An improved soluble polynorbornene support for peptide synthesis

Article abstract of DOI:10.1039/c5ra21668k

Soluble polymers that can be readily isolated via precipitation and possess multiple amino acid attachment sites are highly attractive for peptide synthesis. Polyethylene glycol supports that solubilize the growing peptide chain and can be readily isolated have been widely used for peptide synthesis. However, a stoichiometric amount of the PEG support is required because each PEG support typically has a single attachment site for peptide synthesis. Reported herein is the development of a polynorbornene support containing multiple attachment sites as well as alkyl and oligoether solubilizing/spacer groups. The attachment site is connected to the polymer backbone through a solubilizing oligoether linker. The support was developed after evaluating the effect of the linker and attachment site-spacing on peptide synthesis using suitably designed polynorbornene supports. The high solubility of the support minimizes the equivalents of reagents used for peptide synthesis. The support has been used to synthesize the natural product Leu⁵-enkephalin in 52% overall yield using only 1.2 equivalents of coupling reagents, which is comparable or superior to reported procedures using a large excess of reagents.

Full text of DOI:10.1039/c5ra21668k

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An improved soluble polynorbornene support for
peptide synthesis†
Cite this: RSC Adv., 2015, 5, 93027
*
Nimmashetti Naganna and Nandita Madhavan
Soluble polymers that can be readily isolated via precipitation and possess multiple amino acid attachment
sites are highly attractive for peptide synthesis. Polyethylene glycol supports that solubilize the growing
peptide chain and can be readily isolated have been widely used for peptide synthesis. However,
a stoichiometric amount of the PEG support is required because each PEG support typically has a single
attachment site for peptide synthesis. Reported herein is the development of a polynorbornene support
containing multiple attachment sites as well as alkyl and oligoether solubilizing/spacer groups. The
attachment site is connected to the polymer backbone through a solubilizing oligoether linker. The
support was developed after evaluating the effect of the linker and attachment site-spacing on peptide
synthesis using suitably designed polynorbornene supports. The high solubility of the support minimizes
the equivalents of reagents used for peptide synthesis. The support has been used to synthesize the
natural product Leu5-enkephalin in 52% overall yield using only 1.2 equivalents of coupling reagents,
which is comparable or superior to reported procedures using a large excess of reagents.
Received 15th July 2015
Accepted 26th October 2015
DOI: 10.1039/c5ra21668k
www.rsc.org/advances
have also been explored for peptide synthesis. However, they
show a decrease in solubility aer tripeptide synthesis.15–18
Introduction
Our group is interested in developing soluble polystyrene
and polynorbornene supports with high loading capacities
(comparable to resins) for peptide synthesis.19–21 Such supports
possess the advantages of solid phase peptide synthesis as well
as PEG derived soluble supports. Herein, we report the devel-
opment of a highly efficient second generation polynorbornene
support 4 (Fig. 1), where a solubilizing and exible oligoether
linker is incorporated in the support design. The support 4 was
designed aer determining the effect of linker and spacer/
solubilizing groups using supports 2 and 3 that do not
contain solubilizing oligoether chains as model systems. The
Insoluble polymers have been widely used as supports in
peptide synthesis so that the growing peptides can be readily
isolated by ltration.1 Synthesis using these heterogeneous
supports requires excess reagents to drive the peptide coupling
reactions to completion. Soluble, non-crosslinked polymers
have been utilized for peptide synthesis as they do not require
excess reagents and the growing peptides can be recovered by
precipitation.2–4 Among the soluble polymer supports, poly-
ethylene glycol (PEG) derivatives signicantly enhance the
solubility and reactivity of the growing peptide chain in the
reaction medium.5–11 However, the loading capacity (i.e. mmols
of amino acids attached per gram) of the PEG support is low
because each support contains only one amino acid attachment
site at the end of the polymer chain. Cross-linked resins
incorporated with PEG chains have been developed that have
excellent loading capacities and improved swelling properties.
Examples of such resins include the cross-linked polystyrene
resins (Tentagel),12 completely cross-linked polyethylene glycol
(PEGA) resin13 and polyethylene glycol–polyvinyl methylamine
resins (Chem Matrix).14 Despite, the incorporation of the PEG
linkers, these supports require excess reagents (ꢀ2–10 equiv.)
for peptide synthesis. Non-crosslinked soluble polymer
supports (without PEG) containing multiple attachment sites
Department of Chemistry, Indian Institute of Technology, Chennai, Tamil Nadu-600
036, India. E-mail: nanditam@iitm.ac.in; Fax: +91-44-22574202
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c5ra21668k
Fig. 1 Poly(norbornene) supports for peptide synthesis.
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second generation support 4 containing the solubilizing linker
as well as oligoether chains was utilized to synthesize the
natural product Leu5-enkephalin in 52% overall yield. The yield
obtained with our support using only 1.2 equivalents of
coupling reagents is superior or comparable to previously re-
ported procedures with supports that utilize excess coupling
reagents (1.5–4.5 equivalents).
Results and discussion
Our previously developed support
1 comprised multiple
Scheme 2 Synthesis of supports 2 and peptide synthesis on support
2b (a) Grubbs' third generation catalyst, CH2Cl2, rt, 1 h, (b) Fmoc-AA-
OH, DIC, DMAP, THF (c) 20% pip/DMF, 10 min, rt (d) Fmoc-AA-OH,
HCTU, DIEA, DMF : CH2Cl2 (1 : 1), rt (e) LiOH, THF, rt, 1 h.
attachment sites dispersed with alkyl and oligoether chains as
spacers/solubilizing groups. Support 1 could not be used for
peptide synthesis as it was insoluble in the reaction medium
aer Fmoc deprotection of the rst amino acid attached. We
envisioned that incorporating an oligoether linker between the
attachment site and the support could serve the dual role of
Table 1 Synthesis of polymer supports 2 and peptide synthesis on
improving support solubility and providing exibility to the support 2b
attachment site to access reagents.
Loadinga 2
Loadinga 10
Yieldb 11
(%)
At rst, we wanted to determine whether the linker had an
effect on the support solubility. Therefore the synthesis of
support 2 that has no solubilizing oligoether groups was
pursued. The alkyl groups were retained to function as spacers.
The alkyl monomer was synthesized as described earlier.19,20
The linker incorporated attachment site was synthesized as
shown in Scheme 1. Hydroxyamine 5 was treated with SOCl2 to
afford the 2-(2-chloroethoxy) ethanamine 6 in 94% yield. The
amine 6 was treated with norbornene-exo-acid in the presence
of HBTU and DIEA to give amide 7 in 92% yield. The amide 7
was treated with 4-hydroxybenzaldehyde and K2CO3 to give the
corresponding aldehyde in 96% yield. The monomer 8 was
obtained in 90% yield by reducing the aldehyde using sodium
borohydride in methanol.
Supports 2 were synthesized by polymerizing monomers 8
and 9 (Scheme 2). The ratio of monomers (x : y) as well as the
total monomer : initiator ratio i.e. [x + y] : [Ru] was varied to
obtain supports 2a and b with varying proportion of spacers
(Table 1). The polymerization reaction was carried out in the
presence of Grubbs' third generation initiator. The reaction was
terminated by addition of ethyl vinyl ether and the polymers 2
were isolated by precipitation with diethyl ether. The comple-
tion of polymerization was conrmed by the absence of signals
corresponding to the monomers in the 1H NMR spectra of
polymers 2. The number of attachment sites present per gram
of polymer (loading) was determined by recording the 1H NMR
2
(mmol gꢁ1
)
(mmol gꢁ1
)
11
2a
2b
2b
1.7
1.23
1.23
n.d
0.88
0.94
n.d
11a:MF
11b:IFG
n.d
78
70
Determined using 1H NMR.
purication.
Isolated yield aer RP-HPLC
a
b
spectra of polymers 2 in the presence of a known amount of
1,1,2,2-tetrachloroethane (TCE). The integration of the peak at
d ¼ 6.9 ppm corresponding to TCE was compared with the peak
at d ¼ 4.4 ppm for the benzylic protons in polymers 2a and b to
determine their loading capacities. The integration values of
benzylic protons (d ¼ 4.4), and methyl protons of the alkyl chain
(d ¼ 0.83) were compared to get the x : y ratios.
Support 2a was found to be insoluble, while support 2b was
found to be soluble in solvents such as DCM or THF. Amino
acids were loaded onto these supports using DIC to obtain
amino acid attached polymer 10a and b with loading capacities
of ꢀ0.9 mmol gꢁ1 (Scheme 2 and Table 1). The resulting poly-
mer was treated with 20% piperidine in DMF and stirred for 10
min to deprotect the Fmoc group. The polymer was isolated as
a precipitate with diethyl ether and then coupled with another
amino acid by using HCTU and DIEA in DCM : DMF (1 : 1) to
afford the dipeptides. The deprotection and coupling reactions
were repeated to obtain tripeptides. Finally peptides were
cleaved from the support using LiOH in THF. Di and tri
peptides 11a and b were synthesized in 70 and 78%, respectively
using support 2b. However, a drop in support solubility was
observed aer each coupling step. It is notable that polymer
support 2b could be used for synthesizing tripeptides in good
yields in contrast to the analogous hydroxy support 1 with three
monomers that has a higher proportion of solubilizing groups.
The improved solubility of support 2b despite the absence of
solubilizing oligoether groups indicates that the linker plays
a role in enhancing the solubility of the support.
Scheme 1 Synthesis of monomer (a) SOCl2, toluene, 0 ꢂC, rt, 3 h, 94%
(b) exo-norbornene acid, HBTU, DIEA, DCM, DMAP, rt, 13 h, 92%, (c) 4-
hydroxy benzaldehyde, K2CO3, DMF, 120 ꢂC, 12 h, 96%, (d) NaBH4,
MeOH, 0 ꢂC, rt, 1 h, 90%.
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In the past, we had observed that supports derived from
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monomers preloaded with amino acids were more soluble than
support 1 because the bulky Fmoc group on the amino acid
ensured uniform spacing of attachment sites.20 We wished to
check if the lower solubility of supports 2 aer tripeptide
synthesis could be attributed to this clustering of attachment
sites. Therefore, we synthesized supports 3 from monomers pre-
loaded with amino acids (Scheme 3). Amino acid attached
monomers 12a–c were synthesized in excellent yields by
coupling monomer 8 with the appropriate amino acid in the
Scheme 4 Peptide synthesis on polymer supports 3(a–c) (a) 20% pip/
DMF, 10 min, precipitation with hexane and diethyl ether; (b)
FmocAA2AA1OH, HCTU, DIEA, rt, 3 h, precipitation with diethyl ether;
(c) FmocAAOH, HCTU, DIEA, rt, 3 h, precipitation with diethyl ether; (d)
presence of EDCI. Supports 3a–c were synthesized in 88–96% LiOH, THF, H2O, rt, 1 h, separate supernatant; (e) dil. HCl.
yields from amino acid attached monomers 12 and monomer 9
(Table 2). The polymer supports 3(a–c) had good loading
deprotected.22,23 Aer completion of the reaction, tripeptide
capacities (ꢀ0.9 mmol gꢁ1) and were extremely soluble in DCM,
attached support 14 was obtained as a precipitate with diethyl
ether. The supported tripeptide 14 was dissolved in a few drops
of DMF and re-precipitated using diethyl ether multiple times to
ensure the removal of excess reagents and by-products. Depro-
tection and coupling steps were repeated to get the polymer
supported tetrapeptide. 1.2 equiv. of coupling reagents and
amino acids were used in each coupling step. The peptide 15
was cleaved from the support using base hydrolysis. A variety of
tetrapeptides 15a–f were synthesized from supports 3a–c in 62–
78% yields (Table 3). The effect of the attachment site spacing
on the support properties is evidenced by the fact that supports
3 are more efficient than supports 2 despite having no solubi-
lizing groups. When we attempted using support 3 for synthe-
sizing a peptide of biological importance such as Leu-
enkephalin24 we observed that the solubility of the support
decreased aer tetrapeptide synthesis. We chose Leu-
enkephalin 17 as a model system as there are several reports
for the synthesis of this peptide using soluble supports.24–29 The
diminished solubility of support 3 indicates that incorporation
of solubilizing ether groups is necessary for synthesis of larger
peptides.
THF and DMF. Due to the high solubility of support 3, we were
concerned that diketopiperazine would be readily formed
during peptide synthesis. We had previously observed this
problem during deprotection of dipeptides on our previously
reported soluble supports. To circumvent this problem, we
directly attached a dipeptide synthesized in solution to the
loaded support 3.
Peptide synthesis was initiated by deprotecting the Fmoc
group of support 3 using 20% piperidine in DMF (Scheme 4).
The polymer with the free amine was isolated by precipitation
with hexane and ether. The amine was coupled with Fmoc
protected dipeptide (Fmoc-AA2-AA1-OH) using HCTU and DIEA
in dichloromethane. The support was coupled with a dipeptide
instead of a single amino acid because highly soluble supports
such as ours that maintain the reactivity of the attached amino
acids are prone to form diketopiperazines when the dipeptide is
The studies with supports 2 and 3 illustrated that a support
containing the linker, oligoether chains and regularly spaced
attachment sites would be desirable for peptide synthesis.
Hence, we designed support 4 from pre-loaded monomers that
contains the linker as well as solubilizing groups. Support 4 was
synthesized in 88% yield (loading ¼ 0.72 mmol gꢁ1) from
monomers 12c, 9 and 16 (Scheme 5). The x, y and z ratios
were chosen based on our success with our reported
poly(norbornene) supports derived from three monomers.19,20
Scheme 3 Synthesis of supports 3 (a) Fmoc-AA-OH, EDCI, DCM, rt. (b)
Monomer 9, Grubbs' third generation catalyst, CH2Cl2, rt, 1 h.
Table 3 Synthesis of peptides using polymer supports 3a–c
No.
Polymer
Peptide
Yielda (%)
Table 2 Synthesis of polymer supports 3a–c
1
2
3
4
5
6
3a
3a
3a
3b
3b
3c
15a: Leu–Ala–Phe–Ala
15b: Pro–Val–Trp–Ala
15c: Met–Val–Trp–Ala
15d: Ala–Gly–Phe–Met
15e: Ala–Ser(OtBu)–Phe–Met
15f: Pro–Gly–Phe–Leu
77
78
76
72
62
64
No. Polymer 3 AA R1 na (x : y) Yield (%) Loadingb (mmol gꢁ1
)
1
2
3
3a
3b
3c
Ala Alk 50 (1 : 2) 88
Met Alk 50 (1 : 2) 96
Leu Alk 50 (1 : 2) 94
0.9
0.87
0.88
a
a
Equivalents with respect to [Ru]. b Determined using 1H NMR.
Isolated yield aer purication using RP-HPLC.
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supports require the use of expensive uorinated solvents/
supports and the synthesis requires 4–4.5 equivalents of
coupling reagents/amino acids, we believe our supports are
more practically viable.
Conclusions
A second generation polynorbornene support 4 that contains
a solubilizing oligoether linker between the amino acid
attachment site and the polymer backbone has been developed.
The linker is found to have a signicant effect on the solubility
and efficiency of the supports during peptide synthesis. Support
1 containing no linker is found to be insoluble aer the rst
step of peptide synthesis despite containing solubilizing oli-
goether side chains in contrast with linker containing supports
2 and 3. Supports 3 formed with preloaded monomers were
found to be more efficient than supports 2 and were used for
synthesizing tri to tetrapeptides in 62–78% yields using only 1.2
equivalents of coupling reagents/amino acids. The higher effi-
ciency of supports 3 is explained by the easier access of reagents
in this support due to the uniform spacing of attachment sites.
Presence of bulky Fmoc groups in the pre-loaded monomers
prevents crowding of attachment sites on the polymer support.
However, support 3 could not be used for synthesizing natural
product Leu-enkephalin due to decrease in its solubility aer
tetrapeptide synthesis. The results from supports 2 and 3 sug-
gested that the solubility as well as spacing of the attachment
sites was crucial for peptide synthesis. Therefore, a highly effi-
cient second generation support 4 containing the solubilizing
linker as well as spacers was synthesized. The efficiency of the
support was demonstrated by synthesizing the natural product
Leu5-enkephalin in 52% overall yield. The yield obtained with
our support using only 1.2 equivalents of reagents is superior or
comparable to previously reported procedures with either
expensive supports or supports that use large excess of reagents.
Scheme 5 Synthesis of polymer support 4 and peptide synthesis (a)
Grubbs' third generation catalyst, CH2Cl2, rt, 1 h; (b) 20% pip/DMF, 10
min, precipitation with hexane and diethyl ether; (c) FmocGly-Phe-
OH, HCTU, DIEA, rt, 3 h, precipitation with diethyl ether; (d) Fmo-
cAAOH, HCTU, DIEA, rt, 3 h, precipitation with diethyl ether (repeat); (e)
LiOH, THF, H2O, rt, 1.5 h, separate supernatant; (f) dil. HCl.
Leu-enkephalin 17 was synthesized in 52% overall yield using
support 4 and following the peptide synthesis approach shown
in Scheme 5. The support was soluble during the synthesis in
contrast to support 3. The HPLC prole of the crude penta-
peptide mainly showed the peak corresponding to the desired
product (Fig. 2).
Comparison of the yields for peptide synthesis using support
4 and previously reported examples26–28 are shown in Table 4.
Support 4 affords Leu-enkephalin in superior yields compared
to the ionic liquid and SPPS supports (entries 3 and 4). The yield
for synthesis of 17 using support 4 is lower than the uorous
support (entry 2). However, given the fact that the uorous
Acknowledgements
This research was supported by CSIR (No. 01(2333)/09/EMR-II),
New Delhi, India. N.N. acknowledges CSIR, India for his
research fellowship.
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