D-Fructose-6-phosphate aldolase-catalyzed one-pot synthesis of iminocyclitols

A one-pot chemoenzymatic method for the synthesis of a variety of new iminocyclitols from readily available, non-phosphorylated donor substrates has been developed. The method utilizes the recently discovered fructose-6-phosphate aldolase (FSA), which is functionally distinct from known aldolases in its tolerance of different donor substrates as well as acceptor substrates. Kinetic studies were performed with dihydroxyacetone (DHA), the presumed endogenous substrate for FSA, as well as hydroxy acetone (HA) and 1-hydroxy-2-butanone (HB) as donor substrates, in each case using glyceraldehyde-3-phosphate as acceptor substrate. Remarkably, FSA used the three donor substrates with equal efficiency, with kcat/KMvalues of 33, 75, and 20 M-1 s-1, respectively. This level of donor substrate tolerance is unprecedented for an aldolase. Furthermore, DHA, HA, and HB were accepted as donors in FSA-catalyzed aldol reactions with a variety of azido- and Cbz-amino aldehyde acceptors. The broad substrate tolerance of FSA and the ability to circumvent the need for phosphorylated substrates allowed for one-pot synthesis of a number of known and novel iminocyclitols in good yields, and in a very concise fashion. New iminocyclitols were assayed as inhibitors against a panel of glycosidases. Compounds 15 and 16 were specific alpha-mannosidase inhibitors, and 24 and 26 were potent and selective inhibitors of beta-N-acetylglucosaminidases in the submicromolar range. Facile access to these compounds makes them attractive core structures for further inhibitor optimization.     

Biosynthesis of a Tricyclo[6.2.2.02,7]dodecane System by a Berberine Bridge Enzyme-Like Aldolase

The aldol reaction is one of the most fundamental stereocontrolled carbon–carbon bond-forming reactions and is mainly catalyzed by aldolases in nature. Despite the fact that the aldol reaction has been widely proposed to be involved in fungal secondary metabolite biosynthesis, a dedicated aldolase that catalyzes stereoselective aldol reactions has only rarely been reported in fungi. Herein, we activated a cryptic polyketide biosynthetic gene cluster that was upregulated in the fungal wheat pathogen Parastagonospora nodorum during plant infection; this resulted in the production of the phytotoxic stemphyloxin II ( 1 ). Through heterologous reconstruction of the biosynthetic pathway and in vitro assay by using cell-free lysate from Aspergillus nidulans, we demonstrated that a berberine bridge enzyme (BBE)-like protein SthB catalyzes an intramolecular aldol reaction to establish the bridged tricyclo[6.2.2.0^2,7]dodecane skeleton in the post-assembly tailoring step. The characterization of SthB as an aldolase enriches the catalytic toolbox of classic reactions and the functional diversities of the BBE superfamily of enzymes.     

Fluorogenic polypropionate fragments for detecting stereoselective aldolases

A series of fluorogenic polypropionate fragments has been prepared. These undergo retroaldolization to an intermediate aldehyde that liberates the fluorescent product umbelliferone by a secondary beta-elimination reaction. leading to a >20-fold increase in fluorescence (lambda(em) = 460 +/- 20 nm, lambdaex = 360 +/- 20 nm). By applying the principle of microscopic reversibility to the reversible aldol reaction, we can use these substrates to detect stereoselective aldolases. Test substrates are available to probe the classical cases of syn- and anti-selective aldolization (11a-d), Cram/ anti-Cram-selective aldolization (10a-d), and double stereoselective aldolization (3a-h). The selectivity of aldolase antibody 38C2 for these substrates is demonstrated as an example. The assay is suitable for high-throughput screening for catalysis in microtiter plates, and therefore provides a convenient tool for the isolation of new stereoselective aldolases from catalyst libraries.     

Serine hydroxymethyl transferase from Streptococcus thermophilus and L-threonine aldolase from Escherichia coli as stereocomplementary biocatalysts for the synthesis of beta-hydroxy-alpha,omega-diamino acid derivatives

A novel serine hydroxymethyl transferase from Streptococcus thermophilus (SHMT) and a L-threonine aldolase from Escherichia coli (LTA) were used as stereocomplementary biocatalysts for the aldol addition of glycine to N-Cbz amino aldehydes and benzyloxyacetaldehyde (Cbz=benzyloxycarbonyl). Both threonine aldolases were classified as low-specific L-allo-threonine aldolases, and by manipulating reaction parameters, such as temperature, glycine concentration, and reaction media, SHMT yielded exclusively L-erythro diastereomers in 34-60 % conversion, whereas LTA gave L-threo diastereomers in 30:70 to 16:84 diastereomeric ratios and with 40-68 % conversion to product. SHMT is among the most stereoselective L-threonine aldolases described. This is due, among other things, to its activity-temperature dependence: at 4 degrees C SHMT has high synthetic activity but negligible retroaldol activity on L-threonine. Thus, the kinetic L-erythro isomer was largely favored and the reactions were virtually irreversible, highly stereoselective, and in turn, gave excellent conversion. It was also found that treatment of the prepared N-Cbz-gamma-amino-beta-hydroxy-alpha-amino acid derivatives with potassium hydroxide (1 m) resulted in the spontaneous formation of 2-oxazolidinone derivatives of the beta-hydroxyl and gamma-amino groups in quantitative yield. This reaction might be useful for further chemical manipulations of the products.     

A ribozyme for the aldol reaction

Directed in vitro evolution can create RNA catalysts for a variety of organic reactions, supporting the "RNA world" hypothesis, which proposes that metabolic transformations in early life were catalyzed by RNA molecules rather than proteins. Among the most fundamental carbon-carbon bond-forming reactions in nature is the aldol reaction, mainly catalyzed by aldolases that utilize either an enamine mechanism (class I) or a Zn(2+) cofactor (class II). We report on isolation of a Zn(2+)-dependent ribozyme that catalyzes an aldol reaction at its own modified 5' end with a 4300-fold rate enhancement over the uncatalyzed background reaction. The ribozyme can also act as an intermolecular catalyst that transfers a biotinylated benzaldehyde derivative to the aldol donor substrate, coupled to an external hexameric RNA oligonucleotide, supporting the existence of RNA-originated biosynthetic pathways for metabolic sugar precursors and other biomolecules.     

Chemoenzymatic Platform for Synthesis of Chiral Organofluorines Based on Type II Aldolases

Aldolases are C?C bond forming enzymes that have become prominent tools for sustainable synthesis of complex synthons. However, enzymatic methods of fluorine incorporation into such compounds are lacking due to the rarity of fluorine in nature. Recently, the use of fluoropyruvate as a non‐native aldolase substrate has arisen as a solution. Here, we report that the type II HpcH aldolases efficiently catalyze fluoropyruvate addition to diverse aldehydes, with exclusive (3S)‐selectivity at fluorine that is rationalized by DFT calculations on a mechanistic model. We also measure the kinetic parameters of aldol addition and demonstrate engineering of the hydroxyl group stereoselectivity. Our aldolase collection is then employed in the chemoenzymatic synthesis of novel fluoroacids and ester derivatives in high stereopurity (d.r. 80–98 %). The compounds made available by this method serve as precursors to fluorinated analogs of sugars, amino acids, and other valuable chiral building blocks.     

Creation of a shikimate pathway variant

The competition between the Escherichia coli carbohydrate phosphotransferase system and 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase for phosphoenolpyruvate limits the concentration and yield of natural products microbially synthesized via the shikimate pathway. To circumvent this competition for phosphoenolpyruvate, a shikimate pathway variant has been created. 2-Keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolases encoded by Escherichia coli dgoA and Klebsiella pneumoniae dgoA are subjected to directed evolution. The evolved KDPGal aldolase isozymes exhibit 4-8-fold higher specific activities relative to that for native KDPGal aldolase with respect to catalyzing the condensation of pyruvate and d-erythrose 4-phosphate to produce DAHP. To probe the ability of the created shikimate pathway variant to support microbial growth and metabolism, growth rates and synthesis of 3-dehydroshikimate are examined for E. coli constructs that lack phosphoenolpruvate-based DAHP synthase activity and rely on evolved KDPGal aldolase for biosynthesis of shikimate pathway intermediates and products.     

Synthesis of Branched‐Chain Sugars with a DHAP‐Dependent Aldolase: Ketones are Electrophile Substrates of Rhamnulose‐1‐phosphate Aldolases

Dihydroxyacetone phosphate (DHAP)‐dependent rhamnulose aldolases display an unprecedented versatility for ketones as electrophile substrates. We selected and characterized a rhamnulose aldolase from Bacteroides thetaiotaomicron (RhuABthet) to provide a proof of concept. DHAP was added as a nucleophile to several α‐hydroxylated ketones used as electrophiles. This aldol addition was stereoselective and produced branched‐chain monosaccharide adducts with a tertiary alcohol moiety. Several aldols were readily obtained in good to excellent yields (from 76 to 95 %). These results contradict the general view that aldehydes are the only electrophile substrates for DHAP‐dependent aldolases and provide a new C?C bond‐forming enzyme for stereoselective synthesis of tertiary alcohols.     

Small peptides as modular catalysts for the direct asymmetric aldol reaction: ancient peptides with aldolase enzyme activity

Simple peptides and their analogues having a primary amino group as the catalytic residue mediate the direct asymmetric intermolecular aldol reaction with high stereoselectivity and furnish the corresponding aldol products with up to 99% ee; this intrinsic ability of highly modular peptides may explain the initial molecular evolution of aldolase enzymes.     

Class I fructose-1,6-bisphosphate aldolases as catalysts for asymmetric aldol reactions

The activity of two commercially available bacterial class I fructose-1,6-bisphosphate aldolases (FruA) towards a number of aldehydes has been compared with rabbit muscle aldolase (RAMA), which is the most widely used enzyme for aldol reactions with dihydroxyacetone phosphate. The kinetic properties of the three aldolases are very similar, but the bacterial aldolases were much more stable than RAMA. Reaction of butanal and dihydroxyacetone phosphate catalyzed by FruA from Staphylococcus carnosus was performed on a 5 mmol scale in 53% isolated yield. Enantiomeric and diastereomeric purity of the major reaction product [90% (3S,4R)] was determined by chiral gas chromatography.     

Highly efficient antibody-catalyzed deuteration of carbonyl compounds

Antibody 38C2 efficiently catalyzes deuterium-exchange reactions at the alpha position of a variety of ketones and aldehydes, including substrates that have a variety of sensitive functional groups. In addition to the regio- and chemoselectivity of these reactions, the catalytic rates (kcat) and rate-enhancement values (kcat/kun) are among the highest values ever observed with catalytic antibodies. Comparison of the substrate range of the catalytic antibody with highly evolved aldolase enzymes, such as rabbit-muscle aldolase, highlights the much broader practical scope of the antibody, which accepts a wide range of substrates. The hydrogen-exchange reaction was used for calibration and mapping of the antibody active site. Isotope-exchange experiments with cycloheptanone reveal that the formation of the Schiff base species (as concluded from the 16O/18O exchange rate at the carbonyl oxygen) is much faster than the formation of the enamine intermediate (as concluded from the H/D exchange rate), and both steps are faster than the antibody-catalyzed aldol addition reaction.     

Stereoselective aldol additions catalyzed by dihydroxyacetone phosphate-dependent aldolases in emulsion systems: preparation and structural characterization of linear and cyclic iminopolyols from aminoaldehydes

The potential of dihydroxyacetone phosphate (DHAP)-dependent aldolases to catalyze stereoselective aldol additions is, in many instances, limited by the solubility of the acceptor aldehyde in aqueous/co-solvent mixtures. Herein, we demonstrate the efficiency of emulsion systems as reaction media for the class I fructose-1,6-bisphosphate aldolase (RAMA) and class II recombinant rhamnulose-1-phosphate aldolase from E. coli (RhuA)-catalyzed aldol addition between DHAP and N-benzyloxycarbonyl (N-Cbz) aminoaldehydes. The use of emulsions improved the RAMA-catalyzed aldol conversions by three to tenfold relative to those in conventional DMF/water mixtures. RhuA was more reactive than RAMA towards the N-Cbz aminoaldehydes regardless of the reaction medium. With (S)- or (R)-Cbz-alaninal, RAMA exhibited preference for the R enantiomer, while RhuA had no enantiomeric discrimination. The linear N-Cbz aminopolyols thus obtained were submitted to catalytic intramolecular reductive amination to afford the corresponding iminocyclitols. This reaction was diastereoselective in all cases examined; the face selectivity was controlled by the stereochemistry of the newly formed hydroxyl group originating from the aldehyde. Characterization of the resulting iminocyclitols allowed the assessment of the diastereoselectivity of the enzymatic aldol reactions with respect to the N-protected aminoaldehyde. RAMA formed single diastereoisomers from N-Cbz-glycinal and from both enantiomers of N-Cbz-alaninal, while 14 % of the epimeric product was observed from N-Cbz-3-aminopropanal. Diastereoselectivity from RhuA was lower than that observed from RAMA. Interestingly, a single diastereoisomer was formed from (S)-Cbz-alaninal, whereas only a 34 % diastereomeric excess was observed from its enantiomer (i.e., (R)-Cbz-alaninal).     

A novel genetic selection system for PLP-dependent threonine aldolases

Threonine aldolases are versatile pyridoxal-5′-phosphate (PLP)-dependent enzymes key to glycine, serine and threonine metabolism. Because they catalyze the reversible addition of glycine to an aldehyde to give β-hydroxy-α-amino acids, they are also attractive as biotechnological catalysts for the diastereoselective synthesis of many pharmaceutically useful compounds. To study and evolve such enzymes, we have developed a simple selection system based on the simultaneous inactivation of four genes involved in glycine biosynthesis in Escherichia coli. Glycine prototrophy in the deletion strain is restored by expression of a gene encoding an aldolase that converts β-hydroxy-α-amino acids, provided in the medium, to glycine and the corresponding aldehyde. Combinatorial mutagenesis and selection experiments with a previously uncharacterized l-threonine aldolase from Caulobacter crescentus CB15 (Cc-LTA) illustrate the power of this system. The codons for four active site residues, His91, Asp95, Glu96, and Asp176, were simultaneously randomized and active variants selected. The results show that only His91, which π-stacks against the PLP cofactor and probably serves as the catalytic base in the carbon-carbon bond cleavage step, is absolutely required for aldolase activity. In contrast, Asp176, one of the most conserved residues in this enzyme superfamily, can be replaced conservatively by glutamate, albeit with a >5000-fold decrease in efficiency. Though neither Asp95 nor Glu96 is catalytically essential, they appear to modulate substrate binding and His91 activity, respectively. The broad dynamic range of this novel selection system should make it useful for mechanistic investigations and directed evolution of many natural and artificial aldolases.     

Rational design of stereoselectivity in the class II pyruvate aldolase BphI

BphI, a pyruvate-specific class II aldolase, catalyzes the reversible carbon-carbon bond formation of 4-hydroxy-2-oxoacids up to eight carbons in length. During the aldol addition catalyzed by BphI, the S-configured stereogenic center at C4 is created via attack of a pyruvate enolate intermediate on the si face of the aldehyde carbonyl of acetaldehyde to form 4(S)-hydroxy-2-oxopentanoate. Replacement of a Leu-87 residue within the active site of the enzyme with polar asparagine and bulky tryptophan led to enzymes with no detectable aldolase activity. These variants retained decarboxylase activity for the smaller oxaloacetate substrate, which is not inhibited by excess 4-hydroxy-2-oxopentanoate, confirming the results from molecular modeling that Leu-87 interacts with the C4-methyl of 4(S)-hydroxy-2-oxoacids. Double variants L87N;Y290F and L87W;Y290F were constructed to enable the binding of 4(R)-hydroxy-2-oxoacids by relieving the steric hindrance between the 5-methyl group of these compounds and the hydroxyl substituent on the phenyl ring of Tyr-290. The resultant enzymes were shown to exclusively utilize only 4(R)- and not 4(S)-hydroxy-2-oxopentanoate as the substrate. Polarimetric analysis confirmed that the double variants are able to synthesize 4-hydroxy-2-oxoacids up to eight carbons in length, which were the opposite stereoisomer compared to those produced by the wild-type enzyme. Overall the k(cat)/K(m) values for pyruvate and aldehydes in the aldol addition reactions were affected ≤10-fold in the double variants relative to the wild-type enzyme. Thus, stereocomplementary class II pyruvate aldolases are now available to create chiral 4-hydroxy-2-oxoacid skeletons as synthons for organic reactions.     

A Type 1 Aldolase,NahE, Catalyzes a Stereoselective Nitro-Michael Reaction: Synthesis of β-Aryl-γ-nitrobutyric Acids

Michael addition reactions are highly useful in organic synthesis and are commonly accomplished using organocatalysts. However, the corresponding biocatalytic Michael additions are rare, typically lack synthetically useful substrate scope, and suffer from low stereoselectivity. Herein we report a biocatalytic nitro-Michael addition, catalyzed by NahE, that proceeds with low catalyst loading at room temperature in moderate to excellent enantioselectivity and high yields. A series of β-nitrostyrenes reacted with pyruvate in the presence of NahE to give, after oxidative decarboxylation, β-aryl-γ-nitrobutyric acids in up to 99 % yield without need for chromatography, providing a simple preparative-scale route to chiral GABA analogues. This reaction represents the first example of an aldolase displaying promiscuous Michaelase activity and opens the use of nitroalkenes in place of aldehydes as substrates for aldolases.     

Synthesis of fluorinated materials catalyzed by proline or antibody 38C2 in ionic liquid

The utility of reusable ionic liquid-proline (or aldolase antibody 38C2) reaction system, proceeding the aldol reactions, is described. Further, obtained α-chloro-β-hydroxy compounds were transformed to the optically active α,β-epoxy carbonyl compounds. The aldolase antibody 38C2-ionic liquid system was able to reuse in Michael additions and the reaction of fluoromethylated imines.     

Synthesis of molluscicidal agent cyanolide a macrolactone from D-(-)-pantolactone

An efficient synthesis of potent molluscicidal agent cyanolide A, a glycosidic 16-membered macrolide, starting from D-(-)-pantolactone is reported. Highly stereoselective aldol, oxa-Michael addition, and Yamaguchi macrolactonization are the key steps in the present synthesis.     

One-pot synthesis of L-Fructose using coupled multienzyme systems based on rhamnulose-1-phosphate aldolase

Two methods have been developed for the highly efficient enzymatic synthesis of L-fructose: one is based on rhamnulose-1-phosphate aldolase and acid phosphatase using racemic glyceraldehyde and dihydroxyacetone phosphate as substrates; the other is to generate enantiomerically pure L-glyceraldehyde in situ from glycerol for the aldol reaction, using galactose oxidase catalyzed oxidation of glycerol in the presence of catalase. Using this four-enzyme system, enantiomerically pure L-fructose was obtained. Using the more expensive dihydroxyacetone phosphate, the yield was 55% after purification.     

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

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.