Electrochemical borylation of carboxylic acids

A simple electrochemically mediated method for the conversion of alkyl carboxylic acids to their borylated congeners is presented. This protocol features an undivided cell setup with inexpensive carbon-based electrodes and exhibits a broad substrate scope and scalability in both flow and batch reactors. The use of this method in challenging contexts is exemplified with a modular formal synthesis of jawsamycin, a natural product harboring five cyclopropane rings.

Boronic acids are among the most malleable functional groups in organic chemistry as they can be converted into almost any other functionality (1–3). Aside from these versatile interconversions, their use in the pharmaceutical industry is gaining traction, resulting in approved drugs such as Velcade, Ninlaro, and Vabomere (4). It has been shown that boronic acids can be rapidly installed from simple alkyl halides (5–19) or alkyl carboxylic acids through the intermediacy of redox-active esters (RAEs) (Fig. 1A) (20–24). Our laboratory has shown that both Ni (20) and Cu (21) can facilitate this reaction. Conversely, Aggarwal and coworkers (22) and Li and coworkers (23) demonstrated photochemical variations of the same transformation. While these state-of-the-art approaches provide complementary access to alkyl boronic acids, each one poses certain challenges. For example, the requirement of excess boron source and pyrophoric MeLi under Ni catalysis is not ideal. Although more cost-effective and operationally simple, Cu-catalyzed borylation conditions can be challenging on scale due to the heterogeneity resulting from the large excess of LiOH•H₂O required. In addition to its limited scope, Li and coworkers’ protocol requires 4 equivalence of B₂pin₂ and an expensive Ir photocatalyst. The simplicity of Aggarwal and coworkers’ approach is appealing in this regard and represents an important precedent for the current study.Open in a separate windowFig. 1.(A) Prior approaches to access alkyl boronic esters from activated acids. (B) Inspiration for initiating SET events electrochemically to achieve borylation. (C) Summary of this work.At the heart of each method described above, the underlying mechanism relies on a single electron transfer (SET) event to promote decarboxylation and form an alkyl radical species. In parallel, the related borylation of aryl halides via a highly reactive aryl radical can also be promoted by SET. While numerous methods have demonstrated that light can trigger this mechanism (Fig. 1B) (16, 25–31), simple electrochemical cathodic reduction can elicit the same outcome (32–35). It was postulated that similar electrochemically driven reactivity could be translated to alkyl RAEs. The development of such a transformation would be highly enabling, as synthetic organic electrochemistry allows the direct addition or removal of electrons to a reaction, representing an incredibly efficient way to forge new bonds (36–40). This disclosure reports a mild, scalable, and operationally simple electrochemical decarboxylative borylation (Fig. 1C) not reliant on transition metals or stoichiometric reductants. In addition to mechanistic studies of this interesting transformation, applications to a variety of alkyl RAEs, comparison to known decarboxylative borylation methods, and a formal synthesis of the polycyclopropane natural product jawsamycin [(–)-FR-900848] are presented.     

Tuning a riboswitch response through structural extension of a pseudoknot

Structural and dynamic features of RNA folding landscapes represent critical aspects of RNA function in the cell and are particularly central to riboswitch-mediated control of gene expression. Here, using single-molecule fluorescence energy transfer imaging, we explore the folding dynamics of the preQ₁ class II riboswitch, an upstream mRNA element that regulates downstream encoded modification enzymes of queuosine biosynthesis. For reasons that are not presently understood, the classical pseudoknot fold of this system harbors an extra stem–loop structure within its 3′-terminal region immediately upstream of the Shine–Dalgarno sequence that contributes to formation of the ligand-bound state. By imaging ligand-dependent preQ₁ riboswitch folding from multiple structural perspectives, we reveal that the extra stem–loop strongly influences pseudoknot dynamics in a manner that decreases its propensity to spontaneously fold and increases its responsiveness to ligand binding. We conclude that the extra stem–loop sensitizes this RNA to broaden the dynamic range of the ON/OFF regulatory switch.A variety of small metabolites have been found to regulate gene expression in bacteria, fungi, and plants via direct interactions with distinct mRNA folds (1–4). In this form of regulation, the target mRNA typically undergoes a structural change in response to metabolite binding (5–9). These mRNA elements have thus been termed “riboswitches” and generally include both a metabolite-sensitive aptamer subdomain and an expression platform. For riboswitches that regulate the process of translation, the expression platform minimally consists of a ribosomal recognition site [Shine–Dalgarno (SD)]. In the simplest form, the SD sequence overlaps with the metabolite-sensitive aptamer domain at its downstream end. Representative examples include the S-adenosylmethionine class II (SAM-II) (10) and the S-adenosylhomocysteine (SAH) riboswitches (11, 12), as well as prequeuosine class I (preQ₁-I) and II (preQ₁-II) riboswitches (13, 14). The secondary structures of these four short RNA families contain a pseudoknot fold that is central to their gene regulation capacity. Although the SAM-II and preQ₁-I riboswitches fold into classical pseudoknots (15, 16), the conformations of the SAH (17) and preQ₁-II counterparts are more complex and include a structural extension that contributes to the pseudoknot architecture (14). Importantly, the impact and evolutionary significance of these “extra” stem–loop elements on the function of the SAH and preQ₁-II riboswitches remain unclear.PreQ₁ riboswitches interact with the bacterial metabolite 7-aminomethyl-7-deazaguanine (preQ₁), a precursor molecule in the biosynthetic pathway of queuosine, a modified base encountered at the wobble position of some transfer RNAs (14). The general biological significance of studying the preQ₁-II system stems from the fact that this gene-regulatory element is found almost exclusively in the Streptococcaceae bacterial family. Moreover, the preQ₁ metabolite is not generated in humans and has to be acquired from the environment (14). Correspondingly, the preQ₁-II riboswitch represents a putative target for antibiotic intervention. Although preQ₁ class I (preQ₁-I) riboswitches have been extensively investigated (18–28), preQ₁ class II (preQ₁-II) riboswitches have been largely overlooked despite the fact that a different mode of ligand binding has been postulated (14).The consensus sequence and the secondary structure model for the preQ₁-II motif (COG4708 RNA) (Fig. 1A) comprise ∼80–100 nt (14). The minimal Streptococcus pneumoniae R6 aptamer domain sequence binds preQ₁ with submicromolar affinity and consists of an RNA segment forming two stem–loops, P2 and P4, and a pseudoknot P3 (Fig. 1B). In-line probing studies suggest that the putative SD box (AGGAGA; Fig. 1) is sequestered by pseudoknot formation, which results in translational-dependent gene regulation of the downstream gene (14).Open in a separate windowFig. 1.PreQ₁ class II riboswitch. (A) Chemical structure of 7-aminomethyl-7-deazaguanosine (preQ₁); consensus sequence and secondary structure model for the COG4708 RNA motif (adapted from reference 14). Nucleoside presence and identity as indicated. (B) S. pneumoniae R6 preQ₁-II RNA aptamer investigated in this study. (C) Schematics of an H-type pseudoknot with generally used nomenclature for comparison.Here, we investigated folding and ligand recognition of the S. pneumoniae R6 preQ₁-II riboswitch, using complementary chemical, biochemical, and biophysical methods including selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE), mutational analysis experiments, 2-aminopurine fluorescence, and single-molecule fluorescence resonance energy transfer (smFRET) imaging. In so doing, we explored the structural and functional impact of the additional stem–loop element in the context of its otherwise “classical” H-type pseudoknot fold (29–32) (Fig. 1C). Our results reveal that the unique 3′-stem–loop element in the preQ₁-II riboswitch contributes to the process of SD sequestration, and thus the regulation of gene expression, by modulating both its intrinsic dynamics and its responsiveness to ligand binding.     

El Ni?o?Southern Oscillation frequency cascade

The El Niño−Southern Oscillation (ENSO) phenomenon, the most pronounced feature of internally generated climate variability, occurs on interannual timescales and impacts the global climate system through an interaction with the annual cycle. The tight coupling between ENSO and the annual cycle is particularly pronounced over the tropical Western Pacific. Here we show that this nonlinear interaction results in a frequency cascade in the atmospheric circulation, which is characterized by deterministic high-frequency variability on near-annual and subannual timescales. Through climate model experiments and observational analysis, it is documented that a substantial fraction of the anomalous Northwest Pacific anticyclone variability, which is the main atmospheric link between ENSO and the East Asian Monsoon system, can be explained by these interactions and is thus deterministic and potentially predictable.The El Niño−Southern Oscillation (ENSO) phenomenon is a coupled air−sea mode, and its irregular occurring extreme phases El Niño and La Niña alternate on timescales of several years (1–8). The global atmospheric response to the corresponding eastern tropical Pacific sea surface temperature (SST) anomalies (SSTA) causes large disruptions in weather, ecosystems, and human society (3, 5, 9).One of the main properties of ENSO is its synchronization with the annual cycle: El Niño events tend to grow during boreal summer and fall and terminate quite rapidly in late boreal winter (9–18). The underlying dynamics of this seasonal pacemaking can be understood in terms of the El Niño/annual cycle combination mode (C-mode) concept (19), which interprets the Western Pacific wind response during the growth and termination phase of El Niño events as a seasonally modulated interannual phenomenon. This response includes a weakening of the equatorial wind anomalies, which causes the rapid termination of El Niño events after boreal winter and thus contributes to the seasonal synchronization of ENSO (17). Mathematically, the modulation corresponds to a product between the interannual ENSO phenomenon (ENSO frequency: f_E) and the annual cycle (annual frequency: 1 y^-1), which generates near-annual frequencies at periods of  ～  10 mo (1 + f_E) and  ～  15 mo (1 − f_E) (19).In nature, a wide variety of nonlinear processes exist in the climate system. Atmospheric examples include convection and low-level moisture advection (19). An example for a quadratic nonlinearity is the dissipation of momentum in the planetary boundary layer, which includes a product between ENSO (E) and the annual cycle (A) due to the windspeed nonlinearity: v_E⋅ v_A (17, 19). In the frequency domain, this product results in the near-annual sum (1 + f_E) and difference (1 − f_E) tones (19). The commonly used Niño 3.4 (N3.4) SSTA index (details in SI Appendix, SI Materials and Methods) exhibits most power at interannual frequencies (Fig. 1A). In contrast, the near-annual combination tones (1 ± f_E) are the defining characteristic of the C-mode (Fig. 1B).Open in a separate windowFig. 1.Schematic for the ENSO (E) and combination mode (ExA) anomalous surface circulation pattern and corresponding spectral characteristics. (A) Power spectral density for the normalized N3.4 index of the Hadley Centre Sea Ice and Sea Surface Temperature data set version 1 (HadISST1) 1958–2013 SSTA using the Welch method. (B) As in A but for the theoretical quadratic combination mode (ExA). (C) Regression coefficient of the normalized N3.4 index and the anomalous JRA-55 surface stream function for the same period (ENSO response pattern). (D) Regression coefficient of the normalized combination mode (ExA) index and the anomalous JRA-55 surface stream function (combination mode response pattern). Areas where the anomalous circulation regression coefficient is significant above the 95% confidence level are nonstippled.Physically, the dominant near-annual combination mode comprises a meridionally antisymmetric circulation pattern (Fig. 1D). It features a strong cyclonic circulation in the South Pacific Convergence Zone, with a much weaker counterpart cyclone in the Northern Hemisphere Central Pacific. The most pronounced feature of the C-mode circulation pattern is the anomalous low-level Northwest Pacific anticyclone (NWP-AC). This important large-scale atmospheric feature links ENSO impacts to the Asian Monsoon systems (20–25) by shifting rainfall patterns (SI Appendix, Fig. S1B), and it drives sea level changes in the tropical Western Pacific that impact coastal systems (26). It has been demonstrated using spectral analysis methods and numerical model experiments that the C-mode is predominantly caused by nonlinear atmospheric interactions between ENSO and the warm pool annual cycle (19, 20). Local and remote thermodynamic air−sea coupling amplify the signal but are not the main drivers for the phase transition of the C-mode and its associated local phenomena (e.g., the NWP-AC) (20).Even though ENSO and the C-mode are not independent, their patterns and spectral characteristics are fundamentally different, which has important implications when assessing the amplitude and timing of their regional climate impacts (Fig. 1). Here we set out to study the role of nonlinear interactions between ENSO and the annual cycle (10) in the context of C-mode dynamics. Such nonlinearities can, in principle, generate a suite of higher-order combination modes, which would contribute to the high-frequency variability of the atmosphere—in a deterministic and predictable way.     

Chlorination of arenes via the degradation of toxic chlorophenols

Aryl chlorides are among the most versatile synthetic precursors, and yet inexpensive and benign chlorination techniques to produce them are underdeveloped. We propose a process to generate aryl chlorides by chloro-group transfer from chlorophenol pollutants to arenes during their mineralization, catalyzed by Cu(NO₃)₂/NaNO₃ under aerobic conditions. A wide range of arene substrates have been chlorinated using this process. Mechanistic studies show that the Cu catalyst acts in cooperation with NO_x species generated from the decomposition of NaNO₃ to regulate the formation of chlorine radicals that mediate the chlorination of arenes together with the mineralization of chlorophenol. The selective formation of aryl chlorides with the concomitant degradation of toxic chlorophenol pollutants represents a new approach in environmental pollutant detoxication. A reduction in the use of traditional chlorination reagents provides another (indirect) benefit of this procedure.

Chlorophenols are widely encountered moieties present in herbicides, drugs, and pesticides (1). Owing to the high dissociation energies of carbon‒chloride bonds, chlorophenols biodegrade very slowly, and their prolonged exposure leads to severe ecological and environmental problems (Fig. 1A) (2–4). Several well-established technologies have been developed for the treating of chlorophenols, including catalytic oxidation (5–11), biodegradation (12–15), solvent extraction (16, 17), and adsorption (18–20) Among these methods, adsorption is the most versatile and widely used method due to its high removal efficiency and simple operation, but the resulting products are of no value, and consequently, these processes are not viable.Open in a separate windowFig. 1.Background and reaction design. (A) Examples of chlorophenol pollutants. (B) Examples of aryl chlorides. (C) The chlorination process reported herein was based on chloro-group transfer from chlorophenol pollutants.With the extensive application of substitution reactions (21, 22), transfunctionalizations (23, 24), and cross-coupling reactions (25, 26), aryl chlorides are regarded as one of the most important building blocks widely used in the manufacture of polymers, pharmaceuticals, and other types of chemicals and materials (Fig. 1B) (27–31). Chlorination of arenes is usually carried out with toxic and corrosive reagents (32–34). Less toxic and more selective chlorination reagents tend to be expensive [e.g., chloroamides (35, 36)] and are not atom economic (37–39). Consequently, from the perspective of sustainability, the ability to transfer a chloro group from unwanted chlorophenols to other substrates would be advantageous.Catalytic isofunctional reactions, including transfer hydrogenation and alkene metathesis, have been widely exploited in organic synthesis. We hypothesized that chlorination of arenes also could be achieved by chloro-group transfer, and since stockpiles of chlorophenols tend to be destroyed by mineralization and chlorophenol pollutants may be concentrated by adsorption (18–20), they could be valorized as chlorination reagents via transfer of the chloro group to arene substrates during their mineralization, thereby adding value to the destruction process (Fig. 1C). Although chlorophenol pollutants are not benign, their application as chlorination reagents, with their concomitant destruction to harmless compounds, may be considered as not only meeting the criteria of green chemistry but also potentially surpassing it. Herein, we describe a robust strategy to realize chloro-group transfer from chlorophenol pollutants to arenes and afford a wide range of value-added aryl chlorides.     

Volatile-consuming reactions fracture rocks and self-accelerate fluid flow in the lithosphere

Hydration and carbonation reactions within the Earth cause an increase in solid volume by up to several tens of vol%, which can induce stress and rock fracture. Observations of naturally hydrated and carbonated peridotite suggest that permeability and fluid flow are enhanced by reaction-induced fracturing. However, permeability enhancement during solid-volume–increasing reactions has not been achieved in the laboratory, and the mechanisms of reaction-accelerated fluid flow remain largely unknown. Here, we present experimental evidence of significant permeability enhancement by volume-increasing reactions under confining pressure. The hydromechanical behavior of hydration of sintered periclase [MgO + H₂O → Mg(OH)₂] depends mainly on the initial pore-fluid connectivity. Permeability increased by three orders of magnitude for low-connectivity samples, whereas it decreased by two orders of magnitude for high-connectivity samples. Permeability enhancement was caused by hierarchical fracturing of the reacting materials, whereas a decrease was associated with homogeneous pore clogging by the reaction products. These behaviors suggest that the fluid flow rate, relative to reaction rate, is the main control on hydromechanical evolution during volume-increasing reactions. We suggest that an extremely high reaction rate and low pore-fluid connectivity lead to local stress perturbations and are essential for reaction-induced fracturing and accelerated fluid flow during hydration/carbonation.

Hydration and carbonation reactions in the crust and mantle transport H₂O and CO₂ from Earth’s surface to the interior and control volatile budgets within the Earth (1–6). These reactions are characterized by solid-volume increase, by up to several tens of vol%, which induces stress that may lead to fracturing (7–10). The driving force of such stress generation is the thermodynamic free energy released when metastable anhydrous/noncarbonate minerals react with fluids (7). The stress generated by the reaction has the potential to cause rock fracture and fragmentation (7, 11–13), thereby increasing the reactive surface area and fluid flow and further accelerating the reactions (7, 8, 14). Such chemical breaking of rocks, or reaction-induced fracturing, appears to be important in driving hydration and carbonation reactions to completion (8, 15, 16) in an otherwise self-limiting process where reaction products can clog pores and suppress fluid flow, thereby hindering the reaction (15, 17).Observations of naturally serpentinized and fractured ultramafic rocks indicate a volume increase of 20 to 60% during hydration reactions (13, 18–20), providing evidence of an accelerated supply of fluids during hydration (Fig. 1 A and B). Natural carbonation of ultramafic rocks is also associated with extensive fracture networks, and reaction-induced fracturing is considered a key process in mineral carbonation (Fig. 1C) (7, 8, 21). Numerical simulations indicate a positive feedback between volume-increasing reaction, fracturing, and fluid flow (10, 22–32). Laboratory experiments partially reproduce fracturing during peridotite carbonation, serpentinization, and periclase hydration (29, 33–36); however, hydrothermal flow-through experiments of peridotite serpentinization and carbonation show a decrease in permeability and deceleration of fluid flow and reaction rate (37–42). Observations of the natural carbonation of serpentinized peridotite indicate the decrease in permeability and reduced fluid flow and reaction rate are a consequence of pore clogging related to carbonation (43). Until now, no experimental studies have shown a clear increase in permeability during expansive fluid–rock reactions under confining pressure. As such, despite their geological and environmental importance, the evolution of expansive fluid–rock reactions remains difficult to predict, owing to the complex hydraulic–chemical–mechanical feedbacks underlying these reactions (15, 16, 44). The processes controlling the self-acceleration or deceleration of these reactions remain largely unknown.Open in a separate windowFig. 1.Reaction-induced fractures related to natural hydration/carbonation. (A) Polygonal block of serpentinite cut by planar lizardite veins, extracted from a serpentinite body, San Andreas Lake, California. (B) Photomicrograph of mesh structure in partly serpentinized peridotite, Redwood City serpentinite, California [crossed-polarized light (61)]. (C) Quartz veins in silica–carbonate rocks (i.e., listvenite, a carbonated ultramafic rock) that occur along the boundaries of serpentinite bodies, San Jose, California. ol, olivine; serp, serpentine (lizardite ± antigorite mixture); br, brucite.Here, we use the hydration of periclase to brucite [MgO + H₂O → Mg(OH)₂] as an analog for solid-volume–increasing reactions in the Earth. This reaction produces an extreme solid-volume increase of 119%, with a high reaction rate at 100 to 600 °C (45). Previous experimental studies on periclase hydration have revealed that extensive fracturing occurs under certain conditions (29, 33, 35), yet the links between fracturing experiments (periclase hydration), nonfracturing experiments (peridotite hydration/carbonation), and natural observations are unknown. On the basis of in situ observations of fluid flow during the reactions, we clearly show that fluid flow and associated permeability are strongly enhanced by solid-volume–increasing reactions under confining pressure (i.e., at simulated depth). Based on the experimental results and nondimensional parameterization, we propose that the ratio of the initial fluid flow rate to the reaction rate has a primary control on the self-acceleration and deceleration of fluid flow and reactions during hydration and carbonation within the Earth.     

A nitroaromatic cathode with an ultrahigh energy density based on six-electron reaction per nitro group for lithium batteries

Organic electrode materials have emerged as promising alternatives to conventional inorganic materials because of their structural diversity and environmental friendliness feature. However, their low energy densities, limited by the single-electron reaction per active group, have plagued the practical applications. Here, we report a nitroaromatic cathode that performs a six-electron reaction per nitro group, drastically improving the specific capacity and energy density compared with the organic electrodes based on single-electron reactions. Based on such a reaction mechanism, the organic cathode of 1,5-dinitronaphthalene demonstrates an ultrahigh specific capacity of 1,338 mAh⋅g⁻¹ and energy density of 3,273 Wh⋅kg⁻¹, which surpass all existing organic cathodes. The reaction path was verified as a conversion from nitro to amino groups. Our findings open up a pathway, in terms of battery chemistry, for ultrahigh-energy-density Li-organic batteries.

With the rapid development of social economy, the demand for high-energy-density storage systems is ever increasing, particularly in the fields of military, aerospace, medical, and civilian applications. Lithium-ion batteries (LIBs) have been extensively explored as high-energy-density storage devices (1–4). Commercial LIBs use crystalline transition metal oxide cathodes, such as LiCoO₂, LiMnO₂, and LiNi_xMn_yCo_1-x-yO₂ (5–12). They store energy via insertion/extraction of Li ions, which highly depends on the crystal structure and limits their capacities and energy densities (<300 mAh⋅g⁻¹ with energy density of <1,000 Wh⋅kg⁻¹). Some new battery systems with conversion reaction cathodes, such as Li-S (2,600 Wh⋅kg⁻¹) and Li-O₂ (3,450 Wh⋅kg⁻¹) batteries (13–19), have been extensively investigated due to their high theoretical energy densities. However, they suffer from serious dissolution, interfacial stability, and/or side reaction issues, and the practical energy densities are much lower than their theoretical values.Compared with the aforementioned rechargeable batteries, lithium primary batteries can provide remarkably higher energy densities. For example, lithium-fluorinated carbon (Li-CF_x) batteries use a CF_x cathode that has a high theoretical energy density of 2,180 Wh⋅kg⁻¹, which is the highest value among all commercial cathode materials in lithium batteries (20–22). Recently, it is reported that, different from the formation of crystal LiF in liquid electrolytes, amorphous LiF was produced and uniformly distributed on the carbon matrix when a solid-state electrolyte was used (23). This may enable a reversible electrochemical reactivity of CF_x with lithium and provide a possibility for high-energy-density batteries. Nevertheless, CF_x must be synthesized in harsh conditions with precise control, leading to high cost and hindering the wide application (20). Some liquid and gas cathodes, such as SO₂, SOCl₂, and SF₆ (24–27), can also offer high energy densities, but they tend to be volatile and induce severe safety hazards, especially on the occasion of thermal runaway. Therefore, it is highly desired to develop feasible and reliable battery systems with high energy density.Organic electrode materials offer many merits compared with inorganic electrode materials, including structure diversity and designability, abundance of raw materials, relatively easy synthesis, and environmental friendliness (28–35). Their energy storage processes rely on the uptake of cations or anions on the active groups, such as carbonyl group, quaternary nitrogen, and nitroxyl radical (36–40). Most of them undergo one electron reaction, leading to limited specific capacity and energy density (Fig. 1). For example, one carbonyl group (C=O) can be converted into C-OLi structure via accepting one electron and one lithium ion (36), providing a theoretical specific capacity of 957 mAh⋅g⁻¹ (based on the mass of functional group as marked by dashed circle in Fig. 1). If considering the inactive components, insulating property, and high solubility of organic molecules, most organic electrode materials exhibit inferior specific capacities. Although a high specific capacity of 902 mAh⋅g⁻¹ was gained for cyclohexanehexone in LIBs, its high solubility induces significant challenges in practical applications (41). Recently, a Li-containing organic compound, dilithium 1,4-phenylenebis ((methylsulfonyl) amide) (Li₂-p-PDSA) was synthesized, offering a new design for organic electrode materials, while it still follows the single-electron reaction path per electroactive group and shows a low specific capacity of 194 mAh⋅g⁻¹ (42). Therefore, new battery system needs to be developed to take full advantage of organic electrode materials for high-energy-density LIBs.Open in a separate windowFig. 1.Reaction mechanisms of organic groups in batteries.In this work, we report a battery chemistry of six-electron reduction on nitro group (−NO₂) of nitroaromatic cathode. Based on such a reaction, 1,5-dinitronaphthalene (1,5-DNN; Fig. 2A) as a cathode in LIBs achieves an ultrahigh specific capacity of 1,338 mAh⋅g⁻¹ and energy density of 3,273 Wh⋅kg⁻¹. This is a record for organic electrode materials, even higher than inorganic electrode materials. It is identified that nitro groups are reduced to amino groups through a six-electron transfer reaction via multiple characterization techniques. Our findings provide a path for achieving high-energy-density Li-organic batteries.Open in a separate windowFig. 2.Electrochemical performance of 1,5-DNN. (A) Chemical structure of 1,5-DNN. (B and C) Galvanostatic charge/discharge profiles of 1,5-DNN. (D) Performance comparison of 1,5-DNN with reported organic/inorganic electrode materials in terms of energy density, specific capacity and voltage. Cathodes for comparison (references): 1, p-DNB (43); 2, Li₂C₆O₆ (49); 3, P14AQ (50); 4, PTO (51); 5, 3Q (31); 6, AQ (44); 7, C₆O₆ (41); 8, o-DNB (43); 9, m-DNB (43); 10, 4,5-PhenQ (51); 11, C4Q (52); 12, LiCoO₂ (53); 13, LiFePO₄ (54); 14, NCA (55); 15, NCM-811 (56); 16, Li-rich (57); 17, CF_x (21); 18, MnO₂ (58); 19 SOCl₂ (59), 20 SO₂ (24); 21, S (60).     

Phase transition-induced band edge engineering of BiVO4 to split pure water under visible light

Through phase transition-induced band edge engineering by dual doping with In and Mo, a new greenish BiVO₄ (Bi_1-XIn_XV_1-XMo_XO₄) is developed that has a larger band gap energy than the usual yellow scheelite monoclinic BiVO₄ as well as a higher (more negative) conduction band than H⁺/H₂ potential [0 V_RHE (reversible hydrogen electrode) at pH 7]. Hence, it can extract H₂ from pure water by visible light-driven overall water splitting without using any sacrificial reagents. The density functional theory calculation indicates that In³⁺/Mo⁶⁺ dual doping triggers partial phase transformation from pure monoclinic BiVO₄ to a mixture of monoclinic BiVO₄ and tetragonal BiVO₄, which sequentially leads to unit cell volume growth, compressive lattice strain increase, conduction band edge uplift, and band gap widening.Photocatalytic water splitting with a particulate semiconductor powered by sunlight is an ideal route to cost-effective, large-scale, and sustainable hydrogen production because of its extreme simplicity. However, it is challenging, because it requires a rare photocatalyst that carries a combination of suitable band gap energy, appropriate band positions, and photochemical stability (1–5). Thus, reproducible photocatalytic systems for visible light-driven overall water splitting (OWS) by one-step photoexcitation are also rare, although there were several reports of such systems (4–6). In the best-known successful case, Domen and coworkers (5) reported in 2005 that a solid solution of GaN and ZnO [(Ga_1−xZn_x)(N_1−xO_x)] was a stable photocatalyst that could split water into H₂ and O₂ under visible light when modified with a cocatalyst. This system remains the most active and reproducible one-step OWS photocatalyst responsive to visible light so far (4).Scheelite monoclinic (m-) BiVO₄ is a well-documented photocatalyst having suitable band gap energy (E_g ∼ 2.4 eV) for absorbing visible light (7–10). Also, it is chemically stable in aqueous solution under light irradiation. Thus, it functions as an excellent photocatalyst for O₂ evolution under visible light in the presence of an appropriate electron acceptor (e.g., AgNO₃). However, because the bottom of its conduction band is located at a more positive potential than the potential of water reduction [0 V_RHE (reversible hydrogen electrode) at pH 7], it is incapable of evolving H₂. In addition, it shows poor charge transport characteristics (11) and weak surface adsorption properties (12), causing low photocatalytic activity. To overcome these weaknesses, a variety of strategies, such as heterojunction structure formation (11, 13, 14), loading cocatalysts (8, 15–17), and impurity doping (1, 7, 12, 18–23), has been attempted. These strategies were successful in improving BiVO₄’s oxidation capability for photoelectrochemical water oxidation (1, 11, 13, 15, 19, 24–28) as well as the Z scheme (two-photon excitation) water splitting system (29). Also, cocatalyzed Bi_xY_1-xVO₄ (x ∼ 0.5) (7, 8) and cocatalyzed Bi_0.5La_0.5VO₄ (23) promoted OWS by raising the conduction band edge (CBE) position, but OWS under visible light irradiation over BiVO₄-based photocatalysts has not been fully shown.To meet this challenge, we developed greenish BiVO₄ (GBVO_x; x = atom ratio of In and Mo), Bi_1-xIn_xV_1-xMo_xO₄, by simultaneously substituting In³⁺ for Bi³⁺ and Mo⁶⁺ for V⁵⁺ in the host lattice of m-BiVO₄. The new GBVO_x photocatalyst has a slightly larger band gap energy than the usual yellow scheelite m-BiVO₄ as supported by the unique color change to green and a higher (more negative) conduction band than H⁺/H₂ potential (0 V_RHE at pH 7). Consequently, as depicted in Fig. 1, GBVO_x is able to split water into H₂ and O₂ under visible light irradiation without using any sacrificial reagents (e.g., CH₃OH or AgNO₃). Herein, we report the dual-metal doping effects on the optical absorption behavior, crystal structure, and electronic band structure of BiVO₄, which led to one-photon OWS under visible light irradiation. We elucidate the physical origin of the augmented photoresponse behaviors of GBVO_x through density functional theory (DFT) calculation of electronic structure as well as a variety of physical and electrochemical characterizations.Open in a separate windowFig. 1.OWS reaction mechanism by GBVO_x.     

Partial synthetic models of FeMoco with sulfide and carbyne ligands: Effect of interstitial atom in nitrogenase active site

Nitrogen-fixing organisms perform dinitrogen reduction to ammonia at an Fe-M (M = Mo, Fe, or V) cofactor (FeMco) of nitrogenase. FeMco displays eight metal centers bridged by sulfides and a carbide having the MFe₇S₈C cluster composition. The role of the carbide ligand, a unique motif in protein active sites, remains poorly understood. Toward addressing how the carbon bridge affects the physical and chemical properties of the cluster, we isolated synthetic models of subsite MFe₃S₃C displaying sulfides and a chelating carbyne ligand. We developed synthetic protocols for structurally related clusters, [Tp*M’Fe₃S₃X]ⁿ⁻, where M’ = Mo or W, the bridging ligand X = CR, N, NR, S, and Tp* = Tris(3,5-dimethyl-1-pyrazolyl)hydroborate, to study the effects of the identity of the heterometal and the bridging X group on structure and electrochemistry. While the nature of M’ results in minor changes, the chelating, μ₃-bridging carbyne has a large impact on reduction potentials, being up to 1 V more reducing compared to nonchelating N and S analogs.

Biological dinitrogen conversion to ammonia is performed by nitrogenases, a class of enzymes displaying several complex iron-sulfur clusters (1). The site of N₂ reduction in the most efficient nitrogenase is a heterometallic cluster displaying Fe and Mo, the iron-molybdenum cofactor (FeMoco) (1). Two other nitrogenases are known where Fe or V are found at the Mo position. FeMoco consists of Fe₄S₃C and MoFe₃S₃C cubanes with μ₃-sulfides joined together by a shared interstitial μ₆-carbide and three additional sulfides that bind in μ₂-fashion (Fig. 1) (2). The impact of the carbide ligand on the electronic structure and reactivity of the cofactor, and therefore its role in the catalytic cycle of N₂-to-NH₃ conversion, is unclear (3). The carbide ligand is not lost during catalysis, and it has been suggested that it becomes protonated before N₂ activation (3). To address the effect of carbon-based ligands for N₂ activation, such as providing electronic stabilization and structural flexibility to accommodate multielectron redox processes, synthetic models have included arene (4), N-heterocyclic carbene (5), aryl (6), and alkyl (7, 8) donors in mononuclear iron complexes.Open in a separate windowFig. 1.(Top) Structure of FeMoco in Mo-dependent nitrogenase from the Protein Data Bank structure 3U7Q with a blue circle emphasizing the cubane subsite and its schematic representation highlighting in color the subsite of focus in this study. (Bottom) Carbyne and carbide-containing model complexes (9–11).Bi- and multimetallic synthetic analogs focused on interrogating the role of the interstitial atom and multimetallic effects have been targeted (7, 9–23), but complexes that display bridging carbide (11, 24–27) or even carbyne (9, 10, 28) ligands are rare. Carbide-containing Fe clusters display four to six metal centers but invariably are rich in CO ligands (24, 25, 27). The presence of this strong field donor limits the comparison to FeMoco given the significantly different electronic structure conferred by the weak field sulfides. Moreover, the formal oxidation state of the Fe centers is significantly more reduced, between Fe⁰ and Fe^II, than in the protein, between Fe^II and Fe^III (2). Recent promising advances have been made toward the incorporation of sulfide ligands into carbide-containing iron carbonyl clusters (10, 11). In order to gain a more accurate understanding of the impact of the carbide on the properties of clusters related to FeMoco, metal complexes structurally related to the biological active site that are multimetallic, have multiple sulfide ligands and few CO ligands, and display bridging carbon-based ligands and oxidation states of Fe^II-Fe^III are desirable.Toward developing synthetic methodologies to structures analogous to FeMoco that include a bridging carbon donor, we focus our initial efforts on the cubane subsite, MoFe₃S₃C (Fig. 1, Top Row). Because the nature of the μ₂-bridging ligands in FeMoco is variable, with sulfide, selenide (29), CO (30), or NH (31) (for FeVco) moieties at these positions as characterized by crystallography, the primary target was to match the composition of the cubane core. In this work, we present the preparation of a series of heterometallic iron-sulfur cubane-type clusters containing Mo or W with biologically relevant μ₃ bridging ligands X (X = N, NR, CR, and S) incorporated at the Fe₃ face—including examples bearing a bridging CR ligand. These variations in the bridging ligand result in a large shift in the biologically relevant M′Fe₃¹¹⁺/M′Fe₃¹⁰⁺ redox couple of up to 2 V, with the most reducing system occurring for the cluster bearing a bridging carbyne. These results suggest an important role of the interstitial carbide ligand in FeMoco in modulating the electronic properties of the cluster toward rendering it more reducing and potentially more reactive in N₂ activation and conversion into NH₃.     

Visualizing molecular weights differences in supramolecular polymers

Issues of molecular weight determination have been central to the development of supramolecular polymer chemistry. Whereas relationships between concentration and optical features are established for well-behaved absorptive and emissive species, for most supramolecular polymeric systems no simple correlation exists between optical performance and number-average molecular weight (M_n). As such, the M_n of supramolecular polymers have to be inferred from various measurements. Herein, we report an anion-responsive supramolecular polymer [M1·Zn(OTf)₂]_n that exhibits monotonic changes in the fluorescence color as a function of M_n. Based on theoretical estimates, the calculated average degree of polymerization (DP_cal) increases from 16.9 to 84.5 as the monomer concentration increases from 0.08 mM to 2.00 mM. Meanwhile, the fluorescent colors of M1 + Zn(OTf)₂ solutions were found to pass from green to yellow and to orange, corresponding to a red shift in the maximum emission band (λ_max). Therefore, a relationship between DP_cal and λ_max could be established. Additionally, the anion-responsive nature of the present system meant that the extent of supramolecular polymerization could be regulated by introducing anions, with the resulting change in M_n being readily monitored via changes in the fluorescent emission features.

Issues of molecular weight determination have been central to the development of polymer chemistry and date back, in fact, to the earliest days of the field and the controversies Staudinger faced (1, 2). The number-average molecular weight (M_n) of polymeric materials has a direct effect on their mechanical properties (1–3). Typically, the higher the molecular weight of the polymer, the greater its hardness and strength, as exemplified by polyethylene (4, 5). Therefore, determining the molecular weight of a polymeric material is often a first key step in its characterization. Being able to do so simply could be particularly advantageous in the case of supramolecular polymers where the degree of polymerization, and hence molecular weight, can vary as a function of conditions. Supramolecular polymers, unlike conventional polymers, are polymeric arrays of repeat units that are held together by directional noncovalent interactions (6, 7). Given the reversible noncovalent bonds embodied in supramolecular polymers, they typically exhibit not only polymer-like properties as bulk materials and in solution, but also dynamic characteristics. These latter attributes often have no parallel in the case of covalent polymers and include inter alia reversible formation, recyclability, and stimulus responsiveness (8–26), including to anions whose role in the fields of biology, chemistry, energy, and supramolecular chemistry is well appreciated (27–42). These key features complicate molecular weight determinations, as does the fact that a distribution of molecular weights around an average is generally observed. Moreover, due to the dynamic nature of noncovalent bonds, the monomers used to create supramolecular polymers can assemble in a variety of aggregation modes (7, 39). For instance, when the monomer concentration is low, cyclic species or oligomers are more likely to form. However, at concentrations greater than the so-called critical polymer concentration (CPC), appropriately designed monomers will assemble to form supramolecular polymers, whose M_n typically grows with monomer concentration. As a consequence, the M_n of supramolecular polymers generally reflects the constituent monomer concentration(s).Whereas relationships between concentration and optical features are established for well-behaved absorptive and emissive species (e.g., monomeric compounds that obey the Beer–Lambert or Beer–Lambert–Bouguer laws), for most polymeric systems no simple correlation exists between easy-to-monitor optical properties, such as fluorescence emission color, and the molecular weight. As such, the molecular weights of supramolecular polymers have to be inferred from less-direct measurements (43–45). For example, the average degree of supramolecular polymerization can be calculated (DP_cal) by measuring the equilibrium constant of the interaction between monomers and applying various theoretical models (39, 46, 47). Alternatively, in certain cases the molecular weights of supramolecular polymers can be deduced empirically from the intrinsic viscosities using the Mark–Houwink equation (48, 49). Vapor pressure osmometry measurements, which rely on the relationship between molecular weight and vapor pressure (Raoult’s law), can be used to deduce the molar mass of supramolecular polymers (50, 51). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry has also been exploited to characterize oligomers and various relatively small self-assembled ensembles (52, 53). The molecular weights (M_n) of supramolecular polymers can also be inferred from concentration-dependent chemical shift changes in the corresponding ¹H NMR spectra (54). Separately, single-molecule force spectroscopy experiments have been used to determine the length of the bridges between the tip and substrate formed by supramolecular polymers, thus providing insights into the effective molecular weight of the polymer (55). These techniques notwithstanding, there remains a need for simple methods that would allow changes in the molecular weight of supramolecular polymers to be determined by optical means. Here, we report a system where a simple correlation is established between the fluorescence emission features and the M_n of a supramolecular polymer.The present approach is based on an appreciation that J-type dyes have been identified in a variety of aggregation modes (56–59), including monomer, dimer, and stacked arrangements. Dyes of this type, which include a number of polycyclic and heterocyclic aromatic molecules, typically display fluorescent colors that vary as a function of concentration. Therefore, we postulated that incorporating J-type dyes into appropriately chosen monomers would endow the resulting putative supramolecular polymers with variable fluorescent colors, the specifics of which could be correlated with the degree of polymerization (Fig. 1). With such thinking in mind, we synthesized monomer M1 by modifying a J-type dye, naphthalene diimide (NDI) (60, 61), with two alkylated terpyridine derivatives at both ends (Fig. 1). The NDI-derived M1 interacts with Zn(OTf)₂ in DMF/H₂O (1/4, vol/vol) solution through terpyridine–Zn²⁺ coordination. At low monomer concentrations, these two components were expected to self-assemble into cyclic species or short oligomers characterized by long distances between the NDI subunits. Thus, little stacking would be seen, resulting in a green, monomer-dominated fluorescence for dilute M1 + Zn(OTf)₂ solutions. As the total monomer concentration is increased with the ratio of M1 and Zn(OTf)₂ kept constant at 1:1, the cyclic species would be expected to transform gradually into linear supramolecular polymers, [M1·Zn(OTf)₂]_n. Under these conditions, a limited degree of interchain association (e.g., the formation of double-strand dimers) is expected, leading to a decrease in the distance between NDI groups and a change in the fluorescent color from green to yellow. Upon increasing the monomer concentration, a corresponding increase in the polymer chain length is expected, which will lead to tight entanglement between individual polymer chains, further reducing the average distance between the emissive NDI groups. In the limit, this reduction in inter-NDI spacing would lead to formation of J-aggregates, which in the case of the NDI-containing M1 component would give rise to orange-colored fluorescence. In accord with prior work involving supramolecular systems (27, 38), we also appreciated that anion recognition could be used to adjust the molecular weight of the supramolecular polymers formed from M1 and Zn(OTf)₂. Specifically, we expected that adding tetraethylammonium hydroxide (TBAOH) to [M1·Zn(OTf)₂]_n would lead to depolymerization as the result of complexation between the OH^– anions and Zn²⁺. This would lead to restoration of the initial emission color. As detailed below, these design expectations were realized. Specifically, the M_n of the supramolecular aggregates formed from M1 and Zn(OTf)₂ produced in dimethylformamide (DMF)/H₂O (1/4, vol/vol) solution could be distinguished by monitoring the fluorescent colors as a function of M1 + Zn(OTf)₂ and TBAOH concentration.Open in a separate windowFig. 1.Schematic representation of the anion-responsive supramolecular polymer of this study. Chemical structure of supramolecular monomers and cartoon representations of the proposed supramolecular polymerization process and anion-responsive behavior and the various fluorescent colors expected as the degree of intermonomer interaction increases.     

Electrophilic aromatic substitution reactions of compounds with Craig-Möbius aromaticity

Electrophilic aromatic substitution (EAS) reactions are widely regarded as characteristic reactions of aromatic species, but no comparable reaction has been reported for molecules with Craig-Möbius aromaticity. Here, we demonstrate successful EAS reactions of Craig-Möbius aromatics, osmapentalenes, and fused osmapentalenes. The highly reactive nature of osmapentalene makes it susceptible to electrophilic attack by halogens, thus osmapentalene, osmafuran-fused osmapentalene, and osmabenzene-fused osmapentalene can undergo typical EAS reactions. In addition, the selective formation of a series of halogen substituted metalla-aromatics via EAS reactions has revealed an unprecedented approach to otherwise elusive compounds such as the unsaturated cyclic chlorirenium ions. Density functional theory calculations were conducted to study the electronic effect on the regioselectivity of the EAS reactions.

Aromaticity, a core concept in chemistry, was initially introduced to account for the bonding, stability, reactivity, and other properties of many unsaturated organic compounds. There have been many elaborations and extensions of the concept of aromaticity (1, 2). The concepts of Hückel aromaticity and Möbius aromaticity are widely accepted (Fig. 1A). A π-aromatic molecule of the Hückel type is planar and has 4n + 2 conjugated π-electrons (n = 0 or an integer), whereas a Möbius aromatic molecule has one twist of the π-system, similar to that in a Möbius strip, and 4n π-electrons (3, 4). Since the discovery of naphthalene in 1821, aromatic chemistry has developed into a rich field and with a variety of subdisciplines over the course of its 200-y history, and the concept of aromaticity has been extended to other nontraditional structures with “cyclic delocalization of mobile electrons” (5). For example, benzene-like metallacycles—predicted by Hoffmann et al. as metallabenzenes—in which a metal replaces a C–H group in the benzene ring (6), have garnered extensive research interest from both experimentalists and theoreticians (7–12). As paradigms of the metalla-aromatic family, most complexes involving metallabenzene exhibit thermodynamic stability, kinetic persistence, and chemical reactivity associated with the classical aromaticity concept (13–15). Typically, like benzene, metallabenzene can undergo characteristic reactions of aromatics such as electrophilic aromatic substitution (EAS) reactions (16–18) (Fig. 1B, I) and nucleophilic aromatic substitution reactions (19–21).Open in a separate windowFig. 1.Schematic representations of aromaticity classification (A) and EAS reactions (B) of benzene, metallabenzene, and polycyclic metallacycles with Craig-Möbius aromaticity.The incorporation of transition metals has also led to an increase in the variety of the aromatic families (22–25). We have reported that stable and highly unusual bicyclic systems, metallapentalenes (osmapentalenes), benefit from Craig-Möbius aromaticity (26–30). In contrast to other reported Möbius aromatic compounds with twisted topologies, which are known as Heilbronner-Möbius aromatics (31–34), the involvement of transition metal d orbitals in π-conjugation switches the Hückel anti-aromaticity of pentalene into the planar Craig-Möbius aromaticity of metallapentalene (35–38) (Fig. 1A, III). Both the twisted topology and the planar Craig-Möbius aromaticity are well established and have been accepted as reasonable extensions of aromaticity (39–43). There has been no experimental evidence, however, as to whether these Möbius aromatic molecules can undergo classical aromatic substitution reactions, such as EAS reactions, instead of addition reactions. Given the key role of EAS in aromatic chemistry to obtain various derivatives, we sought to extend the understanding of the reactivity paradigm in the metalla-aromatic family.Our recent synthetic efforts associated with the metallapentalene system prompted us to investigate whether typical EAS reactions could proceed in these Craig-Möbius aromatics. If so, how could substitution be achieved in the same way that it is with traditional Hückel aromatics such as benzenes? In this paper, we present EAS reactions, mainly the halogenation of osmapentalene, osmafuran-fused osmapentalene, and osmabenzene-fused osmapentalene, which follow the classic EAS mechanistic scheme (Fig. 1B). With the aid of density functional theory (DFT) calculations, we characterized the effects on EAS reactivity and regioselectivity.     

Thermodynamic origins of protein folding,allostery, and capsid formation in the human hepatitis B virus core protein

HBc, the capsid-forming “core protein” of human hepatitis B virus (HBV), is a multidomain, α-helical homodimer that aggressively forms human HBV capsids. Structural plasticity has been proposed to be important to the myriad functions HBc mediates during viral replication. Here, we report detailed thermodynamic analyses of the folding of the dimeric HBc protomer under conditions that prevented capsid formation. Central to our success was the use of ion mobility spectrometry–mass spectrometry and microscale thermophoresis, which allowed folding mechanisms to be characterized using just micrograms of protein. HBc folds in a three-state transition with a stable, dimeric, α-helical intermediate. Extensive protein engineering showed thermodynamic linkage between different structural domains. Unusual effects associated with mutating some residues suggest structural strain, arising from frustrated contacts, is present in the native dimer. We found evidence of structural gatekeepers that, when mutated, alleviated native strain and prevented (or significantly attenuated) capsid formation by tuning the population of alternative native conformations. This strain is likely an evolved feature that helps HBc access the different structures associated with its diverse essential functions. The subtle balance between native and strained contacts may provide the means to tune conformational properties of HBc by molecular interactions or mutations, thereby conferring allosteric regulation of structure and function. The ability to trap HBc conformers thermodynamically by mutation, and thereby ablate HBV capsid formation, provides proof of principle for designing antivirals that elicit similar effects.The “protein-folding problem” describes how a polypeptide sequence contains all the information needed for it to adopt a specific 3D structure spontaneously (1). The chemistry and thermodynamic code that causes proteins to fold also underpins protein–protein interactions, allostery, and supramolecular assembly. An emerging trend has been the study of model proteins free from kinetic traps, aggregation, or metal binding, features that can confound experimental execution and data interpretation (2, 3). Consequently, model proteins are small (typically <130 residues), soluble monomers with few proline or cysteine residues and no prosthetic groups (2, 3).Although model proteins have been instrumental in taking the field to its current zenith, there is a paucity of experimental insights into the conformational dynamics of larger, oligomeric proteins, especially those implicated in diseases (3). Such proteins usually have complex behavior refractory to detailed experimental studies. However, the connection between sequence, structure, dynamics, and allostery makes studies of larger proteins central to understanding biological function and aiding drug design (vide infra) (4). One such protein is HBc, the capsid-forming “core protein” of human hepatitis B virus (HBV), a major pathogen that kills 600,000 people annually (5). Although excellent vaccines exist, there are no effective cures for extant chronic infections (5, 6). In addition to capsid formation, HBc plays many essential roles in HBV replication (7–9), making it an attractive drug target (10–15).WT HBc is a 183-residue polypeptide comprising a structured capsid-forming region (residues 1–149; Fig. 1A) and a basic, nucleic acid-binding domain (residues 150–183) (16–18). The structured N-terminal region (hereafter HBc_1–149) spontaneously self-assembles in vitro and in vivo to form icosahedral capsid-like particles (CLPs) identical to nucleocapsids isolated from patient serum (19, 20). X-ray crystallography and cryo-EM have characterized the structure of HBc_1–149 within the context of CLPs, virions, and hexamers (16, 19–23). HBc homodimers comprise two structural domains (Fig. 1A): Helices α₃ and α₄ from opposing monomers pack together and form a disulfide-linked, four-helix bundle dimerization interface (visible as protrusions on the capsid exterior; Fig. 1B), whereas α₁, α₂, and α₅ pack together and around the base of the four-helix bundle to create the hydrophobic core of “contact” domains (19). Weak interdimer interactions between contact domains stabilize HBV capsids (19, 24) (Fig. 1B).Open in a separate windowFig. 1.HBc_1–149 dimer structure within HBV capsids. (A) Four-helix bundle dimerization interface (black) is flanked by contact domains (orange and red). Helices are numbered, and the N and C termini of one monomer are indicated. The disulfide link between C61 of each monomer is indicated (cyan). (B) Exterior surface of a T = 4 capsid HBc_1–149 (PDB ID code 1QGT) (19). Dimers around the threefold and fivefold axes are indicated in blue/green and purple/orange, respectively. (Inset) Interacting quasiequivalent HBc_1–149 dimers from the fivefold (purple and orange) and threefold (blue and green) axes are shown. Hydrophobic contacts between contact domains stabilize capsids. Residues that perturb capsid formation when mutated are indicated.Multiple studies show clearly that HBc has a very malleable structure, with this structural plasticity argued to be functionally important (22, 23). This hypothesis accords well with antivirals that modulate HBc structure (11–15, 22, 23). Studies of HBV capsid assembly have inferred the existence of assembly-active (HBc^Ass) and assembly-incompetent (HBc^Inc) HBc conformations (12, 13, 21, 24, 25). However, there are few detailed insights on the thermodynamic origins of structure, allostery, and dynamics for the dimeric HBc_1–149 protomer, where structural plasticity must originate. This arises from dimeric HBc_1–149 being very challenging to study in vitro (compared with the model proteins described above) because it is a 298-residue disulfide-linked homodimer (containing 6 cysteine and 24 proline residues) that aggregates aggressively and forms capsids.Here, we report detailed folding and stability studies of dimeric HBc_1–149. These show HBc_1–149 folds in a three-state transition with a populated, dimeric, α-helical intermediate. Of 29 “chemically conservative” mutants used to probe folding energetics (26), many had similar effects on the stability of the intermediate and native ensembles. The distribution of these mutations was consistent with the intermediate being stabilized by a significant native-like structure. However, some mutations destabilized the native state (N) much less than the intermediate state (I) relative to the denatured state (D), or significantly increased the free energy of unfolding (ΔG_D–N) relative to WT HBc_1–149. This suggests HBc_1–149 contains structural strain arising from frustrated contacts (27, 28). We found evidence of HBc_1–149 adopting multiple native conformers, where capsid assembly-competent conformers were less stable than those incapable of, or attenuated in, capsid formation. Frustrated regions likely contain structural gatekeepers that (28), when mutated, subtly tuned the folding energy landscape and altered capsid assembly. The presence of multiple native conformations and frustrated regions may explain the origins of allostery reported for HBc. Frustration is likely an evolved tradeoff that balances the conflicting requirements of HBc folding with allosteric regulation of native structure, capsid formation, and diverse functions of different conformers (29). The ability to trap HBc conformers thermodynamically by mutation and ablate capsid formation provides a proof of principle for designing antivirals that elicit similar effects.     

Cross-α/β polymorphism of PSMα3 fibrils

The formation of ordered cross-β amyloid protein aggregates is associated with a variety of human disorders. While conventional infrared methods serve as sensitive reporters of the presence of these amyloids, the recently discovered amyloid secondary structure of cross-α fibrils presents new questions and challenges. Herein, we report results using Fourier transform infrared spectroscopy and two-dimensional infrared spectroscopy to monitor the aggregation of one such cross-α–forming peptide, phenol soluble modulin alpha 3 (PSMα3). Phenol soluble modulins (PSMs) are involved in the formation and stabilization of Staphylococcus aureus biofilms, making sensitive methods of detecting and characterizing these fibrils a pressing need. Our experimental data coupled with spectroscopic simulations reveals the simultaneous presence of cross-α and cross-β polymorphs within samples of PSMα3 fibrils. We also report a new spectroscopic feature indicative of cross-α fibrils.

Amyloids are elongated fibers of proteins or peptides typically composed of stacked cross β-sheets (1, 2). Self-assembling amyloids are notorious for their involvement in human neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases (1, 2). Phenol soluble modulins (PSMs) are amyloid peptides secreted by the bacteria Staphylococcus aureus (S. aureus) (3–5). Of the PSM family, PSMα3 is of recent interest due to its unique secondary structure upon fibrillation. Whereas other PSM variants undergo conformational changes with aggregation, the α-helical PSMα3 peptide retains its secondary structure while stacking in a manner reminiscent of β-sheets, forming what has been termed cross-α fibrils (3, 4, 6). Although “α-sheet” amyloid fibrils have been previously observed in two-dimensional infrared (2DIR) (7) and associated with PSMs (8), the novel cross-α fibril is distinct from that class of structures. To avoid confusion between these two similarly named but distinct secondary structures, a comparison between the α-sheet domain in cytosolic phosphatase A2 (9) (Protein Data Bank [PDB] identification:1rlw) (10) and cross-α fibrils adopted by PSMα3 (PDB ID:5i55) (3) has been highlighted in SI Appendix, Fig. S1. Interestingly, shorter terminations of PSMα3 have been shown to exhibit β-sheet polymorphs (11). The proposed cross-α fibril structure of the full-length PSMα3 peptide has been confirmed with X-ray diffraction and circular dichroism (4). The present study aims to further characterize these fibrils with linear and nonlinear infrared spectroscopies.S. aureus is an infectious human pathogen with the ability to form communities of microorganisms called biofilms that hinder traditional treatment methods (12–14). PSMs contribute to inflammatory response and play a crucial role in structuring and detaching biofilms (11, 12, 14). While biofilm growth requires the presence of multiple PSMs (14, 15), Andreasen and Zaman have demonstrated that PSMα3 acts as a scaffold, seeding the amyloid formation of other PSMs (5). To effectively inhibit S. aureus biofilm growth, a better understanding of PSMα3 aggregation is needed.The α-helical structure of PSMα3 (12) presents a challenge for probing the vibrational modes and secondary structure of both the monomer and the fibrils. While IR spectroscopy has been used extensively to characterize β-sheets (16–19), the spectral features associated with α-helices are difficult to distinguish from those of the random coil secondary structure (20, 21). This limitation has left researchers to date with an incomplete picture of the spectroscopic features unique to cross-α fibers. The present work combines a variety of 2DIR methods to remove these barriers and probe the active infrared vibrational modes of cross-α fibers.The full-length, 22-residue PSMα3 peptide was synthesized and prepared for aggregation studies following reported methods (3, 4, 11). A total of 10 mM PSMα3 was incubated in D₂O at room temperature over 7 d. These data were compared to the monomer treated under similar conditions. Monomeric samples were prepared at a significantly lower concentration of 0.5 mM to prevent aggregation. Fiber formation was confirmed by transmission electron microscopy (see SI Appendix, Fig. S2 for details). Fourier transform infrared (FTIR) spectra were taken for both the fibrils in solution as well as the low concentration monomers. Spectroscopic simulations of the PSMα3 monomer and fibers were performed on previously reported PDB structures (PDB identification: 5i55) (3) (Fig. 1).Open in a separate windowFig. 1.PDB structures of PSMα3 (A) monomers and (B) cross-α fibers extended along the screw axis. (C) FTIR spectra of 0.5 mM monomeric PSMα3 (blue) compared to the 10 mM PSMα3 fibril (red) in D₂O upon aggregation.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Click-to-lead design of a picomolar ABA receptor antagonist with potent activity in vivo

Abscisic acid (ABA) is a key plant hormone that mediates both plant biotic and abiotic stress responses and many other developmental processes. ABA receptor antagonists are useful for dissecting and manipulating ABA’s physiological roles in vivo. We set out to design antagonists that block receptor–PP2C interactions by modifying the agonist opabactin (OP), a synthetically accessible, high-affinity scaffold. Click chemistry was used to create an ∼4,000-member library of C4-diversified opabactin derivatives that were screened for receptor antagonism in vitro. This revealed a peptidotriazole motif shared among hits, which we optimized to yield antabactin (ANT), a pan-receptor antagonist. An X-ray crystal structure of an ANT–PYL10 complex (1.86 Å) reveals that ANT’s peptidotriazole headgroup is positioned to sterically block receptor–PP2C interactions in the 4′ tunnel and stabilizes a noncanonical closed-gate receptor conformer that partially opens to accommodate ANT binding. To facilitate binding-affinity studies using fluorescence polarization, we synthesized TAMRA–ANT. Equilibrium dissociation constants for TAMRA–ANT binding to Arabidopsis receptors range from ∼400 to 1,700 pM. ANT displays improved activity in vivo and disrupts ABA-mediated processes in multiple species. ANT is able to accelerate seed germination in Arabidopsis, tomato, and barley, suggesting that it could be useful as a germination stimulant in species where endogenous ABA signaling limits seed germination. Thus, click-based diversification of a synthetic agonist scaffold allowed us to rapidly develop a high-affinity probe of ABA–receptor function for dissecting and manipulating ABA signaling.

The phytohormone abscisic acid (ABA) controls numerous physiological processes in plants ranging from seed development, germination, and dormancy to responses for countering biotic and abiotic stresses (1). ABA binds to the PYR/PYL/RCAR (Pyrabactin Resistance 1/PYR1-like/Regulatory Component of ABA Receptor) soluble receptor proteins (2, 3) and triggers a conformational change in a flexible “gate” loop flanking the ligand-binding pocket such that the ABA–receptor complex can then bind to and inhibit clade A type II C protein phosphatases (PP2Cs), which normally dephosphorylate and inactivate SNF1-related protein kinase 2 (SnRK2). This, in turn, leads to SnRK2 activation, phosphorylation of downstream targets, and multiple cellular outputs (4, 5).Chemical modulators of ABA perception have been sought as both research tools for dissecting ABA’s role in plant physiology and for their potential agricultural utility (6, 7). Dozens of ABA receptor agonists, which reduce transpiration and water use by inducing guard cell closure, have been developed and are being explored as chemical tools for mitigating the effects of drought on crop yields (7–23), most of them either being analogs of ABA or sulfonamides similar to quinabactin (24). ABA receptor antagonists could conceivably be useful in cases where water is not limiting, for example, to increase transpiration and gas exchange under elevated CO₂ in glasshouse agriculture, as germination stimulators, and for studying the ABA dependence of physiological processes, among other applications (25–31). Thus, both ABA receptor agonists and antagonists have potential uses as research tools and for plant biotechnology.In principle, there are at least two mechanisms for blocking ABA receptor activation: by preventing gate closure, which is necessary for PP2C binding, or by sterically disrupting the activated, closed-gate receptor conformer from binding to PP2Cs. Prior efforts to design antagonists have focused on the latter strategy and include multiple ABA-derived ligands such as AS6 (25), PanMe (26), 3′-alkyl ABA (30–32), 3′-(phenyl alkynyl) ABA (33), or ligands derived from tetralone ABA (34) with varying degrees of conformational restriction (27, 28, 35). With the exception of PanMe, these antagonists have linkers attached to the 3′ carbon of ABA or 11′ carbon of tetralone ABA, which is positioned to disrupt receptor–PP2C interactions by protruding through the 3′ tunnel. PanMe was created by modifying ABA’s C4′ (Fig. 1) with a toluylpropynyl ether substituent designed to occupy the 4′ tunnel, a site of close receptor–PP2C contact (26). Structural studies showed that this 4′ moiety adopts two conformations, one that resides in the 4′ tunnel and another that occupies the adjacent 3′ tunnel (26). Collectively, these elegant studies have demonstrated that antagonists of receptor–PP2C interactions can be designed by modifying agonists at sites situated proximal to the 3′ or 4′ tunnels. Despite these advances, current antagonists have limitations. For example, PanMe, which has low nanomolar affinity for the subfamily II receptor PYL5, is limited by relatively low activity on subfamily I and III ABA receptors, and as we show here, the ABA antagonist AA1 (36) (Fig. 1) lacks detectable antagonist activity in vitro and is, therefore, unlikely to be a true ABA receptor antagonist. Together, these data suggest that higher-affinity pan-antagonists and/or molecules with increased bioavailability will be necessary to more efficiently block endogenous ABA signaling. We set out to address these limitations by modifying the scaffold of the synthetic ABA agonist opabactin (OP), which has an approximately sevenfold increase in both affinity and bioactivity relative to ABA (21). We describe an OP derivative called antabactin (ANT) and show that it is a high-affinity binder and antagonist of ABA receptors that disrupts ABA-mediated signaling in vivo.Open in a separate windowFig. 1.Structures of ABA, PanMe, and AA1.     

Decoupling of rotational and translational diffusion in supercooled colloidal fluids

We use confocal microscopy to directly observe 3D translational and rotational diffusion of tetrahedral clusters, which serve as tracers in colloidal supercooled fluids. We find that as the colloidal glass transition is approached, translational and rotational diffusion decouple from each other: Rotational diffusion remains inversely proportional to the growing viscosity whereas translational diffusion does not, decreasing by a much lesser extent. We quantify the rotational motion with two distinct methods, finding agreement between these methods, in contrast with recent simulation results. The decoupling coincides with the emergence of non-Gaussian displacement distributions for translation whereas rotational displacement distributions remain Gaussian. Ultimately, our work demonstrates that as the glass transition is approached, the sample can no longer be approximated as a continuum fluid when considering diffusion.Rapidly cooling a glass-forming liquid fundamentally changes the nature of fluid transport at a molecular scale (1–7). For a tracer in a continuum fluid, the translational and rotational diffusion coefficients D_T and D_R, respectively, depend on temperature T and viscosity η as D ∝ T/η. Therefore, the ratio D_T/D_R is a constant that is independent of both T and η. However, this relationship breaks down in the deeply supercooled regime near the glass transition, according to experiments with molecular glass formers and also molecular dynamics simulations (1–3, 8–14).Experiments with glass-forming materials find that rotational diffusion remains strongly coupled with viscosity, where D_R ∝ η⁻¹, whereas translational diffusion decouples, developing a fractional dependence on η where D_T ∝ η^−ξ with ξ < 1 (2, 8, 15). Near the glass transition, D_T can be enhanced by two orders of magnitude over what would be calculated from the material’s viscosity. The rotational diffusion coefficients from these experiments are inferred from measurements related to molecular rotations, and are evaluated using the “Debye model” due to an inability to directly observe molecular rearrangements in a material’s bulk (3, 8–10, 16, 17). This experimental limitation has inspired computational studies where diffusion can be calculated using the Debye model and also a complementary method, the “Einstein formulation,” which is more directly related to the trajectories of the diffusing objects. These simulations studied pure systems of water (9), ortho-terphenyl (10), and hard dumbbell particles (11). Intriguingly, the simulations found that decoupling depends qualitatively on the analysis method: They find rotational motion is enhanced over translational motion when quantified with the Einstein formulation, with the opposite being true in the Debye formulation. The results from these simulations raise the need for a critical reexamination of our current understanding of the relationship between translational and rotational diffusion, and only through direct observation can these differences be addressed (10). Unfortunately, there has been no direct experimental observation of diffusive decoupling in a 3D system which would allow for these findings to be tested.We use high-speed confocal microscopy to directly visualize the 3D translational and rotational motion of tetrahedral tracer colloidal clusters in a dense amorphous suspension of colloidal spheres. A glass transition occurs as the colloidal suspension’s volume fraction is increased above the value of ϕ_G ∼ 0.58 (18, 19). We observe that as the glass transition is approached from ϕ < ϕ_G, the long-time rotational diffusion of the tracers decreases proportionally with the bulk viscosity η, whereas long-time translational diffusion decreases by a much lesser extent, similar to observations made with molecular glasses (2, 8). Moreover, we quantify the rotational motion in several ways, and find that all rotational observations remain proportional to η⁻¹. Our results are in contrast with the aforementioned results from computer simulations (9–11), where the details depend on the analysis method, and rotational diffusion was either apparently enhanced or suppressed depending on the analysis. Our results persist regardless of the analysis used to characterize diffusion. Direct comparison between our results and those of computer simulations is problematic, given that they studied pure liquids whereas we study tracers; these differences are evaluated in the Discussion.Colloidal suspensions provide an insightful avenue for experimentally exploring the glass transition (18–22). The key control parameter is the volume fraction ϕ, rather than temperature. As ϕ increases, colloidal microspheres exhibit phase behavior that is in good agreement with the hard-sphere model (18, 19). Hard spheres are arguably the simplest system in which to study the most fundamental features of the glass transition. An important advantage of colloids is that individual particles can be followed in 3D using a confocal microscope, which permits direct observation of the complex dynamical processes of individual particles (22–25) that can be difficult to study with more conventional methods that average over many particles within the sample’s bulk (2, 16).Steric effects are key to understanding the colloidal glass transition: Particles cannot overlap one another, complicating their motion in a dense sample. For one sphere to move, its neighbors must move out of its way, and their neighbors must move out of their way, etc. This leads to large-scale rearrangements involving many particles (22, 25–27). In contrast, rotation of spheres is possible without colliding with neighboring particles. Rotation of optically anisotropic colloidal spheres has been studied (28–31). These rotations do slow near the colloidal glass transition, but due to hydrodynamic interactions rather than steric interactions. For this reason, in dense suspensions of spheres rotational diffusion is faster than translational diffusion (30); this is discussed further in Materials and Methods. The hydrodynamic slowing of rotational diffusion, while interesting in its own right, cannot speak to the question of slowing rotational diffusion in molecular glasses.Therefore, we use a dispersion of isolated nonspherical particles in a suspension of spherical particles. This is a simple physical model system in which to study rotational and translational diffusion near the glass transition. Our nonspherical tracer particles, developed through recent advances in colloidal science (32), are highly ordered clusters of colloidal particles (33). A confocal micrograph of one such cluster with corresponding 3D representations is shown in Fig. 1. The rotational motion of such clusters is hindered by collisions with neighboring spherical particles, and thus is a reasonable model of steric hindrances within molecular glass-forming systems. At short time scales, hydrodynamics are still expected to be important (30, 31); however, we focus on the long-time dynamics, for which the steric hindrance should be most significant (34–37).Open in a separate windowFig. 1.Visual representations of a colloidal tetrahedral cluster. Only the core of each particle is fluorescently labeled, surrounded by a blank undyed shell of PMMA, making each particle visually distinct from its clustered neighbors. (A) Composite, imaged using a confocal microscope and calculated by taking the mean of a 3D image. The composite images allow us to see through the cluster, where the overlap between multiple particles appears black. (B) Three-dimensional reconstruction. (C) Three-dimensional rendering of spheres at the centers of the particle coordinates, as determined by our tracking algorithms (38, 41). Sphere diameters are roughly equal to that of the fluorescent cores.With a recently developed means of tracking motion of such clusters (38), we are in a position to simultaneously study translational and rotational diffusion of anisotropic tracers in glassy and otherwise densely packed systems. Our system can be thought of as a 3D analog of samples used in very recent colloidal experiments, where rotational diffusion was studied in dense 2D samples of colloidal ellipsoids, with aspect ratios of ∼1.1 (39) and ∼6.0 (40). We follow the full 3D motion of our clusters, allowing us to observe rotations around any axis without any ambiguity, as discussed in Materials and Methods.     

A 5,000-year vegetation and fire history for tierra firme forests in the Medio Putumayo-Algodón watersheds,northeastern Peru

This paper addresses an important debate in Amazonian studies; namely, the scale, intensity, and nature of human modification of the forests in prehistory. Phytolith and charcoal analysis of terrestrial soils underneath mature tierra firme (nonflooded, nonriverine) forests in the remote Medio Putumayo-Algodón watersheds, northeastern Peru, provide a vegetation and fire history spanning at least the past 5,000 y. A tree inventory carried out in the region enables calibration of ancient phytolith records with standing vegetation and estimates of palm species densities on the landscape through time. Phytolith records show no evidence for forest clearing or agriculture with major annual seed and root crops. Frequencies of important economic palms such as Oenocarpus, Euterpe, Bactris, and Astrocaryum spp., some of which contain hyperdominant species in the modern flora, do not increase through prehistoric time. This indicates pre-Columbian occupations, if documented in the region with future research, did not significantly increase the abundance of those species through management or cultivation. Phytoliths from other arboreal and woody species similarly reflect a stable forest structure and diversity throughout the records. Charcoal ¹⁴C dates evidence local forest burning between ca. 2,800 and 1,400 y ago. Our data support previous research indicating that considerable areas of some Amazonian tierra firme forests were not significantly impacted by human activities during the prehistoric era. Rather, it appears that over the last 5,000 y, indigenous populations in this region coexisted with, and helped maintain, large expanses of relatively unmodified forest, as they continue to do today.

More than 50 y ago, prominent scholars argued that due to severe environmental constraints (e.g., poor natural resources), prehistoric cultures in the Amazon Basin were mainly small and mobile with little cultural complexity, and exerted low environmental impacts (1, 2). Contentious debates ensued and have been ongoing ever since. Empirical data accumulated during the past 10 to 20 y have made it clear that during the late Holocene beginning about 3,000 y ago dense, permanent settlements with considerable cultural complexity had developed along major watercourses and some of their tributaries, in seasonal savannas/areas of poor drainage, and in seasonally dry forest. These populations exerted significant, sometimes profound, regional-scale impacts on landscapes, including with raised agricultural fields, fish weirs, mound settlements, roads, geometric earthworks called geoglyphs, and the presence of highly modified anthropic soils, called terra pretas or “Amazonian Dark Earths” (Fig. 1) (e.g., refs. 3–15).Open in a separate windowFig. 1.Location of study region (MP-A) and other Amazonian sites discussed in the text. River names are in blue. The black numbers represent major pre-Columbian archaeological sites with extensive human alterations (1, Marajó Island; 2, Santarém; 3, Upper Xingu; 4, Central Amazon Project; 5, Bolivian sites) (3, 5–10, 14, 15). ADE, terra preta locations (e.g., refs. 19 and 20); triangles are geoglyph sites (6, 8). The white circles are terrestrial soil locations previously studied by Piperno and McMichael (29, 31–33, 54) (Ac, Acre; Am, Amacayacu; Ay, Lake Ayauchi; B, Barcelos; GP, lakes Gentry-Parker; Iq, Iquitos to Nauta; LA, Los Amigos; PVM, Porto Velho to Manaus; T, Tefe).An important, current debate that frames this paper centers not on whether some regions of the pre-Columbian Amazon supported large and complex human societies, but rather on the spatial scales, degrees, and types of cultural impacts across this continental-size landscape. Some investigators drawing largely on available archaeological data and studies of modern floristic composition of selected forests, argue that heavily modified “domesticated” landscapes were widespread across Amazonia at the end of prehistory, and these impacts significantly structure the vegetation today, even promoting higher diversity than before (e.g., refs. 14–21). It is believed that widespread forms of agroforestry with planted, orchard-like formations or other forest management strategies involving the care and possible enrichment of several dozens of economically important native species have resulted in long-term legacies left on forest composition (e.g., refs. 14–22). Some (20) propose that human influences played strong roles in the enrichment of “hyperdominant” trees, which are disproportionately common elements in the modern flora (sensu ref. 23). Some even argue that prehistoric fires and forest clearance were so spatially extensive that post-Columbian reforestation upon the tragic consequences of European contact was a principal contributor to decreasing atmospheric CO₂ levels and the onset of the “Little Ice Age” (24, 25).However, modern floristic studies are often located in the vicinity of known archaeological sites and/or near watercourses (26). Many edible trees in these studies are early successional and would not be expected to remain as significant forest elements for hundreds of years after abandonment. Historic-period impacts well-known in some regions to have been profound have been paid little attention and may be mistaken for prehistoric legacies (26–28). Moreover, existing phytolith and charcoal data from terrestrial soils underneath standing tierra firme forest in some areas of the central and western Amazon with no known archaeological occupations nearby exhibit little to no evidence for long-term human occupation, anthropic soils, agriculture, forest clearing or other significant vegetation change, or recurrent/extensive fires during the past several thousand years (Fig. 1) (29–33). Even such analyses of terrestrial soils of lake watersheds in western Amazonia known to have been occupied and farmed in prehistory revealed no spatially extensive deforestation of the watersheds, as significant human impacts most often occurred in areas closest to the lakes (Fig. 1) (34). Furthermore, vast areas have yet to be studied by archaeologists and paleoecologists, particularly the tierra firme forests that account for 95% of the land area of Amazonia.To further inform these issues, we report here a vegetation and fire history spanning 5,000 y derived from phytolith and charcoal studies of terrestrial soils underneath mature tierra firme forest in northeastern Peru. Phytoliths, the silica bodies produced by many Neotropical plants, are well preserved in terrestrial soils unlike pollen, and are deposited locally. They can be used to identify different tropical vegetational formations, such as old-growth forest, early successional vegetation typical of human disturbances including forest clearings, a number of annual seed and root crops, and trees thought to have been cultivated or managed in prehistory (e.g., refs. 29–33 and 35).     

Nonlinear rotational spectroscopy reveals many-body interactions in water molecules

Because of their central importance in chemistry and biology, water molecules have been the subject of decades of intense spectroscopic investigations. Rotational spectroscopy of water vapor has yielded detailed information about the structure and dynamics of isolated water molecules, as well as water dimers and clusters. Nonlinear rotational spectroscopy in the terahertz regime has been developed recently to investigate the rotational dynamics of linear and symmetric-top molecules whose rotational energy levels are regularly spaced. However, it has not been applied to water or other lower-symmetry molecules with irregularly spaced levels. We report the use of recently developed two-dimensional (2D) terahertz rotational spectroscopy to observe high-order rotational coherences and correlations between rotational transitions that were previously unobservable. The results include two-quantum (2Q) peaks at frequencies that are shifted slightly from the sums of distinct rotational transitions on two different molecules. These results directly reveal the presence of previously unseen metastable water complexes with lifetimes of 100 ps or longer. Several such peaks observed at distinct 2Q frequencies indicate that the complexes have multiple preferred bimolecular geometries. Our results demonstrate the sensitivity of rotational correlations measured in 2D terahertz spectroscopy to molecular interactions and complexation in the gas phase.

Water has attracted extensive spectroscopic interest because of its critical implications for theoretical and applied sciences (1, 2). Water shows anomalous properties because of complicated fluxional hydrogen-bonded networks and has been investigated by Raman and infrared spectroscopy (3, 4), sum frequency spectroscopy (5), optical Kerr effect spectroscopy (6), vibration-rotation-tunneling spectroscopy (7), and recently by two-dimensional (2D) infrared spectroscopy (8, 9) and 2D Raman-terahertz (THz) spectroscopy (10). In the gas phase, water is of utmost importance for atmospheric science, astrophysics, combustion research, and fundamental chemistry and physics (1, 11, 12). Although the pure rotation spectrum of water vapor has been well known for decades (13, 14), nonlinear THz spectroscopy of water rotational dynamics has not been previously reported. Nonlinear rotational spectroscopy in the microwave spectral range is well established (15), but because of the small moments of inertia of water, most of its rotational transition frequencies lie in the THz frequency range (Fig. 1). Nonlinear THz rotational spectroscopy was reported only recently (16, 17), and 2D THz rotational spectroscopy (10, 18, 19) more recently still. As in 2D spectroscopy of vibrational, electronic, and other degrees of freedom (9, 20–25), 2D rotational spectroscopy can reveal correlations between rotational states, many-body effects, and distinct multiple-field interactions that cannot be observed by linear spectroscopy (26). The large dipole moment of water, manifest in strong atmospheric absorption in the THz window (27–31), and the existence of water dimers and larger clusters with complex structures and dynamics (7, 32, 33), suggest that 2D spectroscopy of water could generate previously elusive insights (34–37).Open in a separate windowFig. 1.Overview of the experiment. (A) Water molecule in the laboratory frame, showing the dipole moment μ at an angle θ from the Z axis (THz polarization direction). (B) Water molecule in the molecule-fixed frame with the three moments of inertia I_a, I_b, and I_c along the corresponding axes. (C) Relative population distribution as a function of the J rotational quantum number. All relevant K_a and K_c components are included in the population distribution. (Inset) The relative population distribution within the state J = 2. (D) Rotational energy levels of para-H₂O and ortho-H₂O molecules. Red arrows illustrate rotational transitions and transition frequencies involved in this work. (E) Measured THz FID (Top) and Fourier transform (Bottom) showing rotational transitions (marked by dashed vertical lines) of water vapor in ambient air. (F) Schematic illustration of the 2D THz experimental setup. Linear THz spectra (example in E) are measured with only one THz pump pulse.Two-dimensional THz rotational spectroscopy has not been extended previously to water or any asymmetric-top molecules, although such molecules, whose rotational spectra are complicated because all three of their moments of inertia are unequal, are the majority of naturally occurring molecular species. Unlike a linear or symmetric-top molecule, in which the spectroscopic transitions between successive total rotational angular momentum levels denoted by the quantum number J are spaced by even-integer multiples of a common factor, the rotational constant B, the asymmetric-top nature of water molecules leads to irregularly spaced rotational energy levels. These levels are described approximately by quantum numbers J, K_a, and K_c that indicate the total angular momentum and its symmetry-axis projections (1, 2). The spectrum of water vapor consists of many transitions, with ΔJ=−1,0,+1 (P, Q, and R branches) all allowed and, for each, changes ΔKa=±1,ΔKc=±1 (1, 2). The large centrifugal distortion of water and the distinct sets of rotational states occupied by its nuclear spin isomers (even symmetry for para, odd symmetry for ortho) further complicate its rotational spectrum (1, 2, 38). A typical THz time-domain free-induction decay (FID) signal from water vapor at ambient conditions, induced by a weak single-cycle THz pulse, is shown in Fig. 1E. The irregular oscillations arise from more than 15 transitions (Fig. 1D) that contribute significantly to the water vapor absorption spectrum in the 0.1- to 2-THz region.     

Machine learning–based inverse design for electrochemically controlled microscopic gradients of O2 and H2O2

A fundamental understanding of extracellular microenvironments of O₂ and reactive oxygen species (ROS) such as H₂O₂, ubiquitous in microbiology, demands high-throughput methods of mimicking, controlling, and perturbing gradients of O₂ and H₂O₂ at microscopic scale with high spatiotemporal precision. However, there is a paucity of high-throughput strategies of microenvironment design, and it remains challenging to achieve O₂ and H₂O₂ heterogeneities with microbiologically desirable spatiotemporal resolutions. Here, we report the inverse design, based on machine learning (ML), of electrochemically generated microscopic O₂ and H₂O₂ profiles relevant for microbiology. Microwire arrays with suitably designed electrochemical catalysts enable the independent control of O₂ and H₂O₂ profiles with spatial resolution of ∼10¹ μm and temporal resolution of ∼10° s. Neural networks aided by data augmentation inversely design the experimental conditions needed for targeted O₂ and H₂O₂ microenvironments while being two orders of magnitude faster than experimental explorations. Interfacing ML-based inverse design with electrochemically controlled concentration heterogeneity creates a viable fast-response platform toward better understanding the extracellular space with desirable spatiotemporal control.

Ubiquitous spatiotemporal heterogeneity of natural environments fosters the diverse and fascinating biology that our world embraces, and motivates researchers to mimic natural environments with high spatiotemporal resolution (1–5). Given their close relevance in biochemical metabolisms, dioxygen (O₂) and hydrogen peroxide (H₂O₂) as a surrogate of reactive oxygen species (ROS) are two ubiquitous biologically relevant species in extracellular medium (1, 6). Their extracellular spatial and temporal distributions, particularly at the microscopic scale ranging from 1 μm to 100 μm (7–11), are critical for signal transduction, protein expression, biochemical redox balance, and regulation for cellular metabolism with extensive ecological, environmental, and biomedical implications (Fig. 1A) (1, 3, 8–13). A programmable creation of the spatiotemporal concentration profiles of O₂ and H₂O₂ offers the freedom to mimic, control, and perturb the microenvironments of O₂ and H₂O₂ and hence advance our understanding in microbiology.Open in a separate windowFig. 1.AI-based inverse design of electrochemically generated O₂ and H₂O₂ heterogeneities. (A) The ubiquitous spatiotemporal heterogeneities of O₂ and H₂O₂ in microbiology and the challenges posed in this research topic. (B) The combination of electrochemistry and ML-based inverse design offers a viable approach to mimicking and controlling the heterogeneities of O₂ and H₂O₂ in microbiology. O, oxidant; R, reductant; E_appl (t), the time-dependent electrochemical voltages applied on electrodes. (C) The design of the electrochemically active microwire array electrodes for the generation of O₂ and H₂O₂ gradients; 4e⁻ ORR & 2e⁻ ORR, four-electron and two-electron oxygen reduction reaction into H₂O and H₂O₂, respectively. (D and E) The 45°-tilting images of SEM for the representative microwire arrays used for the training of the ML model (D) and the ones inversely designed for targeted O₂ and H₂O₂ gradients (E); k = (P, D, L), the morphological vector that includes the P, D, and L of the synthesized wire arrays in units of micrometers. (Scale bars, 20 μm.)Despite recent progress (14–18), there remain major technical challenges, particularly in the achievable spatiotemporal resolution and high-throughput design of concentration profiles to suit a plethora of scenarios in microbiology. Approaches based on microfluidics and hydrogels have been able to achieve concentration gradients of O₂ and H₂O₂ through the provision of either O₂/H₂O₂ source (14, 19–21), O₂/H₂O₂ scavenging agents (15, 22, 23), or a combination of both (24) across liquid-impermeable barriers such as agar layers or polymeric thin films (25, 26). Yet such approaches, dependent on passive mass transport and diffusion across more than 10² μm, are inherently incapable of achieving spatial features of less than 100 μm and temporal resolution smaller than ∼10¹ s, the prerequisites to investigate microbiology at cluster or single-cell levels (10–12). Moreover, the large variations of extracellular O₂ and H₂O₂ gradients in different microbial systems demand an inverse design strategy, which, with minimal expenditure, quickly programs a desired concentration profile catering to a specific biological scenario (2–5). The current lack of inverse design protocol impedes the adoption of controllable extracellular heterogeneity to mimic and investigate microbial systems that are of environmental, biomedical, and sustainability-related significance.We envision that the integration of electrochemically generated concentration gradients with inverse design based on machine learning (ML) will address the aforementioned challenges (Fig. 1B). Electrochemistry offers a venue for transducing electric signals into microscopic concentration profiles within ∼10⁰ μm to ∼10² μm away from electrodes’ surface, following the specific electrode reaction kinetics and the mass transport governing equations in the liquid phase (27). Proper designs of electrodes’ microscopic spatial arrangement and electrochemical kinetics lead to concentration gradients that are spatiotemporally programmable by time-dependent electric signals of varying voltages (28). Such benefits of electrochemically generated concentration gradients lead us to employ electrochemistry as a tool to spatiotemporally control the concentration profiles in the extracellular medium. In one example, we found that wire arrays electrochemically active toward O₂ reduction create anoxic microenvironment about 20 μm away from the aerobic external bulk environments, modulate the size and extent of O₂ depletion in the anoxic microenvironment by the wire array’s morphology and applied electrochemical potential (E_appl), and hence enable O₂-sensitive rhizobial N₂ fixation in ambient air powered by renewable electricity (29). Moreover, while not reported before as far as we know, electrochemically generated concentration heterogeneity is commensurate with ML-based inverse design (30, 31), thanks to the mathematically well-defined electrochemical processes that can be numerically simulated (32, 33). We recently reported neural networks, trained by numerically simulated data, that explore the influence of electrode geometry on electrochemical N₂ fixation and achieve optimized morphologies of wire array electrodes untenable without such an ML-based strategy (34). An inverse design for the electrochemically generated gradients will quickly program desirable microenvironments of O₂ and ROS with high spatiotemporal resolutions, thanks to the well-reported electrochemical transformation related to O₂ and H₂O₂ with high electrochemical selectivity (35, 36).In this work, we report an inverse design based on neural networks for independent electrochemical creation of O₂ and ROS microscopic gradients that are relevant, and mimic their extracellular heterogeneities in microbial systems. We hypothesize that careful design of electrocatalysis of O₂ reduction reaction (ORR) can either facilitate four-electron ORR on Pt electrocatalyst for a controllable O₂ spatiotemporal profile or promote two-electron ORR on Au electrocatalyst for a programmable generation of H₂O₂ gradient without significantly perturbing the O₂ one, thanks to their concentration differences in biological mediums (∼10⁻¹ μM to ∼10¹ μM for H₂O₂ and ∼10¹ μM to ∼10² μM for O₂) (2, 7–11). Electrochemically active microwire array electrodes as exemplary model systems (Fig. 1C) are experimentally shown to achieve tunable heterogeneities of O₂ and H₂O₂ independently, with spatial resolution of ∼10¹ μm and temporal resolution of ∼10° s, and are suitable as a platform for independently perturbing biologically relevant O₂ and H₂O₂ profiles in microbial systems. We further established and experimentally validated two neural networks that inversely design the wire array electrodes’ morphologies toward targeted microenvironments of O₂ and H₂O₂, respectively, which is at least one order of magnitude faster than trial-and-error numerical simulation and two orders of magnitude faster than experimental explorations. The demonstrated inverse design of electrochemically generated controlled gradients not only demonstrates a full electrochemical control of concentration profiles in an electrode’s proximity but also establishes an approach of spatiotemporally mimicking and perturbing extracellular space guided by artificial intelligence.