Development of an aminotransferase-driven biocatalytic cascade for deracemization of d,l-phosphinothricin

2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO) is the essential precursor keto acid for the asymmetric biosynthesis of herbicide l -phosphinothricin (l -PPT). Developing a biocatalytic cascade for PPO production with high efficiency and low cost is highly desired. Herein, a d -amino acid aminotransferase from Bacillus sp. YM-1 (Ym DAAT) with high activity (48.95 U/mg) and affinity (K_m = 27.49 mM) toward d -PPT was evaluated. To circumvent the inhibition of by-product d -glutamate (d -Glu), an amino acceptor (α-ketoglutarate) regeneration cascade was constructed as a recombinant Escherichia coli (E. coli D), by coupling Ym d -AAT, d -aspartate oxidase from Thermomyces dupontii (TdDDO) and catalase from Geobacillus sp. CHB1. Moreover, the regulation of the ribosome binding site was employed to overcome the limiting step of expression toxic protein TdDDO in E. coli BL21(DE3). The aminotransferase-driven whole-cell biocatalytic cascade (E. coli D) showed superior catalytic efficiency for the synthesis of PPO from d ,l -phosphinothricin (d ,l -PPT). It revealed the production of PPO exhibited high space–time yield (2.59 g L⁻¹ h⁻¹) with complete conversion of d -PPT to PPO at high substrate concentration (600 mM d ,l -PPT) in 1.5 L reaction system. This study first provides the synthesis of PPO from d ,l -PPT employing an aminotransferase-driven biocatalytic cascade.     

Total synthesis of alpha-galactosyl cerebroside

A highly convergent synthetic approach was developed to obtain alpha-galactosyl cerebroside O-(alpha-D-galactopyranosyl)-2-hexacosylamino-D-ribo-1,3,4-octa decantriol, which has previously been demonstrated to have immunostimulatory activity. Known 4,6-O-benzylidene galactose was the starting material for both the required alpha-galactosyl and the phytosphingosine building blocks O-(2,3-di-O-benzyl-4,6-O-benzylidene-D-galactopyranosyl) trichloroacetimidate (4) and 2-O-methanesulfonyl-D-arabino-1,2,3,4-octadecantetrol (5). The key step of the synthetic strategy is the highly regio- and stereoselective O-galactosylation of 1,3,4-O-unprotected phytosphingosine acceptor 5 using known 4 as donor. The total synthesis required only 11 synthetic steps starting from galactose.     

Solid-phase synthesis of 2,4-diaminoquinazolines

A highly efficient and versatile solid-phase synthesis of 2,4-diaminoquinazoline library from 2,4-dichloroquinazolines and amines using 3,5-dimethoxy 4-formylphenoxy-polystyrene resin is described.     

Enzymatic synthesis of oligosaccharides

As the importance of the oligosaccharide moieties of glycoproteins and glycolipids is being increasingly recognized, efforts to synthesize them are expanding. The number of functional groups of carbohydrate monomers and the variety of configurations that oligomers can adopt is greater than with nucleotides/nucleic acids or amino acids/peptides. By reversing the hydrolytic action of glycosidases and by using highly regiospecific glycosyltransferases, enzymatic oligosaccharide synthesis can be performed.     

Total synthesis of gigantetrocin A

A highly efficient synthetic method for the trans-tetrahydrofuran (THF) ring building block was established and the title compound was synthesized in 19 steps from trans-1,4-dichloro-2-butene via a convergent route with a Wittig reaction as the key step.     

Efficient synthesis of pyrenylalanine

An efficient synthesis of L-3-(1'-pyrenyl)alanine (Pya), a highly fluorescent amino acid, is described. The amino acid was obtained by the classical asymmetric hydrogenation of chiral 1-acetyl-3-pyrenemethylidene-6-methyl-piperazine-2,5-dione. In the proposed improved procedure mild conditions of the synthesis were applied and the final product--N-tertbutoxycarbonyl-pyrenylalanine--was obtained in good yield. Pyrenylalanine, due to its interesting photophysical properties, can be applied as a fluorescent probe in numerous biochemical and conformational studies.     

Total synthesis of 12,13-desoxyepothilone B (Epothilone D)

A highly convergent total synthesis of 12,13-desoxyepothilone B (4, Epothilone D) is described involving the coupling of vinyl iodide (5) and olefin (6). Key steps in the synthesis are the introduction of chirality at C15 via highly enantioselective lipase-mediated enzymatic resolution, diastereoselective alkylation at C8, highly diastereoselective Evans aldol reaction to establish C6-C7, and Mukaiyama aldol reaction to introduce chiral center C3. Palladium catalyzed Suzuki coupling of (5) and (6) provided the methyl ester (27), which was converted to 12,13-desoxyepothilone B (4).     

The chemoenzymatic synthesis of usnic acid

Usnic acid, a highly functionalized dibenzofuran, is a polyketide secondary metabolite produced by several species of lichens. Synthesis of usnic acid from commercially available starting material was accomplished in two steps. The synthesis involves the methylation of phloracetophenone followed by oxidation with horseradish peroxidase. This work will lay the foundation for further biosynthetic studies on usnic acid.     

Biotechnological synthesis of functional nanomaterials

Biological systems, especially those using microorganisms, have the potential to offer cheap, scalable and highly tunable green synthetic routes for the production of the latest generation of nanomaterials. Recent advances in the biotechnological synthesis of functional nano-scale materials are described. These nanomaterials range from catalysts to novel inorganic antimicrobials, nanomagnets, remediation agents and quantum dots for electronic and optical devices. Where possible, the roles of key biological macromolecules in controlling production of the nanomaterials are highlighted, and also technological limitations that must be addressed for widespread implementation are discussed.     

Soluble polymer-supported synthesis of benzodiazepinones

Soluble polymer-supported synthetic method provides a highly efficient route for the construction of biologically important 1,5-benzodiazepin-2-ones. A library of N-substituted benzodiazepinones can be readily assembled utilizing S(N)Ar reaction, reduction of nitro group and one-pot cyclization following N-alkylation as the key step in the synthesis. All reactions in the sequence were performed at room temperature to facilitate the generation of libraries in a parallel fashion. The crude products were obtained in 80-95% yield with 60-96% HPLC purity.     

Regulation and cellular localization of ent-kaurene synthesis

The two-step cyclization reaction of ent -kaurene synthesis from geranylgeranyl diphosphate is the first committed step in the biosynthetic pathway of the plant hormone gibberellin. Recent molecular cloning and characterization of the genes encoding the two corresponding enzymes, copalyl diphosphate synthase (CPS) and ent -kau-rene synthase (KS), have demonstrated that ent -kaurene synthesis is localized in the plastids and is highly regulated in specific tissues and cell types during plant development. In addition to occurring in actively growing tissues, ent -kaurene synthesis also takes place in fully expanded leaves. Therefore mature leaves may produce gibberellin intermediates or bioactive gibberellins for transport to responsive tissues. DNA sequence analyses have revealed a conserved aspartate-rich motif, D(I/V)DDTA among CPS and other protonation-initiated terpene cyclases, while KS contains a highly conserved DDXXD motif which was proposed to function as a divalent metal ion-diphos-phate complex binding site in ionization-initiated terpene cyclases and prenyltrans-ferases.     

Screening of various glycosidases for the synthesis of octyl glucoside

Thirteen glycosidases of microbial origin and almond beta-glycosidase were assayed in octanol/DMF (80:20, v/v), using a combination of hydrolysis, transglycosylation, and condensation reactions, in order to assess their potential for the production of alkyl glucosides. The two mesophile enzymes were highly impaired by the organic media. Three of the 11 thermophile enzymes gave interesting results in the hydrolysis and transglycosylation reactions, but they were highly inhibited by glucose. This made their use in a condensation reaction less interesting than the use of almond beta-glucosidase, which has a lower activity but shows less inhibition by the glucose.     

Dipentafluorophenyl carbonate--a reagent for peptide synthesis

Dipentafluorophenylcarbonate, belonging to transesterifiying reagents, has been prepared and used for the synthesis of pentafluorophenyl esters of amino acids. In contrast to many other reagents of the kind, its preparation is simple, it is highly reactive and at the same time stable upon storage.     

The synthesis of chitosan-based silver nanoparticles and their antibacterial activity

Chitosan-based silver nanoparticles were synthesized by reducing silver nitrate salts with nontoxic and biodegradable chitosan. The silver nanoparticles thus obtained showed highly potent antibacterial activity toward both Gram-positive and Gram-negative bacteria, comparable with the highly active precursor silver salts. Silver-impregnated chitosan films were formed from the starting materials composed of silver nitrate and chitosan via thermal treatment. Compared with pure chitosan films, chitosan films with silver showed both fast and long-lasting antibacterial effectiveness against Escherichia coli. The silver antibacterial materials prepared in our present system are promising candidates for a wide range of biomedical and general applications.     

Solid phase synthesis of acylglycine human metabolites

Acylglycines represents a large and important class of human metabolites. They are often used in medicine to identify fatty acid oxidation disorders. A highly efficient solid phase synthesis approach to obtain these clinically important compounds is developed via coupling reaction between glycine-preloaded Wang resin and a set of carboxylic acids. The developed methodology facilitates the preparation of several structurally-diverse acylglycines with high yields and purity.     

Microwave-assisted RCM for the synthesis of carbocyclic peptides.

Microwave irradiation dramatically improves the efficiency of ring closing metathesis (RCM) reactions of resin-attached peptides and the technology is illustrated by the highly selective synthesis of dicarba analogues of alpha-conotoxin IMI.     

Interaction between membrane functions and protein synthesis in reticulocytes. An elongation-stage inhibitor of protein synthesis extracted from the reticulocyte membrane.

A component of the reticulocyte cell membrane was found to inhibit protein synthesis severely in a reticulocyte lysate system. An investigation into the mode of action of the membrane inhibitor revealed the following facts. (1) The binding of the tertiary initiation complex (methionyl-tRNAfMet-Initiation Factor 2-GTP) to the 40S ribosomal subunit was unaffected by the membrane inhibitor. (2) The membrane component did not interfere with the binding of the 40S initiation complex to the AUG initiation codon and subsequent attachment of the 60S ribosomal subunit. (3) Elongation of the peptide chain, as assayed by peptidyl-puromycin formation, was markedly affected by the membrane inhibitor. Surprisingly, the membrane component caused a considerable increase in peptidyl-puromycin formation. (4) Reticulocyte ribosomes that had been reisolated by high-speed centrifugation, after preincubation with the membrane component, were found to be highly defective when assayed in a cell-free protein-synthesizing system. These results indicated that an extract of the reticulocyte cell membrane inhibited protein synthesis by interacting with the ribosome and thus interfered with the correct functions of the elongation stage of protein synthesis. The implications of this conclusion are discussed in the light of data showing that a highly purified preparation of the membrane inhibitor also displayed an endonucleolytic activity highly specific for 28S RNA.     

Biological synthesis of circular polypeptides

Here, we review the use of different biochemical approaches for biological synthesis of circular or backbone-cyclized proteins and peptides. These methods allow the production of circular polypeptides either in vitro or in vivo using standard recombinant DNA expression techniques. Protein circularization can significantly impact protein engineering and research in protein folding. Basic polymer theory predicts that circularization should lead to a net thermodynamic stabilization of a folded protein by reducing the entropy associated with the unfolded state. Protein cyclization also provides a valuable tool for exploring the effects of topology on protein folding kinetics. Furthermore, the biological production of cyclic polypeptides makes possible the production of cyclic polypeptide libraries. The generation of such libraries, which was previously restricted to the domain of synthetic chemists, now offers biologists access to highly diverse and stable molecular libraries for probing protein structure and function.