Enantioselective Synthesis of Pharmaceutically Relevant Bulky Arylbutylamines Using Engineered Transaminases

ATAs engineered for having an enlarged small binding pocket were applied for the synthesis of enantiomerically pure (R)‐benzo[1,3]dioxol‐5‐yl‐butylamine, a chiral component of human leukocyte elastase inhibitor DMP 777 (L‐694,458). Kinetic resolution of the racemic amine was performed by using the L59A variant of the (S)‐selective ATA from Chromobacterium violaceum (Cv‐ATA), providing the residual (R)‐enantiomer in excellent yield and >99% ee. At moderate enzyme loading and absence of co‐solvent, high volumetric productivity of 0.22 mol L⁻¹ h⁻¹ (42.5 g L⁻¹ h⁻¹) was achieved. Complementarily, the (S)‐enantiomer was generated via kinetic resolution using the (R)‐selective ATA‐117‐Rd11 from Arthrobacter sp. with acetone as the amino acceptor. In an alternative approach, we employed ATA‐117‐Rd11 for the asymmetric amination of the prochiral ketone precursor, which at 86% conversion gave the (R)‐benzo[1,3]dioxol‐5‐yl‐butylamine with excellent >99% ee. We further evaluated the utility of Cv‐ATA L59A for the asymmetric synthesis of pharmaceutically relevant (S)‐1‐phenylbutan‐1‐amine, a chiral component of the deubiquitinase inhibitor degrasyn (WP1130). The enzyme showed good tolerance to high concentrations of isopropylamine, producing (S)‐1‐phenylbutan‐1‐amine in enantiomerically pure form (>99% ee)

Mild oxidation and functionalisation of synthetic polymer surfaces

How to extend the lifetime of plastics? This might sound as a somewhat odd question in the age of bio-degradable plastics, but plastics that can withstand extreme conditions can be (re)used more often and thereby thus contribute to an eco-friendly economy. In order to improve these plastics, we need to adjust them and that is not easy! Traditional plastic modification technologies often require a lot of energy or dangerous chemicals. To by-pass these traditional measures, we explored, and pushed, the boundaries of novel eco-friendly technologies. We studied how enzymes, nature’s architects, modified plastic drinking water filtration membranes, which proved to occur in in a completely unprecedented manner. Additionally, a novel eco-friendly chemical tool for modifying plastics opened up a whole new route towards water-repellent materials. We hope that, through our research, we get yet a little closer to a sustainable future. Chapter 1 provides the required background knowledge for any chemically oriented scholar to comprehend the interdisciplinary work presented herein. Crucial topics, such as polymer surface modification and analysis, wetting behaviour and adhesion prevention were introduced. Current methodologies for modifying polymer surfaces typically require harsh chemicals and conditions. The research described herein has therefore been focussed on acquiring a better understanding and increasing the scope of novel tools for mildly modifying polymers. One of these novel tools is the laccase-mediated surface functionalisation of poly(ethersulfone) membranes using 4-hydroxybenzoic acid (4-HBA). The resulting overlayer minimised membrane fouling by several biofoulants. In order to comprehend the underlying functionalisation mechanism, the solution-phase oligomerisation of 4-HBA had to be studied first, which is described in Chapter 2. Initial conversion of 4-HBA proved to occur only slowly and resulted in two main products: a C3-C3’-bound and a C3-O-bound dimer. A plurality of other products were found after 24 h of incubation, which included a C1-C3’-bound and possibly a C1-O-bound dimer. Furthermore, laccase-mediated conversion of these dimers proved to be far more rapid than conversion of 4-HBA itself, and correlated strongly with the abundance of the individual dimers. The influence of dimer reactivity on their abundance was confirmed by quantum chemical calculations. These findings provided us with handles for designing phenols with enhanced reactivity and controlled binding profiles. We used the gained knowledge to synthesise novel positively charged phenolic monomers that were anticipated to, upon laccase-mediated surface functionalisation, introduce anti-bacterial properties to the membrane while allowing it to be used as support membrane for layer-by-layer deposition. As is described in Chapter 3, however, in-situ laccase-mediated conversion of these phenolics did not lead to significant surface functionalisation. In order to understand why functionalisation was achieved for other monomers (i.e. 4-HBA), 4-HBA, laccase and any of several PES model compounds were incubated together and the resultant mixture was studied using LC-MS. However, no covalent bond formation between (oligomeric) 4-HBA and either of the soluble, insoluble or resin-bound PES model compounds could be observed. The use of phenols bearing negatively charged substituents did also not lead to membrane surface modification. Finally, membranes having an overlayer of oligomeric 4-HBA proved to be extensively decolourised upon washing with a detergent solution. Considering all of the above, it was concluded that laccase-mediated surface modification resulted from strong physisorption, rather than from covalent grafting of oligomeric 4-HBA. As it was challenging to reveal the mechanisms underlying our functionalisation strategy, we anticipated that other researchers might also have encountered similar challenges. It is therefore that in Chapter 4 recently published laccase-mediated surface modification strategies are discussed and assessed on whether grafting is likely to have occurred. This assessment was based on five factors: mechanistic rationale, pre-treatment, control experiments, washing/cleaning and the used analytical tools. Generally speaking, laccase-mediated grafting on lignocelluloses proved to be likely. Quite commonly, however, grafting coincided with physical adsorption due to insufficient washing. We concluded that a lack of proper surface analyses and studies towards the mechanisms underlying grafting on polysaccharides, proteins and synthetic polymers regularly hampered achieving covalent grafting on these materials. Apart from enzymatic surface modification, additional chemical strategies for achieving mild polymer functionalisation were assessed too. PMMA activation was accordingly achieved through peroxidative copper catalysis, followed by sodium borohydride reduction to result in surface hydroxylation. As was described in Chapter 5, this offered a platform for the robust growth of SiHCl3-based silicone nanofilaments, while maintaining polymer transparency. Due to their intricate nanostructure, these silicone nanofilaments granted superhydrophobicity (SWCA > 150°, sliding angles Finally, Chapter 6 summarises the highlights of previous chapters, while offering an in-depth discussion on possible improvements and future work.</p

Laccase-Mediated Grafting on Biopolymers and Synthetic Polymers : A Critical Review

Laccase-mediated grafting on lignocelluloses has gained considerable attention as an environmentally benign method to covalently modify wood, paper and cork. In recent decades this technique has also been employed to modify fibres with a polysaccharide backbone, such as cellulose or chitosan, to infer colouration, antimicrobial activity or antioxidant activity to the material. The scope of this approach has been further widened by researchers, who apply mediators or high redox potential laccases and those that modify synthetic polymers and proteins. In all cases, the methodology relies on one- or two-electron oxidation of the surface functional groups or of the graftable molecule in solution. However, similar results can very often be achieved through simple deposition, even after extensive washing. This unintended adsorption of the active substance could have an adverse effect on the durability of the applied coating. Differentiating between actual covalent binding and adsorption is therefore essential, but proves to be challenging. This review not only covers excellent research on the topic of laccase-mediated grafting over the last five to ten years, but also provides a critical comparison to highlight either the lack or presence of compelling evidence for covalent grafting

One-Step Generation of Reactive Superhydrophobic Surfaces via SiHCl₃-Based Silicone Nanofilaments

Superhydrophobic surfaces gain ever-growing attention because of their applicability in many (consumer) products/materials as they often display, among others, antifouling, anti-icing, and/or self-cleaning properties. A simple way to achieve superhydrophobicity is through the growth of silicone nanofilaments. These nanofilaments, however, are very often nonreactive and thus difficult to utilize in subsequent chemistries. In response, we have developed a single-step procedure to grow (SiHCl3-based) silicone nanofilaments with selective reactivity that are intrinsically superhydrophobic. The silicone nanofilaments could be further functionalized via Pt-catalyzed hydrosilylation of exposed Si-H moieties. These surfaces are easily obtained using mild conditions and are stable under hydrolytic conditions (neutral water, 24 h at 80 °C) while remaining highly transparent, which makes them well suited for optical and photochemical experiments.</p

Elucidating the mechanism behind the laccase-mediated modification of poly(ethersulfone)

Laccase-mediated oligomerisation of 4-hydroxybenzoic acid (4-HBA) derivatives and simultaneous in situ surface modification has proven to be a cost-effective, easily applicable and eco-friendly strategy for preventing biofouling of poly(ethersulfone) (PES) water filtration membranes. Modification of the membrane surface has previously been hypothesised to occur through covalent bonding of enzymatically generated phenolic radicals to the polymeric membrane. The current study shows, however, that in situ formation of soluble phenolic oligomers does not result in covalent membrane modification. We studied in situ laccase-mediated oligomerisation of custom-synthesised positively charged and commercially available negatively charged monomeric phenols, and demonstrated that their mode of binding to PES is not covalent. In addition, soluble, non-soluble and on-resin PES model compounds were synthesised and used in the laccase-mediated oligomerisation of 4-HBA. Covalent bond formation between these model compounds and (oligomeric) 4-HBA could not be observed either. Furthermore, extensive washing of PES membranes modified through laccase-mediated oligomerisation of 4-HBA resulted in substantial discolouration of the membrane surface, showing that the layer of oligomerised phenolics could easily be removed. Altogether, it was concluded that laccase-assisted modification of PES membranes resulted from strong physical adsorption of phenolic oligomers and polymers rather than from covalent bonding of those.</p

Enantioselective Synthesis of Pharmaceutically Relevant Bulky Arylbutylamines Using Engineered Transaminases

ATAs engineered for having an enlarged small binding pocket were applied for the synthesis of enantiomerically pure (R)-benzo[1,3]dioxol-5-yl-butylamine, a chiral component of human leukocyte elastase inhibitor DMP 777 (L-694,458). Kinetic resolution of the racemic amine was performed by using the L59A variant of the (S)-selective ATA from Chromobacterium violaceum (Cv-ATA), providing the residual (R)-enantiomer in excellent yield and &gt;99% ee. At moderate enzyme loading and absence of co-solvent, high volumetric productivity of 0.22 mol L-1 h(-1) (42.5 g L-1 h(-1)) was achieved. Complementarily, the (S)-enantiomer was generated via kinetic resolution using the (R)-selective ATA-117-Rd11 from Arthrobacter sp. with acetone as the amino acceptor. In an alternative approach, we employed ATA-117-Rd11 for the asymmetric amination of the prochiral ketone precursor, which at 86% conversion gave the (R)-benzo[1,3]dioxol-5-yl-butylamine with excellent &gt;99% ee. We further evaluated the utility of Cv-ATA L59A for the asymmetric synthesis of pharmaceutically relevant (S)-1-phenylbutan-1-amine, a chiral component of the deubiquitinase inhibitor degrasyn (WP1130). The enzyme showed good tolerance to high concentrations of isopropylamine, producing (S)-1-phenylbutan-1-amine in enantiomerically pure form (&gt;99% ee)