P. Clapés (IQAC-CSIC)

Biocatalytic Construction of Quaternary Centers by Aldol Addition of 3,3-Disubstituted 2-Oxoacid Derivatives to Aldehydes

The congested nature of quaternary carbons hinders their preparation, most notably when stereocontrol is required. Here we report a biocatalytic method for the creation of quaternary carbon centers with broad substrate scope, leading to different compound classes bearing this structural feature. The key step comprises the aldol addition of 3,3-disubstituted 2-oxoacids to aldehydes catalyzed by metal dependent 3-methyl-2-oxobutanoate hydroxymethyltransferase from E. coli (KPHMT) and variants thereof. The 3,3,3-trisubstituted 2-oxoacids thus produced were converted into 2-oxolactones and 3-hydroxy acids and directly to ulosonic acid derivatives, all bearing gem-dialkyl, gem-cycloalkyl, and spirocyclic quaternary centers. In addition, some of these reactions use a single enantiomer from racemic nucleophiles to afford stereopure quaternary carbons. The notable substrate tolerance and stereocontrol of these enzymes are indicative of their potential for the synthesis of structurally intricate molecules.

Aldolase-Catalyzed Asymmetric Synthesis of N-Heterocycles by Addition of Simple Aliphatic Nucleophiles to Aminoaldehydes

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 (DERA_Tma) as catalysts. FSA catalyzed most of the additions of ketones while DERA_Tma 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%.

Nucleophile Promiscuity of Engineered Class II Pyruvate Aldolase from E. Coli, YfaU

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.

Intramolecular Benzoin Reaction catalyzed by benzaldehyde lyase from pseudomonas Fluorescens Biovar I.

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.

Asymmetric assembly of formaldehyde and glycolaldehyde into aldose carbohydrates by tandem biocatalytic aldol reactions

The preparation of multifunctional chiral molecules can be largely simplified by a sequential catalytic assembly of achiral building blocks. In the bottom-up synthesis of aldose sugars, biomimetic routes starting from simple substrates result inconveniently complex, requiring multiple enzymes and cofactors that hamper the efficiency of the process. Pursuing a simpler and more expedient enzymatic approach, we used engineered variants of the D-fructose-6-phosphate aldolase from E. coli (FSA) to prepare a stereochemically consistent series of three to six-carbon aldoses by sequential addition of formaldehyde and glycolaldehyde. Notably, the pertinent selection of the aldolase mutant provides control on the sugar size. The stereochemical outcome of the addition was also altered to allow the one-pot synthesis of L-glucose and derivatives. Engineered biocatalysts offer new avenues to the straightforward synthesis of natural molecules and analogues, which circumvent the intricate enzymatic pathways forged by evolution.

Structure-Guided Engineering of d-Fructose-6-Phosphate Aldolase for Improved Acceptor Tolerance in Biocatalytic Aldol Additions

A structure-guided redesign of d-fructose-6-phosphate aldolase from Escherichia coli (FSA) was devised for improving the acceptor tolerance towards a-substituted and conformationally constrained aldehydes. Two FSA variants, were the most suited biocatalysts for dihydroxyacetone, hydroxyacetone and glycolaldehyde additions to 20 a-substituted N-Cbz-aminoaldehydes (Cbz=benzyloxycarbonyl) including pyrrolidine and piperidine derivatives. Full kinetic stereocontrol for si-si face addition of the aldolase-bound nucleophile to the N-Cbz-aminoaldehyde carbonyl was observed, furnishing the corresponding d-threo configured aldol adduct in >95:5 dr as assessed by NMR. After reductive amination, 47 different iminocyclitols were identified and characterized. In some examples partial racemization of the corresponding aldehyde was observed, which appears to be produced mostly during the aldol addition reactions.

Expedient Synthesis of C-Aryl Carbohydrates by Consecutive Biocatalytic Benzoin and Aldol Reactions

An expedient asymmetric “de novo” synthetic route to new aryl carbohydrate (6-C-aryl-l-sorbose, 6-C-aryl–l-fructose, 6-C-aryl–l-tagatose, and 5-C-aryl-l-xylose) derivatives based on two sequential stereoselectively biocatalytic carboligation reactions is presented.

Sequential Biocatalytic Aldol Reactions in Multistep Asymmetric Synthesis: Pipecolic Acid, Piperidine and Pyrrolidine (Homo)Iminocyclitol Derivatives from Achiral Building Blocks

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 reported.

Aldolase-Catalyzed Synthesis of Conformationally Constrained Iminocyclitols: Preparation of Polyhydroxylated Benzopyrrolizidines and Cyclohexapyrrolizidines

A straightforward chemo-enzymatic synthesis of new polyhydroxylated benzopyrrolizidines and cyclohexapyrrolizidines is developed. The two-step strategy consists of L-fuculose-1-phosphate aldolase variant F131A-catalyzed aldol addition of dihydroxyacetone phosphate to rac-N-benzyloxycarbonylindoline-2-carbaldehyde derivatives and a subsequent one-step catalytic deprotection−reductive amination.

Chemoenzymatic synthesis, structural study and biological activity of novel indolizidine and quinolizidine iminocyclitols

The synthesis, conformational study and inhibitory properties of diverse indolizidine and quinolizidine iminocyclitols are described. The compounds were chemo-enzymatically synthesized by two-step aldol addition and reductive amination reactions. A structural analysis by NMR and in silico density functional theory (DFT) calculations allowed us to determine the population of stereoisomers with the trans or cis ring fusion, as a consequence of the inversion of configuration of the bridgehead nitrogen.Indolizidines were found to be moderate inhibitors of the rat intestinal sucrase and of the exoglucosidase amyloglucosidase from Aspergillus niger.