The mechanism behind enhanced reactivity of unsaturated phosphorus(v) electrophiles towards thiols.

Vinyl- and ethynyl phosphorus(v) electrophiles are a versatile class of thiol-reactive reagents suitable for cysteine-selective peptide and protein modifications, especially for the generation of antibody conjugates. Herein we investigated the reactivity of various P(v) reagents towards thiol addition. Complementing previous studies, we observed that the heteroatoms X (X = S, O, NH) as well as the vinyl- vs. ethynyl-substituent bound to phosphorus greatly influence the overall reactivity. These experimentally observed trends, as well as the high Z-selectivity for thiol additions to ethynyl derivatives, were further elucidated using DFT calculations. Hyperconjugation was a key means of stabilizing the intermediate generated upon the thiol addition, thus determining both the reactivity and stereoselectivity of unsaturated P(v) electrophiles. Specifically, the energetically low-lying σ antibonding orbital of the P-S bond more readily stabilizes the electron density from the lone pair (LP) of the generated carbanion, rendering the phosphonothiolates more reactive compared to the derivatives bearing oxygen and nitrogen. Our studies provide a detailed mechanistic picture for designing P(v)-based electrophiles with fine-tuned reactivity profiles.

Synthesis and Biological Evaluation of Endocannabinoid Uptake Inhibitors Derived from WOBE437.

WOBE437 ((2E,4E)-N-(3,4-dimethoxyphenethyl)dodeca-2,4-dienamide, 1) is a natural product-derived, highly potent inhibitor of endocannabinoid reuptake. In this study, we synthesized almost 80 analogues of 1 with different types of modifications in the dodecadienoyl domain as well as the dimethoxyphenylethyl head group, and we investigated their effects on anandamide uptake into U937 cells. Intriguingly, none of these analogues was a more potent inhibitor of anandamide uptake than WOBE437 (1). At the same time, a number of WOBE437 variants exhibited potencies in the sub-100 nM range, with high selectivity over inhibition of the endocannabinoid-degrading enzyme fatty acid amide hydrolase; two compounds were virtually equipotent with 1. Interestingly, profound activity differences were observed between analogues in which either of the two methoxy substituents in the head group had been replaced by the same bulkier alkoxy group. Some of the compounds described here could be interesting departure points for the development of potent endocannabinoid uptake inhibitors with more drug-like properties.

Chemically Induced Vinylphosphonothiolate Electrophiles for Thiol-Thiol Bioconjugations.

Herein we introduce vinylphosphonothiolates as a new class of cysteine-selective electrophiles for protein labeling and the formation of stable protein-protein conjugates. We developed a straightforward synthetic route to convert nucleophilic thiols into electrophilic, thiol-selective vinylphosphonothiolates: In this protocol, intermediately formed disulfides can be chemoselectively substituted with vinylphosphonites under acidic conditions to yield the desired vinylphosphonothiolates. Notably, this reaction sequence enables the installation of vinylphosphonothiolate electrophiles directly on cysteine side chains within peptides and proteins. In addition to labeling the monoclonal antibody trastuzumab with excellent cysteine-selectivity, we applied our protocol for the site-specific conjugation of two proteins with unique cysteine residues yielding a nonhydrolyzable phosphonothiolate-linked diubiquitin and an ubiquitin-α-synuclein conjugate. The latter was recognized as a substrate in a subsequent enzymatic ubiquitination reaction.

Total Synthesis of the Endocannabinoid Uptake Inhibitor Guineensine and SAR Studies.

Guineensine ((2E,4E,12E)-13-(benzo[d][1,3]dioxol-5-yl)-N-isobutyltrideca-2,4,12-trienamide) is a plant-derived natural product that inhibits reuptake of the endocannabinoid anandamide with sub-micromolar potency. We have established a highly efficient total synthesis of guineensine, which provided the natural product in only five steps from commercially available 3-nonyn-1-ol in 17 % overall yield, relying on the attachment of the benzodioxolyl moiety to the unsaturated fatty acid chain by means of a Suzuki coupling as the key step. Subsequent SAR studies revealed that replacement of the N-isobutyl group in the natural product by various alkyl, arylalkyl, or aryl groups is generally well tolerated, and derivatives could be identified that are slightly more potent anandamide reuptake inhibitors than guineensine itself. In contrast, modifications of the benzodioxolyl moiety led to decreased activity. Intriguingly, a change in the configuration of the C4=C5 double bond from E to Z was found to be very well tolerated, in spite of the associated change in the overall geometry of the molecule.

Tag and release: strategies for the intracellular cleavage of protein conjugates.

Attaching a functional moiety to a protein is required for a wealth of applications, comprising targeted delivery, controlling of enzyme activity, and prodrug-based therapy. Targeting intracellular processes requires the cellular delivery of the protein. While at first, a stable connection between the protein and the modification is desired, once inside the cell, the conjugate might be cleaved again to restore or activate the function of the individual parts. This can be achieved by employing cleavable linkages in conjugates, which are responsive to chemical or enzymatic stimuli inside cells. In this overview we describe strategies, how such entities can be introduced into proteins and how selective intracellular cleavage can be accomplished.

Engineered Peptide Macrocycles Can Inhibit Matrix Metalloproteinases with High Selectivity.

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases at the intersection of health and disease due to their involvement in processes such as tissue repair and immunity as well as cancer and inflammation. Because of the high structural conservation in the catalytic domains and shallow substrate binding sites, selective, small-molecule inhibitors of MMPs have remained elusive. In a tour-de-force peptide engineering approach combining phage-display selections, rational design of enhanced zinc chelation, and d-amino acid screening, we succeeded in developing a first synthetic MMP-2 inhibitor that combines high potency (Ki =1.9±0.5 nm), high target selectivity, and proteolytic stability, and thus fulfills all the required qualities for in cell culture and in vivo application. Our work suggests that selective MMP inhibition is achievable with peptide macrocycles and paves the way for developing specific inhibitors for application as chemical probes and potentially therapeutics.

Modern Ligation Methods to Access Natural and Modified Proteins.

Proteins and peptides are gaining increasing interest as tools and targets in fundamental research and drug discovery. Growing research applications have prompted the need for methodologies that produce homogenous peptide and protein material. The development of efficient, chemoselective ligation reactions using unprotected peptide fragments presents a key solution for this challenging task. This review outlines modern ligation methods that enable the synthesis of both native, and also labelled or post-translationally modified peptides and proteins. The ligation methods herein discussed focus on the formation of the backbone amide bond.