Biocatalytic Generation of o-Quinone Imines in the Synthesis of 1,4-Benzoxazines and Its Comparative Green Chemistry Metrics.

1,4-Benzoxazines are important motifs in many pharmaceuticals and can be formed by a reaction sequence involving the oxidation of o-aminophenols to their corresponding quinone imine followed by an in situ inverse electron demand Diels-Alder (IEDDA) cycloaddition with a suitable dienophile. Reported herein is the development of a reaction sequence that employs horseradish peroxidase to catalyze the oxidation of the aminophenols prior to the IEDDA as a more sustainable alternative to the use of conventional stoichiometric oxidants. The synthesis of 10 example benzoxazines is demonstrated in this "one-pot, two-step" procedure with yields between 42% and 92%. The green chemistry metrics, including the E-factor and generalized reaction mass efficiency, for this biocatalytic reaction were compared against the conventional chemical approach. It was found that the reported biocatalytic route was approximately twice as green by these measures.

Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies.

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive ('additive') top-down fabrication of nanometer-scale features. Historically, a major drawback of scanning probe lithography has been the intrinsically low throughput of single probe systems. This has been tackled by the use of arrays of multiple probes to enable increased nanolithography throughput. In order to implement such parallelized nanolithography, the accurate alignment of probe arrays with the substrate surface is vital, so that all probes make contact with the surface simultaneously when lithographic patterning begins. This protocol describes the utilization of polymer pen lithography to produce nanometer-scale features over centimeter-sized areas, facilitated by the use of an algorithm for the rapid, accurate, and automated alignment of probe arrays. Here, nanolithography of thiols on gold substrates demonstrates the generation of features with high uniformity. These patterns are then functionalized with fibronectin for use in the context of surface-directed cell morphology studies.

Parallelized biocatalytic scanning probe lithography for the additive fabrication of conjugated polymer structures.

Scanning probe lithography (SPL) offers a more accessible alternative to conventional photolithography as a route to surface nanofabrication. In principle, the synthetic scope of SPL could be greatly enhanced by combining the precision of scanning probe systems with the chemoselectivity offered by biocatalysis. This report describes the development of multiplexed SPL employing probes functionalized with horseradish peroxidase, and its subsequent use for the constructive fabrication of polyaniline features on both silicon oxide and gold substrates. This polymer is of particular interest due to its potential applications in organic electronics, but its use is hindered by its poor processability, which could be circumvented by the direct in situ synthesis at the desired locations. Using parallelized arrays of probes, the lithography of polymer features over 1 cm2 areas was achieved with individual feature widths as small as 162 ± 24 nm. The nature of the deposited materials was confirmed by Raman spectroscopy, and it was further shown that the features could be chemically derivatized postlithographically by Huisgen [2 + 3] "click" chemistry, when 2-propargyloxyaniline was used as the monomer in the initial lithography step.

A high-throughput assay for arylamine halogenation based on a peroxidase-mediated quinone-amine coupling with applications in the screening of enzymatic halogenations.

Arylhalides are important building blocks in many fine chemicals, pharmaceuticals and agrochemicals, and there has been increasing interest in the development of more "green" halogenation methods based on enzyme catalysis. However, the screening and development of new enzymes for biohalogenation has been hampered by a lack of high-throughput screening methods. Described herein is the development of a colorimetric assay for detecting both chemical and enzymatic arylamine halogenation reactions in an aqueous environment. The assay is based on the unique UV/Vis spectrum created by the formation of an ortho-benzoquinone-amine adduct, which is produced by the peroxidase-catalysed benzoquinone generation, followed by Michael addition of either a halogenated or non-halogenated arylamine. This assay is sensitive, rapid and amenable to high-throughput screening platforms. We have also shown this assay to be easily coupled to a flavin-dependent halogenase, which currently lacks any convenient colorimetric assay, in a "one-pot" workflow.