Complete (1)H NMR spectral fingerprint of huperzine A

Complete analysis of the (1)H NMR spectrum of huperzine A, 1-amino-13-ethylidene-11-methyl-6-aza-tricyclo[7.3.1.0(2, 7)]trideca-2(7),3,10-trien-5-one, a Lycopodium alkaloid and anti-Alzheimer drug lead containing an ABCD(E)(MN)(OP)X(3)Y(3)-type system of 15 nonexchangeable proton spins, is reported for the first time, and earlier assignments are corrected. The complete (1)H parameter set of 11 chemical shifts clarifies the diastereotopism of both methylene groups, and provides a total of 38 observed H,H-couplings including 31 long-range ((4-6)J) connectivities. The NMR data is consistent with the comparatively rigid alicyclic backbone predicted by molecular mechanics calculations, and forms the basis for (1)H NMR fingerprint analysis for the purpose of dereplication, purity analysis, and elucidation of structural analogs.     

Strategies for organic impurity quantification by H NMR spectroscopy: Constrained total-line-shape fitting

A constrained total-line-shape (CTLS) fitting strategy for organic impurity analysis from ¹H NMR spectra was developed and assessed by studying two examples. In general, total-line-shape fitting allows integration of overlapping lines without suffering from baseline artifacts as much as traditional integration methods. It is shown here that the constrained total-line-shape fitting, where the spectral structures of the multiplets to be fitted are taken into account in form of constraints, allows quantification of seriously overlapping lines and when the signals are close to the root of major signals. Also, a method for removal of ¹³C satellite signals is described. The results indicate that our approach significantly improves the usefulness of qNMR in impurity analysis and that impurity levels of 0.1 mol%, which in some cases means down to 0.01 wt%, can be easily determined with relative standard error smaller than 10%.     

Spectral analysis of 1H coupled 13C spectra of the amino acids: adaptive spectral library of amino acid 13C isotopomers and positional fractional 13C enrichments

The natural abundance 1H-coupled 13C NMR spectra of all proteogenic amino acids were measured in D2O at pH* 1. The accurate 1H,13C spin-spin coupling constants were analyzed using total-line-shape fitting. The obtained spectral parameters can be used to establish a spectral library of amino acid 13C isotopomers. The adaptive spectral library principle is introduced and discussed in this article. The simulated spectra can be applied to quantification of 13C isotopomer mixtures of amino acids and, thus, for exploring metabolic pathways. Also a protocol for amino acid 13C isotopomer metabolomic profiling in 13C labeled glucose feeding experiments is outlined. The approach is suggested to give invaluable information about positional fractional 13C enrichments, which are not easily available by any other method.     

Conformational Analysis of Sulfur-Containing Heterocycles,Part I. Synthesis and Structural Determination of Diastereomerically Pure 4,6-Bis-(phenoxymethyl)-1,2,5-trithiepanes

Some diastereomerically pure 4,6-bis-(phenoxymethyl)-l,2,5-trithiepanes were synthesized and unambiguously assigned. Their conformational properties and dynamic behavior were investigated by various NMR spectroscopic methods and quantum-chemical calculations at the HF/6-31G* level. The ground states of these compounds proved to be twist-chairs. A ring interconversion can occur in the meso-isomers as well as in the (±)-isomers. This interconversion can be described as a simultaneous inversion of the disulfide bridge. In the case of the meso-isomers, both ground states are mirror images of each other and the transition state is a highly symmetrical chair. The barrier heights of interconversion were determined to be in the range of 50 kJ/mol by variable-temperature NMR measurements. The ground states as well as the transition state of the (±)-isomers were found to be nonsymmetrical. However, those dynamic processes that are fast with respect to the NMR time scale lead to averaged NMR spectra at room temperature. A further dynamic process found through the quantum-chemical calculations is a flapping of the meth-ylene groups of the rings. The energy barrier of this flapping was calculated to be very small (< 20 kJ/mol) and could not be observed by low-temperature NMR measurements.     

Automated quantum mechanical total line shape fitting model for quantitative NMR-based profiling of human serum metabolites

An automated quantum mechanical total line shape (QMTLS) fitting model was implemented for quantitative nuclear magnetic resonance (NMR)-based profiling of 42 metabolites in ultrafiltrated human serum samples. Each metabolite was described by a set of chemical shifts, J-couplings, and line widths. These parameters were optimized for each metabolite in each sample by iteratively minimizing the difference between the calculated and the experimental spectrum. In total, 92.0 to 98.1 % of the signal intensities in the experimental spectrum could be explained by the calculated spectrum. The model was validated by comparison to signal integration of metabolites with isolated signals and by means of standard additions. Metabolites present at average concentration higher than 50 μM were quantified with average absolute relative error less than 10 % when using different initial parameters for the fitting procedure. Furthermore, the biological applicability of the QMTLS model was demonstrated on 287 samples from an intervention study in 37 human volunteers undergoing an exercise challenge. Our automated QMTLS model was able to cope with the large dynamic range of metabolite concentrations in serum and proved to be suitable for high-throughput analysis.