Development of a High-Affinity Antibody-Binding Peptide for Site-Specific Modification

Article abstract of DOI:10.1002/cmdc.202000977

Immunoglobulin G (IgG)-binding peptides such as 15-IgBP are convenient tools for the site-specific modification of antibodies and the preparation of homogeneous antibody–drug conjugates. A peptide such as 15-IgBP can be selectively crosslinked to the fragment crystallizable region of human IgG in an affinity-dependent manner via the ?-amino group of Lys8. Previously, we found that the peptide 15-Lys8Leu has a high affinity (K_d=8.19 nM) due to the presence of the γ-dimethyl group in Leu8. The primary amino group required for the crosslinking to the antibodies has, however, been lost. Here, we report the design and synthesis of a novel unnatural amino acid, 4-(2-aminoethylcarbamoyl)leucine (Aecl), which possesses both the γ-dimethyl fragment and a primary amino group. A peptide containing Aecl8 (15-Lys8Aecl) was synthesized and showed a binding affinity ten times higher (K_d=24.3 nM) than that of 15-IgBP (K_d=267 nM). Fluorescein isothiocyanate (FITC)-labeled 15-Lys8Aecl with an N-hydroxy succinimide ester at the side chain of Aecl8 (FITC-15-Lys8Aecl(OSu)) successfully labeled an antibody (trastuzumab, Herceptin) with the fluorophore. This peptide scaffold has both strong binding affinity and crosslinking capability, and could be a useful tool for the selective chemical modification of antibodies with molecules of interest such as drugs.

Full text of DOI:10.1002/cmdc.202000977

Full Papers
doi.org/10.1002/cmdc.202000977
ChemMedChem
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
Development of a High-Affinity Antibody-Binding Peptide
for Site-Specific Modification
[a, b]
[a]
[a]
[c]
[a]
Kyohei Muguruma,
Rento Osawa, Akane Fukuda, Naoto Ishikawa, Konomi Fujita,
[a]
[a, d]
[a]
[c]
Akihiro Taguchi, Kentaro Takayama,
Atsuhiko Taniguchi, Yuji Ito, and
[a]
Yoshio Hayashi*
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
Immunoglobulin G (IgG)-binding peptides such as 15-IgBP are
convenient tools for the site-specific modification of antibodies
and the preparation of homogeneous antibody–drug conju-
gates. A peptide such as 15-IgBP can be selectively crosslinked
to the fragment crystallizable region of human IgG in an
affinity-dependent manner via the ɛ-amino group of Lys8.
Previously, we found that the peptide 15-Lys8Leu has a high
both the γ-dimethyl fragment and a primary amino group. A
peptide containing Aecl8 (15-Lys8Aecl) was synthesized and
showed a binding affinity ten times higher (K =24.3 nM) than
d
that of 15-IgBP (K =267 nM). Fluorescein isothiocyanate (FITC)-
d
labeled 15-Lys8Aecl with an N-hydroxy succinimide ester at the
side chain of Aecl8 (FITC-15-Lys8Aecl(OSu)) successfully labeled
®
an antibody (trastuzumab, Herceptin ) with the fluorophore.
affinity (K =8.19 nM) due to the presence of the γ-dimethyl
This peptide scaffold has both strong binding affinity and
crosslinking capability, and could be a useful tool for the
selective chemical modification of antibodies with molecules of
interest such as drugs.
d
group in Leu8. The primary amino group required for the
crosslinking to the antibodies has, however, been lost. Here, we
report the design and synthesis of a novel unnatural amino
acid, 4-(2-aminoethylcarbamoyl)leucine (Aecl), which possesses
[
3]
Introduction
In the general preparation of ADCs, including Kadcyla,
payloads are randomly conjugated by using their active esters
to bind to lysine residues that are abundant on the surface of
antibodies. Alternatively, inherent disulfides in the antibody are
partially reduced, and resultant thiols are used for the selective
Antibodies modified with a molecule of interest such as a
fluorophore or
a cytotoxic drug (payload) for example,
monomethyl auristatin E or DM-1 are frequently utilized in
research and drug discovery. Antibody-drug conjugates (ADC)
are used clinically to treat cancer patients. An ADC can achieve
the target-specific delivery of a cytotoxic drug. This results in
the improvement of the therapeutic index by suppressing the
adverse effect caused by the payload.
have been approved by Food and Drug Administration (FDA)
for cancer treatment.
conjugation with
completely control the partial reduction. Consequently, most
of the approved ADCs are heterogeneous as a result of their
a payload. However, it is difficult to
[2]
[2]
random modification by payloads. Recent studies have
revealed that the heterogeneity of modification sites affects
in vivo pharmacokinetic, pharmacological efficacy and safety
[1,2]
To date, nine ADCs
[2,4]
profiles,
and homogeneity is required to maintain an
adequate quality of the ADC. Therefore, development of a
method for the site-specific chemical modification of antibody
would be valuable for the preparation of homogeneous ADC.
Known site specific antibody modification methods can be
[
a] Dr. K. Muguruma, R. Osawa, A. Fukuda, K. Fujita, Dr. A. Taguchi,
Dr. K. Takayama, Dr. A. Taniguchi, Prof. Y. Hayashi
Department of Medicinal Chemistry
Tokyo University of Pharmacy and Life Sciences
Hachioji, Tokyo, 192-0392 (Japan)
E-mail: yhayashi@toyaku.ac.jp
b] Dr. K. Muguruma
Present address:
[5]
broadly classified into two categories; a method using the
genetic engineering technique and a method using the site-
specific antibody-binding peptide as a directing unit. In the
former case, unnatural amino acids or sequences, such as p-
[
[6]
6
[7]
acetylphenyl-alanine, N -((2-azidoethoxy)carbonyl)lysine or
Department of Chemical Science and Engineering
School of Materials and Chemical Technology
Tokyo Institute of Technology, Tokyo 152-8552 (Japan)
[8]
an enzyme recognition sequence, is introduced genetically
into the antibody, then the payload is crosslinked to the
genetically introduced structure. In the second case, an anti-
body-binding peptide bearing the small molecule with a
chemical reactivity or catalytic activity induces a crosslinking
reaction near the peptide binding region. In this way, a
molecule of interest can be introduced selectively into a certain
[c] N. Ishikawa, Prof. Y. Ito
Department of Chemistry and Bioscience
Graduate School of Science and Engineering
Kagoshima University
Korimoto, Kagoshima, 890-0065 (Japan)
d] Dr. K. Takayama
[
Present address:
[9]
Department of Environmental Biochemistry
Kyoto Pharmaceutical University
Kyoto 607-8414 (Japan)
site. This site-specific crosslinking depends upon the affinity of
the peptide, and therefore this strategy is classified as ligand-
[11]
[12]
directed or affinity-guided chemistry. Modification by the
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202000977
ChemMedChem 2021, 16, 1–9
1
© 2021 Wiley-VCH GmbH
��
These are not the final page numbers!

Full Papers
doi.org/10.1002/cmdc.202000977
ChemMedChem
second method is applicable to the native antibody, which is a
big advantage over the first method.
The previous study also suggests that the high affinity of
15-Lys8Leu can be attributed to the γ-dimethyl structure of
leucine, which interacts with the hydrophobic region of the
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
Based on the ligand-directed chemistry in the second
concept, Kishimoto et al., developed a method using Fc binding
peptide, for an antibody modification with homogeneity (Fig-
[13a]
antibody.
In this study, we proposed new unnatural amino
acid, 4-(2-aminoethylcarbamoyl)leucine (Aecl), containing both
a primary amino group for the crosslinking and γ-dimethyl
structure necessary for the high binding affinity (Figure 1b). The
novel peptide derivative, 15-Lys8Aecl, was expected to show a
high binding affinity due to the presence of the γ-dimethyl
structure and to be applicable to site-specific crosslinking with
an antibody via the amino group of Aecl by the CCAP method.
[10]
ure 1a). This is referred to as the chemical conjugation by
affinity peptide (CCAP) method. In this method, IgG binding
peptide with the payload attached to the N terminus is
prepared, the side chain of Lys is reacted with the bivalent
crosslinking agent disuccinimidylglutarate (DSG), and then the
reactive succinimidyl ester of the DSG on the peptide forms an
amide bond with Lys248 which is located near the Fc region of
the antibody. As a result, covalent crosslinking between the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
payload and the antibody is selectively accomplished via the Results and Discussion
peptide. In this strategy, the binding affinity of IgG-binding
peptide is important to achieve the efficient site-specific
reaction.
Fmoc-Aecl(Cbz)-OH, a building block for the Fmoc-based solid-
phase peptide synthesis (SPPS), was synthesized as depicted in
Scheme 1. The γ-position of tert-butyl N-Boc-pyroglutamate (3)
was dimethylated with a moderate yield (40%) by lithium bis
(trimethylsilyl)amide (LiHMDS) and methyl iodide. The γ-lactam
ring was then opened by hydrolyzation to give the carboxylic
acid (5), and a subsequent condensation reaction with benzyl
(2-aminoethyl)carbamate afforded compound 6 in a yield of
59%. After deprotection of the Boc and tert-butyl groups under
acidic conditions, the α-amino group was protected with Fmoc-
Cl to give the target amino acid Fmoc-Aecl(Cbz)-OH (7), in a
yield of 20% (5 steps).
With the aim of increasing the binding affinity of peptide 1,
we have studied its structure-activity relationships (SAR), and
found several IgG-binding peptides with potent binding proper-
[13]
ties (Figure 1b). For example, the shortened peptide, 15-IgBP,
possesses a binding affinity (15 residues, K =267 nM) compara-
d
ble to that of peptide 1 (17 residues, Ac-DC*AYHKGELVWC*TFH-
NH , *disulfide bridge, K =225 nM). Substitution of Lys by Leu
2
d
at position 8 improves the binding affinity by a factor of
approximately 32 (15-Lys8Leu, K =8.12 nM). This peptide
d
shows one of the best antibody binding affinities among the
[14]
Fc-binding peptides which have been reported. However, 15-
Lys8Leu cannot be applied to antibody modification using the
CCAP method, because there is no ɛ-amino group of lysine at
position 8 which is required for selective crosslinking to
antibodies.
An IgG binding peptide, 15-Lys8Aecl, containing an Aecl
residue at the position 8 was synthesized by the general Fmoc-
based SPPS method. Fmoc-Aecl(Cbz)-OH (7) was coupled to the
peptide-resin using the HATU/HOAt/DIPEA method. For the
final deprotection and cleavage, the resin-bound peptide was
treated with a solution of trifluoroacetic acid (TFA)/m-cresol/
thioanisole: 1,3-dimethoxybenzene (1,3-DMB) (40:1:2:1). As the
scavenger in this step, 1,3-DMB was used to suppress the side
[
13b]
reaction, p-hydroxybenzylation of the C-terminal amide,
and
soft-nucleophilic scavengers were used to enhance the depro-
tection of the Cbz group by the push–pull mechanism reported
[15]
by Kiso et al. Intramolecular disulfide bond formation in the
crude peptide was performed by the Npys-OMe reagent that
Scheme 1. Synthesis of the unnatural amino acid, 4-(2-aminoethylcarbamoyl)
Figure 1. a) Chemical conjugation by affinity peptide (CCAP) method. b)
leucine (Aecl). a) LiHMDS, MeI, THF, À 78°C, 40%; b) LiOH·H O, THF/
2
Design concept of high-affinity IgG binding peptide (IgBP) applicable to
H
Et
2
O=9:1, quant.; c) benzyl (2-aminoethyl)carbamate, EDC·HCl, HOBt·H
N, DMF, 59%; d) TFA, CH Cl ; e) Fmoc-Cl, NaHCO , H O/THF=1:1, 85% (2
3 2 2 3 2
2
O,
[13a]
[13a]
CCAP method. IgG-binding peptides 15-IgBP,
Lys8Aecl developed by our group.
15-Lys8Leu
and 15-
steps).
ChemMedChem 2021, 16, 1–9
www.chemmedchem.org
2
© 2021 Wiley-VCH GmbH
��
These are not the final page numbers!

Full Papers
doi.org/10.1002/cmdc.202000977
ChemMedChem
[
16]
was developed by our group. After HPLC purification, the
desired 15-Lys8Aecl was obtained in a total yield of 8% and
with a purity of 97%. 15-Lys8Aecl derivatives were also
obtained by the modified protocols for the synthesis of 15-
Lys8Aecl (see the Experimental Section for more detail).
modeling. The CD spectra of 15-IgBP, 15-Lys8Leu and 15-
Lys8Aecl were similar, indicating that these peptides adopt the
similar secondary structures (Figure 2a). The positive maximum
peak around 230 nm is an important signal relating to the
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
[13a]
suitable conformation for the antibody binding.
It suggests
The binding kinetics (k , k and K ) of the peptides were
determined by a surface plasmon resonance (SPR) assay using a
:1 Langmuir fit model (Table 1). As expected, 15-Lys8Aecl
that the binding mode of 15-Lys8Aecl to the Fc region of
antibody may be similar to that of 15-IgBP and other previously
on
off
d
[13]
1
examined derivatives. Molecular modeling of 15-Lys8Aecl was
performed on the basis of the co-crystal structure of Fc region
and Fc-III peptide including Leu residue at the position 8 (PDB
showed tenfold higher binding affinity (entry 2, K =24.3 nM)
d
than parental 15-IgBP (K =267 nM), and both association and
d
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
À 1
À 1
[14e]
dissociation rates were modified (k =1.26 s ·μM , k =
.0306 s ). The introduced γ-dimethyl structure contributed to
ID: 1DN2 ), which has a similar sequence and antibody
on
off
À 1
0
binding mode to peptide 1 (Figure 1b). The dimethyl group of
Aecl in 15-Lys8Aecl is located in a similar place to the methyl
group of Leu in Fc-III peptide that could interact with hydro-
phobic region of the antibody. This interaction could induce
high binding affinity in 15-Lys8Aecl peptide, and might succeed
in directing the primary amino group of Aecl8 to the target
residue, Lys248 in the antibody. The distance of 0.63 nm
between the both amino groups of Aecl8 and Lys248 could be
appropriate for the crosslinking reaction, because the length of
DSG linker is 0.77 nm.
improvement of the binding affinity. The affinity was three
times weaker than that of 15-Lys8Leu with no amino group
(
entry 1, K =8.19 nM). This result is consistent with a previous
d
result that the tertiary carbon structure at γ-position slightly
decreases the affinity in both association and dissociation steps
compared to the secondary carbon structure (15-Lys8Ala
[13a]
(
tBu)).
15-Lys8Aecl exhibited, however, the best affinity
among the reported peptides with the side chain amino group
[13]
at the position 8, and is applicable to the CCAP method.
In the CCAP method, this amino group was acylated by DSG
to attach the reactive moiety to the peptide. To evaluate the
effect of the acyl linker on the binding affinity, 15-Lys8Aecl(Ac)
with an acetylated amino group at position 8 was prepared as a
mimic of DSG-form. Its binding affinity was found to be slightly
To examine the antibody modification using 15-Lys8Aecl, an
FITC-labeled peptide (FITC-15-Lys8Aecl) was prepared. The
labeled peptide showed a similar binding affinity to that of
unlabeled 15-Lys8Aecl (Table 1, entries 4 and 2, K =25.5 vs
d
24.3 nM), suggesting that the Aecl-containing peptide can work
as a good affinity-guide for the site-specific conjugation of the
payload to the Fc region of the antibody. FITC-15-Lys8Aecl was
reacted with DSG in the presence of N-methylmorpholine to
obtain FITC-15-Lys8Aecl(OSu) bearing the reactive succinimidyl
ester (Figure 3a). As extending the reaction time leads to
hydrolysis of the unstable succinimidyl ester moiety under basic
reaction conditions, excess amounts of reagents were used to
complete the reaction before hydrolysis. In HPLC analysis, the
peak of starting peptide FITC-15-Lys8Aecl disappeared and the
desired product, FITC-15-Lys8Aecl(OSu), was produced after a
decreased (entry 3, K =89.8 nM) compared to unacetylated 15-
d
Lys8Aecl (entry 2), mainly due to the slower association step
À 1
À 1
(
8
kon =1.26 vs. 0.478 s ·μM ). The amino group at the position
might contribute partially to the high affinity of 15-Lys8Aecl
[13a]
by interacting with Glu380 of the antibody.
However, 15-
Lys8Aecl(Ac) still retained a higher binding affinity than 15-
Lys8Lys(Ac) (K =268 nM), indicating that 15-Lys8Aecl is a
d
promising peptide for the CCAP method.
To estimate the binding of the Aecl-containing peptide with
antibody, we performed CD spectral analysis and molecular
Table 1. Antibody binding affinity of peptide derivatives.
À 1
À 1
À 1
Peptide
R
X
K
d
[nM]
k
on [s ·μM
]
koff [s ]
a
a
a
1
2
15-Lys8Leu
Ac
8.19�2.1
24.3�0.4
1.47�0.00
1.26�0.00
0.0116�0.0010
15-Lys8Aecl
Ac
0.0306�0.0005
3
4
15-Lys8Aecl(Ac)
FITC-15-Lys8Aecl
Ac
89.8�1.7
25.5�0.7
0.478�0.002
0.815�0.003
0.0429�0.0008
0.0208�0.0006
FITC-Acp
Binding affinity was measured by using trastuzumab with an immobilized amount of 2000 RU. [a] Values from ref. [13a].
ChemMedChem 2021, 16, 1–9
www.chemmedchem.org
3
© 2021 Wiley-VCH GmbH
��
These are not the final page numbers!

Full Papers
doi.org/10.1002/cmdc.202000977
ChemMedChem
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
Figure 2. a) CD spectra of 15-IgBP, 15-Lys8Leu and 15-Lys8Aecl. b) Structural
model of 15-Lys8Aecl binding with antibody on the basis of the X-ray co-
crystal structure of human IgG1 and Fc-III peptide (PDB ID: 1DN2). 15-
Lys8Aecl and antibody are shown as pink and yellow ribbons, respectively.
The Fc-III peptide in the original PDB file (1DN2) is presented as a green
ribbon. Aecl8 of 15-Lys8Aecl, Lys248 of antibody and Leu6 of Fc-III
(
corresponding to the position 8 in 15-Lys8Aecl) are shown as cyan, orange
and gray, respectively.
1
0 min reaction (Figure 3b). FITC-15-Lys8Aecl(OSu) was ob-
tained after HPLC purification with a purity of 98%.
Finally, we examined the crosslinking reaction of FITC-15-
®
Lys8Aecl(OSu) to trastuzumab (Herceptin ; Figure 4a), moni-
tored by hydrophobic interaction chromatography (HIC), gel
filtration chromatography (GFC) and MALDI-TOF MS analysis. In
the HIC analysis, the peak for trastuzumab at a retention time of
8
.0 min disappeared after the crosslinking reaction with 3
equivalents of FITC-15-Lys8Aecl(OSu) for 1 h (Figure 4b and c,
blue lines). A new peak with a retention time of 22.4 min,
detected by both UV absorption and fluorescence, was
observed after the reaction (Figure 4c, blue and orange lines).
These results indicate that the modification of trastuzumab with
FITC succeeded completely. In addition, MALDI-TOF MS showed
that molecular weight of antibody increased by 4.4 kDa after
the reaction, indicating that two peptide molecules are
attached to the antibody because the increase of molecular
mass by conjugation of single peptide molecule would be
Figure 3. a) Synthesis of FITC-15-Lys8Aecl(OSu). i) Solid-phase peptide syn-
thesis (coupling: Fmoc-amino acid, HATU, HOAt and DIPEA; deprotection:
2
0% piperidine/DMF), ii) FITC, DMF; iii) TFA/m-cresol/thioanisole/1,3-DMB
(40:1:2:1); iv) Npys-OMe, 83% aq. CH CN, 9% from resin (HPLC purification);
3
v) DSG, N-methylmorpholine, DMF, 14% (HPLC purification). b) HPLC
chromatograms of the peptide solution before reaction, after reaction of 10
min and after purification. Analysis conditions; Gradient: 0.1% TFA aq./
CH CN=90:10 to 50:50 over 40 min, Flow rate: 0.9 mL/min, λ=230 nm,
3
Column: COSMOSIL 5C18-AR-II.
2
477 Da (Figures 4d, e and S1). This is reasonable because the
antibody possesses two binding sites for the peptide in the
symmetric structure of the Fc region. In the GFC analysis, the
fluorescence intensity was increased with an equivalent of the
peptide reagent, and reached a plateau at 3 equivalents
(Figure S2). This result indicates that no overreaction occurs
even if a large excess amount of peptide reagent was added.
ChemMedChem 2021, 16, 1–9
www.chemmedchem.org
4
© 2021 Wiley-VCH GmbH
��
These are not the final page numbers!

Full Papers
doi.org/10.1002/cmdc.202000977
ChemMedChem
Conclusion
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
We have designed and synthesized a novel IgG binding peptide
containing an unnatural amino acid, Aecl, with both the γ-
dimethyl and primary amino groups at the position 8. The
building block for SPPS, Fmoc-Aecl(Cbz)-OH, was synthesized in
five steps from the commercially available amino acid (3) with a
total yield of 20%. 15-Lys8Aecl shows the best binding affinity
[
13]
(Kd =24.3 nM) among the reported peptides
and can be
applied to antibody modification based on the CCAP method.
Moreover, the FITC-labelled peptide, FITC-15Lys8Aecl, retained
a high binding affinity (K =25.5 nM), and was successfully
applied to the efficient FITC labelling of the antibody. Therefore,
the peptide with Aecl can be a useful tool for the site-specific
antibody modification on the basis of ligand-directed or
affinity-guided chemistry.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
d
Experimental Section
General procedures
Reagents and solvents were purchased from FujiFilm Wako Pure
Chemical Industries (Osaka, Japan), Kanto Chemical Co., Inc. (Tokyo,
Japan), Sigma–Aldrich (St. Louis, MO), Watanabe Chemical Indus-
tries (Hiroshima, Japan), Tokyo Chemical Industries (Tokyo, Japan)
and Nacalai tesque (Kyoto, Japan). All were used as received.
Trastuzumab was purchased from Chugai Pharmaceutical Co., Ltd.
Column chromatography was performed on silica gel 60 N
(
spherical, neutral; 40–50 μm) and thin-layer chromatography (TLC)
was performed on precoated plates (0.25 mm, silica gel Merk
1
Kieselgel 60F245). H NMR spectra were measured in CDCl3 or
CD OD solution and referenced to TMS (0.00 ppm) using a Bruker
3
13
DPX-400 (400 MHz) NMR spectrometer. C NMR spectra were
measured in CDCl or CD OD solution and referenced to a residual
solvent peak of CDCl
3
3
(77.05 ppm) or CD OD (49.00 ppm) using a
3
3
Bruker DPX-400 (100 MHz) NMR spectrometer. When peak multi-
plicities are reported, the following abbreviations are used: s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet. IR spectra were
recorded on JASCO FT/IR 4100 spectrometer. Mass spectra were
obtained on a Waters MICRO MASS LCT-premier (ESI) or Shimadzu
Biotech AXIMA Assurance (MALDI). Melting points were measured
with a Yanaco MP-500D melting point apparatus. Optical rotations
were measured with a JASCO Polarimeter P-1030 at the sodium-D
line (589 nm) at the concentrations (c, in g/dL). The measurements
were carried out at 25°C in a cell with path length of 1 dm.
Figure 4. a) Antibody modification using FITC-labeled 15-Lys8Aecl peptide.
HIC chromatograms of the reaction solution b) before and c) after adding
the peptide reagent, detected by absorbance (230 nm, blue lines) and
fluorescence (λex =490 nm, λem =520 nm, orange lines). MALDI-TOF mass-
spectra of the reaction solution d) before and e) after adding the peptide
reagent.
Benzyl (2-aminoethyl)carbamate
Benzyl chloroformate (1.3 mL, 9 mmol) in CH Cl (25 mL) was added
2
2
dropwise at 0 C to a solution of ethylene diamine (0.6 mL, 9 mmol)
in CH Cl (90 mL). After stirring for 1 h, the reaction solution was
washed with brine, dried over Na SO , filtered and concentrated.
°
2
2
2
4
The product was used in next step without further purification.
White solid, yield 95% (1.57 g). Characterization data matched with
Additionally, the peptide–antibody conjugate was purified by
dialysis with a yield of 60% (calculated on the basis of protein
amount, which is determined by bicinchoninic acid (BCA) assay;
Figure S4). Taken together, the peptide reagent based on 15-
Lys8Aecl sequence could rapidly provide the homogeneously
modified antibody which would be applicable to preparing
homogeneous ADCs.
[17]
previously reported data of this known compound; m.p. 74.3–
1
7
5.1°C; H NMR (400 MHz, CDCl ): δ=7.32-7.27 (m, 5H), 5.82 (s, 1H),
3
5.07 (s, 2H), 3.17 (q, J=5.6 Hz, 2H), 2.73 (t, J=5.6 Hz, 2H), 1.23 (s,
1
3
1H); C NMR (100 MHz, CDCl
): δ=156.7, 136.6, 128.5, 128.1, 66.7,
3
À 1
43.8, 41.7; IR (KBr) cm : 3320, 1692, 1541, 1454, 1266; HRMS (ESI):
+
m/z calcd. for C H N O [M+H] 195.1134, found 195.1128.
1
0
14
2
2
ChemMedChem 2021, 16, 1–9
www.chemmedchem.org
5
© 2021 Wiley-VCH GmbH
��
These are not the final page numbers!

Full Papers
doi.org/10.1002/cmdc.202000977
ChemMedChem
di-tert-Butyl
(S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)
amino)-5-((2-(((benzyloxy)carbonyl)amino)ethyl)
amino)-4,4-dimethyl-5-oxopentanoic acid (7)
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
(S)-4,4-dimethyl-5-oxopyrrolidine-1,2-dicarboxylate (4)
This reaction was performed in oven-dried glassware under an
argon atmosphere. tert-Butyl N-(tert-butoxycarbonyl)-l-pyroguluta-
mate 3 (5 g, 17.5 mmoL) in THF was added dropwise at À 78°C to a
TFA (4.8 mL) was added at 0°C to a solution of compound 6
(220 mg, 0.43 mmol) in CH Cl (2.4 mL). After stirring at RT for 1 h,
2
2
solution of lithium bis(trimethylsilyl)amide (LiHMDS) in THF
the solvent was removed in vacuo to obtain the compound with
(
36.8 mL, 36.8 mmol). After stirring for 15 min at À 78 C, methyl
°
deprotected amino and carboxyl groups. The compound was
iodide (2.3 mL, 36.8 mmol) in THF (25 mL) was added dropwise.
Then, after stirring for 2 h at room temperature (RT), the reaction
dissolved in THF/H O (1:1, 14.4 mL), and NaOH (180 mg,
2
2.15 mmol) and Fmoc-Cl (120 mg, 0.47 mmol) were added to the
solution. After stirring at RT for 2 h, the reaction solution was
acidified to pH 3 by adding 1 M HCl aq. at 0°C. The mixture was
extracted with EtOAc, washed with brine, dried over Na SO , filtered
solution was neutralized with sat. NH Cl aq., and then extracted
with EtOAc. The extract was washed with brine, dried over Na SO ,
filtered, and concentrated. The residual oil was purified by silica gel
chromatography (hexane/EtOAc 5:1), resulting in a white solid
(yield 40%, 2.2 g). Characterization data matched with those in a
4
2
4
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
2
4
and concentrated. The residue was purified by flash column
chromatography (hexane/EtOAc 5:1 to 4:1) to obtain compound 7
[
18]
25
D
25
previous report of this known compound (4); [α] =À 22.30° (c
as a white solid, yield 85% (140 mg). [α] =À 2.09° (c 0.25, CH CN);
D
3
2
0
1
1
.02, MeOH) (lit. [α] =À 26.24° (c 1.0, CHCl ); m.p. 104.6–105.8 °C
m.p. 69.5–70.4°C; H NMR (400 MHz, MeOD): δ=7.75 (d, J=7.5 Hz,
2H), 7.63-7.57 (m, 2H), 7.35 (t, J=7.5 Hz, 2H), 7.29–7.18 (m, 7H), 5.00
(s, 2H), 4.26 (d, J=7.0 Hz, 2H), 4.13 (t, 6.8 Hz, 1H), 4.06 (d, J=8.2 Hz,
1H), 3.28-3.13 (m, 4H), 2.17 (d, J=14.2 Hz, 1H), 1.89–1.78 (m, 1H),
D
3
1
(
lit. 100–102°C); H NMR (400 MHz, CDCl ): δ=4.41 (dd, J=4.3 and
3
9
1
.7 Hz, 1H), 2.19 (dd, J=9.8 and 13.3, 1H), 1.89 (dd, J=4.3 and
13
3.3 Hz, 1H), 1.52 (s, 9H), 1.48 (s, 9H), 1.21 (s, 3H), 1.21 (s, 3H);
C
13
NMR (100 MHz, CDCl ): δ=178.3, 170.7, 149.8, 83.2, 82.2, 58.5, 41.6,
36.7, 27.9, 25.8, 25.3; IR (KBr) cm : 2980, 1784, 1731, 1308, 1164,
1.22–1.08 (m, 6H); C NMR (100 MHz, MeOD): δ=180.3, 175.9,
3
À 1
159.2, 158.3, 145.3, 145.2, 142.6, 142.5, 138.3, 129.4, 129.0, 129.0,
128.9, 128.8, 128.2, 128.1, 126.4, 126.2, 120.9, 68.1, 67.5, 52.9, 42.6,
+
1255; HRMS (ESI): m/z calcd for C16
36.1777.
H
27NO
[M+H] 336.1787, found
5
À 1
3
42.4, 41.5, 41.3, 41.2, 27.1, 25.0; IR (KBr) cm : 3403, 1715, 1525,
+
1
236, 739; HRMS (ESI): m/z calcd for C H N O [M+H] 574.2553,
3
2
35
3
7
found 574.2555.
(
S)-5-(tert-Butoxy)-4-((tert-butoxycarbonyl)
amino)-2,2-dimethyl-5-oxopentanoic acid (5)
Solid-phase peptide synthesis (SPPS)
IgG binding peptides were synthesized by the Fmoc-based solid-
LiOH·H O (370 mg, 8.8 mmol) was added at 0°C to a solution of
2
compound 4 in THF/H O (9:1, 40 mL) After stirring at RT for 4 h, the
2
[19]
reaction solution was acidified to pH 3 with 1 MHCl aq. and
extracted with EtOAc. The extract was washed with brine, dried
phase peptide synthetic method
using an automatic peptide
synthesizer (Prelude). Using the Fmoc-NH-SAL resin (40 μmol), the
peptide chain was elongated with the Fmoc-amino acid (5 equiv),
1-[bis(dimethylamino)-methylene]-1H-1,2,3-triazolo[4,5-b]
pyridinium 3-oxid hexafluorophosphate (HATU, 5 equiv), 1-hydroxy-
7-azabenzotriazole (HOAt, 5 equiv) and N,N-diisopropylethylamine
(DIPEA, 10 equiv) for 30 min. The following amino acid derivatives
were used as the building blocks; Fmoc-Asp(OtBu)-OH, Fmoc-Cys
(Trt)-OH, Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-
Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Trp
over Na
SO , filtered, and concentrated, resulting in a white solid
4
2
(
2.7 g). This compound was used without further purification. m.p.
2
5
1
1
05.2–106.5°C; [α] =À 8.29° (c 0.40, MeOH); H NMR (400 MHz,
D
MeOD): δ=4.11–4.07 (m, 1H), 1.96–1.94 (m, 2H), 1.45 (s, 9H), 1.43 (s,
13
9
H), 1.23 (s, 3H), 1.20 (s, 3H); C NMR (100 MHz, MeOD): δ=181.1,
1
73.8, 157.7, 82.6, 80.5, 53.3, 42.0, 41.9, 28.7, 28.2, 26.8, 24.8; IR (KBr)
cm-1: 3254, 2978, 1737, 1709, 1402, 1369, 1150; HRMS (ESI): m/z
+
calcd for C H NO [M+H] 354.1893, found 354.1885.
16 29
6
(
Boc)-OH, Fmoc-Thr(tBu)-OH and Fmoc-Phe-OH. The Fmoc group
was deprotected with 20% piperidine in DMF for 10 min twice.
These reactions were repeated to lengthen the desired peptide.
The N-terminal amino group was acetylated by acetic anhydride
tert-Butyl (S)-5-((2-(((benzyloxy)carbonyl)amino)ethyl)
amino)-2-((tert-butoxycarbonyl)
amino)-4,4-dimethyl-5-oxopentanoate (6)
(3 equiv) and DIPEA (3 equiv) in DMF for 15 min. For FITC-labelled
peptide, the N-terminal amino group of the peptide-resin was
coupled with Fmoc-aminocaproic acid, the Fmoc group was
deprotected, and then mixed with FITC (2.5 equiv) in DMF.
Cleavage from resin and final deprotection were performed by
EDC·HCl (260 mg, 1.35 mmol), HOBt·H O (210 mg, 1.35 mmol) and
2
benzyl (2-aminoethyl)carbamate (262 mg 1.35 mmol) in DMF were
added at 0°C to a solution in DMF of compound 5 (300 mg,
0.9 mmol). After stirring for 2 h at RT, the solvent was removed in
vacuo, and the residue was extracted with EtOAc. The extract was
washed with brine, filtered and concentrated. Then, the residue
was purified by flash column chromatography (hexane/EtOAc 1:1)
[15]
[13b]
treating with TFA/m-cresol/thioanisole /1,3-DMB
(40:1:2:1) for
7
h at RT. The crude peptide was precipitated with Et O and
2
washed twice. After drying, the residual solid was dissolved in 83%
aq. CH CN, and then methyl 3-nitro-2-pyridinesulfenate (Npys-OMe,
5
2
5
3
to obtain compound 6 as a clear oil, yield 60% (270 mg); [α] =
[16]
D
equiv) was added to form the intramolecular disulfide bond.
1
À 5.47° (c 0.35, MeOH); H NMR (400 MHz, CDCl ): δ=7.36–7.31 (m,
3
After stirring for 7–9 h at RT, the solution was lyophilized and
5
H), 6.42 (s,1H), 5.56 (s, 1H), 5.09 (s, 2H), 5.04 (d, J=9.0 Hz, 1H),
washed with Et O to remove the excess reagent. After drying, the
2
4.13–4.07 (m, 1H), 3.51–3.25 (m, 4H), 2.11–2.05 (m, 1H), 1.82-1.79
residual solid was purified by RP-HPLC [SunFire PrepC18 OBD 19 x
1
3
(
(
m, 1H), 1.44 (s, 9H), 1.38 (s, 9H), 1.21 (s, 3H), 1.18 (s, 3H); C NMR
100 MHz, CDCl ): δ=178.3, 172.0, 157.2, 155.6, 136.4, 128.3, 128.0,
1
50 mm (5 μm)] to give the desired peptide. The purity of
3
synthesized peptides was analyzed by RP-HPLC (Cosmosil 5 C18 AR-
1
28.0, 81.5, 79.7, 66.6, 52.1, 41.4, 40.8, 40.7, 40.4, 28.2, 27.8, 23.3; IR
II, 4.6 i.d. ×150 mm) using a binary solvent system with a linear
À 1
(KBr) cm : 3361, 2977, 1715, 1530, 1367, 1256, 1154; HRMS (ESI): m/
gradient starting from 10% CH CN in 0.1% aqueous TFA to 50%
+
3
z calcd for C H N O [M+H] 530.2842, found 530.2838.
26
41
3
CH CN in 0.1% aqueous TFA at a flow rate of 0.9 mL/min, and
3
detected at UV 230 nm. The yield and analytical data of peptides
are shown in Supporting Information.
ChemMedChem 2021, 16, 1–9
www.chemmedchem.org
6
© 2021 Wiley-VCH GmbH
��
These are not the final page numbers!

Full Papers
doi.org/10.1002/cmdc.202000977
ChemMedChem
Acylation of peptide side chains
20:80 to 100:0 over 40 min at a flow rate of 0.7 mL/min. The signals
were detected by UV absorbance at 230 nm and fluorescence with
excitation of 490 nm and emission of 520 nm.
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
Acylation of side chains of 15-Lys8Aecl and FITC-15-Lys8Aecl was
performed in solution with the previously described conditions.
After disulfide bridging, the purified peptide was dissolved in
DMSO at a concentration of 2.5 μM. Then, the peptide was treated
with N-succinimidyl acetate (10 equiv) or DSG (25 equiv) in the
presence of N-methylmorpholine (10–20 equiv) at RT. After 10–
30 min, the desired peptide was directly purified by RP-HPLC
[
13a]
Gel filtration chromatography (GFC)
For GFC, TSKgel G3000SWXL column (7.8 mm i.d. ×30 cm) was used
in the elution conditions with 0.1 M sodium phosphate buffer
(pH 7.0) at a flow rate of 0.7 mL/min. The signals were detected by
UV absorbance at 230 nm and fluorescence with excitation of
[SunFire PrepC18 OBD 19×150 mm (5 μm)].
490 nm and emission of 520 nm.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
Circular dichroism (CD) spectra
The circular dichroism spectra of the peptides were measured on
the basis of the previous conditions
Mass spectrometry analysis
[
20]
using a Jasco J-1500CD
spectrometer (Jasco, Japan) in a quartz cell with a 0.5 cm path
length. Spectra were collected between 190–250 nm with a scan
speed of 100 nm/min, a response time of 1 s, and a bandwidth of
1 nm at 25°C. The peptides were dissolved in a 10 mM phosphate
buffer (pH 7.4) including 10% 2,2,2-trifluoroethanol at a concen-
tration of 2.5 μM.
Samples were desalted with ZipTip Pipette Tips C4 (Millipore Co.)
and analyzed by MALDI-TOF MS in linear mode using sinapinic acid
as a matrix. The mass value is calibrated with mass of Trastuzumab
as 148.0 kDa
Dialysis
The reaction mixture (100 μL) was diluted with 25 mM phosphate
buffer (pH 6.0, 100 μL), and dialyzed with 25 mM phosphate buffer
(pH 6.0) at RT for 1 day twice using a Pur-A-Lyzer Maxi 3500 Dialysis
Kit (Sigma-Aldrich) according to the manufacturer’s instruction. The
protein concentration after dialysis (25 μL) was measured with
Pierce BCA Protein Assay Kit (Thermo) according to the manufac-
turer’s instruction.
Surface plasmon resonance (SPR) assay
The binding kinetics were determined by a Biacore T-200 system on
the basis of the previous conditions. Trastuzumab (human IgG1),
[20]
human serum albumin or mouse IgG1 was dissolved in 10 mM
acetate buffer (pH 5.5) and immobilized by premixed N-hydroxysuc-
cinimide and EDC·HCl (1-(3-dimethylaminopropyl)-3-ethyl-carbodii-
mide hydrochloride) onto a CM5 censor chip. The analytes (peptide
derivatives) were adjusted to the desired concentration (ranging
from 62.5 to 100 nM) by a serial dilution in a running buffer (HBS-
EP; 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% tween 20,
pH 7.4). The sensorgrams were obtained with an association time of
180 s, dissociation time of 600 s, and flow rate of 50 μL/min. To
Acknowledgements
The authors acknowledge H. Fukaya of the Tokyo University of
Pharmacy and Life Sciences for the mass spectrometry analysis.
This work was supported by the Japan Society for the Promotion
of Science (JSPS), KAKENHI, grants no. 15H04658, 19H03356 and
determine the binding kinetics (k , k and K ), the obtained
sensorgrams were analyzed by the Biacore T200 Evaluation
software Ver.1.0, using 1:1 binding model.
on
off
d
1
5J09551, and by AMED (Japan Agency for Medical Research and
Molecular modelling
Development), Basic Science and Platform Technology Program
for Innovative Biological Medicine, grants no. JP18am0301006.
The crystal structure of the Fc region of human IgG1 in complex
with Fc-III peptide (PDB ID: 1DN2) was obtained from the Protein
Data Bank. The 15-Lys8Aecl peptide was constructed on the basis
of the secondary structure of the Fc-III peptide by using molecular
operating environment (MOE) software. The plausible conformation
of the side chain of the Aecl residue was obtained by scanning a
separately created side chain library. Energy minimization process
was performed using the Amber10:EHT force field.
Conflict of Interest
The authors declare no conflict of interest.
Keywords: IgG-binding peptide · bioconjugation · antibody-
drug conjugate · structure activity relationship · unnatural
amino acid
Antibody modification reaction
Several equivalents of FITC-15-Lys8Aecl(OSu) in DMSO were added
at RT to a solution of trastuzumab in 100 mM sodium acetate buffer
(pH 5.0). The final concentration of trastuzumab was adjusted to
4
μM in 10% DMSO/buffer solution. After 1 h, the reaction was
[1] a) R. V. Chari, M. L. Miller, W. C. Widdison, Angew. Chem. Int. Ed. 2014,
analyzed by hydrophobic interaction chromatography (HIC) or gel
filtration chromatography (GFC).
53, 3796; b) B. E. de Goeij, J. M. Lambert, Curr. Opin. Immunol. 2016, 40,
14.
[
[
2] N. Joubert, A. Beck, C. Dumontet, C. Denevault-Sabourin, Pharmaceut-
icals 2020, 13, 245.
3] G. D. Lewis Phillips, G. Li, D. L. Dugger, L. M. Crocker, K. L. Parsons, E.
Mai, W. A. Blattler, J. M. Lambert, R. V. Chari, R. J. Lutz, et al. Cancer Res.
2008, 68, 9280–9290.
Hydrophobic interaction chromatography (HIC)
For HIC, TSKgel Butyl-NPR column (4.6 mm i.d. ×3.5 cm) was used
in the elution conditions with [25 mM phosphate buffer (pH 7.0)+
1.5 M (NH )SO ]/[25 mM phosphate buffer (pH 7.0)+5% IPA]=
[4] J. R. Junutula, H. Raab, S. Clark, S. Bhakta, D. D. Leipold, S. Weir, Y. Chen,
M. Simpson, S. P. Tsai, M. S. Dennis, Y. Lu, Y. G. Meng, C. Ng, J. Yang,
C. C. Lee, E. Duenas, J. Gorrell, V. Katta, A. Kim, K. McDorman, K. Flagella,
4
4
ChemMedChem 2021, 16, 1–9
www.chemmedchem.org
7
© 2021 Wiley-VCH GmbH
��
These are not the final page numbers!

Full Papers
doi.org/10.1002/cmdc.202000977
ChemMedChem
R. Venook, S. Ross, S. D. Spencer, W. Lee Wong, H. B. Lowman, R.
Vandlen, M. X. Sliwkowski, R. H. Scheller, P. Polakis, W. Mallet, Nat.
Biotechnol. 2008, 26, 925.
5] a) P. Agarwal, C. R. Bertozzi, Bioconjugate Chem. 2015, 26, 176; b) K.
Yamada, Y. Ito, ChemBioChem 2019, 20, 2729–2737.
6] J. Y. Axup, K. M. Bajjuri, M. Ritland, B. M. Hutchins, C. H. Kim, S. A.
Kazane, R. Halder, J. S. Forsyth, A. F. Santidrian, K. Stafin, et al. Proc. Natl.
Acad. Sci. U. S. A. 2012, 109, 16101–16106.
7] M. P. VanBrunt, K. Shanebeck, Z. Caldwell, J. Johnson, P. Thompson, T.
Martin, H. Dong, G. Li, H. Xu, F. D’Hooge, L. Masterson, P. Bariola, A.
Tiberghien, E. Ezeadi, D. G. Williams, J. A. Hartley, P. W. Howard, K. H.
Grabstein, M. A. Bowen, M. Marelli, Bioconjugate Chem. 2015; 26, 2249–
Sun, T. Tamura, K. Kuwata, Z. Song, Y. Takaoka, I. J. Hamachi, Am. Chem.
Soc. 2013, 135, 12252; c) T. Tamura, Z. Song, K. Amaike, S. Lee, S. Yin, S.
Kiyonaka, I. Hamachi, J. Am. Chem. Soc. 2017, 139, 14181–14191.
[13] a) K. Muguruma, K. Fujita, A. Fukuda, S. Kishimoto, S. Sakamoto, R.
Arima, M. Ito, M. Kawasaki, S. Nakano, S. Ito, et al., ACS Omega 2019, 4,
14390–14397; b) K. Muguruma, M. Ito, A. Fukuda, S. Kishimoto, A.
Taguchi, K. Takayama, A. Taniguchi, Y. Ito, Y. Hayashi, MedChemComm
2019, 10, 1789–1795.
[14] a) W. Choe, T. A. Durgannavar, S. J. Chung, Materials. 2016, 9, E994; b) N.
Kruljec, T. Bratkovic, Bioconjugate Chem. 2017, 28, 2009–2030; c) A. C.
Braisted, J. A. Wells, Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 5688–5692;
d) R. L. Dias, R. Fasan, K. Moehle, A. Renard, D. Obrecht, J. A. Robinson, J.
Am. Chem. Soc. 2006, 128, 2726–2732; e) W. L. DeLano, M. H. Ultsch,
A. M. de Vos, J. A. Wells, Science 2008, 287, 1279–1283.
[15] Y. Kiso, K. Ukawa, T. Akita, J. Chem. Soc. Chem. Commun. 1980, 101–102.
[16] a) A. Taguchi, K. Kobayashi, A. Kotani, K. Muguruma, M. Kobayashi, K.
Fukumoto, K. Takayama, H. Hakamata, Y. Hayashi, Chem. Eur. J. 2017, 23,
8262; b) Y. Hayashi, A. Taguchi, K. Fukumoto, WO 2017200109, 2017.
[17] B. Gruber, S. Balk, S. Stadlbauer, B. König, Angew. Chem. Int. Ed. 2012,
51, 10060–10063.
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
[
[
[
2260.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
[
8] P. Strop, S. H. Liu, M. Dorywalska, K. Delaria, R. G. Dushin, T. T. Tran,
W. H. Ho, S. Farias, M. G. Casas, Y. Abdiche, D. Zhou, R. Chandrasekaran,
C. Samain, C. Loo, A. Rossi, M. Rickert, S. Krimm, T. Wong, S. M. Chin, J.
Yu, J. Dilley, J. Chaparro-Riggers, G. F. Filzen, C. J. O’Donnell, F. Wang,
J. S. Myers, J. Pons, D. L. Shelton, A. Rajpal, Chem Biol. 2013, 20, 161–
167.
[
9] a) N. Vance, N. Zacharias, M. Ultsch, G. Li, A. Fourie, P. Liu, J. LaFrance-
Vanasse, J. A. Ernst, W. Sandoval, K. R. Kozak, et al. Bioconjugate Chem.
[18] J.-D. Charrier, J. E. S. Duffy, P. B. Hitchcock, D. W. Young, J. Chem. Soc.
Perkin Trans. 1 2001, 2367–2371.
[19] C. D. Chang, J. Meienhofer, J. Int. J. Pept. Protein Res. 1978, 11, 246–249.
[20] K. Muguruma, F. Yakushiji, R. Kawamata, D. Akiyama, R. Arima, T.
Shirasaka, Y. Kikkawa, A. Taguchi, K. Takayama, T. Fukuhara, T. Watabe,
Y. Ito, Y. Hayashi, Bioconjugate Chem. 2016, 27, 1606–1613.
2019, 30, 148–160; b) J. Ohata, Z. T. Ball, A. J. Am, Chem. Soc. 2017, 139,
12617–12622; c) C. Yu, J. Tang, A. Loredo, Y. Chen, S. Y. Jung, A. Jain, A.
Gordon, H. Xiao, Bioconjugate Chem. 2018, 29, 3522–3526; d) J. Z. Hui, S.
Tamsen, Y. Song, A. Tsourkas, Bioconjugate Chem. 2015, 26, 1456–1460;
e) J. Park, Y. Lee, B. J. Ko, T. H. Yoo, Bioconjugate Chem. 2018, 29, 3240–
3244; f) N. Vance, N. Zacharias, M. Ultsch, G. Li, A. Fourie, P. Liu, J.
LaFrance-Vanasse, J. A. Ernst, W. Sandoval, K. R. Kozak, G. Phillips, W.
Wang, J. Sadowsky, Bioconjugate Chem. 2019, 30, 148–160.
[
[
[
10] S. Kishimoto, Y. Nakashimada, R. Yokota, T. Hatanaka, M. Adachi, Y. Ito,
Bioconjugate Chem. 2019, 30, 698–702.
11] S. Tsukiji, M. Miyagawa, Y. Takaoka, T. Tamura, I. Hamachi, Nat. Chem
Biol. 2009, 5, 341–343.
Manuscript received: December 18, 2020
Revised manuscript received: February 13, 2021
Accepted manuscript online: February 17, 2021
Version of record online: ■■■, ■■■■
12] a) HÀ X. Wang, Y. Koshi, D. Minato, H. Nonaka, S. Kiyonaka, Y. Mori, S.
Tsukiji, I. Hamachi, J. Am. Chem. Soc. 2011, 133, 12220; b) T. Hayashi, Y.
ChemMedChem 2021, 16, 1–9
www.chemmedchem.org
8
© 2021 Wiley-VCH GmbH
��
These are not the final page numbers!

1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
FULL PAPERS
Two in one: A novel unnatural amino
acid 4-(2-aminoethylcarbamoyl)
leucine (Aecl) was synthesized and
used to develop a high-affinity IgG-
binding peptide. Synthesized Aecl-
containing peptides not only showed
their strong binding affinity but also
efficiently labeled trastuzumab. This
suggests that 15-Lys8Aecl is a useful
tool for the site-specific modification
of native antibodies, leading to the
preparation of homogeneous
Dr. K. Muguruma, R. Osawa, A.
Fukuda, N. Ishikawa, K. Fujita, Dr. A.
Taguchi, Dr. K. Takayama, Dr. A.
Taniguchi, Prof. Y. Ito, Prof. Y.
Hayashi*
1
– 9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
Development of a High-Affinity
Antibody-Binding Peptide for Site-
Specific Modification
antibody–drug conjugates.