UNIVERSITI PUTRA MALAYSIA
ENZYMATIC SYNTHESIS OF BETULINIC ACID ESTER
CHEW WON YIN
FSAS 2001 38
ENZYMATIC SYNTHESIS OF BETIILiNIC ACID ESTER
BY
CHEW WON YIN
Thesis Submitted in Fulfilment of the Requirement for the Degree of Master of Science in the Faculty of Science and Enyironmental Studies
U niversiti Putra Malaysia
May 2001
Abstract of the thesis presented to the Senate of the Universiti Putra Malaysia in the fulfilment of the requirement for the degree of Master of Science
ENZYMATIC SYNTHESIS OF BETlJLINIC ACID ESTER
By
CHEW WON YIN
May 2001
Chairman: Associate Professor Dr. Mabiran Basri
Faculty: Science and Environmental Studies
Enzymatic synthesis of betulinic acid ester (3-fJ.hydroxyl-oley-lup-20(29)-en-
28-oic acid) from betulinic acid and oleic acid in chloroform were investigated. Five
commercial lipases (Candida rugosa. A.\pergil/us niger. Penicillium roquerti.
Novozyme 435 and Lipozyme) were tested for their suitability for the reaction. Among
the lipase tested, Novozyme 435 and Lipozyme were chosen for optimization studies
because of their higher specific activity. The effect of various reaction parameters such
as time course, temperature, organic s01vent, amount of enzyme, mole ratio of
substrates, initial water activity (aw) and continuous water activity (a,,) were studied to
detennine optimal condition ofbetulinic acid ester.
The optimal condition for betulinic acid ester synthesis using Novozyme 435
were obtained at incubation period of 13 h; temperature, 40GC� mole ratio of substrates,
6.0; amount of lipase, 120 mg; organic solvent, chloroform, initial water activity (aw),
0.12 and continuous water activity (aw), 0.59. Optimal condition using Lipozyme were
obtained at incubation period of 13 h; temperature, 50GC; mole ratio of substrates, 5.0;
amount of lipase, 80 mg; organic solvent, chloroform; initial water activity (aw), 0.75 and
continuous water activity (aw), 0.59. The maximum conversion for Novozyme 435 and
Lipozyme at optimal condition were 95.15% and 64.55% respectively without removal of
water in the reaction medium. This result clearly demonstrated that Novozyme 435 was
well suited for the preparation ofbetulinic acid ester in organic media (chloroform).
For scale up reaction, betulinic acid ester was easily isolated and purified using
column chromatography with solvent system; anhydrous ether: hexane (20:8.0, v/v). The
percentage conversion obtained was 75.68% when the reaction was scale up to thirteen
folds. This study indicated that enzymatic reaction might be easily scaled up while
maintaining the process selectivity as well as produced high yield of betulinic acid ester.
Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan ijazah Master Sains
SINTESIS ESTER ASID BETlJLINIK DENGAN MENGGUNAKAN ENZIM
Oleh
CHEWWONYlN
Mei2001
Pengerusi: Profesor Madya Dr. Mahiran Basri
Fakulti: Sains dan Pengajian Alam Sekitar
Ester aSld betuhnik (asid 3-,B-hidroksi-olei-lup-20(29)-en-28-oik) boleh
disintesis terus daripada asid betulink dan asid oleik dalam kloroform. Lima jenis lipase
(Candida rugosa, A�pergillus niger, Penicilium roquerli, Novozyme 435 dan
Lipozyme) telah diuji untuk memenuhi kesesuaian dalam tindak balas. Di antara lipase
yang diuji, Novozyme 435 dan Lipozyme dipilih untuk kajian optimum disebabkan oleh
akrtivitinya yang tinggi. Kesan-kesan pelbagai parameter tindak balas seperti masa
tindak balas, suhu, pelarut organik, kuantiti lipase, pecahan mol reaktan (mmol asid betuliniklmol asid oleik), akrtiviti air awaJ (a\\) dan ak1:iviti air berterusan (aw) telah
dikaji untuk menentukan keadaan tindak balas maksimum bagi sintesis tersebut.
4
Sintesis ini diperoleh dengan menggunakan Novozyme 435 pada masa tindak
balas 13 jam: suhu, 40°C; pelarut, kloroform: kuantiti lipase, 120 mg: pecahan mol
reaktan, 6.0; aktiviti air awal (a .... ), 0.]2 dan ak1:iviti air berterusan (av..), 0.59. Keadaan
maksimum diperolehi dengan menggunakan Lipozyme ialah masa tindak balas, 13 jam;
suhu 50°C; pelarut organik, kloroforrn; kuantiti lipase, 120 mg; pecahan mol reaktan, 6.0;
aktiviti air awal (a .... ), 0.75 dan aktiviti air berterusan (aw), 0.59. Penghasilan maksimum
untuk Novozyme 435 dan Lipozyme pada keadaan maksimwn ialah 95.15% dan 64.55%
tanpa menyingkirkan air dari media tindak balas. Keputusan ini dengan jelas
menunjukkan Novozyme 435 adalah enzim yang paling sesuai untuk penyediaan ester
asid betulinik dalam pelarut organik.
Bagi tindak balas skala besar, ester asid betulinik boleh diasing dan ditulenkan
dengan menggunakan kromatografi tuTUs. Pelarut organik yang digunakan ialah
campuran dietil eter: heksana (20:80, v/v) dan penghasilan yang diperoleh adalah 75.66%
apabila tindak balas dalam skala yang besar sehingga tiga belas kali ganda. Kajian ini
menunjukkan tindak balas enzimatik adaIah mudah, berskala serta bo1eh menghasilkan
peratusan tinggi.
ACKNOWLEDGEMENTS
1 wish to express my special appreciation and thanks to my project supervisor,
Associate Professor Dr. Mahiran Basri for her guidance, invaluable kinds words of
wisdom, patience, assistance and encouragement throughout this research. The sharing
of idea had not only been helpful in the progress of my project but also served as useful
principles of life in years to come and these experiences are indeed invaluable.
Special thanks also forward to my co-supervisor Associate Professor Dr. Faujan
Ahmad, Professor Abu Bakar Salleh and members of enzyme and laboratory technology
for their valuable time, effort and support. Their reinforcement made this dissertation
possible.
Last for not least, I wish to convey my deepest gratitude to my support family
member (Siew Kheng, Kok Bien, Bee Ling and Meng Ean) and all my friends,
housemates (Esther, Su Shing, Fei Peng, Yang Wei, Kok Kiong, Wen Yang and Pei
Khim) who help me in one way and another way in the preparation of this project.
I certify than an Examination Committee met on I ill May 200 J to conduct the final
examination of Chew Won Yin on her Master of Science thesis entitled "Enzymatic Synthesis of Betulinic Acid Ester" in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regu lation J 981. The Committee recommends that the candidate be awarded the relevant de!:,'Tee. Members of the Examination Committee are as fo l lows:
Abdul Rahman Manas, Ph.D. Faculty of Science and Environmental Studies Universiti Putra Malaysia (Chairman)
Mahiran Basri, Ph.D. Faculty of Science and Environmental Studies Universiti Putra Malaysia (Member)
Faujan B.H. Ahmad.., Ph.D., CChem. FRSC. Faculty of Science and Environmental Studies Universiti Putra Malaysia (Member)
Abu Bakar Salleh, Ph.D. Faculty of Science and Environmental Studies Universiti Putra Malaysia (Member)
GHAZALI MOHA YlDlN , PH.D. Professor/Deputy Dean of Graduate School Universiti Putra Malaysia
Date: 2 8 JUN 2001
7
This thesis submitted to the Senate of Universiti Putra Malaysia has been accepted as fulfilment of the requirement for the de1:,1fee of Master of Science.
AINI IDERIS, Ph.D. Professor, Dean of Graduate School, Uruversiti Putra Malaysia
Date:
DECLARATION
I hereby declare that the thesis is based on my original work except for quotations and citations, which have been duly acknowledged. ] declare that this thesis has not been previously or concurrently submitted for any other degree at UPM or other institutions.
(CHEW WON YIN)
Date:
TABLE OF CONTENTS
ABSTRACT ABSTRAK ACKNOLEDGEMENTS APPROV AL SHEETS DECLARA TJON FORM LIST OF TABLETS LlST OF FIGURES LIST OF SCHEMES LlST OF ABBREVlA TIONS
CHAPTER
INTRODUCTION
11 LITERATURE REVIEW Natural Products as Drugs Background for the Invention of Betulinic Acid as Plants Derived Anticancer Agents Betulinic Acid
Synthesis of Betulinic Acid Derivatives of Betulinic Acid Pharmacology of Betulinic Acid and Derivatives of Betulinic Acid
Enzymatic Synthesis of Betulinic Acid Lipase
Source of Lipase Lipase Specificity Immobilized Lipase Lipozyme Novozyme 435 Lipase Catalyzed Reaction
Lipase as Catalyzed in Organic Media The Role of Water Content on Enzymatic Reaction
III MA TERlALS AND METHODOLOGY Chemical and Materials Methodology Purification of Betulinic Acid Determination the Solubility of Betulinic Acid Synthesis of Betulinic Acid ESler
J ()
Page
2 4 6 7 9 13 ]4 15 16
17
21 21
22 24 29 32
36 46 46 47 48 49 50 52 53 54 56
59 59 62 62 63 63
IV
Preparation of Betulinic Acid Ester Standard 65 Standard Method of Protein Assay 67
Bradford Method 67
TNBS Method 68
Optimization Studies 70 Screening of Lipase for Activity Assay 70 Effect of Different Reaction Time on the Esterification Reaction 71 Effect of Different Reaction Temperature on the Esterification Reaction 71 Effect of Amount of Enzyme on the Esterification Reaction 71 Effect of Various Organic Solvents on the Esterification Reaction 72 Effect of Initial Water Activity (aw) on the Esterification Reaction 72 Effect of Continuous Water Activity (aw) on the Esterification Reaction 74 Effect of Mole Ratio of Substrates (Betulinic Acid/Oleic Acid) on the Esterification Reaction 74 Optimized Reaction Between Betulinic Acid and Oleic Acid 75 Scale-Up Reaction 76
RESULTS AND DISCUSSION Determination the Solubility of Betulinic Acid Synthesis ofBetulinic Acid Ester Preparation of Betuhnic Acid Ester Optimization Studies
Screening of Lipase for Specific Activity Effect of Different Reaction Time on the
78 78 79 91 93 93
Esterification Reaction 96 Effect of Different Reaction Temperature on the Esterification Reaction 98 Effect of Amount of Enzyme on the Esterification Reaction 102 Effect of Organic Solvents on the Esterification Reaction 104 Effect oflnitial Water Activity (aw) on the Esterification Reaction 107 Effect of Continuous Water Activity (aw) on the Esterification Reaction 1 10 Effect of Mole Ratio of Substrates (Betulinic Acid! Oleic Acid) on the Esterifcation Reaction 112 Optimized Reaction Between Betulinic Acid and Oleic Acid 115
1 1
Scale-Up Reaction 117
V CONCLUS1ON 1 19 Recommendation and Suggestion 120
BIBLIOGRAPHY 122 APPENDICES 133 VITA 141
12
LIST OF TABLES
Table Page
Example of Several Genus of Higher Plants Which Produced 27 Betuhnic Acid
") Examples of Betuhc Acid Derivatives 44
3 Preparation of Bovine Serum Albumin Solution and Samples by 68 Bradford Method
4 Reagent Preparation for Determination the Protein Content using 69 TNBSMethod
5 Preparation of Samples for Protein Determination usmg TNBS 70 Method
6 Log P Values of commonly Used Organic Solvents 73
7 Various Salt Hydrate of Different Water Activity (aw) at 250C 73
8 Relationship between Salt Hydrate Pair with Their Water Activity 74 (aw)
9 Parameters for the Optimization Reaction of Betulinic Acid Ester 75 using Novozyme 435
10 Parameters for the Optimization Reaction of Betulinic Acid Ester 76 using Lipozyrne
11 Quantity of Reactants, Solvent and Amount of Enzyme used In 76 Simple Scaled-up Reactions
12 The Solubility of Betulinic Acid and Oleic Acid in Various Organic 79 Solvents
13 13C_NMR Data for Betulinic Acid 90
14 Specific Activity of Various Lipases on the esterification Reaction 95
LIST 01" FIGllRES
Thin Layer Chromato!,Tfaphy of SubstTates and Products on the 82 Esterification Reaction using Solvent System, Ether Anhydrous: Hexane (1.5/8.5, v/v)
') Thin Layer Chromatography of Substrates and products on the 83 Esterification Reaction using Chloroform as a Solvent System
3 Chromatographic Trace of internal Standard, Betulinic Acid Ester 86 and Oleic Acid
4 IR Spectrum of Betulinic Acid 87
5 IR Spectrum of Reaction Mixture in the Presence of Novozyme 435 88 at 5 h Before Being Separated through Column Chromatography
6 The lH_NMR Spectrum in Deterium Chloroform of the product in 92 the present ofNovozyme 435 After 5 h Incubation
7 IR Spectrum of Reaction Mixture in the Presence of Novozyme 435 94 After Separated through Column Chromatography
8 The Effect of Different Reaction Time on the Esterifcation Reaction 97
9 The Effect of Different Reaction Temperature on the Esterifcation ] 00 Reaction
10 The Effect of Amount of Enzyme on the Esterification Reaction 103
11 The Effect of Various Organic Solvents on the Esterification 105 Reaction
12 The Effect of Initial Water Activity (aw) on the Esterification 108 Reaction
13 The Effect of Continuous Water Activity on the Esterification 1 1 1 Reaction
14 The Effect of Mole Ratio of Substrates (Betulinic Acid/Oleic Acid) 1 14 on the Esterifcation Reaction
15 Optimized of Reaction Between Betulinic Acid and Oleic Acid on 1 16 the Esterification Reaction .
14
LIST OF SCHEMES
Scheme
2
3
4
5
6
7
8
Conversion of Betulin to a and f31somer Betulinic Acid
Conversion of Betuhn to ,B-lsomer Betuhnic Acid
Preparation of Betul inic Acid Ester at the C-3 Position
Preparation ofBetulinic Acid Ester at the C-28 Position
Preparation of Betulinic Acid Amides at the C-28 Position
Conversion of Isopropenyl Group at the C-20 Position to Ketone
Preparation of Ketone Derivatives of Betulinic Acid
The Esterification Between BetuIinic Acid and Oleic Acid to Produce Betulinic Acid Ester.
15
Page
30
31
35
37
38
39
40
81
LIST OF ABBREVIATIONS
Log P logarithm of the partition coefficient
aw water activity
TLC thin layer chromatography
FT-IR Fourier transform infra red
GC gas chromatography
NMR nuclear magnetic resonance
TNBS trinitrobenzene sulfonate
BSA bovine serum albumin
PPTS Pyridium p-toluene sulfonic acid sah
THP T etrahydropyri din
THF Tetrahydrofuran
DHP Dihydropyran
TAG TriacyJglycerol
16
CHAPTER I
INTRODlJCTJON
The lupane type pentacyclic triterpene betulinic acid (1), 3-,B-hydroxy-lup-
20(29)-ene-�8-oic, is widely distributed in nature. Considerable amounts of betulinic
acid (up to 2.5%) are available in the outer bark of a variety of tree species that are
valuable for timber purposes (O'Connell et aI., 1988). A closely related compound,
betulin (2), lup-20(29)-ene-3p,28-diol� is a major constituent of white barked birch tree
with yield up to about 25%. Betulin can be easily converted chemically to betulinic acid
in a high yield (Kim et aI., 1997). Betulinic acid has been shown to exhibit a variety of
biological activities, including inhibition of human immunodeficiency virus (HJV) replication in lymphocyte cells, blockage of mv -1 entry into cells and cytotoxicity
against a variety of cultured human tumor cells (Bringmann et aI., 1997). In addition,
betulinic acid was identified as a melanoma specific cytotoxic agent in both in vitro cell
cultures and in vivo studies (Pisha et aI., 1995).
Betulinic acid derivatives can be used more efficiently in a topically applied
composition to selectively treat or prevent or inhibit a melanoma (Pezzuto et al., 1999).
It could be absorbed more efficiently as compared to their acid counterpart and thus are
more desirable. Derivatives of betulinic acid also have been investigated as specific
inhibitors of ill V -1 and as potential of anti-HIV drug candidates (Kashiwada el al.,
1998). Furthennore, the lower water activity of the betulinic acid can be overcome by
providing an appropriate derivative of betulinic acid. Modirying the parent structure of
23
(1)
(2)
28 COOH
OH
betulinic acid also can further improve antitumor activity against various cancer cells
(Pezzuto ef al., 1999).
There are several studies which reported on the preparation of betulinic acid
derivatives using chemical catalysis. The use of solids acid, clay minerals or inorganic
catalysts was reported (Bringmann el ai., 1997, Li et al., 1998). However, the process
usually carried out at higher temperature (> 100°C) and a produced some impurities
which may caused coloration or toxicity to the product. The chemical reaction is also
tedious and nonselective. Moreover, the products obtained need further purification
either by alkaline washing, stream refining, ultrafiltration or activated carbon treatment.
The isolation of the end product may not be economical.
The increasing emphasis on the use of biocatalysts for their favourable
properties may offer an improvement over these conventional methods. Enzymatic
reaction is carried out at ambient pressure and temperature (40-60oC). The overall cost
is also brought down by the fact that the reaction need not be highly corrosive-resistant
(minerals acids used as catalyst in conventional procedure are very corrosive) (Grandhi
et al. , 1997). The products of such bioprocesses are usually pure (Anonymous, 1981).
Furthermore, the lower temperature reaction employed ensured minimal thermal
degradation (Linfield et aI., 1984, Kosugi et ai., 1988).
In this study, an alternative synthesis of betulinic acid derivatives using lipases
has been investigated. The modification at C-3 position was chosen to yield more potent
In this study, an alternative synthesis of betulinic acid derivatives using hpases
has been investigated. The modification at C-3 position was chosen to yield more potent
of betulinic acid derivatives. To date, there are no published reports on the enzymatic
esterification of betulinic acid at the modification of C-3 position. Therefore, the
objectives of this study are to synthesize betulinic acid ester from betulinic acid and
oleic acid using lipase and to optimize the reaction condition with respect to the effect
of different reaction time, temperature, mole ratio, amount of enzyme, organic solvents,
initial water activity (aw) and continuous water activity (aw). The product was
charaterized using Fourier Transform-Infrared Spectroscopy (FT -IR), stet
chromatography (TLC), gas chromatography (GC) and Proton Nuclear Magnetic
Resonance Spectroscopy eH-NMR).
]0
CHAPTER I I
LITERATURE REVIEW
Natural Products as Drugs
The search for new pharmacologically active agents obtained by screemng
natural sources such as microbial fermentation and plants extracts has led to the
discovery of many clinically useful drugs that play a major role in the treatment of
human diseases (Shu, 1999). Analysis of the number and source of anticancer and
antiinfective agent, reported mainly in Annual Reports oj Medicinal Chemistry from
1984 to 1995, indicates that over 60% of the approved drugs (for the period 1989-1995)
developed in these disease areas are of natural origin. Of the world's 25 best selling
pharmaceutical agents, 12 are natural product origin (O'Neill et a!.. 1993).
The use of traditional medicine is widespread and plants still present a large
source of structurally novel compounds that might serve as leads for the development of
novel drugs (Heras ef aI., 1998). The role played by plants in the provision of novel
agents have potential in the treatment and prevention of many diseases such as cancer,
acquired immunodeficiency syndrome (AIDS) and related infections, and malaria has
been reviewed in an American Chemical Society Series volume (Baker et al., 1995).
Cragg ef al., ( 1 997) also reported that at least 1 1 9 compounds derived from the 90 plant
species can be considered as important drugs and currently was used in one or more
countries. Further evidence of the importance of natural products is provided by the fact
�l
that close to half of the best selling phannaceuticals in 199 1 were either plant drugs or
their derivatives (O'Neill el al .. 1993).
Background for tbe Invention of Betulinic Acid as Plant-Derived Anticancer
Agents
The incidence of melanoma has been increasing at a rate higher than that of any
type of cancer for the past four decades. It is now anticipated that as many as 1 in 90
Caucasian Americans will develop maglinant melanoma in their lifetime (Ries et al.,
1990, Brozena et al., J 993). Although an increasing proportion of melanomas is
diagnosed at a stage that is early enough to be responsive to surgical treatment (10 year
survival rate greater than 90%), it has been estimated that over 7,000 patients will die
with metastatic melanoma in the United States in 1995.
Patients with metastatic melanoma which is not amenable to surgical
extirpation, treatment options are limited. Since DTIC (5-(3,3-dimethyl-l-triazenyl)-1-
H-imidazole-4-carboxamide) (3), also known as dacarbazine is the most efficacious
single chemotherapeutic agent for melanoma having an overall response rate of 24%.
But, the duration of response to DTIC is generally quite poor (Comis et aI., 1 976).
Combination therapy V\�th other synthetic and recombinant agents, including BCND (N.
Y-bis(2-chloroethyl)-N-nitrosourea (4), also known as cannustine, cisplatin (5),
tamoxifen (6), interferon-a (lNF-a) and interleukin-2 (rL-2), has a higher response rate
(example: 30-50%) in some trials, but a durable complete response rate is uncommon
and toxicity is increased (Mc Clay el al . . 1994).
H
CH3 I \ N
CW/·N- N�N --( ) 3 NH2-CO�
(3)
(4)
(5)
(6)
') " --'
Various drugs derived from natural products, such as adriamycin (7)
(doxorubicin) derivatives, bleomycin (8), etoposide (9), and vincristine (10), and their
derivatives have been tested for efficacy against melanoma either as single agents or in
combination therapy. However, similar to the synthetic and recombinant compounds,
these compounds exhibited low response rates, transient complete responses, and high
toxicity (Thompson, et al., ] 992).
Under the auspices of National Co-operative Natural Product Drug Discovery
supported by the National Cancer Institute in United States, the potential antitumor
activity of approximately 2,500 extracts derived from globally collected plants was
evaluated. As a result of their bioassay-guided fractionation studies, compounds isolated
from the stem bark of Ziziphus mauritiana displayed selective cytotoxicity against
human melanoma cells. This led to the isolation of a pentacyclic triterpene, betulinic
acid (1), as the active principle. (Pisha et at., 1995).
Betulinic Acid
Betulinic acid, 3 p-hydroxy-Iup-20(29)-ene-28-oic acid, is a natural product isolated
from several genus of higher plants (Table 1 as example). The isolated active compound
has a molecular formula of C301itg03, as determined by high-resolution mass spectral
analysis (Pezzuto et ai., 1999). Betulinic acid may be crystallised from chloroform
methanol as shining needles with a melting point 294-296° (Fukunaga et al., 1985). The
optical rotation of the compound was measured as ..... 7.3° (c= 1.2: pyridine). Betulinic
24
