A sulfanyl-PEG derivative of relaxin-like peptide utilizable for the conjugation with KLH and the antibody production

Article abstract of DOI:10.1016/j.bmc.2016.05.068

A small peptide–keyhole limpet hemocyanin (KLH) conjugate is generally used as an antigen for producing specific antibodies. However, preparation of a disulfide-rich heterodimeric peptide–KLH conjugates is difficult. In this study, we developed a novel method for preparation of the conjugate, and applied it to the production of specific antibodies against the relaxin-like gonad-stimulating peptide (RGP) from the starfish. In this method, a sulfanyl group necessary for the conjugation with KLH was site-specifically introduced to the peptide after regioselective disulfide bond formation reactions. Using the conjugate, we could obtain specific antibodies with a high antibody titer. This method might also be useful for the production of antibodies against other heterodimeric peptides with disulfide cross-linkages, such as vertebrate relaxins.

Full text of DOI:10.1016/j.bmc.2016.05.068

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A sulfanyl-PEG derivative of relaxin-like peptide utilizable for the
conjugation with KLH and the antibody production
b
Hidekazu Katayama a, , Masatoshi Mita
⇑
a Department of Applied Biochemistry, School of Engineering, Tokai University, 4-1-1 Kitakaname, Hiratsuka, Kanagawa 259-1292, Japan
b Department of Biology, Faculty of Education, Tokyo Gakugei University, 4-1-1 Nukuikita-machi, Koganei, Tokyo 184-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A small peptide–keyhole limpet hemocyanin (KLH) conjugate is generally used as an antigen for produc-
ing specific antibodies. However, preparation of a disulfide-rich heterodimeric peptide–KLH conjugates is
difficult. In this study, we developed a novel method for preparation of the conjugate, and applied it to
the production of specific antibodies against the relaxin-like gonad-stimulating peptide (RGP) from the
starfish. In this method, a sulfanyl group necessary for the conjugation with KLH was site-specifically
introduced to the peptide after regioselective disulfide bond formation reactions. Using the conjugate,
we could obtain specific antibodies with a high antibody titer. This method might also be useful for
the production of antibodies against other heterodimeric peptides with disulfide cross-linkages, such
as vertebrate relaxins.
Received 13 April 2016
Revised 20 May 2016
Accepted 30 May 2016
Available online xxxx
Keywords:
Antigen
PEGylation
Relaxin-like gonad-stimulating peptide
Solid-phase peptide synthesis
Starfish
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
introduced into the synthetic peptide, the disulfide bond might be
randomized by the reducing ability of free sulfanyl group. It is,
Antibodies for peptides/proteins are powerful tools for
biochemical and histochemical analyses, and various anti-peptide/
protein antibodies are available from commercial resources. Since
it is difficult to recognize the peptide by antibodies in immunized
animals, the peptide needs to be conjugated to a large protein, such
as bovine serum albumin (BSA) and keyhole limpet hemocyanin
(KLH), for general use as an antigen.1 Since the sulfanyl group easily
and specifically reacts with a maleimide moiety to form a covalent
bond, this reaction has been widely used for preparing the
peptide–KLH conjugates.
Chemical synthesis of various disulfide-rich peptides has been
achieved by ordinary solid-phase peptide synthesis (SPPS) and
oxidative disulfide bond formation reactions. Chemically, disulfide
bonds can be formed regioselectively by repeating the deprotection
of Cys-protecting groups and the oxidation to form disulfide bonds.
To date, it has been reported that four disulfide bonds could be
formed regioselectively using three or four orthogonal protecting
groups.2–4 Heterodimeric disulfide-rich peptides, such as human
relaxins5 and crustacean insulin-like peptides,3,6 have also been
synthesized by this strategy. On the other hand, to use a synthetic
heterodimeric peptide with disulfide cross-linkages for preparing
a peptide–KLH conjugate, an additional sulfanyl group is needed
in the peptide structure. When an additional Cys residue is
therefore, difficult to prepare the KLH conjugate of a heterodimeric
peptide with the proper disulfide bond arrangement by the
ordinary conjugation method, and the preparation method for such
a conjugate has not yet been reported.
Relaxin-like gonad-stimulating peptide (RGP) from the starfish
Patiria (Asterina) pectinifera has
a relaxin-like heterodimeric
structure that contains one intra-chain and two inter-chain
disulfide bonds (Fig. 1), and is an echinodermatous gonadotropic
hormone responsible for final gamete maturation.7 We have tried
to obtain antibodies against RGP using the A chain or the B chain
conjugated with KLH as an antigen. However, these trials failed
and no antibodies that specifically recognized RGP could be
obtained (unpublished data).
A cDNA encoding RGP from the crown-of-thorns starfish,
Acanthaster planci, has recently been cloned.8 Interestingly, the
deduced amino acid sequence of A. planci RGP was identical to that
of P. pectinifera RGP. A. planci is the major reef coral predator in the
Indo-Pacific region, and it is quite important to investigate the
molecular mechanisms of reproduction in order to avoid A. planci
outbreak. RGP is the key intrinsic factor in the reproduction of
starfishes, and therefore anti-RGP antibodies are desired for
biochemical and histochemical analyses of RGP. In this study, we
tried to prepare KLH conjugated with the whole P. pectinifera
RGP molecule, and to obtain the specific antibodies against
P. pectinifera and A. planci RGPs.
⇑
Corresponding author. Tel./fax: +81 463 50 2075.
E-mail address: katay@tokai-u.jp (H. Katayama).
http://dx.doi.org/10.1016/j.bmc.2016.05.068
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
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2
H. Katayama, M. Mita / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Cys11 was replaced with a 2-pyridylsulfenyl group by 2,20-dipyridyl
disulfide (DPDS) treatment under acidic conditions, the
heterodimerization was performed by mixing with Cys16(Acm)-B
chain under neutral pH, giving CysA24,B16(Acm)-RGP. Finally the
acetamidomethyl (Acm) groups were oxidatively removed by iodine
treatment under acidic pH, giving RGP 1.
Figure 1. Primary structure of P. pectinifera RGP (1).
2.2. Chemical synthesis of RGP derivative with a sulfanyl group
2. Results and discussion
To obtain the RGP–KLH conjugate, we first tried to synthesize
the RGP derivative, which had an additional Cys residue at the
N-terminus of the A chain. Since a free sulfanyl group prevents
the regioselective formation of disulfide bonds, the N-terminal
Cys residue should be protected by a protecting group that is
orthogonal to the other Cys protecting groups used in the RGP
synthesis. For this purpose, we used thiazolidinecarboxylic acid
as the Cys surrogate. A thiazolidine ring at the peptide N-terminus
has been widely used for the protection of N-terminal Cys residue,9
especially in protein chemical syntheses by the native chemical
ligation strategy.10 Since the thiazolidine ring can be opened by
methoxyamine treatment under weakly acidic conditions,9 it was
2.1. Chemical synthesis of RGP
In order to obtain P. pectinifera RGP (1) for use in the comparative
studies with RGP derivatives described below, we synthesized it by
the ordinary 9-fluorenylmethoxycarbonyl (Fmoc)-based SPPS and
regioselective disulfide bond formation reactions, essentially
according to the method for the synthesis of crustacean insulin-like
peptide described previously.6 In brief, Cys11(MeOBn),
Cys24(Acm)-A chain was synthesized by Fmoc-SPPS and dimethyl
sulfoxide (DMSO)-mediated intrachain disulfide bond formation
reactions. After the p-methoxybenzyl (MeOBn) group attached at
Fmoc-Ser(But)-Wang resin
Fmoc-Cys(Acm)-Wang resin
a, b
a
HO
H2N
Acm
H
SEYSGIASYCCLHGCTPSELSVVC-OH
N
O
O
5
O
O
MeOBn
Acm
H-EKYCDDDFHMAVFRTCAVS-OH
c
d
HO
Acm
H
N
SEYSGIASYCCLHGCTPSELSVVC-OH
Acm
O
H2N
O
5
O
O
H-EKYCDDDFHMAVFRTCAVS-OH
e
HO
H
N
SEYSGIASYCCLHGCTPSELSVVC-OH
O
H2N
O
5
O
O
H-EKYCDDDFHMAVFRTCAVS-OH
3
f
H
N
SEYSGIASYCCLHGCTPSELSVVC-OH
O
O
O
5
O
O
H-EKYCDDDFHMAVFRTCAVS-OH
4
H
N
NH2
TrtS
O
g
O
5
O
H
N
SEYSGIASYCCLHGCTPSELSVVC-OH
HS
O
O
N
N
O
5
H
O
O
H-EKYCDDDFHMAVFRTCAVS-OH
2
Scheme 1. Synthetic procedure of the antigen 2. Reaction conditions: (a) 1. Fmoc-SPPS, 2. TFA/H2O/thioanisole/phenol/triisopropylsilane (82.5/5/5/5/2.5), (b) 10% DMSO/6 M
guanidine-HCl/50 mM Pi buffer (pH 7.0), (c) 2,20-dipyridyl disulfide, TfOH/thioanisole/TFA (1/2/20), (d) 40% CH3CN/50 mM NaHCO3/H2O (50%), (e) I2, HCl, H2O/CH3OH (91%),
(f) NaIO4 (1.3 equiv), 50 mM Pi buffer (pH 7.0) (49%), (g) 1. Probe 5, 5% AcOH/N,N-dimethylacetamide, 2. TFA/H2O/triisopropylsilane (96/2/2) (41% in two steps).
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H. Katayama, M. Mita / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
group by glyoxylic acid treatment under the existence of nickel ion
(Ni2+ 12,13 or by pyridoxal 5-phosphate treatment.14 This method
)
has been used for site-specific modification reactions, such as fluo-
rescent-labeling15,16 and biotinylation.17 In our case, however, there
were two N-terminal amino groups at the A and B chains, and it was
difficult to use this method. The other one is the sodium periodate
oxidation method, in which the N-terminal Ser/Thr residue is
specifically oxidized to an aldehyde group.18 Since the N-terminal
residue of the B chain is Glu, when a Ser residue is introduced to
the A chain N-terminus, only the A chain is allowed to react. There-
fore, we selected the sodium periodate oxidation method for the car-
bonylation of the N-terminus. To take apart the antigen peptide from
KLH part, a PEG chain was inserted between RGP and the N-terminal
Ser residue, and we designed the antigen 2 as shown in Scheme 1.
RGP carrying seryl-PEG chain (Ser-PEG-RGP, 3) was synthesized
essentially according to the method for the synthesis of RGP 1 as
described above (Scheme 1). In the Fmoc-SPPS of the A chain,
Fmoc-NH-PEG5-COOH, which was purchased from a commercial
resource, was used as a PEGylation reagent. During SPPS and
regioselective disulfide bond formation reactions, the seryl-PEG
moiety attached at the A chain N-terminus did not affect the reac-
tions, and the desired product 3 was obtained in yields comparable
to those in the case of RGP 1. The N-terminal Ser residue was con-
verted to a glyoxylyl group by sodium periodate oxidation under
neutral pH, giving the N-terminally carbonylated PEG-RGP 4 in
49% yield (Fig. 2).
For introduction of a sulfanyl group into the peptide, a thiol
probe 5 was synthesized as shown in Scheme 2. The sulfanyl group
of cysteamine was protected by a trityl (Trt) group, and condensed
with Boc-NHOCH2COOH. The Boc group was specifically removed
in TFA solution without cation scavengers. During the deprotection
step, no cleavage of Trt group was observed, and the desired com-
pound 5 was obtained in 33% yield. To introduce the probe into
peptide 4, these were mixed under weakly acidic conditions, giving
peptide 6 in 83% yield. The Trt group was finally removed in TFA
solution with triisopropylsilane, giving the desired compound 2
in 49% yield. It is well known that the disulfide exchange reaction
is generally quite slow under acidic conditions. After the removal
of Trt group, acidic conditions were kept until the purified peptide
2 was lyophilized, and no significant disulfide isomerization was
observed in the purification step by reversed-phase HPLC (Fig. 2e).
Figure 2. Reversed-phase HPLC elution profiles in the synthesis of peptide 2. (a)
Gel-filtration-purified peptide 3. (b) The reaction mixture after NaIO4 oxidation. (c)
Purified peptide 4. (d) Reaction mixture after the reaction with thiol probe 5. (e)
Reaction mixture after the deprotection of trityl group. Elution conditions: column,
Mightysil RP-18 (4.6/ ꢀ 150 mm, Kanto Kagaku, Japan) at a flow rate of 1 mL/min.
likely to be orthogonal to the other Cys protecting groups.
However, when the thiazolidinecarboxylic acid-attached A chain
was treated with DPDS in trifluoroacetic acid (TFA)/trifluo-
romethanesulfonic acid (TfOH) solution, the thiazolidine ring was
opened, and the pyridylsulfenylation of the N-terminal Cys residue
was observed. Therefore, it was difficult to use the product in the
next reaction. Since it was difficult to find another Cys protecting
group that would be orthogonal to the other protecting groups,
we changed the strategy in which sulfanyl group used for
conjugation with KLH was introduced to the peptide after the
disulfide bond formation reactions.
For the introduction of the sulfanyl group to the A chain
N-terminus, we used an O-alkyloxime formation reaction between
a carbonyl group and an alkoxyamine. This oxim-based structure
has been used for the preparation of a drug-antibody conjugate,
and has also been shown to be stable in animal serum.11 For prepa-
ration of peptides with an N-terminal carbonyl group, two methods
have been reported. One is the transamination reaction, in which the
N-terminal amino group is specifically converted into a carbonyl
2.3. Evaluation of the synthetic RGP derivatives
In order to confirm biological activity, an in vitro biological assay
was performed according to the method described previously.5
Since antigen 2 had a free sulfanyl group, making it unstable under
a
NH2
NH2
HS
TrtS
H
H
c
b
N
NHBoc
N
NH2
TrtS
O
TrtS
O
O
O
5
Scheme 2. Synthetic procedure of thiol probe 5. Reaction conditions: (a) Trityl
chloride, TFA, (b) Boc-NHOCH2COOH, WSCDꢁHCl, HOSu, CH2Cl2, (c) 2% H2O/50%
TFA/CH2Cl2 (33% in three steps).
Figure 3. Circular dichroism spectra of the synthetic RGPs. Dotted and solid lines
represent the spectra of RGP 1 and Ser-PEG-RGP 3, respectively.
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H. Katayama, M. Mita / Bioorg. Med. Chem. xxx (2016) xxx–xxx
neutral pH, we used the intermediate, Ser-PEG-RGP 3, in the assay.
As a result, the synthetic RGP 1 and Ser-PEG-RGP 3 showed EC50
values of 0.30 0.02 nM and 0.30 0.03 nM (means s.e.), respec-
tively, indicating that the PEGylation of the A chain N-terminus
had no effect on the biological activity of RGP in vitro. Circular
dichroism (CD) spectral analysis of peptides 1 and 3 showed that
these spectral patterns were quite similar to each other (Fig. 3),
demonstrating that the PEGylation did not affect the RGP folding.
Although CD spectrum of the native RGP has not yet been reported,
the spectrum of synthetic RGP 1 was similar to those of other
insulin/relaxin superfamily peptides.3,6,19–23 All of these results
indicated that the PEGylation had no effect on either biological or
chemical aspects of RGP.
indicating that the antibodies with a high titer (>1:100,000 in ELISA)
arose in both rabbits immunized (Fig. 4a). In order to analyze the
specificity of antibodies, immunoblotting analysis was performed
with one of the anti-sera against RGPs from P. pectinifera, Asterias
amurensis24 and Aphelasterias japonica25 and against related pep-
tides (Fig. 4b). P. pectinifera RGP was recognized by the antibodies,
and a clear band was observed on the membrane. On the other hand,
the A chain gave only a faint band, and the B chain was not recog-
nized by the antibodies. These results indicated that the antibodies
were highly specific to the heterodimeric structure of RGP. The RGPs
from other starfishes, bovine insulin and human relaxin-3 also gave
no band on the membrane in the immunoblotting analysis. These
results clearly indicated that the antibodies obtained in this study
were highly specific to P. pectinifera RGP. Since the chemical
structure of A. planci RGP is the same as that of P. pectinifera RGP,8
the antibodies obtained in this study should recognize A. planci
RGP. Using these anti-sera, various analyses of RGP in A. planci and
P. pectinifera, such as the tissue distribution and the measurement
of titers in hemolymph, will be examined.
2.4. Production and evaluation of anti-RGP antibodies
Using antigen 2 (1.8 mg), the conjugate with KLH was prepared
by the maleimide method, which was performed by Eurofins
Genomics (Tokyo, Japan). In brief, KLH was modified with excess
an amount of N-(6-maleimidocaproyloxy)succinimide, and then
antigen 2 was conjugated. The conjugation ratio was 54%. The
RGP–KLH conjugate was immunized against two rabbits. After four
weeks, the sera were collected, and the antibody titers against
P. pectinifera RGP were measured by ELISA. Both of two anti-sera
recognized P. pectinifera RGP at the dilution level of 1.3 ꢀ 106, and
the absorbance reached a maximum at the level of 1.6 ꢀ 105,
3. Conclusion
We have developed a novel method for the preparation of a
disulfide-rich heterodimeric peptide–KLH conjugate, and applied
it to the production of antibodies against RGP from the starfish P.
pectinifera. Specific antibodies with a high titer (>1:100,000 in
ELISA) were obtained. Since the antibodies could not be obtained
when the A or B chains were immunized as an antigen, it was likely
that our novel preparation method for peptide–KLH conjugate suc-
cessfully produced specific antibodies. To our best knowledge, this
is the first report of a preparation method for a heterodimeric pep-
tide–KLH conjugate. This method might be applicable not only to
other heterodimeric peptides with disulfide linkages such as a ver-
tebrate relaxin but also to the single-chain disulfide-rich peptides.
4. Experimental procedure
4.1. General
NMR spectra were recorded by a Bruker AVANCE III HD spec-
trometer (500 MHz in 1H NMR, Bruker, Germany). MALDI-TOF mass
spectra were recorded using an Autoflex spectrometer (Brucker).
High-resolution ESI mass spectra were measured with The Accu-
TOF-plus JMS-T100LP spectrometer (JEOL, Tokyo, Japan). Amino acid
composition was determined using a LaChrom amino acid analyzer
(Hitachi, Tokyo, Japan) after hydrolysis with a 6 M HCl solution at
150 °C for 2 h in a vacuum-sealed tube. Circular dichroism (CD)
spectra were measured with a Jasco J-820 spectropolarimeter
(JASCO, Tokyo, Japan) at room temperature with a 2-mm path length
cell using a phosphate buffer (50 mM, pH 7.0) as a solvent.
4.2. Thiol probe 5
Cysteamine hydrochloride (570 mg, 5.0 mmol) and trityl
chloride (1.4 g, 5.0 mmol) were dissolved in trifluoroacetic acid
(TFA, 20 mL), and stirred at room temperature for 10 min. After
the removal of TFA with an evaporator, the residue was dissolved
in EtOAc, washed with NaHCO3 aqueous solution, H2O and brine,
and dried over Na2SO4. After filtration, the solvent was removed
in vacuo. The residue was dissolved in CH2Cl2 containing
Boc-aminooxyacetic acid succinimidyl ester, which was prepared
by mixing Boc-aminooxyacetic acid (1.15 g, 6.0 mmol), N-hydroxy-
succinimide (0.69 g, 6.0 mmol) and water-soluble carbodiimide
hydrochloride (1.15 g, 6.0 mmol) in CH2Cl2 (15 mL) for 1 h, and
stirred at room temperature for 2 h. The reaction mixture was
Figure 4. Antibody titers and specificity of the anti-sera. (a) Titer curves of anti-
serum in rabbit #1 (d) and #2 (▲) with respect to relaxin-like gonad-stimulating
peptide (RGP) of P. pectinifera. RGP was used for immobilized antigen at
a
concentration of 1 mM. Open circles show control preimmune serum. (b)
Immunoblotting after SDS–PAGE of RGP and related peptides with anti-RGP anti-
serum. 1, P. pectinifera RGP; 2, A chain of P. pectinifera RGP; 3, B chain of P. pectinifera
RGP; 4, Asterias amurensis RGP; 5, Aphelasterias japonica RGP; 6, bovine insulin; 7,
human relaxin-3. Each peptide was used at concentration of 0.1 nmol per well.
MWM indicates molecular weight marker.
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H. Katayama, M. Mita / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
diluted with EtOAc, washed with H2O and brine, and dried over
4.5. Cys11(SPy), Cys24(Acm)-RGP A chain 9
The peptide 8 (3.11 mol) was dissolved in TFA (2.0 mL) and
Na2SO4. After filtration and evaporation, the crude product was
dissolved in 2% H2O/50% TFA/CH2Cl2 (50 mL), and stirred at room
temperature for 30 min. After the solvent was removed with an
evaporator, the residue was chromatographed on silica gel with
CHCl3/CH3OH (19:1) to give the desired compound 5 (650 mg,
1.66 mmol, 33% in 3 steps) as a colorless solid: mp 106–110 °C
(decomp.); Rf 0.55 (CHCl3/CH3OH, 9/1); 1H NMR (CDCl3,
500 MHz): d 7.42–7.20 (m, 15H, Ar), 6.47 (br s, 1H, NH), 4.08
(s, 2H, O-CH2-CO), 3.12 (q, 2H, J = 6.2 Hz, NH-CH2–), 2.43 (t, 2H,
J = 6.5 Hz, S-CH2–); 13C NMR: d 169.7 (C@O), 144.6, 129.5, 128.0,
126.9 (Ar), 74.9 (OCH2), 66.9 (CPh3), 37.5 (NHCH2), 32.1 (SCH2);
ESI mass, found: m/z 415.154, calcd: 415.150 for (M+Na)+.
l
thioanisole (0.2 mL) containing 2,2’-dipyridyl disulfide (DPDS,
22 mg), and cooled to ꢂ10 °C. Trifluoromethanesulfonic acid (TfOH,
100 lL) was added to the solution, and the mixture was kept at
ꢂ10 °C for 5 min. The crude peptide was precipitated with diethyl
ether, washed twice with ether, and dried in vacuo. The residue
was applied to the gel filtration HPLC using a TSKgel G3000PWXL
column (7.8/ ꢀ 300 mm, Tosoh, Japan) with 0.1% TFA/50% acetoni-
trile aqueous solution as a solvent at a flow rate of 0.5 mL/min, to
give peptide 9 (3.07
lmol, 99% yield). MALDI-TOF mass, found:
m/z 2685.9, calcd: 2686.1 for (M+H)+. Amino acid analysis: Thr0.65
Ser3.79Glu1.77Pro1.02Gly2Ala1.09 Val1.76Ile0.96Leu1.99Tyr1.88His0.99
.
4.3. Cys16(Acm)-RGP B chain 7
4.6. CysA24,B16(Acm)-RGP 10
Fmoc-Ser(But)-Wang resin (0.31 mmol/g, 0.81 g, 0.25 mmol) was
swelled in 1-methyl-2-pyrrolidinone (NMP) for 30 min, and was
treated with 20% piperidine/NMP for 5 and 15 min. After washing
with NMP, Fmoc-Val-OBt, which was prepared by mixing
Fmoc-Val-OH (1.0 mmol), 1 M N,N0-dicyclohexylcarbodiimide
(DCC)/NMP (1.5 mL) and 1 M 1-hydroxybenzotriazole (HOBt)/NMP
(1.5 mL) at room temperature for 30 min, was added and the reac-
tion mixture was mixed with vortex at 50 °C for 1 h. The resin was
washed with NMP and 50% dichloromethane/methanol, treated
with 10% acetic anhydride (Ac2O)/5% N,N-diisopropylethylamine
(DIEA)/NMP for 5 min, and washed with NMP. The peptide chain
was elongated in essentially the same manner as described above,
and H-Glu(OBut)-Lys(Boc)-Tyr(But)-Cys(Trt)-Asp(OBut)-Asp(OBut)-
Asp(OBut)-Phe-His(Trt)-Met-Ala-Val-Phe-Arg(Pbf)-Thr(But)-Cys
(Acm)-Ala-Val-Ser(But)-resin (1.57 g) was obtained. A part of the
resin (202 mg) was treated with TFA cocktail (TFA/H2O/thioani-
sole/phenol/triisopropylsilane, 82.5/5/5/5/2.5, 1.5 mL) at room
temperature for 2 h. TFA was removed under an Ar stream and the
peptide was precipitated with diethyl ether. After washing twice
with ether, the precipitate was dried in vacuo. The crude peptide
Peptides 7 (3.07 lmol) and 9 (3.07 lmol) were dissolved in 40%
acetonitrile/50 mM sodium bicarbonate aqueous solution (20 mL)
and the solution was gently stirred at room temperature for 1 h.
The reaction was quenched by adding acetic acid (400 lL), and
the mixture was purified by RP-HPLC on a Mightysil RP-18 column
with a linear gradient of acetonitrile containing 0.1% TFA to give
peptide 10 (1.54
4887.9, calcd: 4885.4 for (M+H)+ (average). Amino acid analysis:
Asp3.24Thr1.59Ser4.82Glu3.02Pro0.87 Gly2Ala3.13Val3.90Met0.84Ile0.99
lmol, 50% yield). MALDI-TOF mass, found: m/z
Leu2.04Tyr3.14Phe2.13Lys1.10His2.11 Arg1.03
.
4.7. RGP 1
Peptide10(1.54
the solution was added dropwise to methanol (20 mL) containing
20 mM I2/methanol (1.2 mL) and conc. HCl (100 L). The mixture
lmol)wasdissolvedindistilledwater(5 mL),and
l
was mixed with vortex at room temperature for 1 h. The reaction
was quenched by adding an ascorbic acid aqueous solution until the
brownish color was abolished. The mixture was purified by RP-HPLC
onaMightysilRP-18columnwithalineargradientofacetonitrilecon-
taining 0.1% TFA to give peptide 1 (904 nmol, 59% yield). MALDI-TOF
mass, found: m/z 4741.8, calcd: 4741.3 for (M+H)+ (average). Amino
acid analysis: Asp3.09Thr1.69Ser4.61Glu2.77Pro1.16Gly2Ala3.23Val3.88
was purified by RP-HPLC on
a Mightysil RP-18 column
(Kanto Kagaku, Tokyo, Japan) with a linear gradient of acetonitrile
containing 0.1% TFA to give peptide 7 (12.9 lmol, 40% yield).
MALDI-TOF mass, found: m/z 2308.7, calcd: 2309.6 for (M+H)+
Met0.51Ile1.04 Leu2.08Tyr2.96Phe2.17Lys1.08His2.07Arg1.04
.
(average). Amino acid analysis: Asp2.97Thr0.53Ser0.71Glu0.90Ala2.14
Val2.01Met0.80Tyr1Phe2.00 Lys1.01His1.01Arg1.02
.
4.8. Ser-PEG-Cys11(MeOBn), Cys24(Acm)-RGP A chain 11
4.4. Cys11(MeOBn), Cys24(Acm)-RGP A chain 8
Starting from Fmoc-Cys(Acm)-Wang resin (0.64 mmol/g, 0.31 g,
0.20 mmol), the peptide chain corresponding to the A chain of RGP
was elongated essentially according to the method for peptide 7
described above. After the removal of the Fmoc group attached at
the N-terminus by 20% piperidine/NMP treatments for 5 and
15 min, the resin was washed with NMP. Fmoc-NH-PEG5-COOBt,
which was prepared by mixing Fmoc-NH-PEG5-COOH (0.40 mmol,
Merck, Germany), 1 M DCC/NMP (0.6 mL) and 1 M HOBt/NMP
(0.6 mL) at room temperature for 30 min, was added and the reaction
mixture was mixed with vortex at 50 °C for 1 h. The resin was washed
with NMP and 50% dichloromethane/methanol, treated with 10%
Ac2O/5% DIEA/NMP for 5 min, and washed with NMP. Fmoc-Ser
(But)-OH was then introduced into the resin by the DCC-HOBt
method, and H-Ser(But)-NH-PEG5-CO-Ser(But)-Glu(OBut)-Tyr(But)-
Ser(But)-Gly-Ile-Ala-Ser(But)-Tyr(But)-Cys(Trt)-Cys(MeOBn)-Leu-
His(Trt)-Gly-Cys(Trt)-Thr(But)-Pro-Ser(But)-Glu(OBut)-Leu-Ser
(But)-Val-Val-Cys(Acm)-resin (1.07 g) was obtained. A part of the
resin (154 mg) was treated with TFA cocktail (1.8 mL) at room
temperature for 2 h. TFA was removed under an Ar stream and the
peptide was precipitated with diethyl ether. After washing twice
with ether, the precipitate was dried in vacuo. The crude peptide
Starting from Fmoc-Cys(Acm)-Wang resin (0.64 mmol/g, 0.16 g,
0.10 mmol), the peptide chain corresponding to the A chain of RGP
was elongated essentially according to the method for the B chain 7
described above, and the protected peptide resin, H-Ser(But)Glu
(OBut)-Tyr(But)-Ser(But)-Gly-Ile-Ala-Ser(But)-Tyr(But)-Cys(Trt)-Cys
(MeOBn)-Leu-His(Trt)-Gly-Cys(Trt)-Thr(But)-Pro-Ser(But)-Glu(OBut)
-Leu-Ser(But)-Val-Val-Cys(Acm)-resin (0.472 g) was obtained. A part
of the resin (101 mg) was treated with TFA cocktail (1.5 mL) at room
temperature for 2 h. TFA was removed under an Ar stream and the
peptide was precipitatedwithdiethyl ether. After washing twice with
ether, the precipitate was dried in vacuo. The crude peptide was dis-
solved in 6 M guanidine-HCl/50 mM phosphate buffer (pH 7.0,
9 mL), and dimethyl sulfoxide (DMSO, 1 mL) was added. The reaction
mixture was gently stirred at room temperature for 2 d, and the crude
peptide was purified by RP-HPLC on a Mightysil RP-18 column with a
linear gradient of acetonitrile containing 0.1% TFA to give peptide 8
(3.11
2697.1 for (M+H)+. Amino acid analysis: Thr0.78Ser3.64Glu1.84
Pro0.83Gly2Ala1.04Val1.78Ile0.97Leu1.99Tyr1.89His0.99
lmol, 15% yield). MALDI-TOF mass, found: m/z 2696.9, calcd:
.
Please cite this article in press as: Katayama, H.; Mita, M. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.05.068

6
H. Katayama, M. Mita / Bioorg. Med. Chem. xxx (2016) xxx–xxx
was dissolved in 6 M guanidine-HCl/50 mM phosphate buffer (pH
7.0, 13.5 mL), and DMSO (1.5 mL) was added. The reaction mixture
wasgentlystirredatroomtemperaturefor2 d, andthecrudepeptide
was purified by RP-HPLC on a Mightysil RP-18 column with a linear
gradient of acetonitrile containing 0.1% TFA to give peptide 11
solution was mixed with vortex at room temperature for 1 h. The
mixture was applied to the gel filtration HPLC using a TSKgel
G3000PWXL column (7.8/ ꢀ 300 mm) with 0.1% TFA/50% acetoni-
trile aqueous solution as a solvent at a flow rate of 0.5 mL/min,
to give peptide 6 (689 nmol, 83% yield). MALDI-TOF mass, found:
m/z 5507.2, calcd: 5507.2 for (M+H)+ (average). Amino acid analy-
sis: Asp2.96Thr1.66Ser4.39Glu2.70Pro0.85 Gly2Ala2.98Val3.79Met0.95
(4.57
3119.4 for (M+H)+. Amino acid analysis: Thr0.81Ser4.30Glu1.77Pro0.90
Gly2Ala1.02Val1.81Ile0.99Leu2.06Tyr2.01His1.01
lmol, 16% yield). MALDI-TOF mass, found: m/z 3119.4, calcd:
.
Ile1.03Leu2.08Tyr3.00Phe2.09Lys1.04His1.99 Arg1.04.
4.9. Ser-PEG-Cys11(SPy), Cys24(Acm)-RGP A chain 12
4.14. Sulfanyl-PEG-RGP 2
The peptide 11 (4.57
thioanisole (0.2 mL) containing DPDS (22 mg), and cooled
to -10 °C. TfOH (100 L) was added to the solution, and the mixture
was kept at ꢂ10 °C for 5 min. The crude peptide was precipitated
with diethyl ether, washed twice with ether, and dried in vacuo.
The residue was applied to the gel filtration HPLC using a TSKgel
G3000PWXL column (7.8/ ꢀ 300 mm) with 0.1% TFA/50% acetoni-
trile aqueous solution as a solvent at a flow rate of 0.5 mL/min, to
lmol) was dissolved in TFA (2.0 mL) and
Peptide 6 (374 nmol) was dissolved in TFA/H2O/triisopropylsi-
lane (96/2/2, 400 L), and the solution was mixed with vortex at
l
l
room temperature for 30 min. The crude peptide was precipitated
with diethyl ether, washed twice with ether, and dried in vacuo.
The crude peptide was purified by RP-HPLC on a Mightysil RP-18
column with a linear gradient of acetonitrile containing 0.1% TFA
to give peptide 2 (183 nmol, 49% yield). MALDI-TOF mass, found:
m/z 5264.9, calcd: 5264.9 for (M+H)+ (average). Amino acid analy-
sis: Asp2.97Thr1.63Ser4.40 Glu2.71Pro1.09Gly2Ala3.07Val3.84Met0.94
give peptide 12 (3.73
lmol, 82% yield). MALDI-TOF mass, found:
m/z 3108.2, calcd: 3108.3 for (M+H)+. Amino acid analysis: Thr0.81
Ile1.01Leu2.12Tyr3.07Phe2.27 Lys1.07His2.02Arg1.11
.
Ser4.37Glu1.93 Pro0.71Gly2Ala1.03Val1.79Ile1.00Leu2.08Tyr2.03His1.00
.
4.15. Biological assay
4.10. Ser-PEG-CysA24,B16(Acm)-RGP 13
RGP activity was biologically assayed using ovarian fragments
of Patiria (Asterina) pectinifera as described previously.7,26 The
ovary of a mature female in P. pectinifera was excised and cut into
small fragments, each of which containing only a few lobes, using
scissors. Ovarian fragments were incubated in artificial seawater
containing synthetic RGP 1 or Ser-PEG-RGP 3 at various concentra-
tions for 1 h. The effective dose for inducing gamete spawning in
50% of ovarian fragments was determined. Four separate assays
using different animals were performed.
Peptides 7 (3.73 lmol) and 12 (3.73 lmol) were dissolved in
40% acetonitrile/50 mM sodium bicarbonate aqueous solution
(30 mL) and the solution was gently stirred at room temperature
for 1 h. The reaction was quenched by adding acetic acid
(500
RP-18 column with a linear gradient of acetonitrile containing 0.1%
TFA to give peptide 13 (1.86 mol, 50% yield). MALDI-TOF mass,
found: m/z 5309.0, calcd: 5307.9 for (M+H)+ (average). Amino acid
analysis: Asp3.11Thr1.55Ser5.33Glu2.92 Pro0.72Gly2Ala3.07Val3.90
lL), and the mixture was purified by RP-HPLC on a Mightysil
l
Met0.94Ile0.99Leu2.08Tyr2.98Phe2.22Lys1.06 His2.05Arg1.09
.
4.16. Production of antibodies
4.11. Ser-PEG-RGP 3
Conjugation with KLH and immunization against two rabbits
were committed to Eurofins Genomics (Tokyo, Japan). KLH
(Thermo Fisher Scientific, Canada) was dissolved in water at the
concentration of 10 mg/mL. N-(6-maleimidocaproyloxy)succin-
imide (EMCS) was dissolved in DMF at a concentration of 10 mg/
mL. KLH and EMCS solutions were mixed at a ratio of 10:1, and
the mixture was gently stirred at room temperature for 1 h. The
solution was applied to a gel-filtration chromatography (PD-10,
GE Healthcare UK Ltd., UK), and the elution was monitored with
absorbance at 280 nm. The protein fraction was collected and con-
centrated with Amicon Ultra-15 (Merck-Millipore, Germany) to
Peptide 13 (1.86
and the solution was added dropwise to methanol (30 mL) contain-
ing iodine (9.2 mg) and conc. HCl (150 L). The mixture was mixed
lmol) was dissolved in distilled water (8 mL),
l
with vortex at room temperature for 1 h. The reaction was quenched
by adding an ascorbic acid aqueous solution until the brownish color
was abolished. The mixture was applied to the gel filtration HPLC
using a TSKgel G3000PWXL column (7.8/ ꢀ 300 mm) with 0.1%
TFA/50% acetonitrile aqueous solution as a solvent at a flow rate of
0.5 mL/min, to give peptide 3 (1.69 lmol, 91% yield). MALDI-TOF
mass, found: m/z 5163.0, calcd: 5163.7 for (M+H)+ (average). Amino
5 mg/mL. Peptide 2 (1.8 mg) was dissolved in water (180
lL), and
acid analysis: Asp3.09 Thr1.68Ser5.09Glu2.86Pro1.04Gly2Ala3.11Val3.92
Met0.96Ile0.99Leu2.03 Tyr2.88Phe2.26Lys1.06His2.04Arg1.09
KLH-EMCS solution (180 L) was added. The mixture was gently
l
.
stirred at room temperature for 2 h. After the reaction, the mixture
was diluted with water at a concentration of 1 mg/mL KLH–pep-
tide conjugate, and used directly for immunization. The reaction
ratio was determined using the Ellman’s reagent27 to be 54%. The
conjugate was immunized against two rabbits. The sera were
collected after four weeks from the immunization.
4.12. Aldehyde-PEG-RGP 4
Peptide 3 (1.69
(pH 7.0, 1.7 mL) containing 1.3 equiv of sodium periodate
(2.2 mol), and the mixture was mixed with vortex at room
lmol) was dissolved in 50 mM phosphate buffer
l
temperature for 1 h. Then, the mixture was purified by RP-HPLC on
a Mightysil RP-18 column with a linear gradient of acetonitrile con-
taining 0.1% TFA to give peptide 4 (834 nmol, 49% yield). MALDI-TOF
mass, found: m/z 5129.3, calcd: 5132.7 for (M+H)+ (average). Amino
acid analysis: Asp2.89Thr1.58 Ser4.01Glu2.48Pro1.05Gly2Ala3.05Val3.64
4.17. Evaluation of antibodies by ELISA
To obtain titer curves of anti-serum against P. pectinifera RGP,
ELISA was conducted. The wells of PVC microtiter plates were
Met0.98Ile0.95Leu1.95Tyr2.61 Phe2.14Lys1.04His1.94Arg0.98
.
coated with peptide solution (1
overnight at 4 °C. After washing and blocking, the plates were dried
at 4 °C. The anti-serum diluted in PBST (100 L) was added to the
lM) in PBS (100 lL) and incubated
4.13. Tritylsulfenyl-PEG-RGP 6
l
wells and incubated overnight at 4 °C. After washing, HRP-anti-
rabbit goat IgG (Zymed Laboratories Inc., CA) diluted in PBST
(100 lL) was added to the wells and incubated for 2 h at room
Peptide 4 (834 nmol) was dissolved in 5% AcOH/N,N-dimethy-
lacetamide (830 L) containing 1% (w/v) of compound 5, and the
l
Please cite this article in press as: Katayama, H.; Mita, M. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.05.068

H. Katayama, M. Mita / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
temperature. After washing, tetramethylbenzine (TMB) was added
References and notes
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Acknowledgments
This work was supported in part by Research and Study
Program of Tokai University Educational System General Research
Organization and in part by JSPS KAKENHI Grant Number
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A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.05.068.
Please cite this article in press as: Katayama, H.; Mita, M. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.05.068