Biocatalytic synthesis of non-standard amino acids by a decarboxylative aldol reaction

Enzymes are renowned for their catalytic efficiency and selectivity, but relatively few carbon-carbon bond forming enzymes have found their way into the biocatalysis toolbox. While engineering can overcome the challenges associated with C-C bond formation for some enzyme systems, the broader synthetic potential of biocatalysis is hindered by the lack of high-quality C-C bond forming transformations. Here we show that the enzyme UstD performs a highly selective decarboxylative aldol addition with diverse aldehyde substrates to make non-standard, γ-hydroxy amino acids. We increased the activity of UstD through three rounds of classic directed evolution and an additional round of computationally-guided engineering. The enzyme that emerged, UstD2.0, is efficient in a whole-cell biocatalysis format, which circumvents the need for enzyme purification, thereby facilitating its use in traditional organic settings. This new, highly stereoselective enzyme represents a unique expansion of the biosynthetic toolbox. The products are highly desirable, functionally rich bioactive γ-hydroxy amino acids that we demonstrate can be prepared stereoselectively on gram-scale. The X-ray crystal structure of UstD2.0 at 2.25 Å reveals the active site and the molecular basis for the remarkably promiscuity of this catalyst. Taking inspiration from the versatile reactivity of enamines in organic synthesis, we hypothesize that the enamine intermediate of UstD can be engineered to react with electrophiles other than aldehydes. The advent of structural information enabled by engineering of UstD2.0 provides a foundation for probing the unique mechanism of UstD and will guide efforts to expand the reactivity of this unique enzyme.

2-Ketoglutarate-Generated In Vitro Enzymatic Biosystem Facilitates Fe(II)/2-Ketoglutarate-Dependent Dioxygenase-Mediated C–H Bond Oxidation for (2s,3r,4s)-4-Hydroxyisoleucine Synthesis

Fe(II)/2-ketoglutarate-dependent dioxygenase (Fe(II)/2-KG DO)-mediated hydroxylation is a critical type of C–H bond functionalization for synthesizing hydroxy amino acids used as pharmaceutical raw materials and precursors. However, DO activity requires 2-ketoglutarate (2-KG), lack of which reduces the efficiency of Fe(II)/2-KG DO-mediated hydroxylation. Here, we conducted multi-enzymatic syntheses of hydroxy amino acids. Using (2s,3r,4s)-4-hydroxyisoleucine (4-HIL) as a model product, we coupled regio- and stereo-selective hydroxylation of l-Ile by the dioxygenase IDO with 2-KG generation from readily available l-Glu by l-glutamate oxidase (LGOX) and catalase (CAT). In the one-pot system, H2O2 significantly inhibited IDO activity and elevated Fe2+ concentrations of severely repressed LGOX. A sequential cascade reaction was preferable to a single-step process as CAT in the former system hydrolyzed H2O2. We obtained 465 mM 4-HIL at 93% yield in the two-step system. Moreover, this process facilitated C–H hydroxylation of several hydrophobic aliphatic amino acids to produce hydroxy amino acids, and C–H sulfoxidation of sulfur-containing l-amino acids to yield l-amino acid sulfoxides. Thus, we constructed an efficient cascade reaction to produce 4-HIL by providing prerequisite 2-KG from cheap and plentiful l-Glu and developed a strategy for creating enzymatic systems catalyzing 2-KG-dependent reactions in sustainable bioprocesses that synthesize other functional compounds.

Sulfur/DABCO Promoted Reductive Coupling/Annulation Cascade Reaction between o-Hydroxy/Amino Nitrobenzenes and Benzaldehydes

Sulfur/DABCO was found to be an efficient reagent in promoting­ the reductive coupling/annulation of o-nitrophenols or o-nitroanilines with benzaldehydes. This method represents a simple, straightforward, and green approach to the construction of benz­oxazoles and benzimidazoles.

Intramolecular hydrogen-bonding-assisted phosphine-catalysed [3 + 2] cyclisation of ynones with o-hydroxy/amino benzaldehydes

A new strategy for the synthesis of functionalized tetrahydrofuran derivatives was developed via a phosphine-catalysed [3 + 2] annulation reaction of aromatic aldehydes with 4-phenylbut-3-yn-2-one.

UV protection and antimicrobial finish on cotton khadi fabric using a mixture of nanoparticles of zinc oxide and poly-hydroxy-amino methyl silicone

A different percentage of nanoparticles of zinc oxide dispersed in a newer amino-silicone binder (poly-hydroxy-amino methyl silicone) were applied to bleached cotton khadi (handloom woven from handspun yarns) fabric to impart both ultraviolet protection and an antimicrobial finish in one step using the pad-dry-cure method, instead of using two processes for two different finishes. Amongst the varying dosages of nanoparticles of zinc oxide (1% to 5%) on the weight of fabric dispersed in poly-hydroxy-amino methyl silicone (2–10%) owf, 1% nanoparticles of zinc oxide and 4% poly-hydroxy-amino methyl silicone show ultraviolet protection factor 10 and 93–95% antibacterial reduction, whereas a 4% poly-hydroxy-amino methyl silicone and 5% nanoparticles of zinc oxide combination yields ultraviolet protection factor 20 and 99% antibacterial reduction. Thus, nanoparticles of zinc oxide at the level of 5% application with 4% poly-hydroxy-amino methyl silicone gives the best antimicrobial (99% bacterial reduction) and ultraviolet protection factor value of 20, balanced with 15–20% loss of fabric tenacity. Fourier transform infrared spectroscopy analysis reveals a complex formation between cellulose/oxy-cellulose and poly-hydroxy-amino methyl silicone that embeds nanoparticles of zinc oxide within it. Supporting reaction mechanisms proposed for both energy dispersive spectroscopy and atomic absorption spectrophotometry results further confirm the presence of zinc, potassium, and silicon on the treated cotton fabric. A wash stability test also shows the stability of the antimicrobial treatment for up to five wash cycles with 96% bacterial reduction and retention of ultraviolet protection factor of 15 after five washes. Thus, this single step combining ultraviolet protective and antimicrobial finishing of cotton fabric may be used for eco-fashion garments to protect the human skin from ultraviolet light and microbes alongside its possible uses in medical textiles to protect human body parts.