Enantiopure Β-amino Acids Research Articles

Enantioenriched γ-Aminoalcohols, β-Amino Acids, β-Lactams, and Azetidines Featuring Tetrasubstituted Fluorinated Stereocenters via Palladacycle-Catalyzed Asymmetric Fluorination of Isoxazolinones.

Enantiopure fluorine containing β-amino acids are of large biological and pharmaceutical interest. Strategies to prepare β-amino acid derivatives possessing a F-containing tetrasubstituted stereocenter at the α-C atom in a catalytic asymmetric sense are rare, in particular using an enantioselective electrophilic C-F bond formation. In the present study, a highly enantioselective palladacycle-catalyzed fluorination of isoxazolinones was developed. It is demonstrated that isoxazolinones are useful precursors toward enantiopure β-amino acid derivatives by diastereo- and chemoselective reduction. The formed γ-aminoalcohols served as valuable precursors toward β-amino acids, β-amino acid esters, and β-lactams, all featuring tetrasubstituted fluorinated stereocenters. In addition, by this work, enantioenriched fluorinated azetidines were accessible for the first time.

β-Aminopeptidases: Insight into Enzymes without a Known Natural Substrate.

β-Aminopeptidases have the unique capability to hydrolyze N-terminal β-amino acids, with varied preferences for the nature of β-amino acid side chains. This unique capability makes them useful as biocatalysts for synthesis of β-peptides and to kinetically resolve β-peptides and amides for the production of enantiopure β-amino acids. To date, six β-aminopeptidases have been discovered and functionally characterized, five from Gram-negative bacteria and one from a fungus, Aspergillus Here we report on the purification and characterization of an additional four β-aminopeptidases, one from a Gram-positive bacterium, Mycolicibacterium smegmatis (BapAMs), one from a yeast, Yarrowia lipolytica (BapAYlip), and two from Gram-negative bacteria isolated from activated sludge identified as Burkholderia spp. (BapABcA5 and BapABcC1). The genes encoding β-aminopeptidases were cloned, expressed in Escherichia coli, and purified. The β-aminopeptidases were produced as inactive preproteins that underwent self-cleavage to form active enzymes comprised of two different subunits. The subunits, designated α and β, appeared to be tightly associated, as the active enzyme was recovered after immobilized-metal affinity chromatography (IMAC) purification, even though only the α-subunit was 6-histidine tagged. The enzymes were shown to hydrolyze chromogenic substrates with the N-terminal l-configurations β-homo-Gly (βhGly) and β3-homo-Leu (β3hLeu) with high activities. These enzymes displayed higher activity with H-βhGly-p-nitroanilide (H-βhGly-pNA) than previously characterized enzymes from other microorganisms. These data indicate that the new β-aminopeptidases are fully functional, adding to the toolbox of enzymes that could be used to produce β-peptides. Overexpression studies in Pseudomonas aeruginosa also showed that the β-aminopeptidases may play a role in some cellular functions.IMPORTANCE β-Aminopeptidases are unique enzymes found in a diverse range of microorganisms that can utilize synthetic β-peptides as a sole carbon source. Six β-aminopeptidases have been previously characterized with preferences for different β-amino acid substrates and have demonstrated the capability to catalyze not only the degradation of synthetic β-peptides but also the synthesis of short β-peptides. Identification of other β-aminopeptidases adds to this toolbox of enzymes with differing β-amino acid substrate preferences and kinetics. These enzymes have the potential to be utilized in the sustainable manufacture of β-amino acid derivatives and β-peptides for use in biomedical and biomaterial applications. This is important, because β-amino acids and β-peptides confer increased proteolytic resistance to bioactive compounds and form novel structures as well as structures similar to α-peptides. The discovery of new enzymes will also provide insight into the biological importance of these enzymes in nature.

From glucose to enantiopure morpholino β-amino acid: a new tool for stabilizing γ-turns in peptides

“Environmentally sustainable” synthesis of a new enantiopure morpholino β-amino acid from glucose: a new tool for exotic peptide architectures.

Enzymatic synthesis of sitagliptin intermediate using a novel ω-transaminase

Enantiopure β-amino acids are essential precursors of various pharmaceuticals, agrochemicals and other industrially important chemicals. In this study, we selected sixteen potential ω-Transaminases (ω-TAs) by BLAST and phylogenetic tree analysis. These ω-TAs were cloned, purified and tested for their reactivity for the synthesis of model β-amino acid (R)-3-amino-4-(2,4,5-triflurophenyl) butanoic acid [3-ATfBA], a key precursor for sitagliptin. In an enzymatic cascade, lipase converted β-ketoester substrate to β-keto acid, which was subsequently aminated by the selected ω-TA to its corresponding β-amino acid. A potent enzyme from Ilumatobacter coccineus (ω-TAIC) was identified for the production of 3-ATfBA. The pH dependency of the product inhibition suggested that lowering the reaction pH to 7.0 can circumvent the inhibition of ω-TAIC by 3-ATfBA and about 92.3% conversion of 100 mM β-keto ester substrate could be achieved. The applicability of this enzymatic system was further evaluated at the scale of 140 mM, wherein 3-ATfBA was generated with excellent conversion (81.9%) and enantioselectivity (99% ee). Furthermore, ω-TAIC was successfully used for the synthesis of various β-amino acids from their corresponding β-keto ester substrates.

An Expedient Synthesis of CMF-019: (S)-5-Methyl-3-{1-(pentan-3-yl)-2- (thiophen-2-ylmethyl)-1H-benzo[d]imidazole-5-carboxamido}hexanoic Acid, a Potent Apelin Receptor (APJ) Agonist.

Apelin receptor (APJ) is a G protein-coupled receptor (GPCR) activated by the endogenous peptide apelin. The apelin-APJ system has emerged as an important regulator of cardiovascular homeostasis. Recently, a potent benzimidazole-derived apelin peptidomimetic, CMF-019, was patented but without a comprehensive description of its synthesis and a complete spectroscopic characterization of the intermediates. Here, a detailed preparation of CMF-019 through a modified and improved synthetic pathway is described. In particular, the benzimidazole ring in 7 was tailored by the condensation of methyl 3- amino-4-(pentan-3-ylamino)benzoate (4) with (thiophene-2-yl)acetimidate salt 6. Saponification of 7 and the subsequent condensation of the free acid 8 with the corresponding enantiopure β-amino acid methyl ester generated methyl (S)-5-methyl-3-{1-(pentan-3-yl)-2-(thiophen-2-ylmethyl)-1Hbenzo[ d]imidazole-5-carboxamido}hexanoate (9). Hydrolysis of the latter with KOH in THF/water, followed by HPLC-purification, afforded the desired product, CMF-019 (potassium salt) 10. The approach reported herein enables preparation of 10 at a total yield of 12% over seven linear steps. Additionally, it does not require applying expensive designated microwave reactors and high-pressure hydrogenators. Thus, the elaborate synthesis provides a latent availability of potent agonist 10 for further exploring the physiologically essential apelin-APJ system.

Purification and characterization of enantioselective N-acetyl-β-Phe acylases from Burkholderia sp. AJ110349.

For the production of enantiopure β-amino acids, enantioselective resolution of N-acyl β-amino acids using acylases, especially those recognizing N-acetyl-β-amino acids, is one of the most attractive methods. Burkholderia sp. AJ110349 had been reported to exhibit either (R)- or (S)-enantiomer selective N-acetyl-β-Phe amidohydrolyzing activity, and in this study, both (R)- and (S)-enantioselective N-acetyl-β-Phe acylases were purified to be electrophoretically pure and determined the sequences, respectively. They were quite different in terms of enantioselectivities and in their amino acids sequences and molecular weights. Although both the purified acylases were confirmed to catalyze N-acetyl hydrolyzing activities, neither of them show sequence similarities to the N-acetyl-α-amino acid acylases reported thus far. Both (R)- and (S)-enantioselective N-acetyl-β-Phe acylase were expressed in Escherichia coli. Using these recombinant strains, enantiomerically pure (R)-β-Phe (>99% ee) and (S)-β-Phe (>99% ee) were obtained from the racemic substrate.

Chemical synthesis and enzymatic, stereoselective hydrolysis of a functionalized dihydropyrimidine for the synthesis of β-amino acids.

A novel substrate, 6-(4-nitrophenyl)dihydropyrimidine-2,4(1H,3H)-dione (pNO2PheDU), was chemically synthesized and analytically verified for the potential biocatalytic synthesis of enantiopure β-amino acids. The hydantoinase (EC 3.5.2.2) from Arthrobacter crystallopoietes DSM20117 was chosen to prove the enzymatic hydrolysis of this substrate, since previous investigations showed activities of this enzyme toward 6-monosubstituted dihydrouracils. Whole cell biotransformations with recombinant Escherichia coli expressing the hydantoinase showed degradation of pNO2PheDU. Additionally, the corresponding N-carbamoyl-β-amino acid (NCarbpNO2βPhe) was chemically synthesized, an HPLC-method with chiral stationary phases for detection of this product was established and thus (S)-enantioselectivity toward pNO2PheDU has been shown. Consequently this novel substrate is a potential precursor for the enantiopure β-amino acid para-nitro-β-phenylalanine (pNO2βPhe).Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-015-0174-8) contains supplementary material, which is available to authorized users.

Whole-cell biocatalytic production of variously substituted β-aryl- and β-heteroaryl-β-amino acids

Biologically-active β-peptides and pharmaceuticals that contain key β-amino acids are emerging as target therapeutics; thus, synthetic strategies to make substituted, enantiopure β-amino acids are increasing. Here, we use whole-cell Escherichia coli (OD600 ∼35) engineered to express a Pantoea agglomerans phenylalanine aminomutase (PaPAM) biocatalyst. In either 5mL, 100mL, or 1L of M9 minimal medium containing α-phenylalanine (20mM), the cells produced ∼1.4mgmL−1 of β-phenylalanine in each volume. Representative pilot-scale 5-mL cultures, fermentation reactions converted 18 variously substituted α-arylalanines to their (S)-β-aryl-β-amino acids in vivo and were not toxic to cells at mid- to late-stage growth. The β-aryl-β-amino acids made ranged from 0.043mg (p-nitro-β-phenylalanine, 4% converted yield) to 1.2mg (m-bromo-β-phenylalanine, 96% converted yield) over 6h in 5mL. The substituted β-amino acids made herein can be used in redox and Stille-coupling reactions to make synthetic building blocks, or as bioisosteres in drug design.

Synthesis of enantiomerically pure δ-benzylproline derivatives

The methodology allows for the preparation of enantiopure δ-substituted l-proline analogues bearing the side chain of proteinogenic residues.

Candida antarctica lipase B-catalyzed ring opening of 4-arylalkyl-substituted β-lactams

The Lipolase-catalyzed ring opening of racemic 4-benzyl- 3 and 4-phenylethyl-2-azetidinone 4 was performed with 0.5equiv of H2O in diisopropyl ether at 45°C. The resulting (S)-β-amino acid 5 or 6 (ee⩾87%) and (R)-β-lactam 7 or 8 (ee>99%) enantiomers could easily be separated. The ring opening of enantiomeric β-lactams with 18% aqueous HCl afforded the corresponding enantiopure β-amino acid hydrochlorides 9 and 10 (ee>99%).

Biocatalysis as a Profound Tool in the Preparation of Highly Enantiopure β‐Amino Acids

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Preparation of Enantiopure β‐Amino Acids by Homologation of α‐Amino Acids

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Biocatalysis as a profound tool in the preparation of highly enantiopure β-amino acids

Enzymatic preparations of highly enantiopure β-amino acids have been reviewed. The report contains 212 references.

Enantioselective Hydrogenation of (E)‐ and (Z)‐Isomeric Ethyl 3‐Acetamidobutenoates with Rh‐Bisphosphine Complexes: Effects of Substrate Geometry and Solvents on Reaction Rates and Catalyst Reusability.

Enantiopure β-amino acids are crucial structural features of numerous biologically active natural products as well as important building blocks for the synthesis of β-peptides and β-lactam antibiotics. Numbers of stoichiometric chiral auxilaries and catalytic methods have been developed to make chiral β-amino acids. One of the most promising processes for the convenient preparation of chiral β-amino acids on large scale is the asymmetric hydrogenation of suitable unsaturated precursors such as β-acetamido acrylates with Rh(I) catalysts bearing chiral phosphine ligands. The requisite prochiral substrates are easily available by treatment of β-keto carboxylates with NH4OAc and subsequent acylation. However, while literally thousands of reports are concerned with the Rh-catalyzed enantioselective hydrogenation of related prochiral α-acetamido acrylates, only a few devoted to the hydrogenation of β-analogues as substrates exist. The main reason for this is probably different behavior in the asymmetric hydrogenation, which had for a long time been attributed to particular substrates such as the isomeric ethyl 3-acetoamido butenoates (E)-1 and (Z)-1.

Enantioselective Hydrogenation of (E)-and (Z)-Isomeric Ethyl 3-Acetamidobutenoates with Rh-Bisphosphine Complexes: Effects of Substrate Geometry and Solvents on Reaction Rates and Catalyst Reusability

Enantiopure β-amino acids are crucial structural features of numerous biologically active natural products as well as important building blocks for the synthesis of β-peptides and β-lactam antibiotics. Numbers of stoichiometric chiral auxilaries and catalytic methods have been developed to make chiral β-amino acids. One of the most promising processes for the convenient preparation of chiral β-amino acids on large scale is the asymmetric hydrogenation of suitable unsaturated precursors such as β-acetamido acrylates with Rh(I) catalysts bearing chiral phosphine ligands. The requisite prochiral substrates are easily available by treatment of β-keto carboxylates with NH4OAc and subsequent acylation. However, while literally thousands of reports are concerned with the Rh-catalyzed enantioselective hydrogenation of related prochiral α-acetamido acrylates, only a few devoted to the hydrogenation of β-analogues as substrates exist. The main reason for this is probably different behavior in the asymmetric hydrogenation, which had for a long time been attributed to particular substrates such as the isomeric ethyl 3-acetoamido butenoates (E)-1 and (Z)-1.

Advanced procedure for the enzymatic ring opening of unsaturated alicyclic β-lactams

Enantiopure β-amino acids 1a– 4a and β-lactams 1b– 4b were prepared simultaneously through the lipolase-catalysed enantioselective ring opening of unsaturated racemic β-lactams (±)- 1-(±)- 4. High enantioselectivities ( E>200) were observed when the reactions were performed with 1 equiv of water in iPr 2O at 70 °C. The resolved (1 R,2 S)-amino acids (yield⩾45%) and (1 S,5 R)-, (1 S,6 R)- and (1 S,8 R)-lactams (yield⩾47%) could be easily separated. The ring opening of lactam enantiomers 1b– 4b with 18% HCl afforded the corresponding β-amino acid hydrochlorides 1c·HCl– 4c·HCl (ee >95%).

Reaction between N-alkylhydroxylamines and chiral enoate esters: more experimental evidence for a cycloaddition-like process, a rationale based on DFT theoretical calculations, and stereoselective synthesis of new enantiopure beta-amino acids.

The reactions between N-benzyl- and N-methylhydroxylamine and chiral enoate esters, derived from D-glyceraldehyde and (-)-verbenone, respectively, have been investigated. Theoretical calculations show that the most favorable mechanism involves the concerted cycloaddition of the hydroxylamine to the substrate. This result is in good agreement with the stereospecificity observed when the trisubstituted olefins are used. The open-chain adducts have been isolated when the processes are carried out at low temperatures and for short reaction times. These compounds evolve to the corresponding isoxazolidinones on standing at room temperature or under acid catalysis. The high pi-facial diastereoselection has been rationalized on the basis of steric effects induced by the dioxolane ring for D-glyceraldehyde derivatives or by the cyclobutane gem-dimethyl substitution for esters prepared from (-)-verbenone. As an application of these reactions, new beta-amino acids have been synthesized in a highly efficient and stereocontrolled manner.

AN IMPROVED SYNTHESIS OF ENANTIOPURE β-AMINO ACIDS

An improved method for the preparation of both the enantiopure β-amino acids is presented. The diastereomer benzyl β-amino esters, obtained by stereoselective reduction of β-enamino esters, were separated and hydrogenolyzed to the free enantiopure β-amino acids.

β-Peptides: a surprise at every turn

β-Peptides, i.e. oligomers of β-amino acids, containing as few as six residues may form surprisingly stable helices, with half-lives for the H/D exchange of the central NH protons of up to several days. Furthermore, these β-peptides (carrying the side chains of familiar α-amino acids such as Ala, Val, Leu, Phe, Lys in the 2- and/or 3-position of their 3-amino carboxylic moieties) have been shown to be stable to common peptidases for at least two days. In this article, a brief account of the results obtained since we started work in this area in early 1995 is given. The synthesis of enantiopure β-amino acids can be achieved by homologation of α-amino acids. The greater structural variability of β-amino acids leads to an even greater multitude of possible β-peptide primary and secondary structures. Circular dichroism, NMR and X-ray investigations have unveiled helical, pleated-sheet and tubular arrangements of linear and cyclic β-peptides composed of up to twelve β-amino acids. The prospects for the use of β-peptides as drugs, the construction of large, enzymatically-active β-proteins and their interaction with the natural, α-peptidic counterparts are discussed.

ChemInform Abstract: Approach to Highly Enantiopure β‐Amino Acid Esters by Using Lipase Catalysis in Organic Media.

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