Structure2010 Speaker Abstracts

What is Structure Elucidation?

Don Richards
 Analytical R & D, Pfizer Global Research & Development, Ramsgate Rd, Sandwich, Kent, CT13 9NJ, UK

This presentation looks at Structure Elucidation in a Pharmaceutical context. Within pharmaceutical analysis there exists a hierarchy of activities related to molecular structure. Structure Characterisation is the collection of primarily spectroscopic data (Infra-red, Ultra violet, Nuclear Magnetic Resonance, Mass Spectrometric and X-ray crystallographic) related to a specific molecular structure. Characterisation does not necessarily involve interpretation of these data in a structural context. Structure Confirmation is the use of some or all of these data to confirm the proposed molecular structure of a chemical substance. The result may be as simple confirmed / not confirmed. Structure Elucidation is the determination of molecular structure without preconception. Structures requiring elucidation may be of a substance related to a known chemical structure such as process related impurities, degradants or metabolites. Alternatively they may be of completely unknown origin such as impurities derived from contamination.

In the Pharmaceutical Industry all process related impurities greater than 0.1 % relative to the active pharmaceutical ingredient (API) must be identified. The Structure Elucidation of such impurities is carried out primarily from MS and NMR experiments often guided by IR, UV and chromatographic ‘clues’. On occasions when one is fortunate enough to have a crystal, x-ray crystallography can be invaluable.

The presentation uses real examples to show how these techniques can be brought together by a team of Structure Elucidation scientists. 

Plant natural products in a drug discovery program, from the ground-up

Mark O’Neil-Johnson1, Jin-Feng Hu1, Courtney Starks1, Russell Williams1, Gary Eldridge1 and Peter Raven2
1Sequoia Sciences, Inc., 1912 Innerbelt Business Center Dr, Saint Louis, MO 63114, Missouri Botanical Garden, 4344 Shaw Blvd., Saint Louis, MO  63110, USA

Natural product chemistry has traditionally been a long and time-consuming process for drug discovery research.  This presentation describes the extraction, isolation, purification and structure elucidation of biologically active compounds. The extraction and isolation method (Sequoia patent) is used to create purified natural product fractions that fit into HTS screening platforms.  Utilizing a CapNMR probe, ACD database and structure elucidation software, it is now possible to routinely elucidate structures on mass limited or microgram quantities of purified natural product compounds.

This methodology has accelerated the discovery of active and novel compounds from plant sources.  In addition, the process has also allowed it to produce focused screening libraries based on selective plant species that may be either underrepresented due to insufficient material or that may produce unique compounds necessary for their survival.  Introduction of new NMR probe technologies has allowed for increase through-put of complete NMR data sets resulting in the discovery of preferred, neglected and novel scaffolds.  A review of our natural product discovery process using working examples of microgram quantities of material to perform isolation and structure elucidation to unravel an active lead compound, as well as the introduction of unique NMR capabilities will be presented.

Hyphenated and hypernated techniques for the structure elucidation of metabolites

Ian Wilson
Clinical Pharmacology, Drug Metabolism and Pharmacokinetics, AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK

 The detection of both endogenous and drug (or xenobiotic) metabolites is relatively easy using modern LC-MS-based strategies, structure elucidation on the other hand is more of a problem. However, with the careful application of accurate mass measurements and fragmentation pathways the search space for novel metabolites, endogenous or exogenous, can be greatly reduced. Indeed it is often possible to unequivocally identify compounds based on this information. Where NMR spectroscopy is also required to confirm identity then LC-NMR represents a viable alternative to tedious and lengthy isolation steps. Examples will be provided that illustrate these points and possible strategies for the structure determination of unknowns in complex biological matrices will be provided.

Using Mobility-Mass Correlation Curves for Structure Identification

Herbert H. Hill, Kimberly Kaplan and William F Siems
Department of Chemistry, Washington State University, Pullman, WA 99163, USA

The value-added information gained by coupling ion mobility spectrometry (IMS) with mass spectrometry (MS) has significantly expanded the use of mass spectrometry with respect to both the qualitative and quantitative analysis chemicals contained in complex mixtures.  Often called ion mobility – mass spectrometry (IM-MS), the addition of IMS to MS offers improved signal to noise ratios, isomer separation, accurate isotope characterization, and enhanced peak capacity.  Because, in ion mobility spectrometry, ions separate according to their size, shape and ion-neutral interaction potential, IMS provides structural information as well.  Empirically, classes of compounds fall along mobility-mass correlation curves (trend lines) which provide additional structural information about a compound not possible by mass spectrometry alone.  For example, proteins, lipids, and nucleic acids all fall along three distinct mobility-mass correlation curves (MMCCs). Multiple charging and polymerization also form unique MMCCs.  With high resolution IMS, structural information can be obtained for compounds within the larger classifications.   Slopes of these MMCCs are related to the densities of the ions – large, floppy singly charged ions have low ion densities while compact, folded singly charged ions have high densities.  Thus each class of compound has its characteristic ion density which can be used to aid structural identification of an unknown compound.  This presentation will discuss the use of ion densities for the identification of various compound structures through mobility-mass measurements.

From data to knowledge: mining the metabolite profile with machine learning

Roy Goodacre
School of Chemistry and Manchester Interdisciplinary Biocentre, University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK

Mass spectrometry-based metabolite profiling is producing bounteous data floods and the extraction of the most meaningful parts of these data is key to the generation of useful new knowledge.  A typical metabolomics experiment is expected to generate thousands of data points (samples times variables) of which only a handful might be needed to describe the problem adequately.  At the moment there are many more metabolite features that are observed compared with those that are known! And to identify these is a monumental but necessary task.

Within the disease diagnostics area it is clearly important in identifying the important metabolite features that are discriminatory for disease stratification, after which detailed identification can be undertaken on the most relevant subset.  This presentation will discuss the road from the metabolite data tsunami to rigorously identified metabolites.  This road has involved the use of QC standards to check data quality, after which a two-stage process of univariate and multivariate analyses is used to identify statistically significant differences.

NMR, MRI & EPR Coming Together in the Aid of Structure Elucidation: Ultrafast, Ultrasensitive Multidimensional NMR

Lucio Frydman
Chemical Physics Department, Weizmann Institute, Israel
 
Although NMR is one of the most powerful analytical tools available, it is also notoriously insensitive.  This is largely due to the low levels of polarization achivable under conventional conditions. Recent developments make it possible to “hyperpolarize” samples under conventional liquid-like conditions.  One of the most promising and generally applicable techniques to achieve hyperpolarization relies on ex situ dynamic nuclear polarization (DNP). In this experiment the hyperpolarization is carried out outside the NMR magnet, and the metastable states thus created are then melted and transferred into the NMR spectrometer.  Given its irreversible nature, DNP-NMR experiments are best suited to the acquisition of a single or at most a small number of free induction decays.  This makes ex situ DNP ill-suited for collecting an array of transients involving complex pulse sequences, of the kind needed in 2D NMR.  To deal with this drawback, we have recently began exploring the combination of DNP with spatially-encoded ultrafast 2D NMR methods, capable of yielding 2D NMR spectra in a single-scan.  Particularly promising are inverse-detected heteronuclear methods exploiting the hyperpolarization of slow-relaxing nuclei, which is well preserved during the sample transfer from the polarizer to the NMR spectrometer, with the higher sensitivity of 1H detection.  Using this approach 2D 13C-1H heteronuclear NMR spectra of small-molecule mixtures can be recorded at very low concentrations and in one scan; e.g., on a 0.1 mM mixture of organic compounds at natural abundance, within 200 ms.

Authentic or Fake? – A mass spectrometric journey to establish what is in your medicine

Jean-Claude Wolff
 GlaxoSmithKline, Analytical Sciences, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, UK

 Pharmaceutical counterfeiting is big business, with great profits to be made, potentially at the expense of patient health. Counterfeit drug products come in many variations. Some do not contain any of the legitimate active pharmaceutical ingredient, or include the ingredient in harmful amounts or from an unacceptable source. Others contain a completely different active pharmaceutical ingredient. Identifying a "wrong" or “substitute” active pharmaceutical ingredient in a drug product presents an interesting analytical challenge. In this paper, Mass Spectrometric strategies are presented for identifying an unknown/substitute active pharmaceutical ingredient in counterfeit drug products. Accurate mass measurement of both MS and MS/MS spectra is very valuable in identifying unknowns. However, one should not forget isotope patterns observed for the protonated molecule to reduce the number of possible elemental compositions or even to decide on the correct elemental composition. In this study examples obtained on state-of-the-art equipment such as quadrupole time-of-flight and orbitrap mass analysers will be shown. Literature searches for compounds of the molecular formula suggested sometimes yield several isobaric compounds (e.g. sulfonamides, steroids). In this paper it is also investigated if the identity of the unknown isobaric compound can be determined/distinguished by mass spectrometry alone (without chromatography) using MSn fragmentation and ion mobility separation. Obviously the ultimate confirmation of the identity of the substitute active is via the analysis of standards of the compound suspected.

Powder NMR Crystallography

Lyndon Emsley
Université de Lyon, Centre de RMN à Très Hauts Champs, CNRS/ENS Lyon/UCB Lyon 1, 5 rue de la Doua, 69100 Villeurbanne, France.

Recent progress in developing new NMR methods to study analytical, structural or dynamic features in a range of solid materials will be presented. Methods for determining spin dynamics in large systems will also be described, and illustrated in the context of spin diffusion and complete structure determination of pharmaceutically relevant polymorphic compounds in powder form at natural isotopic abundance, when coupled with state of the art DFT chemical shift calculations. We will also show how powder structures can be obtained only using NMR chemical shifts. Starting from an ensemble of predicted crystal structures for powdered Thymol, comparison between experimental and calculated 1H solid-state isotropic NMR chemical shifts is sufficient to determine which predicted structure corresponds to the powder under study.

Small Molecule Crystallography: All you ever wanted to know about X-rays but ...

David Watkin
Chemical Crystallography, Chemistry Research Laboratory, Mansfield Road, Oxford OX1 3TA, UK

For my PhD (1964-67) I completed three X-ray crystal structure determinations.  In 2009, all three determinations could be started and brought to publishable conclusions in a single morning. 

 During the last decade improvements in X-ray diffraction equipment, computers and crystallographic software have revolutionised crystal structure analysis.  Once, the technique was rightly regarded as slow, laborious and expensive.  Now it is fast, computer-assisted and inexpensive.

 Significant developments in the technology are:

1) Low-consumption high-power X-ray sources.
2) X-ray focussing optics.
3) High-sensitivity low-noise CCD detectors.
4) Routine use of cooling to 100-150K.
5) Software to reliably solve The Phase Problem for structures with several  hundred non-hydrogen atoms in the molecule.
6) Personal Computers easily able to handle the computations involved.

Factors contributing to a poor take-up of the technology include:

1) Unawareness of recent trends.
2) Time delay and effort required to prepare crystalline samples.
3) Reluctance by some intuitions to teach basic molecular  crystallography.
4) Occasional total failure of the technique.
5) lack of access to facilities.
6) The crystallographic High Priesthood's reluctance to change with the times.

In practical terms, the real bottle neck is preparing suitable crystals. Prior to the development of chromatographic techniques, purification of materials by crystallisation was a standard technique.  Now it has become a lost art.  Perhaps now is the time to try to rediscover it.

Residual dipolar couplings as novel NMR-spectroscopic restraints in organic structure determination

Christina M. Thiele
Technische Universität Darmstadt, Clemens Schöpf Institut, Petersenstr. 22, D-64287 Darmstadt, Germany.

 The determination of conformations and relative configurations by nuclear magnetic resonance usually involves distances from the NOE and dihedral angles from 3J couplings. It is, however, often complicated by either absence of NOE data and/or 3J coupling data, remoteness of the stereocenters or conformational equilibria.

The recently reintroduced residual dipolar couplings[1] provide complementary information to these conventional NMR restraints. In order to demonstrate the utility of residual RDCs for organic structure determination we have applied this methodology to a problem, which is comparable to the determination of relative configurations, namely the differentiation and assignment of diastereo­topic protons in strychnine, which can also readily be solved by conventional methods [2].

The second application that will be shown is the determination of the relative configuration of an a-methylene-g-butyrolactone. For this substance conventional NMR spectroscopic means fail due to the existence of conformational equilibria such that both possible diastereoisomers are in line with the experimental data (3J and NOE). Using RDCs, however, it is possible to unambiguously assign the relative configuration (in this case to be trans) and even to determine conformer populations.[3]

References:

[1]   Reviews: Gschwind, R.M. Angew. Chem. 2005, 117, 4744-4746; Angew. Chem. Int. Ed. Engl. 2005, 44, 4666-4668; Yan, J., Zartler, E.R. Magn. Res. Chem. 2005, 43, 53-64; Thiele, C. M., Conc. Magn. Res. 2007, 30A, 65-80; Thiele, C. M., Eur. J. Org. Chem. 2008, 14, 5465-5481.

[2]   Thiele, C.M., Berger, S., Org. Lett. 2003, 5, 705-708. Thiele, C. M., J. Org. Chem. 2004, 62, 7403-7413.

[3]   Thiele, C. M., Marx, A., Berger, R., Fischer, J., Biel, M., Giannis, A., Angew. Chem. 2006, 118, 4566-4571, Angew. Chem. Int. Ed. 2006, 45, 4455-4460; Thiele, C. M., Schmidts, V., Böttcher, B., Louzao, I., Berger, R.,  Maliniak, A., Stevensson, B., Angew. Chem. 2009, 121, 6836-6840, Angew. Chem. Int. Ed. 2009, 48, 6708-6712.
 

Structural studies of small molecules by ion mobility-mass spectrometry

Colin S. Creaser
Centre for Analytical Science, Department of Chemistry, Loughborough University, Leicestershire, LE11 3TU, UK

The mobility of a gas-phase ion under the influence of a weak electric field and in the presence of a buffer gas is related to the collision cross section (i.e. the size and shape) of the ion. It is therefore possible to correlate measured ion mobilities with structural characteristics of analyte ions and to observe subtle changes in gas-phase conformations using the ion mobility method. This presentation will discuss approaches to the measurement of ion mobility and collision cross section in drift tube ion mobility devices using reference standards. The correlation of the measured collision cross section with modelled data provides information on the gas-phase structures of small molecules and non-covalent complexes.