Synthesis of angiogenesis-targeted peptide and hydrophobized polyethylene glycol conjugate

Article abstract of DOI:10.1016/j.bmcl.2003.11.073

For the purpose of cancer antineovascular therapy, a novel angiogenesis-targeted peptide, Ala-Pro-Arg-Pro-Gly, (APRPG) was attached to hydrophobized polyethylene glycol (distearoylphosphatidylethanolamine [DSPE]-PEG). DSPE-PEG and the 5-mer peptide were condensed with DCC-HOBt method. Liposome modified with this DSPE-PEG-APRPG conjugate highly accumulated in tumor of tumor-bearing mice.

Full text of DOI:10.1016/j.bmcl.2003.11.073

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1015–1017
Synthesis of angiogenesis-targeted peptide and hydrophobized
polyethylene glycol conjugate
Noriyuki Maeda,a,b Yoshito Takeuchi,a Miki Takada,a
Yukihiro Nambab and Naoto Okua,*
aDepartment of Medical Biochemistry and COE Program in the 21st Century, School of Pharmaceutical Sciences,
University of Shizuoka, Shizuoka, Japan
bNippon Fine Chemical Co., Ltd, Takasago, Hyogo, Japan
Received 8 September 2003;accepted 20 November 2003
Abstract—For the purpose of cancer antineovascular therapy, a novel angiogenesis-targeted peptide, Ala-Pro-Arg-Pro-Gly,
(APRPG) was attached to hydrophobized polyethylene glycol (distearoylphosphatidylethanolamine [DSPE]-PEG). DSPE-PEG
and the 5-mer peptide were condensed with DCC-HOBt method. Liposome modified with this DSPE-PEG-APRPG conjugate
highly accumulated in tumor of tumor-bearing mice.
# 2003 Elsevier Ltd. All rights reserved.
Anigiogenesis is a critical event for maintenance, pro-
liferation and metastasis of tumor,1,2 and anti-
neovascular therapy that causes tumor regression
through damaging neovessel endothelial cells is expec-
ted to suppress both primary tumor and metastasis
without aquiring drug resistance. The therapy is also
expected for a broad spectrum of cancers. We pre-
viously isolated a peptide specifically bound to tumor
angiogenic vasculature from a phage-displayed peptide
library. The epitope sequence of the peptide was deter-
mined as APRPG.3 In fact, APRPG-modified liposome
highly accumulated in tumor tissue, and the liposome
encapsulating anticancer drugs strongly suppressed
tumor growth.3,4 On the other hand, it is known that
polyethylene glycol (PEG) conjugate liposome has long-
circulating characteristics through avoidance of trap-
ping by the reticuloendothelial system (RES) such as
liver and spleen.4,5 Furthermore, since liposomes have a
tendency to passively accumulate in tumor tissues due
to their enhanced permeability, long-circulating lipo-
somes are more favorable for tumor targeting than
conventional liposomes. Therefore, we aimed to endow
angiogenesis-targeted liposome with long circulating
character by the PEG conjugation. Conjugate 1 is the
designed compound composed of phospholipid,
polyethylene glycol and APRPG peptide. We synthe-
sized DSPE-PEG-SA (4) and APRPG peptide sepa-
rately, and then condensed them to obtain conjugate 1.
DSPE-PEG-SA was prepared as shown in Scheme 1.
First, distearoylphosphatidylethanolamine (DSPE; 2)
15.0 g and carbonyl diimidazole (CDI) 3.9 g were
dissolved in 70 mL of toluene. Reaction was done at
100 ꢀC for 1 h after addition of triethyl amine 2.0 g.
Then polyethylene glycol (average molecular weight of
2000) 40.0 g dissolved in toluene was added dropwise to
the solution. Solvent was evaporated in vacuo followed
by the reaction, and the product was dissolved in
acetone 500 mL and insoluble materials were filtrated
and solvent was evaporated. The reaction mixture was
exchanged into Na+ salt with ion exchange resin.
Purification by column chromatography on silica gave
11.4 g of the desired product 3 in a 26% yield. In order
to use the PEG-end of obtained DSPE-PEG as a car-
boxylic group (referred to as 4), it was allowed to react
with succinic anhydride 2.1 g in the presence of pyridine
1.7 g in 100 mL of toluene. After powdering with ether,
the yield of 4 was 80%.6
Preparation of APRPG peptide moiety was carried out
by the liquid-phase method as shown in Scheme 2.
N,N0-Dicyclohexylcarbodiimide (DCC, 1.1 equiv based
on peptide) and 1-hydroxybenzotriazol (HOBt, 1.1 equiv
based on peptide) were used for peptide coupling in
DMF. HCl in 1,4-dioxane was used for deprotection of
* Corresponding author. Tel.: +81-54-264-5701;fax: +81-54-264-
5705;e-mail: oku@u-shizuoka-ken.ac.jp
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.11.073

1016
N. Maeda et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1015–1017
Scheme 1. Pathway for synthesis of DSPE-PEG-SA.
the Boc group of N-terminal and NaOH was used for
deprotection of methyl ester group of C-terminal in
water and methanol. In order to avoid racemization,
segment condensation was proceeded between Boc-
Ala-Pro and Arg(NO2)-Pro-Gly-OBz to yield 78% of
Boc-Ala-Pro-Arg(NO2)-Pro-Gly-OBz. Next the Boc
protecting group was deprotected by HCl in 1,4-dioxane
to obtain peptide 5.7
Peptide 5 was condensed with 4 (0.93 equiv based on 5)
in CHCl3 by DCC (1 equiv based on 5) and HOBt (1
equiv based on 5). The progress of the reaction was
monitored by TLC. The reaction was almost complete
overnight without any serious side reactions. It was
purified by column chromatography on silica. The yield
was 83% based on 4. Deprotections of NO2 group
of arginine side chain and benzyl ester group of glycine
C-terminal were carried out by 10% palladium–carbon
catalytic reduction under hydrogen atomosphere in
methanol. It was purified by column chromatography
on silica and ion exchange resin. This compound of
single spot on TLC was in a 43% yield. This conjugate 1
was positive for Sakaguchi reagent, while negative for
UV lamp on TLC. These showed that NO2 protecting
group and benzyl ester protecting group were deprotected
simultaneously (Fig. 1).8
Scheme 2. Pathway for synthesis of conjugate 1.
tumor-bearing model mice. Colon 26 NL-17 carcinoma
cells (1.0ꢁ106 cells/mouse) were subcutaneously
implanted in 5-week-old BALB/c male mice, and bio-
distribution study was performed when the tumor size
had become about 10 mm in diameter. PEG-APRPG
liposome was composed of distearoylphosphatidyl-
choline (DSPC), cholesterol and conjugate 1 (10:5:1 as a
molar ratio) with trace of [3H]cholesterol oleoyl ether
Thus obtained DSPE-PEG-APRPG was used for the
modification of liposome, and we examined the bio-
distribution of PEG-APRPG conjugate liposome in
Figure 1. Structure of peptide-PEG-lipid conjugate 1.

N. Maeda et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1015–1017
1017
Figure 2. Biodistribution of PEG-APRPG modified liposome. Data are presented as the percentage of the injected dose per 100 mg tissue and S.D.
in each tissue (*p<0.05;** p<0.01;*** p<0.001). Open bars, non-modified liposome;shadow bars, PEG liposome;closed bars, PEG-APRPG
liposome.
References and notes
(74 kBq/mouse). PEG liposome composed of DSPC,
cholesterol and DSPE-PEG; 3 (10:5:1) and non-mod-
ified liposome composed of DSPC, cholesterol (10:5)
were also radiolabeled and used as control. Liposomes
were hydrated with 0.3 M glucose solution and freeze-
thawed for three cycles following the thrice extrusion
through polycarbonate membrane filter with a 100 nm
pore size. Size-matched Colon 26 NL-17 carcinoma-
bearing mice were injected with the radiolabeled
liposomes via the tail vein. At 24 h after the injection,
the mice were sacrificed under diethyl ether anesthesia
for obtaining the blood, heart, lung, liver, spleen,
kidney, and tumor. After weighing each organ, the
radioactivity was determined with a liquid scintillation
counter. The results are shown in Figure 2.
1. Folkman, J.;D’Amore, P. A. Cell 1996, 87, 1153.
2. O’Reilly, M. S.;Holmgren, L.;Chen, C.;Folkman, J.
Nat. Med. 1996, 2, 689.
3. Oku, N.;Asai, T.;Watanabe, K.;Kuromi, K.;Nagatsuka,
M.;Kurohane, K.;Kikkawa, H.;Ogino, K.;Tanaka, M.;
Ishikawa, D.;Tsukada, H.;Momose, M.;Nakayama, J.;
Taki, T. Oncogene 2002, 21, 2662.
4. Asai, T.;Shimizu, K.;Kondo, M.;Kuromi, K.;
Watanabe, K.;Ogino, K.;Taki, T.;Shuto, S.;Matsuda,
A.;Oku, N. FEBS Lett. 2002, 520, 167.
5. Woodle, M. C., Storm, G., Eds. Long Circulating
Liposomes: Old Drugs, New Therapeutics;Springer-
Verlag: Berlin, 1998.
6. Selected data for compound 4: 1H NMR (90 MHz,
CDCl3): d 0.78–1.40 (66H, dialkyl H), 2.33 (4H, brt, CO–
CH2), 3.64 (160H, brs, PEG-H), 3.85–4.50 (9H, m, gly-
cerol-H and O–CH2–CH2–N). FAB-MS m/z: 2314+44n
(n=0–13) [M+H]+. Rf=0.45 (CHCl3/CH3OH=4/1, v/v).
7. Selected data for compound 5: 1H NMR (600 MHz,
CD3OD): d 1.52 (3H, d, J=6.90, alanine CH3), 1.75–2.27
(12H, m, proline C–CH2–C and arginine C–CH2–C),
3.22–3.34 (6H, m, proline N–CH2–C and arginine N–
CH2–C), 3.59 (1H, m, arginine CH), 3.73 (2H, m, proline
CH), 4.28 (1H, brq, alanine CH), 4.47 (2H, m, glycine
CH2), 5.15 (2H, dd, J=12.3, 14.9, benzyl CH2), 7.30–7.35
(5H, m, benzyl C6H5). FAB-MS m/z: 632.2 [M+H]+.
8. Selected data for compound 1, FAB-MS m/z: 2912+44n
(n=0–13) [M+H]+. Rf=0.74 (CHCl3/CH3OH/H2O=60/
30/5, v/v/v).
The results indicate that PEG conjugate endowed
liposomes with long-circulating character through
avoidance of RES trapping. Furthermore, PEG-
APRPG conjugate liposome accumulated in tumor
more than PEG liposome at 24 h after the injection,
suggesting that not only PEGylation enhanced passive
targeting to tumor tissue through leaky endothelium of
the tissue but also APRPG conjugation enhanced active
targeting to the tumor angiogenic vasculature.
Therefore, PEG-APRPG conjugate liposome developed
in this study would be useful tools for anti-neovascular
therapy.