A green protocol for peptide bond formation in WEB

Article abstract of DOI:10.1016/j.tetlet.2016.04.041

A simple, efficient, and environmentally friendly approach has been developed for the synthesis of peptides in aqueous medium. In this work, peptides are easily synthesized in water extract of banana (WEB)/ethylene glycol and without using external base u

Full text of DOI:10.1016/j.tetlet.2016.04.041

Accepted Manuscript
A Green Protocol for Peptide Bond Formation in WEB
Manashjyoti Konwar, Abdul Aziz Ali, Diganta Sarma
PII:
S0040-4039(16)30391-4
DOI:
http://dx.doi.org/10.1016/j.tetlet.2016.04.041
TETL 47541
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 March 2016
7 April 2016
13 April 2016
Please cite this article as: Konwar, M., Ali, A.A., Sarma, D., A Green Protocol for Peptide Bond Formation in WEB,
Tetrahedron Letters (2016), doi: http://dx.doi.org/10.1016/j.tetlet.2016.04.041
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
Leave this area blank for abstract info.
A Green Protocol for Peptide Bond Formation in WEB
Manashjyoti Konwar, Abdul Aziz Ali and Diganta Sarma*
O
R1
R2
O
R1
H
EDC.HCl
+
N
COOMe
Ph
N
COOH
H2N
COOMe.HCl
Ph
N
WEB/EG, rt
H
H
O
R2
R1= -H, -CH3, -CH2Ph, -CH(CH3) 2, -CH2CH(CH3) 2
R2= -H, -CH3

1
Tetrahedron Letters
A Green Protocol for Peptide Bond Formation in WEB
Manashjyoti Konwar, Abdul Aziz Ali and Diganta Sarma *
Department of Chemistry, Dibrugarh University, Dibrugarh, Assam, India 786004
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A simple, efficient and environmentally friendly approach has been developed for the synthesis
of peptides in aqueous medium. In this work, peptides are easily synthesized in water extract of
banana (WEB)/ethylene glycol and without using external base under mild condition.
© 2009 Elsevier Ltd. All rights reserved.
Available online
Keywords:
Peptide
water
green chemistry
WEB
Introduction
Results and discussion
In this work, we present a green methodology towards the
formation of peptides in aqueous medium. Peptides are easily
synthesized in water extract of banana (WEB) in presence of
trace amount of ethylene glycol and without using external base
under mild condition in a relatively shorter reaction time with
good to excellent yields. We recently reported the synthesis of
biaryls through Suzuki-Miyaura (SM) cross coupling reaction
performed in novel ‘WEB’ in a highly efficient and green
manner.17 WEB, “water extract of banana” (scientific name:
Musa balbisiana Colla; family: Musaceae; species: Musa
balbisiana), is prepared by first drying the banana peels followed
by burning them to ash (Fig. 1). Water is added to the ash, mixed
well and then filtered. The filtrate is termed as WEB. It is
envisaged that WEB has a very great potential in green chemical
processes from the economic and environmental points of view
in the near future. Here we have chosen WEB as a reaction
medium due to the fact that it has a dual function of green solvent
as well as a base. However, our main objective is to develop a
green and efficient method for the peptide bond formation in
aqueous solvent at room temperature. This solvent system has a
superior catalytic activity than aqueous medium since it has basic
nature. In this method, the yields are relatively high, reaction
proceeds in short time interval, the reaction is highly economical
and most importantly it is eco-friendly leading to green chemistry
protocols. 18-24
The peptide formation reaction between two molecules of amino
acids is one of the important carbon–nitrogen bond formation
reactions in organic chemistry.1 It has a great importance not only
in the field of biology but also in the fields of agriculture,
pharmaceuticals and industrial applications.2-6 However, the
choice of peptide synthesis in water is a relatively challenging
work in the field of organic chemistry. However, in our body
proteins are naturally synthesized in aqueous medium.7-9
Usually, peptide synthesis is carried out in presence of an
activating agent, coupling agent catalyst and a base.8 Traditional
bases such as K2CO3, NaHCO3, triethylamine are generally used
in peptide couplings reactions. The use of bases in peptide
synthesis is proved to be detrimental when the coupling reaction
is carried out in water. Base catalyzes the hydrolysis of amino
acid ester, which is used as a coupling partner; thus inhibiting the
reaction progress. Therefore, development of a basic green
solvent medium will have a great impact on the peptide synthesis
so as to avoid the need of an external base.
Due to inflammability and toxicity of organic solvents, water is
considered as a green and superior solvent for organic
reactions.10-11 Water is easily available and its low cost leads the
reaction more economical towards the green process. Over the
past few years, a lot of experiments have been carried out in the
field of peptide chemistry trying to perform the reaction in the
aqueous media with great efficiency and in a relatively shorter
reaction time.12-14 Literature revels that lot of experiments are
carried out in organic solvents using different ligands but there
are only few reports presenting a relatively resourceful results of
peptide synthesis in aqueous media without use of any kind of
ligands.15-16
The reaction of N-benzoyl glycine with glycine methyl ester
hydrochloride catalyzed in WEB at room temperature in presence
of EDC.HCl afforded 70% yields after 4 h (Table 1, entry 1).
However, the same reaction while carried out in presence of little
amount of ethylene glycol gave almost quantitative yields (95%
isolated yields) after 4 h (Table 1, entry 2) which acts as a
promoter for the reaction. The co-solvent effect on peptide
i
coupling was tested with two other solvents viz. PrOH and
tBuOH (Table 1, entries 5-6); unlike ethylene glycol the yields
i
t
obtained with PrOH and BuOH were relatively low. In absence
of the coupling agent EDC.HCl, the reaction did not proceed at
*Corresponding author. Tel.: +91 373-2370210
E-mail address: dsarma22@gmail.com; dsarma22@dibru.ac.in

2
Tetrahedron
all (Table 1, entry 3) indicating the necessity of the coupling
Table 2: Effect of substituents on peptide synthesisa
agent under our reaction condition. To understand the role of
base, the
O
R1
R2
O
R1
EDC.HCl
H
+
N
COOMe
Ph
N
COOH
H2N
COOMe.HCl
Ph
N
WEB/EG, rt
H
H
O
R2
Entry
1
R1
-H
R2
-H
Yieldb(%)
95
2
3
-CH3
-H
-CH3
-CH3
-H
71
67
76
58
61
76
72
68
77
Dry banana peels
Musa balbisiana Colla
4
-CH3
5
-CH2Ph
-CH2Ph
-H
6
-CH3
-H
7
-CH(CH3) 2
-CH(CH3)2
8
-CH3
-H
9
Water extract of
banana (WEB)
Ashes of
banana peels
-CH2CH(CH3) 2
10
-CH2CH(CH3) 2
-CH3
a Reaction conditions:- benzoyl protected amino acid (1 mmol), amino acid
methyl ester hydrochloride(1.5 mmol), EDC.HCl (1 mmol) in WEB (3 mL)
and ethylene glycol (0.2mL) at room temperature for 4 h.
Fig. 1 Preparation of WEB
b Isolated yields.
reaction of N-benzoyl glycine with glycine methyl ester
hydrochloride was carried out in water in absence of any base,
the reaction did not proceed at all (Table 1, entry 4). In short,
under our optimized conditions, the peptide coupling reaction
precedes in presence of a coupling agent (EDC.HCl), basic
catalytic medium (WEB) and a promoter (Ethylene glycol) at
room temperature.
In order to demonstrate the practical importance and wide
application of the catalytic system consisting of WEB, coupling
agent and ethylene glycol, it is essential to show the efficient
recycling of the catalyst system.25-26 The results obtained in our
experiment confirmed that it was possible to recycle and reuse
the catalytic medium after extraction of products up to at least
three times. For this recyclability study, the reaction between N-
benzoyl glycine and glycine methyl ester hydrochloride was
carried out under our optimized condition, affording the desired
dipeptide in almost quantitative yields (95% isolated yields) after
4 h (Table 3, entry 1) without adding extra ethylene glycol on
successive runs. The resulting mixture was extracted with diethyl
ether (3x10 mL). The ether layer was separated and the catalytic
medium was reused for next two runs (Table 3, entries 2–3). For
every repeated run, homogeneous solution was obtained. The
yields obtained for successive runs were 88% and 85%
respectively.
Table 1: Optimization of reaction conditions for peptide
synthesis.a
O
O
H
EDC.HCl
N
COOMe
Ph
N
Ph
N
COOH
H2N
COOMe.HCl
H
Solvent, r.t.
H
O
Entry Solvent
Coupling
agent
Time
(h)
Yieldb(%)
1
2
3
4
5
WEB
EDC.HCl
EDC.HCl
-
4
4
12
12
70
95
-
WEB/EG
WEB/EG
Water
WEB/ iPrOH
WEB/ tBuOH
EDC.HCl
-
Table 3: Recyclability testa
EDC.HCl
EDC.HCl
4
4
65
60
O
O
H
EDC.HCl
WEB, r.t.
6
N
COOMe
Ph
N
Ph
N
COOH
H2N
COOMe.HCl
H
a
H
O
Reaction conditions: benzoyl protected amino acid (1 mmol), amino acid
methyl ester hydrochloride (1.5 mmol), EDC.HCl (1 mmol) in WEB (3 mL),
Ethylene Glycol (0.2 mL) at room temperature.
b isolated yields
Different benzoyl protected amino acids and various amino acid
methyl ester hydrochloride salts were used to construct the
peptide moieties in a base free environment (Table 2). The
coupling of benzoyl glycine with alanine methyl ester
hydrochloride afforded 67% yields in 4 h (Table 2, entry 3).
Identical results were obtained when Bz-alanine, Bz-
phenylalanine, Bz-valine and Bz-leucine were coupled with the
glycine methyl ester and alanine methyl ester hydrochloride
(Table 2, entries 3-10).

3
11. Hojo K.; Ichikawa H.; Fukumori Y.; Kawasaki K. Int J Pept Res
Ther. 2008, 14, 373–380.
a
Reaction conditions: benzoyl protected amino acid (1 mmol), amino acid
12. Shie J.J.; Fang J.M. J. Org. Chem. 2003, 68, 1158-1160.
13. Nepomniaschiy N.; Grimminger V.; Cohen A.; DiGiovanni S.;
Lashuel H.A.; Brik A. Org. Lett. 2008, 10, 5243-5246.
14. Cui Y.; Wang Y.; Liu R.; Sun Z.; Wei Y.; Zhao Y.; Gao X. ACS
Nano, 2011, 5, 8684–8689.
15. Zajac K.; Kaminski Z.J. Acta Poloniae Pharmaceutica- Drug
Research. 2010, 67, 725-728.
methyl ester hydrochloride (1.5 mmol), EDC.HCl (1 mmol) in WEB (3 mL)
and ethylene glycol (0.2 mL) at room temperature.
bisolated yield.
16. Katz C.; Levy-Beladev L.; Rotem-Bamberger S.; Rito T.; Rudiger
S. G. D.; Friedler A. Chemical Society Reviews, 2011, 40, 2131–
2145.
17. (a) Boruah P.R.; Ali A.A.; Saikia B.; Sarma D. Green Chem.
2015, 17, 1442–1445; (b) Boruah P.R.; Ali A.A.; Chetia M.;
Saikia, B.; Sarma, D. Chem. Commun. 2015, 51, 11489-11492.
18. Stelakatos G.C.; Paganou A.; Zervas L. J. Chem. SOC.(C). 1966,
1191-1199.
19. Meneses C.; Nicoll S.L.; Trembleau L. J. Org. Chem. 2010, 75,
564–569.
20. Sheehan J.C., Hlaviia J.J. A review article from the department of
chemistry, Massachusetts institute o f technology. 1956, 21, 439-
441.
21. Llobet A.L.; lvarez M.A.; Albericio F. Chem. Rev. 2009, 109,
2455–2504.
22. Mitchell A.R. Peptide Science. 2007, 90, 215-233.
23. Galanis A.S.; Albericio F.; Grøtli M. Org. Lett. 2009, 11, 4488-
4491.
24. Pedersen S.L.; Tofteng A.P.; Malik L.; Jensen K.J. Chem. Soc.
Rev. 2012, 41, 1826–1844.
25. Miao W.; Chan T.H. Acc. Chem. Res. 2006, 39, 897-908.
26. Guzman F.; Barberis S.; Illanes A. Electronic Journal of
Biotechnology. 2005, 10, 279-314.
The coupling reaction of two amino acid moieties in WEB has
a great importance as the coupling reaction proceeds in aqueous
media, at room temperature and in absence of external base.
Literature reports reveal that banana peels contain potassium,
sodium, carbonate and chloride as major constituents along with
a host of other trace elements.27 Therefore, it is believed that
carbonates of sodium and potassium of banana peels act as a base
here and chlorides of sodium and potassium act as promoters for
peptide bond forming reactions. Burning of the banana peels may
also cause the decomposition of the carbonates to oxides but this
decomposition takes place at a very high temperature of more
than 12000 C and the heating of Bunsen burner is in the range of
10000 C. Again literature reveals that, potassium carbonate has
the thermal stability near its melting point because the vapor
pressure over K2CO3 suggest a low rate of decomposition near
the melting point (13 mbar is achieved only by 12000C).28 The
effects of salt additives such as LiCl, LiBr, LiClO4, ZnCl2 etc. on
yields, side-product formation, racemisation and reaction rates on
peptide coupling are reported earlier in literature.29
27. Deka D.C.; Talukdar N.N. Indian J. Tradit. Know. 2007, 6, 72–78.
28. Lehmana R. L.; Gentry J. S.; Glumaca N.G. Thermochimica Acta.
1998, 316, 1-9.
Conclusion
29. Thaler A.; Seebach D.; Cardinaux F. Helvetica Chimica Acta.
1991, 74, 617-627.
30. Ingenito R.; Wenschuh H. Org. Lett. 2003, 5, 4587-4590.
31. Lee S.; Valentine S.J.; Reilly J.P.; Clemmer D.E. J. Am. Chem.
Soc. 2011, 133, 15834–15837.
32. Made V.; Els-Heindl S.; Beck-Sickinger A.G. Beilstein J. Org.
Chem. 2014, 10, 1197–1212.
33. Lowe G.; Vilaivan T. J. Chem. Soc. Perkin Trans. 1. 1997; 555-
560.
34. General experimental procedure for peptide synthesis: In a 50 ml
round bottom flask, a mixture of benzoyl protected glycine (1
mmol, 0.179g), glycine methyl ester hydrochloride (1.5 mmol,
0.188g), EDC.HCl (1 mmol, 0.192g ) in WEB (3 mL) and 0.2 ml
ethylene glycol was added and stirred at room temperature for 4
hours. After completion of the reaction (by TLC monitoring); the
reaction mixture was extracted with diethyl ether (3x10 ml) and
the organic layer was washed with distilled water (20 mL), dried
over Na2SO4 and organic layer is evaporated in vacuuo. The
residue was purified by column chromatography on silica gel
using ethyl acetate/hexanes (1:1) as eluent to give the
corresponding peptide. The products were characterized by
1HNMR and ESI-MS spectrometry.
In summary, we have demonstrated the efficient green method
for the peptide bond formation reaction in WEB at room
temperature in presence of ethylene glycol as promoter/additive
and without using external base.30 The field of peptide chemistry
is a very broad subject which also includes the protein synthesis
as the higher branch of peptide coupling reactions. 31-32 By using
this coupling protocol of the two amino acid residues we expect
that it will give a great utility in the synthesis of polypeptides in
near future. 33
Acknowledgments
MK is thankful to UGC for UGC-BSR fellowship. The authors
acknowledge the Department of Science and Technology for
financial assistance under DST-FIST programme and UGC, New
Delhi for Special Assistance Programme (UGC-SAP) to the
Department of Chemistry, Dibrugarh University.
References and notes
1.
Jursic B.S.; Zdravkovdki Z. Synth. Commun. 1993, 23, 2761–
2770.
2.
Mhidia R.; Vallin A.; Ollivier N.; Blanpain A.; Shi G.; Christiano
R.; Johannes L.; Melnyk O. Bioconjugate Chem. 2010, 21, 219–
228.
3.
Schreiner E.; Nair N.N.; Dominik M. J. Am. Chem. Soc. 2009,131,
13668–13675.
4.
5.
Arthur G. Wiley-Interscience. 2000.
Carey J.S.; Laffan D.; Thomson C.; Williams M.T. Org. Biomol.
Chem. 2006, 4, 2337–2347.
6.
Lenstra D.C.; Rutjes F.P.J.T.; Jasmin M. Chem. Commun. 2014,
50, 5763—5766.
7.
8.
9.
Tanga W.; Becker M.L. Chem. Soc. Rev. 2014, 43, 7013—7039.
Michael D. B. Biochemistry, 2009, 48, 6571–6584.
Constable D.J.C.; Dunn P.J.; Hayler J.D.; Humphrey G.R.; Leazer
J.J.L.; Linderman R.J.; Lorenz K.; Manley J.; Pearlman B.A.;
Wells A.; Zaks A.; Zhang T.Y. Green Chem. 2007, 9, 411–420.
10. Chandrudu S.; Simerska P.; Toth I. Molecules, 2013, 18, 4373-
4388.

4
Tetrahedron
HIGHLIGHTS
 Economical and environmentally friendly aqueous system as the medium
 No external base is required for peptide coupling
 Peptides are synthesized in water at room temperature
 Organic solvents are not used