DERA reactions catalyzed

Another promising route was reported in patent and open hterature by both DSM and Diversa [13, 14]. This route employs a 2-deoxy-D-ribose 5-phosphate aldolase (DERA) that catalyzes a tandem aldol addition in which two equivalents of acetaldehyde (AA) are added in sequence to chloroacetaldehyde (CIAA) to produce a lactol derivative that is similar to the 3,5-dihydoxy side chain of synthetic statins (Figure 6.2e). Diversa screened environmental libraries for novel wild-type DERAs and identified an enzyme that was both tolerant to increased substrate concentrations and more active than DERA from E. coli in the target reaction [13]. 

To make the DERA-catalyzed process commercially attractive, improvements were required in catalyst load, reaction time, and volumetric productivity. We undertook an enzyme discovery program, using a combination of activity- and sequence-based screening, and discovered 15 DERAs that are active in the previously mentioned process. Several of these enzymes had improved catalyst load relative to the benchmark DERA from E. coli. In the first step of our process, our new DERA enzymes catalyze the enantioselective tandem aldol reaction of two equivalents of acetaldehyde with one equivalent of chloroacetaldehyde (Scheme 20.6). Thus, in 1 step a 6-carbon lactol with two stereogenic centers is formed from achiral 2-carbon starting materials. In the second step, the lactol is oxidized to the corresponding lactone 7 with sodium hypochlorite in acetic acid, which is crystallized to an exceptionally high level of purity (99.9% ee, 99.8% de). 

Scheme 5.41. Aldol addition reaction catalyzed in vivo by 2-deoxyribose-5-phosphate aldolase (DERA). P = PO32. ...

Reactions Catalyzed by DERA Acceptor Donor Product... 

Figure 14.1-29. Aldol addition reaction catalyzed in iwo by DERA, and reactions F with unnatural sub-...

Figure 14.1-31. Sequential aldol reactions catalyzed by DERA.

Wong and coworkers reported one-pot aldol reactions catalyzed by a deoxyribose-5-phosphate aldolase (DERA), in which 2 eq of acetaldehyde was added in sequence to two-carbon aldehyde acceptors to afford six-membered lactol derivatives 126 (Figure 4.35). The DERA-catalyzed reaction is an equilibrium process the intermediate four-carbon adduct 127 is reversibly formed under the reaction conditions. The second condensation between this intermediate and a second equivalent of acetaldehyde drives the equilibrium favorably due to the stability of the cydized lactol product 126. DERA has been expressed in E. coli [158,159]. 

Aldolases catalyze asymmetric aldol reactions via either Schiff base formation (type I aldolase) or activation by Zn2+ (type II aldolase) (Figure 1.16). The most common natural donors of aldoalses are dihydroxyacetone phosphate (DHAP), pyruvate/phosphoenolpyruvate (PEP), acetaldehyde and glycine (Figure 1.17) [71], When acetaldehyde is used as the donor, 2-deoxyribose-5-phosphate aldolases (DERAs) are able to catalyze a sequential aldol reaction to form 2,4-didexoyhexoses [72,73]. Aldolases have been used to synthesize a variety of carbohydrates and derivatives, such as azasugars, cyclitols and densely functionalized chiral linear or cyclic molecules [74,75]. 

In contrast to transketolase and the DHAP-dependent aldolases, deoxyribose aldolase (DERA) catalyzes the aldol reaction with the simple aldehyde, acetaldehyde. In vivo it catalyzes the formation of 2-deoxyribose-5-phosphate, the building block of DNA, from acetaldehyde and D-glyceraldehyde-3-phosphate, but in vitro it can catalyze the aldol reaction of acetaldehyde with other non-phosphorylated aldehydes. The example shown in Scheme 6.28 involves a tandem aldol reaction... 

Scheme 6.28 DERA-catalyzed double aldol reaction.

This reaction is catalyzed by DERA from E. coli. DERA is the only aldolase known which accepts two aldehydes as substrates, offering a versatile approach to... 

Scheme 6.4 DERA-catalyzed stereoselective tandem aldol reaction, using chloroacetaldehyde and 2 equivalents of acetaldehyde, yielding (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranoside (1).

Scheme 6.5 NMR elucidation of the DERA-catalyzed reaction of CIAA and 2.5 equivalents of AA using on-line monitoring of the reaction mixture by 600MHz NMR.

Figure 6.3 Progress curve of the DERA-catalyzed reaction of ClAA with two equivalents AA (as analyzed by NMR spectroscopy). The y-axis shows the relative molar concentration of the reaction components expressed as arbitrary units (AU). The products and by-products are...

An interesting enzyme-catalyzed three-component aldolization reaction has been described by Gijsen and Wong [18]. Here, acetaldeyde, 2-substituted acetaldehydes, and dihydroxyacetone phosphate react in the presence of the aldolases 2-deoxyribose-5-phosphate aldolase (DERA) and fructose 1,6-diphosphate aldolase (RAMA) forming the corresponding 5-deoxyketose derivatives (Scheme 9.9). 

In comparison to other aldolases, DERA has a rather broad substrate range. DERA-catalyzed aldol reactions were used to get an access to key intermediates for epothilones (Fig. 36) [194]. According to retrosynthetic analysis, both fragments of the molecule could be obtained from aldol building blocks, and two out of seven stereocenters were established enzymatically. For the southern part of epothilone A,... 

S )-3-hydroxy-2-methoxypropanal was successfully converted with acetaldehyde, with the primary open chain aldol product forming the lactol. The aldehyde needed as starting material for the northern part was generated in situ from the acetal a DERA-catalyzed kinetic resolution led to conversion of the (R)-enantiomer in the aldol reaction, only. 

A particularly successful synthesis of Epothilone A is based on two DERA-cata-lyzed steps. In these two of the seven stereocentres of Epothilone A were established. When a racemic aldehyde was released in situ from its acetal, DERA converted only the R-enantiomer into the stable cyclic hemiacetal. This is a combined kinetic resolution and carbon-carbon bond formation yielding a building block with two chiral centers. Since the alcohol function was oxidized, the optical information obtained from the kinetic resolution was lost. Thus, for the overall yield it would have been better if DERA had displayed no stereoselectivity towards the acceptor (Scheme 5.32). In the DERA-catalyzed synthesis of another part of Epothilone A DERA is again highly stereoselective. Fortunately its preference is for the S-enan-tiomer of the acceptor aldehyde, the enantiomer that has to be submitted to the carbon-carbon bond formation in order to obtain the desired building block, again a stable hemiacetal (Scheme 5.32). Indeed, both DERA-catalyzed reactions yield open chain products that form stable cyclic hemiacetals. This ensures that the equilibria of these aldol reactions are shifted towards the desired products. Further synthetic manipulations converted these intermediates into Epothilone A [55]. 

A particularly elegant application of DERA is the sequential synthesis of thermodynamically stable cyclic hemiacetal. Two DERA-catalyzed aldol reactions convert one equivalent of acceptor and two equivalents of acetaldehyde into this stable compound. A mild subsequent oxidation yielded the corresponding lactone in ex-... 

The enzyme DERA, 2-deoxyribose-5-phosphate aldolase (EC 4.1.2.4), is unique among the aldolases in that the donor is an aldehyde. In vivo it catalyzes the reversible aldol reaction of acetaldehyde and D-glyceraldehyde 3-phosphate, forming 2-deoxyribose 5-phosphate, with an equilibrium lying in the synthetic direction (Scheme 5.41). DERA, the only well-characterized member of this type I aldolase, has been isolated from both animal tissue and microorganisms.67... 

When acetaldehyde is used as the donor, the products from the DERA-catalyzed reaction are aldehydes, capable of being acceptor substrates for a second aldol condensation (Fig. 14.1-31)[1871. For example, when a-substituted acetaldehydes were employed as substrates, products of the first aldol condensation could not cyclize to a hemiacetal, and the products reacted with a second molecule of acetaldehyde to form 2,4-dideoxyhexoses. These products could then cyclize to stable... 

A sequential aldol reaction leading to sialic acid derivatives has been also presented [59], Combination of acetaldehyde dependent aldolase (DERA) and Neu5Ac aldolase catalyzed reactions led to (R)-C-4 keto acids. In this case, however, one-pot reaction sequence was not possible due to the incompatibility of the reaction conditions for two enzymes. 

It is the only known member of the group of acetaldehyde-dependent aldolases. In vivo, DERA catalyzes the reversible aldol reaction of acetaldehyde and G3P. The donor substrate specificity of this enzyme is not as strict as with the other aldolases. 

An economically viable alternative to the synthesis of deoxyribonuclosides has been developed as a two stage process involving 2-deoxy-D-ribose 5-phosphate aldolase (DERA) (Fig. 6.5.14) (Tischer et al. 2001). The first step was the aldol addition of G3P to acetaldehyde catalyzed by DERA. G3P was generated in situ by a reverse action of EruA on L-fructose-1,6-diphosphate and triose phosphate isomerase which transformed the DHAP released into G3P. In a second stage, the action of pentose-phosphate mutase (PPM) and purine nucleoside phosphorylase (PNP), in the presence of adenine furnished the desired product. The released phosphate was consumed by sucrose phosphorylase (SP) that converts sucrose to fructose-1-phosphate, shifting the unfavorable equilibrium position of the later reaction. 

Deoxy-D-ribose 5-phosphate aldolase (RibA, or "DERA" EC 4.1.2.4.) uses acetaldehyde as the nucleophile to catalyze enzymatic self- and cross-aldol reactions [165,166]. In vivo, RibA catalyzes the reversible addition of acetaldehyde to D-glyceraldehyde 3-phosphate furnishing 2-deoxy-D-ribose 5-phosphate (Scheme 10.8). 

As with to FSA and GO, the most significant feature of DERA is the ability to catalyze self- and cross-aldol additions of acetaldehyde. Therefore, the first aldol addition furnishes another aldehyde that can be used as acceptor by DERA, or in combination with other aldolases, for cascade aldol reactions (Scheme 10.31) [25-27,195]. 

The attractiveness of this enzyme resides in its potential to catalyze self-aldol, cross-aldol, and cascade aldol reactions (for a review, see Ref. 87). DERA generally presents a stereoselectivity toward the aldehyde acceptor and catalyzes often the conversion of only one enantiomer. However, it often requires the presence of phosphorylated aldehyde acceptors. The most recent efforts were thus focused on directed evolution of the enzyme in view to improve the scope toward unphosphorylated substrates. DeSantis et al., in particular, performed some saturation site-directed mutagenesis in view to improve the reactivity for the unnatural D-glyceraldehyde substrate 37 for the synthesis of 2-deoxy-D-ribose 38. They reached a 2.5-fold improvement in DERA activity toward the unphosphorylated aldehyde with the mutant S238D (Scheme 28.18). 

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