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Chiral 2‐hydroxy‐4‐arylbut‐3‐enoic acid derivatives are important precursors for the synthesis of angiotensin converting enzyme (ACE) inhibitors, such as enalapril, lisinopril, cilapril or benazepril. In this work, we take advantage of the unexplored promiscuous ketoreductase activity of Δ1‐piperidine‐2‐carboxylate/Δ1‐pyrroline‐2‐carboxylate reductase from Pseudomonas syringae pv. tomato DSM 50315 (DpkAPsyrin) for the synthesis of (S,E)‐2‐hydroxy‐4‐arylbut‐3‐enoic acids. The strategy was designed as an enzymatic cascade comprising an aldol condensation between pyruvate with aryl aldehydes, catalyzed by the trans‐o‐hydroxybenzylidene pyruvate hydratase‐aldolase from Pseudomonas putida (HBPAPputida), for the construction of carbon scaffold, and an ensuing asymmetric reduction of the carbonyl group catalyzed by DpkAPsyrin. The enzymatic cascade provided quantitative conversions, with global isolated yields between 57–85%. A total of nine structurally diverse (S,E)‐2‐hydroxy‐4‐arylbut‐3‐enoic acids were prepared in ee between 87–99%. [ABSTRACT FROM AUTHOR]

Thiamine diphosphate (ThDP) dependent enzymes are useful catalysts for asymmetric C−C bond formation through benzoin‐type condensation reactions that result in α‐hydroxy ketones. A wide range of aldehydes and ketones can be used as acceptor substrates; however, the donor substrate range is mostly limited to achiral α‐keto acids and simple aldehydes. By using a unifying retro‐biosynthetic approach towards acyl‐branched sugars, we identified a subclass of (myco)bacterial ThDP‐dependent enzymes with a greatly extended donor substrate range, namely functionalized chiral α‐keto acids with a chain length from C4 to C8. Highly enantioenriched acyloin products were obtained in good to high yields and several reactions were performed on a preparative scale. The newly introduced functionalized α‐keto acids, accessible by known aldolase‐catalyzed transformations, substantially broaden the donor substrate range of ThDP‐dependent enzymes, thus enabling a more general use of these already valuable catalysts. [ABSTRACT FROM AUTHOR]

Nitrogen heterocycles are structural motifs found in many bioactive natural products and of utmost importance in pharmaceutical drug development. In this work, a stereoselective synthesis of functionalized N‐heterocycles was accomplished in two steps, comprising the biocatalytic aldol addition of ethanal and simple aliphatic ketones such as propanone, butanone, 3‐pentanone, cyclobutanone, and cyclopentanone to N‐Cbz‐protected aminoaldehydes using engineered variants of d‐fructose‐6‐phosphate aldolase from Escherichia coli (FSA) or 2‐deoxy‐d‐ribose‐5‐phosphate aldolase from Thermotoga maritima (DERATma) as catalysts. FSA catalyzed most of the additions of ketones while DERATma was restricted to ethanal and propanone. Subsequent treatment with hydrogen in the presence of palladium over charcoal, yielded low‐level oxygenated N‐heterocyclic derivatives of piperidine, pyrrolidine and N‐bicyclic structures bearing fused cyclobutane and cyclopentane rings, with stereoselectivities of 96–98 ee and 97:3 dr in isolated yields ranging from 35 to 79%. [ABSTRACT FROM AUTHOR]

Abstract: Pyruvate‐dependent aldolases exhibit a stringent selectivity for pyruvate, limiting application of their synthetic potential, which is a drawback shared with other existing aldolases. Structure‐guided rational protein engineering rendered a 2‐keto‐3‐deoxy‐ l‐rhamnonate aldolase variant, fused with a maltose‐binding protein (MBP‐YfaU W23V/L216A), capable of efficiently converting larger pyruvate analogues, for example, those with linear and branched aliphatic chains, in aldol addition reactions. Combination of these nucleophiles with N‐Cbz‐alaninal (Cbz=benzyloxycarbonyl) and N‐Cbz‐prolinal electrophiles gave access to chiral building blocks, for example, derivatives of (2S,3S,4R)‐4‐amino‐3‐hydroxy‐2‐methylpentanoic acid (68 %, d.r. 90:10) and the enantiomer of dolaproine (33 %, d.r. 94:6) as well as a collection of unprecedented α‐amino acid derivatives of the proline and pyrrolizidine type. Conversions varied between 6–93 % and diastereomeric ratios from 50:50 to 95:5 depending on the nucleophilic and electrophilic components. [ABSTRACT FROM AUTHOR]

4-Hydroxy-2-keto acid derivatives are versatile building blocks for the synthesis of amino acids, hydroxy carboxylic acids and chiral aldehydes. Pyruvate aldolases are privileged catalysts for a straightforward access to this class of keto acid compounds. In this work, a Class II pyruvate aldolase from Escherichia coli K-12, 2-keto-3-deoxy- l-rhamnonate aldolase (YfaU), was evaluated for the synthesis of amino acid derivatives of proline, pipecolic acid, and pyrrolizidine-3-carboxylic acid. The aldol addition of pyruvate to N-protected amino aldehydes was the key enzymatic aldol addition step followed by catalytic intramolecular reductive amination. The corresponding N-Cbz-amino-4-hydroxy-2-keto acid (Cbz=benzyloxycarbonyl) precursors were obtained in 51-95% isolated yields and enantioselectivity ratios from 26:74 to 95:5, with chiral α-substituted N-Cbz-amino aldehydes. ( S)- N-Cbz-amino aldehydes gave aldol adducts with preferentially ( R)-configuration at the newly formed stereocenter, whereas the contrary is true for ( R)- N-Cbz-amino aldehydes. Addition reactions to achiral amino aldehydes rendered racemic aldol adducts. Molecular models of the pre-reaction ternary complexes YfaU-pyruvate enolate-acceptor aldehyde were constructed to explain the observed stereochemical outcome of the reactions. Catalytic reductive amination of the aldol adducts yielded 4-hydroxy-2-pipecolic acid, and unprecedented C-5 substituted 4-hydroxyproline and pyrrolizidine-3-carboxylic acid derivatives. [ABSTRACT FROM AUTHOR]

Intramolecular benzoin reactions catalyzed by benzaldehyde lyase from Pseudomonas fluorescens biovar I (BAL) are reported. The structure of the substrates envisaged for this reaction consists of two benzaldehyde derivatives linked by an alkyl chain. The structural requirements needed to achieve the intramolecular carbon-carbon bond reaction catalyzed by BAL were established. Thus, a linker consisting of a linear alkyl chain of three carbon atoms connected through ether-type bonds to the 2 and 2′ positions of two benzaldehyde moieties, which could be substituted with either Cl, Br, or OCH3 at either the 3 and 3′ or 5 and 5′ positions, were suitable substrates for BAL. Reactions with 61-84 % yields of the intramolecular product and ee values between 64 and 98 %, were achieved. [ABSTRACT FROM AUTHOR]

Rhamnulose-1-phosphate aldolase from Thermotoga maritima (Rhu1PA Tm) has been recently cloned and characterised. This hyperthermophilic enzyme offers intriguing possibilities for practical catalysis. This is due to its high stability under extreme reaction conditions, such as high temperature or the presence of organic co-solvents. The Rhu1PA Tm potentiality in organic synthesis has been explored focusing on (i) the reaction stereocontrol, (ii) the possibility of combining it with other mesophilic enzymes in multienzyme systems and (iii) its application to the synthesis of imino- and nitrocyclitols. In our study, the diastereoselectivity of Rhu1PA Tm was similar to the one reported for Rhu1PA from Escherichia coli (Rhu1PA Ec). However, we observed significant differences for some aldehyde acceptors. Indeed, the diastereoselectivity control was not complete, since mixtures of L- threo (2 R,3 S; natural stereopreference) and D- erythro (2 R,3 R) diastereoisomers were obtained as described for Rhu1PA Ec. Rhu1PA Tm was able to catalyse aldol reactions using nitroaldehydes and N-Cbz-aminoaldehydes. Conversion of the selected nitroaldehydes was complete, leading to mixtures of L- threo and D -erythro diastereoisomers, in 95:5 and 75:25 ratios. When N-Cbz-aminoaldehydes were used, aldol reaction yields were lower and the stereoselectivity L- threo: D -erythro ranged between 32:68 to>95:5. [ABSTRACT FROM AUTHOR]

The introduction of aromatic residues connected by a CC bond into the non-reducing end of carbohydrates is highly significant for the development of innovative structures with improved binding affinity and selectivity (e.g., Caril-sLex). In this work, an expedient asymmetric 'de novo' synthetic route to new aryl carbohydrate derivatives based on two sequential stereoselectively biocatalytic carboligation reactions is presented. First, the benzoin reaction of aromatic aldehydes to dimethoxyacetaldehyde is conducted, catalyzed by benzaldehyde lyase from Pseudomonas fluorescens biovar I. Then, the α-hydroxyketones formed are reduced by using NaBH4 yielding the anti diol. After acetal hydrolysis, the aldol addition of dihydroxyacetone, hydroxyacetone, or glycolaldehyde catalyzed by the stereocomplementary D-fructose-6-phosphate aldolase and L-rhamnulose-1-phosphate aldolase is performed. Both aldolases accept unphosphorylated donor substrates, avoiding the need of handling the phosphate group that the dihydroxyacetone phosphate-dependent aldolases require. In this way, 6- C-aryl- L-sorbose, 6- C-aryl- L-fructose, 6- C-aryl- L-tagatose, and 5- C-aryl- L-xylose derivatives are prepared by using this methodology. [ABSTRACT FROM AUTHOR]

A multistep chemoenzymatic synthesis for stereodiverse polyhydroxypipecolic acid analogues, homoiminocyclitols and polyhydroxylated piperidine and pyrrolidine derivatives combining glycine-dependent aldolases and both D-fructose-6-phosphate aldolase (FSA) or dihydroxyacetone phosphate (DHAP)-dependent aldolases is presented. The methodology allowed the preparation of known and innovative imine-derived molecules with a great structural diversity from simple achiral substrates. The strategy consisted of two key aldol addition steps: a first aldol addition of glycine to dimethoxyacetaldehyde catalyzed by L- and D-glycine aldolases and a second aldol addition of DHAP, dihydroxyacetone, hydroxyacetone or glycolaldehyde using FSA or DHAP-dependent aldolases as catalysts to a conveniently transformed aldol adduct from the first aldol addition. Catalytic reductive amination on the aldol adducts rendered the polyhydroxypipecolic acid analogues, (homo)iminocyclitols and polyhydroxylated pyrrolidine iminocyclitols. The reported strategy is thus designed to create up to five new stereogenic centers in three steps, four of them being controlled in two enzymatic reactions. Moreover, it allowed the installation of diverse functionalities in the molecules. This was possible by taking the full advantage of using aldolases in a multistep approach by virtue of their stereocomplementarity, stereoselectivity and broad substrate tolerance. [ABSTRACT FROM AUTHOR]

Abstract: A monoaminated dextran derivative was attached to trypsin via a carbodiimide-catalyzed reaction. The modified enzyme contained 3mol of polysaccharide per mol of protein, and retained about 93% and 85% of the native esterolytic and proteolytic activity, respectively. The thermostability was enhanced from 49.7 to 67.4°C for modified trypsin. The activation free energy of thermal inactivation at 55°C was increased by 7.2kJ/mol for the protease after modification with the polymer. The improved conformational stability of trypsin after glycosidation with dextran was confirmed by fluorescence spectroscopy. The glycosidated protease retained 70% of its initial activity after 3h incubation at pH 9.0. [Copyright &y& Elsevier]

Moringa has leaves, flowers and fruit with a high level of vitamins, minerals and proteins of interest for the food and medical industries. This species can be propagated by seeds or cuttings, methods that not suitable to introduce new cultivars. The aim of this research was to establish an in vitro propagation protocol of different cultivars of Moringa oleifera Lam. To disinfect seeds three different disinfection times were evaluated five, seven and nine minutes in 10 gL-1 sodium hipoclorite. Two methods for in vitro micropropagation were used: conventional system (semi-solid culture medium) and temporary immersion bioreactor. In the in vitro rooting phase, four concentrations of the naphthalene acetic acid and Indole butyric acid auxins (0; 2,5; 5,0 and 7,5 μmol L-1) were evaluated and also the kind of explant. The survival rates of the sprout during the acclimatization phase was evaluated in relation to their origin in the rooting phase. Besides the effect of the genotype on in vitro establishment and multiplication was determined. The disinfection of the Supergenius cultivar was better at seven minutes of treatment with sodium hipoclorite, with 54 % of germination. The Temporary Immersion Bioreactor increased the sprouts morphological quality and multiplication coefficient from 6,2 until 16,1 of the Supergenius cultivar. Rooting was successful without auxin and acclimatization survival was around 85 % at 35 days for shoots rooted without growth regulators, regardless of explant origin. It was shown that the genotype influenced the establishment and multiplication of explants. [ABSTRACT FROM AUTHOR]

We present a methodology for improving some enzymatic characteristics of glucose oxidase (GOx) (EC 1.1.3.4). The enzyme was chemically modified with a β-cyclodextrin-branched carboxymethylcellulose polymer (CMC-CD), using carbodiimide as coupling agent. The obtained neoglycoenzyme had 0.78 mol of polysaccharide per mol of GOx and retained 67% of its initial activity. Comparison of some characteristics of the modified and free enzymes showed a higher Km for derivatized GOx and better thermostability, which increased from 45 °C to 51 °C. In addition, derivatization of GOx with CMC-CD increased its resistance to inactivation at 45 °C by 2.2-fold, protected the molecule against inactivation with the anionic surfactant sodium dodecylsulphate to the point that it retained 75% of its activity after an incubation of 3 h, and extended its pH tolerance toward alkaline pH (7.5). Covalent glycosidation of glucose oxidase with CD-branched carboxymethylcellulose polymer constitutes therefore an effective strategy for enhancing the stability of this enzyme. [ABSTRACT FROM AUTHOR]