One-pot biocatalytic amine transaminase/acyl transferase cascade for aqueous formation of amides from aldehydes or ketones†‡

Biocatalytic acylation/amidation is well-known and has been applied for decades but it has often been limited to the use of organic solvents to avoid hydrolysis. The acyl transferase from Mycobacterium smegmatis (MsAcT) is an enzyme that can perform trans-acylations in aqueous solution. Only a few hydrolases can catalyze trans-acylation in water and MsAcT also has the ability to act as a perhydrolase and catalyze the formation of peracids from esters. Since MsAcT can catalyze acylation for the formation of esters in aqueous solution we hypothesized that it could also utilize amines for amidation under the same conditions. Amidation should also in theory be able to reach higher conversions due to the higher nucleophilicity of the amine compared to the alcohol and the more stable amide product which would drive the equilibrium towards product formation. Amine transaminases have been extensively studied during the last decades due to their ability to perform asymmetric synthesis of both chiral and achiral primary amines from the corresponding pro-chiral ketones or aldehydes with excellent yield and enantiomeric excess (ee). There are many examples of cascades involving amine transaminases and in most cases by-product removal to shift the unfavorable equilibrium towards amine synthesis is used. There are also recent reports of synthetic cascades where amine transaminases are involved in longer reactions sequences. There have however been fewer reports of amine transaminase cascades where the reductive amination is followed by amidation in a later stage of the cascade and it has never been performed in a one-pot one-step fashion where one-step means that the reaction is carried out in only one operational step even though the cascade contains several reaction steps working simultaneously. The amide is a common functional group in a wide variety of important compounds such as pharmaceuticals and polymers and an aqueous onepot biocatalytic synthesis route starting from inexpensive aldehydes and ketones would be a cheap and green alternative to existing procedures. The irreversible nature of amide formation in these mild conditions and in the presence of the previously mentioned enzymes would also serve as efficient equilibrium displacement of the thermodynamically challenging reductive amination. One theoretical drawback of a one-pot one-step amine transaminase/acyl transferase cascade is that the amine donor of the transamination will compete with the amine product in the second step of the cascade. This can however be circumvented when MsAcT is used since it does not accept amino acids as substrates for amidation (unpublished results). This opens up the possibility of using alanine as amine donor for the transamination step of the cascade since it is accepted by most amine transaminases. L-alanine (used in the case of an (S)-selective amine transaminase) is cheap and readily available. It also has a high solubility in water and can therefore be used in large excess to displace the equilibrium towards product formation. Since there is a plethora of available amine transaminases that catalyze the desired reaction under a wide range of pHvalues, the optimization of the cascade conditions was focused on MsAcT. The pH-optimum was investigated using a model reaction with benzylamine as acyl acceptor/nucleophile and methyl acetate as acyl donor forming the product N-benzylacetamide (Scheme 1, MsAcT catalyzed step). The optimal pH was found to be around pH 11 (Fig. S1, ESI‡). The reason for the high pH-optimum is believed to be due to the