Kinetic resolution of esters of amino acids in t‐butanol containing 5% water catalyzed by a stable industrial alkaline protease

Abstract

We developed a procedure for the resolution of esters of amino acids in 95% t-butanol, followed by saponification of the unreacted esters to afford both enantiomers with high yield and optical purity. The hydrolysis, catalyzed by alkaline protease, was conducted in a mixture of t-butanol(95%) and water (5%) at 25"C, with a pH controlled at pH 8.5 by the addition of NaOH (2 M). The hydrolyzed L-amino acid, which was insoluble under these conditions, precipitated during the course of hydrolysis. After separation of the precipitate, the pH of the filtrate was adjusted to 11.5 to sapomfy the unreacted ester. The D-antipode precipitated at pH 6.2-6.5. Both optically pure antipodes were obtained with high enantiomeric excesses and yields by simple filtration. o 1994 Wiey-Liss, Inc. KEY WORDS: resolution, D,L-&Oacid esters, alkaline protease, simple separation, high enantiomeric excess and yieldsThe search for proteases for peptide synthesis that are stable in organic solvents is the subject of extensive investigation. 's2 Several studies have demonstrated the possibility of using proteases to catalyze peptide synthesis in organic solvents, but a drawback of those reactions is the poor stability of the enzyme in organic solvents. Many immobilization processes have been developed to overcome this. Here we describe the use of an inexpensive enzyme, alcalase, as a catalyst for resolution of amino acids, some of which have uncommon side chains and are found in many biologically and pharmaceutically important peptides. 35 Most of these unnatural amino acids cannot be obtained by fermentation or recombinant DNA technology. Resolution is an effective way to produce optically pure unnatural amino acids.Alcalase is a proteolytic enzyme prepared from a selective strain of Bacillus lichenifomis. The major enzyme component in alcalase is subtilisin Carlsberg (alkaline protease A), which is a serine protease and which is widely used as additives in detergents as a digesting enzyme. This alcalase can maintain enzymatic activity and stability in organic solvents.6v7 Application of alcalase in organic synthesis has been limited, although it is a potent and inexpensive catalyst.'*' Scheme 1 shows the reaction sequence of the resolution. The hydrolysis catalyzed by alkaline protease was conducted in a mixture of t-butanol (95%) and water (5%) at 25°C with a pH controller at pH 8.5. The hydrolyzed amino acid was insoluble under these conditions and precipitated during the course of hydrolysis. The precipitate was separated by filtration, and the pH of the filtrate was adjusted to 11.5 to saponify the unreacted ester of the D-&O acid.

Enantioselective Synthesis of l‐Homophenylalanine by Whole Cells of Recombinant Escherichia coli Expressing l‐Aminoacylase and N‐Acylamino Acid Racemase Genes from Deinococcus radiodurans BCRC12827

“…The separately expressed whole cell system with the same activity level of NAAAR and LAA is more efficient than coexpressed whole system in the production of L-HPA. The separately expressed whole cell system with NAAAR and LAA activities of 3600 U/L gave a productivity of 10 mmol L-HPA/L h, which was higher than the previously reported methods for L-HPA production (7,(11)(12)(13). These findings have considerable importance for the possible commercial manufacture of L-HPA by an enzymatic process.…”

“…Proteases have been used to enantioselectively hydrolyze racemic HPA ester and N-protected HPA to L-HPA (13,33). Since these processes are based on kinetic resolution, only 50% theoretical yield can be obtained in the reaction.…”

“…Since the B. caldolyticus hydantoinase has very low activity toward L-form HPAH, the space-time yield of the L-hydantoinase process for the desired 100% conversion of HPAH to L-HPA is rather low. Proteases can enantioselectively hydrolyze L-HPA ester to L-HPA, whereas only 50% theoretical yield can be obtained in the processes (13).…”

Benzoylphosphonate‐Based Photoactive Phosphopeptide Mimetics for Modulation of Protein Tyrosine Phosphatases and Highly Specific Labeling of SH2 Domains

“…Next, the methyl ester 14 was saponified enzymatically with subtilisin Carlsberg ("alcalase") at pH 8. [25] The remaining protons in the two acidic positions of the molecule were replaced by sodium ions using sodium-loaded Amberlite IR-120 ion-exchange resin; the resulting N-Fmoc-protected 4-(O-benzylphosphonocar- As the first target protein for photoactive phosphotyrosine peptide mimetics, we selected the STAT5b protein, which contains a phosphotyrosine-recognizing SH2 domain. In the cellular context STAT5 is phosphorylated at a tyrosine residue predominantly by JAK2 kinase, [26] which is associated with several receptors including the erythropoietin [26,27] and interleukin 2 receptors; [26,28,29] this reaction results in the formation and subsequent dimerization of phospho-STAT5.…”

Benzoylphosphonat‐basierte, photoaktive Phosphotyrosinpeptidmimetika zur funktionellen Modulierung von Proteintyrosinphosphatasen und hochspezifischen Markierung von SH2‐Domänen

“…Als Nächstes wurde der Methylester 14 enzymatisch mit Subtilisin Carlsberg ("Alcalase") bei pH 8 verseift. [25] Die beiden Säuregruppen des Moleküls wurden mithilfe eines Natrium-beladenen Amberlite-IR-120-Ionenaustauscherharzes in die Natriumsalze überführt, wodurch der N-Fmoc-ge- [26] phosphoryliert. Das Protein bindet an einige Rezeptoren, darunter die Erythropoietin- [26,27] und Interleukin-2-Rezeptoren, [26,28,29] was die Bildung und Dimerisierung von Phospho-STAT5 zur Folge hat.…”