A unique class of near-infrared functional fluorescent dyes with carboxylic-acid-modulated fluorescence ON/OFF switching: Rational design, synthesis, optical properties, theoretical calculations, and applications for fluorescence imaging in living animals

Article abstract of DOI:10.1021/ja209292b

Fluorescence imaging is one of the most powerful techniques for monitoring biomolecules in living systems. Fluorescent sensors with absorption and emission in the near-infrared (NIR) region are favorable for biological imaging applications in living anima

Full text of DOI:10.1021/ja209292b

Article
pubs.acs.org/JACS
A Unique Class of Near-Infrared Functional Fluorescent Dyes with
Carboxylic-Acid-Modulated Fluorescence ON/OFF Switching:
Rational Design, Synthesis, Optical Properties, Theoretical
Calculations, and Applications for Fluorescence Imaging in Living
Animals
Lin Yuan, Weiying Lin,* Yueting Yang, and Hua Chen
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University,
Changsha 410082, P. R. China
S
* Supporting Information
ABSTRACT: Fluorescence imaging is one of the most powerful techniques for
monitoring biomolecules in living systems. Fluorescent sensors with absorption and
emission in the near-infrared (NIR) region are favorable for biological imaging
applications in living animals, as NIR light leads to minimum photodamage, deep tissue
penetration, and minimum background autofluorescence interference. Herein, we have
introduced a new strategy to design NIR functional dyes with the carboxylic-acid-
controlled fluorescence on−off switching mechanism by the spirocyclization. Based on the design strategy, we have developed a
series of Changsha (CS1−6) NIR fluorophores, a unique new class of NIR functional fluorescent dyes, bearing excellent
photophysical properties including large absorption extinction coefficients, high fluorescence quantum yields, high brightness,
good photostability, and sufficient chemical stability. Significantly, the new CS1−6 NIR dyes are superior to the traditional
rhodamine dyes with both absorption and emission in the NIR region while retaining the rhodamine-like fluorescence ON-OFF
switching mechanism. In addition, we have performed quantum chemical calculations with the B3LYP exchange functional
employing 6-31G* basis sets to shed light on the structure-optical properties of the new CS1−6 NIR dyes. Furthermore, using
CS2 as a platform, we further constructed the novel NIR fluorescent TURN-ON sensor 7, which is capable of imaging
endogenously produced HClO in the living animals, demonstrating the value of our new CS NIR functional fluorescent dyes. We
expect that the design strategy may be extended for development of a wide variety of NIR functional dyes with a suitable
fluorescence-controlled mechanism for many useful applications in biological studies.
process is very challenging.5 In a well-designed work, Murthy’s
group presented a different strategy to turn on the fluorescence
INTRODUCTION
Recently, fluorescence imaging has become one of the most
powerful techniques for monitoring biomolecules in living
■
of cyanine-based NIR sensors with the π-electron deconjuga-
tion.6 Very recently, Shabat et al. introduced an elegant strategy
to switch on the fluorescence of cyanine-based NIR sensors by
analyte-specific unmasking of a protected quinone derivative of
Cy7.7
systems.1 Fluorescent sensors are essential molecular tools for
bioimaging. So far, a large number of fluorescent sensors have
been constructed.2 However, most of them have the absorption
and emission in the visible range (400−650 nm). By contrast,
the fluorescent sensors with absorption and emission in the
near-infrared (NIR) region (650−900 nm) are relatively few,
although they are highly sought due to their useful applications
in chemical biology.1j,k,n,3 When compared to visible light, NIR
light is advantageous to be employed in biological imaging due
to minimum photodamage to biological samples, deep tissue
penetration, and minimum interference from background
autofluorescence by biomolecules in the living systems.3
Rhodamine dyes have good photochemical properties such
as high molar extinction coefficients, large fluorescence
quantum yields, and tolerance to photobleaching.8 Significantly,
the carboxylic acid group of rhodamine dyes can undergo
intramolecular cyclization (Figure 1A). The open form is highly
fluorescent, whereas the spirocyclic form is essentially non-
fluorescent.8a,b At physiological pH, the fluorescent open form
is predominated. In other words, the fluorescence of rhodamine
dyes is on at physiological pH. By sharp contrast, the
fluorescence of rhodamine amide derivatives is off at
physiological pH, as the nonfluorescent spirolactam form is
predominated (Figure 1A). By exploiting the unique
Cyanine dyes are one type of the widely employed NIR
fluorophores for NIR fluorescent sensor design. However, as
pointed out by Nagano and co-workers, on−off switching of
cyanine fluorescence by photoinduced electron transfer (PeT)
is quite difficult, as cyanine dyes have relatively high-lying
occupied molecular orbital (HOMO) energy levels.4 Thus, the
development of cyanine-based NIR sensors by means of a PeT
Received: October 2, 2011
Published: December 15, 2011
© 2011 American Chemical Society
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Figure 1. Design strategy for the new Changsha (CS) NIR functional dyes with a rhodamine-like carboxylic-acid-modulated fluorescence on/off
switching mechanism. (A) Spirocyclization of the classic rhodamine and rhodamine amide derivatives. (B) Structure and the fluorescence emission
spectrum (in CH3OH) of merocyanine dye A. Notably, the emission profile of merocyanine dye A has not been previously disclosed. (C)
Spirocyclization of the new Changsha (CS) and Changsha amide derivatives. The highlighted structures are the putative forms predominantly
present at physiological pH. Significantly, the electronic pulling pathway for the novel CS NIR functional dye spirocyclization highly resembles that
of the traditional rhodamine dye spirocyclization.
fluorescence ON−OFF switching between rhodamine dyes and
their amide derivatives, in a pioneering work, Czarnik’s group
constructed a fluorescence turn-on sensor for copper ions.9 The
specific copper-mediated hydrolysis of nonfluorescent rhod-
amine B hydrazide into fluorescent rhodamine B was employed
in this case. Since then, the rhodamine spirolactam has been
employed as an effective platform for construction of
fluorescence turn-on sensors for a diverse array of targets
including Cu2+, Hg2+, Fe3+, Cr3+, Pb2+, Zn2+, Ag+, Au3+, Pd2+,
Pt2+, HOCl, and NO, based on the unique spirolactam ring-
opening process of rhodamines.8a,b,10 Thus, the rhodamine
spirolactam platform is one of the most robust platform for
fluorescent sensor development. However, the fluorescence
turn-on sensors derived from the classic rhodamine dyes such
as rhodamine B, rhodamine 6G, and rhodamine 101 have
absorption and emission only in the visible region (500−600
nm). This limitation renders them unsuitable for biological
imaging in living animals. Thus, it is highly desirable to develop
rhodamine analogues with absorption and emission in the NIR
region.
800 lacks the unique spirolactone ring-opening process of
rhodamines. In other words, the fluorescence on−off switching
of rhodamine 800 cannot be readily achieved by the
interconversion between the fluorescent open form and the
nonfluorescent spirocyclic form. Thus, rhodamine 800 is only
suitable as NIR fluorescent labels for biomolecules, but not as
the platform for design of analyte-responsive NIR fluorescent
sensors. Recently, in an elegant work, Nagano et al. developed a
Si-rhodamine-based12a fluorescent hypochlorous acid sensor
with absorption and emission located in the far-red to near-
infrared region (Absmax/Emmax = 652/670 nm), and it operates
by the typical spirocyclization-based fluorescence on−off
switching mechanism.12b The red-shift of Si-rhodamine
derivatives compared to the classic rhodamine dyes is believed
due to the relatively low lying LUMO energy levels of silicon.
However, to obtain rhodamine analogues with longer
absorption and emission (>700 nm) while retaining the unique
spirocyclization-based fluorescence on−off switching mecha-
nism of rhodamines, a distinct novel strategy is needed to
modify rhodamine dyes. This appears to be a challenging but
interesting task.
In this regard, some NIR rhodamine analogues have been
constructed.11 For example, rhodamine 800 (Absmax/Emmax
=
It has been reported that merocyanine A (Figure 1B) has
maximal absorption at around 700 nm, in the NIR region.13
However, to the best of our knowledge, the fluorescence
properties of merocyanine A have not been reported in the
680/700 nm) is commercially available (The structure is shown
in Figure S1). However, it has a CN functional group instead of
benzoic acid at the 9-position of rhodamine. Thus, rhodamine
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Figure 2. Comparison of the classical rhodamine dyes and the novel CS NIR dyes. Top: Structures of the classical rhodamine dyes (rhodamines B,
6G, 101). Bottom: Structures of the newly designed NIR dyes CS1−6.
literature. Thus, we decided to synthesize and examine the
fluorescence profiles of merocyanine A. Our studies reveal that
merocyanine A has a strong emission peak at 734 nm (Figure
1B) with a high fluorescence quantum yield of 0.34 in CH3OH.
Obviously, it is difficult to control the optical properties of
merocyanine A, as it lacks of a simple fluorescence switching
mechanism. However, we envisioned that merocyanine A could
be employed as a building block to design new NIR fluorescent
platforms with tunable optical properties, if a handy
fluorescence switching mechanism can be rationally installed.
Toward this end, in this work, based on the above design
strategy, we have developed a new type of NIR functional dyes,
called Changsha (CS) NIR, which is the hybrid of merocyanine
A and benzoic acid (Figure 1C). In principle, the merocyanine
A unit could show the desired NIR optical profiles. We further
hypothesized that the carboxylic acid functional group could
operate as a fluorescence on−off switcher by spirocyclization.
In other words, the novel CS NIR dyes may bear the same
robust fluorescence ON−OFF switching mechanism as the
traditional rhodamine dyes (Figure 1A) while having the
advantage of NIR absorption and emission, as the driving force
for the spirocyclization of the new CS NIR dyes and the
traditional rhodamine dyes is quite similar, an iminium moiety
(notably, the electronic pulling pathway for the CS NIR dye
spirocyclization highly resembles that of the traditional
rhodamine dye spirocyclization; see Figure 1A and C). Since
CS NIR dyes intrinsically bear a handy fluorescence on−off
switching mechanism, they could serve as the effective new
platforms for NIR fluorescent sensor design. Herein, we
described the design, synthesis, and optical properties of the
new class of CS NIR functional dyes. To get insight into the
structure−optical properties of the CS NIR functional dyes, we
further performed quantum chemical calculations with the
B3LYP exchange functional employing 6-31G* basis sets using
a suite of Gaussian 09 programs. Furthermore, based on a novel
CS NIR platform, we constructed a new NIR fluorescent
TURN-ON sensor, which is suitable for fluorescence imaging
of endogenously produced HClO in the living animals.
RESULTS AND DISCUSSION
■
Design and Synthesis of a Series of Changsha NIR
Dyes (CS1−6). It is known that the absorption and emission
wavelengths of traditional rhodamine dyes are highly depend-
ent on the substituents on the amino group. For instance,
rhodamines 6G, B, and 101 emit at 551, 572, and 599 nm,
respectively.8 To provide a series of Changsha NIR dyes with
diverse absorption and emission wavelengths, we first designed
NIR dyes CS 1−3 (Figure 2), in parallel to rhodamines 6G, B,
and 101. On the other hand, it is known that the absorption
and emission wavelengths of traditional cyanine dyes could be
tuned with distinct substituents on the indolium moiety. It has
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a
Scheme 1. Synthesis of NIR Dyes CS1−6
a
Conditions: (a) H2SO4, Δ, then 70% HClO4; (b) acetic anhydride, 25 °C, 10 min; (c) acetic anhydride, 50 °C, 30 min.
been reported that benzo[e]indole-based cyanines have a
bathochromic shift of 20 nm when compared to indole-based
cyanines.14 On the basis of this consideration, we further
designed CS 4−6 to bear the benzo[e]indole moiety, in parallel
to CS 1−3. We anticipated that CS 4−6 may have longer
absorption and emission wavelengths relative to the corre-
sponding NIR dyes CS 1−3. Overall, we should have six new
NIR dyes, CS 1−6, with diverse wavelengths in the NIR region.
A retrosynthesis analysis of compounds CS1−6 is shown in
Scheme S1. Based on the proposed retrosynthesis analysis, we
designed a facile synthetic route to CS1−6 as outlined in
Scheme 1. Fisher aldehyde 3 is commercially available, and the
starting materials 1a−c and 4 were prepared according to the
reported procedures.10h,15,16 Condensation of compound 1a
with cyclohexanone in acidic conditions afforded the critical
intermediate 2a, which was then condensated with aldehyde 3
or 4 under very mild conditions (in acetic anhydride at 25 °C
within 10 min) to give the product CS1 or CS4 as a bright
green solid, respectively. The key to obtain the desired product
CS1 or CS4 is to control the reaction time (less than 10 min),
as prolonged reaction time would lead to the formation of
byproduct 5 or 6, the acetylated derivatives of CS1 or CS4. We
reasoned that this is due to the solvent of the reaction, acetic
anhydride, which is a good acetylating reagent and could
further react with the ethylamino group of CS1 or CS4 at the
prolonged reaction time. The similar synthetic pathway was
employed to synthesize compounds CS2, CS3, CS5, and CS6.
Since the additional acetylation is not impossible, the synthesis
of CS2, CS3, CS5, and CS6 proceeded at 50 °C for 30 min in
acetic anhydride for a good yield. Although compounds 5 and 6
are not the desired products, we envisioned that they could
serve as the control compounds in the process of the
examination of the structure-optical properties of CS1−6.
The structures of the products CS1−6 and controls 5, 6 were
1
fully characterized by H NMR, 13C NMR, MS (ESI), and
HRMS (ESI). Notably, the modular nature of the synthetic
strategy may allow it to be adapted to prepare a wide variety of
Changsha (CS)-type of NIR fluorescent dyes bearing distinct
substituents on the amino group and the indolium moieties.
Furthermore, the synthetic strategy is facile and scalable for
production of the new NIR dyes in large quantity.
Optical Properties. The absorption and emission profiles
of compounds CS1−6 in distinct solvents (EtOH, MeOH,
CH3CN, pH 7.4 PBS/CH3OH (v/v, 8: 2), CHCl3) are shown
in Figures 3 and S2−5, and the corresponding photophysical
data are compiled in Tables 1 and S1−4, respectively. As
designed, in general, compounds CS1−6 display both
absorption and emission peaks well into the NIR region, with
maximum at 688−728 nm and 721−763 nm in EtOH,
respectively (Table 1). Interestingly, CS1−6 tend to exhibit
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was observed for the corresponding rhodamines (rhodamine
101 > rhodamine B > rhodamine 6G),8 further corroborating
our design strategy. As anticipated, the emission bands of CS4,
CS5, and CS6 peak at 741, 750, and 763 nm, respectively,
which have an around 20 nm red-shift when compared to the
corresponding CS1, CS2, and CS3. This bathochromic shift
behavior is consistent with that observed for benzo[e]indole-
based cyanines over indole-based cyanines.14
The molar extinction coefficients (εmax) of the new NIR dyes
CS1−6 are in the range of 110 000 −140000 M−1cm−1, which
are superior or comparable to those of the classic rhodamine
dyes (80 000−120 000 M−1 cm−1). Remarkably, CS1−6 have
the fluorescence quantum yields (Φf) of 0.29−0.56 in EtOH,
which are great values for NIR functional dyes. In addition, the
brightness (εmaxΦf) of CS1−6 is in the range of 37 816−73 640
M−1 cm−1, which is comparable or superior to those of the
classic rhodamine dyes (for example, rhodamine B (52 650 M−1
cm−1) in EtOH.8b Furthermore, the Stokes shifts of CS1−6 are
from 31 to 35 nm, comparable to those of the typical
rhodamine and cyanine dyes.
We then examined the fluorescence profiles of the new dyes
and their amide derivatives at different pH values. As a
representative example, CS 2 (Figure 4A) and its amide
derivative CS2-NH2NH2 (Figure 4B) were studied. As shown
in Figure 4C, CS 2 is highly fluorescent over a wide pH range
of 5−11. However, the fluorescence drops significantly at
higher pH values (>11) likely due to the formation of the
nonfluorescent spirocyclic form (Figure 4A). pKcycl denotes the
pH value at which the emission of the compound decreases to
half the maximum emission due to the spirocyclization.1m The
pKcycl of CS 2 was determined to be over 12. In sharp contrast,
CS2-NH2NH2, the amide derivative of CS 2, has a pKcycl of 4.3
(Figure 4B). These results indicate that, at the physiological pH
of ∼7.4, CS 2 is present mainly as the fluorescent open form,
while CS2-NH2NH2 predominately exists as the nonfluorescent
spirocyclic form. In other words, the fluorescence of CS2 is on
while its amide derivative CS2-NH2NH2 is off at physiological
pH. The fluorescence on−off switching of CS dyes and their
amide derivatives is very similar to that of the classic
rhodamines and their amide derivatives. This implies that,
like the classic rhodamines,8a,b,9,10 the new CS NIR dyes could
be utilized as platforms to design NIR fluorescent sensors.
It is of importance to evaluate both the photostability and
chemical stability of functional fluorescent dyes intended for
bioapplications. The photostability of CS1−6 in EtOH solution
Figure 3. Absorption (A) and emission (B) spectra of compounds
CS1 (dark purple tilted square), CS2 (magenta circle), CS3 (olive-
green triangle), CS4 (green star), CS5 (purple square), and CS6
(maroon open circle) in EtOH. For comparison, the absorption and
emission spectra of rhodamine 6G (black open triangle) are also
shown.
longer absorption and emission in CH3CN. For instance, CS6
has an emission peak with maximum at 780 nm (Table S2),
which further extends to 870 nm in CH3CN (Figure S3B).
Thus, the emission of CS1−6 covers a significant part of the
“biological window (650−900 nm)” for in vivo imaging,
indicating the potential use of these new NIR dyes for
biological imaging in living animals.
CS1 shows an intense absorption band with maximum at 688
nm, assigned to the 0−0 band of the S1←S0 transition, and a
(less pronounced) shoulder peak on the high-energy side (at
around 650 nm), attributed to the 0−1 vibrational band of the
same transition (Figure 3A). In addition, CS1 also bears a very
weak and broad absorption band at around 460 nm, which can
be attributed to the S3←S0 transition. Close examination
reveals that the shape of the absorption spectrum of CS1 is
highly resemble to that of rhodamine 6G, indicating that CS1
may be called the analogue of rhodamine 6G at some sense.
However, significantly, CS1 displays a striking advantage over
rhodamine 6G: The absorption of CS1 has a large red-shift
(160 nm) in comparison to rhodamine 6G (Figure 3A). For the
emission, as shown in Figure 3B, CS1 has an intense emission
peak with maximum at 721 nm, extending to 850 nm. In good
agreement with the findings in the absorption, CS1 also
exhibits a drastic bathochromic shift (160 nm) in emission
while maintaining the similar shape in the emission spectrum
when compared to rhodamine 6G. The same red-shift trend
was observed in CS2 and CS3 relative to rhodamines B and
101, respectively, validating the design strategy. On the other
hand, the emission wavelengths of these new NIR functional
dyes are in the order of CS3 > CS2 > CS1, and the same order
Table 1. Photophysical Data of the Dyes CS1−6, 5, and 6, Rhodamines 6G, B, and 101 in EtOH
a
b
c
d
−1
dye
CS 1
λabs/nm
εmax/M−1 cm
λem/nm
Φf
Stokes shift
fwhm/nm
688
700
131 500
139 500
721
731
745
741
750
763
687
715
551
572
599
0.56
0.41
32
31
43
42
48
45
44
48
90
90
CS 2
CS 3
CS 4
CS 5
CS 6
5
710
125 445
0.34
35
708
135 880
0.34
33
720
125 971
0.30
30
728
130 400
0.29
35
612/656
632/678
530
54 120/49 913
55071/43618
116 000
<0.005
<0.005
0.958c
0.53
75/31
83/37
21
6
Rho 6G
Rho B8b
Rho 1018b
553
117 000
19
574
110 000
0.89
25
a
b
c
The maximal absorption of the dye. The maximal emission of the dye. Φf is the relative fluorescence quantum yield estimated by using ICG (Φf =
0.13 in DMSO) as a fluorescence standard.17,18 The full width at half-maximum height.
d
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chemical calculations with the B3LYP exchange functional
employing 6-31G* basis sets using a suite of Gaussian 09
programs to get insight into this phenomenon (see below).
Although controls 5 and 6 are essentially nonfluorescent, we
envisioned that they can be employed as“latent” fluorophores
for potential applications in chemical biology.19a,b
Thus, the above optical studies indicate that the new
functional NIR dyes CS1−6 have the important parameters
such as molar absorptivity, quantum yields, Stokes shifts, and
brightness superior to or comparable to those of the classic
rhodamine dyes. In addition, CS1−6 possess good photo-
stability and chemical stability. Interestingly, the shape of both
absorption and emission spectra of CS1−6 highly resembles
that of the classic rhodamine dyes. Significantly, like the classic
rhodamine dyes, CS1−6 also intrinsically bear a carboxylic-
acid-controlled fluorescence on−off switching mechanism, as
they have very similar electron pulling systems for spirocycliza-
tion. However, notably, new functional NIR dyes CS1−6 are
advantageous over the classic rhodamine dyes, as CS1−6 have
both absorption and emission in the NIR region (in some case,
extending up to 870 nm). Thus, novel NIR dyes CS1−6 may
have the potential as the NIR surrogates of the classic
rhodamine dyes with promising biological imaging applications
in living animals.
Figure 4. (A,B) Structures of (A) CS2 and (B) CS2-NH2NH2 mainly
present at various pH values. The highlighted structures are the
putative forms predominantly present at the physiological pH. (C)
pH-dependence of the fluorescence intensity of CS2 (black square)
and CS2-NH2NH2 (red circle).
Density Functional Theory (DFT) Calculations. To shed
light on the optical properties of new functional NIR
fluorescent dyes CS1−6 and controls 5 and 6, DFT calculations
with the B3LYP exchange functional employing 6-31G* basis
sets using a suite of Gaussian 09 programs20 were conducted.
Figures 5 and S10−14 show the representative optimized
was measured by continuous irradiation with a Xe lamp (150
W) at 10 nm slit width at the maximal absorption wavelength of
CS1−6, and the results demonstrate that over 97% of the initial
fluorescence intensity was retained after 1 h irradiation (Figure
S8A), indicating that these novel functional NIR dyes have
sufficient photostability for potential biological applications. It
is known that many long wavelength organic dyes are easily
bleached by various oxidants.17 Thus, we decided to investigate
the emission profiles of the new CS NIR dyes in the presence
of biologically relevant oxidants, reactive oxygen species (ROS).
As a representative case, CS2 was treated with 100 μM
hydroxyl radical (OH·), H2O2, HClO, tert-butyl hydroperoxide
(TBHP), and tert-butoxy radical (·OtBu) for 30 min. As
demonstrated in Figure S8B, the emission profile of CS2 is
almost unchanged. Furthermore, over 94% of the initial
fluorescence intensity was still retained after treatment of
CS2 with HClO (a biologically significant ROS) for 70 min
(Figure S8C). Thus, these data suggest that the new functional
NIR dyes have sufficient chemical stability.
We also investigated the optical properties of controls 5 and
6, the acetylated derivatives of compounds CS1 and CS4,
respectively. However, the optical profiles of controls 5 and 6
(Figure S9) are drastically distinct from those of CS1 and CS4.
Controls 5 and 6 have very broad and blue-shifted peaks in the
absorption ranging from 500 to 700 nm. By contrast, the
absorption bands of CS1 and CS4 are relatively sharp in the
NIR region. In addition, controls 5 and 6 are almost
nonfluorescent with fluorescent quantum yields < 0.005,
whereas CS1 and CS4 are highly fluorescent with large
fluorescent quantum yields up to 0.56. The dramatic distinction
in the optical profiles of 5 and 6 relative to CS1 and CS4 can be
attributed to the acetyl group on the nitrogen atom, which
significantly diminishes the electron donating ability of the
nitrogen atom. The similar behavior was observed on the classic
rhodamines or its analogues.19 We further performed quantum
Figure 5. DFT optimized structure of CS 1. In the ball-and-stick
representation, carbon, nitrogen, and oxygen atoms are colored in
gray, blue, and red, respectively. H atoms were omitted for clarity.
structures of CS1−6. The C−C (N or O) bond lengths (in
pm) of CS1−6 determined by DFT calculations are displayed
in Tables 2 and S5−9, and the HOMO/LUMO molecular
orbital plots of CS1−6 and controls 5 and 6 are exhibited in
Figure 6. In the case of CS1, the benzoic acid moiety is highly
twisted and essentially vertical to the 2,3-dihydro-1H-xanthene
core with a dihedral angle of 89.8° (Figure 5). The C9−C27
length is 149.8 pm (Table 2), which is close to the typical
carbon−carbon single bond (154 pm). These findings reveal
that the benzoic acid moiety and the 2,3-dihydro-1H-xanthene
core are essentially electronically decoupled. In other words,
they are orthogonal and almost no electronic conjugation, in
good agreement with the observation that the benzoic acid unit
has essentially no contribution to the HOMO/LUMO of CS1
(Figure 6). Interestingly, this finding is highly resemble to the
case in the classic xanthene-based fluorophores (i.e.,
fluorescein), in which the benzoic acid moiety and the
xanthene core are electronically decoupled.21 On the other
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Table 2. Representative C−C (N or O) Bond Lengths (in pm) of CS 1 Determined by DFT Calculations
C−C bond bond lengths (in pm)
C−C bond
bond lengths (in pm)
C−C bond
bond lengths (in pm) C−N/O bond bond lengths (in pm)
C5−C6
C6−C7
C7−C8
C8−C11
C5−C12
140.9
142.9
138.9
141.4
138.9
C11−C12
C11−C9
C9−C13
C13−C14
C14−C4
140.9
142.8
139.1
141.9
141.8
C4−C15
C15−C16
C16−C17
139.3
140.5
139.1
C14−O10
O10−C12
C6−N26
C17−N18
N18−C19
135.9
136.4
136.7
136.2
141.5
C9−C27
149.8
hand, the indolium moiety is coplanar and perfectly conjugated
with the 2,3-dihydro-1H-xanthene core. Notably, all the C−C
bond lengths of the conjugated 2,3-dihydro-1H-xanthene-
indolium backbone are very similar, at around 140 pm (Table
2). This value is the intermediate between the typical carbon−
carbon single (154 pm) and double (134 pm) bonds, attributed
to the strong electronic delocalization and the partial decrease
in the carbon−carbon bond length alternation (BLA, difference
between consecutive single and double bonds) along the π-
conjugated system.22 This is further confirmed by the analysis
of the terminal C−N and C−O bond lengths in the conjugated
system. The bond lengths of C6−N26 and C17−N18 are 136.7
and 136.2 pm, respectively, which are the intermediates
between the lengths of C−N bond (single bond, around 147
pm) and the iminium CN (double bond, around 128 pm).
The bond lengths of C14−O10 and O10−C12 are 135.9 and
136.4 pm, respectively, which are the intermediates between the
lengths of C−O (single bond, around 144 pm) and CO
(double bond, around 121 pm). Significantly, these results are
in accordance with the absorption spectrum of CS 1 (Figure
3A), which bears a sharp and intense absorption band in the
NIR spectral range resulting from the reduced vibronic
contribution in the nonalternating structure, a characteristic
of cyanine dyes in the “cyanine limit”.22 In addition, time-
dependent DFT (TDDFT) calculations indicate that CS1 has a
strong absorption band at long wavelength (f (oscillator
strength) = 1.275) attributed to the S1←S0 transition and a
weak absorption band at short wavelength (f = 0.435) assigned
to the S3←S0 transition. Thus, the results of TDDFT
calculations are in good agreement with the absorption spectra
of CS1 observed experimentally (Figure 3A).
The π electrons on the HOMO of CS1 is mainly located on
the whole π-conjugated 2,3-dihydro-1H-xanthene-indolium
framework (including the amino group), but the LUMO is
mostly positioned at the center of the conjugated 2,3-dihydro-
1H-xanthene-vinyl bridge (Figure 6), where the BLA is
minimal. When the substituent on the amino group was
changed from monoethylamino (CS1), diethylamino (CS2), to
fused amino (CS3), the energy levels of both HOMO and
LUMO increase relative to those of CS1 due to the enhanced
hyperconjugation between the electrons in the σ bonds of the
substituents on the amino group with the adjacent p and π
orbitals, giving an extended molecular orbital which boosts the
stability of the system (Figure 6). However, the HOMO energy
level raises more pronouncedly than the LUMO level. Thus,
the HOMO−LUMO energy gaps of CS2 and CS3 become
smaller relative to that of CS1. The HOMO−LUMO energy
gaps of these new functional NIR dyes obey the following
order: CS1 > CS2 > CS3, which is in good agreement with the
measured emission wavelengths of them, in the order of CS1 <
CS2 < CS3.
Figure 6. Molecular orbital plots (LUMO and HOMO) and HOMO/
LUMO energy gaps of NIR dyes CS1−6 and controls 5 and 6.
of C17−N18 (135.9 pm) (Table S7), and the electrons in the
fused phenyl moiety only have minor contributions to the
HOMO of CS4 (Figure 6). These data could account for only a
moderate enhancement of the energy levels of both HOMO
and LUMO of CS4 in comparison to those of CS1. However,
the HOMO level increases more markedly than the LUMO
level. Thus, the HOMO−LUMO gap of CS4 is smaller than
that of CS1, consistent with the observation that the emission
wavelength of CS4 has a slight red-shift (20 nm) compared to
CS1. The similar findings are present in CS5 and CS6 relative
to CS2 and CS3.
From the structure point of view, the only difference between
control 5 and CS1 is that there is an acetyl group on N26.
However, this subtle distinction apparently elicits a significant
effect on the molecular structures, which may account for their
drastic difference in the optical properties. The C6−N26 bond
length of compound 5 is 142.4 pm (Figure S15, Table S10),
which is longer than that (136.7 pm) of C6−N26 of CS1 and
that (135.6 pm) of C17−N18 of compound 5. This weakens
the p−π conjugation between the amino group and π-
conjugated xanthene−indolium framework, and the energy
levels of both HOMO and LUMO decrease relative to those of
CS1 (Figure 6). Close examination reveals that the π electrons
on the HOMO of control 5 are mainly located on the π-
conjugated xanthene−indolium framework, while the lone pair
electrons of N26 no longer contributes to the HOMO of
compound 5. By contrast, the lone pair electrons of N26
contribute to the HOMO of CS 1. This suggests that the
acetylation on N26 atom indeed decreases the electron-
The HOMO−LUMO energy gaps and the emission
wavelengths of CS4−6 are also closely correlated. The N18−
C19 bond length (141.4 pm) of CS4 is much longer than that
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donating ability of the nitrogen atom, and compound 5 loses its
“cyanine limit” structure to some extent. In other words, the
acetylation on N26 atom forbids the formation of the
resonance form. This could account for the blue-shift in
fluorescence and much lower quantum yield of control 5
compared to CS1. In addition, the observation that CS 2 and
CS2-NH2NH2 have low fluorescence at strong acidic
conditions could also be explained by the protonation of the
nitrogen atom (Figure 4), which significantly diminishes the
electron donating ability of the nitrogen atom. Raines et al.
pointed out that the decrease of electron-donation capability of
the different nitrogen substituents of the classic rhodamines
contributes to the reduction in quantum yields by decreasing
the C−N bond order and thus enhancing nonradiative decay of
the excited state through vibrational relaxation processes.19a
Thus, our findings in new CS NIR dyes are in good agreement
with those in the classic rhodamines. The drastic distinction
between the optical properties of control 6 (Figure S16, Table
S11) and CS4 can be explained in a similar manner.
Development of a Novel NIR Fluorescent Turn-on
HClO Sensor and Its Application for Biological Imaging
in the Living Animals. New NIR functional fluorescent dyes
CS1−6 bear excellent photochemical properties including large
absorption extinction coefficients, high fluorescence quantum
yields, high brightness, sufficient photostability and chemical
stability, and, most importantly, both absorption and emission
in the NIR region. These desirable features should render the
novel NIR functional fluorescent dyes suitable for fluorescence
staining in the living animals. To demonstrate this possibility, as
a representative example, the new NIR functional fluorescent
dye CS2 was injected into the living mice, and a large
fluorescence signal was noted within a few minutes of injection
(Figure S17). This indicates the potential in vivo imaging
applications of the new NIR dyes.
biologically significant ROS, is produced from peroxidation of
chloride ions catalyzed by the enzyme myeloperoxidase (MPO)
in activated leukocytes.23 However, abnormal levels of HOCl
are implicated with many inflammation-associated diseases
including cardiovascular diseases, rheumatoid arthritis, and
cancer.24 Thus, the detection of endogenously produced HClO
in living systems is very important. Briefly, the design of NIR
fluorescent turn-on sensor 7 was based on the oxidant-
mediated reaction of thiosemicarbazides to 1,3,4-oxadiazoles.25
We hypothesized that the thiosemicarbazide-based 7 is
nonfluorescent as the spirocyclic form is predominated at
physiological pH. However, upon reaction with HClO, the
thiosemicarbazide should be converted to 1,3,4-oxadiazole
(Scheme S2). The resulting 1,3,4-oxadiazole-based dye should
be fluorescent, as the open form is predominated at
physiological pH. Thus, we envisioned that treatment of sensor
7 with HClO could provide a fluorescence turn-on signal under
the physiological conditions.
As designed, the free sensor 7 is essentially nonfluorescent
(Figure 7a). However, addition of HClO elicits a dramatic
Figure 7. (a) Fluorescence spectra of NIR fluorescent sensor 7 (5
μM) in the presence of various concentrations of HClO (0−70 μM) in
PBS buffer (50 mM, pH 7.4, containing 30% CH3CN) with excitation
at 690 nm. Inset: Fluorescence intensity of sensor 7 (5 μM) at 746 nm
vs HClO concentration (0−70 μM). (b) The fluorescence intensity of
sensor 7 at 746 nm excited at 690 nm in the presence of various
biologically relevant analytes (70 μM).
Notably, another important character of the unique NIR
functional dyes CS1−6 is that, like classic rhodamines, they
have the carboxylic-acid-regulated fluorescence on−off switch-
ing mechanism by the spirocyclization. This implies that, like
classic rhodamines, CS1−6 could be employed as robust
platforms for development of NIR fluorescent sensors for
biological imaging applications in living animals.
change in the fluorescence spectra. A significant fluorescence
turn-on response was observed at 746 nm (extending into 870
nm). Mass, NMR, absorption, and emission spectrometry
analysis confirmed that the fluorescence turn-on is indeed due
to the HClO-mediated formation of the open form 1,3,4-
oxadiazole-based fluorescent dye (Figures S18−21). In
addition, as shown in Figure 7b, NIR sensor 7 is highly
selective to HOCl over other various biologically relevant
analytes (reactive oxygen species, reactive nitrogen species, and
other relevant analytes.) represented by hydrogen peroxide
(H2O2), superoxide (O2•‑), hydroxyl radical (OH·), tert-butyl
hydroperoxide (TBHP), tert-butoxy radical (·OtBu), nitric
Toward this end, we constructed a novel NIR fluorescent
turn-on sensor 7 (Scheme 2) for biological imaging of
Scheme 2. Synthesis of NIR Fluorescent Turn-on Sensor 7
a
for HOCl
−
−
oxide (NO), NO3 , NO2 , ascorbic acid (AA), and
dehydroascorbic acid (DHA), suggesting that the new NIR
sensor is promising for applications in biological systems.
The new NIR sensor 7 was preliminarily applied to detect
endogenously produced HClO in living macrophage cells.
When stimulated by lipopolysaccharide (LPS) and phorbol
myristate acetate (PMA), macrophages may produce endoge-
nous HOCl.26 The living RAW264.7 macrophage cells loaded
with only NIR sensor 7 (5 μM) display almost no fluorescence
(Figure 8a). However, after the macrophage cells have been
incubated with LPS (1 μg/mL) for 12 h, and then further
coincubated with PMA (1 μg/mL) and sensor 7 (5 μM) for 30
min, a dramatic enhancement in the NIR emission (Figure 8e)
was observed. These data indicate that the new NIR sensor 7 is
a
Conditions: (a) N-hydroxysuccinimide (HOSu), DCC, CH2Cl2; (b)
NH2NHCSNH2, TEA.
endogenously produced HClO in both the living macrophage
cells and the living mice. Reactive oxygen species (ROS)
mediate a wide variety of biological processes in the healthy and
disease states. Hypochlorous acid (HOCl), one of the
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Figure 9. Representative fluorescent images (pseudocolor) of in vivo
HClO production from the peritoneal cavity of the mice with sensor 7
during an LPS-mediated inflammatory response: (a) negative control,
neither LPS nor sensor 7 was injected; (b) saline was injected in the
intraperitoneal (i.p.) cavity of mice, followed by i.p. injection of sensor
7 (120 nmol); (c) LPS was injected into the peritoneal cavity of the
mice, followed by i.p. injection of sensor 7 (120 nanomoles). Ten
minutes after the sensor 7 injection, the mice were imaged using a
FMT 2500 LX quantitative tomography in vivo imaging system with
an excitation filter 670 nm and an emission range = 690−740 nm. (d)
Quantification of fluorescence emission intensity from the groups a−c.
The total number of photons from the entire peritoneal cavity of the
groups a−c was integrated and plotted as a ratio to the control group
a. The time-dependent and dose-dependent fluorescence images are
shown in Figure S22.
Figure 8. (a) Fluorescence image (red channel) of RAW264.7
macrophage cells incubated with only NIR sensor 7 (5 μM) for 60 min
at 37 °C. (b−f) RAW264.7 macrophage cells pretreated with 1 μg/mL
LPS for 12 h, further coincubated with PMA (1 μg/mL), sensor 7 (5
μM), Hoechst 33258 (4.5 μM), and Mito Tracker Green (5 μM) for
60 min. (b) DIC image; (c) emission from the blue channel (nuclear
staining); (d) emission from the green channel (mitochondria
staining); (e) emission from the red channel; and (f) overlay of the
blue, green, and red channels.
capable of fluorescence imaging of endogenously produced
HOCl in the living RAW264.7 macrophage cells. Notably, the
nuclear staining with Hoechst 33258 (Figure 8c) suggests that
the sensor has no obvious cytotoxicity to the cells. In addition,
the nuclear staining (Figure 8c) and mitochondria staining
(Figure 8d) reveal that sensor 7 associates with the
mitochondria of RAW264.7 macrophage cells (Figure 3c−f).
The major advantage of the new NIR CS functional dyes
over the traditional rhodamines is that our CS dyes have both
absorption and emission in the NIR region while retaining the
rhodamine-like fluorescence on−off switching mechanism by
the spirocyclization. Thus, the unique NIR CS dyes are superior
to the traditional rhodamines in that our CS dyes are favorable
for biological imaging applications in living animals, as NIR
light leads to minimum photodamage to biological samples,
deep tissue penetration, and minimum interference from
background autofluorescence by biomolecules in the living
animals. Thus, we decided to examine the possibility of the new
NIR sensor 7 for biological imaging of endogenously produced
HClO in the living mice. The HClO production in vivo was
generated by activated macrophages and neutrophils in an
lipopolysaccharide (LPS) model of acute inflammation.6 The
ICR mice were divided into three groups. One group was
untreated as the control group. The second group was given
saline (400 μL) in the peritoneal cavity, followed by
intraperitoneal (i.p.) injection with sensor 7 (120 nanomoles).
The third group was given an i.p. injection of LPS (1 mg in 400
μL saline), and followed by i.p. injection with sensor 7 (120
nmol) after 4 h. The mice were anesthetized, and the
abdominal fur was removed. Ten minutes after the sensor 7
injection, the mice were imaged using a FMT 2500 LX
quantitative tomography in vivo imaging system. As shown in
Figure 9c, the mice treated with both LPS and sensor 7
exhibited a significantly higher fluorescence readout (pseudo-
color) than the untreated mice (Figure 9a) or mice treated with
only sensor 7 (Figure 9b). The fluorescent intensity from the
abdominal area of the mice was quantified, and the data
indicate that the mice loaded with LPS and sensor 7 have
approximately 4-fold higher fluorescence intensity than the
mice loaded with saline and sensor 7 (Figure 9d). Thereby,
these results demonstrate that the new NIR sensor 7 can image
endogenously produced HClO in the living animals.
CONCLUSION
■
In summary, we have introduced a new strategy to design NIR
functional dyes with the carboxylic-acid-controlled fluorescence
on−off switching mechanism by the spirocyclization. Based on
the design strategy, we have synthesized a series of Changsha
(CS1−6) NIR fluorophores, a unique class of NIR functional
fluorescent dyes, bearing excellent photophysical properties
while retaining the rhodamine-like fluorescence ON−OFF
switching mechanism. Importantly, CS1−6 NIR dyes are
superior to the traditional rhodamine dyes with both absorption
and emission in the NIR region. In addition, we have
performed quantum chemical calculations with the B3LYP
exchange functional employing 6-31G* basis sets using a suite
of Gaussian 09 programs to provide insights into the structure-
optical properties of CS NIR dyes. Furthermore, using CS2 as a
platform, we further constructed the novel NIR fluorescent
TURN-ON sensor 7, which is capable of imaging endogenously
produced HClO in the living animals, demonstrating the value
of our CS NIR functional fluorescent dyes. We expect that the
design strategy may be extended for development of a wide
variety of NIR functional dyes with a suitable fluorescence-
controlled mechanism, and CS1−6 will be employed as a
molecular tool for numerous biological applications. Future
efforts include development of ratiometric fluorescent sensors
based on the CS NIR functional fluorescent dyes.
EXPERIMENTAL SECTION
■
Raw 264.7 Murine Macrophages Culture and Imaging Using
Sensor 7. Raw 264.7 murine macrophages were obtained from
Xiangya hospital and cultured in DMEM (Dulbecco’s modified Eagle's
medium) supplemented with 10% FBS (fetal bovine serum) in an
atmosphere of 5% CO2 and 95% air at 37 °C. For detection of
endogenously produced HOCl, the living RAW 264.7 macrophages
were treated with LPS (1 μg/mL) for 12 h and then further
coincubated with PMA (1 μg/mL), sensor 7 (5 μM), Hoechst 33258
(4.5 μM), and Mito Tracker Green (5 μM) for 60 min. Prior to
imaging, the cells were washed three times with 1 mL of PBS, and the
fluorescence images were acquired through an Olympus fluorescence
microscopy equipped with a cooled CCD camera.
Fluorescence Imaging in Living Mice. ICR mice (20−25 g)
were given an intraperitoneal (i.p.) injection of LPS (1 mg in 400 μL
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saline). After 4 h, the mice were anesthetized by an i.p. injection of
xylazine (10 mg/kg) and ketamine (80 mg/kg) and the abdominal fur
was removed with an electric shaver. Then, the mice were
intraperitoneally injected with sensor 7 (400 μM in 400 μL of 1:3 =
CH3OH/PBS). As a control, untreated mice (neither treated with LPS
nor sensor 7) or unstimulated mice intraperitoneally injected only with
sensor 7 (400 μM in 400 μL of 1:3 = CH3OH/PBS) were also
prepared. The mice were then imaged (10 min after the injection of
sensor 7) by using a FMT 2500 LX quantitative tomography in vivo
imaging system, with an excitation filter of 670 nm and an emission
filter of 690−740 nm.
3.17−3.20 (t, J = 6.0 Hz, 2H), 3.40−3.43 (t, J = 5.6 Hz, 2H), 3.46−
3.50 (2H), 3.68 (s, 3H), 6.07−6.10 (d, J = 14.0 Hz, 1H), 6.39 (s, 1H),
7.21 (d, J = 7.6 Hz, 1H), 7.44 (t, J = 7.6 Hz, 1H), 7.55 (d, J = 8.8 Hz,
1H), 7.60−7.66 (2H), 7.72 (t, J = 7.6 Hz, 1H), 7.97 (t, J = 8.4 Hz,
2H), 8.19 (d, J = 8.0 Hz, 1H), 8.24 (d, J = 8.4 Hz, 1H), 8.70−8.74 (d, J
= 14.0 Hz, 1H). 13C NMR (125 MHz, CDCl3): 21.89, 22.08, 25.17,
27.65, 28.44, 28.49, 28.74, 30.74, 31.72, 50.79, 51.31, 51.78, 98.65,
106.03, 111.68, 115.16, 116.89, 123.26, 124.50, 125.22, 125.66, 128.67,
129.55, 130.44, 130.67, 131.13, 131.64, 132.28, 133.18, 133.89, 140.50,
163.85. MS (ESI) m/z 633.3 (M-ClO4)+; HRMS (ESI) m/z calcd for
+
C43H41N2O3 (M-ClO4)+: 633.3112. Found 633.3135.
Synthesis of Compounds CS 2−3, CS 5−6, and 5−6. Freshly
distilled cyclohexanone (6.6 mL, 63.7 mmol) was added dropwise to
concentrated H2SO4 (70 mL) and cooled down to 0 °C. Then,
compound 1a, 1b, or 1c (32 mmol) was added in portions with
vigorous stirring. The reaction mixture was heated at 90 °C for 1.5 h,
cooled down, and poured onto ice (300 g). Perchloric acid (70%; 7
mL) was then added, and the resulting precipitate was filtered off and
washed with cold water (100 mL). Compound 2a, 2b, or 2c obtained
as a red solid was used for the next step without further purification.
Compound 2a, 2b, or 2c (0.21 mmol) and Fisher aldehyde 3 or 4
(0.22 mmol) were dissolved in acetic anhydride (8 mL), and the
reaction mixture was heated to 50 °C and further stirred at 50 °C for
30 min. Then, water (8 mL) was added to the reaction mixture to
quench the reaction. The solvent was removed under reduced pressure
to give the crude product, which was purified by silica gel flash
chromatography using CH2Cl2 to CH2Cl2/ethanol (200:1 to 20:1) as
eluent to afford compounds CS 2−3 and CS 5−6 as green solid and
compounds 5−6 as blue solid.
5. 1H NMR (500 MHz, CDCl3):1.14−1.19 (3H), 1.73 (9H), 1.80−
1.82 (3H), 2.02−2.10 (4H), 2.52 (1H), 2.64 (1H), 3.31−3.41 (4H),
4.02 (m, 1H), 5.57 (t, J = 14.4 Hz, 1H), 6.63 (t, J = 2.4 Hz, 1H), 6.76
(t, J = 8.4 Hz, 1H), 6.95−6.99 (2H), 7.20−7.26 (3H), 7.59 (t, J = 7.4
Hz, 1H), 7.66 (d, J = 7.6 Hz, 1H), 7.73 (t, J = 14.4 Hz, 1H), 8.04−8.07
(1H). 13C NMR (100 MHz, CDCl3): 13.11, 17.25, 21.29, 22.44, 27.98,
28.59, 30.76, 32.81, 44.59, 105.46, 113.77, 116.55, 117.32, 123.63,
124.74, 128.38, 128.46, 130.06, 130.14, 130.32, 131.27, 131.56, 134.07,
135.34, 143.48, 143.76, 144.58, 147.54, 152.41, 162.45, 172.35, 179.84.
MS (ESI) m/z 587.3(M-ClO4)+; HRMS (ESI) m/z calcd for
+
C38H39N2O4 (M-ClO4)+: 587.2904. Found 587.2917.
1
6. H NMR (400 MHz, CDCl3): 1.16−1.22 (3H), 1.70−1.79 (m,
3H), 1.82−1.84 (3H), 2.06−2.07 (10H), 2.50−2.70 (2H), 3.33−3.42
(m, 1H), 3.46−3.48 (3H), 4.01−4.10 (m, 1H), 5.67 (t, J = 14.4 Hz,
1H), 6.66 (d, J = 4.0 Hz, 1H), 7.01 (s, 1H), 7.16−7.22 (2H), 7.30−
7.36 (m, 1H), 7.52−7.54 (m, 1H), 7.56 (t, J = 7.6 Hz, 1H), 7.64 (t, J =
7.6 Hz, 1H), 7.80−7.86 (2H), 7.94 (t, J = 14.4 Hz, Hz, 1H), 8.07−8.09
(1H), 7.14−8.16 (d, J = 8.4 Hz, 1H). 13C NMR (100 MHz, CDCl3):
12.96, 17.06, 19.19, 21.45, 22.64, 24.37, 24.77, 27.60, 27.69, 27.82,
30.56, 43.12, 109.50, 116.61, 118.09, 119.99, 121.87, 123.29, 124.73,
127.17, 128.74, 129.42, 129.96, 131.41, 133.82, 143.05, 150.02, 170.05.
MS (ESI) m/z 637.3 (M-ClO4)+; HRMS (ESI) m/z calcd for
CS 2. 1H NMR (400 MHz, CD3OD): 1.24 (t, J = 6.8 Hz, 6H), 1.81
(8H), 2.26−2.36 (m, 2H), 2.68 (t, J = 6.0 Hz, 2H), 3.52−3.57 (q, 4H),
3.63 (s, 3H), 6.16−6.19 (d, J = 13.6 Hz, 1H), 6.73−6.80 (3H), 7.26−
7.32 (3H), 7.42 (t, J = 7.6 Hz, 1H), 7.54 (d, J = 7.2 Hz, 1H), 7.65−
7.69 (td, J = 7.6, 1.2 Hz, 1H), 7.75−7.79 (td, J = 7.6, 1.2 Hz, 1H), 8.24
(d, J = 7, 2 Hz, 1H), 8.66−8.69 (d, J = 14.0 Hz, 1H). 13C NMR (100
MHz, CD3OD): 13.73, 26.19, 28.81, 29.63, 29.64, 32.66, 47.07, 51.49,
97.79, 101.55, 112.84, 114.54, 117.57, 124.37, 127.27, 130.28, 130.79,
131.46, 131.72, 132.43, 133.35, 135.12, 138.35, 143.29, 144.42, 145.33,
158.25, 165.46, 169.60. 176.22. MS (ESI) m/z 559.2 (M-ClO4)+;
+
C42H41N2O4 (M-ClO4)+: 637.3061. Found 637.3080.
Synthesis of Compounds CS 1 and CS 4. Compound 2a (100.1
mg, 0.21 mmol) and Fisher aldehyde 3 or 4 (0.26 mmol) were
dissolved in acetic anhydride (20 mL), and the reaction mixture were
stirred at room temperature for 10 min. Then, the reaction mixture
was poured into saturated NaHCO3 solution (200 mL) and was
extracted with CH2Cl2 (200 mL) for three times. The organic phases
were combined and dried by anhydrous MgSO4. The solution was
then removed under reduced pressure to give the crude product, which
was purified by silica gel flash chromatography using CH2Cl2 to
CH2Cl2/ethanol (200:1 to 20:1) as eluent to afford compound CS 1
or CS 4 as a green solid.
+
HRMS (ESI) m/z calcd for C37H39N2O3 (M-ClO4)+: 559.2955.
Found 559.2967.
1
CS 3. H NMR (500 MHz, CD3OD):1.79−1.83 (8H), 1.89−1.92
(m, 2H), 2.14−2.17 (m, 2H), 2.31−2.34 (m, 2H), 2.62 (t, J = 6.0 Hz,
2H), 2.67 (s, 2H), 3.13 (t, J = 6.0 Hz, 2H), 3.41 (t, J = 5.8 Hz, 2H),
3.48 (t, J = 5.6 Hz, 2H), 3.56 (s, 3H), 6.02−6.05 (d, J = 13.6 Hz, 1H),
6.39 (s, 1H), 7.18−7.22 (3H), 7.38 (t, J = 7.6 Hz, 1H), 7.48 (d, J = 6.8
Hz, 1H), 7.64 (td, J = 7.6, 1.2 Hz, 1H), 7.73 (d, J = 7.6 Hz, 1H), 8.19
(dd, J = 7.0, 1.2 Hz, 1H), 8.59−8.62 (d, J = 13.6 Hz, 1H). 13C NMR
(100 MHz, CDCl3): 22.13, 22.85, 23.01, 26.12, 26.16, 29.73, 29.78,
29.83, 31.72, 36.32, 51.74, 52.30, 99.46, 106.78, 111.92, 116.39,
118.16, 122.27, 124.23, 125.69, 126.20, 126.47, 130.68, 131.32, 133.01,
134.25, 137.95, 141.42, 142.76, 145.65, 150.71, 153.91, 164.98, 173.37.
MS (ESI) m/z 583.3(M-ClO4)+; HRMS (ESI) m/z calcd for
1
CS 1. H NMR (400 MHz, CD3OD): 1.33 (3H), 1.78 (8H), 2.05
(s, 3H), 2.33 (2H), 2.67 (2H), 3.38 (2H), 3.56 (s, 3H), 6.07−6.10 (d,
J = 14.0 Hz, 1H), 6.57 (s, 1H), 6.63−6.68 (m, 1H), 7.24 (3H), 7.40 (t,
J = 7.2 Hz, 1H), 7.50 (t, J = 7.6 Hz, 1H), 7.66 (t, J = 7.2 Hz, 1H), 7.75
(t, J = 7.6 Hz, 1H), 8.22 (d, J = 7.2 Hz, 1H), 8.61 (1H). 13C NMR
(100 MHz, CD3OD): 15.20, 18.60, 22.73, 26.25, 29.77, 32.38, 36.38,
40.13, 50.63, 95.50, 100.60, 112.41, 116.06, 117.66, 123.02, 124.28,
126.01, 126.76, 129.25, 130.73, 131.48, 133.24, 134.81, 138.17, 143.02,
143.14, 145.46, 154.96, 157.89, 165.53, 175.03. MS (ESI) m/z 545.3
+
C39H39N2O3 (M-ClO4)+: 583.2955. Found 583.2969.
1
+
CS 5. H NMR (400 MHz, CD3OD): 1.23 (t, J = 6.8 Hz, 6H),
(M-ClO4)+; HRMS (ESI) m/z calcd for C36H37N2O3 (M-ClO4)+:
1.74−1.80 (m, 2H), 2.02 (6H), 2.42−2.60 (m, 2H), 2.65 (t, J = 6.0
Hz, 2H), 3.49−3.56 (q, 4H), 3.72 (s, 3H), 6.14−6.18 (d, J = 14.0 Hz,
1H), 6.65−6.73 (3H), 7.20−7.22 (d, J = 7.2 Hz, 1H), 7.43 (t, J = 7.6
Hz, 1H), 7.54−7.61 (3H), 7.69−7.739 (td, J = 7.6, 1.2 Hz, 1H), 7.93−
7.98 (2H), 8.17 (d, J = 8.0 Hz, 1H), 8.26 (d, J = 8.4 Hz, 1H), 8.70−
8.74 (d, J = 14.8 Hz, 1H). 13C NMR (125 MHz, CD3OD): 13.80,
22.69, 26.29, 28.86, 29.21, 31.74, 33.09, 46.93, 97.85, 101.46, 112.99,
114.22, 115.60, 117.32, 122.98, 124.49, 127.15, 129.79, 130.22, 130.39,
131.42, 131.76, 132.12, 132.75, 133.35, 134.53, 135.07, 135.92, 138.44,
142.69, 143.81, 154.38, 158.27, 165.21, 169.77, 177.89. MS (ESI) m/z
545.2799. Found 545.2824.
CS 4. H NMR (400 MHz, CD3OD): 1.32 (t, J = 7.2 Hz, 3H),
1
1.80−1.84 (m, 2H), 2.05 (s, 3H), 2.07 (s, 3H), 2.08 (s, 3H), 2.30−
2.35 (q, 2H), 2.69 (t, J = 5.6 Hz, 2H), 3.38−3.38 (m, 2H), 3.68 (s,
3H), 6.11−6.14 (d, J = 14.0 Hz, 1H), 6.55 (s, 1H), 6.69 (s, 1H), 7.23
(d, J = 7.6 Hz, 1H), 7.46 (t, J = 7.6 Hz, 1H), 7.56 (d, J = 9.2 Hz, 1H),
7.61−7.68 (2H), 7.75 (td, J = 6.8, 1.2 Hz, 1H), 7.98 (t, J = 8.8 Hz,
2H), 8.22 (d, J = 7.2 Hz, 1H), 8.31 (d, J = 8.4 Hz, 1H), 8.69−8.72 (d, J
= 14.0 Hz, 1H). 13C NMR (125 MHz, CD3OD): 14.24, 17.56, 21.74,
25.31, 28.42, 31.65, 36.94, 39.19, 52.07, 94.69, 99.52, 111.78, 114.99,
116.40, 122.08, 123.42, 124.69, 125.88, 128.26, 128.70, 129.44, 130.36,
130.50, 131.07, 131.64, 132.14, 133.32, 133.57, 134.43, 137.16, 141.76,
153.77, 154.67, 156.77, 164.24, 175.89. MS (ESI) m/z 595.3 (M-
+
609.3 (M-ClO4)+; HRMS (ESI) m/z calcd for C41H41N2O3 (M-
ClO4)+: 609.3112. Found 609.3125.
1
CS 6. H NMR (500 MHz, CD3OD):1.80−1.86 (m, 2H), 1.90−
+
1.95 (m, 2H), 2.08 (s, 6H), 2.17−2.22 (m, 2H), 2.32−2.35 (t, J = 6.0
Hz, 2H), 2.62−2.65 (t, J = 6.0 Hz, 2H), 2.69−2.72 (t, J = 6.0 Hz, 2H),
ClO4)+; HRMS (ESI) m/z calcd for C40H39N2O3 (M-ClO4)+:
595.2955. Found 595.2963.
1209
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Journal of the American Chemical Society
Article
71−79. (i) Near-Infrared Applications in Biotechnology; Raghavachari,
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Arunkumar, E.; Leevy, W. M.; Gammon, S. T.; Piwnica-Worms, D.;
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(b) Egawa, T.; Hanaoka, K.; Koide, Y.; Ujita, S.; Takahashi, N.;
Ikegaya, Y.; Matsuki, N.; Terai, T.; Ueno, T.; Komatsu, T.; Nagano, T.
J. Am. Chem. Soc. 2011, 133, 14157−14159.
ASSOCIATED CONTENT
* Supporting Information
Experimental procedures, some spectra, and complete ref 20.
This material is available free of charge via the Internet at
http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
weiyinglin@hnu.edu.cn
■
(5) For some examples, see: (a) Ozmen, B.; Akkaya, E. U.
Tetrahedron Lett. 2000, 41, 9185−9188. (b) Sasaki, E.; Kojima, H.;
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ACKNOWLEDGMENTS
■
Funding was partially provided by NSFC (20872032,
20972044, 21172063), NCET (08-0175), the Doctoral Fund
of Chinese Ministry of Education (20100161110008), and the
Fundamental Research Funds for the Central Universities,
Hunan University.
(6) Kundu, K.; Knight, F.; Willett, N.; Lee, S.; Taylor, W. R.; Murthy,
N. Angew. Chem., Int. Ed. 2009, 48, 299−303.
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