Biotechnological production of amino acids and derivatives: current status and prospects.

For almost 50 years now, biotechnological production processes have been used for industrial production of amino acids. Market development has been particularly dynamic for the flavor-enhancer glutamate and the animal feed amino acids L: -lysine, L: -threonine, and L: -tryptophan, which are produced by fermentation processes using high-performance strains of Corynebacterium glutamicum and Escherichia coli from sugar sources such as molasses, sucrose, or glucose. But the market for amino acids in synthesis is also becoming increasingly important, with annual growth rates of 5-7%. The use of enzymes and whole cell biocatalysts has proven particularly valuable in production of both proteinogenic and nonproteinogenic L: -amino acids, D: -amino acids, and enantiomerically pure amino acid derivatives, which are of great interest as building blocks for active ingredients that are applied as pharmaceuticals, cosmetics, and agricultural products. Nutrition and health will continue to be the driving forces for exploiting the potential of microorganisms, and possibly also of suitable plants, to arrive at even more efficient processes for amino acid production.

Membrane reactors at Degussa.

The review covers the development of membrane reactor technologies at Degussa for the synthesis of fine chemicals. The operation of fed-batch or continuous biocatalytic processes in the enzyme membrane reactor (EMR) is well established at Degussa. Degussa has experience of running EMRs from laboratory gram scale up to a production scale of several hundreds of tons per year. The transfer of the enzyme membrane reactor from biocatalysis to chemical catalysis in the chemzyme membrane reactor (CMR) is discussed. Various homogeneous catalysts have been investigated in the CMR, and the scope and limitation of this new technique is discussed.

The first aminoacylase-catalyzed enantioselective synthesis of aromatic beta-amino acids.

The first aminoacylase-catalyzed enantioselective synthesis of aromatic beta-amino acids is reported. The presence of an N-chloroacetyl group as acyl group in the substrate as well as the use of porcine kidney acylase I as a suitable enzyme component are prerequisites for this resolution process whereby optically active beta-amino acids are formed with high enantioselectivities of >98% ee.

Synthesis of a new chiral bisphospholane ligand for the Rh(I)-catalyzed enantioselective hydrogenation of isomeric beta-acylamido acrylates.

The highly stereoselective synthesis of a chiral silylphospholane has been described, which can be advantageously used as a building block under base-free conditions for the construction of diphosphines related to DuPHOS. The utility of silylphospholane is shown in the synthesis of a new bisphospholane ligand 1 (MalPHOS), which is characterized by a maleic anhydride backbone. The ligand forms with Rh(I) a complex with a larger bite angle P-Rh-P than the analogue Me-DuPHOS complex. Both complexes have been tested in the asymmetric hydrogenation of unsaturated alpha- and beta-amino acid precursors of pharmaceutical relevance. In several cases, the new catalyst was superior in comparison to the Me-DuPHOS complex, in particular when (Z)-configured beta-acylamido acrylates were used as substrates.

Melanoma cell CD44 interaction with the alpha 1(IV)1263-1277 region from basement membrane collagen is modulated by ligand glycosylation.

Invasion of the basement membrane is believed to be a critical step in the metastatic process. Melanoma cells have been shown previously to bind distinct triple-helical regions within basement membrane (type IV) collagen. Additionally, tumor cell binding sites within type IV collagen contain glycosylated hydroxylysine residues. In the present study, we have utilized triple-helical models of the type IV collagen alpha1(IV)1263-1277 sequence to (a) determine the melanoma cell receptor for this ligand and (b) analyze the results of single-site glycosylation on melanoma cell recognition. Receptor identification was achieved by a combination of methods, including (a) cell adhesion and spreading assays using triple-helical alpha1(IV)1263-1277 and an Asp(1266)Abu variant, (b) inhibition of cell adhesion and spreading assays, and (c) triple-helical alpha1(IV)1263-1277 affinity chromatography with whole cell lysates and glycosaminoglycans. Triple-helical alpha1(IV)1263-1277 was bound by melanoma cell CD44/chondroitin sulfate proteoglycan receptors and not by the collagen-binding integrins or melanoma-associated proteoglycan. Melanoma cell adhesion to and spreading on the triple-helical alpha1(IV)1263-1277 sequence was then compared for glycosylated (replacement of Lys(1265) with Hyl(O-beta-d-galactopyranosyl)) versus non-glycosylated ligand. Glycosylation was found to strongly modulate both activities, as adhesion and spreading were dramatically decreased due to the presence of galactose. CD44/chondroitin sulfate proteoglycan did not bind to glycosylated alpha1(IV)1263-1277. Overall, this study (a) is the first demonstration of the prophylactic effects of glycosylation on tumor cell interaction with the basement membrane, (b) provides a rare example of an apparent unfavorable interaction between carbohydrates, and (c) suggests that sugars may mask "cryptic sites" accessible to tumor cells with cell surface or secreted glycosidase activities.

Practical asymmetric enzymatic reduction through discovery of a dehydrogenase-compatible biphasic reaction media.

[reaction: see text] An enzyme-compatible biphasic reaction media for the asymmetric biocatalytic reduction of ketones with in situ cofactor regeneration has been developed. In this biphasic reaction media, which is advantageous for reactions at higher substrate concentrations, both enzymes (alcohol dehydrogenase and FDH from Candida boidinii) remain stable. The reductions with poorly water-soluble ketones were carried out at substrate concentrations of 10-200 mM, and the optically active (S)-alcohols were formed with moderate to good conversions and with up to >99% ee.

Synthesis and use of alpha-silyl-substituted alpha-hydroxyacetic acids.

[reaction: see text] Rhodium-catalyzed oxygen transfer was used to generate benzyl 2-silyl-2-oxoacetates in good yields. The hydrogenation of these compounds led to chiral alpha-silyl-substituted alpha-hydroxyacetic acids. Resolution by means of HPLC using a chiral stationary phase afforded an enantiomerically pure representative of this class of compounds, which was successfully applied as a chiral ligand in an asymmetric aldol-type reaction.

On the enantioselective hydrogenation of isomeric methyl 3-acetamidobutenoates with RhI complexes.

The enantioselective hydrogenation of E- and Z-methyl 3-acetamidobutenoate, key intermediates in the synthesis of a pharmaceutically important chiral beta-amino acid, with RhI catalysts in MeOH as solvent has been investigated in detail. As chiral ligands, Et-DuPHOS, Me4-BASPHOS, DI-PAMP, DIOP, HO-DIOP and Et-Ferro-TANE have been employed. The particular role of oxyfunctionalization in some diphosphine catalysts is addressed in relation to the E/Z geometry of the substrate and the dependency of the ee on the H2 pressure. Kinetic investigations with [Rh(diphosphane)(MeOH)2]-BF4, taking into consideration the special nature of the precatalyst [[Rh-(cod)2]BF4/ligand versus [Rh(cod)ligand)]BF4], NMR spectroscopic measurements and the H2 pressure dependence of the observed enantioselectivity provide evidence that the reaction proceeds via an "unsaturated route" mechanism. This mechanism correlates to catalytic features found in the past for the hydrogenation of related unsaturated alpha-amino acid precursors. The influence of the temperature was similarly investigated. A nonlinear dependency of the enantiomeric ratio as a function of the reciprocal of the temperature has been found. The correlation between temperature and H2 pressure and their effects on the enantioselectivity is discussed. In general, the highest enantioselectivities for the hydrogenation of both isomeric substrates can be achieved at room temperature and below, whereas the fastest conversion takes place at 30-50 degrees C.

Synthesis of Optically Active α-Amino- phosphinic Acids by Catalytic Asymmetric Hydrogenation in Organic Solvents and Aqueous Micellar Media.

Enantiomeric excesses of up to 99 % could be obtained in the synthesis of the biologically interesting acylated α-aminophosphinic acids 1 (R1 =Me, Ph; R2 =H, Me, Et; R3 =H, F, iPr) by asymmetric hydrogenation with rhodium complex catalysts and subsequent hydrolysis [Eq. (1)]. cod=1,5-cyclooctadiene.