091
Session Assigned: W&Th P
Title: The Interaction of Methyl Bromide with Soil

Institute: Department of Chemistry, Colorado State University
Keywords: environment; pollutant; soil sterilization;
Abstract:
Methyl bromide is a widely used agricultural fumigant for soil disinfection.^[1] In considering the potential effects that methyl bromide may have on a soil to be disinfected, it is important to know the chemical behavior and fate of methyl bromide as a result of its use for soil treatment.^[2] A solid-state ¹³C NMR study of ¹³CH₃Br-treated soil samples shows that methylation of soil organic matter may be the major pathway for degradation of methyl bromide in soils. Adsorption of methyl bromide on a dried clay like Ca-montmorillonite or kaolinite does not contribute to the degradation of methyl bromide.

[1] P. Morse, C&EN, 76, 46-48, November 9, 1998.
[2] J. H. Shorter, C. E. Kolb, R. C. Harris, Nature, 377, 717-718, 1995.

092
Session Assigned: W&Th P
Title: Solid-State Deuterium MAS NMR Studies of TNT/Soil adsorption

Institute: Louisiana State University, Baton Rouge, LA, USA
Keywords:
Abstract:
TNT binding to soil components is studied by adding deuterated TNT(made by H/D exchange in supercritical D₂O) to soil and soil components with loading levels of 5 mg/g. At this loading, the number density of deuterons in the solid sample is low, yet deuterium MAS NMR shows features from methyl and aromatic ring sites and yields information about the dynamics of these sites. For example, d₃-TNT adsorbed to quartz sand shows methyl group rotation, indicating TNT/quartz bonding suffienciently strong to prevent aromatic ring motion. A soil sample shows two d₃-TNT components; one component is assigned to freely moving TNT while the other has restricted phenyl ring motion similar to TNT/clay samples.

093
Session Assigned: W&Th P
Title: Effects of restricted rotational diffusion on deuteron MAS NMR spectra

Institute: College of William and Mary
Keywords: deuteron MAS NMR
Abstract:
The effects of restricted rotational diffusion on deuteron magic angle spinning (MAS) NMR spectra are investigated. The approach is based on the Smoluchowski model that describes restricted rotational diffusion in an arbitrary ordering potential with an arbitrary diffusion tensor. It is shown that the Smoluchowski model gives a physically more reasonable description of molecular motion than the discrete Markov (jump) model. The models are shown to be consistent for high ordering potentials and (or) low temperatures provided the diffusion coefficient is sufficiently high. However, for low ordering potentials and (or) high temperatures the discrete Markov model is not a useful approximation. This is also the case for small diffusion coefficients independent of the ordering potential and the temperature. The formalism is based on finite difference solutions to the stochastic Liouville-von Neumann equation. This defines a linear homogeneous system of coupled parabolic partial differential equations which includes both first- and second-order spatial derivatives. Numerical solutions are very difficult to obtain and some useful finite difference methods are presented. The results are elaborated for deuteron MAS NMR spectroscopy and include effects of finite pulse width. The method is applied experimentally to investigate ammonium group reorientation in L-alanine. The orientational dependence of the ordering potential and the quadrupole parameters is determined from the Smoluchowski model. The activation energies are found to be temperature dependent. These effects have not previously been observed and give new information on the motion in L-alanine. The diffusion results are compared with the discrete Markov model and it is found that in this case the two models are consistent. The most important difference is that the Markov model does not map out the orientational dependence of the ordering potential and the quadrupole parameters. Another advantage of the rotational diffusion model is that it is physically more reasonable than the Markov model and that the parameters may be interpreted in terms of molecular properties.

094
Session Assigned: W&Th P
Title: High-resolution ¹⁴N NMR in polycrystalline solids

Institute: Department of Chemistry, University of Oklahoma
Keywords:
Abstract:
The ¹⁴N NMR spectra of solids are usually very broad due to the presence of large quadrupole coupling constants. However, even partial excitation of the whole spectrum can give valuable information. With magic angle spinning (MAS), the spectrum consists of a number of peaks, but normally the centerband cannot be readily distinguished from the spinning sidebands. Multiple-pulse methods of sideband elimination, such as TOSS and PASS, cannot be used for ¹⁴N because of its short spin-spin relaxation time. On the other hand, the sidebands can be eliminated by systematic data treatment. First, the signal-to-noise ratio (S/N) is enhanced by co-adding all the peaks in a MAS spectrum in a periodic way. Then, several spectra obtained at different spinning rates are added or multiplied together to identify the centerband. In the centerband region of the spectrum obtained from the addition method, the residual sidebands can be distinguished from the weak signals by the use of logical or digital filtering. Results obtained by using these methods to treat the spectra of two mixtures of KNO₃, Pb(NO₃)₂, and NH₄Cl are shown. The experimental requirements are not very stringent, the S/N ratio is good, and peaks covering a large range of chemical shifts can be readily observed with high-resolution.

095
Session Assigned: W&Th P
Title: Nitrogen-14 Overtone NMR Methods to Determine the Backbone Conformation of Peptides

Institute: The University of Michigan
Keywords:
Abstract:
Theoretical and experimental methods have been developed to determine the 14N overtone-shift and 1H-14N dipolar coupling parameters pertaining to the amide site of peptides. Density matrix theory using the fictitious spin 1/2 operator formalism has been developed to design, and evaluate the efficacy of, rf pulse sequences and to interpret the 14N overtone experimental results. Pulse sequences for optimum cross polarization from 1H to 14N overtone transition and for selective and broadband excitation of 14N overtone transitions have been successfully demonstrated. A simple one-dimensional method has been used to measure 14N overtone-shift and 1H-14N dipolar coupling in order to determine the backbone conformation of single crystalline samples of peptides.

096
Session Assigned: W&Th P
Title: New kind of structure information from resolved single-crystal ¹H-NMR spectra

Institute: MPI for medical research, Heidelberg, Germany
Keywords:
Abstract:
In most cases, it is not possible to obtain resolved ¹H-NMR spectra from solid samples, irrespective of whether they consist of a powder or a single crystal. The reason is that the strong long-range dipolar couplings of the protons lead to broad lines. We will show that with the help of isotopic labelling, i.e., selective deuteration, we get well resolved non-spinning ¹H-NMR spectra that enable us to deduce structural information that appears to be inaccessible even to neutron and x-ray scattering techniques as well as to other NMR, in particular to ²H-NMR, experiments.

The compound we studied is isotopically labelled biphenyl, C₁₂D₈H₂, with one proton in ortho-position with respect to the bridging carbon in each of the two phenyl-rings. A crystal was grown from the melt. In the gas and liquid phases biphenyl is twisted. By contrast it is claimed to be planar in the high-temperature solid phase which is stable above 40K. Accordingly, in our selectively deuterated samples, two configurations of biphenyl should exist: either the two protons lie on one side or on opposite sides of the long axis of the molecule. In the former case the internuclear proton-proton vector is parallel to the long axis and the inter-proton distance is expected to be about 0.18 nm. In the latter case the inter-proton distance is much larger, namely about 0.45 nm. Because the rest of the hydrogen positions are occupied by deuterons with their low dipolar coupling, the pairs of protons in the molecules can be considered as isolated from one another. Therefore, the two kinds of proton-pairs constitute separate ensembles with distinct dipolar couplings. If the crystal is oriented such that the magnetic field is parallel to the long axis of the molecules, a doublet with a splitting of about 60 kHz is expected from the ensemble of close proton pairs, whereas the doublet splitting of the second ensemble is expected to be too small to be resolved.

The ¹H spectrum we observe for such a crystal orientation at ambiant temperature indeed consists of a doublet with well-resolved components plus a central peak. However, the splitting of the doublet is 36 kHz, that is, it is far smaller than expected. On lowering the temperature down to 40 K the splitting increases to 45 kHz. The only explanation for this finding, which is also consistent with the scattering data (not with their interpretation), is that the biphenyl molecules are, in fact, virtually never in a planar configuration but oscillate between two symmetrically displaced twisted configurations. Only the time-average configuration is planar. From relaxation experiments we learn that the frequency of the oscillations must be large compared with the Larmor frequency in our experiment (270 MHz). The measured value of the doublet splitting allows us to estimate the twist angle defined as the angle between the normal vectors of the phenyl-rings. It is 21 degre at room-temperature and decreases to 17 degre at 40K.

097
Session Assigned: W&Th P
Title: Evidence of steric interactions in methyl ¹³chemical shift tensors.

Institute: University of Utah, Salt Lake City, Utah, USA
Keywords: Steric interactions, terpenes, MAT, 13C tensors
Abstract:
Chemical shift tensors have long been known to reflect local geometry in molecules. Establishing useful and general trends of shift to solid-state structure, however, is not trivial. Much of the challenge has previously been in obtaining and assigning all tensors for molecules of known geometry. Recently, 2D magic angle slow turning (MAT) methods have been developed for measuring tensor principal values for all carbons in moderately large molecules. Here, the MAT methods have been used to obtain principal values for all methyl groups in the natural products caryophyllene oxide and parthenolide. Comparison to ab initio computed tensorÕs shows that when a methyl hydrogen is within the Van Der Waals distance of a neighboring hydrogen, a specific principal value moves to lower frequency (or higher field) by up to 30 ppm. When two of the methyl protons are sterically perturbed, two principal values are observed to shift significantly upfield. In all cases the tensor principal value oriented along the C - methyl bond is nearly invariant. These theoretical results indicate that steric repulsion of methyl protonÕs result in electronic variations in the C - methyl bond. These effects appear to be very pronounced in polycyclic compounds such as the natural products described here. In such compounds, methyls are forced into sterically unfavorable structures to accommodate the rings. These results demonstrate that shift tensors can predict such steric interactions and specify the hydrogen at which the steric perturbation is occurring. With time, extension to carbon types other than methyls (i. e., methylene, methine, olefinic etc.) should also allow a wider variety of shift/structure correlations to be established, to more fully realize the predictive potential of the shift tensor.

098
Session Assigned: W&Th P
Title: New solid state NMR methods for structure determination of fully labeled oriented proteins

Institute: Laboratory of Chemical Physics, NIDDK, NIH
Keywords: correlation NMR
Abstract:
Developments of various solid state NMR methods have enabled us to obtain accurate information on local sites of proteins or peptides which are selectively isotopically labeled. On the other hand, extraction of information on global molecular structures of proteins has been extremely difficult, in spite of its importance, because of the low resolution obtained by solid state NMR: typical line widths obtained by MAS (2-4 ppm) are much less than sufficient, considering that the distributions of ¹³C and ¹⁵N chemical shifts are only 10-20 ppm for each chemical species[1]. Solid state NMR spectra of oriented systems can exhibit better resolution, because the range of ¹³C and ¹⁵N NMR frequencies can be 100-200 ppm for large anisotropic shifts. So far, multidimensional correlation NMR methods based on ¹⁵N detection have been developed for ¹⁵N labeled oriented samples[2-3]. However, low sensitivity of ¹⁵N detection and the long mixing time for exchange among ¹⁵N spins have restricted their application to samples of high molecular weight and have made assignments difficult.

In this paper, a new approach using fully ¹³C and ¹⁵N labeled samples is discussed for structure determination of peptides/proteins in oriented systems. Experiments and simulation for model oriented peptides demonstrate that sharp signals from carbonyl or carboxyl carbons (CO) can be obtained by selectively irradiating C_a resonances. Preliminary results for a single crystal of U-¹³C, ¹⁵N-labeled AlaGlyGly show that ¹³CO/¹⁵N correlation spectra using selective ¹³C decoupling enable us to extract structural constraints as well to assign the resonances sequentially.

[1] R. Tycko, J. Biomol. NMR 8 (1996) 239.
[2] A. Ramamoorthy, C. H. Wu, and S. J. Opella, J. Mag. Reson. B 107 (1995) 88.
[3] A. Ramamoorthy, F. M. Marassi, and J. Opella, in Dynamics and the problem of recognition in biological macromolecules, edited by Jardetzky and Lefevre (Plenum Press, New York, 1996), pp. 237.

099
Session Assigned: W&Th P
Title: Novel Strategies for Backbone and Sidechain Assignments in Solid-phase Polypeptides

Institute: FBML, MIT, Cambridge, MA & Brandeis U, Waltham, MA
Keywords:
Abstract:
Hetero- and homonuclear backbone and sidechain correlations are essential for NMR assignments in multiply labeled peptides and proteins in the immobilized state. Usually correlations are retrieved from multi-dimensional (¹H,¹³C,¹⁵N) correlation experiments. We demonstrate that, alternatively, a small number of one-dimensional experiments employing selective and efficient heteronuclear polarization transfer [1] can be used for backbone assignments. We also show how this approach can be extended for sidechain assignments using homonuclear through-bond, through-space or proton-mediated transfer techniques. In particular, we investigate the polarization transfer dynamics in (NH, Calpha, Cbeta) subsystems of a tripeptide and compare our results to theoretical predictions in multi-spin systems. These experimental protocols can be employed at arbitrary MAS frequencies and are very forgiving with respect to the size of the applied r. f. fields.

[1] M. Baldus, A. T. Petkova, J. Herzfeld, and R. G. Griffin, Mol. Phys. (1998), in press.

100
Session Assigned: W&Th P
Title: Relative Orientation between two NH Bond Vectors in Peptides by Solid State NMR in Rotating Solids

Institute: MIT, Cambridge, USA.
Keywords:
Abstract:
In the past few years, MAS solid state NMR has made important contributions to structure elucidation of selectively labeled samples, e.g. membrane or amyloidogenic proteins. In uniformly ¹³C-,¹⁵N- labeled samples however, it is difficult to retrieve long range structural information due to the truncation of the weak dipolar couplings. Here, we present a novel pulse sequence to measure the NH_i-NH_i+1 projection angle in the uniformly ¹⁵N-labeled tripeptide N-Acetyl-MLF. The angle is directly related to the backbone angles phi and psi. The same experiment can be used to restrain side chain torsion angles, e.g. in ¹⁵N-labeled arginine. In larger spin systems like a protein, the projection angles permits the determination of the geometry between different secondary structure elements. The method relies on chemical shift correlation between two nitrogens. The NH dipolar dephasing periods are correlated by a polarization transfer step in an indirect evolution period. The heteronuclear interaction is recoupled using T-MREV, a MREV-8 (1) derived, gamma-encoded TC-5 (2) type pulse sequence.

Literature:

1. P. Mansfield, M.J. Orchard, D.C. Stalker, K.H.B. Richards, Phys. Rev. B 7, 90 (1973); W.K. Rhim, D.D. Elleman, R.W. Vaughan, J. Chem. Phys. 59, 3740 (1973).
2. J.D. Gross, P.R. Costa, R.G. Griffin, J. Chem. Phys. 108, 7286 (1998).

101
Session Assigned: W&Th P
Title: Determination of Torsion Angles in MAS NMR Using CSA Correlation and ¹³C-¹³C Dipolar Measurement

Institute: MIT, Cambridge, MA, USA
Keywords:
Abstract:
We present two MAS solid-state NMR methods aimed at the determination of three-dimensional molecular structure and demonstrate them in the context of secondary structure determination in peptides. The experiments can be used in a complementary fashion to determine the backbone torsion angles .vphi. and .psi.. The first experiment relies on the accurate measurement of the carbonyl-carbonyl distance to determine .vphi. and the second correlates the relative orientation of carbonyl CSA tensors, which depends primarily on .vphi. and .psi.. Both experiments can be performed on a peptide, specifically ¹³C labeled at the carbonyl positions of two adjacent amino acids (the tripeptide L-Ala-L-Gly-L-Gly with ¹³C labels at (Ala-1) and (Gly-2) was employed in our studies). Among important recent applications using specifically carbonyl ¹³C labeled systems are the determination of the conformation of a peptide adsorbed to the surface of an inorganic crystal [1] and the evaluation of the fibril structure in Alzheimers .beta.-amyloid [2].

The determination of the homonuclear ¹³C-¹³C dipolar coupling between two carbonyl spin labels provides a direct measurement of the .vphi. angle in peptides and is very sensitive to this angle in most biologically relevant regions. The use of a .gamma.-encoded dipolar recoupling sequence of the C7-type, combined with the possibility to employ long mixing times (ca. 40 ms), results in large oscillations in dipolar dephasing curves, which significantly enhance the resolution of the experiment. With the proper inclusion of relaxation effects, the dephasing curves can be simulated to yield accurate estimates for ¹³C-¹³C dipole couplings.

The relative orientation of carbonyl CSA tensors in peptides depends on the .vphi., .psi., and .omega. torsion angles (in simulations .omega. is set to 180^o - a reasonable assumption for most peptides). The correlation of CSA tensors is accomplished by evolving a 2Q state between ¹³C nuclei under the sum CSA, where CSA recoupling during MAS is achieved using a constant-time .pi. pulse sequence. Studies on model peptides [3] indicate that carbonyl CSA tensors have a relatively fixed orientation with respect to the amide plane, making them useful in experiments relying on tensor correlation. The experiment can be performed under conditions of high resolution and sensitivity (.omega._r/2.pi. ~ 10 kHz), with 2Q filtration used to suppress the natural abundance ¹³C signals. Numerical simulations indicate that the experiment provides good resolution for .vphi. and .psi. angles in biologically relevant regions, especially for torsion angles commonly observed in helices.

[1] J. R. Long et al., Proc. Natl. Acad. Sci. USA, 95, 12083 (1998).
[2] T. L. S. Benzinger et al., Proc. Natl. Acad. Sci. USA, 95, 13407 (1998).
[3] T. G. Oas et al., J. Am. Chem. Soc., 109, 5956 (1987).

102
Session Assigned: W&Th P
Title: A Direct Transform for the NMR Chemical Shift Anisotropy

Institute: University of Nottingham, Nottingham, U.K.
Keywords:
Abstract:
A direct transform of the NMR chemical shift anisotropy, operating on data acquired under magic angle spinning conditions, yielding rotationally invariant quantities, is potentially a source of increased resolution in solid state NMR. The mathematical basis for such a transform is discussed by exploiting an analogy with projection reconstruction. A specific example of the kind of pulse sequence required is given, and optimal sampling of the data is discussed.

103
Session Assigned: W&Th P
Title: Spin-echo experiments for homonuclear coupled spins under magic-angle sample spinning

Institute: Institute for Molecular Science, Okazaki, Japan
Keywords:
Abstract:
Spin-echo pulse sequence is well-known from the early years of the NMR development. Today it is usually used to get J-resolved spectra in a two-dimensional (2D) NMR experimental scheme. In general, a 2D spin-echo experiment yields sharp resonance lines at .omega₁=0 position on the F₁ axis for a spin system having negligibly small J-coupling constants. However, additional resonance lines emerged along the F₁ axis on a 2D NMR spectrum when the 2D spin-echo pulse sequence was applied to a homonuclear two spin system under magic-angle sample spinning. We found that the intensities of the additional lines are closely related to the homonuclear dipolar interaction and that the positions of the lines are determined by a sample spinning frequency and the difference of the isotropic chemical shifts.

104
Session Assigned: W&Th P
Title: Selective-Exchange Solid-State NMR

Institute: Polymer Science & Eng., UMass, Amherst, MA,USA
Keywords: Segmental orientation; Molecular motion
Abstract:
We will present two exchange NMR techniques that yield NMR spectra exclusively of slowly reorienting segments, suppressing the often dominant signals of the immobile components. The first technique eliminates the diagonal ridge that usually dominates 2D exchange NMR spectra and makes it hard to detect the broad and low off-diagonal exchange patterns. The approach relies on producing a sin²(ω₁-ω₂)τ modulation on the 2D exchange spectrum. In the most efficient version of the experiment, the modulation is generated as 1 - cos(ω₁-ω₂)τ. The method suppresses not only the intensity exactly on the diagonal, but also off-diagonal wings and cut-off wiggles of the diagonal ridge. In the 1D spectrum obtained for vanishing evolution time, the signal of the exchanging site(s) can be observed selectively and thus identified. The second approach, a 1D MAS experiment, is a simple extension of the exchange-induced sideband (EIS) method (Yang et al., CPL 139, 239, 1987). A TOSS spectrum obtained with the same number of pulses and delays, with a simple change in sequence, is subtracted from the EIS spectrum, leaving only the exchange-induced sidebands and negative centerband of the mobile site(s). Both techniques can identify and analyze dynamics of specific groups in solids of moderate to large chemical complexity. The MAS technique has high sensitivity, the 2D static technique good angular resolution. Applications to model compounds and polymers will be presented.

105
Session Assigned: W&Th P
Title: Magnetic Field Gradients in Solid State MAS NMR

Institute: Bruker Instruments, Inc.
Keywords:
Abstract:
Gradient enhanced NMR spectroscopy is widely used in liquid state spectroscopy for coherence pathway selection, solvent suppression, artifact reduction and diffusion weighting and has had a tremendous impact by improving the quality of NMR spectra. To date the same advantages have not been translated to high-resolution solid state NMR.

The recent combination of MAS and gradients (Maas et al. JACS 118, 13085 (1996)) has extended these advantages to high-resolution measurements of semi-solids. These samples exhibit linebroadening due to residual dipole-dipole couplings and susceptibility broadening. MAS alone is sufficient to remove the line-broadening and yield high-resolution spectra.

In solid samples MAS alone is not enough to obtain a high-resolution lineshape, due to the presence of strong dipolar couplings. A combination of MAS and coherent averaging techniques may be employed to increase the effective transverse relaxation time. The challenge in solids is to create a magnetization grating in a time short compared to this decoherence time.

Here we show the first examples in which gradients are applied for coherence pathway selection in dipolarly coupled solids. The results demonstrate that the experiments must be tailored to the spin dynamics of the sample under study. In the case of dipolarly coupled systems, this means that for successful gradient experiments, gradient induced offsets must not interfere with dipolar decoupling. Examples of both homo-nuclear and hetero-nuclear experiments will be shown.

Institute: Indiana University, Bloomington, IN, USA
Keywords:
Abstract:
The polymerization of vinyl chloride has an important economical impact, with a total world production of 19 million tons in 1993. Examples of products made from PVC include pipes, electric wire insulation, packaging materials, medical fittings and filters, gloves, table cloths and shower curtains.[1]

We present proton and carbon NMR studies of the microstructure of pure and plasticized poly(vinyl chloride). A lineshape analysis of the proton spectra of the pure compounds compared to the plasticized materials at various concentrations gives insight into the mobility of the components. Using the Williams-Landel-Ferry equation and the differences in mobility, we conclude that there is an inhomogeneous motional and spatial distribution of the plasticizer in the polymer matrix. We compare the estimated glass transition temperatures with the calculated values derived from the Fox-Flory equation and those observed by dynamic scanning calorimetry. To quantify the heterogeneous distribution of the domains we apply dipolar-filtered proton spectroscopy. The dipole filter consists of a 12-pulse sequence, which is compensated for offset and chemical shift effects. It selectively suppresses magnetization from static protons, while leaving that from mobile protons unaffected. By increasing the inter-pulse spacing we filter out successively more of the mobile species.[2] In this way we map a mobility profile of the polymer at a specific temperature which we compare to model systems.

[1] J. W. Summers, Journal of Vinyl & Additive Technology, 3, 130, (1997)
[2] F. Mellinger, M. Wilhelm, H. W. Spiess, P. Belik, H. Schwind, to be submitted

107
Session Assigned: W&Th P
Title: Identification of Polyester Connectivity in Commercial Polymers at Natural Abundance

Institute: Morton International, Woodstock, IL, USA
Keywords:
Abstract:
Polyesters are key components in a wide range of commercial products from adhesives to food packaging to automotive parts. The deformulation of polyesters is key to understanding how their structures relate to their physical properties. Identifying and quantifying the individual monomer units in a polyester is a relatively simple task for NMR. However, when a polyester contains more than one diacid and/or one diol, determining how the system is structurally ordered (e.g. distinguishing between a random polymer and a block co-polymer) has been non-trivial. Since the physical properties of a given polymer are determined by its monomer connectivity, elucidating the structural order of multi-component polyesters is of high priority. The application of a 2-dimensional PFG enhanced heteronuclear correlated technique (¹H-¹³C HMBC) to quickly determine whether a polyester contains random and/or block segments will be demonstrated. These studies were carried out using industrial polymers at natural abundance.

108
Session Assigned: W&Th P
Title: NMR investigation of AMS Copolymers

Institute: IMA/UFRJ - Rio de Janeiro, Brazil
Keywords:
Abstract:
Two copolymers based on alpha-methylstyrene (AMS) have been investigated by nuclear magnetic resonance. The styrene-co-alpha-methylstyrene (SAMS) and alpha-methylstyrene-co-acrilonitrile (AMSAN) were analysed by solution and solid state NMR techniques. Three copolymers of SAMS with different compositions showed a particular behaviour. The solution results showed the microstructure of the copolymers as well as the AMS content. The carbon-13 spectra showed that the AMS CH3 signal were detected at three distinct chemicals shifts, because the polimerization process of the alpha-methylstyrene monomer is randomly distributed in the molecule and different configurations can be detected. The proton spin-lattice relaxation time in the rotating frame parameter was chosen because it is able to evaluate the changes in the microstructure and the molecular mobility in polymers. It also permits to verify the homogeneity in the polymer systems as it is sensitive to the spatial proximity of chains. The proton spin-lattice relaxation time in the rotating frame parameter, found for the copolymers, confirms the random distribution in the samples. The copolymer with low quantity of AMS (1.7%), when analysed by this relaxation parameter, showed lower values which was interpreted as an antiplasticization effect. The SAMS copolymer with higher AMS quantity showed a plasticization effect. From the results obtained by NMR experiments, we could conclude that the AMS content influenced very much the copolymer distribution sequence. The AMSAN copolymer was prepared by the same way like the SAMS copolymers and the ratio between them was 75/25 AMS/AN. As far as this study is concerned this copolymer showed a particular behaviour because of its chemical characteristics.

109
Session Assigned: W&Th P
Title: ¹H CRAMPS and CP/MAS ¹³C NMR Studies of Hydrogen Bonding of Polymers in the Different States

Institute: Institute for Chemical Research, Kyoto University
Keywords: ¹CRAMPS;CP/MAS ¹³C NMR;Hydrogen Bond
Abstract:
Hydrogen bonding and the conformation of poly(vinyl alcohol) (PVA) with different tacticities have been examined for the films and frozen solutions by ¹H CRAMPS and CP/MAS ¹³C NMR spectroscopy. OH, CH, and CH₂ resonance lines are separately observed in the order of decreasing chemical shift in the ¹H CRAMPS spectra for the films, and additional contribution from OH groups, which should be assigned to OH groups free from hydrogen bonding, is also found to exist between the CH and CH₂ lines by the evaluation with the employment of OH-deuterated PVA. The former OH line is further resolved into two contributions from OH groups associated with the intramolecular and intermolecular hydrogen bonding by the detailed line shape analysis. In addition to these three components, another OH resonance line is found to appear at 4.3 ppm above T_g , which is explicitly confirmed for the main-chain deuterated PVA films. This OH line should be assigned to OH groups that undergo the enhanced molecular motion in the noncrystalline region above T_g. On the basis of the temperature dependencies of the four components, it is concluded that the OH groups free from hydrogen bonding are allowed to exist only in the crystalline region. Similar ¹H CRAMPS analyses are in progress for frozen PVA solutions as well as for different hydrogen-bonded polymers.

It is well known that the CH resonance line splits into three lines due to the formation of two, one, and no intramolecular hydrogen bond(s) in CP/MAS ¹³C NMR spectra for solid PVA. Such splitting of the CH line is found to greatly change for frozen PVA solutions, depending on the differences in solvent and triad tacticity. An statistical treatment is successfully applied to the interpretation of the CH splitting by considering the statistical formations of the intramolecular-intermolecular hydrogen bonds and trans-gauche conformations. Some good correlations will be shown between the results obtained for PVA by ¹H CRAMPS and CP/MAS ¹³C NMR spectroscopy.

110
Session Assigned: W&Th P
Title: Solution and Solid-State Spectroscopy of Poly(lactide)

Institute: University of Minnesota, Minneapolis, MN USA
Keywords: Poly(lactide)
Abstract:
Lactic acid based aliphatic polyesters such as poly(lactide) are increasingly being explored for use in several applications including biodegradable packaging materials, food containers, bioresorbable medical implants, and drug delivery systems. A number of physical properties of poly(lactide) (PLA) are linked to its stereosequence distribution, which is influenced by many factors such as the starting lactide feed composition, polymerization kinetics, and extent of conversion. The polymerization kinetics in turn are influenced by the catalyst, temperature, impurities, batch vs. continuous process, etc. We have been using solution and solid-state NMR spectroscopy to understand important features of PLA such as polymerization kinetics, tacticity, solid-state morphology, and incorporation of stereosequence defects.

We have been using ¹H and ¹³C NMR of PLA in solution to determine the sequence of the lactic acid units from the relationship between the stereogenic centers and their influence on the chemical shift. Recently the accepted stereosequence assignments for poly(lactide) have been called into question based upon the results of two-dimensional ¹H-¹³C HETCOR experiments. One-dimensional ¹H and ¹³C experiments reveal that the previously accepted assignments are correct because the integrated intensities match the expected probabilities from Bernoullian statistics for a sample of 5% L-lactide in D-lactide when the old stereosequence assignments are used. ¹H-¹³C HMQC experiments on this sample reveal correlations between ¹H isi and ¹³C sis resonances (and vice versa), indicating that there is an asymmetric influence on the ¹H and ¹³C chemical shifts, which causes difficulty in directly interpreting two-dimensional HETCOR type experiments of chiral polymers such as poly(lactide).

In the solid state, ¹³C CP/MAS NMR spectra provide information about polymer morphology and crystal structure. Solid-state NMR data suggest that there are five crystallographically inequivalent sites per unit cell. We are using two-dimensional exchange NMR to probe the microstructure of PLA to understand the conformation and arrangement of the polymer chains. The two-dimensional exchange NMR spectrum of 5% L-lactide ¹³C labeled at the carbonyl site in unlabeled L-lactide suggests the presence of up to 10 crystallographically inequivalent sites in highly crystalline poly(lactide). ¹³C CP/MAS NMR spectroscopy is also a powerful technique for probing the incorporation of stereosequence defects into the polymer chain. ¹³C CP/MAS NMR spectra of poly(D-lactide) containing small amounts (<10%) of L-lactide ¹³C labeled at the carbonyl carbon can be deconvoluted to quantitate the relative amounts of stereosequence defects which are incorporated into the crystalline chain versus the amount in the amorphous domain. For a sample containing 3% L-lactide, approximately 50% of the defects are incorporated into the crystalline chain, while the remainder are forced into the amorphous domain.

111
Session Assigned: W&Th P
Title: Fluorine and Carbon Solid-State NMR of Heterogeneous Systems

Institute: Institut für Polymerforschung Dresden e.V.
Keywords: high-speed MAS, fluorine solid-state NMR, spin exchange, double quantum
Abstract:
¹⁹F high-speed MAS NMR has been applied to study the structure of various fluoropolymers. In order to achieve high resolution, high-speed MAS, in excess of 30 kHz, averaging the dipolar coupling and the anisotropy of the chemical shift, and proton decoupling are applied. The dipolar coupling between the fluorine nuclei, which is averaged by high-speed MAS, on the other hand can provide information about spatial proximity. This can be exploited in two-dimensional spin-exchange experiments or in double quantum experiments. In the spin-exchange experiments the dipolar coupling is reintroduced during the mixing time by rotor-synchronized pulses. The rate of spin exchange depends on the strength of the dipolar coupling, hence on the distance, but as well on the frequency difference of the coupling spins. Thus the recoupling sequence must average the chemical shift difference as well, in order to exploit spatial information. In double quantum spectra the double quantum coherences between dipolar coupled fluorine nuclei in t₁ are correlated with single quantum spectra t₂. The intensities of the double quantum signals depend on the strength of the dipolar coupling, thus the distance.The effectivity of the sequences used for the excitation of double quantum coherences strongly depend on the frequency offset, which is again important because of large distribution of chemical shifts for ¹⁹F. In systems containing fluorine and protons, as presented here, the heteronuclear coupling to the protons which often is recoupled in the recoupling experiments as well.

Carbon spectra in addition offer the advantage to detect signals from protonated and fluorinated parts of the sample. However the resolution in the range of the fluorinated carbon is limited and hence the superior resolution from the fluorine spectra can be utilized in a ¹⁹F-¹³C Hetcor experiment for the assignment in the carbon spectra.

Various cross polarization and depolarization experiments between ¹H, ¹⁹F and ¹³C have been applied for the study of interfaces between protonated and fluorinated domains in heterogeneous samples such as blockcopolymers and physical mixtures of fluoropolymers and other polymers.

112
Session Assigned: W&Th P
Title: Fast MAS Proton NMR Studies of Polymers at Surfaces

Institute: Bell Labs, Lucent Technologies
Keywords:
Abstract:
Solid state proton NMR with fast MAS is developed as a method to study the structure and dynamics of polymers at surfaces and interfaces. We have found that high resolution spectra can be obtained for many polymers at temperatures above the glass transition temperature with 15 kHz MAS, and that the signal-to-noise ratio is high enough that both high and low surface area materials can be studied. The dynamics are often restricted in the vicinity of surfaces and multiple-quantum NMR can be used to identify the surface-adsorbed layer. Surface modification dramatically changes the dynamics of polymers at surfaces and we have observed magnetization exchange across the polymer-glass interface that allows us to identify the polymer functional groups interacting with the surface. The goal of these studies is to provide a molecular level understanding of polymer-surface interactions.

113
Session Assigned: W&Th P
Title: In situ deformation of nylon monitored by deuterium NMR

Institute: Massachusetts Institute of Technology,Cambridge MA
Keywords:
Abstract:
Understanding the dynamics of chain motions occurring during tensile deformation of polymers is crucial in the design of such materials to achieve desired mechanical properties such as high Youngs modulus or ductility. Deuterium NMR was used to examine the role that the amorphous phase and plasticizers play in the large strain deformation of semicrystalline and amorphous nylon. Commercially available deuterated plasticizers such as D₂O and phenol-d₅ are easily absorbed into the amorphous phase of nylon. Furthermore, the NH groups in the amorphous regions of nylon can be deuterated readily by exchange with D₂O. Since the deuterium line shapes are sensitive to changes in the orientation and time scales of motion of the XD (where X=C, O or N) bond, monitoring the evolution of the line shapes with strain provides insight into the complex dynamics of the deformation process.

An in situ stretching device was constructed to fit within the NMR probe thereby allowing us to perform NMR acquisition during active deformation of nylon. The device is able to produce elongations of up to 10 mm and withstand a load of 1000 N. The initial length and diameter of the undeformed samples are 10 mm and 2 mm respectively.

For the system consisting of nylon 6 plasticized with phenol-d₅, the elongation rate was 0.25 mm/min and the total time of the experiment was 40 min. A total of 20 spectra were obtained each of which was the sum of 1200 averages over 2 min. The initial motionally narrowed Lorentzian line shape of phenol-d₅ in the amorphous region of the undeformed material gives rise to a splitting as strain increases. The splitting increases linearly with strain indicating that the orientation of the phenol molecules is simply a function of strain. However, the line width increases with strain initially but attains a constant value at moderate to large strains, showing that the motion of the plasticizer becomes more restricted at first but then becomes unaffected by changes in the polymer induced by the deformation process.

Deuterium spectra were also obtained with the solid echo pulse sequence for nylon 6 with deuterated NH groups in the amorphous regions. At an elongation rate of 0.06 mm/min, 6 spectra were obtained each of which was the sum of 6000 averages over 10 min. The sample was stretched up to the yield point, which occurred at an extension of 3 mm. Each spectrum shows the Pake pattern with hardly any changes with strain. Hence over this region of strain it appears that there is little overall orientation and hardly any changes in mobility of the amorphous chains; however, it does not rule out the possibility of localized orientation and changes in mobility during deformation. At an elongation rate of 0.25 mm/min, the sample necked and was extended to 10 mm. A total of 4 spectra were obtained. The spectrum of the necked region in the polymer differs from that of the undeformed sample indicating preferential alignment of the amorphous chains along the tensile axis.

114
Session Assigned: W&Th P
Title: Natural-Abundance two-dimensional 29Si NMR Investigation of the Bonding Connectivities in the Silsesquioxane-Based Polymers

Institute: Dow Corning Corporation, Midland, MI 48686
Keywords: 2D-INADEQUATE NMR, silsesquioxane, polymer
Abstract:
Several novel silsesquioxane-based resins have been synthesised and their potential applications are under intense investigation. Silsesquioxane-based resins are complex systems. To understood structures, then tailored synthesis and understanding structure-process-property relationships, is very important and challenging work. Multidimensional NMR spectroscopy has been widely used to study the detailed structure of macromolecules. We have extended the two-dimensional NMR experiments, such as C7 and 2D-INADEQUTE, that use dipolar interactions to correlate different Si-O-Si sites in silsesquioxane-based polymers. Homo-nuclear correlation sequence and its applications to the model silicone compounds, such as MD3M(H) and MD3M(Vi), will be presented. The potential application of this technique to obtain detailed structural and chemical information of the silsesquioxane resin materials, will also be addressed.

Institute: NIAAA, NIH, Rockville, MD 20852, USA
Keywords:
Abstract:
The good resolution and long spin-lattice relaxation times of ¹H NMR lipid resonances in fluid membranes recorded with magic angle spinning (MAS) enable the study of cross-relaxation by two-dimensional NOESY experiments. Crosspeaks between resonances of polar headgroups and the end of apolar hydrocarbon chains have been reported by several laboratories. They are caused by close approach of these protons, or they reflect magnetization transfer by spin diffusion along the proton network of lipids. Because direct contacts between hydrophobic and hydrophilic molecular groups in membranes appear to be at odds with an arrangement of lipids in bilayers, it was suggested that these crosspeaks are the result of spin diffusion along the proton network of lipids. Evidence of the purely intermolecular nature of these crosspeaks comes from the comparison of cross-relaxation rates in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and a 50/50 (mol/mol) mixture of headgroup perdeuterated DPPC-d₁₃ and chain perdeuterated DPPC-d₆₂. In a third experiment, spin diffusion along protonated lipid hydrocarbon chains was suppressed by mixing DPPC-d₂₁, with protonated palmitic acid chains that contain two deuterated methylene groups in the chains center and a perdeuterated headgroup, and the chain perdeuterated DPPC-d₆₂. Within experimental error, the cross-relaxation rates for headgroup-to-chain interactions did not change - indicating that spin diffusion along lipid hydrocarbon chains is negligible. Intermolecular cross-relaxation may also relay magnetization by a cascade of intermolecular contacts that shuffles magnetization from the lipid headgroup to the end of the hydrocarbon chains in multiple transfer steps. If such a mechanism of magnetization transfer would be important, then the head-to-chain cross-relaxation rates would depend in a non-linear fashion on the DPPC-d₂₁ mole fraction in the DPPC-d₂₁ /DPPC-d₆₂ binary mixture. The experimentally determined cross-relaxation rates are a linear function of the mole fraction indicating that single step, direct contacts between the choline- and chain methyl protons are the sole source of cross-relaxation. For the first time without doubt, the experiments demonstrate that crosspeaks between distant molecular groups in the lipid matrix, like headgroups and hydrocarbon chains, are caused by direct interactions between these protons. The result illustrates the high degree of motional and conformational disorder in fluid lipid bilayers.

117
Session Assigned: W&Th P
Title: High-Resolution MAS NMR Study of Polypeptides in Multilamellar Phospholipid-Membrane

Institute: Institute of Biomedical Engineering (IBMT)
Keywords:
Abstract:
High-Resolution MAS NMR Study of Polypeptides in Multilamellar Phospholipid-Membrane A. Pampel and F. Volke(1) Fakultät für Physik und Geowissenschaften, Universität Leipzig, 04103 Leipzig, Germany (1)Fraunhofer Institut für Biomedizinische Technik, 66386 St. Ingbert, Germany

The 13C/15N-double-labeled Hymenistatin 1 embedded in multilamellar bilayers of phospholipids was investigated with High-Resolution MAS NMR spectroscopy.

Hymenistatin 1, the cyclic octapeptide cyclo(-Pro-Pro-Tyr-Val-Pro-Leu-Ile-Ile-) was isolated from Hymeniacidon sponge [1]. The substance is of interest because of its cytostatic activity. It was synthesized and examined conformationally using NMR and molecular-dynamics simulations some years ago [2], and it was shown, that there are differences in the conformations for different solvents, which was explained in terms of solvent interactions. Furthermore, Hymenistatin 1 is insoluble in water, but we have shown, that Hymenistatin 1 attaches to vesicles in lipid-water dispersions with high excess of water. Therefore, it might be of interest to study the structure of Hymenistatin 1 in an environment that mimics lipid membranes, e.g. SDS-micelles or even multilamellar lipid bilayers.

Here we focus on studies of the peptide embedded in multilamellar bilayers with lower content of water. The multilamellar dispersion under study contained partially deuterated DMPC (Dimyristoyl phosphatidylcholine), Hymenistatin 1 and water (15:1:15 molar ratio). It was proven, by 31P and 2H solid-state NMR, that the dispersion forms a liquid-crystalline phase under the experimental conditions. In this phase the lipid molecules are globally arranged in stacks of multilamellar bilayers but undergo rapid local motions. NMR spectra of such dispersion are rather featureless, since the resonances are broaden by the residual dipolar couplings, chemical shift anisotropy and susceptibility effects. Therefore, the standard high-resolution NMR techniques for structure determination cannot be applied. This problem can be overcome by Magic Angle Spinning (MAS) . The properties of MAS-NMR spectra of phospholipid membranes allow the straight forward application of many standard high-resolution NMR experiments [3-6].

We performed J-coupling based 1H,13C correlation experiments at spectrometers with 400 and 750MHz operating frequency. For spinning speeds up to 14 kHz also the backbone signals of the peptide could be separated. Side chain resonances could be observed at lower spinning speed due to their fast internal motions. The obtained spectra are compared to spectra of Hymenistatin 1 in DMSO, Chloroform and in SDS-micelles. The 13C chemical shift values of the Ca-carbons are different in all environments, indicating a slightly different structure. Such a systematic study will allow to differentiate between different membrane mimetics to choose the most suitable one for the appropriate kind of application.

These studies are currently in progress in our lab.

Acknowledgements

We thank Prof. H. Kessler (TU München) for providing the peptide. This work was supported by the Deutsche Forschungsgemeinschaft in the framework of the Sonderforschungsbereich "Moleküle in Wechselwirkung mit Grenzflächen."

References

[1] G. R. Pettit, P. J. Clewlow, C. Dufresne, D. L. Doubek, R. L. Cerny, K. Rützler, Can. J. Chem. 68 (1990) 708.
[2] R. K. Konat, D. F. Mierke, H. Kessler, B. Kutscher, M. Bernd, R. Voegeli, Helv. Chim. Acta 76 (1993) 1949.
[3] J. H. Davis, M. Auger, R. S. Hodges, Biophys. J. 69 (1995) 1917.
[4] F. Volke, A. Pampel, Biophys. J. 68 (1995) 1960.
[5] A. Pampel, F. Volke, F. Engelke, Bruker Report 145 (1998) 23.
[6] A. Pampel, F. Volke, J. Magn. Reson. Anal. 3 (1997) 222.

118
Session Assigned: W&Th P
Title: 13-C MAS NMR + Lanthanides: A Universal Tool to Probe Membrane Surfaces and Location of Membrane Compounds

Institute: Dept. of Biochemistry
Keywords: membranes; 13-C-MAS-NMR; Peptide-Location
Abstract:
A combination of 13-C MAS NMR spectroscopy and binding of paramagnetic lanthanide ions (e.g. Dyprosium) to the membrane surface of lipid bilayers has been established as a simple method to determine the location of membrane embedded compounds, especially peptides and sterols, relative to the membrane surface. Using MAS NMR the spectral resolution is shown to be sufficient to detect and assign all carbon nuclei in the lipid matrix. Using various lanthanide ion concentrations revealed in the spectra a distance dependent differential paramagnetic quenching of resonance intensities for the various carbon sites with positions at the bilayer surface quenched effectively, and hydrophobic sites in the core of the membrane unaffected. The established method was applied to the 50 residue long M13 coat protein, 13-C labelled at its Val-29 and Val-31 and incorporated into lipid bilayers. The 13-C MAS NMR spectra revealed no paramagnetic quenching for these residues while the lipid resonances at the membrane interface were quenched completely, suggesting that both protein residues are buried deeply inside the hydrophobic core of the membrane. Currently studies are under process on the amyloid-beta peptide to differentiate residues in the extracellular domain from transmembrane residues, a question of high biological significance.

119
Session Assigned: W&Th P
Title: A ²H-NMR study of the chain length effect on lipid phase coexistence in model skin membranes

Institute: University of British Columbia, Vancouver, BC, Can
Keywords:
Abstract:
Specialized lipid layers packed between the dead cells found in the outermost layer of skin, the stratum corneum (SC), are responsible for the existence of a highly effective barrier to water diffusion across the epidermis. Understanding correlations between lipid composition and phase behavior in SC model membranes is a prerequisite to understanding the molecular basis of this highly impermeable structure. The main components of SC intercellular membranes are ceramides (Cer), cholesterol (Chol) and free fatty acids (FFA) in approximately equimolar proportions. Lipid modification occurs during epidermal differentiation, mainly from sphingolipids to Cer and phosphoglycerolipids to FFA. Previous studies by our group^[1] showed that the small headgroup of Cer is the determinant of the complex phase behaviour observed in SC model membranes composed of equal parts bovine brain Cer (BBCer), Chol and palmitic acid at pH 5.2. Besides the unusual lipid classes of which SC membranes are composed, SC lipids also have unusually long hydrocarbon chains. In order to determine the influence of free fatty acid and ceramide chain length on the phase behaviour of SC model membranes, deuterium nuclear magnetic resonance (²H-NMR) was used to study SC model membranes composed of equimolar Cer, Chol and FFA with different fixed chain lengths of Cer and FFA, labeling Cer and FFA respectively. ²H-NMR spectra show that each mixture has complex phase coexistence at room temperature. As temperature rises, all the samples undergo phase transitions from a "solid" dominant phase to a "fluid" phase (spectra characteristic of axially symmetric motion), then to a phase having isotropic lipid motion. The difference in transition temperature between labelled Cer and labelled FFA shows that there is phase separation between them. The transition point of FFA from "solid" to "fluid" phase depends on the FFA chain length but is insensitive to the Cer chain length. The transition behavior of Cer is influenced both by the Cer and the FFA chain length. Our results complement recent X-ray^[2] and FTIR results^{[3] [4]} on similar model SC membranes.

[1]Thewalt, J., N. Kitson, C. Araujo, A. Mackay, and M. Bloom. 1992. Models of stratum corneum intercellular membranes: the sphingolipid headgroup is a determinant of phase behavior in mixed lipid dispersions. Biochem. Biophys. Res. Commun. 188:1247-1252. [2]Bouwstra, J. A., J. Thewalt, G.S. Gooris, N. Kitson. 1997. A model membrane approach to the epidermal permeability barrier: An X-ray diffraction study. Biochem. 36:7717-7725. [3]Moore, D.J., M.E. Rerek, and R. Mendelsohn. 1997. Lipid Domains and Orthorhombic phase in model stratum corneum: evidence from fourier transform infrared spectroscopy studies Biochem. Biophys. Res. Commun. 231:797-801. [4]Lafleur, M. 1998. Phase behaviour of model stratum corneum lipid mixtures: an infrared spectroscopy investigation. Can. J. Chem. (in press)

120
Session Assigned: W&Th P
Title: P-31 NMR Analysis of O-Alkyl and O-Alkenyl Glycerophospholipids in Tissue Extracts

Institute: Harvard Medical School, Boston, MA, USA
Keywords:
Abstract:
High-resolution P-31 NMR spectroscopy has emerged as a viable alternative to chromatography for the analysis of phospholipids in biological extracts. Using a ternary chloroform / methanol / water solvent system, a unique resonance line is obtained for each phospholipid molecular class, permitting the determination of relative composition. Absolute quantitation in micromoles per gram of tissue can be achieved by adding an internal standard during the extraction procedure (Metz and Dunphy, J. Lipid Res., 37, 2251-65 (1996); 38, 1275 (1997).). Although the P-31 NMR peak assignments for the abundant 2,3-diacylglycerophospholipids are well established, the chemical shifts of the important 2-acyl-3-O-alkyl and 2-acyl-3-O-alkenyl (plasmalogen) derivatives are controversial and have inspired considerable speculation in the literature. Recent work has clarified their relative shifts in 50/50/15 chloroform / methanol / water (v/v/v), but since the shifts depend heavily on the solvent and temperature, there remains a need to assign these peaks in other solvent systems. This is especially true in the 100/40/10 mixture which has been most widely used for these analyses. Simple chemical conversions including mild acid hydrolysis and partial catalytic hydrogenation have now been employed to assign the resonance lines unambiguously. The total phospholipid peak separation has also been measured as a function of temperature and solvent composition, and it has been found that the widely-used 100/40/10 system is not optimal.