Hyphenated Techniques in Natural Product Analysis
1. Key Words:
Rapid identification and characterization of known and new natural products without the necessity of isolation and purification, e.g., directly from plant, marine, or microbial sources (see also 'Naphthylisoquinoline Alkaloids', 'Anthraquinones and Knipholones', and 'Marine Natural Products') using our LC triad consisting of the combination of high-performance liquid chromatography with NMR spectroscopy (HPLC-NMR), electrospray ionization tandem mass spectrometry (HPLC-MS/MS), and circular dichroism (CD) spectroscopy (HPLC-CD): online elucidation (by MS/MS and NMR) of the full constitution including the assignment of the relative configuration of compounds, and determination of full absolute stereostructures by HPLC-CD experiments and interpretation of CD spectra by empirical analysis or by quantum chemical CD calculations (see 'Computational Chemistry'); application of capillary electrophoresis (CE) coupled to electrospray ionization time-of-flight mass spectrometry (ESI-oTOF-MS) for the identification of genuine, non-desulfated glucosinolates in Arabidopsis thaliana (see also www.sfb567.uni-wuerzburg.de), and for the analysis of other charged natural products, like e.g., phosphates, also including sulfated phenylanthraquinones and alkaloids like the naphthylisoquinolines.
2. Graphical Abstract:
Absolute Stereostructures of Natural Products Directly from Extracts - by the Analytical Triad, HPLC Coupled to MS/MS, NMR, and CD
Discovery of Novel Natural Products from Tropical Plants by On-Line Metabolite Profiling Using our LC-Triad
Chiroptical Online Stereoanalysis by HPLC-CD for Natural Products Directly from Stereoisomeric Mixtures
Analysis of the Glucosinolate Pattern of Arabidopsis thaliana by Capillary Zone Electrophoresis Coupled to Electrospray Ionization-Mass Spectrometry
3. Brief Description:
The isolation and structural elucidation of new natural products, e.g., from plants or microorganisms, is a rewarding – but often time-consuming – task, since it is a major effort to isolate each compound in a pure form, even the known ones. Furthermore, to obtain the required milligram quantities of all metabolites, even in the case of minor substances, one may need large amounts of the sometimes rare biological material and of expensive tools and supplies, like e.g., adsorbents and eluents. Another problem, in particular when dealing with unstable compounds, is that they may decompose already during the preparative separation and thus may escape the analysis.
The hyphenation of high-performance liquid chromatography (HPLC) with spectroscopic methods such as NMR spectroscopy (HPLC-NMR) or tandem mass spectrometry (HPLC-MS/MS) has led to new strategies that do not only permit to differentiate between known and unknown compounds, but can even establish full constitutions and relative configurations of new natural products directly from the crude extract with a minimum amount of material. The potential of HPLC-NMR for the investigation and structural elucidation of novel natural products has been strongly extended by the advent of powerful solvent suppression schemes, and their combination with a series of homo- and heteronuclear two-dimensional NMR experiments such as total-correlation spectroscopy (TOCSY), nuclear Overhauser enhancement spectroscopy (NOESY), heteronuclear multiple-bond correlation spectroscopy (HMBC), or heteronuclear multiple-quantum correlation spectroscopy (HMQC). The first successful adaption of a 2D rotating frame Overhauser enhancement spectroscopy (ROESY) sequence to stop-flow HPLC-NMR experiments on extracts of Ancistrocladus species [1-4] by our group provided the full constitution of novel naphthylisoquinoline alkaloids and, moreover, a reliable attribution of their relative configurations at the stereogenic centers and axes. Moreover, by hyphenation of HPLC-MS/MS and HPLC-NMR using an NMR cryoprobe it was for the first time possible to generate online-HSQC and online-HMBC spectra and, thus, to elucidate the full constitution of the new, chemically labile secondary metabolite secohyperforin from Hypericum, perforatum without isolation .
Due to the intrinsically nonchiral character of NMR signals and mass fragments, however, no information concerning the full absolute 3-dimensional structure is available by HPLC-NMR or HPLC-MS/MS (unless by the use of chiral chromatographic phases, which requires the availability of both – configurationally known – enantiomers!). A clue to the solution of this problem are the chiroptical properties of chiral analytes, in particular their circular dichroism (CD) data, since two enantiomers are unambiguously characterized by their fully opposite CD spectra. By hyphenation of CD measurements with HPLC techniques, the absolute configuration of the respective stereoisomers can then easily be established – if experimental data from structurally related compounds of known absolute configurations are available or if the structures fit into empirical CD rules. A most efficient alternative for the interpretation of CD spectra is their quantum chemical simulation as established in our group (see also ‘Computational Chemistry’), in particular, for the stereochemical assignment of novel-type structures. In combination with these experimental and computational CD methods, the use of HPLC-CD online coupling experiments for the stereochemical assignment of natural products completes our most efficient ‘LC triad’ (see Figure 1).
An important field of application of the LC triad is, first of all, the rapid identification of known compounds already in crude extracts as presented in Figure 2. Fast dereplication, i.e., recognizing known compounds at an early stage of the screening, is essential for the efficient identification of truly novel natural products from well-examined biological sources such as fungi. For this purpose, extracts of marine fungi were investigated by HPLC-UV, -MS, -NMR, and –CD as a powerful dereplication tool. The spectroscopic data thus gained were then compared to those from natural products databases. Several common fungal metabolites, namely roquefortine C, griseofulvine, and cyclopiazonic acid, were thus easily identified without prior isolation .
Moreover, the combination of the three online coupling methods HPLC-MS/MS, HPLC-NMR, and HPLC-CD has enabled us to successfully determine the full absolute stereostructures also of novel compounds without the necessity of isolation and purification – for the first time in phytochemical analysis! Several examples have been demonstrated in which, by the use of this LC-NMR-LC-MS/MS-LC-CD coupling triad, the complete 3-dimensional structures of novel compounds with stereogenic centers and/or chiral axes have been established directly from crude extracts, among them the phenylanthraquinone knipholone anthrone from the East African torch lily Kniphofia foliosa (see Figure 3) [1,6], ent-knipholone-6'-O-sulfate from Bulbine plants [6,11], and several natural products from marine fungi (see also 'Marine Natural Products') like sorbicillactone A , xestodecalactone B [1,10], and petrosifungin B [1,12] (see Figure 6).
By applying the analytical LC triad, a whole series of naphthylisoquinoline alkaloids were structurally elucidated directly from leaf extracts, i.a., from the two very rare Westafrican species Habropetalum dawei (Dioncophyllaceae) and Ancistrocladus guineënsis (Ancistrocladaceae), and from the previously uninvestigated Asian liana Ancistrocladus griffithii (Ancistrocladaceae). New naphthylisoquinolines thus identified and characterized online, were e.g., habropetaline A (see Figure 1) [1,3], ancistroguineine B , ancistrogriffine B (see Figure 6) and the novel dimer ancistrogriffithine A (see Figure 4) [2,7] as well as some related compounds like the isoquinoline phylline and the tetralone isoshinanolone (see Figure 6), [1,3]. The efficiency of the LC triad has also been demonstrated in the discovery of ancistrocladinium B and its rotational isomer (see Figure 5) with a hitherto unprecedented N,6'-coupling type, and a slow rotation about the hetero biaryl axis at room temperature  (see also ‘The Naphthylisoquinoline Alkaloids’).
In particular, the HPLC-CD hyphenation has become a most valuable tool for the stereochemical analysis of most diverse chiral natural products . For example, coupling of HPLC with CD easily permitted to directly distinguish between the enantiomers of two regioisomers (type I and II) of the ubiquitously occurring plant-derived phytoprostane B1 (see Figure 7), which occur in a racemic form due to the formation by a non-enzymatic free-radical mechanism. These prostaglandin/jasmonate-like compounds are plant effectors that are able to trigger a massive detoxification and defense response. With the aid of LC-CD on a chiral phase the absolute configuration of each enantiomer, peak by peak trapped in the flow cell of the CD spectropolarimeter, was determined . As outlined in Figure 8, LC-CD in combination with quantum chemical calculations again proved to be a powerful and reliable stereoanalytical tool for the elucidation of the absolute configurations of the atropo-enantiomers of parvistemin A, a dimeric phenylethyl benzoquinone from the aerial parts of the Chinese plant Stemona parviflora Wright. Due to the restricted rotation about the central biaryl axis, the compound is chiral, although racemic . Furthermore, with the stereochemical assignment of the atropo-enantiomers of murrastifoline-F by CD calculations, the first determination of a natural enantiomeric ratio of this unsymmetric, N,C-bonded heterobiarylic biscarbazole succeeded by analyzing a root extract of Murraya koenigii by LC-CD (see Figure 9) . For further selected examples of stereochemical assignments by LC-CD coupling combined with quantum chemical CD calculations: see also ‘Computational Chemistry’.
Capillary zone electrophoresis (CZE) in combination with electrospray ionization mass spectrometry (ESI-MS) has proven to be a powerful separation technique for charged compounds (e.g., organic acids, phosphates, sulfates, amino acids, or proteins) including alkaloids like, e.g., naphthylisoquinolines . CZE-ESI-MS coupling combines the advantage of an easy and rapid sample pretreatment, small-volume compatibility, fast analysis, and efficiency of CZE separations with the high selectivity and sensitivity of ESI-MS. Of particular interest is the coupling of CZE to ESI-oTOF-MS. This method combines the benefits of CZE separation with the high selectivity due to mass accuracy of < 5 ppm, which opens the possibility of determining elemental compositions. The analysis of the true isotopic pattern by ESI-oTOF-MS provides an additional analytical dimension for the identification of natural compounds even in complex biological samples. By hyphenation of CZE-ESI-MS and CZE-ESI-oTOF-MS we have developed a sensitive method for the analysis of genuine, still undesulfated, and thus anionic glucosinolates (see Figure 10) for the characterization of these prominent sulfur-containing natural products in Arabidopsis thaliana .
Within the scope of the SFB 567 (see also www.sfb567.uni-wuerzburg.de) our joint research project (B8) with the group of R. Hedrich (Julius von Sachs Institute for Biosciences, Würzburg) focusses on the role of the phloem during the propagation of long-distance signals after infection of Arabidopsis thaliana plants with virulent and avirulent strains of Pseudomonas syringae pv. tomato. Applying CZE-oTOF-MS, the glucosinolate profiles in Arabidopsis thaliana were studied without any complicated work-up procedure directly from crude plant extracts or from phloem exudates on a nL-scale. The sensitivity of the method together with mass accuracy and true isotopic pattern of the oTOF-MS allowed the identification of a broad series of glucosinolates in Arabidopsis thaliana seeds  (see Figure 10) and in phloem exudates .
Summarzing, the presented examples demonstrate that the use of hyphenated techniques like LC-MS/MS-NMR-CD or CZE-oTOF-MS contributes to the solution of various, e.g., sensitivity, selectivity or stereochemical problems in an efficient and elegant manner. Especially the not yet fully utilized potential for the development of LC-NMR with its cryoprobe technique together with the continuous improvement of the other coupled online methods will lead to powerful and indispensable analytical tools, in particular, in the field of natural products chemistry.
4. Selected Publications:
5. Cooperations and Research Grants
a) Within a special research project entitled "A New Class of Active Agents against Infectious Diseases" incorporated into the Collaborative Research Centre „Recognition, Preparation, and Functional Analysis of Agents against Infectious Diseases“ (Sonderforschungsbereich 630, see also www.sfb630.de) sponsored by the Deutsche Forschungsgemeinschaft (DFG).
b) Within a special research project entitled "Role of the phloem in propagation of pathogen-mediated signals: chemical signals and gene activation", in collaboration with Prof. Dr. Rainer Hedrich (Julius-von-Sachs-Institut für Biowissenschaften, Universität Würzburg), incorporated into the collaborative research center "Mechanisms of Interspecific Interactions of Organisms" (Sonderforschungsbereich 567, see also www.sfb567.uni-wuerzburg.de), sponsored by the DFG.
c) Within a special research project entitled "Enantioselective Synthesis of Bisbibenzyl Natural Products of the Isoplagiochin-Type with Combined Axially and Helical Chirality", in collaboration with PD Dr. Andreas Speicher (Universität des Saarlandes, Fachrichtung 8.1. Chemie – Organische Chemie), sponsored by the DFG (Individual Grant Br 699/12);
d) Within a special research project entitled "Natural Products from African Plants ", in collaboration with Prof. Dr. M.G. Peter (Institut für Chemie der Univerisität Potsdam) and Dr. A. Yenesew (Department of Chemistry, University of Nairobi, Kenya), sponsored by the DFG (Individual Grant Br 699/13-5);
e) Within a special research project entitled "Convergence in the Biosynthesis of Acetate- or Prenyl-Derived Natural Products", incorporated into the DFG priority programme 1152 "Evolution of Metabolic Diversity", Individual Grant Br 699/9-3);
f) Within a special research project entitled "Molecular Phylogeny and Chemotaxonomy of the Ancistrocladaceae Plant Family", in collaboration with Prof. Dr. G. Heubl (Institut für Systematische Botanik der LMU München), sponsored by the DFG (Individual Grants Br 699/7 and Br 699/14-2);
g) Within the special research project entitled „BIOTECmarin – Center of Competence: Molecular Biotechnology and New Agents from Marine Sponges and Associated Microorganisms“, sponsored by the Bundesminsterium für Bildung, Forschung, Wissenschaft und Technologie (BMBF).
g) Within the special research project entitled „Molecular Interaction between Targets and Natural Products as a Tool for the Identification of New Agents and Agent Models for Agrochemical and Pharmaceutical Research“, sponsored by the Bundesministerium für Bildung, Forschung, Wissenschaft und Technologie (BMBF) in collaboration with the BASF AG, Ludwigshafen (completed).