Triazoyl-phenyl linker system enhancing the aqueous solubility of a molecular probe and its efficiency in affinity labeling of a target protein for jasmonate glucoside

Article abstract of DOI:10.1016/j.bmcl.2012.10.124

In methods employing molecular probes to explore the targets of bioactive small molecules, long or rigid linker moieties are thought to be critical factors for efficient tagging of target protein. We previously reported the synthesis of a jasmonate glucoside probe with a highly rigid linker consisting of a triazoyl-phenyl (TAzP) moiety, and this probe demonstrated effective target tagging. Here we compare the TAzP probe with other rigid or flexible probes with respect to target tagging efficiency, hydrophobic parameters, aqueous solubility, and dihedral angles around the biaryl linkage by a combination of empirical and calculation methods. The rigid biaryl linkage of the TAzP probe has a skewed conformation that influences its aqueous solubility. Such features that include rigidness and good aqueous solubility resulted in highly efficient target tagging. These findings provide a promising guideline toward designing of better linkers for improving molecular probe performance.

Full text of DOI:10.1016/j.bmcl.2012.10.124

Bioorganic & Medicinal Chemistry Letters 23 (2013) 188–193
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Triazoyl–phenyl linker system enhancing the aqueous solubility
of a molecular probe and its efficiency in affinity labeling of a target protein
for jasmonate glucoside
Satoru Tamura a, Sho Inomata a, Makoto Ebine a, Takahisa Genji a, Izumi Iwakura b, Makoto Mukai a,
Mitsuru Shoji c, Takeshi Sugai c, Minoru Ueda a,
⇑
a Department of Chemistry, Tohoku University, 6-3 Aramaki-aza Aoba, Aoba-ku, Sendai 980-8578, Japan
b Faculty of Engineering, Kanagawa University, 3-27-1 Rokkakubashi, Kanagawa-ku, Yokohama 221-8686, Japan
c Faculty of Pharmacy Keio University, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
In methods employing molecular probes to explore the targets of bioactive small molecules, long or rigid
linker moieties are thought to be critical factors for efficient tagging of target protein. We previously
reported the synthesis of a jasmonate glucoside probe with a highly rigid linker consisting of a tria-
zoyl–phenyl (TAzP) moiety, and this probe demonstrated effective target tagging. Here we compare
the TAzP probe with other rigid or flexible probes with respect to target tagging efficiency, hydrophobic
parameters, aqueous solubility, and dihedral angles around the biaryl linkage by a combination of empir-
ical and calculation methods. The rigid biaryl linkage of the TAzP probe has a skewed conformation that
influences its aqueous solubility. Such features that include rigidness and good aqueous solubility
resulted in highly efficient target tagging. These findings provide a promising guideline toward designing
of better linkers for improving molecular probe performance.
Received 4 September 2012
Revised 4 October 2012
Accepted 29 October 2012
Available online 6 November 2012
Keywords:
Molecular probe
Linker
CuAAC
TAzP
Ó 2012 Elsevier Ltd. All rights reserved.
Target protein
Molecular probes are recognized as powerful tools for exploring
the targets of bioactive small molecules.1–3 An appropriately de-
signed molecular probe affords highly efficient chemical tagging
of target proteins. A molecular probe consists of four essential
components: a pharmacophore, reactive functionality, a molecular
tag, and a linker moiety (Fig. 1). Among these, the selection of a lin-
ker of the appropriate length and structure strongly affects the effi-
ciency of target tagging (Fig. 1).4–7 In general, it is considered that
the role of the linker is to cast the molecular tag away from phar-
macophore, which is an essential unit for binding with a specific
target.8 It is generally accepted that the longer or more rigid the
linker is, the more effective the casting process.9 To date, substan-
tial attention has been paid to the design of the linker. Polymeth-
ylene,9 oligopeptides, including polyglycine10 or polyproline-rod,5
and polyethyleneglycol (PEG)11,12 are widely used as standard link-
ers (Fig. 1). However, a long linker causes serious issues associated
with its hydrophobic nature. An increase in hydrophobicity is
accompanied by a decrease in aqueous solubility as well as an in-
crease in nonspecific binding.6,13 Thus, a linker that combines rig-
idness for effective casting of the molecular tag with high aqueous
solubility is strongly desired.
Recently, we reported the potency of a molecular probe (1) hav-
ing a highly rigid triazoyl–phenyl (TAzP)14 linker designed for effi-
cient affinity tagging of the membrane target of jasmonate
glucoside (216 in Fig. 2) (MTJG).10,15 Surprisingly, even a short TAzP
linker functioned well and provided useful target tagging. The TAzP
probe can be easily constructed by copper-catalyzed azide alkyne
cycloaddition (CuAAC)17–19 between an alkyl azide and aryl acety-
lene, and it provided better results for tagging the corresponding
target protein than the conventional molecular probe. In this study,
we will discuss the chemical basis for the high efficiency of the
TAzP linker and report our findings that the TAzP linker confers rig-
idness as well as good aqueous solubility to a molecular probe be-
cause of its skewed conformation around the biaryl linkage.
TAzP molecular probe 1 possesses a biaryl structure substituted
by a pharmacophore and a large tag at each end of the unit (Fig. 2).
In a previous study, we hypothesized that the reason for the favor-
able performance of the TAzP probe might be attributed to the rig-
idness of the biaryl structure, which functions to cast the tag away
from the pharmacophore. Novel insight regarding the proficiency of
the TAzP linker can contribute to the development of other high-
performance linkers. However, our hypothesis has remained unpro-
ven because of a lack of appropriate control experiments. To resolve
this deficiency, we designed and synthesized alkyne units 9–12
(Schemes S1–S4, Supplementary data), which provide ‘CuAAC’-pre-
pared probes 3, 4, 6, and 7 (Figs. 2 and 3). Each alkyne unit (9–12)
⇑
Corresponding author.
E-mail address: ueda@m.tohoku.ac.jp (M. Ueda).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.124

S. Tamura et al. / Bioorg. Med. Chem. Lett. 23 (2013) 188–193
189
Reactive
functionality
Linker
Pharmacophore
Tag
Figure 1. A ball-and-stick model of 5 consisting of four essential components: a pharmacophore, reactive functionality, a molecular tag, and a linker moiety. Structures of
conventional linkers are also shown (framed rectangle).
was coupled with azide-JAG (13)14,20 under CuAAC conditions using
tris(3-hydroxypropyltriazolylmethyl)amine (THPTA)21,22 as a cata-
lyst. The resulting probes were used as appropriate controls for
TAzP probe 1 in the following experiments.
remaining probes, including TAzQ, did not provide any band corre-
sponding to biotinylated MTJG (Fig. 4).
Because ethyl-tethered probe 3 with the same linker length (14
atoms) as 1 does not show a band for tagged MTJG, it can be con-
cluded that the efficient photoaffinity tagging by 1 is not related to
the length of the linker but depends on the chemical nature of the
biaryl structure in the TAzP linker. This result suggests that the
advantage of the rigid TAzP linker might lie in its ability to effec-
tively cast the large tag away from the pharmacophore.
According to our hypothesis,14 the advantage of the TAzP linker
is lost when its planarity is disrupted by the introduction of meth-
ylene units between the triazole and aryl rings (Fig. 2). Thus, we de-
signed ethyl-tethered probe 3, in which the C2-unit in 1,
encompassing the a,b-positions of the carbonyl group, was shifted
to the position between the triazole and aryl groups. Probe 3 has the
same linker length (14 atoms) as 1 (Fig. 2) and is a useful control for
comparison with 1. We also synthesized alkyne units 11 and 12,
which provided the triazoylquinolyl (TAzQ) probes 6 and 7, respec-
tively. Higher planarity can be anticipated for the biaryl group of 6
and 7 than for the corresponding moiety of 1. The nitrogen atom in
the quinoline ring in 6 and 7 serves to lower the hydrophobicity of
the corresponding probe despite the large size of this aromatic
ring.23 In addition, alkyne unit 10 with a hexa-PEG linker provided
probe 4 by possessing the long PEG fragment, which added flexibil-
ity as compared to the rigid TAzP probe (Fig. 2).
First, we compared the efficiencies in target tagging among
ethyl-tethered probe 3, TAzQ probes (6 and 7), PEG-substituted
probe 4, conventional triglycyl probe 510, and TAzP probe 1. The effi-
ciency of each probe was assessed by the photoaffinity-based target
tagging experiment with MTJG.15 MTJG has been identified as a tar-
get protein for the leaf-closing factor of Samanea saman (2).16,24,25
Photoaffinity labeling by each probe was performed using the motor
cell of S. saman,10,14 and the prepared membrane fractions were sub-
jected to SDS–PAGE analysis with labeled MTJG. The preparation
and subsequent utilization of Samanea protoplasts in photoaffinity
labeling experiments were performed according to the procedures
described in our previous study.14 Unexpectedly, TAzP probe 1 pro-
vided the highest tagging efficiency among all the probes (1 and 3–
7) followed by triglycyl-linked probe 5 (Fig. 4). Conversely, all of the
This advantage of the rigid TAzP linker was also supported by
the fact that hexa-PEG probe 4 equipped with a linker extending
27 atoms in length does not show a band for tagged MTJG. Hydro-
philic and flexible PEG linkers are expected to adopt an extended
conformation, in which the tag is effectively cast away from the
pharmacophore, whereas experimental and theoretical studies
suggest that low molecular weight PEG oligomers/polymers pre-
dominantly adopt a helical conformation in an aqueous environ-
ment.26–28
A helical conformation of the PEG linker would
confine the large tag close to the pharmacophore and sometimes
cause aggregation with the attached pharmacophore.27 Thus, this
drawback of the PEG linker could be responsible for the disap-
pointing results for hexa-PEG probe 4. It is also important to deter-
mine whether nonspecific binding to proteins other than MTJG
occurred using PEG-linked 4 (data not shown). It is reported that
the use of a long PEG linker in affinity-based protein purification
cannot suppress the incorporation of nonspecific proteins.6,13
These results suggest that increasing the length of the linker would
not be an effective strategy.
Interestingly, no biotinylated MTJG was found in photoaffinity
labeling experiments using TAzQ probe 6 or 7. As it is expected that
the TAzQ linker affords planarity and rigidness equal to or greater
than the TAzP linker, we suspected that an unknown chemical
property of TAzP might be responsible for its notable target
affinity.

190
S. Tamura et al. / Bioorg. Med. Chem. Lett. 23 (2013) 188–193
solubility (5 ꢁ 4 > 3 ’ 1 > 7 ’ 6) suggested that the most hydro-
phobic pair (1 and 3) have greater aqueous solubility than that indi-
cated from the hydrophobicity.
The successful tagging of the target by 5 is highly correlated
with its exceptional aqueous solubility as well as its low hydropho-
bicity, which is similar to that of parent compound 2. The three
characterizations, CLogP, retention time in RP-HPLC, and aqueous
solubility, indicated that 5 is the most hydrophilic molecule among
all the synthesized probes. However, PEG probe 4 gave no success-
ful target tagging result in spite of having the second best aqueous
solubility and hydrophobicity. This can possibly be attributed to
the abovementioned helical conformation of PEG in an aqueous
environment, which lowers the affinity of the pharmacophore with
its target. Conversely, the most hydrophobic probe, 1, provided the
best result in target tagging experiments, whereas ethyl tethered
3, which demonstrated a similar CLogP, retention time in RP-HPLC,
and aqueous solubility to that of probe 1, was unsuccessful in tar-
get tagging analyses. These results suggest that the spatial arrange-
ment of each component (pharmacophore, reactive functionality,
molecular tag, and linker) of the probes affected their performance
more strongly than aqueous solubility.
However, TAzQ probes 6 and 7 with higher planarity and rigid-
ness than the TAzP linker do not work as expected. The TAzP linker
may work more effectively in separating the tag from the pharma-
cophore, but the decrease in aqueous solubility, which stems from
the triazoylquinolyl fragment, canceled this advantage. However,
here, we mention a peculiar discrepancy regarding TAzP (1). This
probe was more soluble in aqueous media than TAzQ (6 and 7)
even though it showed higher hydrophobicity than TAzQ (Table
1). Unobvious chemical properties can potentially enhance the
aqueous solubility of 1 more than expected considering the
hydrophobicity.
Aqueous solubility depends on two factors: crystallinity and
interactions with water.32 Hashimoto et al. reported that disrup-
tion of molecular planarity and symmetry increase the solubility
of drugs.33 This phenomenon results from a decrease in the effi-
ciency of crystal packing and was consistently observed in drugs
with a biaryl moiety. The skewed conformation in biaryl structures
confers enhanced solubility in aqueous media because it disrupts
the crystal packing. Thus, it is speculated that a skewed conforma-
tion around the biaryl linkage of the TAzP linker may improve the
aqueous solubility more than expected from the hydrophobicity.
We calculated the dihedral angle of TAzP and triazoylquinolyl
model compounds 14 and 15 from the optimized structures
obtained by density functional theory (DFT) calculations (B3LYP/
6-31G⁄).34 The TAzP linker in 1 confers a skewed conformation
around the biaryl linkage with the dihedral angle (N1–C1–C10–
C20) of 13.1°, which was determined from the most stable confor-
mation of 1 in Ref. 14 (Fig. 5). Similar DFT calculation studies gave
the dihedral angles of the most stable conformation of 14 and 15,
models of 6 and 7, respectively (Fig. 5). Expectedly, no twist was
observed around the biaryl linkages in 14 (h = 0.0°) and 15
(h = 0.5°). Therefore, we suspected that the distinct performance
of 1 among the ‘CuAAC’-prepared probes can be attributed to such
disruption of planarity owing to the skewed conformation around
the biaryl linkage (Fig. 5).
In conclusion, TAzP probe 1 afforded greater efficiency in target
tagging than probes 3–7 because of a combination of high rigidness
and good aqueous solubility. The rigid biaryl linker effectively casts
the molecular tag away from the pharmacophore and provides
improved aqueous solubility in spite of its hydrophobic properties.
The enhanced aqueous solubility is suggested to be attributable to
the skewed conformation around the biaryl linkage identified in
the DFT-calculated structure. Though the aqueous solubility of
triglycyl 5 was higher than that of 1, the flexible triglycyl linker
would not afford adequate spatial separation of the tag and
Figure 2. Structures of synthetic probes (1 and 3–7) and parent ligand JAG (2).
The structural modifications to parent ligand 2 required for its
conversion into various synthetic probes afford dramatic changes
in the physicochemical properties of 2 and cause a decrease in its
target affinity. As parent ligand 2 is highly soluble in aqueous med-
ia, it is expected that the poor aqueous solubility of a ‘CuAAC’-pre-
pared probe might decrease its availability for formation of an
adduct in photoaffinity tagging using living cells. Therefore, the
reason for the differences in target affinity among 1 and 3–7 might
be identified by an assessment of aqueous solubility.
In general, the aqueous solubility of a small molecule depends on
its hydrophobicity. Thus, we examined the hydrophobicity parame-
ters (calculated log P and retention time on reversed phase (RP)-
HPLC) and aqueous solubility of probes (1 and 3–7). Both calculated
logP (CLogP)29–31 and retention time in reversed phase RP-HPLC are
widely used to assess the hydrophobicity of small molecules. All the
CLogP values for probes 1 and 3–7 were much higher than that of
parent ligand 2 (CLogP = ꢀ0.39) (Table 1). Among them, the CLogP
value of TAzP probe 1 was the highest and was equivalent to that of
3. TAzQ probes 6 and 7 have medium values, and the values for 4 and
5
are the lowest. The rank order of the CLogP values
(3 ’ 1 > 6 = 7 > 4 > 5) strongly correlated with that of the retention
times in RP-HPLC (1 > 3 > 7 > 6 > 4 > 5) (Table 1). These data suggest
the following rankorder of hydrophobicity (3 ’ 1 > 6 ’ 7 > 4 > 5).
Interestingly, no clear relationship was found between aqueous
solubility and hydrophobicity (Table 1). The rank order of aqueous

S. Tamura et al. / Bioorg. Med. Chem. Lett. 23 (2013) 188–193
191
Figure 3. CuAAc coupling of alkyne units 8–12 and azide-JAG (13)14 for production of probes 1, 3, 4, 6, and 7, respectively.
pharmacophore. The distinguished performance of our TAzP linker
offers advantages such as high-performance, aqueous solubility,
and synthetic accessibility. The TAzP linker provides a powerful
alternative linker system for molecular probe technology.
Furthermore, our results provide a useful guideline for the
development of improved linkers. For example, it is already known
that meta-fluorination of a biaryl structure increases the dihedral
angle around the biaryl linkage.33 Thus, a m-fluoro-TAzP adduct
can achieve greater aqueous solubility and higher performance as
a molecular probe.
Unless otherwise stated, reactions were performed in flame-
dried glassware under argon or nitrogen atmosphere using dry sol-
vents. Solvents were dried over activated molecular sieves under
argon atmosphere. All starting materials were purchased from
commercial sources and used as received, unless otherwise stated.
Liquids and solutions were transferred via syringe or a positive-
pressure cannula. Brine solutions refer to saturated aqueous so-
dium chloride solutions. Thin-layer chromatography (TLC) was
performed using silica gel 60 F254 precoated plates (0.25 mm)
and visualized by UV fluorescence quenching or anisaldehyde or
H3(PMo12O40) staining. Silica gel 60 N (particle size 63–210 mm)
was used for column chromatography. HPLC purifications were
performed using PU-2089 with a UV-2075 detector (Jasco Ltd)
equipped with Cosmosil 5C18AR (
u
20 ꢂ 250 mm, Nakalai. Tesque
MTJG
3
4
1
1
5
MTJG
6
7
Figure 4. Chemiluminescence detection of MTJG by each probe (1, 3–7): Motor cell
protoplasts from pulvini of S. saman (ꢃ1 ꢂ 104 protoplasts in 0.1 mL) were mixed
with each probe (1 ꢂ 10–4 M). The mixture was incubated for 5 min at 0 °C, and
cross-linking was carried out by UV irradiation (365 nm) for 20 min at 4 °C. After
centrifugation (110 ꢂ g, 5 min, 4 °C), the sediment was homogenized with buffer.
Centrifugations of lysate (1st: 3000 ꢂ g, 15 min, 4 °C; 2nd: 100,000 ꢂ g, 1 h, 4 °C)
gave a crude membrane pellet. This pellet was analyzed by SDS–PAGE (7.5–15%
gradient gel), and protein bands were detected by LAS-4000 Bioimager (Fuji Film
Co., Ltd) using an ECL Advance western blotting detection kit (GE Healthcare UK,
Ltd) and analyzed using MultiGauge software (Fuji Film Co., Ltd).

192
S. Tamura et al. / Bioorg. Med. Chem. Lett. 23 (2013) 188–193
1H NMR (500 MHz, CD3OD) 7.85–7.82 (m, 4 H), 7.79–7.77 (m, 2
H), 7.72 (br, 1 H), 7.68–7.65 (m, 3 H), 7.55–7.52 (m, 2 H), 5.47–5.39
(m, 2 H), 4.66–4.61 (m, 3 H), 4.52–4.48 (m, 3 H), 4.29 (dd, J = 8.0,
4.5 Hz, 1 H), 3.95–3.89 (m, 2 H), 3.85 (br, 1 H), 3.69–3.63 (m, 1
H), 3.52–3.45 (m, 3 H), 3.37–3.34 (m, 2 H), 3.25–3.15 (m, 4 H),
3.07–3.04 (m, 2 H), 2.91 (dd, J = 12.8, 5.0 Hz, 1 H), 2.69 (d,
J = 12.8 Hz, 1 H), 2.62 (dd, J = 15.0, 3.5 Hz, 1 H), 2.45–2.16 (m, 10
H), 2.05–1.94 (m, 2 H), 1.75–1.30 (m, 6 H), 0.94–0.86 (m, 1 H); IR
(film) 3277, 2929, 1672, 1281, 1201, 1178, 1124, 1161, 796, 721,
Table 1
Hydrophobic parameters and solubility of synthetic probes (1 and 3–7)
No
CLogP
Rta (min)
Isocratic
Satd conc (mM)
1
3
4
5
6
7
4.89
5.08
1.14
0.32
3.56
3.56
7.73
5.70
2.20
1.78
2.35
2.47
0.211
0.268
1.057
55.739
0.053
0.082
696 cmꢀ1; ½a 2D7
ꢅ
ꢀ 6:4ꢆ (c 0.06, DMSO); HRMS (ESI, positive) m/z
a
Column: Eclipse plus C18 narrow bore RRHT 2.1 ꢂ 50 mm, flow rate: 0.15 mL/
[M+H]+ calcd for C57H67F6N8O11S, 1185.4554, found 1185.4564.
min, isocratic: 33% aq CH3CN.
To a solution of azide 13 (17.5 mg, 20.7
lmol) and alkyne unit
10 (16.9 mg, 27.4 mol) in DMSO/250 mM HEPES buffer (pH 7)
l
Ltd) at the flow rate of 4.0 mL/min. 1H and 13C NMR spectra were
recorded on ECA400 and Alpha 500 spectrometers (Jeol Ltd).
Chemical shifts are given in parts per million (ppm) relative to
Me4Si (0.0 ppm). Data for 1H NMR spectra are reported as follows:
chemical shift in ppm; integration, multiplicity, and coupling con-
stants in Hz. Multiplicity and qualifier abbreviations are as follows:
s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet,
m = multiplet, br = broad. IR spectra were recorded on an FT/IR-
410 spectrometer (Jasco Co., Ltd) and are reported as frequency
of absorption (cmꢀ1). LR- and HR-MS were recorded in ESI-mode
using MicrOTOF-II and APEX-III spectrometers (Bruker Daltonics
Ltd). The optical rotation was recorded on a DIP-360 spectrometer
using a 100-mm cell. UPLC–MS analysis was performed using an
Agilent 1290 Infinity LC (Agilent Ltd) equipped with a MicrOTOF-
II (Bruker Daltonics Ltd)
(4:1) were added CuSO4ꢄ5H2O–THPTA complex (5 mol %) and
ascorbic acid (50 mol %). After being shaken for 1 h at 30 °C, the
reaction mixture was cooled to rt and successively purified by
HPLC (30% MeCN aq containing 0.1% TFA) to give 4 (20.0 mg,
16.3 lmol, 79%).
1H NMR (500 MHz, CD3OD) 7.86–7.85 (m, 3 H), 7.79–7.77 (m, 2
H), 7.67–7.65 (m, 3 H), 7.56–7.53 (m, 2 H), 5.52–5.43 (m, 2 H),
4.66–4.63 (m, 2 H), 4.54–4.40 (m, 4 H), 4.29 (dd, J = 8.0, 4.5 Hz, 1
H), 4.02–3.99 (m, 1 H), 3.88 (br, 2 H), 3.79–3.72 (m, 3 H), 3.65–
3.60 (m, 22 H), 3.54–3.47 (m, 4 H), 3.22–3.18 (m, 2 H), 2.91 (dd,
J = 13.0, 5.0 Hz, 1 H), 2.68 (d, J = 13.0 Hz, 1 H), 2.65–2.62 (m, 1 H),
2.48–2.45 (m, 2 H), 2.41–2.25 (m, 9 H), 2.22–2.19 (m, 2 H), 2.08–
1.97 (m, 2 H), 1.76–1.30 (m, 6 H), 0.96–0.87 (m, 1 H); IR (film)
3278, 3076, 2873, 1659, 1551, 1450, 1279, 1196, 1049, 1023,
1003, 822, 798, 762, 706 cmꢀ1; ½a D28
ꢅ
ꢀ 32:1ꢆ (c 0.19, MeOH); HRMS
(ESI, positive) m/z [M+H]+ calcd for C60H87N8O17S, 1223.5910,
To a solution of azide 13 (1.0 mg, 1.32
lmol) and alkyne unit 9
(1.0 mg, 1.68 mol) in DMSO/250 mM HEPES buffer (pH 7) (4:1)
l
found 1223.5925.
were added CuSO4ꢄ5H2O–THPTA complex (5 mol %) and ascorbic
acid (50 mol %). After being shaken for 1 h at 30 °C, the reaction
mixture was cooled to rt and successively purified by HPLC (44%
To a solution of azide 13 (3.9 mg, 6.43
lmol) and alkyne unit 11
(3.0 mg, 6.44 mol) in DMSO/250 mM HEPES buffer (pH 7) (4:1)
l
were added CuSO4ꢄ5H2O–THPTA complex (5 mol %) and ascorbic
MeCN aq containing 0.1% TFA) to give 3 (1.1 mg, 0.928 lmol, 70%).
acid (50 mol %). After being shaken for 1 h at 30 °C, the reaction
Figure 5. Dihedral angles around the biaryl linkage in TAzP probe (1) and TAzQ models (14 and 15). Each angle was determined by DFT calculations (B3LYP/6-31G⁄).

S. Tamura et al. / Bioorg. Med. Chem. Lett. 23 (2013) 188–193
193
mixture was cooled to rt and successively purified by HPLC (34%
MeCN aq containing 0.1% TFA) to give 6 (2.4 mg, 2.24 mol, 35%).
and Grants-in-Aid for Scientific Research (No. 23310147), JSPS Bilat-
eral Programs from JSPS, Japan.
l
1H NMR (400 MHz, CD3OD) 9.24 (d, J = 2.0 Hz, 1 H), 8.84 (d,
J = 2.0 Hz, 1 H), 8.57 (s, 1 H), 8.52 (d, J = 2.0 Hz, 1 H), 8.35 (dd,
J = 8.8, 2.0 Hz, 1 H), 8.17 (d, J = 8.8 Hz, 1 H), 7.86–7.84 (m, 2 H),
7.79–7.77 (m, 2 H), 7.67–7.65 (m, 3 H), 7.56–7.52 (m, 2 H), 5.38–
5.27 (m, 2 H), 4.66 (d, J = 7.8 Hz, 1 H), 4.53 (d, J = 13.2 Hz, 1 H),
4.48 (d, J = 13.2 Hz, 1 H), 4.38 (dd, J = 7.8, 4.4 Hz, 1 H), 4.17–4.08
(m, 2 H), 3.99–3.98 (m, 1 H), 3.94 (dd, J = 11.2, 3.2 Hz, 1 H), 3.70–
3.63 (m, 1 H), 3.61–3.58 (m, 2 H), 3.51–3.47 (m, 4 H), 3.34–3.32
(m, 3 H), 3.04–3.00 (m, 1 H), 2.78 (dd, J = 12.8, 4.8 Hz, 1 H), 2.60
(d, J = 12.8 Hz, 1 H), 2.54–2.50 (m, 1 H), 2.39–2.08 (m, 9 H), 1.98–
1.82 (m, 2 H), 1.68–1.28 (m, 7 H); 13C NMR (100 MHz, CD3OD), d
221.5, 197.6, 176.7, 175.9, 168.0, 166.0, 150.0, 149.4, 147.3,
139.8, 138.4, 137.7, 136.9, 134.1, 131.6 (2C), 131.5 (2C), 131.3,
131.0 (2C), 130.6, 130.2, 129.6 (2C), 129.5, 129.3, 128.8, 128.4,
126.1, 124.8, 100.5, 75.2, 70.5, 69.7, 63.3, 61.6, 59.5, 56.8, 55.1,
53.6, 52.1, 51.3, 41.2, 41.0, 40.0, 39.7, 39.0, 38.7, 36.9, 29.7, 29.5,
28.9, 28.2, 26.9, 26.2; HRMS (ESI, positive) m/z [M+H]+ calcd for
Supplementary data
Supplementary data (synthesis of alkyne units and NMR charts
of probes) associated with this article can be found, in the online
version, at http://dx.doi.org/10.1016/j.bmcl.2012.10.124.
References and notes
1. Ueda, M. Chem. Lett. 2012, 41, 658.
2. Leslie, B. J.; Hergenrother, P. J. Chem. Soc. Rev. 2008, 37, 1347.
3. Bottcher, T.; Pitscheider, M.; Sieber, S. A. Angew. Chem., Int. Ed. Engl. 2010, 49,
2680.
4. Hashimoto, M.; Yang, J.; Holman, G. D. ChemBioChem 2001, 2, 52.
5. Sato, S.-I.; Kwon, Y.; Kamisuki, S.; Srivastava, N.; Mao, Q.; Kawazoe, Y.; Uesugi,
M. J. Am. Chem. Soc. 2007, 129, 873.
6. Shiyama, T.; Furuya, M.; Yamazaki, A.; Terada, T.; Tanaka, A. Bioorg. Med. Chem.
2004, 12, 2831.
7. Ueda, M.; Manabe, Y.; Mukai, M. Bioorg. Med. Chem. Lett. 2011, 21, 1359.
8. Sato, S.; Murata, A.; Shirakawa, T.; Uesugi, M. Chem. Biol. 2010, 17, 616.
9. Dean, P. M.; Cambridge University Press: Molecular foundations of drug-
receptor interaction, 1987.
10. Nakamura, Y.; Miyatake, R.; Ueda, M. Angew. Chem., Int. Ed. Engl. 2008, 47, 7289.
11. Abuchowski, A.; Mccoy, J. R.; Palczuk, N. C.; Vanes, T.; Davis, F. F. J. Biol. Chem.
1977, 252, 3582.
12. Harris, J. M. Poly (Ethylene Glycol) Chemistry Biotechnical and Biomedical
Applications; Springer, 1992.
13. Tamura, T.; Terada, T.; Tanaka, A. Bioconjug. Chem. 2003, 14, 1222.
14. Nakamura, Y.; Inomata, S.; Ebine, M.; Manabe, Y.; Iwakura, I.; Ueda, M. Org.
Biomol. Chem. 2011, 9, 83.
15. Nakamura, Y.; Mithöfer, A.; Kombrink, E.; Boland, W.; Hamamoto, S.; Uozumi,
N.; Tohma, K.; Ueda, M. Plant Physiol. 2011, 155, 1226.
16. Ueda, M.; Okazaki, M.; Ueda, K.; Yamamura, S. Tetrahedron 2000, 56, 8101.
17. Meldal, M.; Tornoe, C. W. Chem. Rev. 2008, 108, 2952.
18. Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057.
19. Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. Engl. 2004, 2001,
40.
20. Ueda, M.; Yang, G.; Ishimaru, Y.; Itabashi, T.; Tamura, S.; Kiyota, H.; Kuwahara,
S.; Inomata, S.; Shoji, M.; Sugai, T. Bioorg. Med. Chem. 2012, 20, 5832.
21. Hong, V.; Presolski, S. I.; Ma, C.; Finn, M. G. Angew. Chem., Int. Ed. Engl. 2009, 48,
9879.
22. Chan, T. R.; Hilgraf, R.; Sharpless, K. B.; Fokin, V. V. Org. Lett. 2004, 6, 2853.
23. Fujita, Y.; Yonehara, M.; Tetsuhashi, M.; Noguchi-Yachide, T.; Hashimoto, Y.;
Ishikawa, M. Bioorg. Med. Chem. 2010, 18, 1194.
24. Ueda, M.; Yamamura, S. Angew. Chem., Int. Ed. Engl. 2000, 39, 1400.
25. Ueda, M.; Nakamura, Y. Nat. Prod. Rep. 2006, 23, 548.
C
56H66N9O11S, 1072.4602, found 1072.4588.
To a solution of azide 13 (2.0 mg, 3.27 mol) and alkyne unit 12
(1.3 mg, 2.79 mol) in DMSO/250 mM HEPES buffer (pH 7) (4:1)
l
l
were added CuSO4ꢄ5H2O–THPTA complex (5 mol %) and ascorbic
acid (50 mol %). After being shaken for 1 h at 30 °C, the reaction
mixture was cooled to rt and successively purified by HPLC (34%
MeCN aq containing 0.1% TFA) to give 7 (1.4 mg, 1.31 lmol, 45%).
1H NMR (400 MHz, CD3OD) 9.28 (d, J = 2.0 Hz, 1 H), 8.80 (d,
J = 2.0 Hz, 1 H), 8.64 (s, 1 H), 8.52 (s, 1 H), 8.23–8.15 (m, 2 H),
7.86–7.84 (m, 2 H), 7.79–7.77 (m, 2 H), 7.67–7.65 (m, 3 H), 7.56–
7.52 (m, 2 H), 5.37–5.26 (m, 2 H), 4.67 (d, J = 8.2 Hz, 1 H), 4.53
(d, J = 13.2 Hz, 1 H), 4.48 (d, J = 13.2 Hz, 1 H), 4.38 (dd, J = 8.2,
4.8 Hz, 1 H), 4.15–4.12 (m, 2 H), 3.98–3.98 (m, 1 H), 3.95–3.91
(m, 1 H), 3.69–3.65 (m, 1 H), 3.61–3.58 (m, 2 H), 3.51–3.47 (m, 4
H), 3.34–3.32 (m, 3 H), 3.02–2.96 (m, 1 H), 2.79 (dd, J = 12.8,
5.2 Hz, 1 H), 2.61 (d, J = 12.8 Hz, 1 H), 2.55–2.50 (m, 1 H), 2.26–
2.20 (m, 9 H), 2.00–1.84 (m, 2 H), 1.64–1.28 (m, 6 H), 0.96–0.86
(m, 1 H); 13C NMR (100 MHz, CD3OD), d 221.8, 197.9, 176.7,
174.6, 168.0, 167.8, 150.0, 149.0, 147.4, 139.7, 139.1, 137.9,
135.2, 134.0, 131.5 (2C), 131.3, 131.0 (2C), 131.0 (2C), 130.6,
130.1, 129.6 (2C), 129.4, 129.2, 128.9, 128.6, 125.1, 123.9, 101.4,
75.1, 70.4, 69.7, 63.3, 61.6, 59.6, 56.9, 55.2, 53.5, 52.2, 51.3, 41.1,
41.0, 39.9, 39.7, 38.9, 38.7, 36.8, 29.7, 29.4, 28.9, 28.2, 26.8, 26.1;
HRMS (ESI, positive) m/z [M+H]+ calcd for C56H66N9O11S,
1072.4602, found 1072.4602.
26. Anderson, P. M.; Wilson, M. R. Mol. Phys. 2005, 103, 89.
27. Xue, Y.; O’Mara, M. L.; Surawski, P. P. T.; Trau, M.; Mark, A. E. Langmuir 2011,
27, 296.
28. Wahab, S. A.; Matsuura, H. PCCP 2001, 3, 4689.
29. Hansch, C.; Quinlan, J. E.; Lawrence, G. L. J. Org. Chem. 1968, 33, 347.
30. Valvani, S. C.; Yalkowsky, S. H.; Roseman, T. J. J. Pharm. Sci. 1981, 70, 502.
31. CL0ogP of each probe was estimated from OSIRIS property explorer (http://
www.chemexper.com/tools/propertyExplorer/main.html).
The aqueous solubility of each probe was determined in miliQ
at room temperature. MiliQ (20 lL) was added to an excess
32. Jain, N.; Yalkowsky, S. H. J. Pharm. Sci. 2001, 90, 234.
33. Ishikawa, M.; Hashimoto, Y. J. Med. Chem. 2011, 54, 1539.
34. GAUSSIAN 03 (Revision D.02): Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria,
G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin, K.
N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.;
Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.;
Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda,
Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.;
Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.;
Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.;
Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.;
Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.;
Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford,
S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.;
Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.;
Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez,
C.; Pople, J. A. GAUSSIAN, Inc., Wallingford CT, 2004.
amount of each probe to prepare a saturated solution. The suspen-
sion was kept in an ultrasonic bath for 15 min and vigorously sha-
ken by a vortex mixer for 10 min. Afterwards, the mixture was
allowed to stand for 30 min, and the suspension was then centri-
fuged (4000 rpm, 2 min). The resultant supernatant was trans-
ferred to a new tube and centrifuged (15,000 rpm, 3 min) again.
Finally, the concentration of the probe in this supernatant was ana-
lyzed by UPLC–MS.
Acknowledgments
This work was supported by a Grant-in-Aid for Scientific Research
on Innovative Areas ‘Chemical Biology of Natural Products’ from the
Ministry of Education, Culture, Sports, Science and Technology, Japan,