Multi-nuclear (119Sn, 13C, 1H), fourier-transform,N.M.R. studies of di- and tri-butylstannyl ethers of carbohydrates

¹¹⁹Sn-N.m.r. spectra are reported for toluene solutions of the tributylstannyl ethers of 2,3,4,6-tetra-O-methyl-d)-glucose, 1,2:3,4-di-O-isopropylidene-α-d-galactopyranose, methyl 2,3-di-O-methyl-α-d-glucopyranoside, and methyl 4,6-O-benzylidene-α-d-glucopyranoside, and the dibutylstannyl ether of the last sugar. In the reaction of bis(tributyltin) oxide with methyl 4,6-O-benzylidene-α-d-glucopyranoside in toluene, HO-3 is much more reactive than HO-2. The presence of the various tin-containing species is readily apparent from the ¹¹⁹Sn-spectra. The importance of suppressing the nuclear Overhauser effect is demonstrated. The appearance of ¹¹⁹Sn satellites in the ¹³C-n.m.r. spectra demonstrates couplings of the types, ²J(¹¹⁹Sn-O-¹³C) and ³J(¹¹⁹Sn-O-C-¹³C), forthe first time, and, together with the ¹³C-chemical shifts, facilitates the determination of the site of substitution. The ¹¹⁹Sn-chemical shifts show that different states of coordination may be recognised. However, although different sites of substitution produce separate resonances, no simple relationship between shift and position is found. ¹³C-Chemical shifts are reported for methyl 4,6-O-benzylidene-α-d-glucopyranoside and its tributylstannyl ethers, and substituent effects are discussed.     

Preparation and N.M.R. studies of pyruvic acid and related acetals of pyranosides: configuration at the acetal carbon atoms

Stereoisomeric pairs of pyruvic acid and related acetals linked to the 3,4- and 4,6-positions, respectively, of the anomeric methyl d-galactopyranosides and the corresponding acetals linked to the 4,6-positions of the anomeric methyl d-glucopyranosides have been prepared by conventional methods, and their structures have been assigned. Their ¹H- and ¹³C-n.m.r. spectra have been recorded. The differences in chemical shifts obtained for stereoisomeric pairs of acetalic CH₃ groups are of sufficient magnitude to make possible the unequivocal determination of the stereo-chemistry of pyruvic acid acetals in naturally occurring polysaccharides.     

13C-n.m.r. spectra of xylo-oligosaccharides and their application to the elucidation of xylan structures

¹³C-N.m.r. spectra of thirteen xylo-oligosaccharides [a complete series of α- and β-d-xylopyranosyl derivatives of methyl α-d-xylopyranoside, β-d-xylopyranosyl derivatives of methyl 4-O-β-d-xylopyranosyl-d-xylopyranoside, methyl O-α-d-xylopyranosyl-(1→3)-O-β-d-xylopyranosyl-(1→4)-d-xylopyranoside, and a branched methyl β-xylotetraoside] have been interpreted. The data obtained have been used for the carbon signal assignment in the spectra of a number of red-algal xylans. ¹³C-N.m.r. spectroscopy is shown to be a rapid and convenient method for the structural analysis of xylose-rich polysaccharides.     

Carbon-13 nuclear magnetic resonance spectra of carbohydrate oxirane derivatives

The ¹³C-chemical shifts and ¹J_C,H values of two series of carbohydrate oxirane derivatives, namely methyl 2,3-anhydro-ribo- and -lyxofuranosides and methyl 2,3-anhydro-4,6-O-benzylidene-manno- and -allopyranosides have been determined. The assignment of ¹³C resonances has been established mainly by the examination of the proton-coupled and the selective proton-decoupled spectra. The effect of the oxirane rings on the chemical shifts of β and γ carbon atoms (from the oxirane ring oxygen atom) has been observed. Large ¹J_C,H values associated with cis CH bonds adjacent to the oxirane rings relative to those of trans counterparts have been found.     

Specific 15N, NH correlations for residues in15 N, 13C and fractionally deuterated proteins that immediately follow methyl-containing amino acids

A triple-resonance pulse scheme is described which records¹⁵N, NH correlations of residues that immediately follow amethyl-containing amino acid. The experiment makes use of a¹⁵N, ¹³C and fractionally deuterated proteinsample and selects for CH₂D methyl types. The experiment isthus useful in the early stages of the sequential assignment process as wellas for the confirmation of backbone ¹⁵N, NH chemical shiftassignments at later stages of data analysis. A simple modification of thesequence also allows the measurement of methyl side-chain dynamics. This isparticularly useful for studying side-chain dynamic properties in partiallyunfolded and unfolded proteins where the resolution of aliphatic carbon andproton chemical shifts is limited compared to that of amide nitrogens.     

Proton and carbon-13 nuclear magnetic resonance studies on methyl (methyl d-galactosid)uronates and their per-O-acetyl derivatives

The ¹H- and ¹³C-n.m.r. spectra of the anomeric methyl (methyl d-galactosid)uronates, as well as the ¹H-n.m.r. spectra of their acetyl derivatives, were analyzed. The spectra of the unacetylated d-galactopyranosiduronates showed good correlation with those of the corresponding anomeric d-galactopyranuronic acids and their methyl esters, and with those of the anomeric methyl d-galactopyranosides. From the values of the chemical shifts and coupling constants, it was concluded that the anomeric methyl (methyl d-galactopyranosid)uronates and their corresponding peracetates are in the ⁴C₁(d) conformation. The chemical shifts in the ¹³C-n.m.r. spectra show good correlation with those of the methyl d-galactosides. The signals of the furanose derivatives appear at fields lower than those of the corresponding pyranose compounds.     

Practical use of chemical shift databases for protein solid-state NMR: 2D chemical shift maps and amino-acid assignment with secondary-structure information

We introduce a Python-based program that utilizes the large database of ¹³C and ¹⁵N chemical shifts in the Biological Magnetic Resonance Bank to rapidly predict the amino acid type and secondary structure from correlated chemical shifts. The program, called PACSYlite Unified Query (PLUQ), is designed to help assign peaks obtained from 2D ¹³C–¹³C, ¹⁵N–¹³C, or 3D ¹⁵N–¹³C–¹³C magic-angle-spinning correlation spectra. We show secondary-structure specific 2D ¹³C–¹³C correlation maps of all twenty amino acids, constructed from a chemical shift database of 262,209 residues. The maps reveal interesting conformation-dependent chemical shift distributions and facilitate searching of correlation peaks during amino-acid type assignment. Based on these correlations, PLUQ outputs the most likely amino acid types and the associated secondary structures from inputs of experimental chemical shifts. We test the assignment accuracy using four high-quality protein structures. Based on only the Cα and Cβ chemical shifts, the highest-ranked PLUQ assignments were 40–60 % correct in both the amino-acid type and the secondary structure. For three input chemical shifts (CO–Cα–Cβ or N–Cα–Cβ), the first-ranked assignments were correct for 60 % of the residues, while within the top three predictions, the correct assignments were found for 80 % of the residues. PLUQ and the chemical shift maps are expected to be useful at the first stage of sequential assignment, for combination with automated sequential assignment programs, and for highly disordered proteins for which secondary structure analysis is the main goal of structure determination.     

Chemical shift assignment and structural plasticity of a HIV fusion peptide derivative in dodecylphosphocholine micelles

A “HFPK3” peptide containing the 23 residues of the human immunodeficiency virus (HIV) fusion peptide (HFP) plus three non-native C-terminal lysines was studied in dodecylphosphocholine (DPC) micelles with 2D ¹H NMR spectroscopy. The HFP is at the N-terminus of the gp41 fusion protein and plays an important role in fusing viral and target cell membranes which is a critical step in viral infection. Unlike HFP, HFPK3 is monomeric in detergent-free buffered aqueous solution which may be a useful property for functional and structural studies. H^α chemical shifts indicated that DPC-associated HFPK3 was predominantly helical from I4 to L12. In addition to the highest-intensity crosspeaks used for the first chemical shift assignment (denoted I), there were additional crosspeaks whose intensities were ∼ 10% of those used for assignment I. A second assignment (II) for residues G5 to L12 as well as a few other residues was derived from these lower-intensity crosspeaks. Relative to the I shifts, the II shifts were different by 0.01-0.23 ppm with the largest differences observed for H^N. Comparison of the shifts of DPC-associated HFPK3 with those of detergent-associated HFP and HFP derivatives provided information about peptide structures and locations in micelles.     

A NMR experiment for simultaneous correlations of valine and leucine/isoleucine methyls with carbonyl chemical shifts in proteins

A methyl-detected ‘out-and-back’ NMR experiment for obtaining simultaneous correlations of methyl resonances of valine and isoleucine/leucine residues with backbone carbonyl chemical shifts, SIM-HMCM(CGCBCA)CO, is described. The developed pulse-scheme serves the purpose of convenience in recording a single data set for all Ile^δ1, Leu^δ and Val^γ (ILV) methyl positions instead of acquiring two separate spectra selective for valine or leucine/isoleucine residues. The SIM-HMCM(CGCBCA)CO experiment can be used for ILV methyl assignments in moderately sized protein systems (up to ~100 kDa) where the backbone chemical shifts of ¹³C^α, ¹³C_β and ¹³CO are known from prior NMR studies and where some losses in sensitivity can be tolerated for the sake of an overall reduction in NMR acquisition time.     

13C-N.M.R.-spectroscopic investigation of agarose oligomers

A complete, unambiguous assignment of all of the ¹³C-n.m.r.-spectral signals of agarose oligomers produced by enzymic hydrolysis has been achieved. The ¹J¹³C-H coupling constants are reported, and the chemical shifts and coupling constants of both the agarose polymer and oligomers are compared.     

Studies related to the synthesis of derivatives of 2,6-diamino-2,3,4,6-tetradeoxy-D-erythro-hexose (purpurosamine C), a component of gentamicin C12

Reduction of 1,6-anhydro-3,4-dideoxy-β-D-glycero-hex-3-enopyranos-2-ulose (levoglucosenone) with lithium aluminium hydride afforded principally 1,6-anhydro-3,4-dideoxy-β-D-threo-hex-3-enopyranose (3), which was converted into 3,4-dihydro-2(S)-hydroxymethyl-2H-pyran (8) following acid-catalysed methanolysis and reductive rearrangement of the resulting α-glycoside 4 with lithium aluminium hydride. 1,6-Anhydro-3,4-dideoxy-2-O-toluene-p-sulphonyl-β-D-threo-hexopyranose, prepared from 3, reacted slowly with sodium azide in hot dimethyl sulphoxide to give 1,6-anhydro-2-azido-2,3,4-trideoxy-β-D-erythro-hexopyranose, which was transformed into a mixture of methyl 2-acetamido-6-O-acetyl-2,3,4-trideoxy-α-D-erythro-hexopyranoside (10) and the corresponding β anomer following acid-catalysed methanolysis, catalytic reduction, and acetylation. Acid treatment of methyl 4,6-O-benzylidene-3-deoxy-α-D-erythro-hexopyranosid-2-ulose yielded the enone 15, which was readily transformed into methyl 6-O-acetyl-3,4-dideoxy-α-D-glycero-hexopyranosid-2-ulose (19). Procedures for the conversions of DL-8, 10, and 19 into methyl 2,6-diacetamido-2,3,4,6-tetradeoxy-α-D-erythro-hexopyranoside (methyl N,N′-di-acetyl-α-purpurosaminide C) have already been described.     

Orientation of the axial ligands and magnetic properties of the hemes in the triheme ferricytochrome PpcA from G. sulfurreducens determined by paramagnetic NMR

The geometry of the axial ligands of the hemes in the triheme cytochrome PpcA from Geobacter sulfurreducens was determined in solution for the ferric form using the unambiguous assignment of the NMR signals of the α-substituents of the hemes. The paramagnetic ¹³C shifts of the hemes can be used to define the heme electronic structure, the geometry of the axial ligands, and the magnetic susceptibility tensor. The latter establishes the magnitude and geometrical dependence of the pseudocontact shifts, which are crucial to warrant reliable structural constraints for a detailed structural characterization of this paramagnetic protein in solution.     

Elaeodendrol and elaeodendradiol,new nor-triterpenes from Elaeodendron glaucum

The isolation of six known and two new D:A-friedooleananes is reported from the bark of Elaeodendron glaucum. The structures of the new nor-triterpenes, elaeodendrol and elaeodendradiol, were established respectively as 17β- hydroxy-28-norfriedelan-3-one and l7β,25-dihydroxy-28-norfriedelan-3-one by a study of the methyl chemical shifts in their ¹H NMR spectra.     

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.     

Total synthesis of PGC2 methyl ester. Comparison with enzymatically produced material

The synthesis of PGC₂ methyl ester is described. Comparison of the synthetic material with the methyl ester prepared from natural PGC₂ showed the two to be identical, thus confirming the structure assignment. The physical and biological data of PGC₂ methyl ester are presented.Horton and Jones have recently shown that PGA₁ (Ic) and PGA₂ (Ia) are deactivated on incubation with cats blood (1,2). Jones has proposed that this deactivation involves the enzymic conversion of PGA (I) to an 11,12-double bond isomer (PGC, II) which is subsequently isomerized by base to the inactive PGB (III) (3). Structure assignment of the PGC's in the earlier study were based on chromatographic mobilities and uv and mass spectrometric properties. We report here a total synthesis of PGC₂ methyl ester (IIb). Comparison of the biological and physical properties of this material with those of diazomethane-esterified natural material confirms the earlier structure assignment for PGC₂ (IIa).     

Sequence-specific assignment of methyl groups from the neuronal SNARE complex using lanthanide-induced pseudocontact shifts

Neurotransmitter release depends critically on the neuronal SNARE complex formed by syntaxin-1, SNAP-25 and synaptobrevin, as well as on other proteins such as Munc18-1, Munc13-1 and synaptotagmin-1. Although three-dimensional structures are available for these components, it is still unclear how they are assembled between the synaptic vesicle and plasma membranes to trigger fast, Ca²⁺-dependent membrane fusion. Methyl TROSY NMR experiments provide a powerful tool to study complexes between these proteins, but assignment of the methyl groups of the SNARE complex is hindered by its limited solubility. Here we report the assignment of the isoleucine, leucine, methionine and valine methyl groups of the four SNARE motifs of syntaxin-1, SNAP-25 and synaptobrevin within the SNARE complex based solely on measurements of lanthanide-induced pseudocontact shifts. Our results illustrate the power of this approach to assign protein resonances without the need of triple resonance experiments and provide an invaluable tool for future structural studies of how the SNARE complex binds to other components of the release machinery.     

New 13C-detected experiments for the assignment of intrinsically disordered proteins

NMR assignment of intrinsically disordered proteins (IDPs) by conventional HN-detected methods is hampered by the small dispersion of the amide protons chemical shifts and exchange broadening of amide proton signals. Therefore several alternative assignment strategies have been proposed in the last years. Attempting to seize that dispersion of ¹³C′ and ¹⁵N chemical shifts holds even in IDPs, we recently proposed two ¹³C-detected experiments to directly correlate the chemical shifts of two consecutive ¹³C′–¹⁵N groups in proteins, i.e. without mediation of other nuclei. Main drawback of these experiments is the interruption of the connection at prolines. Here we present new ¹³C-detected experiments to correlate consecutive ¹³C′–¹⁵N groups in IDPs, hacacoNcaNCO and hacaCOncaNCO, that overcome this limitation. Moreover, the experiments provide recognition of glycine residues, thereby facilitating the assignment process.     

Complete backbone and DENQ side chain NMR assignments in proteins from a single experiment: implications to structure–function studies

Resonance assignment is the first and the most crucial step in all nuclear magnetic resonance (NMR) investigations on structure–function relationships in biological macromolecules. Often, the assignment exercise has to be repeated several times when specific interactions with ligands, substrates etc., have to be elucidated for understanding the functional mechanisms. While the protein backbone serves to provide a scaffold, the side chains interact directly with the ligands. Such investigations will be greatly facilitated, if there are rapid methods for obtaining exhaustive information with minimum of NMR experimentation. In this context, we present here a pulse sequence which exploits the recently introduced technique of parallel detection of multiple nuclei, e.g. ¹H and ¹³C, and results in two 3D-data sets simultaneously. These yield complete backbone resonance assignment (¹H^N, ¹⁵N, ¹³CO, ¹Hα/¹³Cα, and ¹Hβ/¹³Cβ chemical shifts) and side chain assignment of D, E, N and Q residues. Such an exhaustive assignment has the potential of yielding accurate 3D structures using one or more of several algorithms which calculate structures of the molecules very reliably on the basis of NMR chemical shifts alone. The side chain assignments of D, E, N, and Q will be extremely valuable for interaction studies with different ligands; D and E side chains are known to be involved in majority of catalytic activities. Utility of this experiment has been demonstrated with Ca²⁺ bound M-crystallin, which contains largely D, E, N and Q residues at the metal binding sites.     

Preparation and 17O NMR Spectra of 17O-Labeled Thymidine 5′-Phosphate Triesters,Alkylphosphonates, Dialkylphosphinates,and Phosphoramidates

Abstract

The ¹⁷O chemical shifts of the title compounds cover a range of values and appear to be useful for the assignment of structure. However, the individual diastereomers of derivatives containing a stereogenic phosphorus center did not display discernably different ¹⁷O chemical shifts.     

A 4D HCC(CO)NNH experiment for the correlation of aliphatic side-chain and backbone resonances in 13C/15N-labelled proteins

Summary We recently proposed a novel four-dimensional (4D) NMR strategy for the assignment of backbone nuclei in spectra of ¹³C/¹⁵N-labelled proteins (Boucher et al. (1992) J. Am. Chem. Soc., 114, 2262–2264 and J. Biomol. NMR, 2, 631–637). In this paper we extend this approach with a new constant time 4D HCC(CO)NNH experiment that also correlates the chemical shifts of the aliphatic sidechain (¹H and ¹³C) and backbone (¹H, ¹³C and ¹⁵N) nuclei. It separates the sidechain resonances, which may heavily overlap in spectra of proteins with large numbers of similar residues, according to the backbone nitrogen and amide proton chemical shifts. When used in conjunction with a 4D HCANNH or HNCAHA experiment it allows, in principle, complete assignment of aliphatic sidechain and backbone resonances with just two 4D NMR experiments.