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History of Natural ProductChemistry and Bioanalytics

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Chemotaxonomy

• Each step inthe biosynthesis is catalysed by one enzyme

• Each enzyme is encoded by one gene

• Related species can biosynthesize the same or structurally close related compounds

ca. 35.000 genen bekend =

35.000 naturstoffen in een organisme !?

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Chemotaxonomy

O

MeO

MeO

OH

OMe

O

O

MeO

MeO

OMe

OMe

O

Arctigenin

Dimethylmatairesinol

O

MeO

HO

OH

OMe

O

O

OH

OMe

OO

O

O

OH

OMe

OO

O

OH

OMe

OHO

MeO

OO

MeO

HO

OH

OMe

O

OH

O

OH

OMe

OO

O

OH

O

OO

O

OO

O

OO

O

OMe

OMe

O

OO

O

OH

OMeMeO

Matairesinol

Hinokinin7'-Hydroxymatairesinol

4'Demethylyatein

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Chemotaxonomy

Different Mentha varieties= different constitutents in number and content

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Ecology

Why produces... Taxus brevifolia paclitaxel?Penicillium Penicillin?Cannabis sativa THC?Streptomyces doxorubicin?

OO

NH OH

HO

O

O

OH

O

O

O

O

O

O

O

OH

COH2

The plant constituent is not made for us!

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Chemical ecology

- communication- Atrackting insects- Protecting against predators- Against spreading plant competitors

(chemical war)- Protection against irradation (e.g. UV)

Chemical war

Juglans regens

Inhibition, allelopathic effect

Hydrojuglon-β-glucoside Hydrojuglon Juglon(not active) (active)

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Chemical ecology

- Tropisch regenwouden- Koraalriffen- Zuid Africa- Ecologische nichen

Knowledge about ecology support selection process

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Biodiversity

Role of Biodiversity in Medicine

• 1.75 million known species of animals, plants, fungi, & micro-organisms

• Estimated to be 10 million species on earth

• Each species contains 100’s -1,000’s of chemicals

• 1 billion - 1 trillion chemicals

Loss of Biodiversity

• ~ 100 species/day is lost

• Loss of potential new medicines

Biological Activity

Major Targets– Infectious Diseases–Cancers–Pesticides

Separation

Isolation

Identification

Extraction

Crude Extract

Extract Fractions

Pure Compounds

Biomass

Active Lead Compound

Determination of Biological Activity

• ecological strategy

• ethno-medicinal strategy

• environmental strategy

• taxonomical strategy

• random strategy

Collection Strategies

Extraction

Biomass

Extraction Methods

• Solvent extraction – at room temperature or higher– flowing or non-flowing

Crude Extract

• A complex mixture of unknown components

obtained from the biomass

• Separation of the mixture done by the

differences in physical or chemical properties

Separation

Extraction

Crude Extract

Biomass

Determination of Biological Activity

(in vitro)

Separation Methods

• Centrifugation• Distillation• Crystallization• Filtration• Chromatography

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Analytical Assays used in Pharmaceutical Industry Labs

for New Chemical Entities

Method 1990 1998 2000 2006

HPLC(UV &Fluorescence) 75% 50-60% 20% 2%

GC/MS12% 3% 2% 0

LC/MS/MS3% 40-50% 60-75% 98%

Immunoassay(ELISA/FPIA etc.) 10% 10% 10% 0

Chromatography- Basic requirements

• Stationary Phase

– paper, glass, plastic, gel

• Mobile Phase– water, other solvent

Gingko biloba Extract

24

Mass Spectrometers

• Separate and measures ions based on their mass-to-charge (m/z) ratio.

• Operate under high vacuum (keeps ions from bumping into gas molecules)

• Key specifications are resolution, mass measurement accuracy, and sensitivity.

• Several kinds exist: for bioanalysis, quadrupole,time-of-flight (TOF) and ion traps are most used.

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Ion source MS-

2MS-1

Mixture of ions

Single ion

Fragments

What is Tandem MS?

• Uses 2 (or more) mass analyzers in a single instrument– One purifies the analyte ion from a mixture

using a magnetic field.– The other analyzes fragments of the analyte

ion for identification and quantification.

26

Applications of Tandem MS

• Biotechnology & Pharmaceutical– To determine chemical structure of drugs and drug

metabolites.– Detection/quantification of impurities, drugs and

their metabolites in biological fluids and tissues.– High through-put drug screening– Analysis of liquid mixtures– Fingerprinting

• Nutraceuticals/herbal drugs/tracing source of natural products or drugs

• Clinical testing & Toxicology– inborn errors of metabolism, cancer, diabetes,

various poisons, drugs of abuse, etc.

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InletInlet DetectDetectMass

AnalyzeMass

AnalyzeIonizeIonize

MSMS

InletInlet FragmentFragmentMass AnalyzeMass

AnalyzeIonizeIonizeMass

AnalyzeMass

AnalyzeDetectDetect

MS1MS1 CollisionCell

CollisionCell

MS2MS2

MS/MSMS/MS

MS vs. MS/MS

GCHPLCCE

Separation Identification

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CH3COCH3CH3COCH3

Sample Inlet

Sample Inlet

CH3+COCH3CH3+COCH3

Ionization& Adsorption

of Excess Energy

Ionization& Adsorption

of Excess Energy

Mass AnalysisMass Analysis

CH3C+OCH3CH3C+OCH3

+COCH3+COCH3

+CH3+CH3

+COH+COH

Fragmentation(Dissociation)

Fragmentation(Dissociation)

DetectionDetection

Mass Spectrometry

29

Multidimensional Analyses

time

response

chromatogram

m/zm/z

m/z

30

Different Types of MS

• Tandem MS–Triple Quatrupole–Hybrid Instruments

• ESI-QTOF– Electrospray ionization source + quadrupole

mass filter + time-of-flight mass analyzer

• MALDI-QTOF– Matrix-assisted laser desorption ionization +

quadrupole + time-of-flight mass analyzer

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LC-MS/MS

32

Analytical Quadrupole

33

Quadrupole TheoryPre-filter Quadrupole Mass Filter Stable Trajectory

Unstable Trajectories

Only ions with the correct m/z values have stable trajectories within an RF/DC quadrupole field.Ions with unstable trajectories collide with the rods, or the walls of the vacuum chamber, and are neutralised.

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Tandem Quadrupole

Collision cellMS1 MS2

35

Components of Tandem Mass Spectrometer

CollisionCell

MassSpectrometer

MassSpectrometer

Detector

Ionization Source

ESIAPPIAPCIMALDI

ArgonXenon

QuatrupoleMagnetic Sector

QuatrupoleMagnetic SectorTime-of-flight

Collision cellMS1 MS2

36

Sample introduction

• Ion Souce– Transforms sample molecules to ions– Soft ionization

• Places positive or negative charge on the analyte without significantly fragmenting the analyte

• M+1 ion (or M-1 ion)• No need to volatilize• Down to fmol detection limits

– Atmospheric Pressure Ionization (API)• Electrospray• MALDI• APCI• APPI

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The Abbé Nollet experimented with electrified liquids in the 18th century !

He observed that when a person was connected to a high-voltage generator he/she would not bleed normally after cutting ...blood “sprayed” from the wound !

F. Lemière, LC•GC Europe “LC-MS Supplement”,December 2001, p29-35

The Macabre History of Electrospray

38J. Zelene, Phys. Rev., 10, 1-6 (1917)

The Electrospray Phenomenon

39

Ionization Source

40

Sample ConeOrifice = 400μmSample ConeOrifice = 400μm

Spraying NeedleSpraying Needle

Vacuum Isolation Valve

Vacuum Isolation Valve

Ionization Source

41

High voltage appliedto metal sheath (~4 kV)

Sample Inlet Nozzle(Lower Voltage)

Charged droplets

++

++

+

+

++

+ +++

++

+ +++ +

++

+++

+++

++++

+

++

+

+

+

+++

+++

+++

MH+

MH3+

MH2+

Pressure = 1 atmInner tube diam. = 100 um

Sample in solution

N2

N2 gas

Partialvacuum

Electrospray ionization:

Ion Sources make ions from sample molecules

ESI and Quadrupol

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SCANNING MODE: The first quadrupole mass analyzer is Scanning over a mass range. The collision cell and the second quadrupole mass analyzer allow all ions to pass to the detector.

SCANNING MODE: The first quadrupole mass analyzer is Scanning over a mass range. The collision cell and the second quadrupole mass analyzer allow all ions to pass to the detector.

MS1 MS2Collision

Cell

Scanning Rf only, pass all masses

Collision cellMS1 MS2

Mass Spectrum: Progesterone

200 220 240 260 280 300 320 340 360 380 400m/z0

100

%

315.1

316.1

[M+H]+

O

O

CH3

CH3CH3

Static (m/z 315.1) Scanning

The first quadrupole mass analyzer is fixed at the mass-to-charge ratio (m/z) of the precursor ion to be interrogated while the second quadrupole is Scanning over a user-defined mass range.

The first quadrupole mass analyzer is fixed at the mass-to-charge ratio (m/z) of the precursor ion to be interrogated while the second quadrupole is Scanning over a user-defined mass range.

Argon gasArgon gas

PrecursorPrecursorProductsProducts

Collision cellMS1 MS2

46

Collision induced dissociation

• Collision conditions (FRAGMENTATION) is controlled by altering:

– The collision energy (speed of the ions as they enter the cell)

– Number of collisions undertaken (collision gas pressure)

Argon gas

O

O

CH3

CH3CH3

Precursor ion Product ions

OCH

2

CH2

CH3

O

CH3

CH3

• In the collision cell, the TRANSLATIONAL ENERGY of the ions is converted to INTERNAL ENERGY.

Product Ion Spectrum: Progesterone

300 305 310 315 320 325 330m/z0

100

%

315.1

316.1

Mass Spectrum from MS1

100 125 150 175 200 225 250 275 300 325m/z0

100

%

109.097.0

Product ion spectrum from MS2Product ions

OCH

2

CH2

CH3

O

CH3

CH3

O

O

CH3

CH3CH3

Precursor ion

MALDI-TOF

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MALDI-TOF

49

Triple Quadrupole

50

Complex Mixture to Single Compound

?Active Compound

O

O

OH

OH

OH

OH

HO

OOH

OH

OH

Determine Molecular Structure

=

Bioactivity-Guided Separation

Plant Crude Extract

Chromatography

Pure Compound

Extraction

Extraction

Bioassay

Bioassay

Repeat

From Plant to Drug

O

O

OH

OH

OH

OH

HO

OOH

OH

OH

Plant Crude Extract

PureCompound

Molecular Structure

Biological Activity

=

Drug Development

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