1. 1
PROJECT NAME:
The Biotransformation of Thujone, Thujaplicin and Derivatives
Project Summary Information
Project start Jan 1 2005
Project end Dec 31 2005
Costs $
Total Employees 312080
Employees specified 100000
Total Contractors 42000
Consumables 85000
Capital Lease Expense 210000
Total costs for year 749080
Employees $ Degree Years Experience
Brown Jeff B.Sc. 3
Chou Song B.Sc. 22
Grigor Spassov Ph.D. 28
Herrington Ted M.Sc. 21
Lazarov Manuela M.Sc. 18
Qian Linda B.Sc. 14
Stoynov Nikolay Ph.D. 22
Todorova Vaska Spasova B.Sc. 20
Wang Chang Qing Ph.D. 24
Zetina Carlos Ph.D. 22
Rose Jamani B.Sc. 18
Total Employees
Employees specified
Wade Reeves B.Sc. 20
Total Specified Employees
Contractors $ Degree
Ellis Consulting (Brian Ellis) B.Sc. 16
ISO Plus (Marino Middleton) M.Sc. 24
UBC Chemistry (various) Ph.D. n/a
Kumi Enterprises (Dr. James Kutney) Ph.D. 35
Total Contractors
Equipment
Lease non-capital
Lease capital
Total Specified Employees
Contractors $ Degree
Ellis Consulting (Brian Ellis) B.Sc. 16
ISO Plus (Marino Middleton) M.Sc. 24
Government Assistance
NRC IRAP government assistance 0
Other data
Total scientist hours employees
&specified

2. 2
Introduction
This project is a continuation of experimental work performed in 2004 by Cavendish Lab.
This discovery work is based on earlier research performed by Dr. James P. Kutney, Professor
Emeritus of Chemistry, University of British Columbia. In this project Dr. Kutney's extensive and
specialized experience was utilized as scientific advisor.
In this project, the essential strategy is to transform abundant natural and inexpensive
phytochemical raw materials into higher value products. The "raw" phytochemicals used in this
project, Thujone and Thujaplicin, are found in abundant quantities in the foliage and heartwood of
British Columbia Western Cedar. From the literature, Thujone, Thujaplicin and known synthetic
derivatives of Thujone have been shown to exhibit antimicrobial, insecticidal, perfumery and other
industrially useful properties (Figure 1,3).
The objective of the project is to produce novel and patentable compounds with increased or
unique activity as compared those known using fermentation (Figure 2.) in combination with
chemical synthesis (Figure 3). To pursue this objective, process development is required in the
areas of biotransformation, chemical synthesis, extraction, separation, purification and analytical
chemistry.
Novel compounds will be studied for usage as insecticides, anti-feedants, insect repellents,
cosmetics, perfumeries, antimicrobial agents among other applications.
Experimental work was performed at Cavendish Lab's specialized research facility which includes
fully equipped analytical, synthetic chemistry, biotransformation, inorganic and microbiology
laboratories. Support facilities were provided by the UBC Department of Chemistry and
Biological Services.
Figure 1. Utilization of Phytochemicals from Western Red Cedar
COMMERCIAL
FOLIAGE
THUJONE
STEAM DISTILLATION
AGRICULTURAL
VALUABLE INTERMEDIATES FOR
PERFUMERY
COSMETICS PHARMACEUTICALS

3. 3
Figure 2. Postulated Biotransformation of Thujone and Synthetic Derivatives
into Novel Products
The Thujone skeleton allows for the chemical modification at the R position from which
many derivatives can be produced. These chemical derivatives are used as substrates
for biotransformation in the attempt to produce novel compounds not possible via
synthetic chemical routes.
R
Thujone
Fungi
(Aspergillus sp., Rhizopus sp.,
Fusarium sp.)
R
OH
R
OH
R
OH
R
HO
Thujone, R =
Thujanol connamoyl ester, R =
Thujanol benzoyl ester, R =
O
O
O H
O
H
O

4. 4
Figure 3. Synthetic Derivatives for Use as Biotransformation Substrates
This illustration describes some known synthetic derivatives of Thujone of industrial importance.
Novel products produced via biotransformation may exhibit similar and enhanced industrial
properties.
O
1
2
3
4
5
6
thujone
OO
HO
O
O
H
OH
H
CHO
CHO
O
O
O
HOOC
OH
geraniol
(perfumery)
2
H C
H
OR
O
pyrethroid insecticides
(agriculture)
ambrox
(perfumery)
KMnO4
3
1
4
O
digitoxigenin
steroids and analogues
(pharmaceutical)
damascones
(rose oil)
(perfumery)
(+)- β-cyperone
sesquiterpenes
(perfumery)
polygodial
(agriculture)
O
MVK
KOH KOH
EVK

5. 5
Abbreviations and special terms used in this report
Abbreviation Definition Use
Biobundle Controlled Fermentation Device Apparatus used to bio-convert substrates
FTIR Fourier Transform Infrared Spectrometer Chemical structure analysis
GCFID Gas Chromatograph Flame Ionization Detector Non polar semi-volatile compound analysis by retention time
GCMS Gas Chromatography with Mass Spectrometer As GCFID with molecular structure information
HRMS High Resolution Mass Spectrometry Determines precisely molecular weight
LCMS Liquid Chromatography Mass Spectrometer Polar and higher molecular weight analysis
LCUV Liquid Chromatography Ultraviolet Detection Polar compound analysis - ketones
NMR Nuclear Magnetic Resonance Functional group analysis - bond orientation
TLC Thin Layer Chromatography Rapid bench-top method to determine product formation

6. 6
A. Scientific or technological objectives
In this project we seek to:
• Biotransform thujaplicin, thujone and chemical derivatives (substrates) to produce
compounds of potential pharmaceutical and industrial importance
• Find fungi, bacteria and/or yeast capable of biotransformation of the selected substrates
• Develop processes for the bioconversion using selected organisms (above)
• Develop methods to monitor the biotransformation process - substrate and products, i.e.
TLC
• Develop methods to isolate and purify biotransformation products
• Develop analytical methods as required for the identification of the purified products using
analytical techniques including FTIR, LCUV, GCMS, LCMS and NMR
• Develop and optimize successful processes for larger scale production for selected
products
• Establish useful fragrance, antibiotic, anti-fungal or insecticidal properties for novel
compounds produced

7. 7
B. Technology or knowledge base level
In the previous period the following conclusions were made:
• The biotransformation of the terpenes, thujone and thujaplicin was possible
• Thujone and thujaplicin synthetic derivatives (such as acetate) were successfully
biotransformed into several polar compounds
• Small-scale processes for the bioconversion of the substrates (above) with identified
organisms were developed
• The biotransformation products were identified as polar terpenoids using in-house
developed TLC and LCUV methods
• Thujaplicin acetate was converted to compounds with antibacterial activity, however the
methods to monitor processes were not successfully developed and work in this area was
discontinued. Nickel, cobalt, manganese and copper chelation led to an inhibition in the
growth of the organisms under study and research in this area was subsequently
abandoned
• Fungi evaluated in the period (2004) for their ability to biotransform the selected
substrates is described below (Table 1)
Table 1. Biotransformation Screening - Product Formation Results
Organisms
Thujaplicin
Acetate
Thujaplicin Fe
Chelate
Pseudomona putida F39/D no products not tested
Nocardioides simplex ATCC 13260 no products not tested
Nocardioides simplex ATCC 6946 no products not tested
Pseudomonas resinovorans ATCC 14235 no products not tested
Streptomyces platensis NRRL 2364 products not tested
Curvularia lunata ATCC 12017. products not tested
Aspergillus niger ATCC 11145 no products not tested
Aspergillus niger ATCC 9642 products products
Aspergillus ochraceus ATCC 18500 products products
Cunninghamella echinulata ATCC 9244 no products not tested
Epicoccum humicola ATCC 12722 products not tested
Epicoccum neglectum ATCC12723 products not tested
Note: Bold indicates organisms selected for further study.

8. 8
Technology or knowledge base level
• The biotransformation products were analyzed throughout the process using methods
developed in-house for TLC, LCUV,LCMS, FTIR and NMR
• Processes were developed and optimized for bioreactor biotransformation of thujaplicin
acetate to produce larger quantities of products
• Data was accumulated for future scale-up (i.e. 50L reactor)
• Products were isolated from the crude extract and several polar extracts were
successfully stabilized using acetylated Thujaplicin
• Products were found to be a variety of Thujaplicin chelates
• Antibiotic activity was established for crude product extracts and several isolated products
• Anti-fungal properties were observed but not studied in detail
• Insecticidal and fragrance properties of biotransformation products remain unstudied
Problem areas and conclusions toward further development
Though several products of interest were produced, thujaplicin and chelates were found to be
toxic to the organisms studied. This toxicity leads to low product yield making commercial
production impossible.
An alternate approach is to reduce the toxicity to organisms under study. To this end, it was
proposed to use other synthetic derivatives which might be less toxic, improve product yield, and
lead to entirely new hydroxylations.

9. 9
C. Scientific or technological advancement
In this project the following technological advancements are sought:
• Biotransform thujaplicin, thujone and chemical derivatives (substrates) to produce novel
compounds of potential pharmaceutical and industrial importance
• Identify fungi, bacteria and/or yeast capable of biotransformation selected substrates into
novel compounds
• Develop new processes as required for the bioconversion using selected organisms
• Develop new methods to monitor the biotransformation process - substrate and products,
i.e. TLC as required
• Develop new methods as required to isolate and purify biotransformation products
• Develop analytical methods as required for the identification novel products using
analytical techniques including FTIR, LCUV, GCMS, LCMS and NMR
• Optimize successful small scale processes
• Scale up processes (above) to produce larger quantities of selected products for
characterization and chemical property study
• Establish useful fragrance, antibiotic, anti-fungal or insecticidal properties for the novel
compounds

10. 10
D. Uncertainties
It was uncertain that:
• Thujaplicin, thujone and chemical derivatives (substrates) would produce novel
compounds with important pharmaceutical and industrial properties
• Fungi, bacteria and/or yeast capable of biotransforming the selected substrates would be
found
• new processes required for the bioconversion of the selected organisms would be found
• new methods to monitor the biotransformation process - substrate and products - would
be effective or reliable given the nature of the product (i.e. polar / non polar / molecular
weight / stability, etc.)
• effective methods to isolate and purify biotransformation products would be found
• analytical methods could be found to identify the novel products using FTIR, GCMS,
LCUV, LCMS and NMR. For example the NMR technique is used to establish the position
of hydroxylation in the product and requires a very pure sample to achieve accurate
results. The best purity achieved within the developed methods might not allow for
successful NMR testing
• successful processes for larger scale production of selected products could be developed.
For example, higher concentrations of substrates might prove to be toxic to the organism
and limit yields to unacceptable levels
• novel compounds would have useful fragrance, antibiotic, anti-fungal or insecticidal
properties

11. 11
E. Description of work done in this taxation year:
Experimentation
Preparation of biotransformation substrates - Chemical synthesis and purification
In the previous period it was well established that micro-organisms are capable of biotransforming
both thujone and chemical thujaplicin derivatives. In this project a number of other chemical
derivatives are investigated. Substrates synthesized for use in biotransformation experimentation
include:
• Thujaplicin acetate
• Thujone
• Thujol-β
• Thujone cinnamoyl ester
• Thujone benzyl ester
The synthesized substrates also require purification as chemical reactions often lead to the
formation of several isomers and other compounds/impurities. Pure substrates are generally
preferred over mixtures because the number of experimental variables is reduced and cause and
effect is more easily deduced.
Thujaplicin acetate
Thujaplicin acetate was prepared from Thujaplicin (99% purity) via a chemical reaction with acetic
anhydride reagent according a known procedure. This reaction was performed in the presence
of an alkaline reagent (triethylamine). The crude Thujaplicin acetate was washed to remove
excess reagent with a mixture of ice-water/dichloromethane. The residual organic solvent was
then removed (evaporated) and the product was dried. Identity and purity of the substrate was
determined by LCMS, FTIR, HRMS and NMR. The molecular weight (206), chemical formula
C12H14O3 and purity (99% +) were confirmed.
This substrate was produced in batches of 5.0 g, 10g and 50.0g for the required
biotransformation experiments.
Thujone-β
Crude red cedar oil contains about 10% Thujone-α, 60% Thujone-β, among other terpenes.
Distillation using a roto-evaporator under semi-vacuum @ 100 -120 0
C yielded Thujone-β with a
purity of 95+% as indicated by TLC, IR, NMR and GCMS analyses.
1000 ml of Thujone-β was prepared for the required biotransformation experiments.
Thujol-β
Crude Thujol-β was prepared from Thujone-β using a known chemical procedure previously
established by Dr. Kutney. Purified Thujone-β was reduced using sodium borohydride in
methanol. Testing revealed that this reaction yields a mixture of Thujol-β along with lesser
quantities of stereo-isomers Thujol-α and Thujol-δ.
A method to separate the Thujol isomers from one another was not described in the literature.
Therefore experiments were conducted to obtain the required separation/purification process. A
number of separation approaches were tested. Experimental variables include solvent
(dichloromethane, chloroform, ethyl acetate, alcohols, etc), stationary phase (various silica gels),
temperature, pH and flow rate (pressure).

12. 12
A rapid test method to monitor the process was developed using TLC. From this experimentation
the following purification process was developed.
Column chromatography with Silica gel proved to be an effective way to separate the isomers.
The crude Thujol isomer mixture was dissolved in dichloromethane/methanol and passed through
the column with controlled positive air pressure. Fractions containing Thujol-β, Thujol-α, and
Thujol-δ were obtained. Solvents were removed using a roto-evaporator from the various
fractions collected.
Fractions were tested and identified as:
• 85% Thujol-β - a solid (crystallized)
• 10% Thujol-α - a liquid (ambient)
• 5% Thujol-δ - a liquid (ambient)
Isomer stereochemistry structures were confirmed by observing MS fragmentation data, proton
shift (NMR) and relative retention times (GC). Thujol-β purity was tested further by GCMS, FTIR,
and HRMS. The molecular weight was found to be 154, chemical formula to be C10H18O and
product purity @98%+.
Using the developed process, batches of 5.0 g, 10g and 50.0g of Thujol-β were produced for use
as a substrate in subsequent biotransformation experiments.
Thujone cinnamoyl ester
Purified Thujol-β was used as a starting material for the synthesis of Thujone cinnamoyl ester.
Using a method described in the literature, Thujol-β was added to cynnamoyl chloride in a base
environment (triethylamine) and dichloromethane (ambient) to produce Thujone cinnamoyl ester.
The Thujone cinnamoyl ester was dried and then re-dissolved in dichloromethane and
subsequently crystallized. This process yielded a colorless product with the desired purity (98+
%), molecular weight and chemical formula as indicated by GCMS, FTIR, NMR, and HRMS
testing.
50 grams of Thujone cinnamoyl ester was prepared for use as a substrate in subsequent
biotransformation experiments.
Thujone benzoyl ester
Thujol-β was also used as a starting material for the synthesis of Thujone benzyl ester.
Using a method described in the literature, Thujol-β was added to benzoylchloride in base
environment (triethylamine), dichloromethane (ambient) to produce thujone benzoyl ester. The
thujone benzoyl ester was dried and then dissolved in dichloromethane and crystallized.
This process yielded a colorless product with the desired purity (98+%), molecular weight and
chemical formula as indicated by GCMS, FTIR, NMR, and HRMS testing.
50 grams of Thujone benzoyl ester was prepared for use as a substrate in subsequent
biotransformation experiments.

13. 13
Experimentation
Organisms tested and chosen to bio-convert the selected substrates in shake flasks on a
small scale (200 mg level)
Organisms tested
• Aspergillus niger ATCC 9642
• Aspergillus ochraceus ATCC 18500
• Aspergillus niger ATCC 11145
• Fusarium oxysporum sp.
• Curvularia lunata ATCC 12017
• Septomyxa affinis ATCC 6737
• Rhizopus rhizopodiformis
• Nocardioides simplex sp.
• Trichothecium roseum
• Cunninghamela echinulata
• Pseudomona putida
• Pseudomonas resinovorans
• Beauveria bassiana
• Epicoccum neglectum
• Epicoccum oryzae
• Epicoccum humicola
Accumulate biomass of the organism
Selected organisms were cultivated according to the following recipe:
• Medium - glucose (1%), yeast extract (0.3%), peptone (1%), malt extract (2%), pH 6.5 not
adjusted
• 1000m Erlenmeyer flasks were filled with 250 ml of medium sterilized in autoclave and
have been rotated on shaker at 26-30 0
C for 72 hours at 150 RPM
• Air supply was not controlled
• Substrate was prepared in a methanol solution
• Amount of the substrate was 0.2 -1.0 g/L and the methanol used was 5 ml/L
• The time of substrate addition is dependent on the growth rate, i.e. the fungus used
• The appropriate biomass was accumulated in approximately 24 hours
These conditions were found to prevent a possible toxic effect of the solvent on the fungal growth
and insured substrate solubility in the broth.

14. 14
Table 2.
Experimental Variables of Biotransformation - The Cultivation of the Microorganism
Variables
Shaker Flasks
(1000 ml total volume)
BioBundle
(3 000 ml total volume)
Substrate concentration (g/L)
0.2-1.0 0.2-1.0
Inoculum volume
Volume/Volume medium (%) 10.0 10.0- 20.0
Nutrient Medium
Medium 1.
Peptone 1%, Yeast extract 0.3%,
Malt extract 2%, Glucose 1%,
pH 6.5
Medium 2.
Corn steep liquor 1%, Glucose 1%,
pH 6.4-6.6
Medium 1.
Peptone 1%, Yeast extract 0.3%,
Malt extract 2%, Glucose 1%,
pH 6.5
Medium 2.
Corn steep liquor 1%, Glucose 1%
pH 6.4.6.6
Volume (working, ml) 250 2000
pH 5.5- 7.5 5.0-7.5
Temperature (
0
C) 24-34 24-34
Aeration (vvm) N/A 0.1-1.0
Impeller rotation speed (rpm) 150-250 300-1000
Incubation time (hours) 24-120 24- 72
Monitoring methods using TLC and GC were developed to monitor the process. The effect on the
hydroxylation process given the various substrates and microorganisms at different pH levels
among other variables (see table 2 below) were studied. The relationship between contact time
for the selected organisms and the bioconversion rate of the substrate was established. Time
parameters for the biotransformation were established.
Methods to separate the mycelia from the liquid phase using separatory funnels and mira-cloth
were developed. Isolation and purification processes to extract the products from both the
mycelia and liquid phases were established.

15. 15
Experimentation
Scale up of Selected Biotransformation Processes
Biobundle reactors - Scale up
Selected biotransformation processes (Table 3) established on a smaller scale was repeated and
further optimized on a larger scale in 3L bioreactors. Methods to isolate, test and purify were
further developed.
The relationship between contact time for the selected organisms and the bioconversion rate of
the substrate was studied from the period of 0 do 72 hours contact time. Contact time was found
to be organism specific. The time parameters for the biotransformation of the substrate were
established empirically. Contact time varied from 60 to 96 hours, but normally 68-72 hours was
sufficient. The bioconversion end time was established by carefully monitoring by TLC and/or
GCMS. The morphology of the vegetative biomass was observed both visually and under a
microscope. The pH was recorded and the amount of the wet biomass was determined using
centrifuge assisted separation in graduated plastic tubes. Lower biomass indicated degradation
which implies a low bioconversion activity and an accumulation of undesired side products.
3-Litre Bioreactor - Scale up of selected Biobundle processes
Experiments in 3L bioreactors were performed. Fungi were cultivated using the media described
above (Table 2). The bioreactor has two impellers with distance 1.0 cm from the bottom of the
reactor wall with 3 cm between. The rotation of the impellers varied from 150 to 1000 RPM
between experiments. After 5-6 hours, the dissolved oxygen sensor read near zero, indicating a
very high oxygen consumption rate by the biomass. The temperature was varied between 24 and
340
C. The most promising value was 28-30 0
C.
Determine the effect of substrate hydroxylation at different pH values
The pH was varied according to the chemical nature of the substrate. For example, basic
conditions may destroy the substrate. Substrates in form of acetates such as thujaplicin acetate
and Thujol acetate were stable in neutral condition or slightly acidic (pH 4-6.5). Thujol was stable
in a broad pH range of 3.0-9.0 and did not require adjustment. The optimum pH was considered
to be in the range of 5.5-6.5. pH was adjusted using 1M nitric acid.
Optimize bioreactor conditions
Optimized bioreactor conditions were determined by the following factors. Optimal biomass
growth was established at a mixing rate of about 150-300 RPM. Higher speed causes the fungal
mycelium to be cut off into smaller threads as seen under microscope. These damaged cells
cannot synthesize the necessary enzymatic complex for the bioconversion process. The
substrate is added to the stationary phase of the fungal growth. Earlier experimentation showed
that aeration was required as the reaction had a very high oxygen demand. The biomass was
suitably aerated through a bottom to top sparger system. The ratio between the medium volume
and the total bioreactor volume was kept at 2:3. Any increase of the ratio led to bad results.
Foaming of the biomass was a sometimes a limited the total volume. In this case an anti-foam
agent (silicon detergent) was added. This led to decreased oxygen solubility in the broth thereby
constricting the biomass's enzymatic hydroxylation activity. The addition of anti-foam was also
required in the product isolation process and interfered with the downstream processes of
purification.

16. 16
Study acetylated biotransformation products from synthetic derivatives
Experimentation performed in this area showed that fungi first hydrolyze and later hydroxylate the
acetylated substrate. The hydroxylated product yield was very low. The hydrolase enzyme
dominated the rate of the hydrolysis of the acetate to acetic acid and was very high in comparison
with the hydroxylation reaction. The hydroxylation reaction requires the cofactor P450 in the
active enzymatic centre, while the hydrolases do not.
Develop isolation and purification processes
Products were extracted efficiently from the broth using ethyl acetate (1:1) in separatory funnels.
Other solvents tested, i.e. isobutyl methyl ketone (IBMK) were more selective but have toxic
properties (for humans) and a higher boiling point, making solvent removal more costly (more
energy to boil). A single extraction was found to recover about 98% of the product.
The extract was combined and reduced to 1/10 volume using a roto-evaporator under reduced
vacuum. The crude extract was purified using open column chromatography with Silica gel (230-
400 mesh). The polarity of the mobile phase was increased from hexane/ethyl acetate stepwise
to Methanol. The separated fractions were monitored with TLC and mobile phase:
chloroform/petroleum ether/water (30:70:18). Chloroform/ methanol as a mobile phase also gave
good results.
Experimentation
Bacteria and Yeast Study
Bacteria Bacillus cereus, Bacillus subtilis and yeast Seccharomyces cerevisiae, streptomyces
platensis were screened as biotransformation candidates.

17. 17
Experimentation
Establish useful Industrial Properties
The product's antibacterial activity was tested using a new variation of the standard agar method
"Bacillus cereus DSM 626".
The classical method uses a round paper disk in which a drop of the antibiotic test substance is
placed in the center. Using this technique, the diffusion of the test compound is limited to the
surface. The method was modified by cutting small holes in the disk allowing a better diffusion
and mobility of the test substance in the entire layer (top and bottom) resulting in a better
measure of activity.
The amount of the compounds was varied from 25 to 1000 mg. The test strain used was not
pathogenic but was assumed to be relevant to the commonly used pathogenic Staphylococcus
aureus. The Petri dishes were incubated for 16 to 24 hours at 350
C. The bacterial cells were
stained with nitro blue tetrasollium solution in ethanol. Zones of inhibition (measured in mm)
remained red while the area of bacterial cultivation is dark blue. The advantage of this improved
method is that the crude extract before and after purification is easily compared and thus
antibiotic activity is established.
This method was further modified. A TLC plate with several products was placed face up on the
bottom of the Petri dish and overplayed with agar. In this way the antibiotic active of the various
fractions was rapidly established.
Figure 4.
TLC Plate before agar placement TLC Plate after agar placement
Red indicates the area of bacteria growth inhibition.
Dark Blue indicates uninhibited bacteria growth.

18. 18
F. Conclusions
• A method to separate and purify the stereo-isomers Thujol β, Thujol α and Thujol δ were
developed using a system of organic solvents (dichloromethane/ethylacetate/methanol),
column chromatography (silica-gel) and crystallization
• Fungi capable of biotransformation of the selected substrates were found
• Bacteria Bacillus cereus, Bacillus subtilis and yeast Seccharomyces cerevisiae,
streptomyces platensis not suitable for use to biotransform Thujone
• Processes for the bioconversion using Fungi were achieved
• Methods to monitor the biotransformation process - substrate and products were
developed for using TLC, GCMS and NMR
• Methods to efficiently isolate and purify biotransformation products were found and
applied
• Analytical methods as required for the identification of the purified products using
analytical techniques including FTIR, LCUV, GCMS, LCMS and NMR were found and
applied
• Processes successful on a small scale were successfully scaled up to produce larger
quantities of product. This allowed for a thorough characterization of the products.
• Basic antibacterial properties for the novel compounds
Refer to Table 3 for details on the above conclusions.

19. 19
Table 3. Summary of Biotransformation Experiments and Results
Organism
Thujaplicin
acetate Thujone Thujol β
Thujone
cinnamoyl
ester
Thujone
cinnamoyl
Aspergillus niger ATCC 9642
May be
TLC spot
May be
TLC spot no no no
Aspergillus ochraceus ATCC 18500
Yes
TLC spots,
MS spectra
Low resol, IR,
NMR spectra
May be
TLC spot
May be
TLC spot
May be
TLC spot,
antibacterial
activity
May be
TLC spot,
antibacterial
activity
Aspergillus niger ATCC 11145
May be
TLC spot no No no test no test
Fusarium oxysporum sp.
May be
TLC spot no no test no test no test
Curvularia lunata ATCC 12017
May be
TLC spot
Yes
TLC spot,
GCFID,
GCMS
Yes
TLC spot,
GCFID,
GCMS no test no test
Septomyxa affinis ATCC 6737
Yes
TLC spot no no test no test no test
Rhizopus rhizopodiformis
Yes
TLC spot
May be
TLC spot
Yes
2 TLC spots
7OH/4OH=3/1,
mass spectra
low and high
res, IR, NMR-
C13
, H1
spetra no test no test
Nocardioides simplex sp. no no no test no test no test
Trichothecium roseum
May be
TLC spot
May be
TLC spot
May be
TLC spot no test no test
Cunninghamela echinulata no
May be
TLC spot
Yes
2 TLC spots
7OH/4OH=1/1,
mass spectra
low and high
res, IR, NMR-
C13, H1
spetra no test no test
Pseudomona putida no no no test no test no test
Pseudomonas resinovorans no no no test no test no test
Beauveria bassiana
Yes
TLC spot
May be
TLC spot
no test
no test no test
Epicoccum neglectum
Yes
TLC spot
May be
TLC spot
no test
no test no test
Epicoccum oryzae
Yes
TLC spot
May be
TLC spot
Yes
TLC spot,
GCFID,
GCMS no test no test
Epicoccum humicola
Yes
TLC spot
May be
TLC spot
Yes, TLC
spots, GCMS,
GCMS no test no test
Products
LEGEND
Description of Biotransformation Results
No test
No tests were performed because preliminary testing showed poor growth of the organism.
None
After a number of experiments no products could be found or found.
Maybe
Evidence for the formation of biotransformation products was achieved. However more promising
organism/substrate combinations (better growth, higher yield) were chosen for a more thorough
investigation.
Yes
The formation of biotransformation products was established. Organism/substrate with the best growth /
highest yields led to novel hydroxylated terpenes

20. 20
Thujaplicin acetate
The purified crude biotransformation extract showed antibacterial activities by testing using an in-
house developed method (Figure 4) in vitro on Petri dishes with solid agar. Four products as
indicated by TLC showed positive antibacterial activity. It was concluded that Thujaplicin acetate
made a complex with metals from the media (Fe, or Al) and with compounds released by the
fungi itself.
This complex shows a relatively high molecular weight (above 400.00) with a high polarity which
leads to problems. For example, instrumental monitoring methods could not be developed for
either GC (too polar) or LC (adheres to column). Additionally yields were poor due to the toxicity
of the substrate.
As a result, further investigation was discontinued in favor of more promising processes.
Thujone-β
Generally speaking, the substrate was consumed within a few hours. For example, a process
using the Fungus Aspergillus ochraceus ATCC 18500 yielded several polar compounds after one
hour as indicated by TLC analysis. After twenty four hours no products were found. It was
assumed that the products were completely oxidized by the organism. It was concluded that free
thujone is not a suitable substrate for biotransformation due to its fast degradation.
Better contact between the substrate and the biomass will improve the rate of the bioconversion.
This could be achieved by increasing the speed of the impellers to perhaps to 1000 RPM.
For example, the distance between the bottom, impellers and the sparger position could be varied
for a better effect. The speed of the impellers was increased up to 1000 RPM (after substrate
additions) and improved the contact between the substrate, the biomass and the air bubbles.
Thujone cinnamoyl ester and Thujone benzoyl ester
Aspergillus ochraceus ATCC 18500 yielded tentative products on a small scale. These products
also showed antibacterial properties. Yields were poor however and the substrate hydrolyzed to a
Thujol isomer mixture. As a result, further work was concentrated on the more promising
substrate Thujol-β.

21. 21
Thujol-β
This substrate proved to be the best tested for biotransformation using Fungi for the following
reasons.
• this substrate was generally non-toxic and well tolerated by most of the organisms tested
• product yields were excellent and reproducible before and after purification
• comprehensive testing confirmed the creation of novel products - hydroxylation at
position 4 and 7 of the Thujol skeleton (Figure 5)
• antibacterial activity was confirmed
• these novel products may be patented as the products and processes are not described in
the available scientific literature or any patent publication
Figure 5. Biotransformation of Thujol-β to Novel Products
G. Work remaining
Future work will concentrate on the production of larger quantities of the novel products illustrated
in Figure 5. Comprehensive testing to establish useful fragrance, antibiotic, anti-fungal or
insecticidal properties will be undertaken.
Should a significant industrial application be found for the novel compound/s, manufacture
processes will be further developed to produce Kg. quantities of material for evaluation by major
corporations such as Merck.
Provided there is sufficient funding, several other products and substrates will be investigated
further as well (Table3).