While peptide macrocycles with rigid conformations have proven to be useful in the design of chemical probes against protein targets, conformational flexibility and rapid conformational interconversion can be equally vital for biological activity. This study introduces the concept of a “structural pin”, which represents an intramolecular hydrogen bond that controls overall ring conformation and can be used to explore macrocyclic conformational energy landscape. Characterization and structural analysis of macrocycles with an endocyclic Brønsted base using NMR and molecular modelling indicates that removal of the structural pin drastically influences the conformation of the entire ring, resulting in novel states with increased conformational heterogeneity. These results suggest that local interactions around structural pins can be effective in controlling overall macrocycle conformation, offering a useful conceptual framework for stabilizing bioactive molecules.

Isoelectronic and isolobal analogies are powerful tools for comparing the electronic structures of molecular fragments, but they do not necessarily capture similarities in chemical reactivity. In this perspective, we introduce the concept of isoreactive relations to address cases in which structurally distinct fragments support chemically productive outcomes despite dissimilar composition. This idea emerged from our studies on boryl migration, where replacing a hydride with a boron-containing substituent unexpectedly unlocked new reaction pathways. More broadly, isoreactive relations should provide a framework for identifying mechanistic parallels that extend beyond the scope of traditional electronic comparisons. By embracing this reactivity-centered perspective, it should become possible to uncover and experimentally validate unconventional fragment substitutions that lead to novel transformations.

Join us for the Thieme Cheminar “Boron in Organic Synthesis” on Thursday, October 16, 2025, at 3:00 PM (CET) This special online event, brought to you by SynOpen Journal, highlights the versatile role of boron in organic synthesis and its importance in advancing organic chemistry. The program will feature outstanding talks from leading researchers in the field: 🔹Prof. Varinder Aggarwal (University of Bristol, UK) 🔹Prof. Andrei Yudin (University of Toronto, Canada) 🔹Prof. Benjamin J. Stokes (Santa Clara University, USA) The session will be chaired by Prof. Thierry Ollevier, Editor-in-Chief of SynOpen. Do not miss this unique opportunity to explore cutting-edge advances in boron chemistry and engage with some of the leading voices defining tomorrow’s organic chemistry! Boron mediated synthesis of tetrasubstituted alkenes Talk by Varinder Aggarwal, Thierry Ollevier Advances in boron chemistry: reagents, mechanisms, and targets Talk by Andrei Yudin Tetrahydroxydiboron in Pd-Catalyzed Transfer Hydrogenations Talk by Ben Stokes

Herein, we apply the principle of iminology to the well-established epoxide ring-closure reaction. The synthesis of tetrasubstituted, nitrile-tethered epoxides can be achieved via activation of iminologous diols followed by fragmentation. Mechanistic study reveals the transformation to be stereoretentive, which is consistent with the concerted nature of the epoxide ring-closure.

Here we describe the design and applications of borindolizine, a novel scaffold with broadly tunable fluorescence and a high Stokes shift. Two classes of emitters were synthesized through rational scaffold modification, resulting in blue-emitting carboxyborindolizines (λ max,em = 431-459 nm) and green-emitting aryl borindolizines (λ max,em = 489-519 nm). Experimental structure-emission trends were used to validate a computational spectral prediction model and to subsequently design a red-emissive borindolizine scaffold. The red-emissive isoquinolyl borinidolizine was prepared, and the experimental emission (λ max,abs = 370 nm, λ max,em = 635 nm) was in excellent agreement with the theoretical emission (λ max,em = 646 nm). These results show how the application of data science can produce fluorophores with desirable spectroscopic properties through the borindolizine scaffold.

A pseudo-ring is a medium-sized cyclic structure, held together by a strong hydrogen bond, that can act as a mimetic of a covalently linked heterocycle. Molecules that contain pseudo-rings can switch between "open" and "closed" forms. This paper examines the utility of pseudo-ring constructs to control conformations and properties of macrocycles. The spectroscopic evidence suggests that the "open" and "closed" pseudo-ring-containing macrocycles differ in their three-dimensional characteristics, which results in markedly different lipophilicities. The dynamic nature of pseudo-rings has led to the emergence of tunable fluorescence that should be useful in environment-sensitive applications.

Chemists need to develop reliable strategies that leverage abundant feedstocks to speed up the production of vital medicines, agrochemicals, and materials. Oligoheterocyclic compounds are one class of molecules that span each of these disciplines. Cross-coupling strategies are commonly used to assemble oligoheterocycles; however, these transformations can be challenging and often rely on costly precious metal catalysts and building blocks derived from unsustainable feedstocks and supply chains. Here, we present a synthetic strategy for iterative construction of oligoheterocycles that leverages sustainable building blocks designed around carbon atoms of high oxidation states. By utilizing feedstocks from the base of the chemical supply chain, we generate bifunctional building blocks that chemoselectively engage with a variety of reaction partners through simple condensation manifolds. Adopting this approach has led to novel scaffolds previously inaccessible by conventional means, which exemplifies sustainable design strategies and enables democratization of chemical synthesis.

ClpXP is a two-component mitochondrial matrix protease. The caseinolytic mitochondrial matrix peptidase chaperone subunit X (ClpX) recognizes and translocates protein substrates into the degradation chamber of the caseinolytic protease P (ClpP) for proteolysis. ClpXP degrades damaged respiratory chain proteins and is necessary for cancer cell survival. Despite the critical role of ClpXP in mitochondrial protein quality control, the specific degrons, or modifications that tag substrate proteins for degradation by human ClpXP, are still unknown. We demonstrated that phosphorylated serine (pSer) targets substrates to ClpX and facilitates their degradation by ClpXP in biochemical assays. In contrast, ClpP hyperactivated by the small-molecule drug ONC201 lost the preference for phosphorylated substrates. Hydrogen deuterium exchange mass spectrometry combined with biochemical assays showed that pSer binds the RKL loop of ClpX. ClpX variants with substitutions in the RKL loop failed to recognize phosphorylated substrates. In intact cells, ClpXP also preferentially degraded substrates with pSer. Moreover, ClpX substrates with the pSer were selectively found in aggregated mitochondrial proteins. Our work uncovers a mechanism for substrate recognition by ClpXP, with implications for targeting acute myeloid leukemia and other disorders involving ClpXP dysfunction.

ClpXP is an AAA+ protease located in mitochondria matrix. In human, ClpXP degrades damaged respiratory chain proteins and is essential for acute myeloid leukemia (AML) cell survival. This complex consists of the tetradecameric ClpP protease and regulatory particles ClpX. In bacteria, ClpXP targets substrates with a co-translationally added SsrA sequence. However, human lacks this tag and the degron for ClpXP remain unidentified. Notably, bacterial ClpXP homologues degrade substrates with phosphorylated arginine (pArg). We hypothesized that phosphorylated amino acids also facilitate substrate degradation by ClpXP in human mitochondria. Here, we demonstrate that ClpXP selectively degraded phosphorylated α-casein and tau proteins over dephosphorylated forms. Replacing ClpX with ONC201 or E. coli ClpA abolished phosphorylation selectivity, indicating ClpX imparts substrate specificity for phosphorylation. Next, we screened a panel of phosphorylated amino acids and peptides for their effect on ClpXP-mediated degradation of α-casein. Phosphorylated serine (pSer) amino acids and peptides inhibited ClpXP protease activity, while phosphorylated tyrosine (pTyr) or pArg did not. Free serine and phosphate also had no effect. Differential scanning fluorimetry showed that pSer stabilized ClpX, consistent with pSer binding to ClpX. We then used hydrogen/deuterium exchange mass spectrometry (HDX-MS) to reveal the ClpX conformational changes upon pSer containing peptides (Ala-pSer-Ala) binding. HDX analysis indicated the ClpX RKL loop as a putative binding site. Mutating this loop in ClpX (ClpXAAL) disrupted its interaction with pSer, resulting in loss of specificity for phosphorylated substrates. This suggests ClpXP selectively recruits phosphorylated protein for degradation through its RKL loop. In AML cells, ClpP knockdown impaired the respiratory chain and increased reactive oxygen species. Through mass spectrometry analysis of ClpXP interactome and their phosphorylation status upon ClpP inhibition, we identified potential ClpXP endogenous substrates SDHA and NDUFA4, components of the OXPHOS complex. Knockdown of ClpX or ClpP increased levels of pSer-SDHA and pSer-NDUFA4 without changing total protein levels. Recombinant ClpXP degraded pSer-SDHA in a dose-dependent manner, with degradation blocked by pSer peptides. ClpXP did not degrade total SDHA, consistent with its preference for phosphorylated substrates. Further analysis showed elevated pSer-SDHA levels only in the insoluble mitochondrial protein fraction after ClpX knockdown, suggesting these might be damaged proteins. In conclusion, ClpXP degrades damaged mitochondrial proteins with serine phosphorylation, maintaining proper mitochondrial function. Yue Feng, Monica M. Goncalves, Yulia Jitkova, Alexander F. Keszei, Yongran Yan, Chaitra Sarathy, Jonathan St-Germain, Tristan M. Kenney, Matthew Tcheng, Vincent Trudel, Ross S. Mancini, Rahul Upadhyay, Rose Hurren, Marcela Gronda, Matthew Schultz, Kaylen Soriano, Kaitlin Lees, Neil C. Pomroy, S. Quinn W. Currie, Gilbert G. Privé, Mark A. Reed, Andrei K. Yudin, Linda Z. Penn, Cheryl H. Arrowsmith, Brian Raught, Mohammad Mazhab-Jafari, Siavash Vahidi, Aaron D. Schimmer. Human mitochondrial ClpXP protease degrades serine phosphorylated substrates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5425.

The bacterial ClpP protease is essential for the virulence and infectivity of many human pathogens and has emerged as a novel antibacterial drug target. Several classes of small molecules dysregulate or activate ClpP, leading to uncontrolled protein degradation and cell death. Here, we investigate the mechanism of ClpP activation by these compounds using an integrative approach combining structural, biochemical, and computational tools. We identified small molecules that activate ClpP through binding at internal catalytic sites where peptide bond hydrolysis occurs. Combined with knowledge of ClpP activation by small molecules that bind to external hydrophobic sites, this work sheds light on the mechanisms governing ClpP allostery and identifies a common molecular pathway utilized by site-specific effectors to achieve allosteric activation. We propose a consensus, bidirectional ClpP activation mechanism causing protease dysregulation.

alpha-Aminoboronic acids and their derivatives are important synthetic targets. Our research interest has been focused on the synthesis and applications of MIDA (N-methyliminodiacetic acid) protected aminoboronates. Herein we report syntheses of regioisomeric beta-borylated azidoalcohols. The geminal azidoboronate represents a rare example of an alpha-azidoalcohol and is produced through trapping of oxonium ions that develop during the rearrangement of alpha-boryl aldehydes. The vicinal azidoboronate can be obtained from alpha-bromoacetyl MIDA boronate and enables the preparation of aziridine MIDA boronate through the Staudinger reaction.

Stabilization of biologically relevant structural motifs has been a long-standing challenge. Here we show that atropisomeric dominant rotors can stabilize rare 310-helices in macrocycles. The target molecules were prepared using solid-phase peptide synthesis and subjected to extensive structural analysis. Molecular dynamics (MD) simulations enabled us to acquire solution structures for the target molecules, which offered evidence for stable 310-helix formation, ordinarily a metastable state. The 310-helices were shown to retain helicity after heating to 100 degrees C for 72 h. Moreover, the crude atropisomeric mixtures could be thermally enriched toward 310-helical macrocycles with selectivities of >20:1.

While peptide macrocycles with rigidified conformations have proven to be useful in the design of chemical probes of protein targets, conformational flexibility and rapid interconversion can be equally vital for biological activity and favorable physicochemical properties. This study introduces the concept of "structural pin", which describes a hydrogen bond that is largely responsible for stabilizing the entire macrocycle backbone conformation. Structural analysis of macrocycles using nuclear magnetic resonance (NMR), molecular modelling and X-ray diffraction indicates that disruption of the structural pin can drastically influence the conformation of the entire ring, resulting in novel states with increased flexibility. This finding provides a new tool to interrogate dynamic behaviour of macrocycles. Identification of structural pins offers a useful conceptual framework to understand positions that can either be modified to give flexible structures or retained to maintain the rigidity of the scaffold.

Abstract ClpXP is a conserved protein degradation system. In human mitochondria, ClpXP degrades damaged respiratory chain proteins and is vital for the survival of acute myeloid leukemia (AML) cells. This system consists of the barrel-like protease ClpP, capped by regulatory particles (RP) ClpX. In bacteria, ClpXP targets substrates with a co-translationally added SsrA sequence. However, in humans, the SsrA tag is absent, and the degradation markers for ClpXP remain unidentified. Notably, bacterial ClpXP homologues degrade substrates with phosphorylated arginine (pArg). We hypothesized that phosphorylated amino acids also mark substrates for ClpXP degradation in human mitochondria. We showed that ClpXP preferentially degraded phosphorylated α-casein substrate over dephosphorylated α-casein. In contrast, ClpP activated by ONC201, without ClpX, degraded phosphorylated and dephosphorylated α-casein with equal efficiency. Next, we screened a panel of phosphorylated amino acids and peptides for their ability to inhibit ClpXP-mediated degradation of α-casein. Phosphorylated serine (pSer) amino acids and peptides inhibited ClpXP protease activity, while phosphorylated tyrosine (pTyr) or pArg did not. Likewise, Ser and free phosphate did not inhibit ClpXP protease activity. Thermal shift assays showed that pSer amino acids and peptides bind to ClpX, not ClpP. Hydrogen/deuterium exchange mass spectrometry revealed that the addition of Ala-pSer-Ala (ApSA) peptide, but not ASA peptide induced conformational changes in the negatively-charged N terminus of ClpX, thus revealing a putative binding site for ApSA. Earlier, we established that ClpP interacts with the respiratory chain complex II subunit SDHA. Knocking down of ClpP in AML cells affected the respiratory chain and increased reactive oxygen species. Therefore, we tested how ClpXP knockdown impacts levels of phospho-serine SDHA (pSer-SDHA) in intact cells. Using shRNA, we knocked down ClpP and ClpX individually in OCI-AML2 cells. After target knockdown, we pulled down pSer proteins and probed for SDHA. Knockdown of both ClpP and ClpX increased the abundance of pSer-SDHA compared to control. We subsequently added recombinant ClpXP protein to total mitochondrial lysates and the pSer immunoprecipitated fraction from OCI-AML2 cells. This led to decreased levels of pSer-SDHA but did not alter levels of total SDHA, consistent with its preference for pSer-marked substrates suggesting that ClpXP degrades pSer-SDHA. Further analysis showed elevated pSer-SDHA levels only in the insoluble mitochondrial protein fraction after ClpX knockdown, suggesting these might be damaged proteins. In conclusion, our findings demonstrate that serine phosphorylation promotes the degradation of damaged mitochondrial proteins by ClpXP protease. Citation Format: Yue Feng, Monica M. Goncalves, Yulia Jitkova, Alexander Keszei, Chaitra Sarathy, Vincent Trudel, Jonathan St-Germain, Matthew Tcheng, Yongran Yan, Rose Hurren, Matthew Schultz, Brian Raught, Andrei Yudin, Mohammad Mazhab-Jafari, Siavash Vahidi, Aaron D. Schimmer. Serine phosphorylation marks proteins for degradation by the mitochondrial matrix protease, ClpXP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7053.

Passive membrane permeability is an important property in drug discovery and biological probe design. To elucidate the cell-penetrating ability of oxadiazole-containing (Odz) peptides, we employed the Chloroalkane Penetration Assay. The present study demonstrates that Odz cyclic peptides can be highly cell-penetrant depending on the position of specific side chains and the chloroalkane tag. Solution NMR shows that Odz cyclic peptides adopt a β-turn conformation. However, despite observing high cell penetration, we observed low passive permeability in experiments with artificial membranes. These findings highlight the complexity of controlling cell penetration for conformationally sensitive macrocycles and suggest that Odz cyclic peptides may provide a framework for designing cell-penetrant cyclic peptides.

Discovered in the 19th century, ethyl acetoacetate has been central to the development of organic chemistry, including its pedagogy and applications. In this study, we present borylated derivatives of this venerable molecule. A boron handle has been installed at either - or -position of acetoacetate by homologation of acyl-MIDA ( N -methyliminodiacetic acid) boronates with diazoacetates. Either alkyl or boryl groups were found to migrate with regiochemistry being a function of the steric bulk of the diazo species. Boryl -ketoesters can be further modified into borylated pyrazolones and oximes, thereby expanding the synthetic toolkit and offering opportunities for additional modifications.

The efficiency of macrocyclization reactions relies on the appropriate conformational preorganization of a linear precursor, ensuring that reactive ends are in spatial proximity prior to ring closure. Traditional peptide cyclization approaches that reduce the extent of terminal ion pairing often disfavor cyclization-conducive conformations and can lead to undesired cyclodimerization or oligomerization side reactions, particularly when they are performed without high dilution. To address this challenge, synthetic strategies that leverage attractive noncovalent interactions, such as zwitterionic attraction between chain termini during macrocyclization, offer a potential solution by reducing the entropic penalty associated with linear peptides adopting precyclization conformations. In this study, we investigate the role of (N-isocyanoimino)triphenylphosphorane (Pinc) in facilitating the cyclization of linear peptides into conformationally rigid macrocycles. The observed moderate diastereoselectivity is consistent with the preferential Si-facial addition of Pinc, where the isocyanide adds to the E-iminium ion on the same face as the l-proline amide group. The resulting peptide chain reveals that the activated phosphonium ylide of Pinc brings the reactive ends close together, promoting cyclization by enclosing the carboxylate within the interior of the pentapeptide and preventing the formation of byproducts. For shorter peptides with modified peptide backbones, the cyclization mechanism and outcome are redirected, as nucleophilic motifs such as thiazole and imidazole can covalently trap nitrilium intermediates. The isolation of the intermediate in the unproductive macrocyclization pathway, along with nuclear magnetic resonance and density functional theory studies, provides insights into heterocycle-dependent selectivity. The Pinc-driven macrocyclization process has generated diverse collections of cyclic molecules, and our models offer a comprehensive understanding of observed trends, facilitating the development of other heterocycle-forming macrocyclization reactions.

Biaryl and heterobiaryl-containing cyclic peptides represent promising scaffolds for the development of bioactive molecules. The incorporation of heterobiaryl motifs continues to pose synthetic challenges, which is partially due to the difficulties in effecting late-stage metal-catalyzed cross-couplings. We report a new strategy to form heterobiaryls that is based on trapping nitrilium ions. The sequence is exemplified using oxadiazole- and oxazole-containing biaryl linkages. NMR analysis and molecular dynamics simulations reveal structural control elements common to each member of the heterobiaryl containing peptide family in this study. Strategic substitutions on the C-terminal aminobenzoic acid moiety paired with installation of oxadiazole or oxazole heterobiaryl backbone linkages allow for the modulation of peptide backbone conformation, which should assist efforts to optimize the biophysical properties of peptide macrocycles.

ClpXP, a serine protease located in the mitochondrial matrix, regulates mitochondrial proteostasis by degrading damaged or aggregated proteins, including respiratory chain subunits. ClpXP is a bipartite protein complex comprised of the ClpX regulatory particle that caps each end of the ClpP degradation chamber. ClpX recognizes substrates, unfolds them, and feeds them into ClpP for proteolysis. Genetic or chemical inhibition of ClpXP leads to impaired oxidative phosphorylation function and leukemic cell death in vitro and in vivo. Importantly, however, the degradation marker of human ClpXP remains elusive. Since the bacterial homologue of ClpXP recognizes phosphorylated arginine (pArg)-tagged protein for degradation, we hypothesized that human ClpXP might also recognize phosphorylated amino acids as degrons. We first investigated whether phosphorylation influences substrate degradation by ClpXP using a model substrate, α-casein. Recombinant human ClpXP preferentially degraded phosphorylated α-casein compared to dephosphorylated α-casein. To further elucidate the effects of phosphorylation, we screened a panel of phosphorylated amino acids for their ability to impede ClpXP-mediated degradation of FITC-tagged α-casein. Phosphorylated serine (pSer) and phosphorylated threonine (pThr) free amino acids, or short peptides containing pSer or pThr, inhibited α-casein degradation by ClpXP in a dose-dependent manner. In contrast, phosphorylated pArg, and phosphorylated tyrosine (pTyr), as well as free unmodified Ser or Thr, did not impact ClpXP protease activity. Next, we applied a thermal shift assay to measure the binding capabilities of pSer and pThr to ClpX. We discovered that these phosphorylated amino acids, whether free or incorporated into short peptides, successfully associated with ClpX. In contrast, their dephosphorylated counterparts did not exhibit the same binding activity. Notably, pSer did not affect enzyme activity of the related LonP1 mitochondrial matrix serine protease, thereby demonstrating the specificity of pSer for ClpXP. Extending our studies to intact cells, we analyzed a proteomic dataset of post-translational modifications in Jurkat cells with and without treatment with bortezomib (1 µM), a proteasome inhibitor that inhibits ClpXP at this concentration. Notably, a global enrichment of phosphorylation was observed for mitochondrial proteins upon bortezomib treatment. We overlaid observed phosphorylated proteins with the ClpXP interactome as determined by BioID, and identified 9 phosphorylated mitochondrial proteins that also interact with ClpXP. Of these proteins, respiratory chain II complex subunit, SDHA, was the top hit, where serine phosphorylation was increased over 4 folds. Next we determined how depletion of ClpXP affected levels of phosphorylated substrate in intact cells. Knockdown of ClpX or ClpP in OCI-AML2 cells increased levels of serine phosphorylated SDHA (pSer-SDHA). We then added recombinant ClpXP protein to mitochondrial lysates and observed selective degradation of pSer-SDHA. To determine if pSer-SDHA are damaged proteins, we partitioned mitochondria into soluble and insoluble fractions using digitonin. pSer-SDHA was enriched in the detergent-insoluble fraction. In addition, we induced mitochondrial proteolytic stress by treating OCI-AML2 cells with antimycin to increase mitochondrial ROS or through heat shock by culturing cells at 42 oC. Both antimycin and heat shock increased pSer-SDHA in the detergent-insoluble fraction, further supporting pSer marking damaged proteins. Finally, we developed small molecule pSer mimics. We showed that these compounds bound ClpX in thermal shift assays. In addition, these compounds inhibited ClpXP protease activity in cell-free enzymatic assays. In summary, ClpXP recognizes serine phosphorylation as a degradation marker for damaged mitochondrial proteins. Small molecules that inhibit ClpX could be leads for novel anti-cancer agents.

We describe a synthetic route to boryl acrylaldehyde, an amphoteric molecule that features BMIDA and aldehyde functionalities attached to an sp2-carbon center. As the project unfolded, conventional protocols based on aldol condensation turned out to be unsuccessful. We eventually zeroed in on oxidative conditions that preserved the BMIDA group and delivered the desired aldehyde functionality. During our investigation, it became clear that boryl alcohol displays hemilabile N–B bonding, which differs dramatically from the previously described congeners with the sp3-carbon center connected to boron. We have attempted to understand the origins of this behavior using DFT calculations. Overall, boryl acrylaldehyde can be considered as an attractive entry point in synthetic methods. These studies are underway in our laboratory.