Versatile soluble oligomeric styrene supports for peptide synthesis

Article abstract of DOI:10.1002/pola.27714

Soluble oligomeric styrene supports are reported here with high loading capacities of 1.5-1.6 mmol/g similar to resins used in solid phase peptide synthesis. Oligoether and alkyl chains are incorporated into the scaffold to improve the support solubility

Full text of DOI:10.1002/pola.27714

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Versatile Soluble Oligomeric Styrene Supports for Peptide Synthesis
Venkataramana Erapalapati, Nandita Madhavan
Department of Chemistry, Indian Institute of Technology Madras, Chennai 600036, Tamil Nadu, India
Correspondence to: N. Madhavan (E-mail: nanditam@iitm.ac.in)
Received 7 May 2015; accepted 27 May 2015; published online 17 June 2015
DOI: 10.1002/pola.27714
ABSTRACT: Soluble oligomeric styrene supports are reported
here with high loading capacities of 1.5–1.6 mmol/g similar to
resins used in solid phase peptide synthesis. Oligoether and
alkyl chains are incorporated into the scaffold to improve the
support solubility and act as spacers between the attachment
sites. Amino acids have been attached to the support in 59–
85% yields and 0.87–1.3 mmol/g loading. The supports have
been used to synthesize tri- to hexapeptides in 38–64% yields
using only 2 equivalents of coupling reagents, which is much
lower than the amount of reagents typically used in solid
phase synthesis. A modular synthetic approach is used to
obtain the supports so that any efficient styrene-based attach-
ment site can be readily incorporated into our soluble support
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2015 Wiley Periodicals, Inc. J. Polym. Sci., Part A:
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KEYWORDS: Oligomers; peptides; supports; synthesis
chains.2(a),4 PEG supports have been used for the synthesis
of a variety of oligopeptides and have also been used for the
combinatorial synthesis of peptides. However, the number of
attachment sites per gram of polymer (i.e., the loading
capacity) of the PEG polymer is typically low (0.1–0.5 mmol/
g) as each polymer unit contains only one attachment site at
the end of the polymer. SPPS resins on the contrary have
loading capacities of ꢀ1 mmol/g on an average, with the
exception of few resins that have lower or higher loading
capacities.1(a) While lower molecular weight hydrophobic,5
fluorous,6 and ionic liquid supports7 have been explored to
improve the loading capacities as well as efficiency of LPPS,
there are not many examples of soluble polymer supports
with high loading capacities for efficient peptide synthesis.
Our group has developed soluble poly(norbornene) supports
appended with oligoether and alkyl chains that have high
loading capacities (0.6–1.1 mmol/g).8 Poly(styrene) derived
LPPS supports have also been reported that possess high
loading capacities, but are not as soluble as the polar PEG
supports.9 The advantage in developing styrene based sup-
ports is that a large number of styrene monomers containing
efficient amino acid attachment sites already exist in the lit-
erature because styrene is the most widely used support in
SPPS. Recently, more soluble poly(styrene) supports have
been developed that have a single amino acid attachment
site for each polymer chain, which lowers their loading
capacity.10 Herein, we report a soluble oligomeric styrene
support 1 (Fig. 1) appended with oligoether and alkyl chains
for peptide synthesis. We have incorporated the oligoether
INTRODUCTION Efficient methods for synthesizing peptides
are essential due to their widespread use as materials and
therapeutic agents. Solid phase peptide synthesis (SPPS),
which utilizes insoluble resins to attach the growing peptide
chain has been an attractive method for synthesizing pep-
tides. SPPS is extremely efficient as the growing peptide can
be readily recovered from the reaction mixture by filtration
and purification is carried out only after the peptide is
cleaved form the resin.1 However, the heterogeneous reaction
medium necessitates the use of a large excess of coupling
reagents (4–5 equivalents) in SPPS. Therefore, soluble poly-
mers with appropriate attachment sites for amino acids have
been explored as supports for what is often referred to as
liquid phase peptide synthesis (LPPS).2 These supports are
attractive alternatives to resins as they are soluble in the
reaction medium and can be readily isolated by precipitation
with a suitable solvent. The design requirements for LPPS
supports are very different from catalyst/reagent supports. A
catalyst support is used multiple times for the same reaction.
Soluble catalyst supports are designed such that they are
soluble, recoverable and reusable.3 In the case of peptide
synthesis, an amino acid is attached to the support and a
peptide is grown on the support using a series of reactions.
The LPPS support properties change with the growing length
of the peptide. Therefore, these supports are designed such
that the support solubility is maintained despite the attach-
ment of polar peptide chains. In LPPS, the most widely used
polymeric supports are derived from polyethylene glycol
(PEG) as the PEG chains solubilize the growing peptide
Additional Supporting Information may be found in the online version of this article.
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solvents are reported as volume percents. Compounds were
visualized using UV light, ninhydrin, and iodine stains. Flash
column chromatography was performed using silica gel
(200–400 mesh) from Acme chemicals. All 1D and 2D NMR
spectra were recorded on Bruker 400 or Bruker 500 spec-
trometers using CDCl3, or DMSO-d6 as solvents. The NMR
spectra were referenced using residual solvent peaks as the
standards. Chemical shifts are denoted in parts per million
(d) and coupling constants (J) are reported in Hertz (Hz).
The spin multiplicities are reported as singlet (s), broad sin-
glet (bs), doublet (d), triplet (t), quartet (q), quintet (quint),
apparent quintet (app. quint.), and multiplet (m). The peaks
corresponding to the alkyl, diethylene glycol and attachment
site monomers are denoted as alk, deg and att, respectively
in the 1H NMR data. High-resolution mass spectra (HRMS)
were recorded on MICRO-Q-TOF mass spectrometer using
the ESI technique. FT-IR spectra were recorded on a JASCO
FT/IR-4100 spectrometer. All IR spectra were recorded in
the form of a KBr pellet for solids or as thin films in chloro-
form for liquids. IR spectra peaks are reported in wavenum-
bers (cm21) as strong (s), medium (m), weak (w), and broad
(br). Semi preparative RP-HPLC was carried out on a Waters
HPLC system using water (0.1% TFA) and methanol (0.1%
TFA) as the mobile phase and a Sunfire prep C 18, 5 lm, 10
3 250 mm column as the stationary phase. Peptides were
injected at a concentration of 10 mg/mL, and a flow rate of
4.1 mL/min was used for semi preparative RP-HPLC. Peptide
elution was monitored at 254 nm with the Waters 2489 UV/
visible detector.
FIGURE 1 Soluble support with multiple attachment sites.
[Color figure can be viewed in the online issue, which is avail-
able at wileyonlinelibrary.com.]
chains in order to provide a polar environment for the grow-
ing peptide chains similar to the PEG based supports. The
alkyl chains and oligoether chains have also been incorpo-
rated to act as spacers between the attachment sites. A mod-
ular synthetic route is proposed to access the supports in
order to facilitate incorporation of any attachment site into
our soluble oligomer scaffold containing the spacer/solubiliz-
ing groups. As a proof of concept the benzylic linker (Wang
resin type) is used to attach the peptides. This linker is com-
patible with Fmoc protected amino acids. The utility of the
support for peptide synthesis has been demonstrated by
loading a variety of amino acids in 59–85% yields and syn-
thesizing hexapeptides in 50% overall yields (using only 2
equiv. of reagents). In contrast to our earlier poly(norbor-
nene) support, these supports are more versatile as they
need not be preloaded with amino acids prior to polymeriza-
tion to improve their solubility. Therefore, peptides with dif-
ferent amino acids at the C-terminus can be readily
synthesized using these supports. Given the modular nature
of the support synthesis, the utility of the support can be
expanded to include other linkers at the attachment site
such that protecting groups other than Fmoc can be used for
peptide synthesis.
1-((heptyloxy)methyl)-4-vinylbenzene (3)
11A suspension of NaH (0.178 g, 7.429 mmol, 2.1 equiv.) in
THF (2 mL) was slowly added to 1-heptanol (2.014 g,
17.339 mmol, 4.9 equiv.) in THF (4 mL) at 0 oC for 2 h. A
solution of p-chloromethylstyrene 2 (0.540 g, 3.538 mmol, 1
equiv.) in THF (3 mL) was slowly added to the reaction mix-
ture at 8C. The mixture was allowed to reflux for 24 h. Sub-
sequently, water (4 mL) was added and the reaction mixture
was extracted with dichloromethane (3 3 35 mL). The
organic layer was concentrated in vacuo to afford the crude
residue. Purification by column chromatography (hexane)
afforded 0.67 g (81%) of ether 3 as colorless oil. TLC Rf 5
0.20 (1% ethyl acetate/hexane). 1H NMR (400 MHz, CDCl3,
25 oC): d 5 7.41 (d, J 5 7.6 Hz, 2H; HAr), 7.32 (d, J 5 7.6
Hz, 2H; HAr), 6.74 (dd, J 5 17.2, 10.8 Hz, 1H; CH@CH2), 5.77
(d, J 5 17.6 Hz, 1H; CH@CH2), 5.25 (d, J 5 10.8 Hz, 1H;
CH@CH2), 4.51 (s, 2H; PhCH2O), 3.52–3.44 (m, 2H; CH2O),
1.69–1.6 (m, 2H; CH2) 1.44–1.27 (8H; 4CH2), 0.96–0.88 (m,
3H; CH3); 13C NMR (100 MHz, CDCl3, 25 8C): d 5 138.5,
136.9, 136.7, 127.9, 126.4, 126.3, 126.2, 113.7, 72.7, 70.6,
31.9, 29.9, 29.3, 26.3, 22.7, 14.2; IR (thin film): 2923 (s),
2856 (s), 1695 (s), 1458 (s), 1373 (s) 1023 (s) cm21; HRMS
(ESI1): calcd. for C16H25O (MH1), 233.1905 found 233.1899.
EXPERIMENTAL
General Methods
All air-sensitive reactions were performed under an inert
atmosphere of nitrogen with magnetic stirring. Syringe or
cannula was used to transfer air-sensitive solvents and solu-
tions. Unless stated otherwise, all the reagents for synthesis
were purchased from commercially available suppliers and
used without further purification. Tetrahydrofurane (THF)
was distilled from sodium benzophenoneketyl; N,N-diisopro-
pylethylamine (DIEA), dichloromethane, and piperidine were
distilled from calcium hydride; N,N-dimethylformamide
(DMF), 20% piperidine-DMF, and N,N-diisopropylcarbodii-
mide (DIC) were dried over 4 Å molecular sieves. All dry sol-
vents were stored over 4 Å molecular sieves prior to use.
Analytical thin-layer chromatography (TLC) was performed
on MERCK precoated silica gel 60 F254TLC plates. Eluting
1-((2-(2-ethoxyethoxy)ethoxy)methyl)-4-vinylbenzene (4)
11A suspension of NaH (0.178 g, 7.429 mmol, 2.1 equiv.) in
THF (3 mL) was slowly added to diethyleneglycol monoethy-
lether (2.326 g, 17.339 mmol, 4.9 equiv.) in THF (20 mL) at
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0 8C for 2 h. A solution of p-chloromethylstyrene 2 (0.540 g,
3.538 mmol, 1 equiv.) in THF (3 mL) was slowly added to
the reaction mixture at 8C. The mixture was allowed to reflux
for 24 h. Subsequently, water (4 mL) was added and the
reaction mixture was extracted with dichloromethane (3 3
30 mL). The organic layer was concentrated in vacuo to
afford the crude residue. Purification by column chromatog-
raphy (10% ethyl acetate/hexane) afforded 0.77 g (87%) of
ether 4 as colorless oil. TLC Rƒ 5 0.30 (20% ethyl acetate/
hexane). H NMR (400 MHz, CDCl3, 25 C): d 5 7.38 (d, J 5
8 Hz, 2H; HAr), 7.29 (d, J 5 8 Hz, 2H; HAr), 6.70 (dd, J 5 18,
11.2 Hz, 1H; CH@CH2), 5.73 (d, J 5 17.6 Hz, 1H; CH@CH2),
5.22 (d, J 5 11.2 Hz, 1H; CH@CH2), 4.55 (s, 2H; PhCH2O),
3.7–3.57 (8H; OCH2), 3.52 (q, J 5 7.2 Hz, 2H; CH2), 1.21 (t,
J 5 7.2, 3H; CH3); 13C NMR (100 MHz, CDCl3, 25 8C): d 5
138.0, 137.1, 136.7, 128.1, 126.3, 113.8, 73.1, 70.81, 70.75,
69.9, 69.5, 66.8, 15.3; IR (thin film): 3065 (bs), 3025 (s)
2921 (s), 2853 (s), 1598 (s), 1491 (s), 1449 (s) 1025 (s)
cm21; HRMS (ESI1): calcd. for C15H23O3 (MH1), 251.1647
found 251.1653.
1H NMR(400) MHz, CDCl3, 25 C): d 5 7.45-7.37 (4H; HAr),
7.29 (d, J 5 8.4 Hz, 2H; HAr), 6.75 (d, J 5 8.4 Hz, 2H; HAr),
6.72 (dd, J 5 17.6, 10.8 Hz, 1H; CH@CH2), 5.76 (d, J 5 18
Hz, 1H; CH@CH2), 5.25 (d, J 5 10.8 Hz, 1H; CH@CH2), 5.06
(s, 2H; PhCH2O), 4.62 (s, 2H; PhCH2OPh); 13C NMR (100
MHz, CDCl3, 25 8C): d 5 158.5, 137.5, 136.6, 136.6, 133.6,
128.8, 127.8, 126.6, 115.1, 114.2, 70.0, 65.2; IR (KBr pellet):
3147 (bs), 2979 (s), 2867 (s), 1604 (bs), 1512 (s), 1405 (s)
1244 (s) cm21; HRMS (ESI1): calcd. for C16H17O2 (MH1)
241.1229, found 241.1230.
1
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General Procedure for Polymerization
A solution containing required amounts of monomers 3, 4,
and 5 in THF was added to a sealed tube and deoxygenated
using a stream of nitrogen gas.12 A deoxygenated solution of
azobisisobutyronitrile (AIBN) in THF was added to this solu-
tion and the reaction mixture was allowed to stir at 80 8C
for 3 days. The reaction mixture was concentrated in vacuo
to a minimum volume. Cold hexane (20 mL) was added to
obtain the polymer support as a precipitate. The precipitate
was dissolved in CH2Cl2 (2 mL) and reprecipitated with cold
hexane (3 3 20 mL). The precipitate was isolated and dried
in vacuo to afford the polymer support 1 as a viscous liquid.
(4-(4-vinylbenzyloxybenzylalcohol) (5)
To a mixture of 4-hydroxybenzaldehyde (1.190 g, 9.745
mmol, 1.3 equiv.) and anhydrous K2CO3 (1.340 g, 9.745
mmol, 1.3 equiv.) in CH3CN (10 mL) was added p-chlorome-
thylstyrene 2 (1.144 g, 7.496 mmol, 1 equiv.) in CH3CN
(10 mL). The mixture was allowed to reflux for 30 h, follow-
ing which it was cooled to room temperature, filtered and
washed with dichloromethane (3 3 20 mL). The filtrate was
concentrated in vacuo to give a yellow oil, which was puri-
fied using column chromatography 10% ethyl acetate/hex-
ane) to yield 1.54 g (86%) of the ether as a white solid. TLC
Rf 5 0.30 (10% ethyl acetate/ hexane). 1H NMR (400 MHz,
CDCl3, 25 8C): d 5 9.89 (s, 1H; CHO), 7.84 (d, J 5 8.4 Hz,
2H; HAr), 7.44 (d, J 5 8 Hz, 2H; HAr), 7.38 (d, J 5 8.4 Hz, 2H;
HAr), 7.07 (d, J 5 8.4 Hz, 2H; HAr), 6.72 (dd, J 5 17.6, 11.2
Hz, 1H; CH@CH2), 5.77 (d, J 5 17.6 Hz, 1H; CH@CH2), 5.27
(d, J 5 10.8 Hz, 1H; CH@CH2), 5.14 (s, 2H; CH2O); 13C NMR
(100 MHz, CDCl3, 25 8C): d 5 191.0, 163.8, 137.8, 136.4,
135.5, 132.2, 130.3, 127.9, 126.7, 115.3, 114.6, 70.2; IR (KBr
pellet): 2932 (s), 2877 (s), 2853 (s), 2726 (s), 1697 (bs),
1596 (s), 1513(s), 1464 (s) cm21; HRMS (ESI1): calcd. for
General Procedure for Determining Loading Capacity of
Support 1 by H NMR
1
The number .of attachment sites present per gram of poly-
mer 1 (loading) was determined by recording the 1H NMR
spectrum of polymer 1 in the presence of a known amount
of 1,1,2,2-tetrachloroethane (TCE). The integration of the
peak at d 5 5.95 ppm for TCE was compared with the peak
at d 5 4.9 ppm for the benzylic protons of the attachment
site in polymer 1 to determine the number of attachment
sites in polymer 1.
General Procedure for Determining the x:y:z Ration of
Supports 1
The x: y: z ratio of the polymer 1 was determined using 1H
NMR spectroscopy. The integration values of benzylic pro-
tons (d 5 4.9 and 4.56–4.20) and methyl protons of the alkyl
chain (d 5 0.87) were used to get the x: y: z ratio.
Polymer 1a
C
16H14O2 Na (MNa1) 261.0891, found 261.0891.
AIBN (0.600 mg, 0.003 mmol, 1 equiv.) in THF (0.5 mL) was
added to monomer 3 (41.76 mg, 0.18 mmol, 60 equiv.),
monomer 4 (45.00 mg, 0.18 mmol, 60 equiv.), and monomer
5 (43.200 mg, 0.180 mmol, 60 equiv.) in THF (1 mL) to
afford 92 mg (71%) of polymer 1a. Loading 5 1.5 mmol/g;
x: y: z 5 1:0.9:1. 1H NMR (400 MHz, CDCl3, 25 oC): d 5
7.3–6.7 (10.7H; HAr), 6.7–6.2 (5.5H; HAr), 4.90 (bs, 2H;
PhOCH2OH), 4.6–4.2 (5.8H; PhCH2O), 3.7–3.2 (11.7H;
OCH2(alk, deg)), 1.8–1.1 (23.0H; PhCHCH2, CH2(alk, deg)), 0.87
(bs, 3H; CH3(alk)).
The above synthesized ether (1.420 g, 5.966 mmol, 1 equiv.)
in methanol (25 mL) and THF (7 mL) at 0 8C was added
sodium borohydride (0.441 g, 11.932 mmol, 2 equiv.). The
reaction mixture was allowed to stir at room temperature
for 3h, following which the volatile solvents were removed
in vacuo. Water (25 mL) was added to the mixture and the
resulting suspension was neutralized with 2N HCl. The reac-
tion mixture was extracted with dichloromethane (3 3
50 mL) and washed with water (20 mL). The combined
organic layers were dried over sodium sulphate, filtered and
concentrated in vacuo to give a white solid. Purification
using column chromatography (15% ethyl acetate/hexane)
gave 1.37 g (96%) of 4-(4-vinylbenzyloxybenzylalcohol) 5 as
a white solid. TLC Rf 5 0.40 (20% ethyl acetate/ hexane).
Polymer 1b
AIBN (2 mg, 0.012 mmol, 1 equiv.) in THF (0.5 mL) was
added to monomer 3 (339.184 mg, 1.462 mmol, 120 equiv.),
monomer 4 (365.500 mg, 1.462 mmol, 120 equiv.) and
monomer 5 (350.880 mg, 1.462 mmol, 120 equiv.) in THF
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(2 mL) to afford 919 mg (87%) of polymer 1b. Loading 5
1.2 mmol/g; x: y: z 5 0.8:0.6:1. 1H NMR (400 MHz, CDCl3,
25 8C): d 5 7.3–6.7 (10.0H; HAr), 6.7–6.2 (4.9H; HAr), 4.90
(bs, 2H; PhOCH2OH), 4.6–4.2 (5.2H; PhCH2O), 3.7–3.2
(9.2H;OCH2(alk, deg)), 2.1–1.1 (21.3H; PhCHCH2, CH2(alk, deg)),
0.87 (s, 2.6H; CH3(alk)).
known amount of TCE. The integration of the peak at d 5
5.9 ppm corresponding to TCE was compared with the peak
at d 5 7.7 ppm for the Fmoc protons of polymers 6(a–j).
6a
Oligomer 1e (0.800 g, 0.904 mmol), Fmoc-L-Ala-OH
(562.288 mg, 1.808 mmol), DMAP (22.057 mg, 0.1808
mmol), DIC (283.955 lL, 1.808 mmol, 2 equiv.), and THF
(10 mL) were used for the reaction. 863 mg (81%) of 6a
was obtained. Loading 5 1.3 mmol/g. 1H NMR (400 MHz,
CDCl3, 25 8C): d 5 7.73 (bs, 2H; HAr(Fmoc)), 7.57 (bs, 2H;
Polymer 1c
AIBN (0.400 mg, 0.002 mmol, 1 equiv.) in THF (0.5 mL) was
added to monomer 3 (111.36 mg, 0.48 mmol, 240 equiv.),
monomer 4 (120.00 mg, 0.48 mmol, 240 equiv.) and mono-
mer 5 (57.600 mg, 0.240 mmol, 120 equiv.) in THF (1 mL)
to afford 251 mg (87%) of polymer 1c. Loading 5 1.3
mmol/g; x: y: z 5 2.2:2.1:1. 1H NMR (400 MHz, CDCl3, 25
oC): d 5 7.3–6.7 (17.4H; HAr), 6.7–6.2 (10.6H; HAr), 4.92 (bs,
2H; PhOCH2OH), 4.6–4.2 (11.1H; PhCH2O), 3.7–3.2 (27.5H;
OCH2(alk, deg)), 2.1–1.5 (16.0H; PhCHCH2,) 1.4–1.1 (m, 35H;
CH2(alk, deg)), 0.88 (s, 7H; CH3(alk)).
HAr(Fmoc)), 7.36 (bs, 2H; HAr(Fmoc)), 7.2–6.7 (13.8H; HAr
and
Ar(Fmoc)), 6.7–6.3 (4.9H; HAr), 5.50 (bs, 0.68H; NH), 5.09 (bs,
2H; PhOCH2OH(Att)), 4.9 (bs, 1.9H; PhOCH2(Att)), 4.5–4.3
(6.3H; PhCH2O, CH2(Fmoc) and CH(Ala)), 4.19 (s, 1.1; CH(Fmoc)),
3.7–3.3 (10.5H; OCH2(alk, deg)), 1.9–1.1 (28.3H; PhCHCH2,
CH3(Ala) and (deg) and CH2(alk)), 0.86 (s, 3.2H; CH3(alk)).
6b
Oligomer 1e (350.000 mg, 0.532 mmol), Fmoc-L-Phe-OH
(411.768 mg, 1.064 mmol), DMAP (12.932 mg, 0.106 mmol),
DIC (167.106 lL, 1.064 mmol) and THF (5 mL) were used
for the reaction. 469 mg (85%) of 6b was obtained. Loading
5 1.13 mmol/g. 1H NMR (400 MHz, CDCl3, 25 8C): d 5 7.74
(s, 2H; HAr(Fmoc)), 7.54 (s, 2H; HAr(Fmoc)), 7.4–6.7 (17.8H; HAr,
Ar(Phe) and Ar(Fmoc)), 6.7–6.2 (5.3H; HAr), 5.36 (bs, 0.7H; NH),
5.2–4.7 (3.7H; PhOCH2OH(Att) and PhOCH2(Att)), 4.67 (s, 0.9H;
CH(Phe)), 4.5–4.2 (5.8H; PhCH2O, CH2(Fmoc)), 4.17 (s, 0.9H;
CH(Fmoc)), 3.7–3.3 (10.6H; OCH2(alk, deg)), 3.09 (bs, 1.5H;
Polymer 1d
AIBN (1.000 mg, 0.006 mmol, 1 equiv) in THF (0.5 mL) was
added to monomer 3 (334.08 mg, 1.44 mmol, 240 equiv.),
monomer 4 (540.00 mg, 2.16 mmol, 360 equiv.) and mono-
mer 5 (172.00 mg, 0.72 mmol, 120 equiv.) in THF (2 mL) to
afford 794 mg (76%) of polymer 1d. Loading 5 0.8 mmol/g;
x: y: z 5 1.8:2.6:1. 1H NMR (400) MHz, CDCl3, 25 oC): d 5
7.3–6.7 (17.8H; HAr), 6.7–6.2 (10.0H; HAr), 4.9 (bs, 2H; PhO-
CH2OH), 4.6–4.2 (10.1H; PhCH2O), 3.7–3.2 (28.7H; OCH2(alk,
deg)), 1.9–1.5 (15.5H; PhCHCH2,) 1.4–1.1 (32.5H; CH2(alk, deg)),
0.88 (s, 6H; CH3(alk)).
CH2(Phe)), 1.8–1.1 (24.9H; PhCHCH2, CH3(deg)
0.87 (s, 3.8H; CH3(alk)).
CH2(alk)),
and
Oligomer 1e
6c
AIBN (30.0 mg, 0.182 mmol, 1 equiv.) in THF (0.5 mL) was
added to monomer 3 (422.24 mg, 1.82 mmol, 10 equiv.),
monomer 4 (455.00 mg, 1.82 mmol, 10 equiv.) and mono-
mer 5 (436.80 mg, 1.82 mmol, 10 equiv.) in THF (2 mL) to
afford 1.077 g (82%) of polymer 1e. Loading 5 1.5 mmol/g;
Oligomer 1e (120 mg, 0.180 mmol), Fmoc-L-Ile-OH
(127.08 mg, 0.36 mmol), DMAP (4.392 mg, 0.036 mmol),
DIC (56.539 lL, 0.360 mmol) and THF (3 mL) were used for
the reaction. 126 mg (70%) of 6c was obtained. Loading 5
1.12 mmol/g. 1H NMR (400 MHz, CDCl3, 25 8C): d 5 7.74
(s, 2H; HAr(Fmoc)), 7.58 (s, 1.8H; HAr(Fmoc)), 7.37 (s, 1.9H;
x: y: z 5 1.3:0.75:1. 1H NMR (400 MHz, CDCl3, 25 C): d 5
o
7.3–6.7 (11.5H; HAr), 6.7–6.3 (5.8H; HAr), 4.91 (bs, 2H; PhO-
CH2OH), 4.7–4.2 (5.7H; PhCH2O), 3.7–3.3 (11.1H;OCH2(alk,
deg)), 1.9–1.1 (26.3H; PhCHCH2, CH2(alk, deg)), 0.88 (s, 3.9H;
CH3(alk)).
HAr(Fmoc)), 7.3–6.7 (16.1H; HAr
Ar(Fmoc)), 6.7–6.2 (6.6H;
and
HAr), 5.42(bs, 0.8H; NH), 5.2–5.00 (2H; PhOCH2OH(Att)), 4.84
(bs, 2.4H; PhOCH2(Att)) 4.5–4.3 (bs, 7.69H; PhCH2O, CH2(Fmoc) and
CH(Ile)), 4.21 (s, 1.1; CH(Fmoc)), 3.7–3.3 (13.4H; OCH2(alk, deg)),
General Procedure for Synthesis of 6
2.17(bs, 1H; CH(Ile)), 1.8–1.1 (30.9H; PhCHCH2, CH3
(deg) and
To a solution of 1e (1 equiv.) in THF was added the required
amino acid (2 equiv.), DMAP (0.2 equiv.), and DIC (2 equiv.).
The reaction mixture was allowed to stir for 2 h. The reac-
tion mixture was filtered to remove diisopropylurea. The fil-
trate was concentrated in vacuo to a minimum volume. Cold
20% IPA/hexane (20 mL) was added to obtain the amino
acid attached polymer as a precipitate. The precipitate was
dissolved in 0.2–0.5 mL of THF and reprecipitated with cold
20% IPA/Hexane (3 3 15 mL) to afford amino acid attached
oligomer 6.
CH2(alk)), 1.0–0.7 (10.7H; CH3(alk) and (Ile)).
6d
Oligomer 1e (106.000 mg, 0.159 mmol), Fmoc-L-Val-OH
(107.929 mg, 0.318 mmol), DMAP (3.782 mg, 0.031 mmol),
DIC (49.943 lL, 0.318 mmol) and THF (3 mL) were used for
the reaction. 129 mg (82%) of 6d was obtained. Loading 5
1.0 mmol/g. 1H NMR (400 MHz, CDCl3, 25 8C): d 5 7.73 (s,
2H; HAr(Fmoc)), 7.57 (s, 2H; HAr(Fmoc)), 7.52 (s, 2H; HAr(Fmoc)),
7.3–6.7 (13.4H; HAr and Ar(Fmoc)), 6.7–6.2 (5.6H; HAr), 5.34(s,
0.81H; NH), 5.09 (s, 2H; PhOCH2OH(Att)), 4.83(s, 1.9H; PhO-
CH2(Att)), 4.5–4.3 (7.69H; PhCH2O, CH2(Fmoc) and CH(Val)), 4.19
(s, 1.1; CH(Fmoc)), 3.7–3.3 (11H; OCH2(alk, deg)), 1.9–1.1
General Procedure for Determination of Amino Acid
Loading
The loading capacities of oligomers 6(a–j) were determined
by recording their 1H NMR spectra in the presence of a
(28.5H; PhCHCH2, CH3
(10.2H; CH3(alk) and (Val)).
,
CH(Val)
CH2(alk)), 1–0.7
and
(deg)
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6e
supernatant was decanted to afford the support containing
the free amine as a precipitate, which was dried in vacuo. A
solution of Fmoc-AA-OH (2 equiv.), HCTU (2 equiv.) in THF
(2 mL) was added to the free amine, following which DIEA
(3 equiv.) was added and the reaction was allowed to stir
for 2 h. The reaction mixture was concentrated to a mini-
mum volume (1 mL) and water (15 mL) was added to it.
The suspension was centrifuged and the supernatant was
decanted to afford the support containing dipeptide as a pre-
cipitate. The precipitate was dissolved in THF (0.5 mL) and
reprecipitated with water (2 3 15 mL). The precipitate was
washed with methanol (3 3 10 mL) and dried in vacuo. The
deprotection/coupling steps were repeated as described
above to get the Fmoc protected tripeptide attached to the
support. The tripeptide was cleaved from the support using
20% TFA in CH2Cl2 for 3 h. The reaction mixture was con-
centrated in vacuo to a minimum volume. Methanol was
added to reaction mixture to precipitate the oligomer. The
supernatant containing the peptide was concentrated and
purified by semi-preparative RP-HPLC to give the pure
tripeptide.
Oligomer 1e (143.000 mg, 0.214 mmol), Fmoc-L-His(Trt)-OH
(265.855 mg, 0.429 mmol), DMAP (5.124 mg, 0.042 mmol)
and DIC (67.376 lL, 0.429 mmol) and THF (3 mL) were
used for the reaction. 186 mg (69%) of 6e was obtained.
Loading 5 0.81 mmol/g. 1H NMR (400 MHz, CDCl3, 25 8C):
d 5 7.72 (s, 1.4H; HAr(Fmoc)), 7.60 (s, 1.34H; HAr(Fmoc)), 7.45
(s, 0.8H; N5CH(His)), 7.3–6.3 (31H; HAr,
5.1–4.1 (9.6H; PhOCH2OH(Att), PhOCH2(Att, alk and deg), CH(His)
CH2(Fmoc) and CH(Fmoc)), 3.7–3.3 (9.3H; OCH2(alk, deg)), 3.09 (s,
1.3H; CH2(His)), 2.2–1.1 (24.5H; PhCHCH2, CH3
Ar(Fmoc)),
Ar(Trt) and
,
(deg) and
CH2(alk)), 1–0.7 (3.7H; CH3(alk)).
6f
Oligomer 1e (112.000 mg, 0.168 mmol), Fmoc-L-Pro-OH
(113.359 mg, 0.336 mmol), DMAP (4.099 mg, 0.033 mmol),
DIC (52 lL, 0.336 mmol) and THF (3 mL) were used for the
reaction. 117 mg (71%) of 6f was obtained. Loading 5 1.02
mmol/g. 1H NMR (400 MHz, CDCl3, 25 8C): d 5 7.8–7.65
(2H; HAr(Fmoc)), 7.6–7.45 (2.1H; HAr(Fmoc)), 7.4–6.7 (16.6H;
HAr
Ar(Fmoc)), 6.7–6.2 (5.13H; HAr), 5.2–4.9 (1.8H; PhO-
and
CH2OH(Att)), 4.85–4.5 (1.9H; PhOCH2(Att)), 4.5–3.8 (7.14H;
PhCH2O, CH2(Fmoc) CH(Pro), CH(Fmoc)), 3.7–3.3 (12.3H;
and
OCH2(alk, deg)), 2.3–1.5 (18.6H; PhCHCH2, 2CH2(Pro)), 1.45–1.1
(16.1H; CH3 (deg) and CH2(alk) &), 0.85 (bs, 3.71H; CH3(alk)).
General Procedure for HPLC Purification and Analysis of
Peptides
All peptides were purified by semi preparative RP-HPLC
(Reverse Phase High Performance Liquid Chromatography)
on a Waters HPLC system with water (0.1% TFA) and meth-
anol (0.1% TFA) as mobile phase solvent. The flow rate used
for analytical RP-HPLC and semipreparative RP-HPLC were
6g
Oligomer 1e (0.800 g, 0.904 mmol), Fmoc-L-Ala-OH
(562.288 mg, 1.808 mmol), DMAP (22.057 mg, 0.1808
mmol), DIC (283.955 lL, 1.808 mmol, 2 equiv.) and THF
(10 mL) were used for the reaction. 863 mg (81%) of 6g
was obtained. Loading 5 1.3 mmol/g. 1H NMR (400 MHz,
CDCl3, 25 8C): d 5 7.73 (bs, 2H; HAr(Fmoc)), 7.57 (bs, 2H;
1
mL/min and 4.1 mL/min, respectively. Peptide was
injected at a concentration of 1.5 mg/mL for analytical HPLC
and 10 mg/mL for semipreparative HPLC. The column used
for semipreparative HPLC was a Sunfire Prep C18, 5 lm.
Peptide elution was monitored at 254 and 220 nm with the
Waters 2489 UV/visible detector. The gradient system shown
in Supporting Information Table S1 was used to separate the
peptides.
HAr(Fmoc)), 7.36 (bs, 2H; HAr(Fmoc)), 7.2–6.7 (13.8H; HAr
and
Ar(Fmoc)), 6.7–6.3 (4.9H; HAr), 5.50 (bs, 0.68H; NH), 5.09 (bs,
2H; PhOCH2OH(Att)), 4.9 (bs, 1.9H; PhOCH2(Att)), 4.5–4.3
(6.3H; PhCH2O, CH2(Fmoc) and CH(Ala)), 4.19 (s, 1.1; CH(Fmoc)),
3.7–3.3 (10.5H; OCH2(alk, deg)), 1.9–1.1 (28.3H; PhCHCH2,
CH3(Ala) and (deg) and CH2(alk)), 0.86 (s, 3.2H; CH3(alk)).
6h
Oligomer 1e (116 mg, 0.174 mmol), Fmoc-L-Gly-OH
(103.467 mg, 0.348 mmol), DMAP (4.148 mg, 0.034 mmol),
DIC (54.655 lL, 0.348 mmol) and THF (10 mL) were used
for the reaction. 141 mg (85%) of 6h was obtained. Loading
5 1.29 mmol/g. 1H NMR (400 MHz, CDCl3, 25 8C): d 5 7.78
(s, 2H; HAr(Fmoc)), 7.74 (s, 2H; HAr(Fmoc)), 7.58 (s, 2H; HAr(F-
Analytical Data for Tripeptides
Fmoc-Phe-Val-Ala-OH (7a)
1H NMR (400 MHz, DMSO-d6, 25 8C): d 5 8.28 (d, J 5 7.2
Hz, 1H; NH), 7.90-7.82 (3H; H(Fmoc) and NH), 7.65–7.58 (3H;
NH and H(Fmoc)), 7.44-7.37 (m, 2H; H(Fmoc)), 7.34-7.14 (7H;
H(Fmoc) and HAr(Phe)), 4.38-4.13 (6H, CH(Val) CH(Phe) CH(Ala)
CHCH2 (Fmoc)), 2.99 (dd, J 5 11.2, 3.6 Hz, 1H; CH2(Phe)), 2.77
(dd, J 5 11.2, 11.2 Hz, 1H; CH2(Phe)), 2.04-1.92 (m, 1H;
CH(Val)), 1.27 (d, J 5 7.6, Hz, 3H; CH3(Ala)) 0.93–0.82 (6H;
CH3(Val)); 13C NMR (100 MHz, DMSO-d6, 25 8C): d 5 173.9,
171.3, 170.5, 155.7, 143.71, 143.69, 140.6, 138.1, 129.2,
128.0, 127.6, 127.0, 126.2,125.3, 125.2, 120.0, 65.6, 57.1,
56.0, 47.5, 46.5, 37.3, 31.0, 19.1, 18.0, 17.0; IR (KBr pellet):
3434 (bs), 3293 (s), 3064 (s), 2960 (s), 2926 (s), 1694 (bs),
moc)), 7.4–6.7 (14.1H; HAr
Ar(Fmoc)), 6.7–6.3 (5.5H; HAr),
and
5.5 (bs, 0.57H; NH), 5.09 (bs, 1.7H; PhOCH2OH(Att)), 4.84 (bs,
2H; PhOCH2(Att)) 4.5–4.3 (5.6H; PhCH2O, CH2(Fmoc)), 4.23 (s,
1.2H; CH(Fmoc)), 3.95 (s, 1.9H; CH2(Gly)), 3.7–3.3 (11.1H;
OCH2(alk, deg)), 1.9–1.1 (27.5H; PhCHCH2, CH3(deg)
and
CH2(alk)), 0.86 (s, 3.6H; CH3(alk)).
General Procedure for Tripeptide Synthesis
Oligomer 6 (0.1 g, 0.1 mmol, 1 equiv.) was treated to a solu-
tion of 20% piperidine in THF (1 mL) for 10 min. Subse-
quently cold hexane (5 mL) was added to the reaction
mixture. The resulting suspension was centrifuged and the
1541 (s), 1448 (s) 1262 (s), 1086 (s), 1041 (s). cm21
HRMS (ESI1): calcd. for C32H35N3O6Na (MNa1) 580.2424,
;
found 580.2417.
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Fmoc-Ala-Phe-Ala-OH (7b)
120.1, 65.7, 57.0, 53.4, 46.6, 43.3, 36.6, 30.9, 29.0, 19.1, 17.8;
IR (KBr pellet): 3372 (bs), 2967 (s), 2931 (s), 1669 (bs),
1591 (s), 1404 (s) 1210 (s), 1053 (s), 1024 (s) cm21; HRMS
(ESI1): calcd. for C31H33N3O6Na (MNa1) 566.2262, found
566.2276.
1H NMR (400 MHz, DMSO-d6, 25 8C): d 5 8.20 (d, J 5 6 Hz,
1H; NH), 7.89 (d, J 5 7.6 Hz, 2H; H(Fmoc)), 7.71 (t, J 5 6.4
Hz, 2H; H(Fmoc)), 7.47 (d, J 5 7.6 Hz, 1H; NH), 7.41 (t, J 5
7.2 Hz, 2H; H(Fmoc)), 7.33 (t, J 5 7.6 Hz, 2H; H(Fmoc)), 7.26-
7.12 (5H; HAr(Phe)), 4.51 (q, J 5 8.8, 1H; CH(Ala)), 4.30–4.15
(4H; CH(Ala), CH2(Fmoc) and CH(Phe)), 3.98 (t, J 5 7.2 Hz, 1H ;
CH(Fmoc)), 3.04 (dd, J 5 14, 4 Hz, 1H; CH2(Phe), 2.8 (dd, J 5
13.6, 9.2 Hz, 1H; CH2(Phe), 1.27 (d, J 5 7.2 Hz, 3H; CH3(Ala)),
1.12 (d, J 5 7.2 Hz, 3H; CH3(Ala)); 13C NMR (100 MHz,
DMSO-d6, 25 8C): d 5 173.8, 172.1, 170.6, 155.7, 143.8,
140.7, 137.6, 129.2, 127.8, 127.6, 126.1, 125.2, 120.0, 65.6,
53.3, 50.2, 47.5, 46.6, 37.3, 18.0, 17.1; IR (KBr pellet): 3388
(bs), 2977 (s), 2933 (s), 1689 (s), 1591 (s), 1405 (s), 1166
Fmoc-Phe-Ile-Phe-OH (7f)
1H NMR (400 MHz, DMSO-d6, 25 8C): d 5 8.20 (bs, 1H; NH),
7.89–7.80 (3H; H(Fmoc)
NH), 7.63–7.57 (3H, H(Fmoc)
and
and
NH), 7.40 (t, J 5 7.6 Hz, 2H; H(Fmoc)), 7.32-7.12 (12H, H(Fmoc)
HAr(Phe)), 4.45-4.38 (m, 1H; CH(Phe)), 4.34-4.20 (2H; CH(Ile)
and
CH(Phe)), 4.18-4.0 (3H; CHCH2 (Fmoc)), 3.05 (dd, J 5 13.6, 5.2
Hz, 1H; CH2(Phe)), 2.95–2.85 (m, 2H; CH2(Phe)), 2.73 (dd, J 5
11.2, 13.6 Hz, 1H; CH2(Phe)), 1.75–1.65 (m, 1H; CH(Ile)), 1.45–
1.30 (m, 1H; CH2(Ile)), 1.09-0.98 (m,1H; CH2(Ile)), 0.90-0.76
(6H, CH3); 13C NMR (100 MHz, DMSO-d6, 25 8C): d 5 171.2,
170.8, 155.7, 143.7, 140.6, 138.2, 137.6, 129.2, 129.0, 128.1,
127.9, 127.6, 127.0, 126.3, 126.1, 125.3, 125.2, 120.0, 65.6,
56.6, 55.9, 53.3, 28.9, 24.0, 15.2, 11.0; IR (KBr pellet): 3316
(s), 3274 (s), 3069 (s), 3029 (s), 2963 (s), 2926 (s), 2877
(s), 1071 (s), 1025 cm21
;
HRMS (ESI1): calcd. for
C30H31N3O6Na (MNa1) 552.2105, found 552.2109.
Fmoc-Ala-Pro-Ala-OH (7c)
1H NMR (400 MHz, DMSO-d6, 25 8C): d 5 8.09 (d, J 5 7.2
Hz, 1H; NH), 7.88 (d, J 5 7.6 Hz, 2H; H(Fmoc)), 7.71 (t, J 5
6.4 Hz, 2H; H(Fmoc)), 7.55 (d, J 5 7.6 Hz, 1H; NH), 7.42 (t, J
5 7.6 Hz, 2H; H(Fmoc)), 7.32 (t, J 5 7.2 Hz, 2H; H(Fmoc)), 4.38-
4.11 (6H; CH(Ala),CHCH2(Fmoc) CH(Pro)), 3.62-3.5 (m, NCH2),
2.05–1.75 (4H; CH2), 1.25 (d, J 5 7.6 Hz, 3H; CH3(Ala)), 1.19
(d, J 5 6.8 Hz, 3H; CH3(Ala)); 13C NMR (100 MHz, DMSO-d6,
25 8C): d 5 174.1, 171.3, 170.8, 143.9, 140.7, 127.6, 127.1,
125.3, 120.1, 65.6, 58.9, 47.9, 47.4, 46.7, 28.9, 24.4, 17.1,
16.8; IR (KBr pellet): 3343(bs), 2934 (s), 1691 (s), 1646 (s),
1590 (s), 1542 (s), 1272 (s), 1025 cm21; HRMS (ESI1):
calcd. for C26H29N3O6Na (MNa1) 502.1954, found 502.1955.
(s), 1714 (s), 1643 (s), 1537 (s), 1450 (s), 1256 (s) cm21
;
HRMS (ESI1): calcd. for C39H41N3O6 (MNa1) 670.2888,
found 670.2901.
General Procedure for Hexapeptide Synthesis
Oligomer 6 (0.1 g, 0.1 mmol, 1 equiv.) was treated to a solu-
tion of 20% piperidine in DMF (1 mL) for 10 min. Hexane
(5 mL) was added and the reaction mixture was allowed to
stir for 1 min. The hexane layer was decanted and this pro-
cess repeated to afford the support containing the free amine
as a precipitate, which was dried in vacuo. A solution of
Fmoc-AA1-AA2-OH13 (2 equiv.), HCTU (2 equiv.) in THF
(2 mL) was added to the free amine, following which DIEA
(3 equiv.) was added and the reaction was allowed to stir
for 4 h. The reaction mixture was concentrated to a mini-
mum volume (1 mL) and water (15 mL) was added to it.
The suspension was centrifuged and the supernatant was
decanted to afford the support containing dipeptide as a pre-
cipitate. The precipitate was dissolved in THF (0.5 mL) and
re-precipitated with water (2 3 15 mL). The precipitate was
washed with methanol (3 3 10 mL) and dried in vacuo. The
deprotection and coupling steps were repeated with Fmoc-
AA-OH as described above to get the Fmoc protected hexa-
peptide attached to the support. The hexapeptide was
cleaved by treating the support with 20% TFA in CH2Cl2 for
3 h. The reaction mixture was concentrated in vacuo to a
minimum volume and methanol was added to reaction mix-
ture to precipitate the oligomer. The supernatant containing
the peptide was concentrated and purified by semi-
preparative RP-HPLC to give the pure hexapeptide.
Fmoc-Phe-Phe-Phe-OH (7d)
1H NMR (400 MHz, DMSO-d6, 25 8C): d 5 8.31 (bs, 1H; NH),
8.04 (d, J 5 8.4 Hz, 1H; NH), 7.87 (d, J 5 7.2 Hz, 2H;
H(Fmoc)), 7.60 (t, J 5 7.6 Hz, 2H, H(Fmoc)), 7.50 (d, J 5 8.8 Hz,
1H; NH), 7.40 (t, J 58, 2H; H(Fmoc)), 7.33-7.10 (17H, H(Fmoc)
HAr(Phe)), 4.62-4.53 (m, 1H; CH(Phe)), 4.50-4.40 (m, 1H;
and
CH(Phe)), 4.23-4.04 (4H; CH(Phe), CHCH2 (Fmoc)), 3.11-2.62 (6H;
CH2(Phe)); 13C NMR (100 MHz, DMSO-d6, 25 8C): d 5 171.2,
171.0, 155.6, 143.8, 143.7, 140.6, 138.1, 137.5, 129.3, 129.1,
128.2, 128.0, 127.6, 127.1, 126.4, 126.23, 126.17, 125.3,
125.2, 120.1, 65.6, 56.0, 53.6, 46.5, 38.5, 37.6, 37.4, 36.7; IR
(KBr pellet): 3286 (bs), 3054 (s), 2922 (s), 2847 (s), 1688
(bs), 1645, 1542 (s), 1256 (s) cm21; HRMS (ESI1): calcd.
for C42H39N3O6Na (MNa1) 704.2737, found 704.2727.
Fmoc-Gly-Val-Phe-OH (7e)
1H NMR (400 MHz, DMSO-d6, 25 8C): d 5 8.29 (d, J 5 7.6
Hz, 1H; NH), 7.89 (d, J 5 7.2 Hz, 2H; H(Fmoc)), 7.73–7.65 (3H;
H(Fmoc) and NH), 7.53 (t, J 5 6 Hz, 1H; NH), 7.42 (t, J 5 7.2
Hz, 2H; H(Fmoc)), 7.32 (t, J 5 7.2 Hz, 2H; H(Fmoc)), 7.28–7.15
(5H; HAr(Phe)), 4.44-4.37 (m, 1H; CH(Val)), 4.29–4.20 (4H;
CH(Phe), CHCH2 (Fmoc)), 3.71–3.59 (m, 2H; CH2(Gly)), 3.01 (dd,
J 5 14, 5.6 Hz, 1H; CH2(Phe)), 2.89 (dd, J 5 14, 9.2 Hz, 1H;
CH2(Phe)), 1.98-1.88 (m, 1H; CH(Val)), 0.82 (d, J 5 6.8 Hz, 3H;
CH3(Val)) 0.76 (d, J 5 6.4 Hz, 3H; CH3(Val)); 13C NMR (100
MHz, DMSO-d6, 25 8C): d 5 172.7, 170.8, 168.7, 156.4,
143.8, 140.7, 137.5, 129.0, 128.1, 127.6, 127.1, 126.4, 125.2,
Fmoc-Ala-Gly-Val-Ile-Pro-Phe-OH (7g)
1H NMR (400 MHz, DMSO-d6, 25 8C): d 5 8.14 (t, J 5 6 Hz,
1H; NH), 8.01 (d, J 5 7.6 Hz, 2H; NH), 7.89 (d, J 5 7.6 Hz,
2H; H(Fmoc)), 7.74–7.68 (2H; NH), 7.59 (d, J 5 7.6 Hz, 2H;
H(Fmoc)), 7.41 (t, J 5 7.2 Hz, 2H; H(Fmoc)), 7.33 (t, J 5 7.2 Hz,
2H; H(Fmoc)), 7.27–7.17 (5H; HAr
(Phe)), 4.42–4.17 (7H;
and
CH(Val), (Ala), (Phe), (pro), (Ile) and CHCH2 (Fmoc)), 4.04 (t, J 5 1H;
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TABLE 2 Attachment of Amino Acids onto Support 1e.
No.
AA1
Yield (%)a
Loading (mmol/g)b
1.
2
3
4
6
7
8
9
6a: Ala
6b: Phe
6c: Ile
81
85
70
59
69
71
86
85
1.3
1.13
1.12
1.0
6d: Val
6e: His (Trt)
6f: Pro
0.81
1.02
1.35
1.29
6g: Ala
6h: Gly
Isolated yield
Determined by 1H NMR.
Fmoc-Ala-Gly-Val-Ile-Pro-Ala-OH (7h)
1H NMR (400 MHz, DMSO-d6, 25 8C): (extra solvent peaks
seen) d 5 8.19–8.08 (2H; NH), 8.00 (d, J 5 8 Hz, 1H; NH),
7.89 (d, J 5 7.6 Hz, 2H; H(Fmoc)), 7.74–7.68 (2H; NH), 7.59
(d, J 5 8 Hz, 2H; H(Fmoc)), 7.41 (t, J 5 7.2 Hz, 2H; H(Fmoc)),
7.33 (t, J 5 7.2 Hz, 2H; H(Fmoc)), 4.34–4.00 (8H; CH(Val), (Ala),
CHCH2 (Fmoc)) 3.83–3.67 (4H; CH2(Gly)
(Phe), (Pro), (Ile) and
and
NCH2(pro)), 1.99–1.71 (6H; CH(Ile), CH(Val) and NCH2(pro)), 1.54–
1.47 (m, 1H; CH2(Ile)), 1.28–1.19 (6H; CH3(Ala)), 1.09–1.0 (m,
SCHEME 1 Modular synthetic approach for obtaining support.
1H; CH2(Ile)), 0.88–0.72 (12H; CH3(Val)
(Ile)); 13C NMR
and
(100 MHz, DMSO-d6, 25 8C): d 5 174.0, 172.8, 171.2, 170.7,
169.8, 168.4, 155.5, 143.9, 140.7, 127.6, 127.1, 125.2, 120.1,
65.7, 58.9, 57.0, 54.6, 50.2, 47.4, 47.1, 46.6, 42.1, 35.8, 30.8,
29.0, 24.4, 24.3, 19.0, 17.9, 17.0, 14.8, 10.7; IR (KBr pellet):
3216 (bs), 2922 (s), 2508 (s), 1664 (bs), 1454 (s) 1193 (bs)
cm21; HRMS (ESI1): calcd. for C39H53N6O9 (M1) 749.3874,
found 749.3882.
CHCH2 (Fmoc)), 3.83–3.67 (3H; CH2(Gly)
NCH2(pro)), 3.55–
and
3.45 (m, 1H; NCH2(pro)), 3.02–2.87 (2H; CH2(Phe)), 1.98-1.67
(6H; CH(Ile), CH(Val) and NCH2(pro)), 1.55–1.46 (m, 1H; CH2(Ile))
1.23 (d, J 5 6.8 Hz, 3H; CH3(Ala)), 1.09–0.98 (m, 1H; CH2(Ile)),
0.86–0.70 (12H; CH3(Val)
(Ile)); 13C NMR (125 MHz,
,
and
DMSO-d6, 25 8C): d 5 172.9 172.8, 171.5, 170.7, 169.9,
168.5, 155.8, 143.9, 143.8, 140.7, 137.3, 129.2,128.2, 127.6,
127.1, 126.5, 125.3, 125.3, 120.1, 65.7, 59.0, 56.9, 54.6, 53.6,
50.2, 47.1, 46.6, 42.1, 36.7, 35.8, 30.8, 29.0, 24.3, 19.0, 18.0,
17.9, 14.9, 10.7; IR (KBr pellet): 3403 (bs), 3303 (bs), 2964
(s), 2929 (s), 1649 (bs), 1628, 1526, (s), 1450 (s) 1198 (bs),
cm21; HRMS (ESI1): calcd. for C45H57N6O9 (M1) 825.4187,
found 825.4169.
RESULTS AND DISCUSSION
Support Synthesis
The monomers for synthesizing the support could be readily
obtained starting from 4-vinylbenzylchloride 2 (Scheme 1).
Treatment of the chloride 2 with sodium hydride and the
requisite alcohol afforded monomers 3 and 4 in 81 and 87%
yields, respectively. Monomer 5 was obtained in 77% yield
by treatment of chloride 2 with 4-hydroxy benzaldehyde in
the presence of K2CO3 and subsequent reduction with
sodium borohydride. Supports 1 were obtained from mono-
mers 3, 4 and 5 via free radical polymerization using AIBN
as the initiator. Such a modular synthetic approach was pro-
posed to facilitate easy incorporation of any styrene based
attachment site into the soluble polymer backbone. The poly-
merization was carried out in THF for 3 days at 80 8C in a
sealed tube. Initially, polymer supports 1a, b (x 1 y 1 z 5 60
and x 1 y 1 z 5 120) with a 1:1:1 monomer ratio was syn-
thesized (Table 1). However, these polymers were sparingly
soluble in organic solvents such as dichloromethane and
THF. Increasing the proportion of the solubilizing groups
did not improve the solubility of the polymer (1c and d,
Table 1). Oligomer 1e was found to be the most optimal
TABLE 1 Supports (Supp.) 1a–e Synthesized for Peptide
Synthesis
Loadingb
Supp.
na(3:4:5)
Yield (%)
(mmol/g)
x:y:zb
1a
1b
1c
1d
1e
60(1:1:1)
120(1:1:1)
120(2:2:1)
120(2:3:1)
10(1:1:1)
71
87
87
67
82
1.5
1.2
1.3
0.8
1.5
1:0.9:1
0.8:0.6:1
2:2.1:1
1.8:2.6:1
1.3:0.8:1
a
n 5 equiv. with respect to AIBN.
Determined by 1H NMR.
b
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SCHEME 2 Peptide synthesis using support 6.
SCHEME
3 Hexapeptide
synthesis
using
combination
approach.
support for peptide synthesis as it was highly soluble in
organic solvents and could be recovered using cold hexane
or water. The supports were found to be partially soluble in
methanol.14 The loading capacity of supports 1e could be
easily determined using 1H NMR spectroscopy in the pres-
ence of 1,1,2,2-tetrachloroethane (TCE) as a standard.
Peptide Synthesis
The loaded supports 6 were used for synthesizing tripepti-
des following the route illustrated in Scheme 2. The Fmoc
group in support 6 was deprotected using 20% piperidine in
DMF. A phase separation was observed when hexane was
added after completion of the reaction.15 The upper layer
was removed with a dropper and after three to four washes
with hexane, the polymer was obtained as a precipitate. Sub-
sequent treatment with the Fmoc protected second amino
acid afforded the supported dipeptide. The supported dipep-
tide was isolated via precipitation with water. The precipitate
was sequentially washed with water and methanol to remove
excess reagents. One can envision continuing the cycle
(n 2 1) times to obtain the peptide of desired length (n). The
support was found to be soluble in the reaction medium dur-
ing peptide synthesis. Two equivalents of coupling reagents
and amino acids were used for each step, which is much
lower that what is typically used for SPPS. After completion
of synthesis, the peptide 7 was cleaved form the support
using 20% TFA. The support was removed as a precipitate in
methanol and the supernatant was purified using reversed
phase HPLC to obtain the Fmoc protected peptide 7. A vari-
ety of tripeptides 7a–f were synthesized in 38–64% yields
using supports 6(a, b & g) (Table 3). The yields are good
considering the fact that we are only using 2 equivalents of
coupling reagents.
Amino Acid Attachment
Nonpolar, polar and basic amino acids amino acids were
loaded onto the supports 1e using DIC and DMAP in 59–
86% yields (eq 1, Table 2). The loaded supports 6 could be
readily characterized using 1H NMR spectroscopy as they
were soluble in organic solvents, such as dichloromethane,
THF, and DMF. The integration values corresponding to TCE
was compared with those of the aromatic protons of Fmoc.
The loading capacities of the supports 6 (0.87–1.3 mmol/g)
were similar to those typically found in the SPPS resins.
TABLE 3 Tripeptides Synthesized Using the Oligomeric
Since peptide synthesis is being carried out from the C- to
N-terminus and the reactivity of amino acids is maintained
in a homogeneous reaction medium, the slightly lower yields
could be attributed to diketopiperazine (DKP) formation
(after deprotection of the dipeptides). One can envisage
improving the yields for peptide synthesis if the DKP forma-
tion is prevented or avoided. Therefore, we used a combina-
tion approach, where dipeptides synthesized in solution12
were loaded onto the oligomeric support for synthesizing
hexapeptides (Scheme 3). We have observed that such an
approach leads to a significant improvement in the yields for
peptide synthesis. The protocols used for the deprotection
and coupling steps were as described before. The
Supports
Isolated
6
Peptide 7
yielda (%)
1.
2.
3.
4.
5.
6.
6g
6a
6g
6b
6b
6b
7a: FmocNHPheValAlaOH
7b: FmocNHAlaPheAlaOH
7c: FmocNHAlaProAlaOH
7d: FmocNHPhePhePheOH
7e: FmocNHGlyValPheOH
7f: FmocNHPheIlePheOH
44
46
64
38
58
53
a
Isolated using semipreparative RP-HPLC.
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2008; (d) N. Madhavan, C. W. Jones, M. Weck, Acc. Chem. Res.
2008, 41, 1153–1165; (e) M. Benaglia (Ed.) Recoverable and
Recyclable Catalysts; Wiley-VCH: Weinheim, 2009; (f) N.
Haraguchi, S. Itsuno, Polymeric Chiral Catalyst Design and
Chiral Polymer Synthesis; Wiley: New York, 2011; (g) V.
Khamatnurova, D. E. Bergbreiter, Polym. Chem. 2013, 4, 1617–
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H. Toy, Pure Appl. Chem. 2014, 86, 1651–1661.
hexapeptides were purified by RP-HPLC after cleavage from
the support. Following the combination approach peptides 7f
and 7g were obtained from supports 6b and 6h in 51 and
45% overall yields, respectively. As before, only 2 equivalents
of coupling reagents were used. Despite the fact that hexa-
peptide synthesis requires six additional steps as compared
to the tripeptide synthesis (Table 3), it is notable that we
are able to get hexapeptides in ꢀ50% yields.
4 (a) V. R. Pillai, M. Mutter, E. Bayer, I. Gatfield, J. Org. Chem.
1980, 45, 5364–5370; (b) P. M. Fischer, D. I. Zheleva, J. Pept.
Sci. 2002, 8, 529–542; (c) M. Roice, I. Johannsen, M. Meldal,
QSAR Comb. Sci. 2004, 23, 662–673; (d) C.-X. Zhang, H.-B.
Tong, C.-G. Yan, J. Comb. Chem. 2007, 9, 9242925.
CONCLUSIONS
In conclusion, oligomeric styrene supports with loading
capacities of 1.5–1.6 mmol/g analogs to styrene-based resins
used in SPPS have been synthesized using a modular
approach. The supports are soluble in organic solvents, such
as DCM, THF or DMF and can be readily precipitated using
cold hexane or water. Nonpolar, polar, basic, and aromatic
amino acids could be loaded on to the supports in 0.87–1.3
mmol/g. The amino acid loading could be determined using
NMR spectroscopy without cleaving the amino acid. Tripepti-
des were synthesized in 38–64% yield using the support,
wherein only 2 equivalents of coupling reagents and amino
acids were used. A combination approach which prevented
DKP formation was used to synthesize larger hexapeptides
with improved overall yields (ca. 50%). The synthetic
approach for formation of our oligomeric supports is modu-
lar. Therefore, one can readily incorporate the efficient sty-
rene monomers with attachment sites used in SPPS resins
into our support scaffold to obtain efficient soluble styrene
supports. Current efforts are focused on expanding the scope
of these supports for peptide synthesis by varying the nature
of the attachment sites.
5 (a) K. Chiba, Y. Kono, S. Kim, K. Nishimoto, Y. Kitano, M.
Tada, Chem. Commun. 2002, 1766–1767; (b) D. Takahashi,
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ACKNOWLEDGMENTS
8 (a) N. Naganna, N. Madhavan, Org. Lett. 2013, 15, 5870–5873; (b)
N. Naganna, N. Madhavan, J. Org. Chem. 2014, 79, 11549–11557.
This research was supported by CSIR (No. 01(2333)/09/EMR-
II), New Delhi, India. V. E. acknowledges CSIR, India for his
research fellowship.
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13 See Supporting Information for solution phase synthesis of
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during this step, which might account for the lowering of yield.
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