Gaucher disease (GD) is a lysosomal storage disorder caused by inherited deficiencies in beta-glucocerebrosidase (GBA). Current treatments require rapid disease diagnosis and a means of monitoring... Show moreGaucher disease (GD) is a lysosomal storage disorder caused by inherited deficiencies in beta-glucocerebrosidase (GBA). Current treatments require rapid disease diagnosis and a means of monitoring therapeutic efficacy, both of which may be supported by the use of GBA-targeting activity-based probes (ABPs). Here, we report the synthesis and structural analysis of a range of cyclophellitol epoxide and aziridine inhibitors and ABPs for GBA. We demonstrate their covalent mechanism-based mode of action and uncover binding of the new N-functionalised aziridines to the ligand binding cleft. These inhibitors became scaffolds for the development of ABPs; the O6-fluorescent tags of which bind in an allosteric site at the dimer interface. Considering GBA's preference for O6- and N-functionalised reagents, a bi-functional aziridine ABP was synthesized as a potentially more powerful imaging agent. Whilst this ABP binds to two unique active site clefts of GBA, no further benefit in potency was achieved over our first generation ABPs. Nevertheless, such ABPs should serve useful in the study of GBA in relation to GD and inform the design of future probes. Show less
Wall teichoic acids (WTAs) are important components of the cell wall of the opportunistic Gram-positive bacterium Staphylococcus aureus. WTAs are composed of repeating ribitol phosphate (RboP)... Show moreWall teichoic acids (WTAs) are important components of the cell wall of the opportunistic Gram-positive bacterium Staphylococcus aureus. WTAs are composed of repeating ribitol phosphate (RboP) residues that are decorated with d-alanine and N-acetyl-d-glucosamine (GlcNAc) modifications, in a seemingly random manner. These WTA-modifications play an important role in shaping the interactions of WTA with the host immune system. Due to the structural heterogeneity of WTAs, it is impossible to isolate pure and well-defined WTA molecules from bacterial sources. Therefore, here synthetic chemistry to assemble a broad library of WTA-fragments, incorporating all possible glycosylation modifications (alpha-GlcNAc at the RboP C4; beta-GlcNAc at the RboP C4; beta-GlcNAc at the RboP C3) described for S. aureus WTAs, is reported. DNA-type chemistry, employing ribitol phosphoramidite building blocks, protected with a dimethoxy trityl group, was used to efficiently generate a library of WTA-hexamers. Automated solid phase syntheses were used to assemble a WTA-dodecamer and glycosylated WTA-hexamer. The synthetic fragments have been fully characterized and diagnostic signals were identified to discriminate the different glycosylation patterns. The different glycosylated WTA-fragments were used to probe binding of monoclonal antibodies using WTA-functionalized magnetic beads, revealing the binding specificity of these WTA-specific antibodies and the importance of the specific location of the GlcNAc modifications on the WTA-chains. Show less
ADP-ribosylation (ADPr), as a post-translational modification, plays a crucial role in DNA-repair, immunity and many other cellular and physiological processes. Serine is the main acceptor for ADPr... Show moreADP-ribosylation (ADPr), as a post-translational modification, plays a crucial role in DNA-repair, immunity and many other cellular and physiological processes. Serine is the main acceptor for ADPr in DNA damage response, whereas the physiological impact of less common ADPr-modifications of cysteine and threonine side chains is less clear. Generally, gaining molecular insights into ADPr recognition and turn-over is hampered by the availability of homogeneous, ADP-ribosylated material, such as mono-ADP-ribosylated (MARylated) peptides. Here, a new and efficient solid-phase strategy for the synthesis of Ser-, Thr- and Cys-MARylated peptides is described. ADP-ribosylated cysteine, apart from being a native post-translational modification in its own right, proved to be suitable as a stabile bioisostere for ADP-ribosylated serine making it a useful tool to further biochemical research on serine ADP-ribosylation. In addition, it was discovered that the Streptococcus pyogenes encoded protein, SpyMacroD, acts as a Cys-(ADP-ribosyl) hydrolase. Show less
Miao, Q.; Zurlo, E.; Bruin, D. de; Wondergem, J.A.J.; Skinner, S.P.; Timmer, M.; ... ; Huber, M.I. 2021
CORRIGENDUM Q. Miao, E. Zurlo, D. de Bruin, J. A. J. Wondergem, S. P. Skinner, M. Timmer, A. Blok, D. Heinrich, M. Overhand, M. Huber,* M. Ubbink* 17128–17133 A Two-Armed Probe for In-Cell DEER... Show moreCORRIGENDUM Q. Miao, E. Zurlo, D. de Bruin, J. A. J. Wondergem, S. P. Skinner, M. Timmer, A. Blok, D. Heinrich, M. Overhand, M. Huber,* M. Ubbink* 17128–17133 A Two-Armed Probe for In-Cell DEER Measurements on Proteins Chem. Eur. J., 2020, 26 DOI: 10.1002/chem.202002743 All authors have agreed that Dr. Simon P. Skinner has made a significant contribution to this work by performing experiments and analyzing data and that his name should have been included in the list of authors. The corrected list of authors therefore reads: Dr. Qing Miao, Dr. Enrico Zurlo, Donny de Bruin, Joeri A. J. Wondergem, Dr. Simon P. Skinner, Monika Timmer, Anneloes Blok, Prof. Dr. Doris Heinrich, Dr. Mark Overhand, Dr. Martina Huber, Prof. Dr. Marcellus Ubbink The relevant affiliations for Dr. Skinner are (1) Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, Einsteinweg 55, 2333, CC Leiden, The Netherlands and (2) School of Molecular and Cellular Biology and Astbury Centre, University of Leeds, Leeds LS2 9JT, UK. The Acknowledgement section should not contain the sentence “and Dr. Simon Skinner for CLaNP5 labeled T4lys NMR data. Show less
Photosystem I (PS I) is a transmembrane protein that assembles perpendicular to the membrane, and performs light harvesting, energy transfer, and electron transfer to a final, water-soluble... Show morePhotosystem I (PS I) is a transmembrane protein that assembles perpendicular to the membrane, and performs light harvesting, energy transfer, and electron transfer to a final, water-soluble electron acceptor. We present here a supramolecular model of it formed by a bicationic oligofluorene 1(2+) bound to the bisanionic photoredox catalyst eosin Y (EY2-) in phospholipid bilayers. According to confocal microscopy, molecular modeling, and time dependent density functional theory calculations, 1(2+) prefers to align perpendicularly to the lipid bilayer. In presence of EY2-, a strong complex is formed (K-a=2.1 +/- 0.1x10(6) m(-1)), which upon excitation of 1(2+) leads to efficient energy transfer to EY2-. Follow-up electron transfer from the excited state of EY2- to the water-soluble electron donor EDTA was shown via UV-Vis absorption spectroscopy. Overall, controlled self-assembly and photochemistry within the membrane provides an unprecedented yet simple synthetic functional mimic of PS I. Show less
The concepts of nucleophilicity and protophilicity are fundamental and ubiquitous in chemistry. A case in point is bimolecular nucleophilic substitution (S(N)2) and base-induced elimination (E2). A... Show moreThe concepts of nucleophilicity and protophilicity are fundamental and ubiquitous in chemistry. A case in point is bimolecular nucleophilic substitution (S(N)2) and base-induced elimination (E2). A Lewis base acting as a strong nucleophile is needed for S(N)2 reactions, whereas a Lewis base acting as a strong protophile (i.e., base) is required for E2 reactions. A complicating factor is, however, the fact that a good nucleophile is often a strong protophile. Nevertheless, a sound, physical model that explains, in a transparent manner, when an electron-rich Lewis base acts as a protophile or a nucleophile, which is not just phenomenological, is currently lacking in the literature. To address this fundamental question, the potential energy surfaces of the S(N)2 and E2 reactions of X-+C2H5Y model systems with X, Y = F, Cl, Br, I, and At, are explored by using relativistic density functional theory at ZORA-OLYP/TZ2P. These explorations have yielded a consistent overview of reactivity trends over a wide range in reactivity and pathways. Activation strain analyses of these reactions reveal the factors that determine the shape of the potential energy surfaces and hence govern the propensity of the Lewis base to act as a nucleophile or protophile. The concepts of "characteristic distortivity" and "transition state acidity" of a reaction are introduced, which have the potential to enable chemists to better understand and design reactions for synthesis. Show less
Doelman, W.; Marqvorsen, M.H.S.; Chiodo, F.; Bruijns, S.C.M.; Marel, G.A. van der; Kooyk, Y. van; ... ; Araman, C. 2020
The protein myelin oligodendrocyte glycoprotein (MOG) is a key component of myelin and an autoantigen in the disease multiple sclerosis (MS). Post‐translational N‐glycosylation of Asn31 of MOG... Show moreThe protein myelin oligodendrocyte glycoprotein (MOG) is a key component of myelin and an autoantigen in the disease multiple sclerosis (MS). Post‐translational N‐glycosylation of Asn31 of MOG seems to play a key role in modulating the immune response towards myelin. This is mediated by the interaction of Lewis‐type glycan structures in the N‐glycan of MOG with the DC‐SIGN receptor on dendritic cells (DCs). Here, we report the synthesis of an unnatural Lewis X (LeX)‐containing Fmoc‐SPPS‐compatible asparagine building block (SPPS=solid‐phase peptide synthesis), as well as asparagine building blocks containing two LeX‐derived oligosaccharides: LacNAc and Fucα1‐3GlcNAc. These building blocks were used for the glycosylation of the immunodominant portion of MOG (MOG31‐55) and analyzed with respect to their ability to bind to DC‐SIGN in different biological setups, as well as their ability to inhibit the citrullination‐induced aggregation of MOG31‐55. Finally, a cytokine secretion assay was carried out on human monocyte‐derived DCs, which showed the ability of the neoglycopeptide decorated with a single LeX to alter the balance of pro‐ and anti‐inflammatory cytokines, inducing a tolerogenic response. Show less
Kim, R.Q.; Misra, M.; Gonzalez, A.; Tomašković, I.; Shin, D.; Schindelin, H.; ... ; Heden- van Noort, G.J. van der 2020
Legionnaires’ disease is caused by infection with the intracellularly replicating Gram‐negative bacterium Legionella pneumophila. This pathogen uses an unconventional way of ubiquitinating host... Show moreLegionnaires’ disease is caused by infection with the intracellularly replicating Gram‐negative bacterium Legionella pneumophila. This pathogen uses an unconventional way of ubiquitinating host proteins by generating a phosphoribosyl linkage between substrate proteins and ubiquitin by making use of an ADPribosylated ubiquitin (UbADPr) intermediate. The family of SidE effector enzymes that catalyze this reaction is counteracted by Legionella hydrolases, which are called Dups. This unusual ubiquitination process is important for Legionella proliferation and understanding these processes on a molecular level might prove invaluable in finding new treatments. Herein, a modular approach is used for the synthesis of triazole‐linked UbADPr, and analogues thereof, and their affinity towards the hydrolase DupA is determined and hydrolysis rates are compared to natively linked UbADPr. The inhibitory effects of modified Ub on the canonical eukaryotic E1‐enzyme Uba1 are investigated and rationalized in the context of a high‐resolution crystal structure reported herein. Finally, it is shown that synthetic UbADPr analogues can be used to effectively pull‐down overexpressed DupA from cell lysate. Show less
We present the synthesis and self‐assembly of a chiral bis(urea) amphiphile and show that chirality offers a remarkable level of control towards different morphologies. Upon self‐assembly in water,... Show moreWe present the synthesis and self‐assembly of a chiral bis(urea) amphiphile and show that chirality offers a remarkable level of control towards different morphologies. Upon self‐assembly in water, the molecular‐scale chiral information is translated to the mesoscopic level. Both enantiomers of the amphiphile self‐assemble into chiral twisted ribbons with opposite handedness, as supported by Cryo‐TEM and circular dichroism (CD) measurements. The system presents thermo‐responsive aggregation behavior and combined transmittance measurements, temperature‐dependent UV, CD, TEM, and micro‐differential scanning calorimetry (DSC) show that a ribbon‐to‐vesicles transition occurs upon heating. Remarkably, chirality allows easy control of morphology as the self‐assembly into distinct aggregates can be tuned by varying the enantiomeric excess of the amphiphile, giving access to flat sheets, helical ribbons, and twisted ribbons. Show less
The inverse electron demand Diels-Alder pyridazine elimination reaction between tetrazines and allylic substituted trans -cyclooctenes (TCOs) is a key player in bioorthogonal bond cleavage... Show moreThe inverse electron demand Diels-Alder pyridazine elimination reaction between tetrazines and allylic substituted trans -cyclooctenes (TCOs) is a key player in bioorthogonal bond cleavage reactions. Determining the rate of elimination on alkylamine substrates has so far proven difficult. Here, we report a fluorogenic tool consisting of a TCO-linked EDANS fluorophore and a DABCYL quencher for accurate detection of both the click and release rates for any tetrazine at physiologically relevant concentrations. Show less
Del Bino, L.; Calloni, I.; Oldrini, D.; Raso, M.M.; Cuffaro, R.; Arda, A.; ... ; Adamo, R. 2019
Group B Streptococcus serotypes Ia and Ib capsular polysaccharides are key targets for vaccine development. In spite of their immunospecifity these polysaccharides share high structural similarity.... Show moreGroup B Streptococcus serotypes Ia and Ib capsular polysaccharides are key targets for vaccine development. In spite of their immunospecifity these polysaccharides share high structural similarity. Both are composed of the same monosaccharide residues and differ only in the connection of the Neu5Acα2-3Gal side chain to the GlcNAc unit, which is a β1-4 linkage in serotype Ia and a β1-3 linkage in serotype Ib. We described development of efficient regioselective routes for GlcNAcβ1-3[Glcβ1-4)]Gal synthons giving access to different GBS Ia and Ib repeating unit frameshifts. These glycans were used to probe the conformation and molecular dynamics of the two polysaccharides, highlighting the different presentation of the protruding Neu5Acα2-3Gal moieties on the polysaccharide backbones and a higher flexibility of Ib polymer compared to Ia which can impact epitope exposure. Show less
Bacterial production of β‐lactamases with carbapenemase activity is a global health threat. The active sites of class D carbapenemases such as OXA‐48, which is of major clinical importance,... Show moreBacterial production of β‐lactamases with carbapenemase activity is a global health threat. The active sites of class D carbapenemases such as OXA‐48, which is of major clinical importance, uniquely contain a carbamylated lysine residue which is essential for catalysis. Although there is significant interest in characterizing this post‐translational modification, and it is a promising inhibition target, protein carbamylation is challenging to monitor in solution. We report the use of 19F NMR spectroscopy to monitor the carbamylation state of 19F‐labelled OXA‐48. This method was used to investigate the interactions of OXA‐48 with clinically used serine β‐lactamase inhibitors, including avibactam and vaborbactam. Crystallographic studies on 19F‐labelled OXA‐48 provide a structural rationale for the sensitivity of the 19F label to active site interactions. The overall results demonstrate the use of 19F NMR to monitor reversible covalent post‐translational modifications. Show less
Phototherapeutic applications of carbon monoxide (CO)‐releasing molecules are limited because they require harmful UV and blue light for activation. We describe two‐photon excitation with NIR light... Show morePhototherapeutic applications of carbon monoxide (CO)‐releasing molecules are limited because they require harmful UV and blue light for activation. We describe two‐photon excitation with NIR light (800 nm)‐induced CO‐release from two MnI tricarbonyl complexes bearing 1,8‐naphthalimide units (1, 2). Complex 2 behaves as a logic OR gate in solution, nonwovens, and in HeLa cells. CO release, indicated by fluorescence enhancement, was detected in solution, nonwoven, and HeLa cells by single‐ (405 nm) and two‐photon (800 nm) excitation. The photophysical properties of 1 and 2 have been measured and supported by DFT and TDDFT quantum chemical calculations. Both photoCORMs are stable in the dark in solution and noncytotoxic, leading to promising applications as phototherapeutics with NIR light. Show less
Vorm, S. van der; Hansen, T.; Rijssel, E.R. van; Dekkers, R.; Madern, J.M.; Overkleeft, H.S.; ... ; Codée, J.D.C. 2019
The 3D shape of glycosyl oxocarbenium ions determines their stability and reactivity and the stereochemical course of SN1-reactions taking place on these reactive intermediates is dictated by the... Show moreThe 3D shape of glycosyl oxocarbenium ions determines their stability and reactivity and the stereochemical course of SN1-reactions taking place on these reactive intermediates is dictated by the conformation of these species. The nature and configuration of functional groups on the carbohydrate ring effect the stability of glycosyl oxocarbenium ions and they control the overall shape of the cations. We here map the stereoelectronic substituent effects of the C2-azide, C2-fluoride and C4-carboxylic acid ester on the stability and reactivity of the complete suite of diastereoisomeric furanoses using a combined computational and experimental approach. Surprisingly all furanosyl donors studied react in a highly stereoselective manner to provide the 1,2-cis products, except for the reactions in the xylose series. The 1,2-cis selectivity in the ribo-, arabino- and lyxo-configured furanosides can be traced back to the lowest energy 3E or E3-conformers of the intermediate oxocarbenium ions. The lack of selectivity of the xylosyl donors is related to the occurrence of oxocarbenium ions, adopting other conformations. Show less
Coubrough, H.; Lubbe, S.C.C. van der; Parashiv, K.; Minard, A.; Pask, C.; Howard, M.; ... ; Wilson, A.J. 2018
A current objective in supramolecular chemistry is to mimic the transitions between complex self‐sorted systems that represent a hallmark of regulatory function in nature. In this work, a self... Show moreA current objective in supramolecular chemistry is to mimic the transitions between complex self‐sorted systems that represent a hallmark of regulatory function in nature. In this work, a self‐sorting network, comprising linear hydrogen motifs, was created. Selecting six hydrogen‐bonding motifs capable of both high‐fidelity and promiscuous molecular recognition gave rise to a complex self‐sorting system, which included motifs capable of both narcissistic and social self‐sorting. Examination of the interactions between individual components, experimentally and computationally, provided a rationale for the product distribution during each phase of a cascade. This reasoning holds through up to five sequential additions of six building blocks, resulting in the construction of a biomimetic network in which the presence or absence of different components provides multiple unique pathways to distinct self‐sorted configurations. Show less
Cyclometallated ruthenium complexes typically exhibit red‐shifted absorption bands and lower photolability compared to their polypyridyl analogues. They also have lower symmetry, which sometimes... Show moreCyclometallated ruthenium complexes typically exhibit red‐shifted absorption bands and lower photolability compared to their polypyridyl analogues. They also have lower symmetry, which sometimes makes their synthesis challenging. In this work, the coordination of four N,S bidentate ligands, 3‐(methylthio)propylamine (mtpa), 2‐(methylthio)ethylamine (mtea), 2‐(methylthio)ethyl‐2‐pyridine (mtep), and 2‐(methylthio)methylpyridine (mtmp), to the cyclometallated precursor [Ru(bpy)(phpy)(CH3CN)2]+ (bpy=2,2′‐bipyridine, Hphpy=2‐phenylpyridine) has been investigated, furnishing the corresponding heteroleptic complexes [Ru(bpy)(phpy)(N,S)]PF6 ([2]PF6–[5]PF6, respectively). The stereoselectivity of the synthesis strongly depended on the size of the ring formed by the Ru‐coordinated N,S ligand, with [2]PF6 and [4]PF6 being formed stereoselectively, but [3]PF6 and [5]PF6 being obtained as mixtures of inseparable isomers. The exact stereochemistry of the air‐stable complex [4]PF6 was established by a combination of DFT, 2D NMR, and single‐crystal X‐ray crystallographic studies. Finally, [4]PF6 was found to be photosubstitutionally active under irradiation with green light in acetonitrile, which makes it the first cyclometallated ruthenium complex capable of undergoing selective photosubstitution of a bidentate ligand. Show less
Artola, M.; Kuo, C.L.; McMahon, S.A.; Oehler, V.; Hansen, T.; Lienden, M.J.C. van der; ... ; Aerts, J.M.F.G. 2018
Cyclophellitol aziridines are potent irreversible inhibitors of retaining glycosidases and versatile intermediates in the synthesis of activity‐based glycosidase probes (ABPs). Direct 3‐amino‐2‐... Show moreCyclophellitol aziridines are potent irreversible inhibitors of retaining glycosidases and versatile intermediates in the synthesis of activity‐based glycosidase probes (ABPs). Direct 3‐amino‐2‐(trifluoromethyl)quinazolin‐4(3H)‐one‐mediated aziridination of l‐ido‐configured cyclohexene has enabled the synthesis of new covalent inhibitors and ABPs of α‐l‐iduronidase, deficiency of which underlies the lysosomal storage disorder mucopolysaccharidosis type I (MPS I). The iduronidase ABPs react covalently and irreversibly in an activity‐based manner with human recombinant α‐l‐iduronidase (rIDUA, Aldurazyme®). The structures of IDUA when complexed with the inhibitors in a non‐covalent transition state mimicking form and a covalent enzyme‐bound form provide insights into its conformational itinerary. Inhibitors 1–3 adopt a half‐chair conformation in solution (4H3 and 3H4), as predicted by DFT calculations, which is different from the conformation of the Michaelis complex observed by crystallographic studies. Consequently, 1–3 may need to overcome an energy barrier in order to switch from the 4H3 conformation to the transition state (2, 5B) binding conformation before reacting and adopting a covalent 5S1 conformation. rIDUA can be labeled with fluorescent Cy5 ABP 2, which allows monitoring of the delivery of therapeutic recombinant enzyme to lysosomes, as is intended in enzyme replacement therapy for the treatment of MPS I patients. Show less
