K
K
K
R1
R1
K
K
R1
A
P
O
P
K
O
O
G
P
N
N
O
O
K
A
P
g
K
4 : R1 = Boc , R2 = Alloc
5 : R1 = H , R2 = Alloc
6 : R1 = CO-CH2-ONHBoc , R2 = Alloc
O
O
O
N
O
K
K
G
A
P
K
K
G
K
N
O
P
K
G
O
R2
a
b
c
d
O
O
K
K
G
O
N
O
f
R
O
O
O
O
O
K
e or f
N
O
K
K
N
N
R1
G
O
K
K
O
O
O
K
K
D
f X
f R
R
f
R
f
R
D
X
D
X
D
X
D
f X
f R
R
f
R
D
X
D
X
D
X
11 : X = G
12 : X = A
N
O
7 : R1 = CO-CH2-ONHBoc , R2 = H
8 : R1 = CO-CH2-ONH2 , R2 = H
S O
O K
+
N
O
S O
O
1 : X = G, namely (Cy5)RAFT(c[-RGDfK-])4
2 : X =A, namely (Cy5)RAFT(c[-RADfK-])4
(a) TFA/CH2Cl2 (1:1) ; (b) BocNHOCH2COOSucc , DIPEA , DMF ; (c) Pd(PPh3)4 , PhSiH3 , CH2Cl2 ;
(d) TFA/TIS/H2O/CH2Cl2 (50:5:5:40) ; (e) 9 , H20/CH3CN (1:1) ; (f) 10 , H20/CH3CN (1:1) ;
(g) Cy5 NHS , DIPEA , DMF
Synthesis.
Materials and equipments. Protected amino acids were obtained from Advanced ChemTech
Europe (Brussels, Belgium), Bachem Biochimie SARL (Voisins-le-Bretonneux, France),
France Biochem SA (Meudon, France), Merck Eurolab (Fontenay-sous-Bois, France) or
Calbiochem-Novabiochem (Merck Biosciences - VWR, Limonest, France). PyBOP® was
purchased from Calbiochem-Novabiochem. Fmoc-Gly-SASRIN® resin was obtained from
Bachem Biochimie SARL and 2-chlorotritylchloride® resin from Advanced ChemTech
Europe. Other reagents were obtained from Aldrich (Saint-Quentin Fallavier, France) and
Acros (Noisy-le-Grand, France). Cy5 NHS ester was purchased from Amersham Biosciences
(Orsay, France). RP-HPLC were performed on Waters equipment consisting of a Waters 600
Controller, a Waters 2487 Dual Absorbance Detector and a Waters In-Line Degasser. The
analytical column (Nucleosil 120 Å 3 µm C18 particles, 30 x 4.6 mm2) was operated at 1.3
mL.min-1 and the preparative column (Delta-Pak™ 300 Å 15 µm C18 particles, 200 x 25 mm2)
at 22 mL.min-1. UV was monitored at 214 nm and either 250 nm or 646 nm. Solvent A
consisted of H2O containing 0.1% TFA and solvent B of CH3CN containing 9.9% H2O and
0.1% TFA. Mass spectra were obtained by electron spray ionization (ES-MS) on an Esquire
3000 (Bruker).
c[-Pro-Gly-Lys(Boc)-Ala-Lys(Boc)-Pro-Gly-Lys(Boc)-Lys(Alloc)-Lys(Boc)-] 4. The cyclic
decapeptide
c[-Pro-Gly-Lys(Boc)-Ala-Lys(Boc)-Pro-Gly-Lys(Boc)-Lys(Alloc)-Lys(Boc)-]
was synthesized as described previously.1 Compound 4 was obtained in 50% overall yield
from the first loading of SPPS. RP-HPLC analysis, RT = 11.8 min (C18, 214 nm, 5-100% B in
15 min). Mass spectrum (ES-MS, positive mode) calcd 1504.8, found 1503.9.
c[-Pro-Gly-Lys(-COCH2ONH2)-Ala-Lys(-COCH2ONH2)-Pro-Gly-Lys(-COCH2ONH2)Lys(H)-Lys(-COCH2ONH2)-] 8. Boc protecting groups from cyclic peptide 4 (180.8 mg,
0.120 mmol) were removed in a solution containing 50% TFA in CH2Cl2 for 1 h at room
temperature. The crude product was concentrated, triturated and washed with ether to yield
compound 5 as a white powder (187.5 mg, 0.120 mmol). To a solution of compound 5 (93.6
mg, 0.060 mmol) in 6 mL anhydrous DMF, were added BocNHOCH2COOSucc (101.8 mg,
0.353 mmol) and DIPEA to adjust the pH at 8.0. The reaction was stirred for 2 h at room
temperature and then concentrated under reduced pressure. The crude product was triturated
and washed with ether to yield compound 6 as a white powder (104.8 mg, 0.058 mmol). Alloc
group was removed using cyclic peptide 6 (104.8 mg, 0.058 mmol) dissolved in 10 mL
1
Boturyn, D.; Coll, J. L.; Garanger, E.; Favrot, M. C.; Dumy, P. Template assembled cyclopeptides as
multimeric system for integrin targeting and endocytosis. J. Am. Chem. Soc. 2004, 126, 5730-5739.
anhydrous CH2Cl2 and 6 mL anhydrous DMF under argon by adding phenylsilane (162.3 mg,
1.540 mmol) for 3 min and then Pd(PPh3)4 (17.7 mg, 15 µmol) for 1 h at room temperature.
The reaction was stopped by adding 5 mL of MeOH and stirred for 30 min at room
temperature. The solvent was removed under reduced pressure. The crude product was
dissolved in the minimum of CH2Cl2 and subsequently precipitated, triturated and washed
three times with ether. Product 7 was obtained as a white powder (92.4 mg, 0.054 mmol). Boc
protecting groups from compound 7 (92.4 mg, 0.054 mmol) were removed in a solution
containing 50% TFA, 5% H2O and 5% TIS in CH2CL2 for 1 h at room temperature. The
crude product was concentrated and dissolved in water. The solution was filtered and
lyophilized. The product was then purified using RP-HPLC to afford compound 8 as a white
powder (13.3 mg, 7 mol, 13% from 5). RP-HPLC analysis, RT = 5.6 min (C18, 214 nm, 560% B in 15 min). Mass spectrum (ES-MS, positive mode) calcd 1312.5, found 1312.5.
c[-Arg-Gly-Asp-D-Phe-Lys(COCHO)-] 9. The cyclic pentapeptide c[-Arg-Gly-Asp-D-PheLys(COCHO)-] 9 was prepared as described previously.2 Compound 9 was obtained in 26%
overall yield from cyclic pentapeptide c[-Arg(Pmc)-Gly-Asp(OtBu)-D-Phe-Lys-]. RP-HPLC
analysis, RT = 5.8 min (C18, 214 nm, 5-100% B in 15 min). Mass spectrum (ES-MS, positive
mode) calcd 659.7, found 659.1.
c[-Arg-Ala-Asp-D-Phe-Lys(COCHO)-] 10. The cyclic pentapeptide c[-Arg-Ala-Asp-DPhe-Lys(COCHO)-] 10 was prepared as described previously.1 Compound 10 was obtained in
17% overall yield from cyclic pentapeptide c[-Arg(Pmc)-Ala-Asp(OtBu)-D-Phe-Lys-]. RPHPLC analysis, RT = 5.7 min (C18, 214 nm, 5-100% B in 15 min). Mass spectrum (ES-MS,
positive mode) calcd 673.7, found 673.1.
(H)RAFT(c[-RGDfK-])4 11. To a solution containing the derivative 8 (6.7 mg, 3.5 µmol) in
175 µL H2O/ CH3CN (1:1) was added a solution of peptide 9 (13.2 mg, 17 µmol) in 800 µL
H2O/CH3CN (1:1). The reaction was stirred for 1 h at room temperature. Conjugate 11 was
isolated after purification by RP-HPLC as a white powder (13.6 mg, 3.1 µmol, 87%). RPHPLC analysis, RT = 10.0 min (C18, 214 nm and 250 nm, 5-60% B in 15 min). Mass
spectrum (ES-MS, positive mode) calcd 3879.3, found 3879.5.
(H)RAFT(c[-RADfK-])4 12. To a solution containing the derivative 8 (6.7 mg, 3.5 µmol) in
175 µL H2O/CH3CN (1:1) was added a solution of peptide 10 (12.9 mg, 16 µmol) in 800 µL
H2O/ CH3CN (1:1). The reaction was stirred for 2 h at room temperature. Conjugate 12 was
isolated after purification by RP-HPLC as a white powder (9.5 mg, 2.1 µmol, 60%). RPBoturyn, D.; Dumy, P. A convenient access to v3/v5 integrin ligand conjugates : regioselective solid-phase
functionalisation of an RGD based peptide. Tetrahedron Lett. 2001, 42, 2787-2790.
2
HPLC analysis, RT = 9.9 min (C18, 214 nm, 5-60% B in 15 min). Mass spectrum (ES-MS,
positive mode) calcd 3935.4, found 3935.3.
c[-Arg-Gly-Asp-D-Phe-Lys-] 13. The cyclic pentapeptide c[-Arg-Gly-Asp-D-Phe-Lys-] 13
was prepared as described in the literature by a combination of SPPS and solution strategy. 2
The compound 13 was obtained in quantitative yield from cyclic pentapeptide c[-Arg(Pmc)Gly-Asp(OtBu)-D-Phe-Lys-]. RP-HPLC analysis, RT = 5.2 min (C18, 214 nm, 5-100% B in
15 min). Mass spectrum (ES-MS, positive mode) calcd 603.7, found 603.3.
(Cy5)RAFT(c[-RGDfK-])4 1. To a solution of compound 11 (12.4 mg, 2.8 µmol) in 279 µL
anhydrous DMF was added 280 µL of a solution of Cyanine 5 N-Hydroxysuccinimide ester
(Cy5 NHS ester) (2.2 mg, 2.8 µmol) in anhydrous DMF and DIPEA to adjust the pH at 9.0.
The reaction was stirred for 4 h at room temperature and purified by RP-HPLC. Conjugate 1
(8.1 mg, 1.6 µmol, 58%) was obtained as a blue powder. RP-HPLC analysis, RT = 11.3 min
(C18, 214 nm and 646 nm, 5-60% B in 15 min). Mass spectrum (ES-MS, positive mode) calcd
4518.2, found 4517.9.
(Cy5)RAFT(c[-RADfK-])4 2. To a solution of compound 12 (4.7 mg, 1.0 µmol) in 105 µL
anhydrous DMF was added 105 µL of a solution of Cy5 NHS ester (0.8 mg, 1.0 µmol) in
anhydrous DMF and DIPEA to adjust the pH at 9.0. The reaction was stirred for 3 h at room
temperature and purified by RP-HPLC. Conjugate 2 (2.2 mg, 0.4 µmol, 43%) was obtained as
a blue powder. RP-HPLC analysis, RT = 11.2 min (C18, 214 nm and 646 nm, 5-60% B in 15
min). Mass spectrum (ES-MS, positive mode) calcd 4574.3, found 4574.5.
c[-Arg-Gly-Asp-D-Phe-Lys(Cy5)-] 3. To a solution of compound 13 (8.4 mg, 10 µmol) in 1
mL anhydrous DMF were added 900 µL of a solution of Cy5 NHS ester (8 mg, 10 µmol) in
dry DMF and DIPEA to adjust the pH at 9.0. The reaction was stirred for 4 h at room
temperature and purified by RP-HPLC. Conjugate 3 (9.5 mg, 6.8 µmol, 68%) was obtained as
a blue powder. RP-HPLC analysis, RT = 7.7 min (C18, 214 nm and 646 nm, 5-100% B in 15
min). Mass spectrum (ES-MS, positive mode) calcd 1241.6, found 1242.3.
Sample preparation. Cy5-conjugated peptides were dissolved in Phosphate Buffer Saline
(PBS, 140 mM NaCl, 10 mM Na2HPO4, 2 mM KCl, pH 7.2) at a final concentration of 0.5
mM.
RP-HPLC spectra.
(Cy5)RAFT(c[-RGDfK-])4 1.
C18, 214 nm and 646 nm, 5-60% B in 15 min : RT = 11.3 min
 = 214 nm
 = 646 nm
(Cy5)RAFT(c[-RADfK-])4 2.
C18, 214 nm and 646 nm, 5-60% B in 15 min : RT = 11.2 min
 = 214 nm
 = 646 nm
c[-Arg-Gly-Asp-D-Phe-Lys(Cy5)-] 3.
C18, 214 nm and 646 nm, 5-100% B in 15 min : RT = 7.7 min
 = 214 nm
 = 646 nm