Bis-Thiourea bearing aryl and amino acids side chains and their antibacterial activities

Article abstract of DOI:10.1080/10426507.2013.858250

(Equation present) A series of symmetrical 1,3-bis thiourea 1a-e and 1,4-bis thiourea derivatives 2a-e have been successfully synthesized from the reactions of amines with 3-acetylbenzoyl isothiocyanate and 4-acetylbenzoyl isothiocyanate, respectively. All the synthesized compounds were characterized by FT-IR spectroscopy and ¹H and ¹³C NMR spectroscopy. The compounds were screened for their antibacterial activity by turbidimetric method using gram-negative bacteria (E. coli ATCC 8739) using turbidimetric method. The newly synthesized bis-thiourea derivatives bearing aryl side chains showed good antibacterial activity against E. coli. The effect of the molecular structure of the synthesized compounds on the antibacterial activity is discussed. 2014 Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/10426507.2013.858250

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Phosphorus, Sulfur, and Silicon and the
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Bis-Thiourea Bearing Aryl and Amino
Acids Side Chains and Their Antibacterial
Activities
Wan Sharifatun Handayani Wan Zullkipleea, Ainaa Nadiah Abd
Halima, Zainab Ngainia, Maya Asyikin Mohd Ariffa & Hasnain Hussainb
a Department of Chemistry, Faculty of Resource Science and
Technology, Universiti Malaysia Sarawak, Kota Samarahan, Sarawak,
Malaysia
b Department of Molecular Biology, Faculty of Resource Science and
Technology, Universiti Malaysia Sarawak, Kota Samarahan, Sarawak,
Malaysia
Accepted author version posted online: 03 Mar 2014.Published
online: 12 Jun 2014.
To cite this article: Wan Sharifatun Handayani Wan Zullkiplee, Ainaa Nadiah Abd Halim, Zainab
Ngaini, Maya Asyikin Mohd Ariff & Hasnain Hussain (2014) Bis-Thiourea Bearing Aryl and Amino
Acids Side Chains and Their Antibacterial Activities, Phosphorus, Sulfur, and Silicon and the Related
Elements, 189:6, 832-838, DOI: 10.1080/10426507.2013.858250
To link to this article: http://dx.doi.org/10.1080/10426507.2013.858250
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Phosphorus, Sulfur, and Silicon, 189:832–838, 2014
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426507.2013.858250
BIS-THIOUREA BEARING ARYL AND AMINO ACIDS SIDE
CHAINS AND THEIR ANTIBACTERIAL ACTIVITIES
Wan Sharifatun Handayani Wan Zullkiplee,1 Ainaa Nadiah
Abd Halim,1 Zainab Ngaini,1 Maya Asyikin Mohd Ariff,1
and Hasnain Hussain2
1Department of Chemistry, Faculty of Resource Science and Technology, Universiti
Malaysia Sarawak, Kota Samarahan, Sarawak, Malaysia
2Department of Molecular Biology, Faculty of Resource Science and Technology,
Universiti Malaysia Sarawak, Kota Samarahan, Sarawak, Malaysia
GRAPHICAL ABSTRACT
5
4
3
6
7
2
9
8
Acetone reflux
Acetone
1
10
1a-e
6
7
5
4
2
9
3
Acetone
Acetone reflux
8
1
NCS
NCS
10
2a-e
= CH (COOH)
(a)
R
2
(b)
(c)
(d)
(e)
R
R
R
R
= CH CH (COOH)
= (CH )CH(COOH)
= (C )CH(COOH)
= C
2
2
3
6H5
6
H5
Abstract A series of symmetrical 1,3-bis thiourea 1a–e and 1,4-bis thiourea derivatives 2a–e
have been successfully synthesized from the reactions of amines with 3-acetylbenzoyl isothio-
cyanate and 4-acetylbenzoyl isothiocyanate, respectively. All the synthesized compounds were
characterized by FT-IR spectroscopy and 1H and 13C NMR spectroscopy. The compounds
were screened for their antibacterial activity by turbidimetric method using gram-negative
bacteria (E. coli ATCC 8739) using turbidimetric method. The newly synthesized bis-thiourea
derivatives bearing aryl side chains showed good antibacterial activity against E. coli. The
effect of the molecular structure of the synthesized compounds on the antibacterial activity is
discussed.
Keywords Antibacterial activity; amino acid; bis-thiourea
Received 15 December 2012; accepted 12 October 2013.
We thank Assoc. Prof. Dr. Wan Mohd Khairul Wan Mohamed Zin from Universiti Malaysia Terengganu for
providing CHN elemental analysis services.
Address correspondence to Assoc. Prof. Dr. Zainab Ngaini, Faculty of Resource Science and Technology,
University Malaysia Sarawak, Chemistry, Unimas, Kota Samarahan, Sarawak, Kuching 94300, Malaysia. E-mail:
nzainab@frst.unimas.my
832

ANTIBACTERIAL STUDY OF BIS-THIOUREA DERIVATIVES
833
INTRODUCTION
Thiourea is an organic compound that consists of carbon, nitrogen, sulfur, and hy-
drogen atoms from the synthesis of amino group and thiocyanate. It is well known that
thioureas and their derivatives play important roles in many biological processes.1,2
The search for new thiourea derivatives with biological activities has intensified.
Most studies reported on compounds with a single thiourea moiety with trivial antimi-
crobial properties.3–5 The quantity of thiourea units in the thiourea backbone contributed
to enhancement of antimicrobial activity.6 For example, the unsymmetrical bis-thiourea
bearing triazole moieties were reported with excellent antimycobacterial activity against
M. tuberculosis in monolayers of mouse bone marrow macrophages. The presence of two
groups of thiourea moieties inhibited 90% of the mycobacterial growth. Other symmetrical
bis-thioureas were also reported for their significant anticancer activities against Calu-6
lung carcinoma cells. The thioureas were able to induce the production of lysine-specific
demethylase, thus, able to control the development of cancer cell.7
In this research, we aimed to synthesize bis-thiourea derivatives which consist of
two differently positioned thiourea groups and study their antibacterial activities. The bis-
thiourea derivatives were synthesized from the reaction of 3-acetylbenzoyl isothiocyanato
and 4-acetylbenzoyl isothiocyanato with various amines groups, such as glycine, beta-
alanine, l-alanine, l-phenylalanine, and aniline. The synthesized thiourea derivatives with
the presence of two thiourea groups were also tested against E. coli (ATCC 8739) to
determine their antibacterial properties.
RESULTS AND DISCUSSION
Chemistry
The preparations of 1a–e and 2a–e are shown in Scheme 1.
5
4
3
6
7
2
9
8
Acetone reflux
Acetone
1
10
1a-e
6
7
5
4
2
9
3
Acetone
Acetone reflux
8
1
NCS
NCS
10
2a-e
= CH (COOH)
(a)
R
2
(b)
(c)
(d)
(e)
R
R
R
R
= CH CH (COOH)
= (CH )CH(COOH)
= (C )CH(COOH)
= C
2
2
3
6H5
6
H5
Scheme 1 The synthesis of 1a–e and 2a–e.
The IR spectra showed the successful formation of 1a–e and 2a–e by the disap-
pearance of v(NCS) at 2500–2000 cm−1 and appearance of v(NH) at 3365–3082 cm−1
.

834
W. S. H. W. ZULLKIPLEE ET AL.
Sharp peaks observed at 1281–1220 cm−1 were attributed to v(C S).3,8 The peaks at
1602–1580 cm−1 and 1730–1691 cm−1 were attributed to v(C N) and v(C O), respec-
tively. v(COOH) were observed at 1693–1611 cm−1 while the aromatic group was repre-
sented by the absorption band at 1557–1509 cm−1
.
The chemical structures of 1a–e and 2a–e were further confirmed by H and 13C
NMR spectroscopy. CONH and CSNH signals were observed at δ 11.33–11.54 and δ
11.01–11.26, respectively. The CONH signal was shifted to higher frequency due to the
downfield effect.5 The presence of peaks at δ 7.69–8.50 region was attributed to aromatic
protons. Four aromatic protons at C3-8 of 2a–e were observed as a duplet peak due to the
symmetrical structures.
1
The 13C NMR spectrum showed the presence of C S at δ 180.0–179.7 whereas
C O resonated at δ 167.4–166.9. The COOH groups were represented by more downfield
absorption peak at δ 173.1–171.3 due to the presence of more electronegative oxygen. The
presence of the aromatic carbons was observed at δ 128.6–133.1.
Antibacterial Activity
The antibacterial activity of bis-thiourea derivatives 1a–e and 2a–e were examined
at the concentration of 50 ppm, 80 ppm, and 100 ppm against bacteria E. coli ATCC
8739 at 37 ◦C. Of all the synthesized bis-thiourea, 2e with aryl side chain exhibited good
antibacterial activities compared to 1a–e and 2a–d. The comparison of the antibacterial
activities for 1e and 2e is shown in Figure S1.
The different effects of the newly synthesized compounds 1a–e and 2a–e at vari-
ous concentrations can be represented by their minimum inhibitory concentration (MIC).
The MIC value for each compound was determined by extrapolating the concentration at
the zero growth rate of E. coli (μ = 0).9 The MIC value of 2e was observed at 175 ppm,
while others were observed to exceed 200 ppm. A compound with a minimum inhibition
value up to 400 ppm is still considered to have inhibition activity against Gram nega-
tive bacteria, however, a compound with the MIC value > 200 ppm is not suitable to be
used for clinical purposes.10 MIC graph for all the synthesized compounds is shown in
Figure S2.
The presence of C S, C O, and N-H functional groups in thiourea derivatives are
reported to give good antibacterial activity as they react with carboxyl and phosphate
groups of the bacterial surface.11 Compounds 1a–e and 2a–d, however, showed no bi-
ological activity against E. coli ATCC 8739. In contrast, 2e shows better antibacterial
activity compare to 1e. It was believed that 1,3-bis thiourea produced more steric hin-
drance compared to 1,4-bisthiourea. The occurrence of steric hindrance creates a force
that obstruct the contact between active sites in the compound with receptor site of the
bacteria.6
Lipophilic characteristic is another important behavior for good antibacterial activ-
ities as it correlates well with bioactivity of chemicals. However, the presence of some
functional groups such as carboxylic acid group might decrease the lipophilic character of
the compound.10 The carboxylic acid groups presence in compounds 1a–d and 2a–d might
contribute to reduction in lipophilic behavior thus gave poor antibacterial activities. On the
other hand, the aryl side chains present in 2e was envisaged to increase the lipophilicity
thus enhance the antibacterial properties.10

ANTIBACTERIAL STUDY OF BIS-THIOUREA DERIVATIVES
835
CONCLUSION
Bis-thiourea derivatives 1a–e and 2a–e were successfully synthesized by the reaction
of various types of amine groups with acetylbenzoyl isothiocyanate. The antibacterial study
of the synthesized compounds discovered that 2e showed good antibacterial activity against
E. coli ATCC8739.
EXPERIMENTAL
Isophthaloyl dichloride, terepthaloyl dichloride, potassium thiocyanate, glycine, beta-
alanine, l-alanine, l-phenylalanine and aniline were obtained from Merck and used without
purification. Acetone was distilled over magnesium sulfate anhydrous. All other reagents
and solvents used as received.
Measurements: Melting points were determined by the open tube capillary method
and are uncorrected. Infra-red (IR) spectra (ν cm−1) were recorded as KBr pellets on a
Perkin Elmer 1605 FTIR spectrophotometer. 1H and 13C NMR spectra were recorded on a
JEOL ECA 500 spectrometer at 500 MHz (1H) and 125 MHz (13C) with the chemical shifts
δ (ppm) reported relative to acetone-d6 and DMSO-d6 as standards.
General Procedure for the Preparation of Bis Thiourea Derivatives 1a–e
and 2a–e
Isophthaloyl dichloride or terepthaloyl dichloride (1 mmol) in dry acetone (15 mL)
was added drop wise to a suspension of potassium thiocyanate (2 mmol) in dry acetone
(15 mL). The mixture was stirred for 1 h at room temperature and the white potassium
chloride (KCl) was filtered. Amines (2 mmol) in dry acetone (15 mL) was added into
the filtrate and heated under reflux for 7 h. The mixture was cooled to room temperature
and filtered. The filtrate was poured into ice in a beaker to form a solid. The crude solid
was filtered, washed with ethanol and recrystallized from ethanol–acetonitrile (1:1). The
general procedure for the preparation of 1a–e and 2a–e with different type of amine groups
(g, mmol) and yields is shown below.
2-[[3-(carboxymethylcarbamothioylcarbamoyl)benzoyl] carbamothioy-
lamino] Acetic Acid (1a). Glycine (0.150 g, 2 mmol). (0.274 g, 73%) as a yellowish
solid, m.p: 226.0-227.0◦C; (Found: C, 42.04; H, 3.36; N, 14.05; S, 15.79. C14H14N4O6S2
Requires C, 42.20; H, 3.50; N, 14.10; S, 16.10%); νmax (KBr/ cm−1) 3233 (NH), 2922
(CH), 1729 (C O), 1679 (COOH), 1602(C N), 1557(Ar C), 1231(C S). δH (500 MHz,
DMSO-d6) 4.23 (4H, d, 2×CH2), 7.69 (1H, t, J = 16.0 Hz, Ar H), 8.14 (2H, d, J =
6.3 Hz, Ar H), 8.47 (1H, s, Ar H), 11.10 (2H, t, 2×NH), 11.40 (2H, s, 2×NH). δC
(125 MHz, DMSO-d6) 47.8, 128.4, 129.0, 131.9, 133.0, 166.9, 169.4, 179.7.
2-[[3-(2-carboxyethylcarbamothioylcarbamoyl)benzoyl]carbamothioyl-
amino]propanoic Acid (1b). Beta-alanine (0.178 g, 2 mmol). (0.183 g, 67%) as a white
solid, m.p: 219.1-220.2◦C; (Found: C, 45.02; H, 4.00; N, 13.05; S, 15.03. C16H18N4O6S2
Requires C, 45.10; H, 4.30; N, 13.10; S, 15.10%); νmax (KBr/ cm−1) 3082 (NH), 2666 (CH),
1691 (C O), 1611 (COOH), 1580(C N), 1520 (Ar C), 1281 (C S). δH (500 MHz,
DMSO-d6) 2.66 (4H, t, 2×CH2), 3.84 (4H, q, 2×CH2), 7.63(1H, t, J = 7.7 Hz, Ar H),
8.15 (2H, d, J = 9.2 Hz, Ar H), 8.48 (1H, s, Ar H), 11.01 (2H, t, 2×NH), 11.33 (2H, s,
2×NH). δC (125 MHz, DMSO- d6) 30.1, 32.5, 128.4, 129.0, 131.8, 132.9, 166.9, 173.1,
179.7.

836
W. S. H. W. ZULLKIPLEE ET AL.
2[[3[(2hydroxy1methyl2oxoethyl)carbamothioylcarbamoyl]benzoyl]car-
bamothioylamino]propanoic Acid (1c). L-alanine (0.178 g, 2 mmol). (0.282 g,
70%) as a white solid, m.p: 207.5-208.3◦C, (Found: C, 45.10; H, 4.27; N, 13.03; S, 15.05.
C16H18N4O6S2 Requires C, 45.10; H, 4.30; N, 13.10; S, 15.10%); νmax (KBr/ cm−1) 3365
(NH), 2992 (CH), 1730 (C O), 1693 (COOH), 1600 (C N), 1514 (Ar C), 1220 (C S).
δH (500 MHz, DMSO-d6) 1.51 (6H, d, 2×CH3), 4.85 (2H, m, 2×CH), 7.69 (1H, t, J =
16.0 Hz, Ar H), 8.14 (2H, d, J = 8.4 Hz, Ar H), 8.49 (1H, s, Ar H), 11.26 (2H, d,
2×NH), 11.49 (2H, s, 2×NH). δC (125 MHz, DMSO-d6) 17.1, 53.2, 128.6, 129.0, 131.8,
133.1, 167.4, 172.8, 179.5.
2-[[3-[(2-hydroxy-2-oxo-1 phenylethyl)carbamothioylcarbamoyl]benzoy-
l]carbamothioylamino]-2-phenyl-acetic Acid (1d). l-phenylalanine (0.302 g,
2 mmol). (0.437 g, 91%) as a yellowish solid, m.p.: 229.0-230.2◦C; (Found: C, 57.72;
H, 4.22; N, 9.68; S, 11.00. C28H26N4O6S2 Requires C, 58.10; H, 4.50; N, 9.70; S,
11.10%); νmax (KBr/ cm−1) 3227(NH),, 2997 (CH), 1718 (C O), 1690 (COOH), 1600
(C N), 1509 (Ar C), 1223 (C S). δH (500 MHz, DMSO-d6) 5.14 (2H, d, 2×CH),
7.21-7.30 (10H, m, J = 7.7 Hz Ar H), 7.70 (1H, t, J = 16.1 Hz, Ar H), 8.10 (2H, d,
J = 7.7 Hz, Ar H), 8.48 (1H, s, Ar H), 11.17 (2H, d, 2×NH), 11.54 (2H, s, 2×NH).
δC (125 MHz, DMSO-d6) 58.7, 126.9, 128.4, 129.2, 131.6, 133.2, 136.1, 167.4, 171.3,
180.0.
N1,N3-bis(phenylcarbamothioyl)benzene-1,3-dicarboxamide (1e). Ani-
line (182 μmL, 2 mmol). (0.390 g, 89%) as a yellow crystalline solid, m.p: 202.0-202.6◦C;
(Found: C, 60.70; H, 4.14; N, 12.75; S, 14.19. C22H18N4O2S2 Requires C, 60.80; H, 4.20;
N, 12.90; S, 14.80%); νmax (KBr/ cm−1) 3087 (NH), 1672 (C O), 1597 (C N), 1520
(Ar C), 1249 (C S). δH (500 MHz, DMSO- d6) 7.30 (2H, t, J = 15.3 Hz, Ar H), 7.44
(4H, t, J = 15.3 Hz, Ar H), 7.70 (4H, d, J = 16.0 Hz, Ar H), 7.74 (1H, t, J = 8.7 Hz,
Ar H), 8.19 (2H, d, J = 7.7 Hz, Ar H), 8.57(1H, s, Ar H), 11.59 (2H, s, 2xNH), 12.55
(2H, s, 2xNH). δC (125 MHz, DMSO-d6) 17.1, 53.2, 128.6, 129.0, 131.8, 133.1, 167.4,
172.8, 179.5.
2-[[4-(carboxymethylcarbamothioylcarbamoyl)benzoyl] carbamothioy-
lamino] acetic Acid (2a). Glycine (0.150 g, 2 mmol). (0.056 g, 14%) as a yellowish
solid, m.p.: 213.2-215.5◦C; (Found: C, 42.10; H, 3.41; N, 13.95; S, 16.09. C14H14N4O6S2
Requires C, 42.20; H, 3.50; N, 14.10; S, 16.10%); νmax (KBr/ cm−1) 3316 (NH), 2927
(CH), 1719 (C O), 1672 (COOH), 1642 (C N), 1552(Ar C), 1242(C S). δH (500 MHz,
DMSO-d6) 4.48 (4H, d, 2×CH2), 7.94 (4H, d, J = 13.8 Hz, Ar H), 9.13 (2H, t, 2×NH),
11.08 (2H, s, 2xNH). δC (125 MHz, DMSO-d6) 39.8, 127.5, 128.6, 134.7, 137.5, 171.20,
169.8, 180.6.
3-[[4-(2-carboxyethylcarbamothioylcarbamoyl)benzoyl]carbamothioyl-
amino]propanoic Acid (2b). Beta-alanine (0. 178 g, 2 mmol). (0.064 g, 15%) as
a white solid, m.p.: 238.1–245.3◦C; (Found: C, 44.61; H, 4.00; N, 12.22; S, 15.00.
C16H18N4O6S2 Requires C, 45.10; H, 4.30; N, 13.10; S, 15.10%); νmax (KBr/ cm−1) 3293
(NH), 3166 (CH), 1707 (C O), 1671 (COOH), 1563 (C N), 1526 (Ar C), 1265 (C S).
δH (500 MHz, DMSO-d6) 2.66 (4H, t, 2×CH2), 3.84 (4H, q, 2×CH2), 7.98 (4H, d, J =
12.3 Hz, Ar H), 10.98 (2H, t, 2×NH), 11.55 (2H, s, 2×NH). δC (125 MHz, DMSO-d6)
39.2, 39.3, 128.5, 135.9, 167.4, 173.0, 180.1.
2-[[4-[(2-hydroxy-1-methyl-2-oxo-ethyl)carbamothioylcarbamoyl]benzo
yl] carbamothioyl Amino]propanoic (2c). l-alanine (0. 178 g, 2 mmol). (0.162 g,
38%) as a yellowish solid, m.p: 235.4-246.4◦C; (Found: C, 44.80; H, 4.17; N, 12.88; S,
14.80. C16H18N4O6S2 Requires C, 45.10; H, 4.30; N, 13.10; S, 15.10%); νmax (KBr/ cm−1
)

ANTIBACTERIAL STUDY OF BIS-THIOUREA DERIVATIVES
837
3356 (NH), 3177 (CH), 1728 (C O), 1675 (COOH), 1451 (C N), 1406 (Ar C), 1259
(C S). δH (500 MHz, DMSO-d6) 3.56 (6H, m, 2×CH3), 4.85 (2H, d, 2×CH), 8.01 (4H, d, J
= 16.3 Hz, Ar H), 11.23 (2H, d, 2×NH), 11.73 (2H, s, 2×NH). δC (125 MHz, DMSO-d6)
17.2, 40.0, 128.6, 135.9, 167.8, 172.9, 179.8.
2-[[4-[(1-benzyl-2-hydroxy-2-oxoethyl) carbamothioylcarbamoyl]benzo-
yl] carbamothioyl amino]-3-phenyl-propanoic Acid (2d). l-phenylalanine
(0.331g, 2 mmol). (0.335 g, 58%) as a white solid, m.p: 220.4-227.1◦C; (Found: C, 57.43;
H, 3.97; N, 10.17; S, 11.70. C28H26N4O6S2 Requires C, 58.10; H, 4.50; N, 9.70; S,
11.10%); νmax (KBr/ cm−1) 3477 (NH), 3026 (CH), 1714 (C O), 1666 (COOH), 1549
(C N), 1515(Ar C), 1232(C S). δH (500 MHz, DMSO-d6) 3.36 (4H, d, 2×CH2), 5.12
(2H, q, 2×CH), 7.30 (10H, t, J = 7.7 Hz, Ar H), 7.98 (4H, d, J = 13.0 Hz, Ar H), 11.14
(2H, d, 2×NH), 11.75 (2H, s, 2×NH). δC (125 MHz, DMSO-d6) 36.0,58.8, 126.9, 128.6,
129.3, 135.8, 136.3, 167.7, 171.4., 180.0.
N1,N4-bis(phenylcarbamothioyl)terephthalamide (2e). Aniline (182 μmL,
2 mmol) (0.10 g, 23%) as a light brown solid, m.p: 215.6-220.2◦C; (Found: C, 60.70; H, 4.18;
N, 12.05; S, 14.59. C22H18N4O2S2 Requires C, 60.80; H, 4.20; N, 12.90; S, 14.80%); νmax
(KBr/ cm−1) 3409 (NH), 3176 (CH), 1673 (C O), 1599 (C N), 1534(Ar C), 1262(C S).
δH (500 MHz, DMSO- d6) 8.0 (6H, t, J = 10.6 Hz, Ar H), 8.1(8H, d, J = 7.7 Hz, Ar H),
11.74 (2H, s, 2×NH), 12.54 (2H, s, 2×NH). δC (125 MHz, DMSO-d6) 120.5, 124.4, 127.5,
128.3, 137.9, 166.9, 179.0.
Antibacterial Screening
The antibacterial activity of synthesized bis-thiourea derivatives were analyzed
against E. coli ATCC 8739 using turbidimetric kinetic method. The E. coli were cul-
tured on a LB plate agar for one day at 37◦C. Then the inoculums were transferred and
allowed to grow in a media containing nutrient broth at 37◦C with permanent stirring at
250 rpm for overnight. And 0.2 mL of inoculums was inoculated with 10 mL of culture
medium that was added with increasing concentration of synthesized compounds dissolved
in DMSO. The mixture was shaken at 230 rpm at 37◦C. Inoculums in the broth medium
containing solvent was used as negative control. One mL aliquots of each replicate were
taken at every 1 h interval for 6 h. The transmittance (T) was recorded using UV-Visible
Spectrophotometer Optima SP-300. The antibacterial activity was determined by plotting
a graph of ln Nt versus time. The ln Nt value was represents the number of colony forming
units/mL which followed the expression of ln Nt = 27.1–8.56T.12
SUPPLEMENTAL MATERIALS
Supplementary data of this article can be accessed on the publisher’s website,
www.tandfonline.com/gpss
FUNDING
We acknowledge support from Universiti Malaysia Sarawak and Ministry of Science,
Technology and Innovation for the financial support through FRGS/01(14)/743/2010 (29)
and FRGS/01(22)/754/2010(40).

838
W. S. H. W. ZULLKIPLEE ET AL.
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