The sporothriolides. A new biosynthetic family of fungal secondary metabolites

The biosynthetic gene cluster of the antifungal metabolite sporothriolide 1 was identified from three producing ascomycetes: Hypomontagnella monticulosa MUCL 54604, H. spongiphila CLL 205 and H. submonticulosa DAOMC 242471. A transformation protocol was established, and genes encoding a fatty acid synthase subunit and a citrate synthase were simultaneously knocked out which led to loss of sporothriolide and sporochartine production. In vitro reactions showed that the sporochartines are derived from non-enzymatic Diels–Alder cycloaddition of 1 and trienylfuranol A 7 during the fermentation and extraction process. Heterologous expression of the spo genes in Aspergillus oryzae then led to the production of intermediates and shunts and delineation of a new fungal biosynthetic pathway originating in fatty acid biosynthesis. Finally, a hydrolase was revealed by in vitro studies likely contributing towards self-resistance of the producer organism.

A new family of fungal biosynthetic pathways is elucidated based on the use of fatty acid and citrate-like intermediates.

Gamma-lactone and alkyl citrate compounds derived from oxaloacetate are widespread natural products in fungi and often possess potent biological activities. Examples include sporothriolide 1,^1,2 piliformic acid 2,³ tyromycin 3⁴ and the cyclic maleidrides including byssochlamic acid 4^5,6 among others (Fig. 1). In some cases, for example those of 4 and squalestatin S1 5,⁷ detailed molecular studies have revealed that a dedicated polyketide synthase (PKS) produces a carbon skeleton that is then condensed with oxaloacetate by a citrate synthase (CS) to give an early alkyl citrate intermediate that is further oxidatively processed. In other cases, such as 1 and the sporochartines 6, the biosynthetic pathways are not yet clear.Open in a separate windowFig. 1Structures of γ-lactone and alkyl citrate metabolites from fungi. Bold bonds show oxaloacetate-derived carbons where known.Sporochartines 6a–6d^8,9 from the fungus Hypoxylon monticulosum CLL 205 (now referred to as Hypomontagnella spongiphila)¹⁰ possesses potent cytotoxicity (IC₅₀: 7.2 to 21.5 μM) vs. human cancer cell lines and are proposed to be Diels Alder (DA) adducts of the furofurandione sporothriolide 1, itself a potent antifungal agent (EC₅₀: 11.6 ± 0.8 μM),¹¹ and trienylfuranol A 7,¹² originally obtained from an endophytic fungus Hypoxylon submonticulosum DAOMC 242471 (now referred to as Hypomontagnella submonticulosa).¹³ Since the biosynthesis of sporothriolide 1 and related compounds is unknown, and biological DA reactions in fungi are currently of high interest,¹⁴ we decided to examine the biosynthesis of the sporochartines 6 in the Hypomontagnella spp. strains MUCL 54604 and CLL 205 (ref. 10 and 13) in detail.     

Hyperpolarisation of weakly binding N-heterocycles using signal amplification by reversible exchange

Signal Amplification by Reversible Exchange (SABRE) is a catalytic method for improving the detection of molecules by magnetic resonance spectroscopy. It achieves this by simultaneously binding the target substrate (sub) and para-hydrogen to a metal centre. To date, sterically large substrates are relatively inaccessible to SABRE due to their weak binding leading to catalyst destabilisation. We overcome this problem here through a simple co-ligand strategy that allows the hyperpolarisation of a range of weakly binding and sterically encumbered N-heterocycles. The resulting ¹H NMR signal size is increased by up to 1400 times relative to their more usual Boltzmann controlled levels at 400 MHz. Hence, a significant reduction in scan time is achieved. The SABRE catalyst in these systems takes the form [IrX(H)₂(NHC)(sulfoxide)(sub)] where X = Cl, Br or I. These complexes are shown to undergo very rapid ligand exchange and lower temperatures dramatically improve the efficiency of these SABRE catalysts.

The scope of the hyperpolarisation method Signal Amplification by Reversible Exchange (SABRE) is dramatically expanded through the use of co-ligands to substrates that weakly interact with the active cataylst.

Hyperpolarised magnetic resonance is receiving increasing attention from both the analytical science and medical communities due to its ability to create signals that are many orders of magnitude higher than those normally detected under Boltzmann control.^1–6 The time and cost benefits associated with this improvement have propelled this area of research forward over the past few decades. Two of the most prominent techniques used to create hyperpolarisation are dissolution Dynamic Nuclear Polarisation (d-DNP) and Para-Hydrogen Induced Polarisation (PHIP),^7,8 which derive their non-Boltzmann spin energy level populations from interactions with unpaired electrons and para-hydrogen (p-H₂, the singlet spin isomer of hydrogen), respectively. Both of these methods have been reviewed in detail.^3–5,9,10Signal Amplification by Reversible Exchange (SABRE) is a PHIP method that does not involve the chemical incorporation of p-H₂ into the target substrate.^11,12 Instead, under SABRE, spin order transfer proceeds catalytically through the temporary formation of a scalar coupling network between p-H₂ derived hydride ligands and the substrate''s nuclei whilst they are located in a transient metal complex. The most common catalysts are of the type [Ir(H)₂(NHC)(sub)₃]Cl (where NHC = N-heterocyclic carbene and sub = the substrate of interest, Fig. 1a),^13,14 although other variants are known.^15–17 For SABRE to be accomplished, the target substrate must be able to reversibly ligate to the metal centre and this limits the methods applicability; although several routes to overcome this have been reported.^18–20 Recently, the use of bidentate ancillary ligands such as NHC-phenolates¹⁶ and phosphine-oxazoles²¹ has been shown to expand the applicability of SABRE for a variety of different ligands and solvents (Fig. 1b). For example, use of the PHOX ligand (PHOX = (2-diphenylphosphanyl)phenyl-4,5-dihydrooxazole) gives ¹H NMR signal gains of up to 132-fold for 2-picoline; a substrate previously shown to be unpolarised under classic SABRE conditions.²²Open in a separate windowFig. 1Development of the SABRE method for hyperpolarisation of a range of substrates.The use of co-ligands to stabilise the active SABRE catalyst has proven successful for substrates that weakly associate to the catalyst (Fig. 1c). Of particular note is the hyperpolarisation of sodium [1,2]-¹³C₂-pyruvate²³ and sodium ¹³C-acetate²⁴ which could be used as in vivo metabolic probes. The importance of co-ligands in breaking the chemical symmetry of the SABRE catalyst is also well established and co-ligands such as acetonitrile,²⁵ sulfoxides,^23,26 1-methyl-1,2,3-triazole²⁷ and substrate isotopologues²⁸ have been employed.We report here on the use of co-ligands to allow the NMR hyperpolarisation of weakly binding N-heterocyclic derived substrates with functionality in the ortho-position that have proven to be routinely inaccessible to the SABRE technique (Fig. 1d). ¹H signal gains of up to 1442 ± 84-fold were obtained for some of these substituted pyridines at 9.4 T and the expansion of this approach to ¹³C and ¹⁵N detection and other N-heterocyclic motifs is also exemplified.     

Correction: Crystal structure and metallization mechanism of the π-radical metal TED

Radical electrons tend to localize on individual molecules, resulting in an insulating (Mott–Hubbard) bandgap in the solid state. Herein, we report the crystal structure and intrinsic electronic properties of the first single crystal of a π-radical metal, tetrathiafulvalene-extended dicarboxylate (TED). The electrical conductivity is up to 30 000 S cm⁻¹ at 2 K and 2300 S cm⁻¹ at room temperature. Temperature dependence of resistivity obeys a T³ power-law above T > 100 K, indicating a new type of metal. X-ray crystallographic analysis clarifies the planar TED molecule, with a symmetric intramolecular hydrogen bond, is stacked along longitudinal (the a-axis) and transverse (the b-axis) directions. The π-orbitals are distributed to avoid strong local interactions. First-principles electronic calculations reveal the origin of the metallization giving rise to a wide bandwidth exceeding 1 eV near the Fermi level. TED demonstrates the effect of two-dimensional stacking of π-orbitals on electron delocalization, where a high carrier mobility of 31.6 cm² V⁻¹ s⁻¹ (113 K) is achieved.

The molecular arrangement that enables metallic conduction in a single-component pure organic crystal is revealed by single-crystal X-ray diffraction.

Organic molecular solids are typically insulating due to their paired electrons in spatially localized s- and p-orbitals. The concept of charge-transfer (CT) between donor and acceptor¹ enabled the development of conducting molecular complexes (salts) including semiconducting perylene-bromine,² metallic tetrathiafulvalene (TTF)-tetracyano-p-quinodimethane (TCNQ),³ and polyacethylene doped with halogen molecules.⁴ A different strategy was proposed in the 1970s based on organic radicals with an open-shell electronic structure.⁵ π-Radicals such as neutral-,⁶ fully-conjugated⁷ and zwitterionic (betainic)⁸ molecules, with an unpaired electron in their singly occupied molecular orbital (SOMO), offered potential candidates. However, all these π-radicals were insulators or semiconductors with a finite bandgap, which is due to the SOMO being localized on an individual molecule. In the Mott–Hubbard model,⁹ the case of the π-radical solids can be described by the on-site Coulomb repulsion U being larger than the electronic bandwidth W (U/W > 1), in contrast to U/W < 1 in molecular metals like CT metal systems (Fig. 1a).Open in a separate windowFig. 1(a) Schematic representation of the electronic band structure of Mott–Hubbard insulators (left) and possible molecular metals (right), respectively. Solids formed from typical π-radicals possess large on-site Coulomb repulsion U compared with the electronic bandwidth W, resulting a finite bandgap (left). This requires a new mechanism to expand W and overcome U to achieve a metallic state at ambient pressure in π-radical crystals. (b) Molecular structure of the zwitterionic radical, tetrathiafulvalene-extended dicarboxylate (TED) with a symmetric intramolecular hydrogen bond.A straightforward approach to realize high conductivity in π-radical systems is to enhance the intermolecular interaction by applying high pressure.¹⁰ Bisdithiazolyl radical crystal achieved W ∼ 1 eV near the Fermi level and the room temperature conductivity σ_RT = 2 S cm⁻¹ under 11 GPa pressure.^10c An alternative route is to decrease the interatomic spacing by incorporating a metal ion. Introduction of a semimetal Se and intermolecular hydrogen bonding in a donor-type radical succeeded to improve a conductivity to σ_RT = 19 S cm⁻¹ but still required high pressure over 1 GPa for breaking its insulating character.^10d An organometallic compound with a transition metal, [Ni(tmdt)₂], by contrast, is known to form a three-dimensional (3D) Fermi surface with W = 0.48 eV and metallic conduction with σ_RT = 400 S cm⁻¹ at ambient pressure.¹¹ A breakthrough concept for expanding W at ambient pressure is desired for achieving metallization in pure organic π-radicals.Tetrathiafulvalene-extended dicarboxylate (TED) is an organic air-stable zwitterionic radical (Fig. 1b),¹² which was designed based on carrier generation induced by a stably-introduced protonic defect (–H⁺) in hydrogen-bonding molecules without adopting CT between multiple molecules.¹³ A polycrystalline film of TED exhibited metallic conduction at ambient pressure, but the mechanism has not been clarified yet due to lack of single crystal information.¹² Herein, we report the first crystal structure and intrinsic electronic properties of the recently grown single crystal TED. Structural analysis and quantum chemical simulations based on the single crystal reveal the origin of its metallic behavior.     

Ferro-self-assembly: magnetic and electrochemical adaptation of a multiresponsive zwitterionic metalloamphiphile showing a shape-hysteresis effect

Metallosurfactants are molecular compounds which combine the unique features of amphiphiles, like their capability of self-organization, with the peculiar properties of metal complexes like magnetism and a rich redox chemistry. Considering the high relevance of surfactants in industry and science, amphiphiles that change their properties on applying an external trigger are highly desirable. A special feature of the surfactant reported here, 1-(Z)-heptenyl-1′-dimethylammonium-methyl-(3-sulfopropyl)ferrocene (6), is that the redox-active ferrocene constituent is in a gemini-position. Oxidation to 6⁺ induces a drastic change of the surfactant''s properties accompanied by the emergence of paramagnetism. The effects of an external magnetic field on vesicles formed by 6⁺ and the associated dynamics were monitored in situ using a custom-made optical birefringence and dual dynamic light scattering setup. This allowed us to observe the optical anisotropy as well as the anisotropy of the diffusion coefficient and revealed the field-induced formation of oriented string-of-pearls-like aggregates and their delayed disappearance after the field is switched off.

The self-organization properties of a stimuli responsive amphiphile can be altered by subjecting the paramagnetic oxidized form to a magnetic field of 0.8 T and monitored in real time by coupling optical birefringence with dynamic light scattering.

Amphiphiles (or surfactants) combine hydrophilic (the so-called headgroups) and lipophilic entities (the so-called tails) as integral parts of their molecular structures. This particular construction principle provides them with the ability to display concentration-dependent self-organization in nonpolar and polar solvents.¹ Amphiphiles with advanced functions that go far beyond the traditional ones as emulsifiers, stabilizing agents for interfaces, or detergents were meanwhile realized by skillful manipulation of any of its constituents.^2–4 Recent examples are micellar LEDs,^5,6 catalysts,^7–9 or batteries.¹⁰ Such applications are important hallmarks on the way to even more sophisticated amphiphiles such as the ones found in nature, e.g. in the pockets of enzymes.^11–18 An important milestone is the advent of (multi-) stimuli-responsive amphiphiles, whose encoded functionalities respond to (different) external triggers. Such systems are capable of adaptive self-assembly, which can be controlled using an external input such as the pH, temperature, ionic strength, or redox state.^19–26Paramagnetic amphiphiles, recently reviewed by Eastoe and coworkers, constitute a fascinating family of stimuli-responsive surfactants.²⁷ Particular attention has been paid to magnetic ionic liquids based on amphiphilic transition metal complexes, as their properties are often superior to those of conventional magnetic fluids (ferrofluids).^28–31 Self-assembly results in high effective concentrations of the paramagnetic metal centers, and this in turn allows us to control their physico-chemical properties and the morphologies of their superstructures through an external magnetic field. Such a scheme has the added advantage that the external stimulus is non-invasive. In many current realizations of such systems, however, the magneto-active (transition) metal ion is only present as a constituent of the counterion of a cationic surfactant, but is not an integral constituent of the surfactant itself.^21,30,31Some of us have previously reported redox-switchable as well as paramagnetic stimuli-responsive amphiphiles of relevance to the current work.^32,33 We thought that ferrocene would be an ideal building block in order to combine both these kinds of stimuli within one single amphiphile.^34–37 On oxidation, the diamagnetic, hydrophobic ferrocene nucleus is transformed into a paramagnetic S = 1/2 ferrocenium ion with a distinct hydrophilic character.^38–41 Oxidation does hence not only generate a magnetic moment, but also transfers the ferrocene nucleus from the lipo- to the hydrophilic part of the amphiphile, thereby changing its entire structure. A 1,1′-disubstitution pattern of the ferrocene scaffold, which is synthetically well accessible,^34,42–44 seemed particularly suited for such an endeavor.Studies on paramagnetic amphiphiles are often thwarted by the non-trivial analytics involved in their characterization. Detailed investigations often rely on small-angle neutron scattering (SANS), which is time-consuming and costly and suffers from poor availability.^{27,30,31,45–47} Moreover, SANS is only of limited value for following kinetically fast processes which would be desirable for the live monitoring of structural changes occurring in solution. Optical birefringence is a well-established method to monitor the dynamic response of materials to external fields.^48–50 Although of high intrinsic value, optical birefringence measurements in magnetic fields were only rarely applied for the study of paramagnetic amphiphiles.²⁹We here report the zwitterionic, ferrocene-based amphiphile FcNMe₂SO₃Heptene 6 (see Fig. 1, Fc = ferrocenyl) with a sultone headgroup. Compound 6 is unique in that its self-assembly properties can be controlled by three different external stimuli, namely the (i) addition of an electrolyte, (ii) addition of an oxidant/reductant, and (iii) exposure to an external magnetic field. We also demonstrate that optical birefringence in combination with dynamic light scattering (DLS) measurements in two orthogonal directions provides detailed insights into the functional response of aggregated magnetic nanoparticles formed by 6⁺ to an external magnetic field in real time. Specifically, we have observed the formation of string-of-pearls-like aggregates of 6⁺ in a magnetic field (0.8 T), the field-induced anisotropy of the diffusion of aggregated nanoparticles, and a hysteresis effect for their disappearance after the magnetic field is switched off. Thus, the anisotropy of larger aggregates persists for more than 5 min, while the structural alignment of smaller ones vanishes at a significantly faster rate.Open in a separate windowFig. 1Synthesis of FcNMe₂SO₃Heptene (6). (a) Synthesis of 6; (b) molecular structure of 6 crystallized from acetonitrile. C; dark grey, N; turquoise, Fe; orange, S; yellow, O; red, H atoms are omitted for clarity.     

Automated linkage of proteins and payloads producing monodisperse conjugates

Controlled protein functionalization holds great promise for a wide variety of applications. However, despite intensive research, the stoichiometry of the functionalization reaction remains difficult to control due to the inherent stochasticity of the conjugation process. Classical approaches that exploit peculiar structural features of specific protein substrates, or introduce reactive handles via mutagenesis, are by essence limited in scope or require substantial protein reengineering. We herein present equimolar native chemical tagging (ENACT), which precisely controls the stoichiometry of inherently random conjugation reactions by combining iterative low-conversion chemical modification, process automation, and bioorthogonal trans-tagging. We discuss the broad applicability of this conjugation process to a variety of protein substrates and payloads.

Controlled protein functionalization holds great promise for a wide variety of applications.

Applications of protein conjugates are limitless, including imaging, diagnostics, drug delivery, and sensing.^1–4 In many of these applications, it is crucial that the conjugates are homogeneous.⁵ The site-selectivity of the conjugation process and the number of functional labels per biomolecule, known as the degree of conjugation (DoC), are crucial parameters that define the composition of the obtained products and are often the limiting factors to achieving adequate performance of the conjugates. For instance, immuno-PCR, an extremely sensitive detection technique, requires rigorous control of the average number of oligonucleotide labels per biomolecule (its DoC) in order to achieve high sensitivity.⁶ In optical imaging, the performance of many super-resolution microscopy techniques is directly defined by the DoC of fluorescent tags.⁷ For therapeutics, an even more striking example is provided by antibody–drug conjugates, which are prescribed for the treatment of an increasing range of cancer indications.⁸ A growing body of evidence from clinical trials indicates that bioconjugation parameters, DoC and DoC distribution, directly influence the therapeutic index of these targeted agents and hence must be tightly controlled.⁹Standard bioconjugation techniques, which rely on nucleophile–electrophile reactions, result in a broad distribution of different DoC species (Fig. 1a), which have different biophysical parameters, and consequently different functional properties.¹⁰Open in a separate windowFig. 1Schematic representation of the types of protein conjugates.To address this key issue and achieve better DoC selectivity, a number of site-specific conjugation approaches have been developed (Fig. 1b). These techniques rely on protein engineering for the introduction of specific motifs (e.g., free cysteines,¹¹ selenocysteines,¹² non-natural amino acids,^13,14 peptide tags recognized by specific enzymes^15,16) with distinct reactivity compared to the reactivity of the amino acids present in the native protein. These motifs are used to simultaneously control the DoC (via chemo-selective reactions) and the site of payload attachment. Both parameters are known to influence the biological and biophysical parameters of the conjugates,¹¹ but so far there has been no way of evaluating their impact separately.The influence of DoC is more straightforward, with a lower DoC allowing the minimization of the influence of payload conjugation on the properties of the protein substrate. The lowest DoC that can be achieved for an individual conjugate is 1 (corresponding to one payload attached per biomolecule). It is noteworthy that DoC 1 is often difficult to achieve through site-specific conjugation techniques due to the symmetry of many protein substrates (e.g., antibodies). Site selection is a more intricate process, which usually relies on a systematic screening of conjugation sites for some specific criteria, such as stability or reactivity.¹⁷Herein, we introduce a method of accessing an entirely new class of protein conjugates with multiple conjugation sites but strictly homogenous DoCs (Fig. 1c). To achieve this, we combined (a) iterative low conversion chemical modification, (b) process automation, and (c) bioorthogonal trans-tagging in one workflow.The method has been exemplified for protein substrates, but it is applicable to virtually any native bio-macromolecule and payload. Importantly, this method allows for the first time the disentangling of the effects of homogeneous DoC and site-specificity on conjugate properties, which is especially intriguing in the light of recent publications revealing the complexity of the interplay between payload conjugation sites and DoC for in vivo efficacy of therapeutic bioconjugates.¹⁸ Finally, it is noteworthy that this method can be readily combined with an emerging class of site-selective bioconjugation reagents to produce site-specific DoC 1 conjugates, thus further expanding their potential for biotechnology applications.¹⁹     

Inhibitors of thiol-mediated uptake

Ellman''s reagent has caused substantial confusion and concern as a probe for thiol-mediated uptake because it is the only established inhibitor available but works neither efficiently nor reliably. Here we use fluorescent cyclic oligochalcogenides that enter cells by thiol-mediated uptake to systematically screen for more potent inhibitors, including epidithiodiketopiperazines, benzopolysulfanes, disulfide-bridged γ-turned peptides, heteroaromatic sulfones and cyclic thiosulfonates, thiosulfinates and disulfides. With nanomolar activity, the best inhibitors identified are more than 5000 times better than Ellman''s reagent. Different activities found with different reporters reveal thiol-mediated uptake as a complex multitarget process. Preliminary results on the inhibition of the cellular uptake of pseudo-lentivectors expressing SARS-CoV-2 spike protein do not exclude potential of efficient inhibitors of thiol-mediated uptake for the development of new antivirals.

Thiol-reactive inhibitors for the cellular entry of cyclic oligochalcogenide (COC) transporters and SARS-CoV-2 spike pseudo-lentivirus are reported.

Thiol-mediated uptake^1–10 has been developed to explain surprisingly efficient cellular uptake of substrates attached to thiol-reactive groups, most notably disulfides. The key step of this mechanism is the dynamic covalent thiol-disulfide exchange between disulfides of the substrates and exofacial thiols on cell surfaces (Fig. 1). The covalently bound substrate then enters the cell either by fusion, endocytosis, or direct translocation across the plasma membrane into the cytosol. Thiol-disulfide exchange has been confirmed to play an essential role in the cellular entry of some viruses^1,11–14 and toxins.² Indeed, diphtheria toxin and HIV were among the first to be recognized to enter cells via thiol-mediated uptake.^1,2 The involvement of cell-surface thiols in cellular uptake is most often probed by inhibition with Ellman''s reagent (DTNB). However, this test is not always reliable, in part due to the comparably poor reactivity of DTNB, and the comparably high reactivity of the disulfide obtained as a product. Thus, the importance of thiol-mediated uptake for viral entry and beyond remains, at least in part, unclear.Open in a separate windowFig. 1In thiol-mediated uptake, dynamic covalent exchange with thiols on the cell surface precedes entry through different mechanisms. Inhibition of thiol-mediated uptake by removal of exofacial thiols and disulfides could thus afford new antivirals.We became interested in thiol-mediated uptake^3–5 while studying the cytosolic delivery of substrates such as drugs, probes and also larger objects like proteins or quantum dots with cell-penetrating poly(disulfide)s.⁶ Our recent focus shifted to cyclic oligochalcogenides (COCs) to increase speed and selectivity of dynamic covalent thiol-oligochalcogenide exchange, and, most importantly, to assure reversibility, i.e., mobility during uptake, with a covalently tethered, intramolecular leaving group.⁷ With increasingly unorthodox COC chemistry, from strained disulfides^7,8 and diselenides⁹ to adaptive dynamic covalent networks produced by polysulfanes,¹⁰ uptake activities steadily increased. Their high activities suggested that the same, or complementary, COCs could also function as powerful inhibitors of thiol-mediated uptake that ultimately might perhaps lead to antivirals. In the following, this hypothesis is developed further.Fluorescently labeled COCs 1⁸ and 2¹⁰ were selected as reporters for the screening of thiol-mediated uptake inhibitors because of their high activity, their destination in the cytosol, and their different characteristics (Fig. 2). The COC in 1 is an epidithiodiketopiperazine (ETP). With a CSSC dihedral angle ∼0°, ETPs drive ring tension to the extreme.^15,16 Ring-opening thiol-disulfide exchange is ultrafast, and the released thiols are acidic enough to continue exchanging in neutral water, including ring closure.⁸ This unique exchange chemistry coincides with efficient cellular uptake and poor retention on thiol affinity columns.⁸Open in a separate windowFig. 2Structure of reporters 1 and 2 and inhibitor candidates 3–30 with their concentrations needed to inhibit by ∼15% (MIC) the uptake of 1 (1 h pre-incubation with inhibitors, 30 min incubation with reporter, filled symbols) and 2 (4 h pre-incubation, empty symbols). Red squares: ETPs; orange circles: BPSs; blue upward triangles: heteroaromatic sulfones; purple diamonds: thiosulfonates; magenta downward triangles: di- and polysulfides; brown hexagons: thiosulfinates. Symbols with upward arrows: MIC not reached at the highest concentration tested. Symbols with downward arrows indicate the lowest concentration tested already exceeds the MIC. (a) Similarly active upon co-incubation of reporters and inhibitor; (b–d) similarly (b), less (c), or more (d) active upon co-incubation in the presence of serum (mostly 6 h); (e) pre-incubation for 15 min; (f) isomerizes into cis22; (g) V-shaped DRC (see Fig. 3f); (h) pre-incubation for 30 min, co-incubation with 2; (i) mixture of regioisomers.The COC in 2 is a benzopolysulfane (BPS). Like ETPs, BPSs occur in natural products and have inspired total synthesis.¹⁷ Unlike ETPs, BPSs are not strained but evolve into adaptive networks of extreme sulfur species for cells to select from. Uptake efficiencies and retention on thiol affinity columns exceed other COCs clearly.^10,18With COCs 1 and 2 as cell-penetrating reporters, a fully automated, fluorescent microscopy image-based high-content high-throughput (HCHT)¹⁹ inhibitor screening assay was developed. HeLa cells in multiwell plates are incubated with a reporter at constant and inhibitors at varying concentrations and incubation times. Hindered reporter uptake then causes decrease of fluorescence inside of cells (Fig. 3a). Automated data analysis¹⁹ was established to extract average fluorescence intensity per cell and, at the same time, cell viability from propidium iodide negative nuclei count (Fig. 3 and S3–S6^†). Standard assay conditions consisted of pre-incubation of HeLa cells with inhibitors for different periods of time, followed by the removal of inhibitors and the addition of reporters, thus excluding possible interactions between the two in the extracellular environment. In alternative co-incubation conditions, inhibitors were not removed before the addition of reporters to allow for eventual interactions between the two.Open in a separate windowFig. 3(a) Fluorescence image of HCHT plates (4 images per well) with HeLa cells pre-incubated with 6 (30 min) followed by co-incubation with 1 (left) and 2 (right, 10 μM each) for constant 30 min. (b–f) HCHT data showing relative fluorescence intensity (filled symbols) and cell viability (empty symbols) of HeLa cells after (b) pre-incubation with 4 for 1 h, followed by washing and incubation with 1 (top), or pre-incubation with 4 for 30 min, followed by co-incubation with 4 and 2 (bottom). (c) As in (b) with 18. (d) As in (b) after incubation for 4 h with 16 followed by incubation with 2. (e) As in (b) after pre-incubation with 11 (circles), 14 (crosses), or 21 (diamonds) for 15 min, followed by washing and incubation with 1. (f) As in (b) after pre-incubation with 20 (30 min), followed by washing and incubation with 1.Among the very high number of thiol-reactive probes, compounds 3–30 were selected based on promise, experience, availability and accessibility. Main focus was on COCs offering increasingly extreme sulfur chemistry because dynamic covalent thiol-oligochalcogenide exchange with different intramolecular leaving groups promises access to different exchange cascades for the intramolecular and, perhaps, also intermolecular crosslinking of the target proteins. More hydrophilic, often anionic COCs were preferred to prevent diffusion into cells and thus minimize toxicity. The expectation was that from such a sketchy outline of an immense chemical space, leads could be identified for future, more systematic exploration. Reporters 1 and 2 and candidates 3–30 were prepared by substantial multistep synthesis (Schemes S1–S11 and Fig. S47–S93,^† commercially available: 20, 25, 30). Inhibitors were numbered in the order of efficiency against reporter 1, evaluated by their minimum inhibitory concentrations (MICs), i.e., concentrations that cause a ∼15% reduction of reporter uptake in cells (Fig. 2 and Tables S1–S37^†). We chose to use MICs because half-maximal inhibitions could not always be reached due to the onset of toxicity, formally anticooperative, or even V-shaped dose–response curves (DRCs, e.g., Fig. 3b–f, all DRCs can be found in the ESI, Fig. S7–S43^†). MICs are usually below the half-maximal cell growth inhibition concentration (GI₅₀, Tables S1–S37^†).Among the most potent inhibitors of ETP reporter 1 were ETPs 4 and 5 (Fig. 2, ​,3b).3b). This intriguing self-inhibition was even surpassed by the expanded cyclic tetrasulfide ETP₄3 (MIC < 0.1 μM), which was of interest because they are much poorer transporters.¹⁰ Further formal ring expansion leads to cyclic pentasulfides BPS₅6 as equally outstanding inhibitors (MIC ≈ 0.3 μM). This trend toward the adaptive networks, reminiscent of elemental sulfur chemistry, did not extend toward inorganic polysulfides 13 (MIC ≈ 20 μM). ETPs 4 and 5 were sensitive to modification of the carboxylate, with the cationic 12 being the worst (MIC ≈ 30 μM) and the neutral glucose hemiacetal 7 the most promising (MIC ≈ 0.5 μM).Although this study focuses on increasingly extreme dynamic covalent COC chemistry, the inclusion of one example for covalent C–S bond formation was of interest for comparison. The classical iodoacetamides⁷ and maleimides⁴ were more toxic than active (not shown). However, nucleophilic aromatic substitution of heteroaromatic sulfones,²⁰ just developed for the efficient bioorthogonal conversion of thiols into sulfides, was more promising. Weaker than dynamic covalent COCs, this irreversible inhibition was best with benzoxazole 11 (MIC ≈ 15 μM) and decreased in accordance with reactivity toward free thiols to oxadiazole 14 and benzothiazole 21 (MIC ≈ 300 μM, Fig. 3e).At constant pH, Ellman''s reagent 20 was confirmed to be erratic also in this assay. The DRC showed minor inhibition up to around 2 mM, which disappeared again at higher concentrations (Fig. 3f). Other cyclic disulfides were inactive as well (28–30). Also disappointing were oxidized disulfides, that is thiosulfinates, including allicin 25, the main odorant component of garlic,^21,22 oxidized cystine 26 and oxidized lipoic acid 27. Thiosulfinates were of interest because they should selectively target the vicinal thiols of reduced disulfides bridges, producing two disulfides.²³ The most active trans dithioerythrol (DTE) thiosulfinate 17 isomerized with time into the less active, hydrogen-bonded cis isomer 22 (Fig. S46^†).Reporter 2 was more difficult to inhibit than 1, as expected from high activity with extreme retention on thiol affinity columns.^10,18 For instance, BPS 6 was very efficient against ETP 1 but much less active against BPS 2 (Fig. 3a), although longer pre-incubation could lower the MIC down to 4 μM (Fig. 2, S41^†). The complementary ETP 4 “self-inhibited” ETP 1 but was also unable to inhibit BPS 2 as efficiently (Fig. 3b). Among the best inhibitors of BPS 2 upon co-incubation were disulfide bridged γ-turn²⁴ peptides 18 and 19 (MIC ≈ 5 μM), both less active against 1 (MIC ≈ 300 μM, Fig. 3c). Disulfide-bridged γ-turn CXC peptides consist of an 11-membered ring with significant Prelog strain. They were introduced by Wu and coworkers as transporters for efficient cytosolic delivery.⁵ The cyclic thiosulfonates 15 and 16 showed promising activities against both 1 and 2, and were tolerant toward the presence of serum (Fig. 2d, S33 and S42^†). Contrary to thiosulfinate 27, the oxidation of lipoic acid to pure thiosulfonates was not successful so far. However, weakly detectable activity of the lipoyl-glutamate conjugate oxidized to the thiosulfinate (MIC ≈ 350 μM, not shown) compared to the inactive thiosulfinate 27 implied that lipoic acid oxidized to the thiosulfonate would also be less active than the glutamate conjugate 15.The oxidized DTE 16^25–28 was particularly intriguing because it was more potent against 2 and could achieve nearly complete inhibition (MIC ∼ 20 μM, Fig. 3d). Highly selective for thiols, the cyclic thiosulfonate 16 was stable for weeks at room temperature, without precaution, in all solvents tested. The disulfides and sulfinates obtained from exchange with thiols were stable as well, and the latter can further react with disulfides²⁷ for intramolecular or eventually intermolecular crosslinking of the target proteins.The overall mismatched inhibition profiles found for reporters 1 and 2 supported that thiol-mediated uptake proceeds through a series of at least partially uncoupled parallel multitarget systems instead of a specific single protein or membrane target. From proteomics studies with cysteine-reactive irreversible probes, it is known that different probes generally target different proteins.^29b Proteomics analysis^29a for asparagusic acid derived transporters supports the involvement of many targets beyond the commonly considered protein disulfide isomerases and the confirmed transferrin receptor.^{12–14,26–30} The unusual, formally anti-cooperative (Hill coefficients < 1) DRCs further supported thiol-mediated uptake as complex multitarget systems.Despite the complexity of these systems, results did not much depend on assay conditions. Compared to the standard protocol of pre-incubation with inhibitors followed by inhibitor removal and incubation with reporters 1 or 2 for detection, the co-incubation protocol, in which pre-incubation with inhibitors is followed by co-incubation with reporters 1 or 2 without inhibitor removal, gave reasonably similar results (Fig. 2). Inhibition characteristics naturally depended on pre-incubation time, with weaker activities at shorter and longer times, reflecting incomplete exchange and cellular response or other ways of inhibitor destruction, respectively. The presence of serum also did not affect the activities much (Fig. 2b–d).Preliminary studies on antiviral activity were performed with pseudo-lentivectors³¹ that express the D614G mutant¹¹ of the SARS-CoV-2 spike protein and code for a luciferase reporter gene, which is expressed by the infected cells.¹² A549 human lung alveolar basal epithelium cell line constitutively overexpressing ACE2 and TMPRSS2 was selected to facilitate the entry of the SARS-CoV-2 spike pseudo-lentivirus. The most significant activities were found for DTE thiosulfonate 16 with an IC₅₀ around 50 μM, while toxicity was detected only at 500 μM (Fig. S44^†). The onset of inhibition could be observed for tetrasulfide ETP 3 at 50 μM, but it coincided with the appearance of cytotoxicity. Protease inhibition is less likely to be the mode of action, as similar activity was found with wild type A549 cells transduced with a standard lentivirus expressing vesicular-stomatitis virus G surface protein VSVG (Fig. S45^†).¹³ Short incubation times of cells and inhibitors before the addition of viruses disfavored contributions from changes in gene expression. More detailed studies are ongoing.The lessons learned from this study are that, firstly, thiol-mediated uptake can be inhibited efficiently by thiol-reactive reagents, confirming that thiol-mediated uptake exists and transporters like ETP 1 and BPS 2 do not simply diffuse into cells; the best inhibitors are more than 5000 times better than Ellman''s reagent. Secondly, inhibitor efficiencies vary with the transporters, supporting that thiol-mediated uptake operates as a complex multitarget system. The best inhibitors are COCs that operate with fast dynamic covalent exchange, suggesting that the reversibility provided by COCs is important. The inhibition of thiol-mediated uptake might contribute to activities of thiol-reactive antivirals such as 16, ETPs or ebselen, although they have been shown to bind to zinc fingers or inhibit proteases.^{16,25,32–34} Finally, the inhibitors reported here could also be of interest for delivery applications and might be worth investigation with regard to antiviral activity. We currently plan to focus more systematically on the most promising leads within COCs, particularly cyclic thiosulfonates, and to expand the screening campaign toward new attractive motifs.^33–35     

Multiscale modeling of molecular structure and optical properties of complex supramolecular aggregates

Supramolecular aggregates of synthetic dye molecules offer great perspectives to prepare biomimetic functional materials for light-harvesting and energy transport. The design is complicated by the fact that structure–property relationships are hard to establish, because the molecular packing results from a delicate balance of interactions and the excitonic properties that dictate the optics and excited state dynamics, in turn sensitively depend on this packing. Here we show how an iterative multiscale approach combining molecular dynamics and quantum mechanical exciton modeling can be used to obtain accurate insight into the packing of thousands of cyanine dye molecules in a complex double-walled tubular aggregate in close interaction with its solvent environment. Our approach allows us to answer open questions not only on the structure of these prototypical aggregates, but also about their molecular-scale structural and energetic heterogeneity, as well as on the microscopic origin of their photophysical properties. This opens the route to accurate predictions of energy transport and other functional properties.

Multiscale modeling resolves the molecular structure of a synthetic light-harvesting complex, unraveling the microscopic origin of its photophysical properties.

Supramolecular structures may self-assemble from a variety of building blocks, resulting in a wide range of advanced materials with attractive biomimetic, sensing, catalytic, optoelectronic and photonic functionalities.^1–10 The close-packed nanoscale organization of the individual molecules within a supramolecular system, held together via noncovalent interactions, gives rise to the aggregate''s (collective) properties. Assemblies consisting of dye molecules often exhibit unique collective optical properties and are of interest for opto-electronic applications as well as artificial light-harvesting complexes that mimic natural antenna systems of photosynthetic bacteria and plants.^11–13 For example, chlorosomal antenna complexes of photosynthetic green sulfur bacteria are self-assembled into multilayer tubular structures having bacteriochlorophyll pigments as building blocks.^14–16 The structure of these antenna complexes and the underlying molecular arrangement ensures that the process of light-harvesting and excitation energy transport is very efficient, even under extremely low light conditions.^17,18 The quest to recreate such efficiency under laboratory conditions has sparked numerous studies of synthetic self-assembled systems mimicking natural chlorosomes, e.g. using porphyrins,¹⁹ zinc chlorin,²⁰ and cyanine dyes.²¹ Of particular interest are the tubular aggregates of 3,3′-bis(2-sulfopropyl)-5,5′,6,6′-tetrachloro-1,1′-dioctylbenzimidacarbocyanine (C8S3).^22–25 Cryo-TEM reveals a hierarchy of supramolecular architectures, including double-walled nanotubes; under certain conditions, bundles of nanotubes arise.²⁶ Thus, this system allows for the occurrence of electronic excitation energy transport at various levels: within one wall, between walls of one tube, and between different tubes, similar to the situation in natural systems.^27,28To understand how such supramolecular systems work, as well as propose design rules for new materials, it is essential to determine the relationship between molecular structure and optical properties. Current experimental techniques, however, are unable to resolve the structure at the molecular level. This, in combination with the sensitivity of spectral properties to the details of the molecular packing, leads to a crucial role for theoretical modeling.²⁹ For example, molecular dynamics (MD) simulations have been used to predict the molecular packing within a variety of supramolecular assemblies.^30–34 However, synthetic amphiphiles with aromatic groups, such as cyanine dyes—often used to prepare aggregates with optical functionality—tend to fall into kinetic traps during spontaneous self-assembly simulations and the packing of the aromatic chromophores remains highly disordered on the accessible time scale, leading to predicted (optical) spectra that are not consistent with experimental data.³⁵ This problem can be overcome by building assemblies based upon proposed architectures and assessing their stability in relatively short MD simulations.^36–38 The drawback of this approach is the requirement of a thorough understanding of what to use as a starting point and how to validate the structure. In any case, proper validation requires the modeling of the optical spectra of the obtained structure, and finally, comparing it to the experiment. The demanding character of such methods explains why an important role is played by phenomenological modeling, in which a molecular packing is guessed and the optics is obtained from parametrizing an exciton model that describes the collective excited states of the assembly with interactions dictated by the guessed packing. By comparing the calculated spectra to experimental ones, the structure and exciton model may be fine-tuned. While this method has been successful in describing spectra,^23,39 it is limited in its predictive power and also lacks access to essential microscopic parameters, such as tuning of the optical excitation energies imposed by the environment, disorder in these energies and structural heterogeneity.In this work, we use an advanced multiscale approach to determine structure–optical property relationships for the C8S3 double-walled nanotubes, guided by comparison to experiments. The optical spectrum of these aggregates, in which multiple exciton peaks may be discerned, suggests a rather complex underlying molecular packing. This fact, combined with their sheer size going up to many thousands of molecules, makes these systems exceptionally challenging to resolve and leaves important questions concerning structure–function relationships unanswered or under debate, for instance the origin of the splitting between the two lowest-energy spectral bands.^23,38 Here, we answer these questions by iteratively combining MD simulations to capture the details of molecular packing and structural disorder, an exciton Hamiltonian approach to calculate optical signatures, and explicit microelectrostatic calculations to estimate energetic disorder and solvent shifts. Previous attempts to reveal the structure of cyanine-based nanotubes were limited to small-scale system sizes,^37,38 modeling optical features phenomenologically rather than using atomistic information³⁸ or featuring simpler, single-walled systems.³⁷ In addition to answering important questions for the C8S3 double-walled nanotubes, our study opens the way to explain and predict at an unprecedented level of detail the functional properties of other highly complex molecular materials.     

Multicolor polymeric carbon dots: synthesis,separation and polyamide-supported molecular fluorescence

Multicolor carbon dots (CDs) have been developed recently and demonstrate great potential in bio-imaging, sensing, and LEDs. However, the fluorescence mechanism of their tunable colors is still under debate, and efficient separation methods are still challenging. Herein, we synthesized multicolor polymeric CDs through solvothermal treatment of citric acid and urea in formamide. Automated reversed-phase column separation was used to achieve fractions with distinct colors, including blue, cyan, green, yellow, orange and red. This work explores the physicochemical properties and fluorescence origins of the red, green, and blue fractions in depth with combined experimental and computational methods. Three dominant fluorescence mechanism hypotheses were evaluated by comparing time-dependent density functional theory and molecular dynamics calculation results to measured characteristics. We find that blue fluorescence likely comes from embedded small molecules trapped in carbonaceous cages, while pyrene analogs are the most likely origin for emission at other wavelengths, especially in the red. Also important, upon interaction with live cells, different CD color fractions are trafficked to different sub-cellular locations. Super-resolution imaging shows that the blue CDs were found in a variety of organelles, such as mitochondria and lysosomes, while the red CDs were primarily localized in lysosomes. These findings significantly advance our understanding of the photoluminescence mechanism of multicolor CDs and help to guide future design and applications of these promising nanomaterials.

Understanding the origin and sensitivity of carbon dot emission will improve their utility in various applications.

Since the accidental discovery of luminescent carbon fragments in 2004,¹ carbon dots (CDs) have attracted great research interest due to the diverse synthetic methods, tunable luminescence, and applicability in a broad range of fields, including bio-imaging,^2–4 sensing,^5,6 and light emitting diodes (LEDs).^7,8 Typically, CDs are fluorescent carbon nanostructures of sizes less than 10 nm, composed of carbon, oxygen, and nitrogen.^9–12 CDs can be produced through bottom-up methods, which involve small molecular precursors like citric acid, malic acid, urea, ethylenediamine, and so on.^13–15 In a high temperature reaction, polymerization and dehydration occur among various functional groups, and the resulting products are usually a mixture of small molecule residues, oligomers, and long chain polymers.¹⁶ The unclear fluorescence mechanisms and poorly understood internal structure of CDs limit the ability to understand, tune, and fully exploit their fluorescence properties.Fortunately, in recent years, breakthrough syntheses of multicolor CDs have been achieved.^17–19 Several different multicolor CDs have been synthesized with aromatic compounds such as phenylenediamine.^4,20–22 However, it should be noted that precursors such as aniline and phenol may have toxic effects on human health and the environment,^23,24 and thus should be avoided where possible. Syntheses of colorful CDs from non-aromatic compounds such as citric acid and urea often employ solvothermal methods. Utilizing different solvents such as formamide and dimethylformamide have been shown to play a significant role in tuning CD emission.^25,26 In addition, chromatographic post-treatment of as-made CDs plays a critical role in obtaining different colored fractions, using techniques such as anion-exchange column chromatography,²⁶ normal phase silica chromatography,²⁷ and reversed phase silica chromatography.¹⁵ Compared with high performance liquid chromatography (HPLC), the aforementioned column chromatography techniques help to separate CDs on a larger scale. These separations are based on charge²⁶ or polarity,²¹ and are efficient in isolating the desired fractions with distinct colors so that detailed structural characterization can be performed.To gain insight into the fluorescence mechanism of these multicolor CDs, researchers have considered three hypotheses: quantum size effects,²⁸ the inclusion of molecular fluorophores,²⁹ and surface state-induced emission.³⁰ For example, Rogach and coworkers developed solid-state CDs with tunable fluorescence via the seeded growth method. They attributed the tunable emission to the size of π-conjugated domains.³¹ Yang and coworkers synthesized CDs by hydrothermal treatment of citric acid and ethylenediamine. They identified a small molecule fluorophore, IPCA (1,2,3,5-tetrahydro-5-oxo-imidazo[1,2-α]pyridiine-7-carboxylic acid) from CD column separation fractions, which contributed to the blue fluorescence.¹³ Xiong and coworkers synthesized CDs from urea and p-phenylenediamine that emitted a range of colors and separated them with silica column chromatography. They found the degree of carbon oxidation increased as the emission redshifted and thus, they endorsed the surface state hypothesis.²¹ In addition to the above mechanisms, computational methods such as density functional theory (DFT) have also been applied to analyze the fluorescence origins of CDs. The charge transfer between functional groups on the polymeric unit of CDs made from citric acid and ethylenediamine was found to facilitate blue emission.¹⁶The goal of present work is to understand the fluorescence origin of multicolor CDs. The model multicolor CDs were obtained by reacting citric acid and urea in formamide via a microwave-assisted hydrothermal treatment. An automated chromatographic apparatus was employed to separate as-made CD mixtures into distinct color fractions. The individual separation process took around 20 minutes, and the obtained CD fractions exhibit discrete illumination-induced emission throughout the visible region of the spectrum. Interestingly, the sizes of separated CD fractions are not statistically different from one another, suggesting that the quantum size effects are not the source of differential emission. Solvatochromism experiments showed that the blue and green fractions have similar fluorescence behavior as a function of solvent polarity, but the red fraction behaved differently. Using computational simulations, three models of the fluorescence origin were constructed and evaluated, showing that the formation of small blue fluorescent molecules is likely and pyrene analogs could be the origins for various emission colors. Moreover, two representative CD fractions, the blue- and red-emitting fractions, were chosen for subsequent cell imaging experiments. The localization pattern for the CD fractions differed: blue-emitting CDs were observed in a wide range of organelles, while red-emitting CDs were primarily enclosed in lysosomes. Understanding the origin and the sensitivity of CD emission will improve their utility in bioimaging applications.     

Nanoscale electrochemistry in a copper/aqueous/oil three-phase system: surface structure–activity-corrosion potential relationships

Practically important metal electrodes are usually polycrystalline, comprising surface grains of many different crystallographic orientations, as well as grain boundaries. In this study, scanning electrochemical cell microscopy (SECCM) is applied in tandem with co-located electron backscattered diffraction (EBSD) to give a holistic view of the relationship between the surface structure and the electrochemical activity and corrosion susceptibility of polycrystalline Cu. An unusual aqueous nanodroplet/oil (dodecane)/metal three-phase configuration is employed, which opens up new prospects for fundamental studies of multiphase electrochemical systems, and mimics the environment of corrosion in certain industrial and automotive applications. In this configuration, the nanodroplet formed at the end of the SECCM probe (nanopipette) is surrounded by dodecane, which acts as a reservoir for oil-soluble species (e.g., O₂) and can give rise to enhanced flux(es) across the immiscible liquid–liquid interface, as shown by finite element method (FEM) simulations. This unique three-phase configuration is used to fingerprint nanoscale corrosion in a nanodroplet cell, and to analyse the interrelationship between the Cu oxidation, Cu²⁺ deposition and oxygen reduction reaction (ORR) processes, together with nanoscale open circuit (corrosion) potential, in a grain-by-grain manner. Complex patterns of surface reactivity highlight the important role of grains of high-index orientation and microscopic surface defects (e.g., microscratches) in modulating the corrosion-properties of polycrystalline Cu. This work provides a roadmap for in-depth surface structure–function studies in (electro)materials science and highlights how small variations in surface structure (e.g., crystallographic orientation) can give rise to large differences in nanoscale reactivity.

Probing Cu corrosion in an aqueous nanodroplet/oil/metal three-phase environment revealed unique patterns of surface reactivity. The electrochemistry of high-index facets cannot be predicted simply from the low-index {001}, {011} and {111} responses.

Corrosion has long been studied, as a significant concern and a costly issue (ca. 3% of the GDP of industrialised countries) for the modern world.^1,2 For metals, in particular, electrochemical techniques, allied to complementary analytical and microscopy methods, play a central role in unveiling corrosion and corrosion protection mechanisms.^3–7 However, a limitation of many experimental approaches is that the electrochemical perturbation (and measurement) is applied globally at a macroscopic electrode immersed in a bulk solution,⁸ but most corrosion processes are initiated and perpetuated at (sub)microscopic surface sites (e.g., grain boundaries, inclusions, microscratches etc.).^9–14 Mismatch between the scale of key corrosion phenomena and conventional electrochemical methods makes it difficult to unambiguously identify the key anodic/cathodic sites driving corrosion. This issue is compounded for the case of atmospheric corrosion,¹⁵ or corrosion in certain automotive/industrial environments (vide infra),^16,17 which take place due to the action of small droplets on the surface in a confined system. Corrosion science needs electrochemical techniques that operate at the (sub)microscale, and allow activity and surface structure to be correlated commensurately at this scale.Among the limited library of electrochemical techniques that can routinely operate at the (sub)microscale,^18,19 scanning electrochemical cell microscopy (SECCM) is attracting significant attention.^20–22 SECCM maps electrochemistry locally and directly via a nanoscale electrochemical meniscus cell (formed at the end of a fluidic probe) that makes measurements over an array of points (typically thousands of discrete areas) on an electrode (or other) surface. For polycrystalline surfaces, SECCM measurements are powerfully combined with co-located electron backscattered diffraction (EBSD), to elucidate nanoscale structure–activity, as exemplified by studies of various electrochemical processes at a range of polycrystalline materials, including Pt,^23–26 Au,²⁷ Pd,²⁸ low carbon steel,^29–31 Zn³² and boron-doped diamond.³³In addition to its high spatiotemporal resolution, the meniscus cell configuration of SECCM facilitates rapid reactant/product exchange with the surrounding environment, mimicking a gas diffusion electrode, with an enhanced flux of gases into the meniscus cell (i.e., at the three-phase boundary).^24,27,34 When operated in air, SECCM emulates the configuration of atmospheric corrosion, with gas exchange (e.g., oxygen, O₂) taking place across the liquid/gas interface of the meniscus in contact with a surface of interest. As recently reported, and expanded upon herein, SECCM can also be operated under oil immersion,^32,35 which not only aids in confinement of the meniscus cell during prolonged measurements,³⁵ but also opens up the possibility of studying the effect of oil-soluble species (e.g., corrosion inhibitors, organic contaminants, redox mediators etc.) on local reactions at the solid/liquid/liquid interface with high spatial-resolution. This configuration is regaining interest for fundamental studies,^36,37 as well as being of great practical importance (e.g., phase-transfer reactions in industrial chemical processes, biology etc.).³⁸A key attribute of SECCM is that a number of conventional dynamic electrochemistry techniques (e.g., potentiometry, amperometry and voltammetry) can be translated readily to the confines of the meniscus cell.^20,22,39 Herein, the versatility of chronopotentiometry for local corrosion and electrochemical measurements is demonstrated. First, it is possible to make meniscus contact at zero applied current, corresponding to open circuit potential (OCP), which is measured. This corresponds to the corrosion (mixed) potential, where the rate of anodic dissolution of the metal (forming metal ions) and the rate of reduction of oxygen are balanced. Surface ion release under this condition is then analysed by subsequent “electrochemical titration” of a portion of the released metal ions, by applying a cathodic current and recording the resulting chronopotentiometric curve.⁴⁰ This allows the evaluation of intrinsic corrosion susceptibility, in situ, with high spatial-resolution, for the entire range of crystallographic orientations of a polycrystalline metal (i.e., revealed through co-located EBSD analysis). Chronopotentiometry measurements with and without O₂ present, and the use of an anodic pulse to induce the anodic dissolution (as well as the cathodic measurements mentioned) allow all of the key electrochemical processes underpinning localised corrosion to be studied. The patterns of surface reactivity establish the intimate link between corrosion susceptibility, electrochemical kinetics and surface structure at the nanoscale.     

Carbohydrate-binding module O-mannosylation alters binding selectivity to cellulose and lignin

Improved understanding of the effect of protein glycosylation is expected to provide the foundation for the design of protein glycoengineering strategies. In this study, we examine the impact of O-glycosylation on the binding selectivity of a model Family 1 carbohydrate-binding module (CBM), which has been shown to be one of the primary sub-domains responsible for non-productive lignin binding in multi-modular cellulases. Specifically, we examine the relationship between glycan structure and the binding specificity of the CBM to cellulose and lignin substrates. We find that the glycosylation pattern of the CBM exhibits a strong influence on the binding affinity and the selectivity between both cellulose and lignin. In addition, the large set of binding data collected allows us to examine the relationship between binding affinity and the correlation in motion between pairs of glycosylation sites. Our results suggest that glycoforms displaying highly correlated motion in their glycosylation sites tend to bind cellulose with high affinity and lignin with low affinity. Taken together, this work helps lay the groundwork for future exploitation of glycoengineering as a tool to improve the performance of industrial enzymes.

Improved understanding of the effect of protein glycosylation is expected to provide the foundation for the design of protein glycoengineering strategies.

The cell walls of terrestrial plants primarily comprise the polysaccharides cellulose, hemicellulose, and pectin, as well as the heterogeneous aromatic polymer, lignin. In nature, carbohydrates derived from plant polysaccharides provide a massive carbon and energy source for biomass-degrading fungi, bacteria, and archaea, which together are the primary organisms that recycle plant matter and are a critical component of the global carbon cycle. Across the various environments in which these microbes break down lignocellulose, a few known enzymatic and chemical systems have evolved to deconstruct polysaccharides to soluble sugars.^1–6 These natural systems are, in several cases, being evaluated for industrial use to produce sugars for further conversion into renewable biofuels and chemicals.From an industrial perspective, overcoming biomass recalcitrance to cost-effectively produce soluble intermediates, including sugars for further upgrading remains the main challenge in biomass conversion. Lignin, the evolution of which in planta provided a significant advantage for terrestrial plants to mitigate microbial attack, is now widely recognized as a primary cause of biomass recalcitrance.⁷ Chemical and/or biological processing scenarios of lignocellulose have been evaluated⁸ and several approaches have been scaled to industrial biorefineries to date. Many biomass conversion technologies overcome recalcitrance by partially or wholly removing lignin from biomass using thermochemical pretreatment or fractionation. This approach enables easier polysaccharide access for carbohydrate-active enzymes and/or microbes. There are however, several biomass deconstruction approaches that employ enzymes or microbes with whole, unpretreated biomass.^9,10 In most realistic biomass conversion scenarios wherein enzymes or microbes are used to depolymerize polysaccharides, native or residual lignin remains.^11,12 It is important to note that lignin can bind and sequester carbohydrate-active enzymes, which in turn can affect conversion performance.¹³Therefore, efforts aimed at improving cellulose binding selectivity relative to lignin have emerged as major thrusts in cellulase studies.^14–25 Multiple reports in the past a few years have made exciting new contributions to our collective understanding of how fungal glycoside hydrolases, which are among the most well-characterized cellulolytic enzymes given their importance to cellulosic biofuels production, bind to lignin from various pretreatments.^15,17 Taken together, these studies have demonstrated that the Family 1 carbohydrate-binding modules (CBMs) often found in fungal cellulases are the most relevant sub-domains for non-productive binding to lignin,^15,17,20,26 likely due to the hydrophobic face of these CBMs that is known to be also responsible for cellulose binding (Fig. 1).²⁷Open in a separate windowFig. 1Model of glycosylated CBM binding the surface of a cellulose crystal. Glycans are shown in green with oxygen atoms in red, tyrosines known to be critical to binding shown in purple, and disulfide bonds Cys8–Cys25 and Cys19–Cys35 in yellow.Furthermore, several studies have been published recently using protein engineering of Family 1 CBMs to improve CBM binding selectivity to cellulose with respect to lignin. Of particular note, Strobel et al. screened a large library of point mutations in both the Family 1 CBM and the linker connecting the catalytic domain (CD) and CBM.^21,22 These studies demonstrated that several mutations in the CBM and one in the linker led to improved cellulose binding selectivity compared to lignin. The emerging picture is that the CBM-cellulose interaction, which occurs mainly as a result of stacking between the flat, hydrophobic CBM face (which is decorated with aromatic residues) and the hydrophobic crystal face of cellulose I, is also likely the main driving force in the CBM-lignin interaction given the strong potential for aromatic–aromatic and hydrophobic interactions.Alongside amino acid changes, modification of O-glycosylation has recently emerged as a potential tool in engineering fungal CBMs, which Harrison et al. demonstrated to be O-glycosylated.^28–31 In particular, we have revealed that the O-mannosylation of a Family 1 CBM of Trichoderma reesei cellobiohydrolase I (TrCel7A) can lead to significant enhancements in the binding affinity towards bacterial microcrystalline cellulose (BMCC).^30,32,33 This observation, together with the fact that glycans have the potential to form both hydrophilic and hydrophobic interactions with other molecules, led us to hypothesize that glycosylation may have a unique role in the binding selectivity of Family 1 CBMs to cellulose relative to lignin and as such, glycoengineering may be exploited to improve the industrial performance of these enzymes. To test this hypothesis, in the present study, we systematically probed the effects of glycosylation on CBM binding affinity for a variety of lignocellulose-derived cellulose and lignin substrates and investigated routes to computationally predict the binding properties of different glycosylated CBMs.     

Enantioselective hydrogenation of annulated arenes: controlled formation of multiple stereocenters in adjacent rings

We report a method for the enantioselective hydrogenation of annulated arenes using 4H-pyrido[1,2-a]pyrimidinones as substrates. The method selectively generates multiple stereocenters in adjacent rings leading to architecturally complex motifs, which resemble bioactive molecules. The mechanistic study of the stereochemical outcome revealed that the catalyst is able to overcome substrate stereocontrol providing all-cis-substituted products predominantly. In a sequential protocol, a matching interaction between catalyst and substrate stereocontrol is achieved that facilitates diastereo- and enantioselective access to trans-products.

We report a method for the enantioselective hydrogenation of annulated arenes using 4H-pyrido[1,2-a]pyrimidinones as substrates.

Chiral, saturated carbo- and heterocycles are important structural elements of secondary metabolites and active ingredients in drugs and agrochemicals.¹ Compared to classical synthetic routes that utilize prefunctionalized precursors,² enantioselective arene hydrogenation provides a more direct route to such motifs from readily accessible (hetero-) aromatic substrates.³ With current hydrogenation technologies, a variety of monocyclic arenes such as pyridines,⁴ furans,⁵ thiophenes,⁶ and annulated arenes such as quinolines and isoquinolines,⁷ indoles,⁸ and naphthalenes⁹ can be hydrogenated with high enantioselectivity. However, in all previous reports on the enantioselective hydrogenation of annulated arenes (1) the resulting products (2) contain only a single saturated ring with at least one aromatic sextet being preserved in the product, and consequently, with stereocenter(s) only being formed in one ring (Fig. 1A).¹⁰ An enantioselective hydrogenation of multiple annulated aromatic rings would enable the generation of multiple stereocenters at adjacent rings in a single step, leading to desirable, architecturally complex and natural product-like motifs. Herein, we report the first such example using N-bridged 4H-pyrido[1,2-a]pyrimidinone (pyrido-pyrimidinone) substrates (3, Fig. 1B).^10a–d Pyrido-pyrimidinones, and their saturated analogs, are featured in bioactive molecules (Fig. 1C). For instance, Roche''s Risdiplam (5), an inhibitor against spinal muscular atrophy, includes a pyrido-pyrimidinone unit¹¹ and a semi-reduced tetrahydropyrido-pyrimidinone is a part of risperidone (6), a blockbuster drug against schizophrenia, while octahydropyrido-pyrimidinones (4) are structurally closely related to quinolizidine alkaloids such as lupanine (7). To date, only a small number of octahydropyrido-pyrimidinones (4) have been accessed with the majority resulting from the hetero-geneous, racemic hydrogenation of tetrahydropyrido-pyrimidinones (8). To the best of our knowledge, enantioenriched octahydropyrido-pyrimidinones have never been accessed by enantioselective catalysis.¹²Open in a separate windowFig. 1(A) Previously: only one ring of annulated arenes reduced by enantioselective hydrogenation. (B) This work: enantioselective hydrogenation of both rings of bicyclic aromatic pyrido-pyrimidinones (3). (C) Biologically active examples/analogues of pyrido-pyrimidinones and their hydrogenated products.We realized that an enantioselective hydrogenation of the readily accessible pyrido-pyrimidinones would in principle offer the most direct approach towards these valuable product motifs, provided that the chemoselectivity could be controlled and potential side products, such as tetrahydro- (8), hexahydropyrido-pyrimidinones 9 and 10, and the presumably unstable hemiaminal 11 resulting from amide reduction, could be avoided (Fig. 2).¹³ Furthermore, we rationalized that up to four different diastereomers could result from the creation of three independent stereocenters, making the simultaneous control of chemo-, diastereo-, and enantioselectivity a daunting challenge. Nevertheless, we were ideally positioned to overcome this challenge given the high reactivity, enantioselectivity, and broad tolerance of functional groups shown by the chiral ruthenium-bisNHC catalyst system developed by our group (12).^14,15 Indeed, we observed that catalytic hydrogenation of model substrate 2,7-dimethylpyrido-pyrimidinone (3a) proceeded with near-complete chemoselectivity under a variety of reaction conditions (Table S1^†). Under the optimized conditions, the desired octahydropyrido-pyrimidinone was obtained in 92% yield as a mixture of just two from the possible four diastereomers (63 : 37 diastereomeric ratio (dr)) and with excellent 96% enantiomeric excess (ee) for the major diastereomer (Table S1,^† entry 4). The evaluation of other substitution patterns of the pyrido- pyrimidinone using the respective dimethyl-substituted substrates revealed that the 2,8-disubstituted product can also be obtained with good chemo-, enhanced diastereo-, and moderate enantioselectivity. However, substrates with 2,6- and 2,9-substitution patterns were less reactive. Only the eastern pyridine ring was reduced in 48% and 82% yield, respectively (Table S2^†). As such we decided to focus on substrates with a 2,7-substitution pattern when exploring the scope of this reaction (Fig. 3). Next, the scope was evaluated (Fig. 3). Substrates without a substituent in the C2-position (3b), and substrates containing sterically more hindering substituents such as a benzyl or an iso-propyl group (3c,d) in the C2-position all delivered the products in high yields as a mixture of just two diastereomers with moderate dr and excellent ee values for the major diastereomer. The dr values increase with the steric size of the C2-substituent. A phenyl substituent in the C2-position rendered the (eastern) pyrimidinone ring inactive, thus chemoselectively providing tetrahydropyrido-pyrimidinone 8e with moderate ee under the standard reaction conditions including the reaction time of 48 h. Contrastingly, the introduction of aryl groups in the 7-position of the (western) pyridine ring was well-tolerated. Consequently, octahydropyrido-pyrimidinones carrying aryl groups such as phenyl (4f), sterically demanding ortho-tolyl (4g), and other aryl groups bearing various synthetically useful functional groups such as NHBoc, OCF₃, or Cl in the para-position (4h–j) can all be accessed in high yields and ee values. Curiously, while 7-para-fluorophenyl-substituted product 4k was obtained in high yield, the enantioselectivity was reduced in comparison to its chlorine-containing counterpart 4j. Products without a substituent in the 7-position (4l–n) were isolated in good yields as single diastereomers with good to high ee.Open in a separate windowFig. 2Chemo- and stereoselectivity challenges.Open in a separate windowFig. 3Scope. Combined isolated yields of both diastereomers are reported. The dr was determined by NMR-spectroscopy. The major diastereomer (and enantiomer) was assigned by X-ray crystallographic analysis of both diastereomers of 4a. The assignment for the other products was conducted by analogy. The ee of the minor diastereomer was measured in all cases (see ESI^†). ^aTraces of an unknown impurity could not be separated from the product. ^bProtection with trifluoroacetic anhydride. ^c80 bar H₂ used. SINpEt: 1,3-bis(1-(naphthalene-1-yl)ethyl)-4,5-dihydroimidazolylidene; TFA: trifluoroacetate.Intrigued by those observations, we proceeded to study the stereochemical mechanism of the reaction. First, we determined the reaction progress of the hydrogenation of model substrate 3a over time (Fig. 4A). After a reaction time of 1.5 h, tetrahydropyrido-pyrimidinone 8a is almost the exclusive species before its relative abundance decreases with a coinciding increase of the proportion of octahydropyrido-pyrimidinone 4a. Notably, 7-methyl- and 7-phenyl-substituted intermediates 8a and 8f were isolated in good yields and with moderate enantiomeric excess (8a: 84% yield, 63% ee; 8f: 50% yield, 62% ee). The reaction is complete after 8 h, leaving octahydrogenated product 4a as the exclusive species. Hence 8a must be an intermediate in the hydrogenation of 3a to 4a. No further signals, other than those allocated to the substrate 3a and tetrahydrointermediate 8a, were observed in the enone or α-ketone regions at any of the time points. Furthermore, the C2 and C9a C–H bonds are cis-aligned in both diastereomers of the 2,7-dimethylproduct 4a (confirmed by X-ray crystallography, see ESI^†). A cis alignment would be enforced by a non-interrupted coordination of the catalyst during the hydrogenation of the (eastern) pyrimidinone ring. The codependence of the configurations of C2 and C9a would result in the formation of only two diastereomers in the hydrogenation of 7-substituted substrates and only a single diastereomer in the hydrogenation of 7-unsubstituted substrates, which is in agreement with the observations made during the exploration of the scope (Fig. 4B).¹⁶ Both observations suggest that enone 9a and imine 10a are not intermediates in this reaction. Finally, we determined that two separate effects cause the (relatively low) diastereomeric ratios of 7-substituted octahydropyrido-pyrimidinones. Firstly, the enantiocontrol over the initially formed C7-stereocenter is only moderate (8a: 63% ee after 1.5 h, Fig. 4B). Secondly, the ee of the intermediates 8 increases with the reaction progress of further hydrogenation towards the octahydroproducts 4 (Fig. 4C). It follows that the minor enantiomer of chiral intermediate 8 is converted into octahydro-product 4 more rapidly than the major enantiomer, indicating a mismatched interaction between substrate and catalyst control. To further test this conclusion, isolated intermediate 8a was submitted to the enantioselective hydrogenation using the opposite enantiomer of the chiral catalyst in the second hydrogenation step (Fig. 4D).¹⁶ In this case, hydrogenation proceeds with a matched interaction between catalyst and substrate control and provides the opposite diastereomer trans–cis-4a with excellent diastereo- and enantiocontrol. Notably even racemic arene hydrogenation rarely produces trans-products.¹⁸ The dr of all–cis-4a derived from the two-step hydrogenation (Fig. 4D, red equation) is higher than that of the standard one-pot protocol (75 : 25 vs 56 : 44 dr). This is a result of the increase of the ee of intermediate 8a as a function of the reaction time (the minor enantiomer is consumed faster to form 4a). The effective ee of 8a in the two-step protocol (isolated after 2 h) was higher than that of 8a in the standard protocol resulting in a higher dr of 4a.Open in a separate windowFig. 4Mechanistic studies. (A) Excerpt of the ¹H NMR spectrum of the reaction mixture as a function of the reaction time. (B) Key observations that suggest a two-step hydrogenation process. (C) ee of 8a as a function of the reaction time. (D) Sequential enantioselective hydrogenation of 3a using opposite enantiomers of the chiral catalyst.¹⁷     

Crystals springing into action: metal–organic framework CUK-1 as a pressure-driven molecular spring

Mercury porosimetry and in situ high pressure single crystal X-ray diffraction revealed the wine-rack CUK-1 MOF as a unique crystalline material capable of a fully reversible mechanical pressure-triggered structural contraction. The near-absence of hysteresis upon cycling exhibited by this robust MOF, akin to an ideal molecular spring, is associated with a constant work energy storage capacity of 40 J g⁻¹. Molecular simulations were further deployed to uncover the free-energy landscape behind this unprecedented pressure-responsive phenomenon in the area of compliant hybrid porous materials. This discovery is of utmost importance from the perspective of instant energy storage and delivery.

Mercury porosimetry and in situ high pressure single crystal X-ray diffraction revealed the wine-rack CUK-1 MOF as a unique crystalline material capable of a fully reversible mechanical pressure-triggered structural contraction.

Reducing the world''s fossil fuel dependence is the focus of many global initiatives,¹ aiming to mitigate the effects of climate change through tapping into sustainable energy resources such as solar and wind power. However, increasing reliance on these renewable energy sources has introduced difficulties due to the offset between power availability and demand peaks. Complementary technologies are necessary to alleviate intermittent supply, such as peaking power plants, demand-side energy management, or large scale energy storage.² The latter is particularly desirable as it can decouple electricity production and consumption, however the lack of a “one size fits all” approach has led the scientific community to envisage unconventional energy storage strategies.One such avenue emerging in recent years is the storage of mechanical energy via the compression of a suitable stimuli-responsive system, either through the intrusion of a non-wetting fluid into hydrophobic porous frameworks,³ or by means of application of an external pressure on flexible materials.⁴The former approach, first pioneered using water intrusion in zeolites and silicas,¹¹ has recently been extended to small pore zeolitic imidazolate frameworks.¹² Unfortunately, besides requiring highly hydrophobic systems, water intrusion achieves a relatively low stored energy density,³ of around 3–25 J g⁻¹. The second strategy takes advantage of the compliant nature of bulk materials. Energy is stored through structural deformations, manifesting as continuous or sudden volume changes under external pressure. The energy stored in flexible materials over a compression/decompression cycle can be an order of magnitude higher compared to the values achieved using fluid intrusion in rigid porous systems.¹³ In theory, three types of pressure-induced structural behaviour can be envisioned for such a responsive system. If the structure contraction is non-reversible, all energy is dissipated and the system is categorized as a nano-shock absorber (Fig. 1b). For structural changes that are reversible upon decompression two families of system can be distinguished, i.e. a nano-damper (Fig. 1c) or an ideal nano-spring (Fig. 1d) when the p–V curves show hysteresis or fully overlap, respectively.¹⁴Open in a separate windowFig. 1(A) Schema of mechanical energy storage in compliant crystalline materials, implying a unit cell volume change between open (op) and contracted (cp) structures, and prototypical pressure-volume curves of stimuli-responsive materials under mechanical pressure for (B) nano-shock absorbers, exemplified by MIL-53(Al),⁵ MIL-53(Ga)-FA⁶ and ZIF-4(Zn),⁷ (C) nano-dampers e.g. MIL-53(Cr),⁸ MIL-47(V)⁹ and MIL-53(Al)-FA¹⁰ and (D) nano-springs, insofar exhibited exclusively by CUK-1 presented herein.Metal–organic frameworks (MOFs), a class of porous, crystalline materials comprised of metal vertices interconnected by organic linkers, are known to exhibit responsiveness to a variety of stimuli,^15,16 including external pressure.¹⁷ Recently, several frameworks of this family of hybrid materials have been shown to act as energy storing nano-dampers or energy dissipative nano-shock absorbers, as is the case for the highly flexible MIL-53(M)^5,8,10 and MIL-47(V)⁹ series and more recently ZIF-4(Zn)⁷ (see Fig. 1b and c for their related structural behaviours). In such flexible crystalline materials compression is associated with a displacive phase transition between distinct structures of differing unit cell volumes, denoted as open (op) and contracted (cp) forms^15,16 and illustrated in Fig. 1a, occurring reversibly or irreversibly for a nano-damper or nano-shock absorber, respectively. The considerable stored energy associated with this transition, in the range of 30–200 J g⁻¹ (up to 4 kJ g⁻¹ for shock absorbers¹⁸) is highly attractive from the perspective of mechanical energy storage. However, the hysteretic compression/decompression curve characterising known nano-damper MOFs leads to a partial loss of work energy, lowering the potential storage efficiency, as well as creating issues through heat dissipation. Insofar, the search for a ideal spring-like crystalline material, capable of reversible pressure-induced structural switching without any hysteresis (Fig. 1d) has been fruitless, precluding their applicability for efficient, high density energy storage applications. Herein, a subtle combination of Hg-porosimetry, high-pressure single crystal X-ray diffraction (SC-XRD) and molecular simulations reveals the 1D-channel CUK-1 (M, M = Co, Mg)¹⁹ MOF as the first compliant hybrid porous material with a spring-back mechanical breathing behaviour.Such unique mechanically-triggered structural response implies a continuous pore contraction/expansion between op and cp forms in a narrow pressure range of 280–290 MPa, accompanied by a unit cell volume change of 20.9%. This optimal scenario paves the way towards fast energy storage/delivery system of about 40 J g⁻¹. The channel-like CUK-1(M) composed of chains of μ₃-OH/O edge and vertex sharing metal octahedra (M = Co,¹⁹ Mg²⁰) coordinated by bidentate 2,4-pyridinedicarboxylic ligands, recently emerged as an attractive porous material owing to its promising sorption performance combined with environmentally-friendly hydrothermal synthesis and high thermal and chemical stability.^20–22 Its wine-rack topology and its relatively rigid behaviour upon guest adsorption are reminiscent to that of MIL-47(V) a MOF which interestingly underwent a hysteretic, reversible structural contraction upon exerting an external pressure of 125 MPa,⁹ associated with a stored/delivered energy of 33 J g⁻¹. Inspired by our previous findings on MIL-47(V), we deliberately explored the pressure-induced structural behaviour of CUK-1 in its isostructural Co and Mg forms. MOF synthesis was performed according to the protocol detailed in ESI.^† Phase purity was confirmed by powder XRD (Fig. S3, S4 and Table S1^†) while their textural features, including BET area and pore volume, were found to match previously reported data.^19,20Mercury intrusion curves were recorded on the powder samples up to a maximum of 413 MPa as shown for CUK-1(Co) in Fig. 2, its Mg variant being reported in Fig. S6, ESI,^† together with full experimental details. A substantial amount of Hg intrudes at low pressure (<10 MPa), due to compaction of the crystals and filling of inter-particle porosity. This is followed by a sudden volume change at 281 MPa where a sharp step is observed (see inset of Fig. 2). By analogy with the conclusions previously drawn for the series of MIL-53(M)/MIL-47(V) frameworks,^5,8–10 this intruded Hg volume increase is associated with a structural contraction of CUK-1(Co), as its channel size (approx. 6.6 Å) is an order of magnitude below the pore dimension where non-wetting mercury can intrude in this pressure range (at 52 Å). The extrusion curve shows a near-perfect overlap, indicating that the framework behaves as an ideal spring, with no hysteresis between the intrusion/extrusion branches.Open in a separate windowFig. 2Sequential mercury intrusion–extrusion curves on CUK-1(Co) powder, in blue line and red, respectively. Line is a guide for eye. Volume below 1 MPa corresponds to powder compaction and intercrystallite void filling. Dotted horizontal lines demarcate contraction lower and upper bounds. Inset highlights the intrusion step in a linear scale with the op/cp contraction marked with an arrow.Moreover, this behaviour is highly repeatable, as confirmed by four consecutive pressure cycles (in Fig. S5^†). Interestingly, the same behaviour also holds true for CUK-1(Mg) (Fig. S6^†), with a similar intrusion pressure of 288 MPa. Since the two metal ions show relatively similar ionic radius (Co²⁺: 1.50(7) Å and Mg²⁺: 1.41(7) Å),²⁴ the averaged metal–oxygen distance is nearly identical in their corresponding coordination: spheres: (Co–O: 2.107(20) Å and Mg–O: 2.073(20) Å). Such analogous metal-linker bond strength is most likely at the origin of the very similar pressure-induced response of the two materials. The high transition pressure of CUK-1(Co) underpins the inability of guest adsorption to induce a breathing effect as observed previously.^20,21 Indeed, the adsorption stresses encountered throughout guest insertion are simply insufficient to overcome the energetic penalty of transition.^14,25 The 0.143 mL g⁻¹ volume change associated with the observed step in the CUK-1(Co) intrusion curve corresponds to a 20.9% change in unit cell volume, lower than in the similar phenyl-based MIL-47(V) of 43%.⁹ However, the stored energy calculated through W = P × ΔV is 40 J g⁻¹, 20% larger than the value reported for MIL-47(V)⁹ of 33 J g⁻¹. Here, the higher pressure of CUK-1(Co) switching, 281 MPa vs. 125 MPa for MIL-47(V) balances out the ΔV term. Moreover, owing to its relatively dense framework, the volumetric energy density of CUK-1(Co) remains attractive when compared to water intrusion systems (Table S4^†).Considering an initial unit cell volume for the CUK-1(Co) op form of 2467 ų from PXRD (see ESI^†), the resulting cp form is estimated to exhibit a unit cell volume of 1950 ų, based on the Hg intruded volume increase at 281 MPa. In order to directly observe the contracted form and identify the mechanism underpinning these intriguing dynamics, high pressure SC-XRD experiments were carried out in a membrane diamond anvil cell (mDAC). Individual CUK-1(Co) crystals were placed in a gasket between the polished diamonds of the mDAC, and immersed in a hydrostatic pressure transmitting medium of silicone oil AP-100, with a gold flake used to monitor inner mDAC pressure (full single crystal synthesis conditions and SC-XRD methodology available in the ESI^†).At ambient pressure, the indexed unit cell volume of the initial op form of CUK-1(Co) is nearly identical (2492 ų) to that of the previously reported²⁰ dehydrated monoclinic phase (2466.72 ų). Upon increasing DAC pressure to around 0.3 GPa, a volume contraction to the cp phase begins, which is in line with Hg porosimetry experiments. Reflections obtained from integrated 2D diffraction images were used to solve the pressure-induced structure through a dual space recycling algorithm in an expanded P1 setting, then further refined on F² using the SHELX suite²⁶ (complete data treatment methodology available in the ESI^†). The structure maintains the same C2/c space group throughout the transition between the two forms, and as such the spring-like dynamics of the framework can be described as a continuous contraction in a narrow pressure range. Above 0.5 GPa, the cp form is attained, with further pressure application leading to a linear decrease of its unit cell volume by 4% up to 1.8 GPa (Fig. 3).Open in a separate windowFig. 3Evolution of the CUK-1(Co) unit cell volume determined through indexation of Bragg reflections as a function of applied pressure as recorded in a DAC. Unit cell parameters corresponding to each pressure point can be found in Table S5, ESI.^†The unit cell dimensions of the solved cp form at 0.5 GPa are provided in Table 1, alongside as-indexed pristine op form parameters with Fig. 4a illustrating the two structures. The anisotropic transition is similar in nature to that of MIL-53(M)/MIL-47(V), characterised by a compaction in the b-direction (from approx. 13 Å to 9 Å) and an elongation along the a-axis (from 18 to nearly 20 Å). The change in the c-parameter is minimal, with only a slight increase, as it lies in the plane of the highly rigid octahedrally coordinated metal chains. A lowering of the angle (from 103 to 99°) is also observed, as the 1D parallel pores are straightened via the linker-induced torsion. A table comparing specific atomic distances, angles and torsions in the two forms is available in Table S7, ESI.^† The unit cell volume of the identified cp phase at 0.5 GPa of 1972 ų is only slightly higher than the value estimated from porosimetry measurements (as 1950 ų). We attribute this offset to the different interactions of the crystal surface with the respective pressure transmitting medium (mercury vs. silicone oil), as observed previously.⁵Crystallographic data of the pristine (op) and high pressure (cp) phases as determined from the CUK-1(Co) SC-XRD

An engineered biosynthetic–synthetic platform for production of halogenated indolmycin antibiotics

Indolmycin is an antibiotic from Streptomyces griseus ATCC 12648 with activity against Helicobacter pylori, Plasmodium falciparum, and methicillin-resistant Staphylococcus aureus. Here we describe the use of the indolmycin biosynthetic genes in E. coli to make indolmycenic acid, a chiral intermediate in indolmycin biosynthesis, which can then be converted to indolmycin through a three-step synthesis. To expand indolmycin structural diversity, we introduce a promiscuous tryptophanyl-tRNA synthetase gene (trpS) into our E. coli production system and feed halogenated indoles to generate the corresponding indolmycenic acids, ultimately allowing us to access indolmycin derivatives through synthesis. Bioactivity testing against methicillin-resistant Staphylococcus aureus showed modest antibiotic activity for 5-, 6-, and 7-fluoro-indolmycin.

A semi-synthetic system for producing indolmycin, an antibiotic, was developed and used to make indole-substituted, halogenated derivatives of indolmycin, some with modest bioactivity against methicillin-resistant Staphylococcus aureus.

Antibiotic-resistant bacteria pose a great threat to human health,^1–4 and the rates of new antibiotic discoveries and clinical approvals have been in a steep decline since the 1980s.¹ Without the discovery and development of new antibiotics, drug-resistant pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA), will become increasingly prevalent.^2–4 One strategy to increase antibiotic development has been to “rediscover” known, but underdeveloped, antibiotics.¹ One such example is indolmycin, which was originally discovered in 1960 from Streptomyces griseus ATCC 12648⁵ but was not originally developed for clinical use because of its narrow spectrum of activity^6–10 and its interference with tryptophan catabolism in the liver.^9,10 However, reignited interest in this old antibiotic led to the discovery of its activity against Helicobacter pylori,⁶Plasmodium falciparum,¹¹ and MRSA.¹² For MRSA, indolmycin was found to be active against mupirocin- and fuscidic acid-resistant MRSA strains, with strains resistant to indolmycin emerging infrequently and with reduced fitness compared to sensitive strains.¹² In addition, indolmycin has been shown to have minimal activity against common members of the human microbiota, suggesting that its narrow spectrum of activity is an asset.⁶ The first indole-substituted derivatives, 5-hydroxy and 5-methoxyindolmycin, were made by precursor-directed feeding of the indolmycin producer, Streptomyces griseus ATCC 12648, and they showed modest improvements in bioactivity against S. aureus and Escherichia coli.¹³ Two practical synthetic routes to indolmycin and some indole-substituted derivatives have been reported more recently,^14,15 which enabled access to a small variety of indole-substituted derivatives. Additionally, a previous patent has described synthetic methods to produce a variety of derivatives; however, these methods do not appear to offer stereochemical control, and some require tailoring steps specific to each analog.¹⁶ Therefore, further development of indolmycin would benefit from a simpler diversification method that could be applied to produce a wider variety of analogs with stereochemical control.Inspired by early biosynthetic studies,^17,18 our group previously identified the indolmycin gene cluster and elucidated the biosynthetic pathway, demonstrating that indolmycin (1) is assembled from tryptophan, arginine and S-adenosylmethionine (SAM) in a three-part process (Fig. 1).¹⁹ In the first part, l-arginine is oxidized by Ind4 in an oxygen- and PLP-dependent reaction to 4,5-dehydro-2-iminoarginine, which is then enantioselectively reduced by imine reductase Ind5 and its chaperone Ind6 to 4,5-dehydro-d-arginine. In the second part, tryptophan (2) is deaminated by PLP-dependent transaminases, giving indole pyruvate (3). Compound 3 is then methylated by SAM-dependent C-methyltransferase Ind1 to 3-methyl-indolepyruvate (4) which is reduced by NADH-dependent ketone reductase Ind2 to form indolmycenic acid (5). Then, in the third part, 4,5-dehydro-d-arginine and 5 are coupled in an ATP-dependent fashion by Ind3 and Ind6, resulting in an oxazolinone-cyclized molecule, N-desmethyl-indolmycin, which is finally N-methylated by Ind7, a SAM-dependent N-methyltransferase, to form 1.Open in a separate windowFig. 1Indolmycin biosynthesis from Streptomyces griseus ATCC 12648. (a) Indolmycin biosynthetic gene cluster. (b) Indolmycin biosynthetic pathway from Streptomyces griseus ATCC 12648. (c) Semi-synthetic scheme towards indolmycin and derivatives using indolmycin biosynthetic genes. The dashed arrow indicates a predicted side-product based on LC-MS analysis.Armed with the elucidated biosynthetic pathway for 1, we set out to create an in vivo system to make 1 in E. coli. We first cloned all necessary genes into four plasmids and co-expressed the genes in E. coli (Fig. S1^†), a strain which we named E. coli I1234670P5 (Table S1^†). We found that the genes needed to produce indolmycin in E. coli were ind1, ind2, ind3, ind4, ind6, ind7, ind0 and pel5, a homologous gene of ind5 from Paenibacillus elgii B69 showing better production of active protein in E. coli.^20,21 We also relied on the activity of endogenous E. coli aminotransferases to catalyze the initial tryptophan deamination step. However, only a small amount of 1 was produced (∼170 μg L⁻¹ of bacterial culture) and the yield could not be improved despite our best efforts (Fig. 2a). However, we found that this construct produced substantial amounts of 5 ([M + H]⁺ = 220 m/z) at 40–50 mg L⁻¹ of culture, along with a shunt product, C-desmethyl-indolmycenic acid (6; [M + H]⁺ = 206 m/z).Open in a separate windowFig. 2Biosynthetic production of 5 and semi-synthetic production of 1. (a) Extracted ion chromatograms show production of 5 with minimal production of 1 from E. coli I1234670P5. (b) Synthetic scheme to 1 from 5, adapted from literature methods.^14,15 (c) Total ion chromatogram of compound 1 isolated after semi-synthesis and final purification by semi-preparative HPLC. Compounds are indicated with coloured boxes and numbered.Compound 5 itself has been a focus of total synthetic efforts toward 1, as it is the key chiral precursor.^{14,15,22–26} Since production of 5 was much higher than that of 1 from E. coli I1234670P5, we pursued a semi-synthetic method of obtaining 1 using our biosynthetic platform to access 5, combined with a three-step chemical transformation (Fig. 2b). We attempted to remove extraneous genes from our biosynthetic platform by only including ind0, ind1, and ind2, but the changes resulted in reduced amounts of 5 (Fig. S2^†). At this time, it is unclear which of the other genes may be contributing to the production of compound 5. Therefore, we employed the full eight-gene construct toward synthesis of 5. We then adapted the three-step synthesis to make indolmycin,^14,15 in which purified 5 was esterified to make the ethyl ester (7; [M + H]⁺ = 248 m/z; Fig. S3a^†), cyclized to give N-desmethyl-indolmycin (8; [M + H]⁺ = 244 m/z; Fig. S3b and S4a^†), and methylated at the exocyclic nitrogen to give 1 ([M + H]⁺ = 258 m/z; Fig. 2, S4b and Table S4^†).Then, we wanted to make derivatives of 1 from indole derivatives, which are more widely accessible than derivatives of 2. The tryptophan synthase (TrpS) from Salmonella enterica has been previously shown to couple a wide variety of indole derivatives to l-serine to generate derivatives of 2.²⁷ We were able to replace pel5 with trpS in our biosynthetic platform without a reduction in the amount of 5 produced (Fig. S1b^†), and we named the resulting strain E. coli I1234670TS. When we fed 5-fluoroindole to E. coli I1234670TS, we observed increased production of fluorinated metabolites, 5-fluoro-indolmycenic acid (5F-5; [M + H]⁺ = 238 m/z) and 5-fluoro-C-desmethyl-indolmycenic acid (5F-6; [M + H]⁺ = 224 m/z) (Fig. 3a). We then optimized the feeding conditions, finding that 5F-5 amounts were optimal when we fed E. coli I1234670TS with 0.5 mM 5-fluoroindole per day over two days (Fig. S5^†).Open in a separate windowFig. 3Addition of the trpS gene to the biosynthetic platform allows incorporation of substituted indoles into 5. (a) LC-MS analysis of strains with and without trpS (E. coli I1234670TS and E. coli I1234670P5, respectively) when fed 5-fluoroindole. Chemical structures of compounds with the corresponding extracted ion chromatogram are shown to the right of the traces. (b) Indole derivatives tested. Dark blue shows indoles that were incorporated into an analog of 5 (>48% of underivatized 5 by LC-MS analysis; Table S5^†) and further purified; medium blue indicates indoles that were incorporated into an analog of 5 at lower levels (6–22% of underivatized 5 by LC-MS analysis; Table S5^†) but were not further verified by purification; and light blue indicates indoles that did not show detectable incorporation into 5.To determine the scope of indole derivatives accepted by our biosynthetic platform, we fed a variety of indoles to E. coli I1234670TS and monitored the production of 5 and its derivatives by LC-MS. Out of the indoles tested, we found that fluorinated and chlorinated indoles substituted at the 5-, 6- and 7-positions were the best accepted by the biosynthetic platform (Fig. 3b, S6 and S7^†). We predict that lower acceptance of indoles substituted at the 4-position may be due to steric hindrance, as 4-fluoroindole was moderately accepted, while 4-chloroindole was not observed at all. Although we observed LC-MS peaks consistent with conversion of some of the azaindoles and hydroxyindoles into 5 derivatives, further work is required to confirm, optimize and scale up the purification of these compounds (Fig. S7^†). Cultures producing derivatives of 5, substituted at 5-, 6- and 7-positions, were further scaled up for purification of the 5-derivatives and downstream synthesis of 1-derivatives (Fig. S8–S10 and Table S4^†). Each purified derivative of 5 and 1 was characterized by HR-MS and NMR (Table S3 and ESI Methods^†). Overall, cultures fed with the fluorinated indoles produced a higher amount of 5-derivatives than the cultures fed with the chlorinated indoles. 1 and its derivatives were tested against MRSA (Fig. S11^†). While the fluorinated derivatives showed bioactivity, the chlorinated derivatives of 1 did not show bioactivity at the maximum amount tested in the disk diffusion assay (30 μg). We determined MIC₅₀ values for each fluorinated compound (Table 1). The MIC₅₀ values demonstrate that 1 is a more potent inhibitor of MRSA than its derivatives, while 6F-1 showed the most potent inhibition of MRSA compared to any of the derivatives, followed by 7F-1 and 5F-1. The lack of bioactivity of the chlorinated compounds may be due to the bulky chlorinated substituent hindering the compounds'' abilities to bind to the tryptophanyl-tRNA synthetase (TrpRS) target, which is supported by docking studies of the analogs into a bacterial TrpRS structure (Fig. S12^†).MIC₅₀ values determined for 1 and its derivatives against MRSA. Values represent the average of three replicates ± the standard deviation. For 1, 5F-1, 6F-1 and 7F-1, concentrations ranging from 50 μg mL⁻¹ to 0.128 ng mL⁻¹ were tested, and for 5Cl-1, 6Cl-1 and 7Cl-1, concentrations ranging from 200 μg mL⁻¹ to 0.512 ng mL⁻¹ were tested. The reported MIC₅₀ value for indolmycin is 0.5 μg mL⁻¹ (range: 0.125–2 μg mL⁻¹) against MRSA¹²

Heparin reversal by an oligoethylene glycol functionalized guanidinocalixarene

Unfractionated heparin (UFH), a naturally occurring anionic polysaccharide, is widely used as an anticoagulant agent in clinical practice. When overdosed or used in sensitive patients, UFH may cause various risks and a UFH neutralizer needs to be administered immediately to reverse heparinization. However, the most common UFH neutralizer, protamine sulfate, often causes various adverse effects, some of which are life-threatening. Herein, we designed a highly biocompatible, oligoethylene glycol functionalized guanidinocalixarene (GC4AOEG) as an antidote against UFH. GC4AOEG and UFH exhibited a strong binding affinity, ensuring specific recognition and neutralization of UFH by GC4AOEG in vitro and in vivo. As a consequence, UFH-induced excessive bleeding was significantly alleviated by GC4AOEG in different mouse bleeding models. Additionally, no adverse effects were observed during these treatments in vivo. Taken together, GC4AOEG, as a strategically designed, biocompatible artificial receptor with strong recognition affinity towards UFH, may have significant clinical potential as an alternative UFH reversal agent.

An oligoethylene glycol functionalized guanidinocalix[4]arene was developed as a safe antidote against heparin, via specific recognition and neutralization of heparin in vitro and in vivo.

Heparin sodium, often referred to as unfractionated heparin (UFH, also known as heparin), is a well-known anionic glycosaminoglycan consisting of long, helical, unbranched chains of repeating sulfonated disaccharide units (Fig. 1).¹ It is currently a gold-standard life-saving drug to overcome blood coagulation by activating antithrombin-III to impede the coagulation process.^2,3 Systemic heparinization is the most common anticoagulation procedure in surgical practice (e.g. open-heart surgery) and extracorporeal therapies such as kidney dialysis. At the end of surgery, excess heparin often needs to be deactivated by using a heparin neutralizer; otherwise patients have risks of low blood pressure and a slow heart rate, and may develop internal bleeding.⁴ Therefore, the neutralization of heparin has been a topic of significant research interest in the biomedical field.Open in a separate windowFig. 1Scheme of heparin reversal by GC4AOEG in the circulatory system.Protamine sulfate, the only FDA-approved neutralizer of UFH, possesses a highly positive charge density due to its polymeric nature and rich arginine residues. Thus, electrostatic interactions are the major driving force in the formation of a UFH–protamine complex, leading to the neutralization and deactivation of UFH.^1,5 However, due to its non-specific interactions, protamine sulfate often causes various adverse effects such as bradycardia, hypotension and pulmonary hypertension, as well as allergic reactions including life-threatening anaphylactic reactions in some patients.⁵ When overdosed, protamine may further impair the intricate balance in the blood and cause coagulopathy.^5–7 Given these issues, there has been a medical need for alternative, safe UFH neutralizers that can specifically counteract UFH without causing serious adverse effects.⁸Discovering and developing new heparin neutralizers has been a popular area of research.^8,9 During the past two decades, a variety of different UFH neutralizers including small molecules,¹⁰ cationic polymers (e.g. polybrene),^11–14 peptides,^11,15 and nanoparticles^16,17 have been designed and evaluated in vitro and/or in vivo. For instance, surfen, as a small-molecule antagonist of UFH, may electrostatically bind with UFH; however only modest neutralizing effects against UFH were observed in rats,^10,18 likely attributed to the lack of strong, specific recognition. On the other hand, polycationic species, including polybrene¹⁹ and poly-dl-lysine,²⁰ exhibited stronger binding with UFH and significant potential as UFH neutralization agents. However, toxicity was still a key concern of these species due to their intrinsic electrostatic interactions with red blood cells (RBC).²¹ Meanwhile, some UFH antagonists have achieved preliminary success in preclinical studies and even moved to clinical evaluations. For instance, ciraparantag (PER977), as a synthetic antidote against several anticoagulants, is currently being evaluated in phase II clinical trials.²² UHRA (Universal Heparin Reversal Agent), a synthetic multivalent dendrimer polymer in the form of nanoparticles with positively charged surfaces, can reverse the activity of all clinically available heparins and it is currently undergoing preclinical studies and will likely move to clinical investigations.²³ However, the oligo- and poly-cationic nature of these species suggests their general tendency towards any negatively charged species, making them “universal” or function against several anticoagulants, implying their low specificity towards heparin.More recently, the sequestration and reversal of toxic agents by supramolecular host molecules have attracted increasing attention, and a typical example of clinical and commercial success is sugammadex, a carboxylated derivative of gamma-cyclodextrin that may specifically reverse the activity of non-depolarizing neuromuscular blocking agents.²⁴ Inspired by this clinical success, several macrocycles were designed and synthesized to selectively bind UFH. For instance, Liu et al. synthesized amphiphilic multi-charged cyclodextrins (AMCD), and AMCD-assembly was utilized for selective heparin binding.¹⁶ Nitz et al. derivatized a cyclodextrin with amide and guanidino groups as a polycationic receptor to recognize and detect UFH.²⁵ Kostiainen and co-workers studied cationic, quaternary ammonium functionalized pillar[5]arene because of its potential complexation with UFH.²⁶ Additionally, cationic calixarene derivatives were designed for UFH binding and guanidinocalixarenes exhibited stronger binding affinity with UFH than their quaternary amine-functionalized counterparts.^27,28 In spite of decent binding affinities and selective recognition of UFH, these macrocycles still possess various limitations such as non-specific toxicity induced mostly by cationic charges, which may disrupt cell membranes and induce blood coagulation.^29,30An ideal UFH neutralizer should full-fill the following three requirements: (1) binding strongly towards UFH in a specific manner; (2) excellent biocompatibility and safety profile, and (3) a clearly defined molecular structure to facilitate batch-to-batch consistency. Thus far, none of the clinical UFH antagonists or previously reported candidates has fulfilled these conditions. Herein we designed an artificial receptor, an oligoethylene glycol functionalized guanidinocalixarene, GC4AOEG, by leveraging the asymmetrical structure of calixarene to strategically add guanidinium groups on one side and oligoethylene glycol (OEG) groups on the other side (Fig. 1). We anticipated that the guanidinium-enriched upper rim would bind strongly with UFH via salt bridges (charge-assisted hydrogen bonds).^28,31 In addition, the biocompatible OEG-functionalized lower rim may help improve the water-solubility and biocompatibility of the host molecule.^32,33GC4AOEG was synthesized in 5 steps starting from the maternal calix[4]arene (Fig. 2). Briefly, p-tert-butylcalix[4]arene 1 was alkylated with tosylate 2³⁴ to obtain compound 3 with a well-defined cone conformation, and replacement of the tert-butyl with nitro groups via an ipso-nitration reaction afforded compound 4.³⁵ Subsequently, compound 4 was hydrogenated in the presence of SnCl₂·2H₂O, affording the tetramine derivative 5. Subsequently, compound 6 was obtained via a reaction between compound 5 and di-Boc-protected thiourea units. The removal of the protecting groups was achieved using SnCl₄ in ethyl acetate, to yield the target GC4AOEG (the characterization of intermediates (Fig. S1 and S2) and GC4AOEG (Fig. S3) are in the ESI^†).Open in a separate windowFig. 2Synthetic route of GC4AOEG and fluorescence titrations. (A(a)) NaH, dry DMF, and 75 °C; (b) HNO₃, AcOH, dry CH₂Cl₂, and r.t.; (c) SnCl₂·2H₂O, C₂H₅OH/AcOEt (1 : 1, v/v), and reflux; (d) 1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea, Et₃N, AgNO₃, dry CH₂Cl₂, and r.t.; (e) SnCl₄, AcOEt, and r.t. (B) Direct fluorescence titration of 0.5 μM EY with different concentrations of GC4AOEG (up to 13.8 μM) in HEPES buffer (10 mM, pH = 7.4), and λ_ex = 517 nm. (Inset) The associated titration curve at λ_em = 537 nm and best fit according to a 1 : 1 binding stoichiometry. (C) Competitive fluorescence titration of GC4AOEG·EY (4.0/0.5 μM) with UFH (up to 8.4 μM in the concentration of monomer units of UFH), and λ_ex = 517 nm. (Inset) The associated titration curve at λ_em = 537 nm and best fit according to a n : 1 competitive binding model, where n = 0.88.The binding affinity between GC4AOEG and UFH was firstly investigated via a competitive titration approach. In this paper, we defined the repeated disaccharide unit as the UFH monomer unit, and the UFH concentration in this paper is the UFH monomer unit concentration. Eosin Y (EY) was selected as the reporter dye, owing to its strong complexation with GC4AOEG and the drastic fluorescence quenching after complexation. The equilibrium association constant (K_a), between GC4AOEG and EY, was determined by direct fluorescence titration and fitted as (2.37 ± 0.12) × 10⁵ M⁻¹ with 1 : 1 binding stoichiometry (Fig. 2B). The displacement of EY from GC4AOEG·EY by gradual addition of UFH resulted in the recovery of the intrinsic emission of EY. The best-fitting of the competitive titration model afforded ca. 1 : 1 binding stoichiometry between GC4AOEG and each monomer unit of UFH, as well as an ultrahigh binding affinity K_a of (1.25 ± 0.13) × 10⁷ M⁻¹ (Fig. 2C).For in vitro analysis of the effectiveness of GC4AOEG against UFH, the activated partial thromboplastin time (aPTT) assay was conducted. The result (Fig. S8^†) indicates that one equivalent of GC4AOEG (to UFH monomer) fully neutralized UFH, similar to protamine. Very importantly, it is obvious that protamine alone negatively influenced the aPTT time. In contrast, GC4AOEG alone did not affect the clotting time, suggesting that GC4AOEG can specifically bind with UFH directly with minimal side influences. The coagulation factor X levels in the plasma analyzed via the enzyme-linked immunosorbent assay (ELISA) further confirmed the safety and reversal effect of GC4AOEG towards UFH (Fig. S9^†).Next, the biocompatibility of GC4AOEG was investigated in vitro. As an alkyl derivative of guanidinocalixarene, GC4A-6C (Fig. S4 and S5^†), which has a similar number of carbons (hexyl groups) at the lower rim to that of GC4AOEG, was also synthesized and examined in this study for comparative purposes. As shown in Fig. 3A and B, GC4AOEG (up to 200 μM) showed remarkably low cytotoxicity in several cell lines via MTT assays, in dramatic contrast to the relatively high cytotoxicity of GC4A-6C (Fig. 3C and D). The cellular toxicity of GC4A-6C was consistent with previous literature.³⁶ In addition, alkyl derivatives of calixarene were generally more toxic than those without alkyl chains,³⁷ likely attributed to their amphiphilic properties that may facilitate cell membrane disruption.^38–40 The results suggested that the much-improved safety profile of GC4AOEG was attributed to oligoethylene glycol functionalization. Meanwhile, it is well known that cationic polymers or oligomers often show poor biocompatibility in the circulation system due to their non-selective binding to negatively charged RBC, resulting in RBC aggregation or hemolysis.⁴¹ Therefore, hemolysis and hemagglutination assays were conducted according to a method previously reported,^42,43 with experimental details described in the method. The percent hemolysis of GC4A-6C (25, 50, 100 and 200 μM, respectively) was over 90%, which would limit its application in the circulatory system (Fig. S6^†), as a hemolysis ratio below 5% is considered safe.⁴⁴ Conversely, GC4AOEG exhibited nearly negligible (less than 3%) hemolytic activity at concentrations of up to 200 μM, and no agglutination was visualized during incubation with RBC (Fig. 3F), implying that OEG functionalization at the lower rim reduced non-specific interactions with the RBC membrane, resulting in less disturbance of the membrane structure and function or cellular aggregations.Open in a separate windowFig. 3Biocompatibility study in cell lines and RBC. Cell viabilities of (A, C) 4T1 and (B, D) 293T, cells treated with different concentrations of GC4AOEG or GC4A-6C for 24 h. Each data point represents the mean ± S.E.M. from a set of experiments (n = 4). (E, G) Hemolysis test of GC4AOEG at different concentrations (NC = negative control; PC = positive control). Each data point represents the mean ± S.E.M. from a set of experiments (n = 3). (F) Agglutination test of RBC incubated with GC4AOEG at 2.0% hematocrit in normal saline.Inspired by the above findings, we further examined whether GC4AOEG may reverse bleeding in different mouse bleeding models under heparinization (with the experimental details described in the method, and the standard curve for the quantification of blood loss volume is showed in Fig. S7^†),⁴⁵ with both the total time of bleeding and total volume of lost blood evaluated for each model. As a proof of concept, 200 U kg⁻¹ UFH and 2.245 mg kg⁻¹ GC4AOEG (molar ratio of GC4AOEG and each monomer unit of UFH = 1 : 1) were respectively used, as representative doses in the study and the dose of UFH was based on a literature report.⁴⁶ In a mouse tail transection model as an external bleeding model, as shown in Fig. 4A–C, after tail transection, the bleeding time and blood loss volume for mice treated with normal saline were 58.9 ± 10.7 min and 72.2 ± 15.8 μL, respectively. As expected, treatment with UFH increased the bleeding time and blood volume to 121.5 ± 20.2 min and 264.0 ± 43.6 μL, respectively. In contrast, the bleeding time was dramatically reduced down to the blank control level, when the mice were treated with GC4AOEG at the same time of, or 30 s after, i.v. administration of UFH (53.8 ± 11.4 min and 89.0 ± 13.3 min, respectively). Accordingly, the blood loss volume of mice successively treated with UFH and GC4AOEG (1 : 1 ratio) reached the control level (72.6 ± 14.3 μL), indicating that the strong binding affinity between GC4AOEG and UFH ensured their recognition in vivo. Of note, there was no significant difference between the GC4AOEG treated group (without heparinization) and the saline treated group, suggesting a decent safety profile of the artificial receptor.Open in a separate windowFig. 4Reversal efficacy in in vivo mouse models. (A–C) Mouse tail transection model. (A) Scheme of the mouse tail transection model. (B) Total time of bleeding and (C) blood loss volume. (D–F & J) Mouse liver injury model. (D) Scheme of the mouse liver injury model. (E) Total time of bleeding and (F) blood loss weight. (J) Pictures exhibiting bleeding in liver injury before and after treatment. (G–I & K) Mouse femoral artery model. (G) Scheme of the mouse femoral artery model. (H) Total time of bleeding and (I) blood loss weight. (K) Pictures exhibiting bleeding in the femoral artery before and after treatment. All of those models were i.v. administration with normal saline (control), GC4AOEG (2.245 mg kg⁻¹), or UFH (200 U kg⁻¹) without and with GC4AOEG (2.245 mg kg⁻¹, 1 : 1 molar stoichiometry of GC4AOEG and the monomer unit of UFH), and UFH–GC4AOEG 1 : 1 successively (GC4AOEG at a dose of 2.245 mg kg⁻¹ 30 s after UFH administration) respectively were quantified. Data presented are the mean ± S.E.M. (n = 6). *p < 0.05, ****p < 0.001, and ns represents “no significant difference” between the experimental group and the control group.In addition to external bleeding, internal bleeding such as liver injury model (Fig. 4D) was established in mice, and GC4AOEG''s reversal of UFH was further evaluated in vivo. Mice were i.v. administered with normal saline (control), GC4AOEG (2.245 mg kg⁻¹), or UFH (200 U kg⁻¹) without and with GC4AOEG (2.245 mg kg⁻¹), and successive UFH–GC4AOEG 1 : 1 (30 s in between), respectively. In 2 minutes, the abdomen was surgically opened to expose the liver. A wound of 0.5 cm length and 2 mm depth, in the left lobe of the liver, was created. Considerable bleeding was immediately observed in the UFH treatment group (Fig. 4J), with the total bleeding time lasting for 450.5 ± 46.8 s, and the total blood loss of 571.0 ± 35.0 mg, in contrast to 143.7 ± 14.7 s total bleeding time and 238.0 ± 45.0 mg total blood loss observed in the saline treated group. Interestingly, the UFH–GC4AOEG treated group showed no significant difference from the normal saline treated group. To simulate the clinical use scenario, GC4AOEG was injected after UFH''s administration, and significantly reduced bleeding (from both time and volume perspectives) was observed, suggesting effective inhibition of the adverse effects of UFH, by GC4AOEG (Fig. 4E and F). GC4AOEG alone (without heparinization) did not exhibit any hematological toxicity in this model. To further evaluate the inhibitory effects of GC4AOEG against UFH in a preclinical model, a more serious internal bleeding model, femoral artery bleeding mouse model, was employed, and the treatment plan followed the previous two models described as above. Upon administration, the skin of the right leg and the overlying muscles were removed to expose the femoral artery and sciatic nerve. After an open injury at the middle segment of the femoral artery was created with a surgical scissor, blood gushed out immediately from the injured site (Fig. 4G and K). As shown in Fig. 4H and I, the longest average bleeding time (16.0 ± 1.9 min) and blood loss weight (103.8 ± 16.9 mg) were observed in the UFH treatment group of mice, in dramatic contrast to the bleeding time and blood loss of 3.9 ± 0.4 min and 24.7 ± 4.5 mg, respectively, in the normal saline treated group of mice. A bleeding time of 3.6 ± 0.4 min and blood loss of 20.8 ± 7.4 mg were recorded in the UFH–GC4AOEG treatment group. When UFH and GC4AOEG (at 1 molar equivalent) were successively injected, a bleeding time of 5.3 ± 0.7 min and blood loss of 27.7 ± 5.8 mg were noted, suggesting the significant reversal effects of GC4AOEG on UFH. Collectively, in all of the three bleeding models including internal and external bleeding models, i.v. administration of GC4AOEG significantly reversed UFH-induced excessive bleeding in external and internal injuries. More importantly, GC4AOEG alone exhibited negligible hematological activity, unlike other previously reported cationic small molecules, polymers, oligomers and macrocycles.Furthermore, in order to further verify the safety profile of GC4AOEG at the effective dose in vivo, acute toxicity evaluation was performed in a mouse model. After the i.v. injection of GC4AOEG in mice at a dose of 2.245 mg kg⁻¹ (i.v. injection of normal saline as the control group), the body weight, behaviors, and overall survival of the treated mice were monitored every day for 3 weeks. All the treated mice remained alive and showed normal behaviors, as well as normal body weight evolvement similar to that of the control group (Fig. 5A). On day 21 post administration, mice were euthanized for blood and organ samples were harvested (for details see the method). The organ indexes of representative major organs including the heart, liver, spleen, lungs, and kidneys isolated from the GC4AOEG treated mice were comparable to those of the mice administered with normal saline, with no significant differences observed (Fig. 5B). Hematological parameters such as the counts of whole blood cells (WBCs), red blood cells (RBCs), platelets (PLTs) and hemoglobin (HGB) (Fig. 5C), as well as the serum concentrations of liver and kidney function biomarkers including blood urea nitrogen (BUN), creatinine (crea), urea alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were all analyzed thoroughly (Fig. 5D and E). These results indicated that the hematological parameters, renal and hepatic functions of the mice treated with GC4AOEG were comparable with those of the mice in the normal saline treated group. Moreover, histopathological examinations of the major organs of the GC4AOEG treated mice showed normal microstructures comparable with those of the control group (Fig. 5F). Collectively, these results suggested that the i.v. administration of GC4AOEG at the therapeutic dose is safe.Open in a separate windowFig. 5Preliminary acute toxicity evaluations on GC4AOEG. (A) Weight changes of mice after i.v. administration with a single dose of GC4AOEG. (B) Major organ indexes of the mice on day 21 post-administration with GC4AOEG. (C) Hematological parameters of the blood samples collected from the mice on day 21 after i.v. administration of GC4AOEG. (D) Renal and (E) hepatic functional biomarkers in the blood samples collected from the mice on day 21 after i.v. administration of GC4AOEG. Data are presented as mean ± S.E.M.; n = 6 for each group. (F) H&E histopathological analysis of the major organs from mice sacrificed 21 days after being injected with saline and GC4AOEG (2.245 mg kg⁻¹). Scale bar = 100 μm.     

Bidirectional triplet exciton transfer between silicon nanocrystals and perylene

Hybrid materials comprised of inorganic quantum dots functionalized with small-molecule organic chromophores have emerged as promising materials for reshaping light''s energy content. Quantum dots in these structures can serve as light harvesting antennas that absorb photons and pass their energy to molecules bound to their surface in the form of spin-triplet excitons. Energy passed in this manner can fuel upconversion schemes that use triplet fusion to convert infrared light into visible emission. Likewise, triplet excitons passed in the opposite direction, from molecules to quantum dots, can enable solar cells that use singlet fission to circumvent the Shockley–Queisser limit. Silicon QDs represent a key target for these hybrid materials due to silicon''s biocompatibility and preeminence within the solar energy market. However, while triplet transfer from silicon QDs to molecules has been observed, no reports to date have shown evidence of energy moving in the reverse direction. Here, we address this gap by creating silicon QDs functionalized with perylene chromophores that exhibit bidirectional triplet exciton transfer. Using transient absorption, we find triplet transfer from silicon to perylene takes place over 4.2 μs while energy transfer in the reverse direction occurs two orders of magnitude faster, on a 22 ns timescale. To demonstrate this system''s utility, we use it to create a photon upconversion system that generates blue emission at 475 nm using photons with wavelengths as long as 730 nm. Our work shows formation of covalent linkages between silicon and organic molecules can provide sufficient electronic coupling to allow efficient bidirectional triplet exchange, enabling new technologies for photon conversion.

We demonstrate that silicon quantum dots can exchange spin triplet excitons with molecules covalently attached to their surface. Such hybrid materials can enable systems that upconvert incoherent far-red light into the visible spectral range.

Hybrid materials comprised of inorganic quantum dots (QDs) interfaced with small-molecule organic chromophores have emerged as a promising platform for materials that convert near-infrared radiation into the visible spectral range.^1–3 In these structures, QDs act as light-harvesting antennas, absorbing long-wavelength photons and passing their energy to organic molecules bound to their surface in the form of spin-triplet excitons. These excitons can then be transferred into a surrounding medium, typically a solution or thin film, where pairs of them can fuse to form a bright spin-singlet state that can emit a short-wavelength photon.^4–8 Due to the long lifetime of molecular triplet excitons, which can range from several microseconds to milliseconds, these materials can operate at low photon flux, enabling their integration into light-harvesting systems that operate under solar flux^9,10 and limiting heat dissipation during their use in biological applications, such as phototherapy,^11,12 live-cell imaging,^13,14 and optogenetics.¹⁵ These hybrid materials can also be used to study interfacial energy transfer processes fundamental to the operation of solar cells that use triplet fusion''s inverse process, singlet fission, to enhance their performance.^9,16–21 The simplest design for a cell of this type is one that interfaces a singlet fission material directly in line with a back-contacted semiconductor solar cell.^22–24 In these structures, the singlet fission material acts as a light sensitizer that captures high-energy photons and uses their energy to generate pairs of triplet excitons that can be passed to the semiconductor to produce photocurrent. As molecules can be readily attached to QDs via a variety of chemical tethers, these materials allow detailed study of how the structure of the organic:inorganic interface impacts the ability of triplet excitons to move from one material to the other.For both triplet fusion-based light upconversion and singlet fission-based light harvesting, silicon represents a key material of interest. While several upconversion systems have been derived using QDs containing toxic elements, such as Cd^5,7,25 or Pb,^6,8,26,27 Si QDs are nontoxic, making them attractive for biological applications.²⁸ Silicon also dominates the solar energy market, accounting for ∼90% of solar power production,^29,30 making Si:organic interfaces that readily transmit triplet excitons a key design target for singlet fission-based solar cells.^18,19,22 Previously, we have shown triplet exciton transfer from Si QDs to surface-bound anthracene molecules can power a photon upconversion system that operates with 7% efficiency.³¹ However, the inverse energy transfer process that is key for singlet fission devices, triplet exciton transfer from surface-bound molecules to Si, was not observed in our prior work.In this report, we address triplet exciton transfer from molecules to Si by demonstrating a hybrid Si QD:perylene system wherein photoexcitation of the Si QD establishes a spin-triplet exciton population that exists in a dynamic equilibrium between the QD and perylene molecules bound to its surface. While such exciton cycling has been reported for other QD:molecule systems,^32–34 our work represents the first observation of this behavior in Si QD based systems. Using nanosecond transient absorption spectroscopy, we find triplet exciton transfer from Si to perylene takes place on a 4.2 μs timescale while energy transfer in the reverse direction occurs more than two orders of magnitude faster, on a 22 ns timescale. We attribute this difference in energy transfer rates to differences in the exciton density of states between perylene molecules and Si QDs. To demonstrate the utility of triplet excitons produced by this system for photon conversion applications, we have constructed a photon upconversion system by interfacing perylene-functionalized Si QDs with a complementary perylene-based triplet fusion annihilator. We find this system performs well, upconverting radiation with a wavelength as long as 730 nm into blue light centered near 475 nm. Under 532 nm illumination, the system upconverts light with an efficiency of 1.5% under incident light fluxes as low as 80 mW cm⁻². This performance is comparable to that recently demonstrated using the same perylene annihilator coupled with a Pd-porphyrin light absorber.³⁵ Our work demonstrates that the introduction of short, chemical linkers between molecules and Si can enable triplet exciton exchange between these materials for the design of new systems for both photon upconversion and light harvesting.