Solid-phase peptide synthesis of dipeptide (histidine-β-Alanine) as a chelating agent by using trityl chloride resin, for removal of Al3+, Cu2+, Hg2+ and Pb2+: Experimental and theoretical study

Article abstract of DOI:10.5935/0103-5053.20160064

Solid-phase peptide synthesis of dipeptide (histidine-β-Alanine) as a chelating agent examined by common N-9-fluorenylmethyloxycarbonyl-N-Trityl-L-histidine and tert-butyloxycarbonyl-β-Alanine-OH amino acid derivatives. Trityl chloride resin was used as a carrier resin. The molecular structure of the dipeptide was definite by using different methods such as ultraviolet visible (UV-Vis), Fourier transform infrared (FTIR), proton (1H) nuclear magnetic ressonance (NMR) and liquid chromatography-mass spectrometry (LC-MS) and the chelating property of synthesized dipeptide was investigated for removing of metal ions Al³⁺, Cu²⁺, Hg²⁺ and Pb²⁺ in vitro. In addition, the pharmacological and biological activities of dipeptide were examined by prediction of activity spectra for substances (PASS) program.

Full text of DOI:10.5935/0103-5053.20160064

http://dx.doi.org/10.5935/0103-5053.20160064
J. Braz. Chem. Soc., Vol. 27, No. 10, 1814-1819, 2016.
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0103 - 5053 $6.00+0.00
Article
Solid-Phase Peptide Synthesis of Dipeptide (Histidine-β-Alanine) as a Chelating
Agent by Using Trityl Chloride Resin, for Removal of Al3+, Cu2+, Hg2+ and Pb2+:
Experimental and Theoretical Study
Rahmatollah Rahimi,*,a Maryam Khosravi,a Mohammd H. H. Tehrani,b Mahboubeh
Rabbania and Ebrahim Safavic
aDepartment of Chemistry, Iran University of Science and Technology, 1684613114 Tehran, Iran
bDepartment of Pharmaceutical Chemistry, School of Pharmacy, Shahid Beheshti University of
Medical Sciences, 1991953381 Tehran, Iran
cWorld Society of the Anti-Aging Medicine and International Hormone Society,
11977634461 Tehran, Iran
Solid-phase peptide synthesis of dipeptide (histidine-β-alanine) as a chelating agent examined
by common N-9-fluorenylmethyloxycarbonyl-N-trityl-L-histidine and tert-butyloxycarbonyl-β-
alanine-OH amino acid derivatives. Trityl chloride resin was used as a carrier resin. The molecular
structure of the dipeptide was definite by using different methods such as ultraviolet visible
(
UV-Vis), Fourier transform infrared (FTIR), proton (1H) nuclear magnetic ressonance (NMR)
and liquid chromatography-mass spectrometry (LC-MS) and the chelating property of
synthesized dipeptide was investigated for removing of metal ions Al3+, Cu2+, Hg2+ and
Pb2+ in vitro. In addition, the pharmacological and biological activities of dipeptide were
examined by prediction of activity spectra for substances (PASS) program
.
Keywords: dipeptide, solid-phase peptide synthesis, trityl chloride resin, chelating property,
PASS program
Introduction
times during the synthesis, and therefore, removal must be
done in mild conditions that do not affect the remaining
protecting groups or even the peptide chain.4 The two most
commonly amine-protecting groups in solid-phase peptide
synthesis are 9-fluorenylmethyloxycarbonyl (Fmoc) and
tert-butyloxycarbonyl (Boc).
The success of the solid-phase method is closely
dependent on the efficiency of the solid support. Trityl
chloride resin is perhaps one of the most useful resins for the
solid-phase synthesis of C-terminal peptide acids. It can be
used for the preparation of both protected and unprotected
peptides. The main advantages of its use are: (i) allows
to cleave the peptide under mild acidic conditions [1%
trifluoroacetic acid (TFA)]; (ii) minimizes the formation
of diketopiperazine (DKP); (iii) minimizes racemization
during the attachment of the first amino acid; and (iv)
minimizes the formation of side chain.5
Peptides are one of the best candidates for drug
development due to their high specificity and low toxicity.
Peptides are mostly obtained by biological technology or
chemical synthesis. The chemical method, especially solid-
phase peptide synthesis (SPPS), is usually used for the
large-scale production of peptides because of its simplified
reaction procedure and easy purification/isolation steps for
the target products.1 SPPS can be defined as a process in
which amino acid bound by its C-terminus to an insoluble
polymer.2 This method has made peptide synthesis simple,
rapid, and easily subject to automation.3 Protection of
α- and β-amino functionality of amino acids is one of the
most important issues in peptide chemistry and is efficient
to prevent polymerization of the amino acid once it is
activated. Because most peptide synthesis, both in solution
and on solid-phase, are carried out in the C to N direction,
α- and β-amino protecting groups are removed several
The ability of dipeptides to chelate of various metal ions
should be investigated.6,7 Complexation of this dipeptide
with numerous cations (such as Ru2+, Zn2+, Co2+, Ni2+, Mn2+,
Cd2+, Mg2+, and Ca2+) have been studied.8-11
*e-mail: rahimi_rah@iust.ac.ir

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Rahimi et al.
1815
In this paper, we report the synthesis of dipeptide
histidine-β-alanine as a chelating agent with chemical
method, solid-phase peptide synthesis. Molecular structure
and chelating property of synthesized dipeptide were
investigated. In addition, the pharmacological and
biological activities of dipeptide were examined by
prediction of activity spectra for substances (PASS)
program.
mixture was shaked for 2 h.After 2 h, the resin was filtered
and washed thoroughly under nitrogen atmosphere with
DMF (5 mL × 3) and after that with DCM (5 mL × 3).
Experimental
Chemicals
Trityl chloride resin (ca. 200-400 mesh) and N-Fmoc-
N-trityl-L-histidine were purchased from Bachem chemical
Company (Budenforf, Switzerland). Boc-β-alanine-OH was
obtained from Aldrich Company (St. Louis, MO, USA).
Coupling reagents: O-(benzotriazol-1-yl)-N,N,N’,N’-
tetramethyluronium tetrafluoroborate (TBTU) was purchased
from Fluka Chemical Company (Buchs, Switzerland).
Scavengers: anisole and phenol; solvents: trifluoroacetic acid
(TFA), piperazine, N,N-di-isopropylethylamine (DIPEA),
diethyl ether, dichloromethane (DCM), N,N-dimethyl
formamide (DMF) and methanol (MeOH); metals: AlCl3,
CuCl2, HgCl2 and Pb(NO3)2 were obtained from Merck
(Darmstadt, Germany).
Scheme 1. Attachment of the first amino acid N-Fmoc-N-trityl-L-histidine
to the resin. DIPEA: N,N-Diisopropylethylamine.
Capping
After the first amino acid be loaded to the resin,
the un-reacted sites must be end-capped with methanol
(MeOH) to ensure that future reactions do not react at those
unloaded sites. The process of methanol capping is fairly
simple and involves a mixture of DCM, MeOH, and DIPEA
in a 80:15:5 ratio added to the resin and shaked for 15 min.
The resin must be washed under nitrogen atmosphere with
DMF (5 mL × 3) and then with DCM (5 mL × 3) to remove
any excess of MeOH and DIPEA before being re-swollen
and coupled with the next amino acid.12
Methods
PG Instruments T80 double beam ultraviolet-visible
(UV-Vis) spectrophotometer (Leicestershire, UK) was
used for UV-Vis measurements. The Fourier transform
infrared spectra (FTIR) were recorded on a Shimadzu
FTIR-8400S spectrophotometer (Kyoto, Japan) in the
range of 400-4000 cm−1. Proton (1H) nuclear magnetic
ressonance (NMR) spectra were recorded on a Bruker
DRX 250 (300 MHz) spectrometer (Billerica, MA, USA) in
water. Mass-spectra was recorded on anAgilent 6410 Triple
Quadrupole liquid chromatography-mass spectrometry
Deprotection of Fmoc group (Scheme 2)
Removal of the Fmoc group (3) was obtained by the
addition of 10% piperazine, shaked for 30 min under nitrogen
atmosphere and after 30 min, washed with DMF (5 mL × 3).
The monitoring of the complete cleavage of the Fmoc was
performed with color detector (acetaldehyde/chloranil), for
detection of free terminal amino groups (4). This detector
will do the transition from white to green if an amino group
is present.13 Solution 1 is composed by 2% acetaldehyde in
DMF, and solution 2 by 2% chloranil in DMF. A few beads
of resin are placed in a small test tube and 2-5 drops of each
solution are added. After a short mixing, the mixture is
left at room temperature for 5 min. The result dark blue to
green beads is positive for Fmoc cleavage, and colorless to
yellowish beads is negative for Fmoc cleavage.
(
LC-MS) using Agilent 1200 series HPLC system
(Santa Clara, CA, USA) column C18 (250 × 4.6 mm,
5 µm); mobile phase A, H2O and B, methanol; flow rate of
1 mL min−1, 20 µL, total run time of 40 min.
Loading of trityl chloride resin (Scheme 1)
Peptide synthesis was carried out using trityl chloride
resin. Trityl chloride resin (2 g) (1) was swelled in
dichloromethane (1 h). N-Fmoc-N-trityl-L-histidine
(2.47 g, 2 equivalents) (2), DIPEA (1 mL), DCM (2.5 mL)
and DMF (5 mL) were added to the reaction vessel. The
Coupling of second amino acid Boc-β-alanine-OH to the
resin (Scheme 3)
After loading the first amino acid on trityl chloride
resin (4), the second amino acid Boc-β-alanine-OH (0.75 g,

1816
Solid-Phase Peptide Synthesis of Dipeptide (Histidine-β-Alanine) as a Chelating Agent
J. Braz. Chem. Soc.
with DCM (5 mL × 3). The filtrate was evaporated under
reduced pressure and the resulting mixture precipitated
by adding diethylether (chemical formula = C9H14N4O3,
molar mass = 226.23 g mol−1, melting point = 258 °C,
yield = 90%; Scheme 4). Boc is acid labile and sensitive to
acid cleavage.A common process used to cleave the Boc off
the amine while on a solid phase resin is to add a solution
of 1:1 TFA in DCM (10 mL) at room temperature for 1 h.
The amine will form a TFA salt and must be washed with
3 successive steps of 1:1 DCM and DIPEA (10 mL g−1
resin) to prepare the deprotected amine product for the
next addition step. If using 2-chlorotritylchloride resin, this
will also cleave it, forming the resin as well as the amine.
It is recommended to use a base labile resin if one needs to
remove the Boc for further alkylation prior to deprotection
from a resin.15 (Yield = 90%, melting point = 258 °C).
Elemental analysis: calculated C (47.78%), H (6.24%),
N (24.77%), O (21.22%). Molecular formula: C9H14N4O3;
formula weight: 226.2324.
Scheme 2. Deprotection of 9-fluorenylmethyloxycarbonyl (Fmoc) group
from the resin.
2 equivalents) (5) was treated with coupling reagents
(TBTU 0.32 g, 2 equivalents in DIPEA 1 mL), DCM
(10 mL) to form a solution, which then was added to the
resin and shaked for 1 h at room temperature. The reaction
was terminated by performing the chloranil test, colorless
to yellowish beads positive for coupling the second amino
acid (6).After coupling, resin was filtered and washed with
DMF (5 mL × 3), after that with DCM (5 mL × 3) and then
with MeOH (5 mL × 3), respectively, and dried in vacuo.
Scheme 4. Deprotection of tert-butyloxycarbonyl (Boc) group and
cleavage from the resin. TFA: Trifluoroacetic acid.
Scheme 3. Coupling of second amino acid Boc-β-alanine-OH to the
resin. DIPEA: N,N-Diisopropylethylamine; TBTU: O-(benzotriazol-1-
yl)-N,N,N’,N”-tetramethyluronium tetrafluoroborate.
Results and Discussion
Characterization of synthesised dipeptide (histidine-β-
Deprotection of Boc group and cleavage from the resin
(Scheme 4)
alanine)
Dipeptide was successfully synthesized via the standard
BOC method. The synthesis of dipeptide (histidine-β-
alanine) was approved by using UV-Vis, FTIR, 1H NMR
and LC-MS analysis. The UV-Vis absorbance spectra of
histidine-β-alanine obtained in water at 25 °C is presented
in Figure 1. The results show that the maximum peaks
were appeared at 214 and 264 nm, which can be assigned
to π→π* and n→π*, respectively.
In the last step of the synthesis, peptide was cleaved
from the resin with a mixture of TFA (1 mL), anizol
(0.3 mL), phenol (0.3 g) and was shaken mechanically at
room temperature for 2 h. One important consideration
when selecting the cleavage cocktail are scavengers, such as
anizol, phenol, triisopropylsilane (TIPS), 1, 2-ethanedithiol
(EDT), thioanisole or water. The reason the scavengers are
often added is because during the course of cleavage, highly
reactive cationic species can be generated, which can cause
damage to the structure. The purpose of a scavenger is to
extinguish any reactive species that may be generated during
revelation from TFA cleavage.14 At the end of 2 h, the resin
was filtered and washed with DMF (5 mL × 3) and after that
FTIR (KBr) n / cm−1 3226 (NH2), 1641 (amide), 1563
(imidazol); 1H NMR (300 MHz, D2O) d 2.60 (m, 2 Hs,
12H), 2.92 (dd, 1 Hs, 6H), 3.08 (dd, 1 Hs, 6H), 3.16 (m,
2 Hs, 11H), 4.40 (dd, 1 Hs, 7H), 6.89 (s, 1 Hs, 4H), 7.66
(dd, 1 Hs, 2H), 7.89 (imidazole ring). The LC-MS analysis
showed a single mass peak in [M + H]+ and [M]−, which

Vol. 27, No. 10, 2016
Rahimi et al.
1817
the metal cation, ligand-to-metal ratios, and the ionic
strength of the supporting solution.11
To study of kinetic reactions between dipeptide
(histidine-β-alanine) and various metal ions, and changes of
absorption in wavelength of 214 nm (maximum wavelength
of dipeptide) were investigated by UV-Vis spectrophotometer
when concentration of dipeptide was kept constant at
10−4 mol L–1 vs. metal ions with different concentrations
(10−1, 10−2, 10−3, 10−4 and 10−5 mol L−1). After interaction of
dipeptide with various metal ions, the maximum absorption
peak of dipeptide reduced differently. For example, at
concentration of 10−4 mol L−1 of metals, Pb2+ ion was shown
the most efficient absorbing by chelating reaction (Figure 3).
Figure 1. Ultraviolet visible (UV-Vis) absorption spectrum of dipeptide
(histidine-β-alanine).
correspond to molecular weight for dipeptide calculated
for C9H14N4O3: 226.23; found m/z [M + H]+: 227.000 and
m/z [M]−: 224.800 (Figure 2).
Figure 3. Ultraviolet visible (UV-Vis) absorption spectra of dipeptide
(10−4 mol L−1) with AlCl3, CuCl2, HgCl2 and Pb(NO3)2.
The results listed in Table 1 show that in initial
times (about 10-15 min), chelating reactions of
dipeptide (concentration of 10−4 mol L–1) were faster
in presence of metals which have same concentration
(10−4 mol L–1) and lower concentration (10−5 mol L–1),
but in higher concentration these reactions carried out
slower (Figures 4-6). As seen in these figures, the order
of the observed strength chelating adsorption in different
concentrations is: (i) 10−5 mol L–1: Pb2+ > Cu2+ > Hg2+ =Al3+;
(ii) 10−4 mol L–1: Pb2+ > Al3+ > Hg2+ > Cu2+; and
(iii) 10−3 mol L–1: Al3+ > Cu2+ > Hg2+ > Pb2+.
Figure 2. Liquid chromatograph-mass spectrometry (LC-MS)
chromatogram of dipeptide (histidine-β-alanine): (a) +scan; and (b) –scan.
Study of chelating property of dipeptide (histidine-β-alanine)
in vitro
At low concentration of metal ions, Pb2+ is more adsorbed
from the solution. It can occur because Pb2+ has a larger
radius rather than other cations; therefore, it can be easily
coordinated to dipeptide.At high concentration of metal ions
(10−3 mol L−1), the adsorption ofAl3+ is the best. Al3+ has the
smallest radius and more charge than the other cations, which
lead to more ability of it for accepting pair electron from N or
O atoms of peptide. However, the coordination of dipeptide
withAl3+ is poor but the higher ability for accepting electron
pairs increase its adsorption.
The bioactivities behaviors of histidine-β-alanine
depend on its complexation of metal cations. This
peptide is a multidentate ligand with five potential
metal-coordinating sites (two N of imidazole ring, one
carboxylate group, an amide linkage and a terminal
amino group). Both tetrahedral and octahedral types of
complexes can be formed, but the accurate configuration
and chelating ability of dipeptide can depend on size of

1818
Solid-Phase Peptide Synthesis of Dipeptide (Histidine-β-Alanine) as a Chelating Agent
J. Braz. Chem. Soc.
Table 1. Kinetic analysis of different concentration of metals with fixed
concentration of dipeptide, at 10−4 mol L–1 after 30 min
Metal
concentration /
(mol L–1)
Al
Cu
Pb
Hg
1 × 10−1
1 × 10−2
1 × 10−3
1 × 10−4
1 × 10−5
2 × 10−5
3 × 10−5
4 × 10−5
5 × 10−5
6 × 10−6
−1 × 10−5
−5 × 10−5
−2 × 10−5
−2 × 10−5
−4 × 10−5
−6 × 10−5
−4 × 10−4
−1 × 10−4
−6 × 10−5
4 × 10−7
−1 × 10−6
−2 × 10−6
6 × 10−6
4 × 10−7
−1 × 10−6
−2 × 10−6
6 × 10−6
1 × 10−8
6 × 10−6
3 × 10−6
−7 × 10−5
−7 × 10−5
−2 × 10−5
−2 × 10−5
−3 × 10−5
−2 × 10−6
1 × 10−6
2 × 10−6
1 × 10−5
−3 × 10−7
−4 × 10−5
−3 × 10−5
9 × 10−7
−5 × 10−6
−6 × 10−6
Figure 5. Kinetic study of dipeptide (histidine-β-alanine, 10−4 mol L–1) with
AlCl3, CuCl2, HgCl2 and Pb(NO3)2 (10−4 mol L–1) in wavelength of 214 nm.
Figure 4. Kinetic study of dipeptide (histidine-β-alanine, 10−4 mol L–1) with
AlCl3, CuCl2, HgCl2 and Pb(NO3)2 (10−5 mol L–1) in wavelength of 214 nm.
Theoretical
Figure 6. Kinetic study of dipeptide (histidine-β-alanine, (10−4 mol L–1)
with AlCl3, CuCl2, HgCl2 and Pb(NO3)2 (10−3 mol L–1) in wavelength of
214 nm.
Theoretical methods
The evaluation of computer system for PASS on
the set of dipeptide using multilevel neighborhoods of
atoms (MNA) descriptor was studied. PASS results for
a compound are presented as a list of activity names and
probability activity (Pa) values in Table 2.
teeth grinding, allergies and colds, snoring, frequent
urination, fibromyalgia, nightmares, etc. that can cause
sleep disturbances, which the treatment depends on the
causes, but dipeptide with 54.2% of pharmacological effect
can be a treatment for sleep disorders. Aminoacylhistidine
dipeptidases (EC 3.4.13.3, also Xaa-His dipeptidase,
X-His dipeptidase and PepD) are zinc-containing
metallopeptidase, which catalyse the cleaving and release
of a N-terminal amino acid, usually neutral or hydrophobic
residue, from Xaa-His dipeptides or polypeptides.16-19
Dipeptide has the role of inhibition by 88.7% PASS activity.
Histidine ammonia lyase (HAL) catalyses the elimination
of ammonia from the substrate to form (E)-urocanate,
which the dipeptide here is the inhibitor through the
molecular mechanisms with 78.2% of biological activity.
Theoretical study of dipeptide (histidine-β-alanine)
Lipotropic compounds are those that help catalyse
the breakdown of fat during metabolism in the body. It
has been predicted that dipeptide (histidine-β-alanine)
produces modest short-term weight loss in some people,
along with the advantages that are the lowest side effects
(1.3 to 6.9%) and good pharmacological activity. As an
illustration, this compound can be introduced as a suitable
in obesity with 56.7% lipotropic effect. There are many
conditions, diseases and disorders, including sleep apnea,

Vol. 27, No. 10, 2016
Rahimi et al.
1819
Table 2. Portion of the predicted probability activity (Pa) spectra for dipeptide (histidine-β-alanine
)
Pharmacological effects
Lipotropic
Pa / %
56.7
54.2
45.4
42.0
40.4
32.2
31.4
Molecular mechanisms
X-his dipeptidase inhibitor
histidine ammonia-lyase inhibitor
histidine-tRNA ligase inhibitor
phytepsin inhibitor
Pa / %
88.7
78.2
77.7
66.1
61.1
53.5
18.4
Side effects and toxicity
ulcerogenic
Pa / %
6.9
5.1
4.9
1.8
1.6
1.4
1.3
Sleep disorders treatment
Bone formation stimulant
Antihypertensive
embryotoxic
vytotoxic
eye irritation
mutagenic
Hematopoietic
acrosin inhibitor
Myelodysplastic syndrome treatment
Restenosis treatment
lysozyme inhibitor
skin irritation
carcinogenic
chelator
Also, dipeptide is the histidine-tRNA ligase, phytepsin,
acrosin and lysozyme inhibitor by 77.7, 66.1, 61.1 and
53.5%, respectively. According to Table 2, it is observed
that controversial point as a dietary supplement seems
to have all the same chelating properties, and it offers a
possibility for an inexpensive oral chelation therapy but
it has 18.4% PASS activity as a chelator, which shows
the chelation chemistry of dipeptide through mixed
complexes. In fact, this dipeptide with drug-likeness of
0.916 and new biological properties presented in this
study can be utilized to identify numerous medicine
applications.
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Conclusions
In this work, dipeptide (histidine-β-alanine) as a
chelating agent was synthesized via chemical method,
SPPS. This method allows performing synthesis much
more quickly compared to solution phase chemistry, which
requires long purification methods after each intermediate
step. The study of chelating property of dipeptide (in vitro)
by UV-Vis absorbance spectra revealed that chelating
activity depends on concentration of metals, molar ratio
and kind of metals. In addition, the pharmacological and
biological activities of this dipeptide were examined
by PASS program. It offers a possibility for an oral
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Acknowledgments
The financial support of this study by Iran University
of Science and Technology is gratefully acknowledged.
Submitted: December 28, 2015
Published online: March 1, 2016