Immacolata Serra

Recent Advances in Lipase-Mediated Preparation of Pharmaceuticals and Their Intermediates.

Biocatalysis offers an alternative approach to conventional chemical processes for the production of single-isomer chiral drugs. Lipases are one of the most used enzymes in the synthesis of enantiomerically pure intermediates. The use of this type of enzyme is mainly due to the characteristics of their regio-, chemo- and enantioselectivity in the resolution process of racemates, without the use of cofactors. Moreover, this class of enzymes has generally excellent stability in the presence of organic solvents, facilitating the solubility of the organic substrate to be modified. Further improvements and new applications have been achieved in the syntheses of biologically active compounds catalyzed by lipases. This review critically reports and discusses examples from recent literature (2007 to mid-2015), concerning the synthesis of enantiomerically pure active pharmaceutical ingredients (APIs) and their intermediates in which the key step involves the action of a lipase.

New active site oriented glyoxyl-agarose derivatives of Escherichia coli penicillin G acylase.

BackgroundImmobilized Penicillin G Acylase (PGA) derivatives are biocatalysts that are industrially used for the hydrolysis of Penicillin G by fermentation and for the kinetically controlled synthesis of semi-synthetic β-lactam antibiotics. One of the most used supports for immobilization is glyoxyl-activated agarose, which binds the protein by reacting through its superficial Lys residues. Since in E. coli PGA Lys are also present near the active site, an immobilization that occurs through these residues may negatively affect the performance of the biocatalyst due to the difficult diffusion of the substrate into the active site. A preferential orientation of the enzyme with the active site far from the support surface would be desirable to avoid this problem.ResultsHere we report how it is possible to induce a preferential orientation of the protein during the binding process on aldehyde activated supports. A superficial region of PGA, which is located on the opposite side of the active site, is enriched in its Lys content. The binding of the enzyme onto the support is consequently forced through the Lys rich region, thus leaving the active site fully accessible to the substrate. Different mutants with an increasing number of Lys have been designed and, when active, immobilized onto glyoxyl agarose. The synthetic performances of these new catalysts were compared with those of the immobilized wild-type (wt) PGA. Our results show that, while the synthetic performance of the wt PGA sensitively decreases after immobilization, the Lys enriched mutants have similar performances to the free enzyme even after immobilization.We also report the observations made with other mutants which were unable to undergo a successful maturation process for the production of active enzymes or which resulted toxic for the host cell.ConclusionThe desired orientation of immobilized PGA with the active site freely accessible can be obtained by increasing the density of Lys residues on a predetermined region of the enzyme. The newly designed biocatalysts display improved synthetic performances and are able to maintain a similar activity to the free enzymes. Finally, we found that the activity of the immobilized enzyme proportionally improves with the number of introduced Lys.

Modulation of the Microenvironment Surrounding the Active Site of Penicillin G Acylase Immobilized on Acrylic Carriers Improves the Enzymatic Synthesis of Cephalosporins

The catalytic properties of penicillin G acylase (PGA) from Escherichia coli in kinetically controlled synthesis of β-lactam antibiotics are negatively affected upon immobilization on hydrophobic acrylic carriers. Two strategies have been here pursued to improve the synthetic performance of PGA immobilized on epoxy-activated acrylic carriers. First, an aldehyde-based spacer was inserted on the carrier surface by glutaraldehyde activation (immobilization yield = 50%). The resulting 3-fold higher synthesis/hydrolysis ratio (vs/vh1 = 9.7 ± 0.7 and 10.9 ± 0.7 for Eupergit® C and Sepabeads® EC-EP, respectively) with respect to the unmodified support (vs/vh1 = 3.3 ± 0.4) was ascribed to a facilitated diffusion of substrates and products as a result of the increased distance between the enzyme and the carrier surface. A second series of catalysts was prepared by direct immobilization of PGA on epoxy-activated acrylic carriers (Eupergit® C), followed by quenching of oxiranes not involved in the binding with the protein with different nucleophiles (amino acids, amines, amino alcohols, thiols and amino thiols). In most cases, this derivatization increased the synthesis/hydrolysis ratio with respect to the non derivatized carrier. Particularly, post-immobilization treatment with cysteine resulted in about 2.5-fold higher vs/vh1 compared to the untreated biocatalyst, although the immobilization yield decreased from 70% (untreated Eupergit® C) to 20%. Glutaraldehyde- and cysteine-treated Eupergit® C catalyzed the synthesis of cefazolin in 88% (±0.9) and 87% (±1.6) conversion, respectively, whereas untreated Eupergit® C afforded this antibiotic in 79% (±1.2) conversion.

Characterization and study of the orientation of immobilized enzymes by tryptic digestion and HPLC-MS: design of an efficient catalyst for the synthesis of cephalosporins.

An innovative approach to determine the orientation of penicillin G acylase (PGA) from Escherichia coli covalently immobilized onto solid supports has been developed. This method is based on tryptic digestion of immobilized PGA followed by HPLC-MS analysis of the released peptides which are supposed to be only those exposed toward the reaction medium and not directly bound to the solid support. To this purpose, PGA was immobilized on Eupergit C (acrylic hydrophobic resin) and glyoxyl-agarose (hydrophilic resin) functionalized with epoxy and aldehyde groups, respectively, both involving the Lys residues of the protein. The peptide maps obtained were analyzed to derive the orientation of immobilized PGA, as the position of the detected Lys gave indication concerning the accessibility of the different areas of the protein. The results indicate that PGA immobilization on both supports involves mainly Lys located near the binding pocket (70%). Some differences in the enzyme orientation on the two supports can be deduced by the presence of different unbound Lys residues in the released peptides, specific to each support (Lys 117alpha for PGA-Eupergit C; Lys 163alpha and Lys 165alpha for PGA-glyoxyl-agarose). These results have been correlated with the data obtained in the kinetically controlled synthesis and indicate that the orientation of PGA on both supports is partially unfavorable, driving the active site near the support surface. This type of orientation of the enzyme enhances the effect of the nature of the support and of the binding chemistry on the catalytic properties. The information obtained indicated the most suitable support and activation strategy to design an immobilized acylase with good synthetic properties for preparative processes. The glyoxyl-Eupergit C support with enhanced porosity synergically combines the mechanical stability and synthetic performances of immobilized PGA and was successfully used in the synthesis of several cephalosporins.

Stereoselective reduction of aromatic ketones by a new ketoreductase from Pichia glucozyma

A new NADPH-dependent benzil reductase (KRED1-Pglu) was identified from the genome of the non-conventional yeast Pichia glucozyma CBS 5766 and overexpressed in E. coli. The new protein was characterised and reaction parameters were optimised for the enantioselective reduction of benzil to (S)-benzoin. A thorough study of the substrate range of KRED1-Pglu was conducted; in contrast to most other known ketoreductases, KRED1-Pglu prefers space-demanding substrates, which are often converted with high stereoselectivity. A molecular modelling study was carried out for understanding the structural determinants involved in the stereorecognition experimentally observed and unpredictable on the basis of steric properties of the substrates. As a result, a new useful catalyst was identified, enabling the enantioselective preparation of different aromatic alcohols and hydroxyketones.

Chemoenzymatic synthesis of neoglycoproteins driven by the assessment of protein surface reactivity

In this paper a series of 2-iminomethoxyethyl mannose-based mono- and disaccharides have been synthesized by a chemoenzymatic approach and used in coupling reactions with e-amino groups of lysine residues in a model protein (ribonuclease A, RNase A) to give semisynthetic neoglycoconjugates. In order to study the influence of structure of the glycans on the conjugation outcomes, an accurate characterization of the prepared neoglycoproteins was performed by a combination of ESI-MS and LC-MS analytical methods. The analyses of the chymotryptic digests of the all neoglycoconjugates revealed six Lys-glycosylation sites with a the following order of lysine reactivity: Lys 1 ≫ Lys 91 ≅ Lys 31 > Lys 61 ≅ Lys 66. A computational analysis of the reactivity of each lysine residue has been also carried out considering several parameters (amino acids surface exposure and pKa, protein flexibility). The in silico evaluation seems to confirm the order in lysine reactivity resulting from proteomic analysis.

Redesigning the synthesis of vidarabine via a multienzymatic reaction catalyzed by immobilized nucleoside phosphorylases

We here report on the enzymatic synthesis of the antiviral drug vidarabine (arabinosyladenine, araA) starting from arabinosyluracil and adenine. To this aim, uridine phosphorylase from Clostridium perfringens (CpUP) and a purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP) were used as covalently immobilized biocatalysts. Upon investigation of the optimal conditions for the enzyme activity (phosphate buffer 25 mM, pH 7.5, 25 °C, DMF 12.5–30%), the synthesis of araA was scaled up (2 L) and the product was isolated in 53% yield (3.5 g L−1) and 98.7% purity. An E-factor comparison between the enzymatic synthesis of araA and the classical chemical procedure clearly highlighted the “greenness” of the enzymatic route over the chemical one (E-factor: 423 vs. 1356, respectively).

Liquid chromatography-mass spectrometry structural characterization of neo glycoproteins aiding the rational design and synthesis of a novel glycovaccine for protection against tuberculosis.

Hereby we describe a pilot study for the rational design and synthesis of a glycoconjugate vaccine against Tuberculosis (TB) by site-specific coupling of well-defined glycans to non-antigenic amino acids in a selected protein carrier. A combination of ESI-MS and LC-MS analytical methods was applied for the systematic characterization of the reactivity of the surface amino acids in the glycosylation reaction with monosaccharides towards 2-iminomethoxyethyl or homobifunctional (4-nitrophenyl ester) linkers, both on the model protein, ribonuclease A (RNase A) and on TB10.4, the simplest antigenic protein isolated from Mycobacterium tuberculosis (MTB). Intact protein analysis was carried out to quantify the glycosylation degree and profile the glycoform composition of all the prepared neo glycoconjugates, while pronase and chymotriptic digests were analyzed to map and rank the reactivity of protein residues. Neo glycopeptides were purified by on-line porous graphitized carbon solid-phase extraction, separated by hydrophilic interaction liquid chromatography and analyzed by electrospray mass spectrometry (ESI-MS(n)). Significantly, different site specificity and glycosylation efficiency were demonstrated for the two linkers, resulting in structurally diverse glycoconjugates. A computational analysis of the amino acids involved in the epitope formation in TB10.4 addressed the choice to 2-iminomethoxyethyl-saccharide activation, that resulted in a more targeted and selective conjugation preserving the protein antigenicity. Additionally, a rational design of experiments lead to the identification of suitable experimental conditions for the preparation of highly pure and homogeneous neo glycoconjugates.

Immobilized purine nucleoside phosphorylase from Aeromonas hydrophila as an on-line enzyme reactor for biocatalytic applications

We described the development of a biochromatographic system which uses a purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP) for the evaluation of the substrate specificity on nucleoside libraries. AhPNP has been covalently immobilized on a fused silica Open Tubular Capillary (OTC) via Schiff base chemistry. The resulting bioreactor has been characterized by the determination of kinetic constants (Km and Vmax) for a natural substrate (inosine) and then assayed versus all natural purine (deoxy)ribonucleosides and a small library of 6-substituted purine ribosides. Characterization of the bioreactor has been carried out through a bidimensional chromatographic system with the sample on-line transfer from the bioreactor to the analytical column for the separation and quantification of substrate and product. Comparison with the soluble enzyme showed that the AhPNP-based bioreactor is reliable as the same ranking order, with respect to the standard activity assay, was obtained. The stability of the IMER was also assessed and the system was found to be stable up to 60 reactions.

Nucleoside phosphorylases from clostridium perfringens in the synthesis of 2',3'-dideoxyinosine.

Four Clostridium perfringens phosphorylases were subcloned, overexpressed and analyzed for their substrate specificity. DeoD(1) and PunA could use a variety of purine substrates, including an antiviral drug 2′,3′-dideoxyinosine (ddI). In one-pot synthesis using Clostridium phosphorylases, 2′,3′-dideoxyuridine and hypoxanthine were converted to ddI at yield of about 30%.