A rink-amide soluble support: High purity conotoxins and other peptides accessed with minimal reagents

Article abstract of DOI:10.1039/c9ob01214a

The use of peptides as therapeutics has been growing due to their biocompatibility. Solid phase peptide synthesis typically used to access these peptides requires excess reagents and/or microwave irradiation to drive reactions to completion because the reaction medium is heterogeneous. Reported herein is a soluble polynorbornene support containing rink amide attached sites for synthesizing oligopeptides and conotoxins in high purity using only 1.2 to 2 equivalents of coupling reagents. The support can be isolated as a precipitate from the reaction medium by adding ether. The loading capacity of the support can be easily determined by spectroscopy and can also be tuned by varying the monomer ratio. This support is promising for accessing peptides as the methodology uses minimal reagents and by-products can be easily separated.

Full text of DOI:10.1039/c9ob01214a

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Page 1 of 9
O rP gl ea na si ce &d Bo i on mo to al e dc juu l sa tr mC ha er mg i ins ts ry
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DOI: 10.1039/C9OB01214A
ARTICLE
A Rink-Amide Soluble Support: High Purity Conotoxins and Other
Peptides Accessed with Minimal Reagents
a
b
a
Babita Bisht, Nimmashetti Naganna and Nandita Madhavan*
Received 00th January 20xx,
Accepted 00th January 20xx
The use of peptides as therapeutics has been growing due to their biocompatibility. Solid phase peptide synthesis typically
used to access these peptides requires excess reagents and/or microwave irradiation to drive reactions to completion
because the reaction medium is heterogeneous. Reported herein is a soluble polynorbornene support containing rink amide
attached sites for synthesizing oligopeptides and conotoxins in high purity using only 1.2 to 2 equivalents of coupling
reagents. The support can be isolated as a precipitate from the reaction medium by adding ether. The loading capacity of
the support can be easily determined by spectroscopy and can also be tuned by varying the monomer ratio. This support is
promising for accessing peptides as the methodology uses minimal reagents and by-products can be easily separated.
DOI: 10.1039/x0xx00000x
there is limited scope for tuning the amino-acid loading on the
support analogous to SPPS. Recently, soluble polystyrene
supports have been used to synthesize pentapeptides,
Introduction
the growing use of oligopeptides as therapeutics.1, 2 Solid phase
peptide synthesis (SPPS) provides quick access to a large variety
of peptides.3 The method is extremely attractive as it can be
automated and does not require chromatographic separation
after each step. The efficiency of solid phase peptide synthesis
has been further enhanced using automated continuous flow
3
1
oligonucleotides and oligosaccharides.
developed soluble polynorbornene
Our group has
and oligostyrene
3
2, 33
34
-5
supports for synthesizing penta-octapeptides in moderate yield.
A competing cyclization reaction was found to lower the yields
for the peptides using these supports. Therefore, the yields for
octapeptides were low and larger oligopeptides were not
synthesized using these supports. We report herein a
polynorbornene support with a rink-amide attachment site
6
-9
techniques that provide peptides in minutes. The only caveat
in using SPPS is that typically excess coupling reagents and
amino acids are used to drive the peptide coupling reactions to
completion in a short span of time. A methodology that
provides high purity peptides without the use of excess
reagents is highly attractive for accessing peptides with minimal
waste. This concept of using soluble supports for peptide
synthesis is often referred to as liquid phase peptide synthesis
(Figure 1b). The support is found to be superior to our earlier
supports in terms of yields for larger peptides and solubility in
the reaction medium after octapeptide synthesis. The support
also provides access to peptides with the amide linkage at the
C-terminus. Peptide synthesis could be accomplished using only
1
.2 to 2 equiv of coupling reagents at room temperature.
1
0
11-13
14-17
(
LPPS). LPPS using salts, fluorous,
ionic liquid,
a)
1
8-20
as well as polymeric supports21-25 that are
hydrophobic
soluble in the reaction medium have led to a considerable
decrease in the equivalents of coupling reagents used for
peptide synthesis. The growing peptide can be readily isolated
from these supports via techniques such as cooling,
precipitation or extraction. This methodology has also been
O
=
HO
n
=
attachment site
b)
m
O
n
2
6
27
l
O
used for oligosachharide and oligonucleotide synthesis.
O
2
8-30
HN
HN
HN
Among the polymeric supports, polyethylene glycol (PEG)
has been found to be highly efficient as it provides a polar
medium for the growing peptide during synthesis. The PEG
supports can load one or two peptides at their termini (Figure
NH
O
=
O
O
O
1
a). Therefore, they are used in stoichiometric amount and
NHFmoc
a. Department of Chemistry, Indian Institute of Technology Bombay, Powai,
Mumbai, Maharasthra, 400076, India.
Department of Chemistry, Indian Institute of Technology Madras, Chennai, Tamil
OMe
1
b.
MeO
Figure 1. Schematic representation of a) PEG support; b) Polynorbornene
Nadu 600036, India.
support for peptide synthesis.
†Electronic Supplementary Information (ESI) available: [Experimental procedures
and characterization of compounds]. See DOI: 10.1039/x0xx00000x
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O rP gl ea na si ce &d Bo i on mo to al e dc juu l sa tr mC ha er mg i ins ts ry
Page 2 of 9
ARTICLE
Journal Name
Scheme 2. a) Synthesis of support and b) attachment of amino acids on support.
Results and Discussion
View Article Online
a)
[Ru], DCM
DOI: 10.1039/C9OB01214A
45 min
The support 1 was designed such that the rink-amide
attachment site is interspersed with alkyl and oligoether chains.
These groups not only act as spacers but also help enhance
solubility of the growing peptide on the support. The rink amide
attached monomer was synthesized from diamine 2, which was
treated with boc anhydride to give monoprotected amine 3 in
+
+
NHFmoc
O
O
O
HN
HN
1a: l:m:n = 1:2:0.7;
HN
0.7 mmol/g; 92%
1
b: l:m:n = 1:3:3;
0.5 mmol/g; 85%
NH
O
O
3
5
8
6% yield (Scheme 1). Amine 3 was coupled with norbornene-
1c: l:m:n = 1:3:5;
.4 mmol/g; 81%
O
O
0
exo-acid 4 using O-(benzotriazole1-yl)-1,1,3,3-tetramethyl-
uroniumhexaflorophosphate (HBTU) to afford the amide 5 in
1d: l:m:n = 1:11:7;
.3 mmol/g; 75%
0
8
5% yield. The boc group was cleaved using TFA and the
resulting amine 6 was coupled with the acid 7 using 1-ethyl- 3-
3-dimethylaminopropyl carbodiimide hydrochloride (EDCI) and
NHFmoc
OMe
Tunable loading
(
MeO
catalytic 4-dimethylaminopyridine (DMAP) to give monomer 8
in 77% yields. Monomers 9 and 10 were prepared following the
procedure reported earlier by our group.
Monomers 8-10 were copolymerized via the ring-opening
metathesis polymerization (ROMP) reaction in the presence of
the Grubbs’ third generation initiator to access the polymer
support (Scheme 2a). The synthesis is highly modular as the
loading capacity (mmol of attachment site/g of polymer) can be
easily varied by changing the monomer ratio during
polymerization. Supports 1a-d with loading capacities of 0.3 –
8
9
10
3
2
b)
1) 20% piperidine-
DMF
H
1a
N
AA-Fmoc
2
) FmocAAOH
HBTU, DIEA
rt, 2h
O
11
t
1
1a: Ser(O Bu)-Fmoc 11b: Arg(Pbf)-Fmoc 11c: Phe-Fmoc 11d: Val-Fmoc
0.4 mmol/g
0.6 mmol/g
0.3 mmol/g
0.5 mmol/g
1
1e: Tyr-Fmoc
.68 mmol/g
11f: Met-Fmoc
0.3 mmol/g
11g: Gln(trt)-Fmoc11h: Gly-Fmoc
0.6 mmol/g 0.5 mmol/g
0
.7 mmol/g, were synthesized, in 75-92% yields. The polymer
0
was highly soluble in solvents such as dichloromethane and
DMF and insoluble in hexane, ethyl acetate and diethyl ether.
Support 1a (0.7 mmol/g) was used for attachment of a variety
of neutral, acidic and basic amino acids (Scheme 2b). After the
reaction, the amino acid attached support was precipitated with
diethyl ether to afford loaded supports 11a-l. The removal of
unreacted amino acids during the washing step was ascertained
by checking the supernatant using thin layer chromatography
1
1i: Ala-Fmoc
0.68 mmol/g
11j: Trp(boc)-Fmoc 11k: Cys(trt)-Fmoc 11l: His(trt)-Fmoc
0.4 mmol/g 0.5 mmol/g
0.5mmol/g
acid, O-(benzotriazole1-yl)-1,1,3,3-tetramethyluroniumhexa-
florophosphate (HBTU) and diisopropylethyl amine (DIEA) in
dichloromethane.
A small volume (2 mL) of dichloromethane was used for the
coupling reactions as the support was highly soluble in the
reaction medium (Figure 2a). The coupling reactions were
monitored by ninhydrin test. After completion of the reaction,
separation of the peptide from the unreacted amino acids and
by-products was very facile as the support was highly insoluble
in diethyl ether (Figure 2b). The sequence of deprotection and
coupling was repeated until the target peptide was obtained.
The peptide was cleaved from the support using a mixture of
TFA, TIS and water. After removal of TFA from the reaction
mixture, water was added to extract peptide 12. The peptides
were purified by semi-preparative HPLC (Table S1).
(
Figure S34). As the support was soluble in chloroform, its
1
loading capacity could be determined using H NMR
spectroscopy in the presence of
a standard 1,1,2,2-
tetrachloroethane (TCE). The NMR spectra of compounds 11a-l
also indicated absence of unreacted amino acids. However, the
peaks corresponding to the base diisopropylethyl amine were
observed.
Peptides were synthesized using support 1 as shown in Scheme
3
. The Fmoc group was deprotected using piperidine/DMF.
Subsequently, the reaction mixture was washed using hexane,
re-dissolved in dichloromethane and precipitated with diethyl
ether. The precipitate was treated with the requisite amino
Scheme 1. Synthesis of rink-amide monomer
Scheme 3. Peptide synthesis
NB-COOH
4
20%
piperidine-
DMF
Fmoc-AA-OH
HBTU, DIEA
DCM, rt, 2 h
Boc2O
DCM, 18h
O
HBTU, DIEA
DCM, 15h
TFA
DCM, 2h
H2N
Boc-HN
NB
N
NHFmoc
NH2
NHCO-(AA) -NHFmoc
86%
85%
H
n
NH2
NH2
NHBoc
99%
15 min
2
3
5
ppt. with ether
ppt. with ether
1
FmocHN OMe
20% pip/DMF
HOOC
FmocHN
OMe
10 min
O
OMe
TFA:TIS:H2O
95:2.5:2.5
7
O
O
H
NHCO-(AA)n-NH2
EDCI, DCM, 12h
NH2CO-(AA)n-NH2
N
NB
NH2
NB
N
O
OMe
2h
N
extracted with water
ppt. with ether
7
7%
H
H
O
12
6
8
n = no. of cycles
NB =
2
| J. Name., 2012, 00, 1-3
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O rP gl ea na si ce &d Bo i on mo to al e dc juu l sa tr mC ha er mg i ins ts ry
Journal Name
a)
ARTICLE
b)
View Article Online
DOI: 10.1039/C9OB01214A
Table 1. Peptides and conotoxins synthesized using support 1
No.
Support
Peptide 10
AA,
eq.
Yield
1
2
3
4
5
6
7
8
9
1a
1a
1a
1a
1a
1a
1a
1a
1a
1a
1b
1d
1d
12a: AFV
12b: GCF
1.2
1.2
1.2
1.2
1.2
1.2
2
85 (70)[a]
72 (65)[a]
69 (64)[a]
85 (56)[a]
65 (57)[a]
69 (56)[a]
88 (54)[a]
65 (56)[a]
63 (53)[a]
41 (29)[a]
44 (31)[a]
49 (43)[a]
35 (14%)
12c: FHL
12d: AFIA
Figure 2. Support 1 a) dissolved in dichloromethane; b) precipitated with diethyl ether.
12e: AFWMHYAF
12f: AFAIAPGF
12g: GCPWQPYC
12h: TCFGCTPCC
12i: CCPPALWCC
12j: WFTTLISTIM
12j: WFTTLISTIM
12j: WFTTLISTIM
12k:GCCGAFACRFGCTPCC
Following the protocol shown in Scheme 3, a variety of
peptides were synthesized (Table 1). The smaller tripeptides
1
2b and 12c were chosen as cysteine and histidine are known
to epimerize during peptide synthesis. The peptides 12d-12f
contain residues such as proline, tryptophan and histidine that
are not easy to couple. Only 1.2 equivalents of amino acids and
coupling reagents were used for synthesis of tri- to
octapeptides 12a-12f in 65-88% yields. The solubility of the
support was found to be good during peptide synthesis. The
HPLC chromatogram of the crude peptides showed the desired
peptide as the major peak (Figures S35 & S36) and minor
impurities. The isolated yields of the peptides after purification
using reversed phase HPLC were also very high (56-70%).
The methodology was extended to synthesize conotoxins 12g-
i that have been previously synthesized on SPPS supports using
2
2
1
0
2
11
2
1
2
2
13
2
a] Isolated yield.
8
automated continuous flow. The synthesis of these cysteine
rich peptides is non-trivial. Our support afforded the desired
peptides in high yields (63-88%) using only 2 equivalents of
coupling reagents and amino acids. None of the coupling steps
were repeated in order to obtain the desired peptide. The HPLC
chromatogram of the crude peptides (Figure 3, S37-S39) were
comparable to those obtained using the automated SPPS
a)
b)
8
method with excess equivalents of coupling reagents. The
isolated yields of the conotoxins after purification was also good
(53-56%). It is notable that the synthesis could be accomplished
using support 1a that has a high loading capacity of 0.7 mmol/g.
In SPPS, typically such large loading capacities are not efficient
for the synthesis of larger peptides because the accessibility of
reagents is reduced. Our support being highly solvated in the
reaction medium shows better reactivity. The synthesis of the Figure 3. HPLC chromatograms of crude conotoxins a) 12g; b) 12h
decapeptide 12j is known to be challenging due to its propensity
8
to aggregate during peptide synthesis. Supports 1a, 1b and 1d
were used to synthesize this peptide The yield for peptide 12j Conclusions
correlated with the loading capacity of the supports (entries 10-
A non-crosslinked polynorbornene support containing the rink-
amide attachment site and oligoether/alkyl chains has been
used for synthesizing peptides. Peptide coupling reactions were
efficiently carried out on the support using significantly lower
equivalents of coupling reagents as compared to solid phase
synthesis. The solubility of the support also enabled
determination of loading capacity using non-destructive
techniques such as NMR spectroscopy. The support was used
for synthesizing a variety of oligopeptides in good isolated
yields. During peptide synthesis, the support could be easily
precipitated using ether. The volumes of solvent required were
1
2, Figure S40). The desired peptide could be obtained in the
best yield (43%) using support 1d with a loading capacity of 0.3
mmol/g. Lastly, the synthesis of hexadecapeptide 12k was
attempted using support 1d and only 2 equiv. of coupling
reagents. As earlier, none of the coupling steps were repeated.
The total synthesis time for the crude hexadecapeptide
including reaction time and work-up is 40 h. Purification by
HPLC afforded the desired peptide in 14% yield.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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O rP gl ea na si ce &d Bo i on mo to al e dc juu l sa tr mC ha er mg i ins ts ry
Page 4 of 9
ARTICLE
Journal Name
not very high. Conotoxins and difficult deca/hexadecapeptides with a 20% aqueous solution of Na CO (30 mL). The organic
2 3
View Article Online
were also synthesized in yields comparable to reported layer was dried over sodium sulphate, filteDrOeId: 1a0n.1d03c9o/nCc9eOnBt0r1a2t1e4dA
methods. Ongoing efforts are focused on automating the in vacuo to give the mono-boc protected amine 3 (3.59 g, 86%)
process to expand the scope of this methodology. Automation as a semi solid, which was used for the next reaction without
1
would not only improve the efficiency of this method, but would further purification. TLC R
also make it a very attractive alternative to SPPS because it (400 MHz, CDCl
minimizes the loss of expensive coupling reagents and amino 2H; CH NH), 2.77 (t, J = 6Hz, 2H; CH
f
= 0.2, CH
2
Cl
2
with Et
3
N (1%). H NMR
3
, 25 C): δ = 5.09 (s, 1H; NHBoc), 3.18-3.10 (m,
NH ), 2.27 (bs, 2H; NH ),
1.41 (s, 9H; CH3(Boc)); CNMR (100 MHz CDCl ,25˚C): δ = 156.4,
9.2, 43.2, 41.8, 28.5; IR (KBr Pellet): ν = 3362 (br), 2985 (s),
2
2
2
2
1
3
acids.
3
7
-1
Experimental
1686 (s), 1595 (m), 1533 (s), 1279 (s), 1176 (s), 986 (s) cm ;
+
+
LRMS (ESI) : calcd for C
7 16 2
H N NaO
2
(MNa ) 183.11, found
General Methods
183.12.
All reagents for synthesis were purchased from commercial
suppliers and used without further purification unless stated Monomer with boc-protected spacer 5: To a solution of
3
2
otherwise. All air-sensitive reactions were performed using norbornene-exo-acid 4 (1.00 g, 0.007 mmol, 1 equiv) and boc-
oven-dried glassware in an inert atmosphere of nitrogen. protected amine 3 (1.40 g, 0.009 mmol, 1.2 equiv) in CH Cl (30
Syringe or cannula was used to transfer air-sensitive solvents mL) at 0 C, was added EDCI (2.0 g, 0.01 mmol, 1.5 equiv), DIEA
and solutions. Dichloromethane and N,N- (2.5 mL, 0.02 mmol, 2 equiv). The reaction mixture was allowed
diisopropylethylamine (DIEA) were distilled over calcium to warm to room temperature and stirred for 15 h.
hydride. All dry solvents were Subsequently, the reaction mixture was concentrated in vacuo.
stored over 4 Å molecular sieves prior to use. All monomers Ethyl acetate (50 mL) was added and the reaction mixture was
were synthesized in solution using HBTU and EDCI (1-Ethyl-3-(3- washed with H O (2 × 100 mL). The organic layer was dried over
2
2
2
dimethylaminopropyl) carbodiimide as coupling reagents and anhydrous sodium sulphate, filtered and concentrated in vacuo
N,N-diisopropylethylamine as a base. Rink amide was to give the crude compound. Purification using flash column
commercially obtained from aapptec chemicals. Analytical thin chromatography (gradient 10-40% EtOAc/Hexane) afforded
layer chromatography (TLC) was performed on Merck amide 5 (1.5 g, 85%) as a white solid. TLC R
f
= 0.3 in 40%
1
precoated silica gel 60 F254TLC plates. Eluting solvents are EtOAc/Hexane. H NMR (400 MHz, CDCl
3
, 25 C): δ = 6.39 (bs,
), 2.95 -
stains. Flash column 2.86 (2H; CHnb), 2.03 (1H; CH(nb)), 1.88(d, J = 9.6Hz, 1H; CH2nb),
chromatography was performed using silica gel (100-200 mesh). 1.66 (d, J = 7.6Hz, 1H; CHnb), 1.43(9H; CH3Boc), 1.37-1.22 (3H;
All NMR spectra were recorded using CDCl , D O or DMSO-d , 25 ˚C): δ = 176.2,
as CH2nb& solvent); 13C NMR (100 MHz CDCl
reported as volume percents. Compounds were visualized using 1H; NH), 6.15-6.05 (2H; CH=CH), 3.38-3.22 (4H; NHCH
UV light and ninhydrin, iodine & KMnO
2
4
3
2
6
3
a solvent. The NMR spectra were referenced using residual 156.8, 138.0, 135.8, 79.5, 46.9, 46.2, 44.5, 41.4, 40.7, 40.1, 30.3,
solvent peaks as the standard. Chemical shifts (δ) are denoted 28.2; IR (KBr Pellet): ν = 3340 (br), 2978 (m), 2939 (m), 1692 (s),
in parts per million, coupling constants (J) are reported in hertz 1649 (s), 1540 (s), 1447 (w), 1365 (m), 1279 (m), 1247 (m), 1176
-
1
+
+
(
(
(
(
Hz), and spin multiplicities are reported as singlet (s), doublet (m) cm ; HRMS (ESI) : calcd for C15
d), triplet (t), quartet (q), quintet (quint), apparent quintet found 303.1676.
app. quint.), multiplet (m). High-resolution mass spectra
24 2 3
H N NaO (MNa ) 303.1679,
HRMS) were recorded on the MICRO-Q-TOF mass Monomer with amine spacer 6: TFA (2.5 mL, 33 mmol, 20
spectrometer using the ESI technique. All IR spectra were equiv) was slowly added over a period of 10 min using a syringe
recorded in the form of KBr pellet. IR spectra peaks are reported to a solution of boc-protected amine 5 (0.46 g, 1.65 mmol, 1
-1
in wavenumbers (cm ) as strong (s), medium (m) and broad equiv) in CH
2 2
Cl (2.5 mL) at 0 C. The reaction mixture was
(br). Semi-preparative RP-HPLC was carried out on an LC-20A allowed to warm to room temperature and stirred for 1h. The
HPLC system using water (0.1%TFA) and acetonitrile (0.1% TFA) excess TFA was removed under a stream of nitrogen to give free
as the mobile phase and a prep C-18,5µm, 10 × 250 mm column amine 6 (0.49 g, 99%). The compound was used in the next step
as the stationary phase. Peptides were injected at a without further purification.
concentration of 10 mg/mL, and a flow rate of 4.5 mL/min was
used for semipreparative RP-HPLC. Peptide elution was Rink amide attached monomer 8: To a solution of amine 6 (0.82
monitored at 190 - 254 nm.
g, 2.8 mmol, 1 equiv) and rink amide 7 (1.5 g, 2.8 mmol, 1.2
equiv) in CH Cl (25 mL) at 0 C, was added EDCI (1.4 g, 3.4
N-tert-butoxycarbonyl-1,2-ethanediamine 3: To a solution of mmol, 1.2 equiv) and DIEA (1.5 mL, 3.4mmol, 3 equiv). The
ethylene diamine 2 (18 mL, 0.26 mmol, 10 equiv) in CH Cl (50 reaction mixture was allowed to warm to room temperature
mL) at 0 C, was added a solution of boc anhydride (5.7 g, 0.03 and stirred for 12 h. Subsequently, the reaction mixture was
mmol, 1 equiv) in CH Cl (10 mL) over a period of 10 min. The diluted with CH Cl (100 mL) and washed with water (2 × 20 mL).
2
2
2
2
2
2
2
2
reaction was allowed to stir at RT under nitrogen for 18 h. The The organic layer was dried over anhydrous sodium sulphate,
reaction mixture was concentrated. A 20% aqueous solution of filtered and concentrated in vacuo to give the crude compound.
Na
2
CO
3
(30 mL) was added to the residue and the solution was Purification using flash column chromatography (5%
Cl (3  20 mL). The organic layer was washed MeOH/DCM) afforded monomer 8 (0.8 g, 67%) as a white solid.
extracted with CH
2
2
4
| J. Name., 2012, 00, 1-3
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Journal Name
TLC R = 0.4 in 5% MeOH/CH
ARTICLE
. 1H NMR (400 MHz,CDCl
, 25 1.71 – 1.30 (7.8H; CH2nb, alk,
&
deg), 1.31 – 1.02 (26.2H; CH2deg, nb,
f
2
Cl
2
3
View Article Online
DOI: 10.1039/C9OB01214A

C): δ = 7.76 (d, J = 7.2Hz, 2H; HArFmoc), 7.58 (d, J = 6.8Hz, 1H; alk CH3deg & solvent), 0.85 (7.7H; CH3alk).
H
Fmoc), 7.45 – 7.27 (4H; ArFmoc), 7.24 – 7.03 (4H; HArFmoc,&Hatt), Polymer 1b: A solution of deoxygenated Grubbs’ third
Cl (4
.05 (3H; CH=CH, & CHFmoc), 5.81 (d, J = 8.8 Hz, 1H; CHFmoc), 4.49 mL) and a solution of monomer 10 (100 mg, 0.42 mmol, 150
4.38 (CH2Fmoc, & O-CH ), 3.80 (s, 3H; O-CH ), 3.73 (s, 3H; O-CH ), equiv), monomer 9 (72 mg, 0.42 mmol, 100 equiv) and
NH), 2.92 – 2.85 (2H; CHnb), 2.01 – 1.94 (m, monomer 8 (100 mg, 0.14 mmol, 50 equiv) in CH Cl were used
6
6
–
3
1
1
1
1
5
.83 (d, J = 8.4Hz, 2H; HAr(att)), 6.51 – 6.42 (m, 2H, HAr(att)), 6.18 – generation initiator (2.4 mg, 0.028 mmol, 1 equiv) in CH
2
2
2
3
3
.52 –2.90 (4H; CH
2
2
2
H; CHnb), 1.90 – 1.83 (m, 1H; CHnb), 1.65 (d, J = 8Hz, 1H; CH2nb), to afford polymer 1b (200mg, 85%). Loading = 0.5mmol/g. l:m:n
.35 – 1.25 (3H; CH2nb), 13C NMR (100 MHz, CDCl
69.6, 160.7, 144.1, 141.5, 138.0, 136.0, 132.3, 129.7, 128.2,
27.8, 127.1, 125.2, 120.1, 114.5, 104.3, 99.6, 67.4, 66.8, 55.62, 7.09 (3.3H; HAr(Fmoc) &HAr(att), 6.84 (2.2H; HAr(att)), 6.47 (2.4H;
5.55, 54.9, 54.9, 50.1, 47.5, 46.3, 44.9, 42.8, 40.0, 39.6, 29.9; Ar(att)), 5.50-5.10 (21.4H; CH=CH), 4.51 - 4.31 (3.8H;
1
3
): δ = 175.4, ratio = 1:3.1:3.2. H NMR (400 MHz, CDCl3, 25 C): δ = 7.75 (2H;
HAr(Fmoc)),7.58 (1.6H; HAr(Fmoc)), 7.43 – 7.34 (2.1H; HFmoc), 7.19 –
H
IR (KBr Pellet): ν = 3327 (br), 3063 (br), 2971 (m), 1660 (s), 1648 CH2(Fmoc),OCH2att), 4.29 – 4.13 (1H; CHFmoc),3.81 – 3.70 (5.6H;
-
1
+
(
C
m), 1537 (s), 1451 (m), 1212 (m) cm ; HRMS (ESI) : calcd for CH
3
O), 3.61 – 2.82 (67.4H; CH2deg, NHatt, CHnb,
(7.9H; CHnb &CH2deg), 2.59 – 1.72 (42.6H; CH2alk, & CHnb), 1.70 –
.34 (25.5H; CH2alk,deg, nb, & solvent), 1.34 – 1.0 (57.5H; CH2nb
&alk), 2.79 – 2.50
+
42
H
43
N
3
NaO
7
(MNa ) 724.2993, found 724.2992.
1
,
General Procedure for polymerization using Grubb’s third CH3deg & solvent), 0.87 (12.3H; CH3alk).
generation initiator
A solution of monomers 8-10 in dichloromethane was Polymer 1c: A solution of deoxygenated Grubbs’ third
deoxygenated using a stream of N gas for 2-3 min. A generation initiator (2.4 mg, 0.028 mmol, 1 equiv) in CH Cl (4
2
2
2
deoxygenated solution of Grubb’s third generation initiator in mL) and a solution of monomer 10 (100 mg, 0.42 mmol, 150
dichloromethane was added to this solution. The reaction equiv), monomer 9 (108 mg, 0.42 mmol, 150 equiv) and
mixture was allowed to stir at room temperature for 45 min. monomer 8 (100 mg, 0.14 mmol, 50 equiv) in CH Cl were used
2 2
Ethylvinyl ether (1 mL) was added and the reaction mixture was to afford polymer 1c in (230mg, 85%). Loading = 0.4 mmol/g. l:
1
allowed to stir for an additional 30 min. The mixture was m:n ratio = 1: 3.2: 4.7. H NMR (400 MHz, CDCl3, 25 C): δ = 7.75
concentrated in vacuo to 1 mL. Diethylether (10 mL) was added (2H;HAr(Fmoc)), 7.58 (1.8H; HAr(Fmoc), 7.51 – 7.42 (2.5H; HAr(Fmoc)),
to precipitate the polymer support. The supernatant was 7.32 – 7.21 (3.3H; HAr(Fmoc)&HAr(att)), 6.83 (2.2H; HAr(att)), HAr(att)),
decanted and the residue was dissolved in dichloromethane (3 6.51 – 6.40 (2H; HAr(att)), 6.09 - 6.00 (2.4H; HAr(att)), 5.48 - 5.05
mL). Re-precipitation with diethyl ether (8 mL) and isolation via (15H; CH=CH), 4.50 – 4.32 (3.5H; CH2Ar(Fmoc) & OCH2(att)), 4.29 -
centrifugation afforded polymer support 1 as a white solid.
4.19 (1H; CHFmoc), 3.82 - 3.59 (5.0H; CH
CH2deg, alk,&solvent), 2.79 –2.50 (6H; CHnb, alk, & CH
3
O), 3.52 - 2.82 (46.1H;
NH), 2.40 –
2
General Procedure for determination of loading capacity and 1.71 (22.7H; CH2deg,& alk), 1.60 – 1.32 (28.8H; CH2alk & solvent),
1
l: m: n ratio of polymers by H NMR Spectroscopy:
1.32 – 1.0 (39.8H; CH3deg, CH2alk,nb &solvent), 0.87 (9.5H; CH3alk).
The number of attachments sites in the polymer was
1
determined by H NMR spectroscopy in presence of a known Polymer 1d: A solution of deoxygenated Grubbs’ third
2 2
amount of a 1,1,2,2-tetrachloroethane (TCE). Integration of the generation initiator (2.4 mg, 0.028 mmol, 1 equiv) in CH Cl (4
peak at δ = 5.95 ppm corresponding to TCE was compared with mL) and a solution of monomer 10 (301 mg, 1 mmol, 450 equiv),
the peak at δ = 7.73 ppm for the Fmoc protons of the monomer 9 (180 mg, 0.71 mmol, 250 equiv) and monomer 8
2 2
attachment site to determine the number of attachment sites (100 mg, 0.14 mmol, 50 equiv) in CH Cl were used to afford
in polymer 10. The l:m:n ratio of monomers in the polymer was polymer 1d in (230mg, 75%). Loading = 0.3 mmol/g. l:m:n ratio
1
determined by the integration of Fmoc protons at the = 1:11.53:7.38. H NMR (400 MHz, CDCl3, 25 C): δ = 7.75
attachment site (δ =7.73 ppm), methyl protons of the alkyl chain (2H;HAr(Fmoc)), 7.58 (1.4H; HAr(Fmoc)), 7.44 – 7.31 (2.8H; HFmoc), 7.2
(
δ =0.85 ppm), and the olefinic protons (δ = 5.27 - 5.16).
– 7.06 (3.5 H; HAr(Fmoc) &HAr(att)), 6.85 (2.3H; HAr(att)), 6.46 (2.6H;
Ar(att)),6.46 (2.5H; HAr), 5.49 - 5.02 (36.6H; CH=CH), 4.50 -4.38
Polymer 1a: A solution of Grubbs’ third generation initiator (2.4 (3.4H; CH2(Fmoc)&CH(Fmoc), 3.82 – 3.70 (5.7H; CH O), 3.69 – 2.89
mg, 0.028 mmol, 1 equiv) in CH Cl (4 mL) and a solution of (109.9H; CHnb, CH2deg, CH NHatt&solvent), 2.66 (12.7H; CHnb
monomer 10 (53 mg, 0.28 mmol, 100 equiv), monomer 9 (29 CH2deg), 2.40 – 1.79 (106.8H; CH2alk, nb, & solvent), 1.70 – 1.32
mg, 0.14 mmol, 50 equiv) and monomer 8 (80 mg, 0.14 mmol, (45.9H; CH2deg solvent), 1.31 1.0 (122.2H;
H
3
2
2
2
&
,
alk
&
–
5
0 equiv) in CH
2
Cl
2
(10 mL) were used to afford polymer 1a (125 CH2nb
,
alk
,deg,CH3deg, &solvent), 0.87 (31.0H; CH3alk)).
1
mg, 92%). Loading = 0.7 mmol/g. l:m:n ratio = 1:2.2:0.7. H NMR
(400 MHz, CDCl3, 25C):δ = 7.74 (2H;HAr(Fmoc), 7.57 (1.6H; General procedure for attachment of first amino acid to
HAr(Fmoc)), 7.41 - 7.21 (2.3H; HAr(Fmoc)), 7.13 (3.1H, HAr(Fmoc)), 6.83 polymer support: Polymer 1a was treated with 20% piperidine
(
(
1.9H, HAr(att)), 6.46 (2.1H, HAr(att)), 6.04 (1.6H, HAr(att)), 5.48 - 5.01 in DMF (1mL) to remove the fmoc group. Subsequently, hexane
8.2H; CH=CH), 4.50 -4.30 (3.3H; CH2(Fmoc),CH(Fmoc(att)), 4.20 (1H; (5 mL) was added to precipitate the support. The precipitate
HFmoc), 3.89 – 3.62 (5.1H; CH
3
O), 3.60 - 2.82 (23.8H; CH2deg, was isolated by centrifugation and dissolved in
CH NHatt, CHnb), 2.63 (2.9H; CHnb), 2.40 – 1.71 ( 19.3H; CH2alk,deg), dichloromethane (1 mL). Diethylether (8 mL) was added to the
2
solution to precipitate the support. The suspension was
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
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O rP gl ea na si ce &d Bo i on mo to al e dc juu l sa tr mC ha er mg i ins ts ry
Page 6 of 9
ARTICLE
Journal Name
centrifuged and the supernatant was decanted. The residue was Compound 11d: Polymer support 1a (15 mg ,0V.i0ew1Amrticmle oOln,lin1e
dried in vacuo to afford the free amine. Fmoc-AA-OH (2 equiv), equiv), Fmoc-ʟ -Val-OH (7.1 mg; 0.02 mDmOIo: 1l,0.2103e9q/uCi9vO),B0H1B21T4UA
HBTU (2equiv) in CH Cl (2-3mL) and DIEA (4 equiv) were added (7.97 mg; 0.02 mmol) and DIEA (7.3 µl, 0.04 mmol) in DCM was
2 2
to the support containing free amine and the reaction was used. The reaction was allowed to stir at room temperature for
allowed to stir for 2 h at room temperature. After completion 2 h to afford compound 11d (10.6 mg, 48%). Loading = 0.34
1
of the reaction, the solvent was concentrated in vacuo to 1 mL. mmol/g. H NMR (400 MHz, CDCl3, 25 C): δ = 7.75 (2H; HAr(Fmoc)),
Diethylether was added to precipitate the amino acid attached 7.61 – 7.50 (2.4H; HAr(Fmoc)), 7.38 (2.2H; HAr(Fmoc) ), 7.09 (2.1H;
support. The supernatant was decanted and the precipitate was
dissolved in dichloromethane (1mL). Re-precipitation with 6.20 (4.4H; HAr(att)), 5.49 – 5.20 (11.2H; CH=CH), 4.49 –4.10
diethylether (8mL) and isolation of the precipitate by (5.5H; ArCH2att, CH2(Fmoc) & CH(Fmoc)), 3.62 - 3.32 (14.4H; CH O,
centrifugation afforded amino acid attached polymer 11a-11l. CH NH), 3.23 - 3.11 (9.1H; solvent), 3.09 – 2.82 (6.9H; CHnb, &
CH2deg), 2.72 - 2.50 (4.3H; CH2deg & CH NH) 2.40 – 2.20 (5.4H;
Serine attached support 11a: Polymer support 1a (30.0 mg CHnb, CH2nb), 2.20 - 1.89 (50.5H; CH2deg, alk & CHval), 1.51 -1.38
0.02 mmol, 1 equiv), Fmoc-ʟ-ser(tbu) (16.1 mg; 0.04 mmol, 2 (49.2H; CH2alk, & solvent), 1.30 – 1.10 (23.3H; CH3deg, val, CH2alk
HFmoc), 6.90 – 6.70 (2.8H; HAr(att)), 6.69 – 6.50 (3H; HAr(att)), 6.49 –
3
2
2
,
,
&
equiv), HBTU (15.9 mg; 0.04 mmol) and DIEA (14.6 µL, 0.08 solvent), 0.86 (9.4H; CH3(alk)).
mmol) in DCM was used. The reaction was allowed to stir at
room temperature for 2h to afford compound 11a (31.5mg, Compound 11e: Polymer 1a (10.0 mg, 0.01 mmol, 1 equiv),
1
6
9%). Loading = 0.49 mmol/g. H NMR (400 MHz, CDCl3, 25C): Fmoc-L-Tyr-OH (9.6mg, 0.02 mmol, 2 equiv), HBTU (7.97 mg;
δ =7.74 (2H;H Fmoc), 7.52 (2.5H; HFmoc), 7.37 (1.6H; Hfmoc), 7.14 - 0.02 mmol) and DIEA (7.3 µl, 0.04 mmol) in DCM was used. The
7
6
.01 (4.5 H; H(Fmoc,&att), 6.89 - 6.68 (4.2H; HFmoc
.30 (4.3H; HAr(att)), 6.23 (2.5H; HAr(att)), 5.49 - 5.13 (15.9H; afford compound 11e (18.2 mg, 94%). Loading 0.64 mmol/g. H
&
Ar(att)), 6.48 – reaction was allowed to stir at room temperature for 2 h to
1
CH=CH), 4.49 – 4.10 (7.9H; CH2Fmoc, CHFmoc, OCH2Ar, CHser&CH2ser), NMR (400 MHz, CDCl3, 25 C): δ = 7.73 (2H; HAr(Fmoc)), 7.60 – 7.42
.60 - 3.33(23.9H; CH O, CHnb), 3.01 – 2.83 (10.7H; CH(nb),CH2alk (2.9H; HAr(Fmoc)), 7.37 (2.7H; HArFmoc), 7.11 – 6.69 (8.3H; HAr(att),
NH), 2.71 – 2.43 (12.7H; CH2deg), 2.39 – 2.20 (14.4H;
3
3
,
&
deg, &CH
CH2alk deg,&nb), 2.20 – 1.50 (113.9H; CH2alk
231.8H; CH2alk
solvent), 0.86 (13.5H; CH3(alk)).
2
H
Ar(Tyr)), 6.51 – 6.12 (9.9H; HAr(att)), 5.49 –5.02 (11.6H; CH = CH),
,
,
deg,&nb), 1.50 – 1.31 4.49 - 4.0 (6.3H; CH2(Tyr), (Fmoc), CHFmoc&Tyr), 3.62 -3.48 (11.8H;
(
,
deg
,
nb & CH3Otbu), 1.31 - 1.21 (55.4H; CH2alk
,
nb,& CH
(4.6H; CHnb), 2.40 – 2.20 (6.2H; CHnb & CH2deg), 2.04 (6.0H; CH2alk
nb), 1.81 - 1.62 (56.8H; CH2alk & deg), 1.50 – 1.30 (108.7H; CH2alk
3 2 ,
O, CH NH & OCH2Ar), 3.10 – 2.80 (9.7H; CHnb & CH2deg), 2.61
&
,
Compound 11b: Polymer support 1a (10 mg ,0.014 mmol, 1 deg & CH3Otbu), 1.30 – 1.0 (31.4H; CH2alk
equiv), Fmoc-ʟ-Arg (Pbf) (8.8 mg; 0.013 mmol, 2 equiv), HBTU (8.4H; CH3(alk)).
, nb, CH3deg & solvent), 0.85
(5.1 mg; 0.013 mmol) and DIEA (4.75 µl, 0.03 mmol) in DCM was
used. The reaction was allowed to stir at room temperature for Compound 11f: Polymer 1a (12.5 mg, 0.008 mmol, 1 equiv),
2
h to afford compound 11b (15.4 mg, 89%). Loading = 0.46 Fmoc-L-Met-OH (6.2 mg, 0.016 mmol, 2 equiv), HBTU (6.3 mg;
1
mmol/g. H NMR (400 MHz, CDCl3, 25 C): δ = 7.83 – 7.63 (2.6H; 0.016 mmol) and DIEA (5.8 µl, 0.033 mmol) in DCM was used.
H
Ar(Fmoc)), 7.62 – 7.40 (2.0H; HAr(Fmoc)), 7.40 – 7.29 (1.6H; The reaction was allowed to stir at room temperature for 2 h to
1
HAr(Fmoc)), 7.19 –7.0 (2.1H; CHAr(Fmoc) & CHAr(att)), 6.78 (2.1H; afford compound 11f (9.3 mg, 50%). Loading 0.34 mmol/g. H
CHAr(att)), 6.42 – 6.20 (3.9H; HAr(att)), 5.44-5.12 (8.3H; CH=CH), NMR (400 MHz, CDCl3, 25 C): δ = 7.73 (2H; HAr(Fmoc)), 7.57 (2.2H;
4
.43 - 4.12 (2.3H; CH2Fmoc
)
,
4.12 – 4.10 (0.7H; CHArg), 3.72 - 3.29
NH), 3.28 – 2.81 (13.4H;
H
H
Ar(Fmoc)), 7.38 (2.3H; HAr(Fmoc)), 7.12 (2.4H; HAr(Fmoc)), 6.83 (3H;
Ar(att)), 6.44 (1.3H; HAr(att)), 6.25 (1.3H; HAr(att)), 5.49 – 5.12 (11H;
(17.6H; CH
3
O, CH2arg, deg, CHFmoc & CH
CH2deg, & CH
.04 (2.5H; CHnb), 2.0 - 1.80 (20.9H; CH2alk, nb
3.4H; CH2alk), 1.49 – 1.32 (20.5H; CH2arg, deg
22.9H; CH2 nb
2
2
NH), 2.70 – 2.40 (5.9H; CHnb), 2.27 (3.3H; CH2deg), CH=CH ), 4.50 –4.13 (5.7H; CH(Fmoc), Met CH2, (Fmoc) & Met), 3.61 -
2
(
(
&
&
deg), 1.75 – 1.50 3.32 (15.7H; CH
alk), 1.30 – 1.12 CH2Met & CH2deg), 2.62 (4.1H; CHnb, & CH2deg), 2.49 – 2.20 (6.5H;
CH2nb & alk), 2.19 –1.71 (33.4H; CH2alk, (deg),(met) & solvent) 1.68 –
.51 (4.2H; CHnb), 1.50 – 1.31 (67.8H; CH2alk, deg, & solvent) 1.30
3 2 2
O, CH NH & OCH2Ar,), 3.09 – 2.79 (7.3H; CH NH,
,
deg, alk, CH3deg, arg & solvent), 0.85 (7.9H; CH3(alk)).
,
1
,
Compound 11c: Polymer support 1a (12.7 mg ,0.009 mmol, 1 – 1.10 (32.4H; CH3met, deg & solvent), 0.86 (7.0H; CH3(alk)).
equiv), Fmoc-L-Phe- OH (6.8 mg; 0.017 mmol, 2 equiv), HBTU
(5.2 mg; 0.017 mmol) and DIEA (6.19 µl, 0.04 mmol) in DCM was Compound 11g: Polymer 1a (14.7 mg, 0.01 mmol, 1 equiv),
used. The reaction was allowed to stir at room temperature for Fmoc-L- Gln (Trt)-OH (12.02 mg, 0.02 mmol, 2 equiv), HBTU (7.4
2
h to afford compound 11c (14.6 mg, 76%). Loading = 0.54 mg; 0.02 mmol) and DIEA (6.8 µl, 0.04 mmol) in DCM was used.
1
mmol/g. H NMR (400 MHz, CDCl3, 25 C): δ = 7.75 (2H; HAr(Fmoc)), The reaction was allowed to stir at room temperature for 2 h to
7
.60 – 7.32 (4.8H; HAr(Fmoc), 7.20 - 6.69 (5.0H; HAr(Fmoc),
&
Phe), 6.38 afford compound 11g (24.2 mg, 90%). Loading = 0.61 mmol/g.
1
(2.1H; HAr), 6.23 – 6.10 (1.8H; HAr(att)), 5.49 - 5.0 (7.1H; CH=CH ),
H NMR (400 MHz, CDCl3, 25 C): δ = 7.73 (2.0H; HAr(Fmoc)), 7.57
4
.50 – 4.09 (4.6H; CH(Fmoc),CH2(Fmoc) & CHPhe), 3.63 – 3.32 (8.8H; (1.8H; (2H; HAr(Fmoc)), 7.28 (2.3H; HAr(Fmoc)), 7.24 – 7.13 (3.6H;
CH3OMe, &CH NH), 3.29 – 2.82 ( 15.6H; CH2deg Phe & solvent) 2.74 Ar(Fmoc) & HAr(att)), 7.11 – 6.90 (0.9H; HAr(att), 6.7(1.7H; HAr(att)), 6.5
2.49 (3.5H; CHnb), 2.49 – 2.20 (4H; CH2deg nb), 2.14 – 1.52 (3.1H; HAr(Trt)), 6.36 (1.3H; HAr(Trt)), 6.19 (0.8H; HAr(Trt)), 5.60 – 5.0
44.3H; CH2deg alk), 1.50 – 1.32 (67.4H; CH2alk, nb & solvent), 1.31 (5.9H; CH=CH ), 4.43 – 4.10 (3.2H; CH(Fmoc), CH2(Fmoc)), 3.60 – 3.42
2
,
H
–
(
&
&
–
1.10 (17.6H; CH2 nb, alk&CH3deg), 0.85 (5.6H; CH3(alk)).
(5.7 H; CH2(Gln)), 3.10 – 2.82 (3.2H; CH2(nb)), 2.70 – 2.50 (1.9H;
6
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
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Page 7 of 9
O rP gl ea na si ce &d Bo i on mo to al e dc juu l sa tr mC ha er mg i ins ts ry
Journal Name
ARTICLE
CHdeg & CH(nb)), 2.28 (2.7H; CH(nb)), 2.10 – 1.70 (17.8H; CHnb
,
7.60–7.42 (1.7H; HAr(Fmoc)),7.40 - 7.30 (5.5H; HAr(Fmoc), &Aratt), 7.21
– 7.10 (6.7H; HArTrt, & att), 7.09 – 6.92 (2.3H; HArTrt), 6.50 – 6.30
(6.8H; HArTrt), 6.20 – 6.08 (1.7H; HArTrt), 5.43 – 5.03 (11.6H;
CH=CH ), 4.41- 4.05 (4H; CHcys, Fmoc & CH2Fmoc), 3.80 – 3.65 (12H;
View Article Online
DOI: 10.1039/C9OB01214A
CH2(alk, deg, & Gln)), 1.50 – 1.02 (65.5H; CH2alk, CH3(deg) & CH2(nb)
solvent), 0.85 (4.7H; CH3(alk)).
&
Compound 11h: Polymer 1a (10.2 mg, 0.007 mmol, 1 equiv), CH2cys, CH
Fmoc-L- Gly-OH (4.0 mg, 0.014 mmol, 2 equiv), HBTU (5.1 mg; 2.61 (5.8H; CHnb), 2.42 – 2.21 (5.8H; CH2deg
.014 mmol) and DIEA (4.7 µl, 0.027 mmol) in DCM was used. (24.4H; CHnb, & CH2deg, alk), 1.52 – 1.31 (82.4H; CH2alk, nb
3
O, & OCH2Ar), 3.64 – 3.83 (43.7H; CH
2
NH, CH2nb&deg),
,
& alk), 2.12 – 1.72
0
&
The reaction was allowed to stir at room temperature for 2 h to solvent), 1.30 – 1.05 (32.6H; CH3deg, CH2nb & solvent)
afford compound 11h (9.2 mg, 65%). Loading = 0.46 mmol/g. H CH3(alk)).
,
0.85 (8.5H;
1
NMR (400 MHz, CDCl3, 25 C): δ = 7.80 – 7.65 (2H; HAr(Fmoc)),
7
,
.62– 7.53 (1.9H; HAr(Fmoc)) 7.41 - 7.32 (2.1H; HAr(Fmoc)), 7.10 Compound 11l: Polymer support 1a (15 mg ,0.01 mmol, 1
(1.9H; HAr(Fmoc)), 6.79 (2.2H; HAr(att)), 6.49 - 6.20 (4H; HAr(att)), 5.42 equiv), Fmoc-L-His (Trt) (13.01 mg; 0.02 mmol, 2 equiv), HBTU
-
5.12 (8.2H; CH=CH ), 4.40 – 4.29 (2.5H; CH(Fmoc), CH2,(Fmoc)), 4.29 (7.97 mg; 0.02 mmol) and DIEA (7.3 µl, 0.042 mmol) in DCM was
-
3 2
4.10 (1.9H; OCH2Ar), 3.51 - 3.49 (8.2H; CH O & CH NH), 3.09 – used. The reaction was allowed to stir at room temperature for
2
2
.82 (5.3H; CH2deg & Gly), 2.72 - 2.50 (4.1H; CH2deg & CHnb), 2.40 – 2 h to afford compound 11l (21 mg, 75%). Loading = 0.46
1
.19 (4.9H; CH(nb), CH2deg), 2.0 – 1.52 (55H; CH2deg, alk, nb
&
mmol/g. H NMR (400 MHz, CDCl3, 25 C):7.73 (2H;HAr(Fmoc)),
solvent), 1.51 – 1.30 (51.5H; CH2alk, nb & solvent), 1.25 (23H; 7.51 (2.6H; HAr(Fmoc)), 7.41 – 7.30 (3.8H; HAr(Fmoc)), 7.12 – 6.93
CH3deg & CH2nb), 0.85 (7.2H; CH3(alk)). (4H; HAr(att)), 6.83 –6.18 (10.9; HAr(att) & Trt), 5.44 - 5.0 (9H; CH=CH),
.52 - 4.49 (1.8H; CHHis, Fmoc), 4.42 – 4.01 (4.3H; CH2Fmoc & His),
Compound 11i: Polymer 1a (10.2 mg, 0.007 mmol, 1 equiv), 3.62 – 3.39 (11.1H; CH2deg, OCH2Ar, & CH
Fmoc-L- Ala-OH (4.2 mg, 0.014 mmol, 2 equiv), HBTU (5.1 mg; CH2nb, & CH NH), 2.75 – 2.50 (4H; CHnb, & CH2deg), 2.42 – 2.10
.014 mmol) and DIEA (4.7 µl, 0.027 mmol) in DCM was used. (8.3H; CHnb, & CH2deg), 2.0 – 1.59 (51.5H; CH2nb alk, & solvent),
The reaction was allowed to stir at room temperature for 2 h to 1.50 – 1.33 (66.3H; CH2alk nb & solvent), 1.30 – 1.05 (CH3deg
4
&
3 ,
O) 3.10 – 2.81 (6.9H;
2
0
,
,
,
afford compound 11i (11.5 mg, 80%). Loading = 0.61 mmol/g. CH2alk & solvent), 0.85 (6.0H; CH3alk).
1
H NMR (400 MHz, CDCl3, 25 C): δ = 7.75 (2H; HAr(Fmoc)), 7.65 –
7
.52 (2.5H; HAr(Fmoc)), 7.43 - 7.34 (2.3H; HAr(Fmoc)), 7.19 - 7.02 General procedure for peptide synthesis
:
The procedure for
(2.9H; HAr(Fmoc) & HAr(att)), 6.91 – 6.70 (3.5H; HAr(att)), 6.49 - attachment of the first amino acid was followed. The process of
6
4
.10(8.9H; CHatt, & solvent), 5.49 - 5.03 (11.8H; CH=CH), 4.50 - deprotection and coupling was repeated until the desired
.09 (7.4H; CH(Ala, Fmoc, CH2Fmoc & OCH2Ar(att)), 3.62 - 3.42 (11.7; peptide sequence was obtained. At the end of synthesis, the
CH
3
O & CH
2
NH), 2.99 (6.4H; CH2(deg) & CH2(nb)), 2.74 – 2.71(5H; peptide was cleaved from the support using a mixture of
CH2deg & CHnb), 2.41 – 1.80 (14.1H; CH2alk, deg, nb), 1.70 – 1.52 95:2.5:2.5 (TFA: TIS: H O). The reaction was allowed to stir at
41.9H;CH2alk,deg & solvent), 1.51 –1.30 (77.7H; CH2alk,deg, room temperature for 2h. After removal of TFA, water was
&
2
(
&
solvent)
CH3(alk)).
,
1.30 – 1.12 (22.9H; CH3deg, Ala & solvent), 0.86 (7.8H; added and the solution was lyophilized to afford the crude
peptide. Purification by semi-preparative HPLC afforded the
desired peptide.
Compound 11j: Polymer 1a (10.2mg, 0.007 mmol, 1 equiv),
Fmoc-L- Trp-(Boc)-OH (7.1 mg, 0.014 mmol, 2 equiv), HBTU (5.1 Characterization of peptides synthesized on support 1
1
mg; 0.014 mmol) and DIEA (4.7 µL, 0.027 mmol) in DCM was Tripeptide AFV 12a: H NMR (500 MHz, D
2
O, 25 °C): δ = 7.37 - 7
used. The reaction was allowed to stir at room temperature for 24 (m, 5H; HAr), 4.62 (app t, J = 8.0 Hz, 1H; CHPhe), 4.16 (q, J =
2
h to afford compound 11j (9.9 mg, 90%). Loading = 0.39 7Hz, 1H; CHAla
)
,
3.75 (d, J = 5.5Hz, 1H; CHVal), 3.16 - 3.03 (17H;
1
mmol/g. H NMR (400 MHz, CDCl3, 25 C): δ = 7.73 (2H; HAr(Fmoc)), CH2(Phe) & solvent), 2.20 - 2.10 (m, 1H; CHVal), 1.25 (d, J = 7.0, Hz,
1
3
7
(
H
4
.60 – 7.30 (5.1H; HAr(Fmoc)), 7.09 - 6.80 (2.5H; HAr(att)), 6.80 – 6.62 3H; CH3Ala
2H; HArTrp), 6.49 – 6.20 (4.4H; HArTrp & att), 6.20 – 6.09 (1.5H; C): 176.8, 172.1, 169.0, 135.8, 129.2, 128.9, 127.4, 58.2, 55.1,
ArTrp), 5.49 – 5.09 (6.8H; CH=CH ), 4.73 - 4.50 (2.1H; CHFmoc & Trp), 49.2, 44.5, 36.8, 22.2, 21.4, 17.5, 16.7, 16.6; IR (KBr Pellet): ν =
.49 – 4.21 (2.7H; CH2Fmoc), 3.60 – 3.32 (9.3H; CH 3854 (br), 3732 (br), 3293 (br), 2925 (m), 2857 (w), 2589 (w),
, 2
) 0.98 – 0.93 (6H; 2CH3Val); C NMR (125MHz, D O, 25
3
O, OCH2Ar, &
-1
2
CH NH), 3.09 – 2.79 (4.9H; CH2Trp & CHnb), 2.70 – 2.50 (2.8H; 1669 (s), 1645 (s), 1202 (w), 1134, (w), 1031 (m), 837 (w) cm ;
+
+
CHnb), 2.39 – 2.19 (3.2H; CH2deg), 2.18 – 1.80 (4.8H; CH2deg
.75 – 1.52 (45.3H; CH2alk, nb & solvent), 1.51 – 1.33 (38.1H; 357.1895.
CH2alk, CH3Boc & CHnb), 1.30 – 1.1 (17.4H; CH3deg, CH2nb & solvent)
.85 (6.2H; CH3(alk)).
26 4 3
&alk), HRMS (ESI ): calcd. for C17H N NaO (MH ) 357.1897, found
1
,
1
0
Tripeptide GCF 12b: H NMR (500 MHz, D
.26 (m, 5.0H; HAr), 4.64 (dd, J = 9Hz, 6.5Hz, 1H; CHCys), 4.49 (app
Compound 11k: Polymer 1a (10.2 mg, 0.007 mmol, 1 equiv), t, J = 6.5Hz, 1H; CHPhe), 3.84 (d, J = 4.5 Hz, 2H; CH2Gly), 3.14 (appt,
2
O, 25 C): δ = 7.41 -
7
1
3
Fmoc-L-Cys-(Trt)-OH (7.1 mg, 0.014 mmol, 2 equiv), HBTU (5.1 J = 9Hz,2H; CHHCys), 3.05- 2.89 (2H; CHHPhe); C NMR (125 MHz,
mg; 0.014 mmol) and DIEA (4.7µl, 0.027 mmol) in DCM was O, 25 C): 171.2, 163.1, 162.9, 136.5, 129.4, 129.3, 128.8,
used. The reaction was allowed to stir at room temperature for 128.7, 127.2, 54.7, 44.6, 44.5, 40.4, 37.1, 22.3, 22.2, 21.5, 21.4;
D
2
2
h to afford compound 11k (12.6 mg, 73%). Loading = 0.49 IR (KBr Pellet): ν = 3773 (br), 3394 (br), 2513 (s), 1645 (s), 1448
1
mmol/g. H NMR (400 MHz, CDCl3, 25 C): δ = 7.73 (2H; HArFmoc), (m), 1305 (w), 1258 (w), 1202 (m), 1148 (m), 810 (w); HRMS
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
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O rP gl ea na si ce &d Bo i on mo to al e dc juu l sa tr mC ha er mg i ins ts ry
Page 8 of 9
ARTICLE
Journal Name
S (MH ) 347.1148, found (m), 2311 (m), 1664 (s), 1425 (s), 1259 (w), 1202 (s), 1137 (w),
+
+
(
ESI ): calcd. for C14
H
20
N
4
NaO
3
View Article Online
DOI: 10.1039/C9OB01214A
-1
+
3
47.1147.
1020 (m), 704 (w) cm ; HRMS (ESI ): calcd. for C40
H
58 9 8
N
O
+
(MH ) 793.4403, found 793.4405.
1
Tripeptide FHL 12c: H NMR (500 MHz, D
2
O, 25 C): δ = 8.60
+
(1.0H; HHis), 7.40 - 7.34 (3.0H; HPhe & His
)
,
7.26 - 7.24, (3.0H; HPhe & Octapeptide GCPWQPYC 12g: HRMS (ESI ): calcd. for
+
His), 4.64(app. t, J = 7Hz, 1.0H; CH(His)), 4.28-4.22 (2H; CH(Phe)
&
C
43
H N O S
48 11 10 2
for (MH ) 952.3804 found 952.3808.
CH(Leu)),
.
3.23- 3.15 (6H; 2CHHHis, 2CHHHPhe, & solvent), 1.80 -
+
1
6
2
1
2
2
.73 (m, 2H; 2CHHH(Leu)
Hz, 3H; CH3(Leu)); 0.92 (d, J = 6Hz, 3H; CH3(Leu)); CNMR (125Hz,
5 C): 176.8, 170.2, 168.7, 133.6, 133.5, 129.2, 129.1, 128.0,
27.7, 117.4, 54.1, 52.3, 52.1, 44.5, 39.8, 36.8, 26.7, 22.2, 22.0, Nonapeptide CCPPALWCC 12i: HRMS (ESI ): calcd. for
1.4, 20.9; IR (KBr Pellet): ν = 3840 (br), 3732 (br), 3447 (br),
)
,
1.60- 1.57 (m, 1H; CH(Leu)
,
) 0.95 (d, J = Nonapeptide TCFGCTPCC 12h: HRMS (ESI ): calcd. for
1
3
+
C
36
H
57
N
10
O
11
S
4
for (MH ) 933.2978 found 933.2971.
+
+
C
42
H
64
N
11
O
9
S
4
for (MH ) 994.3629 found 994.3627.
955 (m), 2864 (w), 2530 (w), 1679 (s), 1601 (w), 1578 (w), 1236,
-1
+
+
(w) 1202 (s), 1135 (m), 1028 (w) cm ; HRMS (ESI ): calcd. for Decapeptide WFTTLISTIM 12j: HRMS (ESI ): calcd. for
+
+
21 31
C H N
6
O
3
(MH ) 415.2452, found 415.2458.
C
58
H
91
N
12
O
14S for (MH ) 1211.6493 found 1211.6494.
1
+
Tetrapeptide AFIA 12d: H NMR (500 MHz, D
2
O, 25 C): δ = Hexadecapeptide GCCGAFACRFGCTPCC 12k: LRMS (ESI ) was
7
.39- 7.26 (m, 5H; HAr), 4.63 (app. t, J = 7.5Hz, 1H; CHPhe), 4.22 found to be 1597.4806
q, J = 7.5Hz, 1.0 H; CHAla 4.16 (d, J = 8.0 Hz, 1H; CHIle), 4.07 (q,
J = 7.0 Hz, 1H; CHAla), 3.13 -3.00 (3H; 2CHHPhe & CHH Ile(a)), 1.82-
(
)
,
Conflicts of interest
There are no conflicts to declare.
1
1
0
1
4
.73 (m, 1H; CHHIle(b)), 1.44 (d, J = 7.0 Hz, 3H; CH3(Ala)), 1.36 –
.29 (5H; CH3(Ala) & solvent), 1.19- 1.09 (m, 1H; CHIle(b)), 0.89 -
1
3
2
.79 (6H; 2CH3(Ile)); C NMR (125 MHz, D O, 25 C): 177.0, 172.9,
72.3, 170.6, 168.7, 136.1, 129.2, 128.8, 127.3, 58.3, 54.8, 49.2,
8.8, 37.0, 36.2, 24.5, 16.7, 14.6, 10.0; IR (KBr pellet):  = 3854 Acknowledgements
(br), 3747 (br), 3421 (br), 3282 (br), 2964 (w), 2925 (w), 1678
This research was supported by CSIR (No. 01(2333)/09/EMR-II),
DST-SERB (EMR/2016/007672), New Delhi, India. B. B.
acknowledges IIT Bombay and IIT Madras for her research
fellowship.
-
(
s), 1543 (m), 1440 (s), 1262 (w), 1206 (w), 1134 (s), 803 (w) cm
;
1
+
+
HRMS (ESI ): calcd. for C21
H N O
34 5 4
(MH ) 420.2605, found
4
20.2604.
1
Octapeptide AFWMHYAF 12e: H NMR (500 MHz, D
δ = 7.60 (d, J = 9.5Hz, 2H; CHHis), 7.41 (d, J = 10Hz, 2H;
ArTyr),7.36- 7.11 (18H; HAr), 4.56 – 4.51 (2H; CHTrp His), 4.47 –
.42 ( 2H; CH(Tyr &Met)), 4.36 – 4.30(2H; CHAla 4.16 – 4.12 (2H;
CHPhe), 4.02 (dd, J = 3.23 -3.16 (4H; CHHPhe & CH 3.30 -
2
O, 25 C):
Notes and references
H
4
&
1
2
3
.
.
.
M. Góngora-Benítez, J. Tulla-Puche and F. Albericio, Chem.
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M. W. Pennington, A. Czerwinski and R. S. Norton, Bioorg.
Med. Chem., 2018, 26, 2738-2758.
)
,
a
CHb(Phe)
CHb(Phe), Trp, His, Tyr & Met)), 1.35 - 1.99 (9H;
CH3(Ala), CH3(Met)); C NMR (125 MHz, D
)
,
2
2
.8 (10H; CH2Met, CH
a
1
3
2
O, 25 C): 177.2, 173.4,
72.7, 171.7, 168.6, 165.0, 163.2, 162.3, 136.1, 135.9, 133.5,
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1
2
7.
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1
4
29.3, 129.3, 129.0, 128.7, 127.9, 127.2, 126.9, 124.5, 122.0, 4.
19.4, 118.1, 117.8, 114.9,111.9, 108.5, 54.6, 54.4, 54.0, 49.3, 5.
4.5, 37.1, 37.0, 36.7, 31.4, 30.2, 27.0, 22.2, 21.5, 16.6; IR (KBr
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pellet):  = 3903, (br), 3772, (br), 3455, (br) 3281, (br), 2918, (s),
2
513 (w), 2308 (m), 1668 (s), 1440 (w), 1202 (w), 1137 (w), 1042
-1
+
(m), 801 (w), 700 (w) cm ; HRMS (ESI ): calcd. For C55
67 12 9
H N O S
8
.
+
(MH ) 1071.4869, found 1071.4866.
Octapeptide AFAIAPGF 12f: (500 MHz, D
7
1
2
O, 25 C): δ = 7.45 -
.25 (10H; HAr), 4.69 – 4.65 (m, 1H; CHPhe), 4.63 - 4.59 (m,
H;CHPhe), 4.37 – 4.31 (m, 1H; CHPro), 4.34 (q, J = 7.0 Hz, 1H;
9
.
CHAla), 4.10 (d, J = 8Hz, 1H CHIle), 4.13 – 4.10 (2H; CH2(Gly)), 4.07 10.
4.0 (m, 1H; CHala), 3.95 - 3.90 (m, 1H; CHala), 3.88 - 3.79 (2H;
CH2(Pro)), 3.78 - 3.65 (4H; CH2Pro & impurity), 3.24 - 3.12 (m, 2H; 11.
–
CH2Phe), 3.11 – 3.0 (2H; CH2Phe), 2.32 -2.26 (m, 1H; CHaHb(Pro)),
1
1
1
2.
3.
4.
2
1
=
.10 - 1.98 (2H; CH2(Ile)), 1.95 – 1.86 (m, 1H; CHaHb(Pro)), 1.87 –
.78 (m, 1H; CHaHb(Pro)), 1.37 (d, J = 7Hz, 3H; CH3(ala)), 1.34 (d, J
7Hz, 3H; CH3(Ala)), 1.25 - 1.17 (m, 1H; CHIle), 0.98 - 0.84 (6H;
CH3(Ile)); IR (KBr pellet):  = 3905 (br), 3783 (br), 3269 (br), 2922
8
| J. Name., 2012, 00, 1-3
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