Peptide Boronic Acids by Late-Stage Hydroboration on the Solid Phase.

Organoboron compounds have a wide range of applications in numerous research fields, and methods to incorporate them in biomolecules are much sought after. Here, on-resin chemical syntheses of aliphatic and vinylogous peptide boronic acids are presented by transition metal-catalyzed late-stage hydroboration of alkene and alkyne groups in peptides and peptoids, for example on allyl- and propargylglycine residues, using readily available chemicals. These methods yield peptide boronic acids with much shorter linkers than previously reported on-resin methods. Furthermore, the methods are regio- and stereoselective, compatible with all canonical amino acid residues and can be applied to short, long, and in part even "difficult" peptide sequences. In a feasibility study, the protected peptide vinylboronic acids are further derivatized by the Petasis reaction using salicylaldehyde derivatives. The ability of the obtained peptide boronic acids to reversibly bind to carbohydrates is demonstrated in a catch-release model experiment using a fluorescently labeled peptide boronic acid on cross-linked dextran beads. In summary, this highlights the potential of the target compounds for drug discovery, glycan-specific target recognition, controlled release, and diagnostics.

Design of Functional Globular ß-Sheet Miniproteins.

The design of discrete ß-sheet peptides is far less advanced than e. g. the design of α-helical peptides. The reputation of ß-sheet peptides as being poorly soluble and aggregation-prone often hinders active design efforts. Here, we show that this reputation is unfounded. We demonstrate this by looking at the ß-hairpin and WW domain. Their structure and folding have been extensively studied and they have long served as model systems to investigate protein folding and folding kinetics. The resulting fundamental understanding has led to the development of hyperstable ß-sheet scaffolds that fold at temperatures of 100 °C or high concentrations of denaturants. These have been used to design functional miniproteins with protein or nucleic acid binding properties, in some cases with such success that medical applications are conceivable. The ß-sheet scaffolds are not always completely rigid, but can be specifically designed to respond to changes in pH, redox potential or presence of metal ions. Some engineered ß-sheet peptides also exhibit catalytic properties, although not comparable to those of natural proteins. Previous reviews have focused on the design of stably folded and non-aggregating ß-sheet sequences. In our review, we now also address design strategies to obtain functional miniproteins from ß-sheet folding motifs.

Rapid On-Resin N-Formylation of Peptides as One-Pot Reaction.

N-formylation is a common pre- and post-translational modification of the N-terminus or the lysine side chain of peptides and proteins that plays a role in the initiation of immune responses, gene expression, or epigenetics. Despite its high biological relevance, protocols for the chemical N-formylation of synthetic peptides are scarce. The few available methods are elaborate in their execution and the yields are highly sequence-dependent. We present a rapid, easy-to-use one-pot procedure that runs at room temperature and can be used to formylate protected peptides at both the N-terminus and the lysine side chain on the resin in near-quantitative yields. Only insensitive, storage-stable standard chemicals - formic acid, acetic anhydride, pyridine and DMF - are used. Formylation works for both short and long peptides of up to 34 amino acids and over the spectrum of canonical amino acids.

An Engineered ß-Hairpin Peptide Forming Thermostable Complexes with ZnII , NiII , and CuII through a His3 Site.

The three-dimensional structure of a peptide, which determines its function, can denature at elevated temperatures, in the presence of chaotropic reagents, or in organic solvents. These factors limit the applicability of peptides. Herein, we present an engineered ß-hairpin peptide containing a His3 site that forms complexes with ZnII , NiII , and CuII . Circular dichroism spectroscopy shows that the peptide-metal complexes exhibit melting temperatures up to 80 °C and remain folded in 6 M guanidine hydrochloride as well as in organic solvents. Intrinsic fluorescence titration experiments were used to determine the dissociation constants of metal binding in the nano- to sub-nanomolar range. The coordination geometry of the peptide-CuII complex was studied by EPR spectroscopy, and a distorted square planar coordination geometry with weak interactions to axial ligands was revealed. Due to their impressive stability, the presented peptide-metal complexes open up interesting fields of application, such as the development of a new class of peptide-metal catalysts for stereoselective organic synthesis or the directed design of extremophilic ß-sheet peptides.

Late-Stage Functionalisation of Peptides on the Solid Phase by an Iodination-Substitution Approach.

The functionalisation of peptides at a late synthesis stage holds great potential, for example, for the synthesis of peptide pharmaceuticals, fluorescent biosensors or peptidomimetics. Here we describe an on-resin iodination-substitution reaction sequence on homoserine that is also suitable for peptide modification in a combinatorial format. The reaction sequence is accessible to a wide range of sulfur nucleophiles with various functional groups including boronic acids, hydroxy groups or aromatic amines. In this way, methionine-like thioethers or thioesters and thiosulfonates are accessible. Next to sulfur nucleophiles, selenols, pyridines and carboxylic acids were successfully used as nucleophiles, whereas phenols did not react. The late-stage iodination-substitution approach is not only applicable to short peptides but also to the more complex 34-amino-acid WW domains. We applied this strategy to introduce 7-mercapto-4-methylcoumarin into a switchable ZnII responsive WW domain to design an iFRET-based ZnII sensor.

Switchable Zinc(II)-Responsive Globular ß-Sheet Peptide.

The natural function of many proteins depends on their ability to switch their conformation driven by environmental changes. In this work, we present a small, monomeric ß-sheet peptide that switches between a molten globule and a folded state through Zn(II) binding. The solvent-exposed hydrophobic core on the ß-sheet surface was substituted by a His3-site, whereas the internal hydrophobic core was left intact. Zn(II) is specifically recognized by the peptide relative to other divalent metal ions, binds in the lower micromolar range, and can be removed and re-added without denaturation of the peptide. In addition, the peptide is fully pH-switchable, has a pKa of about 6, and survives several cycles of acidification and neutralization. In-depth structural characterization of the switch was achieved by concerted application of circular dichroism (CD) and multinuclear NMR spectroscopy. Thus, this study represents a viable approach toward a globular ß-sheet Zn(II) mini-receptor prototype.