Correction: Molecular basis of sulfolactate synthesis by sulfolactaldehyde dehydrogenase from Rhizobium leguminosarum.

[This corrects the article DOI: 10.1039/D3SC01594G.].

Molecular Basis for Inhibition of Heparanases and ß-Glucuronidases by Siastatin B.

Siastatin B is a potent and effective iminosugar inhibitor of three diverse glycosidase classes, namely, sialidases, ß-d-glucuronidases, and N-acetyl-glucosaminidases. The mode of inhibition of glucuronidases, in contrast to sialidases, has long been enigmatic as siastatin B appears too bulky and incorrectly substituted to be accommodated within a ß-d-glucuronidase active site pocket. Herein, we show through crystallographic analysis of protein-inhibitor complexes that siastatin B generates both a hemiaminal and a 3-geminal diol iminosugar (3-GDI) that are, rather than the parent compound, directly responsible for enzyme inhibition. The hemiaminal product is the first observation of a natural product that belongs to the noeuromycin class of inhibitors. Additionally, the 3-GDI represents a new and potent class of the iminosugar glycosidase inhibitor. To substantiate our findings, we synthesized both the gluco- and galacto-configured 3-GDIs and characterized their binding both structurally and kinetically to exo-ß-d-glucuronidases and the anticancer target human heparanase. This revealed submicromolar inhibition of exo-ß-d-glucuronidases and an unprecedented binding mode by this new class of inhibitor. Our results reveal the mechanism by which siastatin B acts as a broad-spectrum glycosidase inhibitor, identify a new class of glycosidase inhibitor, and suggest new functionalities that can be incorporated into future generations of glycosidase inhibitors.

Structural Disorder at the Edges of Rubrene Crystals Enhances Singlet Fission.

Materials that undergo singlet fission are of interest for their use in light-harvesting, photocatalysis, and quantum information science, but their ability to undergo fission can be sensitive to local variations in molecular packing. Herein we employ transient absorption microscopy, molecular dynamics simulations, and electronic structure calculations to interrogate how structures found at the edges of orthorhombic rubrene crystals impact singlet fission. Within a micrometer-scale spatial region at the edges of rubrene crystals, we find that the rate of singlet fission increases nearly 4-fold. This observation is consistent with formation of a region at crystal edges with reduced order that accelerates singlet fission by disrupting the symmetry found in rubrene's orthorhombic crystal structure. Our work demonstrates that structural distortions of singlet fission materials can be used to control fission in time and in space, potentially offering a means of controlling this process in light harvesting and quantum information applications.

ß-l-Arabinofurano-cyclitol Aziridines Are Covalent Broad-Spectrum Inhibitors and Activity-Based Probes for Retaining ß-l-Arabinofuranosidases.

GH127 and GH146 microorganismal retaining ß-l-arabinofuranosidases, expressed by human gut microbiomes, feature an atypical catalytic domain and an unusual mechanism of action. We recently reported that both Bacteroides thetaiotaomicron BtGH146 and Bifidobacterium longum HypBA1 are inhibited by ß-l-arabinofuranosyl cyclophellitol epoxide, supporting the action of a zinc-coordinated cysteine as a catalytic nucleophile, where in most retaining GH families, an aspartate or glutamate is employed. This work presents a panel of ß-l-arabinofuranosyl cyclophellitol epoxides and aziridines as mechanism-based BtGH146/HypBA1 inhibitors and activity-based probes. The ß-l-arabinofuranosyl cyclophellitol aziridines both inhibit and label ß-l-arabinofuranosidase efficiently (however with different activities), whereas the epoxide-derived probes favor BtGH146 over HypBA1. These findings are accompanied by X-ray structural analysis of the unmodified ß-l-arabinofuranosyl cyclophellitol aziridine in complex with both isozymes, which were shown to react by nucleophilic opening of the aziridine, at the pseudoanomeric carbon, by the active site cysteine nucleophile to form a stable thioether bond. Altogether, our activity-based probes may serve as chemical tools for the detection and identification of low-abundance ß-l-arabinofuranosidases in complex biological samples.

Molecular basis of sulfolactate synthesis by sulfolactaldehyde dehydrogenase from Rhizobium leguminosarum.

Sulfolactate (SL) is a short-chain organosulfonate that is an important reservoir of sulfur in the biosphere. SL is produced by oxidation of sulfolactaldehyde (SLA), which in turn derives from sulfoglycolysis of the sulfosugar sulfoquinovose, or through oxidation of 2,3-dihydroxypropanesulfonate. Oxidation of SLA is catalyzed by SLA dehydrogenases belonging to the aldehyde dehydrogenase superfamily. We report that SLA dehydrogenase RlGabD from the sulfoglycolytic bacterium Rhizobium leguminsarum SRDI565 can use both NAD+ and NADP+ as cofactor to oxidize SLA, and indicatively operates through a rapid equilibrium ordered mechanism. We report the cryo-EM structure of RlGabD bound to NADH, revealing a tetrameric quaternary structure and supporting proposal of organosulfonate binding residues in the active site, and a catalytic mechanism. Sequence based homology searches identified SLA dehydrogenase homologs in a range of putative sulfoglycolytic gene clusters in bacteria predominantly from the phyla Actinobacteria, Firmicutes, and Proteobacteria. This work provides a structural and biochemical view of SLA dehydrogenases to complement our knowledge of SLA reductases, and provide detailed insights into a critical step in the organosulfur cycle.

Spatial Heterogeneity of Biexcitons in Two-Dimensional Ruddlesden-Popper Lead Iodide Perovskites.

Quantum confinement in two-dimensional (2D) Ruddlesden-Popper (RP) perovskites leads to the formation of stable quasi-particles, including excitons and biexcitons, the latter of which may enable lasing in these materials. Due to their hybrid organic-inorganic structures and the solution phase synthesis, microcrystals of 2D RP perovskites can be quite heterogeneous, with variations in excitonic and biexcitonic properties between crystals from the same synthesis and even within individual crystals. Here, we employ one- and two-quantum two-dimensional white-light microscopy to systematically study the spatial variations of excitons and biexcitons in microcrystals of a series of 2D RP perovskites BA2MAn-1PbnI3n+1 (n = 2-4, BA= butylammonium, MA = methylammonium). We find that the average biexciton binding energy of around 60 meV is essentially independent of the perovskite layer thickness (n). We also resolve spatial variations of the exciton and biexciton energies on micron length scales within individual crystals. By comparing the one-quantum and two-quantum spectra at each pixel, we conclude that biexcitons are more sensitive to their environments than excitons. These results shed new light on the ways disorder can modify the energetic landscape of excitons and biexcitons in RP perovskites and how biexcitons can be used as a sensitive probe of the microscopic environment of a semiconductor.

Synthesis of Uronic Acid 1-Azasugars as Putative Inhibitors of α-Iduronidase, ß-Glucuronidase and Heparanase.

1-Azasugar analogues of l-iduronic acid (l-IdoA) and d-glucuronic acid (d-GlcA) and their corresponding enantiomers have been synthesized as potential pharmacological chaperones for mucopolysaccharidosis I (MPS I), a lysosomal storage disease caused by mutations in the gene encoding α-iduronidase (IDUA). The compounds were efficiently synthesized in nine or ten steps from d- or l-arabinose, and the structures were confirmed by X-ray crystallographic analysis of key intermediates. All compounds were inactive against IDUA, although l-IdoA-configured 8 moderately inhibited ß-glucuronidase (ß-GLU). The d-GlcA-configured 9 was a potent inhibitor of ß-GLU and a moderate inhibitor of the endo-ß-glucuronidase heparanase. Co-crystallization of 9 with heparanase revealed that the endocyclic nitrogen of 9 forms close interactions with both the catalytic acid and catalytic nucleophile.

4-O-Substituted Glucuronic Cyclophellitols are Selective Mechanism-Based Heparanase Inhibitors.

Degradation of the extracellular matrix (ECM) supports tissue integrity and homeostasis, but is also a key factor in cancer metastasis. Heparanase (HPSE) is a mammalian ECM-remodeling enzyme with ß-D-endo-glucuronidase activity overexpressed in several malignancies, and is thought to facilitate tumor growth and metastasis. By this virtue, HPSE is considered an attractive target for the development of cancer therapies, yet to date no HPSE inhibitors have progressed to the clinic. Here we report on the discovery of glucurono-configured cyclitol derivatives featuring simple substituents at the 4-O-position as irreversible HPSE inhibitors. We show that these compounds, unlike glucurono-cyclophellitol, are selective for HPSE over ß-D-exo-glucuronidase (GUSB), also in platelet lysate. The observed selectivity is induced by steric and electrostatic interactions of the substituents at the 4-O-position. Crystallographic analysis supports this rationale for HPSE selectivity, and computer simulations provide insights in the conformational preferences and binding poses of the inhibitors, which we believe are good starting points for the future development of HPSE-targeting antimetastatic cancer drugs.

Energy cascades in donor-acceptor exciton-polaritons observed by ultrafast two-dimensional white-light spectroscopy.

Exciton-polaritons are hybrid states formed when molecular excitons are strongly coupled to photons trapped in an optical cavity. These systems exhibit many interesting, but not fully understood, phenomena. Here, we utilize ultrafast two-dimensional white-light spectroscopy to study donor-acceptor microcavities made from two different layers of semiconducting carbon nanotubes. We observe the delayed growth of a cross peak between the upper- and lower-polariton bands that is oftentimes obscured by Rabi contraction. We simulate the spectra and use Redfield theory to learn that energy cascades down a manifold of new electronic states created by intermolecular coupling and the two distinct bandgaps of the donor and acceptor. Energy most effectively enters the manifold when light-matter coupling is commensurate with the energy distribution of the manifold, contributing to long-range energy transfer. Our results broaden the understanding of energy transfer dynamics in exciton-polariton systems and provide evidence that long-range energy transfer benefits from moderately-coupled cavities.

Time-Domain Photothermal AFM Spectroscopy via Femtosecond Pulse Shaping.

A time-domain version of photothermal microscopy using an atomic force microscope (AFM) is reported, which we call Fourier transform photothermal (FTPT) spectroscopy, where the delay between two laser pulses is varied and the Fourier transform is computed. An acousto-optic modulator-based pulse shaper sets the delay and phases of the pulses shot-to-shot at 100 kHz, enabling background subtraction and data collection in the rotating frame. The pulse shaper is also used to flatten the pulse spectrum, thereby eliminating the need for normalization by the laser spectrum. We demonstrate the method on 6,13-bis(triisopropylsilylethynyl)pentacene (TIPS-Pn) microcrystals and Mn-phthalocyanine islands, confirming subdiffraction spatial resolution, and providing new spectroscopic insights likely linked to structural defects in the crystals.

Cryo-EM structures of human fucosidase FucA1 reveal insight into substrate recognition and catalysis.

Enzymatic hydrolysis of α-L-fucose from fucosylated glycoconjugates is consequential in bacterial infections and the neurodegenerative lysosomal storage disorder fucosidosis. Understanding human α-L-fucosidase catalysis, in an effort toward drug design, has been hindered by the absence of three-dimensional structural data for any animal fucosidase. Here, we have used cryoelectron microscopy (cryo-EM) to determine the structure of human lysosomal α-L-fucosidase (FucA1) in both an unliganded state and in complex with the inhibitor deoxyfuconojirimycin. These structures, determined at 2.49 Å resolution, reveal the homotetrameric structure of FucA1, the architecture of the catalytic center, and the location of both natural population variations and disease-causing mutations. Furthermore, this work has conclusively identified the hitherto contentious identity of the catalytic acid/base as aspartate-276, representing a shift from both the canonical glutamate acid/base residue and a previously proposed glutamate residue. These findings have furthered our understanding of how FucA1 functions in both health and disease.

Mechanism-based heparanase inhibitors reduce cancer metastasis in vivo.

Heparan sulfate proteoglycans (HSPGs) mediate essential interactions throughout the extracellular matrix (ECM), providing signals that regulate cellular growth and development. Altered HSPG composition during tumorigenesis strongly aids cancer progression. Heparanase (HPSE) is the principal enzyme responsible for extracellular heparan sulfate catabolism and is markedly up-regulated in aggressive cancers. HPSE overactivity degrades HSPGs within the ECM, facilitating metastatic dissemination and releasing mitogens that drive cellular proliferation. Reducing extracellular HPSE activity reduces cancer growth, but few effective inhibitors are known, and none are clinically approved. Inspired by the natural glycosidase inhibitor cyclophellitol, we developed nanomolar mechanism-based, irreversible HPSE inhibitors that are effective within physiological environments. Application of cyclophellitol-derived HPSE inhibitors reduces cancer aggression in cellulo and significantly ameliorates murine metastasis. Mechanism-based irreversible HPSE inhibition is an unexplored anticancer strategy. We demonstrate the feasibility of such compounds to control pathological HPSE-driven malignancies.

Azido Groups Hamper Glycan Acceptance by Carbohydrate Processing Enzymes.

Azido sugars have found frequent use as probes of biological systems in approaches ranging from cell surface metabolic labeling to activity-based proteomic profiling of glycosidases. However, little attention is typically paid to how well azide-substituted sugars represent the parent molecule, despite the substantial difference in size and structure of an azide compared to a hydroxyl. To quantitatively assess how well azides are accommodated, we have used glycosidases as tractable model enzyme systems reflecting what would also be expected for glycosyltransferases and other sugar binding/modifying proteins. In this vein, specificity constants have been measured for the hydrolysis of a series of azidodeoxy glucosides and N-acetylhexosaminides by a large number of glycosidases produced from expressed synthetic gene and metagenomic libraries. Azides at secondary carbons are not significantly accommodated, and thus, associated substrates are not processed, while those at primary carbons are productively recognized by only a small subset of the enzymes and often then only very poorly. Accordingly, in the absence of careful controls, results obtained with azide-modified sugars may not be representative of the situation with the natural sugar and should be interpreted with considerable caution. Azide incorporation can indeed provide a useful tool to monitor and detect glycosylation, but careful consideration should go into the selection of sites of azide substitution; such studies should not be used to quantitate glycosylation or to infer the absence of glycosylation activity.

The structure of Phocaeicola vulgatus sialic acid acetylesterase.

Sialic acids terminate many N- and O-glycans and are widely distributed on cell surfaces. There are a diverse range of enzymes which interact with these sugars throughout the tree of life. They can act as receptors for influenza and specific betacoronaviruses in viral binding and their cleavage is important in virion release. Sialic acids are also exploited by both commensal and pathogenic bacteria for nutrient acquisition. A common modification of sialic acid is 9-O-acetylation, which can limit the action of sialidases. Some bacteria, including human endosymbionts, employ esterases to overcome this modification. However, few bacterial sialic acid 9-O-acetylesterases (9-O-SAEs) have been structurally characterized. Here, the crystal structure of a 9-O-SAE from Phocaeicola vulgatus (PvSAE) is reported. The structure of PvSAE was determined to resolutions of 1.44 and 2.06 Šusing crystals from two different crystallization conditions. Structural characterization revealed PvSAE to be a dimer with an SGNH fold, named after the conserved sequence motif of this family, and a Ser-His-Asp catalytic triad. These structures also reveal flexibility in the most N-terminal α-helix, which provides a barrier to active-site accessibility. Biochemical assays also show that PvSAE deacetylates both mucin and the acetylated chromophore para-nitrophenyl acetate. This structural and biochemical characterization of PvSAE furthers the understanding of 9-O-SAEs and may aid in the discovery of small molecules targeting this class of enzyme.

Water-Accelerated Photooxidation of CH3NH3PbI3 Perovskite.

Halide perovskites have the potential to disrupt the photovoltaics market based on their high performance and low cost. However, the decomposition of perovskites under moisture, oxygen, and light raises concerns about service lifetime, especially because degradation mechanisms and the corresponding rate laws that fit the observed data have thus far eluded researchers. Here, we report a water-accelerated photooxidation mechanism dominating the degradation kinetics of archetypal perovskite CH3NH3PbI3 in air under >1% relative humidity at 25 °C. From this mechanism, we develop a kinetic model that quantitatively predicts the degradation rate as a function of temperature, ambient O2 and H2O levels, and illumination. Because water is a possible product of dry photooxidation, these results highlight the need for encapsulation schemes that rigorously block oxygen ingress, as product water may accumulate beneath the encapsulant and initiate the more rapid water-accelerated photooxidative decomposition.

Synthesis of broad-specificity activity-based probes for exo-ß-mannosidases.

Exo-ß-mannosidases are a broad class of stereochemically retaining hydrolases that are essential for the breakdown of complex carbohydrate substrates found in all kingdoms of life. Yet the detection of exo-ß-mannosidases in complex biological samples remains challenging, necessitating the development of new methodologies. Cyclophellitol and its analogues selectively label the catalytic nucleophiles of retaining glycoside hydrolases, making them valuable tool compounds. Furthermore, cyclophellitol can be readily redesigned to enable the incorporation of a detection tag, generating activity-based probes (ABPs) that can be used to detect and identify specific glycosidases in complex biological samples. Towards the development of ABPs for exo-ß-mannosidases, we present a concise synthesis of ß-manno-configured cyclophellitol, cyclophellitol aziridine, and N-alkyl cyclophellitol aziridines. We show that these probes covalently label exo-ß-mannosidases from GH families 2, 5, and 164. Structural studies of the resulting complexes support a canonical mechanism-based mode of action in which the active site nucleophile attacks the pseudoanomeric centre to form a stable ester linkage, mimicking the glycosyl enzyme intermediate. Furthermore, we demonstrate activity-based protein profiling using an N-alkyl aziridine derivative by specifically labelling MANBA in mouse kidney tissue. Together, these results show that synthetic manno-configured cyclophellitol analogues hold promise for detecting exo-ß-mannosidases in biological and biomedical research.

Ultrafast Fluctuations in PM6 Domains of Binary and Ternary Organic Photovoltaic Thin Films Probed with Two-Dimensional White-Light Spectroscopy.

We present two-dimensional white-light spectroscopy (2DWL) measurements of binary and ternary bulk heterojunctions of the polymer donor PM6 mixed with state-of-the-art nonfullerene acceptors Y6 or IT4F. The ternary film has a shorter lifetime and faster spectral diffusion than either of the binary films. 2D line shape analysis of the PM6 ground state bleach with a Kubo model determines that all three films have similar amplitudes of fluctuations (Δ = 0.29 fs-1) in their transition frequencies, but different relaxation times (ranging from 102 to 24 fs). The ternary film exhibits faster dynamics than either of the binary films. The short lifetime of the ternary blend is consistent with increased photoexcitation transfer and the fast frequency fluctuations are consistent with structural dynamics of aliphatic side chains. These results suggest that the femtosecond fluctuations of PM6 are impacted by the choice of the acceptor molecules. We hypothesize that those dynamics are either indicative, or perhaps the initial source, of structural dynamics that ultimately contribute to solar cell operation.

Activity-Based Protein Profiling of Retaining α-Amylases in Complex Biological Samples.

Amylases are key enzymes in the processing of starch in many kingdoms of life. They are important catalysts in industrial biotechnology where they are applied in, among others, food processing and the production of detergents. In man amylases are the first enzymes in the digestion of starch to glucose and arguably also the preferred target in therapeutic strategies aimed at the treatment of type 2 diabetes patients through down-tuning glucose assimilation. Efficient and sensitive assays that report selectively on retaining amylase activities irrespective of the nature and complexity of the biomaterial studied are of great value both in finding new and effective human amylase inhibitors and in the discovery of new microbial amylases with potentially advantageous features for biotechnological application. Activity-based protein profiling (ABPP) of retaining glycosidases is inherently suited for the development of such an assay format. We here report on the design and synthesis of 1,6-epi-cyclophellitol-based pseudodisaccharides equipped with a suite of reporter entities and their use in ABPP of retaining amylases from human saliva, murine tissue as well as secretomes from fungi grown on starch. The activity and efficiency of the inhibitors and probes are substantiated by extensive biochemical analysis, and the selectivity for amylases over related retaining endoglycosidases is validated by structural studies.

High-Throughput Generation of Product Profiles for Arabinoxylan-Active Enzymes from Metagenomes.

Metagenomics is an exciting alternative to seek carbohydrate-active enzymes from a range of sources. Typically, metagenomics reveals dozens of putative catalysts that require functional characterization for further application in industrial processes. High-throughput screening methods compatible with adequate natural substrates are crucial for an accurate functional elucidation of substrate preferences. Based on DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) analysis of enzymatic-reaction products, we generated product profiles to consequently infer substrate cleavage positions, resulting in the generation of enzymatic-degradation maps. Product profiles were produced in high throughput for arabinoxylan (AX)-active enzymes belonging to the glycoside hydrolase families GH43 (subfamilies 2 [MG432], 7 [MG437], and 28 [MG4328]) and GH8 (MG8) starting from 12 (arabino)xylo-oligosaccharides. These enzymes were discovered through functional metagenomic studies of feces from the North American beaver (Castor canadensis). This work shows how enzyme loading alters the product profiles of all enzymes studied and gives insight into AX degradation patterns, revealing sequential substrate preferences of AX-active enzymes.IMPORTANCE Arabinoxylan is mainly found in the hemicellulosic fractions of rice straw, corn cobs, and rice husk. Converting arabinoxylan into (arabino)xylo-oligosaccharides as added-value products that can be applied in food, feed, and cosmetics presents a sustainable and economic alternative for the biorefinery industries. Efficient and profitable AX degradation requires a set of enzymes with particular characteristics. Therefore, enzyme discovery and the study of substrate preferences are of utmost importance. Beavers, as consumers of woody biomass, are a promising source of a repertoire of enzymes able to deconstruct hemicelluloses into soluble oligosaccharides. High-throughput analysis of the oligosaccharide profiles produced by these enzymes will assist in the selection of the most appropriate enzymes for the biorefinery.

Manno-epi-cyclophellitols Enable Activity-Based Protein Profiling of Human α-Mannosidases and Discovery of New Golgi Mannosidase II Inhibitors.

Golgi mannosidase II (GMII) catalyzes the sequential hydrolysis of two mannosyl residues from GlcNAcMan5GlcNAc2 to produce GlcNAcMan3GlcNAc2, the precursor for all complex N-glycans, including the branched N-glycans associated with cancer. Inhibitors of GMII are potential cancer therapeutics, but their usefulness is limited by off-target effects, which produce α-mannosidosis-like symptoms. Despite many structural and mechanistic studies of GMII, we still lack a potent and selective inhibitor of this enzyme. Here, we synthesized manno-epi-cyclophellitol epoxide and aziridines and demonstrate their covalent modification and time-dependent inhibition of GMII. Application of fluorescent manno-epi-cyclophellitol aziridine derivatives enabled activity-based protein profiling of α-mannosidases from both human cell lysate and mouse tissue extracts. Synthesized probes also facilitated a fluorescence polarization-based screen for dGMII inhibitors. We identified seven previously unknown inhibitors of GMII from a library of over 350 iminosugars and investigated their binding modalities through X-ray crystallography. Our results reveal previously unobserved inhibitor binding modes and promising scaffolds for the generation of selective GMII inhibitors.