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High-throughput quantification of the post-translational modification of many individual protein samples is challenging with current label-based methods. This paper demonstrates an efficient method that addresses this gap by combining Escherichia coli -based cell-free protein synthesis (CFPS) and self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS) to analyze intact proteins. This high-throughput approach begins with polyhistidine-tagged protein substrates expressed from linear DNA templates by CFPS. Here, we synthesized an 87-member library of the E. coli Immunity Protein 7 (Im7) containing an acceptor sequence optimized for glycosylation by the Actinobacillus pleuropneumoniae N -glycosyltransferase (NGT) at every possible position along the protein backbone. These protein substrates were individually treated with NGT and then selectively immobilized to self-assembled monolayers presenting nickel-nitrilotriacetic acid (Ni-NTA) complexes before final analysis by SAMDI-MS to quantify the conversion of substrate to glycoprotein. This method offers new opportunities for rapid synthesis and quantitative evaluation of intact glycoproteins.

This paper presents an enzyme building block for the assembly of megamolecules. The system is based on the inhibition of the human‐derived cellular retinoic acid binding protein II (CRABP2) domain. We synthesized a synthetic retinoid bearing an arylfluorosulfate group, which uses sulfur fluoride exchange click chemistry to covalently inhibit CRABP2. We conjugated both the inhibitor and a fluorescein tag to an oligo(ethylene glycol) backbone and measured a second‐order rate constant for the protein inhibition reaction of approximately 3,600 M−1s−1. We used this new enzyme‐inhibitor pair to assemble multi‐protein structures in one‐pot reactions using three orthogonal assembly chemistries to demonstrate exact control over the placement of protein domains within a single, homogeneous molecule. This work enables a new dimension of control over specificity, orientation, and stoichiometry of protein domains within atomically precise nanostructures. [ABSTRACT FROM AUTHOR]

Wepreviously identified 3-hydroxypyridine-2-thione (3HPT) as anovel zinc binding group for histone deacetylase (HDAC) inhibition.Early structure–activity relationship (SAR) studies led tovarious small molecules possessing selective inhibitory activity againstHDAC6 or HDAC8 but devoid of HDAC1 inhibition. To delineate furtherthe depth of the SAR of 3HPT-derived HDAC inhibitors (HDACi), we haveextended the SAR studies to include the linker region and the surfacerecognition group to optimize the HDAC inhibition. The current effortsresulted in the identification of two lead compounds, 10dand 14e, with potent HDAC6 and HDAC8 activities thatare inactive against HDAC1. These new HDACi possess anticancer activitiesagainst various cancer cell lines including Jurkat J.γ1 forwhich SAHA and the previously disclosed 3HPT-derived HDACi were inactive. [ABSTRACT FROM AUTHOR]