Application of 2D-NMR spectroscopy to metabolomics and identification of metabolites

Transcription

1 Application of 2D-NMR spectroscopy to metabolomics and identification of metabolites November 10 14, 2008 Young Hae Choi Division of Pharmacognosy, Section Metabolomics Institute of Biology, Leiden University Leiden, Then Netherlands

2 Structure elucidation of metabolites in the mixture - Using diverse spectroscopy You have knowledge about metabolites You ve prepared samples for analysis You ve collected data using analytical methods You ve selected interesting and significant signals Then, what is the next step?

3 How can we elucidate the selected metabolites? ? w*c[2] w*c[1]

4 Advantages of NMR in Metabolomics Simultaneous observation of wide range of chemical classes (Mole based detection, non-selective) Powerful structural elucidation tool Non-destructive method High throughput (no sample preparation) Measures molecular interaction and concentration Easy to obtain digitized data for statistical analysis Independent on instruments (the same results from any apparatus)

5 Example : Identification of Differentiating Metabolites General procedure 1 H-NMR analysis Data processing Multivariate data analysis Signal sorting 2D-NMR measurements 1 Fractionation (if necessary) 2 Structure elucidation 2D-NMR measurements 1 J-resolved COSY, TOCSY, LR-COSY HSQC (HMQC) HMBC, HSQC-TOCSY Fractionation (chromatography) 2 HP-20, Sephadex LH-20, C18, Centrifugal partition chromatography Quantitative NMR for target metabolites

6 If you find interesting signal using multivariate data analysis PC ? Return to original spectrum Loading plot (δ 0 δ 10.0) 1 H-NMR spectrum Control Local-infected? 1 H-NMR (δ 6.0 δ 6.8)

7 Two dimensional (2D)-NMR 1D-NMR : Chemical shift and its intensity 2D-NMR : Chemical shift, other variables ( 13 C-chemical shift, or correlation), and its intesity 2D Chemical shift of 13 C-NMR 1D Chemical shift of 1 H-NMR Chemical shift of 1 H-NMR

8 COSY, TOCSY, HSQC, HMBC 1 H-NMR : basic identification, fingerprinting, further statistical analysis 13 C-NMR : second identification for quaternary carbon (e. g. carbonyl group) COSY, TOCSY : elucidation of carbohydrates and organic acids, amino acids HSQC : confirmation of 1 H-NMR elucidation, further statistical analysis for complex plant extract HMBC : confirmation of 1 H-NMR elucidation, elucidation of glycosides NOESY : spatial correlation (stereo chemistry of metabolites)

9 1 H-NMR: all information therein NMR always wants to give a lot of information for compounds! But it s up to you?

10 First approach Using Chemical Shift in 1 H-NMR

11 1 H-NMR for the Mixture of Tobacco 2' 3 N N H N CH 3 N

12 Flavonoids, Phenylpropanoids, Aromatic amino acids, Simple phenolics

13 Carbohydrates, Amino acids, Terpenes, Organic acids

14 Organic acids, Amino acids, Terpenes

15 Methyl of Terpenes (δ 0.5 δ 2.0)

16 Database? ACD, Chenomx, Biorad (KnowItall), AMIX But lack of plant secondary metabolites In-hose database

17 1 H-NMR spectrum of glucose β Η1 α Η1 Monosaccharide has two forms in water solution

18 1 H-NMR spectrum of rhamnose β Η6 HO HO H H 3 C H OH O H OH α Η6 H α Η1 β Η1

19 Differentiation of Diasteromers R 1 O O H R 2 OH H-2 of R- and S-form OH O R and S forms

20 1 H-NMR spectrum of secologanin H-3 H-3 H-3 H-3 has 1-3 signals depending ph (one signal in strong acid condition)

21 Second approach Using J-resolved for splitting pattern

22 How many signals do you need for elucidation? It depends on structures At least 7 phenylpropanoids At least 3 flavonoids Dimeric phenylpropanoids

23 1 H-NMR spectrum of ursolic acid Methyl signals are characteristics for terpenoids in NMR

24 2D J-Resolved : phenylpropanoid and flavonoid R R R COOR phenylpropanboids R R R R R flavonoids Brassica rapa leaves

25 2D J-Resolved :terpenoid HO 18 OH Nicotiana tabacum leaves

26 COSY H-2 H-6 H-5 H-8 H-6 3 Vitis vinifera leaves

27 Total Correlated Spectroscopy (TOCSY) Useful for amino acids and carbohydrates identification

28 TOCSY

29 TOCSY Mixing time is important : low mixing time results in less correlations (the same to COSY) 50 ms 80 ms

30 HSQC (or HMQC) for target 13 C signal Genista tenera leaves

31 HMBC: a wealth of information

32 Flavonoids from Genista tenera : HMBC H-1, C-8 H-1, C-7 H-1, C-8a H-1, C-7

33 Application of HSQC-TOCSY to metabolomics Indirectly, the information of 13 C chemical shift can be obtained using combined 2D-NMR (e.g. HSQC-TOCSY) 1: malic acid 2: aspartic acid Nicotiana plumbaginifolia cell lines

34 Structure Confirmation by Mass Spectrometry m/z m/z m/z

35 Application of SPE for flavonoids in Arabidopsis thaliana Total extract Sephadex LH-20 fraction tyrosine fumaric acid 1 H-NMR (δ 5.0 δ 8.5) 1 H-NMR (δ 5.0 δ 8.5) Sinapoyl malate 1 H-NMR (δ 5.0 δ 8.5) flavonoids R 1 = Glc, Rha, or Glc-Glc R 2 = H, OH 1 H-NMR (δ 5.0 δ 8.5)

36 Complex Spectra: - Projection of J-resolved Spectra - 1 H-NMR 2D-J-resolved Projection of J-resolved Less complex spectra Decoupled spectra like 13 C Higher resolution Better separation in further PCA

37 From a Metabolomids to a real System Biologist Elucidation of Metabolites Snapshot Function of Metabolites Film Metabolomics can just open chemical world of living organisms -First step to confirm chemical changes But there is still something missing -When or where (Time and Space) Fluxomics?

38 Thanks to Dr. Hye Kyong Kim: for Data Processing Dr. Federica Maltese: for flavonids work Mr. Ibrahim Abdelfreed El-Bayoumi: for Brassica spectra Mr. Muhammad Jahangir: for Brassica spectra Ms. Sanimah Simoh: for Brassica spectra Dr. Alexandre T. Cardoso Taketa: for Galphimia glauca work - Universidad Autónoma del Estado de Morelos, Morelos, Mexico