ABSTRACT
To enhance the phosphorylating activity of the bacterial nonspecific acid phosphatase from Salmonella enterica ser. typhimurium LT2 towards dihydroxyacetone (DHA), a mutant library was generated from the native enzyme. Three different variants that showed enhanced activity were identified after one round of epPCR. The single mutant V78L was the most active and showed an increase in the maximal DHAP concentration to 25 % higher than that of the wild-type enzyme at pH 6.0. This variant is 17 times more active than the wild-type acid phosphatase from Salmonella enterica ser. typhimurium LT2 in the acid phosphatase/aldolase cascade reaction at pH 6.0 and is also six times more active than the phosphatase from Shigella flexneri that we previously used.
Subject(s)
Acid Phosphatase/metabolism , Dihydroxyacetone/metabolism , Directed Molecular Evolution , Aldehyde-Lyases/metabolism , Amino Acid Sequence , Dihydroxyacetone/chemistry , Hydrogen-Ion Concentration , Mutagenesis, Site-Directed , Phosphorylation , Protein Conformation , Salmonella enterica/enzymologyABSTRACT
Two complementary strategies for the synthesis of febrifugine are detailed based on previously developed chemoenzymatic approaches to the 3-hydroxypiperidine skeleton. The introduction of the quinazolone-containing side chain in both strategies was based on an N-acyliminium ion-mediated coupling reaction.
Subject(s)
Piperidines/chemical synthesis , Quinazolines/chemical synthesis , Catalysis , Piperidines/chemistry , Quinazolines/chemistry , Stereoisomerism , Substrate SpecificityABSTRACT
A stereoselective synthesis of (+)-epiquinamide is presented in combination with determination of the absolute configuration of the natural product. Key steps in the sequence involved chemoenzymatic formation of an enantiomerically pure cyanohydrin, reductive cyclization to the corresponding cyclic N,N-acetal, and subsequent conversion into a suitable N-acyliminium ion precursor to enable construction of the second ring.
Subject(s)
Acetals/chemistry , Cations/chemistry , Imines/chemistry , Quinolizines/chemical synthesis , Animals , Anura , Biological Products/chemical synthesis , Biological Products/chemistry , Quinolizines/chemistry , Stereoisomerism , Substrate SpecificityABSTRACT
Enzyme-catalyzed reductions have been studied for decades and have been introduced in more than 10 industrial processes for production of various chiral alcohols, alpha-hydroxy acids and alpha-amino acids. The earlier hurdle of expensive cofactors was taken by the development of highly efficient cofactor regeneration methods. In addition, the accessible number of suitable dehydrogenases and therefore the versatility of this technology is constantly increasing and currently expanding beyond asymmetric production of alcohols and amino acids. Access to a large set of enzymes for highly selective C=C reductions and reductive amination of ketones for production of chiral secondary amines and the development of improved D-selective amino acid dehydrogenases will fuel the next wave of industrial bioreduction processes.
Subject(s)
Oxidoreductases/chemistry , Alcohols/chemistry , Amino Acids/chemistry , Catalysis , Coenzymes/chemistry , Keto Acids/chemistry , Ketones/chemistry , Oxidation-ReductionABSTRACT
The nitrile hydrolyzing properties of the bacterium strain Rhodococcus erythropolis NCIMB 11540 have been investigated. Using whole cells of the microorganism, a wide variety of aromatic and aliphatic cyanide-containing substrates was successfully hydrolyzed to the corresponding amide or acid. In the case of dicyanides, selective monohydrolysis took place, which was further explored in the desymmetrization of malononitriles resulting in the corresponding cyano amides in enantiomeric excesses of up to 98%.
Subject(s)
Nitriles/metabolism , Rhodococcus/classification , Rhodococcus/metabolism , Hydrolysis , Species SpecificityABSTRACT
A new enzymatic method for the generation of dihydroxyacetone phosphate (DHAP) using the acid phosphatase from Shigella flexneri (PhoN-Sf) and the cheap phosphate donor pyrophosphate (PPi) is described. The utility of this method was demonstrated in an aldolase-catalyzed condensation carried out in one pot in which DHAP was generated and coupled to propionaldehyde to give a yield of 53% of the isolated dephosphorylated end product.
Subject(s)
Carbohydrate Metabolism , Carbohydrates/chemical synthesis , Dihydroxyacetone Phosphate/chemistry , Dihydroxyacetone Phosphate/metabolism , Phosphates/chemistry , Aldehydes/chemistry , Carbohydrates/chemistry , Magnetic Resonance Spectroscopy , Molecular Structure , Shigella flexneri/enzymologyABSTRACT
An industrially attractive L-specific amidase was purified to homogeneity from Ochrobactrum anthropi NCIMB 40321 wild-type cells. The purified amidase displayed maximum initial activity between pH 6 and 8.5 and was fully stable for at least 1 h up to 60 degrees C. The purified enzyme was strongly inhibited by the metal-chelating compounds EDTA and 1,10-phenanthroline. The activity of the EDTA-treated enzyme could be restored by the addition of Zn2+ (to 80%), Mn2+ (to 400%), and Mg2+ (to 560%). Serine and cysteine protease inhibitors did not influence the purified amidase. This enzyme displayed activity toward a broad range of substrates consisting of alpha-hydrogen- and (bulky) alpha,alpha-disubstituted alpha-amino acid amides, alpha-hydroxy acid amides, and alpha-N-hydroxyamino acid amides. In all cases, only the L-enantiomer was hydrolyzed, resulting in E values of more than 150. Simple aliphatic amides, beta-amino and beta-hydroxy acid amides, and dipeptides were not converted. The gene encoding this L-amidase was cloned via reverse genetics. It encodes a polypeptide of 314 amino acids with a calculated molecular weight of 33,870. Since the native enzyme has a molecular mass of about 66 kDa, it most likely has a homodimeric structure. The deduced amino acid sequence showed homology to a few other stereoselective amidases and the acetamidase/formamidase family of proteins (Pfam FmdA_AmdA). Subcloning of the gene in expression vector pTrc99A enabled efficient heterologous expression in Escherichia coli. Altogether, this amidase has a unique set of properties for application in the fine-chemicals industry.
Subject(s)
Amidohydrolases/genetics , Amidohydrolases/metabolism , Ochrobactrum anthropi/enzymology , Amidohydrolases/antagonists & inhibitors , Amino Acid Sequence , Base Sequence , Consensus Sequence , Enzyme Stability , Hydrogen-Ion Concentration , Kinetics , Molecular Sequence Data , Ochrobactrum anthropi/growth & development , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Restriction Mapping , Sequence Alignment , Sequence Homology, Amino Acid , Stereoisomerism , Substrate Specificity , ThermodynamicsABSTRACT
This article provides an overview of the literature concerning synthetic applications of unsaturated aliphatic amino acids in the period May 2000 to December 2004.
Subject(s)
Amino Acids/chemical synthesis , Chemistry, Organic/methods , Amino Acids, Branched-Chain/chemical synthesis , Indicators and Reagents , Molecular Structure , Protein Binding , Protein Conformation , StereoisomerismABSTRACT
The stereoselective total synthesis of the novel quinolizidine alkaloid (+)-epiquinamide is presented, starting from the amino acid l-allysine ethylene acetal. Key steps in the synthesis involved a highly diastereoselective N-acyliminium ion allylation and a ring-closing metathesis reaction to provide the bicyclic skeleton. [reaction: see text]
Subject(s)
Alkaloids/chemical synthesis , Quinolizines/chemical synthesis , Alkaloids/chemistry , Cyclization , Molecular Conformation , Molecular Structure , Quinolizines/chemistry , StereoisomerismABSTRACT
[reaction: see text] Catalytic Sn(OTf)2-induced cyclization of linear, aryl-containing allylic N,O-acetals produced vinyl-substituted tetrahydroisoquinolines and tetrahydro-1H-beta-carbolines. The usefulness of the vinyl moiety in the resulting products was demonstrated via the synthesis of various key building blocks for alkaloid structures. The alpha-vinyl moiety was utilized in a [2,3] sigmatropic rearrangement, in ring-closing metathesis and a cross-metathesis-based synthesis of vincantril, an antianoxia agent, and a synthetic member of the vincamine type natural products.
Subject(s)
Alkaloids/chemical synthesis , Carbolines/chemical synthesis , Imines/chemistry , Isoquinolines/chemical synthesis , Sulfones/chemistry , Vinyl Compounds/chemistry , Catalysis , Cyclization , Hypoxia , Molecular Structure , StereoisomerismABSTRACT
[reaction: see text] Herein, we report a diastereoselective synthesis of the natural product (2S,5R)-5-hydroxypipecolic acid and 6-substituted derivatives thereof. The key step in the synthetic sequence is a novel highly diastereoselective epoxidation reaction of an enantiomerically pure cyclic enamide intermediate.
Subject(s)
Pipecolic Acids/chemical synthesis , Crystallography, X-Ray , Models, Molecular , Molecular Conformation , Pipecolic Acids/chemistry , StereoisomerismABSTRACT
A palladium-catalyzed amidation of alkoxyallenes has been developed for the construction of linear allylic N,O-acetals under basic conditions involving (cyclic) amides, sulfonamides, carbamates, and amidophosphates. Application of the methodology provided access to the enantiopure 1-ethylquinolizidine structural motif, which is a key synthon in the synthesis of the naturally occurring poisonous frog quinolizidine 233A and derivatives such as the 1-epi-isomer of quinolizidine 207I.
ABSTRACT
A new method to cyclise peptides is reported based on insertion of a salicylaldehyde derived pincer auxiliary in the linear precursor sequence. H-betaAla-Phe-OH and H-Phe-betaAla-OH were chosen as representative model peptides.
Subject(s)
Lactams/chemical synthesis , Oligopeptides/chemistry , Peptides, Cyclic/chemical synthesis , Aldehydes/chemistry , Amino Acid Sequence , Cyclization , Peptides, Cyclic/chemistryABSTRACT
Biocatalysis has emerged as an important tool in the industrial synthesis of bulk chemicals, pharmaceutical and agrochemical intermediates, active pharmaceuticals, and food ingredients. However, the number and diversity of the applications are modest, perhaps in part because of perceived or real limitations of biocatalysts, such as limited enzyme availability, substrate scope, and operational stability. Recent scientific breakthroughs in genomics, directed enzyme evolution, and the exploitation of biodiversity should help to overcome these limitations. As a result, we expect many new industrial applications of biocatalysis to be realized, from single-step enzymatic conversions to customized multistep microbial synthesis by means of metabolic pathway engineering.
Subject(s)
Biotechnology , Catalysis , Chemical Industry , Enzymes/chemistry , Enzymes/metabolism , Carbon/chemistry , Chemistry, Organic , Computational Biology , Directed Molecular Evolution , Ecosystem , Enzyme Stability , Genetic Engineering , Kinetics , Organic Chemistry Phenomena , Oxidation-Reduction , Protein Engineering , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Substrate Specificity , Technology, PharmaceuticalABSTRACT
A stereodivergent route toward both diastereomeric forms of functionalized 4-hydroxypiperidines has been successfully developed. This route involves biocatalytic generation of the enantiopure starting materials followed by functionalization via N-acyliminium ion-mediated CC-bond formation.
ABSTRACT
[reaction: see text] A novel method for the synthesis of medium-sized lactams based on an auxiliary-mediated combined tethered/templated strategy is presented. Cyclization by a tethered ring-closing metathesis reaction was followed by a templated transannular aminolysis reaction to give seven- to ten-membered lactams in good yields.
ABSTRACT
Vanadium haloperoxidases catalyze the oxidation of halides by hydrogen peroxide to produce hypohalous acid. We demonstrate that these enzymes also slowly mediate the enantioselective oxidation of organic sulfides (methyl phenyl sulfide, methyl p-tolyl sulfide, and 1-methoxy-4 (methylthio)benzene) to the corresponding sulfoxides (turnover frequency 1 min(-)(1)). The vanadium bromoperoxidase from the brown seaweed Ascophyllum nodosum converts methyl phenyl sulfide to the (R)-enantiomer of the sulfoxide (55% yield and 85% enantiomeric excess (ee)). At low peroxide concentrations a selectivity of 91% can be attained. The enzyme catalyzes the selective sulfoxidation reaction over a broad pH range with an optimum around pH 5-6 and remains completely functional during the reaction. When the vanadium bromoperoxidase from the red seaweed Corallina pilulifera is used the (S)-enantiomer (18% yield and 55% ee) is formed. In contrast, the vanadium chloroperoxidase from the fungus Curvularia inaequalis catalyzes the production of a racemic mixture (54% yield), which seems to be an intrinsic characteristic of this enzyme.