Streptavidin-hosted organocatalytic aldol addition

In this report, the streptavidin-biotin technology was applied to enable organocatalytic aldol addition. By attaching pyrrolidine to the valeric motif of biotin and introducing it to streptavidin (Sav), a protein-based organocatalytic system was created, and the aldol addition of acetone with p-nitrobenzaldehyde was tested. The conversion of substrate to product can be as high as 93%. Although the observed enantioselectivity was only moderate (33:67 er), further protein engineering efforts can be included to improve the selectivity. These results have proven the concept that Sav can be used to host stereoselective aldol addition

Asparaginyl endopeptidases: enzymology, applications and limitations

Asparaginyl endopeptidases (AEP) are cysteine proteases found in mammalian and plant cells. Several AEP isoforms from plant species were found to exhibit transpeptidase activity which is integral for the key head-to-tail cyclisation reaction during the biosynthesis of cyclotides. Since many plant AEPs exhibit excellent enzyme kinetics for peptide ligation via a relatively short substrate recognition sequence, they have become appealing tools for peptide and protein modification. In this review, research focused on the enzymology of AEPs and their applications in polypeptide cyclisation and labelling will be presented. Importantly, the limitations of using AEPs and opportunities for future research and innovation will also be discussed

Investigating the effects of cyclic topology on the performance of a plastic degrading enzyme for polyethylene terephthalate degradation

Agitation is a commonly encountered stress for enzymes during all stages of production and application, but investigations that aim to improve their tolerance using topological engineering have yet to be reported. Here, the plastic-degrading enzyme IsPETase was cyclized in a range of topologies including a cyclic monomer, cyclic dimer and catenane using SpyTag/SpyCatcher technologies, and their tolerance towards different stresses including mechanical agitation was investigated. The cyclic dimer and catenane topologies were less susceptible to agitation-induced inactivation resulting in enhancement of polyethylene terephthalate (PET) degradation. While contrary to conventional belief, cyclic topologies did not improve tolerance of IsPETase towards heat or proteolytic treatment, the close proximity of active sites in the dimeric and catenane variants was found to enhance PET conversion into small soluble products. Together, these findings illustrate that it is worthwhile to explore the topology engineering of enzymes used in heterogeneous catalysis as it improves factors that are often overlooked in homogeneous catalysis studies

Reactions of biologically inspired hydride sources with B(C6F5)3

The combination of 1-benzyl-1,4-dihydropyridines with the strong Lewis acid, B(C6F5)3, generates a stable pyridinium borohydride species in high yields (94%) in as little as 10 min. This use of biologically inspired hydride sources further builds on the recent work of new hydride donors in the formation of borohydrides. When functionalizing the dihydropyridine with an amide or carboxylic acid moiety, a disproportionation reaction composed of a series of protonation/reduction steps is observed upon the addition of B(C6F5)3. As a result, one equivalent of dihydropyridine undergoes net hydrogenation, whereas the other is dehydrogenated yielding the pyridinium counterpart in a transfer hydrogenation-type mechanism

Reaction Mechanism of Organocatalytic Michael Addition of Nitromethane to Cinnamaldehyde: A Case Study on Catalyst Regeneration and Solvent Effects

The Michael addition of nitromethane to cinnamaldehyde has been computationally studied in the absence of a catalyst and the presence of a biotinylated secondary amine by a combined computational and experimental approach. The calculations were performed at the density functional theory (DFT) level with the M06-2X hybrid functional, and a polarizable continuum model has been employed to mimic the effect of two different solvents: dichloromethane (DCM) and water. Contrary to common assumption, the product-derived iminium intermediate was absent in both of the solvents tested. Instead, hydrating the C1–C2 double bond in the enamine intermediate directly yields the tetrahedral intermediate, which is key for forming the product and regenerating the catalyst. Enamine hydration is concerted and found to be rate-limiting in DCM but segregated into two non-rate-limiting steps when the solvent is replaced with water. However, further analysis revealed that the use of water as solvent also raises the energy barriers for other chemical steps, particularly the critical step of C–C bond formation between the iminium intermediate and nucleophile; this consequently lowers both the reaction yield and enantioselectivity of this LUMO-lowering reaction, as experimentally detected. These findings provide a logical explanation to why water often enhances organocatalysis when used as an additive but hampers the reaction progress when employed as a solvent

Towards controlling activity of a peptide asparaginyl ligase (PAL) by lumazine synthetase compartmentalization

Peptide asparaginyl ligases (PALs) hold significant potential in protein bioconjugation due to their excellent kinetic properties and broad substrate compatibility. However, realizing their full potential in biocatalytic applications requires precise control of their activity. Inspired by nature, we aimed to compartmentalize a representative PAL, OaAEP1-C247A, within protein containers to create artificial organelles with substrate sorting capability. Two encapsulation approaches were explored using engineered lumazine synthases (AaLS). The initial strategy involved tagging the PAL with a super-positively charged GFP(+36) for encapsulation into the super-negatively charged AaLS-13 variant, but it resulted in undesired truncation of the enzyme. The second approach involved genetic fusion of the OaAEP1-C247A with a circularly permutated AaLS variant (cpAaLS) and its co-production with AaLS-13, which successfully enabled compartmentalization of the PAL within a patch-work protein cage. Although the caged PAL retained its activity, it was significantly reduced compared to the free enzyme (~30-40-fold), likely caused by issues related to OaAEP1-C247A stability and folding. Nevertheless, these findings demonstrated feasibility of the AaLS encapsulation approach and encourages further optimization in the design of peptide-ligating artificial organelle in E. coli, aiming for a more effective and stable system for protein modifications

D‐peptide and d‐protein technology: recent advances, challenges, and opportunities

Total chemical protein synthesis provides access to entire D‐protein enantiomers enabling unique applications in molecular biology, structural biology, and bioactive compound discovery. Key enzymes involved in the central dogma of molecular biology have been prepared in their D‐enantiomeric forms facilitating the development of mirror‐image life. Crystallization of a racemic mixture of L‐ and D‐protein enantiomers provides access to high‐resolution X‐ray structures of polypeptides. Additionally, D‐enantiomers of protein drug targets can be used in mirror‐image phage display allowing discovery of non‐proteolytic D‐peptide ligands as lead candidates. This review discusses the unique applications of D‐proteins including the synthetic challenges and opportunities

Towards the use of an amino acid cleavable linker for solid-phase chemical synthesis of peptides and proteins

The synthesis of proteins by solid-phase chemical ligation (SPCL) suffers from the paucity of linkers that can be cleaved under mild conditions. Here, we deployed a spontaneous nickel-assisted cleavage (SNAC) tag, known to undergo spontaneous cleavage in the presence of nickel(ii), as a linker for C-to-N SPCL

Enabling protein-hosted organocatalytic transformations

In this review, the development of organocatalytic artificial enzymes will be discussed. This area of protein engineering research has underlying importance, as it enhances the biocompatibility of organocatalysis for applications in chemical and synthetic biology research whilst expanding the catalytic repertoire of enzymes. The approaches towards the preparation of organocatalytic artificial enzymes, techniques used to improve their performance (selectivity and reactivity) as well as examples of their applications are presented. Challenges and opportunities are also discussed