MA'AT Analysis: Probability Distributions of Molecular Torsion Angles in Solution from NMR Spectroscopy.

ConspectusMonosaccharides adopt multiple conformations in solution, and this structural complexity increases significantly when they are assembled into oligosaccharides and polysaccharides. Characterization of the conformational properties of saccharides in solution by NMR spectroscopy has been hampered by several complicating factors, including difficulty interpreting spectra because of significant signal overlap, population averaging of NMR parameters, and unique properties of the spectra that make accurate measurements of NMR parameters prone to error (e.g., non-first-order effects on J-couplings). Current conformational assignments rely heavily on theoretical calculations, especially molecular dynamics (MD) simulations, to interpret the experimental NMR parameters. While these studies assert that the available experimental data fit the calculated models well, a lack of independent experimental validation of the force fields from which MD models are derived and an inability to test all possible models that might be compatible with the experimental data in an unbiased manner make the approach less than ideal.NMR spin couplings or J-couplings have been used as structure constraints in organic and other types of molecules for more than six decades. The dihedral angle dependence of vicinal (three-bond) 1H-1H spin couplings (3JHH) first described by Karplus led to an explosion of applications for a wide range of conformational problems. Other vicinal J-couplings (e.g., 3JCCOP, 3JHCOP, and 3JCOCH) have been found to exhibit similar dihedral angle dependencies. 3J values have been used to assign the preferred conformation in molecules that are conformationally homogeneous. However, many molecules, particularly those in biological systems, are conformationally flexible, which complicates structural interpretations of J values in solution. Three-state staggered models are often assumed in order to deconvolute the conformationally averaged J values into conformer populations. While widely applied, this approach assumes highly idealized models of molecular torsion angles that are likely to be poor representations of those found in solution. In addition, this treatment often gives negative populations and neglects the presence of librational averaging of molecular torsion angles.Recent work in this research group has focused on the development of a hybrid experimental-computational method, MA'AT analysis, that provides probability distributions of molecular torsion angles in solution that can be superimposed on those obtained by MD. Ensembles of redundant NMR spin couplings, including 3J (vicinal), 2J (geminal), and sometimes 1J (direct) values, are used in conjunction with circular statistics to provide single- and multistate models of these angles. MA'AT analysis provides accurate mean torsion angles and circular standard deviations (CSDs) of each mean angle that describe the librational motion about the angle. Both conformational equilibria and dynamics are revealed by the method. In this Account, the salient features of MA'AT analysis are discussed, including some applications to conformational problems involving saccharides and peptides.

Methyl α-D-galactopyranosyl-(1→3)-ß-D-galactopyranoside and methyl ß-D-galactopyranosyl-(1→3)-ß-D-galactopyranoside: Glycosidic linkage conformation determined from MA'AT analysis.

MA'AT analysis has been applied to two biologically-important O-glycosidic linkages in two disaccharides, α-D-Galp-(1→3)-ß-D-GalpOMe (3) and ß-D-Galp-(1→3)-ß-D-GalpOMe (4). Using density functional theory (DFT) to obtain parameterized equations relating a group of trans-O-glycosidic NMR spin-couplings to either phi (ϕ') or psi (ψ'), and experimental 3JCOCH, 2JCOC, and 3JCOCC spin-couplings measured in aqueous solution in 13C-labeled isotopomers, probability distributions of ϕ' and ψ' in each linkage were determined and compared to those determined by aqueous 1-µs molecular dynamics (MD) simulation. Good agreement was found between the MA'AT and single-state MD conformational models of these linkages for the most part, with modest (approximately <15°) differences in the mean values of ϕ' and ψ', although the envelope of allowed angles (encoded in circular standard deviations or CSDs) is consistently larger for ϕ' determined from MA'AT analysis than from MD for both linkages. The MA'AT model of the α-Galp-(1→3)-ß-Galp linkage agrees well with those determined previously using conventional NMR methods (3JCOCH values and/or 1H-1H NOEs), but some discrepancy was observed for the ß-Galp-(1→3)-ß-Galp linkage, which may arise from errors in the conventions used to describe the linkage torsion angles. Statistical analyses of X-ray crystal structures show ranges of ϕ' and ψ' for both linkages that include the mean angles determined from MA'AT analyses, although both angles adopt a wide range of values in the crystalline state, with ϕ' in ß-Galp-(1→3)-ß-Galp linkages showing greater-than-expected conformational variability.

One-bond 13C-13C spin-coupling constants in saccharides: a comparison of experimental and calculated values by density functional theory using solid-state 13C NMR and X-ray crystallography.

Methyl aldohexopyranosides were 13C-labeled at contiguous carbons, crystallized, and studied by single-crystal X-ray crystallography and solid-state 13C nuclear magnetic resonance (NMR) spectroscopy to examine the degree to which density functional theory (DFT) can calculate one-bond 13C-13C spin-coupling constants (1JCC) in saccharides with sufficient accuracy to permit their use in MA'AT analysis, a newly-reported hybrid DFT/NMR method that provides probability distributions of molecular torsion angles in solution (Zhang et al., J. Phys. Chem. B, 2017, 121, 3042-3058; Meredith et al., J. Chem. Inf. Model., 2022, 62, 3135-3141). Experimental 1JCC values in crystalline samples of the doubly 13C-labeled compounds were measured by solid-state 13C NMR and compared to those calculated from five different DFT models: (1) 1JCC values calculated from single structures identical to those observed in crystalline samples by X-ray crystallography (all atom refinement); (2) 1JCC values calculated from the single structures in (1) but after Hirshfeld atom refinement (HAR); (3) 1JCC values calculated from the single structures in (1) after DFT-optimization of hydrogen atoms only; and (4 and 5) 1JCC values calculated in rotamers of torsion angle θ2 (C1-C2-O2-O2H) or ω (C4-C5-C6-O6) from which either specific or generalized parameterized equations were obtained and used to calculate 1JCC values in the specific θ2 or ω rotamers observed in crystalline samples. Good qualitative agreement was observed between calculated 1JCC values and those measured by solid-state 13C NMR regardless of the DFT model, but in no cases were calculated 1JCC values quantitative, differing (over-estimated) on average by 4-5% from experimental values. These findings, and those reported recently from solution NMR studies (Tetrault et al., J. Phys. Chem. B 2022, 126, 9506-9515), indicate that improvements in DFT calculations are needed before calculated 1JCC values can be used directly as reliable constraints in MA'AT analyses of saccharides in solution.

Radiation damage and dose limits in serial synchrotron crystallography at cryo- and room temperatures.

Radiation damage limits the accuracy of macromolecular structures in X-ray crystallography. Cryogenic (cryo-) cooling reduces the global radiation damage rate and, therefore, became the method of choice over the past decades. The recent advent of serial crystallography, which spreads the absorbed energy over many crystals, thereby reducing damage, has rendered room temperature (RT) data collection more practical and also extendable to microcrystals, both enabling and requiring the study of specific and global radiation damage at RT. Here, we performed sequential serial raster-scanning crystallography using a microfocused synchrotron beam that allowed for the collection of two series of 40 and 90 full datasets at 2- and 1.9-Å resolution at a dose rate of 40.3 MGy/s on hen egg white lysozyme (HEWL) crystals at RT and cryotemperature, respectively. The diffraction intensity halved its initial value at average doses (D1/2) of 0.57 and 15.3 MGy at RT and 100 K, respectively. Specific radiation damage at RT was observed at disulfide bonds but not at acidic residues, increasing and then apparently reversing, a peculiar behavior that can be modeled by accounting for differential diffraction intensity decay due to the nonuniform illumination by the X-ray beam. Specific damage to disulfide bonds is evident early on at RT and proceeds at a fivefold higher rate than global damage. The decay modeling suggests it is advisable not to exceed a dose of 0.38 MGy per dataset in static and time-resolved synchrotron crystallography experiments at RT. This rough yardstick might change for proteins other than HEWL and at resolutions other than 2 Å.

MA'AT Analysis of Aldofuranosyl Rings: Unbiased Modeling of Conformational Equilibria and Dynamics in Solution.

MA'AT analysis has been applied to methyl ß-d-ribofuranoside (3) and methyl 2-deoxy-ß-d-erythro-pentofuranoside (4) to demonstrate the ability of this new experimental method to determine multi-state conformational equilibria in solution. Density functional theory (DFT) was used to obtain parameterized equations for >20 NMR spin-coupling constants sensitive to furanose ring conformation in 3 and 4, and these equations were used in conjunction with experimental spin-couplings to produce unbiased MA'AT models of ring pseudorotation. These models describe two-state north-south conformational exchange consistent with results obtained from traditional treatments of more limited sets of NMR spin-couplings (e.g., PSEUROT). While PSEUROT, MA'AT, and aqueous molecular dynamics models yielded similar two-state models, MA'AT analysis gives more reliable results since significantly more experimental observables are employed compared to PSEUROT, and no assumptions are needed to render the fitting tractable. MA'AT models indicate a roughly equal distribution of north and south ring conformers of 4 in aqueous (2H2O) solution compared to ∼80% north forms for 3. Librational motion about the mean pseudorotation phase angles P of the preferred north and south conformers of 3 in solution is more constrained than that for 4. The greater rigidity of the ß-ribo ring may be caused by synergistic stereoelectronic effects and/or noncovalent (e.g., hydrogen-bonding) interactions in solution that preferentially stabilize north forms of 3. MA'AT analysis of oligonucleotides and other furanose ring-containing biomolecules promises to improve current experimental models of sugar ring behavior in solution and help reveal context effects on ring conformation in more complex biologically important systems.

N-Acetyl Side-Chain Conformation in Saccharides: Solution Models Obtained from MA'AT Analysis.

MA'AT analysis has been applied to model the conformational properties of N-acetyl side-chains in biologically important GlcNAc and ManNAc monosaccharides and in a ßGlcNAc-(1→4)-ßGlcNAc disaccharide. Density functional theory calculations were conducted to obtain parameterized equations that relate the magnitudes and signs of 10 spin-coupling constants to conformations of the C2-N2 bonds of GlcNAc and ManNAc. Six of these equations were used with experimental J-couplings, measured in H2O/2H2O and DMSO-d6 solvents in selectively 13C-labeled compounds, to model the C1-C2-N2-C1' torsion angle (θ1) in GlcNAc and ManNAc residues. MA'AT analysis gave mean values of θ1 of 106° for αGlcNAc and ∼116° for ßGlcNAc residues, with circular standard deviations (CSDs) of 21-22° in aqueous solution, in excellent agreement with those obtained by aqueous molecular dynamics (MD) simulation. Parameter space plots revealed unique MA'AT fits of the data, and root mean squared deviations (<0.2 Hz) were twofold smaller than those back-calculated from MD models, indicating that the MA'AT models better fit the experimental J-couplings. Context effects on both θ1 values were found to be small in a ßGlcNAc-(1→4)-ßGlcNAc disaccharide. MA'AT analysis gave a mean value of θ1 of 249° for αManNAc in H2O/2H2O, with a CSD of ∼19°, with both values in good agreement with MD. MA'AT models of N-acetyl side-chains are similar to those obtained previously for O-acetyl side-chains (J. Phys. Chem. B 2017, 121, 66-77). Both O- and N-acetylation conformationally constrain the C-O or C-N bonds relative to the same bonds in unsubstituted compounds. The present work confirms the ability of MA'AT analysis to reveal relatively small changes in mean molecular torsion angles in solution and provides additional evidence of the method as an experimental tool complementary to MD simulation.

Reactivity of Zn+aq in high-temperature water radiolysis.

Reactivity of transients involving Zn+ in high-temperature water radiolysis has been studied in the temperature range of 25-300 °C. The reduced monovalent zinc species were generated from an electron transfer process between the hydrated electron and Zn2+ ions using pulse radiolysis. The Zn+ species can subsequently be oxidized by the radiolytically-produced oxidizing species: ˙OH, H2O2 and ˙H. We find that the absorption of monovalent zinc is very sensitive to the pH of the medium. An absorption maximum at 306-311 nm is most pronounced at pH 7 and the signal then decreases in acidic media where the reducing electrons are competitively captured by protons. At pH values higher than 7, hydroxo-forms of Zn2+ are created and the maximum of the absorption signal begins to shift to the red spectral region. We find that the optical spectrum of Zn+aq cannot be fully explained in terms of a charge-transfer to solvent (CTTS) process, which was previously proposed. Reaction rates of most of the recombination reactions investigated follow the empirical Arrhenius relationship at temperatures up to 200 °C and have been determined at higher temperatures for the first time. A bimolecular disproportionation reaction of Zn+aq is not observed under the conditions investigated.

OH radical reactions with the hydrophilic component of sphingolipids.

In this work, using the example of model compounds, we studied the reactions resulting from the interaction of OH radicals with the hydrophilic part of sphingolipids. We compared the stopped-flow EPR spectroscopy and pulse radiolysis with optical detection methods to characterize radical intermediates formed in the reaction of OH radicals with glycerol, serinol and N-boc-serinol. Quantum chemical calculations were also performed to help interpret the observed experimental data. It was shown that H-abstraction from the terminal carbon atom is the main process that is realized for all the studied compounds. The presence of the unsubstituted amino group (-NH2) is seen to completely change the reaction properties of serinol in comparison with those observed in glycerol and N-boc serinol.

Two-bond 13C-13C spin-coupling constants in saccharides: dependencies on exocyclic hydroxyl group conformation.

Seven doubly 13C-labeled isotopomers of methyl ß-D-glucopyranoside, methyl ß-D-xylopyranoside, methyl ß-D-galactopyranoside, methyl ß-D-galactopyranosyl-(1→4)-ß-D-glucopyranoside and methyl ß-D-galactopyranosyl-(1→4)-ß-D-xylopyranoside were prepared, crystallized, and studied by single-crystal X-ray crystallography and solid-state 13C NMR spectroscopy to determine experimentally the dependence of 2JC1,C3 values in aldopyranosyl rings on the C1-C2-O2-H torsion angle, θ2, involving the C2 carbon of the C1-C2-C3 coupling pathway. Using X-ray crystal structures to determine θ2 in crystalline samples and by selecting compounds that exhibit a relatively wide range of θ2 values in the crystalline state, 2JC1,C3 values measured in crystalline samples were plotted against θ2 and the resulting plot compared to that obtained from density functional theory (DFT) calculations. For θ2 values ranging from ∼90° to ∼240°, very good agreement was observed between the experimental and theoretical plots, providing strong validation of DFT-calculated spin-coupling dependencies on exocyclic C-O bond conformation involving the central carbon of geminal C-C-C coupling pathways. These findings provide new experimental evidence supporting the use of 2JCCC values as non-conventional spin-coupling constraints in MA'AT conformational modeling of saccharides in solution, and the use of NMR spin-couplings not involving coupled hydroxyl hydrogens as indirect probes of C-O bond conformation. Solvomorphism was observed in crystalline ßGal-(1→4)-ßGlcOCH3 wherein the previously-reported methanol solvate form was found to spontaneously convert to a monohydrate upon air-drying, leading to small but discernible conformational changes in, and a new crystalline form of, this disaccharide.

Pulse Radiolysis Investigation of Radicals Derived from Water-Soluble Cyanine Dyes: Implications for Super-resolution Microscopy.

Light-induced blinking, an inherent feature of many forms of super-resolution microscopy, has been linked to transient reduction of the fluorescent cyanine dye used as an imaging agent. There is, however, only scant literature information related to one-electron reduced cyanine dyes, especially in an aqueous environment. Here, we examine a small series of cyanine dyes, possessing disparate π-conjugation lengths, under selective reducing or oxidizing conditions. The experiment allows recording of both differential absorption spectra and decay kinetics of the resultant one-electron reduced or oxidized transient species in water. Relative to the ground state, absorption transitions for the various radicals are weak and somewhat broadened but do allow correlation with the π-conjugation length. In all cases, absorption maxima lie to the blue of the main ground-state transition. Under anaerobic conditions, the transient species decay on the microsecond to millisecond time scale, with the mean lifetime depending on molecular structure, radiation dose, and dye concentration. The experimental absorption spectra recorded for the one-electron reduced radicals and the presumed dimer cation radical compare well to spectra obtained from time-dependent density functional theory calculations. The results allow conclusions to be drawn regarding the plausibility of the reduced species being responsible for light-induced blinking in direct stochastic optical reconstruction microscopy.

Reconciling MA'AT and molecular dynamics models of linkage conformation in oligosaccharides.

MA'AT conformational models of the phi torsion angles of O-glycosidic linkages differ from those obtained from MD simulation. To determine the source of the discrepancy, MA'AT analyses were performed using DFT-derived equations obtained with and without psi constraints. The resulting phi models were essentially the same, indicating a force-field problem. Circular standard deviations (CSDs) were found to provide reliable estimates of torsional averaging.

Dissociative electron attachment to amide bond containing molecules: N-ethylformamide and N-ethylacetamide.

To advance our quest to understand the role of low energy electrons in biomolecular systems, we performed investigations on dissociative electron attachment (DEA) to gas-phase N-ethylformamide (NEF) and N-ethylacetamide (NEA) molecules. Both molecules contain the amide bond, which is the linkage between two consecutive amino acid residues in proteins. Thus, their electron-induced dissociation can imitate the resonant behavior of the DEA process in more complex biostructures. Our experimental results indicate that in these two molecules, the dissociation of the amide bond results in a double resonant structure with peaks at ∼5 eV and 9 eV. We also determined the energy position of resonant states for several negative ions, i.e., the other dissociation products from NEF and NEA. Our predictions of dissociation channels were supported by density functional theory calculations of the corresponding threshold energies. Our results and those previously reported for small amides and peptides imply the fundamental nature for breakage of the amide bond through the DEA process.

Use of Circular Statistics To Model αMan-(1→2)-αMan and αMan-(1→3)-α/ßMan O-Glycosidic Linkage Conformation in 13C-Labeled Disaccharides and High-Mannose Oligosaccharides.

A new experimental method, MA' AT analysis, has been applied to investigate the conformational properties of O-glycosidic linkages in several biologically important mannose-containing di- and oligosaccharides. Methyl α-d-mannopyranosyl-(1→2)-α-d-mannopyranoside (2), methyl α-d-mannopyranosyl-(1→3)-α-d-mannopyranoside (3), and methyl α-d-mannopyranosyl-(1→3)-ß-d-mannopyranoside (4) were prepared with selective 13C-enrichment to enable the measurement of NMR scalar couplings across their internal O-glycosidic linkages. Density functional theory (DFT) was used to parameterize equations for JCH and JCC values in 2-4 that are sensitive to phi (ϕ) and psi (ψ). The experimental J-couplings and parameterized equations were treated using a circular statistics algorithm encoded in the MA' AT program. Conformations about ϕ and ψ treated using single-state von Mises models gave excellent fits to the ensembles of redundant J-couplings. Mean values and circular standard deviations (CSDs) for each linkage torsion angle ϕ (CSD) and ψ (CSD) in 2, -29° (25°) and 20° (22°); in 3, -36° (36°) and 8° (27°); in 4, -37° (34°) and 10° (26°); ϕ = H1'-C1'-O1'-CX and ψ = C1'-O1'-CX-HX (CX = aglycone carbon) were compared to histograms obtained from 1 µs aqueous molecular dynamics (MD) simulations and X-ray database statistical analysis. MA' AT-derived models of ψ were in very good agreement with the MD and X-ray data, but not those of ϕ, suggesting a need for force field revision. The effect of structural context on linkage conformation was also investigated in four selectively 13C-labeled homomannose tri- and tetrasaccharides using the MA' AT method. In the cases examined, context effects were found to be small.

Radiation-damage investigation of a DNA 16-mer.

In macromolecular crystallography, a great deal of effort has been invested in understanding radiation-damage progression. While the sensitivity of protein crystals has been well characterized, crystals of DNA and of DNA-protein complexes have not thus far been studied as thoroughly. Here, a systematic investigation of radiation damage to a crystal of a DNA 16-mer diffracting to 1.8 Šresolution and held at 100 K, up to an absorbed dose of 45 MGy, is reported. The RIDL (Radiation-Induced Density Loss) automated computational tool was used for electron-density analysis. Both the global and specific damage to the DNA crystal as a function of dose were monitored, following careful calibration of the X-ray flux and beam profile. The DNA crystal was found to be fairly radiation insensitive to both global and specific damage, with half of the initial diffraction intensity being lost at an absorbed average diffraction-weighted dose, D1/2, of 19 MGy, compared with 9 MGy for chicken egg-white lysozyme crystals under the same beam conditions but at the higher resolution of 1.4 Å. The coefficient of sensitivity of the DNA crystal was 0.014 Å2 MGy-1, which is similar to that observed for proteins. These results imply that the significantly greater radiation hardness of DNA and RNA compared with protein observed in a DNA-protein complex and an RNA-protein complex could be due to scavenging action by the protein, thereby protecting the DNA and RNA in these studies. In terms of specific damage, the regions of DNA that were found to be sensitive were those associated with some of the bound calcium ions sequestered from the crystallization buffer. In contrast, moieties farther from these sites showed only small changes even at higher doses.

Dipole-Supported Electronic Resonances Mediate Electron-Induced Amide Bond Cleavage.

Dissociative electron attachment (DEA) plays a key role in radiation damage of biomolecules under high-energy radiation conditions. The initial step in DEA is often rationalized in terms of resonant electron capture into one of the metastable valence states of a molecule followed by its fragmentation. Our combined theoretical and experimental investigations indicate that the manifold of states responsible for electron capture in the DEA process can be dominated by core-excited (shake-up) dipole-supported resonances. Specifically, we present the results of experimental and computational studies of the gas-phase DEA to three prototypical peptide molecules, formamide, N-methylformamide (NMF), and N,N-dimethyl-formamide (DMF). In contrast to the case of electron capture by positively charged peptides in which amide bond rupture is rare compared to N─C_{α} bond cleavage, fragmentation of the amide bond was observed in each of these three molecules. The ion yield curves for ions resulting from this amide bond cleavage, such as NH_{2}^{-} for formamide, NHCH_{3}^{-} for NMF, and N(CH_{3})_{2}^{-} for DMF, showed a double-peak structure in the region between 5 and 8 eV. The peaks are assigned to Feshbach resonances including core-excited dipole-supported resonances populated upon electron attachment based on high-level electronic structure calculations. Moreover, the lower energy peak is attributed to formation of the core-excited resonance that correlates with the triplet state of the neutral molecule. The latter process highlights the role of optically spin-forbidden transitions promoted by electron impact in the DEA process.

Synthesis and O-Glycosidic Linkage Conformational Analysis of 13C-Labeled Oligosaccharide Fragments of an Antifreeze Glycolipid.

NMR studies of two 13C-labeled disaccharides and a tetrasaccharide were undertaken that comprise the backbone of a novel thermal hysteresis glycolipid containing a linear glycan sequence of alternating [ßXyl p-(1→4)-ßMan p-(1→4)] n dimers. Experimental trans-glycoside NMR J-couplings, parameterized equations obtained from density functional theory (DFT) calculations, and an in-house circular statistics package ( MA'AT) were used to derive conformational models of linkage torsion angles ϕ and ψ in solution, which were compared to those obtained from molecular dynamics simulations. Modeling using different probability distribution functions showed that MA'AT models of ϕ in ßMan(1→4)ßXyl and ßXyl(1→4)ßMan linkages are very similar in the disaccharide building blocks, whereas MA'AT models of ψ differ. This pattern is conserved in the tetrasaccharide, showing that linkage context does not influence linkage geometry in this linear system. Good agreement was observed between the MA'AT and MD models of ψ with respect to mean values and circular standard deviations. Significant differences were observed for ϕ, indicating that revision of the force-field employed by GLYCAM is probably needed. Incorporation of the experimental models of ϕ and ψ into the backbone of an octasaccharide fragment leads to a helical amphipathic topography that may affect the thermal hysteresis properties of the glycolipid.

13C-13C spin-coupling constants in crystalline 13C-labeled saccharides: conformational effects interrogated by solid-state 13C NMR spectroscopy.

Solid-state 13C NMR spectroscopy has been used in conjunction with selectively 13C-labeled mono- and disaccharides to measure 13C-13C spin-couplings (JCC) in crystalline samples. This experimental approach allows direct correlation of JCC values with specific molecular conformations since, in crystalline samples, molecular conformation is essentially static and can be determined by X-ray crystallography. JCC values measured in the solid-state in known molecular conformations can then be compared to corresponding JCC values calculated in the same conformations using density functional theory (DFT). The latter comparisons provide important validation of DFT-calculated J-couplings, which is not easily obtained by other approaches and is fundamental to obtaining reliable experiment-based conformational models from redundant J-couplings by MA'AT analysis. In this study, representative 1JCC, 2JCCC and 3JCOCC values were studied as either intra-residue couplings in the aldohexopyranosyl rings of monosaccharides or inter-residue (trans-glycoside) couplings in disaccharides. The results demonstrate that (a) accurate JCC values can be measured in crystalline saccharides that have been suitably labeled with 13C, and (b) DFT-calculated JCC values compare favorably with those determined by solid-state 13C NMR when molecular conformation is a constant in both determinations.

Dissociative electron attachment induced ring opening in five-membered heterocyclic compounds.

Five-membered heterocyclic structures, which exist widely in biological systems and play an active role in various biochemical processes, have been studied extensively from a fundamental perspective. Here, the fragmentation patterns of isoxazole, a representative five-membered heterocycle, upon dissociative electron attachment (DEA) were examined carefully by comparing isoxazole's products with those of its methylated derivatives. It was found that the most dominant DEA pathway occurs through the loss of hydrogen at C(3), which leads to ring opening by O-N bond cleavage at an energy of ∼1.5 eV. The ring opening was investigated further for DEA to other related five-membered ring compounds, i.e., oxazole and thiazole. The DEA-induced hydrogen loss was much less pronounced or quenched completely in these two compounds and simultaneous ring-opening behavior was not detected. This observation is of special interest to applied fields, for example, the pharmaceutical industry, because several drugs that contain isoxazole substructures exhibit extensive ring opening during biotransformation.

OH cleavage from tyrosine: debunking a myth.

During macromolecular X-ray crystallography experiments, protein crystals held at 100 K have been widely reported to exhibit reproducible bond scission events at doses on the order of several MGy. With the objective to mitigate the impact of radiation damage events on valid structure determination, it is essential to correctly understand the radiation chemistry mechanisms at play. OH-cleavage from tyrosine residues is regularly cited as amongst the most available damage pathways in protein crystals at 100 K, despite a lack of widespread reports of this phenomenon in protein crystal radiation damage studies. Furthermore, no clear mechanism for phenolic C-O bond cleavage in tyrosine has been reported, with the tyrosyl radical known to be relatively robust and long-lived in both aqueous solutions and the solid state. Here, the initial findings of Tyr -OH group damage in a myrosinase protein crystal have been reviewed. Consistent with that study, at increasing doses, clear electron density loss was detectable local to Tyr -OH groups. A systematic investigation performed on a range of protein crystal damage series deposited in the Protein Data Bank has established that Tyr -OH electron density loss is not generally a dominant damage pathway in protein crystals at 100 K. Full Tyr aromatic ring displacement is here proposed to account for instances of observable Tyr -OH electron density loss, with the original myrosinase data shown to be consistent with such a damage model. Systematic analysis of the effects of other environmental factors, including solvent accessibility and proximity to disulfide bonds or hydrogen bond interactions, is also presented. Residues in known active sites showed enhanced sensitivity to radiation-induced disordering, as has previously been reported.

Characterization of Neutral Radicals from a Dissociative Electron Attachment Process.

Despite decades of gas-phase studies on dissociative electron attachment (DEA) to various molecules, as yet there has been no direct detection and characterization of the neutral radical species produced by this process. In this study, we performed stepwise electron spectroscopy to directly measure and characterize the neutrals produced upon zero-electron-energy DEA to the model molecule, carbon tetrachloride (CCl_{4}). We observed the direct yield of the trichloromethyl radical (CCl_{3}^{·}) formed by DEA to CCl_{4} and measured the appearance energies of all the other neutral species. By combining these experimental findings with high-level quantum chemical calculations, we performed a complete analysis of both the DEA to CCl_{4} and the subsequent electron-impact ionization of CCl_{3}^{·}. This work paves the way toward a complete experimental characterization of DEA processes, which will lead to a better understanding of the low-energy electron-induced formation of radical species.