Recent progress in stereoselective synthesis with aldolases

doi:10.1016/j.cbpa.2009.11.029

Current Opinion in Chemical Biology

Volume 14, Issue 2, April 2010, Pages 154-167

https://doi.org/10.1016/j.cbpa.2009.11.029 Get rights and content

Aldol reactions constitute a powerful methodology for carbon–carbon bond formation in synthetic organic chemistry. Biocatalysis by means of aldolases offers a unique stereoselective and green tool to perform this transformation. Recent advances in the field, fueled by either protein engineering or screening, greatly improved the number of synthetic opportunities from small chiral polyfunctional molecules to highly complex oligosaccharide analogs with potential pharmaceutical relevance. Furthermore, aldolases have been shown to be particularly valuable for obtaining new types of structures (i.e. generate molecular diversity) accessible for investigations in drug discovery. Extensive knowledge arising from biochemical studies and synthetic applications of natural aldolases has fostered the development of novel catalysts, such as the de novo computational design of aldolase enzymes, aldolase ribozymes, or synthetic peptides and foldamers with aldolase activity, outlining first steps toward the creation of tailor-made (bio)catalysts to suit any desired application.

Introduction

Carbon–carbon bond formation is one of the cornerstone reactions in synthetic organic chemistry. Among the tools used to perform this transformation, aldol reactions offer one of the most powerful strategies toward the synthesis of enantiopure multifunctional molecules. Hence, developing aldol processes with precise control over the stereochemistry at the newly formed C–C bond by catalyst control are of paramount importance. Aldolases catalyze the reversible formation of C–C bonds by the aldol addition of a nucleophilic donor, typically a ketone enolate (or analog), onto an electrophilic aldehyde acceptor. Concomitantly, the stereochemistry at the newly formed stereo center(s) in most cases is strictly controlled by the enzyme. This makes aldolases attractive tools in the synthesis of chiral complex, bioactive compounds, such as carbohydrates, amino acids, and their analogs. Whereas aldolases can typically use a broad range of aldehydes as acceptors, the donor compound is often structurally invariable. Hence, aldolases can be classified according to their donor specificity into firstly, pyruvate/2-oxobutyrate aldolases; secondly, dihydroxyacetone phosphate (DHAP) aldolases; thirdly, DHA aldolases; fourthly, glycine/alanine aldolases; and fifthly, acetaldehyde aldolases. Another discrimination concerns the different enzyme mechanisms to activate the nucleophilic component. Class I aldolases exhibit a conserved lysine residue in the active site which forms a Schiff base intermediate with the donor compound to generate an enamine nucleophile. In Class II aldolases a divalent metal ion promotes the enolization of the donor substrate via Lewis acid complexation. The nucleophilic enamine or enolate then attacks the carbonyl carbon of the acceptor substrate forming the new C–C bond.

During the last two decades, an increasing number of applications of aldolases in stereoselective synthesis have been reported. In this overview, because of space limitations we summarize the major progress of the last four years only; beyond the scope are reports of aldolase applications that lack sufficient specificity, or that are merely mechanistic or structural in focus. For a more comprehensive coverage of the general topic we refer the readers to previous reviews [1•, 2•, 3, 4•].

Section snippets

Pyruvate aldolases

Pyruvate-dependent aldolases are usually Class I aldolases (i.e. Schiff base/enamine formation) that reversibly catalyze the aldol addition of pyruvate to a variety of polyhydroxylated aldehydes yielding α-oxo acids. N-Acetylneuraminic acid lyase (NeuA) and its mutants are among the most studied and have been extensively exploited for the synthesis of sialic acid (1) and its analogs [4•, 5, 6]. These compounds have received a great deal of attention because they are involved in a number of

Related pyruvate aldolases/2-oxobutyrate aldolases

Nikkomycins (e.g. Nikkomycin Z and X 11/12, Figure 2a) are a group of peptidyl nucleoside antibiotics regarded as promising fungicidal agents in agriculture and antifungal drugs for human therapy. For the biosynthesis of 11 and 12 in Streptomyces ansochromogenes two enzymes, SanM and SanN, acting in concert have been discovered [19^•]. SanM is a Class II aldolase (i.e. requires divalent metal ions for its activity) that catalyzes the addition of 2-ketobutyrate 13 to picolinaldehyde 14, produced

Dihydroxyacetone phosphate (DHAP) aldolases

Dihydroxyacetone phosphate (DHAP)-dependent aldolases constitute a family of lyases, which catalyze the reversible aldol addition reaction of DHAP to a large variety of aldehyde acceptors. As a result, two new asymmetric centers are formed whose stereochemical configuration can be controlled by choosing one out of four stereocomplementary DHAP aldolases (Box 1a). A plethora of studies have been reported demonstrating their synthetic applicability (for previous reviews see: [1•, 3, 27•]).

One of

DHA aldolases (d-fructose-6-phosphate aldolase (FSA); transaldolase mutant)

One of the drawbacks of DHAP aldolases is their strict specificity toward the donor substrate. Moreover, in most instances the phosphate group of the product must be removed in a separate reaction disfavoring the atom economy of the process. In this connection, the discovery of a novel d-fructose-6-phosphate aldolase isoenzyme (FSA) from E. coli, [35] which readily accepts unphosphorylated DHA as donor, was highly promising and significant to this field. Even more remarkable was the finding

Glycine/alanine aldolases

The aldol addition of glycine to aldehydes is catalyzed by pyridoxal-5′-phosphate (PLP)-dependent lyases such as threonine aldolases (ThrA). Since two new stereogenic centers are formed, four different products with complementary stereochemistry can be obtained formally from a single aldehyde using either l-threonine and d-threonine or corresponding allo-threonine selective aldolases (Box 1) (for a recent review see [42]).

l-Threonine aldolases are powerful tools in chemo-enzymatic multistep

2-Deoxy-d-ribose 5-phosphate aldolase (DERA)

In vivo, DERA catalyzes the reversible aldol addition of acetaldehyde to d-glyceraldehyde-3-phosphate to furnish 2-deoxy-d-ribose 5-phosphate. Preparatively, the most attractive feature of this enzyme proved to be the unique ability to catalyze self-aldol and cross-aldol additions of acetaldehyde, because the first aldol addition furnishes another aldehyde that can be used by DERA, or in combination with other aldolases, for cascade aldol reactions (for a recent review see [27^•]).

DERA has

Novel catalysts with aldolase activity

Apart from the discovery and development of novel naturally evolved aldolases, many efforts have been devoted to design biologically compatible artificial catalysts that mimic the effectivity and selectivity of natural aldolases, while potentially widening their synthetic scope. The underlying concepts and classes of catalysts cover a broad range, from organocatalysis using small molecules as simple as l-proline that act via covalent enamine intermediates similar to Class I aldolases [61],

Conclusions

This review demonstrates the recent progress in exploiting the potential of aldolases as synthetic tools for stereoselective aldol addition reactions, a strategic reaction in synthetic organic chemistry that allows complex chiral molecules to be rapidly assembled from smaller fragments. It has been shown that diversity-oriented synthesis can also be accomplished with great potential to generate various structures and various stereochemistries. The discovery of novel enzyme activities, or of an

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

• of special interest
•• of outstanding interest

Acknowledgements

This work was supported by the Spanish MCINN CTQ2006-01345/BQU, Generalitat de Catalunya DURSI 2005-SGR-00698. AS/GS and WDF were supported by the DFG through SPP1170 (Sp503/4-2 and Fe244/7-2, respectively). The authors especially acknowledge the financial support from ESF project COST CM0701, which has provided a valuable opportunity for knowledge exchange between the research groups.

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Citation Excerpt :
For synthetic purposes, they can be used as catalysts for the aldol addition of a ketone (donor substrate) to an aldehyde (acceptor substrate) [6,7]. They constitute one of the few groups of biocatalysts that enantioselectively catalyze different type of CC bond formations, so a wide spectrum of synthetic applications involving aldolases at laboratory [1,3,8–14] or industrial [15] scales have been described. Regarding functionality, aldolases can be categorized into four groups, according to the structure of their donor substrate [1].
Stereoselective CC bond formation is one of the most exciting challenges of synthetic organic chemistry. Biocatalysts have emerged as promising tools, with aldolases being among the most employed. Unfortunately, the features of their active sites that modulate the mechanistic details have been poorly assessed. Here, the electronic and structural factors associated with the substrates binding and catalytic mechanism of rhamnulose-1-phosphate aldolase (RhuA) from E. coli are computationally analysed. The results allowed us to furnish a holistic view of the RhuA’s entire turnover cycle. The active site bears three pockets that anchor different moieties of the substrates: l-rhamnulose-1-phosphate (R1P), dihydroxyacetone phosphate (DHAP) and (S)-lactaldehyde (LLA). The results predict a 5-step catalytic mechanism, being the CC bond breaking (E_a =24.2 kcal mol⁻¹) and the DHAP deprotonation (E_a =22.0 kcal mol⁻¹) the rate-determining steps for the retro- and aldolic reactions, respectively. The residues E117, E171′, G31 and N29, and the Zn²⁺ cofactor, play a major role in stabilizing the correct substrates’ orientation, securing the stereocontrol at C4. Our findings suggest the presence of two acid/base catalytic residues, E117 and E171′, the latter of which being also involved in assisting the CC bond formation through a concerted protonation of the transition state.

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Recent progress in stereoselective synthesis with aldolases

Introduction

Section snippets

Pyruvate aldolases

Related pyruvate aldolases/2-oxobutyrate aldolases

Dihydroxyacetone phosphate (DHAP) aldolases

DHA aldolases (d-fructose-6-phosphate aldolase (FSA); transaldolase mutant)

Glycine/alanine aldolases

2-Deoxy-d-ribose 5-phosphate aldolase (DERA)

Novel catalysts with aldolase activity

Conclusions

References and recommended reading

Acknowledgements

Biochem Biophys Res Commun

J Am Chem Soc

Adv Synth Catal

Tetrahedron

Angew Chem Int Ed

Biotechnol J

Chem Biol

Angew Chem Int Ed

Microbial aldolases as C–C bonding enzymes — unknown treasures and new developments

Appl Microbiol Biotechnol

Biotechnological applications of aldolases

Recent advances in aldolase-catalyzed asymmetric synthesis

Adv Synth Catal

Directed evolution of aldolases for exploitation in synthetic organic chemistry

Arch Biochem Biophys

Structure-guided saturation mutagenesis of N-acetylneuraminic acid lyase for the synthesis of sialic acid mimetics

Prot Eng Design Selec

Aldolase-catalyzed synthesis of β-d-Galp-(1–9)-d-KDN: a novel acceptor for sialyltransferases

Org Lett

Disaccharides as sialic acid aldolase substrates: synthesis of disaccharides containing a sialic acid at the reducing end

Angew Chem Int Ed

Highly efficient chemoenzymatic synthesis of naturally occurring and non-natural α-2,6-linked sialosides: a P. damsela α-2,6-sialyltransferase with extremely flexible donor-substrate specificity

Angew Chem Int Ed

High-throughput substrate specificity studies of sialidases by using chemoenzymatically synthesized sialoside libraries

ChemBioChem

Efficient chemo-enzymatic synthesis of biotinylated human serum albumin-sialo-glycoside conjugates containing O-acetylated sialic acids

Org Biomol Chem

Enzymatic synthesis of fluorinated mechanistic probes for sialidases and sialyltransferases

J Am Chem Soc

Chemoenzymatic synthesis of CMP-N-acetyl-7-fluoro-7-deoxyneuraminic acid

Carbohydr Res

Chemoenzymatic synthesis of ring 18O-labeled sialic acid

Can J Chem

Preparation of a fluorous protecting group and its application to the chemoenzymatic synthesis of sialidase inhibitor

Chem Commun

Creation of a pair of stereochemically complementary biocatalysts

J Am Chem Soc

Pasteurella multocida sialic acid aldolase: a promising biocatalyst

Appl Microbiol Biotechnol

Efficient whole-cell biocatalytic synthesis of N-acetyl-d-neuraminic acid

Adv Synth Catal

Characterization of an aldolase–dehydrogenase complex that exhibits substrate channeling in the polychlorinated biphenyls degradation pathway

Biochemistry

Synthesis of 4-hydroxyisoleucine by the aldolase-transaminase coupling reaction and basic characterization of the aldolase from Arthrobacter simplex AKU 626

Biosci Biotechnol Biochem

A novel strategy for enzymatic synthesis of 4-hydroxyisoleucine: identification of an enzyme possessing HMKP (4-hydroxy-3-methyl-2-keto-pentanoate) aldolase activity

FEMS Microbiol Lett

Pyruvate aldolases in chiral carbon–carbon bond formation

Nat Protoc

Characterization and crystal structure of Escherichia coli KDPGal aldolase

Bioorg Med Chem

Mutagenesis of the phosphate-binding pocket of KDPG aldolase enhances selectivity for hydrophobic substrates

Protein Sci

Chemoenzymatic synthesis of ring ¹⁸O-labeled sialic acid