Application of 2D-NMR spectroscopy to metabolomics and ... - CSC

Application of 2D-NMR spectroscopyto metabolomics and identification of metabolitesNovember 10 – 14, 2008Young Hae ChoiDivision of Pharmacognosy, Section MetabolomicsInstitute of Biology, Leiden UniversityLeiden, Then Netherlands

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 signalsThen, what is the next step?

How can we elucidate the selected metabolites?0.20.1?w*c[2]-0.0-0.1-0.2-0.10 -0.05 -0.00 0.05 0.10w*c[1]

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)

Example : Identification of Differentiating MetabolitesGeneral procedure1H-NMR analysisData processingMultivariate data analysisSignal sorting2D-NMR measurements 1Fractionation (if necessary) 2Structure elucidation2D-NMR measurements 1J-resolvedCOSY, TOCSY, LR-COSYHSQC (HMQC)HMBC, HSQC-TOCSYFractionation (chromatography) 2HP-20, Sephadex LH-20, C18,Centrifugal partition chromatographyQuantitative NMR for target metabolites

If you find interesting signal using multivariate data analysisPC30 .2 00 .1 00 .0 0-0 .1 0?-0 .2 0-0 .3 02 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0Return tooriginal spectrumLoading plot (δ 0 – δ 10.0)1H-NMR spectrumControlLocal-infected?1H-NMR (δ 6.0 – δ 6.8)

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 intesity2DChemical shift of 13 C-NMR1DChemical shift of 1 H-NMRChemical shift of 1 H-NMR

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 organicacids, amino acids• HSQC :confirmation of 1 H-NMR elucidation,further statistical analysis for complex plantextract• HMBC :confirmation of 1 H-NMR elucidation,elucidation of glycosides• NOESY :spatial correlation(stereo chemistry of metabolites)

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

First approachUsing Chemical Shift in 1 H-NMR

1H-NMR for the Mixture of Tobacco2'3NNHNCH 3N

Flavonoids, Phenylpropanoids, Aromatic amino acids, Simple phenolics …

Carbohydrates, Amino acids, Terpenes, Organic acids …

Organic acids, Amino acids, Terpenes …

Methyl of Terpenes (δ 0.5 – δ 2.0)

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

1H-NMR spectrum of glucoseβ−Η1α−Η1Monosaccharide has two forms in water solution

1H-NMR spectrum of rhamnoseβ−Η6HOHOHH 3 CHOHOHOHα−Η6Hα−Η1β−Η1

Differentiation of DiasteromersR 1 OOHR 2OHH-2 of R- and S-formOHOR and S forms

1H-NMR spectrum of secologaninH-3H-3H-3H-3 has 1-3 signals depending pH (one signal in strong acid condition)

Second approachUsing J-resolved for splitting pattern

How many signals do you need for elucidation?It depends on structuresAt least 7 phenylpropanoidsAt least 3 flavonoidsDimeric phenylpropanoids

1H-NMR spectrum of ursolic acidMethyl signals are characteristics for terpenoids in NMR

2D J-Resolved : phenylpropanoid and flavonoidRRRCOORphenylpropanboidsRRRRRflavonoidsBrassica rapa leaves

2D J-Resolved :terpenoidHO18OH641619111151720Nicotiana tabacum leaves

COSYH-2’H-6’ H-5’ H-8 H-63Vitis vinifera leaves

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

TOCSY

TOCSYMixing time is important : low mixing time resultsin less correlations (the same to COSY)50 ms 80 ms

HSQC (or HMQC) for target 13 C signalGenista tenera leaves

HMBC: a wealth of information

Flavonoids from Genista tenera : HMBCH-1”, C-8H-1”, C-7H-1”, C-8aH-1”, C-7

Application of HSQC-TOCSY to metabolomics• Indirectly, the information of 13 C chemical shift can be obtainedusing combined 2D-NMR (e.g. HSQC-TOCSY)1: malic acid2: aspartic acid1 12222112 2Nicotiana plumbaginifolia cell lines

Structure Confirmation by Mass Spectrometry295.0325.0200 250 300 350 400 450 500 m/z279.1339.1200 250 300 350 400 450 500 m/z309.1200 250 300 350 400 450 500 m/z

Application of SPE for flavonoids in Arabidopsis thalianaTotal extractSephadex LH-20 fractiontyrosinefumaric acid1H-NMR (δ 5.0 – δ 8.5)1H-NMR (δ 5.0 – δ 8.5)Sinapoyl malate1H-NMR (δ 5.0 – δ 8.5)flavonoidsR 1= Glc, Rha, or Glc-GlcR 2= H, OH1H-NMR (δ 5.0 – δ 8.5)

Complex Spectra: - Projection of J-resolved Spectra -1H-NMR2D-J-resolvedProjection of J-resolved• Less complex spectra• Decoupled spectra like 13 C• Higher resolution• Better separation in further PCA

From a ‘Metabolomids’ to a real ‘System Biologist’Elucidation of MetabolitesSnapshotFunction of MetabolitesFilmMetabolomics can just open chemical world of living organisms-First step to confirm chemical changesBut there is still something missing-When or where (Time and Space)Fluxomics?

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