Spontaneously cleavable glycosylated linker capable of extended release of its conjugated peptide

Article abstract of DOI:10.1248/cpb.c18-00626

Reversibly glycosylated conjugates were developed by adding complex-type N-linked oligosaccharides to peptides through self-cleavable linkers with the aim of increasing the solubility and stability of the peptides in plasma. The amino or carboxyl group of the peptide was connected to a glycosylated Ascendis or ester/thioester-type linker, respectively. Use of the linkers enabled extended release of the peptides depending on the pH and temperature of the buffer according to a first order reaction, and their cleavage rate was also affected by the structure of the peptide-linker coupling. This tunability will allow optimization towards the intended use of the peptides to be released. Furthermore, because glycosylation is a reliable method of greatly increasing the solubility of a peptide, the presented glycosylated linkers are expected to permit the preparation of antibodies in aqueous buffers even in the case of sparingly soluble antigen peptides.

Full text of DOI:10.1248/cpb.c18-00626

Chemical and Pharmaceutical Bulletin Advance Publication by J-STAGE
DOI:10.1248/cpb.c18-00626
Advance Publication
October 10, 2018
Chem. Pharm. Bull.
Regular Article
Spontaneously Cleavable Glycosylated Linker Capable of Extended Release of its
Conjugated Peptide
†
Hirofumi Ochiai, Kenta Yoshida, Hiroshi Shibutani, Akio Kanatani, and Yuji Nishiuchi*
GlyTech, Inc.; Kyoto Research Park #1-109, 134 Chudoji minami-machi, Shimogyo-ku,
Kyoto 600-8813, Japan.
†
Earth Corporation; 2-12-1 Kanda-Tsukasamachi, Chiyoda-ku, Tokyo 101-0048.
*Correspondence e-mail: yuji-nishiuchi@glytech.jp
Ⓒ
2018 The Pharmaceutical Society of Japan

Summary
Reversibly glycosylated conjugates were developed by adding complex-type N-linked
oligosaccharides to peptides through self-cleavable linkers with the aim of increasing the
solubility and stability of the peptides in plasma. The amino or carboxyl group of the peptide
was connected to a glycosylated Ascendis or ester/thioester-type linker, respectively. Use of
the linkers enabled extended release of the peptides depending on the pH and temperature of
the buffer according to a first order reaction, and their cleavage rate was also affected by the
structure of the peptide-linker coupling. This tunability will allow optimization towards the
intended use of the peptides to be released. Furthermore, because glycosylation is a reliable
method of greatly increasing the solubility of a peptide, the presented glycosylated linkers are
expected to permit the preparation of antibodies in aqueous buffers even in the case of
sparingly soluble antigen peptides.
Keywords self-cleavable linker; extended release; chemical glycosylation; bromoacetamide
method; complex-type asparagine-linked oligosaccharide
Chemical and Pharmaceutical Bulletin Advance Publication

Introduction
Peptides are involved in a wide variety of human physiological functions, including
regulation of the immune system, metabolism and protein degradation, defense against
microorganisms, and signal transmission. In general, biologically active peptides can induce a
rapid and specific beneficial response at very low concentrations when they bind to specific
cell surface receptors or ion channels and trigger intracellular effects. In addition, they are
recognized as being relatively safe and well tolerated. Consequently, peptides that possess
attractive pharmacological profiles and intrinsic properties can be potential therapeutic
1
)
candidates. However, parenterally-administered bioactive peptides often have limited
pharmacological efficacy due to their rapid degradation by enzymes, which leads to a short
circulating plasma half-life. To solve these issues, several strategies that improve the plasma
stability of peptides have been examined, such as polyethylene glycosylation (PEGylation),
acylation/lipidation, and glycosylation as well as traditional rational design with the aid of
2
)
peptide technology. One of the most promising methods is to link a glycan to the peptide of
interest, since glycosylation is generally considered to provide peptides and proteins with
prolonged circulating plasma half-life, improved solubility and/or lowered
3
-6)
immunogenicity.
Taking advantage of the beneficial characteristics of glycans, we have
been focusing on covalently attaching human type N-linked glycans to bioactive peptides to
improve not only their pharmacokinetic properties but also their solubility. However,
glycopeptides developed via this approach sometimes suffer from a significant loss of their
biological or pharmacological activity, especially when the attached glycan(s) have altered
their structures and/or shield their active center, interfering with the interaction with the target
receptors or proteins. To overcome this obstacle, in the present study, we attempted to
develop a reversibly glycosylated peptide by coupling the parent peptide and glycan through
a spontaneously cleavable linker. The resulting extended release of the parent peptide upon
Chemical and Pharmaceutical Bulletin Advance Publication

cleavage of the linker in aqueous physiological conditions indicates that the present strategy
should offer a useful method of enhancing the lifetime of peptides in vivo. Furthermore,
because glycosylation is a reliable method of greatly increasing the solubility of peptides, this
approach could be readily applied to antibody preparation even for antigen peptides that are
sparingly soluble in water.
Results and Discussion
The linkers used to connect peptide to glycan needed to meet the following
prerequisite conditions: they must be stable during the peptide synthesis procedures, but
undergo cleavage at 37 ºC under neutral physiological conditions to gradually release the
7
-9)
peptide in its non-modified, native active form.
Asparagine (Asn)-linked complex-type
1
0)
oligosaccharides, readily obtained from hen egg yolk, were used as a glycan building block
in the subsequent peptide synthesis. Using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry,
incorporation of Fmoc-Asn(Glycan) onto the growing peptide chain was performed without
11)
any protection of its hydroxy groups, except for those of the sialic acid carboxyl groups. In
addition, a bromoacetamidyl glycan without protecting groups on its hydroxyl or carboxyl
groups could be coupled with the sulfhydryl group on the linker molecule to preserve the
native glycan structure. This derivative was prepared by coupling bromoacetic acid with the
amino glycan produced by peptide N-glycosidase digestion, which cleaved the amide bond
between the reducing end of the amino glycan and the carbonyl group of the side functional
1
2)
Asn. This bromoacetamide method allows the structure of the conjugated glycan to be
changed easily, facilitating a high-throughput synthesis system, because its attachment can be
13)
performed via a post-synthetic procedure. A spontaneously cleavable conjugate was
designed and prepared by combining a peptide and N-glycan through an ester, thioester or
Ascendis-type linkage (Fig. 1). The utility of the N-glycan bearing cleavable linkers was
Chemical and Pharmaceutical Bulletin Advance Publication

examined by attaching them to human epidermal growth factor receptor 2 (HER2, 8-16)
1
4)
15)
(
RWGLLLALL), which is sparingly soluble in water, or chemerin-9 (YFPGQFAFS).
Ester linkage
Ester linkages can be expected to be not only gradually cleaved under neutral conditions but
also degraded by esterases within living organisms. 4-(Hydroxymethyl)benzoic acid (HMBA)
was adopted as a linkage to connect the C-terminal of HER2(8-16) or chemerin-9 and the
amino group of Cys-NH
that such an HMBA ester is readily cleavable in alkaline solution. The HER2(8-16) conjugate
was assembled by stepwise SPPS using an Fmoc strategy on
HO-CH C H -CO-Cys(Trt)-Rink amide resin. After completion of the chain elongation,
2
through ester and amide bonds, respectively. It has been reported
2
6
4
trifluoroacetic acid (TFA) treatment afforded the product in free peptide form.
Disialoundecasaccharide (disialo) or asialononasaccharide (asialo) was introduced to the SH
group of the C-terminal Cys by the bromoacetamide method to obtain the glycosylated
conjugate (Chart 1). First, we examined the effect of chemical glycosylation on the solubility
of the HER2(8-16) conjugates. The parent peptide is sparingly soluble in water. Upon
introduction of the glycan to the molecule, however, the product was found to have
dramatically increased solubility, as shown in Table 1.
Next, the self-cleavage behavior of the glycosylated conjugate was assessed by
subjecting chemerin-9-HMBA-Cys(Disialo) conjugate 5 to various conditions. The
chemerin-9 conjugate was synthesized using the same procedure as that for the HER2(8-16)
conjugate. After dissolving compound 5 in a buffer, its recovery rate and the regeneration of
the parent peptide were measured over time by RP-HPLC. A complementary relationship was
found between the decrease in the starting material and the increase in the parent peptide. The
half-life of compound 5 at 37 ºC was determined over 45 h in a phosphate buffered saline
Chemical and Pharmaceutical Bulletin Advance Publication

(
PBS, pH 7.4) and over 0.8 h in a boric-acid buffer (pH 9.0). No cleavage was observed in
sodium acetate buffer (pH 4.0) (Fig. 2). Elevation of the pH and temperature of the buffer
was found to increase the cleavage rate. Furthermore, the ester linkage of compound 5 also
could be cleaved by human carboxylesterases (CES) such as CES1b and CES2, which are
1
6)
abundant in the liver but not in the blood. Their cleavage rate was found to be slower than
expected: the CES digestion only slightly shortened the half-life attributable to the
self-cleavage of compound 5 in PBS (data not shown). This may be due to steric hindrance
with the glycan chain. However, the parent peptide generated upon the ester cleavage was
likely to be rapidly degraded by these enzymes.
Thioester linkage
Thioester linkages are considered to be more labile than ester linkages under neutral
conditions. HER2(8-16) was linked through its C-terminal carboxylic acid to the SH group on
the linker bearing the glycan. Ethane dithiol and 4-mercaptophenylacetic acid were
incorporated as linker moieties and were coupled with the acetamidyl asialoglycan and
Asn(Asialo)-Gly through thioether and amide bonds, respectively (Chart 2). The water
solubility of the resulting thioalkylester and thioarylester conjugates (> 75 mg/mL) was found
to be dramatically improved compared with that of the parent peptide (0.71 mg/mL), as in the
case of the ester-type conjugate. However, the cleavage rate of the thioester-type conjugates
was faster than that of the ester-type conjugates (Fig. 3).
Ascendis-type linker
Ascendis-type linkers are cleavable linkers that can be bound to the amino group of
1
7)
peptides. A chemerin-9 conjugate bearing the asialoglycan (14) was designed based on an
Ascendis linker framework (Chart 3). Phthalic anhydride was subjected to aminolysis with
Chemical and Pharmaceutical Bulletin Advance Publication

the N-terminal amino group of the side-chain protected chemerin-9 which had been
assembled on a solid support and the resulting carboxyl group was coupled with the amino
group of N-(2-aminoethyl)piperazine. Subsequently, Fmoc-Asn(Asialo) was introduced to the
N-terminal imino group. The protected peptide resin 12 was treated by TFA to cleave the
α
resin and all of the protecting groups, except for the N -Fmoc group. The Fmoc group on 13
was removed using 20% piperidine in DMF to produce the glycosylated conjugate 14. It was
supposed that the carbonyl group of the phthalic acid amide bound to the peptide could be
attacked by the nitrogen atom of its other amide activated via an intramolecular catalytic
event, resulting in autolysis to release the peptide. The time course of the development of the
parent peptide from glycosylated conjugate 14 measured by RP-HPLC and its half-life
determined under various conditions are summarized in Fig. 4. This linker was found to have
a shorter half-life than the ester and thioester linkers, but it functioned in a similar fashion
from the viewpoint that the cleavage rate of the linker increased with the pH and/or
temperature of the buffer. Usually, immunization is performed using an antigen peptide
emulsified in an adjuvant. For conjugate 14 to be applicable in the development of peptide
vaccines, therefore, it must be self-cleavable not only in aqueous solution but also in
emulsion. We examined the release rate of the parent peptide from conjugate 14 in an
emulsion prepared by mixing equal volumes of the sodium acetate solution (pH 4.0) of
conjugate 14 and an adjuvant based on a light mineral oil, Montanide™ ISA 206VG.
Conjugate 14 was found to release the parent peptide even in this emulsion in accordance
with the same first-order reaction observed in the sodium acetate buffer (pH 4.0). This clearly
indicated that the present glycan-bearing Ascendis-type linkers could be used in antibody
preparation. Next, to clarify the influence of the N-terminal structure in conjugate 14 on its
self-cleavage efficiency, we prepared analogs by replacing the Asn(Asialo) residue as shown
in Table 2. These analogs were synthesized using the same procedure as that for conjugate 14.
Chemical and Pharmaceutical Bulletin Advance Publication

Substitution of Cys(glycan) for Asn(glycan) resulted in alteration of the linkage structure
which connected the glycan and the amino acid residue. This change in linkage structure as
well as changes of the glycan structure showed no effect on the cleavage efficiency of the
Ascendis-type linkers. When the N-terminal amino group was masked by an Fmoc or acetyl
group, however, the half-life of the resulting conjugates in the sodium acetate buffer (pH 4.0)
was significantly prolonged. In contrast, electron-withdrawing chlorine substitution on the
benzene ring of the phthalic acid amide accelerated the cleavage of the linker. Decreasing the
electron density of the carbonyl carbon bonded to the peptide could make it susceptible to
nucleophilic attack by nitrogen. These results imply that modification of the self-cleavable
linker molecule may control its peptide release behavior.
Conclusion
We have demonstrated the utility of ester/thioester and Ascendis-type linkers bearing
biodegradable glycans which can be bound to peptides through their carboxyl and amino
functions, respectively. In addition, the resulting chemical glycosylation efficiently improved
the solubility of the conjugates even when the conjugated peptide itself was sparingly soluble
under physiological conditions. The linkers enabled extended release of the conjugated
peptides but were stable throughout the Fmoc chemistry-based synthetic process. The
self-cleavage rate of the glycosylated linkers was dependent on their coupling structure.
Furthermore, their cleavability could be controlled by modifying the electronic environment
on their linker molecules. These features of the linkers are expected to allow optimization of
the conjugates towards the intended use of the peptides to be released. Especially, such
glycosylated linkers could function as a useful method of preparing the antibodies in aqueous
buffers even for sparingly soluble antigen peptides.
It has been reported that glycan conjugates with certain sugar chain structures can
Chemical and Pharmaceutical Bulletin Advance Publication

18)
target particular organs or tissues. Thus, we are currently aiming to develop conjugates
containing a bioactive peptide and glycosylated self-cleavable linker that can not only be
delivered to a selected target through the guidance of the glycan molecule but also release the
parent peptide at that location.
Chemical and Pharmaceutical Bulletin Advance Publication

Experimental
Materials
Bromoacetamidyl glycan derivatives having disialoundecasaccharide, asialononasaccharide,
and GlcNAc moieties were obtained from GlyTech, Inc. (Kyoto, Japan). All other reagents
and solvents were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan),
Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), Nacalai Tesque, Inc. (Kyoto, Japan),
Watanabe Chemical Industries, Ltd. (Hiroshima, Japan), Merck KGaA (Darmstadt, Germany),
Peptide Institute, Inc. (Osaka, Japan) and Sigma-Aldrich Co. LLC.(St. Louis, MO).
HPLC and MS
Preparative HPLC was carried out on a Hitachi LaChrom Elite HPLC system with a
SHISEIDO CAPCELL PAK C18 UG120 (20 × 250 mm) with a binary mixture of A (0.1%
TFA in H O) and B (MeCN/H O/TFA (90/10/0.09)) mobile phases. Separation was
2
2
performed using the described linear gradient, a flow rate of 7.0 mL/min and detection at 220
nm. Analytical HPLC was performed on a Hitachi LaChrom Elite HPLC system or a
Shimadzu UFLC system with a SHISEIDO CAPCELL PAK C18 UG120 (4.6 × 250 mm)
2 2
using a binary mixture of A (0.1% TFA in H O) and B (0.1% TFA in MeCN/H O (90/10))
mobile phases. The analysis was performed using a linear gradient at a flow rate of 0.7
mL/min and detection at 220 nm. ESI-MS experiments were conducted on a Synapt HDMS
mass spectrometer (Waters).
Solid phase peptide synthesis
Automated peptide synthesis by Fmoc SPPS was carried out on a Prelude automated peptide
synthesizer (Protein Technologies, Inc.). The peptide chain was elongated using the standard
Chemical and Pharmaceutical Bulletin Advance Publication

Fmoc
acid/1-[bis(dimethylamino)methylene]-5-chloro-1H-benzotriazolium
hexafluorophosphate (HCTU)/N-methylmorpholine (5.3/5.0/20
protocol
of
coupling
with
Fmoc-amino
3-oxide
equiv.)
in
α
N,N-dimethylformamide (DMF, 15 min, double coupling). Deprotection of N -Fmoc groups
was performed using 20% piperidine/DMF (5 min × 2). During peptide synthesis, all
washings after couplings and deprotections were performed with DMF. The following side
chain-protecting groups were employed: t-butyl (tBu) for Tyr, t-butyloxycarbonyl (Boc) for
Trp, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for Arg, trityl (Trt) for Cys
and Gln.
Synthesis of glycosylated conjugates
Glycosylated conjugate 2
Rink amide PEGA resin (100 μmol) was placed in a solid-phase synthesis tube and washed
with dichloromethane (DCM) and DMF. After the washing, a DMF (2.5 mL) solution
containing Fmoc-Cys(Trt)-OH (234 mg, 0.399 mmol), HCTU (157 mg, 0.380 mmol), and
2
,4,6-trimethylpyridine (79.6 μL, 0.600 mmol) was added thereto, and the mixture was
1
9)
shaken at room temperature. After 10 min, the resin was washed with DMF, and the
coupling reaction was repeated once again. The Fmoc group was removed by treatment with
2
0% piperidine in DMF to obtain Cys(Trt)-Rink amide PEGA resin, which was then washed
with DMF. Next, a DMF (2.5 mL) solution of 4-hydroxymethyl benzoic acid (HMBA, 61.1
mg, 0.402 mmol), HCTU (157.8 mg, 0.381 mmol), and N,N-diisopropylethylamine (DIPEA,
1
04.5 μL, 0.600 mmol) was added, and the mixture was shaken at room temperature. After 1
h, the resin was washed with DMF and DCM to obtain HMBA-Cys(Trt) on the resin. To the
resin (100 μmol) were added a DCM (5.0 mL) solution containing Fmoc-Leu-OH (176.7 mg,
0
.500 mmol), 1-(2-mesitylenesulfonyl)-3-nitro-1H-1,2,4-triazole (MSNT, 148.2 mg, 0.500
Chemical and Pharmaceutical Bulletin Advance Publication

mmol), and N-methylimidazole (27.9 μL, 0.350 mmol), and the mixture was shaken at room
temperature for 1 h. After the shaking, the resin was washed with DCM and DMF. The
peptide chain was elongated on the Leu-HMBA-Cys(Trt)-Rink amide resin with a Prelude
automated peptide synthesizer using the standard protocols. After the automated synthesis,
the peptide resin was treated with TFA/triisopropylsilane (TIS)/H O/1,2-ethanedithiol (EDT)
2
(
90/5/2.5/2.5) at room temperature for 3 h to give a crude peptide (1a). The aliquot of the
obtained peptide 1a (15.5 mg) was then dissolved in a dimethyl sulfoxide (DMSO)/0.1 M
phosphate buffer (pH 7.4) mixed solution (9/1, v/v, 240 μL) containing 50 mM
1
7
,4-dithiothreitol (DTT). To this solution was added a DMSO/0.1 M phosphate buffer (pH
.4) mixed solution (9/1, v/v, 946 μL) containing 30 mM of asialo-BrAc, and the resulting
mixture was shaken at room temperature for 2 h. The reaction mixture was purified by
preparative HPLC using a linear gradient of 40–60% B in 20 min to obtain a fraction
containing glycosylated conjugate 2. This fraction was further purified by HPLC [column:
SHISEIDO CAPCELL PAK C18 UG120 (5 μm), 20 × 250 mm, flow rate: 7.0 mL/min, eluent
A: 0.1% AcOH in H O, eluent B: 0.09% AcOH/10% H O/90% acetonitrile, A:B =
2
2
7
5:25→60:40 (30 min) linear gradient] to obtain glycosylated conjugate 2 (17.2 mg, 5.79
2
+
3+
204 20
μmol). ESI-MS: m/z calcd for C127H N O58S [M+2H] 1485.7, [M+3H] 990.8,
4
+
[
M+4H] 743.3; found 1485.7, 990.8, 743.3.
Glycosylated conjugate 3
This conjugate was prepared as described above for conjugate 2 to yield 21.8 mg (51%).
2
+
3+
ESI-MS: m/z calcd for
C
149
H
238
N
22
O
74
S
[M+2H] 1776.8, [M+3H] 1184.8,
4
+
[
M+4H] 888.9; found 1776.8, 1184.8, 888.9.
Glycosylated conjugate 5
Chemical and Pharmaceutical Bulletin Advance Publication

The peptide chain was elongated on the Ser(tBu)-HMBA-Cys(Trt)-Rink amide resin using
the same procedure as that for conjugate 2. After the chain assembly was completed, the
peptide resin was treated with TFA/TIS/H O/EDT (90/5/2.5/2.5) at room temperature for 3 h
2
to give the crude peptide (1b). The aliquot of the obtained peptide 1b (14.2 mg), disialo-BrAc
(
41.6 mg, 17.7 μmol), and tris(2-carboxyethyl)phosphine (TCEP, 16.0 mg, 55.8 μmol) were
dissolved in a 0.2 M phosphate buffer solution (pH 6.8, 1.15 mL) containing 7 M guanidine
hydrochloride and reacted at room temperature. After 3 h, the reaction mixture was purified
by HPLC [column: SHISEIDO CAPCELL PAK C18 UG120 (5 μm), 20 × 250 mm, flow
rate: 7.0 mL/min, eluent A: 0.1% AcOH in H O, eluent B: 0.09% AcOH/10% H O/90%
2
2
MeCN, A:B = 70:30→55:45 (10 min) linear gradient] to give glycosylated conjugate 5 (19.0
3
+
217 19
mg, 5.33 μmol, 49%). ESI-MS: m/z calcd for C151H N O77S [M+3H] 1187.8,
4
+
5+
[
M+4H] 891.1, [M+5H] 713.1; found 1187.8, 891.1, 713.1.
Protected HER2(8-16) (6)
The peptide chain was assembled onto Leu-HMPB-PEGA resin (200 μmol) using a Prelude
automated peptide synthesizer. After completion of the chain elongation, the peptide resin
was treated with AcOH/2,2,2-trifluoroethanol (TFE) (v/v, 1/1) for 30 min. This procedure
was repeated and the combined filtrate was concentrated to dryness under reduced pressure to
obtain Boc-Arg(Pbf)-Trp(Boc)-Gly-Leu-Leu-Leu-Ala-Leu-Leu (0.59 g).
2
MPAA-Asn(Asialo)-Gly-NH (7)
Trityl chloride (2.0 g, 7.1 mmol) was reacted with 4-mercaptophenylacetic acid (MPAA, 1.0
g, 6.1 mmol) in DCM (35 mL) at room temperature for 2.5 h. The reaction mixture was
2
washed with H O, and dried over anhydrous sodium sulfate. After removal of the organic
solvent in vacuo, the residue was triturated with ether to give a solid material (2.8 g), which
Chemical and Pharmaceutical Bulletin Advance Publication

was used for the subsequent reaction without further purification. Next, a DMF (2.0 mL)
solution containing the Trt-MPAA (320 μmol), 1-hydroxybenzotriazole (HOBt, 50.7 mg, 375
μmol), and N,N’-diisopropylcarbodiimide (DIC, 54 μL, 522 μmol) was added to
Asn(asialo)-Gly-Rink-Amide-PEGA resin, and the mixture was shaken at room temperature
for 1 h. The obtained peptide resin was washed with DMF and DCM, dried, and then treated
with TFA/TIS/H O (92.5/5/2.5) for 3 h at room temperature. Then, the filtrate was
2
concentrated under reduced pressure to obtain compound 7 (19.6 mg, 10 μmol, 16%).
2
+
ESI-MS: m/z calcd for C76
120 8
H N O49S [M+2H] 981.34; found 981.37.
Glycosylated conjugate 8
The glycosylated linker compound 7 (8.6 mg, 4.4 μmol), protected peptide 6 (35.0 mg, 23.0
μmol), and 1H-benzotriazol-1-yloxy-tri(pyrrolidino)phosphonium hexafluorophosphate
(
PyBOP, 22.8 mg, 43.8 μmol) were dissolved in DMF (0.4 mL) and cooled to −15 °C under
nitrogen atmosphere. To this solution was added DIPEA (7.5 μL, 76.4 μmol), and the mixture
was stirred at −5 °C to −10 °C. After 2.5 h, TFA (50 μL) was added, and the mixture was
concentrated to dryness under reduced pressure. A TFA/H O (95/5) solution (1 mL) was
2
added to the obtained residue, and the mixture was stirred for 3 h. Ether was added to the
resulting solution to obtain the crude peptide as a precipitate, which was purified by
preparative HPLC using a linear gradient of 35–55% B in 30 min to obtain glycosylated
2
+
205 21 58 1
conjugate 8 (2.3 mg, 18%). ESI-MS: m/z calcd for C128H N O S [M+2H] 1500.09,
3
+
[
M+3H] 1000.39; found 1499.67, 1000.08.
Glycosylated conjugate 10
Asialo-BrAc (131.7 mg, 75 μmol) in a phosphate buffer solution (0.1 M, pH 6.72, 4.0 mL)
was added dropwise to EDT (63 μL, 750 μmol, 10 eq.) in a phosphate buffer solution (0.1 M,
Chemical and Pharmaceutical Bulletin Advance Publication

pH 6.72, 4.0 mL), and reacted at room temperature for 40 min. After completion of the
reaction was confirmed by HPLC, the product was purified by preparative HPLC (gradient
A:B = 99:1 (0–1 min)→80:20 (30 min)) to obtain product 9 (118.3 mg, yield: 89%). ESI-MS:
2
+
m/z calcd for C H N O S [M+2H] 888.36; found 888.34.
6
6
111
5
46 2
Next, the protected peptide 6 (63.5 mg, 41.8 μmol), the glycosylated linker 9 (41.5 mg, 23.4
μmol), and PyBOP (121.8 mg, 234 μmol) were dissolved in DMF (1 mL) and cooled to
−
15 °C under nitrogen atmosphere. DIPEA (40.0 μL, 40.7 μmol) was then added to this
solution and the mixture was stirred at −15 °C. After 3 h, TFA (100 μL) was added, and the
mixture was concentrated to dryness under reduced pressure. A TFA/H O (95/5) solution (1
2
2
mL) was added to the obtained residue, and the mixture was stirred for 3 h. Et O was then
added to obtain the crude peptide conjugate as a precipitate. The crude peptide conjugate was
purified by preparative HPLC using a linear gradient of 35–55% B in 30 min to obtain the
thioalkyl-type glycosylated linker-HER2(8-16) (6.9 mg, yield: 10.5%). ESI-MS: m/z calcd for
2
+
3+
118 196 18 55 2
C H N O S [M+2H] 1406.52, [M+3H] 938.01; found 1406.10, 937.73.
Glycosylated conjugate 13
Amino-PEGA resin (200 μmol) was placed in a solid-phase synthesis tube and washed with
DMF. A DMF (5.0 mL) solution containing HMPB (121.2 mg, 0.504 mmol),
2
-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU, 161.7 mg,
.504 mmol), and N-ethylmorpholine (57.3 μL, 0.495 mmol) was added, and the mixture was
0
shaken at room temperature for 3 h. After washing with DMF and DCM, a DCM (10 mL)
solution containing Fmoc-Ser(tBu)-OH (383.1 mg, 1.00 mmol), MSNT (296.4 mg, 1.00
mmol) and N-methylimidazole (55.8 μL, 0.700 mmol) was added, and the mixture was
shaken at room temperature for 3 h. After washing with DCM and DMF, DMF (4 mL),
pyridine (1.2 mL) and acetic anhydride (189 μL, 2.00 mmol) were added sequentially, and the
Chemical and Pharmaceutical Bulletin Advance Publication

mixture was shaken at room temperature. After 1 h, the resin was washed with DMF and
DCM. Fmoc groups in an aliquot (100 μmol) of the resin thus obtained were then removed by
treatment with 20% piperidine in DMF for 15 min. The peptide chain was elongated using the
same procedure as that for conjugate 2. The resin (70 μmol) was treated with 20% piperidine
in DMF to remove the Fmoc group. After washing with DMF and DCM, phthalic anhydride
(
104 mg, 0.702 mmol), DCM (2.1 mL), and pyridine (126 μL) were added sequentially, and
the mixture was shaken at room temperature for 3 h. After washing with DCM and DMF, a
DMF (1.75 mL) solution containing HOBt (47.3 mg, 0.35 mmol) and DIC (51.2 μL, 0.33
mmol) was added, and the mixture was shaken at room temperature. After 15 min,
N-(2-aminoethyl)piperazine (46.1 μL, 0.35 mmol) was added, and the mixture was shaken at
room temperature for 1 h. The coupling reaction was repeated once, and the resin was washed
with DMF to give the peptide resin 11. To an aliquot (20 μmol) of the obtained resin,
Fmoc-Asn(asialo) (75 mg, 38 μmol) in a DMSO-DMF (1/1, v/v, 833 μL) solution, TBTU
(
16.1 mg, 50 μmol) and DIPEA (13.1 μL, 75.2 μmol) were added sequentially, and the
mixture was shaken at room temperature for 4.5 h. The resin was then washed with DMF and
DCM. Next, TFA/TIS/H O/EDT (90/5/2.5/2.5) was added to the resin, and the mixture was
2
2
shaken at room temperature for 3 h. The resulting filtrate was collected and cooled Et O was
added to obtain the target compound as a precipitate. The compound was purified by
preparative HPLC using a linear gradient of 35–60% B in 30 min to obtain the glycosylated
3
+
4+
conjugate 13 (8.6 mg). ESI-MS: m/z calcd for C149
21.1; found 1094.4, 821.1.
201 19
H N O64 [M+3H] 1094.4, [M+4H]
8
Glycosylated conjugate 14
Compound 13 (4.0 mg, 1.2 μmol) was treated with 20% piperidine in DMF (60 μL) for 5 min.
After addition of AcOH (57.5 μL) and 0.1% TFA in H O (1.3 mL), the compound was
2
Chemical and Pharmaceutical Bulletin Advance Publication

purified by preparative HPLC using a linear gradient of 20–50% B in 30 min to yield the
191 19 62
glycosylated conjugate 14 (3.5 mg, 1.1 μmol, 92%). ESI-MS: m/z calcd for C134 N O
H
3
+
4+
[
M+3H] 1020.4, [M+4H] 765.6; found 1020.4, 765.6.
Glycosylated conjugate 15
The peptide resin 12 (15 μmol) was treated with 20% piperidine in DMF. After washing with
DMF, a solution of AcOH (4.3 μL), HOBt (10.1 mg, 75 μmol), and DIC (11.0 μL, 71 μmol)
in DMF (375 μL) was added, and the mixture was shaken at room temperature for 1 h. After
washing with DMF, the coupling reaction was repeated once again. After washing with DMF
and DCM, the peptide resin was treated with TFA/H
2
O/TIS/EDT (90/2.5/5.0/2.5) at room
temperature for 3 h. Cooled Et O was then added to the filtrate to obtain the product as a
2
precipitate. Preparative HPLC of the crude product using a linear gradient of 25–33% B in 8
min to yield 5.0 mg of glycosylated conjugate 15. ESI-MS: m/z calcd for
2
+
3+
C
136 193 19
H N O63 [M+2H] 1551.1, [M+3H] 1034.4; found 1551.1, 1034.4.
Glycosylated conjugate 20
This conjugate was prepared as described above for conjugate 14 to yield 1.1 mg. ESI-MS:
2
+
3+
m/z calcd for C134
065.7.
187 14 19 62 4
H C N O C [M+2H] 1598.0, [M+3H] 1065.7; found 1598.0,
1
Glycosylated conjugate 16
To the peptide resin 11 (50 μmol) was added a DMF (1.25 mL) solution containing
Fmoc-Cys(Trt)-OH (146.8 mg, 0.251 mmol), HOBt (33.8 mg, 0.250 mmol), and DIC (36.6
μL, 0.238 mmol), and the mixture was shaken at room temperature. After washing with DMF,
this procedure was repeated once. After washing with DMF and DCM, the peptide resin was
Chemical and Pharmaceutical Bulletin Advance Publication

2 2
treated with TFA/H O/TIS/EDT (90/2.5/5.0/2.5) at room temperature for 3 h. Cooled Et O
was then added to the filtrate to obtain the product as a precipitate. The crude product was
purified by preparative HPLC using a linear gradient of 50–57.3% B in 22 min to yield 6.6
mg of the Fmoc-Cys-chemerin-9 conjugate connected via the Ascendis-type linker. ESI-MS:
2
+
98 14
m/z calcd for C86H N O18S [M+2H] 824.4; found 824.4.
The obtained peptide (6.6 mg, 4.0 μmol) was treated with 20% piperidine in DMF (1.0 mL).
After addition of AcOH (1.0 mL) and 0.1% TFA in H O (2.0 mL), the resulting compound
2
was purified by preparative HPLC using a linear gradient of 30–50% B in 30 min to yield
Cys-chemerin-9 conjugate connected via the Ascendis-type linker (3.4 mg, 60%). ESI-MS:
2
+
88 14
m/z calcd for C71H N O16S [M+2H] 713.3; found 713.3.
The obtained peptide (2.2 mg) and GlcNAc-BrAc (2.7 mg) were dissolved in 50 mM
phosphate buffer solution (pH 7.4, 316 μL) containing 4.9 mM DTT and reacted at room
temperature for 50 min. The reaction solution was purified by preparative HPLC using a
linear gradient of 25–55% B in 20 min to obtain the glycosylated conjugate 16 (1.8 mg, 73%).
2
+
104 16
ESI-MS: m/z calcd for C81H N O22S [M+2H] 843.4; found 843.4.
Glycosylated conjugate 17
This conjugate was prepared as described above for conjugate 16. ESI-MS: m/z calcd for
2
+
3+
C
135 193 19
H N O62S [M+2H] 1553.1, [M+3H] 1035.7; found 1553.2, 1035.8.
Glycosylated conjugate 18
This conjugate was prepared as described above for conjugate 16 to yield 0.9 mg. ESI-MS:
3
+
4+
m/z calcd for C H N O S [M+3H] 1303.8, [M+4H] 978.1; found 1308.8, 978.1.
1
72 237 21 80
Glycosylated conjugate 19
Chemical and Pharmaceutical Bulletin Advance Publication

This conjugate was prepared as described above for conjugate 16. ESI-MS: m/z calcd for
3
+
4+
C H N O S [M+3H] 1229.8, [M+4H] 922.6; found 1229.8, 922.6.
1
57 227 21 78
General procedure for tracing of hydrolysis behavior in aqueous solution
The hydrolysis behaviors of the glycan-conjugated peptides were traced. The hydrolysis
reaction was started by the addition of a buffer solution (acetate buffer solution (pH 4.0) or
PBS (pH 7.4)) prewarmed to the reaction temperature (25 °C or 37 °C) to each of the
freeze-dried glycan-conjugated peptides. The reaction mixtures were kept at a constant
temperature (25 °C or 37 °C) using a block incubator. A given amount of each solution was
injected into the HPLC at appropriate time intervals to trace the hydrolysis reaction. The
relative starting material concentration was determined from the area of the HPLC peak
corresponding to the starting material. The natural logarithm of relative concentration of the
starting material was plotted against incubation time. A linear plot was obtained, indicating
that the hydrolysis reaction was the first order reaction. Also, the half-life (t ) of the starting
1
/2
material was calculated according to the expression t1/2 = ln(2)/k (wherein k represents the
slope of the linear plot).
General procedure for solubility measurement
Peptide solubilities in aqueous solution were determined as the maximum supernatant
concentration in supersaturated peptide solutions. Samples of supersaturated peptide solution
(
~30 μL) were prepared by adding small aliquots of aqueous solution to approximately 4.5
mg of lyophilized peptide in a microtube. The microtube was shaken at 25 °C for 15 min and
then centrifuged at 16100 ×g at 25 °C for 10 min to separate the insoluble and soluble
fractions. The concentration of peptide in the supernatant was determined from the extinction
coefficient of the solution at 280 nm (ε280).
Chemical and Pharmaceutical Bulletin Advance Publication

The molar extinction coefficients of the glycan-peptide conjugates at 280 nm were
determined by dividing the absorbance of the conjugate peptide at 280 nm by the
concentration determined by amino acid analysis. The molar absorption coefficient ε280 of the
unmodified peptide at 280 nm was calculated according to the following expression:
−
1
−1
ε280 (L·mol ·cm ) = n × 5500 + nTyr × 1490 + n × 125
Trp
ss
wherein nTrp represents the number of tryptophan residues, nTyr represents the number of
2
0)
tyrosine residues, and n represents the number of disulfide bonds.
ss
Acknowledgment
The authors are grateful to their colleague, Dr. Sofia Elouali, for proofreading the English in
the manuscript.
Conflict of Interest
The authors declare no conflict of interest.
Chemical and Pharmaceutical Bulletin Advance Publication

References and Notes
1
2
3
) Fosgerau, K., Hoffmann, T., Drug Discov. Today, 20,122–128 (2015).
) Kang, J. S., DeLuca, P. P., Lee, K. C., Expert Opin. Emerg. Drugs, 14, 363–380 (2009).
) Moradi S.V., Hussein W. M., Varamini P., Simerska P., Toth I., Chem. Sci., 7, 2492–2500
(
2016).
) Tomabechi Y., Krippner G., Rendle P. M., Squire M. A., Fairbanks A. J., Chemistry., 19,
5084–15088 (2013).
4
5
1
) Ueda T., Tomita K., Notsu Y., Ito T., Fumoto M., Takakura T., Nagatome H., Takimoto
A., Mihara S., Togame H., Kawamoto K., Iwasaki T., Asakura K., Oshima T., Hanasaki
K., Nishimura S., Kondo H., J. Am. Chem. Soc., 131, 6237–6245 (2009).
6
7
) Sinclair A. M., Elliott S., J. Pharm. Sci., 94, 1626–1635 (2005).
) Hossain M. A., Belgi A., Lin F., Zhang S., Shabanpoor F., Chan L., Belyea C., Truong H.
T., Blair A. R., Andrikopoulos S., Tregear G. W., Wade J. D., Bioconjug. Chem., 20,
1
390–1396 (2009).
8
9
1
) Schechter, Y., Heldman, E., Sasson, K., Bachar, T., Popov, M., Fridkin, M., ACS Chem.
Neurosci., 1, 399-406 (2010).
) Sasson, K., Marcus, Y., Lev-Goldman, V., Rabinraut, S., Fridkin, M., Schechter, Y., J.
Controlled Release, 142, 214-220 (2010).
0) Seko A., Koketsu M., Nishizono M., Enoki Y., Ibrahim H. R., Juneja L. R., Kim
M., Yamamoto T., Biochim. Biophys. Acta., 1335, 23–32 (1997).
11) Yamamoto N., Ohmori Y., Sakakibara T., Sasaki K., Juneja LR., Kajihara Y., Angew.
Chem, Int. Ed. Engl., 42, 2537–2540 (2003).
1
1
2) Yamamoto N., Sakakibara T., Kajihara Y., Tetrahedron Lett., 45, 3287–3290 (2004).
3) Murase, T., Tsuji, T., Kajihara, Y., Carbohydr Res., 344, 762–770 (2009).
Chemical and Pharmaceutical Bulletin Advance Publication

1
1
4) Tanaka, H., Tsunoda, T., Nukaya, I., Sette, A., Matsuda, K., Umano, Y., Yamaue, H.,
Takesako, K., Tanimura, H., British J. Cancer, 84, 94–99 (2001).
5) Wittamer V., Gregoire F., Robberecht P., Vassart G., Communi D., Parmentier M., J. Biol.
Chem., 279, 9956–9962 (2004).
1
1
6) Imai T., Drug Metab. Pharmacokinet., 21, 173–185 (2006).
7) Cleemann F., Hersel U., Kaden S., Rau H., Wegge, T., Int. Pat. number, WO 2009095479
(2009).
1
8) Ogura A., Tahara T., Nozaki S., Morimoto K., Kizuka Y., Kitazume S., Hara M., Kojima
S., Onoe H., Kurbangalieva A., Taniguchi N., Watanabe Y., Tanaka, K., Sci. Rep., 6,
2
1797 (2016).
1
2
9) Hibino, H., Miki, Y., Nishiuchi, Y., J. Pept. Sci., 20, 30-35 (2014).
0) Pace C. N., Vajdos F., Fee L., Grimsley G., Gray T., Protein Sci., 4, 2411–2423 (1995).
Chemical and Pharmaceutical Bulletin Advance Publication

Legends for Charts and Figures
Chart 1. Synthesis of the glycan-HER2(8-16) conjugates 2, 3, and 5 connected via the ester
linker.
Chart 2. Synthesis of the glycan-HER2(8-16) conjugates 8 and 10 connected via the
thioarylester linker and the thioalkylester linker, respectively. Reagents and conditions: (a)
PyBOP, DIPEA, DMF; (b) TFA/H O (v/v, 95/5).
2
Chart 3. Synthesis of the glycan-chemerin-9 conjugate 14 connected via the Ascendis-type
linker.
Fig. 1. (A) Structures of the glycan-peptide conjugates and their spontaneously cleavable
glycosylated linkers; (B) Structure of the complex-type N-linked oligosaccharide and its
symbolic representation.
Fig. 2. Time course of the recovery rate of glycan-HMBA-chemerin-9 conjugate 5 in acetate
buffer (pH 4.0), PBS (pH 7.4), and borate buffer (pH 9.0). The conversion reaction was
carried out at 25 or 37 °C.
Fig. 3. Time course of the recovery rate of glycan-HER2(8-16) conjugates 8 and 10
containing thioarylester and thioarylester linkers, respectively, in acetate buffer (pH 4.0) and
PBS (pH 7.4). The conversion reaction was carried out at 37 °C.
Fig. 4. Time course of the recovery rate of glycan-chemerin-9 14 containing Ascendis-type
linker in acetate buffer (pH 4.0) and PBS (pH 7.4) at 25 or 37 °C.
Chemical and Pharmaceutical Bulletin Advance Publication

Chart 1.
Chart 2.
Chemical and Pharmaceutical Bulletin Advance Publication

Chart 3.
Chemical and Pharmaceutical Bulletin Advance Publication

Fig. 1.
Fig. 2.
Chemical and Pharmaceutical Bulletin Advance Publication

Fig. 3.
Fig. 4.
Chemical and Pharmaceutical Bulletin Advance Publication

Table 1. Water solubility of HER2 (8-16) peptide and its glycan conjugates.
a)
Conjugate
Glycan
Solubility
(
parent peptide without linker)
0.71 mg/mL (0.68 mM)
> 121 mg/mL (> 41 mM)
> 157 mg/mL (> 44 mM)
2
3
Asialo
Disialo
a)
Determined by measuring the peptide content in the supernatant by UV analysis.
Glycopeptide solutions were clearly homogeneous at the above concentrations.
Chemical and Pharmaceutical Bulletin Advance Publication

Table 2. Cleavage half-life of glycan-chemerin-9 conjugates containing the Ascendis-type
linker.
a)
Half-Life (h)
Conjugate
Xaa(Glycan)
R
X
pH 4.0
4.4
pH 7.4
1.2
1
1
1
1
1
1
1
2
4
6
7
9
3
8
5
0
Asn(Asialo)
Cys(GlcNAc)
Cys(Asialo)
Cys(Disialo)
Asn(Asialo)
Cys(Disialo)
Asn(Asialo)
Asn(Asialo)
H
H
H
H
H
H
H
H
H
4.3
1.4
H
4.5
1.3
H
4.8
1.4
Fmoc
Fmoc
Ac
H
12.2
10.5
12.7
1.3
2.6
2.5
1.4
Cl
0.25
b)
1
4
Asn(Asialo)
H
H
3.8
n.t.
a)
Values were calculated from the respective degradation profiles. Tests were performed in
b)
sodium acetate buffer (pH 4.0) or PBS (pH 7.4) at 37 °C. The emulsion was prepared by
mixing equal volumes of the conjugate solution in sodium acetate buffer (pH 4.0) and the
mineral oil adjuvant.
Chemical and Pharmaceutical Bulletin Advance Publication