School of Biological and Chemical Sciences

The Chemical Analysis of Natural

Products
Produced by Fungi Grown in the
Wild

By

Temitayo M. Odutola

A dissertation in partial fulfilment of MSc Analytical Chemistry,

Birkbeck College

2009

pg. 1
 ABSTRACT

Cells of organisms such as fungi and bacteria produce a wide variety of

natural products, which possess properties of great interest in chemical

research; these properties vary from therapeutic (pharmaceutical)

purposes to having toxins which are detrimental to humans and plants

alike. An unknown compound was extracted from a given fungi specie

which was cultured for about three months at 25oC prior to the extraction

process. This extract was concentrated and subjected to a biological

assay test using bacteria and fungi species. The bioassay showed that the

crude extract displays bioactivity towards 2 of the bacteria species and

none towards the fungi. The extract is then subjected to a purification

process using TLC and column chromatography to obtain pure

compound/s after which another bioassay was carried out to determine

which component in the extract actually possesses the bioactive property.

Analytical techniques and spectroscopic methods such as LCMS and NMR

were used to determine a suggested partial structure for the purified

fungi extract.

pg. 2
 ACKNOWLEDGEMENT

I dedicate this work to almighty God for affording me the wisdom, strength and

perseverance to carry through with this research. Praised be thy name. My

sincere appreciation goes to my project supervisor, Dr Phillip Lowden for the

guidance, tips and advice which were of immense help. I would also wish to

thank the following people; the lab technicians from the microbiology

department for their help in the supply of the raw materials I used for this project

and Mr Frank Baretto for his kind contribution to my work by making sure I have

all the necessities (in form of solvents and equipment) as required by my work.

My heartfelt appreciation and gratitude goes out to my parents, Mr and Mrs

Odutola, for always being there in time of need and their unconditional love and

support (financially and spiritually) towards my growth. I also thank my siblings

Abi, Seun and Enit for their moral support and pep talks.

I would like to say a big thank you to my mates; Derek, Gloria, Jhon, Ola, Mr Kay,

Rae, Gbenga and Banky for their moral support and unwavering friendship.

pg. 3
 DECLARATION

I certify that this thesis contains no materials which has been accepted for the

award of any other degree or diploma in any Institute ,College of University and

that, to the best of my knowledge, it contains no material previously published or

written by another person, except where due reference is made in the text of the

thesis.

Temitayo M. Odutola

pg. 4
 Table of contents

Abstract.................

…………………………………………………………………………........................................

...2

Acknowledgements…………………………………………………………………………..........

................................3

Declaration..............................................................................................................

................4

List of

Tables……………………………………………………………………….................................

.......................8

List of

Diagrams………………………………………………………………………….........................

........................9

Glossary of

Terms…………………………………………………………………………...............................

..............12

Section 1 Introduction

pg. 5
1.1 Natural products................

……………………………………………………............................................13

1.2 Background

1.2.1 Introduction to Fungi

species………………………………………………….........................................13

1.2.2 History of Fungal

Metabolites………………………………………………….........................................14

1.2.3 Botanical background……………………………..............................

….......................................15

1.2.4 Structure of Fungi……………………………………………………............

….........................................16

1.3 Aims and

Objectives……………………………………………..........................................................

.....17

1.4 Analytical techniques

1.4.1 Introduction to

extraction…………………………………..........................................................18

1.4.2 Pre-concentration

step…………………………………..............................................................18

1.4.3 Fractionation and purification step

1.4.3.1. Theory of Thin layer Chromatography

…………………………………………………………..............19

1.4.3.2 Introduction to column

chromatography………………………………………………………………......22

pg. 6
 1.4.3.3 Recombination

step………………………………………………………………......................................23

1.4.3.4 Introduction to

HPLC.................................................................................................23

1.4.4. Structural characterization step

1.4.4.1 Background of Mass

spectrometry…………………………………………………………….................26

1.4.4.2 Introduction to Nuclear-magnetic

Resonance…………………………………………………………….26

1.5. Bioassay test

procedure……………………………………………………………........................................

....30

Section 2 Literature review

2.1

Introduction…………………………………………………………........................................

.......................40

2.2 Aspergillus metabolites

2.2.1. Metabolites from Aspergillus Flavus

……………………………………………….............................40

2.2.2. Metabolites derived from Aspergillus

ustus…………………………….....................................44

2.3. Chaetonium sp.

metabolites……………………...........................................................................47

pg. 7
 2.3.1. Metabolites derived from Chaetonium

spp…………………………………....................................47

2.3.2. Metabolites from Chaetonium brasiliene

…………………………………………….............................49

Section 3 Experimental

3.1.

Instrumentation…………………………….....................................................................

.................54

3.2. List of materials……………………………………………………....

….........................................................54

3.3. Experimental procedures for analytical

methods…………......................................................55

3.4 Computing

……………………………………………………………………...............................................

........61

Section 4 Results and

Discussion...................................................................62

Section 5

Conclusion..........................................................................................................
75

pg. 8
References..........................................................................................................
...................76

Appendices.........................................................................................................
...................78

LIST OF TABLES

pg. 9
Table No. Page

No.

1. Elutropic series ...

……………....................................................................................21

2. Types of stationary phases and the corresponding separation

mechanism…….......24

3. Types of fungus found in papers used for the literature

review.............................40

4. The major components and their bioassay

results…………......................................44

5. Weight of the crude extract………………………..................

……….................................62

6. Rf values for solvent

mixtures..............................................................................62

7. Recombined fractions and their weights………………………..................

………..............63

8. Weight of the dried crude fungi extract before liquid-liquid

partition…………………..65

9. Visual appraisal of the liquid-liquid partition………………………..................

……….........65

10. Weight of the dried chloroform and aqueous extract……….....

……….......................65

pg. 10
11. Bioassay results for the 2nd batch of fungi

samples………………………..................……67

12. Table 12:1H NMR data………………………..................

……............................................73

LIST OF DIAGRAMS

Figure No. Page No.

1 examples of natural

products………………………………..........................................15

2 Two types of fruiting bodies…………………………………………….

……….....................16

3 apparatus used in extraction………………………………………………………….

……….......18

4 BUCHI rotator

evaporator…………………………………………………………....................18

5 Typical TLC separation ……………….…..

…….........................................................20

pg. 11
6 apparatus used in column

chromatography……………………………………………………22

7 separation of components in a column

chromatography………………................23

8 HPLC instrumentation…………….

….....................................................................25

9 Block diagram of a LC-

MS………….....................................................................25

10 Ion formation by ESI …………………….

……............................................................28

11 Schematic diagram of a ion trap mass analyzer………………………….

……..............28

12 Energy diagram for nuclei with spin quantum number of

1/2…......................30

13 NMR signals for different compounds……………...….

…......................................32

14 Typical 1H chemical shift

ranges………………………………......................................32

13
15 Typical C chemical shift ranges…………………………………………….

………...............33

16 NMR integration example ethyl

acetate…………………………………………………………34

pg. 12
17 Schematic diagram of a NMR

spectrometer……………………...............................35

18 FID signal before and after transformation ……………….…..

……..........................36

19 Pascal’s triangle…………………………………………………………….

…..............................37

20 Agar plate showing inhibition

zones ................................................................38

21 Structure of Monorden Analog 1(compound 1)…………….

…..............................41

22 Structures of Monorden and Monocillin IV

respectively ................................42

23 Structures of Cerebrosides C and Cerebrosides D …………………………….

…….…....43

24 Structures of compounds 1 and 2 respectively…………….…….

….........................45

25 General structure/s and respective side chains of compounds 6, 7 and

10......46

26 Structures of Cochliodinol and

Isocochliodinol……...........................................48

27 Structures of Mollicellin K and L (compounds 1 and 2 respectively)

……………...51

pg. 13
28 Structures of Mollicellin M and N (compounds 3 and 4 respectively)

………......51

29 2nd batch of fungi samples used for the analysis……….

….................................55

30 Filtrate of crude fungi extract…………….…….

…...................................................56

31 Crude fungi extract being concentrated using the rotavapor

…………...............57

32 The four fractions obtained after a round of column chromatography

……......63

33 TLC plates for fractions 11-15 and 29-46 respectively….

…................................64

34 TLC plates for fractions 47-49 and 61-79 respectively…………….…….

…...............64

35 TLC plate for aqueous extract showing no signs of separation….

…...................66

36 TLC plate for the extract dissolved in chloroform (10:90 and 90:10)….

…..........66

37 TLC plates viewed under UV light (254 nm).………….

….......................................67

38 Agar plate showing inhibition of the growth of the bacteria, M.Luteus….

….....68

pg. 14
39 Agar plate showing inhibited growth of the bacteria,

B.Megaterium…….........68

40 Chromatogram showing a distinct

peak……......................................................69

41 Positive Ion ESI spectrum of main

peak……........................................................70

42 Expanded ESI spectrum

2…….............................................................................70

43 Expanded ESI spectrum

2…….............................................................................71

pg. 15
 GLOSSARY OF TERMS

COSY – Correlated Spectroscopy

DCM – Dichloromethane

ESI-MS – Electro spray Ionisation Mass Spectrometry

FAB MS- Fast Atom Bombardment Mass Spectrometry

FTICR – Fourier Transform Ion Cyclotron Resonance

HETCOR- Hetero-nuclear Correlation

HMBC- Hetero-nuclear Multiple Bond Correlation

HPLC – High Performance liquid Chromatography

HRESIMS – High Resolution Electro spray Ionisation Mass Spectroscopy

HRESITOFMS- Hi Resolution Time Of Flight Mass spectroscopy

IC50 – half maximal Inhibitory concentration

NMR – Nuclear Magnetic Resonance

PDA – Potato Dextrose Agar

TLC – Thin layer Chromatography

TMS – Tetra Methyl Silane

UV – Ultra Violet

VLC – Vacuum Liquid Chromatography

pg. 16
 CHAPTER ONE

Introduction

1.1 Natural Products

“The term “natural products” refers to materials derived from higher plants,

microorganisms, invertebrates and vertebrates. They may be of interest in the

crude form, in partially purified concentrates, in pure form or as structurally

modified chemicals. They are useful for their ability to prevent or treat diseases

amongst other therapeutic properties. They also serve as raw material for

chemical or biological modification to new products and also useful as reagents

in fundamental metabolic studies which may lead to new therapeutic agents. The

research “explosion” has resulted in increased knowledge regarding the

identification, distribution, and variations of biological species; the development

of new and improved tests useful in the therapeutic evaluation of drugs; new

procedures for the isolation, identification, structural elucidation of natural

products; and new types of organic syntheses. This has lead to developments in

techniques in the chemical field. Techniques such as thin layer, and partition

chromatography; ion exchange fractionation; nuclear magnetic resonance ; mass

spectrometry; rotator dispersion; and X-ray crystallography”.1

Microorganism as stated in the first paragraph generally includes the

bacteria, viruses, fungi (yeasts, moulds and mushrooms), algae, lichens, and

protozoa but due to the scope of this project, I will be focusing entirely on natural

products from fungi.

1.2 Background:

pg. 17
 1.2.1 Introduction to Fungi species: Fungi are widely distributed non-

photosynthetic microorganisms found wherever moisture is present. They play

an essential role in both Nitrogen and carbon cycle by breaking down dead

organic materials which allows nutrients to be cycled through the eco-system,

they are also essential starting materials especially yeast in industrial processes

involving fermentation e.g. Bread baking and in wine production. Fungi are also

used in the manufacture of many antibiotics examples are Griseofulvin,

Penicillin and certain types of drugs2. The study of their metabolites has made

many contributions to the overall development of chemistry 1 and the natural

product derived from these metabolites has been of interest to organic chemists

since the 1800s when pigments were synthesized from fruiting mushroom

bodies.

These metabolites are classified into two main types; primary and

secondary metabolites. Primary metabolites are compounds related to the

synthesis of microbial cells which are necessary for the survival of the organism

examples include Lipids and Nucleic acids. Hence they are directly involved in

normal growth, development and reproduction. Secondary metabolites on the

other hand are compounds which have no direct relationship to the synthesis of

cell materials and normal growth and they usually accumulate during the period

of nutrient limitation or waste product accumulation following the active growth

phase.

Secondary metabolites in particular are compounds (most antibiotics and

mycotoxins fall into this category) which are unique to a specific species or

genus and are often highly biologically active against other organisms; hence

they play ecological roles in nature, deterring potential predators and pathogens.

These bioactivities observed in them also affect humans due to structural

pg. 18
resemblance to innate neurotransmitters or by binding to proteins in a manner

that disrupts normal cellular function.

1.2.2 History of Fungal Metabolites: Research on fungal metabolites date

back to the 1800s but the chemical history of fungi dates back further.

Dioscorides, a Greek physician, described the use of an infusion he called

Agaricium. Also in ancient Chinese medicine, there are records of the use of

Ganoderma lucidum. In the 19th century, Pasteur used the fungus Penicillium

plaucum to degrade an enantiomer of tartaric acid in experiments that laid the

foundations for the study of chirality. He was also one of the first to recognize

the antagonism between microorganisms which led to the coining of the word

“antibiote” by French biologist Vuillemin describing the substances involved. The

20th century witnessed the isolation and characterization of certain natural

products from secondary metabolites driven early on by the discovery of

Penicillin by Fleming in 1928 and its development into a precious antibiotic.

This led to a search for fungal metabolites which can lead to the discovery of

several compounds with useful pharmaceutical benefits. Examples of such

compounds that have achieved commercial importance are Lovastatin from

Asperigillus tereus (used for inhibition of cholesterol biosynthesis) and the

aforementioned Penicillin.3

pg. 19
 Figure 1: examples of natural products

1.2.3 Biological background: Fungi are eukaryotic organisms containing a

distinct nucleus with complex structures enclosed within their membranes. Cell

division in fungi is much different from organisms without a nucleus (prokaryotes

e.g. Bacteria and Archaea) and occurs via two main division types namely

mitosis and meiosis. Most of the species grow as multicellular filaments called

hyphae forming a mycelium while some species grow as unicellular cells e.g.

Yeasts. Some fungi grow in a symbiotic relationship with photosynthetic algae or

cyanobacteria in the form of Lichens4. Fungi feed by absorbing nutrients from

organic matter that they live on. This is achieved by secreting acids and

hydrolytic enzymes to digest such foods. Different fungi have evolved to live on

various types of organic matter, some live on plants e.g. Phytopthora infestans.

Some live on animals e.g. athlete’s foot while others live on insects e.g.

Cordyceps australis.

1.2.4 Structure of Fungi: The basic structural units of most fungi are the

filaments known as hyphae. Accumulation of hyphae produces a felt called

mycelium. The size of a single mycelium is not fixed and as long as nutrients are

available, outward growth by hyphal extension can continue. Fungal mycelia are

usually hidden in a food source like wood and it is only noticeable when they

develop mushrooms or other fruiting bodies which sprout from the mycelium and

produce spores. At the other extreme some fungi only produce microscopic

fruiting bodies and are not visible to the naked eye, a particular example is that

of the unicellular micro fungi, yeast which produces small globular or ellipsoid

cells that are only visible under the microscope. Culture conditions greatly affect

pg. 20
the form which a fungus takes. Under a set of conditions, some fungi may

assume yeast like form and a filamentous form under other conditions.

Fig 2 a Fig2 b
Figure 2: shows two types of fruiting bodies developed from the mycelium of fungus, Fig 2a is the
Clouded Agaric toadstool and Fig2 b is Calocera (adopted from
http://www.countrysideinfo.co.uk/fungi/struct.htm)

1.3. Aims and Objectives:

The aim of this project is to completely isolate organic extracts from fungi

samples; purify those components that appear to be dominant in the extract and

also test those components for any bioactive properties which they may

possess .Using data from previous work and spectroscopic techniques can help

in structural characterization and identification of the novel compounds obtained

after extraction and purification. As easy as it sounds, it should be noted that the

yield, in terms of conversion of the major carbon source into antibiotic, are very

low and greatly influenced by the composition of the medium and by other

cultural conditions. As a matter of fact these compounds to be analysed are

often present at a concentration which is less than 10% of the dry weight of the

pg. 21
organism. The three major steps taken to successfully isolate, purify and

structurally characterize natural products are listed as follows

• Extraction and purification of unknown molecules from Fungi samples

• Bioassay to ascertain the presence of any bioactive properties these

molecule(s) might possess

• Structural determination and characterization to fully identify the unknown

molecule(s)

1.4. Analytical techniques

1.4.1. Introduction to extraction

Organic chemistry employs solid-liquid, liquid-liquid, and acid-base extractions.

In this project I will be focussing on liquid-liquid extractions. The foremost step is

to obtain a crude extract from the fungi material using the common extraction

procedure. This is achieved due to the solubility of secondary metabolites in

organic solvents. A separating funnel is used for this process. “The organic

product will be soluble in the organic solvent used while the inorganic product

will be soluble in water (aqueous layer). Common extraction solvents are diethyl

ether, methylene chloride”.5

pg. 22
Figure 3: Separating funnel used for an extraction procedure (Adopted from Mohrig, pp. 57-64, 72-
77)

1.4.2. Pre-concentration step:

This step besides aiding in the increase in the concentration of the extract to a

desirable level, it also serves as a quantitative measure. The solvent in the

extract is evaporated off using a rotary vacuum evaporator and to obtain the

amount of sample retrieved from the fungi, it is ensured that the solvent is

completely removed from the sample. This can be done more effectively under a

high pressure vacuum pump and a solid filtrate is obtained. The solid is then

weighed to obtain amount of extract from the fungi.

pg. 23
Figure 4: BUCHI rotator evaporator (Adopted from www.jimseven.com/2007/09/10/clarified-coffee/)

1.4.3. Fractionation and purification step:

The dried sample is re-dissolved in a minimal volume of solvent and TLC is

used for preliminary identification purposes such as to determine the number of

components in the mixture, to determine the appropriate conditions for a column

chromatographic separation and to monitor column chromatography.

1.4.3.1. Theory of Thin layer Chromatography (TLC)

Thin layer Chromatography is a solid-liquid technique involving two phases, a

solid stationary phase and a liquid mobile phase. Most commonly used solids are

silica gel and alumina.TLC is a fast, sensitive, simple and cost effective analytical

technique. It is a micro technique; as little as 10-9g of material can be detected,

although the sample size is from 1 to 100x10-6. TLC involves spotting the sample

to be analyzed near one end of a sheet of glass or plastic that is coated with a

thin layer of an adsorbent (silica gel). The sheet, which can be the size of a

microscope slide, is placed on end in a covered jar (TLC developing chamber)

containing a shallow layer of solvent. As the solvent rises by capillary action up

through the adsorbent, different partitioning occurs between the components of

the mixture dissolved in the solvent on the stationary adsorbent phase. The

more strongly a given component of a mixture is adsorbed onto the stationary

phase, the less time it will spend in the mobile phase and the more slowly it will

migrate up the plate5.

pg. 24
 Figure5: A typical TLC separation (adopted from http://www.waters.com/waters/nav.htm?

locale=es_ES&cid=10048919)

Visualisation of the plate: After the plate has been developed (as shown in

figure 5), the plate is removed from the chamber, the solvent front is marked

with a pencil, and the plate is allowed to dry. The position of the components can

be visualized in several ways but the two most common methods are UV light at

254 nm (shortwave UV) and chemical staining to make spots visible.

UV light Visualization: Although many compounds are not colored, they

absorb UV light, and then re-emit colored light which can be viewed easily under

a UV lamp.

Chemical staining: This method involves the application of a reagent to the

plate either by spraying or dipping, usually followed by heating, and observation

of the colored products. It should be noted that not all staining reagents will

visualize the sample zone. An example of a universal reagent is a 10% sulfuric

acid solution. When sprayed on the plate, the plate is heated and the spots are

charred which can be seen by the naked eye.

pg. 25
Rf Values: The symbol Rf is the retardation factor and it is defined as the ratio of

the distance the compound travels to the distance the solvent travels. It is

calculated by measuring the distance the sample zone travels divided by the

distance the developing solvent travels as shown in equation 1

Rf = Distance of centre of spot from the baseline/Distance of solvent front from

baseline

Equation 1

Each compound is defined by its Rf (unitless) which corresponds to its relative

migration compared to the solvent hence the V is constant for a given

compound. The optimum Rf. Value for an analytical TLC is 0.3-0.5.

Solvent choice: The distance which a compound moves up a TLC plate is

dependent on its polarity and the polarity of the solvent. The more polar the

compound the less distance it travels as it is more tightly bound to the silica and

vice versa. In selecting a solvent, there is a fair amount of trial and error but it is

best to start the elution with an equal proportion of the solvent (1:1) and then

evaluate the plate and alter the proportions accordingly. A list of commonly

encountered solvents in a decreasing order of polarity is given in table 1 in what

is generally called the Elutropic series

High Polarity
Water
Acetic Acid (Ethanoic Acid)
Methanol
Ethanol
Propan-1-ol
Acetonitrile (Ethanenitrile)
Ethyl Acetate (Ethyl Ethanoate)
Acetone (Propanone)
Dichloromethane
Chloroform
Diethyl Ether

pg. 26
 Toluene (Methyl Benzene)
Cyclohexane
n-Hexane
Low Polarity

Table 1: Elutropic series (adopted from http://www.rsc-teacher-

fellows.net/labTechniques/ElutropicSeries.htm)

1.4.3.2. Introduction to Column chromatography:

Column chromatography is a useful method for the separation and purification of

both solids and liquids when carrying out small-scale experiments. It is another

solid-liquid technique in which the two phases are solid (stationary phase) and

liquid (mobile phase). The theory of column chromatography is analogous to that

of TLC due to the use of the common adsorbents- silica gel and alumina. The

sample is dissolved in a small amount of solvent (the eluent) and applied on the

column. The eluent this time around flows down through the column filled with

the adsorbent. The mixture to be analyzed by column chromatography is applied

to the top of the column. The liquid solvent (the eluent) is passed through the

column by gravity or by the application of air pressure. Just as in TLC, there is an

equilibrium established between the solute adsorbed on the silica gel or alumina

and the eluting solvent flowing down through the column. Because the different

components in the mixture have different interactions with the stationary and

mobile phases, they will be carried along with the mobile phase to varying

degrees and a separation will be achieved. The individual components, or

elutants, are collected as the solvent drips from the bottom of the column into

test tubes collected in fractions.

pg. 27
 Figure6: shows a chromatographic column (Adopted from wfu.edu/academics/chemistry)

Figure 7: shows what happens during the separating process in a column (adopted from
http://www.chemguide.co.uk/analysis/chromatography/column.html

1.4.3.3. Recombination step:

This step requires combining fractions that gave similar TLC

characteristics. After recombining the appropriate fractions, they are then pre-

concentrated using the evaporator and the weight of each concentrated fraction

can be determined. Another TLC run is usually carried out on the concentrated

fractions to ascertain their purity. After the extract is semi purified using column

chromatography, it is usually subjected to another purification process using

HPLC.

pg. 28
1.4.3.4. Brief introduction to HPLC: HPLC is a chemistry based tool for

quantifying and analyzing mixtures of chemical compounds6. Separation is based

on the analyte’s relative affinity between two phases7. The two phases involved

are

Mobile phase: This is in liquid form and is pumped under high pressure through

the column.

Stationary phase: consist of a finely divided solid held inside the column.

A wide range of stationary phases are usually employed depending on the

property of the analyte being exploited.

Separation
Stationary phase mechanism
Solid Adsorption
Liquid Layer Partition
Ion exchange resin Ion exchange
Microporous beads Size exclusion
Chemically modified
resin Affinity

Table 2: types of stationary phases and the corresponding separation mechanism

Instrumentation: Consists of solvent reservoirs (Mobile phases), pump, sample

injector, columns, detector and data system.

Solvent reservoirs: component solvents/mobile phases make up a gradient in the

reservoir and are pumped at a constant rate through the column and detector.

Pumps: effect solvent delivery by pumping the mobile phase at a steady

accurate rate of as low as a few microlitres/min to as much as tens of ml/min.

Injector: allows sample introduction without disrupting the solvent flow, in this

project the injector used was automated.

pg. 29
 Analytical column: this is where separation takes place. They range in length

from 10 to 30 cm and the most common column currently in use is one that is 25

cm in length, 4.6 mm inside diameter, and packed with 5 µ m particles.

Guard column: A guard column is introduced before the analytical column to

increase the life of the analytical column by removing not only particulate matter

and contaminants from the solvents but also sample components that bind

irreversibly to the stationary phase. The composition of the guard-column

packing is similar to that of the analytical column but the particle size is usually

larger.

Detector: measures response changes between the solvent itself, and the

solvent and sample when passing through it. The electrical response is digitized

and sent to a data system. Unlike gas chromatography, liquid chromatography

has no detectors that are as universally applicable and as reliable as the flame

ionization and thermal conductivity detectors. A major challenge in the

development of liquid chromatography has been in detector improvement. Types

of detectors include bulk property detectors (responding to mobile-phase bulk

property, such as refractive index or dielectric constant and solute property

detectors respond to some property of solutes, such as UV absorbance,

fluorescence, or diffusion current, which is not possessed by the mobile phase.

pg. 30
 Figure 8: Basic HPLC instrumentation (adoptedfromwww.nj.gov/dep/oqa/powerpoint/HPLC
%20Course.ppt)

Figure 9: Block diagram of a hyphenated technique comprising HPLC and MS

1.4.4. Structural characterization stage:

In this step, the concentrated fractions are now prepared for structural

characterization using any of the spectroscopic techniques such as NMR, IR, UV

and analytical technique such as MS and X-ray crystallography. In structure

determination, it is highly desirable to have the molecular weight of the unknown

from its mass spectrum, and ideally to have the molecular formula from a high

pg. 31
resolution measurement, while for the spectroscopic characterization, the

objective is to identify functional groups and possibly other molecular fragments

present. The milligram scale on which these methods work meant that amounts

of material produced by different organisms in the lab scale cultures and that

could be separated by chromatographic methods could be studied and it should

be noted that some of the earliest applications of NMR to organic structure

determination involved fungal metabolites e.g. Gibberellic acid and in correcting

the structure of the Trichothecenes3.

1.4.4.1. Mass spectrometry:

Mass spectrometry is an analytical method of characterizing matter, based on

the determination of atomic or molecular masses of individual species present in

a sample8 .The instrument employed for carrying out mass spectrometry can be

classified into different categories according to the mass separation technique

used. Of the many spectroscopic techniques available, MS provides one of the

few such structural probes of an entire molecule9.In a typical MS procedure, the

sample molecules are introduced in the gas phase or a suitable form,

chromatographic separation may also occur at this stage. The molecules are

converted into ions and they may be vaporised at the same time. The ions then

move through electric and or magnetic fields (mass analyzer) where their

movement depends on their mass to charge ratio, so they arrive at the detector

at different times. The ions then induce an electric current which is proportional

to the number of ions and an electrical response is processed by the computer

and presented as a mass spectrum. This method destroys the compound sample,

but owing to the great sensitivity of the technique, only a tiny quantity is

required. This property makes MS an extremely useful analytical tool and often

pg. 32
some of the necessary structural information is obtained from the spectrum

using only extremely small samples or mixtures9.

Mass spectrometers create and manipulate gas-phase ions hence need to be

operated under a high vacuum system. They consist of three essential parts

namely: an ionization source, a mass selective analyzer and an ion detector as

shown in figure 9. Many methods have been derived to ionise molecules in mass

spectrometry, but the choice of method depends on the mass and structure of

the molecule and the information required by the user. A list of the various

ionisation methods are as follows: Electron ionisation, Chemical ionisation, Field

ionisation, Fast atom bombardment, matrix assisted laser desorption ionisation

and electro-spray ionisation (ESI). The ionisation source I will be focussing on is

electro-spray ionisation which provides the source of ions in the mass

spectrometer used for this project.

Introduction to ESI: ESI is a type of mass spectrometer whereby an electro-

spray is produced by applying a strong electric field, under atmospheric pressure

to a liquid passing through a capillary tube with a weak flux (1-10microlitre/min).

10

Basic principle: Sample in solution is passed through a silica capillary whose

surface has been metalized held at a high positive potential, hence forming very

small droplets of solvent containing the sample of interest. The electric potential

on the capillary charges the surface of the spray droplets. Solvent is removed

through complex mechanisms by heat or by energetic collisions with a dry gas,

usually nitrogen gas. The ions formed are desolvated and pass into the mass

analyzer. This method of ionization is referred to as soft ionization because very

pg. 33
little fragmentation occurs, which makes ESI-MS very important in biological

studies10.

Figure 10: Ion formation by ESI (Adopted from kerbarle and Tang, Anal Chem, 65, 2, 972A-985A
(1993))

ESI is also easily coupled to HPLC and very high molecular weight polar

molecules can be analysed. After formation of ions, the next stage is to observe

how the motions of the ions are affected by electric and/ or magnetic fields. The

five main methods are Magnetic sector, Quadrupole-mass analyzer, Ion trap,

Time of flight and FTICR.

Introduction to Ion Trap mass analyzers: In this mass analyzer, the

ions are contained within three electrodes whose shape appears to be like a

quadrupole wrapped round itself.

pg. 34
 Figure 11: schematic diagram of an ion trap mass analyzer (Adopted from

http://www.matrixscience.com/help/ion_trap_main_help.html)

Ions are introduced at the top and are trapped by repulsion from the end caps. A

radiofrequency voltage is then applied to the annular electrode which confines

these ions in the centre of the trap by forcing them to follow complex trajectories

in the presence of a low pressure of helium (~10-3 torr).

Initially all ions are trapped together, and then the radio frequency amplitude is

scanned upwards to release ions sequentially from the trap starting with the ion

with lightest mass. As the radio frequency amplitude increases the ions oscillate

further from the centre until they are unstable and are emitted from the

analyser.

Spectroscopic analysis is usually carried out using NMR in order to identify

the underlying carbon skeleton of the compound. The objective is to identify

fragments which can then be linked together.

pg. 35
1.4.4.2. Introduction to Nuclear Magnetic Resonance

NMR is a powerful technique used for the determination of organic compounds

and also for certain types of inorganic material. NMR collects information

concerning interactions between the nuclei of certain atoms present in the

sample when they are subjected to a static magnetic field which has a very high

and constant intensity and exposed to a second oscillating magnetic field. The

second magnetic field, around 10,000 times weaker than the first is produced by

a source of electromagnetic radiation in the radio frequency domain.10

Basic principles: NMR spectrometry is concerned with the ability of nuclei to

absorb energy from the electromagnetic radiation in the radiofrequency region

of the spectrum. Nuclei of many, but not all elements are considered to spin

about their own axis. The criteria for a nuclei to be NMR active is that the nuclei

in question must have its mass number and atomic number to be either

odd/even or odd/odd respectively e.g.1H,13C,19F and 13

P. The spinning nuclei,

being charged particles generate a minute magnetic field or moment along their

axis of rotation. The axis of rotation and hence the magnetic moment are

randomly orientated in space. If the nuclei are now subjected to a powerful

external magnetic field, interaction between this field and the nuclear magnetic

moments forces the nuclei to adopt a limited number of orientations. This

orientation(s) are known as spin states, each of which has a different energy.

pg. 36
 Figure 12: Energy diagram for nuclei with spin quantum number of 1/2 (Adopted from
http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/nmr/nmr1.htm)

E represents the energy of the spinning nuclei and delta E is the energy

difference between the spin states, u is the magnetic moment, Bx is the point

where resonance occurs. Bo is the strength of the external magnetic field, while

-1/2 and +1/2 refer to the upper spin state and lower spin state respectively.

From figure 12, it is seen that the energy difference between the spin states

increase linearly with the strength of the external field (Bo). The diagram

represents the two spin states of NMR active nuclei such as 1H, 13
C, 19
F and 13
P

(other nuclei may have two or more spin states). Nuclei irradiated by a radio

frequency source will undergo transitions from one spin state to another by

emission or absorption of radiation of one particular frequency. This process is

called resonance and the corresponding radiation frequency is called the

resonance frequency which is related to the energy difference shown in equation

V=γ /2 π Bo

Equation 3

γ is the magnetogyric ratio whose value is different for each NMR active nuclei .It

is determined by the RAM and magnetic moment(u). Each element therefore has

a different linear relation between Bo and v .

pg. 37
 Methods of generating the NMR spectrum: There are two methods of

generating NMR spectrum namely; Continuous wave-NMR and Pulsed-Fourier

transform-NMR.

Continuous wave NMR: In this method, the sample is irradiated by a fixed

radio frequency and external magnetic field is varied over a given range so that

the net field for each nucleus or group of nuclei corresponds in turn to the field

required for resonance at frequency (v).This method is no longer in use.

FT-NMR: This method involves the sample being irradiated by a high energy

radio frequency pulse of short duration and wide frequency range at a fixed

external field (Bo) thereby causing all the nuclei to undergo resonance at once.

Measurements of resonance peaks in the spectrum are made relative to a

peak from an added reference standard. The compound used for 1H and 13
C

spectra is Tetra Methyl Silane (TMS) due to its highly shielded nuclei which

undergo resonance at a higher field than most of other compounds. The position

of the TMS peak is defined as zero and those of chemically different nuclei in the

sample are assigned chemical shift values relative to TMS. These chemical shift

values are used to indicate the chemical nature of individual nuclei or groups of

nuclei, and of neighbouring atoms in the structure. The scale used is calibrated

in units of parts per million (ppm) that are independent of the operating

frequency of the spectrometer. For 1H resonances, values are from 0 to 12ppm

13
while for C resonances, values are from 0 to 220ppm.

pg. 38
 Figure13: Scale of NMR signals for different compounds (Adopted from
http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/nmr/nmr1.htm)

It is seen from figure 13 that TMS is located at the extreme right of the spectrum

(zero position) due to its highly shielded nuclei (Si(CH 3)4). Using TMS as a

reference point, molecules that give signals at the high frequency end of the

spectrum to the extreme left are described as being downfield (high chemical

shift values).All other signals occurring to the right hand side is said to be upfield

(low chemical shift values)11.

Figure 14: Typical proton chemical shift ranges (Adopted from

http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/nmr/nmr1.htm)

pg. 39
 13
Figure 15: Typical C chemical shift ranges (Adopted from

http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/nmr/nmr1.htm)

Chemical shift is hence defined as the difference in ppm between the

resonance frequency of the protons being observed and that of TMS and its scale

is measured thus

σ sample peak= V sample peak - V TMS / V Spectrometer)106ppm

Equation 4

“σ is a unitless parameter used to measure the position of the observed signal,

it is expressed as fractions of the applied field in ppm.

V Spectrometer corresponds to the operating frequency of the spectrometer.

TMS is widely used as an internal standard for due to its inert, volatile and toxic

nature and it produces only one signal which is observed at the zero position on

the resonance scale. Chemical shift values indicate the chemical nature of

individual nucleus or groups of nuclei, and of neighbouring toms in the structure.

Also the relative numbers of nuclei in each group can be established by

pg. 40
integrating the areas of the corresponding resonance peaks as shown in figure

16.

Figure 16: NMR spectra of ethyl acetate (adopted from

http://www.wfu.edu/~ylwong/chem/nmr/h1/integration.html)

Measuring the area under the NMR resonance gives a value which is

proportional to the relative number of hydrogen atoms which that resonance

represents.

The ratio of hydrogen atoms for the spectrum in figure 16 can be explained thus:

measuring the peak height from the right of the spectrum gives groups of

hydrogen's with ratio ~4.5:~4.5:~3.0 hence it is possible that the number of H's

are 3:3:2, 6:6:4 etc.

In NMR, it is known that within a given molecule, the electronic and steric

environment of each nucleus creates a very weak local magnetic field which

shields it more or less from the action of the external field Bo 8. Thus an element

with nuclei that are chemically different, give rise to different resonances. The

degree of shielding or de-shielding is directly proportional to the electron density

around the nucleus. And hence to the electro negativity of neighbouring atoms,

and is affected by the presence of unsaturated sites in the structure. This

pg. 41
shielding and deshielding of the nuclei is the basis of the exploitation of NMR:

rather than observing all of the nuclei present, spread over a wide range of

frequencies, the study focuses on a single type of nucleus at a time. In other

words the technique zooms over a narrow range of frequencies (e.g.: 1000 Hz) in

order to record the different signals which result from specificities of each

compound. This screening effect is quantified by the shielding constant ( σ) thus

in order to compensate and achieve resonance at frequency(v) Bo must be

increased by Δ Bo to Bo+ Δ Bo, hence the effective field experienced by the

nucleus is given by equation 4.

Beff=Bo (1- σ)

Equation 4

Figure 17: schematic diagram of an NMR spectrometer (Adopted from

http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/nmr/nmr1.htm)

The net absorbance is recorded after electronic and computer processing of the

signal from the detector and usually gives a pattern called FID as shown on the

graph in figure 18 while the right hand side shows a transformed FID signal.

pg. 42
Fig 18: FID signal before and after transformation Adopted from (http://pslc.ws/macrog/nmrsft.htm)

Spin-spin coupling: The FID spectrum after undergoing transformation gives

rise to series of peaks seen in figure 18. Single peaks, double peaks and larger

groups of peaks are observed. The group of peaks observed are due to the

coupling effect of hydrogen atoms on one carbon on another hydrogen atom on

an adjacent carbon atom.11. This is due to the small differences in magnetic field

experienced by each nucleus or group of nuclei as a result of the different spin

states of neighbouring nuclei. This coupling "splits" the signal into the multiple

peaks seen in the spectrum. This splitting follows Pascal’s triangle and the "N

plus one rule", which states that the number of peaks seen for each type of

hydrogen is equal to the number of hydrogen atoms on adjacent nuclei (N) plus

one. For example, the spectrum shown in figure 18 is that of ethyl acetate, the

structure of which is H3C-COOCH2CH3. The peak at 1 corresponds to the hydrogen

atoms on the CH3 group. It is split into three peaks (triplet) by the hydrogen

atoms on the CH2 group (2+1=3). The peak at 4 is the peak for the hydrogen

atoms on the CH2 group. It is split into four peaks (quartet) by the hydrogen

atoms on the CH3 group (3+1=4)11.While the single peak at 2 belongs to the 2nd

CH3 which is not coupled to any other hydrogen atoms.

pg. 43
 Figure 19: Pascal’s triangle (Adopted from

http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/nmr/nmr1.htm)

13 13
C spectra are inherently more complex than proton spectra due to C-H

coupling and the detail of multiplets is less easy to observe. Hence it is common

practice to record these spectra whilst instrumentally decoupling the interacting

13
nuclei so that each C resonance appears as a singlet.

1.5: Bioassay test

A bioassay test is simply a test to determine the relative strength of a

substance by comparing its effect on a test organism with that of a standard

preparation. Bioassay tests are either qualitative or quantitative. For qualitative

bioassays, assessment of the physical effects of a substance is all that is

required while for the quantitative bioassay-the concentration of potency of the

substance is estimated by measurement of the biological response produced. 13

There are various analytical approaches to using bioassays e.g. screening

approach (disk diffusion),dilution approach and toxicology approach 13. Due to the

scope of this project, I will only focus on the agar disk diffusion assay which I

used for all the bioassay tests.

Agar disk diffusion assay is the simplest and most common method used to test

for antimicrobial activity. In this method, a cell free culture broth, culture extract,

or purified compound is applied in solution to a small paper disk (6-7mm

diameter). The disk is allowed to dry and then placed on an agar plate that has

pg. 44
been laced with a test microorganism. The plate (along with the paper disk) is

incubated at conditions appropriate for the microbe's growth. This assay relies

on the diffusion of the test material through the agar where it contacts the test

microbe and, if active, a zone of inhibited microbial growth (clear zone) around

the disk is produced (figure 20). Disk diffusion assays are easy to run, requiring

only small amounts of material and no complex equipment. Their ability to

rapidly identify active components makes them especially useful in the initial

screening for antimicrobial activities and as a means of following activity during

chemical purification.

Figure 20: Agar plate showing inhibition zones as a result of bioactivity of components on the agar

disks (adopted from http://www.answers.com/topic/antibiotic)

To prepare for this assay the medium is typically inoculated either by adding a

liquid microbial culture to molten agar or by directly applying a culture to the

surface of a solidified agar plate with a sterile swab or glass spreader. The

purpose of inoculation is to obtain sufficient and reproducible microbial growth,

pg. 45
so that following incubation, the plate becomes turbid except for any area

around the disk where growth is inhibited. The size and age of the inoculants,

incubation, temperature, growth medium, medium volume and inoculation

method must be modified depending on the microorganism tested. Once

appropriate conditions have been selected, reproducibility will be maximized if a

standard protocol is followed and solvent controls and standard antibiotic disks

are run for each experiment. Antibiotic activities are generally reported as the

diameter of the zone of inhibited microbial growth around a disk.14

CHAPTER TWO

LITERATURE REVIEW

pg. 46
2.1. INTRODUCTION

This literature survey aims to look at the isolation and structure elucidation of

natural products from published research papers. It’s common knowledge that

extraction of a small amount of material for initial biological and chemical

characterization entails a lot of throughput and precision especially during the

preparation stage and sample preparation has to be applied before separation

can take place so as not to damage the chromatographic equipment. Although a

lot of research has been carried out in this field, there are still a multitude of

unknown natural products which are yet to be investigated and the very dynamic

nature of the metabolism of Fungi and Bacteria species give rise to the

opportunity of discovering novel natural products. After a library and internet

search on books on the isolation and characterization of metabolites, I found out

that the fundamentals for isolation are the same in all cases with only a slight

difference in the manner of approach and of course developments in the

chemical techniques used. In this chapter I would be focusing mainly on the

aspects of isolation, bioassay tests and structural characterization of different

fungal species from published papers.

Fungi species

Aspergillus

Chaetonium
Table 3: List of fungi used for the literature review

2.2. Aspergillus metabolites

2.2.1. Metabolites from Aspergillus Flavus15: The general concept of

mycoparasites producing antifungal agents is not new but the number of cases

investigated from a chemical standpoint is quite small. In this study by Wicklow

etal15, the mycoparasite Humicola fuscoatra NRRL 22980 was isolated from a

pg. 47
sclerotium of Asperigillus flavus which had been buried in a cornfield. The

isolation procedure was carried out as described by Wicklow et al16.

Cultivation of the fungus: Prior to Isolation, the fungus was grown on slants

of PDA for two weeks at a temperature of 25oC. A hyphal fragment spore

suspension prepared from the PDA slants served as inoculants to be introduced

to flasks containing fermented autoclaved rice.3 ml of the hyphal fragment spore

suspension was introduced to the contents of the flask and incubated for 40 days

at 25oC.

Isolation procedure: 5 compounds were eventually isolated from the ethyl

acetate extract. Compound 1 was isolated thus: 2g of the ethyl acetate extract

was dissolved in 80:20 water/ methanol mixture and the resulting solution is

extracted sequentially using Hexane and Chloroform (CHCL3), 50ml (two times

each). The CHCL3fractions were combined and evaporated to give a residue

(212mg).The residue is then subjected to a fractionation process on a Sephadex

column, using varying ratios of acetone-CH2CL2 and CH2CL2 -Hexane followed by a

complete 100% Methanol wash. The fraction eluted using 4:1 acetone- CH2CL2

(26mg) was then purified by semi preparative reverse phase HPLC to give

Monorden analog 1(compound 1), whose structure was determined by analysis

of 1H NMR.13C NMR, 2D-NMR and mass spectral data.

HO

CH3
O O

HO

O
Cl
OH

Figure 21: Structure of Monorden Analog 1(compound 1)*

pg. 48
 Compound 2 and 3 were isolated as follows; 7g of the initial ethyl acetate

extract was purified on a silica gel vacuum liquid chromatography column eluting

with 1:9 hexane-CH2CL2, followed by a step gradient of methanol-CH2CL2 in which

methanol ratio is increased subsequently.

The fractions eluted with 1:99 methanol-CH 2CL2 were combined on the

basis of their TLC behaviour with 3:2:1 hexane-CHCL3-Methanol as the eluent.

These combined fractions were further fractionated on a column of silica gel with

a step gradient ethyl acetate-CH2CL2 (5:95, 20:80, 40:60, 20:80), Methanol-

CH2CL2 (1:99) and ethyl acetate-CH2CL2 (10:90). Fractions eluting with ethyl

acetate-CH2CL2 (20:80, 40:60) were combined and 70mg of the resulting material

was purified by semi preparative reverse phase HPLC (dynamax 5 micrometer

particle size C18 column; 40 to 80% acetonitrile in 0.1% HCOOH in 20min) to

produce Monorden (compound2) and Monocillin IV (compound 3).The 2

compounds were distinguished by comparison of their 1H and 13

C NMR chemical

shifts and mass spectral data with published values from Ayer et al17.

CH3 O
O O

HO

O
Cl
OH
Compound 2

CH3
O O

HO

OH Compound 3

pg. 49
Figure 22: structures of Monorden (compound2) and Monocillin IV (compound 3) respectively *

Compounds 4 and 5 were isolated as follows. Using the fraction eluting from the

VLC column with 15:85 Methanol-CH2CL2 (367mg) was fractionated on a silica gel

column with a step gradient of Methanol- CH2CL2 in varying ratios and volumes.

The fraction eluting with 15:85 Methanol- CH2CL2 (200ml) on trituration with

Acetone gave an insoluble portion in acetone which was then purified by semi

preparative reverse-phase HPLC to produce Cerebrosides C and Cerebrosides

D (compound 4 and 5 respectively). The structures of compound 4 and 5 were

elucidated by comparison of their 1H NMR,13C NMR and mass spectral data with

published values from Sitrin Et al18

CH3
HN
HO
OH
O CH3
HO O
HO
OH OH CH3

Compound 4

O
CH3
HN
HO
OH
O CH3
HO O
HO
OH OH CH3

Compound 5

Figure 24: structures of Cerebrosides C and Cerebrosides D (compound 4 and 5 respectively)*

Bioassay tests: Bioassay of the extractable residue: following incubation, the

fermented rice substrate in each fernbach flask was first fragmented using a

spatula and extracted 3 times with ethyl acetate (200ml each time).The

combined ethyl acetate extracts were filtered and evaporated, 6mg of the

pg. 50
residue was re-dissolved in ethyl acetate for anti-fungal activity assays while the

remaining dried extract was stored at -20oC. 1 and 0.5m of extractable residue

are dissolved in methanol and pipetted onto individual analytical grade filter

paper disks in Petri dish lids and dried for 30 minutes in a laminar flow hood.

Four disks were placed on the surface of fresh yeast malt agar containing 22%

glycerol as modified by Nout19. Pure compounds were evaluated for antifungal

activity by placing 0.25mg onto individual paper disks and the observed results

are as follows.

Bioassay Results: Ethyl acetate of the fermented rice cultures inoculated with

Humicola fuscoatra displayed potent antifungal activity on agar plates seeded

with A.flavus. Three of the major components at 250ppm each showed potent

activity as measured by zone of inhibition.

Name of Bioassay test Zone of

compound on A.flavus inhibition
Monorden bioactivity
Compound 1 Analog observed 5mm

bioactivity
Compound 2 Monorden observed 13mm
bioactivity
Compound 3 Monocillin IV observed 13mm
Compound 4 Cerebrosides C No bioactivity Nil
Cerebrosides
Compound 5 D No bioactivity Nil

Table4: The major components and their bioassay results.

2.2.2. Metabolites derived from Aspergillus ustus: In this next paper

by Liu et al20, the isolation and structural elucidation of drimane

sesquiterpenoids obtained from Aspergillus ustus which was isolated from

the marine sponge Suberites domuncula is discussed. Liu et al were able to

isolate seven new drimane sesquiterpenoids along with already known

compounds deoxyuvidin B (compound 4), strobilactone B(compound 5) and RES-

pg. 51
1149-29(compound 10).Cultivation of the fungus: The fungus was cultivated at

22oC for 21 days on both biomalt agar and barley spelt solid media and the

cultures were lyophilized and extracted with ethyl acetate. The dried residues

were defatted by petroleum extraction. This crude extract showed reasonable

cytotoxic activity against murine lymphoma cell line L5176Y which prompted the

research.

Isolation procedure: The crude ethyl acetate extract was fractionated using

VLC on a silica gel using CH2Cl2-MeOH gradient elution which gave 10 fractions.

Four of the fractions (2, 6 ,7 and 8) were subjected to Sephadex LH-20 eluting

with CH2Cl2-MeOH(1:1) and further purified by silica gel column

chromatography(2 and 8) and semi preparative HPLC(6 and 7).20

Structural elucidation of the purified compounds: Spectroscopic analyses

used include 1 and 2D NMR spectroscopy and high resolution MS. Unlike the first

paper reviewed, Liu et al showed the correlation between 1H NMR and 13

C NMR

as used to structurally characterize the compounds of interest but due to time

constraint and also space, I would only discuss in detail one or two structures

elucidated.

OH OH
CH3 CH3
H CH3 HO CH3
OH
OH

HO H
H H
H3C CH3 O H3C CH3 O

Figure 24: Structures of compounds 1 and 2 respectively

Compounds 1 and 2 both have the same molecular formula C 15H24O4 as

assigned on the basis of HRESIMS. The 1H NMR spectrum displayed resonances

for four tertiary methyl groups, an oxymethylene, an olefinic proton and three

pg. 52
exchangeable protons while the only difference between the two is the position
13
of the hydroxyl substituent on the ring in compound 2. C NMR spectrum

showed 15 carbon signals including those assigned to a ketone carbonyl group, 2

olefinic carbons, 3 oxygenated carbons, 4 methyls and 5 sp 3 carbons. The

molecular formula accounted for 4 degrees of unsaturation, so it was suggested

that 2 rings in association with a double bond and a carbonyl group were

present. 1H NMR data of both compounds were compared and found to be similar

to 9, 11-dihydroxy-6-oxodrim-7-ene (Hayes et al21), thus indicating the presence

of drimane sesquiterpenoid skeleton. . It is seen from figure 24 that hydroxyl

substituent in compound2 is located on the 2 nd Carbon of ring 1 while in

compound 1 the OH is on the 3rd carbon. Compounds 6 and 7 have their

molecular formulas to be C21H26O7 and C21H26O6 respectively and on the basis of

13
C NMR compound 7 was found to be similar to 6, except for the presence of a

terminal aldehyde group in the side chain of 7 instead of a carboxyl group. This
1
was supported by H-1H COSY experiments. The NMR data for 6 was also

compared to those for compounds 10 and 5 and it was revealed they all shared

the same drimane sesquiterpene nucleus except for the side chains which are

different.

O
O
CH3
R1
OH

H
H3C CH3 R2

Compound 5: R1= OH, R2= H

O
OH
H3C
Compound 6: R1= H, R2= O

pg. 53
 O
H
H3C
Compound 7: R1= H, R2= O

CH3
Compound 10: R1=H, R2= H3C

Figure 25: General structure/s and the respective side chains of compounds 6-7 and 10*

Bioassay: The same cell line used on the initial crude ethyl acetate extract was

used for the isolated compounds. Only compounds 6, 7 and 10 showed cytotoxic

activity at concentrations 0.6-5.3 ug/mL with compound 7 being the most active.

The bioactivity of these three compounds were explained to be attributed to

their structural features, which included an olefinic ester side chain comprising of

2(compounds 6 and 7) or 3 conjugated olefinic double bonds (compound 10) with

a terminal carboxylic, aldehyde or methyl substituent. Compound 5’s lack of

cytotoxic activity was reported to be due to the lack of an ester side chain as

seen in figure 25.

2.3. Chaetonium spp metabolites: “Chaetomium is a dematiaceous

filamentous fungus found in soil, air, and plant debris. As well as being a

contaminant, Chaetomium spp. are also encountered as causative agents of

infections in humans. Some species are thermophilic and neurotropic in nature”22

2.3.1. Metabolites derived from Chaetonium sp: In the following paper by

Debbab et al23 he and his co workers carried out a study on the bioactive

metabolites extracted from the endophytic fungus Chaetonium sp which was

isolated from Salvia officinalis (a plant that grows in morocco). Prior to

extraction, fresh stems of S.officinalis was rinsed in sterilized distilled water and

pg. 54
subjected to surface sterilization by immersing in 70% ethanol for 2 minutes.

This was followed by rinsing again twice in sterilized distilled water, after which

the stems were cleaved aseptically into small segments and placed on a Petri

dish of malt agar medium containing an antibiotic used to suppress bacterial

growth. The stems were incubated at room temperature and after several days

hyphae growing from the plant material were transferred to other plates,

incubated again for 10 days and periodically checked for culture purity. The

isolated fungus strain was then grown on solid rice medium at room temperature

for 40 days.

Extraction procedure: The culture was extracted with 300 ml ethyl acetate

(twice) and the extract was dried and partitioned between n-hexane and 90%

MeOH. The 90% MeOH fraction was evaporated to yield an extract weighing

220mg. This extract was chromatographed over a sephadex LH-20 column with

100% MeOH as solvent. Based on the TLC characteristics using a solvent system

of MeOH: DCM (5:95), collected fractions were combined and subjected to semi

preparative HPLC using a Eurosphere 100-10 C18 column with a gradient of

acetonitrile and H2O.

Structural elucidation: The identity of the isolated compounds (1 and 2) was

determined through the use of UV, NMR and ESI-MS techniques. The UV

spectrum showed signals at wavelengths (227.7, 279.8 and 471.4 nm) indicating

presence of an indole chromophore. Positive and negative ESI-MS showed

molecular ion peaks at m/z 507.3[M-H]+ and m/z 505.7[M-H]- respectively

indicating a molecular weight of 506g/mol. The 1H NMR spectrum showed pairs

of chemically equivalent groups due to the symmetry of the molecule; also

aromatic proton and carbon resonances had chemical shifts and multiplicities

consistent with the presence of a di-substituted indole residue.

pg. 55
 H 3C
CH 3

CH3
O
NH
O
NH CH3
OH OH

HO
HO
O
H3C
N O
H N
Compound 1:Cochliodinol
H
H3C Compound 2: Isocochliodinol
H 3C CH3

Figure 26- structure of the Cochliodinol and Isocochliodinol

Combination of all the results from the spectrometric methods used led to the

proposal of C32H30N2O4 as the molecular formula and by comparing all the results

to published data for cochliodinol by Jerran et al24 the identity of compound 1

was confirmed to be Cochliodinol. UV spectrum for Compound 2 showed high

similarity to that of compound 1 while the positive and negative ESI-MS had m/z

(507.3, 505.7 respectively) indicating a molecular weight of 506g/mol identical to

compound 1. Proton NMR spectrum showed identical spin systems similar to

compound 1 with the mass spectrum supporting the presence of a 2-methyl-but-

2-enyl group thus compound 2 was proposed to be a symmetrical isomer of

Cochliodinol.

Bioassay: A micro culture Tetrazolium (MTT) assay was used to determine the

cytotoxicity of the isolated compounds against L5178Y mouse lymphoma cell

line. Compound 1 showed high activity against the cancer cell line (EC50 of

7ug/ml) while compound 2 showed weak bioactivity ( EC50 at 71.5ug/ml). The

observed difference in activity between the two compounds was reported to be

attributable to the position of prenyl substituents at the indole rings.

pg. 56
2.3.2. Isolation and structural elucidation of Metabolites from

Chaetonium brasiliense25: In this paper, Khumkomhet et al25reported a study

on antimalarial and cytotoxic depsidones obtained from the fungus Chaetonium

brasiliense (one of various chaetonium species found in Thailand). Ten depsidone

compounds (4 new depsidones and 6 known ones) and two known sterols were

eventually isolated and tested for bioactivity in the study.

Isolation procedure : Air dried Mycelial mat(300g) was ground and extracted

at room temperature with Hexane(700ml*3),Ethyl acetate(700ml*3) and

Methanol(700ml*3).The following crude extracts were obtained;

Hexane(6.8g),Ethyl-acetate(17.8g), Methanol(20.6g).

CH2Cl2-Hexane (35 and 300ml respectively) was added to the hexane extract

which gave a solid, which was re-crystallized from ethyl acetate to give

Mollicellin B (compound 5). The filtrate was evaporated to give a residue. This

residue was subjected to flash column chromatography eluted with a gradient

system of Hexane-ethyl acetate and six fractions were collected. The filtrate

from the second fraction was evaporated and subjected to silica gel flash column

chromatography with gradient system of Hexane-ethyl acetate to give

Ergosterol. The third and fourth fractions were purified using preparative TLC

using 20% ethyl acetate- hexane. Third fraction gave Mollicellin E (compound 7)

while additional Mollicellin B and Mollicellin K (compound 1) were obtained from

the fourth fraction. Fractions five and six were purified using silica gel flash

column chromatography with a gradient system of hexane-ethyl acetate. The

second sub fraction from fraction 5, after being subjected to the same

purification method used on fraction five, gave Mollicellin L (compound 2). The

first and third sub-fractions from fraction six were re-chromatographed using

pg. 57
flash column chromatography with 20% and 40% ethyl acetate-hexane

respectively. The first sub fraction gave additional amounts of Mollicellin B

(compound 5) and Mollicellin K (compound 1) while the third sub fraction gave

additional Mollicellin E (compound 7).

The initial ethyl-acetate extract was subjected to silica gel flash column

chromatography eluted with a gradient system of hexane-ethyl acetate and ten

fractions were obtained. Silica gel flash column chromatography (same gradient

system) was applied on all the fractions. The first fraction gave Mollicellin J, E

and N (compounds 10, 7 and 4 respectively). Fifth fraction yielded Mollicellin C,

B, M,N(compounds 6,5,3 and 4 respectively).The sixth fraction gave three sub-

fractions which were re-chromatographed (40 and 50% ethyl acetate-hexane) to

give Mollicellin L and H(compounds 2 and 9).The seventh fraction also had three

sub fractions. The first sub fraction was purified by preparative TLC using 20%

ethyl acetate-hexane to yield Mollicellin B and the second sub fraction was

subjected to FCC(50% ethyl acetate-hexane) to give Mollicellin F(compound 8).

The initial methanol extract (20.6g) was subjected to silica gel FCC eluted with a

gradient of hexane-ethyl acetate and ethyl acetate-hexane to give four fractions.

The second and third fractions yielded Mollicellin C (compound 6) and Mollicellin

E (compound 7) respectively.

Structural elucidation: Spectroscopic data from IR, 1H and 13

C NMR, 2D NMR

and MS were used along with data from published values. IR was used to

ascertain the types of functional groups (such as hydroxyl, carbonyl ester,

aromatic aldehyde, alpha and beta unsaturated ketone and aromatic groups)

present in the molecule of the isolated compounds. The four new depsidones

were reported to be Mollicellin K-M (compounds 1-4), this was concluded on the

pg. 58
basis of the results from the spectrometric data. 1HNMR data for compound 1

showed 21 signals which are attributable to thirteen sp 2 quaternary, four sp2

13
methine and four methyl carbons while the C NMR revealed the presence of

two aromatic rings in the molecule.NMR data for compound 2 was similar to

compound 1 except for the presence of OCH3 at C7 in compound 2 replacing the

OH group in 1.

O O
CH3 CH3
CH3 CH3
O O
O O
CH3 CH3

HO HO
O O
7 CH3 7 CH3
O H OH O H O CH3
Compound 1 Compound 2

Figure 27: structures of Mollicellin K and L (compounds 1 and 2 respectively)

13
C NMR spectrum of compound 3 showed 21 signals attributable to 13 sp2

quaternary, one sp3 quaternary, two sp3 methine and four methyl carbons. The

proton spectra showed only one aromatic singlet signal and two aromatic methyl

substituents which were different from compound 1. As for compound 4, its

13
reported that the proton and C NMR data were similar to that of compound 3

with four singlet methyl, 1 methylene and 2 methine groups however groups

substituted on the aromatic ring and chromophore units of compound 4 were

located at different positions than in compound 3.

pg. 59
 CH3 O
CH3 O
CH3
Cl O OH
2 H O
O 2

HO
O O
HO CH3
O
O H
O CH3
CH3 O H
H3C
Compound 3 Compound 4
O

Figure 28: structures of Mollicellin M and N (compounds 3 and 4 respectively)

Bioassay test: four different types of assays were carried out on the isolated

compounds which includes antimalarial assay using the parasite Plasmodium

falciparum, antimycobacterial using mycobacterium tuberculosis, antifungal

using Candida albicans and cytotoxic assays against human epidermoid

carcinoma(KB), human breast cancer, human cell lung cancer and

Cholangiocarcinoma cell lines. It was reported that only Mollicellin K-M (1-3), B,

C, E and J showed antimalarial activity with IC50 ranging from 1.2-9.1ug/ml

also only Mollicellin K showed moderate activity against mycobacterium

tuberculosis (12.5ug/ml) and potent activity against Candida albicans

(1.2ug/ml).Only compounds 1-10 showed cytotoxicity against KB cells,BC1,NCI-

H187 and the five Cholangiocarcinoma cells.

Discussion on the literature:

Metabolite from Aspergillus fungi- The Aspergillus specie illustrates a spectrum of

positive and negative aspects of fungi with respect to the environment and

disease. Some Aspergillus species produce enzymes which have important

industrial applications while other Aspergillus can produce mycotoxins – these

pg. 60
are often found in contaminated foodstuff and are hazardous to the consumer.

The 2 papers I presented showed how useful metabolites are isolated from this

fungi specie. Besides using the common analytical techniques, alternative

techniques were employed, example is the use of vacuum liquid chromatography

.Another paper which I found(but not discussed) illustrated how VLC is able to

separate both large and small sample sizes efficiently, rapidly and

inexpensively26. Both papers show the extensive use of sephadex LH-20 for

fractionation of the extract of interest. This made me look for more information

on this gel matrix. The general use of sephadex LH 20 is attributed to its wide

applicability in the fractionation of small bio molecules, lipids, steroids and fatty

acids. It has high reproducibility, chemical and physical robustness26. For the

structure characterization section I believe Liu et al were more thorough in the

manner by which they presented their spectroscopic data. They showed the
13
structural correlation between C and 1H with results obtained from the HRMS

while comparing their findings with that from other related papers.

Metabolites from Chaetonium spp-Both papers showed the extensive use of

methanol and in particularly ethyl acetate:hexane as excellent solvents for

extraction purposes. The isolation process described in the papers were quite

similar: Debbab et al23 made use of sephadex LH-20 column(described in the first

paper I reviewed for this section) and semi prep HPLC while Khumkomhet et al25

made use of FCC leading to a more intense isolation process. For the structural

determination sections in both papers: Should be noted that Debbab et al made

use of only UV, MS and NMR while Khumkomhet et al went a step further using

IR, NMR (both 1H and 13

C).Correlations were made between the aforementioned

spectroscopic methods and also to data from literature to confirm the identity of

the compounds of interest.

pg. 61
 CHAPTER THREE

EXPERIMENTAL

3.1 Instrumentation

LC-MS instrument: Agilent 1100 HPLC system attached to an Esquire 3000

electrospray mass spectrometer. Software –Agilent ChemStation (to operate

HPLC), Bruker Esquire Control (to operate MS), Bruker Data Analysis (to analyse

LCMS data).

Column –HICHROM ACE 3 micron C18 reverse phase column (2.1 mm x 100 mm).

Elution method – Eluted with 77% methanol, 33% water, 5 micro litre injection

Mass spectrometry method –Nebuliser gas – 20 l/min, drying gas – 6 l/min, drying
temp – 330 °C

pg. 62
Automatic tuning on m/z = 500, wide range

UV Lamp: TLC plates were viewed under the Spectroline(R) (model CM-10)

fluorescence analysis cabinet using a short UV wave length (254 nm).

NMR: NMR data was obtained using Bruker AV600 spectrometer located at

School of Biological and Chemical Sciences at Queen Mary, University of London

(QMUL).

3.2 Materials

All solvents used for preparative TLC and column chromatography include

Methanol (LC-MS grade), Ethyl acetate, petrolium ether (40-60) and chloroform.

All solvents were from Fisher scientific

Absorbents used for packing the column includes Silica 60A , 40-63 micron from

Fisher scientific and pure sand,40-100 mesh from Acros-organics.

TLC staining reagent was prepared from a mixture of 18ml Ethanol (96%), 1ml P.

Anisaldehyde (99%) and 1ml, (97%) Sulphuric acid. TLC plates used were Sil Gel

60 with a layer of 0.20mm (20*20cm) pre-coated ILC sheets (ALUGRAM(c)).

3.3 Experimental procedures

pg. 63
 Figure 29: second batch of fungi samples used

Two batches of fungi samples were used for the entire project experiment and a

purified sample from a previous experiment was structurally elucidated. Both

sets of Fungi samples were incubated on potato dextrose agar at a temperature

of 25oC for about 3 months prior to the experiment. The following analyses were

carried out on the fungi samples as follows.

Extraction step:

The foremost step was to obtain a crude extract from the fungi material.

This was achieved due to the solubility of secondary metabolites in organic

solvents and methanol is a very good solvent for this purpose. Five agar plates

containing fungi samples were scraped into a large beaker (2litre) and mixed

with a small quantity of methanol. The beaker was now filled to the 1L mark with

methanol and the contents in the beaker are hand-stirred thoroughly to ensure

that most of the fungi get dissolved into the solvent. The beaker along with its

contents were now placed on a stirrer hotplate (with a bar magnet) for one hour.

The mixture was now filtered into a Buchner flask using filter paper and a

Buchner funnel under a vacuum pump to obtain a clear golden filtrate as shown

in figure 31. About 500ml methanol was then added into the remaining residue

pg. 64
to dissolve any remaining fungi sample which may still be present on the

residue, this was now filtered the same way as mentioned earlier. The residue is

then discarded upon filtration.

Figure 30: Crude fungi extract

pg. 65
 Pre-concentration step:

The golden filtrate was now transferred into a round bottom flask (200ml) and

concentrated using a rotatory evaporator (Buchi rotavapor R200). After

evaporating a large portion of the solvent, the filtrate was now transferred into a

smaller round bottom flask (25ml), whose weight was recorded prior to this. After

most of all the solvent has gone off, the round bottom flask was placed under a

more powerful vacuum pump to get rid of all the solvent in the flask and a solid

residue was obtained. The weight of the dried sample can then be determined by

pg. 66
weighing the flask again.

Figure 31: crude fungi extract being concentrated using the rotavapor

pg. 67
Liquid/Liquid partition: This technique was used only on the 2nd batch of

fungal samples. The dried extract was partitioned between water (50 ml) and

chloroform (50 ml). The aqueous phase was extracted with two more portions of

chloroform (50 ml). The three chloroform portions were mixed together and

concentrated using the evaporator.

Purification step:

Initial TLC analysis on the 1st batch of fungi samples: The dried

sample was now re-dissolved in a minimal volume of methanol (5ml) and TLC is

used to determine the number of components present in the extract. Pre-coated

TLC plates were cut to an appropriate size. The resized TLC plates were now

prepared by using a pencil to draw a faint line at about 0.75cm from the bottom

edge and another at about 10cm to the top edge from the bottom edge. The

plate was now spotted using a prepared capillary tube. The TLC is carried out

using various ratios of Ethyl acetate-petroleum ether solvent system but the

following ratios gave distinct separations (30:70, 50:50, 70:30, 90:10 and 98:2.

10ml of each). After developing the plate in the TLC tank, the plate was now

visualized using either a staining reagent and or UV-light. The staining reagent

used in this experiment is a mixture of p-Anisaldehyde (99+%, MW=136.15), 1ml

Sulphuric acid and 18ml, Ethanol. After dipping the plate into the staining

reagent, a hot air gun is used to dry the plate using heat and hence spots can be

easily visualized.

Initial TLC analysis on the 2nd batch of fungi samples: TLC was

carried using the same solvent system as before. This time the concentrated

extract is dissolved in chloroform (1.5ml).The extract in aqueous solvent was

also analysed to determine if they both contain similar components. The solvent

ratio used is as follows ethyl acetate-petroleum ether (0:100, 10:90, 50:50,

90:10, 100:0).

pg. 68
 Fractionation step: In this stage, a column(1.30cm inner diameter) was set

up using silica gel at a height of about 14.8cm of the length of the column with

about 1.2cm layer of sand placed on top of the silica. Methanol is now used to

condition the column by running it through the column to obtain a homogenous

packing. After running the methanol out of the column, the starting solvent

system (for the 1st batch of fungi samples,30;70,Ethyl acetate-Petrolium ether)

was now added to the column and a hand pump is used to flush it through the

column while ensuring the silica portion of the column doesn’t get dried out. The

fungi extract is concentrated once more using the evaporator and this time, it is

dissolved in methanol (about 5ml). The starting solvent mixture which is still in

the column was now allowed to run through leaving about an inch of the solvent

above the sand layer. The extract dissolved in methanol is now added to the

column using a long pipette to ensure all the extract is successfully transferred

into the column. When this is completed, the remaining solvent system is now

poured back into the column and fractions from the first solvent system is

collected using test tubes. Although volume of fractions to be collected in the

test tubes is not usually specified, 10ml was collected in each test tube. TLC was

run parallel to the collection of the fractions to monitor the collection.

Recombination step: After the fractions from all the listed solvent systems

were collected, the fractions were recombined on the basis of their TLC

characteristics. These recombined fractions were now concentrated respectively

using the evaporator and weighed to determine the weight of the sample.

pg. 69
Bioassay step:

Preparation of the sample: 10 micro litres of each of the four recombined

fractions was drawn using a micropipette and placed on a sterile disk. The same

volume was drawn from the methanol solvent, which is used as a control disk.

Preparation of bacteria samples: 0.5ML of TSB was placed unto a micro

centrifuge tube and one inoculation loop of the stock bacteria sample was

scraped into the TSB container. The contents are now mixed together

thoroughly. 0.1ML of the TSB and bacteria mixture is placed unto the agar plate

and spread evenly on the surface of the plate. The disks containing the

recombined fractions where now placed on the bacteria laced agar plate along

with the disks containing methanol. This process is repeated for all the given

bacteria samples. Four bacteria samples were provided as follows: Pseudomonas

Diminuta, Eschericha Coli, Micrococcus Luteus and Bacillus Megaterium. The

prepared agar plates were now covered and sealed off with paraffin nylon to

prevent contamination.

pg. 70
 Preparation of fungi samples: A metal cork borer was used to sample the

fungal species. The sample is in a plug form which is placed in the centre of the

agar plate. A control disk and a triplicate of each recombined fraction were

placed unto the plate and sealed with parafilm. This process is repeated for all

the fungal species provided, the fungi provided are as follows: Pyremophora

Quenea, Fusarium Oxysporum, Rhizoctonia Solani and Trichoderma Harzianum.

All the prepared bioassay agar plates were now placed in the incubating room

(temperature of 25oC) for about a week.

Structural characterization step: In this step, the concentrated fractions are

now prepared for structural characterization using any of the available

spectroscopic techniques available. For use in LC-MS, the concentrated fractions

are prepared using an equal volume of methanol and water (50microLitres

respectively) with 10microLitres of the sample, all placed inside an HPLC vial.

This method is used to obtain information on the separate functional groups by

establishing the molecular formula and at this stage; it is often possible to

recognize the class of natural product to which the compound belongs.

Spectroscopic analysis was also carried out using NMR in order to identify the

underlying carbon skeleton of the compound.

pg. 71
Computing: For the mass spectrometry data, I made use of an online isotopic
pattern calculator version 4(developed by Junhua Yan*2).

NMR data was processed using a software called SpinWorks_v.255*3

CHAPTER 4

Results and Discussion

The most important step in the analysis of fungi metabolites is the

isolation/purification steps. This involves adding solvent to the fungi sample.

Most bioactive compounds are polar in nature hence the polar solvent draws out

the polar compounds into the solution after which the extracts are subjected to

TLC and column chromatography to isolate different components in the extract.

Both batches of fungi samples were extracted using methanol solvent. On

filtration both gave clear yellow crude extracts without any un-dissolved

particles. The extracts were then concentrated using the evaporator.

pg. 72
The 1st batch of fungi extract was semi purified using column chromatography

alongside TLC.

Weight(g)
Flask + dried
extract 23.0231
Empty flask 22.8238
Mass of extract 0.4252
Table 5: weight of the crude extract after concentration

TLC results on the crude extract using the following solvent system; ethyl

acetate-petrolium ether showed reasonable separations which was confirmed by

the Rf values calculated.

Solvent Mixture
Ethyl Petrolium
Acetate Ether Rf Values
Solvent Ratio 30 70 0.45
50 50 0.58
70 30 0.70
90 10 0.60
Table6: Rf values results for the solvent mixtures used for TLC

From the initial TLC profile, it was observed that the extracts contained mostly

polar components, using a less polar solvent mixture; it is seen as a dark spot at

the baseline of the plate. Trying to obtain optimum Rf values for the extract was

somewhat difficult using the simple TLC set up, Using 50:50(Rf value 0.58)

solvent mixture gave a fairly appreciable separation between the observed spots

but as the more polar solvent is increased(e.g. 98,100),the components get

eluted faster than expected leading to obscured separations.

pg. 73
 Column chromatography afforded the fractionation of the extract into six

different batches. These fractions were now recombined into four sets of

fractions based on their TLC characteristics. These fractions were then

concentrated using the evaporator and high pressure vacuum pump and their

individual weight was determined. This is summarised in table 7.

Recombined
Ethyl Acetate Petrolium Ether Fractions Weight(g)
30 70 11-15 0.0019
50 50 29-46 0.0078
70 30 47-49 0.0345
90 10 61-79 0.4654
Table 7: recombined fractions and their weights after concentration

Figure 32: The four fractions obtained after a round of column chromatography

pg. 74
Figure 33: TLC plates for fractions11-15 and 29-46 respectively

Figure 34: TLC plates for fractions 47-49 and 61-79 respectively

pg. 75
It is seen from figure that fractions 11-15 was clearly purified as shown by the

appearance of a single spot on the TLC plate. The TLC results of the other

fractions showed that further purification is required for a purified compound to

be obtained.

Results for the 2nd batch of Fungi samples: The second batch of fungi

showed more promise as the crude extract showed signs of bioactivity against

the test microorganisms used. The same extraction procedure used on the first

batch was employed in this case also. Liquid-liquid partition is employed here to

reduce amount of contaminants one might encounter during the purification

process and to augment the organic component in the extract.

Crude extract
Weight(g)
Flask + dried extract 23.2565
Empty flask 22.8335
Mass of extract 0.4230

Table 8: weight of the dried crude fungi extract before liquid-liquid partition

The liquid-liquid partition showed a distinct separation of the extract between

the aqueous and chloroform phases.

observation
chloroform
portion 1 yellowish solution
chloroform slightly turbid
portion 2 solution
chloroform
portion 3 colourless solution
brown coloured
water portion solution

Table 9: Visual appraisal of the liquid-liquid partition

The chloroform portions were combined and concentrated using the evaporator
and high pressure vacuum pump.

Chloroform
extract Aqueous extract
Weight(g)
Flask + 22.9296 61.0732

pg. 76
 dried extract
Empty flask 22.8851 60.8307
Mass of
extract 0.0445 0.2425
Table 10: weight of the dried chloroform and aqueous extract

TLC was used to determine if both the aqueous and chloroform extracts have the

same components. The results proved otherwise, as it is seen in figures 35 and

36, that the aqueous extract showed a dark spot which is not separated by ethyl

acetate-pet spirit solvent system(50:50 and 90;10 respectively). On the other

hand the chloroform extract showed 6 spots so we can safely assume that the

two extracts contain different compositions.

Figure 35: TLC plate for aqueous extract showing no signs of separation

pg. 77
 Figure 36: TLC plate for the extract dissolved in chloroform (10:90 and 90:10)

The TLC plates viewed under UV light (254 nm) showed no additional spots as

seen in figure 37

Figure 37: TLC plates viewed under UV light (254 nm).

Bioassay results: In preparation for the bioassay, the dried chloroform

extract was re-dissolved in 3ml chloroform while the aqueous extract was re-

pg. 78
dissolved in 24ml of distilled water. Bioassay results for the aqueous and

chloroform extracts are summarised in table 11.

Chloroform Water
Bacteria extract extract
No
P.diminuta No bioactivity bioactivity
No
E.coli No bioactivity bioactivity
No
M.luteus Bioactivity Observed bioactivity
No
B.megaterium Bioactivity Observed bioactivity
Fungal
species
No
P.quenea No bioactivity bioactivity
No
F.oxysporum No bioactivity bioactivity
No
R.solani No bioactivity bioactivity
No
T.harzianum No bioactivity bioactivity

Table 11: Bioassay results for the 2nd batch of fungi samples

Figure 38: Agar plate showing inhibition of the growth of the bacteria, M.Luteus.

pg. 79
 Figure 39: Agar plate showing inhibited growth of the bacteria, B.Megaterium.

Structural determination step: Fractions from the first batch of fungi samples

gave conflicting LCMS data. This is likely due to the presence of impurities in the

samples used indicating that the purification process might have been less than

perfect. The mass spectra obtained from the fractions were difficult to resolve

but it should also be noted that the equipment used was not in optimal working

conditions at the time which is a technical issue way beyond my control. No MS

analysis was carried out on the 2nd batch of fungi samples. The previously

purified sample was subjected to both LCMS and NMR analysis and the results

are as follows.

pg. 80
Chromatographic data: Base Peak Chromatogram for metabolite after 2

rounds of column chromatography.

Intens.
x10 5

1.5

1.0

0.5

0.0
0 5 10 15 20 25 30 35 Time [min]

Figure 40: Chromatogram showing a distinct peak

Mass spectrometry data: Positive ion ESI spectrum of the main peak shows

dominant ions at m/z 678(corresponding to [M+Na]) which is at a high intensity

and m/z 656.1 (corresponding to [M+H]) and 347.5 at a much lower intensity.

Intens. MS, 21.7-22.8min (#672-#717)

x104

678.2

1.5

1.0

0.5

656.1
347.5
0.0
100 200 300 400 500 600 m/z

Figure 41: Positive ion ESI spectrum of main peak

Expansion of the main spectrum(Figure 42) shows a series of doubly charged

ions of m/z 347.5,348.0 and 348.5.This is caused by ionic species such as H +,

Na+, K+ etc being attached to the neutral analyte in the positive ion mode. The

m/z difference between these peaks is 0.5m.u and taking the reciprocal value,

these doubly charged ions give a z value of 2. Mass of z is obtained from

pg. 81
m=347.5*2, giving a mass of 695.Comparing the mass of the ion and the un-

protonated low intensity peak of m/z 655.1 shows a mass difference of 40 which

corresponds to a calcium ion.

Intens. MS, 21.7-22.8min (#672-#717)

800
347.5

600

400

200

0
345.5 346.0 346.5 347.0 347.5 348.0 348.5 349.0 349.5 350.0 m/z

Figure 42: Expanded ESI spectrum

Looking at the expansion ms of the main peak, isotope peaks can be seen as

follows 678,679 and 680.1.These can then be used to estimate the number of

carbon atoms in the molecule.

Intens. MS, 21.7-22.8min (#672-#717)

x104

678.2

1.5

1.0

679.0
0.5

680.1

0.0
676 677 678 679 680 681 m/z

Figure 43: Expansion of the MS of main peak showing ion isotopes

Taking M peak=679 with an approximate intensity of ~30% and [M+1] peak=6

of intensity of ~10%

Number of C=10/30*100=33

Number of C atoms =33/1.1~ 30 carbon atoms

pg. 82
A list of accurate mass data for the [M+H]+ and [M+Na]+ ions measured by the

UCL Chemistry department MS service were provided along with a list of

potential molecular formulae. I used an online Isotope Pattern Calculator

(Developed by Junhua Yan, v4.0) to try to see if I could find a match between the

formulae given and what was observed. The closest match for the [M+Na] +

adduct was seen from the formulae C30H41N9O8Na which gave similar isotope

peaks to what was observed while for the [M+H] C23H54N9O8SCa gave a similar

peaks.

Now comparing the mass of a protonated C30H41N9O8Na and that of m/z for M+H

measured mass gives a difference of 0.16338.We can conclude that compound

gives rise to the following peaks m/z= 678 is C 30H41N9O8Na and m/z= 656 is

C23H54N9O8SCa, Hence the MW is C30H41N9O8

From this formula, the number of double bond equivalents can be determined

using equation 5

DBE= (2a+2)-(b-d)/2

Equa

tion 5

Where a=Carbon atoms, b= Hydrogen atoms and d= Oxygen atoms

DBE= (2[30]+ 2)-(41-8)/2= 14

This shows the molecule has 14 bond equivalents hence indicating the absence

of ring structures.

Also looking at fragment ions separated by particular values can help give hints

on possible structural information about this unknown compound.

The Tandem MS shows peaks 543 and 515 separated by mass of 28 implies loss

of C2H4 which is from ethyl esters or n-propyl ketones, aromatic ethyl esters are

ruled out as no information on benzene groups was found in the unknown

pg. 83
compound. Peaks 515.1 and 472.1 are separated by 43m.u, corresponds to a

loss ofC3H7 or CH3CO.Both losses imply the presence of propyl-ketone and methyl

ketone respectively so most likely there exist a ketone group within the molecule

of the unknown.

Peaks 472.1 and 430 also give a difference of 42m.u showing losses of CH2CO

and C3H6 accrued to methyl ketone or isobutyl ketone groups respectively.

NMR data: Looking at the overall 1H spectrum, no signal is observed above

8.5ppm so we can safely eliminate the presence of groups such as

-RCOOH(acidic),H-R=O(aldehyde groups) and benzene-rings. Exclusion of

benzene groups is consistent with the result of the double bond equivalent

calculated earlier which showed the presence of 14 double bond equivalents.

Previous UV analysis (which is not presented) showed no significant peaks

relating to the aforementioned groups so confirming the conclusion on that

strength. It should also be noted that the spectrum baseline is not clearly defined

sometimes leading to severe difficulties in assigning peaks to their

corresponding proton groups. To make interpretation a bit easier, the

SpinWorks_255 NMR program was utilized. The program has distinct features

and was able to help in the integration of the areas of the resonance peaks

.Using this version of spinworks also has its cons, as integral ratios calculated

sometimes gave conflicting values and hence extreme care was exercised during

this partial structural elucidation process.

1H NMR
chemical Integ J Value multipli
shift(ppm) ral H (Hz) city

pg. 84
 8.16 ~1 NH 7.5 Doublet
7.71 ~2 RCONH 3.2 Triplet
7.53 ~3 RCONH 3.5 Triplet
R2C=C
5.14 ~2 H2 10.5 Doublet
R2C=C
5.05 ~1 H2 5.3 Singlet
4.30 ~2 RNH2 10.8 Doublet
RCO2C
4.22 ~3 H 5.5 Quartet
3.16 Acetate singlet
3.0 Acetate singlet
2.22 ~4 Ketone 7.6 Triplet
1
Table 12: H NMR data

Looking at the 1H spectrum from the downfield region, I observed a doublet with

J=7.5Hz at around 8.16ppm and we can say it could be a proton from a NH

group. At 7.71ppm, a triplet with another peak conjoined to its base is seen.

Interestingly the outer peaks have similar coupling constant J value of 3.2Hz but

due to the closeness of the peaks a second order perturbation occurs. At 7.53,a

similar peak is seen, with the outer peaks having low J values of 3Hz indicating

identical protons experiencing 2nd order distortion. I believe most of these

signals might be from amide protons (RCONH). The large singlet peaks are

observed around 7.09 and 6.99ppm corresponds to the solvent used (CDCl3). A

doublet is seen at 5.14 with a high coupling constant of 10Hz corresponding to a

cis-proton alkene system, signal at 5.05ppm shows a singlet corresponding to

alkene group.

Upfield signals at 4.30ppm a doublet is seen but due to coupling with close by

protons, it is swamped at the base with undefined peaks with lower intensity.

Around 4.22ppm, a quartet is observed. I assume groups around this chemical

shift positions could be from ester groups. Singlet seen 3.66ppm is due to

impurities(e.g. plastics)2 large singlets are also seen at 3.16 and 3.0ppm

indicating protons from acetates. At 2.22ppm a triplet is seen, these could be

from ketone protons. At 2.02ppm, a quartet is observed with an undefined

pg. 85
baseline. Resonance signals from 1.68ppm to 0 appears to be swamped by

series of undefined peaks giving much distorted signals.

1H COSY spectrum: Up-field signals the signals between 0-1.8ppm seem a bit

daunting due to excessive distortion as shown in the spectrum(attached in

appendices),also cross peaks are difficult to ascertain at this position Downfield

region shows signals at the following positions-8.16, 7.71, 7.53. Cross peaks

between 7.7 and 7.53ppm may indicate coupling between the amide protons in

those signal positions. Signals at 5.3, 5.1 and 4.1ppm, show no cross peaks seem

to appear at this resonance positions. Cross peaks also appear between signals

2.85 and 2.59 showing connectivity between the protons.

The HMBC spectrum doesn’t display much information apart from the peaks at

3.16 and 3.0ppm which are connected to carbonyl groups at 173.7 and

169.8ppm.

pg. 86
 CHAPTER 5

Conclusion

For proper structural characterization of a natural product, certain requirement

has to be met, such as the purity of the compound and its characterization in

terms of elemental composition and molecular formulae. The criteria for purity

used in this project are as follows; Analytical criteria: LC-MS, Chromatographic

criteria: TLC and spectroscopic criteria: 1H NMR, 2D COSY, HMBC. Due to tight

schedule, time constraints, delays and unforeseen factors which are due to no

fault of mine, it seems I ended up not being able to fully maximise the vast

potentials of this particular project. The outcome, though not entire conclusive is

somewhat a starting point for future work. The purification process I embarked

on took more time than I imagined and after much repetition I got quite used to

the multi tasking required. The other thing I would like to point out regarding the

method of extraction I used is though I didn't quite manage to completely purify

the samples in my possession, I wished I had the time to embark on other forms

e.g. acidic, basic extraction which might have yielded different results. The

spectroscopic techniques, at least was able to give hints on types of functional

groups present in the molecule and I was able to rule out other groups which

were not observed. Regardless of all these shortcomings, the highlight of the

project was the positive results from the bioassay tests. I believe future work

could be carried out on fully identifying the components of the crude chloroform

extract that displayed bioactivities against the bacteria-M.luteus and

pg. 87
B.Megaterium. Being that the result from the bioassay was mainly qualitative. A

graded quantitative bioassay could also help in determining the relative potency

of the bioactive components.

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* structure redrawn using ACD Labs Chem-sketch free software, version

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pg. 90