IR4.

Course : Natural Products Biotechnology

Course : SY32402
Credit Hour :2
Lecture : 2 hours/week
Lecturers : Prof. Dr. Jualang Azlan Gansau
Week Lecture Topic Remarks
1 Primary and Secondary Metabolism; Introduction to Natural Products JAG
Chemistry
2-3 Classes and Functions of Secondary Products JAG
4 Biodiversity of Plant, Bacteria and Fungi: Sources of Natural Products JAG
5 Screening for Natural Product Activity JAG
6-7 Modern Methods of Secondary Product Isolation and Analysis JAG
8 Compounds of Microbial Origin SAS
9 Products from Basidiomycetes SAS
10 Secondary Products from Plant Cell Cultures SAS
11 Genomics and Proteomics of Natural Products SAS
12 Natural Products and Drug Discovery SAS
13 Large-Scale Production of Secondary Metabolites SAS
14 Recent Developments in Biosynthesis, Production and Biotechnology SAS
Applications of Natural Products

Assessment Methods and Marks Distribution:

Component Weight Lecturer in charge
Quiz/Assignment 40% JAG/SAS
Mid-Term Exam 20% JAG
Final Exam 40% SAS
Total 100%
 Lectures 6 -7
• Methods of SP Isolation and Analysis
 Microbes:
Isolation, identification and
culturing of
Plants/animals/aquatic
organisms:
• Identification /and
conservation (in situ/ex
situ)
• Ethnobotany vs.
random sampling

Collection, Storage, and Vouchering of Plants

• Collection of Plants in the Field: Do’s and Don’ts
• Storage of Plants at Low Temperatures
• Vouchering of Plants Collected in the Field
1. Keep a Good Logbook
2. Photographic Records of Plants Collected
3. Preparation of Dry Specimens
4. Living Plant Specimens
 Traditional, Analytical, and Preparative Separations of Natural
Products

1. Traditional Methods
• The use of hot water extracts to make teas or natural plant dyes (e.g., the
monoterpenes from mints to make mint tea).
• Salves and decoctions are often made from a single plant source (e.g., the oleoresin
terpenes from pitch of balsam fir used directly to treat burns).
• Mixtures of plants are also used(e.g., many commercial herbal teas that utilize
chamomile, mint, bee balm, lavender, and other plants or upregulators of the
immune system, such as Echinacea and goldseal).

2. Contemporary Methods
• Modern methods of isolation of natural products, utilize principles of extraction
that are based on the polarity (relative solubility in organic solvents), solubility
in water, and various alterational solubilities based on salts and pH (relative
acidity or alkalinity)
 A. Collection, Storage, and Vouchering of Plants

1. Collection of Plants in the Field: Do’s and Don’ts

• Wear field clothes, raincoat, insect repellent.
• Notepad (waterproof paper is excellent) and pencil to record information about the
collecting site location, soil conditions, ecological habitat, date of collection, plant
identity, and who collected the plant(s).
• A portable global positioning system (GPS) unit/altimeter for recording geographical
positions and elevations.
• Take soil samples from each site so as to get information later on soil nutrients, soil
pH, and soil type where each plant grows.
• Take along a pocket-size field guide (with photos, drawings, and good, usable
identification keys) to the local flora and a hand lens to help you identify each plant.
• A good-quality digital camera to keep a photographic record of each plant.
• If you are collecting live plants on a short trip (1 or 2 h) and have access to liquid
nitrogen, take along a large thermos full of liquid nitrogen and some aluminum foil.
The thermos should have a good handle on it to avoid spilling liquid nitrogen on your
skin. The samples can be collected, wrapped in aluminum foil pockets, labeled with
pen, and dropped into the liquid nitrogen. This is by far the best method for
preserving compounds within the plant that may be easily degraded or chemically
altered. (Not to close the cap on the thermos too tightly, or the container will
explode)
• If you are collecting live plants on a longer trip, or you do not have access to liquid
nitrogen, take some Ziploc® plastic bags of various sizes in which to put the samples after
collecting plants on site. Such bags keep the plant materials alive until they can be
frozen. Slips of recycled waterproof paper or flagging tape are good to have so you can
place notes on plant identity with your collected specimens that match up with your field
notes about the respective collections.
• It is important to get representative samples of all parts available: roots, vegetative
shoots, bark from stems (if woody plant), flowers, fruits, and seeds (if mature).
• When collecting plants in the field, do not take every last plant in the population,
especially if the plant is rare, threatened, or endangered.
• In the process of collecting herbaceous perennial plants (plants that come from the same
mother plant year after year), leave some of the original plant intact where it is growing
so that it can reproduce during the current and following years. Many of these plants
take years to produce even a small amount of new biomass every year.
• If you are collecting mushrooms or puffballs in the field, wrap the fruiting bodies in wax
paper and place them in a collecting basket or other suitable container where they will
not become squashed. This will help for later identification and for making spore prints
from the fruiting bodies.
• Do, thank the plant for providing you with substrate for your extracts. While this may not
seem important to some, we all do this in various ways when we collect plants in the
home garden for food or for aesthetic purposes, or when we collect wild edible plants in
the field.
2. Storage of Plants at Low Temperatures

• The best way to do this is by quickly freezing the tissue in liquid nitrogen. Such
samples can then be stored long term in a –20 °C or –80 °C commercial freezer. This
technique prevents almost all degradation of the plant material or any enzymatic
changes that alter or degrade naturally occurring metabolites. Such techniques are
especially important for molecular biological studies, because once a plant is
damaged by being picked, it undergoes a drastic defense response that can greatly
alter the composition of plant compounds, such as RNA and proteins.

• Natural drying of the plant material can also be done if yield of metabolites is not
critical or dried at controlled temperature (40 °C)
3. Vouchering of Plants Collected in the Field

3.1. Keep a Good Logbook

• Always keep a logbook of the plants and the tissues that were collected and frozen.
[collecting site location, soil conditions, ecological habitat, environmental
conditions, as well as the date of collection, plant identity, and who collected the
plant]
• The environmental conditions help control the biosynthesis of various compounds;
so, a record of the collection conditions may help to avoid confusion when
comparing multiple collections of the same type of plant.
• For similar reasons, we recommend taking soil samples and later recording
information on soil nutrients, soil pH, and soil type where each plant grows. It is also
important that proper labels be kept on samples stored in the freezer. Slips of paper
work well inside Ziploc plastic bags at –20 °C.
3.2. Photographic Records of Plants Collected
It is often desirable to keep photographic records of plants that were collected, either
for plant identification or for re-collection of a particular plant. We recommend taking
digital photographs of the live plant in the field in addition to performing high-
resolution scans (using a flatbed computer scanner) back in the laboratory.
3.3. Preparation of Dry Specimens
Dried plant specimens are prepared in order to have them available at any time as voucher
specimens representing typical plants that were collected in the field and used for plant
extracts. They are also called herbarium specimens. Dried plant specimens are prepared in
the traditional way by placing the collected plant between single newspaper sheets and
then placing this in a sandwich consisting of a dry blotter above and below the newspaper
sheets. A piece of corrugated cardboard (with air spaces present) is then placed above and
below each blotter. Successive sandwiches are placed atop one another and then
compressed between two wood-slatted frames and tied together tightly with straps. The
entire assembly is then placed upright on its side over a heat source, such as a radiator or
a plant drier, with the heat on a moderate temperature (e.g., 35 to 40 °C). The specimens
are allowed to dry this way for 48 h or longer. If plant specimens are very high in water
content, it is a good idea to replace the blotters with dry ones in the middle of the drying
process.
3.4. Living Plant Specimens
In our experience, we found it to be a good idea to collect seeds and living
specimens of the plants that are to be used for the extraction of natural
products. We do this in order to have the living plants on hand (e.g., medicinal
plants, dye plants, or culinary herb garden or in a greenhouse) to be used for
later extractions or experimental treatments to enhance metabolite
biosynthesis; this is especially important when access to the original collecting
site is not possible or convenient.

In situ
Ex situ germplasm
In vitro
 4. Grinding and Extraction Protocols

4.1. General Extraction Protocols for Biologically Important Compounds

It is important to note here that many compounds are rapidly degraded once the
extraction process has begun. For example, there are many proteases located inside of
each cell. Normally, these proteases are sequestered into specific regions of the cell,
where they do little damage to important proteins. However, once the cells are ruptured,
the proteases are released, and they begin to destroy potentially important proteins. To
maximally inhibit those proteases, it is important to keep the temperature low (from 0 to
4 °C), and protease inhibitors are commonly added to lysis solutions or buffers used for
protein/enzyme isolation. There is also a wide variety of different solutions used for
compound extraction. It is strongly recommended that specific buffer conditions be
maintained during the extraction and purification of water-soluble compounds because
the structures or activities of many compounds are sensitive to pH changes. Using the
example of proteins, it is essential to use proper buffering, because some proteins or
enzymes may be degraded or lose their enzymatic activities. Such buffer conditions need
to be optimized for specific cells, tissues, and protein types. In addition, extraction and
purification of membrane proteins usually requires detergents to help release them from
the membranes, and many proteins require specific reducing agents, salts, and metal ions
to remain active.
4.1.1. Rupturing Bacterial Cells

Bacterial cells, like plant cells, have very strong cell walls that often make them difficult to
extract. Modern protocols for the extraction of water-soluble compounds make use of
specific enzymes that are able to cleave the protein components of the bacterial cell wall.
In such protocols, cell cultures are pelletized by spinning them in a centrifuge. The pellet is
then resuspended in lysis buffer, to which an enzyme, such as lysozyme , is added. After an
incubation period, the suspension becomes very viscous due to large amounts of released
DNA from inside the cells. This can be reduced through the addition of other enzymes, such
as DNase I, to chop up the DNA and reduce the viscosity of the final extract. Traditional
bacterial extractions make use of a French press so as to break open the cell walls. This
involves using a heavy cylinder with high pressure applied to a piston that compresses the
cells in an extraction solution into a successively smaller volume within the free cylinder. As
the cells leave the cylinder, the rapid drop in pressure causes the cells to lyse and release
their components.
4.1.2. Rupturing Plant Cell Suspension Cultures

Like the bacterial procedures discussed above, similar procedures are used for plant cells
grown in suspension culture or for plant callus tissue. Specific enzymes, such as cellulase
Or pectinase, can be added to lyse the walls of cells in aqueous solutions, or the cells can
be passed through a French press. Many plant cells grown in culture can simply be
ruptured with a glass tissue homogenizer. However, another good method for disrupting
cells in suspension involves the use of special equipment called a sonicator. In this case,
the sonicator releases repeated high-frequency pulses of ultrasonic vibrations that
rupture the cell membranes. For heat-sensitive compounds, such as proteins or RNA, it is
especially important to keep the sample on ice, because the sonicator vibrations
generate a great deal of heat. This is why repeated pulses are used instead of a
continuous discharge; this procedure allows the sample to cool between pulses. Many of
these methods, however, assume that water-soluble compounds are being extracted,
and they may not be applicable to many compounds.
 Techniques of isolation and analysis in the
natural product research
• Sample preparation, isolation techniques
• Identification methods, structure elucidation
• Determination of absolute configuration
(stereochemistry) of natural products
in microscale
• Biological methods of testing activity
of isolated natural products
 Good knowledge of the life cycle
of the selected organism
• does it produce chemical signals?
• when does the production reach its maximum?
• which organ/tissue/gland produces the signal?

female moth calling

 Sample preparation
• hydrodistillation (essential oils)
• solvent extraction (universal)
• „head-space“ techniques (volatile compounds)
• solid-phase microextraction, SPME (volatile
compounds)
• solid sample injection (insect glands)
 Hydrodistillation, steam distillation
• Clevenger apparatus
• plant material cut in pieces,
boils in water
• essential oil distil off together
with steam, forming the upper
layer
 Hydrodistillation, steam distillation
_____________________________________________________
Advantage Disadvantage
_____________________________________________________
large scale possible danger of artefacts (oxidation)
suitable for plants, not insects
_____________________________________________________
 Solvent extraction
_____________________________________________________
Advantage Disadvantage
_____________________________________________________
simple presence of balast compounds
possible to repeat pure solvents needed
analysis sometimes a low concentration
(amounts produced in the moment
of extraction)
_____________________________________________________
 Supercritical fluid extraction
• fluid, which has the ability
of dissolution at the supercritical pressure and the
supercritical temperature
• most often used CO2
• higher dissolving
capability for various
substances
• extraction is fast
 Supercritical fluid extraction
_____________________________________________________
Advantage Disadvantage
_____________________________________________________
good extraction potential expensive apparatus
room temperature CO2 is a greenhouse gas
reuse of solvent
CO2 - green chemistry
safe in food processing
cheap and easy to handle
large scale possible
_____________________________________________________
 „Head-space“ techniques
• static
• dynamic
◆ Trapping volatiles:

◆ sorbents:
◆ charcoal
◆ Porapak Q
◆ Tenax

◆ elution with solvent

◆ freezing out
„Head-space“ techniques
 „Head-space“ techniques
___________________________________________________________________________

Advantage Disadvantage
___________________________________________________________________________
accurate composition apparatus needed
higher concentration pure solvents needed
compared to the extraction danger of „break-through“
possible to repeat danger of contamination
analysis of the loop
closed loop possible
_____________________________________________________________________________
Codling moth (Cydia pomonella)
• gland ◆ head-space
• 10:OH ◆ 10:OH
• 12:OH ◆ 12:OH
• E9-12:OH ◆ E9-12:OH
• E8E10-12:Ald ◆ -
• E8E10-12:Ac ◆ -
• E8E10-12:OH ◆ E8E10-12:OH
• Z8E10-12:OH ◆ Z8E10-12:OH
• E8Z10-12:OH ◆ E8Z10-12:OH
• 14:OH ◆ 14:OH
• 16:OH ◆ 16:OH
• 18:OH ◆ 18:OH
• 18:Ac ◆ -
• 20:Ac ◆ -
 Solid phase microextraction
• SPME
• developed originally for trace analysis of
organic compounds in water
• adsorption on a thin film of polysiloxane
• thermal desorption in GC injector
SPME
 Gas chromatography
• sensitive analytical technique
• potent in separation complex mixtures
• evaporation of the sample in a heated
injector
• capillary silica chromatographic column
(standard 30 m x 0.25 mm)
• siloxane bound to the column walls
• carrier gas – He, H2, N2
• flame ionisation
detector or selective
detectors
 SPME
_____________________________________________________________________
Advantage Disadvantage
_____________________________________________________________________
without solvent only one analysis
high sensitivity equipment needed
simple
use in the field possible
_____________________________________________________________________
Chromatograms - DHS and SPME

head-space

SPME
 Small („light“) molecules discriminated
(preference of „havier“molecules)
head-space

SPME
Solid sample
injection
◆ biological material
(gland) sealed
in a capillary
◆ injection port
adapted
◆ vaporisation
of volatiles
in the injector
directly
 Solid sample injection
__________________________________________________________________________________
Advantage Disadvantage
__________________________________________________________________________________
without solvent only one analysis
no loss of compounds injector port specially adapted
cleaning of injector needed
___________________________________________________________________________________
 Structure elucidation
• classical spectral methods used in organic
chemistry (IR, NMR, MS, UV, CD) – larger
amount of sample (mg)
• derivatisation, degradation, X-ray
• „hyphenated techniques“ (GC-MS, LC-MS, GC-
IR) – small amount of sample (µg)
• GCxGC-MS (2D-GC-MS, latest technique)
• 2D-GC determination of absolute
configuration (standards)
 Stereochemistry
• double bond configuration (geometry)

(E)-But-2-ene (trans) (Z)-But-2-ene (cis)

• absolute configuration (chirality, enantiomer,

antipode)

(S)-Alanine (R)-alanine
Spectroscopy and wavelength
 Infrared (IR) spectroscopy
• wavelength range from 2,500 to 16,000 nm
• vibrational excitation of covalently bonded atoms and
groups
• each functional group has a characteristic frequency

IR spectrum of formaldehyde
 Nuclear magnetic resonance spectroscopy
(NMR)
• excitation in magnetic field
• elements with odd number of protons + neutrons
(1H, 13C, 31P, 19F)
• each type has a characteristic frequency (chemical shift)

1H-NMR 2D-NMR
 Ultraviolet (UV) spectroscopy
• range 200 to 800 nm
• unsaturated compounds absorb UV light
• typical absorption of funcional groups
 Optical rotation, circular dichroism
• chiral compounds, polarised light
• CD = absorption in UV + optical rotation
 Mass spectrometry
• Molecules in high vacuum
• Ion source, Electron ionisation
• Typical fragmentation of a molecule

mass spectrum
of n-decane
 Information from mass spectrum
• Molecular weight – from molecular ion M+.
• Structure – from fragments (hard ionisation, MS/MS)
• Elemental composition – from exact mass
Gas chromatography - mass spectrometry
(GC-MS)
• benchtop instrument, usually without direct
inlet
• silica capillary column, inner diameter 0,25
mm, carrier gas helium
• end of the column introduced into the ion
source
• 2 classical types - quadrupole and ion trap
• new type –Time Of Flight (TOF)
 Quadrupole GC-MS

obrázek
 Quadrupole GC-MS
• ions arise in ion source
• electron ionisation (EI)
• positive and negative ions possible to record
• chemical ionisation (CI) – determination
of molecular weight (reaction gas methane,
ammonia, isobutane)
• change from EI to CI is time-demanding
in some instruments
100 88
100

% 101

55
41 43 7073
60 89
39 45 74 83 102 115 129 143 157 183 185 199
171 228
0
R:931
NIST 29645:
88
DODECANOIC ACID, ETHYL ESTER Hit 3
% 100

% 101

41 43 55 7073
89
45 61
27 29 39 83 102 111 125 143 157 171
183 185 199 228
0
R:911 NIST 46382: NONADECANOIC ACID, ETHYL ESTER
Hit 4
88
100
0 rt 43
10.000 20.000 30.000 40.000 50.000 60.000 70.000 80.000
41
101
% 55
57
100 69 70 89
88 0
45 60
74
83
95 111115
129 143
157
171

R:867 NIST 32384: ETHYL TRIDECANOATE

Hit 5
88
100

43
41 101
%
55

%
101 29
27 39
45
57 7073

67
89
102 115 143
157
197 199 213
54 74 83 129 158 171 242
185
0
R:849 NIST 29640: UNDECANOIC ACID, 2,8-DIMETHYL-, METHYL ESTER
Hit 6
88
55 100

43
41 70 73
57 101
60 89 %
157
39
45
67
83
81
97 102 115
129 143
183 185
228 41 43
55 57 69
51 90 158 171 186 199
139 144 200 213 229 27 39 59
71 83 97 102 111 157
228
0 129 143 171
m/z 185 197 199
30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 0 m/z
20 40 60 80 100 120 140 160 180 200 220 240 260
 Quadrupole GC-MS
• spectra comparable with big sector
spectrometers
• comparison with databases National Institute
of Standards and Technology (over 60 000
spectra) and Wiley Library (230 000 spectra)
• sensitivity can be increased using Selective Ion
Monitoring (registeration of one fragment
only)
Ion trap
 Ion trap
• classical - internal ionisation
• ions arise in the ion trap where they are stored
and analysed
• higher sensitivity than quadrupole GC-MS
• recorded spectra sometimes different from
sector spectrometers
• EI and CI possible in one injection
• tandem technique MS/MS and MS(n)
Comparison quadrupole – ion trap
(selectivity of detection)
100

quadrupole GC-MS
%

0 rt
20.000 22.000 24.000 26.000 28.000 30.000 32.000 34.000

ion trap

17 18 19 20 21
minutes
 Spectrum of hexadec-9-en-1-ol
81
100%

ion trap
75% 67 95

50%
109
124
25% 39 55
222
138
152 166
180 194 241
0% 207
50 100 150 200 250
m/z

55
100 82
67 81
69
41 95 96
% 54
83
56
70 97 109
31 53
66 84 123
32 98 138 152 166 222
180 194 207 224240
0 m/z
40 60 80 100 120 140 160 180 200 220 240

quadrupole GC-MS
 Spectrum of hexadecan-1-ol
97
100%
83
69

ion trap
75%
111

50%
55
125
25% 39
138
152 166 180 196 225
0%
50 100 150 200 250
m/z

55
100 69 83
57
43 97
41 70
% 68 82
71 84 111
67 85 98
31 54 81 112125
58 95 110 139 196
154 168 181 222224
0 m/z
40 60 80 100 120 140 160 180 200 220 240

quadrupole GC-MS
GC-TOF
 Advantage of GC-TOF
• mass determined at high resolution (elemental
composition of ions, slow data collection)

OR

• two-dimensional chromatography possible,

GCxGCxTOF (quick data collection, but nominal
mass only)
Mass spectrum does not differ at different
parts of chromatograhic peak
Difference between scanning techniques
and TOF
Simultaneous Sampling GC Peak Scanning
1000 1000
8000
750 750
Rel. Abund.

6000

Rel. Abund.
Intensity
500 4000 500
250 2000 250

0 0 0
60 80 100 120 140 42.5 43.0 43.5
Time (sec)
60 80 100 120 140
m/z m/z

1000 1000
8000
750 750
Rel. Abund.

Rel. Abund.
6000
Intensity

500 500
4000
250 2000 250

0 0 0
60 80 100 120 140 42.5 43.0 43.5 60 80 100 120 140
1000 Time (sec) 1000
m/z 8000 m/z
750 750
Rel. Abund.

Rel. Abund.
6000
Intensity

500 4000 500

250 2000 250

0 0 0
60 80 100 120 140 42.5 43.0 43.5
60 80 100 120 140
Time (sec)
m/z m/z
 Chromatographic system
• first dimension – classical column, non-polar, 30 m
x 0.25 mm
• secon dimension – short and narrow column (1-2
m x 0,1 mm), polar phase
• separation on the second column is very fast,
therefore data collection must be fast, too
 Fast GC: short time of analysis
Conventional GC Fast GC

187 compounds in 75 min 187 compounds in 5 min

naphtalene derivatives
TOF - fast data collection, very narrow peaks can
be recorded

dodecane
150 ms peak width
10 spectra/s
250 spectra/s
2D-Technique removes the chemical noise and
increases the sensitivity
D9-d29-C16:COOEt
Coelution of analytes of very different
concentration

propylene glycol

furfural

peak area of furfural is 0.001 % of the glycol peak area

Spectra recorded at different parts of the
peak of propylene glycol

At Peak 45 Seconds

35 Seconds
50 Seconds

without deconvolution it is impossible to determine furfural

 Comparison of spectra

manual deconvolution
subtraction
of spectra

library spectrum
 Liquid chromatography – mass
spectrometry (LC-MS)
• large amounts of mobile phase has to be
removed
• particle beam interface
• thermospray (TSP)
• electrospray (ESI)
• chemical ionisation in atmospheric
pressure (APCI)
 LC-MS
• only the technique of „particle beam
interface“ gives spectra comparable with
sector spectrometers
• other techniques give quasimolecular ions
(addition or elimination of particle from
molecular ion)
 GC-FTIR (Fourier transform infrared
spectroscopy)
• flow detection cell covered with a layer
of gold („light pipe“)
• less sensitive compared to GC-MS (~ 100x)
• GC columns of a larger inner diameter
(0,32-0,5 mm)
• carrier gas helium
• spectra in gas phase
(no intermolecular interactions)
Double bond configuration (E,Z)
H H
O O

3012 cm-1
 Derivatisation in microscale
• characterisation of functional groups
in the molecule
• spectra easier to interpret
• better separation
• increased volatility or thermal stability
of compounds for GC analysis
• enantiomeric composition
• improvement of detection properties
 Derivatisation reactions
• methylation of acids with diazomethane CH2N2
(for GC) OH OCH3
R R

O O

• acetylation of alcohols and amines (for GC)

OH O CH3
R R

O
H
NH2 N CH3
R R

• silylation of alcohols (for higher volatility) O

OH O CH3
R R Si
CH3
H3C
 Derivatisation reactions
• dimethylhydrazones NH2N(CH3)2 (CO, CHO)
H H CH3

N
R O R N CH3

• transesterification of triacylglycerols (for GC)

O
CO
O mixture
CO of FAME
CO
O

• catalytic hydrogenation (carbon skeleton chromatography)

 Double bond position
• ozonolysis - pure compound only, formation of
aldehydes
• other oxidative cleavage of C=C bond (RuO4) – formation
of acids
• oxidation with OsO4 – formation of a diol
• methylthiolation of double bond (reaction with dimethyl
disulfide, DMDS, (CH3)2S2) – possible
in mixtures
• epoxidation with m-chlorperoxybenzoic acid (MCPBA)
and following reactions
 Double bond position
CH3S SCH3
R2 CHO OHC R1 R2 C C R1
H H
O3
Me2S2/I2

R2 C C R1
H H
ClC6H4COOOH
OsO4
H H H H
R C C R1
2
R 2
C C R1
HO OH [SiMe3]2NH O

H H
R C C R1
2

Me3SiO OSiMe3
 Fragmentation of DMDS adducts
octadec-9-en-1-yl acetate
S S M+ 404
(CH2)5 (CH2)3
OAc

m/z 173 m/z 231

43
100

173 231
61
81
69
55
% 41
87
79 171
123
95
122
174 232 404
0 m/z
100
55 spectrum DMDS adducts of icosa-11,15-dienal

67
81
41
% 117
95
43 79 123 215
87 97
171 269 291
338 386
0 m/z
50 100 150 200 250 300 350 400

M+. 386

m/z 171 m/z 215

S S
(CH2)8
CHO
S S

m/z 117 m/z 269

Chemical ionisation - methylvinylether
H H
R1 C C R2

H 2C C OCH 3
H

H H H H
R1 C C R2 R1 C C R2

H 2C C OCH 3 H3CO C CH2

H H

H H H H
R2 C C OCH 3 R1 C C OCH 3
 Chemical ionisation with acetonitrile in ion
trap MS
320
100%

75%

50%

25%
97 111 123 137 151 165
180 194 222
250 265 279
302
204 236
0%
100 150 200 250 300 m/z

CI spectrum octadec-11-enal +
m/z 180 M 320
+
[C3H4N]
CH3(CH2)4 (CH2)8CHO
active particle [C3H4N]+
m/z 54
m/z 250
Absolute configuration of natural products

• enantiomers may have different ecological

functions
• enantiomers may have different physiological
effects
• determination of enantiomeric purity
of natural products is important

(S)-Alanine (R)-alanine
 Different species of one genus use
opposite enantiomers

HO HO

(+) (–)
Ips paraconfusus ipsdienol Ips calligraphus
One enantiomer attracts
males, the other one females

O O

O O

(R)-(–)-oleane (S)-(+)-oleane
males females

fruit fly Dacus oleae,

pest on olives
 Biotransformation of resin components to
the aggregation pheromone of bark beetles

OH

(–)-a-pinene (–)-cis-verbenol
spruce bark beetle
(Ips typographus)

OH

(+)-a-pinene (+)-trans-verbenol
 Determination of the absolute
configuration

• separation and measurement of optical

rotation
• chiroptical methods
• NMR with shift reagents
• preparation of diastereoisomers
• enantioselective chromatographic
separation
 Classical methods

• large amount of pure natural product

needed
• separation of the enantiomeric pair from
other sample components is difficult
• measurement of optical rotation –
inaccurate
 Enantioselective chromatographic
separations
• columns based on cyklodextrin, hydroxy groups
substituted with different groups
OH

HO O
OH O O
a-cyclodextrin, 6 units HO

OH
b-cyclodextrin, 7 units O HO O

g-cyclodextrin, 8 units O
OH HO OH

O
HO OH HO
O
OH O
HO

OH
O O HO
O OH

HO
a-Cyclodextrin
Two-dimensional GC
2D-GC, separation examples
 Advantages of 2D-GC
• possible in minute quantities
• presaparation of components no needed
• high accuracy provided good (base-line)
separation of enantiomeric pairs
• high sensitivity, detection of minor impurities
of the opposite enantiomer
• information on enantiomeric purity of several
components in one analysis
standards needed!