Structural Insights into the Recovery of Aldolase Activity in N‐Acetylneuraminic Acid Lyase by Replacement of the Catalytically Active Lysine with γ‐Thialysine by Using a Chemical Mutagenesis Strategy

Chemical modification has been used to introduce the unnatural amino acid γ‐thialysine in place of the catalytically important Lys165 in the enzyme N‐acetylneuraminic acid lyase (NAL). The Staphylococcus aureus nanA gene, encoding NAL, was cloned and expressed in E. coli. The protein, purified in high yield, has all the properties expected of a class I NAL. The S. aureus NAL which contains no natural cysteine residues was subjected to site‐directed mutagenesis to introduce a cysteine in place of Lys165 in the enzyme active site. Subsequently chemical mutagenesis completely converted the cysteine into γ‐thialysine through dehydroalanine (Dha) as demonstrated by ESI‐MS. Initial kinetic characterisation showed that the protein containing γ‐thialysine regained 17 % of the wild‐type activity. To understand the reason for this lower activity, we solved X‐ray crystal structures of the wild‐type S. aureus NAL, both in the absence of, and in complex with, pyruvate. We also report the structures of the K165C variant, and the K165‐γ‐thialysine enzyme in the presence, or absence, of pyruvate. These structures reveal that γ‐thialysine in NAL is an excellent structural mimic of lysine. Measurement of the pH‐activity profile of the thialysine modified enzyme revealed that its pH optimum is shifted from 7.4 to 6.8. At its optimum pH, the thialysine‐containing enzyme showed almost 30 % of the activity of the wild‐type enzyme at its pH optimum. The lowered activity and altered pH profile of the unnatural amino acid‐containing enzyme can be rationalised by imbalances of the ionisation states of residues within the active site when the pK_a of the residue at position 165 is perturbed by replacement with γ‐thialysine. The results reveal the utility of chemical mutagenesis for the modification of enzyme active sites and the exquisite sensitivity of catalysis to the local structural and electrostatic environment in NAL.     

Kinetic Resolution of Aliphatic β‐Amino Acid Amides by β‐Aminopeptidases

Access to enantiopure β‐amino acids : β‐Aminopeptidases are hydrolases that possess the unique ability to cleave N‐terminal β‐amino acids from peptides and amides. Hydrolysis of racemic β‐amino acid amides catalyzed by these enzymes displays enantioselectivity with strong preference for substrates with the L ‐configuration, and gives access to various aliphatic β‐amino acids of high enantiopurity.

     

Identification and Characterization of a Methionine γ‐Lyase in the Calicheamicin Biosynthetic Cluster of Micromonospora echinospora

CalE6 is a previously uncharacterized protein involved in the biosynthesis of calicheamicins in Micromonospora echinospora. It is a pyridoxal‐5′‐phosphate‐dependent enzyme and exhibits high sequence homology to cystathionine γ‐lyases and cystathionine γ‐synthases. However, it was found to be active towards methionine and to convert this amino acid into α‐ketobutyrate, ammonium, and methanethiol. The crystal structure of the cofactor‐bound holoenzyme was resolved at 2.0 Å; it contains two active site residues, Gly105 and Val322, specific for methionine γ‐lyases. Modeling of methionine into the active site allows identification of the active site residues responsible for substrate recognition and catalysis. These findings support that CalE6 is a putative methionine γ‐lyase producing methanethiol as a building block in biosynthesis of calicheamicins.     

Enzymatic Conversion of Unnatural Amino Acids by Yeast D‐Amino Acid Oxidase

Unnatural amino acids, particularly synthetic α‐amino acids, are becoming crucial tools for modern drug discovery research. In particular, this application requires enantiomerically pure isomers. In this work we report on the resolution of racemic mixtures of the amino acids d,l ‐naphthylalanine and d,l ‐naphthylglycine by using a natural enzyme, D ‐amino acid oxidase from the yeast Rhodotorula gracilis. A significant improvement of the bioconversion is obtained using a single‐point mutant enzyme designed by a rational approach. With this D ‐amino acid oxidase variant the complete resolution of all the unnatural amino acids tested was obtained: in this case, the bioconversion requires a shorter time and a lower amount of biocatalyst compared to the wild‐type enzyme. The simultaneous production of the corresponding α‐keto acid, a possible precursor of the amino acid in the L ‐form, improves the significance of the procedure.     

Organocatalytic Enantioselective Michael‐Addition of Malonic Acid Half‐Thioesters to β‐Nitroolefins: From Mimicry of Polyketide Synthases to Scalable Synthesis of γ‐Amino Acids

Highly enantioselective biomimetic Michael addition reactions of malonic acid half thioesters (MAHTs) to a variety of nitroolefins, affording the optically active γ‐amino acid precursors, were developed by employing the Cinchona‐based squaramides (up to >99% ee). Remarkably, this biomimetic process is enantioconvergent, a highly desirable feature of a catalytic asymmetric reaction, whereby E/Z‐isomers of the nitroolefins afford the same product enantiomer. The synthetic utility of this organocatalytic protocol was also demonstrated in the formal synthesis of pharmaceutically important γ‐amino acids such as baclofen. Moreover, a quantum chemical analysis of the catalyst‐substrate complexes is shown to give a detailed and instrumental insight into the origin of the observed catalytic activity.     

Chemoenzymatic Preparation of Enantiopure Homoadamantyl β‐Amino Acid and β‐Lactam Derivatives

Racemic cis‐10‐azatetracyclo[7.2.0.1^2,6.1^4,8]tridecan‐11‐one was prepared from homoadamant‐4‐ene by chlorosulfonyl isocyanate addition. The transformation of the β‐lactam to the corresponding β‐amino ester followed by Candida antarctica lipase A‐catalyzed enantioselective (E>>200) N‐acylation with 2,2,2‐trifluoroethyl butanoate afforded methyl (1R,4R,5S,8S)‐5‐aminotricyclo[4.3.1.1^3,8]undecane‐4‐carboxylate and the (1S,4S,5R,8R)‐butanamide with>99% ee at 50% conversion. Alternatively, transformation of the β‐lactam to the corresponding N‐hydroxymethyl‐β‐lactam and the following Pseudomonas cepacia (currently Burkholderia cepacia) lipase‐catalyzed enantioseletive O‐acylation provided the (1S,4S,6R,9R)‐alcohol (ee=87%) and the corresponding (1R,4R,6S,9S)‐butanoate (ee>99%). In the latter method, competition for the enzyme between the (1R,4R,6S,9S)‐butanoate, 2,2,2‐trifluoroethyl butanoate and the hydrolysis product, butanoic acid, tended to stop the reaction at about 45% conversion and finally gave racemization in the (1S,4S,6R,9R)‐alcohol with time.     

Multi‐Enzymatic Synthesis of Optically Pure β‐Hydroxy α‐Amino Acids

A novel enzymatic production system of optically pure β‐hydroxy α‐amino acids was developed. Two enzymes were used for the system: an N‐succinyl L ‐amino acid β‐hydroxylase (SadA) belonging to the iron(II)/α‐ketoglutarate‐dependent dioxygenase superfamily and an N‐succinyl L ‐amino acid desuccinylase (LasA). The genes encoding the two enzymes are part of a gene set responsible for the biosynthesis of peptidyl compounds found in the Burkholderia ambifaria AMMD genome. SadA stereoselectively hydroxylated several N‐succinyl aliphatic L ‐amino acids and produced N‐succinyl β‐hydroxy L ‐amino acids, such as N‐succinyl‐L ‐β‐hydroxyvaline, N‐succinyl‐L ‐threonine, (2S,3R)‐N‐succinyl‐L ‐β‐hydroxyisoleucine, and N‐succinyl‐L ‐threo‐β‐hydroxyleucine. LasA catalyzed the desuccinylation of various N‐succinyl‐L ‐amino acids. Surprisingly, LasA is the first amide bond‐forming enzyme belonging to the amidohydrolase superfamily, and has succinylation activity towards the amino group of L ‐leucine. By combining SadA and LasA in a preparative scale production using N‐succinyl‐L ‐leucine as substrate, 2.3 mmol of L ‐threo‐β‐hydroxyleucine were successfully produced with 93% conversion and over 99% of diastereomeric excess. Consequently, the new production system described in this study has advantages in optical purity and reaction efficiency for application in the mass production of several β‐hydroxy α‐amino acids.

     

Asymmetric Aza‐Morita–Baylis–Hillman‐Type Reactions: The Highly Enantioselective Reaction between Unmodified α,β‐ Unsaturated Aldehydes and N‐Acylimines by Organo‐co‐catalysis

The highly enantioselective organo‐co‐catalytic aza‐Morita–Baylis–Hillman (MBH)‐type reaction between N‐carbamate‐protected imines and α,β‐unsaturated aldehydes has been developed. The organic co‐catalytic system of proline and 1,4‐diazabicyclo[2.2.2]octane (DABCO) enables the asymmetric synthesis of the corresponding N‐Boc‐ and N‐Cbz‐protected β‐amino‐α‐alkylidene‐aldehydes in good to high yields and up to 99% ee. In the case of aza‐MBH‐type addition of enals to phenylprop‐2‐ene‐1‐imines, the co‐catalytic reaction exhibits excellent 1,2‐selectivity. The organo‐co‐catalytic aza‐MBH‐type reaction can also be performed by the direct highly enantioselective addition of α,β‐unsaturated aldehydes to bench‐stable N‐carbamate‐protected α‐amidosulfones to give the corresponding β‐amino‐α‐alkylidene‐aldehydes with up to 99% ee. The organo‐co‐catalytic aza‐MBH‐type reaction is also an expeditious entry to nearly enantiomerically pure β‐amino‐α‐alkylidene‐amino acids and β‐amino‐α‐alkylidene‐lactams (99% ee). The mechanism and stereochemistry of the chiral amine and DABCO co‐catalyzed aza‐MBH‐type reaction are also discussed.     

Enantiopure N‐Benzyloxycarbonyl‐β2‐amino Acid Allyl Esters from Racemic β‐Lactams by Dynamic Kinetic Resolution using Candida antarctica Lipase B

The dynamic kinetic resolution of α‐substituted racemic β‐lactams by alcoholytic ring‐opening, catalyzed by immobilized lipase B from Candida antarctica is described. With this process, a variety of racemic α‐substituted N‐Cbz‐azetidinones (Cbz=benzyloxycarbonyl) was transformed to the corresponding N‐Cbz‐protected β²‐amino acid allyl esters with high enantioselectivity (up to 99%) and high yields (up to quantitative) at room temperature.

     

Surface free‐energy analysis of poly(N‐vinyl‐2‐pyrrolidone–crotonic acid) copolymers prepared by γ‐ray‐induced polymerization technique

Composite hydrogels of pectin and polyacrylamide were synthesized and evaluated by scanning electron microscopy, atomic force microscopy, light microscopy, and dynamic mechanical analysis. The crosslinking polymerization of acrylamide in pectin solution resulted in a composite having a macroporous pectin domain with an interstitial polyacrylamide domain. This composite had improved mechanical properties compared to those of either polymer alone, and it absorbed and retained more water than crosslinked polyacrylamide alone. Furthermore, crosslinking polymerization of acrylamide in an existing pectinate scaffold resulted in a double‐network architecture, where filamentous polyacrylamide networks penetrated through pores of the pectin scaffold. It was found that pectins dictated the features of microstructure in the composites through regulating the coordination of phase separation of the two components and water partition between the two phases. Results from this study highlight potential new uses of pectins in protecting the physical structure of environmentally sensitive polymers from mechanical damage related to freezing, lyophilization, and other conditions experienced during their use in biomedical and industrial products. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 92: 1893–1901, 2004     

Synthesis,Structure–Activity,and Structure–Stability Relationships of 2‐Substituted‐N‐(4‐oxo‐3‐oxetanyl) N‐Acylethanolamine Acid Amidase (NAAA) Inhibitors

N‐Acylethanolamine acid amidase (NAAA) is a cysteine amidase that preferentially hydrolyzes saturated or monounsaturated fatty acid ethanolamides (FAEs), such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), which are endogenous agonists of nuclear peroxisome proliferator‐activated receptor‐α (PPAR‐α). Compounds that feature an α‐amino‐β‐lactone ring have been identified as potent and selective NAAA inhibitors and have been shown to exert marked anti‐inflammatory effects that are mediated through FAE‐dependent activation of PPAR‐α. We synthesized and tested a series of racemic, diastereomerically pure β‐substituted α‐amino‐β‐lactones, as either carbamate or amide derivatives, investigating the structure–activity and structure–stability relationships (SAR and SSR) following changes in β‐substituent size, relative stereochemistry at the α‐ and β‐positions, and α‐amino functionality. Substituted carbamate derivatives emerged as more active and stable than amide analogues, with the cis configuration being generally preferred for stability. Increased steric bulk at the β‐position negatively affected NAAA inhibitory potency, while improving both chemical and plasma stability.     

Enantioselective Hydrogenation of α‐Dehydroamino Acid Esters Catalyzed by Rhodium Complexes with Chiral Bisaminophosphine Ligands

A highly efficient strategy for the synthesis of a series of chiral bisaminophosphine ligands was well established with several remarkable features. The synthetic utility of these ligands was explored for rhodium‐catalyzed asymmetric hydrogenations of α‐dehydroamino acid esters. Up to 98% ee values were achieved for the enantioselective synthesis of aminocarboxylic acids and their derivatives, which are very important chiral building blocks for the synthesis of a variety of natural products and biologically active molecules.