Immune responses against Lewis y tumor-associated carbohydrate antigen displayed densely on self-assembling nanocarriers

Article abstract of DOI:10.1039/c8ob01955j

Immune responses against Lewis y (LY) displayed on nanocarriers at different surface densities were studied. The high surface density of LY was obtained by the A₂B-type amphiphilic polypeptides having LY at the two terminals [LY-poly(sarcosine)₂-b-(l- or d-Leu-Aib)₆]. The equimolar mixture of these two amphiphilic polypeptides formed interdigitated planar sheet-like molecular assemblies densely displaying LY (G4). G4 seemed to induce the anti-LY IgM upon immunization to BALB/c mice by only a single administration. However, the amount of anti-LY IgM produced was moderate and significantly less than that induced by two administrations of the other molecular assembly (G1) with the average surface density of LY at a 1/4 of that of G4. Further, the anti-LY IgM produced after two administrations of G4 lowered the avidity more than after one administration.

Full text of DOI:10.1039/c8ob01955j

Organic &
Biomolecular Chemistry
View Article Online
View Journal | View Issue
PAPER
Immune responses against Lewis Y tumor-
associated carbohydrate antigen displayed
densely on self-assembling nanocarriers†
Cite this: Org. Biomol. Chem., 2018,
16, 8095
Yuji Yamazaki, a Yukiko Nambu,b Masashi Ohmae, *a Manabu Sugaib and
a
Shunsaku Kimura
Immune responses against Lewis y (LY) displayed on nanocarriers at different surface densities were
studied. The high surface density of LY was obtained by the A2B-type amphiphilic polypeptides having LY
at the two terminals [LY-poly(sarcosine)2-b-(L- or D-Leu-Aib)6]. The equimolar mixture of these two
amphiphilic polypeptides formed interdigitated planar sheet-like molecular assemblies densely displaying
LY (G4). G4 seemed to induce the anti-LY IgM upon immunization to BALB/c mice by only a single admin-
istration. However, the amount of anti-LY IgM produced was moderate and significantly less than that
induced by two administrations of the other molecular assembly (G1) with the average surface density of
LY at a 1/4 of that of G4. Further, the anti-LY IgM produced after two administrations of G4 lowered the
avidity more than after one administration.
Received 10th August 2018,
Accepted 2nd October 2018
DOI: 10.1039/c8ob01955j
rsc.li/obc
not been approved for use in humans to raise adequate
immune responses against the target antigens. Further, the
Introduction
Aberrant glycosylation occurs on tumor cells resulting in them number of antigens introduced to the carriers varied depending
abnormally expressing carbohydrate antigens in quality and/or on their synthesis lots, causing difficulty in their quality
quantity, which is strongly related to tumor cell biology.1 control. Multivalent presentation of TACAs can also induce an
These antigens termed tumor-associated carbohydrate anti- immune response, probably due to reinforced stimulation to
gens (TACAs) are therefore considered as good targets for the the immune cells.13–17 Several intriguing results have been
diagnosis and treatment of cancer. Particularly, cancer immu- reported in relation to the boosted antibody responses against
notherapy using TACAs as the antigens has attracted signifi- TACAs on the basis of the concept of “self-adjuvanting
cant attention as cutting-edge research in the medical field.2 vaccines”.18–20 These vaccine candidates have a unique but
TACAs are self- and T-cell-independent (TI) antigens that make common molecular architecture, that is, a TACA-linker-toll-like
it extremely difficult for the immune system to eradicate receptor (TLR21–23) agonist; however, the detailed mechanism(s)
tumors with TACAs.3 In order for such tricky but attractive for inducing potent antibody production is still unknown.
TACAs to function as practical tumor antigens, they have been
Recently, we reported the potential cancer vaccine formu-
conjugated with highly immunogenic carriers,4–9 or special- lation of densely TACA-presenting amphiphilic polypeptide
ized carriers precisely designed to work in a T-cell-dependent assemblies of ca. 100 nm size.24 The previously prepared mole-
(TD) manner.10–12 These vaccine candidates, however, require cular assemblies had interdigitated, sheet-like monolayer
multiple administrations with adjuvants, such as highly toxic structures formed by equimolar amounts of the right-handed
Freund’s complete adjuvant, and synthetic chemicals that have and the left-handed hydrophobic helical peptides. This system
has several advantages, such as stability of the assemblies pre-
senting dense TACAs, the flexibility of the selection and combi-
nation of the TACAs, and easy control of the density of the
TACAs, which are all difficult to achieve with the traditional
vaccine designs mentioned above. The TACA employed in our
previous study is the Lewis y (LY) blood group antigen consist-
ing of Fucα(1 → 2)Galβ(1 → 4)[Fucα(1 → 3)]GlcNAcβ, which is
overexpressed in a variety of tumor cells.25 Particularly in
breast cancer, monoclonal antibodies (mAbs) against LY have
been reported to non-competitively inhibit the interaction of
aDepartment of Material Chemistry, Graduate School of Engineering,
Kyoto University, Kyoto Daigaku-Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.
E-mail: ohmae.masashi.4n@kyoto-u.ac.jp
bDivision of Molecular Genetics, Department of Biochemistry and Bioinformative
Sciences, School of Medicine, University of Fukui, 23-3 Matsuokashimoaizuki,
Eiheiji-cho, Yoshida-gun, Fukui 910-1193, Japan
†Electronic supplementary information (ESI) available: 1H and 13C NMR spectra
for synthesized compounds as well as additional experimental data for NTA and
the AFM analysis. See DOI: 10.1039/c8ob01955j
This journal is © The Royal Society of Chemistry 2018
Org. Biomol. Chem., 2018, 16, 8095–8105 | 8095

View Article Online
Paper
Organic & Biomolecular Chemistry
the epidermal growth factor (EGF) with EGF receptors expres-
sing LY, resulting in termination of MAPK signaling and pro-
liferation of the tumor cell.26 Our previous vaccine candidates
could elicit anti-LY antibody in mice in a density-dependent
manner of LY by using nanocarriers with planar-sheet mor-
phology. In this study, we aimed to provoke more effective
anti-LY antibody production using the exceedingly LY-present-
ing nanocarriers.
Results and discussion
Design and synthesis of novel amphiphilic polypeptides for
the vaccine candidates with high LY density
In order to increase the surface density of LY on the nanosheet
to more than that in the previous report, we newly designed
two kinds of amphiphilic polypeptides with two hydrophilic
chains carrying LY in each terminal with one hydrophobic
helical peptide, that is, so-called A2B-type amphiphilic poly-
peptides (Fig. 1). The hydrophilic block is composed of poly
(sarcosine) with LY at the terminal, and the hydrophobic one
is either (L-leucine-α-aminoisobutylic acid)6 [(L-Leu-Aib)6] or
(D-Leu-Aib)6; these amphiphilic polypeptides are abbreviated
hereafter as 2(LY-S)-L and 2(LY-S)-D, respectively.
The structures of the hydrophilic and hydrophobic blocks
are the same as those of the AB-type LY-carrying amphiphilic
polypeptides previously used23 (abbreviated as LY-S-L and
LY-S-D, Fig. 1), but the new A2B-type amphiphilic polypeptides
have a branching connector between the two hydrophilic
blocks and the one hydrophobic block. Therefore, 2(LY-S)-L
and 2(LY-S)-D were newly synthesized according to the reac-
tions outlined in Scheme 1.
Serinol (1) was coupled with mono-t-Bu-succinate (2) to
obtain the dihydroxy derivative (3). In order to introduce
amino-termini for the following poly(sarcosine) chain elonga-
tion, the two hydroxy groups in 3 were esterified with the
Fig. 1 Molecular designs of the amphiphilic polypeptides 2(LY-S)-L, 2
Fmoc-protected sarcosine (4) to give the protected branching (LY-S)-D, LY-S-L, LY-S-D, S-L, and S-D.
connector (5). The t-Bu-protection in 5 was cleaved by tri-
fluoroacetic acid (TFA), followed by coupling with both the
L-form-polypeptide (7)27 and the D-form-polypeptide (8),27
which gave rise to the corresponding polypeptides with the
Preparation of the LY-presenting molecular assemblies using
the amphiphilic polypeptides
forked connector (9 and 10, respectively). The Fmoc protec-
tions in 9 and 10 were successfully removed by the addition Four kinds of molecular assemblies of nanosheets displaying
of piperidine, then the secondary amino group initiated the LY (G1–G4) at different surface densities were prepared in
ring-opening polyaddition of sarcosine-N-carboxyanhydride addition to the nanosheet consisting of S-L and S-D without
(Sar-NCA), followed by the terminal alkynylation through LY (G0) as a control28 (Table 1). All the molecular assemblies
coupling with 4-pentynoic acid. After purification by have been prepared using combinations of two kinds of the
Sephadex LH20 column chromatography, the forked block amphiphilic polypeptides of the opposite chirality in the
polypeptides with the two terminal alkynyl groups (11 and hydrophobic helical blocks at a mixing ratio of 1/1 (mol/mol).
12) were obtained. The average degrees of polymerization G0 has no LY, but the other assemblies are designed to have
(DP) of the poly(sarcosine) blocks were determined to be 39 the surface LY as follows: G1 with 100% LY (the AB-type
(11) and 32 (12) by 1H NMR and MALDI-TOF MS measure- amphiphilic polypeptide protruding one LY group) only on
ments (Fig. S1 and S2†). The LY derivative (13)24 was then one face (the same as that previously reported24), G2 with
introduced by Huisgen cycloaddition to provide the target 100% LY on both faces, G3 with 200% LY (the A2B-type amphi-
forked block polypeptides carrying LY at both termini [2 philic polypeptide protruding two LY groups) on one face and
(LY-S)-L and 2(LY-S)-D].
100% LY on the other, and G4 with 200% LY on both faces.
8096 | Org. Biomol. Chem., 2018, 16, 8095–8105
This journal is © The Royal Society of Chemistry 2018

View Article Online
Organic & Biomolecular Chemistry
Paper
These molecular assemblies were filtered through a 0.8 μm
cellulose acetate membrane before use.
Physical characterization of the molecular assemblies was
carried out by nanoparticle tracking analysis29 (NTA) for the
size and particle concentrations and conventional DLS ana-
lyses for the size and polydispersity index. The sizes of all the
molecular assemblies determined by the NTA analysis were
in the range from ca. 80 to 190 nm. Both G0 and G1 were
160–190 nm in size with higher particle concentrations.
These results indicate that stable molecular assemblies were
formed with G0 and G1. The size of G2 was the smallest
among all the molecular assemblies, suggesting that LY-S-L
and LY-S-D are difficult to assemble with each other, prob-
ably because of too many hydrophilic LY groups existing in
the AB-type molecular assembly. In fact, small particles
around ca. 10 nm were detected during the NTA analysis of
G2 (Fig. S3c and Fig. S6(b)†-red line). The particle concen-
tration of G3 was the lowest, but the small particles existing
in G2 were not detected (Fig. S3d†). The low particle concen-
tration of ca. 150 nm in G3 suggests that the nanosheet was
not obtained from a mixture of 2(LY-S)-L and LY-S-D, result-
ing in a lack of control of the surface design of the one with
200% LY and the other with 100% LY. This is probably owing
to the large difference in the molecular shapes between 2
(LY-S)-L and LY-S-D, where the former is predicted to be
planar-like, and the latter to be rod-like.30 The cone-like poly-
peptide tended to self-assemble into a wormlike micelle
while the rod-like polypeptide self-assembled into a poly-
meric micelle. Therefore, G3 was excluded from the following
assays.
Morphology observations of the assemblies
Morphology observations of the molecular assemblies G0–G2
and G4 were carried out by using transmission electron
microscopy (TEM) and atomic force microscopy in liquid
(AFM, Fig. 2). The morphology of G0 with no LY moiety
(control) was consistent with that reported previously,28 that
is, a planar sheet-like shape with ca. 10 nm thickness, which
indicates the formation of the interdigitated monolayer assem-
blies (Fig. S4†). Similarly, G1 formed a planar-sheet monolayer
with ca. 10 nm thickness (Fig. 2a). G2 also showed molecular
assemblies with a flat region in the AFM image, however, with
5 nm thickness (Fig. 2b), suggesting that G2 self-assembled
into nanosheets with a loose molecular packing structure,
which allowed the chain-bending conformation in the poly
(sarcosine) layer to make the thickness thin.31 The chain-
bending may prevent the LY groups from exposure to the
outside. G4 was found to self-assemble into a planar sheet-like
morphology with a little larger thickness (ca. 15 nm) (Fig. 2c).
The large thickness should be owing to the dense molecular
packing leading to the extended conformation of poly(sarco-
sine) blocks. G4, therefore, displays LY groups densely on the
surface. It is notable that the combination of 2(LY-S)-L and 2
(LY-S)-D provided nanosheets that were more stable than the
combination of LY-S-L and LY-S-D, even though the former
combination is more hydrophilic than the latter. The planar-
Scheme 1 Synthesis of 2(LY-S)-L and 2(LY-S)-D: (a) (1) DMT-MM, EtOH/
DMF, r.t. 6 h, 95%, (b) Fmoc-sarcosine, DCC, DMAP, CH2Cl2/DMF, r.t. 17 h,
11%, (c) TFA, CHCl3, 2 h, 83%, (d) HATU, HOAt, DIEA, DMF, 7, r.t., 12 h,
65%, (e) HATU, HOAt, DIEA, DMF, 8, r.t., 15 h, 64%, (f) (1) piperidine,
CH3CN/CH2Cl2, 0 °C, (2) sarcosine-NCA, DMF, r.t., overnight, (3) 4-penty-
noic acid, HATU, HOAt, DIEA, DMF, r.t., overnight, 84% (11), 50% (12), (g)
Cu(I)OAc, DMF, 40 °C, 31 h, 31% [2(LY-S)-L], 39 h, 90% [2(LY-S)-D]. DIEA:
N,N-diisopropylethylamine, DMT-MM: 4-(4,6-dimethoxy-1,3,5-triazin-2-
yl)-4-methylmorpholinium chloride, HATU: 1-[bis(dimethylamino)methyl-
ene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate.
This journal is © The Royal Society of Chemistry 2018
Org. Biomol. Chem., 2018, 16, 8095–8105 | 8097

View Article Online
Paper
Organic & Biomolecular Chemistry
like molecular shape of 2(LY-S)-L and 2(LY-S)-D was pointed Immune responses
out to favor molecular stacking to grow into a worm-like
micelle.30 The planar-like molecular shape is therefore the The molecular assemblies of G0–G2 and G4 were administered
reason for the formation of the stable nanosheets of G4. Thus, intraperitoneally to BALB/c mice (8-week-old) in addition to
both G1 and G4 have similar planar sheet-like morphologies, the monophosphoryl lipid A (MPL) adjuvant according to the
whereas G4 displays 4-fold more LY on the surface compared time schedule including two administrations (Fig. 3a). After
to G1, as we designed.
the first administration (at day 7, Fig. 3b), the anti-LY IgM was
Table 1 Molecular assemblies prepared from the combination of the amphiphilic polypeptides
NTA
DLS
LY-displayb
/%/%
Sample
Contentsa
Size/nm
Conc. (×10−11)/particles per mL
Size/nm
PDI
G0
G1
G2
G3
G4
S-L/S-D
LY-S-L/S-D
LY-S-L/LY-S-D
2(LY-S)-L/LY-S-D
2(LY-S)-L/2(LY-S)-D
0/0
100/0
100/100
200/100
200/200
189
156
78
148
129
3.26
7.04
1.96
0.77
1.92
198
104
74
119
92
0.07
0.13
0.17
0.25
0.15
a Molar ratio is 1 : 1. An aliquot of ethanol solution of the mixed amphiphiles was injected into saline. b Designed surface density of LY.
Fig. 2 Morphology observations of the assemblies (a) G1, (b) G2 and (c) G4. The columns show (1) the TEM images, (2) the AFM images and (3) the
height traces of the assemblies. The AFM images were recorded on an APTES-modified Si-wafer in water. The height profiles were obtained by
tracing along the red and the blue lines.
8098 | Org. Biomol. Chem., 2018, 16, 8095–8105
This journal is © The Royal Society of Chemistry 2018

View Article Online
Organic & Biomolecular Chemistry
Paper
significantly produced only by G4, but not G1 and G2. After
the second administration (at day 14, Fig. 3c), the anti-LY IgM
was produced most effectively by G1, but G2 was insignificant.
Unexpectedly, G4 was not as effective at triggering the immune
response compared to G0.
From the structural viewpoint of the molecular assemblies,
the poly(sarcosine) block also triggered the immune response
in addition to the LY moiety.32,33 After the first administration,
only G4 moderately produced the anti-poly(sarcosine) IgM, but
others showed no significant production of anti-poly(sarco-
sine) IgM (Fig. 3d). This result is consistent with our previous
report that poly(sarcosine) displayed on nanosheets induced
less immune responses than that on polymeric micelles.34 In
contrast, after two administrations the anti-poly(sarcosine)
IgM was significantly produced by all the molecular assem-
blies (Fig. 3e). The IgM production increased in the order of
G4 < G0 < G2 ∼ G1. Taken together, G1 was the most effective
in production of anti-LY IgM and anti-poly(sarcosine) IgM
after two administrations. On the other hand, G4 instantly
triggered the production of both anti-LY and poly(sarcosine)
IgMs, but the responses became moderate after two
administrations.
This suppressive immunity might have been caused via so-
called “carrier-induced epitope suppression”,35 or via regulat-
ory B cell (Breg) generation.36 Recently, suppressive immunity
against excess immune response has been reported, which can
be induced by Bregs. Bregs are believed to derive from various
B cell subsets including mature B cells and plasmablasts,37
and are known to actively secrete interleukin-10 (IL-10), which
is an immune suppressive cytokines.38 B cell activation by the
B cell receptors (BCR) with costimulation through toll-like
receptor 4 (TLR4) can induce Breg generation.39 In the present
study, the combined use of the molecular assemblies with the
MPL adjuvant, which is a TLR4 agonist,40 therefore, could
generate Bregs. Thus, we suppose that the unexpected
immune suppression against G4 was caused through the gene-
ration of Bregs, although the detailed mechanism is still
unknown.
There are some reports that a linker moiety attached to a
carrier protein in the TACA vaccine had an effect on the pro-
duction of antibody against TACA.41,42 It is possible that the
linker/spacer region in the amphiphilic polypeptide could lead
to the immune suppression; however, the IgMs against other
parts of the amphiphilic polypeptides, such as the oligo-ethyl-
ene oxide linker containing the triazole moiety and the helix
peptide (L), were not detected at all (Fig. S8†). Furthermore,
other classes of antibodies than IgM were not detected in any
serum samples (Fig. S9†).
Specificity of the anti-LY IgMs
Fig. 3 Immune responses against the assemblies G0–G2 and G4: (a)
The time schedules for the experiments, (b) IgM amounts by ELISA
against anti-LY at day 7 and (c) day 14, and (d) anti-poly(sarcosine) at day
7 and (e) day 14. The p-value: *p < 0.05, **p < 0.01, ***p < 0.005.
Specificity of anti-LY IgM was studied by competitive inhi-
bition assay using ELISA on LY-L coating plates (Fig. 4). Free
LY [Fucα(1 → 2)Galβ(1 → 4)[Fucα(1 → 3)]GlcNAc-OH] and G1
were employed as the inhibitors. Free LY could weakly inhibit
the binding of the IgM induced after the two administrations
of G1, indicating that the epitope of the anti-LY IgM consists
This journal is © The Royal Society of Chemistry 2018
Org. Biomol. Chem., 2018, 16, 8095–8105 | 8099

View Article Online
Paper
Organic & Biomolecular Chemistry
also points out the instant response by G4 as described
above.
Conclusion
Two kinds of the A2B-type block polypeptides with LY at both
hydrophilic termini [2(LY-S)-L and 2(LY-S)-D] have been suc-
cessfully synthesized for the first time. These two polypeptides
have the chiral blocks of L and D polypeptides, which could
form the interdigitated planar sheet-like monolayer (G4) when
they were mixed in equimolar amounts in saline. G4, to the
best of our knowledge, has the highest surface density of LY
among those prepared as cancer vaccine candidates to date.
The G4 assemblies could induce the production of the IgM
specific for LY through only one administration. This instant
production of anti-LY IgM by G4 is remarkable, but the
immune response became moderate after two administrations
accompanying changes in IgM specificity. The super high
surface density is therefore considered not to be suitable to
trigger the immune response under the condition of multiple
doses, for instance, of the booster effect. Further, the A2B-type
molecular shape contributed to the stabilization of
nanosheets, but resulted in modulation in the immune
responses. It is interesting to know that the immune responses
via B cells are sensitively affected by geometrical arrangements
of LY groups on nanocarriers. In the future, for inducing a
more effective immune response to TACAs, we will prepare
some vaccine candidates with a set of TACAs and T cell epi-
topes as components of the amphiphiles. These molecular
architectures could induce immunological memory and anti-
body class switching. We will also examine adjuvants other
Fig. 4 Competitive inhibition assay of the anti-LY IgMs by ELISA on
LY-L-coating plate with (a) free LY in the sera of the G1 (2 adminis-
trations) and the G4 (2 administrations) assemblies, and with (b) the G1 than MPL for better vaccine formulation.
assemblies in the sera of the G1 (2 administrations), the G2 (2 adminis-
trations) and the G4 (1 and 2 administrations) assemblies. The p-value:
*p < 0.05, **p < 0.01.
Experimental
General
of LY. On the other hand, the anti-LY IgM assay after the two Anhydrous MeOH and CH2Cl2 were purchased from Wako
administrations of G4 was not inhibited by free LY (Fig. 4a). Pure Chemical Industries, Ltd. Other chemicals were pur-
When G1 was used as an inhibitor, IgM in the serum after the chased from commercial sources and were used without
two administrations of G1 or G2 was inhibited to adsorb to the further purification. Silica gel 60 (spherical, neutral) for
ELISA plate at a lower concentration of G1 than that of free LY column chromatography was purchased from Nacalai Tesque.
because of the multivalency of LY on the surface of the self- Transmission electron micrography (TEM) images were taken
assemblies. Notably, G1 showed an inhibitory effect against using a JEM-2000EX II or JEM-1400 (JEOL Ltd, Japan).
anti-LY IgM obtained by one administration of G4, but the Dynamic light scattering (DLS) measurements were taken
inhibition became less with the serum after two adminis- using a DLS-8000KS (Photal Otsuka Electronics). The concen-
trations of G4 (Fig. 4b). Accordingly, the IgM species should be tration of the molecular assembly was determined by nano-
different between one and two administrations. The major particle tracking analysis (NTA 3.2 Dev Build 3.2.16) using
epitope of IgM after the two administrations of G4 differs from NANOSIGHT LM10 (Malvern).29 Atomic force microscopy
LY, however, the detailed mechanism remains to be solved.
(AFM) images were obtained using a MultiMode 8-HR
In the competitive inhibition assay, IgM produced by one (Bruker). TLC was carried out on silica gel 60 F254 (Merck).
administration of G4 was inhibited by the addition of G1 NMR spectra were recorded on a Bruker DPX400 spectrometer.
similarly to IgM after the two administrations of G1. These HRMS (ESI-MS analysis) spectra were obtained on an Exactive
suggest that IgMs have similar avidity between on adminis- Plus spectrometer (Thermo Fischer Scientific). MALDI-TOF
tration of G4 and two administrations of G1. This observation mass spectra were recorded on an Autoflex III plus (Bruker)
8100 | Org. Biomol. Chem., 2018, 16, 8095–8105
This journal is © The Royal Society of Chemistry 2018

View Article Online
Organic & Biomolecular Chemistry
Paper
spectrometer using super-DHB (Sigma-Aldrich) or α-cyano-4- syringe pump. After measuring three times, the concentration
hydroxycinnamic acid (CHCA; Sigma-Aldrich) as the matrix. of the molecular assembly was calculated according to the
Purification by silica gel column chromatography was carried Stokes–Einstein equation as described below:
qffiffiffiffiffiffiffiffiffiffiffiffiffiffi
out by elution of a column with a stepwise gradient elution
procedure or using a CombiFlash Rf 75 system with a linear
gradient elution procedure using standard conditions.
Chemical reactions were monitored by TLC visualized by
immersion in an appropriate stain (10% H2SO4, 500 mL;
H3(PMo12O40)nH2O, 12.5 g; Ce(SO4)2nH2O, 5 g, or 5% ninhy-
drin in ethanol) followed by heating.
2KBT
3rhπη
2
ðx; yÞ ¼
qffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2
where KB is the Boltzmann constant and ðx; yÞ is the mean
squared speed of a particle at a temperature T, in a medium of
viscosity η, with a hydrodynamic radius of rh.
ELISA assay
Preparation of peptide assemblies
An 8-week-old BALB/c mouse (n = 4 per group, Japan SLC, Inc.,
Japan) was administered 100 μL of each sample intraperitone-
ally by subcutaneous injection. The blood was collected from
eyegrounds at 7 and 14 days, and then the serum was obtained
by centrifugation (3000 rpm, 10 min) after the blood had been
left to stand at 4 °C overnight. For coating the 96-well plates,
S-L was dissolved in acetonitrile, and LY-L, PEG-(L-Leu-Aib)6-
OMe and Boc-(L-Leu-Aib)6-OMe were dissolved in MeOH. Each
solution was put into a well (50 μL per well), followed by air-
drying completely at 4 °C overnight. Then, the blocking buffer
[2% BSA in phosphate-buffered saline (PBS)] was added
(150 μL per well) followed by incubation for 2 h at room temp-
erature. For the competitive inhibition assays, the inhibitor
(free LY or G1) was added to the sera prior to incubation. All
the wells were washed three times with PBS-T (PBS containing
0.05% Tween 20). The sera with serial dilution were added to
the wells, incubated for 2 h at room temperature, and washed
three times with PBS-T. Peroxidase-conjugated goat-anti-mouse
IgM (Southern Biotech, USA), IgA (abcam, USA), IgG1 (abcam,
USA), IgG2a (abcam, USA) or IgG3 (Southern Biotech, USA) in
0.1% BSA in PBS (50 μL) was added to the wells as the second-
ary antibody according to the protocol provided by the
suppliers. After incubation for 2 h at room temperature, the
wells were washed again three times with PBS-T.
o-Phenylenediamine (0.5 mg mL−1, Sigma, St Louis, MO) dis-
solved in 0.0003% H2O2–0.1 M citrate phosphate buffer
(pH 5.0) was added to the wells, and the plate was kept stand-
ing at 30 °C for 10 min. 2 M H2SO4 aqueous solution was
added to terminate the reaction, then the OD was determined
by UV measurements (Thermo Scientific, MultiSkan FC
Advance) at 490 nm/reference at 620 nm.
An equimolar mixture of the polypeptides solution (1 mg in
20 μL EtOH) was injected into saline (1 mg/1 mL; Otsuka
Pharmaceutical Co., Ltd), and kept stirring at 4 °C for 30 min.
Each prepared sample was filtered using a 0.80 µm cellulose
acetate syringe filter (Tokyo Roshi).
Transmission electron microscopy (TEM)
A drop of the molecular assembly dispersed in saline was
mounted on a carbon-coated Cu grid and stained negatively
with 2% uranyl acetate, followed by suction of the excess fluid
with filter paper. TEM images were obtained at an accelerating
voltage of 100 kV.
Dynamic light scattering (DLS)
Each prepared sample (1.0 mg in 1.0 mL saline) was filtered
using a 0.80 µm cellulose acetate syringe filter (Tokyo Roshi),
and then measured by DLS8000KS at 25 °C.
Substrate modification for atomic force microscopy (AFM)
A Si wafer was cleaned successively with 2% hydrofluoric acid
and a piranha solution. Then, the Si wafer was treated with 1%
3-aminopropyltriethoxysilane (APTES) solution (toluene) at
60 °C for 10 min. The surface modification was confirmed to
be a monolayer of APTES with a thickness of about 1 nm by
AFM (data not shown).
Imaging by AFM
Topological images of the molecular assemblies were
obtained in saline using a Multimode 8 AFM with Peak force
QNM in an aqueous mode with a gold-coated silicon tip on a
nitride cantilever (SCANASYST-FLUID+, 0.7 N m−1, Bruker)
on an APTES-modified Si wafer. Before measuring the
images, the freshly prepared dispersion of the molecular
assembly in saline was incubated in a fluid liquid cell on an
APTES-modified Si wafer at room temperature for 30 min,
and then the excess molecular assemblies in the fluid liquid
cell were removed gently by replacing with saline using a
syringe.
Statistical analysis
Differences between groups in antibody production and com-
petitive inhibition assays were assessed using the t-test for
independent samples (n = 3). A P value <0.05 is considered
statistically significant. P < 0.05, <0.01 and <0.005 are desig-
nated by *, ** and ***, respectively.
Nanoparticle tracking analysis (NTA)
Ethics
A freshly prepared dispersion of the molecular assembly
(5.0 μg mL−1 saline) was injected into an optical cell using a All of our in vivo animal experiments were approved by the
syringe. Each sample was measured three times for 1 min at Animal Care and Use Committee of the University of Fukui.
room temperature under the flow of the mixture using a Animals were treated humanely.
This journal is © The Royal Society of Chemistry 2018
Org. Biomol. Chem., 2018, 16, 8095–8105 | 8101

View Article Online
Paper
Organic & Biomolecular Chemistry
Synthesis
δ (ppm) 7.75–7.29 (m, 16H, aromatic group of fmoc group),
6.68–6.55 (1H J = 4.8 Hz, NH), 4.50–3.87 (m, 15H, CHCH2CO of
Preparation of LY-S-L, S-L, S-D and the LY derivative (13). Fmoc group, CH2 of Sar, CH2CHNHCH2, CH2CHNHCH2),
LY-S-L, S-L, S-D and the LY derivative (13) were prepared 3.03–2.98 (m, 6H, NCH3 of Sar), 2.60, (m, 2H, COCH2CH2CO),
according to the procedures reported previously.24–26,43
2.46 (m, 2H, COCH2CH2CO), HRMS (ESI) m/z: [M + Na]+, calcd
Butanoic acid, 4-[[2-hydroxy-1-hydroxymethylethyl]amino]-4- for C43H43N3NaO11 800.2795; found, 800.2781.
oxo-tert-buthyl ester (3). To a mixed solution of 2-amino-1,3-
Synthesis of compound 9. To a mixture of 6 (65 mg,
di-propanol (1: 1.0 g, 11.0 mmol) and mono-t-Bu-succinate 83.5 μmol) and H-(LLeuAib)6-OMe (7: 68 mg, 55.7 μmol) in dry
(2: 2.9 g, 19.5 mmol) in an EtOH–dry DMF mixture (30 mL– CH2Cl2–dry DMF (2.0 mL–2.0 mL) was added 2-(1-H-7-azaben-
15 mL) was added 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl- zotriazol-1-yl)-1,1,3,3-tetramethyl
uronium
hexafluoro-
morpholinium chloride (DMT-MM) (3.6 g, 13.2 mmol) at 0 °C. phosphate methanaminium (HATU) (85 mg, 0.223 mmol),
After stirring at room temperature under a dry atmosphere for 1-hydroxy-7-azabenzotriazole (HOAT) (30 mg, 0.223 mmol) and
9 h, the reaction mixture was concentrated under reduced diisopropyl ethyl amine (DIEA) (105 μL, 0.557 mmol) at 0 °C.
pressure. The residue was purified by silica gel column chrom- After stirring at room temperature under an Ar atmosphere for
atography eluting with CHCl3/MeOH (1 : 0 to 6 : 1, v/v, Rf 75 12 h, the reaction mixture was concentrated under reduced
system, linear gradient) to afford 3 (2.6 g, 10.5 μmol, 95%) as a pressure. The residue was purified by silica gel column chrom-
colorless syrup. 1H NMR (400 MHz, CDCl3) δ (ppm) 6.90 (d, atography eluting with CHCl3/MeOH (1 : 0 to 6 : 1, v/v, Rf 75
1H J = 7.6 Hz, NH), 3.94–3.91 (m, 1H, CH2CHNHCH2), system, linear gradient), and then Sephadex LH-20 eluting
3.83–3.69 (m, 4H, CH2CHNHCH2), 2.60, 2.46 (t × 2, 4H, J = 6.8 with CHCl3/MeOH (1 : 1, v/v) to afford 9 (72 mg, 36.3 μmol,
Hz, COCH2CH2CO), 1.44 (s, 9H, t-Bu). 13C NMR (100 MHz, 65%) as a white amorphous powder. 1H NMR (400 MHz,
CDCl3) δ (ppm) 172.90, 172.67 (CvO), 81.15 (t-Bu), 62.31 MeOH-d4) δ (ppm) 8.12–7.24 (m, 28H, aromatic group of fmoc
(CH2CH(NH)CH2), 52.68 (CH2CH(NH)CH2), 31.10, 30.80 group, amide), 4.42–3.86 (m, 21H, LeuCαH, CHCH2CO of
(COCH2CH2CO), 26.04 (t-Bu). HRMS (ESI) m/z: [M + Na]+, calcd Fmoc group, CH2 of Sar, CH2CHNHCH2, CH2CHNHCH2),
for C11H21NNaO5 270.1317; found, 270.1309.
2.98–2.87 (m, 6H, NCH3 of Sar), 2.80–2.76 (m, 1H,
Synthesis of compound 5. To a solution of 3 (2.6 g, COCH2CH2CO), 2.47–2.32 (m, 3H, COCH2CH2CO), 1.8–1.3
10.5 mmol) and Fmoc-Sar-OH (4: 7.8 g, 25.2 mmol) in dry (m, 54H, LeuCH2, LeuCγH, AibCH3), 0.88–0.74 (m, 36H, Leu
CH2Cl2–dry DMF (20 mL–20 mL) was added N,N′-dicyclohexyl- (CH3)2). HRMS (MALDI-TOF MS, CHCA) m/z: [M + Na]+, calcd
carbodiimide (DCC; 8.6 g, 42.1 mmol) and 4-dimethyl- for C104H153N15NaO23 2004.120; found, 2004.893.
aminopyridine (DMAP; 0.6 g, 5.26 mmol) at 0 °C. After stirring
Synthesis of compound 10. To a solution of 6 (110 mg,
at room temperature under an N2 atmosphere for 17 h, the 141.2 μmol) and H-(DLeuAib)6-OMe (8: 115 mg, 94.1 μmol) in
reaction mixture was concentrated under reduced pressure. dry DMF (5.0 mL) was added HATU (143 mg, 0.377 mmol),
The residue was purified by silica gel column chromatography HOAT (48 mg, 0.377 mmol) and DIEA (172 μL, 0.941 mmol) at
eluting with CHCl3/MeOH (1 : 0 to 6 : 1, v/v, Rf 75 system, linear 0 °C. After stirring at room temperature under an Ar atmo-
gradient) and then n-hexane/EtOAc (1 : 0 to 0 : 1, v/v, Rf 75 sphere for 15 h, the reaction mixture was concentrated under
system, linear gradient) to afford 5 (0.94 g, 1.13 μmol, 11%) as a reduced pressure. The residue was diluted with CHCl3, and
1
white amorphous powder. H NMR (400 MHz, CDCl3) δ (ppm) washed successively with 4% aq. KHSO4, saturated aq.
7.78–7.29 (m, 16H, aromatic group of Fmoc group), 6.29–6.15 NaHCO3, and brine. The organic layer was dried over Na2SO4,
(m, 1H, NH), 4.49–3.91 (m, 15H, CHCH2CO of Fmoc group, CH2 filtered through a bed of diatomaceous earth (Celite), and con-
of Sar, CH2CHNHCH2, CH2CHNHCH2), 3.07–2.97 (m, 6H, NCH3 centrated under reduced pressure. The residue was purified by
of Sar), 2.60, (m, 2H, COCH2CH2CO), 2.46 (m, 2H, Sephadex LH-20 eluting with CHCl3/MeOH (1 : 1, v/v) to afford
COCH2CH2CO), 1.42 (s, 9H, t-Bu). 13C NMR (100 MHz, CDCl3) δ 10 (180 mg, 90.8 μmol, 64%) as a white amorphous powder.
(ppm) 172.47, 172.17, 169.64, 157.16 (carbonyl carbons of Fmoc 1H NMR (400 MHz, CDCl3, TMS) δ (ppm) 7.89–7.29 (m, 28H,
and succinate), 144.17, 141.62, 128.05, 127.42, 125.36, 125.15, aromatic group of fmoc group, amide), 4.41–3.93 (m, 21H,
120.32 (aromatic groups of Fmoc), 81.10 (t-Bu), 68.33, 67.98, LeuCαH, CHCH2CO of Fmoc group, CH2 of Sar,
63.52, 51.24, 51.19, 50.51, 47.42 (CHCH2CO of Fmoc group, CH2 CH2CHNHCH2, CH2CHNHCH2), 3.65 (s, 3H, OCH3), 3.09–2.99
of Sar, CH2CHNHCH2), 36.30, 36.05 (NCH3 of Sar), 31.27, 30.83 (m, 6H, NCH3 of Sar), 2.71–2.32 (m, 4H, COCH2CH2CO),
(COCH2CH2CO), 28.38 (t-Bu). HRMS (ESI) m/z: [M + Na]+, calcd 1.84–1.22 (m, 54H, LeuCH2, LeuCγH, AibCH3), 0.89–0.78 (m,
for C47H51N3NaO11 856.3421; found, 856.3424.
Method for removal of t-butyl ester group in compound 5. Na]+, calcd for C104H153N15NaO23 2004.120; found, 2004.113.
To a solution of 5 in CHCl3 (0.5 mL) was added trifluoroacetic Synthesis of compound 11. To a solution of 9 (44 mg,
36H, Leu(CH3)2).HRMS (MALDI-TOF MS, CHCA) m/z: [M +
acid (TFA; 2.0 mL) and anisole (0.2 mL). After stirring at room 22.2 μmol) in anhydrous CH2Cl2 (800 μL) was added 3 M piper-
temperature for 2 h, the reaction mixture was concentrated idine in anhydrous CH2Cl2 (800 μL) at 0 °C. After stirring for
under reduced pressure. Completion of the reaction was deter- 30 min at 0 °C, the reaction mixture was poured into pet-
1
mined by H NMR and ESI-MS. The residue was washed with roleum ether, and the formed precipitate was washed with pet-
diisopropyl ether and dried under reduced pressure to give 6 roleum ether three times, followed by drying in vacuo. To a
(145 mg, 0.186 mmol, 83%). 1H NMR (400 MHz, CDCl3) solution of the residue in DMF–CH2Cl2 (1.5 mL–1.5 mL) was
8102 | Org. Biomol. Chem., 2018, 16, 8095–8105
This journal is © The Royal Society of Chemistry 2018

View Article Online
Organic & Biomolecular Chemistry
Paper
added Sar-NCA (150 mg, 1.46 mmol) under an Ar atmosphere. tion of the Sar-NCA was confirmed, a solution of 4-pentyonic
After complete consumption of Sar-NCA was confirmed, a solu- acid (12 mg, 0.120 mmol), HATU (60 mg, 0.160 mmol), and
tion of 4-pentyonic acid (12 mg, 130.4 μmol), HATU (50 mg, HOAT (21 mg, 0.160 mmol) in anhydrous DMF (500 μL) and
130.4 μmol), HOAT (34 mg, 130.4 μmol) in anhydrous DMF DIEA (68 μL, 0.400 mmol) was added at 0 °C under Ar atmo-
(1.5 mL) and DIEA (60 μL, 326.0 μmol) was added therein at sphere to react with the N-terminal. After stirring for 24 h
0 °C under an Ar atmosphere. After stirring for 15 h under an under Ar atmosphere, the solution was condensed, and the
Ar atmosphere, the solution was condensed, and the residue residue was purified by a Sephadex LH-20 column with MeOH
was purified by Sephadex LH20 column with MeOH as an as an eluent to afford the amphiphilic polypeptide inducing
eluent to afford 11 (107 mg, 84%). The degree of polymeriz- the alkyne function group (84 mg, 24.5 μmol, 61%). The
ation of the poly(sarcosine) block was determined to be 39 by degree of polymerization of the poly(sarcosine) block was
1
1H NMR and MALDI-TOF MS measurements. 1H NMR determined to be 30 from the H NMR spectrum. MALDI-TOF
(400 MHz, MeOH-d4) δ (ppm) 8.15–7.70 (m, 12H, amide), MS analysis also supported the degree of polymerization to be
1
4.56–3.96 (br, 167H, LeuCαH, SarCH2, CH2CHNHCH2, 30. H NMR (400 MHz, MeOH-d4) δ (ppm) 7.98–7.74 (m, 12H,
CH2CHNHCH2), 3.61 (s, 3H, OCH3), 3.01–2.88 (m, 238H, Sar amide), 4.4–4.0 (br, 66H, LeuCαH, SarCH2), 3.65 (s, 3H, OCH3),
N–CH3), 2.65–2.24 (m, 14H, CHCH2CH2CO, COCH2CH2CO), 3.08–2.93 (m, 90H, Sar N–CH3), 2.68–2.66 (m, 1H,
1.8–1.4 (m, 54H, LeuCH2, LeuCγH, AibCH3), 0.96–0.80 (m, CHCH2CH2CO), 2.46–2.42 (m, 3H, CHCH2CH2CO), 2.26 (s, 1H,
36H, Leu(CH3)2). HRMS (MALDI-TOF MS, DHB) m/z: [M + Na]+, CHCH2CH2CO), 1.8–1.4 (m, 54H, LeuCH2, LeuCγH, AibCH3),
calcd for C312H521N91NaO97, 7120.863; found, 7119.821.
1.00–0.85 (m, 36H, Leu(CH3)2). HRMS (MALDI-TOF MS, CHCA)
Synthesis of compound 12. 3 M Piperidine in anhydrous m/z: [M + Na]+, calcd for C156H266N42NaO44, 3455.980; found,
CH2Cl2 (800 μL) was added to a solution of 10 (48 mg, 3456.098.
24.2 μmol) in anhydrous CH2Cl2 (800 μL) at 0 °C. After stirring
Synthesis of 2(LY-S)-L. To a solution of the amphiphilic poly-
for 2 h at 0 °C, the reaction mixture was poured into petroleum peptide having the alkyne functional group (11; 29 mg,
ether, and the formed precipitate was washed with petroleum 4.09 μmol) and 13 (12 mg, 13.7 μmol) in anhydrous DMF
ether three times, followed by drying in vacuo. To a solution of (1.0 mL) was added Cu(I)OAc (2 mg, 16.3 μmol). After stirring
the residue in DMF–CH2Cl2 (1.2 mL–1.2 mL) was added Sar- at 40 °C for 31 h, the solution was condensed, and the residue
NCA (146 mg, 1.27 mmol) under Ar atmosphere. After com- was purified by a Sephadex LH-20 column with MeOH as an
plete consumption of Sar-NCA, a mixture of 4-pentyonic acid eluent to afford 2(LYS)-L (11 mg, 1.2 μmol, 31%). The degree of
(12 mg, 0.130 mmol), HATU (50 mg, 0.13 mmol), and HOAT polymerization of the poly(Sar) block was determined to be 41
1
1
(34 mg, 0.130 mmol) in anhydrous DMF (1.0 mL) and DIEA by H NMR and MALDI-TOF MS analysis. H NMR (400 MHz,
(60 μL, 0.326 mmol) was added therein at 0 °C under an Ar MeOH-d4) δ (ppm) 8.07–7.71 (m, 12H, amide), 5.11 (d, 1H J =
atmosphere. After stirring overnight under an Ar atmosphere, 2.8 Hz, LY-proton), 4.98 (d, 1H J = 4.0 Hz, LY-proton), 4.55–3.35
the reaction mixture was concentrated, and then the residue (m, 258H, LeuCαH, SarCH2, LY-protons, LeuAibOCH3,
was purified by Sephadex LH-20 column chromatography OCH2CH(NH)–CH2O), 3.1–2.9 (m, 246H, Sar N–CH3), 2.6–2.2
eluting with MeOH to afford 12 (76 mg, 12.1 μmol, 50%). The (br, 14H, CH2CH2CvCH, CvOCH2CH2CvO), 1.91 (s, 6H,
degree of polymerization of the poly(sarcosine) block was 33 as LY-NHAc), 1.8–1.5 (m, 54H, LeuCH2, LeuCγH, AibCH3),
determined by the procedures described above. 1H NMR 1.19–1.16 (m, 12H, LY-fucose-H6), 0.96–0.80 (m, 36H, Leu
(400 MHz, MeOH-d4) δ (ppm) 8.12–7.70 (m, 12H, amide), (CH3)2). HRMS (MALDI-TOF MS, super DHB) m/z: [M + Na]+,
4.4–3.9 (br, 143H, LeuCαH, SarCH2, CH2CHNHCH2, calcd for C392H661N103NaO145, 9158.7548; found, 9158.994.
CH2CHNHCH2), 3.62 (s, 3H, OCH3), 3.01–2.89 (m, 198H, Sar
Synthesis of 2(LY-S)-D. To a mixture of 12 (23 mg,
N–CH3), 2.64–2.23 (m, 14H, CHCH2CH2CO, COCH2CH2CO), 3.68 μmol) and 13 (10 mg, 11.4 μmol) in anhydrous DMF
1.8–1.4 (m, 54H, LeuCH2, LeuCγH, AibCH3), 0.96–0.81 (m, (1.0 mL) was added Cu(I)OAc (2 mg, 16.3 μmol). After stirring
36H, Leu(CH3)2). HRMS (MALDI-TOF MS, CHCA) m/z: at 40 °C for 39 h, the mixture was condensed, and then the
[M + Na]+, calcd for C276H461N79NaO8, 6267.414; found, 6267.933. residue was purified by Sephadex LH-20 column chromato-
Synthesis of alkyne-functionalized AB type amphiphile. graphy eluting with MeOH to afford 2(LYS)-D (27 mg, 3.4 μmol,
According to the reported synthetic method,27 Boc-(D-Leu- 90%). The degree of polymerization of the poly(Sar) block was
Aib)6-OMe was obtained. The Boc group of the Boc-(D-Leu- 32, determined similarly as described above. 1H NMR
Aib)6-OMe (58 mg, 49.1 μmol) was removed by treatment with (400 MHz, MeOH-d4) δ (ppm) 8.06–7.65 (m, 12H, amide), 5.06
trifluoroacetic acid (TFA, 1.5 mL) and anisole (0.15 mL). The (d, 1H, J = 4.0 Hz, LY-proton), 4.94–4.87 (m, 2H, LY-proton),
reaction mixture was evaporated and dried in vacuo. The 4.50–3.30 (m, 198H, LeuCαH, SarCH2, LY-protons,
residue was dissolved in chloroform and washed with satu- LeuAibOCH3, OCH2CH(NH)–CH2O), 3.0–2.8 (m, 192H, Sar N–
rated NaHCO3 and saturated NaCl aqueous solutions. The CH3), 2.6–2.1 (br, 14H, CH2CH2CvCH, CvOCH2CH2CvO),
organic layer was dried over anhydrous Na2SO4 and the solvent 1.86 (s, 6H, LY-NHAc), 1.8–1.4 (m, 54H, LeuCH2, LeuCγH,
was removed and dried in vacuo to afford H-(D-Leu-Aib)6-OMe AibCH3), 1.14–1.11 (m, 12H, LY-fucose-H6), 0.96–0.76 (m, 36H,
(49 mg, 0.401 mmol). To a solution of H-(D-Leu-Aib)6-OMe in Leu(CH3)2). HRMS (MALDI-TOF MS, super DHB) m/z:
DMF/CH2Cl2 (2.5 mL, 1/1, v/v) was added Sar-NCA (138 mg, [M + Na]+, calcd for C338H571N85NaO127, 7879.083; found,
1.20 mmol) under an Ar atmosphere. After complete consump- 7879.024.
This journal is © The Royal Society of Chemistry 2018
Org. Biomol. Chem., 2018, 16, 8095–8105 | 8103

View Article Online
Paper
Organic & Biomolecular Chemistry
Synthesis of LY-S-D. To a solution of the terminal alkyne-
functionalized poly(sarcosine)33-(D-Leu-Aib)6-OMe (alkyne-
M. G. Finn and X. Huang, ACS Chem. Biol., 2013, 8, 1253–
1262.
functionalized AB type amphiphile, 31 mg, 10.9 μmol) and 13 10 U. Westerlind, A. Hobel, N. Gaidzik, E. Schmitt and
(11 mg, 12.5 μmol) in anhydrous DMF (1.0 mL) was added Cu H. Kunz, Angew. Chem., Int. Ed., 2008, 47, 7551–7556.
(I)OAc (2 mg, 16.3 μmol). After stirring at 40 °C overnight, the 11 M. Shi, K. A. Kleski, K. R. Trabbic, J. P. Bourgault and
reaction mixture was condensed, and the residue was purified P. R. Andreana, J. Am. Chem. Soc., 2016, 138, 14264–14272.
by Sephadex LH20 column chromatography eluting with 12 A. Kaiser, N. Gaidzik, T. Becker, C. Menge, K. Groh, H. Cai,
MeOH to afford LY-S-D (36 mg, 8.4 μmol, 73%). The degree of
Y. M. Li, B. Gerlitzki, E. Schmitt and H. Kunz, Angew.
Chem., Int. Ed., 2010, 49, 3688–3692.
polymerization of the poly(sarcosine) block was determined as
described above. 1H NMR (400 MHz, MeOH-d4) δ (ppm) 13 M. G. Baek and R. Roy, Bioorg. Med. Chem., 2002, 10, 11–
8.07–7.71 (m, 12H, amide), 5.12 (d, 1H, J = 4.0 Hz, LY-proton),
17.
4.99 (d, 1H, J = 4.0 Hz, LY-proton), 4.57–3.44 (m, 118H, 14 T. C. Shiao and R. Roy, New J. Chem., 2012, 36, 324–339.
LeuCαH, SarCH2, LY-protons, OCH3), 3.1–2.9 (m, 90H, Sar 15 D. Sames, X. T. Chen and S. J. Danishefsky, Nature, 1997,
N–CH3), 1.91 (s, 3H, LY-NHAc), 1.9–1.4 (m, 54H, LeuCH2,
LeuCγH, AibCH3), 1.19–1.16 (m, 6H, LY-fucose-H6), 0.96–0.81 16 H. Cai, Z. Y. Sun, M. S. Chen, Y. F. Zhao, H. Kunz and
(m, 36H, Leu(CH3)2). HRMS (MALDI-TOF MS, super DHB) m/z: Y. M. Li, Angew. Chem., Int. Ed., 2014, 53, 1699–1703.
[M + Na]+, calcd for C199H341N49NaO69 4333.353; found, 17 B. Richichi, B. Thomas, M. Fiore, R. Bosco, H. Qureshi,
389, 587–591.
4332.982.
C. Nativi, O. Renaudet and L. BenMohamed, Angew. Chem.,
Int. Ed., 2014, 53, 11917–11920.
18 Z. Zhou, M. Mondal, G. Liao and Z. Guo, Org. Biomol.
Chem., 2014, 12, 3238–3245.
19 Z. Zhou, G. Liao, S. S. Mandal, S. Suryawanshi and Z. Guo,
Chem. Sci., 2015, 6, 7112–7121.
Conflicts of interest
There are no conflicts to declare.
20 T. Buskas, S. Ingale and G. J. Boons, Angew. Chem., Int. Ed.,
2005, 44, 5985–5988.
21 S. Ingale, M. A. Wolfert, J. Gaekwad, T. Buskas and
G. J. Boons, Nat. Chem. Biol., 2007, 3, 663–667.
22 S. Ingale, M. A. Wolfert, T. Buskas and G. J. Boons,
ChemBioChem, 2009, 10, 455–463.
Acknowledgements
This study was supported by JSPS KAKENHI Grant Number JP
17J09903 for YY.
23 V. Lakshminarayanan, P. Thompson, M. A. Wolfert,
T. Buskas, J. M. Bradley, L. B. Pathangey, C. S. Madsen,
P. A. Cohen, S. J. Gendler and G. J. Boons, Proc. Natl. Acad.
Sci. U. S. A., 2012, 109, 261–266.
24 Y. Yamazaki, N. Watabe, H. Obata, E. Hara, M. Ohmae and
S. Kimura, J. Pept. Sci., 2017, 23, 189–197.
25 D. H. Dube and C. R. Bertozzi, Nat. Rev. Drug Discovery,
2005, 4, 477–488.
26 M. Klinger, H. Farhan, H. Just, H. Drobny, G. Himmler,
H. Loibner, G. C. Mudde, M. Freissmuth and V. Sexl,
Cancer Res., 2004, 64, 1087–1093.
Notes and references
1 M. M. Fuster and J. D. Esko, Nat. Rev. Cancer, 2005, 5, 526–
542.
2 M. M. Wei, Y.-S. Wang and X.-S. Ye, Med. Res. Rev., 2018,
38, 1003–1026.
3 S. J. Danishefsky and J. R. Aleen, Angew. Chem., Int. Ed.,
2000, 39, 836–863.
4 G. Ragupathi, F. Koide, P. O. Livingston, Y. S. Cho, A. Endo,
Q. Wan, M. K. Spassova, S. J. Keding, J. Allen, O. Ouerfelli,
R. M. Wilson and S. J. Danishefsky, J. Am. Chem. Soc., 2006, 27 T. Kanzaki, Y. Horikawa, A. Makino, J. Sugiyama and
128, 2715–2725. S. Kimura, Macromol. Biosci., 2008, 8, 1026–1033.
5 H. Y. Chuang, C. T. Ren, C. A. Chao, C. Y. Wu, 28 M. Ueda, A. Makino, T. Imai, J. Sugiyama and S. Kimura,
S. S. Shivatare, T. J. Cheng, C. Y. Wu and C. H. Wong, J. Am.
Chem. Soc., 2013, 135, 11140–11150.
6 Y. L. Huang, J. T. Hung, S. K. Cheung, H. Y. Lee, K. C. Chu,
Chem. Commun., 2011, 47, 3204–3206.
29 V. Filipe, A. Hawe and W. Jiskoot, Pharm. Res., 2010, 27,
796–810.
S. T. Li, Y. C. Lin, C. T. Ren, T. J. Cheng, T. L. Hsu, A. L. Yu, 30 A. Makino, E. Hara, I. Hara, E. Ozeki and S. Kimura,
C. Y. Wu and C. H. Wong, Proc. Natl. Acad. Sci. U. S. A.,
2013, 110, 2517–2522.
7 S. J. Danishefsky, Y. K. Shue, M. N. Chang and C. H. Wong,
Acc. Chem. Res., 2015, 48, 643–652.
Langmuir, 2014, 30, 669–674.
31 C. J. Kim, S. Kurauchi, T. Uebayashi, A. Fujisaki and
S. Kimura, Bull. Chem. Soc. Jpn., 2017, 90, 568–573.
32 E. Hara, A. Makino, K. Kurihara, F. Yamamoto, E. Ozeki
and S. Kimura, Int. Immunopharmacol., 2014, 14, 261–266.
8 E. Kaltgrad, S. Sen Gupta, S. Punna, C. Y. Huang, A. Chang,
C. H. Wong, M. G. Finn and O. Blixt, ChemBioChem, 2007, 33 C. J. Kim, E. Hara, A. Shimizu, M. Sugai and S. Kimura,
8, 1455–1462. J. Pharm. Sci., 2015, 104, 1839–1847.
9 Z. Yin, M. Comellas-Aragones, S. Chowdhury, P. Bentley, 34 E. Hara, M. Ueda, C. J. Kim, A. Makino, I. Hara, E. Ozeki
K. Kaczanowska, L. Benmohamed, J. C. Gildersleeve,
and S. Kimura, J. Pept. Sci., 2014, 20, 570–577.
8104 | Org. Biomol. Chem., 2018, 16, 8095–8105
This journal is © The Royal Society of Chemistry 2018

View Article Online
Organic & Biomolecular Chemistry
Paper
35 L. A. Herzenberg and T. Tokushima, J. Exp. Med., 1982, 40 C. B. Fox, M. riede, S. G. Reed and G. C. Ireton, in
155, 1730–1740.
36 E. C. Rossoer and C. Mauri, Immunity, 2015, 42, 607–612.
37 W. van de Veen, B. Stanic, O. F. Wirz, K. Jansen,
Endotoxins: Structure, Function, and Recognition. Subcellular
Biochemistry, ed. X. Wang and P. Quinn, Springer,
Dordrecht, 2010, vol. 14, pp. 303–321.
A. Globinska and M. Akdis, J. Allergy Clin. Immunol., 2016, 41 J. Ni, H. Song, Y. Wang, N. M. Stamatos and L. X. Wang,
138, 654–665. Bioconjugate Chem., 2006, 17, 493–500.
38 R. Sabat, G. Grütz, K. Warszawska, S. Kirsch, E. Witte, 42 T. Buskas, Y. Li and G. J. Boons, Chem. – Eur. J., 2004, 10,
K. Wolk and J. Geginat, Cytokine Growth Factor Rev., 2010,
21, 331–344.
39 K. Yanaba, J.-D. Bouaziz, T. Matsushita, T. Tubata and
T. Tedder, J. Immunol., 2009, 182, 7459–7472.
3517–3524.
43 Y. Yamazaki, K. Sezukuri, J. Takada, H. Obata,
S. Kimura and M. Ohmae, Carbohydr. Res., 2016, 422,
34–44.
This journal is © The Royal Society of Chemistry 2018
Org. Biomol. Chem., 2018, 16, 8095–8105 | 8105