Abstract:The l-forms of racemic-N-protected-b,gunsaturated a-amino acid thioesters were found to be substrates for the subtilisin-catalysed hydrolysis to the corresponding acids. The d-enantiomer was continuously racemised in the presence of an organic base. The combined reactions in a biphasic system allowed the deracemisation of the amino acid derivatives based on a dynamic kinetic resolution. Excellent yields and enantioselectivities were achieved.Keywords: amino acids; biotransformations; dynamic kinetic resolution; enzyme catalysis; racemization Hydrolytic enzymes are the most readily available biocatalysts for synthetic applications, particularly in the kinetic resolution of racemic mixtures.[1] Like in any resolution method, a maximum of 50 % yield of enantiomerically pure product can be obtained, based on racemic starting material. When kinetic resolution is coupled with an in situ racemisation of the substrate either chemical or enzymatic, the yield limitation can be overcome leading to a much more efficient process: a deracemisation based on a dynamic kinetic resolution (DKR).[2] A well known example in amino acid chemistry is the industrial process d-hydantoinase-carbamoylase which allows the large scale preparation of relevant d-amino acids. [3] In this process a microbial catalyst containing the two enzymatic activities transforms the R-starting material into the damino acids, while the remaining hydantoin is continuously racemised at slightly basic pH. Requisites for a successful DKR are an enzyme selective for one of the enantiomers, a racemising system (chemical or enzymatic) acting on the substrate but not on the product, and a rate of racemisation of the substrate higher than the rate of conversion to product. These conditions require the design of suitable substrates. In thioesters, the acidity of the hydrogen in the a-position is higher in comparison to the corresponding oxo esters, amides or acids. The enzymatic transformation of a thioester into the corresponding carboxylate with a higher pK a of the a-proton is the basis for a successful DKR, provided that the enzymatic systems resist the basic conditions required for substrate racemisation. This concept has been applied in the DKR of a-alkyl thioesters.[4] Our interest in the synthesis and deracemisation of non-natural amino acids [5] prompted us to design racemic compounds suitable for this novel application. From previous data [6] aryllycines and analogues were considered as candidates. In fact not only p-hydroxyphenylglycine esters have been shown to be the substrate for subtilisin, but also phenylglycine (Phg) has been partly deracemised in the presence of chiral copper catalysts indicating a suitable pKa value for the a-proton. [7] We initially checked the ability of subtilisin Carlsberg to transform amino acid thioesters 1-6 (Scheme 1) and found a good activity on these N-Boc amino acid derivatives with reaction rates comparable although inferior to the one observed with the corresponding oxoesters. [8] Racemisation conditions were s...
An alternative synthesis of 4-amino-3-hydroxy-4,5,6,6a-tetrahydro-3aH-cyclopenta [d]isoxazole-4-carboxylic acid, a conformationally constrained analogue of aspartic acid, is described. The synthetic strategy is based on a regioselective 1,3-dipolar cycloaddition to give the cyclopenta[d]isoxazoline framework; subsequent condensation of this 4-oxocyclopenta[d]isoxazoline with 4-methoxybenzylamine gives a 4-imino derivative, which undergoes a highly stereoselective nucleophilic attack by the cyanide ion. This gives a 4-(methoxybenzylamino)-4-cyano derivative, which is oxidized to the corresponding 4-amino-4-cyano derivative, which itself is transformed into the target 4-carboxylic acid. This amino acid is obtained in 27% overall yield in five steps, whereas the previously described synthetic strategy gave the target derivative in only 0.5% overall yield over 15 steps.
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