Multifunctional ionic liquid-bound polystyrene resin with high loading capacity as support in solid-phase peptide synthesis

Article abstract of DOI:10.1016/j.tetlet.2017.03.014

Polystyrene resin-bound ionic liquids (PSILs) with high loading capacities were prepared by immobilizing multifunctional ionic liquids (ILs) on modified polystyrene (PS) resin and used in the solid phase peptide synthesis. Introduction of hydrophobic anio

Full text of DOI:10.1016/j.tetlet.2017.03.014

Accepted Manuscript
Multifunctional ionic liquid-bound polystyrene resin with high loading capacity
as support in solid-phase peptide synthesis
Tanmoy Patra, Sandip Karmakar, Sreedevi Upadhyayula
PII:
S0040-4039(17)30300-3
DOI:
http://dx.doi.org/10.1016/j.tetlet.2017.03.014
TETL 48715
Reference:
To appear in:
Tetrahedron Letters
Received Date:
Revised Date:
Accepted Date:
28 January 2017
21 February 2017
5 March 2017
Please cite this article as: Patra, T., Karmakar, S., Upadhyayula, S., Multifunctional ionic liquid-bound polystyrene
resin with high loading capacity as support in solid-phase peptide synthesis, Tetrahedron Letters (2017), doi: http://
dx.doi.org/10.1016/j.tetlet.2017.03.014
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Tetrahedron Letters
journal homepage: www.els evier.com
Multifunctional ionic liquid-bound polystyrene resin with high loading capacity
as support in solid-phase peptide synthesis
a
Tanmoy Patra , Sandip Karmakar and Sreedevi Upadhyayula
b
a,∗
a
Department of Chemical Engineering, Indian Institute of Technology Delhi, New Delhi 110016, India.
b
Department of Chemistry, Indian Institute of Technology Delhi, New Delhi 110016, India.
ARTICLE INFO
ABSTRACT
Article history:
Received
Polystyrene resin-bound ionic liquids (PSILs) with high loading capacities were prepared by
immobilizing multifunctional ionic liquids (ILs) on modified polystyrene (PS) resin and used in
the solid phase peptide synthesis. Introduction of hydrophobic anions and functional side chain
containing ILs resulted in high yield (82-98%) and purity (92-98%) of the synthesized peptides.
The coupling kinetic studies of the first and second amino acids to the PSILs were performed to
investigate the effect of IL functionalization on PS supports.
Received in revised form
Accepted
Available online
Keywords:
Ionic liquid
Peptide synthesis
Peptides
©2017 Elsevier Ltd. All rights reserved.
SPPS
Amino acid
*
Corresponding author. Tel.: +91-11-2659-1083; fax: +91-11-2659-1120.

With the invention of a number of commercially important
peptide-based pharmaceutical products as well as synthetic
peptides, the innovative synthesis of peptides have gained a
tremendous interest among the researchers in last few years. With
the decade-long persisting problems in the field of peptide
manufacture, the best method till date is still the solid-phase
methodology can be further explored to improve the loading
capacities, optimize the reaction conditions, improvement of the
kinetics, substitution of toxic imidazole components from the
support, etc.
Recently, diethanolamine (DEA)-based PSILs with high
loading capacities were reported earlier for the chemical fixation
of CO
1
,2
8
with very high efficiency. The immobilization of the –
peptide synthesis (SPPS) prescribed back in 1963. Several
innovative methodologies are also reported in last few years in an
attempt to get rid of the persisting problems in this field like low
loading capacity of the available solid supports, separation
problems in the homogeneous phase synthesis, poor kinetics of
the SPPS, energy and cost-inefficient enzyme mediated
2
OH group containing functionalized ILs on PS changes the
flexibility of the resin moiety. In this study, DEA-based ILs were
supported on the PS resin and used as support in C-terminal
peptide manufacture process. The anion metathesis of the ILs led
to the formation of a series of ILs and their effect on the coupling
kinetics of the amino acid was investigated. The synthetic
efficiency of the PSILs on the peptide manufacture cycle was
also demonstrated by the synthesis of oligopeptides.
3
processes, etc. Most of these methods suffer from the
disadvantages like low loading capacity, poor industrial
4
scalability, costly starting materials, etc. Polystyrene (PS) resins
are most commonly used resin because of their good swelling
property in various solvents and good chemical and mechanical
stability. However, the hydrophobic matrix of these typical solid-
phase resin supports leads to problems like aggregation of the
growing peptides and poor diffusion of the amino acids and
To improve the swelling properties of the support, 1%
divinylbenzene containing PS resins were used. The loading of
diethanolamine on the PS resin was achieved by the substitution
of the –Cl group of chloromethyl PS (PS-Cl) with the N-atom to
obtain PS-DEA. The synthesized PSILs as shown in Scheme 1,
were composed of an additional spacer to stop additional inter-
chain interactions among the amino acids of the growing peptide
chains. Reaction with 2-butyl bromide led to the formation of
4
hence, retarded kinetics. Hence, to improve the swelling
properties of the hydrophobic supports, sustainable flexible
supports with hydrophilic groups are to be developed.
Recently, ionic liquids (ILs) were reported as homogeneous
8
bromide containing PSIL as prescribed in literature. Further,
-
5
phase soluble support for the C-terminal peptide synthesis.
Functionalized ILs were also reported as solvents in synthesis of
-
-
-
6 4 2
substitution of the Br anion with entities like PF , BF and NTf
6
peptides with difficult sequences with high yields. The use of
was achieved by the anion metathesis with their corresponding Li
or K salts in a 1:1 mixture of water and DMF. A similar
triethanolic IL (as shown in Scheme 1) was synthesized by
addition of 2-bromoethanol group to PS-DEA followed by
ILs in the homogeneous phase resulted in more controlled chain
growth without any puckering or agglomeration, no racemization
5
,6
and controlled solubility of the peptides. However, these
methods suffer from the disadvantages like poor recyclability of
the ILs used as homogeneous support, poor kinetics imparted by
viscous ionic liquids along with inherent homogeneous phase
problems like poor industrial scalability, solubility issues for
larger peptides, etc.
2
substitution of the bromide anion with NTf . FT-IR spectroscopic
studies were firstly carried out with PS-Cl, PS-DEA and pure
DEA. Prior to analysis the samples were dried over phosphorus
pentoxide under reduced pressure at 70°C for 24 h. The loading
of DEA on the PS-Cl was confirmed by the presence of a strong
-
1
Hence, to address these issues, efficiency of imidazole-based
alkylamine functionalized PS resin-bound ILs (PSILs) were
investigated using the inherent advantages imparted by ILs in
peak around 3400-3450 cm in both DEA and PS-DEA as shown
in Suppl. Figure 1. Complete DEA loading on the PS-Cl by
removal of –Cl groups was confirmed by the absence of C-Cl
bond stretching (1265 cm ) in the FT-IR spectrum of the resin,
PS-DEA. The loading% of the ILs on PS resin was determined
by the nitrogen content estimated from elemental analysis.
7
-1
solid-phase C-terminal peptide synthesis approach. A new
methodology using PSILs with excellent swelling property were
successfully used for the synthesis of oligo and polypeptides.
However, the reported PSILs suffered with issues like low
loading capacity, requirement of additional linker, etc. Hence, the

Scheme 1. Synthetic pathway of the PSILs.
SEM images and the EDX data of the pure PS and PSILs are
shown in Suppl. Figure 2. The loading of ILs on the PS does not
alter the morphology of the surface as evident from the SEM
images. The anion metathesis of the PSILs was confirmed from
the EDX data showing the presence of exchanged anions.
Swelling property of the resins was estimated in terms of the
swelling volume of the PSILs in different solvents as
summarized in Suppl. Table 1. Swelling property determines the
diffusion tendency of the reactants on the polymer surface and
hence, the overall kinetics and yield in the process. The polymer
supported ILs normally swelled more in polar aprotic solvents
rather than less polar organic solvents which can be attributed to
the amphiphilic nature provided by the hydrophobic anion and
purity: 80-96%) than similar homogeneous phase IL supports
5
(yield: 50-55%, purity: 86-90%). Among the PSILs, PS-
DESBA-NTf was most efficient in terms of yield (95%) and
purity of the pentapeptide (96%). Comparably lower efficiency of
2
-
-
-
6 4
other PSILs composed of PF , BF or Br anions can be
attributed to the inherent lower swelling property leading to less
flexibility of the support matrix. The amphiphilic environment
provided by the anionic component plays important role in the
diffusion of the reagents over the resin surface. As more than one
–OH groups were present in the side chain of the PSILs, almost
two or three equivalent of amino acids could be loaded. Thus,
higher loading efficiency was obtained using these PSILs than
7
,10
similar resin supports. Further, in addition to the swelling
property, the electrostatic environment provided by these ILs
stabilizes the growing peptide chain and the reagents resulting in
higher yield and purity of the obtained peptides.
hydrophilic –OH groups. Among the PSILs, PS-DESBA-NTf
exhibited more swelling in polar aprotic solvents like DMF,
NMP and DCM than the others mainly due to the more
2
-
-
-
-
hydrophobic nature of NTf
2
than PF
6
, BF
4
or Br .
The synthetic efficiency of the PSILs in SPPS was
To demonstrate the electrostatic effects of these ILs, the
coupling kinetics of the first amino acid, Fmoc-Leu-OH, on the
PSILs as well as the second amino acid, Fmoc-Phe-OH, on the
Leu-OH-attached PSILs was investigated and the results are
shown in Figure 1. The progress of the reaction was measured by
collecting resin samples at different time intervals followed by
investigated following the steps shown in Scheme 2. The
dipeptide, Phenylalanyl leucine (Phe-Leu-OH) and pentapeptide,
Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu), was synthesized using
the same coupling-decoupling reaction sequences and
1
11
characterized using H NMR, MALDI-TOF and Circular
9
Dichroism (CD) spectral data (Suppl. Figure 5). Further, the
Fmoc-titration as reported in literature. All these PSILs showed
high loading capacity as well as fast reaction rates proportional to
yield and purity of the obtained peptide were estimated using
HPLC data (Suppl. Figure 3) as shown in Table 1. All the
PSILs showed higher yield and purity of the dipeptide (yield:
2
their swelling properties. PS-DESBA-NTf showed the maximum
Table 1. Yield and purity of the peptides produced via Scheme 2 using
different PSILs.
a,b
9
2-98%, purity 89-98%) and pentapeptides (yield: 82-95%,
c
d
Phe-Leu-OH
Leu-enkephalin
Resins
Yield
%)
Purity (%)
Yield (%)
Purity (%)
(
PS-Cl
7
9
3
5
8
2
7
82
89
96
98
93
95
45
82
89
95
82
94
68
80
95
96
91
94
PS-DESBA-Br
PS-DESBA-PF
9
9
9
9
9
6
PS-DESBA-NTf
2
PS-DESBA-BF
4
e
PS-TEA-NTf
2
a
Reaction conditions (coupling step): Temperature, 27°C; Resin, 0.15 g; Fmoc-amino acid, 1.2 mmol,
bHBTU, 1.2 mmol; DIPEA, 2.4 mmol; 5 mL DMF.
Reaction conditions (Fmoc deblocking step): Temperature, 27°C; 20% (v/v) piperidine in 5 mL DMF.
Reaction conditions (detachment step): Temperature, 27°C, 95% TFA, 5 mL.
c
d
Reaction conditions (detachment step): Temperature, 27°C, TFA/triisopropysilane/H
mL.
2
O (95:2.5:2.5), 5
e
Fmoc-amino acid, 1.8 mmol.
Scheme 2. General outline of the peptide synthesis steps using PSILs.

by Nano research facility (NRF), Indian Institute of Technology
Delhi.
(
a)
Supplementary data
Supplementary data associated with this article can be found
in the online version, at http://dx.doi.org/.
References and notes
1
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9:1–38.
6
.
Figure 1. Kinetics of (a) Fmoc-Leu-OH loading on PSILs and (b) Fmoc-
Phe-OH on Leu-OH loaded PSILs; Reaction conditions: Temperature, 27°C;
Resin, 0.15 g; Fmoc-amino acid, 1.2 mmol, HBTU, 1.2 mmol; DIPEA, 2.4
mmol; 5 mL DMF.
7. H.J. Cho, S.M. Lee, S. Jung, T.K. Lee, H.J. Yoon, Y.S. Lee, Tetrahedron
Lett. 2011; 52:1459–1461.
reaction rate and higher loading efficiency. PS-TEA-NTf
showed higher loading efficiency as it contained three –OH
functionalities. However, the loading efficiency of the PS-TEA-
2
8
.
X. Chen, J. Sun, J. Wang, W. Cheng, Tetrahedron Lett. 2012; 53:2684–
688.
Leu-enkephalin: H NMR (400 MHz, DMSO-d ) δ(ppm): 0.91 [m, 6H,
2
1
9
.
6
2
NTf was not proportional to the number of –OH groups in
CH (Leu)], 1.25 [m, 1H, CH(Leu)], 1.52-1.66 [m, 2H, CH (Leu)], 2.68,
2.70, 3.01-3.16 [m, 4H, CH (Tyr and Phe)], 3.50-3.70 [m, 2H, α-H(Tyr
2
3
2
contrary to the PSILs containing a 2-butyl group as a spacer to
decrease the inter-chain interactions in between the two growing
peptide chains. The growth of the three simultaneous peptide
and Leu)], 3.84 [d, 4H, α-H(Gly
and Gly
.86 [m, 2H, aryl(Tyr)], 7.02 [d, 2H, aryl(Tyr)], 7.14 [m, 2H, aryl(Phe)],
)], 4.18 [m, 1H, α-H(Phe)], 6.54,
3
2
6
7
7
8
.27 [m, 2H, aryl(Phe)], 7.40 [t, 1H, aryl(Phe)], 7.53 [t, 1H, NH(Tyr)],
.70 [m, 1H, NH(Leu)], 7.94 [br, 1H, NH(Gly )], 8.12 [d, 1H, NH(Phe)],
.38 [br, 1H, NH(Gly )]. MALDI-TOF (m/z): calc. as 555.63 for
chains on the PS-TEA-NTf
chain interaction between the amino acids as evident from Figure
. Further, this faster kinetics and efficient loading can be
2
support results in increased inter-
3
2
+
1
C H N O [M+H] , found 555.18.
28 37 5 7
attributed to the flexible nature of these functionalized supports
with –OH groups on the side chains and hydrophobic anions
resulting in efficient biphasic reaction media.
10. M. Kim, Y.S. Park, D.S. Shin, S. Lee, Y.S. Lee, Tetrahedron Lett. 2012;
3:4576–4579.
1. B. Yoo, M.D. Pagel, Bioconjug. Chem. 2008; 18:903–911.
5
1
In conclusion, a series of multifunctional ILs with different
anionic entities were supported on PS resin and used in the
manufacture of di- and pentapeptides. The modification of the
resin supports with ILs containing –OH groups on the side chain
and hydrophobic anions played important role in the swelling
property of the polymer support. The kinetics of the first and
second amino acid attachment on the polymer support was
investigated and a model di- and pentapeptide was synthesized
using these PSILs to demonstrate the synthetic efficiency. The
inherent electrostatic interactions of the ILs was found to play
crucial role in controlling the conformation of the growing
peptide chain resulting in improved overall yield and purity of
the peptides. Overall, a new class of amine based ionic liquid
modified resin support is provided as a solution with new insights
to get rid of the persisting problems in peptide manufacture
processes.
Acknowledgments
Tanmoy Patra thanks the Council of Scientific and Industrial
Research (CSIR), India, for the senior research fellowship. The
authors also acknowledge the instrumentation facility provided

Graphical Abstract
Multifunctional ionic liquid-bound polystyrene resin with
high loading capacity as support in solid-phase peptide
synthesis
a
Tanmoy Patra , Sandip Karmakar and Sreedevi Upadhyayula *
b
a

Highlights:
•
•
•
•
•
Ionic liquid incorporated polystyrene resin
(
PSIL) in peptide synthesis
Improved flexibility and swelling property
of the resin
High yield and purity of the di- and
pentapeptides
Kinetics of first and second amino acid
loading on PSILs
Controlled chain growth on PSILs with the
stabilization of growing peptide chain