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

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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

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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

OHR

OH

R

OH

R

HO

Thujone, R =

Thujanol connamoyl ester, R =

Thujanol benzoyl ester, R =

O

O

O H

O

HO

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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

12

3

4

5

6

thujone

OO

HO

OO

H

O H

H

CHO

CHO

O

O

OHOOC

OH

geraniol(perfumery)

2

H C

H

OR

O

pyrethroid insecticides(agriculture)

ambrox(perfumery)

K M nO 4

31

4

O

digitoxigeninsteroids and analogues

(pharmaceutical)

damascones(rose oil)

(perfumery)

(+)- β-cyperonesesquiterpenes

(perfumery)

polygodial(agriculture)

O

MVK

KOH KOH

EVK

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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

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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

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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.

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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.

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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

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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

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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 0C 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).

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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.

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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 0C 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.

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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 (0C) 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.

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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 340C. The most promising value was 28-30 0C. 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.

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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.

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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 350C. 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.

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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.

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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

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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.

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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).