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

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. 5

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

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

pg. 6

Section 4 Results and Discussion...................................................................62

Section 5 Conclusion..........................................................................................................75

References.............................................................................................................................76

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

pg. 7

LIST OF TABLES

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

11. Bioassay results for the 2nd batch of fungi samples………………………..................……67

12. Table 12:1H NMR data………………………..................……............................................73

pg. 8

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

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

15 Typical 13C chemical shift ranges…………………………………………….………...............33

16 NMR integration example ethyl acetate…………………………………………………………34

17 Schematic diagram of a NMR spectrometer……………………...............................35

pg. 9

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

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

pg. 10

37 TLC plates viewed under UV light (254 nm).………….….......................................67

38 Agar plate showing inhibition of the growth of the bacteria, M.Luteus….….....68

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. 11

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. 12

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:

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

pg. 13

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

chemistry1 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

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

pg. 14

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

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

pg. 15

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

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 bFigure 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)

pg. 16

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 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. 17

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. 18

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. 19

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.

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

pg. 20

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 PolarityWaterAcetic Acid (Ethanoic Acid)MethanolEthanolPropan-1-olAcetonitrile (Ethanenitrile)Ethyl Acetate (Ethyl Ethanoate)Acetone (Propanone)DichloromethaneChloroformDiethyl EtherToluene (Methyl Benzene)Cyclohexanen-Hexane

Low Polarity

Table 1: Elutropic series (adopted from http://www.rsc-teacher-fellows.net/labTechniques/ElutropicSeries.htm)

pg. 21

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.

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

pg. 22

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.

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.

pg. 23

Stationary phaseSeparation mechanism

Solid AdsorptionLiquid Layer PartitionIon exchange resin Ion exchangeMicroporous beads Size exclusionChemically 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.

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 mm 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.

pg. 24

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.

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

pg. 25

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

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:

pg. 26

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 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.

pg. 27

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 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.

pg. 28

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.

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.

pg. 29

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.

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 13P. 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. 30

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, 13C, 19F and 13P (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 3

V=γ /2 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 .

Methods of generating the NMR spectrum: There are two methods of generating NMR spectrum

namely; Continuous wave-NMR and Pulsed-Fourier transform-NMR.

pg. 31

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 13C 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 while

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

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

pg. 32

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(CH3)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. 33

Figure 15: Typical 13C 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 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.

pg. 34

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 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

pg. 35

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.

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

pg. 36

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.

Figure 19: Pascal’s triangle (Adopted from http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/nmr/nmr1.htm)

13C spectra are inherently more complex than proton spectra due to 13C-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 nuclei so that each 13C resonance appears as a singlet.

1.5: Bioassay test

pg. 37

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 approach13. 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 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.

pg. 38

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, 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

pg. 39

CHAPTER TWO

LITERATURE REVIEW

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

pg. 40

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 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.

pg. 41

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

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-CH2CL2 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 13C NMR chemical

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

pg. 42

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- CH 2CL2

(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

pg. 43

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 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 compound

Bioassay test on A.flavus

Zone of inhibition

Compound 1 Monorden Analog bioactivity observed 5mm

Compound 2 Monorden bioactivity observed 13mm

pg. 44

Compound 3 Monocillin IV bioactivity observed 13mmCompound 4 Cerebrosides C No bioactivity NilCompound 5 Cerebrosides 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-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 13C 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.

pg. 45

Figure 24: Structures of compounds 1 and 2 respectively

Compounds 1 and 2 both have the same molecular formula C15H24O4 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 exchangeable protons while the only difference between

the two is the position of the hydroxyl substituent on the ring in compound 2. 13C NMR spectrum

showed 15 carbon signals including those assigned to a ketone carbonyl group, 2 olefinic carbons, 3

oxygenated carbons, 4 methyls and 5 sp3 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 2nd Carbon of ring 1 while in compound 1 the OH is on the 3 rd carbon. Compounds 6

and 7 have their molecular formulas to be C21H26O7 and C21H26O6 respectively and on the basis of 13C

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 was supported by 1H-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.

pg. 46

Compound 5: R1= OH, R2= H

Compound 6: R1= H, R2=

Compound 7: R1= H, R2=

Compound 10: R1=H, R2=

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

pg. 47

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 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

pg. 48

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.

NH

CH3

CH3

NHO

OH

OH

O

CH3

CH3

Compound 2: Isocochliodinol

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. 49

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 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).

pg. 50

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 13C 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 basis of the results from the

spectrometric data. 1HNMR data for compound 1 showed 21 signals which are attributable to

thirteen sp2 quaternary, four sp2 methine and four methyl carbons while the 13C 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.

pg. 51

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

13C 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 reported that the proton and 13C 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.

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

pg. 52

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 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 inexpensively 26.

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

pg. 53

presented their spectroscopic data. They showed the structural correlation between 13C 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 13C).Correlations

were made between the aforementioned spectroscopic methods and also to data from literature to

confirm the identity of the compounds of interest.

pg. 54

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

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)).

pg. 55

3.3 Experimental procedures

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 to dissolve any

pg. 56

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. 57

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 weighing the flask again.

Figure 31: crude fungi extract being concentrated using the rotavapor

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.

pg. 58

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).

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

pg. 59

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. 60

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.

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.

pg. 61

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.

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

pg. 62

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.

The 1st batch of fungi extract was semi purified using column chromatography alongside TLC.

Weight(g)Flask + dried extract 23.0231Empty flask 22.8238Mass 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 Acetate Petrolium 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

pg. 63

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.

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.

Ethyl Acetate Petrolium EtherRecombined

Fractions Weight(g)30 70 11-15 0.001950 50 29-46 0.007870 30 47-49 0.034590 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. 64

Figure 33: TLC plates for fractions11-15 and 29-46 respectively

pg. 65

Figure 34: TLC plates for fractions 47-49 and 61-79 respectively

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 23.2565Empty flask 22.8335Mass 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.

observationchloroform portion 1 yellowish solutionchloroform portion 2 slightly turbid solutionchloroform portion 3 colourless solutionwater portion brown coloured 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 + dried extract 22.9296 61.0732Empty flask 22.8851 60.8307Mass of extract 0.0445 0.2425

Table 10: weight of the dried chloroform and aqueous extract

pg. 66

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. 67

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-dissolved in 24ml of distilled water. Bioassay

results for the aqueous and chloroform extracts are summarised in table 11.

Bacteria Chloroform extractWater extract

P.diminuta No bioactivity No bioactivityE.coli No bioactivity No bioactivityM.luteus Bioactivity Observed No bioactivityB.megaterium Bioactivity Observed No bioactivityFungal species P.quenea No bioactivity No bioactivityF.oxysporum No bioactivity No bioactivityR.solani No bioactivity No bioactivityT.harzianum No bioactivity No bioactivity

pg. 68

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.

Figure 39: Agar plate showing inhibited growth of the bacteria, B.Megaterium.

pg. 69

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.

Chromatographic data: Base Peak Chromatogram for metabolite after 2 rounds of column

chromatography.

0 5 10 15 20 25 30 35 Time [min]

0.0

0.5

1.0

1.5

5x10Intens.

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.

pg. 70

347.5656.1

678.2

MS, 21.7-22.8min (#672-#717)

0.0

0.5

1.0

1.5

4x10Intens.

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

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.

347.5

MS, 21.7-22.8min (#672-#717)

0

200

400

600

800

Intens.

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.

pg. 71

678.2

679.0

680.1

MS, 21.7-22.8min (#672-#717)

0.0

0.5

1.0

1.5

4x10Intens.

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

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 C30H41N9O8Na 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

Equation 5

Where a=Carbon atoms, b= Hydrogen atoms and d= Oxygen atoms

pg. 72

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 C 2H4 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 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

pg. 73

sometimes gave conflicting values and hence extreme care was exercised during this partial

structural elucidation process.

1H NMRchemical

shift(ppm) Integral H J Value (Hz) multiplicity8.16 ~1 NH 7.5 Doublet7.71 ~2 RCONH 3.2 Triplet7.53 ~3 RCONH 3.5 Triplet5.14 ~2 R2C=CH2 10.5 Doublet5.05 ~1 R2C=CH2 5.3 Singlet4.30 ~2 RNH2 10.8 Doublet4.22 ~3 RCO2CH 5.5 Quartet3.16 Acetate singlet3.0 Acetate singlet

2.22 ~4 Ketone 7.6 Triplet

Table 12:1H 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 baseline. Resonance signals from 1.68ppm to 0

appears to be swamped by series of undefined peaks giving much distorted signals.

pg. 74

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. 75

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 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.

pg. 76

REFERENCES

1. Bohonos, N., Piersma H.D., Natural products in pharmaceutical industry. Bioscience, vol 16,oct 1966, 706-729

2. Prescott, L.M., Harley J.P, and Klein D.A., Microbiology.5th edition.(2002)3. Hanson, R.J. Chemistry of fungi. RSC publishing, 20084. http://en.wikipedia.org/wiki/fungi5. Mohrig, J.R., Hammond, C.N, Schalz, P.F. Techniques in organic chemistry.2nd edition, Jan

20066. Targett , N, M, . Kilcoyne, J, P. Green, B. Vacuum liquid chromatography: An alternative to

common chromatographic methods. American chemistry society, Journal of org chem. Volume 44,No 26 pp 4962,jun 8, 1979

7. http://www.ebiotrade.com/GE/AKTAclub7/1.PDFs/LH-20.pdf8. Hoffman, E.D., Charette, J. and Stroobant, V., Mass spectrometry-Principles & applications.

John wiley and sons, 1996. pp 279. Sorrell,N.T., Interpreting spectra of organic molecules. University science books,1988, pp5310. Rouessac, F., Rouessac, A., Modern instrumentation, methods and techniques of chemical

analysis., John wiley.2nd edition,2007 pp 398-39911. Williams, D, H., Fleming , I., Spectroscopic methods in chemistry. Mcgraw hill.5th

edition,1995, pp 22612. http://www.wfu.edu/~ylwong/chem/nmr/h1/integration.html 13. Tortora, G.J., Funke, B.R., Case, C.L. Microbiology, an introduction. Benjamin/Cummings pub

co. 1997. pp 33514. http://en.wikipedia.org/wiki/Bioassay15. Wicklow, D.T., Joshi, B.K., Gamble, W.R., Gloer,J.B.and Dowd,P.F.,Antifungal metabolites

from Humicola fuscoatra Traaen NRRL 22980,Applied and Environmental Microbiology,Nov 1998 pg.4482-4484,Nov.1998

16. Wicklow,D.T,D.M. Wilson and T.C nelsen.1993,survival of Asperigillus flavus sclerotia and conidia buried in soil in Illinois and Georgia. Phytophytology 83:1141-1147).

17. Ayer W.A, S.P. lee, A Tsuneda and Y.Hiratsuka (1980) and Nozawa, K and S. Nakajima (1979).

18. Sitrin, R.D, Chan, G, J. Dingerdissen, C debrosse, R. Mehta, G, Roberts, S. Rottschaeffer, D. Staiger, J. Valenta, K.M. sander, R.J. Stedman, and J.R.E Hoover(1988).Isolation and structure determination of Pachybasium cerebrosides which potentiate the antifungal activity of aculeacin.J. Antibiot. 41:469-480.

19. Nout, M.J.R.(1996).Personal communication

20. Liu, H. Edraba-Ebel, R.Ebel, R.Wang, R. SChulz, B. Draeger, S. Muller W, E,G. Wray, V.Lin ,W.Proksch,P. drimane sesquiterpenoids from the fungus Aspergillus ustus Isolated from the marine sponge suberites domuncula. Journal of natural products(2009)

21. Hayes, M.A,; Wrigley, S.K,; I.; Reynolds, E.E.;Ainsworth, A.M.;Renno, D, V.;Latif, M.A.; Cheng, X.M.; Hupe, D.J; Charlton, P.; Doherty, A. M. J. Antibiot .1996, 505-512

22. Larone, D. H. 1995. Medically Important Fungi - A Guide to Identification, 3rd edition

pg. 77

23. Debbab, A., Aly , A, A., Edrada-Ebel ,R,A., Muller , W,E,G., Mosaddak, M., Hakiki, A,. Ebel, R., Proksch, P. Bioactive metabolites from the endophytic fungus Chaetonium sp. isolated from Salvia officinalis growing in Morocco.Biotechnol.Argon.Soc.Environ.2009 13(2), pp229-234

24. Jerran et al., 1975.The chemistry of Cochliodinol, a metabolite of Chaetonium spp. Can, J. Chem., 53(5), 727-737

25. Khumkomkhet, P, Kanokmedhakul, S. Kanokmedhakul, K,. Hahnvajanawong, C. SOytong, K .Journal of natural products, may 26,2009

26. http://www.800mainstreet.com/e3/e3.html

* structure redrawn using ACD Labs Chem-sketch free software, version 5.0

*2-http://www.geocities.com/junhuayan/pattern.htm

*3-http://www.umanitoba.ca/chemistry/nmr/spinworks/

pg. 78