Efficient assignment of methyl resonances: Enhanced sensitivity by gradient selection in a DE-MQ–(H)CC<Superscript><Emphasis Type="Italic">m</Emphasis></Superscript>Ht <Superscript><Emphasis Type="Italic">m</Emphasis></Superscript>–TOCSY experiment

We present a gradient selected and doubly sensitivity-enhanced DE-MQ–(H)CC ^m H ^m –TOCSY experiment for the sequence-specific assignment of methyl resonances in ¹³C,¹⁵N labeled proteins. The proposed experiment provides improved sensitivity and artifact suppression relative to the phase-cycled experiments. One part of the ¹³Cchemical shift evolution takes place under heteronuclear multiple quantum coherence, whereas the other part occurs under ¹³C single quantum coherence in a semi-constant time fashion. The feasibility of the experiment was assessed using ¹⁵N,¹³C labeled Mus musculus coactosin (16 kDa), having a rotational correlation time of 14.5 ns at 15 °C in D₂O. A 16-h experiment on 600 MHz ¹H yielded good quality data and enabled the assignment of 70 out of 72 methyl groups in coactosin. As well as being an improved approach for methyl resonance assignment, this experiment can also be highly valuable for the rapid assignment of methyl resonances in SAR by NMR studies.     

Synthesis of some derivatives of C-(1-deoxy-1-N-substituted-D-glucopyranosyl)formic acid (D-gluco-hept-2-ulopyranosonic acid) as potential inhibitors of glycogen phosphorylase

Per-O-benzoylated derivatives (amide, methyl ester and glycinamide) of C-(1-azido-1-deoxy-alpha-D-glucopyranosyl)formic acid obtained by azide substitution in the corresponding C-(1-bromo-1-deoxy-beta-D-glucopyranosyl)formic acid derivatives were debenzoylated by the Zemplén-protocol. Per-O-benzoylated C-(1-azido-1-deoxy-alpha-D-glucopyranosyl)formamide was dehydrated by oxalyl chloride-DMF to give the corresponding nitrile, while from its reduction mixture obtained by Raney-nickel or sodium hydrogentelluride C-(1-amino-1-deoxy-beta-D-glucopyranosyl)formamide could be isolated. Acetylation of this amino-amide by Ac2O/Py and subsequent debenzoylation gave C-(1-acetamido-1-deoxy-beta-D-glucopyranosyl)formamide. Applying the same conditions to the crude reduction mixture allowed the alpha-anomer to be isolated as a minor component. An alternative pathway to produce the above beta-anomer appeared in the reaction of C-(1-bromo-1-deoxy-beta-D-glucopyranosyl)formamide with CH3CN in the presence of Ag2CO3 to yield 1-acetamido-2,3,4,6,-tetra-O-benzoyl-1-deoxy-beta-D-glucopyranosyl cyanide, which was hydrated, in the presence of TiCl4, to the formamide. Some of the new compounds were shown to be weak inhibitors of muscle glycogen phosphorylase b.     

Computer-assisted assignment of 2D1H NMR spectra of proteins: Basic algorithms and application to phoratoxin B

Summary A suite of computer programs (CLAIRE) is described which can be of assistance in the process of assigning 2D¹H NMR spectra of proteins. The programs embody a software implementation of the sequential assignment approach first developed by Wüthrich and co-workers (K. Wüthrich. G. Wider, G. Wagner and W. Braun (1982)J. Mol. Biol. 155, 311). After data-abstraction (peakpicking), the software can be used to detect patterns (spin systems), to find cross peaks between patterns in 2D NOE data sets and to generate assignments that are consistent with all available data and which satisfy a number of constraints imposed by the user. An interactive graphics program calledCONPAT is used to control the entire assignment process as well as to provide the essential feedback from the experimental NMR spectra. The algorithms are described in detail and the approach is demonstrated on a set of spectra from the mistletoe protein phoratoxin B, a homolog of crambin. The results obtained compare well with those reported earlier based entirely on a manual assignment process.     

Cytotoxic protobassic acid glycosides from Planchonella obovata leaf

Four triterpenoid glycosides, possessing protobassic acid as common aglycon, together with 16 known compounds were isolated from the leaves of Planchonella obovata. They are 6β-hydroxy-conyzasaponin G (2), 3‴-O-de-β-d-apiofuranosylisoarganin F (3), isoarganin F (4), and 6β-hydroxy-conyzasaponin N (5). The structures of these glycosides were elucidated based on spectroscopic analysis, in particular using 1D TOCSY to confirm the ¹H NMR assignment of each sugar residue. The absolute configuration of each monosaccharide in the glycon part was determined by GC-FID. Compound 5, Mi-saponin A (8), and ursolic acid (10) showed moderate inhibitory activities against HL-60 leukemia cell line with the IC₅₀ values of 16.88, 15.50, and 12.68 μM, respectively.     

一种百合甾体皂苷纯度及糖链结构核磁共振法分析

采用多种NMR分析技术,首次对百合甾体皂苷(25R,26R)-26-甲氧基螺甾烷-5-烯-3β-α-L-鼠李糖-(1→2)-[β-D-葡萄糖-(1→6)]-β-D-葡萄糖苷的1H和13C NMR信号进行了全归属,特别是应用选择性的1D TOCSY和1D NOESY核磁共振分析技术,对该化合物1中的氢谱信号严重重叠的糖链进行了详细的分析,提出了一套对甾体皂苷糖链信号进行全归属的核磁共振法.在确认其结构的基础上,建立了核磁共振法(1H NMR)测定该化合物1的纯度,给出了完整的实验条件,线性回归系数为0.9998,重复性实验RSD为0.58％,稳定性实验RSD为0.24％,操作简单、快速准确,且不需要其它对照品,是中药化学对照品纯度研究的一个有益补充.     

Refinement of 3D models of horseradish peroxidase isoenzyme C: Predictions of 2D NMR assignments and substrate binding sites

In this study, two alternative three-dimensional (3D) models of horseradish peroxidase (HRP-C)—differing mainly in the structure of a long untemplated insertion—were refined, systematically assessed, and used to make predictions that can both guide and be tested by future experimental studies. A key first step in the model-building process was a procedure for multiple sequence alignment based on structurally conserved regions and key conserved residues, including those side chains providing ligands to the two Ca²⁺ binding sites. The model refinements reported here include (1) optimization of side-chain conformations; (3) addition of structural waters using a template-independent procedure; (2) structural refinement of the untemplated 34 amino acid insertion located between the F and G helices, using both energy criteria and NMR data; (4) unconstrained energy optimization of the refined models. Using these procedures, two refined structures of HRP-C were obtained, differing mainly in the conformation of this long insertion. The presence of residues in this insertion that could potentially interact with bound substrates suggests a functional role that may be related to the general ability of class III peroxidases to form stable 1:1 complexes with a variety of substrates. The structural validity of the models was systematically assessed by a variety of criteria. Most notably, the ProsaII z scores and Profiles 3D scores of the two HRP-C models indicated that they are significantly better than would be obtained by simple amino acid replacement, using any of the known structures as a template. These two 3D HRP-C models, were then used to predict candidate residues for the assignment of NOESY cross-peaks previously noted in 2D-NMR studies. Specifically, the residues known as Ile X, Phe A, Phe B, aliphatic residue Q, and Ile T. Candidate substrate binding sites were also identified and compared with experimentally based predictions. This work is timely because new X-ray structures are anticipated that will facilitate the validation of these procedures. © 1996 Wiley-Liss, Inc.     

眼镜王蛇神经毒素CM—11核磁共振氢谱谱峰的完全归属

眼镜王蛇抽提物ＣＭ－１１为含７２个残基的长链神经毒素,对其进行了ＤＱＦ－ＣＯＳＹ,ＴＯＣＳＹ和ＮＯＥＳＹ等一系列２Ｄ－ＮＭＲ谱测定,借助序列专一归属法完成了ＣＭ－１１ＮＭＲ氢谱的完整归属。     

Computer-assisted assignment of peptides with non-standard amino acids

Summary A comprehensive peptide assignment program and its application to a cyclic peptide, cyclosporin A, are presented in this paper. A group of graph theoretical algorithms using fuzzy logic are discussed with the aid of examples from cyclosporin A. The algorithms deal with heavily overlapped peaks, recover disjointed and distorted spin coupling networks, and include strategies for sequence-specific assignment. A procedure to extend the Protein Knowledge Base for automatically assigning non-standard amino acid residues is also presented. The program is capable of completely automated assignment for small peptides (20 residues). For such molecules, it is insensitive to whether the peptide chain is cyclic or acyclic, and to whether amide protons are present or absent. For larger peptides/proteins, more user interaction is required and the sequence-specific assignment step usually must proceed through fragments smaller than the full length to avoid problems due to occurrence of a combinatorial explosion. The program can be applied as a rigorous tool to check manual assignments. The fuzzy graph theoretical concepts built in the program are illustrated with 2D proton spectra of a peptide, but may be extended to higher-dimensional spectra, other biopolymers, natural products and other organic structures.     

Recent progress in heteronuclear long-range NMR of complex carbohydrates: 3D H2BC and clean HMBC

The new NMR experiments 3D H2BC and clean HMBC are explored for challenging applications to a complex carbohydrate at natural abundance of ¹³C. The 3D H2BC experiment is crucial for sequential assignment as it yields heteronuclear one- and two-bond together with COSY correlations for the ¹H spins, all in a single spectrum with good resolution and non-informative diagonal-type peaks suppressed. Clean HMBC is a remedy for the ubiquitous problem of strong coupling induced one-bond correlation artifacts in HMBC spectra of carbohydrates. Both experiments work well for one of the largest carbohydrates whose structure has been determined by NMR, not least due to the enhanced resolution offered by the third dimension in 3D H2BC and the improved spectral quality due to artifact suppression in clean HMBC. Hence these new experiments set the scene to take advantage of the sensitivity boost achieved by the latest generation of cold probes for NMR structure determination of even larger and more complex carbohydrates in solution.     

Quantitative covariance NMR by regularization

The square root of a covariance spectrum, which offers high spectral resolution along both dimensions requiring only few t ₁ increments, yields in good approximation the idealized 2D FT spectrum provided that the amount of magnetization exchanged between spins is relatively small. When this condition is violated, 2D FT and covariance peak volumes may differ. A regularization method is presented that produces a modified covariance spectrum with cross-peak volumes that closely match their 2D FT analogues. The method is demonstrated for TOCSY spectra with variable mixing times.     

Stereoselective synthesis of N-galactofuranosyl amides

α- or β-Galactofuranosyl (Galf) amides can be synthesized with high stereoselectivity by traceless Staudinger ligation starting from unprotected β-galactofuranosyl azide or tetra-O-acetyl-β-galactofuranosyl azide, respectively. The resulting Galf amides are hitherto unknown molecules, with interesting potential as inhibitors of mycobacterial growth.     

新型抗菌多肽LC1的1H—NMR谱峰归属和二级结构分析

LC1是从枯草杆菌A0 14的分泌物中分离出的一种新型抗菌多肽 ,具有很强的抗水稻白叶枯致病菌的能力。应用 2D NMR技术研究LC1的溶液构象 ,通过分析其在水和重水中的DQF COSY、TOCSY和NOESY等1H NMR谱 ,识别了LC1全部 4 7个氨基酸残基的自旋体系 ,并通过分析NOESY谱中dαN、dNN、dβN和dαδ的联系完成了序列专一谱峰归属 ,标定了全部主链质子和绝大部分侧链质子的化学位移。谱峰归属结果和NMR数据分析表明LC1的二级结构主要为伸展构象 ,其中肽段Phe2 5～Asp3 1和Tyr3 6～Glu42 构成反平行 β折叠 ,并由Ser3 2 ～Gly3 5所形成的β转角相连接。LC1不含或仅含少量α螺旋。同时 ,通过对LC1的大量疏水氨基酸残基之间NOE联系的分析 ,推测LC1具有一个以Trp2 3 为中心的疏水核心。     

Synthesis of glycosyl-triazole linked 1,2,4-oxadiazoles

The synthesis of four different types of oxadiazoles containing a terminal acetylenic group is described. Reaction of these oxadiazoles with various azidoglycosides via a copper-catalyzed [3+2] cycloaddition ('click chemistry') afforded the corresponding glycosyl-triazole linked 1,2,4-oxadiazoles in good yields.     

1H, 13C and 15N NMR backbone assignments and secondary structure of the 269-residue protease subtilisin 309 from Bacillus lentus

Summary ¹H, ¹³C and ¹⁵N NMR assignments of the backbone atoms of subtilisin 309, secreted by Bacillus lentus, have been made using heteronuclear 3D NMR techniques. With 269 amino acids, this protein is one of the largest proteins to be sequentially assigned by NMR methods to date. Because of the size of the protein, some useful 3D correlation experiments were too insensitive to be used in the procedure. The HNCO, HN(CO)CA, HNCA and HCACO experiments are robust enough to provide most of the expected correlations for a protein of this size. It was necessary to use several experiments to unambiguously determine a majority of the -protons. Combined use of HCACO, HN(COCA)HA, HN(CA)HA, ¹⁵N TOCSY-HMQC and ¹⁵N NOESY-HMQC experiments provided the H chemical shifts. Correlations for glycine protons were absent from most of the spectra. A combination of automated and interactive steps was used in the process, similar to that outlined by Ikura et al. [(1990) J. Am. Chem. Soc., 112, 9020–9022] in the seminal paper on heteronuclear backbone assignment. A major impediment to the linking process was the amount of overlap in the C and H frequencies. Ambiguities resulting from this redundancy were solved primarily by assignment of amino acid type, using C chemical shifts and TOCSY ladders. Ninety-four percent of the backbone resonances are reported for this subtilisin. The secondary structure was analyzed using 3D ¹⁵N NOESY-HMQC data and C secondary chemical shifts. Comparison with the X-ray structure [Betzel et al. (1992) J. Mol. Biol., 223, 427–445] shows no major differences.Supplementary material available from F.J.M. van de Ven: the source code (PASCAL) for the computer program described in this paper.     

不同坡位对GPS项圈定位性能的影响

GPS项圈已广泛应用于大中型野生动物的野外定位监测,支持野生动物生态学研究和保护管理规划.但由于地形等因素影响,GPS项圈返回的数据可能有定位误差和定位数据缺失偏歧.因此,在进行生境选择等相关生态学研究之前,应对GPS项圈的定位性能进行评估.本研究于2019年11月至2020年10月,在云南大理苍山5个测试点各静态放置...