Amino acid dynamic resolution

Hsu et have cloned two enzymes from Deimcoccus radiodurans for overexpression in E. coli in order to carry out a dynamic kinetic resolution to obtain L-homophenylalanine, frequently required for pharmaceutical synthesis. The starting material is the racemic mixture of A acetylated homophenylalanine, and the two enzymes are an amino acid A -acylase, which specifically removes the acetyl group from the L-enantiomer, and a racemase, which interconverts the D- and L-forms of the A acyl amino acids. The resolution was carried out successfully using whole-cell biocatalysts, with the two enzymes either expressed in separate E. coli strains or coexpressed in the same cells. 

Felten, A.E., Zhu, G., and Aron, Z.D. (2010) Simplifying pyridoxal practical methods for amino acid dynamic kinetic resolution. Org. Lett., 12 (9), 1916-1919. 

Spectrometric Analysis. Remarkable developments ia mass spectrometry (ms) and nuclear magnetic resonance methods (nmr), eg, secondary ion mass spectrometry (sims), plasma desorption (pd), thermospray (tsp), two or three dimensional nmr, high resolution nmr of soHds, give useful stmcture analysis information (131). Because nmr analysis of or N-labeled amino acids enables determiaation of amino acids without isolation from organic samples, and without destroyiag the sample, amino acid metaboHsm can be dynamically analy2ed (132). Proteia metaboHsm and biosynthesis of many important metaboUtes have been studied by this method. Preparative methods for labeled compounds have been reviewed (133). 

Hydantoinases belong to the E.C.3.5.2 group of cyclic amidases, which catalyze the hydrolysis of hydantoins [4,54]. As synthetic hydantoins are readily accessible by a variety of chemical syntheses, including Strecker reactions, enantioselective hydantoinase-catalyzed hydrolysis offers an attractive and general route to chiral amino acid derivatives. Moreover, hydantoins are easily racemized chemically or enzymatically by appropriate racemases, so that dynamic kinetic resolution with potential 100% conversion and complete enantioselectivity is theoretically possible. Indeed, a number of such cases using WT hydantoinases have been reported [54]. However, if asymmetric induction is poor or ifinversion ofenantioselectivity is desired, directed evolution can come to the rescue. Such a case has been reported, specifically in the production of i-methionine in a whole-cell system ( . coli) (Figure 2.13) [55]. 

Figure 6.38 Dynamic kinetic resolution of amino acid amides.

Moreover, it is possible to open racemic azlactones by acyl bond cleavage to form protected amino acids in a dynamic kinetic resolution process. As azlactones suffer a fast racemization under the reaction conditions, eventually all starting material is converted [115]. 

Liang J, Ruble JC, Fu GC (1998) Dynamic kinetic resolutions catalyzed by a planar-chiral derivative of DMAP enantioselective synthesis of protected a-amino acids from racemic azlactones. J Org Chem 63 3154—3155... 

Mixing the additive in the eluent used as a mobile phase can also modify the chromatographic system (dynamic modification), but the use of modified adsorbents has led to an improvement of resolution. Example works include that by Armstrong and Zhou [11], who used a macrocyclic antibiotic as the chiral selector for enantiomeric separations of acids, racemic drugs, and dansyl amino acid on biphenyl-bonded silica. 

Several hundred tons of L-methionine per year are produced by enzymatic conversion in an enzyme membrane reactor. An alternative approach is dynamic resolution, where the unconverted enantiomer is racemized in situ. Starting from racemic /V-acetyl-amino acid, the enantioselective L-acylase is used in combination with an TV-acyl-amino acid racemase to enable nearly total conversion of the substrate. 

Asano, Y. and Yamaguchi, S. (2005) Dynamic kinetic resolution of amino acid amide catalyzed by D-aminopeptidase and a-amino-e-caprolactam racemase. Journal of the American Chemical Society, 127 (21), 7696-7697. 

May O., Verseck, S., Bommarius, A. and Drauz, K. (2002) Development of dynamic kinetic resolution processes for biocatalytic production of natural and nonnatural L-amino acids. Organic Process Research Development, 6 (4), 452-457. 

The complete transformation of a racemic mixture into a single enantiomer is one of the challenging goals in asymmetric synthesis. We have developed metal-enzyme combinations for the dynamic kinetic resolution (DKR) of racemic primary amines. This procedure employs a heterogeneous palladium catalyst, Pd/A10(0H), as the racemization catalyst, Candida antarctica lipase B immobilized on acrylic resin (CAL-B) as the resolution catalyst and ethyl acetate or methoxymethylacetate as the acyl donor. Benzylic and aliphatic primary amines and one amino acid amide have been efficiently resolved with good yields (85—99 %) and high optical purities (97—99 %). The racemization catalyst was recyclable and could be reused for the DKR without activity loss at least 10 times. 

Dynamic Kinetic Resolution Synthesis of a Fluorinated Amino Acid Ester Amide by a Continuous Process Lipase-mediated Ethanolysis of an Azalactone... 

Enzymes may be used either directly for chiral synthesis of the desired enantiomer of the amino acid itself or of a derivative from which it can readily be prepared, or for kinetic resolution. Resolution of a racemate may remove the unwanted enantiomer, leaving the intended product untouched, or else the reaction may release the desired enantiomer from a racemic precursor. In either case the apparent disadvantage is that the process on its own can only yield up to 50% of the target compound. However, in a number of processes the enzyme-catalyzed kinetic resolution is combined with a second process that re-racemizes the unwanted enantiomer. This may be chemical or enzymatic, and in the latter case, the combination of two simultaneous enzymatic reactions can produce a smooth dynamic kinetic resolution leading to 100% yield. 

The resolution was then based on the enzymatic propanolysis of this derivative in dioxane as solvent. Lip Novozyme 435 selectively cleaves the L-form of the oxazolone producing an L-enriched (81-87% ee) 2-acetamido-3-(heteroaryl)propionic acid propyl ester, the dynamic aspect of the process being based on the continual racemization of the residual oxazolone. The propyl group was then removed with alkali and a second selective enzymatic step to remove the acetyl protecting group with Fluka Acylase 1 produced the L-amino acid at better than 99% ee (Scheme 13). 

Novozymes, a subtilisin produced by Bacillus licheniformis, was used by Chen et al ° to carry out a dynamic kinetic resolution of benzyl, butyl, or propyl esters of DL-phenylalanine, tyrosine, and leucine. The hydrolysis was performed at pH 8.5 in 2-methyl-2-propanol/water (19 1) and the freed L-amino acids precipitated. The key feature bringing about continual racemization of the remaining D-amino acid esters was the inclusion of 20 mmol 1 pyridoxal phosphate. 

Figure 2.6 By resolution of df-amino acid esters under conditions of dynamic resolution 100% of a single enantiomer may be produced. Using catalytic amounts of pyiidoxyl-5-phosphate, which forms a Schiff s base with the ester and not the acid, the unreacted D-ester may be racemised in situ and for instance L-tyrosin has been obtained in 97% ee and 95% yield.

The synthesis of jS-hydoxy-a-amino acids is important since these compounds are incorporated into the backbone of a wide range of antibiotics and cyclopeptides such as vancomycins. These highly functional compounds are also subject to dynamic kinetic resolution (DKR) processes, as the stereocenter already present in the substrate epimerizes under the reaction conditions and hence total conversions into single enantiomers are possible. These transformations can be iy -selective ° for N-protected derivatives as shown in Figure 1.27 when using a mthenium-BlNAP catalyzed system and anfi-selective when the jS-keto-a-amino acid hydrochloride salts are reduced by the iridium-MeOBlPHEP catalyst as shown in Figure 1.28. One drawback is that both these reductions use 100 atm hydrogen pressure. 

Makino, K., Hiroki, Y. and Hamada, Y. Dynamic Kinetic Resolution Catalyzed by Ir Axially Chiral Phosphine Catalyst Asymmetric S3mthesis of anti-Aromatic -Hydroxy-ot-amino Acid Esters. J. Am. Chem. Soc. 2005, 127, 5784—5785. 

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