This document summarizes research on synthesizing and testing two peptide models (TBS-md11 and TBS-md16) of the α-amylase inhibitor Tendamistat. The peptides were successfully synthesized using solid phase peptide synthesis. TBS-md11 folded into its correct disulfide bonded form within one minute, while TBS-md16 folded into multiple species with different disulfide connectivities. Testing showed that neither peptide was able to inhibit porcine, fungal, or human α-amylases, suggesting more work is needed to understand their activity and design more effective inhibitors.

1. Proceedings of the URECA@NTU 2013-14
Synthesis and Oxidative Folding of Three β-Stranded α-Amylase
Inhibitors
Muhammad Azhari Bin Mohammad Zain
School of Biological Sciences
Professor James P. Tam
School of Biological Sciences
Abstract – Proteinaceous α-amylase inhibitors found in
plants and bacteria are potential cures for diseases such
as diabetes and obesity due to their role in regulating
starch metabolism. Recently, a 74-amino acid α-
amylase inhibitor Tendamistat has been identified from
Streptomyces tendae to effectively inhibit mammalian
and fungal α-amylases. In this study, we had
successfully engineered and synthesized two three-beta-
stranded peptides modelled from Tendamistat: 27-
amino-acid TBS-md11 and 22-amino-acid TBS-md16.
Here, we performed oxidative folding for both peptides
in different folding conditions and found out that TBS-
md11 was oxidised in one minute, whereas for TBS-
md16 we obtained three types of peptide species with
different disulphide connectivity, each having two
isomers. Further studies to investigate inhibition of
porcine pancreatic α-amylase (PPA), Aspergillus
amylase (AA) and human salivary amylase (HSA) by
both peptides were tested using amylase inhibition
assay. The results from the test showed that both TBS-
md11 and TBS-md16 failed to inhibit any of these α-
amylases. Although more work is needed to understand
the activity and stability of both peptides, our work may
provide insights into the synthesis and oxidative folding
of TBS-md11 and TBS-md16, which could be used as a
platform to design more efficient α-amylase inhibitors.
Keywords – α-amylase inhibitors, Tendamistat,
Streptomyces tendae, TBS-md11, TBS-md16.
1 INTRODUCTION
The α-amylase also known as (α-1,4-glucon-4-
gluconohydrolase) has been in the forefront of research
studies due to its crucial role in regulating starch
metabolism in mammals [1,2]. This enzyme hydrolyses
long sugar polysaccharide at the α-1,4 glycosidic bond
[1,2].
Many diabetic and obese patients tend to have a high
glucose level in the blood [3]. As such, the discovery of
α-amylase inhibitors proves to be an important stepping
stone in tackling diseases such as type II diabetes
mellitus and obesity by reducing the production of
glucose in the blood.
Proteinaceous α-amylase inhibitors are largely found
from microbial sources. Examples include PaimI,
HaimII, Z-2685, AI-409, T-76 and Tendamistat [4]. All
of them were extracted from the same genus
Steptomyces and their amino acid sequences are
homologous [4]. Tendamistat, derived from
Streptomyces tendae, is known to be one of the most
potent α-amylase inhibitors against porcine pancreatic
α-amylase (PPA) and forms a strong stoichiometric 1:1
complex with the enzyme [4].
Tendamistat is 74 amino acids long and contains a total
of six β-sheets and two disulfide bonds [4]. Generally,
the main advantage of small peptidyl drugs is that they
are much easier to synthesize and studied as compared
to large peptidyl drugs. Therefore, in this study, we will
develop a shorter modified version of Tendamistat
while retaining its inhibitory effects on α-amylase.
Here we report the synthesis of two α-amylase
inhibitors from Tendamistat (Table 1): a 27-amino-acid
peptide, named TBS-md11 (Figure 1A) and a 22-
amino-acid peptide named TBS-md16 (Figure 1B). We
also report on their oxidative folding and inhibitory
effect using α-amylase inhibition assay.
A B
Figure 1: (A) Modeled structure of TBS-md11. (B)
Modeled structure of TBS-md16

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Table 1. Sequence comparison of TBS-md11 and TBS-md16 with Tendamistat.
Peptide Sequence m/z
Residue
Number
Species Reference
Tendamistat
TBS-md11
TBS-md16
1 10 20 30
DTTVSEPAPSCVTLYQSWRYSQADNGCAET
VTVKVVYEDDTEGLCYAVAPGQQITTVGDG
YIGSHGHARYLARCL
CQSRYCSWRYSQKQRCNRGISTQGSGC
SCSRYQSWRYCQKQC(dP)GTISTC
8090
3141
2585
74
27
22
Streptomyces tendae
-
-
(G. Wiegand et al., 1995)
URECA work
URECA work
2 OBJECTIVES
This study aims to synthesize and test the inhibitory
effects of two modelled mammalian α-amylase
inhibitors: TBS-md11 and TBS-md16 from
Tendamistat. Our objectives are:
 To chemically synthesize TBS-md11and TBS-
md16 via Solid Phase Peptide Synthesis
(SPPS).
 To optimize the oxidative folding of TBS-
md11 and TBS-md16 in aqueous conditions.
 To test their inhibitory activity on mammalian
α-amylases via α-amylase inhibition assay.
4 MATERIALS AND METHODS
Fmoc-Solid Phase Peptide Synthesis (SPPS)
The solid phase peptide synthesis of TBS-md11 and
TBS-md16 was carried out using Fmoc chemistry.
Wang’s resin with a substitution ratio of 0.34 mmol/g
was used as the solid support. Synthesis of peptides was
carried out using an automated Liberty Microwave
Peptide Synthesizer (CEM, US).
The basic steps of the peptide synthesis process can be
found in Figure 2. Dimethylformamide (DMF) was
first added to swell the resin. Following this, the
coupling reagent, pyBOP (benzotriazol-1-yl-
oxytripyrrolidinophosphonium hexafluorophosphate)
and the base, N,N Diisopropylethylamine (DIEA) in
DMF were added to allow the first amino acid to be
attached to the resin and the subsequent amino acids to
attach to the N-terminus end of the peptide chain. Then
20% piperidine in DMF was added to deprotect the
amino acid by removing the Fmoc group followed by
washing of resin with DMF again. This step is known
as deprotection. The process of coupling and
deprotecting repeats as the amino acids are added to the
elongating peptide chain [5].
After coupling the last residue, the final step involves
cleaving of the peptide from the resin and the removal
of side chain protecting groups. TBS-md11 was cleaved
for 1 hour at room temperature in cleavage buffer A
containing 90% trifluoroacetic acid (TFA), 5%
triisopropysilane (TIS), 2.5% thioanisole and 2.5%
H2O. As for TBS-md16, cleavage buffer B which
contains 82.5% TFA, 15% p-cresol, 15% 1,2-
Ethanedithiol (EDT), 5% TIS and 5% H2O were used.
TBS-md16 was cleaved for 3 hours at room
temperature. Once both peptides had been cleaved, they
were precipitated using diethyl ether and aliquoted into
four tubes to test their solubility in different solvents:
DMSO, 70% acetic acid, isopropanol (iPrOH) and 50%
acetonitrile (MeCN). Mass spectrometry was used to
confirm the complete cleavage of our peptide followed
by purification by HPLC and then lyophilizing the
samples overnight to obtain the purified peptide.
Figure 2: The basic steps involved in Fmoc-Solid
Phase Peptide Synthesis [5].

3. Proceedings of the URECA@NTU 2013-14
Oxidative Folding in Aqueous Conditions
The fully reduced 0SS peptides, with SS denoting
disulphide bond, were folded in various oxidative
conditions. Reduced/oxidised glutathione (GSH/GSSG)
in a peptide:GSH:GSSG ratio of 1:200:10 were used to
provide a redox environment. To maintain the pH, 0.1M
Tris buffers with pH 7.5 and 8.0 were used. To facilitate
correct folding, organic solvents such as isopropanol
(iPrOH), acetonitrile (MeCN) or trifluoroethanol (TFE)
were tested. The oxidative folding products were
collected at different time points (0 hour, 1 min, 15 min
and 24 hours) for TBS-md11 and (0 hour and 2 hours)
for TBS-md16 and analysed using Reverse-Phase High
Performance Liquid Chromatography (RP-HPLC) and
MALDI-TOF Mass Spectrometry (MS).
Purified TBS-md11 and TBS-md16 were dissolved in
DMSO and MeCN respectively, followed by oxidative
folding in presence or absence of GSH/GSSG.
Isopropanol (iPrOH) was used in TBS-md16 but not for
TBS-md11. Equal volume of 4% Trifluoroacetic acid
(TFA) was added to stop the oxidative reaction at each
time point. The samples were then transferred into glass
vials and injected into RP-HPLC. For TBS-md11, the
purification is focused at 15-35% buffer B for 30
minutes, whereas for TBS-md16, the focusing range is
10-30% buffer B for 40 minutes. The products of the
oxidative folding were then analyzed using RP-HPLC
and MALDI-TOF MS.
Amylase Inhibition Assay
In order to determine the activity of the folded TBS-
md11 and TBS-md16, an amylase inhibition assay was
carried out using 96-well microtitre plates based on the
protocol that was developed previously in our
laboratory [5]. The activities of the following enzymes,
porcine pancreatic amylase (PPA), Aspergillus amylase
(AA) and human salivary amylase (HSA) were tested
before the addition of peptides to ensure that these
enzymes were active. Amylase inhibition assay was
then performed by adding 20ul of (PPA, AA and HSA)
with an equal volume of peptides. The mixture was
incubated for 20 minutes at 37°C. Subsequentlly, 60ul
of 1% starch (in 20mM sodium phosphate buffer, pH
6.7) was added and incubated for five minutes at room
temperature. 50ul of colour reagent (3,5-dinitrosaliclic
acid, Sigma and sodium potassium tartrate, Sigma) was
added into each well and incubated for 20 minutes at
100°C in an incubator. Once completed, the plate was
cooled down on ice for 1-2 minutes, followed by adding
100ul of water. To determine the α-amylase activity, the
absorbance at 540 nm was measured.
5 RESULTS
Synthesis of TBS-md11 and TBS-md16 Using Fmoc
Chemistry
Figure 3: RP-HPLC and MS profile of TBS-md11. The
synthesis yield is 75%.
Figure 4: RP-HPLC and MS profile of TBS-md16. The
synthesis yield is 45%.
The automated solid phase peptide synthesis of TBS-
md11 and TBS-md16 were successful. The results taken
from both RP-HPLC and MALDI-TOF mass
spectrometry as seen from Figure 3 & 4 showed the
presence of a linear 0SS TBS-md11 (m/z = 3141.5) and
TBS-md16 (m/z = 2585.0). Purity yield of 75% and
45% was achieved by TBS-md11 and TBS-md16
respectively.
Oxidative folding of TBS-md11 in DMSO with and
without GSH/GSSG
TBS-md11 has the highest solubility in DMSO as
compared to 70% acetic acid, isopropanol (iPrOH) and
50% acetonitrile (MeCN). The oxidative folding with
the reduced and oxidised glutathione (GSH/GSSG)
showed that in one minute, the peptide was completely
oxidised to 2SS as seen from Figure 5b. The m/z of the
product determined by mass spectrometry was found to
be 3137.7 Da ±0.3, which was in agreement with our
calculated m/z of TBS-md11 3137.4 Da. Time points at
15 minutes and 24 hours showed the formation of
degraded products from the peptide.

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Figure 5: Oxidative folding in aqueous conditions. (A) Various oxidative folding conditions were prepared. For each
experiment, 4% TFA was added to stop the reaction at different time points (0 hour to 24 hours). *
Redox environment
carried out using a peptide:GSH:GSSG ratio of 1:200:10. (B) Oxidative folding of 10µM 0SS TBS-md11 in 10% DMSO,
0.1M Tris buffer pH 8.0 in the presence or absence of GSH/GSSG, 25°C. Peak shift represents the folding from 0SS to
2SS. (C) Oxidative folding of 10µM 0SS TBS-md16 in 10% MeCN, 0.1M Tris buffer pH 8.0 in the presence or absence
of GSH/GSSG, 40% iPrOH, 25°C. Peak shift represents the folding from 0SS to 2SS.
A
B C

5. Proceedings of the URECA@NTU 2013-14
Oxidative folding of TBS-md16 in 10% MeCN with
and without GSH/GSSG
TBS-md16 was completely dissolved in 10% MeCN.
The oxidative folding with the reduced and oxidised
glutathione (GSH/GSSG) showed that at 2 hours, the
peptide was completely oxidised to 2SS species as seen
from Figure 5C. However instead of a single peak, the
results from the RP-HPLC showed six distinct peaks.
The m/z of the product determined by mass
spectrometry was found to be 2585.0 Da ±0.3, which
was in agreement with our calculated molecular weight
of TBS-md16 2585.0Da.
Amylase Inhibition Assay
We have successfully purified and lyophilized the
folded TBS-md11 and TBS-md16 using RP-HPLC. To
determine whether the peptide has an inhibitory effect
on amylase enzymes, we carried out an amylase assay
on three different amylases; porcine pancreatic,
Aspergillus and human salivary amylase. The result
showed that both TBS-md11 and TBS-md16 did not
cause any inhibition on the three amylases (Figure 6).
The enzymes in the presence or absence of TBS-md11
or TBS-md16 did not show any significant difference in
absorbance at 540 nm.
Figure 6: Amylase inhibition assay performed for three different species of TBS-md16 (indicated as TBS-md16a, TBS-
md16b and TBS-md16c) using a 96-well microtitre plates. Porcine pancreatic α-amylase (PPA), Aspergillus amylase
(AA) and human salivary amylase (HSA) were used to test the inhibitory effect of the peptide.
6 DISCUSSION
Oxidative folding of TBS-md11 in DMSO with and
without GSH/GSSG
The aqueous conditions that were used are important in
determining the correct folding of the peptide. The
oxidative folding with the reduced and oxidised
glutathione (GSH/GSSG) could serve as disulphide-
exchange reagents during protein folding which will
help to ensure proper folding whereas the presence of
Tris buffer with pH 7.5-8.0 will help to improve the rate
of folding [5,6]. We previously found that the linear
0SS TBS-md11 quickly oxidises to 2SS species in less
than one minute in Tris buffer pH 8.0. This is probably
because TBS-md11 has the tendency to form long β-
strands and thus, due to this molecular structure,
disulphide bond formation will be much easier [7]. In
addition, DMSO provides further oxidative strength.
The presence of degraded peptide products seen from
Figure B was probably due to the instability of the
folded peptide after a certain time.
PPA AA HSA
Tbs-md16a
Tbs-md16b
Tbs-md16c
Control
Blank

6. Proceedings of the URECA@NTU 2013-14
Oxidative folding of TBS-md16 in 10% MeCN with
and without GSH/GSSG
The main issue we encountered with TBS-md16 is the
presence of six peaks in the RP-HPLC profile. This
finding suggests that there is a possibility of the
presence of three types of peptide species,
corresponding to three possible disulphide connectivity,
each having two isomers. More work is to be done to
optimise the folding conditions and determine the
disulphide connectivity of these species.
Amylase Inhibition Assay
The amylase inhibition assay was performed to measure
the extent of inhibition from both TBS-md11 and TBS-
md16 on three different α-amylases. We carried out a
series of tests and found that both peptides failed to
significantly inhibit any of the three amylases.
The reason could be due to the short simulation time (t
= 16 ns) during model validation for both peptides [5].
Thus, more robust and stable modelled structures need
to be prepared and tested.
7 CONCLUSION
Literature precedents previously reported that a 74-
amino acid Tendamistat peptide from Streptomyces
tendae of the Streptomyces family was found to inhibit
α-amylase activity. We designed and engineered two
shorter version of Tendamistat, named TBS-md11 and
TBS-md16 consisting of 27 and 22 amino acids
respectively. In this study, we report on the synthesis
and oxidative folding of both TBS-md11 and TBS-
md16 followed by carrying out an amylase inhibition
assay on three different α-amylases to determine the
effect of inhibition by these two peptides.
Here, we successfully synthesized both peptides using
an automated solid phase peptide synthesizer, tested
their solubility in different solvents and collected the
purified peptides after undergoing several dimensions
of RP-HPLC.
The oxidative folding of TBS-md11 in Tris buffer pH
8.0 showed that the linear 0SS peptide undergone
complete oxidative folding within one minute and
degraded products was seen at 15 minutes.
As for the oxidative folding of TBS-md16, the resulting
RP-HPLC profile showed six peaks which suggest the
presence of three types of peptide species, with
different disulphide arrangements each having two
isomers.
We also report on the extent of inhibition by both
peptides on three different amylases using the amylase
inhibition assay. We found out that both TBS-md11 and
TBS-md16 failed to inhibit any of the three amylases.
Structural study, for example using NMR or X-ray
crystallography, is needed to address the lack of
inhibition of these modelled peptides.
ACKNOWLEDGEMENT
I would like to thank Professor James P. Tam from the
Division of Structural Biology and Biochemistry for
giving me this opportunity to be part of this meaningful
research.
I would also like to thank Dr. Nguyen Quoc Thuc
Phuong for providing me with the guidance and support
throughout this 11-month long Ureca project.
Special thanks to Assoc. Professor Sze Siu Kwan
Newman for his help throughout this study.
We wish to acknowledge the funding support for this
project from Nanyang Technological University under
the Undergraduate Research Experience on CAmpus
(URECA) programme.
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