Problem of DHAP Dependence

The main drawback of the DHAP-dependent aldolases is their strict specificity for the donor substrate. Apart from the scope limitation that this fact represents, DHAP is expensive to be used stoichiometrically in high-scale synthesis, and labile at neutral and basic pH, and therefore its effective concentration decreases over time in enzymatic reaction media, hindering the overall yield of the aldol reaction. In addition, due to the presence of a phosphate group in both DHAP and the 

The synthehc applicability of arsenates is restricted by their toxicity that avoids the green aspect of the enzymatic processes. Wong et al. have shown that the use of inorganic borate buffer allows L-rhamnulose-l-phosphate aldolase (Rha-IPA) to accept DHA as substrate, although the of the reaction is about 50 times lower than with the natural substrate [10]. In spite of this fact, these authors have successfully used this approach for the one-step synthesis of L-fructose and L-rhamnulose, and for the facile two-step synthesis of several L-iminocyclitols. 

we will focus on the enzymatic routes since enzymatic preparation of DHAP is usually coupled with the aldol addition catalyzed by the aldolase representing genuine multi-enzyme systems. 

Fessner and Sinerius, in their seminal work [14], showed that the depicted strategy was adaptable to the synthesis of DHAP analogs modified at the phosphate group and from two of them they could identify the aldol adduct as mixtures with remaining starting material. 

In the multi-enzyme system described by Sheldon et al. [15], the key point is the use of the phosphatase phytase from Aspergillus ficuum, which is a cheap and readily available industrial enzyme. Phytase is active at acid pH and becomes inactive at neutral pH. Thus, the pH can be used to switch on/off the activities of the various enzymes, allowing us to carry out the four-enzyme cascade in one pot. 

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