Synthesis of nitric oxide probes with fluorescence lifetime sensitivity

Article abstract of DOI:10.1039/c3ob41498a

We present the rationale, synthesis and evaluation of the first activatable fluorescent probe that utilizes fluorescence lifetime change for detection of nitric oxide. The new probe DAP-LT1 features a near-infrared polymethine skeleton with a diaminobenzene functionality incorporated into the meso-position. The probe is partially quenched, and upon reaction with nitric oxide shows an increase in the fluorescence lifetime from 1.08 ns to 1.24 ns.

Full text of DOI:10.1039/c3ob41498a

Organic &
Biomolecular Chemistry
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Synthesis of nitric oxide probes with fluorescence
lifetime sensitivity†
Cite this: Org. Biomol. Chem., 2013, 11,
8228
Natalia G. Zhegalova, Garrett Gonzales and Mikhail Y. Berezin*
We present the rationale, synthesis and evaluation of the first activatable fluorescent probe that utilizes
fluorescence lifetime change for detection of nitric oxide. The new probe DAP-LT1 features a near-
infrared polymethine skeleton with a diaminobenzene functionality incorporated into the meso-position.
The probe is partially quenched, and upon reaction with nitric oxide shows an increase in the
fluorescence lifetime from 1.08 ns to 1.24 ns.
Received 21st July 2013,
Accepted 7th October 2013
DOI: 10.1039/c3ob41498a
www.rsc.org/obc
artifacts.10,11 Additional benefits of fluorescence lifetime include
contrast enhancement through alleviation of some of the
Introduction
Reactive nitrogen species (RNS) are known biomarkers of typical problems associated with intensities including scatter-
inflammation and tumour proliferation. They participate in a ing,12 sample turbidity, and autofluorescence.13–15 Recently,
great number of physiological functions from cell signalling to several papers describing lifetime sensitive probes based on
cell damage oxidation. The current methods for assessing NO cyanine dyes,16,17 quantum dots18,19 and fluorescent proteins20
include direct measurements with NO sensitive electrodes,1 have been published.
chemiluminescence through NO reactions with ozone,2 stain-
Herein, we present a first example of the fluorescence life-
ing tissues for nitrosylated proteins with antinitrotyrosine time probe for NO detection. As with other lifetime probes,
specific antibodies, measuring the expression of NOS via two criteria have to be met. First, the change in the lifetime
qPCR3 and analysis of nitrite absorbance with the Griess upon reaction with NO should be substantial enough to over-
reagent.4 With the rise of fluorescent techniques in the last come the sensitivity of the current fluorescence lifetime instru-
two decades a number of activatable fluorescence probes for mentation (50–100 ps). Second, the fluorescence intensity of
measuring nitric oxide and its metabolites in cells and thin the probe before and after reaction with the substrate should
tissues have been developed. Today, a variety of activatable be strong, otherwise an impractically long scanning time will
“OFF–ON” probes that include NBD methylhydrazine, fluore- be needed to measure the lifetime. So far, all published probes
scein based DAF-FM, and rhodamine DAR-2 for nitric oxide according to the corresponding references exhibit a high level
are available commercially and new Cu-based,5 NK-green-2,6 of quenching (>100×) which is ideal for intensity imaging, but
HK-green-3,7 and PyBor8 probes for peroxynitrite detection detrimental to fluorescence lifetime imaging.
have been recently published.
The sensitivity of all up-to-date published and commercially
available “OFF–ON” fluorescent probes for RNS research relies
on the increase of the fluorescence intensity. However, the
fluorescence intensities are difficult to quantify in hetero-
Results and discussion
Probe design and synthesis
geneous systems such as thick tissues and biofluids where the
recorded intensity changes may be due to a concentration gra-
dient instead of differences in RNS expression. In contrast to
intensity, the fluorescence lifetime is an intrinsic property of
the fluorophore9 and has been recently introduced as a comp-
lementary modality to overcome the problem of concentration
The design of the NO lifetime probes was motivated by our
recently published work where amine-containing pyrazoles
were inserted into the meso-position to form fluorescence life-
time pH sensitive dyes.16 The fluorescence lifetime of poly-
methine probes is, in general, sensitive to the environment
and structural modifiers.9–11 Placing electron rich amines in
close proximity to the fluorophore decreases the emission due
to photoinduced electron transfer (PET),21 also known as the
excited state electron transfer (ESET) effect that results in the
lower fluorescence lifetime. This approach was implemented
in the design of NO sensitive lifetime probes where the
NO reactive diaminopolymethine sensor was placed in the
Department of Radiology, Washington University School of Medicine in St. Louis,
St. Louis, MO 63110, USA. E-mail: berezinm@mir.wustl.edu; Fax: +1 314 747 5191;
Tel: +1 314 747 0701
†Electronic supplementary information (ESI) available: Optical spectra, fluore-
scence decays and NMR spectra. See DOI: 10.1039/c3ob41498a
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Fig. 1 Design and principle of DAP-LT fluorescence lifetime NO probes.
meso-position of the heptamethine chain to form diaminopoly-
methine lifetime probes (DAP-LTs) shown in Fig. 1.
Upon reacting with NO in the presence of molecular
oxygen, the diaminobenzene is converted into a benzotriazole,
leading to the restoration of fluorescence.22 This approach has
been successfully used for the preparation of a number of NO
sensitive fluorescent probes based on fluoresceins,23 rhod-
amines,24 spirobifluorenes,25 and polymethines.26 All these
probes were designed to achieve the highest quenching to
maximize the sensitivity of the probe. However, none of them
have been reported to show any fluorescence lifetime
sensitivity.
Scheme 1 Synthesis of diamino-polymethine lifetime probes DAP-LT1 and
DAP-LT2.
The synthesis of DAP-LTs was based on the methodology
developed for selective substitution of meso-positions in poly-
methine dyes via Suzuki coupling.27 Briefly, arylbromide 1 or a
methylated form of 4-bromo-2-nitroaniline 2 was converted to
the boronic esters 4–5 using bis-pinacolato diboron 3 under
Scheme 2 Synthesis of benzotriazole polymethine dye from IR783 via Suzuki
coupling and via the reaction between DAP-LT1 and the NO donor.
Suzuki coupling conditions (Scheme 1). The introduction of hydrogen is transferred from a hydrogen donor ammonium
the methyl group was motivated by the structure of the com- formate to the substrate in the presence of catalysts such as Pt
mercial NO sensor DAF-FM (see below). Subsequent coupling or Pd.29,30 The reduction of the nitro group in 6–7 with excess
of the 4–5 with a commercially available dye-precursor IR783 of ammonium formate in the presence of 10% Pd/C in air-free
led to the formation of C–C bond in dyes 6–7. A standard methanol at room temperature led to the desired product,
method for the reduction of the nitro group with tin chlor- diamino-compounds DAP-LT1 8 and DAP-LT2 9. The reaction
ide26,28 was successful; however, we were unable to purify the was clean and complete within 20 min. To the best of our
products from tin. This was apparently due to a strong com- knowledge, this is the first time that catalytic hydrogen trans-
plexation between the tin chloride and the o-diamino group. fer has been used in the chemistry of polymethine dyes.
The presence of tin after several rounds of purifications includ-
Since the reaction between NO and diaminebenzene leads
ing orthogonal column chromatography (reverse and normal to the formation of a benzotriazole product, a representative
phase), trituration, gel filtration and dialysis was confirmed by dye 11 was synthesized (Scheme 2). The dye was prepared in a
ICP-MS analysis and NMR.
manner similar to DAP-LT1 via Suzuki coupling of IR783 and a
To avoid potential complexation with metals on the corresponding commercially available benzotriazole boronic
reduction step, we utilized a catalytic hydrogen transfer ester 10. Optical properties of the dyes are tabulated in
approach. In this mild and selective reduction method, Table 1.
Table 1 Spectral properties of the probe before and after reaction with NO donor in 10% of serum in PBS
Rate constant,
Binding constant
λabs/λfl
(nm)
τ
ε
K (min−1
)
to BSA (M−1
)
(ns)
(M−1 cm−1
)
Φfl
c
Probe
DAP-LT1
DAP-LT1 + NO
DAP-LT2
DAP-LT2 + NO
Entry 11
DAF-FM
—
92 400
See 11
n/d
760/780
767/782
762/788
767/788
778/795
558/515
488/515
1.08a
1.24a
0.92a
0.96a
1.25a
3.75b
3.72b
247 000
220 000d
240 000
212 000
214 000
n/d
0.037
0.28d
0.040
0.40d
0.367
0.005e
0.81e
0.24 0.03
—
0.33 0.09
No reaction
—
—
142 000
28 10031
n/d
DAF-FM + NO
1.82 0.53
n/d
a Lifetime, ex/em 740/790 nm. b Lifetime, ex/em 460/510 nm. c Quantum yield (QY) measured in DMSO, relative to ICG (QY = 0.22) in DMSO.32,33
d Estimated based on the change in absorption and fluorescence spectra. e Relative to QY of fluorescein.
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Fluorescence intensity of the probe in the presence of NO
donor
Having the starting materials and the products in hand, we
tested DAP-LT1 and DAP-LT2 in the presence of a known NO
donor, DEA NONOate (Scheme 2) in 10% serum/PBS at room
temperature. This compound is relatively stable under basic
conditions but decomposes to produce NO under neutral and
acidic conditions with the half-life of 16 min at room tempera-
ture and pH 7.4.34 The choice of serum was dictated by poten-
tial applications of the probe for clinically relevant
measurements of NO in biological fluids.35,36 The visible
probe DAF-FM with almost complete quenching was utilized
as a positive control.
Fig. 3 Kinetics of DAP-LT1 fluorescence increase after treatment with DEA
NONOate and DEA NONOate decomposition. The half-life of DEA NONOate is
16 min at 22–25 °C.
Both DAP-LT1 and DAP-LT2 showed reasonably good sensi-
tivity to NO, as judged by the fluorescence intensity measure-
ments but were not completely quenched with the QY in the
initial state at ∼2%. Within 15 min after addition of the NO
donor, fluorescence increased ∼5.3 times for DAP-LT1 without
the change of the absorption spectra (Fig. 2). Such behaviour
is typical of PET-type compounds37,38 and confirms the
suggested mechanism of fluorescence quenching.
DAP-LT2, having a methylated amine like in DAF-FM, was
even more sensitive with ∼9 times change of fluorescence
intensity also without the change of the absorption spectra
(Fig. S2, ESI†).
For a biological application it is important for a probe to
react fast to monitor the NO burst in real time. The reaction
rates of DAP-LTs with NO calculated from fluorescence inten-
sity data were similar between the studied DAP-LT probes but
lower than that of DAF-FM (Table 1, Fig. 3). Although the
overall reaction kinetics between the probes and NO are
complex because of the non-stationary conditions (change in
NO concentration with time, Fig. 3), the difference in reaction
rates can be, in part, explained by the steric effects induced by
serum proteins. The measured binding constant of DAP-LT1 to
bovine serum albumin (BSA), the major serum protein, was
found to be 92 400 M−1 (Fig. S4, ESI†), more than three times
higher than that of fluorescein (∼28 100 M−1).31 This compari-
son suggests that probes with lower affinity to plasma proteins,
such as more hydrophilic dyes (PEGylated or conjugated to
nanoparticles), will respond to NO faster.
Fluorescence lifetime of the probe in the presence of NO
donor
Out of the two synthesized probes, only DAP-LT1 showed
appreciable fluorescence lifetime sensitivity to NO growing
from 1.08 ns to 1.24 ns within 30 min (Table 1, Fig. 4A, the
corresponding fluorescence decays are shown in Fig. S3, ESI†).
This increase in the lifetime was in agreement with the life-
time of the dye 11 – the final product of the reaction between
DAP-LT1 and NO. This product of the reaction (entry DAP-LT1
+ NO) and benzotriazole 11 with a lifetime of 1.25 ns showed
almost identical fluorescence decays of the reaction (Fig. 4B).
Similar sensitivity was observed under physiological temp-
erature (37 °C). The compounds retain their fluorescence
lifetime difference although the absolute values of the life-
times were lower due to the higher rate of non-radiative de-
activation at higher temperature9 (decays at 37 °C are shown in
Fig. S4, ESI†).
Despite the larger fluorescence intensity sensitivity of
DAP-LT2 to NO, the compound showed weak lifetime response
with visually indistinguishable fluorescence decays before and
after the reactions (Table 1, Fig. S5, ESI†). Similarly, no
Fig. 2 DAP-LT1 reaction with DEA NONOate. A: absorption change. B: fluore-
scence intensity change (ex. 720 nm).
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(FRET), aggregate formation, and PET. In these cases, the
excited state energy of the fluorophore is deactivated resulting
in an initially low fluorescence intensity of the probe. Upon
removal of the quenching effects, the significant rise of the
fluorescence intensity and, in certain cases, the fluorescence
lifetime increase such as described throughout this work can
be observed.
The lack of direct correlation between strong enhancement
in fluorescence intensity and the change in the fluorescence
lifetime demonstrates the complexity of the photophysical pro-
cesses in the excited state. Understanding such behaviour
requires a larger set of examples and model compounds.
However, the number of publications reporting fluorescence
lifetime changes in activatable probes is still limited to a few
reports such as describing calcium probes,43 fluorescent pro-
teins46,47 and cyanine dyes.11,40 We are optimistic that more
research will be conducted to better understand the under-
lying principles of fluorescence lifetime probe design.
Conclusions
Fig. 4 A: fluorescence lifetimes of DAP-LT1 increase after reaction with DEA
NONOate at different time points. B: fluorescence decays of DAP-LT1 after reac-
tion with DEA NONOate and benzotriazole dye 11 (ex/em 740/790 nm, 10%
serum/PBS buffer).
We synthesized an activatable fluorescent probe for detecting
nitric oxide that is based on the fluorescence lifetime change.
The new probe DAP-LT1 is built on a heptamethine scaffold
with incorporated diaminobenzene functionality in the meso-
position. The probe is only partially quenched, but upon
reaction with a nitric oxide donor shows the increase of the
fluorescence lifetime from 1.08 ns to 1.24 ns. A control
benzotriazole derivative that corresponds to the product of the
reaction showed the lifetime of 1.25 ns confirming the
suggested PET mechanism of quenching. The probe could find
applications in monitoring nitric oxide in a biologically rele-
vant media such as blood serum and other biological fluids.
The observed fluorescence lifetime change is sufficient for NO
monitoring in vitro; however, such a conclusion for in vivo is
yet to be demonstrated. We envision that the probe perform-
ance could be further improved by the modification of the
skeleton of the cyanine molecule and/or by placing the
diamino functionality in a different location of the dye.
Fig. 5 Fluorescence lifetime of DAF-FM in the presence of DEA-NONOate in
10% serum/PBS buffer. Lifetime: ex/em: 460/520 nm.
fluorescence lifetime sensitivity was observed for DAF-FM
during the reaction (Fig. 5) despite the very strong change in
the fluorescence emission. The absence of fluorescence life-
Materials and methods
Materials
time sensitivity for DAF-FM can be explained by the almost Solvents, 10% Pd/C, tin(II) chloride, ammonium formate,
complete quenching of the original fluorophore which as methyl iodide, bovine serum albumin (BSA, grade agarose gel
mentioned violates the second criterion for the lifetime electrophoresis, 99% purity), 6-(4,4,5,5-tetramethyl-1,3,2-
sensitivity.
dioxaborolan-2-yl)-1H-benzo[d][1,2,3]triazole (Sigma-Aldrich),
Activatable probes are a broad class of probes with a non- 1× PBS buffer (Mediatech Inc), Pd(PPh3)4 (Strem Chemicals,
or minimally fluorescent initial state that are only “turned on” Inc.), dye IR783 (American Dye Source Inc.), sodium hydride
under certain conditions.39 These conditions might include (Alfa Aesar), bis-pinacolato diboron (Oakwood Products Inc.),
enzymatic activity,40 change in the pH41,42 or the presence of NMR solvents (Cambridge Isotope Lab), DEA NONOate
metals43 and metabolites such as reactive oxygen species44,45 (Cayman Chemical Company), fetal bovine serum heat in-
and nitric oxide (see ref. above). Several photochemical mecha- activated (Life Technologies) were used without purification,
nisms can be utilized in the activatable probe design, includ- and Millipore water was used throughout this work. All chemi-
ing homo- and hetero-fluorescence resonance energy transfer cals were used without further purification.
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Chemical analysis methods
lifetime changes shown in Fig. 4A the lowest measurable sensi-
tivity was 4 µM of NO. DAF-FM was pre-incubated in 10%
serum for 5 min prior to the addition of the NO donor to
ensure complete deacetylation of the probe by serum pro-
teases. The reactions were monitored by measuring the fluore-
scence intensity or fluorescence lifetime. Kinetics of DEA
NONOate decomposition was modelled according to eqn (2)
assuming a half-life of DEA NONoate of 16 min at 22–25 °C.51
1H and 13C NMR data were recorded on a 400 MHz Varian
spectrometer at ambient temperature in either chloroform-d or
methanol-d4 and referenced to tetramethylsilane (TMS) as an
internal standard unless otherwise stated. Chromatographic
purification was performed using C18 silica gel column.
LC-MS spectra were recorded on a Shimadzu 2010A (ESI, posi-
tive mode) equipped with an UV/Vis detector with up to
900 nm detection limit. Thin layer chromatography (TLC) was
performed using either alumina-backed plates coated with
0.20 mm silica gel Xtra SIL G/UV254 or 0.15 mm RP-18W/UV254
silica gel. ICP-MS for measurements of Sn were conducted
using Elan DRC II ICP-MS (PerkinElmer).
t=t1=2
NðtÞ ¼ N0ð1=2Þ
ð2Þ
where N0 is 100%, t is the reaction time, t1/2 = 16 min, all in
10% serum/PBS buffer.
Synthesis
Optical measurements
General procedure for synthesis of boronic esters (I). To a
solution of the corresponding aryl-bromide in oxygen-free
THF, Pd(PPh3)4 (0.06 mol%) was added under an argon atmos-
phere followed by the bis-pinacolato diboron (2.0 eq.) and
potassium acetate (2.5 eq.) The reaction mixture was left for
overnight reflux, cooled down, and filtered through a small
layer of silica gel using an ethyl acetate–hexane mixture as an
eluent. Solvents were evaporated under reduced pressure; the
residue was subjected to the normal phase column chromato-
graphy on silica gel using gradient ethyl acetate–hexane as
an eluent. Fractions containing the desired product were
collected, combined and solvent evaporated to afford a pure
product.
General procedure for Suzuki coupling of IR783 with
boronic esters (II). Dye IR783 was dissolved in oxygen-free
water purged with argon. Pd(PPh3)4 (0.06 mol%) was added to
the stirred solution of IR783 under an argon atmosphere fol-
lowed by the addition of a solution of an appropriate boronic
ester (2.0 eq.) in a small amount of DMF (water–DMF ratio 5 : 1
by vol.). The reaction mixture was sealed and left for overnight
reflux, analysed by TLC and/or LC-MS. The reaction mixture
was cooled down and solvents were evaporated under reduced
pressure. The residue was subjected to reverse phase column
chromatography using a water–methanol gradient as an
eluent. Fractions containing the desired product (by LS-MS)
were collected, combined, and methanol was evaporated under
reduced pressure. The remaining solution was lyophilized.
General procedure for reduction of the nitro-group with
General spectroscopy methods. UV/Vis spectra of samples
were recorded on a Beckman Coulter DU640 UV-visible spectro-
photometer. The molar absorptivities were obtained using
Beer’s law at 0.1–0.6 µM concentration of the dye. Steady state
fluorescence spectra and fluorescence lifetime were recorded
on a Nanolog spectrofluorometer (Horiba Jobin Yvon). Fluore-
scence quantum yield of the samples was measured using ICG
as a standard.32,33,48 The fluorescence lifetime of dyes was
determined using time-correlated single photon counting
(TCSPC) technique with excitation sources NanoLed-740 nm,
emission 790 nm for NIR dyes or NanoLed-460 nm, emission
520 nm for fluorescein as described previously.10 Temperature
was adjusted using a cuvette holder connected to a thermostat.
Measuring the binding constants to BSA. The details of the
method were described previously.49 Briefly, BSA was dissolved
in 1× PBS buffer to obtain a concentration of 0.08 mg mL−1
and kept as a stock solution. The DAP-LT1 concentration was
varied from 2.5 × 10−7 to 4.0 × 10−6 M. For binding measure-
ments, a solution of albumin (2 mL) was placed in a quartz
cuvette and titrated with the dye in microliter scale incre-
ments. After each addition, the solution was stirred and
allowed to equilibrate for a few minutes, and its emission
spectra of tryptophan residue were recorded (ex/em.
279/294–450 nm). The binding constant was obtained from
eqn (1).
ðF0 ꢀ FÞ=F ¼ K½dyeꢁ
ð1Þ
where F0 is the initial fluorescence, F is the fluorescence after SnCl2 (III). To a solution of the corresponding dye in metha-
addition of the dye, [dye] is dye concentration and K is a nol, SnCl2 (6.0 eq.) was added, and the reaction mixture was
binding constant.
left with stirring at 50 °C. The reaction was monitored by
Sensitivity of the probes to NO. The probe was predissolved LC-MS until the complete conversion of the starting material
in water and added to a quartz cuvette with 10% serum in PBS was confirmed. The reaction mixture was filtered through a
buffer. Serum solution was chosen to maintain conditions layer of Celite and the solvent was evaporated. A cascade of
close to in vivo. The NO donor DEA NONOate (10 µM) was purification techniques was used to remove SnCl2 and Sn-pro-
added to the solution of the probe in 10% serum at room ducts from the reaction mixture. Trituration of the dye with
temperature or at 37 °C in order to mimic the NO level found diethyl ether from methanol solution, dialysis, and column
in biological tissues.50 Direct measurement of the detection chromatography on reverse phase silica gel led to the single
limit was difficult due to a non-steady concentration of the NO compound which contained SnCl2 apparently complexed
donor in the solution. Based on the indirect estimate using with the diamino group. The presence of tin complex was
the rate of DEA NONOate decomposition and kinetics of the confirmed by broader 1H NMR signals and by ICP-MS analysis.
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General procedure for reduction of the nitro-group via cata-
Synthesis of intermediate dye (7). The dye 7 was obtained
lytic hydrogen transfer (IV). To a solution of the corres- according to the standard procedure (II) as a green powder.
ponding dye in methanol thoroughly purged with argon, 10% Purity >95% area LC-MS. 1H NMR (400 MHz, CD3OD) δ 7.91
Pd/C was added followed by the addition of ammonium (d, 1H), 7.32–7.27 (m, 3H), 7.25–7.23 (d, 4H), 7.18–7.16
formate (20 eq.) The reaction mixture was sealed and stirred at (m, 3H), 7.08–7.05 (t, 2H), 6.18–6.14 (d, 2H). 4.04–4.01 (t, 4H),
room temperature. After ca. 40 min, TLC indicated a complete 3.05 (s, 3H), 2.79–2.76 (t, 4H), 2.66–2.63 (t, 4H), 2.59 (s, 1H),
conversion of the starting nitro-derivative into the desired 1.95 (m, 2H), 1.83 (m, 8H), 1.19–1.15 (d, 12H). 13C NMR
product. The reaction mixture was filtered through a layer of (400 MHz, CD3OD) δ 171.79, 160.11, 147.41, 145.82, 142.23,
Celite, the solvent was evaporated and the residue was subjected 140.65, 137.82, 131.82, 131.05, 128.33, 127.24, 124.74, 124.49,
to the column chromatography on reverse phase silica gel using 121.88, 114.05, 110.48, 99.92, 50.39, 48.51, 43.31, 28.76, 26.71,
a water–methanol gradient as an eluent. Fractions containing 26.32, 25.77, 24.43, 24.39, 22.17, 21.08.
the desired product were collected, combined and lyophilized.
Synthesis of DAP-LT1 (8). DAP-LT1 was obtained according
Synthesis of 4-bromo-N-methyl-2-nitroaniline (2). To a solu- to the standard procedure (IV) as a pure green powder with
tion of 4-bromo-2-nitroaniline (500 mg, 2.29 mmol) in dry THF 55% yield. Purity >95% area LC-MS. 1H NMR (400 MHz,
(2.0 mL), sodium hydride (92 mg of 60% suspension in CD3OD) δ 8.30 (s, 1H), 7.51–7.47 (m, 2H), 7.25–7.20 (m, 4H),
mineral oil, 2.29 mmol) was added at 0 °C under an argon 7.14–7.12 (d, 2H), 7.07–7.03 (t, 2H), 6.81–6.79 (d, 1H), 6.51–6.50
atmosphere and stirred for 10 min. Methyl iodide (325 mg, (d, 1H), 6.34–6.31 (dd, 1H), 6.07–6.04 (d, 1H), 4.00–3.96 (t, 4H),
2.29 mmol) was added to the reaction mixture, and the temp- 2.79–2.76 (m, 5H), 2.58–2.56 (m, 5H), 1.91–1.88 (t, 2H), 1.81
erature of the reaction mixture was raised to ambient tempera- (m, 8H), 1.21–1.18 (d, 12H). 13C NMR (400 MHz, CD3OD)
ture and the mixture was stirred for two hours. TLC showed δ 171.46, 164.98, 153.86, 148.83, 142.38, 140.69, 139.02,
complete conversion of the starting material. The reaction 135.08, 133.89, 131.46, 128.92, 128.12, 123.88, 123.15, 121.72,
mixture was washed with 5% HCl(aq), extracted with ethyl 120.28, 117.48, 115.24, 109.95, 107.12, 105.14, 99.01, 50.47,
acetate twice, washed with brine, and dried over sodium sulfate. 48.46, 43.23, 33.85, 28.88, 26.70, 25.75, 24.29, 22.17, 21.24.
The solvents were evaporated under reduced pressure; the
Synthesis of DAP-LT2 (9). DAP-LT2 was obtained according
residue was subjected to column chromatography on silica gel to the standard procedure (IV) as a green powder with 50%
using mixtures of hexane–ethyl acetate as eluents affording yield. Purity >95% area LC-MS. 1H NMR (400 MHz, CD3OD)
530 mg (99%) of pure 4-bromo-N-methyl-2-nitroaniline as a δ 7.48–7.44 (d, 2H), 7.22–7.21 (d, 4H), 7.14–7.12 (d, 2H),
reddish solid. 1H NMR (400 MHz, CDCl3) δ 8.32, 8.32, 8.02, 7.54, 7.06–7.02 (t, 2H), 6.71–6.69 (d, 1H), 6.57 (d, 1H), 6.51–6.48 (dd,
7.53, 7.52, 7.51, 7.26, 6.77, 6.75, 3.03, 3.02. 13C NMR (400 MHz, 1H), 6.08–6.05 (d, 2H), 3.97 (m, 4H), 2.87 (s, 3H), 2.78–2.75
CDCl3) δ 145.23, 138.98, 128.89, 115.11, 110.03, 106.37, 29.85.
(t, 4H), 2.59–2.56 (m, 4H), 1.91–1.88 (t, 2H), 1.81 (m, 9H),
Synthesis of 2-nitro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan- 1.20–1.17 (d, 12H). 13C NMR (400 MHz, CD3OD) δ 171.46,
2-yl)aniline (4). The compound 4 was obtained according 161.07, 148.82, 142.37, 140.64, 132.84, 128.21, 125.42, 124.15,
to the standard procedure (I) as a colorless solid. 1H NMR 122.11, 121.81, 116.52, 110.17, 109.99, 99.01, 50.46, 48.46,
(400 MHz, CDCl3) δ 8.59 (s, 1H), 7.73–7.71 (d, 1H), 6.78–6.76 43.23, 30.01, 26.78, 26.72, 25.75, 24.35, 22.16, 21.29.
(d, 1H), 6.20 (br. s, 2H), 1.33 (s, 12H). 13C NMR (400 MHz,
Synthesis of benzotriazole dye (11). The dye 11 was obtained
CDCl3) δ 146.46, 141.06, 133.72, 132.14, 117.95, 83.98, 24.84.
from commercially available 6-(4,4,5,5-tetramethyl-1,3,2-dioxa-
Synthesis of N-methyl-2-nitro-4-(4,4,5,5-tetramethyl-1,3,2- borolan-2-yl)-1H-benzo[d][1,2,3]triazole 10 and IR783 accord-
dioxaborolan-2-yl)aniline (5). The compound 5 was obtained ing to the standard procedure (II) as a pure green powder with
according to the standard procedure (I) as a colorless solid. 57% (31 mg) yield.
1H NMR (400 MHz, CDCl3) δ 8.64 (s, 1H), 8.19 (br. s, 1H),
7.83–7.81 (d, 1H), 6.82–6.79 (d, 1H), 3.05–3.03 (d, 3H), 1.33
Acknowledgements
(s, 12H). 13C NMR (400 MHz, CDCl3) δ 147.87, 141.72, 134.29,
131.96, 112.54, 83.90, 29.74, 25.03, 24.85.
We gratefully acknowledge the financial support from the
National Cancer Institute of the National Institutes of Health
(R21CA149814), the National Heart, Lung and Blood Institute
Synthesis of intermediate dye (6). The dye 6 was obtained
according to the standard procedure (II) as a green powder.
1
Purity 95% area LC-MS. H NMR (400 MHz, CD3OD) δ 7.81 (s,
as
a
Program
of
Excellence
in
Nanotechnology
1H), 7.61–7.52 (m, 3H), 7.47–7.45 (m, 1H), 7.33–7.30 (m, 2H),
7.25–7.22 (m, 3H), 7.17–7.14 (m, 3H), 7.08–7.04 (t, 2H),
6.16–6.13 (d, 2H), 4.03–4.00 (t, 4H), 2.79–2.76 (t, 4H), 2.63
(m, 3H), 2.59 (s, 2H), 1.93 (br s, 2H), 1.83 (m, 8H), 1.20–1.16
(d, 12H). 13C NMR (400 MHz, CD3OD) δ 171.76, 147.43, 145.88,
142.23, 140.65, 136.98, 134.36, 134.22, 133.11, 132.32, 131.68,
131.58, 130.50, 129.88, 129.83, 129.45, 128.59, 128.47, 128.33,
126.49, 125.85, 125.53, 124.51, 121.89, 121.77, 119.14, 113.66,
110.47, 99.89, 50.40, 48.52, 43.33, 33.95, 26.70, 26.33, 25.78,
25.07, 24.36, 22.17, 21.08.
(HHSN268201000046C). We also thank the Washington Uni-
versity Optical Spectroscopy Core facility (NIH 1S10RR031621-
01) and its manager Steven Wang for help with spectroscopy
measurements.
Notes and references
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