Racemisation kinetics, amino acids

Recently it was reported that an a-amino-e-caprolactam racemase from Achro-mobacter obae can racemise a-amino acid amides efficiently. In combination with a D-amino acid amidase from Ochrobactrum anthropi L-alanine amide could be converted into D-alanine. This tour de force demonstrates the power of the racemase [84]. If racemic amide is used as a starting material the application of this racemase in combination with a d- or L-amidase allows the preparation of 100% d- or L-amino acid, a dynamic kinetic resolution instead of DSM s kinetic resolution (Scheme 6.24). 

Bada, J.L. and Shou, M-Y. (1980). Kinetics and mechanism of amino acid racemisation in aqueous solution and in bones. In Biogeochemistry of Amino Acids, ed. Hare P.E., Hoering T.C. and King K. Jr, John Wiley, New York, pp. 235-255. 

A straightforward approach to avoid low yields is to perform the reaction as a dynamic kinetic resolution. Racemisation can be achieved chemically [33] or enzymatically, indeed a number of N-acyl amino acid racemases have been described and it has been demonstrated that they could be employed together with the l-N-acyl amino acylase for the production of optically pure methionine [81]. 

DSM developed a slightly different approach towards enantiopure amino acids. Instead of performing the Strecker synthesis with a complete hydrolysis of the nitrile to the acid it is stopped at the amide stage. Then a stereoselective amino acid amidase from Pseudomonas putida is employed for the enantioselective second hydrolysis step [83], yielding enantiopure amino acids [34, 77, 78]. Although the reaction is a kinetic resolution and thus the yields are never higher than 50% this approach is overall more efficient. No acylation step is necessary and the atom efficiency is thus much higher. A drawback is that the racemisation has to be performed via the Schiff s base of the D-amide (Scheme 6.23). 

The enzymes of the nucleic acid metabolism are used for several industrial processes. Related to the nucleobase metabolism is the breakdown of hydantoins. The application of these enzymes on a large scale has recently been reviewed [85]. The first step in the breakdown of hydantoins is the hydrolysis of the imide bond. Most of the hydantoinases that catalyse this step are D-selective and they accept many non-natural substrates [78, 86]. The removal of the carbamoyl group can also be catalysed by an enzyme a carbamoylase. The D-selective carbamoylases show wide substrate specificity [85] and their stereoselectivity helps improving the overall enantioselectivity of the process [34, 78, 85]. Genetic modifications have made them industrially applicable [87]. Fortunately hydantoins racemise readily at pH >8 and additionally several racemases are known that can catalyze this process [85, 88]. This means that the hydrolysis of hydantoins is always a dynamic kinetic resolution with yields of up to 100% (Scheme 6.25). Since most hydantoinases are D-selective the industrial application has so far concentrated on D-amino acids. Since 1995 Kaneka Corporation has produced 2000 tons/year of D-p-hydroxyphenylglycine with a D-hydantoinase, a d-carbamoylase [87] and a base-catalysed racemisation [85, 89]. 

Such inferences derive from data on the kinetics of racemisation, measured in the laboratory (described in Section 4.18.2) and there is a good deal of controversy surrounding the dating method since no account is taken of the catalytic influence on racemisation rates of molecular structures that surrounded the amino-acid residue for some or all the years. It is, for example, now known that the rate of racemisation of an amino acid, when it is a residue in a protein, is strongly dependent on the nature of the adjacent amino acids in the sequence the particular amino acid on which measurement is made might have been located in a racemisation-promoting environment for many years after the death of the organism. 

Resolution of amino acids by differential crystallisation with racemisation Differential crystallisation and racemisation when enolisation is impossible Kinetic resolution with racemisation... 

Biocatalytic resolution plays a major role in the industrial scale synthesis of a wide variety of optically pure amino acids. Tanabe uses an L-spe-cific aminoacylase for the manufacture of several L-amino acids, immobilized on DEAE-Sephadex. Degussa on the other hand, uses the free acylase in a membrane bioreactor. The process is highly efficient in enzyme use, and racemisation of the D-isomer is straightforward, thus providing good economics, and virtually no waste (Scheme 7.4). The process can be further refined by the use of racemase enzymes, which makes dynamic kinetic resolution feasible. 

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