Novel proteoglycan glycotechnology: Chemoenzymatic synthesis of chondroitin sulfate-containing molecules and its application

Article abstract of DOI:10.1007/s10719-009-9252-y

Proteoglycans consist of a protein core, with one or more glycosaminoglycan chains (i.e., chondroitin sulfate, dermatan sulfate and heparin sulfate) bound covalently to it. The glycosaminoglycan chains account for many of the functions and properties of p

Full text of DOI:10.1007/s10719-009-9252-y

Glycoconj J (2010) 27:189–198
DOI 10.1007/s10719-009-9252-y
Novel proteoglycan glycotechnology: chemoenzymatic
synthesis of chondroitin sulfate-containing
molecules and its application
Masanori Yamaguchi & Keiichi Takagaki &
Kaoru Kojima & Naohiro Hayashi & Fengchao Chen &
Ikuko Kakizaki & Atsushi Kon & Masahiko Endo
Received: 8 January 2009 /Revised: 12 June 2009 /Accepted: 24 June 2009 /Published online: 9 July 2009
#
Springer Science + Business Media, LLC 2009
Abstract Proteoglycans consist of a protein core, with one
or more glycosaminoglycan chains (i.e., chondroitin sulfate,
dermatan sulfate and heparin sulfate) bound covalently to it.
The glycosaminoglycan chains account for many of the
functions and properties of proteoglycans. The develop-
ment of proteoglycan glycotechnology to exploit the
functionality of glycosaminoglycan chains is an extremely
important aspect of glycobiology. Here we describe an
efficient and widely applicable method for chemoenzymatic
synthesis of conjugate compounds comprising intact long
chondroitin sulfate (ChS) chains. An alkyne containing
ChS was prepared by an enzymatic transfer reaction and
linked with a chemically synthesized core compound
containing an azido group using click chemistry. This
method enabled highly efficient introduction of ChS into
target materials. Furthermore, the ChS-introduced com-
pounds had marked stability against proteolysis, and the
chemically linked ChS chain contributed to the stability of
these core compounds. We believe the present method will
contribute to the development of proteoglycan glycobiology
and technology.
.
.
Keywords Chondroitin sulfate Click chemistry
.
.
Glycosaminoglycan Neo-proteoglycan Proteoglycan
Abbreviations
AMC
Boc
7-amino-4-methylcoumarin
t-butoxycarbonyl
BSA
ChS
bovine serum albumin
chondroitin sulfate
DCC
DMT-MM
dicyclohexylcarbodiimide
4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-
M. Yamaguchi (*)
Department of Organic Chemistry, Faculty of Education,
Wakayama University,
4
-methyl-morpholinium chloride
EDC
1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide
9
30, Sakaedani,
Wakayama 640-8510, Japan
e-mail: masayama@center.wakayama-u.ac.jp
ESI-MS-MS electrospray ionization tandem mass
spectrometry
:
:
:
:
:
GAG
Gal
glycosaminoglycan
galactose
N-acetylgalactosamine
glucuronic acid
high-performance liquid chromatography
N-methylmorpholine
reverse phase
size exclusion chromatography
triethylamine
M. Yamaguchi K. Takagaki K. Kojima F. Chen I. Kakizaki
:
A. Kon M. Endo
Department of Glycotechnology,
Center for Advanced Medical Research,
Hirosaki University Graduate School of Medicine,
GalNAc
GlcA
HPLC
NMM
RP
SEC
TEA
Xyl
5
, Zaifu-cho,
Hirosaki 036-8562, Japan
N. Hayashi
Otsuka Chemical Co., Ltd.,
63, Kagasuno, Kawaguchi-chio,
Tokushima 771-0193, Japan
4
xylose

1
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Glycoconj J (2010) 27:189–198
Introduction
Results
Proteoglycans are present in the tissues of many mamma-
lian species, both at the cell surface and in the extracellular
matrix. These compounds consist of a protein core, which
has one or more glycosaminoglycan side chains bound
covalently to it. The highly sulfated glycosaminoglycan
chondroitin sulfate (ChS) is a composite element of
proteoglycan in cartilage [1]. ChS has various important
biological roles in cell migration, recognition, and morpho-
genesis [2]. ChS has many types of structural domains that
are known to participate in specific physiological functions.
Recently, greater attention has been directed towards the
functions of ChS in the brain, optic nerves and chondro-
cytes [3, 4].
Recent advances in gene engineering have now made it
possible to mass-produce extremely useful proteins, but the
technology involved is not ideal for the creation of
glycoproteins. This is because DNA incorporated by gene
recombination has no direct information about biosynthesis
of the carbohydrate chains. Many reports have described
that those proteins have few biological activities because of
the incompletion of carbohydrate chains [5, 6].
The saccharide moieties of glycoproteins contribute to their
solubility and thermal stability, and to protection against
proteolysis [7]. Therefore, to study the functionality and
applications of ChS in detail, there is a need to develop a
method for probing ChS and attaching it to peptides and
proteins. Unfortunately there are a number of obstacles to
achieving this. Although a method for effective chemical
synthesis of the linkage region tetrasaccharide [i.e.,
GlcAβ(1-3)Galβ(1-3)Galβ(1-4)Xyl] [8] and ChS oligosac-
charides has been reported [9, 10], it is difficult to supply an
intact ChS, including the linkage region tetrasaccharide,
employing organic synthesis. In addition, it is not possible to
synthesize an artificial proteoglycan consisting of an intact
ChS and core protein by organic synthesis alone.
ChS probe design and synthesis strategy
Our strategy in the present study consisted of three steps.
The first step was the preparation of alkyne-containing ChS
1 using an enzymatic method. In order to do this, we
employed peptide chondroitin sulfate (Peptide-ChS) as a
donor, which was prepared from proteoglycan derived from
salmon nose cartilage [26]. Use of proteoglycan derived
from a non-mammalian source reduces the risk of con-
tamination with pathogenic agents. The peptide-ChS was
treated with endo-β-xylosidase [22] in the presence of
propargyl alcohol. This enzyme specifically cleaved the β-
xyloside linkage between the reducing end xylose and the
hydroxyl group of the Ser side chain, and at the same time
introduced the alkyne into the xylose in accordance with its
transglycosylation activity [25]. There are several advan-
tages of using propargyl alcohol as an acceptor, as the
transglycosylation yield is better than that of a peptide or
protein. In our previous study, we had tried direct trans-
glycosylation of ChS to the target peptide and protein of the
Ser residue, but the yield of the target compounds was very
low; when peptide and protein were used as acceptors, the
yields were >5% and 0%, respectively [25]. Furthermore, it
is not necessary to consider degeneration of the acceptor
material and the reaction is easily quenched by boiling.
Also, any residual excess propargyl alcohol can easily be
removed by lyophilization (Scheme 1). The second step
was the synthesis of a core compound containing an azido
group in order to introduce the alkyne-containing ChS. For
synthesis of the compound, we employed 4-azido benzoic
acid (Tokyo Kasei Kogyo Co., Japan) as the azidolation
reagent. The reagent was introduced into the target
compound by forming an ester and an amide using a
carbodiimide reagent (DCC, EDC) and a non-carbodiimide
reagent (DMT-MM; 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-
methyl-morpholinium chloride), as appropriate [25, 27]
(Scheme 2). The last step was the ligation reaction for ChS
1 and the core compound 2, utilizing click chemistry to
afford the target ChS-linked compound 3. The conditions of
click chemistry afford superior regioselectivity, high tolerance
of other functionalities, and almost quantitative transforma-
tion under mild conditions [25, 28–31] (Scheme 2).
Several strategies for glycopeptides and glycoproteins
have been reported [11–19], but these methods are difficult
to use for proteoglycans. Because the molecular weight
4
of ChS ranges from 6.5 to 3.5×10 , and furthermore its
hydroxyl groups are highly and randomly sulfated [2, 20], it
is difficult to artificially reproduce the ChS in homogeneity
that is found in the natural compounds. Therefore, to
address these problems, we have developed original
approaches for proteoglycan glycotechnology [21–24],
and have recently applied it for effective synthesis of neo-
proteoglycans [25].
Synthesis of azido group-containing fluorogenic peptide
substrate 5 for trypsin
Here we report a highly efficient chemoenzymatic method
for synthesizing several types of novel ChS-containing
compounds. Furthermore, with a view to potential application
of the synthesized ChS-containing compounds, we also
describe their proteolytic stabilities.
The title compound 5 was synthesized to investigate the
influence of the introduction of a long ChS chain on trypsin
digestion. The fluorogenic peptide, peptidyl-4-methylcumaryl-
7-amide (MCA), consisted of three amino acid residues: Phe,
Ser and Arg. The N-terminal amino group was acylated with

Glycoconj J (2010) 27:189–198
191
Scheme 1 Enzymatic
preparation of alkne containing
ChS 1. a actinase E, 0.1 M
Tris-HCl buffer (pH 8.0) con-
taining 10 mM CaCl
2
, 40°C,
4
8 h. b endo-β-xylosidase,
propargyl alcohol, 0.1 M
sodium acetate buffer (pH 5.0),
3
7°C, 24 h
4-azido benzoic acid in order to prepare a site for introduction
293.0 indicated that the 4-azido benzoic acid introduced the
of the alkyne containing ChS 1 by click chemistry. The
synthetic starting material, Boc-Phe-Ser-Arg-MCA 4 [32],
was purchased from Peptide Institute Inc., Japan. The Boc
group of this peptide was removed by treatment with TFA :
CH Cl =1 : 3, and the resulting N-terminal amino group was
acylated with 4-azido benzoic acid employing DMT-MM and
N-methylmorpholine (NMM) in MeOH [27]. After purifica-
tion, the target peptide was obtained at 65% yield (2 steps;
Scheme 3). The characterization of 5 was performed by
NMR, amino acid analysis and ESI-MS-MS. The MS-MS
data of the precursor ion that matches the molecular mass of
compound 5 were extensively analyzed to identify the site of
attachment of 4-azido benzoic acid. The fragment ion of m/z
N-terminal of compound 5 (Fig. 1).
Synthesis and analysis of fluorogenic peptide-ChS 6 and 7
The click chemistry reaction to ligate fluorogenic peptide 5
with ChS 1 was carried out under mild conditions (room
temperature, 3 h) using 10 mM CuSO with 10 mM sodium
ascorbate as the in situ reducing agent to generate the Cu (I)
species [30] (Scheme 4). The resulting products were
subjected to HPLC (TSK-gel ODS-100 V; RP-column and
Showdex OH pack SB-804 HQ; SEC column) (Fig. 2a, b).
The RP-column chromatograms showed that the peak of
peptide 5 disappeared completely and that a new peak
2
2
4
Scheme 2 Chemical ligation of
ChS 1 and azido group-
containing core compounds 2

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Glycoconj J (2010) 27:189–198
Scheme 3 Synthesis of azido
group-containing fluorogenic
peptide 5. a TFA, CH
h b DMT-MM, NMM, MeOH,
r.t., 24 h, two steps 65%
2 2
Cl , 0°C,
2
appeared at an early elution time, resulting from binding of
the highly polar ChS 1 to the peptide 5. Judging from these
results, the yield of fluorogenic peptide-ChS 6 was almost
quantitative (Fig. 2a). The molecular weight of the peptide
showed a pronounced increase after the click chemistry
reaction, as judged from the SEC column chromatogram
After exhaustive digestion, the reaction mixture was
subjected to HPLC (Polyamine-II column), and the result-
ing profile is shown in Fig. 2c. Three peaks (I, II and III)
were observed. The fraction corresponding to peak I in
the reaction mixture was isolated by HPLC, desalted by
dialysis, and subjected to ESI mass spectrometry. The
−
(
Fig. 2b). After purification, 6 was subjected to cellulose
spectrum of peak I showed a peak at m/z 1836.0 [M-3H] .
acetate membrane electrophoresis and stained with alcian
blue, which detects glycosaminoglycans. Compound 6 was
clearly stained as a single band (Fig. 3). Furthermore, 6 was
digested with chondroitinase ABC (protease-free), which
acts on the GlcAβ(1–3)GalNAc sulfate linkage and con-
verts ChS to 4,5-unsaturated disaccharides, except for the
reducing-end hexasaccharide (i.e., 4,5-unsaturated GlcA-
The molecular mass of 1836.0 was in accord with the
theoretical value for 4,5-unsaturated GlcA-GalNAc(OSO H)-
3
GlcA-Gal-Gal-Xyl-fluorogenic peptide 7 (Fig. 4). Peaks II
and III were assigned to 4,5-unsaturated GlcAβ(1–3)GalNAc
6-O-sulfate and 4-O-sulfate respectively, using an Unsaturat-
ed Chondro-Disaccharide Kit. These results indicated that
the intact long ChS chain had bound to the core compound
efficiently through the click chemistry reaction.
GalNAc(OSO H)-GlcA-Gal-Gal-Xyl) [33] (Scheme 4).
3
Fig. 1 ESI-MS-MS spectrum of azido group-containing fluorogenic peptide 5

Glycoconj J (2010) 27:189–198
193
Scheme 4 Synthesis of
fluorogenic peptide-ChS 6 and
7
4
. a 10 mM CuSO , 10 mM
sodium ascorbate, r.t., 3 h,
quant. The yield depended on
peptide 5. b chondroitinase
ABC (0.5 U), 0.4 M Tris-HCl
buffer (pH 8.0), 0.4 M sodium
acetate, 37°C, 24 h
Km values of trypsin for the three synthetic compounds
digested by trypsin, but 6, bearing a ChS, showed marked
resistance to trypsin digestion. 7, which contained hexasac-
charide, also showed resistance to trypsin digestion, although
not to such a marked extent as substance 6. Nevertheless, the
resistance was significant in comparison with 5. On the other
hand, the mixed ChS 1 and peptide 5 that were not linked to
each other did not show marked resistance to digestion in
comparison with the ChS-linked peptide (Fig. 6).
5
, 6 and 7
The Km values for the three synthetic compounds 5, 6 and
−
5
−4
7
1
were calculated as 8.4×10 M, 5.7×10 M and 3.8×
−4
0 M, respectively (Table 1). The Km values of trypsin for
these synthetic compounds decreased with the increasing
attached saccharide chain length of the peptide. These
results suggested that introduction of the long ChS into the
peptide largely decreased the affinity for trypsin.
Discussion
Stability of four synthetic compounds 4, 5, 6 and 7 against
protease digestion
We have developed a chemoenzymatic method for intro-
duction of intact ChS into target materials. The method
consists of three simple steps and can efficiently provide
purpose-made ChS conjugate compounds. In our previous
study, we succeeded in constructing ChS-bound polyethylene
glycol and BSA, but did not conduct biological assays [25].
Our present objective is to clarify the relative stabilities of
core materials in relation to the attached long ChS chain and
oligosaccharide. To achieve this purpose, it is necessary to
develop novel ChS-bound probes. Accordingly, we have
synthesized fluorogenic peptide 5, fluorogenic peptide-ChS
We investigated the resistance of the ChS-bound peptide
against protease (trypsin) digestion. To evaluate the stabiliza-
tion of the peptide, we employed four types of substrate for
trypsin (4, 5, 6 and 7) (Scheme 4). Each substrate was
digested by trypsin, and the released 7-amino-4-methylcou-
marin (AMC) was measured on a spectorofluorometer. The
relative activity toward compound 4 is shown in Fig. 5. In
this case, the low activity indicated that the compound was
resistant to trypsin digestion. Compound 5 was promptly

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Glycoconj J (2010) 27:189–198
Fig. 2 HPLC profiles of compound 5, 6 and 7. a Reversed-phase
HPLC (TSKgel ODS 100 V) profile of compound 5 was shown by
the solid line and 6 was shown by the dotted line. b Size exclusion
chromatography HPLC (Shodex OHpak SB-804 HQ) profile of
compound 5 was shown by the dotted line and 6 was shown by the
solid line. c HPLC (YMC Pollyamine II) profile of chondroitinase
ABC digestion products of compound 6. Peaks marked with I, II
and III indicate the compound 7, 4,5-unsaturated GlcAβ(1–3)
GalNAc 6-O-sulfate and 4,5-unsaturated GlcAβ(1–3)GalNAc 4-O-
sulfate respectively
R
6
and 7, whose relative stabilities can be easily evaluated.
The use of these synthetic probes has allowed us for the first
time to clarify the relative stabilities of core materials to
protease digestion. We found that a long ChS chain
decreased the affinity of the substrate to protease. This was
enough to stabilize the peptide against protease digestion,
compared with a short saccharide chain (i.e., hexasaccharide),
and demonstrated the importance of direct binding of the
ChS chain to the peptide for effective protection against
protease digestion. In addition, we prepared a 4,5-unsaturated
GlcA-Gal-Gal-Xyl-fluorogenic peptide by digestion of 7 with
chondroitinase AcII [33]. We then investigated the suscep-
tibility of the fluorogenic peptide-tetrasaccharide against
protease digestion. There was no significant difference in
comparison with 7. These results have important implica-
tions for controlling the stability of core materials.
As another approach, to obtain alkyne-containing gly-
cosaminoglycan (GAG) oligosaccharides, we tried to trans-
glycosylate GAG oligosaccharides to propargyl alcohol
utilizing the transglycosylation activity of bovine testicular
hyaluronidase [34]. However, no alkyne-containing GAG
oligosaccharides were obtained. Therefore it appears that
propargyl alcohol is not an appropriate acceptor for the
transglycosylation reaction of hyaluronidase.
In conclusion, our present method has made it possible
to manipulate long intact glycosaminoglycans with prede-
signed properties. This proteoglycan glycotechnology will
be applicable for comprehensive research on biological
phenomena that involve glycosaminoglycans. For instance,
it would be useful for high-throughput analytical techniques
such as carbohydrate microarrays, surface plasmon reso-
nance analysis (SPR), and quartz crystal microbalance
(QCM). In addition, our results suggest that ChS-conjugated
therapeutic peptides and proteins can be made to acquire
resistance to proteolysis, which would improve their per-
formance as medicines from two viewpoints. First, their
resistance to proteolysis would extend their biological half-
life, and second, their binding to specific tissues would enable
them to deliver drugs precisely to a target site. Thus, addition
of ChS would improve the biopharmaceutical properties of
drugs. The approach proposed here will be broadly applicable
for utilization of ChS.

Glycoconj J (2010) 27:189–198
195
Fig. 3 Electrophoretogram of
compound 6 on cellulose acetate
membrane. The staining of
chondroitin sulfate on the cellu-
lose acetate membrane was
carried out with 0.05% alcian
blue in 70% ethanol. Lane 1;
chondroitin 6-O-sulfate from
shark cartilage. Lane 2;
ChS (donor) and alkyne containing ChS 1 (from salmon
cartilage, average molecular mass, 8100) were prepared
from proteoglycan using the procedure described in our
previous reports [25, 26]. Chondroitinase ABC protease-
free (from Proteus vulgaris), chondroitin 6-O-Sulfate (from
shark cartilage, average molecular mass, 64000) and
unsaturated Chondro-Disaccharide Kit were purchased
from Seikagaku Kogyo Co. (Tokyo Japan). Trypsin (from
bovine pancreas, 10,000 BAEE units/mg protein) was
purchased from Sigma-Aldrich (St. Louis, MO). All
chemicals used were obtained from commercial sources.
compound 6
1
13
H and C NMR spectra were recorded with a Buruker
1
13
AV500 spectrometer H (400 Hz) and C (100 Hz). The
values of δ (ppm) are given relative to Me Si as the internal
4
standard.
ESI-MS and MS-MS spectra were measured using a PE
SCIEX, API 100 and 300. The samples were dissolved in
0.1% formic acid-containing acetonitrile (50 : 50) and
injected at 2µL/min with a micro-HPLC syringe pump.
Amino acids were analyzed by a Hitachi L-8500 amino
acid analyzer.
HPLC A high-performance liquid chromatography (Hitachi
L-6200, Hitachi Co., Tokyo, Japan) connected to a fluores-
cence detector (Hitachi F-1050) or a UV-detector (Hitachi L-
Experimental section
4200) was used. Purification and analysis of the synthetic
General methods
peptide and peptide-ChS were carried out using reverse-phase
chromatography TSKgel ODS-100 V (150×4.6 mm, TOSOH
Co., Tokyo, Japan) and size exclusion chromatography
Shodex OH pack SB-804 HQ (300×8.0 mm, Showa Denko
Endo-β-xylosidase was isolated from Patnopecten mid-gut
gland, as described by our previous report [22]. Peptide-
Fig. 4 ESI-MS spectrum of compound 7

1
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Glycoconj J (2010) 27:189–198
Table 1 Km values of trypsin for three substrates 5, 6 and 7
Substrate
Km (M)
8.4×10−
5.7×10−
3.8×10−
5
4
4
Fluorogenic peptide 5
Fluorogenic peptide-ChS 6
Fluorogenic peptide-hexasaccharide 7
Co., Kawasaki, Japan). The flow rate was 1 ml/min and
column temperature 40°C. The eluates were monitored at
excitation and emission wavelengths of 330 and 400 nm,
respectively. The elution conditions were described as
follows: TSKgel ODS=100 V column, elution buffer A:
0
.1% TFA in H O; elution buffer B: 0.1% TFA in MeCN.
2
Fig. 6 The relative activity of trypsin toward compound 5 in the
presence of different concentrations of ChS 1. 0 w% ChS, open
squares; 0.5 w% ChS, closed triangles; 1 w% ChS, open circles
The linear gradient was A : B=80 : 20 to A : B=50 : 50 after
0 min. SB-804 HQ column was eluted with 0.2 M NaCl :
3
MeOH=80 : 20.
Analysis and purification of chondroitinase ABC diges-
tion products were carried out with a Polyamine II column
in CH Cl (1 mL) was added to the reaction mixture. The
2 2
reaction mixture was concentrated, the residual syrup was
treated with 4-azido benzoic acid (4.67 mg, 28.6µM),
DMT-MM (15.7 mg, 5.71µM) and NMM (4.5µL, 34.3µM)
in MeOH (1.1 mL), and the mixture was stirred for 24 h at
(
250×4.6 mm, YMC Co., Tokyo, Japan) at a flow rate of
ml/min and a column temperature of 40°C. The eluates
were monitored by absorbance at 232 nm. Elution buffer A:
0 mM NaH PO ; elution buffer B: 1 M NaH PO . The
1
1
room temperature under a N atmosphere, and then
2
2
4
2
4
linear gradient was A : B=100 : 0 to A : B=60 : 40 after
concentrated. The obtained residue was purified on HPLC
4
0 min.
(TOSO TSKgel ODS-100 V) to yield 5 (11.0 mg, 65%) as
Cellulose acetate membrane electrophoresis was carried
out using Separax (6×22 cm, Jookoo Co., Tokyo, Japan) in
formic acid-pyridine buffer (0.1 M, pH 3.0) at 1 mA/cm for
an amorphous mass. RP-HPLC: t
solid line) and SEC-HPLC: t =15.62 min (Fig. 2b: dotted
line). H NMR 400 MHz (CD OD): δ=7.84–7.09 (12 H,
R
=23.02 min (Fig. 2a:
R
1
3
1
0 min. Staining of ChS on the cellulose acetate membrane
Ar), 6.27 (s, 1 H), 4.79 (m, 1 H), 4.61 (m, 1 H), 4.44 (m, 1
was carried out with 0.05% alcian blue in 70% ethanol.
H), 3.97 (m 1 H), 3.85 (dd, 1 H, J=4.8 and 8.8 Hz), 3.28–
3
.24 (m, 2 H), 3.14 (dd, 1 H, J=7.6 and 10.8 Hz), 2.47 (s, 3
13
Azido group-containing fluorogenic peptide 5
H), 2.13 (m, 1 H), 1.87 (m, 1 H), 1.76 (m, 2H). C NMR
00 Hz (CD OD): δ=174.45, 172.81, 172.26, 163.10,
1
3
To a solution of 4 (15.4 mg, 23.9µM) in CH Cl (1.2 mL)
was added trifluoro acetic acid (0.4 mL) at 0°C. The
reaction mixture was stirred for 2 h at 0°C, then 10% TEA
155.32, 155.28, 155.21, 143.06, 138.59, 130.40, 130.26,
129.56, 127.90, 126.68, 119.92, 117.46, 117.27, 113.79,
108.98, 108.26, 108.19, 93.29, 62.64, 57.33, 57.22, 54.90,
2
2
4
1.96, 41.60, 38.06, 34.86, 29.78, 26.35, 18.50. ESI MS
+
calcd. for C H N O [M + H] 711.3, found 711.4.
3
5 38 10 7
Amino acid analysis by hydrolysis with 6 M HCl at 150°C
for 2 h: Ser1.01Phe Arg1.14.
1
Fluorogenic peptide-ChS 6
To a solution of 1 (3 mg) in H O (300µL) were added 5
2
(
20µL; 10 mM in DMSO), CuSO (40µL; 10 mM) and
4
sodium ascorbate (40µL; 10 mM). The reaction mixture
was vortexed and allowed to react at 37°C for 3 h under a
N atmosphere. Column chromatography (H O : EtOH=9 : 1)
2
2
of the residue on Sephadex G-25 gave the crude target
molecule, and this was purified using a C18-Sep-Pac
Fig. 5 The relative activity of trypsin toward compound 4, 5, 6 and 7.
The activity shows the relative rate of the 7-amino-4-methylcoumarin
cartridge (MeOH : H O=0 : 100 to 50 : 50) to afford 6
2
(
1
AMC) when the compound 4 was completely digested by trypsin is
00%. Compound 4, open squares; compound 5, closed squares;
compound 6, closed circles; compound 7, closed triangles
(1.6 mg, quant. Based on peptide 5). Compound 6 was
analyzed by RP-HPLC (t =16.31 min, Fig. 2a: dotted line)
R

Glycoconj J (2010) 27:189–198
197
and SEC-HPLC (t =7.18 min, Fig. 2b: solid line). Electro-
phoresis of 6 was carried out on a cellulose acetate membrane
of 0.4 mL. The subsequent procedures were the same as
those used in the prior experiment.
R
and stained with alcian blue. The conditions and results are
Acknowledgements We dedicate this paper to the late Prof. Keiichi
Takagaki. We thank Prof. Hironobu Hojo, University Tokai, for the
amino acid analysis. We also thank Dr. Akiharu Ueki and Dr. Jun
Wada for helpful discussion. This work was partly supported by Japan
Society for the Promotion of Science (Gant-in-Aid for Young
Scientists (B) No. 21710230), by the Found for the Promotion of
International Scientific Research and by the Found for Cooperation for
Innovative Technology and Advanced Research in Evolution Area
summarized in Fig. 3.
1
H NMR 400 MHz (DMSO-d ): δ=7.01 (br–d), 5.48
6
(
4
m), 5.03 (m), 4.88 (m), 4.76 (br–d), 4.56 (d, J=4.4 Hz),
.51 (d, J=4 Hz), 3.65–3.16 (m), 3.06 (m), 2.08 (s, AcN).
Fluorogenic peptide-hexasaccharide 7
(CITY AREA).
To a solution of 6 (0.8 mg) in H O (75µL) were added
2
4
00 mM Tris-HCl buffer (pH 8.0, 10µL), 400 mM AcONa
buffer (pH 5.0, 10µL), 0.1% BSA in H O solution (10µL)
2
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boiling water bath for 3 min, followed by filtration. The
resulting solution was purified by HPLC (YMC-Pack
1
2
3
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