R.B. Leveson-Gower

A tryptophan overproducing Corynebacterium glutamicum strain was metabolically engineered to produce 7-Br/Cl-indole and 7-Br/Cl-tryptamine. For this purpose, tryptophanases and aromatic l-amino acid decarboxylases from different origins were screened and utilized in combination with halogenase RebH. Fermentative production of 7-Br-tryptamine in a bioreactor resulted in a product titer of 0.36 g L⁻¹.

Abstract

The aromatic amino acid l-tryptophan serves as a precursor for many valuable compounds such as neuromodulators, indoleamines and indole alkaloids. In this work, tryptophan biosynthesis was extended by halogenation followed by decarboxylation to the respective tryptamines or cleavage to the respective indoles. Either the tryptophanase genes tnaAs from E. coli and Proteus vulgaris or the aromatic amino acid decarboxylase genes AADCs from Bacillus atrophaeus, Clostridium sporogenes, and Ruminococcus gnavus were expressed in Corynebacterium glutamicum strains producing (halogenated) tryptophan. Regarding indoles, final titers of 16 mg L⁻¹ 7-Cl-indole and 23 mg L⁻¹ 7-Br-indole were attained. Tryptamine production led to a much higher titer of 2.26 g L⁻¹ upon expression of AADC from B. atrophaeus. AADC enzymes were shown to be active with halogenated tryptophan in vitro and in vivo and supported production of 0.36 g L⁻¹ 7-Br-tryptamine with a volumetric productivity of 8.3 mg L⁻¹ h⁻¹ in a fed-batch fermentation.

The discovery of ligands for the “undruggable proteome” is likely to require the development of new chemical matter with a greater “molecular wingspan” than traditional Lipinski-compliant small molecules. A DNA-encoded library (DEL) of non-peptidic thioether macrocycles has been constructed and screened for high affinity protein ligands to a model protein, SA.

Abstract

There is considerable interest in the development of libraries of non-peptidic macrocycles as a source of ligands for difficult targets. We report here the solid-phase synthesis of a DNA-encoded library of several hundred thousand thioether-linked macrocycles. The library was designed to be highly diverse with respect to backbone scaffold diversity and to minimize the number of amide N−H bonds, which compromise cell permeability. The utility of the library as a source of protein ligands is demonstrated through the isolation of compounds that bind Streptavidin, a model target, with high affinity.

Nature Catalysis, Published online: 21 February 2022; doi:10.1038/s41929-022-00743-0

Enantioselective C–C bond-forming reactions are underdeveloped in the biocatalysis toolbox. Now, engineering an efficient and promiscuous decarboxylative aldolase enzyme provides a solution to facilitate the convenient synthesis of non-standard γ-hydroxy amino acids from simple building blocks.

Supported sub-nano clusters hold great promise as economical and highly active catalysts. However, they tend to deactivate rapidly by poisoning and sintering, impeding their widespread use. We find that self-limiting poisoning can stabilize and promote cluster catalysis, i.e., poisoning is not always detrimental, but can sometimes be exploited. Specifically, Pt-Ge alloy clusters supported on alumina undergo slow coking (carbon deposition) under conditions of thermal dehydrogenation, yet preserve strong binding sites. For the case of Pt4Ge/alumina, theory shows a number of thermally populated isomers, one of which catalyzes carbon deposition. Because the clusters are fluxional at high temperatures, this isomer acts as a gateway, slowly converting all the clusters to Pt4GeC2. The surprising result is that Pt4GeC2 is highly catalytically active and selective against further coking, i.e., coking produces functional, stable catalytic clusters. Ge and C2 have synergistic electronic effects, leading to efficient and highly selective catalytic dehydrogenation that stops at alkenes, and improving stability. Thus, under reaction conditions, the clusters develop into a robust catalyst, suggesting an approach to practicable cluster catalysis.

Adding function: A boronate-affinity ligand and an S-aryl thioester have been developed that are capable of labelling native Abs with a functional molecule. Additionally, a photoactivatable crosslinker allows purification of labelled Abs in a reagentless manner, thereby producing homogeneous Ab-drug conjugates.

Abstract

The excellent molecular recognition capabilities of monoclonal antibodies (mAbs) have opened up exciting opportunities for biotherapeutic discovery. Taking advantage of the full potential of this tool necessitates affinity ligands capable of conjugating directly with small molecules to a defined degree of biorthogonality, especially when modifying natural Abs. Herein, a bioorthogonal boronate-affinity-based Ab ligand featuring a 4-(dimethylamino)pyridine and an S-aryl thioester to label full-length Abs is reported. The photoactivatable linker in the acyl donor facilitated purification of azide-labelled Ab (N₃-Ab) was quantitatively cleaved upon brief exposure to UV light while retaining the original Ab activity. Click reactions enabled the precise addition of biotin, a fluorophore, and a pharmacological agent to the purified N₃-Abs. The resulting immunoconjugate showed selectivity against targeted cells. Bioorthogonal traceless design and reagentless purification allow this strategy to be a powerful tool to engineer native antibodies amenable to therapeutic intervention.

BioTrans2021: We created an artificial enzyme consisting of a non-enzymatic protein (LmrR) containing an unnatural catalytic residue with an aniline side chain (LmrR_pAF). Building on our previous work showing how LmrR_pAF can catalyse a Friedel-Crafts alkylation of indoles, here we show that when α-substituted acroleins are applied as substrates the protein scaffold enables enantioselective protonation with good selectivity.

Abstract

The incorporation of organocatalysts into protein scaffolds holds the promise of overcoming some of the limitations of this powerful catalytic approach. Previously, we showed that incorporation of the non-canonical amino acid para-aminophenylalanine into the non-enzymatic protein scaffold LmrR forms a proficient and enantioselective artificial enzyme (LmrR_pAF) for the Friedel-Crafts alkylation of indoles with enals. The unnatural aniline side-chain is directly involved in catalysis, operating via a well-known organocatalytic iminium-based mechanism. In this study, we show that LmrR_pAF can enantioselectively form tertiary carbon centres not only during C−C bond formation, but also by enantioselective protonation, delivering a proton to one face of a prochiral enamine intermediate. The importance of various side-chains in the pocket of LmrR is distinct from the Friedel-Crafts reaction without enantioselective protonation, and two particularly important residues were probed by exhaustive mutagenesis.

A transition state theory-influenced approach on maximum battery cycle-life is outlined, arriving at an ideal model of general validity. The outcome may be understood further as a thermodynamic final regularity reminiscent of Carnot-efficiency. In contrast to the common perception which attributes in blanket fashion the causality of changes in cycle-life to the engineering of battery-specific tangibles, this model allows for a more differentiated picture: That changes to battery-specific tangibles may yield differences of several hundred or more cycles is here the result of them being enhanced by a comparatively long, natural constant-based, logarithmic lever. That way such changes can cause big differences though being comparatively small to the lever base value, which emerges as a quantity of natural constants, temperature(s) and relative capacity margins but independent of battery specific energy and applied power. These are findings suggesting a revision of the current empirics-biased consensus opinion about the matter.