Chemical Shift Differences Research Articles

Investigating the Formation of Giant {Pd72}Prop and {Pd84}Gly Macrocycles Using NMR, HPLC, and Mass Spectrometry.

The formation of giant polyoxometalate (POM) species is relatively underexplored, as their self-assembly process is complex due to the rapid kinetics. Polyoxopalladates (POPds) are a class of POMs based on Pd, the largest of which is the {Pd84}Ac wheel, and its slower kinetics mean the system is more amenable to systematic study. Here, we show that it is possible to follow the assembly of two types of Pd wheels, {Pd84}Gly and the smaller {Pd72}Prop, formed using glycolate and propionate ligands, respectively. We analyzed the formation of {Pd72}Prop and {Pd84}Gly using mass spectrometry (SEC-HPLC-MS and preparative desalting followed by MS). This was accompanied by studies that followed the chemical shift differences between the outer/inner ligands and the free ligand in solution for the {Pd84}Ac, {Pd72}Prop, and {Pd84}Gly species using NMR, which showed it was possible to track the formation of the wheels. Our findings confirm that the macrocycles assemble from smaller building blocks that react together to form the larger species over a period of days. These findings open the way for further structural derivatives and exploration of their host-guest chemistry.

Solvent, structural, quantum chemical study and antioxidative activity of symmetrical 1-methyl-2,6-bis[2-(substituted phenyl)ethenyl]pyridinium iodides

15 symmetric 1-methyl-2,6-bis[2-(substituted phenyl)ethenyl]pyridinium iodides were synthesized in this work. Their structures were characterized using IR, 1H and 13C NMR, and UV–Vis spectroscopy. DFT calculations indicated that s-trans/s-trans conformation prevail in all compounds. The effects of specific and non-specific solvent–solute interactions on the UV–Vis absorption maxima shifts were evaluated using linear solvation-free energy relationships (LSER), i.e., Kamlet–Taft and Catalan models. A linear free energy relationship (LFER) in the form of single substituent parameter equations (SSP) was used to postulate quantitative structure–property relations of substituent effect on NMR data. TD-DFT results showed dependence of electronic transition on the substituent effects. The push–pull character of these compounds was analyzed by differences in 13C chemical shift of the ethylenic double bond in 2- and 6-positions of cross-conjugated with pyridinum central ring. Also, the quotient of the occupations for the bonding π and anti-bonding π* orbitals of this bond was considered. Good correlations of the selected parameter between double bond lengths with π*/π and 13C chemical shift differences of the bridging group proved them to be adequate descriptor of push–pull character. Synthesized compounds were screened for the antioxidant activity, using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical methods, and results demonstrated moderate antioxidant potential.

Identification, chemical synthesis, structure, and function of a new KV1 channel blocking peptide from Oulactis sp.

AbstractRapid progress in transcriptomic and proteomic studies of sea anemones has led to the identification of a large number of new peptide sequences. Some of these peptides have high sequence similarity and identical cysteine frameworks to those of previously reported sequences. One such peptide we have identified from a transcriptomic study of Oulactis sp is OspTx2a, which has a cysteine framework similar to that of ShK (from Stichodactyla helianthus) and BgK (from Bunodosoma granulifera). This peptide was made using solid‐phase peptide synthesis, but, upon oxidative folding, it generated two peptides with identical masses (OspTx2a‐p1 and OspTx2a‐p2) that were distinguishable by high‐performance liquid chromatography. The structures of OspTx2a‐p1 and OspTx2a‐p2 were determined using nuclear magnetic resonance spectroscopy, and voltage‐clamp electrophysiology assays were performed in order to assess the activity against a range of potassium channels. The structures of the two peptides were very similar to each other and to BgK, with the same disulfide bond connectivities, and both had an all‐trans backbone conformation. In functional assays, both OspTx2a‐p1 and OspTx2a‐p2 inhibited KV1.2 and KV1.6 channel currents at low µM concentrations, with similar but not identical IC50 values. Peptides containing a C‐terminal Cys residue are particularly sensitive to racemization at this residue, and the two products obtained for OspTx2a could be a consequence of racemization of the Cys residue at its C‐terminus during synthesis. NMR chemical shift differences between the two products and their structural preferences for a d‐Cys residue were consistent with this interpretation.

Dramatic Decrease in CEST Measurement Times Using Multi-Site Excitation.

Chemical exchange saturation transfer (CEST) has recently evolved into a powerful approach for studying sparsely populated, "invisible" protein states in slow exchange with a major, visible conformer. Central to the technique is the use of a weak, highly selective radio-frequency field that is applied at different frequency offsets in successive experiments, "searching" for minor state resonances. The recording of CEST profiles with enough points to ensure coverage of the entire spectrum at sufficient resolution can be time-consuming, especially for applications that require high static magnetic fields or when small chemical shift differences between exchanging states must be quantified. Here, we show - with applications involving 15 N CEST - that the process can be significantly accelerated by using a multi-frequency irradiation scheme, leading in some applications to an order of magnitude savings in measurement time.

Chirp mixing

In this paper, we develop the theory of chirp mixing in NMR spectroscopy. The working principle is simple, given coupled homonuclear spins with offsets in range [-B,B], we adiabatically sweep through the resonances. This achieves cross polarization between the z magnetization of the coupled spins. We repeat this basic operation many times with a supercycle to achieve appropriate mixing time. When we sweep through the resonances, midway between the resonances of the coupled spin I and S, the effective field seen by two spins is the same and hence they precess at same frequency around their effective fields. This means the coupling, which normally gets averaged out due to the chemical shift difference is no more averaged out for a short time and we get mixing. In this paper, we develop these basic ideas. By virtue of its design, the chirp mixing is much more broadband compared to state of the art methods. The proposed methodology is demonstrated on 13C mixing in a sample of Alanine.

Backbone resonance assignments of complexes of apo human calmodulin bound to IQ motif peptides of voltage-dependent sodium channels NaV1.1, NaV1.4 and NaV1.7.

Human voltage-gated sodium (NaV) channels are critical for initiating and propagating action potentials in excitable cells. Nine isoforms have different roles but similar topologies, with a pore-forming α-subunit and auxiliary transmembrane β-subunits. NaV pathologies lead to debilitating conditions including epilepsy, chronic pain, cardiac arrhythmias, and skeletal muscle paralysis. The ubiquitous calcium sensor calmodulin (CaM) binds to an IQ motif in the C-terminal tail of the α-subunit of all NaV isoforms, and contributes to calcium-dependent pore-gating in some channels. Previous structural studies of calcium-free (apo) CaM bound to the IQ motifs of NaV1.2, NaV1.5, and NaV1.6 showed that CaM binding was mediated by the C-domain of CaM (CaMC), while the N-domain (CaMN) made no detectable contacts. To determine whether this domain-specific recognition mechanism is conserved in other NaV isoforms, we used solution NMR spectroscopy to assign the backbone resonances of complexes of apo CaM bound to peptides of IQ motifs of NaV1.1, NaV1.4, and NaV1.7. Analysis of chemical shift differences showed that peptide binding only perturbed resonances in CaMC; resonances of CaMN were identical to free CaM. Thus, CaMC residues contribute to the interface with the IQ motif, while CaMN is available to interact elsewhere on the channel.

Multiple frequency saturation pulses reduce CEST acquisition time for quantifying conformational exchange in biomolecules.

Exchange between conformational states is required for biomolecular catalysis, allostery, and folding. A variety of NMR experiments have been developed to quantify motional regimes ranging from nanoseconds to seconds. In this work, we describe an approach to speed up the acquisition of chemical exchange saturation transfer (CEST) experiments that are commonly used to probe millisecond to second conformational exchange in proteins and nucleic acids. The standard approach is to obtain CEST datasets through the acquisition of a series of 2D correlation spectra where each experiment utilizes a single saturation frequency to 1H, 15N or 13C. These pseudo 3D datasets are time consuming to collect and are further lengthened by reduced signal to noise stemming from the long saturation pulse. In this article, we show how usage of a multiple frequency saturation pulse (i.e., MF-CEST) changes the nature of data collection from series to parallel, and thus decreases the total acquisition time by an integer factor corresponding to the number of frequencies in the pulse. We demonstrate the applicability of MF-CEST on a Src homology 2 (SH2) domain from phospholipase Cγ and the secondary active transport protein EmrE as model systems by collecting 13C methyl and 15N backbone datasets. MF-CEST can also be extended to additional sites within proteins and nucleic acids. The only notable drawback of MF-CEST as applied to backbone 15N experiments occurs when a large chemical shift difference between the major and minor populations is present (typically greater than ~ 8ppm). In these cases, ambiguity may arise between the chemical shift of the minor population and the multiple frequency saturation pulse. Nevertheless, this drawback does not occur for methyl group MF-CEST experiments or in cases where somewhat smaller chemical shift differences occur are present.

Chiral one-dimensional hydrogen-bonded architectures constructed from single-enantiomer phosphoric triamides.

The two single-enantiomer phosphoric triamides N-(2,6-difluorobenzoyl)-N',N''-bis[(S)-(-)-α-methylbenzyl]phosphoric triamide, [2,6-F2-C6H3C(O)NH][(S)-(-)-(C6H5)CH(CH3)NH]2P(O), denoted L-1, and N-(2,6-difluorobenzoyl)-N',N''-bis[(R)-(+)-α-methylbenzyl]phosphoric triamide, [2,6-F2-C6H3C(O)NH][(R)-(+)-(C6H5)CH(CH3)NH]2P(O), denoted D-1, both C23H24F2N3O2P, have been investigated. In their structures, chiral one-dimensional hydrogen-bonded architectures are formed along [100], mediated by relatively strong N-H...O(P) and N-H...O(C) hydrogen bonds. Both assemblies include the noncentrosymmetric graph-set motifs R22(10), R21(6) and C22(8), and the compounds crystallize in the chiral space group P1. Due to the data collection of L-1 at 120 K and of D-1 at 95 K, the unit-cell dimensions and volume show a slight difference; the contraction in the volume of D-1 with respect to that in L-1 is about 0.3%. The asymmetric units of both structures consist of two independent phosphoric triamide molecules, with the main difference being seen in one of the torsion angles in the OPNHCH(CH3)(C6H5) part. The Hirshfeld surface maps of these levo and dextro isomers are very similar; however, they are near mirror images of each other. For both structures, the full fingerprint plot of each symmetry-independent molecule shows an almost asymmetric shape as a result of its different environment in the crystal packing. It is notable that NMR spectroscopy could distinguish between compounds L-1 and D-1 that have different relative stereocentres; however, the differences in chemical shifts between them were found to be about 0.02 to 0.001 ppm under calibrated temperature conditions. In each molecule, the two chiral parts are also different in NMR media, in which chemical shifts and P-H and P-C couplings have been studied.

An NMR strategy to detect conformational differences in a protein complexed with highly analogous inhibitors in solution.

This manuscript presents an NMR strategy to investigate conformational differences in protein-inhibitor complexes, when the inhibitors tightly bind to a protein at sub-nanomolar dissociation constants and are highly analogous to each other. Using HIV-1 protease (PR), we previously evaluated amide chemical shift differences, ΔCSPs, of PR bound to darunavir (DRV) compared to PR bound to several DRV analogue inhibitors, to investigate subtle but significant long-distance conformation changes caused by the inhibitor's chemical moiety variation [Khan, S. N., Persons, J. D. Paulsen, J. L., Guerrero, M., Schiffer, C. A., Kurt-Yilmaz, N., and Ishima, R., Biochemistry, (2018), 57, 1652-1662]. However, ΔCSPs are not ideal for investigating subtle PR-inhibitor interface differences because intrinsic differences in the electron shielding of the inhibitors affect protein ΔCSPs. NMR relaxation is also not suitable as it is not sensitive enough to detect small conformational differences in rigid regions among similar PR-inhibitor complexes. Thus, to gain insight into conformational differences at the inhibitor-protein interface, we recorded 15N-half filtered NOESY spectra of PR bound to two highly analogous inhibitors and assessed NOEs between PR amide protons and inhibitor protons, between PR amide protons and hydroxyl side chains, and between PR amide protons and water protons. We also verified the PR amide-water NOEs using 2D water-NOE/ROE experiments. Differences in water-amide proton NOE peaks, possibly due to amide-protein hydrogen bonds, were observed between subunit A and subunit B, and between the DRV-bound form and an analogous inhibitor-bound form, which may contribute to remote conformational changes.

An Efficient Method for Determining the Relative Configuration of Furofuran Lignans by 1H NMR Spectroscopy.

An efficient 1H NMR spectroscopic approach for determining the relative configurations of lignans with a 7,9':7',9-diepoxy moiety has been established. Using the chemical shift differences of H2-9 and H2-9' (ΔδH-9 and ΔδH-9'), the configurations of 8-H and 8-OH furofuran lignans can be rapidly and conveniently determined. The rule is applicable for data acquired in DMSO- d6, methanol- d4, or CDCl3. Notably, the rule should be applied carefully when the C-2 or C-6 substituent of the aromatic rings may alter the dominant conformers of the furofuran moiety.

Confirmation of the Structure of Trans‐Cyclic Azobenzene by X‐Ray Crystallography and Spectroscopic Characterization of Cyclic Azobenzene Analogs

AbstractThe photoisomerization properties of a series of cyclic azobenzene (cAb) analogs, including non‐substituted, Br‐, CN‐, carboxyl‐, and amino cAb, were compared. While the trans‐form of these analogs show different degrees of thermal stability, no spectral changes in the absorption wavelengths were observed for amino cAb upon exposure to purple light. The 15N NMR chemical shifts of cAb in both cis‐ and trans‐forms were found to show a large difference from those of aromatic azobenzene; however, the trend of difference in chemical shift is in agreement with that obtained from quantum chemical calculations. More importantly, the structure of the photoisomerization product from cis‐cAb was confirmed to be, indeed, the trans‐cAb by X‐ray diffraction experiments. The 15NMR data of the cAb isomers and confirmation of trans‐cAb through crystal structure will be of importance for future studies in the design and applications of cAb as a photoswitch.

Measuring the signs of the methyl 1H chemical shift differences between major and 'invisible' minor protein conformational states using methyl 1H multi-quantum spectroscopy.

Carr-Purcell-Meiboom-Gill (CPMG) type relaxation dispersion experiments are now routinely used to characterise protein conformational dynamics that occurs on the μs to millisecond (ms) timescale between a visible major state and 'invisible' minor states. The exchange rate(s) ([Formula: see text]), population(s) of the minor state(s) and the absolute value of the chemical shift difference [Formula: see text] (ppm) between different exchanging states can be extracted from the CPMG data. However the sign of [Formula: see text] that is required to reconstruct the spectrum of the 'invisible' minor state(s) cannot be obtained from CPMG data alone. Building upon the recently developed triple quantum (TQ) methyl [Formula: see text] CPMG experiment (Yuwen in Angew Chem 55:11490-11494, 2016) we have developed pulse sequences that use carbon detection to generate and evolve single quantum (SQ), double quantum (DQ) and TQ coherences from methyl protons in the indirect dimension to measure the chemical exchange-induced shifts of the SQ, DQ and TQ coherences from which the sign of [Formula: see text] is readily obtained for two state exchange. Further a combined analysis of the CPMG data and the difference in exchange induced shifts between the SQ and DQ resonances and between the SQ and TQ resonances improves the estimates of exchange parameters like the population of the minor state. We demonstrate the use of these experiments on two proteins undergoing exchange: (1) the ~18kDa cavity mutant of T4 Lysozyme ([Formula: see text]) and (2) the [Formula: see text]kDa Peripheral Sub-unit Binding Domain (PSBD) from the acetyl transferase of Bacillus stearothermophilus ([Formula: see text]).

Heteroassembly Ability of Dicationic Ionic Liquids and Neutral Active Pharmaceutical Ingredients

Extensive investigation of interactions and aggregation properties of IL + API systems is necessary to apply ionic liquids (ILs) with different hydrophobic characteristics to drug delivery or in active pharmaceutical ingredient (API) formulations. Therefore, this study aims to investigate the heteroassembly between dicationic ILs ([BisOct(MIM)2][2X], in which X is Br or BF4, and [BisOct(BnIM)2][2Br]), both in the absence and the presence of neutral APIs (salicylic acid, ibuprofen, and paracetamol) with different functional groups. Isothermal titration calorimetry results demonstrate that IL–API associations occur at very low concentrations of IL. These results were reinforced by electrospray ionization mass spectrometry with variable collision-induced dissociation, in which the IL dication interactions with APIs were detected. The strength of the dication–API interaction was determined from Ecm,1/2 data. The aggregation parameters (cac, ΔGagg°, and K) between ILs and APIs were evaluated by conductivity. The 1H NMR data showed that differences in chemical shifts provided relevant insights about interaction sites in both components.

Hydroxy protons as structural probes to reveal hydrogen bonding properties of polyols in aqueous solution by NMR spectroscopy

The solution properties of ethylene glycol (ethane-1,2-diol), glycerol (propane-1,2,3-triol), erythritol ((2R,3S)-butane-1,2,3,4-tetraol), D-xylitol ((2R,3r,4S)-pentane-1,2,3,4,5-pentaol), D-mannitol ((2R,3R,4R,5R)-hexane-1,2,3,4,5,6-hexaol), and D-sorbitol ((2S,3R,4R,5R)-hexane-1,2,3,4,5,6-hexaol), constituting a subgroup of polyalcohols/polyols of maximum six carbon atoms have been investigated using 1H NMR chemical shifts, coupling constants, temperature coefficients, and chemical exchange rates of hydroxy protons in aqueous medium.Relative within a molecule, minimum two-fold difference in rate of exchange values and higher temperature dependence of chemical shifts of the hydroxy protons on terminal carbon atoms confirm that sustainable hydrogen bonding interactions is accentuated for the hydroxyl groups on secondary carbons. Compared to the primary carbons i.e. terminal ones, the hydroxy protons on second and third carbon atoms exhibit much lower rate of exchange and smaller temperature coefficients, indicating that they are further involved in transient hydrogen bonding interactions. Scalar 3JOH,CH-couplings ranging between 3.9 and 7.2 Hz imply that the hydroxyl groups are practically in free rotation regime. Examination of the chemical shift differences with respect to the shift of glycol hydroxy proton reveals that the disparity between terminal and inner hydroxyl groups disclosed by the exchange rates and temperature coefficients is sustained with the exception of 0.003 and 0.053 ppm for O(3)H of mannitol and O(5)H of sorbitol respectively. The experimental findings have been augmented by quantum chemical calculations targeting theoretical NMR chemical shifts, as well as the conformational analysis of the structures.

Referenced MR thermometry using three-echo phase-based fat water separation method

A three-point image reconstruction method for internally referenced MR thermometry was developed. The technique exploits the fact that temperature-induced changes in the water resonance frequency are small relative to the chemical shift difference between water and fat signals. This property enabled the use of small angle approximations to derive an analytic phase-based fat-water separation method for MR thermometry. Ethylene glycol and cream cool-down experiments were performed to validate measurement technique. Over a cool-down temperature range of 20 °C, maximum deviation between probe and MR measurement (averaged over 1.3 cm3 region surrounding probe) was 0.6 °C and 1.1 °C for ethylene glycol and cream samples, respectively.

A simple method to determine the water content in organic solvents using the 1H NMR chemical shifts differences between water and solvent

A simple and general method which relies on chemical shift difference between water and solvent peaks in 1H NMR spectroscopy has been developed for the determination of water content in organic solvent. As the water content is increased, the water proton peak is shifted to down-field because the water is involved in the more hydrogen bonding. The linear relation between water content and chemical shift difference was used to assess the water content in organic solvent. The intrinsic water and solvent proton peak was used, thus no additional reagents was necessary. The developed method has been applied to six organic solvents having various chemical properties. Furthermore, temperature effect was investigated and correction factor was evaluated. We propose that this new method will be useful for wide range of applications requiring accurate and convenient assessment of water content in organic solvents.

Intramolecular hydrogen bond directed stable conformations of benzoyl phenyl oxalamides: unambiguous evidence from extensive NMR studies and DFT-based computations.

A number of benzoyl phenyl oxalamide derivatives have been synthesized and characterized by the extensive utility of one- and two-dimensional NMR experimental techniques. The manifestation of intramolecular hydrogen bonds in all of the synthesized molecules, convincingly established using NMR studies, governs the stable conformations of the molecules. In the fluorine substituted molecules, the coupling between two NMR active nuclei mediated through hydrogen bonds has been detected. The measured chemical shift difference of an NH proton has been employed to calculate the energy of the HBs. NMR analysis revealed the electrostatic nature of the hydrogen bonds in all of the molecules. The NMR experimental findings have been validated using Density Functional Theory (DFT)-based Non Covalent Interactions (NCIs) and Quantum Theory of Atoms In Molecules (QTAIM) computations.

Direct detection of lysine side chain NH3+ in protein-heparin complexes using NMR spectroscopy.

Two NMR observables, the NζH3+ peak in the HISQC spectrum and Nζ chemical shift difference between the free and heparin-bound forms, can identify binding-interface lysines in protein-heparin complexes. Unlike backbone chemical shifts, these direct probes are stringent and are less prone to either false positives or false negatives.

Microsecond Timescale Protein Dynamics: a Combined Solid-State NMR Approach.

Conformational exchange in proteins is a major determinant in protein functionality. In particular, the μs-ms timescale is associated with enzymatic activity and interactions between biological molecules. We show here that a comprehensive data set of R1ρ relaxation dispersion profiles employing multiple effective fields and tilt angles can be easily obtained in perdeuterated, partly back-exchanged proteins at fast magic-angle spinning and further complemented with chemical-exchange saturation transfer NMR experiments. The approach exploits complementary sources of information and enables the extraction of multiple exchange parameters for μs-ms timescale conformational exchange, most notably including the sign of the chemical shift differences between the ground and excited states.

The Rich Solid-State Phase Behavior of dl-Aminoheptanoic Acid: Five Polymorphic Forms and Their Phase Transitions.

The rich landscape of enantiotropically related polymorphic forms and their solid-state phase transitions of dl-2-aminoheptanoic acid (dl-AHE) has been explored using a range of complementary characterization techniques, and is largely exemplary of the polymorphic behavior of linear aliphatic amino acids. As many as five new polymorphic forms were found, connected by four fully reversible solid-state phase transitions. Two low temperature forms were refined in a high Z′ crystal structure, which is a new phenomenon for linear aliphatic amino acids. All five structures consist of two-dimensional hydrogen-bonded bilayers interconnected by weak van der Waals interactions. The single-crystal-to-single-crystal phase transitions involve shifts of bilayers and/or conformational changes in the aliphatic chain. Compared to two similar phase transitions of the related amino acid dl-norleucine, the enthalpies of transition and NMR chemical shift differences are notably smaller in dl-aminoheptanoic acid. This is explained to be a result of both the nature of the conformational changes and the increased chain length, weakening the interactions between the bilayers.