Preparation and biological activities of a bivalent poly(ethylene glycol) hybrid containing an active site and its synergistic site of fibronectin.

Article abstract of DOI:10.1248/cpb.50.1229

A bivalent poly(ethylene glycol) or PEG hybrid of fibronectin-related peptides was prepared. An active site peptide (RGD) and its synergistic site peptide (PHSRN) of fibronectin were conjugated with an amino acid-type PEG (aaPEG) to form PHSRN-aaPEG-RGD.

Full text of DOI:10.1248/cpb.50.1229

September 2002
Chem. Pharm. Bull. 50(9) 1229—1232 (2002)
1229
Preparation and Biological Activities of a Bivalent Poly(Ethylene Glycol)
Hybrid Containing an Active Site and Its Synergistic Site of Fibronectin1)
Yuichi SUSUKI,a Keiko HOJO,a Ikuko OKAZAKI,b Haruhiko KAMATA,c Masahiko SASAKI,a
Mitsuko MAEDA,a Motoyoshi NOMIZU,b Yo ko Y AMAMOTO,c Shinsaku NAKAGAWA,c
c
,a
*
Tadanori MAYUMI, and Koichi KAWASAKI
a Faculty of Pharmaceutical Sciences and High Technology Research Center, Kobe Gakuin University; Ikawadani-cho,
Nishi-ku, Kobe 651–2180, Japan: b Graduate School of Environmental Earth Science, Hokkaido University; Sapporo
060–0810, Japan: and c Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka
University; 1–6 Yamadaoka, Suita, Osaka 565–0871, Japan. Received April 15, 2002; accepted June 8, 2002
A bivalent poly(ethylene glycol) or PEG hybrid of fibronectin-related peptides was prepared. An active site
peptide (RGD) and its synergistic site peptide (PHSRN) of fibronectin were conjugated with an amino acid-type
PEG (aaPEG) to form PHSRN–aaPEG–RGD. A moderate spatial array between RGD and PHSRN in fi-
bronectin may be required for synergic activity. The bivalent hybrid exhibited potent cell spreading activity and
exhibited potent anti-metastatic activity in a model of experimental metastasis with B16–BL6 cells in mice. PEG
may serve as a spacer for maintaining the desired spatial array.
Key words poly(ethylene glycol); fibronectin; hybrid; spreading activity; anti-metastatic activity
Bioactive peptides and proteins are the focus of many lar matrix protein. It plays an important biological role in
medical applications, but they have some disadvantages as many cell-surface interactions, mediating cell adhesion, em-
drugs. They are rapidly hydrolyzed with loss of biological bryonic cell migration, and wound healing.9,10) Among the
activities. Furthermore, proteins derived from non-human many active sites in extracellular matrix proteins, the cell-ad-
species are often antigenic. To overcome these disadvan- hesive domain of fibronectin has been well studied.11) An
tages, protein hybrids with natural and artificial polymers RGD sequence located in the 10th type III repeating unit is a
have been studied. Among various polymers, poly(ethylene critical cell-adhesive site for the cell-surface receptor inte-
glycol) (PEG) has been used for preparation of hybrids with grin,11,12) and a PHSRN sequence in the 9th type III repeating
biologically-active proteins, since PEG has low toxicity, low unit, although itself not biologically active, enhances the cell-
immunogenicity and good solubility in both aqueous and or- adhesive activity of RGD.13—15) However, the cell-adhesive
ganic solvents. Pegylation (hybrid formation with PEG) of activity of a recombinant protein corresponding to the 10th
proteins has been well documented and pegylated adenosine type III repeating unit of fibronectin is not stimulated by a
deamidase2) and interferon3) are used in clinical therapeutics. PHSRN peptide. Based upon this evidence, the spatial array
In contrast, relatively few studies have focused on the pegy- between PHSRN and RGD is important for a synergistic in-
lation of small peptides. This may be based upon the assump- teraction.15)
tion that modification of a small bioactive peptide with a
We designed a bivalent PEG hybrid, PHSRN–aaPEG–
large molecule such as PEG would result in a loss of activity. RGD, in which aaPEG functions as a spacer between
However, we prepared PEG hybrids of laminin- and fibro- two functional peptides. aaPEG [H2NCH2CH2NHCOCH2-
nectin-related peptides and proved that PEG is also a promis- (OCH2CH2)nOCH2COOH] was prepared from commercial
ing drug carrier for bioactive small peptides.4,5) These PEG poly(oxyethylene)diglycolic acid 3000 (carboxymethylated
hybrids were pegylated at the carboxyl terminal of the pep- PEG, cmPEG. average MW 2500—3400).16) The flexible
tides. Site-specific pegylation of large proteins is difficult but conformation of PEG may allow the two functional groups to
site-specific pegylation of relatively small peptides is possi- bind properly. As shown in Fig. 2, the PEG hybrid, PHSRN–
ble by careful selection of synthetic strategies. Lu and Felix6) aaPEG–RGD, was prepared on a Rink amide resin17) using a
succeeded in the site-specific pegylation of an interleukin-re- Fmoc-based solid-phase strategy.18) The Fmoc groups were
lated peptide. Furthermore, PEG can function as a flexible removed by treatment with 20% piperidine/dimethylform-
spacer, linking 2 functional groups that allow a structural amide (DMF) for 20 min. Coupling reactions were performed
conformation for optimal biological activity. We designed an by the diisopropylcarbodiimide (DIC)/1-hydroxybenzotria-
amino acid type PEG (aaPEG) as a novel peptide carrier to zole (HOBt) method.19) The side chains of amino acids were
prepare multivalent PEG-peptide hybrids. We were success- protected as follows: His and Asn by a trityl (Trt) group; Ser
ful in forming bivalent PEG hybrids of fibronectin- and
laminin-related peptides (Arg–Gly–Asp–aaPEG–Glu–Ile–
Leu–Asp–Val and Pro–Asp–Ser–Gly–Arg–aaPEG–Tyr–Ile–
Gly–Ser–Arg)7,8) which have two active sites in each protein.
Here, we report the preparation and biological activity of a
bivalent poly(ethylene glycol) hybrid containing an active
site (Arg–Gly–Asp, RGD) and its synergistic site (Pro–His–
Ser–Arg–Asn, PHSRN) of fibronectin.
Fig. 1. Structure of Fibronectin
, Type I module; , type II module; , type III module.
Fibronectin, a glycoprotein, is a multifunctional extracellu-
To whom correspondence should be addressed. e-mail: kawasaki@pharm.kobegakuin.ac.jp
© 2002 Pharmaceutical Society of Japan

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Vol. 50, No. 9
Fig. 2. Synthetic Scheme for PHSRN–aaPEG–RGD
and Asp by a tert-butyl (But) group; and Arg by a 2,2,5,7,8-
pentamethylchroman-6-sulfonyl (Pmc) group.20) Since the
coupling reaction of Fmoc–aaPEG–OH was slow, it was pre-
incubated with DIC and HOBt at 0 °C for 1 h and allowed
to be used in a coupling reaction. The reaction was repeat-
ed once to completion. Fmoc–aaPEG–Arg(Pmc)–Gly–
Asp(OBut)–Rink amide resin (prepared by the manual
method) was treated with 20% piperidine/DMF to form the
H–aaPEG–Arg(Pmc)–Gly–Asp(OBut)–Rink amide resin,
followed by treatment with trifluoroacetic acid (TFA)/thio-
anisole/ethanedithiol (94/3/3). The resulting aaPEG–RGD
was purified by HPLC on a DAISOPAK SP-120-5-ODS-B
column (DAISO Co., Ltd.).
Fig. 3. Cell Spreading Activity of Synthetic Fibronectin-Related Peptide–
PEG Hybrids
Various amounts of synthetic PEG hybrids and fibronectin were coated on 96-well
dishes and dried overnight. After blocking with BSA, BHK cells (5ϫ103/well) were
added and incubated for 45 min. The percentage of cells that had spread (%) was
counted under a microscope. Each value represents the mean of five separate determi-
nations. Triplicate experiments gave similar results.
After introduction of Fmoc–aaPEG–OH on the resin, the
deprotection reaction of the Fmoc group with piperidine be-
came slow and quantitative measurement of deprotection
could not be determined by the Kaiser test (ninhydrin test).21)
Therefore, synthesis of PHSRN–aaPEG–RGD was per- TFA/thioanisole/ethanedithiol (94/3/3). The crude PHSRN–
formed by an automatic peptide synthesizer (ABI 433A) PEG was purified by HPLC on a DAISOPAK SP-120-5-
which facilitated monitoring the deprotection reaction after ODS-B column. Molecular weight of the product (measured
introduction of Fmoc–aaPEG on the resin. The deprotection by TOF-MS) was 4127. Since molecular weight of PHSRN
reaction could be monitored by the peptide synthesizer, mea- portion is 593, the average molecular weight of PEG portion
suring the conductivity of the solvent from the deprotection of TentaGel NH2 was approximately 3500. The molecular
reaction mixture. Synthetic Fmoc–Pro–His(Trt)–Ser(But)– weight of the PEG portion in the resin was larger than what
Arg(Pmc)–Asn(Trt)–aaPEG–Arg(Pmc)–Gly–Asp(OBut)– the manufacturer specified.
Rink amide resin was treated first with 20% piperidine/DMF
The cell spreading activity of the PEG hybrids (aaPEG–
and then with TFA/thioanisole/ethanedithiol (94/3/3) at room RGD, PHSRN–PEG, and PHSRN–aaPEG–RGD) was exam-
temperature for 2 h. The resulting product was purified by ined, using Baby Hamster Kidney (BHK) cells as described
HPLC using a DAISOPAK SP-120-5-ODS-B column fol- previously.13) Fibronectin was used as a positive control.
lowed by an ASAHIPAK GS-320P (ASAHI CHEMICAL
Fibronectin was most effective in terms of cell spreading
INDUSTRY Co., Ltd.) column. The average molecular activity (Fig. 3). The aaPEG–RGD hybrid displayed cell
weight of the purified PHSRN–aaPEG–RGD (measured by spreading activity but PHSRN–PEG lacked significant activ-
time of flight mass spectrometer (TOF-MS)) was 4007. ity. These findings are comparable to previous results ob-
PHSRN–PEG was prepared on TentaGel–NH2 resin (RAPP tained using synthetic peptides.15) The PHSRN–aaPEG–RGD
POLYMER GmbH). The TentaGel–NH2 resin contains PEG peptide had a greater cell spreading activity than aaPEG–
and liberates a peptide–PEG hybrid by TFA treatment after RGD, suggesting that PHSRN synergistically enhances the
the completion of peptide synthesis. According to the manu- cellular activity of RGD in the PEG–bridged compounds.
facturer, the average molecular weight of the PEG portion of These observations are comparable to previous results ob-
TentaGel–NH2 is approximately 3000. Synthetic Fmoc–Pro– tained using recombinant proteins.15) Thus, aaPEG is poten-
His(Trt)–Ser(But)–Arg(Pmc)–Asn(Trt)–TentaGel–NH2 resin tially useful as a spacer and support matrix that allows the
was treated first with 20% piperidine/DMF and then with binding of functional peptides.

September 2002
1231
Fig. 4. Viability of B16—BL6 Melanoma with PEG Hybrids
Fig. 5. In Vivo Anti-metastatic Activity of PEG Hybrids
Cells in MEM(Ϫ) containing 0.1% BSA (2ϫ104/ml) and samples (10 mg/ml) were
admixed at the ratio of 1 to 1, and incubated at room temperature. After 60 min, the
cells were seeded onto culture dishes, and colonies were counted after 1 week. Each
value represents the meanϮS.E.
B16–BL6 melanoma cells (1ϫ105/0.1 ml) and a synthetic PEG hybrid (1 mg/0.1 ml/
mouse) were intravenously injected separately into C57BL/6 mice. The mice were
killed at 14 d after tumor inoculation, and the lungs were removed. The number of sur-
face melanoma colonies on the lungs was counted under a stereoscopic microscope.
Each value represents the meanϮS.E.
Recently many biologically active sequences have been
identified in extra-cellular matrix proteins.22) Some of the ac-
tive peptides may synergistically interact with cellular recep-
tors,23) thus, the bivalent aaPEG hybrid method may be use-
ful for enhancing the synergistic interactions of these pep-
tides.
Since peptides containing the RGD sequence were re-
ported to be inhibitors of experimental metastasis,12) RGD–
peptides are a focus for development of anti-cancer drugs.
The anti-metastatic effect of PHSRN–aaPEG–RGD on ex-
perimental metastasis was examined in mice. Prior to the
metastasis assay, the viability of B16–BL6 incubated with
synthetic hybrids was examined and the results are shown in
Fig. 4. Previous studies indicated that aaPEG was not cyto-
toxic8) and the data in Fig. 4 indicates that the synthetic hy-
brids are not cytotoxic.
hybrid formation may be in part due to a longer half-life in
blood.24) Stability of RGD to enzymatic degradation in blood
may be increased by PEG-hybrid formation.
Based upon these studies, aaPEG will be a useful material
not only to prepare a bivalent PEG-hybrid, but also to prepare
a multivalent PEG-hybrid and a multifunctional PEG-hybrid
of biological peptides.
Experimental
Acid hydrolyses were performed in constant-boiling HCl at 110 °C for
24 h in evacuated tubes. Amino acid compositions of acid hydrolysates were
determined with a Waters Pico·TAG amino acid analyzer and reversed
phase (RP)-HPLC was performed using a Waters 600 with a DAISOPAK
column and gradient systems of CH3CN/water containing 0.05% TFA. TOF-
mass spectra were obtained from a SHIMADZU/KRATOS KOMPACT
MALDI IV spectrometer. 1H-NMR spectra were obtained on a Brucker
DPX-400 (400 MHz) spectrometer. Rink amide resin, protected amino acids
and coupling reagents were purchased from Watanabe Chemical Industries,
Ltd. TentaGel–NH2 resin was purchased from Bio-Medical System, Shi-
madzu Scientific Research Inc. According to the manufacturer, the average
molecular weight of the PEG portion of TentaGel–NH2 is approximately
3000.
The inhibitory effect of the hybrids on experimental
metastasis in mice was examined and the results are shown in
Fig. 5. B16–BL6 cells and the hybrids were intravenously ad-
ministered as separate injections to mice. Separate injections
may increase the variability in response, but will best isolate
General Procedure for Peptide Synthesis by the Manual Solid
the anti-metastatic effect of the sample, avoiding other anti- Phase Method Peptides and hybrids were synthesized on Rink amide or
TentaGel–NH2 resin by a manual method according to the procedure
metastatic factors. The mice were sacrificed 14 d after tumor
inoculation, and the lungs were removed. The number of sur-
face melanoma colonies on the lungs was counted with a
shown below. Coupling reactions were checked by the Kaiser test (nin-
hydrin test).21)
Step
1
Reagents
Reaction time
1 h
stereoscopic microscope. As shown in Fig. 5, when com-
pared to control values, hybrids appear to have a dose-depen-
dent inhibitory effect. Although RGD, which is an inhibitor
of experimental metastasis, did not show an inhibitory effect
at a dose of 3 mg(5.33 mmol)/mouse, PHSRN–aaPEG–RGD
and aaPEG–RGD did show a similar inhibitory effect at 3
mg/mouse. Since each 3 mg of PHSRN–aaPEG–RGD and
aaPEG–RGD corresponds to 0.67 mmol and 0.78 mmol, the
inhibitory effect of RGD is potentiated by the PEG-hybrid
formation. These results are comparable to previous studies
of PEG-hybrid formation with RGD.5) Although significant
difference in cell spreading activity was observed, a sig-
nificant difference in the anti-metastatic effect between
PHSRN–aaPEG–RGD and aaPEG–RGD was not observed.
The relationship between the anti-metastatic effect and cell
spreading activity of RGD is not explained by these results.
Fmoc–amino acid (3 eq) in DMF
1 M DIC/DMF (3 eq) in DMF
1 M HOBt (3 eq) in DMF
DMF
2
3
2 minϫ7
3 minϫ3
20 minϫ1
2 minϫ7
20% piperidine/DMF
4
DMF
H–aaPEG–Arg–Gly–Asp–NH2 Fmoc–aaPEG–Arg(Pmc)–Gly–
Asp(OBut)–Rink amide resin was prepared from 330 mg of Rink amide
resin (amino content 0.11 mmol) following the above procedure (manual
method). Fmoc–aaPEG–OH was preincubated with DIC and HOBt at 0 °C
for 1 h and allowed to be used in a coupling reaction. Since reaction of
Fmoc–aaPEG–OH (3 eq) was slow, it was reacted with H–Arg(Pmc)–Gly–
Asp(OBut)–Rink amide resin for 10 h and the reaction was repeated once.
Then the Fmoc group was removed with 20% piperidine/DMF and the
resulting resin (580 mg) was treated with a mixture of TFA/thioanisole/
ethanedithiol (94/3/3) at room temperature for 2 h. The resin was removed
by filtration and the TFA was removed by evaporation. The residue was
The potentiation of anti-metastatic effect of RGD by PEG- washed with ether (3 times) and ethyl acetate (3 times), and dried. The mate-

1232
Vol. 50, No. 9
thetic PEG hybrid (1 mg/0.1 ml/mouse) were intravenously injected sepa-
rately into C57BL/6 mice. The mice were killed at 14 d after tumor inocula-
tion, and the lungs were removed. The number of surface melanoma
colonies on the lungs was counted under a stereoscopic microscope.
rial was purified by HPLC on a DAISOPAK SP-120-ODS column. Yield
123 mg (33%, calculated from NH2 content of the used resin). Amino acid
ratios in an acid hydrolysate: Arg 0.99, Gly 1.00, Asp 1.06 (average recovery
98%). TOF-MS m/z 3355(average).
H–Pro–His–Ser–Arg–Asn–PEG Peptide was constructed on TentaGel–
NH2 resin (500 mg, amino content 0.11 mmol) according to the above proce-
dure (manual method). The synthetic Fmoc–Pro–His(Trt)–Ser(But)–
Arg(Pmc)–Asn(Trt)–TentaGel–NH2 resin (610 mg) was treated with 20%
piperidine/DMF, followed by treatment with a mixture of TFA/thioanisole/
ethanedithiol (94/3/3). The resulting crude H–Pro–His–Ser–Arg–Asn–PEG
was purified by HPLC on a DAISOPAK SP-120-ODS column. Yield 220 mg
(45% , calculated from NH2 content of the used resin). Amino acid ratios in
an acid hydrolysate: Pro 1.00, His 1.01, Ser 0.87, Arg 0.93, Asp 1.00 (aver-
age recovery 96%). TOF-MS m/z 4127 (average).
References and Notes
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The Cell Spreading Activity The activity was examined using Baby
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plastic tissue culture plates were coated with various amounts of PEG-pep-
tides or fibronectin, and dried overnight. The plates were subsequently
blocked by 3% bovine serum albumin (BSA, Sigma) in Dulbecco’s modified
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the coated wells at 5ϫ103 cells/well. After a 45 min incubation at 37 °C, the
cells were fixed with 3% formaldehyde and the percentage of spreading
BHK cells was counted under a phase contrast microscopy.
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Viability of B16-BL6 Melanoma Admixed with PEG Hybrids Cells
in MEM(Ϫ) containing 0.1% BSA (2ϫ104/ml) and samples (10 mg/ml)
were mixed in the ratio of 1 to 1, and incubated at room temperature. After
60 min, the cells were seeded onto culture dishes, and colonies were counted
after 1 week. Each value represents the meanϮS.E.
Metastasis Assay B16–BL6 melanoma cells (1ϫ105/0.1 ml) and a syn-