Analysis of Intractable Biological Samples by Solids NMR - ACS

Chapter 2

Analysis of Intractable Biological Samples by Solids NMR 1

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Karl J. Kramer , Theodore L. Hopkins , and Jacob Schaefer 1

Grain Marketing and Production Research Center, Agricultural Research Service, U.S. Department of Agriculture, Manhattan, KS 66502 Department of Entomology, Kansas State University, Manhattan, KS 66506 Department of Chemistry, Washington University, St. Louis, MO 63130 2

Downloaded by UNIV LAVAL on July 11, 2016 | http://pubs.acs.org Publication Date: June 29, 1998 | doi: 10.1021/bk-1998-0707.ch002

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Solids N M R is a noninvasive analytical method that can be used to investigate the chemical compositions and covalent interactions that occur in biological samples intractable to conventional analytical approaches. Solids N M R techniques such as cross polarization, dipolar decoupling, magic angle spinning, magnetization dephasing, and isotopic enrichment have been used to obtain high resolution spectra that provided information about the relative concentrations and internuclear distances between atoms in complex biological solids. Levels of proteins, chitin, catechols, lipids, pigments, and other organic constituents in composite materials were estimated. Covalent interactions between specific carbons and nitrogens have been detected by isotopic enrichment with labeled precursor molecules. We have used solids N M R in studies of polymeric and analytically intractable samples such as insect cuticular exoskeletons, egg cases, egg shells, silk cocoons, and marine coral skeletons. Evidence was obtained for stabilization mechanisms occurring primarily when quinones derived from catechol-containing compounds including catecholamines and o-diphenols with acid, aldehyde and alcohol side chains form adducts with functional groups of structural proteins and perhaps chitin. Solids N M R can be utilized for probing the compositions and covalent interactions of many other types of biological samples found in the biosphere and geosphere and, when combined with other analytical techniques, provides a powerful approach for elucidating the complex structures of biopolymeric materials. Many invertebrates stabilize and strengthen skeletal structures by crosslinking of structural proteins, dehydration, and impregnation with chitin, minerals, and phenolic compounds (1-6). Some of the skeletal components are relatively stable, and they can be preserved during decay in the biosphere and geosphere. For example, Bass et al. (7) reported the selective preservation of 2

Address correspondence to K. J. Kramer, GMPRC-ARS-USDA, 1515 College Avenue, Manhattan, KS 66502, U . S. A. Phone: 785-776-2711. Fax: 785-5375584. Email: [email protected].

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©1998 American Chemical Society

Stankiewicz and van Bergen; Nitrogen-Containing Macromolecules in the Bio- and Geosphere ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

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chitin during the anoxic decay of shrimp in a marine environment. Analysis using solid-state C nuclear magnetic resonance (NMR) revealed that, whereas other organic components were highly degraded after only 8 weeks, chitin remained the major constituent of the biomass. Longer-term decay of chitinous tissues, however, did occur, with replacement of chitin by more resistant organic matter such as alkanes and alkenes derived from other sources. Therefore, depending on the type of sample and environmental conditions, solids N M R can monitor the levels of some N- and C-containing macromolecules and provide information about their physical states and decay processes. This chapter reviews the results of solids N M R research conducted in our laboratories on biological sclerotized structures, such as insect cuticles, egg cases, egg shells, cocoons and marine coral skeletons, describes some of the progress made on problems in insect and coral biochemistry, and previews a few of the experiments that we hope to conduct in the future. Solids N M R also has been used to investigate the compositions of other types of complex organic materials found in plants, animals, and microbes and offers great potential for future development of more powerful analytical techniques. We have also used solids N M R to probe the types of covalent interactions that arise when some of these materials are assembled and stabilized. Because of the intractable nature of sclerotized structures, there is little quantitative data available about the structural composition or cross-linking chemistry. This review focuses on compositional and developmental information sought by those interested in the chemistry of organic materials found in nature, rather than on details of the analytical methods. N M R techniques were developed initially (8) to investigate nuclear properties of several different elements (e.g. 9). Today, N M R is a powerful tool for solving problems in structural chemistry and, most recently, medical imaging. With the development of line-narrowing and gradient-imaging methods in the last 20 years, N M R spectroscopy has blossomed into a major technique in solid-state materials science and now provides a noninvasive approach to investigate detailed structures of heterogeneous materials such as C - and N-containing macromolecules and other polymeric composites. Sclerotization is a complex process used by insects and other invertebrate animals to confer stability and mechanical versatility to their cuticular exoskeletons and certain other proteinaceous structures. Cross polarization (CP), dipolar decoupling, magic angle spinning (MAS), magnetization dephasing, and isotopic enrichment provide high resolution C and **N N M R spectra that yield information about the types and relative concentrations of carbon and nitrogen atoms as well as internuclear distances and covalent bonding between specific carbons and nitrogens. Relative amounts of protein, chitin, catechols, lipids, pigment, and oxalate have been estimated, and covalent interactions between protein nitrogens and catechol carbons detected. The results of these solids N M R studies together with those of traditional chemical analyses support the hypothesis that sclerotization of protective structures in insects, corals, and other invertebrates occurs primarily when quinones derived from various compounds containing the catechol moiety (including catecholamines and o-diphenols with acid, aldehyde, or alcohol side chains) form cross-links and adducts with functional groups of proteins incorporated into these structures.


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Solids N M R methodology N M R measures the radio frequencies emitted and the rates of realignment of nuclear spins in a magnetic field after atomic nuclei absorb energy from radio frequency (rf) pulses. The single-resonance, natural abundance, C N M R spectrum of a solid usually consists of a single, broad, featureless line, with a width of approximately 20 kHz (Fig. 1, top). The major source of line broadening in this situation is the static dipolar interaction between carbons and nearby protons (10). These protons include covalently bonded methine, methylene, or methyl protons, together with more distant, indirectly bonded protons. Dipolar interactions depend upon the orientations of internuclear vectors with the applied magnetic field. In a crystal powder or an amorphous material, all orientations occur, resulting in a broad distribution of dipolar splittings. Dipolar decoupling removes this broadening in a straightforward way. If the C resonance is observed in the presence of a strong rf field at the Larmor resonancefrequencyof the protons, the protons undergo rapid transitions or spin flips, which cause the time-averaged dipolarfieldgenerated by the protons at the carbon nucleus to disappear. This process is analogous to the more familiar scalar decoupling used to remove spin-spin splitting from high resolution C N M R of liquids (77). The only difference is the strength of the rf fields used in the two experiments. Dipolar decoupling requires rf fields greater than the local fields experienced by the protons arising from H- H and Ή - ^ Ο interactions. For a typical solid, this might require an rf field of 40 kHz, or about 10 times as large as that necessary to perform ordinary scalar decoupling (10). With dipolar decoupling, the C N M R spectrum of a solid begins to show signs of improved resolution (Fig. 1, middle). A typical spectrum now has a width of approximately 15 kHz at a C Larmor frequency of 50 M H z with a few spectral features clearly evident. The spectrum is not, however, of liquidlike high-resolution quality. The remaining broadening is due to chemical shift anisotropy (CSA). The magnetic field at a carbon nucleus depends upon the shielding or screening afforded by the surrounding electron density. In general, the surrounding electron density is not symmetric. Thus, the chemical shift of, for example, a carbonyl carbon in an ester group, depends upon whether the C - 0 carbonyl is lined along the magnetic field, is perpendicular to it, or is in some other orientation. In a liquid with rapid molecular motion, only an average or isotropic chemical shift is observed. In a single crystal, a single chemical shift may be observed, but its value depends upon the orientation of the crystal relative to the magneticfield(72). In an amorphous solid or crystal powder, on the other hand, a complicated C S A line shape is observed that arises from the sum of all possible chemical shifts. For a typical solid with a variety of chemically different carbons, the C N M R spectrum is a sum of different C S A patterns, having somewhat different shapes and, most importantly, having different isotropic centers. Overlapping C S A patterns for carbonyl carbons, aromatic carbons, and aliphatic carbons destroy the resolution that was expected from dipolar decoupling (Fig. 1. middle). Fortunately, a method is available to regain the lost resolution. The broadening arises from restrictions placed on molecular motion in the solid. In the laboratory, we can supply a kind of molecular motion by mechanically


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C r t m r of solids smqk resonance


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Figure 1. Schematic C N M R lineshapes for a disordered sample in the solid state. Single-resonance spectra are broadened by strong, orientationdependent Ή - ^ Ο dipolar interactions (top). These are removed by resonant irradiation of the protons, which reveals overlapping asymmetric lineshapes (middle) arising from the orientation dependence of the C chemical shift. High-speed mechanical rotation of the solid at the magic angle produces liquid-like lineshapes (bottom). Adapted from references 10 and 23. 1 3



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rotating the sample about the diagonal of a cube whose edges are provided by the rectilinear coordinate system defined by the applied static magnetic field (75). The rotation axis is at half the tetrahedral angle relative to the applied field, the so-called "magic angle" of 54.7%. This mechanical rotation interchanges axes and internuclear directions relative to the magnetic field and, therefore, has many of the same averaging properties as isotropic motion (Fig. 1, bottom). Fast M A S is achieved using the same kind of gas-supported bearings that are used in high speed centrifuges. The combination of dipolar decoupling and fast M A S affords resolution that usually is limited only by variations of bulk susceptibility within the sample, typically about 0.5 ppm. Solids N M R experiments combine cross polarization for sensitivity enhancement of the signal to noise ratio (70) with dipolar decoupling to remove dipolar interactions from protons and M A S for high resolution of chemical shifts (14-17). Assignments of carbon chemical shifts are made by comparison with literature values or chemical shifts of standard compounds. Table 1 lists the chemical shift assignments in the C CP-MAS N M R spectra of organic materials. For example, carbon chemical shifts for chitin were assigned by comparison to carbon solution and solid C N M R spectra of 2-acetamido-2deoxyglucopyranoside and crab chitin (Fukamizo et al., 1986). In general, signals from chitin, protein, catechol, and lipid carbon dominate the natural abundance C solid-state N M R spectra. Concentrations of carbons can be estimated by extrapolation of signal intensity to zero contact time and comparison with the signal from an external standard compound. Difference spectra from single and double cross-polarization (DCP) experiments allow measurement of heteronuclear coupling between two stable isotopes that are within approximately 2 Â of each other (18). A relatively recent technique for M A S solid-state NMR, rotational echo double resonance (REDOR; 19, 20), is more sensitive than D C P in measuring long-range heteronuclear interactions up to about 5 Â for C - N , 8 Â for C- P and 12 A for C - F . For REDOR analysis, magnetization on one rare spin rf channel is dephased by rotor-synchronized pulses on a second isotope channel, while interactions with protons are suppressed by dipolar decoupling. The extent of dephasing has a simple relation to the strength of the dipolar coupling and the internuclear distance. REDOR provides a direct measure of heteronuclear dipolar coupling between isolated pairs of labeled nuclei. In a solid with a C - N labeled pair, for example, the C rotational echoes that form each rotor period following a Ή - ^ Ο cross polarization transfer can be prevented from reaching full intensity by insertion of a N π pulse each half rotor period. The REDOR difference (the difference between a C N M R spectrum obtained under these conditions and one obtained with no N π pulses) has a strong dependence on the C - N dipolar coupling and, hence, the C - N internuclear distance. R E D O R is described as double resonance even though three radio frequencies (for example, ^ C and N) are used, because the protons are removed from the important evolution part of the experiment by resonant decoupling. The dephasing of magnetization in REDOR arises from a local dipolar C - N field gradient and involves no polarization transfer. R E D O R has no dependence on C or N chemical shift tensors and does not require resolution of a C - N coupling in the chemical shift dimension. Another technique that enables high-resolution N M R analysis of specific 1 3

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172 170 155 144 129 116 104 82 75 72 60 55 44 33 23 19

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C NMR spectra of insect support structures.

Carbonyl carbons of chitin, protein, lipid, and catechol acyl groups Carbonyl carbons of oxalate Phenoxy carbon of tyrosine, guanidino carbons in arginine Phenoxy carbons of catechols Aromatic carbons Tyrosine carbons 3 and 5, imidazole carbon 4, catechol carbons 2 and 5 GlcNAc carbon 1 GlcNAc carbon 4 GlcNAc carbon 5 GlcNAc carbon 3 GlcNAc carbon 6, amino acid α-carbons GlcNAc carbon 2, amino acid α-carbons Aliphatic carbons of amino acids, catechols, and lipids Aliphatic carbons of amino acids, catechols, and lipids Methyl carbons of chitin, protein, lipid, and catechol acyl groups; amino acid methyne carbons Methyl carbons of amino acids and lipids

Assignment

*Values relative to external tetramethylsilane reference.

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Resonance no. Values (ppm)*

Table 1. Chemical shift assignments of resonances in the CP-MAS


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atoms in compounds is selective isotopic enrichment, which helps to rniriirnize the background of both isotopic natural abundance and similar chemical shifts that occur in rather complex molecular assemblies or mixtures. Labeling has been used to examine the chemical compositions and structures of a variety of sclerotized proteinaceous materials from insects (21) and marine mussels (22), all of them not easily amenable to analysis by conventional solution-based techniques. We have employed labeling with detection by M A S solids N M R for measuring distances between pairs of spin-1/2 heteronuclei. In particular, our method of choice determines specific internuclear separations between dilute C , N spin pairs that can be incorporated into samples by isotopic enrichment. The low natural abundance of C and N (1.11 and 0.37 atom %, respectively) makes these spins ideal candidates for site-specific isotopic labeling. Internuclear distances between spin pairs can be determined from the 1/r distance dependence of the dipolar interaction. A sample is needed that can efficiently incorporate labels into specific atom pairs, while at the same time not scrambling the labels into other types of bonding. Solids N M R experiments can not only be difficult to conduct but also expensive. Major costs are incurred from construction of spectrometers and probes, and, to a lesser degree, the synthesis or purchase of compounds with specific atoms enriched with NMR-active nuclei. Although a number of commercial instruments are available, many of them are primarily solution-based instruments adapted for solid samples rather than designed solely for the analysis of solids, which limits their performance. Also, rather large samples (~1 micromole of label) are needed for solids N M R analysis, and in the case of isotopic enrichment, a biological system that incorporates micromolar amounts of a label into appropriate chemical structures usually is required. 1 3

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Solids N M R applications 1 3

Schaefer and Stejskal (23) first obtained natural abundance C N M R spectra of good resolution from completely solid amorphous samples. Comparable natural abundance N N M R spectra are generally not of usuable quality because signal levels are reduced by an order of magnitude. In addition, N is four times less abundant than C and three times less sensitive as an N M R nucleus. Peter et al. (24) first used solids N M R to study the compositions of sclerotized insect cuticles. Similar natural abundance C N M R spectra were collected for pupal cases and exuviae of several insect species, which generally indicated comparable compositions of protein, chitin, and catechols (Fig. 2). Those results were interpreted to be consistent with the hypothesis that the sclerotization of cuticle occurs by the denaturation of structural proteins by polyphenols compounds, but the actual mechanism of sclerotization was not determined. Subsequently, Schaefer et al. (18) and Merritt et al. (25) reported the results of a study on the composition and several heteronuclear interactions of pupal and adult moth cuticles from the tobacco hornworm, Manduca sexta. Selective C and N isotopic enrichment of cuticle by injection of appropriately labeled precursor amino acids and the catecholamine dopamine (Fig. 3) enabled the detection of covalent linkages between ring nitrogens of protein histidyl residues and ring carbons of the catecholamine, which is a precursor of quinonoid tanning 1 5

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Figure 2. Cross-polarization magic-angle spinning C N M R spectra of Manduca sexta chitin (top) and pupal exuviae (bottom). Adapted from ref. 18.


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β-[

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Analysis of Intractable Biological Samples by Solids NMR - ACS

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