Design, Synthesis, and Evaluation of Lipopeptide Conjugates of Mercaptoundecahydrododecaborate for Boron Neutron Capture Therapy

Article abstract of DOI:10.1002/cmdc.201800793

We developed new ¹⁰B carriers for boron neutron capture therapy (BNCT) that can effectively transport and accumulate boron clusters into cells. These carriers consist of a lipopeptide, mercaptoundecahydrododecaborate (BSH), and a disulfide linker. The carriers were conceived according to the structure of pepducin, a membrane-penetrating lipopeptide targeting protease-activated receptor 1 (PAR1). To improve the membrane permeability of BSH, the structure was optimized using various lipopeptides possessing different peptides and lipid moieties. These synthesized lipopeptides were conjugated with BSH and evaluated for intracellular uptake using T98G glioblastoma cells. Among them, the most effectively incorporated and accumulated in the cells was compound 5 a, which contains a peptide of 13 residues derived from the intracellular third loop of PAR1 and a palmitoyl group. For further improvement of ¹⁰B accumulation in cells, the introduction of an amine linker was investigated; intracellular uptake similar to that of 5 a was observed for compound 14, which has a piperazine linker. Both compounds 5 a and 14 showed a stronger radiosensitizing effect than BSH along on T98G cells under mixed-neutron beam irradiation. The results demonstrate that lipopeptide conjugation is effective for enhancing intracellular delivery and accumulation of BSH and improving the cytotoxic effect of BNCT.

Full text of DOI:10.1002/cmdc.201800793

Accepted Article
Title: Design, synthesis, and evaluation of lipopeptide conjugates of
mercaptoundecahydrododecaborate for boron neutron capture
therapy
Authors: Aoi Isono, Mieko Tsuji, Yu Sanada, Akari Matsushita,
Shinichiro Masunaga, Tasuku Hirayama, and Hideko
Nagasawa
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To be cited as: ChemMedChem 10.1002/cmdc.201800793
Link to VoR: http://dx.doi.org/10.1002/cmdc.201800793
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ChemMedChem
Design, synthesis, and evaluation of lipopeptide conjugates of
mercaptoundecahydrododecaborate for boron neutron capture therapy
Aoi Isonoa, Mieko Tsujia, Yu Sanadab, Akari Matsushita a, Shinichiro Masunagab, Tasuku Hirayamaa,
Hideko Nagasawaa*
a Laboratory of Pharmaceutical and Medicinal Chemistry, Gifu Pharmaceutical University
b Institute for Integrated Radiation and Nuclear Science, Kyoto University
* To whom correspondence should be addressed. E-mail: hnagasawa@gifu-pu.ac.jp
Abstract:
We developed new 10B carriers for boron neutron capture therapy (BNCT) that can effectively
transport and accumulate boron clusters into cells. These carriers consist of lipopeptide,
mercaptoundecahydrododecaborate (BSH) and a disulfide linker. The carriers were conceived
according to the structure of pepducine, a membrane-penetrating lipopeptide targeting protease-
activated receptor 1 (PAR1). To improve the membrane permeability of BSH, the structure was
optimised using various lipopeptides possessing different peptides and lipid moieties. These
synthesised lipopeptides were conjugated with BSH and evaluated for intracellular uptake using T98G
glioblastoma cells. Among them, the most effectively incorporated and accumulated in the cells was
compound 5a, which has a peptide of 13 residues derived from the intracellular third loop of PAR1
10
and a palmitoyl group. For further improvement of B accumulation in cells, the introduction of an
amine linker was investigated; an intracellular uptake comparable to 5a was found for compound 14,
which has a piperazine linker. Compound 5a and compound 14 both showed a stronger radio-
sensitising effect than BSH on T98G cells under mixed-neutron beam irradiation. The results
demonstrate that lipopeptide conjugation is apparently effective for enhancing intracellular delivery
and accumulation of BSH and improving the cytotoxic effect of BNCT.
Introduction
Boron neutron capture therapy (BNCT) [1] is a binary radiotherapeutic modality utilising the nuclear
reaction of boron-10 (10B) with low-energy thermal neutrons to produce high-energy alpha particles
and lithium-7 nuclei with a short path length (5 to 9 m) equal to approximately one tumour cell
diameter. BNCT has been clinically evaluated for locally invasive malignant tumours such as
melanomas, high-grade gliomas and advanced head and neck cancers. Furthermore, accelerator-
based BNCT has become feasible in recent years, using a compact accelerator that can be installed in
hospitals as an alternative neutron source instead of the reactor.[2] Against this backdrop, the
development of practical boron carriers is an urgent task for the success of BNCT. These carriers can
1
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10.1002/cmdc.201800793
ChemMedChem
selectively and efficiently deliver 10B to cancer cells and accumulate it. Although not considered
optimal as boron carriers, only two boron-containing agents, sodium mercaptoundecahydodecaborate
(BSH) and p-bromophenylalanine (BPA), are currently used in clinical trials [3], based on their low
toxicity to normal cells and tissues. Due to the limitations of BSH and BPA, much effort has been
devoted to developing more effective boron delivery agents for practical use in BNCT. [4]
Polyhedral boron clusters such as BSH and carboranes are useful building blocks and possess
particular advantages as pharmacophores for the development of boron delivery agents for BNCT. In
particular, they contain a plurality of boron atoms in every molecule, making for efficient delivery of
10B atoms.[5] Among them, BSH is the more promising candidate, given its low toxicity and its current
clinical use. However, it has a significant drawback: it does not penetrate the cell membrane directly,
due to its highly hydrophilic and anionic properties. Therefore, to enhance the intracellular uptake and
tumour selectivity of BSH, various derivatives of BSH have been developed by peptide or amino acid
conjugation. [6]
In the present study, a novel, membrane-permeable boron cluster is developed using the cell-
penetrating lipopeptide pepducin as the vehicle for intracellular delivery of boron clusters. Pepducins
are membrane-permeable, lipidated peptides, developed as allosteric modulators of the G-protein-
[7]
coupled receptor (GPCR). The lipopeptide can flip across the lipid bilayer and can tether to the
inner leaflet of the membrane to exert an allosteric effect. We have previously developed fluorescence
resonance energy transfer (FRET) -based pepducin probes to demonstrate the direct membrane
penetration of a pepducin by fluorescence imaging in living cells. [8] In this report, the lipid moiety of
pepducins was revealed to play an important role in flipping across the bilayer and tethering to the
inner surface of the cell membrane. Pepducin has also been shown to be incorporated into cells even
when labelled with a number of anionic dyes, such as fluorescein.[7a] Therefore, we envisioned that
anionic boron clusters could be delivered into cells through direct penetration utilising the unique cell
membrane-penetrating ability of pepducins. Therefore, we designed and synthesised lipopeptide
conjugates of BSH, performed structural optimisation to improve cellular uptake and evaluated in vitro
radio-sensitising effects with thermal neutron exposure to the selected compounds.
Results and discussion
Design of boron carriers, lipopeptide conjugates of BSH
The new boron carriers consist of BSH, a linker and a lipopeptides derived from pepducin (Fig. 1).
Covic et al. invented P1pal-13(N-pal-AVANRSKKSRALF-NH2) as a pepducin for protease-activated
receptor 1 (PAR1).[9] In our previous report, we developed a fluorescent probe composed of FRET
moiety and a lipopeptide derived from P1pal-13 and showed that this fluorescent probe was able to
flip across the membrane to penetrate cells, even MCF-7 cells without PAR1 expression[10] (see also
Fig. S4B). [8] Those results suggested that the peptide sequence of P1pal-13 derived from C-terminal
2
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13 residues of the intracellular third loop of PAR1 can be used for intracellular delivery with or without
expression of the target GPCR on the cell membrane. Therefore, the palmitoyl peptide of P1pal-13
was selected as the initial structure for structural optimisation of the boron carrier. As Fig. 1 shows,
lysine was inserted between the peptide and the lipid moiety of the pepducin-type vehicle and
connected to the boron cluster via a disulfide linker. Disulfide bonds are useful for tumuor drug
delivery because they are stable in the human body and readily cleaved into reduced forms by
glutathione (GSH) in cells.[11] Moreover, it is known that GSH levels in tumour tissues are significantly
higher than those in normal tissues.[12] Once the boron carrier enters the cell, the cargo moiety
containing BSH is thought to be rapidly released by the reductive cleavage of the disulfide bond by
the cytosolic GSH, and then, accumulate in the cell due to its anionic charge.
Figure 1. A) Molecular design of lipopeptide conjugates of BSH as boron carriers. B) Putative
mechanism of intracellular delivery of the lipopeptide conjugate by flipping across a membrane.
Syntheses of lipopeptide conjugates of BSH
At first, compounds 5a–f, with different amino acid sequences, were synthesised as shown in Scheme
1. BSH was converted into S-(3-carboxypropyl)-S-(2-cyanoethyl)-sulfonio-undecahydro-closo-
dodecaborate (1-) sodium salt (2) in good yield and subsequent conjugation with (2-
pyridyldithio)ethylamine gave pyridyl disulfide-activated BSH 3. Pepducin derivatives 4a–j,
containing N6-(3-sulfanylpropanoyl)-lysine were synthesised by Fmoc solid-phase synthesis (SPPS)
and were coupled with 3 to produce pepducin-BSH conjugates 5a–j. BSH-sulfide derivative 6a was
obtained by basic treatment. Next, 5a derivatives 8a-e were similarly prepared by SPPS (Scheme 2),
with different lipid moieties.
Furthermore, for modification of the linker structure, piperazine linker derivative 14 and linear
diamine linker derivative 24 were synthesised. Since these linkers become cationic under
3
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physiological conditions, they were expected to improve cell membrane affinity. The synthesis route
of 14 is shown in Scheme 3. The pyridyldithio-activated piperazine linker 13 was coupled to pepducin
derivative 4a to give 14. The linear diamine linker 22 was prepared from S-triphenylmethyl-2-
mercaptoethanol (15) in five steps using Fukuyama’s procedure,[13] then conjugated with the pepducin
moiety and 2-chloroacetyl chloride on the resin (Scheme 4). Finally, deprotection of the side chain and
cleavage from the resin gave 23, which was conjugated with BSH to produce 24. All conjugates of
BSH were purified by RP-HPLC and identified by electrospray ionization mass spectrometry (ESI-
MS).
Scheme 1. Synthesis of 5 and 6a. Reagents and conditions: (a) 3-bromopropionitrile, Na2CO3 in
MeCN, 65 °C, 1 day; (b) NaOH in 15% acetone aq., rt, 2 h; (c) 4-bromobutyric acid in DMF, rt, 5 h
4
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then 40 °C, 1.5 h; (d) 2-(pyridyldithio)ethylamine hydrochloride, HBTU, DIPEA in DMF, rt, 1 day;
(e) (i) Fmoc-AA-OH, HOBt·H2O, DIPCI in DMF, 75 °C, 5 min, W; (ii) 20% piperidine in DMF,
40 °C, 10 min, W; (i), (ii) repeat; (f) Fmoc-Lys(COCH2CH2STrt)-OH, HBTU, DIPEAin DMF, 40 °C,
10 min, W; (g) 20% piperidine in DMF, 40 °C, 10 min, W; (h) palmitic acid, HBTU, DIPEA in
DMF, 40 °C, 10 min, W; (i) TFA/thioanisole/m-cresol/EDT/H2O (80:5:5:5:5), rt, 3 h; (j) compound
3 in MeCN/H2O/CH3COOH (2:1:0.15), rt, 8 h; (k) NaOH in MeCN/H2O (1:2), rt, 5 h.
-
CN
Na+
O
S+
3
O
O
S
Fmoc-based SPPS
a - e
HN
S
NH
HN
SH
4TFA
f
H2N
4TFA
O
O
AVANRSKKSRALF NH2
Novasyn
TGR resin
AVANRSKKSRALF NH2
R
N
R
N
H
H
O
O
7
8
compound
R
regents
yield of 7
yield of 8
55%
8a
8b
8c
8d
8e
CH3
Ac2O, pyridine
59%
17%
29%
docosahexaenoic acid, HBTU, DIPEA
myristic acid, HBTU, DIPEA
35%
C12H27
59%
R' = H
19%
H
lithocholic acid, HBTU, DIPEA
13%
H
H
R'O
R' = COCF3
18%
H
Scheme 2. Synthesis of 8a–e. Reagents and conditions: (a) (i) Fmoc-AA-OH, HOBt·H2O, DIPCI in
DMF, 75 °C, 5 min, W; (ii) 20% piperidine in DMF, 40 °C, 10 min, W; (i), (ii) repeat; (b) Fmoc-
Lys(COCH2CH2STrt)-OH, HBTU, DIPEA in DMF, 40 °C, 10 min, W; (c) 20% piperidine in DMF,
40 °C, 10 min, W; (d) reagents in DMF, 40 °C, 10 min, W; (e) TFA/thioanisole/m-cresol/EDT/H2O
(80:5:5:5:5), rt, 3 h; (f) compound 4 in MeCN/H2O/CH3COOH (2:1:0.15), rt, 3-12 h.
5
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Scheme 3. Synthesis of 14. Reagents and conditions: (a) (i) MsCl, Et3N in CH2Cl2, 0 °C, 30 min; (ii)
compound 10, DIPEA in MeCN, rt, overnight; (b) (i) 2 M HCl in MeOH, rt, 1.5 h; (ii) 2-
chloroacetylchloride, Et3N in CH2Cl2, 0 °C, 15 min; (c) compound 1 in DMF, 50 °C, 1 h; (d) 4a in
MeCN/H2O/CH3COOH (2:1:0.15), rt, 9 h.
Scheme 4. Synthesis of 24. Reagents and conditions: (a) (i) MsCl, Et3N in CH2Cl2, 0 °C, 15 min; (ii)
compound 16, Cs2CO3 in DMF, 50 °C, 1 day; (b) (i) MsCl, Et3N in CH2Cl2, 0 °C, 30 min; (ii)
compound 18, Cs2CO3 in DMF, 50 °C, overnight; (c) (i) TFA, TIPS in CH2Cl2, rt, 3 h; (ii) Dmt-Cl,
Et3N in CH2Cl2, rt, 1 h; (d) (i) thiophenol, K2CO3 in MeCN, rt, overnight; (ii) Boc2O, Et3N in CH2Cl2,
rt, 3 h; (e) 2,2'-dipyridyl disulfide, TFA, TIPS in CH2Cl2 rt, 3 h; (f) (i) TFA/TIPS/CH2Cl2 (10:5:85), rt,
5 min × 3; (ii) compound 22 in 1% CH3COOH in MeOH, rt, overnight; (iii) 2-chloroacetyl chloride,
DIPEA in DMF, 40 °C, W; (iv) m-cresol/H2O/TFA (5:5:90), rt, 3 h; (g) BSH·2Na in DMF, 50 °C, 2
6
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h.
Structural optimisation based on intracellular uptake
Table 1 shows structures of the new boron carriers. At first, for structural optimisation of the pepducin
moiety, the sequence and length of the peptide and the lipid structure were modified. All compounds
have lysine for conjugation to a disulfide linker between the palmitoyl moiety and the peptide fragment
shown in Table 1. Next to be synthesised were compound 5a with the original peptide sequence of
P1pal-13, and 5b with truncated peptide lacking the N-terminal 6 residues. T98G glioblastoma cells
were used to evaluate these compounds’ intracellular uptake. T98G cells were treated with the boron
carrier at a concentration of 10 or 20 µM and the intracellular 10B concentrations were measured
through time by inductively coupled plasma-atomic emission spectroscopy (ICP-AES).
7
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Table 1. Structures of new boron carriers.
8
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Figure 2. Time series of 10B uptake into T98G cells treated with compound 5a or 5b at a concentration
of 10 or 20 M.
Treatment with 5a increased intracellular boron concentration to 544 ng B per 106 cells, dose-
dependently with time, but 5b was hardly incorporated into the cells. (Fig. 2)
Even though 5b possesses the pepducin moiety known as PZ-128 which contains the C-terminal 7
residues of P1pal-13 and is under clinical trial as a PAR-1 inhibitor for coronary artery disease, [14] 5b
showed no effect on the delivery of boron clusters into T98G glioblastoma cells. When treated with
5a, the concentration of intracellular boron continued to increase over 24 hours, suggesting that it
existed stably in the culture medium and gradually accumulated in the cells. Therefore, the stability of
5a in the culture medium containing 10% fetal bovine serum (FBS) was examined using HPLC
analysis. Even after incubation at 37 °C for 24 hours, the recovery rate of 5a was more than 70%. (Fig.
S3) In general, acylation of peptides by long chain lipids improves stability by facilitating binding
with serum albumin and self-aggregation. [15] Palmitoylation effects may also contribute to the stability
of 5a.
Then, intracellular uptake of each compound was investigated by treatment at a concentration of 10
μM for 12 hours. (Fig. 3A) As with 5b, no effect was apparent with compound 5c with the C-terminal
10 residues of P1pal-13, mild effects were observed for 5d with the N-terminal 7 residues and 5e with
the N-terminal 10 residues. The same effect was obtained using 5f in which the C-terminal 3
hydrophobic amino acid residues were placed at the N-terminus. Neither 5g with scrambled peptide
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nor 5h with reverse sequence peptide was effective at all. These results suggest that the N-terminal
sequence is required for membrane penetration. To induce a β-turn conformation, Ser-Lys in the central
region of the peptide was replaced with a Pro-Gly segment in 5i, while in 5j, Ser in the central region
of the peptide was replaced with Pro.[16] Consequently, although water solubility improved,
intracellular uptake was significantly lower than that of 5a. The S-cyanoethyl on the sulfonium group
in the above compound was introduced for synthetic reasons to prevent excessive alkylation of the
BSH group; therefore, the charge of the cargo moiety containing BSH decreased from −2 to −1. In the
case of deprotected sulfide 6a, boron uptake was significantly lower than for 5a. In sum, it was
revealed that 5a was the most effectively incorporated into the cell, having a lipopeptide moiety with
the original 13-residue amino acid sequence (AVANRSKKSRALF) derived from the PAR1 loop
domain.
Next, intracellular uptake of compounds 8a–e was evaluated to optimise the structure of the lipid
moiety. (Fig. 3B) In the case of acetyl group (8a) and steroid type lipids (8d and 8e), membrane
permeability vanished. Even DHA (8b) and myristoyl (8c) groups showed some membrane
permeability; however, ultimately the palmitoyl (5a) group was found to be the most effective.
10
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Figure 3. Intracellular boron uptake using various boron carriers.
A) Boron uptake in T98G cells treated with 5a–i and 6a at a concentration of 10 M for 12 h. B)
Boron uptake treated with 8a–e at a concentration of 10 M for 12 h. C) Boron uptake treated with
5a, 14 and 24 at a concentration of 10 or 20 M for 12 or 24 h. n=3, * p<0.05, ** p<0.01 (compared
with 5a)
For further improvement of 10B accumulation in cells, the linker between the cargo moiety and the
disulfide bond was modified. Piperazine linker 12 and linear diamine liker 22, both amine linkers,
were prepared and conjugated to produce 14 (piperazine linker) and 24 (linear diamine linker). Since
these amine linkers have positive charges under physiological conditions, we expected then to increase
affinity for membranes. As show in Fig. 3B, when treated with 14 at a concentration of 10 M for 12
hours, boron uptake comparable to 5a evident. However, when treated with 24, boron uptake was
11
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significantly reduced. When treated at a concentration of 20 μM for 24 hours, the boron accumulation
for 14 was comparable to or higher than that for 5a.
Since we had detected PAR1 expression on T98G by flow cytometry analysis (Fig. S4A), we
separately synthesised lipopeptide P1pal-13 targeting PAR1 for a competition experiment. In this
experiment, T98G cells were treated with 5a (10 M) in the presence of P1pal-13 (10 M) for 12
hours, but no significant difference was observed. (Fig. S5) Next, after incubating the cells with 5a
(10 M) for 12 hours, the cells were treated at a concentration of 10 mM for 10 min with dithiothreitol
(DTT). With DTT treatment, there was no significant difference in boron uptake. (Fig. S5) From these
results, we conclude that the lipopeptide conjugate of BSH did not remain on the external surface of a
membrane, but rather moved inside to accumulate the BSH-containing moiety in cells. Furthermore,
this phenomenon appeared not to be related to the expression of PAR1.
In vitro radio-sensitising effect under neutron beam irradiation
Using the promising boron carriers 5a and 14, the radio-sensitisation effect was evaluated through
mixed-neutron beam irradiation. In this experiment, T98G cells were treated with 20 M of 5a, 14 or
BSH for 24 hours, then washed and suspended in the fresh medium, before being irradiated with a
mixed-neutron beam at the Heavy Water Facility of the Kyoto University Research Reactor (KUR).
Survival rate was determined by a colony formation assay. The D10 values were calculated by linear
regression analysis from the survival curves shown in Fig. 4, and represent the radiation dose that
produces a surviving fraction of 10%. Table 2 lists the D10 dose parameters and the plating efficiencies
at 0 Gy. The enhancing effect of treatment with the boron carrier is 7.60 for 5a, 5.79 for 14 and 0.97
for BSH. The effect of the piperidine linker was not observed, since no significant differences in
survival curves and in intracellular uptake were found for 5a and 14. There was no significant
difference in survival between the non-treated and BSH-treated groups, suggesting that BSH hardly
penetrated into the cells. On the other hand, 5a and 14, the lipopeptide conjugates of BSH, both showed
remarkable sensitising effects under the present experimental conditions. The results are consistent
with the concept that the lipopeptide conjugates of BSH are apparently incorporated and accumulated
in the cells more efficiently than BSH, resulting in substantial cytotoxicity by BNCT.
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Radiation dose (Gy)
Figure 4. Cell survival following irradiation with neutron beams for T98G cells with or without a
boron carrier as a function of the physical radiation dose of the external beams.
D10 value (Gy)a The enhancing effectc
PE without irradiation (%)
19.0±4.4b
32.9±3.6b
51.2±5.6b
20.0±4.5b
control
BSH
5a
4.17
4.32
0.55
0.72
-
0.97
7.60
5.79
14
aEach D10 calculated from a survival curve shows in Fig.4.
b95% confidence limits determined using mean values, standard deviations and the numbers of
observations on which the means and standard deviations were based.
cEnhancing effect is D10 for control / D10 for tested compound.
13
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Table 2. Plating efficiencies without irradiation, D10 values and the enhancing effects of boron carriers.
Conclusions
In this paper, we focused on the intracellular uptake function of pepducins and developed a novel
boron carrier by exploiting the lipopeptide as an intracellular delivery vehicle. We designed and
synthesised boron carriers composed of lipopeptide, BSH and disulfide linker, and performed
structural optimisation for each moiety while evaluating intracellular uptake. The best uptake to cells
was shown by the lipopeptide consisting of the palmitoyl group and a peptide fragment comprising an
amino acid sequence (AVANRSKKSRALF) derived from P1pal-13. Compound 5a, the BSH
conjugate of the lipopeptide efficiently accumulated intracellularly in a dose- and time-dependent
manner. For both 5a and for 14, which has the same lipopeptide as 5a but a piprazine linker, a dramatic
reduction in survival of T98 G cells resulted from treatment at a concentration of 20 μM for 24 h
followed by neutron beam irradiation. In vitro radio-sensitizing effects of these compounds under
mixed-neutron irradiation were significantly higher than that of BSH. Taken together, these results
reveal that by conjugating with the lipopeptide, it is possible to efficiently transport and accumulate
an anionic boron cluster such as BSH into cells and to significantly enhance the radiation effect of
neutrons. This lipopeptide conjugation methodology may be a promising strategy for drug delivery of
membrane-impermeable compounds. Our future work will investigate the tumour-targeting
capabilities of these compounds.
Experimental Section
General
All chemicals used in this study were commercial products and were purified further by standard
methods, if necessary. Microwave reactions were conducted using an Initiator 2.5 (Biotage). High-
resolution mass spectra (HRMS) were measured on a JMS-SX102A (JEOL) or a LCMS-IT-TOF
(Shimadzu). RP-HPLC analyses were performed on a 1260 infinity series (Agilent) equipped with a
Waters Symmetry C18 analytical column (Waters, 3.5 m, 4.6 × 75 mm, flow rate 0.5 mL/min). For
analysis, a solvent system containing 0.05% formic acid in ultra-pure water (v/v, solvent A) and 0.05%
formic acid in MeCN (v/v solvent B) or 0.1% TFA in ultra-pure water (v/v, solvent C) and 0.1% TFA
in MeCN (v/v solvent D) was used; the eluting products were detected by light absorption at 190, 195,
or 220 nm. Medium-pressure liquid chromatography (MPLC) separation was performed on a C18
Ultrapack column (YAMAZEN) with a YFLC W-Prep 2XY (YAMAZEN) or a SNAP Bio C18 and a
C4 column (Biotage) with an Isolera One 3.0 (Biotage) using solvent C and D; the eluting products
were detected by UV light absorption at 190 or 220 nm. HPLC separation was performed on a Bensil
5-C18 preparative column (Bentech, 20 × 250 nm, flow rate 5 mL/min) or a Cosmosil 5C18-AR II
14
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preparative column (Nacalai Tesque, 10 × 250 nm, flow rate 3 mL/min) with a 1260 infinity series
(Agilent) using solvent A and B or C and D; the eluting products were detected by UV light absorption
at 190 or 220 nm. ICP-AES analysis of the boron concentration was performed on an ULTIMA2
(HORIBAJobin Yvon) detecting at 249.773 nm. Irradiation was performed at the Heavy Water Facility
KUR. [27]
Syntheses
Preparation of peptide
Peptidyl-resins were synthesised by Fmoc-based SPPS on a NovaSyn TGR resin. For side-chain
protection, Trt was used for L-Asn, Boc, Trt or Dmt for L-Lys, Pbf for L-Arg and t-Bu for L-Ser.
Solid-phase peptide synthesis by microwave
The protected peptidyl resin was manually constructed at 0.10 mmol or 0.05 mmol scale on a NovaSyn
TGR resin (0.24 mmol/g). Fmoc-amino acids (Phe, Leu, Ala, Arg, Ser, Lys, Val, Asn) (5 eq.) were
coupled using DIPCI (5.0 eq.) and HOBt·H2O (5.0 eq.) in DMF at 75 °C for 5 min, or at 40 °C for 10
min (microwave heating). For the coupling of Fmoc-Lys(COC2H4STrt)-OH (28), Fmoc-
Lys(COC2H4SDmt)-OH (29), or each fatty acid, three equivalents of each reagent was reacted using
HBTU (2.9 eq.) and DIPEA (10 eq.) in DMF at 40 °C for 10 min (microwave heating). Acetylation
was performed using acetic anhydride (5 eq.) and pyridine (5 eq.) at 40 °C for 5 min (microwave
heating). The Fmoc group was deprotected with 20% piperidine in DMF at 40 °C for 10 min
(microwave heating).
Cleavage and deprotection of peptides
The protected peptide resin was treated with thioanisole/m-cresol/EDT/H2O/TFA (5: 5: 5: 5: 80 (v/v),
0.2 mL/20 mg resin) at room temperature for 3 h, and then the reaction mixture was filtered. The
filtrate was added to cold Et2O and the resulting precipitate was collected by centrifugation. The
precipitate was washed three times with cold Et2O then purified by RP-HPLC to produce peptide as a
freeze-dried powder to be analysed by HPLC and HRMS (ESI).
General procedure A (Preparation of lipopeptide conjugates of BSH)
Peptide (5 to 50 mol) and linker moiety (1 eq.) were dissolved in MeCN/H2O (2:1, 0.5 to 1 mL), then
CH3COOH was added to the mixture to a final concentration of 5%. After stirring for 3 to 12 h, the
reaction mixture was purified by RP-HPLC or RP-MPLC. The obtained main fractions were
lyophilized to give TFA salts. The purity of the lyophilized material was determined by analytical RP-
HPLC followed by UV detection at 220 nm according to the conventional method [17] (page S16-S24
in Supporting Information). The lyophilized material was further analysed by high resolution mass
15
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spectrometry analysis using LCMS-IT-TOF or direct infusion ESI-MS (see page S25 in Supporting
Information). The purities of lipopeptide conjugates of BSH for irradiation experiment were ≥95% as
confirmed by analytical RP-HPLC.
S-(cyanoethyl)-S-{4-oxo-{2-{3-oxo-{[6-oxo-(L-Ala-L-Val-L-Ala-L-Asn-L-Arg-L-Ser-L-Lys-L-Lys-L-
Ser-L-Arg-L-Ala-L-Leu-L-Phe-NH2)-5-N-palmitoylamino]hexylamino}propyldithio}ethylamino}
butyl}sulfonioundecahydro-closo-dodecaborate (1-) tetratrifluorate sodium salt (5a)
Compound 5a was prepared according to general procedure A using 4a (24 mg, 10.0 mol) and 3 (1
eq.) to produce 5a (13.5 mg, 50%) as a white solid. The peak integration ratio of 5a was 97.1% by
HPLC analysis (Fig. S1). Analytical HPLC conditions: linear gradient of solvent D into C, 20 to 80%
10
D over 20 min. r.t. = 16.6 min; O.D. 220 nm; HRMS (ESI) m/z: calcd. for C97H184 B12N27O20S3
[M+2H]2+ 1132.2496, found 1132.2520.
S-(cyanoethyl)-S-{4-oxo-{2-{3-oxo-{[6-oxo-(L-Lys-L-Lys-L-Ser-L-Arg-L-Ala-L-Leu-L-Phe-NH2)-5-
N-palmitoylamino]hexylamino}propyldithio}ethylamino}butyl}sulfonioundecahydro-closo-
dodecaborate (1-) tritrifluorate sodium salt (5b)
Compound 5b was prepared according to general procedure A using 4b (52.8 mg, 32.1 mol) and 3
(1 eq.) to produce 5b (12.4 mg, 15%) as a white solid. Analytical HPLC conditions: linear gradient of
solvent D into C, 20 to 95% D over 20 min, r.t. = 18.6 min; O.D. 220 nm; HRMS (ESI) m/z: calcd.
10
for C73H143 B12N17O12S3 [M+2H]2+ 833.0903, found 833.0918.
S-(cyanoethyl)-S-{4-oxo-{2-{3-oxo-{[6-oxo-(L-Asn-L-Arg-L-Ser-L-Lys-L-Lys-L-Ser-L-Arg-L-Ala-L-
Leu-L-Phe-NH2)-5-N-palmitoylamino]hexylamino}propyldithio}ethylamino}
butyl}sulfonioundecahydro-closo-dodecaborate (1-) tetratrifluorate sodium salt (5c)
Compound 5c was prepared according to general procedure A using 4c (9.8 mg, 4.6 mol) and 3 (1
eq.) to produce 5c (1.6 mg, 14%) as a white solid. Analytical HPLC conditions: linear gradient of
solvent D into C, 20 to 80% D over 20 min. r.t. = 14.9 min; O.D. 220 nm; HRMS (ESI) m/z: calcd.
10
for C86H167 B12N24O17S3 [M+3H]3+ 674.7880, found 674.7861.
S-(cyanoethyl)-S-{4-oxo-{2-{3-oxo-{[6-oxo-(L-Ala-L-Val-L-Ala-L-Asn-L-Arg-L-Ser-L-Lys--NH2)-
5-N-palmitoylamino]hexylaminopropyldithio}ethylamino}butyl}sulfonioundecahydro-closo-
dodecaborate (1-) ditrifluorate sodium salt (5d)
Compound 5d was prepared according to general procedure A using 4d (9.9 mg, 6.9 mol) and 3 (1
eq.) to produce 5d (1.3 mg, 10%) as a white solid. Analytical HPLC conditions: linear gradient of
solvent D into C, 20 to 80% D over 20 min. r.t. = 18.8 min; O.D. 220 nm; HRMS (ESI) m/z: calcd.
10
for C64H131 B12N17O13S3 [M+2H]2+ 781.0408, found 781.0395.
16
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S-(cyanoethyl)-S-{4-oxo-{2-{3-oxo-{[6-oxo-(L-Ala-L-Val-L-Ala-L-Asn-L-Arg-L-Ser-L-Lys-L-Lys-L-
Ser-L-Arg-NH2)-5-N-palmitoylamino]hexylamino}propyldithio}ethylamino}
butyl}sulfonioundecahydro-closo-dodecaborate (1-) tetratrifluorate sodium salt (5e)
Compound 5e was prepared according to general procedure A using 4e (15 mg, 7.4 mol) and 3 (1
eq.) to produce 5e (10.4 mg, 59%) as a white solid. Analytical HPLC conditions: linear gradient of
solvent D into C, 20 to 95% D over 20 min. r.t. = 14.3 min; O.D. 220 nm; HRMS (ESI) calcd. for
10
C79H161 B12N24O17S3 [M+3H]3+ 644.7723, found 644.7722.
S-(cyanoethyl)-S-{4-oxo-{2-{3-oxo-{[6-oxo-(L-Ala-L-Leu-L-Phe-L-Ala-L-Val-L-Ala-L-Asn-L-Arg-
L-Ser-L-Lys-L-Lys-L-Ser-L-Arg-NH2)-5-N-palmitoylamino]hexylamino}propyldithio}
ethylamino}butyl}sulfonioundecahydro-closo-dodecaborate (1-) tetratrifluorate sodium salt (5f)
Compound 5f was prepared according to general procedure A using 4f (8.0 mg, 3.4 mol) and 3 (1
eq.) to produce 5f (4.3 mg, 46%) as a white solid. Analytical HPLC conditions: linear gradient of
solvent D into C, 20 to 95% D over 20 min. r.t. = 16.6 min; O.D. 220 nm; HRMS (ESI) calcd. for
10
C97H186 B12N27O20S3 [M+3H]3+ 755.1689, found 755.1721.
S-(cyanoethyl)-S-{4-oxo-{2-{3-oxo-{[6-oxo-(L-Ala-L-Arg-L-Val-L-Ser-L-Ala-L-Lys- L-Asn-L-Lys-L-
Ala-L-Ser-L-Leu-L-Arg-L-Phe-NH2)-5-N-palmitoylamino]hexylamino}propyldithio}
ethylamino}butyl}sulfonioundecahydro-closo-dodecaborate (1-) tetratrifluorate sodium salt
(5g)
Compound 5g was prepared according to general procedure A using 4g (4.9 mg, 1.8 mol) and 3 (1
eq.) to produce 5g (1.4 mg, 30%) as a white solid. Analytical HPLC conditions: linear gradient of
solvent D into C, 20 to 80% D over 20 min. r.t. = 17.4 min; O.D. 220 nm; HRMS (ESI) calcd. for
10
C97H185 B12N27O20S32 [M+2H]2+ 1132.2496, found 1132.2471.
S-(cyanoethyl)-S-{4-oxo-{2-{3-oxo-{[6-oxo-(L-Phe-L-Leu-L-Ala-L-Arg-L-Ser-L-Lys- L-Lys-L-Ser-
L-Arg-L-Asn-L-Ala-L-Val-L-Ala-NH2)-5-N-palmitoylamino]hexylamino}propyldithio}
ethylamino}butyl}sulfonioundecahydro-closo-dodecaborate (1-) tetratrifluorate sodium salt
(5h)
Compound 5h was prepared according to general procedure A using 4h (13.8 mg, 3.4 mol) and 3 (1
eq.) to produce 5h (9.9 mg, 61%) as a white solid. Analytical HPLC conditions: linear gradient of
solvent D into C, 20 to 80% D over 20 min. r.t. = 15.8 min; O.D. 220 nm; HRMS (ESI) calcd. for
10
C97H186 B12N27O20S3 [M+3H]3+ 755.1689, found 755.1791.
S-(cyanoethyl)-S-{4-oxo-{2-{3-oxo-{[6-oxo-(L-Ala-L-Val-L-Ala-L-Asn-L-Arg-L-Pro- L-Gly-L-Lys-
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L-Ser-L-Arg-L-Ala-L-Leu-L-Phe-NH2)-5-N-palmitoylamino]hexylaminopropyldithio}
ethylamino}butyl}sulfonioundecahydro-closo-dodecaborate (1-) tritrifluorate sodium salt (5i)
Compound 5i was prepared according to general procedure A using 4i (10.2 mg, 4.7 mol) and 3 (1
eq.) to produce 5i (3.2 mg, 27%) as a white solid. Analytical HPLC conditions: linear gradient of
solvent D into C, 20 to 95% D over 20 min. r.t. = 17.9 min. O.D. 220 nm; HRMS (ESI) calcd. for
10
C95H179 B12N26O19S3 [M+3H]3+ 734.8179, found 734.8163.
S-(cyanoethyl)-S-{4-oxo-{2-{3-oxo-{[6-oxo-(L-Ala-L-Val-L-Ala-L-Asn-L-Arg-L-Pro- L-Lys-L-Lys-
L-Ser-L-Arg-L-Ala-L-Leu-L-Phe-NH2)-5-N-palmitoylamino]hexylamino}propyldithio}
ethylamino}butyl}sulfonioundecahydro-closo-dodecaborate (1-) tetratrifluorate sodium salt (5j)
Compound 5j was prepared according to general procedure A using 4j (11.8 mg, 5.0 mol) and 3 (1
eq.) to produce 5j (1.3 mg, 10%) as a white solid. Analytical HPLC conditions: linear gradient of
solvent D into C, 20 to 80% D over 20 min. r.t. = 16.6 min. O.D. 220 nm; HRMS (ESI) calcd. for
10
C99H188 B12N27O19S3 [M+3H]3+ 758.5091, found 758.5065.
S-(cyanoethyl)-S-{4-oxo-{2-{3-oxo-{[6-oxo-(L-Ala-L-Val-L-Ala-L-Asn-L-Arg-L-Ser- L-Lys-L-Lys-L-
Ser-L-Arg-L-Ala-L-Leu-L-Phe-NH2)-5-N-palmitoylamino]hexylamino}propyldithio}
ethylamino}butyl}sulfonioundecahydro-closo-dodecaborate (1-) tetratrifluorate sodium salt
(6a).
To a solution of 5a (3.7 mg, 1.58 mol) in acetone (50 L) and H2O (110 L), 2 M NaOH aq. (87 L)
was added. After stirring for 5 hours at room tempareture, the reaction mixture was acidified with TFA
and purified by RP-MPLC (column Ultra Pack AM, 11 x 100 mm, flow rate 10 mL min−1, linear
gradient of solvent D into C, 10 to 80% D over 20 min, then 80% over 10 min) to produce 6a (1.2 mg,
29%). Analytical HPLC conditions: linear gradient of solvent D into C, 20 to 80% over 20 min. r.t. =
16.8 min. O.D. 220 nm; HRMS (ESI) m/z: calcd. for C94H18010B12N26O20S3 [M-Na+3H]2+
1105.7364, found 1105.7376.
S-(cyanoethyl)-S-{4-oxo-{2-{3-oxo-{[5-N-acetylamino-6-oxo-(L-Ala-L-Val-L-Ala-L-Asn-L-Arg-L-
Ser-L-Lys-L-Lys-L-Ser-L-Arg-L-Ala-L-Leu-L-Phe-NH2)]hexylamino}propyldithio}ethylamino}
butyl}sulfonioundecahydro-closo-dodecaborate (1-) tetratrifluorate sodium salt (8a)
Compound 8a was prepared according to general procedure A using 7a (10.0 mg, 4.6 mol) and 3 (1
eq.) to produce 8a (6.3 mg, 55%) as a white solid. Analytical HPLC conditions: linear gradient of
solvent D into C, 15 to 60% D over 20 min. r.t. = 13.4 min; O.D. 220 nm; HRMS (ESI) calcd. for
10
C83H158 B12N27O20S3 [M+3H]3+ 689.7625, found 689.7612.
S-(cyanoethyl)-S-{4-oxo-{2-{3-oxo-{[5-N-docosahexaenoylamino-6-oxo-(L-Ala-L-Val-L-Ala-L-
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Asn-L-Arg-L-Ser-L-Lys-L-Lys-L-Ser-L-Arg-L-Ala-L-Leu-L-Phe-NH2)]hexylamino}propyldithio}
ethylamino}butyl}sulfonioundecahydro-closo-dodecaborate (1-) tetratrifluorate sodium salt
(8b)
Compound 8b was prepared according to general procedure A using 7b (10.6 mg, 4.4 mol) and 3 (1
eq.) to produce 8b (4.2 mg, 35%) as a white solid. Analytical HPLC conditions: linear gradient of
solvent D into C, 10 to 80% D over 20 min. r.t. = 17.3 min; O.D. 220 nm; HRMS (ESI) calcd. for
10
C103H186 B12N27O20S3 [M+3H]3+ 779.1689, found 779.1671.
S-(cyanoethyl)-S-{4-oxo-{2-{3-oxo-{[-5-N-myristoylamino-6-oxo-(L-Ala-L-Val-L-Ala-L-Asn-L-
Arg-L-Ser-L-Lys-L-Lys-L-Ser-L-Arg-L-Ala-L-Leu-L-Phe-NH2)]hexylamino}propyldithio}
ethylamino}butyl}sulfonioundecahydro-closo-dodecaborate (1-) tetratrifluorate sodium salt (8c)
Compound 8c was prepared according to general procedure A using 7c (10.0 mg, 4.3 mol) and 3 (1
eq.) to afford 8c (6.9 mg, 59%) as a white solid. Analytical HPLC conditions: linear gradient of solvent
D into C, 20 to 80% D over 20 min. r.t. = 15.3 min; O.D. 220 nm; HRMS (ESI) calcd. for
10
C95H182 B12N27O20S3 [M+3H]3+ 745.8251, found 745.8253.
S-(cyanoethyl)-S-{4-oxo-{2-{3-oxo-{[5-N-(3α-hydroxy-5β-cholan-24-oyl)amino-6-oxo-(L-Ala-L-
Val-L-Ala-L-Asn-L-Arg-L-Ser-L-Lys-L-Lys-L-Ser-L-Arg-L-Ala-L-Leu-L-Phe-NH2)]hexylamino}
propyldithio}ethylamino}butyl}sulfonioundecahydro-closo-dodecaborate (1-) tetratrifluorate
sodium salt (8d)
S-(cyanoethyl)-S-{4-oxo-{2-{3-oxo-{6-oxo-(L-Ala-L-Val-L-Ala-L-Asn-L-Arg-L-Ser-L-Lys-L-Lys-L-
Ser-L-Arg-L-Ala-L-Leu-L-Phe-NH2)-5-N-[3α-(2,2,2-trifluoro-acetoxy)-5β-cholan-24-oyl]amino-
hexylamino}propyldithio}ethylamino}butyl}sulfonioundecahydro-closo-dodecaborate
(1-)
tetratrifluorate sodium salt (8e)
Compound 8d and 8e was prepared according to general procedure A using mixture of 7d and 7e (10.0
mg, 4.0 mol) and 3 (1 eq.) to produce 8d (4.7 mg, 41%) and 8e (2.2 mg, 18%) as a white solid.
Analytical HPLC conditions: linear gradient of solvent D into C, 20 to 80% over 20 min. r.t. = 14.2
10
min (8d) and r.t. =17.3 min (8e). O.D. 220 nm; HRMS (ESI) calcd. for C105H193 B12N27O21S3
10
[M+2H]2+ 1192.2784, found 1192.2786 (8d) and calcd. for C107H193 B12F3N27O22S3 [M+3H]3+
827.1821, found 827.1821 (8e). Trifluoroacetylation of LCA was previously reported.[18]
S-(cyanoethyl)-S-{2-oxo-{2-{4-{2-{3-oxo-{[6-oxo-(L-Ala-L-Val-L-Ala-L-Asn-L-Arg-L-Ser-L-Lys-L-
Lys-L-Ser-L-Arg-L-Ala-L-Leu-L-Phe-NH2)-5-N-palmitoylamino]hexylamino}propyldithio}
ethyl}-1-piperazinyl}ethylamino}ethyl}sulfonioundecahydro-closo-dodecaborate
(1-)
tetratrifluorate sodium salt (14)
Compound 14 was prepared according to general procedure A using 4a (11.4 mg, 4.8 mol) and 13 (1
19
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eq.) to produce 14 (3.5 mg, 26%) as a white solid. The peak integration ratio of 14 was 95% by HPLC
analysis (Fig. S2). Analytical HPLC conditions: linear gradient of solvent D into C, 30 to 95% D over
10
20 min. r.t. = 15.2 min. O.D. 220 nm; HRMS(ESI) calcd. C101H195 B12N29O20S3 [M+4H]4+ 587.6456,
found 587.6435.
S-{2-oxo-{2-{3-{2-{3-oxo-{[6-oxo-(L-Ala-L-Val-L-Ala-L-Asn-L-Arg-L-Ser-L-Lys-L-Lys-L-Ser-L-
Arg-L-Ala-L-Leu-L-Phe-NH2)-5-N-palmitoylamino]hexylamino}propyldithio}
ethylamino}propylamino}ethylamino}ethyl}thioundecahydro-closo-dodecaborate(2-)
tetratrifluorate disodium salt (24)
23 (3.6 mg, 1.27 mol) and BSH·2Na (2.7 mg, 12.7 mol) were dissolved in DMF (0.25 mL). After
stirring at 50 °C for 2 h, the reaction mixture was diluted with H2O (0.5 mL) and purified by RP-HPLC
(Cosmosil 5C18-AR II preparative column, 10 × 250 nm, flow rate 3 mL/min, linear gradient of
solvent D into C, 30 to 60% D over 30 min) to produce 24 (1.6 mg, 42%). Analytical HPLC conditions:
linear gradient of solvent D into C, 20 to 80% D over 20 min, r.t. = 18.0 min. O.D. 220 nm; HRMS
10
(ESI) m/z: calcd. for C97H190 B12N28O20S3 [M+2H]2+ 1163.7527, found 1163.7497.
Biology
Cell culture
T98G cells were grown in EMEM containing 10% FBS with 50 U/mL penicillin and 0.05%
streptomycin and kanamycin in a free gas exchange with 5% CO2 at 37 °C.
Evaluation of intracellular boron concentration
T98G cells (1 × 106 cells) were plated onto a 100 mm dish. After 1 day, the cells were treated with a
boron carrier (10 μM, 1% DMSO as a cosolvent) at 37 °C for 12 h in growth medium (10% FBS) and,
then, washed with PBS three times, 2.5 mL of 0.05w/v% trypsin and 0.53 mM EDTA·4Na were added,
quenched by 2.5 mL of growth medium. After centrifugation, the collected cells were dissolved in
HNO3 (200 μL) for boron detection (wako) at room temperature for 1 h, and diluted with water to 5
mL. After passing through a filter (Dismic 13HP045AN, ADVANTEC), the boron concentration was
measured by ICP-AES.
Irradiation experiment and colony formation assay
T98G cells were seeded into 100 mm dishes in a compound-free medium. After pre-incubation for 24
hours, the cells were incubated for 25 h in a medium containing 5a, 14 and BSH (20 μM, 1% DMSO
as a cosolvent) in growth medium and then trypsinised. The single-cell suspensions were placed into
a Teflon tube and irradiated at room temperature by mixed-neutron beams at the Heavy Water Facility
of the KUR, 1 MW. Neutron fluence was measured by the radioactivation of gold foils on the front
20
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10.1002/cmdc.201800793
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and back of the tubes and, then, converted to a physical dose as described in previous studies.[19]
Contaminating gamma rays, including secondary gamma rays, were measured with
thermoluminescence dosimeter (TLD) powder. The average neutron flux of the employed beam was
1.0×109 n/cm2s. Total and gamma-ray dose rates were 0.04 Gy/min and approximately 0.01 Gy/min,
respectively. After irradiation, the cell suspensions were properly diluted, and cells were seeded in cell
culture dishes and incubated for 2 weeks. The cells were fixed with 70% ethanol and stained with
crystal violet solution for counting colonies.
Statistical analysis
Data are presented as mean ± S.D. and were analysed using Welch’s t-test to compare the two
conditions. A p value of <0.05 was considered statistically significant.
Acknowledgments
This research was supported by JSPS Grant-in-Aid for Challenging Exploratory Research (No.
26670059 for H.N.). The authors thank Prof. Dr. Osamu Sakurada (Gifu University) for his kind help
in ICP-AES measurement.
Conflict of interest
The authors declare no conflict of interest.
Keywords: Boron neutron capture therapy, boron carriers, antitumor therapy, membrane permeable
lipopeptides.
References
[1]
R. F. Barth, J. A. Coderre, M. G. Vicente, T. E. Blue, Clin. Cancer Res. 2005,
11(11), 3987-4002.
[2]
M. Suzuki, H. Tanaka, Y. Sakurai, G. Kashino, L. Yong, S. Masunaga, Y. Kinashi,
T. Mitsumoto, S. Yajima, H. Tsutsui, T. Sato, A. Maruhashi, K. Ono, Radiother. Oncol. 2009,
92(1), 89-95.
[3]
(a)K. Yokoyama, S. Miyatake, Y. Kajimoto, S. Kawabata, A. Doi, T. Yoshida, T.
Asano, M. Kirihata, K. Ono, T. Kuroiwa, J. Neurooncol. 2006, 78(3), 227-232; (b)S.
Miyatake, S. Kawabata, R. Hiramatsu, T. Kuroiwa, M. Suzuki, N. Kondo, K. Ono, Neurol.
Med. Chir. (Tokyo) 2016, 56(7), 361-371.
[4]
(a)R. F. Barth, P. Mi, W. Yang, Cancer Commun (Lond) 2018, 38(1), 35; (b)N. S.
Hosmane, Handbook of boron science : with applications in organometallics, catalysis,
21
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201800793
ChemMedChem
materials and medicine, 2019; (c)W. Sauerwein, A. Wittig, R. Moss, Y. Nakagawa, Neutron
capture therapy : principles and applications, 2015; (d)N. S. Hosmane, J. A. Maguire, Y.
Zhu, Boron and gadolinium neutron capture therapy for cancer treatment, World Scientific,
2012.
[5]
(a)B. P. Dash, R. Satapathy, J. A. Maguire, N. S. Hosmane, NEW. J. CHEM. 2011,
35(10), 1955-1972; (b)F. Issa, M. Kassiou, L. M. Rendina, Chem. Rev. 2011, 111(9), 5701-
5722.
[6]
(a)H. Michiue, Y. Sakurai, N. Kondo, M. Kitamatsu, F. Bin, K. Nakajima, Y.
Hirota, S. Kawabata, T. Nishiki, I. Ohmori, K. Tomizawa, S. Miyatake, K. Ono, H. Matsui,
Biomaterials 2014, 35(10), 3396-3405; (b)S. Kimura, S. Masunaga, T. Harada, Y.
Kawamura, S. Ueda, K. Okuda, H. Nagasawa, Bioorg. Med. Chem. 2011, 19(5), 1721-1728;
(c)G. Futamura, S. Kawabata, N. Nonoguchi, R. Hiramatsu, T. Toho, H. Tanaka, S. I.
Masunaga, Y. Hattori, M. Kirihata, K. Ono, T. Kuroiwa, S. I. Miyatake, Radiat. Oncol. 2017,
12(1), 26.
[7]
(a)L. Covic, M. Misra, J. Badar, C. Singh, A. Kuliopulos, Nat. Med. 2002, 8(10),
1161-1165; (b)K. E. Carlson, T. J. McMurry, S. W. H. III, Drug. Discov. Today Technol. 2012,
9(1), e1-e70.
[8]
Org. Biomol. Chem. 2013, 11(18), 3030-3037.
[9] L. Covic, A. L. Gresser, J. Talavera, S. Swift, A. Kuliopulos, Proc. Natl. Acad. Sci.
U. S. A. 2002, 99(2), 643-648.
M. Tsuji, S. Ueda, T. Hirayama, K. Okuda, Y. Sakaguchi, A. Isono, H. Nagasawa,
[10]
A. Boire, L. Covic, A. Agarwal, S. Jacques, S. Sherifi, A. Kuliopulos, Cell 2005,
120(3), 303-313.
[11]
[12]
G. Saito, J. A. Swanson, K. D. Lee, Adv Drug Deliv Rev 2003, 55(2), 199-215.
M. P. Gamcsik, M. S. Kasibhatla, S. D. Teeter, O. M. Colvin, Biomarkers 2012,
17(8), 671-691.
[13]
(a)Y. Hidai, T. Kan, T. Fukuyama, Tetrahedron Lett. 1999, 40(25), 4711-4714; (b)T.
Kan, T. Fukuyama, Chem. Commun. (Camb.) 2004(4), 353-359.
[14]
(a)P. A. Gurbel, K. P. Bliden, S. E. Turner, U. S. Tantry, M. G. Gesheff, T. P. Barr,
L. Covic, A. Kuliopulos, Arterioscler. Thromb. Vasc. Biol. 2016, 36(1), 189-197; (b)L. Covic,
A. Kuliopulos, Int J Mol Sci 2018, 19(8).
[15]
(a)B. P. Ward, N. L. Ottaway, D. Perez-Tilve, D. Ma, V. M. Gelfanov, M. H. Tschop,
R. D. Dimarchi, Mol Metab 2013, 2(4), 468-479; (b)J. Han, Y. Huang, X. Chen, F. Zhou, Y.
Fei, J. Fu, Eur. J. Pharm. Sci. 2018, 123, 111-123; (c)J. Ni, L. Qu, H. Yang, M. Wang, Y.
Huang, Int. J. Biochem. Cell Biol. 2006, 38(12), 2209-2218.
[16]
(a)S. K. Brahmachari, V. S. Ananthanarayanan, Proc. Natl. Acad. Sci. U. S. A.
22
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201800793
ChemMedChem
1979, 76(10), 5119-5123; (b)S. Pujals, E. Giralt, Adv Drug Deliv Rev 2008, 60(4-5), 473-484.
[17] (a)S. Stalmans, B. Gevaert, F. Verbeke, M. D'Hondt, N. Bracke, E. Wynendaele, B.
De Spiegeleer, J. Pharm. Biomed. Anal. 2016, 117, 289-297; (b)A. Swietlow, R. Lax,
CHOGGI 2004, 22(7/8), 22-25; (c)K. Takayama, A. Nakamura, C. Rentier, Y. Mino, T. Asari,
Y. Saga, A. Taguchi, F. Yakushiji, Y. Hayashi, ChemMedChem 2016, 11(8), 845-849.
[18]
J. C. Jimenez, A. Lopez-Macia, C. Gracia, S. Varon, M. Carrascal, J. M. Caba, M.
Royo, A. M. Francesch, C. Cuevas, E. Giralt, F. Albericio, J. Med. Chem. 2008, 51(16), 4920-
4931.
[19]
S. Masunaga, K. Ono, A. Takahashi, Y. Sakurai, K. Ohnishi, T. Kobayashi, Y.
Kinashi, M. Takagaki, T. Ohnishi, Jpn. J. Cancer Res. 2002, 93(12), 1366-1377.
23
This article is protected by copyright. All rights reserved.