Modulation of intramolecular heterodimer-induced fluorescence quenching of tricarbocyanine dye for the development of fluorescent sensor

Article abstract of DOI:10.1039/c0ob00207k

Various approaches have been used to modulate the fluorescence changes of sensors in the presence of target analytes, including intramolecular interaction between fluorophores or between fluorophore and other molecular species, like resonance energy trans

Full text of DOI:10.1039/c0ob00207k

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Modulation of intramolecular heterodimer-induced fluorescence quenching of
tricarbocyanine dye for the development of fluorescent sensor†
Tomoya Hirano,*a Jun Akiyama,b Shuichi Morib and Hiroyuki Kagechika*a,b
Received 4th June 2010, Accepted 31st August 2010
DOI: 10.1039/c0ob00207k
Various approaches have been used to modulate the fluorescence changes of sensors in the presence of
target analytes, including intramolecular interaction between fluorophores or between fluorophore and
other molecular species, like resonance energy transfer (RET). Here, we focus on fluorescence
quenching by intramolecular heterodimer complex formation, which can be modulated over a shorter
distance range than RET. We synthesized several conjugates of tricarbocyanine, which is a
near-infrared fluorophore, with several quencher candidates via flexible short linker structure, and
examined their fluorescence properties. Of our synthesized compounds, the dabcyl group proved to be
the best quencher via heterodimer complex formation. The fluorescence of tricarbocyanine–dabcyl
conjugates in aqueous media was almost completely quenched, and there was a dramatic fluorescence
enhancement when heterodimer formation was blocked. These results suggested a design approach to
develop fluorescence sensors for probing proximity relationships and structural transitions.
fluorescence of the carbocyanine dye was quenched by homo-
Introduction
aggregation in the polymer, but on cleavage of the peptide chain
Fluorescent sensors, whose fluorescence properties change upon
by tumor-associated peptidase, carbocyanine is released and its
binding or reacting with an analyte, have been widely used in
fluorescence is recovered. In the case of heterodimer formation,
various fields, including cell biology and clinical diagnosis.1,2
several interactions, such as static quenching by the ground state
Various empirical or theoretical approaches have been used to
complex and dynamic interactions like RET and PeT, can occur
obtain a sufficient fluorescence change in the presence of the
simultaneously. In the case of molecular beacons, which can
analyte. For example, resonance energy transfer (RET) between
sense specific sequences of nucleobases, two fluorophores or a
two fluorophores or between a fluorophore and another molecular
fluorophore and a quencher are placed close together, where
RET and/or static quenching can operate.16–20 Upon binding to
analyte oligonucleotides, the two chromophores are separated,
resulting in an increase of fluorescence intensity. In recently
reported fluorescent sensors for tyrosine kinase, pyrene and the
hydroxyphenyl group of substrate tyrosine form a heterodimer,
resulting in fluorescence quenching via both static and dynamic
quenching.21 Phosphate ester formation by the analyte kinase
hinders the formation of this heterodimer, resulting in recovery
of fluorescence.
Generally, the efficiency of RET depends on the distance
between donor and acceptor, which is typically in the range of
2–10 nm.3,4 Compared with RET, the change of fluorescence prop-
erties by the formation of homodimer or heterodimer complex
requires contact formation, like van der Waals contact, which
means that it can be modulated over a shorter distance range than
RET, and those characteristic are useful for sensing the proximity
relationship.22–24 For example, Sauer’s group reported that oxadine
derivative MR121, which was used as a fluorophore, could form
a non-fluorescent complex with amino acid tryptophan, and this
pair could be used to study conformational dynamics of some
proteins in the sub-nanometre range.25–28 In this paper, we focused
on tricarbocyanine dye, which has near-infrared fluorescence and
is potentially attractive for in vivo fluorescence imaging,29 as a
fluorophore, and examined the structural requirements for in-
tramolecular heterodimer formation and fluorescence quenching.
Although some compounds such as NIRQ750, BHQ-3 and Cy7Q,
which can quench the fluorescence of tricarbocyanine dye mainly
via RET,30–32 have already been reported, we tried to find the
species has been utilized to develop fluorescent sensors for
enzymatic activity or for sensing structural changes of proteins.3,4
Its efficiency depends mainly on the distance between the donor
and acceptor moieties, and when this is changed by interaction
of the sensor with the analyte, there is a change of fluorescence
intensity and wavelength. Photo-induced electron transfer (PeT)
has also been utilized in sensors. A change of the distance between
PeT donor and acceptor, or the change of the oxidation or
reduction potential of these substructures in the sensor modulates
the extent of PeT, resulting in a change of fluorescence intensity.5,6
Based on this mechanism, fluorescent sensors for proton,7,8 metal
cations,9–11 and nitric oxide12 have been developed.
The control of intermolecular or intramolecular complex
formation between two fluorophores or between a fluorophore
and another molecular species has also been utilized for the
development of fluorescent sensors.13,14 For example, a carbocya-
nine dye-conjugated graft copolymer consisting of poly-L-lysine
and methoxypolyethylene glycol succinate was reported to be
useful as a fluorescent sensor for some tumors in vivo.15 The
aInstitutes of Biomaterials and Bioengineering, Tokyo Medical and Dental
University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan.
E-mail: hiraomc@tmd.ac.jp, kage.omc@tmd.ac.jp; Fax: +81-3-5280-8127;
Tel: +81-3-5280-8128
bGraduate School of Biomedical Sciences, Tokyo Medical and Dental
University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan
† Electronic supplementary information (ESI) available: Synthesis and
characterization data for compounds 1a–1d, 2a–2c and their intermediates.
See DOI: 10.1039/c0ob00207k
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best quencher compound, whose absorbance has no significant
spectral overlap with tricarbocyanine fluorescence. It is expected
to quench the fluorescence of tricarbocyanine by heterodimer
complex formation not by RET. We synthesized some conjugates
of tricarbocyanine with several quencher candidates via various
flexible short linker structures, and examined their fluorescence
properties under various conditions. We anticipated that the
fluorescence of compounds with appropriate quencher and linker
structures would be quenched by intramolecular heterodimer
formation via hydrophobic interaction in aqueous media, while
disruption of this dimer formation, e.g., by solvent polarity change
or protein binding, would restore the fluorescence (Fig. 1). We
considered that this might be an effective design strategy for
fluorescent sensors.
Fig. 1 Fluorescence quenching of intramolecular heterodimer formation
and recovery of fluorescence in response to environmental change.
Results and discussion
Fig. 2 Structures of quencher–linker–fluorophore (tricarbocyanine) con-
Design and synthesis
jugated compounds.
Based on the strategy shown in Fig. 1, we designed tricarbocyanine
derivatives that were expected not to show fluorescence until their
structure was unfolded owing to disruption of intramolecular
hydrophobic interaction. The designed compounds consisted of
tricarbocyanine, a hydrophobic aromatic structure as the quencher
candidate, and a linking group between them. As quencher candi-
dates, benzene (1a), naphthalene (1b), biphenyl (1c), diphenyl ether
(1d) and 4-(4-dimethylaminophenylazo)benzoic acid (dabcyl, 1e),
whose absorbance spectra have no significant spectral overlap with
tricarbocyanine fluorescence, were selected (Fig. 2). Further, we
examined the effect of linker structure by modifying compound
1e to have a hydrophobic, longer, or shorter linking group, i.e.,
compounds 2a, 2b, and 2c, respectively.
Synthesis of compounds 1a–1e is illustrated in Scheme 1.
Condensation of monoprotected bis(2-aminoethoxy)ethane 4 with
p-hydroxyphenylpropionic acid 3 afforded compound 5. Tricar-
bocyanine 633 was reacted with 5 to obtain linker–fluorophore
compound 7. After deprotection under acidic conditions, the
amine 8 was reacted with succinimidyl esters of quencher can-
didates (9a–9e), yielding compounds 1a–1e, respectively (Scheme
1). Compounds 2a–2c were synthesized similarly to 1e by using
10a, 10b, or 11c as the starting material, respectively (Scheme 2).
solution, and their quantum yields were 0.19, 0.15, 0.13 and 0.13
respectively (Table 1, Fig. 3(a)). Compound 1e showed strong
fluorescence quenching in aqueous solution, and its quantum yield
was less than 0.01 (Table 1, Fig. 3(a)). In the absorption spectra
(Fig. 3(b)), compounds 1a–1d showed almost no significant shift in
the absorption maximum from that of the control compound 7 and
decreased absorption around 770 nm was observed, except for 1a.
However, in the case of compound 1e, its absorption maximum
(663 nm) was blue-shifted from that of compound 7 (766 nm).
Absorption maximum shift was also observed around the region
of 350 nm–550 nm, where the dabcyl group, the quencher moiety
of 1e, has absorption. 1e showed blue shift, compared with
the control dabcyl derivative, dabcyl N-(2-hydroxyethyl)amide
(Dabcyl) (Fig. 3(c)). Those spectral shapes did not change in the
concentration range from 0.2 mM to 4 mM (data not shown).
This spectral change indicates the presence of intramolecular
heterodimeric folding structures involving tricarbocyanine and the
dabcyl group. In methanol solution, the partially or completed
quenched fluorescence was almost completely restored, becoming
similar to that of 7 (Fig. 4(a), Table 1). The absorption spectra
also became similar (Fig. 4(b)) even in the case of the largely blue-
shifted spectra of 1e, which also gave a similar absorption spectrum
as the control dabcyl derivative, “Dabcyl” (Fig. 4(c)). Our results
indicate that the dabcyl group is the best quencher moiety of our
quencher candidates for linking to tricarbocyanine dye, affording
the largest enhancement of fluorescence in methanol compared
with aqueous solution.
Fluorescence properties in aqueous solution and methanol
Fluorescence and absorption spectra of compounds 1a–1e in
aqueous solution (10 mM sodium phosphate buffer, pH 7.4) are
shown in Fig. 3. Compared with control compound 7 without
a quencher moiety, whose quantum yield (U) was estimated to
be 0.22, 1a–1d showed partial fluorescence quenching in aqueous
Among the compounds with various linker structures, com-
pound 2b (absorption maximum at 776 nm) with a longer linker
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Scheme 1 Synthesis of compounds 1a–1e
Scheme 2 Synthesis of compounds 2a–2c
group showed an absorption spectrum slightly red-shifted from
that of 7 (absorption maximum at 766 nm) in aqueous solution.
Compound 2a with a hydrophobic linker and 2c with a shorter
linker group showed broad spectra, with maxima at 710 nm
and 693 nm, respectively, so those spectra seemed intermediates
between those of 2b and 1c (Fig. 5(a), Table 1). Every compound
showed sufficient fluorescence quenching in aqueous media, with
quantum yields of 0.02 for 2b and less than 0.01 for 2a and 2c,
compared with control compound 12c without a quencher moiety,
whose quantum yield is 0.17 (Table 1, Fig. 5(b)). This quenched
fluorescence could be restored by increasing the percentage of
methanol in the solvent (aqueous methanol). Compound 2c with
a shorter linking group showed almost similar values to 12c in
50% aqueous methanol solution, whereas 1e, 2a and 2b showed
56%, 74%, and 61% as a percentage of the value of 12c (taken
as 100%) under same condition (Fig. 5(b)). In methanol solution,
every compound showed a similar fluorescence spectrum to the
control compound 12c.
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Table 1 Fluorescent properties of the synthesized compounds
Absorption
max/nm
Emission
max/nm
Compound
Solventa
Quantum yieldc
1a
1b
1c
1d
1e
7
Water
Methanol
Water
Methanol
Water
Methanol
Water
Methanol
Water
Methanol
Water
Methanol
Water
Methanol
Water
768
767
769
767
770
768
768
767
663
768
766
767
710
767
776
769
693
767
783
783
784
782
784
782
784
782
n.d.b
783
783
782
n.d.b
782
784
781
n.d.b
782
0.19
0.44
0.15
0.44
0.13
0.44
0.13
0.45
<0.01
0.41
0.22
0.46
<0.01
0.44
0.02
0.42
<0.01
0.45
2a
2b
2c
Methanol
Water
Methanol
a All data were measured in 10 mM sodium phosphate buffer (pH
7.4) or methanol. b Not determined. c Quantum yields of fluorescence
were determined using that of indocyanine green (0.13) in DMSO as a
standard.34
Fluorescence change in the presence of cyclodextrins or various
proteins
Cyclodextrins (CDs) are torus-shaped cyclic oligosaccharides
composed of D-glucopyranose units, and can include a variety
of organic compounds in their cavities.35 Fluorescence quenching
by heterodimer formation, e.g., in the case of the coumarin–
fluorescein pair, is reported to be hindered by the inclusion of
substructure within cyclodextrin.36 Therefore, the fluorescence
intensity of our compounds was examined in the presence of
cyclodextrins of various sizes. Whereas the fluorescence intensity
of 7 without a quencher part almost did not change by the addition
of cyclodextrins, the quenched fluorescence of compound 1e in
aqueous solution was restored by the addition of 10 mM g-
cyclodextrin (g-CD) (Fig. 6(a)). The recovery of the fluorescence
depended on the cavity size of CD, and the addition of b-
CD or a-CD with a smaller cavity gave less recovery. In the
case of compounds 1b–1d, whose fluorescence was partially
quenched in aqueous solution, similar recovery was observed by
the addition of g-CD. b-CD also induced a relatively large recovery
of fluorescence for compounds 1c and 1d, compared with 1e. Thus,
the size of the quencher part is significant for the efficiency of
fluorescence recovery by the addition of b-CD. The absorption
spectra of compound 1e at various concentrations of g-CD showed
concentration-dependent change (Fig. 6(b)). As the concentration
of g-CD increased (0–10 mM), the absorption at 663 nm was
decreased, while that at 772 nm was increased. The absorption
of dabcyl moiety around 350 nm ~ 550 nm was also changed.
The absorption at 407 nm was decreased and that around 440 nm
was increased, and the spectral shape became similar to that of
control compound, dabcyl N-(2-hydroxyethyl)amide (Fig. 6(c)).
These data suggested that the fluorescence recovery in the presence
of cyclodextrins is presumably a consequence of the inclusion of
the substructure of each compound, probably the quencher, into
the cyclodextrins. Thus, the hindrance to heterodimer formation
in aqueous media could recover the quenched fluorescence as
Fig. 3 Absorption and fluorescence spectra of compounds 1a–1e and 7
(2 mM) in 10 mM sodium phosphate buffer (pH 7.4). (a) Fluorescence
spectra (excitation at 745 nm), (b) and (c) absorption spectra are shown.
In (c), the absorption spectrum of the control dabcyl compound (dabcyl
N-(2-hydroxyethyl)amide, designated as “Dabcyl”) is also shown. Each
solution contained 0.2% DMSO as a cosolvent.
Since the spectral overlap between the absorption of the dabcyl
group and the fluorescence of the tricarbocyanine group was
estimated to be almost zero, the fluorescence quenching would not
arise from RET. Among possible mechanisms, contact-mediated
quenching was thought to be most likely. From absorption spectra,
1e showed blue-shifted spectra, 2a showed spectra a little red-
shifted, and 2b and 2c showed those intermediate. Such differences
in absorption spectra would be derived from a difference in
the quenching mechanism. One is static quenching via ground
state complex formation between tricarbocyanine and dabcyl
group, and similar spectral changes have been reported for
heterodimer pairs such as Cy5-BHQ1.18–20 Another one is dynamic
quenching, like photo-induced electron transfer (PeT). The length
and constituents of the linker structure were thought to cause these
differences. For elucidation of such mechanisms, time-resolved
fluorescence measurement, fluorescence correlation spectroscopy
and molecular dynamics simulation would be necessary, as for
the previously reported case of oxadine dyes MR121, MR113 or
Rhodamine 6G in formations with amino acid tryptophan, and
such work is in progress.
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Fig. 5 (a) Absorption spectra of 1e, 2a, 2b, 2c and 12c in 10 mM sodium
phosphate buffer (pH 7.4). (b) Changes of fluorescence intensity (excitation
at 745 nm, emission at 790 nm) of 1e, 2a, 2b, 2c and 12c in various
percentages of methanol in 10 mM sodium phosphate buffer (pH 7.4). All
spectra were measured at a concentration of 2 mM in the presence of 0.2%
DMSO as a cosolvent.
based on the amino acid composition39 is BSA > Oval > Lyso
> RNase), and compound 1e might be more sensitive than ANS.
In addition, specific interaction of substructure of 1e also might
cause such difference, so the optimization of the substructure or
conjugation with other molecular species, such as ligand molecules
for receptor proteins, should enable us to develop fluorescent
sensors with selectivity for particular proteins.
Fig. 4 Absorption and fluorescence spectra of compounds 1a–1e and 7
(2 mM) in methanol. (a) Fluorescence spectra (excitation at 745 nm), (b) and
(c) absorption spectra are shown. In (c), the absorption spectrum of the
control dabcyl compound (dabcyl N-(2-hydroxyethyl)amide, designated
as “Dabcyl”) is also shown. Each solution contained 0.2% DMSO as a
cosolvent.
From the fluorescence intensity of compound 7, which does
not have dabcyl group, with various concentrations of BSA (Fig.
7(c)), the fluorescence of tricarbocyanine was also increased by
the binding with a relatively high concentration of BSA. So
for the fluorescence enhancement of 1e by binding with BSA,
various mechanisms including the hindrance of intramolecular
heterodimer formation and the change of the environment around
tricarbocyanine, both of which could induce the fluorescence
enhancement, were thought to simultaneously operate, whereas by
introduction of dabcyl group, the detection limit of 1e for sensing
BSA was greatly improved.
well as change of solvent polarity as shown in Fig. 3–5. Even
in the extended form of these compounds, the distance between
fluorophore and quencher could be relatively short, and therefore
this pair could be utilized for sensing proximity relationships.
In order to explore the suitability of our compounds for biologi-
cal applications, the fluorescence change of 1e was measured in the
presence of various proteins such as bovine serum albumin (BSA),
Ovalbumin (Oval), Chicken egg lysozyme (Lyso) or Ribonuclease
A (RNase), and these results are shown in Fig. 7 along with that
of 1-(anilino)naphthalene-8-sulfonate (ANS), that is reported as
a fluorescent sensor of protein surface hydrophobicity.37,38 The
fluorescence of 1e increased in the presence of BSA or high
concentration of Oval, and showed no significant enhancement by
Lyso or RNase. In the case of ANS, BSA also induced fluorescence
enhancement, whereas Oval, Lyso or RNase induced little increase
of fluorescence, which is coincident with the reported fluorescence
quantum yields of ANS in binding to those proteins, 0.12 for BSA,
0.008 for Oval, 0.008 for Lyso and 0.005 for RNase, respectively.37
The difference between the fluorescence response of 1e and ANS
would be partly derived from the sensitivity to the protein surface
hydrophobicity, (the calculated average hydrophobicity values
Conclusions
Our data indicate that molecules consisting of tricarbocyanine
dye and dabcyl group linked via short and flexible linker
form intramolecular heterodimers, resulting in almost complete
quenching of fluorescence in aqueous media. The fluorescence is
restored by disruption of the heterodimer under various condi-
tions, e.g., change of solvent polarity, inclusion of substructure
within cyclodextrin or protein binding. Even in the extended form
of our compounds, the distance between tricarbocyanine and the
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Fig. 6 (a) Fluorescence spectra (excitation at 745 nm) of 7 and 1b–1e on
the addition of 10 mM of cyclodextrin (a-CD (alpha), b-CD (beta) or g-CD
(gamma)). (b) Absorption spectra of 1e at various concentrations of g-CD.
The absorption spectrum of 7 is also shown. (c) Absorption spectrum of
1e and the control dabcyl compound (dabcyl N-(2-hydroxyethyl)amide,
designated as “Dabcyl”) on the addition of 10 mM g-CD. All spectra were
measured at 2 mM of sensor in the presence of 0.2% DMSO as a cosolvent
in 10 mM sodium phosphate buffer (pH 7.4).
Fig. 7 Fluorescence intensity of (a) 1e (0.5 mM, excitation at 745 nm,
emission at 790 nm) or (b) ANS (0.5 mM, excitation at 370 nm, emission at
460 nm) in 10 mM sodium phosphate buffer (pH 7.4) in the presence
of 1–100 mM of Bovine serum albumin (BSA), Ovalbumin (Oval),
Chicken egg lysozyme (Lyso) or Ribonuclease A (RNase). (c) Fluorescence
intensities of 1e and 7 (0.5 mM, excitation at 745 nm, emission at 790 nm)
with various concentrations of BSA.
dabcyl group could be short enough for RET to generally occur.
However, such interactions would not occur efficiently for the
tricarbocyanine–dabcyl pair, partly because there is no significant
spectral overlap between tricarbocyanine fluorescence and dabcyl
absorption. This characteristic should make these molecules useful
for sensing changes of distance in the nm range, as well as recently
reported “short-distance” RET pairs.40,41 These insights should
be useful in developing new fluorescent sensors as tools to probe
relative proximity relationships and structural transitions.
Materials and Reagents
All reagents were purchased from Sigma-Aldrich Chemical Co.,
Tokyo Kasei Kogyo Co, Wako Pure Chemical Industries, and
Kanto Kagaku Co., Inc. Silica gel for column chromatography was
purchased from Kanto Kagaku Co., Inc. 1H and 13C NMR spectra
were recorded on a Bruker Advance 500 spectrometer. Mass
spectral data was obtained on Bruker Daltonics microTOF-2focus
in the positive and negative ion detection modes. Melting points
were taken on a Yanagimoto micro melting point apparatus and
are uncorrected. Elemental analyses were carried out by Yanaco
MT-6 CHN CORDER spectrometer. UV spectra were recorded
with JASCO V-550, and fluorescence spectra were recorded with
JASCO FP-6600.
Experimental
See the ESI† for the synthesis and characterization data for
compounds 1a–1d, 2a–2c and their intermediates.
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Preparation of 5
d, J = 14.2 Hz), 4.06 (4 H, t, J = 7.4 Hz), 3.67 (2 H, t, J = 5.0 Hz),
3.64–3.58 (4 H, m), 3.48 (2 H, t, J = 5.8 Hz), 3.34 (2 H, m), 3.01
(2 H, t, J = 5.0 Hz), 2.84 (2 H, t, J = 7.9 Hz), 2.72 (4 H, t, J = 6.0
Hz), 2.43 (2 H, t, J = 7.9 Hz), 2.03 (2 H, quintet, J = 6.0 Hz), 1.82
(4 H, sextet, J = 7.4 Hz), 1.33 (12 H, s) 1.00 (6 H, t, J = 7.4 Hz).
3-(p-Hydroxyphenyl)propionic acid (3, 0.12 g, 1.0 mmol), triethy-
lamine (0.21 ml, 1.5 mmol), 1-hydroxybenzotriazole monohydrate
(0.18 g, 1.2 mmol) and 1-ethyl-3-(3-dimethylaminopropyl) car-
bodiimide hydrochloride (0.23 g, 1.2 mmol) were added to a
solution of 4 (0.30 g, 1.2 mmol) in DMF (10 ml). After 15 h,
the reaction mixture was evaporated. The residue was diluted
with dichloromethane, and the organic layer was washed with
saturated NaHCO3 and brine, dried over Na2SO4 and evaporated.
The crude was purified by silica gel column chromatography (5%
MeOH–CH2Cl2) and 0.22 g of 5 (0.58 mmol, 57%) was obtained
Preparation of 1e
Compound 9e (8.8 mg, 24 mmol) was added to a solution of 8
(20 mg, 19 mmol) in DMF/pyridine (3 : 1, 4 ml), and the mixture
was stirred overnight. The reaction mixture was evaporated,
and the residue was diluted with dichloromethane. The organic
layer was washed with water and brine, dried over Na2SO4
and evaporated. The crude was purified by silica gel column
chromatography (10% MeOH–CH2Cl2) and 12 mg of 1e (16 mmol,
84%) was obtained as a green powder. 1H NMR (500 MHz, CDCl3)
d 8.16 (1 H, br s), 8.10 (2 H, d, J = 8.5 Hz), 7.91 (2 H, d, J = 14.2
Hz), 8.09 (1 H, br s), 7.88 (2 H, d, J = 9.2 Hz), 7.84 (2 H, d, J = 8.5
Hz), 7.32–7.29 (6 H, m), 7.17 (2 H, dd, J = 7.6, 7.2 Hz), 7.00 (2 H,
d, J = 7.9 Hz), 6.90 (2 H, d, J = 8.6 Hz), 6.74 (2 H, d, J = 9.2 Hz),
5.90 (2 H, d, J = 14.2 Hz), 3.91 (4 H, t, J = 7.3 Hz), 3.71 (2 H, t, J
= 5.3 Hz), 3.64–3.55 (8 H, m), 3.46 (2 H, m), 3.09 (6 H, s), 2.92 (2
H, t, J = 7.8 Hz), 2.63 (4 H, t, J = 5.8 Hz), 2.58 (2 H, t, J = 7.8 Hz),
2.00 (2 H, quintet, J = 5.8 Hz), 1.82 (4 H, sextet, J = 7.4 Hz), 1.30
(12 H, s), 1.01 (6 H, t, J = 7.4 Hz); 13C NMR (125 MHz, CDCl3) d
173.1, 172.3, 167.1, 165.1, 158.2, 154.6, 152.6, 143.8, 142.5, 142.0,
141.0, 136.3, 134.8, 130.5, 128.5, 128.5, 125.2, 122.5, 122.0, 121.9,
114.3, 111.5, 110.3, 99.4, 70.3, 70.2, 69.9, 69.8, 49.2, 45.8, 40.3,
39.9, 39.2, 38.3, 31.1, 27.9, 24.2, 21.1, 20.7, 11.6; HRMS (ESI+)
Calcd for C66H80N7O5 (M - I-): 1050.6215; Found: 1050.6229.
1
as colorless liquid. H NMR (500 MHz, CDCl3) d 7.03 (2 H, d,
J = 8.3 Hz), 6.75 (2 H, d, J = 8.3 Hz), 6.59 (1 H, br s), 5.89 (1 H,
br s), 5.00 (1 H, br s), 3.60–3.49 (6 H, m), 3.45 (2 H, t, J = 4.6
Hz), 3.41 (2 H, m), 3.30 (2 H, m), 2.87 (2 H, t, J = 7.2 Hz), 2.43
(2 H, t, J = 7.2 Hz), 1.45 (9 H, s); 13C NMR (125 MHz, CDCl3)
d 172.8, 156.2, 154.86, 131.9, 129.3, 115.4, 79.6, 70.3, 70.1, 70.0,
69.9, 69.9, 40.2, 39.2, 38.6, 30.9, 28.4; HRMS (ESI+) Calcd for
C20H32N2NaO6 (M + Na+): 419.2153; Found: 419.2147.
Preparation of 7
Sodium hydride (2.7 mg, 0.11 mmol) was added to a solution of
5 (44 mg, 0.11 mmol) in anhydrous DMF (5 ml) at 0 ◦C under an
argon atmosphere. After 30 min, 6 (67 mg 0.10 mmol) was added to
the reaction mixture, and the whole was stirred overnight at 50 ◦C.
The mixture was quenched with saturated aqueous ammonium
chloride, and the solvent was evaporated. The residue was diluted
with dichloromethane, and the organic layer was washed with
water and brine, dried over Na2SO4, filtered and evaporated.
The crude was purified by silica gel column chromatography (3%
MeOH–CH2Cl2 then 8% MeOH–CH2Cl2) and 49 mg of 7 (48
Acknowledgements
1
mmol, 47%) was obtained as a green solid. H NMR (500 MHz,
The work described in this paper was partially supported by
Grants-in-Aid for Scientific Research from The Ministry of Edu-
cation, Science, Sports and Culture, Japan (Grant Nos 19201044
to HK, 19790090 and 22790106 to TH). TH also thanks the
Mochida Memorial Foundation and Astellas Foundation for
financial support.
CDCl3) d 7.91 (2 H, d, J = 14.1 Hz), 7.85 (1 H, br s), 7.31 (2 H,
dd, J = 7.9, 7.5 Hz), 7.29 (2 H, d, J = 7.4 Hz), 7.24 (2 H, d, J = 8.6
Hz), 7.17 (2 H, dd, J = 7.5, 7.4 Hz), 7.04 (2 H, d, J = 7.9 Hz), 6.91
(2 H, d, J = 8.6 Hz), 5.97 (2 H, d, J = 14.1 Hz), 5.14 (1 H, br s),
3.97 (4 H, t, J = 7.3 Hz), 3.56 (6 H, m), 3.50 (2 H, t, J = 5.2 Hz),
3.39 (2 H, m), 3.26 (2 H, m), 2.90 (2 H, t, J = 8.0 Hz), 2.67 (4 H,
t, J = 5.8 Hz), 2.55 (2 H, t, J = 8.0 Hz), 2.02 (2 H, quintet, J = 5.8
Hz), 1.83 (4 H, sextet, J = 7.4 Hz), 1.40 (9 H, s), 1.31 (12 H, s),
1.02 (6 H, t, J = 7.4 Hz); 13C NMR (125 MHz, CDCl3) d 172.8,
172.2, 164.9, 158.1, 156.1, 142.4, 142.1, 140.9, 136.0, 130.3, 128.5,
125.1, 122.3, 121.9, 114.2, 110.3, 99.5, 78.9, 70.3, 70.2, 70.0, 69.6,
49.0, 45.8, 40.3, 38.9, 38.1, 30.9, 28.4, 27.8, 24.2, 21.0, 20.7, 11.6;
HRMS (ESI+) Calcd for C56H75N4O6 (M - I-): 899.5681; Found:
899.5658.
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Preparation of 8
A solution of 7 (20 mg, 0.020 mmol) in trifluoroacetic acid (1 ml)
was stirred at room temperature for 1 h, and was evaporated. The
residue was purified by silica gel column chromatography (eluent:
10% MeOH–CH2Cl2 then 20% MeOH–CH2Cl2) and 22 mg of
8 was obtained as green solid, which was used in next reaction
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