Conformational flexibility of xanthene‐based covalently linked dimers

The conformational flexibility of three covalently linked dimers consisting of two xanthene‐based moieties connected by a diphenyl ether linker was studied using NMR spectroscopy, X‐ray crystallography, and density functional theory (DFT) calculations. The three dimers interconvert as a function of pH: the doubly cationic dimer (Xan⁺)₂ exists in acidic solutions (pH < 0.5), the mono‐alcohol monocation Xan⁺–Xan‐OH at intermediate pH values (pH = 1–3), and the neutral diol at the highest pH‐values (pH > 3). Each dimer exhibits conformational degrees of freedom associated with rotations of either the xanthene moiety or of the diphenyl ether (DPE) linker. The barriers for rotation of the xanthylium moiety were evaluated using DFT calculations, yielding values of 23 kcal/mol for (Xan⁺)₂ and 11 kcal/mol for (Xan‐OH)₂, respectively. The rotational barrier for the diphenyl ether linker in Xan⁺–Xan‐OH (15 kcal/mol) was experimentally determined using variable temperature NMR measurements. The relative orientation of the two –OH groups in (Xan‐OH)₂ diol was investigated in solution and the solid state using NMR spectroscopy and X‐ray crystallography. The conformer observed in the solid state was found to be the In–Out conformer, while free rotation of the xanthenol units is thought to occur on the NMR timescale at room temperature. These studies are relevant for the design of linkers for efficient water oxidation catalysts. Copyright © 2016 John Wiley & Sons, Ltd.     

pH‐controlled reaction divergence of decarboxylation versus fragmentation in reactions of dihydroxyfumarate with glyoxylate and formaldehyde: parallels to biological pathways

The reactions of dihydroxyfumarate with glyoxylate and formaldehyde exhibit a unique pH‐controlled mechanistic divergence leading to different product suites by two distinct pathways. The divergent reactions proceed via a central intermediate (2,3‐dihydroxy‐oxalosuccinate, 3 , in the reaction with glyoxylate and 2‐hydroxy‐2‐hydroxymethyl‐3‐oxosuccinate, 14 , in the reaction with formaldehyde). At pH 7–8, products ( 7 , 8 , and 15 ) exclusively from a decarboxylation of the intermediate are observed, while at pH 13–14, products ( 9 , 10 , and 16 ) solely derived from a hydroxide‐promoted fragmentation of the intermediate are formed. The decarboxylative and fragmentation pathways are mutually exclusive and do not appear to coexist under the range of pH (7–14) conditions investigated. Herein, we employ a combination of quantitative ¹³C NMR measurements and density functional theory calculations to provide a rationale for this pH‐driven reaction divergence. These rationalizations also hold true for the reactions of dihydroxyfumarate produced in situ by the catalytic cyanide‐mediated dimerization of glyoxylate. In addition, the non‐enzymatic decarboxylation and fragmentation transformations of these central intermediates ( 3 and 14 ) appear to have intriguing parallels to the enzymatic reactions of oxalosuccinate and formation of glyceric acid derivatives in extant metabolism – the high and low pH mimicking the precise control exerted by the enzymes over reaction pathways. Copyright © 2016 John Wiley & Sons, Ltd.     

Detection of NMR signals with a radio-frequency atomic magnetometer

We demonstrate detection of proton NMR signals with a radio-frequency (rf) atomic magnetometer tuned to the NMR frequency of 62 kHz. High-frequency operation of the atomic magnetometer makes it relatively insensitive to ambient magnetic field noise. We obtain magnetic field sensitivity of 7 fT/Hz1/2 using only a thin aluminum shield. We also derive an expression for the fundamental sensitivity limit of a surface inductive pick-up coil as a function of frequency and find that an atomic rf magnetometer is intrinsically more sensitive than a coil of comparable size for frequencies below about 50 MHz.     

Biselective refocusing pulses and the SERF experiment

The SERF experiment is a variant of the homonuclear J-resolved experiment, in which a single coupling constant is measured. It consists of a single chemical shift selective excitation that is followed by a biselective spin echo. Recent articles mention the existence of artefacts in SERF spectra that are supposedly related to pulse imperfections. This article presents a detailed study of the biselective refocusing pulses. It also reports a method for predicting the position and amplitude of the expected and unexpected 2D spectral peaks in SERF spectra. Artefacts can be partially eliminated by phase cycling or by the introduction of static field gradient pulses in the acquisition sequence. A procedure to obtain of pure absorption peaks in SERF spectra is proposed.     

Trehalose-protected lipid membranes for determining membrane protein structure and insertion

Trehalose preserves lipid bilayers during dehydration and rehydration by replacing water to form hydrogen bonds between its own OH groups and lipid headgroups. We compare the lipid conformation and dynamics between trehalose-protected lyophilized membranes and hydrated membranes, to assess the suitability of the trehalose-containing membrane as a matrix for membrane protein structure determination. (31)P spectra indicate that the lipid headgroup of trehalose-protected dry POPC membrane (TRE-POPC) have an effective phase transition temperature that is approximately 50K higher than that of the hydrated POPC membrane. In contrast, the acyl chains have similar transition temperatures in the two membranes. Intramolecular lipid (13)C'-(31)P distances are the same in TRE-POPC and crystalline POPC, indicating that the lipid headgroup and glycerol backbone conformation is unaffected by trehalose incorporation. Intermolecular (13)C-(31)P distances between a membrane peptide and the lipid headgroups are 10% longer in the hydrated membrane at 226 K than in the trehalose-protected dry membrane at 253 K. This is attributed to residual motions in the hydrated membrane, manifested by the reduced (31)P chemical shift anisotropy, even at the low temperature of 226 K. Thus, trehalose lyoprotection facilitates the study of membrane protein structure by allowing experiments to be conducted at higher temperatures than possible with the hydrated membranes.     

Precision and sensitivity optimization of quantitative measurements in solid state NMR

This work presents a methodology for optimizing the precision, accuracy and sensitivity of quantitative solid state NMR measurements based on the external reference method. It is shown that the sample must be exclusively located within and completely span the coil region where the NMR response is directly proportional to the sample amount. We describe two methods to determine this "quantitative" coil volume, based on whether the probe is equipped or not with a gradient coil. In addition, to improve the sensitivity and the accuracy, an optimum rotor packing design is described, which allows the sample volume of the rotor to be matched to the quantitative coil volume. Experiments conducted on adamantane and NaCl, which are representative of a soft and hard material, respectively, show that one order of magnitude increase in experimental precision can be achieved with this methodology. Interestingly, the precision can be further improved by using the ERETIC method in order to compensate for most instrumental instabilities.     

A method for C(alpha) direct-detection in protonless NMR

Attempts are made to efficiently decouple (13)C nuclei without significant loss of coherence during the application of the decoupling package. Such attempts are based on the S(3)E spin-state selection method. A newly developed double S(3)E (DS(3)E) is particularly efficient for C(alpha) detection for proteins as large as 480 kDa.     

Experimental evidence of chiral crown ether complexation with aromatic amino acids

The complexation of L ‐ and D ‐enantiomers of phenylglycine, phenylalanine, and tryptophan with D ‐mannonaphto‐crown‐6‐ether in methanol solution was studied by NMR and isothermal titration calorimetry (ITC) at 298.15 K. The total heat effects attributed to the binding phenomena were measured in the range of 1.8 to 7.7 mJ, and the complexation was found stereo‐specific. The binding topologies were estimated basing on ¹H 2D‐ROESY experiments. The analysis of Job plots obtained from ¹H NMR‐monitored titrations proved the coexistence of 1:1 and 1:2 (crown ether:amino acids) complexes, which thermodynamic parameters, K_s, ΔG, ΔH°, and TΔS were determined with the aid of ITC. The 1:1 complexes were found enthalpically stabilized, generally by electrostatic interactions between the charged NH group of amino acid and crown ether macrocyclic moiety, while the binding of the second amino acid molecule was driven entropically due to solvatophobic effect. Strong enthalpy–entropy compensation points towards the uniform binding mode of all complexes studied. The mode of complex formation was found solvent dependent. For phenylalanine guest studied in various solvent systems, in contrast to the aqueous media, the noticeable chiral recognition is observed in methanol solution, and the complex stoichiometry (1:2 ether:Phe) differs from the 2:1 one, determined previously for the same host‐guest system in water (J. Thermal. Anal. Cal. 2006; 83: 575–578). Copyright © 2007 John Wiley & Sons, Ltd.     

DOUBLESLICING: a non-iterative single profile multi-exponential curve resolution procedure. Application to time-domain NMR transverse relaxation data

A new non-iterative curve resolution technique for resolving single decay profiles is proposed. The new technique, called DoubleSlicing, is based on the Decra (Direct Exponential Curve Resolution Algorithm) principle. While the original Decra was designed to resolve several decay curves simultaneously and thus fitting common pure exponentials, DoubleSlicing can resolve single decay profiles by a simple double data transformation followed by an analytical and unique three-way decomposition. The new approach is successfully demonstrated on experimental NMR CPMG relaxation data, measured on combinations of unmixed paramagnetic CuSO(4) solutions. Decay signals of the water component were acquired following an innovative experimental design that ensured no interaction between the components present in each sample under observation. DoubleSlicing proved to be accurate in estimating relaxation times differing in one order of magnitude (range: 19.6-159.4ms). Its performance was comparable to discrete exponential fitting with the advantage of being much faster - in terms of computation time, DoubleSlicing outperformed exponential fitting by a factor of four.