- Creation of a broad-range and highly stereoselective D-amino acid dehydrogenase for the one-step synthesis of D-amino acids
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Using both rational and random mutagenesis, we have created the first known broad substrate range, nicotinamide cofactor dependent, and highly stereoselective D-amino acid dehydrogenase. This new enzyme is capable of producing D-amino acids via the reductive amination of the corresponding 2-keto acid with ammonia. This biocatalyst was the result of three rounds of mutagenesis and screening performed on the enzyme meso-diaminopimelate D-dehydrogenase. The first round targeted the active site of the wild-type enzyme and produced mutants that were no longer strictly dependent on the native substrate. The second and third rounds produced mutants that had an increased substrate range including straight-and branched-aliphatic amino acids and aromatic amino acids. The very high selectivity toward the D-enantiomer (95 to >99% ee) was shown to be preserved even after the addition of the five mutations found in the three rounds of mutagenesis and screening. This new enzyme could complement and improve upon current methods for D-amino acid synthesis.
- Vedha-Peters, Kavitha,Gunawardana, Manjula,Rozzell, J. David,Novick, Scott J.
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p. 10923 - 10929
(2007/10/03)
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- Resolution of non-protein amino acids via the microbial protease-catalyzed enantioselective hydrolysis of their N-unprotected esters
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In the Aspergillus oryzae protease-catalyzed ester hydrolysis, substitution of N-unprotected amino acid esters for the corresponding N-protected amino acid esters resulted in a large enhancement of the hydrolysis rate, while the enantioselectivity was deteriorated strikingly when the substrates employed were the conventional methyl esters. This difficulty was overcome by employing esters bearing a longer alkyl chain such as the isobutyl ester. Utilizing this ester, amino acids carrying an aromatic side chain were resolved with excellent enantioselectivities (E=50 to >200). With amino acids bearing an aliphatic side chain also, good results in terms of the hydrolysis rate and enantioselectivity were obtained by employing such an ester as the isobutyl ester. Moreover, the enantioselectivity proved to be enhanced further by conducting the reaction at low temperature. This procedure was applicable to the case where the enantioselectivity was not high enough even by the use of the isobutyl ester.
- Miyazawa, Toshifumi,Imagawa, Kiwamu,Minowa, Hiroe,Miyamoto, Toyoko,Yamada, Takashi
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p. 10254 - 10261
(2007/10/03)
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- Conversion of Serine β-Lactones to Chiral α-Amino Acids by Copper-Containing Organolithium and Organomagnesium Reagents
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A method for the synthesis of optically pure α-amino acids has been developed.Mono- and di-N-protected α-amino-β-lactones 3a (L, R1=H, R2=COOCH2Ph (Z)), 3b (D, R1=H, R2=Z), 3c (L, R1=CH2Ph, R2=Z), and 3d (D, R1=CH2Ph, R2=Z) are readily produced by cyclization of the corresponding serine derivatives 2 under modified Mitsunobu conditions without loss of optical purity.Stereochemical integrity was demonstrated by conversion of 3 to 2-methoxy-2-(trifluoromethyl)phenylacetate esters 6 and analysis by HPLC, (19)F NMR, and (1)H NMR.Reaction of 3 with organolithium-derived cuprate reagents (R2CuLi or R2Cu(CN)Li2) at low temperature produces N-protected α-amino acids by attack at the β-methylene group.Yields of di-N-protected amino acids are generally higher (ca. 50-75percent), but some decrease in enantiomeric excess (ee) can occur (0-27percent).In contrast, the mono-N-protected β-lactones 3a and 3b give slightly lower yields (ca. 44-62percent) but negligible decrease in ee (0-1.7percent) with the exception of Ph2Cu(CN)Li2 (67percent loss of ee).However, the use of Cu(I)-catalyzed Grignard (RMgCl) additions gives better yields (44-83percent), complete retention of optical purity ( 99.4percent), and fewer side products.Reductive removal of the protecting groups in a single step (H2/Pd-C or Na/NH3) affords the free α-amino acids in 91-99percent yield.Their stereochemical purity was determined by conversion to the corresponding N-(-)-camphanoyl methyl esters and analysis by gas chromatography and (1)H NMR spectroscopy.
- Arnold, Lee D.,Drover, John C. G.,Vederas, John C.
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p. 4649 - 4659
(2007/10/02)
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