High-resolution solid-state NMR studies on uniformly [13C,15N]-labeled ubiquitin

Understanding of the effects of intermolecular interactions, molecular dynamics, and sample preparation on high-resolution magic-angle spinning NMR data is currently limited. Using the example of a uniformly [13C,15N]-labeled sample of ubiquitin, we discuss solid-state NMR methods tailored to the construction of 3D molecular structure and study the influence of solid-phase protein preparation on solid-state NMR spectra. A comparative analysis of 13C', 13Calpha, and 13Cbeta resonance frequencies suggests that 13C chemical-shift variations are most likely to occur in protein regions that exhibit an enhanced degree of molecular mobility. Our results can be refined by additional solid-state NMR techniques and serve as a reference for ongoing efforts to characterize the structure and dynamics of (membrane) proteins, protein complexes, and other biomolecules by high-resolution solid-state NMR.     

Backbone and side-chain 13C and 15N signal assignments of the alpha-spectrin SH3 domain by magic angle spinning solid-state NMR at 17.6 Tesla

The backbone and side-chain 13C and 15N signals of a solid 62-residue (u-13C,15N)-labelled protein containing the alpha-spectrin SH3 domain were assigned by two-dimensional (2D) magic angle spinning (MAS) 15N-13C and 13C-13C dipolar correlation spectroscopy at 17.6 T. The side-chain signal sets of the individual amino acids were identified by 2D 13C-13C proton-driven spin diffusion and dipolar recoupling experiments. Correlations to the respective backbone nitrogen signals were established by 2D NCACX (CX=any carbon atom) experiments, which contain a proton-nitrogen and a nitrogen-carbon cross-polarisation step followed by a carbon-carbon homonuclear transfer unit. Interresidue correlations leading to sequence-specific assignments were obtained from 2D NCOCX experiments. The assignment is nearly complete for the SH3 domain residues 7-61, while the signals of the N- and C-terminal residues 1-6 and 62, respectively, outside the domain boundaries are not detected in our MAS spectra. The resolution observed in these spectra raises expectations that receptor-bound protein ligands and slightly larger proteins (up to 20 kDa) can be readily assigned in the near future by using three-dimensional versions of the applied or analogous techniques.     

Structure and Energetics of Ligand–Fluorine Interactions with Galectin-3 Backbone and Side-Chain Amides: Insight into Solvation Effects and Multipolar Interactions

Multipolar fluorine–amide interactions with backbone and side-chain amides have been described as important for protein–ligand interactions and have been used to improve the potency of synthetic inhibitors. In this study, fluorine interactions within a well-defined binding pocket on galectin-3 were investigated systematically using phenyltriazolyl-thiogalactosides fluorinated singly or multiply at various positions on the phenyl ring. X-ray structures of the C-terminal domain of galectin-3 in complex with eight of these ligands revealed potential orthogonal fluorine–amide interactions with backbone amides and one with a side-chain amide. The two interactions involving main-chain amides seem to have a strong influence on affinity as determined by fluorescence anisotropy. In contrast, the interaction with the side-chain amide did not influence affinity. Quantum mechanics calculations were used to analyze the relative contributions of these interactions to the binding energies. No clear correlation could be found between the relative energies of the fluorine–main-chain amide interactions and the overall binding energy. Instead, dispersion and desolvation effects play a larger role. The results confirm that the contribution of fluorine–amide interactions to protein–ligand interactions cannot simply be predicted, on geometrical considerations alone, but require careful consideration of the energetic components.     

Stabilization of local structures by {pi}-CH and aromatic-backbone amide interactions involving prolyl and aromatic residues

Weakly polar interactions between the side-chain aromatic ringsand hydrogens of backbone amides (Ar–HN) and CHn of aliphaticgroups (     

Manifestations of stereoelectronic interactions in 1JC-H one-bond coupling constants

Two-electron/two-orbital hyperconjugative interactions depend on the relative orientation of bonds and lone pairs in a molecule and are also inversely proportional to the energy difference between the interacting orbitals. Spectroscopic manifestations of stereoelectronic interactions are particularly useful experimental signatures of these effects which can be utilized for testing molecular models. Empirical observations together with theoretical interpretations in cyclohexane and six-membered heterocycles confirm the relevance of sigma C-H ax --> sigma* C-H ax , n X --> sigma* C-H ax (X = O or N), sigma C-S --> sigma* C-H eq , beta-n O --> sigma* C-H eq , sigma C(2)-H ax --> pi* CY (Y = O, S, or CH 2), and sigma C(2)-H ax --> sigma* S-O ax two-electron/two-orbital stereoelectronic interactions that weaken the acceptor (or donor) C-H bonds and attenuate the Fermi contribution to the one-bond (13)C- (1)H coupling constants.     

N,N-Dialkylamide der 2-Hydroxy-2-phenyl-cyclopropan-1-carbonsäure – photochemische Darstellung und Strukturuntersuchung

N,N-Dialkylamides of 2-Hydroxy-2-phenyl-cyclopropan-1-carboxylic Acid – Photochemical Synthesis and Determination of the Structure Irradiation of N,N-dialkylamides of β-benzoylpropionic acid 2a , b in ether by n,π*-excitation leads to the corresponding amides of the hitherto unknown 2-hydroxy-2-phenyl-cyclo-propancarboxylic acid 3a , b , 4a , b . The photocyclization yields diastereomeric mixtures E:Z = 2.5–3:1. The determination of the relative configurations is based on the different tendency of dimerization of the E and Z diastereomers.     

1,2,3-Triazabutadiene. XII. Abhängigkeit der 13C-15N-Kopplungskonstanten von der Z/E-Isomerie der 1-Phenyl-3-[3-ethylbenzthiazolinyliden-(2)]-triazene und 1-Phenyl-1-ethyl-3-[3-ethylbenzthiazolinyliden-(2)]-triazenium- tetrafluoroborate

1,2,3-Triazabutadienes. XII. Dependence of the ¹³C- ¹⁵N Coupling Constants on the Z/E-Isomerism of the 1-Phenyl-3-[3-ethylbenzthiazolinyden-(2)]-triazenes and 1-Phenyl-1-ethyl-3-[3-ethylbenzthiazolinyliden-(2)]-triazenium-tetrafluoroborates ¹³C- and ¹⁵N spectra have been measured for the ¹⁵N-labelled triazene 1 and triazenium salt 2 . The ¹³C-¹⁵N coupling constants are discussed in the light of the Z/E-isomerism of the compounds. A significant nitrogen lone pair effect of the Z/E isomers on the magnitude of the coupling constant ¹J(¹⁵N₁ ¹³C₁·) was observed and discussed.     

Nitrogen-15 Kinetic Isotope Effects in the Menschutkin-Type Reaction of Benzyl Benzenesulfonates with N,N-Dimethylanilines

Nitrogen-15 kinetic isotope effects were measured for the Menschutkin-type reaction of Z-substituted benzyl X-substituted benzenesulfonates with Y-substituted N,N-dimethylanilines (Eq. 1) in acetone at 35°C. The values of ¹⁴k/¹⁵k for six combinations of X, Y, and Z fell in the range of 1.002–1.004. These small and almost constant nitrogen isotope effects are in contrast to the large and extensively varying carbon-14 and small but distinctly changing α-tritium isotope effects observed for this reaction series. The results are discussed in connection with the reactivity–selectivity relationships.     

The preparation and characterization of the methoxy derivatives of polyunsaturated fatty acids: Stabilized product analogs of lipoxygenase catalysis

The products from the action of soybean lipoxygenase on a series of polyunsaturated fatty acids have been converted in 2 steps into the corresponding methoxy derivatives. The product hydroperoxides were reduced in situ to alcohols with sodium borohydride. The ethers were generated by treatment of the alcohols with sodium hydride and methyl iodide in tetrahydrofuran. 13-Methoxy-9(Z),11(E)-octadecadienoic, 15-methoxy-11(Z),13(E)-eicosadienoic and 15-methoxy-5(Z),8(Z),11(Z), 13(E)-eicosatetraenoic acids were thus prepared. The methyl ethers were analyzed and the structures established by high performance liquid chromatography, nuclear magnetic resonance and infrared spectrometry, polarimetry and mass spectroscopy (as methyl esters). The methylation reaction proceeds without molecular rearrangements or racemiziation.     

Synthesis of 3-oxalinolenic acid and β-oxidation-resistant 3-oxa-oxylipins

3-Oxalinolenic acid (3-oxa-9(Z), 12(Z), 15(Z)-octadecatrienoic acid or (6(Z), 9(Z), 12(Z)-pentadecatrienyloxy)acetic acid) was synthesized from 5(Z), 8(Z), 11(Z), 14(Z), 17(Z)-eicosapentaenoic acid by a sequence involving the C15 aldehyde 3(Z), 6(Z), 9(Z), 12(Z)-pentadecatetraenal as a key intermediate. Conversion of the aldehyde by isomerization and two steps of reduction afforded 6(Z), 9(Z), 12(Z)-pentadecatrienol, which was coupled to bromoacetate to afford after purification by HPLC >99%-pure 3-oxalinolenic acid in 10–15% overall yield. 3-Oxalinolenic acid was efficiently oxygenated by soybean lipoxygenase-1 into 3-oxa-13(S)-hydroperoxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid, and this hydroperoxide could be further converted chemically into 3-oxa-13(S)-hydroxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid and 3-oxa-13-oxo-9(Z), 11(E), 15(Z)-octadecatrienoic acid. The 3-oxa-hydroperoxide also served as the substrate for the plant enzymes allene oxide synthase, divinyl ether synthase, and hydroperoxide lyase to produce 3-oxa-12-oxo-10, 15(Z)-phytodienoic acid and other 3-oxa-oxylipins that were characterized by MS, 3-Oxalinolenic acid was not oxygenated by 9-lipoxygenase from tomato but was converted at a slow rate into 3-oxa-9(S)-hydroperoxy-10(E), 12(Z), 15(Z)-octadecatrienoic acid by recombinant maize 9-lipoxygenase. Recombinant α-dioxygenase-1 from Arabidopsis thaliana catalyzed the conversion of 3-oxalinolenic acid into a 2-hydroperoxide, which underwent spontaneous degradation into a mixture of 6,9,12-pentadecatrienol and 6,9,12-pentadecatrienyl formate. A novel α-dioxygenase from the moss Physcomitrella patens was cloned and expressed and was found to display the same activity with 3-oxalinolenic acid as Arabidopsis thaliana α-dioxygenase-1. Lipoxygenase-generated 3-oxa-oxylipins are resistant toward β-oxidation and have the potential for displaying enhanced biological activity in situations where activity is limited by metabolic degradation.     

Crystallization kinetics modeling of high density and linear low density polyethylene resins

This paper describes isothermal and nonisothermal crystallization kinetics of a Ziegler‐Natta catalyzed high density polyethylene (HDPE) and linear low density polyethylene (LLDPE) resins. Standard techniques such as differential scanning calorimetry (DSC) and light depolarization microscopy (LDM) techniques were used to measure isothermal kinetics at low supercoolings. DSC was also used to measure nonisothermal crystallization kinetics at low cooling rates. Extrapolation of isothermal crystallization half‐times of Z‐N catalyzed LLDPE resin using the isothermal half‐time analysis led to erroneous predictions, possibly due to Z‐N LLDPE consisting of a mixture of molecules having different amounts of short chain branching (comonomer). However, predicted reciprocal half‐times at high supercoolings, using isothermal half‐time analysis and using nonlinear regression of nonisothermal crystallization kinetics measured at low cooling rates using the differential Nakamura model, of the HDPE were similar to measured reciprocal half times at high supercoolings of a similar HDPE by Patki and Phillips. It is also shown that the differential Nakamura model can be effectively used to model nonisothermal crystallization kinetics of HDPE resins. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

~(13)C、~(15)N双标记氨基脲的同位素丰度测定

采用微量高温灼烧法制备样品气,运用同位素质谱测定稳定性同位素13 C、15 N双标记氨基脲(13C、15 N3-SEM)产品的13C、15 N同位素丰度。通过实验对影响测定结果准确度的相关因素进行了考察,确定了最佳质谱转化条件为:称取1mg 13C、15 N3-SEM.HCl样品,10mg微粒状氧化铜,放入玻璃样品管,于真空状态下熔封样品管。将样品管放入马弗炉,530℃反应4h。实验表明:测定结果具有良好的精密度、重复性及再现性,满足了稳定性同位素13 C、15 N双标记氨基脲产品的同位素丰度测定的要求。     

PEGylation and polyPEGylation of nanodiamond

Polyethylene glycol (PEG) and poly(PEGMA) conjugated nanodiamond (ND) have been synthesized via “grafting to” and “grafting from” methods, respectively. In “grafting to” method, hydroxyl groups on ND surface were firstly oxidized to carboxyl groups, and then reacted with thionyl chloride to form acyl chloride groups. The acyl chloride functionalized ND (ND–COCl) was subsequently reacted with poly(ethylene glycol) monomethyl ether (mPEG) in the presence of triethylamine to generate mPEG conjugated ND (ND–mPEG). On the other hand, in “grafting from” method, ND–OH was modified with 2-bromoisobutyryl bromide (ND–Br), and then poly(PEG methyl ether methacrylate) (Poly(PEGMA)) chains were linked on the ND surface through surface-initiated atom transfer radical polymerization (ATRP) using ND–Br as the initiator and Cu(Br)/N,N,N′,N″,N″-pentmethyl diethylenetriamine (PMDETA) as the catalyst and ligand. The polymer conjugated ND particles were characterized using transmission electron microscopy (TEM), Fourier transform infrared (FT-IR) spectroscopy, thermal gravimetric analysis (TGA) and X-ray photoelectron spectroscopy (XPS). TGA analyses demonstrated that the polymer weight ratios through “grafting to” and “grafting from” methods were 29.8% and 34.4%, respectively. The mPEG and poly(PEGMA) conjugated ND nanoparticles exhibited enhanced dispersibility in organic media. More importantly, due to the relative high graft ratios and molecular weight, poly(PEGMA) functionalized ND was also dispersed well in water. Given the excellent physicochemical and biological properties of PEG and ND, the methods described in current work might be useful for the preparation of functional ND nanoparticles for potential biomedical applications.     

一维链状磺酸银配合物[Ag_2(2-Pyr)(H_2O)_4]L_2的合成与晶体结构

由Ag2CO3、2,5-二氯-4-氨基苯磺酸(HL)和2-甲基吡嗪(2-Pyr)反应,得了一种新型磺酸银配合物[Ag2(2-Pyr)(H2O)4]L2(1),通过元素分析、红外光谱和X-单晶衍射等对配合物的结构进行了表征。结果表明:1属单斜晶系,空间群P21/c,晶胞参数a=0.92748(19),b=0.77156(15),c=2.0044(4)nm,β=91.34(3)°,V=1.4340(5)nm3,Z=2,F(000)=850,Dc=1.999g/cm3,R1=0.0867,wR2=0.1717[I>2σ(I)]。配合物中银离子被2-Pyr配体和配位水分子连接成一维的链状结构,进一步通过分子间氢键作用扩展为三维的超分子结构。并且得出了第二配体(2-Pyr)的存在使磺酸银化合物的结构发生了根本的改变。     

SEPARATION OF STABLE NITROGEN ISOTOPES BY ION EXCHANGE CHROMATOGRAPHY

ABSTRACT

The ion exchange chromatography technique was used for the enrichment of nitrogen 15 (¹⁵N). A set of columns filled with the ion exchange resin Wofatit KPS (medium porosity type) with 5.4?cm I.D. and 1.5?m height was set up. Ammonia NH₄ ⁺/NH₃ aq. was chosen as the isotopic exchange system. The ammonium bands formed in the columns were eluted by a solution of sodium hydroxide. The isotope separation factor, ?, was found to decrease with increasing temperature or ammonia concentration. Operational conditions of temperature and ammonium concentration were proposed as the optimum for the production of the enriched isotope by using the present process. Results showed that: (1) the rear part of the band is enriched in ¹⁵N, while the frontal part is depleted; (2) it is possible to obtain 7.6?at. % of ¹⁵N after a 60?m displacement of the ammonium band. The analytical determination of the samples was made by emission spectrometry (NOI-6E).     

Photolyse von 13(S)-Hydroperoxy-9(Z), 11(E)-octadecadiensäuremethylester in Gegenwart von Sauerstoff — Analyse der niedermolekularen Reaktionsprodukte

Photolysis of methyl 13(S)-Hydroperoxy-9(Z),11(E)-Octadecadienoate in the Presence of Oxygen — Analysis of the Low Molecular Reaction Products A solution of methyl 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoate in n-pentane was irradiated in the presence of oxygen at 15°C with light at wavelengths greater than 230 nm. After 2 hours 3.5 mol-% volatile aldehydes and esters and in addition 5 mol-% low molecular acids were isolated. Four major products were identified: hexanal, caproic acid, azelaic acid monomethyl ester and methyl 6-heptenoate.     

Three three-dimensional supramolecular networks formed by one-dimensional chains of Ag(I) and N,N′-di(3-pyridyl)succinamide via weak intermolecular interactions

Reactions of N,N′-di(3-pyridyl)succinamide (L) with AgOTf, AgTFA and AgNO₃ (OTf = triflate, TFA = trifluoroacetate) produced three coordination polymers {Ag(L)(OTf)·DMF·MeCN}_n (1·DMF·MeCN), {Ag(L)(TFA)·2H₂O}_n (2·2H₂O) and {[Ag(L)]₂(NO₃)₂}_n (3), respectively. In 1, two one-dimensional (1D) [Ag(L)] chains are joined by the Ag⋯Ag, π⋯π and hydrogen-bonding interactions to form a 1D double chain, which is further linked to other equivalent ones via the hydrogen bonding interactions to yield a three-dimensional (3D) structure. In 2, the 1D [Ag(L)] helical chains are connected by the Ag⋯O, π⋯π, CH⋯π and hydrogen-bonding interactions to afford a 3D framework. In 3, two kinds of 1D [Ag(L)] chains are linked by the Ag⋯O and hydrogen-bonding interactions to form a 3D structure. The luminescent properties of 1–3 along with L were also investigated.     

Isolation and structures of two divinyl ether fatty acids from <Emphasis Type="Italic">Clematis vitalba</Emphasis>

[1-14C]Linolenic acid was incubated with a homogenate of leaves of Clematis vitalba, a plant belonging to the Ranunculaceae family. Analysis of the reaction product by reversed-phase high-performance liquid radiochromatography demonstrated the presence of the following labeled oxylipins: 12-oxo-10, 15(Z)-phytodienoic acid, 9(S)-hydroxy-10(E), 12(Z), 15(Z)-octadecatrienoic acid, omega5(Z)-etherolenic acid, and 9-[1'(E), 3'(Z),6'(Z)-nonatrienyloxy]-8(Z)-nonenoic acid [8(Z)-colnelenic acid]. The last compound was a new divinyl ether FA, and an analogous compound, i.e., 9-[1'(E),3'(Z)-nonadienyloxy]-8(Z)-nonenoic acid [8(Z)-colneleic acid], was obtained following incubation of linoleic acid with the Clematis homogenate. Structures of the two divinyl ethers were assigned by spectral and chromatographic comparison with authentic compounds prepared synthetically using previously described methodology. Separate incubation of the 9- and 13-hydroperoxides of linolenic acid demonstrated that the first hydroperoxide served as the precursor of 8(Z)-colnelenic acid and indicated the presence in C. vitalba of a new divinyl ether synthase acting on 9-lipoxygenase-generated hydroperoxides. A close structural relationship between this enzyme and the well-studied divinyl ether synthase in the potato and tomato seems likely.     

Triplex-Forming Peptide Nucleic Acids with Extended Backbones

Peptide nucleic acid (PNA) forms a triple helix with double-stranded RNA (dsRNA) stabilized by a hydrogen-bonding zipper formed by PNA's backbone amides (N−H) interacting with RNA phosphate oxygens. This hydrogen-bonding pattern is enabled by the matching ∼5.7 Å spacing (typical for A-form dsRNA) between PNA's backbone amides and RNA phosphate oxygens. We hypothesized that extending the PNA's backbone by one −CH₂− group might bring the distance between PNA amide groups closer to 7 Å, which is favourable for hydrogen bonding to the B-form dsDNA phosphate oxygens. Extension of the PNA backbone was expected to selectively stabilize PNA-DNA triplexes compared to PNA-RNA. To test this hypothesis, we synthesized triplex-forming PNAs that had the pseudopeptide backbones extended by an additional −CH₂− group in three different positions. Isothermal titration calorimetry measurements of the binding affinity of these extended PNA analogues for the matched dsDNA and dsRNA showed that, contrary to our structural reasoning, extending the PNA backbone at any position had a strong negative effect on triplex stability. Our results suggest that PNAs might have an inherent preference for A-form-like conformations when binding double-stranded nucleic acids. It appears that the original six-atom-long PNA backbone is an almost perfect fit for binding to A-form nucleic acids.     

15N- und 13 C-NMR-chemische Verschiebungen sowie 13C-1H-, 13C-15N- und 15N-15N-Spin-Spin-Kopplungskonstanten para-substituierter Benzendiazoniumsalze

Nitrogen-15 and Carbon-13 Chemical Shifts and ¹³C-¹H, ¹³C-¹⁵N and ¹⁵N-¹⁵N Coupling Constants of Parasubstituted Arene Diazonium Salts Nitrogen-15 and carbon-13 chemical shifts and ¹³C-¹H,¹³C-¹⁵N and ¹⁵N-¹⁵N coupling constants of ¹⁵N-enriched p-substituted arene diazonium salts are reported and within the ΔE-approximation investigated. It is shown that changes of ΔE should be taken into account when considering variation of the nitrogen-15 chemical shifts. The influence of substituent effects was discussed within the DSP (dual substituent parameter)-approach.