A conjugate from a laminin-related peptide, Tyr-IIe-Gly-Ser-Arg, and chitosan: Efficient and regioselective conjugation and significant inhibitory activity against experimental cancer metastasis

Article abstract of DOI:10.1039/a908145c

A laminin-related antimetastatic peptide was conjugated with chitosan, and antimetastatic activity of the peptide-chitosan conjugate was assayed. Chitosan was converted to its organosoluble derivative, 6-O-trityl-chitosan, in 3 steps, and then coupled wit

Full text of DOI:10.1039/a908145c

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A conjugate from a laminin-related peptide, Tyr-Ile-Gly-Ser-Arg,
and chitosan: efficient and regioselective conjugation and
significant inhibitory activity against experimental cancer
metastasis†,1
1
Yasuhiro Nishiyama,*a Tomoko Yoshikawa,a Nobumichi Ohara,a Keisuke Kurita,a Keiko Hojo,b
Haruhiko Kamada,c Yasuo Tsutsumi,c Tadanori Mayumic and Koichi Kawasaki*b
a Department of Industrial Chemistry, Faculty of Engineering, Seikei University,
Musashino-shi, Tokyo 180-8633, Japan
b Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Nishi-ku, Kobe 651-2180,
Japan
c Graduate School of Pharmaceutical Sciences, Osaka University, Yamadaoka, Suita-shi,
Osaka 565-0871, Japan
Received (in Cambridge, UK) 11th October 1999, Accepted 18th February 2000
A laminin-related antimetastatic peptide was conjugated with chitosan, and antimetastatic activity of the peptide–
chitosan conjugate was assayed. Chitosan was converted to its organosoluble derivative, 6-O-trityl-chitosan, in
3 steps, and then coupled with the peptide portion, Ac-Tyr-Ile-Gly-Ser-Arg-βAla-OH (βAla; β-alanine), which
contains a spacer amino acid at the carboxy-terminus. The product was treated with CHCl2CO2H to afford the
desired conjugate, Ac-Tyr-Ile-Gly-Ser-Arg-βAla–chitosan. The peptide was introduced to every 6.3 glucosamine
residues. The conjugate proved to have higher inhibitory activity against experimental lung metastasis of B16BL6
melanoma cells in mice than did the parent peptide.
aceans, by simple deacetylation.9,10 Owing to their low toxicity,
Introduction
biodegradability, and abundance, their applications in various
fields including their use as drug carriers are being studied
extensively.11 Since chitosan has an amino function in every
glucosamine residue, peptide molecules introduced at these
amino groups would be ordered in a comb-like fashion, whereas
the peptide–PEG conjugate is a linear molecule. A comb-like
arrangement might be more advantageous than a linear one to
increase the local concentration of the peptide. Furthermore,
the degree of substitution (d.s.) of the chitosan-conjugate
would be controlled over a wider range than that of linear con-
jugates on demand. The biodegradability and extremely low
toxicity of chitosan are also attractive in view of its possible use
as a therapeutic agent for humans. Under these circumstances,
we have focused our attention on the potential of chitosan for
use in conjugation with the YIGSR peptide. Chitosan is, how-
ever, insoluble in common reaction media, and is thus rather
intractable. We report here the efficient and regioselective
preparation of the YIGSR–chitosan conjugate, as shown in
Fig. 1, via organosoluble chitosan derivatives, and its enhanced
inhibitory activity on experimemtal cancer metastasis.
Laminin, a 900 kDa trimeric glycoprotein in the basement
membrane, is known to be involved in the invasion and
metastasis of tumour cells.2–4 A synthetic pentapeptide corre-
sponding to a partial sequence of the laminin β1 chain, H-Tyr-
Ile-Gly-Ser-Arg-NH2, was reported to inhibit experimental
metastasis formation.5 It was also reported that peptides con-
taining the Tyr-Ile-Gly-Ser-Arg (YIGSR) sequence inhibited
angiogenesis and tumour growth.6 YIGSR-related peptides
have thus attracted attention as potential candidates for the
development of anticancer and antimetastatic agents, and a
number of modifications have been attempted to enhance their
activity and clinical utility. In particular, conjugation of the
YIGSR peptide with polymeric materials appears to offer
promise in developing highly active antimetastatic agents. For
instance, Kawasaki et al.7,8 reported that hybrid compounds of
the YIGSR peptide and a bioinert polymer, PEG, exhibited a
much higher antimetastatic activity and stability to enzymic
degradation than did the parent peptide.
An amino polysaccharide, chitosan, is highly attractive as
a polymeric carrier for bioactive peptides. Chitosan can be
readily obtained from chitin, which consists of N-acetyl--
glucosamine and occurs to a great extent in shells of crust-
Results and discussion
Preparation of YIGSR–chitosan conjugates
† Amino acids used in this study are of -configuration, except
β-alanine. Abbreviations used in this report for amino acids, peptides,
and their derivatives are those recommended by the IUPAC–IUB
Commission on Biochemical Nomenclature: Biochem. J., 1984,
219, 345. The following additional abbreviations are used: AcOEt,
ethyl acetate; β-Ala, β-alanine; BOP, benzotriazol-1-yloxytris(dimethyl-
amino)phosphonium hexafluorophosphate; 2,6-Cl2Bzl, 2,6-dichloro-
benzyl; DPPA, diphenylphosphoryl azide; d.s., degree of substitution;
GPC, gel-permeation chromatography; TFMSA, trifluoromethane-
sulfonic acid; Tos, p-tolylsulfonyl; Trt, trityl; WSCI, 1-[3-(dimethyl-
amino)propyl]-3-ethylcarbodiimide hydrochloride.
To avoid two possible side-reactions in the direct conjugation of
YIGSR with chitosan, i.e., epimerization and δ-lactam form-
ation of the carboxy-terminal Arg,12 βAla was employed as a
spacer molecule. The peptide moiety containing the spacer
amino acid, Ac-Tyr-Ile-Gly-Ser-Arg-βAla-OH 1, was syn-
thesized by the conventional solution method as outlined in
Scheme 1. Protecting groups employed were as follows: Boc for
α-amino functions; benzyl (Bzl) for the carboxy group of βAla
and the hydroxy function of Ser; p-tolylsulfonyl (Tos) for the
DOI: 10.1039/a908145c
J. Chem. Soc., Perkin Trans. 1, 2000, 1161–1165
This journal is © The Royal Society of Chemistry 2000
1161

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Fig. 1 Structure of the YIGSR–chitosan conjugate.
Table 1 D.s. of conjugates 2a–c
Coupling method
Peptide/-NH2
D.s.
2a
2b
2c
WSCI
DPPA
DPPA
1.0
0.5
1.0
0.10
0.13
0.16
carbodiimide hydrochloride (WSCI, 1.2 equiv.); (2) peptide 1
(0.5 equiv.), DPPA (0.5 equiv.), EtNPri2 (0.5 equiv.); (3) peptide
1 (1 equiv.), DPPA (1.2 equiv.), EtNPri2 (1 equiv.). All coupling
reactions were carried out in a homogeneous DMA solution.
The products were then treated with CHCl2CO2H to remove the
Trt group. The IR spectra of the products, 2a (WSCI method),
2b (DPPA method, 0.5 equiv.), and 2c (DPPA method, 1
equiv.), demonstrated the regioselective introduction of the
peptide at the amino function of chitosan; clear amide bands
and no ester bands were observed. The d.s.-values of conju-
gates were determined by amino acid analysis of their acid
hydrolysates, and are listed in Table 1. The DPPA-mediated
condensation at an equivalent ratio gave the highest d.s.
so far examined. Recently, it was reported that either of
DPPA, diisopropylcarbodiimide–HOBT, WSCI, and 2-(1H-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluorobo-
rate18 failed to conjugate the YIGSR peptide with water-
soluble chitosan in a mixture of DMF and water.19 Our data
clearly indicate that the efficient and regioselective conjugation
was accomplished under mild reaction conditions, proving the
effectiveness of our conjugation strategy using 6-O-protected
chitosan.
Scheme 1 Synthetic scheme for peptide 1. Reagents and conditions: (i)
DPPA, Et3N, DMF; (ii) TFA, anisole; (iii) BOP, Et3N, DMF; (iv) 4 mol
dmϪ3 HCl in AcOEt; (v) BOP, HOBT, Et3N, DMF; (vi) Ac2O, DMF;
(vii) 1 mol dmϪ3 TFMSA–thioanisole in TFA, m-cresol.
guanidino function of Arg; 2,6-dichlorobenzyl (2,6-Cl2Bzl) for
the hydroxy function of Tyr. Boc-Arg(Tos)-OH was coupled
with H-βAla-OBzl by diphenylphosphoryl azide13 (DPPA) to
minimize δ-lactam formation.14 The other couplings were per-
formedwiththeaidofbenzotriazol-1-yloxytris(dimethylamino)-
phosphonium hexafluorophosphate (BOP)15 or BOP–HOBT.
After acetylation of the terminal amino function with acetic
anhydride, all protecting groups were removed with 1 mol dmϪ3
trifluoromethanesulfonic acid (TFMSA)–thioanisole in TFA
containing m-cresol.16 After purification with HPLC, peptide 1
was converted to its hydrochloride to prevent the undesirable
acylation of the guanidino function of Arg during the coupling
with chitosan.
DCC-mediated coupling of peptide 1 with chitosan sus-
pended in DMA failed to yield the peptide–chitosan conjugate,
due to the heterogeneous conditions used. However, regio-
selective introduction of the peptide at the amino function
of chitosan has been accomplished using an organosoluble
chitosan derivative, 6-O-trityl (Trt)-chitosan,17 which has a pro-
tective group at the reactive C-6 hydroxy function. 6-O-Trt-
chitosan was prepared as reported previously;17 chitosan was
treated with phthalic anhydride and then chlorotriphenyl-
methane to give N-phthaloyl-6-O-Trt-chitosan, whose phthaloyl
group was selectively removed with hydrazine hydrate to afford
6-O-Trt-chitosan. The synthetic scheme for the chitosan conju-
gate from 6-O-Trt-chitosan is shown in Scheme 2. To couple
peptide 1 with 6-O-Trt-chitosan, the following three reaction
conditions were attempted: (1) peptide 1 (1 equiv. for -NH2 of
the chitosan derivative), 1-ethyl-3-[3-(dimethylamino)propyl]-
Antimetastatic activity
The inhibitory activity of Ac-Tyr-Ile-Gly-Ser-Arg-βAla–
chitosan 2c, as well as those of chitosan 3‡ and Ac-Tyr-Ile-Gly-
Ser-Arg-βAla-OH 1, on experimental lung metastasis was
examined with B16BL6 melanoma cells in mice. As shown in
Table 2, conjugate 2c showed a significant inhibitory activity at
a dose of 0.08 mg mouseϪ1, whereas chitosan 3 did not show
any inhibition even at 1.0 mg mouseϪ1. Injection of 1.0 mg
mouseϪ1 of peptide 1 also decreased the number of colonies in
the lung to ≈50% of the control. Previously, 0.3 mg mouseϪ1 of
the YIGSR–PEG conjugate was reported to decrease the num-
ber of colonies to ≈50% of the control.7,8 Based on the d.s., 0.08
mg of chitosan-conjugate 2c contains 0.04 µmol of the YIGSR
peptide, while 0.3 mg of the PEG-conjugate contains 0.05 µmol
peptide, and 1.0 mg of peptide 1 corresponds to 1.2 µmol. Thus
‡ To obtain chitosan with a molecular mass similar to that of the con-
jugate, chitosan 3 was regenerated from 6-O-Trt-chitosan with CHCl2-
CO2H as described in the Experimental section.
1162
J. Chem. Soc., Perkin Trans. 1, 2000, 1161–1165

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Scheme 2 Synthetic scheme for conjugates 2a–c. Reagents and conditions: (i) 1 (1 equiv. for -NH2 of chitosan), WSCI (1.2 equiv.), DMA (rt,
overnight); (ii) 1 (0.5 equiv.), DPPA (0.5 equiv.), EtNPri2 (0.5 equiv.), DMA (rt, overnight); (iii) 1 (1 equiv.), DPPA (1.2 equiv.), EtNPri2 (1 equiv.),
DMA (rt, overnight); (iv) CHCl2CO2H (rt, 20 min × 2); (v) Sephadex G-25 column chromatography (3% aq. AcOH) or dialysis (3% aq. AcOH).
βA = βAla.
Table 2 Antimetastatic activity of peptide 1, conjugate 2c, and
chitosan 3
the utilization of peptide–chitosan conjugates in medicinal
applications.
Dose
Experimental
mg
µmol peptide
Number of
colonies
%
mouseϪ1 mouseϪ1
Inhibition
Rf-values of TLC (Kieselgel 60 F254, Merck) refer to CHCl3–
MeOH–AcOH (90:8:2), and spots were detected with UV
light and/or staining with 0.1% ninhydrin in acetone. Optical
rotations were measured with a JASCO DIP-370 polarimeter,
and [α]D-values are in units of 10Ϫ1 deg cm2 gϪ1. HPLC was
conducted with a WATERS 600 system, and A and B in the
mobile phase system refer to 0.05% TFA aq. and 0.05% TFA
in MeCN, respectively. The amino acid compositions of acid
hydrolysates were determined with a WATERS PicoTAG
amino acid analyser. The IR spectra were recorded on a JASCO
IRS-700 instrument. Gel-permeation chromatography (GPC)
was carried out with a JASCO 880-PU connected with a
Shodex RI detector SE-61 [column TSK-GMPWXK × 2,
solvent 0.1% lactic acid (1.0 cm3 minϪ1)] using pullulan as
reference.
Silica gel (WAKO Gel C-200) for column chromatography
was purchased from WAKO Pure Chemical Industries, Ltd.
(Osaka, Japan). DMF, DMA, and EtNPri2 were distilled from
ninhydrin before use. HOBT used was its hydrate. Boc-Arg-
(Tos)-OHؒ4/5AcOEt, DPPA, TFA, anisole, and 4 mol dmϪ3
HCl in AcOEt were purchased from Watanabe Chemical
Industries, Ltd. (Hiroshima, Japan). Chitosan was prepared
from shrimp chitin in the usual manner.21 WSCI was purchased
from Peptide Institute, Inc. (Osaka, Japan). Other reagents were
of reagent grade and used without purification.
Control
Chitosan 3
Peptide 1
220 33.6
265 37.0
141 47.2
108 24.7
104 35.5
77.8 27.2
1.0
0.1
1.0
0.08
0.24
0.12
1.2
0.04
0.12
36
51
53
65
Conjugate 2c
chitosan-conjugate 2c has been confirmed to have a more
potent antimetastatic activity than free peptide 1 and a
comparable activity with the PEG-conjugate.
It is noteworthy that conjugate 2c exhibited higher activity
than did free peptide 1. Generally, conjugation of small bio-
active peptides with polymers has a possible drawback; a
large polymer portion may hinder the peptide–receptor inter-
action. The results of an antimetastatic assay in this study
clearly indicate that the chitosan molecule does not severely
hinder the YIGSR portion from the interaction with the lam-
inin receptor. In previous reports,8,20 the YIGSR–PEG conju-
gate was demonstrated to be much more stable against
enzymic degradation than the free peptide. This might be
one reason for the high antimetastatic activity of the PEG-
conjugate. Although the chitosan-conjugate is structurally dif-
ferent from the PEG-conjugate, a similar effect of protection
against enzymic digestion is expected and may be partly
responsible for the high activity of 2c. It is also notable that
chitosan-conjugate 2c with d.s. 0.16 showed an antimetastatic
potency comparable to that of the PEG-conjugate, whose two
termini are completely substituted with the peptide.8 Chito-
san, unlike PEG, may be conjugated with the YIGSR peptide at
a higher d.s. than that of 2c, if reaction conditions are appro-
priately controlled. The chitosan conjugate with a high d.s. is
undoubtedly favourable for the increase in the local concentra-
tion of the peptide, and might possibly inhibit cancer metastasis
more potently. Appropriate reaction conditions to control the
d.s. of the peptide–chitosan conjugate are now under examin-
ation in our laboratories.
Boc-Arg(Tos)-ꢀAla-OBzl
To an ice-cooled solution of Boc-Arg(Tos)-OHؒ4/5AcOEt (5.3
g, 10 mmol) and H-βAla-OBzlؒTosOH (3.5 g, 10 mmol) in
DMF containing Et3N (1.4 cm3, 10 mmol) were added DPPA
(2.2 cm3, 10 mmol) and Et3N (1.4 cm3, 10 mmol). The mixture
was stirred for 4 h under cooling with ice. After removal of the
solvent, the residue was extracted with AcOEt. The extract was
washed successively with 5% aq. NaHCO3 and water, dried over
Na2SO4, and evaporated. The oily residue in CHCl3 (10 cm3)
was applied to a silica gel (100 g) column, which was equili-
brated with CHCl3 and eluted with CHCl3 (500 cm3) and then
with 1.5% MeOH in CHCl3 (1.9 dm3). The solvent of the efflu-
ent (0.9–2.4 dm3) was removed by evaporation to give a slightly
yellowish amorphous powder (4.6 g, 78%); [α]D25 ϩ3.5 (c 0.5 in
DMF); Rf 0.43 (Found: C, 54.6; H, 6.49; N, 10.9. C28H39-
N5O7Sؒ1.6H2O requires C, 54.4; H, 6.88; N, 11.3%). The amino
acid ratio of the acid hydrolysate was βAla:Arg 1.00:1.09
(average recovery 108%).
Conclusions
The Ac-Tyr-Ile-Gly-Ser-Arg-βAla–chitosan conjugate has been
successfully synthesized on the basis of regioselective modifi-
cation strategy of chitosan. Organosoluble 6-O-Trt-chitosan
was useful to enable the regioselective introduction of the
YIGSR peptide at the amino function of chitosan under mild
conditions, and would be applicable to conjugation with
other bioactive peptides. Conjugate 2c has been shown to
exhibit higher antimetastatic activity than the parent peptide.
This indicates that chitosan is promising as a polymeric carrier
for bioactive peptides. The results obtained here would facilitate
Boc-Ser(Bzl)-Arg(Tos)-ꢀAla-OBzl
To an ice-cooled solution of H-Arg(Tos)-βAla-OBzlؒTFA [pre-
pared from Boc-Arg(Tos)-βAla-OBzl (4.0 g, 6.8 mmol), TFA
(8.0 cm3), and anisole (2.0 cm3) in the usual manner] in DMF
J. Chem. Soc., Perkin Trans. 1, 2000, 1161–1165
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(100 cm3) were added Boc-Ser(Bzl)-OH (2.4 g, 8.1 mmol), BOP
(3.6 g, 8.1 mmol), and Et3N (3.2 cm3, 23 mmol). The mixture
was stirred at rt for 2 h. After removal of the solvent, the resi-
due was extracted with AcOEt. The extract was washed succes-
sively with 5% aq. NaHCO3 and water, dried over Na2SO4, and
evaporated. The oily residue in CHCl3 (30 cm3) was applied to a
silica gel (100 g) column, which was equilibrated with CHCl3
and eluted with CHCl3 (0.6 dm3) and then with 1% MeOH in
CHCl3 (2.8 dm3). The solvent of the effluent (1.4–3.4 dm3) was
removed by evaporation to give a slightly yellowish amorphous
powder (3.3 g, 63%); [α]D25 ϩ1.7 (c 0.5 in DMF); Rf 0.50 (Found:
C, 57.4; H, 6.48; N, 10.4. C38H50N6O9Sؒ1.5H2O requires C,
57.5; H, 6.73; N, 10.6%). The amino acid proportions of the
acid hydrolysate were βAla:Arg:Ser 1.00:0.89:0.83 (average
recovery 90.7%).
in DMF); Rf 0.58 (Found: C, 58.9; H, 6.23; N, 9.68. C62H77-
Cl2N9O13Sؒ0.2H2O requires C, 59.0; H, 6.18; N, 9.68%). βAla:
Arg:Ser:Gly:Ile:Tyr 1.00:0.81:0.57:0.94:0.91:0.95 (average
recovery 96.0%).
Ac-Tyr(Cl2Bzl)-Ile-Gly-Ser(Bzl)-Arg(Tos)-ꢀAla-OBzl
To an ice-cooled solution of H-Tyr(Cl2Bzl)-Ile-Gly-Ser(Bzl)-
Arg(Tos)-βAla-OBzlؒHCl [prepared from Boc-Tyr(Cl2Bzl)-Ile-
Gly-Ser(Bzl)-Arg(Tos)-βAla-OBzl (1.08 g, 0.86 mmol), TFA
(4.0 cm3), and anisole (1.0 cm3) in the usual manner] in DMF
(50 cm3) containing Et3N (0.12 cm3, 0.86 mmol) was added
acetic anhydride (0.10 g, 0.94 mmol). The mixture was stirred
at rt overnight. After removal of the solvent, water was added
to the residue to afford a precipitate, which was collected by
filtration and washed successively with water, EtOH, and
n-hexane; yield 0.76 g (74%); mp range 216–221 ЊC; [α]D25 Ϫ1.6
(c 0.5 in DMF); Rf 0.58 (Found: C, 58.5; H, 5.96; N, 10.1.
C59H71Cl2N9O12Sؒ0.6H2O requires C, 58.5; H, 6.00; N, 10.4%).
Boc-Gly-Ser(Bzl)-Arg(Tos)-ꢀAla-OBzl
To an ice-cooled solution of H-Ser(Bzl)-Arg(Tos)-βAla-
OBzlؒHCl [prepared from Boc-Ser(Bzl)-Arg(Tos)-βAla-OBzl
(3.3 g, 4.25 mmol) and 4 mol dmϪ3 HCl in AcOEt (11 cm3) in
the usual manner] in DMF (80 cm3) were added Boc-Gly-OH
(0.89 g, 5.1 mmol), BOP (2.3 g, 5.1 mmol), and Et3N (2.0 cm3,
14.45 mmol). The mixture was stirred at rt for 2 h. After
removal of the solvent, the residue was extracted with AcOEt.
The extract was washed successively with 5% aq. NaHCO3 and
water, dried over Na2SO4, and evaporated. The oily residue
in CHCl3 (10 cm3) was applied to a silica gel (80 g) column,
which was equilibrated with CHCl3 and eluted successively with
CHCl3 (0.2 dm3), 1% MeOH in CHCl3 (0.5 dm3), and then 2%
MeOH in CHCl3 (1.8 dm3). The solvent of the effluent (1.0–2.5
dm3) was removed by evaporation to give a slightly yellowish
amorphous powder (2.9 g, 83%); [α]D25 ϩ2.5 (c 0.5 in DMF); Rf
0.35 (Found: C, 56.1; H, 6.36; N, 11.0. C40H53N7O10Sؒ2H2O
requires C, 55.9; H, 6.68; N, 11.4%). The amino acid propor-
tions of the acid hydrolysate were βAla:Arg:Ser:Gly 1.00:
0.97:0.80:1.19 (average recovery 99.0%).
Ac-Tyr-Ile-Gly-Ser-Arg-ꢀAla-OHؒHCl 1
To an ice-cooled solution of Ac-Tyr(Cl2Bzl)-Ile-Gly-Ser(Bzl)-
Arg(Tos)-βAla-OBzl (0.24 g, 0.20 mmol) in TFA (3.8 cm3) con-
taining thioanisole (0.59 cm3, 5.0 mmol) and m-cresol (0.17
cm3) was added TFMSA (0.44 cm3, 5.0 mmol). The mixture was
stirred at an ice-bath temperature for 30 min, and then at rt for
90 min. Diethyl ether (50 cm3) and water (20 cm3) were added to
the reaction mixture. The water layer was washed with diethyl
ether (50 cm3 × 5), neutralized with NaHCO3, and applied to a
Sephadex G-10 column (2.8 × 48 cm), which was equilibrated
and eluted with 3% aq. AcOH. Individual fractions (11 cm3)
were collected, and the desired fractions (# 11–20) were com-
bined and lyophilized to give a white fluffy powder (174 mg).
After purification by preparative HPLC [column YMC-D-ODS
(20 × 250 mm), A:B 90:10 to 40:60 in 60 min (10 cm3 minϪ1)],
successive lyophilization from 0.01 mol dmϪ3 aq. HCl gave the
title compound as a white powder (145 mg, 98%); tR 15.87 min
[99.4%, column YMC-R-ODS (4.6 × 250 mm), A:B 95:5 to
20:80 in 45 min (1.0 cm3 minϪ1)]; m/z (FAB) 708 (MHϩ).
Boc-Ile-Gly-Ser(Bzl)-Arg(Tos)-ꢀAla-OBzl
To an ice-cooled solution of H-Gly-Ser(Bzl)-Arg(Tos)-βAla-
OBzlؒTFA [prepared from Boc-Gly-Ser(Bzl)-Arg(Tos)-βAla-
OBzl (2.9 g, 3.6 mmol), TFA (4.0 cm3), and anisole (1.0 cm3) in
the usual manner] in DMF (80 cm3) were added Boc-Ile-OHؒ
1/2H2O (0.94 g, 3.9 mmol), HOBT (0.6 g, 3.9 mmol), BOP (1.7
g, 3.9 mmol), and Et3N (1.6 cm3, 11.4 mmol). The mixture was
stirred at rt for 2 h. After removal of the solvent, the residue
was extracted with AcOEt. The extract was washed successively
with 5% NaHCO3 and water, dried over Na2SO4, and evapor-
ated. Diethyl ether was added to the gelatinous residue, which
was collected by filtration, washed with diethyl ether, and repre-
cipitated from EtOH–diethyl ether (1:10 v/v; 110 cm3); yield
2.8 g (83%); mp range 65–73 ЊC; [α]D25 Ϫ1.6 (c 0.5 in DMF);
Rf 0.47 (Found: C, 58.9; H, 7.04; N, 11.5. C46H64N8O11Sؒ
0.25H2O requires C, 58.7; H, 6.90; N, 11.9%). The amino acid
proportions of the acid hydrolysate were βAla:Arg:Ser:
Gly:Ile 1.00:0.90:0.63:0.96:1.00 (average recovery 102%).
Coupling of peptide 1 with 6-O-Trt-chitosan
WSCI method. To an ice-cooled solution of 6-O-Trt-chitosan
(200 mg, 0.50 mmol equiv. NH2-) and 1 (372 mg, 0.50 mmol) in
DMA (5 cm3) was added WSCI (0.12 g, 0.60 mmol). The mix-
ture was stirred at rt overnight, and then poured into ice-cooled
diethyl ether (400 cm3) to give an off-white precipitate, which
was collected by filtration and washed successively with water
(10 cm3 × 5) and acetone (10 cm3 × 5); yield 267 mg. The IR
spectrum of the product was almost identical with that of the
product obtained by the DPPA method (1 equiv.) (see below).
DPPA method (0.5 equiv.). To a solution of 6-O-Trt-chitosan
(40 mg, 0.10 mmol equiv. NH2-) and 1 (37 mg, 0.05 mmol) in
DMA (2 cm3) were added DPPA (11 mm3, 0.05 mmol) and
1 mol dmϪ3 EtNPri2 in DMF (50 mm3, 0.05 mmol). The mix-
ture was stirred at rt overnight. After removal of the solvent,
the residue was dissolved in MeOH (10 cm3). The solution was
poured into ice-cooled diethyl ether (200 cm3) to give an off-
white precipitate, which was collected by centrifugation and
washed successively with diethyl ether (50 cm3) and water (15
cm3 × 5); yield 38 mg. The IR spectrum of the product was
almost identical with that of the product by the DPPA method
(1 equiv.).
Boc-Tyr(Cl2Bzl)-Ile-Gly-Ser(Bzl)-Arg(Tos)-ꢀAla-OBzl
To an ice-cooled solution of H-Ile-Gly-Ser(Bzl)-Arg(Tos)-
βAla-OBzlؒTFA [prepared from Boc-Ile-Gly-Ser(Bzl)-Arg-
(Tos)-βAla-OBzl (1.00 g, 1.1 mmol), TFA (4.0 cm3), and anisole
(1.0 cm3) in the usual manner] in DMF (50 cm3) were added
Boc-Tyr(Cl2Bzl)-OH (0.56 g, 1.3 mmol), HOBT (0.20 g, 1.3
mmol), BOP (0.57 g, 1.3 mmol), and Et3N (0.5 cm3, 3.6 mmol).
The mixture was stirred at rt for 2 h. After removal of the
solvent, EtOH–water (1:1 v/v; 50 cm3) was added to the residue
to afford a precipitate, which was collected by filtration, washed
with EtOH–water (1:1 v/v), and recrystallized from EtOH (30
cm3); yield 1.1 g (79%); mp range 185–190 ЊC; [α]D25 Ϫ0.1 (c 0.5
DPPA method (1 equiv.). To a solution of 6-O-Trt-chitosan
(200 mg, 0.50 mmol equiv. NH2-) and 1 (372 mg, 0.50 mmol) in
DMA (5 cm3) were added DPPA (0.13 cm3, 0.60 mmol) and
1 mol dmϪ3 EtNPri2 in DMF (0.50 cm3, 0.50 mmol). The mix-
ture was stirred at rt overnight. After removal of the solvent,
1164
J. Chem. Soc., Perkin Trans. 1, 2000, 1161–1165

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the residue was dissolved in MeOH (10 cm3). The solution was
poured into ice-cooled diethyl ether (200 cm3) to give an off-
white precipitate, which was collected by filtration, washed suc-
cessively with diethyl ether (200 cm3) and water (200 cm3), and
reprecipitated from MeOH–diethyl ether (1:10 v/v; 220 cm3);
yield 245 mg; νmax(KBr)/cmϪ1 3386, 1655, 1517, 1489, 1447,
1207, 1156–1000, 914, 761, 700.
Acknowledgements
We are grateful to Asahi Chemical Industry Co. Ltd. for FAB-
MS measurements.
References
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Ac-Tyr-Ile-Gly-Ser-Arg-ꢀAla–chitosan (2a–c)
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2c. The 6-O-Trt-chitosan-conjugate obtained by the DPPA
method (1 equiv.) (240 mg) was dissolved in CHCl2CO2H (10
cm3) and the solution was stirred at rt for 1 h. The solution was
poured into ice-cooled diethyl ether (200 cm3) to give an off-
white precipitate, which was collected by centrifugation and
washed with diethyl ether (20 cm3 × 5). This was treated again
with CHCl2CO2H (10 cm3) as described above. The crude prod-
uct in 3% aq. AcOH (15 cm3) was applied to a Sephadex G-25
column (2.8 × 90 cm), which was equilibrated and eluted with
3% aq. AcOH. Individual fractions (7 cm3) were collected, and
the desired fractions (# 30–46) were combined and lyophilized
to give an off-white fluffy powder (140 mg), νmax(KBr)/cmϪ1
3388, 1654, 1541, 1406, 1251 and 1151–1000. Number-average
molecular mass (determined by GPC): 22.2 × 103. The amino
acid proportions in the acid hydrolysate were Tyr:Ile:Gly:Ser:
Arg:βAla 0.76:0.87:1.00:1.00:0.97:0.78 (peptide content
0.49 mmol gϪ1).
Conjugates 2a and 2b were prepared by similar methods to
that of 2c. 2a: Yield 160 mg. The amino acid proportions in the
acid hydrolysate were Tyr:Ile:Gly:Ser:Arg:βAla 0.83:0.97:
1.00:0.97:0.98:1.01 (peptide content 0.35 mmol gϪ1). 2b: Yield
20 mg. The amino acid proportions in the acid hydrolysate were
Tyr:Ile:Gly:Ser:Arg 0.94:0.90:1.00:1.12:0.92 (peptide con-
tent 0.40 mmol gϪ1). βAla was not determined. The IR spectra
of 2a and 2b were almost identical with that of 2c.
12 R. Geiger and W. König, in The Peptides: Analysis, Synthesis, and
Biology. Vol. 3. Protection of Functional Groups in Peptide Synthesis,
ed. E. Gross and J. Meienhofer, Academic Press, New York, 1981,
pp. 60–70.
Chitosan regenerated from 6-O-Trt-chitosan 3
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A solution of 6-O-Trt-chitosan (500 mg) in CHCl2CO2H (20
cm3) was sitirred at rt for 1 h, and poured into ice-cooled diethyl
ether (200 cm3) to give an off-white precipitate, which was col-
lected by centrifugation and washed with diethyl ether (20
cm3 × 5). The crude product in 3% aq. AcOH (15 cm3) was
applied to a Sephadex G-25 column (2.8 × 90 cm), which was
equilibrated and eluted with 3% aq. AcOH. Individual fractions
(7 cm3) were collected, and the desired fractions (# 31–46) were
combined and lyophilized to give an off-white fluffy powder
(240 mg), νmax(KBr)/cmϪ1 1643, 1384, 1151–1000. Number-
average molecular mass (determined by GPC): 13.8 × 103.
Antimetastatic activity
B16BL6 melanoma cells (1.5 × 105/100 mm3) were inoculated
intravenously into C57BL6 mice, and then 1, 2c, or 3 was
injected intravenously. Mice were killed 2 weeks after the
tumour inoculation and tumour colonies of lungs were counted
with a stereoscopic microscope. Each value represents the
mean S.E. (n = 5). Details of the assay procedure were
described previously.7,8,20
21 K. Kurita, T. Sannan and Y. Iwakura, Makromol. Chem., 1977, 178,
3197.
Paper a908145c
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