Microscale Parallel Synthesis of Acylated Aminotriazoles Enabling the Development of Factor XIIa and Thrombin Inhibitors.

Herein we report a microscale parallel synthetic approach allowing for rapid access to libraries of N-acylated aminotriazoles and screening of their inhibitory activity against factor XIIa (FXIIa) and thrombin, which are targets for antithrombotic drugs. This approach, in combination with post-screening structure optimization, yielded a potent 7 nM inhibitor of FXIIa and a 25 nM thrombin inhibitor; both compounds showed no inhibition of the other tested serine proteases. Selected N-acylated aminotriazoles exhibited anticoagulant properties in vitro influencing the intrinsic blood coagulation pathway, but not extrinsic coagulation. Mechanistic studies of FXIIa inhibition suggested that synthesized N-acylated aminotriazoles are covalent inhibitors of FXIIa. These synthesized compounds may serve as a promising starting point for the development of novel antithrombotic drugs.

Automated Chiral Analysis of Amino Acids Based on Chiral Derivatization and Trapped Ion Mobility-Mass Spectrometry.

A fast and fully automated method for chiral analysis has been developed by combining a chiral derivatization approach with high-resolution trapped ion mobility separation. Although the presented approach can be potentially applied to diverse types of chiral compounds, several benchmark amino acids were used as model compounds, focusing on the smallest amino acid alanine. An autosampler with an integrated chromatography system was used for inline chiral derivatization with (S)-naproxen chloride and subsequent preseparation. Afterwards, derivatized amino acids were directly introduced into the electrospray interface of a trapped ion mobility-mass spectrometer for rapid diastereomer separation in the gas phase. This unique combination of preseparation and trapped ion mobility spectrometry separation in the negative ion mode enabled rapid chiral analysis within 3 min per sample. Furthermore, the diastereomer separation proved to be independent of alkali salts or other metal ions, offering robustness with regard to samples containing high amounts of salts. Highly sensitive detection of amino acid diastereomers was possible down to the lower nanomolar concentration range, and enantiomeric ratios could be readily determined from the recorded mobilograms with excellent reproducibility and precision. To demonstrate the general applicability of our method, alanine and other amino acids were analyzed from soy sauces and seasonings, which revealed extraordinarily high d-Ala contents of up to 99% in all samples.

Acylated 1H-1,2,4-Triazol-5-amines Targeting Human Coagulation Factor XIIa and Thrombin: Conventional and Microscale Synthesis, Anticoagulant Properties, and Mechanism of Action.

We herein report the conventional and microscale parallel synthesis of selective inhibitors of human blood coagulation factor XIIa and thrombin exhibiting a 1,2,4-triazol-5-amine scaffold. Structural variations of this scaffold allowed identifying derivative 21i, a potent 29 nM inhibitor of FXIIa, with improved selectivity over other tested serine proteases and also finding compound 21m with 27 nM inhibitory activity toward thrombin. For the first time, acylated 1,2,4-triazol-5-amines were proved to have anticoagulant properties and the ability to affect thrombin- and cancer-cell-induced platelet aggregation. Performed mass spectrometric analysis and molecular modeling allowed us to discover previously unknown interactions between the synthesized inhibitors and the active site of FXIIa, which uncovered the mechanistic details of FXIIa inhibition. Synthesized compounds represent a promising starting point for the development of novel antithrombotic drugs or chemical tools for studying the role of FXIIa and thrombin in physiological and pathological processes.

A mass spectrometry-based approach gives new insight into organotin-protein interactions.

In this study, the combination of speciation analysis and native mass spectrometry is presented as a powerful tool to gain new insight into the diverse interactions of environmentally relevant organotin compounds (OTCs) with proteins. Analytical standards of model proteins, such as ß-lactoglobulin A (LGA), were thereby incubated with different phenyl- and butyltins. For adduct identification and characterization, the incubated samples were analyzed by inductively coupled plasma-mass spectrometry (ICP-MS) and electrospray ionization-mass spectrometry (ESI-MS) in combination with size exclusion chromatography (SEC). It allowed for a mild separation, which was most crucial to preserve the acid-labile organotin-protein adducts during their analyses. The binding of triorganotin compounds, such as triphenyltin, was shown to be sulfhydryl-directed by using cysteine-specific protein labeling. However, the sole availability of reduced cysteine residues in proteins did not automatically enable adduct formation. This observation complements previous studies and indicates the necessity of a highly specific binding pocket, which was identified for the model protein LGA via enzymatic digestion experiments. In contrast to triorganotins, their natural di- and mono-substituted degradation products, such as dibutyltin, revealed to be less specific regarding their binding to several proteins. Further, it also did not depend on reduced cysteine residues within the protein. In this context, they can probably act as linker molecules, interconnecting proteins, and leading to dimers and probably to higher oligomers. Furthermore, dibutyltin was observed to induce hydrolysis of the protein's peptide backbone at a specific site. Concerning unknown long-term toxic effects, our studies emphasize the importance of future studies on di- and mono-substituted OTCs.