Oligosaccharides are ubiquitous in molecular biology and are used for functions ranging from governing protein folding to intercellular communication. Perhaps paradoxically, the exact role of the glycan in most of these settings is not well understood. One reason for this contradiction concerns the fact that carbohydrates often appear in heterogeneous form in nature. These mixtures complicate the isolation of pure material and characterization of structure–activity relationships. As a result, a major bottleneck in glycoscience research is the synthesis and modification of pure materials. While synthetic and chemoenzymatic methods have enabled access to homogeneous tool compounds, a central problem, particularly for newer synthetic chemists, is the matter of problem selection. This outlook aims to provide an entry level overview of fundamental principles in carbohydrate chemistry with an eye toward enabling solutions to frontier challenges.

This IBM surface scientist wants to tease out the mechanisms that drive chemical reactions, one molecule at a time

The Escherichia coli (E. coli) ribosome can incorporate a variety of non-l-α-amino acid monomers into polypeptide chains in vitro but with poor efficiency. Although these monomers span a diverse set of compounds, there exists no high-resolution structural information regarding their positioning within the catalytic center of the ribosome, the peptidyl transferase center (PTC). Thus, details regarding the mechanism of amide bond formation and the structural basis for differences and defects in incorporation efficiency remain unknown. Within a set of three aminobenzoic acid derivatives─3-aminopyridine-4-carboxylic acid (Apy), ortho-aminobenzoic acid (oABZ), and meta-aminobenzoic acid (mABZ)─the ribosome incorporates Apy into polypeptide chains with the highest efficiency, followed by oABZ and then mABZ, a trend that does not track with the nucleophilicity of the reactive amines. Here, we report high-resolution cryo-EM structures of the ribosome with each of these three aminobenzoic acid derivatives charged on tRNA bound in the aminoacyl-tRNA site (A-site). The structures reveal how the aromatic ring of each monomer sterically blocks the positioning of nucleotide U2506, thereby preventing rearrangement of nucleotide U2585 and the resulting induced fit in the PTC required for efficient amide bond formation. They also reveal disruptions to the bound water network that is believed to facilitate formation and breakdown of the tetrahedral intermediate. Together, the cryo-EM structures reported here provide a mechanistic rationale for differences in reactivity of aminobenzoic acid derivatives relative to l-α-amino acids and each other and identify stereochemical constraints on the size and geometry of non-monomers that can be accepted efficiently by wild-type ribosomes.

Interfacial electron-transfer (ET) reactions underpin the interconversion of electrical and chemical energy. It is known that the electronic state of electrodes strongly influences ET rates because of differences in the electronic density of states (DOS) across metals, semimetals, and semiconductors. Here, by controlling interlayer twists in well-defined trilayer graphene moirés, we show that ET rates are strikingly dependent on electronic localization in each atomic layer and not the overall DOS. The large degree of tunability inherent to moiré electrodes leads to local ET kinetics that range over 3 orders of magnitude across different constructions of only three atomic layers, even exceeding rates at bulk metals. Our results demonstrate that beyond the ensemble DOS, electronic localization is critical in facilitating interfacial ET, with implications for understanding the origin of high interfacial reactivity typically exhibited by defects at electrode–electrolyte interfaces.

Biomolecular condensate is an emerging structural entity that regulates various cellular processes. Recent studies have discovered the phase-separation (PS) capability of several transcription factors (TFs) including YAP/TAZ upon biological stimuli, which provide new mechanisms of gene regulation. However, it remains mostly unanswered as to whether PS from a diffuse state to a phase-separated state promotes gene transcription. To address this question, we have designed a chemogenetic tool, dubbed SPARK-ON, which manipulates the PS of YAP and TAZ without a biological stimulus, forming condensates that are transcriptionally active, containing the DNA-binding partner TEAD, genomic DNA, transcriptional machinery, and nascent RNA. Most importantly, PS of TAZ increases the transcription of its target genes. Therefore, our data indicate that PS promotes gene transcription of TAZ. SPARK-ON is advantageous to current mutagenesis-based approaches that are often problematic when mutagenesis affects the transcriptional activity of a TF. Furthermore, protein abundance levels also affect gene transcription, but PS depends on protein abundance because PS occurs only when the protein level is above a saturation concentration. SPARK-ON decouples PS from protein abundance levels without introducing mutations and thus will find important applications in understanding the biological roles of PS for many TFs and other biomolecular condensates.

Molecularly targeted radionuclide therapies (TRTs) struggle with balancing efficacy and safety, as current strategies to increase tumor absorption often alter drug pharmacokinetics to prolong circulation and normal tissue irradiation. Here we report the first covalent protein TRT, which, through reacting with the target irreversibly, increases radioactive dose to the tumor without altering the drug’s pharmacokinetic profile or normal tissue biodistribution. Through genetic code expansion, we engineered a latent bioreactive amino acid into a nanobody, which binds to its target protein and forms a covalent linkage via the proximity-enabled reactivity, cross-linking the target irreversibly in vitro, on cancer cells, and on tumors in vivo. The radiolabeled covalent nanobody markedly increases radioisotope levels in tumors and extends tumor residence time while maintaining rapid systemic clearance. Furthermore, the covalent nanobody conjugated to the α-emitter actinium-225 inhibits tumor growth more effectively than the noncovalent nanobody without causing tissue toxicity. Shifting the protein-based TRT from noncovalent to covalent mode, this chemical strategy improves tumor responses to TRTs and can be readily scaled to diverse protein radiopharmaceuticals engaging broad tumor targets.

Scanning transmission electron microscopy tomography with ChromEM staining (ChromSTEM), has allowed for the three-dimensional study of genome organization. By leveraging convolutional neural networks and molecular dynamics simulations, we have developed a denoising autoencoder (DAE) capable of postprocessing experimental ChromSTEM images to provide nucleosome-level resolution. Our DAE is trained on synthetic images generated from simulations of the chromatin fiber using the 1-cylinder per nucleosome (1CPN) model of chromatin. We find that our DAE is capable of removing noise commonly found in high-angle annular dark field (HAADF) STEM experiments and is able to learn structural features driven by the physics of chromatin folding. The DAE outperforms other well-known denoising algorithms without degradation of structural features and permits the resolution of α-tetrahedron tetranucleosome motifs that induce local chromatin compaction and mediate DNA accessibility. Notably, we find no evidence for the 30 nm fiber, which has been suggested to serve as the higher-order structure of the chromatin fiber. This approach provides high-resolution STEM images that allow for the resolution of single nucleosomes and organized domains within chromatin dense regions comprising of folding motifs that modulate the accessibility of DNA to external biological machinery.

Fission of micron-size charged droplets has been observed using optical methods, but little is known about fission dynamics and breakup of smaller nanosize droplets that are important in a variety of natural and industrial processes. Here, spontaneous fission of individual aqueous nanodrops formed by electrospray is investigated using charge detection mass spectrometry. Fission processes ranging from formation of just two progeny droplets in 2 ms to production of dozens of progeny droplets over 100+ ms are observed for nanodrops that are charged above the Rayleigh limit. These results indicate that Rayleigh fission is a continuum of processes that produce progeny droplets that vary widely in charge, mass, and number.

No current methods can selectively elicit an antibody response to a specific conformational epitope in a whole antigen in vivo. Here, we incorporated Nε-acryloyl-l-lysine (AcrK) or Nε-crotonyl-l-lysine (Kcr) with cross-linking activities into the specific epitopes of antigens and immunized mice to generate antibodies that can covalently cross-link with the antigens. By taking advantage of antibody clonal selection and evolution in vivo, an orthogonal antibody–antigen cross-linking reaction can be generated. With this mechanism, we developed a new approach for facile elicitation of antibodies binding to specific epitopes of the antigen in vivo. Antibody responses were directed and enriched to the target epitopes on protein antigens or peptide-KLH conjugates after mouse immunization with the AcrK or Kcr-incorporated immunogens. The effect is so prominent that the majority of selected hits bind to the target epitope. Furthermore, the epitope-specific antibodies effectively block IL-1β from activating its receptor, indicating its potential for the development of protein subunit vaccines.

Despite genetic perturbations resulting in embryo lethality for most mitotic kinases, loss of the histone H3 mitotic kinase HASPIN reveals no adverse effect in mice models, establishing HASPIN as a promising target for anticancer therapy. However, developing a HASPIN inhibitor from conventional pharmacophores poses a technical challenge as this atypical kinase shares slight similarities with eukaryotic protein kinases. Chemically modifying a cytotoxic 4′-thioadenosine analogue through high genotoxicity yielded several novel nongenotoxic kinase inhibitors. In silico apporoaches utilizing transcriptomic and chemical similarities with known compounds and KINOMEscan profiles unveiled the HASPIN inhibitor LJ4827. LJ4827’s specificity and potency as a HASPIN inhibitor were verified through in vitro kinase assay and X-ray crystallography. HASPIN inhibition by LJ4827 reduced histone H3 phosphorylation and impeded Aurora B recruitment in cancer cell centromeres but not in noncancer cells. Through transcriptome analysis of lung cancer patients, PLK1 was determined as a druggable synergistic partner to complement HASPIN inhibition. Chemical or genetic PLK1 perturbation with LJ4827 effectuated pronounced lung cancer cytotoxicity in vitro and in vivo. Therefore, LJ4827 is a novel anticancer therapeutic for selectively impeding cancer mitosis through potent HASPIN inhibition, and simultaneous HASPIN and PLK1 interference is a promising therapeutic strategy for lung cancer.

The long-term stability of an active-pharmaceutical ingredient and its drug products plays an important role in the licensing process of new pharmaceuticals and for the application of the drug at the patient. It is, however, difficult to predict degradation profiles at early stages of the development of new drugs, making the entire process very time-consuming and costly. Forced mechanochemical degradation under controlled conditions can be used to realistically model long-term degradation processes naturally occurring in drug products, avoiding the use of solvents, thus excluding irrelevant solution-based degradation pathways. We present the forced mechanochemical oxidative degradation of three platelet inhibitor drug products, where the drug products contain thienopyridine. Model studies using clopidogrel hydrogen sulfate (CLP) and its drug formulation Plavix show that the controlled addition of excipients does not affect the nature of the main degradants. Experiments using drug products Ticlopidin-neuraxpharm and Efient show that significant degradation occurs after short reaction times of only 15 min. These results highlight the potential of mechanochemistry for the study of degradation processes of small molecules relevant to the prediction of degradation profiles during the development of new drugs. Furthermore, these data provide exciting insights into the role of mechanochemistry for chemical synthesis in general.

Tumor cells adapt to diverse survival strategies defying our pursuit of multimodal cancer therapy. Prostate cancer (PCa) is an example that is resistant to one of the most potent chemotherapeutics, cisplatin. PCa cells survive and proliferate using fatty acid oxidation (FAO), and the dependence on fat utilization increases as the disease progresses toward a resistant form. Using a pool of patient biopsies, we validated the expression of a key enzyme carnitine palmitoyltransferase 1 A (CPT1A) needed for fat metabolism. We then discovered that a cisplatin prodrug, Platin-L, can inhibit the FAO of PCa cells by interacting with CPT1A. Synthesizing additional cisplatin-based prodrugs, we documented that the presence of an available carboxylic acid group near the long chain fatty acid linker on the Pt(IV) center is crucial for CPT1A binding. As a result of fat metabolism disruption by Platin-L, PCa cells transition to an adaptive glucose-dependent chemosensitive state. Potential clinical translation of Platin-L will require a delivery vehicle to direct it to the prostate tumor microenvironment. Thus, we incorporated Platin-L in a biodegradable prostate tumor-targeted orally administrable nanoformulation and demonstrated its safety and efficacy. The distinctive FAO inhibitory property of Platin-L can be of potential clinical relevance as it offers the use of cisplatin for otherwise resistant cancer.

Platinum(IV) prodrug overcomes acquired resistance to cisplatin in prostate cancer by inhibiting fatty acid oxidation.

Macroautophagy is one of two major degradation systems in eukaryotic cells. Regulation and control of autophagy are often achieved through the presence of short peptide sequences called LC3 interacting regions (LIR) in autophagy-involved proteins. Using a combination of new protein-derived activity-based probes prepared from recombinant LC3 proteins, along with protein modeling and X-ray crystallography of the ATG3-LIR peptide complex, we identified a noncanonical LIR motif in the human E2 enzyme responsible for LC3 lipidation, ATG3. The LIR motif is present in the flexible region of ATG3 and adopts an uncommon β-sheet structure binding to the backside of LC3. We show that the β-sheet conformation is crucial for its interaction with LC3 and used this insight to design synthetic macrocyclic peptide-binders to ATG3. CRISPR-enabled in cellulo studies provide evidence that LIRATG3 is required for LC3 lipidation and ATG3∼LC3 thioester formation. Removal of LIRATG3 negatively impacts the rate of thioester transfer from ATG7 to ATG3.

Systematic binding free energy analysis reveals a dual binding mechanism as a protein-protein interaction stabilizer design strategy.

Sodium-ion batteries (SIBs) have been deemed to be a promising energy storage technology in terms of cost-effectiveness and sustainability. However, the electrodes often operate at potentials beyond their thermodynamic equilibrium, thus requiring the formation of interphases for kinetic stabilization. The interfaces of the anode such as typical hard carbons and sodium metals are particularly unstable because of its much lower chemical potential than the electrolyte. This creates more severe challenges for both anode and cathode interfaces when building anode-free cells to achieve higher energy densities. Manipulating the desolvation process through the nanoconfining strategy has been emphasized as an effective strategy to stabilize the interface and has attracted widespread attention. This Outlook provides a comprehensive understanding about the nanopore-based solvation structure regulation strategy and its role in building practical SIBs and anode-free batteries. Finally, guidelines for the design of better electrolytes and suggestions for constructing stable interphases are proposed from the perspective of desolvation or predesolvation.

Recreating the proteins and sugars that most mouths make effortlessly is proving to be a challenge for chemists.

Fluoromethyl, difluoromethyl, and trifluoromethyl groups are present in numerous pharmaceuticals and agrochemicals, where they play critical roles in the efficacy and metabolic stability of these molecules. Strategies for late-stage incorporation of fluorine-containing atoms in molecules have become an important area of organic and medicinal chemistry as well as synthetic biology. Herein, we describe the synthesis and use of Te-adenosyl-L-(fluoromethyl)homotellurocysteine (FMeTeSAM), a novel and biologically relevant fluoromethylating agent. FMeTeSAM is structurally and chemically related to the universal cellular methyl donor S-adenosyl-L-methionine (SAM) and supports the robust transfer of fluoromethyl groups to oxygen, nitrogen, sulfur, and some carbon nucleophiles. FMeTeSAM is also used to fluoromethylate precursors to oxaline and daunorubicin, two complex natural products that exhibit antitumor properties.

Deciphering the conformations and interactions of peptides in their assemblies offers a basis for guiding the rational design of peptide-assembled materials. Here we report the use of scanning tunneling microscopy (STM), a single-molecule imaging method with a submolecular resolution, to distinguish 18 types of coexisting conformational substates of the β-strand of the 8-37 segment of human islet amyloid polypeptide (hIAPP 8-37). We analyzed the pairwise peptide–peptide interactions in the hIAPP 8-37 assembly and found 82 interconformation interactions within a free energy difference of 3.40 kBT. Besides hIAPP 8-37, this STM method validates the existence of multiple conformations of other β-sheet peptide assemblies, including mutated hIAPP 8-37 and amyloid-β 42. Overall, the results reported in this work provide single-molecule experimental insights into the conformational ensemble and interpeptide interactions in the β-sheet peptide assembly.

Cell entry by SARS-CoV-2 is accomplished by the S2 subunit of the spike S protein on the virion surface by capture of the host cell membrane and fusion with the viral envelope. Capture and fusion require the prefusion S2 to transit to its potent fusogenic form, the fusion intermediate (FI). However, the FI structure is unknown, detailed computational models of the FI are unavailable, and the mechanisms and timing of membrane capture and fusion are not established. Here, we constructed a full-length model of the SARS-CoV-2 FI by extrapolating from known SARS-CoV-2 pre- and postfusion structures. In atomistic and coarse-grained molecular dynamics simulations the FI was remarkably flexible and executed giant bending and extensional fluctuations due to three hinges in the C-terminal base. The simulated configurations and their giant fluctuations are quantitatively consistent with SARS-CoV-2 FI configurations measured recently using cryo-electron tomography. Simulations suggested a host cell membrane capture time of ∼2 ms. Isolated fusion peptide simulations identified an N-terminal helix that directed and maintained binding to the membrane but grossly underestimated the binding time, showing that the fusion peptide environment is radically altered when attached to its host fusion protein. The large configurational fluctuations of the FI generated a substantial exploration volume that aided capture of the target membrane, and may set the waiting time for fluctuation-triggered refolding of the FI that draws the viral envelope and host cell membrane together for fusion. These results describe the FI as machinery that uses massive configurational fluctuations for efficient membrane capture and suggest novel potential drug targets.