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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, Apergillus 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, 40C; 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 50C; 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 (> 100C) 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- NN --( )

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