Soluble and reusable poly(norbornene) supports with high loading capacities for peptide synthesis

Article abstract of DOI:10.1021/ol4029435

Poly(norbornene) supports comprising solubilizing ethylene glycol units and multiple amino acid attachment sites have been developed for peptide synthesis. A variety of amino acids have been efficiently loaded (0.6-1.1 mmol/g) onto the support in high yields (83-98%). Several tripeptides have been synthesized in moderate-to-good overall yields (41-66%) using only 1.2 equiv of coupling reagents/amino acids, and the support could be efficiently recycled up to 3 times.

Full text of DOI:10.1021/ol4029435

ORGANIC
LETTERS
2013
Vol. 15, No. 22
5870–5873
Soluble and Reusable Poly(norbornene)
Supports with High Loading Capacities
for Peptide Synthesis
Nimmashetti Naganna and Nandita Madhavan*
Department of Chemistry, Indian Institute of Technology Madras, Chennai - 600036,
Tamil Nadu, India
nanditam@iitm.ac.in
Received October 11, 2013
ABSTRACT
Poly(norbornene) supports comprising solubilizing ethylene glycol units and multiple amino acid attachment sites have been developed for peptide
synthesis. A variety of amino acids have been efficiently loaded (0.6ꢀ1.1 mmol/g) onto the support in high yields (83ꢀ98%). Several tripeptides have
been synthesized in moderate-to-good overall yields (41ꢀ66%) using only 1.2 equiv of coupling reagents/amino acids, and the support could be
efficiently recycled up to 3 times.
The growing number of oligopeptide-derived therapeu-
tic agents, and biomaterials, necessitates the development
of efficient methods for peptide synthesis. Solid phase
peptide synthesis (SPPS), where the growing peptide is
attached to a resin, is extremely convenient for laboratory-
scale peptide synthesis, as it facilitates peptide recovery
via filtration.1 However, the amino acids attached to
insoluble SPPS supports are less reactive and excess cou-
pling reagents are typically required to ensure completion
of reactions. Liquid phase peptide synthesis (LPPS), which
employs soluble supports for attaching amino acids, pro-
vides an attractive alternative to SPPS, as the amino acid
reactivity is not compromised and the growing peptide can
be readily isolated by precipitation or extraction.2 LPPS
has been achieved using fluorous,3 polymeric,2 hydro-
phobic,4 and ionic liquid supports.5 Poly(ethylene glycol)
supports2b,6 are among the most efficient polymeric LPPS
supports, asthey provide a localized polar environment for
solubilizing and improving the reactivity of the growing
peptides. Despite the versatility of PEG supports their
widespread use for peptide synthesis is limited by their
low loading capacities (0.1ꢀ0.5 mmol/g). Polymeric LPPS
supports derived from poly(styrene) and other hydrophilic
polymers typically have higher loading capacities than PEG,
but are not as efficient for peptide synthesis due to their
lower solubility.2c,7 Herein, we present poly(norbornene) 1
(1) (a) Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149. (b) Chan,
W. C.; White, P. D. Fmoc Solid Phase Peptide Synthesis: A Practical
Approach; Oxford University Press Inc.: New York, 2000. (c) Martin, F. G.;
Albericio, F. Chim. Oggi 2008, 26, 29.
(2) (a) Bayer, E.; Mutter, M. Nature 1972, 237, 512. (b) Pillai, V. R.;
Mutter, M.; Bayer, E.; Gatfield, I. J. Org. Chem. 1980, 45, 5364. (c)
Gravert, D. J.; Janda, K. D. Chem. Rev. 1997, 97, 489.
(4) (a) Chiba, K.; Kono, Y.; Kim, S.; Nishimoto, K.; Kitano, Y.;
Tada, M. Chem. Commun. 2002, 1766. (b) Okada, Y.; Suzuki, H.; Nakae,
T.; Fujita, S.; Abe, H.; Nagano, K.; Yamada, T.; Ebata, N.; Kim, S.;
Chiba, K. J. Org. Chem. 2013, 78, 320. (c) Takahashi, D.; Yano, T.;
Fukui, T. Org. Lett. 2012, 14, 4514. (d) Takahashi, D.; Inomata, T. Pept.
Sci. 2013, 49, 83.
(5) (a) Miao, W.; Chan, T. H. Acc. Chem. Res. 2006, 39, 897. (b)
Miao, W.; Chan, T.-H. J. Org. Chem. 2005, 70, 3251. (c) He, X.; Chan,
T. H. Org. Lett. 2007, 9, 2681.
(3) (a) Mizuno, M.; Goto, K.; Miura, T.; Hosaka, D.; Inazu, T.
Chem. Commun. 2003, 972. (b) Zhang, W. Curr. Opin. Drug Discovery
Dev. 2004, 7, 784. (c) Mizuno, M.; Goto, K.; Miura, T.; Inazu, T. QSAR
Comb. Sci. 2006, 25, 742. (d) Fustero, S.; Sancho, A. G.; Chiva, G.; Sanz-
(6) (a) Zhang, C.-X.; Tong, H.-B.; Yan, C.-G. J. Comb. Chem. 2007,
9, 924. (b) Fischer, P. M.; Zheleva, D. I. J. Pept. Sci. 2002, 8, 529. (c)
Janda, K. D.; Han, H. Methods Enzymol. 1996, 267, 234.
~
Cervera, J. F.; del Pozo, C.; Acena, J. L. J. Org. Chem. 2006, 71, 3299.
r
10.1021/ol4029435
Published on Web 11/07/2013
2013 American Chemical Society

comprising multiple solubilizing diethylene glycol groups as
well as attachment sites as efficient polymeric LPPS supports
with high loading capacities (Figure 1).
aluminum hydride in THF, and the hydroxy group was
protected using tert-butyldimethylsilyl (TBS) chloride and
imidazole to afford the amine 12 in 86% yield. The amine 12
was coupled with norbornene-exo-acid 8 using 1-Ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDCI) and DMAP and
subsequently deprotected to afford the monomer 13 contain-
ing the attachment site in 86% yield.
Scheme 1. Synthesis of Monomers
Figure 1. Poly(norbornene) LPPS support.
Poly(norbornene) was chosen as the support, as its highly
controlled synthesis via ring-opening metathesis polymeriza-
tion (ROMP) affords polymers with relatively uniform mo-
lecular weight distributions.8 Alkyl and oligoether chains have
been incorporated to increase support solubility and spacing
the attachment sites apart. The synthesis of support 1 can be
envisioned by the random copolymerization of the requisite
monomers. Such an approach provides a modular support for
peptide synthesis wherein the properties of the support can be
tuned by varying the monomer ratios during ROMP.
The monomers containing the heptyl and diethylene
glycol solubilizing groups were synthesized starting from
the alcohols 2 and 3, respectively (Scheme 1a). The alco-
hols were tosylated and converted to the corresponding
azides 4 and 5 using sodium azide and DMF. Treatment of
the azides with triphenyl phosphine in THF, followed by
an aqueous workup, gave the corresponding amines 6 and
7. The alkyl amine 6 was coupled with norbornene-exo-
acid 8 in the presence of N,N,N0,N0-Tetramethyl-O-(1H-
benzotriazol-1-yl)uronium hexafluorophosphate (HBTU),
Ndiisopropylethylamine (DIEA) and N,N-dimethyamino-
pyridine (DMAP) to give alkyl monomer 9 in 98% yield.
The oligoether amine 7 was reacted with acid 8 in the
presence of EDCI and DMAP to give the diethylene glycol
monomer 10 in 94% yield. The monomer containing
the attachment site was synthesized starting from commer-
cially available p-cyano benzaldehyde 11 as shown in
Scheme 1b. Compound 11 was reduced using lithium
Poly(norbornene) support1 was synthesized byadding a
Grubbs third generation initiator to a solution of mono-
mers 9, 10, and 13 (eq 1).9 Polymers 1aꢀe with varying l, m,
and n ratios and polymer lengths were synthesized (Table 1).
The yields for lower molecular weight polymers 1a and
b were less due to their higher solubility in solvents used to
precipitate the polymers. In all other cases, the polymers
were obtained in excellent yields. The loading capacities
of the supports were determined using 1H NMR spectro-
scopy in the presence of TCE as a standard, as the protons
corresponding to the benzylic position of the attachment site
were distinctly seen in the NMR spectrum.10 The loading
capacities (1.3ꢀ2.6 mmol/g) of the polymers were
(7) (a) Narita, M.; Hirata, M.; Kusano, K.; Itsuno, S.; Ue, M.;
Okawara, M. Pept. Chem. 1980, 17th, 107. (b) Meszynska, A.; Badi,
N.; Boerner, H. G.; Lutz, J.-F. Chem. Commun. 2012, 48, 3887.
(8) (a) Choi, T. L.; Grubbs, R. H. Angew. Chem., Int. Ed. 2003, 42,
€
1743. (b) Barrett, A. G. M.; Hopkins, B. T.; Kobberling, J. Chem. Rev.
2002, 102, 3301. (c) Dickerson, T. J.; Reed, N. N.; Janda, K. D. Chem.
Rev. 2002, 102, 3325. (d) Harned, A. M.; Mukherjee, S.; Flynn, D. L.;
Hanson, P. R. Org. Lett. 2003, 5, 15. (e) Harned, A. M.; Zhang, M.;
Vedantham, P.; Mukherjee, S.; Herpel, R. H.; Flynn, D. L.; Hanson,
P. R. Aldrichimica Acta 2005, 38, 3. (f) Lee, B. S.; Mahajan, S.; Clapham,
B.; Janda, K. D. J. Org. Chem. 2004, 69, 3319. (g) Harned, A. M.; Probst,
D. A.; Hanson, P. R. The Use of Olefin Metathesis in Combinatorial
Chemistry: Supported and Chromatography-Free Syntheses. In Hand-
book of Metathesis: Catalyst Development; Wiley-VCH Verlag GmbH:
Weinheim, Germany, 2008, doi: 10.1002/9783527619481.ch25.
(9) Grubbs first generation initiator was also surveyed for ROMP.
However, the l:m:n ratios of the resultant polymers varied significantly
due to different rates of polymerization for the three monomers.
Org. Lett., Vol. 15, No. 22, 2013
5871

comparable to those seen for SPPS supports. The solubility
of supports 1cꢀe in solvents used for peptide synthesis as
well as the ease of their recovery from solution was inves-
tigated. Polymer 1e (entry 5, Table 1) with an l, m, n ratio of
1:1:2 and a loading capacity of 1.35 mmol/g was found to be
the optimal support for peptide synthesis, as it was readily
soluble in dichloromethane/THF/DMF and could be easily
recovered from solution via precipitation with ether.
Table 2. Attachment of Amino Acids onto Support 1e
amino acid
(AA)
time
(h)
yield
(%)
loadinga
(mmol/g)
1
2
14a: Ala
3
6
2
4
3
3
3
3
6
6
87
96
92
96
98
95
97
93
83
98
1.1
14b: Phe
0.9
3
14c: Leu
0.92
0.96
1.1
4
14d: Pro
5
14e: Met
6
14f: Tyr
0.87
0.84
0.77
0.6
7
14g: Asp(OtBu)
14h: Lys (Boc)
14i: His (Trt)
14j: Arg (Pbf)c
8
9
10
0.75
1
a Determined using H NMR. c Pbf = (2,2,4,6,7-pentamethyldihy-
drobenzofuran-5-sulfonyl)-.
Such an approach, while not as versatile as the first
approach, ensures that there are no free hydroxyl groups
in the support. The polymer 16 containing FmocAla was
obtained in 95% yield using this approach. The overall
yield for obtaining 16 from monomer 13 is 86%, which is
slightly higher than that for obtaining 14a (83%).
Table 1. Supports Synthesized by ROMP
yield
(%)
loadingb
(mmol/g)
1
x (13:10:9)a
l:m:nb
1
2
3
4
5
1a
1b
1c
1d
1e
31(1:1:1)
41(1:1:1)
54 (1:1:1)
66 (1:1:1)
50 (1:1:2)
36
42
95
95
95
1.5
2.6
1.6
1.7
1.35
1:0.6:0.5
1:0.6:0.4
1:1:1.1
1:1.1:1.1
1:1:2
a x = equivalents with respect to [Ru]. b Determined by 1H NMR.
The first approach to obtain supports containing amino
acids involved attachment of amino acids to support 1e
using diisopropyl carbodiimide (DIC) and DMAP in THF
(eq 2). Using this approach, a variety of polar, nonpolar,
acidic, and basic amino acids could be attached in excellent
yield to support 1e (Table 2). The solubility of the support
facilitated efficient loading of amino acids onto support 1e,
using only a slight excess of Fmoc amino acids (1.2ꢀ1.5
equiv). Furthermore, the loading of amino acids onto the
polymer support could be determined without cleaving the
amino acids, using 1H NMR spectroscopy.10
As a proof of concept, alanine attached support 16 was
used to synthesize several tripeptides as shown in Scheme 2
and Table 3. The polymer 16 was treated with a solution of
20% piperidine in DMF to remove Fmoc. The resultant
polymer with the free amine was isolated as a precipitate
with hexane and ether. The amine was coupled with Fmoc
protected amino acid (AA1) using 1-[bis(dimethylamino)-
methylene]-6-chloro-1H-benzotriazolium hexafluoropho-
sphate 3-oxide (HCTU) and DIEA in dichloromethane to
give the dipeptide, which was isolated by precipitation with
diethyl ether. The precipitate was dissolved in DMF and
reprecipitated with diethyl ether multiple times to ensure
removal of byproducts and excess coupling reagents. The
dipeptide was subjected to the deprotection and coupling
steps as described above to gain polymer attached tripeptides.
After obtaining the requisite peptides, the Fmoc protecting
group was removed and the peptides were cleaved from the
support via base mediated hydrolysis. The supernatant con-
tained the peptides in their salt form, while support 1e was
recovered as a precipitate in 92ꢀ98% yield. Neutralization
A second approach involves ROMP of monomer 15
containing an amino acid along with monomers 9 and 10
using the before mentioned optimalmonomer ratios(eq 3).
(10) See Supporting Information for details.
5872
Org. Lett., Vol. 15, No. 22, 2013

The support 1e could be recovered in high yield and
purity after peptide synthesis. Alanine was loaded onto 1e
using eq 2 to give support 16, which was used for synthesiz-
ing tripeptide 17e following Scheme 2. The isolated yields
for 17e did not vary significantly from the first cycle
(Table 4), indicating that amino acid attachment and
peptide synthesis were equally efficient with the recovered
polymer.
Scheme 2. Peptide Synthesis on Support
Table 4. Reuse of Support for Synthesis of Peptide 17e
isolated yielda
support
cycle
(%)
1
2
3
16
1
2
3
43
44
41
16b
16b
a Isolated using semipreparative RP-HPLC. b Synthesized from re-
cycled polymer 1e.
Table 3. Synthesis of Peptides Using Support 16
peptide 17
isolated yielda
(%)
In conclusion, soluble poly(norbornene) supports with
high loading capacities of attachment sites (>1 mmol/g)
have been developed for peptide synthesis. The support
contains solubilizing ethylene glycol and alkyl units which
improves its efficiency for peptide synthesis. A variety of
amino acids have been efficiently loaded (0.6ꢀ1.1 mmol/g)
onto the support in excellent yields (83ꢀ98%). Several
tripeptides have been synthesized in good to moderate
yields (41ꢀ66%) without using excess amino acids
or coupling reagents. Lastly, the cleaved support could
be readily isolated in high purity and recycled up to
2 times to synthesize tripeptides. Efforts are currently
underway to improve the efficiency of peptide synthe-
sis using our poly(norbornene) supports via modified
linkers and attachment sites, for obtaining more complex
oligopeptides.
1
2
3
4
5
17a: H2N-TyrPheAla-OH
17b: H2N-PheMetAla-OH
17c: H2N-ProPheAla-OH
17d: H2N-TrpLeuAla-OH
17e: H2N-LeuPheAla-OH
46
41
52
66
43
a Isolated using semipreparative RP-HPLC.
of the supernatant with dil. HCl afforded peptides 17aꢀe.
Since we wished to probe the reactivity of amino acids on
the support, only 1.2 equiv of Fmoc-amino acids were used
for the coupling reactions. Crude HPLC and NMR of the
tripeptides were relatively clean and indicatedformation of
a few byproducts in relatively small amounts.10,11 Pure
tripeptides were isolated by semipreparative reversed
phase HPLC in 41ꢀ66% yields (Table 3), which is notable
considering the fact that we were not using an excess of
amino acids or coupling reagents. We believe that the
lower isolated yields could be due to the higher reactivity
of amino acids on our supports, which could result in the
formation of diketopiperazines during deprotection of the
dipeptide.
Acknowledgment. This research was supported by CSIR
(No. 01(2333)/09/EMR-II), New Delhi, India. N.N
acknowledges CSIR, India for his research fellowship.
Supporting Information Available. Detailed experimen-
tal procedures, characterization of compounds, HPLC
traces of crude tripeptides. This material is available free
of charge via the Internet at http://pubs.acs.org.
(11) The yield for H2N-LeuPheAla-OH prior to HPLC (determined
using H NMR spectroscopy in the presence of TCE as a standard) is
73%, which is comparable to the overall yield (77%) for tripeptide
GlyValGly-Fmoc synthesized on the mPEG LPPS supports using
2 equiv of amino acids.6a
1
The authors declare no competing financial interest.
Org. Lett., Vol. 15, No. 22, 2013
5873