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.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Chirality-matched catalyst-controlled macrocyclization reactions

Macrocycles, formally defined as compounds that contain a ring with 12 or more atoms, continue to attract great interest due to their important applications in physical, pharmacological, and environmental sciences. In syntheses of macrocyclic compounds, promoting intramolecular over intermolecular reactions in the ring-closing step is often a key challenge. Furthermore, syntheses of macrocycles with stereogenic elements confer an additional challenge, while access to such macrocycles are of great interest. Herein, we report the remarkable effect peptide-based catalysts can have in promoting efficient macrocyclization reactions. We show that the chirality of the catalyst is essential for promoting favorable, matched transition-state relationships that favor macrocyclization of substrates with preexisting stereogenic elements; curiously, the chirality of the catalyst is essential for successful reactions, even though no new static (i.e., not “dynamic”) stereogenic elements are created. Control experiments involving either achiral variants of the catalyst or the enantiomeric form of the catalyst fail to deliver the macrocycles in significant quantity in head-to-head comparisons. The generality of the phenomenon, demonstrated here with a number of substrates, stimulates analogies to enzymatic catalysts that produce naturally occurring macrocycles, presumably through related, catalyst-defined peripheral interactions with their acyclic substrates.

Macrocyclic compounds are known to perform a myriad of functions in the physical and biological sciences. From cyclodextrins that mediate analyte separations (1) to porphyrin cofactors that sit in enzyme active sites (2, 3) and to potent biologically active, macrocyclic natural products (4) and synthetic variants (5–7), these structures underpin a wide variety of molecular functions (Fig. 1A). In drug development, such compounds are highly coveted, as their conformationally restricted structures can lead to higher affinity for the desired target and often confer additional metabolic stability (8–13). Accordingly, there exists an entire synthetic chemistry enterprise focused on efficient formation and functionalization of macrocycles (14–18).Open in a separate windowFig. 1.(A) Examples of macrocyclic compounds with important applications. HCV, hepatitis C virus. (B) Use of chiral ligands in metal-catalyzed or mediated stereoselective macrocyclization reactions. (C) Remote desymmetrization using guanidinylated ligands via Ullmann coupling. (D) This work: use of copper/peptidyl complexes for macrocyclization and the exploration of matched and mismatched effect.In syntheses of macrocyclic compounds, the ring-closing step is often considered the most challenging step, as competing di- and oligomerization pathways must be overcome to favor the intramolecular reaction (14). High-dilution conditions are commonly employed to favor macrocyclization of linear precursors (19). Substrate preorganization can also play a key role in overcoming otherwise high entropic barriers associated with multiple conformational states that are not suited for ring formation. Such preorganization is most often achieved in synthetic chemistry through substrate design (14, 20–22). Catalyst or reagent controls that impose conformational benefits that favor ring formation are less well known. Yet, critical precedents include templating through metal-substrate complexation (23, 24), catalysis by foldamers (25) or enzymes (26–29), or, in rare instances, by small molecules (discussed below). Characterization of biosynthetic macrocyclization also points to related mechanistic issues and attributes for efficient macrocyclizations (30–34). Coupling macrocyclization reactions to the creation of stereogenic elements is also rare (35). Metal-mediated reactions have been applied toward stereoselective macrocyclizations wherein chiral ligands transmit stereochemical information to the products (Fig. 1B). For example, atroposelective ring closure via Heck coupling has been applied in the asymmetric total synthesis of isoplagiochin D by Speicher and coworkers (36–40). Similarly, atroposelective syntheses of (+)-galeon and other diarylether heptanoid natural products were achieved via Ullman coupling using N-methyl proline by Salih and Beaudry (41). Finally, Reddy and Corey reported the enantioselective syntheses of cyclic terpenes by In-catalyzed allylation utilizing a chiral prolinol-based ligand (42). While these examples collectively illustrate the utility of chiral ligands in stereoselective macrocyclizations, such examples remain limited.We envisioned a different role for chiral catalysts when addressing intrinsically disfavored macrocyclization reactions. When unfavorable macrocyclization reactions are confronted, we hypothesized that a catalyst–substrate interaction might provide transient conformational restriction that could promote macrocyclization. To address this question, we chose to explore whether or not a chiral catalyst-controlled macrocyclization might be possible with peptidyl copper complexes. In the context of the medicinally ubiquitous diarylmethane scaffold, we had previously demonstrated the capacity for remote asymmetric induction in a series of bimolecular desymmetrizations using bifunctional, tetramethylguanidinylated peptide ligands. For example, we showed that peptidyl copper complexes were able to differentiate between the two aryl bromides during C–C, C–O, and C–N cross-coupling reactions (Fig. 1C) (43–45). Moreover, in these intermolecular desymmetrizations, a correlation between enantioselectivity and conversion was observed, revealing the catalyst’s ability to perform not only enantiotopic group discrimination but also kinetic resolution on the monocoupled product as the reaction proceeds (44). This latter observation stimulated our speculation that if an internal nucleophile were present to undergo intramolecular cross-coupling to form a macrocycle, stereochemically sensitive interactions (so-called matched and mismatched effects) (46) could be observed (Fig. 1D). Ideally, we anticipated that transition state–stabilizing interactions might even prove decisive in matched cases, and the absence of catalyst–substrate stabilizing interactions might account for the absence of macrocyclization for these otherwise intrinsically unfavorable reactions. Herein, we disclose the explicit observation of these effects in chiral catalyst-controlled macrocyclization reactions.     

Conjugated polymers based on metalla-aromatic building blocks

Conjugated polymers usually require strategies to expand the range of wavelengths absorbed and increase solubility. Developing effective strategies to enhance both properties remains challenging. Herein, we report syntheses of conjugated polymers based on a family of metalla-aromatic building blocks via a polymerization method involving consecutive carbyne shuttling processes. The involvement of metal d orbitals in aromatic systems efficiently reduces band gaps and enriches the electron transition pathways of the chromogenic repeat unit. These enable metalla-aromatic conjugated polymers to exhibit broad and strong ultraviolet–visible (UV–Vis) absorption bands. Bulky ligands on the metal suppress π–π stacking of polymer chains and thus increase solubility. These conjugated polymers show robust stability toward light, heat, water, and air. Kinetic studies using NMR experiments and UV–Vis spectroscopy, coupled with the isolation of well-defined model oligomers, revealed the polymerization mechanism.

Conjugated polymers are macromolecules usually featuring a backbone chain with alternating double and single bonds (1–3). These characteristics allow the overlapping p-orbitals to form a system with highly delocalized π-electrons, thereby giving rise to intriguing chemical and physical properties (4–6). They have exhibited many applications in organic light-emitting diodes, organic thin film transistors, organic photovoltaic cells, chemical sensors, bioimaging and therapies, photocatalysis, and other technologies (7–10). To facilitate the use of solar energy, tremendous efforts have been devoted in recent decades to developing previously unidentified conjugated polymers exhibiting broad and strong absorption bands (11–13). The common strategies for increasing absorption involve extending π-conjugation by incorporating conjugated cyclic moieties, especially fused rings; modulating the strength of intramolecular charge transfer between donor and acceptor units (D–A effect); increasing the coplanarity of π conjugation through weak intramolecular interactions (e.g., hydrogen bonds); and introducing heteroatoms or heavy atoms into the repeat units of conjugated polymers (11–16). Additionally, appropriate solubility is a prerequisite for processing and using polymers and is usually achieved with the aid of long alkyl or alkoxy side chains (12, 17).Aromatic rings are among the most important building blocks for conjugated polymers. In addition to aromatic hydrocarbons, a variety of aromatic heterocycles composed of main-group elements have been used as fundamental components. These heteroatom-containing conjugated polymers show unique optical and electronic properties (4–10). However, while metalla-aromatic systems bearing a transition metal have been known since 1979 due to the pioneering work by Thorn and Hoffmann (18), none of them have been used as building blocks for conjugated polymers. The HOMO–LUMO gaps (E_g) of metalla-aromatics are generally narrower (Fig. 1) than those of their organic counterparts (19–22). We reasoned that this feature should broaden the absorption window if polymers stemming from metalla-aromatics are achievable.Open in a separate windowFig. 1.Comparison of traditional organic skeletons with metalla-aromatic building blocks (the computed energies are in eV). (A) HOMO–LUMO gaps of classic aromatic skeletons. (B) Carbolong frameworks as potential building blocks for novel conjugated polymers with broad absorption bands and improved solubility.In recent years, we have reported a series of readily accessible metal-bridged bicyclic/polycyclic aromatics, namely carbolong complexes, which are stable in air and moisture (23–25). The addition of osmium carbynes (in carbolong complexes) and alkynes gave rise to an intriguing family of d_π–p_π conjugated systems, which function as excellent electron transport layer materials in organic solar cells (26, 27). These observations raised the following question: Can this efficient addition reaction be used to access metalla-aromatic conjugated polymers? It is noteworthy that incorporation of metalla-aromatic units into conjugated polymers is hitherto unknown. In this contribution, we disclose a polymerization reaction involving M≡C analogs of C≡C bonds, which involves a unique carbyne shuttling strategy (Fig. 2A). This led to examples of metalla-aromatic conjugated polymers (polycarbolongs) featuring metal carbyne units in the main chain. On the other hand, the development of polymerization reactions plays a crucial role in involving certain building blocks in conjugated polymers (28–32). These efficient, specific, and feasible polymerizations could open an avenue for the synthesis of conjugated polymers.Open in a separate windowFig. 2.Design of polymers and synthesis of monomers. (A) Schematic illustration of the polymerization strategy. (B) Preparation of carbolong monomers. Insert: X-ray molecular structure for the cations of complex 3. Ellipsoids are shown at the 50% probability level; phenyl groups in PPh₃ are omitted for clarity.     

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.     

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.     

Variation in Rapa Nui (Easter Island) land use indicates production and population peaks prior to European contact

Many researchers believe that prehistoric Rapa Nui society collapsed because of centuries of unchecked population growth within a fragile environment. Recently, the notion of societal collapse has been questioned with the suggestion that extreme societal and demographic change occurred only after European contact in AD 1722. Establishing the veracity of demographic dynamics has been hindered by the lack of empirical evidence and the inability to establish a precise chronological framework. We use chronometric dates from hydrated obsidian artifacts recovered from habitation sites in regional study areas to evaluate regional land-use within Rapa Nui. The analysis suggests region-specific dynamics including precontact land use decline in some near-coastal and upland areas and postcontact increases and subsequent declines in other coastal locations. These temporal land-use patterns correlate with rainfall variation and soil quality, with poorer environmental locations declining earlier. This analysis confirms that the intensity of land use decreased substantially in some areas of the island before European contact.There is ongoing debate about the demographic trajectory of Rapa Nui (or Easter Island) from its settlement around AD 1200 (1–4) until the arrival of Jesuit missionaries in the 1860s (Fig. 1) (5). The central issue is whether the Rapa Nui population experienced significant demographic decline before European contact in AD 1722. Proponents of this “pre-contact collapse” scenario suggest that environmental degradation reduced food production, and a number of researchers have elaborated a chronological model (6) that argues for a period of warfare, population reduction, and political fragmentation in the AD 1500s (7–13) or late AD 1600s (14–16). Alternately, other researchers view the archaeological evidence as favoring socio-political continuity until Western smallpox, syphilis, and tuberculosis pathogens decimated the population after European contact (17–22).Open in a separate windowFig. 1.A map of Rapa Nui showing the three study areas (pink squares) and locations mentioned in the text. Rainfall isohyets demonstrate the rain shadow effect. Note that the area in the northwest of the island appears to be quite dry, as does the area immediately west of Poike, the peninsula on the extreme east of the island. Both these areas are, in fact, drier than elevation alone would predict. Solid purple dots represent field weather stations. The red dot represents the weather station at the Mataveri airport.There is archaeological evidence for societal change on Rapa Nui, including the manufacture of obsidian spear points, the destruction of elite dwellings, habitation in refuge caves, cannibalism, a change in burial practice, and a marked ideological shift away from ceremonial platform (ahu) structures to the formation of the Birdman (tangata manu) cult centered at Orongo (see ref. 23 for a summary). These changes have been associated with the abandonment of inland field systems and houses and decreased population levels (14–16, 24). The question is when these changes occurred. Poor chronological control over the timing of past events in all these cases makes it difficult to draw firm conclusions as to whether these changes occurred before or after European contact. Oral histories recorded in the early 20th century by Routledge (25) reflect a period of precontact societal upheaval but are shrouded in mythology.Empirical evidence for societal collapse, extensive environmental degradation (other than deforestation), or warfare that could have caused such a collapse before European contact is minimal (20). A recent analysis of radiocarbon dates from throughout Rapa Nui noted the inherent (and severe) ambiguities in radiocarbon calibrations in the time period of interest but concluded that there was demographic continuity into the postcontact era as opposed to population decline during the late precontact period (19). Additional analysis of ¹⁴C and obsidian hydration dates from a smaller study region in Hanga Ho‘onu on the northeast coast also reported continuity of settlement and agricultural activity into the period of European contact (18). For environmental degradation, there is substantial evidence for deforestation (26–30) but its timing and causes have been debated (2, 31–34). Soil erosion occurred in limited areas (i.e., on the older Poike peninsula (35–37), along a small section of the northwest coast, and on the slopes of some of the smaller volcanic cones); however, there is no evidence of widespread soil erosion that could have interfered with agricultural production (38). The assertion that the proliferation of obsidian spear points is an indicator of endemic violence is challenged by lithic use-wear analysis that shows the artifacts to be used extensively in processing vegetation (39, 40).Here we identify spatial and temporal variation in the intensity of land use across portions of Rapa Nui and relate these observations to new data on spatial variation in climate and soil fertility. We base our analysis of land use on obsidian hydration dating (OHD) of tools and flakes, using 428 obsidian hydration dates developed under revised calibrations and protocols (SI Appendix, Table S1) (41, 42). Obsidian nodules were fashioned into everyday working tools and are plentiful at many archaeological sites. We use the quantity of hydration dates as a measure of the amount of discarded material over time and as a proxy for land-use intensity.     

Coevolution can reverse predator–prey cycles

A hallmark of Lotka–Volterra models, and other ecological models of predator–prey interactions, is that in predator–prey cycles, peaks in prey abundance precede peaks in predator abundance. Such models typically assume that species life history traits are fixed over ecologically relevant time scales. However, the coevolution of predator and prey traits has been shown to alter the community dynamics of natural systems, leading to novel dynamics including antiphase and cryptic cycles. Here, using an eco-coevolutionary model, we show that predator–prey coevolution can also drive population cycles where the opposite of canonical Lotka–Volterra oscillations occurs: predator peaks precede prey peaks. These reversed cycles arise when selection favors extreme phenotypes, predator offense is costly, and prey defense is effective against low-offense predators. We present multiple datasets from phage–cholera, mink–muskrat, and gyrfalcon–rock ptarmigan systems that exhibit reversed-peak ordering. Our results suggest that such cycles are a potential signature of predator–prey coevolution and reveal unique ways in which predator–prey coevolution can shape, and possibly reverse, community dynamics.Population cycles, e.g., predator–prey cycles, and their ecological drivers have been of interest for the last 90 y (1–4). Classical models of predator–prey systems, developed first by Lotka (5) and Volterra (6), share a common prediction: Prey oscillations precede predator oscillations by up to a quarter of the cycle period (7). When plotted in the predator–prey phase plane, these cycles have a counterclockwise orientation (4). These cycles are driven by density-dependent interactions between the populations. When predators are scarce, prey increase in abundance. As their food source increases, predators increase in abundance. When the predators reach sufficiently high densities, the prey population is driven down to low numbers. With a scarcity of food, the predator population crashes and the cycle repeats.While many cycles, like the classic lynx–hare cycles (Fig. 1A) (3), exhibit the above characteristics, predator–prey cycles with different characteristics have also been observed. For example, antiphase cycles where predator oscillations lag behind prey oscillations by half of the cycle period (Fig. 1B) (8) and cryptic cycles where the predator population oscillates while the prey population remains effectively constant (Fig. 1C) (9) have been observed in experimental systems. This diversity of cycle types motivates the question, “Why do cycle characteristics differ across systems?”Open in a separate windowFig. 1.Examples of different kinds of predator–prey cycles. (A) Counterclockwise lynx–hare cycles (3). (B) Antiphase rotifer–algal cycles (8). (C) Cryptic phage-bacteria cycles (9). In all time series, red and blue correspond to predator and prey, respectively. See SI Text, section C for data sources.In Lotka–Volterra and other ecological models, predator and prey life history traits are assumed to be fixed. However, empirical studies across taxa have shown that prey (9–16) and predators (17–20) can evolve over ecological time scales. That is, changes in allele frequencies (and associated phenotypes) can occur at the same rate as changes in population densities or spatial distributions and alter the ecological processes driving the changes in population densities or distributions; this phenomenon has been termed “eco-evolutionary dynamics” (21, 22). Furthermore, predator–prey coevolution is important for driving community composition and dynamics (16, 19, 20, 23–26). This body of work suggests that the interaction between ecological and evolution processes has the potential to alter the ecological dynamics of communities.Experimental (8, 9, 13, 14) and theoretical studies (13, 27, 28) have shown that prey or predator evolution alone can alter the characteristics of predator–prey cycles and drive antiphase (Fig. 1B) and cryptic (Fig. 1C) cycles. Additional theoretical work has shown that predator–prey coevolution can also drive antiphase and cryptic cycles (29). Thus, evolution in one or both species is one mechanism through which antiphase or cryptic predator–prey cycles can arise. However, it is unclear if coevolution can drive additional kinds of cycles with characteristics different from those in Fig. 1.The main contribution of this study is to show that predator–prey coevolution can drive unique cycles where peaks in predator abundance precede peaks in prey abundance, the opposite of what is predicted by classical ecological models. We refer to these reversed cycles as “clockwise cycles.” The theoretical and empirical finding of clockwise cycles represents an example of how evolution over ecological time scales can alter community-level dynamics.     

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.     

An image-computable model of how endogenous and exogenous attention differentially alter visual perception

Attention alters perception across the visual field. Typically, endogenous (voluntary) and exogenous (involuntary) attention similarly improve performance in many visual tasks, but they have differential effects in some tasks. Extant models of visual attention assume that the effects of these two types of attention are identical and consequently do not explain differences between them. Here, we develop a model of spatial resolution and attention that distinguishes between endogenous and exogenous attention. We focus on texture-based segmentation as a model system because it has revealed a clear dissociation between both attention types. For a texture for which performance peaks at parafoveal locations, endogenous attention improves performance across eccentricity, whereas exogenous attention improves performance where the resolution is low (peripheral locations) but impairs it where the resolution is high (foveal locations) for the scale of the texture. Our model emulates sensory encoding to segment figures from their background and predict behavioral performance. To explain attentional effects, endogenous and exogenous attention require separate operating regimes across visual detail (spatial frequency). Our model reproduces behavioral performance across several experiments and simultaneously resolves three unexplained phenomena: 1) the parafoveal advantage in segmentation, 2) the uniform improvements across eccentricity by endogenous attention, and 3) the peripheral improvements and foveal impairments by exogenous attention. Overall, we unveil a computational dissociation between each attention type and provide a generalizable framework for predicting their effects on perception across the visual field.

Endogenous and exogenous spatial attention prioritize subsets of visual information and facilitate their processing without concurrent eye movements (1–3). Selection by endogenous attention is goal-driven and adapts to task demands, whereas exogenous attention transiently and automatically orients to salient stimuli (1–3). In most visual tasks, both types of attention typically improve visual perception similarly [e.g., acuity (4–6), visual search (7, 8), perceived contrast (9–11)]. Consequently, models of visual attention do not distinguish between endogenous and exogenous attention (e.g., refs. 12–19). However, stark differences also exist. Each attention type differentially modulates neural responses (20, 21) and fundamental properties of visual processing, including temporal resolution (22, 23), texture sensitivity (24), sensory tuning (25), contrast sensitivity (26), and spatial resolution (27–34).The effects of endogenous and exogenous attention are dissociable during texture segmentation, a visual task constrained by spatial resolution [reviews (1–3)]. Whereas endogenous attention optimizes spatial resolution to improve the detection of an attended texture (32–34), exogenous attention reflexively enhances resolution even when detrimental to perception (27–31, 34). Extant models of attention do not explain these well-established effects.Two main hypotheses have been proposed to explain how attention alters spatial resolution. Psychophysical studies ascribe attentional effects to modulations of spatial frequency (SF) sensitivity (30, 33). Neurophysiological (13, 35, 36) and neuroimaging (37, 38) studies bolster the idea that attention modifies spatial profiles of neural receptive fields (RFs) (2). Both hypotheses provide qualitative predictions of attentional effects but do not specify their underlying neural computations.Differences between endogenous and exogenous attention are well established in segmentation tasks and thus provide an ideal model system to uncover their separate roles in altering perception. Texture-based segmentation is a fundamental process of midlevel vision that isolates regions of local structure to extract figures from their background (39–41). Successful segmentation hinges on the overlap between the visual system’s spatial resolution and the levels of detail (i.e., SF) encompassed by the texture (39, 41, 42). Consequently, the ability to distinguish between adjacent textures varies as resolution declines toward the periphery (43–46). Each attention type differentially alters texture segmentation, demonstrating that their effects shape spatial resolution [reviews (1–3)].Current models of texture segmentation do not explain performance across eccentricity and the distinct modulations by attention. Conventional models treat segmentation as a feedforward process that encodes the elementary features of an image (e.g., SF and orientation), transforms them to reflect the local structure (e.g., regions of similarly oriented bars), and then pools across space to emphasize texture-defined contours (39, 41, 47). Few of these models account for variations in resolution across eccentricity (46, 48, 49) or endogenous (but not exogenous) attentional modulations (18, 50). All others postulate that segmentation is a “preattentive” (42) operation whose underlying neural processing is impervious to attention (39, 41, 46–49).Here, we develop a computational model in which feedforward processing and attentional gain contribute to segmentation performance. We augment a conventional model of texture processing (39, 41, 47). Our model varies with eccentricity and includes contextual modulation within local regions in the stimulus via normalization (51), a canonical neural computation (52). The defining characteristic of normalization is that an individual neuron is (divisively) suppressed by the summed activity of neighboring neurons responsive to different aspects of a stimulus. We model attention as multiplicative gains [attentional gain factors (15)] that vary with eccentricity and SF. Attention shifts sensitivity toward fine or coarse spatial scales depending on the range of SFs enhanced.Our model is image-computable, which allowed us to reproduce behavior directly from grayscale images used in psychophysical experiments (6, 26, 27, 29–33). The model explains three signatures of texture segmentation hitherto unexplained within a single computational framework (Fig. 1): 1) the central performance drop (CPD) (27–34, 43–46) (Fig. 1A), that is, the parafoveal advantage of segmentation over the fovea; 2) the improvements in the periphery and impairments at foveal locations induced by exogenous attention (27–32, 34) (Fig. 1B); and 3) the equivalent improvements across eccentricity by endogenous attention (32–34) (Fig. 1C).Open in a separate windowFig. 1.Signatures of texture segmentation. (A) CPD. Shaded region depicts the magnitude of the CPD. Identical axis labels are omitted in B and C. (B) Exogenous attention modulation. Exogenous attention improves segmentation performance in the periphery and impairs it near the fovea. (C) Endogenous attention modulation. Endogenous attention improves segmentation performance across eccentricity.Whereas our analyses focused on texture segmentation, our model is general and can be applied to other visual phenomena. We show that the model predicts the effects of attention on contrast sensitivity and acuity, i.e., in tasks in which both endogenous and exogenous attention have similar or differential effects on performance. To preview our results, model comparisons revealed that normalization is necessary to elicit the CPD and that separate profiles of gain enhancement across SF (26) generate the effects of exogenous and endogenous attention on texture segmentation. A preferential high-SF enhancement reproduces the impairments by exogenous attention due to a shift in visual sensitivity toward details too fine to distinguish the target at foveal locations. The transition from impairments to improvements in the periphery results from exogenous attentional gain gradually shifting to lower SFs that are more amenable for target detection. Improvements by endogenous attention result from a uniform enhancement of SFs that encompass the target, optimizing visual sensitivity for the attended stimulus across eccentricity.     

Self-assembly of amphiphilic Janus dendrimers into uniform onion-like dendrimersomes with predictable size and number of bilayers

A constitutional isomeric library synthesized by a modular approach has been used to discover six amphiphilic Janus dendrimer primary structures, which self-assemble into uniform onion-like vesicles with predictable dimensions and number of internal bilayers. These vesicles, denoted onion-like dendrimersomes, are assembled by simple injection of a solution of Janus dendrimer in a water-miscible solvent into water or buffer. These dendrimersomes provide mimics of double-bilayer and multibilayer biological membranes with dimensions and number of bilayers predicted by the Janus compound concentration in water. The simple injection method of preparation is accessible without any special equipment, generating uniform vesicles, and thus provides a promising tool for fundamental studies as well as technological applications in nanomedicine and other fields.Most living organisms contain single-bilayer membranes composed of lipids, glycolipids, cholesterol, transmembrane proteins, and glycoproteins (1). Gram-negative bacteria (2, 3) and the cell nucleus (4), however, exhibit a strikingly special envelope that consists of a concentric double-bilayer membrane. More complex membranes are also encountered in cells and their various organelles, such as multivesicular structures of eukaryotic cells (5) and endosomes (6), and multibilayer structures of endoplasmic reticulum (7, 8), myelin (9, 10), and multilamellar bodies (11, 12). This diversity of biological membranes inspired corresponding biological mimics. Liposomes (Fig. 1) self-assembled from phospholipids are the first mimics of single-bilayer biological membranes (13–16), but they are polydisperse, unstable, and permeable (14). Stealth liposomes coassembled from phospholipids, cholesterol, and phospholipids conjugated with poly(ethylene glycol) exhibit improved stability, permeability, and mechanical properties (17–20). Polymersomes (21–24) assembled from amphiphilic block copolymers exhibit better mechanical properties and permeability, but are not always biocompatible and are polydisperse. Dendrimersomes (25–28) self-assembled from amphiphilic Janus dendrimers and minidendrimers (26–28) have also been elaborated to mimic single-bilayer biological membranes. Amphiphilic Janus dendrimers take advantage of multivalency both in their hydrophobic and hydrophilic parts (23, 29–32). Dendrimersomes are assembled by simple injection (33) of a solution of an amphiphilic Janus dendrimer (26) in a water-soluble solvent into water or buffer and produce uniform (34), impermeable, and stable vesicles with excellent mechanical properties. In addition, their size and properties can be predicted by their primary structure (27). Amphiphilic Janus glycodendrimers self-assemble into glycodendrimersomes that mimic the glycan ligands of biological membranes (35). They have been demonstrated to be bioactive toward biomedically relevant bacterial, plant, and human lectins, and could have numerous applications in nanomedicine (20).Open in a separate windowFig. 1.Strategies for the preparation of single-bilayer vesicles and multibilayer onion-like vesicles.More complex and functional cell mimics such as multivesicular vesicles (36, 37) and multibilayer onion-like vesicles (38–40) have also been discovered. Multivesicular vesicles compartmentalize a larger vesicle (37) whereas multibilayer onion-like vesicles consist of concentric alternating bilayers (40). Currently multibilayer vesicles are obtained by very complex and time-consuming methods that do not control their size (39) and size distribution (40) in a precise way. Here we report the discovery of “single–single” (28) amphiphilic Janus dendrimer primary structures that self-assemble into uniform multibilayer onion-like dendrimersomes (Fig. 1) with predictable size and number of bilayers by simple injection of their solution into water or buffer.     

Arenium ions are not obligatory intermediates in electrophilic aromatic substitution

Our computational and experimental investigation of the reaction of anisole with Cl₂ in nonpolar CCl₄ solution challenges two fundamental tenets of the traditional S_EAr (arenium ion) mechanism of aromatic electrophilic substitution. Instead of this direct substitution process, the alternative addition–elimination (AE) pathway is favored energetically. This AE mechanism rationalizes the preferred ortho and para substitution orientation of anisole easily. Moreover, neither the S_EAr nor the AE mechanisms involve the formation of a σ-complex (Wheland-type) intermediate in the rate-controlling stage. Contrary to the conventional interpretations, the substitution (S_EAr) mechanism proceeds concertedly via a single transition state. Experimental NMR investigations of the anisole chlorination reaction course at various temperatures reveal the formation of tetrachloro addition by-products and thus support the computed addition–elimination mechanism of anisole chlorination in nonpolar media. The important autocatalytic effect of the HCl reaction product was confirmed by spectroscopic (UV-visible) investigations and by HCl-augmented computational modeling.Interest in the chemistry of electrophilic aromatic substitution reactions continues because of their widespread application for the production of a great variety of chemicals and materials (1–4). Electrophilic substitution, considered to be the most characteristic reaction of aromatic systems, is typically described in textbooks, monographs, and reviews by the two-stage S_EAr mechanism depicted in Fig. 1 (5–11). Arenium ion (σ-complex) intermediates are often ascribed to Wheland (9) inaccurately, since Pfeiffer and Wizinger (10) laid out the principles of such species for bromination in 1928. Following Brown and Pearsall (11), they are widely believed to have σ-complex structures. Arenium ions (σ-complexes) (9–11) are widely accepted to be obligatory intermediates and are used to rationalize ortho/para vs. meta position orientation preferences (6–11).Open in a separate windowFig. 1.Typical depiction of the arenium ion mechanism for S_EAr reactions.We now reinforce our challenges (12, 13) of this conventional “reaction mechanism paradigm” (14) by a combined computational and experimental study of the facile chlorination of anisole (methoxybenzene) with Cl₂ in CCl₄ solution (15, 16). We find that Fig. 1 is not the favored pathway. Instead, addition reactions of Cl₂ to anisole have the lowest activation energies (Fig. 2). Ready HCl elimination from the initially formed adducts leads to ortho- and para-chloroanisole as the predominate products. This addition–elimination (AE) mechanism (the historical antecedent to Fig. 1) (17–26) predicts the same positional orientation as the usually assumed direct substitution (“S_EAr”) alternative. Instead of this classic S_EAr mechanism (Fig. 1), we find that direct concerted substitution, not involving an arenium ion, σ-complex (“Wheland”) (9–11) intermediate, competes energetically with the AE route. Like some earlier computational studies on aromatic substitution (12, 13, 27, 28) (Rzepa H, www.ch.imperial.ac.uk/rzepa/blog/?p=2423, accessed March 10, 2013), our study finds no such intermediates in the direct substitution of anisole by Cl₂. A concerted mechanism without an arenium ion intermediate was computed at some levels for the related arene nitrosation, but reaction medium and counter ion effects were not considered. Gwaltney et al. (28) reported a single concerted transition state after reoptimizing all saddle points at CCSD(T)/6-31G(d,p) and modeling bulk solvation by the Onsager approximation, and Rzepa (www.ch.imperial.ac.uk/rzepa/blog/?p=2423, accessed March 10, 2013) also found a concerted transition state including a trifluoroacetate counterion. Instead, one-step reactions via single transition states take place (Fig. 2). Our experimental investigations of the chlorination of anisole in CCl₄ solution revealed tetrachloro by-products, which must have arisen by further reaction of intermediate dichloro-adducts. Both our UV-visible (UV-VIS) spectroscopic investigation and our theoretical modeling of this reaction clearly verified the autocatalytic effect of the HCl by-product, in harmony with Andrews and Keefer’s (29, 30) early experimental kinetic studies of the chlorination of arenes, which found that HCl reduces the activation barriers significantly.Open in a separate windowFig. 2.The HCl-catalyzed concerted and addition–elimination pathways of para-chlorination of anisole in nonpolar media.We also applied reliable theoretical methods to model a typical experimental example of the highly investigated S_EAr electrophilic aromatic halogenations, the electrophilic chlorination of anisole by molecular chlorine in simulated CCl₄ solution (15, 16). Although the elucidation of the classic S_EAr mechanism [Fig. 1, involving the initial formation of a π-complex, followed by a transition state leading to a σ-complex (arenium) intermediate in the rate-controlling stage, and, finally, proton loss from the ipso-position leading to the reaction product] is considered to be a triumph of physical organic chemistry (1, 31–37), an alternative addition–elimination pathway leading to substitution products has been discussed since the 19th century (19–26, 38, 39). Nevertheless, it is commonly believed that the classic multistep S_EAr mechanism involving the formation of a σ-complex intermediate in the rate-controlling stage is the only mechanistic route to aromatic substitution products. Our present and previous (12, 13) results challenge the generality of such traditional interpretations. Although the initial stages of the alternative AE route seem unattractive because aromaticity is lost, many arenes are known experimentally to give addition products in considerable amounts (19–26, 38, 39). Thus, de la Mare (21, 25, 38, 39) demonstrated the formation of halogen adduct intermediates. Polybenzenoid hydrocarbons (PBHs) react with halogens to give isolable addition products, which then give substitution products easily by hydrogen halide elimination (23). Our computational investigations of arene bromination with molecular bromine (12) and sulfonation with SO₃ (13) provided clear evidence that the mechanisms of the inherent substitution reactions (i.e., uncatalyzed, gas phase, or weakly solvated) are concerted and do not involve the conventional σ-complex (or any other) intermediates. Moreover, the energetics of the bromination processes document the significance of competition between AE and direct substitution mechanisms leading to the same substitution products. Thus, the computed barrier in a simulated nonpolar (CCl₄) medium is 4 kcal/mol lower for Br₂ addition to benzene (followed by HBr elimination) than that for the direct substitution pathway to bromobenzene (12).Previous theoretical studies of electrophilic aromatic halogenation processes have been based on the classic S_EAr mechanism, involving arenium ion intermediates (Fig. 1). Osamura et al.’s (40) Hartree-Fock computations of the AlCl₃-catalyzed electrophilic aromatic chlorination mechanism found an initial π-complex, a transition state preceding the intermediate σ-complex, and a second transition state leading to final products. Aluminum chloride was important as a Lewis acid catalyst throughout the process. AlCl₃ coordination polarizes Cl₂ and thereby assists its reaction with the arene. Rasokha and Kochi (41) considered the interaction of Br₂ with benzene and toluene in detail in their survey of theoretical and experimental data on the prereactive charge-transfer complexes in electrophilic aromatic substitutions. They argued that the structures and properties of the prereactive complexes provide important mechanistic insights for the S_EAr reactions. Wei et al.’s (42) theoretical study of the iodination of anisole by iodine monochloride at the B3LYP/6-311G* and MP2//B3LYP/6-311G* levels (B3LYP, Becke''s three parameter hybrid functional, using the Lee-Yang-Parr correlation functional; MP2, second order Møller-Plesset perturbation theory computations) found that the highest energy transition state precedes the formation of an intermediate, which they interpreted to be a σ-complex. Instead, the structure of this complex represents a protonated iodobenzene. Volkov et al.’s MP2/LANL2DZ(d)+ study (43) of the chlorination of benzene established that dimers of group 13 metal halides catalyzed the processes more effectively. Optimized geometries of π- and σ-complexes as well as transition structures were reported. Theoretical investigations by Ben-Daniel et al. (44) and by Filimonov et al. (45) of the chlorination of benzene with Cl₂ (and other related processes) reported structural details of transition states purported to lead to the chlorobenzene product. Our reinvestigations revealed errors in major suppositions of both these studies. Our IRC computations show clearly that the transition states in question lead to 1,2 Cl₂–benzene addition products (rather than to chlorobenzene). Zhang and Lund (46) investigated the neat chlorination of toluene by Cl₂ experimentally and theoretically at B3LYP/cc-pVTZ(-f) [cc-pVTZ(-f), correlation consistent polarized triple-zeta without f-functions basis set]. Although we verified their reported geometry of the concerted transition state (figure 6 in ref. 46), our stability check revealed that its wavefunction is unstable. This casts doubt on their conclusions because of the homolysis vs. heterolysis issues. In contrast, all wavefunctions in our paper were checked and all are stable. Most prior theoretical studies of S_EAr halogenations did not consider the connections between transition states, intermediates, and products explicitly, as we have done.Experimental findings not always have been in accord with the prevailing mechanistic assumption for aromatic halogenation: that arenium ion formation is the rate-limiting step. Thus, Olah et al. (47), Kochi and coworkers (48), and Fukuzumi and Kochi (49) have emphasized that substrate and positional selectivity are inconsistent (e.g., low toluene/benzene reactivity ratios but high toluene ortho–para vs. meta regiospecificity) for some electrophiles under certain conditions. This disparity indicates the existence of at least one other mechanistic pathway. It has been suggested that π-complexes may control product formation. Olah et al.’s (47) kinetics of the ferric chloride-catalyzed bromination of benzene and alkyl benzenes provided strong evidence for low substrate selectivity in the rate-determining step, which precedes the formation of a σ-complex intermediate (Fig. 1). High positional selectivity is governed by the transition state associated with the second step of the reaction.However, our earlier study (50) examined the possible participation of π-complexes in the key mechanistic steps of S_EAr bromination reactions in detail but found no link between the energy of formation of these complexes and the overall reactivity. Although there is no doubt that π-complexes form easily (via essentially barrierless processes) in most S_EAr reactions after mixing the electrophile and the aromatic substrate, it is unlikely that these low-energy “bystander” structures influence rates of S_EAr reactions significantly. Thus, the lack of accord between substrate and positional selectivity, established by Olah et al. (47), Kochi and coworkers (48), and Fukuzumi and Kochi (49) may be due to other mechanistic differences. De la Mare and Bolton (21) and de la Mare (51) have stressed the plurality of aromatic substitution mechanisms, depending on the substrate and the conditions.Reactive substrates are known to undergo uncatalyzed aromatic substitution in nonpolar solvents at room temperature. Thus, our computational investigations modeled Watson’s careful experiments on the chlorination of anisole in CCl₄ at 25 °C (15, 16). His low conversion (25%) conditions for chlorophenol permitted more accurate determination of the initial product ratios (and avoided further Cl₂ additions to 4-chloroanisole, which ultimately gave 1,3,4,5,6-pentachloro-4-methoxycyclohexene). After introduction of gaseous Cl₂ into a CCl₄ solution of anisole for 1 h, the products were 4-chloroanisole (76%), 2-chloroanisole (13.6%), 2,6-dichloro anisole (2.1%), 2,4-dichloroanisole (3.0%), and 2,4,6-trichloroanisole (0.4%).Analogous chlorinations of phenol, 2-methylphenol, and 2-chlorophenol in CCl₄ also have been carried out with high conversion rates at the reflux temperature (79 °C) (16). Chlorination of phenol with Cl₂ in CCl₄ has been reported by other groups (52, 53).     

Inside-out regulation of E-cadherin conformation and adhesion

Cadherin cell–cell adhesion proteins play key roles in tissue morphogenesis and wound healing. Cadherin ectodomains bind in two conformations, X-dimers and strand-swap dimers, with different adhesive properties. However, the mechanisms by which cells regulate ectodomain conformation are unknown. Cadherin intracellular regions associate with several actin-binding proteins including vinculin, which are believed to tune cell–cell adhesion by remodeling the actin cytoskeleton. Here, we show at the single-molecule level, that vinculin association with the cadherin cytoplasmic region allosterically converts weak X-dimers into strong strand-swap dimers and that this process is mediated by myosin II–dependent changes in cytoskeletal tension. We also show that in epithelial cells, ∼70% of apical cadherins exist as strand-swap dimers while the remaining form X-dimers, providing two cadherin pools with different adhesive properties. Our results demonstrate the inside-out regulation of cadherin conformation and establish a mechanistic role for vinculin in this process.

E-cadherins (Ecads) are essential, calcium-dependent cell–cell adhesion proteins that play key roles in the formation of epithelial tissue and in the maintenance of tissue integrity. Ecad adhesion is highly plastic and carefully regulated to orchestrate complex movement of epithelial cells, and dysregulation of adhesion is a hallmark of numerous cancers (1). However, little is known about how cells dynamically regulate the biophysical properties of individual Ecads.The extracellular region of Ecads from opposing cells bind in two distinct trans orientations: strand-swap dimers and X-dimers (Fig. 1 A and B). Strand-swap dimers are the stronger cadherin adhesive conformation and are formed by the exchange of conserved tryptophan (Trp) residues between the outermost domains of opposing Ecads (2–4). In contrast, X-dimers, which are formed by extensive surface interactions between opposing Ecads, are a weaker adhesive structure and serve as an intermediate during the formation and rupture of strand-swap dimers (5–7). Using cell-free, single-molecule experiments we previously showed that X-dimers and strand-swap dimers can be distinguished based on their distinctly different response to mechanical force. When a strand-swap dimer is pulled, its lifetime decreases with increasing force, resulting in the formation of a slip bond (8, 9) (Fig. 1B). In contrast, an X-dimer responds to pulling force by forming a catch bond, where bond lifetime initially increases up to a threshold force and then subsequently decreases (8, 10) (Fig. 1B). It has also been shown that wild-type Ecad ectodomains in solution can interconvert between X-dimer and strand-swap dimer conformations (9, 11). However, the biophysical mechanisms by which Ecad conformations (and adhesion) are regulated on the cell surface are unknown.Open in a separate windowFig. 1.Overview of experiment. (A) The extracellular region of Ecad from opposing cells mediates adhesion. The cytoplasmic region of Ecad associates either directly or indirectly with p120 catenin, β-catenin, α-catenin, vinculin, and F-actin. (B) Strand-swap dimers form slip bonds (blue) and X-dimers form catch bonds (red). Ecads interconvert between these two dimer conformations. Structures were generated from the crystal structure of mouse Ecad (PDB ID code 3Q2V); the X-dimer was formed by alignment to an X-dimer crystal structure (PDB ID code 3LNH). (C) Graphics showing the cell lines used in experiments and Western blot analysis of corresponding cell lysates.The cytoplasmic region of Ecad associates with the catenin family of proteins, namely, p120-catenin, β-catenin, and α-catenin. The Ecad–catenin complex, in turn, links to filamentous actin (F-actin) either by the direct binding of α-catenin and F-actin or by the indirect association of α-catenin and F-actin via vinculin (12) (Fig. 1A). Adhesive forces transmitted across intercellular junctions by Ecad induce conformational changes in α-catenin (13, 14), strengthen F-actin binding (15), and recruit vinculin to the sites of force application (16, 17). However, vinculin and α-catenin do not merely serve as passive cytoskeletal linkers; they also dynamically modulate cytoskeletal rearrangement and recruit myosin to cell–cell junctions (13, 18–20). Studies show that α-catenin and vinculin play important roles in strengthening and stabilizing Ecad adhesion: bead-twisting experiments show force-induced stiffening of Ecad-based junctions and cell doublet stretching experiments demonstrate reinforcement of cell–cell adhesion in vinculin- and α-catenin–dependent manners (18, 19, 21).Currently, actin anchorage and cytoskeletal remodeling are assumed to be the exclusive mechanisms by which α-catenin and vinculin strengthen Ecad adhesion (22–24). Here, we directly map the allosteric effects of cytoplasmic proteins on Ecad ectodomain conformation and demonstrate, at the single-molecule level, that vinculin association with the Ecad cytoplasmic region switches X-dimers to strand-swap dimers. We show that cytoskeletal tension, due to vinculin-mediated recruitment of myosin II, regulates Ecad ectodomain structure and adhesion. Finally, we demonstrate that only ∼50% of Ecads are linked to the underlying cytoskeleton and that while about 70% of Ecads form strand-swap dimers the remaining form X-dimers, which provides cells with two Ecad pools with different adhesive properties.     

Opacity and conductivity measurements in noble gases at conditions of planetary and stellar interiors

The noble gases are elements of broad importance across science and technology and are primary constituents of planetary and stellar atmospheres, where they segregate into droplets or layers that affect the thermal, chemical, and structural evolution of their host body. We have measured the optical properties of noble gases at relevant high pressures and temperatures in the laser-heated diamond anvil cell, observing insulator-to-conductor transformations in dense helium, neon, argon, and xenon at 4,000–15,000 K and pressures of 15–52 GPa. The thermal activation and frequency dependence of conduction reveal an optical character dominated by electrons of low mobility, as in an amorphous semiconductor or poor metal, rather than free electrons as is often assumed for such wide band gap insulators at high temperatures. White dwarf stars having helium outer atmospheres cool slower and may have different color than if atmospheric opacity were controlled by free electrons. Helium rain in Jupiter and Saturn becomes conducting at conditions well correlated with its increased solubility in metallic hydrogen, whereas a deep layer of insulating neon may inhibit core erosion in Saturn.Noble gases play important roles in the evolution and dynamics of planets and stars, especially where they appear in a condensed, purified state. In gas giant planets, helium and neon can precipitate as rain in metallic hydrogen envelopes, leading to planetary warming and specifically the anomalously slow cooling of Saturn (1–8). In white dwarf stars cooling can be especially fast due to the predicted low opacity of dense helium atmospheres, affecting the calibration of these objects as cosmological timekeepers (9–12). In these systems, the transformation of dense noble gases (particularly He) from optically transparent insulators to opaque electrical conductors is of special importance (2, 9, 11, 12).Dense noble gases are expected to show systematic similarities in their properties at extreme conditions (13–17); however, a general understanding of their insulator–conductor transformation remains to be established. Xe is observed to metallize near room temperature under pressures similar to those at Earth’s core–mantle boundary (18, 19). Ar and He are observed to conduct only at combined high pressure and temperature (12, 13, 17). Ne is predicted to have the highest metallization pressure of all known materials—10³ times that of Xe and 10 times that of He (14, 18, 20, 21)—and has never been documented outside of its insulating state. Experimental probes of extreme densities and temperatures in noble gases have previously relied on dynamic compression by shock waves (12, 13, 17, 22–24). However, in such adiabatic experiments, light and compressible noble gases heat up significantly and can ultimately reach density maxima (12, 13, 17, 21, 24, 25), so that conditions created often lie far from those deep within planets (7, 8) and stars (9).Here we report experiments in the laser-heated diamond anvil cell (15, 16, 26–29) on high-density and high-temperature states of the noble gases Xe, Ar, Ne, and He (Fig. 1). Rapid heating and cooling of compressed samples using pulsed laser heating (26, 27) is coupled with time domain spectroscopy of thermal emission (26) to determine sample temperature and transient absorption to establish corresponding sample optical properties (Figs. S1 and S2). A sequence of heat cycles to increasing temperature documents optical changes in these initially transparent insulators.Open in a separate windowFig. 1.Creating and probing extreme states of noble gases. (A) Configuration of laser heating and transient absorption probing of the diamond anvil cell, with probe beams transmitted through the cell into the detection system. (B) Microscopic view of the diamond cell cavity, which contains a noble gas sample and a metal foil (Ir) which converts laser radiation to heat and has small hole at the heated region through which probe beams are transmitted to test optical character of samples. (C) Finite element model (26) (Fig. S3) of the temperature distribution in heated Ar at 51 GPa (Fig. 2), with solid–melt (16) and insulator–conductor (α = 0.1 μm⁻¹) boundaries in the sample marked dashed and dotted, respectively. (D) Schematic of time domain probing during transient heating. Temperature is determined from thermal emission (red) and absorption from transmitted probe beams: a continuous laser (cw; green) and pulsed supercontinuum broadband (bb; blue).     

Entropic factors provide unusual reactivity and selectivity in epoxide-opening reactions promoted by water

Despite the myriad of selective enzymatic reactions that occur in water, chemists have rarely capitalized on the unique properties of this medium to govern selectivity in reactions. Here we report detailed mechanistic investigations of a water-promoted reaction that displays high selectivity for what is generally a disfavored product. A combination of structural and kinetic data indicates not only that synergy between substrate and water suppresses undesired pathways but also that water promotes the desired pathway by stabilizing charge in the transition state, facilitating proton transfer, doubly activating the substrate for reaction, and perhaps most remarkably, reorganizing the substrate into a reactive conformation that leads to the observed product. This approach serves as an outline for a general strategy of exploiting solvent-solute interactions to achieve unusual reactivity in chemical reactions. These findings may also have implications in the biosynthesis of the ladder polyether natural products, such as the brevetoxins and ciguatoxins.Given its simple structure and low molecular weight, water is a remarkably complex substance. Several landmark investigations (1) have revealed the ability of water to self-assemble to form sophisticated dynamic hydrogen bond networks, which accounts for its unusual properties, such as its high boiling point and high surface tension (2). Despite the unique properties that it offers and that nature has exploited, water is generally eschewed in synthetic chemistry largely because of chemical incompatibility with many commonly used reagents and the low aqueous solubility of many organic molecules coupled with the attendant assumption that homogeneity is required for reactivity. Although several important examples of remarkable reactivity have been documented for reactions carried out in water (3–5), on the surface of water (6, 7), or in micelles suspended in water (8, 9), utilization of the “biological solvent” in organic reactions remains uncommon.We became acutely aware of the remarkable properties of water when we discovered that neutral aqueous solutions of epoxy alcohol 1a underwent a spontaneous and selective “endo” cyclization reaction to form 6,6-fused bicyclic product 2a (Fig. 1) (10–12). Exceptional reactivity and selectivity were observed only when two criteria were satisfied: The substrate contained a six-membered tetrahydropyran ring, and the solvent used was water at pH 7.0. These surprising results were counter to a set of general empirical rules put forth by Baldwin for cyclization reactions, which state that the smaller ring product resulting from what is commonly called an exo cyclization is favored for similar reactions (13). The tetrahydropyranol ring, therefore, appeared to “template” the endo cyclization pathway to form the larger ring product with water playing a critical yet heretofore unknown role.Open in a separate windowFig. 1.Endo selective cyclization of epoxy alcohols templated by a tetrahydropyran ring(s) and promoted by water.Moreover, 2a is a substructure found in a large family of natural products commonly referred to as the ladder polyethers (e.g., brevetoxin B, S) (14–16). These constituents of harmful algal blooms (a.k.a., red tide) have attracted significant attention (17) because of the remarkable structural regularities that temper their apparent complexity. Several biogenetic pathways for the construction of rings of these natural products have been proposed and generally involve a cascade of cyclizations by way of epoxide-opening reactions (Fig. 2) (18–23). However, this hypothesis requires that all of the epoxide ring-opening events proceed with atypical endo regioselectivity.Open in a separate windowFig. 2.Biosynthetic proposal for the formation of the ladder polyether natural product brevetoxin B, a potent neurotoxin.Our discovery that reactions of 1a in water preferentially lead to the endo product 2a rather than exo product 3a provided a possible means to overcome this obstacle. Further support was provided when we demonstrated that cascade reactions akin to those proposed for the biosynthesis of the natural products was possible with the selective endo cyclization of di- and triepoxide analogs, such as 4 and 6, respectively (Fig. 1). Once again selective reactions were only observed when reactions were carried out in neutral water.These striking results and their possible relevance to the biogenesis of the ladder polyether natural products prompted us to investigate the mechanism of the cyclization reaction. Here we report the culmination of these efforts. They reveal an intimate connection between water and the tetrahydropyran ring (template) of 1a, provide support for the feasibility of the biosynthetic proposal for the ladder polyether natural products, and demonstrate the importance of solvent–substrate interactions for promoting selectivity. We believe that this last concept may have broad implications and a different means for chemists to attain unusual selectivity for a variety of chemical reactions.     

Submersed micropatterned structures control active nematic flow,topology, and concentration

Coupling between flows and material properties imbues rheological matter with its wide-ranging applicability, hence the excitement for harnessing the rheology of active fluids for which internal structure and continuous energy injection lead to spontaneous flows and complex, out-of-equilibrium dynamics. We propose and demonstrate a convenient, highly tunable method for controlling flow, topology, and composition within active films. Our approach establishes rheological coupling via the indirect presence of fully submersed micropatterned structures within a thin, underlying oil layer. Simulations reveal that micropatterned structures produce effective virtual boundaries within the superjacent active nematic film due to differences in viscous dissipation as a function of depth. This accessible method of applying position-dependent, effective dissipation to the active films presents a nonintrusive pathway for engineering active microfluidic systems.

Active fluids are inherently out of equilibrium; they locally transform internal energy into material stresses that can result in spontaneous hydrodynamic motion. An increasing number of biophysical systems, including colonies of bacilliform microbes (1–4), cellular monolayers (5–9), and subcellular filaments (10–12), display such collective active motion, orientational order, and topological singularities. Controlling active dynamics is essential not only to fully understanding how such biological systems employ self-generated stresses but also, in order to develop active microfluidic devices.To this end, recent work considers how confining walls (13–15), arrangements of obstacles (16, 17), and the dynamics of topological defects (18) dictate active nematic flow. Control of active material concentration has been studied from the perspectives of coexistence of phases in self-propelled rods (19–21) and motility-induced phase separation (22–24). Controlled accumulation and depletion of active matter have been engineered in bacterial systems to concentrate cells (25, 26) and to drive bacterial-ratchet motors (27–29). Similarly, substrate gradients modify cellular motility, driving density variation (30) and directed migration (31, 32).In addition to varying concentration and flow, topology has been controlled by including externally driven flows (33–35) and curvature (36, 37). Recent work shows that locally altering activity modifies defect populations (38–41), and anisotropic smectic sublayers below active nematic sheets can constrain orientation (42). Such studies demonstrate how underlying sublayer properties have pronounced effects on active dynamics and suggest approaches for engineering control of active matter.We propose a micropattern-based method for controlling active nematic dynamics without contiguous contact with active films. By patterning oil-submersed solid substrates below two-dimensional (2D) active nematic films with geometrical structures of differing height, we achieve effective virtual boundaries within active films that control topological defect populations, collective flow, and concentration of active nematic material without penetrating the film. By implementing underlying submersed patterned microstructures, we tune the depth of the oil layer to adjust dissipation within the superjacent film and thereby, generate a highly tunable technique for controlling the active dynamics. Presently, we introduce four initial submersed structures: micropatterned trenches (Fig. 1 A–C), undulated substrates (SI Appendix, Fig. S1), stairways (Fig. 1 D–F), and pillars (Fig. 1 G–I).Open in a separate windowFig. 1.Submersed micropatterns control active nematic dynamics. (A–C) Trench setup. An active film resides at the oil–water interface above different substrate depths. The active flows drag the underlying oil layer, but viscous dissipation is depth dependent, affecting active nematic film dynamics. (B) Fluorescence microscope image of the active nematic bundled microtubule film above a submersed trench. (Scale bar: 250 μm.) (C) Simulation results for the vorticity field within the superjacent active nematic layer. The flow behaviors within the low-friction region (between the dashed lines) are distinct from the behavior in the high friction region (beyond the dashed lines). Plus-half (minus-half) defects denoted by dark green (magenta) symbols behave differently in the two regions. (D–F) Stairway setup. (E) Fluorescence microscope image of the micromilled stairway and the superjacent bundled microtubule film. Step location is indicated by dashed lines. The oil depth increases from left to right. The differences in oil depth alter the length scale of the active turbulence above each step. (Scale bar: 250 μm.) (F) Simulations results for discrete steps in the effective friction (dashed lines). The effective friction coefficient decreases from left to right. The color bar is shared with C. (G–I) Pillar setup. (H) Fluorescence microscope image of the bundled microtubule film above the SU-8 micropillar. (Scale bar: 100 μm.) (I) Simulation results show that the active nematic concentration ϕ is depleted within the high-friction region encircled by the pillar perimeter (dashed line).     

Fluid flows created by swimming bacteria drive self-organization in confined suspensions

Concentrated suspensions of swimming microorganisms and other forms of active matter are known to display complex, self-organized spatiotemporal patterns on scales that are large compared with those of the individual motile units. Despite intensive experimental and theoretical study, it has remained unclear the extent to which the hydrodynamic flows generated by swimming cells, rather than purely steric interactions between them, drive the self-organization. Here we use the recent discovery of a spiral-vortex state in confined suspensions of Bacillus subtilis to study this issue in detail. Those experiments showed that if the radius of confinement in a thin cylindrical chamber is below a critical value, the suspension will spontaneously form a steady single-vortex state encircled by a counter-rotating cell boundary layer, with spiral cell orientation within the vortex. Left unclear, however, was the flagellar orientation, and hence the cell swimming direction, within the spiral vortex. Here, using a fast simulation method that captures oriented cell–cell and cell–fluid interactions in a minimal model of discrete particle systems, we predict the striking, counterintuitive result that in the presence of collectively generated fluid motion, the cells within the spiral vortex actually swim upstream against those flows. This prediction is then confirmed by the experiments reported here, which include measurements of flagella bundle orientation and cell tracking in the self-organized state. These results highlight the complex interplay between cell orientation and hydrodynamic flows in concentrated suspensions of microorganisms.In the wide variety of systems termed “active matter” (1, 2), one finds the spontaneous appearance of coherent dynamic structures on scales that are large compared with the individual motile units. Examples range from polar gels (3, 4), bacterial suspensions (5–10), and microtubule bundles (11) to cytoplasmic streaming (12, 13). At high concentrations, suspensions of rod-like bacteria are known to arrange at the cellular scale with parallel alignment as in nematic liquid crystals (5, 14), but with local order that is polar, driven by motility (15, 16). At meso- and macroscopic scales, coherent structures such as swirls, jets, and vortices at scales 10 μm to 1 mm have been experimentally observed (5–10). Many studies have focused on how complex cell interactions can give rise to macroscopic organization and ordering, and the role of self-generated fluid flows in the dynamics of dense suspensions is still under debate (8, 10, 17–21). This controversy is due in part to the inherent complexity of the systems under investigation and the difficulty in making faithful mathematical models.Microswimmers such as Escherichia coli, Bacillus subtilis, and Chlamydomonas rheinhardtii produce dipolar fluid flows through the combined action of their flagella and cell body on the fluid. In the far field, they are well described as “pusher” or “puller” stresslets (22–24), corresponding to the case of flagella behind or in front of the cell body. These fluid flows affect passive tracers (25, 26), as well as swimmers: their motion is subject to convection and shear reorientation induced by neighboring organisms, which can lead to complex collective organization. Macroscopic fluid flows emerge from the collective motion of a colony of motile bacteria, and the suspension can exhibit a quasi-turbulent dynamics (5). Microorganisms like B. subtilis live in porous environments, such as soil, where contact with surfaces is inevitable as mesoscale obstacles and confinement are the norm. Recent experiments give insight into the interactions of single microorganisms with surfaces (24, 27–29), yet suspension dynamics in confinement has only begun to be investigated (30), and the role of the collectively generated fluid flows in the macroscopic organization has yet to be fully understood.Recently, Wioland et al. (30) showed that a dense suspension of B. subtilis, confined into a flattened drop, can self-organize into a spiral vortex, in which a boundary layer of cells at the drop edge moves in the opposite direction to the bulk circulation. This spatiotemporal organization is driven by the presence of the circular boundary and the interactions of bacteria with it. At the interface, the packed cells move at an angle to the tangential that is dictated by the drop curvature, swimmer size, and shape. This macroscopic nonequilibrium pattern and double circulation were not anticipated by theory and have not been seen in any simulations of discrete particle systems due to the computational difficulty of capturing both confinement and complex interactions between elongated swimmers. Although previous simulations have demonstrated the importance of hydrodynamics in populations of spherical squirmers (31) and rod-shaped swimmers (32), they do not consider boundary effects and the elongated shape of the swimmers in the steric interactions. On the other hand, continuum models of motile suspensions that include fluid dynamics and have been successful in explaining large-scale patterns (32, 33), have either ignored confinement or interactions with surfaces, or, if addressing confinement (34), have imposed boundary conditions that generally do not resolve the orientations of the bacteria at the interface. Thus, the conditions at boundaries and microscopic interactions between cells warrant careful consideration in the modeling of these suspensions so that the macroscopic dynamics and organization are correctly captured.Here, we elucidate the origin and nature of the spontaneous emergence of the spiral vortex and cellular organization in a confined motile suspension. A computational model is described for bacterial suspensions in which the direct and hydrodynamic interactions between the swimmers and the confining circular interface can be tuned. The cells are represented as oriented circles or ellipses subject to cell–cell and cell–fluid interaction, whereas the fluid flow is the total of the pusher dipolar fluid flows produced by each swimmer’s locomotion. It is shown that, although some circulation under conditions of confinement may arise with direct interactions only, hydrodynamics are necessary and crucial to reproduce and explain the double circulation that is observed experimentally. Simulations (Fig. 1 A–C) are able to reproduce the emergence of the spiral vortex from an isotropic state (Fig. 1 D–F) and give insights into the origin of the microscopic organization of the bacteria in the drop. The computational results show the remarkable feature that cells in the bulk of the drop swim against the stronger colony-generated fluid flow and thus have a net backward motion. We confirm this observation by measuring the orientation of the cells and of their flagella through suitable fluorescent labeling methods.Open in a separate windowFig. 1.Snapshots of the bacterial suspension self-organization from simulations (A–C) and experiments (D–F). (A–C) An initially isotropic suspension of microswimmers inside a circle with diameter 12ℓ (ℓ = individual swimmer length). Black dots indicate the swimming direction. The swimmer-generated fluid flow is shown superimposed in each plot (blue arrows). (D–F) A dense suspension of B. subtilis in a drop, 70 μm in diameter. (Upper) Bright field. (Lower) Images processed by edge-detection filtering. Initial disordered state is obtained by shining a blue laser that causes cells to tumble. In both simulations and experiments, the suspension organization initiates at the boundary, as seen in B and E. See also Movie S1.     

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.     

Studies on enmetazobactam clarify mechanisms of widely used β-lactamase inhibitors

β-Lactams are the most important class of antibacterials, but their use is increasingly compromised by resistance, most importantly via serine β-lactamase (SBL)-catalyzed hydrolysis. The scope of β-lactam antibacterial activity can be substantially extended by coadministration with a penicillin-derived SBL inhibitor (SBLi), i.e., the penam sulfones tazobactam and sulbactam, which are mechanism-based inhibitors working by acylation of the nucleophilic serine. The new SBLi enmetazobactam, an N-methylated tazobactam derivative, has recently completed clinical trials. Biophysical studies on the mechanism of SBL inhibition by enmetazobactam reveal that it inhibits representatives of all SBL classes without undergoing substantial scaffold fragmentation, a finding that contrasts with previous reports on SBL inhibition by tazobactam and sulbactam. We therefore reinvestigated the mechanisms of tazobactam and sulbactam using mass spectrometry under denaturing and nondenaturing conditions, X-ray crystallography, and NMR spectroscopy. The results imply that the reported extensive fragmentation of penam sulfone–derived acyl–enzyme complexes does not substantially contribute to SBL inhibition. In addition to observation of previously identified inhibitor-induced SBL modifications, the results reveal that prolonged reaction of penam sulfones with SBLs can induce dehydration of the nucleophilic serine to give a dehydroalanine residue that undergoes reaction to give a previously unobserved lysinoalanine cross-link. The results clarify the mechanisms of action of widely clinically used SBLi, reveal limitations on the interpretation of mass spectrometry studies concerning mechanisms of SBLi, and will inform the development of new SBLi working by reaction to form hydrolytically stable acyl–enzyme complexes.

β-Lactamases are a major mechanism of resistance to the clinically vital β-lactam antibiotics, with >2,000 different β-lactamases reported (1). β-Lactamases are grouped into classes A, C, and D, which employ a nucleophilic serine in catalysis (serine β-lactamases, SBLs), and class B, which employ metal ions in catalysis (2). Presently, SBLs are the most important β-lactamases from a clinical perspective. SBL inhibitors (SBLi) have been developed for use in combination with a β-lactam antibiotic, with tazobactam (3), sulbactam (4), and clavulanic acid (5) being the most widely used SBLi. These SBLi all contain a β-lactam ring which reacts with SBLs to produce an acyl–enzyme complex (AEC) intermediate, as is also the case for efficient SBL substrates (Fig. 1A). With efficient substrates the β-lactam–derived AEC is readily hydrolyzed. With SBLi the reaction bifurcates at the AEC stage; in addition to hydrolysis, reaction of the AEC via opening of the β-lactam fused five-membered ring occurs to give one or more relatively hydrolytically stable species (Figs. 1B and ​and2).2). The nature of these species is central to SBLi inhibition and has been studied by crystallography (6–11) and ultraviolet-visible (UV/Vis) (10, 12) and Raman (6, 7, 9, 12–15) spectroscopy, as well as different types of mass spectrometry (MS) (10, 16–22).Open in a separate windowFig. 1.Sulfone derivatives of penicillins are potent clinically used mechanism-based inhibitors of SBLs. (A) Outline mechanism for penicillin hydrolysis as catalyzed by SBLs; reaction proceeds via an AEC, which is efficiently hydrolyzed. (B) Sulfone derivatives of penicillins are SBLi that react to give one or more hydrolytically stable complex(es), the nature of which was the focus of our work.Open in a separate windowFig. 2.Pathways for reactions of penam sulfones with SBLs. Following initial acyl–enzyme 2 formation the main transient inactivation pathway occurs via thiazolidine ring opening to give species 3-5 which are relatively stable to hydrolysis. Fragmentation of 3-5 can occur in rare cases and is promoted by acid to give 6-8 or heat to give 11. In rare cases fragmentation of 2-5 can result in irreversible inactivation of the SBL to give 9 and 10. Efficient hydrolysis of the β-lactam occurs to give a β-amino acid product 12, which in solution fragments to give 13-16. Our results imply biologically relevant inhibition involves 3-5, or equivalent mass species.The structures of tazobactam and sulbactam are closely related to those of the penicillins; they differ by lack of a C-6 side chain, functionalization of the pro-S methyl group (in case of tazobactam), and by oxidation of the thiazolidine to a sulfone. These differences result in a loss of useful antibacterial activity but a gain of potent SBL inhibition. Although the presence of sulfur in drugs is common [e.g., sulfonamide antibiotics (23)] and there is growing interest in covalently acting drugs (24, 25), sulfones are rare in drugs and, as far as we are aware, sulbactam and tazobactam are the only clinically approved sulfone-containing drugs working by covalent reaction with their targets (26–28).Since the clinical introduction of the pioneering SBLi, β-lactamases have evolved and SBLi use is increasingly compromised by extended spectrum β-lactamases (ESBLs) and inhibitor-resistant SBLs (29). Efforts have been made to develop new SBLi, including those with and without a β-lactam. The latter include diazabicyclooctanes (30) and cyclic boronates (31, 32). However, β-lactam–containing SBLi remain of most clinical importance. Among SBLi in clinical development, enmetazobactam (formerly AAI-101; Fig. 1) is of particular interest because it is a “simple” N-methylated derivative of the triazole ring of tazobactam (33). In combination with cefepime, enmetazobactam is reported to manifest substantially better antimicrobial properties against class A ESBL-producing strains than the commonly used piperacillin/tazobactam combination (20, 33, 34).We report studies on the mechanism of SBL inhibition by enmetazobactam using denaturing and nondenaturing (native) MS methods, NMR spectroscopy, and crystallography. The results led us to reevaluate the mechanisms of SBL inhibition by the clinically important sulfone-containing SBLi, i.e., tazobactam and sulbactam, and reveal limitations on the interpretation of MS studies concerning SBL inhibition.