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.     

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.     

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.     

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.     

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.     

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     

A cofacial metal–organic framework based photocathode for carbon dioxide reduction

Innovative and robust photosensitisation materials play a cardinal role in advancing the combined effort towards efficient solar energy harvesting. Here, we demonstrate the photocathode functionality of a Metal–Organic Framework (MOF) featuring cofacial pairs of photo- and electro-active 1,4,5,8-naphthalenediimide (NDI) ligands, which was successfully applied to markedly reduce the overpotential required for CO₂ reduction to CO by a well-known rhenium molecular electrocatalyst. Reduction of [Cd(DPNDI)(TDC)]_n (DPNDI = N,N′-di(4-pyridyl)-1,4,5,8-naphthalenediimide, H₂TDC = thiophene-2,5-dicarboxylic acid) to its mixed-valence state induces through-space Intervalence Charge Transfer (IVCT) within cofacial DPNDI units. Irradiation of the mixed-valence MOF in the visible region generates a DPNDI photoexcited radical monoanion state, which is stabilised as a persistent species by the inherent IVCT interactions and has been rationalised using Density Functional Theory (DFT). This photoexcited radical monoanion state was able to undergo charge transfer (CT) reduction of the rhenium molecular electrocatalyst to effect CO generation at a lower overpotential than that required by the discrete electrocatalyst itself. The exploitation of cofacial MOFs opens new directions for the design philosophy behind light harvesting materials.

The photocathode functionality of a Metal–Organic Framework (MOF) featuring cofacial photo- and electro-active ligands provides a new approach to CO₂ reduction via charge transfer with a rhenium electrocatalyst.

The development of photocathode materials for CO₂ reduction and hydrogen evolution catalyses has traditionally focussed on photosensitising transition metal complexes or nanostructured solid state semiconductors.^1,2 At the nascent frontier between robust solid state semiconductors and synthetically protean metal complexes are photo-/electro-active Metal–Organic Frameworks (MOFs) that consolidate the flexibility of homogeneous systems into the robust heterogeneous phase.³ Contrasting with reported MOF examples, natural photosynthesis remains one of the most efficient light harvesting systems.⁴ One common reaction centre adopted in photosynthesis features a redox-active cofacial dimer of chlorophyll pigment molecules.⁵ This cofacial moiety stabilises the photoexcited charge separated state through intra-dimer Intervalence Charge Transfer (IVCT) interactions, enabling the trapping and conversion of light to chemical energy. Recently, we characterised IVCT interactions upon reduction to the mixed-valence state in the MOF [Zn₂(TDC)₂(DPPTzTz)₂]_n (DPPTzTz = 2,5-bis(4-(4-pyridyl)phenyl)thiazolo[5,4-d]thiazole and H₂TDC = thiophene-2,5-dicarboxylic acid) featuring cofacial dimers of the thiazolothiazole redox-active core, and probed its structure–activity dependence computationally and experimentally.^6–9 Subsequently, we sought design a new MOF featuring cofacial pairs of the photo- and redox-active N,N′-di(4-pyridyl)-1,4,5,8-naphthalenediimide (DPNDI) ligand, as a conceptually neoteric photosensitiser for incorporation into systems relevant towards artificial photosynthesis.The naphthalene diimide (NDI) core was selected for its photoactive radical monoanion state.¹⁰ For a number of discrete systems, Wasielewski and coworkers have computationally and experimentally demonstrated the ability to photoexcite the easily accessible NDI radical monoanion using visible light, facilitating its transient photoelectrochemical reduction of Re based catalytic CO₂ reduction sites.^2,11–14 Recently, Goswami et al. synthesised a Zr NDI-based MOF, applying this as a radical state heterogeneous photosensitiser to decompose dichloromethane.¹⁵Here, we describe the synthesis of a new photo- and redox-active MOF [Cd(DPNDI)(TDC)]_n, denoted csiMOF-6 (cofacial stacked IVCT), featuring cofacial dimers of the DPNDI ligand. Cofacial DPNDI MOFs have been reported previously by Takashima et al.¹⁶ and Sikdar et al.,¹⁷ where guest dependent charge transfer (CT) and neutral state photoexcitation behaviours were examined. Dinolfo et al. also incorporated DPNDI into a rhenium based cofacial complex, where its mixed-valence IVCT behaviour was probed using electrochemical and spectroelectrochemical (SEC) techniques.¹⁸ We envisaged that the cofacial NDI units in csiMOF-6 would stabilise its photoexcited radical monoanion state by IVCT interactions, akin to cofacial moieties in natural photosynthsesis processes. This strengthens the persistence of the NDI photoexcited radical monoanion state, thereby improving its efficacy at photoelectrochemical reduction of catalytically active sites. Effectiveness of the cofacial design principle behind csiMOF-6 photocathodes was verified using a combined experimental and computational approach. The successful photocathode performance of csiMOF-6 under broad band visible light irradiation encompassed its photoelectrochemical reduction of the [Re(bipy-tBu)(CO)₃Cl] (bipy-tBu = 4,4′-di-tert-butyl-2,2′-bipyridine, developed by Smieja et al.¹⁹) CO₂ reduction electrocatalyst, resulting in CO generation at reduced overpotential requirements.     

Achieving high circularly polarized luminescence with push–pull helicenic systems: from rationalized design to top-emission CP-OLED applications

While the development of chiral molecules displaying circularly polarized luminescence (CPL) has received considerable attention, the corresponding CPL intensity, g_lum, hardly exceeds 10⁻² at the molecular level owing to the difficulty in optimizing the key parameters governing such a luminescence process. To address this challenge, we report here the synthesis and chiroptical properties of a new family of π-helical push–pull systems based on carbo[6]helicene, where the latter acts as either a chiral electron acceptor or a donor unit. This comprehensive experimental and theoretical investigation shows that the magnitude and relative orientation of the electric (μ_e) and magnetic (μ_m) dipole transition moments can be tuned efficiently with regard to the molecular chiroptical properties, which results in high g_lum values, i.e. up to 3–4 × 10⁻². Our investigations revealed that the optimized mutual orientation of the electric and magnetic dipoles in the excited state is a crucial parameter to achieve intense helicene-mediated exciton coupling, which is a major contributor to the obtained strong CPL. Finally, top-emission CP-OLEDs were fabricated through vapor deposition, which afforded a promising g_El of around 8 × 10⁻³. These results bring about further molecular design guidelines to reach high CPL intensity and offer new insights into the development of innovative CP-OLED architectures.

A CPL intensity of up to 3 × 10⁻² is achieved in π-extended 6-helicene derivatives, owing to an intense helicene-mediated exciton coupling. Corresponding top-emission CP-OLEDs afforded a promising g_El of around 8 × 10⁻³.

The design of chiral emitters displaying intense circularly polarized luminescence (CPL) has attracted significant interest, thanks to the potential of CP light in a diverse range of applications going from chiroptoelectronics (organic light-emitting diodes (OLEDs), optical information processing, etc.) to bio-imaging and chiral sensing.¹ Recently, designing OLEDs with CP electroluminescence (CP-OLEDs) has emerged as an interesting approach to improve high-resolution display performance. Namely, using unpolarised OLEDs, up to 50% of the emitted light can be lost due to the use of antiglare polarized filters.² In CP-OLEDs, the electro-generated light can pass these filters with less attenuation owing to its circular polarization and thus lead to an increase of the image brightness with lower power consumption.³ To develop CP-OLED devices, the main approach relies on the doping of the device''s emitting layer by a CPL emitter, which should ensure simultaneously high exciton conversion and a high degree of circular polarization. The harvesting of both singlet and triplet excitons has been successfully addressed using either chiral phosphorescent materials or thermally activated delayed fluorescence (CP-TADF) emitters with device efficiencies of up to 32%.⁴ However, the intensity of circularly polarized electroluminescence (CPEL), evaluated by the corresponding dissymmetry factor g_El, remains inefficient and typically falls within the range of 10⁻³ with limited examples reaching g_El > 10⁻² based on polymeric materials and lanthanide complexes.⁵ For CP-OLEDs using a molecular chiral emissive dopant, g_El, defined as the ratio between the intensity difference of left- and right-CPEL, and the total generated electroluminescence, 2(El_L − El_R)/(El_L + El_R), can be generally related to the luminescence dissymmetry factor g_lum measured in diluted solution.² Accordingly, it is of crucial importance to design luminescent molecules with high g_lum values,^3,28a–d,29 in order to reach strong CP electro-luminescence when going to practical devices. However, structural and electronic factors that govern the CPL of chiral compounds are still poorly understood even if a few studies have recently tried to rationalize and establish molecular guidelines to obtain high g_lum values.⁶Our team has contributed to the research in this area by developing extended π-helical molecular architectures resulting from the association of carbo[6]helicene and achiral dyes,⁷ which afforded enhanced chiroptical properties, with notably a g_lum up to 10⁻², owing to an uncommon chiral exciton coupling process mediated by the chiral helicenic unit.⁸ In addition, we also described an unusual solvent effect on the intensity of CPL of π-helical push–pull helicene–naphthalimide derivatives,^7b which showed a decrease of g_lum from 10⁻² to 10⁻³ upon increasing the polarity of solvent.^7b This solvatochromism effect was shown to be related to a symmetry breaking of the chiral excited state before emission,⁹ which modifies the relative intensity of the magnetic (μ_m) and electric (μ_e) dipole transition moments, and the angle, θ, between them (Fig. 1), ultimately impacting g_lum. The latter is well approximated as 4|m|cos θ/(|μ|) for an electric dipole-allowed transition.¹⁰Open in a separate windowFig. 1Chemical structures of “push–pull” 2,15-diethynylhexahelicene-based emitters with their polarized luminescence characteristics including their calculated electric and magnetic transition dipole moments and the angle between them corresponding to the S₁ → S₀ transition.While these results highlight interesting aspects regarding the key parameters influencing the CPL of organic emitters, this type of “helical push–pull design” remains limited to only one example, which render the systematic rationalization of these findings difficult. Accordingly, we decided to develop a complete family of new chiral push–pull compounds to explore the structural and electronic impact of the grafted substituents on the helical π-conjugated system. In addition, we went a step further and incorporated the designed chiral emitter into proof-of-concept CP-OLEDs using a top-emission architecture,¹¹ which remains scarcely explored for CP-light generation despite its considerable potential for micro-display applications. To the best of our knowledge, only one example of such type of electroluminescent device has been reported, using a CP-TADF emitter, affording a modest g_El of 10⁻³.^11aHerein, we report the synthesis and chiroptical properties of a new family of π-helical push–pull systems based on chiral carbo[6]helicene, functionalized by either electron donor or acceptor units. Interestingly, the chiral π-conjugated system of the helicene may act as either an electron acceptor or a donor, depending on the nature of the attached substituents, thereby impacting the chiroptical properties, notably the resulting CPL. By optimizing the chiral exciton coupling process through the modulation of the magnitude and relative orientation of the electric (μ) and magnetic (m) dipoles, the chiroptical properties of classical carbo[6]helicene-based emitters can be dramatically enhanced and reach high g_lum values at the molecular level, i.e. up to 3–4 × 10⁻². Experimental and theoretical investigations revealed that the mutual orientation of the electric and magnetic dipoles in the excited-state is a crucial parameter and is optimal when the substituents attached to the helicene core possess a rather weak electron withdrawing or donating ability. Finally, proof of concept top-emission CP-OLEDs were fabricated through vapor deposition of π-helical push–pull derivatives and afforded a g_El of around 8 × 10⁻³, which represents a significant improvement for the polarization of electroluminescence emitted using this device architecture.     

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.     

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     

Serine is the molecular source of the NH(CH2)2 bridgehead moiety of the in vitro assembled [FeFe] hydrogenase H-cluster

The active site of [FeFe] hydrogenase, the H-cluster, consists of a canonical [4Fe–4S]_H subcluster linked to a unique binuclear [2Fe]_H subcluster containing three CO, two CN⁻ and a bridging azadithiolate (adt, NH(CH₂S⁻)₂) ligand. While it is known that all five diatomic ligands are derived from tyrosine, there has been little knowledge as to the formation and installation of the adt ligand. Here, by using a combination of a cell-free in vitro maturation approach with pulse electronic paramagnetic resonance spectroscopy, we discover that serine donates the nitrogen atom and the CH₂ group to the assembly of the adt ligand. More specifically, both CH₂ groups in adt are sourced from the C3 methylene of serine.

The CH₂NHCH₂ bridgehead moiety of the [FeFe] hydrogenase H-cluster is derived from serine as revealed by isotope labeling and EPR spectroscopy.

Hydrogenases catalyze the reversible reactions of H₂ oxidation and proton reduction, and are involved in many microbial metabolic pathways.¹ [FeFe] hydrogenases in particular are hyper-efficient, with turnover rates up to 10⁴/s.² This has led to intense focus on [FeFe] hydrogenases for sustainable production of H₂ and the design of fuel cells.³ The active site of [FeFe] hydrogenases is a six-iron cofactor called the H-cluster (Scheme 1), which consists of a canonical cuboid [4Fe–4S]_H subcluster linked through a bridging cysteine (Cys) residue to a binuclear [2Fe]_H subcluster in which the two iron ions are coordinated by three CO, two CN⁻ and an azadithiolate (adt, NH(CH₂S⁻)₂) bridging ligand. The [2Fe]_H subcluster has been proposed to be the site for H₂ binding and hydride formation,^4,5 which serves as a natural blueprint for designing small molecule catalysts for hydrogen evolution reactions.⁶ The unique structure and catalytic activity has thus raised much interest in the biosynthesis of the H-cluster, which poses a great challenge in cofactor assembly that involves toxic ligands, oxygen sensitivity and an organic adt ligand that has little inherent stability.Open in a separate windowScheme 1Bioassembly of the H-cluster highlighting the source of each moiety.While the [4Fe–4S]_H subcluster in the H-cluster can be formed by the housekeeping gene products that are used to assemble such standard Fe–S clusters, the in vivo bioassembly of the unique [2Fe]_H subcluster requires three special Fe–S “maturase” proteins: HydE, HydF, and HydG.^7,8 Although the functions of HydE and HydF have not been fully elucidated,^9–12 recent studies indicate that HydG is a bifunctional 4Fe–4S radical S-adenosyl-l-methionine (SAM) enzyme which lyses tyrosine to generate CO and CN⁻ and forms a [(Cys)Fe(CO)₂(CN)] organometallic precursor to the H-cluster on a dangler Fe(Cys) site in HydG.^13–16 More recently, by using a synthetic [(Cys)Fe(CO)₂(CN)] carrier we have shown that the two sulfur atoms in the adt ligand are derived from the precursor-bound Cys, but that the CH₂NHCH₂ component is not.¹⁷ Taken together, the biosynthetic origins of the [Fe₂S₂(CO)₃(CN)₂] part of the [2Fe]_H subcluster are depicted in Scheme 1: all five diatomic ligands are tailored from tyrosine by HydG;¹⁸ the two sulfur atoms and the two Fe atoms are from the dangler Fe(Cys) site in HydG (which can be reconstituted with Fe²⁺ and free Cys in solution¹⁹). Remarkably, these components are all delivered to the binuclear cluster assembly in the form of the [(Cys)Fe(CO)₂(CN)] product of HydG. Given these recent advances, the only missing part of the puzzle is the crucial NH(CH₂)₂ moiety: what are its molecular precursors? It has been hypothesized that HydE, which is also a 4Fe–4S radical SAM enzyme, may be involved in the formation of adt, though its physiological substrate and reaction mechanism remains under investigation.^9,10 As for any enzymatic reaction, knowing the actual substrate(s) for the reaction is crucial for unraveling the ultimate mechanism. Therefore, determining of molecular sourcing of the CH₂NHCH₂ component of the adt bridge, currently unknown, is the focus of this work.Assembly of the H-cluster in the lab can be achieved by semi-synthetic and biochemical approaches other than directly co-expressing hydA, hydE, hydF and hydG genes in cells. One very useful method alleviates the need for HydG, HydE, and in some cases, HydF, by using a synthetic [Fe₂(adt)(CO)₄(CN)₂] complex as a direct donor to the [2Fe]_H subcluster assembly.^20–22 Another “cell free synthesis” approach uses HydE/F/G in an in vitro H-cluster maturation reaction developed by the Swartz group.^23,24 The specific in vitro maturation reaction used in our current investigation contains a mixture of E. coli cell lysate containing separately overexpressed HydE, HydF, HydG (all from Shewanella oneidensis), apo-HydA1 (from Chlamydomonas reinhardtii) that harbors the [4Fe-4S]_H subcluster, and a cocktail of low molecular weight cofactors and precursors.²³ This biochemical approach gives us the opportunity to use the same set of enzymes that build the H-cluster in cells, but also enables us to determine the molecular source of each of the components in the H-cluster by using isotope-labeled cofactors/precursors, a procedure that would be very difficult to carry out and fully control in vivo. For example, by supplementing 1-¹³C-Tyr or 2-¹³C-Tyr into the in vitro maturation reaction, the CO or CN⁻ ligands to the diiron subcluster of the maturated HydA1 are respectively labeled with ¹³C.^25,26 The presence of these ¹³C labels can then in turn be detected and analyzed by using advanced electron paramagnetic resonance (EPR) spectroscopy to measure the hyperfine couplings between the magnetic ¹³C nuclei and the unpaired electron spin distributed over the H-cluster in its redox-poised paramagnetic states. In this work, we now search for the source(s) of the CH₂NHCH₂ moiety by using a similar strategy of in vitro maturation coupled to high resolution EPR to screen the assembly products formed with various isotopically labeled small molecule candidates. The presence of nitrogen element in the CH₂NHCH₂ fragment suggests an amino acid origin as one possibility. A systematic screening by pulse EPR of the in vitro maturation products generated with ¹³C, ¹⁵N, and ²H-labeled amino acids reveals that serine (Ser) serves as a molecular source for the NH(CH₂)₂ moiety of the H-cluster.     

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

Nitric oxide monooxygenation (NOM) reaction of cobalt-nitrosyl {Co(NO)}8 to CoII-nitrito {CoII(NO2−)}: base induced hydrogen gas (H2) evolution

Here, we report the nitric oxide monooxygenation (NOM) reactions of a Co^III-nitrosyl complex (1, {Co-NO}⁸) in the presence of mono-oxygen reactive species, i.e., a base (OH⁻, tetrabutylammonium hydroxide (TBAOH) or NaOH/15-crown-5), an oxide (O²⁻ or Na₂O/15-crown-5) and water (H₂O). The reaction of 1 with OH⁻ produces a Co^II-nitrito complex {3, (Co^II-NO₂⁻)} and hydrogen gas (H₂), via the formation of a putative N-bound Co-nitrous acid intermediate (2, {Co-NOOH}⁺). The homolytic cleavage of the O–H bond of proposed [Co-NOOH]⁺ releases H₂via a presumed Co^III-H intermediate. In another reaction, 1 generates Co^II-NO₂⁻ when reacted with O²⁻via an expected Co^I-nitro (4) intermediate. However, complex 1 is found to be unreactive towards H₂O. Mechanistic investigations using ¹⁵N-labeled-¹⁵NO and ²H-labeled-NaO²H (NaOD) evidently revealed that the N-atom in Co^II-NO₂⁻ and the H-atom in H₂ gas are derived from the nitrosyl ligand and OH⁻ moiety, respectively.

Base-induced hydrogen (H₂) gas evolution in the nitric oxide monoxygenation reaction.

As a radical species, nitric oxide (NO) has attracted great interest from the scientific community due to its major role in various physiological processes such as neurotransmission, vascular regulation, platelet disaggregation and immune responses to multiple infections.¹ Nitric oxide synthase (NOS),² and nitrite reductase (NiR)³ enzymes are involved in the biosynthesis of NO. NOSs produce NO by the oxidation of the guanidine nitrogen in l-arginine.⁴ However, in mammals and bacteria, NO₂⁻ is reduced to NO by NiRs in the presence of protons, i.e., NO₂⁻ + e⁻ + 2H⁺ → NO + H₂O.⁵ Biological dysfunctions may cause overproduction of NO, and being radical it leads to the generation of reactive nitrogen species (RNS), i.e., peroxynitrite (PN, OONO⁻)⁶ and nitrogen dioxide (˙NO₂),⁷ upon reaction with reactive oxygen species (ROS) such as superoxide (O₂˙⁻),⁸ peroxide (H₂O₂),⁹ and dioxygen (O₂).¹⁰ Hence, it is essential to maintain an optimal level of NO. In this regard, nitric oxide dioxygenases (NODs)¹¹ are available in bio-systems to convert excess NO to biologically benign nitrate (NO₃⁻).¹²NO₂⁻ + Fe^II + H⁺ ↔ NO + Fe^III + OH⁻1[M–NO]ⁿ + 2OH⁻ → [M–NO₂]⁽ⁿ⁻²⁾ + H₂O2NOD enzymes generate NO₃⁻ from NO;^11b,12−13 however, the formation of NO₂⁻ from NO is still under investigation. Clarkson and Bosolo reported NO₂⁻ formation in the reaction of Co^III-NO and O₂.¹⁴ Nam and co-workers showed the generation of Co^II-NO₂⁻ from Co^III-NO upon reaction with O₂˙⁻.¹⁵ Recently, Mondal and co-workers reported NO₂⁻ formation in the reaction of Co^II-NO with O₂.¹⁶ Apart from cobalt, the formation of Cu^II-NO₂⁻ was also observed in the reaction of Cu^I-NO and O₂.¹⁷ For metal-dioxygen adducts, i.e., Cr^III-O₂˙⁻ and Mn^IV-O₂²⁻, NOD reactions led to the generation of Cr^III-NO₂⁻ (ref. 18) and Mn^V O + NO₂⁻,¹⁹ respectively. However, the NOD reaction of Fe^III-O₂˙⁻ and Fe^III-O₂²⁻ with NO and NO⁺, respectively, generated Fe^III-NO₃⁻via Fe^IV O and ˙NO₂.²⁰ Ford suggested that the reaction of ferric-heme nitrosyl with hydroxide leads to the formation of NO₂⁻ and H⁺.¹² Lehnert and co-workers reported heme-based Fe-nitrosyl complexes²¹ showing different chemistries due to the Fe^II-NO⁺ type electronic structures. On the other hand, Bryan proposed that the one-electron reduction of NO₂⁻ to NO in ferrous heme protein is reversible (eqn (1)).²² Also, it is proposed that excess NO in biological systems is converted to NO₂⁻ and produces one equivalent of H⁺ upon reaction with ˙OH.²³ Previously reported reactivity of M–NOs of Fe²⁴ with OH⁻ suggested the formation of NO₂⁻ and one equivalent of H⁺, where H⁺ further reacts with one equivalent of OH⁻ and produces H₂O (eqn (2)).²⁵Here in this report, we explore the mechanistic aspects of nitric oxide monooxygenation (NOM) reactions of the Co^III-nitrosyl complex, [(12TMC)Co^III(NO⁻)]²⁺/{Co(NO)}⁸ (1),^15,26 bearing the 12TMC ligand (12TMC = 1,4,7,10-tetramethyl-1,4,7,10-tetraazacyclododecane) with mono-oxygen reactive species (O²⁻, OH⁻ and H₂O) (Scheme 1). Complex 1 reacts with the base (OH⁻, tetrabutylammonium hydroxide (TBAOH)/or NaOH in the presence of 15-crown-5 as the OH⁻ source) and generates the corresponding Co^II-nitrito complex, [(12TMC)Co^II(NO₂⁻)]⁺ (3), with the evolution of hydrogen gas (H₂) via the formation of a plausible N-bound Co-nitrous acid intermediate ([Co-NOOH]⁺, 2) in CH₃CN at 273 K (Scheme 1, reaction (I)). Also, when 1 reacts with the oxide (O²⁻ or Na₂O in the presence of 15-crown-5), it generates the Co^II-nitrito complex (3) via a probable Co^I-nitro, [(12TMC)Co^I(NO₂⁻)] (4), intermediate (Scheme 1, reaction (II)); however, 1 does not react with water (Scheme 1, reaction (III)). Mechanistic investigations using ¹⁵N-labeled-¹⁵NO, D-labeled-NaOD and ¹⁸O-labelled-¹⁸OH⁻ demonstrated, unambiguously, that the N and O-atoms in the NO₂⁻ ligand of 3 resulted from NO and OH⁻ moieties; however, the H-atoms of H₂ are derived from OH⁻. To the extent of our knowledge, the present work reports the very first systematic study of Co^III-nitrosyl complex reactions with H₂O, OH⁻ and O²⁻. This new finding presents an alternative route for NO₂⁻ generation in biosystems, and also illustrates a new pathway of H₂ evolution, in addition to the reported literature.^12,27Open in a separate windowScheme 1Nitric oxide monooxygenation (NOM) reactions of cobalt-nitrosyl complex (1) in the presence of a base (OH⁻), sodium oxide (Na₂O) and water (H₂O).To further explore the chemistry of [(12TMC)Co^III(NO⁻)]²⁺ (1),^15,26 and the mechanistic insights of NOM reactions, we have reacted it with a base (OH⁻), an oxide (O²⁻), and water (H₂O). When complex 1 was reacted with TBAOH in CH₃CN, the color of complex 1 changed to light pink from dark pink. In this reaction, the characteristic absorption band of 1 (370 nm) disappears within 2 minutes (Fig. 1a; ESI, Experimental section (ES) and Fig. S1a^†), producing a Co^II-nitrito complex, [(12TMC)Co^II(NO₂⁻)]⁺ (3), with H₂ (Scheme 1, reaction (Ib)), in contrast to the previous reports on base induced NOM reactions (eqn (2)).^12,25,28 The spectral titration data confirmed that the ratio-metric equivalent of OH⁻ to 1 was 1 : 1 (ESI, Fig. S1b^†). 3 was determined to be [(12TMC)Co^II(NO₂⁻)](BF₄) based on various spectroscopic and structural characterization experiments (vide infra).^15,26bOpen in a separate windowFig. 1(a) UV-vis spectral changes of 1 (0.50 mM, black line) upon addition of OH⁻ (1 equiv.) in CH₃CN under Ar at 273 K. Black line (1) changed to red line (3) upon addition of OH⁻. Inset: IR spectra of 3-¹⁴NO₂⁻ (blue line) and 3-¹⁵NO₂⁻ (red line) in KBr. (b) ESI-MS spectra of 3. The peak at 333.2 is assigned to [(12TMC)Co^II(NO₂)]⁺ (calcd m/z 333.1). Inset: isotopic distribution pattern for 3-¹⁴NO₂⁻ (red line) and 3-¹⁵NO₂⁻ (blue line).The FT-IR spectrum of 3 showed a characteristic peak for nitrite stretching at 1271 cm⁻¹ (Co^II-14NO₂⁻) and shifted to 1245 cm⁻¹ (Co^II-15NO₂⁻) when 3 was prepared by reacting ¹⁵N-labeled NO (Co^III-15NO) with OH⁻ (Inset, Fig. 1a and Fig. S2^†). The shifting of NO₂⁻ stretching (Δ = 30 cm⁻¹) indicates that the N-atom in the NO₂⁻ ligand is derived from Co^III-15NO. The ESI-MS spectrum of 3 showed a prominent peak at m/z 333.2, [(12TMC)Co^II(¹⁴NO₂⁻)]⁺ (calcd m/z 333.2), which shifted to 334.2, [(12TMC)Co^II(¹⁵NO₂⁻)]⁺ (calcd m/z 334.2), when the reaction was performed with Co^III-15NO (Inset, Fig. 1b; ESI, Fig. S3a^†); indicating clearly that NO₂⁻ in 3 was derived from the NO moiety of 1. In addition, we have reacted 1 with Na¹⁸OH (ES and ESI^†), in order to follow the source of the second O-atom in 3-NO₂⁻. The ESI-MS spectrum of the reaction mixture, obtained by reacting 1 with Na¹⁸OH, showed a prominent peak at m/z 335.2, [(12TMC)Co^II(¹⁸ONO⁻)]⁺ (calcd m/z 335.2), (SI, Fig. S3b^†) indicating clearly that NO₂⁻ in 3 was derived from ¹⁸OH⁻. The ¹H NMR spectrum of 3 did not show any signal for aliphatic protons of the 12TMC ligand, suggesting a bivalent cobalt center (Fig. S4^†).^26b Furthermore, we have determined the magnetic moment of 3, using Evans'' method, and it was found to be 4.62 BM, suggesting a high spin Co(ii) metal center with three unpaired electrons (ESI^† and ES).²⁹ The exact conformation of 3 was provided by single-crystal X-ray crystallographic analysis (Fig. 2b, ESI, ES, Fig. S5, and Tables T1 and T2^†) and similar to that of previously reported Co^II-NO₂⁻/M^II-NO₂⁻.^15,26b Also, we have quantified the amount of nitrite (90 ± 5%), formed in the above reaction, using the Griess reagent (ESI, ES, and Fig. S6^†).Open in a separate windowFig. 2Displacement ellipsoid plot (20% probability) of 3 at 100 K. Disordered C-atoms of the TMC ring, anion and H-atoms have been removed for clarity.As is known from the literature, a metal-nitrous acid intermediate may form either by the reaction of a metal-nitrosyl with a base²⁷ or by the metal-nitrite reaction with an acid (nitrite reduction chemistry);^26b however, the products of both the reactions are different. Here, for the first time, we have explored the reaction of Co^III-nitrosyl (1) with a base. In this reaction, it is clear that the formation of Co^II-nitrito would be accomplished by the release of H₂ gas via the generation of a transient N-bound [Co-(NOOH)]⁺ intermediate (Scheme 2, reaction (II)). The formation of Co^II-NO₂⁻ (3) from the [Co-(NOOH)]⁺ intermediate is likely to proceed by either (i) homolytic cleavage of the O–H bond and release of H₂via the proposed Co^III-H transient species (Co^III-H = Co^II + 1/2H₂)³⁰ (Scheme 2, reaction (III)), as reported in previous literature where the reduced cobalt, in a number of different ligand environments, is a good H⁺ reduction catalyst and generates H₂ gas via a Co^III-H intermediate³¹ or (ii) heterolytic cleavage of the O–H bond and the formation of Co^I-NO₂⁻ + H⁺.²⁷ In the present study, we observed the formation of 3 and H₂via the plausible homolytic cleavage of the NOO–H moiety of 2 as shown in Scheme 2, in contrast to the previous reports on base-induced reactions on metal-nitrosyls (eqn (3)).²⁷ Taking together both possibilities, (i) is the most reasonable pathway for the NOM reaction of complex 1 in the presence of a base (as shown in Scheme 2, reaction (III)). And the reaction is believed to go through a Co^III-H intermediate as reported previously in Co^I-induced H⁺ reduction in different ligand frameworks and based on literature precedence, we believe that complex 1 acts in a similar manner.³¹Open in a separate windowScheme 2NOM reaction of complex 1 in the presence of OH⁻, showing the generation of Co^II-nitrito (3) and H₂via a Co(iii)-hydrido intermediate.In contrast to an O-bound Co^II-ONOH intermediate, where N–O bond homolysis of the ON-OH moiety generates H₂O₂ (Scheme 2, reaction (IV)),^26b the N-bound [Co-(NOOH)]⁺ intermediate decomposes to form NO₂⁻ and a Co(iii)-H transient species, arising from β-hydrogen transfer from the NOO–H moiety to the cobalt-center (Scheme 2, reaction (II)).^30a,c,32 The Co(iii)-hydrido species may generate H₂ gas either (a) by its transformation to the Co(ii)-nitrito complex (2) and H₂ gas as observed in the case of Co^III-H intermediate chemistry^30a,c,e−g as proposed in the chemistry of the Co^I complex with H⁺ reduction³¹ and other metal-hydrido intermediates³² and also explained in O₂ formation in PN chemistry^17,33 or (b) by the reacting with another [Co-(NOOH)]⁺ intermediate (Scheme 2, reaction (III)).Furthermore, we have confirmed the H₂ formation in the NOM reaction of 1 with OH⁻ by headspace gas mass spectrometry (Fig. 3a). Also, carrying out the reaction of 1 with NaOD leads to the formation of the [Co-(NOOD)]⁺ intermediate, which then transforms to a Co^III-D transient species. Further, as described above, the Co^III-D species releases D₂ gas, detected by headspace gas mass spectrometry (Fig. 3b), which evidently established that H₂ gas formed in the reaction of 1 with OH⁻. In this regard, we have proposed that in the first step of this reaction, the nucleophilic addition of OH⁻ to {Co-NO}⁸ generates a transient N-bound [Co-(NOOH)]⁺ intermediate that is generated by an internal electron transfer to Co^III (Scheme 2, reaction (I)). By following the mechanism proposed in the case of Co^III-H,^30a−c O₂,¹⁵ and H₂O₂(ref. 26b) formation, we have proposed the sequences of the NOM reaction of 1, which leads to the generation of Co^II-nitrito and H₂ (Scheme 2, reaction (I)–(III) and Scheme 3). In the second step, O–H bond homolytic cleavage generates a Co^III-H transient species + NO₂⁻via a β-hydrogen elimination reaction of the [Co-(NOOH)]⁺ intermediate.³² The Co^III-H intermediate may undergo the following reactions to generate H₂ gas and Co^II-nitrito either (a) by the natural decomposition of the Co^III-H transient species to generate H₂,^30a,c,e−g or (b) by the H-atom abstraction from another [Co-(NOOH)]⁺ intermediate (Scheme 3). Also, to validate our assumption that the reaction goes through a plausible N-bound [Co-(NOOH)]⁺ intermediate followed by its transformation to the Co^III-H species (vide supra), we have performed the reaction of 1 with NaOH/NaOD (in 1 : 1 ratio). In this reaction, we have observed the formation of a mixture of H₂, D₂, and HD gases, which indicates clearly that the reaction goes through the formation of Co^III-H and Co^III-D transient species via the aforementioned mechanism (Fig. 3c). This is the only example where tracking of the H atoms has confirmed the H₂ generation from an N-bound NOO–H moiety as proposed for H₂ formation from Co^III-H.³⁰Open in a separate windowFig. 3Mass spectra of formation of (a) H₂ in the reaction of 1 (5.0 mM) with NaOH (5.0 mM), (b) D₂ in the reaction of 1 (5.0 mM) with NaOD (5.0 mM), (c) D₂, HD, and H₂ in the reaction of 1 (5.0 mM) with NaOD/NaOH (1 : 1), and (d) H₂ in the reaction of 1 (5.0 mM) with NaOH in the presence of 2,4 DTBP (50 mM).Open in a separate windowScheme 3NOM reaction of complex 1 in the presence of OH⁻, showing the different steps of the reaction.While, we do not have direct spectral evidence to support the formation of the transient N-bound [Co-(NOOH)]⁺ intermediate and its decomposition to the Co^III-H transient species via β-hydrogen transfer from the NOOH moiety to the cobalt center, support for its formation comes from our finding that the reactive hydrogen species can be trapped by using 2,4-di-tert-butyl-phenol (2,4-DTBP).³⁴ In this reaction, we observed the formation of 2,4-DTBP-dimer (2,4-DTBP-D, ∼67%) as a single product (ESI, ES, and Fig. S7^†). This result can readily be explained by the H-atom abstraction reaction of 2,4-DTBP either by [Co-(NOOH)]⁺ or Co^III-H, hence generating a phenoxyl-radical and 3 with H₂ (Fig. 3d and Scheme 2, reaction (a)). Also, we have detected H₂ gas formation in this reaction (ESI,^† ES, and Fig. 3d). In the next step, two phenoxyl radicals dimerized to give 2,4-DTBP-dimer (Scheme 2c, reaction (II)). Thus, the observation of 2,4-DTBP-dimer in good yield supports the proposed reaction mechanism (Scheme 2, reaction (a) and (b)). Further, the formation of 2,4 DTBP as a single product also rules out the formation of the hydroxyl radical as observed in the case of an O-bound nitrous acid intermediate.^26bFurthermore, we have explored the NOM reactivity of 1 with Na₂O/15-crown-5 (as the O²⁻ source) and observed the formation of the Co^II-nitrito complex (3) via a plausible Co^I-nitro (4) intermediate (Scheme 1, reaction (IIa); also see the ESI^† and ES); however, 1 was found to be inert towards H₂O (Scheme 1, reaction (III); also see the ESI, ES and Fig. S8^†). The product obtained in the reaction of 1 with O²⁻ was characterized by various spectroscopic measurements.^15,26b The UV-vis absorption band of 1 (λ_max = 370 nm) disappears upon the addition of 1 equiv. of Na₂O and a new band (λ_max = 535 nm) forms, which corresponds to 3 (ESI, Fig. S9^†). The FT-IR spectrum of the isolated product of the above reaction shows a characteristic peak for Co^II-bound nitrite at 1271 cm⁻¹, which shifts to 1245 cm⁻¹ when exchanged with ¹⁵N-labeled-NO (¹⁵N¹⁶O) (ESI, ES, and Fig. S10^†), clearly indicating the generation of nitrite from the NO ligand of complex 1.^26b The ESI-MS spectrum recorded for the isolated product (vide supra) shows a prominent ion peak at m/z 333.1, and its mass and isotope distribution pattern matches with [(12-TMC)Co^II(NO₂)]⁺ (calc. m/z 333.1) (ESI, Fig. S11^†). Also, we quantified the amount of 3 (85 ± 5%) by quantifying the amount of nitrite (85 ± 5%) using the Griess reagent test (ESI, ES, and Fig. S6^†).In summary, we have demonstrated the reaction of Co^III-nitrosyl, [(12-TMC)Co^III(NO⁻)]²⁺/{CoNO}⁸ (1), with mono-oxygen reactive species (O²⁻, OH⁻ and H₂O) (Scheme 1). For the first time, we have established the clear formation of a Co^II-nitrito complex, [(12TMC)Co^II(NO₂⁻)]⁺ (3), and H₂ in the reaction of 1 with one equivalent of OH⁻via a transient N-bound [Co-(NOOH)]⁺ (2) intermediate. This [Co-(NOOH)]⁺ intermediate undergoes the O–H bond homolytic cleavage and generates a Co^III-H transient species with NO₂⁻, via a β-hydrogen elimination reaction of the [Co-(NOOH)]⁺ intermediate, which upon decomposition produces H₂ gas. This is in contrast to our previous report, where acid-induced nitrite reduction of 3 generated 1 and H₂O₂via an O-bound Co^II-ONOH intermediate.^26b Complex 1 was found to be inert towards H₂O; however, we have observed the formation of 3 when reacted with O²⁻. It is important to note that H₂ formation involves a distinctive pathway of O–H bond homolytic cleavage in the [Co-(NOOH)]⁺ intermediate, followed by the generation of the proposed Co^III-H transient species (Co^II + 1/2H₂)³⁰ prior to H₂ evolution as described in Co^I chemistry with H⁺ in many different ligand frameworks.³¹ The present study is the first-ever report where the base induced NOM reaction of Co^III-nitrosyl (1) leads to Co^II-nitrito (3) with H₂ evolution via an N-bound [Co-(NOOH)]⁺ intermediate, in contrast to the chemistry of O-bound Co^II-ONOH^26b, hence adding an entirely new mechanistic insight of base induced H₂ gas evolution and an additional pathway for NOM reactions.