Dipolar Chemical Shift Correlation Research Articles

Evaluation of chitosan crystallinity: A high-resolution solid-state NMR spectroscopy approach

We propose a novel approach relied on high-resolution solid-state 13C NMR spectroscopy to quantify the crystallinity index of chitosans (Ch) prepared with variable average degrees of acetylation (DA¯) from 5% to 60 % and average weight molecular weight (M¯w) ranged in 0.15 × 106 g mol−1–1.2 × 106 g mol−1. The Dipolar Chemical Shift Correlation (DIPSHIFT) curve of the C(6)OH segment revealed increased mobility dynamic, which induced different distribution from trans-to-gauche conformations in relation to C(4). Indeed, 1H-13C Heteronuclear Correlation (2D HETCOR) showed that distinguished C4 chemical shifts correlates with the same aliphatic protons. The short-range ordering can be assigned to C4/C6 signals on 13C CPMAS and, for our case, the deconvolution procedure between disordered and ordered phases revealed increasing crystallinity with DA¯, as confirmed by SVD multivariate analysis. This work extended the knowledge regarding the use of 13C CPMAS technique to predict the crystallinity of chitosans without the use of amorphous standards.

MAS NMR Investigation of Molecular Order in an Ionic Liquid Crystal.

The structure and molecular order in the thermotropic ionic liquid crystal (ILC), [choline][geranate(H)octanoate], an analogue of Choline And GEranate (CAGE), which has potential for use as a broad-spectrum antimicrobial and transdermal and oral delivery agent, were investigated by magic-angle spinning (MAS) nuclear magnetic resonance (NMR), polarizing optical microscopy, small-angle X-ray scattering (SAXS), and mass spectrometry. Mass spectrometry and the 1H NMR chemical shift reveal that CAGE-oct is a dynamic system, with metathesis (the exchange of interacting ions) and hydrogen exchange occurring between hydrogen-bonded/ionic complexes such as [(choline)(geranate)(H)(octanoate)], [(choline)(octanoate)2(H)], and [(choline)(geranate)2(H)]. These clusters, which are shown by mass spectrometry to be significantly more stable than expected for typical electrostatic ion clusters, involve hydrogen bonding between the carboxylic acid, carboxylate, and hydroxyl groups, with rapid hydrogen bond breaking and re-formation observed to average the 1H chemical shifts. The formation of a partial bilayer liquid crystal (LC) phase was identified by SAXS and polarizing optical microscopy at temperatures below ∼293 K. The occurrence of this transition close to room temperature could be utilized as a potential temperature-induced “switch” of the anisotropic properties for particular applications. The presence of an isotropic component of approximately 23% was observed to coexist with the LC phase, as detected by polarizing optical microscopy and quantified by both 1H–13C dipolar-chemical shift correlation (DIPSHIFT) and 1H double-quantum (DQ) MAS NMR experiments. At temperatures above the LC-to-isotropic transition, intermediate-range order (clustering of polar and nonpolar domains), a feature of many ILs, persists. Site-specific order parameters for the LC phase of CAGE-oct were obtained from the MAS NMR measurement of the partially averaged 13C–1H dipolar couplings (DCH) by cross-polarization (CP) build-up curves and DIPSHIFT experiments, and 1H–1H dipolar couplings (DHH) by double-quantum (DQ) build-up curves. The corresponding order parameters, SCH and SHH, are in the range 0–0.2 and are lower compared to those for smectic (i.e., layered) phases of conventional nonionic liquid crystals, resembling those of lamellar phases formed by lyotropic surfactant–solvent systems.

UV Pretreatment Impairs the Enzymatic Degradation of Polyethylene Terephthalate.

The biocatalytic degradation of polyethylene terephthalate (PET) emerged recently as a promising alternative plastic recycling method. However, limited activity of previously known enzymes against post-consumer PET materials still prevents the application on an industrial scale. In this study, the influence of ultraviolet (UV) irradiation as a potential pretreatment method for the enzymatic degradation of PET was investigated. Attenuated total reflection Fourier transform infrared (ATR-FTIR) and 1H solution nuclear magnetic resonance (NMR) analysis indicated a shortening of the polymer chains of UV-treated PET due to intra-chain scissions. The degradation of UV-treated PET films by a polyester hydrolase resulted in significantly lower weight losses compared to the untreated sample. We also examined site-specific and segmental chain dynamics over a time scale of sub-microseconds to seconds using centerband-only detection of exchange, rotating-frame spin-lattice relaxation (T1ρ), and dipolar chemical shift correlation experiments which revealed an overall increase in the chain rigidity of the UV-treated sample. The observed dynamic changes are most likely associated with the increased crystallinity of the surface, where a decreased accessibility for the enzyme-catalyzed hydrolysis was found. Moreover, our NMR study provided further knowledge on how polymer chain conformation and dynamics of PET can mechanistically influence the enzymatic degradation.

Solid-State NMR Approaches to Internal Dynamics of Proteins: From Picoseconds to Microseconds and Seconds

Solid-state nuclear magnetic resonance (NMR) spectroscopy has matured to the point that it is possible to determine the structure of proteins in immobilized states, such as within microcrystals or embedded in membranes. Currently, researchers continue to develop and apply NMR techniques that can deliver site-resolved dynamic information toward the goal of understanding protein function at the atomic scale. As a widely-used, natural approach, researchers have mostly measured longitudinal (T1) relaxation times, which, like in solution-state NMR, are sensitive to picosecond and nanosecond motions, and motionally averaged dipolar couplings, which provide an integral amplitude of all motions with a correlation time of up to a few microseconds. While overall Brownian tumbling in solution mostly precludes access to slower internal dynamics, dedicated solid-state NMR approaches are now emerging as powerful new options. In this Account, we give an overview of the classes of solid-state NMR experiments that have expanded the accessible range correlation times from microseconds to many milliseconds. The measurement of relaxation times in the rotating frame, T1ρ, now allows researchers to access the microsecond range. Using our recent theoretical work, researchers can now quantitatively analyze this data to distinguish relaxation due to chemical-shift anisotropy (CSA) from that due to dipole-dipole couplings. Off-resonance irradiation allows researchers to extend the frequency range of such experiments. We have built multidimensional analogues of T2-type or line shape experiments using variants of the dipolar-chemical shift correlation (DIPSHIFT) experiment that are particularly suited to extract intermediate time scale motions in the millisecond range. In addition, we have continuously improved variants of exchange experiments, mostly relying on the recoupling of anisotropic interactions to address ultraslow motions in the ms to s ranges. The NH dipolar coupling offers a useful probe of local dynamics, especially with proton-depleted samples that suppress the adverse effect of strong proton dipolar couplings. We demonstrate how these techniques have provided a concise picture of the internal dynamics in a popular model system, the SH3 domain of α-spectrin. T1-based methods have shown that large-amplitude bond orientation fluctuations in the picosecond range and slower 10 ns low-amplitude motions coexist in these structures. When we include T1ρ data, we observe that many residues undergo low amplitude motions slower than 100 ns. On the millisecond to second scale, mostly localized but potentially cooperative motions occur. Comparing different exchange experiments, we found that terminal NH2 groups in side chains can even undergo a combination of ultraslow large-angle two-site jumps accompanied by small-angle fluctuations that occur 10 times more quickly.

Correlations between Conjugation Length, Macromolecular Dynamics, and Photophysics of Phenylene-Vinylene/Aliphatic Multiblock Copolymers

This work reports a detailed spectroscopy study of a series of multiblock conjugated nonconjugated copolymers built by p-phenylene vinylene type units (PV) and octamethylene spacers, namely, poly(1,8-octanedioxy-2,6-dimethoxy-1,4-phenylene-1,2-ethenylene) (LaPPS18). The relative proportions of the PV and aliphatic segments were estimated on the basis of solid-state NMR and Raman spectroscopy. The overall structure was characterized by wide angle X-ray diffraction; (1)H wide-line dipolar chemical shift correlation (DIPSHIFT), and centerband-only detection of exchange (CODEX) NMR data, that together with glass transition temperatures allowed us to identify the groups involved in the molecular dynamics. These different structural properties were used to explain the photoluminescence properties in terms of peak position and spectral profile.

Intermediate motions as studied by solid-state separated local field NMR experiments

In this report, the application of a class of separated local field NMR experiments named dipolar chemical shift correlation (DIPSHIFT) for probing motions in the intermediate regime is discussed. Simple analytical procedures based on the Anderson-Weiss (AW) approximation are presented. In order to establish limits of validity of the AW based formulas, a comparison with spin dynamics simulations based on the solution of the stochastic Liouville-von-Neumann equation is presented. It is shown that at short evolution times (less than 30% of the rotor period), the AW based formulas are suitable for fitting the DIPSHIFT curves and extracting kinetic parameters even in the case of jumplike motions. However, full spin dynamics simulations provide a more reliable treatment and extend the frequency range of the molecular motions accessible by DIPSHIFT experiments. As an experimental test, molecular jumps of imidazol methyl sulfonate and trimethylsulfoxonium iodide, as well as the side-chain motions in the photoluminescent polymer poly[2-methoxy-5-(2'-ethylhexyloxy)-1,4-phenylenevinylene], were characterized. Possible extensions are also discussed.

Dipolar Chemical Shift Correlation Spectroscopy for Homonuclear Carbon Distance Measurements in Proteins in the Solid State: Application to Structure Determination and Refinement

High-resolution solid-state NMR spectroscopy has become a promising tool for protein structure determination. Here, we describe a new dipolar-chemical shift correlation experiment for the measurement of homonuclear 13C-13C distances in uniformly 13C,15N-labeled proteins and demonstrate its suitability for protein structure determination and refinement. The experiments were carried out on the beta1 immunoglobulin binding domain of protein G (GB1). Both intraresidue and interresidue distances between carbonyl atoms and atoms in the aliphatic side chains were collected using a three-dimensional chemical shift correlation spectroscopy experiment that uses homogeneously broadened rotational resonance recoupling for carbon mixing. A steady-state approximation for the polarization transfer function was employed in data analysis, and a total of 100 intramolecular distances were determined, all in the range 2.5-5.5 A. An additional 41 dipolar contacts were detected, but the corresponding distances could not be accurately quantified. Additional distance and torsional restraints were derived from the proton-driven spin diffusion measurements and from the chemical shift analysis, respectively. Using all these restraints, it was possible to refine the structure of GB1 to a root-mean square deviation of 0.8 A. The approach is of general applicability for peptides and small proteins and can be easily incorporated into a structure determination and refinement protocol.

Solid state NMR at high magic angle spinning frequencies: Dipolar chemical shift correlation with adiabatic inversion pulse based RF pulse schemes

The efficacy of hetero- and homonuclear dipolar recoupling employing tanh/tan adiabatic inversion pulse based RF pulse schemes has been examined at high magic angle spinning (MAS) frequencies via numerical simulations and experimental measurements. An approach for minimising the recoupling RF power level is presented, taking into consideration the spinning speed, the range of resonance offsets and H(1) inhomogeneities and the available RF field strength. This involves the tailoring of the frequency and amplitude modulation profiles of the inversion pulses. The applicability of tanh/tan pulse based dipolar recoupling schemes to spinning speed regimes where the performance with conventional rectangular pulses may not be satisfactory is demonstrated.

Photoluminescence and Relaxation Processes in MEH−PPV

A detailed description of the thermal relaxation processes in MEH−PPV is reported. Bulk methods such as DMTA were employed in conjunction with other techniques that probe molecular motions, such as fluorescence spectroscopy, thermal stimulated current, and 13C NMR. From the two main transitions observed (glass transition process at 340 K and β-relaxation between 200 and 220 K), it was demonstrated that the first is strongly correlated with the dissociation of a fluorescent emissive interchain complex and that the second relaxation involves movements of the lateral substituents of the polymer backbone and, more specifically, their CH2 groups. NMR dipolar chemical shift correlation experiments pointed an increasing gain in mobility through the side chain, the lateral carbons close to the aromatic ring being more rigid than those located more distant from the main polymer chain. A kinetic model involving the dissociation of interchains to re-form intrachain excitons was proposed to explain the profiles of the photoluminescence spectra at higher temperatures.

TEDOR with adiabatic inversion pulses: Resonance assignments of 13C/15N labelled RNAs

We have examined via numerical simulations the performance characteristics of different 15N RF pulse schemes employed in the transferred echo double resonance (TEDOR) experimental protocol for generating 13C-15N dipolar chemical shift correlation spectra of isotopically labelled biological systems at moderate MAS frequencies (omega(r) approximately 10 kHz). With an 15N field strength of approximately 30-35 kHz that is typically available in 5 mm triple resonance MAS NMR probes, it is shown that a robust TEDOR sequence with significant tolerance to experimental imperfections sa as H1 inhomogeneity and resonance offsets can be effectively implemented using adiabatic heteronuclear dipolar recoupling pulse schemes. TEDOR-based 15N-13C and 15N-13C-13C chemical shift correlation experiments were carried out for obtaining 13C and 15N resonance assignments of an RNA composed of 97 (CUG) repeats which has been implicated in the neuromuscular disease myotonic dystrophy.

Identification of NH...N hydrogen bonds by magic angle spinning solid state NMR in a double-stranded RNA associated with myotonic dystrophy.

RNA plays a central role in biological processes and exhibits a variety of secondary and tertiary structural features that are often stabilized via hydrogen bonds. The distance between the donor and acceptor nitrogen nuclei involved in NH...N hydrogen bonds in nucleic acid base pairs is typically in the range of 2.6-2.9 A. Here, we show for the first time that such spatial proximity between 15N nitrogen nuclei can be conveniently monitored via magic angle spinning solid state NMR on a uniformly 15N-labelled RNA. The presence of NH.N hydrogen bonds is reflected as cross-peaks between the donor and acceptor nitrogen nuclei in 2D 15N dipolar chemical shift correlation spectra. The RNA selected for this experimental study was a CUG repeat expansion implicated in the neuromuscular disease myotonic dystrophy. The results presented provide direct evidence that the CUG repeat expansion adopts a double-stranded conformation.