3, CYP86A7, CYP86A8, CYP89A2, CYP90A1, CYP94A1) and five putative UGTs (UGT73B4, UGT76B1, UGT74F2, UGT91C1 and UGT91A1) were selected as candidate structural modifiers of triterpenoid saponins, which were cloned for gene expression analysis. Comprehensive analysis of RNA sequencing and proteome sequencing showed that some CYPs and UGTs were found at both the transcription and translation levels. In this study, an expression analysis of 7 CYPs and 5 UGTs that contributed to gypenoside biosynthesis and distribution in G. pentaphyllum was performed, providing consistent results that will inspire more future research on vital genes/proteins involved in gypenoside biosynthesis.

Genetic evidence for the requirements of antroquinonol biosynthesis by Antrodia camphorata during liquid-state fermentation.

The solid-state fermentation of Antrodia camphorata could produce a variety of ubiquinone compounds, such as antroquinonol (AQ). However, AQ is hardly synthesized during liquid-state fermentation (LSF). To investigates the mechanism of AQ synthesis, three precursors (ubiquinone 0 UQ0, farnesol and farnesyl diphosphate FPP) were added in LSF. The results showed that UQ0 successfully induced AQ production; however, farnesol and FPP could not induce AQ synthesis. The precursor that restricts the synthesis of AQ is the quinone ring, not the isoprene side chain. Then, the Agrobacterium-mediated transformation system of A. camphorata was established and the genes for quinone ring modification (coq2-6) and isoprene synthesis (HMGR, fps) were overexpressed. The results showed that overexpression of genes for isoprene side chain synthesis could not increase the yield of AQ, but overexpression of coq2 and coq5 could significantly increase AQ production. This is consistent with the results of the experiment of precursors. It indicated that the A. camphorata lack the ability to modify the quinone ring of AQ during LSF. Of the modification steps, prenylation of UQ0 is the key step of AQ biosynthesis. The result will help us to understand the genetic evidence for the requirements of AQ biosynthesis in A. camphorata.

Minimal mitochondrial respiration is required to prevent cell death by inhibition of mTOR signaling in CoQ-deficient cells

Coenzyme Q (CoQ) is a lipid-like mobile electron transporter of the mitochondrial respiratory chain. Patients with partial loss-of-function mutations in the CoQ biosynthesis pathway suffer from partial primary CoQ deficiency (MIM 607426). This leads to mitochondrial dysfunction, which presents like mitochondrial disease syndrome (MDS). In addition, many other conditions, including MDS itself, lead to secondary CoQ deficiency. We sought to identify drugs that can alleviate the consequences of the mitochondrial dysfunction that is associated with CoQ deficiency. Loss of the CoQ-biosynthetic enzyme COQ7 prevents CoQ synthesis but leads to the accumulation of the biosynthetic intermediate demethoxyubiquinone (DMQ). Coq7-knockout mouse embryonic fibroblasts (MEFs) die when rapid ATP generation from glycolysis is prevented. We screened for drugs that could rescue cell death under these conditions. All compounds that were identified inhibit mTOR signaling. In the CoQ-deficient cells, the beneficial action mTOR inhibition appears to be mediated by inhibition of protein translation rather than by stimulation of autophagy. We further studied the Coq7-knockout cells to better determine under which conditions mTOR inhibition could be beneficial. We established that Coq7-knockout cells remain capable of a low level of mitochondrial respiration mediated by DMQ. To obtain more profound mitochondrial dysfunction, we created double-knockout mutant MEFs lacking both Coq7, as well as Pdss2, which is required for sidechain synthesis. These cells make neither CoQ nor DMQ, and their extremely small residual respiration depends on uptake of CoQ from the culture medium. Although these cells are healthy in the presence of sufficient glucose for glycolysis and do not require uridine or pyruvate supplementation, mTOR inhibitors were unable to prevent their death in the absence of sufficient glycolysis. We conclude that, for reasons that remain to be elucidated, the energy-sparing benefits of the inhibition of mTOR signaling require a minimally functional respiratory chain.

Molecular Brush Surfactants: Versatile Emulsifiers for Stabilizing and Structuring Liquids

AbstractUsing amphiphilic molecular brushes to stabilize emulsions usually requires the synthesis of specific side chains, which can be a time‐consuming and difficult challenge to meet. By taking advantage of the electrostatic interactions between water‐soluble molecular brushes and oil‐soluble oligomeric ligands, the in situ formation, assembly and jamming of molecular brush surfactants (MBSs) at the oil‐water interface is described. With MBSs, stable emulsions including o/w, w/o and o/w/o can be easily prepared by varying the molar ratios of the molecular brushes to the ligands. Moreover, when jammed, the binding energy of MBSs at the interface is sufficiently strong to allow the stabilization of liquids in nonequilibrium shapes, i.e., structuring liquids, producing an elastic film at the interface with exceptional mechanical properties. These structured liquids have numerous potential applications, including chemical biphasic reactions, liquid electronics, and all‐liquid biomimetic system.

Molecular Brush Surfactants: Versatile Emulsifiers for Stabilizing and Structuring Liquids.

Using amphiphilic molecular brushes to stabilize emulsions usually requires the synthesis of specific side chains, which can be a time-consuming and difficult challenge to meet. By taking advantage of the electrostatic interactions between water-soluble molecular brushes and oil-soluble oligomeric ligands, the in situ formation, assembly and jamming of molecular brush surfactants (MBSs) at the oil-water interface is described. With MBSs, stable emulsions including o/w, w/o and o/w/o can be easily prepared by varying the molar ratios of the molecular brushes to the ligands. Moreover, when jammed, the binding energy of MBSs at the interface is sufficiently strong to allow the stabilization of liquids in nonequilibrium shapes, i.e., structuring liquids, producing an elastic film at the interface with exceptional mechanical properties. These structured liquids have numerous potential applications, including chemical biphasic reactions, liquid electronics, and all-liquid biomimetic system.

Report of two siblings with spondylodysplastic Ehlers-Danlos syndrome and B4GALT7 deficiency

BackgroundThe spondylodysplastic Ehlers-Danlos subtype (OMIM #130070) is a rare connective tissue disorder characterized by a combination of connective tissue symptoms, skeletal features and short stature. It is caused by variants in genes encoding for enzymes involved in the proteoglycan biosynthesis or for a zinc transporter.Presentation of casesWe report two brothers with a similar phenotype of short stature, joint hypermobility, distinct craniofacial features, developmental delay and severe hypermetropia indicative for a spondylodysplastic Ehlers-Danlos subtype. One also suffered from a recurrent pneumothorax. Gene panel analysis identified two compound heterozygous variants in the B4GALT7 gene: c.641G > A and c.723 + 4A > G. B4GALT7 encodes for galactosyltransferase I, which is required for the initiation of glycosaminoglycan side chain synthesis of proteoglycans.ConclusionsThis is a first full report on two cases with spondylodysplastic Ehlers-Danlos syndrome and the c.723 + 4A > G variant of B4GALT7. The recurrent pneumothoraces observed in one case expand the variable phenotype of the syndrome.

Synthesis and Characterization of Urethane Side Chain Substituted Diketopyrrolopyrrole

The synthesis of Diketopyrrolopyrrole (DPP) having secondary interaction in the side chain explores its possibility to use in electronic and sensing applications. Herein we report easy method to engineer side chains of DPP. The hydrogen bonding is introduced on the side chain by substitution of urethane side chains on Diketopyrrolopyrrole (DPPurethane).The urethane side chain comprises a branched alkyl chain with good yields and purities. The DPPurethane characterized by NMR and IR, optical properties along with energy minimized structure were studied.

Generation of polyclonal antibodies against recombinant Agrobacterium tumefaciens decaprenyl diphosphate synthase produced in Escherichia coli

Coenzyme Q (CoQ), an electron carrier of the mitochondrial respiratory chain and a potent antioxidant, consists of a benzoquinone head and an isoprenoid side chain of varying numbers of isoprene units, 10 in humans, animals and some microbes including Agrobacterium tumefaciens (CoQ10), 9 in Arabidopsis and cereals (CoQ9) and 8 in Escherichia coli (CoQ8). The synthesis of CoQ10 side chain is catalyzed by a single side chain length determining enzyme, decaprenyl diphosphate synthase (DPS) which is encoded by a dps gene. We report here, the isolation and sequencing of a dps gene from A. tumefaciens strain LBA4404 and its cloning in pET28a expression vector of E. coli BL21 (DE3) strain to produce and purify recombinant DPS protein for the production of its antibodies in rabbit. The dps gene isolated from A. tumefaciens consists of 1077 bp efficient for encoding a polypeptide of 359 amino acids with a molecular mass of around 39 kDa. DPS protein expression was induced by adding isopropyl β-D-1-thiogalactopyranoside in bacterial culture to a final concentration of 1.0 mM. Recombinant protein tagged with histidine at N-terminal was purified by immobilized metal-affinity chromatography. The purified DPS protein was capable of producing polyclonal antibodies in rabbit whose specificity to DPS enzyme was confirmed by the western blot analysis. Expression of dps gene in E. coli extended the side chain length of CoQ8 to 10 isopenoid units and produced CoQ10 in addition to CoQ8. This is the first report on the production of polyclonal antibodies against recombinant DPS protein and demonstration of their utility.

Benzoisothiazolone (BIT): A Fast, Efficient, and Recyclable Redox Reagent for Solid Phase Peptide Synthesis

Solid‐phase peptide synthesis (SPPS), a preferred synthetic procedure, generates by‐products and effluents in multiple equivalents for one equivalent of desired product. Presented herein is the use of a fast and efficient coupling protocol for SPPS using a benzoisothiazolone (BIT), which can be fully recycled. The BIT, as redox activator, works under very mild conditions and generates minimal amount of waste. As a case study, the BIT coupling protocol is applied to the synthesis of side chain of the recently discovered antibiotic, teixobactin.

Synthesis and properties of side chain fluorinated polyurethanes and evaluation of changes in microphase separation

To develop marketable new waterproof and breathable membranes, this study successfully applied a simple synthesis method to combine pentadecafluorooctanoyl chloride and 2-Amino-2-methyl-1,3-propanediol into a novel long-segment side-chain fluorinated chain extender (AMPF), the structure was examined using nuclear magnetic resonance (NMR) spectroscopy. To promote environmental-friendliness, biodegradable polycaprolactone (PCL) was chosen to supply the soft segment of polyurethane (PU), methylene diphenyl diisocyanate to supply the hard segment, The successful introduction of AMPF into the PCL-based PU enabled the production of AMPF/PUs. To investigate the effect of AMPF on PCL-based PU, NMR and gel permeation chromatography were performed for the preliminary analysis of AMPF/PUs; curve fitting was applied in Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. The atomic force microscopy to determine the difference in van der Waals forces and microphase separation in AMPF/PUs; a thermal gravimetric analysis and dynamic mechanical analysis were conducted to determine the thermal properties of AMPF/PUs, and tensile test were conducted to determine the mechanical properties of AMPF/PUs. The results reveal that AMPF/PUs have a high degree of microphase separation, and the heat resistance of AMPF/PUs can be improved by 4–5 %, while the tensile strength can be increased by about 28 MPa with the increase of AMPF content. From the contact angle test, it is known that the increase of AMPF content can convert the hydrophilic polyurethane (77.8 ± 2.7°) into the hydrophobic (95.2 ± 2.1°) polyurethane. Therefore, it is shown that AMPF can not only improve the strength of polyurethane but also provide hydrophobic properties. This fluorinated chain extender shows considerable potential for the coatings materials and waterproof and breathable fabrics.

Gram-Scale Solution-Phase Synthesis of Heptapeptide Side Chain of Teixobactin

We report herein a scalable synthesis of linear heptapeptide side chain of the depsipeptide natural product teixobactin through solution phase. The synthesis of heptapeptide was achieved through an efficient coupling of suitably protected tripeptide and tetrapeptide comprising of three d-amino acids and four usual l-amino acid subunits.

A concise synthesis of isoxazole-based side chain of Micafungin

A concise synthesis of key isoxazole-based side chain of Micafungin, an USFDA approved anti-fungal agent, has been delineated. The route design notably involves a one pot regioselective isoxazole construction from the corresponding aryl aldehyde and alkyne intermediates.

Synthesis and Properties of Side-Chain Functionalized Polytetrahydrofuran Derivatives via the Blue-Light Photocatalytic Thiol-Ene Reaction.

A series of side-chain functionalized polytetrahydrofuran (PTHF) derivatives were synthesized via the blue-light photocatalytic thiol-ene “click” reaction. Firstly, unsaturated polytetrahydrofuran (UPTHF) as a new unsaturated polyether was synthesized via condensation polymerization of cis-2-butene-1,4-diol and trans-1,4-dibromo-2-butene using potassium hydroxide (KOH) as a catalyst. Then, double bonds in the backbone of UPTHF were modified into different pendant functionality side groups by blue-light photocatalytic thiol-ene “click” reaction using Ru(bpy)3Cl2 as a photoredox catalyst, obtaining different side-chain functionalized PTHF derivatives. The structure and the morphology of the side-chain functionalized PTHF derivatives was characterized via Fourier-transform infrared spectra (FTIR), nuclear magnetic resonance (NMR), size exclusion chromatography/multi-angle laser light scattering (SEC/MALLS), and differential scanning calorimeter (DSC). The results showed that the blue-light photocatalytic thiol-ene reaction exhibited high efficiency, and all the unsaturated bonds were modified. Different branch units bestowed different performance of PTHF derivatives; we systematically investigated the thermal properties, pH-triggered and temperature-triggered, self-assembly behaviors of different PTHF derivatives.

Synthesis of Paclitaxel Side Chain via Multi-Component Reaction and Its Application to the Synthesis of Paclitaxel Analogues

Synthesis of Paclitaxel Side Chain via Multi-Component Reaction and Its Application to the Synthesis of Paclitaxel Analogues

Upregulation of glycosaminoglycan synthesis by Neurotropin in nucleus pulposus cells via stimulation of chondroitin sulfate N-acetylgalactosaminyltransferase 1: A new approach to attenuation of intervertebral disc degeneration.

It is suggested that most cases of low back pain are related to degeneration of intervertebral discs. Disc degeneration is a chronic and progressive disease and the search for effective medical treatments continues. Neurotropin is widely used in Japan and China to treat low back pain and neck–shoulder–arm syndrome. The present study aimed to investigate the effect of Neurotropin on glycosaminoglycan synthesis in nucleus pulposus cells. Cultured human nucleus pulposus cells were treated with Neurotropin every second day for two weeks. Production of glycosaminoglycan was assessed using a dimethyl-methylene blue assay and PicoGreen was used to measure DNA content. Microarray analysis, real-time PCR, and western blotting were performed to assess the biological processes related to Neurotropin-stimulated glycosaminoglycan synthesis. The results showed that the level of glycosaminoglycan normalized to DNA content was significantly upregulated by the addition of Neurotropin. Gene expression profiling showed over two-fold upregulation of 697 genes in response to Neurotropin treatment. Among these genes, ontological analysis suggested significant implication of phosphatidylinositol 3-kinase signaling, and analysis focused on this pathway demonstrated marked upregulation of angiopoietin 1 and insulin-like growth factor 1. Activation of phosphorylation of the signal transducer protein AKT was detected by western blotting. Of the genes related to sulfated glycosaminoglycan synthesis, the greatest increase in mRNA levels was observed for chondroitin sulfate N-acetylgalactosaminyltransferase 1, an enzyme initiating synthesis of chondroitin sulfate side chains attached to a core protein of aggrecan, which is a predominant disc matrix component. These findings suggest that Neurotropin may activate the phosphatidylinositol 3-kinase–AKT pathway and stimulate glycosaminoglycan synthesis through upregulation of expression of mRNA for chondroitin sulfate N-acetylgalactosaminyltransferase 1. Because there was no cytotoxic cellular growth inhibition, Neurotropin treatment might offer an accessible therapeutic strategy for intervertebral disc degeneration.

Improvement of carbonyl reductase activity for the bioproduction of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate

tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate ((3R,5S)-CDHH) is a key chiral intermediate for the side chain synthesis of rosuvastatin. In this study, random mutagenesis, site-saturation mutagenesis and combinatorial mutagenesis methods were applied to improve the activity of a synthesized stereoselective short chain carbonyl reductase (SCR) to prepare (3R,5S)-CDHH. After screened by high-throughput screening method and high-performance liquid chromatography, mut-Phe145Met/Thr152Ser and mut-Phe145Tyr/Thr152Ser, were obtained, and the enzyme activities of mutants were improved by 1.60- and 1.91-fold compared with parent enzyme, respectively. The catalytically efficiencies (kcat/Km) of mut-Phe145Met/Thr152Ser and mut-Phe145Tyr/Thr152Ser exhibited 5.11- and 8.07-fold improvements in initial activity toward (S)-6-chloro-5-hydroxy-3-oxohexanoate ((S)-CHOH), respectively. In the asymmetric reduction, mut-Phe145Tyr/Thr152Ser catalyzed 500 g L−1 of (S)-CHOH to produce (3R,5S)-CDHH with >99% yield and >99% e.e., and the highest space-time yield achieved at 752.76 mmol L−1 h−1 g−1 wet cell weight within 8 h bioconversion. This study provides a foundation for the preparation of (3R,5S)-CDHH by carbonyl reductase.