Coumarin- and rhodamine-fused deep red fluorescent dyes: Synthesis, photophysical properties, and bioimaging in vitro

Article abstract of DOI:10.1021/jo400783x

A series of deep red fluorescent dyes (CR1 to CR3) was developed via introduction of a coumarin moiety into the rhodamine molecular skeleton. The novel dyes possessed the individual advantages of coumarin and rhodamine derivatives, and the emission wavelength was extended to the deep red region (>650 nm) due to the extension of fused-ring conjugate structure simultaneously. To illustrate its value, we designed and conveniently synthesized a series of novel deep red bioimaging dyes (CR1E to CR3E) by esterification of CR1 to CR3, which could selectively stain mitochondria. They were superior to the MitoTrackers for mitochondrial staining in terms of large Stokes shift, excellent contrast for imaging, high photostability, and low cytotoxicity. Furthermore, the fluorescence of the coumarin moiety and rhodamine-like fluorophore could be switched like classical rhodamine. Thus, they could be used as an effective platform in constructing fluorescence sensors. Based on this fact, we constructed a novel ratiometric sensor (CR1S) for Hg²⁺ with good selectivity that could be successfully applied to the imaging of Hg²⁺ in living A549 cells. This design strategy is straightforward and adaptable to various deep red dyes and sensing platforms by simply introducing different fluorophores.

Full text of DOI:10.1021/jo400783x

Subscriber access provided by NORTH CAROLINA STATE UNIV
Article
Coumarin- and rhodamine-fused deep red fluorescent dyes:
synthesis, photophysical properties and bioimaging in vitro
Jianhong Chen, Weimin Liu, Bingjiang Zhou, Guangle Niu, Hongyan
Zhang, Jiasheng Wu, Ying Wang, Weigang Ju, and Pengfei Wang
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/jo400783x • Publication Date (Web): 24 May 2013
Downloaded from http://pubs.acs.org on May 30, 2013
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
The Journal of Organic Chemistry is published by the American Chemical Society.
1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 31
The Journal of Organic Chemistry
1
2
3
4
5
6
7
Coumarinꢀ and rhodamineꢀfused deep red fluorescent
dyes: synthesis, photophysical properties and bioimaging
8
9
in vitro
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Jianhong Chen,†,‡ Weimin Liu,*,† Bingjiang Zhou,†,‡ Guangle Niu,†,‡ Hongyan Zhang,† Jiasheng Wu,† Ying
Wang,† Weigang Ju,† and Pengfei Wang*,†
†Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of
Physics and Chemistry, Chinese Academy of Sciences, Beijing, 100190, China
‡University of Chinese Academy of Sciences, Beijing, 100049, China
* wmliu@mail.ipc.ac.cn; wangpf@mail.ipc.ac.cn
RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required
according to the journal that you are submitting your paper to)
A series of deep red fluorescent dyes (CR1 to CR3) was developed via introducing a coumarin moiety
into the rhodamine molecular skeleton. The novel dyes possessed the individual advantages of coumarin
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 2 of 31
1
2
3
4
5
6
7
8
and rhodamine derivatives, and the emission wavelength was extended to deep red region (>650 nm) due
to the extension of fusedꢀring conjugate structure simultaneously. To illustrate its value, we designed and
conveniently synthesized a series of novel deep red bioimaging dyes (CR1E to CR3E) by esterification of
CR1 to CR3, which could selectively stain mitochondria. They were superior to the MitoTrackers for
mitochondrial staining in terms of large Stokes shift, excellent contrast for imaging, high photostability,
and low cytotoxicity. Furthermore, the fluorescence of the coumarin moiety and rhodamineꢀlike
fluorophore could be switched like classical rhodamine. Thus, they could be used as an effective platform
in constructing fluorescence sensors. Based on this fact, we constructed a novel ratiometric sensor (CR1S)
for Hg2+ with good selectivity which could be successfully applied to the imaging of Hg2+ in living A549
cells. This design strategy is straightforward and adaptable to various deep red dyes and sensing platforms
by simply introducing different fluorophores.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Introduction
Due to the virtue of its highly sensitive and highꢀspeed spatial analysis of cells, fluorescence bioimaging
has become a facile and powerful tool for visualizing morphological details and monitoring biomolecules
in living systems.1 This imaging technique is highly dependent on the photophysical and photochemical
properties of the applied fluorescent dyes. Among the commonly used fluorescent dyes, rhodamine
derivatives are highly favored in bioimaging and biosensor because of their excellent photophysical
properties, such as high molar extinction coefficients, excellent fluorescence quantum yields, and great
photostability.2 For example, Rhodamine 123 has been described to selectively sequester in active
mitochondria and be used as a marker for mitochondrial membrane potentials (MMP).3 In addition,
rhodamine derivatives can undergo equilibrium between spirocyclic and openꢀring forms. The spirocyclic
forms of these dyes are colorless and nonfluorescent due to the small πꢀconjugated systems, while the
ACS Paragon Plus Environment

Page 3 of 31
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
openꢀring forms show intensive spectroscopic signals in the absorption and fluorescence spectra by
generating the large πꢀconjutated systems.4 Thus, they can be used as an effective platform based on
spirocyclic/openꢀring switching mechanism in constructing fluorescence turnꢀon sensors for heavy metal
ions and biomolecules.1a, 2, 4ꢀ5
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
However, most rhodamineꢀbased sensors only show a single emission, which is perturbed by
environmental factors, such as sensor molecular concentration, pH, temperature, solvent polarity, and so
on.6 To eliminate these influences, ratio measurements are favorable, because the simultaneous
observation of the absorption or emission intensities at two different wavelengths can increase the
selectivity and sensitivity. Thus a lot of rhodamine sensors based on fluorescence resonance energy
transfer (FRET) have been designed by introducing additional fluorophore as a donor, such as
naphthalimine, dansyl, coumarin, fluorescein, or BODIPY derivatives.6 The FRET process will occur only
when there is a substantial overlap between the donor emission spectrum and the acceptor absorption
spectrum. In addition, the emission of this energy transfer system is still confined to the characteristic
emission band of the acceptor (rhodamine; <600 nm).
In contrast to visible and above all ultraviolet, fluorescence imaging with deep red light is advantageous
because of its minimum photodamage to biological samples, deep tissue penetration, and negligible
interference from the background autofluorescence of biomolecules in the organism.7 Recently, several
studies have aimed to obtain long wavelength dyes.1a These efforts include the extension of the conjugate
system2b, 7a, 8 and the introduction of donor and acceptor groups into the chromophore,9 as well as the
replacement of the oxygen bridge atom of rhodamine derivatives by other elements.2b, 10 However, there
are some disadvantages of these methods including their difficult synthesis, chemical instability, and/or
high sensitivity to photobleaching. Moreover, rhodamine derivatives exhibit very small Stokes shift
(Rhodamine B: 19 nm vs 600 cmꢀ1)7a. The large Stokes shift could reduce selfꢀabsorption as well as obtain
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 4 of 31
1
2
3
4
5
6
7
high resolution and low detection limits. Therefore, it remains highly desirable to explore new design
strategies for the development of deep red fluorescent dye with great photostability and large Stokes shift
while being used as ratiometric sensing platforms.
8
9
Scheme 1. The structural changes of CR1 to CR3 between spirocyclic and openꢀring forms
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
COOH
O
O
H+
O
O
O
OH-
O
N
O
N+
N
N
spirocyclic form
N
ring-open form
N
CR1:
CR2:
CR3:
N
In this work, we constructed a series of novel deep red fluorescent dyes by hybridizing coumarin and
rhodamine moieties (CR1, CR2, and CR3, Scheme 1). The new dyes not only retained the advantages of
coumarin and rhodamine derivatives themselves, but also demonstrated following expected advantages: (i)
the emission wavelength was extended to the deep red region (>650 nm in H2O) when the fusedꢀring
conjugate structure was extended during its facile synthesis; (ii) the fluorescence of the coumarin moiety
and rhodamineꢀlike fluorophore was switched by the spirocyclic/openꢀring process of the carboxylic acid
functional group (ꢀCOOH) at the 2'ꢀposition. Considering their good photophysical properties, these deep
red fluorescent dyes were promising candidates for biological applications, such as biosensor, biomarker,
and bioimaging. Based on these new dyes, we first developed deep red mitochondriaꢀspecific fluorescent
dyes (CR1E, CR2E, and CR3E). Cellꢀimaging experiments indicated that these dyes were superior over
MitoTrackers because of their deep red emission with large Stokes shift, excellent contrast for imaging,
high photostability, and low cytotoxicity. To illustrate its value as a ratiometric platform, we further
constructed a ratiometric sensor for Hg2+ (CR1S) with good selectivity and “turnꢀon” response in the deep
red region which was different from all previously reported rhodamineꢀbased FRET systems.
ACS Paragon Plus Environment

Page 5 of 31
The Journal of Organic Chemistry
1
2
Results and Discussion
3
4
5
6
7
8
9
Design and Synthesis. The absorption and emission wavelengths of traditional coumarin dyes were
highly dependent on the substituents of the amino group because of intramolecular charge transfer (ICT)
character. Thus, the coumarinꢀ and rhodamineꢀfused compounds CR1 to CR3 were designed using
different amino substituents. The detailed synthetic routes and methods were shown in Scheme 2.
Compounds CR1 to CR3 were synthesized from 3ꢀalkylaminophenol in two steps. Compounds 1, 2, 3, 8,
and 9 were commercially available. Compound 2 was added to 85% phosphoric acid and heated to reflux
for 24 h to obtain compound 5. The condensation of compounds 1 and 8 in toluene for 4 h afforded the
intermediate 4, which was heated to reflux with compound 9 in 1,1,2,2ꢀtetrachloroethane with P2O5 for 4 h
to produce CR1. Similar synthetic pathways were used to synthesize CR2 and CR3.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
To demonstrate its properties as fluorescent bioimaging agents, we designed a series of deep red
fluorescent dyes (CR1E to CR3E) by esterification of CR1 to CR3 in ethanol with concentrated H2SO4
for 2 h, which can selectively stain mitochondria. Mitochondria function as the “powerhouses” of cells by
generating ATP through oxidative phosphorylation.11 These organelles are related to several diseases,
ranging from metabolic disorders to cancer.11c, 12 The mitochondria of cancer cells are structurally and
functionally different from their normal counterparts. To understand the cellular processes and molecular
mechanisms of mitochondrial function, several fluorescent dyes have been developed to selectively stain
mitochondria by electrostatic interactions due to its highly negative MMP (about ꢀ180 mV).13 For example,
lipophilic and positively charged dyes, such as Rhodamine 1233 and MitoTracker Red CMXRos (MTR),14
can selectively accumulate in mitochondria. However, most of these dyes are limited to wavelengths
below 600 nm and have small Stokes shift or poor photostability. Compound CR1E to CR3E
demonstrated the long wavelength emissions in the deep red region and large Stokes shift due to the ICT
of the coumarin moiety.
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 6 of 31
1
2
3
4
5
6
7
8
In these new dyes, the fluorescence of the coumarin moiety and rhodamineꢀlike fluorophore could be
switched like classical rhodamine. Different from all previously reported rhodamineꢀbased sensing
systems, these dyes CR1 to CR3 have the advantage of deep red emission and very large Stokes shift. To
validate our molecular design strategy, we developed a ratiometric sensor (CR1S) for Hg2+ based on the
wellꢀknown desulfurization reaction, which can be used in fluorescence imaging of Hg2+ in the living
A549 (human lung adenocarcinoma apithelial) cells. Compound CR1S was synthesized from CR1 which
was heated to reflux with POCl3 in 1,2ꢀdichloroethane for 4 h and then reacted with saturated Na2S.15
Details of the synthesis were given in the Experimental Section. The structures of the intermediate
compounds (4ꢀ7) and their respective products (CR1 to CR3, CR1E to CR3E and CR1S) were
characterized by 1H NMR, 13C NMR, and HRMS.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 2. Synthesis of deep red dyes CR1 to CR3, CR1E to CR3E and the ratiometric sensor CR1S
O
O
O
COOH
O
O
O
COOC2H5
OH
O
-
-
ClO4
N+
ClO4
O
O
O
O
O
O
(c)
8
(a)
9
(b)
O
N+
O
N
OH
N
O
N
N
N
N
CR1
1
CR1E
4
COOH
ClO4
COOC2H5
OH
O
-
-
(c)
ClO4
N+
(d)
8
9
(a)
OH
(b)
O
N+
O
N
O
N
NH2
N
N
CR2
CR2E
5
6
2
COOH
ClO4
OH
COOC2H5
-
-
ClO4
N+
8
(a)
9
(b)
(c)
O
N+
N
OH
N
O
O
O
N
3
7
CR3
CR3E
O
O
N
COOH
8=
9=
O
O
O
O
OH
O
O
COOH
S
(e), (f)
-
ClO4
N+
O
O
O
O
N
N
N
CR1
CR1S
ACS Paragon Plus Environment

Page 7 of 31
The Journal of Organic Chemistry
Conditions: (a) toluene, reflux, 4 h; (b) P2O5, 1,1,2,2ꢀtetrachloroethane, reflux, 4 h; (c) C2H5OH, H2SO4,
reflux, 2 h; (d) 85% phosphoric acid, reflux, 24 h; (e) POCl3, ClCH2CH2Cl, reflux, 4 h; (f) excess Na2S
saturated aqueous solution.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. Intermolecular hydrogen bonds (IHB) of compounds CR2 and CR3 with ellipsoids shown at the
50% probability level (other hydrogen atoms are omitted for clarity).
X-ray Structures. The molecular structures of CR2, CR3, CR2E, CR3E and CR1S were elucidated by
Xꢀray crystallography, which confirmed a coumarinꢀ and rhodamineꢀfused skeleton. Single crystals of
compounds CR2, CR3, CR2E, CR3E, and CR1S suitable for Xꢀray crystallographic analysis were
obtained by slow evaporation of CH3OH and H2O solutions of the compounds at ambient temperature.
Details of Xꢀray experimental conditions, cell data, and refinement data of CR2, CR3, CR2E, CR3E, and
CR1S were summarized in Table S1. These compounds were all racemic mixtures, given the helix
structure and the presence of both enantiomers in unit cells. Compounds CR2, CR3, CR2E, and CR3E
formed a monoclinic unit cell and the P2(1)/n space group, whereas CR1S formed a triclinic unit cell and
the Pꢀ1 space group. The CR2 and CR3 crystals were observed capturing one water molecule in the
crystal lattice with IHB (Figure 1). The water molecule was located between the dye and perchlorate. The
lengths and angles of the IHB were presented in Table 1. The IHB between the dye and water molecule
was stronger than that between the water molecule and perchlorate. For example, the distances of H1ꢁꢁꢁO10
and H10bꢁꢁꢁO9 for CR2 were 1.780 Å and 2.086 Å, respectively.
Table 1. IHB Data of Compounds CR2 and CR3
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 8 of 31
OaꢀHa HaꢁꢁꢁOb OaꢀOb
OaꢀHaꢁꢁꢁ
Ob(deg)
1
2
3
4
5
6
7
8
OaꢀHaꢁꢁꢁOb
O1ꢀH1ꢁꢁꢁO10
(Å)
(Å)
(Å)
0.841 1.780 2.586 159.97
CR2
CR3
O10ꢀH10bꢁꢁꢁO9 0.840 2.086 2.926 178.62
O6ꢀH6ꢁꢁꢁO10 0.840 1.815 2.618 159.35
O10ꢀH10aꢁꢁꢁO1 0.841 2.073 2.892 164.69
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The representative CꢀN bond lengths were listed in Table 2 (for atomic labels, see Figure 2). The Xꢀray
crystallographic data showed that the representative CꢀN bond length r values of CR2 (1.348 Å and 1.351
Å), CR3 (1.348 Å and 1.353 Å), and CR2E (1.352 Å and 1.346 Å) were almost equal. Therefore,
πꢀconjugate interactions existed between the two CꢀN bonds. The large discrepancy for CR3E (1.342 Å
and 1.371 Å) may have been caused by intermolecular interactions of its crystalline state. These
conclusions were consistent with the results of density functional theory (DFT) calculations (error ≤ 0.015
Å; see Table 2). All benzoyl moieties were nearly vertical to the tetracyclic molecular skeletons by their
rotation around the bridged single bond. The torsion angles θ of CR2, CR3, CR2E, and CR3E were listed
in Table 2 (for atomic labels, see Figure 2). The θ values of CR2 and CR3 were closer to a vertical angle
than those of CR2E and CR3E because of the small steric hindrance of benzoyl moieties, which were
further confirmed by DFT calculations except for CR3E. The C−N bond of CR1S (1.374 and 1.369) was a
single bond, which was longer than that of CR2 and CR3. The benzo[c]thiophenꢀ1(3H)ꢀone moiety was
nearly vertical to the tetracyclic molecular skeleton as shown in Figure 2.
ACS Paragon Plus Environment

Page 9 of 31
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Figure 2. Crystal structures of compounds CR2, CR3, CR2E, CR3E and CR1S with ellipsoids shown at
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
the 50% probability level (hydrogen atoms are omitted for clarity).
Table 2. Crystallographic Data and Theoretical Calculation of Compounds CR2, CR3, CR2E, CR3E and
CR1S
ra/rb (Å)
1.348/1.353 1.351/1.356 88.31/82.69
N1ꢀC7 N2ꢀC30 C19ꢀC11ꢀC12ꢀC18
1.348/1.353 1.353/1.357 85.34/83.05
N1ꢀC7 N2ꢀC33 C19ꢀC11ꢀC12ꢀC17
1.352/1.356 1.346/1.358 98.20/105.14
θa/θb (deg)
CR2
CR3
CR2E
CR3E
CR1S
N1ꢀC16
1.342/1.357 1.371/1.359 104.65/90.41
N1ꢀC14 N2ꢀC27 C22ꢀC20ꢀC9ꢀC8
1.374/1.396 1.369/1.388
N2ꢀC27
C11ꢀC12ꢀC9ꢀC8
ꢀ
N1ꢀC24
N2ꢀC13
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 10 of 31
aThe bond data determined from crystal structure.
1
2
3
4
bThe bond data determined from theoretical
calculation.
5
6
7
8
9
Theoretical Basis. To investigate the geometric and electronic properties of novel deep red dyes,
quantum calculations using the Gaussian 03 program16 were performed. The calculated typical CꢀN bond
lengths and torsion angles were summarized in Table 2. As expected, the results of geometry optimizations
were consistent with the experimental data from single crystal structures. The calculated data for CR3
(1.353 Å, 1.357 Å, and 83.05°) agreed with the experimental data (1.348 Å, 1.353 Å, and 85.34°).
Conversely, the calculated data for CR3E (1.357 Å, 1.359 Å, and 90.41°) slightly deviated from the
corresponding experimental data (1.342 Å, 1.371 Å, and 104.65°). The discrepancy between calculation
results and experimental data may have been caused by intermolecular interactions in the actual
experiments, such as πꢀπ stacking or the interaction with counterion. The calculated HOMOs and LUMOs
of compounds CR1 to CR3 and CR1E to CR3E were shown in Figure 3. The electron density of these
dyes in terms of the HOMOs and LUMOs was predominantly localized in the tetracyclic structures. These
dyes exhibited ICT characteristics of the coumarin moiety. The benzoyl moieties had little influence on
photophysical properties because only a small portion of the electron density was localized in their
structures. This result was further confirmed by the photophysical properties of the dyes.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Page 11 of 31
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Figure 3. Spatial distributions of calculated HOMOs and LUMOs of compounds CR1 to CR3 and CR1E
to CR3E.
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Photophysics. To study the structureꢀproperty relationships, we investigated the photophysical
properties of CR1 to CR3 and CR1E to CR3E. Compounds CR1 to CR3 were sensitive to environmental
pH because of the ꢀCOOH group at the 2'ꢀposition. We first examined the absorption profiles of CR1 to
CR3 and CR1E to CR3E at different pH levels (Figure 4). The absorption of CR1 to CR3 significantly
decreased within the pH range from 2 to 4, whereas their absorption was stable at pH > 4 because of the
occurrence of their spirocyclic forms. Consequently, the spirocyclic and openꢀring forms of CR1 to CR3
were in equilibrium, which was similar to the properties of other rhodamine derivatives. To ensure the
formation of openꢀring forms, the photophysics of CR1 to CR3 was investigated in different solvents with
0.5% trifluoroacetic acid. The absorption of CR1E to CR3E was independent of the solution pH (from 2
to 8) because these compounds lack the unique spirocyclic/openꢀring process of rhodamines. However, the
absorption of CR1E to CR3E was remarkably decreased at the pH range of 8 to 10 because of the open
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 12 of 31
1
2
ring of coumarin moieties in basic solutions. Thus, compounds CR1E to CR3E were suitable deep red
3
4
fluorescence imaging reagents under the physiological condition.
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. pHꢀdependence of the absorption intensity of compounds CR1 to CR3 and CR1E to CR3E (c =
5 ꢂM).
The absorption and fluorescence spectra of CR1 to CR3 and CR1E to CR3E were investigated in
CHCl3, C2H5OH, and H2O (Figures S1, 5, and S2, respectively). The corresponding photophysical data
was summarized in Table 3. The absorption and fluorescence peaks in C2H5OH were redꢀshifted in both
the CR1→CR2→CR3 and CR1E→CR2E→CR3E series, with the increasing donor property of the
amino group in the coumarin moiety. Similar results were obtained in CHCl3 and H2O because of the
increased ICT characteristics of the coumarin moiety. Compounds CR1 to CR3 and CR1E to CR3E
showed a larger Stokes shift than the classic rhodamine dyes. For example, CR3E exhibited a Stokes shift
about 1549 cmꢀ1 (65 nm) in C2H5OH, which was more than twice that of Rhodamine B (600 cmꢀ1, 19
nm).7a The molar extinction coefficients of CR1 to CR3 in CHCl3 and C2H5OH were ~70,000 Mꢀ1 cmꢀ1,
whereas those of CR1E to CR3E in CHCl3, C2H5OH, and H2O ranged from ~64,000 Mꢀ1 cmꢀ1 to
~108,000 Mꢀ1 cmꢀ1. The fluorescence quantum yields of CR1 and CR1E in CHCl3 were 0.507 and 0.494,
respectively, which are significant for deep red fluorescent dyes. However, the fluorescence quantum
yields of CR1E to CR3E in H2O only ranged from 0.002 to 0.014. Thus, biological imaging using CR1E
to CR3E could provide strong contrast, and almost no background fluorescence would be observed. The
ACS Paragon Plus Environment

Page 13 of 31
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
fluorescence quantum yields decreased in both the CR1→CR2→CR3 and CR1E→CR2E→CR3E series
as the energy gap was decreased.17 This result is consistent with the literature.7a, 18 The ꢀCOOH and
ꢀCOOC2H5 groups had little influence on the photophysical properties, as compared with CR1 and CR1E
in different solvents. These results were previously predicted by the DFT calculations.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5. Absorption and fluorescence spectra of a) compounds CR1 to CR3 in C2H5OH with 0.5%
trifluoroacetic acid and b) compounds CR1E to CR3E in C2H5OH (c = 5 ꢂM).
Table 3. Photophysical Properties of Compounds CR1 to CR3 and CR1E to CR3E
Compound Solvent λmax (nm) λem (nm) Stokes shift (nm/ cmꢀ1) εb (Mꢀ1cmꢀ1)
Фc
τ(ns)
CHCl3
C2H5OH
H2O
602
591
594
610
598
601
623
612
612
601
595
596
608
636
637
646
647
651
658
664
674
684
640
642
652
647
34/888
46/1222
52/1355
37/937
77400
70800
26800
76600
64800
44800
75000
69200
38600
99000
64000
71600
108000
0.51 4.16
0.21 2.29
0.01 0.31
0.27 3.79
0.05 0.98
<0.01 0.15
0.09 2.87
0.01 0.47
<0.01 0.16
0.49 4.31
0.20 2.12
0.01 0.27
0.33 4.07
CR1a
CR2a
CR3a
CHCl3
C2H5OH
H2O
53/1361
57/1441
41/991
CHCl3
C2H5OH
H2O
62/1503
72/1720
39/1014
47/1230
56/1441
39/991
CHCl3
C2H5OH
H2O
CR1E
CR2E
CHCl3
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 14 of 31
C2H5OH
H2O
602
602
625
616
616
658
666
669
681
687
56/1414
64/1596
44/1052
65/1549
71/1678
77800
76200
107800
87600
66200
0.04 0.86
1
2
3
4
5
6
7
8
<0.01 0.16
0.11 3.44
0.01 0.43
<0.01 0.13
CHCl3
C2H5OH
H2O
CR3E
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
aThe photophysics are investigated in different solvents with 0.5% trifluoroacetic acid. bThe molar
extinction coefficients at maximum absorption. cUsing cresyl violet in methanol as the reference (Φ =
0.54).19
Intracellular imaging for mitochondria (CR1E to CR3E). The molecular structures of CR1E to
CR3E contain a positive charge and are highly similar to those of the mitochondriaꢀspecific fluorescent
dyes MTR and Rhodamine 123.3 Thus, these positively charged deep red dyes may preferentially
accumulate in mitochondria by electrostatic interactions. The electrostatic interactions can accumulate and
increase by a 100ꢀfold to 500ꢀfold, as described by the Nernst equation.13a To demonstrate this possibility,
novel deep red fluorescent dyes CR1E to CR3E were incubated with living A549 cells (Figures 6). The
localization of dyes’ accumulation could be confirmed by dualꢀchannel laser scanning confocal
microscopy in the coꢀstaining experiments. The extensive overlapping with MitoTracker Green FM (MTG)
indicated that these deep red dyes were able to efficiently penetrate the cell membrane and specifically
label mitochondria. These results revealed the potential imaging and therapeutic applications of these
novel deep red dyes. To confirm their mechanism of accumulation in mitochondria, carbonyl cyanide
3ꢀchlorophenylhydrazone (CCCP) was used to change MMP before the staining procedure. Upon
treatment with CCCP, the MMP was lost because of the rapid acidification of mitochondria and the
dysfunction of ATP synthase.20 After treatment with 30 ꢂM CCCP, fluorescence intensity in mitochondria
became faint, because the decreasing MMP affected the accumulation of the cationic dye (CR1E to
CR3E) in mitochondria (Figure S3).
ACS Paragon Plus Environment

Page 15 of 31
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Figure 6. Confocal fluorescence microscopy images of A549 incubated with MTG (10 nM) and
compounds CR1E to CR3E (50 nM).
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 7. Photostability of compounds CR1E to CR3E and MitoTrackers (5 ꢂM) in PBS buffer (pH =
7.4).
As is well known, photostability is very important for fluorescent dyes due to the temporal monitoring
of dynamic events inside cells. To evaluate the practical utility of the novel deep red dyes, their
photostability in PBS buffer was performed by continuous irradiation of compounds CR1E to CR3E
(compared with MitoTrackers) using a 500 W Xe lamp as the light source. After 30 min continuous
irradiation, the absorption intensity of CR1E to CR3E decreased to 76% to 84%, whereas that of MTR
dropped to 64% as shown in Figure 7. It is significant that the photostability of CR1E to CR3E was much
higher than that of cyanine dyes, such as MTG (7%) and MitoTracker Deep Red FM (MTDR, 12%). The
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 16 of 31
1
2
3
4
5
6
7
8
cytotoxicity of CR1E to CR3E in A549 cells was examined using the MTT
(3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenylꢀ2Hꢀtetrazolium bromide) assay. The cytotoxicity of MTG,
MTR, and MTDR were tested as the controls. Compounds CR1E to CR3E exhibited low cytotoxicity
(cell viability > 90%) at concentrations up to 100 nM after incubated with A549 cells for 24 h (Figure 8).
However, MTG and MTDR reduced the cell viability below 90% even at 10 nM.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 8. The cytotoxicity of compounds CR1E to CR3E and MitoTrackers in A549 cells.
Figure 9. Confocal fluorescence microscopy images of A549 cells incubated with Hoechst 33342 (1
mg/mL) and compounds CR1E to CR3E (50 nM).
These deep red emissive properties helped reduce the interference of autofluorescence from
biomolecules in the organism and obtained a higher signalꢀtoꢀnoise ratio. To evaluate the potential
ACS Paragon Plus Environment

Page 17 of 31
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
imaging and diagnostic applications of novel deep red dyes, CR1E to CR3E were incubated with different
cell lines (cancer cells, A549 and HeLa; normal cells, L929). As shown in Figure S4ꢀS6, the strong
intracellular fluorescence may be attributed to the distribution of dyes in the inner mitochondrial
membrane, where a relatively organicꢀlike mitochondrial matrix of cancer and normal cells exists.
Although the fluorescence quantum yields of CR1E to CR3E were relatively low (0.002 to 0.014) in a
polar solvent (H2O), the fluorescence intensity in mitochondria was sufficient to provide strong contrast
for imaging due to its enrichment inside mitochondria. The morphologies of mitochondria in cancer and
normal cells can be clearly visualized. Furthermore, almost no background fluorescence could be
observed, especially in the nucleus. To demonstrate the compatibility of CR1E to CR3E with other dyes
such as Hoechst 33342, coꢀstaining experiments were performed. We were able to clearly visualize the
blue nuclear staining in the DAPI channel and the red mitochondrial staining by the novel dyes in the Cy5
channel (Figure 9). The superimposition patterns between CR1E to CR3E and Hoechst 33342 allowed for
the simultaneous imaging of mitochondria and DNA.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Ratiometric bioimaing for Hg2+ (CR1S). Based on the wellꢀknown desulfurization reaction, we
developed a new ratiometric sensor (CR1S, Scheme 1) for Hg2+ in this work. With compound CR1S in
hand, we firstly investigated its absorption response to Hg2+ at different pH levels (Figure S7). The
intensities of compound CR1S solutions in a citric acid (0.1 M)ꢀphosphate (0.2 M) buffer (H2O/CH3OH =
1:1, v/v) within pH 2 to 8 were found to be stable. However, the addition of Hg2+ resulted in a distinct
increasing in the absorption intensity at 596 nm with increasing acidity (from pH 8 to 2), which was
similar with the reported Hg2+ sensor.15 According to reference, we chose pH = 4 as the measuring
conditions.
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 18 of 31
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 10. Absorption (a) and fluorescence spectra (b) of CR1S (5 ꢃM) with different Hg2+
concentrations in a citric acid (0.1 M)ꢀphosphate (0.2 M) buffer (pH = 4, H2O/CH3OH = 1:1, v/v). λex=
380 nm. The insets show the color changes and fluorescence changes in the absence and presence of Hg2+.
Figure 10 showed the spectroscopic properties of CR1S at various Hg2+ concentrations in a citric acid
(0.1 M)ꢀphosphate (0.2 M) buffer (pH = 4, H2O/CH3OH = 1:1, v/v). The UV/Vis spectra of CR1S only
showed the absorption profile from the coumarin moiety of the spirocyclic form with a maximum at 381
nm (Figure 10a). The addition of Hg2+ immediately decreased the absorption peak at 381 nm with
redꢀshifted and a new absorption band at 596 nm appeared, which corresponded to the absorption of
rhodamineꢀlike moiety. The spectral shifts were about 215 nm, and two isosbestic points at 340 nm and
400 nm were formed. They exhibited a clear colour change from colourless to blue and colorimetric
performance could be easily monitored by nakedꢀeye for instantaneous visual sensing (inset, Figure 10a).
Similar ratio changes can also be observed from the fluorescence spectra (Figure 10b). The intensity at 430
nm from the coumarin moiety decreased and that at 653 nm from rhodamineꢀlike moiety gradually
ACS Paragon Plus Environment

Page 19 of 31
The Journal of Organic Chemistry
increased with increasing Hg2+ concentration. Indeed, this coumarinꢀ and rhodamineꢀfused πꢀconjugated
system had very large spectral shifts of up to 223nm, which were much larger than those of TBET or
FRETꢀbased rhodamine systems.21 In addition, such fluorescence performance could be detected by
nakedꢀeye as depicted in the inset of Figure 10b, where photos of solutions of compound CR1S before and
after addition of Hg2+ under UV light irradiation were displayed.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
As shown in Figure S8, the linear fitting can be obtained between Hg2+ concentrations ranging from 0
ꢃM to 100 ꢃM and the absorption ratio (A596/A381) as well as the square root of fluorescence intensity ratio
(I1/2653/I1/2430) of CR1S, respectively. The response to Hg2+ of CR1S was also very fast and spectral data
were recorded within 10 seconds after the addition of Hg2+. The selectivity of CR1S for different metal
ions was also investigated. As shown in Figure S9, the fluorescence response of CR1S displayed excellent
selectivity to Hg2+ over all other tested ions. We proposed that the high thiophilicity of Hg2+ resulted in the
formation of the openꢀring complex, and then hydrolyzed to CR1. This result can be confirmed by the
ESIꢀMS signals at m/z 511.2 (corresponding to CR1) in the solution of CR1S treated with excess Hg2+
(Figure S10).
Figure 11. Confocal fluorescence imaging of Hg2+ in A549 cells with (a) CR1S (2 ꢃm) for 10 min and (b)
CR1S (2 ꢃm) for 10 min and then Hg2+ (20 ꢃm) for 10 min.
Taking into account of the advantages of ratiometric fluorescence changes, compound CR1S was used
for in vitro Hg2+ detection in living A549 cells. As shown in Figure 11a, A549 cells incubated with CR1S
(2 ꢃM) for 10 min showed a clear blue fluorescence, whereas weak red fluorescence was observed in the
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 20 of 31
red channel, which indicated that CR1S mainly existed in the spirolactam form. After Hg2+ (20 ꢃm)
treatment for 10 min, the blue fluorescence intensity decreased and the red fluorescence intensity
significantly increased (Figure 11b). As shown in the ratio imaging (Red/Blue) in Figure 11, distinct
changes in ratio fluorescence responses in A549 cells were observed. This indicated the potential of
biological imaging application of the new ratiometric deep red sensor.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Conclusions
We have introduced a new strategy to design deep red fluorescent dyes (CR1 to CR3) containing a
coumarinꢀ and rhodamineꢀfused conjugated skeleton. The novel deep red fluorescent dyes have excellent
photophysical properties, such as high molar extinction coefficients, excellent fluorescence quantum yields
in CHCl3, great photostability and large Stokes shift. Importantly, these dyes CR1 to CR3 could undergo
equilibrium between the coumarin moiety and rhodamineꢀlike fluorophore switched by the
spirocyclic/openꢀring process of the ꢀCOOH, which can be used as versatile sensing platform for designing
ratiometric fluorescent sensors. Considering their good photophysical properties, we designed a series of
deep red fluorescent bioimaging agents (CR1E to CR3E) by esterification of CR1 to CR3, which can
selectively stain mitochondria. Cellꢀimaging experiments indicated that these dyes were superior over
MitoTrackers because of their deep red emission with large Stokes shift, excellent contrast for imaging,
high photostability, and low cytotoxicity. Furthermore, using CR1 as a platform, we developed a novel
ratiometric sensor (CR1S) for mercury ions with the decreased fluorescence of coumarin moiety and large
turnꢀon deep red fluorescence of rhodamineꢀlike moiety. CR1S could be successfully applied to the
ratiometric imaging of Hg2+ in living A549 cells. This design strategy may be extended for development of
new deep red fluorescent dyes by simply introducing different fluorophores, and dyes CR1 to CR3 will be
good candidates for biosensor, biomarker, and bioimaging.
Experimental Section
ACS Paragon Plus Environment

Page 21 of 31
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
Materials and Methods. All commercial chemicals were used without further purification. Compound
2 (7ꢀaminoꢀ1ꢀmethylꢀ1,2,3,4ꢀtetrahydroquinoline hydrochloride) was purchased from HangZhou Trylead
Chemical Technology Co., Ltd. MTG, MTR, MTDR and Hoechst were purchased from Invitrogen (USA).
CCCP was purchased from Alfa Aesar. Crystallographic data of CR2, CR3 and CR2E were done on a
Saturn724+ CCD with Mo Kα radiation (λ = 0.71073 Å) at 173 K. Crystallographic data of CR3E was
done on a Rigaku raxis Rapid IP Area Detector with Cu Kα radiation (λ = 1.54178 Å) at 173 K.
Crystallographic data of CR1S was done on an Image Plate XꢀRay Diffractameter with Mo Kα radiation
(λ = 0.71073 Å) at 293 K. The quantum chemical calculations were optimized using the restricted B3LYP
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
13
functions with a 6ꢀ31 G (d) basis set at the DFT level. H NMR and C NMR spectra were recorded at
400 and 100 MHz respectively. The HRMS were measured with Fourier Transform Ion Cyclotron
Resonance Mass Spectrometry (FTICRꢀMS). Absorption and fluorescence spectra of all compounds were
obtained in an absorption spectrometer and a fluorescence spectrometer at room temperature, respectively.
The fluorescence lifetimes were measured in FLS920 Combined Steady State & Time Resolved
Fluorescence Spectrometer. A549, HeLa and L929 cells (gifted from the center of cells, Peking Union
Medical College) were cultured in confocal dishes in culture media (McCoy's 5A, DMEM or RPM1640
respectively, supplemented with 10% FBS, 50 unit/mL penicillin, and 50 mg/mL of streptomycin) under
5% carbon dioxide/air at 37 °C in a humidified incubator for 24 h. The cells were washed with PBS for
three times before fluorescent imaging. Confocal microscopy and image analysis was acquired with a laser
scanning confocal microscopy (for Hoechst 33342, excited at 405 nm, DAPI channel; for MTG, excited at
488 nm, FITC channel; for CR1E to CR3E, excited at 640 nm, Cy5 channel). The microscope was
equipped with a liveꢀcell incubation chamber maintaining a humidified atmosphere of 37 °C and 5% CO2.
Images were processed by NISꢀElements Viewer 3.20.
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 22 of 31
1
2
3
4
5
6
7
8
Synthesis of 7-diethylamino-4-hydroxycoumarin (4). 3ꢀDiethylaminophenol 1 (12.8 g, 77.5 mmol)
and diphenyl malonate 8 (18.8 g, 73.4 mmol) were added into anhydrous toluene (50 mL). The mixture
was heated to reflux for 4 h and compound 4 was obtained by filtration as grey solid (5.2 g, 30%).22 mp
233ꢀ235 °C; 1H NMR (400 MHz, DMSO) δ (ppm): 11.98 (s, 1H), 7.55ꢀ7.53 (d, 1H, J = 9.0 Hz), 6.67ꢀ6.64
(dd, 1H, J1 = 2.2 Hz, J2 = 9.0 Hz), 6.45ꢀ6.44 (d, 1H, J = 2.2 Hz), 5.23 (s, 1H), 3.43ꢀ3.38 (q, 4H, J = 7.0
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13
Hz), 1.13ꢀ1.09 (t, 6H, J = 7.0 Hz); C NMR (100 MHz, DMSO) δ (ppm): 166.7, 162.8, 156.1, 150.8,
124.1, 108.1, 103.6, 96.4, 86.1, 44.0, 12.3; ESIꢀHRMS m/z calcd for [C13H15NO3+H]+ 234.11247, found
234.11205.
Synthesis of 7-hydroxy-1-methyl-1,2,3,4-tetrahydroquinolin (5). Compound 2 (3.2 g, 1.6 mmol) was
added into 85% phosphoric acid (15 mL) and heated to reflux for 24 h.23 After cooling to room
temperature, the reaction solution was neutralized with 200 mL 40% NaOH aqueous solution. Plenty of
Na3PO4 was precipitated and filtrated. The filtrate was extracted with CH2Cl2 (total 60 mL for three times).
The organic phase was concentrated under vacuum and purified by column chromatography to afford
compound 5 as white solid using v(CH3COOC2H5)/v(petroleum ether) = 1:3 as the eluent (1.3g, 48%). mp
1
103ꢀ104 °C; H NMR (400 MHz, DMSO) δ (ppm): 8.74 (s, 1H), 6.64ꢀ6.62 (d, 1H, J = 7.9 Hz), 5.98ꢀ5.97
(d, 1H, J = 2.1 Hz), 5.95ꢀ5.93 (dd, 1H, J1 = 2.2 Hz, J2 = 7.9 Hz), 3.13ꢀ3.10 (t, 2H, J = 5.6 Hz), 2.75 (s,
3H), 2.57ꢀ2.54 (t, 2H, J = 6.4 Hz), 1.86ꢀ1.80 (m, 2H). 13C NMR (100MHz, DMSO) δ (ppm): 156.4, 147.3,
128.9, 112.9, 102.9, 98.2, 50.5, 38.7, 26.5, 22.3. ESIꢀHRMS m/z calcd for [C10H13NO+H]+ 164.10699,
found 164.10657.
Synthesis of 4-hydroxy-9-methyl-6,7,8,9-tetrahydropyrano[3,2-g]quinolin-2-one (6). Compound 5
(1.0 g, 3.9 mmol) and diphenyl malonate 8 (0.64 g, 3.9 mmol) were added into anhydrous toluene (8 mL).
The mixture was heated to reflux for 6 h and compound 6 was obtained by filtration as grey solid (0.8 g,
1
88%).22 mp 255ꢀ257 °C; H NMR (400 MHz, DMSO) δ (ppm): 11.83 (s, 1H), 7.26 (s, 1H), 6.35 (s, 1H),
ACS Paragon Plus Environment

Page 23 of 31
The Journal of Organic Chemistry
1
2
3
4
5
6
7
5.23 (s, 1H), 3.33ꢀ3.31 (t, 2H, J = 5.6 Hz), 2.92 (s, 3H), 2.73ꢀ2.70 (t, 2H, J = 6.1 Hz), 1.87ꢀ1.84 (t, 2H).
13C NMR (100 MHz, DMSO) δ (ppm): 166.4, 162.7, 154.7, 149.7, 121.7, 118.8, 103.2, 95.5, 86.0, 50.0,
38.5, 26.6, 21.2. ESIꢀHRMS m/z calcd for [C13H13NO3+H]+ 232.09682, found 232.09648.
8
9
Synthesis of 4-hydroxypyrano[3,2-g]julolidine-2-one (7). 8ꢀHydroxyjulolidine 3 (3.7 g, 19.3 mmol)
and diphenyl malonate 8 (4.1 g, 15.9 mmol) were added into anhydrous toluene (10 mL). The mixture was
heated to reflux for 4 h and compound 7 was obtained by filtration as straw yellow solid (3.6 g, 88%).22
mp 268ꢀ270 °C; 1H NMR (400 MHz, DMSO) δ (ppm): 11.73 (s, 1H), 7.15 (s, 1H), 5.21 (s, 1H), 3.24ꢀ3.20
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13
(m, 4H), 2.70 (s, 4H), 1.91ꢀ1.83 (m, 4H). C NMR (100 MHz, DMSO) δ (ppm): 166.5, 162.8, 151.0,
146.0, 119.8, 117.3, 105.3, 103.1, 85.8, 49.2, 48.7, 26.9, 21.0, 20.1, 20.0. ESIꢀHRMS m/z calcd for
[C15H15NO3+H]+ 258.11247, found 258.11210.
Synthesis
of
compound
CR1.
Compound
4
(440
mg,
1.9
mmol)
and
2ꢀ(4ꢀdiethylaminoꢀ2ꢀhydroxy)ꢀbenzoylbenzoic acid 9 (629 mg, 2.0 mmol) were heated to reflux in 15 mL
1,1,2,2ꢀtetrachloroethane until the substances have dissolved completely. A total of 2.8 g phosphorus
pentoxide was then added in portions and the reaction mixture was heated to reflux for 4 h.24 After
cooling, the mixture was dissolved using water (25 mL) and chloroform (25 mL), and the water phase was
extracted with chloroform (total 100 mL for three times). The combined organic phases were concentrated
under vacuum and purified by column chromatography using v(CH2Cl2)/v(CH3OH) = 100:1 as the eluent.
The dye fractions are concentrated under vacuum, and the residue is dissolved in 2 mL ethanol. After
addition of 3 mL perchloric acid (note: the operation should be executed very carefully due to the
instability of perchlorate salts), the solution was precipitated by dropwise addition of water (100 mL).
1
Compound CR1 was obtained by filtration as dark green solid (682 mg, 59%). mp 287ꢀ289 °C; H NMR
(400 MHz, DMSO) δ (ppm): 8.02ꢀ7.94 (m, 2H), 7.71ꢀ7.60 (m, 2H), 7.31ꢀ7.29 (d, 1H), 6.90ꢀ6.87 (m, 2H),
6.68 (s, 1H), 6.60ꢀ6.55 (m, 2H), 3.52ꢀ3.44 (m, 8H), 1.16ꢀ1.11 (m, 12H). 13C NMR (100 MHz,
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 24 of 31
1
2
3
4
5
6
7
8
CD3OD/CD2Cl2) δ (ppm): 166.9, 163.6, 163.4, 158.7, 158.4, 158.0, 157.9, 156.6, 136.8, 133.7, 132.9,
131.4, 130.4, 129.6, 128.8, 127.8, 116.9, 116.7, 112.7, 104.6, 101.3, 98.0, 97.9, 52.9, 47.2, 46.4, 12.7.
ESIꢀHRMS m/z calcd for [C31H31N2O5]+ 511.22275, found 511.22229. Elem. anal. calcd for
C31H31ClN2O9·1/2H2O: C, 60.05; H, 5.20; N, 4.52, found: C, 60.11; H, 5.14; N, 4.33.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthesis
of
compound
CR2.
Compound
6
(659
mg,
2.9
mmol)
and
2ꢀ(4ꢀdiethylaminoꢀ2ꢀhydroxy)ꢀbenzoylbenzoic acid 9 (1.1 g, 3.5 mmol) were heated to reflux in 20 mL
1,1,2,2ꢀtetrachloroethane until the substances have dissolved completely. A total of 4.0 g phosphorus
pentoxide was then added in portions and the reaction mixture was heated to reflux for 4 h.24 After
cooling, the mixture was dissolved using water (25 mL) and chloroform (25 mL), and the water phase was
extracted with chloroform (total 100 mL for three times). The combined organic phases were concentrated
under vacuum and purified by column chromatography using v(CH2Cl2)/v(CH3OH) = 100:1 as the eluent.
The dye fractions are concentrated under vacuum, and the residue is dissolved in 3 mL ethanol. After
addition of 5 mL perchloric acid (note: the operation should be executed very carefully due to the
instability of perchlorate salts), the solution was precipitated by dropwise addition of water (100 mL).
1
Compound CR2 was obtained by filtration as dark green solid (720 mg, 42%). mp 289ꢀ290 °C; H NMR
(400 MHz, DMSO) δ (ppm): 7.97ꢀ7.96 (m, 1H), 7.74ꢀ7.60 (m, 3H), 7.29ꢀ7.27 (m, 1H), 6.87 (s, 1H), 6.69
(s, 1H), 6.57 (s, 1H), 6.49 (s, 1H), 3.44 (s, 6H), 3.01 (s, 3H), 2.84ꢀ2.81 (t, 2H, J = 5.8 Hz), 1.92 (s, 2H),
1.15ꢀ1.12 (t, 6H, J = 6.8 Hz). 13C NMR (100 MHz, CD3OD/CD2Cl2) δ (ppm): 168.2, 162.9, 158.2, 157.9,
157.5, 155.7, 137.5, 133.5, 132.8, 131.7, 130.4, 130.3, 128.5, 124.3, 124.2, 116.4, 104.4, 101.2, 98.0, 96.7,
52.4, 47.2, 40.0, 27.8, 21.8, 12.8. ESIꢀHRMS m/z calcd for [C31H29N2O5]+ 509.20710, found 509.20627.
Elem. anal. calcd for C31H29ClN2O9·3/2H2O: C, 58.54; H, 5.07; N, 4.40, found: C, 58.33; H, 4.80; N, 4.49.
Synthesis
of
compound
CR3.
Compound
7
(1.2
g,
4.7
mmol)
and
2ꢀ(4ꢀdiethylaminoꢀ2ꢀhydroxy)ꢀbenzoylbenzoic acid 9 (1.8 g, 5.7 mmol) were heated to reflux in 50 mL
ACS Paragon Plus Environment

Page 25 of 31
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
1,1,2,2ꢀtetrachloroethane until the substances have dissolved completely. A total of 7.8 g phosphorus
pentoxide was then added in portions and the reaction mixture was heated to reflux for 4 h.24 After
cooling, the mixture was dissolved using water (25 mL) and chloroform (25 mL), and the water phase was
extracted with chloroform (total 100 mL for three times). The combined organic phases were concentrated
under vacuum and purified by column chromatography using v(CH2Cl2)/v(CH3OH) = 100:1 as the eluent.
The dye fractions are concentrated under vacuum, and the residue is dissolved in 5 mL ethanol. After
addition of 10 mL perchloric acid (note: the operation should be executed very carefully due to the
instability of perchlorate salts), the solution was precipitated by dropwise addition of water (150 mL).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
Compound CR3 was obtained by filtration as dark green solid (982 mg, 33%). mp 290ꢀ292 °C; H NMR
(400 MHz, DMSO) δ (ppm): 8.04ꢀ8.03 (m, 1H), 7.74ꢀ7.62 (m, 3H), 7.28ꢀ7.26 (m, 1H), 6.92ꢀ6.64 (m, 3H),
3.49ꢀ3.36 (m, 8H), 2.81ꢀ2.78 (m, 2H), 2.65ꢀ2.64 (m, 2H), 1.94ꢀ1.83 (m, 4H), 1.16ꢀ1.13 (t, 6H, J = 6.9 Hz).
13C NMR (100 MHz, CD3OD/CD2Cl2) δ (ppm): 167.1, 161.8, 157.4, 157.3, 156.5, 152.3, 152.0, 136.3,
132.5, 131.8, 130.9, 129.4, 129.3, 127.6, 122.3, 122.1, 115.2, 115.1, 106.3, 103.5, 100.0, 96.9, 50.8, 50.3,
46.2, 27.2, 20.6, 19.6, 19.5, 11.9. ESIꢀHRMS m/z calcd for [C33H31N2O5]+ 535.22275, found 535.22213.
Elem. anal. calcd for C33H31ClN2O9: C, 62.41; H, 4.92; N, 4.41, found: C, 63.28; H, 5.46; N, 4.14.
Synthesis of compound CR1E. Concentrated H2SO4 (0.5 mL) was added to an ethanol solution of
compound CR1 (120 mg, 0.2 mmol), and the solution was heated to reflux for 2 h.25 After cooling, ethanol
was removed under vacuum and the residue was neutralized with 20 mL 10% NaOH aqueous solution to
collect the resulting precipitate. After drying, it was purified by silica gel chromatography using
v(CH2Cl2)/v(CH3OH) = 100:1 as the eluent to give CR1E as dark green solid (39 mg, 31%). mp 272ꢀ274
1
°C; H NMR (400 MHz, DMSO) δ (ppm): 8.20ꢀ8.17 (m, 2H), 7.83ꢀ7.71 (m, 2H), 7.40ꢀ7.31 (m, 2H),
7.20ꢀ7.07 (m, 2H), 6.90ꢀ6.75 (m, 2H), 4.10ꢀ4.08 (q, 2H, J = 7.1 Hz), 3.71ꢀ3.60 (m, 8H), 1.23ꢀ1.18 (m,
13
12H), 1.07ꢀ1.04 (t, 3H, J = 7.1 Hz). C NMR (100 MHz, DMSO) δ (ppm): 164.8, 161.8, 161.2, 157.2,
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 26 of 31
1
2
3
4
5
6
7
8
156.9, 156.6, 156.4, 155.1, 135.6, 133.0, 132.9, 131.4, 130.1, 129.5, 128.3, 126.9, 116.7, 115.4, 111.8,
103.6, 99.9, 97.2, 96.9, 61.1, 54.9, 45.8, 45.0, 13.6, 12.4. ESIꢀHRMS m/z calcd for [C33H35N2O5]+
539.25405, found 539.25309. Elem. anal. calcd for C33H35ClN2O9·1/2H2O: C, 61.16; H, 5.60; N, 4.32,
found: C, 61.29; H, 5.61; N, 4.23.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthesis of compound CR2E. Concentrated H2SO4 (1 mL) was added to an ethanol solution of
compound CR2 (505 mg, 0.8 mmol), and the solution was heated to reflux for 2 h.25 After cooling, ethanol
was removed under vacuum and the residue was neutralized with 40 mL 10% NaOH aqueous solution to
collect the resulting precipitate. After drying, it was purified by silica gel chromatography using
v(CH2Cl2)/v(CH3OH) = 100:1 as the eluent to give CR2E as dark green solid (350 mg, 66%). mp 279ꢀ280
1
°C; H NMR (400 MHz, DMSO) δ (ppm): 8.20ꢀ8.18 (m, 1H), 7.90ꢀ7.71 (m, 3H), 7.33ꢀ7.31 (m, 2H),
7.16ꢀ7.13 (m, 1H), 6.88ꢀ6.84 (m, 1H), 6.64 (s, 1H), 4.12ꢀ4.06 (q, 2H, J = 7.0 Hz), 3.69ꢀ3.68 (m, 4H),
3.58ꢀ3.55 (m, 2H), 3.15 (s, 3H), 2.87ꢀ2.84 (t, 2H, J = 5.7 Hz), 1.96ꢀ1.94 (m, 2H), 1.23 (s, 6H), 1.07ꢀ1.03
(t, 3H, J = 7.1 Hz). 13C NMR (100 MHz, DMSO) δ (ppm): 164.9, 161.3, 161.1, 156.8, 156.5, 156.4, 156.2,
154.3, 135.6, 132.9, 131.3, 130.1, 129.5, 128.4, 128.3, 123.1, 122.9, 116.3, 115.1, 103.5, 99.6, 97.0, 96.0,
61.1, 54.9, 51.0, 45.8, 26.4, 20.5, 13.7, 12.5. ESIꢀHRMS m/z calcd for [C33H33N2O5]+ 537.23840, found
537.23773. Elem. anal. calcd for C33H33ClN2O9·1/2H2O: C, 61.35; H, 5.30; N, 4.34, found: C, 61.46; H,
5.35; N, 4.30.
Synthesis of compound CR3E. Concentrated H2SO4 (1 mL) was added to an ethanol solution of
compound CR3 (430 mg, 0.7 mmol), and the solution was heated to reflux for 2 h.25 After cooling, ethanol
was removed under vacuum and the residue was neutralized with 40 mL 10% NaOH aqueous solution to
collect the resulting precipitate. After drying, it was purified by silica gel chromatography using
v(CH2Cl2)/v(CH3OH) = 100:1 as the eluent to give CR3E as dark green solid (3908mg, 68%). mp
1
273ꢀ274 °C; H NMR (400 MHz, DMSO) δ (ppm): 8.20ꢀ8.18 (m, 1H), 7.83ꢀ7.70 (m, 3H), 7.32ꢀ7.30 (m,
ACS Paragon Plus Environment

Page 27 of 31
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
2H), 7.12ꢀ7.10 (m, 1H), 6.84ꢀ6.82 (m, 1H), 4.09ꢀ4.07 (m, 2H), 3.68ꢀ3.66 (m, 4H), 3.51ꢀ3.48 (m, 4H), 2.86
(s, 2H), 2.69 (s, 2H), 1.95ꢀ1.90 (m, 4H), 1.23 (s, 6H), 1.06ꢀ1.03 (t, 3H, J = 7.0 Hz). 13C NMR (100 MHz,
DMSO) δ (ppm): 164.8, 161.0, 160.9, 156.8, 156.5, 156.0, 151.6, 151.3, 135.7, 132.8, 131.2, 130.1, 129.4,
128.2, 122.0, 121.6, 115.9, 114.8, 105.7, 103.3, 99.3, 97.0, 61.0, 50.3, 49.7, 45.6, 26.7, 20.3, 19.4, 19.1,
13.6, 12.5. ESIꢀHRMS m/z calcd for [C35H35N2O5]+ 563.25405, found 563.25315. Elem. anal. calcd for
C35H35ClN2O9·1/2H2O: C, 62.54; H, 5.40; N, 4.17, found: C, 62.59; H, 5.37; N, 4.15.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthesis of compound CR1S. Compounds CR1 (150 mg, 0.25 mmol) and phosphorus oxychloride (1
mL) was added into 1,2ꢀdichloromethane (5 mL) and heated to reflux for 4 h.15 After cooling, the reaction
mixture was concentrated under vacuum to obtain crude acyl chloride. 5 mL of saturated Na2S aqueous
solution (1.2g) was then added to the crude acyl chloride and the mixture was stirred for 3 h at room
temperature. Following extraction with ethyl acetate (total 100 mL for three times), the organic phase was
purified by column chromatography using CH2Cl2 as the eluent to afford the pale yellow solid CR1S in
56% yield (72 mg). mp 301ꢀ302 °C; 1H NMR (400 MHz, CDCl3) δ (ppm): 7.88ꢀ7.85 (m, 2H), 7.51ꢀ7.41
(m, 2H), 7.21ꢀ7.19 (m, 1H), 6.72ꢀ6.64 (m, 2H), 6.44ꢀ6.38 (m, 2H), 3.45ꢀ3.33 (m, 8H), 1.23ꢀ1.17 (m, 12H).
13C NMR (100 MHz, CDCl3) δ (ppm): 197.4, 159.7, 158.0, 156.8, 155.6, 151.4, 149.8, 148.5, 136.5,
133.8, 130.4, 128.0, 125.4, 124.4, 123.0, 110.2, 108.9, 108.8, 102.0, 97.4, 97.1, 95.8, 58.5, 44.9, 44.5,
12.7, 12.5. ESIꢀHRMS m/z calcd for [C31H30N2O4S+H]+ 527.19990, found 527.20044. Elem. anal. calcd
for C31H30N2O4S·1/4H2O: C, 70.10; H, 5.79; N, 5.27, found: C, 70.19; H, 5.91; N, 5.19.
Acknowledgment. This work was supported by the NNSF of China (Grant Nos. 20903110, 61227008,
21073213, and 20673132) and the Main Direction Program of Knowledge Innovation of Chinese
Academy of Sciences.
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 28 of 31
1
2
Supporting Information Available: Xꢀray structure details of compounds CR2, CR3, CR2E, CR3E
3
4
13
and CR1S (CIF); photophysical data; imaging data; sensor data and 1HꢀNMR and CꢀNMR spectra of
5
6
7
8
9
new compounds. This material is available free of charge via the Internet at http://pubs.acs.org.
References
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(1) (a)Yuan, L.; Lin, W.; Zheng, K.; He, L.; Huang, W. Chem. Soc. Rev. 2013, 42, 622; (b)Schäferling,
M. Angew. Chem., Int. Ed. 2012, 51, 3532; (c)Chan, J.; Dodani, S. C.; Chang, C. J. Nat. Chem. 2012, 4,
973.
(2) (a)Zheng, H.; Zhan, X.ꢀQ.; Bian, Q.ꢀN.; Zhang, X.ꢀJ. Chem. Commun. 2013, 49, 429; (b)Sun,
Y.ꢀQ.; Liu, J.; Lv, X.; Liu, Y.; Zhao, Y.; Guo, W. Angew. Chem., Int. Ed. 2012, 51, 7634; (c)Chen, X.;
Pradhan, T.; Wang, F.; Kim, J. S.; Yoon, J. Chem. Rev. 2012, 112, 1910.
(3) (a)Summerhayes, I. C.; Lampidis, T. J.; Bernal, S. D.; Nadakavukaren, J. J.; Nadakavukaren, K.
K.; Shepherd, E. L.; Chen, L. B. Proc. Natl. Acad. Sci. U. S. A. 1982, 79, 5292; (b)Darzynkiewicz, Z.;
Staianocoico, L.; Melamed, M. R. Proc. Natl. Acad. Sci. U. S. A. 1981, 78, 2383; (c)Johnson, L. V.; Walsh,
M. L.; Chen, L. B. Proc. Natl. Acad. Sci. U. S. A. 1980, 77, 990.
(4) Shi, W.; Ma, H. Chem. Commun. 2012, 48, 8732.
(5) Kim, H. N.; Ren, W. X.; Kim, J. S.; Yoon, J. Chem. Soc. Rev. 2012, 41, 3210.
(6) Fan, J.; Hu, M.; Zhan, P.; Peng, X. Chem. Soc. Rev. 2013, 42, 29.
(7) (a)Yuan, L.; Lin, W.; Yang, Y.; Chen, H. J. Am. Chem. Soc. 2012, 134, 1200; (b)Kolmakov, K.;
Belov, V. N.; Bierwagen, J.; Ringemann, C.; Müller, V.; Eggeling, C.; Hell, S. W. Chem.ꢀꢀEur. J. 2010, 16,
158; (c)Weissleder, R.; Ntziachristos, V. Nat. Med. 2003, 9, 123.
(8) Qian, G.; Wang, Z. Y. Chem.ꢀꢀAsian J. 2010, 5, 1006.
(9) KartonꢀLifshin, N.; Albertazzi, L.; Bendikov, M.; Baran, P. S.; Shabat, D. J. Am. Chem. Soc.
2012, 134, 20412.
ACS Paragon Plus Environment

Page 29 of 31
The Journal of Organic Chemistry
1
2
3
4
5
(10)(a)Koide, Y.; Urano, Y.; Hanaoka, K.; Piao, W.; Kusakabe, M.; Saito, N.; Terai, T.; Okabe, T.;
Nagano, T. J. Am. Chem. Soc. 2012, 134, 5029; (b)Koide, Y.; Urano, Y.; Hanaoka, K.; Terai, T.; Nagano,
T. J. Am. Chem. Soc. 2011, 133, 5680.
6
7
8
9
(11)(a)Glancy, B.; Balaban, R. S. Biochemistry 2012, 51, 2959; (b)Lee, S.; Chen, X. Chembiochem
2011, 12, 2120; (c)Abeliovich, A. Nature 2010, 463, 744.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(12)(a)Wallace, D. C. Nat. Rev. Cancer 2012, 12, 685; (b)Manji, H.; Kato, T.; Di Prospero, N. A.;
Ness, S.; Beal, M. F.; Krams, M.; Chen, G. Nat. Rev. Neurosci. 2012, 13, 293; (c)Goldberg, J. A.; Guzman,
J. N.; Estep, C. M.; Ilijic, E.; Kondapalli, J.; SanchezꢀPadilla, J.; Surmeier, D. J. Nat. Neurosci. 2012, 15,
1414; (d)Zhou, R.; Yazdi, A. S.; Menu, P.; Tschopp, J. Nature 2011, 469, 221; (e)Blanco, F. J.; Rego, I.;
RuizꢀRomero, C. Nat. Rev. Rheumatol. 2011, 7, 161.
(13)(a)Hoye, A. T.; Davoren, J. E.; Wipf, P.; Fink, M. P.; Kagan, V. E. Acc. Chem. Res. 2008, 41, 87;
(b)Murphy, M. P.; Smith, R. A. J. Annu. Rev. Pharmacol. Toxicol. 2007; 47, 629.
(14)(a)Li, L.; Ge, J.; Wu, H.; Xu, Q.ꢀH.; Yao, S. Q. J. Am. Chem. Soc. 2012, 134, 12157; (b)Qian, F.;
Zhang, C.; Zhang, Y.; He, W.; Gao, X.; Hu, P.; Guo, Z. J. Am. Chem. Soc. 2009, 131, 1460; (c)Kim, Y. K.;
Ha, H.ꢀH.; Lee, J.ꢀS.; Bi, X.; Ahn, Y.ꢀH.; Hajar, S.; Lee, J.ꢀJ.; Chang, Y.ꢀT. J. Am. Chem. Soc. 2009, 132,
576.
(15)Zhan, X.ꢀQ.; Qian, Z.ꢀH.; Zheng, H.; Su, B.ꢀY.; Lan, Z.; Xu, J.ꢀG. Chem. Commun. 2008, 44,
1859.
(16)Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.;
Montgomery, J. A. Jr.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.;
Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.;
Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai,
H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.;
ACS Paragon Plus Environment