Overcoming a Conceptual Limitation of Industrial ε-Caprolactone Production via Chemoenzymatic Synthesis in Organic Medium.

The multi-10.000 tons scale manufactured chemical ε-caprolactone attracts high industrial interest due to its favorable biodegradability properties. However, besides being of petrochemical origin yet, its industrial production has a conceptual limitation that is the difficult extraction of this highly water-soluble monomer from the water phase resulting from the aqueous solution of H2O2 applied as reagent. In this contribution, we report a chemoenzymatic cascade starting from bio-based phenol, which makes use of O2 instead of H2O2 and runs in pure organic medium, thus requiring only simply decantation and distillation as work-up. In a first step, phenol is hydrogenated quantitatively to cyclohexanol under solvent-free conditions with a Ru-catalyst. After simple removal of the heterogenous catalyst, cyclohexanol is converted to ε-caprolactone in a biocatalytic double oxidation with very high yields just requiring O2 as reagent. This biocatalytic process proceeds in pure organic medium, thus avoiding tedious extraction to isolate the highly water-soluble ε-caprolactone and enabling a dramatically simplified work-up by only centrifugal separation of lyophilized whole cells and solvent removal. This oxidation is accomplished using a tailor-made recombinant whole-cell catalyst containing an alcohol dehydrogenase and a cyclohexanone monooxygenase mutant.

Enantioselective Synthesis of Secondary Amines by Combining Oxidative Rearrangement and Biocatalysis in a One-Pot Process.

This contribution describes the development of chemoenzymatic one-pot processes, which combine an oxidative rearrangement and a biotransformation catalyzed by an imine reductase (IRED), for the synthesis of highly enantiomerically enriched secondary amines, such as an aryl-substituted pyrrolidine and a benzazepine. The benefits of this chemoenzymatic one-pot approach include high overall conversions (up to >99%), high enantiomeric excesses (up to >99% ee), and a straightforward synthetic approach toward secondary amines without the need to isolate the formed intermediate. For the initial chemical reaction, namely, the oxidative rearrangement, PhI(OAc)2 in methanol is used as a non-natural reagent, whereas the enzymatic step requires only stoichiometric amounts of d-glucose along with catalytic amounts of IRED, glucose dehydrogenase (GDH), and the cofactor NADPH. This methodology, demonstrating the compatibility of a "classic" organic synthesis using a non-natural, highly reactive reagent and a subsequent biocatalytic step, can be applied for different amines as substrates, thus making this concept a versatile tool in synthetic organic chemistry in general and for enantioselective synthesis of heterocyclic secondary amines in particular.

Intervertebral disc microbiome in Modic changes: Lack of result replication underscores the need for a consensus in low-biomass microbiome analysis.

Introduction: The emerging field of the disc microbiome challenges traditional views of disc sterility, which opens new avenues for novel clinical insights. However, the lack of methodological consensus in disc microbiome studies introduces discrepancies. The aims of this study were to (1) compare the disc microbiome of non-Modic (nonMC), Modic type 1 change (MC1), and MC2 discs to findings from prior disc microbiome studies, and (2) investigate if discrepancies to prior studies can be explained with bioinformatic variations. Methods: Sequencing of 16S rRNA in 70 discs (24 nonMC, 25 MC1, and 21 MC2) for microbiome profiling. The experimental setup included buffer contamination controls and was performed under aseptic conditions. Methodology and results were contrasted with previous disc microbiome studies. Critical bioinformatic steps that were different in our best-practice approach and previous disc microbiome studies (taxonomic lineage assignment, prevalence cut-off) were varied and their effect on results were compared. Results: There was limited overlap of results with a previous study on MC disc microbiome. No bacterial genera were shared using the same bioinformatic parameters. Taxonomic lineage assignment using "amplicon sequencing variants" was more sensitive and detected 48 genera compared to 22 with "operational taxonomic units" (previous study). Increasing filter cut-off from 4% to 50% (previous study) reduced genera from 48 to 4 genera. Despite these differences, both studies observed dysbiosis with an increased abundance of gram-negative bacteria in MC discs as well as a lower beta-diversity. Cutibacterium was persistently detected in all groups independent of the bioinformatic approach, emphasizing its prevalence. Conclusion: There is dysbiosis in MC discs. Bioinformatic parameters impact results yet cannot explain the different findings from this and a previous study. Therefore, discrepancies are likely caused by different sample preparations or true biologic differences. Harmonized protocols are required to advance understanding of the disc microbiome and its clinical implications.

Fluorogenic Diversification of Unprotected Bromotryptophan by Aqueous Mizoroki-Heck Cross-Coupling.

Fluorescent styryl-tryptophans have been synthesized by a Mizoroki-Heck cross-coupling from unprotected bromotryptophan in aqueous medium showing promising spectrophotometric properties for possible application in fluorescence labelling of biomolecules. Moreover, this strategy permits a modular combination of biocatalytic halogenation by using immobilized FAD-dependent tryptophan halogenases and Pd-mediated chemocatalysis in a multistep one-pot process.

Boost and loss of immune responses against tumor-associated antigens in the course of pregnancy as a model for allogeneic immunotherapy.

Donor-derived immunity against tumor-associated antigens (TAAs) may exert selective antileukemic activity reprieving the allogeneic recipient from graft-versus-host disease. As TAAs are highly expressed in placental tissues we hypothesized that pregnancy could drive respective immunity in healthy individuals. Thus, we investigated the frequency and level of immune responses against clinically relevant TAAs in 114 blood donors and 44 women during their first pregnancy. Quantitative reverse-transcription polymerase chain reaction was employed to detect low levels of interferon-γ after primary peptide stimulation of CD8(+) T lymphocytes. In blood donors, primary immune responses of low and/or high avidity were found against WT1 (15%), MUC1 (14%), PRAME (7%), and HER2/neu (5%) and exerted killing functions against leukemic cells. Men had higher responses than women, likely due to gonadal cancer-testis-antigen expression. Interestingly, a history of prior delivery was not associated with increased responses, whereas the strongest responses during pregnancy were found in early trimesters to disappear after delivery. This boost and loss of TAA-specific immunity suggests that virtually every donor harbors the potential to mount antileukemic immune responses in a recipient. However, in the absence of the driving target and a permissive environment, they are short-lived and thus require supplemental strategies such as vaccination or immunomodulation to facilitate their persistence.