NMR Chemical Shift Analysis Research Articles

Solution structure, aggregation behavior, and flexibility of human relaxin-2.

Relaxin is a member of the relaxin/insulin peptide hormone superfamily and is characterized by a two-chain structure constrained by three disulfide bonds. Relaxin is a pleiotropic hormone and involved in a number of physiological and pathogenic processes, including collagen and cardiovascular regulation and tissue remodelling during pregnancy and cancer. Crystallographic and ultracentrifugation experiments have revealed that the human form of relaxin, H2 relaxin, self-associates into dimers, but the significance of this is poorly understood. Here, we present the NMR structure of a monomeric, amidated form of H2 relaxin and compare its features and behavior in solution to those of native H2 relaxin. The overall structure of H2 relaxin is retained in the monomeric form. H2 relaxin amide is fully active at the relaxin receptor RXFP1 and thus dimerization is not required for biological activity. Analysis of NMR chemical shifts and relaxation parameters identified internal motion in H2 relaxin at the pico-nanosecond and milli-microsecond time scales, which is commonly seen in other relaxin and insulin peptides and might be related to function.

A Tool Set to Map Dynamic Allosteric Networks through the NMR Chemical Shift Covariance Analysis (CHESCA)

Allostery is an essential regulatory mechanism of biological function and allosteric sites are also pharmacologically relevant, as they are typically targeted with higher selectivity than orthosteric sites. However, obtaining a comprehensive map of allosteric sites poses experimental challenges because allostery is driven not only by structural changes, but also by modulations in dynamics that often remain elusive to classical structure determination methods. An avenue to overcome these challenges is provided by the covariance analysis of NMR chemical shift [1, 2], which are exquisitely sensitive to redistributions in dynamic conformational ensembles. Here, we propose a set of complementary algorithms for the NMR chemical shift covariance analysis (CHESCA) designed to reliably detect allosteric networks with minimal occurrences of false positives or negatives. The proposed CHESCA toolset was tested for two allosteric proteins (Protein Kinase A, PKA, and the Exchange Protein directly Activated by cAMP, EPAC) and is expected to complement traditional comparative structural analyses in the comprehensive identification of functionally relevant allosteric sites, including those in otherwise elusive partially unstructured regions.[1] Signaling through dynamic linkers as revealed by PKA. Akimoto M, Selvaratnam R, McNicholl ET, Verma G, Taylor SS, Melacini G. Proc Natl Acad Sci U S A. 2013;110(35):14231-6.[2] Mapping allostery through the covariance analysis of NMR chemical shifts. Selvaratnam R, Chowdhury S, VanSchouwen B, Melacini G. Proc Natl Acad Sci U S A. 2011;108(15):6133-8.

A Fuzzy DNA Binding Region in MBD2 Recruits the Histone Deacetylase Core Complex of NuRD and Modifies Kinetics of DNA Binding

The MBD2 protein recruits and assembles the Nucleosome Remodeling and Deacetylase (NuRD) complex and thereby uniquely combines binding specificity for methylated DNA with histone deacetylation and chromatin remodeling. This complex has been implicated in methylation dependent silencing of genes during development and aberrant silencing of tumor suppressor genes during carcinogenesis. We have focused on structural and biophysical analyses of MBD2 with the long-term goal of developing methods to disrupt formation of the MBD2-NuRD complex. Along these lines, we have previously characterized and determined the structures of the coiled-coil and methylcytosine binding (MBD) domains of MBD2. More recently we have characterized an intrinsically disordered region (IDR) of ∼120 amino acids linking the MBD and coiled-coil. NMR chemical shift analyses, CD, and AUC show that this region, MBD2(IDR), does not adopt a regular structure in isolation or in the context of full-length protein. Yet the MBD2(IDR) stably binds three proteins that form the histone deacetylase core of NuRD. We show that the first two-thirds of the MBD2IDR are necessary and sufficient to bind the histone deacetylase core while mutating two consecutive within this region is sufficient to abrogate binding to the core complex and disrupt the function of MBD2 in cells. At the same time, adding the MBD2(IDR) to the MBD2(MBD) in vitro modifies DNA binding primarily by reducing the observed off rate and increasing affinity by ∼100 fold. We find that the MBD2(IDR) does not adopt a regular fold in the presence of DNA thereby functioning as a fuzzy DNA binding region. Together our studies show that the IDR of MBD2 plays a dual role both augmenting DNA binding affinity and recruiting a large portion of the NuRD complex.

Aromaticities of Five Membered Heterocycles through Dimethyldihydropyrenes Probe by Magnetic and Geometric Criteria

Aromaticities of five membered heterocycles, containing up to three heteroatoms, are quantified through the dimethyldihydropyrene (DHP) probe. Bond fixation caused by the fusion of heterocycles to the dimethyldihydropyrene nucleus (DHPN) was measured by changes in the1H NMR chemical shifts (magnetic) and bond lengths alterations (structural criterion). Chemical shifts of dihydropyrenes were calculated at GIAO HF/6-31G(d)//B3LYP/6-31+G(d). For1H NMR chemical shift analysis, two nonaromatic reference models are studied. Among the studied heterocycles, pyrazole and triazole are about 80–85% aromatic relative to benzene, through both magnetic and geometric criteria. Thiazole and oxazoles are found least aromatic where quantitative estimates of aromaticities are about 34–42%, relative to benzene. These quantitative estimates of aromaticities of five membered heterocycles are also comparable to those from aromatic stabilization energies. The quantification of aromaticity through energetic, magnetic, and structural criteria can deliver the similar inferences provided that suitable reference systems are chosen.

The H50Q Mutation Induces a 10-fold Decrease in the Solubility of α-Synuclein

The conversion of α-synuclein from its intrinsically disordered monomeric state into the fibrillar cross-β aggregates characteristically present in Lewy bodies is largely unknown. The investigation of α-synuclein variants causative of familial forms of Parkinson disease can provide unique insights into the conditions that promote or inhibit aggregate formation. It has been shown recently that a newly identified pathogenic mutation of α-synuclein, H50Q, aggregates faster than the wild-type. We investigate here its aggregation propensity by using a sequence-based prediction algorithm, NMR chemical shift analysis of secondary structure populations in the monomeric state, and determination of thermodynamic stability of the fibrils. Our data show that the H50Q mutation induces only a small increment in polyproline II structure around the site of the mutation and a slight increase in the overall aggregation propensity. We also find, however, that the H50Q mutation strongly stabilizes α-synuclein fibrils by 5.0 ± 1.0 kJ mol(-1), thus increasing the supersaturation of monomeric α-synuclein within the cell, and strongly favors its aggregation process. We further show that wild-type α-synuclein can decelerate the aggregation kinetics of the H50Q variant in a dose-dependent manner when coaggregating with it. These last findings suggest that the precise balance of α-synuclein synthesized from the wild-type and mutant alleles may influence the natural history and heterogeneous clinical phenotype of Parkinson disease.

The s2D Method: Simultaneous Sequence-Based Prediction of the Statistical Populations of Ordered and Disordered Regions in Proteins

Extensive amounts of information about protein sequences are becoming available, as demonstrated by the over 79 million entries in the UniProt database. Yet, it is still challenging to obtain proteome-wide experimental information on the structural properties associated with these sequences. Fast computational predictors of secondary structure and of intrinsic disorder of proteins have been developed in order to bridge this gap. These two types of predictions, however, have remained largely separated, often preventing a clear characterization of the structure and dynamics of proteins. Here, we introduce a computational method to predict secondary-structure populations from amino acid sequences, which simultaneously characterizes structure and disorder in a unified statistical mechanics framework. To develop this method, called s2D, we exploited recent advances made in the analysis of NMR chemical shifts that provide quantitative information about the probability distributions of secondary-structure elements in disordered states. The results that we discuss show that the s2D method predicts secondary-structure populations with an average error of about 14%. A validation on three datasets of mostly disordered, mostly structured and partly structured proteins, respectively, shows that its performance is comparable to or better than that of existing predictors of intrinsic disorder and of secondary structure. These results indicate that it is possible to perform rapid and quantitative sequence-based characterizations of the structure and dynamics of proteins through the predictions of the statistical distributions of their ordered and disordered regions.

Relationship between nonlinear pressure-induced chemical shift changes and thermodynamic parameters.

NMR chemical shift analysis is a powerful method to investigate local changes in the environment of the observed nuclear spin of a polypeptide that are induced by application of high hydrostatic pressure. Usually, in the fast exchange regime, the pressure dependence of chemical shifts is analyzed by a second order Taylor expansion providing the first- and second-order pressure coefficient B1 and B2. The coefficients then are interpreted in a qualitative manner. We show here that in a two-state model, the ratio of B2/B1 is related to thermodynamic parameters, namely the ratio of the difference of compressibility factors Δβ' and partial molar volumes ΔV. The analysis is applied to the random-coil model peptides Ac-Gly-Gly-Xxx-Ala-NH2, with Xxx being one of the 20 proteinogenic amino acids. The analysis gives an average Δβ'/ΔV ratio of 1.6 GPa(-1) provided the condition |ΔG(0)| ≪ 2RT holds for the difference of the Gibbs free energies (ΔG(0)) of the two states at the temperature (T0) and the pressure (p0). The amide proton and nitrogen B2/B1 of a given amino acid Xxx are strongly correlated, indicating that their pressure-dependent chemical shift changes are due to the same thermodynamic process. As a possible physical mechanism providing a two-state model, the hydrogen bonding of water with the corresponding amide protein was simulated for isoleucine in position Xxx. The obtained free energy could satisfy the relation |ΔG(0)| ≪ 2RT. The derived relation was applied to the β-amyloid peptide Aβ and the phosphocarrier protein HPr from S. carnosus. For the transition of state 1 to state 2' of Aβ, the derived relation of B2/B1 to Δβ'/ΔV can be confirmed experimentally. The HPr protein is characterized by substantially higher negative B2/B1 values than those found in the tetrapeptides with an average value of approximately -5.1 GPa(-1) (Δβ'/ΔV of 5.1 GPa(-1) provided |ΔG(0)| ≪ 2RT holds). Qualitatively, the B2/B1 ratio can be used to predict regions of the HPr protein involved in the interaction with enzyme I or HPr-kinase/phosphatase.

Finding Order in Disorder: Probing Transient Functional States in the Amyloidogenic Alzheimer's Aβ Peptide Using the NMR Chemical Shift Covariance Analysis (CHESCA)

Prior to self-association into toxic soluble oligomers, amyloidogenic peptides are often unstructured or only partially structured and they are best described by an heterogeneous conformational ensemble, in which multiple discrete structures are populated. Despite the complexity in the ensemble of conformations accessible to amyloidogenic peptides, it has been suggested that a key determinant of the propensity to aggregate is the transient access to a subset of functional conformations, i.e. conformers that are either aggregation competent or are incompetent but compete with aggregation competent states. However, due to the conformational ‘noise’ generated by the highly degenerate free-energy landscape typical of amyloidogenic peptides, the identification of minor populations of functional conformers relevant for aggregation remains experimentally challenging. Here, we propose a method to identify which residues within amyloidogenic peptides are involved in functional states based on the covariance analysis of NMR chemical shifts (CHESCA) [1]. CHESCA relies on linear correlations between residue-specific chemical shifts measured for the peptide system of interest subject to a set of perturbations that modulate a given functional property, e.g. the aggregation propensity in the case of amyloidogenic peptides. Such linear chemical shift correlations are useful to identify the residues involved in transient and minimally populated partially folded conformers. In addition, a distinct advantage of the chemical shift covariance method is that the relative order in which the perturbed states appear in the inter-residue correlations defines the functional relevance of the partially folded conformers. This general approach will be illustrated through its application to the Aβ peptide, which self-associates into insoluble β-sheet structured aggregates linked to Alzheimer's disease.1) Selvaratnam R, Chowdhury S, VanSchouwen B, Melacini G. Proc Natl Acad Sci U S A. 2011;108(15):6133.

The C-Terminal V5 Domain of Protein Kinase Cα is a Multi-Functional Intrinsically Disordered Protein Module

Protein Kinase C (PKC) family of isozymes regulate a multitude of signaling pathways that control cell growth, differentiation, and apoptosis. The extreme C-terminal V5 domain has been identified as a key player in maturation, activation and down-regulation of PKCs, but the molecular basis of these events remains poorly understood. We report the first large-scale purification and NMR characterization of the V5 domain from PKCα (V5α) and its phosphorylation-mimicking variant. The combined analysis of NMR chemical shifts and circular dichroism data revealed that both V5α constructs are intrinsically disordered protein domains. Unexpectedly, we found that V5α has a propensity to partition into membrane mimetics acquiring a partial helical structure. Our data suggest that V5α anchors its parent enzyme to membranes during the maturation process. Using NMR techniques, we obtained direct evidence that V5α interacts with another domain of PKCα -- the C2 regulatory domain. This interaction is mediated by the phosphorylated hydrophobic motif of V5α and is enhanced by Ca2+. Similarly, the affinity of C2 to Ca2+ is enhanced in the presence of the phosphorylated hydrophobic motif. These findings indicate that V5α may function as an intra-molecular protein interaction module that sensitizes PKCα to Ca2+ ions. The third aspect of V5α function pertains to its interactions with Pin1, a peptidyl-prolyl isomerase that has been implicated in the down-regulation of conventional PKCs. Pin1 catalyzes proline isomerization of the phosphorylated Ser/Thr-Pro motif. Our preliminary NMR data demonstrate that Pin1 interacts with the hydrophobic motif of V5α, which is a non-canonical site that lacks a proline after the phosphorylated serine. Our data support the hypothesis that V5α serves as a phosphorylation-dependent docking site for Pin1. Acknowledgments: This work was supported by the Welch Foundation (A-1784).

The Family of Ferrocene‐Stabilized Silylium Ions: Synthesis, 29Si NMR Characterization, Lewis Acidity, Substituent Scrambling, and Quantum‐Chemical Analyses

The purpose of this systematic experimental and theoretical study is to deeply understand the unique bonding situation in ferrocene-stabilized silylium ions as a function of the substituents at the silicon atom and to learn about the structure parameters that determine the (29)Si NMR chemical shift and electrophilicity of these strong Lewis acids. For this, ten new members of the family of ferrocene-stabilized silicon cations were prepared by a hydride abstraction reaction from silanes with the trityl cation and characterized by multinuclear (1)H and (29)Si NMR spectroscopy. A closer look at the NMR spectra revealed that additional minor sets of signals were not impurities but silylium ions with substitution patterns different from that of the initially formed cation. Careful assignment of these signals furnished experimental proof that sterically less hindered silylium ions are capable of exchanging substituents with unreacted silane precursors. Density functional theory calculations provided mechanistic insight into that substituent transfer in which the migrating group is exchanged between two silicon fragments in a concerted process involving a ferrocene-bridged intermediate. Moreover, the quantum-chemical analysis of the (29)Si NMR chemical shifts revealed a linear relationship between δ((29)Si) values and the Fe···Si distance for subsets of silicon cations. An electron localization function and electron localizability indicator analysis shows a three-center two-electron bonding attractor between the iron, silicon, and C'(ipso) atoms, clearly distinguishing the silicon cations from the corresponding carbenium ions and boranes. Correlations between (29)Si NMR chemical shifts and Lewis acidity, evaluated in terms of fluoride ion affinities, are seen only for subsets of silylium ions, sometimes with non-intuitive trends, indicating a complicated interplay of steric and electronic effects on the degree of the Fe···Si interaction.

Conformational change of Sos-derived proline-rich peptide upon binding Grb2 N-terminal SH3 domain probed by NMR

Growth factor receptor-bound protein 2 (Grb2) is a small adapter protein composed of a single SH2 domain flanked by two SH3 domains. The N-terminal SH3 (nSH3) domain of Grb2 binds a proline-rich region present in the guanine nucleotide releasing factor, son of sevenless (Sos). Using NMR relaxation dispersion and chemical shift analysis methods, we investigated the conformational change of the Sos-derived proline-rich peptide during the transition between the free and Grb2 nSH3-bound states. The chemical shift analysis revealed that the peptide does not present a fully random conformation but has a relatively rigid structure. The relaxation dispersion analysis detected conformational exchange of several residues of the peptide upon binding to Grb2 nSH3.

α-Hemoglobin-stabilizing Protein (AHSP) Perturbs the Proximal Heme Pocket of Oxy-α-hemoglobin and Weakens the Iron-Oxygen Bond

α-Hemoglobin (αHb)-stabilizing protein (AHSP) is a molecular chaperone that assists hemoglobin assembly. AHSP induces changes in αHb heme coordination, but how these changes are facilitated by interactions at the αHb·AHSP interface is not well understood. To address this question we have used NMR, x-ray absorption spectroscopy, and ligand binding measurements to probe αHb conformational changes induced by AHSP binding. NMR chemical shift analyses of free CO-αHb and CO-αHb·AHSP indicated that the seven helical elements of the native αHb structure are retained and that the heme Fe(II) remains coordinated to the proximal His-87 side chain. However, chemical shift differences revealed alterations of the F, G, and H helices and the heme pocket of CO-αHb bound to AHSP. Comparisons of iron-ligand geometry using extended x-ray absorption fine structure spectroscopy showed that AHSP binding induces a small 0.03 Å lengthening of the Fe-O2 bond, explaining previous reports that AHSP decreases αHb O2 affinity roughly 4-fold and promotes autooxidation due primarily to a 3-4-fold increase in the rate of O2 dissociation. Pro-30 mutations diminished NMR chemical shift changes in the proximal heme pocket, restored normal O2 dissociation rate and equilibrium constants, and reduced O2-αHb autooxidation rates. Thus, the contacts mediated by Pro-30 in wild-type AHSP promote αHb autooxidation by introducing strain into the proximal heme pocket. As a chaperone, AHSP facilitates rapid assembly of αHb into Hb when βHb is abundant but diverts αHb to a redox resistant holding state when βHb is limiting.

High-affinity Cyclic Peptide Matriptase Inhibitors

Sunflower trypsin inhibitor-1 (SFTI-1) and Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II) are potent protease inhibitors comprising a cyclic backbone. Elucidation of structure-activity relationships for SFTI-1 and MCoTI-II was used to design inhibitors with enhanced inhibitory activity. An analog of MCoTI-II is one of the most potent inhibitors of matriptase. These results provide a solid basis for the design of selective peptide inhibitors of matriptase with therapeutic potential. The type II transmembrane serine protease matriptase is a key activator of multiple signaling pathways associated with cell proliferation and modification of the extracellular matrix. Deregulated matriptase activity correlates with a number of diseases, including cancer and hence highly selective matriptase inhibitors may have therapeutic potential. The plant-derived cyclic peptide, sunflower trypsin inhibitor-1 (SFTI-1), is a promising drug scaffold with potent matriptase inhibitory activity. In the current study we have analyzed the structure-activity relationships of SFTI-1 and Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II), a structurally divergent trypsin inhibitor from Momordica cochinchinensis that also contains a cyclic backbone. We show that MCoTI-II is a significantly more potent matriptase inhibitor than SFTI-1 and that all alanine mutants of both peptides, generated using positional scanning mutagenesis, have decreased trypsin affinity, whereas several mutations either maintain or result in enhanced matriptase inhibitory activity. These intriguing results were used to design one of the most potent matriptase inhibitors known to date with a 290 pm equilibrium dissociation constant, and provide the first indication on how to modulate affinity for matriptase over trypsin in cyclic peptides. This information might be useful for the design of more selective and therapeutically relevant inhibitors of matriptase.

Structural Characterization by NMR of a Double Phosphorylated Chimeric Peptide Vaccine for Treatment of Alzheimer’s Disease

Rational design of peptide vaccines becomes important for the treatment of some diseases such as Alzheimer’s disease (AD) and related disorders. In this study, as part of a larger effort to explore correlations of structure and activity, we attempt to characterize the doubly phosphorylated chimeric peptide vaccine targeting a hyperphosphorylated epitope of the Tau protein. The 28-mer linear chimeric peptide consists of the double phosphorylated B cell epitope Tau229-237[pThr231/pSer235] and the immunomodulatory T cell epitope Ag85B241-255 originating from the well-known antigen Ag85B of the Mycobacterium tuberculosis, linked by a four amino acid sequence -GPSL-. NMR chemical shift analysis of our construct demonstrated that the synthesized peptide is essentially unfolded with a tendency to form a β-turn due to the linker. In conclusion, the -GPSL- unit presumably connects the two parts of the vaccine without transferring any structural information from one part to the other. Therefore, the double phosphorylated epitope of the Tau peptide is flexible and accessible.

On relative electronic effects of polyhedral boron hydrides

Relative electron-donating properties of anionic polyhedral boron hydrides were estimated on analysis of 1H NMR chemical shifts of the β-hydrogens in their derivatives. The electron donating properties of boron clusters were found to decrease in the following order [2-B10H9]2− > [B12H11]2− ∼ [12-CB11H11]− ∼ [10-nido-7,8-C2B9H10]− > [8-3,3′-Co(1,2-C2B9H10)(1,2-C2B9H11)]− > [1-B10H9]2−.

Synthesis and characterization of some novel tetrazole liquid crystals

The synthesis and characterization of new 4-[(2-alkyl)-2H-tetrazol-5-yl]phenyl 4-alkyloxybenzoates (5a–i) and 5-{4-[(4-substituted)benzyloxy]phenyl}-2-alkyl-2H-tetrazoles (7a–e) are reported. The reported tetrazoles contain two alkyl chains (six to ten carbon atoms) except for 5h and 7e which have a chiral citronellyl group linked at the N-2 nitrogen atom of the tetrazole ring and 5i having a bromine atom at the para position of benzoate. The regiochemistry in the alkylation step was established unequivocally by single-crystal X-ray analysis for 5a and 5f. The regioselectivity in the alkylation step was also confirmed by analysis of 13C NMR chemical shifts of the C5 carbon atom in the tetrazole ring and the methylene carbon atom vicinal to the nitrogen atom. The mesogenic behavior of the liquid crystals was characterized by polarizing optical microscopy (POM), differential scanning calorimetry (DSC) and X-ray diffraction (XRD) techniques. The tetrazolyl benzoates 5a–h show a nematic mesophase. The lengthening of alkyl chains favors the appearance of the smectic mesophase. For instance, 5g displayed nematic and smectic C mesophases, while 5h which has the chiral citronellyl group displayed nematic and smectic A mesophases. Tetrazoles 7a–e did not show mesomorphic behavior mainly due to replacement of the carbonyl by a methylene group.

A Small Molecule Inhibitor of Pot1 Binding to Telomeric DNA

Chromosome ends are complex structures, consisting of repetitive DNA sequence terminating in an ssDNA overhang with many associated proteins. Because alteration of the regulation of these ends is a hallmark of cancer, telomeres and telomere maintenance have been prime drug targets. The universally conserved ssDNA overhang is sequence-specifically bound and regulated by Pot1 (protection of telomeres 1), and perturbation of Pot1 function has deleterious effects for proliferating cells. The specificity of the Pot1/ssDNA interaction and the key involvement of this protein in telomere maintenance have suggested directed inhibition of Pot1/ssDNA binding as an efficient means of disrupting telomere function. To explore this idea, we developed a high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) screen for inhibitors of Pot1/ssDNA interaction. We conducted this screen with the DNA-binding subdomain of Schizosaccharomyces pombe Pot1 (Pot1pN), which confers the vast majority of Pot1 sequence-specificity and is highly similar to the first domain of human Pot1 (hPOT1). Screening a library of ∼20 000 compounds yielded a single inhibitor, which we found interacted tightly with sub-micromolar affinity. Furthermore, this compound, subsequently identified as the bis-azo dye Congo red (CR), was able to competitively inhibit hPOT1 binding to telomeric DNA. Isothermal titration calorimetry and NMR chemical shift analysis suggest that CR interacts specifically with the ssDNA-binding cleft of Pot1, and that alteration of this surface disrupts CR binding. The identification of a specific inhibitor of ssDNA interaction establishes a new pathway for targeted telomere disruption.

Using NMR chemical shifts to calculate the propensity for structural order and disorder in proteins

NMR spectroscopy offers the unique possibility to relate the structural propensities of disordered proteins and loop segments of folded peptides to biological function and aggregation behaviour. Backbone chemical shifts are ideally suited for this task, provided that appropriate reference data are available and idiosyncratic sensitivity of backbone chemical shifts to structural information is treated in a sensible manner. In the present paper, we describe methods to detect structural protein changes from chemical shifts, and present an online tool [ncSPC (neighbour-corrected Structural Propensity Calculator)], which unites aspects of several current approaches. Examples of structural propensity calculations are given for two well-characterized systems, namely the binding of α-synuclein to micelles and light activation of photoactive yellow protein. These examples spotlight the great power of NMR chemical shift analysis for the quantitative assessment of protein disorder at the atomic level, and further our understanding of biologically important problems.

Investigation of the Electronic Origin of Asymmetric Induction in Palladium-Catalyzed Allylic Substitutions with Phosphinooxazoline (PHOX) Ligands by Hammett and Swain–Lupton Analysis of the13C NMR Chemical Shifts of the (π-Allyl)palladium Intermediates

Isomeric 4′- and 5′-substituted phosphinooxazoline (PHOX) ligands are used to probe the electronic origins of enantioselective nucleophilic additions to (1,3-diphenylallyl)palladium PHOX ligand complexes. Hammett analysis of the 13C NMR chemical shifts of the allyl C-1 and C-3 carbons shows that the major exo diastereomer is less susceptible to differential changes at C-1 and C-3 and that the location of the substituent has a smaller impact on these changes. In contrast, the minor endo diastereomer is more susceptible to differential 13C NMR changes at C-1 and C-3 and the location of the substituent has a greater impact on these changes. The endo diastereomer exhibits a pronounced “cis effect” by the ligating nitrogen and phosphorus atoms across the palladium center that explains its lower reactivity and, therefore, how the enantioselectivity typically obtained with PHOX ligands exceeds the approximately 8/1 ratio of exo to endo intermediates. Swain–Lupton analysis reveals the importance of both resonance and field effects by the substituents regardless of their location and supports the overall electronic control model for enantioselection by PHOX ligands. For rational chiral ligand design and electronic tuning of ligand properties, these results suggest that the overall electronic impact of a remote substituent generally depends more on its identity than on its location within the ligand.

Characterization of the microvoids of a tetramethyl polycarbonate/polystyrene blend system using Xe sorption measurements and 129Xe NMR spectroscopy

Xe sorption properties of tetramethyl bisphenol A polycarbonate (TMPC)/polystyrene (PS) blends indicated the reduction of microvoids by blending. From the analysis of 129Xe NMR chemical shifts of the 129Xe in these blends, the mean volume of individual microvoids (v) was calculated. The v values for these blends were smaller than those of predicted by a simple additive rule, as well as the cases of variation of density and Xe sorption properties. This fact indicates that the volume contraction induced by blending for TMPC/PS blends is mainly attributed to contraction volume of individual microvoids.