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Enzymatic C─C Bond Formation by Aldol Reactions

Aldolases

In order to synthesise/metabolise carbohydrates Nature has invented the aldol reaction. Proteins catalysing this reaction between a ketone such as 1 as a nucleophilic donor and an electrophilic aldehyde acceptor 2 leading to a ß-hydroxy ketone 3 are called aldolases (Figure 1.1). In general, this reaction is reversible; however, for aldolases, the equilibrium constant can approach 104?M-1 in favour of the aldol product.

These enzymes are ubiquitous in Nature and have been isolated from mammals, plants, fungi, bacteria, and even thermophylic archaebacteria [1]. Though proteins from different sources are divers with respect to amino acid sequences and global structure they share (a/ß)8 barrels that contain active sites displaying two modes of activation of the ketone donor: (i) Typ I aldolases employ organocatalysis and (ii) Typ II aldolases use metalorganic activation. Accordingly, aldolases circumvent the use of strong bases - pK a>18 - that are required for the formation of solvated metal enolates under conventional chemical conditions.

Class I Aldolases

Class I aldolases have been identified more than 80?years ago [2]. Most of these proteins have a molecular weight close to 158?kDa and high sequence homology within one species. The structures of several members of this enzyme family have been determined by X-ray crystallography at high resolution [3] revealing the association of four identical subunits, each containing an active site with a catalytically important lysine. Investigations of aldolases substrate specificity showed a clear preference for DHAP-like nucleophilic ketones, whereas the acceptance of aldehydes is quite broad. However, in all cases the overall stereospecificity of C─C bond formation is the same as explained in the following example.

Fructose 1,6-diphosphate aldolase catalyses the reversible reaction of dihydroxyacetone phosphate 4 (DHAP) with D-glyceraldehyde 3-phosphate5 (G3P) in favour of the product D-fructose 1,6-diphosphate6 (FDP) (Figure 1.2).

Figure 1.1 The aldol reaction.

Figure 1.2 Aldolase reaction.

DHAP 4 binds first to the enzyme [4] and reacts with a lysine in the active site to form the Schiff base 7 (Figure 1.3). It is important to note that the pK a value of the Schiff base-forming lysine must be significantly perturbed in the active site because at physiological pH values lysine is protonated and hence not nucleophilic. For aldolases, two factors adjust the lysin's pK a value to around pH 7: a hydrophobic, low dielectric environment disfavours charged residues, and a positively charged residue in close proximity further decreases the pK a of the reactive lysine. A second protonated lysine has been identified indeed in a particular aldolase (DERA) [5].

Figure 1.3 Type I aldolase mechanism, 14C label.

Evidence for the formation of 7 derives from two experiments (Figure 1.3): (i) on incubation of 18O=C-labelled DHAP the 18O label is released into the medium [6] and (ii) reduction of 14C-labelled 7 yields a catalytically inactive, radioactive enzyme 8 from which the amine 9 can be isolated after hydrolysis of the protein [7]. When incubation of 4 is pursued in the absence of 5 in D2O [8] or tritiated water only, the pro-3S proton at C-3 of 7 is exchanged by means of the equilibrium between Schiff base 7 and enamine 10.

It follows that protonation/deuteration of 10 occurs stereospecific from the si-face of the enamine. Interestingly, this is the same face where C─C bond formation occurs. That is the aldehyde 5 approaches the enamine 10 diastereospecifically to give the S-configuration at C-3 and the R-configuration at C-4 of 11 and 6, respectively (Figure 1.4).

Figure 1.4 Approach of G3P 5 towards the enzyme-bound DAHP 10.

Due to the stereospecificity of Typ I aldolases and the possibility generating several new stereocentres these enzymes are of synthetic value [9, 10], and many are commercially available and used in industrial processes. Besides the above-mentioned keto aldolases a very interesting aldolase, DERA [5], accepts aldehydes as donors and acceptors for the aldol reaction; even reactions with three to four substrate aldehydes can be performed [11] yielding sugar analogues and key intermediates for the preparation of, e.g. statins and epothilones.

Bioorganic Class I Aldolase Reactions

Since Class I aldolase reactions proceed via enantioface-selective addition of an enamine ketone to an aldehyde, it was obvious that chemists would envisage mimicking this process by preparing chiral enamines in situ expecting on addition of an aldehyde chirality transfer to the aldol product. For this purpose, the amino acid proline 12 was the first catalyst of choice due to the presence of a secondary amine for Schiff base formation and a COOH group that could help to organise a stereospecific approach of the acceptor aldehyde/ketone to the enamine by means of H-bonding activation.

The first promising example of a proline-assisted intramolecular aldol reaction was published some 50 years ago [12] using 3?mol% (S)-proline 12 to convert the triketone 13 into the ketol 14 and subsequently into the Wieland-Miescher ketone 15 that has been a key intermediate for steroid synthesis at his time (Figure 1.5).

Figure 1.5 Hajos-Perrish-Eder-Sauer-Wiechert (HPESW) reaction.

This conversion proved to be extremely efficient generating the diketone 15 in 84% yield and 94% ee. Ever since, the mechanism of this conversion has been a matter of debate. Analysis of stereoelectronic and steric factors that govern the reaction [13] and DFT calculations [14] led to the conclusion that at least for the HPESW reaction the enamine pathway is the most likely route. This was confirmed by an experiment in the presence of H2 18O leading to 18 O-14 and 18 O-15 labelled at the keto group that formed the enamine/imine (Figure 1.6) [15].

Figure 1.6 Mechanism of the HPESW reaction.

More than 30 years later, stimulated by first results regarding an intermolecular 'direct' aldol reaction [16] catalysed by proline (Figure 1.7), the research field of 'organocatalysis' exploded and hundreds of (S)-proline derivatives were tested for catalytic efficiency.

Figure 1.7 (S)-Prolin-assisted intermolecular aldol reaction.

The most effective proline-derived catalysts belong to the class of diaryl prolinols such as 16 that assisted, for example, in the cross-aldol reaction of o-chloro benzaldehyde 17 and acetaldehyde 18 to furnish after reduction the diol 19 in 85% yield and 99% ee (Figure 1.8) [17]. In the proposed transition (TS) state 20, the aldehyde 17 reacts on the more hindered face of the intermediate enamine due to H-bonding between the aldehyde and the OH group of the prolinol subunit.

Figure 1.8 (S???)-Prolin-assisted intermolecular aldol reaction.

The concept of H-bonding-assisted organocatalysis has been exploited using, for example, chiral binaphthyl derivatives 21 and diamino cyclohexanes such as 22. Both classes of compounds contain two different amine functions of which one forms the enamine and the other being acidic or protonated to provide H-bonding. Using 21 in a cross-aldol reaction of aromatic aldehyde 23 and aldehyde 24 excellent diastereo- and enantioselectivity, see 25, were observed at relatively low catalyst loading (Figure 1.9) [18].

Figure 1.9 Cross-aldol reaction catalysed by sulfonamide 21.

When the same aromatic aldehyde 23 was reacted with ketone 26 in the presence of diamine 22 the hydroxy ketones 27 were obtained in very good diastereo- and enantioselectivity (Figure 1.10) [19]. However, catalyst loading of =10?mol% is required to furnish products in good yield. This unfortunately is a general phenomenon in organocatalysis that catalyst loading is often between 10 and 30?mol% which in principle questions the term catalysis as the turnover is rather low and rarely approaches the corresponding figures of metalorganic catalysis.

Figure 1.10 Direct aldol reaction catalysed by diamine 22.

Several other very successful organocatalysts...