Comparative Nucleosomal Reactivity of 5-Formyl-Uridine and 5-Formyl-Cytidine.

5-Formyl-deoxyuridine (fdU) and 5-formyl-deoxycytidine (fdC) are formyl-containing nucleosides that are created by oxidative stress in differentiated cells. While fdU is almost exclusively an oxidative stress lesion formed from deoxythymidine (T), the situation for fdC is more complex. Next to formation as an oxidative lesion, it is particularly abundant in stem cells, where it is more frequently formed in an epigenetically important oxidation reaction performed by α-ketoglutarate dependent TET enzymes from 5-methyl-deoxycytidine (mdC). Recently, it was shown that genomic fdC and fdU can react with the ϵ-aminogroups of nucleosomal lysines to give Schiff base adducts that covalently link nucleosomes to genomic DNA. Here, we show that fdU features a significantly higher reactivity towards lysine side chains compared with fdC. This result shows that depending on the amounts of fdC and fdU, oxidative stress may have a bigger impact on nucleosome binding than epigenetics.

A Click-Chemistry-Based Enrichable Crosslinker for Structural and Protein Interaction Analysis by Mass Spectrometry.

Mass spectrometry is the method of choice for the characterisation of proteomes. Most proteins operate in protein complexes, in which their close association modulates their function. However, with standard MS analysis, information on protein-protein interactions is lost and no structural information is retained. To gain structural and interactome data, new crosslinking reagents are needed that freeze inter- and intramolecular interactions. Herein, the development of a new reagent, which has several features that enable highly sensitive crosslinking MS, is reported. The reagent enables enrichment of crosslinked peptides from the majority of background peptides to facilitate efficient detection of low-abundant crosslinked peptides. Due to the special cleavable properties, the reagent can be used for MS2 and potentially for MS3 experiments. Thus, the new crosslinking reagent, in combination with high-end MS, should enable sensitive analysis of interactomes, which will help researchers to obtain important insights into cellular states in health and diseases.

Azobenzene-based inhibitors of human carbonic anhydrase II.

Aryl sulfonamides are a widely used drug class for the inhibition of carbonic anhydrases. In the context of our program of photochromic pharmacophores we were interested in the exploration of azobenzene-containing sulfonamides to block the catalytic activity of human carbonic anhydrase II (hCAII). Herein, we report the synthesis and in vitro evaluation of a small library of nine photochromic sulfonamides towards hCAII. All molecules are azobenzene-4-sulfonamides, which are substituted by different functional groups in the 4´-position and were characterized by X-ray crystallography. We aimed to investigate the influence of electron-donating or electron-withdrawing substituents on the inhibitory constant K i. With the aid of an hCAII crystal structure bound to one of the synthesized azobenzenes, we found that the electronic structure does not strongly affect inhibition. Taken together, all compounds are strong blockers of hCAII with K i = 25-65 nM that are potentially photochromic and thus combine studies from chemical synthesis, crystallography and enzyme kinetics.