We study the hyperactivation of α‐chymotrypsin (α‐ChT) using the acrylate polymer poly(2‐carboxyethyl) acrylate (PCEA) in comparison to the commonly used poly(acrylic acid) (PAA). The polymers are added during the enzymatic cleavage reaction of the substrate N‐glutaryl‐L‐phenylalanine p‐nitroanilide (GPNA). Enzyme activity assays reveal a pronounced enzyme hyperactivation capacity of PCEA, which reaches up to 950% activity enhancement, and is significantly superior to PAA (revealing an activity enhancement of approx. 450%). In a combined experimental and computational study, we investigate α‐ChT/polymer interactions to elucidate the hyperactivation mechanism of the enzyme. Isothermal titration calorimetry reveals a pronounced complexation between the polymer and the enzyme. Docking simulations reveal that binding of polymers significantly improves the binding affinity of GPNA to α‐ChT. Notably, a higher binding affinity is found for the α‐ChT/PCEA compared to the α‐ChT/PAA complex. Further molecular dynamics (MD) simulations reveal changes in the size of the active site in the enzyme/polymer complexes, with PCEA inducing a more pronounced alteration compared to PAA, facilitating an easier access for the substrate to the active site of α‐ChT.

Rational re-design of the substrate pocket of phenylpropanoid-flavonoid O-methyltransferase (PFOMT) from Mesembryanthe-mum crystallinum, an enzyme that selectively methylates the 3’-position (= meta-position) in catechol-moieties of flavonoids to guiacol-moieties, provided the basis for the generation of variants with opposite, i. e. 4’- (para-) regioselectivity and enhanced catalytic efficiency. A double variant (Y51R/N202W) identified through a newly developed colorimetric assay efficiently modified the para-position in flavanone and flavano-nol substrates, providing access to the sweetener molecule hesperetin and other rare plant flavonoids having an isovanil-loid motif.

In contrast to the myriad of methods available to produce α‐helices and antiparallel β‐sheets in synthetic peptides, just a few are known for the construction of stable, non‐cyclic parallel β‐sheets. Herein, we report an efficient on‐resin approach for the assembly of parallel β‐sheet peptides in which the N‐alkylated turn moiety enhances the stability and gives access to a variety of functionalizations without modifying the parallel strands. The key synthetic step of this strategy is the multicomponent construction of an N‐alkylated turn using the Ugi reaction on varied isocyano‐resins. This four‐component process assembles the orthogonally protected turn fragment and incorporates handles serving for labeling/conjugation purposes or for reducing peptide aggregation. NMR and circular dichroism analyses confirm the better‐structured and more stable parallel β‐sheets in the N‐alkylated peptides compared to the non‐functionalized variants.

Bioinspired, synthetic porphyrin complexes are important catalysts in organic synthesis and play a pivotal role in efficient carbene transfer reactions. The advances in this research area stimulated recent, “chemo‐inspired” developments in biocatalysis. Today, both synthetic iron complexes and enzymes play an important role to conduct carbene transfer reactions. The advances and potential developments in both research areas are discussed in this concept article.

Catalyst discovery and development requires the screening of large reaction sets necessitating analytic methods with the potential for high‐throughput screening. These techniques often suffer from substrate dependency or the requirement of expert knowledge. Chromatographic techniques (GC/LC) can overcome these limitations but are generally hampered by long analysis time or the need for special equipment. The herein developed multiple injections in a single experimental run (MISER) GC‐MS technique allows a substrate independent 96‐well microtiter plate analysis within 60 min. This method can be applied to any laboratory equipped with a standard GC‐MS. With this concept novel, unspecific peroxygenase (UPO) chimeras, could be identified, consisting of subdomains from three different fungal UPO genes. The GC‐technique was additionally applied to evaluate an YfeX library in an E. coli whole‐cell system for the carbene‐transfer reaction on indole, which revealed the thus far unknown axial heme ligand tryptophan.

For the first time, the Petasis (borono‐Mannich) reaction is employed for the multicomponent labeling and stapling of peptides. The report includes the solid‐phase derivatization of peptides at the N‐terminus, Lys, and Nϵ‐MeLys side‐chains by an on‐resin Petasis reaction with variation of the carbonyl and boronic acid components. Peptides were simultaneously functionalized with aryl/vinyl substituents bearing fluorescent/affinity tags and oxo components such as dihydroxyacetone, glyceraldehyde, glyoxylic acid, and aldoses, thus encompassing a powerful complexity‐generating approach without changing the charge of the peptides. The multicomponent stapling was conducted in solution by linking Nϵ‐MeLys or Orn side‐chains, positioned at i, i+7 and i, i+4, with aryl tethers, while hydroxy carbonyl moieties were introduced as exocyclic fragments. The good efficiency and diversity oriented character of these methods show prospects for peptide drug discovery and chemical biology.

Die Funktionalisierung von C‐H‐Bindungen mit Nichtedelmetallkatalysatoren ist ein wichtiges Forschungsgebiet für die Entwicklung effizienter und nachhaltiger Synthesemethoden. In diesem Artikel beschreiben wir die Entwicklung Eisenporphyrin‐katalysierter Reaktionen von Diazoacetonitril mit N‐Heterocyclen um so einen Zugang zu wertvollen Vorläufern zu Tryptaminen zu erhalten. Darüberhinaus berichten wir über experimentelle mechanistische Studien sowie über konzeptionelle Studien zu einer enzymatischen Synthese mit dem Enzym YfeX. Mit dem leicht zugänglichen FeTPPCl‐Katalysator konnten wir hoch effiziente C‐H‐Funktionalisierungsreaktionen von Indol und Indazol‐Heterocyclen zeigen. Diese Reaktionen können unter milden Reaktionsbedingungen, mit exzellenten Ausbeuten und großer Toleranz funktioneller Gruppen inklusive Anwendungen im Grammmaßstab durchgeführt werden und eröffnen so einen einzigartigen, effizienten Zugang zu Tryptaminen.

An important development in the field of macrocyclization strategies towards molecular cages is described. The approach comprises the utilization of a double Ugi four‐component macrocyclization for the assembly of macromulticycles with up to four different tethers, that is, hybrid cages. The innovation of this method rests on setting up the macromulticycle connectivities not through the tethers but through the bridgeheads, which in this case involve N‐substituted amino acids. Both dilution and metal‐template‐driven macrocyclization conditions were implemented with success, enabling the one‐pot formation of cryptands and cages including steroidal, polyether, heterocyclic, peptidic, and aryl tethers. This method demonstrates substantial complexity‐generating character and is suitable for applications in molecular recognition and catalysis.

The reduction of activated C=C double bonds is an important reaction in synthetic chemistry owing to the potential formation of up to two new stereogenic centers. Artificial nicotinamide cofactors were recently presented as alternative suppliers of hydride equivalents needed for alkene reduction. To study the effect of cofactors on the reduction of activated alkenes, a set of N‐substituted synthetic nicotinamide cofactors with differing oxidation potentials were synthesized and their electrochemical and kinetic behavior was studied. The effects of the synthetic cofactors on enzyme activity of four ene reductases are outlined in this study, where the cofactor mimic with an N‐substituted 4‐hydroxy‐phenyl residue led to a sixfold higher vmax relative to the natural cofactor NADH.

Three different reductases have been fused to CYP153 monooxygenase from Marinobacter aquaeolei. The most promising candidate has been analysed in terms of its linker part, which connects the reductase with the haem domain through sequence alignment of the corresponding reductase family CYP116B. To improve the artificial fusion construct, the linker length has been varied, thereby only altering the non‐conserved middle part of the linker. This way seven artificial fusion constructs have been engineered, which varied in linker length between 11 and 32 amino acids (“natural” is 16). These variations showed a substantial impact on the fusion construct. The best mutant, extended by two amino acids, showed an improved activity (67 %), higher stability (67 % more active haem domain after 2 h) and a coupling efficiency of 94 % (55 % higher than before). Presented in this paper is an approach to find and optimise artificial fusion constructs for P450 monooxygenases.