Basics of Primary and Secondary Metabolites

Transcription

1 Basics of Primary and Secondary Metabolites March 27, 207 Young ae hoi atural Products Laboratory Institute of Biology, Leiden University Leiden, The etherlands Difficulty of at. Prod. Res.: Diversity of metabolites Metabolites in organisms - onstitutive metabolites : primary and secondary metabolites - on constitutive metabolites : phytoalexin - Exogenous metabolites : by other organism or chemicals about 250,000 natural products are known more than 3,500 metabolites in single plant about 4000 new ones are reported every year about 5% of all plants have been studied to some extend for their constituents

2 ADES Workshop - The st day (Introduction) atural Products Research: What to do and Where to go? Green Technology: DES, ILs and SFE Basic Phytochemistry - The 2nd day (Application of ADES) Sample Preparation and Analysis Application of ADES to atural Products Extraction - The 3rd day (Practice) ADES extraction of Flavonoid from Sophora japonica flowers The st day atural Products Research Green Technology Basic Phytochemistry ow, you have samples for metabolomics! 2

3 Basics of plant metabolites The st step of research to know about metabolites present in living organisms hemical characteristics of group of metabolites (carbohydrates, amino acids, flavonoids, terpenoids, alkaloids ) detected by diverse analytical methods (UV, MS, MR) ommon metabolites (usual suspect) detected in plants Exceptional metabolites which show different analytical features from others Are you afraid of huge number of metabolites? Plant Metabolites Glucose Tannin Phosphenol pyruvic acid Erythrose-4- phosphate Shikimic acid Aromatic amino acid Phenyl propanoid Lignan, Lignin Pyruvic acid Krebs cycle Aliphatic amino acid Acetyl oa Malonyl oa MEP xaloacetic acid a-ketoglutaric acid Succinyl oa Purine, Pyrimidine d-aminolevulinic acid Flavonoid Alkaloid Peptide Terpenoid Steroid arotenoid Fatty acid Prostanoid Aliphatic and aromatic polyketide ucleotide, ucleoside holine, Porphine Quinone 3

4 Fatty Acid and Lipid () Simple and complex Simple lipid: fatty acid, glyceride (ester of fatty acid and glycerol), wax (ester of higher fatty acid and higher alcohol) omplexed: glycolipid (sphingo, glycero), phospholipid (sphingo, glycero) o. of carbon atom 8:2 (9c, 2c) o. of double bonds Saturated Position of double bonds Stereochemistry of double bonds (c=cis/z; t=trans/e) butyric 3 ( 2 ) 2 (4:0) stearic 3 ( 2 ) 6 (8:0) caproic 3 ( 2 ) 4 (6:0) arachidic 3 ( 2 ) 8 (20:0) caprylic 3 ( 2 ) 6 (8:0) behenic 3 ( 2 ) 20 (22:0) capric 3 ( 2 ) 8 (0:0) lignoceric 3 ( 2 ) 22 (24:0) lauric 3 ( 2 ) 0 (2:0) cerotic 3 ( 2 ) 24 (26:0) myristic 3 ( 2 ) 2 (4:0) montanic 3 ( 2 ) 26 (28:0) palmitic 3 ( 2 ) 4 (6:0) melissic 3 ( 2 ) 28 (30:0) Fatty Acid and Lipid (2) palmitoleic oleic cis-vaccenic linoleic a-linolenic g-linolenic gadoleic arachidonic eicosapentanoic (EPA) erucic docosapentaenoic (DPA) docosahexaenoic (DA) nervonic Abundant in the seeds Unsaturated holine, a-linolenic acid 3 ( 2 ) 5 =( 2 ) 7 Difficult identify each lipid by MR -> G-MS 3 ( 2 ) 7 =( 2 ) 7 3 ( 2 ) 5 =( 2 ) 9 3 ( 2 ) 4 = 2 =( 2 ) = 2 = 2 = ( 2 ) 7 3 ( 2 ) 4 = 2 = 2 = ( 2 ) 4 3 ( 2 ) 9 =( 2 ) 7 3 ( 2 ) 4 = 2 = 2 = 2 =( 2 ) = 2 = 2 = 2 = 2 =( 2 ) 3 3 ( 2 ) 7 =( 2 ) 3 2 = 2 = 2 = 2 = 2 =( 2 ) = 2 = 2 = 2 = 2 = 2 =( 2 ) 2 3 ( 2 ) 7 =( 2 ) 3 4

5 -MR spectrum of a-linolenic acid Terminal 3 might be a characteristic in MR -MR spectrum of glycerol moiety of lipid 2 R 2 Most fatty acids exist as conjugated form with glycerol 5

6 G analysis of fatty acids (methylation): lipidomics. Add ml of 0.5 M a solution (0 g/500 ml in Me) to extract 2. React for 30 min at 75 o 3. Transfer to 0 ml test tube 4. Extract with n-hexane (2 ml x 3) and transfer to 25 ml evaporating flask 5. Evaporate n-hexane extract 6. Add 0.5 ml of BF 3 in Me and transfer to.5 ml-glass vial 7. React for 20 min at 75 o 8. Add 0.5 ml of n-hexane 9. Injection of µl of n-hexane solution to G 0. G condition is the same to nonpolar metabolites analysis arbohydrate () Polyhydroxy aldehyde or polyhydroxy ketone Stereoisomer having n ( 2 ) n Aldose (Aldehyde functional group), Ketose (Ketone functional group) Pentose: 5, exose: 6 Sugars - Monosaccharides (aldoses and ketoses) Trioses, Tetroses, exoses - ligosaccharides Disaccharides, Trisaccharides - Sugar derivatives Alcohols, Acids, Esters, Glycosides - Polysaccharides (glycans) exosans (Glucans, Fructans, Galactans, Mannans, Glucomannans, Galactomannans) Pentosans (Xylans, Arabans) Glycouronans (Glucouranans, Galactouronans) 6

7 arbohydrate (2) D-Glucose D-Mannose D-Arabinose D-fructose 2 2 a-d-glucopyranose a-d-glucofuranose b-d-fructofuranosyl-(2 )-a-d-glucopyroanoside (Sucrose) arbohydrate (3) D- and L- form: Fischer-Rosanoff s rule * 2 (+)-D-glyceraldehyde 2 D-glucose : natural form 7

8 arbohydrate (4) Anomeric configuration : a- and b form equitorial axial a-d-glucopyranose b-d-glucopyranose a form- 60 o : J=2-3 z b form- 80 o : J=7-8 z Ratio of glucose (a:b)= 36:64 -MR spectrum of glucose b- a- Monosaccharide has two forms in water solution 8

9 -MR spectrum of rhamnose b-6 3 a-6 a- b- -MR spectrum of sucrose If at - position attach with other molecules there is only one form 9

10 arbohydrate (5) Low sensitivity in L-MS (derivatization needed for increasing sensitivity) Good sensitivity and resolution in G-MS after TMS-derivatization Several signals in G-MS analysis ligosaccharides make MR signals broadening In MR analysis PMG pulse needed -MR spectrum of cannabis cell line ormal cell line Polysaccharide-treated ligo- or polysaccharide-rich sample cause signal broadening 0

11 Amino acid () Alanine (Ala) 3 (d.48, d, J=7.2) Leucine (Leu) Glutamic acid (Glu) 2 2 Serine (Ser) Arginine (Arg) Glutamine (Gln) Threonine (Thr) (d.32, d, J=6.6) 2 Asparagine (Asn) 2 S 3 2 Glycine (Gly) Lysine (Lys) Methionine (Met) Tryptophan (Trp) Progress in uclear Magnetic Resonance Spectroscopy, 996, 28: Amino acid (2) Aspartic acid (Asp) istidine (is) Phenylalanine (Phe) Tyrosine (Tyr) S 2 3 ysteine (ys) 3 Isoleucine (Ile) Proline (Pro) 3 Valine (Val) (d 0.98,.06, d, J=6.6)

12 ucleic acid Adenine Guanine ytosine Thymine -MR spectrum of adenine and cytosine 2 Adenine 2 ytosine Signals are very p sensitive 2

13 rganic acid 3 itramalic acid itric acid Fumaric acid Isocitric acid Malic acid Succinic acid Tartaric acid -MR spectrum of citric acid and malic acid Malic acid itric acid p effect: nalic acid in plants oncentration effect: citric acid and malic acid p 6 (acetate) 0 mg/ml p 6 (phosphate) p 7 (phosphate) 5 mg/ml p 8 (phosphate) mg/ml -MR spectra of Senecio jacobea (d 2.5 d 3.0) -MR spectra of malic- and citric acid (d 2.5 d 3.0) 3

14 Monoterpenoid (0) omponent of plant essential oil G-MS targeted analysis PP nerylprophosphate menthane limonene a-pinene carnene thujene -MR spectrum of secologanin has -3 signals depending p (one signal in strong acid condition) 4

15 Sesquiterpenoid (5) Most diverse group of compounds (more than 00 skeletons) ytotoxic: sesquiterpene lactone Phytoalexins in Solanaceae o o o o o o o Ac o o o o o Diterpenoid (20) Labdane leordane Pimarane Isopimarane Abietane Podocarpane Totarane assane Kaurane Beyerane Gibberellane Atisane stevioside Aconane Taxane taxol Specific in some plant family (Abietane diterpene: Labiate) 5

16 Triterpenoid (30) Generally present in plant/tetracyclic, pentacyclic leanane: oleanolic acid, b-amyrin Ursolic acid: ursolic acid, a-amyrin Glycoside (3, 28); saponin leanane Ursane Friedelane Prostane Lanostane ycloartane urcurbitane Steroid (27) Sterol: steroid alcohol (3-) b-sitosterol: most abundant phytosterol mostly present with stigmasterol, campesterol steroid glycoside: saponin holesterol Stigmasterol Ergosterol b-sitosterol 6

17 arotenoid (40) 8 isoprenoid color (yellow, red) trans (natural form)/cis (light) abundant in carrot b-carotene Analysis of terpenoid and steroid MS-based method is more powerful than MR for individual terpenoid or steroid G-MS for aglycones and L-MS for glycosides Methyl and anomeric proton of sugar are characteristic features in MR spectra Many terpenoids exist as glycosides triterpenoids and steroids glycosides : saponin 7

18 ESI-MS spectrum of stevioside Intensity x [M--Glc] - -Glc-Glc Glc [M-] m/z MW peak of glycoside is not always base (the highest) signal in ESI or API API-MS spectrum of glycyrrhizin x [M-glc-glc] [M-glc] R m/z R= Glucose-Glucose Sometimes MW peak of glycosides can not be detected 8

19 -MR spectrum of ursolic acid Methyl signals are characteristics for terpenoids in MR -MR spectrum of a saponin * * * Anomeric proton of sugars in saponin 9

20 Phenylpropanoid 6-3 (phenyl + propane) 3 : carboxyl acid, aldehyde, alcohol or olefin igh vapor pressure: Essential oil hlorogenic acid : Ester of quinic acid and caffeic acid innamic acid : innamonum, volitile oniferin, syringin, anethole, eugenol, safrole, myristicin R R 3' ' 7' Me R 2 5' 8' R=, safrole R=ome, myristicin eugenol R = 3, R 2 =, Ferulic acid R =, R 2 =, affeic acid chlorogenic acid R =, R 2 =, oumaric acid R = 3, R 2 = 3, Sinapic acid -MR spectrum of caffeic acid MR: most powerful method for structure elucidation Large coupling constant (6z): trans form Mostly conjugated with organic acid or sugars

21 -MR spectrum of dicaffeoyl quinic acid isomerization from trans forms to cis forms artifact by light, solvent, temperature trans -8 cis -8 -MR spectrum of a stilbenoid (resveratol) Stilbenoid: 6-2 The same coupling constant but higher chemical shift compared to phenylpropanoid 2

22 Lignan xidative coupling of 6-3 : 8 Generally b coupling podophyllotoxin : Podophyllum, Juniperus species Precursor of etoposide, tenuposide gomisin: Schisandra species, hepatoprotective enterodiol, enterolactone: from human and animal,980 pinoresinol, sesamin: polarity (+) (Sesamum indicum) neolignan: not b coupling, magnol, honokiol norlignan: 7 Me Me Me Me R Me gomisin A R 2 R 3 Me Me Me R =, R 2 =, R 3 =Me, podphyllotoxin R =, R 2 =R 3 =, a-peltatin R =, R 2 =, R 3 =Me, b=peltatin sesamin oumarin 2--Benzopyran-2-one Umbeliferae, Ranunculaceae, Leguminosae, ompositae simple coumarin, furanocoumarin, pyroanocoumarin, biscoumarin pen cycle by alkali dicoumarol: Medicago sativa, Melilotus officinalis inhibition vitamin K inhibition coumarin novobiocin (gram negative infection, Streptomyces niveus), umbelliferone, scopoletin Me Me 2 Me Umbeliferone dicoumarol novobiocin 22

23 -MR spectrum of umbelliferone (d, J= 9.5 z) -3 (d, J= 9.5 z) -3 and -4 are characteristics in MR Flavonoid (6-3-6) B A b 5 a 6 flavone flavonol chalcone flavanone + dihydroflavonol 5 isoflavone R 2 R R =, R 2 =, catechin anthocyanidin R =R 2 =, leucoanthocyanidin neoflavonoid aurone 23

24 UV spectrum of flavone & flavonol nm region Band I: nm (B ring) Band II: nm (A ring). A ring: more group, Band II bathochromic shift 2. B ring: more group, Band I bathochromic shift A 5 Benzoyl B innamoyl Flavone A-ring pattern Band II flavone flavone ,7- flavone 5,7 268 baicalein 5,6,7 274 norwogonin 5,7,8 28 Flavonol B-ring pattern Band I galangin kaempferol quercetin 3,4 370 myricetin 3,4,5 374 Flavonoid type Band I Flavone nm Flavonol (3-) nm Flavonol (sub. 3-) nm UV shift reagent for flavonoid. ame: 3- (band I nm bathochromic shift) 4 - (band I nm shift, intensity decrease) 2. aac: 7- (band II 5-20 bathochromic shift) 4 -, no 3,7- (band I shoulder) 3. aac/ 3 B 3 : B ring ortho di (band I 2-30nm bathochromic shift) 4. All 3 & All 3 /l : 3 or 5- (acid stable complex), ortho-di (acid unstable complex) -> B ring di: All 3 & All 3 /l nm shift tri 20 nm shift 5-: nm 3-: 60 nm 3,5-di: nm (Ref: Systemic identification of Flavonoids, T.J.Mabry, K.R.Markham & M.B.Thomas, Springer-Verlag, 970) 24

25 -MR spectrum of flavonoid A-ring proton: -6, -8 5,7-di: ppm, d, J=2.5 z compound ,7-di flavone, isoflavone , 7-glycosyl flavone, isoflavone ,7-di flavanone, dihydroflavonol , 7-glycosyl flavanone, dihydroflavonol B-ring proton: downfield than A-ring, ppm -ring proton: ppm -MR spectrum of quercetin and -8 are characteristics in MR 25

26 -MR spectrum of naringenin and naringin Diasteromeric exchange in naringin - aringenin might have two enantiomers which are not separated by MR - Sugar attachment change enantiomers to diastereomers separted by MR (aringin) R 2-2 of naringenin -2 of naringin R=, naringenin R=rhamnoglucose (neohesperidose), naringin -MR spectrum of vitexin Rotamers in -glycoside - Sugar attachment on -8 makes rotation of B-ring slower 26

27 Quinone Animal, plant, microrganismn Anthraquinone, naphthoquinone, benzoquinone Ubiquinone, vitamin K, mitomycin, sennoside, alkannin, alizarin Diverse color:,4-quinone (yellow),,2-quinone (red), makes color darker m 8.m m 8.07m 6.95 Benzoquinone aphtoquinone Anthraquinone Emodin -MR spectrum of tectoquinone , -8-6,

28 Alkaloid () eterocyclic itrogen containing compound from plant, Basic, Intensive biological activity There is no exact definition ow, just nitrogen containing natural products Very plant-specific Pyridine alkaloid icotine 5% in dried leaves of icotiana tabacum Insectcidal Anabasine rnithine Lysine Glycine, Aspartic acid icotine Glycine, Aspartic acid Anabasine Alkaloid (2) Tropane alkaloid Atropine: 833, Solanaceane Atropa belladonna, isolated by Mein ocaine: from oca leaves Scopolamine Atropine Scopolamine ocaine 28

29 Alkaloid (3) Pyrrolizidine alkaloid ytotoxic, carcinogenic ompositae senecio species Senecionine, monocrotaline Indolizidine alkaloid minor Elaeocarpine, tylophorine Quinolizidine alkaloid Lupinane group alkaloid Matrine, nuphridine, sparteine Benzylamine alkaloid Ephedrine group alkaloid 887, agai isolated from Ephedra species many enantiomer 3 3 Ephedrine Alkaloid (4) Isoquinoline alkaloid Morphine, berberine Mostly benzylisoquinoline Indole alkaloid Biosynthesized from Tryptophan, secologanin Reserpine (Rauwolfia serpentiana) Yohimbine, vincristine, vinblastine Piperidine alkaloids Arecoline Areca catechu Pyrrolizidine Alkaloid Widespread occurrence Diverse range of biological activities epatotoxic

30 -MR spectrum of nicotine 3 Signals are very p sensitive -MR spectrum of trigonelline 3 ommon in Solanaceae plants 30

31 -MR spectrum of tryptophan (for indole alkaloids) 2 2 Typical pattern of indole moiety -MR spectrum of arecoline 3 3 igher chemical shift because of ketone group 3

32 Glucosinolates Mostly occur in ruciferae (Arabidopsis, Brassica) b-thioglucoside--hydroxysulfates R S Glc S 3 - Sulfur-linked b-d-glucopyranose R = side chain aliphatic (w-methylthioalkyl) most abundant aromatic (benzyl) heterocyclic (indole) -MR spectrum of sinigrin S S of glucosinolate has big coupling constants (0 z) 32

33 yanogenic glycoside Producing gas by treatment of dil. acid or alkali Mostly monoglucoside Diglycoside: amygdalin, vicianin, lucumin, linustatin, neolinustatin containing gentiobiose [glucose-glucose ( 6)] 2 Amygdalin Tomorrow Sample Preparation and Analysis Application of ADES to atural Products Research Preparation of ADES 33