Tricolor emission of a fluorescent heteroditopic ligand over a concentration gradient of zinc(II) ions

Article abstract of DOI:10.1021/jo3016659

The internal charge transfer (ICT) type fluoroionophore arylvinyl-bipy (bipy = 2,2′-bipyridyl) is covalently tethered to the spirolactam form of rhodamine to afford fluorescent heteroditopic ligand 4. Compound 4 can be excited in the visible region, the e

Full text of DOI:10.1021/jo3016659

Article
pubs.acs.org/joc
Tricolor Emission of a Fluorescent Heteroditopic Ligand over a
Concentration Gradient of Zinc(II) Ions
Kesavapillai Sreenath, Ronald J. Clark, and Lei Zhu*
Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306-4390, United States
S
* Supporting Information
ABSTRACT: The internal charge transfer (ICT) type fluoroionophore
arylvinyl-bipy (bipy = 2,2′-bipyridyl) is covalently tethered to the
spirolactam form of rhodamine to afford fluorescent heteroditopic
ligand 4. Compound 4 can be excited in the visible region, the emission
of which undergoes sequential bathochromic shifts over an increasing
concentration gradient of Zn(ClO4)2 in acetonitrile. Coordination of
Zn2+ stabilizes the ICT excited state of the arylvinyl-bipy component of
4, leading to the first emission color shift from blue to green. At
sufficiently high concentrations of Zn(ClO4)2, the nonfluorescent
spirolactam component of 4 is transformed to the fluorescent
rhodamine, which turns the emission color from green to orange via
intramolecular fluorescence resonance energy transfer (FRET) from the
Zn2+-bound arylvinyl-bipy fluorophore to rhodamine. While this work
offers a new design of ratiometric chemosensors, in which sequential analyte-induced emission band shifts result in the sampling
of multiple colors at different concentration ranges (i.e., from blue to green to orange as [Zn2+] increases in the current case), it
also reveals the nuances of rhodamine spirolactam chemistry that have not been sufficiently addressed in the published literature.
These issues include the ability of rhodamine spirolactam as a fluorescence quencher via electron transfer, and the slow kinetics
of spirolactam ring-opening effected by Zn2+ coordination under pH neutral aqueous conditions.
acetonitrile. A limitation of the selected rhodamine spirolactam
ring-opening chemistry in the new design is also described, which
at this point prevents the reported prototype structure from
being applied as an indicator in aqueous-based systems.
INTRODUCTION
■
Developing selective and sensitive detection tools for Zn2+ in
biological specimens has attracted much interest during the past
decade.1−11 The motivation for this line of research is the need to
characterize the roles of Zn2+ in disease-related processes.12−14
Our group has focused on developing indicators for Zn2+ with a
concentration coverage appropriate for the uniquely broad range
of “free” Zn2+ ions15 in mammalian cellular systems.16,17 Most
known indicators for Zn2+ report concentration variations within
3 orders of magnitude under physiological conditions. This
rather limited range originates from the species distribution
makeup of a monotopic (one binding site) indicator, and it is
inadequate to cover the entire physiological range of Zn2+
observed in mammalian cells.
Our strategy to achieve broad concentration coverage entails
the development of a fluorescent heteroditopic ligand, which
contains two Zn2+ binding sites of different affinities.18−20 As
shown in Figure 1, a fluorescent heteroditopic ligand binds two
Zn2+ ions in a sequential manner over a concentration gradient. If
the three coordination states of the ligand (free, mono-, and
dicoordinated) have distinct fluorescence spectra, the emission
intensity and/or wavelength of the ligand upon single-wave-
length excitation is a function of [Zn2+] over the collective
response range of two coordination sites.16 In this paper, a new
design of fluorescent heteroditopic ligands is reported, which
shows a continuous emission color change from blue to orange
over a concentration gradient of Zn2+ in the organic solvent
RESULTS AND DISCUSSION
■
Design. To design a tricolor fluorescent heteroditopic ligand,
we employ a bichromophoric system where the emissions of
both fluorophores are susceptible to the presence of Zn2+, but in
different concentration regimes. To achieve the sequential
emission band shifts over an increasing [Zn2+] gradient depicted
in Figure 1 under excitation in the visible region, the arylvinyl-
bipy-containing compound 121,22 (Scheme 1A) is tethered with
rhodamine spirolactam 2 (Scheme 1B) to afford compound 4
(Scheme 1). The emission color of compound 1, which
undergoes excited state internal charge transfer (ICT),23,24
changes from blue to green upon Zn2+ coordination (Scheme
1A).21 The nonfluorescent 2 was designed by the addition of a
1,2,3-triazolyl ring on the spirolactam ligand developed by
Shiraishi et al.25 The triazolyl ligand acts as both an additional
metal coordination ligand for enhancing the coordination affinity
and a linker for tethering other functional groups (as in
compound 4).26−29 The spirolactam precursor of rhodamine
has been incorporated in fluorescent sensor structures targeting
Received: August 4, 2012
Published: August 27, 2012
© 2012 American Chemical Society
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Figure 1. Cartoons depicting tricolor emission upon single-wavelength excitation resulting from sequential binding of two Zn2+ ions to a fluorescent
heteroditopic ligand.
a
Scheme 1. Zn2+-Dependent Fluorescence Changes of 1 and 2, and the Structure of Ditopic Ligand 4
a
(A) From blue to green; (B) from non-fluorescent to orange. Oct = n-octyl. L: counter ion or solvent.
various substances,30−33 including Zn2+.25,34−38 The analyte-
induced spirolactam ring-opening reaction affords rhodamine of
a bright orange emission.
Compound 1 is susceptible to Zn2+-coordination at a lower
concentration than that applied to compound 2. Therefore, in
compound 4, we expect that the increase of [Zn2+] will first lead
to a blue to green emission color change as a result of binding at
the bipy position. As [Zn2+] grows further, the spirolactam ring
opens to afford the orange-emitting rhodamine. The emission
spectrum of Zn2+-bound arylvinyl-bipy component in 4 overlaps
well with the absorption spectrum of rhodamine (Figure 2),
which shall lead to an efficient intramolecular FRET from Zn2+-
bound arylvinyl-bipy to rhodamine, and consequently a color
change from green to orange.20,38,39 Both ICT and FRET
processes result in emission band shifts. Therefore, this design
shall afford a ratiometric indicator for Zn2+ over two
Figure 2. Normalized emission spectra of 1 (blue), [Zn(1)](ClO4)2
concentration regimes (low and high) under the excitation of
arylvinyl-bipy fluorophore.
(green), absorption (purple), and emission (orange) spectra of
[Zn(2)](ClO4)2. The shaded area represents the spectral overlap
between the emission of [Zn(1)](ClO4)2 and absorption of [Zn(2)]-
(ClO4)2.
Synthesis. The syntheses of monotopic rhodamine spiro-
lactam ligands 2 and 3 are depicted in Scheme 2. Briefly, 2-
amino-6-ethynylpyridine reacts with rhodamine B under POCl3-
mediated dehydration conditions to afford spirolactam 7 in 62%
yield. Compounds 2 and 3 were synthesized via the copper(I)-
catalyzed azide−alkyne cycloaddition (CuAAC) reaction39
between alkyne 7 and n-octyl azide or 4-bromobenzyl azide in
85% and 79% yields, respectively. These two compounds were
used to characterize the structural and fluorescence changes of
the spirolactam tridentate ligand motif (carbonyl-pyridyl-
triazolyl) upon binding to Zn2+.
The synthesis of heteroditopic compound 4 (Scheme 3)
started from the alkylation of 4-hydroxybenzaldehyde by 1,4-
dibromobutane to afford 8. Horner-Wadsworth-Emmons
olefination between 8 and phosphonate 9 resulted in alkene 10
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a
Scheme 2. Syntheses of Monotopic Compounds 2 and 3
a
NaAsc = sodium ascorbate; TBTA = tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine.
a
Scheme 3. Synthesis of Heteroditopic Compound 4
a
NaAsc = sodium ascorbate; TBTA = tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine; KHMDS = potassium hexamethyldisilazide; TBAI =
tetrabutylammonium iodide.
in the trans form,40 which, upon conversion to azide 11 via an SN2
substitution, underwent the CuAAC reaction with alkyne 7 to
reach compound 4. Rhodamine 101-derived monotopic
compound 5 and ditopic compound 6 (Scheme 4) were
prepared via similar routes to those leading to compounds 2−
4. Compounds 5 and 6 were used to study the impact of
structural variation at the xanthene moiety to H+- and Zn2+-
mediated the ring-opening chemistry of the spirolactam.
conversion of the colorless spirolactam to the ring-opened amide
form of rhodamine. The dissociation constant of a presumed 1:1
complex [Zn(2)](ClO4)2 is 0.1 mM via curve fitting using a 1:1
binding isotherm equation (Figure S2).41
Absorption and fluorescence responses of spirolactam 2 (10
μM) to other divalent metal ions (Cd2+, Ca2+, Co2+, Cu2+, Fe2+,
Mg2+, Mn2+, and Pb2+, in 5 equiv each as perchlorate salts) in
CH3CN are shown in Figure 4. Similar to Zn2+, additions of Cu2+,
Fe2+, and Pb2+ salts result in the development of a pink color of
the solution, characteristic of the conversion from spirolactam to
rhodamine. Among these metal ions, Cu2+ leads to a distinctive
absorption enhancement (549 nm, apparent molar absorptivity =
59 × 103 mol L−1 cm−1). Fe2+ under the same conditions also
leads to a more pronounced absorbance change (561 nm, 15 ×
103 mol L−1 cm−1) than those of Zn2+ (562 nm, 6 × 103 mol L−1
cm−1), Pb2+ (558 nm, 4 × 103 mol L−1 cm−1), and Co2+ (547 nm,
0.7 × 103 mol L−1 cm−1). All other metal ions fail to manage
detectable changes in the absorption spectrum of 2.
The fluorescence responses of these metal ions are shown in
Figure 4b. Consistent with the formation of rhodamine, the
additions of Fe2+ and Pb2+ lead to intense orange emission upon
excitation at 530 nm (ϕf = 0.08 and 0.26, respectively), similar to
that of Zn2+ (ϕf = 0.21). Even though Cu2+ opens spirolactam
ring effectively as shown by the absorption data (Figure 4a), the
Zn2+-Dependent Absorption and Fluorescence of
Monotopic Ligands 1 and 2. As previously reported,21
compound 1 (Scheme 1A) shows an absorption maximum at 347
nm (ε = 34 × 103 L mol−1 cm−1) and an emission maximum at
431 nm (ϕf = 0.28) in CH3CN. Upon complexation with
Zn(ClO4)2, as a result of stabilizing the charge-transfer excited
state, both maxima of absorption and emission of 1 shift
bathochromically to 374 nm (ε = 28 × 103 L mol−1 cm−1) and
528 nm (ϕf = 0.46, Table 1), respectively.
A solution of spirolactam 2 (Scheme 1B) in CH3CN is
colorless. The absorption and emission spectra of 2 in CH3CN in
the presence of varying concentrations of Zn(ClO4)2 (1 nM to 1
M) are shown in Figure 3. Upon increasing [Zn(ClO4)2] from 1
nM to 10 μM, the solution remains colorless. Further increasing
the concentration above 10 μM results in the development of a
pink color and orange fluorescence (Figure S1), signaling the
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a
Scheme 4. Syntheses of Rhodamine 101-Derived Compounds 5 and 6
a
NaAsc = sodium ascorbate; TBTA = tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine.
Table 1. Absorption Maximum (λabs), Extinction Coefficient (ε), Emission Maximum (λem), and Fluorescence Lifetime (τ) of Zn2+
Complexes of Model Systems (1, 2, and 5) and Heteroditopic Ligands (4 and 6) in CH3CN
compound
λabs/nm
ε/103 mol L−1 cm−1
λem/nm
ϕf
τ/ns (rel. ampl. %)
ab
,
[Zn(1)]2+
[Zn(2)]2+
[Zn(4)]2+
[Zn(4)]2+
[Zn(5)]2+
[Zn(6)]2+
[Zn(6)]2+
374
562
28
107
32
528
587
528
587
607
516
0.46
0.30
0.04
0.11
0.72
0.05
0.13
3.1
c
1.5
a
d
c
374
0.2
376, 567
584
35, 45
72
1.6 (70), 3.7 (30)
5.06
e
366
25
0.4 (56), 1.9 (44)
5.4 (92), 1.4 (8)
d
373, 588
34, 31
608
a
b
c
d
e
Of the monozinc complex. Value taken from ref 21. Zn(ClO4)2 saturated condition. In presence of 10 equiv of Zn(ClO4)2 in acetonitrile. In
presence of 0.6 equiv of Zn(ClO4)2.
Figure 3. (a) Effect of addition of Zn(ClO4)2 from 1 nM to 1 M on the absorption spectrum of 2 (10 μM) in CH3CN. Inset shows the variation of
absorbance at 562 nm vs [Zn(ClO4)2]. (b) Corresponding emission changes. Inset shows the variation of fluorescence intensity at 587 nm vs
[Zn(ClO4)2]. The solutions contain 0.14% DMSO.
emission is almost entirely quenched. All other metal ions show
negligible changes in the emission spectra, in line with the
absorption data. Overall, the metal ion selectivity of compound 2
with regard to absorption can be understood based on the Irving-
Williams series42 and the HSAB theory, which are typically
applicable to polyaza-type ligands. On the emission side,
paramagnetic metal ions, such as Cu2+, tend to mask the strong
affinities to the ligand by effectively quenching the fluorescence.
Zn2+-Dependent Structural Changes of Monotopic
Spirolactam Ligand 3. The single crystals of 3 were grown
by slow evaporation of a CH2Cl2/hexanes solution to space
group P21/c. As depicted in Figure 5, the lactam ring and
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Figure 4. (a) Absorption spectra of 2 (10 μM) obtained with 5 equiv of perchlorate salts of Zn2+, Cd2+, Ca2+, Co2+, Cu2+, Fe2+, Mg2+, Mn2+, and Pb2+ in
CH3CN. (b) Corresponding fluorescence spectra. λex = 530 nm. The solutions contain 0.14% DMSO.
spectrum of spirolactam 3 shows the absorption of an amide
carbonyl at 1682 cm−1. Upon coordination with Zn2+, the
carbonyl stretching frequency was lowered to 1584 cm−1 (Figure
S3), which is consistent with the hypothesis that carbonyl oxygen
participates in binding to result in a reduction of CO bond
order (see structure in Figure 6), hence the force constant of the
stretching vibration of the carbonyl.25
The titration of 3 with Zn(ClO4)2 was monitored via 1H NMR
spectroscopy in CD3CN (Figure 6). The 1H NMR spectrum of 3
shows the triazolyl proton (Hb) at 8.19 ppm and the pyridyl
protons at 7.70−7.78 ppm (Hc and Hd), and 8.59 ppm (He). The
Figure 5. Chemdraw (left) and ORTEP diagram (30% ellipsoids, right)
of single crystal X-ray structure of 3. Carbon and hydrogen: black;
nitrogen: blue; oxygen: red.
addition of Zn(ClO4)2 from 0 to 1 equiv results in broadening of
the peaks, which upon further addition of Zn2+ sharpen at
different chemical shifts. At 2 equiv, the triazolyl Hb proton and
the pyridyl proton Hd that is para to the nitrogen atom shift
downfield to 8.38 and 7.95 ppm, respectively. The other pyridyl
protons, Hc and He, shift to upfield. The benzylic protons of 3,
Ha, which appear as a single peak at 5.65 ppm in the free ligand,
shift to 5.17 ppm as two doublets in an AB system. The splitting is
explained by the diastereotopicity of the benzylic protons in the
axially chiral Zn2+ complex. These structural data are consistent
with the assignment that Zn2+ binds at the tridentate
coordination site containing carbonyl oxygen, pyridyl, and
triazolyl26,46,47 nitrogen atoms, as shown in Scheme 1B and
Figure 6.
xanthene core of 3 are almost orthogonal to each other. Similar
rhodamine spirolactam systems with different substituents have
recently been reported.35,43,44 The pyridyl and triazolyl groups
are in a typical transoid arrangement45 with a dihedral angle of
13.6° for minimizing the dipole interactions between the two
heterocycles.
Attempts to obtain single crystals of the Zn2+ complexes of 3
suitable for X-ray diffraction were not successful in several trials.
Therefore, IR and 1H NMR data of the complex were acquired to
study the structural changes of 3 upon Zn2+ binding. The IR
Figure 6. 1H NMR spectrum of 3 (500 MHz, CD3CN): (a) no Zn(ClO4)2 added, and (b) in the presence of 2 equiv of Zn(ClO4)2.
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Figure 7. (a) Effect of Zn(ClO4)2 (0−30 μM) on the absorption spectrum of compound 4 (3.0 μM) in CH3CN. (b) Corresponding changes of the
emission spectrum (λex = 370 nm). Blue: free ligand; green: molar ratio of 4 and Zn2+ is 1:1; red: Zn2+ is in 10-fold excess relative to 4.
Zn2+-Dependent Absorption and Fluorescence of
Heteroditopic Ligand 4. The absorption spectra of hetero-
ditopic ligand 4 over a [Zn(ClO4)2] gradient (0−10 equiv) are
shown in Figure 7a. The absorption maximum of 4 in CH3CN at
347 nm (the blue trace) is assigned to the S0→S1 transition of 5-
(4-methoxystyryl)-5′-methyl-2,2′-bipyridine. The absence of any
absorbance band above 400 nm indicates the existence of
rhodamine moiety in the ring-closed spirolactam form. Addition
of Zn(ClO4)2 from 0 to 3 μM results in a bathochromic shift of
the absorption maximum to 376 nm. Further addition of
Zn(ClO4)2 results in the development of a pink color (Figure 8,
top) and appearance of the band corresponding to the ring-
opened amide form of rhodamine at 567 nm.
The corresponding changes of the emission spectrum of 4
upon excitation at 370 nm are shown in Figure 7b. The addition
of Zn(ClO4)2 from 0 to 5 μM leads to a shift of emission from
blue (λem = 420 nm) to green (λem = 528 nm), which results from
Zn2+-coordination at the arylvinyl-bipy component. Upon
further addition of Zn(ClO4)2 beyond 5 μM, the spirolactam
converts to the ring-opened amide form of rhodamine. The
spectral overlap between the absorption of the ring-opened
amide form of rhodamine and the emission of Zn2+-arylvinyl-
bipy complex (Figure 2) leads to FRET, which results in the
second emission shift from green to orange (587 nm). The
fluorescence decay at 587 nm upon excitation at 370 nm, where
the Zn2+-arylvinyl-bipy absorbs, has a longer lifetime (1.6 and 3.7
ns; see Table 1 and Figure 11) than that obtained via direct
excitation of the rhodamine fluorophore (λex = 560 nm, the
lifetime of [Zn(2)]2+ is 1.5 ns; see Figure 11, inset). This
observation is consistent with the hypothesis that the orange
emission from the Zn2+-bound 4 shown in Figure 7b results from
a FRET mechanism. We further verified the sensitized excitation
of the Zn2+-rhodamine moiety in 4 using the following control
experiment.20,39 When excited at 370 nm, titration of Zn(ClO4)2
into an equimolar mixture of monotopic compounds 1 and 2
results in an intense green emission (Figure S4) instead of the
orange emission observed with ditopic compound 4, where
compounds 1 and 2 are covalently tethered.
The emission color transition of 4 over an increasing
[Zn(ClO4)2] gradient was examined visually. Fluorescence
emission of an CH3CN solution of 4 (10 μM) upon illumination
using a hand-held UV lamp in the presence of various equiv of
Zn(ClO4)2 is shown in Figure 8. In the absence of Zn(ClO4)2, a
blue emission was observed. The addition of 1.5 equiv of
Zn(ClO4)2 leads to a green color, indicative of the formation of
the Zn2+-arylvinyl-bipy moiety. Further addition of 1.5−3.0 equiv
Zn(ClO4)2 results in a yellowish emission; and in the presence of
10 equiv Zn(ClO4)2, the fluorescence of the solution is bright
orange, typical of that of rhodamine. Comparing to the color of
the solution of 4 in the presence of Zn(ClO4)2 at various
concentrations under the ambient light (Figure 8, top), the
emission of 4 carries a much higher visual sensitivity to the
change of [Zn(ClO4)2].
The sequential emission band shifts of 4 over an increasing
[Zn(ClO4)2] gradient offer an opportunity to correlate emission
intensity and [Zn(ClO4)2] ratiometrically. As shown in Figure
9a, the ratio of fluorescence intensity at 420 nm (blue channel)
over that of 528 nm (green channel) decreases steadily as
Figure 8. Photographs show the color of a CH3CN solution of 4 (top, 10
μM) and the fluorescence emission (bottom) upon illuminating the
same sample using a hand-held UV lamp (λex = 365 nm) in the presence
of various equiv of Zn(ClO4)2, from left to right: 0, 1.5, 3, 10 equiv.
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Figure 9. Emission intensity ratios of compound 4 vs [Zn(ClO4)2] in CH3CN (λex = 370 nm). (A) [Zn(ClO4)2] = 0−5 μM; inset: [Zn(ClO4)2] = 2.5−
5.0 μM; (B) [Zn(ClO4)2] = 5.0−30 μM.
[Zn(ClO4)2] increases up to 3 μM. The inverse intensity ratio of
528 nm over 420 nm (Figure 9a, inset) tracks the growth of
[Zn(ClO4)2] in the 3−5 μM range. When [Zn(ClO4)2] is over 5
μM, the orange emission channel (587 nm) is open. The
intensity ratio at 587 nm over 528 nm increases as [Zn(ClO4)2]
grows in the high concentration regime (Figure 9b). In
conjunction with the visual inspection in estimating [Zn-
(ClO4)2] (Figure 8), the observed ratiometric correlation
between fluorescence intensity and [Zn(ClO4)2], the “analyte”
in this case, over a range in acetonitrile offers promises in
developing multicolor fluorescent indicator for Zn2+ for
quantitative analyses based on this fluorescent heteroditopic
scaffold.
rhodamine component quenches the excited Zn2+-arylvinyl-bipy
complex in 4 (Figure 10).
Effect of Pb2+ and Fe2+ on the Fluorescence of
Heteroditopic Ligand 4. The influence of Pb2+ and Fe2+,
which induce fluorescence turn-on of the monotopic spirolactam
2, on the spectral response of heteroditopic ligand 4 was also
analyzed in CH3CN. The addition of Pb(ClO4)2 leads to a
bathochromic shift of bipy absorption toward 379 nm, along with
a concomitant increase of absorbance at 561 nm (Figure S5).
This observation suggests that the two binding sites in
compound 4 have comparable affinities to Pb2+, leading to
simultaneous rather than sequential binding. As a result, the
emission spectra collected during the Pb2+ titration experiment
include only one emission band of the Pb2+/4 complex, which is
centered at 583 nm assignable to the amide form of rhodamine.
A clear piece of evidence for FRET-mediated sensitization of
rhodamine moiety in 4 was observed upon titrating with
Fe(ClO4)2 (Figure S6). The addition of 0−10 equiv of Fe2+
results in sequential emergence of absorption bands at 370 and
564 nm. However, due to the inability of the nonemissive
Fe(bipy)2+ to act as the FRET donor, no emission bands were
observed from the solution upon excitation at 370 nm, even
though Fe2+ induces ring-opening of the spirolactam effectively,
and the complex of which fluoresces under direction excitation
(Figure 4b).
Figure 10. PET and FRET pathways in heteroditopic ligand 4. L:
counterion or solvent.
To verify the PET quenching process in the mononuclear
[Zn(4)L2] shown in Figure 10, we estimated the Gibbs free
energy (ΔG°) of electron transfer from the rhodamine
spirolactam moiety to the excited Zn2+-arylvinyl-bipy moiety in
4 using the Rehm−Weller eq 148
e2
dε
ΔG° = Eox − Ered − E0,0
−
(1)
where Eox is the oxidation potential of the donor (i.e., rhodamine
spirolactam moiety in 4, 0.48 V), Ered is the reduction potential of
the acceptor (i.e., Zn2+-arylvinyl-bipy moiety in 4, −1.77 V), E0,0
is the excitation energy of the Zn2+-arylvinyl-bipy complex (as
determined for [Zn(1)]2+ at 2.82 eV), d is the center-to-center
distance between the electron donor and the acceptor, and ε is
the dielectric constant of the solvent. Eox and Ered of model
compounds 1 and 2 and heteroditopic compound 4 were
determined using cyclic voltammetry (Table S1). The cyclic
voltammograms are shown in Figure S12. With the large
dielectric constant of the CH3CN solvent (ε = 37), the work term
e2/dε is omitted in the calculation. The application of the Rehm−
Intramolecular Photoinduced Electron Transfer in
Monozinc Complex [Zn(4)]2+. Comparing to the Zn2+
complex of compound 1 (ϕf = 0.46), the weak fluorescence of
the monozinc complex of heteroditopic ligand 4 (ϕf = 0.04,
Table 1), which arises from the same arylvinyl-bipy moiety in 1,
caught our attention. We hypothesized that the intramolecular
photoinduced electron transfer (PET) from the two electron-
rich N,N-diethylanilinyl groups in the spirolactam form of
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Weller equation gave a value of ΔG° = −0.57 eV, which indicates
that the intramolecular photoinduced reduction of Zn2+-
arylvinyl-bipy moiety from the spirolactam component in 4 is
spontaneous.
To further investigate the intramolecular PET quenching
process in [Zn(4)]2+, we measured fluorescence lifetime (τ) of
the monozinc complex of 4 in CH3CN using the nanosecond
time-correlated single photon counting technique, and com-
pared it with that of the model system, [Zn(1)]2+ complex. The
decay profiles along with the instrument response function (IRF)
are shown in Figure 11. Complex [Zn(1)]2+ experiences a
Figure 12. (a) Emission changes of 2 (10 μM, λex = 570 nm) at 588 nm
at various pH values (pH 1.2 to 11.4) in a mixed buffer (HEPES − 27
mM, EPPS − 9 mM, CHES − 9 mM, MES − 27 mM, NaOAc − 18 mM,
EGTA − 9 mM) solution containing KNO3 (90 mM). 20% DMSO was
added as cosolvent. Inset: emission spectra of 2 collected at various pH
values.
component as described earlier. ZnCl2 is unable to effect the ring-
opening of the spirolactam moiety, based on the absence of
absorption band of rhodamine centering near 588 nm.
A survey of the reported examples in which rhodamine
spirolactam chemistry is applied in developing fluorescent
indicators for Zn2+ under physiological conditions35−38 reveals
that similar predicaments of slow Zn2+-mediated spirolactam
ring-opening kinetics are probably encountered in all cases, to
various degrees. To the best of our knowledge, two rhodamine
spirolactam ligands for Zn2+ that operate in entirely aqueous
media have been reported thus far.35,36 This is an astonishingly
small number relative to the volume of reports on targeting other
metal ions using the rhodamine ring-opening chemistry.33
Lippard et al. developed probe ZRL1 (Chart 1) by utilizing
the strong chelating ability of dipicolylamino49 group to hold
Zn2+ close to the carbonyl of the spirolactam moiety, but the ring-
opening rate is slow as evidenced by the long wait time (2 h)
between Zn2+ additions in the titration experiment.35 The
Nagano group utilized iminodiacetate as the Zn2+ chelator on a
similar scaffold (e.g., 13 in Chart 1), and studied the role of
xanthene core on the ring-opening kinetics of rhodamine
spirolactam. The study by Nagano et al. showed that under
physiological conditions the spirolactams derived from rhod-
amine B and 6G are hardly opened upon addition of Zn2+, while
the derivatives from rhodamine 101, as in compound 13, show
much more sensitive responses.
▲
Figure 11. Fluorescence decay profiles monitored at 520 nm ( ) and
□
580 nm ( ) by exciting samples using a 370 nm nanoLED excitation
source: (a) IRF ( ), (b) the monozinc complex of 4 ( , blue), (c) 4 in
the presence of 10 equiv of Zn(ClO4)2 ( , wine). Inset: (d) 2 in the
presence of 10 equiv of Zn(ClO4)2 ( , red) in CH3CN excited at 560
●
▲
□
□
nm.
monoexponential decay with τ = 3.1 ns.25 The fluorescence
lifetime of the monozinc complex of 4 (blue triangles) is much
shorter and close to the IRF (ca. 0.2 ns). These data support an
efficient intramolecular PET relaxation pathway available to the
Zn2+-arylvinyl-bipy complex in 4, which shortens its lifetime.
Zn2+-Dependent Fluorescence of 4 under Physiolog-
ical Conditions. One of the potential uses of the heteroditopic
ligands is as indicators for Zn2+ under physiological conditions.
The bipy moiety in 4 has a pKa value ∼4.4.17 To assess the pKa of
the rhodamine spirolactam moiety of 4, pH titration of
compound 2 was carried out in the range of pH 1.2 to 11.4.
The pH-dependent emission intensity change at 588 nm is
plotted in Figure 12. Assuming that protonation (likely of the
carbonyl group) leads to the ring-opening of the spirolactam, the
pKa of the spirolactam moiety is ∼2.8, as estimated using the
Henderson-Hasselbach equation (Figure S7). Based on the pKa
values of both Zn2+ binding sites in 4, we conclude that the
fluorescence of 4 is not sensitive to the fluctuations of the pH
value within the physiological range (e.g., 6−8).
Based on the work from Lippard35 and Nagano groups,36 two
strategies to enhance the rate of Zn2+-coordination effected ring-
opening of spirolactam emerge: (1) to include a strong-binding
ligand that directs Zn2+ to interact with the carbonyl of the
spirolactam; (2) to increase the electronic conjugation of the
xanthene amino groups to the spiro carbon as in compound 13.
In this work, we synthesized the monotopic 5 and ditopic
compound 6 by replacing rhodamine B in 2 and 4 with
rhodamine 101 (Scheme 4), to see whether this structural
modification (Strategy #2) accelerates the ring-opening kinetics
to allow for the application of this heteroditopic design under
physiological conditions.
The Zn2+-dependent spectroscopic characteristics of hetero-
ditopic ligand 4 were studied under simulated physiological
conditions. Compound 4 (5 μM) upon titrating with ZnCl2 (0−
98 μM) in a 1:1 mixture of CH3CN and a pH neutral aqueous
buffer solution (50 mM HEPES, 50 mM NaCl, pH 7.2) (Figure
13) shows only the bathochromic absorption and emission
spectral shifts characteristic of the formation of the Zn2+-
arylvinyl-bipy complex. The fluorescence intensity from the
Zn2+-arylvinyl-bipy component of 4 is relatively weak (red
spectrum in Figure 13b), which can be attributed to the
quenching by the diethylanilinyl group in the spirolactam
Spectral response of monotopic spirolactam 5 to Zn2+
coordination was studied in CH3CN. Compound 5 remains
colorless in the presence of 1 nM up to 1 μM of Zn(ClO4)2.
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Figure 13. (a) Effect of addition of ZnCl2 (0−98 μM) on the absorption spectrum of compound 4 (5 μM) in a 1:1 CH3CN/HEPES buffer (50 mM
HEPES, 50 mM NaCl, pH 7.2). (b) Corresponding changes of the emission spectrum (λex = 370 nm).
Chart 1. Structures of Zn2+ Indicators ZRL1 (by Lippard et
608 nm were observed (Figure 14). As observed in the case of 4,
al.)35 and 13 (by Nagano et al.)36
at ratios of [Zn2+]/[6] less than 1:1, the samples exhibit a weak
green fluorescence. However, in contrast to 4, no clear transition
of emission color from green to orange was observed due to a
more competitive binding between the two coordination sites, as
a result of the increased affinity of the rhodamine 101-derived
spirolactam pyridyl-triazolyl secondary site to Zn2+.
Spirolactam 5 derived from rhodamine 101 shows a more
sensitive response to pH change than rhodamine B-derived
compound 2. Fluorescence of 5 increases as the pH value drops
from 6 to 3 to result in a sigmoidal pH-fluorescence profile
(Figure S10). The higher pKa value of 5 (5.0) than that of 2
(∼2.8) suggests that the ring-opening reaction of 5 is more
susceptible to a Brønsted or a Lewis acid than that of 2, which is
consistent with the conclusion of Nagano et al.36
Further increasing [Zn(ClO4)2] above 1 μM results in the
development of a pink color and orange fluorescence of the
solution (Figure S8). The dissociation constant of complex
[Zn(5)](ClO4)2 is 20 μM (Figure S9), comparing to the 0.1 mM
observed for [Zn(2)](ClO4)2.41 Therefore, the replacement of
rhodamine B in compound 2 by rhodamine 101 increases the
affinity to Zn2+ in CH3CN by 5-fold.
A solution of heteroditopic 6 (2.1 μM) in CH3CN is colorless
and shows only absorption corresponding to the bipy moiety.
The addition of Zn(ClO4)2 from 0 to 0.6 equiv leads to an
increase of absorbance near 360 nm, as a result of the formation
of Zn2+-bipy complex. Further addition of Zn2+ leads to the
increase of the band centered at 580 nm. Fluorescence change
over the Zn(ClO4)2 titration experiment also suggests the two-
stage binding process of 6, in which emission bands at 520 and
Fluorescence responses of ditopic 6 to Zn2+ were studied
under simulated physiological conditions (Figure S13). Similarly
to 4, addition of Zn2+ results in a green emission, corresponding
to the binding at bipy site. Further addition of Zn2+ is unable to
elicit the rhodamine emission. These observations emphasize the
reluctance of the spirolactam components in both 4 and 6 to
undergo Zn2+-effected ring-opening under physiological con-
ditions, which can be attributed to the weak affinity of pyridyl-
triazolyl moiety to Zn2+ in an aqueous environment. Extending
from this work, the next logical step is to undertake structural
alterations of the Zn2+ binding moiety for enhancing the
interaction between Zn2+ and the carbonyl of the spirolactam in
an aqueous environment (Strategy #1).
Figure 14. (a) Effect of addition of Zn(ClO4)2 (0−10 μM) on the absorption spectrum of 6 (2.1 μM) in CH3CN. (b) Corresponding changes of the
emission spectrum (λex = 370 nm). Blue: free ligand; green: ratio of [6]/[Zn2+] is 1:0.6; red: Zn2+ is in 10-fold excess relative to 6.
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compartment cell with a Pt disk working electrode, a Pt wire counter
electrode, and a Ag/AgCl reference electrode. The cyclic voltammo-
grams at a scan rate of 100 mV/s are reported.
CONCLUSION
■
A fluorescent heteroditopic ligand (4) undergoing sequential
emission bathochromic shifts over an increasing [Zn(ClO4)2]
gradient in CH3CN is demonstrated. Compound 4 can be
excited in the visible region. Its emission color spans from blue
continuously to orange depending on [Zn(ClO4)2]. The initial
emission band shift from blue to green is attributed to the Zn2+-
coordination stabilized internal charge transfer (ICT) compo-
nent in 4. At a sufficiently high [Zn(ClO4)2], the spirolactam
component in 4 transforms to the highly fluorescent rhodamine,
which turns the emission color from green to orange via
intramolecular FRET from the ICT fluorophore to rhodamine.
This work represents a new fluorescent heteroditopic ligand
design where sequential target-induced emission band shifts
result in a drastic emission color change (i.e., from blue to orange
in the case of compound 4).
In the current demonstration, the second step emission color
change of heteroditopic compounds 4 and 6 from green to
orange, which shall result from Zn2+-induced spirolactam ring-
opening, fails to occur under physiological conditions. From this
observation, the nuances of rhodamine spirolactam chemistry
that have not been sufficiently addressed are revealed, which
include the ability of the spirolactam as a fluorescence quencher
via electron transfer, and more significantly, the slow kinetics of
spirolactam ring-opening effected by Zn2+-coordination. Given
the ever-growing number of reports of metal ion sensor
development utilizing the ring-opening chemistry of rhodamine
spirolactam,33 we feel it is of importance to report the limitations
of this system, so that they can be addressed by the collective
attention and effort from the relevant research communities. Our
ongoing efforts focus on structural modification to overcome the
slow Zn2+-dependent ring-opening kinetics, so that fluorescent
heteroditopic indicators based on this design can be applied in
aqueous solutions.
2-Amino-6-[(trimethylsilyl)ethynyl]pyridine. 2-Amino-6-bro-
mopyridine (100 mg, 0.58 mmol), Pd(PPh3)2Cl2 (12 mg, 0.017
mmol, 3 mol %), and CuI (5 mg, 0.028 mmol, 5 mol %) were added to
dry THF (10 mL) in a flame-dried round-bottom flask. The mixture was
purged by argon for 30 min. The reaction mixture was cooled on ice.
Triethylamine (2 mL) was added and stirred for 5 min. Ethynyl-
trimethylsilane (100 μL, 0.69 mmol) was subsequently added and
stirred at rt. After 12 h, the reaction mixture was filtered through a short
pad of alumina and washed with THF. Solvent was removed under
reduced pressure, and the obtained crude product was purified by silica
column chromatography using a THF-CH2Cl2 (1:9) mixture as a white
1
solid. Mp: 125−126 °C. The yield was 90 mg (82%). H NMR (300
MHz, CDCl3): δ/ppm 7.36 (t, J = 7.8 Hz, 1H), 6.85 (d, J = 7.8 Hz, 1H),
6.45 (d, J = 8.4 Hz, 1H), 4.5 (bs, 2H), 0.25 (s, 9H); 13C NMR (125
MHz, CDCl3): δ/ppm 158.4, 140.8, 137.8, 117.9, 108.9, 104.2, 93.5,
−0.11; HRMS (CI) (m/z): [M+H]+ calcd for C10H15N2Si 191.1005,
found 191.1006.
2-Amino-6-ethynylpyridine. 2-Amino-6-[(trimethylsilyl)-
ethynyl]pyridine (90 mg, 0.47 mmol) was stirred in CH3OH (10 mL)
containing 20% KOH. After 1 h, water (20 mL) was added to dilute the
mixture, which was subsequently extracted with ethyl acetate (3 × 30
mL). The combined organic phases were dried over anhydrous Na2SO4.
Solvent was removed under reduced pressure to afford the analytically
pure product a pale brown solid. Mp: 117−120 °C. The yield was 55 mg
(99%). 1H NMR (300 MHz, CDCl3): δ/ppm 7.38 (t, J = 7.8 Hz, 1H),
6.86 (d, J = 7.2 Hz, 1H), 6.47 (d, J = 8.4 Hz, 1H), 4.5 (bs, 2H), 3.06 (s,
1H); 13C NMR (125 MHz, CDCl3): δ/ppm 158.5, 140.1, 137.9, 117.9,
109.4, 83.2, 76.1; HRMS (CI) (m/z): [M+H]+ calcd for C7H7N2
119.0609, found 119.0604.
Compound 7. Rhodamine B (400 mg, 0.84 mmol) and 2-amino-6-
ethynylpyridine (120 mg, 1.01 mmol) were refluxed with a catalytic
amount of POCl3 in CH3CN (20 mL). After 1 h, the reaction mixture
was cooled to rt, and the solvent was removed under reduced pressure.
The crude product was purified as an amorphous pale orange solid by
column chromatography on basic alumina using CH2Cl2 as eluent. The
yield of 7 was 280 mg (62%). 1H NMR (300 MHz, CDCl3): δ/ppm 8.38
(d, J = 8.4 Hz, 1H), 7.99 (d, J = 8.4 Hz, 1H), 7.42−7.53 (m, 3H), 7.17 (d,
J = 7.2 Hz, 1H), 6.97 (d, J = 7.2 Hz, 1H), 6.40 (m, 4H), 6.13 (dd, J = 8.4,
2.4 Hz, 2H), 3.29 (m, 8H), 2.97 (s, 1H), 1.12 (t, J = 7.2 Hz, 12H); 13C
NMR (125 MHz, CDCl3): δ/ppm 168.3, 154.3, 153.6, 150.5, 148.5,
139.5, 137.1, 133.8, 130.8, 128.3, 127.9, 124.7, 123.3, 122.9, 115.6, 108.3,
107.0, 97.9, 82.7, 75.5, 66.7, 44.5, 12.8; HRMS (CI) (m/z): [M+H]+
calcd for C35H35N4O2 543.2760, found 543.2744.
Compound 2. Compound 7 (20 mg, 0.037 mmol) and n-octyl azide
(10 mg, 0.037 mmol) were dissolved in a CH2Cl2−CH3OH (1:3)
mixture (4 mL). Aqueous solutions of sodium ascorbate (0.5 M, 25 μL)
and Cu(OAc)2·H2O (0.1 M, 25 μL) were mixed to produce an orange
suspension containing the copper(I) catalytic species, which was
subsequently added to the stirring solution. TBTA (catalytic amount)
was added, and the mixture was stirred for 12 h at rt. It was then
partitioned between CH2Cl2 and a basic EDTA (pH = 10) solution. The
organic fraction was washed with basic brine (pH > 10) two more times
before drying over K2CO3. The solvent was removed, and compound 2
was isolated by alumina chromatography as a pale orange amorphous
solid using CH3OH in CH2Cl2 (gradient 0−1%) as eluent. The yield was
22 mg (85%). 1H NMR (300 MHz, CDCl3): δ/ppm 8.47 (d, J = 7.8 Hz,
1H), 8.00 (d, J = 8.4 Hz,1H), 7.99 (s, 1H), 7.62−7.73 (m, 2H), 7.45−
7.49 (m, 2H), 7.08 (d, J = 7.8 Hz, 1H), 6.53 (d, J = 9.0 Hz, 2H), 6.31 (d, J
= 2.4 Hz, 2H), 6.13−6.17 (dd, J = 8.4 2.4 Hz, 2H), 4.38 (t, J = 7.2 Hz,
2H), 3.26 (q, J = 7.2 Hz, 8H), 2.01 (t, J = 6.6 Hz, 2H), 1.23−1.43 (m,
10H), 1.09 (t, J = 7.2 Hz, 12H), 0.90 (t, J = 6.6 Hz, 3H); 13C NMR (125
MHz, CDCl3): δ/ppm 168.9, 154.5, 152.5, 150.4, 148.6, 148.4, 148.1,
138.0, 133.8, 129.5, 128.2, 127.9, 124.3, 123.5, 122.6, 115.4, 115.3, 108.8,
107.9, 97.5, 66.0, 50.5, 44.5, 32.0, 30.7, 29.3, 26.8, 22.8, 14.3, 12.8;
HRMS (CI) (m/z): [M+H]+ calcd for C43H52N7O2 698.4182, found
698.4171.
EXPERIMENTAL SECTION
■
Materials and General Methods. Reagents and solvents were
purchased from various commercial sources and used without further
purification unless otherwise stated. All reactions were carried out in
oven- or flame-dried glassware. Analytical thin-layer chromatography
(TLC) was performed using precoated TLC plates with silica gel 60
F254 or with aluminum oxide 60 F254 neutral. Flash column
chromatography was performed using 40−63 μm (230−400 mesh
ASTM) silica gel or alumina (80−200 mesh, pH 9−10) as the stationary
phases. Silica and alumina gel was flame-dried under vacuum to remove
adsorbed moisture before use. 1H and 13C NMR spectra were recorded
at 300 and 125 MHz (on two different instruments), respectively. All
chemical shifts were reported in δ units relative to tetramethylsilane.
Spectrophotometric and fluorometric titrations were conducted on a
UV−visible spectrophotometer and a fluorescence spectrophotometer,
respectively, with a 1 cm semimicro quartz cell. The fluorescence
quantum yields were determined by comparison of the integrated area of
corrected emission spectrum with those of quinine bisulphate (ϕf = 0.54
in 1 N H2SO4) and rhodamine 101 inner salt (ϕf = 1.0 in ethanol) using
the literature methods.50−52 Fluorescence lifetimes were determined
using a nanosecond single-photon counting system employing a 370 and
560 nm nanoLED excitation source. Photon count was set at 10 000, the
TAC range was 100 ns, the sync delay was 70 ns, and coaxial delay was 65
ns. The fluorescence lifetime values were determined by deconvoluting
the instrument response function with exponential decay using a decay
analysis software. The quality of the fit was judged by χ2 value (<1.2) and
the visual inspection of the residuals. The cyclic voltammograms were
acquired in CH3CN (spectroscopic grade) containing Bu4NPF6 (100
mM) as supporting electrolyte using an electrochemical analyzer. The
data were collected at a concentration of 1.0 mM in a single
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Compound 3. Synthesized by the same procedure as above. 4-
bromobenzyl azide was used instead of n-octyl azide. The product was
isolated as a crystalline pale orange solid. The yield was 22 mg (79%).
Mp: 216−217 °C. 1H NMR (300 MHz, CDCl3): δ/ppm 8.54 (dd, J =
8.4, 1.2 Hz, 1H), 8.12 (s, 1H), 7.99 (dd, J = 6.6, 1.8 Hz, 1H), 7.61−7.73
(m, 4H), 7.45−7.49 (m, 2H), 7.27 (d, J = 9.0 Hz, 2H), 7.05 (dd, J = 5.4,
2.4 Hz, 1H), 6.47 (dd, J = 6.6, 3.0 Hz, 2H), 6.12 (m, 4H), 5.53 (s, 2H),
3.26 (m, 8H), 1.09 (t, J = 7.2 Hz, 12H); 13C NMR (125 MHz, CDCl3):
δ/ppm 168.9, 154.4, 152.4, 150.5, 149.2, 148.3, 147.7, 137.9, 134.2,
133.8, 132.4, 129.7, 129.4, 128.2, 127.9, 124.2, 123.4, 122.9, 122.8, 115.4,
115.1, 108.6, 107.7, 97.3, 65.8, 53.5, 44.3, 12.7. HRMS (ESI-TOF) (m/
z): [M+Na]+ calcd for C42H40N7BrO2Na 776.2324, found 776.2313.
Compound 8. 4-Hydroxybenzaldehyde (1.0 g, 8.2 mmol), 1,4-
dibromobutane (1.8 g, 8.2 mmol) and K2CO3 (2.2 g, 16 mmol) were
refluxed in CH3CN (20 mL). After 12 h, the solution was cooled to rt
before water (50 mL) was added. The aqueous solution was extracted
with ethyl acetate (3 × 30 mL). The combined organic phases were
dried over anhydrous Na2SO4, concentrated in vacuo, and purified by
flash chromatography on silica. Unreacted 1,4-dibromobutane was
collected by eluting with hexanes. Compound 8 was isolated using ethyl
acetate−hexanes mixture (1:19) as eluent. The product was an
(d, J = 17.0 Hz, 1H), 7.09 (d, J = 4.2 Hz, 1H), 6.98 (d, J = 17.0 Hz, 1H),
6.94 (d, J = 8.4 Hz, 2H), 6.53 (d, J = 9.0 Hz, 2H), 6.33 (s, 2H), 6.15 (d, J
= 8.4 Hz, 2H), 4.49 (t, J = 6.9 Hz, 2H), 4.09 (t, J = 5.7 Hz, 2H), 3.23 (q, J
= 7.2 Hz, 8H), 2.39 (s, 3H), 2.26 (m, 2H), 1.91 (m, 2H), 1.06 (t, J = 6.9
Hz, 12H); 13C NMR (125 MHz, CDCl3): δ/ppm 168.9, 159.1, 154.9,
154.5, 153.6, 152,5, 150.5, 149.8, 148.8, 148.4, 148.0, 147.9, 138.1, 137.7,
133.9, 133.5, 133.2, 133.1, 130.3, 129.9, 129.4, 128.2, 127.9, 124.3, 123.5,
122.9, 122.7, 120.8, 120.7, 115.4, 114.9, 108.8, 107.9, 97.5, 66.9, 66.0,
50.0, 44.4, 31.1, 27.4, 26.4, 18.6, 12.8; HRMS (ESI-TOF) (m/z): [M
+H]+ calcd for C58H58N9O3 928.4663, found 928.4669.
Compound 12. Synthesized by the same procedure as described for
7. Rhodamine 101 inner salt was used in place of rhodamine B. The yield
of 12 was 296 mg (60%) as an amorphous pale red solid. 1H NMR (300
MHz, CDCl3): δ/ppm 8.47 (d, J = 8.4 Hz, 1H), 7.98 (d, J = 8.4 Hz, 1H),
7.42−7.53 (m, 3H), 7.19 (d, J = 7.2 Hz, 1H), 6.95 (d, J = 7.2, 1H), 5.80
(s, 2H), 2.91−3.11 (m, 13H), 2.39 (t, J = 6.6 Hz, 4H), 2.02 (t, J = 5.4 Hz,
4H), 1.81 (t, J = 5.4 Hz, 4H); 13C NMR (125 MHz, CDCl3): δ/ppm
168.4, 153.7, 150.4, 149.4, 143.2, 139.2, 136.9, 133.7, 131.0, 128.2, 125.0,
124.2, 123.0, 122.6, 115.8, 115.2, 108.3, 107.5, 83.3, 75.1, 67.6, 50.1,
49.7, 27.1, 22.2, 21.9, 21.4. HRMS (ESI-TOF) (m/z): [M+H]+ calcd for
C39H35N4O2 591.2760, found 591.2756.
1
amorphous white solid. The yield was 1.42 g (68%). H NMR (300
Compound 5. Synthesized by the same procedure as described for 2.
Compound 12 was used in place of 7 and 1-azido-2-(2-(2-
methoxyethoxy)ethoxy)ethane was used in place of n-octyl azide. The
residue was purified by column chromatography on alumina using ethyl
acetate in CH2Cl2 (gradient 0−80%). The yield of 5 was 17 mg (59%) as
an amorphous pale reddish solid. 1H NMR (300 MHz, CDCl3): δ/ppm
8.46 (dd, J = 8.4, 1.2 Hz, 1H), 7.98 (dd, J =, 6.0, 2.4 Hz, 1H), 7.79 (s,
1H), 7.62−7.73 (m, 2H), 7.40−7.48 (m, 2H), 7.04 (dd, J = 5.4, 1.8 Hz,
1H), 6.06 (s, 2H), 4.54 (t, J = 6.0 Hz, 2H), 3.98 (t, J = 6.0 Hz, 2H), 3.34−
3.67 (m, 8H), 3.29 (s, 3H), 2.98−3.08 (m, 10H), 2.75−2.83 (m, 2H),
2.38 (t, J = 6.6 Hz, 4H), 1.98−2.06 (m, 4H), 1.74−1.82 (m, 4H); 13C
NMR (125 MHz, CDCl3): δ/ppm 169.2, 154.9, 150.5, 148.6, 147.9,
147.3, 143.1, 137.9, 133.8, 129.2, 127.9, 124.5, 124.2, 123.7, 123.4, 116.9,
115.4, 115.3, 108.7, 106.9, 72.0, 71.1, 70.7, 70.6, 69.8, 66.8, 59.2, 50.4,
50.0, 49.5, 27.2, 22.1, 21.8, 21.7; HRMS (ESI-TOF) (m/z): [M+H]+
calcd for C46H50N7O5 780.3873, found 780.3873.
Compound 6. Synthesized by the same procedure as described
above. The yield of 6 was 18 mg (52%) as an amorphous pale reddish
solid. 1H NMR (300 MHz, CDCl3): δ/ppm 8.72 (s, 1H), 8.36−8.53 (m,
2H), 8.34 (d, J = 8.4 Hz, 1H), 8.28 (d, J = 8.1 Hz, 1H), 7.92−8.00 (m,
2H), 7.78 (s, 1H), 7.61−7.73 (m, 3H), 7.40−7.48 (m, 4H), 7.15 (d, J =
16.8 Hz, 1H), 7.02−7.06 (m, 1H), 6.97 (d, J = 16.2 Hz, 1H), 6.88 (d, J =
9.0 Hz, 2H), 6.08 (s, 2H), 4.48 (t, J = 7.2 Hz, 2H), 4.05 (t, J = 5.4 Hz,
2H), 2.98−3.08 (m, 10H), 2.75−2.85 (m, 2H), 2.36−2.39 (m, 7H), 2.23
(t, J = 7.2 Hz, 2H), 1.92−2.04 (m, 6H), 1.77 (t, J = 6.0 Hz, 4H); 13C
NMR (125 MHz, CDCl3): δ/ppm 169.3, 159.0, 155.0, 154.9, 153.6,
150.5, 149.8, 148.9, 148.0, 147.8, 147.2, 143.1, 137.9, 137.7, 133.9, 133.5,
133.2, 133.1, 130.4, 129.9, 129.1, 128.2, 128.0, 124.4, 124.2, 123.4, 122.9,
122.7, 120.8, 120.7, 117.0, 115.4, 115.3, 114.8, 108.7, 106.8, 67.6, 66.7,
56.2, 50.3, 50.0, 49.6, 29.9, 27.8, 27.2, 26.6, 22.1, 21.9, 21.8, 18.6; HRMS
(ESI-TOF) (m/z): [M+H]+ calcd for C62H58N9O3 976.4663, found
976.4693.
MHz, CDCl3): δ/ppm 9.88 (s, 1H), 7.83 (d, J = 9.0 Hz, 2H), 6.98 (d, J =
9.0 Hz, 2H), 4.08 (t, J = 5.7 Hz, 2H), 3.49 (t, J = 6.1 Hz, 2H), 1.95−2.14
(m, 4H); 13C NMR (125 MHz, CDCl3): δ/ppm 190.7, 163.9, 131.9,
129.9, 114.7, 67.3, 33.5, 29.3, 27.7; HRMS (CI) (m/z): [M+H]+ calcd
for C11H14BrO2 257.0177, found 257.0176.
Compound 10. In a flame-dried flask, compounds 940 (200 mg, 0.62
mmol) and 7 (160 mg, 0.62 mmol) were dissolved in dry THF (20 mL)
and cooled to 0 °C. Potassium hexamethyldisilazide (1.5 mL, 0.5 M in
toluene, 0.74 mmol) was added dropwise. Upon completing the
addition, the stirring was continued for 3 h while the temperature rose to
rt. The reaction mixture was then partitioned between CH2Cl2 and
water. The aqueous layer was washed with CH2Cl2 (3 × 50 mL). The
organic portions were combined, and dried over Na2SO4 followed by
solvent removal under vacuum. The product was isolated via silica
chromatography using a 1:1 mixture of CH2Cl2−hexanes as a pale
yellowish amorphous solid. The yield was 162 mg (62%). 1H NMR (300
MHz, CDCl3): δ/ppm 8.73 (s, 1H), 8.51 (s, 1H), 8.34 (d, J = 8.4 Hz,
1H), 8.29 (d, J = 7.8 Hz, 1H), 7.94 (d, J = 8.4 Hz, 1H), 7.62 (d, J = 8.4
Hz, 1H), 7.49 (d, J = 9.0 Hz, 2H), 7.17 (d, J = 16.2 Hz, 1H), 6.99 (d, J =
16.2 Hz, 1H), 6.90 (d, J = 8.1 Hz, 2H), 4.03 (t, J = 6.0 Hz, 2H), 3.50 (t, J
= 6.6 Hz, 2H), 2.40 (s, 3H), 1.94−2.13 (m, 4H); 13C NMR (125 MHz,
CDCl3): δ/ppm 159.0, 154.7, 153.5, 149.7, 147.9, 137.5, 133.3, 133.1,
133.0, 130.2, 129.6, 128.0, 122.7, 120.7, 120.5, 114.8, 66.9, 33.5, 29.5,
27.9, 18.5; HRMS (ESI-TOF) (m/z): [M+H]+ calcd for C23H24BrN2O
423.1072, found 423.1063.
Compound 11. Compound 10 (100 mg, 0.24 mmol), NaN3 (30 mg,
0.47 mmol), 18-crown-6 (catalytic amount), and tetrabutylammonium
iodide (catalytic amount) were heated in DMF (5 mL) at 55 °C. After 12
h, the solution was cooled to rt before water (15 mL) was added. The
yellow solid was filtered and dried to afford the analytically pure product
1
as a pale yellowish amorphous solid. The yield was 86 mg (94%). H
NMR (300 MHz, CDCl3): δ/ppm 8.72 (s, 1H), 8.51 (s, 1H), 8.34 (d, J =
8.4 Hz, 1H), 8.28 (d, J = 7.5 Hz, 1H), 7.93 (d, J = 8.4 Hz, 1H), 7.62 (d, J
= 7.8 Hz, 1H), 7.49 (d, J = 9.0 Hz, 2H), 7.17 (d, J = 16.2 Hz, 1H), 6.99
(d, J = 16.2 Hz, 1H), 6.90 (d, J = 8.7 Hz, 2H), 4.03 (t, J = 6.0 Hz, 2H),
3.38 (t, J = 6.6 Hz, 2H), 2.40 (s, 3H), 1.80−1.93 (m, 4H); 13C NMR
(125 MHz, CDCl3): δ/ppm 159.0, 154.6, 153.4, 149.7, 147.9, 137.5,
133.3, 133.0, 130.2, 129.6, 128.0, 122.5, 120.6, 120.5, 114.7, 67.2, 51.2,
26.5, 25.8, 18.4; HRMS (ESI-TOF) (m/z): [M+Na]+ calcd for
C23H23N5ONa 408.1800, found 408.1795.
ASSOCIATED CONTENT
■
S
* Supporting Information
Copies of 1H and 13C NMR spectra for all reported compounds,
additional spectra, and .cif file of compound 3. This material is
available free of charge via the Internet at http://pubs.acs.org/.
Compound 4. Synthesized by the same procedure as described for 2.
Compound 11 (14 mg, 0.037 mmol) was used in place of n-octyl azide.
The residue was purified by column chromatography on silica using
CH3OH in CH2Cl2 (gradient 0−2%) as eluent. The product was
obtained as a pale orange amorphous solid. The yield was 24 mg (69%).
1H NMR (300 MHz, CDCl3): δ/ppm 8.72 (s, 1H), 8.49 (s, 1H), 8.48 (d,
J = 8.4 Hz, 1H), 8.35 (d, J = 8.4 Hz, 1H), 8.29 (d, J = 7.8 Hz, 1H), 8.01 (s,
1H), 7.92−8.00 (m, 2H), 7.61−7.72 (m, 3H), 7.44−7.50 (m, 4H), 7.17
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: lzhu@chem.fsu.edu
Notes
The authors declare no competing financial interest.
8278
dx.doi.org/10.1021/jo3016659 | J. Org. Chem. 2012, 77, 8268−8279

The Journal of Organic Chemistry
Article
(37) Xu, L.; Xu, Y.; Zhu, W.; Zeng, B.; Yang, C.; Wu, B.; Qian, X. Org.
Biomol. Chem. 2011, 9, 8284−8287.
ACKNOWLEDGMENTS
■
This work was supported by the National Institute of General
Medical Sciences (R01GM081382).
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