Time-saving methods for heteronuclear multidimensional NMR of (13C, 15N) doubly labeled proteins

Summary Heteronuclear 2D (¹³C, ¹H) and (¹⁵N, ¹H) correlation spectra of (¹³C, ¹⁵N) fully enriched proteins can be acquired simultaneously with virtually no sensitivity loss or increase in artefact levels. Three pulse sequences are described, for 2D time-shared or TS-HSQC, 2D TS-HMQC and 2D TS-HSMQC spectra, respectively. Independent spectral widths can be sampled for both heteronuclei. The sequences can be greatly improved by combining them with field-gradient methods. By applying the sequences to 3D and 4D NMR spectroscopy, considerable time savings can be obtained. The method is demonstrated for the 18 kDa HU protein.Abbreviations HMQC heteronuclear multiple-quantum coherence spectroscopy - HSQC heteronuclear single-quantum coherence spectroscopy - HSMQC heteronuclear single- and multiple-quantum coherence spectroscopy - NOESY nuclear Overhauser enhancement spectroscopy     

Rat brain slices oxidize glucose at high rates: a (13)C NMR study

Since glucose is the main cerebral substrate, we have characterized the metabolism of various ¹³C glucose isotopomers in rat brain slices. For this, we have used our cellular metabolomic approach that combines enzymatic and carbon 13 NMR techniques with mathematical models of metabolic pathways. We identified the fate and the pathways of the conversion of glucose carbons into various products (pyruvate, lactate, alanine, aspartate, glutamate, GABA, glutamine and CO₂) and determined absolute fluxes through pathways of glucose metabolism. After 60 min of incubation, lactate and CO₂ were the main end-products of the metabolism of glucose which was avidly metabolized by the slices. Lactate was also used at high rates by the slices and mainly converted into CO₂. High values of flux through pyruvate carboxylase, which were similar with glucose and lactate as substrate, were observed. The addition of glutamine, but not of acetate, stimulated pyruvate carboxylation, the conversion of glutamate into succinate and fluxes through succinate dehydrogenase, malic enzyme, glutamine synthetase and aspartate aminotransferase. It is concluded that, unlike brain cells in culture, and consistent with high fluxes through PDH and enzymes of the tricarboxylic acid cycle, rat brain slices oxidized both glucose and lactate at high rates.     

C, N CP MAS and high resolution multinuclear NMR study of methyl

Standard chemical methods involving the use of O-acetylated glycosyl trichloroacetimidates as glycosylating agents were used to prepare the five 1,3-dideoxynojirimycin-3-yl beta-(1-->3)-linked oligo-glucosides (1-5) and also the beta-(1-->6)-bonded glucobiose (gentiobiose)-based analogue 6 as potential fungicides. In the course of the work, the beta-(1-->6), beta-(1-->6)-linked analogue 8 of 6 and 6-O- and 4-O-beta-glucopyranosyl-deoxynojirimycins 7 and 9, respectively, were also produced.     

1,3-Dideoxynojirimycin-3-yl glycosides of beta-(1-->3)- and beta-(1-->6)-linked gluco-oligosaccharides

Standard chemical methods involving the use of O-acetylated glycosyl trichloroacetimidates as glycosylating agents were used to prepare the five 1,3-dideoxynojirimycin-3-yl beta-(1-->3)-linked oligo-glucosides (1-5) and also the beta-(1-->6)-bonded glucobiose (gentiobiose)-based analogue 6 as potential fungicides. In the course of the work, the beta-(1-->6), beta-(1-->6)-linked analogue 8 of 6 and 6-O- and 4-O-beta-glucopyranosyl-deoxynojirimycins 7 and 9, respectively, were also produced.     

Characterization of O-acetyl-(4-O-methylglucurono)xylan isolated from birch and beech

The structures of water-soluble birch and beech xylans, extracted from holocellulose using dimethyl sulfoxide, were determined employing 1H and 13C NMR spectroscopy together with chemical analysis. These polysaccharides were found to be O-acetyl-(4-O-methylglucurono)xylans containing one 4-O-methylglucuronic acid substituent for approximately every 15 D-xylose residues. The average degree of acetylation of the xylose residues in these polymers was 0.4. The presence of the structural element -->4)[4-O-Me-alpha-D-GlcpA-(1-->2)][3-O-Ac]-beta-D-Xylp-(1--> was demonstrated. Additional acetyl groups were present as substituents at C-2 and/or C-3 of the xylopyranosyl residues. Utilizing size-exclusion chromatography in combination with mass spectroscopy, the weight-average molar masses (and polydispersities) were shown to be 8000 (1.09) and 11,100 (1.08) for birch and beech xylan, respectively.     

Conformational studies of a novel cationic glycolipid,glyceroplasmalopsychosine, from bovine brain by NMR spectroscopy

A novel glycosphingolipid containing a long chain aldehyde conjugated to galactose and glycerol, Gro1(3)-O-CH((CH(2))(n)CH(3))-O-6Galbeta-sphingosine (glyceroplasmalopsychosine) has been studied by NMR spectroscopy (Hikita et al. J. Biol. Chem. 2001, 276, 23084-23091). We further report here on the conformation showing the galactose and the glycerol at the end of two parallel hydrophobic chains, i.e. the sphingosine and the fatty aldehyde. This is proposed based on the interproton distances derived from ROESY experiments and 3 J (H,H) coupling constants. The absence of any intraresidual NOEs between protons in the glycerol residue suggested that the C-C-2 and C-C-3 bonds in the glycerol may be rotating freely, supporting the proposed conformation in which the unique terminal glycerol is in an environment with a minimal steric hindrance. The present study proposes a conformation of glyceroplasmalopsychosine greatly different from the two conventional plasmalopsychosines possessing a fatty aldehyde chain oriented in an opposite direction to the sphingosine.     

New class of quaternary ammonium salts, derivatives of methyl D-glucopyranosides

Reactions of two aromatic and two aliphatic amines with methyl 6-O-p-toluenesulfonyl-alpha-D-glucopyranoside or methyl 6-O-p-toluenesulfonyl-beta-D-glucopyranoside were performed on a micro-scale. The synthesis and preparative isolation methods have been developed for quaternary N-(methyl 2,3,4-tri-O-acetyl-6-deoxy-alpha- and -beta-D-glucopyranoside-6-yl)ammonium salts derived from three amines: trimethylamine, 2-methylpyridine, and pyridine. The reaction products were examined with 1H, 13C NMR spectroscopy. N-(Methyl 2,3,4-tri-O-acetyl-6-deoxy-beta-d-glucopyranoside-6-yl)trimethylammonium tosylate was additionally analyzed with X-ray crystallography.     

Practical one-pot conversion of 17beta-estradiol to 10beta-hydroxy- (p-quinol) and 10beta-chloro-17beta-hydroxyestra-1,4-dien-3-one

An efficient one-pot procedure for the preparation of 10beta,17beta-dihydroxyestra-1,4-dien-3-one (p-quinol, 1, 75%) is reported, involving oxidation of 17beta-estradiol with potassium permanganate. Similar treatment of 17beta-estradiol with sodium chlorite led to 10beta-chloro-17beta-hydroxyestra-1,4-dien-3-one (2) in 44% yield along with smaller amounts 4-chloro-10beta,17beta-dihydroxyestra-1,4-dien-3-one (3), 2,10beta-dichloro-17beta-hydroxyestra-1,4-dien-3-one (4), and 4,10beta-dichloro-17beta-hydroxyestra-1,4-dien-3-one (5).     

Conformationally Restricted 2-Substituted Wyosine Derivatives. 1H, 13C,and 15N NMR Study

Abstract

It was found by ¹H, ¹³C and ¹⁵N NMR study that substitution of 4,9-dihydro-4, 6-dimethyl-9-oxo-3-(2′,3′,5′-tri-O-acetyl-β-D-ribofuranosyl) imidazo [1,2-a]purine (wyosine triacetate, 1) at C2 position with electronegative groups CH₃O and C₆H₅CH₂O results in a noticeable electron distribution disturbance in the “left-hand” imidazole ring and a significant increase in the North conformer population of the sugar moiety.     

H2BC: a new technique for NMR analysis of complex carbohydrates

It is demonstrated that the H2BC NMR pulse sequence (J. Am. Chem. Soc.2005, 127, 6154, Magn. Reson. Chem.2005, 43, 971-974) offers unambiguous assignments and significant simplification of NMR spectra of large and complex carbohydrates compared to other techniques for the establishment of correlations over more than one bond. H2BC almost exclusively correlates protons and proton-bearing carbon spins separated by two covalent bonds and is independent of occasionally vanishing (2)J(CH) coupling constants, which alleviates the problem of missing two-bond correlations in HMBC spectra. H2BC also solves the problem of distinguishing two- and three-bond correlations in HSQC-TOCSY or HMBC. It is a further asset of H2BC that the experiment is significantly shorter than HMBC and HSQC-TOCSY, and hence less sensitive to transverse relaxation. The H2BC experiment is demonstrated on an approximately 30-residue oligosaccharide from Francisella victoria.     

NMR and modelling studies of disaccharide conformation

Long-range heteronuclear coupling constants were measured across the glycosidic linkages for a series of eight alpha- or beta-linked disaccharides in aqueous solution. Multiple 13C site-selective excitation experiments using 1H decoupling in conjunction with pulsed field gradient-enhanced spectroscopy were used to determine 3J(C,H) values. These were subsequently compared with the respective couplings calculated, using a Karplus relationship, from molecular dynamics simulations with the explicit inclusion of water.     

Assigning stereodiversity of the 27-Me group of furostane-type steroidal saponins via NMR chemical shifts

Applicability of (13)C and (1)H NMR chemical shifts for the assignment of the 25R/25S configuration of the 27-methyl group in the case of furostane-type steroidal saponins has been investigated. A comparative study of (13)C NMR data suggest that chemical shift values for C-20, C-21, C-22, C-23, C-24, C-25, C-26 and C-27 resonances were not much influenced by R/S configuration of the 27-Me group, thus reflecting limited application of (13)C NMR chemical shifts for such stereochemical determinations. In contrast, (1)H NMR chemical shifts (delta(a), delta(b)) for geminal protons of glycosyloxy methylene (H(2)-26) exhibit pronounced dependence and the difference (Delta(ab)=delta(a)-delta(b)) among their chemical shifts [Delta(ab)= or <0.48 for 25R; Delta(ab)= or >0.57 for 25S] seems to be of general applicability for ascertaining 25R/25S orientation of the 27-methyl group of furostane-type steroidal saponins.     

Oxytocin solution structure changes upon protonation of the N-terminus in dimethyl sulfoxide

Summary With the combined use of various two-dimensional (2D) NMR techniques, a complete assignment of the ¹H and ¹³C resonances of oxytocin, , for two molecular states, protonated and unprotonated at the N-terminal group, was performed in dimethyl sulfoxide. A small but distinct change in the backbone conformation of the six-residue cyclic moiety, associated with the protonation, was first suggested from those NMR parameters relevant to conformation, such as change with temperature in the chemical shifts of the peptide amide protons and changes in chemical shifts and homonuclear as well as heteronuclear three-bond coupling constants. The solution structures of oxytocin for the protonated and unprotonated forms were then calculated using distance analysis in dihedral-angle space, based on a relaxation matrix evaluated from quantitative NOE intensities at different mixing times. Total amounts of 93 and 105 distances were determined for the protonated and the unprotonated forms, respectively. There were 25 interresidue distances relevant to the structure of the cyclic moiety for the protonated form of oxytocin and 43 for the unprotonated form. Overall structures with the lowest target penalty function were similar between the two forms, having a -turn structure at the endocyclic residues of the Tyr-Ile-Gln-Asn moiety. The local backbone conformations near the N-terminus, however, were significantly different between the two forms. This was found to be due to a change in the dihedral angle of the disulfide bridge (^ss around C-S-S-C), which closes the ring in the cyclic peptide. The dihedral angle was about +90° for the unprotonated form and an intermediate value of about +45° for the protonated form.     

1H, 13C and 15N random coil NMR chemical shifts of the common amino acids. I. Investigations of nearest-neighbor effects

Summary In this study we report on the ¹H, ¹³C and ¹⁵N NMR chemical shifts for the random coil state and nearest-neighbor sequence effects measured from the protected linear hexapeptide Gly-Gly-X-Y-Gly-Gly (where X and Y are any of the 20 common amino acids). We present data for a set of 40 peptides (of the possible 400) including Gly-Gly-X-Ala-Gly-Gly and Gly-Gly-X-Pro-Gly-Gly, measured under identical aqueous conditions. Because all spectra were collected under identical experimental conditions, the data from the Gly-Gly-X-Ala-Gly-Gly series provide a complete and internally consistent set of ¹H, ¹³C and ¹⁵N random coil chemical shifts for all 20 common amino acids. In addition, studies were also conducted into nearest-neighbor effects on the random coil shift arising from a variety of X and Y positional substitutions. Comparisons between the chemical shift measurements obtained from Gly-Gly-X-Ala-Gly-Gly and Gly-Gly-X-Pro-Gly-Gly reveal significant systematic shift differences arising from the presence of proline in the peptide sequence. Similarly, measurements of the chemical shift changes occurring for both alanine and proline (i.e., the residues in the Y position) are found to depend strougly on the type of amino acid substituted into the X position. These data lend support to the hypothesis that sequence effects play a significant role in determining peptide and protein chemical shifts.     

A Karplus equation for (3)J(HCCN) in amino sugar derivatives

The (1)H-(15)N coupling constants of a suite of organic-soluble amino sugar derivatives have been measured by one-dimensional and two-dimensional (1)H/(15)N heteronuclear single quantum, multiple bond correlation (HSQMBC), and the values so obtained are compared with those measured by analysis of (1)H spectra of (15)N-labeled amino sugar derivatives. A number of bicyclic amino sugar models have been studied, including methyl 2- (and 3-)amino-4,6-O-benzylidene-2- (and 3-)deoxy-alpha-D-hexopyranosides in chair or skew conformations, and methyl 2,6-anhydro-3-deoxy-3-phthalimido-alpha-d-mannopyranoside in a locked, almost classical boat conformation. The magnitudes of the vicinal (1)H-(15)N coupling constants (3)J(HCCN) have been correlated with (1)H/(15)N dihedral angles phi computed for the favored conformations by molecular dynamics with molecular mechanics energy minimization. Non-linear regression of the coupling constants on the dihedral angles has yielded a Karplus equation: (3)J(HCCN)=3.1 cos(2) phi-0.6 cos phi+0.4. The coefficients of the terms in this equation have been compared with those reported for 15 other pairs of nuclei, and the coefficient of the important cos(2)phi term found to be numerically smallest for (3)J(HCCN).     

Physicochemical characterization of carrageenan fromGigartina teedii (Rooth) Lamouroux (Gigartinales,Rhodophyta)

The phycocolloids of female gametophytes ofGigartina teedii (Roth) Lamouroux harvested in Roscoff (Brittany, France) are a hybrid carrageenan resulting from juxtaposition of fragments of kappa-, iota-and nu-carrageenan. They represent 70% of the dry matter of the alga in summer. After alkaline transformation the proportion of iota-carrageenan increased to 76%, demonstrating the presence of nu-carrageenan. Absence of mu-carrageenan, the precursor of kappa-carrageenan, suggests that iota-carrageenan is desulfated enzymically to kappa-carrageenan.     

Synthesis and solid state 13C and 1H NMR analysis of new oxamide derivatives of methyl 2-amino-2-deoxy-alpha-D-glucopyranoside and ester of amino acids or dipeptides

The syntheses of new oxamide derivatives of methyl 2-amino-2-deoxy-alpha-D-glucopyranoside and amino acid or peptide esters are presented. The reaction of methyl 3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-alpha-D-glucopyranoside and oxalyl chloride gave N-(methyl 3,4,6-tri-O-acetyl-2-deoxy-alpha-D-glucopyranosid-2-yl) oxamic acid chloride which on reaction with the ester of Gly, L-Ala, L-Phe, GlyGly, Gly-L-Phe and Gly-L-Ala afforded N-(methyl 3,4,6-tri-O-acetyl-2-deoxy-alpha-D-glucopyranosid-2-yl), N'-oxalyl-amino acid or dipeptide esters. The structure of the oxamides was studied using 1H, 13C NMR in solution and solid state.     

Synthesis of a novel N[bond]O-interglycosidic disaccharide

The carbohydrate subunits carrying an N-O-interglycosidic bond play a very important role in the biological activity of the enediyne antibiotics. Condensation of O-(alpha- and beta-D-glucopyranosyl)hydroxylamine (5a and 5b) with the hex-3-ulopyranoside (6) furnished methyl 4,6-O-benzylidene-2,3-dideoxy-3-(2,3,4,6-tetra-O-benzyl-alpha-D-glucopyranosyloxy)imino-alpha- and beta-D-erythro-hexopyranoside (7a and 7b). Stereoselective reduction of the Cz.dbnd6;N bond of 7a and 7b with sodium cyanoborohydride resulted in the formation of the required protected N-O-interglycosidic disaccharides (8a and 8b). Finally, catalytic hydrogenation of 8a afforded methyl 2,3-dideoxy-3-(alpha-D-glucopyranosyloxy)amino-alpha-D-ribo-hexopyranoside (9a). Under similar conditions the beta anomer 8b underwent decomposition.     

[H,N] NMR studies of the aquation of cis-diamine platinum(II) complexes

Two ¹⁵N-labelled cis-Pt(II) diamine complexes with dimethylamine (¹⁵N-dma) and isopropylamine (¹⁵N-ipa) ligands have been prepared and characterised. [¹H,¹⁵N] HSQC NMR spectroscopy is used to obtain the rate and equilibrium constants for the aquation of cis-[PtCl₂(¹⁵N-dma)₂] at 298 K in 0.1 M NaClO₄ and to determine the pK_a values of cis-[PtCl(H₂O)(¹⁵N-dma)₂]⁺ (6.37) and cis-[Pt(H₂O)₂(¹⁵N-dma)₂]²⁺ (pK_a1 = 5.17, pK_a2 = 6.47). The rate constants for the first and second aquation steps (k₁ = (2.12 ± 0.01) × 10⁻⁵ s⁻¹, k₂ = (8.7 ± 0.7) × 10⁻⁶ s⁻¹) and anation steps (k₋₁ = (6.7 ± 0.8) × 10⁻³ M⁻¹ s⁻¹, k₋₂ = 0.043 ± 0.004 M⁻¹ s⁻¹) are very similar to those reported for cisplatin under similar conditions, and a minor difference is that slow formation of the hydroxo-bridged dimer is observed. Aquation studies of cis-[PtCl₂(¹⁵N-ipa)₂] were precluded by the close proximity of the NH proton signal to the ¹H₂O resonance.     

Synthesis, crystal structure and DNA interaction studies on mononuclear zinc complexes

A new family of mononuclear Zn(II) complexes [Zn(Pyimpy)₂](ClO₄)₂ (1), [Zn(Pyimpy)(Cl)₂] (2), [Zn(Pyimpy)(SCN)₂] (3) and [Zn(Pyimpy)(N₃)₂] (4) were synthesized using designed tridentate ligand Pyimpy having NNN donors (Pyimpy: (2-((2-phenyl-2-(pyridin-2-l)hydazono)methyl)pyridine)). Complexes were characterized by different spectroscopic studies and it has been found out that all complexes exhibited strong fluorescent emission at room temperature. Molecular structures of [Zn(Pyimpy)₂](ClO₄)₂·C₆H₅CH₃·0.5H₂O (1·C₆H₅CH₃·0.5H₂O) and [Zn(Pyimpy)(Cl)₂]·CH₃CN (2·CH₃CN) were determined by X-ray crystallography and ligand coordinated Zn(II) ions was described as distorted octahedral and distorted square pyramidal, respectively. DNA binding properties of these complexes were investigated by absorption spectral, fluorescence quenching and circular dichroism spectral studies.