Efficient solid-phase synthesis of cyclic RGD peptides under controlled microwave heating

Article abstract of DOI:10.1016/j.tetlet.2011.12.069

Cyclic RGD peptides are potent antagonists for the α _vβ₃ integrin receptor. In this Letter, microwave-assisted solid-phase synthesis of cyclic RGD peptides is described. In a coupling reaction between Fmoc-Arg(Pbf)-OH and high-loadin

Full text of DOI:10.1016/j.tetlet.2011.12.069

Tetrahedron Letters 53 (2012) 1066–1070
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Tetrahedron Letters
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Efficient solid-phase synthesis of cyclic RGD peptides under controlled
microwave heating
Keiichi Yamada a,b, , Izuru Nagashima b, Masakazu Hachisu b, , Ichiro Matsuo a, Hiroki Shimizu b,
⇑
⇑
a Department of Chemistry and Chemical Biology, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan
b Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo 062-8517, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Cyclic RGD peptides are potent antagonists for the
a
vb3 integrin receptor. In this Letter, microwave-
Received 21 November 2011
Accepted 16 December 2011
Available online 23 December 2011
assisted solid-phase synthesis of cyclic RGD peptides is described. In a coupling reaction between
Fmoc-Arg(Pbf)-OH and high-loading H-Gly-Trt(2-Cl) resin, multiple coupling reactions were required
for completion under the conventional HBTU activation. We found that the use of COMU, a new coupling
reagent, under microwave heating to 50 °C accelerated the reaction even inside the resin. This method
was applicable to the synthesis of linear pentapeptides, H-Asp(OtBu)-Xxx-Yyy-Arg(Pbf)-Gly-OH
Keywords:
Microwave-assisted synthesis
Solid-phase peptide synthesis
Cyclic RGD peptides
COMU
(Xxx =
D-Phe(p-Br) or D-Tyr, Yyy = Lys(Boc) or MeVal). Cyclization of these peptides followed by deprotec-
tion gave the desired cyclic RGD peptides with high purity.
Ó 2011 Elsevier Ltd. All rights reserved.
Cyclic RGD peptides are potent antagonists for the avb3 integrin
NH
receptor.1 To date, various cyclic RGD peptides conjugated to a
radioactive tracer or fluorophore have been developed for the
O
H2N
N
H
O
O
N
H
vb3 receptor-overexpressed tumors.2,3
O
NH
NH
non-invasive imaging of
a
HN
O
Our research interest has been aimed to develop new radiohalo-
gen-labeled cyclic RGD peptides for positron emission tomography
(PET) imaging of tumor in vivo. For this purpose, we were pursuing
H
N
OH
O
an efficient synthetic protocol for 1, cyclo(Asp-D-Phe(p-Br)-Lys-
H2N
Arg-Gly (Fig. 1), an useful precursor and non-radioactive standard
of radiobromine-labeled PET tracer. According to the improved
synthetic protocol for cyclic RGD peptides,4 side-chain protected
linear peptides could be prepared using acid-labile 2-chlorotrityl
resin (Trt(2-Cl)-resin). In our attempt for synthesizing a linear
RGD peptide using commercially available high-loading H-Gly-
Br
Figure 1. Structure of 1.
Trt(2-Cl)-resin (100–200 mesh, substitution: 0.74 mmol/g resin),
we were suffering from the contamination of many impurities in
the desired peptide. (RP-HPLC chromatogram of the crude of linear
RGD peptide was shown in Figure S1 in the Supplementary data.) It
is well known that conjugation of Arg-containing peptides is diffi-
cult due to the steric hindrance of Pbf in a side-chain as a protect-
ing group, resulting in low peptide yield or even synthesis failure.5
Exactly, we found that triple coupling reactions between Fmoc-
Arg(Pbf)-OH and the resin were required for completion of the
reaction when using the conventional uronium-based reagent
HBTU or the more potent coupling reagent HATU. Although micro-
wave (MW) heating is an efficient tool for accelerating coupling
reactions in solid-phase peptide synthesis (SPPS),6–15 activation
Abbreviations: Trt(2-Cl)-resin, 2-chlorotrityl resin; Pbf, 2,2,4,6,7-pentame-
thyldihydrobenzofuran-5-sulfonyl; PET, positoron-emission tomography; RP-HPLC,
reversed-phase high performance liquid chromatography; HATU, 2-(1H-7-aza-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HBTU, 2-
(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HOBt,
1-hydroxy-benzotriazole; COMU, (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)
dimethylamino-morpholinocarbenium hexafluorophosphate; MW, microwave;
a
SPPS, solid-phase peptide synthesis; MeVal, N -methyl-
L-valine; Boc, tert-butoxy-
carbonyl;
Fmoc,
9-fluorenylmethyloxycarbonyl;
tBu, tert-butyl; TIS,
triisopropylsilane.
⇑
Corresponding authors. Tel./fax: +81 277 30 1345 (K.Y.); tel.: +81 11 857 8497;
fax: +81 11 857 8435 (H.S.).
E-mail addresses: kyamada@gunma-u.ac.jp (K. Yamada), hiroki.shimizu@
aist.go.jp (H. Shimizu).
Current address: ERATO, Japan Science and Technology Agency, 2-1 Hirosawa,
Wako, Saitama 351-0198, Japan.
of Fmoc-Arg(Pbf)-OH under MW irradiation causes c-lactam for-
mation.5a To avoid this side reaction, Fmoc-Arg(Pbf)-OH should
be activated under mild conditions, that is, low power of MW
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.12.069

K. Yamada et al. / Tetrahedron Letters 53 (2012) 1066–1070
1067
up to a 95% yield.7b Therefore, the coupling reaction in this study
was conducted under the same condition. H-Gly-Trt(2-Cl) resin
(50 mg, substitution: 0.74 mmol/g) was swollen in DMF for over
2 h before use. Each Fmoc-amino acid (0.185 mmol, 5 equiv) was
coupled by a coupling reagent (0.185 mmol, 5 equiv) and DIEA
(0.37 mmol, 10 equiv) in DMF with MW heating (50 °C, 10 min).
The Fmoc group was removed using 20% piperidine in DMF with
MW heating (50 °C, 3 min). Each coupling and deprotection was
conducted only once except Fmoc-Arg(Pbf)-OH and was monitored
by chloranil test.17 Following completion of the sequence, the linear
pentapeptide was cleaved from resin by the treatment with AcOH/
TFE/DCM = 1:1:4 (v/v/v) for 90 min at room temperature. The solu-
tion was filtered and concentrated in vacuo, followed by lyophiliza-
tion to afford the crude product of the protected linear precursor 2,
O
O
NC
X
OEt
N
N
N
N
N
PF6
O
N
PF6
N
N
HBTU (X=C)
HATU (X=N)
COMU
Figure 2. Structure of COMU, HBTU and HATU.
irradiation at room temperature.5b,c Very recently, a novel coupling
reagent COMU (Fig. 2) was shown to be more effective than either
HBTU or HATU for MW-assisted SPPS of hindered peptides.16 Fur-
thermore, COMU had advantage that it was free from significant
side reactions such as N-terminal capping,16b leading to reduce
the by-products and so on. Therefore, we considered to evaluate
the performance of COMU in MW-assisted SPPS of Arg-containing
peptides. In this paper, we describe the synthesis of cyclic RGD
peptides using COMU under controlled MW heating.
Scheme 1 shows MW-assisted SPPS of 1. According to our previ-
ous studies on MW-assisted SPPS of an acetylated Core2 glycoside-
dipeptide conjugation, coupling reactions proceeded smoothly
under MW irradiation only at 50 °C for 10 min to afford a product
H-Asp(OtBu)-D-Phe(p-Br)-Lys(Boc)-Arg(Pbf)-Gly-OH. For compari-
son, the synthesis using HBTU as the coupling reagent was
conducted similarly. The yields and purity of 2 after chromato-
graphic purification are shown in Table 1. Reversed-HPLC analysis
of the crude product shown in Figure 3 indicated that some minor
by-products were formed but not quantified. On contrary, MW
irradiation made the amount of the by-products reduced as to com-
pare to the reaction without MW irradiation. Especially, the synthe-
sis using COMU afforded 2 in good yield and purity. When using
HBTU under MW irradiation, a major by-product was observed
(tR = 4.7 min in RP-HPLC), which was estimated to be the deletion
Fmoc-Arg(Pbf)-OH
Pbf
HN
Pbf
(5 equiv)
NH
O
1) 20% piperidine/DMF
MW(+), 50 °C, 3 min
HN
COMU (5 equiv)
O
NH
HN
O
N
H
DIEA (10 equiv)
N
O
O
H
O
NH
NH
O
H2N
N
H
2) Fmoc-Lys(Boc)-OH
(5 equiv)
O
DMF, MW(+),
50°C, 10 min
(Double coupling)
NH
O
O
Fmoc
COMU (5 equiv)
DIEA (10 equiv)
DMF, MW(+),
Fmoc
0.037 mmol
Boc
N
H
50 °C, 10 min
Pbf
HN
NH
O
Pbf
NH
O
1) 20% piperidine/DMF
MW(+), 50 °C, 3 min
O
1) 20% piperidine/DMF
MW(+), 50 °C, 3 min
HN
N
H
N
N
H
N
H
H
O
NH
O
2) Fmoc-Asp(OtBu)-OH
O
NH
NH
O
2) Fmoc-D-Phe(p-Br)-OH
(5 equiv)
Fmoc
(5 equiv)
NH
HN
H
N
COMU (5 equiv)
DIEA (10 equiv)
DMF, MW(+),
H
N
COMU (5 equiv)
DIEA (10 equiv)
DMF, MW(+),
O
O
Fmoc
Boc
NH
O
N
H
O
OtBu
Boc
N
H
50 oC, 10 min
50 °C, 10 min
Br
Br
Pbf
HN
Pbf
NH
O
O
N
H
N
H
OH
PyBOP (1.2 equiv.)
DIEA (2.4 equiv.)
N
H
O
1) 20% piperidine/DMF
MW(+), 50 °C, 3 min
N
H
N
H
NH
NH
O
O
NH
NH
O
HN
O
H
DCM (conc. <1 mM)
0°C to r.t., 22 h
2) AcOH/TFE/DCM
r.t., 90 min
NH2
N
H
N
OtBu
O
O
O
Boc
O
Boc
N
H
O
OtBu
N
H
Br
3 (87%)
2 (84%)
NH
Br
O
H2N
N
H
O
N
H
TFA:TIS:H2O
NH
O
=92.5:2.5:2.5 (v/v/v)
HN
H
NH
O
N
r.t., 2 h
OH
O
O
H2N
Br
1 (85%)
Scheme 1. MW-SPPS of the linear and cyclic RGD peptides (1–3).

1068
K. Yamada et al. / Tetrahedron Letters 53 (2012) 1066–1070
high dilution in DCM18 ([2]: <1 mM) afforded the desired fully-
protected cyclic RGD peptide cyclo(Asp(OtBu)- -Phe(p-Br)-Lys
Table 1
Isolated yield and purity of 2 after chromatographic purificationa
D
(Boc)-Arg(Pbf)-Gly) (3) in a good yield. Characterization of 3 by
MALDI-TOF-MS and RP-HPLC (Fig. 4) showed the product to be
highly pure, with no detectable dimerized side-product, cyclo
COMU
HBTU
MW(À) (rt, 30 min)a
73% (>95%)b
84% (>95%)b
69% (>95%)b
39% (>95%)b
MW(+) (50 °C, 10 min)a
(Asp(OtBu)-D-Phe(p-Br)-Lys(Boc)-Arg(Pbf)-Gly)2. Final deprotec-
a
H-Gly-(Cl-Trt)-resin (50 mg, substitution: 0.74 mmol/g resin) was used. Cou-
tion by TFA-triisopropylsilane (TIS)-H2O afforded 1 in an 85% yield.19
High-yielding synthesis of the linear RGD peptide 2 was accom-
plished by the combination of MW irradiation and COMU. In order
to evaluate the effectiveness of the activation of Fmoc-Arg(Pbf)-OH
by COMU, microscopic images of the resin beads after the coupling
between Fmoc-Arg(Pbf)-OH and H-Gly-Trt(2-Cl) resin was
observed (Fig. 5). Aliquots of the beads were stained with 2%
chloranil/acetaldehyde solution in DMF to detect amino groups.
Single coupling of Fmoc-Arg(Pbf)-OH using HBTU or COMU
(5 equiv) without MW irradiation (MW(À)) was insufficient
(Fig. 5: upper panel)., and double coupling with HBTU with MW
irradiation (MW(+)) provided some improvement but was
pling reaction was carried out using Fmoc-AA (5 equiv), coupling reagent (5 equiv)
and DIEA (10 equiv).
The value in the parentheses indicates the purity of 2 estimated by RP-HPLC
peak area%, UV absorbance at 220 nm.
b
peptide as H-D-Phe(p-Br)-Lys(Boc)-Arg(Pbf)-Gly-OH by spectro-
scopic analyses (ESI-MS: m/z 939.0 [M+H]+, 1H NMR spectra of 2
and the by-product were shown in Figure S2 in Supplementary
data). Subsequently, isolated yield of 2 was significantly decreased
(39%).
The linear pentapeptide 2 was subjected to head-to-tail cycliza-
tion in solution. The reaction using PyBOP under the conditions of
Figure 3. Comparison of reversed-phase HPLC profiles between the crude products obtained using COMU and HBTU. The peak marked by asterisk corresponds to the desired
linear pentapeptide 2. Left panel: using COMU; right panel: using HBTU. The following conditions were applied; column: YMC-pack Pro C18 (4.6 Â 150 mm); eluent: 40–70%
MeCN aq containing 0.1% TFA (30 min analysis); flow rate: 1 mL/min; detection, k 220 nm.
Figure 4. Analysis of the crude cyclic peptide 3. Left panel: comparison of reversed-phase HPLC profiles between 3 and its linear peptide, 2. Following conditions were
applied; Column: Inertsil ODS-3 (4.6 Â 250 mm), eluent: 40–70% MeCN aq containing 0.1% TFA (30 min analysis), flow rate: 1 mL/min, detection: k 220 nm. Right panel:
MALDI-TOF-MS spectrum, calcd. for C49H72BrN9O12S 1089.42, mass found m/z 1090.2 [M+H]+, 1112.2 [M+Na]+, 1128.2 [M+K]+.

K. Yamada et al. / Tetrahedron Letters 53 (2012) 1066–1070
1069
Figure 5. Microscopic images of the resin beads after coupling of Fmoc-Arg(Pbf)-OH to H-Gly-Trt(2-Cl) resin. Upper panel: coupling without MW irradiation (MW(À)) using
HBTU (left) and COMU (right); lower panel: coupling with MW irradiation (MW(+)) using HBTU (left) and COMU (right). The resin beads were stained by chloranil/
acetaldehyde in DMF for 2 min.
inadequate for complete acylation (Fig. 5: left and lower panels). In
contrast, the use of COMU (5 equiv) under MW irradiation was
more effective than HBTU even in a single coupling (Fig. 5: right
and lower panels), and double coupling resulted essentially to
completion (Fig. 5: right and lower panels). In the case of incom-
plete acylation, microscopic images of the beads showed staining
of the inside of the resin but not the surface (Fig. 5), which sug-
gested that the incomplete acylation between Fmoc-Arg(Pbf)-OH
and the resin was mainly due to insufficient permeation of the acti-
vated species into the resin beads.
Although the effectiveness of MW irradiation has not been com-
prehensively understood at present, we conjecture the MW effect
for this improvement of poor resin penetration of the activated spe-
cies as follow. As our previous report of a MW-assisted glycosyla-
tion at low temperature,20 Shimizu et al. found that MW
irradiation was effective for glycosylation reactions at low temper-
ature (À10 °C) to afford oligosaccharides though no MW irradiation
at the same temperature had led only to side reactions, and which
indicated that MW plays a more significant roles than heating. They
considered that a glycosyl acceptor might exist as clusters such as
micelle formation and therefore reactive hydroxyl groups on the
acceptor would be hidden inside the cluster.21 As a result, an acti-
vated glycosyl donor could not react with the hydroxyl groups. In
our case, Fmoc-Arg(Pbf) molecules might also exist as micelle-like
clusters due to the bulky and hydrophobic side chain protecting
group (Pbf) and Fmoc group. The clustered molecules would be
too large to permeate into the resin beads under the normal condi-
tion but were broken by MW irradiation, enabling the activated
Fmoc-Arg(Pbf) molecules to permeate and react with the amino
groups in the beads. The efficacy of COMU would be due to in-
creased resin permeability of the activated species derived from
Fmoc-Arg(Pbf)-OH and COMU. Little is known about MW irradia-
tion effects on the diffusion of organic molecules in cross-linked
polystyrene, but MW irradiation may change the swelling charac-
teristics of the beads, allowing the COMU-activated Arg(Pbf) mole-
cules to permeate the beads effectively. Another several
experiments will be required to confirm this hypothesis, that is,
studying the coupling between Fmoc-Arg(Pbf)-OH and resins with
excellent swelling properties under MW irradiation will provide
useful information.
To address further usefulness and limitation of the MW/COMU
method for synthesizing cyclic RGD peptides, we synthesized
side-chain protected derivative of N-methylated cyclic peptide
cilengetide (4, cyclo(Asp(OtBu)-D
-Tyr(tBu)-MeVal-Arg(Pbf)-Gly)).1d
In general, acylation to N-terminal MeVal residue resulted in a low
yield, which is mainly due to its steric hindrance.22 In this study,
coupling reactions using HATU were also conducted to compare
the reactivity of COMU with HATU. The synthetic protocol for 4
is shown in Figure 6. The reaction between Fmoc-Arg(Pbf)-OH
and H-Gly-Trt(2-Cl) resin by COMU was completed after double
coupling. On contrary, triple coupling reactions were required for
completion when using HATU. MeVal residue was successfully
introduced by a single coupling. The coupling of Fmoc-D-Tyr
(tBu)-OH, however, required triple coupling, indicating that both
COMU and HATU activations under MW irradiation did not
significantly accelerate the acylation to MeVal residue. After the
a
b
COMU x 1
or
HATU x 1
COMU x 1
or
HATU x 1
NH
O
HN
Pbf
N
H
O
O
N
NH
H
O
HN
O
Arg(Pbf) Gly
D-Tyr(tBu)
MeVal
Asp(OtBu)
H
N
O
N
OtBu
COMU x 2
HATU x 1
COMU x 2
or
HATU x 3
or
OtBu
HATU x 3
Figure 6. (a) Structure of 4, (b) graphical synthesis outline for H-Asp(OtBu)-D-Tyr(OtBu)-MeVal-Arg(Pbf)-Gly-OH (5). Each coupling reaction was carried out under MW
irradiation at 50 °C for 10 min.

1070
K. Yamada et al. / Tetrahedron Letters 53 (2012) 1066–1070
5. (a) White, P.; Collins, J.; Cox, Z. In Poster presentation at 19th American peptide
Symposium, San Diego, CA, USA, 2005.; (b) Friligou, I.; Papadimitriou, E.; Gatos,
D.; Matsoukas, J.; Tselios, T. Amino acids 2011, 40, 1431–1440; (c) Rizzolo, F.;
Testa, C.; Lambardi, D.; Chorev, M.; Chelli, M.; Rovero, P.; Papini, A. M. J. Pept.
Sci. 2011, 17, 708–714.
elongation was completed, the peptide was cleaved by the same
procedure described above (AcOH/TFE/DCM = 1:1:4 (v/v/v) for
90 min at room temperature) to afford H-Asp(OtBu)-D-Tyr(OtBu)-
MeVal-Arg(Pbf)-Gly-OH (5). RP-HPLC analysis revealed that the
syntheses by both protocols did not form any significant impuri-
ties. Finally, cyclization of 5 by PyBOP in DMF afforded 4 in a good
yield (see Fig. S3 in the Supplementary data).
In summary, we accomplished the efficient solid-phase synthe-
sis of cyclic RGD peptides by utilizing COMU under controlled MW
heating. We demonstrate that the use of COMU is effective for the
coupling of Fmoc-Arg(Pbf)-OH under MW irradiation at 50 °C for
10 min. In general, coupling reaction in SPPS is monitored by color-
ation of the beads using the amine-reactive dyes such as ninhydrin
and chloranil. In our study, microscopic analysis of the stained
beads enabled us to evaluate the MW effect for the coupling reac-
tion. The purity of the crude pentapeptide was over 90%, and no
significant side-reactions or amino acid residue deletions were ob-
served. Our method should greatly contribute to the development
of RGD-based pharmaceutics and molecular probes for detecting
6. Kappe, C. O.; Stadler, A. Microwaves in Organic and Medicinal Chemistry; Wiley-
VCH, 2005.
7. (a) Matsushita, T.; Hinou, H.; Kurogochi, M.; Shimizu, H.; Nishimura, S.-I. Org.
Lett. 2005, 7, 877–880; (b) Matsushita, T.; Hinou, H.; Fumoto, M.; Kurogochi,
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9. Bacsa, B.; Desai, B.; Dibó, G.; Kappe, C. O. J. Pept. Sci. 2006, 12, 633–638.
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808–817.
12. Bacsa, B.; Horváti, K.; Bõsze, S.; Andreae, F.; Kappe, C. O. J. Org. Chem. 2008, 73,
7532–7542.
13. Katrizky, A. R.; Haase, D. N.; Johnson, J. V.; Chung, A. J. Org. Chem. 2009, 74,
2028–2032.
14. Pedersen, S. L.; Sørensen, K. K.; Jensen, K. J. Biopolymers (Pept. Sci.) 2009, 94,
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avb3 integrin-overexpressed tumors in vivo.
17. Vojkovsky, T. Pept. Res. 1995, 8, 236–237.
18. Procedure for cyclization: To a stirred solution of PyBOP (6.2 mg, 0.022 mmol)
Supplementary data
and DIEA (6.1 lL, 0.036 mmol) in DCM (20 mL) at 0 °C was added 1 (20 mg,
0.018 mmol) in DCM (4 mL) dropwise. The reaction mixture was stirred for 1 h.
The solvent was evaporated off and the residue was lyophilized to afford 2.
(17 mg, 87% yield).
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2011.12.069.
19. Procedure for the final deprotection: 2 (6.2 mg, 0.0057 mmol) was dissolved
in TFA-TIS-H2O (95:2.5:2.5 (v/v/v), 5 mL) and the solution was placed at room
temperature for 2 h. The solution was concentrated in vacuo and the residue
was lyophilized to afford the crude. Gel filtration chromatography (Sephadex
LH-20, DMF) gave the desired cyclic RGD peptide 3 as a TFA salt (3.9 mg, 85%
yield). ESI-MS: calcd. for C27H41BrN9O7 682.2, mass found m/z 681.9 [M+H]+.
20. Shimizu, H.; Yoshimura, Y.; Hinou, H.; Nishimura, S-. I. Tetrahedron 2008, 64,
10091–10096.
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