Supramolecular DNA-based catalysis in organic solvents.

The distinct folding accompanied by its polymorphic character renders DNA G-quadruplexes promising biomolecular building blocks to construct novel DNA-based and supramolecular assemblies. However, the highly polar nature of DNA limits the use of G-quadruplexes to water as a solvent. In addition, the archetypical G-quadruplex fold needs to be stabilized by metal-cations, which is usually a potassium ion. Here, we show that a noncovalent PEGylation process enabled by electrostatic interactions allows the first metal-free G-quadruplexes in organic solvents. Strikingly, incorporation of an iron-containing porphyrin renders the self-assembled metal-free G-quadruplex catalytically active in organic solvents. Hence, these "supraG4zymes" enable DNA-based catalysis in organic media. The results will allow the broad utilization of DNA G-quadruplexes in nonaqueous environments.

Parahydrogen-Induced Polarization of a Labeled, Cancer-Targeting DNA Aptamer.

Enhancing NMR signals of biomacromolecules by hyperpolarization offers exciting opportunities for diagnostic applications. However, their hyperpolarization via parahydrogen remains challenging as specific catalytic interactions are required, which are difficult to tune due to the large size of the biomolecule and its insolubility in organic solvents. Herein, we show the unprecedented hyperpolarization of the cancer-targeting DNA aptamer AS1411. By screening different molecular motifs for an unsaturated label in nucleosides and in DNA oligomers, we were able to identify structural prerequisites for the hyperpolarization of AS1411. Finally, adjusting the polarity of AS1411 by complexing the DNA backbone with amino polyethylene glycol chains allowed the hydrogenation of the label with parahydrogen while the DNA structure remains stable to maintain its biological function. Our results are expected to advance hyperpolarized molecular imaging technology for disease detection in the future.

Mapping Cell Viability Quantitatively and Independently From Cell Density in 3D Gels Noninvasively.

OBJECTIVE: In biomanufacturing there is a need for quantitative methods to map cell viability and density inside 3D bioreactors to assess health and proliferation over time. Recently, noninvasive MRI readouts of cell density have been achieved. However, the ratio of live to dead cells was not varied. Herein we present an approach for measuring the viability of cells embedded in a hydrogel independently from cell density to map cell number and health. METHODS: Independent quantification of cell viability and density was achieved by calibrating the 1H magnetization transfer- (MT) and diffusion-weighted NMR signals to samples of known cell density and viability using a multivariate approach. Maps of cell viability and density were generated by weighting NMR images by these parameters post-calibration. RESULTS: Using this method, the limits of detection (LODs) of total cell density and viable cell density were found to be 3.88 ×108 cells · mL -1· Hz -1/2 and 2.36 ×109 viable cells · mL -1· Hz -1/2 respectively. CONCLUSION: This mapping technique provides a noninvasive means of visualizing cell viability and number density within optically opaque bioreactors. SIGNIFICANCE: We anticipate that such nondestructive readouts will provide valuable feedback for monitoring and controlling cell populations in bioreactors.

Automated tangential-flow diafiltration device.

Tangential flow filtration (TFF) is a chemical unit operation used to purify and concentrate liquid suspensions of colloids, proteins, or cells. The solution flows tangentially across a membrane, such that a selective part of the fluid permeates the membrane while the filtrated matter is retained, increasing its concentration. TFF is a mild mechanical purification method that does not interact chemically with the filtrate. It is applied in sensitive separation tasks in protein chemistry, microbiology, or immunology. It is a fast alternative for dialysis applications, also applicable in the field of colloid purification. However, the costs of automated lab-scale devices (30,000 €) and the consumable membrane modules (100-600 €) make TFF currently hardly accessible for lab-scale polymer researchers. Therefore, we built a low-cost TFF system (2400 €) partly automated by an Arduino microcontroller and optimized for diafiltration buffer exchange and concentration processes in soft matter colloid research. We use medical hemodialysis membrane modules that only cost a share (20-50 €) of alternative TFF modules, and we demonstrate the functionality of the system for an exemplary colloidal microgel purification process.

Stimuli-Responsive Zwitterionic Core-Shell Microgels for Antifouling Surface Coatings.

Fouling on filtration membranes is induced by the nonspecific interactions between the membrane surface and the foulants, and effectively hinders their efficient use in various applications. Here, we established a facile method for the coating of membrane surface with a dual stimuli-responsive antifouling microgel system enriched with a high polyzwitterion content. Different poly(sulfobetaine) (PSB) zwitterionic polymers with defined molecular weights and narrow dispersities were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization and integrated onto poly(N-vinylcaprolactam) (PVCL) microgels via a controlled dosage of a cross-linker, adapting a precipitation polymerization technique to obtain a core-shell microstructure. Increasing the PSB macro-RAFT concentration resulted in a shift of both upper critical solution temperature and lower critical solution temperature toward higher temperatures. Cryogenic transmission electron microscopy at different temperatures suggested the formation of a core-shell morphology with a PVCL-rich core and a PSB-rich shell. On the other hand, the significant variations of different characteristic proton signals and reversible phase transitions of the microgel constituents were confirmed by temperature-dependent 1H NMR studies. Utilizing a quartz crystal microbalance with dissipation monitoring, we have been able to observe and quantitatively describe the antipolyelectrolyte behavior of the zwitterionic microgels. The oscillation frequency of the sensor proved to change reversibly according to the variations of the NaCl concentration, showing, in fact, the effect of the interaction between the salt and the opposite charges present in the microgel deposited on the sensor. Poly(ethersulfone) membranes, chosen as the model surface, when functionalized with zwitterionic microgel coatings, displayed protein-repelling property, stimulated by different transition temperatures, and showed even better performances at increasing NaCl concentration. These kinds of stimuli-responsive zwitterionic microgel can act as temperature-triggered drug delivery systems and as potential coating materials to prevent bioadhesion and biofouling as well.

Selective Hydrogenation and Hydrodeoxygenation of Aromatic Ketones to Cyclohexane Derivatives Using a Rh@SILP Catalyst.

Rhodium nanoparticles immobilized on an acid-free triphenylphosphonium-based supported ionic liquid phase (Rh@SILP(Ph3 -P-NTf2 )) enabled the selective hydrogenation and hydrodeoxygenation of aromatic ketones. The flexible molecular approach used to assemble the individual catalyst components (SiO2 , ionic liquid, nanoparticles) led to outstanding catalytic properties. In particular, intimate contact between the nanoparticles and the phosphonium ionic liquid is required for the deoxygenation reactivity. The Rh@SILP(Ph3 -P-NTf2 ) catalyst was active for the hydrodeoxygenation of benzylic ketones under mild conditions, and the product distribution for non-benzylic ketones was controlled with high selectivity between the hydrogenated (alcohol) and hydrodeoxygenated (alkane) products by adjusting the reaction temperature. The versatile Rh@SILP(Ph3 -P-NTf2 ) catalyst opens the way to the production of a wide range of high-value cyclohexane derivatives by the hydrogenation and/or hydrodeoxygenation of Friedel-Crafts acylation products and lignin-derived aromatic ketones.

Production of highly concentrated and hyperpolarized metabolites within seconds in high and low magnetic fields.

Hyperpolarized metabolites are very attractive contrast agents for in vivo magnetic resonance imaging studies enabling early diagnosis of cancer, for example. Real-time production of concentrated solutions of metabolites is a desired goal that will enable new applications such as the continuous investigation of metabolic changes. To this end, we are introducing two NMR experiments that allow us to deliver high levels of polarization at high concentrations (50 mM) of an acetate precursor (55% 13C polarization) and acetate (17% 13C polarization) utilizing 83% para-state enriched hydrogen within seconds at high magnetic field (7 T). Furthermore, we have translated these experiments to a portable low-field spectrometer with a permanent magnet operating at 1 T. The presented developments pave the way for a rapid and affordable production of hyperpolarized metabolites that can be implemented in e.g. metabolomics labs and for medical diagnosis.

Heterogeneity of Network Structures and Water Dynamics in κ-Carrageenan Gels Probed by Nanoparticle Diffusometry.

A set of functionalized nanoparticles (PEGylated dendrimers, d = 2.8-11 nm) was used to probe the structural heterogeneity in Na+/K+ induced κ-carrageenan gels. The self-diffusion behavior of these nanoparticles as observed by 1H pulsed-field gradient NMR, fluorescence recovery after photobleaching, and raster image correlation spectroscopy revealed a fast and a slow component, pointing toward microstructural heterogeneity in the gel network. The self-diffusion behavior of the faster nanoparticles could be modeled with obstruction by a coarse network (average mesh size <100 nm), while the slower-diffusing nanoparticles are trapped in a dense network (lower mesh size limit of 4.6 nm). Overhauser dynamic nuclear polarization-enhanced NMR relaxometry revealed a reduced local solvent water diffusivity near 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO)-labeled nanoparticles trapped in the dense network, showing that heterogeneity in the physical network is also reflected in heterogeneous self-diffusivity of water. The observed heterogeneity in mesh sizes and in water self-diffusivity is of interest for understanding and modeling of transport through and release of solutes from heterogeneous biopolymer gels.

Non-Pairwise Interactions in Parahydrogen Experiments: Nuclear Exchange of Single Protons Enables Bulk Water Hyperpolarization.

Hyperpolarization with parahydrogen (p-H2 ) is a fast developing field in NMR, which enables overcoming the inherent low sensitivity of this important technique. The hyperpolarization of solvents, particularly of water, offers a wide range of applications for structural investigations of macromolecules and biomedical imaging. Until lately, only organic solvents could be polarized by means of parahydrogen via coherent redistribution of polarization (SABRE mechanism). In this study, we investigate in detail the mechanism of the recently reported bulk water hyperpolarization with a combination of theoretical and experimental methods, finally showing a chemical exchange pathway of single protons as basis for the enhancement. The prerequisites for preserving hyperpolarization upon separation of the two hydrogen atoms of p-H2 are demonstrated by theoretical examinations of the boundary conditions for the hyperpolarization experiments in accordance with the OneH-PHIP theory. These findings yielded the proposal of the novel NEPTUN mechanism (Nuclear Exchange Polarization by Transposing Unattached Nuclei) as the non-hydrogenative equivalent to the established OneH-PHIP and thus the missing link in parahydrogen hyperpolarization theory.

Hyperpolarizing Water with Parahydrogen.

Studies of water-based systems are of fundamental interest for nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) as water is the most abundant and important medium for global living. Hence, increasing the polarization of water and dissolved compounds is particularly attractive for biomedical applications such as investigations of intermolecular interactions and metabolite structures as well as for imaging purposes. In this work, we show a new approach based on para enriched hydrogen (p-H2 ) that enables the hyperpolarization of bulk water if a suitable catalytic system is employed. The results indicate that the polarization is transferred by a new exchange mechanism.

Direct Hyperpolarization of Nitrogen-15 in Aqueous Media with Parahydrogen in Reversible Exchange.

Signal amplification by reversible exchange (SABRE) is an inexpensive, fast, and even continuous hyperpolarization technique that uses para-hydrogen as hyperpolarization source. However, current SABRE faces a number of stumbling blocks for translation to biochemical and clinical settings. Difficulties include inefficient polarization in water, relatively short-lived 1H-polarization, and relatively limited substrate scope. Here we use a water-soluble polarization transfer catalyst to hyperpolarize nitrogen-15 in a variety of molecules with SABRE-SHEATH (SABRE in shield enables alignment transfer to heteronuclei). This strategy works in pure H2O or D2O solutions, on substrates that could not be hyperpolarized in traditional 1H-SABRE experiments, and we record 15N T1 relaxation times of up to 2 min.

A New Ir-NHC Catalyst for Signal Amplification by Reversible Exchange in D2 O.

NMR signal amplification by reversible exchange (SABRE) has been observed for pyridine, methyl nicotinate, N-methylnicotinamide, and nicotinamide in D2 O with the new catalyst [Ir(Cl)(IDEG)(COD)] (IDEG=1,3-bis(3,4,5-tris(diethyleneglycol)benzyl)imidazole-2-ylidene). During the activation and hyperpolarization steps, exclusively D2 O was used, resulting in the first fully biocompatible SABRE system. Hyperpolarized (1) H substrate signals were observed at 42.5 MHz upon pressurizing the solution with parahydrogen at close to the Earth's magnetic field, at concentrations yielding barely detectable thermal signals. Moreover, 42-, 26-, 22-, and 9-fold enhancements were observed for nicotinamide, pyridine, methyl nicotinate, and N-methylnicotinamide, respectively, in conventional 300 MHz studies. This research opens up new opportunities in a field in which SABRE has hitherto primarily been conducted in CD3 OD. This system uses simple hardware, leaves the substrate unaltered, and shows that SABRE is potentially suitable for clinical purposes.

Para-hydrogen induced polarization of amino acids, peptides and deuterium-hydrogen gas.

Signal Amplification by Reversible-Exchange (SABRE) is a method of hyperpolarizing substrates by polarization transfer from para-hydrogen without hydrogenation. Here, we demonstrate that this method can be applied to hyperpolarize small amounts of all proteinogenic amino acids and some chosen peptides down to the nanomole regime and can be detected in a single scan in low-magnetic fields down to 0.25 mT (10 kHz proton frequency). An outstanding feature is that depending on the chemical state of the used catalyst and the investigated amino acid or peptide, hyperpolarized hydrogen-deuterium gas is formed, which was detected with (1)H and (2)H NMR spectroscopy at low magnetic fields of B(0) = 3.9 mT (166 kHz proton frequency) and 3.2 mT (20 kHz deuterium frequency).

Selective drug trace detection with low-field NMR.

Advances with para-hydrogen induced polarization open up new fields of applications for portable low-field NMR. Here we report the possibility of tracing drugs down to the micromolar regime. We could selectively polarize nicotine quantities similar to those found in one cigarette. Also less than 1 mg of harmine, a drug used for treatment of Parkinson's disease, and morphine extracted from an opium solution were detectable after polarization with para-hydrogen in single-scan (1)H-experiments. Moreover, we demonstrate the possibility to selectively enhance and detect the (1)H-signal of drug molecules with PHIP in proton rich standard solutions that would otherwise mask the (1)H NMR signal of the drug.