A novel approach for preparing disulfide-rich peptide-KLH conjugate applicable to the antibody production

Article abstract of DOI:10.1080/09168451.2019.1618696

To produce the antiserum against a small peptide, the target peptide-keyhole limpet hemocyanine (KLH) conjugate is generally used as an antigen, although the disulfide-rich peptide-KLH conjugate is still difficult to prepare. In our previous study, we hav

Full text of DOI:10.1080/09168451.2019.1618696

Bioscience, Biotechnology, and Biochemistry
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: https://www.tandfonline.com/loi/tbbb20
A novel approach for preparing disulfide-rich
peptide-KLH conjugate applicable to the antibody
production
Hidekazu Katayama, Ryo Mizuno & Masatoshi Mita
To cite this article: Hidekazu Katayama, Ryo Mizuno & Masatoshi Mita (2019): A novel approach
for preparing disulfide-rich peptide-KLH conjugate applicable to the antibody production,
Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2019.1618696
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BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY
https://doi.org/10.1080/09168451.2019.1618696
A novel approach for preparing disulfide-rich peptide-KLH conjugate
applicable to the antibody production
Hidekazu Katayamaa, Ryo Mizunoa and Masatoshi Mitab,c
b
aDepartment of Applied Biochemistry, School of Engineering, Tokai University, Kanagawa, Japan; Center for Advanced Biomedical
Sciences, TWIns, Research Institute for Science and Engineering, Waseda University, Tokyo, Japan; cDepartment of Biochemistry, Showa
University School of Medicine, Tokyo, Japan
ABSTRACT
ARTICLE HISTORY
Received 21 February 2019
Accepted 8 May 2019
To produce the antiserum against a small peptide, the target peptide-keyhole limpet hemocyanine
(KLH) conjugate is generally used as an antigen, although the disulfide-rich peptide-KLH conjugate
is still difficult to prepare. In our previous study, we have developed a preparation method of the
disulfide-rich peptide-KLH conjugate, and this method was applied to produce the antiserum
against a relaxin-like peptide. However, this method is limited to the synthetic peptide antigen,
and is not applicable to a native or a recombinant peptide. In this study, to expand the applicability
of this method to wide variety of peptides, we newly designed a novel thiol probe enabling the
conjugation between various peptides and KLH, and applied it to produce the antiserum against
relaxin-like peptide of a starfish Asterias amurensis. The antiserum obtained here showed high
antibody-titer and good specificity, strongly suggesting that the method developed in this study is
applicable to various peptides.
KEYWORDS
Relaxin-like gonad
stimulating peptide;
disulfide-rich peptide;
peptide conjugate
Antibodies against peptides/proteins are widely
used in biochemical and histochemical studies. To
obtain specific antibodies against proteins, proteins
or peptides with partial sequence of the target pro-
teins are generally immunized as an antigen to
animals. Since it is difficult to recognize a small
peptide by antibodies in immunized animals, the
peptide needs to enlarge its molecular structure by
the oligomerization [1] or by the conjugation with
a large protein, such as bovine serum albumin
(BSA) and keyhole limpet hemocyanin (KLH) [2],
for the use as an antigen.
The production of peptide-KLH conjugate is gen-
erally performed using a specific reaction between
a cysteinyl peptide and KLH modified with maleimide
groups. On the other hand, since a sulfanyl group of
the Cys side chain has an ability to reduce disulfide
bond(s) denaturing the disulfide-containing peptides,
it is difficult to apply this method to disulfide-
containing peptides. Thus, the preparation of disul-
fide-rich peptide-KLH conjugate is still challenging
issue. Recently, we have developed a method to pre-
pare the disulfide-rich peptide-KLH conjugate, and
this method was applied to produce the antisera
against relaxin-like gonad-stimulating peptide (RGP)
from a starfish Patiria pectinifera (Ppe-RGP) (Figure
1) [3,4]. In this method, RGP having Ser residue at the
N-terminus of A chain was chemically synthesized
with the ordinary solid-phase peptide synthesis
(SPPS) and the regioselective disulfide bond formation
reactions, and a chemical probe having both sulfanyl
and aminooxy groups was finally introduced to the
N-terminal aldehyde group which was generated
from N-terminal Ser residue with sodium periodate
oxidation [4]. To minimize the undesirable side reac-
tion, we thought that the additional sulfanyl group
should be taken apart from the antigen peptide, and
polyethylene glycol (PEG) chain was inserted between
the N-terminal Ser residue and the peptide part. Since
the antisera obtained by this method showed high
antibody titers and high specificity against Ppe-RGP,
these antisera could be used for the development of
a specific and sensitive radioimmunoassay (RIA) [5]
and enzyme-linked immuno-sorbent assay (ELISA) [6]
for the measurement of Ppe-RGP. However, due to the
necessity of PEG chain insertion between the reactive
N-terminal Ser residue and the antigen peptide por-
tion, our preparation method of peptide-KLH conju-
gate was not applicable to native peptides or
recombinant peptides.
To produce the carbonyl group in the target peptide
in our previous study, we used the sodium periodate
oxidation of N-terminal Ser residue [4]. In the prepara-
tion of recombinant peptides with bacterial expression
system, since the N-terminal initial Met residue
attached to Ser is automatically cleaved with methio-
nine aminopeptidase in bacterial cells [7], recombinant
peptides carrying Ser residue at the N-terminus can be
easily prepared by the conventional bacterial expression
systems, and therefore the problem on PEG insertion is
CONTACT Hidekazu Katayama
katay@tokai-u.jp
© 2019 Japan Society for Bioscience, Biotechnology, and Agrochemistry

2
H. KATAYAMA ET AL.
Results and discussion
Design and synthesis of pegylated thiol probe
Pentaethylene glycol was inserted into the structure of
previously designed thiol probe [4], and the new probe
was designed as compound 1 and was synthesized as
shown in Scheme 1. One of two hydroxyl groups of
pentaethylene glycol was protected with tert-
butyldiphenylsilyl (TBDPS) group, giving mono-
O-TBDPS-pentaethylene glycol 2 in 40% yield. On the
other hand, the sulfanyl group of 2-aminoethanethiol
was protected with trityl (Trt) group, and the product
was then condensed with diglycolic anhydride, giving
compound 3 in 34% yield. Compounds 2 and 3 was
condensed using 1,3-diisopropylcarbodiimide (DIC)
and 4-(dimethylamino)pyridine (DMAP) as condensa-
tion reagents, giving compound 4 in 70% yield. After
TBDPS group was removed with tetra-n-butylammo-
nium fluoride (TBAF) to give compound 5, tert-
butoxycarbonyl (Boc)-aminooxyacetic acid was con-
densed with DIC-DMAP, giving compound 6 in 34%
yield. Finally, Boc group was removed with trifluoroace-
tic acid (TFA), affording the desired thiol probe 1 in 69%
yield.
Figure 1. Structures of starfish relaxin-like gonad-stimulating
peptides (RGPs). (a) Primary structure of Asterias amurensis
RGP. Solid lines represent the disulfide bonds. (b) Amino acid
sequence alignment of starfish RGPs. Aam-RGP, Asterias
amurensis RGP; Aja-RGP, Aphelasterias japonica RGP; Ppe-
RGP, Patiria pectinifera RGP. Characters with a gray back-
ground represent identical residues.
likely to be overcome by reconsideration of the thiol
probe design.
On the other hand, it is well known that the
N-terminal amino group can be converted with
carbonyl group with transamination reaction,
which is performed with glyoxylic acid in the pre-
sence of nickel ion (Ni2+) [8,9] or with pyridoxal
5-phosphate [10]. This method has been widely
used for site-specific modification reactions, such
as fluorescent labeling [11,12] and biotinylation
[13]. The combination of transamination reaction
and the newly designed thiol probe might also
enable the preparation of the peptide-KLH conju-
gate using native disulfide-rich peptides. In this
paper, we describe the design and synthesis of the
new thiol probe with PEG chain and its application
to produce the antisera against RGP of the starfish
Asterias amurensis (Aam-RGP) among RGP ortho-
logs (Figure 1) [14].
Chemical synthesis of the antigen
To introduce an aldehyde group using the reaction with
the thiol probe 1, Aam-RGP having an additional Ser
residue at the N-terminus of A chain was chemically
synthesized at first. The N-terminal Ser residue of this
peptide, however, was not converted to aldehyde with
NaIO4 oxidation. Since the undesirable side reaction in
which the aldehyde group was immediately hindered
via the cyclization with the carbonyl group of Pro
residue was found (Scheme 2), the introduction of
thiol probe 1 did not proceed. This side reaction is likely
to occur at the N-terminal aldehyde group directly
attached the Pro residue, because the side reaction can
proceed when peptide bond has the cis configuration as
shown in Scheme 2, and only the peptide bond at
Scheme 1. Synthetic procedure of the PEGylated thiol probe 1.Reaction conditions: a, (1) Trityl chloride, TFA; (2) Diglycolic
anhydride, CH2Cl2, DIEA; b, TBDPS-Cl, imidazole, DMF; c, 1,3-diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBt),
4-(dimethylamino)pyridine (DMAP), DMF; d, TBAF, acetic acid, THF; e, Boc-aminooxyacetic acid, DIC, HOBt, DMAP, CH2Cl2; f, TFA,
CH2Cl2.

PREPARATION METHOD FOR DISULFIDE-RICH PEPTIDE-KLH CONJUGATE
3
Scheme 2. Possible mechanism of side reaction in NaIO4 oxidation at the N-terminal Ser-Pro sequence.
Scheme 3. Synthetic procedure of the antigen 14. Reaction conditions: a, solid-phase peptide synthesis; b, DMSO, 6 M
guanidine-HCl/50 mM phosphate buffer (pH 7.0); c, Dipyridyl disulfide, thioanisole, TFA, TfOH; d, 30% acetonitrile/50 mM
sodium bicarbonate; e, iodine, HCl, methanol/H2O; f, NaIO4, 50 mM phosphate buffer (pH 7.0); g, 1, 5% acetic acid/DMA; h, TIS,
H2O, TFA.
N-terminal side of Pro residue can have cis configura-
tion. To take apart the aldehyde from Pro residue, a Gly
residue was inserted between Ser-Pro, and we synthe-
sized Ser-Gly-Aam-RGP as shown in Scheme 3.
Ser-Gly-A chain was prepared by the ordinary
9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase
peptide synthesis (SPPS) method. To enable the regiose-
lective formation of disulfide bonds, S-methoxybenzyl
(MeOBn)-cysteine and S-acetamidomethyl (Acm)-
cysteine derivatives were used at Cys12 and Cys25, respec-
tively. After the cleavage of crude peptide from the resin
with TFA treatment, an intrachain disulfide bond at

4
H. KATAYAMA ET AL.
Cys11-Cys16 was formed with dimethyl sulfoxide
(DMSO) oxidation in a neutral buffer solution, giving
partially protected Ser-Gly-A chain 7 in 13% yield.
MeOBn group at Cys12 was then cleaved with triflic
acid (TfOH), and simultaneously S-pyridylsulfenylated
with 2,2ʹ-dipyridyl disulfide (DPDS), giving SPy-attached
Ser-Gly-A chain 8 quantitatively.
B chain was also prepared by the ordinary Fmoc-
SPPS, and Cys17(Acm)-B chain 9 was obtained in 15%
yield. Equimolar amounts of peptides 8 and 9 were
mixed in an aqueous solution to form the interchain
disulfide bond at CysA12-CysB5 with thiolysis reaction,
giving the heterodimeric peptide 10 in 43% yield. The
third disulfide bond was regioselectively formed with
iodine oxidation in an acidic solution, giving Ser-Gly-
Aam-RGP 11 in 40% yield.
The N-terminal Ser residue at the A-chain was con-
verted to aldehyde group with sodium periodate oxida-
tion, giving aldehyde-Gly-Aam-RGP 12 in 69% yield.
Then, peptide 12 was treated with the thiol probe 1
under weakly acidic conditions, giving peptide 13 in
88% yield. Finally, the Trt group was removed in TFA
solution containing triisopropylsilane, giving the
designed antigen 14 in 45% yield. It is well known that
the disulfide exchange reaction is quite slow under acidic
conditions. After the removal of Trt group, acidic condi-
tions were kept until the antigen 14 was purified and
lyophilized, and no significant disulfide isomerization
was observed on the HPLC chromatogram in purifica-
tion step of peptide 14.
The physiological conditions in animal are at a neutral
pH, although peptide 14 was likely to be labile under
these conditions. Therefore, we used peptide 13 instead
of peptide 14 for the conformational analysis. The con-
formation of peptide 13 was confirmed by circular
dichroism (CD) spectral measurement. Aam-RGP with
native form which was also synthesized by the Fmoc-
SPPS and the regioselective disulfide bond formation
reactions [15] showed the spectral pattern typical for α-
helical peptides (Figure 2). This spectral pattern is similar
to those of other insulin-family peptides reported
previously [4,16–23], strongly suggesting that Aam-
RGP has a tertiary structure similar to the other insulin-
family peptides. Peptide 13 showed the CD spectrum
essentially the same as that of Aam-RGP (Figure 2).
The α-helix contents of peptide 13 and Aam-RGP were
calculated based on the α values at 208 nm [24] to be
36.0% and 37.5%, respectively. These results strongly
suggest that peptide 13 possess the same conformation
as Aam-RGP, and that the attachment of thiol probe 1
does not affect the peptide conformation.
Production and evaluation of anti-Aam-RGP
antiserum
Using antigen 14, the conjugation with KLH was car-
ried out with the maleimide method, which was per-
formed in Eurofins Genomics (Tokyo, Japan). The
conjugate was immunized against a rabbit. After four
weeks, the serum was collected, and the antibody titer
against Aam-RGP was measured by enzyme-linked
immuno-sorbent assay (ELISA). As shown in Figure 3,
Aam-RGP was recognized with the antiserum in the
dilution level of 10−5. This antibody titer was compar-
able to that of the anti-Ppe-RGP antisera obtained in
our previous study [4].
To confirm the specificity of antiserum against Aam-
RGP, Western blotting analysis using several insulin/
relaxin-family peptides was performed, and the result
was shown in Figure 4. Aam-RGP gave a band on the
membrane (lane 2), indicating that the antisera obtained
in this study recognized Aam-RGP properly. RGP from
Aphelasterias japonica (Aja-RGP) (Figure 1(b)) [25] also
gave a faint band on the membrane (lane 3), whereas
RGP from Patiria pectinifera (Ppe-RGP) [3] did not (lane
1). These results are coincided well with that Aam-RGP
shows the higher sequence identity to Aja-RGP (87%)
than to Ppe-RGP (67%) (Figure 1) [25,26]. In the lanes of
Figure 3. Titer-curves of anti-serum against Aam-RGP and its
chains. Peptides were used for immobilized antigens at
a concentration of 1 mM. Circles, crosses and squares indicate
Aam-RGP, Aam-RGP A-chain and Aam-RGP B-chain,
respectively.
Figure 2. Circular dichroism spectra of Aam-RGP (black line)
and TrtS-PEG-Gly-Aam-RGP 13 (gray line).

PREPARATION METHOD FOR DISULFIDE-RICH PEPTIDE-KLH CONJUGATE
5
1H-NMR, Bruker, Germany). MALDI-TOF mass
spectra were recorded using an Autoflex spectrometer
(Brucker). High-resolution ESI mass spectra were
measured with The AccuTOF-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 150ºC for 2 h
in a vacuum-sealed tube. Circular dichroism (CD)
spectra were measured with a Jasco J-820 spectro-
polarimeter (JASCO, Tokyo, Japan) at room tempera-
ture with a 2-mm path length cell using a phosphate
buffer (50 mM, pH 7.0) as a solvent. Helix contents
were calculated by the method as described pre-
viously [24].
Figure 4. Western blotting analysis using an anti-Aam-RGP
antiserum against various insulin/relaxin family peptides.
Ppe-RGP (lane 1), Aam-RGP (lane 2), Aja-RGP (lane 3), Aam-
RGP A-chain (lane 4), Aam-RGP B-chain (lane 5), bovine
insulin (lane 6), and human relaxin-3 (lane 7) at the concen-
trations of 0.1 nmol/well were separated by SDS-PAGE and
used for Western blotting. Arrows (a) and (b) indicate the
positions of RGPs and A and B chains, respectively.
Mono-o-(tert-butyldiphenylsilyl)pentaethylene
glycol 2
A and B chains of Aam-RGP (lanes 4 and 5), thick bands
were found at the proper molecular weight position,
indicating that the antibodies also recognized each
chain as an antigen. A chain of Aam-RGP gave signals
at high molecular weight region. It may due in part to
a low solubility of A chain in an aqueous solution, and
aggregate of A chain may gave some signals. B chain also
gave a several bands with high molecular weights. It was
likely that these were oligomers of B chain connected
with disulfide bond(s). Since B chain easily underwent
oxidation under air atmosphere and a reducing reagent
could not be used in this experiment, these bands could
not be disappeared. Neither human relaxin-3 (lane 6) nor
bovine insulin (lane 7) gave a band in the Western
blotting analysis, illuminating that the antiserum
obtained here had a high specificity against Aam-RGP.
These results indicated that the preparation
method of antigen developed in this study is useful
at the same level as previously developed one in
which only the synthetic peptides could be applicable.
Pentaethylene glycol (0.53 g, 2.2 mmol) and imida-
zole (0.30 g, 4.4 mmol) were dissolved in DMF
(10
mL),
and
tert-butylchlorodiphenylsilane
(0.58 mL, 2.2 mmol) was added dropwisely under
Ar atmosphere. The reaction mixture was stirred at
room temperature for 2.5 h. The solution was diluted
with EtOAc, washed with 1 M HCl, distilled water
and brine, and dried over Na2SO4. After filtration and
evaporation, the residue was chromatographed on
silica gel with toluene/methanol (19:1) to give the
desired compound 2 (0.42 g, 0.88 mmol, 40%) as
a colorless oil: Rf 0.55 (toluene/methanol, 9/1);
1H-NMR (CDCl3, 500 MHz): d 7.73–7.33 (m, 10H,
Ar), 3.81–3.60 (m, 20H, O-CH2-CH2-), 1.49 (s, 9H,
But); 13C-NMR: δ 135.6, 133.7, 129.6, 127.7 (Ar),
72.6, 72.4, 70.8, 70.7, 70.6, 70.6, 70.3, 63.4, 61.3
(OCH2), 26.8 [C(CH3)3], 19.2 [C(CH3)3]; ESI mass,
found: m/z 499.248, calcd: 499.248 for (M+ Na)+.
O-[n-(tritylthioethyl)carbamoyl]methoxyacetic
acid 3
Conclusion
In this study, we designed and synthesized the novel
thiol probe for utilizing the antibody production
against Aam-RGP. The synthetic thiol probe might
be applicable not only to the synthetic peptides but
also to native and recombinant peptides. The anti-
Aam-RGP antiserum obtained here was confirmed to
have high antibody titer with high specificity, and
therefore it must be useful for the studies on starfish
reproduction.
Trityl chloride (0.93 g, 3.3 mmol) and 2-aminoatha-
nethiol hydrochloride (0.36 g, 3.2 mmol) were dis-
solved in trifluoroacetic acid (TFA, 10 mL), and the
solution was stirred at room temperature for 10 min.
After the evaporation, the residue was dissolved in
dichloromethane (DCM, 10 mL), and diglycolic
anhydride (0.43 g, 3.7 mmol) and N,
N-diisopropylethylamine (DIEA, 1.1 mL, 6.3 mmol)
were added. The reaction mixture was stirred at room
temperature for 20 h. The solution was diluted with
EtOAc, washed with 1 M HCl, distilled water and
brine, and dried over Na2SO4. After filtration and
evaporation, the residue was chromatographed on
silica gel with toluene/methanol/acetic acid (90:10:1)
to give the desired compound 3 (0.49 g, 1.1 mmol,
34%) as a colorless solid: Rf 0.37 (toluene/methanol/
Experimental procedure
General
Aam-RGP was chemically synthesized as described
[15]. NMR spectra were recorded by a Bruker
AVANCE III HD spectrometer (500 MHz in

6
H. KATAYAMA ET AL.
acetic acid, 90/10/1); 1H-NMR (DMSO-d6, 500 MHz):
δ 7.96 (t, 1H, J = 6.0 Hz, NH), 7.38–7.23 (m, 15H,
Ar), 4.10 (s, 2H, -O-CH2-CO-), 3.94 (s, 2H, -O-CH2-
CO-), 3.08 (q, 2H, J = 6.7 Hz, -NH-CH2-), 2.24 (t, 2H,
J = 7.0 Hz, -S-CH2-); 13C-NMR: d 171.9, 169.1
(C = O), 145.1, 129.5, 128.5, 127.2 (Ar), 70.4, 68.2
(OCH2), 66.4 (CPh3), 49.1 (-CH2-NH-), 40.1 (-CH2
-S-); ESI mass, found: m/z 458.142, calcd: 458.140 for
(M+ Na)+.
O-CH2-CH2-), 3.13 (q, 2H, J = 6.5 Hz, -NH-CH2-),
2.41 (t, 2H, J = 6.8 Hz, -S-CH2-); 13C-NMR: d 169.8,
168.8 (C = O), 144.4, 129.6, 128.0, 126.8 (Ar), 70.5,
68.8 (OCH2CO-), 72.6, 71.0, 70.6, 70.6, 70.5, 70.2,
64.2, 61.7 (OCH2, Ph3C-), 37.7 (-CH2-NH-), 31.8 (-
CH2-S-); ESI mass, found: m/z 678.268, calcd: 678.271
for (M+ Na)+.
O-(tert-Butoxycarbonylaminooxyacetyl)
pentaethylene glycolyl O-[N-(Tritylthioethyl)
carbamoyl]methoxyacetate 6
Mono(tert-butyldiphenylsilyl)pentaethylene
glycolyl O-[N-(Tritylthioethyl)carbamoyl]
methoxyacetate 4
Compound 5 (0.20 g, 0.30 mmol) was dissolved in
DCM (1 mL), and HOBt (62 mg, 0.46 mmol) and
DIC (70 µL, 0.46 mmol) were added. The solution
was stirred at room temperature for 30 min. Boc-
aminooxyacetic acid (87 mg, 0.46 mmol) and
DMAP (5.6 mg, 46 µmol) were added to the solution,
and the mixture was stirred for further 20 h. The
solution was diluted with EtOAc, washed with 1 M
HCl, distilled water and brine, and dried over Na2
SO4. After filtration and evaporation, the residue was
chromatographed on silica gel with CHCl3/methanol
(19:1) to give the desired compound 6 (0.16 g,
0.18 mmol, 61%) as a colorless oil: Rf 0.49 (CHCl3
Compound 3 (0.31 g, 0.72 mmol) was dissolved in
DMF (3 mL), and 1-hydroxybenzotriazole (HOBt,
0.12 g, 0.86 mmol) and 1,3-diisopropylcarbodiimide
(DIC, 0.13 mL, 0.86 mmol) were added. The solution
was stirred at room temperature for 30 min.
Compound 2 (0.34 g, 0.72 mmol) and 4-(dimethyla-
mino)pyridine (DMAP, 18 mg, 0.14 mmol) were
added to the solution, and the mixture was stirred
for further 20 h. The solution was diluted with
EtOAc, washed with 1 M HCl, distilled water and
brine, and dried over Na2SO4. After filtration and
evaporation, the residue was chromatographed on
silica gel with toluene/EtOAc (1:1) to give the desired
compound 4 (0.43 g, 0.51 mmol, 70%) as a colorless
1
/methanol, 9/1); H-NMR (CDCl3, 500 MHz): δ 8.18
(brs, 1H, ONH), 7.41–7.19 (m, 15H, Ar), 7.01 (brs,
1H, CH2NH), 4.46 (s, 2H, -NHO-CH2-CO-), 4.33 (t,
2H, J = 2.7 Hz, -COO-CH2-), 4.29 (t, 2H, J = 3.0 Hz, -
COO-CH2-), 4.18 (s, 2H, -O-CH2-CO-), 4.04 (s, 2H,
-O-CH2-CO-), 3.72–3.58 (m, 16H, O-CH2-CH2-),
3.13 (q, 2H, J = 6.5 Hz, -NH-CH2-), 2.42 (t, 2H,
J = 6.8 Hz, -S-CH2-), 1.47 (s, 9H, But); 13C-NMR:
d 169.8, 169.7, 169.1, 156.4 (C = O), 144.6, 129.5,
128.0, 126.8 (Ar), 82.1 [C(CH3)3], 70.5, 66.8 (OCH2
CO-), 72.6, 70.9, 70.5, 68.8, 68.8, 68.5, 64.2, 64.1, 61.6
(OCH2, Ph3C-), 37.8 (-CH2-NH-), 31.2 (-CH2-S-),
28.2 [C(CH3)3]; ESI mass, found: m/z 851.345, calcd:
851.340 for (M+ Na)+.
1
oil: Rf 0.56 (toluene/EtOAc, 1/1); H-NMR (CDCl3,
500 MHz): δ 7.70–7.20 (m, 25H, Ar), 6.93 (brs, 1H,
NH), 4.28 (t, 2H, J = 4.8 Hz, -COO-CH2-), 4.15 (s,
2H, -O-CH2-CO-), 4.01 (s, 2H, -O-CH2-CO-),
3.81–3.59 (m, 18H, O-CH2-CH2-), 3.13 (q, 2H,
J = 6.5 Hz, -NH-CH2-), 2.41 (t, 2H, J = 6.7 Hz, -S-
CH2-), 1.05 (s, 9H, But); 13C-NMR: d 169.8, 168.2
(C = O), 144.7, 135.6, 133.7, 129.7, 129.6, 128.0, 127.7,
126.8 (Ar), 70.6, 66.8 (OCH2CO-), 72.5, 71.1, 70.8,
70.7, 68.9, 68.6, 64.2, 63.5 (OCH2, Ph3C-), 37.7 (-CH2
-NH-), 31.9 (-CH2-S-), 26.9 [C(CH3)3], 19.2
[C(CH3)3]; ESI mass, found: m/z 916.387, calcd:
916.389 for (M+ Na)+.
O-(aminooxyacetyl)pentaethylene glycolyl
o-[n-(tritylthioethyl)carbamoyl]methoxyacetate 1
Pentaethylene glycolyl o-[n-(tritylthioethyl)
carbamoyl]methoxyacetate 5
Compound 6 (0.16 g, 0.18 mmol) was dissolved in
DCM (5 mL) and TFA (5 mL), and the solution was
stirred at room temperature for 10 min. After con-
centration under vacuum, the residue was chromato-
graphed on silica gel with toluene/methanol (17:3) to
give the desired compound 1 (0.090 g, 0.12 mmol,
69%) as a colorless oil: Rf 0.19 (CHCl3/methanol, 97/
Compound 4 (0.50 g, 0.56 mmol) was dissolved in
tetrahydrofuran (THF, 2 mL), and 1.0 M tetrabuty-
lammonium fluoride (TBAF)/THF (0.64 mL) was
added. The solution was stirred at room temperature
for 20 h. After concentration under vacuum, the
residue was chromatographed on silica gel with
CHCl3/methanol (19:1) to give the desired compound
5 (0.20 g, 0.30 mmol, 55%) as a colorless oil: Rf 0.52
(CHCl3/methanol, 9/1); 1H-NMR (CDCl3, 500 MHz):
δ 7.42–7.19 (m, 15H, Ar), 6.97 (brs, 1H, NH), 4.30 (t,
2H, J = 2.3 Hz, -COO-CH2-), 4.18 (s, 2H, -O-CH2-
CO-), 4.01 (s, 2H, -O-CH2-CO-), 3.71–3.58 (m, 18H,
1
3); H-NMR (CDCl3, 500 MHz): δ 7.42–7.10 (m,
15H, Ar), 6.92 (brs, 1H, CH2NH), 4.33–4.27 (m, 6H,
H2NO-CH2-CO-, -COO-CH2-), 4.18 (s, 2H, -O-CH2-
CO-), 4.02 (s, 2H, -O-CH2-CO-), 3.72–3.58 (m, 16H,
O-CH2-CH2-), 3.13 (q, 2H, J = 6.4 Hz, -NH-CH2-),
2.41 (t, 2H, J = 6.7 Hz, -S-CH2-); 13C-NMR: d 170.7,
169.8, 168.8 (C = O), 144.6, 129.6, 128.0, 126.8 (Ar),

PREPARATION METHOD FOR DISULFIDE-RICH PEPTIDE-KLH CONJUGATE
7
70.5, 66.8 (OCH2CO-), 70.6, 70.6, 70.5, 70.2, 68.9,
68.8, 68.7, 68.5, 64.2, 64.1, 63.8, 61.6 (OCH2, Ph3
C-), 37.7 (-CH2-NH-), 31.9 (-CH2-S-); ESI mass,
found: m/z 729.310, calcd: 729.305 for (M + H)+.
300 mm, Tosoh, Japan) with 0.1% TFA/50% acetonitrile
aqueous solution as a solvent at a flow rate of 0.5 mL/
min, to give peptide 8 (2.41 µmol, quant.). MALDI-TOF
mass, found: m/z 3036.0, calcd: 3036.5 for (M + H)+
(average). Amino acid analysis: Asp1.08Thr1.76Ser2.49
Glu3.03 Pro0.91Gly4Val3.04Met0.89Leu2.11Tyr1.84Arg1.09
.
Cys12(meobn), cys25(acm)-ser-gly-a chain 7
Fmoc-Cys(Acm)-Wang resin (0.64 mmol/g, 0.234 g,
0.15 mmol) was swelled in 1-methyl-2-pyrrolidinone
(NMP) for 30 min, and was treated with 20% piper-
idine/NMP for 5 and 15 min. After washing with
NMP, Fmoc-Val-OBt, which was prepared by mix-
Cys17(acm)-b chain 9
Starting from H-His(Trt)-Trt(2-Cl) resin (0.68 mmol/g,
0.368 g, 0.25 mmol), the peptide chain corresponding
to the B chain of RGP was elongated essentially accord-
ing to the method for the A chain 7 described above,
and the protected peptide resin, H-Ala-Glu(OBut)-Lys
(Boc)-Tyr(But)-Cys(Trt)-Asp(OBut)-Glu(OBut)-Asp
(OBut)-Phe-His(Trt)-Met-Ala-Val-Tyr(But)-Arg(Pbf)-
Thr(But)-Cys(Acm)-Thr(But)-Glu(OBut)-His(Trt)-
resin (1.11 g) was obtained. A part of the resin (102 mg)
was treated with TFA cocktail (1.5 mL) at room tem-
perature 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 was purified by RP-
HPLC on a Mightysil RP-18 column with a linear
gradient of acetonitrile containing 0.1% TFA to give
peptide 9 (3.11 µmol, 15% yield). MALDI-TOF mass,
found: m/z 2519.2, calcd: 2519.0 for (M + H)+. Amino
acid analysis: Asp1.96Thr1.52Ser2.78Ala2.12Val0.98Met0.90
ing Fmoc-Val-OH (0.60 mmol),
1 M N,N’-
dicyclohexylcarbodiimide (DCC)/NMP (0.9 mL)
and 1 M 1-hydroxybenzotriazole (HOBt)/NMP
(0.9 mL) at room temperature for 30 min, was
added and the reaction mixture was mixed with
vortex at 50ºC 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 elon-
gated in essentially the same manner as described
above, and H-Ser(But)-Gly-Pro-Glu(OBut)-Thr(But)-
Tyr(But)-Val-Gly-Met-Gly-Ser(But)-Tyr(But)-Cys
(Trt)-Cys(MeOBn)-Leu-Val-Gly-Cys(Trt)-Thr(But)-
Arg(Pbf)-Asp(OBut)-Gln(Trt)-Leu-Ser(But)-Gln
(Trt)-Val-Cys(Acm)-resin (0.839 g) was obtained.
A part of the resin (104 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 precipitated with diethyl ether. After washing
twice with ether, the precipitate was dried in vacuo.
The crude peptide was dissolved 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 tem-
perature for 2 d, and the crude peptide was purified
by RP-HPLC on a Mightysil RP-18 column (Kanto
Kagaku, Japan) with a linear gradient of acetonitrile
containing 0.1% TFA to give peptide 7 (2.41 µmol,
13% yield). MALDI-TOF mass, found: m/z 3047.0,
calcd: 3047.5 for (M + H)+ (average). Amino acid
analysis: Asp1.08Thr1.77Ser2.46Glu3.02Pro0.83Gly4
Tyr2.03 Phe1Lys0.99His2.00Arg1.01
.
Cysa25, b17(acm)-ser-gly-rgp 10
Peptides 8 (2.56 µmol) and 9 (2.56 µmol) were dis-
solved in 30% acetonitrile/50 mM sodium bicarbonate
aqueous solution (12.5 mL) and the solution was gently
stirred at room temperature for 1 h. The reaction was
quenched by adding acetic acid (240 µL), and 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 10 (1.10 µmol, 43%
yield). MALDI-TOF mass, found: m/z 5444.9, calcd:
5445.0 for (M + H)+ (average). Amino acid analysis:
Asp3.09Thr3.56Ser2.62Glu6.04Pro0.94Gly4Ala2.12Val4.01
Met1.65Leu2.06Tyr4.03 Phe1.10Lys1.10His2.09Arg2.11
.
Val3.08Met0.80Leu2.18Tyr1.89 Arg1.09
.
Aldehyde-gly-rgp 12
Cys12(spy), cys25(acm)-ser-gly-a chain 8
Peptide 11 (371 nmol) was dissolved in 50 mM phos-
phate buffer (pH 7.0, 370 µL) containing 1.3 equiva-
lents of sodium periodate (480 nmol), and the
mixture was mixed with vortex at room temperature
for 1 h. Then, 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
12 (257 nmol, 69% yield). MALDI-TOF mass,
found: m/z 5270.3, calcd: 5269.9 for (M + H)+ (aver-
age). Amino acid analysis: Asp3.04Thr3.54Ser1.68Glu6.04
The peptide 7 (2.41 µmol) was dissolved in TFA
(2.0 mL) and thioanisole (0.2 mL) containing 2,2ʹ-
dipyridyl disulfide (DPDS, 22 mg), and cooled to
−10ºC. Trifluoromethanesulfonic acid (TfOH, 100 µL)
was added to the solution, and the mixture was kept at
−10ºC 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 (7.8φ ×

8
H. KATAYAMA ET AL.
Pro0.95Gly4Ala2.05Val3.96Met1.58Leu2.02Tyr3.90Phe1.09
Lys1.14His2.02Arg2.08
Immunoblotting analysis
Ppe-RGP, Aam-RGP,
.
Aja-RGP,
Aam-RGP
A-chain, Aam-RGP B-chain, bovine insulin
(Sigma), and human relaxin-3 (PeproTech, NJ)
were dissolved in gel sample buffer without 2-mer-
captoethanol. Aliquots (0.1 nmoles/well) were
loaded into the lanes of a sodium dodecylsulfate–
polyacrylamide (SDS–PAGE) mini slab (15% gel)
(Cosmobio, Tokyo, Japan) and resolved by electro-
phoresis. Peptides separated by SDS–PAGE were
Trts-peg-gly-rgp 13
Peptide 12 (78.7 nmol) was dissolved in 5% AcOH/N,
N-dimethylacetamide (100 µL) containing 1% (w/v)
of compound 1, and the solution was mixed with
vortex at room temperature for 5 h. The mixture
was applied to the gel filtration HPLC using
a TSKgel G3000PWXL column (7.8φ x 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
13 (69.5 nmol, 88% yield). MALDI-TOF mass,
found: m/z 5979.7, calcd: 5980.6 for (M + H)+ (aver-
age). Amino acid analysis: Asp2.96Thr3.49Ser1.90Glu5.71
Pro0.94Gly4Ala2.05Val3.79Met1.63Leu2.17 Tyr3.86Phe1.08
transferred
to
an
Immobilon
membrane
(Millipore, Billerica, MA, USA) by electro-
blotting, as described previously [27]. The mem-
brane was rinsed in Tris-buffered saline (TBS)
consisting of 20 mM Tris–HCl (pH 7.5) and
150 mM NaCl, blocked with 5% nonfat dry milk
in TBS containing 0.1% Tween-20 (TTBS), and
incubated with a 1:20,000 dilution of anti-serum
against Aam-RGP in TTBS overnight at 4ºC. After
three washes with TTBS, the membrane was incu-
bated with a 1:30,000 dilution of alkaline phospha-
tase-conjugated goat anti-rabbit IgG (Sigma-
Aldrich, MO). After three further washes with
TTBS, phosphatase activity was visualized by treat-
ing the membrane with 0.2 mM 5-bromo-
4-chloro-3-indolyl phosphate p-toluidine salt and
nitroblue tetrazolium in 100 mM diethanolamine
buffer (pH 9.5) containing 5 mM MgCl2.
Lys1.12His2.08Arg1.98
.
Hs-peg-gly-rgp 14
Peptide 13 (69.2 nmol) was dissolved in TFA/H2O/
triisopropylsilane (96/2/2, 100 µL), and the solution
was mixed with vortex at 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
14 (31.4 nmol, 45% yield). MALDI-TOF mass,
found: m/z 5738.3, calcd: 5738.3 for (M + H)+ (aver-
age). Amino acid analysis: Asp2.85Thr3.33Ser1.54Glu5.61
Pro1.37Gly4Ala2.06Val3.92Met1.64Leu2.07 Tyr3.82Phe1.04
Author contributions
H. Katayama and M. Mita conceived and designed the
study, and wrote the manuscript. H. Katayama,
R. Mizuno, and M. Mita performed experiments.
Lys1.07His2.04Arg1.98
.
Acknowledgments
Evaluation of antibodies by ELISA
This work was supported in part by JSPS KAKENHI, Grant
number 18K05113.
To obtain titer curves of anti-serum against Aam-
RGP, ELISA was conducted. The wells of PVC micro-
titer plates were coated with 1 µM Aam-RGP, Aam-
RGP A-chain, or Aam-RGP B-chain in PBS (100 µL)
and incubated overnight at 4ºC. After washing and
blocking, the plates were dried at 4ºC. The anti-serum
diluted in PBST (100 µL) was added to the wells and
incubated overnight at 4ºC. After washing, horserad-
ish peroxidase-conjugated goat anti-rabbit immuno-
globulin (IgG) (Zymed Laboratories Inc., CA) diluted
in PBST (100 µL) was added to the wells and incu-
bated for 2 h at room temperature. After washing,
tetramethylbenzine (TMB)/hydrogen peroxidase
solution (100 µL) was added to the wells and incu-
bated for 30 min at room temperature. The reaction
was stopped by adding 1 M sulfuric acid (100 µL),
and absorbance at 450 nm was measured by a micro-
plate reader Model 680 (Bio-Rad Laboratories, Inc.,
Hercules, CA).
Disclosure statement
No potential conflict of interest was reported by the
authors.
References
[1] Van de Vijver P, Schmitt M, Suylen D, et al.
Incorporation of disulfide containing protein mod-
ules into multivalent antigenic conjugates: genera-
tion of antibodies against the thrombin-sensitive
region of murine protein S. J Am Chem Soc.
2012;134:19318–19321.
[2] Adrian TE. Production of antisera using peptide
conjugates. Methods Mol Biol. 1977;73:239–249.
[3] Mita M, Yoshikuni M, Ohno K, et al. A relaxin-like
peptide purified from radial nerves induces oocyte
maturation and ovulation in the starfish, Asterina

PREPARATION METHOD FOR DISULFIDE-RICH PEPTIDE-KLH CONJUGATE
9
pectinifera.
Proc
Natl
Acad
Sci
USA.
stepwise and selective formation of three disulfide
2009;106:9507–9512.
bridges. J Protein Chem 1992;11:1–12.
[4] Katayama H, Mita M. A sulfanyl-PEG derivative of
relaxin-like peptide utilizable for the conjugation
with KLH and the antibody production. Bioorg
Med Chem. 2016;24:3596–3602.
[5] Yamamoto K, Kiyomoto M, Katayama H, et al.
Radioimmunoassay of relaxin-like gonad-stimulating
peptide in the starfish Patiria (= Asterina) pectinifera.
Gen Comp Endocrinol. 2017;243:84–88.
[6] Mita M, Katayama H. Enzyme-linked immunosor-
bent assay of relaxin-like gonad-stimulating peptide
in the starfish Patiria (Asterina) pectinifera. Gen
Comp Endocrinol. 2018;258:157–162.
[17] Hua QX, Jia W, Frank BH, et al. A protein caught in
a kinetic trap: structures and stabilities of insulin dis-
ulfide isomers. Biochemistry. 2002;41:14700–14715.
[18] Hua QX, Mayer JP, Jia W, et al. The folding nucleus
of the insulin superfamily: A flexible peptide model
foreshadows the native state.
J
Biol Chem.
2006;281:28131–28142.
[19] Hossain MA, Rosengren KJ, Zhang S, et al. Solid
phase synthesis and structural analysis of novel
A-chain dicarba analogs of human relaxin-3
(INSL7) that exhibit full biological activity. Org
Biomol Chem. 2009;7:1547–1553.
[7] Ben-Bassat A, Bauer K. Amino-terminal processing
of proteins. Nature. 1987;326:315.
[8] Dixon HBF. Transamination of peptides. Biochem J.
1964;92:661–666.
[20] Katayama H, Hojo H, Ohira T, et al. Correct dis-
ulfide pairing is required for the biological activity of
crustacean androgenic gland hormone (AGH): syn-
thetic
studies
of
AGH.
Biochemistry.
[9] Dixon HBF N-terminal modification of proteins-A
review. J Protein Chem 1984;3:99–108.
[10] Gilmore JM, Scheck RA, Esser-Kahn AP, et al.
2010;49:1798–1807.
[21] Shabanpoor F, Bathgate RA, Belgi A, et al. Site-
specific conjugation of a lanthanide chelator and its
effects on the chemical synthesis and receptor bind-
ing affinity of human relaxin-2 hormone. Biochem
Biophys Res Commun. 2012;420:253–256.
N-terminal
protein
modification
through
a
biomimetic transamination reaction. Angew
Chem Int Ed. 2006;45:5307–5311.
[11] Duan X, Zhao Z, Ye J, et al. Donor-donor
energy-migration measurements of dimeric DsbC
labeled at its N-terminal amines with fluorescent
probes: a study of protein unfolding. Angew Chem
Int Ed. 2004;43:4216–4219.
[12] Dong SY, Ma HM, Duan XJ, et al. Detection of local
polarity of a-lactalbumin by N-terminal specific
labeling with a new tailor-made fluorescent probe.
J Proteome Res. 2005;4:161–166.
[22] Katayama H, Kubota N, Hojo H, et al. Direct evi-
dence for the function of crustacean insulin-like
androgenic gland factor (IAG): total chemical synth-
esis of IAG. Bioorg Med Chem. 2014;22:5783–5789.
[23] Katayama H, Mukainakano T, Kogure J, et al.
Chemical synthesis of the crustacean insulin-like
peptide with four disulfide bonds. J Pept Sci.
2018;24:e3132.
[24] Greenfield N, Fasman GD. Computated circular
dichroism spectra for the evaluation of protein
conformation. Biochemistry. 1969;8:4108–4116.
[13] Sasaki T, Kodama K, Suzuki H, et al. N-terminal
labeling of proteins by the Pictet-Spengler reaction.
Bioorg Med Chem Lett. 2008;18:4550–4553.
[25] Mita
M,
Katayama
H.
A
relaxin-like
[14] Mita M, Daiya M, Haraguchi S, et al. A new
relaxin-like gonad-stimulating peptide identified in
the starfish Asterias amurensis. Gen Comp
Endocrinol. 2015;222:144–149.
[15] Mita M, Nakamura K, Tsutsui K, et al. Interaction of
starfish gonadotropin with its receptor: effect of chimeric
relaxin-like gonad-stimulating peptides. Gen Comp
Endocrinol. In press. DOI:10.1016/j.ygcen.2019.02.019
[16] Maruyama K, Nagata K, Tanaka M, et al. (1992)
Synthesis of bombyxin-IV, and insulin superfamily
peptide from the silkworm, Bombyx mori, by
gonad-stimulating peptide from the starfish
Aphelasterias japonica. Gen Comp Endocrinol.
2016;229:56–61.
[26] Mita M. Starfish gonadotropic hormone: relaxin-like
gonad-stimulating peptides. Gen Comp Endocrinol.
2016;230-231:166–169.
[27] Mita M, Yamamoto K, Nakamura M, et al.
Participation of Gs-proteins in the action of
relaxin-like gonad-stimulating substance (GSS) for
1-methyladenine production in starfish ovarian fol-
licle cells. Gen Comp Endocrinol. 2012;176:432–437.