An Efficient Method for the Conjugation of Hydrophilic and Hydrophobic Components by Solid-Phase-Assisted Disulfide Ligation

Article abstract of DOI:10.1002/anie.201712324

Chemical conjugation between hydrophilic and hydrophobic components is difficult because of their extremely different solubility. Herein, we report a new versatile method with a solid-phase-assisted disulfide ligation to overcome the difficulty of conjugation attributed to solubility. The method involves two steps in a one-pot process: 1) loading of a hydrophobic molecule onto a resin in an organic solvent, and 2) release of the solid-supported hydrophobic molecule as a conjugate with a hydrophilic molecule into an aqueous solvent. This strategy allows the use of a suitable solvent system for the substrates in each step. Conjugates of a water-insoluble drug, plinabulin, with hydrophilic carriers that could not be prepared by solution-phase reactions were obtained in moderate yields (29–45 %). This strategy is widely applicable to the conjugation of compounds with solubility problems.

Full text of DOI:10.1002/anie.201712324

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: An efficient conjugation method between hydrophilic and
hydrophobic components using a solid-phase assisted disulfide
ligation
Authors: Kyohei Muguruma, Takuya Shirasaka, Daichi Akiyama,
Kentarou Fukumoto, Akihiro Taguchi, Kentaro Takayama,
Atsuhiko Taniguchi, and Yoshio Hayashi
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201712324
Angew. Chem. 10.1002/ange.201712324
Link to VoR: http://dx.doi.org/10.1002/anie.201712324
http://dx.doi.org/10.1002/ange.201712324

10.1002/anie.201712324
Angewandte Chemie International Edition
COMMUNICATION
An efficient conjugation method between hydrophilic and
hydrophobic components using a solid-phase assisted disulfide
ligation
Kyohei Muguruma, Takuya Shirasaka, Daichi Akiyama, Kentarou Fukumoto, Akihiro Taguchi, Kentaro
Takayama, Atsuhiko Taniguchi, Yoshio Hayashi*
Abstract: Chemical conjugation between hydrophilic and
hydrophobic components is a difficult challenge due to their extremely
different solubilities. We here report a new versatile method with a
“solid-phase assisted disulfide ligation” for overcoming the difficulty of
conjugation attributed to the solubility. The method involves two steps
in one-pot: 1) loading a hydrophobic molecule on the resin in an
organic solvent, and 2) releasing the solid-supported hydrophobic
molecule as the conjugate with a hydrophilic molecule into an
aqueous solvent. This strategy allows the use of a suitable solvent
system for the substrates in each step. Indeed, the conjugates of a
water-insoluble drug, plinabulin, with hydrophilic carriers which were
impossible to be prepared by general solution phase reaction were
obtained in moderate yields (29-45%). This strategy is widely
applicable to the conjugation of compounds with solubility problems.
component is conjugated with the loaded hydrophobic component
to generate the desired product. This procedure allows use of two
suitable solvent systems, which can solubilize the respective
reaction components in each step. In this way, the procedure
overcomes the difficulty in the conjugation of hydrophobic and
hydrophilic components.
A) conventional method
: solid support
: hydrophobic
compound
organic solvent
aqueous solvent
: hydrophilic
compound
Conjugation of functional molecules with carrier molecules
is an attractive strategy for the efficient delivery of the former to
target biomolecules or cells in chemical biology1 or medicinal
chemistry2. For the preparation of such conjugates, a selective
crosslinking reaction of the two components is necessary. For
example, a copper-catalyzed alkyne azide cycloaddition (CuAAC)
is widely used because linkage with 1,2,3-triazole is easily created
by mixing the azide with an alkyne and a copper (I) catalyst in a
solvent.3 However, general conjugation reactions, including click
chemistry are hampered when the reaction solvent is not suitable
to solubilize two components which may have extremely different
solubilities.4 This problem becomes serious when a hydrophobic
functional molecule is linked with a hydrophilic carrier in an
attempt to improve water solubility or targeting ability (Figure 1A).
As an alternative method, we report a one-pot solid-phase
conjugation method consisting of two reactions (Figure 1B). The
first step involves a hydrophobic component loaded on the solid
support in an organic solvent. Then, after the exchange of the
organic solvent for an aqueous solvent, the hydrophilic
B) this method
first step
second step
solvent exchange
solid-supported
intermediate
desired product
Figure 1. Conjugation reaction between hydrophobic and hydrophilic
components: A) in solution-phase and B) via solid-phase.
Our group previously reported an anti-microtubule agent,
plinabulin (1, Phase III) for treatment of non-small cell lung
cancer.5,6 Plinabulin has an extremely poor water-solubility (< 0.1
µg/mL) notwithstanding its use in injections, and it is an
appropriate model drug for the conjugation with a water-soluble
carrier. As a carrier, we adopted a highly water-soluble2c and cell
penetrating7 octaarginine peptide that is often used as a carrier
peptide for drug delivery2d. We attempted the conjugation using a
CuAAC reaction, which we had previously used successfully to
prepare a water-soluble plinabulin prodrug (2) with a hydrophilic
amino acid moiety (Figure 2).8 However, the reaction failed to
proceed because no solvent could sufficiently dissolve both
plinabulin and peptide moieties simultaneously (Scheme S1).
Consequently, we performed a two-step solid-phase conjugation
using our recently developed “solid-phase assisted disulfide
ligation (SPDSL)”9,10 whose details are described below. The
ligation has been successfully applied to the synthesis of a
[*]
K. Muguruma, T. Shirasaka, D. Akiyama, Dr. K. Fukumoto, Dr. A.
Taguchi, Dr. K. Takayama, Dr. A. Taniguchi, Prof. Y. Hayashi.
Department of Medicinal Chemistry, Tokyo University of Pharmacy
and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392,
Japan.
E-mail: yhayashi@toyaku.ac.jp
Dr. K. Fukumoto
Kokusan Chemical Co., Ltd, 3-1-3 Nihonbashihoncho, Chuo-ku,
Tokyo 103-0023, Japan.
Supporting information for this article is given via a link at the end of
the document
This article is protected by copyright. All rights reserved.

10.1002/anie.201712324
Angewandte Chemie International Edition
COMMUNICATION
plinabulin-Z33 (IgG-binding peptide)11 conjugate for
a
thiol-containing peptide (4). As a result, the drug-component on
the resin (7) was transferred to the peptide by a disulfide
exchange reaction. Subsequently, the desired product (8), a
disulfide-bridged peptide-drug conjugate (PDC), was released
into the aqueous phase. We adopted an amphiphilic solid-support,
ChemMatrix® resin12 as the resin, which swells in both organic
and aqueous solvents. The SPDSL is a useful one-pot method to
prepare the conjugate between materials possessing different
solubilities, for the following reasons: 1) the solid-phase assisted
method allows a simple solvent exchange by simply washing the
resin, 2) the disulfide formation enables a selective linkage
between thiol groups of both components with no need for
protecting groups, and 3) the conjugation reaction occurs
simultaneously with the removal process of the plinabulin
segment from the resin. The resultant conjugate can produce the
active plinabulin via enzymatic degradation of the linker part.8
To investigate the reaction conditions in the first step of our
method, the sulfide 3 was mixed with resin 6 in various organic
solvents and the loading of 3 was monitored by HPLC which
tracks the decrease of 3 in the reaction solution. When CH2Cl2,
CHCl3 or CH3CN were used as solvent, 3 disappeared from the
reaction solution within 1 h (Table 1), indicating that it was
completely loaded on the resin to afford the Npys-assisted active
disulfide 7. It was found that for each solvent, the disappearance
level of 3 was inversely correlated (correlation coefficient (R) = –
noncovalent-type antibody-drug conjugate.9 It is a versatile
method for the chemical conjugation between two molecules with
very different solubilities. We synthesized conjugates of plinabulin
with several water-soluble carriers by this method, through
optimization of the reaction conditions.
O
N
HN
O
O
HN
N
O
N
HN
HN
O
COONa
COONa
NH
N
N
N
O
Plinabulin (1)
Prodrug 2
Figure 2. Structure of plinabulin and a water-soluble prodrug.
As starting materials, two types of building blocks, the
sulfide derivative of plinabulin (3) and a cysteine-containing
octaarginine peptide, Ac-Arg8-aminocaproic acid (Acp)-Cys-NH2
(4) were prepared according to a previously reported method8,9
and by general Fmoc-based solid-phase peptide synthesis,
respectively. As depicted in Scheme 1, the solid-supported 3-
nitro-2-pyridine sulfenyl (Npys) group was activated from its
benzyl form (5) to a chloride form (6) by chlorosulfenylation with
2% SO2Cl2 in 1,2-dichloroethane.10 The Npys-Cl resin (6)
prepared in this way was allowed to react with the hydrophobic
sulfide (3) in an organic solvent. This resulted in the formation of
drug-supported resin (7) through an active disulfide. The resin
was washed twice with CH3CN and four times with water to
quench the residual Npys-Cl. In a second step, the organic
solvent was replaced by an aqueous solvent of the hydrophilic
0.96, Figure 3) with Gutmann’s donor number (DN)13,
a
quantitative measure of Lewis basicity (see Supporting
Information for the details).
The sulfide 3 in CH3CN was almost completely (98%)
loaded even when the amount of resin 6 (corresponding to resin
5) was decreased to 2 eq. (Table 1, entry 8 and Figure 4A),
although the reaction was slower when using 1.1 eq. of 6 (80%,
entry 9). We previously reported that the 5 eq. of resin 6 were
required for the complete reaction when using 90% formic
acid/H2O as solvent.10 This difference suggests that the water
O
O
N
HN
HN
O2N
N
O
N
tBu
H
Table 1. Reaction conditions for the first step.
R
S
O
S
+
N
R = Bn (5)
Cl (6)
activation9
O
Resin
(eq.)[a]
Disappearance
DN[c]
(kcal/mol)
N
3
Entry
Solvent
N
N
level of 3 (%)[b]
O
3
O
O
O2N
1
2
3
4
5
6
7
8
9
CH2Cl2
CHCl3
CH3CN
MeOH
THF
5
5
100
100
100
64
1
N
H
N
HN
HN
N
S
S
N
4
solvents
Conditions in Table 1
O
N
3
5
14.1
19
N
N
O
Plinabulin-supported resin 7
5
O
O
5
74
20
N
HN
HN
N
O
O
peptide 4
(0.9 eq.)
DMF
5
42
26.6
30.9
14.1
14.1
O2N
+
N
H
CH3CN/buffer (2:3)
S
N
H
DMSO
CH3CN
CH3CN
5
80
3
N
Conditions in Table 2
9
N
N
O
S
S
2
98
Ac (Arg)8 Acp Cys NH2
Plinabulin-SS-Arg8 (8)
1.1
80
: ChemMatrix resin
[a] Eq. of resin 5 against sulfide 3, [b] The disappearance level was
calculated by the following formula; (1-[peak area of 3 after 1h]/[peak area
of 3 at 0 h] X 100), [c] Donor number.13
Scheme 1. Synthesis of plinabulin-SS-Arg8 8 by SPDSL.
This article is protected by copyright. All rights reserved.

10.1002/anie.201712324
Angewandte Chemie International Edition
COMMUNICATION
When using non-buffered water (Table 2, entry 1), the reaction
was very slow and incomplete even after 5 days (data not shown).
In addition, the parent drug (1) was produced by hydrolysis of the
linker under these conditions. To enhance the reaction, we used
aqueous buffer (pH 3.8–7.4) instead of water. At pH 5.0 (entry 4),
the reaction proceeded efficiently and the peptide 4 completely
disappeared within 3 h (Figure 4B). Subsequently, a disulfide-
bridged PDC, plinabulin-SS-Arg8 (8) was produced in a good
HPLC yield (91%). The disulfide dimer of peptide 4 was produced
at higher pH values (Table 2, entry 4, 5 and Figure S2). In addition,
the loss of peptide 4 was observed when using pH 7.4 buffer
(entry 6) due to a non-specific adsorption of Arg8 by a weakly
acidic thiopyridone moiety of resin 9 that was produced (Figure
S3). Thus, this reaction is sensitive to pH. The tendency was
remarkable in the requirement for 5 eq. of resin 5 (Table S1 and
Figure S4). Moreover, an improved yield was observed in 1 M
sodium acetate (43%) compared to a 50 mM solution (13%)
(Table S1, entry 1 vs 5). This indicated that higher concentrations
of buffer salt promote the reaction.
120
CH2Cl2
CH3CN
100
CHCl3
THF
80
60
DMSO
MeOH
40
20
0
DMF
0
10
20
30
Donor number (kcal/mol)
Figure 3. Correlationship between disappearance of 3 in the first step and
DN13 of solvent. The correlation coefficient (R) of approximation straight line
between CH3CN and DMF is –0.96.
Table 2. Reaction optimization in the second step.
molecule reacted with 6 to form an inactive form, while in CH3CN,
the reactivity of Npys-Cl resin (6) was maintained. Accordingly,
we selected the conditions indicated in Table 1, entry 8 for the first
step reaction.
In the second step, the reaction with a Cys-containing Arg8
peptide (4) (0.9 eq. with respect to sulfide 3) was performed using
40% aqueous acetonitrile as solvent, and the formation of
conjugate 8 was tracked by HPLC analysis of the reaction solution.
Dimer[b]
Yield
Entry Aqueous solvent
(%)[a]
1
2
3
4
5
6
non-buffered water
7
n.d.[c]
n.d.[c]
0.8
50 mM sodium acetate (pH3.8)
50 mM sodium acetate (pH4.5)
50 mM sodium acetate (pH5.0)
50 mM sodium acetate (pH5.6)
50 mM sodium phosphate (pH7.4)
82
81
91
75
7
1
1.3
3
A)
n.d.[c]
[a] HPLC yields are calculated from the area of the peak of 8 in the analytical
HPLC measured after 3 h on the basis of the amount of peptide 4, [b] The
values are reported as a relative to the condition in entry 4, [c] not detected.
0 h
1 h
Finally, conjugate 8 was obtained in 44% isolated yield
(Table 3, entry 1). The isolated yield was lower than the HPLC
yield (Table 2, entry 4), as a result of the generally observed non-
specific adsorption of octaarginine. Next, we applied the solid-
phase assisted reaction to coupling with other functional
hydrophilic peptides as shown in Table 3. The PDC, including D-
octaarginine peptide, that has an ability to accumulate in tumors,2d
was obtained in 37% isolated yield (entry 2). On the other hand,
the non-ionized peptide, (Gly-Ser)4, a repeated sequence, which
is used as a control for cell-penetrating octaarginine peptide7b,
was conjugated with plinabulin in moderate yield (45%, entry 3).
Although some by-products were observed in the conjugation of
Z33 peptide in which the disulfide homo-dimer was the main by-
product, the reaction with Z33 peptide was drastically accelerated
(5 h, 32%: entry 4) by change of solvent, compared with the
previously reported conditions which used 50% DMF/H2O (20 h,
28%)9. In addition, we successfully synthesized a plinabulin-
galactose conjugate with a yield of 29 % (entry 5). This suggests
0
20
30
40
10
Retention time (min)
B)
4
0 h
8
Dimer of 4
3 h
0
20
Retention time (min)
30
40
10
Figure 4. HPLC chromatograms of A) the first step reaction (Table 1, entry 8),
and B) the second step reaction (Table 2, entry 4).
This article is protected by copyright. All rights reserved.

10.1002/anie.201712324
Angewandte Chemie International Edition
COMMUNICATION
that the reaction can be applied to conjugations not only with
peptides but also with other hydrophilic functional materials such
as sugars. In Table 3, entries 4 and 5, the buffer at pH 4.5 was
preferred to the one at pH 5.0, because of the slow homo-
dimerization at lower pH values, resulting in higher isolated yields.
versatile system for the conjugation of materials with extremely
different solubilities.
Acknowledgements
This work was supported by the Japan Society for the Promotion
of Science (JSPS), KAKENHI, a Grant-in-Aid for JSPS Fellows
15J09551, Basic Science and Platform Technology Program for
Innovative Biological Medicine.
Table 3. Application of SPDSL to the production of other conjugates.
Entry Thiol containing hydrophilic components[a]
Isolated
yield (%)
1
2
3
4
5
Ac-(L-Arg)8-Acp-Cys-NH2 (Table 2, Entry 4)
Ac-(D-Arg)8-Acp-Cys-NH2
Ac-(GS)4-Acp-Cys-NH2
44
37
Keywords: Peptide-drug conjugate • Solubility • Synthetic
methods • Solid-phase synthesis
45
[1]
[2]
T. Hayashi, Y. Yasueda, T. Tamura, Y. Takaoka, I. Hamachi, J. Am.
Chem. Soc. 2015, 137, 5372.
H-CG-Z33-NH2 (pH 4.5)
32[b]
29
a) D. Böhme, A. G. Beck-Sickinger, J. Pept. Sci. 2015, 21, 186; b) P.
Agarwal, C. R. Bertozzi, Bioconjugate Chem. 2015, 26, 176; c) H. Choi,
Galactose-SH (pH4.5)
M. T. Jeena, L. Palanikumar, Y. Jeong, S. Park, E. Lee, J. H. Ryu, Chem.
29c
Commun. 2016, 52, 5637; d) I. Nakase, Y. Konishi, M. Ueda, H. Saji, S.
[a] The sequence of the peptides and products are shown in SI, [b] 0.75 eq.
of peptide was used to directly compare with the previous condition9.
Futaki, J. Control. Release. 2012, 159, 181.
[3]
[4]
a) V. V. Rostovtsev, L. G. Green, V. V. Fokin, K. B. Sharpless, Angew.
Chem. 2002, 114, 2708; Angew. Chem. Int. Ed. 2002, 41, 2596; b) H. C.
Kolb, K. B. Sharpless, Drug Discov. Today 2003, 8, 1128.
a) S. B. H. Kent, Chem. Soc. Rev. 2009, 38, 338; b) H. Hojo, Org. Biomol.
Chem. 2016, 14, 6368; c) A. Delgado, J. Clardy, J. Org. Chem. 1993, 58,
2862.
In conclusion, we successfully synthesized the conjugates
of the hydrophobic plinabulin with several hydrophilic carriers
using SPDSL. This method involves two steps: 1) loading a
hydrophobic moiety on the resin in an organic solvent, and 2)
conjugating the loaded hydrophobic moiety and a hydrophilic
moiety in an aqueous solvent. This strategy allows the use of
different solvent systems in each step, and the difficulty in
conjugation between materials possessing opposite solubilities
was overcome. The solubility problem in organic synthesis is not
limited to peptides but also natural products,4c and our strategy
would be productively applied to such instances. This study
established the benefit of the solid-phase assisted disulfide
coupling reaction. Optimisation of the reaction conditions
revealed that the first step prefers low DN solvents and that a pH
below 5.0 and high salt concentrations are important in the
second step. High yields of the conjugations were obtained, which
is difficult in solution-phase reactions. This strategy will provide a
[5]
[6]
http://www.beyondspringpharma.com/plinabulin/. (accessed November,
2017)
a) B. Nicholson, G. K. Lloyd, B. R. Miller, M. A. Palladino, Y. Kiso, Y.
Hayashi, S. T. Neuteboom, Anticancer Drugs 2006, 17, 25; b) Y.
Yamazaki et al., J. Med. Chem. 2012, 55, 1056.
[7]
a) S. Futaki, T. Suzuki, W. Ohashi, T. Yagami, S. Tanaka, K. Ueda, Y.
Sugiura, J. Biol. Chem. 2001, 276, 5836; b) M. M. Fretz, N. A. Penning,
S. Al-Taei, S. Futaki, T. Takeuchi, I. Nakase, G. Storm, A. T. Jones,
Biochem. J. 2007, 403, 335.
[8]
[9]
F. Yakushiji et al., Bioorg. Med. Chem. 2017, 25, 3623.
K. Muguruma et al., Bioconjugate Chem. 2016, 27, 1606.
[10] A. Taguchi et al., Org. Biomol. Chem. 2015, 13, 3186.
[11] A. C. Braisted, J. A. Wells, Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 5688.
[12] F. García-Martín, M. Quintanar-Audelo, Y. García-Ramos, L. J. Cruz, C.
Gravel, R. Furic, S. Côté, J. Tulla-Puche, F. Albericio, ACS Comb. Sci.
2006, 8, 213
[13] a) V. Gutmann, Coordin. Chem. Rev. 1976, 18, 225; b) F. Cataldo, Eur.
Chem. Bull. 2015, 4, 92.
This article is protected by copyright. All rights reserved.

10.1002/anie.201712324
Angewandte Chemie International Edition
COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
Kyohei Muguruma, Takuya Shirasaka,
Daichi Akiyama, Kentarou Fukumoto,
Akihiro Taguchi, Kentaro Takayama,
Atsuhiko Taniguchi, Yoshio Hayashi*
organic solvent
aqueous solvent
: hydrophobic
compound
solvent exchange
: hydrophilic
compound
desired product
solid-supported
intermediate
solid support
Page No. – Page No.
An efficient conjugation method
between hydrophilic and hydrophobic
components using a solid-phase
assisted disulfide ligation
We report a new method using a “solid-phase assisted disulfide ligation” for
overcoming the difficulty of conjugation attributed to the solubility. In the optimized
conditions, the conjugates of a poorly water-soluble model drug, plinabulin, with
various hydrophilic carriers, which are difficult to prepare by general solution phase
reaction, were obtained in moderate yields.
This article is protected by copyright. All rights reserved.