Exploring the Chemical Properties and Medicinal Applications of Tetramethylthiocycloheptyne Sulfoximine Used in Strain-Promoted Azide-Alkyne Cycloaddition Reactions.

The recently developed compound, tetramethylthiocycloheptyne sulfoximine (TMTHSI), has shown to be a promising strained alkyne for strain-promoted azide-alkyne cycloaddition (SPAAC), metal-free click chemistry. This research explores the properties of TMTHSI-based compounds via three aspects: (1) large-scale production, (2) unique stability in acidic conditions and its subsequent use in peptide synthesis, and (3) the functionalization of antibodies. Here, it is shown that (1) scale-up is achieved on a scale of up to 100 g. (2) TMTHSI is remarkably stable against TFA allowing for the site-specific functionalization of peptides on resin. Finally, (3) the functionalization of an antibody with a model payload is very efficient, with antibody conjugation demonstrating more beneficial features such as a high yield and limited hydrophobicity as compared to other alkyne reagent conjugates. These results illustrate the high potential of TMTHSI for diverse bioconjugation applications, with production already being GMP-compatible and a highly efficient conversion resulting in attractive costs of goods.

Directed evolution of carbon-hydrogen bond activating enzymes.

As industrial biocatalysis is maturing, access to enzymatic activities beyond chiral resolutions, asymmetric ketone reductions and reductive aminations is gradually becoming reality. Especially the utilization of carbon-hydrogen bond (C-H) activating enzymes is very attractive as they catalyze a variety of chemically extremely challenging transformations. Because of their intrinsic complexity, the use of these enzymes in manufacturing has been limited. However, recent advances in enzyme engineering and bioinformatics have led to activity improvements for native and non-native substrates, the introduction of new-to-nature chemistries and the identification of promising novel enzyme families. Looking forward, the use of automation and advanced computer algorithms will help to streamline the evolution process of C-H activating enzymes leading to more robust and active biocatalysts.

Enzyme-mediated polymerization inside engineered protein cages.

Engineered variants of the capsid-forming enzyme lumazine synthase, AaLS, were used as nanoreactors for an enzyme-mediated polymerization. Oxidation of 3,3-diaminobenzidine (DAB) by the engineered ascorbate peroxidase APEX2 encapsulated in AaLS capsids resulted in templated formation of polyDAB-capsid nanoparticles of homogeneous size and shape.

Bottom-up Construction of a Primordial Carboxysome Mimic.

We have constructed a synthetic mimic of the carboxysome, a cyanobacterial carbon-fixing organelle. Using an electrostatic tagging system, we coencapsulated the two key carboxysomal enzymes, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and carbonic anhydrase (CA), in an engineered protein cage based on lumazine synthase. A statistically significant kinetic effect of coencapsulated CA on RuBisCO activity was not observed under ambient or oxygen saturated conditions, suggesting that enzyme proximity alone may not be the key determinant in carboxysome function. The capsid shell protected the enzyme from proteolytic damage, a factor that could have provided early cyanobacteria with an evolutionary benefit. Our strategy to coencapsulate different proteins can easily be extended to other sequentially acting enzymes and lays down principles for developing artificial organelles to control biosynthetic pathways in vivo.

Iodobenzene-catalyzed oxabicyclo[3.2.1]octane and [4.2.1]nonane synthesis via cascade C-O/C-C formation.

Iodobenzene-catalyzed 1,2-olefin functionalization via C-C and C-O bond formation has been achieved with electron rich aromatic groups and vinylogous esters acting as independent nucleophiles. The reaction provides oxabicyclo[3.2.1]octanes and [4.2.1]nonanes from commercially available 3-alkoxy cycohexen-2-ones in three steps.

Orthogonal assembly of a designed ankyrin repeat protein-cytotoxin conjugate with a clickable serum albumin module for half-life extension.

The generation of drug conjugates for safe and effective tumor targeting requires binding proteins tolerant to functionalization by rational engineering. Here, we show that Designed Ankyrin Repeat Proteins (DARPins), a novel class of binding proteins not derived from antibodies, can be used as building blocks for facile orthogonal assembly of bioconjugates for tumor targeting with tailored properties. DARPin Ec1, which targets the Epithelial Cell Adhesion Molecule (EpCAM), was genetically modified with a C-terminal cysteine for conjugation of the small molecule cytotoxin monomethylauristatin F (MMAF). In addition, it was N-terminally functionalized by metabolic introduction of the non-natural amino acid azidohomoalanine to enable linkage of site-specifically dibenzocyclooctyne-modified mouse serum albumin (MSA) for half-life extension using Cu(I)-free click chemistry. The conjugate MSA-Ec1-MMAF was assembled to obtain high yields of a pure and stable drug conjugate as confirmed by various analytical methods and in functional assays. The orthogonality of the assembly led to a defined reaction product and preserved the functional properties of all modules, including EpCAM-specific binding and internalization, FcRn binding mediated by MSA, and cytotoxic potency. Linkage of MMAF to the DARPin increased receptor-specific uptake of the drug while decreasing nonspecific uptake, and further coupling of the conjugate to MSA enhanced this effect. In mice, albumin conjugation increased the serum half-life from 11 min to 17.4 h, resulting in a more than 22-fold increase in the area-under-the-curve (AUC). Our data demonstrate the promise of the DARPin format for facile modular assembly of drug conjugates with improved pharmacokinetic performance for tumor targeting.

Iodobenzene catalysed synthesis of spirofurans and benzopyrans by oxidative cyclisation of vinylogous esters.

Vinylogous esters bearing para or meta methoxy benzyl groups undergo oxidative cyclisation with 5-20 mol% iodobenzene and m-CPBA to give spirofuran or benzopyran containing heterocycles. The reaction allows rapid generation of skeletal complexity in good to excellent yields via a novel oxidative cyclisation.