New strategy towards the efficient solid phase synthesis of cyclopeptides

Article abstract of DOI:10.1016/j.tet.2005.10.080

Synthetic cyclopeptides, and particularly those designed from VEGF structure, present considerable interest for the development of nanodevices devoted to tumor imaging or drug delivery. In order to obtain functionalizable cyclopeptides, we herein present

Full text of DOI:10.1016/j.tet.2005.10.080

Tetrahedron 62 (2006) 1124–1130
New strategy towards the efficient solid phase synthesis
of cyclopeptides
a
Thomas Berthelot, Mario Gonc¸alves, Georges Laın, Karine Estieu-Gionnet
a
a
a,b
¨
a,
*
´ ´ ´
and Gerard Deleris
a
´ ´
CNRS UMR 5084 CNAB, Groupe de Chimie Bio Organique, Universite Victor Segalen Bordeaux 2, 146 rue Leo Saignat,
33076 Bordeaux Cedex, France
´
b
´ ` ´
INSERM E0113, Laboratoire des Mecanismes Moleculaires de l’Angiogenese, Universite Bordeaux 1,
33405 Talence, France
Received 9 August 2005; revised 28 October 2005; accepted 28 October 2005
Available online 28 November 2005
Abstract—Synthetic cyclopeptides, and particularly those designed from VEGF structure, present considerable interest for the development
of nanodevices devoted to tumor imaging or drug delivery. In order to obtain functionalizable cyclopeptides, we herein present a novel
efficient way based on Fmoc-Lys-ODmab use, which was applied to the synthesis of c(PHGRIK) cyclopeptide.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
Angiogenesis is a key process of cancer development and
metastasis. Targeting and/or imaging of this phenomenom
are of great interest in order to develop or follow-up new
therapeutic approaches.
In this way, vascular endothelial growth factor (VEGF),
which is the major tumoral angiogenic factor, is a promising
target. Its biological properties are mediated through
dimerizing interactions with several receptors among
which Type 2 VEGF receptor is the most efficient towards
tumoral neoangiogenesis. These interactions are mainly
achieved through three basic (R,K,H) residues. We have
Figure 1. Structure of c(PHGRIK) 4.
previously demonstrated that synthetic cyclopeptides,
As often cyclic structure by far increase metabolic
stability4–9 but within these series they require synthetic
designed from the structural requirements for the activity
of VEGF, present a very high affinity for VEGFR2 and
subsequently a high level of antitumoral in vivo activity.1–3
One of them, cycloVEGI is presently under preclinical
development as anticancer agent and studies to use it as
nanodevice for tumor delivery of anticancer drug, gene
delivery system or imaging agent are in progress. Within
this series, we synthesized, by solution ‘head-to-tail’ cyclic
peptide synthesis, the smaller deprotected 6-mer
cyclopeptide c(PHGRIK) 4 (Fig. 1) designed from VEGF.
improvements as they embody only a few, but biologically
essential basic residus as functional amino acids. Thus, we
focused our approach on the synthesis of cyclopeptide 4
which only contents alkyl and basic amino acids. One of
them appeared suitable for functionalization, namely lysine.
2. Results and discussion
Actually, on resin preparation of head-to-tail-cyclic
peptides requires temporary protection of the a-carboxyl
group of resin bound amino acid.10–14 To date, general
synthesis of these peptides has mainly used allyl group as
Keywords: Nanodevice; Cyclopeptides; On-resin cyclization; Dmab ester;
Lysine.
*
Corresponding author. Tel.: C33 5 57 57 10 01; fax: C33 5 57 57 10 02;
e-mail: gerard.deleris@u-bordeaux2.fr
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.10.080

T. Berthelot et al. / Tetrahedron 62 (2006) 1124–1130
1125
orthogonal a-carboxyl protection15–21 as Fmoc-Lys-OAll
for side-chain anchoring for instance. This strategy presents
some disadvantages for peptide synthesis: (i) the selective
removal of allyl esters requires palladium chemistry, which
results on partial deprotection of a-carboxyl group22 (ii)
acetylation of peptide N-terminal group by acetic acid used
for allyl deprotection has been observed and prevents
cyclization step.20 To overcome these drawbacks, we
focused our approach on the use of a recently described
carboxyl protective group, Dmab (4{N-[1-(4,4-dimethyl-
2,6-dioxocyclohexylidene)-3-methylbutyl]-amino} benzyl
alcohol) (Fig. 2), based on aminobenzylester moiety. To
date, the use of this group has been reported as a temporary
a-carboxyl protecting group of acidic (Asp, Glu) and
hydroxyl (Tyr) amino acids23–26 but never for basic amino
acid such as lysine.
purification, treatment of Fmoc-Lys(Boc)-ODmab 1 with
trifluoroacetic acid (TFA) in DCM gave the required Fmoc-
Lys-ODmab 2 with 92% yield (Scheme 2). The structure and
purity of these original compounds were confirmed by NMR
and mass spectrometry for 1 and by NMR, IR, mass and
analytical reversed-phase HLPC (RP-HPLC) for 2.
Synthetic usefulness of our approach was highlighted by (on
resin) synthesis of 4. Solid-phase synthesis of the 6-mer
cyclic peptide was performed on a 2-chloro chlorotrityl
resin (1.5 mmol/g). Loading of the resin was achieved with
2 and an excess of diisopropyldiethylamine (DIEA) in
tetrahydrofurane (THF) (Scheme 3). Reaction was stopped
after 5 h, resin was filtered off and washed successively with
DMF, DCM and methanol. After solvents removal under
vacuum, a substitution level of 0.1 mmol/g was estimated
for this novel preloaded resin 3 by UV determination of the
concentration of liberated dibenzofulvene, resulting from
the cleavage of the Fmoc group with piperidine.27 Low
substitution level favors cyclization by pseudodilution, and
therefore favors intramolecular reactions over intermole-
cular side reactions.28 This resin (0.1 mmol) was then used
for linear peptide synthesis on a ABI-431A continuous-flow
automated peptide synthesizer using dicyclohexylcarbo-
diimide (DCC)/N-hydroxy-benzotriazole (HOBt) activa-
tion. After completion of peptide assembly, resin was
treated three times with 2% hydrazine/DMF at room
temperature for 3 min. The complete removal of the
Dmab group was monitored by UV at 300 nm for each
treatment. After the third, no absorbance was detected,
which demonstrated that a-COOH group was totally
deprotected. Resin was then washed with DMF.
Figure 2. Dmab structure.
Removal of this protecting group does not require any metal
catalyst, which avoids side reactions or further biological
peptide poisoning. Dmab group is removed using brief
treatment with 2% hydrazine in dimethylformamide (DMF),
giving the free acid and by-products that are easily washed
away from the solid phase (Scheme 1).
Moreover, Dmab protection of the carboxyl groups is
orthogonal to the Fmoc/Boc strategy as it is stable to
piperidine and TFA used for peptide synthesis.
For the intramolecular cyclization, PyBOP and HOBt
(5 mol equiv for both) were used along with 10 mol equiv
of DIEA for 48 h. Then, the peptide was cleaved from resin
using a TFA–triisopropylsilane (TIS)–H2O (95/2.5/2.5)
solution affording totally unprotected cyclic peptide 4
(Scheme 4).
Thus, the use of such orthogonal protecting group for the
synthesis of Dmab ester of a Fmoc-lysine yields a novel
building block for ‘on-resin’ head-to-tail peptide
cyclization.
Peptide cyclic structure was confirmed on crude product
with MALDI TOF mass spectrometry analysis. Crude
product was purified by a semi-preparative RP-HPLC on a
C18 column coupled with UV–vis detection (l amide bond
Z214 nm) for separation. Purification gave 17 mg of 4
(25%). The structure of cyclic peptide 4 was confirmed by
For this purpose, esterification of the Fmoc-Lys(Boc)-OH
with Dmab-OH was carried out by activation with diisopro-
pylcarbodiimide (DCI) and dimethylaminopyridine (DMAP)
inamixtureofdichloromethane(DCM)/acetonitrinile(ACN):
1:1 to yield Fmoc-Lys(Boc)-ODmab 1 (75%). After
Scheme 1. Hydrazine-mediated cleavage of Dmab group.

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T. Berthelot et al. / Tetrahedron 62 (2006) 1124–1130
Scheme 2. Synthesis of Fmoc-Lys-ODmab. (a) DmabOH, DMAP, DCI, DCM–ACN (1/1), rt, 24 h; (b) TFA–DCM (1/1), rt, 3 h.
Fmoc
HN
Cl
Cl
O
c
NH
C
Cl
C
Fmoc-Lys-ODmab
ODmab
3
0.1 mmol/g
Scheme 3. Anchoring of Fmoc-Lys-ODmab to the 2-chloro chlorotrityl resin. (c) DIEA, THF, rt, 5 h.
Scheme 4. Solid phase peptide synthesis of c(PHGRIK) 4. (d) See Section 3: general procedure; (e) 2% H4N2/DMF; (f) PyBOP, HOBt, DIEA, NMP, rt, 48 h;
(f) TFA–TIS–H20 (9.5/0.25/0.25), rt, 3 h.
MALDI mass spectrometry analysis and its purity by
analytical RP-HPLC.
Thus, our results highlight that Dmab a carboxyl protection of
lysine and further coupling on trityl resin offers an efficient
waystosynthesizepoorlyfunctionalizedbasiccyclicpeptides,
via Fmoc chemistry. As we had previously demonstrated that
such derivatives exhibited high affinity for VEGFR2 and were
of a great interest towards the generation of VEGF targeting
nanodevices, the synthesis of their conjugates is of a great
importance. With facile access to further functionalization via
the liberated amine function, lysine appeared to be a good
candidate. Subsequent functionalization on the side chain
amine function resulting in a neutral amide bond, introduction
of a supplementary lysine within an active cyclopeptide has to
be considered as a key-step to apply this strategy.
Synthesized peptide
4 was then studied for its
properties as in vitro inhibitor of the binding of
VEGF165 to its receptor. To this purpose, Chinese
hamster ovary (CHO) cells expressing VEGFR2 were
used. Competition between 125I-VEGF165 and
4
revealed a fairly high level of inhibition, that is,
IC50Z1.9 mM (Fig. 3). Therefore, when compound with
other peptidic inhibitory derivatives29 our family
behaves as one of them, low molecular weight most
active derivatives.

T. Berthelot et al. / Tetrahedron 62 (2006) 1124–1130
1127
100 mM, 4 scans, 2 cmK1, transmission mode). Solid
phase peptide synthesis was carried out on an Applied
Biosystems 431A automated peptide synthesizer using
dicyclohexylcarbodiimide (DCC)/N-hydroxybenzotriazole
(HOBt) activation. Reversed phase high pressure liquid
chromatography (RP-HPLC) was performed on a Shimadzu
instrument equipped with a SCL-10 AVP system controller,
LC8A HPLC pumps and SPD-10 AVP UV–vis detector
probing at 214, 267 and 254 nm on a SATISFACTION
RP18AB 5 mM 250!4.6 mm C18 column (C.L.I Cluzeau).
The following solvent systems were used for the elution in a
linear gradient mode at a flow rate of 1 or 4 ml/min (for
analytical and semi preparative HPLC, respectively): (A)
0.1% aqueous trifluoroacetic acid (TFA) and (B) 0.1% TFA
in 70% aqueous acetonitrile (ACN). MALDI-TOF mass
spectrometry was performed on a Reflex III Brucker
apparatus.
Figure 3. Inhibition of 125I-VEGF165 binding to CHO-VEGFR2 cells by
cyclo(PHGRIK) 4.
3. Experimental
3.1. General methods
3.1.1. Synthesis of Fmoc-Lys(Boc)ODmab (1). To a
solution of Fmoc-lys(Boc)OH (1.3 g, 2.77 mmol) in a
mixture of ACN–DCM (1/1.16 ml) was added successively
Dmab-OH (0.61 g, 1.85 mmol) and dimethylamino pyridine
(DMAP) (52 mg, 0.42 mmol). After complete dissolution,
diisopropylcarbodiimide (DIC) (485 ml, 3.1 mmol) was
added. Thereafter the solution was stirred at room tempera-
ture for 24 h. Then the reaction mixture was filtered, and the
solvent was removed under vacuum. The crude product was
dissolved in DCM (50 ml) and washed with water
(3!50 ml). The organic layer was dried over MgSO4 and
filtered. The solvent is evaporated under vacuum to give
greenish oil, which was purified by silica gel chromatography
(DCM/ethyl acetate, 90:10). Then 1.1 g (1.4 mmol, RF 0.53
(DCM–ethyl acetate, (80/20)) (75%) of compound 1 were
obtained as a yellowish oil. MALDI-TOF mass spectrometry
analysis gave the expected result (m/z calculated: 779.97
found (MCNa)C: 802.46, (MCK)C: 818.42). 1H NMR
(CDCl3) d ppm: 15.18 (s, 1H, NH Dmab), 7.72 (d, 2H,
All standard chemicals and solvents were of analytical grade
and purchased from Sigma Aldrich. 2-Chloro chlorotrityl
resin and all the Fmoc-amino acids were purchased from
Advanced Chemtech. Dmab-OH was purchased from
Novabiochem. All chemical reactions were carried out
under nitrogen with dry solvents. N,N-dimethyl formamide
(DMF) was refluxed over CaH2 overnight and distilled, and
methylene chloride (DCM) was distilled before use. Thin-
layer chromatography (TLC) was performed on aluminium
plates precoated with silica gel 60F254 (Merck) with
detection by UV light (365 nm), and column chromatog-
raphy was performed on silica gel 60, 70–230 mesh (SDS).
1H and 13C NMR spectra were recorded at 298 K on a
Brucker Avance 300 MHz spectrometer at 300.13 and
75.46 MHz, respectively. Chemical shifts (d) are given in
parts per million (ppm). IR spectra were recorded on a
Perkin-Elmer Spotlight 300 and Spectrum one (point mode
Figure 4. Analytical RP-HPLC of compound 2. tRZ22.45 min.

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T. Berthelot et al. / Tetrahedron 62 (2006) 1124–1130
3
CHArFmoc, JH–HZ7.5 Hz), 7.56 (d, 2H, CHArFmoc,
compound 2 was determinated by RP-HPLC (Fig. 4) mp
92–94 8C; [a]2D0 C5.95 (c 0.84 in DCM); HRMS gave the
expected result (m/z (mC1) calcd for C41H50N3O6
680.3699 found 680.3673); IR (cmK1): 1045, 1550, 1590,
1638, 1687, 2920, 2959; 1H NMR (CDCl3) d ppm: 15.19 (s,
1H, NH Dmab), 7.97 (br s, 2H, NH2), 7.71 (d, 2H,
3JH–HZ7.32 Hz), 7.28–7.36 (m, 4H, CHArFmoc), 7.25 (d,
3
2H, CHArBzl, JH–HZ7.20 Hz), 7.06 (d, 2H, CHArBzl,
3JH–HZ8.25), 5.68 (d, 1H, NaH, JH–HZ7.8 Hz), 5.14 (s,
2H, CH2Bzl), 4.6 (br s, 1H, NH-Boc), 4.34–4.37 (m, 1H,
3
3
CaH), 4.19 (t, 1H, CH-Fmoc, JH–HZ14.3), 4.04–4.13 (m,
2H, CH2-Fmoc), 3.15–3.02 (m, 2H, CH2–NH), 2.94 (d, 2H,
3
CHArFmoc, JH–HZ7.3 Hz), 7.54 (d, 2H, CHArFmoc,
3
CH2 Dmab, JH–HZ5.73 Hz), 2.49 (s, 4H, 2 CH2 Dmab),
3JH–HZ6.87 Hz), 7.28–7.34 (m, 4H, CHArFmoc), 7.25 (d,
3
1.39–1.99 (m, 7H, 3 CH2, CH ipropyl), 1.38 (s, 9H, 3 CH3
(Boc)), 1.03 (s, 6H, 2 CH3), 0.75 (d, 6H, (CH3)2CH,
3JH–HZ6.66 Hz); 13C NMR (CDCl3) d ppm: 198.82,
176.99, 172.27, 176.10, 156.52, 143.85, 141.50, 137.10,
135.13, 129.32, 128.00, 127.29, 126.88, 125.22, 120.25,
107.95, 78.87, 67.47, 66.55, 53.84, 53.00, 47.27, 39.71,
38.66, 31.96, 30.24, 29.76, 28.42, 27.00, 22.74, 22.27,
13.98.
2H, CHArBzl, JH–HZ7.47 Hz), 7.05 (d, 2H, CHArBzl,
3
3JH–HZ8.88 Hz), 5.68 (d, 1H, NaH, JH–HZ7.8 Hz), 5.14
(s, 2H, CH2Bzl), 4.30–4.35 (m, 3H, CH2-Fmoc, CH-Fmoc),
4.14–4.17 (m, 1H, CaH), 2.88–2.96 (m, 4H, CH2–N, CH2
Dmab), 2.42 (s, 4H, CH2 Dmab), 1.94–1.37 (m, 7H, 3 CH2,
CH ipropyl), 1.04 (s, 6H, 2 CH3), 0.71 (d, 6H, (CH3)2CH,
3JH–HZ6.51 Hz); 13C NMR (CDCl3) d ppm: 198.80,
176.98, 172.21, 176.04, 143.79, 141.36, 137.05, 135.18,
129.28, 128.10, 127.23, 126.88, 125.29, 120.20, 107.98,
67.50, 66.49, 53.91, 53.08, 47.31, 39.76, 38.61, 31.99,
30.27, 29.76, 27.05, 22.79, 22.25, 13.91.
3.1.2. Synthesis of Fmoc-Lys-ODmab (2). To a solution of
compound 1 (1.1 g, 1.4 mmol) in DCM (30 ml) was added
trifluoroacetic acid (TFA) (30 ml). The reaction mixture
was stirred at room temperature for 3 h. The solution was
concentrated to dryness in vacuo, followed by repeated
washings and evaporations with diethylether. Then it was
dried in dessicator under high vacuum over KOH to give
0.9 g (1.3 mmol, RF 0.43 (DCM–methanol (90/10)) (92%)
of the compound 2 as a pale yellow solid. Purity of
3.1.3. Synthesis of Fmoc-Lys-(Trityl resin)-ODmab (3).
Fmoc-Lys-ODmab 2 (1 g, 1.5 mmol) was dissolved in
dry tetrahydrofuran (THF) (5 ml). After complete
dissolution, diisopropylethylamine (DIEA) (600 ml,
3.46 mmol) was added. After 5 min, 2-chloro chorotrityl
resin (500 mg, 1.5 mmol/g) was added. The resulting
Figure 5. Analytical RP-HPLC of crude compound 4.

T. Berthelot et al. / Tetrahedron 62 (2006) 1124–1130
1129
suspension was stirred at room temperature for 5 h. The
resin was filtered off and successively washed with DMF
(1!20 ml), DCM–methanol–DIEA (17:2:1) (2!20 ml),
DMF (1!10 ml) and DCM (2!20 ml). The new
preloaded resin was dried in dessicator under high
vacuum over KOH. The substitution level was deter-
minated spectrophotometrically by Fmoc cleavage: Fmoc-
Lys-(Trityl resin)-ODmab (4.3 mg) was introduced into a
test tube and a solution of 20% piperidine in DMF was
added (0.5 ml). 20% piperidine in DMF (0.5 ml) was also
added to an empty test tube to serve as a blank. Over the
next 15 min, the test tube with the resin was swirled to
make sure all the resin has come in contact with the
cleavage solution. DMF was added to both tubes to bring
a volume of 50 ml. The blank was used to zero the UV
spectrophotometer at 301 nm. The absorbance of the
solution is 0.07. The substitution level was calculated
from the formule: (Abs301 nm!vol(ml))/(7800!m(g))
and was determinated to be 0.1 mmol/g.
allowed to equilibrate for 5 min in DMF (20 ml/g of
resin). The solvent was removed from the resin by
applying a nitrogen pressure and the residue was
resuspended in a solution of 2% hydrazine monohydrate
in DMF (20 ml/g of resin). Reaction was allowed to
proceed for 3 min with gentle manual agitation and the
hydrazine treatement was repeated a further two times to
ensure complete reaction. The resin was washed
successively with DMF (2!20 ml), DCM (2!20 ml),
methanol (1!20 ml) and DCM (1!20 ml) and dried.
Peptidyl resin (1.21 mg) was mixed with a solution of
PyBOP (260 mg, 0,5 mmol), HOBt (69 mg, 0.51 mmol)
and DIEA (180 ml, 1 mmol) in N-methyl-pyrrolidone
(NMP) (10 ml). The mixture was swelled at room
temperature for 48 h. The peptidyl resin was washed
with 30 ml of each DMF, DCM and methanol and was
dried over vacuum. Final cleavage from the resin and
total deprotection of cyclo(PHGRIK) was performed with
TFA–H2O–Triisopropylsilane (TIS) (95/2.5/2.5) mixture
for 2.5 h at room temperature. Then the filtrate was
evaporated under reduced pressure to 5% of the volume.
The product was precipitated from cold diethyl ether,
filtered to give 30 mg of crude product (Fig. 5). MALDI-
TOF mass spectrometry analysis of a sample of crude
product gave the expected result (m/z calculated: 687.83
found (MCH)C: 689.34) (Fig. 6). Then it was purified
by semi-preparative HPLC to yield 17 mg of pure
compound 4 (Fig. 6). MALDI-TOF mass spectrometry
analysis of the pure product gave the expected result (m/z
calculated: 687.83 found (MCH)C: 689.34).
3.1.4. Synthesis of cyclo(PHGRIK) (4). Cyclo(PHGRIK)
was synthesized by Fmoc/tBu batch solid phase synthesis
on an Applied Biosystems 431A automated peptide
synthesizer. Preloaded Fmoc-Lys-(Trityl resin)-ODmab
(1 g, 0.1 mmol) was used for the linear chain assembly.
Subsequent Fmoc amino acids were coupled using a
10-fold excess of amino acids activated as HOBt ester.
Removal of the Dmab group was performed after the
N-terminal automated Fmoc deprotection. The peptidyl
resin (1.28 g) was placed on a frit syringe barrel and
Figure 6. Analytical RP-HPLC of compound 4. tRZ10.5 min.

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T. Berthelot et al. / Tetrahedron 62 (2006) 1124–1130
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CHO-VEGFR2 cells were grown in DMEM supplemented
with 10% fetal calf serum, 2 mM L-glutamine, 1% non-
essential amino acids and antibiotics (Invitrogen).
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3.3. Binding assays
VEGF165 was labeled with Na125I using IODO-GEN
(Pierce) as coupling agent according to the manufacturer’s
instructions, CHO-VEGFR2 cells were seeded at 2.5!105
density in gelatin-coated 6-well plates and cultured in
complete medium for 2 days. Cells were washed twice with
ice-cold PBS and incubated with 10 ng/ml 125I-VEGF and
cyclo(PHGRIK) 4 at indicated concentrations in binding
medium (DMEM; 20 mM Hepes, pH 7.4; 0.15% gelatin) on
a shaker at 4 8C. After 2 h, cells were washed three times
with PBS and solubilized by the addition of 2% Triton, 10%
glycerol, and 1 mg/ml bovine serum albumin prior to
g-counting. Each condition was tested in duplicate and
repeated at least two times. Data are expressed as
percentage of total radioactivity.
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Acknowledgements
´
Financial support is acknowledged from ‘Conseil Regional
d’Aquitaine’ and ‘Ligue Nationale contre le cancer. Comite
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´
UMR 5144 MOBIOS, Universite Bordeaux 1 for mass
spectra acquisitions and Xavier Canron, INSERM E0113,
´
universite Bordeaux 1 for binding assays.
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