We previously demonstrated that osmium tetroxide and TMEDA generate stable crystalline adducts with alkenes that facilitate X-ray analysis and structure assignments. Alternatively, osmate esters can be prepared from diols, potassium osmate, and TMEDA·2TsOH in a nonoxidative condensation reaction. This new approach provides a convenient route to form stable, crystalline osmate(VI) esters for X-ray analysis. Because it is redox neutral, it works with a variety of diol substrates, including 1,3-diols, that cannot be prepared from alkenes. α-Hydroxy acids also form stable osmate esters in reasonable yields and readily crystallize. An alternative ligand screen was performed to assess the improved crystallinity from substituted TMEDA analogues. The enhanced crystallinity of osmate esters and the incorporation of a heavy atom make a reliable determination of structure and absolute configuration routine.

Cross-linking mass spectrometry (XL-MS) is a powerful technology for mapping protein-protein interactions (PPIs) at the systems level. While bivalent cross-links are effective for defining protein interactions and structures, multivalent cross-links offer enhanced spatial resolution to facilitate characterization of heterogeneous protein complexes. However, their identification remains challenging due to fragmentation complexity and the vast expansion of database search space. Here, we present tris-succinimidyl trioxane (TSTO), a novel trioxane-based, MS-cleavable homotrifunctional cross-linker capable of targeting three proximal lysines simultaneously. TSTO's unique MS-cleavability enables concurrent release of cross-linked peptide constituents during collision-induced dissociation, permitting their unambiguous identification. The TSTO-based XL-MS platform is effective for mapping cellular networks from intact cells and tissues, illustrating its versatility for complex biological systems. Trimeric interactions captured by TSTO reveal structural details inaccessible to bifunctional reagents, enhancing modeling accuracy and precision. Furthermore, this development opens a new avenue for designing multifunctional MS-cleavable cross-linkers to further advance structural systems biology.

Sulfuric acid in the atmosphere can participate in acid-catalyzed and acid-driven reactions, including those within secondary organic aerosols (SOA). Previous studies have observed enhanced absorption at visible wavelengths and significant changes in the chemical composition when SOA was exposed to sulfuric acid. However, the specific chromophores responsible for these changes could not be identified. The goals of this study are to identify the chromophores and determine the mechanism of browning in highly acidified alpha-pinene SOA by following the behavior of specific common alpha-pinene oxidation products, namely, cis-pinonic acid and cis-pinonaldehyde, when they are exposed to highly acidic conditions. The products of these reactions were analyzed with ultra-performance liquid chromatography coupled with photodiode array spectrophotometry and high-resolution mass spectrometry, UV-vis spectrophotometry, and nuclear magnetic resonance spectroscopy. cis-Pinonic acid (2) was found to form homoterpenyl methyl ketone (4), which does not absorb visible radiation, while cis-pinonaldehyde (3) formed weakly absorbing 1-(4-(propan-2-ylidene)cyclopent-1-en-1-yl)ethan-1-one (5) and 1-(4-isopropylcyclopenta-1,3-dien-1-yl)ethan-1-one (6) via an acid-catalyzed aldol condensation. This chemistry could be relevant for environments characterized by high sulfuric acid concentrations, for example, during the transport of organic compounds from the lower to the upper atmosphere by fast updrafts.

The development of MS-cleavable cross-linking mass spectrometry (XL-MS) has enabled the effective capture and identification of endogenous protein-protein interactions (PPIs) and their residue contacts at the global scale without cell engineering. So far, only lysine-reactive cross-linkers have been successfully applied for proteome-wide PPI profiling. However, lysine cross-linkers alone cannot uncover the complete PPI map in cells. Previously, we have developed a maleimide-based cysteine-reactive MS-cleavable cross-linker (bismaleimide sulfoxide (BMSO)) that is effective for mapping PPIs of protein complexes to yield interaction contacts complementary to lysine-reactive reagents. While successful, the hydrolysis and limited selectivity of maleimides at physiological pH make their applications in proteome-wide XL-MS challenging. To enable global PPI mapping, we have explored an alternative cysteine-labeling chemistry and thus designed and synthesized a sulfoxide-containing MS-cleavable haloacetamide-based cross-linker, Dibromoacetamide sulfoxide (DBrASO). Our results have demonstrated that DBrASO cross-linked peptides display the same fragmentation characteristics as other sulfoxide-containing MS-cleavable cross-linkers, permitting their unambiguous identification by MSn. In combination with a newly developed two-dimensional peptide fractionation method, we have successfully performed DBrASO-based XL-MS analysis of HEK293 cell lysates and demonstrated its capability to complement lysine-reactive reagents and expand PPI coverage at the systems-level.

Cross-linking mass spectrometry (XL-MS) is an emergent technology for studying protein-protein interactions (PPIs) and elucidating architectures of protein complexes. The development of various MS-cleavable cross-linkers has facilitated the identification of cross-linked peptides, enabling XL-MS studies at the systems level. However, the scope and depth of cellular networks revealed by current XL-MS technologies remain limited. Due to the inherently broad dynamic range and complexity of proteomes, interference from highly abundant proteins impedes the identification of low-abundance cross-linked peptides in complex samples. Thus, peptide enrichment prior to MS analysis is necessary to enhance cross-link identification for proteome-wide studies. Although chromatographic techniques including size exclusion (SEC) and strong cation exchange (SCX) have been successful in isolating cross-linked peptides, new fractionation methods are still needed to further improve the depth of PPI mapping. Here, we present a two-dimensional (2D) separation strategy by integrating peptide SEC with tip-based high pH reverse-phase (HpHt) fractionation to expand the coverage of proteome-wide XL-MS analyses. Combined with the MS-cleavable cross-linker DSSO, we have successfully mapped in vitro PPIs from HEK293 cell lysates with improved identification of cross-linked peptides compared to existing approaches. The method developed here is effective and can be generalized for cross-linking studies of complex samples.

We report the first total syntheses of strasseriolide A and B. Strasseriolide B shows potent activity against the wild-type malaria parasite Plasmodium falciparum and good activity against a chloroquine-resistant strain. A convergent strategy was envisioned with an aldehyde-acid fragment and a vinyl iodide-alcohol fragment. Both fragments were prepared using chiral pool starting materials. They were combined with a Yamaguchi esterification and cyclized with a Nozaki-Hiyama-Kishi reaction. Strasseriolide B was assembled in a 16-step LLS.

We report the first total synthesis of (2R)-hydroxynorneomajucin, a norsesquiterpene derived from the Illicium genus. This natural product displays neurotrophic properties. Small molecule neurotrophins have potential as therapeutics in neurodegenerative diseases. Key steps of our synthesis include a Tsuji-Trost reaction, a Pauson-Khand cyclization, and a Nagata hydrocyanation. A simple sequence of reductions and a Mukaiyama hydration introduce the A-ring substituents with the correct configurations. The overall synthesis was completed in 17 steps (longest linear sequence, LLS).

The tumor suppressor p53 is the most frequently mutated protein in human cancer. The majority of these mutations are missense mutations in the DNA binding domain of p53. Restoring p53 tumor suppressor function could have a major impact on the therapy for a wide range of cancers. Here we report a virtual screening approach that identified several small molecules with p53 reactivation activities. The UCI-LC0023 compound series was studied in detail and was shown to bind p53, induce a conformational change in mutant p53, restore the ability of p53 hotspot mutants to associate with chromatin, reestablish sequence-specific DNA binding of a p53 mutant in a reconstituted in vitro system, induce p53-dependent transcription programs, and prevent progression of tumors carrying mutant p53, but not p53null or p53WT alleles. Our study demonstrates feasibility of a computation-guided approach to identify small molecule corrector drugs for p53 hotspot mutations.

Defining protein-protein interactions (PPIs) in their native environment is crucial to understanding protein structure and function. Cross-linking-mass spectrometry (XL-MS) has proven effective in capturing PPIs in living cells; however, the proteome coverage remains limited. Here, we have developed a robust in vivo XL-MS platform to facilitate in-depth PPI mapping by integrating a multifunctional MS-cleavable cross-linker with sample preparation strategies and high-resolution MS. The advancement of click chemistry-based enrichment significantly enhanced the detection of cross-linked peptides for proteome-wide analyses. This platform enabled the identification of 13,904 unique lysine-lysine linkages from in vivo crosslinked HEK 293 cells, permitting construction of the largest in vivo PPI network to date, comprising 6,439 interactions among 2,484 proteins. These results allowed us to generate a highly detailed yet panoramic portrait of human interactomes associated with diverse cellular pathways. The strategy presented here signifies a technological advancement for in vivo PPI mapping at the systems level and can be generalized for charting protein interaction landscapes in any organisms.

Cross-linking mass spectrometry (XL-MS) is a powerful tool for studying protein-protein interactions and elucidating architectures of protein complexes. While residue-specific XL-MS studies have been very successful, accessibility of interaction regions nontargetable by specific chemistries remain difficult. Photochemistry has shown great potential in capturing those regions because of nonspecific reactivity, but low yields and high complexities of photocross-linked products have hindered their identification, limiting current studies predominantly to single proteins. Here, we describe the development of three novel MS-cleavable heterobifunctional cross-linkers, namely SDASO (Succinimidyl diazirine sulfoxide), to enable fast and accurate identification of photocross-linked peptides by MSn. The MSn-based workflow allowed SDASO XL-MS analysis of the yeast 26S proteasome, demonstrating the feasibility of photocross-linking of large protein complexes for the first time. Comparative analyses have revealed that SDASO cross-linking is robust and captures interactions complementary to residue-specific reagents, providing the foundation for future applications of photocross-linking in complex XL-MS studies.

The utility of the dioxasiline ring as a π-facial directing group in the intramolecular Diels-Alder cyclization is explored. An initial investigation of substrate scope demonstrates that the rigidity of this directing group delivers robust stereocontrol across a number of substrates, affording single diastereomers in moderate to good yields. A mechanistic investigation reveals that the reactive diene is formed through γ deprotonation followed by [1,5] hydride shifts.

The competing enantioselective conversion (CEC) method is a quick and reliable means to determine absolute configuration. Previously, Bode's chiral acylated hydroxamic acids were used to determine the stereochemistry of primary amines, as well as cyclic and acyclic secondary amines. The enantioselective acylation has been evaluated for 4-, 5-, and 6-membered cyclic secondary amines, including medicinally relevant compounds. The limitations of the method were studied through computational analysis and experimental results. Piperidines with substituents at the 2-position did not behave well unless the axial conformer was energetically accessible, which is consistent with the transition state geometries proposed by Bode and Kozlowski. Control experiments were performed to investigate the cause of degrading selectivity under the CEC reaction conditions. The present study expands the scope of the CEC method for secondary amines and provides a better understanding of the reaction profile.

Cross-linking mass spectrometry (XL-MS) has become a powerful structural tool for defining protein-protein interactions (PPIs) and elucidating architectures of large protein assemblies. To advance XL-MS studies, we have previously developed a series of sulfoxide-containing MS-cleavable cross-linkers to facilitate the detection and identification of cross-linked peptides using multistage mass spectrometry (MSn). While current sulfoxide-based cross-linkers are effective for in vivo and in vitro XL-MS studies at the systems-level, new reagents are still needed to help expand PPI coverage. To this end, we have designed and synthesized six variable-length derivatives of disuccinimidyl sulfoxide (DSSO) to better understand the effects of spacer arm modulation on MS-cleavability, fragmentation characteristics, and MS identification of cross-linked peptides. In addition, the impact on cross-linking reactivity was evaluated. Moreover, alternative MS2-based workflows were explored to determine their feasibility for analyzing new sulfoxide-containing cross-linked products. Based on the results of synthetic peptides and a model protein, we have further demonstrated the robustness and predictability of sulfoxide chemistry in designing MS-cleavable cross-linkers. Importantly, we have identified a unique asymmetric design that exhibits preferential fragmentation of cross-links over peptide backbones, a desired feature for MSn analysis. This work has established a solid foundation for further development of sulfoxide-containing MS-cleavable cross-linkers with new functionalities.

Cytotoxic protein aggregation-induced impairment of cell function and homeostasis are hallmarks of agerelated neurodegenerative pathologies. As proteasomal degradation represents the major clearance pathway for oxidatively damaged proteins, a detailed understanding of the molecular events underlying its stress response is critical for developing strategies to maintain cell viability and function. Although the 26S proteasome has been shown to disassemble during oxidative stress, its conformational dynamics remains unclear. To this end, we have developed a new quantitative cross-linking mass spectrometry (QXL-MS) workflow to explore the structural dynamics of proteasome complexes in response to oxidative stress. This strategy comprises SILAC-based metabolic labeling, HB tag-based affinity purification, a 2-step cross-linking reaction consisting of mild in vivo formaldehyde and on-bead DSSO cross-linking, and multi-stage tandem mass spectrometry (MSn) to identify and quantify cross-links. This integrated workflow has been successfully applied to explore the molecular events underlying oxidative stress-dependent proteasomal regulation by comparative analyses of proteasome complex topologies from treated and untreated cells. Our results show that H2O2 treatment weakens the 19S-20S interaction within the 26S proteasome, along with reorganizations within the 19S and 20S subcomplexes. Altogether, this work sheds light on the mechanistic response of the 26S to acute oxidative stress, suggesting an intermediate proteasomal state(s) before H2O2-mediated dissociation of the 26S. The QXL-MS strategy presented here can be applied to study conformational changes of other protein complexes under different physiological conditions. Molecular & Cellular Proteomics 18: 954-967, 2019. DOI: 10.1074/ mcp. TIR119.001323.

Illisimonin A was isolated from Illicium simonsii and has a previously unreported tricyclic carbon framework. It displayed neuroprotective effects against oxygen-glucose deprivation-induced cell injury in SH-SY5Y cells. It incorporates a highly strained trans-pentalene ring system. We report the first synthesis of illisimonin A. Notable steps in the route include a 1,3-dioxa-2-silacyclohexenetemplated Diels-Alder cycloaddition and type-3 semipinacol rearrangement to generate the trans-pentalene. The final step is an iron-catalyzed C-H oxidation. The synthetic route is robust, with 94 mg prepared in a single pass.

Abstract(−)‐Himeradine A is a complex lycopodium alkaloid with seven rings and ten stereogenic centers that shows anticancer activity against lymphoma L1210 cells. A total synthesis has been developed that builds off prior work on (+)‐fastigiatine. A 2,4,6‐trisubstitited piperidine ring forms the core of the quinolizidine segment, and was prepared by diastereoselective reduction of a pyridine and classic resolution of an intermediate. The remaining secondary amine was introduced with a catalyst‐controlled Overman rearrangement. The piperidine segment was coupled in a B‐alkyl Suzuki reaction with a bicyclic bromoenone, which was a key intermediate for the synthesis of (+)‐fastigiatine. The final transformation featured a transannular Mannich reaction and cyclization to complete the quinolizidine. Five bonds and four new rings were generated in this one‐pot procedure. (−)‐Himeradine A was prepared in 17 steps in the longest linear sequence.

Osmium tetroxide and TMEDA form stable crystalline adducts with alkenes. The structure of liquid alkenes can be determined through X-ray analysis of these derivatives. Osmium, a heavy atom, facilitates the crystallographic analysis and the determination of the absolute configuration using common Mo X-ray sources. The utility of this method for assigning structures and absolute configurations was demonstrated on a number of unsaturated substrates that include simple alkenes, enones, enol ethers, and silyl enol ethers.