Improving Binding Affinity and Stability of Peptide Ligands by Substituting Glycines with D‐Amino Acids

Improving the binding affinity and/or stability of peptide ligands often requires testing of large numbers of variants to identify beneficial mutations. Herein we propose a type of mutation that promises a high success rate. In a bicyclic peptide inhibitor of the cancer‐related protease urokinase‐type plasminogen activator (uPA), we observed a glycine residue that has a positive ? dihedral angle when bound to the target. We hypothesized that replacing it with a D ‐amino acid, which favors positive ? angles, could enhance the binding affinity and/or proteolytic resistance. Mutation of this specific glycine to D ‐serine in the bicyclic peptide indeed improved inhibitory activity (1.75‐fold) and stability (fourfold). X‐ray‐structure analysis of the inhibitors in complex with uPA showed that the peptide backbone conformation was conserved. Analysis of known cyclic peptide ligands showed that glycine is one of the most frequent amino acids, and that glycines with positive ? angles are found in many protein‐bound peptides. These results suggest that the glycine‐to‐D ‐amino acid mutagenesis strategy could be broadly applied.     

Probing the Outstanding Local Hydrophobicity Increases in Peptide Sequences Induced by Incorporation of Trifluoromethylated Amino Acids

In order to achieve accurate determination of the local hydrophobicity increases in peptide sequences produced by incorporation of trifluoromethylated amino acids (TfmAAs), the chromatographic hydrophobicity indexes (?₀) of three series of tripeptides containing three unnatural trifluoromethylated amino acids have been measured and compared with those of their non‐fluorinated analogues. The hydrophobic contribution of each fluorinated amino acid was quantified by varying the position and the protection of (R)‐ and (S)‐α‐trifluoromethylalanine (TfmAla), (R)‐trifluoromethylcysteine (TfmCys), and (S)‐trifluoromethionine (TFM) in a short peptide sequence. As a general trend, strong increases in hydrophobicity were precisely measured, even exceeding the high hydrophobic contribution of the natural amino acid isoleucine. This study validates the incorporation of trifluoromethylated amino acids into peptide sequences as a rational strategy for the fine‐tuning of hydrophobic peptide–protein interactions.     

Synthesis of thymosin β4Xen and its comparison with the natural tritetraconpeptide

Thymosin β₄^Xen, a 43 residue peptide recently isolated from Xenopus laevis, was synthesized by automatic solid phase procedure and compared with the natural product, isolated from the ovaries of Xenopus laevis For the synthesis N-methylpyrrolidone was chosen as solvent instead of the commonly used dimethylformamide because this solvent seems to be superior for solid phase peptide synthesis due to the favorable swelling properties of the polystyrene resin in this solvent and its dissolving power against the resin-bound peptide which reduces intermolecular aggregation. With acetic anhydride/pyridine and hydroxysuccinimide acetate two different acetylation reagents were tested for the final acetylation step, which gave both comparable results as shown by analytical HPLC investigations. The crude synthetic product was purified by HPLC, confirmed by ASA and LD-MS and was identical compared with the natural thymosin β₄^Xen     

聚丙烯熔喷驻极过滤材料表面静电势的研究

将未经过驻极处理的聚丙烯熔喷过滤材料进行电晕驻极处理,通过测定聚丙烯熔喷驻极滤料的过滤效率和表面静电势,分析滤料表面正负电荷的分布以及表面静电势与滤料过滤效率的关系.结果表明:滤料表面的静电势呈现随机的正负分布,并且随着滤料表面平均静电势的提高,滤料的过滤效率提高.     

Pharmacokinetic Studies around the Mono‐ and Difunctionalization of a Bioavailable Cyclic Decapeptide Scaffold

We previously reported the design of several cyclic decapeptides based on a generic scaffold that achieved favorable oral bioavailability and exposure. With the goal to further investigate the potential of this approach, we describe herein the effect of mono‐ and difunctionalization of this scaffold. A series of cyclic decapeptides were therefore subjected to a range of in vitro assays and pharmacokinetic (PK) studies to investigate whether the introduction of polar or charged groups could be tolerated by the “engineered” scaffold while maintaining good PK profiles. Whereas the introduction of charged amino acids proved—besides maintaining low clearance—to conceal the inherent PK properties of the scaffold, the introduction of polar amino acids (i.e., threonine and pyridyl alanine) led to several cyclic decapeptides exhibiting excellent PK profiles together with a solubility that was significantly improved relative to that of previously reported cyclic decapeptides.     

Electronic and Steric Control of n→π* Interactions: Stabilization of the α-Helix Conformation without a Hydrogen Bond

The preferred conformations of peptides and proteins are dependent on local interactions that bias the conformational ensemble. The n→π* interaction between consecutive carbonyls promotes compact conformations, including the α-helix and polyproline II helix. In order to further understand the n→π* interaction and to develop methods to promote defined conformational preferences through acyl N-capping motifs, a series of peptides was synthesized in which the electronic and steric properties of the acyl group were modified. Using NMR spectroscopy, van't Hoff analysis of enthalpies, X-ray crystallography, and computational investigations, we observed that more electron-rich donor carbonyls (pivaloyl, iso-butyryl, propionyl) promote stronger n→π* interactions and more compact conformations than acetyl or less electron-rich donor carbonyls (methoxyacetyl, fluoroacetyl, formyl). X-ray crystallography indicates a strong, electronically tunable preference for the α-helix conformation, as observed directly on the φ and ψ torsion angles. Electron-donating acyl groups promote the α-helical conformation, even in the absence of the hydrogen bonding that stabilizes the α-helix. In contrast, electron-withdrawing acyl groups led to more extended conformations. More sterically demanding groups can promote trans amide bonds independent of the electronic effect on n→π* interactions. Chloroacetyl groups additionally promote n→π* interactions through the interaction of the chlorine lone pair with the proximal carbonyl π*. These data provide additional support for an important role of n→π* interactions in the conformational ensemble of disordered or unfolded proteins. Moreover, this work suggests that readily incorporated acyl N-capping motifs that modulate n→π* interactions may be employed rationally to promote conformational biases in peptides, with potential applications in molecular design and medicinal chemistry.     

Potential-controlled chromatography for the separation of amino acids and peptides

Potential-controlled chromatography is introduced as a new technique for the separation of amino acids and peptides. The principle of potential-controlled chromatography depends on the use of electrically conductive material as the stationary phase of the chromatographic column. Thus from an electrochemical point of view the packed column can be regarded as a packed-bed electrode. The electrical potential of this stationary phase can be controlled by a potentiostat. The separation of amino acid and peptide molecules during their migration through the column depends on their own electric charge on the one hand and on the electrical potential of the stationary phase on the other. The chromatographic separation of some amino acids could be demonstrated.     

Combination of allyl protection and HYCRAMTM-linker technology for the synthesis of peptides with problematical amino acids and sequences

Analogues of both the nonapeptides, bradykinin and bradykinin potentiating nonapeptide BPP_9α, were synthesized using HYCRAM^TM-technique. The bradykinin analogues were assembled by the Boc-, Ddz- and Fmoc-strategy starting with Boc-Arg(Aloc)₂-OCr–OH, Ddz-Arg(Mtr)-OCr–OH and Fmoc–Arg(Mtr)-OCr–OH. While Boc- and Ddz-strategy provide peptides in good yield and purity, the Fmoc-strategy leads to a loss of peptide from resin. For simultaneous cleavage from HYCRAM^TM-resin and removal of Aloc-side chain protection optimized conditions for catalytic cleavage with Pd° were developed. As shown by the synthesis of BPP_9αanalogues the HYCRAM^TM-linker and the chlorotrityl resin allow the assembly of peptides with the C-terminal sequence Pro-Pro by preventing dioxopiperazine formation. Since the BPP_9α sequence contains the tripeptide Trp-X-Arg an intramolecular migration of the N^G-protecting group to the indole ring under conditions used for its removal had to be avoided. By the use of HYCRAM^TM-linker in combination with Aloc protection for the guanidino group and Ddz for N^αno modification of Trp occurred. HYCRAM^TM-technology in combination with Boc-, Ddz- or Aloc/All-protecting groups facilitates the synthesis of peptides with such very labile amino acids like cis-4-hydroxyproline.     

Peptides and proteins as a continuing exciting source of inspiration for peptidomimetics

Despite their enormous diversity in biological function and structure, peptides and proteins are endowed with properties that have induced and stimulated the development of peptidomimetics. Clearly, peptides can be considered as the "stem" of a phylogenetic molecular development tree from which branches of oligomeric peptidomimetics such as peptoids, peptidosulfonamides, urea peptidomimetics, as well as β-peptides have sprouted. It is still a challenge to efficiently synthesize these oligomeric species, and study their structural and biological properties. Combining peptides and peptidomimetics led to the emergence of peptide-peptidomimetic hybrids in which one or more (proteinogenic) amino acid residues have been replaced with these mimetic residues. In scan-like approaches, the influence of these replacements on biological activity can then be studied, to evaluate to what extent a peptide can be transformed into a peptidomimetic structure while maintaining, or even improving, its biological properties. A central issue, especially with the smaller peptides, is the lack of secondary structure. Important approaches to control secondary structure include the introduction of α,α-disubstituted amino acids, or (di)peptidomimetic structures such as the Freidinger lactam. Apart from intra-amino acid constraints, inter-amino acid constraints for formation of a diversity of cyclic peptides have shaped a thick branch. Apart from the classical disulfide bridges, the repertoire has been extended to include sulfide and triazole bridges as well as the single-, double- and even triple-bond replacements, accessible by the extremely versatile ring-closing alkene/alkyne metathesis approaches. The latter approach is now the method of choice for the secondary structure that presents the greatest challenge for structural stabilization: the α-helix.     

A Newcomer′s Guide to Peptide Crystallography

Herein we provide a guide for adapting the tools developed for protein X-ray crystallography to study the structures and supramolecular assembly of peptides. Peptide crystallography involves selecting a suitable peptide, crystallizing the peptide, collecting X-ray diffraction data, processing the diffraction data, determining the crystallographic phases and generating an electron density map, building and refining models, and depositing the crystallographic structure in the Protein Data Bank (PDB). Advances in technology make this process easy for a newcomer to adopt. This paper describes techniques for determining the X-ray crystallographic structures of peptides: incorporation of amino acids containing heavy atoms for crystallographic phase determination, commercially available kits to crystallize peptides, modern techniques for X-ray crystallographic data collection, and free user-friendly software for data processing and producing a crystallographic structure.