Efficient synthesis of NIR emitting bis[2-(2′-hydroxylphenyl)benzoxazole] derivative and its potential for imaging applications

Article abstract of DOI:10.1016/j.bioorg.2020.103585

Unassymetric bis[2-(2′-hydroxyphenylbenzoxole)] bis(HBO) derivatives with a DPA functionality for zinc binding have been developed with an efficient synthetic route, using the retrosynthetic analysis. Comparison of bis(HBO) derivatives with different substitution patterns allows us to verify and optimize their unique fluorescence properties. Upon binding zinc cation, bis(HBO) derivatives give a large fluorescence turn-on in both visible (λ_em ≈ 536 nm) and near-infrared (NIR) window (λ_em ≈ 746 nm). The probes are readily excitable by a 488 nm laser, making this series of compounds a suitable imaging tool for in vitro and in vivo study on a confocal microscope. The application of zinc binding-induced fluorescence turn-on is successfully demonstrated in cellular environments and thrombus imaging.

Full text of DOI:10.1016/j.bioorg.2020.103585

Journal Pre-proofs
Efficient Synthesis of NIR Emitting Bis[2-(2´-hydroxylphenyl)benzoxazole]
Derivative and Its Potential for Imaging Applications
Junfeng Wang, Hannah Baumann, Xiaoman Bi, Leah P. Shriver, Zhaoda
Zhang, Yi Pang
PII:
S0045-2068(19)31265-9
DOI:
https://doi.org/10.1016/j.bioorg.2020.103585
YBIOO 103585
Reference:
To appear in:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
2 August 2019
12 January 2020
13 January 2020
Please cite this article as: J. Wang, H. Baumann, X. Bi, L.P. Shriver, Z. Zhang, Y. Pang, Efficient Synthesis of
NIR Emitting Bis[2-(2´-hydroxylphenyl)benzoxazole] Derivative and Its Potential for Imaging Applications,
Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103585
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Efficient Synthesis of NIR Emitting Bis[2-(2´-
hydroxylphenyl)benzoxazole] Derivative and Its Potential for
Imaging Applications
Junfeng Wang,a,c,d Hannah Baumann,a Xiaoman Bi,a Leah P. Shriver,a Zhaoda Zhang,d and Yi Pang*ab
a
b
Department of Chemistry & Maurice Morton Institute of Polymer Science, The University of Akron,
Akron, OH, USA, 44325
c Gordon Center for Medical Imaging, Massachusetts General Hospital and Harvard Medical School, 125
Nashua Street, Boston, MA, 02114
d Martinos Center for Biomedical Imaging, Massachusetts General Hospital and Harvard Medical School,
149, 13th st, Charlestown, MA, 02129
* Email: yp5@uakron.edu
Abstract: Unassymetric bis[2-(2‘-hydroxyphenylbenzoxole)] bis(HBO) derivatives with a DPA
functionality for zinc binding have been developed with an efficient synthetic route, using the
retrosynthetic analysis. Comparison of bis(HBO) derivatives with different substitution patterns
allows us to verify and optimize their unique fluorescence properties. Upon binding zinc cation,
bis(HBO) derivatives give a large fluorescence turn-on in both visible (em536 nm) and near-
infrared (NIR) window (em746 nm). The probes are readily excitable by a 488 nm laser, making
this series of compounds a suitable imaging tool for in vitro and in vivo study on a confocal
microscope. The application of zinc binding-induced fluorescence turn-on is successful
demonstrated in cellular environments and thrombus imaging.
Keywords: Fluorescent probe; Near Infrared; Benzoxazole; ESIPT; Zinc; Thrombus imaging
Introduction
In recent years, fluorescent sensors are emerging as powerful tools for molecular imaging.1 In this
attractive field, there are increasing interests to develop optical probes that give emission in the
near-infrared (NIR) region (650-900 nm). This is because the NIR photons can penetrate deeply
through biological tissues, thereby enabling detection of molecular activity for in vivo
applications.2-7 An ideal NIR probe should also exhibit a large Stokes´ shift, in order to have
minimum interference between excitation and emission signals. Additionally, a qualified NIR
probe should be integrated with a suitable chemical event that can generate a large optical response
when responding to a specific analyte of interest.8
2-(2´-Hydroxyphenyl)benzoxazole (HBO) derivatives represent an interesting class of dyes, which
exhibit a large Stokes’ shift (> 150 nm) arising from the excited-state intramolecular proton
transfer (ESIPT). 9,10 The photochemical event is dependent on the presence of the phenolic proton,
whose migration to the “nitrogen” allows the formation of its corresponding keto tautomer with
emission at a longer wavelength. The metal chelation to mono HBO system, however, removes
the phenolic proton, thereby eliminating the ESIPT.11,12 In an effort to overcome the barrier and
enable the ESIPT from a metal-HBO complex, we recently show that 2, 5-bis(bebzoxazole-2-
yl)benzene-1,4-diol derivatives 1, bis(HBO), can exhibit a remarkable optical response upon zinc
binding, giving the turn-on emission in both visible (at ~550 nm) and NIR regions (at ~750 nm).13-
15
The chemical event for the sensor response is dependent on the formation of 2, where the zinc
complexation acts as an effective switch to turn-on the ESIPT emission (corresponding to the
1

emission in NIR region). In the molecular design of 1, a 2, 2´-dipicolylamine (DPA) group is
attached to selectively bind Zn2+ cation, while a remaining HBO is used to enable the ESIPT. The
choice of DPA ligand is based on its high selectivity to Zn2+ cation. Despite the impressive sensor
performance of 1b (low toxicity and large response to Zn2+ binding),15 the synthesis of 1 suffers
from low yields, which hamper its broad application. The previous synthesis was based on a linear
consecutive sequence consisting of 8 steps,15,16 which makes it difficult to access this class of
compounds for extensive studies. In an continuing effort to overcome the barrier, we now report a
highly efficient synthesis of 2, 5-bis(HBO) derivatives with a DPA functionality, by adopting a
convergent synthetic strategy. In the synthesis design, the target molecule was disconnected in the
middle to give two fragments A and B (Scheme 1), thereby leading to improved yields.
remaining HBO
R1
H
O
3
4
R2
5
O
N
N
R1
R1
H
O
H
O
R2
R3
O
N
2
R2
R3
O
N
O
O
1
R3
6
HO
N
O
hv
N
N
Zn2+
O
N
Zn
O
Zn
ESIPT
N
enol tautomer
keto tautomer
N
N
N
1
N
N
N
Zinhbo
3
2
1a:
R
1 = CH3; R2 = R3 = H;
2+
Zinhbo
"
+ Zn "
1b:
1c:
1d:
t
R
1 = R3 = H; R2 = -Bu
R1 = R2 = R3 = H;
R
1 = R2 = H; R3 = Me
Figure 1. Chemical structure of Zinhbo compounds 1 as well as the enol and keto tautomer
structures of the corresponding zinc complex.
OH
OH
OMe
OMe
O
N
N
O
N
O
O
H2N
HO
NH2
R
R
R
+
+
O
HO
N
N
HO
MeO
MeO
B
N
N
D
C
N
N
1
A
Scheme 1. Retrosynthetic analysis of the bis(HBO) derivatives.
In the previous synthesis of 1b,15 the low yield was associated with the introduction of the DPA
group, which was accomplished by using a sequence of low-yielding reactions to accomplish the
transformation Ar-CH3  Ar-CH2-N(CH2-py)2. Although an improved methodology was
developed to synthesize Zinhbo compounds with higher yields, the reduction of the carboxylic
ester group in the synthesis greatly limited the diversity of these derivatives.16 Herein we report a
new strategy for efficient synthesis of bis(HBO) derivatives, which is based on a retrosynthetic
analysis involving the bond disconnections in the middle of the target to give two fragments A and
B (Scheme 1). The synthetic direction can be outlined as follows: (1) preparation of the key
intermediate A, which incorporates the DPA group in the early stage; (2) efficient synthesis of
benzoxazole B from aldehyde C and 2-amino-phenol D; (3) reaction of A and B to give the
bis(HBO) products.
Results and Discussion
Scheme 2 presents the construction of the key intermediate A. Thus compound 5 was prepared
conveniently by the nitration of 4 with nitric acid at room temperature, which precipitated from
2

the solution and was recrystallized from EtOAc/hexane as large colorless crystals (50-60% yield).
Selective reduction of ester 5 by NaBH4 in THF/MeOH17,18 proceeded cleanly to furnish alcohol
6, which then underwent tosylation or bromination to provide corresponding 7 or 8 in almost
quantitative yields.19,20 Reaction of 7 or 8 with 2, 2’-dipicolylamine (DPA) in the presence of
K2CO3 and KI gave compound 9 in good yields.21 Deprotection of the methoxy group proceeded
smoothly at room temperature by using AlCl3 to give 10,22 which was then reduced by H2 over
Pd/C to afford the intermediate A in almost quantitative yield. Although the procedure was quite
satisfactory on a small scale (e.g., 1 mmol), the deprotection of 9 by AlCl3 was found to be
troublesome for a larger scale reaction (e.g., 5 mmol). The reaction was not completed even by
using a large excess of AlCl3. This is because some syrup-like residues precipitated out during the
reaction, possibly due to (a) chelation of AlCl3 with DPA, and (b) encapsulation of unreacted 9. In
order to overcome the deficiency in the deprotection of 9, our attention was directed to seek
deprotection of the methyl phenol ether at an earlier stage, i.e. before introduction of DPA.
Attempts to deprotect the methyl from the phenol ether 6 gave low yield by using either AlCl3 or
BBr3. However, the methyl group of 8 was removed effectively by using AlCl3 to give 3-
(bromomethyl)-2-nitrophenol, which could be converted to 10 in high yield.
OMe
OMe
OMe
OMe
TsCl, NaH, THF
or CH3CN, PBr3
NO2
NO2
OH
NO2
NaBH4
97%
HNO3, rt
60%
COOMe
95-99%
COOMe
7
8
l2
R = OTs
R= Br
C
6
H2
5
C
4
R
,
l3
C
l
%
6
A
9
OH
DPA,K2CO3, KI
91%
NO2
t
r
,
A
P
OH
D
OH
OMe
%
3
Br
9
NH2
N
NO2
N
NO2
N
H2/Pd/C
98%
AlCl3, CH2Cl2
95%
A
10
9
N
N
N
N
N
N
Scheme 2. Synthesis of key intermediate A.
O
O
R2
R3
O
R2
R3
Pb(OAc)4
or DDQ
N
+
H2O
HO
O
HO
N
HO
11
O
O
12
[O]
O
O
O
CHO
R2
R2
H2N
O
O
1) EtOH, reflux
HO
+
OHC
+
HO
N
O
R3 2) CH2Cl2, PCC/silica
R3
O
O
D
B
13
C
b:
c
d
R2=t-Bu, R3=H; : R2=R3=H; : R2=H, R3=CH3;
Scheme 3. Synthesis of key intermediate B.
The construction of the benzoxazole fragment B from intermediate C and 2-aminophenols D
(Scheme 3) was also significantly improved with a one-pot reaction. In the synthesis of
benzoxazole compounds, most reported methods require the isolation of the imine intermediate
11, followed by an oxidative cyclization to form the benzoxazole intermediate 12.23-29 Although
DDQ and Pb(OAc)4 worked well for oxidative cyclization of 11,13-15 the reaction typically gave
3

the alcohol 12 without further oxidation. An additional oxidation step was thus required to further
oxidize the alcohol 12 with another oxidant, in order to obtain the aldehyde B. In other words, the
reaction sequence 1112B could be accomplished by involving two steps of oxidation. An
intriguing question is whether one can find a suitable oxidant that can execute both “oxidative
cyclization” and “oxidation of the alcohol to the aldehyde”, thereby greatly simplifying the
reaction procedure. Bearing this in mind, we decided to examine PCC which is known to oxidize
primary alcohols to aldehydes and to oxidize imine to benzoxazoles as well.30 Simple mixing of C
and D with PCC in CH2Cl2 led to B in 26% yield, and a large amount of byproduct was identified
as 13. The initial result indicated that PCC was effective for both cyclization and oxidation.
Interestingly, when using TLC to monitor the reaction, we found C and D reacted quickly on TLC
plate, forming imine intermediate 11. This observation encouraged us to optimize the reaction
conditions with silica gel and PCC. After extensive trials, a one-pot multistep method for the
synthesis of B (by direct reaction between C and D) was developed in 60-80% yield with high
reproducibility by silica gel and PCC.
R1
OH
OMe
R1
OMe
OH
R2
R3
N
O
R2
R3
NH2
N
O
N
reflux
N
O
MeO
N
MeO
EtOH or toluene
B
N N
14
N
N
A
Activated carbon
O2, 120oC; 60-75%
R1
OH
R1
OMe
R2
R3
O
N
O
R2
R3
O
N
O
N
N
HO
AlCl3 or BBr3; CH2Cl2
MeO
N
1b (Zinhbo5):
R1 = R3 = H; R2 = t-Bu
N
15
b:
c:
d:
1c (Zinhbo7):
1d (Zinhbo6):
R1 = R2 = R3 = H;
R1 = R3 = H; R2 = t-Bu
1 = R2 = R3 = H;
R1 = R2 = H; R3 = Me
N N
N N
R
1 = R2 = H; R3 = Me
R
Zinhbo1:
R
1 = Me; R2 = R3 = H;
Scheme 4. Construction of bis(HBO) system (Zinhbo dyes).
Finally, the coupling of A and B was studied (Scheme 4). Unfortunately, all the reported catalysts
and reagents such as Mn(OAc)3,26 KAl(SO4)2,27 Pb(OAc)4,28 Ba(MnO4)2,29 DDQ and even the
PCC/silica gel method failed to give any desirable 15 (Scheme 4). It was found that the imine 14
was not formed by refluxing A and B in EtOH or toluene overnight, probably due to the steric
hindrance. After exhaustive exploration, fortunately, we found that Tagawa’s method using O2
with activated carbon gave 15 in up to 72% yield.31 The final product 1b could be easily prepared
by the deprotection of methoxy groups catalyzed by AlCl3 or BBr3 in anhydrous CH2Cl2. With the
availability of the intermediate A, the products (1b-1d) could be effectively synthesized in 2 steps
in high yields.
4

Zinhbo5
Zinhbo5+Zn2+
Zinhbo7
536
7x105
6x105
5x105
4x105
3x105
2x105
746 nm
Zinhbo7+Zn2+
Zinhbo6
Zinhbo6+Zn2+
Zinhbo1
Zinhbo1+Zn2+
607
1x105
0
500
550
600
650
700
750
800
850
Wavelength (nm)
Figure 2 Normalized fluorescence spectra of Zinhbo compounds (5 µM; excitation at 414 nm)
and Zinhbo-Zn2+ complexes (formed upon addition of 1 equiv Zn2+) in CH2Cl2 (excitation at 480
nm). The spectra are normalized at 607 nm for Zinhbo, and at 536 nm for Zinhbo-Zn2+ complexes.
R1
4
Substituent
O
H
2
3
R2
5
O
N
R1
CH3
H
R2
H
-Bu
H
R3
H
H
N
O
R3
6
1
Zinhbo1:
Zinhbo5:
Zinhbo6:
Zinhbo7:
O
Zn
N
keto tautomer
t
N
H
Me
H
N
3
H
H
2+
Zinhbo-
"
Zn "
Scheme 5. Chemical structures of keto tautomer of Zinhbo-Zn complexes.
Fluorescent properties and imaging application. The newly developed synthetic strategy
allowed us to obtain Zinhbo-6 and Zinhbo-7, in addition to the previously reported Zinhbo-113 and
Zinhbo-5.15 Optical properties of these Zinhbo compounds (Scheme 5) allowed us to examine the
possible substituent effect. Fluorescence spectra of these Zinhbo compounds revealed one
emission peak (em607 nm) (Figure 2). Upon binding Zn2+, the resulting Zinhbo-Zn2+ complexes
gave two characteristic emission peaks (em536, 746 nm), corresponding to their enol and keto
tautomers (2 and 3, respectively). The ability of using Zn2+ binding to generate two well separated
emission is a unique property of Zinhbo compounds. In the study, one fundamental question is
how the structure can be modified to affect the NIR emission wavelength and its intensity. In order
to make comparison, the fluorescence spectra were normalized at the green emission peak
(em536, Figure 2). The alkyl substituent on 4-, 5-, and 6-position exhibited essentially no effect
on the emission wavelength. However, the positions of alkyl substituents significantly influenced
the relative NIR emission intensity, which could be attributed to the electronic perturbation by the
substituents. The position of the alkyl substituents was found to affect not only the NIR emission,
but also the quantum yield (ESI, Table S1). However, Zinhbo-5 remained to be the best among
them in terms of the strong NIR emission (Figure 2 and ESI, Figure S2).
The ability of Zinhbo-5 to give simultaneous emission in both green and NIR channels, when
responding to Zn2+ binding,15 encouraged us to use the probe on confocal microscope study, which
has not been examined before. Since the probe-Zn2+ complex can be excited readily by a 488 nm
laser, it would be a useful tool for imaging exogenous and endogenous Zn2+ ions in living cells
5

(Figure 3). Thus, Hela cells were stained with Zinhbo-5 (10 µM) in culture medium with prolonged
incubation time (60 min) at 37℃, we were delighted to find observable green and NIR signal,
which reveals the great sensitivity of this sensor. With exogenous Zn2+ ions (10 µM), very strong
green and NIR signals were obtained on the confocal image throughout the cytoplasm. This further
proved the great sensitivity and bio-compatibility of the sensor. As far as we know, this is the first
successful in vitro zinc selective NIR turn-on imaging with ESIPT property (> 270 nm stokes'
shift).
Green channel
Overlay
NIR channel
Figure 3. Top: Fluorescence images of Hela cells incubated with 10 μM Zinhbo-5 for 60 min at
37℃ in cell culture medium; Bottom: Hela cells were first treated with 10 μM Zn2+ for 30 mins,
washed 3 times with PBS, the cells were further incubated with 10 μM Zinhbo-5 for another 60
min at 37℃ in cell culture medium. The experiments were carried out on an Olympus FV1000
Confocal Microscopy, excited with a 488 nm laser, and the images were collected in both green
channel (535-565 nm) and NIR channel (700-750 nm).
In order to examine the probe’s general utility, Zinhbo-5 was further used to stain oligodendrocytes
that are myelinating glial cells of the central nervous system. Thus, to the MO3.13 cells media
(oligodendrocytes) were treated with TPEN (N,N,N’,N’-tetrakis(2-pyridylmethyl)ethylenediamine)
that is a high-affinity zinc chelator, washed with cell medium and added Zinhbo-5 (5 M). The
fluorescence confocal imaging revealed that Zinhbo-5 readily stained the cells (Figure 4). While
the fluorescence signal of Zinhbo-5 was very weak in the absence of Zn2+ (Figure 4d), addition of
Zn2+ induced a large fluorescence turn-on (Figure 4e and 4f). Similar results were also obtained
when staining Fibroblast cells. These results illustrated that Zinhbo-5 could be a useful probe for
monitoring cellular zinc level.
6

Figure 4. Confocal imaging of fibroblast (top row) and oligodendrocyte cells (bottom row) treated
with TPEN (50 M), washed with cell medium, and incubated with Zinhbo-5 (5 M) for 30 min
(images a & d; Ex/Em= 488/525 nm), followed by treatment with50 μM Zn2+ for 30 mins in green
channel (b and e, Ex/Em= 488/525 nm) and NIR channel (c and f; Ex/Em= 488/700 nm).
Human plasma
Human plasma
+ Zinhbo-5
+ Zinhbo-5
+ Thrombin
Bright
field
0.8 cm
Green
channel
0.8 cm
NIR
channel
0.8 cm
Figure 5. In vitro NIR imaging of thrombus by using Zinhbo-5. Top row: Human plasma (0.5 mL)
containing 1.0 μM of Zinhbo-5 (left well); Human plasma containing 1.0 μM of Zinhbo-5,
followed by addition of 1.0 μL of 1Unit/mL thrombin (right well, from Millipore Sigma, Cat. No.
605160). Middle and bottom rows: Human plasma and resulting clots were imaged with Ex/Em =
465/560 nm (Green channel) or Ex/Em = 465/760 nm (NIR channel) using an IVIS® Spectrum
imaging system.
Cell viability of Zinhbo-5 was checked with HEK293 cells in DMEM medium, by using alamar
blue assay. No significant cell toxicity was observed up to 48 hours of the cells with up to 100 µM
concentration, when the DMSO stock solution (10 mM) was diluted to cell culture medium (ESI
Figure S5). The ability of Zinhbo-5 in responding to cellular Zn2+, in addition to its low toxicity,
encouraged us to seek its further application. An interesting problem is blood clot, which is formed
when responding to the blood vessel injury (e.g. resulting from cuts and bruises) in order to prevent
bleeding. However, blood clot in the absence of injury (called thrombus) can be dangerous, as it
can block a key blood vessel to impede blood flow, leading to cardiovascular diseases such as
heart attack, stroke, pulmonary embolism, and deep vein thrombosis. It is known that zinc is
involved in thrombosis and thrombolysis, where Zn2+ concentration is delicately controlled in the
blood.32 When Zn2+ cations circulate in plasma at a concentration of 10–20 μM, most cations are
bound to plasma proteins such as albumin (forming a labile, exchangeable pool), but some (about
0.1–2 μM) are in a free unbound state. Because the platelets in thrombi, which respond to blood
vessel damage, are 50-100 folds higher than those found in the circulating blood. Upon activation,
Zn2+ stored in platelets are released, thereby increasing the free zinc concentrations.32-37 Therefore,
7

the high Zn2+ concentrations in thrombus may enable the zinc sensors for thrombus imaging. To
test this hypothesis, the in vitro blood clot model was generated by addition of thrombin into the
human plasma according to a reported protocol.38 The clear blood clots could be seen within 10-
15 min (Figure 5, right well of top row). The significant fluorescence turn-on signals within the
clots were displayed in both green and NIR channel (Figure 5, right well). There was no
fluorescence signal generated by Zinhbo-5 in the plasma (Figure 5, left well). This result
demonstrates that the NIR fluorescence zinc turn-on dyes such as Zinhbo-5 are promising probes
for thrombus imaging. To our best knowledge, this is the first thrombus imaging evidenced by an
NIR zinc sensor.
In summary, an efficient synthetic route has been developed for the synthesis of bis(HBO)
compounds with the zinc-chelating DPA functionality. When using together with silica gel, PCC
is found to be effective for accomplishing both “oxidative cyclization” and “oxidation of alcohol”
steps, thereby greatly simplifying the reaction sequence. The finding opens a path to access this
class of compounds for imaging applications, which are known to exhibit low toxicity and
attractive fluorescence15 but are rather difficult to synthesize. The availability of the bis(HBO)
sensor 1 allows us to further examine its potential imaging applications for zinc detection, since it
exhibits attractive photophysical properties including large fluorescence turn-on in the desirable
NIR region upon zinc binding. The successful in vitro NIR imaging for the detection of exogenous
and endogenous Zn2+ ions in living cells makes this sensor a promising tool for imaging
applications. This is further demonstrated by observing significant fluorescence turn-on signals
within the blood clots. Study by using the improved synthesis to access other new NIR-emitting
zinc sensors is in progress, and their potential applications for imaging zinc-related
disease/diagnosis will be reported in another forthcoming manuscript.
Acknowledgements. YP acknowledge support from Coleman endowment (from the University
of Akron) and National Institute of Health (Grant No: 1R15GM126438-01A1) and ZZ
acknowledge support from NIH National Cancer Institute (NCI) (Grant No:
1R01CA197401).
Appendix A. Supplementary data associated with this article can be found in the online version
at https://doi.org/xxxxxx.
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Graphics Abstract
OH
OH
OMe
O
N
N
O
N
O
NH2
R
R
+
O
N
N
HO
MeO
B
N
N
N
N
Zinhbo
A
An efficient synthesis route is found to give Zinhbo compounds that give NIR-emission upon
binding Zn2+ in cellular environments.
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Highlights
 Efficient synthesis for construction of a zinc probe
 Zinc binding to induce NIR emission from excited state intramolecular proton transfer
 NIR-emission with large Stokes shift
 Zinc binding response for thrombus imaging
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Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships that
could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:
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