Elucidation of the Enantiodiscrimination Properties of a Nonracemic Chiral Alignment Medium through Gel-based Capillary Electrochromatography: Separation of the Mefloquine Stereoisomers.

Enantiodiscrimination and enantioseparation are two highly important processes in chemistry, often performed by using NMR spectroscopy and chromatography. For a better understanding of the mechanistic details, the same system should be studied by both methods. In addition, isotropic and anisotropic NMR parameters should be obtained, the latter using alignment media so that residual dipolar couplings and chemical-shift anisotropies can be measured. Consequently, a chiral alignment medium was used for the first time in chiral gel-based capillary electrochromatography with the four stereoisomers of the antimalaria drug mefloquine as test compounds. Chromatographic data verify that enantiodiscrimination obtained with this alignment gel is caused by differences in the equilibrium constants related to associate formation. Hence, the chromatographic separation provides physicochemical data that form a basis for the understanding and optimization of alignment processes, and vice versa.

Trapped in misbelief for almost 40 years: selective synthesis of the four stereoisomers of mefloquine.

Here we report the synthesis of all four stereoisomers of mefloquine. Mefloquine (Lariam) is an important anti-malaria drug that is applied as a racemate of the erythro form. However, the (-)-isomer induces psychosis, while the (+)-enantiomer does not have this undesired side effect. There are six syntheses of which five lead to the wrong enantiomer without the authors of these syntheses noting that they had synthesized the wrong compound. At the same time physical chemistry investigations had assigned the absolute configuration correctly and the last enantioselective synthesis that took these results into account delivered the correct absolute configuration. Since various synthetic approaches failed to provide the correct stereoisomers in previous syntheses, we submit here a synthetic approach with a domino Sonogashira-6π-electrocyclisation as key step that confirmed synthetically the correct absolute configuration of all four isomers.

The absolute configuration of (+)- and (-)-erythro-mefloquine.

The controversy over the absolute configuration of (+)-erythro-mefloquine, the less psychosis-causing enantiomer of the anti-malarial drug Lariam, has been resolved by Mosher ester crystallization. The configuration determined previously by physical methods is correct, whereas the configuration determined by three enantioselective syntheses is wrong.

Predicting the structure of cyclic lipopeptides by bioinformatics: structure revision of arthrofactin.

Arthrofactin, a bioactive cyclic lipopeptide from Pseudomonas sp. MIS38, was reinvestigated for its structural and stereochemical features due to discrepancies between the genetics-based sequence prediction and the currently suggested structure. The structure of arthrofactin and its derivatives was reassigned on the basis of chiral HPLC analysis and extensive NMR and MS experiments. Furthermore, derivatives of arthrofactin were discovered.

Chiral discrimination of amines by anisotropic NMR parameters using chiral polyacrylamide-based gels.

A new chiral alignment medium for dimethyl sulfoxide, methanol, and water as solvents was developed. Because both enantiomers of the gel are available, it is possible to enantiodiscriminate natural products such as strychnine HCl that naturally occurs as single enantiomer. With the two methods of achieving anisotropy, namely stretching and confinement, the degree of alignment can be adjusted, and the director changed from horizontal to vertical. This increases the applicability. Three compounds were enantiodiscriminated on the basis of residual dipolar coupling data: mefloquine HCl, strychnine HCl, and menthylamine HCl.

Determining the absolute configuration of (+)-mefloquine HCl, the side-effect-reducing enantiomer of the antimalaria drug Lariam.

Even though the important antimalaria drug rac-erythro-mefloquine HCl has been on the market as Lariam for decades, the absolute configurations of its enantiomers have not been determined conclusively. This is needed, since the (-) enantiomer is believed to cause adverse side effects in malaria treatment resulting from binding to the adenosine receptor in the human brain. Since there are conflicting assignments based on enantioselective synthesis and anomalous X-ray diffraction, we determined the absolute configuration using a combination of NMR, optical rotatory dispersion (ORD), and circular dichroism (CD) spectroscopy together with density functional theory calculations. First, structural models of erythro-mefloquine HCl compatible with NMR-derived (3)J(HH) scalar couplings, (15)N chemical shifts, rotational Overhauser effects, and residual dipolar couplings were constructed. Second, we calculated ORD and CD spectra of the structural models and compared the calculated data with the experimental values. The experimental results for (-)-erythro-mefloquine HCl matched our calculated chiroptical data for the 11R,12S model. Accordingly, we conclude that the assignment of 11R,12S to (-)-erythro-mefloquine HCl is correct.

Challenge of large-scale motion for residual dipolar coupling based analysis of configuration: the case of fibrosterol sulfate A.

Fibrosterol sulfate A is a polysulfated bis-steroid with an atypical side chain. Due to the flexibility of the linker, large-scale motions that change dramatically the shape of the entire molecule are expected. Such motions pose major challenges to the structure elucidation and the correct determination of configuration. In this study, we will describe the determination of the relative configuration of fibrosterol sulfate A through a residual dipolar coupling based multiple alignment tensor analysis complemented by molecular dynamics. For completeness, we applied also the single tensor approach which is unreliable due to the large-scale motions and compare the results.

The structure elucidation of isomalyngamide K from the marine cyanobacterium Lyngbya majuscula by experimental and DFT computational methods.

The 2Z-isomer of malyngamide K has been isolated along with the known compounds malyngamide C, deoxy-C and K, and characterized from a Papua New Guinea field collection of the cyanobacterium Lyngbya majuscula. The planar structure was deduced by 1D and 2D NMR spectroscopic and mass spectral data interpretation. The absolute configurations were determined on the basis of spectroscopic techniques, chemical degradation and DFT theoretical calculations.

Bijvoet in solution reveals unexpected stereoselectivity in a Michael addition.

The absolute configuration of small crystallizable molecules can be determined with anomalous X-ray diffraction as shown by Bijvoet in 1951. For the majority of compounds that can neither be crystallized nor easily be converted into crystallizable derivatives, stereocontrolled organic synthesis is still required to establish their absolute configuration. In this contribution, a new fundamental methodology for resolving the absolute configuration will be presented that does not require crystallization. With residual dipolar coupling enhanced NMR spectroscopy, ensembles of a limited number of structures are created reflecting the correct conformations and relative configuration. Subsequently, from these ensembles, optical rotation dispersion (ORD) spectra are predicted by DFT calculations and compared to experimental results. The combination of these two steps reveals the absolute configuration of a flexible molecule in solution, which is a big challenge to chiroptical methods and DFT in the absence of NMR spectroscopy. Here the absolute stereochemistry of the product of a new Michael addition, synthesized via a niobium(V) chiral enolate, will be elucidated by using the new methodology.

Accelerated Hydrolysis of Aspirin Using Alternating Magnetic Fields.

The major problem of current drug-based therapy is selectivity. As in other areas of science, a combined approach might improve the situation decisively. The idea is to use the pro-drug principle together with an alternating magnetic field as physical stimulus, which can be applied in a spatially and temporarily controlled manner. As a proof of principle, the neutral hydrolysis of aspirin in physiological phosphate buffer of pH 7.5 at 40 degrees C was chosen. The sensor and actuator system is a commercially available gold nanoparticle (NP) suspension which is approved for animal usage, stable in high concentrations and reproducibly available. Applying the alternating magnetic field of a conventional NMR magnet system accelerated the hydrolysis of aspirin in solution.

Synthesis and structural model of an alpha(2,6)-sialyl-t glycosylated MUC1 eicosapeptide under physiological conditions.

To study the effect of O-glycosylation on the conformational propensities of a peptide backbone, a 20-residue peptide (GSTAPPAHGVTSAPDTRPAP) representing the full length tandem repeat sequence of the human mucin MUC1 and its analogue glycosylated with the (2,6)-sialyl-T antigen on Thr11, were prepared and investigated by NMR and molecular modeling. The peptides contain both the GVTSAP sequence, which is an effective substrate for GalNAc transferases, and the PDTRP fragment, a known epitope recognized by several anti-MUC1 monoclonal antibodies. It has been shown that glycosylation of threonine in the GVTSAP sequence is a prerequisite for subsequent glycosylation of the serine at GVTSAP. Furthermore, carbohydrates serve as additional epitopes for MUC1 antibodies. Investigation of the solution structure of the sialyl-T glycoeicosapeptide in a H(2)O/D(2)O mixture (9:1) under physiological conditions (25 degrees C and pH 6.5) revealed that the attachment of the saccharide side-chain affects the conformational equilibrium of the peptide backbone near the glycosylated Thr11 residue. For the GVTSA region, an extended, rod-like secondary structure was found by restrained molecular dynamics simulation. The APDTR region formed a turn structure which is more flexibly organized. Taken together, the joined sequence GVTSAPDTR represents the largest structural model of MUC1 derived glycopeptides analyzed so far.

Helical structure of tercyclopropanedimethanol in solution.

Oligocyclopropanes with repetitive stereochemistry occur in two unusual natural products with interesting bioactivity. X-ray crystal structures are available for these compounds but with partially contradicting results. Because the 1H and 13C NMR spectra of oligocyclopropanes are far from trivial to be assigned even at highest magnetic fields, we have prepared a specifically deuterated sample and have applied high field NMR spectroscopy and DFT calculations to determine its conformation. The helix with equal handedness shown in the stereopicture was found for tercyclopropanedimethanol. A dihedral angle of around +40 degrees is the best representation of the experimental data and characterizes, therefore, the dominating helical conformation of tercyclopropanedimethanol with a single repetitive (+)-gauche interunit dihedral angle. This is in full agreement with the crystal structure of the all syn,trans-quinquecyclopropanedimethanol with an R configuration at the termini that also adopted an all (+)-gauche conformation. However, the crystal structure of the title compound and the solution structure are different.

Mild hydrolysis of 2-trifluoromethylphenol: kinetics, mechanism and environmental relevance.

2-Trifluoromethylphenol was hydrolysed in a phosphate buffer at neutral pH. At mild temperatures ranging from 34 degrees C to 69 degrees C this compound liberates consecutively fluorine anions to form salicylic acid. This process is energetically driven by the hydration of the fluorine anions. No intermediates have been detected by HPLC and (19)F-NMR and this was confirmed by computer calculations which favor the first step in the whole reaction sequence being rate-limiting. Accordingly, the reaction energy of the first dehalogenation of the trifluoromethyl anion is 28.4 kcal mol(-1) higher than for the second dehalogenation. The pseudo-first-order kinetic was determined and from an Arrhenius diagram an activation energy of E(a)=25.1 kcal mol(-1) has been estimated. At 37 degrees C and a pH of 7.4 the half-life was 6.9 h. The rate of hydrolysis was favored at higher pH and it was not influenced by oxygen, sunlight or trace elements found in natural water. The latter was shown by incubations with lake water instead of distilled water.

Characterization of the binding epitope of ciprofloxacin bound to human serum albumin.

Aqueous solutions of ciprofloxacin in phosphate buffer were measured by NMR under physiological conditions. The chemical shifts differ substantially compared to earlier investigations at low pH or in DMSO. Protein binding experiments using saturation transfer were optimized to measure proton resonances of ciprofloxacin that are in close proximity to human serum albumin. The relative intensities were mapped on the molecule to define the binding epitope. According to this methodology the cyclopropane ring and the chinolon ring constitute the binding epitope. Competition experiments with increasing amounts of salicylic acid did not change the saturation transfer to the ciprofloxacin protons indicating at least two different binding sites.

Effect of the solvent on the conformation of a depsipeptide: NMR-derived solution structure of hormaomycin in DMSO from residual dipolar couplings in a novel DMSO-compatible alignment medium.

The macrocyclic compound hormaomycin has been investigated by NMR spectroscopy and by restrained molecular-dynamics simulations. Measurement of residual dipolar couplings induced by dissolving the depsipeptide in a polyacrylamide gel compatible with DMSO and their incorporation into the structure calculation of the title compound improved the precision of the family of structures. In DMSO the macrocyclic ring shows two beta-turns, whose positions in the sequence differ from those found in the CDCl3 solution structure and in the crystal structure obtained from hexylene glycol/H2O (50:50). The bulky side chain consisting of a 3-(2-nitrocyclopropyl)alanine and a chlorinated N-hydroxypyrrole moiety is flexible in DMSO.

Direct determination of ciprofloxacin in admixtures with metronidazol and ampicillin by NMR.

Methods to analyse mixtures of pharmaceutical compounds are often based on chromatographic separations coupled to UV detectors. These procedures typically have to verify with other methods that the specificity is sufficient because structural information is sometimes too limited. NMR methods can be particularly useful in the analysis of admixtures of antibiotics such as ciprofloxacin because of the inherent high selectivity. This can even be extended by the measurement of diffusion coefficients that help to identify the relevant signal for quantification. Taking advantage of the resolving power of NMR an analytical procedure to directly measure ciprofloxacin without separation steps is presented. The diffusion coefficients of the three components were measured. Tests of precision (repeatability) and recovery of ciprofloxacin without any separation steps in the presence of metronidazol and ampicillin were conducted.

The structure of hormaomycin and one of its all-peptide aza-analogues in solution: syntheses and biological activities of new hormaomycin analogues.

Four new aza-analogues of hormaomycin 1, a secondary metabolite with interesting biological activities produced by Streptomyces griseoflavus, were synthesized and subjected to preliminary tests of their antibiotic activity to provide new insights into the structure-activity relationship studies of this class of compounds. The solution structures of hormaomycin 1 and its aza-analogue 2 a were determined by NMR spectroscopy. The data exhibited a reasonably rigid conformation for both molecules, stabilized by stacking interactions between the aromatic moieties attached to the ring and the side chain. According to NMR-spectral data the aza-analogue epi-2 a has a rather different conformation and indeed shows no antibacterial activity whatsoever.