Detection and Characterization of Rapidly Equilibrating Glycosylation Reaction Intermediates Using Exchange NMR.

The stereoselective introduction of glycosidic bonds (glycosylation) is one of the main challenges in the chemical synthesis of carbohydrates. Glycosylation reaction mechanisms are difficult to control because, in many cases, the exact reactive species driving product formation cannot be detected and the product outcome cannot be explained by the primary reaction intermediate observed. In these cases, reactions are expected to take place via other low-abundance reaction intermediates that are in rapid equilibrium with the primary reaction intermediate via a Curtin-Hammett scenario. Despite this principle being well-known in organic synthesis, mechanistic studies investigating this model in glycosylation reactions are complicated by the challenge of detecting the extremely short-lived reactive species responsible for product formation. Herein, we report the utilization of the chemical equilibrium between low-abundance reaction intermediates and the stable, readily observed α-glycosyl triflate intermediate in order to infer the structure of the former species by employing exchange NMR. Using this technique, we enabled the detection of reaction intermediates such as ß-glycosyl triflates and glycosyl dioxanium ions. This demonstrates the power of exchange NMR to unravel reaction mechanisms as we aim to build a catalog of kinetic parameters, allowing for the understanding and eventual prediction of glycosylation reactions.

Readily Accessible Strained Difunctionalized trans-Cyclooctenes with Fast Click and Release Capabilities.

The click reaction between a functionalized trans-cyclooctene (TCO) and a tetrazine (Tz) is a compelling method for bioorthogonal conjugation in combination with payload releasing capabilities. However, the synthesis of difunctionalized TCOs remains challenging. As a result, these compounds are poorly accessible, which impedes the development of novel applications. In this work, the scalable and accessible synthesis of a new bioorthogonal difunctionalized TCO is reported in only four single selective high yielding steps starting from commercially available compounds. The TCO-Tz click reaction was assessed and revealed excellent kinetic rates and subsequently payload release was shown with various functionalized derivatives. Tetrazine triggered release of carbonate and carbamate payloads was demonstrated up to 100 % release efficiency and local drug release was shown in a cellular toxicity study which revealed a >20-fold increase in cytotoxicity.

113Cd as a Probe in NMR Studies of Allosteric Host-Guest-Ligand Complexes of Porphyrin Cage Compounds.

Cadmium porphyrin cage compounds Cd1 and 113 Cd1 have been synthesized from the free base porphyrin cage derivative H21 and Cd(OAc)2 ⋅ 2 H2O or 113Cd(OAc)2 ⋅ 2 H2O, respectively. The compounds form allosteric complexes with the positively charged guests N,N'-dimethylimidazolium hexafluorophosphate (DMI) and N,N'-dimethylviologen dihexafluorophosphate (Me2V), which bind in the cavity of the cage, and tbupy, which coordinates as an axial ligand to the outside of the cage. In the presence of tbupy, the binding of DMI in Cd1 is enhanced by a factor of ∼31, while the presence of DMI or Me2V in the cavity of Cd1 enhances the binding of tbupy by factors of 55 and 85, respectively. The X-ray structures of the coordination complexes of Cd1 with acetone, acetonitrile, and pyridine, the host-guest complex of Cd1 with a bound viologen guest, and the ternary allosteric complex of Cd1 with a bound DMI guest and a coordinated tbupy ligand, were solved. These structures revealed relocations of the cadmium center in and out of the porphyrin plane, depending on whether a guest or a ligand is present. 113Cd NMR could be employed as a tool to quantify the binding of guests and ligands to 113 Cd1. 1D EXSY experiments on the ternary allosteric system Cd1-tbupy-Me2V revealed that the coordination of tbupy significantly slowed down the dissociation of the Me2V guest. Eyring plots of the dissociation process revealed that this kinetic allosteric effect is entropic in nature.

Characterization of Mannosyl Dioxanium Ions in Solution Using Chemical Exchange Saturation Transfer NMR Spectroscopy.

The stereoselective introduction of the glycosidic bond remains one of the main challenges in carbohydrate synthesis. Characterizing the reactive intermediates of this reaction is key to develop stereoselective glycosylation reactions. Herein we report the characterization of low-populated, rapidly equilibrating mannosyl dioxanium ions that arise from participation of a C-3 acyl group using chemical exchange saturation transfer (CEST) NMR spectroscopy. Dioxanium ion structure and equilibration kinetics were measured under relevant glycosylation conditions and highly α-selective couplings were observed suggesting glycosylation took place via this elusive intermediate.

Amide-derived lysine analogues as substrates and inhibitors of histone lysine methyltransferases and acetyltransferases.

Histone lysine methyltransferases and acetyltransferases are two classes of epigenetic enzymes that play pivotal roles in human gene regulation. Although they both recognise and posttranslationally modify lysine residues in histone proteins, their difference in histone peptide-based substrates and inhibitors remains to be firmly established. Here, we have synthesised lysine mimics that posses an amide bond linker in the side chain, incorporated them into histone H3 tail peptides, and examined synthetic histone peptides as substrates and inhibitors for human lysine methyltransferases and acetyltransferases. This work demonstrates that histone lysine methyltransferases G9a and GLP do catalyse methylation of the most similar lysine mimic, whereas they typically do not tolerate more sterically demanding side chains. In contrast, histone lysine acetyltransferases GCN5 and PCAF do not catalyse acetylation of the same panel of lysine analogues. Our results also identify potent H3-based inhibitors of GLP methyltransferase, providing a basis for development of peptidomimetics for targeting KMT enzymes.

Host-Guest Exchange of Viologen Guests in Porphyrin Cage Compounds as Studied by Selective Exchange Spectroscopy (1D EXSY) NMR.

Dynamics in complexes of porphyrin cage compounds and viologen-derived guest molecules are investigated by selective exchange NMR spectroscopy (1D EXSY). Exchange rates were found to be independent of excess guest concentration, revealing a dissociative exchange mechanism, which is accompanied by negative activation entropies, indicating significant reorganization of the host-guest complex during dissociation. Nonsymmetric viologen guests with bulky head groups had more unidirectional binding and slower exchange rates than guests with less-bulky head groups. Thermodynamic and kinetic studies revealed that the exchange process is primarily driven by the thermodynamics of binding and that guest binding can be influenced by introducing steric and electronic groups on the host . Exchange studies with guests bearing a polymer chain revealed that both slippage and full dissociation takes place and the rate constants for both processes were determined. The slippage rate constant revealed that for smaller guests exchange takes place nearly exclusively under thermodynamic control.

C-2 auxiliaries for stereoselective glycosylation based on common additive functional groups.

The stereoselective introduction of the glycosidic bond is one of the main challenges in chemical oligosaccharide synthesis. Stereoselective glycosylation can be achieved using neighbouring group participation of a C-2 auxiliary or using additives, for example. Both methods aim to generate a defined reactive intermediate that reacts in a stereoselective manner with alcohol nucleophiles. This inspired us to develop new C-2 auxiliaries based on commonly used additive functionalities such as ethers, phosphine oxides and tertiary amides. Good 1,2-trans-selectivity was observed for the phosphine oxide and amide-based auxiliaries expanding the toolbox with new auxiliaries for stereoselective glycosylation reactions.

Ligand-Promoted Palladium-Catalyzed Aerobic Oxidation Reactions.

Palladium-catalyzed aerobic oxidation reactions have been the focus of industrial application and extensive research efforts for nearly 60 years. A significant transition occurred in this field approximately 20 years ago, with the introduction of catalysts supported by ancillary ligands. The ligands play crucial roles in the reactions, including promotion of direct oxidation of palladium(0) by O2, bypassing the typical requirement for Cu salts or related redox cocatalysts to facilitate oxidation of the reduced Pd catalyst; facilitation of key bond-breaking and bond-forming steps during substrate oxidation; and modulation of chemo-, regio-, or stereoselectivity of a reaction. The use of ligands has contributed to significant expansion of the scope of accessible aerobic oxidation reactions. Increased understanding of the role of ancillary ligands should promote the development of new synthetic transformations, enable improved control over the reaction selectivity, and improve catalyst activity and stability. This review surveys the different ligands that have been used to support palladium-catalyzed aerobic oxidation reactions and, where possible, describes mechanistic insights into the role played by the ancillary ligand.

Double Porphyrin Cage Compounds.

The synthesis and characterization of double porphyrin cage compounds are described. They consist of two porphyrins that are each attached to a diphenylglycoluril-based clip molecule via four ethyleneoxy spacers, and are linked together by a single alkyl chain using "click"-chemistry. Following a newly developed multistep synthesis procedure we report three of these double porphyrin cages, linked by spacers of different lengths, i.e. 3, 5, and 11 carbon atoms. The structures of the double porphyrin cages were fully characterized by NMR, which revealed that they consist of mixtures of two diastereoisomers. Their zinc derivatives are capable of forming sandwich-like complexes with the ditopic ligand 1,4-diazabicyclo[2,2,2]octane (dabco).

Multicore Liquid Perfluorocarbon-Loaded Multimodal Nanoparticles for Stable Ultrasound and 19F MRI Applied to In Vivo Cell Tracking.

Ultrasound is the most commonly used clinical imaging modality. However, in applications requiring cell-labeling, the large size and short active lifetime of ultrasound contrast agents limit their longitudinal use. Here, 100 nm radius, clinically applicable, polymeric nanoparticles containing a liquid perfluorocarbon, which enhance ultrasound contrast during repeated ultrasound imaging over the course of at least 48 h, are described. The perfluorocarbon enables monitoring the nanoparticles with quantitative 19F magnetic resonance imaging, making these particles effective multimodal imaging agents. Unlike typical core-shell perfluorocarbon-based ultrasound contrast agents, these nanoparticles have an atypical fractal internal structure. The nonvaporizing highly hydrophobic perfluorocarbon forms multiple cores within the polymeric matrix and is, surprisingly, hydrated with water, as determined from small-angle neutron scattering and nuclear magnetic resonance spectroscopy. Finally, the nanoparticles are used to image therapeutic dendritic cells with ultrasound in vivo, as well as with 19F MRI and fluorescence imaging, demonstrating their potential for long-term in vivo multimodal imaging.

Effect of Chirality on the Binding of Viologen Guests in Porphyrin Macrocycles.

As part of a project aimed at the development of chiral processive catalysts that can write information on a polymer chain we describe the synthesis of two optically active porphyrin macrocycles, which are prepared in 3 steps from an achiral precursor compound. Fluorescence and 1H-NMR studies show that one of the macrocycles displays selectivity in the binding of chiral viologen guest molecules.

Biodegradable, Drug-Loaded Nanovectors via Direct Hydration as a New Platform for Cancer Therapeutics.

The stabilization and transport of low-solubility drugs, by encapsulation in nanoscopic delivery vectors (nanovectors), is a key paradigm in nanomedicine. However, the problems of carrier toxicity, specificity, and producibility create a bottleneck in the development of new nanomedical technologies. Copolymeric nanoparticles are an excellent platform for nanovector engineering due to their structural versatility; however, conventional fabrication processes rely upon harmful chemicals that necessitate purification. In engineering a more robust (copolymeric) nanovector platform, it is necessary to reconsider the entire process from copolymer synthesis through self-assembly and functionalization. To this end, a process is developed whereby biodegradable copolymers of poly(ethylene glycol)-block-poly(trimethylene carbonate), synthesized via organocatalyzed ring-opening polymerization, undergo assembly into highly uniform, drug-loaded micelles without the use of harmful solvents or the need for purification. The direct hydration methodology, employing oligo(ethylene glycol) as a nontoxic dispersant, facilitates rapid preparation of pristine, drug-loaded nanovectors that require no further processing. This method is robust, fast, and scalable. Utilizing parthenolide, an exciting candidate for treatment of acute lymphoblastic leukemia (ALL), discrete nanovectors are generated that show strikingly low carrier toxicity and high levels of specific therapeutic efficacy against primary ALL cells (as compared to normal hematopoietic cells).

Lewis Acid Enabled Copper-Catalyzed Asymmetric Synthesis of Chiral ß-Substituted Amides.

Here we report that readily available silyl- and boron-based Lewis acids in combination with chiral copper catalysts are able to overcome the reactivity issues of unactivated enamides, known as the least reactive carboxylic acid derivatives, toward alkylation with organomagnesium reagents. Allowing unequaled chemo-reactivity and stereocontrol in catalytic asymmetric conjugate addition to enamides, the method is distinguished by its unprecedented reaction scope, allowing even the most challenging and synthetically important methylations to be accomplished with good yields and excellent enantioselectivities. This catalytic protocol tolerates a broad temperature range (-78 °C to ambient) and scale up (10 g), while the chiral catalyst can be reused without affecting overall efficiency. Mechanistic studies revealed the fate of the Lewis acid in each elementary step of the copper-catalyzed conjugate addition of Grignard reagents to enamides, allowing us to identify the most likely catalytic cycle of the reaction.

Redox-Sensitive Stomatocyte Nanomotors: Destruction and Drug Release in the Presence of Glutathione.

The development of artificial nanomotor systems that are stimuli-responsive is still posing many challenges. Herein, we demonstrate the self-assembly of a redox-responsive stomatocyte nanomotor system, which can be used for triggered drug release under biological reducing conditions. The redox sensitivity was introduced by incorporating a disulfide bridge between the hydrophilic poly(ethylene glycol) block and the hydrophobic polystyrene block. When incubated with the endogenous reducing agent glutathione at a concentration comparable to that within cells, the external PEG shells of these stimuli-responsive nanomotors are cleaved. The specific bowl-shaped stomatocytes aggregate after the treatment with glutathione, leading to the loss of motion and triggered drug release. These novel redox-responsive nanomotors can not only be used for remote transport but also for drug delivery, which is promising for future biomedical applications.

Structure and Isotope Effects of the ß-H Agostic (α-Diimine)Nickel Cation as a Polymerization Intermediate.

Single-crystal X-ray characterization of cationic (α-diimine)Ni-ethyl and isopropyl ß-agostic complexes, which are key intermediates in olefin polymerization and oligomerization, are presented. The sharp Ni-Cα -Cß angles (75.0(3)° and 74.57(18)°) and short Cα -Cß distances (1.468(7) and 1.487(5) Å) provide unambiguous evidence for a ß-agostic interaction. An inverse equilibrium isotope effect (EIE) for ligand coordination upon cleavage of the agostic bond highlights the weaker bond strength of Ni-H relative to the C-H bond. An Eyring plot for ß-hydride elimination-olefin rotation-reinsertion is constructed from variable-temperature NMR spectra with 13 C-labeled agostic complexes. The enthalpy of activation (ΔH≠ ) for ß-H elimination is 13.2 kcal mol-1 . These results offer important mechanistic insight into two critical steps in polymerization: ligand association upon cleavage of the ß-agostic bonds and chain-migration via ß-H elimination.

Structurally Diverse Diazafluorene-Ligated Palladium(II) Complexes and Their Implications for Aerobic Oxidation Reactions.

4,5-Diazafluoren-9-one (DAF) has been identified as a highly effective ligand in a number of Pd-catalyzed oxidation reactions, but the mechanistic basis for its utility has not been elucidated. Here, we present the complex coordination chemistry of DAF and palladium(II) carboxylate salts. Multiple complexes among an equilibrating mixture of species have been characterized by (1)H and (15)N NMR spectroscopy and X-ray crystallography. These complexes include monomeric and dimeric Pd(II) species, with monodentate (κ(1)), bidentate (κ(2)), and bridging (µ:κ(1):κ(1)) DAF coordination modes. Titration studies of DAF and Pd(OAc)2 reveal the formation of two dimeric DAF/Pd(OAc)2 complexes at low [DAF] and four monomeric species at higher [DAF]. The dimeric complexes feature two bridging acetate ligands together with either a bridging or nonbridging (κ(1)) DAF ligand coordinated to each Pd(II) center. The monomeric structures consist of three isomeric Pd(κ(1)-DAF)2(OAc)2 complexes, together with Pd(κ(2)-DAF)(OAc)2 in which the DAF exhibits a traditional bidentate coordination mode. Replacing DAF with the structurally related, but more-electron-rich derivative 9,9-dimethyl-4,5-diazafluorene (Me2DAF) simplifies the equilibrium mixture to two complexes: a dimeric species in which the Me2DAF bridges the two Pd centers and a monomeric species with a traditional κ(2)-Me2DAF coordination mode. The use of DAF in combination with other carboxylate ligands (CF3CO2(-) or tBuCO2(-)) also results in a simplified collection of equilibrating Pd(II)-DAF complexes. Collectively, the results highlight the ability of DAF to equilibrate rapidly among multiple coordination modes, and provide valuable insights into the utility of DAF as a ligand in Pd-catalyzed oxidation reactions.

Multicolor Photoluminescence Including White-Light Emission by a Single Host-Guest Complex.

Achieving multicolor photoluminescence, especially white-light emission, under mild conditions based on a single fluorescent compound is a great challenge. Herein, we report a novel colorful-emission host-guest complex BPCY, which is composed of a two-arm fluorescent guest molecule (BPC) and γ-cyclodextrin (γ-CD) as the host molecule. BPC bears a unique asymmetrical donor-acceptor-donor (D1-A+∼D2)-type structure, where D1, A+, and D2 stand for the binaphthol electron donor, pyridinium electron acceptor, and coumarin electron donor, respectively. The luminescence property of BPC shows dual-sensitivity, i.e., toward the excitation wavelength and the cyclodextrin host molecule. Under certain conditions, the complex shows three different emission wavelengths, allowing the realization of multicolor photoluminescence, including red (R), green (G), and blue (B) as well as various intermediate colors by orthogonally modulating these two stimuli. In this way, nearly pure white-light emission with CIE coordinates (0.33, 0.34) could be generated. A combination of structural, spectroscopic, and computational simulation studies revealed the occurrence of synergetic mechanistic processes for the stimuli-responsive multicolor luminescence of the BPCY complex, namely, host-enhanced intramolecular charge-transfer (ICT) and host-induced restriction of intramolecular rotation (RIR). This new supramolecular complex with superior multicolor emission abilities may find wide applications in the fields of information processing and display media. Furthermore, the molecular design rationale presented here may provide a new design strategy for the development of high performance optical materials using a single supramolecular platform.

Kinetic and thermodynamic characterization of C-N bond rotation by N-methylacetohydroxamic acid in aqueous media.

Hydroxamic acids (HAs) perform tasks in medicine and industry that require bidentate metal binding. The two favored conformations of HAs are related by rotation around the C(=O)-N bond. The conformations are unequal in stability. Recently, we reported that the most stable conformation of a small secondary HA in water places the oxygen atoms anti to one another. The barrier to C-N bond rotation may therefore modulate metal binding by secondary HAs in aqueous media. We have now determined the activation barrier to C-N rotation from major to minor conformation of a small secondary HA in D2O to be 67.3 kJ/mol. The HA rotational barrier scales with solvent polarity, as is observed in amides, although the HA barrier is less than that of a comparable tertiary amide in aqueous solution. Successful design of new secondary HAs to perform specific tasks requires solid understanding of rules governing HA structural behavior. Results from this work provide a more complete foundation for HA design efforts.

Stereoselective ß-Mannosylation by Neighboring-Group Participation.

The stereoselective synthesis of glycosidic bonds is the main challenge of oligosaccharide synthesis. Neighboring-group participation (NGP) of C2 acyl substituents can be used to provide 1,2-trans-glycosides. Recently, the application of NGP has been extended to the preparation of 1,2-cis-glycosides with the advent of C2 chiral auxiliaries. However, this methodology has been strictly limited to the synthesis of 1,2-cis-gluco-type sugars. Reported herein is the design and synthesis of novel mannosyl donors which provide 1,2-cis-mannosides by NGP of thioether auxiliaries. A key element in the design is the use of (1) C4 locked mannuronic acid lactones to enable NGP of the C2 auxiliary. In addition to C2 participation a new mode of remote participation of the C4 benzyl group was identified and provides 1,2-cis-mannosides.

Water-polysaccharide interactions in the primary cell wall of Arabidopsis thaliana from polarization transfer solid-state NMR.

Polysaccharide-rich plant cell walls are hydrated under functional conditions, but the molecular interactions between water and polysaccharides in the wall have not been investigated. In this work, we employ polarization transfer solid-state NMR techniques to study the hydration of primary-wall polysaccharides of the model plant, Arabidopsis thaliana. By transferring water (1)H polarization to polysaccharides through distance- and mobility-dependent (1)H-(1)H dipolar couplings and detecting it through polysaccharide (13)C signals, we obtain information about water proximity to cellulose, hemicellulose, and pectins as well as water mobility. Both intact and partially extracted cell wall samples are studied. Our results show that water-pectin polarization transfer is much faster than water-cellulose polarization transfer in all samples, but the extent of extraction has a profound impact on the water-polysaccharide spin diffusion. Removal of calcium ions and the consequent extraction of homogalacturonan (HG) significantly slowed down spin diffusion, while further extraction of matrix polysaccharides restored the spin diffusion rate. These trends are observed in cell walls with similar water content, thus they reflect inherent differences in the mobility and spatial distribution of water. Combined with quantitative analysis of the polysaccharide contents, our results indicate that calcium ions and HG gelation increase the amount of bound water, which facilitates spin diffusion, while calcium removal disrupts the gel and gives rise to highly dynamic water, which slows down spin diffusion. The recovery of spin diffusion rates after more extensive extraction is attributed to increased water-exposed surface areas of the polysaccharides. Water-pectin spin diffusion precedes water-cellulose spin diffusion, lending support to the single-network model of plant primary walls in which a substantial fraction of the cellulose surface is surrounded by pectins.