Synthesis of biologically important angiotensin-II and angiotensin-IV peptides by using 4-(2',4'-dimethoxypenyl-fmoc-aminomethyl)phenoxy resin

Article abstract of DOI:10.14233/ajchem.2013.14998

In peptide chemistry, there is the goal to produce pure biologically active peptides with the highest possible efficiency, even its possible by chemical synthesis purity and yield as a major problem. In this work, we focus to overcome above difficulty by novel synthetic approach. Angiotensin an oligopeptide is a hormone and a powerful dipsogen, which exhibits a wide range of biological activities, the 8-amino acid sequence Angiotensin-II and 6-amino acid sequence. Angiotensin-IV were synthesized by using 4-(2',4'- dimethoxyphenyl- Fmoc-aminomethyl)phenoxy resin, the modified Fmoc-chemistry and effective hydroxybenzenetriazole, N,N'-diisopropylcarbodiimide coupling and activation methods was used. The yield and chromatographic purities were compared.

Full text of DOI:10.14233/ajchem.2013.14998

Asian Journal of Chemistry; Vol. 25, No. 15 (2013), 8673-8676
http://dx.doi.org/10.14233/ajchem.2013.14998
Synthesis of Biologically Important Angiotensin-II and Angiotensin-IV Peptides by
Using 4-(2',4'-Dimethoxypenyl-Fmoc-Aminomethyl)phenoxy Resin
1,*
2
3
2,*
R. SELVAM , K.C. ROHINI , C. ARUNAN and K.P. SUBASHCHANDRAN
1Research and Development Centre, Bharathiar University, Coimbatore-641 046, India
2Research and PG Department of Chemistry, Sri Vyasa NSS College, Thrissur-680 623, India
3School of Chemical Science, Mahatma Gandhi University, Kottayam, India
*Corresponding authors: E-mail: selvachemist@rediffmail.com; kpsubhash@rediffmail.com
(Received: 18 December 2012;
Accepted: 26 August 2013)
AJC-14024
In peptide chemistry, there is the goal to produce pure biologically active peptides with the highest possible efficiency, even its possible
by chemical synthesis purity and yield as a major problem. In this work, we focus to overcome above difficulty by novel synthetic
approach. Angiotensin an oligopeptide is a hormone and a powerful dipsogen, which exhibits a wide range of biological activities, the
8-amino acid sequence Angiotensin-II and 6-amino acid sequence. Angiotensin-IV were synthesized by using 4-(2',4'-dimethoxyphenyl-
Fmoc-aminomethyl)phenoxy resin, the modified Fmoc-chemistry and effective hydroxybenzenetriazole, N,N'-diisopropylcarbodiimide
coupling and activation methods was used. The yield and chromatographic purities were compared.
Key Words: Peptide, Angiotensin, 4-(2',4'-Dimethoxyphenyl-Fmoc-aminomethyl)phenoxy resin.
Here in this proposed research work we successfully
synthesized Angiotensin II and Angiotensin IV. Angiotensin
was independently isolated in Indian apolis and Argentina in
the late 1930s (as ‘angiotonin’and ‘hypertensin’, respectively)
and subsequently characterized and synthesized by groups at
the Cleveland Clinic and Ciba laboratories in Basel, Switzer-
land. Angiotensin is a peptide hormone that causes vasocon-
striction and a subsequent increase in blood pressure8,9. It is
part of the renin-angiotensin system, which is a major target
for drugs that lower blood pressure.Angiotensin also stimulates
the release of aldosterone, another hormone, from the adrenal
cortex. Aldosterone promotes sodium retention in the distal
nephron, in the kidney, which also drives blood pressure up.
Angiotensin II acts on the adrenal cortex, causing it to release
aldosterone, a hormone that causes the kidneys to retain sodium
and lose potassium. Elevated plasma angiotensin II levels are
responsible for the elevated aldosterone levels present during
the luteal phase of the menstrual cycle8,9. In this work we con-
cerned about the purity (hygienic) for biological research and
yield for further development.
INTRODUCTION
Nowadays, large biotechnology-based initiatives, like the
Human Genome Project1,2, as well as the improved understan-
ding of fundamental biological processes, provides a huge
number of new protein sequences. This leads to a rapid
increase in the number of novel or important targets for drugs
applications2. Therefore, there is a high demand of these new
targets in at least micro multimilligram quantities. Obviously,
access to these proteins should be provided within the
shortest possible time frame, mainly used to fulfill this
requirement is chemical synthesis3. Synthetic peptides find
application in all areas of biomedical research including
immunology, neurobiology, pharmacology, enzymology and
molecular biology4. The chemical synthesis of peptides with
the naturally occurring structure is possible, it was used for
the development of artificial vaccines and potent drugs that
can substitute the conventional drugs having various side
effects. Investigation of structure-activity relationship of
biologically active peptides also demands the synthesis of many
analogues of a given peptide. But in peptide chemistry there
is a goal to synthesize a peptide with highest purity and yield,
it should be a considerable factor5,6. For biological research
the recommended peptide purity should be needed. Nowa-
days advanced purification process are applicable to purify
the peptide, but purification process will reduce the peptide
yield3,7.
The chemical structure ofAngiotensinII andAngiotensin
IV peptide were shown in Figs. 1 and 2.
EXPERIMENTAL
Amino acids: Fmoc-Asp(otBu)-OH, Fmoc-Arg(pbf)-OH,
Fmoc-Tyr-OH and Fmoc-Phe-OH where purchased from
Aldrich, Switzerland, Fmoc-His(Trt)-OH, Fmoc-Val-OH and

8674 Selvam et al.
Asian J. Chem.
Fmoc-Ile-OH were purchased from Alfa-easer, England,
Fmoc-Pro-OH were purchased from Novabiochem, Germany,
all above amino acids are biochemical reagent were of the
highest purity available and tested.
of ethanol. C. 1 mL of 0.001 N aqueous KCN in 15 mL of
pyridine.
Fmoc deprotection: 3 mL of the mixture added to the
resin and shake it for 10 min × 2 times filtrate were removed
and washed.
Solid support: 4-(2',4'-Dimethoxyphenyl-Fmoc-amino-
methyl)phenoxy resin 100-200 mesh (loading 0.71 mmol/g)
were purchased from Novabiochem, Germany.
Washing: Minimum amount of DMF × 4 times and NMP
× 2 times shake well and filtrate were removed.
Ninhydrin test for free amino group: A small pinch of
resin beads with one drop of A, B and C were taken in a small
fusion tube and heated to 100 ºC blue colour shows presence
Special reagents: N,N′-Diisopropylcarbodiimide (DIC),
DIPEA, hydroxybenzenetriazole (HOBt), triisopropyl silane
(TIS), trifluoroacetic acid (TFA), m-cresol, aceticanhydride,
piperidine, pyridine, ninhydrin and dichlorodimethylsilane
were purchased from Sigma-Aldrich China, Alfa-Asser
England and Novabiochem Germany.
of free amino group7,10,11
.
TLC plates were coated with silica gel G, basic solvent
system suspended CHCl3-MeOH were prepared and iodine
vapours were used as visualizing agent for monitoring the
coupling process and purity10.
Solvents: N-methyl-2-pyrrolidone (NMP), dimethyl
formamide (DMF), dichloromethane (DCM), diethyl ether,
methanol and acetonitrile are of HPLC grade and were
purchased from E. Merck (India) and Lobachemi, Mumbai.
Solvents were purified by standard procedures.
Swelling: 150 mg of a 4-(2',4'-dimethoxyphenyl-Fmoc-
aminomethyl)phenoxy resin were transferred to well clean
sililated and sterilized peptide synthesizer, to that sufficient
amount of N-methyl pyrolidone were added and swelled for
1 h and removed the filtrate under vacuum. Fmoc group from
The HPLC system specially made for peptide and biolo-
gical samples (Make: M/s Shimadzu Corporation, Japan) RP-
C18 column diameter 250 mm × 4.6 mm, 25 cm length, 5 µm
particle size, run time 0.5 h, injection volume: 20 µL, linear
gradient 5 % acetonitrile: 95 % water at 0 min, 100 % acetoni-
trile : 0 % water at 0.5 h, flow rate 1 mL/min.
resin were removed and washed7,10,11
.
Coupling: Fmoc amino acid dissolved in minimum
quantity of NMP in well closed 25 mL round bottom flask to
that hydroxybenzenetriazole were added and dissolved and
N,N′-diisopropylcarbodiimide were added and shake it well
for 3 min and immediately the content was transferred in to
the resin with moisture free atmosphere and shake it well for
5 min, to that N,N′-diisopropylethylamine were added and
shake well for 45 min. Reaction progress were monitored by
Reagents were prepared(freshly) under hygienic
condition: 20 % piperidine in dimethylformamide for Fmoc
deproduction.
Ninhydrin solutions to check free amino group:A. 0.5 g
of ninhydrin in 10 mL of ethanol, B. 10 g of phenol in 10 mL
(NH2)Asp-Arg-Val-Tyr-Ile-His-Pro-Phe(CONH2)
H3C
H3C
H3C
O
CH3
NH2
O
NH2
O
O
O
O
NH
NH
NH
N
NH
NH
NH
HO
O
O
O
N
H
N
NH
OH
NH2
HN
Fig. 1. Chemical composition and structure of Angiotensin II contains 8 amino acid residue (m.wt :1046 g/mol)
(NH2)Val-Tyr-Ile-His-Pro-Phe(CONH2)
H3C
H3C
H2N
H3C
O
CH3
NH2
O
O
O
NH
NH
N
NH
NH
O
O
N
H
N
OH
Fig. 2. Chemical composition and structure of Angiotensin IV contains 6 amino acid residue (m.wt: 775 g/mol)

Vol. 25, No. 15 (2013)
Synthesis of Biologically Important Angiotensin-II and Angiotensin-IV Peptides 8675
uV
TLC. Small pinch of the resin were taken and washed,
ninhydrin were tested in case positive means same amino acid
coupling was repeated, in case negative means washed,
deprotected, remaining amino acids coupling were done by
above method.
500000
250000
0
The detailed synthetic strategy, time duration of reaction
process and conditions are given in Tables 1 and 2.
Cleavage of crude peptide from resin: After synthesis
the resin were washed with hexane, DCM, CHCl3 and MeOH
and dried. The cleavage were done with 95 % TFA: 2.5 %
TIS: 2 % water: 0.5 % m-crosal at 3 h under nitrogen atmos-
phere and the resin were washed 4 times with TFA, the filtrate
were collected in 50 mL RB-Flash and all the traces of TFA
0
5
10
15
min
20
25
30
Fig. 3. HPLC of Angiotensin-2 (NH2)Asp-Arg-Val-Tyr-Ile-His-Pro-
Phe(CONH2). The single sharp peak at ret. time 7.24 min shows
our target peptide purity. Crude peptide yield: 83.67 %
uV
was evaporated by using Rota vacuum evaporator10,12
.
2000000
The peptide were isolated with excess of peroxide free
pure cold diethyl ether and the peptide were washed 7 times
with diethyl ether and centrifuged. The clear white powder
form of peptide were collected in a small tubes and sealed5.
1000000
0
0
5
10
15
min
20
25
30
RESULTS AND DISCUSSION
Fig. 4. HPLC of (NH2)Val-Tyr-Ile-His-Pro-Phe(CONH2), the single sharp
peak at ret. time 7.14 min shows our target peptide purity. Crude
peptide yield: 85.78 %
In this present work, three valuable factors are focused.
The hygienic purity of peptide were recorded, expected yield
should be achieved and the observations of this work
compared with earlier methods. A very important part of this
research was that the biologically important crude peptide with
> 90 % purity (hygienic) should be achieved. In earlier methods
the main factor is purification process reduce the peptide yield,
so crude peptide with maximum purity should be needed for
further development. The most essential biologically important
Angiotensin II andAngiotensin IV crude peptide with > 90 %
purity were achieved.
The given HPLC analysis report gives clear evidence for
the purity of the crude Angiotensin II and Angiotensin IV
peptide.
Comparison: The success of this modified synthetic
approach was compared with other known methods (Fig. 5).
Here maximum crude peptide purity and yield was obtained
in new modified fmoc synthetic approach when compared to
other approach, the result and observations of this work is
compared with earlier reported peptide synthetic procedures.
The following graphical evidence clearly reveals the success
of this methods.
The accuracy of HPLC assay method was assessed by
standard method (the known peptide sample purity were
recorded and compared with standard reference). The purity
of both crude peptides were shown in Figs. 3 and 4.
TABLE-1
SYNTHESIS OF ANGIOTENSIN-II (NH2)Asp-Arg-Val-Tyr-Ile-His-Pro-Phe(CONH2)
Coupling(min)
Deprotection
15 min (× 2)
Amino acid (Fmoc-)
Fmoc-Phe-OH
Ninhydrin
Washing
Washing
1st
45
30
30
30
30
30
30
30
2nd
40
35
30
30
40
35
35
40
3rd
–
-ve
-ve
Done
Done
Done
Done
Done
Done
Done
Done
Done
Done
Done
Done
Done
Done
Done
–
Done
Done
Done
Done
Done
Done
Done
–
Fmoc-Pro-OH
–
Fmoc-His(trt)-OH
Fmoc-Ile-OH
–
-ve
45
–
-ve
Fmoc-Tyr(tbu)-OH
Fmoc-Val-OH
-ve
40
–
+ve*
-ve
Fmoc-Arg(pbf)-OH
Boc-Asp(tbu)-OH
–
-ve
*In case ninhydrin is +ve after 3rd coupling acetylation were done.
TABLE-2
SYNTHESIS OF ANGIOTENSIN-IV (NH2)Val-Tyr-Ile-His-Pro-Phe(CONH2)
Coupling (min)
Deprotection
15 min (× 2)
Amino acid (Fmoc-)
Fmoc-Phe-OH
Ninhydrin
Washing
Washing
1st
45
30
30
30
30
30
2nd
40
30
30
40
35
35
3rd
–
-ve
-ve
-ve
-ve
-ve
+ve
Done
Done
Done
Done
Done
Done
Done
Done
Done
Done
Done
–
Done
Done
Done
Done
Done
–
Fmoc-Pro-OH
Fmoc-His(trt)-OH
Fmoc-Ile-OH
–
–
45
–
Fmoc-Tyr(tbu)-OH
Boc-Val-OH
40

8676 Selvam et al.
Asian J. Chem.
100
ACKNOWLEDGEMENTS
Purity (%)
90
Yield (%)
The authors thankful to University Grant Commison,
Newdelhi, India for providing financial support to this research
work under major research scheme.
80
70
60
50
40
30
20
10
REFERENCES
1. URL, http://www.ornl.gov/TechResources/Human_Genome/home.html
2. R. Rudolph and H. Lilie, FASEB J., 10, 49 (1996).
3. G.B. Fields, R.H. Angeletti, S.A. Carr, A.J. Smith, J.T. Stults, L.C.
Williams and J.D. Young, In ed.: J.W. Crabb, Techniques in Protein
Chemistry V: pp. 501-507 (1994).
4. J. Galpin, Amino Acids, Peptides and Proteins; The Royal Society
ofChemistry, Burlington House, London, Vo1. 16, pp. 272- 341 (1985).
5. B. Merrifield, Science, 232, 341 (1986).
0
A
B
B*
6. L.P. Miranda and P.F. Alewood, Accelerated Chemical Synthesis of
Peptides and Small Proteins, Proceedings of the National Academy of
Sciences of the United States of America, 96, pp. 1181-1186 (1999).
7. G.B. Fields, R.H. Angeletti, L.F. Bonewald, W.T. Moore, A.J. Smith,
J.T. Stults and L.C. Williams, In ed.: J.W. Crabb, Techniques in Pro-
tein Chemistry VI, pp. 539-546 (1995).
Fig. 5. Purity and yield of the biologically active peptide achieved from a
modified Fmoc solid phase synthesis method were compared with
standard reference of solution phase and earlier solid phase synthetic
methods. A. Solution phase peptide synthesis. B. Fmoc peptide
chemistry. B*. Modified Fmoc peptide chemistry
8. E.L. Boulpaep and W.F. Boron, Medical Physiology: a Cellular and
Molecular Approach. St. Louis, Mo: Elsevier Saunders, p. 771 (2005).
9. N. Basso and N.A. Terragno, Hypertension, 38, 1246 (2001).
10. M. Bodanszky, Principles of Peptide Synthesis; Springer Verlag: New
York (1984).
Conclusion
The present modified fmoc chemistry method has been
found to enhance the biologicaly active peptide purity and
yield. Here biologically activeAngiotensin II andAngiotensin
IV peptide synthesized and hygienic purity was absorbed and
the success of this method were compared. The main goal of
the proposed modified fmoc peptide synthetic method was
enhance the biologically important peptide purity and yield.
The coupling method is also relatively fast as compared with
previously reported procedures. It will helpful for further
research and improvement.
11. G. Barany and R.B. Merrifield, In eds.: E. Gross and J. Meienhoferm,
In: The Peptides: Analysis, Synthesis and Biology, Academic Press:
New York, pp. 1-228 (1980).
12. E. Atherton and R.C. Shepard, Solid Phase Peptide Synthesis-A Practical
Approachusing the Fmoc-Polyamide Techinique, IRL Press, Oxford
(1984).