HMQC NMR Spectroscopy Research Articles

Influence of dT20 and [d(AT)10]2 on Cisplatin Hydrolysis Studied by Two‐Dimensional [1H,15N] HMQC NMR Spectroscopy

The influence of the presence of DNA on the kinetics of cisplatin (cis-[PtCl2(NH3)2]) aquation (replacement of Cl- by H2O) and anation (replacement of H2O by Cl-) involved in the hydrolysis of cisplatin have been determined by two-dimensional [1H,15N] HMQC NMR spectroscopy. Single-stranded dT20 and double-stranded [d(AT)10]2 oligonucleotides were used as DNA models, avoiding guanines which are known to react rapidly with aquated cisplatin forms. Reactions starting from cis-[PtCl2(15NH3)2], or from a stoichiometric mixture of cis-[Pt(15NH3)2(H2O)2]2+ and Cl- (all 0.5 mM Pt(II); in ionic strength, adjusted to 0.095 M or 0.011 M with NaClO4, pH between 3.0 and 4.0) were followed in an NMR tube in both the absence and presence of 0.7 mM dT20 or [d(AT)10]2. In the presence of dT20, we observed a slight and ionic-strength-independent decrease (15-20 %) of the first aquation rate constant, and a more significant decrease of the second anation rate constant. The latter was more important at low ionic strength, and can be explained by efficient condensation of cis-[Pt(15NH3)2(H2O)2]2+ on the surface of single-stranded DNA, in a region depleted of chloride anions. At low ionic strength, we observed an additional set of [1H,15N] HMQC spectral signals indicative of an asymmetric species of PtN2O2 coordination, and we assigned them to phosphate-bound monoadducts of cis-[Pt(15NH3)2(H2O)2]2+. Double-stranded [d(AT)10]2 slowed down the first aquation step also by approximately 15 %; however, we could not determine the influence on the second hydrolysis step because of a significant background reaction with cis-[Pt(NH3)2(H2O)2]2+.

Demonstration of co-polymerization in melamine–urea–formaldehyde reactions using 15N NMR correlation spectroscopy

Melamine–urea–formaldehyde resins are very important commercially, yet little is known about their structures, and the techniques that have been available to examine this are quite limited. In this manuscript we describe the use of 1H– 15N HMQC NMR spectroscopy and the development of a doubly-selective 1H– 15N– 13C CP INEPT NMR experiment, to establish for the first time that melamine and urea co-polymerize with formaldehyde and to identify the 1H, 15N and 13C chemical shifts of the bridging units. This methodology makes it possible to examine co-condensation in industrial preparations of melamine–urea–formaldehyde resins, in order to correlate structure and function and optimize the manufacturing process.

Synthesis and characterization of a new {RuNO} 6 complex, [Ru(NO)(bpb)Cl] (bpb = N, N′-bis(2-pyridinecarboxamide)-1,2-benzene dianion)

As part of our efforts to develop ruthenium nitrosyl compounds for use as nitric oxide delivery agents, a new six coordinate {RuNO} 6 compound, [Ru(NO)(bpb)Cl] (bpb = N, N′-bis(2-pyridinecarboxamide)-1,2-benzene dianion) was prepared from the reaction between [Ru(NO)Cl(H 2O) 2] and N, N′-bis(2-pyridine carboxamide)-1,2-benzene. We report here the characterization of this new compound by 1H, 13C, HH COSY, HMQC and HMBC NMR spectroscopy, IR spectroscopy ( ν NO = 1867 cm −1) and ESI-MS (M + Na + 505.9).

Cytotoxic trans-oriented platinum complexes only form adducts with single-stranded oligodeoxynucleotides.

The reactions of the anticancer complex trans-[PtCl(2)[(E)-HN==C(OMe)Me](2)] (trans-EE) with both single-stranded and double-stranded deoxyribonucleotides have been studied by HPLC and 2D [(1)H,(15)N] HMQC NMR spectroscopy and compared with those of cis-[PtCl(2)(NH(3))(2)] (cis-DDP). Reactions of trans-EE with the single-stranded oligonucleotides d(CCTCGCTCTC) and d(CCTGGTCC) proceed rapidly through solvolysis of the starting substrate and subsequent formation of G-N7/monochloro trans-EE adducts. The rate of reaction is comparable to that of formation of an adduct from trans-EE and the dinuclotide d(ApG). Quite unexpectedly, the double-helical duplexes, d(TATGGTACCATA)(2) and d(TATGGCCATA)(2), with no terminal G residues, are practically inert towards trans-EE, and only minor species (< 5 % as estimated from HPLC traces) appear during 24 h reaction time. However, the duplexes d[(CCTCGCTCTC). (GAGAGCGAGG)] and d(GATAGGCCTATC)(2), which contain both terminal and central G residues, undergo platination only at the terminal, solvent-exposed, G residues, thereby confirming that the interior of the duplex is not accessible to trans-EE due to steric hindrance. In contrast, cis-DDP was found to bind exclusively to the central GG pair in d(GATAGGCCTATC)(2).

Beta-galactofuranose-containing O-linked oligosaccharides present in the cell wall peptidogalactomannan of Aspergillus fumigatus contain immunodominant epitopes.

O-linked oligosaccharide groups ranging from di- to hexasaccharide were beta-eliminated by mild alkaline treatment under reducting conditions from the peptidogalactomannan of Aspergillus fumigatus mycelial cell wall. The resulting reduced oligosaccharides, which were the minor components of the peptidogalactomannan fraction, were fractionated to homogeneity by successive gel filtration and high-performance liquid chromatography. Their primary structures were determined based on a combination of techniques including gas chromatography, ESI-QTOF-MS, 1H COSY and TOCSY, and 1H-13C HMQC NMR spectroscopy and methylation analysis, to be: alpha-Glcp-(1 --> 6)-Man-ol, beta-Galf-(1 --> 6)-alpha-Manp-(1 --> 6)-Man-ol, beta-Galf-(1 --> 5)-beta-Galf-(1 --> 6)-alpha-Manp-(1 --> 6)-Man-ol and beta-Galf-(1 --> 5)-[beta-Galf-(1 --> 5]3-beta-Galf-(1 --> 6)-Man-ol. The beta-Galf containing oligosaccharides have not been previously described as fungal O-linked oligosaccharides. The peptidogalactomannan is antigenic and was recognized by human sera of patients with aspergillosis when probed by ELISA, but de-O-glycosylation rendered a 50% decrease in its reactivity. Furthermore, when tested in a hapten inhibition test, the isolated oligosaccharide alditols were able to block, on a dose-response basis, recognition between human sera and the intact peptidogalactomannan. The immunodominant epitopes were present in the tetra- and hexasaccharide, which contain a beta-Galf-(1 --> 5)-beta-Galf terminal group. These results suggest that the O-glycosidically linked oligosaccharide chains, despite being the less abundant carbohydrate component of the A. fumigatus peptidogalactomannan, may account for a significant part of its antigenicity, other than the known activity associated with the galactomannan component.

Stable Rh2(MTPA)4 Phosphane Adducts − The First Example of P-Chirality Recognition by 103Rh NMR Spectroscopy

The optimal parameters for 31P-detected 103Rh HMQC NMR spectroscopy of AMX spin systems (A = 31P; M and X = 103Rh) have been derived and applied to adducts of Rh2[(R)-MTPA]4 with PPh3 (1) and a chiral phosphane (2). It is shown that all nuclei (1H, 13C, 31P, and 103Rh) reflect the diastereomerism of the latter complex. (© Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002)

Reinvestigation of Reaction of (2‐Ethoxyvinyl)stannanes with Acetyl Bromide.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Formation of adenine-N3/guanine-N7 cross-link in the reaction of trans-oriented platinum substrates with dinucleotides.

The reactions of the anticancer complex trans-[PtCl2{(E)-HN=C(OMe)Me}2] (trans-EE) with a series of ribo and deoxyribodinucleotides have been studied by HPLC and 2D [1H, 15N] HMQC NMR spectroscopy and compared with those of the inactive trans isomer of cisplatin, trans-[PtCl2(NH3)2] (trans-DDP). Reactions of trans-EE with r(ApG) and d(ApG) take place through solvolysis of the starting substrate and subsequent formation of trans G-N7/monochloro and G-N7/monoaqua adducts. Slowly, the monofunctional adducts evolve to a bifunctional adduct forming an unprecedented and unexpected A-N3/G-N7 platinum cross-link spanning two trans positions. For stereochemical reasons, trans platinum complexes cannot form N7/N7 cross-links between adjacent purines in di- or polynucleotides. For the reverse sequence r(GpA), no chelate structure was formed even after a two-week reaction. The reaction of trans-DDP with r(ApG) produces many more products than the analogous reaction with trans-EE. One of these products was identified as the A-N3/G-N7 trans-chelate.

A partially 3- O-methylated (1→4)-linked α- D-galactan and α- D-mannan from Pleurotus ostreatoroseus Sing

The two main water-soluble extracellular polysaccharides produced by the basidiomycete fungus Pleurotus ostreatoroseus Sing were isolated and purified. They were characterized using 13C, 1H, and 1H, 13C HMQC NMR spectroscopy, methylation analysis, and Smith degradation. One was a mannan having a main chain of (1→6)-linked α- D-mannopyranosyl residues, almost all of which were branched at O-2 with side chains of different lengths, containing 2- O- and 3- O-linked mannopyranosyl units. The other was a partially 3- O-methylated (1→4)-linked α- D-galactopyranan, a structure that has not been previously described.

A partially 3-O-methylated (1-->4)-linked alpha-D-galactan and alpha-D-mannan from Pleurotus ostreatoroseus Sing.

The two main water-soluble extracellular polysaccharides produced by the basidiomycete fungus Pleurotus ostreatoroseus Sing were isolated and purified. They were characterized using 13C, 1H, and 1H, 13C HMQC NMR spectroscopy, methylation analysis, and Smith degradation. One was a mannan having a main chain of (1-->6)-linked alpha-D-mannopyranosyl residues, almost all of which were branched at O-2 with side chains of different lengths, containing 2-O- and 3-O-linked mannopyranosyl units. The other was a partially 3-O-methylated (1-->4)-linked alpha-D-galactopyranan, a structure that has not been previously described.

Kinetic study of the reaction between an antitumor 15N labeled trans-platinum iminoether complex and GMP by [1H, 15N] HMQC NMR

The kinetics and mechanism of binding of the 15N-labeled anticancer compound trans-[PtCl2{E-HNC(OMe)Me}2] (trans-EE) to guanosine 5′-monophosphate (5′-GMP) and the tripeptide glutathione (GSH) have been investigated by 2D [1H, 15N] HMQC NMR spectroscopy. The different reaction products can easily be identified as crosspeaks in the 2D NMR spectra. The integrated peak intensities vs. time are used to obtain rate constants by a non-linear optimization procedure. The calculated rate constants (forward and back reaction) for hydrolysis of the first (k1 and k−1) and second chloride (k2 and k−2) are: k1 = 2.1 × 10−4 s−1, k−1 = 890 × 10−4 M−1 s−1; k2 = 0.025 × 10−4 s−1, and k−2 = 4.6 × 10−4 M−1 s−1 at 298 K. The corresponding literature values for the inactive trans-[PtCl2(NH3)2] compound exhibit a large dispersion. The formation of the mono-chloro GMP adduct takes place via a bimolecular process involving the mono-chloro mono-aqua species and the entering nucleotide (k3 = 2700 × 10−4 M−1 s−1), the concentration of this adduct reaches a maximum value after about 6 hours at 298 K. Subsequently, mono-aqua GMP (k4 = 0.131 × 10−4 s−1, k−4 = 10.0 × 10−4 M−1 s−1) and bifunctional GMP adducts (k5 = 99.6 × 10−4 M−1 s−1) are formed in successive steps. Competition reactions of trans-EE with GSH and GMP show a clear preference for GSH adduct formation. Some of the reactions were run both with and without phosphate buffer to assess the degree of platinum–phosphate binding.

Reinvestigation of Reaction of (2-Ethoxyvinyl)stannanes with Acetyl Bromide

Four analogous (2-ethoxyvinyl)stannanes (E/Z)-Bu3SnC(R)=CHOEt (R = Bu, H) were prepared and characterised using 1H, 13C, 119Sn, 1H-13C HMQC, 1H-13C HMBC, and 1H-119Sn HMQC NMR spectroscopy. The course of their reactions with acetyl bromide was studied by NMR spectroscopy. Although tributyltin bromide, ethyl acetate and the coresponding alkyne were identified as reaction products, this present reinvestigation showed unambiguously that heterolytic fragementation reactions, as stated previously, did not take place. Acetyl bromide cleaves the Sn-C= bond yielding tributyltin bromide and vinyl ethers. Subsequent decomposition of vinyl ethers and impurities in the starting stannane is the source of ethyl acetate and the alkyne, respectively.

Unusual C−H⋅⋅⋅Se=C Interactions in Aldols of ChiralN-Acyl Selones Detected by Gradient-Selected1H–77Se HMQC NMR Spectroscopy and X-ray Crystallography

A fundamental new type of non-opportunistic hydrogen bond was detected between a proximal C−H group and the selenium atom of a selenocarbonyl group in aldols of chiral N-acyl selones (see structure) by gradient-selected 1H–77Se HMQC NMR spectroscopy and X-ray crystallography. The C−H⋅⋅⋅Se=C interaction provides a possible explanation for the ability of chiral selone groups to interrogate remotely disposed chiral centers by 77Se NMR spectroscopy.

Unusual C−H⋅⋅⋅Se=C Interactions in Aldols of Chiral N-Acyl Selones Detected by Gradient-Selected 1H–77Se HMQC NMR Spectroscopy and X-ray Crystallography

Unusual C−H⋅⋅⋅Se=C Interactions in Aldols of Chiral N-Acyl Selones Detected by Gradient-Selected 1H–77Se HMQC NMR Spectroscopy and X-ray Crystallography

The roles of Glu-327 and His-446 in the bisphosphatase reaction of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase probed by NMR spectroscopic and mutational analyses of the enzyme in the transient phosphohistidine intermediate complex.

The bisphosphatase domain derived from the rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was studied by 1H-13C HMQC NMR spectroscopy of the histidine C2' and H2' nuclei. The bacterially expressed protein was specifically labeled with 13C at the ring C2' position of the histidines. Each of the seven histidine residues gave rise to a single cross-peak in the HMQC spectra, and these were assigned by use of a series of histidine-to-alanine point mutants. His-304, His-344, and His-469 exhibit 13C and 1H resonances that titrated with pH, while the remaining histidine-associated resonances did not. The 13C and 1H chemical shifts indicate that at neutral pH, His-304 and His-446 are deprotonated, while His-469 is protonated. The pKa of His-344 was determined to be 7.04. The 13C chemical shifts suggest that the deprotonated His-258 exists as the N1' tautomer, while His-392 and His-419 are protonated in the resting, wild-type enzyme. Mutation of the remaining member of the catalytic triad, Glu-327, to alanine in the resting enzyme caused an upfield shift of 1.58 and 1.30 ppm in the 1H and 13C dimensions, respectively, and significant narrowing of the His-258 cross-peak. Mutation of His-446 to alanine produced perturbations of the His-258 cross-peak that were similar to those detected in the E327A mutant. The His-392 resonances were also shifted by the E327A and H446A mutations. These observations strongly suggest that residues His-258, Glu-327, His-392, and His-446 exist within a network of interacting residues that encompasses the catalytic site of the bisphosphatase and includes specific contacts with the C-terminal regulatory region of the enzyme. The specifically 13C-labeled bisphosphatase was monitored during turnover by HMQC spectra acquired from the transient N3' phosphohistidine intermediate complex in the wild-type enzyme, the E327A mutant, and the H446A mutant. These complexes were formed during reaction with the physiological substrate fructose-2, 6-bisphosphate. Upon formation of the phosphohistidine at His-258, the 13C and 1H resonances of this residue were shifted downfield by 1.7 and 0.31 ppm, respectively, in the wild-type enzyme. The upfield shifts of the His-258 resonances in the E327A and H446A mutant resting enzymes were reversed when the phosphohistidine was formed, generating spectra very similar to that of the wild-type enzyme in the intermediate complex. In contrast, the binding of fructose-6-phosphate, the reaction product, to the resting enzyme did not promote significant changes in the histidine-associated resonances in either the wild-type or the mutant enzymes. The interpretation of these data within the context of the X-ray crystal structures of the enzyme is used to define the role of Glu-327 in the catalytic mechanism of the bisphosphatase and to identify His-446 as a putative link in the chain of molecular events that results in activation of the bisphosphatase site by cAMP-dependent phosphorylation of the hepatic bifunctional enzyme.

Steric and Electronic Effects on the 103Rh NMR Chemical Shifts of RhI(cyclooctadiene) Compounds Bearing N-Donor Ligands

In order to study electronic and steric effects on δ(103Rh), the103Rh-NMR spectra of a series of [Rh(cod)L2] complexes (with L = nitrogen, oxygen, or chloride) have been measured by gradient-enhanced HMQC (1H,103Rh) NMR spectroscopy. Density functional computations of representative examples have been performed at the GIAO-B3LYP level of theory. The obtained experimental values of 103Rh shifts fit the generally observed trend of coordinating ligands in the first coordination sphere of the metal: P < N < O. Calculated 103Rh shifts agreed to within 8 % with experimental values. The computational results indicate that the Rh–N bond distance has a larger influence on the chemical shift than the N–Rh–N angle.

Transition-metal complexes of isatin-β-thiosemicarbazone. X-ray crystal structure of two nickel complexes

Manganese, iron, cobalt, nickel, copper and zinc complexes of isatin-β-thiosemicarbazone (H 2L) have been synthesized and spectroscopically characterized. The X-ray crystal structures of two nickel complexes, namely [Ni(HL) 2] -EtOH( 1) and [Ni(HL) 2] · 2DMF ( 2), reveal a distorted octahedral coordination with the monodeprotonated ligand that behaves as an O,N,S terdentate. Different packing interactions are determined by the presence of different crystallization solvents, i.e. ethanol in 1 and dimethylformamide (DMF) in 2. 1H and 13C NMR studies of the ligand and zinc complexes in solution were carried out and a complete assignment for the ligand was made by homodecoupling, gradient assisted 2D 1H- 13C HMQC and HMBC NMR spectroscopy. Biological studies, carried out in vitro on human leukaemic cell lines U937, have shown that the free ligand and the copper(II) complex are more active in the inhibition of cell proliferation than the nickel complexes. No compound was able to induce apoptosis.

Steric and Electronic Effects on the103Rh NMR Chemical Shifts of RhI(cyclooctadiene) Compounds Bearing N-Donor Ligands

In order to study electronic and steric effects on δ(103Rh), the103Rh-NMR spectra of a series of [Rh(cod)L2] complexes (with L = nitrogen, oxygen, or chloride) have been measured by gradient-enhanced HMQC (1H,103Rh) NMR spectroscopy. Density functional computations of representative examples have been performed at the GIAO-B3LYP level of theory. The obtained experimental values of 103Rh shifts fit the generally observed trend of coordinating ligands in the first coordination sphere of the metal: P < N < O. Calculated 103Rh shifts agreed to within 8 % with experimental values. The computational results indicate that the Rh–N bond distance has a larger influence on the chemical shift than the N–Rh–N angle.

183W NMR study of alkenylcarbyne- and alkenylvinylidene-tungsten complexes

183W NMR data are reported for a series of alkenylvinylidene– and alkenylcarbyne–tungsten complexes of the type [(dppe)(CO)3WCCHR], [(dppe)(CO)3WC−CHCR2]+BF4- and [(dppe)(CO)2LWC−CHCR2] [dppe=1,2-bis(diphenylphosphino)ethane]. Spectra were recorded using inverse 2D 1H,183W- and 31P,183W{1H}-HMQC NMR spectroscopy without 183W decoupling. In some examples of the alkenylcarbyne series minor amounts of the propenylcarbyne isomer were present and the corresponding 183W chemical shifts could also be characterized. For a given complex and measuring time the proton-detected correlation spectra based on the existing long-range coupling to the metal affords a higher signal-to-noise ratio than the phosphorus-detected correlation via 1J(183W,31P). Additionally, the tilt of cross peaks in the 1H,183W HMQC spectra reveals the relative sign of the coupling of these nuclei with phosphorus. 183W chemical shift variations for compounds [(dppe)(CO)2LWC−CHCR2] (L=CO, PMe3, CH3CN or halogen) are rationalized in terms of the π-acceptor ability of the ligand. Halogen derivatives show shielding decreases in the order F<Cl<Br<I. For carbyne complexes a deshielding of the metal is observed with increase in the size of the carbocyclic ring substituent as a result of steric effects. For complexes [(dppe)(CO)2LWC−CHCR2] (L=CO, PMe3, CH3CN) the phosphorus–tungsten scalar coupling is also determined by the σ-donor/π-acceptor ability and bulkiness of the ligands. © 1998 John Wiley & Sons, Ltd.

X-ray Diffraction and Gradient-Assisted 2D 1H−119Sn HMQC NMR Studies on the Structure of Condensation Complexes of Salicylaldoxime and Dimethyltin(IV) Oxide

Reaction of dimethyltin(IV) oxide with salicylaldoxime in the molar ratio 3:2 or 1:1 in refluxing toluene/ethanol (4/1) affords reaction mixtures which, upon crystallization from alcohols TOH (T = Me, Et, n-Pr, i-Pr), generate compounds with three dimethyltin and two salicylaldoximate moieties. These compounds obey the formula [(Me(2)Sn)(Me(2)SnO)(Me(2)-SnOT)(HONZO)(ONZO)], where HONZOH represents salicylaldoxime, o-HON=CHC6H4OH. X-ray diffraction analysis of the compounds at room temperature (T = Me, compound 3; T = Et, 4; T = n-Pr, 5; T = i-Pr, 6) reveals that all structures contain one seven-coordinate tin atom bound to two nitrogen and three oxygen atoms in the equatorial plane of a pentagonal bipyramid, the two methyl groups being in apical positions. The two other tin atoms are five-coordinate with a trigonal-bipyramidal geometry having the two methyl groups and a common oxygen atom in the equatorial positions and, in apical positions, two further oxygen atoms, of which the trifurcated alkoxy oxygen atom bridges these tin atoms. Reaction of dimethyltin(IV) oxide with salicylaldoxime in the molar ratio 1:1 in refluxing benzene generates, after solvent evaporation, compound 2b, obeying the formula [(Me(2)Sn)(2)(Me(2)SnO)-(HONZO)(ONZO)(HONZO)]. The solution-state structures of the alkoxy-bridged compounds 3-6, investigated by gradient-assisted 2D H-1-Sn-119 HMQC as well as H-1-C-13 HMQC and HMBC NMR spectroscopy, are the same as those in the solid state. The bonding mode of the third ligand (i.e. HOZNO) of compound 2b has been elucidated.