Reversible Linkage Research Articles

Photolysis of Isoelectronic Ruthenium Nitrosyl and Diazonium Complexes in Frozen PVC Matrices: Retention of Dinitrogen on Ruthenium Following Photochemical Phenyl Radical Loss

AbstractPhotolysis of RuCl3(η1‐NO)(PPh3)2 in poly(vinyl chloride) (PVC) matrices at 85 K results in reversible linkage isomerism of the nitrosyl ligand to form the isonitrosyl complex RuCl3(η1‐ON)(PPh3)2. Metal‐to‐ligand charge‐transfer (MLCT) excitation of the isoelectronic phenylazo complex, RuCl3(η1‐N2Ph)(PPh3)2, has previously been shown to result in generation of the phenyl radical, presumably through decomposition a photogenerated diazenyl radical. Examination of this photolysis in a PVC matrix at cryogenic temperatures has permitted direct observation of an isotopically sensitive product band that may be assigned to a 17‐electron ruthenium dinitrogen species, suggesting that this photochemical decomposition does not proceed through formation of a diazenyl intermediate but through homolytic cleavage of the parent diazonium complex to give the phenyl radical and the 17‐electron RuCl3(η1‐N2)(PPh3)2.

Competition-driven selection in covalent dynamic networks and implementation in organic reactional selectivity.

Competition among reagents in dynamic combinatorial libraries of increased complexity leads to reactional self-sorting (improved regioselectivity) in mixtures of aldehydes and oligoamines. High selectivity of a given library component is transferred to a different reacting component of low selectivity through a network of underlying equilibrating reactions which provide component exchange between all species. The selectivity of various carbonyl compounds in reactions with amines was also assessed towards the formation of defined sequences of residues along oligoamine chains. The approach was further exploited for defining selective dynamic protecting groups (DPGs), based on the reversible linkage between the substrate and the protecting group. They represent an intermediate approach between the conventional protecting groups and the protecting-group-free approach in organic synthesis. Removal of the protecting group is effected via dynamic exchange trapping by formation of a more stable product. The establishment of equilibrium eliminates the need for isolation and purification of the dynamically protected intermediate(s) and enables as well the selective sequential derivatisation of oligoamines. The DPG concept can be generalised to other reversible reactions and can thus represent a valuable alternative in the design of total synthesis of complex molecules.

Photo-induced linkage NO isomers in the dinitrosyl complex [Ru(NO)2(PCy3)2Cl](BF4) identified by photocrystallography and IR-spectroscopy

Photoinduced metastable linkage NO isomers have been known for a long time in mono-nitrosyl ruthenium or iron transition metal complexes [MLxLy(NO)]n for which appropriate temperature and excitation conditions allow a controlled generation of different metastable states. Dinitrosyl complexes offer attractive possibilities owing to the potential switching of the two nitrosyl ligands, possibly in a selective manner, leading to a complicated and intricated population of metastable states. [Ru(NO)2(PCy3)2Cl]BF4 belongs to a complete series of bis-phosphane complex salts of general formula [Ru(NO)2(PR3)2X](BF4) (X = Cl, Br, I). It presents a square pyramidal geometry with both a linear and a bent NO ligand, and an Ru(d6) electron configuration. Multiple light-induced reversible metastable NO linkage isomers (PLIs) are detected by a combination of photocrystallographic and infrared (IR) analysis. The IR signature of three PLI states has been clearly identified. The structural configuration of the major component (PLI-1) has been derived by X-ray photocrystallography. After irradiation with 405 nm, the orientation of the bent nitrosyl is changed by rotation towards the Cl ligand, opposite to the linear NO. The minor second metastable state (PLI-2) is proposed to result from a side-bonded (similar to MS2 in mono-nitrosyl complexes) state of the linear nitrosyl.

Abstract B43: Reversible linkage of two distinct small molecule inhibitors of MYC generates a more potent and selective dimeric inhibitor that is active in cancer cell lines over-expressing MYC

Abstract Up to 70% of all human cancers have deregulated or elevated levels of MYC, making MYC a high profile cancer drug target. The disordered structure of the MYC protein has made identifying small molecule inhibitors of this target very challenging, however recent reports have identified molecules that bind to MYC and block its function. Here we describe the successful application of a novel approach for generating selective dimeric MYC inhibitors by modifying and reversibly linking two previously described small molecule inhibitors of MYC (10058-F4 and 10074-G5) that bind to distinct sites in the bHLHZip domain of the protein. We synthesized two directed libraries of monomers based on each of these molecules to identify the optimal dimer configuration required to inhibit MYC activity. Each monomer contains one of the respective ligands, a connector of varying length, and a bioorthogonal linker element that allows reversible dimerization. We identified specific combinations of monomers (termed active dimers), which displayed synergistic inhibition of MYC in biochemical and cellular assays. Through SPR, ELISA and EMSA we demonstrate that these dimers directly bind to MYC, block its interaction with Max and thus its binding to DNA, respectively. These dimeric inhibitors have superior anti-proliferative activity in MYC over-expressing cell lines compared to their monomeric components, and this activity is correlated with a decrease in MYC protein levels and an inhibition of MYC-dependent gene expression. Control combinations that lack a linker element, and thus are unable to form a dimer, do not show any synergistic effects in these assay systems. Collectively, these data validate our new approach to generate selective inhibitors of MYC assembled from smaller, lower affinity components. This approach provides an opportunity for developing novel therapeutics against MYC and other challenging intracellular drug target classes. Citation Format: Jutta Wanner, Darlene Romashko, Douglas S. Werner, Earl W. May, Yue Peng, Ryan Schulz, Kenneth W. Foreman, Suzanne Russo, Lee D. Arnold, Maneesh Pingle, Donald E. Bergstrom, Francis Barany, Stuart Thomson. Reversible linkage of two distinct small molecule inhibitors of MYC generates a more potent and selective dimeric inhibitor that is active in cancer cell lines over-expressing MYC. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B43.

Novel bioluminescent receptor-binding assays for peptide hormones: using ghrelin as a model.

Peptide hormones perform important biological functions by binding specific cell membrane receptors. For hormone-receptor interaction studies, receptor-binding assays are widely used. However, conventional receptor-binding assays rely on radioactive tracers that have drawbacks. In recent studies, we established novel non-radioactive receptor-binding assays for some recombinant protein hormones based on the ultrasensitive bioluminescence of a newly developed nanoluciferase (NanoLuc) reporter. In the present work, we extended the novel bioluminescent receptor-binding assay to peptide hormones that have small size and can be conveniently prepared by chemical synthesis. Human ghrelin, a 28-amino acid peptide hormone carrying a special O-fatty acid modification, was used as a model. To prepare a bioluminescent ghrelin tracer, a chemically synthesized ghrelin analog with a unique cysteine residue at the C-terminus was site-specifically conjugated with an engineered NanoLuc with a unique exposed cysteine residue at the C-terminus via a reversible disulfide linkage. The NanoLuc-conjugated ghrelin retained high binding affinity with the ghrelin receptor GHSR1a (K d=1.14±0.13nM, n=3) and was able to sensitively monitor the receptor-binding of various GHSR1a ligands. The novel bioluminescent receptor-binding assay will facilitate the interaction studies of ghrelin with its receptor. We also proposed general procedures for convenient conjugation of other peptide hormones with NanoLuc for novel bioluminescent receptor-binding assays.

Reversible linkage of two distinct small molecule inhibitors of Myc generates a dimeric inhibitor with improved potency that is active in myc over-expressing cancer cell lines.

We describe the successful application of a novel approach for generating dimeric Myc inhibitors by modifying and reversibly linking two previously described small molecules. We synthesized two directed libraries of monomers, each comprised of a ligand, a connector, and a bioorthogonal linker element, to identify the optimal dimer configuration required to inhibit Myc. We identified combinations of monomers, termed self-assembling dimeric inhibitors, which displayed synergistic inhibition of Myc-dependent cell growth. We confirmed that these dimeric inhibitors directly bind to Myc blocking its interaction with Max and affect transcription of MYC dependent genes. Control combinations that are unable to form a dimer do not show any synergistic effects in these assays. Collectively, these data validate our new approach to generate more potent and selective inhibitors of Myc by self-assembly from smaller, lower affinity components. This approach provides an opportunity for developing novel therapeutics against Myc and other challenging protein:protein interaction (PPI) target classes.

Multiple light-induced NO linkage isomers in the dinitrosyl complex [RuCl(NO)2(PPh3)2]BF4 unravelled by photocrystallographic and IR analysis.

Multiple light-induced reversible metastable NO linkage isomers (PLIs) have been detected in the dinitrosyl compound [RuCl(NO)2(PPh3)2]BF4 by a combination of photocrystallographic and IR analysis. The IR signature of three PLI states has been clearly identified, with estimated populations of 59% (PLI-1), 8% (PLI-2) and 5% (PLI-3) for a total population of the metastable state of 72%. The structural configuration of the major component (PLI-1) has been derived by X-ray photocrystallography. In the ground state, the structure is characterized by a bent and a linear nitrosyl, the bent one being oriented towards the linear equatorial nitrosyl with an Ru-N-O angle of 133.88 (9)°. X-ray Fourier difference maps indicate a selectivity of the photo-isomerization process in PLI-1: only the bent NO ligand changes its position, while the linear NO is unaffected. After irradiation at 405 nm, the orientation is changed by rotation towards the Cl ligand opposite the linear NO, with an Ru-N-O angle in this new position of 109 (1)°. The photocrystallographic analysis provides evidence that, in the photo-induced metastable state, the bent NO group is attached to the Ru atom through the N atom (Ru-N-O), rather than in an isonitrosyl Ru-O-N binding mode. In the IR spectra, the asymmetric NO vibrational band shifts by -33 cm(-1) to a lower value, whereas the symmetric band splits and shifts by 5 cm(-1) to a higher value and by -8 cm(-1) to a lower value. The down shift is a clear indication of the structural change, and the small upward shift in response to the new electronic configuration of the metastable structure. Variable-temperature IR kinetic measurements in the range 80-114 K show that the decay of the PLI-1 state follows an Arrhenius behaviour with an activation energy of 0.22 eV.

Chlorotoxin-conjugated onconase as a potential anti-glioma drug.

Gliomas are rarely curable malignant brain tumors arising from normal glial cells. The scorpion-derived small peptide, chlorotoxin (CTX), can selectively bind malignant gliomas. In the present study, a CTX-conjugated onconase (Onc), a small cytotoxic ribonuclease, was prepared as a potential anti-glioma drug. In this conjugate, recombinant CTX was covalently linked with recombinant Onc by reversible disulfide linkage. The chemically conjugated CTX-Onc showed much higher cytotoxicity to the cultured glioma U251 and SHG-44 cells than the physical mixture of CTX and Onc (CTX + Onc). In the nude mouse models bearing subcutaneous U251 or SHG-44 tumors, the CTX-Onc conjugate also showed improved anti-tumor effects than the CTX + Onc control. These results suggested that the reversible chemical-conjugated CTX promoted the tumor targeting of Onc, and thus the present CTX-Onc conjugate could be further developed as a potential targeted anti-glioma drug.

SO2 Phototriggered Crystalline Nanomechanical Transduction of Aromatic Rotors in Tosylates: Rationalization via Photocrystallography of [Ru(NH3)4SO2X]tosylate2 (X = pyridine, 3-Cl-pyridine, 4-Cl-pyridine)

Thermally reversible solid-state linkage SO2 photoisomers of three complexes in the [Ru(NH3)4SO2X]tosylate2 family are captured in their metastable states using photocrystallography, where X = pyridine (1), 3-Cl-pyridine (2), and 4-Cl-pyridine (3). This photoisomerism exists only in the single-crystal form; accordingly, the nature of the crystalline environment surrounding the photoactive species controls its properties. In particular, the structural role of the tosylate anion needs to be understood against possible chemical influences due to varying the trans ligand, X. The photoexcited geometries, photoconversion levels, and thermal stabilities of the photoisomers that form in 1–3 are therefore studied. 1 and 2 yield two photoisomers at 100 K: the O-bound end-on η1-SO2 (MS1) configuration and the side-bound η2-SO2 (MS2); 3 exhibits only the more thermally stable MS2 geometry. The decay kinetics of the MS2 geometry for 1–3 demonstrate that the greater the free volume of the GS SO2 ligand for a given coun...

Metal Switch-controlled Myosin II from Dictyostelium discoideum Supports Closure of Nucleotide Pocket during ATP Binding Coupled to Detachment from Actin Filaments

G-proteins, kinesins, and myosins are hydrolases that utilize a common protein fold and divalent metal cofactor (typically Mg(2+)) to coordinate purine nucleotide hydrolysis. The nucleoside triphosphorylase activities of these enzymes are activated through allosteric communication between the nucleotide-binding site and the activator/effector/polymer interface to convert the free energy of nucleotide hydrolysis into molecular switching (G-proteins) or force generation (kinesins and myosin). We have investigated the ATPase mechanisms of wild-type and the S237C mutant of non-muscle myosin II motor from Dictyostelium discoideum. The S237C substitution occurs in the conserved metal-interacting switch-1, and we show that this substitution modulates the actomyosin interaction based on the divalent metal present in solution. Surprisingly, S237C shows rapid basal steady-state Mg(2+)- or Mn(2+)-ATPase kinetics, but upon binding actin, its MgATPase is inhibited. This actin inhibition is relieved by Mn(2+), providing a direct and experimentally reversible linkage of switch-1 and the actin-binding cleft through the swapping of divalent metals in the reaction. Using pyrenyl-labeled F-actin, we demonstrate that acto·S237C undergoes slow and weak MgATP binding, which limits the rate of steady-state catalysis. Mn(2+) rescues this effect to near wild-type activity. 2'(3')-O-(N-Methylanthraniloyl)-ADP release experiments show the need for switch-1 interaction with the metal cofactor for tight ADP binding. Our results are consistent with strong reciprocal coupling of nucleoside triphosphate and F-actin binding and provide additional evidence for the allosteric communication pathway between the nucleotide-binding site and the filament-binding region.

Thermoresponsive Dynamic Covalent Polymers with Tunable Properties

A bisaldehyde-containing trithiocarbonate chain transfer agent was prepared and mediated the synthesis of polymers with bisaldehyde-functionalized α-termini by reversible addition–fragmentation chain transfer (RAFT) radical polymerization. The α-termini of RAFT-derived thermoresponsive poly(N-isopropylacrylamide) was conjugated with hydrazides via reversible acylhydrazone bonds. Introduction of hydrophilic end-groups generated a dynamic covalent polymer with “isothermal” lower critical solution temperature (LCST) response to medium pH and dynamic chain exchange character. A biofunctional dynamic covalent polymer was prepared by conjugation with biotin hydrazide. Under appropriate reaction conditions, a dynamic covalent block copolymer with an aldehyde group at the junction point was constructed through a reversible arylhydrazone linkage by coupling with acylhydrazide-terminated poly(ethylene glycol). The dynamic covalent block copolymer exhibited pH-dependent LCST. The remaining aldehyde group was used to react with amino-containing molecules. Bioconjugated dynamic covalent block copolymers containing reversible imine and arylhydrazone linkages were constructed by conjugation of the block copolymer to glucosamine and protein.

Stimuli‐Responsive Polymer Networks with β‐Cyclodextrin and Ferrocene Reversible Linkage Based on Linker Chemistry

SummaryDifferent from the chemical structure and self‐assembly behavior of traditional block copolymer, a homopolymer self‐assembly approach with reversible linkage based on linker chemistry has been developed. The strategy could be used for the preparation of stimuli‐responsive polymer networks with reversible linkers. A redox‐responsive supramolecular hydrogel was obtained by two hydrophilic copolymers (poly(N, N'‐dimethylacrylamide‐r‐glycidyl methacrylateglycidyl methacrylate) and poly(N, N'‐dimethylacrylamide‐r‐hydroxyethyl methacrylateglycidyl methacrylate)) with β‐CD and ferrocene pendent groups, respectively. The hydrogel exhibited gel‐sol transition after the addition of an oxidizing agent FeCl3 and returned to its gel state when a reducing agent ascorbic acid was added. SEM observation showed that the hydrogel had a porous network structure. The host‐guest interaction between β‐CD and ferrocene was a dominant driving force for the formation of the hydrogel.

A reversible flux linkage model of the switched reluctance motor with rotor position computable

PurposeThe purpose of this paper is to present a reversible flux linkage model of the switched reluctance motor so that the rotor position can be computed analytically.Design/methodology/approachThe presented flux linkage model uses a Fermi function like equation with four coefficients to fit flux linkage characteristics. In this work, the coefficients are calculated from flux‐current‐angle data using numerical curve fitting with the least squares method. A rotor position model is then derived by inverting the flux linkage model. With a simple estimation scheme based on the rotor position model, the rotor position is estimated continuously. Simulation and experiments are then performed to verify the proposed method.FindingsThe presented flux linkage model agrees well with flux linkage characteristics. The average absolute relative error (AARE) of the model varies between 0.3 per cent and 5.3 per cent. With the derived rotor position model, rotor position can be estimated conveniently for either steady or dynamic operations.Practical implicationsThe simulation and experimental results indicate that the presented model is an eligible candidate for applications such as rotor position estimation, performance simulation and other model based controls.Originality/valueUnlike previously reported methods, the presented flux linkage model is reversible so that a rotor position model can be derived and the rotor position can be computed analytically.

Boronic acid-based autoligation of nucleic acids: influence of the nature of the 3′-end ribonucleotidic strand

The development of synthetic systems displaying dynamic and adaptive characteristics is a formidable challenge with wide applications from biotechnology to therapeutics. Recently, we described a dynamic and programmable nucleic acid-based system relying on the formation of reversible boronate internucleosidic linkages. The DNA- or RNA-templated system comprises a 5′-ended boronic acid probe connecting a 3′-ended ribonucleosidic oligonucleotide partner. To explore the dominant factors that control the reversible linkage, we synthesized a series of 3′-end modified ribonucleotidic strands. Evidence suggests that geometric and steric factors are key features for controlling the equilibria.

Introducing a Reversible Linkage to Block Copolymer Self-Assembly: Towards Controlling Nanopore Chemistry

Introducing a Reversible Linkage to Block Copolymer Self-Assembly: Towards Controlling Nanopore Chemistry

Synthesis and self-assembly of dynamic covalent block copolymers: towards a general route to pore-functionalized membranes

A diblock copolymer is designed to have incompatible blocks, unsymmetrical block lengths, and a reversible linkage. This copolymer self-assembles into nanostructured cylindrical morphology in thin films. Removal of the nanosized cylinders by breaking the reversible linkage then affords nanoporous membranes featuring a chemically reactive functionality in the pores.

Global profiling of dynamic protein palmitoylation

The reversible thioester linkage of palmitic acid on cysteines is known as protein S-palmitoylation, which facilitates the membrane association and proper subcellular localization of proteins. Here we report the metabolic incorporation of the palmitic acid analogue 17-octadecynoic acid (17-ODYA) in combination with stable-isotope labeling of cells (SILAC) and pulse-chase methods to generate a global quantitative map of dynamic protein palmitoylation events in cells. We distinguished stably palmitoylated proteins from those that show rapid turnover. Treatment with a serine lipase-selective inhibitor identified a special pool of dynamically palmitoylated proteins regulated by palmitoyl-protein thioesterases. This subset was enriched in oncogenes and other proteins linked to aberrant cell growth, migration, and cancer. Our method provides a straightforward way to characterize global palmitoylation dynamics in cells and confirms enzyme-mediated depalmitoylation as a critical regulatory mechanism for a specific subset of rapidly cycling palmitoylated proteins.

Minor Groove Orientation of the KWKK Peptide Tethered via the N-Terminal Amine to the Acrolein-Derived 1,N2-γ-Hydroxypropanodeoxyguanosine Lesion with a Trimethylene Linkage,

DNA−protein conjugates are potentially repaired via proteolytic digestion to DNA−peptide conjugates. The latter have been modeled with the amino-terminal lysine of the peptide KWKK conjugated via a trimethylene linkage to the N2-dG amine positioned in 5′-d(GCTAGCXAGTCC)-3′·5′-d(GGACTCGCTAGC)-3′ (X = N2-dG−trimethylene link−KWKK). This linkage is a surrogate for the reversible linkage formed by the γ-OH-1,N2-propanodeoxyguanosine (γ-OH-PdG) adduct. This conjugated KWKK stabilizes the DNA. Amino acids K26, W27, K28, and K29 are in the minor groove. The W27 indolyl group does not intercalate into the DNA. The G7N2 amine and the K26 N-terminal amine nitrogens are in the trans configuration with respect to the Cα or Cγ of the trimethylene tether, respectively. The structure of this DNA−KWKK conjugate is discussed in the context of its biological processing.

Ultrafast reversible ligand isomerisation in Na2[Fe(CN)5NO]·2H2O single crystals

We present a time-resolved absorption study on the light-induced generation of reversible linkage NO isomers in single crystals of Na(2)[Fe(CN)(5)NO] x 2 H(2)O using laser pulses of 160 fs width. Using the pump wavelength lambda = 500 nm the singlet-singlet (1)A(1)-->(1)E excitation induces the NO rotation by about 90 degrees from the linear Fe-N-O configuration to a side-on configuration [structure: see text]. The formation of the isomer is monoexponential with a characteristic time of tau = 300(20) fs and proceeds along a diabatic potential surface without occupation of further intermediate states. The side-on structure has a lifetime of 270(30) ns at T = 23 degrees C.

Using Metallo‐Supramolecular Block Copolymers for the Synthesis of Higher Order Nanostructured Assemblies

Many research groups have explored the properties and solution self-assembly of main chain metallo-supramolecular multiblock copolymers. Until recently, these metal complexes have been used to prepare mainly micelle type structures. However, the self-assembly of such copolymers has been exploited further to create more advanced architectures which utilize the reversible supramolecular linkage of their building blocks as a key component in their synthesis. Furthermore, the incorporation of multiple orthogonal interactions and stimuli responsive polymers into their design, enables more precise external control of their properties. This feature article discusses recent developments and provides an insight into their potential exploitation and development for the creation of novel, smart, and responsive nanostructures.