Nuclear Overhauser Enhancement Studies Research Articles

Synthesis and Spectral Study of Some Novel Oxime Esters Derived from 3-azabicyclo[3.3.1]nonan-9-one Oxime

Ten substituted 3-azabicyclo[3.3.1]nonan-9-one oxime ester derivatives 3-12 were synthesized from the starting material 3-azabicyclo[3.3.1]nonan-9-one oxime 2 with activated carboxylic acids in POCl3/pyridine. A wide range of carboxylic acids were used to produce the corresponding oxime esters up to 85% yield. The synthesized compounds were confirmed by FT-IR, NMR (1H, 13C and NOESY) and mass spectrometry. The chemical shift values were assigned unambiguously using one- and two-dimensional NMR spectroscopic data. The nuclear overhauser enhancement study suggests that the compounds exist in twin-chair conformation with equatorial orientation of the bulky substituents. The single crystal structure of compound 4 was determined to elucidate the structure unambiguously. Keywords: Azabicyclo[3.3.1]nonane, carboxylic acid, Mannich reaction, NOESY, ring conformation.

Steric and electronically biasing substituent effects on the Photoreversibility of novel, 3′-, 5′- and 3-substituted indolospirobenzopyrans. Thermal evaluation using 1H NMR spectroscopy and Overhauser enhancement studies

Several, novel, 3′-, 5- and 3-substituted indolospirobenzopyrans were prepared and their reversible thermochromic equilibria were investigated by synthesising derivatives with appropriately placed “electronically” modifying substituents in both the indole and benzopyran rings as well as by synthesising structural variants that contained sterically hindering (with regard to spiropyran-opening ↔ closing) groups, within the spirocyclic ring-system. In addition, structural combinations of the above, involving the syntheses of sterically restricted and electronically biasing substituents were prepared and their effects upon the spiropyran ring-opening ↔ closing process investigated. The influence of these steric and electronic substituent effects on the relative quantities of the open- and closed-forms was established using 1H NMR at six temperature intervals (298, 320, 340, 360, 380 and 410 K). Further elucidation of the stereochemical conformation in these systems was undertaken using nuclear Overhauser enhancement (nOe) studies.

Dissection Study on the Severe Acute Respiratory Syndrome 3C-like Protease Reveals the Critical Role of the Extra Domain in Dimerization of the Enzyme: DEFINING THE EXTRA DOMAIN AS A NEW TARGET FOR DESIGN OF HIGHLY SPECIFIC PROTEASE INHIBITORS

The severe acute respiratory syndrome (SARS) 3C-like protease consists of two distinct folds, namely the N-terminal chymotrypsin fold containing the domains I and II hosting the complete catalytic machinery and the C-terminal extra helical domain III unique for the coronavirus 3CL proteases. Previously the functional role of this extra domain has been completely unknown, and it was believed that the coronavirus 3CL proteases share the same enzymatic mechanism with picornavirus 3C proteases, which contain the chymotrypsin fold but have no extra domain. To understand the functional role of the extra domain and to characterize the enzyme-substrate interactions by use of the dynamic light scattering, circular dichroism, and NMR spectroscopy, we 1) dissected the full-length SARS 3CL protease into two distinct folds and subsequently investigated their structural and dimerization properties and 2) studied the structural and binding interactions of three substrate peptides with the entire enzyme and its two dissected folds. The results lead to several findings; 1) although two dissected parts folded into the native-like structures, the chymotrypsin fold only had weak activity as compared with the entire enzyme, and 2) although the chymotrypsin fold remained a monomer within a wide range of protein concentrations, the extra domain existed as a stable dimer even at a very low concentration. This observation strongly indicates that the extra domain contributes to the dimerization of the SARS 3CL protease, thus, switching the enzyme from the inactive form (monomer) to the active form (dimer). This discovery not only separates the coronavirus 3CL protease from the picornavirus 3C protease in terms of the enzymatic mechanism but also defines the dimerization interface on the extra helical domain as a new target for design of the specific protease inhibitors. Furthermore, the determination of the preferred solution conformation of the substrate peptide S1 together with the NMR differential line-broadening and transferred nuclear Overhauser enhancement study allows us to pinpoint the bound structure of the S1 peptide.

FT-IR and nuclear overhauser enhancement study of the state of urea confined in AOT-reversed micelles

Abstract The system urea/sodium-bis(2-ethylhexyl)sulfosuccinate (AOT) dispersed in CCl 4 and benzene-d6 was investigated by viscosimetry, FT-IR and high-resolution 1 H NMR spectroscopy, as a function of urea/AOT molar ratio ( R urea ) at fixed AOT molal concentration (0.3467 mol kg −1 ) and as a function of AOT concentration at fixed R urea (0.5) at 25 °C. The experimental data are consistent with the hypothesis that urea is encapsulated as small-size hydrogen-bonded cluster in the hydrophilic micellar core of the AOT-reversed micelles and that this structure is maintained well above the volume fraction of the dispersed phase, where a percolative transition occurs. Intermolecular nuclear overhauser enhancements (NOEs) were observed between urea amidic protons and AOT protons located in the proximity of the sulfonate group, thus proving that the solubilizate/surfactant interaction is mainly localized around the polar head of AOT within the micellar core. Long-range NOEs were also detected between urea NH 2 and methyl group of the β-alkyl chain of AOT, suggesting that the two alkyl chains of the surfactant play different roles in the interaction with urea and the bulk solvent. Some changes to the structural and dynamical properties were observed during solubilization in AOT-reversed micelles.

Hantzsch 1,4-dihydropyridines containing a nitrooxyalkyl ester moiety to study calcium channel antagonist structure-activity relationships and nitric oxide release

A group of 3-nitrooxyalkyl 5-alkyl 1,4-dihydro-2,6-dimethyl-4-(pyridyl)-3,5-pyridinedicarboxylates were prepared using a modified Hantzsch reaction that involved the condensation of a nitrooxyalkyl acetoacetate with an alkyl 3-aminocrotonate and a pyridinecarboxaldehyde. 1H NMR nuclear Overhauser enhancement (nOe) studies for 3-(3-nitrooxypropyl) 5-isopropyl 1,4-dihydro-2,6-dimethyl-4-(2-pyridyl)-3,5-pyridinedicarboxylate (17) indicates a predominant rotamer exists in solution where the pyridyl nitrogen atom is orientated above the 1,4-DHP ring system, and the pyridyl nitrogen atom is antiperiplanar to the 1,4-DHP ring H-4 proton. Variable temperature 1H NMR studies (–30 to +60°C) showed the 1,4-DHP NH proton in 17 is H-bonded in CHCl3 solution. This interaction is believed to be due to intermolecular H-bonding between the pyridyl nitrogen free electron pair and the 1,4-DHP NH proton. In vitro calcium channel antagonist (CCA) activities were determined using a muscarinic-receptor-mediated Ca+2-dependent contraction of guinea pig ileal longitudinal smooth muscle assay. This class of compounds exhibited lower CCA activity (IC50 = 5.3 × 10–6 to 3.5 × 10–8 M range) than the reference drug nifedipine (IC50 = 1.4 × 10–8 M). For compounds having C-3 –CH2CH2ONO2 and C-4 pyridyl substituents, the C-5 alkyl was a determinant of CCA (i-Pr > the approximately equipotent i-Bu, t-Bu, and Et analogs). The point of attachment of the isomeric C-4 pyridyl substituent was a determinant of CCA when C-3 –CH2CH2ONO2 and C-5 i-Pr substituents were present providing the potency profile 2-pyridyl ≥ 3-pyridyl > 4-pyridyl. CCA with respect to the C-3 nitrooxyalkyl substituent was inversely dependent on the length of the alkyl spacer. The percent nitric oxide (·NO) released in vitro by this group of compounds (range of 0.03–0.43%/ONO2 group), quantified as nitrite by reaction with the Griess reagent, was lower than that for the reference drug glycerol trinitrate (3.81%/ONO2 group). Nitric oxide release studies showed that the %·NO released was dependent on the number of ONO2 groups/molecule. A QSAR study for this group of compounds showed a correlation between the specific polarizability descriptor (SpPol) and %·NO release. Drug Dev. Res. 51:233–243, 2000. © 2001 Wiley-Liss, Inc.

NMR study of the stereochemistry of 2,4,6,8‐tetraaryl‐3,7‐diazabicyclo[3.3.1]nonan‐9‐ones

The 1H and 13C NMR spectra of 2,4,6,8-tetraaryl-3,7-diazabicyclo[3.3.1]nonan-9-ones were measured at 360 and 90 MHz, respectively. The chemical shifts were assigned unambiguously using one- and two-dimensional NMR spectroscopic data and nuclear Overhauser enhancement studies. These results clearly indicate a chair–boat conformation for these compounds with (i) all aryl groups orientated equatorially and (ii) the aryl groups of the boat lying in the shielding zone of the aryl groups of the chair. Literature assignments of carbon chemical shifts were also revised. Copyright © 2000 John Wiley & Sons, Ltd.

NMR study of the stereochemistry of 2,4,6,8-tetraaryl-3,7-diazabicyclo[3.3.1]nonan-9-ones

The H-1 and C-13 NMR spectra of 2,4,6,8-tetraaryl-3,7-diazabicyclo 3.3.1]nonan-9-ones were measured at 360 and 90 MHz, respectively. The chemical shifts were assigned unambiguously using one- and two-dimensional NMR spectroscopic data and nuclear Overhauser enhancement studies. These results clearly indicate a chair-boat conformation for these compounds with (i) all aryl groups orientated equatorially and (ii) the aryl groups of the boat lying in the shielding zone of the aryl groups of the chair. Literature assignments of carbon chemical shifts were also revised. Copyright (C) 2000 John Wiley & Sons, Ltd.

5387865 Permeability determination from NMR relaxation measurements for fluids in porous media

The proton nuclear magnetic resonance (NMR) spin-lattice relaxation of all six amides of deferriferrichrome and of various alumichromes dissolved in hexadeutero-dimethylsulfoxide have been investigated at 100, 220, and 360 MHz. We find that, depending on the type of residue (glycyl or ornithyl), the amide proton relaxation rates are rather uniform in the metal-free cyclohexapeptide. In contrast, the 1H spinlattice relaxation times (T1's) are distinct in the Al3+-coordination derivative. Similar patterns are observed in a number of isomorphic alumichrome homologues that differ in single-site residue substitutions, indicating that the spin-lattice relaxation rate is mainly determined by dipole-dipole interactions within a rigid molecular framework rather than by the specific primary structures. Analysis of the data in terms of 1H—1H distances (r) calculated from X-ray coordinates yields a satisfactory linear fit between T1-1 and Σr-6 at the three magnetic fields. Considering the very sensitive r-dependence of T1, the agreement gives confidence, at a quantitative level, both on the fitness of the crystallographic model to represent the alumichromes' solution conformation and on the validity of assuming isotropic rotational motion for the globular metallopeptides. An extra contribution to the amide proton T1-1 is proposed to mainly originate from the 1H-14N dipolar interaction: this was supported by comparison with measurements on an 15N-enriched peptide. The nitrogen dipolar contribution to the peptide proton relaxation is discussed in the context of {1H}—1H nuclear Overhauser enhancement (NOE) studies because, especially at high fields, it can be dominant in determining the amide proton relaxation rates and hence result in a decreased effectiveness for the 1H—1H dipolar mechanism to cause NOE's. From the slope and intersect values of T1-1 vs. Σr-6 linear plots, a number of independent estimates of τr, the rotational correlation time, were derived. These and the field-dependence of the T1's yield a best estimate <τr> ≈ 0.37 ns, in good agreement with 0.38 ns [unk] <τr> [unk] 0.41 ns, previously determined from 13C and 15N spin-lattice relaxation data.

Gas chromatography/mass spectrometric analysis of 24R,25-dihydroxyvitamin D 3 using 24R,25-dihydroxy[6,19,19- 2H]vitamin D 3 as internal standard

Gas chromatography/mass spectrometric (GC/MS) analysis of 24R,25-dihydroxyvitamin D 3 (24,25-(OH) 2D 3, 1a) in rat serum is examined using 6,19,19-trideuterated derivative (24,25-(OH) 2[6,19,19- 2H]D 3, 1c) as an internal standard. Pyro- and isopyro-24,25-(OH) 2D 3 ( 2a and 3a) are synthesized and the structures are determined unambiguously by their spectral data including nuclear overhauser enhancement study of the NMR spectra. The two peaks which appear on the total ion chromatogram of 24,25-cyclic n-butyl boronate-3-trimethylsilyl derivative of 24,25-(OH) 2D 3 are identified to be those of the pyro and isopyro isomers ( 2a and 3a) by direct comparison with the synthetic standards. Analysis of the GC/MS spectrum of 24,25-(OH) 2[6,19,19- 2H]D 3 ( 1c) indicated that the CH 3(19) is preferentially eliminated in the fragmentation giving rise to a base peak (M-TMSOH-Me) +. Thus the molecular ion peak, which is about 20% of the base peak, rather than the base peak is used for GC/MS assay of 24,25-(OH) 2D 3 in serum.

Solid-state and solution conformations of isotiazofurin: crystallographic, computational and 1H NMR studies.

Isotiazofurin (C9H12N2O5S, NSC363223) is a thiazole nucleoside analogue of the antitumour agent tiazofurin. The conformation of this analogue has been studied using a variety of experimental and computational techniques. The crystal and molecular structure of isotiazofurin has been determined using single-crystal X-ray diffraction techniques and refined to a conventional R value of 0.030 for all data. Conformational features conserved in other thiazole nucleoside structures are also observed in the solid-state structure of isotiazofurin. The C-glycosidic torsion angle remains in the anti conformation and the carboxamide amino group remains cis-planar to the ring nitrogen. Ab initio calculations at the RHF/321G*@321G* level and natural bond orbital analysis of the results suggest that the carboxamide cis-planar conformation observed in the solid state is maintained in solution. However, semiempirical calculations suggest that a syn conformation about the C-glycosidic bond is energetically favored. This is supported by 1H nuclear Overhauser enhancement (NOE) studies. Analyses of NOE results using both slow- and rapid-exchange models indicate a preference for the syn conformation in solution. Thus, the anti conformation observed in the crystal structures of isotiazofurin is not maintained by isotiazofurin in solution.

Nuclear magnetic resonance studies of cyclodextrin complexes of linoleic acid and arachidonic acid

Structures of a- and /3-cyclodextrin complexes of linoleic and arachidonic acids were investigated by detailed 1H spin-lattice relaxation time (7'1) measurements and one-dimensional difference nuclear Overhauser enhancement (NOE) studies. On the basis of the observed NOE proximities and 2'1 values, it is concluded that the carboxyl arms of both linoleic and arachidonic acids are present within the cyclodextrin cavity with double bonds partly exposed or buried. While the double bond at 9-10 in linoleic acid is buried, the one at 12-13 is exposed, the molecule from position 11 being present in an inclined position, with the methyl end pointing away from the cavity laterally. The double bond in arachidonic acid at position 5-6 is buried; the other at 8-9 lies exposed. While the carboxyl arm is present within the cavity, the methyl arm lies parallel to the carboxyl arm outside the cavity.

A comparative NMR study between the macrolide antibiotic roxithromycin and erythromycin A with different biological properties

1H nuclear Overhauser enhancement studies and 1H NMR 3J analysis establish the similarity between the major solution-state conformation of roxithromycin (1) and the erythromycin (2). A major difference between the structure of antibiotics 1 and 2 is the replacement of the 9-keto group in 2 by a 9-[O-(2,5-dioxahexyl)oxime] group. The NOE studies show that this oxime chain is oriented above the macrocyclic lactone ring and that the oxygen atoms of this chain are engaged in tight hydrogen bonding with a water molecule and with the 6- and 11-hydroxyl groups of the macrocycle. It results in a globular form of the whole roxithromycin molecule. These data explain also a relative hydrophobicity of this antibiotic. Erythromycin A (2), which presents a less rigid macrocycle with two free hydroxyl groups (6-OH and 11-OH), forms a dimer detected by FAB mass spectroscopy. 1H and 13C NMR relaxation measurements (T1) for both antibiotics show that interresidue hydrogen bonds in roxithromycin reduce the rotational freedom of the macrocyclic lactone ring and consequently the motions of desosamine and cladinose sugars. In another way, an ionization of the amino function occurs in the various media according to the nature of the antibiotic. This would allow the reactivity modification of the desosamine unit. In the biological study, the modifications of the 455-nm metabolite-cytochrome P-450 complex formation are observed.

Structure assignment of some 3,4′‐diquinolinyl sulfides studied by Nuclear Overhauser Effects, X‐ray analysis and reactions with sodium methoxide

Abstract1H NMR spectral assignment and nuclear Overhauser enhancement studies of 3‐[[3‐(methylthio)‐4‐quinolinyl]thio]‐4‐methoxyquinoline (2) and ‐4(1H)‐quinolinone (3) were carried out. The structure of compound 2a (R = R′ = Me) was also confirmed by X‐ray analysis. Reactions of compounds of types 2 and 3 with sodium methoxide proceeded with the cleavage of 4‐quinolinyl‐sulfur bond yielding two molecules of 3‐(methylthio)quinolines of type 4 or types 4 and 5.

NMR studies of the Escherichia coli trp aporepressor. Sequence-specific assignment of the aromatic proton resonances.

The resonances in the aromatic region of the 1H-NMR spectrum of the Escherichia coli trp aporepressor have been assigned to amino acid type by two-dimensional correlated spectroscopy (COSY), homonuclear Hartmann-Hahn (HOHAHA) spectroscopy and nuclear Overhauser enhancement spectroscopy (NOESY) techniques and studies of the pH dependence of the chemical shifts, in combination with selective deuteration of the protein. Complete sequence-specific assignments of the aromatic resonances have been made by comparing the observed inter-residue NOEs with those expected on the basis of the crystal structure of the protein [Zhang, R.-G., Joachimiak, A., Lawson, C.L., Shevitz, R.W., Otwinowski, Z. & Sigler, P.B. (1987) Nature 327, 591-597]. The latter experiments have also permitted the sequence-specific assignment of some of the high-field methyl resonances. The complete assignment of the aromatic region of the spectrum, in particular of resonances from residues at the dimer interface, opens the way to detailed studies of the conformational effects of corepressor and operator binding.

On the structure of mucus glycoproteins: 1H n.m.r. nuclear Overhauser enhancement study

Glycoproteins stemming from gastric intestinal mucus are investigated by means of 2D nuclear Overhauser enhancement measurements. Thereby, the results are best understood, if one assumes that the glycoprotein is arranged in well defined tertiary structures. The lifetimes of these structures are unknown, but have to be at least in the range necessary to build up a cross relaxation. Moreover, this structural behaviour also depends on the actual pH values, as the n.m.r. results change with a variation of the pH.

Use of the nuclear Overhauser effect in the determination of the orientation of aromatic substitution in tricyclic quinoxalinones

1 H N.m.r. nuclear Overhauser enhancement studies involving the amide NH of 7,8,9,10-tetrahydropyrido[1,2-a]quinoxalin-6-ones have been used to identify the aromatic proton signals of the quinoxalin-6-ones and to show that bromination with bromine in glacial acetic acid takes place at C-3 whilst nitration with potassium nitrate–concentrated sulphuric acid takes place at C-2.

Selective 1H–13C and 1H–1H nuclear overhauser enhancement studies of adenosine–thymidine interaction in solution

Selective proton–carbon nuclear Overhauser effect (NOE) measurements have been widely used to analyse through-space proton–carbon interactions. Intermolecular interactions between adenosine and thymidine have been studied by means of selective {H}C-NOE spectroscopy. Moreover, the role of proton chemical exchange in determining {H}C-NOE has been considered. A combined use of homo- and hetero-nuclear selective NOE to account the chemical exchange contribution to the experimental {H}C-NOE has been proposed.

Apotrisporin-E: A new sesquiterpenoid isolated from Phycomyces blakesleeanus and Blakeslea trispora

A new sesquiterpenoid has been identified in two species of Mucorales. It is apotrisporin-E: 2,4,4-trimethyl-3-[5′-hydroxyl-3′-methylpenta-(1′ E,3′ E)-dienyl]-(5 S)-hydroxylcyclohex-2-enone. Nuclear Overhauser enhancement studies indicated that the cis-trans isomeric assignments at carbon atom 3′ based upon the NMR signals for the hydrogens on carbon atoms 1′ and 2′ were valid. The existence of apotrisporin-E implies that trisporin-D and trisporin-E, two putative pheromones, are made by the Mucorales.

An examination of the solution conformations of bicuculline using NMR and theoretical methods

The temperature-dependence of the H1/H9 coupling constant and of the H6′ and H8 chemical shifts have been used to examine the solution conformation of the phthalide isoquinoline alkaloid, bicuculline, in terms of the central C1C9 dihedral angle (γ). The results indicate more than one low-energy conformation. A model with three low-energy conformations within ca. 10 kJ mol −1 of each other is suggested, similar to theoretical gas-phase results. Nuclear Overhauser enhancement studies support two of these minima. An estimate of 40 kJ mol −1 is derived as an upper limit for the barrier to internal conversation between the minima. A barrier to total rotation, of ca. 100 kJ mol −1 found in the gas-phase and due to steric hindrance, exists at γ ≌ 340°.

Conformation of the DNA undecamer 5′d(A-A-G-T-G-T-G-A-T-A-T) bound to the single-stranded DNA binding protein of Escherichia coli: A time-dependent transferred nuclear overhauser enhancement study

A time-dependent transferred nuclear Overhauser enhancement study of the conformation of the single-stranded DNA 11mer 5′d(A-A-G-T-G-T-G-A-T-A-T) bound to the single-stranded DNA binding protein of Escherichia coli (SSB) is presented. It is shown that the conformation of the bound 11mer is that of a right-handed B-type helix similar to that of the free 11mer. The observation of internucleotide transferred nuclear Overhauser enhancements for every base step excludes the possibility of intercalation by aromatic protein residues. In addition, it is shown that the effective correlation time of the bases (80 ns) corresponds to that of a complex of molecular weight $ ̃ 170,000, containing two SSB tetramers. The sugars, on the other hand, exhibit a shorter effective correlation time (40 ns), indicating the presence of internal motion. This suggests that the bases are anchored to the protein surface, possibly by hydrophobic interactions, whereas the sugarphosphate groups are directed outwards towards the solvent.